WO2023186063A1 - 抗pvrig抗体、其药物组合物及用途 - Google Patents

抗pvrig抗体、其药物组合物及用途 Download PDF

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Publication number
WO2023186063A1
WO2023186063A1 PCT/CN2023/085357 CN2023085357W WO2023186063A1 WO 2023186063 A1 WO2023186063 A1 WO 2023186063A1 CN 2023085357 W CN2023085357 W CN 2023085357W WO 2023186063 A1 WO2023186063 A1 WO 2023186063A1
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seq
amino acid
acid sequence
antibody
chain variable
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French (fr)
Inventor
翟天航
戴爽
黄威峰
曾竣玮
布考斯基约翰
舒茨凯文
黑默莱恩梅根
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Biotheus Inc
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Biotheus Inc
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Priority to EP23778428.5A priority Critical patent/EP4506360A4/en
Priority to JP2024558263A priority patent/JP2025511288A/ja
Priority to US18/852,907 priority patent/US20250223353A1/en
Priority to CA3256036A priority patent/CA3256036A1/en
Priority to KR1020247036668A priority patent/KR20240168451A/ko
Priority to AU2023243878A priority patent/AU2023243878A1/en
Priority to CN202380031274.4A priority patent/CN118946588A/zh
Publication of WO2023186063A1 publication Critical patent/WO2023186063A1/zh
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IG], 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
    • 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
    • A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
    • A61K39/39558—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against tumor tissues, cells, antigens
    • 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 [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IG], 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
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IG], 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
    • C07K16/2818—Immunoglobulins [IG], 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 CD28 or CD152
    • 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
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • 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
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A61K2039/507—Comprising a combination of two or more separate antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
    • 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
    • C07K2317/55—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
    • 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
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C07K2317/732—Antibody-dependent cellular cytotoxicity [ADCC]
    • 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
    • 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
    • C07K2317/94—Stability, e.g. half-life, pH, temperature or enzyme-resistance

Definitions

  • the invention belongs to the field of biomedicine and relates to an anti-PVRIG antibody, its pharmaceutical composition and uses.
  • Poliovirus receptor related immunoglobulin domain-containing protein is a member of the PVR family. Its extracellular region has an IgV domain and its intracellular region contains an ITIM ( Immunoreceptor tyrosine-based inhibitory motif) domain is a new type of immunosuppressive receptor expressed on natural killer (NK) cells and T cells.
  • ITIM Immunoreceptor tyrosine-based inhibitory motif
  • PVRL2 Poliovirus receptor-related 2, CD112, Nectin-2
  • PVRL2 is an adhesion molecule involved in cell-cell junctions and overexpressed in a variety of cancers.
  • PVRL2 is currently identified as the only functional ligand of PVRIG, which can bind to PVRIG with high affinity and inhibit immune activation of T and NK cells.
  • PVRL2 is also a ligand for the co-activating receptor DNAX accessory molecule 1 (DNAM-1, CD226) and binds weakly to another inhibitory receptor, the T cell immune receptor with Ig and ITIM domains (TIGIT). Therefore, similar to the immunosuppressive mechanism of TIGIT, a member of the same family, PVRIG can also exert an immunosuppressive effect by competitively binding to its ligand PVRL2 with CD226 and blocking the activation signal transmitted by CD226.
  • DNAX accessory molecule 1 DNAX accessory molecule 1
  • TAGIT T cell immune receptor with Ig and ITIM domains
  • PVRIG or its ligand PVRL2 is highly expressed in lung cancer, renal cancer, endometrial cancer, breast cancer, skin cancer and their tumor microenvironment, and PVRIG is also highly expressed on NK cells of prostate cancer patients (Whelan S, et al. Cancer Immunol Res. 2019;7(2):257-268.).
  • PVRIG co-expressed with exhausted CD8+ T cell markers TIGIT and PD-1 molecules, suggesting that the expression of PVRIG has a certain tumor specificity and may be related to the activation/exhaustion status of TIL in the tumor (Whelan S, et al. Cancer Immunol Res. 2019;7(2):257-268.).
  • WO2021180205A1 reports that anti-PVRIG antibodies can be used for the prevention and treatment of lung cancer, breast cancer, ovarian cancer, kidney cancer, gastric cancer, endometrial cancer, head and neck cancer, etc.
  • PVRIG antibodies can effectively stimulate anti-tumor immune responses in certain cancers, and have been found to have a certain synergistic effect with PD-1/L1 and TIGIT antibodies, which can enhance the function of T/NK cells and improve anti-tumor responses. , slow down the growth of tumors.
  • Using PVRIG as a therapeutic target is expected to improve the therapeutic effect of existing antibody drugs (such as anti-TIGIT and anti-PD-1/L1 antibodies), presenting advantages that are different from other drug targets and clinical combination therapies.
  • COM701 developed by Compugen is the world's first clinically approved PVRIG antibody.
  • the inventors obtained new anti-PVRIG antibodies through in-depth research and creative efforts.
  • the inventors surprisingly found that the anti-PVRIG antibody of the present invention has good affinity and biological activity, and has anti-tumor potential.
  • the following invention is thereby provided:
  • One aspect of the invention relates to an anti-PVRIG antibody or antigen-binding fragment thereof, wherein,
  • the antibody comprises a heavy chain variable region comprising HCDR1 to HCDR3 and a light chain variable region comprising LCDR1 to LCDR3, wherein:
  • amino acid sequence of HCDR1 is shown in SEQ ID NO:9
  • amino acid sequence of HCDR2 is shown in SEQ ID NO: 10
  • amino acid sequence of HCDR3 are shown in SEQ ID NO:11
  • amino acid sequence of HCDR1 is shown in SEQ ID NO:15
  • amino acid sequence of HCDR2 is shown in SEQ ID NO:16
  • amino acid sequence of HCDR3 is shown in SEQ ID NO:17
  • amino acid sequence of HCDR1 is set forth in SEQ ID NO:21
  • amino acid sequence of HCDR2 is set forth in SEQ ID NO:22
  • amino acid sequence of HCDR3 is set forth in SEQ ID NO:23, or
  • amino acid sequence of HCDR1 is shown in SEQ ID NO:27
  • amino acid sequence of HCDR2 is shown in SEQ ID NO:28
  • amino acid sequence of HCDR3 is shown in SEQ ID NO:29;
  • the amino acid sequence of LCDR1 is shown in SEQ ID NO:12
  • the amino acid sequence of LCDR1 is shown in SEQ ID NO:13
  • the amino acid sequence of LCDR1 is shown in SEQ ID NO:14
  • the amino acid sequence of LCDR1 is shown in SEQ ID NO:18, the amino acid sequence of LCDR1 is shown in SEQ ID NO:19 and the amino acid sequence of LCDR1 is shown in SEQ ID NO:20,
  • the amino acid sequence of LCDR1 is shown in SEQ ID NO:24, the amino acid sequence of LCDR1 is shown in SEQ ID NO:25, and the amino acid sequence of LCDR1 is shown in SEQ ID NO:26, or
  • the amino acid sequence of LCDR1 is shown in SEQ ID NO:30, the amino acid sequence of LCDR1 is shown in SEQ ID NO:31 and the amino acid sequence of LCDR1 is shown in SEQ ID NO:32.
  • the anti-PVRIG antibody or antigen-binding fragment thereof wherein,
  • the amino acid sequence of HCDR1 is shown in SEQ ID NO:9
  • the amino acid sequence of HCDR2 is shown in SEQ ID NO:10
  • the amino acid sequence of HCDR3 is shown in SEQ ID NO:11
  • the amino acid sequence of LCDR1 is shown in SEQ ID NO:12
  • the amino acid sequence of LCDR2 is shown in SEQ ID NO:13
  • the amino acid sequence of LCDR3 is shown in SEQ ID NO:14;
  • the amino acid sequence of HCDR1 is shown in SEQ ID NO:15
  • the amino acid sequence of HCDR2 is shown in SEQ ID NO:16
  • the amino acid sequence of HCDR3 is shown in SEQ ID NO:17
  • the amino acid sequence of LCDR1 is shown in SEQ ID NO:18
  • the amino acid sequence of LCDR2 is shown in SEQ ID NO:19
  • the amino acid sequence of LCDR3 is shown in SEQ ID NO:20;
  • the amino acid sequence of HCDR1 is shown in SEQ ID NO:21
  • the amino acid sequence of HCDR2 is shown in SEQ ID NO:22
  • the amino acid sequence of HCDR3 is shown in SEQ ID NO:23
  • the amino acid sequence of LCDR1 is shown in SEQ ID NO:24 It is shown that the amino acid sequence of LCDR2 is shown in SEQ ID NO:25 and the amino acid sequence of LCDR3 is shown in SEQ ID NO:26;
  • the amino acid sequence of HCDR1 is shown in SEQ ID NO:27
  • the amino acid sequence of HCDR2 is shown in SEQ ID NO:28
  • the amino acid sequence of HCDR3 is shown in SEQ ID NO:29
  • the amino acid sequence of LCDR1 is shown in SEQ ID NO:30
  • the amino acid sequence of LCDR2 is shown in SEQ ID NO:31
  • the amino acid sequence of LCDR3 is shown in SEQ ID NO:32.
  • variable regions of the light chain and heavy chain determine the binding of antigens; the variable region of each chain contains three hypervariable regions, called complementarity-determining regions (CDRs) (the CDRs of the heavy chain (H) include HCDR1, HCDR2, and HCDR3 , the CDR of the light chain (L) includes LCDR1, LCDR2, and LCDR3.
  • CDRs complementarity-determining regions
  • H the CDRs of the heavy chain
  • L includes LCDR1, LCDR2, and LCDR3.
  • HCDR1-HCDR3 and LCDR1-LCDR3 refer to U.S. Patent Publication US20210380669A1 or the document Lu. et al (Deamidation and isomerization liability analysis of 131 clinical-stage antibodies, MABS, 2019, VOL.11, NO.1, 45–57, DOI: 10.1080/19420862.2018.1548233) to be defined or numbered.
  • the anti-PVRIG antibody or antigen-binding fragment thereof wherein the amino acid sequence of the heavy chain variable region is selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO :5 and SEQ ID NO:7;
  • amino acid sequence of the light chain variable region is selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6 and SEQ ID NO:8.
  • the anti-PVRIG antibody or antigen-binding fragment thereof wherein,
  • amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:1, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:2;
  • amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:3, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:4;
  • amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:5, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:6;
  • amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO:7
  • amino acid sequence of the light chain variable region is set forth in SEQ ID NO:8.
  • the anti-PVRIG antibody or antigen-binding fragment thereof wherein the heavy chain constant region of the antibody is Ig gamma-1 chain C region or Ig gamma-4 chain C region; the light chain The constant region is the Ig kappa chain C region;
  • amino acid sequence of the heavy chain constant region of the antibody is as shown in SEQ ID NO: 33, SEQ ID NO: 34 or SEQ ID NO: 35, and the amino acid sequence of the light chain constant region of the antibody is as shown in SEQ ID NO: 36 shown.
  • the anti-PVRIG antibody or antigen-binding fragment thereof wherein, according to the EU numbering system, the heavy chain constant region of the antibody contains L234A mutation and L235A mutation (referred to as LALA).
  • LALA L234A mutation and L235A mutation
  • the letters before the site represent the amino acid before mutation
  • the letters after the site represent the amino acid after mutation.
  • the anti-PVRIG antibody or antigen-binding fragment thereof wherein the anti-PVRIG antibody or antigen-binding fragment thereof is selected from Fab, Fab', F(ab')2, Fd, Fv , dAb, complementarity determining region fragment, single chain antibody, humanized antibody, chimeric antibody or diabody.
  • the anti-TIGIT antibody or antigen-binding fragment thereof wherein,
  • the antibody includes non-CDR regions, and the non-CDR regions are from a species other than murine, such as from a human antibody.
  • the anti-PVRIG antibody or antigen-binding fragment thereof, wherein the EC 50 of the antibody binding to human PVRIG protein overexpressed on the surface of CHO cells is less than 3 nM, less than 2.9 nM, or less than 2.8 nM; preferably, the EC 50 is measured by flow cytometry detection.
  • Another aspect of the invention relates to an isolated nucleic acid molecule encoding an anti-TIGIT antibody or antigen-binding fragment thereof according to any one of the invention.
  • a further aspect of the invention relates to a recombinant vector comprising an isolated nucleic acid molecule of the invention.
  • Yet another aspect of the invention relates to a host cell comprising an isolated nucleic acid molecule of the invention, or a recombinant vector of the invention.
  • Another aspect of the present invention relates to a conjugate, which includes an antibody and a coupling part, wherein the antibody is an anti-PVRIG antibody or an antigen-binding fragment thereof according to any one of the present invention, and the coupling part is a detectable label; preferably, the coupling moiety is a radioactive isotope, a fluorescent substance, a colored substance or an enzyme.
  • kits which includes the anti-PVRIG antibody or antigen-binding fragment thereof according to any one of the present invention, or the conjugate of the present invention;
  • the kit further includes a second antibody that specifically recognizes the antibody; optionally, the third Secondary antibodies also include detectable labels such as radioisotopes, fluorescent substances, colored substances, or enzymes.
  • detectable labels such as radioisotopes, fluorescent substances, colored substances, or enzymes.
  • Another aspect of the present invention relates to a pharmaceutical composition, which contains the anti-PVRIG antibody or antigen-binding fragment thereof or the conjugate of the present invention according to any one of the present invention; optionally, the pharmaceutical composition further Includes one or more pharmaceutically acceptable excipients.
  • the pharmaceutical composition further comprises one or more anti-TIGIT antibodies
  • the pharmaceutical composition further comprises one or more anti-PD-1 antibodies or one or more anti-PD-L1 antibodies.
  • the pharmaceutical composition wherein,
  • the molar ratio of anti-PVRIG antibody or its antigen-binding fragment to anti-TIGIT antibody is (1:5)-(5:1), such as 1:5, 1:4, 1:3, 1:2, 1:1, 2 :1, 3:1, 4:1 or 5:1;
  • the molar ratio of anti-PVRIG antibody or antigen-binding fragment thereof to anti-PD-1 antibody or anti-PD-L1 antibody is (1:5)-(5:1), such as 1:5, 1:4, 1:3, 1 :2, 1:1, 2:1, 3:1, 4:1 or 5:1.
  • the pharmaceutical composition wherein the anti-TIGIT antibody includes a heavy chain variable region and a light chain variable region, and the heavy chain variable region includes HCDR1 to HCDR3, the light chain variable region comprising LCDR1 to LCDR3, wherein:
  • the amino acid sequence of HCDR1 is shown in SEQ ID NO:42
  • the amino acid sequence of HCDR2 is shown in SEQ ID NO:43
  • the amino acid sequence of HCDR3 is shown in SEQ ID NO:44
  • the amino acid sequence of LCDR1 is shown in SEQ ID NO:45
  • the amino acid sequence of LCDR2 is shown in SEQ ID NO:46
  • the amino acid sequence of LCDR3 is shown in SEQ ID NO:47;
  • amino acid sequence of the heavy chain variable region of the anti-TIGIT antibody is shown in SEQ ID NO:40, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:41.
  • the pharmaceutical composition wherein the anti-PD-1 antibody includes a heavy chain variable region and a light chain variable region, and the heavy chain variable region The region includes HCDR1 to HCDR3, and the light chain variable region includes LCDR1 to LCDR3, wherein:
  • the amino acid sequence of HCDR1 is shown in SEQ ID NO:50
  • the amino acid sequence of HCDR2 is shown in SEQ ID NO:51
  • the amino acid sequence of HCDR3 is shown in SEQ ID NO:52
  • the amino acid sequence of LCDR1 is shown in SEQ ID NO:53
  • the amino acid sequence of LCDR2 is shown in SEQ ID NO:54
  • the amino acid sequence of LCDR3 is shown as Shown in SEQ ID NO:55;
  • amino acid sequence of the heavy chain variable region of the anti-PD-1 antibody is shown in SEQ ID NO: 48, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 49.
  • the pharmaceutical composition wherein the unit dose of the pharmaceutical composition, calculated based on the mass of the anti-PVRIG antibody or antigen-binding fragment thereof, is 100 mg- 1000mg, 200mg-800mg, 200mg-500mg, 300mg-600mg, 400mg-500mg or 450mg.
  • the active ingredient of the pharmaceutical composition consists of an anti-PVRIG antibody or an antigen-binding fragment thereof.
  • the active ingredient of the pharmaceutical composition consists of an anti-PVRIG antibody or an antigen-binding fragment thereof and an anti-TIGIT antibody.
  • the active ingredient of the pharmaceutical composition consists of an anti-PVRIG antibody or an antigen-binding fragment thereof, an anti-TIGIT antibody and an anti-PD-1 antibody.
  • Yet another aspect of the invention relates to a combination product comprising a first product and a second product individually packaged, wherein,
  • the first product includes the anti-PVRIG antibody or antigen-binding fragment thereof according to any one of the present invention or the conjugate of the present invention;
  • the second product includes at least one anti-TIGIT antibody
  • the combination product further includes a third product, the third product comprising at least one anti-PD-1 antibody or at least one anti-PD-L1 antibody;
  • the first product, the second product and the third product also independently contain one or more pharmaceutically acceptable excipients;
  • the combination product also contains product instructions.
  • the molar ratio of anti-PVRIG antibody or its antigen-binding fragment to anti-TIGIT antibody is (1:5)-(5:1), such as 1:5, 1:4, 1:3, 1:2, 1:1, 2 :1, 3:1, 4:1 or 5:1;
  • the molar ratio of anti-PVRIG antibody or antigen-binding fragment thereof to anti-PD-1 antibody or anti-PD-L1 antibody is (1:5)-(5:1), such as 1:5, 1:4, 1:3, 1 :2, 1:1, 2:1, 3:1, 4:1 or 5:1.
  • the combination product wherein the anti-TIGIT antibody includes a heavy chain variable region and a light chain variable region, and the heavy chain variable region includes HCDR1 to HCDR3,
  • the light chain variable region includes LCDR1 to LCDR3, wherein:
  • the amino acid sequence of HCDR1 is shown in SEQ ID NO:42
  • the amino acid sequence of HCDR2 is shown in SEQ ID NO:43
  • the amino acid sequence of HCDR3 is shown in SEQ ID NO:44
  • the amino acid sequence of LCDR1 is shown in SEQ ID NO:45
  • the amino acid sequence of LCDR2 is shown in SEQ ID NO:46
  • the amino acid sequence of LCDR3 is shown in SEQ ID NO:47;
  • amino acid sequence of the heavy chain variable region of the anti-TIGIT antibody is shown in SEQ ID NO:40, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:41.
  • the combination product wherein the anti-PD-1 antibody includes a heavy chain variable region and a light chain variable region, the heavy chain variable region includes HCDR1 to HCDR3, the light chain variable region includes LCDR1 to LCDR3, wherein:
  • the amino acid sequence of HCDR1 is shown in SEQ ID NO:50
  • the amino acid sequence of HCDR2 is shown in SEQ ID NO:51
  • the amino acid sequence of HCDR3 is shown in SEQ ID NO:52
  • the amino acid sequence of LCDR1 is shown in SEQ ID NO:53
  • the amino acid sequence of LCDR2 is shown in SEQ ID NO:54
  • the amino acid sequence of LCDR3 is shown in SEQ ID NO:55;
  • amino acid sequence of the heavy chain variable region of the anti-PD-1 antibody is shown in SEQ ID NO: 48, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 49.
  • Another aspect of the present invention relates to the use of the antibody or antigen-binding fragment thereof, the conjugate of the present invention, or the pharmaceutical composition of any one of the present invention in the preparation of drugs for treating or preventing tumors. the use of;
  • the tumor is selected from colon cancer, melanoma, lung cancer, kidney cancer, endometrial cancer, breast cancer, skin cancer, ovarian cancer, gastric cancer, head and neck cancer, liver cancer, brain tumor, urothelial cancer, bone tumor , one or variety;
  • the lung cancer is non-small cell lung cancer or small cell lung cancer.
  • the tumor is selected from colon cancer, melanoma, lung cancer, kidney cancer, endometrial cancer, breast cancer, skin cancer, ovarian cancer, gastric cancer, head and neck cancer, liver cancer, brain tumor, urothelial cancer, bone tumor , cholangiocarcinoma, rectum
  • the lung cancer is non-small cell lung cancer or small cell lung cancer.
  • Another aspect of the present invention relates to a method for treating or preventing tumors, which includes administering an effective amount of the antibody or antigen-binding fragment thereof or the conjugate of the present invention to a subject in need. Or the steps of the pharmaceutical composition according to any one of the present invention;
  • the tumor is selected from colon cancer, melanoma, lung cancer, kidney cancer, endometrial cancer, breast cancer, skin cancer, ovarian cancer, gastric cancer, head and neck cancer, liver cancer, brain tumor, urothelial cancer, bone tumor , one or variety;
  • the lung cancer is non-small cell lung cancer or small cell lung cancer.
  • the single administration dose of the anti-PVRIG antibody is 0.1-100 mg per kilogram of body weight, preferably 1-10 mg (such as 1 mg, 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg or 10 mg); alternatively, the anti-PVRIG antibody A single administration dose is 10-1000 mg per subject (e.g., about 100 mg, about 150 mg, about 200 mg, about 250 mg, about 300 mg, about 350 mg, about 400 mg, about 450 mg, about 500 mg, about 600 mg, about 700 mg, about 800mg, about 900mg or about 1000mg), preferably 50-500mg, 100-400mg, 150-300mg, 150-250mg or 200mg;
  • it is administered every 3 days, 4 days, 5 days, 6 days, 10 days, 1 week, 2 weeks or 3 weeks;
  • the administration method is intravenous drip or intravenous injection.
  • the anti-PVRIG antibody is administered in a 2-week (14-day) or 3-week (21-day) cycle, and the anti-PVRIG antibody is preferably administered intravenously on the first day (D1) of each cycle.
  • the anti-PVRIG antibody is administered once every two weeks (q2w) or once every three weeks (q3w).
  • PVRIG when referring to the amino acid sequence of PVRIG (NCBI GenBank ID: NP_076975.2), it includes the full length or extracellular fragment of the PVRIG protein; also includes fusion proteins of PVRIG, For example, a fragment fused to the Fc protein fragment (mFc or hFc) of mouse or human IgG.
  • fusion proteins of PVRIG For example, a fragment fused to the Fc protein fragment (mFc or hFc) of mouse or human IgG.
  • mutations or variations including but not limited to substitutions, deletions and/or additions
  • PVRIG protein or “PVRIG” shall include all such sequences, including the sequences shown and natural or artificial variants thereof.
  • a sequence fragment of a PVRIG protein when describing a sequence fragment of a PVRIG protein, it includes not only the sequence fragment, but also the corresponding sequence fragment in its natural or artificial variant.
  • TIGIT when referring to the amino acid sequence of TIGIT (NCBI GenBank ID: NP_776160.2), it includes the full length or functional fragments of the TIGIT protein; also includes fusion proteins of TIGIT, such as with mouse or human IgG Fc protein fragment (mFc or hFc) fusion fragment.
  • mFc or hFc mouse or human IgG Fc protein fragment
  • a sequence fragment of a TIGIT protein when describing a sequence fragment of a TIGIT protein, it includes not only the sequence fragment, but also the corresponding sequence fragment in its natural or artificial variant.
  • EC50 refers to the concentration for 50% of maximal effect, the concentration that causes 50% of the maximal effect.
  • antibody refers to an immunoglobulin molecule typically composed of two pairs of polypeptide chains, each pair having a "light” (L) chain and a “heavy” (H) chain.
  • Antibody light chains can be classified into kappa and lambda light chains.
  • Heavy chains can be classified as mu, delta, gamma, alpha, or epsilon, and define the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively.
  • the variable and constant regions are connected by a "J" region of approximately 12 or more amino acids, and the heavy chain also contains a "D" region of approximately 3 or more amino acids.
  • Each heavy chain consists of a heavy chain variable region ( VH ) and a heavy chain constant region ( CH ).
  • the heavy chain constant region consists of 3 domains ( CH1 , CH2 and CH3 ).
  • Each light chain consists of a light chain variable region (V L ) and a light chain constant region ( CL ).
  • the light chain constant region consists of one domain, CL .
  • the constant region of an antibody may mediate binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (eg, effector cells) and the first component (Clq) of the classical complement system.
  • the VH and VL regions can also be subdivided into regions of high variability called complementarity determining regions (CDRs), interspersed with more conservative regions called framework regions (FRs).
  • CDRs complementarity determining regions
  • FRs framework regions
  • Each VH and VL consists of 3 CDRs and 4 FRs arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • the variable regions ( VH and VL ) of each heavy chain/light chain pair respectively form the antigen-binding site.
  • the assignment of amino acids to regions or domains follows the Kabat Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda MD (1987and 1991)), or Chothia & Lesk J. Mol. Biol. 1987;196:901-917; Chothia et al.
  • IMGT/3Dstructure-DB and IMGT/DomainGapAlign a database and a tool for immunoglobulins or antibodies, T cell receptors, MHC, IgSF and MhcSF.” Nucleic acids research 2009;38(suppl_1):D301-D307. definition.
  • antibody is not limited to any particular method of producing the antibody. For example, this includes, inter alia, recombinant antibodies, monoclonal antibodies and polyclonal antibodies.
  • the antibodies may be of different isotypes, for example, IgG (eg, IgGl, IgG2, IgG3 or IgG4 subtypes), IgA1, IgA2, IgD, IgE or IgM antibodies.
  • IgG eg, IgGl, IgG2, IgG3 or IgG4 subtypes
  • IgA1, IgA2, IgD, IgE or IgM antibodies may be of different isotypes, for example, IgG (eg, IgGl, IgG2, IgG3 or IgG4 subtypes), IgA1, IgA2, IgD, IgE or IgM antibodies.
  • the term "antigen-binding fragment" of an antibody refers to a polypeptide comprising a fragment of a full-length antibody that retains the ability to specifically bind to the same antigen that the full-length antibody binds, and/or competes with the full-length antibody Specific binding to an antigen, which is also called an "antigen-binding moiety.”
  • an antigen-binding moiety which is also called an "antigen-binding moiety.
  • the antigen-binding fragments include Fab, Fab', F(ab') 2 , Fd, Fv, dAb, and complementarity determining regions (CDRs) Fragments, single chain antibodies (eg, scFv), chimeric antibodies, diabodies, and polypeptides containing at least a portion of an antibody sufficient to confer specific antigen-binding ability to the polypeptide.
  • CDRs complementarity determining regions
  • the term “Fd fragment” means an antibody fragment consisting of the VH and CH1 domains;
  • the term “Fv fragment” means a fragment consisting of the VL and VH domains of a single arm of an antibody Antibody fragments;
  • the term “dAb fragment” means an antibody fragment consisting of a VH domain (Ward et al., Nature 341:544-546 (1989));
  • the term “Fab fragment” means an antibody fragment consisting of VL , VH , C Antibody fragment consisting of L and CH 1 domains;
  • the term “F(ab') 2 fragment” means an antibody fragment containing two Fab fragments connected by a disulfide bridge on the hinge region.
  • the antigen-binding fragment of the antibody is a single chain antibody (e.g., scFv) in which the V L and V H domains are paired to form a monovalent molecule by a linker that enables production of a single polypeptide chain (see, e.g., Bird et al., Science 242:423-426 (1988) and Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988)).
  • scFv molecules may have the general structure: NH2 - VL -linker- VH -COOH or NH2 - VH -linker- VL -COOH.
  • Suitable prior art linkers consist of repeated GGGGS amino acid sequences or variants thereof.
  • a linker having the amino acid sequence (GGGGS) 4 can be used, but variants thereof can also be used (Holliger et al. (1993), Proc. Natl. Acad. Sci. USA 90:6444-6448).
  • Other linkers useful in the present invention are provided by Alfthan et al. (1995), Protein Eng. 8:725-731, Choi et al. (2001), Eur. J. Immunol. 31:94-106, Hu et al. (1996), Cancer Res. 56:3055-3061, Kipriyanov et al. (1999), J. Mol. Biol. 293:41-56 and Roovers et al. (2001), Cancer Immunol.
  • the antigen-binding fragment of an antibody is a diabody, i.e., a diabody in which the V H and V L domains are expressed on a single polypeptide chain, but a linker that is too short is used to allow the use of two linkers on the same chain. Two domains pair together, thereby forcing the domain to pair with the complementary domain of the other chain and creating two antigen-binding sites (see, e.g., Holliger P. et al., Proc. Natl. Acad. Sci. USA 90:6444 -6448(1993), and Poljak RJ et al., Structure 2:1121-1123(1994)).
  • the antigen-binding fragment of an antibody is a "bispecific antibody", which refers to a conjugate formed by a first antibody (fragment) and a second antibody (fragment) or antibody analog through a coupling arm.
  • Methods of linkage include, but are not limited to, chemical reactions, gene fusion, and enzymatic processes.
  • Antigen-binding fragments of antibodies can be "multispecific antibodies” including, for example: trispecific antibodies and tetraspecific antibodies, the former being antibodies with three different antigen-binding specificities, and the latter being antibodies with four different antigen-binding specificities. of antibodies.
  • ankyrin repeat proteins linked to IgG antibodies, scFv-Fc antibody fragments or combinations thereof, such as CN104341529A.
  • Anti-IL-17a fynomer is combined with anti-IL-6R antibody, such as WO2015141862A1.
  • a given antibody (such as the monoclonal antibodies ADI-56127, ADI-56204, ADI-56249 provided by the invention) can be obtained using conventional techniques known to those skilled in the art (for example, recombinant DNA technology or enzymatic or chemical fragmentation methods). and ADI-56145) are obtained and screened for specificity in the same manner as for intact antibodies.
  • the terms “monoclonal antibody” and “monoclonal antibody” refer to an antibody or a fragment of an antibody from a group of highly homologous antibody molecules, that is, except for natural mutations that may occur spontaneously. A group of identical antibody molecules. Monoclonal antibodies are highly specific for a single epitope on the antigen. Polyclonal antibodies are relative to monoclonal antibodies, which usually contain at least two or more different antibodies, and these different antibodies usually recognize different epitopes on the antigen. Monoclonal antibodies can usually be obtained using hybridoma technology first reported by Kohler et al. ( G, Milstein C. Continuous cultures of fused cells secreting antibody of predefined specificity [J]. nature, 1975; 256 (5517): 495), but can also be obtained using recombinant DNA technology (eg, see US Patent 4,816,567).
  • humanized antibody refers to a product in which all or part of the CDR regions of a human immunoglobulin (recipient antibody) are replaced by the CDR regions of a non-human antibody (donor antibody).
  • Antibodies or antibody fragments wherein the donor antibody may be a non-human (eg, mouse, rat or rabbit) antibody with desired specificity, affinity or reactivity.
  • some amino acid residues in the framework region (FR) of the recipient antibody can also be replaced by amino acid residues of the corresponding non-human antibody, or by amino acid residues of other antibodies, for further improvement or optimization.
  • Antibody performance is provided.
  • the term “isolated” or “isolated” means obtained from the natural state by artificial means. If an "isolated" substance or ingredient occurs in nature, it may be that the natural environment in which it is located has changed, or that the substance has been separated from its natural environment, or both. For example, a certain unisolated polynucleotide or polypeptide naturally exists in a living animal, and the high purity of the same polynucleotide or polypeptide isolated from this natural state is called isolation. of.
  • isolation a certain unisolated polynucleotide or polypeptide naturally exists in a living animal, and the high purity of the same polynucleotide or polypeptide isolated from this natural state is called isolation. of.
  • the term “isolated” or “isolated” does not exclude the admixture of artificial or synthetic substances, nor does it exclude the presence of other impure substances that do not affect the activity of the substance.
  • the term "vector” refers to a nucleic acid delivery vehicle into which a polynucleotide can be inserted.
  • the vector can express the protein encoded by the inserted polynucleotide, the vector is called an expression vector.
  • the vector can be introduced into the host cell through transformation, transduction or transfection, so that the genetic material elements it carries can be expressed in the host cell.
  • Vectors are well known to those skilled in the art, including but not limited to: plasmids; phagemids; cosmids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) or P1-derived artificial chromosomes (PAC) ; Phages such as lambda phage or M13 phage and animal viruses, etc.
  • Animal viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, papillomaviruses, Polyoma vacuolating viruses (such as SV40).
  • retroviruses including lentiviruses
  • adenoviruses such as herpes simplex virus
  • poxviruses such as herpes simplex virus
  • baculoviruses such as herpes simplex virus
  • baculoviruses such as baculoviruses
  • papillomaviruses papillomaviruses
  • Polyoma vacuolating viruses such as SV40.
  • a vector can contain a variety of expression-controlling elements, including, but not limited to,
  • the term "host cell” refers to a cell that can be used to introduce a vector, which includes, but is not limited to, prokaryotic cells such as Escherichia coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, etc. Insect cells such as S2 Drosophila cells or Sf9, or animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, GS cells, BHK cells, HEK 293 cells or human cells.
  • prokaryotic cells such as Escherichia coli or Bacillus subtilis
  • fungal cells such as yeast cells or Aspergillus
  • Insect cells such as S2 Drosophila cells or Sf9
  • animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, GS cells, BHK cells, HEK 293 cells or human cells.
  • amino acids are generally represented by one-letter and three-letter abbreviations well known in the art.
  • alanine can be represented by A or Ala.
  • the term "pharmaceutically acceptable excipient” refers to substances that are pharmacologically and/or physiologically compatible with the recipient.
  • Carriers and/or excipients that are compatible with the subject and the active ingredient are well known in the art (see, e.g., Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995) and include, but are not Limited to: pH adjusters, surfactants, adjuvants, ionic strength enhancers.
  • pH adjusters include but are not limited to phosphate buffer; surfactants include but are not limited to cationic, anionic or nonionic surfactants such as Tween-80; ionic strength enhancers include but are not limited to sodium chloride.
  • an effective amount refers to an amount sufficient to obtain, at least in part, the desired effect.
  • an effective amount to prevent a disease is an amount that is sufficient to prevent, prevent, or delay the occurrence of a disease (such as a tumor);
  • an effective amount to treat a disease is an amount that is sufficient to cure or at least partially prevent the occurrence of a disease in a patient who already has the disease. The amount of disease and its complications.
  • hybridoma and “hybridoma cell line” are used interchangeably, and when the terms “hybridoma” and “hybridoma cell line” are referred to, it also includes subclones of the hybridoma and progeny cells.
  • the "first” for example, the first product
  • the "second” for example, the second product
  • the "third” for example, the third product
  • the present invention achieves one or more of the technical effects described in the following items (1) to (4):
  • the antibody of the present invention can bind to PVRIG with high affinity.
  • the antibody of the present invention can effectively treat and/or prevent tumors, such as colon cancer, melanoma, lung cancer, kidney cancer, endometrial cancer, breast cancer, skin cancer, ovarian cancer, gastric cancer, head and neck cancer, liver cancer, Brain tumors, urothelial cancer, bone tumors, cholangiocarcinoma, rectal cancer, pancreatic cancer, cervical tumors, multiple myeloma, Hodgkin lymphoma, non-Hodgkin lymphoma, B lymphocyte tumor, plasma cell carcinoma, Prostate cancer and testicular cancer, among others.
  • tumors such as colon cancer, melanoma, lung cancer, kidney cancer, endometrial cancer, breast cancer, skin cancer, ovarian cancer, gastric cancer, head and neck cancer, liver cancer, Brain tumors, urothelial cancer, bone tumors, cholangiocarcinoma, rectal cancer, pancreatic cancer, cervical tumors, multiple myeloma, Hodgkin lymphoma, non-
  • the antibody of the present invention has low toxic and side effects.
  • Figures 1A to 1B Binding curves of anti-PVRIG antibodies to human PVRIG overexpressed on CHO cells.
  • Figures 2A to 2B Binding curves of anti-PVRIG antibodies to cynomolgus monkey PVRIG overexpressed on CHO cells. picture.
  • Figures 3A to 3B Binding curves of anti-PVRIG antibodies to mouse PVRIG overexpressed on CHO cells.
  • Figures 4A to 4B Graphs of anti-PVRIG antibodies blocking the binding of human PVRIG to human CD112 overexpressed on CHO cells.
  • Figures 5A to 5B Graphs of anti-PVRIG antibodies blocking the binding of mouse CD112 to mouse PVRIG protein.
  • Figure 6 Binding curve of anti-PVRIG antibody to PVRIG on activated human primary T cells.
  • Figure 7 Curve chart of anti-PVRIG antibody and anti-TIGIT antibody combined to block the PVRIG/CD112 and TIGIT/CD155 signaling pathways.
  • Figure 8 Curve chart of anti-PVRIG antibody combined with anti-TIGIT and anti-PD-L1 antibodies to block the PVRIG/CD112, TIGIT/CD155 and PD-1/PD-L1 signaling pathways.
  • FIGS. 9A to 9C Anti-PVRIG antibodies sequentially mediate ADCC test results in CHO cells overexpressing human PVRIG, overexpressing cynomolgus PVRIG, and overexpressing mouse PVRIG.
  • Figure 10 Pharmacodynamic diagram of anti-PVRIG antibody in wild-type mouse CT-26 tumor model.
  • Figure 11 Pharmacodynamic diagram of anti-PVRIG antibody in NDG mouse model mixed with A375 and human PBMC.
  • Figure 12 The efficacy of anti-PVRIG antibody combined with anti-TIGIT antibody in the NDG mouse model mixed with A375 and human PBMC.
  • Figure 13 The efficacy of anti-PVRIG antibody combined with anti-TIGIT and anti-PD-1 antibodies in the NDG mouse model mixed with A375 and human PBMC.
  • Figure 14 Half-life curve of anti-PVRIG antibody in mice.
  • Antibody Atezolizumab (ATE): anti-PD-L1 monoclonal antibody, trade name Tecentriq, Roche.
  • control antibodies used in the examples were expressed and purified from HEK293 cells:
  • COM701 is an anti-human PVRIG antibody from Compugen expressed in HEK293 cells. Its light chain variable region sequence and heavy chain variable region sequence are consistent with SEQ ID NO: 1372 and SEQ ID NO. 1372 in US patent US10227408B2. SEQ ID NO:1380 is consistent;
  • Mab46 is an anti-human PVRIG antibody from Surface Oncology expressed by HEK293 cells. Its light chain variable region and heavy chain variable region sequences are consistent with SEQ ID NO:912 and SEQ ID NO:918 in the US patent publication US20200040081A1.
  • Tiragolumab is an anti-human TIGIT antibody from Genentech expressed by HEK293 cells. Its light chain and heavy chain sequences are derived from WHO Drug Information, Vol. 31, No. 2, 2017, Proposed INN: List 117, CAS number: 1918185-84 -8.
  • yeast antibody display library (Adimab)
  • amplification and optimization were performed according to existing methods (see patent applications WO2009036379, WO2010105256 and WO2012009568).
  • the yeast cells obtained through screening were shaken and induced at 30°C for 48 hours to secrete and express the target anti-PVRIG antibody (full-length IgG). After induction, the yeast cells were removed by centrifugation at 1300 rpm for 10 minutes, and the supernatant was harvested.
  • Use Protein A to purify the anti-PVRIG antibody in the supernatant, elute with pH 2.0 acetic acid solution, and harvest the anti-PVRIG antibody.
  • anti-PVRIG antibodies ADI-56127, ADI-56204, ADI-56249 and ADI-56145 were obtained.
  • the antibodies ADI-56127, ADI-56204, ADI-56249 and ADI-56145 were digested with papain and purified with KappaSelect (GE Life Healthcare) to obtain the corresponding antibody Fab fragments.
  • variable region sequence information of the four antibodies ADI-56127, ADI-56204, ADI-56249 and ADI-56145 is as follows.
  • the CDRs of each of the above antibodies are as follows:
  • LCDR2 DASSLHT(SEQ ID NO:19)
  • LCDR3 QQLSHHPLT(SEQ ID NO:32)
  • control antibody COM701 its heavy chain variable region was recombined into the human IgG4 heavy chain constant region (the amino acid sequence is shown in SEQ ID NO: 35).
  • the light chain variable regions (VL) of the above antibodies were recombined into the human kappa light chain constant region (the amino acid sequence is shown in SEQ ID NO: 36).
  • Transient expression and purification via HEK293 expression system The specific operation is as follows: use chemical transfection method to transfer the pcDNA3.1 vector with antibody heavy chain and light chain into HEK293 cells, and culture at 37°C, 8% CO2 for 7 days. Collect the cell fluid and centrifuge at 13,000 rpm for 20 minutes.
  • the antibodies prepared were named ADI-56127-G1, ADI-56127-G1LALA, ADI-56204-G1, ADI-56204-G1LALA, ADI-56249-G1LALA, ADI-56145-G1LALA, Mab46-G1, Mab46-G1LALA. and COM701-G4.
  • Biofilm layer optical interference technology (ForteBio) was used to determine the binding dissociation constant (K D ) of the antibody and its variants or Fab fragments obtained in Example 1 and human, cynomolgus monkey, and mouse PVRIG. Fortebio affinity measurement was performed according to existing methods (Este, P et al. High throughput solution-based measurement of antibody-antigen affinity and epitope binning. Mabs, 2013.5(2):p.270-8). The extracellular amino acid sequences of human, cynomolgus monkey and mouse PVRIG are shown in SEQ ID NOs: 37-39 respectively.
  • the sensor is equilibrated offline in the assay buffer for 20 minutes, then detected online for 120 seconds to establish a baseline, and the intact PVRIG antibody is loaded To the AHQ sensor to a thickness of 1nm for affinity detection.
  • the antibody-loaded sensor was incubated in 100 nM PVRIG-his antigen to plateau, and then the sensor was transferred to assay buffer for at least 2 minutes for off-rate measurement.
  • Kinetic analysis was performed using a 1:1 binding model.
  • the sensor is equilibrated offline in the assay buffer for 20 minutes, and then placed online Detect for 120 seconds to establish a baseline, load the PVRIG-Fc antigen to the AHQ sensor to a thickness of 1nm for affinity detection.
  • the antigen-loaded sensor was incubated in 100 nM Fab solution until the plateau phase, and then the sensor was transferred to the assay buffer for at least 2 minutes for off-rate measurement.
  • Kinetic analysis was performed using a 1:1 binding model.
  • the sensor is equilibrated offline in the analysis buffer for 20 minutes, and then detected online for 120 seconds to establish a baseline, and the complete PVRIG antibody is loaded onto the AHQ sensor to a thickness of 1nm.
  • Affinity testing Load the antibody-loaded sensor in a high-concentration irrelevant intact antibody solution for 10 minutes to saturate and block the Fc binding site on the AHQ sensor. Place the blocked sensor in 100nM PVRIG-Fc antigen until it reaches the plateau, and then transfer the sensor. into assay buffer for at least 2 minutes for dissociation rate measurements. Kinetic analysis was performed using a 1:1 binding model.
  • ADI-56127 and ADI-56249 have cross-binding activity with mouse PVRIG.
  • Example 3 Binding activity of anti-PVRIG antibodies to overexpressing human/cynomolgus/mouse PVRIG CHO cells and blocking activity
  • CHO cells overexpressing human PVRIG were generated through pressure screening by transfecting the pCHO1.0 vector (purchased from Invitrogen) of human PVRIG, cynomolgus PVRIG, and mouse PVRIG cDNA cloned into MCS, CHO cells overexpressing cynomolgus PVRIG (CHO-cynoPVRIG cells) and CHO cells overexpressing mouse PVRIG (CHO-muPVRIG cells). Adjust the expanded cultured overexpressing cells to a suitable cell density and add them to a 96-well flow cytometry plate. After centrifugation, add gradient dilutions of the test sample and incubate at 4°C for 30 minutes.
  • pCHO1.0 vector purchased from Invitrogen pressurized screening to generate CHO cells overexpressing human CD112 (CHO-huCD112 cells). Adjust the cell density of the expanded cultured CHO-huCD112 cells to 2 ⁇ 10 6 cells/mL, add 100 ⁇ L/well to a 96-well flow plate, and centrifuge for later use. Use PBS to gradiently dilute the purified monoclonal antibody, add 60 ⁇ L/well of the diluted sample into a blank 96-well flow cytometry plate, then add 60 ⁇ L/well of 1ug/mL human PVRIG protein with Mouse IgG2a Fc Tag, and mix Incubate at 4°C for 30 minutes.
  • mouse CD112-his protein was diluted with 1 ⁇ coating buffer to a final concentration of 1 ⁇ g/mL, 100 ⁇ L/well was added to the enzyme plate, covered with film, and left to coat at 4°C overnight. Discard the coating solution in the enzyme plate, wash three times with 1 ⁇ PBST, and add 200 ⁇ L/well of 5% BSA/PBS to block for 2 hours at room temperature. During the blocking period, the sample to be tested was diluted with 1% BSA/PBS gradient to a final volume of 60 ⁇ L/well.
  • Table 2 Summary table of overexpressed cell binding blocking activities of anti-PVRIG candidate molecules and their variant molecules Note: “NB”: no binding; “NA”: not available (not detected).
  • Some anti-PVRIG antibody molecules (ADI-56127-G1 and ADI-56127-G1LALA) that bind to mouse PVRIG protein overexpressed on the surface of CHO cells can significantly block the binding of mouse PVRIG to mouse CD112 protein. And the blocking activity is better than that of the control molecules Mab46-G1 and Mab46-G1LALA.
  • Example 4 Anti-PVRIG antibody binds to PVRIG on the surface of primary T cells
  • the anti-PVRIG antibody of the invention is detected and the PVRIG binding on the surface of activated T cells is detected. combined activity.
  • human PBMC were sorted according to the experimental protocol provided by STEMCELL (stemcell, product number: #17951C) to obtain human total T cells. Adjust the T cell concentration to 1.0 ⁇ 10 6 cells/mL with to-cell), add CD3/CD28 Dynabeads (purchased from gibco, product number: 11132D), and culture it in a 37°C, 5% CO2 incubator for 48 hours. Adjust the activated T cells to a suitable cell density and add them to a 96-well flow cytometry plate. After centrifugation, add gradient dilutions of the test sample and incubate at 4°C for 30 minutes.
  • STEMCELL stemcell, product number: #17951C
  • the results are shown in Figure 6.
  • the results show that the anti-PVRIG antibodies ADI-56127-G1 and ADI-56127-G1LALA of the present invention can bind to PVRIG molecules on the surface of activated T cells, and the binding activity is better than the control molecules Mab46-G1, Mab46-G1LALA and COM701-G4.
  • Example 5 Luciferase reporter gene system detects anti-PVRIG antibody and anti-TIGIT antibody combined blockade effect
  • this example constructed the following luciferase reporter gene system, which is simply described as follows: lentivirus was used to transfect cells to construct overexpressed human CD155, human CHO-K1 cell line (CHO-K1-CD155-CD112) of CD112 and OKT-3scFv, and a Jurkat cell line (Jurkat-TIGIT-PVRIG-luc) overexpressing human TIGIT, human PVRIG and NF-AT luciferase reporter genes was constructed. , and subsequent experiments will be carried out using this reporter gene system.
  • CHO-K1-CD155-CD112 functional cells were obtained by digestion, the cell density was adjusted, 100 ⁇ L/well was added to a 96-well white bottom plate, and adhered and cultured overnight. The next day, prepare a Jurkat-TIGIT-PVRIG-luc effector cell suspension, and serially dilute the sample to be tested with reaction medium. Take out the white bottom plate, aspirate the culture supernatant, add 40 ⁇ L/well of the above diluted sample to the white bottom plate, and add 40 ⁇ L/well of Jurkat-TIGIT-PVRIG-luc effector cell suspension, and incubate at 37°C in a 5% CO 2 incubator. Incubate for 6 hours. After the culture is completed, add 80 ⁇ L/well of Bio-Glo TM reagent, and use a multifunctional microplate reader to read the fluorescence signal value.
  • Example 6 Luciferase reporter gene system detects anti-PVRIG antibodies, anti-TIGIT and anti-PD-L1 antibodies body joint blocking effect
  • this example constructed the following luciferase reporter gene system, which is briefly described as, based on Example 5, lentivirus Infect CHO-K1-CD155-CD112 to overexpress PD-L1 to obtain CHO-K1-CD155-CD112-PD-L1 functional cells, and infect Jurkat-TIGIT-PVRIG-luc with lentivirus to overexpress PD-1 to obtain Jurkat-TIGIT -PVRIG-PD-1-luc effector cell suspension, and subsequent experiments will be carried out using this reporter gene system.
  • CHO-K1-CD155-CD112-PD-L1 functional cells were obtained by digestion, the cell density was adjusted, 100 ⁇ L/well was added to a 96-well white bottom plate, and the cells were adherent and cultured overnight.
  • a Jurkat-TIGIT-PVRIG-PD-1-luc effector cell suspension was prepared, and the sample to be tested was serially diluted with reaction medium. Take out the white bottom plate, aspirate the culture supernatant, add 40 ⁇ L/well of the above diluted sample to the white bottom plate, and add 40 ⁇ L/well of Jurkat-TIGIT-PVRIG-PD-1-luc effector cell suspension at 37°C for 5 % CO2 incubator for 6 hours. After the culture is completed, add 80 ⁇ L/well of Bio-Glo TM reagent, and use a multifunctional microplate reader to read the fluorescence signal value.
  • Example 7 In vitro ADCC activity detection of anti-PVRIG antibodies
  • Jurkat-NFAT-Luciferase-CD16ADCC effector cells purchased from Promega
  • 1640 medium containing 10% low IgG FBS to 4 ⁇ 10 6 cells/mL.
  • the results are shown in Figures 9A to 9C.
  • the results show that the anti-PVRIG antibodies ADI-56204-G1 and ADI-56127-G1 of the present invention can mediate the ADCC activity on CHO-huPVRIG cells, CHO-cynoPVRIG cells, and CHO-muPVRIG cells in vitro in the luciferase reporter gene system.
  • the ADCC activity mediated by ADI-56204-G1 on CHO-huPVRIG cells and CHO-cynoPVRIG cells was stronger than that of the control molecule Mab46-G1.
  • the ADCC activity mediated by ADI-56127-G1 on CHO-huPVRIG cells, CHO-cynoPVRIG cells, and CHO-muPVRIG cells was comparable to that of the control molecule Mab46-G1.
  • a CT-26 cell suspension was first prepared, and 0.1 mL of approximately 5 ⁇ 10 5 cells was subcutaneously injected into the right abdominal groin of the mouse to establish a CT-26 tumor-bearing mouse model.
  • the average tumor volume reaches about 100 mm, they are divided into groups and treated with PBS or antibodies of different doses and the same volume by intraperitoneal injection, with 6 mice in each group. Monitor the changes in tumor volume and body weight of mice in each group.
  • the monitoring frequency is 2-3 days/time, and continuous monitoring is performed for 2 to 3 weeks.
  • the dosage and method of administration are as shown in Table 3.
  • Example 9 In vivo pharmacodynamic study of anti-PVRIG antibody in NDG mice mixed with A375 and human PBMC study
  • B-NDG mice were mixedly inoculated with A375 (purchased from Addexbio, C0020004, a human malignant melanoma cell) and human PBMC cells (Shanghai Miaoshun, A10S033014/PB100C) model (A375huPBMC Model) to determine the anti-tumor effect of the anti-PVRIG antibody of the present invention.
  • A375 purchased from Addexbio, C0020004, a human malignant melanoma cell
  • human PBMC cells Sthanghai Miaoshun, A10S033014/PB100C model
  • humanized tumor mouse models that locally restructure the human immune system are generated by inoculating human immune cells (PBMC) into immunodeficient mice.
  • A375 cells and human PBMC were first mixed into 0.1 mL cell suspension at a 1:1 equal volume, and the A375huPBMC model was established by subcutaneous injection in the right abdominal groin of mice.
  • the average tumor volume reaches about 30-50 mm, they are divided into groups, and different doses and the same volume of PBS or antibody treatment are administered intraperitoneally, with 6 mice in each group.
  • the changes in tumor volume and body weight of mice in each group were monitored.
  • the frequency of monitoring was 2-3 days/time, and continuous monitoring was performed for 2 to 3 weeks.
  • the dosage and method of administration are as shown in Table 4.
  • Example 10 Anti-PVRIG antibody combined with anti-TIGIT antibody was mixedly inoculated with A375 and human in NDG mice In vivo pharmacodynamic study of PBMC
  • A375 and human PBMC tumor-bearing mouse models were established by subcutaneous mixed inoculation (the modeling steps are the same as those in Example 9).
  • the modeling steps are the same as those in Example 9).
  • the average tumor volume grew to about 60 mm, they were divided into groups, and different doses and the same dose were administered intraperitoneally.
  • drug volume of PBS or antibody treatment 6 mice per group. Monitor the changes in tumor volume and body weight of mice in each group. The monitoring frequency is 2-3 days/time, and continuous monitoring is performed for 2 to 3 weeks.
  • the dosage and method of administration are as shown in Table 5.
  • the sequence of the anti-TIGIT monoclonal antibody used in this example is as follows:
  • Anti-TIGIT antibody VL amino acid sequence is amino acid sequence:
  • the CDRs of the above anti-TIGIT antibodies are as follows:
  • the anti-TIGIT monoclonal antibody heavy chain constant region used in this example uses wild-type human IgG1 heavy chain constant region (SEQ ID NO: 33).
  • the anti-TIGIT monoclonal antibody light chain constant region used in this example uses the human kappa light chain constant region (SEQ ID NO: 36).
  • Example 11 Mixed vaccination of anti-PVRIG antibody with anti-PD-1 and anti-TIGIT antibodies in NDG mice In vivo pharmacodynamic study of A375 and human PBMC
  • A375 and human PBMC tumor-bearing mouse models were first established by subcutaneous mixed inoculation (the modeling steps were the same as those in Example 9).
  • the modeling steps were the same as those in Example 9).
  • the average tumor volume grew to about 200 mm, they were divided into groups and intraperitoneally injected with different doses of the same mice.
  • Administration volume of PBS or antibody treatment 6 mice per group. Monitor the changes in tumor volume and body weight of mice in each group. The monitoring frequency is 2-3 days/time, and continuous monitoring is performed for 2 to 3 weeks.
  • the dosage and method of administration are as shown in Table 6.
  • the sequence of the anti-PD-1 monoclonal antibody used in this example is as follows:
  • the 6 CDRs of the above-mentioned anti-PD-1 antibodies are as follows:
  • the anti-PD-1 monoclonal antibody heavy chain constant region used in this example uses the IgG1 heavy chain constant region modified by L234A and L235A (SEQ ID NO: 34)
  • the anti-PD-1 monoclonal antibody light chain constant region used in this example uses human kappa light chain constant region (SEQ ID NO: 36)
  • the tail vein single injection method was used to detect the half-life of the anti-PVRIG antibody ADI-56127-G1 of the present invention in mice.
  • the experiment used Balb/c mice, 3 males and 3 males, with 12/12 hours of light/dark regulation, a temperature of 24°C ⁇ 2°C, a humidity of 40%-70%, and free access to water and food.
  • Balb/c mice were given a single tail vein injection of monoclonal antibody molecules at a dose of 10 mg/kg.
  • Blood collection time points Collect blood from the mouse orbits 5 minutes, 0.5 hours, 2 hours, 6 hours, 24 hours, 48 hours, 96 hours, 168 hours, 336 hours, and 504 hours after administration.
  • Whole blood samples were placed at 2°C-8°C for 30 minutes, centrifuged at 12,000 rpm for 5 minutes to collect serum. The resulting serum was centrifuged at 2°C-8°C, 12,000 rpm for 5 minutes, and stored at -80°C.
  • the content of monoclonal antibody molecules in the serum was detected by ELISA.

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Abstract

本发明提供了一种抗PVRIG抗体或其抗原结合片段，其中，所述抗体包含重链可变区和轻链可变区，所述重链可变区包含HCDR1至HCDR3，所述轻链可变区包含LCDR1至LCDR3，其中：所述抗体的重链可变区，HCDR1的氨基酸序列如SEQ ID NO:9所示、HCDR2的氨基酸序列如SEQ ID NO:10所示和HCDR3的氨基酸序列如SEQ ID NO:11所示，并且所述抗体的轻链可变区，LCDR1的氨基酸序列如SEQ ID NO:12所示、LCDR2的氨基酸序列如SEQ ID NO:13所示和LCDR3的氨基酸序列如SEQ ID NO:14所示。

Description

抗PVRIG抗体、其药物组合物及用途 技术领域
本发明属于生物医药领域，涉及一种抗PVRIG抗体、其药物组合物及用途。
背景技术
脊髓灰质炎病毒受体相关免疫球蛋白结构域(poliovirus receptor related immunoglobulin domain-containing protein，PVRIG，又称CD112R)属于PVR家族成员，其胞外区具有一个IgV结构域，胞内区含有一个ITIM(Immunoreceptor tyrosine-based inhibitory motif)结构域，是一种表达在自然杀伤(NK)细胞和T细胞上的新型免疫抑制受体。
PVRL2(poliovirus receptor-related 2，CD112，Nectin-2)是一种粘附分子，参与细胞-细胞连接，并在多种癌症中过度表达。PVRL2目前被鉴定为PVRIG唯一的功能性配体，可以高亲和力与PVRIG结合，抑制T和NK细胞免疫激活。PVRL2也是共激活受体DNAX附属分子1(DNAM-1，CD226)的配体，并且与另一个抑制性受体—具有Ig和ITIM结构域的T细胞免疫受体(TIGIT)存在弱性结合。因此，与同一家族成员TIGIT发挥免疫抑制的作用机制类似，PVRIG也可以通过与CD226竞争性结合其配体PVRL2而阻断CD226传递的激活信号，来发挥免疫抑制的作用。
在肺癌、肾癌、子宫内膜癌、乳腺癌、皮肤癌及其肿瘤微环境中，PVRIG或其配体PVRL2均高表达，且PVRIG在前列腺癌患者的NK细胞上也高表达(Whelan S,et al.Cancer Immunol Res.2019；7(2):257-268.)。此外，PVRIG与耗竭性CD8+T细胞标志TIGIT和PD-1分子呈共表达，提示了PVRIG的表达有着一定的肿瘤特异性，并且可能与肿瘤内的TIL的激活/耗竭状态有关(Whelan S,et al.Cancer Immunol Res.2019；7(2):257-268.)。WO2021180205A1报道了抗PVRIG抗体能够用于肺癌、乳腺癌、卵巢癌、肾癌、胃癌、子宫内膜癌、头颈癌等的防治。
在临床前小鼠肿瘤模型中的研究表明，PVRIG缺失或阻断可以恢复T/NK细胞活性，促进抗肿瘤免疫反应。从Pmel-1TCR-CD112R-缺陷小鼠分离的CD8+T细胞表现出增强的脱颗粒和细胞因子分泌效应。与野生型小鼠中相比，CD112R缺陷小鼠中MC38肿瘤的生长减缓，CD8+TILs表现出增强的免疫效应和炎症/细胞毒性相关基因上调的转录组特征。文献同时报道，PVRIG抗体或PVRIG基因敲除小鼠与PD-L1 抗体在MC38及CT26小鼠肿瘤模型中联用比PD-L1抗体单药具有更好的治疗效果(Murter B,et al.Cancer Immunology Research,2019.)。Feng Xu等人证明用抗体阻断PVRIG与CD112的相互作用可增强NK细胞对肿瘤细胞的细胞毒性，此外与TIGIT阻断单抗联用可进一步增强Trastuzumab介导的NK细胞对乳腺癌细胞的杀伤效果(Xu F,et al.Cancer Immunology Immunotherapy Cii,2017.)。在基因敲除小鼠模型中，PVRIG抗体可减少TIGIT-/-小鼠肿瘤的生长，并且PVRIG与TIGIT的双敲除小鼠中的肿瘤生长控制，与单敲除小鼠相比也有进一步提高(RJ Sullivan,et al,ASCO-SITC Clinical Immuno-Oncology Symposium,2020)。
临床前数据表明，PVRIG抗体能有效刺激某些癌症的抗肿瘤免疫反应，并发现其和PD-1/L1和TIGIT抗体具有一定的协同作用，能够增强T/NK细胞的功能，提高抗肿瘤反应，减缓肿瘤的生长。以PVRIG作为治疗靶点，有望提高现有抗体药物(如抗TIGIT和抗PD-1/L1抗体)的治疗效果，呈现出不同于其他药物靶点和临床中组合疗法的优势。Compugen公司开发的COM701是全球首个获批临床的PVRIG抗体，目前正在进行与TIGIT单抗(COM902)联用Ⅰ期研究，并与BMS达成合作进行COM701+BMS-986207+nivolumab三联Ⅰ期试验。除此之外，Surface Oncology授权给GSK的GSK4381562以及国内君实生物的JS009已于2021年年底前递交了临床申请，Amgen公司也布局了PVRIG单抗，目前处于临床前阶段。
因此，本领域存在开发新的高亲合力、高选择性、高生物活性的抗PVRIG抗体需求，用于疾病尤其是癌症的治疗。
发明内容
本发明人经过深入的研究和创造性的劳动，得到了新的抗PVRIG抗体。本发明人惊奇地发现，本发明的抗PVRIG抗体具有良好的亲和力和生物活性，具有抗肿瘤的潜力。由此提供了下述发明：
本发明的一个方面涉及一种抗PVRIG抗体或其抗原结合片段，其中，
所述抗体包含重链可变区和轻链可变区，所述重链可变区包含HCDR1至HCDR3，所述轻链可变区包含LCDR1至LCDR3，其中：
所述抗体的重链可变区，
HCDR1的氨基酸序列如SEQ ID NO:9所示、HCDR2的氨基酸序列如SEQ ID NO: 10所示和HCDR3的氨基酸序列如SEQ ID NO:11所示，
HCDR1的氨基酸序列如SEQ ID NO:15所示、HCDR2的氨基酸序列如SEQ ID NO:16所示和HCDR3的氨基酸序列如SEQ ID NO:17所示，
HCDR1的氨基酸序列如SEQ ID NO:21所示、HCDR2的氨基酸序列如SEQ ID NO:22所示和HCDR3的氨基酸序列如SEQ ID NO:23所示，或者
HCDR1的氨基酸序列如SEQ ID NO:27所示、HCDR2的氨基酸序列如SEQ ID NO:28所示和HCDR3的氨基酸序列如SEQ ID NO:29所示；
并且
所述抗体的轻链可变区，
LCDR1的氨基酸序列如SEQ ID NO:12所示、LCDR1的氨基酸序列如SEQ ID NO:13所示和LCDR1的氨基酸序列如SEQ ID NO:14所示，
LCDR1的氨基酸序列如SEQ ID NO:18所示、LCDR1的氨基酸序列如SEQ ID NO:19所示和LCDR1的氨基酸序列如SEQ ID NO:20所示，
LCDR1的氨基酸序列如SEQ ID NO:24所示、LCDR1的氨基酸序列如SEQ ID NO:25所示和LCDR1的氨基酸序列如SEQ ID NO:26所示，或者
LCDR1的氨基酸序列如SEQ ID NO:30所示、LCDR1的氨基酸序列如SEQ ID NO:31所示和LCDR1的氨基酸序列如SEQ ID NO:32所示。
在本发明的一些实施方式中，所述的抗PVRIG抗体或其抗原结合片段，其中，
HCDR1的氨基酸序列如SEQ ID NO:9所示，HCDR2的氨基酸序列如SEQ ID NO:10所示，HCDR3的氨基酸序列如SEQ ID NO:11所示，LCDR1的氨基酸序列如SEQ ID NO:12所示，LCDR2的氨基酸序列如SEQ ID NO:13所示和LCDR3的氨基酸序列如SEQ ID NO:14所示；
HCDR1的氨基酸序列如SEQ ID NO:15所示，HCDR2的氨基酸序列如SEQ ID NO:16所示，HCDR3的氨基酸序列如SEQ ID NO:17所示，LCDR1的氨基酸序列如SEQ ID NO:18所示，LCDR2的氨基酸序列如SEQ ID NO:19所示和LCDR3的氨基酸序列如SEQ ID NO:20所示；
HCDR1的氨基酸序列如SEQ ID NO:21所示，HCDR2的氨基酸序列如SEQ ID NO:22所示，HCDR3的氨基酸序列如SEQ ID NO:23所示，LCDR1的氨基酸序列如SEQ ID NO:24所示，LCDR2的氨基酸序列如SEQ ID NO:25所示和LCDR3的氨基酸序列如SEQ ID NO:26所示；
或者
HCDR1的氨基酸序列如SEQ ID NO:27所示，HCDR2的氨基酸序列如SEQ ID NO:28所示，HCDR3的氨基酸序列如SEQ ID NO:29所示，LCDR1的氨基酸序列如SEQ ID NO:30所示，LCDR2的氨基酸序列如SEQ ID NO:31所示和LCDR3的氨基酸序列如SEQ ID NO:32所示。
轻链和重链的可变区决定抗原的结合；每条链的可变区均含有三个高变区，称互补决定区(CDR)(重链(H)的CDR包含HCDR1、HCDR2、HCDR3，轻链(L)的CDR包含LCDR1、LCDR2、LCDR3。在本发明的一些实施方式中，HCDR1-HCDR3以及LCDR1-LCDR3参照美国专利公开US20210380669A1或者文献Lu.et al(Deamidation and isomerization liability analysis of 131 clinical-stage antibodies,MABS,2019,VOL.11,NO.1,45–57,DOI:10.1080/19420862.2018.1548233)进行定义或者编号。
在本发明的一些实施方式中，所述的抗PVRIG抗体或其抗原结合片段，其中，所述重链可变区的氨基酸序列选自SEQ ID NO:1、SEQ ID NO:3、SEQ ID NO:5和SEQ ID NO:7；
并且
所述轻链可变区的氨基酸序列选自SEQ ID NO:2、SEQ ID NO:4、SEQ ID NO:6和SEQ ID NO:8。
在本发明的一些实施方式中，所述的抗PVRIG抗体或其抗原结合片段，其中，
重链可变区的氨基酸序列如SEQ ID NO:1所示，并且轻链可变区的氨基酸序列如SEQ ID NO:2所示；
重链可变区的氨基酸序列如SEQ ID NO:3所示，并且轻链可变区的氨基酸序列如SEQ ID NO:4所示；
重链可变区的氨基酸序列如SEQ ID NO:5所示，并且轻链可变区的氨基酸序列如SEQ ID NO:6所示；
或者
重链可变区的氨基酸序列如SEQ ID NO:7所示，并且轻链可变区的氨基酸序列如SEQ ID NO:8所示。
在本发明的一些实施方式中，所述的抗PVRIG抗体或其抗原结合片段，其中，所述抗体的重链恒定区为Ig gamma-1 chain C region或Ig gamma-4 chain C region；轻链 恒定区为Ig kappa chain C region；
优选地，所述抗体的重链恒定区氨基酸序列如SEQ ID NO：33、SEQ ID NO：34或SEQ ID NO：35所示，所述抗体的轻链恒定区氨基酸序列如SEQ ID NO：36所示。
在本发明的一些实施方式中，所述的抗PVRIG抗体或其抗原结合片段，其中，按照EU编号系统，所述抗体的重链恒定区包含L234A突变和L235A突变(简称为LALA)。本发明中，如果没有特别说明，位点之前的字母表示突变前的氨基酸，位点之后的字母表示突变后的氨基酸。
在本发明的一些实施方式中，所述的抗PVRIG抗体或其抗原结合片段，其中，所述抗PVRIG抗体或其抗原结合片段选自Fab、Fab'、F(ab')2、Fd、Fv、dAb、互补决定区片段、单链抗体、人源化抗体、嵌合抗体或双抗体。
在本发明的一些实施方式中，所述的抗TIGIT抗体或其抗原结合片段，其中，
所述的抗体包括非-CDR区，且所述非-CDR区来自不是鼠类的物种，例如来自人抗体。
在本发明的一些实施方式中，所述的抗PVRIG抗体或其抗原结合片段，其中，所述抗体与CHO细胞表面过表达的人PVRIG蛋白的结合的EC50小于3nM、小于2.9nM或小于2.8nM；优选地，所述EC50通过流式细胞术检测法测得。
本发明的另一方面涉及一种分离的核酸分子，其编码本发明中任一项所述的抗TIGIT抗体或其抗原结合片段。
本发明的再一方面涉及一种重组载体，其包含本发明的分离的核酸分子。
本发明的再一方面涉及一种宿主细胞，其包含本发明的分离的核酸分子，或者本发明的重组载体。
本发明的再一方面涉及一种偶联物，其包括抗体以及偶联部分，其中，所述抗体为本发明中任一项所述的抗PVRIG抗体或其抗原结合片段，所述偶联部分为可检测的标记；优选地，所述偶联部分为放射性同位素、荧光物质、有色物质或酶。
本发明的再一方面涉及一种试剂盒，其包括本发明中任一项所述的抗PVRIG抗体或其抗原结合片段，或者包括本发明的偶联物；
优选地，所述试剂盒还包括第二抗体，其特异性识别所述抗体；任选地，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、有色物质或酶。
本发明的再一方面涉及一种药物组合物，其包含本发明中任一项所述的抗PVRIG抗体或其抗原结合片段或者本发明的偶联物；可选地，所述药物组合物还包括一种或多种药学上可接受的辅料。
在本发明的一些实施方式中，所述的药物组合物，其还包含一种或多种抗TIGIT抗体；
优选地，所述药物组合物还包含一种或多种抗PD-1抗体或者一种或多种抗PD-L1抗体。
在本发明的一些实施方式中，所述的药物组合物，其中，
抗PVRIG抗体或其抗原结合片段与抗TIGIT抗体的摩尔比为(1：5)-(5：1)，例如1：5、1：4、1：3、1：2、1：1、2：1、3：1、4：1或5：1；
和/或
抗PVRIG抗体或其抗原结合片段与抗PD-1抗体或者抗PD-L1抗体的摩尔比为(1：5)-(5：1)，例如1：5、1：4、1：3、1：2、1：1、2：1、3：1、4：1或5：1。
在本发明的一个或多个实施方式中，所述的药物组合物，其中，所述的抗TIGIT抗体，其包含重链可变区和轻链可变区，所述重链可变区包含HCDR1至HCDR3，所述轻链可变区包含LCDR1至LCDR3，其中：
HCDR1的氨基酸序列如SEQ ID NO:42所示，HCDR2的氨基酸序列如SEQ ID NO:43所示，HCDR3的氨基酸序列如SEQ ID NO:44所示，LCDR1的氨基酸序列如SEQ ID NO:45所示，LCDR2的氨基酸序列如SEQ ID NO:46所示和LCDR3的氨基酸序列如SEQ ID NO:47所示；
优选地，所述的抗TIGIT抗体，其重链可变区的氨基酸序列如SEQ ID NO:40所示，并且轻链可变区的氨基酸序列如SEQ ID NO:41所示。
在本发明的一个或多个实施方式中，所述的药物组合物，其中，所述的抗PD-1抗体，其包含重链可变区和轻链可变区，所述重链可变区包含HCDR1至HCDR3，所述轻链可变区包含LCDR1至LCDR3，其中：
HCDR1的氨基酸序列如SEQ ID NO:50所示，HCDR2的氨基酸序列如SEQ ID NO:51所示，HCDR3的氨基酸序列如SEQ ID NO:52所示，LCDR1的氨基酸序列如SEQ ID NO:53所示，LCDR2的氨基酸序列如SEQ ID NO:54所示和LCDR3的氨基酸序列如 SEQ ID NO:55所示；
优选地，所述的抗PD-1抗体，其重链可变区的氨基酸序列如SEQ ID NO:48所示，并且轻链可变区的氨基酸序列如SEQ ID NO:49所示。
在本发明的一个或多个实施方式中，所述的药物组合物，其中，所述药物组合物的单位剂量，按照其中的所述抗PVRIG抗体或其抗原结合片段的质量计算，为100mg-1000mg、200mg-800mg、200mg-500mg、300mg-600mg、400mg-500mg或者450mg。
在本发明的一些实施方式中，所述的药物组合物，其活性成分由抗PVRIG抗体或其抗原结合片段组成。
在本发明的一些实施方式中，所述的药物组合物，其活性成分由抗PVRIG抗体或其抗原结合片段和抗TIGIT抗体组成。
在本发明的一些实施方式中，所述的药物组合物，其活性成分由抗PVRIG抗体或其抗原结合片段、抗TIGIT抗体和抗PD-1抗体组成。
本发明的再一方面涉及一种组合产品，其包含独立包装的第一产品和第二产品，其中，
所述第一产品包含其包含本发明中任一项所述的抗PVRIG抗体或其抗原结合片段或者本发明的偶联物；
所述第二产品包含至少一种抗TIGIT抗体；
优选地，所述组合产品还包括第三产品，所述第三产品包含至少一种抗PD-1抗体或者至少一种抗PD-L1抗体；
优选地，所述第一产品、所述第二产品和第三产品还独立地包含一种或多种药学上可接受的辅料；
优选地，所述组合产品还包含产品说明书。
在本发明的一些实施方式中，所述的组合产品，其中，
抗PVRIG抗体或其抗原结合片段与抗TIGIT抗体的摩尔比为(1：5)-(5：1)，例如1：5、1：4、1：3、1：2、1：1、2：1、3：1、4：1或5：1；
和/或
抗PVRIG抗体或其抗原结合片段与抗PD-1抗体或者抗PD-L1抗体的摩尔比为(1：5)-(5：1)，例如1：5、1：4、1：3、1：2、1：1、2：1、3：1、4：1或5：1。
在本发明的一些实施方式中，所述的组合产品，其中，所述的抗TIGIT抗体，其包含重链可变区和轻链可变区，所述重链可变区包含HCDR1至HCDR3，所述轻链可变区包含LCDR1至LCDR3，其中：
HCDR1的氨基酸序列如SEQ ID NO:42所示，HCDR2的氨基酸序列如SEQ ID NO:43所示，HCDR3的氨基酸序列如SEQ ID NO:44所示，LCDR1的氨基酸序列如SEQ ID NO:45所示，LCDR2的氨基酸序列如SEQ ID NO:46所示和LCDR3的氨基酸序列如SEQ ID NO:47所示；
优选地，所述的抗TIGIT抗体，其重链可变区的氨基酸序列如SEQ ID NO:40所示，并且轻链可变区的氨基酸序列如SEQ ID NO:41所示。
在本发明的一些实施方式中，所述的组合产品，其中，所述的抗PD-1抗体，其包含重链可变区和轻链可变区，所述重链可变区包含HCDR1至HCDR3，所述轻链可变区包含LCDR1至LCDR3，其中：
HCDR1的氨基酸序列如SEQ ID NO:50所示，HCDR2的氨基酸序列如SEQ ID NO:51所示，HCDR3的氨基酸序列如SEQ ID NO:52所示，LCDR1的氨基酸序列如SEQ ID NO:53所示，LCDR2的氨基酸序列如SEQ ID NO:54所示和LCDR3的氨基酸序列如SEQ ID NO:55所示；
优选地，所述的抗PD-1抗体，其重链可变区的氨基酸序列如SEQ ID NO:48所示，并且轻链可变区的氨基酸序列如SEQ ID NO:49所示。
本发明再一方面涉及本发明中任一项所述的抗体或其抗原结合片段、本发明的偶联物或者本发明中任一项所述的药物组合物在制备治疗或预防肿瘤的药物中的用途；
优选地，所述肿瘤选自结肠癌、黑色素瘤、肺癌、肾癌、子宫内膜癌、乳腺癌、皮肤癌、卵巢癌、胃癌、头颈癌、肝癌、脑瘤、尿路上皮癌、骨肿瘤、胆管癌、直肠癌、胰腺癌、宫颈瘤、多发性骨髓瘤、霍奇金淋巴瘤、非霍奇金淋巴瘤、B淋巴细胞瘤、浆细胞癌、前列腺癌和睾丸癌中的一种或多种；
优选地，所述肺癌为非小细胞肺癌或小细胞肺癌。
根据本发明中任一项所述的抗体或其抗原结合片段、本发明的偶联物或者本发明中任一项所述的药物组合物，其用于治疗或预防肿瘤；
优选地，所述肿瘤选自结肠癌、黑色素瘤、肺癌、肾癌、子宫内膜癌、乳腺癌、皮肤癌、卵巢癌、胃癌、头颈癌、肝癌、脑瘤、尿路上皮癌、骨肿瘤、胆管癌、直肠 癌、胰腺癌、宫颈瘤、多发性骨髓瘤、霍奇金淋巴瘤、非霍奇金淋巴瘤、B淋巴细胞瘤、浆细胞癌、前列腺癌和睾丸癌中的一种或多种；
优选地，所述肺癌为非小细胞肺癌或小细胞肺癌。
本发明的再一方面涉及一种治疗或预防肿瘤的方法，包括给予有需求的受试者以有效量的本发明中任一项所述的抗体或其抗原结合片段、本发明的偶联物或者本发明中任一项所述的药物组合物的步骤；
优选地，所述肿瘤选自结肠癌、黑色素瘤、肺癌、肾癌、子宫内膜癌、乳腺癌、皮肤癌、卵巢癌、胃癌、头颈癌、肝癌、脑瘤、尿路上皮癌、骨肿瘤、胆管癌、直肠癌、胰腺癌、宫颈瘤、多发性骨髓瘤、霍奇金淋巴瘤、非霍奇金淋巴瘤、B淋巴细胞瘤、浆细胞癌、前列腺癌和睾丸癌中的一种或多种；
优选地，所述肺癌为非小细胞肺癌或小细胞肺癌。
在本发明的一个或多个实施方式中，所述的方法，其中，
抗PVRIG抗体的单次给药剂量为每千克体重0.1-100mg，优选1-10mg(例如1mg、2mg、3mg、4mg、5mg、6mg、7mg、8mg、9mg或10mg)；或者，抗PVRIG抗体的单次给药剂量为每位受试者10-1000mg(例如大约100mg、大约150mg、大约200mg、大约250mg、大约300mg、大约350mg、大约400mg、大约450mg、大约500mg、大约600mg、大约700mg、大约800mg、大约900mg或大约1000mg)，优选50-500mg、100-400mg、150-300mg、150-250mg或200mg；
优选地，每3天、4天、5天、6天、10天、1周、2周或3周给药一次；
优选地，给药方式为静脉滴注或静脉注射。
在一些方案中，抗PVRIG抗体的施用治疗以2周(14天)或3周(21天)为一个周期，优选在每个周期第一天(D1)静脉给予抗PVRIG抗体。例如，所述抗PVRIG抗体以每两周一次(q2w)或者每三周一次(q3w)的频率施用。
在本发明中，除非另有说明，否则本文中使用的科学和技术名词具有本领域技术人员所通常理解的含义。并且，本文中所用的细胞培养、分子遗传学、核酸化学、免疫学实验室操作步骤均为相应领域内广泛使用的常规步骤。同时，为了更好地理解本发明，下面提供相关术语的定义和解释。
如本文中所使用的，当提及PVRIG(NCBI GenBank ID:NP_076975.2)的氨基酸序列时，其包括PVRIG蛋白的全长或者细胞外片段；还包括PVRIG的融合蛋白， 例如与小鼠或人IgG的Fc蛋白片段(mFc或hFc)进行融合的片段。然而，本领域技术人员理解，在PVRIG蛋白的氨基酸序列中，可天然产生或人工引入突变或变异(包括但不限于置换，缺失和/或添加)，而不影响其生物学功能。因此，在本发明中，术语“PVRIG蛋白”或“PVRIG”应包括所有此类序列，包括所示的序列以及其天然或人工的变体。并且，当描述PVRIG蛋白的序列片段时，其不仅包括的序列片段，还包括其天然或人工变体中的相应序列片段。
如本文中所使用的，当提及TIGIT(NCBI GenBank ID:NP_776160.2)的氨基酸序列时，其包括TIGIT蛋白的全长或功能片段；还包括TIGIT的融合蛋白，例如与小鼠或人IgG的Fc蛋白片段(mFc或hFc)进行融合的片段。然而，本领域技术人员理解，在TIGIT蛋白的氨基酸序列中，可天然产生或人工引入突变或变异(包括但不限于置换，缺失和/或添加)，而不影响其生物学功能。因此，在本发明中，术语“TIGIT蛋白”或“TIGIT”应包括所有此类序列，包括所示的序列以及其天然或人工的变体。并且，当描述TIGIT蛋白的序列片段时，其不仅包括的序列片段，还包括其天然或人工变体中的相应序列片段。
如本文中所使用的，术语EC50是指半最大效应浓度(concentration for 50％of maximal effect)，是指能引起50％最大效应的浓度。
如本文中所使用的，术语“抗体”是指通常由两对多肽链(每对具有一条“轻”(L)链和一条“重”(H)链)组成的免疫球蛋白分子。抗体轻链可分类为κ和λ轻链。重链可分类为μ、δ、γ、α或ε，并且分别将抗体的同种型定义为IgM、IgD、IgG、IgA和IgE。在轻链和重链内，可变区和恒定区通过大约12或更多个氨基酸的“J”区连接，重链还包含大约3个或更多个氨基酸的“D”区。各重链由重链可变区(VH)和重链恒定区(CH)组成。重链恒定区由3个结构域(CH1、CH2和CH3)组成。各轻链由轻链可变区(VL)和轻链恒定区(CL)组成。轻链恒定区由一个结构域CL组成。抗体的恒定区可介导免疫球蛋白与宿主组织或因子，包括免疫系统的各种细胞(例如，效应细胞)和经典补体系统的第一组分(C1q)的结合。VH和VL区还可被细分为具有高变性的区域(称为互补决定区(CDR))，其间散布有较保守的称为构架区(FR)的区域。各VH和VL由按下列顺序：FR1、CDR1、FR2、CDR2、FR3、CDR3、FR4从氨基末端至羧基末端排列的3个CDR和4个FR组成。各重链/轻链对的可变区(VH和VL)分别形成抗原结合部位。氨基酸至各区域或结构域的分配遵循Kabat Sequences of Proteins of Immunological Interest(National Institutes of Health,Bethesda M.d.(1987and  1991))，或Chothia & Lesk J.Mol.Biol.1987；196:901-917；Chothia等人Nature 1989；342:878-883或者IMGT编号系统定义，见Ehrenmann,Francois,Quentin Kaas,and Marie-Paule Lefranc."IMGT/3Dstructure-DB and IMGT/DomainGapAlign:a database and a tool for immunoglobulins or antibodies,T cell receptors,MHC,IgSF and MhcSF."Nucleic acids research 2009；38(suppl_1):D301-D307.的定义。术语“抗体”不受任何特定的产生抗体的方法限制。例如，其包括，特别地，重组抗体、单克隆抗体和多克隆抗体。抗体可以是不同同种型的抗体，例如，IgG(例如，IgG1，IgG2，IgG3或IgG4亚型)，IgA1，IgA2，IgD，IgE或IgM抗体。
如本文中所使用的，术语抗体的“抗原结合片段”是指包含全长抗体的片段的多肽，其保持特异性结合全长抗体所结合的相同抗原的能力，和/或与全长抗体竞争对抗原的特异性结合，其也被称为“抗原结合部分”。通常参见，Fundamental Immunology,Ch.7(Paul,W.,ed.,第2版，Raven Press,N.Y.(1989)，其以其全文通过引用合并入本文，用于所有目的。可通过重组DNA技术或通过完整抗体的酶促或化学断裂产生抗体的抗原结合片段。在一些情况下，抗原结合片段包括Fab、Fab'、F(ab')2、Fd、Fv、dAb和互补决定区(CDR)片段、单链抗体(例如，scFv)、嵌合抗体、双抗体(diabody)和这样的多肽，其包含足以赋予多肽特异性抗原结合能力的抗体的至少一部分。
如本文中所使用的，术语“Fd片段”意指由VH和CH1结构域组成的抗体片段；术语“Fv片段”意指由抗体的单臂的VL和VH结构域组成的抗体片段；术语“dAb片段”意指由VH结构域组成的抗体片段(Ward等人,Nature 341:544-546(1989))；术语“Fab片段”意指由VL、VH、CL和CH1结构域组成的抗体片段；术语“F(ab')2片段”意指包含通过铰链区上的二硫桥连接的两个Fab片段的抗体片段。
在一些情况下，抗体的抗原结合片段是单链抗体(例如，scFv)，其中VL和VH结构域通过使其能够产生为单个多肽链的连接体配对形成单价分子(参见，例如,Bird等人,Science 242:423-426(1988)和Huston等人,Proc.Natl.Acad.Sci.USA85:5879-5883(1988))。此类scFv分子可具有一般结构：NH2-VL-接头-VH-COOH或NH2-VH-接头-VL-COOH。合适的现有技术接头由重复的GGGGS氨基酸序列或其变体组成。例如，可使用具有氨基酸序列(GGGGS)4的接头，但也可使用其变体(Holliger等人(1993)，Proc.Natl.Acad.Sci.USA 90:6444-6448)。可用于本发明的其他接头由Alfthan等人(1995)，Protein Eng.8:725-731，Choi等人(2001)，Eur.J.Immunol.31:94-106，Hu等人(1996)，Cancer Res.56:3055-3061，Kipriyanov等人(1999)，J.Mol.Biol. 293:41-56和Roovers等人(2001)，Cancer Immunol.描述。
在一些情况下，抗体的抗原结合片段是双抗体，即，双价抗体，其中VH和VL结构域在单个多肽链上表达，但使用太短的连接体以致不允许在相同链的两个结构域之间配对，从而迫使结构域与另一条链的互补结构域配对并且产生两个抗原结合部位(参见，例如,Holliger P.等人,Proc.Natl.Acad.Sci.USA 90:6444-6448(1993)，和Poljak R.J.等人,Structure 2:1121-1123(1994))。
在另一些情况下，抗体的抗原结合片段是“双特异性抗体”，指由第一抗体(片段)和第二抗体(片段)或抗体类似物通过偶联臂所形成的偶联物，偶联的方式包括但不限于化学反应、基因融合和酶促。抗体的抗原结合片段可以是“多特异性抗体”包括例如：三特异性抗体和四特异性抗体，前者是具有三种不同抗原结合特异性的抗体，而后者是具有四种不同抗原结合特异性的抗体。例如，经设计的锚蛋白重复蛋白(DARPin)，与IgG抗体，scFv-Fc抗体片段相连或其组合，如CN104341529A。抗IL-17a的fynomer与抗IL-6R抗体结合，如WO2015141862A1。
可使用本领域技术人员已知的常规技术(例如，重组DNA技术或酶促或化学断裂法)从给定的抗体(例如本发明提供的单克隆抗体ADI-56127、ADI-56204、ADI-56249和ADI-56145)获得抗体的抗原结合片段(例如，上述抗体片段)，并且以与用于完整抗体的方式相同的方式就特异性筛选抗体的抗原结合片段。
如本文中所使用的，术语“单抗”和“单克隆抗体”是指，来自一群高度同源的抗体分子中的一个抗体或抗体的一个片段，也即除可能自发出现的自然突变外，一群完全相同的抗体分子。单抗对抗原上的单一表位具有高特异性。多克隆抗体是相对于单克隆抗体而言的，其通常包含至少2种或更多种的不同抗体，这些不同的抗体通常识别抗原上的不同表位。单克隆抗体通常可采用Kohler等首次报道的杂交瘤技术获得(G,Milstein C.Continuous cultures of fused cells secreting antibody of predefined specificity[J].nature,1975；256(5517):495)，但也可采用重组DNA技术获得(如参见U.S.Patent 4,816,567)。
如本文中所使用的，术语“人源化抗体”是指，人源免疫球蛋白(受体抗体)的全部或部分CDR区被一非人源抗体(供体抗体)的CDR区替换后得到的抗体或抗体片段，其中的供体抗体可以是具有预期特异性、亲和性或反应性的非人源(例如，小鼠、大鼠或兔)抗体。此外，受体抗体的构架区(FR)的一些氨基酸残基也可被相应的非人源抗体的氨基酸残基替换，或被其他抗体的氨基酸残基替换，以进一步完善或优化 抗体的性能。关于人源化抗体的更多详细内容，可参见例如，Jones et al.,Nature 1986；321:522-525；Reichmann et al.,Nature 1988；332:323-329；Presta,Curr.Op.Struct.Biol.,1992；2:593-596；和Clark M.Antibody humanization:a case of the‘Emperor’s new clothes’？[J].Immunol.Today,2000；21(8):397-402。
如本文中所使用的，术语“分离的”或“被分离的”指的是，从天然状态下经人工手段获得的。如果自然界中出现某一种“分离”的物质或成分，那么可能是其所处的天然环境发生了改变，或从天然环境下分离出该物质，或二者情况均有发生。例如，某一活体动物体内天然存在某种未被分离的多聚核苷酸或多肽，而从这种天然状态下分离出来的高纯度的相同的多聚核苷酸或多肽即称之为分离的。术语“分离的”或“被分离的”不排除混有人工或合成的物质，也不排除存在不影响物质活性的其它不纯物质。
如本文中所使用的，术语“载体(vector)”是指，可将多聚核苷酸插入其中的一种核酸运载工具。当载体能使插入的多核苷酸编码的蛋白获得表达时，载体称为表达载体。载体可以通过转化，转导或者转染导入宿主细胞，使其携带的遗传物质元件在宿主细胞中获得表达。载体是本领域技术人员公知的，包括但不限于：质粒；噬菌粒；柯斯质粒；人工染色体，例如酵母人工染色体(YAC)、细菌人工染色体(BAC)或P1来源的人工染色体(PAC)；噬菌体如λ噬菌体或M13噬菌体及动物病毒等。可用作载体的动物病毒包括但不限于，逆转录酶病毒(包括慢病毒)、腺病毒、腺相关病毒、疱疹病毒(如单纯疱疹病毒)、痘病毒、杆状病毒、乳头瘤病毒、乳头多瘤空泡病毒(如SV40)。一种载体可以含有多种控制表达的元件，包括但不限于，启动子序列、转录起始序列、增强子序列、选择元件及报告基因。另外，载体还可含有复制起始位点。
如本文中所使用的，术语“宿主细胞”是指，可用于导入载体的细胞，其包括但不限于，如大肠杆菌或枯草杆菌等的原核细胞，如酵母细胞或曲霉菌等的真菌细胞，如S2果蝇细胞或Sf9等的昆虫细胞，或者如纤维原细胞，CHO细胞，COS细胞，NSO细胞，HeLa细胞，GS细胞，BHK细胞，HEK 293细胞或人细胞等的动物细胞。
如本文中所使用的，术语“单克隆抗体”和“单抗”具有相同的含义且可互换使用；术语“多克隆抗体”和“多抗”具有相同的含义且可互换使用；术语“多肽”和“蛋白质”具有相同的含义且可互换使用。并且在本发明中，氨基酸通常用本领域公知的单字母和三字母缩写来表示。例如，丙氨酸可用A或Ala表示。
如本文中所使用的，术语“药学上可接受的辅料”是指在药理学和/或生理学上与受 试者和活性成分相容的载体和/或赋形剂，其是本领域公知的(参见例如Remington's Pharmaceutical Sciences.Edited by Gennaro AR,19th ed.Pennsylvania:Mack Publishing Company,1995)，并且包括但不限于：pH调节剂，表面活性剂，佐剂，离子强度增强剂。例如，pH调节剂包括但不限于磷酸盐缓冲液；表面活性剂包括但不限于阳离子，阴离子或者非离子型表面活性剂，例如Tween-80；离子强度增强剂包括但不限于氯化钠。
如本文中所使用的，术语“有效量”是指足以获得或至少部分获得期望的效果的量。例如，预防疾病(例如肿瘤)有效量是指，足以预防，阻止，或延迟疾病(例如肿瘤)的发生的量；治疗疾病有效量是指，足以治愈或至少部分阻止已患有疾病的患者的疾病和其并发症的量。
如本文中所使用的，术语“杂交瘤”和“杂交瘤细胞株”可互换使用，并且当提及术语“杂交瘤”和“杂交瘤细胞株”时，其还包括杂交瘤的亚克隆和后代细胞。
在本发明中，如果没有特别说明，所述“第一”(例如第一产品)、“第二”(例如第二产品)和“第三”(例如第三产品)是为了指代上的区分或表述上的清楚，并不具有典型的次序上的含义。
发明的有益效果
本发明实现了如下的(1)至(4)项中所述技术效果中的一项或多项：
(1)本发明的抗体能够与PVRIG结合，亲和力较高。
(2)本发明的抗体与抗TIGIT抗体或者抗PD-1抗体或者抗PD-L1抗体之间很可能具有协同作用。
(3)本发明的抗体能够有效地治疗和/或预防肿瘤，例如结肠癌、黑色素瘤、肺癌、肾癌、子宫内膜癌、乳腺癌、皮肤癌、卵巢癌、胃癌、头颈癌、肝癌、脑瘤、尿路上皮癌、骨肿瘤、胆管癌、直肠癌、胰腺癌、宫颈瘤、多发性骨髓瘤、霍奇金淋巴瘤、非霍奇金淋巴瘤、B淋巴细胞瘤、浆细胞癌、前列腺癌和睾丸癌等。
(4)本发明的抗体具有较低的毒副作用。
附图说明
图1A至1B：抗PVRIG抗体与CHO细胞上过表达的人PVRIG的结合曲线图。
图2A至2B：抗PVRIG抗体与CHO细胞上过表达的食蟹猴PVRIG的结合曲线 图。
图3A至3B：抗PVRIG抗体与CHO细胞上过表达的小鼠PVRIG的结合曲线图。
图4A至4B：抗PVRIG抗体阻断人PVRIG与CHO细胞上过表达的人CD112结合的曲线图。
图5A至5B：抗PVRIG抗体阻断小鼠CD112与小鼠PVRIG蛋白结合的曲线图。
图6：抗PVRIG抗体与激活的人原代T细胞上PVRIG的结合曲线图。
图7：抗PVRIG抗体与抗TIGIT抗体联合阻断PVRIG/CD112、TIGIT/CD155信号通路的曲线图。
图8：抗PVRIG抗体与抗TIGIT及抗PD-L1抗体联合阻断PVRIG/CD112、TIGIT/CD155和PD-1/PD-L1信号通路的曲线图。
图9A至9C：抗PVRIG抗体依次介导过表达人PVRIG、过表达食蟹猴PVRIG、过表达小鼠PVRIG CHO细胞ADCC试验结果。
图10：抗PVRIG抗体在野生型小鼠CT-26肿瘤模型的药效图。
图11：抗PVRIG抗体在混合接种A375和人PBMC的NDG小鼠模型的药效图。
图12：抗PVRIG抗体联合抗TIGIT抗体在混合接种A375和人PBMC的NDG小鼠模型的药效图。
图13：抗PVRIG抗体联合抗TIGIT及抗PD-1抗体在混合接种A375和人PBMC的NDG小鼠模型的药效图。
图14：抗PVRIG抗体在小鼠体内半衰期曲线。
具体实施方式
下面将结合实施例对本发明的实施方案进行详细描述，但是本领域技术人员将会理解，下列实施例仅用于说明本发明，而不应视为限定本发明的范围。实施例中未注明具体条件者，按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者，均为可以通过市购获得的常规产品。
抗体Atezolizumab(ATE)：抗PD-L1单抗，商品名Tecentriq，罗氏Roche。
由HEK293细胞表达并纯化实施例中使用的下述对照抗体：
COM701是由HEK293细胞表达的来自Compugen公司的抗人PVRIG抗体，其轻链可变区序列和重链可变区序列与美国专利US10227408B2中的SEQ ID NO:1372、 SEQ ID NO:1380一致；
Mab46是由HEK293细胞表达的来自Surface Oncology公司的抗人PVRIG抗体，其轻链可变区和重链可变区序列与美国专利公开US20200040081A1中的SEQ ID NO:912、SEQ ID NO:918一致。
Tiragolumab由HEK293细胞表达的来自Genentech公司的抗人TIGIT抗体，其轻链和重链序列来源于WHO Drug Information,Vol.31,No.2,2017，Proposed INN:List 117，CAS号：1918185-84-8。
实施例1：抗体的序列设计、表达和纯化
1.抗体的序列设计
基于酵母抗体展示文库(Adimab)，按照现有的方法(参见专利申请WO2009036379、WO2010105256和WO2012009568)进行扩增和优化。通过筛选获得的酵母细胞在30℃下震荡诱导48小时以分泌表达目标抗PVRIG抗体(全长IgG)。诱导结束之后，1300rpm离心10分钟去除酵母细胞，收获上清液。使用Protein A对上清液中的抗PVRIG抗体进行纯化，pH2.0醋酸溶液洗脱，收获抗PVRIG抗体。其中获得抗PVRIG抗体ADI-56127、ADI-56204、ADI-56249和ADI-56145。同时，将抗体ADI-56127、ADI-56204、ADI-56249和ADI-56145分别使用木瓜蛋白酶酶切并用KappaSelect(GE生命医疗集团)进行纯化获得相应抗体Fab片段。
4个抗体ADI-56127、ADI-56204、ADI-56249和ADI-56145的可变区序列信息如下。
ADI-56127的重链可变区：
ADI-56127的轻链可变区：
ADI-56204的重链可变区：
ADI-56204的轻链可变区：
ADI-56249的重链可变区：
ADI-56249的轻链可变区：
ADI-56145的重链可变区：
ADI-56145的轻链可变区：
上面各抗体的CDR如下：
(1)ADI-56127
HCDR1：GTESVDAIS(SEQ ID NO:9)
HCDR2：DIIPFFDTDYAQKFQG(SEQ ID NO:10)
HCDR3：AREGGTSWTHFFDL(SEQ ID NO:11)
LCDR1：RASQSVSSSYLA(SEQ ID NO:12)
LCDR2：GASSRAT(SEQ ID NO:13)
LCDR3：QQYGSSPIT(SEQ ID NO:14)
(2)ADI-56204
HCDR1：YTFTHYGIY(SEQ ID NO:15)
HCDR2：HIYAYSGDTDYAQKLQG(SEQ ID NO:16)
HCDR3：ARGSPTYEHFNWFDP(SEQ ID NO:17)
LCDR1：QASDDISDYLN(SEQ ID NO:18)
LCDR2：DASSLHT(SEQ ID NO:19)
LCDR3：QQYGNLPIT(SEQ ID NO:20)
(3)ADI-56249
HCDR1：GTFSTFAIS(SEQ ID NO:21)
HCDR2：DIIPIFGSNYAQKFQG(SEQ ID NO:22)
HCDR3：ARGPLEGKVSPFDI(SEQ ID NO:23)
LCDR1：RSSQSLLHSTGYNYLD(SEQ ID NO:24)
LCDR2：VGSTRAS(SEQ ID NO:25)
LCDR3：MQERQFPIT(SEQ ID NO:26)
(4)ADI-56145
HCDR1：YTFTSYGIS(SEQ ID NO:27)
HCDR2：WISSYDASTKYSQELQG(SEQ ID NO:28)
HCDR3：ARGSPIYEHFNWFDP(SEQ ID NO:29)
LCDR1：RASQSVSSYLA(SEQ ID NO:30)
LCDR2：DSSNRAT(SEQ ID NO:31)
LCDR3：QQLSHHPLT(SEQ ID NO:32)
1.2抗体及其变体的表达与纯化
对于抗体ADI-56127、ADI-56204和对照抗体Mab46，均分别将其重链可变区(VH)重组到野生型人IgG1重链恒定区(氨基酸序列如SEQ ID NO:33所示)。此外，对于抗体ADI-56127、ADI-56204、ADI-56249、ADI-56145和对照抗体Mab46，均分别将其重链可变区(VH)重组到经L234A、L235A改造的重链恒定区(氨基酸序列如SEQ ID NO:34所示)中，构成完整的“VH-CH1-CH2-CH3”抗体重链。对于对照抗体COM701，将其重链可变区重组到人IgG4重链恒定区(氨基酸序列如SEQ ID NO:35所示)中。此外，将上述抗体的轻链可变区(VL)均重组到人kappa轻链恒定区(氨基酸序列如SEQ ID NO:36所示)。经由HEK293表达系统瞬时表达纯化。具体操作如下：使用化学转染的方法将带有抗体重链和轻链的pcDNA3.1载体转入HEK293细胞中，在37℃，8％CO2条件下，培养7天。收集细胞液，13000rpm离心20分钟。取上清液，Protein A纯化上清液，SEC检测抗体纯度，同时控制内毒素含量。制得的抗体分别命名为ADI-56127-G1、ADI-56127-G1LALA、ADI-56204-G1、ADI-56204-G1LALA、ADI-56249-G1LALA、ADI-56145-G1LALA、Mab46-G1、Mab46-G1LALA和COM701-G4。
野生型人IgG1重链恒定区：
经L234A、L235A改造的重链恒定区：

人IgG4重链恒定区：
人kappa轻链恒定区：
实施例2：抗体亲和力检测
采用生物膜层光学干涉技术(ForteBio)测定实施例1获得的抗体及其变体或Fab片段与人、食蟹猴、小鼠PVRIG的结合解离常数(KD)。Fortebio亲和力测定按照现有方法(Este,P等人High throughput solution-based measurement of antibody-antigen affinity and epitope binning.Mabs,2013.5(2):p.270-8)进行。所述人、食蟹猴、小鼠PVRIG的胞外段氨基酸序列分别如SEQ ID NOs:37-39所示。
人PVRIG胞外段氨基酸序列:
食蟹猴PVRIG胞外段氨基酸序列：
小鼠PVRIG胞外段氨基酸序列：
测量完整抗体(即，通过Adimab原始获得的全长IgG)与人的PVRIG-his蛋白的单价亲和力：传感器在分析缓冲液中线下平衡20分钟，然后线上检测120s建立基线，加载完整的PVRIG抗体至AHQ传感器至厚度1nm进行亲和力检测。将已加载抗体的传感器于100nM PVRIG-his抗原中作用至平台期，然后将传感器转移到分析缓冲液中至少2分钟用于解离速率测量。使用1:1结合模型进行动力学分析。
测量候选抗体Fab片段(即全长IgG经木瓜蛋白酶处理后得到的Fab片段)与人、食蟹猴、小鼠PVRIG-Fc蛋白的单价亲和力：传感器在分析缓冲液中线下平衡20分钟，然后线上检测120s建立基线，加载PVRIG-Fc抗原至AHQ传感器至厚度1nm进行亲和力检测。将已加载抗原的传感器于100nM Fab液中作用至平台期，然后将传感器转移到分析缓冲液中至少2分钟用于解离速率测量。使用1:1结合模型进行动力学分析。
测量完整抗体与食蟹猴、小鼠PVRIG-Fc蛋白的双价亲和力：传感器在分析缓冲液中线下平衡20分钟，然后线上检测120s建立基线，加载完整的PVRIG抗体至AHQ传感器至厚度1nm进行亲和力检测。将已加载抗体的传感器于高浓度无关完整抗体液中继续加载10分钟，饱和封闭AHQ传感器上Fc结合位点，将封闭完成的传感器于100nM PVRIG-Fc抗原中作用至平台期，然后将传感器转移到分析缓冲液中至少2分钟用于解离速率测量。使用1:1结合模型进行动力学分析。
在如上测定方法中，候选分子及其变体分子的KD值如下表1所示。
表1：抗PVRIG候选分子及其变体分子的KD值

注："N.B.":no binding(无结合)；"NA":not available(未检测)。
由表1结果可见：
ADI-56127和ADI-56249与小鼠PVRIG有交叉结合活性。
实施例3：抗PVRIG抗体与过表达人/食蟹猴/小鼠PVRIG CHO细胞的结合活性 及阻断活性
3.1基于流式细胞术检测法检测抗PVRIG抗体及其变体与过表达在CHO细胞上人/食蟹猴/小鼠PVRIG的结合活性。
具体地，通过转染克隆到MCS的人PVRIG、食蟹猴PVRIG、小鼠PVRIG cDNA的pCHO1.0载体(购自Invitrogen)加压筛选产生过表达人PVRIG的CHO细胞(CHO-huPVRIG细胞)，过表达食蟹猴PVRIG的CHO细胞(CHO-cynoPVRIG细胞)以及过表达小鼠PVRIG的CHO细胞(CHO-muPVRIG细胞)。将扩大培养的过表达细胞调整至合适细胞密度加入96孔流式板，离心后加入梯度稀释的待测样品，4℃孵育30分钟。PBS清洗两次，加入对应稀释至合适浓度的荧光二抗，4℃孵育30分钟，PBS清洗两次。加入PBS重悬细胞，在CytoFlex流式细胞仪上进行检测并计算对应的MFI。采用Graphpad软件作图分析获得EC50数值。结果如表2、图1A至图1B、图2A至图2B以及图3A至图3B所示。
3.2基于流式细胞术检测法检测抗PVRIG抗体及其变体，阻断人PVRIG与过表达在CHO细胞上人CD112结合的活性。
具体地，通过转染克隆到MCS的人CD112 cDNA的pCHO1.0载体(购自 Invitrogen)加压筛选产生过表达人CD112的CHO细胞(CHO-huCD112细胞)。将扩大培养的CHO-huCD112细胞调整细胞密度至2×106细胞/mL，100μL/孔加入96孔流式板，离心备用。用PBS梯度稀释纯化的单克隆抗体，将稀释好的样品60μL/孔加入空白96孔流式板中，然后60μL/孔加入1ug/mL的带有Mouse IgG2a Fc Tag的人PVRIG蛋白，混匀后4℃孵育30分钟。将上述孵育完毕的样品100μL/孔加入含有CHO-huCD112细胞的96孔流式板中，4℃共孵育30分钟，PBS清洗两次。100μL/孔加入用PBS稀释100倍的APC羊抗鼠IgG抗体，4℃孵育30分钟，PBS清洗两次。100μL/孔加入PBS重悬细胞，在CytoFlex流式细胞仪上进行检测并计算对应的MFI。结果如表2、图4A至图4B所示。
3.3基于ELISA检测法检测抗PVRIG抗体及其变体，阻断小鼠PVRIG蛋白与小鼠CD112蛋白结合的活性。
具体地，用1×包被缓冲液稀释mouse CD112-his蛋白至终浓度为1μg/mL，100μL/孔加入酶标板中，覆膜，4℃静置包被过夜。弃去酶标板中包被液，用1×PBST清洗3次，200μL/孔加入5％BSA/PBS室温封闭2小时。封闭期间，取待测样品用1％BSA/PBS梯度稀释，终体积为60μL/孔。取生物素标记的Mouse PVRIG huFc蛋白用1％BSA/PBS稀释至2μg/mL，60μL/孔加入上述样品孔中，混匀后室温孵育1小时。弃去酶标板中封闭液，100μL/孔加上上述样品混合液，室温孵育2小时。弃去上述共孵育样品，用1×PBST清洗3次，100μL/孔加入1％BSA/PBS稀释的SA-HRP，室温孵育1小时。弃去SA-HRP稀释液，1×PBST清洗3次，100μL/孔加入ELISA显色液室温反应1-3分钟，50μL/孔加入ELISA终止液，读取450nm处吸光度数值。采用Graphpad软件作图获得浓度-吸光度值的结合曲线图，结果如表2、图5A至图5B所示。
表2：抗PVRIG候选分子及其变体分子过表达细胞结合阻断活性汇总表


注："N.B.":no binding(无结合)；"NA":not available(未检测)。
由表2、图1A至图1B、图2A至图2B以及图3A至图3B可以得出如下结论：
(1)ADI-56249-G1LALA、ADI-56145-G1LALA、ADI-56204-G1、ADI-56204-G1LALA、ADI-56127-G1、ADI-56127-G1LALA与CHO细胞表面过表达的人PVRIG蛋白的结合活性优于对照分子Mab46-G1、Mab46-G1LALA和COM701-G4；
(2)ADI-56249-G1LALA、ADI-56204-G1、ADI-56204-G1LALA、ADI-56127-G1、ADI-56127-G1LALA与CHO细胞表面过表达的食蟹猴PVRIG蛋白的结合活性优于对照分子Mab46-G1、Mab46-G1LALA和COM701-G4；
(3)ADI-56127-G1、ADI-56127-G1LALA与CHO细胞表面过表达的小鼠PVRIG蛋白有明显结合，结合活性与对照分子Mab46-G1、Mab46-G1LALA相当。
由表2和图4A至图4B、图5A至图5B可以得出如下结论：
(1)ADI-56145-G1LALA、ADI-56204-G1、ADI-56204-G1LALA、ADI-56127-G1、ADI-56127-G1LALA阻断人PVRIG与CHO细胞表面过表达的人CD112蛋白结合的活性与对照分子Mab46-G1、Mab46-G1LALA和COM701-G4相当；
(2)部分与CHO细胞表面过表达的小鼠PVRIG蛋白有结合的抗PVRIG抗体分子(ADI-56127-G1和ADI-56127-G1LALA)能够明显阻断小鼠PVRIG与小鼠CD112蛋白的结合，且阻断活性优于对照分子Mab46-G1、Mab46-G1LALA。
实施例4：抗PVRIG抗体与原代T细胞表面PVRIG结合
基于流式细胞术检测法检测发明抗PVRIG抗体与激活T细胞表面的PVRIG结 合活性。
具体地，将人PBMC按照STEMCELL公司提供的试验方案(stemcell,货号：#17951C)进行分选获得human total T细胞。用X-VIVO15培养基(购自Lonza，货号：04-418Q)调整T细胞浓度至1.0×106cells/mL，加入1μL IL-2储备液(100万IU)，同时1:1(bead-to-cell)加入CD3/CD28Dynabeads(购自gibco，货号：11132D)，于37℃，5％CO2培养箱内培养48小时。将活化后的T细胞调整至合适细胞密度加入96孔流式板，离心后加入梯度稀释的待测样品，4℃孵育30分钟。PBS清洗两次，加入对应稀释至合适浓度的荧光二抗，4℃孵育30分钟，PBS清洗两次。加入PBS重悬细胞，在CytoFlex流式细胞仪上进行检测并计算对应的MFI。
结果如图6所示。结果表明，本发明抗PVRIG抗体ADI-56127-G1、ADI-56127-G1LALA能够结合激活T细胞表面的PVRIG分子，且结合活性优于对照分子Mab46-G1、Mab46-G1LALA、COM701-G4。
实施例5：荧光素酶报告基因系统检测抗PVRIG抗体与抗TIGIT抗体联合阻断 效应
为检测抗PVRIG抗体与抗TIGIT抗体在细胞水平的联合阻断活性，本实施例构建了如下荧光素酶报告基因系统，简单描述为，利用慢病毒转染细胞，构建了过表达人CD155、人CD112和OKT-3scFv的CHO-K1细胞株(CHO-K1-CD155-CD112)，构建了过表达人TIGIT、人PVRIG和NF-AT luciferase报告基因的Jurkat细胞株(Jurkat-TIGIT-PVRIG-luc)，后续利用这一报告基因系统开展相关试验。
具体地，消化获取CHO-K1-CD155-CD112功能细胞，调整细胞密度，100μL/孔加入96孔白底板中，贴壁培养过夜。第二天，制备Jurkat-TIGIT-PVRIG-luc效应细胞悬液，将待测样品用反应培养基梯度稀释。取出白底板，吸去培养上清，将上述稀释好的样品40μL/孔加入白底板，同时40μL/孔加入Jurkat-TIGIT-PVRIG-luc效应细胞悬液，于37℃，5％CO2培养箱培养6h。培养完成后80μL/孔加入Bio-GloTM reagent，并使用多功能酶标仪读取荧光信号值。
结果如图7所示。结果表明，本发明抗PVRIG抗体ADI-56127-G1LALA、ADI-56145-G1LALA、ADI-56204-G1LALA、ADI-56249-G1LALA分别按照摩尔比1：1联合抗TIGIT抗体Tiragolumab均能够解除CD112介导的PVRIG下游抑制信号通路，以及CD155介导的TIGIT下游抑制信号通路，更大程度增强报告基因荧光素酶 表达，且活性和对照分子COM701-G4相当。
实施例6：荧光素酶报告基因系统检测抗PVRIG抗体与抗TIGIT及抗PD-L1抗 体联合阻断效应
为检测抗PVRIG抗体与抗TIGIT及抗PD-L1抗体在细胞水平的联合阻断活性，本实施例构建了如下荧光素酶报告基因系统，简略描述为，在实施例5的基础上，慢病毒感染CHO-K1-CD155-CD112以过表达PD-L1获得CHO-K1-CD155-CD112-PD-L1功能细胞，慢病毒感染Jurkat-TIGIT-PVRIG-luc以过表达PD-1，获得Jurkat-TIGIT-PVRIG-PD-1-luc效应细胞悬液，后续利用这一报告基因系统开展相关试验。
具体地，消化获取CHO-K1-CD155-CD112-PD-L1功能细胞，调整细胞密度，100μL/孔加入96孔白底板中，贴壁培养过夜。第二天，制备Jurkat-TIGIT-PVRIG-PD-1-luc效应细胞悬液，将待测样品用反应培养基梯度稀释。取出白底板，吸去培养上清，将上述稀释好的样品40μL/孔加入白底板，同时40μL/孔加入Jurkat-TIGIT-PVRIG-PD-1-luc效应细胞悬液，置于37℃，5％CO2培养箱培养6小时。培养完成后80μL/孔加入Bio-GloTM reagent，使用多功能酶标仪读取荧光信号值。
结果如图8所示。结果表明，本发明抗PVRIG抗体ADI-56127-G1LALA、ADI-56145-G1LALA、ADI-56204-G1LALA、ADI-56249-G1LALA分别按照1：1：1的摩尔比与抗TIGIT抗体Tiragolumab及抗PD-L1抗体Atezolizumab(ATE)联合均能够通过分别阻断PVRIG/CD112、TIGIT/CD155和PD-1/PD-L1介导的下游抑制信号，进一步增强报告基因荧光素酶表达，且活性和对照分子COM701-G4相当。
实施例7：抗PVRIG抗体体外ADCC活性检测
基于荧光素酶报告基因系统，检测发明抗PVRIG抗体体外ADCC活性。
具体地，扩大培养Jurkat-NFAT-Luciferase-CD16ADCC效应细胞(购自Promega)，用含10％low IgG FBS的1640培养基重悬细胞至4×106细胞/mL。用含10％low IgG FBS的1640培养基重悬CHO-huPVRIG细胞，CHO-cynoPVRIG细胞，CHO-muPVRIG细胞稀释至1.6×106细胞/mL。将上述3种细胞悬液分别与Jurkat-NF-AT-Luciferase-CD16细胞1:1混合均匀，50μL/孔加入无菌96孔白底板内， 加入含10％low IgG FBS的1640培养基梯度稀释的待检测抗体样品。37℃，5％CO2共孵育6小时。培养完成，取出细胞，室温平衡5分钟，100μL/孔加入Bio-Glo TM reagent，使用多功能酶标仪读取荧光信号值。
结果如图9A至图9C所示。结果表明，本发明抗PVRIG抗体ADI-56204-G1、ADI-56127-G1在荧光素酶报告基因系统中可以体外介导对CHO-huPVRIG细胞、CHO-cynoPVRIG细胞、CHO-muPVRIG细胞的ADCC活性，且ADI-56204-G1介导对CHO-huPVRIG细胞、CHO-cynoPVRIG细胞的ADCC活性强于对照分子Mab46-G1。而ADI-56127-G1介导对CHO-huPVRIG细胞、CHO-cynoPVRIG细胞、CHO-muPVRIG细胞的ADCC活性与对照分子Mab46-G1相当。
实施例8：抗PVRIG抗体在野生型Balb/c小鼠体内药效学研究
本实验采用在野生型Balb/c小鼠体内接种CT-26小鼠结肠癌细胞(购自江苏集萃药康生物科技股份有限公司)测定本发明抗PVRIG抗体的抗肿瘤作用。
具体地，首先制备CT-26细胞悬液，在小鼠右腹沟皮下注射0.1mL约5×105细胞建立CT-26荷瘤小鼠模型。待平均肿瘤体积长至约100mm3时进行分组，腹腔注射给予不同剂量、相同给药体积的PBS或抗体进行治疗，每组6只小鼠。监测各组小鼠瘤体积和体重变化，监测频率均为2-3天/次，连续监测2到3周，给药剂量和方式如表3。
表3：抗PVRIG抗体肿瘤抑制活性试验方案
结果如图10所示。结果表明，本发明抗PVRIG抗体ADI-56127-G1能够显著抑制小鼠CT26肿瘤的生长。
实施例9：抗PVRIG抗体在NDG小鼠混合接种A375和人PBMC体内药效学研 究
本实验在B-NDG小鼠混合接种A375(购自Addexbio,C0020004，一种人恶性黑色素瘤细胞)，人PBMC细胞(上海妙顺,A10S033014/PB100C)的模型(A375huPBMC 模型)中测定本发明的抗PVRIG抗体的抗肿瘤作用。其中，通过将人的免疫细胞(PBMC)接种至免疫缺陷小鼠中，产生局部重组人类免疫系统的人源化肿瘤小鼠模型。
具体地，首先A375细胞与人PBMC 1：1等体积混合成0.1mL细胞悬液，在小鼠右腹沟以皮下注射的方式建立A375huPBMC模型。待平均肿瘤体积长至30-50mm3左右时进行分组，腹腔注射给予不同剂量、相同给药体积的PBS或抗体治疗，每组6只小鼠。监测各组小鼠瘤体积和体重变化，监测频率均为2-3天/次，连续监测2到3周，给药剂量和方式如表4。
表4：抗PVRIG抗体A375PBMC模型给药方案
结果如图11所示。结果表明，本发明的抗PVRIG抗体ADI-56127-G1可以显著抑制小鼠A375肿瘤的生长，且抗肿瘤活性优于对照分子Mab46-G1和COM701-G4。
实施例10：抗PVRIG抗体联合抗TIGIT抗体在NDG小鼠混合接种A375和人 PBMC体内药效学研究
本实验采用皮下混合接种的方式建立A375，人PBMC荷瘤小鼠模型(建模步骤与实施例9相同)，待平均肿瘤体积长至60mm3左右时进行分组，腹腔注射给予不同剂量、相同给药体积的PBS或抗体治疗，每组6只小鼠。监测各组小鼠瘤体积和体重变化，监测频率均为2-3天/次，连续监测2到3周，给药剂量和方式如表5。
表5：抗PVRIG抗体与抗TIGIT抗体A375模型给药方案
结果如图12所示。结果表明，联合施用本发明的抗PVRIG抗体ADI-56127-G1与抗TIGIT单抗相较于两单药治疗具有更显著的抑瘤作用。
本实施例中用到的抗TIGIT单抗的序列如下：
抗TIGIT抗体VH氨基酸序列：
抗TIGIT抗体VL氨基酸序列：
上述抗TIGIT抗体的CDR如下：
HCDR1：GSISSYDHYWT(SEQ ID NO:42)
HCDR2：TVYYSGSTFHNPSLKS(SEQ ID NO:43)
HCDR3：ARVGPDVSHPPFDY(SEQ ID NO:44)
LCDR1：RASQSISSYLN(SEQ ID NO:45)
LCDR2：AASSLQS(SEQ ID NO:46)
LCDR3：QQSYSTPIT(SEQ ID NO:47)
本实施例中用到的抗TIGIT单抗重链恒定区采用野生型人IgG1重链恒定区(SEQ ID NO:33)。
本实施例中用到的抗TIGIT单抗轻链恒定区采用人kappa轻链恒定区(SEQ ID NO:36)。
实施例11：抗PVRIG抗体联合抗PD-1及抗TIGIT抗体在NDG小鼠混合接种 A375和人PBMC体内药效学研究
本实验首先采用皮下混合接种的方式建立A375，人PBMC荷瘤小鼠模型(建模步骤与实施例9相同)，待平均肿瘤体积长至200mm3左右时进行分组，腹腔注射给予不同剂量、相同给药体积的PBS或抗体治疗，每组6只小鼠。监测各组小鼠瘤体积和体重变化，监测频率均为2-3天/次，连续监测2到3周，给药剂量和方式如表6。
表6：抗PVRIG抗体与抗PD-1及抗TIGIT抗体A375模型给药方案
结果如图13所示，结果表明联合施用本发明的抗PVRIG抗体ADI-56127-G1、抗TIGIT单抗和抗PD-1抗体能够有效地抑制肿瘤生长，且对A375的肿瘤抑制活性明显优于其它给药组。
本实施例中用到的抗TIGIT单抗的序列与实施例10一致。
本实施例中用到的抗PD-1单抗的序列如下：
抗PD-1抗体VH氨基酸序列
抗PD-1抗体VL氨基酸序列
上述抗PD-1抗体的6个CDR如下：
HCDR1：YTFTEYYIY(SEQ ID NO:50)
HCDR2：GINPSNGGTNFNEKFKP(SEQ ID NO:51)
HCDR3：TVRDFRFDKGFKY(SEQ ID NO:52)
LCDR1：RASKSVSTSGLNYVH(SEQ ID NO:53)
LCDR2：LGSYLDS(SEQ ID NO:54)
LCDR3：QQSWELPLT(SEQ ID NO:55)
本实施例中用到的抗PD-1单抗重链恒定区采用经L234A、L235A改造的IgG1重链恒定区(SEQ ID NO:34)
本实施例中用到的抗PD-1单抗轻链恒定区采用人kappa轻链恒定区(SEQ ID NO:36)
实施例12：抗PVRIG抗体与小鼠体内半衰期研究
采用尾静脉单次注射法，检测本发明抗PVRIG抗体ADI-56127-G1在小鼠体内半衰期。
具体地，实验用Balb/c小鼠，雌雄各3只，12/12小时光/暗调节，温度24℃±2℃，湿度40％-70％，自由进水饮食。实验当天对Balb/c小鼠单次尾静脉注射单克隆抗体分子，注射剂量为10mg/kg。取血时间点：给药后5分钟、0.5小时、2小时、6小时、24小时、48小时、96小时、168小时、336小时、504小时于小鼠眼眶采血。全血样品2℃-8℃放置30分钟，12000rpm离心5分钟收集血清，所得血清再于2℃-8℃，12000rpm离心5分钟，-80℃保存，ELISA检测血清中单克隆抗体分子含量。
结果如图14所示。结果表明，本发明抗PVRIG抗体ADI-56127-G1单次注射在小鼠体内半衰期为160小时。
尽管本发明的具体实施方式已经得到详细的描述，本领域技术人员将会理解。根据已经公开的所有教导，可以对那些细节进行各种修改和替换，这些改变均在本发明的保护范围之内。本发明的全部范围由所附权利要求及其任何等同物给出。

Claims (26)

  1. 抗PVRIG抗体或其抗原结合片段，其中，
    所述抗体包含重链可变区和轻链可变区，所述重链可变区包含HCDR1至HCDR3，所述轻链可变区包含LCDR1至LCDR3，其中：
    所述抗体的重链可变区，
    HCDR1的氨基酸序列如SEQ ID NO:9所示、HCDR2的氨基酸序列如SEQ ID NO:10所示和HCDR3的氨基酸序列如SEQ ID NO:11所示，
    HCDR1的氨基酸序列如SEQ ID NO:15所示、HCDR2的氨基酸序列如SEQ ID NO:16所示和HCDR3的氨基酸序列如SEQ ID NO:17所示，
    HCDR1的氨基酸序列如SEQ ID NO:21所示、HCDR2的氨基酸序列如SEQ ID NO:22所示和HCDR3的氨基酸序列如SEQ ID NO:23所示，或者
    HCDR1的氨基酸序列如SEQ ID NO:27所示、HCDR2的氨基酸序列如SEQ ID NO:28所示和HCDR3的氨基酸序列如SEQ ID NO:29所示；
    并且
    所述抗体的轻链可变区，
    LCDR1的氨基酸序列如SEQ ID NO:12所示、LCDR1的氨基酸序列如SEQ ID NO:13所示和LCDR1的氨基酸序列如SEQ ID NO:14所示，
    LCDR1的氨基酸序列如SEQ ID NO:18所示、LCDR1的氨基酸序列如SEQ ID NO:19所示和LCDR1的氨基酸序列如SEQ ID NO:20所示，
    LCDR1的氨基酸序列如SEQ ID NO:24所示、LCDR1的氨基酸序列如SEQ ID NO:25所示和LCDR1的氨基酸序列如SEQ ID NO:26所示，或者
    LCDR1的氨基酸序列如SEQ ID NO:30所示、LCDR1的氨基酸序列如SEQ ID NO:31所示和LCDR1的氨基酸序列如SEQ ID NO:32所示。
  2. 根据权利要求1所述的抗PVRIG抗体或其抗原结合片段，其中，
    HCDR1的氨基酸序列如SEQ ID NO:9所示，HCDR2的氨基酸序列如SEQ ID NO:10所示，HCDR3的氨基酸序列如SEQ ID NO:11所示，LCDR1的氨基酸序列如SEQ ID NO:12所示，LCDR2的氨基酸序列如SEQ ID NO:13所示和LCDR3的氨基酸序列如SEQ ID NO:14所示；
    HCDR1的氨基酸序列如SEQ ID NO:15所示，HCDR2的氨基酸序列如SEQ ID NO:16所示，HCDR3的氨基酸序列如SEQ ID NO:17所示，LCDR1的氨基酸序列如SEQ ID NO:18所示，LCDR2的氨基酸序列如SEQ ID NO:19所示和LCDR3的氨基酸序列如SEQ ID NO:20所示；
    HCDR1的氨基酸序列如SEQ ID NO:21所示，HCDR2的氨基酸序列如SEQ ID NO:22所示，HCDR3的氨基酸序列如SEQ ID NO:23所示，LCDR1的氨基酸序列如SEQ ID NO:24所示，LCDR2的氨基酸序列如SEQ ID NO:25所示和LCDR3的氨基酸序列如SEQ ID NO:26所示；
    或者
    HCDR1的氨基酸序列如SEQ ID NO:27所示，HCDR2的氨基酸序列如SEQ ID NO:28所示，HCDR3的氨基酸序列如SEQ ID NO:29所示，LCDR1的氨基酸序列如SEQ ID NO:30所示，LCDR2的氨基酸序列如SEQ ID NO:31所示和LCDR3的氨基酸序列如SEQ ID NO:32所示。
  3. 根据权利要求1至2中任一权利要求所述的抗PVRIG抗体或其抗原结合片段，其中，所述重链可变区的氨基酸序列选自SEQ ID NO:1、SEQ ID NO:3、SEQ ID NO:5和SEQ ID NO:7；
    并且
    所述轻链可变区的氨基酸序列选自SEQ ID NO:2、SEQ ID NO:4、SEQ ID NO:6和SEQ ID NO:8。
  4. 根据权利要求1至3中任一权利要求所述的抗PVRIG抗体或其抗原结合片段，其中，
    重链可变区的氨基酸序列如SEQ ID NO:1所示，并且轻链可变区的氨基酸序列如SEQ ID NO:2所示；
    重链可变区的氨基酸序列如SEQ ID NO:3所示，并且轻链可变区的氨基酸序列如SEQ ID NO:4所示；
    重链可变区的氨基酸序列如SEQ ID NO:5所示，并且轻链可变区的氨基酸序列如SEQ ID NO:6所示；
    或者
    重链可变区的氨基酸序列如SEQ ID NO:7所示，并且轻链可变区的氨基酸序列如SEQ ID NO:8所示。
  5. 根据权利要求1至4中任一权利要求所述的抗PVRIG抗体或其抗原结合片段，其中，所述抗体的重链恒定区为Ig gamma-1 chain C region或Ig gamma-4 chain C region；轻链恒定区为Ig kappa chain C region；
    优选地，所述抗体的重链恒定区氨基酸序列如SEQ ID NO：33、SEQ ID NO：34或SEQ ID NO：35所示，所述抗体的轻链恒定区氨基酸序列如SEQ ID NO：36所示。
  6. 根据权利要求1至5中任一权利要求所述的抗PVRIG抗体或其抗原结合片段，其中，按照EU编号系统，所述抗体的重链恒定区包含L234A突变和L235A突变。
  7. 根据权利要求1至6中任一权利要求所述的抗PVRIG抗体或其抗原结合片段，其中，所述抗PVRIG抗体或其抗原结合片段选自Fab、Fab'、F(ab')2、Fd、Fv、dAb、互补决定区片段、单链抗体、人源化抗体、嵌合抗体或双抗体。
  8. 根据权利要求1至7中任一权利要求所述的抗TIGIT抗体或其抗原结合片段，其中，
    所述的抗体包括非-CDR区，且所述非-CDR区来自不是鼠类的物种，例如来自人抗体。
  9. 根据权利要求1至8中任一权利要求所述的抗PVRIG抗体或其抗原结合片段，其中，所述抗体与CHO细胞表面过表达的人PVRIG蛋白的结合的EC50小于3nM、小于2.9nM或小于2.8nM；优选地，所述EC50通过流式细胞术检测法测得。
  10. 分离的核酸分子，其编码权利要求1至9中任一权利要求所述的抗TIGIT抗体或其抗原结合片段。
  11. 一种重组载体，其包含权利要求10所述的分离的核酸分子。
  12. 一种宿主细胞，其包含权利要求10所述的分离的核酸分子，或者权利要求11所述的重组载体。
  13. 偶联物，其包括抗体以及偶联部分，其中，所述抗体为权利要求1至9中任一权利要求所述的抗PVRIG抗体或其抗原结合片段，所述偶联部分为可检测的标记；优选地，所述偶联部分为放射性同位素、荧光物质、有色物质或酶。
  14. 试剂盒，其包括权利要求1至9中任一权利要求所述的抗PVRIG抗体或其抗原结合片段，或者包括权利要求13所述的偶联物；
    优选地，所述试剂盒还包括第二抗体，其特异性识别所述抗体；任选地，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、有色物质或酶。
  15. 一种药物组合物，其包含权利要求1至9中任一权利要求所述的抗PVRIG抗体或其抗原结合片段或者权利要求13所述的偶联物；可选地，所述药物组合物还包括一种或多种药学上可接受的辅料。
  16. 根据权利要求15所述的药物组合物，其还包含一种或多种抗TIGIT抗体；
    优选地，所述药物组合物还包含一种或多种抗PD-1抗体或者一种或多种抗PD-L1抗体。
  17. 根据权利要求15至16中任一权利要求所述的药物组合物，其中，
    抗PVRIG抗体或其抗原结合片段与抗TIGIT抗体的摩尔比为(1：5)-(5：1)；
    和/或
    抗PVRIG抗体或其抗原结合片段与抗PD-1抗体或者抗PD-L1抗体的摩尔比为(1：5)-(5：1)；
  18. 根据权利要求16至17中任一权利要求所述的药物组合物，其中，所述的抗TIGIT抗体，其包含重链可变区和轻链可变区，所述重链可变区包含HCDR1至HCDR3，所述轻链可变区包含LCDR1至LCDR3，其中：
    HCDR1的氨基酸序列如SEQ ID NO:42所示，HCDR2的氨基酸序列如SEQ ID NO: 43所示，HCDR3的氨基酸序列如SEQ ID NO:44所示，LCDR1的氨基酸序列如SEQ ID NO:45所示，LCDR2的氨基酸序列如SEQ ID NO:46所示和LCDR3的氨基酸序列如SEQ ID NO:47所示；
    优选地，所述的抗TIGIT抗体，其重链可变区的氨基酸序列如SEQ ID NO:40所示，并且轻链可变区的氨基酸序列如SEQ ID NO:41所示。
  19. 根据权利要求16至18中任一权利要求所述的药物组合物，其中，所述的抗PD-1抗体，其包含重链可变区和轻链可变区，所述重链可变区包含HCDR1至HCDR3，所述轻链可变区包含LCDR1至LCDR3，其中：
    HCDR1的氨基酸序列如SEQ ID NO:50所示，HCDR2的氨基酸序列如SEQ ID NO:51所示，HCDR3的氨基酸序列如SEQ ID NO:52所示，LCDR1的氨基酸序列如SEQ ID NO:53所示，LCDR2的氨基酸序列如SEQ ID NO:54所示和LCDR3的氨基酸序列如SEQ ID NO:55所示；
    优选地，所述的抗PD-1抗体，其重链可变区的氨基酸序列如SEQ ID NO:48所示，并且轻链可变区的氨基酸序列如SEQ ID NO:49所示。
  20. 一种组合产品，其包含独立包装的第一产品和第二产品，其中，
    所述第一产品包含其包含权利要求1至9中任一权利要求所述的抗PVRIG抗体或其抗原结合片段或者权利要求13所述的偶联物；
    所述第二产品包含至少一种抗TIGIT抗体；
    优选地，所述组合产品还包括第三产品，所述第三产品包含至少一种抗PD-1抗体或者至少一种抗PD-L1抗体；
    优选地，所述第一产品、所述第二产品和第三产品还独立地包含一种或多种药学上可接受的辅料；
    优选地，所述组合产品还包含产品说明书。
  21. 根据权利要求20所述的组合产品，其中，
    抗PVRIG抗体或其抗原结合片段与抗TIGIT抗体的摩尔比为(1：5)-(5：1)；
    和/或
    抗PVRIG抗体或其抗原结合片段与抗PD-1抗体或者抗PD-L1抗体的摩尔比为(1： 5)-(5：1)。
  22. 根据权利要求20至21中任一权利要求所述的组合产品，其中，所述的抗TIGIT抗体，其包含重链可变区和轻链可变区，所述重链可变区包含HCDR1至HCDR3，所述轻链可变区包含LCDR1至LCDR3，其中：
    HCDR1的氨基酸序列如SEQ ID NO:42所示，HCDR2的氨基酸序列如SEQ ID NO:43所示，HCDR3的氨基酸序列如SEQ ID NO:44所示，LCDR1的氨基酸序列如SEQ ID NO:45所示，LCDR2的氨基酸序列如SEQ ID NO:46所示和LCDR3的氨基酸序列如SEQ ID NO:47所示；
    优选地，所述的抗TIGIT抗体，其重链可变区的氨基酸序列如SEQ ID NO:40所示，并且轻链可变区的氨基酸序列如SEQ ID NO:41所示。
  23. 根据权利要求20至22中任一权利要求所述的组合产品，其中，所述的抗PD-1抗体，其包含重链可变区和轻链可变区，所述重链可变区包含HCDR1至HCDR3，所述轻链可变区包含LCDR1至LCDR3，其中：
    HCDR1的氨基酸序列如SEQ ID NO:50所示，HCDR2的氨基酸序列如SEQ ID NO:51所示，HCDR3的氨基酸序列如SEQ ID NO:52所示，LCDR1的氨基酸序列如SEQ ID NO:53所示，LCDR2的氨基酸序列如SEQ ID NO:54所示和LCDR3的氨基酸序列如SEQ ID NO:55所示；
    优选地，所述的抗PD-1抗体，其重链可变区的氨基酸序列如SEQ ID NO:48所示，并且轻链可变区的氨基酸序列如SEQ ID NO:49所示。
  24. 权利要求1至9中任一权利要求所述的抗体或其抗原结合片段或者权利要求13所述的偶联物在制备治疗或预防肿瘤的药物中的用途；
    优选地，所述肿瘤选自结肠癌、黑色素瘤、肺癌、肾癌、子宫内膜癌、乳腺癌、皮肤癌、卵巢癌、胃癌、头颈癌、肝癌、脑瘤、尿路上皮癌、骨肿瘤、胆管癌、直肠癌、胰腺癌、宫颈瘤、多发性骨髓瘤、霍奇金淋巴瘤、非霍奇金淋巴瘤、B淋巴细胞瘤、浆细胞癌、前列腺癌和睾丸癌中的一种或多种；
    优选地，所述肺癌为非小细胞肺癌或小细胞肺癌。
  25. 根据权利要求1至9中任一权利要求所述的抗体或其抗原结合片段或者权利要求13所述的偶联物，其用于治疗或预防肿瘤；
    优选地，所述肿瘤选自结肠癌、黑色素瘤、肺癌、肾癌、子宫内膜癌、乳腺癌、皮肤癌、卵巢癌、胃癌、头颈癌、肝癌、脑瘤、尿路上皮癌、骨肿瘤、胆管癌、直肠癌、胰腺癌、宫颈瘤、多发性骨髓瘤、霍奇金淋巴瘤、非霍奇金淋巴瘤、B淋巴细胞瘤、浆细胞癌、前列腺癌和睾丸癌中的一种或多种；
    优选地，所述肺癌为非小细胞肺癌或小细胞肺癌。
  26. 一种治疗或预防肿瘤的方法，包括给予有需求的受试者以有效量的权利要求1至9中任一权利要求所述的抗体或其抗原结合片段或者权利要求13所述的偶联物的步骤；
    优选地，所述肿瘤选自结肠癌、黑色素瘤、肺癌、肾癌、子宫内膜癌、乳腺癌、皮肤癌、卵巢癌、胃癌、头颈癌、肝癌、脑瘤、尿路上皮癌、骨肿瘤、胆管癌、直肠癌、胰腺癌、宫颈瘤、多发性骨髓瘤、霍奇金淋巴瘤、非霍奇金淋巴瘤、B淋巴细胞瘤、浆细胞癌、前列腺癌和睾丸癌中的一种或多种；
    优选地，所述肺癌为非小细胞肺癌或小细胞肺癌。
PCT/CN2023/085357 2022-04-02 2023-03-31 抗pvrig抗体、其药物组合物及用途 Ceased WO2023186063A1 (zh)

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