WO2021037007A1 - 抗pd-l1抗体及其应用 - Google Patents

抗pd-l1抗体及其应用 Download PDF

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WO2021037007A1
WO2021037007A1 PCT/CN2020/110935 CN2020110935W WO2021037007A1 WO 2021037007 A1 WO2021037007 A1 WO 2021037007A1 CN 2020110935 W CN2020110935 W CN 2020110935W WO 2021037007 A1 WO2021037007 A1 WO 2021037007A1
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amino acid
seq
antibody
acid sequence
cancer
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French (fr)
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房健民
姜静
李慎军
赵国锐
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Remegen Co Ltd
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Remegen Co Ltd
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Priority to JP2021549665A priority Critical patent/JP2022521958A/ja
Priority to EP20859087.7A priority patent/EP3858862A4/en
Priority to US17/312,884 priority patent/US12404333B2/en
Priority to KR1020217038569A priority patent/KR20220012856A/ko
Priority to CN202080013686.1A priority patent/CN113795510A/zh
Priority to BR112021016512A priority patent/BR112021016512A2/pt
Priority to AU2020339478A priority patent/AU2020339478A1/en
Priority to CA3114467A priority patent/CA3114467C/en
Priority to SG11202103221QA priority patent/SG11202103221QA/en
Publication of WO2021037007A1 publication Critical patent/WO2021037007A1/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
    • C07K16/2827—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 B7 molecules, e.g. CD80, CD86
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/30—Cellular immunotherapy characterised by the recombinant expression of specific molecules in the cells of the immune system
    • A61K40/36—Immune checkpoint inhibitors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A61K47/6807—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug or compound being a sugar, nucleoside, nucleotide, nucleic acid, e.g. RNA antisense
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • 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
    • A61K2121/00—Preparations for use in therapy
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2300/00—Mixtures or combinations of active ingredients, wherein at least one active ingredient is fully defined in groups A61K31/00 - A61K41/00
    • 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
    • C07K2317/70—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
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
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    • C12N2510/00—Genetically modified cells

Definitions

  • the present invention relates to the field of biomedicine, in particular to an anti-PD-L1 antibody or antigen binding fragment thereof and its medical use.
  • Tumor immunotherapy is the most promising research direction in the current tumor treatment field, including cellular immunotherapy, tumor vaccines, passive tumor-targeting passive immunotherapy, and immune checkpoint inhibitors, and other research hotspots, and have achieved many applications. Prospective research results.
  • Immune checkpoint refers to a signal pathway that controls the intensity of T cell immune response by balancing costimulatory and cosuppressive signals (Reference 1). Under normal circumstances, immune checkpoints can maintain immune tolerance by adjusting the intensity of autoimmune responses. However, when the body is invaded by tumors, the activation of immune checkpoints can inhibit autoimmunity, which is conducive to the growth and escape of tumor cells.
  • CTLA-4, programmed death receptor-1 (PD-1)/programmed death ligand-1 (PD-L1), TIM-3, etc. are immune checkpoints The key negative regulatory molecules in the tumor play an important role in tumor immune escape.
  • PD-1/PD-L1 has received high attention in tumor immune research and treatment.
  • PD-1 programmed death 1, programmed death receptor 1, CD279
  • CD279 is an important immunosuppressive molecule and a member of the CD28 superfamily.
  • PD-1 is expressed in activated T cells, B cells, NK cells, monocytes and some tumor cells. It is a transmembrane protein coded by the PDCD1 gene and composed of 288 amino acids.
  • the structure of PD-1 mainly includes the extracellular domain-immunoglobulin variable domain (IgV)-like domain, transmembrane domain and intracellular domain.
  • IgV extracellular domain-immunoglobulin variable domain
  • the intracellular region includes C-terminal and N-terminal amino acid residues, and contains two independent phosphorylation sites, namely immunoreceptor tyrosine-based inhibitorymotif (ITIM) and immunoreceptor tyrosine conversion Motif (immunore-ceptor tyrosine based switch motif, ITSM).
  • ITIM immunoreceptor tyrosine-based inhibitorymotif
  • ITSM immunoreceptor tyrosine conversion Motif
  • PD-L1 Programmed death ligand 1, CD274, B7-H1
  • CD274 Programmed death ligand 1, CD274, B7-H1
  • CD274 is a 40kDa transmembrane protein, encoded by the CD274 gene, which is induced to express on T cells, B cells, dendritic cells, macrophages, mesenchymal stem cells, bone marrow Sourced mast cells and non-hematopoietic cells may be rapidly upregulated in tumor tissues and other tissues that respond to interferon and other inflammatory factors (Reference 4).
  • the PD-1/PD-L1 pathway After the PD-1/PD-L1 pathway is activated, it suppresses the immune system in cancer, pregnancy, tissue transplantation, and autoimmune diseases.
  • PD-L2 Programmed death ligand 1, CD273, B7-DC
  • the expression range of PD-L2 is relatively narrow, and the expression is mainly up-regulated in activated macrophages, dendritic cells, and mast cells (Reference 5).
  • PD-L1 and PD-L2 have 37% homologous sequences, but due to the difference in the main expression vector, the regulation effect is different.
  • PD-L1 is expressed in a variety of tumor cells, making PD-L1 the main ligand for the study of PD-1/PD-L pathway in the field of tumor immunotherapy.
  • PD-L1 can also bind to CD80 (belonging to the immunoglobulin superfamily, its ligands are CD28 and CTLA4, which play an important role in autoimmune monitoring, humoral immune response, and transplantation response) (Ref. 6) to inhibit PD-L1 Or it can relieve the interference to CD80, thereby enhancing T cell activity; from the perspective of drug safety, because PD-1 has another receptor PD-L2, and the affinity of PD-L2 to PD-1 is that of PD-L1 Three times (document 7), and PD-L1 blockers do not bind to PD-L2.
  • the PD-1 antibody blocks the association between PD-1 and PD-L1 and PD-L2 at the same time, while the PD-L1 antibody only blocks the association between PD-1 and PD-L1, retaining PD-L1.
  • PD-L2 is very important for maintaining the immune tolerance of the lungs and gastrointestinal tract. Therefore, PD-L1 antibody may have less side effects on lung and gastrointestinal tract than PD-1 antibody. Compared with PD-1 blockers, PD-L1 blockers may have a better performance in terms of effectiveness and safety (Reference 8).
  • Atezolizumab was first approved by the US FDA in May 2016, and was approved for multiple indications in the following two years (as shown in Table 2).
  • Atezolizumab was approved for the first-line treatment of three major refractory advanced cancer indications, including the EU-approved Atezolizumab+beva+ chemotherapy for the first-line treatment of advanced non-squamous non-small cell lung cancer; Atezolizumab combined with chemotherapy approved in the United States , Used for the first-line treatment of PD-L1 positive advanced triple-negative breast cancer and for the first-line treatment of advanced small cell lung cancer.
  • Avelumab and Durvalumab are also approved for the treatment of metastatic MERKEL cell carcinoma, locally advanced or metastatic urothelial carcinoma, or unresectable stage III non-small cell lung cancer, etc., see details Table 3-4.
  • Patent application CN102245640A also discloses a PD-L1 antibody and its use for enhancing T cell function so as to up-regulate cell-mediated immune response and providing treatment for T cell dysfunction disorders, including infections (such as acute and Chronic) and tumor immunity.
  • Cancers targeted by tumor immunity include: breast cancer, lung cancer, colon cancer, ovarian cancer, melanoma, bladder cancer, kidney cancer ( kidney cancer, liver cancer, salivary cancer, stomach cancer, glioma, thyroid cancer, thymic cancer, epithelial cancer ), head and neck cancer (head and neck cancers), gastric cancer and pancreatic cancer (gastric and pancreatic cancer).
  • the present invention provides an anti-PD-L1 antibody, as well as nucleotides, polynucleotide combinations, expression vectors and expression vector combinations encoding these antibodies.
  • the present invention also provides a conjugate or pharmaceutical composition containing the above-mentioned anti-PD-L1 antibody.
  • the present invention also provides the use of the above-mentioned anti-PD-L1 antibody nucleotide, polynucleotide combination, expression vector, expression vector combination, conjugate or pharmaceutical composition in medicine for treating or preventing cancer.
  • the present invention provides an isolated anti-PD-L1 antibody or antigen-binding fragment thereof.
  • the anti-PD-L1 antibody or antigen-binding fragment thereof includes a heavy chain variable region and a light chain variable region.
  • the heavy chain variable The CDR of the region and/or the CDR of the variable region of the light chain has the same CDR sequence as the antibody defined by the following sequence, or 1-2 amino acid substitutions are made on the CDR of the antibody defined by the following sequence, the antibody defined by the sequence for:
  • variable region of the heavy chain is shown in SEQ ID NO: 31; and/or
  • the complementarity determining regions CDR 1-3 of the corresponding heavy chain and light chain variable regions are shown in Table 5.
  • the present invention also provides an isolated anti-PD-L1 antibody or antigen-binding fragment thereof.
  • it includes heavy chain and light chain variable region sequences, wherein:
  • the CDR1 amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 1, 7, 13, 19 or 25 or after 1 or 2 amino acid substitutions are made to SEQ ID NO: 1, 7, 13, 19 or 25
  • the amino acid sequence of CDR2; the amino acid sequence of CDR2 is shown in SEQ ID NO: 2, 8, 14, 20 or 26 or the amino acid sequence of SEQ ID NO: 2, 8, 14, 20 or 26 after 1 or 2 amino acid substitutions
  • the CDR3 amino acid sequence is as shown in SEQ ID NO: 3, 9, 15, 21 or 27 or the amino acid sequence of SEQ ID NO: 3, 9, 15, 21 or 27 after 1 or 2 amino acid substitutions; and/or
  • the CDR1 amino acid sequence of the light chain variable region is shown in SEQ ID NO: 4, 10, 16, 22 or 28 or after 1 or 2 amino acid substitutions are made to SEQ ID NO: 4, 10, 16, 22 or 28
  • the amino acid sequence of CDR2 is shown in SEQ ID NO: 5, 11, 17, 23 or 29, and the amino acid sequence of SEQ ID NO: 5, 11, 17, 23 or 29 after 1 or 2 amino acid substitutions
  • the CDR3 amino acid sequence is shown in SEQ ID NO: 6, 12, 18, 24, or 30 or the amino acid sequence of SEQ ID NO: 6, 12, 18, 24, or 30 after 1 or 2 amino acid substitutions.
  • isolated anti-PD-L1 antibody or antigen-binding fragment thereof provided by the present invention, in some specific embodiments, wherein:
  • the amino acid sequence of the CDR 1-3 of the heavy chain variable region is SEQ ID NO: 1-3 or the amino acid sequence of SEQ ID NO: 1-3 after 1 or 2 amino acid substitutions; and/or the light chain is variable
  • the CDR 1-3 amino acid sequence of the region is SEQ ID NO: 4-6 or the amino acid sequence of SEQ ID NO: 4-6 with 1 or 2 amino acid substitutions; or
  • the CDR 1-3 amino acid sequence of the heavy chain variable region is SEQ ID NO: 7-9 or the amino acid sequence of SEQ ID NO: 7-9 after 1 or 2 amino acid substitutions; and/or the light chain is variable
  • the CDR 1-3 amino acid sequence of the region is SEQ ID NO: 10-12 or the amino acid sequence of SEQ ID NO: 10-12 with 1 or 2 amino acid substitutions; or
  • the CDR 1-3 amino acid sequence of the heavy chain variable region is SEQ ID NO: 13-15 or the amino acid sequence of SEQ ID NO: 13-15 after 1 or 2 amino acid substitutions; and/or the light chain variable
  • the CDR 1-3 amino acid sequence of the region is SEQ ID NO: 16-18 or the amino acid sequence of SEQ ID NO: 16-18 with 1 or 2 amino acid substitutions; or
  • the amino acid sequence of the CDR 1-3 of the heavy chain variable region is SEQ ID NO: 19-21 or the amino acid sequence of SEQ ID NO: 19-21 after 1 or 2 amino acid substitutions; and/or the light chain is variable
  • the CDR 1-3 amino acid sequence of the region is SEQ ID NO: 22-24 or the amino acid sequence of SEQ ID NO: 22-24 after 1 or 2 amino acid substitutions; or
  • the CDR 1-3 amino acid sequence of the heavy chain variable region is SEQ ID NO: 25-27 or the amino acid sequence of SEQ ID NO: 25-27 after 1 or 2 amino acid substitutions; and/or the light chain is variable
  • the CDR1-3 amino acid sequence of the region is SEQ ID NO: 28-30 or the amino acid sequence of SEQ ID NO: 28-30 after 1 or 2 amino acid substitutions.
  • the antibody or antigen-binding fragment thereof provided by the present invention has an amino acid sequence of CDR 1-3 in the variable region of the heavy chain as SEQ ID NO: 1-3; CDR 1-3 in the light chain variable region
  • the amino acid sequence is SEQ ID NO: 4-6.
  • the antibody or antigen-binding fragment thereof provided by the present invention includes a variable region selected from the following group:
  • variable region of the heavy chain is shown in SEQ ID NO: 31, or has the same CDR1-3 as SEQ ID NO: 31 and the identity is greater than 80%, 85%, compared with SEQ ID NO: 31, 90%, 95%, 96%, 97%, 98%, 99% sequence; and/or
  • the sequence of the light chain variable region is shown in SEQ ID NO: 32, or it has the same CDR 1-3 as SEQ ID NO: 32 and the identity is greater than 80%, 85% compared with SEQ ID NO: 32 , 90%, 95%, 96%, 97%, 98%, 99% sequence.
  • amino acid sequence of the heavy chain variable region of the anti-PD-L1 antibody involved in the present invention (SEQ ID NO: 31):
  • amino acid sequence of the variable region of the light chain of the anti-PD-L1 antibody involved in the present invention (SEQ ID NO: 32):
  • the antibody or antigen-binding fragment thereof provided by the present invention wherein: (1) the sequence of the variable region of the heavy chain is shown in SEQ ID NO: 31; and/or (2) the light chain can be The sequences of the variable regions are shown in SEQ ID NO: 32 respectively.
  • the antibody or antigen-binding fragment thereof provided by the present invention (1) the amino acid sequence of the heavy chain is shown in SEQ ID NO: 33; and/or (2) the amino acid sequence of the light chain They are shown in SEQ ID NO: 34 respectively.
  • the antibodies provided by the present invention can be monoclonal antibodies, chimeric antibodies, humanized antibodies, bispecific antibodies, multispecific antibodies or Fab fragments, F(ab') fragments, F(ab') 2 fragments, Fv Fragment, dAb, Fd, single chain antibody (scFv). Further, the antibody is a humanized monoclonal antibody.
  • the antibody provided by the present invention further includes a human or murine constant region, and the constant region may further be selected from IgG1, IgG2, IgG3, and IgG4.
  • the IgG2 includes IgG2A and IgG2B.
  • the present invention also provides an isolated polynucleotide encoding the antibody or antigen-binding fragment thereof or a polynucleotide combination encoding the heavy chain or partial fragment of the heavy chain and the light chain or partial fragment of the light chain of the antibody or antigen-binding fragment.
  • the present invention also provides a nucleic acid construct or vector, which comprises the above-mentioned polynucleotide encoding the anti-PD-L1 antibody or antigen-binding fragment thereof.
  • the present invention further provides a host cell, which comprises the aforementioned nucleic acid construct or vector.
  • the cells may be prokaryotic cells, eukaryotic cells, yeast cells, mammalian cells, E. coli cells or CHO cells, NSO cells, Sp2/0 cells, BHK cells.
  • the present invention further provides an antibody-drug conjugate, which contains the anti-PD-L1 antibody or its antigen-binding portion of the present invention and a drug toxin.
  • the drug toxins can be selected from chemicals, toxins, polypeptides, enzymes, isotopes, cytokines that can directly or indirectly inhibit cell growth or kill cells, or inhibit or kill cells by activating the body's immune response, so as to treat tumors.
  • Antibodies or other biologically active substances or mixtures preferably interleukins, tumor necrosis factor, chemokines, nanoparticles, MMAE, MMAF, DM1, DM4, Calicheamicin, duocarmycin, adriamycin Su (Doxorubicin).
  • the present invention further provides a pharmaceutical composition comprising the anti-PD-L1 antibody or antigen-binding fragment thereof and/or the above-mentioned conjugate of the present invention, and a pharmaceutically acceptable carrier.
  • the present invention further provides a method for preparing an anti-PD-L1 antibody, which comprises culturing the above-mentioned host cell under conditions suitable for expressing a vector encoding the anti-PD-L1 antibody or antigen-binding fragment, and recovering the antibody or fragment.
  • the present invention further provides a method for enhancing T cell function, which comprises administering an effective amount of the above-mentioned pharmaceutical composition of the present invention to the dysfunctional T cell.
  • the present invention provides a method for treating or preventing cancer, which comprises administering to a subject in need thereof a therapeutically effective amount of the antibody, polynucleotide, polynucleotide combination, expression vector, and coupling according to the present invention. And/or pharmaceutical composition.
  • the present invention provides the anti-PD-L1 antibody or antigen-binding fragment thereof, the encoding polynucleotide, polynucleotide combination, corresponding nucleic acid construct or carrier, antibody-drug conjugate, or drug combination of the present invention In the preparation of drugs for the treatment or prevention of cancer.
  • the present invention provides antibodies, polynucleotides, polynucleotide combinations, expression vectors, conjugates and/or pharmaceutical compositions according to the present invention, which are used for the treatment or prevention of cancer.
  • the cancer is a solid tumor.
  • the solid tumor is lung cancer, colorectal cancer, breast cancer, ovarian cancer, melanoma, bladder cancer, urothelial cancer, kidney cancer, liver cancer, salivary gland cancer, gastric cancer, glioma, thyroid cancer, Thymic cancer, epithelial cancer, head and neck cancer, stomach cancer and pancreatic cancer.
  • lung cancer is non-small cell lung cancer.
  • the ovarian cancer is triple-negative breast cancer.
  • Yet another aspect of the present invention provides a method for treating a disorder of T cell dysfunction, which comprises administering a therapeutically effective amount of the above-mentioned pharmaceutical composition of the present invention to a patient suffering from a disorder of T cell dysfunction.
  • Diseases include infection, tumor immunity.
  • the tumor immunity is caused by cancers selected from the following: breast cancer, lung cancer, colon cancer, ovarian cancer, melanoma, bladder cancer, kidney cancer, liver cancer, salivary gland cancer, gastric cancer, glioma, thyroid cancer, thymus Cancer, epithelial cancer, head and neck cancer, stomach cancer and pancreatic cancer.
  • the present invention further provides the preparation of the anti-PD-L1 antibody or its antigen-binding fragment, its encoding polynucleotide, polynucleotide combination, corresponding nucleic acid construct or carrier, antibody drug conjugate, or pharmaceutical composition of the present invention Use in medicines for the treatment or prevention of cancer.
  • the anti-PD-L1 monoclonal antibody provided by the present invention is a brand-new monoclonal antibody targeting PD-L1, which has brand-new CDR sequences and amino acid sequences.
  • the anti-PD-L1 monoclonal antibody provided by the present invention has surprising affinity and specificity with human PD-L1, and has significant advantages in competing with human PD-1 to bind to human PD-L1.
  • the anti-PD-L1 monoclonal antibody provided by the present invention exhibits unexpected tumor inhibition effects in non-small cell lung cancer and colorectal cancer in vitro animal models, and provides a better choice for inhibiting tumor progression.
  • Figure 1 shows the affinity binding and dissociation curve of hAAG5D8 and human PD-L1.
  • the curve numbered 1 indicates that the concentration of hAAG5D8 is 100nM
  • the curve numbered 2 indicates that the concentration of hAAG5D8 is 50nM
  • the curve numbered 3 indicates the concentration of hAAG5D8 The concentration is 25nM.
  • Figure 2 shows the competitive binding curve of hAAG5D8 and human PD-1.
  • Figure 3 shows the binding ability of hAAG5D8 with a variety of B7 family proteins (PD-L1, PD-L2, B7-H3, PD-1 and CD80).
  • Figure 4 shows the tumor growth after administration of hAAG5D8 (5mg/kg), MPDL3820A (5mg/kg, positive control), Human IgG1 (5mg/kg, negative control) in a human non-small cell lung cancer subcutaneous xenograft tumor model Graph.
  • Figure 5 shows the human colorectal cancer subcutaneous xenograft tumor model, after administration of hAAG5D8 (1mg/kg, 3mg/kg, 9mg/kg), MPDL3820A (10mg/kg, positive control), PBS (negative control) Trend of tumor volume change.
  • CDRs complementarity determining regions
  • the "identity”, “identity” or “similarity” between two nucleic acid or amino acid sequences refers to the two to be compared obtained after the best alignment (optimal alignment).
  • the percentage of identical nucleotides or identical amino acid residues between sequences This percentage is purely statistical and the differences between the two sequences are randomly distributed and cover their entire length.
  • Sequence comparisons between two nucleic acid or amino acid sequences are usually performed by comparing these sequences after they are matched in an optimal manner, and the comparison can be performed through segments or through a "comparison window".
  • the optimal alignment used to compare sequences can also be passed through the local homology algorithm of Smith and Waterman (1981) [Ad.App.Math.
  • Antibody as used herein is used in the broadest sense and encompasses various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, and multispecific antibodies (such as bispecific antibodies); as used herein, "antigen binding "Fragment” refers to an antibody fragment consisting of or containing partial sequences of the heavy or light variable chain of the antibody from which it is derived.
  • the partial sequence is sufficient to retain the same binding specificity and sufficient affinity as the antibody from which it is derived, preferably at least equal to the antibody from which it is derived 1/100 of the affinity is at least equal to 1/10 in a more preferred manner.
  • This functional fragment will contain a minimum of 5 amino acids, preferably 10, 15, 25, 50 and 100 consecutive amino acids of the antibody sequence from which it is derived.
  • a heavy chain includes a heavy chain variable region (abbreviated as VH) and a heavy chain constant region.
  • the heavy chain constant region contains three domains, CH1, CH2 and CH3.
  • a light chain contains a light chain variable region (abbreviated as VL) and a light chain constant region.
  • the light chain constant region contains one domain, CL.
  • the VH and VL regions can be further subdivided into multiple regions with high variability, called complementarity determining regions (CDR), interspersed with more conservative regions called framework regions (FR).
  • CDR complementarity determining regions
  • FR framework regions
  • Each VH and VL are composed of three CDRs and four FRs, arranged in the following order from the amino terminal to the carboxy terminal: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • These variable regions of the heavy and light chains contain binding domains that interact with antigens.
  • the constant regions of antibodies can mediate the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (such as effector cells) and the first component (Clq) of the classical complement system.
  • Chimeric or humanized antibodies are also encompassed by the antibodies according to the invention.
  • humanized antibody refers to an antibody that contains CDR regions derived from a non-human antibody, and other parts of the antibody molecule are derived from one (or several) human antibodies. Moreover, in order to retain the binding affinity, some residues of the backbone (called FR) segment can be modified (Jones et al., Nature, 321:522-525, 1986; Verhoeyen et al., Science, 239: 1534-1536, 1988; Riechmann et al., Nature, 332: 323-327, 1988).
  • the humanized antibodies or fragments thereof according to the present invention can be prepared by techniques known to those skilled in the art (for example, described in the document Singer et al., J. Immun. 150: 2844-2857, 1992; Mountain et al., Biotechnol. Genet. Eng. Rev., 10: 1-142, 1992; or Bebbington et al., Bio/Technology, 10: 169-175, 1992).
  • chimeric antibody refers to an antibody in which the variable region sequence is derived from one species and the constant region sequence is derived from another species, for example, the variable region sequence is derived from a mouse antibody and the constant region sequence is derived from a human antibody.
  • the chimeric antibody or fragment thereof according to the present invention can be prepared by using gene recombination technology.
  • the chimeric antibody can be produced by cloning recombinant DNA comprising a promoter and a sequence encoding the variable region of a non-human, particularly murine monoclonal antibody according to the present invention, and a sequence encoding the constant region of a human antibody .
  • the chimeric antibody of the present invention encoded by this recombinant gene will be, for example, a mouse-human chimera whose specificity is determined by the variable region derived from murine DNA, and its isotype is determined by the variable region derived from human DNA. The constant region is determined.
  • the document Verhoeyn et al. BioEssays, 8:74, 1988
  • Verhoeyn et al. BioEssays, 8:74, 1988
  • the term "monoclonal antibody” refers to a preparation of antibody molecules of single molecular composition.
  • the monoclonal antibody composition shows a single binding specificity and affinity for a specific epitope.
  • isolated nucleic acid molecule is a nucleic acid molecule that is identified and separated from at least one contaminant nucleic acid molecule, which is generally associated with the contaminant nucleic acid molecule in the natural source of antibody nucleic acid.
  • An isolated nucleic acid molecule differs in its form or environment from when it is found in nature. The isolated nucleic acid molecule is therefore different from the nucleic acid molecule that it exists in natural cells.
  • an isolated nucleic acid molecule includes a nucleic acid molecule contained in a cell that normally expresses an antibody in which, for example, the nucleic acid molecule is located on a chromosomal position different from that of a natural cell.
  • a candidate mouse antibody mAAG5D8 was determined. Through further comparison in the antibody variable region database, we determined the human IgG1 framework region with high homology to the murine PD-L1 antibody mAAG5D8. For mAAG5D8, we further designed a variety of humanized antibodies and compared their affinity, and finally determined the candidate humanized anti-PD-L1 antibody hAAG5D8.
  • Amino acid sequence of heavy chain variable region of anti-PD-L1 humanized antibody hAAG5D8 (SEQ ID NO: 31):
  • Amino acid sequence of light chain variable region of anti-PD-L1 humanized antibody hAAG5D8 (SEQ ID NO: 32):
  • amino acid sequences of the heavy chain and light chain of the anti-PD-L1 humanized antibody hAAG5D8 are shown in sequence 33 (SEQ ID NO: 33) and sequence 34 (SEQ ID NO: 34), respectively.
  • the biological layer interference (BLI) method was used to detect the affinity of hAAG5D8 to human PD-L1.
  • Column 1, 3, and 5 of the black 96-well plate AD were added with PBS solution as baseline 1, baseline 2 and dissociation solution respectively, column 2 was added with PD-L1 solution (R&D company), column 4 was added with hAAG5D8 solution (concentrations are as follows: 100nM-50nM-25nM-0nM), add imidazole solution in column 10, add water in column 11, and add nickel sulfate solution in column 12.
  • Ni-NTA probe (Fortebio) was used.
  • the probe was immersed in AD column 1 and kept for 180s to stabilize the base; the Ni-NTA probe was immersed in AD column 2 and kept for 300s to cure PD-L1 to On the probe; dip the Ni-NTA probe into the third column of AD for 120s to stabilize the base; dip the Ni-NTA probe into the fourth column of AD for 600s to make different concentrations of hAAG5D8 and the cured PD on the probe -L1 protein binding; immerse the Ni-NTA probe in AD column 5 for 600s to allow the antibody to dissociate naturally; then immerse the Ni-NTA probe in AD column 10, 11, and 12 to solidify on the probe The PD-L1 is forcibly dissociated from the probe.
  • Use Data analysis 7.0 for data analysis to obtain the binding and dissociation equilibrium constant K D.
  • the ELISA method was used to detect the ability of hAAG5D8 to compete with PD-1 to bind PD-L1.
  • hAAG5D8 The affinity of hAAG5D8 with a variety of other B7 family proteins (PD-L1, PD-L2, B7-H3, PD-1 and CD80) was detected by ELISA. Dilute hAAG5D8 to 100 ng/mL with coating buffer (6mM Na 2 CO 3 , 14 mM NaHCO 3 ), add 100 ⁇ L to each well, and incubate overnight at 4°C.
  • wash the plate with PBST add 250 ⁇ L of blocking solution to each well (3%BSA/PBST) and block for 2h at 25°C, wash the plate with PBST, add 100 ⁇ L of protein sample (rhPD-L1, rhPD-L2, rhB7- H3, rhPD-1 and rhCD80) incubate at 25°C for 2h, wash the plate with PBST, add 100 ⁇ L of enzyme-labeled secondary antibody (diluted in the ratio of 1:5000) to each well, incubate for 1h at 25°C, wash the plate with PBST, and develop color , Reading at 450nm.
  • protein sample rhPD-L1, rhPD-L2, rhB7- H3, rhPD-1 and rhCD80
  • Example 5 The curative effect of hAAG5D8 on human non-small cell lung cancer HCC827NCG mice subcutaneously transplanted tumor
  • mice Twelve male NCG mice (purchased from the Model Animal Research Center of Nanjing University), aged 4-5 weeks, weighing 20-26g, were taken from 12 weeks old, 4-5 weeks old, and 100 ⁇ l of human peripheral blood mononuclear cells PBMC (1 ⁇ 10 7 cells/100 ⁇ l, isolated from the blood of healthy donors), 3 days after tail vein injection, mice were subcutaneously inoculated with human non-small cell lung cancer HCC827 cells (ATCC) on the right side of the mouse, 3 days after inoculation (average tumor volume was 57mm 3 ), The mice were randomly divided into 3 groups (A-1 group, A-2 group, A-3 group), 4 mice in each group, and they were administered according to the dosing schedule shown in Table 9.
  • PBMC peripheral blood mononuclear cells
  • the positive control is MPDL3820A (namely Atezolizumab)
  • the negative control is Human IgG1 (China American Crown Branch (Taicang) Co., Ltd., batch number is AB160083).
  • TGI RTV tumor inhibition rate
  • TGI ⁇ TV tumor growth inhibition rate
  • TGI RTV 1-T RTV /C RTV (%).
  • TRTV is the relative tumor volume (TV) of the positive control group or treatment group at a specific time point
  • C RTV is the relative tumor volume of the negative control group at a specific time point
  • T RTV /C RTV is positive
  • RTV V t /V 0
  • V 0 is the tumor volume of the animal at the time of grouping
  • V t is the tumor volume of the animal after treatment.
  • TGI ⁇ TV % (1- ⁇ T/ ⁇ C) ⁇ 100%.
  • ⁇ T the average tumor volume of the positive control group or treatment group at a specific time point-the average tumor volume of the positive control group or the treatment group at the beginning of administration
  • ⁇ C the average tumor volume of the negative control group at a specific time point -Average tumor volume at the start of administration of the negative control group.
  • Table 10 shows the relative tumor inhibition rate and tumor growth inhibition rate of hAAG5D8 on human non-small cell lung cancer cells, Figure 4 It is a graph of tumor growth after administration. Experimental results show that hAAG5D8 has a significant inhibitory effect on human non-small cell lung cancer tumors.
  • Table 9 The dosage regimen of hAAG5D8 on the tumor suppressor activity of human non-small cell lung cancer
  • the first group is the negative control group
  • the second group is the positive control group
  • the third group is the treatment group.
  • mice 30 transgenic female C57 mice of human PD-1 (Tongji University Animal Experiment Center), each mouse implanted 3 ⁇ 10 6 mouse colorectal cancer cells expressing human PD-L1 (Tongji University, cell code MC38-hPD-L1), when the tumor volume grows to about 100mm 3 , the above mice are randomly divided into 5 groups: C-1 group (negative control group, PBS, 6 mice), C-2 Group (positive control group, Atezolizumab, 3mg/kg, 6 mice), C-3 group (administration group, hAAG5D8, 1mg/kg, 6 mice), C-4 group (administration group, hAAG5D8, 3mg/kg, 6 mice), C-5 group (administration group, hAAG5D8, 9mg/kg, 6 mice), intraperitoneal administration 2 times a week, 5 times in total. The tumor volume was measured twice a week until the 16th day after the start of the administration.
  • T/C(%) T RTV /C RTV ⁇ 100%
  • T RTV represents the RTV of the treatment group or positive control group
  • C RTV represents the RTV of the negative control group
  • TGI RTV (%) (1-T RTV /C RTV ) ⁇ 100%.
  • the day of administration is defined as D0; *P ⁇ 0.05, **P ⁇ 0.01 vs PBS

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Abstract

本发明提供了一种PD-L1免疫抑制剂，具体提供了一种能够靶向PD-L1的单克隆抗体以及编码这些抗体的核苷酸、多核苷酸组合、表达载体以及表达载体组合。本发明还提供了包含上述抗PD-L1抗体的偶联物或者药物组合物。本发明还提供了上述抗PD-L1抗体核苷酸、多核苷酸组合、表达载体、表达载体组合、偶联物或者药物组合物在治疗或预防癌症的药物中的用途。

Description

抗PD-L1抗体及其应用 技术领域
本发明涉及生物医药领域，具体涉及一种抗PD-L1抗体或其抗原结合片段及其医药用途。
背景技术
近年来，在肿瘤治疗领域，越来越多地人们致力于利用自身免疫系统来抵御肿瘤，这种通过调动机体免疫系统，抑制和杀伤肿瘤细胞的方法称为肿瘤免疫治疗。肿瘤免疫治疗是当前肿瘤治疗领域中最具前景的研究方向，其中包括细胞免疫疗法、肿瘤疫苗、靶向肿瘤的被动免疫治疗，以及免疫检查点抑制剂等研究热点，并已取得了很多具有应用前景的研究成果。
免疫检查点是指通过平衡共刺激和共抑制信号来控制T细胞免疫应答强度的信号通路(文献1)。机体在正常情况下，免疫检查点可以通过调节自身免疫反应的强度来维持免疫耐受。然而，机体在受到肿瘤侵袭时，免疫检查点的激活可抑制自身免疫，有利于肿瘤细胞的生长和逃逸。CTLA-4、程序性死亡受体-1(programmed death receptor-1，PD-1)/程序性死亡配体-1(programmed death ligand-1，PD-L1)、TIM-3等是免疫检查点中关键负向调控分子，在肿瘤免疫逃逸中起重要作用。用特异性抗体阻断免疫检查点的负向调控通路，重建机体免疫系统对肿瘤细胞的识别和杀伤能力，在肿瘤免疫治疗中取得了较好的疗效。在已知的免疫检查点中，PD-1/PD-L1在肿瘤免疫研究和治疗中受到高度关注。
PD-1(programmed death 1，程序性死亡受体1，CD279)是一种重要的免疫抑制分子，CD28超家族成员。PD-1表达于活化的T细胞、B细胞、NK细胞、单核细胞以及部分肿瘤细胞，是由PDCD1基因编码，288个氨基酸组成的跨膜蛋白。PD-1结构主要包括胞外区─免疫球蛋白可变区(IgV)样结构域、跨膜区以及胞内区。胞内区包括C端和N端氨基酸残基，含有2个独立的磷酸化作用位点，分别为免疫受体酪氨酸抑制基(immunoreceptor tyrosine based inhibitorymotif，ITIM)和免疫受体酪氨酸转换基序(immunore-ceptor tyrosine based switch motif，ITSM)。PD-1胞外IgV样结构与其配体结合，ITSM改变，募集SHP2信号，激活下游通路(文献2)。
PD-1的天然配体有两个，分别为PD-L1和PD-L2(文献3)。PD-L1(Programmed death ligand 1，CD274，B7-H1)是40kDa的跨膜蛋白，由CD274基因编码，诱导表达于T细胞、B细胞、树突细胞、巨噬细胞、间充质干细胞、骨髓来源的肥大细胞和非造血细胞，在干扰素及其他炎症因子刺激应答的肿瘤组织和其他组织中都可能会迅速上调(文献4)。PD-1/PD-L1通路激活后，在癌症、妊娠、组织移植以及自身免疫病中抑制免疫系统。PD-L2(Programmed death ligand 1，CD273，B7-DC)表达范围较窄，主要在活化的巨噬细胞、树突细胞、肥大细胞中表达上调(文献5)。PD-L1与PD-L2有37％的同源序列，但由于主要表达载体的不同，导致调节作用是不同的。PD-L1不同于PD-L2，表达在多种肿瘤细胞中，使PD-L1成为研究PD-1/PD-L通路在肿瘤免疫治疗领域中的主要配体。
由于PD-L1还可以与CD80(属于免疫球蛋白超家族，其配体是CD28和CTLA4，在自身免疫监控、体液免疫应答及移植反应中发挥重要作用)结合(文献6)，抑制PD-L1或可解除对CD80的干扰，从而增强T细胞活性；从药物安全性方面考虑，由于PD-1还有另外一个受体PD-L2，且PD-L2对PD-1的亲和力是PD-L1的三倍(文献7)，而PD-L1阻断剂不与PD-L2结合。从理论上而言，PD-1抗体同时阻断了PD-1与PD-L1、PD-L2的关联，而PD-L1抗体只阻断PD-1与PD-L1的关联，保留了PD-1与PD-L2的相互作用。而PD-L2对于维持肺部、胃肠道的免疫耐受非常重要。因此，PD-L1抗体可能比PD-1抗体，在肺和胃肠道方面的副作用上，可能更小。相对PD-1阻断剂，PD-L1阻断剂从有效性和安全性方面可能会有一个更好的表现(文献8)。
目前，美国FDA已批准上市了3种PD-L1抗体药物(如表1所示)。
表1 FDA批准的PD-L1免疫检查点抑制剂
Figure PCTCN2020110935-appb-000001
其中，Atezolizumab在2016年5月首次获美国FDA批准，此后两年又获批多种适应症(如表2所示)。
表2 Atezolizumab获批适应症
Figure PCTCN2020110935-appb-000002
在2019年，Atezolizumab又获批了3大难治性晚期癌症一线治疗的适应症，包括欧盟批准的Atezolizumab+贝伐+化疗，用于晚期非鳞非小细胞肺癌一线治疗；美国批准的Atezolizumab联合化疗，用于PD-L1阳性的晚期三阴性乳腺癌的一线治疗以及用于晚期小细胞肺癌的一线治疗。
另外两种上市的PD-L1抗体药物Avelumab和Durvalumab也陆续被批准用于治疗转移性MERKEL细胞癌、局部晚期或转移性尿路上皮癌或无法手术切除的III期非小细胞肺癌等，具体见表3-4。
表3 Avelumab获批适应症
Figure PCTCN2020110935-appb-000003
Figure PCTCN2020110935-appb-000004
表4 Durvalumab获批适应症
Figure PCTCN2020110935-appb-000005
专利申请CN102245640A中也公开了一种PD-L1抗体以及其用于增强T细胞功能从而上调细胞介导的免疫应答的用途和提供治疗T细胞功能障碍病症，所述病症包括感染(例如急性的和慢性的)和肿瘤免疫。肿瘤免疫针对的癌症包括：乳腺癌(breast cancer)、肺癌(lung cancer)、结肠癌(colon cancer)、卵巢癌(ovarian cancer)、黑色素瘤(melanoma)、膀胱癌(bladder cancer)、肾癌(kidney cancer)、肝癌(liver cancer)、唾液腺癌(salivary cancer)、胃癌(stomach cancer)、神经胶质瘤(gliomas)、甲状腺癌(thyroid cancer)、胸腺癌(thymic cancer)、上皮癌(epithelial cancer)、头和颈癌(head and neck cancers)、胃癌和胰腺癌(gastric and pancreatic cancer)。
从目前各PD-L1抗体药物批准的适应症及现有技术文献来看，尽管同为PD-L1抗体，但所针对的适应症还存在各方面的差异，已上市的三种抗体药物还存在多个三期临床失败进程，如Bavencio针对卵巢癌适应症的3项试验：JAVELIN Ovarian 100、JAVELIN Ovarian 200和JAVELIN Ovarian PARP 100。这表明目前还存在大量的重大临床需求未得到满足。因此临床上还亟需开发更多药效更好且针对更多适应症有效的PD-L1类抑制剂，尤其是靶向PD-L1的单克隆抗体。
发明内容
本发明提供了一种抗PD-L1抗体，以及编码这些抗体的核苷酸、多核苷酸组合、表达载体以及表达载体组合。本发明还提供了包含上述抗PD-L1抗体的偶联物或者药物组合物。本发明还提供了上述抗PD-L1抗体核苷酸、多核苷酸组合、表达载体、表达载体组合、偶联物或者药物组合物在治疗或预防癌症的药物中的用途。
本发明提供一种分离的抗PD-L1抗体或其抗原结合片段，所述的抗 PD-L1抗体或其抗原结合片段包括重链可变区和轻链可变区，所述重链可变区的CDR和/或所述轻链可变区的CDR与如下序列限定的抗体具有相同的CDR序列或在如下序列限定的抗体的CDR上进行1-2个氨基酸替换，所述序列限定的抗体为：
(1)重链可变区氨基酸序列为SEQ ID NO:31所示；和/或
(2)轻链可变区氨基酸序列为SEQ ID NO:32所示。
在特定实施方案中，根据采用不同的测定方法或系统认定，相应重链和轻链可变区的互补决定区CDR 1-3如表5所示。
表5 重链和轻链可变区CDR 1-3氨基酸序列
Figure PCTCN2020110935-appb-000006
Figure PCTCN2020110935-appb-000007
进一步的，本发明还提供了一种分离的抗PD-L1抗体或其抗原结合片段，在某些具体实施方案中，其包括重链和轻链可变区序列，其中：
(1)重链可变区CDR1氨基酸序列如SEQ ID NO:1、7、13、19或25所示或对SEQ ID NO:1、7、13、19或25进行1或2个氨基酸替换后的氨基酸序列；CDR2氨基酸序列序列如SEQ ID NO:2、8、14、20或26所示或对SEQ ID NO:2、8、14、20或26进行1或2个氨基酸替换后的氨基酸序列CDR3氨基酸序列如SEQ ID NO:3、9、15、21或27所示或对SEQ ID NO:3、9、15、21或27进行1或2个氨基酸替换后的氨基酸序列；和/或
(2)轻链可变区CDR1氨基酸序列如SEQ ID NO:4、10、16、22或28所示或对SEQ ID NO:4、10、16、22或28进行1或2个氨基酸替换后的氨基酸序列；、CDR2氨基酸序列如SEQ ID NO:5、11、17、23或29所示对SEQ ID NO:5、11、17、23或29进行1或2个氨基酸替换后的氨基酸序列；CDR3氨基酸序列如SEQ ID NO:6、12、18、24或30所示或对SEQ ID NO:6、12、18、24或30进行1或2个氨基酸替换后的氨基酸序列。
进一步，本发明提供的分离的抗PD-L1抗体或其抗原结合片段，在某些具体实施方案中，其中：
(1)重链可变区CDR 1-3氨基酸序列为SEQ ID NO：1-3或对SEQ ID NO:1-3进行1或2个氨基酸替换后的氨基酸序列；和/或轻链可变区CDR 1-3氨基酸序列为SEQ ID NO：4-6或对SEQ ID NO：4-6进行1或2个氨基酸替换后的氨基酸序列；或
(2)重链可变区CDR 1-3氨基酸序列为SEQ ID NO：7-9或对SEQ ID NO:7-9进行1或2个氨基酸替换后的氨基酸序列；和/或轻链可变区CDR 1-3氨基酸序列为SEQ ID NO：10-12或对SEQ ID NO：10-12进行1或2个氨基酸替换后的氨基酸序列；或
(3)重链可变区CDR 1-3氨基酸序列为SEQ ID NO：13-15或对SEQ ID  NO:13-15进行1或2个氨基酸替换后的氨基酸序列；和/或轻链可变区CDR 1-3氨基酸序列为SEQ ID NO：16-18或对SEQ ID NO：16-18进行1或2个氨基酸替换后的氨基酸序列；或
(4)重链可变区CDR 1-3氨基酸序列为SEQ ID NO：19-21或对SEQ ID NO:19-21进行1或2个氨基酸替换后的氨基酸序列；和/或轻链可变区CDR 1-3氨基酸序列为SEQ ID NO：22-24或对SEQ ID NO：22-24进行1或2个氨基酸替换后的氨基酸序列；或
(5)重链可变区CDR 1-3氨基酸序列为SEQ ID NO：25-27或对SEQ ID NO:25-27进行1或2个氨基酸替换后的氨基酸序列；和/或轻链可变区CDR1-3氨基酸序列为SEQ ID NO：28-30或对SEQ ID NO：28-30进行1或2个氨基酸替换后的氨基酸序列。
在某些具体实施方案中，本发明所提供的抗体或其抗原结合片段，其重链可变区CDR 1-3氨基酸序列为SEQ ID NO：1-3；轻链可变区CDR 1-3氨基酸序列为SEQ ID NO：4-6。
在某些具体实施方案中，本发明所提供的抗体或其抗原结合片段，其包括选自如下一组的可变区：
(1)重链可变区的序列如SEQ ID NO:31所示，或与SEQ ID NO:31具有相同的CDR1-3且与SEQ ID NO:31相比同一性大于80％、85％、90％、95％、96％、97％、98％、99％的序列；和/或
(2)轻链可变区的序列如SEQ ID NO:32所示，或与SEQ ID NO:32具有相同的CDR 1-3且与SEQ ID NO:32相比同一性大于80％、85％、90％、95％、96％、97％、98％、99％的序列。
本发明涉及的抗PD-L1抗体重链可变区氨基酸序列(SEQ ID NO:31):
Figure PCTCN2020110935-appb-000008
本发明涉及的抗PD-L1抗体轻链可变区氨基酸序列(SEQ ID NO:32):
Figure PCTCN2020110935-appb-000009
在某些具体实施方案中，本发明所提供的抗体或其抗原结合片段，其中：(1)重链可变区的序列如SEQ ID NO:31所示；和/或(2)轻链可变区的序列分别SEQ ID NO:32所示。
在某些具体实施方案中，本发明所提供的抗体或其抗原结合片段，其：(1)重链的氨基酸序列如SEQ ID NO:33所示；和/或(2)轻链的氨基酸序列分别SEQ ID NO:34所示。
本发明所提供的抗体可以是单克隆抗体、嵌合抗体、人源化抗体、双特异性抗体、多特异性抗体或Fab片段、F(ab')片段、F(ab') 2片段、Fv片段、dAb、Fd、单链抗体(scFv)。进一步地，所述抗体是人源化单克隆抗体。
本发明所提供的抗体进一步包括人或鼠恒定区，所述恒定区进一步可选自IgG1,IgG2,IgG3,IgG4。所述IgG2包括IgG2A，IgG2B。
本发明还提供分离的编码上述抗体或其抗原结合片段的多核苷酸或编码上述抗体或抗原结合片段重链或重链部分片段以及轻链或轻链部分片段的多核苷酸组合。
本发明还提供一种核酸构建体或载体，其包含了上述编码抗PD-L1抗体或其抗原结合片段的多核苷酸。
本发明进一步提供了一种宿主细胞，其包含上述核酸构建体或载体。所述细胞可以是原核细胞、真核细胞、酵母细胞、哺乳动物细胞、大肠杆菌细胞或CHO细胞、NS0细胞、Sp2/0细胞、BHK细胞。
本发明进一步提供了一种抗体药物偶联物，其含有本发明中的抗PD-L1抗体或其抗原结合部分及药物毒素。所述药物毒素可以选自可直接或间接抑制细胞生长或杀灭细胞、或通过激活机体免疫反应从而抑制或杀死细胞，从而达到治疗肿瘤的化学物质、毒素、多肽、酶、同位素、细胞因子、抗体或其他具有生物活性的但以物质或混合物质，优选白介素类、肿瘤坏死因子、趋化因子、纳米颗粒、MMAE、MMAF、DM1、DM4、卡奇霉素(Calicheamicin)、duocarmycin、阿霉素(Doxorubicin)。
本发明进一步提供了一种药物组合物，包含了本发明中的抗PD-L1抗体或其抗原结合片段和/或上述偶联物，以及药学上可接受的载体。
本发明进一步提供了一种制备抗PD-L1抗体的方法，其包括在适合表达编码抗PD-L1抗体或抗原结合片段的载体的条件下培养上述宿主细胞，并且回收所述抗体或片段。
本发明进一步提供了一种增强T细胞功能的方法，其包括向功能障碍的T细胞施用有效量本发明中的上述药用组合物。
又一方面，本发明提供了一种治疗或预防癌症的方法，其包括向有此 需要的对象施用治疗有效量的根据本发明的抗体、多核苷酸、多核苷酸组合、表达载体、偶联物和/或药物组合物。
又一方面，本发明提供了本发明中抗PD-L1抗体或其抗原结合片段、其编码多核苷酸、多核苷酸组合、相应的核酸构建体或载体、抗体药物偶联物、或药物组合物在制备用于治疗或预防癌症的药物中的用途。
又一方面，本发明提供了根据本发明的抗体、多核苷酸、多核苷酸组合、表达载体、偶联物和/或药物组合物，其用于治疗或预防癌症。
进一步地，其中所述癌症为实体瘤。
进一步地，其中所述实体瘤为肺癌、结直肠癌、乳腺癌、卵巢癌、黑色素瘤、膀胱癌、尿路上皮癌、肾癌、肝癌、唾液腺癌、胃癌、神经胶质瘤、甲状腺癌、胸腺癌、上皮癌、头和颈癌、胃癌和胰腺癌。
进一步地，其中所述肺癌为非小细胞肺癌。
进一步地，其中所述卵巢癌为三阴性乳腺癌。
本发明又一方面提供了治疗T细胞功能障碍的病症的方法，其包括向患有T细胞功能障碍的病症的患者施用治疗有效量本发明中的上述药用组合物，所述T细胞功能障碍的病症包括感染、肿瘤免疫。所述肿瘤免疫由选自下述各项癌症导致：乳腺癌，肺癌、结肠癌、卵巢癌、黑色素瘤、膀胱癌、肾癌、肝癌、唾液腺癌、胃癌、神经胶质瘤、甲状腺癌、胸腺癌、上皮癌、头和颈癌、胃癌和胰腺癌。
本发明进一步提供了本发明中抗PD-L1抗体或其抗原结合片段、其编码多核苷酸、多核苷酸组合、相应的核酸构建体或载体、抗体药物偶联物、或药物组合物在制备用于治疗或预防癌症的药物中的用途。
本发明提供的抗PD-L1单克隆抗体是一种全新的靶向PD-L1的单克隆抗体，其具有全新的CDR序列及氨基酸序列。本发明提供的抗PD-L1单克隆抗体与人PD-L1具有惊人的亲和力和特异性，在与人PD-1竞争结合人PD-L1上具有显著优势。本发明提供的抗PD-L1单克隆抗体在非小细胞肺癌和结直肠癌的体外动物模型中均表现出意料不到的抑制肿瘤效果，为抑制肿瘤进程提供了更佳的选择。
附图说明
图1显示了hAAG5D8与人PD-L1的亲和力结合解离曲线，其中编号为1的曲线表示hAAG5D8的浓度为100nM，编号为2的曲线表示hAAG5D8 的浓度为50nM，编号为3的曲线表示hAAG5D8的浓度为25nM。
图2显示了hAAG5D8与人PD-1的竞争结合曲线。
图3显示了hAAG5D8与多种B7家族蛋白(PD-L1、PD-L2、B7-H3、PD-1及CD80)的结合能力。
图4显示了在人非小细胞肺癌皮下异种移植肿瘤模型中，给药hAAG5D8(5mg/kg)、MPDL3820A(5mg/kg，阳性对照)、Human IgG1(5mg/kg，阴性对照)后的肿瘤生长曲线图。
图5显示了在人结直肠癌皮下异种移植肿瘤模型中，给药hAAG5D8(1mg/kg、3mg/kg、9mg/kg)、MPDL3820A(10mg/kg，阳性对照)、PBS(阴性对照)后的肿瘤体积变化趋势。
具体实施方式
定义
除非另有定义，本文使用的所有科技术语具有本领域普通技术人员所理解的相同含义。关于本领域的定义及术语，专业人员具体可参考Current Protocols in Molecular Biology(Ausubel)。氨基酸残基的缩写是本领域中所用的指代20个常用L-氨基酸之一的标准3字母和/或1字母代码。
在本发明中，抗体可变域的互补决定区(CDR)测定或编号方法包括本领域熟知的IMGT、Kabat、Chothia、AbM以及Contact法。
对本发明来说，两种核酸或者氨基酸序列间的“一致性”、“同一性”或“相似性”是指，在最佳比对(最优比对)后所获得的、待比较的两序列之间相同核苷酸或相同氨基酸残基的百分数，该百分数是纯粹统计学的并且两种序列间的差异随机分布并覆盖其全长。两种核酸或者氨基酸序列之间的序列比较通常是在以最优方式使它们匹配以后，通过比较这些序列而进行，所述比较能够通过区段或者通过“比较窗”实施。除了能够手工实施外，用于比较序列的最优比对，还能够通过Smith和Waterman(1981)[Ad.App.Math.2：482]的局部同源性算法、通过Neddleman和Wunsch的(1970)[J.MoI.Biol.48：443]局部同源性算法、通过Pearson和Lipman的(1988)[Proc.Natl.Acad.Sci.USA 85：2444)相似性搜索方法、通过使用这些算法的计算机软件实施(GAP、BESTFIT、FASTA和TFASTA in the Wisconsin Genetics Software Package，Genetics Computer Group，575Science Dr.，Madison，WI，或者通过BLAST N or BLAST P比较软件)。
本文所使用的“抗体”以最广义使用，并且涵盖各种抗体结构，包括但不限于单克隆抗体、多克隆抗体、多特异性抗体(例如双特异性抗体)；本文所使用的“抗原结合片段”是指抗体片段由其来源抗体的重或轻可变链的部分序列构成或者包含它们，所述部分序列足以保留与其来源抗体相同的结合特异性和充分的亲和力，优选至少等于其来源抗体亲和力的1/100，在更优选方式中至少等于1/10。这种功能片段将包含最少5个氨基酸，优选其来源的抗体序列的10、15、25、50和100个连续氨基酸。包括(特别是)Fab、F(ab')、F(ab')2、Fv、dAb、Fd、互补决定区(CDR)片段、单链抗体(scFv)、二价单链抗体，其含有至少一个足以使得特异性抗原结合于该多肽的免疫球蛋白片段。上述片段可经合成、或酶法、或通过对完整免疫球蛋白的化学切割而制备，或通过重组DNA技术进行遗传工程改造。其生产的方法在本领域中众所周知。一条重链包含一重链可变区(缩写为VH)和一重链恒定区。重链恒定区包含三个域(domain)，CH1、CH2和CH3。一条轻链包含一轻链可变区(缩写为VL)和一轻链恒定区。该轻链恒定区包含一个域，CL。VH和VL区域还可再细分为具有高可变性的多个区，被称为互补决定区(CDR)，其间散布有更为保守的被称为框架区(FR)的多个区域。每个VH和VL均由三个CDR和四个FR构成，按照以下顺序从氨基端至羧基端排布：FR1、CDR1、FR2、CDR2、FR3、CDR3、FR4。重链和轻链的这些可变区包含与抗原相互作用的结合域。抗体的恒定区可介导免疫球蛋白与宿主的组织或因子结合，包括免疫系统的各种细胞(如效应细胞)和经典补体系统的第一成分(Clq)。嵌合或人源化抗体也涵盖在根据本发明的抗体中。
术语“人源化抗体”是指一种抗体，其包含来源于非人源抗体的CDR区、并且该抗体分子的其他部分来源于一种(或几种)人抗体。而且，为了保留结合亲和力，可以修饰骨架(称为FR)区段的一些残基(Jones et al.，Nature，321：522-525，1986；Verhoeyen et al.，Science，239：1534-1536，1988；Riechmann et al.，Nature，332：323-327，1988)。通过本领域技术人员已知的技术可以制备根据本发明的人源化抗体或其片段(例如，描述于文件Singer et al.，J.Immun.150：2844-2857，1992；Mountain et al.，Biotechnol.Genet.Eng.Rev.，10：1-142，1992；或Bebbington et al.，Bio/Technology，10：169-175，1992)。
术语“嵌合抗体”系指以下抗体，其中的可变区序列来自一物种而恒定区序列来自另一物种，例如可变区序列来自小鼠抗体而恒定区序列来自人抗体的抗体。通过使用基因重组技术可以制备根据本发明的嵌合抗体或其 片段。例如，所述嵌合抗体可以通过克隆重组DNA来生产，所述重组DNA包含启动子和编码根据本发明的非人尤其是鼠单克隆抗体可变区的序列、以及编码人抗体恒定区的序列。由这种重组基因编码的本发明嵌合抗体将是，例如，鼠-人嵌合体，该抗体的特异性由来源于鼠DNA的可变区确定，并且其同种型由来源于人DNA的恒定区来确定。对于制备嵌合抗体的方法，例如，可以参考文件Verhoeyn et al.(BioEssays，8：74，1988)。
术语“单克隆抗体”系指具有单一分子组成的抗体分子的制备物。单克隆抗体组合物显示出对于特定表位的单一结合特异性和亲和性。
术语“分离地”核酸分子是从至少一个污染物核酸分子中鉴别与分离的核酸分子，在抗体核酸的天然来源中其一般地与该污染物核酸分子有联系。分离的核酸分子在其形式或环境上与其在自然界中发现时不同。分离的核酸分子因此不同于其在自然细胞中存在的核酸分子。然而，分离的核酸分子包括在通常表达抗体的细胞中加以包含的核酸分子，在该细胞中例如核酸分子位于与自然细胞不同的染色体位置上。
通常，为了制备单克隆抗体或其功能片段，尤其是鼠源的单克隆抗体或其功能片段，可以参考尤其描述在手册“Antibodies”中的技术(Harlow and Lane，Antibodies：A Laboratory Manual，Cold Spring Harbor Laboratory，Cold Spring Harbor NY，pp.726，1988)或者参考Kohler和Milstein描述的从杂交瘤细胞制备的技术(Nature，256：495-497，1975)。
实施例
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，下面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1 抗PD-L1抗体的产生
通过对免疫小鼠后获得的抗PD-L1抗体的大量筛选，从中确定了一个侯选鼠源抗体mAAG5D8。通过进一步在抗体可变区数据库中比对，我们确定了与鼠源PD-L1抗体mAAG5D8具有很高同源性的人IgG1框架区。针对mAAG5D8，我们进一步设计了多种人源化抗体并进行亲和力比较，最终确定候选的人源化抗PD-L1抗体hAAG5D8。
表6.抗PD-L1人源化抗体hAAG5D8重链可变区和轻链可变区CDR 1-3(IMGT法测定)的氨基酸序列
Figure PCTCN2020110935-appb-000010
抗PD-L1人源化抗体hAAG5D8重链可变区氨基酸序列(SEQ ID NO:31):
Figure PCTCN2020110935-appb-000011
抗PD-L1人源化抗体hAAG5D8轻链可变区氨基酸序列(SEQ ID NO:32):
Figure PCTCN2020110935-appb-000012
抗PD-L1人源化抗体hAAG5D8重链和轻链氨基酸序列分别如序列33(SEQ ID NO:33)和序列34(SEQ ID NO:34)所示。
实施例2 hAAG5D8与人PD-L1的亲和力比较
采用生物层干涉(BLI)法检测hAAG5D8对人PD-L1的亲和力。黑色96孔板A-D第1、3、5列分别加入PBS溶液分别作为基线1、基线2和解离液，第2列加入PD-L1溶液(R&D公司)，第4列加入hAAG5D8溶液(浓度依次为100nM-50nM-25nM-0nM)，第10列加入咪唑溶液，第11列加入水，第12列加入硫酸镍溶液。实验采用Ni-NTA探针(Fortebio公司)，首先将探针浸入A-D第1列，保持180s，使基数稳定；将Ni-NTA探针浸入A-D第2列，保持300s，使PD-L1固化到探针上；将Ni-NTA探针浸入A-D第3列，保持120s，使基数稳定；将Ni-NTA探针浸入A-D第4列，保持600s，使不同浓度的hAAG5D8与探针上固化的PD-L1蛋白结合；将Ni-NTA探针浸入A-D第5列，保持600s，使抗体自然解离；之后将Ni-NTA探针依次浸入A-D第10、11、12列，使固化在探针上的PD-L1从探针上强制解离。使用Data analysis 7.0进行数据分析获得结合解离平衡常数K D。
结果及结论：hAAG5D8与人PD-L1的结合解离平衡常数K D值和亲和 力结合解离曲线分别如表7和图1所示。实验结果表明，hAAG5D8亲和力与人PD-L1亲和力远高于PD-L1与PD-1的亲和力8.2μM(Molecular Interactions of Antibody Drugs Targeting PD-1,PD-L1,and CTLA-4in Immuno-Oncology，Hyun Tae Lee等，Molecules 2019,24,1190；doi:10.3390/molecules24061190，2019年3月26日，第4页最后1段)。
表7 抗PD-L1抗体结合PD-L1的亲和动力学常数
Figure PCTCN2020110935-appb-000013
实施例3 hAAG5D8与人PD-1的竞争结合
采用ELISA法检测hAAG5D8竞争PD-1结合PD-L1的能力。用包被缓冲液(6mM Na 2CO 3，14mM NaHCO 3)稀释PD-L1(Fc Tag)至1μg/mL，每孔加入100μL，4℃孵育过夜。PBST洗板，每孔加入250μL封闭液封闭(3％BSA/PBST)25℃封闭2h，PBST洗板，每孔加入50μL浓度为6μg/mL的人PD-1，同时加入50μL梯度稀释的hAAG5D8溶液(浓度为8000ng/mL、2000ng/mL、500ng/mL、250ng/mL、125ng/mL、62.5ng/mL、31.25ng/mL、6.25ng/mL、1.25ng/mL)混匀后25℃孵育2h，PBST洗板，每孔加入100μL酶标二抗(1：5000比例稀释的羊抗人IgG-Fc-HRP)25℃孵育1h，显色，在450nm处读数，并用四参数方程进行曲线拟合，计算EC 50值。
结果及结论：hAAG5D8竞争结合人PD-L1的EC 50值和竞争结合曲线分别如表8和图2所示。实验结果表明，hAAG5D8能够有效阻断PD-1与PD-L1的结合。
表8 hAAG5D8与人PD-1的竞争结合(pM，Mean±SD)
Figure PCTCN2020110935-appb-000014
实施例4 hAAG5D8与多种其他B7家族蛋白的亲和力
采用ELISA法检测了hAAG5D8与多种其他B7家族蛋白的亲和力(PD-L1、PD-L2、B7-H3、PD-1及CD80)。用包被缓冲液(6mM Na 2CO 3，14mM NaHCO 3)稀释hAAG5D8至100ng/mL，每孔加入100μL，4℃孵育过夜。PBST洗板，每孔加入250μL封闭液封闭(3％BSA/PBST)25℃封闭 2h，PBST洗板，每孔加入100μL浓度为5μg/mL的蛋白样品(rhPD-L1，rhPD-L2，rhB7-H3，rhPD-1及rhCD80)25℃孵育2h，PBST洗板，每孔加入100μL酶标二抗(1：5000比例稀释二抗Anti-His-tag)25℃孵育1h，PBST洗板，显色，在450nm处读数。
结果及结论：hAAG5D8与B7家族蛋白(PD-L1、PD-L2、B7-H3、PD-1及CD80)的结合如图3所示。结果表明，hAAG5D8能够特异性的结合PD-L1不结合PD-L2，hAAG5D8也不与同家族具有相关功能的其他蛋白结合(B7-H3，PD-1，CD80)，由此可知hAAG5D8与PD-L1的结合具有极高的特异性。
实施例5 hAAG5D8对人非小细胞肺癌HCC827NCG小鼠皮下移植瘤的疗效研究
取12只周龄4-5周，体重20-26g，高度免疫缺陷的雄性NCG小鼠(购自南京大学模式动物研究中心)，分别自尾静脉注射100μl人外周血单个核细胞PBMC(1×10 7细胞/100μl，分离自健康捐献者血液)，尾静脉注射3天后于小鼠右侧皮下接种人非小细胞肺癌HCC827细胞(ATCC)，接种后3天(平均瘤体积为57mm 3)，将小鼠随机分为3个组(A-1组、A-2组、A-3组)，每组4只，按照表9所示的给药方案给药。实验同时设置阳性对照和阴性对照，阳性对照为MPDL3820A(即Atezolizumab)，阴性对照为Human IgG1(中美冠科(太仓)有限公司，批号为AB160083)。第4次给药后当天(即肿瘤细胞接种后第13天)，将实验小鼠CO 2安乐死后取瘤，分别计算相对肿瘤抑制率(TGI RTV)和肿瘤增长抑制率(TGI ΔTV)进行药效评价。
相对肿瘤抑制率的计算公式为：TGI RTV＝1-T RTV/C RTV(％)。(其中，T RTV为阳性对照组或治疗组在某一特定时间点的相对肿瘤体积(TV)；C RTV为阴性对照组在某一特定时间点的相对肿瘤体积；T RTV/C RTV为阳性对照组或治疗组和阴性对照组相对肿瘤体积的百分比值；RTV＝V t/V 0，V 0为分组时该动物的瘤体积，V t为治疗后该动物的瘤体积。)；
肿瘤增长抑制率的计算公式为：TGI ΔTV％＝(1-ΔT/ΔC)×100％。(其中，ΔT＝阳性对照组或治疗组某一特定时间点的平均肿瘤体积-阳性对照组或治疗组给药起始的平均肿瘤体积，ΔC＝阴性对照组某一特定时间点的平均肿瘤体积-阴性对照组给药起始的平均肿瘤体积。)
结果及结论：hAAG5D8对人非小细胞肺癌模型肿瘤抑制作用分别如 表10和图4所示，其中表10为hAAG5D8对人非小细胞肺癌细胞的相对肿瘤抑制率和肿瘤增长抑制率，图4为给药后的肿瘤生长曲线图。实验结果表明，hAAG5D8对人非小细胞肺癌肿瘤有显著的抑制作用。
表9 hAAG5D8对人非小细胞肺癌肿瘤抑制活性研究给药方案
Figure PCTCN2020110935-appb-000015
注：第1组为阴性对照组，第2组为阳性对照组，第3组为治疗组。
表10 hAAG5D8对人非小细胞肺癌肿瘤细胞的相对肿瘤抑制率和肿瘤增长抑制率
Figure PCTCN2020110935-appb-000016
实施例6 hAAG5D8对小鼠结直肠癌MC38转基因小鼠皮下移植瘤疗效的研究
人PD-1的转基因雌性C57小鼠30只(同济大学动物实验中心)，每只小鼠在左侧腹皮下植入3×10 6个表达人PD-L1的小鼠结直肠癌细胞(同济大学，细胞代号MC38-hPD-L1)，待肿瘤体积生长至约100mm 3左右时，将上述小鼠随机分为5组：C-1组(阴性对照组、PBS，6只)、C-2组(阳性对照组、Atezolizumab、3mg/kg，6只)、C-3组(给药组、hAAG5D8、1mg/kg，6只)、C-4组(给药组、hAAG5D8、3mg/kg，6只)、C-5组(给药组、hAAG5D8、9mg/kg，6只)，每周腹腔给药2次，共5次。每周测两次肿瘤体积，直至给药开始后第16天。
计算公式：
肿瘤体积：TV＝D1×D2 2/2，其中D1、D2分别表示瘤长径、瘤短径；
相对肿瘤体积：RTV＝T VT/T V1，其中T V1为给药前肿瘤体积，T VT为每次测量时肿瘤体积；
相对肿瘤增殖率：T/C(％)＝T RTV/C RTV×100％，T RTV表示给药组或阳性对照组RTV，C RTV表示阴性对照组RTV；
相对肿瘤抑制率：TGI RTV(％)＝(1-T RTV/C RTV)×100％。
结果及结论：hAAG5D8对小鼠结直肠癌肿瘤抑制作用分别如表11和图5所示，结果表明，在浓度为3mg/kg及以上剂量hAAG5D8对小鼠结直肠癌皮下移植瘤具有显著的抑瘤作用。在同样3mg/kg剂量时，抗PD-L1抗体的抑瘤作用已经显著优于Atezolizumab的抑瘤效果。
表11 给药后荷瘤鼠肿瘤体积变化
Figure PCTCN2020110935-appb-000017
注：给药当天定义为D0；*P<0.05,**P<0.01vs PBS
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Claims (30)

  1. 分离的抗PD-L1抗体或其抗原结合片段，所述抗体或其抗原结合片段包括重链可变区和轻链可变区，所述重链可变区的CDR和/或所述轻链可变区的CDR与如下序列限定的抗体具有相同的CDR序列或在如下序列限定的抗体的CDR上进行1-2个氨基酸替换，所述序列限定的抗体为：
    (1)重链可变区氨基酸序列为SEQ ID NO:31所示；和/或
    (2)轻链可变区氨基酸序列为SEQ ID NO:32所示。
  2. 根据权利要求1所述的抗体或其抗原结合片段，其中：
    (1)重链可变区CDR1氨基酸序列如SEQ ID NO:1、7、13、19或25所示或对SEQ ID NO:1、7、13、19或25进行1或2个氨基酸替换后的氨基酸序列；CDR2氨基酸序列如SEQ ID NO:2、8、14、20或26所示或对SEQ ID NO:2、8、14、20或26进行1或2个氨基酸替换后的氨基酸序列；CDR3氨基酸序列如SEQ ID NO:3、9、15、21或27所示或对SEQ ID NO:3、9、15、21或27进行1或2个氨基酸替换后的氨基酸序列；和/或
    (2)轻链可变区CDR1氨基酸序列如SEQ ID NO:4、10、16、22或28所示或对SEQ ID NO:4、10、16、22或28进行1或2个氨基酸替换后的氨基酸序列；CDR2氨基酸序列如SEQ ID NO:5、11、17、23或29所示或对SEQ ID NO:5、11、17、23或29进行1或2个氨基酸替换后的氨基酸序列；CDR3氨基酸序列如SEQ ID NO:6、12、18、24或30所示或对SEQ ID NO:6、12、18、24或30进行1或2个氨基酸替换后的氨基酸序列。
  3. 根据权利要求2所述的抗体或其抗原结合片段，其中，
    (1)重链可变区CDR 1-3氨基酸序列为SEQ ID NO：1-3或对SEQ ID NO:1-3进行1或2个氨基酸替换后的氨基酸序列；和/或轻链可变区CDR 1-3氨基酸序列为SEQ ID NO：4-6或对SEQ ID NO：4-6进行1或2个氨基酸替换后的氨基酸序列；或
    (2)重链可变区CDR 1-3氨基酸序列为SEQ ID NO：7-9或对SEQ ID NO:7-9进行1或2个氨基酸替换后的氨基酸序列；和/或轻链可变区CDR 1-3氨基酸序列为SEQ ID NO：10-12或对SEQ ID NO：10-12进行1或2个氨基酸替换后的氨基酸序列；或
    (3)重链可变区CDR 1-3氨基酸序列为SEQ ID NO：13-15或对SEQ ID NO:13-15进行1或2个氨基酸替换后的氨基酸序列；和/或轻链可变区CDR 1-3氨基酸序列为SEQ ID NO：16-18或对SEQ ID NO：16-18进行1或 2个氨基酸替换后的氨基酸序列；或
    (4)重链可变区CDR 1-3氨基酸序列为SEQ ID NO：19-21或对SEQ ID NO:19-21进行1或2个氨基酸替换后的氨基酸序列；和/或轻链可变区CDR 1-3氨基酸序列为SEQ ID NO：22-24或对SEQ ID NO：22-24进行1或2个氨基酸替换后的氨基酸序列；或
    (5)重链可变区CDR 1-3氨基酸序列为SEQ ID NO：25-27或对SEQ ID NO:25-27进行1或2个氨基酸替换后的氨基酸序列；和/或轻链可变区CDR1-3氨基酸序列为SEQ ID NO：28-30或对SEQ ID NO：28-30进行1或2个氨基酸替换后的氨基酸序列。
  4. 根据权利要求1-3中任一项所述的抗体或其抗原结合片段，其特征在于，重链可变区CDR 1-3氨基酸序列为SEQ ID NO：1-3；和/或轻链可变区CDR 1-3氨基酸序列为SEQ ID NO：4-6。
  5. 根据权利要求1-4中任一项所述的抗体或其抗原结合片段，其特征在于：
    (1)重链可变区的序列如SEQ ID NO:31所示，或与SEQ ID NO:31具有相同的CDR 1-3且与SEQ ID NO:31相比同一性大于80％、85％、90％、95％、96％、97％、98％、99％的序列；和/或
    (2)轻链可变区的序列如SEQ ID NO:32所示，或与SEQ ID NO:32具有相同的CDR 1-3且与SEQ ID NO:32相比同一性大于80％、85％、90％、95％、96％、97％、98％、99％的序列。
  6. 根据权利要求5所述的抗体或其抗原结合片段，其中：
    (1)重链可变区的氨基酸序列如SEQ ID NO:31所示；和/或
    (2)轻链可变区的氨基酸序列分别SEQ ID NO:32所示。
  7. 根据权利要求6所述的抗体或其抗原结合片段，其中：
    (1)重链的氨基酸序列如SEQ ID NO:33所示；和/或
    (2)轻链的氨基酸序列分别SEQ ID NO:34所示。
  8. 根据权利要求1-7任一项所述的抗体或其抗原结合片段，其中所述抗体是单克隆抗体、嵌合抗体、人源化抗体、双特异性抗体、多特异性抗体或Fab片段、F(ab')片段、F(ab') 2片段、Fv片段、dAb、Fd、单链抗体(scFv)。
  9. 根据权利要求1-8任一项所述的抗体或其抗原结合片段，其中所述 抗体是人源化单克隆抗体。
  10. 根据权利要求1-9任一项所述的抗体或其抗原结合片段，其进一步包括人或鼠恒定区。
  11. 根据权利要求10所述的抗体或其抗原结合片段，所述恒定区选自IgG1,IgG2,IgG3,IgG4。
  12. 根据权利要求11所述的抗体或其抗原结合片段，所述恒定区是IgG1，IgG2A，IgG2B。
  13. 分离的多核苷酸，其编码根据权利要求1-12中任一项所述的抗体或其抗原结合片段。
  14. 分离的多核苷酸的组合，其包括：编码权利要求1-12中任一项所述抗体或其抗原结合片段之重链的多核苷酸和编码权利要求1-12中任一项所述抗体或其抗原结合片段之轻链的多核苷酸。
  15. 一种核酸构建体，其包含根据权利要求13的多核苷酸。
  16. 权利要求15所述的核酸构建体，所述核酸构建体为载体。
  17. 一种宿主细胞，其包含权利要求15所述的核酸构建体或权利要求16的载体。
  18. 权利要求17所述的宿主细胞，所述细胞是原核细胞、真核细胞、酵母细胞、哺乳动物细胞、大肠杆菌细胞或CHO细胞、NS0细胞、Sp2/0细胞、BHK细胞。
  19. 一种抗体药物偶联物，其含有权利要求1-12任一项中所述的抗体或其抗原结合部分及药物毒素。
  20. 根据权利要求19所述的抗体药物偶联物，所述药物毒素选自可直接或间接抑制细胞生长或杀灭细胞、或通过激活机体免疫反应从而抑制或杀死细胞，从而达到治疗肿瘤的化学物质、毒素、多肽、酶、同位素、细胞因子、抗体或其他具有生物活性的但以物质或混合物质，优选白介素类、肿瘤坏死因子、趋化因子、纳米颗粒、MMAE、MMAF、DM1、DM4、卡奇霉素(Calicheamicin)、duocarmycin、阿霉素(Doxorubicin)。
  21. 药物组合物，包含权利要求1-12中任一项所述的抗体或其抗原结合片段和/或权利要求19或20中所述的偶联物，以及药学上可接受的载体。
  22. 制备抗PD-L1抗体的方法，其包括在适合表达编码抗PD-L1抗体或抗原结合片段的载体的条件下培养权利要求17或18所述的宿主细胞，并且回收所述抗体或片段。
  23. 增强T细胞功能的方法，其包括向功能障碍的T细胞施用有效量的权利要求21所述的药物组合物。
  24. 治疗T细胞功能障碍的病症的方法，其包括向患有T细胞功能障碍的病症的患者施用治疗有效量的权利要求21所述的药物组合物，所述T细胞功能障碍的病症包括感染、肿瘤免疫。
  25. 权利要求24的方法，其中所述肿瘤免疫由选自下述各项癌症导致：乳腺癌、肺癌、结肠癌、卵巢癌、黑色素瘤、膀胱癌、肾癌、肝癌、唾液腺癌、胃癌、神经胶质瘤、甲状腺癌、胸腺癌、上皮癌、头和颈癌、胃癌和胰腺癌。
  26. 权利要求1-12中任一项所述的抗体或其抗原结合片段、权利要求13的多核苷酸、权利要求14的多核苷酸组合、权利要求15的核酸构建体、权利要求16的载体、权利要求19或20的抗体药物偶联物、或权利要求21的药物组合物在制备用于治疗或预防癌症的药物中的用途。
  27. 权利要求26所述的用途，其中所述癌症为实体瘤。
  28. 权利要求27所述的用途，其中所述实体瘤为肺癌、结直肠癌、乳腺癌、卵巢癌、黑色素瘤、膀胱癌、尿路上皮癌、肾癌、肝癌、唾液腺癌、胃癌、神经胶质瘤、甲状腺癌、胸腺癌、上皮癌、头和颈癌、胃癌、胰腺癌、Merkel细胞癌。
  29. 权利要求28所述的用途，其中所述肺癌为非小细胞肺癌。
  30. 权利要求29所述的用途，其中所述卵巢癌为三阴性乳腺癌。
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