WO2022068775A1 - 抗pd-l1抗体及其用途 - Google Patents

抗pd-l1抗体及其用途 Download PDF

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WO2022068775A1
WO2022068775A1 PCT/CN2021/121022 CN2021121022W WO2022068775A1 WO 2022068775 A1 WO2022068775 A1 WO 2022068775A1 CN 2021121022 W CN2021121022 W CN 2021121022W WO 2022068775 A1 WO2022068775 A1 WO 2022068775A1
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antibody
active fragment
spd
cells
seq
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PCT/CN2021/121022
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French (fr)
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周军
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锋宏生物医药科技(昆山)有限公司
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Priority to JP2023520184A priority Critical patent/JP2023545394A/ja
Priority to US18/247,252 priority patent/US20230406936A1/en
Priority to EP21874435.7A priority patent/EP4223776A1/en
Publication of WO2022068775A1 publication Critical patent/WO2022068775A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A61P31/06—Antibacterial agents for tuberculosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
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    • 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
    • A61K47/6835—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 the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6849—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 the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a receptor, a cell surface antigen or a cell surface determinant
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
    • A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
    • A61K51/04—Organic compounds
    • A61K51/08—Peptides, e.g. proteins, carriers being peptides, polyamino acids, proteins
    • A61K51/10—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody
    • A61K51/1027—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody against receptors, cell-surface antigens or cell-surface determinants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/70532—B7 molecules, e.g. CD80, CD86
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2827—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against B7 molecules, e.g. CD80, CD86
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/531—Production of immunochemical test materials
    • G01N33/532—Production of labelled immunochemicals
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6854—Immunoglobulins
    • 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
    • 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
    • C07K2317/73—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
    • C07K2317/90—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
    • C07K2319/00—Fusion polypeptide
    • C07K2319/20—Fusion polypeptide containing a tag with affinity for a non-protein ligand
    • C07K2319/21—Fusion polypeptide containing a tag with affinity for a non-protein ligand containing a His-tag
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/70503—Immunoglobulin superfamily, e.g. VCAMs, PECAM, LFA-3
    • G01N2333/70532—B7 molecules, e.g. CD80, CD86
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/70596—Molecules with a "CD"-designation not provided for elsewhere in G01N2333/705

Definitions

  • the present invention relates to the field of tumor immunology, and more particularly to a class of antibodies that are anti-PD-L1 pan-IgC segment and induce ADCC/CDC functional effects.
  • tumor immune checkpoint antibody blockers such as ipilimumab (CTLA4 blocking antibody), pembrolizumab/nivolumab (PD- 1 blocking antibody) and atezolizumab (PD-L1 blocking antibody), which have made a milestone breakthrough in clinical application. Therefore, American scientist James Allison first proposed the generally accepted concept of immune checkpoint blockade. In different clinical indications, the treatment response rate of patients varies widely. In the most common solid tumor patient population, the response rate to this treatment is limited, probably around 20%.
  • PD-L1 programmed death ligand 1
  • PD-1 programmed death protein receptor 1
  • T cells B cells, dendritic and monocytes.
  • T cells recognize and bind tissue-associated antigens, T cells are activated to express PD-1.
  • the combination of PD-L1 and PD-1 inhibits the activation of T cells and leads to immunocompromised. Studies have found that various types of tumors also express PD-L1.
  • the study further showed that the interaction between PD-1 and PD-L1 is achieved by binding to the IgV functional domain (amino acids 19-127) of PD-L1.
  • the heavy chain of atezolizumab binds PD-L1 epitopes E58, Q66, V111, R113, and R125 by hydrogen bonding; and epitopes I54, Y56, N63, V111, M115, S117, A121, and Y123 Binds to heavy chain complementarity determining regions (HCDRs) within the CC'FG plasmid of PD-L1 via van der Waals forces.
  • HCDRs heavy chain complementarity determining regions
  • PD-L1 antibodies such as atezolizumab, durvalumab, BMS-963559 and avelumab recognize different epitopes, they all act on the IgV of PD-L1 Functional Area. They compete with PD-1 for the same epitope, and both aim to block the interaction of PD-L1 with PD-1.
  • the development of domestic PD-L1 antibodies is also based on the concept of immune checkpoint blockade, but its clinical application has not yet been approved.
  • the therapeutic response elicited by immune checkpoint blockade depends to some extent on tumor characteristics, such as the generation of tumor-specific immune responses, the formation of an intratumoral immunosuppressive microenvironment, and tumor susceptibility to immune responses.
  • soluble PD-L1 sPD-L1
  • sPD-L1 soluble PD-L1
  • High levels of sPD-L1 are present in aggressive renal cell carcinoma, multiple myeloma, and diffuse large B-cell lymphoma, and are associated with poor survival.
  • Recent studies have found that skin melanoma cells have four PD-L1 splice variants that are responsible for sPD-L1 secretion.
  • the secretion of sPD-L1 is associated with splicing activity, cytokine stimulation, cellular stress response, and cellular injury and death.
  • the expression of membranous PD-L1 was proportional to the secretion of sPD-L1.
  • various types of tumor cells generally have PD-L1 splicing activity and the function of secreting sPD-L1, among which PD-L1-3/12 and PD-L1-9 are the most common or most expressed.
  • the full amino acid lengths of PD-L1-3/12 and PD-L1-9 are amino acids 19-178 and 19-243, respectively.
  • secretory sPD-L1 contains IgV, which is the recognition region of existing blocking PD-L1 antibodies.
  • sPD-L1 splice variants secreted by tumors can be used as "bait" to bind PD-L1 antibodies and generate drug resistance. Only 1% of sPD-L1 secreted by tumor cells can completely abolish the antitumor effect of PD-L1 antibody. In PD-L1 antibody therapy, sPD-L1 secretion is associated with recurrence in tumor patients. This suggests that tumor-secreted sPD-L1 may be a barrier to PD-L1 blocking antibody therapy for tumors, or one of its important drug resistance mechanisms.
  • the purpose of the present invention is to provide a class of anti-PD-L1 anti-pan-IgC segment of PD-L1 and induce ADCC/CDC functional effect, anti-tumor and anti-sPD-L1 secretory drug resistance PD-L1 antibody.
  • a first aspect of the present invention provides a PD-L1 antibody or an active fragment thereof, the antibody does not block the binding of PD-1 to PD-L1, and the antibody specifically binds to the pan-IgC of PD-L1 segment, and has the activity of inducing ADCC/CDC functional effects.
  • the antibody does not bind to the IgV functional region of PD-L1 (ie, amino acids 19-127).
  • the antibody does not compete with PD-1 for binding to the IgV functional region of PD-L1.
  • the antibody does not compete with PD-1 for binding to the same epitope of the IgV functional region of PD-L1.
  • the antibody does not bind or has low affinity to soluble PD-L1 (sPD-L1) protein.
  • the antibody does not bind to the soluble PD-L1 protein PD-L1-3.
  • the binding ability P 1 of the antibody to PD-L1-9 is much lower than the binding ability P 0 of the antibody 130021 (reference antibody) to PD-L1-9, preferably P 1 /P 0 ⁇ 1/5, more preferably ⁇ 1/10, more preferably ⁇ 1/100.
  • the antibodies with low affinity for soluble PD-L1 protein include: PD-L1-1 and PD-L1-9.
  • the antibody does not bind or partially binds to soluble PD-L1 (sPD-L1) protein.
  • the soluble PD-L1 protein that the antibody does not bind to includes: PD-L1-3.
  • the soluble PD-L1 proteins bound by the antibody portion include: PD-L1-1 and PD-L1-9.
  • the antibody is a non-blocking antibody and does not bind to any amino acid site of PD-L1:
  • the described PD-L1 antibody or its active fragment comprises:
  • the antibody specifically binds to the pan-IgC segment of PD-L1.
  • the pan-IgC segment is the IgC segment of the PD-L1 protein and the region connecting it to the cell membrane, wherein the region consists of amino acid residues 132-238 of the PD-L1 protein.
  • the PD-L1 antibody or its active fragment induces ADCC/CDC functional effects to kill tumors.
  • any one of the amino acid sequences in the above amino acid sequence also includes a derivative sequence that is optionally added, deleted, modified and/or substituted by at least one amino acid and can retain the binding affinity of PD-L1.
  • the amino acid sequence of any of the above CDRs comprises a derived CDR sequence that has been added, deleted, modified and/or substituted by 1, 2 or 3 amino acids, and the VH and VL of the derived CDR sequence are made to contain
  • the derivatized antibody was constructed to retain affinity for binding to PD-L1.
  • the number of added, deleted, modified and/or substituted amino acids is 1-5 (eg 1-3, preferably 1-2, more preferably 1).
  • the derivative sequence that has undergone addition, deletion, modification and/or substitution of at least one amino acid and can retain the binding affinity of PD-L1 is an amino acid with homology or sequence identity of at least 96% sequence.
  • the PD-L1 antibody or its active fragment comprises a heavy chain variable region or a light chain variable region
  • the polypeptide sequence of the heavy chain variable region is the same as that of SEQ ID NOs: 11, 20, 21 , 22, 23, 24 or 25 are at least 95% identical and the polypeptide sequence of the light chain variable region is at least 95% identical to SEQ ID NO: 15, 26, 27, 28, 29 or 30.
  • the PD-L1 antibody or its active fragment comprises: a heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 11, 20, 21, 22, 23, 24 or 25, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 15, 26, 27, 28, 29 or 30.
  • the PD-L1 antibody or its active fragment comprises a heavy chain variable region and a light chain variable region selected from the group consisting of:
  • the amino acid sequence of the heavy chain variable region is at least 80%, 85%, 90% of the amino acid sequence shown in SEQ ID NO. 11, 20, 21, 22, 23, 24 or 25 , 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence homology or sequence identity.
  • the amino acid sequence of the light chain variable region is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence homology or sequence identity.
  • the PD-L1 antibody or its active fragment is chimeric.
  • the PD-L1 antibody or its active fragment is humanized.
  • a second aspect of the present invention provides a recombinant protein, the recombinant protein has:
  • the tag sequence includes a 6His tag.
  • the recombinant protein is specific for anti-PD-L1 protein.
  • the third aspect of the present invention provides a polynucleotide encoding the PD-L1 antibody or its active fragment according to the first aspect of the present invention, or the recombinant protein according to the second aspect of the present invention.
  • the fourth aspect of the present invention provides a vector, which contains the polynucleotide according to the third aspect of the present invention.
  • the vector includes: bacterial plasmid, bacteriophage, yeast plasmid, plant cell virus, mammalian cell virus such as adenovirus, retrovirus, or other vectors.
  • the fifth aspect of the present invention provides a genetically engineered host cell comprising the vector of the fourth aspect of the present invention or the polynucleotide of the third aspect of the present invention integrated into the genome.
  • the sixth aspect of the present invention provides an immunoconjugate, the immunoconjugate contains:
  • a conjugation moiety selected from the group consisting of a detectable label, drug, toxin, cytokine, radionuclide, or enzyme.
  • a seventh aspect of the present invention provides a pharmaceutical composition comprising:
  • the pharmaceutical composition further contains: additional active ingredients, preferably the active ingredients include: small molecule compounds, cytokines, antibodies (such as anti-PD-1 antibody, anti-OX40 antibody) , anti-CD137 antibody, anti-CD47 antibody, ADC, CAR-immune cells).
  • additional active ingredients include: small molecule compounds, cytokines, antibodies (such as anti-PD-1 antibody, anti-OX40 antibody) , anti-CD137 antibody, anti-CD47 antibody, ADC, CAR-immune cells).
  • the pharmaceutical composition is in the form of injection.
  • the pharmaceutical composition is used to prepare a medicine for treating tumors, and the tumor is selected from the group consisting of bladder cancer, brain cancer such as glioma, such as low-grade glioma, glioblastoma Cell tumor, cervical cancer, breast cancer, colon cancer, rectal cancer, endometrial cancer, kidney cancer, renal cell cancer, renal pelvis cancer, leukemia, lung cancer, non-small cell lung cancer, melanoma, non-Hodgkin lymphoma, pancreas carcinoma, prostate cancer, ovarian cancer, fibrosarcoma, acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), multiple myeloma, bone marrow-derived tumor, thyroid cancer.
  • bladder cancer such as glioma, such as low-grade glioma, glioblastoma Cell tumor, cervical cancer, breast cancer, colon cancer, rectal cancer,
  • the pharmaceutical composition is used to prepare a medicine for treating infectious diseases, and the infectious diseases include infectious viral diseases, infectious bacterial diseases or infectious fungal diseases.
  • the infection focus or the infected host cell expresses PD-L1 (HIV, hepatitis virus, Mycobacterium tuberculosis).
  • the infectious viral disease is caused by a pathogen selected from the group consisting of HIV, hepatitis virus (A, B, C), herpes virus, adenovirus, influenza virus, arbovirus, echo virus, Rhinovirus, coxsackie virus, coronavirus, respiratory syncytial virus, parotid virus, rotavirus, measles virus, rubella virus, parvovirus, vaccinia virus, HTLV virus, dengue virus, papilloma virus, molluscum virus , poliovirus, rabies virus, JC virus, arbovirus, and encephalitis virus.
  • a pathogen selected from the group consisting of HIV, hepatitis virus (A, B, C), herpes virus, adenovirus, influenza virus, arbovirus, echo virus, Rhinovirus, coxsackie virus, coronavirus, respiratory syncytial virus, parotid virus, rotavirus, measles virus
  • the infectious bacterial disease is caused by a pathogen selected from the group consisting of Mycobacterium tuberculosis, Chlamydia, Rickettsia, Staphylococcus, Streptococcus, Pneumococcus, Meningococcus, Neisseria gonorrhoeae, Klebsiella, Proteus, Ralstonia, Pseudomonas, Legionella, Diphtheria, Salmonella, Bacillus, Cholera, Tetanus, Botox, Anthrax, Yersinia pestis, Leptospirosis, and Lyme disease bacteria.
  • a pathogen selected from the group consisting of Mycobacterium tuberculosis, Chlamydia, Rickettsia, Staphylococcus, Streptococcus, Pneumococcus, Meningococcus, Neisseria gonorrhoeae, Klebsiella
  • the infectious fungal disease is caused by a pathogen selected from the group consisting of Candida, Cryptococcus neoformans, Aspergillus, Mucor, Sporothrix schenckii, Blastomyces dermatitidis, Paracoccus brasiliensis bacteria, Coccidioides spp., and Histoplasma capsularis.
  • the infectious disease is caused by a pathogen selected from the group consisting of HIV, hepatitis virus, influenza virus, herpes virus, Giardia, Plasmodium, Leishmania, Staphylococcus aureus, Pseudomonas aeruginosa Bacillus.
  • the infectious disease is caused by a pathogen selected from the group consisting of HIV, hepatitis virus (A, B, C) and Mycobacterium tuberculosis.
  • the composition can be used in combination with another tumor immunotherapy, including but not limited to: chemotherapy, anti-CD20 mAb, anti-TIM-3 mAb, anti-LAG-3 mAb, anti-CD73 mAb, Anti-CD47 mAb, anti-DLL3 mAb, anti-FRmAb mAb, anti-CTLA-4 antibody, anti-OX40 antibody, anti-CD137 antibody, anti-PD-1 antibody, PD-1/PD-L1 therapy, other immuno-oncology drugs, anti-angiogenic agents , radiotherapy, antibody-drug conjugates (ADC), targeted therapy or other anticancer drugs.
  • another tumor immunotherapy including but not limited to: chemotherapy, anti-CD20 mAb, anti-TIM-3 mAb, anti-LAG-3 mAb, anti-CD73 mAb, Anti-CD47 mAb, anti-DLL3 mAb, anti-FRmAb mAb, anti-CTLA-4 antibody, anti-OX40 antibody, anti-CD137 antibody, anti-PD-1 antibody,
  • the method for targeting PD-L1 on the surface of cancer cells of a subject in need of the pharmaceutical composition, or for enhancing the effect of ADCC/CDC comprises administering the seventh aspect of the present invention to the subject.
  • the pharmaceutical composition of the aspect comprises administering the seventh aspect of the present invention to the subject.
  • the eighth aspect of the present invention provides a method for detecting PD-L1 protein in a sample, the method comprising the steps of:
  • the method is non-therapeutic and non-diagnostic.
  • the method is in vitro detection.
  • the ninth aspect of the present invention provides a kit for detecting PD-L1, the kit contains one or more selected from the group consisting of the PD-L1 antibody according to the first aspect of the present invention or its The active fragment, or the immunoconjugate according to the sixth aspect of the present invention, the pharmaceutical composition according to the seventh aspect of the present invention, and the instructions.
  • the description describes that the kit is used to detect the PD-L1 expression of the subject to be tested.
  • the kit is used for the detection of tumors expressing PD-L1 protein (ie, PD-L1 positive).
  • an immune cell expressing or exposing the antibody of the first aspect of the present invention outside the cell membrane.
  • the immune cells include NK cells and T cells.
  • the immune cells are derived from human or non-human mammals (eg, mice).
  • the eleventh aspect of the present invention provides a method for treating a disease associated with abnormal PD-L1 expression or function, the method comprising administering to a subject in need thereof a pharmaceutically effective amount of the PD according to the first aspect of the present invention -L1 antibody, the immunoconjugate of the sixth aspect of the present invention, or the pharmaceutical composition of the seventh aspect of the present invention, the immune cell of the tenth aspect of the present invention, or a combination thereof.
  • the disease related to abnormal PD-L1 expression or function is a tumor or an infectious disease.
  • the PD-L1 antibody can be used alone or in combination with other therapeutic agents.
  • the other therapeutic agents include immune checkpoint blockers, adjuvants or vaccines.
  • Panel A in Figure 1 shows the full length of PD-L1 and its splice variants sPD-L1-3 or 12, the amino acid region of sPD-L1-9, the known recognition region of PD-L1 antibody and PD-L1 mouse Epitopes of monoclonal antibody mAb #19 and its humanized antibodies.
  • Panel B shows the amino acid sequence of human PD-L1 IgC and its junction with the cell membrane.
  • the underlined dot segment represents the signal peptide segment
  • the underlined double-line segment represents IgV (amino acids 19-127)
  • the underlined single-line segment represents IgC and its connecting segment with the cell membrane (amino acids 132-238)
  • the underlined dash is underlined
  • the dotted line segment represents the transmembrane domain (amino acids 239-259) and the underlined wavy line segment represents the intracellular domain (amino acids 260-290).
  • Figure 2 shows a schematic representation of human PD-L1 splice variants.
  • Figure A shows that the full length of PD-L1 contains six exons, PD-L1-1, PD-L1-3, PD-L1-9 and PD-L1-12 are splice variants, respectively, and the transmembrane region is located in the first Four exons are indicated in pink, snips are indicated in brackets, and Stop stands for stop codon.
  • Panel B shows the nucleic acid and amino acid sequences of full-length membrane-type PD-L1, splice variants PD-L1-1, PD-L1-3, PD-L1-9 and PD-L1-12, respectively, in which the transmembrane region is expressed in the
  • the sub-4 segments are indicated in pink, the spliced segment is indicated in red brackets, the amino acid sequence of each splice variant is indicated in blue, and additional different amino acids are underlined.
  • Figure 3 shows the binding ability of PD-L1 murine monoclonal antibody to human PD-L1 and pan-IgC.
  • Figure A shows the flow cytometry detection of PD-L1 murine monoclonal antibodies 130021 (R&D systems, USA) and 29E.2A3 (Biolegend, USA) for the binding of human PD-L1 and pan-IgC.
  • antibody 130021 binds PD-L1 and pan-IgC;
  • antibody 29E.2A3 only binds PD-L1 and does not recognize pan-IgC segment, indicating that its recognition region is only in IgV;
  • clone 24F.10C12 (Biolegend) is a human PD-L2 mouse Monoclonal antibody;
  • Panel B shows the flow cytometry chart of PD-L1 mouse monoclonal antibody, in which 5 PD-L1 mouse monoclonal antibodies mAb#4, mAb#11, mAb#15, mAb#19 and mAb were screened #23 can both bind PD-L1 and recognize pan-IgC.
  • PD-L1PE represents PE fluorescently labeled PD-L1 antibody.
  • Figure 4 shows the affinity of PD-L1 murine monoclonal antibody to recombinant protein rPD-L1.
  • Panel A shows the affinity of five PD-L1 mouse monoclonal antibodies mAb#4, mAb#11, mAb#15, mAb#19 and mAb#23 to recombinant protein PD-L1 detected by ELISA; Cytometry detection of recombinant proteins rPD-L1-3-HIS and rPD-L1-9-HIS against five PD-L1 mouse monoclonal antibodies mAb#4, mAb#11, mAb#15, mAb#19 and mAb#23 Blocking effect;
  • Panel C shows the effect of different concentrations of recombinant protein rPD-L1-9-HIS on the binding of murine monoclonal antibody #19 to PD-L1 by flow cytometry.
  • Figure 5 shows the affinity of PD-L1 murine mAb #19 for sPD-L1 in its native form by ELISA.
  • Picture A shows the affinity of mouse monoclonal antibody mAb#19 to sPD-L1-3 and sPD-L1-9 in the cell culture supernatant
  • picture B shows the affinity of mouse monoclonal antibody mAb#19 to sPD-L1-9 in the cell culture supernatant - Affinity of L1-3
  • Panel C shows the affinity of murine monoclonal antibody mAb #19 for sPD-L1-9 in cell culture supernatant.
  • Blank represents the negative control of culture medium.
  • Figure 6 shows the anti-tumor effect of PD-L1 murine monoclonal antibody #19 on the MC38 mouse tumor model expressing human PD-L1 and sPD-L1-9, in which Panel A represents the tumor size of mice at different days , expressed as the mean ⁇ standard error of tumor volume; Panel B shows the survival rate of mice on different days.
  • Figure 7 shows humanization of murine mAb #19. Different heavy (A) light (B) chain Fab amino acid sequences are shown. mVH and mVL: murine mAb heavy and light chain variable regions. VH and VL: Humanized antibody heavy and light chain variable regions. The CDR regions of the heavy and light chains are shown in bold red and underlined.
  • Figure 8 shows the affinity of murine mAb #19 humanized, different heavy (H) light (L) chain combinations for membrane and secreted PD-L1, and for pan-IgC.
  • Figure 9 shows the affinity of murine mAb #19 humanized, different heavy (H) light (L) chain combinations for membrane-type PD-L2 (human).
  • the affinity of clone #19 humanized antibody for membrane-type PD-L2 was detected by flow cytometry.
  • C CHO-K1 parental cells, as a negative control.
  • Clone 24F.10C12 human PD-L2 mouse mAb.
  • Secondary antibody PE-labeled goat anti-mouse antibody (BD biosciences, USA).
  • Figure 10 shows the affinity of murine mAb #19 humanized, different heavy (H) light (L) chain combinations for membrane-type CD80 (human).
  • the affinity of clone #19 humanized antibody for membrane-type CD80 was detected by flow cytometry.
  • C CHO-K1 parental cells, as a negative control.
  • Clone 2D10 human CD80 mouse monoclonal antibody clone (Elabscience, Wuhan). Secondary antibody: PE-labeled goat anti-mouse antibody (BD biosciences).
  • Figure 11 shows the affinity of humanized antibody H6L2 for membrane-type PD-L1 in the absence or presence of rPD-L1-9-HIS.
  • Figure 12 shows the affinity of humanized antibody H6L2 for membrane-type PD-L1 in the presence or rPD-L1-3-HIS.
  • the effect of different concentrations of rPD-L1-3-HIS on H6L2 affinity was detected by flow cytometry (A, shown by MFI, and B, shown by dots), and at the same time, compared with Avelumab (Avelumab) and Ate Atezolizumab.
  • Figure 13 shows the blocking effect of humanized antibody H6L2 on the binding of PD-1 to PD-L1.
  • Figure 14 shows the ADCC activity of the humanized antibody H6L2 and the effect of secreted sPD-L1 on its ADCC effect.
  • Detection of induction of humanized antibody #19 under the condition of Jurkat-CD16-NFAT-Luciferase platform without (A) and with (B) secreted sPD-L1 (recombinant protein rPDL1-3-HIS or rPDL1-9-HIS) The activity of Luciferase (luciferase), meanwhile, compared with Avelumab.
  • Figure 15 shows the antitumor effect of humanized antibody H6L2.
  • A Tumor size, expressed as the mean ⁇ standard error of tumor volume in each group.
  • B Survival rate. PBS and Avelumab were negative and positive controls, respectively.
  • Figure 16 shows the binding of the anti-human PD-L1 antibody atezolizumab to sPD-L1 in its native form.
  • the binding ability of anti-human PD-L1 antibody atezolizumab to sPD-L1 in natural form was detected by ELISA.
  • BL means no sPD-L1 supernatant, which is used as a background control;
  • ate treated means treated with atezolizumab;
  • hIgG1 treated means treated with human hIgG1; sPD-L1-3 or sPD-L1-9 respectively means containing Supernatant of sPD-L1-3 or sPD-L1-9
  • Figure 17 shows the blocking effect of human PD-L1 antibodies avelumab and atezolizumab on the binding of PD-1 to PD-L1, and the effect of secreted sPD-L1 on its blocking effect.
  • a and B Effects of Avelumab (alikuzumab) and Atezolizumab (atezolizumab) on intercellular PD-1 and PD-L1 interaction;
  • C and D Effects of sPD-L1 on Avelumab and Atezolizumab blockade. The amount of antibody was 100ng/data point, and the amount of rPD-L1-9-HIS was shown in the figure.
  • non-blocking PD-L1 antibody which does not block the binding of PD-1 and PD-L1, but this type of non-blocking PD-L1 antibody Antibodies have excellent antitumor activity.
  • the non-blocking PD-L1 antibody of the present invention can specifically target the pan-IgC region of PD-L1, can avoid sPD-L1 to the greatest extent, and induce ADCC/CDC activity at the same time.
  • the present invention has been completed on this basis.
  • the inventors constructed five specific anti-human PD-L1 mouse monoclonal antibodies, none of which bind to sPD-L1-3, and one of them can partially circumvent the extracellular full-length sPD-L1 of PD-L1.
  • L1-9 the humanized antibody of which was significantly better than the control anti-human PD-L1 Atezoli and Aliku antibodies in this regard.
  • non-blocking PD-L1 antibody of the present invention refers to an antibody that specifically binds to the PD-L1 protein.
  • the non-blocking PD-L1 antibody of the present invention does not block the combination of PD-1 and PD-L1, can specifically target the pan-IgC region of PD-L1, and has the activity of inducing ADCC/CDC functional effects.
  • a heavy chain as shown in the amino acid sequences of SEQ ID NOs: 11, 20, 21, 22, 23, 24 and 25
  • a light chain as shown in SEQ ID NOs: 15, 26, 27, 28, 29 and 30
  • proteins or polypeptide may or may not contain the starting methionine.
  • the junction of the IgC region with the cell membrane is denoted as the "pan-IgC segment", which is the segment of amino acids 132-238 in SEQ ID No: 19 of the PD-L1 protein (A and B in Figure 1).
  • the present invention provides PD-L1 antibodies with high affinity to human PD-L1 protein.
  • the tested antibodies exhibit potent binding and tumor killing activities and can be used for therapeutic and diagnostic applications.
  • PD-L1 protein is a 40 kDa type I transmembrane protein. Its extracellular portion contains an N-terminal immunoglobulin V (IgV) domain (amino acids 19-127) and a C-terminal immunoglobulin C (IgC) domain (amino acids 132-225).
  • IgV immunoglobulin V
  • IgC immunoglobulin C domain
  • the present invention discloses a discovery that the non-blocking PD-L1 antibody of the present invention can effectively utilize the PD-L1 target after binding to the pan-IgC domain of the PD-L1 protein, resulting in a further therapeutic effect. improvement. Therefore, one embodiment of the present disclosure provides an anti-PD-L1 antibody or an active fragment thereof that can specifically bind to pan-immunoglobulin C of human programmed death ligand 1 (PD-L1) protein (IgC) domain.
  • PD-L1 human programmed death ligand 1
  • Antibodies of the invention possess one or more desirable functional properties including, but not limited to, high affinity binding to PD-L1, high specificity for PD-L1, ability to stimulate complement-dependent cytotoxicity (CDC), antibody Dependent phagocytosis (ADPC) and/or antibody-dependent cell-mediated cytotoxicity (ADCC) against PD-L1-expressing cells, and in subjects and animal models when used alone or in combination with other anticancer therapies Ability to inhibit tumor growth.
  • CDC complement-dependent cytotoxicity
  • ADPC antibody Dependent phagocytosis
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • the pan-IgC domain consists of amino acid residues 132-238.
  • the antibody or active fragment thereof can bind to amino acid residues of PD-L1 protein.
  • amino acid sequence of PD-L1 is:
  • position 132-238 in SEQ ID No: 19 is the pan-IgC region.
  • amino acid segments and positions of PD-L1 are shown in the table below:
  • the present inventors discovered for the first time secretory PD-L1 splice variants and their relationship with sPD-L1 secretion.
  • the secretion of sPD-L1 is proportional to the expression of membranous PD-L1, and is also related to its splicing activity, cytokine stimulation, cellular stress response, and cell injury and death.
  • the human PD-L1 splice variants PD-L1-1, PD-L1-3, PD-L1-9 and PD-L1-12 and their sequences are shown in Figure 2 (Note: PD-L1-12 and PD-L1-12 L1-3 amino acid sequence is the same).
  • secretory PD-L1 secretory PD-L1
  • soluble PD-L1 secretory sPD-L1
  • sPD-L1 secretory PD-L1 splice variants
  • secretory sPD-L1 in natural form is expressed and secreted by MC38 cells;
  • rPD-L1-HIS is a His-tagged secreted sPD-L1 recombinantly expressed by HEK293 cells, including rPD-L1- 3-HIS, rPD-L1-9-HIS.
  • PD-L1 mouse monoclonal antibodies were successfully selected, which recognize PD-L1 and IgC expressed on cells. None of the five mAbs bind to sPD-L1-3, and one mAb can partially circumvent sPD-L1-9, which is the full-length extracellular portion of PD-L1, including IgV and IgC functional segment. In vivo experiments in a mouse tumor model secreting sPD-L1-9 showed that it has anti-drug and anti-tumor effects.
  • PD-L1 mouse monoclonal antibody #19 was selected.
  • Antibody 130021 (reference antibody), i.e. murine monoclonal antibody 130021 (R&D systems), binds to the pan-IgC segment of PD-L1, does not bind to sPD-L1-3 in soluble PD-L1 protein, and binds to sPD-L1-9 .
  • antibody or "immunoglobulin” is a heterotetraglycan protein of about 150,000 Daltons having the same structural characteristics, consisting of two identical light (L) chains and two identical heavy chains (H) Composition. Each light chain is linked to the heavy chain by a covalent disulfide bond, and the number of disulfide bonds varies between heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bridges. At one end of each heavy chain is a variable region (VH) followed by a number of constant regions.
  • VH variable region
  • Each light chain has a variable domain (VL) at one end and a constant domain at the other end; the constant domain of the light chain is opposite the first constant domain of the heavy chain, and the variable domain of the light chain is opposite the variable domain of the heavy chain .
  • VL variable domain
  • Particular amino acid residues form the interface between the variable regions of the light and heavy chains.
  • variable means that certain portions of the variable regions of an antibody differ in sequence that contribute to the binding and specificity of each particular antibody for its particular antigen. However, the variability is not evenly distributed throughout the antibody variable region. It is concentrated in three segments called complementarity determining regions (CDRs) or hypervariable regions in the light and heavy chain variable regions. The more conserved parts of the variable regions are called the framework regions (FRs).
  • CDRs complementarity determining regions
  • FRs framework regions
  • the variable regions of native heavy and light chains each contain four FR regions, which are roughly in a ⁇ -sheet configuration, connected by three CDRs that form linking loops, and in some cases can form part of a ⁇ -sheet structure.
  • the CDRs in each chain are tightly packed together by the FR regions and together with the CDRs of the other chain form the antigen-binding site of the antibody (see Kabat et al., NIH Publ. No. 91-3242, Vol. 1, pp. 647-669 (1991)).
  • the constant regions are not directly involved in the binding of the antibody to the antigen, but they exhibit different effector functions, such as involvement in antibody-dependent cytotoxicity of the antibody.
  • immunoglobulins can be grouped into one of two distinct classes (called kappa and lambda) based on the amino acid sequence of their constant regions. Immunoglobulins can be classified into different classes according to the amino acid sequence of their heavy chain constant region. There are five main classes of immunoglobulins: IgA, IgD, IgE, IgG and IgM, some of which can be further divided into subclasses (isotypes) such as IgG1, IgG2, IgG3, IgG4, IgA and IgA2.
  • the heavy chain constant regions corresponding to the different classes of immunoglobulins are called alpha, delta, epsilon, gamma, and mu, respectively.
  • the subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known to those in the art.
  • the term "monoclonal antibody (monoclonal antibody)” refers to an antibody obtained from a substantially homogeneous population, ie, the individual antibodies contained in the population are identical except for a few naturally occurring mutations that may be present. Monoclonal antibodies are highly specific to a single antigenic site. Also, unlike conventional polyclonal antibody preparations, which typically have different antibodies directed against different determinants, each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, the benefit of monoclonal antibodies is that they are synthesized by hybridoma culture without contamination by other immunoglobulins. The modifier "monoclonal" indicates that the antibody is characteristically obtained from a substantially homogeneous population of antibodies, which should not be construed as requiring any particular method to produce the antibody.
  • the present invention also includes a monoclonal antibody having the corresponding amino acid sequence of the anti-PD-L1 protein monoclonal antibody, a monoclonal antibody having the variable region chain of the anti-PD-L1 protein monoclonal antibody, and these chains of other proteins or protein conjugates and fusion expression products.
  • the present invention includes any protein or protein conjugate and fusion expression product (ie, immunoconjugate and fusion expression product) having light and heavy chains containing hypervariable regions (complementarity determining regions, CDRs), as long as the The hypervariable regions are identical or at least 90% homologous, preferably at least 95% homologous, to the hypervariable regions of the light and heavy chains of the invention.
  • immunoconjugates and fusion expression products include: drugs, toxins, cytokines, radionuclides, enzymes and other diagnostic or therapeutic molecules and the anti-PD-L1 protein monoclonal Conjugates formed by binding of antibodies or fragments thereof.
  • the present invention also includes cell surface markers or antigens that bind to the anti-PD-L1 protein monoclonal antibody or fragment thereof.
  • the present invention includes not only complete monoclonal antibodies, but also immunologically active antibody fragments, such as Fab or (Fab') 2 fragments; antibody heavy chains; antibody light chains.
  • immunologically active antibody fragments such as Fab or (Fab') 2 fragments; antibody heavy chains; antibody light chains.
  • variable region is used interchangeably with “complementarity determining region (CDR)”.
  • variable region of the heavy chain of the antibody includes three complementarity determining regions CDR1, CDR2, and CDR3.
  • the heavy chain of the antibody includes the above-mentioned heavy chain variable region and heavy chain constant region, and the heavy chain constant region may be of murine or human origin.
  • the antibody is a mouse or human-mouse chimeric monoclonal antibody against PD-L1 protein, and its heavy chain constant region and/or light chain constant region can be a humanized heavy chain constant region region or light chain constant region. More preferably, the humanized heavy chain constant region or light chain constant region is the heavy chain constant region or light chain constant region of human IgG1, IgG2 and the like.
  • the present invention also provides other protein or fusion expression products with the antibodies of the present invention.
  • the present invention includes any proteins or protein conjugates and fusion expression products (ie, immunoconjugates and fusion expression products) having variable region-containing heavy and light chains, as long as the variable region is compatible with the antibody of the invention
  • the variable regions of the heavy and light chains are identical or at least 90% homologous, preferably at least 95% homologous.
  • variable regions which are separated into four framework regions (FRs), four
  • FRs framework regions
  • the amino acid sequence of FR is relatively conservative and does not directly participate in the binding reaction.
  • CDRs form a circular structure, and the ⁇ -sheets formed by the FRs in between are spatially close to each other, and the CDRs on the heavy chain and the CDRs on the corresponding light chain constitute the antigen-binding site of the antibody.
  • Which amino acids make up the FR or CDR regions can be determined by comparing the amino acid sequences of antibodies of the same type.
  • variable regions of the heavy and/or light chains of the antibodies of the invention are of particular interest because at least some of them are involved in binding antigen.
  • the present invention includes those molecules having CDR-bearing monoclonal antibody light and heavy chain variable regions, as long as their CDRs are greater than 90% (preferably greater than 95%, optimally 98%) of the CDRs identified herein above) homology.
  • the present invention includes not only complete monoclonal antibodies, but also fragments of immunologically active antibodies or fusion proteins formed by antibodies and other sequences. Accordingly, the present invention also includes fragments, derivatives and analogs of said antibodies.
  • fragment refers to polypeptides that retain substantially the same biological function or activity of an antibody of the invention.
  • a polypeptide fragment, derivative or analog of the present invention may be (i) a polypeptide having one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) substituted, and such substituted amino acid residues may or may not be encoded by the genetic code, or (ii) a polypeptide having a substituent group in one or more amino acid residues, or (iii) a mature polypeptide with another compound (such as a compound that prolongs the half-life of a polypeptide, e.g.
  • polyethylene glycol polyethylene glycol
  • an additional amino acid sequence fused to the polypeptide sequence such as a leader sequence or a secretory sequence or a sequence used to purify the polypeptide or a proprotein sequence, or with 6His-tagged fusion protein.
  • an additional amino acid sequence fused to the polypeptide sequence such as a leader sequence or a secretory sequence or a sequence used to purify the polypeptide or a proprotein sequence, or with 6His-tagged fusion protein.
  • fragments, derivatives and analogs are well known to those skilled in the art.
  • the antibody of the present invention refers to a polypeptide comprising the above-mentioned CDR region having PD-L1 protein-binding activity.
  • the term also includes variant forms of the polypeptides comprising the above-mentioned CDR regions having the same function as the antibodies of the present invention. These variants include (but are not limited to): deletion of one or more (usually 1-50, preferably 1-30, more preferably 1-20, most preferably 1-10) amino acids , insertion and/or substitution, and addition of one or several (usually within 20, preferably within 10, more preferably within 5) amino acids at the C-terminus and/or N-terminus. For example, in the art, substitution with amino acids of similar or similar properties generally does not alter the function of the protein. As another example, the addition of one or more amino acids to the C-terminus and/or N-terminus generally does not alter the function of the protein.
  • the term also includes active fragments and active derivatives of the antibodies of the invention.
  • variant forms of the polypeptide include: homologous sequences, conservative variants, allelic variants, natural mutants, induced mutants, proteins capable of hybridizing with the DNA encoding the antibody of the present invention under high or low stringency conditions, and polypeptides or proteins obtained by using antiserum against the antibodies of the present invention.
  • the invention also provides other polypeptides, such as fusion proteins comprising human antibodies or fragments thereof.
  • the present invention also includes fragments of the antibodies of the present invention.
  • the fragment has at least about 50 contiguous amino acids, preferably at least about 50 contiguous amino acids, more preferably at least about 80 contiguous amino acids, and most preferably at least about 100 contiguous amino acids of an antibody of the invention.
  • the present invention also provides polynucleotide molecules encoding the above-mentioned antibodies or fragments or fusion proteins thereof.
  • the polynucleotides of the present invention may be in the form of DNA or RNA.
  • DNA forms include cDNA, genomic DNA or synthetic DNA.
  • DNA can be single-stranded or double-stranded.
  • DNA can be the coding or non-coding strand.
  • Polynucleotides encoding the mature polypeptides of the present invention include: coding sequences encoding only the mature polypeptides; coding sequences and various additional coding sequences for the mature polypeptides; coding sequences (and optional additional coding sequences) for the mature polypeptides and non-coding sequences .
  • polynucleotide encoding a polypeptide may include a polynucleotide encoding the polypeptide or a polynucleotide that also includes additional coding and/or non-coding sequences.
  • the present invention also relates to polynucleotides that hybridize to the above-mentioned sequences and have at least 50%, preferably at least 70%, more preferably at least 80% identity between the two sequences.
  • the present invention relates to polynucleotides that are hybridizable under stringent conditions to the polynucleotides of the present invention.
  • stringent conditions refer to: (1) hybridization and elution at lower ionic strength and higher temperature, such as 0.2 ⁇ SSC, 0.1% SDS, 60°C; or (2) adding a There are denaturing agents, such as 50% (v/v) formamide, 0.1% calf serum/0.1% Ficoll, 42°C, etc.; or (3) only the identity between the two sequences is at least 90% or more, more Hybridization occurs when it is above 95%.
  • the polypeptide encoded by the hybridizable polynucleotide has the same biological function and activity as the mature polypeptide.
  • the full-length nucleotide sequence of the antibody of the present invention or its fragment can usually be obtained by PCR amplification method, recombinant method or artificial synthesis method.
  • a feasible method is to use artificial synthesis to synthesize the relevant sequences, especially when the fragment length is short. Fragments with very long sequences are usually obtained by first synthesizing multiple small fragments and then ligating them.
  • the coding sequence of the heavy chain and the expression tag (such as 6His) can also be fused together to form a fusion protein.
  • Biomolecules nucleic acids, proteins, etc.
  • Biomolecules include biomolecules in isolated form.
  • DNA sequences encoding the proteins of the present invention can be obtained entirely by chemical synthesis.
  • This DNA sequence can then be introduced into various existing DNA molecules (or eg vectors) and cells known in the art.
  • mutations can also be introduced into the protein sequences of the invention by chemical synthesis.
  • the present invention also relates to vectors comprising suitable DNA sequences as described above together with suitable promoter or control sequences. These vectors can be used to transform appropriate host cells so that they can express proteins.
  • Host cells can be prokaryotic cells, such as bacterial cells; or lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as mammalian cells.
  • prokaryotic cells such as bacterial cells
  • lower eukaryotic cells such as yeast cells
  • higher eukaryotic cells such as mammalian cells.
  • Representative examples are: Escherichia coli, Streptomyces; bacterial cells of Salmonella typhimurium; fungal cells such as yeast; insect cells of Drosophila S2 or Sf9; animal cells of CHO, COS7, 293 cells, etc.
  • Transformation of host cells with recombinant DNA can be performed using conventional techniques well known to those skilled in the art.
  • competent cells capable of uptake of DNA can be harvested after exponential growth phase and treated with the CaCl2 method using procedures well known in the art. Another method is to use MgCl 2 .
  • transformation can also be performed by electroporation.
  • DNA transfection methods can be used: calcium phosphate co-precipitation method, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.
  • the obtained transformants can be cultured by conventional methods to express the polypeptides encoded by the genes of the present invention.
  • the medium used in the culture can be selected from various conventional media depending on the host cells used. Cultivation is carried out under conditions suitable for growth of the host cells. After the host cells have grown to an appropriate cell density, the promoter of choice is induced by a suitable method (eg, temperature switching or chemical induction), and the cells are cultured for an additional period of time.
  • recombinant polypeptide in the above method can be expressed intracellularly, or on the cell membrane, or secreted outside the cell.
  • recombinant proteins can be isolated and purified by various isolation methods utilizing their physical, chemical and other properties. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to: conventional renaturation treatment, treatment with protein precipitants (salting-out method), centrifugation, osmotic disruption, ultratreatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption layer chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC) and various other liquid chromatography techniques and combinations of these methods.
  • Antibodies of the invention may be used alone, or may be conjugated or conjugated to a detectable label (for diagnostic purposes), a therapeutic agent, a PK (protein kinase) modifying moiety, or a combination of any of the above.
  • Detectable labels for diagnostic purposes include, but are not limited to, fluorescent or luminescent labels, radiolabels, MRI (magnetic resonance imaging) or CT (computed tomography) contrast agents, or those capable of producing detectable products. enzymes.
  • Therapeutic agents that can be combined or conjugated with the antibodies of the present invention include but are not limited to: 1. Radionuclides; 2. Biotoxicity; 3. Cytokines such as IL-2, etc.; 4. Gold nanoparticles/nanorods; 5. Viruses 6. Liposomes; 7. Nanomagnetic particles; 8. Prodrug-activating enzymes (eg, DT-diaphorase (DTD) or biphenyl hydrolase-like protein (BPHL)); 10. chemotherapeutic agents ( For example, cisplatin) or any form of nanoparticles, etc.
  • DTD DT-diaphorase
  • BPHL biphenyl hydrolase-like protein
  • the present invention also provides an immunoconjugate based on the antibody of the present invention, preferably, the immunoconjugate is an antibody-drug conjugate (ADC).
  • ADC antibody-drug conjugate
  • the antibody-drug conjugate includes the antibody, and an effector molecule, and the antibody is conjugated to the effector molecule, preferably chemically conjugated.
  • the effector molecule is preferably a drug with therapeutic activity.
  • the effector molecule can be one or more of a toxin, a chemotherapeutic drug, a small molecule drug, or a radionuclide.
  • the antibody of the present invention and the effector molecule can be coupled through a coupling agent.
  • the coupling agent may be any one or more of non-selective coupling agents, coupling agents utilizing carboxyl groups, peptide chains, and coupling agents utilizing disulfide bonds.
  • the non-selective coupling agent refers to a compound that forms a covalent bond between the effector molecule and the antibody, such as glutaraldehyde and the like.
  • the coupling agent utilizing the carboxyl group may be any one or more of a cis-aconitic anhydride type coupling agent (such as cis-aconitic anhydride) and an acyl hydrazone type coupling agent (the coupling site is an acyl hydrazone).
  • antibodies are used to link with various functional groups, including imaging reagents (such as chromophores and fluorophores), diagnostic reagents (such as MRI contrast agents and radioisotopes) , stabilizers (eg ethylene glycol polymers) and therapeutic agents.
  • imaging reagents such as chromophores and fluorophores
  • diagnostic reagents such as MRI contrast agents and radioisotopes
  • stabilizers eg ethylene glycol polymers
  • therapeutic agents eg, antibodies, antibodies, antibodies, antibodies can be conjugated to functional agents to form antibody-functional agent conjugates.
  • Functional agents eg, drugs, detection reagents, stabilizers
  • the functional agent can be attached to the antibody either directly or indirectly through a linker.
  • Antibodies can be conjugated to drugs to form antibody drug conjugates (ADCs).
  • ADC antibody drug conjugates
  • the ADC contains a linker between the drug and the antibody.
  • Linkers can be degradable or non-degradable linkers. Degradable linkers are typically susceptible to degradation in the intracellular environment, eg, at the target site, where the linker is degraded, thereby releasing the drug from the antibody.
  • Suitable degradable linkers include, for example, enzymatically degradable linkers, including peptidyl-containing linkers that can be degraded by intracellular proteases (eg, lysosomal or endosomal proteases), or sugar linkers that, for example, can be degraded by glucuronides Enzymatically degraded glucuronide-containing linkers.
  • Peptidyl linkers can include, for example, dipeptides such as valine-citrulline, phenylalanine-lysine, or valine-alanine.
  • degradable linkers include, for example, pH sensitive linkers (eg, linkers that hydrolyze at pH less than 5.5, eg, hydrazone linkers) and linkers that degrade under reducing conditions (eg, disulfide linkers).
  • Non-degradable linkers typically release the drug under conditions where the antibody is hydrolyzed by proteases.
  • the drug can be any cytotoxic, cytostatic or immunosuppressive drug.
  • the linker connects the antibody and the drug, and the drug has a functional group that can form a bond with the linker.
  • the drug can have an amino, carboxyl, sulfhydryl, hydroxyl, or keto group that can form a bond with the linker.
  • the drug is directly attached to the linker, the drug has a reactive reactive group prior to attachment to the antibody.
  • Useful drug classes include, for example, antitubulin drugs, DNA minor groove binding agents, DNA replication inhibitors, alkylating agents, antibiotics, folate antagonists, antimetabolites, chemosensitizers, topoisomerase inhibitors , Vinca alkaloids, etc.
  • particularly useful cytotoxic drugs include, for example, DNA minor groove binding agents, DNA alkylating agents, and tubulin inhibitors.
  • Typical cytotoxic drugs include, for example, auristatins, camptothecins camptothecins, duocarmycins, etoposides, maytansines and maytansinoids (eg DM1 and DM4), taxanes ( taxanes), benzodiazepines, or benzodiazepine-containing drugs (eg, pyrrolo[1,4]benzodiazepines (PBDs), indoline benzodiazepines) indolinobenzodiazepines and oxazolidinobenzodiazepines) and vinca alkaloids.
  • auristatins e.g, camptothecins camptothecins, duocarmycins, etoposides
  • maytansines and maytansinoids eg DM1 and DM4
  • taxanes taxanes
  • benzodiazepines eg, pyrrolo[1,4]benzodiazepines (PBDs)
  • drug-linkers can be used to form ADCs in one simple step.
  • bifunctional linker compounds can be used to form ADCs in a two- or multi-step process. For example, a cysteine residue reacts with the reactive moiety of the linker in the first step, and in a subsequent step, the functional group on the linker reacts with the drug to form the ADC.
  • the present invention also provides a method for preparing an ADC, which may further include: combining the antibody with the drug-linker compound under conditions sufficient to form an antibody conjugate (ADC).
  • the methods of the invention comprise binding the antibody to a bifunctional linker compound under conditions sufficient to form the antibody-linker conjugate. In these embodiments, the methods of the invention further comprise: conjugating the antibody linker conjugate to the drug moiety under conditions sufficient to covalently link the drug moiety to the antibody via the linker.
  • the antibody drug conjugate ADC is represented by the following molecular formula:
  • Ab is an antibody
  • D is a drug
  • the present invention also provides a composition.
  • the composition is a pharmaceutical composition, which contains the above-mentioned antibody or its active fragment or its fusion protein, and a pharmaceutically acceptable carrier.
  • these materials can be formulated in a non-toxic, inert and pharmaceutically acceptable aqueous carrier medium, usually at a pH of about 5-8, preferably at a pH of about 6-8, although the pH may vary depending on the This will vary depending on the nature of the formulation material and the condition to be treated.
  • the formulated pharmaceutical compositions can be administered by conventional routes including, but not limited to, intratumoral, intraperitoneal, intravenous, or topical administration.
  • the pharmaceutical composition of the present invention can be directly used to bind PD-L1 protein molecules, and thus can be used to prevent and treat tumors.
  • other therapeutic agents may also be used concomitantly.
  • the pharmaceutical composition of the present invention contains the above-mentioned monoclonal antibody (or its conjugate) of the present invention in a safe and effective amount (eg, 0.001-99 wt %, preferably 0.01-90 wt %, more preferably 0.1-80 wt %) and a pharmaceutical an acceptable carrier or excipient.
  • a pharmaceutical an acceptable carrier or excipient include, but are not limited to, saline, buffers, dextrose, water, glycerol, ethanol, and combinations thereof.
  • the drug formulation should match the mode of administration.
  • the pharmaceutical composition of the present invention can be prepared in the form of injection, for example, prepared by conventional methods with physiological saline or an aqueous solution containing glucose and other adjuvants.
  • compositions such as injections and solutions are preferably manufactured under sterile conditions.
  • the active ingredient is administered in a therapeutically effective amount, eg, about 1 microgram/kg body weight to about 5 mg/kg body weight per day.
  • the polypeptides of the present invention may also be used with other therapeutic agents.
  • the pharmaceutical composition further contains: additional active ingredients, preferably the active ingredients include: small molecule compounds, cytokines, antibodies (such as anti-PD-1 antibody, anti-OX40 antibody, anti- CD137 antibody, anti-CD47 antibody, ADC, CAR-immune cells).
  • additional active ingredients include: small molecule compounds, cytokines, antibodies (such as anti-PD-1 antibody, anti-OX40 antibody, anti- CD137 antibody, anti-CD47 antibody, ADC, CAR-immune cells).
  • composition for use in the treatment of cancer an inflammatory disease, disorder or condition, an immune disease, disorder or condition, an autoimmune disease, disorder or condition or an infectious disease, disorder or condition may be In combination with another therapy, including but not limited to: chemotherapy, anti-CD20 mAb, anti-TIM-3 mAb, anti-LAG-3 mAb, anti-CD73 mAb, anti-CD47 mAb, anti-DLL3 mAb, anti-FRmAb mAb, anti-CTLA- 4 Antibodies, anti-PD-1 antibodies, PD-1/PD-L1 therapy, other immuno-oncology drugs, anti-angiogenic agents, radiotherapy, antibody-drug conjugates (ADC), targeted therapy or other anti-cancer drugs.
  • chemotherapy including but not limited to: chemotherapy, anti-CD20 mAb, anti-TIM-3 mAb, anti-LAG-3 mAb, anti-CD73 mAb, anti-CD47 mAb, anti-DLL3 mAb, anti-FRmAb mAb, anti-CTLA
  • Anti-PD-L1 antibodies can be co-constructed with partner mAbs against the following targets to construct bispecific antibodies to treat cancers/tumors expressing PD-L1 and specific tumor-associated antigens: PD-1, OX40, CD137, LAG3, TIM-3, CTLA-4, EGFR, HER-2, CD19, CD20, CD33, CD73, CD47, DLL-3, CLDN18.2, folate receptor alpha (FOLR1), and/or other tumor surface antigens.
  • FOLR1 folate receptor alpha
  • a safe and effective amount of the immunoconjugate is administered to the mammal, wherein the safe and effective amount is generally at least about 10 micrograms/kg body weight, and in most cases no more than about 8 mg/kg body weight, Preferably the dose is about 10 micrograms/kg body weight to about 1 mg/kg body weight.
  • the specific dosage should also take into account the route of administration, the patient's health and other factors, which are all within the skill of the skilled physician.
  • Another aspect of the present invention provides a method of preventing and/or treating a tumor or infectious disease in a subject, comprising administering to the subject a PD-L1 binding molecule of the present invention such that the tumor or infectious disease in the subject is Diseases are prevented and/or treated.
  • the pharmaceutical composition is used to prepare a medicine for treating tumors, and the tumor is selected from the group consisting of bladder cancer, brain cancer such as glioma, such as low-grade glioma, glioblastoma Cell tumor, cervical cancer, breast cancer, colon cancer, rectal cancer, endometrial cancer, kidney cancer, renal cell cancer, renal pelvis cancer, leukemia, lung cancer, non-small cell lung cancer, melanoma, non-Hodgkin lymphoma, pancreas carcinoma, prostate cancer, ovarian cancer, fibrosarcoma, acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), multiple myeloma, bone marrow-derived tumor, thyroid cancer.
  • bladder cancer such as glioma, such as low-grade glioma, glioblastoma Cell tumor, cervical cancer, breast cancer, colon cancer, rectal cancer,
  • the PD-L1 antibodies of the invention can be used alone or as an adjuvant in combination with vaccines to stimulate immune responses to pathogens, toxins and self-antigens.
  • pathogens to which this method of treatment is particularly applicable include pathogens for which there is currently no effective vaccine, or pathogens for which conventional vaccines are not fully effective. These include, but are not limited to, H1V, hepatitis virus (A, B, C), influenza virus, herpes virus, giardia, malaria, Leishmania, Staphylococcus aureus, Pseudomonas aeruginosa.
  • PD-L1 blockade is particularly useful against established infections by pathogens such as H1V, which present altered antigens during infection.
  • pathogens such as H1V
  • these novel epitopes were recognized as foreign substances, resulting in strong T-cell responses unaffected by negative PD-L1 signaling.
  • the antibodies of the present invention can be used for the treatment of infectious diseases.
  • infectious disease treated by the antibody of the present invention may be selected from the group consisting of HIV, hepatitis (A, B, C), Mycobacterium tuberculosis.
  • the PD-L1 antibodies of the invention may be combined with other forms of immunotherapy such as cytokine therapy (eg interferon, GM-CSF, G-CSF, IL-2) or bispecific antibody therapy, Bispecific antibody therapy provides enhanced presentation of tumor antigens.
  • cytokine therapy eg interferon, GM-CSF, G-CSF, IL-2
  • bispecific antibody therapy provides enhanced presentation of tumor antigens.
  • the present invention also provides a hybridoma cell line capable of producing the monoclonal antibody against PD-L1 protein of the present invention; preferably, the present invention provides a hybridoma cell line with a high titer of monoclonal antibody against PD-L1 protein.
  • the hybridoma producing the PD-L1 protein monoclonal antibody of the present invention After obtaining the hybridoma producing the PD-L1 protein monoclonal antibody of the present invention, those skilled in the art can conveniently use the hybridoma cell line to prepare the antibody. In addition, those skilled in the art can easily know the structure of the antibody of the present invention (such as the heavy chain variable region and light chain variable region of the antibody), and then the monoclonal antibody of the present invention can be prepared by recombinant methods.
  • the present invention relates to a method for the detection of tumors in a cell and/or tissue lysed sample.
  • the method steps are roughly as follows: obtaining a cell and/or tissue sample; lysing the sample in a medium; detecting the level of PD-L1 protein in the lysed sample.
  • the sample to be used is not particularly limited, and a representative example is a cell-containing sample existing in a cell preservation solution.
  • the present invention also provides a kit containing the antibody (or its fragment) of the present invention or the detection plate of the present invention.
  • the kit further includes a container, an instruction manual, a buffer agent, etc.
  • the present invention further designs a detection kit for detecting PD-L1 level, the kit includes an antibody that recognizes PD-L1 protein, a lysis medium for dissolving the sample, general reagents and buffers required for detection, such as various buffers solution, detection label, detection substrate, etc.
  • the detection kit may be an in vitro diagnostic device.
  • the anti-PD-L1 protein antibody of the present invention has a unique antigenic epitope and targets the pan-IgC functional segment. Different from known PD-L1 monoclonal antibodies that target the IgV functional segment, such as atezolizumab, durvalumab, BMS-963559, and aliculumab.
  • the anti-PD-L1 protein antibody of the present invention can specifically recognize membrane-type PD-L1 and avoid secretory-type sPD-L1 to the greatest extent.
  • the anti-PD-L1 protein antibody of the present invention is resistant to sPD-L1-secreting tumors.
  • the anti-PD-L1 protein antibody of the present invention can enhance ADCC function.
  • the anti-PD-L1 protein antibody of the present invention has a tumor-killing effect.
  • the PD-L1 antibody of the present invention provides an alternative method for tumors resistant to blocking PD-L1 antibodies, thereby improving the efficacy of immunotherapy.
  • h indicates human origin
  • s indicates secretory or soluble.
  • MC38 is a mouse colon cancer cell and HEK293 is a human embryonic kidney cell, both obtained from Shanghai Gaining Biotechnology Co., Ltd. and grown in DMEM+10% fetal bovine serum medium.
  • CHO-K1 is a Chinese hamster ovary cell, provided by Hangzhou Huaan Biotechnology Co., Ltd., grown in RPMI1640+10% fetal bovine serum medium.
  • MC38/CHO-K1 cells express PD-L1 and pan-IgC respectively as positive detection cells.
  • the MC38/CHO-K1 cells were wild-type and expressing PD-L2 as negative controls.
  • the cells were labeled with PE-conjugated goat anti-mouse antibody (BD biosciences), and incubated at 4°C for 15 min in the dark. After washing with PBS, the cells were detected by flow cytometry (NovoCyte, Aceabio, Hangzhou).
  • PD-L1 cell ELISA direct PD-L1 protein ELISA, and secreted sPD-L1 indirect ELISA
  • PD-L1 antibody subclones were screened by an established cellular ELISA. Plate 5000 positive cells per well on a 96-well cell culture plate and culture in a 37°C CO 2 cell incubator for 2 days. Add PBS to wash the cells, centrifuge the culture plate at 1500 rpm for 3 minutes, and wash twice in total. After immunization, serum or antibody clones were added to the culture supernatant of tumor cells and incubated at 4°C for 1 hour. Washed twice with PBS, added HRP-conjugated goat anti-mouse secondary antibody (Proteintech, Wuhan, 1:1000 dilution) and incubated at 4°C for 30 minutes.
  • HRP-conjugated goat anti-mouse secondary antibody Proteintech, Wuhan, 1:1000 dilution
  • Whether the PD-L1 antibody binds to sPD-L1 was analyzed by an established direct protein ELISA. As done in the past, coat 50ng PD-L1-3-HIS or PD-L1-9-HIS per well on a 96-well plate with protein high affinity and let stand overnight at 4°C. The supernatant was discarded, and 100ul of PBS+Tween 20 (0.05%) was added to each well to wash the 96-well plate three times. Add 100 ⁇ l of PBS+1% BSA to each well and let stand for 4 hours at room temperature. After discarding the supernatant, add 50 ⁇ l of the antibody cloned hybridoma cell culture supernatant to each well, and incubate at 4°C overnight.
  • sPD-L1 (native form) ELISA was used to further clarify antibody affinity for sPD-L1.
  • Native form sPD-L1 was expressed and secreted by MC38 cells as previously done. Cell culture supernatants containing sPD-L1 were used to test the affinity of PD-L1 antibodies.
  • Purified PD-L1 monoclonal antibody (0.2 ⁇ g/well) was coated overnight at 4°C. Add 100 ⁇ l/well to the supernatant of MC38 cells expressing sPD-L1-3 and/or sPD-L1-9.
  • MC38 and CHO-K1 cells were transfected and expressed human PD-L1 and PD-1, respectively.
  • MC38 and CHO-K1 cells expressing PD-L1 were labeled with PKH67
  • MC38 and CHO-K1 cells expressing PD-1 were labeled with PKH26.
  • Two differently labeled cells were co-cultured for one hour with or without PD-L1 or isotype antibodies.
  • the effect of PD-L1 antibody on the interaction between PD-L1 and PD-1 was detected by flow cytometry.
  • Double-positively labeled cell clusters indicate that PD-L1 has bound to PD-1.
  • MC38 and CHO-K1 cells expressing human PD-L1 were marked by PKH26, while MC38 and CHO-K1 cells expressing human PD-1 were marked by PKH67.
  • Co-culture of MC38 cells expressing PD-L1 but different markers co-culture of MC38 cells expressing PD-1 but different markers
  • double-positively labeled cell clusters indicate non-specific binding (background control).
  • Jurkat NFAT-Luciferase-CD16 was provided by Ikant Bio (Suzhou) to measure the activation of CD16 induced by antibody Fc and the activation of intracellular NF-AT signaling pathway and the production of reporter gene luciferase, and quantitatively detect the function of antibody Fc effector.
  • About 1.25 ⁇ 10 4 /well of PD-L1-expressing CHO-K1 target cells and humanized Fc ⁇ RIIIa-expressing effector cells were mixed at a ratio of 1:10 under different concentrations of humanized PD-L1 antibody. Incubate for 10 hours in a cell incubator.
  • Antibodies with high affinity that bind to the pan-IgC segment of PD-L1 were obtained by performing preliminary antibody production, purification and validation.
  • VH heavy chain
  • VH-CDR heavy chain variable region
  • VL light chain
  • VL-CDR light chain variable region
  • H6L2 refers to an anti-PD-L1 humanized antibody in which the variable region of the heavy chain is VH6, and the variable region of the light chain is VL2.
  • Step 1 Establishment of PD-L1/PD-1 and PD-L1-pan-IgC fragment expressing cell lines
  • Human or murine PD-L1, PD-1 and PD-L2 gene templates were obtained from Changsha Youbao Bio. Each gene and gene fragment were amplified by PCR using gene-specific primers (as shown in Table 1).
  • the amplified PCR product was inserted into the same digested pcDNA4 plasmid (Thermofisher, USA) by enzyme digestion and T4 DNA ligase.
  • the pcDNA4 plasmid vectors expressing genes and gene fragments were transfected into different expression host cells, and resistance screening was carried out.
  • plasmid vectors containing PD-L1, pan-IgC, PD-L2, or PD-1 gene fragments were transfected into CHO-K1 and MC38 cells, respectively; plasmids containing sPD-L1-3 and sPD-L1-9 gene fragments The vectors were respectively transfected into MC38 cells; the plasmid vectors containing sPD-L1-3-HIS and sPD-L1-9-HIS gene fragments were transfected into HEK293 cells respectively.
  • the host cells and uses for PD-L1, pan-IgC, PD-L2, or PD-1 gene expression are listed in Table 2.
  • mice were immunized in two ways with a plasmid vector expressing hPD-L1 (amino acids 19-290) and pan-IgC high (amino acids 132-290) expressing CHO-K1 cell lines. Serum was diluted 1:100, 1:1000, 1:10000, and MC38-expressing PD-L1 cells (positive cells), MC38 wild-type and PD-L2-expressing cells (negative cells) were used to detect mouse serum by flow cytometry Titer of specific antibody. Mice with positive serum were selected for cell fusion. The specificity and titer of antibodies in culture supernatants were tested by direct PD-L1 protein ELISA and positive and negative cell flow cytometry.
  • Monoclonal antibodies were obtained by intraperitoneal injection of monoclonal hybridoma cells, preparation of ascites, and purification by protein A affinity chromatography.
  • Step 4 DNA sequencing of heavy and light chain variable regions of anti-human PD-L1 mouse monoclonal antibody, as well as its humanization, expression and purification
  • RNA will be obtained from hybridoma cells producing murine monoclonal antibodies.
  • the heavy chain and corresponding light chain variable region DNAs were obtained by reverse transcription and PCR reaction, and the target gene was further inserted into the pCR TM vector plasmid by TOPO TA cloning method and sequenced.
  • Humanization of murine monoclonal antibody variable region includes CDR transplantation and back mutation (Back mutation), and transplantation into human IgG1, ⁇ .
  • humanized heavy and light chain antibody expression vectors pcDNA3.1 (Thermofisher) were constructed respectively; at the same time, a human-mouse chimeric antibody expression vector was constructed as a control.
  • the heavy and light chain expression plasmids were co-transfected into HEK293 cells. Antibody-secreting cell lines were further expanded. The antibody-containing culture supernatant was purified by protein A affinity chromatography.
  • the PD-L1 mouse monoclonal antibody with strong binding to human PD-L1 and pan-IgC was screened by flow cytometry.
  • MC38 cells express PD-L1 and pan-IgC respectively as positive detection cells.
  • MC38 cells express human PD-L2, and MC38 parental cells were used as negative controls.
  • PD-L1 murine mAb clone 130021 (R&D systems) binds PD-L1 and pan-IgC.
  • PD-L1 mouse monoclonal antibody clone 29E.2A3 (Biolegend) only binds to PD-L1 and does not recognize the pan-IgC segment, indicating that its recognition region is only in IgV.
  • 5 monoclonal antibodies that can both bind to PD-L1 and recognize pan-IgC were screened from 33 PD-L1 mouse monoclonal antibodies by flow cytometry, namely mAb#4, mAb #11, mAb #15, mAb #19 and mAb #23.
  • Example 3 Affinity of PD-L1 mouse monoclonal antibody to PD-L1, and the effect of sPD-L1 on its affinity
  • the affinity of PD-L1 antibody to recombinant protein PD-L1 was detected by direct PD-L1 protein ELISA.
  • Recombinant proteins PD-L1-3-HIS and PD-L1-9-HIS were expressed by HEK293 cells, and the culture supernatant was passed through Ni column to purify rPD-L1-HIS.
  • PD-L1 murine monoclonal antibodies mAb#4, mAb#11, mAb#15, mAb#19 and mAb#23 The binding of five PD-L1 murine monoclonal antibodies mAb#4, mAb#11, mAb#15, mAb#19 and mAb#23 to recombinant protein PD-L1 (rPD-L1) was detected by direct ELISA.
  • recombinant proteins PD-L1-3-HIS rPD-L1-3-HIS
  • PD-L1-9-HIS PD-L1 mouse monoclonal antibodies
  • 200ng recombinant protein PD-L1-3-HIS (14 times the moles of the antibody) or recombinant protein PD-L1-9-HIS (10 times the moles of the antibody) and 100ng PD-L1 mouse monoclonal antibody mAb#4, mAb #11, mAb #15, mAb #19 and mAb #23 were pre-incubated with PD-L1 expressing MC38 cells for 30 min after pre-incubation at 4°C for 2 h.
  • PD-L1 mouse monoclonal antibody #19 was selected from the screened 5 mouse monoclonal antibodies, and the effect of recombinant protein PD-L1-9 on the binding of mouse monoclonal antibody #19 to PD-L1 was detected. 100ng of antibody #19 was pre-incubated with various concentrations (as shown) of recombinant protein rPD-L1-9-HIS at 4°C for 2 hours, and then incubated with PD-L1-expressing MC38 cells for 30 minutes. The effect of rPD-L1-9-HIS on the binding function of PD-L1 murine monoclonal antibody #19 was analyzed by flow cytometry.
  • PD-L1 murine monoclonal antibody #19 for native sPD-L1 was detected by secreted sPD-L1 indirect ELISA.
  • MC38 cells were transfected and expressed native form of sPD-L1. Its cell culture supernatant contains the native form of sPD-L1.
  • Purified PD-L1 monoclonal antibody (0.2 ⁇ g/well) was coated overnight at 4°C. Add 100 ⁇ l/well of culture supernatant from MC38 cells expressing sPD-L1-3 or sPD-L1-9.
  • the experimental results are shown in Figure 5.
  • the results show that: PD-L1 mouse monoclonal antibody #19 specifically recognizes the human membrane-type PD-L1 on cells, but does not bind to the natural form sPD-L1-3.
  • the affinity of -9 is much lower than that of clone 130021 (it is worth noting that this antibody has a very low affinity for membrane-type hPD-L1 in the flow assay in Figures 3A and 3B, much weaker than PD-L1 murine mAb #19), namely Maximum circumvention of secreted sPD-L1 (sPD-L1-3 and sPD-L1-9).
  • Mouse mAb #19 was humanized to obtain its humanized antibody.
  • Murine mAb #19 and its humanized amino acid sequence, different heavy (A) light (B) chain Fab amino acid sequences are shown in Figure 7 and Table 4.
  • VH1, VH2, VH3, VH4, VH5, VH6 are the heavy chain variable regions of the mouse mAb #19 humanized antibody
  • VL1, VL2, VL3, VL4, VL5 are the mouse mAb #19 humanized antibody. light chain variable region.
  • H6L2, H6L5, H4L4, H5L4, H6L4, H5L5, and H4L5 had the best affinity.
  • humanized different heavy (H) light (L) chain combinations (H6L2, H6L5, H4L4, H5L4) of murine mAb #19 have affinity detection for membrane-type and secretory-type PD-L1, as well as for pan-IgC. 8 shown.
  • MC38 cells were transfected and secreted the cell culture supernatants of native sPDL1-3 and sPDL1-9, respectively, compared with mouse mAb #19 and its chimeric antibody (Chimeric), 130021, and anti-PD-L1 total , detected by ELISA Binding of murine mAb #19 humanized antibody to secreted sPD-L1 (C and D).
  • the experimental results showed that the humanized heavy and light chain combination of murine mAb #19 (H6L2, H6L5, H4L4, H5L4) bound PD-L1 and pan-IgC, and they did not bind sPD-L1-3, and did not bind sPD-L1 -9 has low affinity.
  • H6L2 and H6L5 did not bind PD-L2, while H5L4 had weak binding to PD-L2 and CHO-K1 parental cells.
  • the EC50 of H6L2 was 0.62 ⁇ g/ml
  • the EC50 of Avelumab was 0.17 ⁇ g/ml
  • the EC50 of Atezolizumab was 0.21 ⁇ g/ml.
  • CHO-K1 cells were transfected and expressed human PD-L1 and PD-1, respectively.
  • CHO-K1 cells expressing PD-L1 were marked by PKH67, while CHO-K1 cells expressing PD-1 were marked by PKH26.
  • Two differently expressed cells were co-cultured in the presence or absence of H6L2 for one hour. Different antibody concentrations are shown in the figure. The effect of this antibody on the interaction between PD-L1 and PD-1 between cells was detected by flow cytometry, and the double-positively labeled cell clusters indicated that PD-L1 had bound to PD-1.
  • CHO-K1 cells expressing PD-L1 were also marked by PKH26, and CHO-K1 cells expressing PD-1 were also marked by PKH67.
  • CHO-K1 cells expressing PD-L1 with different markers were co-cultured, CHO-K1 cells expressing PD-1 with different markers were co-cultured, and the double-positively labeled cell population indicated non-specific binding (background control). The results are shown in Figure 13.
  • 3 to 5 ⁇ 10 5 /mouse of MC38 cells expressing human PD-L1 were injected subcutaneously into C57BL/6J mice, 5 mice per group. Tumor formation was observed after 7 days. On the 7th, 10th, and 13th days after tumor cell injection, 100 ⁇ g/mouse of humanized antibody H6L2 or Avelumab, or PBS control group was injected intraperitoneally for three times.
  • the experimental results show that the humanized antibody H6L2 has a statistically significant anti-tumor effect, and is significantly better than Avelumab.
  • Comparative Example 1 Indirect ELISA to detect the binding ability of anti-human PD-L1 antibody atezolizumab to sPD-L1 in its natural form
  • the binding ability of anti-human PD-L1 antibody atezolizumab to native sPD-L1 was detected by indirect ELISA of secreted sPD-L1.
  • MC38 cells expressed sPD-L1-3 or sPD-L1-9, respectively.
  • the binding capacity of atezolizumab was analyzed by measuring the concentration of sPD-L1 in the supernatant of cells incubated with atezolizumab by ELISA.
  • Serum-free cell culture supernatants containing sPD-L1-3 or sPD-L1-9 were incubated with 1.5 ⁇ g/ml atezolizumab or human IgG1 overnight respectively (ate treated or hIgG1 treated in the figure), followed by protein G agarose (Thermo fisher) and centrifugation to remove atezolizumab.
  • Supernatants containing sPD-L1-3 or sPD-L1-9 (sPD-L1-3 or sPD-L1-9 shown) without any pre-incubation served as positive controls.
  • BL means supernatant without sPD-L1, as background control.
  • CHO-K1 cells were transfected and expressed human Source PD-L1 and PD-1.
  • CHO-K1 cells expressing PD-L1 were marked by PKH67, while CHO-K1 cells expressing PD-1 were marked by PKH26.
  • Two differently expressed cells were co-cultured for one hour in the presence of avelumab or atezolizumab, respectively. Different antibody concentrations are shown in Figure 17. The effect of the two antibodies on the interaction between PD-L1 and PD-1 between cells was detected by flow cytometry, and the double-positively labeled cell clusters indicated that PD-L1 had bound to PD-1.
  • a and B show the effects of Avelumab and Atezolizumab on the intercellular PD-1 and PD-L1 interaction.
  • C and D show the effect of sPD-L1 blockade with Avelumab and Atezolizumab.
  • the amount of antibody was 100ng/data point, and the amount of rPD-L1-9-HIS was shown in the figure.
  • CHO-K1 cells expressing PD-L1 were also marked by PKH26, and CHO-K1 cells expressing PD-1 were also marked by PKH67.
  • CHO-K1 cells expressing PD-L1 with different markers were co-cultured
  • CHO-K1 cells expressing PD-1 with different markers were co-cultured
  • the double-positively labeled cell population indicated non-specific binding (background control).
  • Fc region of an antibody binds to its receptor Fc ⁇ R.
  • Fc ⁇ Rs are expressed on a range of immune cells such as natural killer cells, monocytes, neutrophils and megakaryocytes.
  • the interaction between Fc and Fc ⁇ R plays an irreplaceable role in regulating ADCC, CDC, antibody metabolism and titer.
  • DEL amino acid substitution in the Fc CH2 segment by molecular bioengineering can significantly increase the function of natural killer cells.
  • 14D8 antibody with IgG2a Fc can specifically bind and activate Fc receptors, which have the function of inhibiting tumor growth; 14D8/IgG1 antibodies that bind inhibitory Fc ⁇ R receptors, and lose Fc receptors
  • the binding capacity of the 14D8/IgG1-D265A antibody was statistically significant for both antibodies.
  • the antitumor effect of another blocking PD-L1 antibody (clone 10F.9G2) was significantly attenuated in a mouse tumor model lacking activating Fc ⁇ Rs but retaining inhibitory Fc ⁇ RIIb relative to wild-type mice . In murine tumor models, the antibody has been widely used in anti-tumor mechanism research.
  • NK cells are innate immune effector cells that lyse histocompatibility complex (MHC class I)-negative tumor cells and virus-infected cells and coordinate the innate immune response. NK cells continuously express the lysis system. Therefore, NK cells do not require pre-activation to kill target cells and play an important role in immune surveillance of mutant cells or tumor cells.
  • MHC class I lyse histocompatibility complex
  • ADCC mainly depends on the killing activity of NK cells. Unlike T cells that express PD-1, NK cells carry various agonistic or inhibitory biomarker states, and they are persistently deficient by different detection methods, such as cell flow cytometry, qRT-PCR, and RNA-seq Express PD-1. This feature is beneficial to bypass the PD-1/PD-L1 pathway and kill tumor cells that express PD-L1. Moreover, tumor-targeting antibodies, such as rituximab, daratumumab, trastuzumab, and cetuximab, have been successfully developed that induce ADCC and are clinically effective in treating cancer patients.
  • our antibody Compared with avelumab, our antibody has strong induction of NK cell activity and significant anti-tumor effect. In summary, it is not limited to blocking the function of PD-L1 and the recovery of T cell function and indirect killing. ADCC and NK cells can be used to target tumor cells expressing PD-L1 to achieve direct killing. Therefore, there are Potential increase in response rate.
  • the inventors Based on the drug resistance mechanism of secreted sPD-L1 and its amino acid sequence characteristics, as well as the key role played by Fc and NK cells of PD-L1 antibody in anti-tumor, the inventors designed a new epitope and used Do immunogens and screen PD-L1-specific monoclonal antibodies, which target the IgC functional segment of PD-L1. Moreover, it maximally circumvents sPD-L1, enhances the ADCC/CDC activity of Fc, and has antitumor effects. This provides an alternative approach or regimen for the treatment of tumors that secrete sPD-L1 and are resistant to blocking PD-L1 antibodies, and has potential implications for improving the efficacy of tumor immunotherapy.

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Abstract

本发明提供了PD-L1抗体及其用途，该抗体或其活性片段不阻断PD-1与PD-L1的结合，并且特异性靶向PD-L1的泛IgC区段。本发明的非阻断型PD-L1抗体能最大限度地规避sPD-L1，且具有诱导ADCC/CDC功能效应的活性，同时具有优异的抗肿瘤活性。

Description

抗PD-L1抗体及其用途 技术领域
本发明涉及肿瘤免疫学领域，更具体地涉及一类抗PD-L1泛IgC区段又诱导ADCC/CDC功能效应的抗体。
背景技术
在过去的十年里，各种肿瘤免疫检查点抗体阻断剂，如伊匹单抗(ipilimumab)(CTLA4阻断抗体)、派姆单抗/纳武单抗(pembrolizumab/nivolumab)(PD-1阻断抗体)和阿特珠单抗(atezolizumab)(PD-L1阻断抗体)，在临床上的运用有了里程碑的突破。因此，美国科学家James Allison首先提出免疫检查点阻断(Check-point blockade)这一个普遍接受的概念。在不同的临床指征中，病人的治疗反应率差异很大。在最常见的实体瘤患者群中，这种治疗反应率有限，大概在20％左右。
PD1/PD-L1通路是重要的免疫检查点之一。PD-L1(程序性死亡配体1)是膜蛋白，它主要通过树突状细胞和单核细胞表达。它的受体是PD-1(程序性死亡蛋白受体1)，通过激活的T细胞、B细胞、树突状和单核细胞表达。当T细胞识别并结合组织相关抗原时，T细胞被激活而表达PD-1。PD-L1结合PD-1抑制T细胞的活化而导致免疫功能低下。研究发现各种不同类型的肿瘤也表达PD-L1。
研究进一步表明，PD-1与PD-L1的相互作用是通过结合PD-L1的IgV功能区域(氨基酸19-127)来实现的。阿特珠单抗的重链通过氢键结合PD-L1抗原决定基团E58、Q66、V111、R113和R125；并且抗原决定基团I54、Y56、N63、V111、M115、S117、A121、和Y123在PD-L1的CC'FG质片内通过范德瓦尔斯力(van der Waals)与重链互补决定区(HCDRs)结合。尽管不同PD-L1抗体如阿特珠单抗、德瓦鲁单抗(durvalumab)、BMS-963559和阿利库单抗(avelumab)识别不同抗原决定基团，但它们都作用于PD-L1的IgV功能区域。它们与PD-1竞争相同的抗原表位，其目的都是阻断PD-L1与PD-1的相互作用。目前，国内PD-L1抗体的研发也是基于免疫检查点阻断概念，但临床应用还没有被批准。
由免疫检查点阻断引起的治疗反应在某种程度上取决于肿瘤的特性，如肿瘤特异性免疫反应的产生、肿瘤内免疫抑制性微环境的形成和肿瘤对免疫反应的敏感度。研究发现肿瘤病人血清中存在可溶性PD-L1(sPD-L1)。高水平sPD-L1 存在于侵袭性肾细胞癌、多发性骨髓瘤和弥漫性大B细胞淋巴瘤，而且与低存活率相关。最近的研究发现，皮肤黑色瘤细胞有四个PD-L1剪接变异体，它们是导致sPD-L1分泌的原因。sPD-L1的分泌与剪接活性、细胞因子的刺激、细胞的应激反应、以及细胞的损伤和死亡有关。膜性PD-L1的表达与sPD-L1的分泌成正比关系。而且，各种不同类型肿瘤细胞普遍具有PD-L1剪接活性和分泌sPD-L1的功能，其中最为常见或表达量最多的是PD-L1-3/12和PD-L1-9。PD-L1-3/12和PD-L1-9氨基酸全长分别是氨基酸19-178和氨基酸19-243。很显然分泌型sPD-L1都含有IgV，即为现有阻断型PD-L1抗体识别区。
目前，研究进一步发现肿瘤分泌的sPD-L1剪接变异体可做为“诱饵”来结合PD-L1抗体并产生耐药性。仅1％的肿瘤细胞分泌的sPD-L1就可完全废除PD-L1抗体的抗肿瘤作用。在PD-L1抗体治疗中，sPD-L1的分泌与肿瘤患者的复发有关。这表明肿瘤分泌的sPD-L1可能是PD-L1阻断抗体***的障碍，或是其重要的耐药机制之一。
综上所述，本领域迫切需要开发一类更有效的抗sPD-L1分泌型耐药的治疗剂，其能最大限度地规避sPD-L1，增强Fc段诱导的ADCC/CDC活性，并具有抗肿瘤作用的PD-L1特异性单克隆抗体。
发明内容
本发明的目的是提供一类抗PD-L1的泛IgC区段又诱导ADCC/CDC功能效应的、抗肿瘤和抗sPD-L1分泌型耐药的PD-L1抗体。
本发明的第一方面，提供了一种PD-L1抗体或其活性片段，所述抗体不阻断PD-1与PD-L1的结合，并且所述抗体特异性结合于PD-L1的泛IgC区段，且具有诱导ADCC/CDC功能效应的活性。
在另一优选例中，所述的抗体不结合于PD-L1的IgV功能区域(即氨基酸19-127)。
在另一优选例中，所述抗体不与PD-1竞争结合PD-L1的IgV功能区域。
在另一优选例中，所述抗体不与PD-1竞争结合PD-L1的IgV功能区域的相同的抗原表位。
在另一优选例中，所述的抗体对可溶性PD-L1(sPD-L1)蛋白不结合或亲和力低下。
在另一优选例中，所述的抗体不结合于可溶性PD-L1蛋白PD-L1-3。
在另一优选例中，所述的抗体对PD-L1-9的结合力P 1远低于抗体130021(参照抗体)对PD-L1-9的结合力P 0，较佳地P 1/P 0≤1/5，更佳地≤1/10，更佳地≤1/100。
在另一优选例中，所述对可溶性PD-L1蛋白亲和力低下的抗体包括：PD-L1-1和PD-L1-9。
在另一优选例中，所述抗体对可溶性PD-L1(sPD-L1)蛋白不结合或部分结合。
在另一优选例中，所述抗体不结合的可溶性PD-L1蛋白包括：PD-L1-3。
在另一优选例中，所述抗体部分结合的可溶性PD-L1蛋白包括：PD-L1-1、和PD-L1-9。
所述的抗体是非阻断型抗体，不结合于PD-L1的任一氨基酸位点：
(i)E58、Q66、V111、R113、R125；和
(ii)I54、Y56、N63、V111、M115、S117、A121、和Y123。
在另一优选例中，所述的一种PD-L1抗体或其活性片段，包含：
(a)氨基酸序列如SEQ ID NO：16所示的LCDR1、SEQ ID NO：17所示的LCDR2、SEQ ID NO：18所示的LCDR3；以及
(b)氨基酸序列如SEQ ID NO：12所示的HCDR1、SEQ ID NO：13所示的HCDR2和SEQ ID NO：14所示的HCDR3。
其中，所述抗体特异性结合于PD-L1的泛IgC区段。
在另一优选例中，泛IgC区段为PD-L1蛋白的IgC区段和其与细胞膜的连接段区域，其中所述的区域由PD-L1蛋白的氨基酸残基132～238组成。
在另一优选例中，所述PD-L1抗体或其活性片段诱导ADCC/CDC功能效应进而杀伤肿瘤。
其中，上述氨基酸序列中任意一种氨基酸序列还包括任选地经过添加、缺失、修饰和/或取代至少一个氨基酸的，并能够保留PD-L1结合亲和力的衍生序列。
在另一优选例中，上述任一CDR的氨基酸序列中包含经过添加、缺失、修饰和/或取代1、2或3个氨基酸的衍生CDR序列，并且使得含有所述衍生CDR序列的VH和VL所构成的衍生抗体能够保留与PD-L1结合的亲和力。
在另一优选例中，所述添加、缺失、修饰和/或取代的氨基酸数量为1-5个(如1-3个，较佳地1-2个，更佳地1个)。
在另一优选例中，所述的经过添加、缺失、修饰和/或取代至少一个氨基 酸的，并能够保留PD-L1结合亲和力的衍生序列为同源性或序列相同性为至少96％的氨基酸序列。
在另一优选例中，所述PD-L1抗体或其活性片段包含重链可变区域或轻链可变区域，所述重链可变区的多肽序列与SEQ ID NO：11、20、21、22、23、24或25至少95％相同，所述轻链可变区的多肽序列与SEQ ID NO：15、26、27、28、29或30至少95％相同。
在另一优选例中，所述的PD-L1抗体或其活性片段，包含：具有SEQ ID NO：11、20、21、22、23、24或25所示多肽序列的重链可变区，和具有SEQ ID NO：15、26、27、28、29或30所示多肽序列的轻链可变区。
在另一优选例中，所述PD-L1抗体或其活性片段包含选自下组的重链可变区和轻链可变区：
(1)如SEQ ID NO：25所示的重链可变区；和如SEQ ID NO：27所示的轻链可变区；
(2)如SEQ ID NO：25所示的重链可变区；和如SEQ ID NO：30所示的轻链可变区；
(3)如SEQ ID NO：23所示的重链可变区；和如SEQ ID NO：29所示的轻链可变区；
(4)如SEQ ID NO：24所示的重链可变区；和如SEQ ID NO：29所示的轻链可变区；
(5)如SEQ ID NO：25所示的重链可变区；和如SEQ ID NO：29所示的轻链可变区；
(6)如SEQ ID NO：24所示的重链可变区；和如SEQ ID NO：30所示的轻链可变区；和
(7)如SEQ ID NO：23所示的重链可变区；和如SEQ ID NO：30所示的轻链可变区。
在另一优选例中，所述重链可变区的氨基酸序列与SEQ ID NO.11、20、21、22、23、24或25所示的氨基酸序列至少有80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％的序列同源性或序列相同性。
在另一优选例中，所述轻链可变区的氨基酸序列与如序列表中SEQ ID NO.15、26、27、28、29或30所示的氨基酸序列至少有80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％的序列同源性或序列相同性。
在另一优选例中，所述PD-L1抗体或其活性片段是嵌合的。
在另一优选例中，所述PD-L1抗体或其活性片段是人源化的。
本发明的第二方面，提供了一种重组蛋白，所述的重组蛋白具有：
(i)如本发明的第一方面所述的PD-L1抗体或其活性片段；以及
(ii)任选的协助表达和/或纯化的标签序列。
在另一优选例中，所述的标签序列包括6His标签。
在另一优选例中，所述的重组蛋白特异性抗PD-L1蛋白。
本发明的第三方面，提供了一种多核苷酸，编码本发明第一方面所述的PD-L1抗体或其活性片段，或本发明第二方面所述的重组蛋白。
本发明的第四方面，提供了一种载体，它含有本发明的第三方面所述的多核苷酸。
在另一优选例中，所述的载体包括：细菌质粒、噬菌体、酵母质粒、植物细胞病毒、哺乳动物细胞病毒如腺病毒、逆转录病毒、或其他载体。
本发明的第五方面，提供了一种遗传工程化的宿主细胞，它含有本发明的第四方面所述的载体或基因组中整合有本发明的第三方面所述的多核苷酸。
本发明的第六方面，提供了一种免疫偶联物，该免疫偶联物含有：
(a)如本发明第一方面所述的PD-L1抗体或其活性片段；和
(b)选自下组的偶联部分：可检测标记物、药物、毒素、细胞因子、放射性核素、或酶。
本发明的第七方面，提供了一种药物组合物，所述药物组合物含有：
(a)编码本发明的第一方面所述的PD-L1抗体或其活性片段，或如本发明第二方面所述的重组蛋白，或如本发明的第六方面所述的免疫偶联物；以及
(b)药学上可接受的载体。
在另一优选例中，所述的药物组合物还含有：额外的活性成分，较佳地所述的活性成分包括：小分子化合物、细胞因子、抗体(如抗PD-1抗体、抗OX40抗体、抗CD137抗体、抗CD47抗体、ADC、CAR-免疫细胞)。
在另一优选例中，所述的药物组合物为注射剂型。
在另一优选例中，所述的药物组合物用于制备***的药物，所述的肿瘤选自下组：膀胱癌、脑癌如胶质瘤，如低度胶质瘤、胶质母细胞瘤、***、乳腺癌、结肠癌、直肠癌、子宫内膜癌、肾癌、肾细胞癌、肾盂癌症、白血病、肺癌、非小细胞肺癌、黑色素瘤、非霍奇金淋巴瘤、胰腺癌、***癌、卵巢癌、纤维肉瘤、急性淋巴细胞白血病(ALL)、急性髓系白血病(AML)、慢性淋巴细胞白血病(CLL)、慢性粒细胞白血病(CML)、多发性骨髓瘤、骨髓衍生肿瘤、甲状腺癌。
在另一优选例中，所述药物组合物用于制备治疗感染性疾病的药物，所述感染性疾病包括感染性病毒疾病、感染性细菌疾病或感染性真菌疾病。
在另一优选例中，所述感染性疾病中，感染病灶或被感染的宿主细胞表达PD-L1(HIV、肝炎病毒、结核杆菌)。
在另一优选例中，所述感染性病毒疾病由选自以下的病原体引起：HIV、肝炎病毒(甲、乙、丙)、疱疹病毒、腺病毒、流感病毒、虫媒病毒、埃可病毒、鼻病毒、柯萨奇病毒、冠状病毒、呼吸道合胞病毒、流行性腮腺病毒、轮状病毒、麻疹病毒、风疹病毒、细小病毒、痘苗病毒、HTLV病毒、登革热病毒、***瘤病毒、软疣病毒、脊髓灰质炎病毒、狂犬病毒、JC病毒、虫媒病毒、和脑炎病毒。
在另一优选例中，所述的感染性细菌疾病由选自以下的病原体引起：结核杆菌、衣原体、立克次氏体菌、葡萄球菌、链球菌、肺炎球菌、脑膜炎球菌、***、克雷伯氏杆菌、变形菌、雷氏菌、假单胞菌、军团菌、白喉杆菌、沙门氏菌、芽孢杆菌、霍乱杆菌、破伤风杆菌、肉毒杆菌、炭疽杆菌、鼠疫杆菌、钩端螺旋体、和莱姆病细菌。
在另一优选例中，所述的感染性真菌疾病由选自以下的病原体引起：假丝酵母、新型隐球菌、曲霉菌、毛霉菌、申克孢子丝菌、皮炎芽生菌、巴西副球孢子菌、粗球孢子菌、和夹膜组织胞浆菌。在另一优选例中，所述感染性疾病由选自以下的病原体引起：HIV、肝炎病毒、流感病毒、疱疹病毒、贾第虫、疟原虫、利什曼原虫、金黄色葡萄球菌、绿脓杆菌。
在另一优选例中，所述感染性疾病由选自以下的病原体引起：HIV、肝炎病毒(甲、乙、丙)和结核杆菌。
在另一优选例中，所述的组合物可与另一种肿瘤免疫治疗联用，包括但不限于：化疗、抗CD20 mAb、抗TIM-3 mAb、抗LAG-3 mAb、抗CD73 mAb、抗CD47 mAb、抗DLL3 mAb、抗FRmAb mAb、抗CTLA-4抗体、抗OX40抗体、抗CD137 抗体、抗PD-1抗体、PD-1/PD-L1治疗、其他免疫肿瘤药物、抗血管生成剂、放射治疗、抗体-药物偶联物(ADC)、靶向治疗或其他抗癌药物。
在另一优选例中，所述的药物组合物，靶向有需要的受试者的癌细胞表面PD-L1、或增强ADCC/CDC作用的方法，包括给所述对象施用本发明的第七方面所述的药物组合物。
本发明的第八方面，提供了一种检测样品中PD-L1蛋白的方法，所述方法包括步骤：
(1)从受试者获得样品；
(2)使样品与本发明第一方面所述的PD-L1抗体或其活性片段接触；
(3)确定受试者中PD-L1的水平。
在另一优选例中，所述方法是非治疗和非诊断的。
在另一优选例中，所述方法为体外检测。
本发明的第九方面，提供了一种检测PD-L1的试剂盒，所述试剂盒含有选自下组的一种或多种：如本发明第一方面所述的PD-L1抗体或其活性片段、或如本发明的第六方面所述的免疫偶联物，如本发明的第七方面所述的药物组合物，以及说明书。
在另一优选例中，所述的说明书记载，所述的试剂盒用于检测待测对象的PD-L1表达。
在另一优选例中，所述的试剂盒用于表达PD-L1蛋白(即PD-L1阳性)的肿瘤的检测。
在本发明的第十方面，提供了一种免疫细胞，所述免疫细胞表达或在细胞膜外暴露有本发明的第一方面所述的抗体。
在另一优选例中，所述的免疫细胞包括NK细胞、T细胞。
在另一优选例中，所述的免疫细胞来自人或非人哺乳动物(如鼠)。
本发明的第十一方面，提供了一种治疗PD-L1表达或功能异常相关的疾病的方法，所述方法包括向有需要的对象施用药学有效量的如本发明第一方面所述的PD-L1抗体，本发明的第六方面所述的免疫偶联物，或如本发明的第七方面所述的药物组合物，本发明的第十方面所述的免疫细胞，或其组合。
在另一优选例中，所述PD-L1表达或功能异常相关的疾病为肿瘤或感染性 疾病。
在另一优选例中，所述的PD-L1抗体可单独使用、或与其他治疗剂联合施用。
在另一优选例中，所述其他治疗剂包括免疫检查点阻断剂、佐剂或疫苗。
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
附图说明
图1中A图显示了PD-L1的全长和它的剪接变异体sPD-L1-3或12，sPD-L1-9的氨基酸区域，已知PD-L1抗体的识别区域和PD-L1鼠单克隆抗体mAb#19和其人源化抗体的抗原表位。B图显示了人源PD-L1 IgC及其与细胞膜的连接段氨基酸序列。其中下划点段表示信号肽段，下划双线段表示IgV(氨基酸19-127)，下划单线段表示IgC及其与细胞膜的连接段(氨基酸132-238)，下划短线加下划点线段表示跨膜结构域(氨基酸239-259)，下划波浪线段表示胞内结构域(氨基酸260-290)。
图2显示了人源PD-L1剪接变异体的示意图。其中A图表示PD-L1全长含有六个外显子，PD-L1-1、PD-L1-3、PD-L1-9和PD-L1-12分别为剪接变异体，跨膜区位于第四外显子并以粉红色标示，剪除段以括弧标示，Stop代表终止密码子。B图分别表示全长膜型PD-L1、剪接变异体PD-L1-1、PD-L1-3、PD-L1-9和PD-L1-12的核酸和氨基酸序列，其中跨膜区在外显子4区段内以粉红色标示，剪除段以红色括弧标示，每一个剪接变异体的氨基酸序列以蓝色标示，下划线表示额外不同的氨基酸。
图3显示了PD-L1鼠单克隆抗体对人源PD-L1和泛IgC的结合力。其中A图显示了流式细胞术检测PD-L1鼠单克隆抗体130021(R&D systems,美国)和29E.2A3(Biolegend，美国)对人源PD-L1和泛IgC的结合力。其中，抗体130021结合PD-L1和泛IgC；抗体29E.2A3仅结合PD-L1而不识别泛IgC段，表明它的识别区只在IgV；克隆24F.10C12(Biolegend)为人源PD-L2鼠单抗；B图显示了PD-L1鼠单克隆抗体的细胞流式筛选图，其中筛选的5个PD-L1鼠单克隆抗体mAb#4、mAb#11、mAb#15、mAb#19和mAb#23既能结合PD-L1也能识别泛IgC。其中，PD-L1PE表示PE萤光标记PD-L1抗体。
图4显示了PD-L1鼠单克隆抗体对重组蛋白rPD-L1的亲和力。其中A图显示了ELISA检测5个PD-L1鼠单克隆抗体mAb#4、mAb#11、mAb#15、mAb#19和mAb#23与重组蛋白PD-L1的亲和力；B图显示了流式细胞术检测重组蛋白rPD-L1-3-HIS和rPD-L1-9-HIS对5个PD-L1鼠单克隆抗体mAb#4、mAb#11、mAb#15、mAb#19和mAb#23的阻断作用；C图显示了流式细胞术检测不同浓度的重组蛋白rPD-L1-9-HIS对鼠单克隆抗体#19结合PD-L1的影响。
图5显示了ELISA检测PD-L1鼠单克隆抗体#19对自然形态sPD-L1的亲和力。其中A图显示了鼠单克隆抗体mAb#19对细胞培养上清中sPD-L1-3与sPD-L1-9的亲和力，B图显示了鼠单克隆抗体mAb#19对细胞培养上清中sPD-L1-3的亲和力，C图显示了鼠单克隆抗体mAb#19对细胞培养上清中sPD-L1-9的亲和力。其中，Blank表示培养液阴性对照。
图6显示了PD-L1鼠单克隆抗体#19在表达人源PD-L1和sPD-L1-9的MC38小鼠肿瘤模型上的抗肿瘤作用，其中A图表示不同天数下小鼠的肿瘤大小，以肿瘤容积平均值±标准误表示；B图表示不同天数下小鼠的存活率。
图7显示了鼠单抗#19人源化。不同重(A)轻(B)链Fab氨基酸序列如图所示。mVH和mVL:鼠单抗重和轻链可变区。VH和VL:人源化抗体重和轻链可变区。重轻链的CDR区以红色粗体字和下划线表示。
图8显示了鼠单抗#19人源化，不同重(H)轻(L)链组合对膜型和分泌型PD-L1，以及对泛IgC的亲和力。
图9显示了鼠单抗#19人源化，不同重(H)轻(L)链组合对膜型PD-L2(人源)的亲和力。通过细胞流式检测克隆#19人源化抗体对膜型PD-L2(A，以MFI图示；B，以点图示)的亲合力。C:CHO-K1亲本细胞，为阴性对照。克隆24F.10C12：人源PD-L2鼠单抗。二抗：PE标记的羊抗鼠抗体(BD biosciences，美国)。
图10显示了鼠单抗#19人源化，不同重(H)轻(L)链组合对膜型CD80(人源)的亲和力。通过细胞流式检测克隆#19人源化抗体对膜型CD80(A，以MFI图示；B，以点图示)的亲合力。C:CHO-K1亲本细胞，为阴性对照。克隆2D10：人源CD80鼠单抗克隆(Elabscience，武汉)。二抗：PE标记的羊抗鼠抗体(BD biosciences)。
图11显示了人源化抗体H6L2，在无或有rPD-L1-9-HIS条件下，对膜型PD-L1的亲和力。通过细胞流式检测人源化抗体H6L2对膜型PD-L1亲合力(A)，和不同浓度的rPD-L1-9-HIS对H6L2亲和力的影响(B，以MFI图示，和C，以 点图示)，同时，对比阿利库单抗(Avelumab)和阿特珠单抗(Atezolizumab)。
图12显示了人源化抗体H6L2，在有或rPD-L1-3-HIS条件下，对膜型PD-L1的亲和力。通过细胞流式检测不同浓度的rPD-L1-3-HIS对H6L2亲和力的影响(A，以MFI图示，和B，以点图示)，同时，对比阿利库单抗(Avelumab)和阿特珠单抗(Atezolizumab)。
图13显示了人源化抗体H6L2对PD-1与PD-L1结合的阻断作用。
图14显示了人源化抗体H6L2的ADCC活性和分泌型sPD-L1对其ADCC作用的影响。在Jurkat-CD16-NFAT-Luciferase平台，无(A)和有(B)分泌型sPD-L1(重组蛋白rPDL1-3-HIS或rPDL1-9-HIS)条件下，检测#19人源化抗体诱导Luciferase(萤光素酶)的活性能力，同时，对比阿利库单抗(Avelumab)。
图15显示了人源化抗体H6L2的抗肿瘤作用。A.肿瘤大小，以各组肿瘤容积的平均值±标准误表示。B.生存率。PBS和阿利库单抗(Avelumab)分别为阴和阳性对照组。
图16显示了抗人源PD-L1抗体阿特珠单抗对自然形态的sPD-L1的结合力。ELISA检测抗人源PD-L1抗体阿特珠单抗与自然形态的sPD-L1的结合力。其中，BL表示无sPD-L1上清，做为背景对照；ate treated表示通过阿特珠单抗处理；hIgG1 treated表示通过人源hIgG1处理；sPD-L1-3或sPD-L1-9分别表示含有sPD-L1-3或sPD-L1-9的上清液
图17显示了人源PD-L1抗体avelumab和atezolizumab对PD-1与PD-L1结合的阻断作用，以及分泌型sPD-L1对其阻断作用的影响。A和B.Avelumab(阿利库单抗)和Atezolizumab(阿特珠单抗)对细胞间PD-1与PD-L1相互作用的影响；C和D.sPD-L1对Avelumab和Atezolizumab阻断影响。抗体用量为100ng/数据点,rPD-L1-9-HIS用量如图所示。
具体实施方式
本发明人经过广泛而深入地研究，首次发现一类非阻断型PD-L1抗体，所述的抗体不阻断PD-1与PD-L1的结合，然而这类非阻断型PD-L1抗体居然具有优异的抗肿瘤活性。研究表明，本发明的非阻断型PD-L1抗体可特异性靶向PD-L1的泛IgC区间，能最大限度地规避sPD-L1，同时诱导ADCC/CDC活性。在此基础上完成了本发明。
具体地，本发明人构建了五个特异性抗人源PD-L1鼠单克隆抗体，均不结合sPD-L1-3，且其中一个单抗能部分规避PD-L1的胞外全长sPD-L1-9，其人 源化抗体在此方面明显优于对照抗人源PD-L1阿特珠和阿利库抗体。
术语
为了可以更容易地理解本公开，首先定义某些术语。如本申请中所使用的，除非本文另有明确规定，否则以下术语中的每一个应具有下面给出的含义。在整个申请中阐述了其它定义。
在本发明中，术语“本发明的非阻断型PD-L1抗体”、“本发明的PD-L1抗体”、“本发明的特异性抗体”、“本发明抗体”、“本发明蛋白”、或“本发明多肽”可互换使用，都指特异性结合PD-L1蛋白的抗体。本发明的非阻断型PD-L1抗体不阻断PD-1与PD-L1的结合，可特异性靶向PD-L1的泛IgC区间，且具有诱导ADCC/CDC功能效应的活性。例如具有重链(如SEQ ID NO:11、20,21,22,23,24和25所示的氨基酸序列)和/或轻链(如SEQ ID NO:15、26，27，28，29和30所示的氨基酸序列)的蛋白或多肽。它们可含有或不含起始甲硫氨酸。
泛IgC区段
如本文所用，IgC区与细胞膜的连接段记为“泛IgC区段”，为PD-L1蛋白SEQ ID No:19中的氨基酸132-238区段(图1中的A和B)。
PD-L1抗体
本发明提供了对人PD-L1蛋白具有高亲和力的PD-L1抗体。受测抗体表现出有效的结合和杀伤肿瘤活性，并可用于治疗和诊断用途。PD-L1蛋白是40kDa的I型跨膜蛋白。其胞外部分包含N-末端免疫球蛋白V(IgV)结构域(氨基酸19～127)和C末端免疫球蛋白C(IgC)结构域(氨基酸132～225)。如同抗体的IgV结构域和T细胞受体的相互作用一样，PD-1和PD-L1通过它们的IgV结构域的保守的正面和侧面相互作用。目前的抗PD-L1抗体全都结合到IgV结构域，从而阻断PD-1和PD-L1之间的结合(图1A和B)。因此，本发明揭示了一个发现：即本发明的非阻断型PD-L1抗体其与PD-L1蛋白的泛IgC结构域结合后可以有效地利用PD-L1靶点，导致治疗效果能够更进一步的改善。因此，本发明公开的一个实施方案提供了抗PD-L1抗体或其活性片段，该抗体或其活性片段可以特异性结合人程序性死亡配体1(PD-L1)蛋白的泛免疫球蛋白C(IgC)结构域。本发明的抗体具有一种或多种期望的功能特性，包括但不限于与PD-L1的 高亲和力结合、对PD-L1的高特异性、刺激补体依赖性细胞毒性(CDC)的能力、抗体依赖性吞噬作用(ADPC)和/或针对表达PD-L1细胞的抗体依赖性细胞介导的细胞毒性(ADCC)，以及在受试者和动物模型中单独或与其他抗癌疗法联合使用时具有抑制肿瘤生长的能力。
在一些实施方案中，泛IgC结构域由氨基酸残基132～238组成。在一些实施方案中，所述抗体或其活性片段可以结合至PD-L1蛋白的氨基酸残基。
在本发明的一个优选的实施方式中，所述PD-L1的氨基酸序列为：
Figure PCTCN2021121022-appb-000001
其中，SEQ ID No:19中第132-238位为泛IgC区。
Figure PCTCN2021121022-appb-000002
PD-L1的氨基酸区段及位置如下表所示：
Figure PCTCN2021121022-appb-000003
分泌型sPD-L1剪接异体
本发明人首次发现分泌型PD-L1剪接异体，以及它们与sPD-L1分泌的关 系。sPD-L1的分泌与膜型PD-L1的表达成正比关系，而且也与它剪接活性、细胞因子的刺激、细胞的应激反应、以及细胞的损伤和死亡有关。人源PD-L1剪接变异体PD-L1-1、PD-L1-3、PD-L1-9和PD-L1-12及其序列如图2所示(注：PD-L1-12与PD-L1-3氨基酸序列相同)。其中，“分泌型PD-L1”、“可溶性PD-L1”、“分泌型sPD-L1”、“sPD-L1”可互换使用，均指分泌型PD-L1剪接变异体。
在一个具体实施例中，自然形态的分泌型sPD-L1通过MC38细胞表达和分泌；rPD-L1-HIS为通过HEK293细胞重组表达的带有His标签的分泌型sPD-L1，包括rPD-L1-3-HIS、rPD-L1-9-HIS。
在另一优选例中，成功挑选出五个特异性抗人源PD-L1鼠单克隆抗体，它们识别表达在细胞上的PD-L1和IgC。这5个单抗都不结合sPD-L1-3，其中1个单抗还能部分规避sPD-L1-9，而sPD-L1-9是整个PD-L1的胞外全长，包括IgV和IgC功能段。分泌sPD-L1-9的小鼠肿瘤模型中的体内实验显示它具有抗耐药和抗肿瘤作用。
在另一优选例中，挑选出PD-L1鼠单克隆抗体#19。
抗体130021(参照抗体),即鼠单抗体130021(R&D systems)，结合PD-L1的泛IgC区段，不与可溶性PD-L1蛋白中的sPD-L1-3结合，与sPD-L1-9结合。
如本文所用，术语“抗体”或“免疫球蛋白”是有相同结构特征的约150000道尔顿的异四聚糖蛋白，其由两个相同的轻链(L)和两个相同的重链(H)组成。每条轻链通过一个共价二硫键与重链相连，而不同免疫球蛋白同种型的重链间的二硫键数目不同。每条重链和轻链也有规则间隔的链内二硫键。每条重链的一端有可变区(VH)，其后是多个恒定区。每条轻链的一端有可变区(VL)，另一端有恒定区；轻链的恒定区与重链的第一个恒定区相对，轻链的可变区与重链的可变区相对。特殊的氨基酸残基在轻链和重链的可变区之间形成界面。
如本文所用，术语“可变”表示抗体中可变区的某些部分在序列上有所不同，它形成了各种特定抗体对其特定抗原的结合和特异性。然而，可变性并不均匀地分布在整个抗体可变区中。它集中于轻链和重链可变区中称为互补决定区(CDR)或超变区中的三个片段中。可变区中较保守的部分称为构架区(FR)。天然重链和轻链的可变区中各自包含四个FR区，它们大致上呈β-折叠构型，由形成连接环的三个CDR相连，在某些情况下可形成部分β折叠结构。每条链中的CDR通过FR区紧密地靠在一起并与另一链的CDR一起形成了抗体的抗原 结合部位(参见Kabat等,NIH Publ.No.91-3242,卷I，647-669页(1991))。恒定区不直接参与抗体与抗原的结合，但是它们表现出不同的效应功能，例如参与抗体的依赖于抗体的细胞毒性。
脊椎动物抗体(免疫球蛋白)的“轻链”可根据其恒定区的氨基酸序列归为明显不同的两类(称为κ和λ)中的一类。根据其重链恒定区的氨基酸序列，免疫球蛋白可以分为不同的种类。主要有5类免疫球蛋白：IgA、IgD、IgE、IgG和IgM，其中一些还可进一步分成亚类(同种型)，如IgG1、IgG2、IgG3、IgG4、IgA和IgA2。对应于不同类免疫球蛋白的重链恒定区分别称为α、δ、ε、γ、和μ。不同类免疫球蛋白的亚单位结构和三维构型是本领域人员所熟知的。
如本文所用，术语“单克隆抗体(单抗)”指从一类基本均一的群体获得的抗体，即该群体中包含的单个抗体是相同的，除少数可能存在的天然发生的突变外。单克隆抗体高特异性地针对单个抗原位点。而且，与常规多克隆抗体制剂(通常是具有针对不同决定簇的不同抗体)不同，各单克隆抗体是针对抗原上的单个决定簇。除了它们的特异性外，单克隆抗体的好处还在于它们是通过杂交瘤培养来合成的，不会被其它免疫球蛋白污染。修饰语“单克隆”表示了抗体的特性，是从基本均一的抗体群中获得的，这不应被解释成需要用任何特殊方法来生产抗体。
本发明还包括具有所述的抗PD-L1蛋白单克隆抗体的相应氨基酸序列的单克隆抗体、具有所述的抗PD-L1蛋白单克隆抗体可变区链的单克隆抗体，以及具有这些链的其他蛋白质或蛋白质偶联物及融合表达产物。具体地，本发明包括具有含超变区(互补决定区，CDR)的轻链和重链的任何蛋白质或蛋白质偶联物及融合表达产物(即免疫偶联物及融合表达产物)，只要该超变区与本发明的轻链和重链的超变区相同或至少90％同源性，较佳地至少95％同源性。
如本领域技术人员所知，免疫偶联物及融合表达产物包括：药物、毒素、细胞因子(cytokine)、放射性核素、酶和其他诊断或治疗分子与所述的抗PD-L1蛋白单克隆抗体或其片段结合的而形成的偶联物。本发明还包括与所述的抗PD-L1蛋白单克隆抗体或其片段结合的细胞表面标记物或抗原。
本发明不仅包括完整的单克隆抗体，还包括具有免疫活性的抗体片段，如Fab或(Fab’) 2片段；抗体重链；抗体轻链。
如本文所用，术语“重链可变区”与“VH”可互换使用。
如本文所用，术语“可变区”与“互补决定区(complementarity determining region,CDR)”可互换使用。
在另一个优选例中，所述抗体的重链可变区包括包括三个互补决定区CDR1、CDR2、和CDR3。
在另一个优选例中，所述抗体的重链包括上述重链可变区和重链恒定区，所述重链恒定区可以为鼠源或人源。
在另一优选例中，所述的抗体为抗PD-L1蛋白的鼠或人鼠嵌合单克隆抗体，它的重链恒定区和/或轻链恒定区可以是人源化的重链恒定区或轻链恒定区。更优选地，所述的人源化的重链恒定区或轻链恒定区为人IgG1、IgG2等的重链恒定区或轻链恒定区。
本发明还提供了具有本发明抗体的其他蛋白质或融合表达产物。具体地，本发明包括具有含可变区的重链和轻链的任何蛋白质或蛋白质偶联物及融合表达产物(即免疫偶联物及融合表达产物)，只要该可变区与本发明抗体的重链和轻链的可变区相同或至少90％同源性，较佳地至少95％同源性。
一般，抗体的抗原结合特性可由位于重链和轻链可变区的3个特定的区域来描述，称为可变区域(CDR)，将该段间隔成4个框架区域(FR)，4个FR的氨基酸序列相对比较保守，不直接参与结合反应。这些CDR形成环状结构，通过其间的FR形成的β折叠在空间结构上相互靠近，重链上的CDR和相应轻链上的CDR构成了抗体的抗原结合位点。可以通过比较同类型的抗体的氨基酸序列来确定是哪些氨基酸构成了FR或CDR区域。
本发明抗体的重链和/或轻链的可变区特别令人感兴趣，因为它们中至少部分涉及结合抗原。因此，本发明包括那些具有带CDR的单克隆抗体轻链和重链可变区的分子，只要其CDR与此处鉴定的CDR具有90％以上(较佳地95％以上，最佳地98％以上)的同源性。
本发明不仅包括完整的单克隆抗体，还包括具有免疫活性的抗体的片段或抗体与其他序列形成的融合蛋白。因此，本发明还包括所述抗体的片段、衍生物和类似物。
如本文所用，术语“片段”、“衍生物”和“类似物”是指基本上保持本发明抗体相同的生物学功能或活性的多肽。本发明的多肽片段、衍生物或类似物可以是(i)有一个或多个保守或非保守性氨基酸残基(优选保守性氨基酸残基)被取代的多肽，而这样的取代的氨基酸残基可以是也可以不是由遗传密码编码的，或(ii)在一个或多个氨基酸残基中具有取代基团的多肽，或(iii)成熟多肽与另一个化合物(比如延长多肽半衰期的化合物，例如聚乙二醇)融合所形成的多肽，或(iv)附加的氨基酸序列融合到此多肽序列而形成的多肽(如前 导序列或分泌序列或用来纯化此多肽的序列或蛋白原序列，或与6His标签形成的融合蛋白)。根据本文所用，这些片段、衍生物和类似物属于本领域熟练技术人员公知的范围。
本发明抗体指具有PD-L1蛋白结合活性的、包括上述CDR区的多肽。该术语还包括具有与本发明抗体相同功能的、包含上述CDR区的多肽的变异形式。这些变异形式包括(但并不限于)：一个或多个(通常为1-50个，较佳地1-30个，更佳地1-20个，最佳地1-10个)氨基酸的缺失、***和/或取代，以及在C末端和/或N末端添加一个或数个(通常为20个以内，较佳地为10个以内，更佳地为5个以内)氨基酸。例如，在本领域中，用性能相近或相似的氨基酸进行取代时，通常不会改变蛋白质的功能。又比如，在C末端和/或N末端添加一个或数个氨基酸通常也不会改变蛋白质的功能。该术语还包括本发明抗体的活性片段和活性衍生物。
该多肽的变异形式包括：同源序列、保守性变异体、等位变异体、天然突变体、诱导突变体、在高或低的严紧度条件下能与本发明抗体的编码DNA杂交的蛋白、以及利用抗本发明抗体的抗血清获得的多肽或蛋白。
本发明还提供了其他多肽，如包含人抗体或其片段的融合蛋白。除了几乎全长的多肽外，本发明还包括了本发明抗体的片段。通常，该片段具有本发明抗体的至少约50个连续氨基酸，较佳地至少约50个连续氨基酸，更佳地至少约80个连续氨基酸，最佳地至少约100个连续氨基酸。
本发明还提供了编码上述抗体或其片段或其融合蛋白的多核苷酸分子。本发明的多核苷酸可以是DNA形式或RNA形式。DNA形式包括cDNA、基因组DNA或人工合成的DNA。DNA可以是单链的或是双链的。DNA可以是编码链或非编码链。
编码本发明的成熟多肽的多核苷酸包括：只编码成熟多肽的编码序列；成熟多肽的编码序列和各种附加编码序列；成熟多肽的编码序列(和任选的附加编码序列)以及非编码序列。
术语“编码多肽的多核苷酸”可以是包括编码此多肽的多核苷酸，也可以是还包括附加编码和/或非编码序列的多核苷酸。
本发明还涉及与上述的序列杂交且两个序列之间具有至少50％，较佳地至少70％，更佳地至少80％相同性的多核苷酸。本发明特别涉及在严格条件下与本发明所述多核苷酸可杂交的多核苷酸。在本发明中，“严格条件”是指：(1)在较低离子强度和较高温度下的杂交和洗脱，如0.2×SSC，0.1％SDS，60℃； 或(2)杂交时加有变性剂，如50％(v/v)甲酰胺，0.1％小牛血清/0.1％Ficoll，42℃等；或(3)仅在两条序列之间的相同性至少在90％以上，更好是95％以上时才发生杂交。并且,可杂交的多核苷酸编码的多肽与成熟多肽有相同的生物学功能和活性。
本发明的抗体的核苷酸全长序列或其片段通常可以用PCR扩增法、重组法或人工合成的方法获得。一种可行的方法是用人工合成的方法来合成有关序列，尤其是片段长度较短时。通常通过先合成多个小片段，然后再进行连接可获得序列很长的片段。此外，还可将重链的编码序列和表达标签(如6His)融合在一起，形成融合蛋白。
一旦获得了有关的序列，就可以用重组法来大批量地获得有关序列。这通常是将其克隆入载体，再转入细胞，然后通过常规方法从增殖后的宿主细胞中分离得到有关序列。本发明所涉及的生物分子(核酸、蛋白等)包括以分离的形式存在的生物分子。
目前，已经可以完全通过化学合成来得到编码本发明蛋白(或其片段，或其衍生物)的DNA序列。然后可将该DNA序列引入本领域中已知的各种现有的DNA分子(或如载体)和细胞中。此外，还可通过化学合成将突变引入本发明蛋白序列中。
本发明还涉及包含上述的适当DNA序列以及适当启动子或者控制序列的载体。这些载体可以用于转化适当的宿主细胞，以使其能够表达蛋白质。
宿主细胞可以是原核细胞，如细菌细胞；或是低等真核细胞，如酵母细胞；或是高等真核细胞，如哺乳动物细胞。代表性例子有：大肠杆菌，链霉菌属；鼠伤寒沙门氏菌的细菌细胞；真菌细胞如酵母；果蝇S2或Sf9的昆虫细胞；CHO、COS7、293细胞的动物细胞等。
用重组DNA转化宿主细胞可用本领域技术人员熟知的常规技术进行。当宿主为原核生物如大肠杆菌时，能吸收DNA的感受态细胞可在指数生长期后收获，用CaCl 2法处理，所用的步骤在本领域众所周知。另一种方法是使用MgCl 2。如果需要，转化也可用电穿孔的方法进行。当宿主是真核生物，可选用如下的DNA转染方法：磷酸钙共沉淀法，常规机械方法如显微注射、电穿孔,脂质体包装等。
获得的转化子可以用常规方法培养，表达本发明的基因所编码的多肽。根据所用的宿主细胞，培养中所用的培养基可选自各种常规培养基。在适于宿主细胞生长的条件下进行培养。当宿主细胞生长到适当的细胞密度后，用合适的 方法(如温度转换或化学诱导)诱导选择的启动子，将细胞再培养一段时间。
在上面的方法中的重组多肽可在细胞内、或在细胞膜上表达、或分泌到细胞外。如果需要，可利用其物理的、化学的和其它特性通过各种分离方法分离和纯化重组的蛋白。这些方法是本领域技术人员所熟知的。这些方法的例子包括但并不限于：常规的复性处理、用蛋白沉淀剂处理(盐析方法)、离心、渗透破菌、超处理、超离心、分子筛层析(凝胶过滤)、吸附层析、离子交换层析、高效液相层析(HPLC)和其它各种液相层析技术及这些方法的结合。
本发明的抗体可以单独使用，也可与可检测标记物(为诊断目的)、治疗剂、PK(蛋白激酶)修饰部分或任何以上这些物质的组合结合或偶联。
用于诊断目的可检测标记物包括但不限于：荧光或发光标记物、放射性标记物、MRI(磁共振成像)或CT(电子计算机X射线断层扫描技术)造影剂、或能够产生可检测产物的酶。
可与本发明抗体结合或偶联的治疗剂包括但不限于：1.放射性核素；2.生物毒；3.细胞因子如IL-2等；4.金纳米颗粒/纳米棒；5.病毒颗粒；6.脂质体；7.纳米磁粒；8.前药激活酶(例如，DT-心肌黄酶(DTD)或联苯基水解酶-样蛋白质(BPHL))；10.化疗剂(例如，顺铂)或任何形式的纳米颗粒等。
免疫偶联物
本发明还提供了基于本发明抗体的免疫偶联物，较佳地，所述免疫偶联物为抗体偶联药物(antibody-drug conjugate，ADC)。
典型地，所述抗体偶联药物包括所述抗体、以及效应分子，所述抗体与所述效应分子偶联，并优选为化学偶联。其中，所述效应分子优选为具有治疗活性的药物。此外，所述效应分子可以是毒蛋白、化疗药物、小分子药物或放射性核素中的一种或多种。
本发明抗体与所述效应分子之间可以是通过偶联剂进行偶联。所述偶联剂的例子可以是非选择性偶联剂、利用羧基的偶联剂、肽链、利用二硫键的偶联剂中的任意一种或几种。所述非选择性偶联剂是指使效应分子和抗体形成共价键连接的化合物，如戊二醛等。所述利用羧基的偶联剂可以是顺乌头酸酐类偶联剂(如顺乌头酸酐)、酰基腙类偶联剂(偶联位点为酰基腙)中的任意一种或几种。
抗体上某些残基(如Cys或Lys等)用于与多种功能基团相连，其中包括成像试剂(例如发色基团和荧光基团)，诊断试剂(例如MRI对比剂和放射性同位素)， 稳定剂(例如乙二醇聚合物)和治疗剂。抗体可以被偶联到功能剂以形成抗体-功能剂的偶联物。功能剂(例如药物，检测试剂，稳定剂)被偶联(共价连接)至抗体上。功能剂可以直接地、或者是通过接头间接地连接于抗体。
抗体可以偶联药物从而形成抗体药物偶联物(ADCs)。典型地，ADC包含位于药物和抗体之间的接头。接头可以是可降解的或者是不可降解的接头。可降解的接头典型地在细胞内环境下容易降解，例如在目标位点处接头发生降解，从而使药物从抗体上释放出来。合适的可降解的接头包括，例如酶降解的接头，其中包括可以被细胞内蛋白酶(例如溶酶体蛋白酶或者内体蛋白酶)降解的含有肽基的接头，或者糖接头例如，可以被葡糖苷酸酶降解的含葡糖苷酸的接头。肽基接头可以包括，例如二肽，例如缬氨酸-瓜氨酸，苯丙氨酸-赖氨酸或者缬氨酸-丙氨酸。其它合适的可降解的接头包括，例如，pH敏感接头(例如pH小于5.5时水解的接头，例如腙接头)和在还原条件下会降解的接头(例如二硫键接头)。不可降解的接头典型地在抗体被蛋白酶水解的条件下释放药物。
药物可以是任何细胞毒性，抑制细胞生长或者免疫抑制的药物。在实施方式中，接头连接抗体和药物，而药物具有可以和接头成键的功能性基团。例如，药物可以具有可以和连接物成键的氨基，羧基，巯基，羟基，或者酮基。在药物直接连接到接头的情况下，药物在连接到抗体之前，具有反应的活性基团。
有用的药物类别包括，例如，抗微管蛋白药物、DNA小沟结合试剂、DNA复制抑制剂、烷化试剂、抗生素、叶酸拮抗物、抗代谢药物、化疗增敏剂、拓扑异构酶抑制剂、长春花生物碱等。特别有用的细胞毒性药物类的例子包括，例如，DNA小沟结合试剂、DNA烷基化试剂、和微管蛋白抑制剂、典型的细胞毒性药物包括、例如奥瑞他汀(auristatins)、喜树碱(camptothecins)、多卡霉素/倍癌霉素(duocarmycins)、依托泊甙(etoposides)、美登木素(maytansines)和美登素类化合物(maytansinoids)(例如DM1和DM4)、紫杉烷(taxanes)、苯二氮卓类(benzodiazepines)或者含有苯二氮卓的药物(benzodiazepine containing drugs)(例如吡咯并[1,4]苯二氮卓类(PBDs)，吲哚啉苯并二氮卓类(indolinobenzodiazepines)和噁唑烷并苯并二氮卓类(oxazolidinobenzodiazepines))和长春花生物碱(vinca alkaloids)。
在本发明中，药物-接头可以用于在一个简单步骤中形成ADC。在其它实施方式中，双功能连接物化合物可以用于在两步或多步方法中形成ADC。例如，半胱氨酸残基在第一步骤中与接头的反应活性部分反应，并且在随后的步骤中，接头上的功能性基团与药物反应，从而形成ADC。
本发明还提供了制备ADC的方法，可进一步地包括：将抗体与药物-接头化合物，在足以形成抗体偶联物(ADC)的条件下进行结合。
在某些实施方式中，本发明方法包括：在足以形成抗体-接头偶联物的条件下，将抗体与双功能接头化合物进行结合。在这些实施方式中，本发明方法还进一步地包括：在足以将药物部分通过接头共价连接到抗体的条件下，将抗体接头偶联物与药物部分进行结合。
在一些实施方式中，抗体药物偶联物ADC如下分子式所示：
Figure PCTCN2021121022-appb-000004
其中：
Ab是抗体，
LU是接头；
D是药物；
而且下标p是选自1到8的值。
药物组合物
本发明还提供了一种组合物。在优选例中，所述的组合物是药物组合物，它含有上述的抗体或其活性片段或其融合蛋白，以及药学上可接受的载体。通常，可将这些物质配制于无毒的、惰性的和药学上可接受的水性载体介质中，其中pH通常约为5-8，较佳地pH约为6-8，尽管pH值可随被配制物质的性质以及待治疗的病症而有所变化。配制好的药物组合物可以通过常规途径进行给药，其中包括(但并不限于)：瘤内、腹膜内、静脉内、或局部给药。
本发明的药物组合物可直接用于结合PD-L1蛋白分子，因而可用于预防和***。此外，还可同时使用其他治疗剂。
本发明的药物组合物含有安全有效量(如0.001-99wt％,较佳地0.01-90wt％，更佳地0.1-80wt％)的本发明上述的单克隆抗体(或其偶联物)以及药学上可接受的载体或赋形剂。这类载体包括(但并不限于)：盐水、缓冲液、葡萄糖、水、甘油、乙醇、及其组合。药物制剂应与给药方式相匹配。本发明的药物组合物可以被制成针剂形式，例如用生理盐水或含有葡萄糖和其他辅剂的水溶液通过常规方法进行制备。药物组合物如针剂、溶液宜在无菌条件下制造。活性成分的给药量是治疗有效量，例如每天约1微克/千克体重-约5毫克/千克体重。此外，本发明的多肽还可与其他治疗剂一起使用。
在另一具体实施方式中，药物组合物还含有：额外的活性成分，较佳地所述的活性成分包括：小分子化合物、细胞因子、抗体(如抗PD-1抗体、抗OX40抗体、抗CD137抗体、抗CD47抗体、ADC、CAR-免疫细胞)。
在另一具体实施方式中，在癌症，炎性疾病、病症或病况，免疫疾病、病症或病况，自身免疫性疾病、病症或病况或感染性疾病、病症或病况的治疗中使用的组合物可与另一种治疗联用，包括但不限于：化疗、抗CD20 mAb、抗TIM-3 mAb、抗LAG-3 mAb、抗CD73 mAb、抗CD47 mAb、抗DLL3 mAb、抗FRmAb mAb、抗CTLA-4抗体、抗PD-1抗体、PD-1/PD-L1治疗、其他免疫肿瘤药物、抗血管生成剂、放射治疗、抗体-药物偶联物(ADC)、靶向治疗或其他抗癌药物。抗PD-L1抗体可与针对以下靶点的伴侣mAb共同构建双特异性抗体以治疗表达PD-L1和特定肿瘤相关抗原的癌症/肿瘤：PD-1、OX40、CD137、LAG3、TIM-3、CTLA-4、EGFR、HER-2、CD19、CD20、CD33、CD73、CD47、DLL-3、CLDN18.2、叶酸受体α(FOLR1)，和/或其他肿瘤表面抗原。
使用药物组合物时，是将安全有效量的免疫偶联物施用于哺乳动物，其中该安全有效量通常至少约10微克/千克体重，而且在大多数情况下不超过约8毫克/千克体重，较佳地该剂量是约10微克/千克体重-约1毫克/千克体重。当然，具体剂量还应考虑给药途径、病人健康状况等因素，这些都是熟练医师技能范围之内的。
应用
本发明的其他方法用于治疗暴露于特定毒素或病原体的患者。因此，本发明的另一方面提供一种预防和/或治疗对象中的肿瘤或感染性疾病的方法，包括给该对象施用本发明的PD-L1结合分子，使得所述对象的肿瘤或感染性疾病得到预防和/或治疗。
在另一优选例中，所述的药物组合物用于制备***的药物，所述的肿瘤选自下组：膀胱癌、脑癌如胶质瘤，如低度胶质瘤、胶质母细胞瘤、***、乳腺癌、结肠癌、直肠癌、子宫内膜癌、肾癌、肾细胞癌、肾盂癌症、白血病、肺癌、非小细胞肺癌、黑色素瘤、非霍奇金淋巴瘤、胰腺癌、***癌、卵巢癌、纤维肉瘤、急性淋巴细胞白血病(ALL)、急性髓系白血病(AML)、慢性淋巴细胞白血病(CLL)、慢性粒细胞白血病(CML)、多发性骨髓瘤、骨髓衍生肿瘤、甲状腺癌。
类似于对于如上所述的肿瘤的应用，本发明的PD-L1抗体可以单独使用，或者作为佐剂与疫苗组合使用来刺激对病原体、毒素和自身抗原的免疫应答。特别可 以应用该治疗方法的病原体的实例包括当前没有有效疫苗的病原体，或常规疫苗不完全有效的病原体。其中包括但不限于H1V、肝炎病毒(甲、乙、丙)、流感病毒、疱疹病毒、贾第虫、疟疾、利什曼原虫、金黄色葡萄球菌、绿脓杆菌。PD-L1阻断剂特别可用于对抗诸如H1V等病原体已建立的感染，其在感染过程中呈现改变的抗原。在抗人PD-L1抗体给药时，这些新的表位被作为外源物识别，从而引起不受PD-L1的负信号影响的强T细胞应答。
较佳地，本发明的抗体可以用于感染性疾病的治疗。本发明的抗体治疗的感染性疾病可选自下组：HIV、肝炎(甲、乙、丙)、结核杆菌。
在所有上述的方法中，本发明的PD-L1抗体可以与其他形式的免疫疗法如细胞因子治疗(例如干扰素、GM-CSF、G-CSF、IL-2)或双特异性抗体治疗联合，双特异性抗体治疗提供增强的肿瘤抗原的呈递。
杂交瘤细胞株
本发明还提供了可生产本发明针对PD-L1蛋白单克隆抗体的杂交瘤细胞株；优选的，本发明提供了高效价的针对PD-L1蛋白单克隆抗体的杂交瘤细胞株。
在获得生产本发明的PD-L1蛋白单克隆抗体的杂交瘤之后，本领域技术人员可以方便地利用该杂交瘤细胞株制备抗体。此外，本领域技术人员还可很方便地获知本发明的抗体的结构(比如抗体的重链可变区和轻链可变区)，然后可通过重组方法来制备本发明的单克隆抗体。
方法和样本
本发明涉及用于在以细胞和/或组织溶解的样本检测肿瘤的方法。该方法步骤大致如下：获得细胞和/或组织样本；将样本溶解在介质中；检测在所述溶解的样本中PD-L1蛋白的水平。在本发明的检测方法中，所使用的样本没有特别限制，代表性的例子是存在于细胞保存液中的含细胞的样本。
试剂盒
本发明还提供了一种指含有本发明的抗体(或其片段)或本发明的检测板的试剂盒，在本发明的一个优选例中，所述的试剂盒还包括容器、使用说明书、缓冲剂等。
本发明进一步设计用于检测PD-L1水平的检测试剂盒，该试剂盒包括识别 PD-L1蛋白的抗体，用于溶解样本的裂解介质，检测所需的通用试剂和缓冲液，如各种缓冲液、检测标记、检测底物等。该检测试剂盒可以是体外诊断装置。
本发明的主要优点包括
(a)本发明抗PD-L1蛋白的抗体，具有独特的抗原表位，靶向泛IgC功能段。不同于已知的针对的是IgV功能段的PD-L1单克隆抗体，如：阿特珠单抗、德瓦鲁单抗、BMS-963559和阿利库单抗。
(b)本发明抗PD-L1蛋白的抗体能够特异性识别膜型PD-L1，最大限度地规避分泌型sPD-L1。
(c)本发明抗PD-L1蛋白的抗体对分泌sPD-L1的肿瘤具有抗耐药性。
(d)本发明抗PD-L1蛋白的抗体能够增强ADCC功能。
(e)本发明抗PD-L1蛋白的抗体具有肿瘤杀伤作用。
(f)本发明的PD-L1抗体，或与其它抗肿瘤药的联合用药，为阻断型PD-L1抗体耐药的肿瘤提供一可选择的方法，进而提高免疫治疗的疗效。
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件，例如Sambrook等人，分子克隆：实验室手册(New York:Cold Spring Harbor Laboratory Press,1989)中所述的条件，或按照制造厂商所建议的条件。除非另外说明，否则百分比和份数是重量百分比和重量份数。
引物的序列及表达宿主细胞
表1.人或鼠源PD-L1/PD-1特异性引物
Figure PCTCN2021121022-appb-000005
Figure PCTCN2021121022-appb-000006
注：h表示人源
基因表达宿主细胞和试验用途
表2.基因表达宿主细胞和试验用途
Figure PCTCN2021121022-appb-000007
注：h表示人源，s表示分泌型或可溶性。
表3.本发明中涉及的抗体
抗体名称 特异性
阿特珠单抗(atezolizumab) 抗PD-L1(结合IgV)
鼠单克隆抗体130021 抗PD-L1(结合泛IgC)
鼠单克隆抗体29E.2A3 抗PD-L1(结合IgV)
鼠单克隆抗体24F.10C12 抗PD-L2
鼠单克隆抗体2D10 抗CD80
鼠单克隆抗体Ab#18 阴性对照
鼠单克隆抗体Ab#4 抗PD-L1(结合泛IgC)
鼠单克隆抗体Ab#11 抗PD-L1(结合泛IgC)
鼠单克隆抗体Ab#15 抗PD-L1(结合泛IgC)
鼠单克隆抗体Ab#19 抗PD-L1(结合泛IgC)
鼠单克隆抗体Ab#23 抗PD-L1(结合泛IgC)
mIgG 阴性对照
注：m表示鼠源
细胞系以及细胞培养条件
MC38是一种小鼠结肠癌细胞，HEK293是一种人胚肾细胞，两者从上海盖宁生物科技有限公司获得，生长在DMEM+10％胎牛血清培养液中。CHO-K1是一种中国仓鼠卵巢细胞，由杭州化安生物科技有限公司提供，生长在RPMI1640+10％胎牛血清培养液中。
细胞荧光抗体标记与细胞流式检测
MC38/CHO-K1细胞分别表达PD-L1和泛IgC做为阳性检测细胞。而MC38/CHO-K1细胞野生型和表达PD-L2做为阴性对照。加小鼠免疫后血清(1:100、1:1000、1:10000)或抗体克隆杂较瘤细胞培养上清液(50μl)，或鼠单抗(浓度在图释中标出),和其人源化抗体(浓度在图释中标出)标记大约1×10 5阴性和阳性检测细胞，在4℃下孵育15min。加PBS清洗后，再加PE结合的羊抗鼠抗体(BD biosciences)标记，在4℃避光下孵育15min。再加PBS清洗后，进行细胞流式检测(NovoCyte,Aceabio,杭州)。
PD-L1细胞ELISA、直接PD-L1蛋白ELISA、和分泌型sPD-L1间接ELISA
通过已建立的细胞ELISA来筛选PD-L1抗体亚克隆。在96孔细胞培养板上，每孔铺5000个阳性细胞，在37℃CO 2细胞培养箱里培养2天。加PBS清洗细胞，离心培养板，每分钟1500转,3分钟，共清洗二遍。加小鼠免疫后血清或抗体克隆杂较瘤细胞培养上清液，在4℃下孵育1小时。再用PBS清洗2遍，加HRP结合的羊抗鼠二抗(Proteintech,武汉，1:1000稀释)并在4℃下孵育30分钟。PBS清洗2遍后，加TMB显色，再用1M HCl终止显色反应。在一酶标仪上(Synergy LX，Biotek，美国)，通过450波长的吸收值来检测上清液中有或无PD-L1抗体。
通过已建立的直接蛋白ELISA来分析PD-L1抗体是否结合sPD-L1。如过去所做，包被50ng PD-L1-3-HIS或PD-L1-9-HIS每孔在具有蛋白高亲和力的96孔板上，在4℃静置过夜。弃上清液，每孔加100ul PBS+吐温20(0.05％)清洗96孔板三次。每孔再加100μl PBS+1％BSA，室温静置4小时。弃上清液后，每孔加50μl抗体克隆杂交瘤细胞培养上清液，在4℃下孵育过夜。弃上清液后，每孔加100μl PBS+吐温20(0.05％)清洗96孔板五次。加HRP结合的羊抗鼠二抗(Proteintech，1:1000稀释)并在室温下孵育两小时。加TMB显色后，通过450波长的吸收值来检测抗体是否结合sPD-L1。
分泌型sPD-L1(自然形态)ELISA用来进一步澄清抗体对sPD-L1的亲和力。如以前所做，自然形态sPD-L1通过MC38细胞表达和分泌。含有sPD-L1的细胞培养上清被用于检测PD-L1抗体的亲合力。纯化的PD-L1单克隆抗体(0.2μg/孔)4℃过夜包被。表达sPD-L1-3和/或sPD-L1-9的MC38细胞培养上清,加100μl/孔。4℃过夜和清洗后，加100μl/0.1μg/孔生物素化的PD-L1抗体(Biolegend,美国)，室温下孵育两小时。清洗后加链霉亲和素-HRP(1:20,000，Jackson ImmunoResearch，美国)，室温下孵育两小时。加70μl/孔TMB底物成色，随后加35μl/孔1M HCl终止反应。抗体与sPD-L1的结合力通过450波长的吸收值来检测。
细胞结合试验
为了分析PD-L1抗体对PD-L1与PD-1相互作用的影响，MC38和CHO-K1细胞分别被转染和表达人源PD-L1和PD-1。按照美国EMD公司的细胞萤光染色标记方法，表达PD-L1的MC38和CHO-K1细胞被PKH67标记，而表达PD-1的MC38和CHO-K1细胞被PKH26标记。两个不同标记的细胞在有或无PD-L1或同型抗体的条件下共培养一小时。PD-L1抗体对PD-L1与PD-1的相互作用的影响，通过细胞流式进行检测。双阳标记的细胞集群表示PD-L1已与PD-1结合。此外，表达人源PD-L1的MC38和CHO-K1细胞被PKH26标记，而表达人源PD-1的MC38和CHO-K1细胞被PKH67标记。表达PD-L1而不同标记的MC38细胞共培养，表达PD-1而不同标记的MC38细胞共培养，双阳标记的细胞集群表示非特异性结合(背景对照)。
ADCC检测
通过爱康德生物(苏州)提供Jurkat NFAT-Luciferase–CD16测量抗体 Fc诱导CD16活化和细胞内NF-AT信号通路的激活和报告基因萤光素酶的产生，定量检测抗体Fc效应因子功能。在不同浓度的人源化PD-L1抗体条件下，大约1.25×10 4/孔表达PD-L1的CHO-K1靶细胞与表达人源FcγRIIIa的效应细胞按1:10的比例混合。在细胞培养箱里培养10小时。加75μl/孔Bright Lite TM萤光素酶底物(诺唯赞生物，南京)并在常温下孵育5至30分钟。通过光度计(Synergy LX)检测发光强度来定量分析抗体Fc诱导的ADCC功能。
抗PD-L1抗体的制备
与PD-L1的泛IgC区段结合的具有高亲和力的抗体通过进行初步抗体生产、纯化和验证获得。
表4本发明的抗PD-L1抗体的重链可变区、HCDR、轻链可变区和LCDR的氨基酸序列
Figure PCTCN2021121022-appb-000008
Figure PCTCN2021121022-appb-000009
注：VH：重链，VH-CDR：重链可变区；VL：轻链；VL-CDR：轻链可变区。如本文所用，H6L2即代表重链可变区为VH6，和轻链可变区为VL2的抗PD-L1人源化抗体。
实施例1 抗PD-L1抗体的制备
步骤一：PD-L1/PD-1和PD-L1-泛IgC片段表达细胞株的建立
人源或鼠源PD-L1、PD-1和PD-L2基因模板从长沙优宝生物获得。各基因与基因片段通过基因特异性引物分别进行PCR扩增(如表1所示)。
扩增的PCR产物通过酶切和T4DNA联结酶***相同酶切的pcDNA4质粒 (Thermofisher，美国)。表达基因与基因片段的pcDNA4质粒载体分别转染入不同的表达宿主细胞，并进行抗性筛选。其中，含PD-L1、泛IgC、PD-L2、或PD-1基因片段的质粒载体分别转染入CHO-K1和MC38细胞；含sPD-L1-3和sPD-L1-9基因片段的质粒载体分别转染入MC38细胞；含sPD-L1-3-HIS和sPD-L1-9-HIS基因片段的质粒载体均分别转染入HEK293细胞。PD-L1、泛IgC、PD-L2、或PD-1基因表达的宿主细胞和用途如表2所列。
步骤二：单克隆杂交瘤细胞的制备
以一质粒载体表达hPD-L1(氨基酸19-290)和泛IgC高(氨基酸132-290)表达的CHO-K1细胞系两种方式各免疫小鼠。将血清1:100、1:1000、1:10000稀释，用MC38表达PD-L1细胞(阳性细胞)，MC38野生型和表达PD-L2细胞(阴性细胞)通过细胞流式来检测小鼠血清中特异性抗体的滴度。选择阳性血清的小鼠进行细胞融合。用直接PD-L1蛋白ELISA和阳性和阴性细胞流式来检测培养上清抗体的特异性和滴定度。选择合格的母克隆和亚克隆，并进一步获得单克隆杂交瘤细胞。将进一步用sPD-L1蛋白检测这些抗体的结合能力，筛选出对sPD-L1亲和力缺失或低下的人源PD-L1鼠单克隆抗体，特别是具有ADCC和CDC功能的鼠IgG2a和IgG2b亚型。
步骤三：抗PD-L1单克隆抗体的制备
单克隆抗体通过单克隆杂交瘤细胞腹腔注摄，腹水制备，蛋白A亲和层析纯化获得。
步骤四：抗人源PD-L1鼠单克隆抗体重轻链可变区DNA测序，以及其人源化和表达与提纯
简而言之，总RNA将从产生鼠单克隆抗体的杂交瘤细胞获取。利用基因特异的引物并通过逆转录和PCR反应获得重链和对应的轻链可变区DNA，并进一步以TOPO TA克隆方法将目的基因***pCR TM载体质粒并进行测序。鼠单抗可变区人源化包括CDR移植和回复突变(Back mutation)，并移植入人源IgG1，к。合成人源化抗体序列后，分别构建人源化重和轻链抗体表达载体pcDNA3.1(Thermofisher)；同时构建一个人鼠嵌合抗体表达载体作为对照。重轻链表达质粒共转染进入HEK293细胞。分泌抗体的细胞株进一步扩增。含抗体的培养上清通过蛋白A亲和层析纯化获得。
实施例2：PD-L1小鼠单克隆抗体的筛选
通过流式细胞仪筛选与人源PD-L1和泛IgC的结合力强的PD-L1鼠单克隆抗体。MC38细胞分别表达PD-L1和泛IgC做为阳性检测细胞。MC38细胞表达人源PD-L2，和MC38亲本细胞，用做阴性对照。
用抗体克隆杂交瘤细胞培养上清液(50μl)或鼠单抗(100ng)标记大约1×10 5阴性和阳性检测细胞，在4℃下孵育15min。加PBS清洗后，再加PE结合的羊抗鼠抗体(BD biosciences)标记，在4℃避光下孵育15min。加PBS清洗后，进行细胞流式检测。
如图3A所示，PD-L1鼠单抗体克隆130021(R&D systems)结合PD-L1和泛IgC。PD-L1鼠单抗体克隆29E.2A3(Biolegend)仅结合PD-L1而不识别泛IgC段，表明它的识别区只在IgV。如图3B所示，通过细胞流式检测，从33个PD-L1鼠单克隆抗体中筛选出5个既能结合PD-L1也能识别泛IgC的单克隆抗体，分别是mAb#4、mAb#11、mAb#15、mAb#19和mAb#23。
实施例3：PD-L1鼠单克隆抗体对PD-L1的亲和力，以及sPD-L1对其亲和力的影响
通过直接PD-L1蛋白ELISA检测PD-L1抗体与重组蛋白PD-L1的亲和力。重组蛋白PD-L1-3-HIS和PD-L1-9-HIS由HEK293细胞表达,其培养上清液通过Ni柱来纯化rPD-L1-HIS。包被50ng PD-L1-3-HIS或PD-L1-9-HIS每孔在具有蛋白高亲和力的96孔板上，在4℃静置过夜。弃上清液，每孔加100ul PBS+吐温20(0.05％)清洗96孔板三次。每孔再加100μl PBS+1％BSA，室温静置4小时。弃上清液后，每孔加100μl(100ng)单克隆抗体，在4℃下孵育过夜。弃上清液后，每孔加100μl PBS+吐温20(0.05％)清洗96孔板五次。加HRP结合的羊抗鼠二抗(Proteintech，1:1000稀释)并在室温下孵育两小时。加TMB显色后，通过450波长的吸收值来检测抗体是否结合sPD-L1。通过直接ELISA来检测5个PD-L1鼠单克隆抗体mAb#4、mAb#11、mAb#15、mAb#19和mAb#23对重组蛋白PD-L1(rPD-L1)的结合力。
实验结果如图4A所示，5个PD-L1鼠单克隆抗体都不与PD-L1-3-HIS结合；鼠单克隆抗体mAb#19与PD-L1-9-HIS结合能力最小。
通过细胞流式检测重组蛋白PD-L1-3-HIS(rPD-L1-3-HIS)和PD-L1-9-HIS对5个PD-L1鼠单克隆抗体的阻断作用。200ng重组蛋白PD-L1-3-HIS(摩尔数14倍于抗体)或重组蛋白PD-L1-9-HIS(摩尔数10倍于抗体)分别与100ng PD-L1鼠单克隆抗体mAb#4、mAb#11、mAb#15、mAb#19和mAb#23在4℃下预先 孵育2小时后，再与表达PD-L1的MC38细胞预孵育30分钟。
实验结果如图4B所示，结果表明：rPD-L1-3-HIS不能阻断5个PD-L1鼠单克隆抗体与PD-L1结合，rPD-L1-9-HIS能阻断mAb#4、mAb#15、和mAb#23，但仅部分抑制PD-L1鼠单克隆抗体mAb#11、mAb#19与细胞上的PD-L1结合。说明鼠单克隆抗体能够规避分泌型的PD-L1-3-HIS，且能够部分规避PD-L1-9-HIS
从筛选的5个鼠单克隆抗体中挑选PD-L1鼠单克隆抗体#19，检测重组蛋白PD-L1-9对鼠单克隆抗体#19结合PD-L1的影响。100ng抗体#19与不同浓度(如图所示)的重组蛋白rPD-L1-9-HIS预先在4℃下孵育2小时后，再与表达PD-L1的MC38细胞孵育30分钟。通过细胞流式分析rPD-L1-9-HIS对PD-L1鼠单克隆抗体#19结合功能的影响。
实验结果如图4C所示，结果表明：重组蛋白PD-L1-9的摩尔数是鼠单克隆抗体mAb#19的40倍时，鼠单克隆抗体mAb#19仍然能和PD-L1结合。进一步说明鼠单克隆抗体能够部分规避rPD-L1-9-HIS。
通过分泌型sPD-L1间接ELISA检测PD-L1鼠单克隆抗体#19对自然形态sPD-L1的亲和力。MC38细胞被转染和表达自然形态sPD-L1。其细胞培养上清含有自然形态sPD-L1。纯化的PD-L1单克隆抗体(0.2μg/孔)4℃过夜包被。加100μl/孔来自于表达sPD-L1-3或sPD-L1-9的MC38细胞培养上清。4℃过夜和清洗后，加100μl/0.1μg/孔生物素化的PD-L1抗体(Biolegend)，室温下孵育两小时。清洗后加链霉亲和素-HRP(1:20,000，Jackson ImmunoResearch)，室温下孵育两小时。加70μl/孔TMB底物成色，随后加35μl/孔1M HCl终止反应。抗体与sPD-L1的结合力通过450波长的吸收值来检测。
ELISA检测#19单抗对细胞培养上清含sPD-L1-3与sPD-L1-9(A)、sPD-L1-3(B)，或sPD-L1-9(C)的亲和力。Anti-PD-L1 total识别sPD-L1-3和sPD-L1-9，而克隆130021只结合sPD-L1-9。
实验结果如图5所示，结果表明：PD-L1鼠单克隆抗体#19特异性识别细胞上人源膜型PD-L1，但不结合自然形态sPD-L1-3,对自然形态sPD-L1-9的亲和力远低于克隆130021(值得注意在图3A和3B的流式检测中显示此抗体对膜型hPD-L1亲和力非常低下，远弱于PD-L1鼠单克隆抗体#19)，即最大限度的规避分泌型sPD-L1(sPD-L1-3和sPD-L1-9)。
实施例4：小鼠MC38肿瘤模型检测抗体活性
MC38细胞表达人源PD-L1与MC38细胞分泌人源sPD-L1-9按20:1比例混和，大约5×10 5细胞皮下注射入C57BL/6J小鼠,每组6只。7天后可见肿瘤形成，腹腔注射100μg/鼠同种型抗体或单克隆抗体#19(anti-PD-L1treatment)，抗体治疗分别在肿瘤细胞注射后的第7、10、和15天，共三次。定期检测肿瘤大小和小鼠生存率。肿瘤容积＝(长×宽 2)/2。T检验(Student’s T test)和时序检验(Log-rank test)分别用于检验肿瘤大小和生存率实验中实验组间有无显著性差异。
实验结果如图6所示。结果表明：在表达人源PD-L1和sPD-L1的MC38小鼠肿瘤模型上，PD-L1特异性抗体具有抗耐药和抗肿瘤作用。
实施例5：鼠单抗#19人源化抗体活性
将鼠单抗#19人源化，获得其人源化抗体。鼠单抗#19和其人源化氨基酸序列，不同重(A)轻(B)链Fab氨基酸序列如图7和表4所示。其中，VH1、VH2、VH3、VH4、VH5、VH6为鼠单抗#19人源化抗体的重链可变区；VL1、VL2、VL3、VL4、VL5为鼠单抗#19人源化抗体的轻链可变区。
5.1.鼠单抗#19人源化抗体对膜型和分泌型PD-L1，以及泛IgC的亲和力
通过检测不同的重(H)轻(L)链组合对膜型和分泌型PD-L1，以及对泛IgC的亲和力，筛选出有高亲和力的重(H)轻(L)链组合。其中，H6L2、H6L5、H4L4、H5L4、H6L4、H5L5，以及H4L5的亲和力最佳。
其中，鼠单抗#19人源化的不同重(H)轻(L)链组合(H6L2、H6L5、H4L4、H5L4)对膜型和分泌型PD-L1，以及对泛IgC的亲和力检测如图8所示。
通过细胞流式检测鼠单抗#19人源化抗体对膜型PD-L1(A)和PD-L1泛IgC(B)亲合力。
MC38细胞分别转染和分泌自然形态sPDL1-3和sPDL1-9的细胞培养上清，对比鼠单抗#19和其嵌合抗体(Chimeric)，130021，和anti-PD-L1 total，通过ELISA检测鼠单抗#19人源化抗体对分泌型sPD-L1(C和D)的结合力。
实验结果显示：鼠单抗#19人源化的重轻链组合(H6L2、H6L5、H4L4、H5L4)结合PD-L1和泛IgC，并且，它们不结合sPD-L1-3,并且对sPD-L1-9的亲和力低下。
5.2鼠单抗#19人源化抗体对膜型PD-L2(人源)的亲和力
通过细胞流式检测鼠单抗#19人源化不同重(H)轻(L)链组合对膜型PD-L2的亲合力。
实验结果如图9所示：H6L2和H6L5不结合PD-L2，而H5L4对PD-L2和CHO-K1亲本细胞有微弱的结合。
5.3鼠单抗#19人源化抗体对膜型CD80(人源)的亲和力
通过细胞流式检测不同重(H)轻(L)链组合对膜型CD80(人源)的亲和力。
实验结果如图10所示：H6L2和H6L5不结合CD80。
5.4鼠单抗#19人源化抗体H6L2对膜型PD-L1的亲和力
5.4.1人源化抗体H6L2，在有或rPD-L1-9-HIS条件下，对膜型PD-L1的亲和力
通过细胞流式检测，与Avelumab和Atezolizumab相比，人源化抗体H6L2对膜型PD-L1的亲和力；和与Avelumab和Atezolizumab相比，不同浓度的rPD-L1-9-HIS对H6L2亲和力的影响。
实验结果如图11A所示，测得H6L2的EC50为0.62μg/ml，Avelumab的EC50为0.17μg/ml，Atezolizumab的EC50为0.21μg/ml。
实验结果如图11B和C显示：人源化抗体H6L2亲合力对抗sPD-L1-9强于Avelumab和Atezolizumab。
5.4.2人源化抗体H6L2，在有或rPD-L1-3-HIS条件下，对膜型PD-L1的亲合力
通过细胞流式检测，与Avelumab和Atezolizumab相比，不同浓度的rPD-L1-3-HIS对H6L2亲和力的影响。
如图12所示，实验结果显示：人源化抗体H6L2亲合力对抗sPD-L1-3强于Avelumab和Atezolizumab。
5.5人源化抗体H6L2对PD-1与PD-L1结合的阻断作用
为分析PD-L1与PD-1相互作用，CHO-K1细胞分别被转染和表达人源PD-L1和PD-1。表达PD-L1的CHO-K1细胞被PKH67标记，而表达PD-1的CHO-K1细胞被PKH26表记。两个不同表记的细胞在有或无H6L2的条件下共培养一小时。 不同抗体浓度如图所示。通过细胞流式进行检测此抗体对细胞间PD-L1和PD-1相互作用的影响，双阳标记的细胞集群表示PD-L1已与PD-1结合。同时，表达PD-L1的CHO-K1细胞也被PKH26标记，而表达PD-1的CHO-K1细胞也被PKH67表记。表达PD-L1而不同标记的CHO-K1细胞共培养，表达PD-1而不同标记的CHO-K1细胞共培养，双阳标记的细胞集群表示非特异性结合(背景对照)。结果如图13所示。
实验结果显示：H6L2不能阻断细胞间PD-L1与PD-1的结合。
5.6人源化抗体H6L2的ADCC活性和分泌型sPD-L1对其ADCC作用的影响
如图14所示，在Jurkat-CD16-NFAT-Luciferase平台，无(A)和有(B)分泌型sPD-L1(重组蛋白rPDL1-3-HIS或rPDL1-9-HIS)条件下，检测#19人源化抗体诱导Luciferase(萤光素酶)的活性能力，同时，对比Avelumab。
实验结果显示：人源化抗体H6L2诱导ADCC活性和其对抗sPD-L1的能力优于Avelumab。
实施例6：鼠单抗#19人源化抗体H6L2的抗肿瘤作用
3至5×10 5/鼠的表达人源PD-L1的MC38细胞被皮下注射入C57BL/6J小鼠，每组5只。7天后可见肿瘤形成，分别在肿瘤细胞注射后的第7、10、和13天，腹腔注射100μg/鼠人源化抗体H6L2或Avelumab、或PBS对照组，共三次。
结果如图15所示，其中A.肿瘤大小，以各组肿瘤大小的平均值±标准误表示。B.生存率。T检验和时序检验分别用于检验肿瘤大小和生存率实验中实验组间有无显著性差异。
实验结果显示：人源化抗体H6L2具有统计学意义的抗肿瘤作用，并显著性的优于Avelumab。
对比例1：间接ELISA检测抗人源PD-L1抗体阿特珠单抗与自然形态的sPD-L1的结合力
通过分泌型sPD-L1的间接ELISA检测抗人源PD-L1抗体阿特珠单抗与自然形态的sPD-L1的结合力，MC38细胞分别表达sPD-L1-3或sPD-L1-9。通过ELISA检测阿特珠单抗孵育过的细胞培养上清液中sPD-L1浓度来分析对其结合力。含有sPD-L1-3或sPD-L1-9无血清细胞培养上清液先分别与1.5μg/ml阿特珠单抗或人源IgG1过夜孵育(图示ate treated或hIgG1 treated)，再加 蛋白G琼脂糖(Thermo fisher)和离心来去除阿特珠单抗。含有sPD-L1-3或sPD-L1-9的上清液(图示sPD-L1-3或sPD-L1-9)无任何预先孵育作为阳性对照。BL表示无sPD-L1上清，做为背景对照。
实验结果如图16所示，结果表明：阿特珠单抗均能与sPD-L1-3和sPD-L1-9结合。
对比例2 人源PD-L1抗体avelumab和atezolizumab对PD-1与PD-L1结合的阻断作用，以及分泌型sPD-L1对其阻断作用的影响
为检测人源PD-L1抗体avelumab和atezolizumab对PD-1与PD-L1结合的阻断作用，以及分泌型sPD-L1对其阻断作用的影响，CHO-K1细胞分别被转染和表达人源PD-L1和PD-1。表达PD-L1的CHO-K1细胞被PKH67标记，而表达PD-1的CHO-K1细胞被PKH26表记。两个不同表记的细胞分别在有avelumab或atezolizumab的条件下共培养一小时。不同抗体浓度如图17所示。通过细胞流式进行检测两个抗体对细胞间PD-L1和PD-1相互作用的影响，双阳标记的细胞集群表示PD-L1已与PD-1结合。
其中A和B显示了Avelumab和Atezolizumab对细胞间PD-1与PD-L1相互作用的影响。C和D显示了sPD-L1对Avelumab和Atezolizumab阻断影响。抗体用量为100ng/数据点,rPD-L1-9-HIS用量如图所示。同时，表达PD-L1的CHO-K1细胞也被PKH26标记，而表达PD-1的CHO-K1细胞也被PKH67表记。表达PD-L1而不同标记的CHO-K1细胞共培养，表达PD-1而不同标记的CHO-K1细胞共培养，双阳标记的细胞集群表示非特异性结合(背景对照)。
实验结果显示：大于1μg/ml浓度的Avelumab和Atezolizumab才能达到完全阻断细胞间PD-L1与PD-1的结合，sPD-L1可使Avelumab和Atezolizumab的阻断作用减弱或丧失，而不能达到完全阻断。
对比例的结果表明高浓度(大于1μg/ml)的阿利库和阿特珠单抗才能完全阻断PD-L1与PD-1的相互作用，但因能与自然形态的sPD-L1的结合，降低这两个单抗的阻断能力，而产生耐药性。
讨论
抗体Fc段与它的受体FcγR相结合。FcγR表达于一系列免疫细胞，如自然杀伤细胞、单核细胞、中性粒细胞和巨核细胞。Fc与FcγR的相互作用对调控ADCC、CDC、抗体代谢和效价起着不可替代的作用。通过分子生物工程在Fc  CH2段进行DEL氨基酸置换能显著地增加自然杀伤细胞的功能。
对于阻断型PD-L1抗体Fc在抗肿瘤中所起的作用，研究显示一个PD-L1阻断型抗体(克隆14D8)被置换为不同功能的小鼠Fc段，它们的识别特异性、亲和力、以及代谢动力学特征没有改变，但抗肿瘤作用有统计学意义的差别。如14D8抗体带有IgG2a的Fc(即14D8/IgG2a)可特异性地结合和激活Fc受体，其具有抑制肿瘤生长的功能；结合抑制性FcγR受体的14D8/IgG1抗体，和失去Fc受体的结合能力的14D8/IgG1-D265A抗体，这两种抗体在统计学上丧失了肿瘤抑制作用。另外，相对于野生型小鼠，在缺失激活性FcγRs而保留抑制性FcγRIIb的小鼠肿瘤模型中，另一阻断型PD-L1抗体(克隆10F.9G2)的抗肿瘤作用有显著意义的减弱。在鼠肿瘤模型中，该抗体被广泛应用于抗肿瘤的机理研究。这些研究表明，在抗肿瘤方面，阻断型PD-L1抗体的关键作用可能在于其Fc诱导的ADCC/CDC活性；另一方面也表明阻断型PD-L1抗体的通过阻断PD-L1与PD-1相互作用来达到抗肿瘤作用非常有限。我们的研究也表明只有较高浓度的阻断型PD-L1抗体才能达到完全阻断。
NK细胞是先天性免疫效应细胞，它能裂解组织相容复合体(MHC class I)阴性的肿瘤细胞和病毒感染的细胞，并协调先天免疫应答。NK细胞持续性地表达裂解***。因此，NK细胞不需要预先激活来杀伤靶细胞，并在免疫监控变异细胞或肿瘤细胞起重要作用。
ADCC主要依赖于NK细胞的杀伤活性。不同于T细胞表达PD-1，NK细胞带有各种激动性或抑制性生物标记状态下，通过不同的检测手段，如细胞流示、qRT-PCR、和RNA-seq，它们持续性地缺乏表达PD-1。这一特点有利于越过PD-1/PD-L1通路和杀伤具有表达PD-L1的肿瘤细胞。而且，已成功研发出靶向肿瘤的抗体，它们能诱导ADCC并临床上有效地治疗癌症病人，如rituximab,daratumumab,trastuzumab,和Cetuximab。对比avelumab，我们的抗体有强诱导NK细胞活性和显著地抗肿瘤作用。综上所述，不局限于阻断PD-L1的功能和T细胞功能恢复和间接杀伤作用，可利用ADCC和NK细胞靶向具有表达PD-L1的肿瘤细胞，达到直接杀伤作用，因此，有潜在增加反应率的可能。
单抗130021、#4、#15和#23结合细胞上膜型PD-L1的能力弱于#19，但结合sPD-L1-9(胞外全长)的能力强于#19。这表明与细胞上膜型PD-L1的亲和力不决定对sPD-L1的泛IgC区的结合力，反之亦然。此外，细胞上的膜型与分泌型蛋白的三级结构可能不完全一样。实验结果提示，抗体对这两种类型的目标蛋白的亲和力产生差异，可能为治疗sPD-L1分泌型耐药性肿瘤提供有利机 会。
基于分泌型sPD-L1这一耐药机理和它们的氨基酸序列特点，以及PD-L1抗体的Fc和NK细胞在抗肿瘤中所起的的关键性作用，发明人设计一个全新的抗原表位并用做免疫原和筛选PD-L1特异性单克隆抗体，它针对PD-L1的IgC功能区段。而且，它最大限度地规避sPD-L1，增强Fc的ADCC/CDC活性，并具有抗肿瘤作用。这对治疗分泌sPD-L1而产生对阻断型PD-L1抗体耐药的肿瘤和联合用药提供一可选择的途径或方案，并对改善肿瘤免疫治疗的疗效有潜在意义。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (17)

  1. 一种PD-L1抗体或其活性片段，其特征在于，所述抗体不阻断PD-1与PD-L1的结合，并且所述抗体特异性结合于PD-L1的泛IgC区段，且具有诱导ADCC/CDC功能效应的活性。
  2. 如权利要求1所述的一种PD-L1抗体或其活性片段，其特征在于，包含：
    (a)氨基酸序列如SEQ ID NO：16所示的LCDR1、SEQ ID NO：17所示的LCDR2、SEQ ID NO：18所示的LCDR3；以及
    (b)氨基酸序列如SEQ ID NO：12所示的HCDR1、SEQ ID NO：13所示的HCDR2和SEQ ID NO：14所示的HCDR3；
    其中，所述抗体特异性结合于PD-L1的泛IgC区段。
  3. 如权利要求1或2所述的PD-L1抗体或其活性片段，其特征在于，所述PD-L1抗体或其活性片段包含重链可变区域或轻链可变区域，所述重链可变区的多肽序列与SEQ ID NO：11、20、21、22、23、24或25所示多肽序列至少95％相同，所述轻链可变区的多肽序列与SEQ ID NO：15、26、27、28、29或30所示多肽序列至少95％相同。
  4. 如权利要求1-3中任一项所述的PD-L1抗体或其活性片段，包含：具有SEQ ID NO：11、20、21、22、23、24或25所示多肽序列的重链可变区，和具有SEQ ID NO：15、26、27、28、29或30所示多肽序列的轻链可变区。
  5. 如权利要求1-4任一项所述的PD-L1抗体或其活性片段，其特征在于，所述PD-L1抗体或其活性片段是嵌合的。
  6. 如权利要求1-5任一项所述的PD-L1抗体或其活性片段，其特征在于，所述PD-L1抗体或其活性片段是人源化的。
  7. 一种重组蛋白，其特征在于，所述的重组蛋白具有：
    (i)如权利要求1-6任一项所述的PD-L1抗体或其活性片段；以及
    (ii)任选的协助表达和/或纯化的标签序列。
  8. 一种多核苷酸，其特征在于，编码权利要求1-6中任一项所述的PD-L1抗体或其活性片段，或权利要求7所述的重组蛋白。
  9. 一种载体，其特征在于，它含有权利要求8所述的多核苷酸。
  10. 一种遗传工程化的宿主细胞，其特征在于，它含有如权利要求9所述的载体或基因组中整合有权利要求8所述的多核苷酸。
  11. 一种免疫偶联物，其特征在于，该免疫偶联物含有：
    (a)如权利要求1所述的PD-L1抗体或其活性片段；和
    (b)选自下组的偶联部分：可检测标记物、药物、毒素、细胞因子、放射性核素、或酶。
  12. 一种药物组合物，其特征在于，所述药物组合物含有：
    (a)如权利要求1所述的PD-L1抗体或其活性片段，或如权利要求2所述的重组蛋白，或如权利要求11所述的免疫偶联物；以及
    (b)药学上可接受的载体。
  13. 一种检测样品中PD-L1蛋白的方法，其特征在于,所述方法包括步骤：
    (1)从受试者获得样品；
    (2)使样品与权利要求1所述的PD-L1抗体或其活性片段接触；
    (3)确定受试者中PD-L1的水平。
  14. 一种免疫细胞，其特征在于，所述免疫细胞表达或在细胞膜外暴露有如权利要求1-4任一项所述的PD-L1抗体或其活性片段。
  15. 一种治疗PD-L1表达或功能异常相关的疾病的方法，其特征在于，所述方法包括向有需要的对象施用药学有效量的如权利要求1所述的PD-L1抗体或其活性片段、如权利要求11所述的免疫偶联物，如权利要求12所述的药物组合物，或如权利要求14所述的免疫细胞，或其组合。
  16. 如权利要求15所述的方法，其特征在于，所述的PD-L1表达或功能异常相关的疾病为肿瘤或感染性疾病。
  17. 如权利要求16所述的方法，其特征在于，所述感染性疾病由选自以下的病原体引起：HIV、肝炎病毒(甲、乙、丙)和结核杆菌。
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