WO2019109974A1 - Anti-pd-l1 antibody and antigen-binding fragment thereof - Google Patents

Anti-pd-l1 antibody and antigen-binding fragment thereof Download PDF

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WO2019109974A1
WO2019109974A1 PCT/CN2018/119536 CN2018119536W WO2019109974A1 WO 2019109974 A1 WO2019109974 A1 WO 2019109974A1 CN 2018119536 W CN2018119536 W CN 2018119536W WO 2019109974 A1 WO2019109974 A1 WO 2019109974A1
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homology
fragment
seq
amino acid
binding protein
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PCT/CN2018/119536
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French (fr)
Chinese (zh)
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张正平
徐宏江
杨玲
张喜全
应树松
施伟
赵凯迪
宋伟
张颖
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正大天晴药业集团南京顺欣制药有限公司
正大天晴药业集团股份有限公司
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Priority to CN201880076255.2A priority Critical patent/CN111356702B/en
Publication of WO2019109974A1 publication Critical patent/WO2019109974A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/10—Antimycotics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention relates to the field of isolated monoclonal antibodies, and in particular to an antibody that binds to PD-L1, an antigen-binding fragment thereof, a mouse antibody comprising the CDR region of the PD-L1 antibody, a chimeric antibody, a humanized antibody, and a coding thereof Nucleic acids, compositions comprising the same, and methods involving the use of such antibodies and antigen-binding fragments.
  • Tumor immunotherapy is an innovative method in the field of cancer treatment, in order to maximize the immune system response of patients to tumors, and to eliminate the inhibition of T cells by tumor cells.
  • killer T cells play a major role in killing tumor cells.
  • Activation of T cells requires two signals: the MHC-antigen peptide presentation signal and the costimulatory signal.
  • one of the most critical signaling pathways is the programmed death receptor 1 (PD-1)/programmed death receptor ligand 1 (PD-L1) signaling pathway.
  • Programmed death receptor ligand 1 is a ligand for programmed death receptor 1 (PD-1).
  • PD-L1 is also known as differentiation cluster 274 (CD274) or B7 homolog 1 (B7-H1) and is a type 1 transmembrane protein of 40 kDa encoded by the CD274 gene.
  • Both PD-L1 and PD-1 belong to the immunoglobulin superfamily and are composed of two extracellular Ig domains (ie, an N-terminal V domain and a C-terminal constant domain).
  • PD-L1 binds to its receptor PD-1 present on activated T cells, B cells and myeloid cells to regulate activation or inhibition of immune cells.
  • PD-L1 In a variety of tumors, such as melanoma, lung cancer, bladder cancer and other cancer cells, the expression of PD-L1 was significantly up-regulated. PD-L1 inhibits T cell activation, cytokine production, and T cell proliferation by binding to PD-1 on the surface of T cells (Fife et al. (2011) Nature Immunology 10: 1185-1193); induction of homologous antigen specificity Failure or non-reactivity in T cells (Hofmeyer et al (2011) Journal of Biomedicine and Biotechnology 2011: 1-9); and induces apoptosis of effector T cells. Destruction of the PD-L1 gene results in up-regulated T cell responses and production of autoreactive T cells (Latchman et al.
  • WO2016061142 discloses that antibody blockade of PD-L1 leads to an increase in tumor infiltrating lymphocytes, an increase in T cell receptor mediated proliferation, and a reduction in immune escape of cancer cells;
  • WO2016022630 demonstrates that a class of anti-PD-L1 antibodies and fragments thereof are in regulating T The ability of effector T cells to proliferate and/or produce cytokines is restored in the presence of cells. Therefore, blocking the binding between PD-L1/PD-1 by PD-L1 antibody has become a very effective method in the field of tumor immunotherapy.
  • the present invention provides a novel sequence of high affinity, highly selective, highly biologically active PD-L1 binding antibodies and antigen binding fragments thereof.
  • the invention provides an isolated antigen binding protein that binds to PD-L1 or an antigen binding fragment thereof.
  • the invention provides an isolated anti-PD-L1 antibody or antigen-binding fragment thereof that binds to human PD-L1 and exhibits superior properties.
  • the antibody or antigen-binding fragment thereof binds to PD-L1 and blocks binding of PD-1 to PD-L1.
  • the antibody or antigen-binding fragment thereof is capable of blocking the interaction between cells producing PD-1 and PD-L1, respectively.
  • the invention provides a method of using such antibodies and antigen-binding fragments, for example, the use of such antibodies and antigen-binding fragments to treat cancer and/or infectious diseases, increase T cell activation, or reduce tumors in a subject A method of immune escape, reduction of tumors, or inhibition of tumor cell growth.
  • the present invention provides a polynucleotide encoding such an antigen binding protein (eg, an antibody) and an antigen-binding fragment thereof, an expression vector comprising the polynucleotide, a recombinant cell comprising the expression vector, and a recombinant protein comprising the same (eg, an antibody), a composition thereof and an antigen-binding fragment thereof, and the like.
  • the invention provides an isolated anti-PD-L1 antibody or antigen-binding fragment thereof comprising one selected from the group consisting of SEQ ID NOs: 12, 14-22 and/or SEQ ID NOs: 27, 29-38 or Multiple CDR regions (antibody complementarity determining regions) sequences or mutant sequences thereof.
  • the anti-PD-L1 antibody or antigen-binding fragment thereof of the invention comprises an antibody heavy chain HCDR selected from the group consisting of SEQ ID NOs: 12, 17-22 and/or SEQ ID NOs: 29-34 A sequence of regions or a sequence thereof.
  • the anti-PD-L1 antibody or antigen-binding fragment thereof of the invention comprises an antibody light chain LCDR selected from the group consisting of SEQ ID NOs: 14-16 and/or SEQ ID NOs: 27, 35-38 A sequence of regions or a sequence thereof.
  • the anti-PD-L1 antibody or fragment thereof comprises a heavy chain HCDR1 sequence having at least 80% homology to an amino acid sequence selected from SEQ ID NO: 17, 18, 29 or 30 , at least 81% homology, at least 82% homology, at least 83% homology, at least 84% homology, at least 85% homology, at least 86% homology, at least 87% homology At least 88% homology, at least 89% homology, at least 90% homology, at least 91% homology, at least 92% homology, at least 93% homology, at least 94% homology, At least 95% homology, at least 96% homology, at least 97% homology, at least 98% homology, or at least 99% homology.
  • the anti-PD-L1 antibody or fragment thereof comprises a heavy chain HCDR2 sequence having at least 80% homology to an amino acid sequence selected from SEQ ID NO: 12, 31 or 32, at least 81% homology, at least 82% homology, at least 83% homology, at least 84% homology, at least 85% homology, at least 86% homology, at least 87% homology, at least 88 % homology, at least 89% homology, at least 90% homology, at least 91% homology, at least 92% homology, at least 93% homology, at least 94% homology, at least 95% Homology, at least 96% homology, at least 97% homology, at least 98% homology, or at least 99% homology.
  • the anti-PD-L1 antibody or fragment thereof comprises a heavy chain HCDR3 sequence that is at least 80% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 19-22 or 33-34 Source, at least 81% homology, at least 82% homology, at least 83% homology, at least 84% homology, at least 85% homology, at least 86% homology, at least 87% homology , at least 88% homology, at least 89% homology, at least 90% homology, at least 91% homology, at least 92% homology, at least 93% homology, at least 94% homology At least 95% homology, at least 96% homology, at least 97% homology, at least 98% homology or at least 99% homology.
  • the anti-PD-L1 antibody or fragment thereof comprises a light chain LCDR1 sequence having at least 80% homology to an amino acid sequence selected from SEQ ID NO: 14, 35 or 36, At least 81% homology, at least 82% homology, at least 83% homology, at least 84% homology, at least 85% homology, at least 86% homology, at least 87% homology, at least 88% homology, at least 89% homology, at least 90% homology, at least 91% homology, at least 92% homology, at least 93% homology, at least 94% homology, at least 95 % homology, at least 96% homology, at least 97% homology, at least 98% homology or at least 99% homology.
  • the anti-PD-L1 antibody or fragment thereof comprises a light chain LCDR2 sequence having at least 80% homology to an amino acid sequence selected from SEQ ID NO: 15 or 27, at least 81 % homology, at least 82% homology, at least 83% homology, at least 84% homology, at least 85% homology, at least 86% homology, at least 87% homology, at least 88% Homology, at least 89% homology, at least 90% homology, at least 91% homology, at least 92% homology, at least 93% homology, at least 94% homology, at least 95% identical Source, at least 96% homology, at least 97% homology, at least 98% homology, or at least 99% homology.
  • the anti-PD-L1 antibody or fragment thereof comprises a light chain LCDR3 sequence, the light chain LCDR3 sequence having at least 80% homology to an amino acid sequence selected from SEQ ID NO: 16, 37 or 38, At least 81% homology, at least 82% homology, at least 83% homology, at least 84% homology, at least 85% homology, at least 86% homology, at least 87% homology, at least 88% homology, at least 89% homology, at least 90% homology, at least 91% homology, at least 92% homology, at least 93% homology, at least 94% homology, at least 95 % homology, at least 96% homology, at least 97% homology, at least 98% homology or at least 99% homology.
  • the anti-PD-L1 antibody or antigen-binding fragment thereof comprises heavy chain HCDR1, HCDR2 and HCDR3 comprising at least 80% of the amino acid sequence of SEQ ID NO: 17, 18, 29 or 30 a sequence of homology, said heavy chain HCDR2 comprising a sequence at least 80% homologous to the amino acid sequence of SEQ ID NO: 12, 31 or 32 and said heavy chain HCDR3 comprising SEQ ID NO: 19-22 or 33 a sequence of at least 80% homology of the amino acid sequence of -34; and a light chain LCDR1, LCDR2 and LCDR3 comprising a sequence at least 80% homologous to the amino acid sequence of SEQ ID NO: 14, 35 or 36
  • the light chain LCDR2 comprises a sequence at least 80% homologous to the amino acid sequence of SEQ ID NO: 15 or 27 and the light chain LCDR3 comprises at least 80% identical to the amino acid sequence of SEQ ID NO: 16, 37 or 38 Source sequence.
  • the anti-PD-L1 antibody or antigen-binding fragment thereof comprises heavy chain HCDR1, HCDR2 and HCDR3 and light chain LCDR1, LCDR2 and LCDR3, said heavy chain HCDR1, HCDR2 and HCDR3 and light chain LCDR1, LCDR2 and The LCDR3 is selected from the following sequences or sequences having at least 80% homology thereto:
  • X 1 is selected from N or S
  • X 2 is selected from Y, C, A or V
  • X 3 is selected from S or F
  • X 4 is selected from R or W
  • X 5 is selected from S or N
  • X 6 is selected from T.
  • S is selected from Q or H
  • X 8 is selected from E or N
  • X 9 is selected from S or A.
  • the anti-PD-L1 antibody or antigen-binding fragment thereof comprises heavy chain HCDR1, HCDR2 and HCDR3; the heavy chain HCDR1 sequence selected from the group consisting of SEQ ID NO: 17, 18, 29 or 30 An amino acid sequence; the heavy chain HCDR2 sequence selected from the group consisting of the amino acid sequence set forth in SEQ ID NO: 12, 31 or 32; and the heavy chain HCDR3 sequence selected from the group consisting of SEQ ID NO: 19-22 or 33-34 The amino acid sequence shown.
  • the anti-PD-L1 antibody or antigen-binding fragment thereof comprises the light chain LCDR1, LCDR2 and LCDR3; the light chain LCDR1 sequence selected from the group consisting of the amino acid sequences set forth in SEQ ID NO: 14, 35 or 36
  • the anti-PD-L1 antibody or antigen-binding fragment thereof comprises heavy chain HCDR1, HCDR2 and HCDR3, the heavy chain HCDR1 being selected from the amino acid sequence set forth in SEQ ID NO: 17 or 18 or at least 80 thereof a sequence of % homology, said heavy chain HCDR2 being selected from the amino acid sequence set forth in SEQ ID NO: 12 or a sequence of at least 80% homology thereof, said heavy chain HCDR3 being selected from the group consisting of SEQ ID NOs: 19-22 Any one of the indicated amino acid sequences or sequences thereof having at least 80% homology; and light chain LCDR1, LCDR2 and LCDR3, respectively selected from the group consisting of SEQ ID NOs: 14, 15 and 16, respectively Amino acid sequence or a sequence thereof that is at least 80% homologous.
  • the anti-PD-L1 antibody or antigen-binding fragment thereof comprises heavy chain HCDR1, HCDR2 and HCDR3, the heavy chain HCDR1 being selected from the amino acid sequence set forth in SEQ ID NO: 29 or 30 or at least 80 thereof a sequence of % homology, said heavy chain HCDR2 being selected from the amino acid sequence set forth in SEQ ID NO: 31 or 32, or a sequence of at least 80% homology thereof, said heavy chain HCDR3 being selected from SEQ ID NO: 33 or An amino acid sequence of 34 or a sequence of at least 80% homology thereof; and a light chain LCDR1, LCDR2 and LCDR3 selected from the amino acid sequence set forth in SEQ ID NO: 35 or 36 or at least 80% thereof a homologous sequence, wherein the light chain LCDR2 is selected from the amino acid sequence set forth in SEQ ID NO: 27 or a sequence thereof of at least 80% homology, and the light chain LCDR3 is selected from the group consisting of SEQ ID NO: 37 or
  • the anti-PD-L1 antibody or antigen-binding fragment thereof comprises heavy chain HCDR1, HCDR2 and HCDR3 comprising amino acids according to SEQ ID NOs: 17, 12 and 19, respectively.
  • the sequence has at least 80% homology, at least 85% homology, at least 90% homology, at least 91% homology, at least 92% homology, at least 93% homology, at least 94% homology.
  • the light chains LCDR1, LCDR2 and LCDR3 comprise at least 80% homology, at least 85% homology, at least 90% homology, at least 91% homology to the amino acid sequences according to SEQ ID NOs: 14, 15 and 16, respectively.
  • an amino acid sequence of at least 99% homology is 99% homology.
  • the anti-PD-L1 antibody or antigen-binding fragment thereof comprises the heavy chain HCDR1, HCDR2 and HCDR3 according to SEQ ID NOs: 17, 12 and 19, respectively, and according to SEQ ID NOs: 14, 15 and 16, respectively.
  • the anti-PD-L1 antibody or antigen-binding fragment thereof comprises heavy chain HCDR1, HCDR2 and HCDR3 comprising amino acids according to SEQ ID NOs: 18, 12 and 20, respectively.
  • the sequence has at least 80% homology, at least 85% homology, at least 90% homology, at least 91% homology, at least 92% homology, at least 93% homology, at least 94% homology.
  • the light chains LCDR1, LCDR2 and LCDR3 comprise at least 80% homology, at least 85% homology, at least 90% homology, at least 91% homology to the amino acid sequences according to SEQ ID NOs: 14, 15 and 16, respectively.
  • an amino acid sequence of at least 99% homology is 99% homology.
  • the anti-PD-L1 antibody or antigen-binding fragment thereof comprises the heavy chain HCDR1, HCDR2 and HCDR3 according to SEQ ID NOs: 18, 12 and 20, respectively, and according to SEQ ID NOs: 14, 15 and 16, respectively.
  • the anti-PD-L1 antibody or antigen-binding fragment thereof comprises heavy chain HCDR1, HCDR2 and HCDR3 comprising amino acids according to SEQ ID NOs: 17, 12 and 20, respectively.
  • the sequence has at least 80% homology, at least 85% homology, at least 90% homology, at least 91% homology, at least 92% homology, at least 93% homology, at least 94% homology.
  • the light chains LCDR1, LCDR2 and LCDR3 comprise at least 80% homology, at least 85% homology, at least 90% homology, at least 91% homology to the amino acid sequences according to SEQ ID NOs: 14, 15 and 16, respectively.
  • an amino acid sequence of at least 99% homology is 99% homology.
  • the anti-PD-L1 antibody or antigen-binding fragment thereof comprises the heavy chain HCDR1, HCDR2 and HCDR3 according to SEQ ID NOs: 17, 12 and 20, respectively, and according to SEQ ID NOs: 14, 15 and 16, respectively.
  • the anti-PD-L1 antibody or antigen-binding fragment thereof comprises heavy chain HCDR1, HCDR2 and HCDR3 comprising amino acids according to SEQ ID NOs: 17, 12, and 21, respectively.
  • the sequence has at least 80% homology, at least 85% homology, at least 90% homology, at least 91% homology, at least 92% homology, at least 93% homology, at least 94% homology.
  • the light chains LCDR1, LCDR2 and LCDR3 comprise at least 80% homology, at least 85% homology, at least 90% homology, at least 91% homology to the amino acid sequences according to SEQ ID NOs: 14, 15 and 16, respectively.
  • an amino acid sequence of at least 99% homology is 99% homology.
  • the anti-PD-L1 antibody or antigen-binding fragment thereof comprises the heavy chain HCDR1, HCDR2 and HCDR3 according to SEQ ID NOs: 17, 12 and 21, respectively, and according to SEQ ID NOs: 14, 15 and 16, respectively.
  • the anti-PD-L1 antibody or antigen-binding fragment thereof comprises heavy chain HCDR1, HCDR2 and HCDR3 comprising amino acids according to SEQ ID NOs: 17, 12, and 22, respectively.
  • the sequence has at least 80% homology, at least 85% homology, at least 90% homology, at least 91% homology, at least 92% homology, at least 93% homology, at least 94% homology.
  • the light chains LCDR1, LCDR2 and LCDR3 comprise at least 80% homology, at least 85% homology, at least 90% homology, at least 91% homology to the amino acid sequences according to SEQ ID NOs: 14, 15 and 16, respectively.
  • an amino acid sequence of at least 99% homology is 99% homology.
  • the anti-PD-L1 antibody or antigen-binding fragment thereof comprises the heavy chain HCDR1, HCDR2 and HCDR3 according to SEQ ID NOs: 17, 12 and 22, respectively, and according to SEQ ID NOs: 14, 15 and 16, respectively.
  • the anti-PD-L1 antibody or antigen-binding fragment thereof comprises heavy chain HCDR1, HCDR2 and HCDR3 comprising amino acids according to SEQ ID NOs: 29, 31 and 33, respectively.
  • the sequence has at least 80% homology, at least 85% homology, at least 90% homology, at least 91% homology, at least 92% homology, at least 93% homology, at least 94% homology.
  • the light chains LCDR1, LCDR2 and LCDR3 comprise at least 80% homology, at least 85% homology, at least 90% homology, at least 91% homology to the amino acid sequences according to SEQ ID NOs: 35, 27 and 37, respectively.
  • an amino acid sequence of at least 99% homology is 99% homology.
  • the anti-PD-L1 antibody or antigen-binding fragment thereof comprises the heavy chain HCDR1, HCDR2 and HCDR3 according to SEQ ID NOs: 29, 31 and 33, respectively, and according to SEQ ID NOs: 35, 27 and 37, respectively.
  • the anti-PD-L1 antibody or antigen-binding fragment thereof comprises heavy chain HCDR1, HCDR2 and HCDR3 comprising amino acids according to SEQ ID NOs: 30, 32 and 34, respectively.
  • the sequence has at least 80% homology, at least 85% homology, at least 90% homology, at least 91% homology, at least 92% homology, at least 93% homology, at least 94% homology.
  • the light chains LCDR1, LCDR2 and LCDR3 comprise at least 80% homology, at least 85% homology, at least 90% homology, at least 91% homology to the amino acid sequences according to SEQ ID NOs: 36, 27 and 38, respectively.
  • an amino acid sequence of at least 99% homology is 99% homology.
  • the anti-PD-L1 antibody or antigen-binding fragment thereof comprises the heavy chain HCDR1, HCDR2 and HCDR3 according to SEQ ID NOs: 30, 32 and 34, respectively, and according to SEQ ID NOs: 36, 27 and 38, respectively.
  • the anti-PD-L1 antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising and selected from SEQs ID NO: 5, 7, 9, 45, 47, 48
  • the amino acid sequences of the groups consisting of 49 and 50 have at least 80% homology, at least 85% homology, at least 90% homology, at least 91% homology, at least 92% homology, at least 93% identical Amino acid sequence of at least 94% homology, at least 95% homology, at least 96% homology, at least 97% homology, at least 98% homology or at least 99% homology; a chain variable region comprising at least 80% homology, at least 85% homology to an amino acid sequence selected from the group consisting of SEQ ID NOs: 6, 8, 10, 46, 51 and 52 , at least 90% homology, at least 91% homology, at least 92% homology, at least 93% homology, at least 94% homology, at least 95% homology, at least 96% homology An amino acid sequence of at least 97% homology
  • the anti-PD-L1 antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising, consisting essentially of, or consisting of: SEQ ID NOs: amino acid sequences of the group consisting of 5, 7, 9, 45, 47, 48, 49 and 50; and a light chain variable region comprising the following sequences consisting essentially of the following sequences Or consisting of the amino acid sequence selected from the group consisting of SEQ ID NOs: 6, 8, 10, 46, 51 and 52.
  • the invention provides an anti-PD-L1 antibody or antigen-binding fragment thereof comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 5 and a light comprising the amino acid sequence of SEQ ID NO: a chain variable region; a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 7 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 8; a heavy chain comprising the amino acid sequence of SEQ ID NO: 9 A variable region and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 10.
  • the invention provides a humanized anti-PD-L1 antibody comprising: selected from the group consisting of hu-Chi1-2.3, hu-Chi1-3.3, hu-Chi1-3.4, hu-Chi1-3.5, and hu-Chi1 -3.6 a variable heavy chain of the antibody of the group consisting of an antibody selected from the group consisting of hu-Chi1-2.3, hu-Chi1-3.3, hu-Chi1-3.4, hu-Chi1-3.5, and hu-Chi1-3.6 Variable light chain.
  • the invention provides a humanized anti-PD-L1 antibody, wherein the antibody comprises a heavy chain variable region having a composition selected from the group consisting of SEQ ID NOs: 45, 47, 48, 49, and 50
  • the amino acid sequence of the group has at least 80% homology, at least 85% homology, at least 90% homology, at least 91% homology, at least 92% homology, at least 93% homology, at least 94% Amino acid sequences of homology, at least 95% homology, at least 96% homology, at least 97% homology, at least 98% homology, or at least 99% homology.
  • the invention provides a humanized anti-PD-L1 antibody, wherein the antibody comprises a light chain variable region having an amino acid selected from the group consisting of SEQ ID NOs: 46, 51, and 52
  • the sequence has at least 80% homology, at least 85% homology, at least 90% homology, at least 91% homology, at least 92% homology, at least 93% homology, at least 94% homology.
  • the invention provides a humanized anti-PD-L1 antibody, wherein the antibody comprises a heavy chain variable region that is at least 80% homologous to SEQ ID NO: 45, at least 85% homolog , at least 90% homology, at least 91% homology, at least 92% homology, at least 93% homology, at least 94% homology, at least 95% homology, at least 96% homology At least 97% homology, at least 98% homology, or at least 99% homology; and a light chain variable region having at least 80% homology to SEQ ID NO: 46, at least 85% homology At least 90% homology, at least 91% homology, at least 92% homology, at least 93% homology, at least 94% homology, at least 95% homology, at least 96% homology, At least 97% homology, at least 98% homology, or at least 99% homology.
  • the invention provides a humanized anti-PD-L1 antibody, wherein the antibody comprises a heavy chain variable region that is at least 80% homologous to SEQ ID NO: 47, at least 85% identical Source, at least 90% homology, at least 91% homology, at least 92% homology, at least 93% homology, at least 94% homology, at least 95% homology, at least 96% homology , at least 97% homology, at least 98% homology or at least 99% homology; and a light chain variable region having at least 80% homology, at least 85% homology to SEQ ID NO: 51 , at least 90% homology, at least 91% homology, at least 92% homology, at least 93% homology, at least 94% homology, at least 95% homology, at least 96% homology At least 97% homology, at least 98% homology or at least 99% homology.
  • the invention provides a humanized anti-PD-L1 antibody, wherein the antibody comprises a heavy chain variable region that is at least 80% homologous to SEQ ID NO: 48, at least 85% identical Source, at least 90% homology, at least 91% homology, at least 92% homology, at least 93% homology, at least 94% homology, at least 95% homology, at least 96% homology , at least 97% homology, at least 98% homology or at least 99% homology; and a light chain variable region having at least 80% homology, at least 85% homology to SEQ ID NO: 52 , at least 90% homology, at least 91% homology, at least 92% homology, at least 93% homology, at least 94% homology, at least 95% homology, at least 96% homology At least 97% homology, at least 98% homology or at least 99% homology.
  • the invention provides a humanized anti-PD-L1 antibody, wherein the antibody comprises a heavy chain variable region that is at least 80% homologous to SEQ ID NO: 49, at least 85% identical Source, at least 90% homology, at least 91% homology, at least 92% homology, at least 93% homology, at least 94% homology, at least 95% homology, at least 96% homology , at least 97% homology, at least 98% homology or at least 99% homology; and a light chain variable region having at least 80% homology, at least 85% homology to SEQ ID NO: 51 , at least 90% homology, at least 91% homology, at least 92% homology, at least 93% homology, at least 94% homology, at least 95% homology, at least 96% homology At least 97% homology, at least 98% homology or at least 99% homology.
  • the invention provides a humanized anti-PD-L1 antibody, wherein the antibody comprises a heavy chain variable region that is at least 80% homologous to SEQ ID NO: 50, at least 85% homolog , at least 90% homology, at least 91% homology, at least 92% homology, at least 93% homology, at least 94% homology, at least 95% homology, at least 96% homology At least 97% homology, at least 98% homology or at least 99% homology; and a light chain variable region having at least 80% homology to SEQ ID NO: 51, at least 85% homology At least 90% homology, at least 91% homology, at least 92% homology, at least 93% homology, at least 94% homology, at least 95% homology, at least 96% homology, At least 97% homology, at least 98% homology, or at least 99% homology.
  • the invention provides a humanized anti-PD-L1 antibody comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 45 and a light chain comprising the amino acid sequence of SEQ ID NO: 46 a variable region; or a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 47 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 51; or a heavy chain comprising the amino acid sequence of SEQ ID NO: 48 a variable region and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 52; or a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 49 and a light chain variable comprising the amino acid sequence of SEQ ID NO: 51 a region; or a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 50 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 51.
  • the invention provides a chimeric anti-PD-L1 antibody, wherein the antibody comprises a heavy chain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 39, 41, and 43 At least 80% homology, at least 85% homology, at least 90% homology, at least 91% homology, at least 92% homology, at least 93% homology, at least 94% homology, at least An amino acid sequence of 95% homology, at least 96% homology, at least 97% homology, at least 98% homology, or at least 99% homology; and a light chain having and selected from the group consisting of SEQ ID NO:
  • the amino acid sequence of the group consisting of 40, 42 and 44 has at least 80% homology, at least 85% homology, at least 90% homology, at least 91% homology, at least 92% homology, Amino acids having at least 93% homology, at least 94% homology, at least 95% homology, at least 96% homology, at least 97% homology, at least 99
  • the invention provides a humanized anti-PD-L1 antibody, wherein the antibody comprises an entire heavy chain having an amino acid selected from the group consisting of SEQ ID NO: 53, 57, 61, and 62
  • the sequence has at least 80% homology, at least 85% homology, at least 90% homology, at least 91% homology, at least 92% homology, at least 93% homology, at least 94% homology.
  • the invention provides a humanized anti-PD-L1 antibody, wherein the antibody comprises an intact light chain having at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 55 and 59 80% homology, at least 85% homology, at least 90% homology, at least 91% homology, at least 92% homology, at least 93% homology, at least 94% homology, at least 95 Amino acid sequence of % homology, at least 96% homology, at least 97% homology, at least 98% homology or at least 99% homology.
  • the invention provides a humanized anti-PD-L1 antibody, wherein the antibody comprises a heavy chain according to SEQ ID NO: 53 and a light chain according to SEQ ID NO: 55.
  • the invention provides a humanized anti-PD-L1 antibody, wherein the antibody comprises a heavy chain according to SEQ ID NO: 57 and a light chain according to SEQ ID NO: 59.
  • the invention provides a humanized anti-PD-L1 antibody, wherein the antibody comprises a heavy chain according to SEQ ID NO: 61 and a light chain according to SEQ ID NO: 55.
  • the invention provides a humanized anti-PD-L1 antibody, wherein the antibody comprises a heavy chain according to SEQ ID NO: 62 and a light chain according to SEQ ID NO: 55.
  • the invention provides a humanized anti-PD-L1 antibody or fragment thereof, preferably, the humanized antibody is a humanized antibody hu-Chi1, said humanized antibody heavy chain variable region
  • the heavy chain FR region sequence is derived from the human germline heavy chain, specifically from the combined sequence of the human germline heavy chain IGHV4-30-4*01 and IGHJ1*01.
  • the heavy chain variable region sequence of the humanized antibody hu-Chi1 described above has a back mutation of 0-10 amino acids, preferably one or more selected from the group consisting of S35N, Q39R, K43N, G44K, W47Y, I48M Amino acid back mutation of V71R; more preferably one or more amino acid back mutations selected from the group consisting of S35N, W47Y and V71R; most preferably a back mutation comprising S35N, W47Y, V71R.
  • the humanized antibody hu-Chi1 heavy chain variable region further comprises an amino acid mutation of C102Y.
  • the light chain FR region sequence of the humanized antibody hu-Chi1 light chain variable region is derived from human germline light chain, specifically derived from human germline light chain IGKV1-39*01 and IGKJ2 A combined sequence of *02.
  • the light chain variable region sequence of the humanized antibody hu-Chi1 has a 0-10 amino acid back mutation, preferably one or more selected from the group consisting of I2F, P8T, G41D, P44V, Y87F, and Q100R Amino acid back mutation; more preferably one or more amino acid back mutations selected from the group consisting of I2F, G41D and P44V; most preferably amino acid back mutations comprising I2F, G41D and P44V.
  • the invention provides an anti-PD-Ll antibody or fragment thereof that binds to the same epitope on PD-L1 as any of the exemplary antibodies provided herein.
  • the antibody or fragment thereof competes with any of the exemplary antibodies provided herein for binding to PD-L1. Binding to PD-L1 can be measured by ELISA, flow cytometry, surface plasmon resonance (SPR) assay, or any other method known in the art.
  • the PD-L1 antibody or fragment thereof is capable of binding to a human PD-L1 protein and binding to a cynomolgous PD-L1 protein.
  • the present invention provides a binding affinity of from about 15nM to about 0.05nM K D of binding with the anti-PD-L1 antibody, and PD-L1 fragments thereof.
  • an anti-PD-L1 antibodies and fragments thereof binding to a binding affinity of from about 0.1nM to about 10nM K D and PD-L1.
  • an anti-PD-L1 antibodies and fragments thereof binding to a binding affinity of from about 0.2nM to about 8nM K D and PD-L1.
  • provided herein is an anti-PD-L1 antibodies and fragments thereof binding to a binding affinity of from about 0.3nM to about 6nM K D and PD-L1.
  • an anti-PD-L1 antibodies and fragments of about 0.05nM or less binding affinity K D of binding to PD-L1.
  • the anti-PD-L1 antibodies and fragments thereof provided herein inhibit the binding of PD-L1/PD-1, wherein the IC50 is from about 1 ng/mL to about 1500 ng/mL. In another embodiment, an anti-PD-Ll antibody and fragment thereof provided herein inhibits binding of PD-L1/PD-1, wherein the IC50 is from about 10 ng/mL to about 1200 ng/mL. In another embodiment, an anti-PD-Ll antibody and fragment thereof provided herein inhibits binding of PD-L1/PD-1, wherein the IC50 is from about 20 ng/mL to about 800 ng/mL.
  • an anti-PD-Ll antibody and fragment thereof provided herein inhibits binding of PD-L1/PD-1, wherein the IC50 is from about 50 ng/mL to about 500 ng/mL. In another embodiment, an anti-PD-Ll antibody and fragment thereof provided herein inhibits binding of PD-L1/PD-1, wherein the IC50 is from about 50 ng/mL to about 200 ng/mL.
  • the anti-PD-L1 antibodies and fragments thereof provided herein inhibit binding of PD-L1/PD-1, wherein the IC50 is about 1200 ng/mL or less, about 1000 ng/mL or less, about 800 ng/ mL or less, about 400 ng/mL or less, about 300 ng/mL or less, about 250 ng/mL or less, about 200 ng/mL or less, about 150 ng/mL or less, about 100 ng/mL or Smaller, about 75 ng/mL or less, about 60 ng/mL or less, about 50 ng/mL or less, about 40 ng/mL or less, about 30 ng/mL or less, about 20 ng/mL or less. Or about 10 ng/mL or less.
  • the IC50 of the anti-PD-L1 antibodies and fragments provided herein are measured by ELISA.
  • anti-PD-L1 antibodies and fragments thereof that inhibit interaction between cells expressing PD-L1 and PD-1 proteins, respectively, on the surface.
  • the anti-PD-L1 antibodies and fragments thereof provided herein inhibit interactions between cells expressing PD-L1 and PD-1 proteins, respectively, wherein the IC50 is from about 1 ng/mL to about 500 ng. /mL.
  • the anti-PD-L1 antibodies and fragments thereof provided herein inhibit interactions between cells expressing PD-L1 and PD-1 proteins, respectively, wherein the IC50 is from about 10 ng/mL to about 400 g. /mL.
  • the anti-PD-L1 antibodies and fragments thereof provided herein inhibit interactions between cells expressing PD-L1 and PD-1 proteins, respectively, wherein the IC50 is from about 20 ng/mL to about 300 ng. /mL. In another embodiment, the anti-PD-L1 antibodies and fragments thereof provided herein inhibit interactions between cells expressing PD-L1 and PD-1 proteins, respectively, wherein the IC50 is from about 50 ng/mL to about 200 ng. /mL.
  • the anti-PD-L1 antibodies and fragments thereof provided herein inhibit interactions between cells expressing PD-L1 and PD-1 proteins, respectively, wherein the IC50 is about 500 ng/mL or less, About 450 ng/mL or less, about 400 ng/mL or less, about 350 ng/mL or less, about 300 ng/mL or less, about 250 ng/mL or less, about 200 ng/mL or less, about 150 ng. /mL or less, about 100 ng/mL or less, or about 50 ng/mL or less.
  • the IC50 of the anti-PD-L1 antibodies and fragments thereof provided herein are measured by FACS.
  • an anti-PD-L1 antibody provided herein is a chimeric antibody having a heavy chain variable region according to the amino acid sequence of SEQ ID NO: 5 and a light chain according to the amino acid sequence of SEQ ID NO: a variable region; or a heavy chain variable region having an amino acid sequence according to SEQ ID NO: 7 and a light chain variable region according to the amino acid sequence of SEQ ID NO: 8; or having a weight according to the amino acid sequence of SEQ ID NO: a chain variable region and a light chain variable region according to the amino acid sequence of SEQ ID NO: 10; wherein the anti-PD-L1 antibody has the following PD-L1 binding EC50 as measured by ELISA or FACS: about 200 ng/mL Or smaller, or about 150 ng/mL or less, or about 100 ng/mL or less, or about 80 ng/mL or less, or about 60 ng/mL or less, or about 50 ng/mL or less.
  • the anti-PD-L1 antibody or antigen-binding fragment thereof of the invention is a chimeric antibody or antibody fragment.
  • An anti-PD-L1 chimeric antibody or fragment thereof according to the invention further comprising a heavy chain constant region of human IgG1, IgG2, IgG3 or IgG4 or variants thereof.
  • the humanized antibody light chain further comprises a constant region of a human kappa, lambda chain or variant thereof. Accordingly, in other embodiments, the anti-PD-L1 antibody or antigen-binding fragment thereof is a humanized antibody or antibody fragment.
  • the anti-PD-L1 antibody or fragment thereof is a monoclonal antibody, scFv, Fab fragment, Fv fragment, F(ab)' fragment, F(ab')2 fragment, bispecific antibody, immunoconjugate Or a combination thereof.
  • an isolated anti-PD-L1 antibody or fragment thereof is provided, wherein the antibody is produced by a hybridoma selected from the group consisting of hybridomas referred to herein as Chi1, Chi2-1, and Chi2-2 . Accordingly, the invention also encompasses hybridomas Chi1, Chi2-1, and Chi2-2, as well as any hybridomas that produce the antibodies disclosed herein.
  • the invention also provides an isolated polynucleotide encoding an antibody and fragment thereof provided herein, eg, a nucleotide sequence encoding a humanized antibody hu-Chi1-3.4 heavy chain, as set forth in SEQ ID NO: 54, encoding a light chain
  • a nucleotide sequence is set forth in SEQ ID NO: 56; the nucleotide sequence encoding the humanized antibody hu-Chi1-2.3 heavy chain is set forth in SEQ ID NO: 58, and the nucleotide sequence encoding the light chain is SEQ. ID NO: 60 is shown.
  • the invention provides an expression vector comprising an isolated polynucleotide, such as a pMD-19T vector, a pcDNA3.1 vector, and the like.
  • the invention provides an expression vector comprising a nucleotide sequence encoding a humanized anti-PD-L1 antibody, eg, an expression vector encoding a hu-Chi1-2.3 heavy and light chain nucleotide sequence.
  • the invention also provides a host cell comprising the expression vector, which may be a eukaryotic cell or a prokaryotic cell, for example, the host cell includes, but is not limited to, a mammalian cell, an insect cell, a yeast cell, a bacterium, etc.
  • the invention provides a method of producing the humanized anti-PD-L1 antibody or fragment thereof, the method comprising culturing a host cell comprising the expression vector.
  • the invention provides an anti-PD-L1 antibody immunoconjugate. Accordingly, the invention provides an antibody or fragment thereof that binds to PD-L1 and is linked or conjugated to a therapeutic agent.
  • Therapeutic agents that can be linked or conjugated to an anti-PD-Ll antibody can include, but are not limited to, cytotoxic drugs, radioisotopes, signaling pathway inhibitors, immunomodulators, or antibodies.
  • an antibody or fragment thereof provided herein is an immunoconjugate comprising an anti-PD-L1 antibody or fragment thereof, and further comprising an agent selected from the group consisting of additional therapeutic agents, cytotoxic agents, An agent in the group of immunoadhesion molecules and imaging agents.
  • the imaging agent is selected from the group consisting of a radioactive label, an enzyme, a fluorescent label, a luminescent label, a bioluminescent label, a magnetic label, and biotin.
  • the imaging agent is a radioactive label selected from the group consisting of: 3 H, 14 C, 35 S, 62 Cu, 64 Cu, 89 Zr, 90 Y, 99 Tc, 111 In, 125 I , 131 I, 177 Lu, 166 Ho and 153 Sm.
  • the therapeutic agent or cytotoxic agent is selected from the group consisting of a chemotherapeutic agent, an immunosuppressive agent, an immunostimulant, an antimetabolite, an alkylating agent, an antibiotic, a growth factor, a cytokine, an anti-angiogenesis Agents, anti-mitotic agents, anthracyclines, toxins and apoptotic agents.
  • the antigen binding protein is directly conjugated to an agent.
  • the antigen binding protein is conjugated to the reagent via a linker. Suitable linkers include, but are not limited to, the amino acid and polypeptide linkers reported in the literature. The linker can be cleavable or non-cleavable.
  • the invention provides a bispecific or multispecific antibody specific for PD-L1 and at least one other antigen or epitope. Binding of the anti-PD-L1 antibodies and fragments thereof provided herein to PD-L1 can be tested using the binding assays provided herein or any other binding assay known in the art.
  • the invention provides a composition comprising one or more of the anti-PD-L1 antibodies or fragments thereof provided herein and a pharmaceutically acceptable carrier.
  • the pharmaceutical compositions of the present invention further comprise a pharmaceutically acceptable stabilizer, buffer or excipient.
  • the invention provides a method for modulating an immune response in a subject, the method comprising administering to the subject a therapeutically effective amount of an anti-PD-L1 antibody or fragment thereof provided herein.
  • the invention provides a method for increasing T cell activation, the method comprising contacting a T cell with an anti-PD-Ll antibody or fragment thereof provided herein.
  • the invention provides a method for treating or preventing a disease or condition in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of an anti-PD-L1 antibody provided herein Or a fragment thereof.
  • the invention provides a method for enhancing an anti-tumor response in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of an anti-PD-L1 antibody of the invention or Fragment.
  • the invention provides a method for reducing tumor immune escape, reducing tumors, or inhibiting growth of tumor cells in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount An anti-PD-L1 antibody of the invention or a fragment thereof.
  • the invention provides a method for treating cancer in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of an anti-PD-L1 antibody or fragment thereof of the invention .
  • the cancer is selected from the group consisting of lymphoma, leukemia, melanoma, glioma, breast cancer, lung cancer, colon cancer, bone cancer, ovarian cancer, bladder cancer, kidney cancer, Liver cancer, stomach cancer, testicular cancer, salivary gland cancer, thyroid cancer, thymic cancer, epithelial cancer, head or neck cancer, gastric cancer, pancreatic cancer, or a combination thereof.
  • the invention provides a method for treating an infectious disease in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of an anti-PD-L1 antibody or fragment thereof of the invention .
  • the infectious disease is selected from the group consisting of: candidiasis, candidemia, aspergillosis, streptococcal pneumonia, streptococcal skin and oropharyngeal conditions, Gram-negative pus Toxic, tuberculosis, mononucleosis, influenza, respiratory diseases caused by respiratory syncytial virus, malaria, schistosomiasis and trypanosomiasis.
  • the antibodies and fragments thereof provided herein are useful for treating diseases mediated by T helper 2 (Th2) T cells, such as, for example, asthma, allergic reactions, or graft versus host disease.
  • Th2 T helper 2
  • the antibodies and fragments thereof provided herein can be used to stimulate an immune response in a subject in need thereof.
  • an anti-PD-Ll antibody and fragments thereof can be administered with an antigen of interest for eliciting an immune response to the antigen.
  • the antigen of interest may be an antigen associated with a pathogen, such as a virus or a bacterium.
  • the invention provides a vaccine comprising an anti-PD-Ll antibody and an antigen, wherein the vaccine elicits an antigen-specific immune response.
  • the antibodies and fragments thereof disclosed herein can be administered to a subject in need thereof in combination with one or more additional therapeutic agents.
  • the antibody and fragments thereof can be administered to the subject before, during, and/or after administration of the additional therapeutic agent to the subject.
  • the additional therapeutic agent is a chemotherapeutic agent, a radiotherapeutic agent, a cytokine, an antibody or fragment thereof, or any other additional therapeutic agent indicative of the condition to be treated.
  • the anti-PD-Ll antibody and the additional therapeutic agent exhibit a therapeutic synergy when administered together, whether administered simultaneously or sequentially.
  • the anti-PD-L1 antibody and the additional therapeutic agent are administered in separate formulations.
  • the anti-PD-Ll antibody and the additional therapeutic agent are administered in the same formulation.
  • the anti-PD-Ll antibodies and fragments provided herein enhance the immunomodulatory effects of one or more additional therapeutic agents.
  • one or more additional therapeutic agents enhance the effect of the anti-PD-Ll antibody or fragment thereof.
  • the invention provides isolated antibodies and antigen-binding fragments thereof, as well as nucleic acids encoding the antibodies and fragments, and compositions comprising the isolated antibodies, fragments or nucleic acids.
  • isolated refers to a compound of interest (eg, an antibody or nucleic acid) that has been isolated from its natural environment.
  • the invention also provides a pharmaceutical composition comprising an isolated antibody or fragment thereof, or a nucleic acid encoding the antibody or fragment, and further comprising one or more pharmaceutically acceptable carriers.
  • Pharmaceutically acceptable carriers include, for example, excipients, diluents, encapsulating materials, fillers, buffers, or other agents.
  • antibody refers to an antigen binding protein having at least one antigen binding domain.
  • the antibodies and fragments thereof of the invention may be the entire antibody or any fragment thereof.
  • antibodies and antigen-binding fragments of the invention include monoclonal antibodies or fragments thereof and antibody variants or fragments thereof, as well as immunoconjugates.
  • antibody fragments include Fab fragments, Fab' fragments, F(ab')2 fragments, Fv fragments, isolated CDR regions, single chain Fv molecules (scFv), and other antibody fragments known in the art.
  • Antibodies and fragments thereof can also include recombinant polypeptides, fusion proteins, and bispecific antibodies.
  • the anti-PD-L1 antibodies and fragments thereof disclosed herein can be of the IgGl, IgG2, IgG3 or IgG4 isotype.
  • the term "isotype" refers to the type of antibody encoded by the heavy chain constant region gene.
  • an anti-PD-L1 antibody and fragments thereof disclosed herein are of the IgGl or IgG4 isotype.
  • the PD-L1 antibodies and fragments thereof of the invention can be derived from any species including, but not limited to, mice, rats, rabbits, primates, llamas, and humans.
  • the anti-PD-L1 antibody and fragments thereof can be chimeric antibodies, humanized antibodies or intact human antibodies.
  • the anti-PD-L1 antibody is an antibody produced by a mouse-derived hybridoma cell line.
  • the anti-PD-L1 antibody is a murine antibody.
  • the anti-PD-L1 antibody is a chimeric antibody.
  • the chimeric antibody is a mouse-human chimeric antibody.
  • the antibody is a humanized antibody.
  • the antibody is derived from a murine antibody and is humanized.
  • a "Fab fragment” consists of a light chain and a heavy chain CH1 domain and a variable region.
  • the heavy chain of a Fab molecule cannot form a disulfide bond with another heavy chain molecule.
  • a "Fab' fragment” contains a light chain and a portion of a heavy chain comprising a VH domain and a CH1 domain and a region between the CH1 domain and the CH2 domain, whereby two heavy chains of the two Fab' fragments are available An interchain disulfide bond is formed to form a F(ab')2 molecule.
  • F(ab')2 fragment contains two light chains and two heavy chains comprising a portion of the constant region between the CH1 domain and the CH2 domain, thereby forming an interchain disulfide bond between the two heavy chains .
  • the F(ab')2 fragment consists of two Fab' fragments held together by a disulfide bond between the two heavy chains.
  • the "Fv region” contains variable regions from both heavy and light chains, but lacks a constant region.
  • a “single-chain Fv antibody” refers to an antibody fragment comprising the VH and VL domains of an antibody, wherein these domains are present in a single polypeptide chain.
  • Fv polypeptides additionally comprise a polypeptide linker between the VH and VL domains that enables the scFv to form the desired structure for antigen binding.
  • a “diabody”, also known as a “bispecific antibody,” is a small antibody fragment having two antigen binding sites.
  • the fragment comprises a heavy chain variable domain (VH) (VH-VL or VL-VH) linked to a light chain variable domain (VL) in the same polypeptide chain.
  • VH heavy chain variable domain
  • VL light chain variable domain
  • a “chimeric antibody” is an antibody having at least a portion of a heavy chain variable region derived from a species and at least a portion of a variable region of a light chain; and at least a portion of a constant region derived from another species .
  • a chimeric antibody can comprise a murine variable region and a human constant region.
  • a “humanized antibody” is an antibody comprising a complementarity determining region (CDR) derived from a non-human antibody; and a framework region derived from a human antibody and a constant region.
  • CDR complementarity determining region
  • an anti-PD-L1 antibody provided herein can comprise a CDR derived from one or more murine antibodies, as well as a human framework region and a constant region.
  • a humanized antibody provided herein binds to the same epitope on PD-L1 as the murine antibody from which the CDR of the antibody is derived.
  • Exemplary humanized antibodies are provided herein.
  • framework sequences suitable for use in the present invention include those framework sequences that are structurally similar to the framework sequences provided herein. Additional modifications can be made in the framework regions to improve the properties of the antibodies provided herein. Such additional framework modifications may include chemical modifications; point mutations to reduce immunogenicity or removal of T cell epitopes; or reversion of the mutation to residues in the original germline sequence. In some embodiments, such modifications include those corresponding to the mutations exemplified herein, including back mutations to germline sequences.
  • one or more amino acids in the human framework regions of the VH and/or VL of the humanized antibodies provided herein are back-mutated to the corresponding amino acids in the parent murine antibody.
  • the amino acid back mutation at position 35 and/or 39 and/or 43 and/or 44 and/or 48 and/or 71 of the humanized antibody hu-Chi1 heavy chain variable region is in mouse Chi1 The corresponding amino acid found at the position of the heavy chain variable region.
  • amino acids at positions 2 and/or 8 and/or 41 and/or 44 and/or 87 and/or 100 of the light chain variable region are back-mutated to be variable in the mouse Chi1 light chain
  • the amino acid back mutation of S35N, Q39R, K43N, G44K, W47Y, I48M, V71R is the corresponding amino acid in the corresponding mouse Chi antibody.
  • the humanized hu-Chi1-3.4 antibody comprises a heavy chain variable region, wherein the amino acid at position 47 is mutated from Trp (W) to Try (Y), and the amino acid at position 35 is from Ser (S) is mutated to Asn(N), and the amino acid at position 71 is mutated from Val(V) to Arg(R); and the light chain variable region, wherein the amino acid at position 2 is mutated from Ile(I) to Phe(F), the amino acid at position 41 is mutated from Gly(G) to Asp(D), and the amino acid at position 44 is mutated from Pro(P) to Val(V).
  • the invention also encompasses a humanized antibody that binds to PD-L1 and comprises a framework modification corresponding to an exemplary modification of any suitable framework sequence described herein, and otherwise improves the antibody
  • Other framework modifications of the property for example, the amino acid at position 102 of the humanized antibody hu-Chi1 heavy chain variable region is mutated from Cys (C) to Try (Y), Ala (A) or Val (V).
  • human PD-L1 refers to the effective concentration, the 50% maximal response of an antibody.
  • IC50 refers to the concentration of inhibition, the 50% maximal response of an antibody. Both EC50 and IC50 can be measured by ELISA or FACS analysis or any other method known in the art.
  • the anti-PD-L1 antibodies disclosed herein having one or more amino acid substitutions, insertions, deletions or combinations thereof in the CDR or light chain variable region or heavy chain variable region retain a corresponding absence of amino acid substitutions, insertions or deletions Biological activity of anti-PD-L1 antibody.
  • the variant anti-PD-L1 antibodies provided herein retain binding to PD-L1.
  • percent homology refers to the number of identical amino acid sequences shared by two reference sequences divided by the total number of amino acid positions, multiplied by 100.
  • the variant of the CDR or light chain variable region or heavy chain variable region amino acid sequence wherein the variant comprises 1, 2, 3, 4, 5, 6, 7, 8, 9 , 10 or more amino acid substitutions, insertions or deletions, or a combination thereof.
  • the amino acid substitution is a conservative substitution.
  • the invention provides methods for treating a disease or condition in a subject that is responsive to enhancing, stimulating or eliciting an immune response.
  • treatment or “treating” refers to both therapeutic treatment as well as preventative or preventative measures.
  • Subjects in need of treatment include those already with the disease or condition, as well as subjects who may have the disease or condition and whose purpose is to prevent, delay or attenuate the disease or condition.
  • subject refers to mammals, such as rodents, felines, canines, and primates.
  • the subject according to the invention is a human.
  • terapéuticaally effective amount is the amount of a compound or composition necessary to provide a therapeutic and/or prophylactic benefit to a subject.
  • mutation sequence and “mutant sequence” are used interchangeably herein to refer to a sequence that has been altered by substitution, deletion or insertion, and the "mutant sequence” and “mutant sequence” are before the mutation.
  • the sequence may have more than 60% homology, for example 70% or more homology, and further, for example, more than 80% homology.
  • the term "about” as used herein is an index of values within an acceptable tolerance of a particular value as determined by one of ordinary skill in the art, depending in part on how the measurement or measurement is made (ie, the limits of the measurement system). For example, “about” in each implementation of the art may mean a standard deviation of one or more than one. Alternatively, “about” or “substantially encompasses” may mean a range of up to 20%. Moreover, particularly for biological systems or processes, the term can mean up to an order of magnitude or up to 5 times the value. The meaning of "about” or “substantially encompasses” should be assumed to be within the acceptable tolerance of the particular value, unless stated otherwise.
  • Figure 1 Detection of binding of chimeric anti-PD-L1 antibody to PD-L1 over a range of concentrations by ELISA;
  • Figure 2 Blocking of PD-1/PD-L1 binding by chimeric anti-PD-L1 antibodies over a range of antibody concentrations by ELISA;
  • Figure 3 Detection of PD-1/PD-L1 binding by chimeric anti-PD-L1 antibodies over a range of antibody concentrations by FACS;
  • Figure 4 Blocking effect of chimeric anti-PD-L1 antibody on PD-1/PD-L1 binding by a range of antibody concentrations by cell activity assay;
  • Figure 5 Blocking effect of humanized anti-PD-L1 antibody on PD-1/PD-L1 binding by a range of antibody concentrations by cell activity assay.
  • the human PD-L1 full-length gene (Hypothesis Shenzhou Biotechnology Co., Ltd., HG10084-M) of UniProt Programmed Cell Death 1Ligand 1 (PD-L1) isoform 1 (SEQ ID NO: 1) was used as the PD-of the present invention.
  • the template of L1 obtains the gene sequence encoding the antigen of the present invention and the protein for detection, and the PD-L1 extracellular domain (ECD) can be recombined with the antibody heavy chain Fc fragment (such as mouse IgG2a) to form PD-L1.
  • -mFc, or recombinantly linked to His Tag to form PD-L1-his for immunization or post-screening detection in mice.
  • PD-L1 extracellular domain recombinant protein hPD-L1(ECD)-mFc, SEQ ID NO: 2) containing a mouse antibody heavy chain Fc tag and histidine-tagged PD-L1 extracellular domain recombinant protein (hPD) -L1-His, SEQ ID NO: 3
  • hPD-1-mFc cDNA of the PD-1 extracellular domain recombinant protein containing the mouse antibody heavy chain Fc tag was obtained by PCR, and They were subcloned into the expression vector pcDNA3.1 (Invitrogen, V-790), respectively.
  • the vector constructed above was transfected into Exi-CHO cells (ThermoFisher A29133) for transient expression.
  • hPD-L1-HisTag protein was purified using an NTA column (GE healthcare), and hPD-L1-mFc and hPD1-mFc recombinant proteins were purified using a Protein A column (GE healthcare).
  • the expression fragment is stably expressed in cells such as CHO-S (Thermo Fisher, A1155701) or CHO-DG44 (Thermo Fisher, A1100001), and purified.
  • mice The purified hPD-L1(ECD)-mFc recombinant protein (according to 100 ⁇ g/mouse) was thoroughly mixed and emulsified with an equal volume of complete Freund's adjuvant (first boost) or incomplete Freund's adjuvant (enhanced immunization).
  • BALB/c mice were immunized subcutaneously every 2 weeks for 8 weeks. Splenocyte fusion was performed in mice with high antibody titers in serum and titers that tended to plateau. Three days before the fusion, mice were immunized by intraperitoneal injection of an adjuvant-free hPD-L1 (ECD)-mFc antigen (50 ⁇ g/mouse).
  • the spleen lymphocytes were fused with myeloma cell Sp2/0 cells using an optimized PEG-mediated fusion step to obtain hybridoma cells.
  • Splenocytes (1 x 10 8 cells) from immunized mice were fused with SP2/0 myeloma cells (2 x 10 7 cells).
  • the cells were resuspended in HAT complete medium, dispensed into 96-well plates at 0.1 ml/well, and cultured in a 37 ° C, 5% CO 2 incubator.
  • the half-liquid exchange was carried out with fresh HAT complete medium, and the cultivation was continued at 37 ° C, 5% CO 2 .
  • the whole medium was changed according to the cell growth density, and the medium used was HT complete medium, 200 ⁇ l/well, and culture was continued at 37 ° C, 5% CO 2 .
  • the 14th day after the fusion it was detected by the ELISA method using PD-L1 binding according to the cell growth density (Test Method 1.1).
  • the detected positive well cells were subjected to blocking ELISA detection of PD-L1/PD-1 binding (detection method 1.2), and the pores capable of binding PD-L1 and blocking binding to PD-1 were selected, and according to cell density Promptly expand into 24-well plates.
  • the cell line transferred into the 24-well plate was subjected to retesting and then subjected to seed conservation and first subcloning.
  • the first subcloning screen was positive for conservation and the second subcloning was performed.
  • the second subcloning was positive for conservation and protein expression.
  • PD-L1 binding screening and analysis of hybridoma antibodies was performed by ELISA method using PD-L1-his protein.
  • PD-L1 antigen 100 ⁇ L, 2 ⁇ g/ml
  • a high-adsorption 96-well plate (Costar, 9018) at 4 ° C overnight.
  • the non-specific binding site was blocked with blocking buffer (PBS containing 2% bovine serum albumin).
  • PBS with 0.05% (v/v) Tween 20) 100 ⁇ L/well of the sample to be tested was added, and incubated at room temperature for 1 hour.
  • HRP horseradish peroxidase
  • PD-L1 antigen 100 ⁇ L, 2 ⁇ g/ml was coated in a highly adsorbed 96-well plate at 4 ° C overnight. After washing the unadsorbed antigen sufficiently, the non-specific binding site was blocked with blocking buffer (PBS containing 2% bovine serum albumin). After washing the plate three times with washing buffer (PBS with 0.05% Tween 20), 100 ⁇ L/well of the sample to be tested was added while 100 ⁇ l of biotin-labeled PD-1-mFc (0.1 ⁇ g/ml) was added to each well. And incubated for 2 h at 37 °C.
  • blocking buffer PBS containing 2% bovine serum albumin
  • Example 3 cDNA acquisition of anti-human PD-L1 antibody and construction of chimeric antibody
  • the PCR mixture was electrophoretically separated in a 1% agarose/Tris-borate gel containing 0.5 ⁇ g/ml ethidium bromide.
  • a DNA fragment of the expected size (about 500 bp of heavy and light chains) was excised from the gel and purified.
  • the purified PCR product was cloned into the pMD-19T vector (Takara, 6013) and transformed into DH5 ⁇ competent E. coli cells (Takara, 9057). Two colonies were picked from LB solid culture plates for DNA sequencing.
  • the heavy chain variable region sequence and the light chain variable region sequence of the antibody are obtained (Chi1 variable region is shown in SEQ ID NOs: 5-6, and Chi2 variable region is shown in SEQ ID NOs: 7-10).
  • These antibodies show specific functions, such as high affinity binding to PD-L1, and are capable of blocking the binding of PD-L1 to PD-1.
  • Chi1 Construction and expression of chimeric antibody 1 (Chi1) and chimeric antibody 2 (Chi2): Chi1 was constructed by ligating a mouse VL region gene synthesis fragment to a nucleotide sequence encoding a human kappa chain constant region by double digestion reaction.
  • the coding sequence for the chi2-1, Chi2-2 chimeric light chain (SEQ ID NOs: 40, 42 and 44, respectively).
  • the gene synthesis fragment of the mouse VH region was ligated into the nucleotide sequence encoding the human IgG1 constant region by double restriction enzyme reaction to construct Chi1, Chi2-1, Chi2-2 chimeric heavy chains (SEQ ID NOs: 39, 41 and 43) The coding sequence.
  • the DNA vector encoding the coding sequence of the above chimeric antibody was transfected into Expi CHO cells (50 mL system, 6 ⁇ 10 6 cells/mL, DNA 1 ⁇ g/ml) for transient expression and cultured for 7 days.
  • the chimeric antibody in the supernatant was then purified using a Protein A column (GE healthcare).
  • the binding of the chimeric antibody to PD-L1 was measured by the following ELISA, Biacore and flow cytometry, and it was verified at the cellular level whether the above chimeric antibody could block the binding of PD-1 to PD-L1.
  • the affinity of the PD-L1 chimeric antibody to the PD-L1 antigen was determined by Biacore, and the anti-human capture antibody was covalently coupled using the anti-human antibody capture kit (GE, BR-1008-39) according to the method described in the specification.
  • the CM5 biochip (GE, BR-1000-12) was used to capture the PD-L1 antibody of the present invention. Then, a series of concentrations of human PD-L1 antigen (hPD-L1-his, Sino biological 10084-H08H-200) or cynomolgus PD-L1 antigen (Cyno-PD-L1-his, Sino biologica1 90251) were flowed on the surface of the chip.
  • Anti-human PD-L1 chimeric antibody binds to PD-L1 activity
  • the PD-L1 binding assay of the chimeric antibody was carried out by the ELISA method, referring to the detection method 1.1 in Example 2, using hPD-L1-mFc instead of the PD-L1-his protein. Finally, the colorimetric signal read by the microplate reader at 450 nm was analyzed using GraphPad Prism5 and the EC50 was calculated. See Table 2, chimeric antibody 1 (Chi1) and chimeric antibody 2-1 (Chi2-1) and control antibody. Chimeric antibody 1 was slightly superior compared to having comparable PD-L1 binding activity.
  • Anti-human PD-L1 chimeric antibody blocks PD-L1/PD-1 binding activity
  • the binding ability of the anti-PD-L1 chimeric antibody to the naturally expressed PD-L1 was analyzed by analyzing binding experiments with the U2OS cell line (PD-L1-U2OS) stably expressing PD-L1.
  • PD-L1-U2OS U2OS cell line
  • 2 ⁇ 10 5 PD-L1-U2OS cells were added to each well of a 96-well culture plate, and a gradient-diluted anti-PD-L1 antibody was added to the cell suspension while biotin-labeled PD-1 protein was added thereto. Competition combines with PD-L1. After incubation at 4 ° C for 120 minutes, the cells were washed 3 times with PBS and incubated with avidin-FITC (eBioscience, 11-4317-87) for 60 minutes at 4 °C.
  • avidin-FITC eBioscience, 11-4317-87
  • engineered Jurkat cells stably expressing PD-1 (DiscoverX, 93-1104C19) and U2OS cells stably expressing PD-L1 (DiscoverX, 93-1066C3) were used, respectively.
  • PD-L1 on the surface of U2OS cells binds to PD-1 on the surface of Jurkat
  • the engineered Jurkat cells induce a downstream pathway and react with the substrate to produce a chemiluminescent signal. Therefore, when an anti-PD-L1 antibody is present in the system and is capable of blocking the binding of PD-1 to PD-L1, there is no chemical signal or a weak chemical signal.
  • recombinant proteins were used as screens in which both PD-L1 and PD-1 were expressed in the native conformation on the cell surface.
  • the ligand cells in the exponential growth phase (U2OS PD-L1 cells, 9.6 ⁇ 10 5 cells/ml) were added to the 384-well plate using an automatic dispenser, 12.5 ⁇ L/well; Gradiently diluted antibody; at the same time, the recipient cells (Jurkat PD-1 cells) in good exponential growth phase, 6.4 ⁇ 10 5 /ml, were inoculated on a 384-well plate using an automatic dispenser, 12.5 ⁇ L/well.
  • the optimal Chi1 was selected for humanization.
  • IMGT human antibody heavy light chain variable region germline gene database http://www.imgt.org/3Dstructure-DB/cgi/DomainGapAlign.cgi
  • the Germline sequence is used as a template to transplant the CDR regions of the murine antibody into the corresponding human template by CDR grafting, and the order of formation is FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.
  • Variable region sequences (where amino acid residues are determined and annotated by the Kabat numbering system).
  • the heavy chain humanized template of the murine antibody Chi1 is a combination of IGHV4-30-4*01 and IGHJ1*01; the light chain humanized template is a combination of IGKV1-39*01 and IGKJ2*02, after direct CDR grafting
  • the sequences are SEQ ID NOs: 45 and 46.
  • the affinity of the antibody after direct humanization generally decreases to a large extent, and further design of the back mutation is needed to restore affinity and function, that is, several sites of the framework amino acid of the above human template antibody are back-mutated into the mouse Chi1 antibody.
  • the amino acid sequence in the corresponding framework region is a combination of IGHV4-30-4*01 and IGHJ1*01.
  • sequences encoding VH and VL of the humanized hu-Chi1 antibody are SEQ ID NOS: 47 and 51, respectively, and ligated to the human antibody constant region to construct the complete antibody form, SEQ ID NOs: 53 and 55, respectively.
  • the DNA sequences encoding the light and heavy chains of the humanized hu-Chi1-3.4 antibody described above (SEQ ID NOs: 54 and 56).
  • the vector was cloned into the expression vector pcDNA3.1 (Invitrogen, V-790).
  • the above DNA vector was transfected into Expi CHO cells (50 mL system, 5 ⁇ 10 6 /mL cells, DNA 1 ⁇ g/ml), transiently expressed, and cultured for 7 days.
  • the humanized antibody in the supernatant was then purified using a Protein A column (GE healthcare).
  • Grafted indicates that the mouse CDR is directly transplanted into the human FR.
  • the antibody numbering rule mutates the W at position 47 to Y according to the Kabat nomenclature, such as W47Y.
  • Humanized PD-L1 antibodies were numbered according to Table 6, as shown in Table 7.
  • Biacore measures the affinity of anti-PD-L1 humanized antibody for PD-L1 antigen.
  • the specific steps are as follows: Example 4.1, Table 8 below is humanized hu-Chi1, hu-Chi1-3.3, hu-Chi1-3.4 and chimeric Affinity data for antibody Chi1 indicated that the humanized PD-L1 antibody maintained a good affinity for PD-L1 during the back mutation.
  • the blocking effect of the anti-PD-L1 humanized antibody was analyzed by cell/cell interaction, and the specific procedure was as described in Example 4.5.
  • the results in Table 9 indicate that the humanized antibody has a comparable blocking activity as the maternal chimeric antibody.

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Abstract

Provided are a humanized anti-PD-L1 antibody and an antigen-binding fragment thereof, a corresponding nucleotide, an expression vector comprising the polynucleotide, a cell comprising the expression vector, and a pharmaceutical composition comprising the antibody or antigen-binding fragment thereof, which can be used for reducing tumors or inhibiting tumor cell growth.

Description

抗PD-L1抗体及其抗原结合片段anti-PD-L1 antibody and antigen-binding fragment thereof 技术领域Technical field
本发明涉及分离的单克隆抗体领域，具体涉及结合PD-Ll的抗体、其抗原结合片段、包含所述PD-Ll抗体CDR区的鼠源抗体、嵌合抗体、人源化抗体、编码其的核酸、包含其的组合物以及涉及使用这类抗体和抗原结合片段的方法。The present invention relates to the field of isolated monoclonal antibodies, and in particular to an antibody that binds to PD-L1, an antigen-binding fragment thereof, a mouse antibody comprising the CDR region of the PD-L1 antibody, a chimeric antibody, a humanized antibody, and a coding thereof Nucleic acids, compositions comprising the same, and methods involving the use of such antibodies and antigen-binding fragments.
背景技术Background technique
肿瘤免疫治疗是肿瘤治疗领域的革新性方法，是为了最大限度的提高患者对肿瘤的免疫系统反应，解除肿瘤细胞对T细胞的抑制作用。其中，杀伤性T细胞是发挥杀伤肿瘤细胞的主要作用细胞，然而肿瘤微环境中存在着极其复杂的免疫调节机制，随着肿瘤的发展，杀伤性T细胞的肿瘤杀伤活性被抑制，肿瘤不断生长恶化。T细胞的活化需要两个信号：MHC-抗原肽呈递信号和共刺激信号。同时，在调控T细胞活化的过程中还存在共抑制信号，防止T细胞的过度活化。其中，最关键的信号通路之一即为程序性死亡受体1(PD-1)/程序性死亡受体配体1(PD-L1)信号通路。Tumor immunotherapy is an innovative method in the field of cancer treatment, in order to maximize the immune system response of patients to tumors, and to eliminate the inhibition of T cells by tumor cells. Among them, killer T cells play a major role in killing tumor cells. However, there are extremely complex immunoregulatory mechanisms in the tumor microenvironment. With the development of tumors, the tumor killing activity of killer T cells is inhibited, and tumors continue to grow. deterioration. Activation of T cells requires two signals: the MHC-antigen peptide presentation signal and the costimulatory signal. At the same time, there is also a co-suppression signal during the regulation of T cell activation to prevent excessive activation of T cells. Among them, one of the most critical signaling pathways is the programmed death receptor 1 (PD-1)/programmed death receptor ligand 1 (PD-L1) signaling pathway.
程序性死亡受体配体1(PD-L1)是程序性死亡受体1(PD-1)的配体。PD-L1也称为分化簇274(CD274)或B7同系物1(B7-H1)，并且是由CD274基因编码的分子量为40kDa的1型跨膜蛋白。PD-L1和PD-1两者都属于免疫球蛋白超家族并且都由两个细胞外Ig结构域(即N末端V结构域和C末端恒定结构域)组成。PD-L1与活化的T细胞、B细胞和髓样细胞上存在的其受体PD-1结合，以调节免疫细胞的活化或抑制。在多种肿瘤中，如黑色素瘤、肺癌、膀胱癌等多种癌细胞上，PD-L1的表达均显著上调。PD-L1通过与T细胞表面的PD-1结合，抑制T细胞的活化、细胞因子的产生和T细胞的增殖(Fife等(2011)Nature Immunology 10:1185-1193)；诱导同源抗原特异性T细胞中的衰竭或无反应性(Hofmeyer等(2011)Journal of Biomedicine and Biotechnology 2011:1-9)；并诱导效应T细胞的凋亡。PD-L1基因的破坏导致上调的T细胞应答和自身反应性T细胞的产生(Latchman等(2004)PNAS 101:10691-10696)。WO2016061142公开了PD-L1的抗体阻断导致肿瘤浸润型淋巴细胞增加、T细胞受体介导的增殖增加以及癌细胞的免疫逃避减少；WO2016022630证明一类抗PD-L1抗体和其片段在调节T细胞的存在下恢复效应T细胞增殖和/或产生细胞因子的能力。因此通过PD-L1抗体阻断PD-L1/PD-1之间的结合成为了肿瘤免疫治疗领域一个非常有效的方法。Programmed death receptor ligand 1 (PD-L1) is a ligand for programmed death receptor 1 (PD-1). PD-L1 is also known as differentiation cluster 274 (CD274) or B7 homolog 1 (B7-H1) and is a type 1 transmembrane protein of 40 kDa encoded by the CD274 gene. Both PD-L1 and PD-1 belong to the immunoglobulin superfamily and are composed of two extracellular Ig domains (ie, an N-terminal V domain and a C-terminal constant domain). PD-L1 binds to its receptor PD-1 present on activated T cells, B cells and myeloid cells to regulate activation or inhibition of immune cells. In a variety of tumors, such as melanoma, lung cancer, bladder cancer and other cancer cells, the expression of PD-L1 was significantly up-regulated. PD-L1 inhibits T cell activation, cytokine production, and T cell proliferation by binding to PD-1 on the surface of T cells (Fife et al. (2011) Nature Immunology 10: 1185-1193); induction of homologous antigen specificity Failure or non-reactivity in T cells (Hofmeyer et al (2011) Journal of Biomedicine and Biotechnology 2011: 1-9); and induces apoptosis of effector T cells. Destruction of the PD-L1 gene results in up-regulated T cell responses and production of autoreactive T cells (Latchman et al. (2004) PNAS 101:10691-10696). WO2016061142 discloses that antibody blockade of PD-L1 leads to an increase in tumor infiltrating lymphocytes, an increase in T cell receptor mediated proliferation, and a reduction in immune escape of cancer cells; WO2016022630 demonstrates that a class of anti-PD-L1 antibodies and fragments thereof are in regulating T The ability of effector T cells to proliferate and/or produce cytokines is restored in the presence of cells. Therefore, blocking the binding between PD-L1/PD-1 by PD-L1 antibody has become a very effective method in the field of tumor immunotherapy.
本发明提供了一种全新序列的高亲和力、高选择性、高生物活性的结合PD-L1的抗体及其抗原结合片段。The present invention provides a novel sequence of high affinity, highly selective, highly biologically active PD-L1 binding antibodies and antigen binding fragments thereof.
发明内容Summary of the invention
一方面，本发明提供一种分离的结合PD-L1的抗原结合蛋白或其抗原结合片段。在一些实施方案中，本发明提供一种分离的抗PD-L1抗体或其抗原结合片段，其能与人PD-L1结合，并表现出优良的特性。在一些实施方案中，所述抗体或其抗原结合片段能结合PD-L1并阻断PD-1与PD-L1的结合。在另一实施方案中，所述抗体或其抗原结合片段能阻断分别产生PD-1与PD-L1的细胞之间的相互作用。另一方面，本发明提供了一种使用这类抗体和抗原结合片段的方法，例如使用这类抗体和抗原结合片段治疗癌症和/或传染病、增加T细胞活化或者在受试者中降低肿瘤免疫逃逸、减少肿瘤或抑制肿瘤细胞生长的方法。又一方面，本发明提供编码这类抗原结合蛋白(例如抗体)及其抗原结合片段的多核苷酸、包含该多核苷酸的表达载体、包含该表达载体的重组细胞以及包含这类抗原结合蛋白(例如抗体)及其抗原结合片段的组合物等。In one aspect, the invention provides an isolated antigen binding protein that binds to PD-L1 or an antigen binding fragment thereof. In some embodiments, the invention provides an isolated anti-PD-L1 antibody or antigen-binding fragment thereof that binds to human PD-L1 and exhibits superior properties. In some embodiments, the antibody or antigen-binding fragment thereof binds to PD-L1 and blocks binding of PD-1 to PD-L1. In another embodiment, the antibody or antigen-binding fragment thereof is capable of blocking the interaction between cells producing PD-1 and PD-L1, respectively. In another aspect, the invention provides a method of using such antibodies and antigen-binding fragments, for example, the use of such antibodies and antigen-binding fragments to treat cancer and/or infectious diseases, increase T cell activation, or reduce tumors in a subject A method of immune escape, reduction of tumors, or inhibition of tumor cell growth. In still another aspect, the present invention provides a polynucleotide encoding such an antigen binding protein (eg, an antibody) and an antigen-binding fragment thereof, an expression vector comprising the polynucleotide, a recombinant cell comprising the expression vector, and a recombinant protein comprising the same (eg, an antibody), a composition thereof and an antigen-binding fragment thereof, and the like.
在一方面，本发明提供的分离的抗PD-L1抗体或其抗原结合片段，其包含选自SEQ ID NOs：12、14-22和/或SEQ ID NOs：27、29-38中的一个或多个CDR区(抗体互补决定区)序列或其突变体序列。In one aspect, the invention provides an isolated anti-PD-L1 antibody or antigen-binding fragment thereof comprising one selected from the group consisting of SEQ ID NOs: 12, 14-22 and/or SEQ ID NOs: 27, 29-38 or Multiple CDR regions (antibody complementarity determining regions) sequences or mutant sequences thereof.
在一些方案中，本发明所述的抗PD-L1抗体或其抗原结合片段，其包含任选自SEQ ID NOs:12、17-22和/或SEQ ID NOs:29-34的抗体重链HCDR区序列或其突变序列。In some embodiments, the anti-PD-L1 antibody or antigen-binding fragment thereof of the invention comprises an antibody heavy chain HCDR selected from the group consisting of SEQ ID NOs: 12, 17-22 and/or SEQ ID NOs: 29-34 A sequence of regions or a sequence thereof.
在一些方案中，本发明所述的抗PD-L1抗体或其抗原结合片段，其包含任选自SEQ ID NOs:14-16和/或SEQ ID NOs:27、35-38的抗体轻链LCDR区序列或其突变序列。In some embodiments, the anti-PD-L1 antibody or antigen-binding fragment thereof of the invention comprises an antibody light chain LCDR selected from the group consisting of SEQ ID NOs: 14-16 and/or SEQ ID NOs: 27, 35-38 A sequence of regions or a sequence thereof.
在一些方案中，所述抗PD-L1抗体或其片段包含重链HCDR1序列，所述重链HCDR1序列与选自SEQ ID NO:17、18、29或30的氨基酸序列具有至少80％同源性、至少81％同源性、至少82％同源性、至少83％同源性、至少84％同源性、至少85％同源性、至少86％同源性、至少87％同源性、至少88％同源性、至少89％同源性、至少90％同源性、至少91％同源性、至少92％同源性、至少93％同源性、至少94％同源性、至少95％同源性、至少96％同源性、至少97％同源性、至少98％同源性或至少99％同源性。In some embodiments, the anti-PD-L1 antibody or fragment thereof comprises a heavy chain HCDR1 sequence having at least 80% homology to an amino acid sequence selected from SEQ ID NO: 17, 18, 29 or 30 , at least 81% homology, at least 82% homology, at least 83% homology, at least 84% homology, at least 85% homology, at least 86% homology, at least 87% homology At least 88% homology, at least 89% homology, at least 90% homology, at least 91% homology, at least 92% homology, at least 93% homology, at least 94% homology, At least 95% homology, at least 96% homology, at least 97% homology, at least 98% homology, or at least 99% homology.
在一个实施方案中，抗PD-L1抗体或其片段包含重链HCDR2序列，所述重链HCDR2序列与选自SEQ ID NO:12、31或32的氨基酸序列具有至少80％同源性、至少81％同源性、至少82％同源性、至少83％同源性、至少84％同源性、至少85％同源性、至少86％同源性、至少87％同源性、至少88％同源性、至少89％同源性、至少90％同源性、至少91％同源性、至少92％同源性、至少93％同源性、至少94％同源性、至少95％同源性、至少96％同源性、至少97％同源性、至少98％同源性或至少99％同源性。In one embodiment, the anti-PD-L1 antibody or fragment thereof comprises a heavy chain HCDR2 sequence having at least 80% homology to an amino acid sequence selected from SEQ ID NO: 12, 31 or 32, at least 81% homology, at least 82% homology, at least 83% homology, at least 84% homology, at least 85% homology, at least 86% homology, at least 87% homology, at least 88 % homology, at least 89% homology, at least 90% homology, at least 91% homology, at least 92% homology, at least 93% homology, at least 94% homology, at least 95% Homology, at least 96% homology, at least 97% homology, at least 98% homology, or at least 99% homology.
在一个实施方案中，所述抗PD-L1抗体或其片段包含重链HCDR3序列，所述重链HCDR3序列与选自SEQ ID NO:19-22或33-34的氨基酸序列具有至少80％同源性、至少81％同源性、至少82％同源性、至少83％同源性、至少84％同源性、至少85％同源性、至少86％同源性、至少87％同源性、至少88％同源性、至少89％同源性、至少90％同源性、至少91％同源性、至少92％同源性、至少93％同源性、至少94％同源性、至少95％同源性、至少96％同源性、至少97％同源性、至少98％同源性或至少99％同源性。In one embodiment, the anti-PD-L1 antibody or fragment thereof comprises a heavy chain HCDR3 sequence that is at least 80% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 19-22 or 33-34 Source, at least 81% homology, at least 82% homology, at least 83% homology, at least 84% homology, at least 85% homology, at least 86% homology, at least 87% homology , at least 88% homology, at least 89% homology, at least 90% homology, at least 91% homology, at least 92% homology, at least 93% homology, at least 94% homology At least 95% homology, at least 96% homology, at least 97% homology, at least 98% homology or at least 99% homology.
在一些方案中，所述抗PD-L1抗体或其片段包含轻链LCDR1序列，所述轻链LCDR1序列与选自SEQ ID NO:14、35或36的氨基酸序列具有至少80％同源性、至少81％同源性、至少82％同源性、至少83％同源性、至少84％同源性、至少85％同源性、至少86％同源性、至少87％同源性、至少88％同源性、至少89％同源性、至少90％同源性、至少91％同源性、至少92％同源性、至少93％同源性、至少94％同源性、至少95％同源性、至少96％同源性、至少97％同源性、至少98％同源性或至少99％同源性。In some embodiments, the anti-PD-L1 antibody or fragment thereof comprises a light chain LCDR1 sequence having at least 80% homology to an amino acid sequence selected from SEQ ID NO: 14, 35 or 36, At least 81% homology, at least 82% homology, at least 83% homology, at least 84% homology, at least 85% homology, at least 86% homology, at least 87% homology, at least 88% homology, at least 89% homology, at least 90% homology, at least 91% homology, at least 92% homology, at least 93% homology, at least 94% homology, at least 95 % homology, at least 96% homology, at least 97% homology, at least 98% homology or at least 99% homology.
在一些方案中，所述抗PD-L1抗体或其片段包含轻链LCDR2序列，所述轻链LCDR2序列与选自SEQ ID NO:15或27的氨基酸序列具有至少80％同源性、至少81％同源性、至少82％同源性、至少83％同源性、至少84％同源性、至少85％同源性、至少86％同源性、至少87％同源性、至少88％同源性、至少89％同源性、至少90％同源性、至少91％同源性、至少92％同源性、至少93％同源性、至少94％同源性、至少95％同源性、至少96％同源性、至少97％同源性、至少98％同源性或至少99％同源性。In some embodiments, the anti-PD-L1 antibody or fragment thereof comprises a light chain LCDR2 sequence having at least 80% homology to an amino acid sequence selected from SEQ ID NO: 15 or 27, at least 81 % homology, at least 82% homology, at least 83% homology, at least 84% homology, at least 85% homology, at least 86% homology, at least 87% homology, at least 88% Homology, at least 89% homology, at least 90% homology, at least 91% homology, at least 92% homology, at least 93% homology, at least 94% homology, at least 95% identical Source, at least 96% homology, at least 97% homology, at least 98% homology, or at least 99% homology.
在一些方案中，所述抗PD-L1抗体或其片段包含轻链LCDR3序列，所述轻链LCDR3序列与选自SEQ ID NO:16、37或38的氨基酸序列具有至少80％同源性、至少81％同源性、至少82％同源性、至少83％同源性、至少84％同源性、至少85％同源性、至少86％同源性、至少87％同源性、至少88％同源性、至少89％同源性、至少90％同源性、至少91％同源性、至少92％同源性、至少93％同源性、至少94％同源性、至少95％同源性、至少96％同源性、至少97％同源性、至少98％同源性或至少99％同源性。In some embodiments, the anti-PD-L1 antibody or fragment thereof comprises a light chain LCDR3 sequence, the light chain LCDR3 sequence having at least 80% homology to an amino acid sequence selected from SEQ ID NO: 16, 37 or 38, At least 81% homology, at least 82% homology, at least 83% homology, at least 84% homology, at least 85% homology, at least 86% homology, at least 87% homology, at least 88% homology, at least 89% homology, at least 90% homology, at least 91% homology, at least 92% homology, at least 93% homology, at least 94% homology, at least 95 % homology, at least 96% homology, at least 97% homology, at least 98% homology or at least 99% homology.
在一些方案中，所述抗PD-L1抗体或其抗原结合片段包含重链HCDR1、HCDR2和HCDR3，所述重链HCDR1包含与SEQ ID NO:17、18、29或30的氨基酸序列至少80％同源性的序列，所述重链HCDR2包含与SEQ ID NO:12、31或32的氨基酸序列至少80％同源性的序列和所述重链HCDR3包含与SEQ ID NO:19-22或33-34的氨基酸序列至少80％同源性的序列；以及轻链LCDR1、LCDR2和LCDR3，所述轻链LCDR1包含与SEQ ID NO:14、35或36的氨基酸序列至少80％同源性的序列，所述轻链LCDR2包含与SEQ ID NO:15或27的氨基酸序列至少80％同源性的序列以及所述轻链LCDR3包含与SEQ ID NO:16、37或38的氨基酸序列至少80％同源性的序列。In some embodiments, the anti-PD-L1 antibody or antigen-binding fragment thereof comprises heavy chain HCDR1, HCDR2 and HCDR3 comprising at least 80% of the amino acid sequence of SEQ ID NO: 17, 18, 29 or 30 a sequence of homology, said heavy chain HCDR2 comprising a sequence at least 80% homologous to the amino acid sequence of SEQ ID NO: 12, 31 or 32 and said heavy chain HCDR3 comprising SEQ ID NO: 19-22 or 33 a sequence of at least 80% homology of the amino acid sequence of -34; and a light chain LCDR1, LCDR2 and LCDR3 comprising a sequence at least 80% homologous to the amino acid sequence of SEQ ID NO: 14, 35 or 36 The light chain LCDR2 comprises a sequence at least 80% homologous to the amino acid sequence of SEQ ID NO: 15 or 27 and the light chain LCDR3 comprises at least 80% identical to the amino acid sequence of SEQ ID NO: 16, 37 or 38 Source sequence.
在一些方案中，所述抗PD-L1抗体或其抗原结合片段包含重链HCDR1、HCDR2和HCDR3及轻链LCDR1、LCDR2和LCDR3，所述重链HCDR1、HCDR2和HCDR3及轻链LCDR1、LCDR2和LCDR3选自以下序列或与其具有至少80％同源性的序列：In some embodiments, the anti-PD-L1 antibody or antigen-binding fragment thereof comprises heavy chain HCDR1, HCDR2 and HCDR3 and light chain LCDR1, LCDR2 and LCDR3, said heavy chain HCDR1, HCDR2 and HCDR3 and light chain LCDR1, LCDR2 and The LCDR3 is selected from the following sequences or sequences having at least 80% homology thereto:
Figure PCTCN2018119536-appb-000001
Figure PCTCN2018119536-appb-000001
Figure PCTCN2018119536-appb-000002
Figure PCTCN2018119536-appb-000002
其中X 1选自N或S，X 2选自Y、C、A或V，X 3选自S或F，X 4选自R或W，X 5选自S或N，X 6选自T或S，X 7选自Q或H，X 8选自E或N，X 9选自S或A。 Wherein X 1 is selected from N or S, X 2 is selected from Y, C, A or V, X 3 is selected from S or F, X 4 is selected from R or W, X 5 is selected from S or N, and X 6 is selected from T. Or S, X 7 is selected from Q or H, X 8 is selected from E or N, and X 9 is selected from S or A.
在一些方案中，所述抗PD-L1抗体或其抗原结合片段包含重链HCDR1、HCDR2和HCDR3；所述重链HCDR1序列，其选自SEQ ID NO:17、18、29或30所示的氨基酸序列；所述重链HCDR2序列，其选自SEQ ID NO:12、31或32所示的氨基酸序列；所述重链HCDR3序列，其选自SEQ ID NO:19-22或33-34所示的氨基酸序列。在一些方案中，所述抗PD-L1抗体或其抗原结合片段包含轻链LCDR1、LCDR2和LCDR3；所述轻链LCDR1序列，其选自SEQ ID NO:14、35或36所示的氨基酸序列；所述轻链LCDR2序列，其选自SEQ ID NO:15或27所示的氨基酸序列；所述轻链LCDR3序列，其选自SEQ ID NO:16、37或38所示的氨基酸序列。In some embodiments, the anti-PD-L1 antibody or antigen-binding fragment thereof comprises heavy chain HCDR1, HCDR2 and HCDR3; the heavy chain HCDR1 sequence selected from the group consisting of SEQ ID NO: 17, 18, 29 or 30 An amino acid sequence; the heavy chain HCDR2 sequence selected from the group consisting of the amino acid sequence set forth in SEQ ID NO: 12, 31 or 32; and the heavy chain HCDR3 sequence selected from the group consisting of SEQ ID NO: 19-22 or 33-34 The amino acid sequence shown. In some embodiments, the anti-PD-L1 antibody or antigen-binding fragment thereof comprises the light chain LCDR1, LCDR2 and LCDR3; the light chain LCDR1 sequence selected from the group consisting of the amino acid sequences set forth in SEQ ID NO: 14, 35 or 36 The light chain LCDR2 sequence selected from the amino acid sequence set forth in SEQ ID NO: 15 or 27; and the light chain LCDR3 sequence selected from the amino acid sequence set forth in SEQ ID NO: 16, 37 or 38.
在一些实施方案中，所述抗PD-L1抗体或其抗原结合片段包含重链HCDR1、HCDR2和HCDR3，所述重链HCDR1选自SEQ ID NO:17或18所示的氨基酸序列或其至少80％同源性的序列，所述重链HCDR2选自SEQ ID NO:12所示的氨基酸序列或其至少80％同源性的序列，所述重链HCDR3选自SEQ ID NOs:19-22中任意一个所示的氨基酸序列或其至少80％同源性的序列；以及轻链LCDR1、LCDR2和LCDR3，所述轻链LCDR1、LCDR2和LCDR3分别选自SEQ ID NOs:14、15和16所示的氨基酸序列或其至少80％同源性的序列。In some embodiments, the anti-PD-L1 antibody or antigen-binding fragment thereof comprises heavy chain HCDR1, HCDR2 and HCDR3, the heavy chain HCDR1 being selected from the amino acid sequence set forth in SEQ ID NO: 17 or 18 or at least 80 thereof a sequence of % homology, said heavy chain HCDR2 being selected from the amino acid sequence set forth in SEQ ID NO: 12 or a sequence of at least 80% homology thereof, said heavy chain HCDR3 being selected from the group consisting of SEQ ID NOs: 19-22 Any one of the indicated amino acid sequences or sequences thereof having at least 80% homology; and light chain LCDR1, LCDR2 and LCDR3, respectively selected from the group consisting of SEQ ID NOs: 14, 15 and 16, respectively Amino acid sequence or a sequence thereof that is at least 80% homologous.
在一些实施方案中，所述抗PD-L1抗体或其抗原结合片段包含重链HCDR1、HCDR2和HCDR3，所述重链HCDR1选自SEQ ID NO:29或30所示的氨基酸序列或其至少80％同源性的序列，所述重链HCDR2选自SEQ ID NO:31或32所示的氨基酸序列或其至少80％同源性的序列，所述重链HCDR3选自SEQ ID NO:33或34所示的氨基酸序列或其至少80％同源性的序列；以及轻链LCDR1、LCDR2和LCDR3，所述轻链LCDR1选自SEQ ID NO:35或36所示的氨基酸序列或其至少80％同源性的序列，所述轻链LCDR2选自SEQ ID NO:27所示的氨基酸序列或其至少80％同源性的序列，且所述轻链LCDR3选自SEQ ID NO:37或38所示的氨基酸序列或其至少80％同源性的序列。In some embodiments, the anti-PD-L1 antibody or antigen-binding fragment thereof comprises heavy chain HCDR1, HCDR2 and HCDR3, the heavy chain HCDR1 being selected from the amino acid sequence set forth in SEQ ID NO: 29 or 30 or at least 80 thereof a sequence of % homology, said heavy chain HCDR2 being selected from the amino acid sequence set forth in SEQ ID NO: 31 or 32, or a sequence of at least 80% homology thereof, said heavy chain HCDR3 being selected from SEQ ID NO: 33 or An amino acid sequence of 34 or a sequence of at least 80% homology thereof; and a light chain LCDR1, LCDR2 and LCDR3 selected from the amino acid sequence set forth in SEQ ID NO: 35 or 36 or at least 80% thereof a homologous sequence, wherein the light chain LCDR2 is selected from the amino acid sequence set forth in SEQ ID NO: 27 or a sequence thereof of at least 80% homology, and the light chain LCDR3 is selected from the group consisting of SEQ ID NO: 37 or 38 A sequence of the indicated amino acid sequence or at least 80% homology thereof.
在一些方案中，所述抗PD-L1抗体或其抗原结合片段包含重链HCDR1、HCDR2和HCDR3，所述重链HCDR1、HCDR2和HCDR3包含分别与根据SEQ ID NOs:17、12和19的氨基酸序列具有至少80％同源性、至少85％同源性、至少90％同源性、至少91％同源性、至少92％同源性、至少93％同源性、至少94％同源性、至少95％同源性、至少96％同源性、至少97％同源性、至少98％同源性或至少99％同源性的氨基酸序列；以及轻链LCDR1、LCDR2和LCDR3，所述轻链LCDR1、LCDR2和LCDR3包含分别与根据SEQ ID NOs:14、15和16的氨基酸序列具有至少80％同源性、至少85％同源性、至少90％同源性、至少91％同源性、至少92％同源性、至少93％同源性、至少94％同源性、至少95％同源性、至少96％同源性、至少97％同源性、至少98％同源性或至少99％同源性的氨基酸序列。在另一个实施方案中，抗PD-L1抗体或其抗原结合片段包含分别根据SEQ ID NOs:17、12和19的重链HCDR1、HCDR2和HCDR3，以及分别根据SEQ ID NOs:14、15和16的轻链LCDR1、LCDR2和LCDR3。In some embodiments, the anti-PD-L1 antibody or antigen-binding fragment thereof comprises heavy chain HCDR1, HCDR2 and HCDR3 comprising amino acids according to SEQ ID NOs: 17, 12 and 19, respectively. The sequence has at least 80% homology, at least 85% homology, at least 90% homology, at least 91% homology, at least 92% homology, at least 93% homology, at least 94% homology. An amino acid sequence of at least 95% homology, at least 96% homology, at least 97% homology, at least 98% homology, or at least 99% homology; and light chain LCDR1, LCDR2, and LCDR3, The light chains LCDR1, LCDR2 and LCDR3 comprise at least 80% homology, at least 85% homology, at least 90% homology, at least 91% homology to the amino acid sequences according to SEQ ID NOs: 14, 15 and 16, respectively. , at least 92% homology, at least 93% homology, at least 94% homology, at least 95% homology, at least 96% homology, at least 97% homology, at least 98% homology Or an amino acid sequence of at least 99% homology. In another embodiment, the anti-PD-L1 antibody or antigen-binding fragment thereof comprises the heavy chain HCDR1, HCDR2 and HCDR3 according to SEQ ID NOs: 17, 12 and 19, respectively, and according to SEQ ID NOs: 14, 15 and 16, respectively. Light chain LCDR1, LCDR2 and LCDR3.
在一些方案中，所述抗PD-L1抗体或其抗原结合片段包含重链HCDR1、HCDR2和HCDR3，所述重链HCDR1、HCDR2和HCDR3包含分别与根据SEQ ID NOs:18、12和20的氨基酸序列具有至少80％同源性、至少85％同源性、至少90％同源性、至少91％同源性、至少92％同源性、至少93％同源性、至少94％同源性、至少95％同源性、至少96％同源性、至少97％同源性、至少98％同源性或至少99％同源性的氨基酸序列；以及轻链LCDR1、LCDR2和LCDR3，所述轻链LCDR1、LCDR2和LCDR3包含分别与根据SEQ ID NOs:14、15和16的氨基酸序列具有至少80％同源性、至少85％同源性、至少90％同源性、至 少91％同源性、至少92％同源性、至少93％同源性、至少94％同源性、至少95％同源性、至少96％同源性、至少97％同源性、至少98％同源性或至少99％同源性的氨基酸序列。在另一个实施方案中，抗PD-L1抗体或其抗原结合片段包含分别根据SEQ ID NOs:18、12和20的重链HCDR1、HCDR2和HCDR3，以及分别根据SEQ ID NOs:14、15和16的轻链LCDR1、LCDR2和LCDR3。In some embodiments, the anti-PD-L1 antibody or antigen-binding fragment thereof comprises heavy chain HCDR1, HCDR2 and HCDR3 comprising amino acids according to SEQ ID NOs: 18, 12 and 20, respectively. The sequence has at least 80% homology, at least 85% homology, at least 90% homology, at least 91% homology, at least 92% homology, at least 93% homology, at least 94% homology. An amino acid sequence of at least 95% homology, at least 96% homology, at least 97% homology, at least 98% homology, or at least 99% homology; and light chain LCDR1, LCDR2, and LCDR3, The light chains LCDR1, LCDR2 and LCDR3 comprise at least 80% homology, at least 85% homology, at least 90% homology, at least 91% homology to the amino acid sequences according to SEQ ID NOs: 14, 15 and 16, respectively. , at least 92% homology, at least 93% homology, at least 94% homology, at least 95% homology, at least 96% homology, at least 97% homology, at least 98% homology Or an amino acid sequence of at least 99% homology. In another embodiment, the anti-PD-L1 antibody or antigen-binding fragment thereof comprises the heavy chain HCDR1, HCDR2 and HCDR3 according to SEQ ID NOs: 18, 12 and 20, respectively, and according to SEQ ID NOs: 14, 15 and 16, respectively. Light chain LCDR1, LCDR2 and LCDR3.
在一些方案中，所述抗PD-L1抗体或其抗原结合片段包含重链HCDR1、HCDR2和HCDR3，所述重链HCDR1、HCDR2和HCDR3包含分别与根据SEQ ID NOs:17、12和20的氨基酸序列具有至少80％同源性、至少85％同源性、至少90％同源性、至少91％同源性、至少92％同源性、至少93％同源性、至少94％同源性、至少95％同源性、至少96％同源性、至少97％同源性、至少98％同源性或至少99％同源性的氨基酸序列；以及轻链LCDR1、LCDR2和LCDR3，所述轻链LCDR1、LCDR2和LCDR3包含分别与根据SEQ ID NOs:14、15和16的氨基酸序列具有至少80％同源性、至少85％同源性、至少90％同源性、至少91％同源性、至少92％同源性、至少93％同源性、至少94％同源性、至少95％同源性、至少96％同源性、至少97％同源性、至少98％同源性或至少99％同源性的氨基酸序列。在另一个实施方案中，抗PD-L1抗体或其抗原结合片段包含分别根据SEQ ID NOs:17、12和20的重链HCDR1、HCDR2和HCDR3，以及分别根据SEQ ID NOs:14、15和16的轻链LCDR1、LCDR2和LCDR3。In some embodiments, the anti-PD-L1 antibody or antigen-binding fragment thereof comprises heavy chain HCDR1, HCDR2 and HCDR3 comprising amino acids according to SEQ ID NOs: 17, 12 and 20, respectively. The sequence has at least 80% homology, at least 85% homology, at least 90% homology, at least 91% homology, at least 92% homology, at least 93% homology, at least 94% homology. An amino acid sequence of at least 95% homology, at least 96% homology, at least 97% homology, at least 98% homology, or at least 99% homology; and light chain LCDR1, LCDR2, and LCDR3, The light chains LCDR1, LCDR2 and LCDR3 comprise at least 80% homology, at least 85% homology, at least 90% homology, at least 91% homology to the amino acid sequences according to SEQ ID NOs: 14, 15 and 16, respectively. , at least 92% homology, at least 93% homology, at least 94% homology, at least 95% homology, at least 96% homology, at least 97% homology, at least 98% homology Or an amino acid sequence of at least 99% homology. In another embodiment, the anti-PD-L1 antibody or antigen-binding fragment thereof comprises the heavy chain HCDR1, HCDR2 and HCDR3 according to SEQ ID NOs: 17, 12 and 20, respectively, and according to SEQ ID NOs: 14, 15 and 16, respectively. Light chain LCDR1, LCDR2 and LCDR3.
在一些方案中，所述抗PD-L1抗体或其抗原结合片段包含重链HCDR1、HCDR2和HCDR3，所述重链HCDR1、HCDR2和HCDR3包含分别与根据SEQ ID NOs:17、12和21的氨基酸序列具有至少80％同源性、至少85％同源性、至少90％同源性、至少91％同源性、至少92％同源性、至少93％同源性、至少94％同源性、至少95％同源性、至少96％同源性、至少97％同源性、至少98％同源性或至少99％同源性的氨基酸序列；以及轻链LCDR1、LCDR2和LCDR3，所述轻链LCDR1、LCDR2和LCDR3包含分别与根据SEQ ID NOs:14、15和16的氨基酸序列具有至少80％同源性、至少85％同源性、至少90％同源性、至少91％同源性、至少92％同源性、至少93％同源性、至少94％同源性、至少95％同源性、至少96％同源性、至少97％同源性、至少98％同源性或至少99％同源性的氨基酸序列。在另一个实施方案中，抗PD-L1抗体或其抗原结合片段包含分别根据SEQ ID NOs:17、12和21的重链HCDR1、HCDR2和HCDR3，以及分别根据SEQ ID NOs:14、15和16的轻链LCDR1、LCDR2和LCDR3。In some embodiments, the anti-PD-L1 antibody or antigen-binding fragment thereof comprises heavy chain HCDR1, HCDR2 and HCDR3 comprising amino acids according to SEQ ID NOs: 17, 12, and 21, respectively. The sequence has at least 80% homology, at least 85% homology, at least 90% homology, at least 91% homology, at least 92% homology, at least 93% homology, at least 94% homology. An amino acid sequence of at least 95% homology, at least 96% homology, at least 97% homology, at least 98% homology, or at least 99% homology; and light chain LCDR1, LCDR2, and LCDR3, The light chains LCDR1, LCDR2 and LCDR3 comprise at least 80% homology, at least 85% homology, at least 90% homology, at least 91% homology to the amino acid sequences according to SEQ ID NOs: 14, 15 and 16, respectively. , at least 92% homology, at least 93% homology, at least 94% homology, at least 95% homology, at least 96% homology, at least 97% homology, at least 98% homology Or an amino acid sequence of at least 99% homology. In another embodiment, the anti-PD-L1 antibody or antigen-binding fragment thereof comprises the heavy chain HCDR1, HCDR2 and HCDR3 according to SEQ ID NOs: 17, 12 and 21, respectively, and according to SEQ ID NOs: 14, 15 and 16, respectively. Light chain LCDR1, LCDR2 and LCDR3.
在一些方案中，所述抗PD-L1抗体或其抗原结合片段包含重链HCDR1、HCDR2和HCDR3，所述重链HCDR1、HCDR2和HCDR3包含分别与根据SEQ ID NOs:17、12和22的氨基酸序列具有至少80％同源性、至少85％同源性、至少90％同源性、至少91％同源性、至少92％同源性、至少93％同源性、至少94％同源性、至少95％同源性、至少96％同源性、至少97％同源性、至少98％同源性或至少99％同源性的氨基酸序列；以及轻链LCDR1、LCDR2和LCDR3，所述轻链LCDR1、LCDR2和LCDR3包含分别与根据SEQ ID NOs:14、15和16的氨基酸序列具有至少80％同源性、至少85％同源性、至少90％同源性、至少91％同源性、至少92％同源性、至少93％同源性、至少94％同源性、至少95％同源性、至少96％同源性、至少97％同源性、至少98％同源性或至少99％同源性的氨基酸序列。在另一个实施方案中，抗PD-L1抗体或其抗原结合片段包含分别根据SEQ ID NOs:17、12和22的重链HCDR1、HCDR2和HCDR3，以及分别根据SEQ ID NOs:14、15和16的轻链LCDR1、LCDR2和LCDR3。In some embodiments, the anti-PD-L1 antibody or antigen-binding fragment thereof comprises heavy chain HCDR1, HCDR2 and HCDR3 comprising amino acids according to SEQ ID NOs: 17, 12, and 22, respectively. The sequence has at least 80% homology, at least 85% homology, at least 90% homology, at least 91% homology, at least 92% homology, at least 93% homology, at least 94% homology. An amino acid sequence of at least 95% homology, at least 96% homology, at least 97% homology, at least 98% homology, or at least 99% homology; and light chain LCDR1, LCDR2, and LCDR3, The light chains LCDR1, LCDR2 and LCDR3 comprise at least 80% homology, at least 85% homology, at least 90% homology, at least 91% homology to the amino acid sequences according to SEQ ID NOs: 14, 15 and 16, respectively. , at least 92% homology, at least 93% homology, at least 94% homology, at least 95% homology, at least 96% homology, at least 97% homology, at least 98% homology Or an amino acid sequence of at least 99% homology. In another embodiment, the anti-PD-L1 antibody or antigen-binding fragment thereof comprises the heavy chain HCDR1, HCDR2 and HCDR3 according to SEQ ID NOs: 17, 12 and 22, respectively, and according to SEQ ID NOs: 14, 15 and 16, respectively. Light chain LCDR1, LCDR2 and LCDR3.
在一些方案中，所述抗PD-L1抗体或其抗原结合片段包含重链HCDR1、HCDR2和HCDR3，所述重链HCDR1、HCDR2和HCDR3包含分别与根据SEQ ID NOs:29、31和33的氨基酸序列具有至少80％同源性、至少85％同源性、至少90％同源性、至少91％同源性、至少92％同源性、至少93％同源性、至少94％同源性、至少95％同源性、至少96％同源性、至少97％同源性、至少98％同源性或至少99％同源性的氨基酸序列；以及轻链LCDR1、LCDR2和LCDR3，所述轻链LCDR1、LCDR2和LCDR3包含分别与根据SEQ ID NOs:35、27和37的氨基酸序列具有至少80％同源性、至少85％同源性、至少90％同源性、至少91％同源性、至少92％同源性、至少93％同源性、至少94％同源性、至少95％同源性、至少96％同源性、至少97％同源性、至少98％同源性或至少99％同源性的氨基酸序列。在另一个实施方案中，抗PD-L1抗体或其抗原结合片段包含分别根据SEQ ID NOs:29、31和33的重链HCDR1、HCDR2和HCDR3，以及分别根据SEQ ID NOs:35、27和37的轻链LCDR1、LCDR2和LCDR3。In some embodiments, the anti-PD-L1 antibody or antigen-binding fragment thereof comprises heavy chain HCDR1, HCDR2 and HCDR3 comprising amino acids according to SEQ ID NOs: 29, 31 and 33, respectively. The sequence has at least 80% homology, at least 85% homology, at least 90% homology, at least 91% homology, at least 92% homology, at least 93% homology, at least 94% homology. An amino acid sequence of at least 95% homology, at least 96% homology, at least 97% homology, at least 98% homology, or at least 99% homology; and light chain LCDR1, LCDR2, and LCDR3, The light chains LCDR1, LCDR2 and LCDR3 comprise at least 80% homology, at least 85% homology, at least 90% homology, at least 91% homology to the amino acid sequences according to SEQ ID NOs: 35, 27 and 37, respectively. , at least 92% homology, at least 93% homology, at least 94% homology, at least 95% homology, at least 96% homology, at least 97% homology, at least 98% homology Or an amino acid sequence of at least 99% homology. In another embodiment, the anti-PD-L1 antibody or antigen-binding fragment thereof comprises the heavy chain HCDR1, HCDR2 and HCDR3 according to SEQ ID NOs: 29, 31 and 33, respectively, and according to SEQ ID NOs: 35, 27 and 37, respectively. Light chain LCDR1, LCDR2 and LCDR3.
在一些方案中，所述抗PD-L1抗体或其抗原结合片段包含重链HCDR1、HCDR2和HCDR3，所述重 链HCDR1、HCDR2和HCDR3包含分别与根据SEQ ID NOs:30、32和34的氨基酸序列具有至少80％同源性、至少85％同源性、至少90％同源性、至少91％同源性、至少92％同源性、至少93％同源性、至少94％同源性、至少95％同源性、至少96％同源性、至少97％同源性、至少98％同源性或至少99％同源性的氨基酸序列；以及轻链LCDR1、LCDR2和LCDR3，所述轻链LCDR1、LCDR2和LCDR3包含分别与根据SEQ ID NOs:36、27和38的氨基酸序列具有至少80％同源性、至少85％同源性、至少90％同源性、至少91％同源性、至少92％同源性、至少93％同源性、至少94％同源性、至少95％同源性、至少96％同源性、至少97％同源性、至少98％同源性或至少99％同源性的氨基酸序列。在另一个实施方案中，抗PD-L1抗体或其抗原结合片段包含分别根据SEQ ID NOs:30、32和34的重链HCDR1、HCDR2和HCDR3，以及分别根据SEQ ID NOs:36、27和38的轻链LCDR1、LCDR2和LCDR3。In some embodiments, the anti-PD-L1 antibody or antigen-binding fragment thereof comprises heavy chain HCDR1, HCDR2 and HCDR3 comprising amino acids according to SEQ ID NOs: 30, 32 and 34, respectively. The sequence has at least 80% homology, at least 85% homology, at least 90% homology, at least 91% homology, at least 92% homology, at least 93% homology, at least 94% homology. An amino acid sequence of at least 95% homology, at least 96% homology, at least 97% homology, at least 98% homology, or at least 99% homology; and light chain LCDR1, LCDR2, and LCDR3, The light chains LCDR1, LCDR2 and LCDR3 comprise at least 80% homology, at least 85% homology, at least 90% homology, at least 91% homology to the amino acid sequences according to SEQ ID NOs: 36, 27 and 38, respectively. , at least 92% homology, at least 93% homology, at least 94% homology, at least 95% homology, at least 96% homology, at least 97% homology, at least 98% homology Or an amino acid sequence of at least 99% homology. In another embodiment, the anti-PD-L1 antibody or antigen-binding fragment thereof comprises the heavy chain HCDR1, HCDR2 and HCDR3 according to SEQ ID NOs: 30, 32 and 34, respectively, and according to SEQ ID NOs: 36, 27 and 38, respectively. Light chain LCDR1, LCDR2 and LCDR3.
在一些方案中，所述抗PD-L1抗体或其抗原结合片段包含重链可变区，所述重链可变区包含与选自由SEQs ID NO:5、7、9、45、47、48、49和50组成的组的氨基酸序列具有至少80％同源性、至少85％同源性、至少90％同源性、至少91％同源性、至少92％同源性、至少93％同源性、至少94％同源性、至少95％同源性、至少96％同源性、至少97％同源性、至少98％同源性或至少99％同源性的氨基酸序列；以及轻链可变区，所述轻链可变区包含与选自由SEQ ID NOs:6、8、10、46、51和52组成的组的氨基酸序列具有至少80％同源性、至少85％同源性、至少90％同源性、至少91％同源性、至少92％同源性、至少93％同源性、至少94％同源性、至少95％同源性、至少96％同源性、至少97％同源性、至少98％同源性或至少99％同源性的氨基酸序列。在另一些方案中，所述抗PD-L1抗体或其抗原结合片段包含重链可变区，所述重链可变区包含以下序列、基本上由以下序列组成或由以下序列组成：选自由SEQ ID NOs:5、7、9、45、47、48、49和50组成的组的氨基酸序列；以及轻链可变区，所述轻链可变区包含以下序列、基本上由以下序列组成或由以下序列组成：选自由SEQ ID NOs:6、8、10、46、51和52组成的组的氨基酸序列。In some embodiments, the anti-PD-L1 antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising and selected from SEQs ID NO: 5, 7, 9, 45, 47, 48 The amino acid sequences of the groups consisting of 49 and 50 have at least 80% homology, at least 85% homology, at least 90% homology, at least 91% homology, at least 92% homology, at least 93% identical Amino acid sequence of at least 94% homology, at least 95% homology, at least 96% homology, at least 97% homology, at least 98% homology or at least 99% homology; a chain variable region comprising at least 80% homology, at least 85% homology to an amino acid sequence selected from the group consisting of SEQ ID NOs: 6, 8, 10, 46, 51 and 52 , at least 90% homology, at least 91% homology, at least 92% homology, at least 93% homology, at least 94% homology, at least 95% homology, at least 96% homology An amino acid sequence of at least 97% homology, at least 98% homology, or at least 99% homology. In other embodiments, the anti-PD-L1 antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising, consisting essentially of, or consisting of: SEQ ID NOs: amino acid sequences of the group consisting of 5, 7, 9, 45, 47, 48, 49 and 50; and a light chain variable region comprising the following sequences consisting essentially of the following sequences Or consisting of the amino acid sequence selected from the group consisting of SEQ ID NOs: 6, 8, 10, 46, 51 and 52.
在一些实施方案中，本发明提供了抗PD-L1抗体或其抗原结合片段，其包含含有SEQ ID NO:5的氨基酸序列的重链可变区和含有SEQ ID NO:6的氨基酸序列的轻链可变区；含有SEQ ID NO:7的氨基酸序列的重链可变区和含有SEQ ID NO:8的氨基酸序列的轻链可变区；含有SEQ ID NO:9的氨基酸序列的重链可变区和含有SEQ ID NO:10的氨基酸序列的轻链可变区。In some embodiments, the invention provides an anti-PD-L1 antibody or antigen-binding fragment thereof comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 5 and a light comprising the amino acid sequence of SEQ ID NO: a chain variable region; a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 7 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 8; a heavy chain comprising the amino acid sequence of SEQ ID NO: 9 A variable region and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 10.
在一个实施方案中，本发明提供了人源化抗PD-L1抗体，其包含选自由hu-Chi1-2.3、hu-Chi1-3.3、hu-Chi1-3.4、hu-Chi1-3.5以及hu-Chi1-3.6组成的组的抗体的可变重链和选自由hu-Chi1-2.3、hu-Chi1-3.3、hu-Chi1-3.4、hu-Chi1-3.5以及hu-Chi1-3.6组成的组的抗体的可变轻链。在一些实施方案中，本发明提供了人源化抗PD-L1抗体，其中所述抗体包含重链可变区，其具有与选自由SEQ ID NOs:45、47、48、49和50组成的组的氨基酸序列具有至少80％同源性、至少85％同源性、至少90％同源性、至少91％同源性、至少92％同源性、至少93％同源性、至少94％同源性、至少95％同源性、至少96％同源性、至少97％同源性、至少98％同源性或至少99％同源性的氨基酸序列。在另一个实施方案中，本发明提供了人源化抗PD-L1抗体，其中所述抗体包含轻链可变区，其具有与选自由SEQ ID NOs:46、51和52组成的组的氨基酸序列具有至少80％同源性、至少85％同源性、至少90％同源性、至少91％同源性、至少92％同源性、至少93％同源性、至少94％同源性、至少95％同源性、至少96％同源性、至少97％同源性、至少98％同源性或至少99％同源性的氨基酸序列。In one embodiment, the invention provides a humanized anti-PD-L1 antibody comprising: selected from the group consisting of hu-Chi1-2.3, hu-Chi1-3.3, hu-Chi1-3.4, hu-Chi1-3.5, and hu-Chi1 -3.6 a variable heavy chain of the antibody of the group consisting of an antibody selected from the group consisting of hu-Chi1-2.3, hu-Chi1-3.3, hu-Chi1-3.4, hu-Chi1-3.5, and hu-Chi1-3.6 Variable light chain. In some embodiments, the invention provides a humanized anti-PD-L1 antibody, wherein the antibody comprises a heavy chain variable region having a composition selected from the group consisting of SEQ ID NOs: 45, 47, 48, 49, and 50 The amino acid sequence of the group has at least 80% homology, at least 85% homology, at least 90% homology, at least 91% homology, at least 92% homology, at least 93% homology, at least 94% Amino acid sequences of homology, at least 95% homology, at least 96% homology, at least 97% homology, at least 98% homology, or at least 99% homology. In another embodiment, the invention provides a humanized anti-PD-L1 antibody, wherein the antibody comprises a light chain variable region having an amino acid selected from the group consisting of SEQ ID NOs: 46, 51, and 52 The sequence has at least 80% homology, at least 85% homology, at least 90% homology, at least 91% homology, at least 92% homology, at least 93% homology, at least 94% homology. An amino acid sequence of at least 95% homology, at least 96% homology, at least 97% homology, at least 98% homology, or at least 99% homology.
在一些实施方案中，本发明提供了人源化抗PD-L1抗体，其中所述抗体包含重链可变区，其与SEQ ID NO:45具有至少80％同源性、至少85％同源性、至少90％同源性、至少91％同源性、至少92％同源性、至少93％同源性、至少94％同源性、至少95％同源性、至少96％同源性、至少97％同源性、至少98％同源性或至少99％同源性；以及轻链可变区，其与SEQ ID NO:46具有至少80％同源性、至少85％同源性、至少90％同源性、至少91％同源性、至少92％同源性、至少93％同源性、至少94％同源性、至少95％同源性、至少96％同源性、至少97％同源性、至少98％同源性或至少99％同源性。在另一些实施方案中，本发明提供了人源化抗PD-L1抗体，其中所述抗体包含重链可变区，其与SEQ ID NO:47具有至少80％同源性、至少85％同源性、至少90％同源性、至少91％同源性、至少92％同源性、至少93％同源性、至少94％同源性、至少95％同源性、至少96％同源性、至少97％同源性、至少98％同源性或至少99％同源性；以及轻链可变区，其与SEQ ID NO:51具有至少80％同源性、至少85％同源性、至少90％同源性、至少91％ 同源性、至少92％同源性、至少93％同源性、至少94％同源性、至少95％同源性、至少96％同源性、至少97％同源性、至少98％同源性或至少99％同源性。在另一些实施方案中，本发明提供了人源化抗PD-L1抗体，其中所述抗体包含重链可变区，其与SEQ ID NO:48具有至少80％同源性、至少85％同源性、至少90％同源性、至少91％同源性、至少92％同源性、至少93％同源性、至少94％同源性、至少95％同源性、至少96％同源性、至少97％同源性、至少98％同源性或至少99％同源性；以及轻链可变区，其与SEQ ID NO:52具有至少80％同源性、至少85％同源性、至少90％同源性、至少91％同源性、至少92％同源性、至少93％同源性、至少94％同源性、至少95％同源性、至少96％同源性、至少97％同源性、至少98％同源性或至少99％同源性。在另一些实施方案中，本发明提供了人源化抗PD-L1抗体，其中所述抗体包含重链可变区，其与SEQ ID NO:49具有至少80％同源性、至少85％同源性、至少90％同源性、至少91％同源性、至少92％同源性、至少93％同源性、至少94％同源性、至少95％同源性、至少96％同源性、至少97％同源性、至少98％同源性或至少99％同源性；以及轻链可变区，其与SEQ ID NO:51具有至少80％同源性、至少85％同源性、至少90％同源性、至少91％同源性、至少92％同源性、至少93％同源性、至少94％同源性、至少95％同源性、至少96％同源性、至少97％同源性、至少98％同源性或至少99％同源性。在一些实施方案中，本发明提供了人源化抗PD-L1抗体，其中所述抗体包含重链可变区，其与SEQ ID NO:50具有至少80％同源性、至少85％同源性、至少90％同源性、至少91％同源性、至少92％同源性、至少93％同源性、至少94％同源性、至少95％同源性、至少96％同源性、至少97％同源性、至少98％同源性或至少99％同源性；以及轻链可变区，其与SEQ ID NO:51具有至少80％同源性、至少85％同源性、至少90％同源性、至少91％同源性、至少92％同源性、至少93％同源性、至少94％同源性、至少95％同源性、至少96％同源性、至少97％同源性、至少98％同源性或至少99％同源性。In some embodiments, the invention provides a humanized anti-PD-L1 antibody, wherein the antibody comprises a heavy chain variable region that is at least 80% homologous to SEQ ID NO: 45, at least 85% homolog , at least 90% homology, at least 91% homology, at least 92% homology, at least 93% homology, at least 94% homology, at least 95% homology, at least 96% homology At least 97% homology, at least 98% homology, or at least 99% homology; and a light chain variable region having at least 80% homology to SEQ ID NO: 46, at least 85% homology At least 90% homology, at least 91% homology, at least 92% homology, at least 93% homology, at least 94% homology, at least 95% homology, at least 96% homology, At least 97% homology, at least 98% homology, or at least 99% homology. In other embodiments, the invention provides a humanized anti-PD-L1 antibody, wherein the antibody comprises a heavy chain variable region that is at least 80% homologous to SEQ ID NO: 47, at least 85% identical Source, at least 90% homology, at least 91% homology, at least 92% homology, at least 93% homology, at least 94% homology, at least 95% homology, at least 96% homology , at least 97% homology, at least 98% homology or at least 99% homology; and a light chain variable region having at least 80% homology, at least 85% homology to SEQ ID NO: 51 , at least 90% homology, at least 91% homology, at least 92% homology, at least 93% homology, at least 94% homology, at least 95% homology, at least 96% homology At least 97% homology, at least 98% homology or at least 99% homology. In other embodiments, the invention provides a humanized anti-PD-L1 antibody, wherein the antibody comprises a heavy chain variable region that is at least 80% homologous to SEQ ID NO: 48, at least 85% identical Source, at least 90% homology, at least 91% homology, at least 92% homology, at least 93% homology, at least 94% homology, at least 95% homology, at least 96% homology , at least 97% homology, at least 98% homology or at least 99% homology; and a light chain variable region having at least 80% homology, at least 85% homology to SEQ ID NO: 52 , at least 90% homology, at least 91% homology, at least 92% homology, at least 93% homology, at least 94% homology, at least 95% homology, at least 96% homology At least 97% homology, at least 98% homology or at least 99% homology. In other embodiments, the invention provides a humanized anti-PD-L1 antibody, wherein the antibody comprises a heavy chain variable region that is at least 80% homologous to SEQ ID NO: 49, at least 85% identical Source, at least 90% homology, at least 91% homology, at least 92% homology, at least 93% homology, at least 94% homology, at least 95% homology, at least 96% homology , at least 97% homology, at least 98% homology or at least 99% homology; and a light chain variable region having at least 80% homology, at least 85% homology to SEQ ID NO: 51 , at least 90% homology, at least 91% homology, at least 92% homology, at least 93% homology, at least 94% homology, at least 95% homology, at least 96% homology At least 97% homology, at least 98% homology or at least 99% homology. In some embodiments, the invention provides a humanized anti-PD-L1 antibody, wherein the antibody comprises a heavy chain variable region that is at least 80% homologous to SEQ ID NO: 50, at least 85% homolog , at least 90% homology, at least 91% homology, at least 92% homology, at least 93% homology, at least 94% homology, at least 95% homology, at least 96% homology At least 97% homology, at least 98% homology or at least 99% homology; and a light chain variable region having at least 80% homology to SEQ ID NO: 51, at least 85% homology At least 90% homology, at least 91% homology, at least 92% homology, at least 93% homology, at least 94% homology, at least 95% homology, at least 96% homology, At least 97% homology, at least 98% homology, or at least 99% homology.
在一些实施方案中，本发明提供了人源化抗PD-L1抗体，其包含含有SEQ ID NO:45的氨基酸序列的重链可变区和含有SEQ ID NO:46的氨基酸序列的轻链可变区；或含有SEQ ID NO:47的氨基酸序列的重链可变区和含有SEQ ID NO:51的氨基酸序列的轻链可变区；或含有SEQ ID NO:48的氨基酸序列的重链可变区和含有SEQ ID NO:52的氨基酸序列的轻链可变区；或含有SEQ ID NO:49的氨基酸序列的重链可变区和含有SEQ ID NO:51的氨基酸序列的轻链可变区；或或含有SEQ ID NO:50的氨基酸序列的重链可变区和含有SEQ ID NO:51的氨基酸序列的轻链可变区。In some embodiments, the invention provides a humanized anti-PD-L1 antibody comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 45 and a light chain comprising the amino acid sequence of SEQ ID NO: 46 a variable region; or a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 47 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 51; or a heavy chain comprising the amino acid sequence of SEQ ID NO: 48 a variable region and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 52; or a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 49 and a light chain variable comprising the amino acid sequence of SEQ ID NO: 51 a region; or a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 50 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 51.
在一些实施方案中，本发明提供了嵌合抗PD-L1抗体，其中所述抗体包含重链，所述重链具有与选自由SEQ ID NO:39、41和43组成的组的氨基酸序列具有至少80％同源性、至少85％同源性、至少90％同源性、至少91％同源性、至少92％同源性、至少93％同源性、至少94％同源性、至少95％同源性、至少96％同源性、至少97％同源性、至少98％同源性或至少99％同源性的氨基酸序列；以及轻链，所述轻链具有与选自由SEQ ID NO:40、42和44组成的组的氨基酸序列具有至少80％同源性、至少85％同源性、至少90％同源性、至少91％同源性、至少92％同源性、至少93％同源性、至少94％同源性、至少95％同源性、至少96％同源性、至少97％同源性、至少98％同源性或至少99％同源性的氨基酸序列。In some embodiments, the invention provides a chimeric anti-PD-L1 antibody, wherein the antibody comprises a heavy chain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 39, 41, and 43 At least 80% homology, at least 85% homology, at least 90% homology, at least 91% homology, at least 92% homology, at least 93% homology, at least 94% homology, at least An amino acid sequence of 95% homology, at least 96% homology, at least 97% homology, at least 98% homology, or at least 99% homology; and a light chain having and selected from the group consisting of SEQ ID NO: The amino acid sequence of the group consisting of 40, 42 and 44 has at least 80% homology, at least 85% homology, at least 90% homology, at least 91% homology, at least 92% homology, Amino acids having at least 93% homology, at least 94% homology, at least 95% homology, at least 96% homology, at least 97% homology, at least 98% homology, or at least 99% homology sequence.
在一些实施方案中，本发明提供了人源化抗PD-L1抗体，其中所述抗体包含完整的重链，其具有与选自由SEQ ID NO:53、57、61和62组成的组的氨基酸序列具有至少80％同源性、至少85％同源性、至少90％同源性、至少91％同源性、至少92％同源性、至少93％同源性、至少94％同源性、至少95％同源性、至少96％同源性、至少97％同源性、至少98％同源性或至少99％同源性的氨基酸序列。在另一个实施方案中，本发明提供了人源化抗PD-L1抗体，其中所述抗体包含完整的轻链，其具有与选自由SEQ ID NO:55和59组成的组的氨基酸序列具有至少80％同源性、至少85％同源性、至少90％同源性、至少91％同源性、至少92％同源性、至少93％同源性、至少94％同源性、至少95％同源性、至少96％同源性、至少97％同源性、至少98％同源性或至少99％同源性的氨基酸序列。In some embodiments, the invention provides a humanized anti-PD-L1 antibody, wherein the antibody comprises an entire heavy chain having an amino acid selected from the group consisting of SEQ ID NO: 53, 57, 61, and 62 The sequence has at least 80% homology, at least 85% homology, at least 90% homology, at least 91% homology, at least 92% homology, at least 93% homology, at least 94% homology. An amino acid sequence of at least 95% homology, at least 96% homology, at least 97% homology, at least 98% homology, or at least 99% homology. In another embodiment, the invention provides a humanized anti-PD-L1 antibody, wherein the antibody comprises an intact light chain having at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 55 and 59 80% homology, at least 85% homology, at least 90% homology, at least 91% homology, at least 92% homology, at least 93% homology, at least 94% homology, at least 95 Amino acid sequence of % homology, at least 96% homology, at least 97% homology, at least 98% homology or at least 99% homology.
在一个实施方案中，本发明提供了人源化抗PD-L1抗体，其中所述抗体包含根据SEQ ID NO:53的重链和根据SEQ ID NO:55的轻链。在另一个实施方案中，本发明提供了人源化抗PD-L1抗体，其中所述抗体包含根据SEQ ID NO:57的重链和根据SEQ ID NO:59的轻链。在另一个实施方案中，本发明提供了人源化抗PD-L1抗体，其中所述抗体包含根据SEQ ID NO:61的重链和根据SEQ ID NO:55的轻链。在另一个实施方案中，本发明提供了人源化抗PD-L1抗体，其中所述抗体包含根据SEQ ID NO:62的重链和根据 SEQ ID NO:55的轻链。In one embodiment, the invention provides a humanized anti-PD-L1 antibody, wherein the antibody comprises a heavy chain according to SEQ ID NO: 53 and a light chain according to SEQ ID NO: 55. In another embodiment, the invention provides a humanized anti-PD-L1 antibody, wherein the antibody comprises a heavy chain according to SEQ ID NO: 57 and a light chain according to SEQ ID NO: 59. In another embodiment, the invention provides a humanized anti-PD-L1 antibody, wherein the antibody comprises a heavy chain according to SEQ ID NO: 61 and a light chain according to SEQ ID NO: 55. In another embodiment, the invention provides a humanized anti-PD-L1 antibody, wherein the antibody comprises a heavy chain according to SEQ ID NO: 62 and a light chain according to SEQ ID NO: 55.
在一些实施方案中，本发明提供了人源化抗PD-L1抗体或其片段，优选地，所述人源化抗体为人源化抗体hu-Chi1，所述人源化抗体重链可变区上的重链FR区序列来源于人种系重链，具体来源于人种系重链IGHV4-30-4*01和IGHJ1*01的组合序列。在一些实施方案中，上述人源化抗体hu-Chi1的重链可变区序列有0-10个氨基酸的回复突变，优选为一个或多个选自S35N、Q39R、K43N、G44K、W47Y、I48M、V71R的氨基酸回复突变；更优选一个或多个选自S35N、W47Y和V71R的氨基酸回复突变；最优选为包含S35N、W47Y、V71R的回复突变。在一些实施方案中，所述人源化抗体hu-Chi1重链可变区还包含C102Y的氨基酸突变。在一些实施方案中，所述人源化抗体hu-Chi1轻链可变区上的轻链FR区序列来源于人种系轻链，具体来源于人种系轻链IGKV1-39*01和IGKJ2*02的组合序列。在一些实施方案中，人源化抗体hu-Chi1的轻链可变区序列有0-10个氨基酸的回复突变，优选为一个或多个选自I2F、P8T、G41D、P44V、Y87F和Q100R的氨基酸回复突变；更优选为一个或多个选自I2F、G41D和P44V的氨基酸回复突变；最优选为包含I2F、G41D和P44V的氨基酸回复突变。In some embodiments, the invention provides a humanized anti-PD-L1 antibody or fragment thereof, preferably, the humanized antibody is a humanized antibody hu-Chi1, said humanized antibody heavy chain variable region The heavy chain FR region sequence is derived from the human germline heavy chain, specifically from the combined sequence of the human germline heavy chain IGHV4-30-4*01 and IGHJ1*01. In some embodiments, the heavy chain variable region sequence of the humanized antibody hu-Chi1 described above has a back mutation of 0-10 amino acids, preferably one or more selected from the group consisting of S35N, Q39R, K43N, G44K, W47Y, I48M Amino acid back mutation of V71R; more preferably one or more amino acid back mutations selected from the group consisting of S35N, W47Y and V71R; most preferably a back mutation comprising S35N, W47Y, V71R. In some embodiments, the humanized antibody hu-Chi1 heavy chain variable region further comprises an amino acid mutation of C102Y. In some embodiments, the light chain FR region sequence of the humanized antibody hu-Chi1 light chain variable region is derived from human germline light chain, specifically derived from human germline light chain IGKV1-39*01 and IGKJ2 A combined sequence of *02. In some embodiments, the light chain variable region sequence of the humanized antibody hu-Chi1 has a 0-10 amino acid back mutation, preferably one or more selected from the group consisting of I2F, P8T, G41D, P44V, Y87F, and Q100R Amino acid back mutation; more preferably one or more amino acid back mutations selected from the group consisting of I2F, G41D and P44V; most preferably amino acid back mutations comprising I2F, G41D and P44V.
在一个实施方案中，本发明提供了与本文提供的任何示例性抗体结合PD-L1上的相同表位的抗PD-L1抗体或其片段。在一个实施方案中，抗体或其片段与本文提供的用于结合PD-L1的任何示例性抗体竞争。可以通过ELISA、流式细胞术、表面等离子体共振(SPR)测定或本领域已知的任何其它方法测量与PD-L1的结合。在本发明的一些实施方案中，所述PD-L1抗体或其片段能结合人PD-L1蛋白，并结合食蟹猴(cynomolgous)PD-L1蛋白。In one embodiment, the invention provides an anti-PD-Ll antibody or fragment thereof that binds to the same epitope on PD-L1 as any of the exemplary antibodies provided herein. In one embodiment, the antibody or fragment thereof competes with any of the exemplary antibodies provided herein for binding to PD-L1. Binding to PD-L1 can be measured by ELISA, flow cytometry, surface plasmon resonance (SPR) assay, or any other method known in the art. In some embodiments of the invention, the PD-L1 antibody or fragment thereof is capable of binding to a human PD-L1 protein and binding to a cynomolgous PD-L1 protein.
在一些实施方案中，本发明提供以约15nM至约0.05nM的结合亲和力K D与PD-L1结合的抗PD-L1抗体和其片段。在另一个实施方案中，本文提供的抗PD-L1抗体和其片段以约10nM至约0.1nM的结合亲和力K D与PD-L1结合。在另一个实施方案中，本文提供的抗PD-L1抗体和其片段以约8nM至约0.2nM的结合亲和力K D与PD-L1结合。在另一个实施方案中，本文提供的抗PD-L1抗体和其片段以约6nM至约0.3nM的结合亲和力K D与PD-L1结合。在另一个实施方案中，本文提供的抗PD-L1抗体和其片段以约15nM或更小的结合亲和力K D与PD-L1结合。在另一个实施方案中，本文提供的抗PD-L1抗体和其片段以约10nM或更小的结合亲和力K D与PD-L1结合。在另一个实施方案中，本文提供的抗PD-L1抗体和其片段以约6nM或更小的结合亲和力K D与PD-L1结合。在另一个实施方案中，本文提供的抗PD-L1抗体和其片段以约4nM或更小的结合亲和力K D与PD-L1结合。在另一个实施方案中，本文提供的抗PD-L1抗体和其片段以约1nM或更小的结合亲和力K D与PD-L1结合。在另一个实施方案中，本文提供的抗PD-L1抗体和其片段以约0.70nM或更小的结合亲和力K D与PD-L1结合。在另一个实施方案中，本文提供的抗PD-L1抗体和其片段以约0.50nM或更小的结合亲和力K D与PD-L1结合。在另一个实施方案中，本文提供的抗PD-L1抗体和其片段以约0.30nM或更小的结合亲和力K D与PD-L1结合。在另一个实施方案中，本文提供的抗PD-L1抗体和其片段以约0.10nM或更小的结合亲和力K D与PD-L1结合。在另一个实施方案中，本文提供的抗PD-L1抗体和其片段以约0.05nM或更小的结合亲和力K D与PD-L1结合。在一些实施方案中，通过Biacore测定来测量本文提供的抗PD-L1抗体和片段的结合亲和力K D。 In some embodiments, the present invention provides a binding affinity of from about 15nM to about 0.05nM K D of binding with the anti-PD-L1 antibody, and PD-L1 fragments thereof. In another embodiment, provided herein is an anti-PD-L1 antibodies and fragments thereof binding to a binding affinity of from about 0.1nM to about 10nM K D and PD-L1. In another embodiment, provided herein is an anti-PD-L1 antibodies and fragments thereof binding to a binding affinity of from about 0.2nM to about 8nM K D and PD-L1. In another embodiment, provided herein is an anti-PD-L1 antibodies and fragments thereof binding to a binding affinity of from about 0.3nM to about 6nM K D and PD-L1. In another embodiment, provided herein is an anti-PD-L1 antibodies and fragments of about 15nM or less binding affinity K D of binding to PD-L1. In another embodiment, provided herein is an anti-PD-L1 antibodies and fragments of about 10nM or less binding affinity K D of binding to PD-L1. In another embodiment, provided herein is an anti-PD-L1 antibodies and fragments of about or less 6nM binding affinity K D of binding to PD-L1. In another embodiment, provided herein is an anti-PD-L1 antibodies and fragments of about 4nM or less binding affinity K D of binding to PD-L1. In another embodiment, provided herein is an anti-PD-L1 antibodies and fragments of about 1nM or less binding affinity K D of binding to PD-L1. In another embodiment, provided herein is an anti-PD-L1 antibodies and fragments of about 0.70nM or less binding affinity K D of binding to PD-L1. In another embodiment, provided herein is an anti-PD-L1 antibodies and fragments of about 0.50nM or less binding affinity K D of binding to PD-L1. In another embodiment, provided herein is an anti-PD-L1 antibodies and fragments of about 0.30nM or less binding affinity K D of binding to PD-L1. In another embodiment, provided herein is an anti-PD-L1 antibodies and fragments of about 0.10nM or less binding affinity K D of binding to PD-L1. In another embodiment, provided herein is an anti-PD-L1 antibodies and fragments of about 0.05nM or less binding affinity K D of binding to PD-L1. In some embodiments, to measure the binding of anti-PD-L1 antibodies and fragments provided herein determined by Biacore affinity K D.
在一些实施方案中，本文提供的抗PD-L1抗体和其片段抑制PD-L1/PD-1的结合，其中IC50为约1ng/mL至约1500ng/mL。在另一个实施方案中，本文提供的抗PD-L1抗体和其片段抑制PD-L1/PD-1的结合，其中IC50为约10ng/mL至约1200ng/mL。在另一个实施方案中，本文提供的抗PD-L1抗体和其片段抑制PD-L1/PD-1的结合，其中IC50为约20ng/mL至约800ng/mL。在另一个实施方案中，本文提供的抗PD-L1抗体和其片段抑制PD-L1/PD-1的结合，其中IC50为约50ng/mL至约500ng/mL。在另一个实施方案中，本文提供的抗PD-L1抗体和其片段抑制PD-L1/PD-1的结合，其中IC50为约50ng/mL至约200ng/mL。在一些实施方案中，本文提供的抗PD-L1抗体和其片段抑制PD-L1/PD-1的结合，其中IC50为约1200ng/mL或更小、约1000ng/mL或更小、约800ng/mL或更小、约400ng/mL或更小、约300ng/mL或更小、约250ng/mL或更小、约200ng/mL或更小、约150ng/mL或更小、约100ng/mL或更小、约75ng/mL或更小、约60ng/mL或更小、约50ng/mL或更小、约40ng/mL或更小、约30ng/mL或更小、约20ng/mL或更小、或约10ng/mL或更小。在一些实施方案中，通过ELISA来测量本文提供的抗PD-L1抗体和片段的IC50。In some embodiments, the anti-PD-L1 antibodies and fragments thereof provided herein inhibit the binding of PD-L1/PD-1, wherein the IC50 is from about 1 ng/mL to about 1500 ng/mL. In another embodiment, an anti-PD-Ll antibody and fragment thereof provided herein inhibits binding of PD-L1/PD-1, wherein the IC50 is from about 10 ng/mL to about 1200 ng/mL. In another embodiment, an anti-PD-Ll antibody and fragment thereof provided herein inhibits binding of PD-L1/PD-1, wherein the IC50 is from about 20 ng/mL to about 800 ng/mL. In another embodiment, an anti-PD-Ll antibody and fragment thereof provided herein inhibits binding of PD-L1/PD-1, wherein the IC50 is from about 50 ng/mL to about 500 ng/mL. In another embodiment, an anti-PD-Ll antibody and fragment thereof provided herein inhibits binding of PD-L1/PD-1, wherein the IC50 is from about 50 ng/mL to about 200 ng/mL. In some embodiments, the anti-PD-L1 antibodies and fragments thereof provided herein inhibit binding of PD-L1/PD-1, wherein the IC50 is about 1200 ng/mL or less, about 1000 ng/mL or less, about 800 ng/ mL or less, about 400 ng/mL or less, about 300 ng/mL or less, about 250 ng/mL or less, about 200 ng/mL or less, about 150 ng/mL or less, about 100 ng/mL or Smaller, about 75 ng/mL or less, about 60 ng/mL or less, about 50 ng/mL or less, about 40 ng/mL or less, about 30 ng/mL or less, about 20 ng/mL or less. Or about 10 ng/mL or less. In some embodiments, the IC50 of the anti-PD-L1 antibodies and fragments provided herein are measured by ELISA.
在另一些实施方案中，本文提供了抗PD-L1抗体和其片段抑制在表面上分别表达PD-L1和PD-1蛋白 的细胞之间的相互作用。在另一个实施方案中，本文提供的抗PD-L1抗体和其片段抑制在表面上分别表达PD-L1和PD-1蛋白的细胞之间的相互作用，其中IC50为约1ng/mL至约500ng/mL。在另一个实施方案中，本文提供的抗PD-L1抗体和其片段抑制在表面上分别表达PD-L1和PD-1蛋白的细胞之间的相互作用，其中IC50为约10ng/mL至约400g/mL。在另一个实施方案中，本文提供的抗PD-L1抗体和其片段抑制在表面上分别表达PD-L1和PD-1蛋白的细胞之间的相互作用，其中IC50为约20ng/mL至约300ng/mL。在另一个实施方案中，本文提供的抗PD-L1抗体和其片段抑制在表面上分别表达PD-L1和PD-1蛋白的细胞之间的相互作用，其中IC50为约50ng/mL至约200ng/mL。在一些实施方案中，本文提供的抗PD-L1抗体和其片段抑制在表面上分别表达PD-L1和PD-1蛋白的细胞之间的相互作用，其中IC50为约500ng/mL或更小、约450ng/mL或更小、约400ng/mL或更小、约350ng/mL或更小、约300ng/mL或更小、约250ng/mL或更小、约200ng/mL或更小、约150ng/mL或更小、约100ng/mL或更小、或约50ng/mL或更小。在一些实施方案中，通过FACS来测量本文提供的抗PD-L1抗体和其片段的IC50。In other embodiments, provided herein are anti-PD-L1 antibodies and fragments thereof that inhibit interaction between cells expressing PD-L1 and PD-1 proteins, respectively, on the surface. In another embodiment, the anti-PD-L1 antibodies and fragments thereof provided herein inhibit interactions between cells expressing PD-L1 and PD-1 proteins, respectively, wherein the IC50 is from about 1 ng/mL to about 500 ng. /mL. In another embodiment, the anti-PD-L1 antibodies and fragments thereof provided herein inhibit interactions between cells expressing PD-L1 and PD-1 proteins, respectively, wherein the IC50 is from about 10 ng/mL to about 400 g. /mL. In another embodiment, the anti-PD-L1 antibodies and fragments thereof provided herein inhibit interactions between cells expressing PD-L1 and PD-1 proteins, respectively, wherein the IC50 is from about 20 ng/mL to about 300 ng. /mL. In another embodiment, the anti-PD-L1 antibodies and fragments thereof provided herein inhibit interactions between cells expressing PD-L1 and PD-1 proteins, respectively, wherein the IC50 is from about 50 ng/mL to about 200 ng. /mL. In some embodiments, the anti-PD-L1 antibodies and fragments thereof provided herein inhibit interactions between cells expressing PD-L1 and PD-1 proteins, respectively, wherein the IC50 is about 500 ng/mL or less, About 450 ng/mL or less, about 400 ng/mL or less, about 350 ng/mL or less, about 300 ng/mL or less, about 250 ng/mL or less, about 200 ng/mL or less, about 150 ng. /mL or less, about 100 ng/mL or less, or about 50 ng/mL or less. In some embodiments, the IC50 of the anti-PD-L1 antibodies and fragments thereof provided herein are measured by FACS.
在一个实施方案中，本文提供的抗PD-L1抗体是嵌合抗体，其具有根据SEQ ID NO:5的氨基酸序列的重链可变区和根据SEQ ID NO:6的氨基酸序列的轻链可变区；或具有根据SEQ ID NO：7的氨基酸序列的重链可变区和根据SEQ ID NO:8的氨基酸序列的轻链可变区；或具有根据SEQ ID NO：9的氨基酸序列的重链可变区和根据SEQ ID NO:10的氨基酸序列的轻链可变区；其中所述抗PD-L1抗体具有如通过ELISA或FACS所测量的下述PD-L1结合EC50：约200ng/mL或更小、或约150ng/mL或更小、或约100ng/mL或更小、或约80ng/mL或更小、或约60ng/mL或更小、或约50ng/mL或更小。In one embodiment, an anti-PD-L1 antibody provided herein is a chimeric antibody having a heavy chain variable region according to the amino acid sequence of SEQ ID NO: 5 and a light chain according to the amino acid sequence of SEQ ID NO: a variable region; or a heavy chain variable region having an amino acid sequence according to SEQ ID NO: 7 and a light chain variable region according to the amino acid sequence of SEQ ID NO: 8; or having a weight according to the amino acid sequence of SEQ ID NO: a chain variable region and a light chain variable region according to the amino acid sequence of SEQ ID NO: 10; wherein the anti-PD-L1 antibody has the following PD-L1 binding EC50 as measured by ELISA or FACS: about 200 ng/mL Or smaller, or about 150 ng/mL or less, or about 100 ng/mL or less, or about 80 ng/mL or less, or about 60 ng/mL or less, or about 50 ng/mL or less.
在一些实施方案中，本发明所述抗PD-L1抗体或其抗原结合片段为嵌合抗体或抗体片段。根据本发明提供的抗PD-L1嵌合抗体或其片段，其进一步包含人源IgG1、IgG2、IgG3或IgG4或其变体的重链恒定区。所述人源化抗体轻链进一步包含人源κ、λ链或其变体的恒定区。因此，在另一些实施方案中，所述抗PD-L1抗体或其抗原结合片段为人源化抗体或抗体片段。In some embodiments, the anti-PD-L1 antibody or antigen-binding fragment thereof of the invention is a chimeric antibody or antibody fragment. An anti-PD-L1 chimeric antibody or fragment thereof according to the invention further comprising a heavy chain constant region of human IgG1, IgG2, IgG3 or IgG4 or variants thereof. The humanized antibody light chain further comprises a constant region of a human kappa, lambda chain or variant thereof. Accordingly, in other embodiments, the anti-PD-L1 antibody or antigen-binding fragment thereof is a humanized antibody or antibody fragment.
在一个实施方案中，抗PD-L1抗体或其片段是单克隆抗体、scFv、Fab片段、Fv片段、F(ab)'片段、F(ab')2片段、双特异性抗体、免疫缀合物或其组合。In one embodiment, the anti-PD-L1 antibody or fragment thereof is a monoclonal antibody, scFv, Fab fragment, Fv fragment, F(ab)' fragment, F(ab')2 fragment, bispecific antibody, immunoconjugate Or a combination thereof.
在一方面，提供了分离的抗PD-L1抗体或其片段，其中所述抗体由杂交瘤产生，所述杂交瘤选自由本文称为Chi1、Chi2-1以及Chi2-2的杂交瘤组成的组。因此，本发明还包括杂交瘤Chi1、Chi2-1以及Chi2-2，以及产生本文公开的抗体的任何杂交瘤。本发明还提供了编码本文提供的抗体和其片段的分离的多核苷酸，例如编码人源化抗体hu-Chi1-3.4重链的核苷酸序列如SEQ ID NO:54所示，编码轻链的核苷酸序列如SEQ ID NO:56所示；编码人源化抗体hu-Chi1-2.3重链的核苷酸序列如SEQ ID NO:58所示，编码轻链的核苷酸序列如SEQ ID NO:60所示。在另一方面，本发明提供了包含分离的多核苷酸的表达载体，例如pMD-19T载体以及pcDNA3.1载体等。在一个具体实施例中，本发明提供了包含编码人源化抗PD-L1抗体的核苷酸序列的表达载体，例如编码hu-Chi1-2.3重链和轻链核苷酸序列的表达载体。本发明还提供了包含所述表达载体的宿主细胞，所述宿主细胞可以是真核细胞或原核细胞，例如所述宿主细胞包括但不限于哺乳动物细胞、昆虫细胞、酵母细胞和细菌等，可优选选自中国仓鼠卵巢(CHO)细胞、ExpiCHO细胞、CHO-S细胞以及CHO-DG44细胞、毕赤酵母和大肠杆菌等。在一个实施方案中，本发明提供了生产所述人源化抗PD-L1抗体或其片段的方法，所述方法包括对包含所述表达载体的宿主细胞进行培养。In one aspect, an isolated anti-PD-L1 antibody or fragment thereof is provided, wherein the antibody is produced by a hybridoma selected from the group consisting of hybridomas referred to herein as Chi1, Chi2-1, and Chi2-2 . Accordingly, the invention also encompasses hybridomas Chi1, Chi2-1, and Chi2-2, as well as any hybridomas that produce the antibodies disclosed herein. The invention also provides an isolated polynucleotide encoding an antibody and fragment thereof provided herein, eg, a nucleotide sequence encoding a humanized antibody hu-Chi1-3.4 heavy chain, as set forth in SEQ ID NO: 54, encoding a light chain The nucleotide sequence is set forth in SEQ ID NO: 56; the nucleotide sequence encoding the humanized antibody hu-Chi1-2.3 heavy chain is set forth in SEQ ID NO: 58, and the nucleotide sequence encoding the light chain is SEQ. ID NO: 60 is shown. In another aspect, the invention provides an expression vector comprising an isolated polynucleotide, such as a pMD-19T vector, a pcDNA3.1 vector, and the like. In a specific embodiment, the invention provides an expression vector comprising a nucleotide sequence encoding a humanized anti-PD-L1 antibody, eg, an expression vector encoding a hu-Chi1-2.3 heavy and light chain nucleotide sequence. The invention also provides a host cell comprising the expression vector, which may be a eukaryotic cell or a prokaryotic cell, for example, the host cell includes, but is not limited to, a mammalian cell, an insect cell, a yeast cell, a bacterium, etc. Preferably, it is selected from the group consisting of Chinese hamster ovary (CHO) cells, ExpiCHO cells, CHO-S cells, and CHO-DG44 cells, Pichia pastoris, Escherichia coli, and the like. In one embodiment, the invention provides a method of producing the humanized anti-PD-L1 antibody or fragment thereof, the method comprising culturing a host cell comprising the expression vector.
在一个实施方案中，本发明提供了抗PD-L1抗体免疫缀合物。因此，本发明提供了结合PD-L1并与治疗剂连接或缀合的抗体或其片段。可以与抗PD-L1抗体连接或缀合的治疗剂可以包括但不限于细胞毒性药物、放射性同位素、信号通路抑制剂、免疫调节剂或抗体。在一个实施方案中，本文提供的抗体或其片段是免疫缀合物，所述免疫缀合物包含抗PD-L1抗体或其片段，并且还包含选自包括另外的治疗剂、细胞毒性剂、免疫粘附分子和显像剂的组中的试剂。在一些实施方案中，显像剂选自由放射性标记、酶、荧光标记、发光标记、生物发光标记、磁性标记和生物素组成的组。在一些实施方案中，显像剂是选自由以下组成的组中的放射性标记： 3H、 14C、 35S、 62Cu、 64Cu、 89Zr、 90Y、 99Tc、 111In、 125I、 131I、 177Lu、 166Ho和 153Sm。在一些实施方案中，治疗剂或细胞毒性剂选自包括以下的组：化学治疗剂、免疫抑制剂、免疫刺激剂、抗代谢物、烷化剂、抗生素、生长因子、细胞因子、抗血管生成剂、抗有丝分裂剂、蒽环霉素、毒素和细胞凋亡剂。在一些实施方案中，抗原结合蛋白直接与试剂缀合。在其它实施方案中，抗原结合蛋白通 过接头与试剂缀合。合适的接头包括但不限于文献报道的氨基酸和多肽接头。接头可以是可裂解的或不可裂解的。 In one embodiment, the invention provides an anti-PD-L1 antibody immunoconjugate. Accordingly, the invention provides an antibody or fragment thereof that binds to PD-L1 and is linked or conjugated to a therapeutic agent. Therapeutic agents that can be linked or conjugated to an anti-PD-Ll antibody can include, but are not limited to, cytotoxic drugs, radioisotopes, signaling pathway inhibitors, immunomodulators, or antibodies. In one embodiment, an antibody or fragment thereof provided herein is an immunoconjugate comprising an anti-PD-L1 antibody or fragment thereof, and further comprising an agent selected from the group consisting of additional therapeutic agents, cytotoxic agents, An agent in the group of immunoadhesion molecules and imaging agents. In some embodiments, the imaging agent is selected from the group consisting of a radioactive label, an enzyme, a fluorescent label, a luminescent label, a bioluminescent label, a magnetic label, and biotin. In some embodiments, the imaging agent is a radioactive label selected from the group consisting of: 3 H, 14 C, 35 S, 62 Cu, 64 Cu, 89 Zr, 90 Y, 99 Tc, 111 In, 125 I , 131 I, 177 Lu, 166 Ho and 153 Sm. In some embodiments, the therapeutic agent or cytotoxic agent is selected from the group consisting of a chemotherapeutic agent, an immunosuppressive agent, an immunostimulant, an antimetabolite, an alkylating agent, an antibiotic, a growth factor, a cytokine, an anti-angiogenesis Agents, anti-mitotic agents, anthracyclines, toxins and apoptotic agents. In some embodiments, the antigen binding protein is directly conjugated to an agent. In other embodiments, the antigen binding protein is conjugated to the reagent via a linker. Suitable linkers include, but are not limited to, the amino acid and polypeptide linkers reported in the literature. The linker can be cleavable or non-cleavable.
在一个实施方案中，本发明提供对PD-L1和至少一种其它抗原或表位具有特异性的双特异性或多特异性抗体。可以使用本文提供的结合测定或本领域已知的任何其它结合测定来测试本文提供的抗PD-L1抗体和其片段与PD-L1的结合。In one embodiment, the invention provides a bispecific or multispecific antibody specific for PD-L1 and at least one other antigen or epitope. Binding of the anti-PD-L1 antibodies and fragments thereof provided herein to PD-L1 can be tested using the binding assays provided herein or any other binding assay known in the art.
在一方面，本发明提供了包含本文提供的一种或多种抗PD-L1抗体或其片段和药学上可接受的载体的组合物。在一些实施方案中，本发明的药物组合物还包括药学上可接受的稳定剂、缓冲剂或赋形剂。In one aspect, the invention provides a composition comprising one or more of the anti-PD-L1 antibodies or fragments thereof provided herein and a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical compositions of the present invention further comprise a pharmaceutically acceptable stabilizer, buffer or excipient.
在一方面，本发明提供了用于在受试者中调节免疫应答的方法，所述方法包括向受试者施用治疗有效量的本文提供的抗PD-L1抗体或其片段。在一个实施方案中，本发明提供了用于增加T细胞活化的方法，所述方法包括使T细胞与本文提供的抗PD-L1抗体或其片段接触。在一个实施方案中，本发明提供了用于在有需要的受试者中治疗或预防疾病或病症的方法，所述方法包括向受试者施用治疗有效量的本文提供的抗PD-L1抗体或其片段。在一个实施方案中，本发明提供了用于在有需要的受试者中增强抗肿瘤应答的方法，所述方法包括向受试者施用治疗有效量的本发明的抗PD-L1抗体或其片段。在另一个实施方案中，本发明提供了用于在有需要的受试者中降低肿瘤免疫逃逸、减少肿瘤或抑制肿瘤细胞的生长的方法，所述方法包括向受试者施用治疗有效量的本发明的抗PD-L1抗体或其片段。在另一个实施方案中，本发明提供了用于在有需要的受试者中治疗癌症的方法，所述方法包括向受试者施用治疗有效量的本发明的抗PD-L1抗体或其片段。在另一个实施方案中，所述癌症选自由以下组成的组：淋巴瘤、白血病、黑色素瘤、神经胶质瘤、乳腺癌、肺癌、肠癌、骨癌、卵巢癌、膀胱癌、肾癌、肝癌、胃癌(stomach cancer)、睾丸癌、唾液腺癌、甲状腺癌、胸腺癌、上皮癌、头或颈癌、胃癌(gastric cancer)、胰腺癌或其组合。In one aspect, the invention provides a method for modulating an immune response in a subject, the method comprising administering to the subject a therapeutically effective amount of an anti-PD-L1 antibody or fragment thereof provided herein. In one embodiment, the invention provides a method for increasing T cell activation, the method comprising contacting a T cell with an anti-PD-Ll antibody or fragment thereof provided herein. In one embodiment, the invention provides a method for treating or preventing a disease or condition in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of an anti-PD-L1 antibody provided herein Or a fragment thereof. In one embodiment, the invention provides a method for enhancing an anti-tumor response in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of an anti-PD-L1 antibody of the invention or Fragment. In another embodiment, the invention provides a method for reducing tumor immune escape, reducing tumors, or inhibiting growth of tumor cells in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount An anti-PD-L1 antibody of the invention or a fragment thereof. In another embodiment, the invention provides a method for treating cancer in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of an anti-PD-L1 antibody or fragment thereof of the invention . In another embodiment, the cancer is selected from the group consisting of lymphoma, leukemia, melanoma, glioma, breast cancer, lung cancer, colon cancer, bone cancer, ovarian cancer, bladder cancer, kidney cancer, Liver cancer, stomach cancer, testicular cancer, salivary gland cancer, thyroid cancer, thymic cancer, epithelial cancer, head or neck cancer, gastric cancer, pancreatic cancer, or a combination thereof.
在一些实施方案中，本发明提供了用于在有需要的受试者中治疗传染病的方法，所述方法包括向受试者施用治疗有效量的本发明的抗PD-L1抗体或其片段。在另一个实施方案中，所述传染病选自由以下组成的组：念珠菌病、念珠菌血症、曲霉菌病、链球菌性肺炎、链球菌性皮肤和口咽病状、革兰氏阴性脓毒症、结核病、单核细胞增多症、流行性感冒、由呼吸道合胞病毒引起的呼吸道疾病、疟疾、血吸虫病和锥虫病。In some embodiments, the invention provides a method for treating an infectious disease in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of an anti-PD-L1 antibody or fragment thereof of the invention . In another embodiment, the infectious disease is selected from the group consisting of: candidiasis, candidemia, aspergillosis, streptococcal pneumonia, streptococcal skin and oropharyngeal conditions, Gram-negative pus Toxic, tuberculosis, mononucleosis, influenza, respiratory diseases caused by respiratory syncytial virus, malaria, schistosomiasis and trypanosomiasis.
在一些实施方案中，本文提供的抗体和其片段可用于治疗由T辅助型2(Th2)T细胞介导的疾病，例如像哮喘、过敏反应或移植物抗宿主病。In some embodiments, the antibodies and fragments thereof provided herein are useful for treating diseases mediated by T helper 2 (Th2) T cells, such as, for example, asthma, allergic reactions, or graft versus host disease.
在一些实施方案中，本文提供的抗体和其片段可用于在有需要的受试者中刺激免疫应答。例如，在一些实施方案中，抗PD-L1抗体和其片段可以与目标抗原一起施用，以便用于引发对所述抗原的免疫应答。目标抗原可以是与病原体(诸如病毒或细菌)相关联的抗原。因此，在一些实施方案中，本发明提供了包含抗PD-L1抗体和抗原的疫苗，其中所述疫苗引发抗原特异性免疫应答。In some embodiments, the antibodies and fragments thereof provided herein can be used to stimulate an immune response in a subject in need thereof. For example, in some embodiments, an anti-PD-Ll antibody and fragments thereof can be administered with an antigen of interest for eliciting an immune response to the antigen. The antigen of interest may be an antigen associated with a pathogen, such as a virus or a bacterium. Accordingly, in some embodiments, the invention provides a vaccine comprising an anti-PD-Ll antibody and an antigen, wherein the vaccine elicits an antigen-specific immune response.
在一些实施方案中，本文公开的抗体和其片段可以与一种或多种另外的治疗剂组合施用至有需要的受试者。在一些实施方案中，可以在向受试者施用另外的治疗剂之前、期间和/或之后将抗体和其片段施用至受试者。在一个实施方案中，另外的治疗剂是化学治疗剂、放射治疗剂、细胞因子、抗体或其片段或指示待治疗的疾病的任何其它另外的治疗剂。在一些实施方案中，当一起施用时，无论是同时施用还是依序施用，抗PD-L1抗体和另外的治疗剂均显示出治疗性协同作用。在一些实施方案中，抗PD-L1抗体和另外的治疗剂以单独的制剂施用。在另一个实施方案中，抗PD-L1抗体和另外的治疗剂以相同的制剂施用。在一个实施方案中，本文提供的抗PD-L1抗体和片段增强一种或多种另外的治疗剂的免疫调节作用。在另一些实施方案中，一种或多种另外的治疗剂增强抗PD-L1抗体或其片段的作用。In some embodiments, the antibodies and fragments thereof disclosed herein can be administered to a subject in need thereof in combination with one or more additional therapeutic agents. In some embodiments, the antibody and fragments thereof can be administered to the subject before, during, and/or after administration of the additional therapeutic agent to the subject. In one embodiment, the additional therapeutic agent is a chemotherapeutic agent, a radiotherapeutic agent, a cytokine, an antibody or fragment thereof, or any other additional therapeutic agent indicative of the condition to be treated. In some embodiments, the anti-PD-Ll antibody and the additional therapeutic agent exhibit a therapeutic synergy when administered together, whether administered simultaneously or sequentially. In some embodiments, the anti-PD-L1 antibody and the additional therapeutic agent are administered in separate formulations. In another embodiment, the anti-PD-Ll antibody and the additional therapeutic agent are administered in the same formulation. In one embodiment, the anti-PD-Ll antibodies and fragments provided herein enhance the immunomodulatory effects of one or more additional therapeutic agents. In other embodiments, one or more additional therapeutic agents enhance the effect of the anti-PD-Ll antibody or fragment thereof.
本发明提供分离的抗体和其抗原结合片段，以及编码所述抗体和片段的核酸，以及包含所述分离的抗体、片段或核酸的组合物。术语“分离的”是指已经从其天然环境中分离的目标化合物(例如，抗体或核酸)。本发明还提供了药物组合物，所述药物组合物包含分离的抗体或其片段，或编码所述抗体或片段的核酸，并且还包含一种或多种药学上可接受的载体。药学上可接受的载体包括，例如，赋形剂、稀释剂、包封材料、填充剂、缓冲剂或其它试剂。The invention provides isolated antibodies and antigen-binding fragments thereof, as well as nucleic acids encoding the antibodies and fragments, and compositions comprising the isolated antibodies, fragments or nucleic acids. The term "isolated" refers to a compound of interest (eg, an antibody or nucleic acid) that has been isolated from its natural environment. The invention also provides a pharmaceutical composition comprising an isolated antibody or fragment thereof, or a nucleic acid encoding the antibody or fragment, and further comprising one or more pharmaceutically acceptable carriers. Pharmaceutically acceptable carriers include, for example, excipients, diluents, encapsulating materials, fillers, buffers, or other agents.
如本发明所述，术语“抗体”是指具有至少一个抗原结合结构域的抗原结合蛋白。本发明的抗体和其片段可以是整个抗体或其任何片段。因此，本发明的抗体和抗原结合片段包括单克隆抗体或其片段和抗体变体或其片段，以及免疫缀合物。抗体片段的实例包括Fab片段、Fab’片段、F(ab’)2片段、Fv片段、分离 的CDR区、单链Fv分子(scFv)和本领域已知的其它抗体片段。抗体和其片段还可以包括重组多肽、融合蛋白和双特异性抗体。本文公开的抗PD-L1抗体和其片段可以是IgG1、IgG2、IgG3或IgG4同种型。术语“同种型”是指由重链恒定区基因编码的抗体种类。在一个实施方案中，本文公开的抗PD-L1抗体和其片段是IgG1或IgG4同种型。本发明的PD-L1抗体和其片段可以衍生自任何物种，其包括但不限于小鼠、大鼠、兔、灵长类动物、美洲驼和人。抗PD-L1抗体和其片段可以是嵌合抗体、人源化抗体或完整的人抗体。在一些实施方案中，抗PD-L1抗体是由源自小鼠的杂交瘤细胞系产生的抗体。因此，在一些实施方案中，抗PD-L1抗体是鼠类抗体。在另一些实施方案中，抗PD-L1抗体是嵌合抗体。在另一些实施方案中，嵌合抗体是小鼠-人嵌合抗体。在另一些实施方案中，抗体是人源化抗体。在另一些实施方案中，抗体衍生自鼠类抗体并且是人源化的。As used herein, the term "antibody" refers to an antigen binding protein having at least one antigen binding domain. The antibodies and fragments thereof of the invention may be the entire antibody or any fragment thereof. Thus, antibodies and antigen-binding fragments of the invention include monoclonal antibodies or fragments thereof and antibody variants or fragments thereof, as well as immunoconjugates. Examples of antibody fragments include Fab fragments, Fab' fragments, F(ab')2 fragments, Fv fragments, isolated CDR regions, single chain Fv molecules (scFv), and other antibody fragments known in the art. Antibodies and fragments thereof can also include recombinant polypeptides, fusion proteins, and bispecific antibodies. The anti-PD-L1 antibodies and fragments thereof disclosed herein can be of the IgGl, IgG2, IgG3 or IgG4 isotype. The term "isotype" refers to the type of antibody encoded by the heavy chain constant region gene. In one embodiment, an anti-PD-L1 antibody and fragments thereof disclosed herein are of the IgGl or IgG4 isotype. The PD-L1 antibodies and fragments thereof of the invention can be derived from any species including, but not limited to, mice, rats, rabbits, primates, llamas, and humans. The anti-PD-L1 antibody and fragments thereof can be chimeric antibodies, humanized antibodies or intact human antibodies. In some embodiments, the anti-PD-L1 antibody is an antibody produced by a mouse-derived hybridoma cell line. Thus, in some embodiments, the anti-PD-L1 antibody is a murine antibody. In other embodiments, the anti-PD-L1 antibody is a chimeric antibody. In other embodiments, the chimeric antibody is a mouse-human chimeric antibody. In other embodiments, the antibody is a humanized antibody. In other embodiments, the antibody is derived from a murine antibody and is humanized.
“Fab片段”由一条轻链和一条重链的CH1结构域及可变区组成。Fab分子的重链不能与另一个重链分子形成二硫键。A "Fab fragment" consists of a light chain and a heavy chain CH1 domain and a variable region. The heavy chain of a Fab molecule cannot form a disulfide bond with another heavy chain molecule.
“Fab’片段”含有一条轻链和包含VH结构域和CH1结构域以及CH1结构域和CH2结构域之间区域的一条重链的部分，由此可在两个Fab’片段的两条重链之间形成链间二硫键以形成F(ab’)2分子。A "Fab' fragment" contains a light chain and a portion of a heavy chain comprising a VH domain and a CH1 domain and a region between the CH1 domain and the CH2 domain, whereby two heavy chains of the two Fab' fragments are available An interchain disulfide bond is formed to form a F(ab')2 molecule.
“F(ab’)2片段”含有两条轻链和两条包含CH1结构域和CH2结构域之间的恒定区的部分的重链，由此在两条重链间形成链间二硫键。因此，F(ab’)2片段由通过两条重链间的二硫键保持在一起的两个Fab’片段组成。"F(ab')2 fragment" contains two light chains and two heavy chains comprising a portion of the constant region between the CH1 domain and the CH2 domain, thereby forming an interchain disulfide bond between the two heavy chains . Thus, the F(ab')2 fragment consists of two Fab' fragments held together by a disulfide bond between the two heavy chains.
“Fv区”包含来自重链和轻链二者的可变区，但缺少恒定区。The "Fv region" contains variable regions from both heavy and light chains, but lacks a constant region.
“单链Fv抗体”(或“scFv抗体”)是指包含抗体的VH和VL结构域的抗体片段，其中这些结构域存在于单个多肽链中。一般而言，Fv多肽另外在VH和VL结构域之间包含多肽接头，该接头使得scFv能形成用于抗原结合的所需结构。对于scFv的综述，可参见Pluckthun(1994)THE PHARMACOLOGY OF MONOCLONAL ANTIBODIES(单克隆抗体药理学)，第113卷，Rosenburg和Moore主编，Springer-Verlag，New York，第269-315页。还参见国际专利申请公开号WO88/01649和美国专利第4,946,778号和第5,260,203号。以引用的方式将上述文献的内容并入本文作为参考。A "single-chain Fv antibody" (or "scFv antibody") refers to an antibody fragment comprising the VH and VL domains of an antibody, wherein these domains are present in a single polypeptide chain. In general, Fv polypeptides additionally comprise a polypeptide linker between the VH and VL domains that enables the scFv to form the desired structure for antigen binding. For a review of scFv, see Pluckthun (1994) THE PHARMACOLOGY OF MONOCLONAL ANTIBODIES, Vol. 113, Rosenburg and Moore, ed., Springer-Verlag, New York, pp. 269-315. See also International Patent Application Publication No. WO 88/01649 and U.S. Patent Nos. 4,946,778 and 5,260,203. The contents of the above documents are incorporated herein by reference.
“双抗体”也称为“双特异性抗体”，为具有两个抗原结合位点的小抗体片段。所述片段包含在相同的多肽链中与轻链可变结构域(VL)连接的重链可变结构域(VH)(VH-VL或VL-VH)。通过使用短至不能在同一链的两个结构域之间配对的接头，迫使所述结构域与另一条链的互补结构域配对并形成两个抗原结合位点。A "diabody", also known as a "bispecific antibody," is a small antibody fragment having two antigen binding sites. The fragment comprises a heavy chain variable domain (VH) (VH-VL or VL-VH) linked to a light chain variable domain (VL) in the same polypeptide chain. By using a linker that is short enough to be able to pair between two domains of the same chain, the domains are forced to pair with the complementary domains of the other chain and form two antigen-binding sites.
“嵌合抗体”是下述抗体：所述抗体具有衍生自一种物种的重链可变区的至少一部分和轻链可变区的至少一部分；以及衍生自另一物种的恒定区的至少一部分。例如，在一个实施方案中，嵌合抗体可以包含鼠类可变区和人恒定区。A "chimeric antibody" is an antibody having at least a portion of a heavy chain variable region derived from a species and at least a portion of a variable region of a light chain; and at least a portion of a constant region derived from another species . For example, in one embodiment, a chimeric antibody can comprise a murine variable region and a human constant region.
“人源化抗体”是下述抗体：所述抗体含有衍生自非人抗体的互补决定区(CDR)；和衍生自人抗体的框架区以及恒定区。例如，本文提供的抗PD-L1抗体可以包含衍生自一种或多种鼠类抗体的CDR以及人框架区和恒定区。因此，在一个实施方案中，本文提供的人源化抗体与衍生出所述抗体的CDR的鼠类抗体结合PD-L1上的相同表位。本文提供了示例性人源化抗体。包含本文提供的重链CDR和轻链CDR的另外的抗PD-L1抗体或其变体可以使用任何人框架序列产生，并且也包括在本发明中。在一个实施方案中，适用于在本发明中使用的框架序列包括在结构上与本文提供的框架序列类似的那些框架序列。可以在框架区中进行另外修饰以改进本文提供的抗体的特性。此类另外的框架修饰可以包括化学修饰；点突变以降低免疫原性或去除T细胞表位；或使突变回复为原始种系序列中的残基。在一些实施方案中，此类修饰包括对应于本文示例的突变的那些修饰，包括对种系序列的回复突变。例如，在一个实施方案中，本文提供的人源化抗体的VH和/或VL的人框架区中的一个或多个氨基酸被回复突变为亲本鼠类抗体中对应的氨基酸。例如，对于人源化hu-Chi的VH和VL，上述模板人抗体的框架氨基酸的几个位点被回复突变为小鼠Chi抗体中对应的氨基酸序列。在一个实施方案中，人源化抗体hu-Chi1重链可变区位置35和/或39和/或43和/或44和/或48和/或71处的氨基酸回复突变为在小鼠Chi1重链可变区所述位置处发现的对应的氨基酸。在另一个实施方案中，轻链可变区的位置2和/或8和/或41和/或44和/或87和/或100处的氨基酸被回复突变为在小鼠Chi1轻链可变区中的所述位置处发现的对应的氨基酸。S35N、Q39R、K43N、G44K、 W47Y、I48M、V71R的氨基酸回复突变为相应小鼠Chi抗体中对应的氨基酸。在一个实施方案中，人源化的hu-Chi1-3.4抗体包含重链可变区，其中在位置47处的氨基酸从Trp(W)突变为Try(Y)，在位置35处的氨基酸从Ser(S)突变为Asn(N)，并且在位置71处的氨基酸从Val(V)突变为Arg(R)；以及轻链可变区，其中在位置2处的氨基酸从Ile(I)突变为Phe(F)，在位置41处的氨基酸从Gly(G)突变为Asp(D)，在位置44处的氨基酸从Pro(P)突变为Val(V)。另外的或另选的回复突变可以在本文提供的人源化抗体的框架区中进行以改进抗体的特性。本发明还包括下述人源化抗体，所述人源化抗体结合PD-L1并且包含对应于本文所述的相对于任何合适的框架序列的示例性修饰的框架修饰，以及以其它方式改进抗体特性的其它框架修饰，例如在人源化抗体hu-Chi1重链可变区的位置102处的氨基酸从Cys(C)突变为Try(Y)、Ala(A)或Val(V)。A "humanized antibody" is an antibody comprising a complementarity determining region (CDR) derived from a non-human antibody; and a framework region derived from a human antibody and a constant region. For example, an anti-PD-L1 antibody provided herein can comprise a CDR derived from one or more murine antibodies, as well as a human framework region and a constant region. Thus, in one embodiment, a humanized antibody provided herein binds to the same epitope on PD-L1 as the murine antibody from which the CDR of the antibody is derived. Exemplary humanized antibodies are provided herein. Additional anti-PD-L1 antibodies or variants thereof comprising the heavy chain CDRs and light chain CDRs provided herein can be produced using any human framework sequences and are also included in the present invention. In one embodiment, framework sequences suitable for use in the present invention include those framework sequences that are structurally similar to the framework sequences provided herein. Additional modifications can be made in the framework regions to improve the properties of the antibodies provided herein. Such additional framework modifications may include chemical modifications; point mutations to reduce immunogenicity or removal of T cell epitopes; or reversion of the mutation to residues in the original germline sequence. In some embodiments, such modifications include those corresponding to the mutations exemplified herein, including back mutations to germline sequences. For example, in one embodiment, one or more amino acids in the human framework regions of the VH and/or VL of the humanized antibodies provided herein are back-mutated to the corresponding amino acids in the parent murine antibody. For example, for the VH and VL of humanized hu-Chi, several sites of the framework amino acid of the above template human antibody are back-mutated to the corresponding amino acid sequence in the mouse Chi antibody. In one embodiment, the amino acid back mutation at position 35 and/or 39 and/or 43 and/or 44 and/or 48 and/or 71 of the humanized antibody hu-Chi1 heavy chain variable region is in mouse Chi1 The corresponding amino acid found at the position of the heavy chain variable region. In another embodiment, amino acids at positions 2 and/or 8 and/or 41 and/or 44 and/or 87 and/or 100 of the light chain variable region are back-mutated to be variable in the mouse Chi1 light chain The corresponding amino acid found at the position in the region. The amino acid back mutation of S35N, Q39R, K43N, G44K, W47Y, I48M, V71R is the corresponding amino acid in the corresponding mouse Chi antibody. In one embodiment, the humanized hu-Chi1-3.4 antibody comprises a heavy chain variable region, wherein the amino acid at position 47 is mutated from Trp (W) to Try (Y), and the amino acid at position 35 is from Ser (S) is mutated to Asn(N), and the amino acid at position 71 is mutated from Val(V) to Arg(R); and the light chain variable region, wherein the amino acid at position 2 is mutated from Ile(I) to Phe(F), the amino acid at position 41 is mutated from Gly(G) to Asp(D), and the amino acid at position 44 is mutated from Pro(P) to Val(V). Additional or alternative back mutations can be made in the framework regions of the humanized antibodies provided herein to improve the properties of the antibodies. The invention also encompasses a humanized antibody that binds to PD-L1 and comprises a framework modification corresponding to an exemplary modification of any suitable framework sequence described herein, and otherwise improves the antibody Other framework modifications of the property, for example, the amino acid at position 102 of the humanized antibody hu-Chi1 heavy chain variable region is mutated from Cys (C) to Try (Y), Ala (A) or Val (V).
术语“人PD-L1”、“hPD-L1”和“hu-PD-L1”等在本文中可互换使用，并且是指人PD-L1和人PD-L1的变体或同种型。“特异于”意指抗体和其片段以比任何其它靶标更大的亲和力结合PD-L1。如本文所用，术语“EC50”是指有效浓度，抗体的50％最大应答。如本文所用，术语“IC50”是指抑制浓度，抗体的50％最大应答。EC50和IC50两者均可以通过ELISA或FACS分析或本领域已知的任何其它方法进行测量。The terms "human PD-L1", "hPD-L1" and "hu-PD-L1" and the like are used interchangeably herein and refer to variants or isoforms of human PD-L1 and human PD-L1. "Specific" means that the antibody and its fragments bind to PD-L1 with greater affinity than any other target. As used herein, the term "EC50" refers to the effective concentration, the 50% maximal response of an antibody. As used herein, the term "IC50" refers to the concentration of inhibition, the 50% maximal response of an antibody. Both EC50 and IC50 can be measured by ELISA or FACS analysis or any other method known in the art.
本文公开的在CDR或轻链可变区或重链可变区具有一个或多个氨基酸取代、插入、缺失或其组合的抗PD-L1抗体保留了不具有氨基酸取代、插入或缺失的对应的抗PD-L1抗体的生物活性。因此，本文提供的变体抗PD-L1抗体保留与PD-L1的结合。如本文所用，同源性百分比是指两个参考序列共有的相同氨基酸序列的数目除以氨基酸位置的总数，乘以100。在一些实施方案中，所述CDR或轻链可变区或重链可变区氨基酸序列的变体，其中所述变体包含1、2、3、4、5、6、7、8、9、10或更多个氨基酸取代、插入或缺失或其组合。在另一个实施方案中，氨基酸取代为保守取代。The anti-PD-L1 antibodies disclosed herein having one or more amino acid substitutions, insertions, deletions or combinations thereof in the CDR or light chain variable region or heavy chain variable region retain a corresponding absence of amino acid substitutions, insertions or deletions Biological activity of anti-PD-L1 antibody. Thus, the variant anti-PD-L1 antibodies provided herein retain binding to PD-L1. As used herein, percent homology refers to the number of identical amino acid sequences shared by two reference sequences divided by the total number of amino acid positions, multiplied by 100. In some embodiments, the variant of the CDR or light chain variable region or heavy chain variable region amino acid sequence, wherein the variant comprises 1, 2, 3, 4, 5, 6, 7, 8, 9 , 10 or more amino acid substitutions, insertions or deletions, or a combination thereof. In another embodiment, the amino acid substitution is a conservative substitution.
在一方面，本发明提供了用于治疗受试者的响应于增强、刺激或引发免疫应答的疾病或病状的方法。如本文所用，术语“治疗(treatment或treating)”是指治疗性治疗以及防范性或预防性措施。需要治疗的受试者包括那些已经患有疾病或病状的受试者，以及可能患疾病或病状并且其目的是预防、延迟或减弱疾病或病状的受试者。如本文所用，术语“受试者”表示哺乳动物，诸如啮齿动物、猫科动物、犬科动物和灵长类动物。优选地，根据本发明的受试者是人。In one aspect, the invention provides methods for treating a disease or condition in a subject that is responsive to enhancing, stimulating or eliciting an immune response. As used herein, the term "treatment" or "treating" refers to both therapeutic treatment as well as preventative or preventative measures. Subjects in need of treatment include those already with the disease or condition, as well as subjects who may have the disease or condition and whose purpose is to prevent, delay or attenuate the disease or condition. As used herein, the term "subject" refers to mammals, such as rodents, felines, canines, and primates. Preferably, the subject according to the invention is a human.
如本文所用，术语“治疗有效量”是指向受试者提供治疗性和/或预防性益处所必需的化合物或组合物的量。As used herein, the term "therapeutically effective amount" is the amount of a compound or composition necessary to provide a therapeutic and/or prophylactic benefit to a subject.
本文中的术语“突变序列”和“突变体序列”可互换使用，表示通过取代、缺失或插入等方式发生改变后的序列，并且所述“突变序列”和“突变体序列”与突变前的序列相比可具有60％以上的同源性、例如70％以上的同源性、进一步例如80％以上的同源性。The terms "mutation sequence" and "mutant sequence" are used interchangeably herein to refer to a sequence that has been altered by substitution, deletion or insertion, and the "mutant sequence" and "mutant sequence" are before the mutation. The sequence may have more than 60% homology, for example 70% or more homology, and further, for example, more than 80% homology.
除非另外特别说明，否则单数的使用包括复数。除非另外特别说明，否则词语“一个(a)”或“一个(an)”意指“至少一个”。除非另外说明，否则“或”的使用意指“和/或”。短语“至少一个”的含义等同于短语“一个或多个”的含义。此外，术语“包括/包含/含有(including)”以及其它形式诸如“包括/包含/含有(includes)”和“包括/包含/含有(included)”的使用不是限制性的。此外，除非另外特别说明，否则术语诸如“要素”或“组分”包括包含一个单元的元素或组分以及包含多于一个单元的元素和组分。The singular use includes the plural unless specifically stated otherwise. The word "a" or "an" means "at least one" unless specifically stated otherwise. The use of "or" means "and/or" unless stated otherwise. The meaning of the phrase "at least one" is equivalent to the meaning of the phrase "one or more." Furthermore, the use of the terms "including" or "including" and "includes" and "includes" and "includes" are not limiting. In addition, terms such as “element” or “component” include elements or components that comprise one unit and elements and components that comprise more than one unit, unless specifically stated otherwise.
本文所用术语“约”是指数值在由本领域一般技术人员所测定的具体值的可接受误差范围内，所述数值部分取决于怎样测量或测定(即测量体系的限度)。例如，在本领域每一次实行中“约”可意味着在1内或超过1的标准差。或者，“约”或“基本上包含”可意味着至多20％的范围。此外，特别对于生物学系统或过程而言，该术语可意味着至多一个数量级或数值的至多5倍。除非另外说明，否则当具体值在本申请和权利要求中出现时，“约”或“基本上包含”的含义应该假定为在该具体值的可接受误差范围内。The term "about" as used herein is an index of values within an acceptable tolerance of a particular value as determined by one of ordinary skill in the art, depending in part on how the measurement or measurement is made (ie, the limits of the measurement system). For example, "about" in each implementation of the art may mean a standard deviation of one or more than one. Alternatively, "about" or "substantially encompasses" may mean a range of up to 20%. Moreover, particularly for biological systems or processes, the term can mean up to an order of magnitude or up to 5 times the value. The meaning of "about" or "substantially encompasses" should be assumed to be within the acceptable tolerance of the particular value, unless stated otherwise.
尽管为了清楚理解的目的，已经通过举例说明和实施例相当详细地描述了前述发明，但是根据本发明的教义，本领域的普通技术人员将显而易见的是，可另外对本发明进行某些改变和修改而不背离所附权利要求的精神和范围。以下实施例仅以说明方式提供，而并不起限制作用。本领域的技术人员将容易地识别多种非关键性参数，所述参数可发生改变或修改以产生基本上类似的结果。Although the foregoing invention has been described in considerable detail, by way of illustration and exemplary embodiments of the embodiments of the present invention Without departing from the spirit and scope of the appended claims. The following examples are provided by way of illustration only and are not limiting. Those skilled in the art will readily recognize a variety of non-critical parameters that may be altered or modified to produce substantially similar results.
附图说明DRAWINGS
图1：通过ELISA检测在一定浓度范围内嵌合抗PD-L1抗体与PD-L1的结合；Figure 1: Detection of binding of chimeric anti-PD-L1 antibody to PD-L1 over a range of concentrations by ELISA;
图2：通过ELISA检测在一定抗体浓度范围内嵌合抗PD-L1抗体对PD-1/PD-L1结合的阻断作用；Figure 2: Blocking of PD-1/PD-L1 binding by chimeric anti-PD-L1 antibodies over a range of antibody concentrations by ELISA;
图3：通过FACS检测在一定抗体浓度范围内嵌合抗PD-L1抗体对PD-1/PD-L1结合的阻断作用；Figure 3: Detection of PD-1/PD-L1 binding by chimeric anti-PD-L1 antibodies over a range of antibody concentrations by FACS;
图4：通过细胞活性检测在一定抗体浓度范围内嵌合抗PD-L1抗体对PD-1/PD-L1结合的阻断作用；Figure 4: Blocking effect of chimeric anti-PD-L1 antibody on PD-1/PD-L1 binding by a range of antibody concentrations by cell activity assay;
图5：通过细胞活性检测在一定抗体浓度范围内人源化抗PD-L1抗体对PD-1/PD-L1结合的阻断作用。Figure 5: Blocking effect of humanized anti-PD-L1 antibody on PD-1/PD-L1 binding by a range of antibody concentrations by cell activity assay.
具体实施方式Detailed ways
实施例1 PD-L1抗原及检测蛋白制备Example 1 PD-L1 antigen and detection protein preparation
以UniProt Programmed Cell Death 1Ligand 1(PD-L1)同种型1(SEQ ID NO:1)的人PD-L1全长基因(义翘神州生物技术有限公司，HG10084-M)，作为本发明PD-L1的模板，获得编码本发明抗原及检测用蛋白的基因序列，可选择PD-L1胞外区(Extra Cellular Domain，ECD)与抗体重链Fc片段(如小鼠IgG2a)重组连接形成PD-L1-mFc，或者与His Tag重组连接形成PD-L1-his，用于小鼠的免疫或后期筛选检测。编码含小鼠抗体重链Fc标签的PD-L1胞外区重组蛋白(hPD-L1(ECD)-mFc，SEQ ID NO:2)与组氨酸标签的PD-L1胞外区重组蛋白(hPD-L1-His，SEQ ID NO:3)、以及含小鼠抗体重链Fc标签的PD-1胞外区重组蛋白(hPD-1-mFc，SEQ ID NO:4)的cDNA通过PCR获得，并且分别被亚克隆到表达载体pcDNA3.1(Invitrogen，V-790)中。将上述构建的载体转染进Exi-CHO细胞(ThermoFisher A29133)，进行瞬时表达。转染时传代细胞至细胞密度为6*10 6个细胞/ml，按转染试剂:DNA＝3:1的比例制备转染复合物，室温孵育5min，缓慢加入到细胞中，20-24h后按比例添加ExpiCHO Enhancer及ExpiCHO Feed。在ExpiCHO细胞中瞬时表达7天-14天后，用NTA柱(GE healthcare)纯化hPD-L1-HisTag蛋白，用Protein A柱(GE healthcare)纯化hPD-L1-mFc和hPD1-mFc重组蛋白。或将表达片段构建于CHO-S(Thermo Fisher，A1155701)或CHO-DG44(Thermo Fisher，A1100001)等细胞稳定表达，并进行纯化。 The human PD-L1 full-length gene (Hypothesis Shenzhou Biotechnology Co., Ltd., HG10084-M) of UniProt Programmed Cell Death 1Ligand 1 (PD-L1) isoform 1 (SEQ ID NO: 1) was used as the PD-of the present invention. The template of L1 obtains the gene sequence encoding the antigen of the present invention and the protein for detection, and the PD-L1 extracellular domain (ECD) can be recombined with the antibody heavy chain Fc fragment (such as mouse IgG2a) to form PD-L1. -mFc, or recombinantly linked to His Tag to form PD-L1-his for immunization or post-screening detection in mice. PD-L1 extracellular domain recombinant protein (hPD-L1(ECD)-mFc, SEQ ID NO: 2) containing a mouse antibody heavy chain Fc tag and histidine-tagged PD-L1 extracellular domain recombinant protein (hPD) -L1-His, SEQ ID NO: 3), and the cDNA of the PD-1 extracellular domain recombinant protein (hPD-1-mFc, SEQ ID NO: 4) containing the mouse antibody heavy chain Fc tag was obtained by PCR, and They were subcloned into the expression vector pcDNA3.1 (Invitrogen, V-790), respectively. The vector constructed above was transfected into Exi-CHO cells (ThermoFisher A29133) for transient expression. The cells were transfected to a cell density of 6*10 6 cells/ml, and the transfection complex was prepared according to the ratio of transfection reagent: DNA=3:1. Incubate for 5 min at room temperature and slowly add to the cells, after 20-24 h. Add ExpiCHO Enhancer and ExpiCHO feeds proportionally. After transient expression in ExpiCHO cells for 7 days to 14 days, hPD-L1-HisTag protein was purified using an NTA column (GE healthcare), and hPD-L1-mFc and hPD1-mFc recombinant proteins were purified using a Protein A column (GE healthcare). Alternatively, the expression fragment is stably expressed in cells such as CHO-S (Thermo Fisher, A1155701) or CHO-DG44 (Thermo Fisher, A1100001), and purified.
实施例2 抗人PD-L1单克隆抗体杂交瘤的制备Example 2 Preparation of anti-human PD-L1 monoclonal antibody hybridoma
将表达纯化的hPD-L1(ECD)-mFc重组蛋白(按照100μg/只小鼠)与等体积的完全弗氏佐剂(首免)或不完全弗氏佐剂(加强免疫)充分混合并乳化，每2周皮下免疫BALB/c小鼠，持续8周。选择血清中抗体滴度高并且滴度趋于平台期的小鼠进行脾细胞融合。融合前3天，通过腹腔注射不含佐剂的hPD-L1(ECD)-mFc抗原(50μg/只小鼠)来冲刺免疫小鼠。采用优化的PEG介导的融合步骤将脾淋巴细胞与骨髓瘤细胞Sp2/0细胞进行融合得到杂交瘤细胞。将来自免疫小鼠的脾细胞(1×10 8个细胞)与SP2/0骨髓瘤细胞(2×10 7个细胞)融合。融合后，将细胞用HAT完全培养基重悬，以0.1ml/孔分配到96孔板中，并且在37℃、5％CO 2的培养箱中进行培养。融合后的第5天，用新鲜的HAT完全培养基进行对半换液，37℃、5％CO 2继续培养。融合后第7天～8天，根据细胞生长密度进行全换液，采用的培养基为HT完全培养基，200μ1/孔，37℃、5％CO 2继续培养。融合后第14天左右，根据细胞生长密度，使用PD-L1结合的ELISA方法检测(检测方法1.1)。并将检测的阳性孔细胞进行PD-L1/PD-1结合的阻断的ELISA检测(检测方法1.2)，挑选能够结合PD-L1并能阻断与PD-1结合的孔，并根据细胞密度及时扩大至24孔板中。移入24孔板的细胞株经过复测后进行保种和第一次亚克隆。第一次亚克隆筛选为阳性的进行保种，并进行第二次亚克隆。第二次亚克隆为阳性的进行保种和蛋白表达。 The purified hPD-L1(ECD)-mFc recombinant protein (according to 100 μg/mouse) was thoroughly mixed and emulsified with an equal volume of complete Freund's adjuvant (first boost) or incomplete Freund's adjuvant (enhanced immunization). BALB/c mice were immunized subcutaneously every 2 weeks for 8 weeks. Splenocyte fusion was performed in mice with high antibody titers in serum and titers that tended to plateau. Three days before the fusion, mice were immunized by intraperitoneal injection of an adjuvant-free hPD-L1 (ECD)-mFc antigen (50 μg/mouse). The spleen lymphocytes were fused with myeloma cell Sp2/0 cells using an optimized PEG-mediated fusion step to obtain hybridoma cells. Splenocytes (1 x 10 8 cells) from immunized mice were fused with SP2/0 myeloma cells (2 x 10 7 cells). After the fusion, the cells were resuspended in HAT complete medium, dispensed into 96-well plates at 0.1 ml/well, and cultured in a 37 ° C, 5% CO 2 incubator. On the fifth day after the fusion, the half-liquid exchange was carried out with fresh HAT complete medium, and the cultivation was continued at 37 ° C, 5% CO 2 . On the 7th to 8th day after the fusion, the whole medium was changed according to the cell growth density, and the medium used was HT complete medium, 200 μl/well, and culture was continued at 37 ° C, 5% CO 2 . On the 14th day after the fusion, it was detected by the ELISA method using PD-L1 binding according to the cell growth density (Test Method 1.1). The detected positive well cells were subjected to blocking ELISA detection of PD-L1/PD-1 binding (detection method 1.2), and the pores capable of binding PD-L1 and blocking binding to PD-1 were selected, and according to cell density Promptly expand into 24-well plates. The cell line transferred into the 24-well plate was subjected to retesting and then subjected to seed conservation and first subcloning. The first subcloning screen was positive for conservation and the second subcloning was performed. The second subcloning was positive for conservation and protein expression.
检测方法1.1结合PD-L1的ELISA检测Detection method 1.1 combined with PD-L1 ELISA detection
通过ELISA方法，使用PD-L1-his蛋白进行杂交瘤抗体的PD-L1结合筛选和分析。将PD-L1抗原(100μL，2μg/ml)包被在高吸附96孔板(Costar，9018)中，4℃，过夜。充分洗去未吸附抗原后，使用封闭缓冲液(含2％牛血清白蛋白的PBS)封闭非特异性结合位点。用洗涤缓冲液(具有0.05％(v/v)吐温20的PBS)洗涤平板三次后，添加100μL/孔的待测样品，室温下孵育1小时。洗涤平板，加入偶联辣根过氧化物酶(HRP)的二抗继续孵育60分钟。洗涤平板后，添加100μL/孔底物TMB溶液(eBioscience，00-4201-56)并将所述平板在室温下温育2min。添加100μL/孔的终止溶液(2NH 2SO 4)以停止反应。产生比色信号并且使用酶标仪(Thermo Fisher，MK3)在450nm处读取所述比色信号，扣除背景之后比空白血清读值高2倍即为阳性孔。 PD-L1 binding screening and analysis of hybridoma antibodies was performed by ELISA method using PD-L1-his protein. PD-L1 antigen (100 μL, 2 μg/ml) was coated in a high-adsorption 96-well plate (Costar, 9018) at 4 ° C overnight. After washing the unadsorbed antigen sufficiently, the non-specific binding site was blocked with blocking buffer (PBS containing 2% bovine serum albumin). After washing the plate three times with washing buffer (PBS with 0.05% (v/v) Tween 20), 100 μL/well of the sample to be tested was added, and incubated at room temperature for 1 hour. The plates were washed and incubated with a horseradish peroxidase (HRP) secondary antibody for 60 minutes. After washing the plates, 100 μL/well of substrate TMB solution (eBioscience, 00-4201-56) was added and the plates were incubated for 2 min at room temperature. 100 μL/well of stop solution (2NH 2 SO 4 ) was added to stop the reaction. A colorimetric signal was generated and the colorimetric signal was read at 450 nm using a microplate reader (Thermo Fisher, MK3), which was a positive well after subtracting the background from the blank serum reading by a factor of two.
检测方法1.2 PD-L1/PD-1结合的阻断的ELISA检测Detection method 1.2 Blocked ELISA detection of PD-L1/PD-1 binding
在高吸附96孔板中包被PD-L1抗原(100μL，2μg/ml)，4℃，过夜。充分洗去未吸附抗原后，使用封闭缓冲液(含2％牛血清白蛋白的PBS)封闭非特异性结合位点。用洗涤缓冲液(具有0.05％吐温20的PBS)洗涤平板三次后，添加100μL/孔的待测样品，同时向每个孔添加100μl生物素标记的PD-1-mFc(0.1μg/ml)，并在37℃下孵育2h。将所述平板洗涤3次后，加入按照1:500稀释的二抗Avidin HRP(eBioscience E07418-1632)，100μl/孔，并在室温孵育1小时。洗涤所述平板后，添加100μL/孔的底物溶液TMB(eBioscience，00-4201-56)，并将所述平板在室温下温育3min。产生比色信号并且使用酶标仪(Thermo Fisher，MK3)在450nm处读取所述比色信号，选取读值较低的孔。PD-L1 antigen (100 μL, 2 μg/ml) was coated in a highly adsorbed 96-well plate at 4 ° C overnight. After washing the unadsorbed antigen sufficiently, the non-specific binding site was blocked with blocking buffer (PBS containing 2% bovine serum albumin). After washing the plate three times with washing buffer (PBS with 0.05% Tween 20), 100 μL/well of the sample to be tested was added while 100 μl of biotin-labeled PD-1-mFc (0.1 μg/ml) was added to each well. And incubated for 2 h at 37 °C. After washing the plate 3 times, secondary antibody Avidin HRP (eBioscience E07418-1632) diluted 1:500 was added, 100 μl/well, and incubated for 1 hour at room temperature. After washing the plate, 100 μL/well of substrate solution TMB (eBioscience, 00-4201-56) was added, and the plate was incubated at room temperature for 3 min. A colorimetric signal was generated and the colorimetric signal was read at 450 nm using a microplate reader (Thermo Fisher, MK3), and wells with lower readings were selected.
实施例3抗人PD-L1抗体的cDNA获取和嵌合抗体构建Example 3 cDNA acquisition of anti-human PD-L1 antibody and construction of chimeric antibody
使用TRIzol从上述具有结合和阻断功能的杂交瘤细胞中分离总RNA作为模板，按照说明书使用superscript II逆转录酶(Life Technology，18064-14)合成第一链cDNA。然后使用简并小鼠IgG引物通过PCR反应扩增抗体的可变区序列。Total RNA was isolated from the above hybridoma cells with binding and blocking functions using TRIzol as a template, and the first strand cDNA was synthesized using superscript II reverse transcriptase (Life Technology, 18064-14) according to the instructions. The variable region sequences of the antibodies were then amplified by PCR reaction using degenerate mouse IgG primers.
将PCR混合物在含有0.5μg/ml溴化乙锭的1％琼脂糖/Tris-硼酸盐凝胶中进行电泳分离。从凝胶上切下具有预期大小(重链和轻链大约500bp)的DNA片段并且对其进行纯化。将纯化的PCR产物克隆到pMD-19T载体(Takara，6013)中，并且转化到DH5α感受态大肠杆菌细胞(Takara，9057)中。从LB固体培养平板上挑取2个菌落进行DNA测序。得到抗体的重链可变区序列和轻链可变区序列(Chi1可变区如SEQ ID NOs:5-6所示，Chi2可变区如SEQ ID NOs:7-10所示)。这些抗体显示出特定的功能，诸如高亲和力结合PD-L1，并且能够阻断PD-L1与PD-1的结合。The PCR mixture was electrophoretically separated in a 1% agarose/Tris-borate gel containing 0.5 μg/ml ethidium bromide. A DNA fragment of the expected size (about 500 bp of heavy and light chains) was excised from the gel and purified. The purified PCR product was cloned into the pMD-19T vector (Takara, 6013) and transformed into DH5α competent E. coli cells (Takara, 9057). Two colonies were picked from LB solid culture plates for DNA sequencing. The heavy chain variable region sequence and the light chain variable region sequence of the antibody are obtained (Chi1 variable region is shown in SEQ ID NOs: 5-6, and Chi2 variable region is shown in SEQ ID NOs: 7-10). These antibodies show specific functions, such as high affinity binding to PD-L1, and are capable of blocking the binding of PD-L1 to PD-1.
嵌合抗体1(Chi1)、嵌合抗体2(Chi2)的构建和表达：将小鼠VL区基因合成片段通过双酶切反应连接到编码人κ链恒定区的核苷酸序列来构建Chi1、Chi2-1、Chi2-2嵌合轻链(分别为SEQ ID NOs:40、42和44)的编码序列。将小鼠VH区的基因合成片段通过双酶切反应连接到编码人IgG1恒定区的核苷酸序列来构建Chi1、Chi2-1、Chi2-2嵌合重链(SEQ ID NOs:39、41和43)的编码序列。Construction and expression of chimeric antibody 1 (Chi1) and chimeric antibody 2 (Chi2): Chi1 was constructed by ligating a mouse VL region gene synthesis fragment to a nucleotide sequence encoding a human kappa chain constant region by double digestion reaction. The coding sequence for the chi2-1, Chi2-2 chimeric light chain (SEQ ID NOs: 40, 42 and 44, respectively). The gene synthesis fragment of the mouse VH region was ligated into the nucleotide sequence encoding the human IgG1 constant region by double restriction enzyme reaction to construct Chi1, Chi2-1, Chi2-2 chimeric heavy chains (SEQ ID NOs: 39, 41 and 43) The coding sequence.
将上述嵌合抗体的编码序列的DNA载体转染Expi CHO细胞(50mL体系，6×10 6个细胞/mL，DNA1μg/ml)进行瞬时表达，培养7天。然后用Protein A柱(GE healthcare)纯化上清液中的嵌合抗体。 The DNA vector encoding the coding sequence of the above chimeric antibody was transfected into Expi CHO cells (50 mL system, 6 × 10 6 cells/mL, DNA 1 μg/ml) for transient expression and cultured for 7 days. The chimeric antibody in the supernatant was then purified using a Protein A column (GE healthcare).
实施例4抗人PD-L1嵌合抗体活性评价Example 4 Evaluation of anti-human PD-L1 chimeric antibody activity
通过以下ELISA、Biacore和流式细胞仪法测量嵌合抗体与PD-L1的结合，并在细胞水平上验证上述嵌合抗体能否阻断PD-1与PD-L1的结合。The binding of the chimeric antibody to PD-L1 was measured by the following ELISA, Biacore and flow cytometry, and it was verified at the cellular level whether the above chimeric antibody could block the binding of PD-1 to PD-L1.
4.1抗PD-L1嵌合抗体的亲和力4.1 Affinity of anti-PD-L1 chimeric antibody
采用Biacore测定PD-L1嵌合抗体对PD-L1抗原的亲和力，使用抗人抗体捕获试剂盒(GE,BR-1008-39)，按照说明书中所述的方法将抗人捕获抗体共价偶联于CM5生物芯片(GE，BR-1000-12)上，用以捕获本发明的PD-L1抗体。然后于芯片表面流经一系列浓度的人PD-L1抗原(hPD-L1-his，Sino biological 10084-H08H-200)或食蟹猴PD-L1抗原(Cyno-PD-L1-his，Sino biologica1 90251-C08H-200)，利用Biacore仪器(GE，BiacoreT200)实时检测反应信号从而获得结合和解离曲线，通过软件拟合得到亲和力数值。在实验中的每个循环解离完成后，使用再生溶液将生物芯片再生，继而进行下一次捕获，循环往复完成不同抗体对PD-L1亲和力的测定。最后使用GE BIAevaluation软件以1:1(Langmuir)结合模型分析所得数据，以此法测定ka(kon)、kd(koff)和K D值。通过K D＝kd/ka计算平衡解离常数K D。下表是嵌合抗体对人PD-L1抗原、食蟹猴PD-L1抗原的亲和力数据。 The affinity of the PD-L1 chimeric antibody to the PD-L1 antigen was determined by Biacore, and the anti-human capture antibody was covalently coupled using the anti-human antibody capture kit (GE, BR-1008-39) according to the method described in the specification. The CM5 biochip (GE, BR-1000-12) was used to capture the PD-L1 antibody of the present invention. Then, a series of concentrations of human PD-L1 antigen (hPD-L1-his, Sino biological 10084-H08H-200) or cynomolgus PD-L1 antigen (Cyno-PD-L1-his, Sino biologica1 90251) were flowed on the surface of the chip. -C08H-200), the reaction signal was detected in real time using a Biacore instrument (GE, Biacore T200) to obtain binding and dissociation curves, and the affinity values were obtained by software fitting. After each cycle of dissociation in the experiment was completed, the biochip was regenerated using the regeneration solution, followed by the next capture, and the determination of the affinity of the different antibodies for PD-L1 was performed cyclically. Finally GE BIAevaluation software using 1: 1 (Langmuir) binding model analysis data obtained, this determination of ka (kon), kd (koff ) and K D values. The equilibrium dissociation constant K D is calculated by K D =kd/ka. The following table is the affinity data of chimeric antibodies against human PD-L1 antigen, cynomolgus PD-L1 antigen.
表1：抗PD-L1抗体与PD-L1的亲和力数据Table 1: Affinity data of anti-PD-L1 antibody and PD-L1
Figure PCTCN2018119536-appb-000003
Figure PCTCN2018119536-appb-000003
Figure PCTCN2018119536-appb-000004
Figure PCTCN2018119536-appb-000004
ND：未测试ND: not tested
*：WO2016022630中公开的一种人源化的PD-L1抗体*: A humanized PD-L1 antibody disclosed in WO2016022630
4.2抗人PD-L1嵌合抗体结合PD-L1的活性4.2 Anti-human PD-L1 chimeric antibody binds to PD-L1 activity
通过ELISA方法，具体操作参照实施例2中的检测方法1.1，使用hPD-L1-mFc代替PD-L1-his蛋白进行嵌合抗体的PD-L1结合分析。最后，酶标仪在450nm处读取的比色信号使用GraphPad Prism5分析数据，并计算EC50，见表2，嵌合抗体1(Chi1)和嵌合抗体2-1(Chi2-1)与对照抗体相比具有相当的PD-L1结合活性，其中嵌合抗体1略优。The PD-L1 binding assay of the chimeric antibody was carried out by the ELISA method, referring to the detection method 1.1 in Example 2, using hPD-L1-mFc instead of the PD-L1-his protein. Finally, the colorimetric signal read by the microplate reader at 450 nm was analyzed using GraphPad Prism5 and the EC50 was calculated. See Table 2, chimeric antibody 1 (Chi1) and chimeric antibody 2-1 (Chi2-1) and control antibody. Chimeric antibody 1 was slightly superior compared to having comparable PD-L1 binding activity.
表2：嵌合抗体Chi1和Chi2的抗原结合数据Table 2: Antigen binding data of chimeric antibodies Chi1 and Chi2
PD-L1结合PD-L1 combination Chi1Chi1 Chi2-1Chi2-1 TecentriqTecentriq
EC50(ng/ml)EC50 (ng/ml) 107.3107.3 145.2145.2 142.3142.3
4.3抗人PD-L1嵌合抗体阻断PD-L1/PD-1的结合活性4.3 Anti-human PD-L1 chimeric antibody blocks PD-L1/PD-1 binding activity
基于ELASA方法，检测嵌合抗体是否能够阻断PD-1与PD-L1的结合。具体操作参照实施例1中的检测方法1.2，最后酶标仪在450nm处读取的比色信号使用GraphPad Prism5分析数据，并计算IC50，见表3，嵌合抗体1、嵌合抗体2-1与对照抗体相比具有相当的阻断PD-1与PD-L1结合的活性，并且嵌合抗体1、嵌合抗体2-1活性略优。Based on the ELASA method, it was detected whether the chimeric antibody could block the binding of PD-1 to PD-L1. For specific operation, refer to the detection method 1.2 in Example 1. Finally, the colorimetric signal read by the microplate reader at 450 nm was analyzed using GraphPad Prism5, and the IC50 was calculated. See Table 3, chimeric antibody 1, chimeric antibody 2-1. The activity of blocking PD-1 binding to PD-L1 was comparable to that of the control antibody, and the chimeric antibody 1 and chimeric antibody 2-1 were slightly more active.
表3：嵌合抗体Chi1和Chi2阻断PD-1结合的IC50数据Table 3: IC50 data for chimeric antibodies Chi1 and Chi2 blocking PD-1 binding
PD-L1阻断PD-L1 blocking Chi1Chi1 Chi2-1Chi2-1 Chi2-2Chi2-2 TecentriqTecentriq
IC50(ng/ml)IC50 (ng/ml) 514.5514.5 462.1462.1 14361436 553.2553.2
4.4抗PD-L1嵌合抗体基于细胞的结合分析4.4 Cell-based binding analysis of anti-PD-L1 chimeric antibodies
通过分析与稳定表达PD-L1的U2OS细胞系(PD-L1-U2OS)的结合实验，分析抗PD-L1嵌合抗体与天然表达的PD-L1的结合能力。将2×10 5个PD-L1-U2OS细胞加入到96孔培养板的各孔中，将梯度稀释的抗PD-L1抗体添加到细胞悬浮液中，同时加入生物素标记的PD-1蛋白与其竞争结合PD-L1。于4℃下孵育120分钟后，将细胞用PBS洗涤3次，加入亲和素-FITC(eBioscience，11-4317-87)4℃下孵育60分钟。然后通过使用流式细胞术(BD，Accuri C6)分析检测荧光信号。通过染色的平均荧光强度(MFI)来测量抗PD-L1抗体与PD-1对PD-L1的竞争结合。使用GraphPad Prism5分析数据，计算的IC50在表4中示出，嵌合抗体1、嵌合抗体2-1与对照抗体相比具有相当的阻断PD-1与细胞表面PD-L1结合的活性，并且嵌合抗体1、嵌合抗体2-1活性略优。 The binding ability of the anti-PD-L1 chimeric antibody to the naturally expressed PD-L1 was analyzed by analyzing binding experiments with the U2OS cell line (PD-L1-U2OS) stably expressing PD-L1. 2×10 5 PD-L1-U2OS cells were added to each well of a 96-well culture plate, and a gradient-diluted anti-PD-L1 antibody was added to the cell suspension while biotin-labeled PD-1 protein was added thereto. Competition combines with PD-L1. After incubation at 4 ° C for 120 minutes, the cells were washed 3 times with PBS and incubated with avidin-FITC (eBioscience, 11-4317-87) for 60 minutes at 4 °C. Fluorescence signals were then detected by flow cytometry (BD, Accuri C6) analysis. Competitive binding of anti-PD-L1 antibody to PD-1 to PD-L1 was measured by mean fluorescence intensity (MFI) of staining. The data were analyzed using GraphPad Prism5, and the calculated IC50 is shown in Table 4. Chimeric antibody 1 and chimeric antibody 2-1 have comparable activities for blocking PD-1 binding to cell surface PD-L1 compared to the control antibody. Furthermore, chimeric antibody 1 and chimeric antibody 2-1 were slightly more active.
表4：嵌合抗体Chi1和Chi2阻断PD-1结合的IC50数据Table 4: IC50 data for chimeric antibodies Chi1 and Chi2 blocking PD-1 binding
U2OS PD-L1阻断U2OS PD-L1 blocking Chi1Chi1 Chi2-1Chi2-1 Chi2-2Chi2-2 TecentriqTecentriq
IC50(ng/ml)IC50 (ng/ml) 195.3195.3 248.3248.3 21672167 342.1342.1
4.5抗PD-L1嵌合抗体阻断细胞/细胞相互作用的分析4.5 Analysis of anti-PD-L1 chimeric antibody blocking cell/cell interaction
在该检测体系中，分别使用了经过改造的稳定表达PD-1的Jurkat细胞(DiscoverX，93-1104C19)和稳定表达PD-L1的U2OS细胞(DiscoverX，93-1066C3)。当U2OS细胞表面的PD-L1与Jurkat表面的PD-1结合后，经过改造的Jurkat细胞会诱导下游通路并与检测底物反应产生化学发光信号。因此，当体系中存在抗PD-L1抗体，并且能够阻断PD-1与PD-L1的结合时，会没有化学信号或较弱的化学信号产生。区别于前述实例中使用重组蛋白作为筛选，该检测方法中PD-L1和PD-1均以天然的构象表达于细胞表面。实验中，取处于指数生长期状态良好的配体细胞(U2OS PD-L1细胞，9.6×10 5个细胞/ml)，使用自动分液器加入384孔板，12.5μL/孔；每孔中加入梯度稀释的抗体；同时，取处于指数生长期状态良好的受体细胞 (Jurkat PD-1细胞)，6.4×10 5个/ml，使用自动分液器接种于384孔板上，12.5μL/孔，混匀，室温孵育120min后，每孔加入2.5μL Bioassay Reagent 1(DiscoverX，93-0933)，混匀室温避光孵育15min。然后每孔加入10μL Bioassay Reagent 2(DiscoverX，93-0933)，混匀室温避光孵育60min。酶标仪读取化学发光信号。使用GraphPad Prism5分析数据，并计算IC50值。表5的结果表明，嵌合抗体Chi1与对照抗体相比，具有相当的阻断U2OS表面PD-L1与Jurkat表面PD-1结合的活性，其中Chi1的阻断活性较优。 In this assay system, engineered Jurkat cells stably expressing PD-1 (DiscoverX, 93-1104C19) and U2OS cells stably expressing PD-L1 (DiscoverX, 93-1066C3) were used, respectively. When PD-L1 on the surface of U2OS cells binds to PD-1 on the surface of Jurkat, the engineered Jurkat cells induce a downstream pathway and react with the substrate to produce a chemiluminescent signal. Therefore, when an anti-PD-L1 antibody is present in the system and is capable of blocking the binding of PD-1 to PD-L1, there is no chemical signal or a weak chemical signal. In contrast to the previous examples, recombinant proteins were used as screens in which both PD-L1 and PD-1 were expressed in the native conformation on the cell surface. In the experiment, the ligand cells in the exponential growth phase (U2OS PD-L1 cells, 9.6×10 5 cells/ml) were added to the 384-well plate using an automatic dispenser, 12.5 μL/well; Gradiently diluted antibody; at the same time, the recipient cells (Jurkat PD-1 cells) in good exponential growth phase, 6.4×10 5 /ml, were inoculated on a 384-well plate using an automatic dispenser, 12.5 μL/well. After mixing for 120 min at room temperature, 2.5 μL of Bioassay Reagent 1 (DiscoverX, 93-0933) was added to each well, and the mixture was incubated at room temperature for 15 min in the dark. Then, 10 μL of Bioassay Reagent 2 (DiscoverX, 93-0933) was added to each well, and the mixture was incubated at room temperature for 60 minutes in the dark. The microplate reader reads the chemiluminescent signal. Data was analyzed using GraphPad Prism5 and IC50 values were calculated. The results in Table 5 indicate that the chimeric antibody Chi1 has comparable activity to blocking PD-1 binding of PD-L1 on the surface of U2OS to PD-1 on the surface of Jurkat, and the blocking activity of Chi1 is superior.
表5：嵌合抗体Chi1阻断细胞/细胞结合的IC50数据Table 5: IC50 data for chimeric antibody Chi1 blocking cell/cell binding
Figure PCTCN2018119536-appb-000005
Figure PCTCN2018119536-appb-000005
实施例5抗人PD-L1杂交瘤单克隆抗体的人源化Example 5 Humanization of anti-human PD-L1 hybridoma monoclonal antibody
根据上述嵌合抗体的活性，选择最优的Chi1进行人源化。通过搜索对比IMGT人类抗体重轻链可变区种系基因数据库(http://www.imgt.org/3Dstructure-DB/cgi/DomainGapAlign.cgi)，挑选与Chi1同源性高的重轻链可变区种系基因(Germline sequence)作为模板，将鼠源抗体的CDR区通过CDR移植的方法移植到相应的人源模板中，形成次序为FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4的可变区序列(其中氨基酸残基由Kabat编号系统确定并注释)。鼠源抗体Chi1的重链人源化模板为IGHV4-30-4*01和IGHJ1*01的组合；轻链人源化模板为IGKV1-39*01和IGKJ2*02的组合，CDR直接移植后的序列为SEQ ID NOs:45和46。直接人源化后的抗体的亲和力一般会有很大程度下降，需要进一步设计回复突变以恢复亲和力和功能，即将上述人源模板抗体的框架氨基酸的几个位点回复突变为小鼠Chi1抗体中对应的框架区中的氨基酸序列。对于人源化Chi1抗体的重链可变区，将35位(按照Kabat编号，下同)的Ser(S)突变回Asn(N)，47位的Trp(W)突变回Tyr(Y)，以及71位的Val(V)突变回Arg(R)；对于人源化Chi1抗体的轻链可变区，将第2位的Ile(I)突变回Phe(F)，将第41位的Gly(G)突变为Asp(D)，以及将第44位的Pro(P)突变成Val(V)。编码人源化hu-Chi1抗体的VH和VL的序列分别为SEQ ID NO:47和51，并与人抗体恒定区连接构建成完整的抗体形式，分别为SEQ ID NOs:53和55。Based on the activity of the chimeric antibody described above, the optimal Chi1 was selected for humanization. By searching for the IMGT human antibody heavy light chain variable region germline gene database (http://www.imgt.org/3Dstructure-DB/cgi/DomainGapAlign.cgi), we can select heavy and light chains with high homology to Chi1. The Germline sequence is used as a template to transplant the CDR regions of the murine antibody into the corresponding human template by CDR grafting, and the order of formation is FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. Variable region sequences (where amino acid residues are determined and annotated by the Kabat numbering system). The heavy chain humanized template of the murine antibody Chi1 is a combination of IGHV4-30-4*01 and IGHJ1*01; the light chain humanized template is a combination of IGKV1-39*01 and IGKJ2*02, after direct CDR grafting The sequences are SEQ ID NOs: 45 and 46. The affinity of the antibody after direct humanization generally decreases to a large extent, and further design of the back mutation is needed to restore affinity and function, that is, several sites of the framework amino acid of the above human template antibody are back-mutated into the mouse Chi1 antibody. The amino acid sequence in the corresponding framework region. For the heavy chain variable region of the humanized Chi1 antibody, the Ser(S) at position 35 (according to Kabat numbering, the same below) was mutated back to Asn(N), and the Trp(W) at position 47 was mutated back to Tyr(Y). And the Val(V) mutation at position 71 back to Arg(R); for the light chain variable region of the humanized Chi1 antibody, the second Ile(I) is mutated back to Phe(F), and the 41st position is Gly (G) is mutated to Asp (D), and the 44th Pro (P) is mutated to Val (V). The sequences encoding VH and VL of the humanized hu-Chi1 antibody are SEQ ID NOS: 47 and 51, respectively, and ligated to the human antibody constant region to construct the complete antibody form, SEQ ID NOs: 53 and 55, respectively.
将上述编码人源化hu-Chi1-3.4抗体的轻链和重链的DNA序列(SEQ ID NOs:54和56)。克隆到表达载体pcDNA3.1(Invitrogen，V-790)中。将上述DNA载体转染进入Expi CHO细胞(50mL体系，5×10 6/mL细胞，DNA 1μg/ml)中，进行瞬时表达，培养7天。然后用Protein A柱(GE healthcare)纯化上清液中的人源化抗体。 The DNA sequences encoding the light and heavy chains of the humanized hu-Chi1-3.4 antibody described above (SEQ ID NOs: 54 and 56). The vector was cloned into the expression vector pcDNA3.1 (Invitrogen, V-790). The above DNA vector was transfected into Expi CHO cells (50 mL system, 5 × 10 6 /mL cells, DNA 1 μg/ml), transiently expressed, and cultured for 7 days. The humanized antibody in the supernatant was then purified using a Protein A column (GE healthcare).
表6：Chi1抗体的人源化回复突变位点Table 6: Humanized back-mutation sites for Chi1 antibodies
Figure PCTCN2018119536-appb-000006
Figure PCTCN2018119536-appb-000006
Grafted:表示将鼠源CDR直接移植到人FR中。Grafted: indicates that the mouse CDR is directly transplanted into the human FR.
抗体编号规则按照Kabat命名法，如W47Y表示将47位的W突变为Y。The antibody numbering rule mutates the W at position 47 to Y according to the Kabat nomenclature, such as W47Y.
根据表6对人源化PD-L1抗体进行编号，如表7所示。Humanized PD-L1 antibodies were numbered according to Table 6, as shown in Table 7.
表7：人源化PD-L1抗体编号Table 7: Humanized PD-L1 antibody numbering
Figure PCTCN2018119536-appb-000007
Figure PCTCN2018119536-appb-000007
Figure PCTCN2018119536-appb-000008
Figure PCTCN2018119536-appb-000008
实施例6人源化抗PD-L1抗体活性评价Example 6 Evaluation of humanized anti-PD-L1 antibody activity
6.1抗PD-L1人源化抗体的亲和力6.1 Affinity of anti-PD-L1 humanized antibody
Biacore测定抗PD-L1人源化抗体对PD-L1抗原的亲和力，具体步骤参照实施例4.1，下表8是人源化hu-Chi1、hu-Chi1-3.3、hu-Chi1-3.4以及嵌合抗体Chi1的亲和力数据，表明在回复突变过程中人源化的PD-L1抗体较好的维持了对PD-L1的亲和力。Biacore measures the affinity of anti-PD-L1 humanized antibody for PD-L1 antigen. The specific steps are as follows: Example 4.1, Table 8 below is humanized hu-Chi1, hu-Chi1-3.3, hu-Chi1-3.4 and chimeric Affinity data for antibody Chi1 indicated that the humanized PD-L1 antibody maintained a good affinity for PD-L1 during the back mutation.
表8：人源化Chi1抗体的亲和力数据Table 8: Affinity data for humanized Chi1 antibodies
抗PD-L1抗体anti-PD-L1 antibody 重链可变区Heavy chain variable region 轻链可变区Light chain variable region K D(人源化) K D (humanization)
hu-Chi1hu-Chi1 VH.1VH.1 VL.lVL.l 3.14E-093.14E-09
hu-Chi1-3.3hu-Chi1-3.3 Chi1_VL.1CChi1_VL.1C Chi1_VH.1CChi1_VH.1C 8.15E-108.15E-10
hu-Chi1-3.4hu-Chi1-3.4 Chi1_VL.1CChi1_VL.1C Chi1_VH.1DChi1_VH.1D 7.99E-107.99E-10
Chi1Chi1 VHVH VLVL 6.74E-106.74E-10
6.2抗PD-L1人源化抗体阻断细胞/细胞相互作用的分析6.2 Analysis of anti-PD-L1 humanized antibody blocking cell/cell interaction
通过细胞/细胞相互作用分析抗PD-L1人源化抗体的阻断作用，具体步骤参照实施例4.5。表9的结果表明，人源化抗体与母本嵌合抗体有相当的阻断作用活性。The blocking effect of the anti-PD-L1 humanized antibody was analyzed by cell/cell interaction, and the specific procedure was as described in Example 4.5. The results in Table 9 indicate that the humanized antibody has a comparable blocking activity as the maternal chimeric antibody.
表9：人源化抗体与嵌合抗体阻断细胞相互作用的IC50数据比较Table 9: Comparison of IC50 data for humanized antibody and chimeric antibody blocking cell interaction
Figure PCTCN2018119536-appb-000009
Figure PCTCN2018119536-appb-000009

Claims (35)

  1. 一种分离的结合PD-L1的抗原结合蛋白或其片段，其中所述抗原结合蛋白或其片段包含以下序列：重链HCDR1序列，其与选自SEQ ID NO:17、18、29或30的氨基酸序列具有至少80％同源性；重链HCDR2，其与选自SEQ ID NO:12、31或32的氨基酸序列具有至少80％同源性；重链HCDR3序列，其与选自SEQ ID NO:19-22或33-34的氨基酸序列具有至少80％同源性；轻链LCDR1序列，其与选自SEQ ID NO:14、35或36的氨基酸序列具有至少80％同源性；轻链LCDR2序列，其与选自SEQ ID NO:15或27的氨基酸序列具有至少80％同源性；轻链LCDR3序列，其与选自SEQ ID NO:16、37或38的氨基酸序列具有至少80％同源性。An isolated PD-L1 binding antigen binding protein or fragment thereof, wherein the antigen binding protein or fragment thereof comprises the sequence: a heavy chain HCDR1 sequence selected from the group consisting of SEQ ID NO: 17, 18, 29 or 30 The amino acid sequence has at least 80% homology; a heavy chain HCDR2 having at least 80% homology to an amino acid sequence selected from the group consisting of SEQ ID NO: 12, 31 or 32; a heavy chain HCDR3 sequence selected from the group consisting of SEQ ID NO The amino acid sequence of 19-22 or 33-34 has at least 80% homology; the light chain LCDR1 sequence which has at least 80% homology to the amino acid sequence selected from SEQ ID NO: 14, 35 or 36; light chain An LCDR2 sequence having at least 80% homology to an amino acid sequence selected from the group consisting of SEQ ID NO: 15 or 27; a light chain LCDR3 sequence having at least 80% of an amino acid sequence selected from SEQ ID NO: 16, 37 or 38 Homology.
  2. 如权利要求1所述的抗原结合蛋白或其片段，其中所述抗原结合蛋白或其片段包含以下序列：重链The antigen binding protein of claim 1 or a fragment thereof, wherein the antigen binding protein or fragment thereof comprises the following sequence: heavy chain
    HCDR1序列，其选自SEQ ID NO:17、18、29或30所示的氨基酸序列；重链HCDR2，其选自SEQ ID NO:12、31或32所示的氨基酸序列；重链HCDR3序列，其选自SEQ ID NO:19-22或33-34所示的氨基酸序列；轻链LCDR1序列，其选自SEQ ID NO:14、35或36所示的氨基酸序列；轻链LCDR2序列，其选自SEQ ID NO:15或27所示的氨基酸序列；轻链LCDR3序列，其选自SEQ ID NO:16、37或38所示的氨基酸序列。An HCDR1 sequence selected from the group consisting of the amino acid sequence set forth in SEQ ID NO: 17, 18, 29 or 30; the heavy chain HCDR2 selected from the amino acid sequence set forth in SEQ ID NO: 12, 31 or 32; the heavy chain HCDR3 sequence, It is selected from the amino acid sequence set forth in SEQ ID NO: 19-22 or 33-34; the light chain LCDR1 sequence selected from the amino acid sequence set forth in SEQ ID NO: 14, 35 or 36; the light chain LCDR2 sequence, selected The amino acid sequence set forth in SEQ ID NO: 15 or 27; the light chain LCDR3 sequence selected from the amino acid sequences set forth in SEQ ID NO: 16, 37 or 38.
  3. 如权利要求1所述的抗原结合蛋白或其片段，其中所述结合PD-L1的抗原结合蛋白或其片段包含重链HCDR1、HCDR2和HCDR3及轻链LCDR1、LCDR2和LCDR3，所述重链HCDR1、HCDR2和HCDR3及轻链LCDR1、LCDR2和LCDR3选自以下序列或与其具有至少80％同源性的序列：The antigen binding protein or fragment thereof according to claim 1, wherein the PD-L1 binding antigen binding protein or fragment thereof comprises heavy chain HCDR1, HCDR2 and HCDR3 and light chain LCDR1, LCDR2 and LCDR3, said heavy chain HCDR1 , HCDR2 and HCDR3 and light chain LCDR1, LCDR2 and LCDR3 are selected from the following sequences or sequences having at least 80% homology thereto:
    HCDR1选自：SDYWX 1                           SEQ ID NO:11 HCDR1 is selected from the group consisting of: SDYWX 1 SEQ ID NO: 11
    或        X 3YWMN                            SEQ ID NO:23 Or X 3 YWMN SEQ ID NO: 23
    HCDR2选自：YISYTGSTYYNPSLKS                 SEQ ID NO:12HCDR2 is selected from: YISYTGSTYYNPSLKS SEQ ID NO: 12
    或         MIHPSDSETX 4LNQKFKD               SEQ ID NO:24 Or MIHPSDSETX 4 LNQKFKD SEQ ID NO: 24
    HCDR3选自：GEAWDRRTLDX 2                     SEQ ID NO:13 HCDR3 is selected from: GEAWDRRTLDX 2 SEQ ID NO: 13
    或         X 5MVWX 6RX 7VMDY                   SEQ ID NO:25 Or X 5 MVWX 6 RX 7 VMDY SEQ ID NO: 25
    LCDR1选自：RASQDITNYLN                      SEQ ID NO:14LCDR1 is selected from: RASQDITNYLN SEQ ID NO: 14
    或         KSSQSLLDSDGX 8TYLN                SEQ ID NO:26 Or KSSQSLLDSDGX 8 TYLN SEQ ID NO:26
    LCDR2选自：YTSRLHS                          SEQ ID NO:15LCDR2 is selected from: YTSRLHS SEQ ID NO: 15
    或         LVSKLDS                          SEQ ID NO:27Or LVSKLDS SEQ ID NO:27
    LCDR3选自：QQVYTLPWT                        SEQ ID NO:16LCDR3 is selected from: QQVYTLPWT SEQ ID NO: 16
    或         LQX 9THFPYT                       SEQ ID NO:28； Or LQX 9 THFPYT SEQ ID NO: 28;
    其中X 1选自N或S，X 2选自Y、C、A或V，X 3选自S或F，X 4选自R或W，X 5选自S或N，X 6选自T或S，X 7选自Q或H，X 8选自E或N，X 9选自S或A。 Wherein X 1 is selected from N or S, X 2 is selected from Y, C, A or V, X 3 is selected from S or F, X 4 is selected from R or W, X 5 is selected from S or N, and X 6 is selected from T. Or S, X 7 is selected from Q or H, X 8 is selected from E or N, and X 9 is selected from S or A.
  4. 如权利要求1所述的抗原结合蛋白或其片段，其中所述结合PD-L1的抗原结合蛋白或其片段包含重链HCDR1、HCDR2和HCDR3，所述重链HCDR1、HCDR2和HCDR3包含分别与根据SEQ ID NO:17、12和19的氨基酸序列具有至少80％同源性的氨基酸序列；以及轻链LCDR1、LCDR2和LCDR3，所述轻链LCDR1、LCDR2和LCDR3包含分别与根据SEQ ID NO:14、15和16的氨基酸序列具有至少80％同源性的氨基酸序列。The antigen-binding protein or fragment thereof according to claim 1, wherein the PD-L1 binding antigen binding protein or fragment thereof comprises a heavy chain HCDR1, HCDR2 and HCDR3, the heavy chain HCDR1, HCDR2 and HCDR3 comprising The amino acid sequences of SEQ ID NOS: 17, 12 and 19 have an amino acid sequence of at least 80% homology; and the light chain LCDR1, LCDR2 and LCDR3 comprising, respectively, according to SEQ ID NO: 14 The amino acid sequences of 15 and 16 have an amino acid sequence of at least 80% homology.
  5. 如权利要求1所述的抗原结合蛋白或其片段，其中所述结合PD-L1的抗原结合蛋白或其片段包含重链HCDR1、HCDR2和HCDR3，所述重链HCDR1、HCDR2和HCDR3包含分别与根据SEQ ID NO:18、12和20的氨基酸序列具有至少80％同源性的氨基酸序列；以及轻链LCDR1、LCDR2和LCDR3，所述轻链LCDR1、LCDR2和LCDR3包含分别与根据SEQ ID NO:14、15和16的氨基酸序列具有至少80％同源性的氨基酸序列。The antigen-binding protein or fragment thereof according to claim 1, wherein the PD-L1 binding antigen binding protein or fragment thereof comprises a heavy chain HCDR1, HCDR2 and HCDR3, the heavy chain HCDR1, HCDR2 and HCDR3 comprising The amino acid sequences of SEQ ID NOS: 18, 12 and 20 have an amino acid sequence of at least 80% homology; and the light chain LCDR1, LCDR2 and LCDR3 comprising, respectively, according to SEQ ID NO: 14 The amino acid sequences of 15 and 16 have an amino acid sequence of at least 80% homology.
  6. 如权利要求1所述的抗原结合蛋白或其片段，其中所述结合PD-L1的抗原结合蛋白或其片段包含重链HCDR1、HCDR2和HCDR3，所述重链HCDR1、HCDR2和HCDR3包含分别与根据SEQ ID NO:17、12和20的氨基酸序列具有至少80％同源性的氨基酸序列；以及轻链LCDR1、LCDR2和LCDR3，所述轻链LCDR1、LCDR2和LCDR3包含分别与根据SEQ ID NO:14、15和16的氨基酸序列具有至少80％同源性 的氨基酸序列。The antigen-binding protein or fragment thereof according to claim 1, wherein the PD-L1 binding antigen binding protein or fragment thereof comprises a heavy chain HCDR1, HCDR2 and HCDR3, the heavy chain HCDR1, HCDR2 and HCDR3 comprising The amino acid sequences of SEQ ID NOS: 17, 12 and 20 have an amino acid sequence of at least 80% homology; and light chain LCDR1, LCDR2 and LCDR3 comprising, respectively, according to SEQ ID NO: 14 The amino acid sequences of 15 and 16 have an amino acid sequence of at least 80% homology.
  7. 如权利要求1所述的抗原结合蛋白或其片段，其中所述结合PD-L1的抗原结合蛋白或其片段包含重链HCDR1、HCDR2和HCDR3，所述重链HCDR1、HCDR2和HCDR3包含分别与根据SEQ ID NO:17、12和21的氨基酸序列具有至少80％同源性的氨基酸序列；以及轻链LCDR1、LCDR2和LCDR3，所述轻链LCDR1、LCDR2和LCDR3包含分别与根据SEQ ID NO:14、15和16的氨基酸序列具有至少80％同源性的氨基酸序列。The antigen-binding protein or fragment thereof according to claim 1, wherein the PD-L1 binding antigen binding protein or fragment thereof comprises a heavy chain HCDR1, HCDR2 and HCDR3, the heavy chain HCDR1, HCDR2 and HCDR3 comprising The amino acid sequences of SEQ ID NOS: 17, 12 and 21 have an amino acid sequence of at least 80% homology; and the light chain LCDR1, LCDR2 and LCDR3 comprising, respectively, according to SEQ ID NO: 14 The amino acid sequences of 15 and 16 have an amino acid sequence of at least 80% homology.
  8. 如权利要求1所述的抗原结合蛋白或其片段，其中所述结合PD-L1的抗原结合蛋白或其片段包含重链HCDR1、HCDR2和HCDR3，所述重链HCDR1、HCDR2和HCDR3包含分别与根据SEQ ID NO:17、12和22的氨基酸序列具有至少80％同源性的氨基酸序列；以及轻链LCDR1、LCDR2和LCDR3，所述轻链LCDR1、LCDR2和LCDR3包含分别与根据SEQ ID NO:14、15和16的氨基酸序列具有至少80％同源性的氨基酸序列。The antigen-binding protein or fragment thereof according to claim 1, wherein the PD-L1 binding antigen binding protein or fragment thereof comprises a heavy chain HCDR1, HCDR2 and HCDR3, the heavy chain HCDR1, HCDR2 and HCDR3 comprising The amino acid sequences of SEQ ID NOS: 17, 12 and 22 have an amino acid sequence of at least 80% homology; and the light chain LCDR1, LCDR2 and LCDR3 comprising, respectively, according to SEQ ID NO: 14 The amino acid sequences of 15 and 16 have an amino acid sequence of at least 80% homology.
  9. 如权利要求1所述的抗原结合蛋白或其片段，其中所述结合PD-L1的抗原结合蛋白或其片段包含重链HCDR1、HCDR2和HCDR3，所述重链HCDR1、HCDR2和HCDR3包含分别与根据SEQ ID NO:29、31和33的氨基酸序列具有至少80％同源性的氨基酸序列；以及轻链LCDR1、LCDR2和LCDR3，所述轻链LCDR1、LCDR2和LCDR3包含分别与根据SEQ ID NO:35、27和37的氨基酸序列具有至少80％同源性的氨基酸序列。The antigen-binding protein or fragment thereof according to claim 1, wherein the PD-L1 binding antigen binding protein or fragment thereof comprises a heavy chain HCDR1, HCDR2 and HCDR3, the heavy chain HCDR1, HCDR2 and HCDR3 comprising The amino acid sequences of SEQ ID NOS: 29, 31 and 33 have an amino acid sequence of at least 80% homology; and the light chain LCDR1, LCDR2 and LCDR3 comprising, respectively, according to SEQ ID NO: 35 The amino acid sequences of 27 and 37 have an amino acid sequence of at least 80% homology.
  10. 如权利要求1所述的抗原结合蛋白或其片段，其中所述结合PD-L1的抗原结合蛋白或其片段包含重链HCDR1、HCDR2和HCDR3，所述重链HCDR1、HCDR2和HCDR3包含分别与根据SEQ ID NO:30、32和34的氨基酸序列具有至少80％同源性的氨基酸序列；以及轻链LCDR1、LCDR2和LCDR3，所述轻链LCDR1、LCDR2和LCDR3包含分别与根据SEQ ID NO:36、27和38的氨基酸序列具有至少80％同源性的氨基酸序列。The antigen-binding protein or fragment thereof according to claim 1, wherein the PD-L1 binding antigen binding protein or fragment thereof comprises a heavy chain HCDR1, HCDR2 and HCDR3, the heavy chain HCDR1, HCDR2 and HCDR3 comprising The amino acid sequences of SEQ ID NOS: 30, 32 and 34 have an amino acid sequence of at least 80% homology; and the light chain LCDR1, LCDR2 and LCDR3 comprising, respectively, according to SEQ ID NO: 36 The amino acid sequences of 27 and 38 have an amino acid sequence of at least 80% homology.
  11. 如权利要求1-10中任一项所述的分离的抗原结合蛋白或其片段，其中所述抗原结合蛋白或其片段是抗体或其片段，优选是嵌合的或人源化的抗体或其片段。The isolated antigen-binding protein or fragment thereof according to any one of claims 1 to 10, wherein the antigen-binding protein or fragment thereof is an antibody or a fragment thereof, preferably a chimeric or humanized antibody or Fragment.
  12. 一种分离的结合PD-L1的抗原结合蛋白或其片段，其中所述抗原结合蛋白或其片段包含重链可变区，所述重链可变区包含与选自由SEQ ID NO:5、7、9、45、47、48、49和50组成的组中的氨基酸序列具有至少80％同源性的氨基酸序列；以及轻链可变区，所述轻链可变区包含与选自由SEQ ID NO:6、8、10、46、51和52组成的组中的氨基酸序列具有至少80％同源性的氨基酸序列。An isolated PD-L1 binding antigen binding protein or fragment thereof, wherein the antigen binding protein or fragment thereof comprises a heavy chain variable region comprising and selected from SEQ ID NOs: 5, 7 , the amino acid sequence of the group consisting of 9, 45, 47, 48, 49 and 50 having an amino acid sequence of at least 80% homology; and a light chain variable region comprising and selected from the group consisting of SEQ ID NO: The amino acid sequence of the group consisting of 6, 8, 10, 46, 51 and 52 has an amino acid sequence of at least 80% homology.
  13. 如权利要求12所述的分离的抗原结合蛋白或其片段，其中所述抗原结合蛋白或其片段包含重链可变区，所述重链可变区包含选自由SEQ ID NO:5、7、9、45、47、48、49和50组成的组中的氨基酸序列；以及轻链可变区，所述轻链可变区包含选自由SEQ ID NO:6、8、10、46、51和52组成的组中的氨基酸序列。The isolated antigen-binding protein or fragment thereof of claim 12, wherein the antigen-binding protein or fragment thereof comprises a heavy chain variable region comprising SEQ ID NOs: 5, 7, An amino acid sequence in the group consisting of 9, 45, 47, 48, 49, and 50; and a light chain variable region comprising SEQ ID NOS: 6, 8, 10, 46, 51 and The amino acid sequence in the group consisting of 52.
  14. 一种分离的结合PD-L1的抗原结合蛋白或其片段，其中所述抗原结合蛋白或其片段包含根据SEQ ID NO:45的重链可变区和根据SEQ ID NO:46的轻链可变区。An isolated PD-L1 binding antigen binding protein or fragment thereof, wherein the antigen binding protein or fragment thereof comprises a heavy chain variable region according to SEQ ID NO: 45 and a light chain variable according to SEQ ID NO: 46 Area.
  15. 一种分离的结合PD-L1的抗原结合蛋白或其片段，其中所述抗原结合蛋白或其片段包含根据SEQ ID NO:47的重链可变区和根据SEQ ID NO:51的轻链可变区。An isolated PD-L1 binding antigen binding protein or fragment thereof, wherein the antigen binding protein or fragment thereof comprises a heavy chain variable region according to SEQ ID NO: 47 and a light chain variable according to SEQ ID NO: 51 Area.
  16. 一种分离的结合PD-L1的抗原结合蛋白或其片段，其中所述抗原结合蛋白或其片段包含根据SEQ ID NO:48的重链可变区和根据SEQ ID NO:52的轻链可变区。An isolated PD-L1 binding antigen binding protein or fragment thereof, wherein the antigen binding protein or fragment thereof comprises a heavy chain variable region according to SEQ ID NO: 48 and a light chain variable according to SEQ ID NO: 52 Area.
  17. 一种分离的结合PD-L1的抗原结合蛋白或其片段，其中所述抗原结合蛋白或其片段包含根据SEQ ID NO:49的重链可变区和根据SEQ ID NO:51的轻链可变区。An isolated PD-L1 binding antigen binding protein or fragment thereof, wherein the antigen binding protein or fragment thereof comprises a heavy chain variable region according to SEQ ID NO: 49 and a light chain variable according to SEQ ID NO: 51 Area.
  18. 一种分离的结合PD-L1的抗原结合蛋白或其片段，其中所述抗原结合蛋白或其片段包含根据SEQ ID NO:50的重链可变区和根据SEQ ID NO:51的轻链可变区。An isolated PD-L1 binding antigen binding protein or fragment thereof, wherein the antigen binding protein or fragment thereof comprises a heavy chain variable region according to SEQ ID NO: 50 and a light chain variable according to SEQ ID NO: 51 Area.
  19. 一种分离的抗原结合蛋白或其片段，其中所述抗原结合蛋白或其片段与如权利要求1至18中任一项所述的抗原结合蛋白或其片段结合相同表位。An isolated antigen binding protein or fragment thereof, wherein the antigen binding protein or fragment thereof binds to the same epitope as the antigen binding protein of any one of claims 1 to 18 or a fragment thereof.
  20. 一种分离的抗原结合蛋白或其片段，其中所述抗原结合蛋白或其片段与如权利要求1至18中任一项所 述的抗原结合蛋白或其片段竞争结合PD-L1，其中所述竞争通过ELISA或流式细胞术测量。An isolated antigen-binding protein or a fragment thereof, wherein the antigen-binding protein or a fragment thereof competes with the antigen-binding protein of any one of claims 1 to 18 or a fragment thereof for binding to PD-L1, wherein the competition Measured by ELISA or flow cytometry.
  21. 如权利要求1-18中任一项的抗原结合蛋白或其片段，其中所述抗原结合蛋白或其片段选自单克隆抗体、scFv、Fab片段、Fv片段、F(ab)'片段、F(ab')2片段、双特异性抗体、免疫缀合物或其组合。The antigen-binding protein or fragment thereof according to any one of claims 1 to 18, wherein the antigen-binding protein or fragment thereof is selected from the group consisting of a monoclonal antibody, a scFv, a Fab fragment, an Fv fragment, a F(ab)' fragment, and F ( Ab') 2 fragment, bispecific antibody, immunoconjugate or a combination thereof.
  22. 如权利要求1-18中任一项所述的抗原结合蛋白或其片段，其中所述抗原结合蛋白或其片段与治疗剂连接或缀合。The antigen binding protein or fragment thereof of any one of claims 1 to 18, wherein the antigen binding protein or fragment thereof is linked or conjugated to a therapeutic agent.
  23. 如权利要求22所述的抗原结合蛋白或其片段，其中所述治疗剂是细胞毒性药物、放射性同位素、免疫调节剂或抗体。The antigen binding protein or fragment thereof according to claim 22, wherein the therapeutic agent is a cytotoxic drug, a radioisotope, an immunomodulator or an antibody.
  24. 一种分离的结合PD-L1的抗原结合蛋白或其片段，其中所述抗原结合蛋白或其片段对PD-L1具有约10nM至约0.1nM的亲和力。An isolated antigen-binding protein or fragment thereof that binds to PD-L1, wherein the antigen-binding protein or fragment thereof has an affinity for PD-L1 of from about 10 nM to about 0.1 nM.
  25. 一种分离的结合PD-L1的抗原结合蛋白或其片段，其中所述抗原结合蛋白抑制PD-L1与PD-1的结合，其抑制PD-L1与PD-1结合的IC50为约20ng/mL至约800ng/mL。An isolated PD-L1 binding antigen binding protein or fragment thereof, wherein the antigen binding protein inhibits binding of PD-L1 to PD-1, and the IC50 for inhibiting PD-L1 binding to PD-1 is about 20 ng/mL Up to about 800 ng/mL.
  26. 一种分离的结合PD-L1的抗原结合蛋白或其片段，其中所述抗原结合蛋白或其片段抑制在表面上分别表达PD-L1和PD-1蛋白的细胞之间的相互作用。An isolated antigen-binding protein or fragment thereof that binds to PD-L1, wherein the antigen-binding protein or fragment thereof inhibits interaction between cells expressing PD-L1 and PD-1 proteins, respectively, on the surface.
  27. 如权利要求26所述的抗原结合蛋白或其片段，其中所述抗原结合蛋白或其片段抑制在表面上分别表达PD-L1和PD-1蛋白的细胞之间的相互作用的IC50为约10ng/mL至约400ng/mL。The antigen-binding protein or fragment thereof according to claim 26, wherein the antigen-binding protein or a fragment thereof inhibits an IC50 of about 10 ng/interaction between cells expressing PD-L1 and PD-1 proteins, respectively. mL to about 400 ng/mL.
  28. 一种组合物，其包含根据权利要求1-27中任一项所述的抗原结合蛋白或其片段以及药学上可接受的载体。A composition comprising the antigen binding protein of any one of claims 1-27, or a fragment thereof, and a pharmaceutically acceptable carrier.
  29. 一种分离的多核苷酸，其编码根据权利要求1-27中任一项所述的抗原结合蛋白或其片段。An isolated polynucleotide encoding the antigen binding protein of any one of claims 1-27 or a fragment thereof.
  30. 一种表达载体，其包含根据权利要求29所述的分离的多核苷酸。An expression vector comprising the isolated polynucleotide of claim 29.
  31. 一种宿主细胞，其包含根据权利要求30所述的表达载体。A host cell comprising the expression vector of claim 30.
  32. 一种用于增加T细胞活化的方法，所述方法包括使T细胞与根据权利要求1-27中任一项所述的抗原结合蛋白或其片段接触。A method for increasing T cell activation, the method comprising contacting a T cell with an antigen binding protein of any one of claims 1-27 or a fragment thereof.
  33. 一种用于在受试者中减少肿瘤或抑制肿瘤细胞生长的方法，所述方法包括向所述受试者施用治疗有效量的根据权利要求1-27中任一项所述的分离的抗原结合蛋白或其片段。A method for reducing tumor growth or inhibiting tumor cell growth in a subject, the method comprising administering to the subject a therapeutically effective amount of the isolated antigen of any one of claims 1-27 Binding protein or fragment thereof.
  34. 一种用于在有需要的受试者中治疗癌症的方法，所述方法包括向所述受试者施用治疗有效量的根据权利要求1-27中任一项所述的分离的抗原结合蛋白或其片段，其中，所述癌症选自由以下组成的组：淋巴瘤、白血病、黑色素瘤、神经胶质瘤、乳腺癌、肺癌、肠癌、骨癌、卵巢癌、膀胱癌、肾癌、肝癌、胃癌、睾丸癌、唾液腺癌、甲状腺癌、胸腺癌、上皮癌、头或颈癌、胰腺癌或其组合。A method for treating cancer in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the isolated antigen binding protein of any one of claims 1-27 Or a fragment thereof, wherein the cancer is selected from the group consisting of lymphoma, leukemia, melanoma, glioma, breast cancer, lung cancer, intestinal cancer, bone cancer, ovarian cancer, bladder cancer, kidney cancer, liver cancer , gastric cancer, testicular cancer, salivary gland cancer, thyroid cancer, thymic cancer, epithelial cancer, head or neck cancer, pancreatic cancer or a combination thereof.
  35. 一种用于在有需要的受试者中治疗传染病的方法，所述方法包括向所述受试者施用治疗有效量的根据权利要求1-27中任一项所述的分离的抗原结合蛋白或其片段，其中，所述传染病优选选自由以下组成的组：念珠菌病、念珠菌血症、曲霉菌病、链球菌性肺炎、链球菌性皮肤和口咽病状、革兰氏阴性脓毒症、结核病、单核细胞增多症、流行性感冒、由呼吸道合胞病毒引起的呼吸道疾病、疟疾、血吸虫病和锥虫病。A method for treating an infectious disease in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the isolated antigen binding according to any one of claims 1-27 a protein or a fragment thereof, wherein the infectious disease is preferably selected from the group consisting of: candidiasis, candidemia, aspergillosis, streptococcal pneumonia, streptococcal skin and oropharyngeal condition, gram-negative Sepsis, tuberculosis, mononucleosis, influenza, respiratory diseases caused by respiratory syncytial virus, malaria, schistosomiasis and trypanosomiasis.
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CN111356702B (en) 2022-07-26

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