WO2019109974A1 - Anticorps anti-pd-l1 et fragment de liaison à l'antigène de celui-ci - Google Patents

Anticorps anti-pd-l1 et fragment de liaison à l'antigène de celui-ci 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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English (en)
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/zh
Publication of WO2019109974A1 publication Critical patent/WO2019109974A1/fr

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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

L'invention concerne un anticorps anti-PD-L1 humanisé et un fragment de liaison à l'antigène de celui-ci, un nucléotide correspondant, un vecteur d'expression comprenant le polynucléotide, une cellule comprenant le vecteur d'expression, et une composition pharmaceutique comprenant l'anticorps ou le fragment de liaison à l'antigène de celui-ci, qui peut être utilisé pour réduire des tumeurs ou inhiber la croissance de cellules tumorales.
PCT/CN2018/119536 2017-12-06 2018-12-06 Anticorps anti-pd-l1 et fragment de liaison à l'antigène de celui-ci WO2019109974A1 (fr)

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CN101248089A (zh) * 2005-07-01 2008-08-20 米德列斯公司 抗程序性死亡配体1（pd-l1）的人单克隆抗体
CN105461808A (zh) * 2015-12-24 2016-04-06 长春金赛药业有限责任公司 单克隆抗体及其应用
CN107298713A (zh) * 2017-08-15 2017-10-27 联合益康（北京）生物科技有限公司 一种抗pd‑l1抗体及应用、制备方法、试剂盒和药物

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JP2023500156A (ja) * 2019-11-08 2023-01-04 チャンスー シムサー ファーマシューティカル カンパニー リミテッド 抗－ヒトプログラム細胞死リガンド－１（ｐｄ－ｌ１）の抗体及びその用途

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