WO2021088904A1 - 抗人程序性死亡配体-1(pd-l1)的抗体及其用途 - Google Patents

抗人程序性死亡配体-1(pd-l1)的抗体及其用途 Download PDF

Info

Publication number
WO2021088904A1
WO2021088904A1 PCT/CN2020/126656 CN2020126656W WO2021088904A1 WO 2021088904 A1 WO2021088904 A1 WO 2021088904A1 CN 2020126656 W CN2020126656 W CN 2020126656W WO 2021088904 A1 WO2021088904 A1 WO 2021088904A1
Authority
WO
WIPO (PCT)
Prior art keywords
antibody
sequence
variable region
antigen
seq
Prior art date
Application number
PCT/CN2020/126656
Other languages
English (en)
French (fr)
Inventor
赵新燕
邓婧
卢士强
李鑫鑫
任晋生
Original Assignee
先声生物医药科技有限公司
江苏先声药业有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 先声生物医药科技有限公司, 江苏先声药业有限公司 filed Critical 先声生物医药科技有限公司
Priority to CA3157516A priority Critical patent/CA3157516A1/en
Priority to US17/755,741 priority patent/US20230002492A1/en
Priority to CN202080077362.4A priority patent/CN115038718B/zh
Priority to JP2022527056A priority patent/JP2023500156A/ja
Priority to EP20884635.2A priority patent/EP4056592A4/en
Publication of WO2021088904A1 publication Critical patent/WO2021088904A1/zh
Priority to JP2023144842A priority patent/JP2023179450A/ja

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2827—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against B7 molecules, e.g. CD80, CD86
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/54—Medicinal preparations containing antigens or antibodies characterised by the route of administration
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/545—Medicinal preparations containing antigens or antibodies characterised by the dose, timing or administration schedule
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C07K2317/732—Antibody-dependent cellular cytotoxicity [ADCC]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

  • the present invention relates to antibodies against human programmed death ligand-1 (PD-L1) or antigen-binding fragments thereof, which encode nucleic acids, expression vectors and expression cells, preparation methods, pharmaceutical compositions, and their use to enhance T cells Function, up-regulation of T cell-mediated immune response and for the treatment of diseases related to abnormal PD-L1 expression and abnormal T cell function, such as tumors.
  • PD-L1 programmed death ligand-1
  • Immunotherapy has become one of the fastest-growing and most promising research fields in tumor therapy, and the use of immune checkpoint inhibitors, such as PD-1/PD-L1 monoclonal antibody, CTLA-4 monoclonal antibody, is a revolution in tumor immunotherapy Sexual treatment has greatly improved the survival time of patients with malignant tumors.
  • immune checkpoint inhibitors such as PD-1/PD-L1 monoclonal antibody, CTLA-4 monoclonal antibody
  • T cell-mediated immune response is strictly regulated by costimulation and co-suppression mechanisms, maintaining the best balance between antigen immune response and maintaining self-tolerance. This balance is participated by a variety of activating and inhibitory proteins. Inhibitory proteins, also known as immune checkpoint proteins, regulate the activation and effector functions of cytotoxic T lymphocytes (CTL) to maintain self-tolerance. Immune checkpoint inhibitory proteins play a key role in tumor regulatory pathways. One of the important immune checkpoint protein PD-1, after binding to its ligand PD-L1, will transmit immunosuppressive signals and reduce the activity of T cells.
  • CTL cytotoxic T lymphocytes
  • tumor cells can also inhibit the activation and proliferation of T cells by expressing PD-L1 on the cell surface, thereby evading the attack and killing of CTL.
  • PD-1 or PD-L1 monoclonal antibody to prevent the binding and interaction of PD-1/PD-L1 can partially restore the function of T cells, thereby enhancing the ability to kill tumor cells.
  • Ipilimumab the first immune checkpoint inhibitor, as an anti-CTLA-4 monoclonal antibody, became a tumor immunotherapy successfully used to treat melanoma. Many patients treated so far have obtained comparisons with traditional The treatment method has a better 5-year survival period. Since then, the FDA has successively approved 3 PD-1 monoclonal antibodies and 3 PD-L1 monoclonal antibodies, which have been successfully used in immunotherapy for more than a dozen tumors other than melanoma, and have become the first-line treatment for a variety of cancers. Such as non-small cell lung cancer (NSCLC), renal cell carcinoma (RCC) and bladder or urothelial cancer.
  • NSCLC non-small cell lung cancer
  • RNC renal cell carcinoma
  • bladder or urothelial cancer urothelial cancer.
  • the present invention provides antibodies and antigen-binding fragments that specifically bind to human programmed death ligand-1 (PD-L1), nucleic acids encoding these antibodies and antigen-binding fragments, including the antibodies and antigen-binding fragments, and pharmaceutical compositions and reagents Cassettes, and their use for enhancing the function of T cells, up-regulating T cell-mediated immune responses, and for treating disorders related to abnormal PD-L1 expression and abnormal T cell function, such as tumor immunity.
  • the antibody can not only bind to human and cynomolgus PD-L1 protein, but also block the interaction between human PD-L1 and human PD-1.
  • the isolated antibody or antigen-binding fragment that specifically binds to human programmed death ligand-1 comprises a combination of heavy chain CDRs and a combination of light chain CDRs:
  • the heavy chain CDRs combination includes: CDR1-VH, CDR2-VH, and CDR3-VH; the CDR1-VH, CDR2-VH, and CDR3-VH have any combination of sequences selected from the following or in combination with the sequence Compared with sequence combinations with 1, 2, 3 or more amino acid insertions, deletions and/or substitutions:
  • the light chain CDRs combination includes: CDR1-VL, CDR2-VL, and CDR3-VL, and the CDR1-VL, CDR2-VL, and CDR3-VL have any sequence combination selected from the following or are in combination with the sequence combination.
  • Each of CDR1-VH, CDR2-VH, CDR3-VH, CDR1-VL, CDR2-VL and CDR3-VL is coded according to the common analysis method of KABAT, Chothia or IMGT.
  • the isolated humanized antibody or antigen-binding fragment that specifically binds to human programmed death ligand-1 comprises a combination of heavy chain CDRs and a combination of light chain CDRs:
  • the heavy chain CDRs combination includes: CDR1-VH, CDR2-VH, and CDR3-VH; the CDR1-VH, CDR2-VH, and CDR3-VH have any combination of sequences selected from the following or in combination with the sequence Compared with sequence combinations with 1, 2, 3 or more amino acid insertions, deletions and/or substitutions:
  • the light chain CDRs combination includes: CDR1-VL, CDR2-VL, and CDR3-VL, and the CDR1-VL, CDR2-VL, and CDR3-VL have any sequence combination selected from the following or are in combination with the sequence combination.
  • Each of CDR1-VH, CDR2-VH, CDR3-VH, CDR1-VL, CDR2-VL and CDR3-VL is coded according to the common analysis method of KABAT, Chothia or IMGT.
  • the antibody or antigen-binding fragment thereof of the present invention comprises a combination of heavy chain CDRs and light chain CDRs selected from: VH1+VL1, VH2+VL2, VH3+VL3, VH4+VL4, VH5+VL5, VH6+VL6 , VH7+VL7, VH8+VL8, VH9+VL9, VH10+VL10, VH11+VL11, VH12+VL12, VH13+VL13, VH14+VL14, VH15+VL15, VH16+VL16, VH17+VL17, VH18+VL18, VH19 +VL19, VH20+VL20, VH21+VL21, VH22+VL22, VH23+VL23, VH24+VL24, VH25+VL25, or VH26+VL26, and has 1, compared with the sequence of the heavy chain
  • the present invention provides such antibodies or antigen-binding fragments thereof, wherein:
  • variable region of the heavy chain and the variable region of the light chain have the sequence shown in SEQ ID NO:1 and SEQ ID NO: 2, or 70%, 75%, 80%, 85%, 90% of the sequence shown , 95%, 96%, 97%, 98%, 99% or higher identity sequence;
  • the heavy chain variable region and the light chain variable region have the sequence shown in SEQ ID NO: 3 and SEQ ID NO: 4, or 70%, 75%, 80%, 85%, 90% of the sequence shown , 95%, 96%, 97%, 98%, 99% or higher identity sequence;
  • the heavy chain variable region and the light chain variable region have the sequence shown in SEQ ID NO: 5 and SEQ ID NO: 6, or 70%, 75%, 80%, 85%, 90% of the sequence shown , 95%, 96%, 97%, 98%, 99% or higher identity sequence;
  • the heavy chain variable region and the light chain variable region have the sequence shown in SEQ ID NO: 7 and SEQ ID NO: 8, or 70%, 75%, 80%, 85%, 90% of the sequence shown , 95%, 96%, 97%, 98%, 99% or higher identity sequence;
  • the heavy chain variable region and the light chain variable region have the sequence shown in SEQ ID NO: 9 and SEQ ID NO: 10, or 70%, 75%, 80%, 85%, 90% of the sequence shown , 95%, 96%, 97%, 98%, 99% or higher identity sequence;
  • the heavy chain variable region and the light chain variable region have the sequence shown in SEQ ID NO: 11 and SEQ ID NO: 12, or 70%, 75%, 80%, 85%, 90% of the sequence shown , 95%, 96%, 97%, 98%, 99% or higher identity sequence;
  • the heavy chain variable region and the light chain variable region have the sequence shown in SEQ ID NO: 13 and SEQ ID NO: 14, respectively, or 70%, 75%, 80%, 85%, 90% of the sequence shown , 95%, 96%, 97%, 98%, 99% or higher identity sequence;
  • the heavy chain variable region and the light chain variable region have the sequence shown in SEQ ID NO: 15 and SEQ ID NO: 16, or 70%, 75%, 80%, 85%, 90% of the sequence shown , 95%, 96%, 97%, 98%, 99% or higher identity sequence;
  • the heavy chain variable region and the light chain variable region have the sequence shown in SEQ ID NO: 17 and SEQ ID NO: 18, or 70%, 75%, 80%, 85%, 90% of the sequence shown , 95%, 96%, 97%, 98%, 99% or higher identity sequence;
  • the heavy chain variable region and the light chain variable region have the sequence shown in SEQ ID NO: 127 and SEQ ID NO: 128, or 70%, 75%, 80%, 85%, 90% of the sequence shown , 95%, 96%, 97%, 98%, 99% or higher identity sequence;
  • the heavy chain variable region and the light chain variable region have the sequence shown in SEQ ID NO: 129 and SEQ ID NO: 130, or 70%, 75%, 80%, 85%, 90% of the sequence shown , 95%, 96%, 97%, 98%, 99% or higher identity sequence;
  • the heavy chain variable region and the light chain variable region have the sequence shown in SEQ ID NO: 131 and SEQ ID NO: 132, or 70%, 75%, 80%, 85%, 90% of the sequence shown , 95%, 96%, 97%, 98%, 99% or higher identity sequence; or,
  • the heavy chain variable region and the light chain variable region have the sequence shown in SEQ ID NO: 133 and SEQ ID NO: 134, or 70%, 75%, 80%, 85%, 90% of the sequence shown , 95%, 96%, 97%, 98%, 99% or higher identity sequence.
  • the antibody or antigen-binding fragment thereof of the present invention is chimeric or humanized or fully human.
  • the antibody or antigen-binding fragment thereof of the present invention has a dissociation constant (KD) that binds to human programmed death ligand-1 (PD-L1) not greater than 10 nM, which is comparable to that of cynomolgus monkeys.
  • the dissociation constant (KD) of death ligand-1 (PD-L1) binding is not greater than 100 nM.
  • the antibody or antigen-binding fragment thereof of the present invention comprises the sequence of the constant region of any one of human or murine antibodies IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE or IgD; preferably comprising human or The sequence of the constant region of the murine antibody IgG1, IgG2, IgG3 or IgG4; or the sequence of the constant region of the human or murine antibody IgG1, IgG2, IgG3 or IgG4 carrying mutations.
  • the antigen-binding fragment of the present invention is selected from one or more of F(ab)2, Fab', Fab, Fv, scFv, bispecific antibody, nanobody, and the smallest recognition unit of antibody.
  • the antibody or antigen-binding fragment thereof of the present invention can competitively bind to PD-L1 with an antibody selected from the group consisting of 34, 50, 90, 130, 156, 370, 373, 413, or 794, and It has the following characteristics:
  • ADCC antibody-dependent cell killing
  • the present invention provides an isolated nucleic acid molecule that encodes the above-mentioned antibody, antigen-binding fragment, or any combination thereof of the present invention.
  • the present invention provides an expression vector comprising the aforementioned isolated nucleic acid molecule of the present invention.
  • the present invention provides a host cell, which comprises the aforementioned isolated nucleic acid molecule or expression vector of the present invention.
  • the host cell is a eukaryotic cell or a prokaryotic cell; more preferably, the host cell is derived from mammalian cells, yeast cells, insect cells, Escherichia coli and/or Bacillus subtilis; more preferably, the host cells are derived from mammalian cells, yeast cells, insect cells, E. coli and/or Bacillus subtilis; The host cell is selected from Chinese hamster ovary cells (CHO).
  • the present invention provides a method for preparing an antibody or antigen-binding fragment, culturing the above-mentioned host cell of the present invention under appropriate conditions, and isolating the antibody or antigen-binding fragment.
  • the present invention provides a pharmaceutical composition, the composition comprising the above-mentioned antibody or antigen-binding fragment of the present invention, the above-mentioned isolated nucleic acid molecule of the present invention, the above-mentioned expression vector of the present invention, and the above-mentioned expression vector of the present invention.
  • the above-mentioned cells, or the products prepared by the above-mentioned methods of the present invention (such as antibodies and antigen-binding fragments), and a pharmaceutically acceptable carrier.
  • the pharmaceutical composition further comprises an additional anti-tumor agent.
  • the present invention provides a method for preventing and/or treating diseases related to abnormal PD-L1 expression and/or abnormal T cell function, which comprises administering the above-mentioned antibody of the present invention to patients in need thereof or Antigen-binding fragments, the above-mentioned isolated nucleic acid molecules of the present invention, the above-mentioned expression vectors of the present invention, the above-mentioned cells of the present invention, and products prepared by the above-mentioned methods of the present invention (e.g., antibodies and antigen-binding fragments) Or the above-mentioned pharmaceutical composition of the present invention;
  • the disease is preferably a tumor; the tumor is preferably colorectal cancer.
  • the present invention provides the above-mentioned antibody or antigen-binding fragment, the above-mentioned isolated nucleic acid molecule of the present invention, the above-mentioned expression vector of the present invention, the above-mentioned cell of the present invention, and the above-mentioned cell of the present invention.
  • the use of the product prepared by the method (for example, antibody and antigen-binding fragment) or the pharmaceutical composition of the present invention in the preparation of drugs for preventing and/or treating diseases related to abnormal PD-L1 expression are preferably Tumor; the tumor is preferably colorectal cancer.
  • the present invention provides a kit comprising the above-mentioned antibody or antigen-binding fragment of the present invention, the above-mentioned isolated nucleic acid molecule of the present invention, the above-mentioned expression vector of the present invention, and the above-mentioned expression vector of the present invention.
  • the above-mentioned cells, or products prepared by the above-mentioned methods of the present invention (such as antibodies and antigen-binding fragments), and instructions for use.
  • antibody refers to an immunoglobulin molecule that specifically binds to a target antigen or has immunoreactivity, including polyclonal, monoclonal, genetically engineered and other modified forms of antibodies (including but not Limited to chimeric antibodies, humanized antibodies, fully human antibodies, heteroconjugate antibodies (such as bispecific, trispecific and tetraspecific antibodies, diabodies, tribodies and tetrabodies), antibody conjugates) And antigen-binding fragments of antibodies (including, for example, Fab', F(ab')2, Fab, Fv, rIgG, and scFv fragments).
  • mAb monoclonal antibody
  • mAb monoclonal antibody
  • antigen-binding fragment refers to one or more antibody fragments that retain the ability to specifically bind to a target antigen.
  • the antigen-binding function of antibodies can be performed by fragments of full-length antibodies.
  • Antibody fragments can be Fab, F(ab')2, scFv, SMIP, diabody, triabody, affibody, nanobody, aptamer or domain antibody.
  • binding fragments that encompass the "antigen-binding fragment" of the term antibody include, but are not limited to: (i) Fab fragment, a monovalent fragment composed of VL, VH, CL and CH1 domains; (ii) F(ab)2 Fragment, a bivalent fragment containing two Fab fragments connected by a disulfide bond in the hinge region; (iii) Fd fragment composed of VH and CH1 domains; (iv) VL and VH domains of one arm of an antibody (V) a dAb consisting of VH and VL domains; (vi) a dAb fragment consisting of VH domains (Ward et al., Nature 341:544-546, 1989); (vii) a VH or VL structure A domain composed of dAb; (viii) an isolated complementarity determining region (CDR); and (ix) a combination of two or more isolated CDRs, which may optionally be joined by a synthetic linker.
  • Fab fragment a mono
  • the two domains VL and VH of the Fv fragment are encoded by independent genes, these two domains can be joined through a linker using a recombination method, which can be made into which the VL and VH regions are paired to form A single protein chain of a monovalent molecule (called single-chain Fv (scFv); see, for example, Bird et al., Science 242:423-426, 1988 and Huston et al., Proc. Natl. Acad. Sci. USA 85: 5879-5883, 1988).
  • scFv single-chain Fv
  • These antibody fragments can be obtained using conventional techniques known to those skilled in the art, and these fragments are screened for use in the same manner as intact antibodies.
  • Antigen-binding fragments can be produced by recombinant DNA technology, enzymatic or chemical cleavage of intact immunoglobulins, or in some embodiments by chemical peptide synthesis procedures known in the art.
  • PD-L1 refers to programmed death ligand-1, also known as CD279 (cluster of differentiation 279), which is an important immunosuppressive molecule.
  • CD279 cluster of differentiation 279
  • the PD-L1 is preferably human PD-L1.
  • anti-programmed death ligand-1 antibody As used herein, the terms “anti-programmed death ligand-1 antibody”, “programmed death ligand-1 antibody”, “anti-PD-L1 antibody”, “PD-L1 antibody”, “anti-PD-L1 "Antibody portion” and/or “anti-PD-L1 antibody fragment” and the like refer to any immunoglobulin molecule that contains at least a portion (such as but not limited to at least one complementarity determination of a heavy chain or a light chain) capable of specifically binding PD-L1 Region (CDR) or its ligand binding portion, heavy or light chain variable region, heavy or light chain constant region, framework region or any part thereof) protein or peptide-containing molecule.
  • CDR PD-L1 Region
  • the PD-L1 antibody also includes an antibody-like protein scaffold (such as the tenth fibronectin type III domain (10Fn3)), which contains BC, DE, and FG structural loops that are similar in structure and solvent accessibility to the antibody CDR.
  • the tertiary structure of the 10Fn3 domain is similar to the tertiary structure of the IgG heavy chain variable region, and the BC, DE, and FG loop residues of 10Fn3 are used from the PD-L1 monoclonal antibody CDR-H1, CDR-H2 Or substitution of residues in the CDR-H3 region, those skilled in the art can graft, for example, the CDR of the PD-L1 monoclonal antibody onto the fibronectin scaffold.
  • the term "bispecific antibody” refers to antibodies with monoclonal binding specificities for at least two different antigens, which are usually human or humanized antibodies.
  • one of the binding specificities can be detected against an epitope of PD-L1
  • the other can be detected against another epitope of PD-L1 or any other antigen, such as for cell surface proteins, receptors, Receptor subunits, tissue-specific antigens, virus-derived proteins, virus-encoded envelope proteins, bacterial-derived proteins or bacterial surface proteins, etc. are detected.
  • chimeric antibody refers to antibodies that have variable sequences derived from immunoglobulins derived from one source organism (such as rat or mouse) and those derived from different organisms (such as human) The constant region of immunoglobulin.
  • Methods for producing chimeric antibodies are known in the art. See, for example, Morrison, 1985, Science 229(4719):1202-7; Oi et al., 1986, BioTechniques 4:214-221; Gillies et al., 1985 J Immunol Methods 125:191-202; the above are incorporated by reference Into this article.
  • CDR complementarity determining region
  • FR framework region
  • the amino acid position representing the hypervariable region of an antibody can vary according to the context and various definitions known in the art. Some positions within the variable domain can be considered as heterozygous hypervariable positions, because these positions can be considered as being within a set of criteria (such as IMGT or KABAT) within the hypervariable region, but considered as being in a different set of criteria (Such as KABAT or IMGT) outside the hypervariable region. One or more of these locations can also be found in extended hypervariable regions.
  • variable domains of the natural heavy chain and light chain each contain four framework regions that mainly adopt a sheet-layer configuration, which are connected by three CDRs (CDR1, CDR2, and CDR3), and these three CDRs form a loop that connects the sheet-layer structure. , And in some cases form part of the lamellar structure.
  • CDRs in each chain are closely held together in the order FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 by the FR region, and together with the CDRs from other antibody chains, they contribute to the formation of the antigen-binding site of the antibody (see Kabat Et al., Sequences of Protein sof Immunological Interest, National Institute of Health, Bethesda, Md. 1987; it is incorporated herein by reference).
  • CDR1-VH, CDR2-VH and CDR3-VH refer to the first CDR, the second CDR and the third CDR of the variable region of the heavy chain (VH) respectively. These three CDRs constitute the heavy chain variable region (VH).
  • CDR1-VL, CDR2-VL and CDR3-VL refer to the first CDR, second CDR and the first CDR of the light chain variable region (VL), respectively Three CDRs, which constitute the CDR combination (VLCDR combination) of the light chain (or its variable region).
  • antibody conjugate refers to a conjugate/conjugate formed by chemically bonding an antibody molecule to another molecule directly or through a linker.
  • ADC antibody-drug conjugates
  • the term "monoclonal antibody” refers to an antibody derived from a single clone (including any eukaryotic, prokaryotic, or phage clone), and is not limited to the method of producing the antibody.
  • VH refers to the variable region of the immunoglobulin heavy chain (including the heavy chain of Fv, scFv, or Fab) of an antibody.
  • VL refers to the variable region of an immunoglobulin light chain (including the light chain of Fv, scFv, dsFv, or Fab).
  • percent (%) sequence identity refers to aligning sequences and introducing gaps (if necessary) in order to achieve the maximum percent sequence identity (for example, for optimal alignment, the candidate and reference After introducing a gap in one or two of the sequence, and for the purpose of comparison, non-homologous sequences can be ignored), the amino acid (or nucleotide) residues of the candidate sequence and the amino acid (or nucleotide) of the reference sequence ) The percentage of residues that are the same.
  • percent sequence identity the alignment can be achieved in a variety of ways known to those skilled in the art, for example, using publicly available computer software, such as BLAST, ALIGN or Megalign (DNASTAIi) software.
  • a reference sequence used for comparison with a candidate sequence can show that the candidate sequence exhibits from 50% of the total length of the candidate sequence or a selected portion of consecutive amino acid (or nucleotide) residues of the candidate sequence.
  • the length of the candidate sequence aligned for comparison purposes may be, for example, at least 30% (e.g., 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100%) of the length of the reference sequence. .
  • a position in the candidate sequence is occupied by the same amino acid (or nucleotide) residue as the corresponding position in the reference sequence, then the molecules are the same at that position.
  • the term "specific binding” refers to a binding reaction that determines the presence of an antigen in a heterogeneous population of proteins and other biological molecules, such as antibodies or their antigens. Specific recognition of binding fragments.
  • the antibody or antigen-binding fragment thereof that specifically binds to the antigen will bind to the antigen with a KD less than 100 nM.
  • an antibody or antigen-binding fragment thereof that specifically binds to an antigen will bind to an antigen with a KD of up to 100 nM (for example, between 1 pM and 100 nM).
  • Antibodies or antibodies that do not specifically bind to a specific antigen or its epitope will show a KD greater than 100 nM (for example, greater than 500 nM, 1 ⁇ M, 100 ⁇ M, 500 ⁇ M, or 1 mM) for the specific antigen or its epitope.
  • a variety of immunoassay methods can be used to select specific proteins or carbohydrates.
  • Antibodies for sexual immune response For example, solid-phase ELISA immunoassays are routinely used to select antibodies that specifically immunoreact with proteins or carbohydrates.
  • vector includes nucleic acid vectors, such as DNA vectors (such as plasmids), RNA vectors, viruses, or other suitable replicons (such as viral vectors).
  • DNA vectors such as plasmids
  • RNA vectors such as RNA vectors
  • viruses or other suitable replicons
  • viral vectors Various vectors have been developed for delivery of polynucleotides encoding foreign proteins into prokaryotic or eukaryotic cells.
  • the expression vector of the present invention contains polynucleotide sequences and, for example, additional sequence elements for expressing proteins and/or integrating these polynucleotide sequences into the genome of mammalian cells.
  • Certain vectors that can be used to express the antibodies and antibody fragments of the present invention include plasmids containing regulatory sequences (such as promoter and enhancer regions) that direct gene transcription.
  • kits for expressing antibodies and antibody fragments contain polynucleotide sequences that enhance the rate of translation of these genes or improve the stability or nuclear output of mRNA produced by gene transcription. These sequence elements include, for example, 5'and 3'untranslated regions, internal ribosome entry sites (IRES), and polyadenylation signal sites to direct efficient transcription of genes carried on the expression vector.
  • the expression vector of the present invention may also contain the following polynucleotide, which encodes a marker for selecting cells containing such a vector. Examples of suitable markers include genes encoding resistance to antibiotics such as ampicillin, chloramphenicol, kanamycin, or nourseothricin.
  • the terms “subject”, “subject” and “patient” refer to organisms that receive treatment for a particular disease or condition (such as cancer or infectious disease) as described herein.
  • subjects and patients include mammals, such as humans, primates, pigs, goats, rabbits, hamsters, cats, dogs, receiving treatment for diseases or disorders (e.g., cell proliferative disorders, such as cancer or infectious diseases).
  • Guinea pigs, members of the bovid family such as cattle, bison, buffalo, elk and yak, etc.
  • treatment refers to surgical or therapeutic treatment, the purpose of which is to prevent, slow down (reduce) undesired physiological changes or pathologies in the subject to be treated, such as cell proliferative disorders (such as cancer). Or infectious disease).
  • Beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, reduced disease severity, stable disease state (i.e., no worsening), delay or slowing of disease progression, improvement or alleviation of disease state, and alleviation (whether Partial or complete remission), whether detectable or undetectable.
  • the subjects in need of treatment include those who have already suffered from a disease or disease, as well as those who are prone to suffering from a disease or disease or who intend to prevent the disease or disease.
  • mitigation, mitigation, weakening, mitigation, mitigation, etc. its meaning also includes elimination, disappearance, and non-occurrence.
  • Figure 1 The titers of mouse serum combined with human PD-L1-mFc (A) and PD-L1-His (B) recombinant proteins determined after the last immunization;
  • FIG. 1 Flow cytometric staining and sorting (FACS) and circle gate strategy diagram of PD-L1 specific B cells;
  • Figure 3 Competitive ELISA method to determine that anti-PD-L1 antibody blocks the binding of PD-L1 protein to PD-1 protein;
  • Anti-PD-L1 antibody increases the expression and activity of reporter genes in the Jurkat-PD-1-CHO-PD-L1-NFAT system
  • Anti-PD-L1 antibody promotes the secretion of IFN- ⁇ in the mixed lymphocyte reaction
  • Murine anti-PD-L1 antibody inhibits MC38-hPD-L1 colon cancer tumor growth in human PD-1/PD-L1 transgenic mice;
  • Figure 9 Humanized anti-PD-L1 antibody inhibits MC38-hPD-L1 colon cancer tumor growth in human PD-L1 transgenic mice.
  • mice For 6-8 weeks old female SJL mice (purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd.) or Balb/c mice (purchased from Shanghai Slack Laboratory Animal Co., Ltd.), use mouse Fc fusion Human PD-L1 protein (PD-L1-mFc, Novoprotein, Cat.CM06, or Sino Biological, Cat.10084-H05H) or human PD-L1-His (Novoprotein, Cat.C315 or Sino Biological, Cat:.10084- H08H) and Freund's complete adjuvant (CFA, Sigma, Cat.F5881) for the first immunization; using the above-mentioned PD-L1-mFc or human PD-L1-His and Freund's incomplete adjuvant (incomplete freund's adjuvant, IFA, Sigma, Cat.F5506) and unmethylated cytosine guanine dinucleotide (CpGODN1826, synthesized from Shanghai Shengg
  • the foot pad and back after the first and second immunization injections, and the third and fourth immunization injections under the skin and back of the tail to obtain high-titer, high-affinity, high-specific antiserum and specific immunity cell.
  • the mice were euthanized and the spleen was aseptically removed, and the lymphocytes of the mouse spleen were aseptically separated and extracted, aliquoted into cryovials, and stored in liquid nitrogen. .
  • mice After the second immunization, 10 days after the third immunization, and the day of euthanasia, the mice were subjected to blood collection operations, the serum was separated, and the titers of anti-PD-L1 specific antibodies in the serum were determined by the enzyme-linked immunosorbent assay (ELISA) method.
  • ELISA enzyme-linked immunosorbent assay
  • mice immunized with PD-L1 protein were treated with the antigen PD-L1-His protein (Novoprotein, Cat.C315 or Sino Biological, Cat.10084-H08H) and the indirect labeled antibody anti-His-APC (R&D Systems, Cat.
  • IC050A antibodies against specific markers on the surface of mouse B cells (anti-mouse B220-Pacfic Blue, R&D Systems, Cat.553089; anti-mouse IgD-PE, R&D Systems, Cat.558597; anti-mouse IgM-PE Cy7 , R&D Systems, Cat.552867), and add the dye 7-AAD (R&D Systems, Cat.51-68981E) to distinguish between dead and live cells before sorting, using AriaIII (BD company) flow cytometer Sort PD-L1 specific single B cells (7AAD - B220 + IgD - IgM - PDL1-His + ) into PCR wells containing cell lysate and RNase inhibitor, and collect one cell in each well.
  • AriaIII BD company
  • Example 1 Using the method of Example 1 in the patent "A combination primer for nested amplification and its application” patent application number: 201811618134.4, single cell mRNA was reverse transcribed into cDNA. Then, nested PCR was performed using cDNA as a template to perform antibody heavy chain and light chain amplification respectively. The antibody heavy chain variable region and light chain variable region are amplified and cloned into the heavy chain expression vector and the light chain expression vector by homologous recombination. The constant regions of both the heavy chain expression vector and the light chain expression vector are derived from human IgG1.
  • the complete heavy chain expression sequence is signal peptide-VH-CH1-hinge region-CH2-CH3, and the complete light chain expression sequence is signal peptide-V ⁇ -C ⁇ .
  • the cloning and expression of the above-mentioned single B cell antibodies are all in a 96-well plate to achieve rapid identification and discovery of antibodies in a high-throughput manner. After a series of physical, chemical and functional screening of 324 pairs of cloned antibody heavy and light chains, a total of 9 candidate murine antibody molecules with physical, chemical and functional activities equivalent to or better than those of the listed PD-L1 antibody Avelumab or Atezolizumab were obtained.
  • Tables 1 to 2 The CDRs of its sequence were analyzed by IMGT and KABAT software, and the corresponding sequence information is shown in Tables 1 to 2.
  • Table 1 shows the VH and VL sequences of the mouse antibody molecule
  • Table 2 shows the IMGT and VL sequences of the mouse antibody molecule. KABAT analysis results.
  • CDRs transplantation the classic "CDRs transplantation" method is adopted for antibody humanization, that is, the human antibody with the highest homology is selected through sequence to provide antibody framework regions (FRs), and the complementarity determining regions of the antigen-binding fragments of the target antibody based on the Kabat naming method are selected. (CDRs), transplanted to the former to form humanized antibodies.
  • CDRs transplanted to the former to form humanized antibodies.
  • MOE software antibody structural modeling analysis
  • the CDRs of the sequence of the preferred candidate antibody molecule after the humanization of the PDL1-156 antibody are as follows, and they are analyzed with IMGT and KABAT software respectively.
  • the corresponding sequence information is shown in Table 3 and Table 4 below.
  • Table 3 shows the human source.
  • Table 4 shows the IMGT and KABAT analysis results of the humanized antibody molecule).
  • Tskgel G3000SWXL chromatographic column (TOSOH, 0008541) and pre-column Tskgel guard column SWXL (TOSOH, Cat.0008543) were used for molecular exclusion chromatography to determine antibody purity.
  • the mobile phase is phosphate buffer (NaH 2 PO 4 -Na 2 HPO 4 ), and the preparation: Weigh 8.88 g of NaH 2 PO 4 ⁇ 2H 2 O, and 33.33 g of Na 2 HPO 4 ⁇ 12H 2 O.
  • the mobile phase equilibrates the column with a flow rate of 1 mL/min. After the baseline becomes flat, the sample is injected.
  • the injection volume is 10 ⁇ L
  • the UV detection wavelength is 280 nm
  • the bandwidth is 16 nm
  • the reference wavelength is turned off.
  • Table 5 The measurement results are shown in Table 5.
  • Biacore T200 (GE Healthcare) was used to determine the binding affinity of PD-L1 antibody to human and cynomolgus PD-L1-His protein.
  • Immobilize anti-human IgG Fc (Genway, Cat.GWB-20A705) on a CM5 chip (GE Healthcare, Cat.BR-1005-30) at 25°C.
  • Dilute anti-human Fc (Genway, Cat.GWB-20A705) with Acetate pH5.0 (GE Healthcare, BR-1003-51) to 20 ⁇ g/mL.
  • a multi-cycle kinetics method is used to determine the affinity between the antibody and the antigen at 25°C.
  • the antibody to be tested is first captured on the fixed CM5 chip, and then injected into recombinant human PD-L1-His (Novoprotein, Cat.315) ) And cynomolgus monkey PD-L1-His protein (Sino Biological, Cat.90251-C08H), and finally regenerated with Glycine pH1.5.
  • the mobile phase is HBS-EP+Buffer (GE Healthcare, Cat.BR-1006-69), the flow rate is 30 ⁇ L/min, and the binding time is 300 seconds. The regeneration flow rate is 30 ⁇ L/min, and the time is 30 seconds.
  • Biacore T200 Evaluation Software version 3.0
  • the tested PD-L1 antibodies have an affinity of nM or higher for the binding of human PD-L1 recombinant protein, and their affinity with the PD-L1 recombinant protein of cynomolgus monkeys ranges from 62.5 nM to 0.375 nM Between, see Table 6 below for details.
  • Table 7 shows that the humanized antibodies 156-1H, 156-7H, 156-10H and 156-11H derived from the murine antibody PDL1-156 bind to the human PD-L1 protein, showing an affinity comparable to that of PDL1-156.
  • Example 7 IC50 determination of antibody blocking the interaction of PD-L1 and PD-1
  • the IC50 of anti-PD-L1 antibody blocking the binding of PD-L1 protein and PD-1 protein was determined by competitive ELISA method.
  • the competition binding curve of the test antibody can be drawn, and the IC50 value can be calculated.
  • Figure 3 shows the competitive binding curve of anti-PD-L1 antibody and human PD-L1 recombinant protein.
  • mice PD-L1-156 The IC50 of mouse PD-L1-156 is 221.3ng/ml, the positive control Atezolizumab (produced by Biotech) is 446.4ng/ml, and Avelumab (Pfizer, lot AU020322) is 190.3ng/ml.
  • Example 8 FACS determination of the EC 50 of PD-L1 antibody binding to cell surface PD-L1
  • the gradient concentration of the antibody to be detected (final antibody concentration: 10000ng/ml-0.1ng/ml, 10-fold serial dilution) is combined with the CHO-PD-L1 cells with high expression of PD-L1 on the cell surface (Nanjing Yongshan Biotechnology Co., Ltd., 10 5 /well), incubated together at 4°C for 30 min. After incubation, add 1:250 diluted anti-human IgG PE fluorescent antibody (eBioscience, Cat.12-4998-8), and incubate for 30 minutes at 4°C. The fluorescent antibody will specifically bind to the Fc section of the antibody to be detected.
  • FACS Fluorescence Activated Cell Sorting
  • the EC50s of the tested humanized antibodies 156-1H, 156-7H, 156-10H and 156-11H are 49.42ng/ml, 78.37ng/ml, 63.5ng/ml and 49.97ng/ml, respectively , which is similar to the positive control Avelumab (EC50 of 56.88ng/ml) in this experiment.
  • the test quantitatively confirmed the ability of each PD-L1 antibody to be tested to bind to the PD-L1 target on the cell surface in a dose-dependent manner.
  • MFI fold MFI value of experimental group/MFI value of control group without drug.
  • Example 9 PD-1/PD-L1-NFAT reporter gene test Anti-PD-L1 antibody inhibits PD-1:PD-L1 binding and signal transduction
  • PD-1 cell line and the PD-L1 antibody to be tested are incubated together.
  • the sample amount of PD-1 cells per well is 16000/well, and the antibody is diluted in a stepwise manner. Each dose is 3 multiple wells, and the incubation volume is 100 ⁇ l/ Well, the incubation time is 6 hours. When the incubation is completed, remove the well plate, add an equal volume (100 ⁇ l) to the luminescence detection reagent, and read the value.
  • FIG. 5A The EC50 value of the tested mouse PD-L1 antibody is the same as the EC50 value of the positive control antibodies Avelumab and Atezolizumab ( 222.9ng/ml, 321.6ng/ml) are similar.
  • Figures 5B and 5C show the four humanized PD-L1 antibodies 156-1H, 156-7H and 156-10H.
  • the EC50 of 156-11H is 342ng/ml, respectively.
  • the EC50 of 313.7ng/ml, 357.1ng/ml and 282.2ng/ml is similar to the positive control Avelumab.
  • Example 10 ELISA detection of IFN- ⁇ secreted by T cells in the mixed lymphocyte reaction
  • the PD-L1 monoclonal antibody enhances the activity of T cells by mixed lymphocyte reaction (MLR).
  • MLR mixed lymphocyte reaction
  • Isolate CD14 + monocytes from peripheral blood mononuclear cells (PBMC) of healthy human donor 1, and apply recombinant human granulocyte-macrophage colony stimulating factor (GM-CSF, Peprotech, Cat. 300) -03) and recombinant human interleukin 4 (rhIL-4, Peprotech, Cat. 200-04) were induced to differentiate into dendritic cells (DC) in vitro, and LPS (Sigma, Cat: L4516) was added on the 6th day of culture. ) Stimulate mature DC.
  • PBMC peripheral blood mononuclear cells
  • rhIL-4 recombinant human interleukin 4
  • DC cells from donor 1 and CD4 + T cells enriched from PBMC from healthy donor 2 are mixed and co-cultured.
  • the ratio of DC:CD4 + T cells is 1:10, and the test is added Antibody or positive control antibody Avelumab or Atezolizumab (antibody concentration is 1000ng/ml, 100ng/ml, 10ng/ml, 1ng/ml), cultured for 4 days. After 4 days, the cell culture supernatant was collected, and the content of IFN- ⁇ in the supernatant was detected by the ELISA method.
  • the anti-PD-L1 monoclonal antibody-mediated antibody-dependent cell killing activity was determined through the killing experiment of natural killer (NK) cells in isolated and purified normal human PBMC against A431 cells that highly express human PD-L1.
  • NK natural killer
  • PBMC cells (Stemexpress), RPMI1640 medium (Invitrogen, Cat. 200-02) supplemented with 100IU/ml recombinant human interleukin 2 (rhIL-2, Peprotech, Cat. 11835030) incubate overnight. After that, the cells were collected and the viable cell count was performed. Then use the NK cell magnetic bead separation kit (Stemcell, Cat. 17955) to purify NK cells from PBMC, and resuspend the NK cells with DMEM (Invitrogen, Cat. 11965084) supplemented with 10% inactivated fetal bovine serum Count and use as effector cells.
  • DMEM Invitrogen, Cat. 11965084
  • target cell A431 was cultured in DMEM medium containing 10% fetal bovine serum. The day before the ADCC experiment, the final concentration of 500IU interferon IFN- ⁇ (Peprotech, Cat.300-02) was added to stimulate the culture overnight. Standby later.
  • DMEM medium containing 10% fetal bovine serum Dilute the anti-PD-L1 antibody with DMEM medium containing 10% fetal bovine serum, divide the diluted antibody into a white wall 96-well plate (Corning, Cat.3610) at 25 ⁇ l/well, and add the target cells to the well (25 ⁇ l /Well, 10000 cells/well). The plate was incubated in a 37°C, 5% CO 2 incubator for 30 minutes. Subsequently, the effector cells were added to the wells (25 ⁇ l/well, 40,000 cells/well, the effective target ratio NK:A431 was 4:1), the total volume was 100 ⁇ l, and the plate was incubated in a 37°C, 5% CO 2 incubator 4 hours.
  • the target cell spontaneous death release hole that is, the hole only has target cells
  • the effector cell spontaneous death release hole that is, there are only NK cells in the hole
  • the target cell The maximum death-release wells (ie, target cells plus the lysis buffer provided in the CytoTox-Glo TM Cytotoxicity kit (Promega, Cat.G9291), the volume of all control wells was finally replenished to 100 ⁇ l with medium.
  • B-hPD-1/hPD-L1 transgenic mice (Beijing Biocytogene Biotechnology Co., Ltd.) were used to establish MC38-hPD-L1 colon cancer animal model and conduct PD-L1 antibody efficacy experiments. Mice were inoculated subcutaneously with 5 ⁇ 10 5 MC38-hPD-L1 colon cancer cells on the right side.
  • mice with moderate individual tumor volume into the group select mice with moderate individual tumor volume into the group, and assign the animals to 3 experimental groups according to the tumor volume using random grouping software: anti-Hel hIgG isotype control group and anti-PDL1-156 antibody group , Positive drug Avelumab group (Pfizer, lotAU020322), 8 rats in each group, start the administration on the day of grouping (defined as study day 0).
  • Each group was administered by intraperitoneal injection twice a week at a dose of 10 mg/kg, a total of 7 times.
  • TGITV(%) [1-(Ti -T0)/(Vi-V0)] ⁇ 100%;
  • Ti mean tumor volume of the treatment group on day i of administration
  • T0 mean tumor volume of the treatment group on day 0 of administration
  • Vi the solvent control group
  • V0 the mean tumor volume on the 0 day of the solvent control group).
  • the tumor volume was statistically analyzed. P ⁇ 0.05 was considered as a significant difference.
  • a Mean ⁇ standard error
  • b The tumor volume of the administration group and the hIgG control tumor volume were statistically analyzed on the 21st day after group administration, Two-way ANOVA test*p ⁇ 0.05**p ⁇ 0.01* **P ⁇ 0.001****p ⁇ 0.0001.
  • a Mean ⁇ standard error
  • b The body weight of the administration group and the hIgG control body weight are statistically compared on the 21st day of group administration, T-test.
  • MC38-hPD-L1 cells were inoculated subcutaneously on the right side of female B-hPD-L1 transgenic mice (Biocytogen Jiangsu Gene Biotechnology Co., Ltd.) for 6-8 weeks at a concentration of 5 ⁇ 10 5 cells/0.1mL, waiting for tumor growth
  • 24 mice were randomly selected according to the tumor volume, 8 mice in each group, a total of 3 groups, namely: normal saline, Avelumab (5mg/kg), 156-10H (5mg/kg).
  • the route of administration in all groups was intraperitoneal injection, twice a week for 6 consecutive times, and the experiment was terminated 5 days after the last administration.
  • the mouse body weight and tumor volume were measured 3 times a week, and the measured values were recorded, and the tumor volume (long diameter x short diameter 2 /2) and growth inhibition rate (TGI TV (%)) were calculated.
  • the positive drug Avelumab group and the PD-L1 antibody 156-10H group had significant and similar inhibitory effects on tumor volume, and there was a statistical difference (P ⁇ 0.05).
  • a Mean ⁇ standard error
  • b The tumor volume of the administration group and the normal saline control group were statistically compared on the 21st day of group administration, T-test analysis, *P ⁇ 0.05, **P ⁇ 0.01.
  • a Mean ⁇ standard error
  • b The weight of the administration group and the vehicle control group were statistically compared on the 21st day of group administration, and analyzed by T-test.
  • the humanized PD-L1 antibody 156-10H has a significant inhibitory effect on the growth of MC38-hPD-L1 tumor subcutaneously transplanted tumors and shows high safety. Compared with the positive control antibody Avelumab, the TGI levels of the two are equivalent, and 156-10H shows a more uniform anti-tumor effect.

Landscapes

  • Health & Medical Sciences (AREA)
  • Immunology (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Genetics & Genomics (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Peptides Or Proteins (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)

Abstract

涉及特异性结合人程序性死亡配体-1(PD-L1)的抗体或抗原结合片段，所述抗体或抗原结合片段能够增强T细胞的功能，上调T细胞介导的免疫应答；还涉及所述抗体或抗原结合片段用于治疗PD-L1表达异常相关和/或T细胞功能异常的疾病，例如肿瘤的用途。

Description

抗人程序性死亡配体-1(PD-L1)的抗体及其用途
本申请要求于2019年11月08日提交中国专利局、申请号为201911088643.5、发明名称为“抗人程序性死亡配体-1(PD-L1)的抗体及其用途”的中国专利申请的优先权，其全部内容通过引用结合在本申请中。
技术领域
本发明涉及抗人程序性死亡配体-1(PD-L1)的抗体或其抗原结合片段，其编码核酸、表达载体和表达细胞、制备方法、药物组合物、以及它们用于增强T细胞的功能，上调T细胞介导的免疫应答和用于治疗PD-L1表达异常和T细胞功能异常相关的疾病，如肿瘤的用途。
背景技术
免疫治疗已成为肿瘤治疗中发展最为迅速、最具前景的研究领域之一，而运用免疫检查点抑制剂，如PD-1/PD-L1单抗，CTLA-4单抗则是肿瘤免疫疗法革命性的治疗方法，大大地提高了恶行肿瘤患者地生存期。
T细胞介导的免疫反应受到共刺激和共抑制机制的严格调控，在抗原免疫反应和维持自我耐受之间保持最佳平衡。该平衡由多种激活性和抑制性蛋白参与。抑制性蛋白，也被称为免疫检查点类蛋白，调节杀伤性T细胞(cytotoxic T lymphocyte,CTL)的激活和效应功能，以维持自我耐受性。免疫检查点类抑制性蛋白在肿瘤的调节通路中起着关键作用。其中一个重要的免疫检查点蛋白PD-1，与其配体PD-L1结合后，会传导免疫抑制信号，降低T细胞的活性。同时肿瘤细胞也可以通过在细胞表面表达PD-L1来抑制T细胞的激活和增殖，从而逃避CTL的攻击和杀伤。利用PD-1或PD-L1单抗，阻止PD-1/PD-L1的结合和相互作用，可以部分恢复T细胞的功能，从而增强杀伤肿瘤细胞的能力。
2011年，第一个免疫检测点抑制剂伊普利单抗作为一种抗CTLA-4单克隆抗体，成为成功用于治疗黑色素瘤的肿瘤免疫疗法，至今治疗的很多患者已获得相比与传统治疗方法更好的5年生存期。之后，FDA又先后批准了3个PD-1单抗和3个PD-L1单抗，成功地用于免疫治疗除黑色素瘤之外的十几种肿瘤，并成为多种癌症的一线治疗手段，如非小细胞肺癌(NSCLC)、肾细胞癌(RCC)和膀胱或尿路上皮癌等。中国至 今已批准了2个进口PD-1抗体和3个国产PD-1抗体上市，但是还没有PD-L1抗体获批，并且鉴于PD-L1抗体与PD-1抗体治疗机理上及目前临床试验联用药物和适用适应症的不同，研发新的PD-L1单抗和基于PD-L1的双抗仍然有重大的社会和经济意义。
发明内容
本发明提供特异性结合人程序性死亡配体-1(PD-L1)的抗体及抗原结合片段，编码这些抗体及抗原结合片段的核酸，包含所述抗体及抗原结合片段和药物组合物和试剂盒，以及它们用于增强T细胞的功能，上调T细胞介导的免疫应答和用于治疗PD-L1表达异常和T细胞功能异常相关的病症，例如肿瘤免疫的用途。该抗体不仅可以与人和食蟹猴PD-L1蛋白结合，还可以阻断人PD-L1和人PD-1之间的相互作用。
在一些实施方案中，特异性结合人程序性死亡配体-1(PD-L1)的分离的抗体或抗原结合片段，包含重链CDRs组合和轻链CDRs组合：
(1)所述重链CDRs组合包含：CDR1-VH、CDR2-VH和CDR3-VH；所述CDR1-VH、CDR2-VH和CDR3-VH具有选自以下的任意序列组合或者与所述序列组合相比具有1、2、3或更多个氨基酸插入、缺失和/或替换的序列组合：
Figure PCTCN2020126656-appb-000001
Figure PCTCN2020126656-appb-000002
和，
(2)所述轻链CDRs组合包含：CDR1-VL、CDR2-VL和CDR3-VL，所述CDR1-VL、CDR2-VL和CDR3-VL具有选自以下任意序列组合或者与所述序列组合相比具有1、2、3或更多个氨基酸插入、缺失和/或替换的序列组合：
Figure PCTCN2020126656-appb-000003
Figure PCTCN2020126656-appb-000004
各个CDR1-VH、CDR2-VH、CDR3-VH、CDR1-VL、CDR2-VL和CDR3-VL为根据KABAT、Chothia或IMGT的通行分析方法编码。
在一些实施方案中，特异性结合人程序性死亡配体-1(PD-L1)的分离的人源化抗体或抗原结合片段，包含重链CDRs组合和轻链CDRs组合：
(1)所述重链CDRs组合包含：CDR1-VH、CDR2-VH和CDR3-VH；所述CDR1-VH、CDR2-VH和CDR3-VH具有选自以下的任意序列组合或者与所述序列组合相比具有1、2、3或更多个氨基酸插入、缺失和/或替换的序列组合：
Figure PCTCN2020126656-appb-000005
和，
(2)所述轻链CDRs组合包含：CDR1-VL、CDR2-VL和CDR3-VL，所述CDR1-VL、CDR2-VL和CDR3-VL具有选自以下任意序列组合或者与所述序列组合 相比具有1、2、3或更多个氨基酸插入、缺失和/或替换的序列组合：
Figure PCTCN2020126656-appb-000006
各个CDR1-VH、CDR2-VH、CDR3-VH、CDR1-VL、CDR2-VL和CDR3-VL为根据KABAT、Chothia或IMGT的通行分析方法编码。
特别地，例如本发明的抗体或其抗原结合片段包含选自以下的重链CDRs和轻链CDRs组合：VH1+VL1、VH2+VL2、VH3+VL3、VH4+VL4、VH5+VL5、VH6+VL6、VH7+VL7、VH8+VL8、VH9+VL9、VH10+VL10、VH11+VL11、VH12+VL12、VH13+VL13、VH14+VL14、VH15+VL15、VH16+VL16、VH17+VL17、VH18+VL18、VH19+VL19、VH20+VL20、VH21+VL21、VH22+VL22、VH23+VL23、VH24+VL24、VH25+VL25、或VH26+VL26，以及与所述重链和轻链CDRs组合之序列相比具有1、2、3或更多个氨基酸插入、缺失和/或替换的CDRs组合。
在另一个具体实施方案中，本发明提供这样的抗体或其抗原结合片段，其中：
1)重链可变区和轻链可变区分别具有SEQ ID NO:1和SEQ ID NO:2所示序列，或者与所示序列具有70％、75％、80％、85％、90％、95％、96％、97％、98％、99％或更高一致性的序列；
2)重链可变区和轻链可变区分别具有SEQ ID NO:3和SEQ ID NO:4所示序列，或者与所示序列具有70％、75％、80％、85％、90％、95％、96％、97％、98％、99％或更高一致性的序列；
3)重链可变区和轻链可变区分别具有SEQ ID NO:5和SEQ ID NO:6所示序列， 或者与所示序列具有70％、75％、80％、85％、90％、95％、96％、97％、98％、99％或更高一致性的序列；
4)重链可变区和轻链可变区分别具有SEQ ID NO:7和SEQ ID NO:8所示序列，或者与所示序列具有70％、75％、80％、85％、90％、95％、96％、97％、98％、99％或更高一致性的序列；
5)重链可变区和轻链可变区分别具有SEQ ID NO:9和SEQ ID NO:10所示序列，或者与所示序列具有70％、75％、80％、85％、90％、95％、96％、97％、98％、99％或更高一致性的序列；
6)重链可变区和轻链可变区分别具有SEQ ID NO:11和SEQ ID NO:12所示序列，或者与所示序列具有70％、75％、80％、85％、90％、95％、96％、97％、98％、99％或更高一致性的序列；
7)重链可变区和轻链可变区分别具有SEQ ID NO:13和SEQ ID NO:14所示序列，或者与所示序列具有70％、75％、80％、85％、90％、95％、96％、97％、98％、99％或更高一致性的序列；
8)重链可变区和轻链可变区分别具有SEQ ID NO:15和SEQ ID NO:16所示序列，或者与所示序列具有70％、75％、80％、85％、90％、95％、96％、97％、98％、99％或更高一致性的序列；
9)重链可变区和轻链可变区分别具有SEQ ID NO:17和SEQ ID NO:18所示序列，或者与所示序列具有70％、75％、80％、85％、90％、95％、96％、97％、98％、99％或更高一致性的序列；
10)重链可变区和轻链可变区分别具有SEQ ID NO:127和SEQ ID NO:128所示序列，或者与所示序列具有70％、75％、80％、85％、90％、95％、96％、97％、98％、99％或更高一致性的序列；
11)重链可变区和轻链可变区分别具有SEQ ID NO:129和SEQ ID NO:130所示序列，或者与所示序列具有70％、75％、80％、85％、90％、95％、96％、97％、98％、99％或更高一致性的序列；
12)重链可变区和轻链可变区分别具有SEQ ID NO:131和SEQ ID NO:132所示序列，或者与所示序列具有70％、75％、80％、85％、90％、95％、96％、97％、98％、99％或更高一致性的序列；或，
13)重链可变区和轻链可变区分别具有SEQ ID NO:133和SEQ ID NO:134所示序列，或者与所示序列具有70％、75％、80％、85％、90％、95％、96％、97％、98％、99％或更高一致性的序列。
在一个优选实施方案中，本发明的抗体或其抗原结合片段是嵌合的或人源化的或全人源的。
在一个优选实施方案中，本发明的抗体或其抗原结合片段，其与人程序性死亡配体-1(PD-L1)结合的解离常数(KD)不大于10nM，与食蟹猴程序性死亡配体-1(PD-L1)结合的解离常数(KD)不大于100nM。
在一个优选实施方案中，本发明的抗体或其抗原结合片段，包含人或鼠抗体IgG1、IgG2、IgG3、IgG4、IgA、IgM、IgE或IgD任何其中之一恒定区的序列；优选包含人或鼠抗体IgG1、IgG2、IgG3或IgG4的恒定区的序列；或携带突变的人或鼠抗体IgG1、IgG2、IgG3或IgG4的恒定区的序列。
在一个优选实施方案中，本发明所述抗原结合片段选自F(ab)2、Fab’、Fab、Fv、scFv、双特异抗体、纳米抗体和抗体最小识别单位中的一种或多种。
在一个优选实施方案中，本发明的抗体或其抗原结合片段可与选自编号34、50、90、130、156、370、373、413、或794的抗体竞争性的结合PD-L1，并且具备以下特性：
1)特异性结合PD-L1重组蛋白及表达PD-L1的细胞；
2)阻断PD-L1与PD-1蛋白的结合；
3)抑制PD-1与细胞表面表达的PD-L1的结合；
4)增强T细胞活性；
5)介导抗体依赖的细胞杀伤(ADCC)活性；或/和
6)抑制肿瘤生长。
在一些实施方案中，本发明提供一种分离的核酸分子，所述核酸分子编码本发明上述所述的抗体、抗原结合片段、或其任意组合。
在一些实施方案中，本发明提供一种表达载体，其包含本发明上述所述分离的核酸分子。
在一些实施方案中，本发明提供一种宿主细胞，其包含本发明上述所述分离的核酸分子或表达载体。
在一个优选实施方案中，所述宿主细胞是真核细胞或原核细胞；更优选，所述宿主细胞来源于哺乳动物细胞、酵母细胞、昆虫细胞、大肠杆菌和/或枯草杆菌；更优选，所述宿主细胞选自中国仓鼠卵巢细胞(CHO)。
在一些实施方案中，本发明提供一种抗体或抗原结合片段的制备方法，在适当的条件下培养本发明上述所述的宿主细胞，并分离抗体或抗原结合片段。
在一些实施方案中，本发明提供一种药物组合物，组合物包含本发明上述所述的抗体或抗原结合片段、本发明上述所述分离的核酸分子、本发明上述所述表达载体、本发明上述所述细胞，或本发明上述所述方法制备的产品(例如抗体和抗原结合片段)，以及药学上可接受的载体。
在一个优选实施方案中，所述药物组合物还包含额外的抗肿瘤剂。
在一些实施方案中，本发明提供一种预防和/或治疗PD-L1表达异常和/或T细胞功能异常相关的疾病的方法，包含向有此需要的患者施用本发明上述所述的抗体或抗原结合片段、本发明上述所述的分离的核酸分子、本发明上述所述的表达载体、本发明上述所述的细胞、本发明上述所述的方法制备的产品(例如抗体和抗原结合片段)、或本发明上述所述药物组合物；所述疾病优选肿瘤；所述肿瘤优选结直肠癌。
在一些实施方案中，本发明提供上述所述的抗体或抗原结合片段、本发明上述所述的分离的核酸分子、本发明上述所述的表达载体、本发明上述所述的细胞、本发明上述所述的方法制备的产品(例如抗体和抗原结合片段)、或本发明上述所述药物组合物在制备预防和/或治疗PD-L1表达异常相关的疾病的药物中的用途，所述疾病优选肿瘤；所述肿瘤优选结直肠癌。
在一些实施方案中，本发明提供一种试剂盒，其包含本发明上述所述的抗体或抗原结合片段、本发明上述所述的分离的核酸分子、本发明上述所述的表达载体、本发明上述所述的细胞、或本发明上述所述的方法制备的产品(例如抗体和抗原结合片段)，以及使用说明。
术语和定义：
除非另外说明，本文所用术语具有所属技术领域普通技术人员通常理解的含义。对于本文中明确定义的术语，则该术语的含义以所述定义为准。
如本文所用，术语“抗体”(Ab)是指与目标抗原特异性结合或具有免疫反应性的免疫球蛋白分子，包括抗体的多克隆、单克隆、基因工程化和其他修饰形式(包括但 不限于嵌合抗体，人源化抗体，全人源抗体，异源偶联抗体(例如双特异性、三特异性和四特异性抗体，双抗体，三抗体和四抗体)，抗体缀合物)以及抗体的抗原结合片段(包括例如Fab’、F(ab’)2、Fab、Fv、rIgG和scFv片段)。此外，除非另有说明，否则术语“单克隆抗体”(mAb)意指包括能够特异性结合靶蛋白的完整抗体分子以及不完整的抗体片段(例如Fab和F(ab’)2片段，它们缺少完整抗体的Fc片段(从动物循环中更快地清除)，因此缺乏Fc介导的效应功能(effector function)(参见Wahl等人，J.Nucl.Med.24:316,1983；其内容援引加入本文))。
如本文所用，术语“抗原结合片段”是指保留特异性结合靶抗原的能力的一个或更多个抗体片段。抗体的抗原结合功能可以由全长抗体的片段执行。抗体片段可以是Fab、F(ab’)2、scFv、SMIP、双抗体、三抗体、亲和体(affibody)、纳米抗体、适体或结构域抗体。涵盖术语抗体的“抗原结合片段”的结合片段的实例包括但不限于：(i)Fab片段，一种由VL、VH、CL和CHl结构域组成的单价片段；(ii)F(ab)2片段，一种包含由二硫键在铰链区连接的两个Fab片段的双价片段；(iii)由VH和CHl结构域组成的Fd片段；(iv)由抗体单臂的VL和VH结构域组成的Fv片段；(V)包含VH和VL结构域的dAb；(vi)由VH结构域组成的dAb片段(Ward等人，Nature341:544-546,1989)；(vii)由VH或VL结构域组成的dAb；(viii)分离的互补决定区(CDR)；以及(ix)两个或更多个分离的CDR的组合，所述CDR可以任选地由合成接头连接。此外，虽然Fv片段的两个结构域VL和VH是通过独立的基因编码的，但是这两个结构域可以使用重组方法通过接头接合，该接头能够使其制成其中VL和VH区配对以形成单价分子的单蛋白质链(称为单链Fv(scFv)；参见例如，Bird等人，Science 242:423-426,1988以及Huston等人，Proc.Natl.Acad.Sci.USA85:5879-5883,1988)。这些抗体片段可以使用本领域技术人员已知的常规技术获得，并且这些片段被筛选用于与完整抗体相同的方式使用。可以通过重组DNA技术、完整免疫球蛋白的酶促或化学裂解、或在一些实施方式中通过本领域已知的化学肽合成程序来产生抗原结合片段。
如本文所用，术语“PD-L1”是指程序性死亡配体-1，也称为CD279(分化簇279)，是一种重要的免疫抑制分子。所述PD-L1优选地是人PD-L1。
如本文所用，术语“抗-程序性死亡配体-1抗体”、“程序性死亡配体-1抗体”、“抗PD-L1抗体”、“PD-L1抗体”、“抗-PD-L1抗体部分”和/或“抗-PD-L1抗体片段”等是指 任何包含能够特异性结合PD-L1的免疫球蛋白分子的至少一部分(例如但不限于重链或轻链的至少一个互补决定区(CDR)或其配体结合部分、重链或轻链可变区、重链或轻链恒定区、框架区或其任何部分)的含蛋白质或肽的分子。PD-L1抗体还包括抗体样蛋白支架(如第十纤连蛋白III型结构域(10Fn3))，其含有与抗体CDR在结构和溶剂可及性上相似的BC、DE和FG结构环。10Fn3结构域的三级结构类似于IgG重链可变区的三级结构，并且通过将10Fn3的BC、DE和FG环的残基用来自PD-L1单克隆抗体的CDR-H1、CDR-H2或CDR-H3区的残基替换，本领域技术人员可以将例如PD-L1单克隆抗体的CDR接枝到纤连蛋白支架上。
如本文所用，术语“双特异性抗体”是指对至少两种不同的抗原具有单克隆结合特异性的抗体，其通常是人或人源化的抗体。在本发明中，结合特异性之一可以针对PD-L1的抗原表位而被检测，另一个可以针对PD-L1的另一个抗原表位或任何其他抗原，例如针对细胞表面蛋白、受体、受体亚基、组织特异性抗原、病毒来源蛋白、病毒编码的包膜蛋白、细菌来源蛋白或细菌表面蛋白等而被检测。
如本文所用，术语“嵌合”抗体是指以下抗体，其具有源自一种来源生物(如大鼠或小鼠)的免疫球蛋白的可变序列以及源自不同生物体(例如人)的免疫球蛋白的恒定区。用于生产嵌合抗体的方法是本领域已知的。参见例如，Morrison,1985,Science 229(4719):1202-7；Oi等人，1986,Bio Techniques 4:214-221；Gillies等人，1985 J Immunol Methods 125:191-202；以上通过援引加入并入本文。
如本文所用，术语“互补决定区”(CDR)指在轻链和重链可变结构域中均发现的高变区。可变结构域中更高保守性的部分称为框架区(FR)。如本领域所理解的，表示抗体的高变区的氨基酸位置可以根据上下文和本领域已知的各种定义而变化。可变结构域内的一些位置可以被视为杂合高变位置，因为这些位置可以被认为是在一组标准(如IMGT或KABAT)下的高变区之内，而被认为在不同组的标准(如KABAT或IMGT)下的高变区之外。这些位置中的一个或更多个也可以在延伸的高变区中找到。本发明包括在这些杂合高变的位置中包含修饰的抗体。天然重链和轻链的可变结构域各自包含主要采用片层构型的四个框架区，其通过三个CDR(CDR1、CDR2和CDR3)连接，这三个CDR形成连接片层结构的环，并且在一些情况下形成片层结构的一部分。每条链中的CDR通过FR区按顺序FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4紧密保持在一起，并且与来自其他抗体链的CDR促成了抗体的抗原结合位点的形成 (参见Kabat等人，Sequences of Protein sofImmunological Interest,National Institute of Health,Bethesda,Md.1987；其通过援引加入并入本文)。例如在本文中，CDR1-VH、CDR2-VH和CDR3-VH分别是指重链可变区(VH)的第一个CDR、第二个CDR和第三个CDR，这三个CDR构成了重链(或其可变区)的CDR组合(VHCDR组合)；CDR1-VL、CDR2-VL和CDR3-VL分别是指轻链可变区(VL)的第一个CDR、第二个CDR和第三个CDR，这三个CDR构成了轻链(或其可变区)的CDR组合(VLCDR组合)。
如本文所用，术语“抗体缀合物”是指抗体分子直接或者通过连接接头与另一个分子化学键合而形成的偶联体/缀合物。例如抗体-药物缀合物(ADC)，其中药物分子就是所述的另一个分子。
如本文所用，术语“单克隆抗体”是指来源于单个克隆(包括任何真核、原核、或噬菌体克隆)的抗体，而不限于该抗体的产生方法。
如本文所用，术语“VH”是指抗体的免疫球蛋白重链(包括Fv、scFv或Fab的重链)的可变区。术语“VL”是指免疫球蛋白轻链(包括Fv、scFv、dsFv或Fab的轻链)的可变区。
如本文所用，术语“百分比(％)序列一致性”是指在为达到最大百分比序列一致性而比对序列和引入空位(如果需要)(例如，为了最佳比对，可以在候选和参比序列中的一个或两个中引入空位，并且出于比较的目的，可以忽略非同源序列)之后，候选序列的氨基酸(或核苷酸)残基与参比序列的氨基酸(或核苷酸)残基相同的百分比。出于确定百分比序列一致性的目的，可以用本领域技术人员熟知的多种方式来实现比对，例如使用公众可得的计算机软件，如BLAST、ALIGN或Megalign(DNASTAIi)软件。本领域技术人员可以确定用于测量比对的适当参数，包括需要在被比较序列的全长范围实现最大比对的任何算法。例如，用于与候选序列进行比较而比对的参比序列可以显示候选序列在候选序列的全长或候选序列的连续氨基酸(或核苷酸)残基的选定部分上表现出从50％至100％的序列同一性。出于比较目的而比对的候选序列的长度可以是例如参比序列的长度的至少30％(例如30％、40％、50％、60％、70％、80％、90％或100％)。当候选序列中的位置被与在参比序列中的相应位置相同的氨基酸(或核苷酸)残基占据时，则这些分子在那个位置是相同的。
如本文所用，术语“特异性结合”是指一种结合反应，其决定抗原在蛋白质和其他生物分子的一个异质性群体中的存在状况，所述蛋白质和其他生物分子例如被抗体或其抗原结合片段特异性识别。与抗原特异性结合的抗体或其抗原结合片段将以小于100nM的KD与抗原结合。例如，与抗原特异性结合的抗体或其抗原结合片段将以高达100nM((例如，1pM至100nM之间)的KD与抗原结合。不显示与特定抗原或其表位特异性结合的抗体或其抗原结合片段将显示对该特定抗原或其表位的大于100nM(例如，大于500nM、1μM、100μΜ、500μΜ或1mM)的KD。可以使用多种免疫测定方式来选择与特定蛋白或碳水化合物进行特异性免疫反应的抗体。例如，常规地使用固相ELISA免疫测定法来选择与蛋白质或碳水化合物进行特异性免疫反应的抗体。参见，Harlow&Lane，Antibodies,ALaboratory Manual,Cold Spring Harbor Press,NewYork(1988)以及Harlow&Lane,Using Antibodies，A Laboratory Manual,Cold Spring Harbor Press,NewYork(1999)，其描述了可以用于确定特异免疫反应性的免疫测定方式和条件。
如本文所用，术语“载体”包括核酸载体，例如DNA载体(如质粒)，RNA载体，病毒或其他适合的复制子(例如病毒载体)。已经开发了多种载体用于将编码外源蛋白质的多核苷酸递送到原核或真核细胞中。本发明的表达载体含有多核苷酸序列以及例如用于表达蛋白质和/或将这些多核苷酸序列整合到哺乳动物细胞基因组中的附加序列元件。可以用于表达本发明的抗体和抗体片段的某些载体包括含有指导基因转录的调控序列(如启动子和增强子区域)的质粒。用于表达抗体和抗体片段的其他有用的载体含有多核苷酸序列，其增强这些基因的翻译速率或改善由基因转录产生的mRNA的稳定性或核输出。这些序列元件包括例如5’和3’非翻译区、内部核糖体进入位点(IRES)和聚腺苷酸化信号位点，以便指导表达载体上携带的基因的有效转录。本发明的表达载体还可以含有以下多核苷酸，该多核苷酸编码用于选择含有这种载体的细胞的标记。适合的标记的实例包括编码抗生素(如氨苄青霉素、氯霉素、卡那霉素或诺尔丝菌素)抗性的基因。
如本文所用，术语“受试者”、“对象”和“患者”是指接受对如本文所述的特定疾病或病症(如癌症或传染性疾病)的治疗的生物体。对象和患者的实例包括接受疾病或病症(例如细胞增殖性病症，如癌症或传染性疾病)的治疗的哺乳动物，如人、灵长 类动物、猪、山羊、兔、仓鼠、猫、狗、豚鼠、牛科家族成员(如家牛、野牛、水牛、麋鹿和牦牛等)、牛、绵羊、马和野牛等。
如本文所用，术语“治疗”是指外科手术或药物处理(surgical or therapeutic treatment)，其目的是预防、减缓(减少)治疗对象中不希望的生理变化或病变，如细胞增殖性病症(如癌症或传染性疾病)的进展。有益的或所希望的临床结果包括但不限于症状的减轻、疾病程度减弱、疾病状态稳定(即，未恶化)、疾病进展的延迟或减慢、疾病状态的改善或缓和、以及缓解(无论是部分缓解或完全缓解)，无论是可检测的或不可检测的。需要治疗的对象包括已患有病症或疾病的对象以及易于患上病症或疾病的对象或打算预防病症或疾病的对象。当提到减缓、减轻、减弱、缓和、缓解等术语时，其含义也包括消除、消失、不发生等情况。
附图说明
下面通过对本发明的详细描述以及附图来清楚地说明本发明前面叙述的方面以及其他方面。本文中附图是为了举例说明本发明的一些优选的实施方案，然而，可以理解，本发明并不限于所公开的特定实施方案。
图1、末次免疫后测定的小鼠血清结合人PD-L1-mFc(A)和PD-L1-His(B)重组蛋白的滴度；
图2、PD-L1特异性B细胞的流式细胞染色分选(FACS)和圈门策略图；
图3、竞争性ELISA方法测定抗PD-L1抗体阻断PD-L1蛋白与PD-1蛋白结合；
图4、FACS测定抗PD-L1抗体结合细胞表面PD-L1蛋白的EC50；
图5、抗PD-L1抗体增加Jurkat-PD-1-CHO-PD-L1-NFAT体系中报告基因的表达和活性；
图6、抗PD-L1抗体促进混合淋巴细胞反应中IFN-γ的分泌；
图7、抗PD-L1抗体对A431细胞的抗体依赖的细胞杀伤(ADCC)活性；
图8、鼠源抗PD-L1抗体抑制人PD-1/PD-L1转基因小鼠体内MC38-hPD-L1结肠癌肿瘤生长；
图9、人源化抗PD-L1抗体抑制人PD-L1转基因小鼠体内MC38-hPD-L1结肠癌肿瘤生长。
具体实施方式
下面结合实施例和附图对本发明进行详细描述，本文中附图是为了举例说明本发明的一些优选的实施方案，然而，可以理解，本发明并不限于所公开的特定实施方案或看作对本发明范围的限制。实施例中未注明具体条件者，按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者，均为可以通过市售购买获得的常规产品。
实施例1小鼠免疫产生PD-L1抗体
对6-8周龄的雌性SJL小鼠(购自北京维通利华实验动物技术有限公司)或者Balb/c小鼠(购自上海斯莱克实验动物有限公司)，使用融合了小鼠Fc的人PD-L1蛋白(PD-L1-mFc,Novoprotein,Cat.CM06,或Sino Biological,Cat.10084-H05H)或人PD-L1-His(Novoprotein,Cat.C315或Sino Biological，Cat：.10084-H08H)与弗氏完全佐剂(complete freund's adjuvant,CFA,Sigma，Cat.F5881)进行第一次免疫；使用上述PD-L1-mFc或人PD-L1-His与弗氏不完全佐剂(incomplete freund's adjuvant，IFA,Sigma，Cat.F5506)和加未甲基化的胞嘧啶鸟嘌呤二核苷酸(CpGODN1826，合成自上海生工生物)进行后三次免疫，免疫时注射50μg/只/次通过乳化操作形成的均一稳定的乳剂。特别地，第一次和第二次免疫注射后足垫和背部，第三次和第四次免疫注射尾部皮下及背部，以获得高滴度高亲和力高特异性的抗血清及特异性的免疫细胞。在末次免疫(第四次免疫)后的第5-7天，安乐死小鼠并无菌取出脾脏，无菌分离提取小鼠脾脏淋巴细胞，分装至冻存管中，冻存于液氮中。分别在二次免疫，三次免疫后10天及安乐死小鼠当天进行小鼠的采血操作，分离血清，使用酶联免疫吸附(ELISA)方法测定血清中抗PD-L1特异性抗体的滴度。
实验结果如图1，显示经过四次免疫后，所免疫小鼠的血清与人PD-L1-mFc和PD-L1-His结合的滴度都很高。说明了使用此方法进行小鼠的免疫，可以使小鼠产生高滴度的抗PD-L1抗体。
实施例2 PD-L1特异性单个B细胞的流式细胞荧光分选(Fluorescence-activated cell sorting,FACS)
PD-L1蛋白免疫的小鼠脾脏细胞，经抗原PD-L1-His蛋白(Novoprotein,Cat.C315或Sino Biological，Cat.10084-H08H)及间接标记抗体anti-His-APC(R&D Systems, Cat.IC050A)和针对小鼠B细胞表面特有标志的抗体(anti-mouse B220-Pacfic Blue，R&D Systems,Cat.553089；anti-mouse IgD-PE，R&D Systems,Cat.558597；anti-mouse IgM-PE Cy7,R&D Systems,Cat.552867)染色，并在分选前加入区分死细胞和活细胞的染料7-AAD(R&D Systems,Cat.51-68981E)，用AriaIII(BD公司)流式细胞分选仪分选PD-L1特异性的单个B细胞(7AAD -B220 +IgD -IgM -PDL1-His +)至含有细胞裂解液，RNA酶抑制剂的PCR孔中，每个孔收集一个细胞。结果显示(图2)空白对照未免疫的小鼠脾脏(A)，或使用带His的不相关蛋白CREG-His染色的PD-L1免疫的小鼠脾脏(B)均未检测到明显的PD-L1 +抗原特异性B细胞亚群，而使用PD-L1-His染色的PD-L1免疫的小鼠脾脏(C)则检测到一群PD-L1 +B细胞，每10 6个脾脏细胞中约有114个PD-L1 +B细胞。
实施例3单克隆抗体的扩增和高通量表达
采用专利“一种用于巢式扩增的组合引物及其应用”专利申请号：201811618134.4中实施例1的方法，将单细胞的mRNA反转录成cDNA。然后以cDNA为模板进行巢式PCR，分别进行抗体重链和轻链扩增。扩增得到抗体重链可变区和轻链可变区，分别通过同源重组方法克隆到重链表达载体和轻链表达载体。重链表达载体和轻链表达载体的恒定区都来自于人IgG1。完整重链表达序列是信号肽-VH-CH1-铰链区-CH2-CH3，完整轻链表达序列是信号肽-Vκ-Cκ。以上所述单B细胞抗体克隆和表达皆在96孔板内以高通量方式达到抗体的快速鉴定和发现。经过一系列理化和功能筛选324对克隆表达的抗体重链和轻链后，共获得9个与已上市的PD-L1抗体Avelumab或Atezolizumab理化和功能活性相当或更好的候选鼠源抗体分子，其序列的CDRs分别用IMGT和KABAT软件分析，对应的序列信息如下表1至2所示，其中表1示出鼠源抗体分子的VH和VL序列，表2示出鼠源抗体分子的IMGT和KABAT分析结果。
表1.鼠源抗PD-L1抗体重链可变区和轻链可变区的具体序列信息
Figure PCTCN2020126656-appb-000007
Figure PCTCN2020126656-appb-000008
Figure PCTCN2020126656-appb-000009
Figure PCTCN2020126656-appb-000010
分别使用KABAT和IMGT软件分析各抗体的CDRs，具体的序列信息如下表2：
表2.KABAT和IMGT软件分析各鼠源抗体的CDRs具体序列信息
Figure PCTCN2020126656-appb-000011
Figure PCTCN2020126656-appb-000012
Figure PCTCN2020126656-appb-000013
Figure PCTCN2020126656-appb-000014
实施例4抗体人源化
首先采用经典“CDRs移植”方法进行抗体人源化，即通过序列挑选同源性最高的人源性抗体提供抗体骨架区(FRs)，把目标抗体的基于Kabat命名方法的抗原结合片段互补决定区(CDRs)，移植到前者形成人源化抗体。其次，为更好保持抗体活性和亲和力，基于抗体结构建模分析(MOE软件)：1).选择抗体骨架区位于VH-VL界面、靠近或与CDRs有直接相互作用等氨基酸残基进行回复突变，这类氨基酸残基对保持CDRs区构象多较重要；2).考虑到免疫原性，尽量选择包埋在蛋白内部的氨基酸进行回复突变；3).考虑到抗体稳定性和表达水平，优先考虑分子能量降低突变；4).在人源化过程中通过CDRs区的氨基酸定点突变尝试进一步提升人源化抗体亲和力。通过测试含有不同突变的人源化抗体与人PD-L1的亲和力以及和表面表达PD-L1的细胞的结合，筛选与鼠源PD-L1抗体亲和力、抗体表征和活性功能相当或更好的人源化抗体。
其中，PDL1-156抗体经过人源化后的优选候选抗体分子的序列的CDRs如下，分别用IMGT和KABAT软件分析，对应的序列信息如下表3和表4所示，其中表3示出人源化抗体分子的VH和VL序列，表4示出人源化抗体分子的IMGT和KABAT分析结果)。
表3.人源化抗PD-L1抗体重链可变区和轻链可变区的具体序列信息
Figure PCTCN2020126656-appb-000015
Figure PCTCN2020126656-appb-000016
分别使用KABAT和IMGT软件分析各人源化抗体的CDRs，具体的序列信息如下：
表4.KABAT和IMGT软件分析各人源化PD-L1抗体的CDRs具体序列信息
Figure PCTCN2020126656-appb-000017
Figure PCTCN2020126656-appb-000018
实施例5分子排阻色谱法测定抗体纯度
采用TSKgel G3000SWXL色谱柱(TOSOH，0008541)，预柱Tskgel guard column SWXL(TOSOH，Cat.0008543)进行分子排阻色谱法测定抗体纯度。流动相为磷酸盐缓冲液(NaH 2PO 4-Na 2HPO 4)，配制：称取8.88g的NaH 2PO 4·2H 2O，33.33g的Na 2HPO 4·12H 2O。流动相平衡色谱柱，流速为1mL/min。待基线走平后进样，其中进样体积10μL，紫外检测波长280nm，带宽16nm，参比波长关闭。测定结果如表5所示。
表5.人源化抗PD-L1抗体的纯度
Figure PCTCN2020126656-appb-000019
Figure PCTCN2020126656-appb-000020
实施例6抗体与人以及食蟹猴PD-L1重组蛋白结合的KD测定
使用Biacore T200(GE Healthcare)测定PD-L1抗体对于人和食蟹猴PD-L1-His蛋白的结合亲和力。25℃下在CM5芯片(GE Healthcare，Cat.BR-1005-30)上固定anti-human IgG Fc(Genway，Cat.GWB-20A705)。将anti-human Fc(Genway,Cat.GWB-20A705)用Acetate pH5.0(GE Healthcare，BR-1003-51)稀释至20μg/mL。使用Immobilization method中Amine方法进行固定。或者使用商品化ProteinA(GE Healthcare，Cat.29127556)芯片进行检测。25℃下采用多循环动力学法测定抗体与抗原的亲和力，在每一个循环中，首先将待测抗体捕获到固定好的CM5芯片，然后注入重组人PD-L1-His(Novoprotein,Cat.315)和食蟹猴PD-L1-His蛋白(Sino Biological，Cat.90251-C08H)，最后用Glycine pH1.5再生。流动相为HBS-EP+Buffer(GE Healthcare，Cat.BR-1006-69)，流速30μL/min，结合时间为300秒。再生流速30μL/min，时间为30秒。应用Biacore T200 Evaluation Software(version 3.0)，以1：1结合模型，分析试验数据，拟合抗体抗原的平衡解离常数KD，确定结合速率常数ka和解离速率常数kd。
从结果可知所测试的PD-L1抗体对人PD-L1重组蛋白的结合，都表现出nM或更高的亲和力，且都与食蟹猴的PD-L1重组蛋白的亲和力在62.5nM到0.375nM之间，详见下表6。
表6.鼠源PD-L1抗体Biacore结合亲和力KD测定结果(M)
抗体编号 人PD-L1 食蟹猴PD-L1
PDL1-156 2.91E-10 3.75E-10
PDL1-794 1.25E-09 8.83E-10
PDL1-130 2.18E-09 1.95E-09
PDL1-34 4.58E-10 5.43E-09
PDL1-50 6.99E-10 3.31E-08
PDL1-90 8.70E-10 6.25E-08
PDL1-370 2.42E-09 2.18E-09
PDL1-373 2.45E-09 3.64E-09
PDL1-413 2.09E-09 2.76E-09
表7则显示鼠源抗体PDL1-156来源的人源化抗体156-1H、156-7H、156-10H和156-11H对人PD-L1蛋白的结合，表现出与PDL1-156相当的亲和力。
表7.人源化PD-L1抗体Biacore结合亲和力测定结果
Figure PCTCN2020126656-appb-000021
实施例7抗体阻断PD-L1和PD-1相互作用的IC50测定
通过竞争性ELISA方法确定抗PD-L1抗体阻断PD-L1蛋白与PD-1蛋白结合的IC50。
使用碳酸盐缓冲液稀释人PD-L1重组蛋白(Sino Biological,Cat.10084-H05H)，加入96孔酶标板，终浓度为1μg/ml。用含3％BSA的PBS溶液封闭，加入梯度稀释的抗PD-L1抗体(6000ng/ml～2ng/ml)以及人PD-1-His重组蛋白(Sino Biological,Cat.10377-H08H)进行共孵育后，加入HRP标记的抗His标签抗体(MBL,Cat.D291-7)，TMB显色，1M硫酸终止后读取OD值(双波长450nm-630nm)。将抗体浓度与OD值对应即可绘制出测试抗体的竞争结合曲线，计算出IC50值。图3显示了抗PD-L1抗体与人PD-L1重组蛋白的竞争结合曲线。结果表明，图中所示与没有任何阻断作用的抗体同型阴性对照anti-Hel(百英生物制备)相比，被测试的9个鼠源抗PD-L1抗体(A)和4个人源化抗体(B)均可以有效的阻断人PD-L1蛋白与人PD-1蛋白的相互作用，且人源化抗体与人源化之前的鼠源PDL1-156的抑制活性相当(B)，IC50分别为197.0ng/mL(156-1H)、230.5ng/mL(156-7H)、250.1ng/mL(156-10H)、207.2ng/mL(156-11H)。鼠源PD-L1-156的IC50则为221.3ng/ml,阳性对照Atezolizumab(百英生物制备)为446.4ng/ml、Avelumab(Pfizer,lot AU020322)为190.3ng/ml。
实施例8 FACS测定PD-L1抗体对细胞表面PD-L1结合的EC 50
将梯度浓度的待检测抗体(抗体终浓度：10000ng/ml-0.1ng/ml，10倍系列稀释)与细胞表面高表达PD-L1的CHO-PD-L1细胞(南京勇山生物科技有限公司，10 5个/孔)，4℃共同孵育30min。孵育结束后，加入1:250稀释的anti-human IgG PE荧光抗体(eBioscience，Cat.12-4998-8)，4℃下共同孵育30min，荧光抗体与待检测抗体的Fc段产生特异性结合，通过FACS检测PE荧光强度的高低而对待检测抗体的结合细胞表面高表达的PD-L1蛋白的能力进行分析。图4结果显示，待测的鼠源PD-L1抗体EC50均与本次实验的阳性对照Avelumab(EC50为58.2ng/ml)和Atezolizumab(～99.73ng/ml)相近，其中PDL1-156和PDL1-370的EC50最低，被检测的人源化抗体156-1H，156-7H，156-10H和156-11H的EC50分别为49.42ng/ml，78.37ng/ml，63.5ng/ml和49.97ng/ml，与本次实验的阳性对照Avelumab(EC50为56.88ng/ml)相近。该检测定量地证实了待测各PD-L1抗体对细胞表面上PD-L1靶点剂量依赖性结合的能力。MFI fold＝实验组MFI值/未加药物的对照组MFI值。
实施例9 PD-1/PD-L1-NFAT报告基因测试抗PD-L1抗体阻抑PD-1:PD-L1结合和信号传导
利用稳定转染PD-1的Jurkat细胞株(GenScript，Cat.00612)和稳定转染PD-L1的CHO细胞株(GenScript，Cat.M00613)比较PD-L1抗体对PD-1/PD-L1蛋白相互作用及其信号通路的拮抗作用。当抑制信号通路阻抑，NFAT控制的发光报告基因表达增强，发光信号值增加。通过发光读值的强弱(relative light units,RLU)反应抗体对PD-L1的阻断作用强弱。
将稳转PD-L1的CHO细胞株种在96孔白底板上，每孔40000个细胞，100μl/孔，放回培养箱过夜；第二天，取出孔板，吸去培养基，加入稳转PD-1的细胞株及待测的PD-L1抗体共孵育，PD-1细胞每孔加样量为16000个/孔，抗体则做梯度稀释，每个剂量3复孔，孵育体积为100μl/孔，孵育时长为6小时，待孵育完成时，取出孔板，等体积(100μl)加入发光检测试剂，读值。根据检测值用Graphpad进行4参数分析做回归曲线，得到各抗体的EC50值，结果详见图5A，被测的鼠源PD-L1抗体的EC50值均与阳性对照抗体Avelumab和Atezolizumab的EC50值(222.9ng/ml，321.6ng/ml)相近，图5B和5C则显示4个人源化PD-L1抗体156-1H，156-7H和156-10H，156-11H的EC50分别为：342ng/ml，313.7ng/ml，357.1ng/ml和282.2ng/ml, 和阳性对照Avelumab的EC50相近。该检测定量地证实了鼠源和人源化的抗PD-L1抗体对细胞表面PD-1：PD-L1相互作用导致的T细胞活性抑制呈现剂量依赖性的阻抑能力，从而剂量依赖地增强Jurkat细胞内报告基因的活性。
实施例10 ELISA检测混合淋巴细胞反应中T细胞分泌的IFN-γ
通过混合淋巴细胞反应(mixed lymphocyte reaction，MLR)来测定PD-L1单抗增强T细胞的活性。从健康人供体1的外周血单核细胞(peripheral blood mononuclear cells,PBMC)中分离CD14 +单核细胞，应用重组人粒细胞-巨噬细胞集落刺激因子(GM-CSF，Peprotech，Cat.300-03)和重组人白介素4(rhIL-4,Peprotech，Cat.200-04)进行体外诱导分化为树突状细胞(dendritic cell,DC)，于培养第6天加入LPS(Sigma，Cat：L4516)刺激成熟DC，第7天将供体1的DC细胞与从健康供体2的PBMC富集的CD4 +T细胞混合共培养，DC：CD4 +T细胞数比例为1:10，加入待测抗体或阳性对照抗体Avelumab或Atezolizumab(抗体浓度为1000ng/ml，100ng/ml，10ng/ml，1ng/ml)，共培养4天。4天后收集细胞培养上清，用ELISA方法检测上清中IFN-γ的含量如图6显示，所有测试的鼠源抗体及阳性对照抗体Avelumab、Atezolizumab相比anti-HEL单抗和no treatment(即没有给药)阴性对照组，均可明显增强MLR实验中CD4 +T细胞分泌IFN-γ的能力，并且随着PD-L1抗体药物浓度降低，增加分泌IFN-γ的活性也降低。该结果表明PD-L1抗体可增强T细胞的功能，且具有剂量依赖性。T-test，*P<0.05，**P<0.01，***P<0.001，****P<0.0001.
实施例11抗PD-L1抗体的抗体依赖的细胞杀伤(Antibody Dependent Cell-mediated Cytotoxicit，ADCC)活性检测
通过分离纯化的正常人PBMC中的天然杀伤(natural killer,NK)细胞对高表达人PD-L1的A431细胞的杀伤实验来测定抗PD-L1单抗介导的抗体依赖的细胞杀伤活性。
效应细胞的制备：复苏冻存的人PBMC细胞(Stemexpress公司)后，在补充有100IU/ml重组人白介素2(rhIL-2,Peprotech,Cat.200-02)的RPMI1640培养基(Invitrogen，Cat.11835030)中孵育过夜。其后，收集细胞后进行活细胞计数。然后使用NK细胞磁珠分离试剂盒(Stemcell,Cat.17955)，从PBMC中纯化NK细胞，将NK细胞用补充有10％灭活的胎牛血清的DMEM(Invitrogen,Cat.11965084)重悬后 进行计数，用作效应细胞。
靶细胞的制备：靶细胞A431用含10％胎牛血清的DMEM培养基培养，ADCC实验前一天，加上终浓度为500IU干扰素IFN-γ(Peprotech,Cat.300-02)进行刺激培养过夜后待用。
用含有10％胎牛血清的DMEM培养基稀释抗PD-L1抗体，稀释好的抗体以25μl/孔分至白壁96孔板(Corning，Cat.3610)中，并且将靶细胞加入孔中(25μl/孔，10000细胞/孔)。使板在37℃，5％CO 2培养箱中孵育30分钟。随后，将效应细胞加入孔中(25μl/孔，40000细胞/孔，即效靶比NK:A431为4:1)，总体积为100μl，使板在37℃，5％CO 2培养箱中孵育4个小时。
需同时设置培养基背景对照孔(即孔中加入100μl培养基)，靶细胞自发死亡释放孔(即孔中只有靶细胞)，效应细胞自发死亡释放孔(即孔中只有NK细胞)，靶细胞最大死亡释放孔(即靶细胞加CytoTox-Glo TMCytotoxicity试剂盒(Promega，Cat.G9291)中提供的lysis buffer，所有对照孔的体积最后用培养基统一补充至100μl。培养4小时后，将板取出，每孔加入50μl CytoTox-Glo TMCytotoxicity Assay Reagent，放于摇床上混匀后，室温放置15分钟后进行读数化学发光(luminescence,RLU)。
根据下面步骤进行计算由ADCC活性引起的细胞裂解率：先将所有孔的读值减去培养基背景对照孔的平均读值，然后计算裂解率％＝(实验孔读值-靶细胞自发死亡释放孔读值-效应细胞自发死亡释放孔)/(靶细胞最大死亡释放孔读值-靶细胞自发死亡释放孔读值)*100
结果显示于图7中，实验结果显示所测试的3个鼠源PD-L1抗体和FDA已批准上市的阳性对照抗PD-L1药物Avelumab均显示出对靶细胞A431细胞的抗体浓度依赖性的裂解杀伤活性和相近的EC50数值，同时，阴性同型对照抗体hIgG1,K(Abdserotec,PHP010)即使在最高浓度时也显示没有ADCC活性。
实施例12鼠源PD-L1抗体PDL1-156的小鼠体内药效检测
利用B-hPD-1/hPD-L1转基因小鼠(北京百奥赛图基因生物技术有限公司)建立MC38-hPD-L1结肠癌动物模型并进行PD-L1抗体药效实验。小鼠右侧皮下接种5×10 5MC38-hPD-L1结肠癌细胞。当肿瘤体积达到～110mm 3时，挑选个体肿瘤体积适中的小鼠入组，将动物按肿瘤体积使用随机分组软件分配到3个实验组：anti-Hel hIgG同型对照组、抗PDL1-156抗体组、阳性药Avelumab组(Pfizer，lotAU020322)，每组 8只，分组当天开始给药(定义为研究第0天)。各组均按照10mg/kg剂量，每周腹腔注射给药两次,共7次。每周两次测量肿瘤体积和小鼠体重，并记录测量值，计算肿瘤体积(长径x短径2/2)和生长抑制率(tumor growth inhibition％,TGITV(％)＝[1-(Ti-T0)/(Vi-V0)]×100％；Ti：治疗组在给药第i天的肿瘤体积均值，T0：治疗组在给药第0天的肿瘤体积均值；Vi：溶剂对照组在给药第i天的肿瘤体积均值，V0：溶剂对照组在给药第0天的肿瘤体积均值)，同时对肿瘤体积进行统计学分析，P<0.05认为有显著性差异。
在分组给药第21天，与对照组相比，阳性药Avelumab 组和PDL1-156鼠源抗PD-L1抗体组在肿瘤体积上有显著且相近的抑制效果，且具有统计学差异(P<0.05)。见图8A、B、C，表8。
表8.受试品对B-hPD-1/hPD-L1人源化小鼠MC38-hPD-L1肿瘤体积的影响
Figure PCTCN2020126656-appb-000022
注：a：平均数±标准误；b：给药组肿瘤体积与hIgG对照肿瘤体积在分组给药后第21天进行统计学分析，Two-way ANOVA test*p<0.05**p<0.01***P<0.001****p<0.0001。
此外，在实验过程中，除hIgG对照组有1只小鼠提前异常死亡。其余实验动物在给药期间活动和进食状态良好，体重均有一定程度的上升，结果说明该抗体安全性较高，见图8D、表9。
表9.受试品对MC38-hPD-L1肿瘤细胞移植B-hPD-1/hPD-L1人源化小鼠体重的影响
Figure PCTCN2020126656-appb-000023
Figure PCTCN2020126656-appb-000024
注：a：平均数±标准误；b：给药组体重与hIgG对照体重在分组给药第21天做统计学比较，T-test。
实施例13人源化抗PD-L1抗体的小鼠体内药效检测
MC38-hPD-L1细胞以5×10 5个/0.1mL浓度接种于雌性6-8周B-hPD-L1转基因小鼠(百奥赛图江苏基因生物技术有限公司)的右侧皮下，待肿瘤生长到大约108mm 3时按肿瘤体积挑选24只随机分组，每组8只，共3组，分别为：生理盐水、Avelumab(5mg/kg)、156-10H(5mg/kg)。所有组给药途径均为腹腔注射，每周给药2次，连续给药6次，末次给药5天后结束实验。给药和观察期间每周测量3次小鼠体重和肿瘤体积，并记录测量值，计算肿瘤体积(长径x短径 2/2)和生长抑制率(TGI TV(％),在分组给药第21天，与生理盐水对照组相比，阳性药Avelumab组和PD-L1抗体156-10H组在肿瘤体积上有显著且相近的抑制效果，且具有统计学差异(P<0.05)。见图9A、B、C，表10。
表10.受试物对B-hPD-L1人源化小鼠MC38-hPD-L1肿瘤体积的影响
Figure PCTCN2020126656-appb-000025
注：a：平均数±标准误；b：给药组肿瘤体积与生理盐水对照组肿瘤体积在分组给药第21天进行统计学比较，T-test分析，*P<0.05,**P<0.01。
实验动物在给药期间活动和进食状态良好，体重均有一定程度的上升，见图9D、 表11。
表11受试物对MC38-hPD-L1细胞移植B-hPD-L1人源化小鼠体重的影响
Figure PCTCN2020126656-appb-000026
注：a：平均数±标准误；b：给药组体重与Vehicle对照组体重在分组给药第21天进行统计学比较，T-test分析。
以上结果表明人源化的PD-L1抗体156-10H对MC38-hPD-L1肿瘤皮下移植瘤生长具有显著抑制作用且表现出较高安全性。与阳性对照抗体Avelumab相比，两者TGI水平相当，且156-10H显示更均一抗肿瘤效果。
以上对本发明所提供的抗人程序性死亡配体-1(PD-L1)的抗体及其用途进行了详细介绍。本文应用了具体个例对本发明的原理及实施方式进行了阐述，以上实施例的说明只是用于帮助理解本发明的方法及其核心思想。应当指出，对于本技术领域技术人员来说，在不脱离本发明原理的前提下，还可以对本发明进行若干改进和修饰，这些改进和修饰也落入本发明权利要求的保护范围内。

Claims (17)

  1. 特异性结合人程序性死亡配体-1(PD-L1)的分离的抗体或抗原结合片段，其特征在于，所述抗体或抗原结合片段包含重链CDRs组合和轻链CDRs组合：
    (1)所述重链CDRs组合包含：CDR1-VH、CDR2-VH和CDR3-VH；所述CDR1-VH、CDR2-VH和CDR3-VH具有选自以下的任意序列组合或者与所述序列组合相比具有1、2、3或更多个氨基酸插入、缺失和/或替换的序列组合：
    Figure PCTCN2020126656-appb-100001
    Figure PCTCN2020126656-appb-100002
    和，
    (2)所述轻链CDRs组合包含：CDR1-VL、CDR2-VL和CDR3-VL，所述CDR1-VL、CDR2-VL和CDR3-VL具有选自以下任意序列组合或者与所述序列组合相比具有1、2、3或更多个氨基酸插入、缺失和/或替换的序列组合：
    Figure PCTCN2020126656-appb-100003
    各个CDR1-VH、CDR2-VH、CDR3-VH、CDR1-VL、CDR2-VL和CDR3-VL为 根据KABAT、Chothia或IMGT的通行分析方法编码。
  2. 特异性结合人程序性死亡配体-1(PD-L1)的分离的人源化抗体或抗原结合片段，其特征在于，所述抗体或抗原结合片段包含重链CDRs组合和轻链CDRs组合：
    (1)所述重链CDRs组合包含：CDR1-VH、CDR2-VH和CDR3-VH；所述CDR1-VH、CDR2-VH和CDR3-VH具有选自以下的任意序列组合或者与所述序列组合相比具有1、2、3或更多个氨基酸插入、缺失和/或替换的序列组合：
    Figure PCTCN2020126656-appb-100004
    和，
    (2)所述轻链CDRs组合包含：CDR1-VL、CDR2-VL和CDR3-VL，所述CDR1-VL、CDR2-VL和CDR3-VL具有选自以下任意序列组合或者与所述序列组合相比具有1、2、3或更多个氨基酸插入、缺失和/或替换的序列组合：
    Figure PCTCN2020126656-appb-100005
    Figure PCTCN2020126656-appb-100006
    各个CDR1-VH、CDR2-VH、CDR3-VH、CDR1-VL、CDR2-VL和CDR3-VL为根据KABAT、Chothia或IMGT的通行分析方法编码。
  3. 权利要求1或2所述的抗体或抗原结合片段，其特征在于，其包含选自以下的重链CDRs和轻链CDRs组合：VH1+VL1、VH2+VL2、VH3+VL3、VH4+VL4、VH5+VL5、VH6+VL6、VH7+VL7、VH8+VL8、VH9+VL9、VH10+VL10、VH11+VL11、VH12+VL12、VH13+VL13、VH14+VL14、VH15+VL15、VH16+VL16、VH17+VL17、VH18+VL18、VH19+VL19、VH20+VL20、VH21+VL21、VH22+VL22、VH23+VL23、VH24+VL24、VH25+VL25、或VH26+VL26，以及与所述重链和轻链CDRs组合之序列相比具有1、2、3或更多个氨基酸插入、缺失和/或替换的CDRs组合。
  4. 权利要求1-3任一项所述的抗体或抗原结合片段，其特征在于，
    1)重链可变区和轻链可变区分别具有SEQ ID NO:1和SEQ ID NO:2所示序列，或者与所示序列具有70％、75％、80％、85％、90％、95％、96％、97％、98％、99％或更高一致性的序列；
    2)重链可变区和轻链可变区分别具有SEQ ID NO:3和SEQ ID NO:4所示序列，或者与所示序列具有70％、75％、80％、85％、90％、95％、96％、97％、98％、99％或更高一致性的序列；
    3)重链可变区和轻链可变区分别具有SEQ ID NO:5和SEQ ID NO:6所示序列，或者与所示序列具有70％、75％、80％、85％、90％、95％、96％、97％、98％、99％或更高一致性的序列；
    4)重链可变区和轻链可变区分别具有SEQ ID NO:7和SEQ ID NO:8所示序列，或者与所示序列具有70％、75％、80％、85％、90％、95％、96％、97％、98％、99％或更高一致性的序列；
    5)重链可变区和轻链可变区分别具有SEQ ID NO:9和SEQ ID NO:10所示序列，或者与所示序列具有70％、75％、80％、85％、90％、95％、96％、97％、98％、99％或更高一致性的序列；
    6)重链可变区和轻链可变区分别具有SEQ ID NO:11和SEQ ID NO:12所示序列，或者与所示序列具有70％、75％、80％、85％、90％、95％、96％、97％、98％、99％或更高一致性的序列；
    7)重链可变区和轻链可变区分别具有SEQ ID NO:13和SEQ ID NO:14所示序列，或者与所示序列具有70％、75％、80％、85％、90％、95％、96％、97％、98％、99％或更高一致性的序列；
    8)重链可变区和轻链可变区分别具有SEQ ID NO:15和SEQ ID NO:16所示序列，或者与所示序列具有70％、75％、80％、85％、90％、95％、96％、97％、98％、99％或更高一致性的序列；
    9)重链可变区和轻链可变区分别具有SEQ ID NO:17和SEQ ID NO:18所示序列，或者与所示序列具有70％、75％、80％、85％、90％、95％、96％、97％、98％、99％或更高一致性的序列；
    10)重链可变区和轻链可变区分别具有SEQ ID NO:127和SEQ ID NO:128所示序列，或者与所示序列具有70％、75％、80％、85％、90％、95％、96％、97％、98％、99％或更高一致性的序列；
    11)重链可变区和轻链可变区分别具有SEQ ID NO:129和SEQ ID NO:130所示序列，或者与所示序列具有70％、75％、80％、85％、90％、95％、96％、97％、98％、99％或更高一致性的序列；
    12)重链可变区和轻链可变区分别具有SEQ ID NO:131和SEQ ID NO:132所示序列，或者与所示序列具有70％、75％、80％、85％、90％、95％、96％、97％、98％、99％或更高一致性的序列；或，
    13)重链可变区和轻链可变区分别具有SEQ ID NO:133和SEQ ID NO:134所示序列，或者与所示序列具有70％、75％、80％、85％、90％、95％、96％、97％、98％、99％或更高一致性的序列。
  5. 权利要求1-4任一项的抗体或抗原结合片段，其特征在于，其与人程序性死亡配体-1(PD-L1)结合的解离常数(KD)不大于10nM，与食蟹猴程序性死亡配体-1(PD-L1)结合的解离常数(KD)不大于100nM。
  6. 权利要求1-5任一项的抗体或抗原结合片段，其特征在于，所述抗体或抗原结合片段为：
    (1)嵌合抗体或其片段；
    (2)人源化抗体或其片段；
    (3)全人源抗体或其片段。
  7. 权利要求1-6任一项的抗体或抗原结合片段，其特征在于，所述抗体包含人或鼠抗体IgG1、IgG2、IgG3、IgG4、IgA、IgM、IgE或IgD任何其中之一恒定区的序列；优选包含人或鼠抗体IgG1、IgG2、IgG3或IgG4的恒定区的序列。
  8. 权利要求1-7任一项的抗体或抗原结合片段，其特征在于，所述抗原结合片段选自F(ab)2、Fab’、Fab、Fv、scFv、双特异抗体、纳米抗体和抗体最小识别单位中的一种或多种。
  9. 一种抗体或抗原结合片段，其特征在于，所述抗体或抗原结合片段与权利要求1-8任一项所述的抗体或抗原结合片段竞争性地结合PD-L1或其抗原表位，并且具备以下特性：
    1)特异性结合PD-L1重组蛋白及表达PD-L1的细胞；
    2)阻断PD-L1与PD-1蛋白的结合；
    3)抑制PD-1与细胞表面表达的PD-L1的结合；
    4)增强T细胞活性；
    5)介导抗体依赖的细胞杀伤(ADCC)活性；或/和
    6)抑制肿瘤生长。
  10. 一种分离的核酸分子，其特征在于，所述核酸分子编码权利要求1-9任一项所述的抗体、抗原结合片段、或其任意组合。
  11. 包含权利要求10所述分离的核酸分子的表达载体。
  12. 包含权利要求10所述的分离的核酸分子、或权利要求11所述的表达载体的分离的宿主细胞；优选，所述宿主细胞是真核细胞或原核细胞；更优选，所述宿主细胞来源于哺乳动物细胞、酵母细胞、昆虫细胞、大肠杆菌和/或枯草杆菌；更优选，所述宿主细胞选自中国仓鼠卵巢细胞(CHO)。
  13. 一种抗体或抗原结合片段的制备方法，其特征在于，在适当的条件下培养权利要求12所述的宿主细胞，并分离抗体或抗原结合片段。
  14. 一种药物组合物，其特征在于，所述组合物包含权利要求1-9任一项所述的抗体或抗原结合片段、权利要求10的分离的核酸分子、权利要求11的表达载体、权 利要求12的细胞，或权利要求13方法制备的产品，以及药学上可接受的载体；优选，所述药物组合物还包含额外的抗肿瘤剂。
  15. 权利要求1-9任一项所述的抗体或抗原结合片段、权利要求10的分离的核酸分子、权利要求11的表达载体、权利要求12的细胞、权利要求13方法制备的产品、或权利要求14所述的药物组合物在制备预防和/或治疗PD-L1表达异常相关的疾病的药物中的用途，所述疾病优选肿瘤。
  16. 一种预防和/或治疗PD-L1表达异常和/或T细胞功能异常相关的疾病的方法，包含向有此需要的患者施用权利要求1-9任一项所述的抗体或抗原结合片段、权利要求10的分离的核酸分子、权利要求11的表达载体、权利要求12的细胞、权利要求13方法制备的产品、或权利要求14所述药物组合物；所述疾病优选肿瘤；所述肿瘤优选结直肠癌。
  17. 一种试剂盒，其包含权利要求1-9任一项的抗体或其抗原结合片段、权利要求10的分离的核酸分子、权利要求11的表达载体、权利要求12的细胞、权利要求13方法制备的产品，以及使用说明。
PCT/CN2020/126656 2019-11-08 2020-11-05 抗人程序性死亡配体-1(pd-l1)的抗体及其用途 WO2021088904A1 (zh)

Priority Applications (6)

Application Number Priority Date Filing Date Title
CA3157516A CA3157516A1 (en) 2019-11-08 2020-11-05 Anti-human programmed cell death ligand-1 (pd-l1) antibody and use thereof
US17/755,741 US20230002492A1 (en) 2019-11-08 2020-11-05 Anti-human programmed cell death ligand-1 (pd-l1) antibody and use thereof
CN202080077362.4A CN115038718B (zh) 2019-11-08 2020-11-05 抗人程序性死亡配体-1（pd-l1）的抗体及其用途
JP2022527056A JP2023500156A (ja) 2019-11-08 2020-11-05 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
EP20884635.2A EP4056592A4 (en) 2019-11-08 2020-11-05 ANTIBODY AGAINST HUMAN PROGRAMMED CELL DEATH LIGAND 1 (PD-L1) AND USE THEREOF
JP2023144842A JP2023179450A (ja) 2019-11-08 2023-09-06 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

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201911088643 2019-11-08
CN201911088643.5 2019-11-08

Publications (1)

Publication Number Publication Date
WO2021088904A1 true WO2021088904A1 (zh) 2021-05-14

Family

ID=75849768

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2020/126656 WO2021088904A1 (zh) 2019-11-08 2020-11-05 抗人程序性死亡配体-1(pd-l1)的抗体及其用途

Country Status (6)

Country Link
US (1) US20230002492A1 (zh)
EP (1) EP4056592A4 (zh)
JP (2) JP2023500156A (zh)
CN (1) CN115038718B (zh)
CA (1) CA3157516A1 (zh)
WO (1) WO2021088904A1 (zh)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2023001025A1 (zh) * 2021-07-23 2023-01-26 南京吉盛澳玛生物医药有限公司 Pd-l1抗体及其用途
WO2024118814A3 (en) * 2022-11-30 2024-08-02 Development Center For Biotechnology Anti-human pd-l1 antibodies and their uses

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2024017362A1 (zh) * 2022-07-22 2024-01-25 上海先博生物科技有限公司 靶向gprc5d和/或bcma的嵌合抗原受体及其应用

Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2016024021A1 (en) * 2014-08-15 2016-02-18 Merck Patent Gmbh Sirp-alpha immunoglobulin fusion proteins
CN106243225A (zh) * 2015-06-11 2016-12-21 智翔（上海）医药科技有限公司 新型抗-pd-l1抗体
WO2017031458A2 (en) * 2015-08-20 2017-02-23 Abbvie Stemcentrx Llc Anti-dll3 antibody drug conjugates and methods of use
WO2017118321A1 (en) * 2016-01-04 2017-07-13 Harbour Biomed Limited Anti-pd-l1 antibodies and uses thereof
WO2017205742A1 (en) * 2016-05-27 2017-11-30 Abbvie Biotherapeutics Inc. Anti-cd40 antibodies and their uses
WO2017215590A1 (en) * 2016-06-13 2017-12-21 I-Mab Anti-pd-l1 antibodies and uses thereof
CN108276492A (zh) * 2018-01-31 2018-07-13 中国药科大学 抗pd－l1单克隆抗体及其应用
CN109563503A (zh) * 2016-07-26 2019-04-02 静冈县 抗b7-h4抗体

Family Cites Families (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
TWI680138B (zh) * 2014-01-23 2019-12-21 美商再生元醫藥公司 抗ｐｄ-ｌ１之人類抗體
CN107001463B (zh) * 2014-08-05 2020-01-17 中美冠科生物技术（太仓）有限公司 抗pd-l1抗体
CN107922470A (zh) * 2015-08-07 2018-04-17 皮里斯制药有限公司 对lag‑3和pd‑1特异性的新型融合多肽
CN105461808B (zh) * 2015-12-24 2019-03-19 长春金赛药业股份有限公司 单克隆抗体及其应用
EP3309177B1 (en) * 2016-03-04 2020-05-13 Sichuan Kelun-Biotech Biopharmaceutical Co., Ltd. Pdl-1 antibody, pharmaceutical composition thereof, and uses thereof
RU2744959C2 (ru) * 2016-03-23 2021-03-17 Мэбспейс Байосайнсиз (Сучжоу) Ко., Лтд Новые анти-pd-l1 антитела
CN108341871A (zh) * 2017-01-24 2018-07-31 三生国健药业（上海）股份有限公司 抗pd-1单克隆抗体及其制备方法和应用
CN106939049B (zh) * 2017-04-20 2019-10-01 苏州思坦维生物技术股份有限公司 拮抗抑制人pd-1抗原与其配体结合的单克隆抗体及其制备方法与应用
WO2019109974A1 (zh) * 2017-12-06 2019-06-13 正大天晴药业集团南京顺欣制药有限公司 抗pd-l1抗体及其抗原结合片段
CN107973854B (zh) * 2017-12-11 2021-05-04 苏州银河生物医药有限公司 Pdl1单克隆抗体及其应用
WO2019129136A1 (zh) * 2017-12-27 2019-07-04 信达生物制药(苏州)有限公司 抗pd-l1抗体及其用途

Patent Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2016024021A1 (en) * 2014-08-15 2016-02-18 Merck Patent Gmbh Sirp-alpha immunoglobulin fusion proteins
CN106243225A (zh) * 2015-06-11 2016-12-21 智翔（上海）医药科技有限公司 新型抗-pd-l1抗体
WO2017031458A2 (en) * 2015-08-20 2017-02-23 Abbvie Stemcentrx Llc Anti-dll3 antibody drug conjugates and methods of use
WO2017118321A1 (en) * 2016-01-04 2017-07-13 Harbour Biomed Limited Anti-pd-l1 antibodies and uses thereof
WO2017205742A1 (en) * 2016-05-27 2017-11-30 Abbvie Biotherapeutics Inc. Anti-cd40 antibodies and their uses
WO2017215590A1 (en) * 2016-06-13 2017-12-21 I-Mab Anti-pd-l1 antibodies and uses thereof
CN109563503A (zh) * 2016-07-26 2019-04-02 静冈县 抗b7-h4抗体
CN108276492A (zh) * 2018-01-31 2018-07-13 中国药科大学 抗pd－l1单克隆抗体及其应用

Non-Patent Citations (11)

* Cited by examiner, † Cited by third party
Title
BIRD ET AL., SCIENCE, vol. 242, 1988, pages 423 - 426
GILLIES ET AL., J IMMUNOL METHODS, vol. 125, 1985, pages 191 - 202
HARLOWLANE: "Antibodies, ALabortory Manual", 1988, COLD SPRING HARBOR PRESS
HARLOWLANE: "Using Antibodies, A Laboratory Manual", 1999, COLD SPRING HARBOR PRESS
HUSTON ET AL., PROC. NATL. ACAD. SCI. USA, vol. 85, 1988, pages 5879 - 5883
KABAT ET AL.: "Sequences of Protein of Immunological Interest", 1987, NATIONAL INSTITUTE OF HEALTH
MORRISON, SCIENCE, vol. 229, no. 4719, 1985, pages 1202 - 7
OI ET AL., BIO TECHNIQUES, vol. 4, 1986, pages 214 - 221
See also references of EP4056592A4
WAHL ET AL., J. NUCL. MED., vol. 24, 1983, pages 316
WARD ET AL., NATURE, vol. 341, 1989, pages 544 - 546

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2023001025A1 (zh) * 2021-07-23 2023-01-26 南京吉盛澳玛生物医药有限公司 Pd-l1抗体及其用途
WO2024118814A3 (en) * 2022-11-30 2024-08-02 Development Center For Biotechnology Anti-human pd-l1 antibodies and their uses

Also Published As

Publication number Publication date
CA3157516A1 (en) 2021-05-14
EP4056592A4 (en) 2024-03-20
US20230002492A1 (en) 2023-01-05
JP2023179450A (ja) 2023-12-19
EP4056592A1 (en) 2022-09-14
CN115038718B (zh) 2024-07-02
CN115038718A (zh) 2022-09-09
JP2023500156A (ja) 2023-01-04

Similar Documents

Publication Publication Date Title
US20220017630A1 (en) Anti-tnfr2 antibody and use thereof
WO2020135201A1 (zh) 一种抗体及其用途
WO2021088904A1 (zh) 抗人程序性死亡配体-1(pd-l1)的抗体及其用途
US11525005B2 (en) Anti-CD40 antibody, antigen binding fragment thereof and medical use thereof
WO2020168555A1 (zh) Cd3抗原结合片段及其应用
WO2022161425A1 (zh) 抗tnfr2人源化抗体及其用途
EP4101867A1 (en) Anti-cd3 and anti-cd123 bispecific antibody and use thereof
IL293138A (en) Antibodies to cd3 and bcma and bispecific binding proteins produced from them
EP4292611A1 (en) Anti-cd112r antibody and use thereof
EP4378954A1 (en) Anti-pvrig/anti-tigit bispecific antibody and application
WO2021143914A1 (zh) 一种激活型抗ox40抗体、生产方法及应用
WO2019076277A1 (zh) 抗pd-1抗体和抗lag-3抗体联合在制备治疗肿瘤的药物中的用途
CN112041347A (zh) 结合人il-4r的抗体、其制备方法和用途
TWI842044B (zh) 抗pvrig/抗tigit雙特異性抗體和應用
WO2023125888A1 (zh) 一种gprc5d抗体及其应用
CN116209680A (zh) 与人CD3ε结合的新型人抗体
CN114106173A (zh) 抗ox40抗体、其药物组合物及应用
EP4410840A1 (en) Bispecific antibody and application thereof
WO2021115404A1 (zh) 一种抗cd38的抗体及其用途
TWI816616B (zh) 抗人程序性死亡配體-1 (pd-l1) 的抗體及其用途
TWI789679B (zh) 抗人程序性死亡配體-1（pd-l1）的抗體及其用途
US20240343803A1 (en) Anti-Pvrig/Anti-Tigit Bispecific Antibodies And Applications Thereof
RU2793165C1 (ru) Антитело против tnfr2 и его применение
WO2024165049A1 (zh) 抗cdh6的抗体及其用途
WO2021013061A1 (zh) 一种人源化抗vegfr2抗体及其应用

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 20884635

Country of ref document: EP

Kind code of ref document: A1

ENP Entry into the national phase

Ref document number: 3157516

Country of ref document: CA

ENP Entry into the national phase

Ref document number: 2022527056

Country of ref document: JP

Kind code of ref document: A

NENP Non-entry into the national phase

Ref country code: DE

ENP Entry into the national phase

Ref document number: 2020884635

Country of ref document: EP

Effective date: 20220608