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 PDFInfo
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- 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
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
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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
- A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
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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
- A61K2039/54—Medicinal preparations containing antigens or antibodies characterised by the route of administration
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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
- A61K2039/545—Medicinal preparations containing antigens or antibodies characterised by the dose, timing or administration schedule
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- 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
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- 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]
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- 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]
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- 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
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- 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.
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Abstract
Description
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 |
Claims (17)
- 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: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: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: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: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。
- 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。
- 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。
- 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。
- 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。
- 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。
- 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。
- 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。
- 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。
- 包含权利要求10所述分离的核酸分子的表达载体。
- 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)。
- 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。
- 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。
- 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。
- 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。
- 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。
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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 |
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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 |
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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 |
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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 |
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