WO2021027851A1 - Trop2 antibody, preparation method therefor, and conjugate and use thereof - Google Patents

Trop2 antibody, preparation method therefor, and conjugate and use thereof Download PDF

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WO2021027851A1
WO2021027851A1 PCT/CN2020/108720 CN2020108720W WO2021027851A1 WO 2021027851 A1 WO2021027851 A1 WO 2021027851A1 CN 2020108720 W CN2020108720 W CN 2020108720W WO 2021027851 A1 WO2021027851 A1 WO 2021027851A1
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amino acid
seq
antibody
acid sequence
sequence
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Chinese (zh)
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彭菲
曲宝源
张利
盛其然
严玉萍
陈海霞
阮文静
杨达志
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凯惠科技发展(上海)有限公司
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    • 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
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56966—Animal cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/35—Valency
    • 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
    • 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
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/94—Stability, e.g. half-life, pH, temperature or enzyme-resistance

Definitions

  • the invention relates to the field of antibodies, in particular to a TROP2 antibody, a preparation method thereof, a conjugate and application thereof.
  • the TROP2 gene belongs to the TACSTD gene family. Another member of this family encodes human trophoblast cell surface antigen 1 (hTROP1), which has about 50% homology with TROP2.
  • hTROP2 is a single-transmembrane type I cell membrane protein, which contains an intracellular domain composed of 26 amino acid residues at the N-terminal, an extracellular domain composed of 248 amino acids at the C-terminus, and a transmembrane composed of 23 amino acid residues. The domain has a total length of 323 amino acid residues.
  • hTROP2 has 4 N glycosylation sites at amino acid residues 33, 120, 168 and 208, and the apparent molecular weight is about 10 kilodaltons larger than the theoretical molecular weight of 35 kilodaltons.
  • hTROP2 protein kinase C
  • the phosphorylation of serine residue at position 303 will cause changes in the intracellular conformation of hTROP2, resulting in the exposure of PIP 2 that binds to it and is hydrolyzed by phospholipase C (PLC) to IP 3 (inositol triphosphate) and DAG (diacylglycerol) , Thereby regulating intracellular calcium signal transduction (Miha Pavsic et al., Scientific Reports, 2015; 5: 10324).
  • PLC phospholipase C
  • IP 3 inositol triphosphate
  • DAG diacylglycerol
  • hTROP2 is only expressed in limited epithelial cells of certain tissues.
  • hTROP2 is found in breast cancer, cervical cancer, colorectal cancer, and esophagus. Cancer, gastric cancer, lung cancer, ovarian cancer, prostate cancer, kidney cancer, pancreatic cancer and other cancer types are overexpressed, and reports have shown that hTROP2 expression levels are closely related to tumor malignancy, tumor invasion ability, and patient prognosis (Anna Shvartsur et al., Genes & Cancer, 2015; 6: 84-105).
  • Antibody drug conjugate (hereinafter referred to as ADC) is formed by coupling an antibody and a high-efficiency small molecule drug through a linker, which enables highly toxic small molecule drugs to specifically recognize target proteins on cancer cells, thereby Kill cancer cells specifically.
  • ADC Antibody drug conjugate
  • Antibody-based immunotherapy and chemical drug-based chemotherapy have always been two major strategies for the treatment of cancer in the clinic.
  • Antibodies target tumor cells specifically expressed or overexpressed antigens, and a variety of therapeutic monoclonal antibodies have achieved great clinical success.
  • therapeutic antibodies have good targeting properties, their killing effects have limitations; although small molecule chemical drugs have high-efficiency killing effects on cancer cells, they can also cause the same damage to non-cancer cells.
  • ADCs Antibody drugs and small molecule drugs have different clinical limitations, which put forward new requirements for drug development.
  • a new generation of ADCs uses the specific binding ability of antibodies to target cells to deliver highly cytotoxic chemical drugs to achieve targeted and efficient killing of cancer cells.
  • ADCs began to enter clinical research in the late 1980s, and 4 ADCs have been approved by the FDA for marketing.
  • TROP2 as a protein specifically and highly expressed in cancer cells, is an excellent candidate target for ADC.
  • the technical problem to be solved by the present invention is to overcome the shortcomings of the current TROP2 antibody, and provide a TROP2 antibody with high affinity and strong specificity and its preparation method and application.
  • the TROP2 antibody is compatible with human or cynomolgus source or small
  • the mouse-derived TROP2 protein has high affinity.
  • the present invention also provides an antibody-drug conjugate, which comprises the TROP2 antibody and a small molecule compound with anti-tumor function coupled to the TROP2 antibody.
  • the antibody-drug conjugate can enter cells and is positive for TROP2 expression.
  • the cells perform targeted cytotoxic killing and can be used in the preparation of drugs for the treatment of tumors and other diseases.
  • After preparing the humanized antibody of the TROP2 antibody of the present invention it can still have a high affinity with human or cynomolgus TROP2 protein, and can well inhibit the growth of tumor cells without significant influence on body weight.
  • the present invention uses hTROP2 protein or a recombinant cell strain overexpressing hTROP2 protein as the immunogen, adopts traditional hybridoma preparation technology (Kohler and Milstein, Nature, 1975; 256:495), and obtains anti-hTROP2 through a series of adjustments and improvements.
  • the lead antibody Through the preliminary production, purification and verification of the lead antibody, TROP2 antibodies with high affinity to hTROP2 or cTROP2 or mTROP2 proteins are obtained. Then the amino acid sequences of the heavy chain variable region and the light chain variable region of the obtained TROP2 antibody are obtained by sequencing by molecular biology methods.
  • the antibody is coupled with a small molecule compound such as MMAF to obtain an antibody-drug conjugate, which can enter cells and has an excellent cytotoxic killing effect on TROP2-positive cells.
  • the present invention provides an isolated protein comprising the heavy chain variable region and/or light chain variable region of the TROP2 antibody, the heavy chain variable region comprising heavy chain CDR1, heavy chain CDR2 and One or more of the heavy chain CDR3, and/or, the light chain variable region includes one or more of the light chain CDR1, light chain CDR2 and light chain CDR3, wherein the heavy chain CDR1
  • the amino acid sequence is shown in SEQ ID NO: 2 or SEQ ID NO: 10 in the sequence list;
  • the amino acid sequence of the heavy chain CDR2 is shown in SEQ ID NO: 3 or SEQ ID NO: 11 in the sequence list;
  • the amino acid of the heavy chain CDR3 The sequence is shown in SEQ ID NO: 4 or SEQ ID NO: 12 in the sequence list;
  • the amino acid sequence of the light chain CDR1 is shown in SEQ ID NO: 6 or SEQ ID NO: 14 in the sequence list; the amino acid sequence of the light chain CDR2 is shown in SEQ ID NO: 7 or SEQ ID NO: 15 in the sequence list.
  • the amino acid sequence of the light chain CDR3 is shown in SEQ ID NO: 8 or SEQ ID NO: 16 in the sequence table;
  • the amino acid sequence of the heavy chain CDR1 is at least 80%, 85%, 90%, 92%, 94%, 95% of the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 10 in the sequence table. , 96%, 97%, 98%, or 99% sequence homology amino acid sequence; and/or, the amino acid sequence of the heavy chain CDR2 is as shown in SEQ ID NO: 3 or SEQ ID NO:
  • the amino acid sequence shown in 11 is shown in an amino acid sequence with at least 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98% or 99% sequence homology; and/ Or, the amino acid sequence of the heavy chain CDR3 is at least 80%, 85%, 90%, 92%, 94%, 95% of the amino acid sequence shown in SEQ ID NO: 4 or SEQ ID NO: 12 in the sequence table.
  • amino acid sequence of the light chain CDR1 is as shown in SEQ ID NO: 6 or SEQ ID NO:
  • the amino acid sequence shown in 14 is shown in an amino acid sequence with at least 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98% or 99% sequence homology; and/ Or, the amino acid sequence of the light chain CDR2 is at least 80%, 85%, 90%, 92%, 94%, 95% of the amino acid sequence shown in SEQ ID NO: 7 or SEQ ID NO: 15 in the sequence table.
  • amino acid sequence of the light chain CDR3 is the same as SEQ ID NO: 8 or SEQ ID NO:
  • the amino acid sequence shown in 16 has at least 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, or 99% sequence homology.
  • Said has at least 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98% or 99% sequence homology to the amino acid sequence described in the sequence listing...
  • a sexual amino acid sequence is "a substitution, deletion or insertion of one or more amino acid residues in the amino acid sequence of the CDR, and the function of the protein is maintained or improved.
  • the amino acid sequence of the heavy chain CDR2 is shown in the amino acid sequence after the G at position 7 and/or the G at position 13 of the amino acid sequence shown in SEQ ID NO: 3 in the sequence listing. More preferably, the amino acid sequence of the heavy chain CDR2 is shown in the amino acid sequence in which the G at position 7 and/or the G at position 13 is mutated to A in the amino acid sequence shown in SEQ ID NO: 3 in the sequence table; The sequence is shown in SEQ ID NO: 44, SEQ ID NO: 30 or SEQ ID NO: 39.
  • amino acid sequence of the heavy chain CDR1 is shown in SEQ ID NO: 2 in the sequence table
  • amino acid sequence of the heavy chain CDR2 is shown in SEQ ID NO: 3 in the sequence table
  • amino acid of the heavy chain CDR3 The sequence is shown in SEQ ID NO: 4 in the Sequence Listing.
  • amino acid sequence of the heavy chain CDR1 is shown in SEQ ID NO: 2 in the sequence listing
  • amino acid sequence of the heavy chain CDR2 is shown in SEQ ID NO: 44 in the sequence listing
  • amino acid sequence of the heavy chain CDR3 is The sequence is shown in SEQ ID NO: 4 in the Sequence Listing.
  • amino acid sequence of the heavy chain CDR1 is shown in SEQ ID NO: 2 in the sequence table
  • amino acid sequence of the heavy chain CDR2 is shown in SEQ ID NO: 30 in the sequence table
  • amino acid of the heavy chain CDR3 The sequence is shown in SEQ ID NO: 4 in the Sequence Listing.
  • amino acid sequence of the heavy chain CDR1 is shown in SEQ ID NO: 2 in the sequence listing
  • amino acid sequence of the heavy chain CDR2 is shown in SEQ ID NO: 39 in the sequence listing
  • amino acid sequence of the heavy chain CDR3 is The sequence is shown in SEQ ID NO: 4 in the Sequence Listing.
  • amino acid sequence of the heavy chain CDR1 is shown in SEQ ID NO: 10 in the sequence table
  • amino acid sequence of the heavy chain CDR2 is shown in SEQ ID NO: 11 in the sequence table
  • amino acid of the heavy chain CDR3 The sequence is shown in SEQ ID NO: 12 in the sequence list;
  • amino acid sequence of the light chain CDR1 is shown in SEQ ID NO: 6 in the sequence list
  • amino acid sequence of the light chain CDR2 is shown in SEQ ID NO: 7 in the sequence list
  • amino acid sequence of the light chain CDR3 The sequence is shown in SEQ ID NO: 8 in the sequence table.
  • amino acid sequence of the light chain CDR1 is shown in SEQ ID NO: 14 in the sequence listing
  • amino acid sequence of the light chain CDR2 is shown in SEQ ID NO: 15 in the sequence listing
  • amino acid sequence of the light chain CDR3 is The sequence is shown in SEQ ID NO: 16 in the Sequence Listing.
  • the amino acid sequence of the heavy chain CDR1 is shown in SEQ ID NO: 2 in the sequence listing, and the amino acid sequence of the heavy chain CDR2 is shown in SEQ ID NO: 3 in the sequence listing, and the heavy chain
  • the amino acid sequence of CDR3 is shown in SEQ ID NO: 4 in the Sequence Listing; and the amino acid sequence of the light chain CDR1 is shown in SEQ ID NO: 6 in the Sequence Listing, and the amino acid sequence of the light chain CDR2 is shown in SEQ ID NO: in the Sequence Listing.
  • the amino acid sequence of the light chain CDR3 is shown in SEQ ID NO: 8 in the sequence listing.
  • the amino acid sequence of the heavy chain CDR1 is shown in SEQ ID NO: 2 in the sequence listing, and the amino acid sequence of the heavy chain CDR2 is shown in SEQ ID NO: 44 in the sequence listing, and the heavy chain
  • the amino acid sequence of CDR3 is shown in SEQ ID NO: 4 in the Sequence Listing; and the amino acid sequence of the light chain CDR1 is shown in SEQ ID NO: 6 in the Sequence Listing, and the amino acid sequence of the light chain CDR2 is shown in SEQ ID NO: in the Sequence Listing.
  • the amino acid sequence of the light chain CDR3 is shown in SEQ ID NO: 8 in the sequence listing.
  • the amino acid sequence of the heavy chain CDR1 is shown in SEQ ID NO: 2 in the sequence listing, and the amino acid sequence of the heavy chain CDR2 is shown in SEQ ID NO: 30 in the sequence listing, and the heavy chain
  • the amino acid sequence of CDR3 is shown in SEQ ID NO: 4 in the sequence list; and the amino acid sequence of light chain CDR1 is shown in SEQ ID NO: 6 in the sequence list, and the amino acid sequence of light chain CDR2 is shown in SEQ ID NO: in the sequence list.
  • the amino acid sequence of the light chain CDR3 is shown in SEQ ID NO: 8 in the sequence listing.
  • the amino acid sequence of the heavy chain CDR1 is shown in SEQ ID NO: 2 in the sequence listing, and the amino acid sequence of the heavy chain CDR2 is shown in SEQ ID NO: 39 in the sequence listing, and the heavy chain
  • the amino acid sequence of CDR3 is shown in SEQ ID NO: 4 in the Sequence Listing; and the amino acid sequence of the light chain CDR1 is shown in SEQ ID NO: 6 in the Sequence Listing, and the amino acid sequence of the light chain CDR2 is shown in SEQ ID NO: in the Sequence Listing.
  • the amino acid sequence of the light chain CDR3 is shown in SEQ ID NO: 8 in the sequence listing.
  • the amino acid sequence of the heavy chain CDR1 is shown in SEQ ID NO: 10 in the sequence listing, and the amino acid sequence of the heavy chain CDR2 is shown in SEQ ID NO: 11 in the sequence listing, and the heavy chain
  • the amino acid sequence of CDR3 is shown in SEQ ID NO: 12 in the Sequence Listing; and the amino acid sequence of the light chain CDR1 is shown in SEQ ID NO: 14 in the Sequence Listing, and the amino acid sequence of the light chain CDR2 is shown in SEQ ID NO: in the Sequence Listing. 15 is shown, and the amino acid sequence of the light chain CDR3 is shown in SEQ ID NO: 16 in the sequence listing.
  • the heavy chain variable region further includes a heavy chain variable region framework region.
  • the heavy chain variable region framework region is the heavy chain variable region framework region of a mouse antibody or the heavy chain variable region framework region of a human antibody or a back mutation thereof; preferably IGHV1-69*08/JH6C( The sequence is shown in SEQ ID NO: 25) or IGHV3-7*01/JH4D (the sequence is shown in SEQ ID NO: 35) or a back mutation based on it.
  • the light chain variable region further includes a light chain variable region framework region.
  • the light chain variable region framework region is the light chain variable region framework region of a mouse antibody or the light chain variable region framework region of a human antibody or its back mutation; preferably IGKV1-39*01/JK4( The sequence is shown in SEQ ID NO: 31) or IGKV1-39*01/JK1 (the sequence is shown in SEQ ID NO: 40) or a back mutation based on it.
  • amino acid sequence of the variable region of the heavy chain is shown in any one of SEQ ID NO: 1, 9, 20, 23, 24, 26-29, 36-38 or its mutant amino acid sequence in the sequence list.
  • amino acid sequence of the light chain variable region is as shown in any one of SEQ ID NO: 5, 13, 32-34, 41-43 or its mutant amino acid sequence in the sequence listing.
  • the mutation has one or more amino acid residue substitutions, deletions or insertions in the amino acid sequence of the heavy chain variable region or the light chain variable region, and maintains or improves the function of the protein; preferably, The amino acid sequence of the mutation and the amino acid sequence of the heavy chain variable region or the light chain variable region have at least 80%, 85%, 90%, 90%, 95%, 96%, 97%, 98% more preferably At least 99% sequence homology.
  • the protein when the protein is a bispecific antibody and one of the protein functional regions is an immunoglobulin, the other protein functional region is an scFv, and the scFv is connected to the C-terminus of the two heavy chains of the immunoglobulin When, the C-terminus of the heavy chain can be mutated from K to A.
  • amino acid sequence of the heavy chain variable region is as shown in any one of SEQ ID NO: 1, 20, 23, 24, 26-29 in the sequence list, and the amino acid sequence of the light chain variable region is as follows The list SEQ ID NO: 5, 32-34 is shown in any one.
  • amino acid sequence of the heavy chain variable region is shown in any one of SEQ ID NO: 9 and 36-38 in the sequence listing, and the amino acid sequence of the light chain variable region is shown in the sequence listing SEQ ID NO: 13 , 41-43 shown in any one.
  • the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 1 in the sequence listing, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 5 in the sequence listing.
  • the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 9 in the sequence listing, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 13 in the sequence listing.
  • the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 20 in the sequence listing, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 5 in the sequence listing.
  • the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 23 in the sequence listing, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 5 in the sequence listing.
  • the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 24 in the sequence listing, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 5 in the sequence listing.
  • the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 26 in the sequence listing, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 32 in the sequence listing.
  • the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 27 in the sequence listing, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 32 in the sequence listing.
  • the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 28 in the sequence listing, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 32 in the sequence listing.
  • the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 29 in the sequence listing, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 32 in the sequence listing.
  • the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 26 in the sequence listing, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 33 in the sequence listing.
  • the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 27 in the sequence listing, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 33 in the sequence listing.
  • the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 28 in the sequence listing, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 33 in the sequence listing.
  • the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 29 in the sequence listing, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 33 in the sequence listing.
  • the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 26 in the sequence listing, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 34 in the sequence listing.
  • the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 27 in the sequence listing, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 34 in the sequence listing.
  • the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 28 in the sequence listing, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 34 in the sequence listing.
  • the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 29 in the sequence listing, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 34 in the sequence listing.
  • the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 36 in the sequence listing, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 41 in the sequence listing.
  • the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 37 in the sequence listing, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 41 in the sequence listing.
  • the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 38 in the sequence listing, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 41 in the sequence listing.
  • the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 36 in the sequence listing, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 42 in the sequence listing.
  • the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 37 in the sequence listing, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 42 in the sequence listing.
  • the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 38 in the sequence listing, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 42 in the sequence listing.
  • the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 36 in the sequence listing, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 43 in the sequence listing.
  • amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 37 in the sequence listing, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 43 in the sequence listing.
  • amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 38 in the sequence listing, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 43 in the sequence listing.
  • the numbers of the above amino acid sequences may be as shown in Table 1:
  • the numbers in Table 1 are the sequence numbers in the sequence list.
  • the amino acid sequence of the heavy chain variable region of 11E8E6D11 is SEQ ID NO:1
  • the amino acid sequence of CDR1 in the heavy chain variable region of 11E8E6D11 is SEQ ID NO: 2
  • the amino acid sequence of the heavy chain CDR2 is SEQ ID NO: 3
  • the amino acid sequence of the heavy chain CDR3 is SEQ ID NO: 4.
  • the amino acid sequence of the light chain variable region is SEQ ID NO: 5
  • the amino acid sequence of light chain CDR1 is SEQ ID NO: 6
  • the amino acid sequence of light chain CDR2 is SEQ ID NO: 7
  • the amino acid sequence of light chain CDR3 is SEQ ID NO: 8.
  • the isolated protein is a humanized antibody, which includes the framework regions of the variable region of a human antibody.
  • Partial mutations occur on the basis of these CDRs; the mutations have one or more amino acid residue substitutions, deletions or insertions in their amino acid sequence, and maintain or improve the function of the protein; the mutated amino acid sequence
  • the amino acid sequence of the heavy chain variable region or the light chain variable region has at least 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98% or 99% Sequence homology.
  • the C-terminus of the heavy chain can be mutated from K to A.
  • the framework region of the variable region of the human antibody includes the framework region of the variable region of the human antibody heavy chain and the framework region of the variable region of the human antibody light chain.
  • the isolated protein also includes an antibody heavy chain constant region and an antibody light chain constant region.
  • the antibody heavy chain constant region is preferably a mouse antibody heavy chain constant region or a human antibody heavy chain constant region; the antibody light chain constant region is preferably a mouse light chain antibody constant region or a human antibody light chain constant region.
  • the antibody heavy chain constant region is more preferably a human antibody heavy chain constant region, further preferably a human IgG1, IgG2, IgG3 or IgG4 antibody heavy chain constant region; the antibody light chain constant region is preferably a human antibody light chain ⁇ Or lambda chain constant region.
  • the heavy chain variable region and the light chain variable region of the isolated protein, the human heavy chain constant region and the human light chain constant region constitute a chimeric antibody full-length protein.
  • the full-length antibody protein is preferably IgG1, IgG2, IgG3 or IgG4.
  • the isolated protein may be a conventional protein in the art, preferably full-length antibody protein, antigen-binding fragment, bispecific antibody, multispecific antibody, single chain antibody fragment (scFv), single chain antibody One or more of single domain antibody (sdAb) and single-domain antibody (single-domain antibody).
  • the isolated protein may also preferably be a monoclonal antibody or a polyclonal antibody prepared from the above antibody, such as the TROP2 antibody; the monoclonal antibody can be developed by a variety of approaches and technologies, including hybridoma technology, Phage display technology, single lymphocyte gene cloning technology, etc., the mainstream is to prepare monoclonal antibodies from wild-type or transgenic mice through hybridoma technology.
  • the isolated protein may also preferably be a superhumanized antibody.
  • the superhumanized antibody is an antibody obtained by a preparation method of humanized antibody. This method does not rely on the human framework sequence as the analysis point, but relies on the comparison of the canonical CDR structure type of non-human antibody and the CDR structure type of human antibody, especially the human antibody encoded by human germline sequence.
  • Candidate human antibody sequences of suitable human framework sequences are obtained. For example, human residues can replace non-human residues in CDRs.
  • a prerequisite for veneering is that the immunogenicity of the murine antibody variable region originates from its surface residues, and the mobility of residues and solvent accessibility are the basic conditions for it to become an antigenic determinant.
  • the residues are different from those in the same position in the framework region of the human antibody
  • the corresponding position in the human antibody Replace the identified residues with the amino acids for veneering. That is, the surface residues of the veneered antibody are mainly human-derived sequences, while the interior residues are mainly the original murine-derived sequences.
  • the surface residues of the veneered antibody are mainly human-derived sequences
  • the interior residues are mainly the original murine-derived sequences.
  • human framework regions having substantial sequence homology with each framework region of related non-human antibodies are identified, and the CDRs of the non-human antibody are grafted onto complexes of these different human framework regions.
  • the above methods can be combined to generate anti-TROP2 antibodies of any desired sequence.
  • the protein when it is a bispecific antibody, it may include a first protein functional region and a second protein functional region.
  • the first protein functional region may be the aforementioned protein, which targets to bind TROP2; the second protein functional region is a protein that does not target to bind TROP2 or is a protein that also targets to bind to TROP2 but is not described in the present invention.
  • the first protein functional domain may be an immunoglobulin, and the second protein functional domain may be one or more scFv; or, the second protein functional domain may be an immunoglobulin, and the first protein The functional area can be one or more scFv.
  • the antibody full-length protein may be a conventional antibody full-length protein in the art, which includes a heavy chain variable region, a light chain variable region, a heavy chain constant region, and a light chain constant region.
  • the single-chain antibody may be a conventional single-chain antibody in the art, which includes a heavy chain variable region, a light chain variable region, and a short peptide of 15-20 amino acids.
  • the antigen-binding fragment can be a conventional antigen-binding fragment in the art, which includes the Fd segment of the light chain variable region, the light chain constant region and the heavy chain constant region.
  • the antigen-antibody binding domain protein fragments are Fab and F(ab') 2 .
  • the single domain antibody may be a conventional single domain antibody in the art, which includes a heavy chain variable region and a heavy chain constant region.
  • the single-domain antibody may be a conventional single-domain antibody in the art, which only includes the heavy chain variable region.
  • the present invention also provides a nucleic acid, which encodes the above-mentioned isolated protein.
  • the preparation method of the nucleic acid is a conventional preparation method in the art, preferably, it includes the following steps: obtaining a nucleic acid molecule encoding the above-mentioned protein through gene cloning technology, or obtaining a nucleic acid molecule encoding the above-mentioned protein through a method of artificial full sequence synthesis .
  • polynucleotide homologues of the present invention can be prepared by replacing, deleting or adding one or more bases of the gene encoding the protein sequence within the scope of maintaining antibody activity.
  • the present invention also provides a recombinant expression vector containing the nucleic acid.
  • the recombinant expression vector can be obtained by conventional methods in the art, that is, the nucleic acid molecule of the present invention is connected to various expression vectors to be constructed.
  • the expression vector is a variety of conventional vectors in the field, as long as it can hold the aforementioned nucleic acid molecule.
  • Said vectors preferably include: various plasmids, cosmids, phage or virus vectors and the like.
  • the present invention also provides a transformant which contains the above-mentioned recombinant expression vector in a host cell.
  • the preparation method of the recombinant expression transformant is a conventional preparation method in the art, preferably: the recombinant expression vector is transformed into a host cell.
  • the host cell is a variety of conventional host cells in the field, as long as the recombinant expression vector can replicate itself stably and the nucleic acid carried can be effectively expressed.
  • the host cell is E. coli TG1 or BL21 cell (expressing single-chain antibody or Fab antibody), or CHO-K1 cell (expressing full-length IgG antibody).
  • the aforementioned recombinant expression plasmid is transformed into a host cell to obtain the preferred recombinant expression transformant of the present invention.
  • the transformation method is a conventional transformation method in the field, preferably a chemical transformation method, a heat shock method or an electrotransformation method.
  • the isolated protein can be used to prepare a chimeric antigen receptor (CAR) and the like to modify it on cells such as T cells or NK cells.
  • the chimeric antigen receptor may be a conventional chimeric antigen receptor in the art, including, for example, a chimeric antigen receptor that uses the scFv of the above-mentioned TROP2 antibody as an extracellular antigen binding domain. Therefore, the present invention also provides a genetically modified cell comprising the aforementioned isolated protein.
  • the genetically modified cell is a eukaryotic cell, preferably an isolated human cell; more preferably an immune cell such as a T cell ( For example, in the form of CAR-T), or NK cells.
  • the present invention also provides a method for preparing an isolated protein, which includes the following steps: culturing the above-mentioned transformant to obtain the isolated protein from the culture.
  • the present invention provides an antibody drug conjugate comprising the above-mentioned isolated protein covalently attached to a cytotoxic agent.
  • the above-mentioned 1 equivalent of the isolated protein is connected to the y equivalent of the cytotoxic agent through an x equivalent linker, and has the structure shown in Formula 1,
  • Ab is the aforementioned isolated protein; L is a linker; D is a cytotoxic agent; the x is a conventional degree of cross-linking in the art, x is a natural number, preferably an integer of 1-20; y is a natural number, preferably 1-20 X and y are each independently an integer of 2-8, for example, 3 or 4; the ratio of x and y is preferably 1:1.
  • the L is a conventional linker (also called a crosslinking agent or coupling agent) in the art.
  • the L includes two functional groups, that is, a group that reacts with antibodies, and a group that reacts with drugs (for example, aldehydes or ketones).
  • the drug is coupled to the aforementioned protein via a linker molecule.
  • the L is released after entering the cell, which includes but is not limited to the following functional groups, active esters, carbonates, carbamates, phosphoimidites, oximes, hydrazones, acetals, orthoesters, and amino groups Class, small peptide or nucleotide fragment.
  • said L mainly contains the structure represented by formula 2, which is the remaining part corresponding to the leaving group in L;
  • Sp 2 does not exist, or -S- or -O-, provided that when Alk 2 does not exist, Sp 2 does not exist;
  • Z 1 is H, (C 1 -C 5 )alkyl, or consists of (C 1 -C 5 )alkyl, (C 1 -C 5 )alkoxy, (C 1 -C 4 )thioalkoxy , Halogen, nitro, -COOR', -CONHR', -O(CH 2 ) n COOR', -S(CH 2 ) n COOR', -O(CH 2 ) n CONHR' or -S(CH 2 )
  • Sp is a straight or branched divalent or trivalent (C 1 -C 18 ) group, a divalent or trivalent aryl or heteroaryl group, a divalent or trivalent (C 3 -C 18 ) cycloalkane Group or heterocycloalkyl group, divalent or trivalent aryl or heteroaryl-aryl (C 1 -C 18 ) group, divalent or trivalent cycloalkyl or heterocycloalkyl-alkyl ( C 1 -C 18 ) group, or divalent or trivalent (C 2 -C 18 ) unsaturated alkyl group, wherein the heteroaryl group is preferably furyl, thienyl, N-methylpyrrolyl, pyridine Group, N-methylimidazolyl, oxazolyl, pyrimidinyl.
  • Alk 1 is a branched or unbranched (C 1 -C 5 ) alkylene chain, Sp 1 is not present, or is -S-, -O-, -CONH-, -NHCO- or -NR', Wherein R'is as defined above, and the condition is that when Alk 1 does not exist, Sp 1 does not exist;
  • Ar is composed of (C 1 -C 6 ) alkyl, (C 1 -C 5 ) alkoxy, (C 1 -C 4 ) thioalkoxy, halogen, nitro, -COOR', -CONHR', -O(CH 2 ) n COOR', -S(CH 2 ) n COOR', -O(CH 2 ) n CONHR' or -S(CH 2 ) n CONHR' 1, 2 or 3 groups are optional Substituted 1,2-, 1,3- or 1,4-phenylene, wherein n and R'are as defined above, or Ar is each composed of C 1 -C 6 )alkyl, (C 1 -C 5 ) Alkoxy, (C 1 -C 4 )thioalkoxy, halogen, nitro, -COOR', -CONHR', -O(CH 2 ) n COOR', -S(CH 2 ) n COOR', 1, 2, 3
  • Z 1 is (C 1 -C 5 ) alkyl, or consists of (C 1 -C 5 ) alkyl, (C 1 -C 4 ) alkoxy, (C 1 -C 4 ) thioalkoxy, halogen , Nitro, -COOR', -CONHR', -O(CH 2 ) n COOR', -S(CH 2 ) n COOR', -O(CH 2 ) n CONHR' or -S(CH 2 ) n CONHR
  • the 1, 2, or 3 groups of ' are optionally substituted phenyl; Alk 2 and Sp 2 are not present; and Sp and Q are as defined only above.
  • the L is preferably maleimidocaproyl (maleimidocaproyl, MC), maleimidocaproyl-L-valine-L-citrulline-aminobenzyl alcohol (MC-VC-PAB) Or 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid succinimide ester (SMCC).
  • maleimidocaproyl maleimidocaproyl, MC
  • MC-VC-PAB maleimidocaproyl-L-valine-L-citrulline-aminobenzyl alcohol
  • SMCC 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid succinimide ester
  • Said D is a conventional cytotoxic agent in the art, preferably selected from cytotoxins, chemotherapeutic agents, radioisotopes, therapeutic nucleic acids, immunomodulators, anti-angiogenesis agents, anti-proliferation and pro-apoptotic agents or cytolytic enzymes.
  • the cytotoxin is a conventional cytotoxin in the art, and generally refers to an active agent that inhibits or prevents cell function and/or causes cell destruction.
  • an active agent that inhibits or prevents cell function and/or causes cell destruction.
  • antibiotics antibiotics, inhibitors of tubulin polymerization, alkylating agents, protein synthesis inhibitors, protein kinase inhibitors, phosphatase inhibitors, topoisomerase inhibitors, protein kinases, phosphatases, topoisomers Enzymes or cyclins.
  • doxorubicin More preferably selected from doxorubicin, daunorubicin, idarubicin, arubicin, zorubicin, mitoxantrone, epirubicin, carrubicin, nogamycin, Menoril, Pirubicin, Valrubicin, Cytarabine, Gemcitabine, Trifluridine, Ancitabine, Enoxabine, Azacitidine, Deoxyfluridine, Pentrestat Butyl, bromouridine, capecitabine, cladribine, decitabine, fluridine, fludarabine, glutamicin, puromycin, tegafur, thiazole carboxamide nucleoside, adriamycin Vitamins, cisplatin, carboplatin, cyclophosphamide, dacarbazine, vinblastine, vincristine, bleomycin, nitrogen mustard, prednisone, procarbazine, methotrexate, fluorouracil
  • the chemotherapeutic agent is a conventional chemotherapeutic agent in the art, preferably selected from alkylating agents, alkyl sulfonate chemotherapeutics, aziridine chemotherapeutics, vinyl amides and methyl methacrylates.
  • Amine chemotherapeutics nitrogen mustard, nitrourea chemotherapeutics, antibiotics, antimetabolites, folic acid chemotherapeutics, purine analogs, pyrimidine analogs, androgens, antiadrenaline, folic acid supplements, maytans Alcohols, polysaccharide complexes, taxanes, platinum analogs, or retinoids, or pharmaceutically acceptable salts, acids and derivatives thereof.
  • the alkylating agent is a conventional alkylating agent in the art, preferably selected from thiotepa or cyclophosphamide.
  • the alkyl sulfonate chemotherapeutic agent is a conventional alkyl sulfonate chemotherapeutic agent in the art, and is preferably selected from busulfan, inprosufane or piposifan.
  • the aziridine chemotherapeutic agent is a conventional aziridine chemotherapeutic agent in the art, and is preferably selected from aziridine, carbachol, metotepa or uritepa.
  • the vinylamide and methylmelamine chemotherapeutics are conventional vinylamide and methylmelamine chemotherapeutics in the art, and are preferably selected from hexamethylmelamine, triethylenemelamine, and triethylene Phosphoramide, triethylene thiophosphoramide or trimethylol melamine.
  • the nitrogen mustard is a conventional nitrogen mustard, preferably selected from chlorambucil, chlorambucil, estramustine, ifosfamide, nitrogen mustard, chlorambucil hydrochloride, Phenylalanine mustard, new mustard, phenyl mustard cholesterol, prednisone, trifosamine or uracil mustard.
  • the nitrourea chemotherapeutic agent is a conventional nitrourea chemotherapeutic agent in the art, preferably selected from carmustine, chlorurea, formustine, lomustine, nimomus Or ramustine.
  • the antibiotic is a conventional antibiotic in the art, preferably selected from the group consisting of aclarithromycin, actinomycin, abramycin, azaserine, bleomycin, actinomycin c, calicheamicin, Carrubicin, carcinomycin, carcinogen, chromomycin, dactinomycin, daunorubicin, ditorubicin, 6-diazo-5-oxo-L-norleucine, more Ruubicin, epirubicin, esorubicin, idarubicin, flabomycin, mitomycin, mycophenolic acid, nogamycin, olivemycin, pelomycin, porphyrin , Puromycin, tri-iron doxorubicin,
  • the antimetabolites are conventional antimetabolites in the art, preferably selected from methotrexate or 5-fluorouracil (5-FU).
  • the folic acid chemotherapeutic agent is a conventional folic acid chemotherapeutic agent in the art, preferably selected from dimethyl folic acid, pterorin or trimethoate.
  • the purine analogues are conventional purine analogues in the art, preferably selected from fludarabine, 6-mercaptopurine, thiomipurine or thioguanine.
  • the pyrimidine analogues are conventional pyrimidine analogues in the art, preferably selected from the group consisting of ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, Deoxyfluridine, enoxabine, fluridine or 5-EU.
  • the androgens are conventional androgens in the art, and are preferably selected from the group consisting of captestosterone, menandrotanone propionate, thiosterol, meandrosterol, or testosterone.
  • the anti-adrenaline is a conventional anti-adrenaline in the art, and is preferably selected from anlumit, mitotane, or tripostane.
  • the folic acid supplement is a conventional folic acid supplement in the art, preferably selected from the group consisting of leucovorin, acetoglucone, aldophosphamide glycoside, aminolevulinic acid, amsacrine, amustine, and bisham Trident, edatrexate, dessfamide, colchicine, diacrquinone, eflornithine, elliptic ammonium, epotoxylon, etoglu, gallium nitrate, hydroxyurea, lentinan or clonida Bright.
  • Said maytansinol is a conventional maytansinol in the art, preferably selected from maytansine, ansamicin, mitoguanidine hydrazone, mitoxantrone, mupendol, diamine nitroacridine, Pentastatin, chlorambucil, pirarubicin, losoxantrone, podophyllic acid, 2-ethylhydrazide or procarbazine.
  • the polysaccharide complex is a conventional polysaccharide complex in the art, and is preferably selected from the group consisting of razosan, rhizopromycin, cizoran, germanspiramine, alternaria tenuinic acid, triimine quinone 2,2' , 2′′-trichlorotriethylamine, trichothecenes, urethane, vindesine, dacarbazine, mannmostine, dibromomannitol, dulcitol dibromide, piper bromide, gacytosine, cytarabine, cyclophosphamide or thiotepa.
  • Taxane is a conventional violet in the art Taxane, preferably selected from paclitaxel, non-hydrogenated castor oil, paclitaxel albumin engineered nanoparticle formulations (American Pharmaceutical Partners, Schaumberg, Illinois), docetaxel, chlorambucil, gemcitabine, 6-sulfur Substitute guanine, mercaptopurine or methotrexate.
  • the platinum analogues are conventional platinum analogues in the art, preferably selected from cisplatin, carboplatin, vinblastine, etoposide, ifosfamide, Mitoxantrone, vincristine, noanto, teniposide, edatrexate, daunomycin, aminopterin, capecitabine ibandronate, CPT-11, topoisomerase Inhibitor RFS 2000 or difluoromethylornithine.
  • Said retinoid is a retinoid in the art, preferably retinoic acid.
  • the radioisotope is a conventional radioisotope in the art, preferably, it is directly bound to the above-mentioned protein, or is bound to the above-mentioned protein through a chelating agent. More preferably, it directly binds to the cysteine residue of the protein.
  • the radioisotope is selected from ⁇ -emitters, ⁇ -emitters and Auger electrons suitable for radiotherapy, and positron emitters or ⁇ -emitters suitable for diagnosis. More preferably, the radioisotope is selected from 18 fluorine, 64 copper, and 65 copper.
  • the therapeutic nucleic acid is a conventional nucleic acid in the art, preferably a gene encoding an immunomodulator, an anti-angiogenic agent, an anti-proliferative agent or a pro-apoptotic agent.
  • the therapeutic agent includes the therapeutic agent, its derivatives, and pharmaceutically acceptable salts, acids and derivatives of the therapeutic agent.
  • the immunomodulator is a conventional immunomodulator in the art, that is, an agent that triggers an immune response, including humoral immune response (such as the production of antigen-specific antibodies) and cell-mediated immune response (such as lymphocyte proliferation) . It is preferably selected from cytokines, growth factors, hormones, antihormones, immunosuppressants or corticosteroids.
  • the cytokine is a conventional cytokine in the art, preferably selected from xanthine, interleukin or interferon.
  • the growth factor is a conventional growth factor in the art, preferably selected from TNF, CSF, GM-CSF or G-CSF.
  • the hormones are conventional hormones in the art, preferably selected from estrogen, androgen or progesterone.
  • the estrogen is diethylstilbestrol or estradiol. More preferably, the androgen is testosterone or fluoxymesterone. More preferably, the progestin is megestrol acetate or medroxyprogesterone acetate.
  • the corticosteroids are conventional corticosteroids in the art, preferably selected from prednisone, dexamethasone or cortisone.
  • the antihormonal drug is a conventional antihormonal drug in the art, which can block the effect of hormones on tumors, inhibit the production of cytokines, down-regulate the expression of self-antigens, or mask the immunosuppressive agents of MHC antigens.
  • the anti-estrogen is selected from the group consisting of tamoxifen, raloxifene, aromatase-inhibiting 4(5)-imidazoles, 4-hydroxytamoxifen, travoxifen or toremi Fen.
  • the anti-androgen is selected from flutamide, nilutamide, bicalutamide, leuprolide or goserelin.
  • the immunosuppressive agent is a conventional immunosuppressant in the art, preferably selected from 2-amino-6 aryl-5 substituted pyrimidines, azathioprine, cyclophosphamide, bromocriptine, danazol, and ammonia Phenylsulfone, glutaraldehyde, anti-idiotypic antibodies against MHC antigens and MHC fragments, cyclosporin A, steroids such as glucocorticoids, streptokinase, TGFb, rapamycin, T cell receptor, T cell receptor Fragment, cytokine receptor antagonist or T cell receptor antibody. More preferably, the cytokine receptor antagonist is selected from an anti-interferon antibody, an anti-IL10 antibody, an anti-TNFa antibody or an anti-IL2 antibody.
  • the anti-angiogenesis agent is a conventional anti-angiogenesis agent in the art, preferably selected from farnesyl transferase inhibitors, COX-2 inhibitors, VEGF inhibitors, bFGF inhibitors, steroid sulfatase Inhibitors, interleukin-24, thromboxane, metallospondin protein, class I interferon, interleukin-12, protamine, angiostatin, laminin, endostatin or prolactin fragments. More preferably, it is 2-Methoxyestradiol disulfamate (2-MeOE2bisMATE).
  • the anti-proliferation and pro-apoptosis agent is a conventional anti-proliferation and pro-apoptosis agent, preferably selected from PPAR- ⁇ activator, retinoid, triterpenoid, EGF receptor inhibitor, terminal Granulose Inhibitors, Iron Chelators, Apoptosis Proteins, Inhibitors of Bcl-2 and Bcl-X(L), TNF- ⁇ /FAS Ligand/TNF-related Apoptosis Inducing Ligand and Its Signal Transduction Activator or PI3K-Akt survival pathway signaling inhibitor.
  • the PPAR- ⁇ activator is a conventional PPAR- ⁇ activator in the art, preferably cyclopentenone prostaglandins (cyPGs).
  • the triterpenoid compound is a conventional triterpenoid compound in the art, preferably selected from the group consisting of cyclic jackfruit, lupinane, ursane, zidonorane, xylane, dammarane, cucurbitacin, lemon Picrine analogs or triterpenoids.
  • the EGF receptor inhibitor is a conventional EGF receptor inhibitor in the art, preferably selected from HER4, rapamycin, or 1,25-dihydroxycholecalciferol (vitamin D).
  • the iron chelate is a conventional iron chelate in the art, preferably 3-aminopyridine-2-carboxaldehyde thiosemicarbazone.
  • the apoptotic protein is a conventional apoptotic protein in the art, preferably the viral protein 3-VP3 of chicken anemia virus.
  • the PI3K-Akt survival pathway signal inhibitor is a conventional PI3K-Akt survival pathway signal inhibitor in the art, and is preferably UCN-01 or geldanamycin.
  • the cytolytic enzyme is a conventional cytolytic enzyme in the art, preferably RNase.
  • -(L) x -(D) y is:
  • n 1-10, preferably m is 5.
  • -(L) x -(D) y is:
  • -(L) x -(D) y is:
  • the D is a tubulin synthase inhibitor-methyl auristatin F (MMAF)
  • the linker L is maleimidocaproyl (MC)
  • the structure of the drug conjugate is shown in formula 3.
  • the L is 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid succinimidyl ester; D is N2'-deacetyl-N2'-3-mercapto-1 Oxopropyl-maytansine (DM1), the structure of the antibody drug conjugate is shown in formula 4.
  • L is maleimidohexanoyl-L-valine-L-citrulline p-aminobenzyl alcohol
  • D is methyl auristatin E (MMAE)
  • n is a natural number, preferably an integer of 1-20, more preferably an integer of 2-8, for example 3 or 4.
  • the preparation method of the antibody-drug conjugate can be conventional in the art, preferably the preparation method described in Doronina, 2006, Bioconjugate Chem. 17, 114-124.
  • the preparation method produces antibody-drug conjugates with a minimum low coupling fraction (LCF) of less than 10%.
  • the present invention also provides the above-mentioned preparation method of the antibody-drug conjugate, which includes the following steps:
  • reaction solution A After the above-mentioned separated protein is dialyzed with sodium borate buffer, tris (2-carboxyethyl) phosphine is added and reduced at room temperature to obtain reaction solution A;
  • reaction solution B Elute reaction solution A to remove excess protein to obtain reaction solution B;
  • the pH of the sodium borate buffer is 6.5-8.5; the molar ratio of the tris(2-carboxyethyl)phosphine (TCEP) to the isolated protein is 2 to 10; the reduction time is 1 to 4 hours; and/or, in the step c, the molar ratio of -(L) x -(D) y to the isolated protein is 5 to 20
  • the temperature of the reaction is 10-37°C, and the time of the reaction is 4 hours.
  • the preparation method includes the following steps: after the above-mentioned protein is dialyzed with a sodium borate buffer of pH 6.5-8.5, tris(2-carboxyethyl)phosphine (TCEP) is added, wherein the TCEP and the above-mentioned protein The molar ratio is 2-10, and the reaction solution A is obtained by reducing at room temperature for 1-4 hours. The reaction solution A is eluted to remove the excess protein mentioned above to obtain the reaction solution B. MC-MMAF is added to the reaction solution B, wherein the molar ratio of MC-MMAF to the purified TROP2 antibody is 5-20, and the reaction is carried out at 10-37°C for 4 hours.
  • TCEP tris(2-carboxyethyl)phosphine
  • the antibody-drug conjugate can exist in any physical form known in the art, preferably a clear solution.
  • the present invention also provides a pharmaceutical composition, which includes the above-mentioned isolated protein and/or the above-mentioned antibody drug conjugate, and a pharmaceutically acceptable carrier.
  • the pharmaceutical composition preferably also includes other anti-tumor antibodies as active ingredients.
  • the pharmaceutically acceptable carrier may be a conventional carrier in the art, and the carrier may be any suitable physiologically or pharmaceutically acceptable pharmaceutical excipient.
  • the pharmaceutical excipients are conventional pharmaceutical excipients in the field, and preferably include pharmaceutically acceptable excipients, fillers or diluents. More preferably, the pharmaceutical composition comprises 0.01-99.99% of the above-mentioned protein and/or the above-mentioned antibody drug conjugate, and 0.01-99.99% of the pharmaceutical carrier, and the percentage is based on the percentage of the pharmaceutical composition. Mass percentage.
  • the pharmaceutical composition is an anti-tumor drug. More preferably, anti-squamous/adenomatous lung cancer (non-small cell lung cancer), invasive breast cancer, colon cancer, rectal cancer, gastric cancer, squamous cervical cancer, invasive endometrial adenocarcinoma, invasive pancreatic cancer, Drugs for ovarian cancer, squamous bladder cancer, choriocarcinoma, bronchial cancer, breast cancer, cervical cancer, pancreatic cancer or seminal vesicle cancer.
  • anti-squamous/adenomatous lung cancer non-small cell lung cancer
  • invasive breast cancer colon cancer
  • rectal cancer gastric cancer
  • squamous cervical cancer invasive endometrial adenocarcinoma
  • pancreatic cancer Drugs for ovarian cancer
  • squamous bladder cancer choriocarcinoma
  • bronchial cancer breast cancer
  • cervical cancer cervical cancer
  • pancreatic cancer pancreatic cancer
  • the administration route of the pharmaceutical composition of the present invention is preferably parenteral administration, injection administration or oral administration.
  • the injection administration preferably includes intravenous injection, intramuscular injection, intraperitoneal injection, intradermal injection, or subcutaneous injection.
  • the pharmaceutical composition is a variety of conventional dosage forms in the art, preferably in the form of solid, semi-solid or liquid, that is, in the form of an aqueous solution, non-aqueous solution or suspension, more preferably tablets, capsules, and granules. Medicine, injection or infusion, etc. More preferably, it is administered via intravascular, subcutaneous, intraperitoneal or intramuscular administration.
  • the pharmaceutical composition can also be administered as an aerosol or coarse spray, that is, nasal administration; or, intrathecal, intramedullary or intraventricular administration. More preferably, the pharmaceutical composition can also be administered transdermally, transdermally, topically, enterally, intravaginally, sublingually or rectally.
  • the dosage level of the pharmaceutical composition of the present invention can be adjusted according to the amount of the composition to achieve the desired diagnosis or treatment result.
  • the administration schedule can also be a single injection or multiple injections, or be adjusted.
  • the selected dosage level and schedule depend on the activity and stability (ie, half-life) of the pharmaceutical composition, formulation, route of administration, combination with other drugs or treatments, diseases or disorders to be detected and/or treated, And various factors such as the health status and previous medical history of the subject to be treated can be reasonably adjusted.
  • the therapeutically effective dose of the pharmaceutical composition of the present invention can be estimated initially in cell culture experiments or animal models such as rodents, rabbits, dogs, pigs and/or primates. Animal models can also be used to determine the appropriate concentration range and route of administration. It can then be used to determine useful dosages and routes of administration in humans. Generally, the determination and adjustment of an effective amount or dosage for administration and the evaluation of when and how to make such adjustments are known to those skilled in the art.
  • the above-mentioned protein, the above-mentioned antibody-drug conjugate, and/or another therapeutic or diagnostic agent can each be used as a single agent and used in any time frame suitable for performing the intended treatment or diagnosis. Therefore, these single agents can be administered substantially simultaneously (ie, as a single formulation or within minutes or hours) or sequentially and consecutively. For example, these single agents can be administered within one year, or within 10, 8, 6, 4, or 2 months, or within 4, 3, 2, or 1 week, or within 5, 4, 3, 2, or 1 day.
  • the present invention provides a kit of medicines, which comprises a medicine box A and a medicine box B, the medicine box A is the above-mentioned protein, and/or the above-mentioned antibody-drug conjugate, and/or In the above-mentioned pharmaceutical composition, the kit B is another anti-tumor antibody or a pharmaceutical composition containing the other anti-tumor antibody.
  • the medicine box A and the medicine box B can be used at the same time, or the medicine box A can be used first and then the medicine box B, or the medicine box B can be used first and then the medicine box A can be determined according to the actual needs of the specific application. .
  • the present invention provides an application of the above isolated protein in the preparation of antitumor drugs.
  • the present invention provides an application of the above antibody-drug conjugate in the preparation of anti-tumor drugs.
  • the present invention provides an application of the above-mentioned pharmaceutical composition in the preparation of anti-tumor drugs.
  • the present invention provides an application of the above-mentioned kit in the preparation of anti-tumor drugs.
  • the present invention provides an application of the above isolated protein in the treatment of tumors.
  • the present invention provides an application of the above antibody drug conjugate in the treatment of tumors.
  • the present invention provides an application of the above pharmaceutical composition in the treatment of tumors.
  • the present invention provides an application of the above-mentioned kit medicine box in the treatment of tumors.
  • the anti-tumor drug is a drug for a disease related to TROP2 expression or abnormal function
  • the tumor is preferably a tumor related to TROP2 expression or abnormal function
  • said TROP2 expression or abnormal function Diseases or tumors related to dysfunction are: autoimmune diseases, inflammatory diseases, infectious diseases or proliferative diseases, preferably non-small cell lung cancer, invasive breast cancer, colon cancer, rectal cancer, gastric cancer, squamous cervical cancer, invasive Endometrial adenocarcinoma, invasive pancreatic cancer, ovarian cancer, squamous bladder cancer, choriocarcinoma, bronchial cancer, breast cancer, cervical cancer, pancreatic cancer or seminal vesicle cancer.
  • the present invention also provides a method for detecting cells overexpressing TROP2 protein, which includes the following steps: the above-mentioned protein is contacted with the test cell sample in vitro, and the above-mentioned protein and the test cell are detected.
  • the sample can be combined.
  • the meaning of said overexpression is conventional in the art.
  • the cells in the sample to be tested are subjected to flow cytometry, and the average fluorescence density (MFI) value of the above-mentioned protein is 3 times or more of the MFI value of subtype IgG .
  • MFI average fluorescence density
  • the detection method of the binding is a conventional detection method in the art, preferably FACS detection.
  • the cell sample to be tested is generally an isolated cell sample to be tested.
  • the "TROP2-positive” cells described in the present invention are cells that overexpress the TROP2 protein, such as the MDA-MB-468 cell line; on the contrary, they are called “TROP2-negative” cells, such as the tumor cell line HCC1395.
  • the reagents and raw materials used in the present invention are all commercially available.
  • the positive progress effect of the present invention is that the TROP2 antibody of the present invention has a high affinity with hTROP2 or cTROP2 or mTROP2 protein, and can bind to the extracellular region of the TROP2 protein at the protein level and the cell level.
  • the TROP2 antibody is coupled with a small molecule compound such as MC-MMAF to obtain an antibody-drug conjugate (ADC), which can effectively kill TROP2-positive cells.
  • ADC antibody-drug conjugate
  • the TROP2 antibody can bring small molecule compounds, such as MMAF, into cells through endocytosis, and degrade and release small molecule compounds in the cells, thereby playing a cytotoxic and killing effect.
  • the TROP2 antibody drug conjugate can effectively kill tumor cells and treat tumors.
  • the humanized antibody of the TROP2 antibody of the present invention After preparing the humanized antibody of the TROP2 antibody of the present invention, it can still have a high affinity with human or cynomolgus TROP2 protein, and can well inhibit the growth of tumor cells without significant influence on body weight.
  • Figure 1 shows the results of FACS screening and detection of HEK293 cells transfected with human TROP2 protein.
  • Figure 2 shows the results of FACS screening and detection of CHOK1 cells transfected with human TROP2 protein.
  • Figure 3 shows the results of FACS screening and detection of CHOK1 cells transfected with cynomolgus monkey TROP2 protein.
  • FIG. 4 shows the results of FACS screening and detection of CHOK1 cells transfected with mouse TROP2 protein.
  • Figure 5 shows the antibody titer of mice immunized with TROP2 by ELISA.
  • Figure 6 shows the detection of the binding reaction between TROP2 antibody and human TROP2-hFc protein by ELISA; the clone numbers of TROP2 antibody are 11E8E6D11 and 44A5F9, respectively.
  • Figure 7 shows the FACS detection of the binding reaction between TROP2 antibody and CHOK1-hTROP2; the clone numbers of TROP2 antibody are 11E8E6D11 and 44A5F9, respectively.
  • Figure 8 shows the FACS detection of the binding reaction between TROP2 antibody and CHOK1-cTROP2; the clone numbers of TROP2 antibody are 11E8E6D11 and 44A5F9, respectively.
  • Figure 9 shows the FACS detection of the binding reaction between TROP2 antibody and CHOK1-mTROP2; the clone numbers of TROP2 antibody are 11E8E6D11 and 44A5F9, respectively.
  • Figure 10 shows the FACS detection of the binding reaction between TROP2 antibody and CHOK1; the clone numbers of TROP2 antibody are 11E8E6D11 and 44A5F9, respectively.
  • Figure 11 shows the cell killing effect of the TROP2 antibody-MMAF antibody drug conjugate on the TROP2 positive triple-negative breast cancer cell line MAD-MB-468; the clone numbers of the TROP2 antibody are 11E8E6D11 and 44A5F9, respectively.
  • Figure 12 shows the cell killing effect of the TROP2 antibody-MMAF antibody drug conjugate on the TROP2 expression-negative breast cancer cell line HCC1395; the clone numbers of the TROP2 antibody are 11E8E6D11 and 44A5F9, respectively.
  • Figure 13 shows the cell killing effect of the TROP2 chimeric antibody drug conjugate coupled with MMAF on the TROP2-positive tumor cell line MDA-MB-468.
  • the clone numbers of the TROP2 chimeric antibodies are 11E8E6D11 and 44A5F9, respectively.
  • Figure 14A shows the cell killing effect of the TROP2 chimeric antibody drug conjugate conjugated with MMAF on the TROP2 positive tumor cell line BxPC-3.
  • the clone numbers of the TROP2 chimeric antibodies are 11E8E6D11 and 44A5F9, respectively.
  • Figure 14B shows the cell killing effect of the TROP2 chimeric antibody drug conjugate coupled with MMAF on the TROP2-positive tumor cell line COLO 205.
  • the clone numbers of the TROP2 chimeric antibodies are 11E8E6D11 and 44A5F9, respectively.
  • Figure 15 shows the cell killing effect of the TROP2 chimeric antibody drug conjugate coupled with MMAE on the TROP2-positive tumor cell line MDA-MB-468.
  • the clone numbers of TROP2 chimeric antibodies are 11E8E6D11 and 44A5F9 respectively.
  • Figure 16 shows the cell killing effect of the TROP2 chimeric antibody drug conjugate coupled with MMAE on the TROP2 positive tumor cell line COLO 205.
  • the clone numbers of the TROP2 chimeric antibodies are 11E8E6D11 and 44A5F9, respectively.
  • Figure 17 is a graph of tumor volume changes after treatment with a TROP2 chimeric antibody drug conjugate coupled to MMAE.
  • the clone numbers of TROP2 chimeric antibodies are 11E8E6D11 and 44A5F9 respectively.
  • Figure 18 is a graph showing the body weight changes of mice after treatment with MMAE-conjugated TROP2 chimeric antibody drug conjugate.
  • the clone numbers of TROP2 chimeric antibodies are 11E8E6D11 and 44A5F9 respectively.
  • Figure 19 is a pharmacokinetic diagram of plasma clearance after a single administration of TROP2 chimeric antibody in rats.
  • Figure 20 is a pharmacokinetic chart of plasma clearance after a single administration of hRS7 in rats.
  • Figure 21 is a graph showing the result of FACS identification of the binding activity of the mutated chimeric antibody.
  • Figure 22A shows the result of a flow cytometry experiment (FACS) detecting the binding of 11E8 humanized antibody to human TROP2 cells.
  • Figure 22B shows the result of a flow cytometry assay (FACS) detecting the binding of 44A5F9 humanized antibody to cells expressing human TROP2.
  • FACS flow cytometry assay
  • Figure 23A shows the results of flow cytometry (FACS) testing of the binding of 11E8 humanized antibody to cynomolgus monkey-derived TROP2 cells.
  • Figure 23B shows the result of flow cytometry (FACS) detection of the binding of 44A5F9 humanized antibody to cynomolgus monkey-derived TROP2 cells.
  • Figure 24A shows a graph of tumor volume change after treatment.
  • Figure 24B shows a graph of the weight change of rats after treatment.
  • the room temperature described in the examples is a conventional room temperature in the art, and is generally 10-30°C.
  • the PBS described in the examples is PBS phosphate buffer, pH 7.2.
  • the nucleotide sequence containing the amino acid sequence of the extracellular region at positions 88-274 (Thr88-Thr274) of the human TROP2 protein full-length amino acid sequence was cloned into a human IgG Fc Fragment (hFc) pCpC vector (purchased from Invitrogen, V044-50) and prepare plasmids according to established standard molecular biology methods. For specific methods, see Sambrook, J., Fritsch, EF, and Maniatis, T. (1989). Molecular Cloning: A Laboratory Manual, Second Edition (Plainview, New York: Cold Spring Harbor Laboratory Press).
  • HEK293 cells purchased from Invitrogen
  • polyetherimide PEI polyetherimide PEI
  • FreeStyle TM 293 purchased from Invitrogen
  • the cell culture fluid was collected, and the cell components were removed by centrifugation to obtain the culture supernatant containing the extracellular domain of hTROP2 protein.
  • the culture supernatant was loaded onto a protein A affinity chromatography column (Mabselect Sure, purchased from GE Healthcare), and the change in ultraviolet absorption (A280nm) was monitored with an ultraviolet (UV) detector.
  • UV ultraviolet
  • Immunogen A needs to undergo a series of quality control tests before use, such as testing its protein concentration, purity, molecular weight, biological activity, etc. It turns out that immunogen A has good indicators and can be used as an antigen for subsequent preparation of TROP2 antibodies.
  • the nucleotide sequence encoding the full-length amino acid sequence of human TROP2 was cloned into the pIRES vector (purchased from Clontech) and a plasmid was prepared.
  • the HEK293 cell line and CHOK1 cell line both purchased from Invitrogen) were transfected with plasmids (PEI, purchased from Polysciences), and then used in fetal cattle containing 0.5 ⁇ g/mL puromycin (puromycin) containing 10% (w/w) Serum was selectively cultured in DMEM medium for 2 weeks, and subcloned in 96-well culture plates by the limiting dilution method, and cultured at 37°C and 5% (v/v) CO 2.
  • the amplified clones were screened by flow cytometry with known TROP2 antibody. Select a cell line with better growth, higher fluorescence intensity, and monoclonal cell line, continue to expand and culture and freeze in liquid nitrogen to obtain immunogen B (ie, 293F-TROP2 stable cell line containing human TROP2).
  • immunogen B ie, 293F-TROP2 stable cell line containing human TROP2.
  • the specific selection results are shown in Table 2 and Figure 1.
  • the IgG subtype control is a mouse IgG control. Table 2 shows that a series of HEK293 cell lines expressing positive hTROP2 have been prepared.
  • Figure 1 is the result of the clone number 293F-TROP2 6F6, where the abscissa is the cell fluorescence intensity and the ordinate is the number of cells.
  • the results in Table 2 and Figure 1 indicate that 6F6 is a cell line expressing high levels of hTROP2, in which the average cell fluorescence density of TROP2 antibody-labeled cells is 3224.98, and the migration rate is 99.69%.
  • the immunogen A (hTROP2-hFc) obtained in step (1) was emulsified with Freund's complete adjuvant and injected intraperitoneally with 0.25 mL, that is, 50 ⁇ g of immunogen A protein was injected per mouse.
  • immunogen A was emulsified with Freund's incomplete adjuvant and injected intraperitoneally with 0.25 mL, that is, 50 micrograms of immunogen A was injected per mouse.
  • the immunogen B obtained in step (2) of Example 1 ie, the 293F-TROP2 stable cell line containing human TROP2
  • the immunogen B obtained in step (2) of Example 1 was expanded to 90% confluence in a T-75 cell culture flask, and the medium was aspirated , Washed twice with DMEM basal medium (Invitrogen), and then treated with enzyme-free cell dissociation solution (Invitrogen) at 37°C until the cells could be detached from the wall of the culture dish, and the cells were collected.
  • the cells are diluted with phosphate buffer to 2 ⁇ 10 7 cells per mL.
  • Each mouse was intraperitoneally injected with 0.5 mL of cell suspension during each immunization.
  • the interval between the first and second immunizations is 2 weeks, and the interval between subsequent immunizations is 3 weeks.
  • blood was collected one week after each immunization, and the antibody titer and specificity in the serum were detected by FACS. After the second booster immunization, the serum antibody titer detected by FACS reached more than 1:1000.
  • each selected mouse will be immunized by intraperitoneal injection of 100 micrograms of purified immunogen A (mouse immune response to immunogen A) or HEK293 containing human TROP2 stable Cell line (mice that reacted to immunogen B). After 5 days, the mice were sacrificed and splenocytes were collected. Add NH 4 OH to a final concentration of 1% (v/v) to lyse the red blood cells adulterated in the spleen cells to obtain a spleen cell suspension.
  • DMEM basal medium purchased from invitrogen
  • mouse myeloma cells SP2/0 purchased from ATCC
  • the fused cells were diluted into DMEM medium containing 20% (v/v) fetal bovine serum and 1 ⁇ HAT. Then add 1 ⁇ 10 5 cells/200 microliters per well to a 96-well cell culture plate, and place it in a 5% (v/v) CO 2 , 37°C incubator for culture.
  • ELISA and Acumen microwell plate cell detection method
  • the positive clones with OD450nm>1.0 in ELISA and MFI value>100 in Acumen were amplified to 24-well plates, and the cells contained 10% (v /v) In DMEM medium of HT fetal bovine serum, expand culture at 37°C and 5% (v/v) CO 2 . After 3 days of culture, the culture solution expanded in the 24-well plate was centrifuged, the supernatant was collected, and the supernatant was analyzed for antibody subtypes.
  • ELISA and FACS were used to determine its binding activity to TROP2 protein and TROP2 positive cells (for the detection methods of binding activity, please refer to the relevant content in Example 3A and Example 3B, respectively), and indirect cytotoxic killing experiment with mouse TROP2 antibody-MMAF Evaluation of indirect cytotoxic activity (see related content in Example 4 for the detection method of indirect cytotoxic activity).
  • the results of the 24-well plate screening select the hybridoma cells whose OD450nm>1.0 in the ELISA experiment, the MFI value>50 in the FACS experiment, and the hybridoma cell culture supernatant in the indirect cytotoxicity experiment to 50% of the TROP2 positive cells.
  • Condition positive clones select qualified hybridoma cells and subclone them in 96-well plates by limiting dilution method, in DMEM medium containing 10% (v/v) fetal bovine serum, 37°C, 5% (v/v) v) Cultivation under CO 2 conditions.
  • Ten days after subcloning, ELISA and Acumen were used for preliminary screening, and a single positive single clone was selected and amplified to a 24-well plate to continue culture.
  • the indirect cytotoxic killing test of mouse-derived TROP2 antibody-MMAF was used to evaluate the indirect cytotoxic killing activity (the evaluation criteria are OD450nm>1.0 in the ELISA test, MFI value>50 in the FACS test, and indirect cytotoxicity
  • the killing rate of hybridoma cell culture supernatant on TROP2 positive cells reached 50% and above).
  • the best clones were selected and placed in DMEM medium containing 10% (v/v) fetal bovine serum at 37°C and 5% (v/v) CO 2
  • the optimal clone is expanded and cultured and frozen in liquid nitrogen to obtain the hybridoma cells of the present invention, which can be used for subsequent lead antibody production and purification.
  • the antibody concentration produced by hybridoma cells is low, only about 1-10 ⁇ g/mL, the concentration varies greatly, and the various proteins produced by the cell culture in the medium and the fetal bovine serum components contained in the medium have many biological activity analysis
  • the methods have different degrees of interference, so small-scale (1-5mg) antibody production and purification is required.
  • the hybridoma cells obtained in Example 1 were inoculated into T-75 cell culture flasks and used production medium (Hybridoma serum free medium, purchased from Invitrogen) for domestication and passage for 3 generations. When it grows well, inoculate the cell culture spinner flask. Add 200mL production medium to each 2L culture spinner flask, and the inoculation cell density is 1.0 ⁇ 10 5 /mL. Tighten the cap, and place the spinner on the spinner in the 37°C incubator at a speed of 3 revolutions/min.
  • used production medium Hybridoma serum free medium, purchased from Invitrogen
  • the cell culture solution was collected, filtered to remove the cells, and filtered with a 0.45 ⁇ m filter membrane until the culture supernatant was clarified to obtain a clarified hybridoma cell culture supernatant.
  • the culture supernatant of the clarified hybridoma cells can be purified immediately or frozen at -30°C.
  • the TROP2 antibody in the obtained culture supernatant (200 mL) was purified with a 2 mL protein A column (GE Healthcare).
  • the protein A column was first equilibrated with equilibration buffer (PBS phosphate buffer, pH 7.4), and then the culture supernatant was loaded onto the protein A column, and the flow rate was controlled at 3 mL/min. After loading the sample, wash the protein A column with equilibration buffer.
  • the volume of the equilibration buffer is 4 times the volume of the protein A column bed.
  • the TROP2 antibody bound to the protein A column was eluted with an eluent (0.1M sodium citrate buffer, pH 3.5), and the elution was monitored with an ultraviolet detector (A280nm ultraviolet absorption peak). Collect the eluted antibodies, add 10% (v/v) 1.0M Tris-HCl buffer to neutralize the pH, and then immediately dialyze with PBS phosphate buffer overnight, change the medium once the next day and continue dialysis for 3 hours. Collect the dialyzed TROP2 antibody, perform aseptic filtration with a 0.22 ⁇ m filter, and store aseptically to obtain purified TROP2 antibody.
  • an eluent 0.1M sodium citrate buffer, pH 3.5
  • an ultraviolet detector A280nm ultraviolet absorption peak
  • the purified TROP2 antibody was tested and analyzed for protein concentration (A280nm/1.4), purity, and endotoxicity (Lonza kit). The results are shown in Table 4. Table 4 shows that the endotoxin concentration of the final antibody product is within 1.0EU/mg .
  • ELISA Enzyme-linked immunosorbent assay
  • the purified TROP2 antibody obtained in Example 2 was reacted with human TROP2-hFc protein (immunogen A).
  • the purified immunogen A obtained in Example 1 [see step (1) of Example 1 for the preparation method] was diluted with PBS to a final concentration of 1.0 ⁇ g/mL, and then 100 ⁇ L per well was added to a 96-well ELISA plate. Cover with plastic film and incubate overnight at 4°C. Wash the plate twice with plate washing solution [PBS containing 0.01% (v/v) Tween 20] on the second day, and add blocking solution [containing 0.01% (v/v) Tween 20 and 1 %(W/w)BSA in PBS] for 2 hours at room temperature. The blocking solution was discarded, and 100 ⁇ L of the purified TROP2 antibody obtained in Example 2 was added to each well.
  • Figure 6 and Table 5 show that the purified TROP2 antibody binds to the TROP2 recombinant protein at the ELISA level.
  • the IgG control (produced by Shanghai Ruizhi Chemical Research Co., Ltd.) in Table 5 is a control mouse IgG, the data in the table is the OD 450nm value, and the meaning of Blank is the OD 450nm value when there is only PBS buffer in the plate.
  • the hRS7 sequence comes from the patent US8758752, which is expressed and produced by Shanghai Ruizhi Chemical Research Co., Ltd. according to the preparation method in the patent.
  • the pIRES plasmid containing the nucleotide sequence encoding the full-length amino acid sequence of human TROP2 described in step (2) of Example 1 was transfected into the CHOK1 cell line to obtain a CHOK1 stable cell line containing human TROP2 (herein referred to as CHOK1- hTROP2 stable cell line).
  • the pIRES plasmid with the full-length gene of cynomolgus monkey TROP2 (its amino acid sequence is shown in SEQ ID NO: 18 in the sequence list) (the preparation method is the same as that of Example 1 step (1) "Preparation of immunogen A"
  • the preparation method of the pCpC vector carrying the human IgG Fc fragment (hFc) is the same.
  • the CHOK1 cell line was transfected to obtain the CHOK1 stable cell line containing the cynomolgus TROP2 (herein referred to as the CHOK1-cTROP2 stable cell line).
  • the pIRES plasmid with the mouse-derived TROP2 full-length gene (its amino acid sequence is shown in SEQ ID NO: 19 in the sequence list) (the preparation method is the same as that of Example 1 step (1) "Preparation of immunogen A" with human The preparation method of the pCpC vector derived from the IgG Fc fragment (hFc) is the same.)
  • the CHOK1 cell line is transfected to obtain the CHOK1 stable cell line containing mouse TROP2 (herein referred to as the CHOK1-mTROP2 stable cell line).
  • FACS was used to detect the protein expression in the above stable cell lines.
  • the detection method refer to the method for identifying the HEK293-TROP2 stable cell line in step (2) "Preparation of immunogen B" in Example 1.
  • the test results are shown in Table 6 and Figures 2 to 4.
  • the abscissa is the cell fluorescence intensity, and the ordinate is the number of cells.
  • CHOK1-hTROP2 2B2 is the human TROP2 expression cell line used for screening, and the FACS screening test results are shown in Figure 2.
  • CHOK1-cTROP2 2B2C2 is the cynomolgus monkey TROP2 expression cell line used for screening, and the FACS screening test results As shown in Figure 3; CHOK1-mTROP2 6F6 is the mouse TROP2 expressing cell line used for screening, and the FACS screening test results are shown in Figure 4.
  • the results in Table 6 indicate that the cell membranes of the CHOK1-hTROP2 stable cell line, the CHOK1-cTROP2 stable cell line, and the CHOK1-mTROP2 stable cell line respectively overexpress the human, cynomolgus monkey or mouse TROP2 protein, which can be used to screen TROP2 antibody.
  • CHOK1-hTROP2 stable cell line CHOK1-cTROP2 stable cell line
  • CHOK1-mTROP2 stable cell line i.e. CHOK1-hTROP2 2B2, CHOK1-cTROP2 2B2C2 and CHOK1-mTROP2 6F6 shown in Table 6) and CHOK1 cells
  • CHOK1 cells were placed in T -75 cell culture flasks were expanded to 90% confluence, the medium was aspirated, washed twice with HBSS buffer (Hanks Balanced Salt Solution, purchased from Invitrogen), and then with enzyme-free cell dissociation solution (Versene solution, purchased From Life Technology Corporation) process and collect cells. Wash the cells twice with HBSS buffer.
  • HBSS buffer Horts Balanced Salt Solution, purchased from Invitrogen
  • enzyme-free cell dissociation solution Versene solution, purchased From Life Technology Corporation
  • Example 2 After counting the cells, dilute the cells with HBSS buffer to 2 ⁇ 10 6 cells/mL, add 10% goat serum blocking solution, the percentage is the mass percentage, and incubate on ice for 30 minutes , And then washed twice with HBSS buffer by centrifugation. Suspend the collected cells in FACS buffer (HBSS+1%BSA, the percentage is mass percentage) to 2 ⁇ 10 6 cells/mL, add 100 ⁇ l per well to a 96-well FACS reaction plate, and add The purified TROP2 antibody test sample obtained in Example 2 was 100 microliters per well and incubated on ice for 2 hours.
  • FACS buffer HBSS+1%BSA, the percentage is mass percentage
  • Example 2 After the purified TROP2 antibody obtained in Example 2 was dialyzed with sodium borate buffer of pH 6.5 ⁇ 8.5, tris(2-carboxyethyl)phosphine (TCEP) was added, wherein the molar ratio of TCEP to the purified TROP2 antibody was 2 , Reduce at room temperature for 1 hour to obtain reaction solution A.
  • the reaction solution A is desalted through a G25 column (purchased from GE), and excess TCEP is removed to obtain reaction solution B.
  • MC-MMAF (purchased from Nanjing Lianning) was added to the reaction solution B, where the molar ratio of MC-MMAF to the purified TROP2 antibody was 5, and the reaction was carried out at room temperature for 4 hours.
  • the purified TROP2 antibody drug conjugate is obtained (refer to Doronina, 2006, Bioconjugate Chem. 17, 114-124 for coupling method).
  • the cytotoxic activity is analyzed.
  • the drug cross-linking rate (DAR) of all antibody-drug conjugates is about 8 (the method of determining DAR is conventional in the art).
  • DAR drug antibody ratio
  • HPLC-HIC high-density lipoprotein
  • HPL-SEC low-density lipoprotein
  • LC-Ms the analysis conditions of HPLC-HIC, HPL-SEC and LC-Ms are as follows:
  • Mobile phase B 0.025M sodium phosphate aqueous solution, 25% (v/v) isopropanol aqueous solution
  • Mobile phase A 0.1% formic acid, 25% acetonitrile aqueous solution.
  • the purified TROP2 antibody-drug conjugates obtained were serially diluted with complete medium, and 90 microliters of TROP2-positive MDA-MB-468 cell line (purchased from ATCC, catalog number) was added to a 96-well cell culture plate at 5000 cells/well. #HTB-132) After the cell suspension was cultured overnight, add 10 microliters of different concentrations of purified TROP2 antibody-drug conjugate dilutions to each well. After culturing for 5 days, use the CellTiter-Glo kit (purchased from Promega, For the method of use, refer to the product manual) to detect cell viability.
  • the TROP2-negative tumor cell line HCC1395 (purchased from ATCC, catalog #CRL-2324) was used for cell killing activity detection, and the method was the same as above.
  • Table 8 and Figures 11-12 where the EC50 in Table 8 refers to the half-effective amount that inhibits cell activity after drug action, which can reflect cell killing activity by detecting cell activity.
  • Figure 11 shows the cell killing activity of the purified TROP2 antibody drug conjugate against TROP2-positive tumor cell line MDA-MB-468
  • Figure 12 shows the purified TROP2 antibody drug conjugate against TROP2-negative breast cancer tumor cells Detection of cell killing activity of line HCC1395.
  • the results indicate that the purified TROP2 antibody drug conjugate obtained above has a killing effect on TROP2-positive cells.
  • the purified antibody to be tested (ie the purified TROP2 antibody obtained in Example 2, the clone numbers of which are 11E8E6D11 and 44A5F9, respectively) were diluted to 1 ⁇ g/mL with PBS, and a 96-well high-absorption ELISA plate was coated with 50 ⁇ L/well at 4°C After overnight coating, use 250 microliters of blocking solution [PBS containing 0.01% (v/v) Tween 20 and 1% (w/w) BSA] for one hour at room temperature, and add 0.05 ⁇ g/mL biotin-labeled to each well Recombinant TROP2 protein.
  • blocking solution [PBS containing 0.01% (v/v) Tween 20 and 1% (w/w) BSA] for one hour at room temperature, and add 0.05 ⁇ g/mL biotin-labeled to each well Recombinant TROP2 protein.
  • the first column from the left is the coated antibody, with a concentration of 1 ⁇ g/mL; the first column above is the competing antibody, with a concentration of 5 ⁇ g/mL.
  • Total RNA isolation Collect 5 ⁇ 10 7 hybridoma cells obtained in Example 1 corresponding to the lead antibody selected in Example 2 by centrifugation, add 1 mL Trizol to mix and transfer to a 1.5 mL centrifuge tube, and let stand at room temperature 5 minute. Add 0.2mL chloroform, shake for 15 seconds, let stand for 10 minutes, centrifuge at 4°C, 12000g for 5 minutes, take the supernatant and transfer to a new 1.5mL centrifuge tube. Add 0.5 mL of isopropanol, gently mix the liquid in the tube, let it stand at room temperature for 10 minutes and centrifuge at 12000g for 15 minutes at 4°C, and discard the supernatant.
  • Reverse transcription and PCR Take 1 ⁇ g of total RNA, configure a 20 ⁇ L system, add reverse transcriptase and react at 42°C for 60 minutes, and at 7°C for 10 minutes to stop the reaction.
  • Configure 50 ⁇ L PCR system including 1 ⁇ L cDNA, 25pmol of each primer, 1 ⁇ L DNA polymerase and matching buffer system, 250 ⁇ mol dNTPs; set PCR program, 95°C pre-denaturation for 3 minutes, 95°C denaturation for 30 seconds, 55°C annealing for 30 seconds, Extend at 72°C for 35 seconds, and then extend at 72°C for an additional 5 minutes after 35 cycles to obtain a PCR product.
  • the kit used for reverse transcription is PrimeScript RT Master Mix, purchased from Takara, item number RR036; the kit used for PCR includes Q5 ultra-fidelity enzyme, purchased from NEB, item number M0492.
  • Cloning and sequencing Take 5 ⁇ L of PCR product for agarose gel electrophoresis detection, and use the column recovery kit to purify the positive samples, where the recovery kit is Gel&PCR Clean-up, purchased from MACHEREY-NAGEL, catalog number 740609. Carry out ligation reaction: sample 50ng, T carrier 50ng, ligase 0.5 ⁇ L, buffer 1 ⁇ L, reaction system 10 ⁇ L, react at 16°C for half an hour to obtain the ligation product.
  • the ligation kit is T4DNA ligase, purchased from NEB, item number M0402; take 5 ⁇ L of the ligation product and add 100 ⁇ L of competent cells (Ecos 101competent cells, purchased from Yeastn, item number FYE607), ice bath for 5 minutes, and then at 42°C Heat shock in a water bath for 1 minute, put it back on ice for 1 minute, add 650 ⁇ L of antibiotic-free SOC medium, and resuscitate on a shaker at 37°C at 200 RPM for 30 minutes. Take out 200 ⁇ L and spread it on LB solid medium containing antibiotics and incubate overnight at 37°C in an incubator.
  • Antibody purification For continuous production of endotoxin-free chromatography columns and Protein A fillers, use 0.1M NaOH for 30 minutes or 5 column volumes of 0.5M NaOH for flushing; for column materials and chromatography columns that have not been used for a long time, use at least Soak in 1M NaOH for 1 hour, rinse with non-endotoxic water to neutrality, and wash the column material with 10 times the column volume of 1% Triton X100. Equilibrate with 5 column volumes of PBS, put the filtered cell supernatant on the column, and collect the flow-through if necessary. After loading the column, wash with 5 times the column volume of PBS.
  • the first paragraph of the chimeric antibody naming uses the first 3 to 5 characters of the corresponding lead antibody clone number.
  • the lead antibody clone number corresponding to the chimeric antibody drug conjugate 11E8-MMAF is 11E8E6D11, chimeric
  • the corresponding lead antibody clone number of the antibody-drug conjugate 44A5-MMAF is 44A5F9 and so on.
  • the purified TROP2 chimeric antibody obtained in Example 7 was coupled to MC-MMAF in the same manner as in Example 4.
  • TCEP tris(2-carboxyethyl)phosphine
  • the reaction solution A is obtained by reducing at room temperature for 1 hour.
  • MC-MMAF was added to the reaction solution B, where the molar ratio of MC-MMAF to the purified chimeric TROP2 antibody was 5, and the reaction was carried out at room temperature for 4 hours.
  • the purified TROP2 chimeric antibody drug conjugate was obtained (for coupling method, see Doronina, 2006, Bioconjugate Chem. 17, 114-124). After analyzing the cross-linking rate of the drug by HIC and the purity of the antibody-drug conjugate by SEC, the cytotoxic activity is analyzed.
  • the drug crosslinking rate (DAR) of all antibody-drug conjugates is 3.0-5.0, as shown in Tables 10 and 11. Among them, DAR (drug antibody ratio) refers to the average number of small molecule drugs carried on an antibody molecule after antibody coupling.
  • the obtained purified TROP2 chimeric antibody drug conjugates were serially diluted with complete medium, and 90 microliters of TROP2-positive MDA-MB-468 cell line (purchased from ATCC) was added to a 96-well cell culture plate at 5000 cells/well. , Item #HTB-132) After the cell suspension is cultured overnight, add 10 microliters of different concentrations of purified TROP2 chimeric antibody drug conjugate dilutions to each well. After continuing the culture for 5 days, use the CellTiter-Glo kit ( (Purchased from Promega, the use method refers to the product manual) to detect cell viability.
  • IC50 in Table 10 refers to the half-effective amount that inhibits cell activity after drug action, which can reflect cell killing activity by detecting cell activity.
  • Figure 13 shows the cell killing activity of the purified TROP2 chimeric antibody drug conjugate against the TROP2 positive tumor cell line MDA-MB-468.
  • TROP2-positive tumor cell lines BxPC-3 purchased from ATCC, catalog number #CRL-1687
  • COLO 205 purchased from ATCC, catalog number #CCL-222
  • FIG. 11 The results are shown in Table 11 and Figures 14A and 14B, where the IC50 in Table 11 refers to the half-effective amount that inhibits cell activity after the drug is applied, which can reflect cell killing activity by detecting cell activity.
  • Figure 14A and Figure 14B show the cell killing activity of the purified TROP2 chimeric antibody drug conjugate against TROP2-positive tumor cell lines BxPC-3 and COLO 205. The results show that the purified TROP2 chimeric antibody drug conjugate is effective against TROP2 Positive cells have a killing effect.
  • Table 10 Cell killing test to detect the specific killing effect of purified TROP2 chimeric antibody drug conjugate on TROP2-positive MDA-MB-468 cells
  • the purified TROP2 chimeric antibody obtained in Example 7 was coupled with MC-VC-PAB-MMAE (Kay Technology Development (Shanghai) Co., Ltd.), the method was the same as that in Example 4, after pH 6.5-8.5 sodium borate buffer After liquid dialysis, tris(2-carboxyethyl)phosphine (TCEP) was added, wherein the molar ratio of TCEP to purified TROP2 antibody was 2, and the reaction solution A was obtained by reducing at room temperature for 1 hour. The reaction solution A is desalted through a G25 column (purchased from GE), and excess TCEP is removed to obtain reaction solution B.
  • TCEP tris(2-carboxyethyl)phosphine
  • MC-VC-PAB-MMAE was added to the reaction solution B, wherein the molar ratio of MC-VC-PAB-MMAE to the purified TROP2 antibody was 5, and the reaction was carried out at room temperature for 4 hours. Then add cysteine to neutralize the excess MC-VC-PAB-MMAE, and pass the G25 column desalting to remove the excess small molecules.
  • the purified TROP2 antibody drug conjugate is obtained (refer to Doronina, 2006, Bioconjugate Chem. 17, 114-124 for coupling method).
  • the cytotoxic activity is analyzed.
  • the drug cross-linking rate (DAR) of all antibody-drug conjugates is 3.0-5.0. Among them, DAR (drug antibody ratio) refers to the average number of small molecule drugs carried on an antibody molecule after antibody coupling.
  • the purified TROP2 antibody-drug conjugates obtained were serially diluted with complete medium, and 90 microliters of TROP2-positive MDA-MB-468 cell line (purchased from ATCC, catalog number) was added to a 96-well cell culture plate at 5000 cells/well. #HTB-132) After the cell suspension was cultured overnight, add 10 microliters of different concentrations of purified TROP2 chimeric antibody-drug conjugate diluent to each well. After culturing for 5 days, use the CellTiter-Glo kit (purchased from Promega, the method of use refers to the product manual) to detect cell viability.
  • the TROP2-positive tumor cell line COLO205 (purchased from ATCC, item #CCL-222) was selected for cell killing activity detection, and the method was the same as above.
  • the results are shown in Table 12 and Figure 15 and Figure 16, where the IC50 in Table 12 refers to the half effective amount that inhibits cell activity after drug action, which can reflect cell killing activity by detecting cell activity.
  • Figures 15 and 16 show the cell killing activity of the purified TROP2 chimeric antibody drug conjugate against TROP2-positive tumor cell lines MDA-MB-468 and COLO 205. The results indicate that the purified TROP2 antibody drug conjugate has a killing effect on TROP2-positive cells.
  • Sprague-Dawley rats (6-8 weeks old, 210-235g each, SLAC Laboratory Animal Co. LTD) were injected into the tail vein of one side of the chimeric antibody.
  • chimeric antibody 11E8, chimeric antibody 44A5, and hRS7 were administered to the animals through the dorsal foot vein at a volume dose of 3 mL/kg.
  • Approximately 150 ⁇ L of whole blood was collected through the tail vein at each time point of 10, and 30 minutes; 1, 4, 8, and 24 hours; and 2, 4, 7, 14, 21, and 28 days after administration, and serum analysis was performed.
  • the experimental program is shown in Table 14.
  • the total clearance (CL) of the chimeric antibody 11E8E6D11 was 5.62 mL/day/kg
  • the steady-state apparent volume of distribution (Vss) was 112 mL/kg
  • the clearance half-life was 13.6 days.
  • the total clearance (CL) of the chimeric antibody 44A5F9 was 9.27 mL/day/kg
  • the steady-state apparent volume of distribution (Vss) was 121 mL/kg
  • the clearance half-life was 9.33 days.
  • the total clearance (CL) of the control antibody hRS7 was 8.19 mL/day/kg
  • the steady-state apparent volume of distribution (Vss) was 110 mL/kg
  • the clearance half-life was 9.48 days.
  • the NG of the chimeric antibody 11E8 antibody heavy chain variable region (SEQ ID NO: 1) CDR2 (SEQ ID NO: 3) may have deamidation reaction, and DG may have different The possibility of conformation, in order to inhibit deamidation, isomerization and hydrolysis, after calculation and analysis, the NG located in the CDR2 of the chimeric antibody 11E8 antibody was mutated to NA.
  • the DG located in CDR2 is mutated to SG; or DG is mutated to EG, or DG is mutated to DA.
  • amino acid sequence of the CDR2 region of the heavy chain variable region of the chimeric antibody 11E8 antibody is shown in SEQ ID NO: 20 -23 shown. It is proposed to implement amino acid modification to remove asparagine residues and asparagyl residues as sites for deamidation by the above-mentioned site-directed mutagenesis.
  • FIG. 21 shows the mutant antibody chimeric antibody 11E8 after the NG of the chimeric antibody 11E8 antibody heavy chain variable region CDR2 was mutated to NA, and DG was mutated to SG, EG or DA. 1. Detection of the binding activity of chimeric antibody 11E8-2, chimeric antibody 11E8-3, chimeric antibody 11E8-4 and wild-type antibody chimeric antibody 11E8 to CHOK-TROP2 cells. Table 15 summarizes the binding activity of the chimeric antibody 11E8 wild-type antibody and mutant antibody in CHOK-TROP2 cells.
  • NG in the CDR2 of the heavy chain variable region of the chimeric antibody 11E8 is mutated to NA (sequence is shown in SEQ ID NO: 44), and DG is mutated to DA (sequence is shown in SEQ ID NO:
  • the mutant antibody chimeric antibody 11E8-1 and mutant chimeric antibody 11E8-4 shown in 30) have close binding activity with CHOk1-TROP2 cells to that of wild-type antibody chimeric antibody 11E8, indicating that NG is mutated into NA and DG mutations.
  • DA does not affect the binding of antibodies to CHOK-TROP2 cells.
  • the DG located in CDR2 is mutated into EG or SG to a certain extent, and the maximum average fluorescence intensity after the binding of the antibody to CHOK-TROP2 is reduced by 53% and 23%, respectively.
  • the chimeric antibody obtained by mutating the NG of CDR2 to NA and the heavy chain CDR2 of DG to DA (sequence shown in SEQ ID NO: 39) will be used for subsequent research.
  • the germline gene sequence with the highest homology of the variable region of the light chain with the candidate antibody 11E8E6D11 heavy chain variable region and the light chain variable region was selected by sequence alignment (NCBI-Igblast) as the variable region transplantation skeleton: GHV1-69*08 (66.0%) And IGKV1-39*01 (65.7%).
  • sequence alignment NCBI-Igblast
  • GHV1-69*08 66.0%
  • IGKV1-39*01 65.7%
  • homology modeling was used to predict the key amino acids that may determine the structure in the mouse anti-constant region, and the grafted framework region was designed with back mutations, as shown in Table 16.
  • the candidate antibody 44A5F9 heavy chain variable region and light chain variable region have no important hotspots.
  • sequence alignment NCBI-Igblast
  • the germline gene sequence with the highest homology to the variable region of the heavy chain of the candidate antibody 44A5F9 and the variable region of the light chain is selected as the variable region transplantation skeleton: IGHV3-7*01 (69.4%) And IGKV1-39*01 (61.6%).
  • the human antibody framework through homology modeling, the key amino acids that may determine the structure in the mouse anti-constant region are predicted, and the grafted framework region is designed for back mutation, as shown in Table 18.
  • 3 heavy chain variable region sequences (humanized 44A5VH.g0, humanized 44A5VH.g1, humanized 44A5VH.g2) and 3 light chain variable region sequences (humanized 44A5VL. g0, humanized 44A5VL.g1, humanized 44A5VL.g2).
  • cross-combination was performed for expression, and the following 9 humanized antibodies were obtained, as shown in Table 19.
  • the amplification primers are synthesized by Genewiz, and then the variable regions of the light chain and the heavy chain are amplified separately by PCR. Configure a 50 ⁇ L reaction system, including 50-100ng heavy chain variable region, 1ul light chain variable region, forward and reverse primers, 1ul pfxD enzyme (purchased from invitrogen, 12344-012), 10*pfx buffer 5ul ( The supplier has the same enzyme as pfx) and add water to make up to 50 ⁇ L.
  • Preparation of humanized antibody perform ligation reaction: insert 20-40ng, digested expression vector 60-100ng, recombinase Exnase (purchased from Vazyme, catalog number C112-01/02) 1 ⁇ L, buffer 2 ⁇ L, reaction system 10 ⁇ L, react at 37°C for half an hour to obtain the ligation product, which is the constructed recombinant vector.
  • the buffer is the buffer used for the purchase of the recombinase; the heavy chain variable region is directionally cloned into an expression vector containing the signal peptide and the human antibody heavy chain IgG1 constant region (the expression vector is purchased from Invitrogen, and the recombination step is a conventional step ), the light chain variable region was directional cloned into an expression vector containing a signal peptide and a human antibody light chain kappa constant region (where the expression vector was purchased from Invitrogen, and the recombination step was a conventional step).
  • the recombinant antibody heavy and light chain expression vectors with the correct sequence are amplified, and then transiently transfected into FreeStyle TM 293-F cells (purchased from Invitrogen) to produce antibodies.
  • the density of 293-F cells should be 1-1.2 ⁇ 10 6 cells/mL, and 100 mL cells need 100 ⁇ g of the above-mentioned constructed recombinant vector and 200 ⁇ g of transfection reagent polyethyleneimine (PEI).
  • PEI transfection reagent polyethyleneimine
  • the mixture of recombinant vector and PEI was allowed to stand at room temperature for 15 minutes. Then the above mixture was slowly added to the cells, and cultured in a 37°C, 8% (v/v) CO 2 incubator at 130 rpm. The culture supernatant and cell pellet are taken every day to detect antibody expression. After 5 days, the cell culture solution was centrifuged at 3000 g for 30 minutes, the supernatant was collected, and filtered with a 0.22 ⁇ m filter. The monoclonal antibody in 200 mL of the clear supernatant was purified with 1 mL MabSelect TM SuRe TM column (purchased from GE Healthcare).
  • MabSelect TM SuRe TM column is first equilibrated with equilibration buffer (PBS phosphate buffer, pH 7.2), MabSelect TM SuRe TM column. After loading the sample, wash the MabSelect TM SuRe TM column with the equilibration buffer. The volume of the equilibration buffer is 5 times the volume of the protein A column bed.
  • the monoclonal antibody bound to the MabSelect TM SuRe TM column was eluted with an eluent (0.1M glycine hydrochloride buffer, pH 3.0). Collect the eluted antibodies, add 10% (v/v) 1.0M Tris-HCl buffer to neutralize the pH. Then immediately dialyzed against PBS phosphate buffer overnight. Collect the dialyzed monoclonal antibodies, filter them aseptically with a 0.22 ⁇ m filter, and store them aseptically to obtain purified TROP2 humanized antibodies.
  • equilibration buffer P
  • FIG. 22A Flow cytometry (FACS) detects the binding of antibodies to human TROP2 cells.
  • the results are shown in Figure 22A.
  • the resulting 11E8 humanized antibodies can all bind to human Trop2 on the cell surface.
  • the obtained 44A5F9 humanized antibodies can all bind to human Trop2 on the cell surface.
  • the isotype control is human IgG1, and the data in the figure is the average fluorescence intensity value (MFI) of the measured cell population.
  • MFI average fluorescence intensity value
  • FIG. 23A Flow cytometry (FACS) to detect the binding of antibodies to TROP2 cells expressing cynomolgus monkey origin.
  • the results are shown in Figure 23A.
  • the obtained 11E8 humanized antibodies can all bind to the cynomolgus monkey-derived Trop2 on the cell surface.
  • the obtained 44A5F9 humanized antibodies can bind to the cynomolgus monkey-derived Trop2 on the cell surface.
  • the isotype control is human IgG1, and the data in the figure is the average fluorescence intensity value (MFI) of the measured cell population.
  • Table 21 shows that the obtained purified humanized TROP2 antibodies can bind well to the cynomolgus monkey TROP2 on the cell surface.
  • the hTINA1-H1L1 sequence comes from the patent WO2015098099A1, which is expressed and produced by Shanghai Ruizhi Chemical Research Co., Ltd. according to the preparation method in the patent.
  • the affinity comparison of different humanized antibodies is performed through Biacore. Some data are shown in Table 22. The specific operations and methods are based on the instrument instructions and detailed methods provided by the manufacturer. Specifically: according to the method described in the manual of the human Fab capture kit (Cat.#28-9583-25, GE), the human Fab capture molecule is covalently coupled to the CM5 biosensor chip (Cat.#BR-1000 -12, GE) to affinity capture the antibody to be tested. Then flow through the human TROP2-his (CAT#10428-H08H-100, Sino biological) antigen on the surface of the chip, and use the Biacore instrument to detect the reaction signal in real time to obtain the binding and dissociation curves. The affinity values are obtained by fitting, see Table 22 below . After each cycle of dissociation in the experiment is completed, the biochip is washed and regenerated with the regeneration solution configured in the human Fab capture kit.
  • the resulting purified humanized anti-TROP2 antibody was conjugated with MC-GGFG-Dxd (Kay Technology Development (Shanghai) Co., Ltd.), after dialysis with 4 ⁇ PBS buffer pH 5.5 ⁇ 8.5, an appropriate amount of pH7 was added. 0 to 9.0 ethylenediaminetetraacetic acid (EDTA) and 7-10 times molar equivalent of tris(2-carboxyethyl)phosphine (TCEP) are reduced for two hours at 37 degrees Celsius to obtain reaction solution A.
  • the reaction solution A is desalted through a G25 column (purchased from GE), and excess TCEP is removed to obtain reaction solution B.
  • MC-GGFG-Dxd is added to the reaction solution, wherein the molar ratio of MC-GGFG-Dxd to reduced TROP2 antibody is 8-12, and the reaction is carried out at room temperature for 4 hours. Finally, N-ethylmaleimide (N-EM) was added to neutralize the unreacted sulfhydryl groups. And through the G25 column desalting to remove the excess small molecules, the purified TROP2 antibody drug conjugate (GGFG-Dxd) was obtained. After analyzing the cross-linking rate of the drug by LC-MS and the purity of the antibody-drug conjugate by SEC, the cytotoxic activity was analyzed.
  • the drug crosslinking rate (DAR) of all antibody drug conjugates is 6.0-8.0. Among them, DAR (drug antibody ratio) refers to the average number of small molecule drugs carried on an antibody molecule after antibody coupling.

Abstract

A TROP2 antibody, a preparation method therefor, and a conjugate and a use thereof. The TROP2 antibody comprises a heavy-chain variable region and a light-chain variable region, the heavy-chain variable region comprising a heavy-chain CDR1, a heavy-chain CDR2, and a heavy-chain CDR3, and/or, the light-chain variable region comprising a light-chain CDR1, a light-chain CDR2, and a light-chain CDR3. The TROP2 antibody features a high affinity and a strong specificity, and a conjugate produced from coupling with a small molecule drug toxin such as MMAF is able to have a cytotoxic effect on TROP2-positive cells, and therefore can be used in the preparation of a drug for treating tumors and the like.

Description

一种TROP2抗体及其制备方法、其偶联物和应用A TROP2 antibody and its preparation method, its conjugate and application
本申请要求申请日为2019/8/12的中国专利申请2019107465360的优先权。本申请引用上述中国专利申请的全文。This application claims the priority of the Chinese patent application 2019107465360 whose filing date is 2019/8/12. This application quotes the full text of the aforementioned Chinese patent application.
技术领域Technical field
本发明涉及抗体领域，具体涉及一种TROP2抗体及其制备方法、其偶联物和应用。The invention relates to the field of antibodies, in particular to a TROP2 antibody, a preparation method thereof, a conjugate and application thereof.
背景技术Background technique
在研究正常人体及癌变的滋养层细胞时，M.Lipinski等人首次发现了人滋养层细胞表面抗原2(Human trophoblast antigen 2，以下表示为hTROP2，Lipinski等，PNAS.1981；78：5147-5150)。随后，该分子也被其他研究者发现，虽然当时也被称为能被将胃癌细胞株免疫而得到的小鼠单克隆抗体GA733识别的肿瘤抗原GA733-1(Linnenbach A J.等，PNAS.1989；86：27-31)、通过非小细胞肺癌细胞免疫而得到的小鼠单克隆抗体RS7-3G11识别的表皮糖蛋白(EGP-1，Basu A.等，Int.J.Cancer，1995；62:472-479)，但在1995年hTROP2基因被克隆后，确认它们为相同的分子(Fornaro M.等，Int.J.Cancer，1995；62：610-618)。When studying normal human trophoblast cells and cancerous trophoblast cells, M. Lipinski et al. first discovered the human trophoblast cell surface antigen 2 (Human trophoblast antigen 2, hereinafter referred to as hTROP2, Lipinski et al., PNAS.1981; 78: 5147-5150) ). Subsequently, the molecule was also discovered by other researchers, although it was also known as the tumor antigen GA733-1 recognized by the mouse monoclonal antibody GA733 obtained by immunizing gastric cancer cell lines (Linnenbach A J. et al., PNAS.1989) ; 86: 27-31), epidermal glycoprotein recognized by mouse monoclonal antibody RS7-3G11 obtained by non-small cell lung cancer cell immunization (EGP-1, Basu A. et al., Int. J. Cancer, 1995; 62 :472-479), but after the hTROP2 gene was cloned in 1995, it was confirmed that they were the same molecule (Fornaro M. et al., Int. J. Cancer, 1995; 62:610-618).
TROP2基因属于TACSTD基因家族，此家族的另一个成员编码与TROP2有约50％同源性的人滋养层细胞表面抗原1(hTROP1)。hTROP2为单次跨膜的I型细胞膜蛋白，其包含N末端26个氨基酸残基组成的胞内结构域，C末端248个氨基酸组成的胞外结构域，以及23个氨基酸残基组成的跨膜结构域，全长共323个氨基酸残基。已知hTROP2在第33、120、168和208位氨基酸残基处有4个N糖基化位点，表观分子量比理论分子量35千道尔顿大10千道尔顿左右。The TROP2 gene belongs to the TACSTD gene family. Another member of this family encodes human trophoblast cell surface antigen 1 (hTROP1), which has about 50% homology with TROP2. hTROP2 is a single-transmembrane type I cell membrane protein, which contains an intracellular domain composed of 26 amino acid residues at the N-terminal, an extracellular domain composed of 248 amino acids at the C-terminus, and a transmembrane composed of 23 amino acid residues. The domain has a total length of 323 amino acid residues. It is known that hTROP2 has 4 N glycosylation sites at amino acid residues 33, 120, 168 and 208, and the apparent molecular weight is about 10 kilodaltons larger than the theoretical molecular weight of 35 kilodaltons.
迄今为止，hTROP2蛋白的配体未有明确鉴定，其分子功能还不十分明确。有报道显示hTROP2胞内结构域具有PIP 2(4,5-二磷酸磷脂酰肌醇)结合序列，同时其第303位丝氨酸残基可被蛋白激酶C(PKC)磷酸化。第303位丝氨酸残基的磷酸化会造成hTROP2胞内构象的变化，导致与其结合的PIP 2暴露后被磷脂酶C(PLC)水解为IP 3(三磷酸肌醇)和DAG(二酰甘油)，进而调控细胞内钙信号传导(Miha Pavsic等，Scientific Reports，2015；5：10324)。 So far, the ligand of hTROP2 protein has not been clearly identified, and its molecular function is not very clear. Reports have shown that the intracellular domain of hTROP2 has a PIP 2 (phosphatidylinositol 4,5-bisphosphate) binding sequence, and its serine residue at position 303 can be phosphorylated by protein kinase C (PKC). The phosphorylation of serine residue at position 303 will cause changes in the intracellular conformation of hTROP2, resulting in the exposure of PIP 2 that binds to it and is hydrolyzed by phospholipase C (PLC) to IP 3 (inositol triphosphate) and DAG (diacylglycerol) , Thereby regulating intracellular calcium signal transduction (Miha Pavsic et al., Scientific Reports, 2015; 5: 10324).
通过使用临床标本进行的免疫组织化学分析表明，在正常组织中hTROP2仅在某些组织的上皮细胞有有限表达，与此形成鲜明对比的是，hTROP2在乳腺癌、宫颈癌、结直肠癌、食道癌、胃癌、肺癌、卵巢癌、前列腺癌、肾癌、胰腺癌等多种癌症种类中表 达过量，且有报道显示hTROP2表达水平与肿瘤恶性程度，肿瘤侵袭能力和病人预后有密切相关性(Anna Shvartsur等，Genes&Cancer，2015；6：84-105)。Immunohistochemical analysis using clinical specimens showed that in normal tissues, hTROP2 is only expressed in limited epithelial cells of certain tissues. In sharp contrast, hTROP2 is found in breast cancer, cervical cancer, colorectal cancer, and esophagus. Cancer, gastric cancer, lung cancer, ovarian cancer, prostate cancer, kidney cancer, pancreatic cancer and other cancer types are overexpressed, and reports have shown that hTROP2 expression levels are closely related to tumor malignancy, tumor invasion ability, and patient prognosis (Anna Shvartsur et al., Genes & Cancer, 2015; 6: 84-105).
抗体药物偶联物(Antibody drug conjugate,以下称为ADC)，是由抗体与高效小分子药物通过连接物偶联而成，能够使高毒性小分子药物特异识别癌细胞上的靶点蛋白，从而特异性杀死癌细胞。基于抗体的免疫疗法与基于化学药物的化学疗法，一直是临床上治疗癌症的两大策略。抗体以肿瘤细胞特异表达或过度表达的抗原为靶点，多种治疗性单抗已经在临床上取得了巨大成功。在临床实践中，治疗性抗体虽然具有很好的靶向性，但是杀伤作用存在局限性；小分子化学药物虽然具备对癌细胞的高效杀伤作用，但是对非癌细胞也会造成同样的伤害。抗体药物和小分子药物在临床上各自不同的局限性，对药物研发提出了新的要求。新一代ADC，利用抗体对靶细胞的特异结合能力，输送高细胞毒的化学药物，实现对癌细胞的靶向高效杀伤。随着新型化学连接技术的出现，ADC在八十年代末开始进入临床研究，目前已经有4个ADC经FDA批准上市。Antibody drug conjugate (hereinafter referred to as ADC) is formed by coupling an antibody and a high-efficiency small molecule drug through a linker, which enables highly toxic small molecule drugs to specifically recognize target proteins on cancer cells, thereby Kill cancer cells specifically. Antibody-based immunotherapy and chemical drug-based chemotherapy have always been two major strategies for the treatment of cancer in the clinic. Antibodies target tumor cells specifically expressed or overexpressed antigens, and a variety of therapeutic monoclonal antibodies have achieved great clinical success. In clinical practice, although therapeutic antibodies have good targeting properties, their killing effects have limitations; although small molecule chemical drugs have high-efficiency killing effects on cancer cells, they can also cause the same damage to non-cancer cells. Antibody drugs and small molecule drugs have different clinical limitations, which put forward new requirements for drug development. A new generation of ADCs uses the specific binding ability of antibodies to target cells to deliver highly cytotoxic chemical drugs to achieve targeted and efficient killing of cancer cells. With the emergence of new chemical connection technologies, ADCs began to enter clinical research in the late 1980s, and 4 ADCs have been approved by the FDA for marketing.
ADC的开发涉及：药物靶点的筛选、重组抗体的制备、连接物技术开发以及高细胞毒性化合物的筛选优化等几个方面。TROP2作为癌细胞特异高表达的蛋白，是一个极佳的ADC候选靶点。The development of ADC involves several aspects: the screening of drug targets, the preparation of recombinant antibodies, the development of linker technology, and the optimization of screening of highly cytotoxic compounds. TROP2, as a protein specifically and highly expressed in cancer cells, is an excellent candidate target for ADC.
发明内容Summary of the invention
本发明所要解决的技术问题是为了克服目前TROP2抗体的不足，提供一种亲和力高、特异性强的TROP2抗体及其制备方法和应用，所述的TROP2抗体与人源或食蟹猴源或小鼠源的TROP2蛋白具有高度亲和力。本发明还提供一种抗体药物偶联物，其包括所述的TROP2抗体和与其偶联的、具有抗肿瘤功能的小分子化合物，所述的抗体药物偶联物能够进入细胞，对TROP2表达阳性的细胞进行靶向性细胞毒杀伤作用，能够运用于治疗肿瘤等疾病的药物制备中。将本发明所述的TROP2抗体制备成人源化抗体后，依然能够与人源或食蟹猴源的TROP2蛋白具有高度亲和力，且能够很好的抑制肿瘤细胞的生长，对体重无显著影响。The technical problem to be solved by the present invention is to overcome the shortcomings of the current TROP2 antibody, and provide a TROP2 antibody with high affinity and strong specificity and its preparation method and application. The TROP2 antibody is compatible with human or cynomolgus source or small The mouse-derived TROP2 protein has high affinity. The present invention also provides an antibody-drug conjugate, which comprises the TROP2 antibody and a small molecule compound with anti-tumor function coupled to the TROP2 antibody. The antibody-drug conjugate can enter cells and is positive for TROP2 expression. The cells perform targeted cytotoxic killing and can be used in the preparation of drugs for the treatment of tumors and other diseases. After preparing the humanized antibody of the TROP2 antibody of the present invention, it can still have a high affinity with human or cynomolgus TROP2 protein, and can well inhibit the growth of tumor cells without significant influence on body weight.
本发明以hTROP2蛋白或者过表达hTROP2蛋白的重组细胞株作为免疫原，采用传统的杂交瘤制备技术(Kohler and Milstein，Nature，1975；256：495)，通过一系列的调整和改进，获得抗hTROP2的先导抗体。再通过对先导抗体的初步生产、纯化和检定，获得具备与hTROP2或cTROP2或mTROP2蛋白具有高度亲和力的TROP2抗体。然后通过分子生物学方法测序获知所得的TROP2抗体的重链可变区和轻链可变区的氨基酸序列。该抗体与小分子化合物如MMAF偶联得到抗体药物偶联物，所述抗体药物偶联物能够进入细胞，对TROP2表达阳性细胞有优异的细胞毒杀伤作用。The present invention uses hTROP2 protein or a recombinant cell strain overexpressing hTROP2 protein as the immunogen, adopts traditional hybridoma preparation technology (Kohler and Milstein, Nature, 1975; 256:495), and obtains anti-hTROP2 through a series of adjustments and improvements. The lead antibody. Through the preliminary production, purification and verification of the lead antibody, TROP2 antibodies with high affinity to hTROP2 or cTROP2 or mTROP2 proteins are obtained. Then the amino acid sequences of the heavy chain variable region and the light chain variable region of the obtained TROP2 antibody are obtained by sequencing by molecular biology methods. The antibody is coupled with a small molecule compound such as MMAF to obtain an antibody-drug conjugate, which can enter cells and has an excellent cytotoxic killing effect on TROP2-positive cells.
为解决上述技术问题，本发明提供一种分离的蛋白质，其包括TROP2抗体的重链可变区和/或轻链可变区，所述重链可变区包括重链CDR1、重链CDR2和重链CDR3中的一种或多种，和/或，所述轻链可变区包括轻链CDR1、轻链CDR2和轻链CDR3中的一种或多种，其中，所述重链CDR1的氨基酸序列如序列表中SEQ ID NO:2或SEQ ID NO:10；所述重链CDR2的氨基酸序列如序列表SEQ ID NO:3或SEQ ID NO:11所示；所述重链CDR3的氨基酸序列如序列表中SEQ ID NO:4或SEQ ID NO:12所示；In order to solve the above technical problems, the present invention provides an isolated protein comprising the heavy chain variable region and/or light chain variable region of the TROP2 antibody, the heavy chain variable region comprising heavy chain CDR1, heavy chain CDR2 and One or more of the heavy chain CDR3, and/or, the light chain variable region includes one or more of the light chain CDR1, light chain CDR2 and light chain CDR3, wherein the heavy chain CDR1 The amino acid sequence is shown in SEQ ID NO: 2 or SEQ ID NO: 10 in the sequence list; the amino acid sequence of the heavy chain CDR2 is shown in SEQ ID NO: 3 or SEQ ID NO: 11 in the sequence list; the amino acid of the heavy chain CDR3 The sequence is shown in SEQ ID NO: 4 or SEQ ID NO: 12 in the sequence list;
所述轻链CDR1的氨基酸序列如序列表中SEQ ID NO:6或SEQ ID NO:14所示；所述轻链CDR2的氨基酸序列如序列表中SEQ ID NO:7或SEQ ID NO:15所示；所述轻链CDR3的氨基酸序列如序列表中SEQ ID NO:8或SEQ ID NO:16所示；The amino acid sequence of the light chain CDR1 is shown in SEQ ID NO: 6 or SEQ ID NO: 14 in the sequence list; the amino acid sequence of the light chain CDR2 is shown in SEQ ID NO: 7 or SEQ ID NO: 15 in the sequence list. The amino acid sequence of the light chain CDR3 is shown in SEQ ID NO: 8 or SEQ ID NO: 16 in the sequence table;
或者，所述重链CDR1的氨基酸序列与如序列表中SEQ ID NO:2或SEQ ID NO:10所示的氨基酸序列至少有80％、85％、90％、92％、94％、95％、96％、97％、98％或99％的序列同源性的氨基酸序列所示；和/或，所述重链CDR2的氨基酸序列与如序列表中SEQ ID NO:3或SEQ ID NO:11所示的氨基酸序列至少有80％、85％、90％、92％、94％、95％、96％、97％、98％或99％的序列同源性的氨基酸序列所示；和/或，所述重链CDR3的氨基酸序列与如序列表中SEQ ID NO:4或SEQ ID NO:12所示的氨基酸序列至少有80％、85％、90％、92％、94％、95％、96％、97％、98％或99％的序列同源性的氨基酸序列所示；和/或，所述轻链CDR1的氨基酸序列与如序列表中SEQ ID NO:6或SEQ ID NO:14所示的氨基酸序列至少有80％、85％、90％、92％、94％、95％、96％、97％、98％或99％的序列同源性的氨基酸序列所示；和/或，所述轻链CDR2的氨基酸序列与如序列表中SEQ ID NO:7或SEQ ID NO:15所示的氨基酸序列至少有80％、85％、90％、92％、94％、95％、96％、97％、98％或99％的序列同源性的氨基酸序列所示；和/或，所述轻链CDR3的氨基酸序列与如序列表中SEQ ID NO:8或SEQ ID NO:16所示的氨基酸序列至少有80％、85％、90％、92％、94％、95％、96％、97％、98％或99％的序列同源性的氨基酸序列所示。所述“与如序列表中……所述的氨基酸序列至少有80％、85％、90％、92％、94％、95％、96％、97％、98％或99％的序列同源性的氨基酸序列为”在所述CDR的氨基酸序列上具有一个或多个氨基酸残基的取代、缺失或插入，并且保持或改善了所述蛋白质的功能。Alternatively, the amino acid sequence of the heavy chain CDR1 is at least 80%, 85%, 90%, 92%, 94%, 95% of the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 10 in the sequence table. , 96%, 97%, 98%, or 99% sequence homology amino acid sequence; and/or, the amino acid sequence of the heavy chain CDR2 is as shown in SEQ ID NO: 3 or SEQ ID NO: The amino acid sequence shown in 11 is shown in an amino acid sequence with at least 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98% or 99% sequence homology; and/ Or, the amino acid sequence of the heavy chain CDR3 is at least 80%, 85%, 90%, 92%, 94%, 95% of the amino acid sequence shown in SEQ ID NO: 4 or SEQ ID NO: 12 in the sequence table. , 96%, 97%, 98%, or 99% sequence homology; and/or, the amino acid sequence of the light chain CDR1 is as shown in SEQ ID NO: 6 or SEQ ID NO: The amino acid sequence shown in 14 is shown in an amino acid sequence with at least 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98% or 99% sequence homology; and/ Or, the amino acid sequence of the light chain CDR2 is at least 80%, 85%, 90%, 92%, 94%, 95% of the amino acid sequence shown in SEQ ID NO: 7 or SEQ ID NO: 15 in the sequence table. , 96%, 97%, 98%, or 99% sequence homology of the amino acid sequence; and/or, the amino acid sequence of the light chain CDR3 is the same as SEQ ID NO: 8 or SEQ ID NO: The amino acid sequence shown in 16 has at least 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, or 99% sequence homology. Said "has at least 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98% or 99% sequence homology to the amino acid sequence described in the sequence listing... A sexual amino acid sequence is "a substitution, deletion or insertion of one or more amino acid residues in the amino acid sequence of the CDR, and the function of the protein is maintained or improved.
较佳地，所述重链CDR2的氨基酸序列如序列表中SEQ ID NO:3所示氨基酸序列的第7位的G和/或第13位的G突变后的氨基酸序列所示。更佳地，所述重链CDR2的氨基酸序列如序列表中SEQ ID NO:3所示氨基酸序列的第7位的G和/或第13位的G突变为A的氨基酸序列所示；其氨基酸序列如SEQ ID NO:44、SEQ ID NO:30或SEQ ID NO:39所示。Preferably, the amino acid sequence of the heavy chain CDR2 is shown in the amino acid sequence after the G at position 7 and/or the G at position 13 of the amino acid sequence shown in SEQ ID NO: 3 in the sequence listing. More preferably, the amino acid sequence of the heavy chain CDR2 is shown in the amino acid sequence in which the G at position 7 and/or the G at position 13 is mutated to A in the amino acid sequence shown in SEQ ID NO: 3 in the sequence table; The sequence is shown in SEQ ID NO: 44, SEQ ID NO: 30 or SEQ ID NO: 39.
较佳地，所述重链CDR1的氨基酸序列如序列表SEQ ID NO:2所示，所述重链CDR2 的氨基酸序列如序列表SEQ ID NO:3所示，且所述重链CDR3的氨基酸序列如序列表SEQ ID NO:4所示。Preferably, the amino acid sequence of the heavy chain CDR1 is shown in SEQ ID NO: 2 in the sequence table, the amino acid sequence of the heavy chain CDR2 is shown in SEQ ID NO: 3 in the sequence table, and the amino acid of the heavy chain CDR3 The sequence is shown in SEQ ID NO: 4 in the Sequence Listing.
较佳地，所述重链CDR1的氨基酸序列如序列表SEQ ID NO:2所示，所述重链CDR2的氨基酸序列如序列表SEQ ID NO:44所示，且所述重链CDR3的氨基酸序列如序列表SEQ ID NO:4所示。Preferably, the amino acid sequence of the heavy chain CDR1 is shown in SEQ ID NO: 2 in the sequence listing, the amino acid sequence of the heavy chain CDR2 is shown in SEQ ID NO: 44 in the sequence listing, and the amino acid sequence of the heavy chain CDR3 is The sequence is shown in SEQ ID NO: 4 in the Sequence Listing.
较佳地，所述重链CDR1的氨基酸序列如序列表SEQ ID NO:2所示，所述重链CDR2的氨基酸序列如序列表SEQ ID NO:30所示，且所述重链CDR3的氨基酸序列如序列表SEQ ID NO:4所示。Preferably, the amino acid sequence of the heavy chain CDR1 is shown in SEQ ID NO: 2 in the sequence table, the amino acid sequence of the heavy chain CDR2 is shown in SEQ ID NO: 30 in the sequence table, and the amino acid of the heavy chain CDR3 The sequence is shown in SEQ ID NO: 4 in the Sequence Listing.
较佳地，所述重链CDR1的氨基酸序列如序列表SEQ ID NO:2所示，所述重链CDR2的氨基酸序列如序列表SEQ ID NO:39所示，且所述重链CDR3的氨基酸序列如序列表SEQ ID NO:4所示。Preferably, the amino acid sequence of the heavy chain CDR1 is shown in SEQ ID NO: 2 in the sequence listing, the amino acid sequence of the heavy chain CDR2 is shown in SEQ ID NO: 39 in the sequence listing, and the amino acid sequence of the heavy chain CDR3 is The sequence is shown in SEQ ID NO: 4 in the Sequence Listing.
较佳地，所述重链CDR1的氨基酸序列如序列表SEQ ID NO:10所示，所述重链CDR2的氨基酸序列如序列表SEQ ID NO:11所示，且所述重链CDR3的氨基酸序列如序列表SEQ ID NO:12所示；Preferably, the amino acid sequence of the heavy chain CDR1 is shown in SEQ ID NO: 10 in the sequence table, the amino acid sequence of the heavy chain CDR2 is shown in SEQ ID NO: 11 in the sequence table, and the amino acid of the heavy chain CDR3 The sequence is shown in SEQ ID NO: 12 in the sequence list;
较佳地，所述轻链CDR1的氨基酸序列如序列表SEQ ID NO:6所示，所述轻链CDR2的氨基酸序列如序列表SEQ ID NO:7所示，且所述轻链CDR3的氨基酸序列如序列表SEQ ID NO:8所示。Preferably, the amino acid sequence of the light chain CDR1 is shown in SEQ ID NO: 6 in the sequence list, the amino acid sequence of the light chain CDR2 is shown in SEQ ID NO: 7 in the sequence list, and the amino acid sequence of the light chain CDR3 The sequence is shown in SEQ ID NO: 8 in the sequence table.
较佳地，所述轻链CDR1的氨基酸序列如序列表SEQ ID NO:14所示，所述轻链CDR2的氨基酸序列如序列表SEQ ID NO:15所示，且所述轻链CDR3的氨基酸序列如序列表SEQ ID NO:16所示。Preferably, the amino acid sequence of the light chain CDR1 is shown in SEQ ID NO: 14 in the sequence listing, the amino acid sequence of the light chain CDR2 is shown in SEQ ID NO: 15 in the sequence listing, and the amino acid sequence of the light chain CDR3 is The sequence is shown in SEQ ID NO: 16 in the Sequence Listing.
在某一实施方案中，所述重链CDR1的氨基酸序列如序列表SEQ ID NO:2所示，所述重链CDR2的氨基酸序列如序列表SEQ ID NO:3所示，且所述重链CDR3的氨基酸序列如序列表SEQ ID NO:4所示；以及所述轻链CDR1的氨基酸序列如序列表SEQ ID NO:6所示，所述轻链CDR2的氨基酸序列如序列表SEQ ID NO:7所示，且所述轻链CDR3的氨基酸序列如序列表SEQ ID NO:8所示。在某一实施方案中，所述重链CDR1的氨基酸序列如序列表SEQ ID NO:2所示，所述重链CDR2的氨基酸序列如序列表SEQ ID NO:44所示，且所述重链CDR3的氨基酸序列如序列表SEQ ID NO:4所示；以及所述轻链CDR1的氨基酸序列如序列表SEQ ID NO:6所示，所述轻链CDR2的氨基酸序列如序列表SEQ ID NO:7所示，且所述轻链CDR3的氨基酸序列如序列表SEQ ID NO:8所示。在某一实施方案中，所述重链CDR1的氨基酸序列如序列表SEQ ID NO:2所示，所述重链CDR2的氨基酸序列如序列表SEQ ID NO:30所示，且所述重链CDR3的氨基酸序列如序列表SEQ ID NO:4所示；以及所述轻链CDR1的氨基酸序列如序列表SEQ ID NO:6 所示，所述轻链CDR2的氨基酸序列如序列表SEQ ID NO:7所示，且所述轻链CDR3的氨基酸序列如序列表SEQ ID NO:8所示。在某一实施方案中，所述重链CDR1的氨基酸序列如序列表SEQ ID NO:2所示，所述重链CDR2的氨基酸序列如序列表SEQ ID NO:39所示，且所述重链CDR3的氨基酸序列如序列表SEQ ID NO:4所示；以及所述轻链CDR1的氨基酸序列如序列表SEQ ID NO:6所示，所述轻链CDR2的氨基酸序列如序列表SEQ ID NO:7所示，且所述轻链CDR3的氨基酸序列如序列表SEQ ID NO:8所示。在某一实施方案中，所述重链CDR1的氨基酸序列如序列表SEQ ID NO:10所示，所述重链CDR2的氨基酸序列如序列表SEQ ID NO:11所示，且所述重链CDR3的氨基酸序列如序列表SEQ ID NO:12所示；以及所述轻链CDR1的氨基酸序列如序列表SEQ ID NO:14所示，所述轻链CDR2的氨基酸序列如序列表SEQ ID NO:15所示，且所述轻链CDR3的氨基酸序列如序列表SEQ ID NO:16所示。In a certain embodiment, the amino acid sequence of the heavy chain CDR1 is shown in SEQ ID NO: 2 in the sequence listing, and the amino acid sequence of the heavy chain CDR2 is shown in SEQ ID NO: 3 in the sequence listing, and the heavy chain The amino acid sequence of CDR3 is shown in SEQ ID NO: 4 in the Sequence Listing; and the amino acid sequence of the light chain CDR1 is shown in SEQ ID NO: 6 in the Sequence Listing, and the amino acid sequence of the light chain CDR2 is shown in SEQ ID NO: in the Sequence Listing. The amino acid sequence of the light chain CDR3 is shown in SEQ ID NO: 8 in the sequence listing. In a certain embodiment, the amino acid sequence of the heavy chain CDR1 is shown in SEQ ID NO: 2 in the sequence listing, and the amino acid sequence of the heavy chain CDR2 is shown in SEQ ID NO: 44 in the sequence listing, and the heavy chain The amino acid sequence of CDR3 is shown in SEQ ID NO: 4 in the Sequence Listing; and the amino acid sequence of the light chain CDR1 is shown in SEQ ID NO: 6 in the Sequence Listing, and the amino acid sequence of the light chain CDR2 is shown in SEQ ID NO: in the Sequence Listing. The amino acid sequence of the light chain CDR3 is shown in SEQ ID NO: 8 in the sequence listing. In a certain embodiment, the amino acid sequence of the heavy chain CDR1 is shown in SEQ ID NO: 2 in the sequence listing, and the amino acid sequence of the heavy chain CDR2 is shown in SEQ ID NO: 30 in the sequence listing, and the heavy chain The amino acid sequence of CDR3 is shown in SEQ ID NO: 4 in the sequence list; and the amino acid sequence of light chain CDR1 is shown in SEQ ID NO: 6 in the sequence list, and the amino acid sequence of light chain CDR2 is shown in SEQ ID NO: in the sequence list. The amino acid sequence of the light chain CDR3 is shown in SEQ ID NO: 8 in the sequence listing. In a certain embodiment, the amino acid sequence of the heavy chain CDR1 is shown in SEQ ID NO: 2 in the sequence listing, and the amino acid sequence of the heavy chain CDR2 is shown in SEQ ID NO: 39 in the sequence listing, and the heavy chain The amino acid sequence of CDR3 is shown in SEQ ID NO: 4 in the Sequence Listing; and the amino acid sequence of the light chain CDR1 is shown in SEQ ID NO: 6 in the Sequence Listing, and the amino acid sequence of the light chain CDR2 is shown in SEQ ID NO: in the Sequence Listing. The amino acid sequence of the light chain CDR3 is shown in SEQ ID NO: 8 in the sequence listing. In a certain embodiment, the amino acid sequence of the heavy chain CDR1 is shown in SEQ ID NO: 10 in the sequence listing, and the amino acid sequence of the heavy chain CDR2 is shown in SEQ ID NO: 11 in the sequence listing, and the heavy chain The amino acid sequence of CDR3 is shown in SEQ ID NO: 12 in the Sequence Listing; and the amino acid sequence of the light chain CDR1 is shown in SEQ ID NO: 14 in the Sequence Listing, and the amino acid sequence of the light chain CDR2 is shown in SEQ ID NO: in the Sequence Listing. 15 is shown, and the amino acid sequence of the light chain CDR3 is shown in SEQ ID NO: 16 in the sequence listing.
较佳地，所述重链可变区还包括重链可变区框架区。更佳地，所述重链可变区框架区为鼠抗体的重链可变区框架区或人抗体的重链可变区框架区或其回复突变；优选为IGHV1-69*08/JH6C(序列如SEQ ID NO:25所示)或IGHV3-7*01/JH4D(序列如SEQ ID NO:35所示)或在其基础上的回复突变。Preferably, the heavy chain variable region further includes a heavy chain variable region framework region. More preferably, the heavy chain variable region framework region is the heavy chain variable region framework region of a mouse antibody or the heavy chain variable region framework region of a human antibody or a back mutation thereof; preferably IGHV1-69*08/JH6C( The sequence is shown in SEQ ID NO: 25) or IGHV3-7*01/JH4D (the sequence is shown in SEQ ID NO: 35) or a back mutation based on it.
较佳地，所述轻链可变区还包括轻链可变区框架区。更佳地，所述轻链可变区框架区为鼠抗体的轻链可变区框架区或人抗体的轻链可变区框架区或其回复突变；优选为IGKV1-39*01/JK4(序列如SEQ ID NO:31所示)或IGKV1-39*01/JK1(序列如SEQ ID NO:40所示)或在其基础上的回复突变。Preferably, the light chain variable region further includes a light chain variable region framework region. More preferably, the light chain variable region framework region is the light chain variable region framework region of a mouse antibody or the light chain variable region framework region of a human antibody or its back mutation; preferably IGKV1-39*01/JK4( The sequence is shown in SEQ ID NO: 31) or IGKV1-39*01/JK1 (the sequence is shown in SEQ ID NO: 40) or a back mutation based on it.
较佳地，所述重链可变区的氨基酸序列如序列表SEQ ID NO:1、9、20、23、24、26-29、36-38或其突变的氨基酸序列中任一所示。Preferably, the amino acid sequence of the variable region of the heavy chain is shown in any one of SEQ ID NO: 1, 9, 20, 23, 24, 26-29, 36-38 or its mutant amino acid sequence in the sequence list.
较佳地，所述轻链可变区的氨基酸序列如序列表SEQ ID NO:5、13、32-34、41-43或其突变的氨基酸序列中任一所示。Preferably, the amino acid sequence of the light chain variable region is as shown in any one of SEQ ID NO: 5, 13, 32-34, 41-43 or its mutant amino acid sequence in the sequence listing.
所述突变在所述重链可变区或轻链可变区的氨基酸序列上具有一个或多个氨基酸残基的取代、缺失或插入，并且保持或改善了所述蛋白质的功能；优选地，所述突变的氨基酸序列与所述重链可变区或轻链可变区的氨基酸序列具有至少80％、85％、90％、90％、95％、96％、97％、98％更优选至少99％的序列同源性。例如，当所述的蛋白质为双特异性抗体且其中一个蛋白功能区为免疫球蛋白，另一个蛋白功能区为scFv，且所述scFv连接在所述免疫球蛋白的两条重链的C末端时，所述重链的C末端可以由K突变为A。The mutation has one or more amino acid residue substitutions, deletions or insertions in the amino acid sequence of the heavy chain variable region or the light chain variable region, and maintains or improves the function of the protein; preferably, The amino acid sequence of the mutation and the amino acid sequence of the heavy chain variable region or the light chain variable region have at least 80%, 85%, 90%, 90%, 95%, 96%, 97%, 98% more preferably At least 99% sequence homology. For example, when the protein is a bispecific antibody and one of the protein functional regions is an immunoglobulin, the other protein functional region is an scFv, and the scFv is connected to the C-terminus of the two heavy chains of the immunoglobulin When, the C-terminus of the heavy chain can be mutated from K to A.
较佳地，所述重链可变区的氨基酸序列如序列表SEQ ID NO:1、20、23、24、26-29任一所示，且所述轻链可变区的氨基酸序列如序列表SEQ ID NO:5、32-34任一所示。Preferably, the amino acid sequence of the heavy chain variable region is as shown in any one of SEQ ID NO: 1, 20, 23, 24, 26-29 in the sequence list, and the amino acid sequence of the light chain variable region is as follows The list SEQ ID NO: 5, 32-34 is shown in any one.
较佳地，所述重链可变区的氨基酸序列如序列表SEQ ID NO:9、36-38任一所示，且 所述轻链可变区的氨基酸序列如序列表SEQ ID NO:13、41-43任一所示。Preferably, the amino acid sequence of the heavy chain variable region is shown in any one of SEQ ID NO: 9 and 36-38 in the sequence listing, and the amino acid sequence of the light chain variable region is shown in the sequence listing SEQ ID NO: 13 , 41-43 shown in any one.
在某一实施方案中，所述重链可变区的氨基酸序列如序列表SEQ ID NO:1所示，且所述轻链可变区的氨基酸序列如序列表SEQ ID NO:5所示。在某一实施方案中，所述重链可变区的氨基酸序列如序列表SEQ ID NO:9所示，且所述轻链可变区的氨基酸序列如序列表SEQ ID NO:13所示。在某一实施方案中，所述重链可变区的氨基酸序列如序列表SEQ ID NO:20所示，且所述轻链可变区的氨基酸序列如序列表SEQ ID NO:5所示。在某一实施方案中，所述重链可变区的氨基酸序列如序列表SEQ ID NO:23所示，且所述轻链可变区的氨基酸序列如序列表SEQ ID NO:5所示。在某一实施方案中，所述重链可变区的氨基酸序列如序列表SEQ ID NO:24所示，且所述轻链可变区的氨基酸序列如序列表SEQ ID NO:5所示。在某一实施方案中，所述重链可变区的氨基酸序列如序列表SEQ ID NO:26所示，且所述轻链可变区的氨基酸序列如序列表SEQ ID NO:32所示。在某一实施方案中，所述重链可变区的氨基酸序列如序列表SEQ ID NO:27所示，且所述轻链可变区的氨基酸序列如序列表SEQ ID NO:32所示。在某一实施方案中，所述重链可变区的氨基酸序列如序列表SEQ ID NO:28所示，且所述轻链可变区的氨基酸序列如序列表SEQ ID NO:32所示。在某一实施方案中，所述重链可变区的氨基酸序列如序列表SEQ ID NO:29所示，且所述轻链可变区的氨基酸序列如序列表SEQ ID NO:32所示。在某一实施方案中，所述重链可变区的氨基酸序列如序列表SEQ ID NO:26所示，且所述轻链可变区的氨基酸序列如序列表SEQ ID NO:33所示。在某一实施方案中，所述重链可变区的氨基酸序列如序列表SEQ ID NO:27所示，且所述轻链可变区的氨基酸序列如序列表SEQ ID NO:33所示。在某一实施方案中，所述重链可变区的氨基酸序列如序列表SEQ ID NO:28所示，且所述轻链可变区的氨基酸序列如序列表SEQ ID NO:33所示。在某一实施方案中，所述重链可变区的氨基酸序列如序列表SEQ ID NO:29所示，且所述轻链可变区的氨基酸序列如序列表SEQ ID NO:33所示。在某一实施方案中，所述重链可变区的氨基酸序列如序列表SEQ ID NO:26所示，且所述轻链可变区的氨基酸序列如序列表SEQ ID NO:34所示。在某一实施方案中，所述重链可变区的氨基酸序列如序列表SEQ ID NO:27所示，且所述轻链可变区的氨基酸序列如序列表SEQ ID NO:34所示。在某一实施方案中，所述重链可变区的氨基酸序列如序列表SEQ ID NO:28所示，且所述轻链可变区的氨基酸序列如序列表SEQ ID NO:34所示。在某一实施方案中，所述重链可变区的氨基酸序列如序列表SEQ ID NO:29所示，且所述轻链可变区的氨基酸序列如序列表SEQ ID NO:34所示。在某一实施方案中，所述重链可变区的氨基酸序列如序列表SEQ ID NO:36所示，且所述轻链可变区的氨基酸序列如序列表SEQ ID NO:41所示。在某一实施方案中，所述重链可变区的氨基酸序列如序列表SEQ ID NO:37所示， 且所述轻链可变区的氨基酸序列如序列表SEQ ID NO:41所示。在某一实施方案中，所述重链可变区的氨基酸序列如序列表SEQ ID NO:38所示，且所述轻链可变区的氨基酸序列如序列表SEQ ID NO:41所示。在某一实施方案中，所述重链可变区的氨基酸序列如序列表SEQ ID NO:36所示，且所述轻链可变区的氨基酸序列如序列表SEQ ID NO:42所示。在某一实施方案中，所述重链可变区的氨基酸序列如序列表SEQ ID NO:37所示，且所述轻链可变区的氨基酸序列如序列表SEQ ID NO:42所示。在某一实施方案中，所述重链可变区的氨基酸序列如序列表SEQ ID NO:38所示，且所述轻链可变区的氨基酸序列如序列表SEQ ID NO:42所示。在某一实施方案中，所述重链可变区的氨基酸序列如序列表SEQ ID NO:36所示，且所述轻链可变区的氨基酸序列如序列表SEQ ID NO:43所示。在某一实施方案中，所述重链可变区的氨基酸序列如序列表SEQ ID NO:37所示，且所述轻链可变区的氨基酸序列如序列表SEQ ID NO:43所示。在某一实施方案中，所述重链可变区的氨基酸序列如序列表SEQ ID NO:38所示，且所述轻链可变区的氨基酸序列如序列表SEQ ID NO:43所示。In a certain embodiment, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 1 in the sequence listing, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 5 in the sequence listing. In a certain embodiment, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 9 in the sequence listing, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 13 in the sequence listing. In a certain embodiment, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 20 in the sequence listing, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 5 in the sequence listing. In a certain embodiment, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 23 in the sequence listing, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 5 in the sequence listing. In a certain embodiment, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 24 in the sequence listing, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 5 in the sequence listing. In a certain embodiment, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 26 in the sequence listing, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 32 in the sequence listing. In a certain embodiment, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 27 in the sequence listing, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 32 in the sequence listing. In a certain embodiment, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 28 in the sequence listing, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 32 in the sequence listing. In a certain embodiment, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 29 in the sequence listing, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 32 in the sequence listing. In a certain embodiment, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 26 in the sequence listing, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 33 in the sequence listing. In a certain embodiment, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 27 in the sequence listing, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 33 in the sequence listing. In a certain embodiment, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 28 in the sequence listing, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 33 in the sequence listing. In a certain embodiment, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 29 in the sequence listing, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 33 in the sequence listing. In a certain embodiment, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 26 in the sequence listing, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 34 in the sequence listing. In a certain embodiment, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 27 in the sequence listing, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 34 in the sequence listing. In a certain embodiment, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 28 in the sequence listing, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 34 in the sequence listing. In a certain embodiment, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 29 in the sequence listing, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 34 in the sequence listing. In a certain embodiment, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 36 in the sequence listing, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 41 in the sequence listing. In a certain embodiment, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 37 in the sequence listing, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 41 in the sequence listing. In a certain embodiment, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 38 in the sequence listing, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 41 in the sequence listing. In a certain embodiment, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 36 in the sequence listing, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 42 in the sequence listing. In a certain embodiment, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 37 in the sequence listing, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 42 in the sequence listing. In a certain embodiment, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 38 in the sequence listing, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 42 in the sequence listing. In a certain embodiment, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 36 in the sequence listing, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 43 in the sequence listing. In a certain embodiment, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 37 in the sequence listing, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 43 in the sequence listing. In a certain embodiment, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 38 in the sequence listing, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 43 in the sequence listing.
在某些实施例方案中，上述氨基酸序列的编号可以是分别如表1所示：In some embodiments, the numbers of the above amino acid sequences may be as shown in Table 1:
表1 TROP2抗体蛋白序列编号Table 1 Sequence number of TROP2 antibody protein
Figure PCTCN2020108720-appb-000001
Figure PCTCN2020108720-appb-000001
Figure PCTCN2020108720-appb-000002
Figure PCTCN2020108720-appb-000002
其中，表1中的数字即为序列表中的序列号，如11E8E6D11的重链可变区的氨基酸序列为SEQ ID NO:1，而11E8E6D11的重链可变区中CDR1的氨基酸序列为SEQ ID NO:2，重链CDR2的氨基酸序列为SEQ ID NO:3，重链CDR3的氨基酸序列为SEQ ID NO:4。其轻链可变区的氨基酸序列为SEQ ID NO:5，轻链CDR1的氨基酸序列为SEQ ID NO:6，轻链CDR2的氨基酸序列为SEQ ID NO:7，轻链CDR3的氨基酸序列为SEQ ID NO:8。Among them, the numbers in Table 1 are the sequence numbers in the sequence list. For example, the amino acid sequence of the heavy chain variable region of 11E8E6D11 is SEQ ID NO:1, and the amino acid sequence of CDR1 in the heavy chain variable region of 11E8E6D11 is SEQ ID NO: 2, the amino acid sequence of the heavy chain CDR2 is SEQ ID NO: 3, and the amino acid sequence of the heavy chain CDR3 is SEQ ID NO: 4. The amino acid sequence of the light chain variable region is SEQ ID NO: 5, the amino acid sequence of light chain CDR1 is SEQ ID NO: 6, the amino acid sequence of light chain CDR2 is SEQ ID NO: 7, and the amino acid sequence of light chain CDR3 is SEQ ID NO: 8.
较佳地，所述分离的蛋白质为人源化抗体，其包括人抗体可变区的构架区。所述的人源化抗体中包含的上述重链CDR1、重链CDR2、和重链CDR3中的一种或多种和/或上述的轻链CDR1、轻链CDR2、和轻链CDR3时，可以在这些CDR的基础上发生部分突变；所述突变在其氨基酸序列上具有一个或多个氨基酸残基的取代、缺失或插入，并且保持或改善了所述蛋白质的功能；所述突变的氨基酸序列优选与所述重链可变区或轻链可变区的氨基酸序列具有至少80％、85％、90％、92％、94％、95％、96％、97％、98％或99％的序列同源性。例如双特异性抗体中，当单链抗体(scFv)连接在免疫球蛋白的重链的C末端时，所述重链的C末端可以由K突变为A。更佳地，所述的人抗体可变区的构架区包括人抗体重链可变区的构架区和人抗体轻链可变区的构架区。Preferably, the isolated protein is a humanized antibody, which includes the framework regions of the variable region of a human antibody. When one or more of the aforementioned heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 and/or the aforementioned light chain CDR1, light chain CDR2, and light chain CDR3 contained in the humanized antibody, Partial mutations occur on the basis of these CDRs; the mutations have one or more amino acid residue substitutions, deletions or insertions in their amino acid sequence, and maintain or improve the function of the protein; the mutated amino acid sequence Preferably, the amino acid sequence of the heavy chain variable region or the light chain variable region has at least 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98% or 99% Sequence homology. For example, in a bispecific antibody, when a single-chain antibody (scFv) is attached to the C-terminus of the heavy chain of an immunoglobulin, the C-terminus of the heavy chain can be mutated from K to A. More preferably, the framework region of the variable region of the human antibody includes the framework region of the variable region of the human antibody heavy chain and the framework region of the variable region of the human antibody light chain.
较佳地，所述的分离的蛋白质还包括抗体重链恒定区和抗体轻链恒定区。所述的抗体重链恒定区优选为小鼠源抗体重链恒定区或人源抗体重链恒定区；所述的抗体轻链恒定区优选为小鼠源轻链抗体恒定区或人源抗体轻链恒定区。所述的抗体重链恒定区更优选为人源抗体重链恒定区，进一步优选人源IgG1、IgG2、IgG3或IgG4抗体重链恒定区；所述的抗体轻链恒定区优选为人源抗体轻链κ或λ链恒定区。所述的分离的蛋白质的重链可变区和轻链可变区与人源重链恒定区和人源轻链恒定区构成嵌合抗体全长蛋白。所述的抗体全长蛋白优选为IgG1、IgG2、IgG3或IgG4。Preferably, the isolated protein also includes an antibody heavy chain constant region and an antibody light chain constant region. The antibody heavy chain constant region is preferably a mouse antibody heavy chain constant region or a human antibody heavy chain constant region; the antibody light chain constant region is preferably a mouse light chain antibody constant region or a human antibody light chain constant region. Chain constant region. The antibody heavy chain constant region is more preferably a human antibody heavy chain constant region, further preferably a human IgG1, IgG2, IgG3 or IgG4 antibody heavy chain constant region; the antibody light chain constant region is preferably a human antibody light chain κ Or lambda chain constant region. The heavy chain variable region and the light chain variable region of the isolated protein, the human heavy chain constant region and the human light chain constant region constitute a chimeric antibody full-length protein. The full-length antibody protein is preferably IgG1, IgG2, IgG3 or IgG4.
所述的分离的蛋白质可为本领域常规的蛋白质，较佳地为抗体全长蛋白、抗原结合片段、双特异性抗体、多特异性抗体、单链抗体(single chain antibody fragment，scFv)、单域抗体(single domain antibody，sdAb)和单区抗体(single-domain antibody)中的一种或多种。所述的分离的蛋白质还可以较佳地为上述抗体所制得的单克隆抗体或多克隆 抗体，例如TROP2抗体；所述单克隆抗体可以由多种途径和技术进行研制，包括杂交瘤技术、噬菌体展示技术、单淋巴细胞基因克隆技术等，主流是通过杂交瘤技术从野生型或转基因小鼠制备单克隆抗体。所述的分离的蛋白质还可以较佳地为超人源化抗体。The isolated protein may be a conventional protein in the art, preferably full-length antibody protein, antigen-binding fragment, bispecific antibody, multispecific antibody, single chain antibody fragment (scFv), single chain antibody One or more of single domain antibody (sdAb) and single-domain antibody (single-domain antibody). The isolated protein may also preferably be a monoclonal antibody or a polyclonal antibody prepared from the above antibody, such as the TROP2 antibody; the monoclonal antibody can be developed by a variety of approaches and technologies, including hybridoma technology, Phage display technology, single lymphocyte gene cloning technology, etc., the mainstream is to prepare monoclonal antibodies from wild-type or transgenic mice through hybridoma technology. The isolated protein may also preferably be a superhumanized antibody.
所述的超人源化抗体为一种通过人源化抗体的制备方法所得的抗体。此方法不依赖于将人构架序列作为分析点，而是依赖于比较非人抗体的规范CDR结构类型和人抗体的CDR结构类型，尤其是人胚系序列所编码的人抗体，从中识别出可以得到适宜的人构架序列的候选的人抗体序列。例如，人残基可以置换CDRs中的非人残基。镶饰(veneering)的一个前提是鼠源抗体可变区的免疫原性起源于它的表面残基，且残基的运动性和溶剂的可及性是其成为抗原决定簇的基本条件。根据对现有的抗体晶体结构数据的分析结果统计，在序列配对位置上，人和鼠的抗体可变区残基的相对溶剂可及性分布的保真度达98％，这说明在异种间诱导免疫反应的残基是由其余的种特异性溶剂可及表面残基引起的。因此将鼠特异性表面残基换成人源性的，就可以模拟人源抗体的表面轮廓，逃避人体免疫系统的识别，达到人源化的目的。简单来说，镶饰基于通过用人氨基酸序列重建抗体的溶剂可及的表面来减少在啮齿类动物或其他非人抗体中的潜在免疫原性的氨基酸序列。参见Padlan(1991)Mol.Immunol.28:489-980。通过鉴定非人抗体中暴露在表面的溶剂可及性残基的外部构架区残基(所述残基不同于人抗体的构架区中相同位置上的那些残基)，并用人抗体中相应位置的氨基酸替换所鉴定的残基，以进行镶饰。即，镶饰的抗体，其表面残基主要是人源序列，而包裹在内部的残基主要是最初的鼠源序列。例如，根据Frankenstein方法，鉴定与相关的非人抗体的各构架区具有实质上的序列同源性的人构架区，并且将非人抗体的CDRs移植到这些不同的人构架区的复合物上。可以结合上述方法，以产生任何所需序列的抗TROP2抗体。The superhumanized antibody is an antibody obtained by a preparation method of humanized antibody. This method does not rely on the human framework sequence as the analysis point, but relies on the comparison of the canonical CDR structure type of non-human antibody and the CDR structure type of human antibody, especially the human antibody encoded by human germline sequence. Candidate human antibody sequences of suitable human framework sequences are obtained. For example, human residues can replace non-human residues in CDRs. A prerequisite for veneering is that the immunogenicity of the murine antibody variable region originates from its surface residues, and the mobility of residues and solvent accessibility are the basic conditions for it to become an antigenic determinant. According to the statistics of the analysis of the existing antibody crystal structure data, the fidelity of the relative solvent accessibility distribution of the variable region residues of the human and mouse antibodies in the sequence matching position reached 98%, which indicates that the fidelity of the heterogeneous The residues that induce the immune response are caused by the remaining species-specific solvent accessible surface residues. Therefore, replacing the mouse-specific surface residues with human-derived ones can simulate the surface contours of human antibodies, avoid recognition by the human immune system, and achieve the purpose of humanization. In simple terms, veneering is based on reducing the potential immunogenicity of amino acid sequences in rodent or other non-human antibodies by reconstructing the antibody's solvent accessible surface with human amino acid sequences. See Padlan (1991) Mol. Immunol. 28:489-980. By identifying the outer framework region residues of the solvent accessible residues exposed on the surface of the non-human antibody (the residues are different from those in the same position in the framework region of the human antibody), and using the corresponding position in the human antibody Replace the identified residues with the amino acids for veneering. That is, the surface residues of the veneered antibody are mainly human-derived sequences, while the interior residues are mainly the original murine-derived sequences. For example, according to the Frankenstein method, human framework regions having substantial sequence homology with each framework region of related non-human antibodies are identified, and the CDRs of the non-human antibody are grafted onto complexes of these different human framework regions. The above methods can be combined to generate anti-TROP2 antibodies of any desired sequence.
当所述的蛋白质为双特异性抗体时，其可以包括第一蛋白功能区和第二蛋白功能区。所述第一蛋白功能区可以为上述的蛋白质，其靶向结合TROP2；所述第二蛋白功能区为非靶向结合TROP2的蛋白质或者为同样靶向结合TROP2但非本发明所述的蛋白质。其中，所述第一蛋白功能区可以为免疫球蛋白，所述第二蛋白功能区可以为一个或多个scFv；或者，所述第二蛋白功能区可以为免疫球蛋白，所述第一蛋白功能区可以为一个或多个scFv。When the protein is a bispecific antibody, it may include a first protein functional region and a second protein functional region. The first protein functional region may be the aforementioned protein, which targets to bind TROP2; the second protein functional region is a protein that does not target to bind TROP2 or is a protein that also targets to bind to TROP2 but is not described in the present invention. Wherein, the first protein functional domain may be an immunoglobulin, and the second protein functional domain may be one or more scFv; or, the second protein functional domain may be an immunoglobulin, and the first protein The functional area can be one or more scFv.
所述的抗体全长蛋白可为本领域常规的抗体全长蛋白，其包括重链可变区、轻链可变区、重链恒定区和轻链恒定区。The antibody full-length protein may be a conventional antibody full-length protein in the art, which includes a heavy chain variable region, a light chain variable region, a heavy chain constant region, and a light chain constant region.
所述的单链抗体可为本领域常规的单链抗体，其包括重链可变区、轻链可变区和15～20个氨基酸的短肽。The single-chain antibody may be a conventional single-chain antibody in the art, which includes a heavy chain variable region, a light chain variable region, and a short peptide of 15-20 amino acids.
所述的抗原结合片段可为本领域常规的抗原结合片段，其包括轻链可变区、轻链恒 定区和重链恒定区的Fd段。较佳地，所述的抗原抗体结合域蛋白质片段为Fab和F(ab’) 2。 The antigen-binding fragment can be a conventional antigen-binding fragment in the art, which includes the Fd segment of the light chain variable region, the light chain constant region and the heavy chain constant region. Preferably, the antigen-antibody binding domain protein fragments are Fab and F(ab') 2 .
所述的单域抗体可为本领域常规的单域抗体，其包括重链可变区和重链恒定区。The single domain antibody may be a conventional single domain antibody in the art, which includes a heavy chain variable region and a heavy chain constant region.
所述的单区抗体可为本领域常规的单区抗体，其仅包括重链可变区。The single-domain antibody may be a conventional single-domain antibody in the art, which only includes the heavy chain variable region.
为解决上述技术问题，本发明还提供一种核酸，其编码上述的分离的蛋白质。In order to solve the above technical problems, the present invention also provides a nucleic acid, which encodes the above-mentioned isolated protein.
所述核酸的制备方法为本领域常规的制备方法，较佳地，包括以下的步骤：通过基因克隆技术获得编码上述蛋白质的核酸分子，或者通过人工全序列合成的方法得到编码上述蛋白质的核酸分子。The preparation method of the nucleic acid is a conventional preparation method in the art, preferably, it includes the following steps: obtaining a nucleic acid molecule encoding the above-mentioned protein through gene cloning technology, or obtaining a nucleic acid molecule encoding the above-mentioned protein through a method of artificial full sequence synthesis .
本领域技术人员知晓，编码上述蛋白质的氨基酸序列的碱基序列可以适当引入替换、缺失、改变、插入或增加来提供一个多聚核苷酸的同系物。本发明中多聚核苷酸的同系物可以通过对编码该蛋白序列基因的一个或多个碱基在保持抗体活性范围内进行替换、缺失或增加来制得。Those skilled in the art know that the base sequence encoding the amino acid sequence of the above-mentioned protein can be replaced, deleted, changed, inserted or added appropriately to provide a polynucleotide homolog. The polynucleotide homologues of the present invention can be prepared by replacing, deleting or adding one or more bases of the gene encoding the protein sequence within the scope of maintaining antibody activity.
为解决上述技术问题，本发明还提供一种包含所述核酸的重组表达载体。To solve the above technical problems, the present invention also provides a recombinant expression vector containing the nucleic acid.
其中所述重组表达载体可通过本领域常规方法获得，即：将本发明所述的核酸分子连接于各种表达载体上构建而成。所述的表达载体为本领域常规的各种载体，只要其能够容载前述核酸分子即可。所述载体较佳地包括：各种质粒、粘粒、噬菌体或病毒载体等。The recombinant expression vector can be obtained by conventional methods in the art, that is, the nucleic acid molecule of the present invention is connected to various expression vectors to be constructed. The expression vector is a variety of conventional vectors in the field, as long as it can hold the aforementioned nucleic acid molecule. Said vectors preferably include: various plasmids, cosmids, phage or virus vectors and the like.
为解决上述技术问题，本发明还提供一种转化体，其在宿主细胞中包含上述的重组表达载体。In order to solve the above technical problems, the present invention also provides a transformant which contains the above-mentioned recombinant expression vector in a host cell.
其中，所述重组表达转化体的制备方法为本领域常规的制备方法，较佳地为：将上述重组表达载体转化至宿主细胞中制得。所述的宿主细胞为本领域常规的各种宿主细胞，只要能满足使上述重组表达载体稳定地自行复制，且所携带所述的核酸可被有效表达即可。较佳地，所述宿主细胞为E.coli TG1或BL21细胞(表达单链抗体或Fab抗体)，或者CHO-K1细胞(表达全长IgG抗体)。将前述重组表达质粒转化至宿主细胞中，即可得本发明优选的重组表达转化体。其中所述转化方法为本领域常规转化方法，较佳地为化学转化法，热激法或电转法。Wherein, the preparation method of the recombinant expression transformant is a conventional preparation method in the art, preferably: the recombinant expression vector is transformed into a host cell. The host cell is a variety of conventional host cells in the field, as long as the recombinant expression vector can replicate itself stably and the nucleic acid carried can be effectively expressed. Preferably, the host cell is E. coli TG1 or BL21 cell (expressing single-chain antibody or Fab antibody), or CHO-K1 cell (expressing full-length IgG antibody). The aforementioned recombinant expression plasmid is transformed into a host cell to obtain the preferred recombinant expression transformant of the present invention. The transformation method is a conventional transformation method in the field, preferably a chemical transformation method, a heat shock method or an electrotransformation method.
本发明中，所述的分离的蛋白质可以用于制备成嵌合抗原受体(CAR)等从而将其修饰在例如T细胞或NK细胞等细胞上面。所述嵌合抗原受体可为本领域常规的嵌合抗原受体，包括例如利用上述TROP2抗体的scFv作为胞外抗原结合结构域的嵌合抗原受体。因此，本发明还提供一种基因修饰的细胞，其包含上述的分离的蛋白质，较佳地，所述基因修饰的细胞为真核细胞，优选分离的人细胞；更优选免疫细胞如T细胞(例如以CAR-T的形式)，或NK细胞。In the present invention, the isolated protein can be used to prepare a chimeric antigen receptor (CAR) and the like to modify it on cells such as T cells or NK cells. The chimeric antigen receptor may be a conventional chimeric antigen receptor in the art, including, for example, a chimeric antigen receptor that uses the scFv of the above-mentioned TROP2 antibody as an extracellular antigen binding domain. Therefore, the present invention also provides a genetically modified cell comprising the aforementioned isolated protein. Preferably, the genetically modified cell is a eukaryotic cell, preferably an isolated human cell; more preferably an immune cell such as a T cell ( For example, in the form of CAR-T), or NK cells.
为解决上述技术问题，本发明还提供一种分离的蛋白质的制备方法，其包括如下步 骤：培养上述的转化体，从培养物中获得分离的蛋白质。In order to solve the above technical problem, the present invention also provides a method for preparing an isolated protein, which includes the following steps: culturing the above-mentioned transformant to obtain the isolated protein from the culture.
为解决上述技术问题，本发明提供一种抗体药物偶联物，其包括共价附着至细胞毒剂的上述的分离的蛋白质。In order to solve the above technical problems, the present invention provides an antibody drug conjugate comprising the above-mentioned isolated protein covalently attached to a cytotoxic agent.
较佳地，所述的抗体药物偶联物中，上述的1当量所述的分离的蛋白质通过x当量接头与y当量的细胞毒剂相连，具有如式1所示的结构，Preferably, in the antibody drug conjugate, the above-mentioned 1 equivalent of the isolated protein is connected to the y equivalent of the cytotoxic agent through an x equivalent linker, and has the structure shown in Formula 1,
Ab-(L) x-(D) y Ab-(L) x -(D) y
式1 Formula 1
其中，Ab为上述的分离的蛋白质；L为接头；D为细胞毒剂；所述x为本领域常规的交联度，x为自然数，优选1-20的整数；y为自然数，优选1-20的整数；x和y各自独立地优选为2～8的整数，例如为3或4；x和y的比例优选为1:1。Wherein, Ab is the aforementioned isolated protein; L is a linker; D is a cytotoxic agent; the x is a conventional degree of cross-linking in the art, x is a natural number, preferably an integer of 1-20; y is a natural number, preferably 1-20 X and y are each independently an integer of 2-8, for example, 3 or 4; the ratio of x and y is preferably 1:1.
所述L是本领域常规的接头(或称交联剂或偶联剂)。所述L包含2个官能团，即与抗体反应的基团，和与药物反应的基团(例如，醛或酮)。The L is a conventional linker (also called a crosslinking agent or coupling agent) in the art. The L includes two functional groups, that is, a group that reacts with antibodies, and a group that reacts with drugs (for example, aldehydes or ketones).
药物经由接头分子与上述的蛋白质偶联。所述L进入细胞后释放，其包括但不限于如下的官能团，活性酯、碳酸盐类、氨基甲酸酯类、亚胺磷酸酯、肟类、腙类、缩醛类、原酸酯类、氨基类、小肽段或核苷酸片段。The drug is coupled to the aforementioned protein via a linker molecule. The L is released after entering the cell, which includes but is not limited to the following functional groups, active esters, carbonates, carbamates, phosphoimidites, oximes, hydrazones, acetals, orthoesters, and amino groups Class, small peptide or nucleotide fragment.
较佳地，所述L主要含有式2所示结构，其为L中离去基团离去后对应的剩余部分；Preferably, said L mainly contains the structure represented by formula 2, which is the remaining part corresponding to the leaving group in L;
(CO-Alk 1-Sp 1-Ar-Sp 2-Alk 2-C(Z 1)＝Q-Sp) (CO-Alk 1 -Sp 1 -Ar-Sp 2 -Alk 2 -C(Z 1 )=Q-Sp)
式2 Formula 2
其中，Alk 1和Alk 2不存在或独立地为分支的或不分支的(C 1-C 10)亚烷基链；Sp 1不存在，或独立地为-S-、-O-、-CONH-、-NHCO-、-NR’-、-N(CH 2CH 2) 2N-、或-X-Ar’-Y-(CH 2) n-Z，其中X、Y和Z不存在，或独立地为-NR’-、-S-或-O-，条件是当n＝0时，Y和Z中的至少一个不存在，且Ar’是由(C 1-C 5)烷基、(C 1-C 4)烷氧基、(C 1-C 4)硫代烷氧基、卤素、硝基、-COOR’、-CONHR’、-(CH 2) nCOOR’、S(CH 2) nCOOR’、-O(CH 2) nCONHR’或-S(CH 2) nCONHR’的1、2或3个基团任选取代的基团，当其为2个基团任选取代时其为1，2-、1，3-或1，4-亚苯基，n是0-5的整数，条件是当Alk 1不存在时，Sp 1不存在；R’是由-OH、(C 1-C 4)烷氧基、(C 1-C 4)硫代烷氧基、卤素、硝基、(C 1-C 3)二烷基氨基、或(C 1-C 3)三烷基铵-A的一个或2个基团任选取代的分支的或不分支的(C 1-C 5)链，其中A是完成盐的药学上可接受的阴离子；Ar是由(C 1-C 6)烷基、(C 1-C 5)烷氧基、(C 1-C 4)硫代烷氧基、卤素、硝基、-COOR’、-CONHR’、-O(CH 2) nCOOR’、-S(CH 2) nCOOR’、-O(CH 2) nCONHR’或-S(CH 2) nCONHR’的1、2或3个基团任选取代的1,2-、1,3-或1,4-亚苯基，其中n和R’如上述的定义，或Ar是1,2-、1,3-、1,4-、1,5-、1,6-、1,7-、1,8-、2,3-、2,6-或2,7-亚萘基，其中亚萘基或吩噻嗪各任选 地由(C 1-C 6)烷基、(C 1-C 5)烷氧基、(C 1-C 4)硫代烷氧基、卤素、硝基、-COOR’、-CONHR’、-O(CH 2) nCOOR’、-S(CH 2) nCOOR’、或-S(CH 2) nCONHR’的1、2、3或4个基团取代，其中n和R’如上文定义，条件是当Ar是吩噻嗪时，Sp 1是仅与氮连接的键；所述的键的含义为共价键。 Among them, Alk 1 and Alk 2 do not exist or are independently branched or unbranched (C 1 -C 10 ) alkylene chains; Sp 1 does not exist, or independently is -S-, -O-, -CONH -, -NHCO-, -NR'-, -N(CH 2 CH 2 ) 2 N-, or -X-Ar'-Y-(CH 2 ) n -Z, where X, Y and Z are not present, or Independently -NR'-, -S- or -O-, provided that when n=0, at least one of Y and Z does not exist, and Ar' is composed of (C 1 -C 5 )alkyl, ( C 1 -C 4 )alkoxy, (C 1 -C 4 )thioalkoxy, halogen, nitro, -COOR', -CONHR', -(CH 2 ) n COOR', S(CH 2 ) n COOR', -O(CH 2 ) n CONHR' or -S(CH 2 ) n CONHR' group optionally substituted by 1, 2 or 3 groups, when 2 groups are optionally substituted It is 1,2-, 1,3- or 1,4-phenylene, n is an integer of 0-5, provided that when Alk 1 does not exist, Sp 1 does not exist; R'is composed of -OH, ( C 1 -C 4 )alkoxy, (C 1 -C 4 )thioalkoxy, halogen, nitro, (C 1 -C 3 )dialkylamino, or (C 1 -C 3 )trialkane A branched or unbranched (C 1 -C 5 ) chain optionally substituted by one or two groups of ammonium-A, wherein A is the pharmaceutically acceptable anion of the complete salt; Ar is composed of (C 1- C 6 )alkyl, (C 1 -C 5 )alkoxy, (C 1 -C 4 )thioalkoxy, halogen, nitro, -COOR', -CONHR', -O(CH 2 ) n 1, 2 or 3 groups of COOR', -S(CH 2 ) n COOR', -O(CH 2 ) n CONHR' or -S(CH 2 ) n CONHR' optionally substituted 1,2-, 1,3- or 1,4-phenylene, where n and R'are as defined above, or Ar is 1,2-, 1,3-, 1,4-, 1,5-, 1,6- , 1,7-, 1,8-, 2,3-, 2,6-or 2,7-naphthylene, wherein naphthylene or phenothiazine is each optionally composed of (C 1 -C 6 ) alkyl Group, (C 1 -C 5 )alkoxy, (C 1 -C 4 )thioalkoxy, halogen, nitro, -COOR', -CONHR', -O(CH 2 ) n COOR',- S(CH 2 ) n COOR' or -S(CH 2 ) n CONHR' is substituted by 1, 2, 3 or 4 groups, where n and R'are as defined above, provided that Ar is phenothiazine , Sp 1 is a bond connected only to nitrogen; the meaning of the bond is a covalent bond.
Sp 2不存在，或为-S-或-O-，条件是当Alk 2不存在时，Sp 2不存在； Sp 2 does not exist, or -S- or -O-, provided that when Alk 2 does not exist, Sp 2 does not exist;
Z 1是H、(C 1-C 5)烷基、或由(C 1-C 5)烷基、(C 1-C 5)烷氧基、(C 1-C 4)硫代烷氧基、卤素、硝基、-COOR’、-CONHR’、-O(CH 2) nCOOR’、-S(CH 2) nCOOR’、-O(CH 2) nCONHR’或-S(CH 2) nCONHR’的1、2、或3个基团任选取代的苯基，其中n和R’如上文定义； Z 1 is H, (C 1 -C 5 )alkyl, or consists of (C 1 -C 5 )alkyl, (C 1 -C 5 )alkoxy, (C 1 -C 4 )thioalkoxy , Halogen, nitro, -COOR', -CONHR', -O(CH 2 ) n COOR', -S(CH 2 ) n COOR', -O(CH 2 ) n CONHR' or -S(CH 2 ) An optionally substituted phenyl group of 1, 2, or 3 groups of n CONHR', wherein n and R'are as defined above;
Sp是直链或支链二价或三价(C 1-C 18)基团，二价或三价芳基或杂芳基基团，二价或三价(C 3-C 18)环烷基或杂环烷基基团，二价或三价芳基或杂芳基-芳基(C 1-C 18)基团，二价或三价环烷基或杂环烷基-烷基(C 1-C 18)基团，或二价或三价(C 2-C 18)不饱和的烷基基团，其中杂芳基优选是呋喃基、噻吩基，N-甲基吡咯基、吡啶基、N-甲基咪唑基、噁唑基、嘧啶基。喹啉基、异喹啉基、N-甲基咔唑基、氨基豆素基、或吩嗪基、并且其中如果Sp是三价基团，那么Sp还可以由低级(C 1-C 5)二烷基氨基、低级(C 1-C 5)烷氧基、羟基、或低级(C 1-C 5)烷硫基任选取代；且，Q是＝NHNCO-、＝NHNCS-、＝NHNCONH-、＝NHNCSNH-或＝NHO-。 Sp is a straight or branched divalent or trivalent (C 1 -C 18 ) group, a divalent or trivalent aryl or heteroaryl group, a divalent or trivalent (C 3 -C 18 ) cycloalkane Group or heterocycloalkyl group, divalent or trivalent aryl or heteroaryl-aryl (C 1 -C 18 ) group, divalent or trivalent cycloalkyl or heterocycloalkyl-alkyl ( C 1 -C 18 ) group, or divalent or trivalent (C 2 -C 18 ) unsaturated alkyl group, wherein the heteroaryl group is preferably furyl, thienyl, N-methylpyrrolyl, pyridine Group, N-methylimidazolyl, oxazolyl, pyrimidinyl. Quinolinyl, isoquinolinyl, N-methylcarbazolyl, amino stigmainyl, or phenazinyl, and if Sp is a trivalent group, then Sp can also be composed of lower (C 1 -C 5 ) Dialkylamino, lower (C 1 -C 5 )alkoxy, hydroxy, or lower (C 1 -C 5 )alkylthio are optionally substituted; and, Q is =NHNCO-, =NHNCS-, =NHNCONH- , =NHNCSNH- or =NHO-.
优选地，Alk 1是分支或不分支的(C 1-C 5)亚烷基链，Sp 1不存在，或为-S-、-O-、-CONH-、-NHCO-或-NR’，其中R’如上文定义，条件是当Alk 1不存在时，Sp 1不存在； Preferably, Alk 1 is a branched or unbranched (C 1 -C 5 ) alkylene chain, Sp 1 is not present, or is -S-, -O-, -CONH-, -NHCO- or -NR', Wherein R'is as defined above, and the condition is that when Alk 1 does not exist, Sp 1 does not exist;
Ar是由(C 1-C 6)烷基、(C 1-C 5)烷氧基、(C 1-C 4)硫代烷氧基、卤素、硝基、-COOR’、-CONHR’、-O(CH 2) nCOOR’、-S(CH 2) nCOOR’、-O(CH 2) nCONHR’或-S(CH 2) nCONHR’的1、2或3个基团任选取代的1,2-、1,3-或1,4-亚苯基，其中n和R’如上文定义，或Ar是各自由C 1-C 6)烷基、(C 1-C 5)烷氧基、(C 1-C 4)硫代烷氧基、卤素、硝基、-COOR’、-CONHR’、-O(CH 2) nCOOR’、-S(CH 2) nCOOR’、-O(CH 2) nCONHR’或-S(CH 2) nCONHR’的1、2、3或4个基团任选取代的1,2-、1,3-、1,4-、1,5-、1,6-、1,7-、1,8-、2,3-、2,6-或2,7-亚萘基。 Ar is composed of (C 1 -C 6 ) alkyl, (C 1 -C 5 ) alkoxy, (C 1 -C 4 ) thioalkoxy, halogen, nitro, -COOR', -CONHR', -O(CH 2 ) n COOR', -S(CH 2 ) n COOR', -O(CH 2 ) n CONHR' or -S(CH 2 ) n CONHR' 1, 2 or 3 groups are optional Substituted 1,2-, 1,3- or 1,4-phenylene, wherein n and R'are as defined above, or Ar is each composed of C 1 -C 6 )alkyl, (C 1 -C 5 ) Alkoxy, (C 1 -C 4 )thioalkoxy, halogen, nitro, -COOR', -CONHR', -O(CH 2 ) n COOR', -S(CH 2 ) n COOR', 1, 2, 3 or 4 groups of -O(CH 2 ) n CONHR' or -S(CH 2 ) n CONHR' optionally substituted 1,2-, 1,3-, 1,4-, 1 ,5-, 1,6-, 1,7-, 1,8-, 2,3-, 2,6-or 2,7-naphthylene.
Z 1是(C 1-C 5)烷基、或由(C 1-C 5)烷基、(C 1-C 4)烷氧基、(C 1-C 4)硫代烷氧基、卤素、硝基、-COOR’、-CONHR’、-O(CH 2) nCOOR’、-S(CH 2) nCOOR’、-O(CH 2) nCONHR’或-S(CH 2) nCONHR’的1、2、或3个基团任选取代的苯基；Alk 2和Sp 2均不存在；且Sp和Q如仅在上文中所定义的。 Z 1 is (C 1 -C 5 ) alkyl, or consists of (C 1 -C 5 ) alkyl, (C 1 -C 4 ) alkoxy, (C 1 -C 4 ) thioalkoxy, halogen , Nitro, -COOR', -CONHR', -O(CH 2 ) n COOR', -S(CH 2 ) n COOR', -O(CH 2 ) n CONHR' or -S(CH 2 ) n CONHR The 1, 2, or 3 groups of 'are optionally substituted phenyl; Alk 2 and Sp 2 are not present; and Sp and Q are as defined only above.
所述L优选为马来酰亚胺基己酰(maleimidocaproyl，MC)、马来酰亚胺基己酰-L-缬氨酸-L-瓜氨酸对氨基苄醇(MC-VC-PAB)或4-(N-马来酰亚胺基甲基)环己烷-1-羧酸琥 珀酰亚胺酯(SMCC)。The L is preferably maleimidocaproyl (maleimidocaproyl, MC), maleimidocaproyl-L-valine-L-citrulline-aminobenzyl alcohol (MC-VC-PAB) Or 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid succinimide ester (SMCC).
所述D为本领域常规的细胞毒剂，较佳地选自细胞毒素、化学治疗剂、放射性同位素、治疗性核酸、免疫调节剂、抗血管生成剂、抗增殖促凋亡剂或细胞溶解酶。Said D is a conventional cytotoxic agent in the art, preferably selected from cytotoxins, chemotherapeutic agents, radioisotopes, therapeutic nucleic acids, immunomodulators, anti-angiogenesis agents, anti-proliferation and pro-apoptotic agents or cytolytic enzymes.
其中，所述细胞毒素为本领域常规的细胞毒素，一般指抑制或阻止细胞功能和/或导致细胞破坏的活性剂。较佳地选自抗生素、微管蛋白聚合的抑制剂、烷化剂、蛋白合成抑制剂、蛋白激酶抑制剂、磷酸酶抑制剂、拓扑异构酶抑制剂、蛋白激酶、磷酸酶、拓扑异构酶或细胞周期蛋白。更佳地选自多柔比星、柔红霉素、依达比星、阿柔比星、佐柔比星、米托蒽醌、表柔比星、卡柔比星、诺加霉素、美诺立尔、吡柔比星、戊柔比星、阿糖胞苷、吉西他滨、曲氟尿苷、安西他滨、依诺他滨、阿扎胞苷、去氧氟尿苷、喷司他丁、溴尿苷、卡培他滨、克拉屈滨、地西他滨、氟尿苷、氟达拉滨、谷氏菌素、嘌呤霉素、替加氟、噻唑羧胺核苷、阿霉素、顺铂、卡铂、环磷酰胺、达卡巴嗪、长春碱、长春新碱、博来霉素、氮芥、强的松、甲基苄肼、氨甲喋呤、氟尿嘧啶、依托泊苷、泰素、泰素类似物、铂类(如顺铂和卡铂)、丝裂霉素、噻替派、紫杉烷、道诺红菌素、放线菌素、安曲霉素、氮丝氨酸、它莫西芬、多拉司他汀、奥瑞他汀及其衍生物、哈米特林、埃斯波霉素或美登素类化合物，最佳地选自甲基奥瑞他汀E(MMAE)、甲基奥瑞他汀F(MMAF)或N2’-脱乙酰-N2’-3-巯基-1氧代丙基-美登素(DM1)。Wherein, the cytotoxin is a conventional cytotoxin in the art, and generally refers to an active agent that inhibits or prevents cell function and/or causes cell destruction. Preferably selected from antibiotics, inhibitors of tubulin polymerization, alkylating agents, protein synthesis inhibitors, protein kinase inhibitors, phosphatase inhibitors, topoisomerase inhibitors, protein kinases, phosphatases, topoisomers Enzymes or cyclins. More preferably selected from doxorubicin, daunorubicin, idarubicin, arubicin, zorubicin, mitoxantrone, epirubicin, carrubicin, nogamycin, Menoril, Pirubicin, Valrubicin, Cytarabine, Gemcitabine, Trifluridine, Ancitabine, Enoxabine, Azacitidine, Deoxyfluridine, Pentrestat Butyl, bromouridine, capecitabine, cladribine, decitabine, fluridine, fludarabine, glutamicin, puromycin, tegafur, thiazole carboxamide nucleoside, adriamycin Vitamins, cisplatin, carboplatin, cyclophosphamide, dacarbazine, vinblastine, vincristine, bleomycin, nitrogen mustard, prednisone, procarbazine, methotrexate, fluorouracil, etoposide, taxol , Taxol analogues, platinums (such as cisplatin and carboplatin), mitomycin, thiatepa, taxane, daunorubicin, actinomycin, antoxin, azserine, it Moxifen, Dolastatin, Auristatin and its derivatives, Hamitlin, Espamycin or maytansine compounds, preferably selected from methyl auristatin E (MMAE), methyl Auristatin F (MMAF) or N2'-deacetyl-N2'-3-mercapto-1oxopropyl-maytansine (DM1).
其中，所述化学治疗剂为本领域常规的化学治疗剂，较佳地选自烷化剂、烷基磺酸酯类化学治疗剂、氮丙啶类化学治疗剂、乙烯酰胺类和甲基密胺类化学治疗剂、氮芥、硝基脲类化学治疗剂、抗生素、抗代谢物、叶酸类化学治疗剂、嘌呤类似物、嘧啶类似物、雄激素、抗肾上腺素、叶酸补充剂、美登醇、多糖复合物、紫杉烷、铂类似物或类视黄醇，或者，其在药学上可接受的盐、酸和衍生物。Wherein, the chemotherapeutic agent is a conventional chemotherapeutic agent in the art, preferably selected from alkylating agents, alkyl sulfonate chemotherapeutics, aziridine chemotherapeutics, vinyl amides and methyl methacrylates. Amine chemotherapeutics, nitrogen mustard, nitrourea chemotherapeutics, antibiotics, antimetabolites, folic acid chemotherapeutics, purine analogs, pyrimidine analogs, androgens, antiadrenaline, folic acid supplements, maytans Alcohols, polysaccharide complexes, taxanes, platinum analogs, or retinoids, or pharmaceutically acceptable salts, acids and derivatives thereof.
所述的烷化剂为本领域常规的烷化剂，较佳地选自噻替派或环磷酰胺。所述的烷基磺酸酯类化学治疗剂为本领域常规的烷基磺酸酯类化学治疗剂，较佳地选自白消安、英丙舒凡或哌泊舒凡。所述的氮丙啶类化学治疗剂为本领域常规的氮丙啶类化学治疗剂，较佳地选自氮丙唳如、卡巴醌、美妥替哌或乌瑞替派。所述乙烯酰胺类和甲基密胺类化学治疗剂为本领域常规的乙烯酰胺类和甲基密胺类化学治疗剂，较佳地选自六甲蜜胺、三乙撑蜜胺、三亚乙基磷酰胺，三亚乙基硫代磷酰胺或三羟甲蜜胺。所述的氮芥为本领域常规的氮芥，较佳地选自苯丁酸氮芥、萘氮芥、雌氮芥(estramustine)、异环磷酰胺、氮芥、氧氮芥盐酸盐、苯丙氨酸氮芥、新氮芥、苯芥胆甾醇、泼尼氮芥、曲磷胺或尿嘧啶氮芥。所述硝基脲类化学治疗剂为本领域常规的硝基脲类化学治疗剂，较佳地选自卡莫司汀、氯脲菌素、福莫司汀、洛莫司汀、尼莫司汀或雷莫司汀。所述抗生素为本领域常规的抗生素，较佳地选自阿克拉霉素、放线菌素、安曲霉素、氮丝氨酸、博来霉素、 放线菌素c、加力车霉素、卡柔比星、洋红霉素、嗜癌素、色霉素、更生霉素、柔红霉素、地托比星、6-重氮基-5-氧代-L-正亮氨酸、多柔比星、表柔比星、依索比星、依达比星、发波霉素、丝裂霉素、霉酚酸、诺加霉素、橄榄霉素、培洛霉素、紫菜霉素、嘌呤霉素、三铁阿霉素、罗多比星、链黑菌素、链脲菌素、杀结核菌素、乌苯美司、静司他丁或佐柔比星。所述的抗代谢物为本领域常规的抗代谢物，较佳地选自氨甲喋呤或5-氟尿嘧啶(5-FU)。所述的叶酸类化学治疗剂为本领域常规的叶酸类化学治疗剂，较佳地选自二甲叶酸、蝶罗呤或三甲曲沙。所述的嘌呤类似物为本领域常规的嘌呤类似物，较佳地选自氟达拉滨、6-巯嘌呤、硫咪嘌呤或硫鸟嘌呤。所述的嘧啶类似物为本领域常规的嘧啶类似物，较佳地选自安西他滨、阿扎胞苷、6-阿扎尿苷、卡莫氟、阿糖胞苷、二脱氧尿苷、去氧氟尿苷、依诺他滨、氟尿苷或5-EU。所述的雄激素为本领域常规的雄激素，较佳地选自卡普睾酮、丙酸甲雄烷酮、环硫雄醇、美雄烷或睾内酯。所述的抗肾上腺素为本领域常规的抗肾上腺素，较佳地选自安鲁米特、米托坦或曲洛司坦。所述的叶酸补充剂为本领域常规的叶酸补充剂，较佳地选自亚叶酸、醋葡全内酯、醛磷酰胺糖苷、氨基酮戊酸、安吖啶、阿莫司汀、比生群、依达曲沙、地磷酰胺、秋水仙胺、地吖醌、依氟鸟氨酸、依利醋铵、埃坡西龙、依托格鲁、硝酸镓、羟基脲、香菇多糖或氯尼达明。所述的美登醇为本领域常规的美登醇，较佳地选自美登素、安丝菌素、米托胍腙、米托蒽醌、莫哌达醇、二胺硝吖啶、喷司他丁、蛋氨氮芥、吡柔比星、洛索蒽醌、鬼臼酸、2-乙基酰肼或丙卡巴肼。所述的多糖复合物为本领域常规的多糖复合物，较佳地选自雷佐生、根霉素、西佐喃、锗螺胺、细交链孢菌酮酸、三亚胺醌2，2′，2″-三氯三乙胺、单端孢霉烯族毒素、乌拉坦、长春地辛、达卡巴嗪、甘露莫司汀、二溴甘露醇、二溴卫矛醇、哌泊溴烷、gacytosine、阿糖胞苷、环磷酰胺或噻替派。更佳地选自T-2毒素、疣孢菌素A、杆孢菌素A或anguidine。所述紫杉烷为本领域常规的紫杉烷，较佳地选自紫杉醇、无氢化蓖麻油、紫杉醇的白蛋白工程化纳米颗粒制剂(American Pharmaceutical Partners，Schaumberg，Illinois)、多西他赛、苯丁酸氮芥、吉西他滨、6-硫代鸟嘌呤、巯嘌呤或甲氨蝶呤。所述的铂类似物为本领域常规的铂类似物，较佳地选自顺铂、卡铂、长春碱、依托泊苷、异环磷酰胺、米托蒽醌、长春新碱、诺安托、替尼泊苷、依达曲沙、道诺霉素、氨基蝶呤、卡培他滨伊班膦酸盐、CPT-11、拓扑异构酶抑制剂RFS 2000或二氟甲基鸟氨酸。所述的类视黄醇为本领域的类视黄醇，较佳地为视黄酸。The alkylating agent is a conventional alkylating agent in the art, preferably selected from thiotepa or cyclophosphamide. The alkyl sulfonate chemotherapeutic agent is a conventional alkyl sulfonate chemotherapeutic agent in the art, and is preferably selected from busulfan, inprosufane or piposifan. The aziridine chemotherapeutic agent is a conventional aziridine chemotherapeutic agent in the art, and is preferably selected from aziridine, carbachol, metotepa or uritepa. The vinylamide and methylmelamine chemotherapeutics are conventional vinylamide and methylmelamine chemotherapeutics in the art, and are preferably selected from hexamethylmelamine, triethylenemelamine, and triethylene Phosphoramide, triethylene thiophosphoramide or trimethylol melamine. The nitrogen mustard is a conventional nitrogen mustard, preferably selected from chlorambucil, chlorambucil, estramustine, ifosfamide, nitrogen mustard, chlorambucil hydrochloride, Phenylalanine mustard, new mustard, phenyl mustard cholesterol, prednisone, trifosamine or uracil mustard. The nitrourea chemotherapeutic agent is a conventional nitrourea chemotherapeutic agent in the art, preferably selected from carmustine, chlorurea, formustine, lomustine, nimomus Or ramustine. The antibiotic is a conventional antibiotic in the art, preferably selected from the group consisting of aclarithromycin, actinomycin, abramycin, azaserine, bleomycin, actinomycin c, calicheamicin, Carrubicin, carcinomycin, carcinogen, chromomycin, dactinomycin, daunorubicin, ditorubicin, 6-diazo-5-oxo-L-norleucine, more Ruubicin, epirubicin, esorubicin, idarubicin, flabomycin, mitomycin, mycophenolic acid, nogamycin, olivemycin, pelomycin, porphyrin , Puromycin, tri-iron doxorubicin, rhodoubicin, streptozotocin, streptozotocin, tuberculin, ubiquitin, venomstatin or zorubicin. The antimetabolites are conventional antimetabolites in the art, preferably selected from methotrexate or 5-fluorouracil (5-FU). The folic acid chemotherapeutic agent is a conventional folic acid chemotherapeutic agent in the art, preferably selected from dimethyl folic acid, pterorin or trimethoate. The purine analogues are conventional purine analogues in the art, preferably selected from fludarabine, 6-mercaptopurine, thiomipurine or thioguanine. The pyrimidine analogues are conventional pyrimidine analogues in the art, preferably selected from the group consisting of ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, Deoxyfluridine, enoxabine, fluridine or 5-EU. The androgens are conventional androgens in the art, and are preferably selected from the group consisting of captestosterone, menandrotanone propionate, thiosterol, meandrosterol, or testosterone. The anti-adrenaline is a conventional anti-adrenaline in the art, and is preferably selected from anlumit, mitotane, or tripostane. The folic acid supplement is a conventional folic acid supplement in the art, preferably selected from the group consisting of leucovorin, acetoglucone, aldophosphamide glycoside, aminolevulinic acid, amsacrine, amustine, and bisham Trident, edatrexate, dessfamide, colchicine, diacrquinone, eflornithine, elliptic ammonium, epotoxylon, etoglu, gallium nitrate, hydroxyurea, lentinan or clonida Bright. Said maytansinol is a conventional maytansinol in the art, preferably selected from maytansine, ansamicin, mitoguanidine hydrazone, mitoxantrone, mupendol, diamine nitroacridine, Pentastatin, chlorambucil, pirarubicin, losoxantrone, podophyllic acid, 2-ethylhydrazide or procarbazine. The polysaccharide complex is a conventional polysaccharide complex in the art, and is preferably selected from the group consisting of razosan, rhizopromycin, cizoran, germanspiramine, alternaria tenuinic acid, triimine quinone 2,2' , 2″-trichlorotriethylamine, trichothecenes, urethane, vindesine, dacarbazine, mannmostine, dibromomannitol, dulcitol dibromide, piper bromide, gacytosine, cytarabine, cyclophosphamide or thiotepa. More preferably selected from T-2 toxin, verrucosporin A, bacillus sporine A or anguidine. The taxane is a conventional violet in the art Taxane, preferably selected from paclitaxel, non-hydrogenated castor oil, paclitaxel albumin engineered nanoparticle formulations (American Pharmaceutical Partners, Schaumberg, Illinois), docetaxel, chlorambucil, gemcitabine, 6-sulfur Substitute guanine, mercaptopurine or methotrexate. The platinum analogues are conventional platinum analogues in the art, preferably selected from cisplatin, carboplatin, vinblastine, etoposide, ifosfamide, Mitoxantrone, vincristine, noanto, teniposide, edatrexate, daunomycin, aminopterin, capecitabine ibandronate, CPT-11, topoisomerase Inhibitor RFS 2000 or difluoromethylornithine. Said retinoid is a retinoid in the art, preferably retinoic acid.
其中，所述放射性同位素为本领域常规的放射性同位素，较佳地，其与上述蛋白质直接结合，或者通过螯合剂与上述蛋白质结合。更佳地，其与所述蛋白质的半胱氨酸残基直接结合。较佳地，所述放射性同位素选自适于放射治疗的α-发射体、β-发射体和俄歇电子以及适于诊断的正电子发射体或γ-发射体。更佳地，所述放射性同位素选自 18氟、 64铜、 65铜。 67镓、 68镓、 77溴、 80m溴、 95钌、 97钌、 103钌、 105钌、 99m锝、 107汞、 203汞、 123碘、 124碘、 125碘、 126碘、 131碘、 133碘、 111铟、 113铟、 99m铼、 105铼、 101铼、 186铼、 188铼、 121m碲、 99锝、 122m碲、 125m碲、 165铥、 167铥、 168铥、 90钇、 213铋、 213铅或 225锕，或者其衍生的氮化物或氧化物。 Wherein, the radioisotope is a conventional radioisotope in the art, preferably, it is directly bound to the above-mentioned protein, or is bound to the above-mentioned protein through a chelating agent. More preferably, it directly binds to the cysteine residue of the protein. Preferably, the radioisotope is selected from α-emitters, β-emitters and Auger electrons suitable for radiotherapy, and positron emitters or γ-emitters suitable for diagnosis. More preferably, the radioisotope is selected from 18 fluorine, 64 copper, and 65 copper. 67 gallium, 68 gallium, 77 bromine, 80m bromine, 95 ruthenium, 97 ruthenium, 103 ruthenium, 105 ruthenium, 99m technetium, 107 mercury, 203 mercury, 123 iodine, 124 iodine, 125 iodine, 126 iodine, 131 iodine, 133 iodine , 111 indium, 113 indium, 99m rhenium, 105 rhenium, 101 rhenium, 186 rhenium, 188 rhenium, 121m tellurium, 99 technetium, 122m tellurium, 125m tellurium, 165 thulium, 167 thulium, 168 thulium, 90 yttrium, 213 bismuth, 213 Lead or 225 actinium, or its derived nitride or oxide.
其中，所述治疗性核酸为本领域常规的核酸，较佳地为编码免疫调节剂、抗血管生成剂、抗增殖剂或促凋亡剂的基因。所述治疗剂包括所述治疗剂、其衍生物和所述治疗剂在药学上可接受的盐、酸及衍生物。Wherein, the therapeutic nucleic acid is a conventional nucleic acid in the art, preferably a gene encoding an immunomodulator, an anti-angiogenic agent, an anti-proliferative agent or a pro-apoptotic agent. The therapeutic agent includes the therapeutic agent, its derivatives, and pharmaceutically acceptable salts, acids and derivatives of the therapeutic agent.
其中，所述的免疫调节剂为本领域常规的免疫调节剂，即引发免疫应答，包括体液免疫应答(例如抗原特异性抗体的产生)和细胞介导的免疫应答(例如淋巴细胞增殖)的试剂。较佳地选自细胞因子、生长因子、激素、抗激素药、免疫抑制剂或皮质类固醇。所述细胞因子为本领域常规的细胞因子，较佳地选自黄嘌呤、白介素或干扰素。所述的生长因子为本领域常规的生长因子，较佳地选自TNF、CSF、GM-CSF或G-CSF。所述的激素为本领域常规的激素，较佳地选自雌激素、雄激素或孕激素。更佳地，所述的雌激素为已烯雌酚或雌二醇。更佳地，所述的雄激素为睾酮或氟甲睾酮。更佳地，所述的孕激素为乙酸甲地孕酮或乙酸甲羟孕酮。所述的皮质类固醇为本领域常规的皮质类固醇，较佳地选自强的松、地塞米松或化可的松。所述抗激素药为本领域常规的抗激素药，其能阻断激素对肿瘤的作用，抑制细胞因子生产，下调自身抗原表达、或掩蔽MHC抗原的免疫抑制剂。较佳地选自抗雌激素药、抗雄激素药或抗肾上腺素药。更佳地，所述抗雌激素药选自它莫西芬、雷洛昔芬、芳香酶抑制性4(5)-咪唑类、4-羟基它莫西芬、曲沃昔芬或托瑞米芬。所述抗雄激素药选自氟他胺、尼鲁米特、比卡鲁胺、亮丙瑞林或戈舍瑞林。所述免疫抑制剂为本领域常规的免疫抑制剂，较佳地选自2-氨基-6芳基-5取代的嘧啶类、硫唑嘌呤、环磷酰胺、溴隐亭、达那唑、氨苯砜、戊二醛、针对MHC抗原和MHC片段的抗独特型抗体、环孢菌素A、类固醇例如糖皮质类固醇、链激酶、TGFb、雷帕霉素、T细胞受体、T细胞受体片段、细胞因子受体拮抗剂或T细胞受体抗体。更佳地，所述细胞因子受体拮抗剂选自抗干扰素抗体、抗IL10抗体、抗TNFa抗体或抗IL2抗体。Wherein, the immunomodulator is a conventional immunomodulator in the art, that is, an agent that triggers an immune response, including humoral immune response (such as the production of antigen-specific antibodies) and cell-mediated immune response (such as lymphocyte proliferation) . It is preferably selected from cytokines, growth factors, hormones, antihormones, immunosuppressants or corticosteroids. The cytokine is a conventional cytokine in the art, preferably selected from xanthine, interleukin or interferon. The growth factor is a conventional growth factor in the art, preferably selected from TNF, CSF, GM-CSF or G-CSF. The hormones are conventional hormones in the art, preferably selected from estrogen, androgen or progesterone. More preferably, the estrogen is diethylstilbestrol or estradiol. More preferably, the androgen is testosterone or fluoxymesterone. More preferably, the progestin is megestrol acetate or medroxyprogesterone acetate. The corticosteroids are conventional corticosteroids in the art, preferably selected from prednisone, dexamethasone or cortisone. The antihormonal drug is a conventional antihormonal drug in the art, which can block the effect of hormones on tumors, inhibit the production of cytokines, down-regulate the expression of self-antigens, or mask the immunosuppressive agents of MHC antigens. It is preferably selected from anti-estrogens, anti-androgens or anti-adrenergics. More preferably, the anti-estrogen is selected from the group consisting of tamoxifen, raloxifene, aromatase-inhibiting 4(5)-imidazoles, 4-hydroxytamoxifen, travoxifen or toremi Fen. The anti-androgen is selected from flutamide, nilutamide, bicalutamide, leuprolide or goserelin. The immunosuppressive agent is a conventional immunosuppressant in the art, preferably selected from 2-amino-6 aryl-5 substituted pyrimidines, azathioprine, cyclophosphamide, bromocriptine, danazol, and ammonia Phenylsulfone, glutaraldehyde, anti-idiotypic antibodies against MHC antigens and MHC fragments, cyclosporin A, steroids such as glucocorticoids, streptokinase, TGFb, rapamycin, T cell receptor, T cell receptor Fragment, cytokine receptor antagonist or T cell receptor antibody. More preferably, the cytokine receptor antagonist is selected from an anti-interferon antibody, an anti-IL10 antibody, an anti-TNFa antibody or an anti-IL2 antibody.
其中，所述的抗血管生成剂为本领域常规的抗血管生成剂，较佳地选自法尼基转移酶抑制剂、COX-2抑制剂、VEGF抑制剂、bFGF抑制剂、类固醇硫酸酯酶抑制剂、白介素-24、凝血栓蛋白、metallospondin蛋白质、I类干扰素、白介素12、鱼精蛋白、血管他丁、层粘连蛋白、内皮他丁或催乳激素片段。更佳地为2-甲氧基雌二醇二氨基磺酸酯(2-MeOE2bisMATE)。Wherein, the anti-angiogenesis agent is a conventional anti-angiogenesis agent in the art, preferably selected from farnesyl transferase inhibitors, COX-2 inhibitors, VEGF inhibitors, bFGF inhibitors, steroid sulfatase Inhibitors, interleukin-24, thromboxane, metallospondin protein, class I interferon, interleukin-12, protamine, angiostatin, laminin, endostatin or prolactin fragments. More preferably, it is 2-Methoxyestradiol disulfamate (2-MeOE2bisMATE).
其中，所述抗增殖促凋亡剂为本领域常规的抗增殖促凋亡剂，较佳地选自PPAR-γ 激活剂、类视黄醇、三萜类化合物、EGF受体抑制剂、端粒末端转移酶抑制剂、铁螯合剂、凋亡蛋白、Bcl-2和Bcl-X(L)的抑制剂、TNF-α/FAS配体/TNF相关的凋亡诱导配体及其信号传导的激活物或PI3K-Akt存活途径信号抑制剂。所述PPAR-γ激活剂为本领域常规的PPAR-γ激活剂，较佳地为环戊烯酮前列腺素(cyPGs)。所述三萜类化合物为本领域常规的三萜类化合物，较佳地选自环菠萝蜜烷、羽扇豆烷、乌苏烷、齐敦果烷、木栓烷、达玛烷、葫芦素、柠檬苦素类似物或三萜类化合物。所述EGF受体抑制剂为本领域常规的EGF受体抑制剂，较佳地选自HER4、雷帕霉素或1，25-二羟基胆钙化醇(维生素D)。所述的铁螯合物为本领域常规的铁螯合物，较佳地为3-氨基吡啶-2-甲醛硫代缩氨基脲。所述的凋亡蛋白为本领域常规的凋亡蛋白，较佳地为鸡贫血病病毒的病毒蛋白质3-VP3。所述PI3K-Akt存活途径信号抑制剂为本领域常规的PI3K-Akt存活途径信号抑制剂，较佳地为UCN-01或格尔德霉素。Wherein, the anti-proliferation and pro-apoptosis agent is a conventional anti-proliferation and pro-apoptosis agent, preferably selected from PPAR-γ activator, retinoid, triterpenoid, EGF receptor inhibitor, terminal Granulose Inhibitors, Iron Chelators, Apoptosis Proteins, Inhibitors of Bcl-2 and Bcl-X(L), TNF-α/FAS Ligand/TNF-related Apoptosis Inducing Ligand and Its Signal Transduction Activator or PI3K-Akt survival pathway signaling inhibitor. The PPAR-γ activator is a conventional PPAR-γ activator in the art, preferably cyclopentenone prostaglandins (cyPGs). The triterpenoid compound is a conventional triterpenoid compound in the art, preferably selected from the group consisting of cyclic jackfruit, lupinane, ursane, zidonorane, xylane, dammarane, cucurbitacin, lemon Picrine analogs or triterpenoids. The EGF receptor inhibitor is a conventional EGF receptor inhibitor in the art, preferably selected from HER4, rapamycin, or 1,25-dihydroxycholecalciferol (vitamin D). The iron chelate is a conventional iron chelate in the art, preferably 3-aminopyridine-2-carboxaldehyde thiosemicarbazone. The apoptotic protein is a conventional apoptotic protein in the art, preferably the viral protein 3-VP3 of chicken anemia virus. The PI3K-Akt survival pathway signal inhibitor is a conventional PI3K-Akt survival pathway signal inhibitor in the art, and is preferably UCN-01 or geldanamycin.
其中，所述细胞溶解酶为本领域常规的细胞溶解酶，较佳地为RNA酶。Wherein, the cytolytic enzyme is a conventional cytolytic enzyme in the art, preferably RNase.
本发明优选地，式1中x＝y＝n；由此In the present invention, preferably, x=y=n in formula 1; thus
在一个优选实施例中，-(L) x-(D) y为： In a preferred embodiment, -(L) x -(D) y is:
Figure PCTCN2020108720-appb-000003
Figure PCTCN2020108720-appb-000003
其中m为1～10，优选m为5。Wherein m is 1-10, preferably m is 5.
在一个优选实施例中，-(L) x-(D) y为： In a preferred embodiment, -(L) x -(D) y is:
Figure PCTCN2020108720-appb-000004
Figure PCTCN2020108720-appb-000004
在一个优选实施例中，-(L) x-(D) y为： In a preferred embodiment, -(L) x -(D) y is:
Figure PCTCN2020108720-appb-000005
Figure PCTCN2020108720-appb-000005
最佳地，所述D为微管蛋白合成酶抑制剂——甲基奥瑞他汀F(MMAF)，且所述接头L为马来酰亚胺基己酰(maleimidocaproyl，MC)，所述抗体药物偶联物的结构如式3所示，Most preferably, the D is a tubulin synthase inhibitor-methyl auristatin F (MMAF), and the linker L is maleimidocaproyl (MC), the antibody The structure of the drug conjugate is shown in formula 3.
Figure PCTCN2020108720-appb-000006
Figure PCTCN2020108720-appb-000006
或者，所述L为4-(N-马来酰亚胺基甲基)环己烷-1-羧酸琥珀酰亚胺酯；D为N2’-脱乙酰-N2’-3-巯基-1氧代丙基-美登素(DM1)，所述抗体药物偶联物的结构如式4所示，Alternatively, the L is 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid succinimidyl ester; D is N2'-deacetyl-N2'-3-mercapto-1 Oxopropyl-maytansine (DM1), the structure of the antibody drug conjugate is shown in formula 4.
Figure PCTCN2020108720-appb-000007
Figure PCTCN2020108720-appb-000007
或者，L为马来酰亚胺基己酰-L-缬氨酸-L-瓜氨酸对氨基苄醇，D为甲基奥瑞他汀E(MMAE)，所述抗体药物偶联物的结构如式5所示，Or, L is maleimidohexanoyl-L-valine-L-citrulline p-aminobenzyl alcohol, D is methyl auristatin E (MMAE), the structure of the antibody drug conjugate As shown in Equation 5,
Figure PCTCN2020108720-appb-000008
Figure PCTCN2020108720-appb-000008
其中，n为自然数，优选为1～20的整数，更优选为2-8的整数，例如为3或4。Among them, n is a natural number, preferably an integer of 1-20, more preferably an integer of 2-8, for example 3 or 4.
所述的抗体药物偶联物的制备方法可为本领域常规，较佳地采用Doronina，2006，Bioconjugate Chem.17,114-124所记载的制备方法。较佳地，所述的制备方法产生具有最低限度的低偶联级分(LCF)小于10％的抗体药物偶联物。The preparation method of the antibody-drug conjugate can be conventional in the art, preferably the preparation method described in Doronina, 2006, Bioconjugate Chem. 17, 114-124. Preferably, the preparation method produces antibody-drug conjugates with a minimum low coupling fraction (LCF) of less than 10%.
为解决上述技术问题，本发明还提供上述的抗体药物偶联物的制备方法，其包括以下步骤：In order to solve the above-mentioned technical problems, the present invention also provides the above-mentioned preparation method of the antibody-drug conjugate, which includes the following steps:
a.将上述的分离的蛋白质经过硼酸钠缓冲液透析后，加入三(2-羧乙基)膦，室温下还原得反应液A；a. After the above-mentioned separated protein is dialyzed with sodium borate buffer, tris (2-carboxyethyl) phosphine is added and reduced at room temperature to obtain reaction solution A;
b.将反应液A洗脱去除多余的蛋白质得反应液B；b. Elute reaction solution A to remove excess protein to obtain reaction solution B;
c.向反应液B中加入上述的-(L) x-(D) y反应即得。 c. Add the above-(L) x -(D) y reaction to the reaction solution B.
较佳地，所述步骤a中，所述硼酸钠缓冲液的pH值为6.5～8.5；所述三(2-羧乙基)膦(TCEP)与所述的分离的蛋白质的摩尔比比率为2～10；所述还原的时间为1～4小时；和/或，所述步骤c中，-(L) x-(D) y与所述的分离的蛋白质的摩尔比比率为5～20，所述反应的温度为10～37℃，所述反应的时间为4小时。 Preferably, in the step a, the pH of the sodium borate buffer is 6.5-8.5; the molar ratio of the tris(2-carboxyethyl)phosphine (TCEP) to the isolated protein is 2 to 10; the reduction time is 1 to 4 hours; and/or, in the step c, the molar ratio of -(L) x -(D) y to the isolated protein is 5 to 20 The temperature of the reaction is 10-37°C, and the time of the reaction is 4 hours.
更佳地，所述的制备方法包括以下的步骤：将上述蛋白质经过pH 6.5～8.5的硼酸钠缓冲液透析后，加入三(2-羧乙基)膦(TCEP)，其中TCEP与上述蛋白质的摩尔比比率为2～10，室温下还原1～4小时，得反应液A。将反应液A洗脱去除多余的上述蛋白质得反应液B。向反应液B中加入MC-MMAF，其中MC-MMAF与纯化的TROP2抗体的摩尔比比率为5～20，10～37℃下反应4小时。More preferably, the preparation method includes the following steps: after the above-mentioned protein is dialyzed with a sodium borate buffer of pH 6.5-8.5, tris(2-carboxyethyl)phosphine (TCEP) is added, wherein the TCEP and the above-mentioned protein The molar ratio is 2-10, and the reaction solution A is obtained by reducing at room temperature for 1-4 hours. The reaction solution A is eluted to remove the excess protein mentioned above to obtain the reaction solution B. MC-MMAF is added to the reaction solution B, wherein the molar ratio of MC-MMAF to the purified TROP2 antibody is 5-20, and the reaction is carried out at 10-37°C for 4 hours.
所述的抗体药物偶联物能够以本领域所知的任何物理形态而存在，较佳地为澄清溶液。The antibody-drug conjugate can exist in any physical form known in the art, preferably a clear solution.
为解决上述技术问题，本发明还提供一种药物组合物，其包括上述的分离的蛋白质和/或上述的抗体药物偶联物，和药学可接受的载体。所述的药物组合物较佳地还包括其他抗肿瘤抗体作为活性成分。In order to solve the above technical problems, the present invention also provides a pharmaceutical composition, which includes the above-mentioned isolated protein and/or the above-mentioned antibody drug conjugate, and a pharmaceutically acceptable carrier. The pharmaceutical composition preferably also includes other anti-tumor antibodies as active ingredients.
所述的药学可接受的载体可为本领域常规的载体，所述的载体可以为任意合适的生理学或药学上可接受的药物辅料。所述的药物辅料为本领域常规的药物辅料，较佳地包括药学上可接受的赋形剂、填充剂或稀释剂等。更佳地，所述的药物组合物包括0.01～99.99％ 的上述蛋白质和/或上述的抗体药物偶联物，和0.01～99.99％的药用载体，所述百分比为占所述药物组合物的质量百分比。The pharmaceutically acceptable carrier may be a conventional carrier in the art, and the carrier may be any suitable physiologically or pharmaceutically acceptable pharmaceutical excipient. The pharmaceutical excipients are conventional pharmaceutical excipients in the field, and preferably include pharmaceutically acceptable excipients, fillers or diluents. More preferably, the pharmaceutical composition comprises 0.01-99.99% of the above-mentioned protein and/or the above-mentioned antibody drug conjugate, and 0.01-99.99% of the pharmaceutical carrier, and the percentage is based on the percentage of the pharmaceutical composition. Mass percentage.
较佳地，所述的药物组合物是抗肿瘤的药物。更佳地为抗鳞状/腺瘤性肺癌(非小细胞肺癌)、浸润性乳腺癌、结肠癌、直肠癌、胃癌、鳞状宫颈癌、浸润性子宫内膜腺癌、浸润性胰腺癌、卵巢癌、鳞状膀胱癌、绒毛膜癌、支气管癌、乳腺癌、子宫颈癌、胰腺癌或精囊癌的药物。Preferably, the pharmaceutical composition is an anti-tumor drug. More preferably, anti-squamous/adenomatous lung cancer (non-small cell lung cancer), invasive breast cancer, colon cancer, rectal cancer, gastric cancer, squamous cervical cancer, invasive endometrial adenocarcinoma, invasive pancreatic cancer, Drugs for ovarian cancer, squamous bladder cancer, choriocarcinoma, bronchial cancer, breast cancer, cervical cancer, pancreatic cancer or seminal vesicle cancer.
本发明所述的药物组合物的给药途径较佳地为肠胃外施用、注射给药或口服给药。所述注射给药较佳地包括静脉注射、肌肉注射、腹腔注射、皮内注射或皮下注射等途径。所述的药物组合物为本领域常规的各种剂型，较佳地为固体、半固体或液体的形式，即可以为水溶液、非水溶液或混悬液，更佳的为片剂、胶囊、颗粒剂、注射剂或输注剂等。更佳地为经由血管内、皮下、腹膜内或肌内施用。较佳地，所述药物组合物还可以作为气雾剂或粗喷雾剂施用，即经鼻施用；或者，鞘内、髓内或心室内施用。更佳地，所述的药物组合物还可以透皮、经皮、局部、肠内、阴道内、舌下或经直肠施用。The administration route of the pharmaceutical composition of the present invention is preferably parenteral administration, injection administration or oral administration. The injection administration preferably includes intravenous injection, intramuscular injection, intraperitoneal injection, intradermal injection, or subcutaneous injection. The pharmaceutical composition is a variety of conventional dosage forms in the art, preferably in the form of solid, semi-solid or liquid, that is, in the form of an aqueous solution, non-aqueous solution or suspension, more preferably tablets, capsules, and granules. Medicine, injection or infusion, etc. More preferably, it is administered via intravascular, subcutaneous, intraperitoneal or intramuscular administration. Preferably, the pharmaceutical composition can also be administered as an aerosol or coarse spray, that is, nasal administration; or, intrathecal, intramedullary or intraventricular administration. More preferably, the pharmaceutical composition can also be administered transdermally, transdermally, topically, enterally, intravaginally, sublingually or rectally.
本发明所述的药物组合物的给药剂量水平可以根据达到所需诊断或治疗结果的组合物量而调整。施用方案也可以为单次注射或多次注射，或进行调整。所选择的剂量水平和方案依赖于包括所述药物组合物的活性和稳定性(即，半衰期)、制剂、施用途径、与其他药物或治疗的组合、待检测和/或治疗的疾病或病症、以及待治疗的受试者的健康状况和先前医疗史等各种因素而进行合理地调整。The dosage level of the pharmaceutical composition of the present invention can be adjusted according to the amount of the composition to achieve the desired diagnosis or treatment result. The administration schedule can also be a single injection or multiple injections, or be adjusted. The selected dosage level and schedule depend on the activity and stability (ie, half-life) of the pharmaceutical composition, formulation, route of administration, combination with other drugs or treatments, diseases or disorders to be detected and/or treated, And various factors such as the health status and previous medical history of the subject to be treated can be reasonably adjusted.
对于本发明的所述药物组合物的治疗有效剂量可以最初在细胞培养实验或动物模型例如啮齿类动物、兔、犬、猪和/或灵长类动物中进行估计。动物模型也可以用于测定合适的施用浓度范围和途径。随后可以用于确定在人中施用的有用剂量和途径。一般地，施用有效量或剂量的确定和调整以及何时和如何进行此类调整的评估为本领域技术人员已知。The therapeutically effective dose of the pharmaceutical composition of the present invention can be estimated initially in cell culture experiments or animal models such as rodents, rabbits, dogs, pigs and/or primates. Animal models can also be used to determine the appropriate concentration range and route of administration. It can then be used to determine useful dosages and routes of administration in humans. Generally, the determination and adjustment of an effective amount or dosage for administration and the evaluation of when and how to make such adjustments are known to those skilled in the art.
对于组合疗法，上述蛋白质、上述抗体药物偶联物和/或另外的治疗或诊断剂可以各自作为单一药剂，在适合于执行预期治疗或诊断的任何时间范围内进行使用。因此，这些单一药剂可以基本上同时(即作为单一制剂或在数分钟或数小时内)或以按顺序连续施用。例如，这些单一药剂可以在一年内，或10、8、6、4或2个月内，或4、3、2、或1周内，或5、4、3、2或1天内施用。For combination therapy, the above-mentioned protein, the above-mentioned antibody-drug conjugate, and/or another therapeutic or diagnostic agent can each be used as a single agent and used in any time frame suitable for performing the intended treatment or diagnosis. Therefore, these single agents can be administered substantially simultaneously (ie, as a single formulation or within minutes or hours) or sequentially and consecutively. For example, these single agents can be administered within one year, or within 10, 8, 6, 4, or 2 months, or within 4, 3, 2, or 1 week, or within 5, 4, 3, 2, or 1 day.
关于制剂、剂量、施用方案和可测量的治疗结果的另外指导，参见Berkow等人(2000)The Merck Manual of Medical Information(Merck医学信息手册)和Merck&Co.Inc.,Whitehouse Station,New Jersey；Ebadi(1998)CRC Desk Reference of Clinical Pharmacology(临床药理学手册)等著作。For additional guidance on formulations, dosages, administration schedules, and measurable treatment results, see Berkow et al. (2000) The Merck Manual of Medical Information and Merck & Co. Inc., Whitehouse Station, New Jersey; Ebadi (2000) The Merck Manual of Medical Information (Merck Medical Information Manual) 1998) CRC Desk Reference of Clinical Pharmacology (Clinical Pharmacology Manual) and other works.
为解决上述技术问题，本发明提供一种套装药盒，其包含药盒A和药盒B，所述的药盒A为上述的蛋白质、和/或上述的抗体药物偶联物、和/或上述的药物组合物，所述的药盒B为其他抗肿瘤抗体或者包含所述其他抗肿瘤抗体的药物组合物。所述的药盒A和药盒B可以同时使用，也可以先使用药盒A再使用药盒B，还可以先使用药盒B再使用药盒A，可以根据具体应用时的实际需求而定。In order to solve the above technical problems, the present invention provides a kit of medicines, which comprises a medicine box A and a medicine box B, the medicine box A is the above-mentioned protein, and/or the above-mentioned antibody-drug conjugate, and/or In the above-mentioned pharmaceutical composition, the kit B is another anti-tumor antibody or a pharmaceutical composition containing the other anti-tumor antibody. The medicine box A and the medicine box B can be used at the same time, or the medicine box A can be used first and then the medicine box B, or the medicine box B can be used first and then the medicine box A can be determined according to the actual needs of the specific application. .
为解决上述技术问题，本发明提供一种上述的分离的蛋白质在制备抗肿瘤药物中的应用。In order to solve the above technical problems, the present invention provides an application of the above isolated protein in the preparation of antitumor drugs.
为解决上述技术问题，本发明提供一种上述的抗体药物偶联物在制备抗肿瘤药物中的应用。In order to solve the above technical problems, the present invention provides an application of the above antibody-drug conjugate in the preparation of anti-tumor drugs.
为解决上述技术问题，本发明提供一种上述的药物组合物在制备抗肿瘤药物中的应用。To solve the above technical problems, the present invention provides an application of the above-mentioned pharmaceutical composition in the preparation of anti-tumor drugs.
为解决上述技术问题，本发明提供一种上述的套装药盒在制备抗肿瘤药物中的应用。In order to solve the above technical problems, the present invention provides an application of the above-mentioned kit in the preparation of anti-tumor drugs.
为解决上述技术问题，本发明提供一种上述的分离的蛋白质在治疗肿瘤中的应用。In order to solve the above technical problems, the present invention provides an application of the above isolated protein in the treatment of tumors.
为解决上述技术问题，本发明提供一种上述的抗体药物偶联物在治疗肿瘤中的应用。In order to solve the above technical problems, the present invention provides an application of the above antibody drug conjugate in the treatment of tumors.
为解决上述技术问题，本发明提供一种上述的药物组合物在治疗肿瘤中的应用。In order to solve the above technical problems, the present invention provides an application of the above pharmaceutical composition in the treatment of tumors.
为解决上述技术问题，本发明提供一种上述的套装药盒在治疗肿瘤中的应用。In order to solve the above technical problems, the present invention provides an application of the above-mentioned kit medicine box in the treatment of tumors.
较佳地，本发明中，所述抗肿瘤药物为与TROP2表达或功能异常相关的疾病的药物，所述肿瘤优选与TROP2表达或功能异常相关的肿瘤；更佳地，所述与TROP2表达或功能异常相关的疾病或肿瘤为：自体免疫疾病、炎症性疾病、感染性疾病或增殖性疾病，优选非小细胞肺癌、浸润性乳腺癌、结肠癌、直肠癌、胃癌、鳞状宫颈癌、浸润性子宫内膜腺癌、浸润性胰腺癌、卵巢癌、鳞状膀胱癌、绒毛膜癌、支气管癌、乳腺癌、子宫颈癌、胰腺癌或精囊癌。Preferably, in the present invention, the anti-tumor drug is a drug for a disease related to TROP2 expression or abnormal function, and the tumor is preferably a tumor related to TROP2 expression or abnormal function; more preferably, said TROP2 expression or abnormal function Diseases or tumors related to dysfunction are: autoimmune diseases, inflammatory diseases, infectious diseases or proliferative diseases, preferably non-small cell lung cancer, invasive breast cancer, colon cancer, rectal cancer, gastric cancer, squamous cervical cancer, invasive Endometrial adenocarcinoma, invasive pancreatic cancer, ovarian cancer, squamous bladder cancer, choriocarcinoma, bronchial cancer, breast cancer, cervical cancer, pancreatic cancer or seminal vesicle cancer.
为解决上述技术问题，本发明还提供一种检测过表达TROP2蛋白的细胞的方法，其包括如下的步骤：上述的蛋白质与待检细胞样品在体外接触，检测上述的蛋白质与所述待检细胞样品的结合即可。In order to solve the above technical problems, the present invention also provides a method for detecting cells overexpressing TROP2 protein, which includes the following steps: the above-mentioned protein is contacted with the test cell sample in vitro, and the above-mentioned protein and the test cell are detected. The sample can be combined.
所述的过表达的含义为本领域常规，较佳地为待检样品中，细胞经过流式检测，上述的蛋白质的平均荧光密度(MFI)值是亚型IgG的MFI值的3倍及以上。The meaning of said overexpression is conventional in the art. Preferably, the cells in the sample to be tested are subjected to flow cytometry, and the average fluorescence density (MFI) value of the above-mentioned protein is 3 times or more of the MFI value of subtype IgG .
所述结合的检测方式是本领域常规的检测方式，较佳地为FACS检测。The detection method of the binding is a conventional detection method in the art, preferably FACS detection.
本发明中，所述待测细胞样品一般为离体的待测细胞样品。In the present invention, the cell sample to be tested is generally an isolated cell sample to be tested.
本发明所述的“TROP2阳性”的细胞即为过表达TROP2蛋白的细胞，如MDA-MB-468细胞株；反之，则称为“TROP2阴性”的细胞，如肿瘤细胞系HCC1395。The "TROP2-positive" cells described in the present invention are cells that overexpress the TROP2 protein, such as the MDA-MB-468 cell line; on the contrary, they are called "TROP2-negative" cells, such as the tumor cell line HCC1395.
在符合本领域常识的基础上，上述各优选条件，可任意组合，即得本发明各较佳实 例。On the basis of conforming to common knowledge in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain preferred examples of the present invention.
本发明所用试剂和原料均市售可得。The reagents and raw materials used in the present invention are all commercially available.
本发明的积极进步效果在于：本发明所述的TROP2抗体与hTROP2或cTROP2或mTROP2蛋白具有高度亲和力，能够在蛋白水平和细胞水平结合所述TROP2蛋白的胞外区。所述的TROP2抗体与如MC-MMAF的小分子化合物偶联后得到一抗体药物偶联物(ADC)，所述抗体药物偶联物能够有效地对TROP2阳性细胞进行细胞毒杀伤作用。此外，本发明的抗体药物偶联物中，TROP2抗体能把小分子化合物，如MMAF，通过内吞作用带入细胞，并在细胞内降解释放小分子化合物，从而起到细胞毒杀伤作用。因此所述的TROP2抗体药物偶联物，能够有效杀伤肿瘤细胞，治疗肿瘤。将本发明所述的TROP2抗体制备成人源化抗体后，依然能够与人源或食蟹猴源的TROP2蛋白具有高度亲和力，且能够很好的抑制肿瘤细胞的生长，对体重无显著影响。The positive progress effect of the present invention is that the TROP2 antibody of the present invention has a high affinity with hTROP2 or cTROP2 or mTROP2 protein, and can bind to the extracellular region of the TROP2 protein at the protein level and the cell level. The TROP2 antibody is coupled with a small molecule compound such as MC-MMAF to obtain an antibody-drug conjugate (ADC), which can effectively kill TROP2-positive cells. In addition, in the antibody-drug conjugate of the present invention, the TROP2 antibody can bring small molecule compounds, such as MMAF, into cells through endocytosis, and degrade and release small molecule compounds in the cells, thereby playing a cytotoxic and killing effect. Therefore, the TROP2 antibody drug conjugate can effectively kill tumor cells and treat tumors. After preparing the humanized antibody of the TROP2 antibody of the present invention, it can still have a high affinity with human or cynomolgus TROP2 protein, and can well inhibit the growth of tumor cells without significant influence on body weight.
附图说明Description of the drawings
图1为人TROP2蛋白转染的HEK293细胞FACS筛选检测结果。Figure 1 shows the results of FACS screening and detection of HEK293 cells transfected with human TROP2 protein.
图2为人TROP2蛋白转染的CHOK1细胞FACS筛选检测结果。Figure 2 shows the results of FACS screening and detection of CHOK1 cells transfected with human TROP2 protein.
图3为食蟹猴TROP2蛋白转染的CHOK1细胞FACS筛选检测结果。Figure 3 shows the results of FACS screening and detection of CHOK1 cells transfected with cynomolgus monkey TROP2 protein.
图4为小鼠TROP2蛋白转染的CHOK1细胞FACS筛选检测结果。Figure 4 shows the results of FACS screening and detection of CHOK1 cells transfected with mouse TROP2 protein.
图5为ELISA检测TROP2免疫后小鼠血清抗体效价情况。Figure 5 shows the antibody titer of mice immunized with TROP2 by ELISA.
图6为ELISA检测TROP2抗体与人TROP2-hFc蛋白的结合反应；TROP2抗体的克隆号分别为11E8E6D11和44A5F9。Figure 6 shows the detection of the binding reaction between TROP2 antibody and human TROP2-hFc protein by ELISA; the clone numbers of TROP2 antibody are 11E8E6D11 and 44A5F9, respectively.
图7为FACS检测TROP2抗体与CHOK1-hTROP2的结合反应；TROP2抗体的克隆号分别为11E8E6D11和44A5F9。Figure 7 shows the FACS detection of the binding reaction between TROP2 antibody and CHOK1-hTROP2; the clone numbers of TROP2 antibody are 11E8E6D11 and 44A5F9, respectively.
图8为FACS检测TROP2抗体与CHOK1-cTROP2的结合反应；TROP2抗体的克隆号分别为11E8E6D11和44A5F9。Figure 8 shows the FACS detection of the binding reaction between TROP2 antibody and CHOK1-cTROP2; the clone numbers of TROP2 antibody are 11E8E6D11 and 44A5F9, respectively.
图9为FACS检测TROP2抗体与CHOK1-mTROP2的结合反应；TROP2抗体的克隆号分别为11E8E6D11和44A5F9。Figure 9 shows the FACS detection of the binding reaction between TROP2 antibody and CHOK1-mTROP2; the clone numbers of TROP2 antibody are 11E8E6D11 and 44A5F9, respectively.
图10为FACS检测TROP2抗体与CHOK1的结合反应；TROP2抗体的克隆号分别为11E8E6D11和44A5F9。Figure 10 shows the FACS detection of the binding reaction between TROP2 antibody and CHOK1; the clone numbers of TROP2 antibody are 11E8E6D11 and 44A5F9, respectively.
图11为TROP2抗体-MMAF抗体药物偶联物对TROP2表达阳性三阴性乳腺癌细胞株MAD-MB-468的细胞杀伤作用；TROP2抗体的克隆号分别为11E8E6D11和44A5F9。Figure 11 shows the cell killing effect of the TROP2 antibody-MMAF antibody drug conjugate on the TROP2 positive triple-negative breast cancer cell line MAD-MB-468; the clone numbers of the TROP2 antibody are 11E8E6D11 and 44A5F9, respectively.
图12为TROP2抗体-MMAF抗体药物偶联物对TROP2表达阴性乳腺癌细胞株HCC1395的细胞杀伤作用；TROP2抗体的克隆号分别为11E8E6D11和44A5F9。Figure 12 shows the cell killing effect of the TROP2 antibody-MMAF antibody drug conjugate on the TROP2 expression-negative breast cancer cell line HCC1395; the clone numbers of the TROP2 antibody are 11E8E6D11 and 44A5F9, respectively.
图13为偶联MMAF的TROP2嵌合抗体药物偶联物对TROP2阳性的肿瘤细胞系MDA-MB-468的细胞杀伤作用。TROP2嵌合抗体的克隆号分别为11E8E6D11和44A5F9。Figure 13 shows the cell killing effect of the TROP2 chimeric antibody drug conjugate coupled with MMAF on the TROP2-positive tumor cell line MDA-MB-468. The clone numbers of the TROP2 chimeric antibodies are 11E8E6D11 and 44A5F9, respectively.
图14A为偶联MMAF的TROP2嵌合抗体药物偶联物对TROP2阳性的肿瘤细胞系BxPC-3的细胞杀伤作用。TROP2嵌合抗体的克隆号分别为11E8E6D11和44A5F9。Figure 14A shows the cell killing effect of the TROP2 chimeric antibody drug conjugate conjugated with MMAF on the TROP2 positive tumor cell line BxPC-3. The clone numbers of the TROP2 chimeric antibodies are 11E8E6D11 and 44A5F9, respectively.
图14B为偶联MMAF的TROP2嵌合抗体药物偶联物对TROP2阳性的肿瘤细胞系COLO 205的细胞杀伤作用。TROP2嵌合抗体的克隆号分别为11E8E6D11和44A5F9。Figure 14B shows the cell killing effect of the TROP2 chimeric antibody drug conjugate coupled with MMAF on the TROP2-positive tumor cell line COLO 205. The clone numbers of the TROP2 chimeric antibodies are 11E8E6D11 and 44A5F9, respectively.
图15为偶联MMAE的TROP2嵌合抗体药物偶联物对TROP2阳性的肿瘤细胞系MDA-MB-468的细胞杀伤作用。TROP2嵌合抗体的克隆号分别为11E8E6D11、44A5F9。Figure 15 shows the cell killing effect of the TROP2 chimeric antibody drug conjugate coupled with MMAE on the TROP2-positive tumor cell line MDA-MB-468. The clone numbers of TROP2 chimeric antibodies are 11E8E6D11 and 44A5F9 respectively.
图16为偶联MMAE的TROP2嵌合抗体药物偶联物对TROP2阳性的肿瘤细胞系COLO 205的细胞杀伤作用。TROP2嵌合抗体的克隆号分别为11E8E6D11、44A5F9。Figure 16 shows the cell killing effect of the TROP2 chimeric antibody drug conjugate coupled with MMAE on the TROP2 positive tumor cell line COLO 205. The clone numbers of the TROP2 chimeric antibodies are 11E8E6D11 and 44A5F9, respectively.
图17为偶联MMAE的TROP2嵌合抗体药物偶联物治疗后的肿瘤体积变化图。TROP2嵌合抗体的克隆号分别为11E8E6D11、44A5F9。Figure 17 is a graph of tumor volume changes after treatment with a TROP2 chimeric antibody drug conjugate coupled to MMAE. The clone numbers of TROP2 chimeric antibodies are 11E8E6D11 and 44A5F9 respectively.
图18为偶联MMAE的TROP2嵌合抗体药物偶联物治疗后的小鼠体重变化图。TROP2嵌合抗体的克隆号分别为11E8E6D11、44A5F9。Figure 18 is a graph showing the body weight changes of mice after treatment with MMAE-conjugated TROP2 chimeric antibody drug conjugate. The clone numbers of TROP2 chimeric antibodies are 11E8E6D11 and 44A5F9 respectively.
图19为TROP2嵌合抗体在大鼠中单次施用后血浆清除药动学图。Figure 19 is a pharmacokinetic diagram of plasma clearance after a single administration of TROP2 chimeric antibody in rats.
图20为hRS7在大鼠中单次施用后血浆清除药动学图。Figure 20 is a pharmacokinetic chart of plasma clearance after a single administration of hRS7 in rats.
图21为突变后的嵌合抗体通过FACS进行结合活性鉴定的结果图。Figure 21 is a graph showing the result of FACS identification of the binding activity of the mutated chimeric antibody.
图22A显示了流式细胞实验(FACS)检测11E8人源化抗体与表达人源TROP2细胞的结合的结果图。Figure 22A shows the result of a flow cytometry experiment (FACS) detecting the binding of 11E8 humanized antibody to human TROP2 cells.
图22B显示了流式细胞实验(FACS)检测44A5F9人源化抗体与表达人源TROP2细胞的结合的结果图。Figure 22B shows the result of a flow cytometry assay (FACS) detecting the binding of 44A5F9 humanized antibody to cells expressing human TROP2.
图23A显示了流式细胞实验(FACS)检测11E8人源化抗体与表达食蟹猴源TROP2细胞的结合的结果图。Figure 23A shows the results of flow cytometry (FACS) testing of the binding of 11E8 humanized antibody to cynomolgus monkey-derived TROP2 cells.
图23B显示了流式细胞实验(FACS)检测44A5F9人源化抗体与表达食蟹猴源TROP2细胞的结合的结果图。Figure 23B shows the result of flow cytometry (FACS) detection of the binding of 44A5F9 humanized antibody to cynomolgus monkey-derived TROP2 cells.
图24A显示了治疗后肿瘤的体积变化图。Figure 24A shows a graph of tumor volume change after treatment.
图24B显示了治疗后的鼠重变化图。Figure 24B shows a graph of the weight change of rats after treatment.
具体实施方式detailed description
下面通过实施例的方式进一步说明本发明，但并不因此将本发明限制在所述的实施例范围之中。下列实施例中未注明具体条件的实验方法，按照常规方法和条件，或按照商品说明书选择。The present invention will be further explained by way of examples below, but the present invention is not limited to the scope of the described examples. In the following examples, the experimental methods without specific conditions are selected according to conventional methods and conditions, or according to the product specification.
实施例中所述的室温为本领域常规的室温，一般为10～30℃。The room temperature described in the examples is a conventional room temperature in the art, and is generally 10-30°C.
若无特别说明，实施例中所述的PBS为PBS磷酸缓冲液，pH7.2。Unless otherwise specified, the PBS described in the examples is PBS phosphate buffer, pH 7.2.
实施例1杂交瘤细胞的制备Example 1 Preparation of hybridoma cells
(一)、免疫原A的制备(1) Preparation of immunogen A
将含有编码人源TROP2蛋白全长氨基酸序列(如序列表SEQ ID NO:17所示)的第88-274位(Thr88-Thr274)胞外区氨基酸序列的核苷酸序列克隆到带有人IgG Fc片段(hFc)的pCpC载体(购自Invitrogen，V044-50)并按已建立的标准分子生物学方法制备质粒。具体方法参见Sambrook,J.,Fritsch,E.F.,and Maniatis,T.(1989).Molecular Cloning:A Laboratory Manual,Second Edition(Plainview,New York:Cold Spring Harbor Laboratory Press)。对HEK293细胞(购自Invitrogen)进行瞬时转染(聚醚酰亚胺PEI，购自Polysciences)并使用FreeStyle TM 293(购自Invitrogen)在37℃下进行扩大培养。4天后收集细胞培养液，离心去除细胞成分，得含hTROP2蛋白胞外区的培养上清液。将培养上清液上样到蛋白A亲和层析柱(Mabselect Sure，购自GE Healthcare)，同时用紫外(UV)检测仪监测紫外吸收值(A280nm)的变化。上样后用PBS磷酸盐缓冲液(pH7.2)清洗蛋白A亲和层析柱直到紫外吸收值回到基线，然后用0.1M甘氨酸盐酸(pH2.5)洗脱，收集从蛋白A亲和层析柱上洗脱下来的带hFc标签的TROP2蛋白(即人源TROP2-hFc)。用PBS磷酸盐缓冲液(pH7.2)在4℃冰箱透析过夜。透析后的蛋白经0.22微米无菌过滤后分装于-80℃保存，即获得纯化的免疫原A(即hTROP2-hFc)。 The nucleotide sequence containing the amino acid sequence of the extracellular region at positions 88-274 (Thr88-Thr274) of the human TROP2 protein full-length amino acid sequence (as shown in SEQ ID NO: 17 in the sequence listing) was cloned into a human IgG Fc Fragment (hFc) pCpC vector (purchased from Invitrogen, V044-50) and prepare plasmids according to established standard molecular biology methods. For specific methods, see Sambrook, J., Fritsch, EF, and Maniatis, T. (1989). Molecular Cloning: A Laboratory Manual, Second Edition (Plainview, New York: Cold Spring Harbor Laboratory Press). HEK293 cells (purchased from Invitrogen) were transiently transfected (polyetherimide PEI, purchased from Polysciences) and expanded using FreeStyle ™ 293 (purchased from Invitrogen) at 37°C. After 4 days, the cell culture fluid was collected, and the cell components were removed by centrifugation to obtain the culture supernatant containing the extracellular domain of hTROP2 protein. The culture supernatant was loaded onto a protein A affinity chromatography column (Mabselect Sure, purchased from GE Healthcare), and the change in ultraviolet absorption (A280nm) was monitored with an ultraviolet (UV) detector. After loading the sample, wash the protein A affinity chromatography column with PBS phosphate buffer (pH 7.2) until the UV absorption value returns to the baseline, and then eluted with 0.1M glycine hydrochloric acid (pH 2.5), and collected from the protein A affinity The hFc-tagged TROP2 protein eluted from the chromatography column (ie, human TROP2-hFc). Dialysis was performed overnight with PBS phosphate buffer (pH 7.2) in a refrigerator at 4°C. The dialyzed protein was sterile filtered by 0.22 micron and stored at -80°C to obtain purified immunogen A (namely hTROP2-hFc).
免疫原A在使用前需要进行一系列质控检测，如检测其蛋白浓度、纯度、分子量、生物活性等，结果发现免疫原A各项指标良好，能够作为抗原进行后续制备TROP2抗体的试验。Immunogen A needs to undergo a series of quality control tests before use, such as testing its protein concentration, purity, molecular weight, biological activity, etc. It turns out that immunogen A has good indicators and can be used as an antigen for subsequent preparation of TROP2 antibodies.
(二)、免疫原B的制备(2) Preparation of immunogen B
编码人源TROP2全长氨基酸序列的核苷酸序列被克隆到pIRES载体(购自Clontech)并制备质粒。对HEK293细胞系和CHOK1细胞系(均购自Invitrogen)进行质粒转染(PEI，购自Polysciences)后，在含0.5μg/mL puromycin(嘌呤霉素)的含10％(w/w)胎牛血清的DMEM培养基中选择性培养2周，用有限稀释法在96孔培养板中进行亚克隆，并置于37℃、5％(v/v)CO 2培养，大约2周后选择部分单克隆孔扩增到6孔板中。对扩增后的克隆用已知的TROP2抗体经流式细胞分析法进行筛选。选择长势较好、荧光强度较高、单克隆的细胞系继续扩大培养并液氮冻存，即获得免疫原B(即，含人源TROP2的293F-TROP2稳定细胞系)。具体选择结果如表2和图1所示，IgG亚型对照为小鼠IgG对照。表2说明，已经制得一系列hTROP2阳性表达的HEK293细胞系。图1是克隆号为293F-TROP2 6F6的结果，其中，横坐标为细胞荧光强度，纵坐标为细胞数。表2和图 1的结果说明，6F6为hTROP2高水平表达细胞株，其中TROP2抗体标记的细胞平均细胞荧光密度为3224.98，迁移率为99.69％。 The nucleotide sequence encoding the full-length amino acid sequence of human TROP2 was cloned into the pIRES vector (purchased from Clontech) and a plasmid was prepared. The HEK293 cell line and CHOK1 cell line (both purchased from Invitrogen) were transfected with plasmids (PEI, purchased from Polysciences), and then used in fetal cattle containing 0.5μg/mL puromycin (puromycin) containing 10% (w/w) Serum was selectively cultured in DMEM medium for 2 weeks, and subcloned in 96-well culture plates by the limiting dilution method, and cultured at 37°C and 5% (v/v) CO 2. After about 2 weeks, a part of the single The cloned wells are expanded into 6-well plates. The amplified clones were screened by flow cytometry with known TROP2 antibody. Select a cell line with better growth, higher fluorescence intensity, and monoclonal cell line, continue to expand and culture and freeze in liquid nitrogen to obtain immunogen B (ie, 293F-TROP2 stable cell line containing human TROP2). The specific selection results are shown in Table 2 and Figure 1. The IgG subtype control is a mouse IgG control. Table 2 shows that a series of HEK293 cell lines expressing positive hTROP2 have been prepared. Figure 1 is the result of the clone number 293F-TROP2 6F6, where the abscissa is the cell fluorescence intensity and the ordinate is the number of cells. The results in Table 2 and Figure 1 indicate that 6F6 is a cell line expressing high levels of hTROP2, in which the average cell fluorescence density of TROP2 antibody-labeled cells is 3224.98, and the migration rate is 99.69%.
表2 hTROP2蛋白转染的HEK293细胞FACS筛选检测结果Table 2 FACS screening test results of HEK293 cells transfected with hTROP2 protein
Figure PCTCN2020108720-appb-000009
Figure PCTCN2020108720-appb-000009
(三)、杂交瘤细胞的制备和抗体筛选(3) Preparation of hybridoma cells and antibody screening
A、免疫原A免疫A, immunogen A immunization
采用6～8周龄BALB/cAnNCrl小鼠或SJL/JorllcoCrl小鼠(上海斯莱克公司)，小鼠在SPF条件下饲养。初次免疫时，将步骤(一)获得的免疫原A(即hTROP2-hFc)用弗氏完全佐剂乳化后腹腔注射0.25mL，即每只小鼠注射50μg免疫原A蛋白。加强免疫时，免疫原A用弗氏不完全佐剂乳化后腹腔注射0.25mL，即每只小鼠注射50微克免疫原A。初次免疫与第一次加强免疫之间间隔2周，以后每次加强免疫之间间隔3周。每次加强免疫1周后采血，用ELISA和FACS检测血清中免疫原A的抗体效价和特异性，结果如图5和表3所示。图5和表3说明，经免疫原A免疫的小鼠的免疫后血清对免疫原A均有不同程度的结合，呈现抗原抗体反应，其中最高稀释度在一百万左右。其中空白对照为1％(w/v)BSA，其中批次指第二次加强免疫后第七天的小鼠血清(Test Blood 2，TB2)，表中的数据为OD450nm值。6 to 8 weeks old BALB/cAnNCrl mice or SJL/JorllcoCrl mice (Shanghai Slack Company) were used, and the mice were raised under SPF conditions. During the first immunization, the immunogen A (hTROP2-hFc) obtained in step (1) was emulsified with Freund's complete adjuvant and injected intraperitoneally with 0.25 mL, that is, 50 μg of immunogen A protein was injected per mouse. When boosting immunization, immunogen A was emulsified with Freund's incomplete adjuvant and injected intraperitoneally with 0.25 mL, that is, 50 micrograms of immunogen A was injected per mouse. There is an interval of 2 weeks between the initial immunization and the first booster, and an interval of 3 weeks between each subsequent booster. Blood was collected one week after each boost, and the antibody titer and specificity of immunogen A in the serum were detected by ELISA and FACS. The results are shown in Figure 5 and Table 3. Figure 5 and Table 3 show that the immunized serum of mice immunized with immunogen A binds to immunogen A to varying degrees, showing an antigen-antibody response, and the highest dilution is about one million. The blank control is 1% (w/v) BSA, and the batch refers to the mouse serum (Test Blood 2, TB2) on the seventh day after the second booster immunization. The data in the table is the OD450nm value.
表3 ELISA检测TROP2蛋白免疫后Balb/c小鼠血清抗体效价Table 3 ELISA to detect serum antibody titer of Balb/c mice after immunization with TROP2 protein
Figure PCTCN2020108720-appb-000010
Figure PCTCN2020108720-appb-000010
Figure PCTCN2020108720-appb-000011
Figure PCTCN2020108720-appb-000011
B、免疫原B免疫B, immunogen B immunization
采用6～8周龄BALB/cAnNCrl小鼠或SJL/JorllcoCrl小鼠(均购自上海斯莱克公司)，小鼠在SPF条件下饲养。按实施例1步骤(二)中的得到的免疫原B(即，含人源TROP2的293F-TROP2稳定细胞系)在T-75细胞培养瓶中扩大培养至90％汇合度，吸尽培养基，用DMEM基础培养基(Invitrogen)洗涤2次，然后用无酶细胞解离液(Invitrogen)37℃处理直至细胞从培养皿壁上可脱落，收集细胞。用DMEM基础培养基洗涤2次，进行细胞计数后将细胞用磷酸盐缓冲液稀释至2×10 7细胞每mL。每只小鼠每次免疫时腹腔注射0.5mL细胞悬液。第一次与第二次免疫之间间隔2周，以后每次免疫间隔3周。除第一次免疫以外，每次免疫1周后采血，用FACS检测血清中抗体效价和特异性。在第二次加强免疫后，FACS检测血清抗体效价达到1:1000以上。 6-8 weeks old BALB/cAnNCrl mice or SJL/JorllcoCrl mice (both purchased from Shanghai Slack Company) were used, and the mice were raised under SPF conditions. The immunogen B obtained in step (2) of Example 1 (ie, the 293F-TROP2 stable cell line containing human TROP2) was expanded to 90% confluence in a T-75 cell culture flask, and the medium was aspirated , Washed twice with DMEM basal medium (Invitrogen), and then treated with enzyme-free cell dissociation solution (Invitrogen) at 37°C until the cells could be detached from the wall of the culture dish, and the cells were collected. Wash twice with DMEM basal medium, and after cell counting, the cells are diluted with phosphate buffer to 2×10 7 cells per mL. Each mouse was intraperitoneally injected with 0.5 mL of cell suspension during each immunization. The interval between the first and second immunizations is 2 weeks, and the interval between subsequent immunizations is 3 weeks. Except for the first immunization, blood was collected one week after each immunization, and the antibody titer and specificity in the serum were detected by FACS. After the second booster immunization, the serum antibody titer detected by FACS reached more than 1:1000.
C、杂交瘤细胞的制备和筛选C. Preparation and screening of hybridoma cells
A～B步骤在各自完成前，将所选择的每只小鼠进行最后一次免疫腹腔注射100微克纯化的免疫原A(针对免疫原A进行免疫反应的小鼠)或含有人源TROP2的HEK293稳定细胞系(针对免疫原B进行免疫反应的小鼠)，5天后处死小鼠，收集脾细胞。加入NH 4OH至终浓度1％(v/v)，裂解脾细胞中掺杂的红细胞，获得脾细胞悬液。用DMEM基础培养基(购自invitrogen)1000转每分钟离心清洗细胞3次，然后按照活细胞数目5:1比率与小鼠骨髓瘤细胞SP2/0(购自ATCC)混合，采用高效电融合方法(参见METHODS IN ENZYMOLOGY,VOL.220)进行细胞融合。融合后的细胞稀释到含20％(v/v)胎牛血清、1×HAT的DMEM培养基中。然后按1×10 5细胞/200微升每孔加入到96孔细胞培养板中，放入5％(v/v)CO 2、37℃培养箱中培养。14天后用ELISA和Acumen(微孔板细胞检测法)筛选细胞融合板上清，将ELISA中OD450nm>1.0和Acumen中MFI值>100的阳性克隆扩增到24孔板，在含10％(v/v)HT胎牛血清的DMEM培养基中，于37℃、5％(v/v)CO 2条件下扩大培养。培养3天后取24孔板中扩大培养的培养液进行离心，收集上清液，对上清液进行抗体亚型分析。用ELISA、FACS确定其对TROP2蛋白和TROP2阳性细胞的结合活性(结合活性的检测方法分别参见实施例3A和实施例3B中的相关内容)，用小鼠源TROP2抗体-MMAF间接细胞毒杀伤实验评估间接细胞毒杀伤活性(间接细胞毒杀伤活性检测方法参见实施例4中的相关内容)。根据24 孔板筛选结果，挑选ELISA实验中OD450nm>1.0、FACS实验中MFI值>50和间接细胞毒杀伤实验中杂交瘤细胞培养上清对TROP2阳性细胞杀伤率达到50％的杂交瘤细胞为符合条件的阳性克隆，选择符合条件的杂交瘤细胞用有限稀释法在96孔板进行亚克隆，在含10％(v/v)胎牛血清的DMEM培养基中，37℃、5％(v/v)CO 2条件下培养。亚克隆后10天用ELISA和Acumen进行初步筛选，挑选单个阳性单克隆扩增到24孔板继续培养。3天后用FACS确定抗原结合活性，并用小鼠源TROP2抗体-MMAF间接细胞毒杀伤实验评估间接细胞毒杀伤活性(评估标准为ELISA实验中OD450nm>1.0、FACS实验中MFI值>50和间接细胞毒杀伤实验中杂交瘤细胞培养上清对TROP2阳性细胞杀伤率达到50％及以上)。根据24孔板样品检测结果，挑选出最优的克隆，并于含10％(v/v)胎牛血清的DMEM培养基中，在37℃、5％(v/v)CO 2条件下将该最优的克隆进行扩大培养，液氮冻存即得本发明杂交瘤细胞，并可用于后续的先导抗体生产和纯化。 Before each step is completed, each selected mouse will be immunized by intraperitoneal injection of 100 micrograms of purified immunogen A (mouse immune response to immunogen A) or HEK293 containing human TROP2 stable Cell line (mice that reacted to immunogen B). After 5 days, the mice were sacrificed and splenocytes were collected. Add NH 4 OH to a final concentration of 1% (v/v) to lyse the red blood cells adulterated in the spleen cells to obtain a spleen cell suspension. Wash the cells with DMEM basal medium (purchased from invitrogen) by centrifugation at 1000 rpm for 3 times, and then mix with mouse myeloma cells SP2/0 (purchased from ATCC) at a ratio of 5:1 for the number of viable cells, using a highly efficient electrofusion method (Refer to METHOD IN ENZYMOLOGY, VOL. 220) for cell fusion. The fused cells were diluted into DMEM medium containing 20% (v/v) fetal bovine serum and 1×HAT. Then add 1×10 5 cells/200 microliters per well to a 96-well cell culture plate, and place it in a 5% (v/v) CO 2 , 37°C incubator for culture. After 14 days, ELISA and Acumen (microwell plate cell detection method) were used to screen the supernatant of the cell fusion plate. The positive clones with OD450nm>1.0 in ELISA and MFI value>100 in Acumen were amplified to 24-well plates, and the cells contained 10% (v /v) In DMEM medium of HT fetal bovine serum, expand culture at 37°C and 5% (v/v) CO 2 . After 3 days of culture, the culture solution expanded in the 24-well plate was centrifuged, the supernatant was collected, and the supernatant was analyzed for antibody subtypes. ELISA and FACS were used to determine its binding activity to TROP2 protein and TROP2 positive cells (for the detection methods of binding activity, please refer to the relevant content in Example 3A and Example 3B, respectively), and indirect cytotoxic killing experiment with mouse TROP2 antibody-MMAF Evaluation of indirect cytotoxic activity (see related content in Example 4 for the detection method of indirect cytotoxic activity). According to the results of the 24-well plate screening, select the hybridoma cells whose OD450nm>1.0 in the ELISA experiment, the MFI value>50 in the FACS experiment, and the hybridoma cell culture supernatant in the indirect cytotoxicity experiment to 50% of the TROP2 positive cells. Condition positive clones, select qualified hybridoma cells and subclone them in 96-well plates by limiting dilution method, in DMEM medium containing 10% (v/v) fetal bovine serum, 37°C, 5% (v/v) v) Cultivation under CO 2 conditions. Ten days after subcloning, ELISA and Acumen were used for preliminary screening, and a single positive single clone was selected and amplified to a 24-well plate to continue culture. After 3 days, FACS was used to determine the antigen binding activity, and the indirect cytotoxic killing test of mouse-derived TROP2 antibody-MMAF was used to evaluate the indirect cytotoxic killing activity (the evaluation criteria are OD450nm>1.0 in the ELISA test, MFI value>50 in the FACS test, and indirect cytotoxicity In the killing experiment, the killing rate of hybridoma cell culture supernatant on TROP2 positive cells reached 50% and above). According to the test results of the 24-well plate samples, the best clones were selected and placed in DMEM medium containing 10% (v/v) fetal bovine serum at 37°C and 5% (v/v) CO 2 The optimal clone is expanded and cultured and frozen in liquid nitrogen to obtain the hybridoma cells of the present invention, which can be used for subsequent lead antibody production and purification.
实施例2先导抗体的生产和纯化Example 2 Production and purification of lead antibody
由于杂交瘤细胞产生的抗体浓度较低，大约仅1-10μg/mL，浓度变化较大，且培养基中细胞培养所产生的多种蛋白和培养基所含胎牛血清成分对很多生物活性分析方法都有不同程度的干扰，因此需要进行小规模(1-5mg)抗体生产纯化。Because the antibody concentration produced by hybridoma cells is low, only about 1-10μg/mL, the concentration varies greatly, and the various proteins produced by the cell culture in the medium and the fetal bovine serum components contained in the medium have many biological activity analysis The methods have different degrees of interference, so small-scale (1-5mg) antibody production and purification is required.
将实施例1所得的杂交瘤细胞接种到T-75细胞培养瓶并用生产培养基(Hybridoma serum free medium，购自Invitrogen)驯化传代3代。待其生长状态良好，接种细胞培养转瓶。每个2升的培养转瓶中加入200mL生产培养基，接种细胞密度为1.0×10 5/mL。盖紧瓶盖，将转瓶置于37℃培养箱中的转瓶机上，转速3转/分钟。连续旋转培养14天后，收集细胞培养液，过滤去除细胞，并用0.45μm的滤膜过滤至培养上清液澄清，得澄清的杂交瘤细胞的培养上清液。澄清的杂交瘤细胞的培养上清液可立即进行纯化或于-30℃冻存。 The hybridoma cells obtained in Example 1 were inoculated into T-75 cell culture flasks and used production medium (Hybridoma serum free medium, purchased from Invitrogen) for domestication and passage for 3 generations. When it grows well, inoculate the cell culture spinner flask. Add 200mL production medium to each 2L culture spinner flask, and the inoculation cell density is 1.0×10 5 /mL. Tighten the cap, and place the spinner on the spinner in the 37°C incubator at a speed of 3 revolutions/min. After continuous rotation culture for 14 days, the cell culture solution was collected, filtered to remove the cells, and filtered with a 0.45 μm filter membrane until the culture supernatant was clarified to obtain a clarified hybridoma cell culture supernatant. The culture supernatant of the clarified hybridoma cells can be purified immediately or frozen at -30°C.
将获得的培养上清液(200mL)中的TROP2抗体用2mL蛋白A柱(GE Healthcare)纯化。蛋白A柱先用平衡缓冲液(PBS磷酸缓冲液，pH7.4)平衡，然后将培养上清液上样到蛋白A柱，控制流速在3mL/分钟。上样完毕后用平衡缓冲液清洗蛋白A柱，平衡缓冲液的体积为蛋白A柱柱床体积的4倍。用洗脱液(0.1M柠檬酸钠缓冲液，pH3.5)洗脱结合在蛋白A柱上的TROP2抗体，用紫外检测器监测洗脱情况(A280nm紫外吸收峰)。收集洗脱的抗体，加入10％(v/v)1.0M Tris-HCl缓冲液中和pH，然后立即用PBS磷酸缓冲液透析过夜，第二天换液1次并继续透析3小时。收集透析后的TROP2抗体，用0.22μm的滤器进行无菌过滤，无菌保存，即得纯化的TROP2抗体。将纯化的TROP2抗体进行蛋白浓度(A280nm/1.4)、纯度、内毒(Lonza试剂盒)等检测分析，结果如表4所示，表4说明，抗体最终产品内毒素浓度在1.0EU/mg以内。The TROP2 antibody in the obtained culture supernatant (200 mL) was purified with a 2 mL protein A column (GE Healthcare). The protein A column was first equilibrated with equilibration buffer (PBS phosphate buffer, pH 7.4), and then the culture supernatant was loaded onto the protein A column, and the flow rate was controlled at 3 mL/min. After loading the sample, wash the protein A column with equilibration buffer. The volume of the equilibration buffer is 4 times the volume of the protein A column bed. The TROP2 antibody bound to the protein A column was eluted with an eluent (0.1M sodium citrate buffer, pH 3.5), and the elution was monitored with an ultraviolet detector (A280nm ultraviolet absorption peak). Collect the eluted antibodies, add 10% (v/v) 1.0M Tris-HCl buffer to neutralize the pH, and then immediately dialyze with PBS phosphate buffer overnight, change the medium once the next day and continue dialysis for 3 hours. Collect the dialyzed TROP2 antibody, perform aseptic filtration with a 0.22μm filter, and store aseptically to obtain purified TROP2 antibody. The purified TROP2 antibody was tested and analyzed for protein concentration (A280nm/1.4), purity, and endotoxicity (Lonza kit). The results are shown in Table 4. Table 4 shows that the endotoxin concentration of the final antibody product is within 1.0EU/mg .
表4纯化的TROP2抗体检测分析Table 4 Purified TROP2 antibody detection analysis
Figure PCTCN2020108720-appb-000012
Figure PCTCN2020108720-appb-000012
实施例3先导抗体的检定Example 3 Assay of lead antibody
A、酶联免疫吸附实验(ELISA)检测TROP2抗体与TROP2蛋白的结合A. Enzyme-linked immunosorbent assay (ELISA) detects the binding of TROP2 antibody to TROP2 protein
对实施例2所得的纯化的TROP2抗体进行与人源TROP2-hFc蛋白(免疫原A)进行反应。The purified TROP2 antibody obtained in Example 2 was reacted with human TROP2-hFc protein (immunogen A).
将实施例1获得的纯化的免疫原A[其制备方法参见实施例1步骤(一)]用PBS稀释到终浓度1.0μg/mL，然后以100μL每孔加到96孔ELISA板。用塑料膜封好4℃孵育过夜，第二天用洗板液[含0.01％(v/v)Tween20的PBS]洗板2次，加入封闭液[含0.01％(v/v)Tween20和1％(w/w)BSA的PBS]室温封闭2小时。倒掉封闭液，加入实施例2所得的纯化的TROP2抗体100μL每孔。37℃孵育2小时后，用洗板液[含0.01％(v/v)Tween20的PBS]洗板3次。加入HRP(辣根过氧化物酶)标记的二抗(购自Sigma，CatA0168)，37℃孵育2小时后，用洗板液[含0.01％(v/v)Tween20的PBS]洗板3次。加入TMB底物100μL每孔，室温孵育30分钟后，加入终止液(1.0N HCl)100μL每孔。用ELISA读板机(SpectraMax 384plus，购自Molecular Device)读取A 450nm数值，结果如图6和表5所示，图6和表5说明，纯化的TROP2抗体与TROP2重组蛋白在ELISA水平结合。表5中IgG对照(上海睿智化学研究有限公司生产)为对照小鼠IgG，表中的数据为OD 450nm值，Blank的含义为板中只有PBS缓冲液时的OD 450nm值。其中，hRS7序列来自专利US8758752，根据专利中制备方法由上海睿智化学研究有限公司表达生产。 The purified immunogen A obtained in Example 1 [see step (1) of Example 1 for the preparation method] was diluted with PBS to a final concentration of 1.0 μg/mL, and then 100 μL per well was added to a 96-well ELISA plate. Cover with plastic film and incubate overnight at 4°C. Wash the plate twice with plate washing solution [PBS containing 0.01% (v/v) Tween 20] on the second day, and add blocking solution [containing 0.01% (v/v) Tween 20 and 1 %(W/w)BSA in PBS] for 2 hours at room temperature. The blocking solution was discarded, and 100 μL of the purified TROP2 antibody obtained in Example 2 was added to each well. After incubating for 2 hours at 37°C, the plate was washed 3 times with a plate washing solution [PBS containing 0.01% (v/v) Tween20]. Add HRP (horseradish peroxidase) labeled secondary antibody (purchased from Sigma, CatA0168), after incubating at 37°C for 2 hours, wash the plate 3 times with a plate washing solution [PBS containing 0.01% (v/v) Tween20] . Add 100μL of TMB substrate per well, after incubating at room temperature for 30 minutes, add 100μL of stop solution (1.0N HCl) per well. The value of A 450nm was read with an ELISA plate reader (SpectraMax 384plus, purchased from Molecular Device). The results are shown in Figure 6 and Table 5. Figure 6 and Table 5 show that the purified TROP2 antibody binds to the TROP2 recombinant protein at the ELISA level. The IgG control (produced by Shanghai Ruizhi Chemical Research Co., Ltd.) in Table 5 is a control mouse IgG, the data in the table is the OD 450nm value, and the meaning of Blank is the OD 450nm value when there is only PBS buffer in the plate. Among them, the hRS7 sequence comes from the patent US8758752, which is expressed and produced by Shanghai Ruizhi Chemical Research Co., Ltd. according to the preparation method in the patent.
表5 ELISA检测TROP2抗体与人TROP2-hFc蛋白的结合反应Table 5 ELISA detection of the binding reaction between TROP2 antibody and human TROP2-hFc protein
Figure PCTCN2020108720-appb-000013
Figure PCTCN2020108720-appb-000013
B、流式细胞实验(FACS)检测TROP2抗体与TROP2表达细胞的结合B. Flow cytometry (FACS) to detect the binding of TROP2 antibody to TROP2 expressing cells
将实施例1步骤(二)中所述含有编码人源TROP2全长氨基酸序列的核苷酸序列的pIRES质粒转染CHOK1细胞株得含人源TROP2的CHOK1稳定细胞株(此处称为CHOK1-hTROP2稳定细胞株)。类似地，将带有食蟹猴TROP2全长基因(其氨基酸序列 如序列表SEQ ID NO:18所示)的pIRES质粒(其制备方法与实施例1步骤(一)“免疫原A的制备”中带有人源IgG Fc片段(hFc)的pCpC载体的制备方法相同)转染CHOK1细胞株得含食蟹猴TROP2的CHOK1稳定细胞株(此处称为CHOK1-cTROP2稳定细胞株)。将带有小鼠源TROP2全长基因(其氨基酸序列如序列表SEQ ID NO:19所示)的pIRES质粒(其制备方法与实施例1步骤(一)“免疫原A的制备”中带有人源IgG Fc片段(hFc)的pCpC载体的制备方法相同)转染CHOK1细胞株得含小鼠TROP2的CHOK1稳定细胞株(此处称为CHOK1-mTROP2稳定细胞株)。The pIRES plasmid containing the nucleotide sequence encoding the full-length amino acid sequence of human TROP2 described in step (2) of Example 1 was transfected into the CHOK1 cell line to obtain a CHOK1 stable cell line containing human TROP2 (herein referred to as CHOK1- hTROP2 stable cell line). Similarly, the pIRES plasmid with the full-length gene of cynomolgus monkey TROP2 (its amino acid sequence is shown in SEQ ID NO: 18 in the sequence list) (the preparation method is the same as that of Example 1 step (1) "Preparation of immunogen A" The preparation method of the pCpC vector carrying the human IgG Fc fragment (hFc) is the same.) The CHOK1 cell line was transfected to obtain the CHOK1 stable cell line containing the cynomolgus TROP2 (herein referred to as the CHOK1-cTROP2 stable cell line). The pIRES plasmid with the mouse-derived TROP2 full-length gene (its amino acid sequence is shown in SEQ ID NO: 19 in the sequence list) (the preparation method is the same as that of Example 1 step (1) "Preparation of immunogen A" with human The preparation method of the pCpC vector derived from the IgG Fc fragment (hFc) is the same.) The CHOK1 cell line is transfected to obtain the CHOK1 stable cell line containing mouse TROP2 (herein referred to as the CHOK1-mTROP2 stable cell line).
用FACS检测上述稳定细胞株中的蛋白表达情况，检测方法参见实施例1步骤(二)“免疫原B的制备”中鉴定HEK293-TROP2稳定细胞株的方法。检测结果如表6和图2～4所示，图2～4中横坐标为细胞荧光强度，纵坐标为细胞数。其中，CHOK1-hTROP2 2B2为用来筛选的人TROP2表达细胞株，其FACS筛选检测结果如图2所示；CHOK1-cTROP2 2B2C2为用来筛选的食蟹猴TROP2表达细胞株，其FACS筛选检测结果如图3所示；CHOK1-mTROP2 6F6为用来筛选的小鼠TROP2表达细胞株，其FACS筛选检测结果如图4所示。表6的结果说明，CHOK1-hTROP2稳定细胞株、CHOK1-cTROP2稳定细胞株和CHOK1-mTROP2稳定细胞株的细胞膜上分别过表达人、食蟹猴或小鼠的TROP2蛋白，其可以用于筛选TROP2抗体。FACS was used to detect the protein expression in the above stable cell lines. For the detection method, refer to the method for identifying the HEK293-TROP2 stable cell line in step (2) "Preparation of immunogen B" in Example 1. The test results are shown in Table 6 and Figures 2 to 4. In Figures 2 to 4, the abscissa is the cell fluorescence intensity, and the ordinate is the number of cells. Among them, CHOK1-hTROP2 2B2 is the human TROP2 expression cell line used for screening, and the FACS screening test results are shown in Figure 2. CHOK1-cTROP2 2B2C2 is the cynomolgus monkey TROP2 expression cell line used for screening, and the FACS screening test results As shown in Figure 3; CHOK1-mTROP2 6F6 is the mouse TROP2 expressing cell line used for screening, and the FACS screening test results are shown in Figure 4. The results in Table 6 indicate that the cell membranes of the CHOK1-hTROP2 stable cell line, the CHOK1-cTROP2 stable cell line, and the CHOK1-mTROP2 stable cell line respectively overexpress the human, cynomolgus monkey or mouse TROP2 protein, which can be used to screen TROP2 antibody.
表6人/猴/小鼠TROP2转染的CHOK1细胞FACS筛选检测结果Table 6 FACS screening test results of CHOK1 cells transfected with human/monkey/mouse TROP2
Figure PCTCN2020108720-appb-000014
Figure PCTCN2020108720-appb-000014
将上述CHOk1-hTROP2稳定细胞株、CHOK1-cTROP2稳定细胞株、CHOK1-mTROP2稳定细胞株(即表6所示的CHOK1-hTROP2 2B2、CHOK1-cTROP2 2B2C2和CHOK1-mTROP2 6F6)以及CHOK1细胞分别在T-75细胞培养瓶中扩大培养至90％汇合度，吸尽培养基，用HBSS缓冲液(Hanks Balanced Salt Solution，购自Invitrogen)洗涤2次，然后用无酶细胞解离液(Versene solution，购自Life technology公司)处理和收集细胞。用HBSS缓冲液洗涤细胞2次，进行细胞计数后将细胞用HBSS缓冲液稀释至2×10 6个细胞/mL，加入10％山羊血清封闭液，所述百分比为质量百分比，冰上孵育30 分钟，然后用HBSS缓冲液离心洗涤2次。将收集的细胞用FACS缓冲液(HBSS+1％BSA，所述百分比为质量百分比)悬浮至2×10 6个细胞/mL，按每孔100微升加入到96孔FACS反应板中，加入实施例2所得的纯化的TROP2抗体待测样品每孔100微升，冰上孵育2小时。用FACS缓冲液离心洗涤2次，加入每孔100微升荧光(Alexa 488)标记的二抗(购自Invitrogen)，冰上孵育1小时。用FACS缓冲液离心洗涤3次，加入每孔100微升固定液[4％(v/v)多聚甲醛]重悬细胞，10分钟后用FACS缓冲液离心洗涤2次。用100微升FACS缓冲液悬浮细胞，用FACS(FACS Calibur，购自BD公司)检测和分析结果。通过软件(CellQuest)进行数据分析，得到细胞的平均荧光密度(MFI)。再通过软件(GraphPad Prism5)分析，进行数据拟合，计算EC50值。分析结果如表7以及图7～10所示，图7～10的数据为细胞的平均荧光密度(MFI)。表7中的数据为根据MFI计算得到的EC50值。表7说明，实施例2所得的纯化的TROP2抗体可结合细胞表面的hTROP2和cTROP2蛋白，但基本不结合mTROP2蛋白。这些抗体的效果均优于hRS7或者与之相当。 The above-mentioned CHOk1-hTROP2 stable cell line, CHOK1-cTROP2 stable cell line, CHOK1-mTROP2 stable cell line (i.e. CHOK1-hTROP2 2B2, CHOK1-cTROP2 2B2C2 and CHOK1-mTROP2 6F6 shown in Table 6) and CHOK1 cells were placed in T -75 cell culture flasks were expanded to 90% confluence, the medium was aspirated, washed twice with HBSS buffer (Hanks Balanced Salt Solution, purchased from Invitrogen), and then with enzyme-free cell dissociation solution (Versene solution, purchased From Life Technology Corporation) process and collect cells. Wash the cells twice with HBSS buffer. After counting the cells, dilute the cells with HBSS buffer to 2×10 6 cells/mL, add 10% goat serum blocking solution, the percentage is the mass percentage, and incubate on ice for 30 minutes , And then washed twice with HBSS buffer by centrifugation. Suspend the collected cells in FACS buffer (HBSS+1%BSA, the percentage is mass percentage) to 2×10 6 cells/mL, add 100 μl per well to a 96-well FACS reaction plate, and add The purified TROP2 antibody test sample obtained in Example 2 was 100 microliters per well and incubated on ice for 2 hours. Wash with FACS buffer by centrifugation twice, add 100 microliters of fluorescent (Alexa 488) labeled secondary antibody (purchased from Invitrogen) per well, and incubate on ice for 1 hour. The cells were washed 3 times by centrifugation with FACS buffer, 100 microliters of fixative [4% (v/v) paraformaldehyde] was added to each well to resuspend the cells, and then washed twice with FACS buffer by centrifugation after 10 minutes. The cells were suspended in 100 microliters of FACS buffer, and the results were detected and analyzed by FACS (FACS Calibur, purchased from BD). Data analysis was performed by the software (CellQuest) to obtain the average fluorescence density (MFI) of the cells. Then analyze by software (GraphPad Prism5), perform data fitting, and calculate EC50 value. The analysis results are shown in Table 7 and Figs. 7-10. The data in Figs. 7-10 are the average fluorescence density (MFI) of the cells. The data in Table 7 are EC50 values calculated based on MFI. Table 7 shows that the purified TROP2 antibody obtained in Example 2 can bind to hTROP2 and cTROP2 proteins on the cell surface, but does not substantially bind to mTROP2 protein. The effects of these antibodies are better than or comparable to hRS7.
表7 FACS分析TROP2抗体与人/猴/小鼠TROP2表达细胞株结合活性Table 7 FACS analysis of the binding activity of TROP2 antibody to human/monkey/mouse TROP2 expressing cell lines
Figure PCTCN2020108720-appb-000015
Figure PCTCN2020108720-appb-000015
实施例4 TROP2抗体药物偶联物的细胞杀伤活性实验Example 4 Cell killing activity experiment of TROP2 antibody drug conjugate
将实施例2所得的纯化的TROP2抗体经过pH 6.5～8.5的硼酸钠缓冲液透析后，加入三(2-羧乙基)膦(TCEP)，其中TCEP与纯化的TROP2抗体的摩尔比比率为2，室温下还原1小时，得反应液A。将反应液A经过G25柱脱盐(购自GE)，去除多余的TCEP，得反应液B。向反应液B中加入MC-MMAF(购自南京联宁)，其中MC-MMAF与纯化的TROP2抗体的摩尔比比率为5，室温下反应4小时。再加入半胱氨酸用以中和多余的MC-MMAF，并通过G25柱脱盐除去多余的小分子。得到纯化的TROP2抗体药物偶联物(偶联方法参见Doronina，2006，Bioconjugate Chem.17,114-124)。通过HPLC-HIC或LC-MS分析药物的交联率、纯度等参数后，进行细胞毒活性的分析。所有抗体药物偶联物的药物交联率(DAR)约为8(测定DAR的方法为本领域常规)。其中，DAR(drug antibody ratio)指抗体偶联后一个抗体分子上携带的小分子药物的平均数量(参考文献mAbs 3:2,161-172，DOI:10.4161/mabs.3.2.14960)。其中，HPLC-HIC,HPL-SEC和LC-Ms 的分析条件如下：After the purified TROP2 antibody obtained in Example 2 was dialyzed with sodium borate buffer of pH 6.5～8.5, tris(2-carboxyethyl)phosphine (TCEP) was added, wherein the molar ratio of TCEP to the purified TROP2 antibody was 2 , Reduce at room temperature for 1 hour to obtain reaction solution A. The reaction solution A is desalted through a G25 column (purchased from GE), and excess TCEP is removed to obtain reaction solution B. MC-MMAF (purchased from Nanjing Lianning) was added to the reaction solution B, where the molar ratio of MC-MMAF to the purified TROP2 antibody was 5, and the reaction was carried out at room temperature for 4 hours. Then add cysteine to neutralize the excess MC-MMAF, and pass through the G25 column desalting to remove the excess small molecules. The purified TROP2 antibody drug conjugate is obtained (refer to Doronina, 2006, Bioconjugate Chem. 17, 114-124 for coupling method). After analyzing the cross-linking rate, purity and other parameters of the drug by HPLC-HIC or LC-MS, the cytotoxic activity is analyzed. The drug cross-linking rate (DAR) of all antibody-drug conjugates is about 8 (the method of determining DAR is conventional in the art). Among them, DAR (drug antibody ratio) refers to the average number of small molecule drugs carried on an antibody molecule after antibody coupling (reference mAbs 3:2, 161-172, DOI: 10.4161/mabs.3.2.14960). Among them, the analysis conditions of HPLC-HIC, HPL-SEC and LC-Ms are as follows:
A.HPLC-HIC分析条件A. HPLC-HIC analysis conditions
色谱柱：Thermol MabPac,HIC-ButylColumn: ThermoMabPac, HIC-Butyl
HPLC：E2695,WatersHPLC: E2695, Waters
流动相A：1.5M硫酸铵，0.025M磷酸钠水溶液，pH＝6.95Mobile phase A: 1.5M ammonium sulfate, 0.025M sodium phosphate aqueous solution, pH=6.95
流动相B：0.025M磷酸钠水溶液，25％(v/v)异丙醇水溶液Mobile phase B: 0.025M sodium phosphate aqueous solution, 25% (v/v) isopropanol aqueous solution
流速：0.5mL/minFlow rate: 0.5mL/min
梯度：0～2min:80％流动相A+20％流动相BGradient: 0～2min: 80% mobile phase A+20% mobile phase B
2～15min:80％～60％流动相A+20％～40％流动相B2～15min: 80%～60% mobile phase A+20%～40% mobile phase B
15～18min:60％～30％流动相A+40％～70％流动相B15～18min: 60%～30% mobile phase A+40%～70% mobile phase B
18～20min:70％流动相B+30％流动相A18～20min: 70% mobile phase B+30% mobile phase A
B.HPLC-SEC分析条件B. HPLC-SEC analysis conditions
色谱柱：TSKgel 3000SWxlColumn: TSKgel 3000SWxl
HPLC:FTN-R,WatersHPLC: FTN-R, Waters
流动相A：1×PBS缓冲液+10％异丙醇Mobile phase A: 1×PBS buffer+10% isopropanol
流速：0.5mL/minFlow rate: 0.5mL/min
梯度：0～20min:100％流动相AGradient: 0～20min: 100% mobile phase A
C.LC-MS分析条件C. LC-MS analysis conditions
色谱柱：Acquity UPLC BEH200,SEC 1.7μm,4.6×300mmColumn: Acquity UPLC BEH200, SEC 1.7μm, 4.6×300mm
HPLC:Acquity UPLC H-Class Bio,WatersHPLC: Acquity UPLC H-Class Bio, Waters
MS:Triple TOF 5600,TOF 5600,AB SciexMS: Triple TOF 5600, TOF 5600, AB Sciex
流动相A：0.1％甲酸，25％乙腈水溶液。Mobile phase A: 0.1% formic acid, 25% acetonitrile aqueous solution.
流速：0.2mL/minFlow rate: 0.2mL/min
梯度：0～25min:流动相AGradient: 0～25min: mobile phase A
将获得的纯化的TROP2抗体药物偶联物分别用完全培养基进行梯度稀释，96孔细胞培养板以5000细胞/孔加入90微升TROP2阳性的MDA-MB-468细胞株(购自ATCC，货号#HTB-132)细胞悬液过夜培养后，每孔分别加入10微升不同浓度的纯化的TROP2抗体药物偶联物的稀释液，继续培养5天后，用CellTiter-Glo试剂盒(购自Promega，使用方法参照产品说明书)检测细胞活力。同时选用TROP2阴性的肿瘤细胞系HCC1395(购自ATCC，货号#CRL-2324)进行细胞杀伤活性检测，方法同上。结果如表8以及图11-12所示，其中表8的EC50指药物作用后，细胞的活性受到抑制的半数有效量，能够通过检测细胞的活性从而反映细胞杀伤活性。其中，图11为纯化的TROP2抗体药物偶 联物对TROP2阳性的肿瘤细胞系MDA-MB-468的细胞杀伤活性检测，图12为纯化的TROP2抗体药物偶联物对TROP2阴性的乳腺癌肿瘤细胞系HCC1395的细胞杀伤活性检测。结果说明，上述所得纯化的TROP2抗体药物偶联物对TROP2阳性的细胞有杀伤作用。The purified TROP2 antibody-drug conjugates obtained were serially diluted with complete medium, and 90 microliters of TROP2-positive MDA-MB-468 cell line (purchased from ATCC, catalog number) was added to a 96-well cell culture plate at 5000 cells/well. #HTB-132) After the cell suspension was cultured overnight, add 10 microliters of different concentrations of purified TROP2 antibody-drug conjugate dilutions to each well. After culturing for 5 days, use the CellTiter-Glo kit (purchased from Promega, For the method of use, refer to the product manual) to detect cell viability. At the same time, the TROP2-negative tumor cell line HCC1395 (purchased from ATCC, catalog #CRL-2324) was used for cell killing activity detection, and the method was the same as above. The results are shown in Table 8 and Figures 11-12, where the EC50 in Table 8 refers to the half-effective amount that inhibits cell activity after drug action, which can reflect cell killing activity by detecting cell activity. Among them, Figure 11 shows the cell killing activity of the purified TROP2 antibody drug conjugate against TROP2-positive tumor cell line MDA-MB-468, and Figure 12 shows the purified TROP2 antibody drug conjugate against TROP2-negative breast cancer tumor cells Detection of cell killing activity of line HCC1395. The results indicate that the purified TROP2 antibody drug conjugate obtained above has a killing effect on TROP2-positive cells.
表8细胞杀伤实验检测纯化的TROP2抗体药物偶联物对TROP2阳性细胞的特异性杀伤作用Table 8 Cell killing experiment detects the specific killing effect of purified TROP2 antibody drug conjugate on TROP2 positive cells
Figure PCTCN2020108720-appb-000016
Figure PCTCN2020108720-appb-000016
实施例5竞争性ELISA检测分析TROP2抗体与抗原的表位分布Example 5 Competitive ELISA detection and analysis of the epitope distribution of TROP2 antibody and antigen
为了鉴定抗体对抗原的结合位点，采用竞争性ELISA的方法对TROP2抗体进行分组。In order to identify the binding site of the antibody to the antigen, a competitive ELISA method was used to group the TROP2 antibodies.
纯化的待测抗体(即实施例2所得的纯化的TROP2抗体，其克隆号分别为11E8E6D11、44A5F9)用PBS稀释至1μg/mL，以50μL/孔包被96孔高吸附酶标板，4℃过夜包被后用250微升封闭液[含有0.01％(v/v)Tween20和1％(w/w)BSA的PBS]进行室温一小时封闭，每孔加入0.05μg/mL的生物素标记的重组TROP2蛋白。同时加入5μg/mL的竞争抗体，即实施例2所得的纯化的TROP2抗体，其克隆号分别为11E8E6D1144A5F9，并于25-37℃孵育1-2小时。用洗板液[含有0.01％(v/v)Tween20的PBS]洗板3次，加入HRP(辣根过氧化物酶)标记的链亲和素(购自Sigma)。37℃孵育0.5小时后，用洗板液[含有0.01％(v/v)Tween20的PBS]洗板3次。加入TMB底物100μL每孔，室温孵育30分钟后，加入终止液(1.0N HCl)100μL每孔。用ELISA读板机(SpectraMax 384plus，购自Molecular Device)读取A 450nm数值。根据A 450nm数值，计算出抗体相互之间的竞争率，结果如表9所示。竞争率的数值越高，表示两个抗体的抗原表面越是接近。 The purified antibody to be tested (ie the purified TROP2 antibody obtained in Example 2, the clone numbers of which are 11E8E6D11 and 44A5F9, respectively) were diluted to 1μg/mL with PBS, and a 96-well high-absorption ELISA plate was coated with 50μL/well at 4°C After overnight coating, use 250 microliters of blocking solution [PBS containing 0.01% (v/v) Tween 20 and 1% (w/w) BSA] for one hour at room temperature, and add 0.05 μg/mL biotin-labeled to each well Recombinant TROP2 protein. At the same time, 5 μg/mL of competitive antibody, namely the purified TROP2 antibody obtained in Example 2, with clone numbers 11E8E6D1144A5F9 were added, and incubated at 25-37°C for 1-2 hours. The plate was washed 3 times with a plate washing solution [PBS containing 0.01% (v/v) Tween20], and HRP (horseradish peroxidase) labeled streptavidin (purchased from Sigma) was added. After incubating at 37°C for 0.5 hours, the plate was washed 3 times with a plate washing solution [PBS containing 0.01% (v/v) Tween20]. Add 100μL of TMB substrate per well, after incubating at room temperature for 30 minutes, add 100μL of stop solution (1.0N HCl) per well. The A 450nm value was read with an ELISA plate reader (SpectraMax 384plus, purchased from Molecular Device). According to the value of A 450nm , the competition rate between the antibodies was calculated. The results are shown in Table 9. The higher the value of the competition rate, the closer the antigen surfaces of the two antibodies are.
表9 TROP2抗体相互之间的竞争率Table 9 Competition rate between TROP2 antibodies
 To 11E8E6D1111E8E6D11 44A5F944A5F9
11E8E6D1111E8E6D11 83％83% 11％11%
44A5F944A5F9 93％93% 62％62%
其中，左第一列为包被抗体，浓度为1μg/mL；上第一行为竞争抗体，浓度为5μg/mL。Among them, the first column from the left is the coated antibody, with a concentration of 1μg/mL; the first column above is the competing antibody, with a concentration of 5μg/mL.
结果说明，11E8E6D11可以竞争44A5F9，但是44A5F9不可以竞争11E8E6D11，44A5F9和11E8E6D11为不同表位。The results show that 11E8E6D11 can compete with 44A5F9, but 44A5F9 cannot compete with 11E8E6D11. 44A5F9 and 11E8E6D11 are different epitopes.
实施例6轻重链可变区氨基酸序列测定Example 6 Determination of amino acid sequence of variable region of light and heavy chain
总RNA分离：通过离心搜集实施例2所选的先导抗体所对应的实施例1所得的杂交瘤细胞5×10 7个，加入1mL Trizol混匀并转移到1.5mL离心管中，室温静置5分钟。加0.2mL氯仿，振荡15秒，静置10分钟后于4℃，12000g离心5分钟，取上清转移到新的1.5mL离心管中。加入0.5mL异丙醇，将管中液体轻轻混匀，室温静置10分钟后于4℃，12000g离心15分钟，弃上清。加入1mL 75％(v/v)乙醇，轻轻洗涤沉淀，4℃，12000g离心5分钟后弃上清，将沉淀物晾干，加入DEPC处理过的H 2O溶解(55℃水浴促进溶解10分钟)，即得总RNA。 Total RNA isolation: Collect 5×10 7 hybridoma cells obtained in Example 1 corresponding to the lead antibody selected in Example 2 by centrifugation, add 1 mL Trizol to mix and transfer to a 1.5 mL centrifuge tube, and let stand at room temperature 5 minute. Add 0.2mL chloroform, shake for 15 seconds, let stand for 10 minutes, centrifuge at 4°C, 12000g for 5 minutes, take the supernatant and transfer to a new 1.5mL centrifuge tube. Add 0.5 mL of isopropanol, gently mix the liquid in the tube, let it stand at room temperature for 10 minutes and centrifuge at 12000g for 15 minutes at 4°C, and discard the supernatant. Add 1 mL of 75% (v/v) ethanol, gently wash the precipitate, centrifuge at 12000g at 4°C for 5 minutes, discard the supernatant, dry the precipitate, add DEPC-treated H 2 O to dissolve (55°C water bath promotes the dissolution 10 Minutes), the total RNA is obtained.
逆转录与PCR：取1μg总RNA，配置20μL体系，加入逆转录酶后于42℃反应60分钟，于7℃反应10分钟终止反应。配置50μL PCR体系，包括1μL cDNA、每种引物25pmol、1μL DNA聚合酶以及相配的缓冲体系、250μmol dNTPs；设置PCR程序，95℃预变性3分钟，95℃变性30秒，55℃退火30秒，72℃延伸35秒，35个循环后再额外于72℃延伸5分钟，得PCR产物。其中逆转录所用的试剂盒为PrimeScript RT Master Mix，购自Takara，货号RR036；PCR所用的试剂盒包括Q5超保真酶，购自NEB，货号M0492。Reverse transcription and PCR: Take 1μg of total RNA, configure a 20μL system, add reverse transcriptase and react at 42°C for 60 minutes, and at 7°C for 10 minutes to stop the reaction. Configure 50μL PCR system, including 1μL cDNA, 25pmol of each primer, 1μL DNA polymerase and matching buffer system, 250μmol dNTPs; set PCR program, 95℃ pre-denaturation for 3 minutes, 95℃ denaturation for 30 seconds, 55℃ annealing for 30 seconds, Extend at 72°C for 35 seconds, and then extend at 72°C for an additional 5 minutes after 35 cycles to obtain a PCR product. The kit used for reverse transcription is PrimeScript RT Master Mix, purchased from Takara, item number RR036; the kit used for PCR includes Q5 ultra-fidelity enzyme, purchased from NEB, item number M0492.
克隆与测序：取5μL PCR产物进行琼脂糖凝胶电泳检测，将检测阳性样品使用柱回收试剂盒纯化，其中回收试剂盒为
Figure PCTCN2020108720-appb-000017
Gel&PCR Clean-up，购自MACHEREY-NAGEL，货号740609。进行连接反应：样品50ng，T载体50ng，连接酶0.5μL，缓冲液1μL，反应体系10μL，于16℃反应半小时得连接产物。其中连接的试剂盒为T4DNA连接酶，购自NEB，货号M0402；取5μL连接产物加入100μL的感受态细胞(Ecos 101competent cells，购自Yeastern，货号FYE607)中，冰浴5分钟，而后于42℃水浴热激1分钟，放回冰上1分钟后加入650μL无抗生素SOC培养基，于37℃摇床上以200RPM的速度复苏30分钟。取出200μL涂布于含抗生素的LB固体培养基上于37℃孵箱过夜培养。次日，使用T载体上引物M13F和M13R配置30μLPCR体系，进行菌落PCR，用移液器枪头蘸取菌落于PCR反应体系中吹吸，并吸出0.5μL点于另一块含100nM氨苄青霉素的LB固体培养皿上以保存菌株。PCR反应结束后，取出5μL进行琼脂糖凝胶电泳检测，将阳性样品进行测序和分析[参见Kabat，“Sequences of Proteins of Immunological Interest，”National Institutes of Health，Bethesda，Md.(1991)]。测序结果如表1所示，具体序列编号对应的氨基酸序列内容请参见序列表。 Cloning and sequencing: Take 5μL of PCR product for agarose gel electrophoresis detection, and use the column recovery kit to purify the positive samples, where the recovery kit is
Figure PCTCN2020108720-appb-000017
Gel&PCR Clean-up, purchased from MACHEREY-NAGEL, catalog number 740609. Carry out ligation reaction: sample 50ng, T carrier 50ng, ligase 0.5μL, buffer 1μL, reaction system 10μL, react at 16°C for half an hour to obtain the ligation product. The ligation kit is T4DNA ligase, purchased from NEB, item number M0402; take 5μL of the ligation product and add 100μL of competent cells (Ecos 101competent cells, purchased from Yeastn, item number FYE607), ice bath for 5 minutes, and then at 42°C Heat shock in a water bath for 1 minute, put it back on ice for 1 minute, add 650 μL of antibiotic-free SOC medium, and resuscitate on a shaker at 37°C at 200 RPM for 30 minutes. Take out 200 μL and spread it on LB solid medium containing antibiotics and incubate overnight at 37°C in an incubator. The next day, use the primers M13F and M13R on the T vector to configure a 30μL PCR system to perform colony PCR. Use a pipette tip to dip the colony into the PCR reaction system and pipette, and aspirate 0.5μL onto another piece of LB containing 100nM ampicillin On a solid petri dish to preserve the strain. After the PCR reaction, 5 μL was taken out for agarose gel electrophoresis detection, and the positive samples were sequenced and analyzed [see Kabat, "Sequences of Proteins of Immunological Interest," National Institutes of Health, Bethesda, Md. (1991)]. The sequencing results are shown in Table 1. Please refer to the sequence table for the content of the amino acid sequence corresponding to the specific sequence number.
实施例7鼠-人嵌合抗体构建、以及抗体的生产和纯化Example 7 Construction of Mouse-Human Chimeric Antibodies, and Production and Purification of Antibodies
1.质粒构建与准备：根据实施例6的测序结果明确了TROP2抗体重链可变区和轻链可变区序列。将实施例2和实施例3所得的先导抗体的重链可变区序列重组到包含信号肽和人源重链抗体IgG1恒定区的表达载体(其中表达载体购买自Invitrogen，重组步 骤也由上海睿智化学完成)中，将TROP2抗体的轻链可变区序列重组到包含信号肽和人源抗体轻链kappa恒定区的表达载体当中，得重组质粒并经测序验证(测序方法与实施例6中测序方法相同)。使用碱裂解法试剂盒(购自MACHEREY-NAGEL)中量抽提高纯度的重组质粒，质量为500μg以上，经0.22μm滤膜(购自Millopore)过滤，供转染使用。1. Plasmid construction and preparation: According to the sequencing results of Example 6, the sequences of the heavy chain variable region and the light chain variable region of the TROP2 antibody were clarified. The heavy chain variable region sequence of the lead antibody obtained in Example 2 and Example 3 was recombined into an expression vector containing the signal peptide and the constant region of the human heavy chain antibody IgG1 (the expression vector was purchased from Invitrogen, and the recombination step was also performed by Shanghai Ruizhi Chemical completion), the light chain variable region sequence of the TROP2 antibody was recombined into an expression vector containing the signal peptide and the human antibody light chain kappa constant region, and the recombinant plasmid was obtained and verified by sequencing (the sequencing method is the same as that in Example 6. Same method). Use an alkaline lysis kit (purchased from MACHEREY-NAGEL) to pump the recombinant plasmid with a mass of 500 μg or more and filter through a 0.22 μm filter membrane (purchased from Millopore) for transfection.
2.细胞转染：在培养基Freestyle 293 expression medium(购自Invitrogen)培养293E细胞(购自Invitrogen)。摇床设置为37℃、130RPM和8％CO 2(v/v)。Freestyle 293 expression medium在转染时添加10％(v/v)F68(购自Invitrogen)至F68终浓度为0.1％(v/v)，得含0.1％(v/v)F68的Freestyle 293表达培养基，即培养基A。取5mL培养基A和200μg/mL PEI(购自Sigma)混匀，得培养基B。取5mL培养基A和100μg/mL步骤(1)所得的重组质粒混匀，得培养基C。5分钟后将培养基B和培养基C合并混匀，静置15分钟，得混合液D。将10mL混合液D缓缓加入100mL含293E细胞的培养基Freestyle 293 expression medium中至293E的细胞密度为1.5×10 6个/mL，边加边振荡，避免PEI过度集中，放入摇床培养。第二天加入蛋白胨至终浓度为0.5％(w/v)。第5～7天，测培养液抗体效价。第6～7天，离心(3500RPM，30分钟)收集上清，经0.22μm滤膜过滤，得滤好的细胞上清液，以供纯化。 2. Cell transfection: culture 293E cells (purchased from Invitrogen) in Freestyle 293 expression medium (purchased from Invitrogen). The shaker was set to 37°C, 130 RPM and 8% CO 2 (v/v). Freestyle 293 expression medium is added with 10% (v/v) F68 (purchased from Invitrogen) during transfection to a final concentration of 0.1% (v/v) to obtain a Freestyle 293 expression medium containing 0.1% (v/v) F68 Base, that is, medium A. Take 5mL medium A and 200μg/mL PEI (purchased from Sigma) and mix well to obtain medium B. Take 5 mL of medium A and 100 μg/mL of the recombinant plasmid obtained in step (1) and mix well to obtain medium C. After 5 minutes, the medium B and medium C are combined and mixed, and the mixture is allowed to stand for 15 minutes to obtain a mixture D. Slowly add 10mL mixture D to 100mL Freestyle 293 expression medium containing 293E cells until the cell density of 293E is 1.5×10 6 cells/mL, and shake while adding to avoid excessive concentration of PEI. Place it in a shaker for culture. Peptone was added the next day to a final concentration of 0.5% (w/v). On days 5-7, measure the antibody titer of the culture medium. On days 6-7, the supernatant was collected by centrifugation (3500RPM, 30 minutes) and filtered through a 0.22μm filter membrane to obtain a filtered cell supernatant for purification.
3.抗体纯化：对于连续生产的无内毒素的层析柱和Protein A填料，使用0.1M NaOH处理30min或者5个柱体积0.5M NaOH冲洗；对于长期未使用的柱料和层析柱至少使用1M NaOH浸泡1h，用无内毒的水冲洗至中性，用10倍柱体积的1％Triton X100对柱料清洗。使用5个柱体积的PBS进行平衡，将过滤好的细胞上清上柱，必要时收集流穿液。上柱完成后，使用5倍柱体积PBS清洗。用5倍柱体积的0.1M pH3.0的Glycine-HCl进行洗脱，收集洗脱液，并用1/10体积的pH8.5的1M Tris-HCl(1.5M NaCl)中和。收获抗体后，在1×PBS中透析过夜，避免内毒素污染。透析结束后，使用分光光度或试剂盒测定浓度，使用HPLC-SEC测定抗体纯度，使用内毒素检测试剂盒(购自Lonza)检测抗体内毒素含量。3. Antibody purification: For continuous production of endotoxin-free chromatography columns and Protein A fillers, use 0.1M NaOH for 30 minutes or 5 column volumes of 0.5M NaOH for flushing; for column materials and chromatography columns that have not been used for a long time, use at least Soak in 1M NaOH for 1 hour, rinse with non-endotoxic water to neutrality, and wash the column material with 10 times the column volume of 1% Triton X100. Equilibrate with 5 column volumes of PBS, put the filtered cell supernatant on the column, and collect the flow-through if necessary. After loading the column, wash with 5 times the column volume of PBS. Elution was performed with 5 column volumes of 0.1M pH3.0 Glycine-HCl, and the eluate was collected and neutralized with 1/10 volume of 1M Tris-HCl (1.5M NaCl) with pH 8.5. After harvesting the antibodies, dialyze overnight in 1×PBS to avoid endotoxin contamination. After the end of dialysis, use spectrophotometry or a kit to determine the concentration, use HPLC-SEC to determine the purity of the antibody, and use an endotoxin detection kit (purchased from Lonza) to detect the content of antibody endotoxin.
下述实施例中嵌合抗体命名中首段字符选用对应的先导抗体克隆号的前3～5位字符，例如嵌合抗体药物偶联物11E8-MMAF对应的先导抗体克隆号为11E8E6D11，嵌合抗体药物偶联物44A5-MMAF对应的先导抗体克隆号为44A5F9等等。In the following examples, the first paragraph of the chimeric antibody naming uses the first 3 to 5 characters of the corresponding lead antibody clone number. For example, the lead antibody clone number corresponding to the chimeric antibody drug conjugate 11E8-MMAF is 11E8E6D11, chimeric The corresponding lead antibody clone number of the antibody-drug conjugate 44A5-MMAF is 44A5F9 and so on.
实施例8偶联MMAF的嵌合抗体药物偶联物的体外药效实验Example 8 In vitro pharmacodynamic experiment of chimeric antibody drug conjugate coupled to MMAF
将实施例7所得的纯化的TROP2嵌合抗体与MC-MMAF进行偶联，方法同实施例4，经过pH 6.5～8.5的硼酸钠缓冲液透析后，加入三(2-羧乙基)膦(TCEP)，其中TCEP与纯化的TROP2抗体的摩尔比比率为2，室温下还原1小时，得反应液A。将反应液A 经过G25柱脱盐(购自GE)，去除多余的TCEP，得反应液B。向反应液B中加入MC-MMAF，其中MC-MMAF与纯化的嵌合TROP2抗体的摩尔比比率为5，室温下反应4小时。再加入半胱氨酸用以中和多余的MC-MMAF，并通过G25柱脱盐除去多余的小分子。得到纯化的TROP2嵌合抗体药物偶联物(偶联方法参见Doronina，2006，Bioconjugate Chem.17,114-124)。通过HIC分析药物的交联率、通过SEC分析抗体药物偶联物的纯度等参数后，进行细胞毒活性的分析。所有抗体药物偶联物的药物交联率(DAR)为3.0-5.0，具体如表10和11所示。其中，DAR(drug antibody ratio)指抗体偶联后一个抗体分子上携带的小分子药物的平均数量。The purified TROP2 chimeric antibody obtained in Example 7 was coupled to MC-MMAF in the same manner as in Example 4. After dialysis with a pH 6.5-8.5 sodium borate buffer, tris(2-carboxyethyl)phosphine ( TCEP), wherein the molar ratio of TCEP to purified TROP2 antibody is 2, and the reaction solution A is obtained by reducing at room temperature for 1 hour. Desalting reaction solution A through a G25 column (purchased from GE), removing excess TCEP, and obtaining reaction solution B. MC-MMAF was added to the reaction solution B, where the molar ratio of MC-MMAF to the purified chimeric TROP2 antibody was 5, and the reaction was carried out at room temperature for 4 hours. Then add cysteine to neutralize the excess MC-MMAF, and pass through the G25 column desalting to remove the excess small molecules. The purified TROP2 chimeric antibody drug conjugate was obtained (for coupling method, see Doronina, 2006, Bioconjugate Chem. 17, 114-124). After analyzing the cross-linking rate of the drug by HIC and the purity of the antibody-drug conjugate by SEC, the cytotoxic activity is analyzed. The drug crosslinking rate (DAR) of all antibody-drug conjugates is 3.0-5.0, as shown in Tables 10 and 11. Among them, DAR (drug antibody ratio) refers to the average number of small molecule drugs carried on an antibody molecule after antibody coupling.
将获得的纯化的TROP2嵌合抗体药物偶联物分别用完全培养基进行梯度稀释，96孔细胞培养板以5000细胞/孔加入90微升TROP2阳性的MDA-MB-468细胞株(购自ATCC，货号#HTB-132)细胞悬液过夜培养后，每孔分别加入10微升不同浓度的纯化的TROP2嵌合抗体药物偶联物的稀释液，继续培养5天后，用CellTiter-Glo试剂盒(购自Promega，使用方法参照产品说明书)检测细胞活力。结果如表10以及图13所示，其中表10的IC50指药物作用后，细胞的活性受到抑制的半数有效量，能够通过检测细胞的活性从而反映细胞杀伤活性。图13为纯化的TROP2嵌合抗体药物偶联物对TROP2阳性的肿瘤细胞系MDA-MB-468的细胞杀伤活性检测。此外，选用TROP2阳性的肿瘤细胞系BxPC-3(购自ATCC，货号#CRL-1687)和COLO 205(购自ATCC，货号#CCL-222)进行细胞杀伤活性检测，方法同上。结果如表11以及图14A和图14B所示，其中表11的IC50指药物作用后，细胞的活性受到抑制的半数有效量，能够通过检测细胞的活性从而反映细胞杀伤活性。图14A和图14B为纯化的TROP2嵌合抗体药物偶联物对TROP2阳性的肿瘤细胞系BxPC-3和COLO 205的细胞杀伤活性检测，结果说明，纯化的TROP2嵌合抗体药物偶联物对TROP2阳性的细胞有杀伤作用。The obtained purified TROP2 chimeric antibody drug conjugates were serially diluted with complete medium, and 90 microliters of TROP2-positive MDA-MB-468 cell line (purchased from ATCC) was added to a 96-well cell culture plate at 5000 cells/well. , Item #HTB-132) After the cell suspension is cultured overnight, add 10 microliters of different concentrations of purified TROP2 chimeric antibody drug conjugate dilutions to each well. After continuing the culture for 5 days, use the CellTiter-Glo kit ( (Purchased from Promega, the use method refers to the product manual) to detect cell viability. The results are shown in Table 10 and Figure 13, where IC50 in Table 10 refers to the half-effective amount that inhibits cell activity after drug action, which can reflect cell killing activity by detecting cell activity. Figure 13 shows the cell killing activity of the purified TROP2 chimeric antibody drug conjugate against the TROP2 positive tumor cell line MDA-MB-468. In addition, TROP2-positive tumor cell lines BxPC-3 (purchased from ATCC, catalog number #CRL-1687) and COLO 205 (purchased from ATCC, catalog number #CCL-222) were selected for cell killing activity detection, and the method was the same as above. The results are shown in Table 11 and Figures 14A and 14B, where the IC50 in Table 11 refers to the half-effective amount that inhibits cell activity after the drug is applied, which can reflect cell killing activity by detecting cell activity. Figure 14A and Figure 14B show the cell killing activity of the purified TROP2 chimeric antibody drug conjugate against TROP2-positive tumor cell lines BxPC-3 and COLO 205. The results show that the purified TROP2 chimeric antibody drug conjugate is effective against TROP2 Positive cells have a killing effect.
表10细胞杀伤实验检测纯化的TROP2嵌合抗体药物偶联物对TROP2阳性MDA-MB-468细胞的特异性杀伤作用Table 10: Cell killing test to detect the specific killing effect of purified TROP2 chimeric antibody drug conjugate on TROP2-positive MDA-MB-468 cells
Figure PCTCN2020108720-appb-000018
Figure PCTCN2020108720-appb-000018
表11细胞杀伤实验检测纯化的TROP2嵌合抗体药物偶联物对TROP2阳性COLO 205细胞、BxPC-3细胞的特异性杀伤作用Table 11: Cell killing experiment detects the specific killing effect of purified TROP2 chimeric antibody drug conjugate on TROP2-positive COLO 205 cells and BxPC-3 cells
Figure PCTCN2020108720-appb-000019
Figure PCTCN2020108720-appb-000019
实施例9偶联MMAE的嵌合抗体药物偶联物的体外药效实验Example 9 In vitro pharmacodynamic experiment of chimeric antibody drug conjugate coupled to MMAE
将实施例7所得的纯化的TROP2嵌合抗体与MC-VC-PAB-MMAE(凯恵科技发展(上海)有限公司)进行偶联，方法同实施例4，经过pH 6.5～8.5的硼酸钠缓冲液透析后，加入三(2-羧乙基)膦(TCEP)，其中TCEP与纯化的TROP2抗体的摩尔比比率为2，室温下还原1小时，得反应液A。将反应液A经过G25柱脱盐(购自GE)，去除多余的TCEP，得反应液B。向反应液B中加入MC-VC-PAB-MMAE，其中MC-VC-PAB-MMAE与纯化的TROP2抗体的摩尔比比率为5，室温下反应4小时。再加入半胱氨酸用以中和多余的MC-VC-PAB-MMAE，并通过G25柱脱盐除去多余的小分子。得到纯化的TROP2抗体药物偶联物(偶联方法参见Doronina，2006，Bioconjugate Chem.17,114-124)。通过HIC分析药物的交联率、通过SEC分析抗体药物偶联物的纯度等参数后，进行细胞毒活性的分析。所有抗体药物偶联物的药物交联率(DAR)为3.0-5.0。其中，DAR(drug antibody ratio)指抗体偶联后一个抗体分子上携带的小分子药物的平均数量。The purified TROP2 chimeric antibody obtained in Example 7 was coupled with MC-VC-PAB-MMAE (Kay Technology Development (Shanghai) Co., Ltd.), the method was the same as that in Example 4, after pH 6.5-8.5 sodium borate buffer After liquid dialysis, tris(2-carboxyethyl)phosphine (TCEP) was added, wherein the molar ratio of TCEP to purified TROP2 antibody was 2, and the reaction solution A was obtained by reducing at room temperature for 1 hour. The reaction solution A is desalted through a G25 column (purchased from GE), and excess TCEP is removed to obtain reaction solution B. MC-VC-PAB-MMAE was added to the reaction solution B, wherein the molar ratio of MC-VC-PAB-MMAE to the purified TROP2 antibody was 5, and the reaction was carried out at room temperature for 4 hours. Then add cysteine to neutralize the excess MC-VC-PAB-MMAE, and pass the G25 column desalting to remove the excess small molecules. The purified TROP2 antibody drug conjugate is obtained (refer to Doronina, 2006, Bioconjugate Chem. 17, 114-124 for coupling method). After analyzing the cross-linking rate of the drug by HIC and the purity of the antibody-drug conjugate by SEC, the cytotoxic activity is analyzed. The drug cross-linking rate (DAR) of all antibody-drug conjugates is 3.0-5.0. Among them, DAR (drug antibody ratio) refers to the average number of small molecule drugs carried on an antibody molecule after antibody coupling.
将获得的纯化的TROP2抗体药物偶联物分别用完全培养基进行梯度稀释，96孔细胞培养板以5000细胞/孔加入90微升TROP2阳性的MDA-MB-468细胞株(购自ATCC，货号#HTB-132)细胞悬液过夜培养后，每孔分别加入10微升不同浓度的纯化的TROP2嵌合抗体药物偶联物的稀释液，继续培养5天后，用CellTiter-Glo试剂盒(购自Promega，使用方法参照产品说明书)检测细胞活力。此外，选用TROP2阳性的肿瘤细胞系COLO205(购自ATCC，货号#CCL-222)进行细胞杀伤活性检测，方法同上。结果如表12以及图15和图16所示，其中表12的IC50指药物作用后，细胞的活性受到抑制的半数有效量，能够通过检测细胞的活性从而反映细胞杀伤活性。图15和图16为纯化的TROP2嵌合抗体药物偶联物对TROP2阳性的肿瘤细胞系MDA-MB-468和COLO 205的细胞杀伤活性检测。结果说明，纯化的TROP2抗体药物偶联物对TROP2阳性的细胞有杀伤作用。The purified TROP2 antibody-drug conjugates obtained were serially diluted with complete medium, and 90 microliters of TROP2-positive MDA-MB-468 cell line (purchased from ATCC, catalog number) was added to a 96-well cell culture plate at 5000 cells/well. #HTB-132) After the cell suspension was cultured overnight, add 10 microliters of different concentrations of purified TROP2 chimeric antibody-drug conjugate diluent to each well. After culturing for 5 days, use the CellTiter-Glo kit (purchased from Promega, the method of use refers to the product manual) to detect cell viability. In addition, the TROP2-positive tumor cell line COLO205 (purchased from ATCC, item #CCL-222) was selected for cell killing activity detection, and the method was the same as above. The results are shown in Table 12 and Figure 15 and Figure 16, where the IC50 in Table 12 refers to the half effective amount that inhibits cell activity after drug action, which can reflect cell killing activity by detecting cell activity. Figures 15 and 16 show the cell killing activity of the purified TROP2 chimeric antibody drug conjugate against TROP2-positive tumor cell lines MDA-MB-468 and COLO 205. The results indicate that the purified TROP2 antibody drug conjugate has a killing effect on TROP2-positive cells.
表12细胞杀伤实验检测纯化的TROP2嵌合抗体药物偶联物对TROP2阳性MDA-MB-468和COLO 205细胞的特异性杀伤作用Table 12: Cell killing experiment detects the specific killing effect of purified TROP2 chimeric antibody drug conjugate on TROP2-positive MDA-MB-468 and COLO 205 cells
Figure PCTCN2020108720-appb-000020
Figure PCTCN2020108720-appb-000020
Figure PCTCN2020108720-appb-000021
Figure PCTCN2020108720-appb-000021
实施例10偶联MMAE的嵌合抗体药物偶联物的体内药效实验Example 10 In vivo pharmacodynamic experiment of chimeric antibody drug conjugate coupled to MMAE
将MDA-MB-468(三阴性乳腺癌细胞株，ATCC，HTB-132)(1×10 7个)200μl细胞悬液(base DMEM:Matrigel＝1:1)接种于CB17SCID小鼠(上海灵畅生物科技有限公司提供，接种时6-8周)右后背皮下，接种后3-4周待肿瘤长至约250mm 3后，去除体重、肿瘤过大和过小的，按肿瘤体积将小鼠随机分为6组，每组7只。D0尾静脉注射抗体，给药1次，每周测2次瘤体积，称鼠重，记录数据。肿瘤体积(V)计算公式为：V＝1/2×a×b 2；其中a、b分别表示肿瘤的长径和短径。分组如表13。 MDA-MB-468 (triple negative breast cancer cell line, ATCC, HTB-132) (1×10 7 cells) 200 μl cell suspension (base DMEM: Matrigel=1:1) was inoculated into CB17SCID mice (Shanghai Lingchang) Provided by Biotechnology Co., Ltd., 6-8 weeks for inoculation) Right back subcutaneously, 3-4 weeks after inoculation, after the tumor grows to about 250mm 3 , remove the body weight, tumors that are too large and small, and randomize the mice according to the tumor volume Divided into 6 groups with 7 animals in each group. At D0, the antibody was injected into the tail vein once, the tumor volume was measured twice a week, the mouse was weighed, and the data was recorded. The calculation formula of tumor volume (V) is: V=1/2×a×b 2 ; where a and b represent the long diameter and short diameter of the tumor, respectively. The grouping is shown in Table 13.
表13 TROP2嵌合抗体及其抗体药物偶联物的体内药效实验Table 13 In vivo efficacy experiments of TROP2 chimeric antibodies and their antibody-drug conjugates
Figure PCTCN2020108720-appb-000022
Figure PCTCN2020108720-appb-000022
结果见图17：治疗后肿瘤的体积变化图，和图18：治疗后的鼠重变化图。结果显示，从处理(即治疗)10天起，上述几种偶联MMAE的嵌合抗体药物偶联物即显示出较对照组明显小约200～300mm 3的肿瘤体积；当治疗21天时，44A5-MMAE治疗的小鼠肿瘤体积仅有微弱的增长，其比对照组治疗的肿瘤体积少400mm 3；更令人惊奇的是，11E8-MMAE治疗的小鼠肿瘤体积与0天时相比几乎没有变化，21天时比对照组的肿瘤体积少约500mm 3，说明这几种偶联MMAE的嵌合抗体药物偶联物可以很好的抑制肿瘤MDA-MB-468的生长，而且对小鼠的体重没有显著的影响。 The results are shown in Figure 17: tumor volume change graph after treatment, and Figure 18: mouse weight change graph after treatment. The results showed that from 10 days of treatment (ie treatment), the above-mentioned chimeric antibody-drug conjugates conjugated to MMAE showed a tumor volume that was significantly smaller than the control group by about 200-300 mm 3 ; when treated for 21 days, 44A5 -The tumor volume of the mice treated with MMAE only increased slightly, which was 400mm 3 less than the tumor volume treated in the control group; more surprisingly, the tumor volume of the mice treated with 11E8-MMAE almost did not change compared with the 0 day At 21 days, the tumor volume was about 500mm 3 less than that of the control group, indicating that these chimeric antibody drug conjugates conjugated to MMAE can inhibit the growth of tumor MDA-MB-468 well, and have no effect on the body weight of mice. Significant impact.
实施例11嵌合抗体在大鼠体内的血清稳定性Example 11 Serum stability of chimeric antibody in rats
将嵌合抗体一侧尾静脉注入Sprague-Dawley大鼠(6～8周龄，每只210-235g，SLAC Laboratory Animal Co.LTD)。于第0天，以3mL/kg的体积剂量通过足背静脉分别给动物施用嵌合抗体11E8、嵌合抗体44A5和hRS7。在给药后10、和30分钟；1、4、8和24小时；和2、4、7、14、21、28天的每个时间点通过尾静脉收集大约150μL全血，进行血清分析。实验方案如表14。Sprague-Dawley rats (6-8 weeks old, 210-235g each, SLAC Laboratory Animal Co. LTD) were injected into the tail vein of one side of the chimeric antibody. On day 0, chimeric antibody 11E8, chimeric antibody 44A5, and hRS7 were administered to the animals through the dorsal foot vein at a volume dose of 3 mL/kg. Approximately 150 μL of whole blood was collected through the tail vein at each time point of 10, and 30 minutes; 1, 4, 8, and 24 hours; and 2, 4, 7, 14, 21, and 28 days after administration, and serum analysis was performed. The experimental program is shown in Table 14.
表14 TROP2嵌合抗体大鼠体内PK实验Table 14 In vivo PK experiment of TROP2 chimeric antibody in rats
Figure PCTCN2020108720-appb-000023
Figure PCTCN2020108720-appb-000023
结果见图19和图20。单次施用11E8E6D11、嵌合抗体44A5F9后，血清中抗体随时间清除。其中嵌合抗体11E8E6D11、嵌合抗体44A5F9表现出比hRS7更好或相当的血清稳定性。The results are shown in Figure 19 and Figure 20. After a single administration of 11E8E6D11 and chimeric antibody 44A5F9, the antibody in the serum cleared over time. Among them, the chimeric antibody 11E8E6D11 and the chimeric antibody 44A5F9 showed better or comparable serum stability than hRS7.
具体地，给药后，嵌合抗体11E8E6D11的总清除率(CL)为5.62mL/day/kg，稳态表观分布容积(Vss)为112mL/kg,清除半衰期为13.6天。嵌合抗体44A5F9的总清除率(CL)为9.27mL/day/kg，稳态表观分布容积(Vss)为121mL/kg,清除半衰期为9.33天。对照抗体hRS7的总清除率(CL)为8.19mL/day/kg，稳态表观分布容积(Vss)为110mL/kg，清除半衰期为9.48天。Specifically, after administration, the total clearance (CL) of the chimeric antibody 11E8E6D11 was 5.62 mL/day/kg, the steady-state apparent volume of distribution (Vss) was 112 mL/kg, and the clearance half-life was 13.6 days. The total clearance (CL) of the chimeric antibody 44A5F9 was 9.27 mL/day/kg, the steady-state apparent volume of distribution (Vss) was 121 mL/kg, and the clearance half-life was 9.33 days. The total clearance (CL) of the control antibody hRS7 was 8.19 mL/day/kg, the steady-state apparent volume of distribution (Vss) was 110 mL/kg, and the clearance half-life was 9.48 days.
实施例12抑制脱酰胺化、异构化、水解反应的突变的导入Example 12 Introduction of mutations that inhibit deamidation, isomerization, and hydrolysis reactions
通过对嵌合抗体11E8的抗体序列分析，发现嵌合抗体11E8抗体重链可变区(SEQ ID NO:1)CDR2(SEQ ID NO:3)的NG存在脱酰胺化反应的可能，DG存在异构化的可能，为了抑制脱酰胺化、异构化和水解，经过计算和分析，将位于嵌合抗体11E8抗体CDR2的NG突变成NA。另外，将位于CDR2的DG突变成SG；或将DG突变成EG，或将DG突变成DA，突变后嵌合抗体11E8抗体重链可变区CDR2区氨基酸序列如SEQ ID NO:20-23所示。拟通过上述定点突变实施去除作为接受脱酰胺化的位点的天冬酰胺残基和天冬酰残基的氨基酸修饰。Through the analysis of the antibody sequence of the chimeric antibody 11E8, it was found that the NG of the chimeric antibody 11E8 antibody heavy chain variable region (SEQ ID NO: 1) CDR2 (SEQ ID NO: 3) may have deamidation reaction, and DG may have different The possibility of conformation, in order to inhibit deamidation, isomerization and hydrolysis, after calculation and analysis, the NG located in the CDR2 of the chimeric antibody 11E8 antibody was mutated to NA. In addition, the DG located in CDR2 is mutated to SG; or DG is mutated to EG, or DG is mutated to DA. After the mutation, the amino acid sequence of the CDR2 region of the heavy chain variable region of the chimeric antibody 11E8 antibody is shown in SEQ ID NO: 20 -23 shown. It is proposed to implement amino acid modification to remove asparagine residues and asparagyl residues as sites for deamidation by the above-mentioned site-directed mutagenesis.
嵌合抗体11E8抗体重链可变区定点突变序列经基因合成后，按实施例7所述进行质粒构建、细胞转染和抗体纯化，突变后的嵌合抗体通过FACS进行结合活性鉴定，鉴定 结果如图21及表15所示，其中图21是嵌合抗体11E8抗体重链可变区CDR2的NG突变成NA，和DG突变成SG、EG或DA后的突变抗体嵌合抗体11E8-1、嵌合抗体11E8-2、嵌合抗体11E8-3和嵌合抗体11E8-4及野生型抗体嵌合抗体11E8与CHOK-TROP2细胞结合活性检测。表15总结了中嵌合抗体11E8野生型抗体及突变抗体在CHOK-TROP2细胞中的结合活性。After gene synthesis of the site-directed mutation sequence of the variable region of the chimeric antibody 11E8 antibody heavy chain, plasmid construction, cell transfection and antibody purification were performed as described in Example 7. The mutant chimeric antibody was identified by FACS for binding activity, and the identification results As shown in Figure 21 and Table 15, Figure 21 shows the mutant antibody chimeric antibody 11E8 after the NG of the chimeric antibody 11E8 antibody heavy chain variable region CDR2 was mutated to NA, and DG was mutated to SG, EG or DA. 1. Detection of the binding activity of chimeric antibody 11E8-2, chimeric antibody 11E8-3, chimeric antibody 11E8-4 and wild-type antibody chimeric antibody 11E8 to CHOK-TROP2 cells. Table 15 summarizes the binding activity of the chimeric antibody 11E8 wild-type antibody and mutant antibody in CHOK-TROP2 cells.
表15.FACS检测嵌合抗体11E8嵌合抗体突变体与CHOk1-TROP2细胞的结合反应Table 15. FACS detection of the binding reaction of chimeric antibody 11E8 chimeric antibody mutant with CHOk1-TROP2 cells
Figure PCTCN2020108720-appb-000024
Figure PCTCN2020108720-appb-000024
从表15和图21来看，位于嵌合抗体11E8重链可变区CDR2的NG突变成NA(序列如SEQ ID NO:44所示)，DG突变成DA(序列如SEQ ID NO:30所示)的突变抗体嵌合抗体11E8-1和突变体嵌合抗体11E8-4与CHOk1-TROP2细胞的结合活性与野生型抗体嵌合抗体11E8接近，说明NG突变成NA，DG突变成DA不影响抗体与CHOK-TROP2细胞的结合。而位于CDR2的DG是突变成EG或SG后一定程度上影响了抗体与对CHOK-TROP2的结合后的最大平均荧光强度分别下降53％和23％。From Table 15 and Figure 21, NG in the CDR2 of the heavy chain variable region of the chimeric antibody 11E8 is mutated to NA (sequence is shown in SEQ ID NO: 44), and DG is mutated to DA (sequence is shown in SEQ ID NO: The mutant antibody chimeric antibody 11E8-1 and mutant chimeric antibody 11E8-4 shown in 30) have close binding activity with CHOk1-TROP2 cells to that of wild-type antibody chimeric antibody 11E8, indicating that NG is mutated into NA and DG mutations. DA does not affect the binding of antibodies to CHOK-TROP2 cells. The DG located in CDR2 is mutated into EG or SG to a certain extent, and the maximum average fluorescence intensity after the binding of the antibody to CHOK-TROP2 is reduced by 53% and 23%, respectively.
因此，后续将选用CDR2的NG突变成NA以及DG突变为DA的重链CDR2(序列如SEQ ID NO:39所示)所得的嵌合抗体进行后续研究。Therefore, the chimeric antibody obtained by mutating the NG of CDR2 to NA and the heavy chain CDR2 of DG to DA (sequence shown in SEQ ID NO: 39) will be used for subsequent research.
实施例13人源化TROP2抗体的制备Example 13 Preparation of humanized TROP2 antibody
通过序列比对(NCBI-Igblast)选择与候选抗体11E8E6D11重链可变区，轻链可变区同源性最高的胚系基因序列作为可变区移植骨架：GHV1-69*08(66.0％)和IGKV1-39*01(65.7％)。在选定人抗体骨架后，通过同源建模，预测在鼠抗恒定区中可能决定结构的关键氨基酸，对嫁接的骨架区进行回复突变设计，如表16。The germline gene sequence with the highest homology of the variable region of the light chain with the candidate antibody 11E8E6D11 heavy chain variable region and the light chain variable region was selected by sequence alignment (NCBI-Igblast) as the variable region transplantation skeleton: GHV1-69*08 (66.0%) And IGKV1-39*01 (65.7%). After selecting the human antibody framework, homology modeling was used to predict the key amino acids that may determine the structure in the mouse anti-constant region, and the grafted framework region was designed with back mutations, as shown in Table 16.
表16 11E8E6D11克隆的人源化骨架和回复突变设计Table 16 Humanized backbone and back mutation design of 11E8E6D11 clone
Figure PCTCN2020108720-appb-000025
Figure PCTCN2020108720-appb-000025
根据以上原则，设计4个重链可变区序列(人源化11E8VH.g0，人源化11E8VH.g1，人源化11E8VH.g2,人源化11E8VH.g3)和3个轻链可变区序列(人源化11E8VL.g0,人源化11E8VL.g1，人源化11E8VL.g2)。随后做交叉组合进行表达，共得到下列12个人源化抗体，见表17。According to the above principles, 4 heavy chain variable region sequences (humanized 11E8VH.g0, humanized 11E8VH.g1, humanized 11E8VH.g2, humanized 11E8VH.g3) and 3 light chain variable regions were designed Sequence (humanized 11E8VL.g0, humanized 11E8VL.g1, humanized 11E8VL.g2). Then, cross-combination was performed for expression, and the following 12 humanized antibodies were obtained, as shown in Table 17.
表17 11E8人源化抗体表达组合Table 17 11E8 humanized antibody expression combination
Figure PCTCN2020108720-appb-000026
Figure PCTCN2020108720-appb-000026
Figure PCTCN2020108720-appb-000027
Figure PCTCN2020108720-appb-000027
另外，经过序列分析，候选抗体44A5F9重链可变区和轻链可变区均无重要的hotspot。通过序列比对(NCBI-Igblast)选择与候选抗体44A5F9重链可变区，轻链可变区同源性最高的胚系基因序列作为可变区移植骨架：IGHV3-7*01(69.4％)和IGKV1-39*01(61.6％)。在选定人抗体骨架后，通过同源建模，预测在鼠抗恒定区中可能决定结构的关键氨基酸，对嫁接的骨架区进行回复突变设计，如表18所示。In addition, after sequence analysis, the candidate antibody 44A5F9 heavy chain variable region and light chain variable region have no important hotspots. Through sequence alignment (NCBI-Igblast), the germline gene sequence with the highest homology to the variable region of the heavy chain of the candidate antibody 44A5F9 and the variable region of the light chain is selected as the variable region transplantation skeleton: IGHV3-7*01 (69.4%) And IGKV1-39*01 (61.6%). After selecting the human antibody framework, through homology modeling, the key amino acids that may determine the structure in the mouse anti-constant region are predicted, and the grafted framework region is designed for back mutation, as shown in Table 18.
表18 44A5F9克隆的人源化骨架和回复突变设计Table 18 Humanized backbone and back mutation design of 44A5F9 clone
Figure PCTCN2020108720-appb-000028
Figure PCTCN2020108720-appb-000028
Figure PCTCN2020108720-appb-000029
Figure PCTCN2020108720-appb-000029
根据以上原则，设计3个重链可变区序列(人源化44A5VH.g0，人源化44A5VH.g1，人源化44A5VH.g2)和3个轻链可变区序列(人源化44A5VL.g0,人源化44A5VL.g1，人源化44A5VL.g2)。随后做交叉组合进行表达，共得到下列9个人源化抗体，见表19。According to the above principles, 3 heavy chain variable region sequences (humanized 44A5VH.g0, humanized 44A5VH.g1, humanized 44A5VH.g2) and 3 light chain variable region sequences (humanized 44A5VL. g0, humanized 44A5VL.g1, humanized 44A5VL.g2). Subsequently, cross-combination was performed for expression, and the following 9 humanized antibodies were obtained, as shown in Table 19.
表19 44A5F9人源化抗体表达组合Table 19 44A5F9 humanized antibody expression combination
Figure PCTCN2020108720-appb-000030
Figure PCTCN2020108720-appb-000030
Figure PCTCN2020108720-appb-000031
Figure PCTCN2020108720-appb-000031
载体构建：扩增引物由Genewiz合成，随后通过PCR方法分别扩增轻链和重链的可变区。配置50μL反应体系，包括50-100ng的重链可变区，轻链可变区、正向反向引物各1ul、1ul pfxD酶(购自invitrogen，12344-012)、10*pfx缓冲液5ul(供应商同pfx同酶)以及加水补足至50μL。设置PCR程序，预变性95℃5分钟，变性95℃30秒，退火56℃30秒，延伸68℃30秒，25个循环后再额外68℃延伸10min，得到PCR产物。取5μL PCR产物进行琼脂糖凝胶电泳检测，将检测阳性样品使用回收试剂盒纯化，其中回收试剂盒为PureLink Quick Gel extraction kit，购自Qiagen，货号28706。Vector construction: The amplification primers are synthesized by Genewiz, and then the variable regions of the light chain and the heavy chain are amplified separately by PCR. Configure a 50μL reaction system, including 50-100ng heavy chain variable region, 1ul light chain variable region, forward and reverse primers, 1ul pfxD enzyme (purchased from invitrogen, 12344-012), 10*pfx buffer 5ul ( The supplier has the same enzyme as pfx) and add water to make up to 50μL. Set up the PCR program, pre-denaturation at 95°C for 5 minutes, denaturation at 95°C for 30 seconds, annealing at 56°C for 30 seconds, extension at 68°C for 30 seconds, and after 25 cycles, an additional extension at 68°C for 10 minutes to obtain PCR products. Take 5 μL of the PCR product for agarose gel electrophoresis detection, and use the recovery kit to purify the positive samples. The recovery kit is PureLink Quick Gel extraction kit, purchased from Qiagen, catalog number 28706.
人源化抗体的制备：进行连接反应：插入片段20-40ng，酶切过的表达载体60-100ng，重组酶Exnase(购自Vazyme，货号C112-01/02)1μL，缓冲液2μL，反应体系10μL，于37℃反应半小时得到连接产物，即构建好的重组载体。缓冲液为该重组酶配套购买使用的缓冲液；将重链可变区定向克隆到包含信号肽和人源抗体重链IgG1恒定区的表达载体(其中表达载体购买自Invitrogen，重组步骤为常规步骤)，将轻链可变区定向克隆到包含信号肽和人源抗体轻链kappa恒定区的表达载体(其中表达载体购买自Invitrogen，重组步骤为常规步骤)中。将10μL连接产物加入100μL的感受态细胞(Ecos 101competent cells，购自Yeastern，货号FYE607)中，42℃水浴热激60秒，放回冰上3分钟，取出80μL涂布于含氨苄青霉素的LB固体培养基上，于37℃孵箱过夜培养。次日，使用表达载体上引物pEF1A和pSV40，配置30μL PCR体系，进行菌落PCR。菌落PCR的体系为：引物各1μL，15μL的PCR预混液(购自Novoprotein)，补足至30μL。用移液器枪头蘸取菌落于PCR反应体系中吹吸，并吸出0.5μL点于另一块含100μg/mL氨苄青霉素的LB固体培养皿上以保存菌株。PCR反应结束后，取出4.5μL进行琼脂糖凝胶电泳检测，将阳性样品进行测序。Preparation of humanized antibody: perform ligation reaction: insert 20-40ng, digested expression vector 60-100ng, recombinase Exnase (purchased from Vazyme, catalog number C112-01/02) 1μL, buffer 2μL, reaction system 10μL, react at 37°C for half an hour to obtain the ligation product, which is the constructed recombinant vector. The buffer is the buffer used for the purchase of the recombinase; the heavy chain variable region is directionally cloned into an expression vector containing the signal peptide and the human antibody heavy chain IgG1 constant region (the expression vector is purchased from Invitrogen, and the recombination step is a conventional step ), the light chain variable region was directional cloned into an expression vector containing a signal peptide and a human antibody light chain kappa constant region (where the expression vector was purchased from Invitrogen, and the recombination step was a conventional step). Add 10μL of the ligation product to 100μL of competent cells (Ecos 101competent cells, purchased from Yeastern, catalog number FYE607), heat shock in a 42℃ water bath for 60 seconds, put it back on ice for 3 minutes, and take out 80μL of LB solid coated with ampicillin On the medium, incubate overnight at 37°C in an incubator. The next day, use the primers pEF1A and pSV40 on the expression vector to configure a 30μL PCR system for colony PCR. The colony PCR system is: 1μL of primers, 15μL of PCR master mix (purchased from Novoprotein), make up to 30μL. Use a pipette tip to dip the colony into the PCR reaction system and pipette, and aspirate 0.5 μL onto another LB solid petri dish containing 100 μg/mL ampicillin to preserve the strain. After the PCR reaction was completed, 4.5 μL was taken out for agarose gel electrophoresis detection, and the positive samples were sequenced.
将序列正确的重组抗体重、轻链的表达载体进行扩增，随后瞬时转染FreeStyle TM293-F细胞(购自Invitrogen)以生产抗体。转染时，293-F细胞的密度应为1-1.2×10 6个/mL，100mL细胞需要100μg上述已构建好的重组载体和200μg的转染试剂聚乙烯亚胺(PEI)。将重组载体和PEI分别加入到5mL培养基中，室温静置5分钟，0.22μm滤膜过滤后，将重组载体和PEI的混合物于室温静置15分钟。然后将上述混合物缓慢地加入到细胞中，在37℃、8％(v/v)CO 2培养箱中以130rpm的转速培养。每天采取培养上清和细胞沉淀检测抗体的表达。5天后，3000g离心细胞培养液30分钟，收集上清液，0.22μm滤器过滤。用1mL MabSelect TMSuRe TMcolumn(购自GE Healthcare)纯化200mL澄清上清液中 的单克隆抗体。MabSelect TMSuRe TMcolumn先用平衡缓冲液(PBS磷酸缓冲液，pH7.2)平衡，MabSelect TMSuRe TMcolumn。上样完毕后用平衡缓冲液清洗MabSelect TMSuRe TMcolumn，平衡缓冲液的体积为蛋白A柱柱床体积的5倍。用洗脱液(0.1M甘氨盐酸缓冲液，pH3.0)洗脱结合在MabSelect TMSuRe TMcolumn上的单克隆抗体。收集洗脱的抗体，加入10％(v/v)1.0M Tris-HCl缓冲液中和pH。然后立即用PBS磷酸缓冲液透析过夜。收集透析后的单克隆抗体，用0.22μm的滤器进行无菌过滤，无菌保存，即得纯化的TROP2人源化抗体。 The recombinant antibody heavy and light chain expression vectors with the correct sequence are amplified, and then transiently transfected into FreeStyle TM 293-F cells (purchased from Invitrogen) to produce antibodies. During transfection, the density of 293-F cells should be 1-1.2×10 6 cells/mL, and 100 mL cells need 100 μg of the above-mentioned constructed recombinant vector and 200 μg of transfection reagent polyethyleneimine (PEI). The recombinant vector and PEI were added to 5 mL culture medium, and the mixture was allowed to stand at room temperature for 5 minutes. After filtration with a 0.22 μm filter, the mixture of recombinant vector and PEI was allowed to stand at room temperature for 15 minutes. Then the above mixture was slowly added to the cells, and cultured in a 37°C, 8% (v/v) CO 2 incubator at 130 rpm. The culture supernatant and cell pellet are taken every day to detect antibody expression. After 5 days, the cell culture solution was centrifuged at 3000 g for 30 minutes, the supernatant was collected, and filtered with a 0.22 μm filter. The monoclonal antibody in 200 mL of the clear supernatant was purified with 1 mL MabSelect TM SuRe TM column (purchased from GE Healthcare). MabSelect TM SuRe TM column is first equilibrated with equilibration buffer (PBS phosphate buffer, pH 7.2), MabSelect TM SuRe TM column. After loading the sample, wash the MabSelect TM SuRe TM column with the equilibration buffer. The volume of the equilibration buffer is 5 times the volume of the protein A column bed. The monoclonal antibody bound to the MabSelect TM SuRe TM column was eluted with an eluent (0.1M glycine hydrochloride buffer, pH 3.0). Collect the eluted antibodies, add 10% (v/v) 1.0M Tris-HCl buffer to neutralize the pH. Then immediately dialyzed against PBS phosphate buffer overnight. Collect the dialyzed monoclonal antibodies, filter them aseptically with a 0.22 μm filter, and store them aseptically to obtain purified TROP2 humanized antibodies.
实施例14人源化TROP2抗体的鉴定Example 14 Identification of humanized TROP2 antibody
A.流式细胞实验(FACS)检测抗体与表达人源TROP2细胞的结合。结果如图22A所示，所得11E8人源化抗体均可结合细胞表面的人源Trop2。如图22B所示，所得44A5F9人源化抗体均可结合细胞表面的人源Trop2。其中同型对照为人IgG1，图中的数据为所测细胞群的平均荧光强度值(MFI)。表20说明，所得的纯化的TROP2人源化抗体都可以较好的结合细胞表面的人源TROP2A. Flow cytometry (FACS) detects the binding of antibodies to human TROP2 cells. The results are shown in Figure 22A. The resulting 11E8 humanized antibodies can all bind to human Trop2 on the cell surface. As shown in Figure 22B, the obtained 44A5F9 humanized antibodies can all bind to human Trop2 on the cell surface. The isotype control is human IgG1, and the data in the figure is the average fluorescence intensity value (MFI) of the measured cell population. Table 20 shows that the purified humanized TROP2 antibodies can bind well to human TROP2 on the cell surface.
表20.人源化TROP2抗体对CHOK1-hTROP2细胞表面的人源TROP2的结合反应Table 20. Binding response of humanized TROP2 antibodies to human TROP2 on the surface of CHOK1-hTROP2 cells
Figure PCTCN2020108720-appb-000032
Figure PCTCN2020108720-appb-000032
B.流式细胞实验(FACS)检测抗体与表达食蟹猴源TROP2细胞的结合。结果如图23A所示，所得11E8人源化抗体均可结合细胞表面的食蟹猴源Trop2。如图23B所示，所得44A5F9人源化抗体均可结合细胞表面的食蟹猴源Trop2。其中同型对照为人IgG1，图中的数据为所测细胞群的平均荧光强度值(MFI)。表21说明，所得的纯化的TROP2人源化抗体都可以较好的结合细胞表面的食蟹猴TROP2。其中，hTINA1-H1L1序列来自专利WO2015098099A1，根据专利中制备方法由上海睿智化学研究有限公司表达生产。B. Flow cytometry (FACS) to detect the binding of antibodies to TROP2 cells expressing cynomolgus monkey origin. The results are shown in Figure 23A. The obtained 11E8 humanized antibodies can all bind to the cynomolgus monkey-derived Trop2 on the cell surface. As shown in Figure 23B, the obtained 44A5F9 humanized antibodies can bind to the cynomolgus monkey-derived Trop2 on the cell surface. The isotype control is human IgG1, and the data in the figure is the average fluorescence intensity value (MFI) of the measured cell population. Table 21 shows that the obtained purified humanized TROP2 antibodies can bind well to the cynomolgus monkey TROP2 on the cell surface. Among them, the hTINA1-H1L1 sequence comes from the patent WO2015098099A1, which is expressed and produced by Shanghai Ruizhi Chemical Research Co., Ltd. according to the preparation method in the patent.
表21.人源化TROP2抗体对CHOK1-cTROP2细胞表面的食蟹猴TROP2的结合反应Table 21. Binding response of humanized TROP2 antibody to cyno TROP2 on the surface of CHOK1-cTROP2 cells
Figure PCTCN2020108720-appb-000033
Figure PCTCN2020108720-appb-000033
C.人源化抗TROP2抗体的结合亲和力检测C. Humanized anti-TROP2 antibody binding affinity detection
通过Biacore进行不同人源化抗体的亲和力比较，部分数据如表22所示，具体操作和方法根据仪器说明书和厂家提供的详细方法。具体为：按照人Fab捕获试剂盒(Cat.#28-9583-25，GE)说明书中所述的方法，将人Fab捕获分子共价偶联于CM5生物传感芯片(Cat.#BR-1000-12，GE)上，从而亲和捕获待测抗体。然后于芯片表面流经人TROP2-his(CAT#10428-H08H-100,Sino biological)抗原，利用Biacore仪器实时检测反应 信号，从而获得结合和解离曲线，通过拟合得到亲和力数值，见下表22。在实验中每个循环解离完成后，用人Fab捕获试剂盒里配置的再生溶液将生物芯片洗净再生。The affinity comparison of different humanized antibodies is performed through Biacore. Some data are shown in Table 22. The specific operations and methods are based on the instrument instructions and detailed methods provided by the manufacturer. Specifically: according to the method described in the manual of the human Fab capture kit (Cat.#28-9583-25, GE), the human Fab capture molecule is covalently coupled to the CM5 biosensor chip (Cat.#BR-1000 -12, GE) to affinity capture the antibody to be tested. Then flow through the human TROP2-his (CAT#10428-H08H-100, Sino biological) antigen on the surface of the chip, and use the Biacore instrument to detect the reaction signal in real time to obtain the binding and dissociation curves. The affinity values are obtained by fitting, see Table 22 below . After each cycle of dissociation in the experiment is completed, the biochip is washed and regenerated with the regeneration solution configured in the human Fab capture kit.
表22.本公开的TROP2抗体对人TROP2-his抗原的亲和力Table 22. Affinity of TROP2 antibodies of the present disclosure to human TROP2-his antigen
配体Ligand ka(1/Ms)ka(1/Ms) kd(1/s)kd(1/s) KD(M)KD(M)
RS7RS7 2.25E+052.25E+05 2.71E-042.71E-04 1.21E-091.21E-09
hTINA1-H1L1hTINA1-H1L1 1.41E+051.41E+05 2.93E-032.93E-03 2.07E-082.07E-08
嵌合抗体11E8(DA,NA)Chimeric antibody 11E8 (DA, NA) 1.05E+051.05E+05 1.07E-031.07E-03 1.03E-081.03E-08
人源化抗体11E8-1Humanized antibody 11E8-1 7.10E+047.10E+04 1.05E-031.05E-03 1.49E-081.49E-08
人源化抗体11E8-2Humanized antibody 11E8-2 7.76E+047.76E+04 1.32E-031.32E-03 1.70E-081.70E-08
人源化抗体11E8-3Humanized antibody 11E8-3 8.18E+048.18E+04 1.48E-031.48E-03 1.80E-081.80E-08
人源化抗体11E8-5Humanized antibody 11E8-5 1.00E+051.00E+05 1.09E-031.09E-03 1.08E-081.08E-08
人源化44A5-1Humanized 44A5-1 4.46E+054.46E+05 8.02E-038.02E-03 1.80E-081.80E-08
人源化44A5-2Humanized 44A5-2 4.59E+054.59E+05 4.50E-034.50E-03 9.81E-099.81E-09
人源化44A5-4Humanized 44A5-4 4.26E+054.26E+05 6.80E-036.80E-03 1.60E-081.60E-08
人源化44A5-7Humanized 44A5-7 6.17E+056.17E+05 9.03E-039.03E-03 1.46E-081.46E-08
实施例15偶联Dxd的人源化抗体药物偶联物的体内药效实验Example 15 In vivo pharmacodynamic experiment of humanized antibody drug conjugate coupled to Dxd
将所得的纯化的人源化抗TROP2抗体与MC-GGFG-Dxd(凯恵科技发展(上海)有限公司)进行偶联,经过pH 5.5～8.5的4×PBS缓冲液透析后，加入适量pH7.0至9.0的乙二胺四乙酸(EDTA)和7-10倍摩尔当量三(2-羧乙基)膦(TCEP)，在37摄氏度下还原反应两小时，得反应液A。将反应液A经过G25柱脱盐(购自GE)，去除多余的TCEP，得反应液B。向反应液中加入MC-GGFG-Dxd，其中MC-GGFG-Dxd与还原的TROP2抗体的摩尔比比率为8～12，室温下反应4小时。最后加入N-乙基顺丁烯二酰亚胺(N-EM)中和未反应的巯基。并通过G25柱脱盐除去多余的小分子，得到纯化的TROP2抗体药物偶联物(GGFG-Dxd)。通过LC-MS分析药物的交联率、通过SEC分析抗体药物偶联物的纯度等参数后，进行细胞毒活性的分析。所有抗体药物偶联物的药物交联率(DAR)为6.0-8.0。其中，DAR(drug antibody ratio)指抗体偶联后一个抗体分子上携带的小分子药物的平均数量。The resulting purified humanized anti-TROP2 antibody was conjugated with MC-GGFG-Dxd (Kay Technology Development (Shanghai) Co., Ltd.), after dialysis with 4×PBS buffer pH 5.5～8.5, an appropriate amount of pH7 was added. 0 to 9.0 ethylenediaminetetraacetic acid (EDTA) and 7-10 times molar equivalent of tris(2-carboxyethyl)phosphine (TCEP) are reduced for two hours at 37 degrees Celsius to obtain reaction solution A. The reaction solution A is desalted through a G25 column (purchased from GE), and excess TCEP is removed to obtain reaction solution B. MC-GGFG-Dxd is added to the reaction solution, wherein the molar ratio of MC-GGFG-Dxd to reduced TROP2 antibody is 8-12, and the reaction is carried out at room temperature for 4 hours. Finally, N-ethylmaleimide (N-EM) was added to neutralize the unreacted sulfhydryl groups. And through the G25 column desalting to remove the excess small molecules, the purified TROP2 antibody drug conjugate (GGFG-Dxd) was obtained. After analyzing the cross-linking rate of the drug by LC-MS and the purity of the antibody-drug conjugate by SEC, the cytotoxic activity was analyzed. The drug crosslinking rate (DAR) of all antibody drug conjugates is 6.0-8.0. Among them, DAR (drug antibody ratio) refers to the average number of small molecule drugs carried on an antibody molecule after antibody coupling.
将MDA-MB-468(三阴性乳腺癌细胞株，ATCC，HTB-132)(1×10 7个)200μl细胞悬液(base DMEM:Matrigel＝1:1)接种于CB17SCID小鼠(上海灵畅生物科技有限公司 提供，接种时6-8周)右后背皮下，接种后3-4周待肿瘤长至约250mm 3后，去除体重、肿瘤过大和过小的，按肿瘤体积将小鼠随机分为6组，每组7只。D0尾静脉注射抗体，给药1次，每周测2次瘤体积，称鼠重，记录数据。肿瘤体积(V)计算公式为：V＝1/2×a×b 2；其中a、b分别表示肿瘤的长径和短径。分组如表23。 MDA-MB-468 (triple negative breast cancer cell line, ATCC, HTB-132) (1×10 7 cells) 200 μl cell suspension (base DMEM: Matrigel=1:1) was inoculated into CB17SCID mice (Shanghai Lingchang) Provided by Biotechnology Co., Ltd., 6-8 weeks for inoculation) Right back subcutaneously, 3-4 weeks after inoculation, after the tumor grows to about 250mm 3 , remove the body weight, tumors that are too large and small, and randomize the mice according to the tumor volume Divided into 6 groups with 7 animals in each group. At D0, the antibody was injected into the tail vein once, the tumor volume was measured twice a week, the mouse was weighed, and the data was recorded. The calculation formula of tumor volume (V) is: V=1/2×a×b 2 ; where a and b represent the long diameter and short diameter of the tumor, respectively. The grouping is shown in Table 23.
表23 TROP2嵌合抗体及其抗体药物偶联物的体内药效实验Table 23 In vivo efficacy experiments of TROP2 chimeric antibodies and their antibody-drug conjugates
Figure PCTCN2020108720-appb-000034
Figure PCTCN2020108720-appb-000034
结果见图24A：治疗后肿瘤的体积变化图，和图24B：治疗后的鼠重变化图。结果 显示，对照hIgG1-GGFG-Dxd给药的小鼠肿瘤体积一直和溶剂对照组无明显差异。但是，从处理(即治疗)7天起，上述几种偶联Dxd的人源化抗体药物偶联物即显示出较对照组明显小约100～200mm 3；直至治疗28天时，上述几种偶联Dxd的人源化抗体药物偶联物治疗的小鼠肿瘤体积与0天时相比几乎没有变化，说明这几种偶联Dxd的人源化抗体药物偶联物可以很好的抑制肿瘤MDA-MB-468的生长，而且对小鼠的体重没有显著的影响。 The results are shown in Figure 24A: the tumor volume change graph after treatment, and Figure 24B: the mouse weight change graph after treatment. The results showed that the tumor volume of mice administered with the control hIgG1-GGFG-Dxd has not been significantly different from that of the solvent control group. However, after 7 days of treatment (ie treatment), the above-mentioned humanized antibody-drug conjugates conjugated to Dxd have been shown to be significantly smaller than the control group by about 100-200 mm 3 ; until 28 days of treatment, the above-mentioned several The tumor volume of mice treated with the humanized antibody drug conjugate of Dxd hardly changed compared with that at day 0, indicating that these humanized antibody drug conjugates of Dxd can inhibit tumor MDA- The growth of MB-468 has no significant effect on the weight of mice.
虽然以上描述了本发明的具体实施方式，但是本领域的技术人员应当理解，这些仅是举例说明，在不背离本发明的原理和实质的前提下，可以对这些实施方式做出多种变更或修改。因此，本发明的保护范围由所附权利要求书限定。Although the specific embodiments of the present invention are described above, those skilled in the art should understand that these are merely examples, and various changes or modifications can be made to these embodiments without departing from the principle and essence of the present invention. modify. Therefore, the protection scope of the present invention is defined by the appended claims.

Claims (19)

  1. 一种分离的蛋白质，其特征在于，其包括TROP2抗体的重链可变区和轻链可变区，所述重链可变区包括重链CDR1、重链CDR2和重链CDR3，和/或，所述轻链可变区包括轻链CDR1、轻链CDR2和轻链CDR3，An isolated protein, characterized in that it comprises the heavy chain variable region and the light chain variable region of a TROP2 antibody, the heavy chain variable region comprising heavy chain CDR1, heavy chain CDR2 and heavy chain CDR3, and/or , The light chain variable region includes light chain CDR1, light chain CDR2 and light chain CDR3,
    其中，所述重链CDR1的氨基酸序列如SEQ ID NO:2或SEQ ID NO:10所示，或与如序列表中SEQ ID NO:2或SEQ ID NO:10所示的氨基酸序列至少有80％、85％、90％、92％、94％、95％、96％、97％、98％或99％的序列同源性的氨基酸序列所示；所述重链CDR2的氨基酸序列如SEQ ID NO:3或SEQ ID NO:11所示，或与如序列表中SEQ ID NO:3或SEQ ID NO:11所示的氨基酸序列至少有80％、85％、90％、92％、94％、95％、96％、97％、98％或99％的序列同源性的氨基酸序列所示；和/或，所述重链CDR3的氨基酸序列如SEQ ID NO:4或SEQ ID NO:12所示，或与如序列表中SEQ ID NO:4或SEQ ID NO:12所示的氨基酸序列至少有80％、85％、90％、92％、94％、95％、96％、97％、98％或99％的序列同源性的氨基酸序列所示；Wherein, the amino acid sequence of the heavy chain CDR1 is shown in SEQ ID NO: 2 or SEQ ID NO: 10, or is at least 80% with the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 10 in the sequence list. %, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, or 99% sequence homology shown in the amino acid sequence; the heavy chain CDR2 amino acid sequence is shown in SEQ ID NO: 3 or SEQ ID NO: 11, or at least 80%, 85%, 90%, 92%, 94% of the amino acid sequence shown in SEQ ID NO: 3 or SEQ ID NO: 11 in the sequence list , 95%, 96%, 97%, 98%, or 99% sequence homology; and/or, the amino acid sequence of the heavy chain CDR3 is as SEQ ID NO: 4 or SEQ ID NO: 12 As shown in the sequence table, or with the amino acid sequence shown in SEQ ID NO: 4 or SEQ ID NO: 12 at least 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97% , 98% or 99% sequence homology shown in the amino acid sequence;
    其中，所述轻链CDR1的氨基酸序列如序列表中SEQ ID NO:6或SEQ ID NO:14所示，或与如序列表中SEQ ID NO:6或SEQ ID NO:14所示的氨基酸序列至少有80％、85％、90％、92％、94％、95％、96％、97％、98％或99％的序列同源性的氨基酸序列所示；所述轻链CDR2的氨基酸序列如序列表中SEQ ID NO:7或SEQ ID NO:15所示，或与如序列表中SEQ ID NO:7或SEQ ID NO:15所示的氨基酸序列至少有80％、85％、90％、92％、94％、95％、96％、97％、98％或99％的序列同源性的氨基酸序列所示；和/或，所述轻链CDR3的氨基酸序列如序列表中SEQ ID NO:8或SEQ ID NO:16所示，或与如序列表中SEQ ID NO:8或SEQ ID NO:16所示的氨基酸序列至少有80％、85％、90％、92％、94％、95％、96％、97％、98％或99％的序列同源性的氨基酸序列所示。Wherein, the amino acid sequence of the light chain CDR1 is shown in SEQ ID NO: 6 or SEQ ID NO: 14 in the sequence list, or is similar to the amino acid sequence shown in SEQ ID NO: 6 or SEQ ID NO: 14 in the sequence list. The amino acid sequence with at least 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, or 99% sequence homology is shown; the amino acid sequence of the light chain CDR2 As shown in SEQ ID NO: 7 or SEQ ID NO: 15 in the sequence list, or at least 80%, 85%, 90% of the amino acid sequence shown in SEQ ID NO: 7 or SEQ ID NO: 15 in the sequence list , 92%, 94%, 95%, 96%, 97%, 98%, or 99% sequence homology amino acid sequence; and/or, the light chain CDR3 amino acid sequence is as SEQ ID in the sequence table NO: 8 or SEQ ID NO: 16, or at least 80%, 85%, 90%, 92%, 94% of the amino acid sequence shown in SEQ ID NO: 8 or SEQ ID NO: 16 in the sequence list , 95%, 96%, 97%, 98% or 99% sequence homology shown in the amino acid sequence.
  2. 如权利要求1所述的分离的蛋白质，其特征在于，所述重链CDR2的氨基酸序列如序列表中SEQ ID NO:3所示氨基酸序列的第7位的G和/或第13位的G突变后的氨基酸序列所示；The isolated protein of claim 1, wherein the amino acid sequence of the heavy chain CDR2 is G at position 7 and/or G at position 13 of the amino acid sequence shown in SEQ ID NO: 3 in the sequence listing. The amino acid sequence after mutation is shown;
    较佳地，所述重链CDR2的氨基酸序列如序列表中SEQ ID NO:3所示氨基酸序列的第7位的G和/或第13位的G突变为A的氨基酸序列所示；其氨基酸序列如SEQ ID NO:44、30、39任一所示。Preferably, the amino acid sequence of the heavy chain CDR2 is shown in the amino acid sequence in which G at position 7 and/or G at position 13 is mutated to A in the amino acid sequence shown in SEQ ID NO: 3 in the sequence list; The sequence is shown in SEQ ID NO: 44, 30, 39.
  3. 如权利要求1或2所述的分离的蛋白质，其特征在于，The isolated protein according to claim 1 or 2, wherein:
    所述重链CDR1的氨基酸序列如序列表SEQ ID NO:2所示，所述重链CDR2的氨基酸序列如序列表SEQ ID NO:3、SEQ ID NO:44、30、39任一所示，且所述重链CDR3 的氨基酸序列如序列表SEQ ID NO:4所示；或，所述重链CDR1的氨基酸序列如序列表SEQ ID NO:10所示，所述重链CDR2的氨基酸序列如序列表SEQ ID NO:11所示，且所述重链CDR3的氨基酸序列如序列表SEQ ID NO:12所示；The amino acid sequence of the heavy chain CDR1 is shown in SEQ ID NO: 2 in the sequence table, and the amino acid sequence of the heavy chain CDR2 is shown in any one of SEQ ID NO: 3, SEQ ID NO: 44, 30, 39 in the sequence table, And the amino acid sequence of the heavy chain CDR3 is shown in SEQ ID NO: 4 in the sequence table; or, the amino acid sequence of the heavy chain CDR1 is shown in SEQ ID NO: 10 in the sequence table, and the amino acid sequence of the heavy chain CDR2 is shown in The sequence listing SEQ ID NO: 11 is shown, and the heavy chain CDR3 amino acid sequence is shown in the sequence listing SEQ ID NO: 12;
    所述轻链CDR1的氨基酸序列如序列表SEQ ID NO:6所示，所述轻链CDR2的氨基酸序列如序列表SEQ ID NO:7所示，且所述轻链CDR3的氨基酸序列如序列表SEQ ID NO:8所示；或，所述轻链CDR1的氨基酸序列如序列表SEQ ID NO:14所示，所述轻链CDR2的氨基酸序列如序列表SEQ ID NO:15所示，且所述轻链CDR3的氨基酸序列如序列表SEQ ID NO:16所示；The amino acid sequence of the light chain CDR1 is shown in SEQ ID NO: 6 in the sequence table, the amino acid sequence of the light chain CDR2 is shown in SEQ ID NO: 7 in the sequence table, and the amino acid sequence of the light chain CDR3 is shown in the sequence table SEQ ID NO: 8; or, the amino acid sequence of the light chain CDR1 is shown in SEQ ID NO: 14 in the sequence listing, and the amino acid sequence of the light chain CDR2 is shown in SEQ ID NO: 15 in the sequence listing, and The amino acid sequence of the light chain CDR3 is shown in SEQ ID NO: 16 in the sequence table;
    较佳地：Preferably:
    所述重链CDR1的氨基酸序列如序列表SEQ ID NO:2所示，所述重链CDR2的氨基酸序列如序列表SEQ ID NO:3、SEQ ID NO:44、30、39任一所示，且所述重链CDR3的氨基酸序列如序列表SEQ ID NO:4所示；以及所述轻链CDR1的氨基酸序列如序列表SEQ ID NO:6所示，所述轻链CDR2的氨基酸序列如序列表SEQ ID NO:7所示，且所述轻链CDR3的氨基酸序列如序列表SEQ ID NO:8所示；The amino acid sequence of the heavy chain CDR1 is shown in SEQ ID NO: 2 in the sequence table, and the amino acid sequence of the heavy chain CDR2 is shown in any one of SEQ ID NO: 3, SEQ ID NO: 44, 30, 39 in the sequence table, And the amino acid sequence of the heavy chain CDR3 is shown in SEQ ID NO: 4 in the sequence list; and the amino acid sequence of the light chain CDR1 is shown in SEQ ID NO: 6 in the sequence list, and the amino acid sequence of the light chain CDR2 is shown in sequence The list SEQ ID NO: 7 is shown, and the amino acid sequence of the light chain CDR3 is shown in the sequence list SEQ ID NO: 8;
    或，所述重链CDR1的氨基酸序列如序列表SEQ ID NO:10所示，所述重链CDR2的氨基酸序列如序列表SEQ ID NO:11所示，且所述重链CDR3的氨基酸序列如序列表SEQ ID NO:12所示；以及所述轻链CDR1的氨基酸序列如序列表SEQ ID NO:14所示，所述轻链CDR2的氨基酸序列如序列表SEQ ID NO:15所示，且所述轻链CDR3的氨基酸序列如序列表SEQ ID NO:16所示。Or, the amino acid sequence of the heavy chain CDR1 is shown in SEQ ID NO: 10 in the sequence table, the amino acid sequence of the heavy chain CDR2 is shown in SEQ ID NO: 11 in the sequence table, and the amino acid sequence of the heavy chain CDR3 is shown in The amino acid sequence of the light chain CDR1 is shown in SEQ ID NO: 14 in the sequence listing, and the amino acid sequence of the light chain CDR2 is shown in SEQ ID NO: 15 in the sequence listing, and The amino acid sequence of the light chain CDR3 is shown in SEQ ID NO: 16 in the sequence listing.
  4. 如权利要求1或2所述的分离的蛋白质，其特征在于，所述重链可变区还包括重链可变区框架区，和/或，所述轻链可变区还包括轻链可变区框架区；The isolated protein of claim 1 or 2, wherein the heavy chain variable region further comprises a heavy chain variable region framework region, and/or the light chain variable region further comprises a light chain variable region Variable area frame area;
    较佳地，所述重链可变区框架区为鼠抗体的重链可变区框架区或人抗体的重链可变区框架区或其回复突变；所述轻链可变区框架区为鼠抗体的轻链可变区框架区或人抗体的轻链可变区框架区或其回复突变；所述人抗体的重链可变区框架区优选为IGHV1-69*08/JH6C或IGHV3-7*01/JH4D，所述人抗体的轻链可变区框架区优选为IGKV1-39*01/JK4或IGKV1-39*01/JK1；Preferably, the heavy chain variable region framework region is the heavy chain variable region framework region of a murine antibody or the heavy chain variable region framework region of a human antibody or a back mutation; the light chain variable region framework region is The light chain variable region framework region of a murine antibody or the light chain variable region framework region of a human antibody or its back mutation; the heavy chain variable region framework region of the human antibody is preferably IGHV1-69*08/JH6C or IGHV3- 7*01/JH4D, the light chain variable region framework region of the human antibody is preferably IGKV1-39*01/JK4 or IGKV1-39*01/JK1;
    更佳地，所述重链可变区的氨基酸序列如序列表SEQ ID NO:1、9、20、23、24、26-29、36-38或其突变的氨基酸序列中任一所示；和/或，所述的轻链可变区的氨基酸序列如序列表SEQ ID NO:5、13、32-34、41-43或其突变的氨基酸序列中任一所示；所述突变在所述重链可变区或轻链可变区的氨基酸序列上具有一个或多个氨基酸残基的取代、缺失或插入，并且保持或改善了所述蛋白质的功能；所述突变的氨基酸序列优选与所述重链可变区或轻链可变区的氨基酸序列具有至少80％更优选至少99％的序列同源性；More preferably, the amino acid sequence of the heavy chain variable region is as shown in any one of SEQ ID NO: 1, 9, 20, 23, 24, 26-29, 36-38 or its mutant amino acid sequence in the sequence list; And/or, the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 5, 13, 32-34, 41-43 of the sequence list or any of its mutant amino acid sequences; The amino acid sequence of the heavy chain variable region or the light chain variable region has one or more amino acid residue substitutions, deletions or insertions, and maintains or improves the function of the protein; the mutant amino acid sequence is preferably The amino acid sequence of the heavy chain variable region or the light chain variable region has a sequence homology of at least 80%, more preferably at least 99%;
    进一步更佳地：Further and better:
    所述重链可变区的氨基酸序列如序列表SEQ ID NO:1、20、23、24、26-29任一所示，且所述轻链可变区的氨基酸序列如序列表SEQ ID NO:5、32-34任一所示；或，所述重链可变区的氨基酸序列如序列表SEQ ID NO:9、36-38任一所示，且所述轻链可变区的氨基酸序列如序列表SEQ ID NO:13、41-43任一所示。The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 1, 20, 23, 24, 26-29 in the sequence list, and the amino acid sequence of the light chain variable region is shown in the sequence list SEQ ID NO : 5, 32-34; or, the amino acid sequence of the heavy chain variable region is shown in any one of SEQ ID NO: 9, 36-38 in the sequence list, and the amino acid of the light chain variable region The sequence is shown in any one of SEQ ID NO: 13, 41-43 in the sequence table.
  5. 如权利要求1-4任一项所述的分离的蛋白质，其特征在于，所述的分离的蛋白质还包括抗体重链恒定区和抗体轻链恒定区；较佳地，所述的抗体重链恒定区为小鼠源抗体重链恒定区或人源抗体重链恒定区；所述的抗体轻链恒定区为小鼠源轻链抗体恒定区或人源抗体轻链恒定区；更佳地，所述的抗体重链恒定区为人源抗体重链恒定区，优选人源IgG1、IgG2、IgG3或IgG4抗体重链恒定区；所述的抗体轻链恒定区为人源抗体轻链κ或λ链恒定区。The isolated protein of any one of claims 1-4, wherein the isolated protein further comprises an antibody heavy chain constant region and an antibody light chain constant region; preferably, the antibody heavy chain The constant region is a mouse-derived antibody heavy chain constant region or a human antibody heavy chain constant region; the antibody light chain constant region is a mouse-derived light chain antibody constant region or a human antibody light chain constant region; more preferably, The antibody heavy chain constant region is a human antibody heavy chain constant region, preferably a human IgG1, IgG2, IgG3 or IgG4 antibody heavy chain constant region; the antibody light chain constant region is a human antibody light chain κ or λ chain constant Area.
  6. 如权利要求1-5任一项所述的分离的蛋白质，其特征在于，所述的分离的蛋白质选自抗体全长蛋白、抗原结合片段、双特异性抗体、多特异性抗体、单链抗体、单域抗体和单区抗体中的一种或多种；The isolated protein of any one of claims 1-5, wherein the isolated protein is selected from the group consisting of antibody full-length protein, antigen-binding fragment, bispecific antibody, multispecific antibody, single chain antibody , One or more of single domain antibodies and single domain antibodies;
    或者，所述的分离的蛋白质为单克隆抗体或多克隆抗体；Alternatively, the isolated protein is a monoclonal antibody or a polyclonal antibody;
    或者，所述的分离的蛋白质为超人源化抗体。Alternatively, the isolated protein is a superhumanized antibody.
  7. 一种核酸，其特征在于，其编码如权利要求1-6任一项所述的分离的蛋白质。A nucleic acid, characterized in that it encodes the isolated protein according to any one of claims 1-6.
  8. 一种包含如权利要求7所述的核酸的重组表达载体；较佳地，所述重组表达载体为质粒、粘粒、噬菌体或病毒载体。A recombinant expression vector containing the nucleic acid of claim 7; preferably, the recombinant expression vector is a plasmid, cosmid, phage or virus vector.
  9. 一种转化体，其在宿主细胞中包含如权利要求8所述的重组表达载体；较佳地，所述宿主细胞为E.coli TG1、BL21细胞，或者CHO-K1细胞。A transformant which contains the recombinant expression vector according to claim 8 in a host cell; preferably, the host cell is E. coli TG1, BL21 cell, or CHO-K1 cell.
  10. 一种基因修饰的细胞，其特征在于，其包含如权利要求6中所述的分离的蛋白质；较佳地，所述基因修饰的细胞为真核细胞，优选分离的人细胞；更优选免疫细胞如T细胞，或NK细胞。A genetically modified cell, characterized in that it contains the isolated protein as described in claim 6; preferably, the genetically modified cell is a eukaryotic cell, preferably an isolated human cell; more preferably an immune cell Such as T cells, or NK cells.
  11. 一种分离的蛋白质的制备方法，其包括如下步骤：培养如权利要求9所述的转化体，从培养物中获得分离的蛋白质。A method for preparing an isolated protein, which comprises the following steps: culturing the transformant according to claim 9 to obtain the isolated protein from the culture.
  12. 一种抗体药物偶联物，其特征在于，其包括共价附着至细胞毒剂的如权利要求1-6任一项所述的分离的蛋白质。An antibody drug conjugate, characterized in that it comprises the isolated protein according to any one of claims 1 to 6 covalently attached to a cytotoxic agent.
  13. 如权利要求12所述的抗体药物偶联物，其特征在于，每1当量所述的分离的蛋白质通过x当量接头与y当量的细胞毒剂相连，其具有如式1所示的结构，The antibody-drug conjugate according to claim 12, wherein every 1 equivalent of the isolated protein is connected to y equivalents of the cytotoxic agent through an x equivalent linker, and it has the structure shown in formula 1,
    Ab-(L) x-(D) y Ab-(L) x -(D) y
    式1Formula 1
    其中，Ab为如权利要求1-6中任一项所述的分离的蛋白质；L为接头；D为细胞毒剂；x和y各自独立地为自然数，优选1-20的整数，更优选2-8的整数，例如3或4；x和y的比例优选为1:1。Wherein, Ab is the isolated protein according to any one of claims 1-6; L is a linker; D is a cytotoxic agent; x and y are each independently a natural number, preferably an integer of 1-20, more preferably 2- An integer of 8, such as 3 or 4; the ratio of x and y is preferably 1:1.
  14. 如权利要求13所述的抗体药物偶联物，其特征在于，所述L为活性酯、碳酸盐类、氨基甲酸酯类、亚胺磷酸酯、肟类、腙类、缩醛类、原酸酯类、氨基类、小肽段或核苷酸片段；和/或，所述D为细胞毒素、化学治疗剂、放射性同位素、治疗性核酸、免疫调节剂、抗血管生成剂、抗增殖促凋亡剂或细胞溶解酶；The antibody-drug conjugate according to claim 13, wherein the L is an active ester, carbonate, carbamate, phosphoimidite, oxime, hydrazone, acetal, ortho acid Ester, amino, small peptide or nucleotide fragment; and/or, said D is cytotoxin, chemotherapeutic agent, radioisotope, therapeutic nucleic acid, immunomodulator, anti-angiogenesis agent, anti-proliferation and anti-apoptosis Death agent or cytolytic enzyme;
    较佳地，所述L主要含有式2所示结构，其为L中离去基团离去后对应的剩余部分；Preferably, said L mainly contains the structure represented by formula 2, which is the remaining part corresponding to the leaving group in L;
    (CO-Alk 1-Sp 1-Ar-Sp 2-Alk 2-C(Z 1)＝Q-Sp) (CO-Alk 1 -Sp 1 -Ar-Sp 2 -Alk 2 -C(Z 1 )=Q-Sp)
    式2；Formula 2;
    更佳地，所述L为马来酰亚胺基己酰、马来酰亚胺基己酰-L-缬氨酸-L-瓜氨酸对氨基苄醇或4-(N-马来酰亚胺基甲基)环己烷-1-羧酸琥珀酰亚胺酯；和/或，所述D为甲基奥瑞他汀F、甲基奥瑞他汀E或N2’-脱乙酰-N2’-3-巯基-1氧代丙基)-美登素。More preferably, the L is maleimidohexanoyl, maleimidohexanoyl-L-valine-L-citrulline p-aminobenzyl alcohol or 4-(N-maleyl Iminomethyl) cyclohexane-1-carboxylic acid succinimide ester; and/or, said D is methyl auristatin F, methyl auristatin E or N2'-deacetyl-N2' -3-Mercapto-1 (oxopropyl)-maytansine.
  15. 如权利要求12-14任一项所述的抗体药物偶联物，其特征在于，所述式1中x＝y＝n；所述抗体药物偶联物的结构如式3或者如式4或者如式5所示，The antibody-drug conjugate according to any one of claims 12-14, wherein x=y=n in the formula 1; the structure of the antibody-drug conjugate is as formula 3 or as formula 4 or As shown in Equation 5,
    Figure PCTCN2020108720-appb-100001
    Figure PCTCN2020108720-appb-100001
    式3中，m为1～10，优选m为5，L为马来酰亚胺基己酰；D为甲基奥瑞他汀F；In formula 3, m is 1-10, preferably m is 5, L is maleimidohexanoyl; D is methyl auristatin F;
    Figure PCTCN2020108720-appb-100002
    Figure PCTCN2020108720-appb-100002
    式4中，L为4-(N-马来酰亚胺基甲基)环己烷-1-羧酸琥珀酰亚胺酯；D为N2’-脱乙酰-N2’-3-巯基-1氧代丙基-美登素；In formula 4, L is 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid succinimide ester; D is N2'-deacetyl-N2'-3-mercapto-1 Oxopropyl-maytansine;
    Figure PCTCN2020108720-appb-100003
    Figure PCTCN2020108720-appb-100003
    式5中，L为马来酰亚胺基己酰-L-缬氨酸-L-瓜氨酸对氨基苄醇，D为甲基奥瑞他汀E；In formula 5, L is maleimidohexanoyl-L-valine-L-citrulline p-aminobenzyl alcohol, and D is methyl auristatin E;
    其中，n为自然数，优选为1-20的整数，更优选为2-8的整数，例如为3或4。Wherein, n is a natural number, preferably an integer of 1-20, more preferably an integer of 2-8, for example, 3 or 4.
  16. 如权利要求12-15中任一项所述的抗体药物偶联物的制备方法，其包括以下步骤：The preparation method of the antibody drug conjugate according to any one of claims 12-15, which comprises the following steps:
    a.将如权利要求1-6中任一项所述的分离的蛋白质经过硼酸钠缓冲液透析后，加入三(2-羧乙基)膦，室温下还原得反应液A；a. After the separated protein of any one of claims 1-6 is dialyzed with sodium borate buffer, tris(2-carboxyethyl)phosphine is added, and the reaction solution A is obtained by reducing at room temperature;
    b.将反应液A洗脱去除多余的蛋白质得反应液B；b. Elute reaction solution A to remove excess protein to obtain reaction solution B;
    c.向反应液B中加入如权利要求13-15中任一项所述的-(L) x-(D) y反应即得； c. Add the -(L) x -(D) y reaction as described in any one of claims 13-15 to the reaction solution B.
    较佳地，所述步骤a中，所述硼酸钠缓冲液的pH值为6.5～8.5；所述三(2-羧乙基)膦(TCEP)与所述的分离的蛋白质的摩尔比比率为2～10；所述还原的时间为1～4小时；和/或，所述步骤c中，-(L) x-(D) y与所述的分离的蛋白质的摩尔比比率为5～20，所述反应的温度为10～37℃，所述反应的时间为4小时。 Preferably, in the step a, the pH of the sodium borate buffer is 6.5-8.5; the molar ratio of the tris(2-carboxyethyl)phosphine (TCEP) to the isolated protein is 2 to 10; the reduction time is 1 to 4 hours; and/or, in the step c, the molar ratio of -(L) x -(D) y to the isolated protein is 5 to 20 The temperature of the reaction is 10-37°C, and the time of the reaction is 4 hours.
  17. 一种药物组合物，其特征在于，其包括如权利要求1-6中任一项所述的分离的蛋白质和/或如权利要求12-15中任一项所述的抗体药物偶联物，和药学可接受的载体；所述的药物组合物较佳地还包括其他抗肿瘤抗体作为活性成分；较佳地，所述的药物组合物包括0.01～99.99％的如权利要求1-6中任一项所述的分离的蛋白质和/或如权利要求12-15中任一项所述的抗体药物偶联物，和0.01～99.99％的药用载体，所述百分比为占所述药物组合物的质量百分比。A pharmaceutical composition, characterized in that it comprises the isolated protein according to any one of claims 1-6 and/or the antibody drug conjugate according to any one of claims 12-15, And a pharmaceutically acceptable carrier; the pharmaceutical composition preferably further includes other anti-tumor antibodies as active ingredients; preferably, the pharmaceutical composition includes 0.01-99.99% of any of claims 1-6 One of the isolated protein and/or the antibody drug conjugate of any one of claims 12-15, and 0.01-99.99% of the pharmaceutical carrier, and the percentage is based on the pharmaceutical composition The percentage of mass.
  18. 一种如权利要求1-6中任一项所述的分离的蛋白质、或者如权利要求12-15中任一项所述的抗体药物偶联物、或者如权利要求17所述的药物组合物在制备抗肿瘤药物中的应用；An isolated protein according to any one of claims 1 to 6, or an antibody drug conjugate according to any one of claims 12-15, or a pharmaceutical composition according to claim 17 Application in the preparation of anti-tumor drugs;
    较佳地，所述抗肿瘤药物为与TROP2表达或功能异常相关的疾病的药物；更佳地，所述与TROP2表达或功能异常相关的疾病为自体免疫疾病、炎症性疾病、感染性疾病或增殖性疾病，优选非小细胞肺癌、浸润性乳腺癌、结肠癌、直肠癌、胃癌、鳞状宫颈癌、 浸润性子宫内膜腺癌、浸润性胰腺癌、卵巢癌、鳞状膀胱癌、绒毛膜癌、支气管癌、乳腺癌、子宫颈癌、胰腺癌或精囊癌。Preferably, the anti-tumor drug is a drug for a disease related to abnormal expression or function of TROP2; more preferably, the disease related to abnormal expression or function of TROP2 is an autoimmune disease, an inflammatory disease, an infectious disease or Proliferative diseases, preferably non-small cell lung cancer, invasive breast cancer, colon cancer, rectal cancer, gastric cancer, squamous cervical cancer, invasive endometrial adenocarcinoma, invasive pancreatic cancer, ovarian cancer, squamous bladder cancer, villi Membrane cancer, bronchial cancer, breast cancer, cervical cancer, pancreatic cancer or seminal vesicle cancer.
  19. 一种检测过表达TROP2蛋白的细胞的方法，其特征在于，其包括以下的步骤：如权利要求1-6中任一项所述的分离的蛋白质与待检细胞样品在体外接触，检测所述的分离的蛋白质与所述待检细胞样品的结合即可；优选地，所述检测为FACS检测，和/或，所述待测细胞样品为离体的待测细胞样品。A method for detecting cells overexpressing TROP2 protein, characterized in that it comprises the following steps: the isolated protein according to any one of claims 1 to 6 is contacted with a sample of cells to be tested in vitro, and the The isolated protein can be combined with the cell sample to be tested; preferably, the detection is FACS detection, and/or the cell sample to be tested is an isolated cell sample to be tested.
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