[go: up one dir, main page]

WO2021027851A1 - Anticorps trop2, son procédé de préparation, conjugué et utilisation associée - Google Patents

Anticorps trop2, son procédé de préparation, conjugué et utilisation associée Download PDF

Info

Publication number
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
Authority
WO
WIPO (PCT)
Prior art keywords
amino acid
seq
antibody
acid sequence
sequence
Prior art date
Application number
PCT/CN2020/108720
Other languages
English (en)
Chinese (zh)
Inventor
彭菲
曲宝源
张利
盛其然
严玉萍
陈海霞
阮文静
杨达志
Original Assignee
凯惠科技发展(上海)有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 凯惠科技发展(上海)有限公司 filed Critical 凯惠科技发展(上海)有限公司
Publication of WO2021027851A1 publication Critical patent/WO2021027851A1/fr

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material
    • 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.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Immunology (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Organic Chemistry (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Molecular Biology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Biomedical Technology (AREA)
  • Cell Biology (AREA)
  • Biochemistry (AREA)
  • Hematology (AREA)
  • Urology & Nephrology (AREA)
  • Genetics & Genomics (AREA)
  • Microbiology (AREA)
  • Biotechnology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Zoology (AREA)
  • Food Science & Technology (AREA)
  • Pathology (AREA)
  • Epidemiology (AREA)
  • General Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Biophysics (AREA)
  • Oncology (AREA)
  • Communicable Diseases (AREA)
  • Pain & Pain Management (AREA)
  • Rheumatology (AREA)
  • Wood Science & Technology (AREA)
  • Virology (AREA)

Abstract

L'invention concerne un anticorps TROP2 et son procédé de préparation ainsi qu'un conjugué et une utilisation associée. L'anticorps TROP2 comprend une région variable de chaîne lourde et une région variable de chaîne légère, la région variable de chaîne lourde comprenant un CDR1 de chaîne lourde, un CDR2 de chaîne lourde et un CDR3 de chaîne lourde, et/ou, la région variable de chaîne légère comprenant un CDR1 de chaîne légère, un CDR2 de chaîne légère et un CDR3 de chaîne légère. L'anticorps TROP2 présente une affinité élevée et une forte spécificité, et un conjugué produit à partir d'un couplage avec une toxine médicamenteuse à petites molécules comme MMAF est capable d'avoir un effet cytotoxique sur les cellules positives à TROP2, et peut par conséquent être utilisé dans la préparation d'un médicament pour le traitement de tumeurs et analogues.
PCT/CN2020/108720 2019-08-12 2020-08-12 Anticorps trop2, son procédé de préparation, conjugué et utilisation associée WO2021027851A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201910746536 2019-08-12
CN201910746536.0 2019-08-12

Publications (1)

Publication Number Publication Date
WO2021027851A1 true WO2021027851A1 (fr) 2021-02-18

Family

ID=74570531

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2020/108720 WO2021027851A1 (fr) 2019-08-12 2020-08-12 Anticorps trop2, son procédé de préparation, conjugué et utilisation associée

Country Status (2)

Country Link
CN (1) CN112390885B (fr)
WO (1) WO2021027851A1 (fr)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2024178310A1 (fr) * 2023-02-23 2024-08-29 Ambrx, Inc. Conjugués anticorps-médicament dirigés contre trop2 et utilisations associées
WO2024194775A1 (fr) * 2023-03-17 2024-09-26 Zydus Lifesciences Limited Conjugués médicamenteux de sacituzumab et leur préparation

Families Citing this family (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115536747B (zh) * 2021-06-30 2024-08-27 益科思特(北京)医药科技发展有限公司 一种结合trop2的抗体及靶向trop2和cd3的双特异性抗体及其制备方法与应用
CN115558026A (zh) * 2021-07-02 2023-01-03 和迈生物科技有限公司 抗trop2单域抗体及其应用
CN114652851B (zh) * 2021-09-29 2024-06-07 石药集团巨石生物制药有限公司 一种抗trop2蛋白的抗体缀合物
CN114634574B (zh) * 2022-04-18 2022-12-20 先进生物(苏州)有限公司 抗B7H6的scFv抗体、其编码基因及其应用

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101679526A (zh) * 2007-05-30 2010-03-24 霍夫曼-拉罗奇有限公司 介导癌细胞细胞毒性的人源化和嵌合抗trop-2抗体
CN104053672A (zh) * 2011-11-11 2014-09-17 瑞纳神经科学公司 Trop-2特异性抗体及其用途
CN105849126A (zh) * 2013-12-25 2016-08-10 第三共株式会社 抗trop2抗体－药物偶联物
CN107446050A (zh) * 2017-08-11 2017-12-08 百奥泰生物科技(广州)有限公司 Trop2阳性疾病治疗的化合物及方法
WO2019039483A1 (fr) * 2017-08-23 2019-02-28 第一三共株式会社 Préparation de conjugué anticorps-médicament et lyophilisation associée

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9770517B2 (en) * 2002-03-01 2017-09-26 Immunomedics, Inc. Anti-Trop-2 antibody-drug conjugates and uses thereof
WO2015047510A1 (fr) * 2013-09-27 2015-04-02 Immunomedics, Inc. Conjugués anticorps anti-trop-2-médicament et leurs utilisations
CN113603703A (zh) * 2017-12-15 2021-11-05 四川科伦博泰生物医药股份有限公司 生物活性物偶联物及其制备方法和用途

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101679526A (zh) * 2007-05-30 2010-03-24 霍夫曼-拉罗奇有限公司 介导癌细胞细胞毒性的人源化和嵌合抗trop-2抗体
CN104053672A (zh) * 2011-11-11 2014-09-17 瑞纳神经科学公司 Trop-2特异性抗体及其用途
CN105849126A (zh) * 2013-12-25 2016-08-10 第三共株式会社 抗trop2抗体－药物偶联物
CN107446050A (zh) * 2017-08-11 2017-12-08 百奥泰生物科技(广州)有限公司 Trop2阳性疾病治疗的化合物及方法
WO2019039483A1 (fr) * 2017-08-23 2019-02-28 第一三共株式会社 Préparation de conjugué anticorps-médicament et lyophilisation associée

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
BARDIA ADITYA, MAYER INGRID A., DIAMOND JENNIFER R., MOROOSE REBECCA L., ISAKOFF STEVEN J., STARODUB ALEXANDER N., SHAH NIKITA C.,: "Efficacy and Safety of Anti-Trop-2 Antibody Drug Conjugate Sacituzumab Govitecan (IMMU-132) in Heavily Pretreated Patients With Metastatic Triple-Negative Breast Cancer", JOURNAL OF CLINICAL ONCOLOGY, vol. 35, no. 19, 1 July 2017 (2017-07-01), American Society of Clinical Oncology, US, pages 2141 - 2148, XP055781665, ISSN: 0732-183X, DOI: 10.1200/JCO.2016.70.8297 *
JOYCE VARUGHESE; EMILIANO COCCO; STEFANIA BELLONE; ELENA RATNER; DAN-ARIN SILASI; MASOUD AZODI; PETER E. SCHWARTZ; THOMAS J. RUTHE: "Cervical carcinomas overexpress human trophoblast cell-surface marker (Trop-2) and are highly sensitive to immunotherapy with hRS7, a humanized monoclonal anti-Trop-2 antibody", AMERICAN JOURNAL OF OBSTETRICS & GYNECOLOGY, vol. 205, no. 6, 29 June 2011 (2011-06-29), MOSBY, ST LOUIS, MO, US, pages 567.e1 - 567.e7, XP028116550, ISSN: 0002-9378, DOI: 10.1016/j.ajog.2011.06.093 *
LIANG JIE, LIU QIONG-QIONG, ZHANG HUI-LIN, LIN HONG, TANG QI, LIU YU, SU YI-PING, FENG ZHEN-QING, ZHU JIN: "Preparation and immunologic characterization of a monoclonal antibody against human trophoblast cell-surface antigen 2", ACTA UNIVERSITATIS MEDICINALIS NANJING(NATURAL SCIENCE), NANJING UNIVERSITY, CN, vol. 31, no. 5, 15 May 2011 (2011-05-15), CN, pages 645 - 650, XP055781662, ISSN: 1007-4368 *
LIU JINRONG, BAI LUYUE; TANG QI; WANG YIQUAN; YE CHUNPING; YIN ZHENGNA; ZHU JIN; FENG ZHENQING; TONG HUA; ZHANG HUILIN: "Preparation of a full human anti-Trop-2 IgG and its effect on ovarian cancer cells", ACTA UNIVERSITATIS MEDICINALIS NANJING(NATURAL SCIENCE), NANJING UNIVERSITY, CN, 15 March 2016 (2016-03-15), CN, pages 280 - 286, XP055781679, ISSN: 1007-4368 *
P. STROP, T.-T. TRAN, M. DORYWALSKA, K. DELARIA, R. DUSHIN, O. K. WONG, W.-H. HO, D. ZHOU, A. WU, E. KRAYNOV, L. ASCHENBRENNER, B.: "RN927C, a Site-Specific Trop-2 Antibody-Drug Conjugate (ADC) with Enhanced Stability, Is Highly Efficacious in Preclinical Solid Tumor Models", MOLECULAR CANCER THERAPEUTICS, vol. 15, no. 11, 1 November 2016 (2016-11-01), pages 2698 - 2708, XP055658796, ISSN: 1535-7163, DOI: 10.1158/1535-7163.MCT-16-0431 *
STARODUB ALEXANDER N., OCEAN ALLYSON J., SHAH MANISH A., GUARINO MICHAEL J., PICOZZI VINCENT J., VAHDAT LINDA T., THOMAS SAJEVE S.: "First-in-Human Trial of a Novel Anti-Trop-2 Antibody-SN-38 Conjugate, Sacituzumab Govitecan, for the Treatment of Diverse Metastatic Solid Tumors", CLINICAL CANCER RESEARCH, vol. 21, no. 17, 1 September 2015 (2015-09-01), American Association for Cancer Research, US, pages 3870 - 3878, XP055781655, ISSN: 1078-0432, DOI: 10.1158/1078-0432.CCR-14-3321 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2024178310A1 (fr) * 2023-02-23 2024-08-29 Ambrx, Inc. Conjugués anticorps-médicament dirigés contre trop2 et utilisations associées
WO2024194775A1 (fr) * 2023-03-17 2024-09-26 Zydus Lifesciences Limited Conjugués médicamenteux de sacituzumab et leur préparation

Also Published As

Publication number Publication date
CN112390885B (zh) 2024-01-19
CN112390885A (zh) 2021-02-23

Similar Documents

Publication Publication Date Title
WO2021027851A1 (fr) Anticorps trop2, son procédé de préparation, conjugué et utilisation associée
JP6010241B2 (ja) Ｄｌｌ３モジュレーター及び使用方法
RU2708075C2 (ru) Конъюгаты анти-ртк7 антитело-лекарственное средство
JP6401060B2 (ja) 抗ｓｅｚ６抗体及び使用方法
TWI402078B (zh) 抗ｃｓｆ－１ｒ抗體
US20230338565A1 (en) Antibody drug conjugate, preparation method therefor and use thereof
WO2018113136A1 (fr) Anticorps anti-tpbg, procédé de préparation, conjugué et utilisation de celui-ci
WO2019114804A1 (fr) Anticorps egfrviii et conjugué, procédé de préparation associé et utilisation correspondante
WO2012057328A1 (fr) Anticorps anti-cdh3 présentant une capacité élevée d'internalisation
US11608384B2 (en) Humanized anti-TPBG antibody, preparation method therefor, conjugate thereof, and applications
JP2016540826A (ja) 抗ｅｆｎａ４抗体−薬物コンジュゲート
TW202432186A (zh) 配體-細胞毒藥物綴合物及其醫藥用途

Legal Events

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

Ref document number: 20852930

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 20852930

Country of ref document: EP

Kind code of ref document: A1