WO2021190480A1 - Conjugué anticorps-médicament et son utilisation médicale - Google Patents

Conjugué anticorps-médicament et son utilisation médicale Download PDF

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WO2021190480A1
WO2021190480A1 PCT/CN2021/082294 CN2021082294W WO2021190480A1 WO 2021190480 A1 WO2021190480 A1 WO 2021190480A1 CN 2021082294 W CN2021082294 W CN 2021082294W WO 2021190480 A1 WO2021190480 A1 WO 2021190480A1
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seq
antibody
group
heavy chain
variable region
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PCT/CN2021/082294
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English (en)
Chinese (zh)
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花海清
包如迪
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上海翰森生物医药科技有限公司
江苏豪森药业集团有限公司
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Priority to CN202180021931.8A priority Critical patent/CN115298220A/zh
Publication of WO2021190480A1 publication Critical patent/WO2021190480A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/47—Quinolines; Isoquinolines
    • A61K31/4738—Quinolines; Isoquinolines ortho- or peri-condensed with heterocyclic ring systems
    • A61K31/4745—Quinolines; Isoquinolines ortho- or peri-condensed with heterocyclic ring systems condensed with ring systems having nitrogen as a ring hetero atom, e.g. phenantrolines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • 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

Definitions

  • the present invention relates to an anti-TROP-2 antibody-drug conjugate which is specifically immunoreactive to human TROP-2 receptor and its pharmaceutical composition, as well as its use as an anti-cancer drug and its use in detecting or diagnosing tumors .
  • Molecular targeted therapy of tumors is a new treatment model that is different from traditional surgery, radiotherapy, and chemotherapy. Its advantage is that drugs usually only bind to the corresponding target, and directly affect the function of its target molecule or carry Physical or chemical effector molecules to achieve the effect of killing or inhibiting target cells. Because the target site is clear, this type of drug usually has a high selectivity, which can effectively kill or inhibit the target cells, but also produces no or only minor toxic side effects on normal tissue cells. Therefore, the development of molecular targeted drugs has become a hot spot in tumor clinical research.
  • TROP-2 Human trophoblast cell surface antigen 2
  • TROP-2 consists of 323 amino acids, including 26 amino acids in signal peptide, 248 amino acids in extracellular region, 23 amino acids in transmembrane region, and 26 amino acids in cytoplasmic region.
  • the extracellular domain has a characteristic thyroglobulin (TY) sequence, which is generally believed to be related to the proliferation, infiltration and metastasis of cancer cells.
  • TY thyroglobulin
  • TROP-2 is overexpressed in a variety of epithelial cancers such as gastric cancer, lung cancer, colorectal cancer, ovarian cancer, breast cancer, prostate cancer, pancreatic cancer, liver cancer, and esophageal cancer.
  • TROP-2 is rarely expressed or not expressed in normal adult tissues, and is limited to a small amount of expression in cells in the epithelial area, and the expression level is lower than that in cancer, indicating that TROP-2 is related to tumor formation.
  • the overexpression of TROP-2 in tumor tissues is closely related to the poor prognosis of patients and the metastasis of cancer cells, and at the same time affects the overall survival rate of patients. Therefore, TROP-2 has become an attractive target in tumor molecular targeted therapy.
  • US Patent No. 5840854 reports the cytotoxicity of anti-hTROP-2 monoclonal antibody (BR110) bound to cytotoxin on human cancer cell lines H3619, H2987, MCF-7, H3396 and H2981.
  • U.S. Patent No. 6653104 discloses an antibody (RS7), which was tested in an in vivo model using an antibody labeled with a radioactive substance. It showed anti-tumor activity in a nude mouse xenograft model, but there is no report on the naked antibody. Timely anti-tumor effect.
  • US Patent No. 7420040 also reported that the isolated monoclonal antibody produced by the hybridoma cell line AR47A6.4.2 or AR52A301.5 obtained from human ovarian cancer tissue immunized mice was bound to hTROP-2, and was used in a nude mouse xenograft model Shows anti-tumor activity in.
  • CN102827282A discloses a human anti-TROP-2 genetically engineered antibody IgG and its application. In vitro test results show that the anti-TROP-2 antibody IgG has a significant inhibitory effect on the proliferation of pancreatic cancer cells.
  • CN104114580A discloses an antibody (especially a humanized antibody) that specifically reacts with hTROP-2 and has anti-tumor activity in the body, as well as hybridomas that produce the antibody, a complex of the antibody and a drug, and diagnostic applications for tumors. Or therapeutic pharmaceutical composition, tumor detection method, tumor detection or diagnostic kit.
  • an antibody-drug conjugate or a pharmaceutically acceptable salt or solvent compound thereof is provided.
  • the antibody-drug conjugate is represented by the general formula (A):
  • D is a cytotoxic drug
  • L 1 is selected from -O-(CR a R b ) m -CR 5 R 6 -C(O)-, -O-CR 5 R 6 -(CR a R b ) m -, -O-CR 5 R 6 -, -NH-(CR a R b ) m -CR 5 R 6 -C(O)- or -S-(CR a R b ) m -CR 5 R 6 -C(O)-;
  • R a and R b are the same or different, and are each independently selected from a hydrogen atom, a deuterium atom, a halogen, an alkyl group, a halogenated alkyl group, a deuterated alkyl group, an alkoxy group, a hydroxyl group, an amino group, a cyano group, a nitro group, a hydroxyalkyl group Group, cycloalkyl or heterocyclic group;
  • R a and R b together with the carbon atom to which they are attached form a cycloalkyl group or a heterocyclic group;
  • R 5 is selected from halogen, haloalkyl, deuterated alkyl, cycloalkyl, cycloalkylalkyl, alkoxyalkyl, heterocyclyl, aryl or heteroaryl;
  • R 6 is selected from hydrogen atom, halogen, haloalkyl, deuterated alkyl, cycloalkyl, cycloalkylalkyl, alkoxyalkyl, heterocyclyl, aryl or heteroaryl;
  • R 5 and R 6 together with the carbon atom to which they are connected form a cycloalkyl group or a heterocyclic group;
  • R a and R 6 together with the carbon atom to which they are connected form a cycloalkyl group or a heterocyclic group;
  • n is selected from an integer from 0 to 4.
  • y is a number selected from 1 to 10, y is a decimal or an integer;
  • L 2 is the joint unit
  • Ab is an anti-TROP-2 antibody or an antigen-binding fragment thereof, which comprises an antibody light chain variable region and an antibody heavy chain variable region, and the antibody heavy chain variable region includes at least one HCDR selected from the following sequences : SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5; the antibody light chain variable region includes at least one LCDR selected from the following sequence: SEQ ID NO: 6, SEQ ID NO :7, SEQ ID NO: 8.
  • the antibody-drug conjugate or a pharmaceutically acceptable salt or solvent compound thereof according to the present invention wherein the antibody heavy chain variable region comprises:
  • the antibody-drug conjugate or a pharmaceutically acceptable salt or solvent compound thereof according to the present invention wherein the antibody light chain variable region comprises:
  • the antibody-drug conjugate or a pharmaceutically acceptable salt or solvent compound thereof according to the present invention wherein the antibody heavy chain variable region comprises:
  • the antibody light chain variable region comprises:
  • the anti-TROP-2 antibody or antigen-binding fragment thereof is selected from murine antibodies or Antigen-binding fragments, chimeric antibodies or antigen-binding fragments thereof, human antibodies or antigen-binding fragments thereof, humanized antibodies or antigen-binding fragments thereof.
  • the anti-TROP-2 antibody or antigen-binding fragment thereof further comprises human IgG1, The heavy chain constant region of IgG2, IgG3, or IgG4 or variants thereof.
  • the anti-TROP-2 antibody or antigen-binding fragment thereof further comprises a heavy chain constant region derived from human IgG1, IgG2 or IgG4 or a variant thereof.
  • the anti-TROP-2 antibody or antigen-binding fragment thereof further comprises a heavy chain constant region as shown in SEQ ID NO: 48 or SEQ ID NO: 49.
  • the anti-TROP-2 antibody or antigen-binding fragment thereof further comprises a human antibody ⁇ Chain, the light chain constant region of the lambda chain, or a variant thereof.
  • the anti-TROP-2 antibody or antigen-binding fragment thereof further comprises a light chain constant region derived from a human antibody kappa chain;
  • the anti-TROP-2 antibody or antigen-binding fragment thereof further comprises a light chain constant region as shown in SEQ ID NO:50.
  • the antibody-drug conjugate or a pharmaceutically acceptable salt or solvent compound thereof according to the present invention, and the anti-TROP-2 antibody or antigen-binding fragment thereof comprises one selected from the group consisting of The heavy chain variable region shown in the sequence, or a heavy chain variable region with at least 70%, 75%, 80%, 85%, 90%, 95% or 99% identity compared with the following sequence: SEQ ID NO :9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23 or SEQ ID NO: 25 .
  • the antibody-drug conjugate or a pharmaceutically acceptable salt or solvent compound thereof according to the present invention wherein the anti-TROP-2 antibody or antigen-binding fragment thereof is selected from the group consisting of The light chain variable region shown in the sequence, or a light chain variable region with at least 70%, 75%, 80%, 85%, 90%, 95% or 99% identity compared with the following sequence: SEQ ID NO :10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, or SEQ ID NO: 26 .
  • the anti-TROP-2 antibody or antigen-binding fragment thereof contains a sequence selected from the following The heavy chain shown, or a heavy chain with at least 80%, 85%, 90%, 95% or 99% identity compared with the following sequence: SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31. SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, or SEQ ID NO: 47.
  • the anti-TROP-2 antibody or antigen-binding fragment thereof contains a sequence selected from The light chain shown, or a light chain with at least 80%, 85%, 90%, 95% or 99% identity compared with the following sequence: SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42 or SEQ ID NO: 44.
  • the antibody-drug conjugate or a pharmaceutically acceptable salt or solvent compound thereof according to the present invention, wherein the anti-TROP-2 antibody or antigen-binding fragment thereof comprises:
  • the antibody-drug conjugate or a pharmaceutically acceptable salt or solvent compound thereof according to the present invention, wherein the anti-TROP-2 antibody comprises:
  • L 1 is represented by general formula (B):
  • M 1 is -CR 1 R 2 -;
  • R 1 and R 2 are the same or different, and R 1 and R 2 are each independently selected from hydrogen, alkyl, halogen, hydroxyl, or amino;
  • n is selected from an integer of 0-5, preferably 1, 2 or 3.
  • L 2 is represented by general formula (C):
  • M 2 is -CR 4 R 5 -;
  • R 3 is selected from hydrogen, halogen, hydroxyl, amino, alkyl, alkoxy and cycloalkyl:
  • R 4 and R 5 are the same or different, and are independently selected from hydrogen, alkyl, halogen, hydroxyl or amino;
  • n is selected from an integer of 0-5, preferably 1, 2 or 3.
  • the O end of the L 1 is connected to the joint unit L 2 .
  • the O end of the L 1 is connected to the S end of the joint unit L 2.
  • the antibody-drug conjugate or a pharmaceutically acceptable salt or solvent compound thereof according to the present invention, wherein:
  • L 1 is -O-(CR a R b ) m -CR 5 R 6 -C(O)-;
  • R a and R b are the same or different, and are each independently selected from a hydrogen atom, a deuterium atom, a halogen, or an alkyl group;
  • R 5 is haloalkyl or C 3-6 cycloalkyl
  • R 6 is selected from a hydrogen atom, a halogenated alkyl group or a C 3-6 cycloalkyl group;
  • R 5 and R 6 together with the carbon atom to which they are attached form a C 3-6 cycloalkyl group
  • n is selected from 0 or 1.
  • the L 1 is represented by the general formula (E):
  • R 5 is haloalkyl or cycloalkyl
  • R 6 is selected from hydrogen, haloalkyl or cycloalkyl
  • R 5 and R 6 together with the carbon atom to which they are attached form a cycloalkyl group
  • R 5 is selected from C 1-6 haloalkyl or C 3-6 cycloalkyl
  • R 6 is selected from hydrogen, C 1-6 haloalkyl or C 3-6 cycloalkyl,
  • R 5 and R 6 together with the carbon atom to which they are attached form a C 3-6 cycloalkyl group
  • n is selected from an integer from 0 to 4.
  • the L 2 is represented by the following general formula (D):
  • K 1 is s is selected from an integer from 2 to 8;
  • K 2 is selected from -NR 1 (CH 2 CH 2 O) p CH 2 CH 2 C(O)-, -NR 1 (CH 2 CH 2 O) p CH 2 C(O)-, -S(CH 2 ) p C(O)- or a single bond, p is selected from an integer from 1 to 20, preferably an integer from 1 to 6;
  • R 1 is selected from hydrogen, deuterium, hydroxyl, amino, alkyl, halogen, haloalkyl, deuterated alkyl and hydroxyalkyl;
  • K 3 is a tetrapeptide residue, preferably, the tetrapeptide residue is selected from two or more of phenylalanine, glycine, valine, lysine, citrulline, serine, glutamic acid, and Peptide residues formed by amino acids in aspartic acid; more preferably tetrapeptide residues of GGFG;
  • K 4 is -NR 2 (CR 3 R 4 )t-, R 2 , R 3 or R 4 are each independently hydrogen, deuterium, hydroxyl, amino, alkyl, halogen, haloalkyl, deuterated alkyl and hydroxyalkane Base, t is selected from 1 or 2.
  • the antibody-drug conjugate or a pharmaceutically acceptable salt or solvent compound thereof according to the present invention wherein the linker unit -L 2 -, its K 1 end is connected to Ab Connected, K 4 terminal is connected to L 1 .
  • the antibody-drug conjugate or a pharmaceutically acceptable salt or solvent compound thereof according to the present invention wherein the -L 2 -L 1 -has the following structure:
  • K 2 is a key
  • K 3 is the tetrapeptide residue of GGFG
  • R 5 is selected from haloalkyl or C 3-6 cycloalkyl
  • R 6 is selected from hydrogen, haloalkyl or C 3-6 cycloalkyl
  • R 5 and R 6 together with the carbon atom to which they are attached form a C 3-6 cycloalkyl group
  • R 2 , R 3 or R 4 are each independently selected from hydrogen or alkyl
  • s is selected from an integer from 2 to 8;
  • n is selected from an integer from 0 to 4.
  • the -L 2 -L 1 - is selected from the following structures:
  • the antibody-drug conjugate or a pharmaceutically acceptable salt or solvent compound thereof according to the present invention wherein the cytotoxic drug is selected from the group consisting of toxins, chemotherapeutics, antibiotics, radioactive Isotope and nucleolytic enzyme.
  • the cytotoxic drug is selected from tubulin inhibitors or DNA topoisomerase inhibitors that inhibit cell division; preferably camptothecin derivatives, DM1, DM3, DM4, SN- 38.
  • MMAF or MMAE more preferably exenotecan or exenotecan derivatives, SN-38, MMAE or MMAF.
  • the cytotoxic drug is selected from:
  • the cytotoxic drug is selected from exenotecan derivatives, preferably, the exenotecan derivative is compound 2-A:
  • the antibody-drug conjugate or a pharmaceutically acceptable salt or solvent compound thereof according to the present invention is a compound represented by general formula (I) or a pharmaceutically acceptable compound thereof Salt or solvent compound:
  • L 1 and L 2 are joint units
  • y is a number selected from 1-10, preferably a number from 2-8, more preferably a number from 2-4;
  • Ab is selected from the TROP-2 antibody or antigen-binding fragment thereof as described above.
  • L 2 is as defined in the foregoing; preferably, L 2 is as defined in the foregoing general formula (C).
  • L 1 is as defined in the foregoing; preferably, L2 is as defined in the foregoing general formula (B).
  • the antibody-drug conjugate or a pharmaceutically acceptable salt or solvent compound thereof according to the present invention is a compound represented by general formula (II) or a pharmaceutically acceptable compound thereof Salt or solvent compound:
  • L 1 and L 2 are joint units
  • y is a number selected from 1-10, preferably a number from 2-8, more preferably a number from 2-4;
  • Ab is selected from the aforementioned anti-TROP-2 antibody or antigen-binding fragment thereof;
  • L 2 is as defined in the foregoing; preferably, L 2 is as defined in the foregoing general formula (C).
  • the antibody-drug conjugate or a pharmaceutically acceptable salt or solvent compound thereof according to the present invention is a compound represented by general formula (III) or a pharmaceutically acceptable compound thereof Salt or solvent compound:
  • L 1 and L 2 are joint units
  • y is a number selected from 1-10, preferably a number selected from 2-8, more preferably a number selected from 4-8;
  • Ab is selected from the aforementioned TROP-2 antibody or antigen-binding fragment thereof.
  • the antibody-drug conjugate or a pharmaceutically acceptable salt or solvent compound thereof described in the general formula (III), and the -L 2 -L 1 - is selected from The following structure:
  • K 2 is the key
  • K 3 is the tetrapeptide residue of GGFG
  • R 5 is selected from haloalkyl or C 3-6 cycloalkyl
  • R 6 is selected from hydrogen, haloalkyl or C 3-6 cycloalkyl
  • R 5 and R 6 together with the carbon atom to which they are attached form a C 3-6 cycloalkyl group
  • R 2 , R 3 or R 4 are each independently hydrogen or alkyl
  • s is selected from an integer from 2 to 8;
  • n is selected from an integer from 0 to 4.
  • -L 2 -L 1 - is selected from the following structures:
  • the antibody-drug conjugate or a pharmaceutically acceptable salt or solvent compound thereof according to the present invention is an antibody-drug conjugate represented by general formula (IV) or Its pharmaceutically acceptable salt or solvate:
  • W is selected from a C 1-8 alkyl group, a C 1-8 alkyl-cycloalkyl group or a linear heteroalkyl group of 1 to 8 atoms, and the heteroalkyl group contains 1 to 3 selected from N, O or S
  • K 2 is selected from -NR 1 (CH 2 CH 2 O) p1 CH 2 CH 2 C(O)-, -NR 1 (CH 2 CH 2 O) p1 CH 2 C(O)-, -S(CH 2 ) p1 C(O) -or bond,
  • R 1 is selected from a hydrogen atom, an alkyl group, a haloalkyl group, a deuterated alkyl group and a hydroxyalkyl group, and p 1 is an integer from 1 to 20;
  • K 3 is selected from peptide residues consisting of 2 to 7 amino acids.
  • the amino acids can be substituted or unsubstituted.
  • the substituents can be substituted at any available point of attachment, and the substituents are one Or more are independently selected from halogen, hydroxy, cyano, amino, alkyl, chloroalkyl, deuterated alkyl, alkoxy and cycloalkyl;
  • R 2 is independently selected from a hydrogen atom, an alkyl group, a haloalkyl group, a deuterated alkyl group and a hydroxyalkyl group;
  • R 3 and R 4 are each independently selected from a hydrogen atom, a halogen, an alkyl group, a haloalkyl group, a deuterated alkyl group, and a hydroxyalkyl group;
  • R 5 is selected from halogen, haloalkyl, deuterated alkyl, cycloalkyl, heterocyclyl, aryl or heteroaryl;
  • R 6 is selected from hydrogen atom, halogen, haloalkyl, deuterated alkyl, cycloalkyl, heterocyclic, aryl or heteroaryl;
  • R 5 and R 6 together with the carbon atom to which they are attached form a cycloalkyl group or a heterocyclic group;
  • n is selected from an integer from 0 to 4.
  • y is a number selected from 1 to 10, y is a decimal or an integer;
  • Ab is selected from the anti-TROP-2 antibody or antigen-binding fragment thereof of the present invention.
  • the antibody-drug conjugate or a pharmaceutically acceptable salt or solvent compound thereof according to the present invention is an antibody-drug represented by general formula (IV-A) Conjugate or its pharmaceutically acceptable salt or solvent compound:
  • the antibody-drug conjugate or pharmaceutically acceptable salt or solvent compound thereof according to the present invention is selected from the following compounds:
  • Ab is selected from the anti-TROP-2 antibody or antigen-binding fragment thereof of the present invention; preferably, Ab is selected from HU1-HU10, HU6DL of the present invention.
  • the antibody-drug conjugate or a pharmaceutically acceptable salt or solvent compound thereof according to the present invention is selected from the following compounds:
  • y is selected from 2-10, preferably 4-8, more preferably 6-8, still more preferably 7-8, most preferably 8, y is a decimal or an integer.
  • the present invention provides a method for preparing a ligand-drug conjugate represented by general formula (IV) or a pharmaceutically acceptable salt or solvate thereof, which comprises the following steps:
  • Ab is selected from the aforementioned anti-TROP-2 antibody or antigen-binding fragment thereof;
  • W, K 2 , K 3 , R 2 to R 6 , m and y are as defined in the general formula (IV).
  • the general formula (F) is a compound represented by the general formula (F-1) or its tautomerism Forms, meso, racemates, enantiomers, diastereomers, or mixtures thereof, or pharmaceutically acceptable salts thereof:
  • K 2 , K 3 , R 2 to R 6 , s and m are as defined in the general formula (IV).
  • the compound represented by general formula (F) or general formula (F-1) according to the present invention is selected from:
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising the antibody-drug conjugate of the present invention or a pharmaceutically acceptable salt or solvent compound of the antibody-drug conjugate, and one or A variety of pharmaceutically acceptable excipients, diluents or carriers.
  • the present invention also provides the antibody-drug conjugate of the general formula (A) or the pharmaceutically acceptable salt or solvent compound of the antibody-drug conjugate or its pharmaceutical composition in preparation for Application in medicines for the treatment of diseases related to human TROP-2.
  • the disease related to human TROP-2 is an application in the preparation of a medicament for the treatment of cancers with high TROP-2 expression
  • the cancer is selected from the group consisting of triple-negative breast cancer, small Cell lung cancer, urothelial carcinoma, human brain astroblastoma, human pharynx cancer, adrenal tumors, AIDS-related cancers, alveolar soft tissue sarcoma, astrocytoma, bladder cancer, bone cancer, brain And spinal cord cancer, metastatic brain tumor, breast cancer, carotid body tumor, cervical cancer, chondrosarcoma, chordoma, renal chromophobe cell carcinoma, clear cell carcinoma, colon cancer, colorectal cancer, connective tissue proliferation small circle Cell tumor, ependymoma,meaning tumor, extraosseous mucinoid chondrosarcoma, bone fibrous hypoplasia, osteofibrous dysplasia, gallbladder or cholangiocarcinoma, gastric cancer,
  • the antibody-drug conjugate or its pharmaceutically acceptable salt or solvent compound of the present invention can specifically bind to the target antigen, has high endocytosis efficiency, and has a long in vivo half-life time, and it can significantly kill tumors while ensuring safety.
  • Figure 1 ELISA in vitro binding experiment of antibodies, showing the binding activity of 11 humanized anti-TROP-2 antibodies to human TROP-2 antigen.
  • antibody in the present invention refers to an immunoglobulin, which is a tetrapeptide chain structure composed of two identical heavy chains and two identical light chains connected by interchain disulfide bonds.
  • the amino acid composition and sequence of the constant region of the immunoglobulin heavy chain are different, so their antigenicity is also different.
  • immunoglobulins can be divided into five categories, or isotypes of immunoglobulins, namely IgM, IgD, IgG, IgA, and IgE.
  • the corresponding heavy chains are ⁇ chain, ⁇ chain, and ⁇ chain. , ⁇ chain and ⁇ chain.
  • IgG can be divided into IgG1, IgG2, IgG3, and IgG4.
  • the light chain is divided into a kappa chain or a lambda chain by the difference of the constant region.
  • Each of the five types of Ig can have a kappa chain or a lambda chain.
  • the antibody light chain variable region of the present invention may further include a light chain constant region, and the light chain constant region includes human or murine ⁇ , ⁇ chains or variants thereof.
  • the antibody heavy chain variable region of the present invention may further comprise a heavy chain constant region, and the heavy chain constant region comprises human or murine IgG1, IgG2, IgG3, IgG4 or its variants. body.
  • variable region The sequence of about 110 amino acids near the N-terminus of antibody heavy and light chains varies greatly and is the variable region (V region); the remaining amino acid sequences near the C-terminus are relatively stable and are the constant region (C region).
  • the variable region includes 3 hypervariable regions (HVR) and 4 framework regions (FR) with relatively conserved sequences. Three hypervariable regions determine the specificity of the antibody, also known as complementarity determining regions (CDR).
  • CDR complementarity determining regions
  • Each light chain variable region (VL) and heavy chain variable region (VH) is composed of 3 CDR regions and 4 FR regions.
  • the sequence from the amino terminus to the carboxy terminus is FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • the 3 CDR regions of the light chain refer to LCDR1, LCDR2, and LCDR3; the 3 CDR regions of the heavy chain refer to HCDR1, HCDR2, and HCDR3.
  • the number and position of the CDR amino acid residues of the VL and VH regions of the antibody or antigen-binding fragment of the present invention comply with the known Kabat numbering rules and Kabat or ABM definition rules (http://bioinf.org.uk/abs /).
  • APC antigen presenting cell
  • DC dendritic cells
  • PBMC topical blood mononuclear cells
  • monocytes B lymphoblasts
  • monocyte-derived dendritic cells monocyte-derived dendritic cells
  • antigen presentation refers to the process by which APC captures antigens and enables them to be recognized by T cells, for example as a component of MHC-I/MHC-II conjugates.
  • TROP-2 includes any variant or isoform of TROP-2 that is naturally expressed by the cell.
  • the antibodies of the present invention can cross-react with TROP-2 derived from non-human species.
  • the antibody may also be human TROP-2 specific, and may not show cross-reactivity with other species.
  • TROP-2 or any variants or isoforms thereof can be isolated from cells or tissues that naturally express them, or can be produced by recombinant techniques using techniques commonly used in the art and those described herein.
  • the anti-TROP-2 antibody targets human TROP-2 with a normal glycosylation pattern.
  • recombinant human antibody includes human antibodies prepared, expressed, created or isolated by recombinant methods, and the techniques and methods involved are well known in the art, such as:
  • Antibodies isolated from host cells transformed to express antibodies such as transfectomas;
  • Antibodies prepared, expressed, created or isolated by methods such as splicing human immunoglobulin gene sequences to other DNA sequences.
  • Such recombinant human antibodies contain variable and constant regions, which utilize specific human germline immunoglobulin sequences encoded by germline genes, but also include subsequent rearrangements and mutations such as those that occur during antibody maturation.
  • murine antibody in the present invention refers to a monoclonal antibody to human TROP-2 prepared according to the knowledge and skills in the art. During preparation, the test subject is injected with TROP-2 antigen, and then hybridomas expressing antibodies with the desired sequence or functional properties are isolated.
  • the murine TROP-2 antibody or antigen-binding fragment thereof may further comprise the light chain constant region of murine kappa, lambda chains or variants thereof, or further comprise murine IgG1 , IgG2, IgG3 or IgG4 or variants of the heavy chain constant region.
  • human antibody includes antibodies having variable and constant regions of human germline immunoglobulin sequences.
  • the human antibodies of the present invention may include amino acid residues that are not encoded by human germline immunoglobulin sequences (such as mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo).
  • the term “human antibody” does not include antibodies in which CDR sequences derived from the germline of another mammalian species (such as a mouse) have been grafted onto human framework sequences (ie, "humanized antibodies”) .
  • humanized antibody also known as CDR-grafted antibody (CDR-grafted antibody)
  • CDR-grafted antibody refers to an antibody produced by grafting mouse CDR sequences into a human antibody variable region framework.
  • Humanized antibodies can overcome the shortcomings of strong immune responses induced by chimeric antibodies that carry a large amount of mouse protein components.
  • the variable region of the human antibody can be subjected to minimal reverse mutation to maintain activity.
  • chimeric antibody refers to an antibody formed by fusing the variable region of a murine antibody with the constant region of a human antibody, which can reduce the immune response induced by the murine antibody.
  • To establish a chimeric antibody it is necessary to select a hybridoma that secretes a murine-specific monoclonal antibody, and then clone the variable region gene from the mouse hybridoma cell, and then clone the constant region gene of the human antibody as needed, and change the mouse variable region gene.
  • the region gene and the human constant region gene are connected to form a chimeric gene and then inserted into a human vector, and finally the chimeric antibody molecule is expressed in a eukaryotic industrial system or a prokaryotic industrial system.
  • the constant region of a human antibody can be selected from the heavy chain constant region of human IgG1, IgG2, IgG3 or IgG4 or variants thereof, preferably comprising human IgG1, IgG2 or IgG4 heavy chain constant region, or using amino acid mutations to enhance ADCC (antibody -dependent cell-mediated cytotoxicity, antibody-dependent cell-mediated cytotoxicity) toxic IgG1 heavy chain constant region.
  • ADCC antibody -dependent cell-mediated cytotoxicity, antibody-dependent cell-mediated cytotoxicity
  • antigen-binding fragment refers to antigen-binding fragments and antibody analogs of antibodies, which usually include at least part of the antigen-binding region or variable region (for example, one or more CDRs) of a parental antibody.
  • Antibody fragments retain at least some of the binding specificity of the parent antibody. Generally, when the activity is expressed on a mole basis, the antibody fragment retains at least 10% of the parental binding activity. Preferably, the antibody fragment retains at least 20%, 50%, 70%, 80%, 90%, 95% or 100% or more of the binding affinity of the parent antibody to the target.
  • antigen-binding fragments include, but are not limited to: Fab, Fab', F(ab')2, Fv fragments, linear antibodies, single-chain antibodies, nanobodies, domain antibodies, and multispecific antibodies.
  • Engineered antibody variants are reviewed in Holliger and Hudson, 2005, Nat. Biotechnol. 23:1126-1136.
  • the "Fab fragment” consists of the CH1 and variable regions of one light chain and one heavy chain.
  • the heavy chain of the Fab molecule cannot form a disulfide bond with another heavy chain molecule.
  • the "Fc" region contains two heavy chain fragments containing the CH2 and CH3 domains of the antibody.
  • the two heavy chain fragments are held together by two or more disulfide bonds and through the hydrophobic interaction of the CH3 domain.
  • the "Fab' fragment” contains a light chain and a portion of a heavy chain that contains the VH domain, the CH1 domain, and the region between the CH1 and CH2 domains, so that it can be between the two heavy chains of the two Fab' fragments The formation of interchain disulfide bonds to form F(ab')2 molecules.
  • the "F(ab')2 fragment” contains two light chains and two heavy chains containing a portion of the constant region between the CH1 and CH2 domains, thereby forming an interchain disulfide bond between the two heavy chains. Therefore, the F(ab')2 fragment is composed of two Fab' fragments held together by the disulfide bond between the two heavy chains.
  • the "Fv region” contains variable regions from both the heavy and light chains, but lacks the constant region.
  • multispecific antibody is used in its broadest sense to encompass antibodies with polyepitope specificity.
  • These multispecific antibodies include, but are not limited to: antibodies comprising a heavy chain variable region VH and a light chain variable region VL, wherein the VH-VL unit has polyepitope specificity; having two or more VL and VH regions Antibodies, each VH-VL unit binds to a different target or a different epitope of the same target; an antibody with two or more single variable regions, each single variable region with a different target Or binding to different epitopes of the same target; full-length antibodies, antibody fragments, diabodies, bispecific diabodies and triabodies, antibody fragments that have been covalently or non-covalently linked together Wait.
  • single-chain antibody is a single-chain recombinant protein formed by connecting the heavy chain variable region VH and the light chain variable region VL of an antibody through a connecting peptide. It is the smallest antibody fragment with a complete antigen-binding site.
  • domain antibody fragment is an immunoglobulin fragment with immunological functions that only contains a heavy chain variable region or a light chain variable region chain.
  • two or more VH regions are covalently linked to a peptide linker to form a bivalent domain antibody fragment.
  • the two VH regions of the bivalent domain antibody fragment can target the same or different antigens.
  • binding to TROP-2 refers to the ability to interact with human TROP-2.
  • antigen-binding site refers to a three-dimensional site recognized by the antibody or antigen-binding fragment of the present invention.
  • epitope refers to a site on an antigen that specifically binds to an immunoglobulin or antibody.
  • Epitopes can be formed by adjacent amino acids or non-adjacent amino acids that are juxtaposed by tertiary folding of the protein. Epitopes formed by adjacent amino acids are usually maintained after exposure to a denaturing solvent, while epitopes formed by tertiary folding are usually lost after treatment with the denaturing solvent.
  • Epitopes usually include at least 3-15 amino acids in a unique spatial conformation. Methods to determine what epitope is bound by a given antibody are well known in the art, including immunoblotting and immunoprecipitation detection analysis. Methods for determining the spatial conformation of an epitope include the techniques in the art and the techniques described herein, such as X-ray crystal analysis and two-dimensional nuclear magnetic resonance.
  • specific binding and “selective binding” as used in the present invention refer to the binding of an antibody to an epitope on a predetermined antigen.
  • the antibody dissociates at an equilibrium of about less than 10 -7 M or even smaller when measured by surface plasmon resonance (SPR) technology in the instrument.
  • SPR surface plasmon resonance
  • K D The constant binds to a predetermined antigen, and its binding affinity to the predetermined antigen is at least twice its binding affinity to non-specific antigens (such as BSA, etc.) other than the predetermined antigen or closely related antigens.
  • antibody that recognizes an antigen can be used interchangeably with the term “antibody that specifically binds” herein.
  • cross-reactivity refers to the ability of the antibodies of the present invention to bind to TROP-2 from different species.
  • the antibody of the present invention that binds to human TROP-2 can also bind to TROP-2 of another species.
  • Cross-reactivity is measured by detecting specific reactivity with purified antigens in binding assays such as SPR and ELISA, or by binding or functional interaction with cells that physiologically express TROP-2. Methods of determining cross-reactivity include standard binding assays as described herein, such as surface plasmon resonance (SPR) analysis, or flow cytometry.
  • SPR surface plasmon resonance
  • Inhibition or “blocking” are used interchangeably and encompass both partial and complete inhibition/blocking. Inhibition/blocking of the ligand preferably reduces or alters the normal level or type of activity that occurs when ligand binding occurs without inhibition or blocking. Inhibition and blocking are also intended to include any measurable decrease in ligand binding affinity when contacted with an anti-TROP-2 antibody compared to a ligand not contacted with an anti-TROP-2 antibody.
  • inhibition of growth is intended to include any measurable decrease in cell growth.
  • inducing an immune response and “enhancing an immune response” are used interchangeably and refer to the stimulation (ie, passive or adaptive) of the immune response to a specific antigen.
  • induction for inducing CDC or ADCC refers to stimulating a specific direct cell killing mechanism.
  • ADCC namely antibody-dependent cell-mediated cytotoxicity, antibody-dependent cell-mediated cytotoxicity
  • Fc receptors are directly killed by recognizing the Fc segment of antibodies and are coated with antibodies.
  • the target cell The ADCC effect function of the antibody can be enhanced or reduced by modifying the Fc segment of IgG.
  • the modification refers to mutations in the constant region of the heavy chain of the antibody.
  • mice can be immunized with human TROP-2 or fragments thereof, and the obtained antibodies can be renatured, purified, and amino acid sequencing can be performed by conventional methods.
  • Antigen-binding fragments can also be prepared by conventional methods.
  • the antibodies or antigen-binding fragments of the invention are genetically engineered to add one or more human FR regions to the non-human CDR regions.
  • the human FR germline sequence can be obtained from the website http://imgt.cines.fr of ImmunoGeneTics (IMGT), or from the Journal of Immunoglobulin, 2001ISBN012441351.
  • the engineered antibody or antigen-binding fragment of the present invention can be prepared and purified by conventional methods.
  • the cDNA sequence of the corresponding antibody can be cloned and recombined into a GS expression vector.
  • the recombinant immunoglobulin expression vector can be stably transfected into CHO cells.
  • mammalian expression systems can lead to glycosylation of antibodies, especially at the highly conserved N-terminus of the FC region.
  • Stable clones are obtained by expressing antibodies that specifically bind to human antigens. Positive clones are expanded in the serum-free medium of the bioreactor to produce antibodies.
  • the antibody-secreted culture medium can be purified and collected by conventional techniques.
  • the antibody can be filtered and concentrated by conventional methods. Soluble mixtures and polymers can also be removed by conventional methods, such as molecular sieves and ion exchange.
  • the resulting product needs to be frozen immediately, such as -70°C, or lyophilized.
  • the antibody of the present invention refers to a monoclonal antibody.
  • the monoclonal antibody (mAb) of the present invention refers to an antibody obtained from a single cloned cell line, and the cell line is not limited to a eukaryotic, prokaryotic or phage cloned cell line.
  • Monoclonal antibodies or antigen-binding fragments can be obtained by recombination using, for example, hybridoma technology, recombination technology, phage display technology, synthesis technology (such as CDR-grafting), or other existing technologies.
  • administering when applied to animals, humans, experimental subjects, cells, tissues, organs or biological fluids refer to exogenous drugs, therapeutic agents, diagnostic agents or compositions and animals , Human, subject, cell, tissue, organ or biological fluid contact.
  • administering can refer to, for example, treatment, pharmacokinetics, diagnosis, research, and experimental methods.
  • the treatment of cells includes contact of reagents with cells, and contact of reagents with fluids, where the fluids are in contact with cells.
  • administering “administration” and “treatment” also mean the treatment of, for example, cells by reagents, diagnostics, binding compositions, or by another cell in vitro and ex vivo.
  • Treatment when applied to human, veterinary or research subjects, refers to therapeutic treatment, preventive or preventive measures, research and diagnostic applications.
  • Treatment means administering an internal or external therapeutic agent, such as containing any one of the antibodies of the present invention, to a patient who has one or more disease symptoms, and the therapeutic agent is known to have a therapeutic effect on these symptoms.
  • the therapeutic agent is administered in the treated patient or population in an amount effective to alleviate one or more symptoms of the disease, whether by inducing the regression of such symptoms or inhibiting the development of such symptoms to any clinically measured extent.
  • the amount of the therapeutic agent effective to alleviate the symptoms of any particular disease can vary depending on various factors, such as the patient's disease state, age, and weight, and the ability of the drug to produce the desired therapeutic effect in the patient.
  • any clinical testing methods commonly used by doctors or other professional health care professionals to evaluate the severity or progression of the symptoms it can be evaluated whether the symptoms of the disease have been alleviated.
  • the embodiments of the present invention may be ineffective in alleviating the symptoms of the target disease that each patient has, according to any statistical test methods known in the art such as Student's t test, chi-square test, and basis Mann and Whitney's U test, Kruskal-Wallis test (H test), Jonckheere-Terpstra test, and Wilcoxon test determined that it should reduce the symptoms of the target disease in a statistically significant number of patients.
  • naturally occurring refers to the fact that the object can be found in nature.
  • polypeptide sequences or polynucleotide sequences that exist in organisms (including viruses) that can be isolated from natural sources and have not been intentionally modified artificially in the laboratory are naturally occurring.
  • Effective amount includes an amount sufficient to improve or prevent the symptoms or conditions of medical conditions.
  • An effective amount also means an amount sufficient to allow or facilitate diagnosis.
  • the effective amount for a particular patient or veterinary subject can vary depending on factors such as the condition to be treated, the patient's general health, the method of administration and dosage, and the severity of side effects.
  • the effective amount can be the maximum dose or dosing schedule that avoids significant side effects or toxic effects.
  • Exogenous refers to substances that are produced outside organisms, cells, or humans according to the background.
  • Endogenous refers to a substance produced in a cell, organism, or human body according to the background.
  • “Homology” refers to the sequence similarity between two polynucleotide sequences or between two polypeptides.
  • the positions in the two comparison sequences are occupied by the same base or amino acid monomer subunit, for example, if each position of the two DNA molecules is occupied by adenine, then the molecules are homologous at that position .
  • the percent homology between two sequences is a function of the number of matching or homologous positions shared by the two sequences divided by the number of positions compared ⁇ 100%. For example, in the optimal sequence alignment, if there are 6 matches or homology in 10 positions in the two sequences, then the two sequences are 60% homologous. Generally speaking, the comparison is made when two sequences are aligned to obtain the greatest percentage of homology.
  • the expressions "cell”, “cell line” and “cell culture” are used interchangeably, and all such names include their progeny. Therefore, the words “transformant” and “transformed cell” include primary test cells and cultures derived therefrom, regardless of the number of transfers. It should also be understood that due to deliberate or unintentional mutations, all offspring cannot be exactly the same in terms of DNA content. Including mutant progeny with the same function or biological activity as screened in the original transformed cell. Where a different name is meant, it is clearly visible from the context.
  • “Pharmaceutical composition” means containing one or more antibodies or antigen-binding fragments thereof described herein, and other components such as physiological/pharmaceutically acceptable carriers and excipients.
  • the purpose of the pharmaceutical composition is to promote the administration to the organism, which is beneficial to the absorption of the active ingredient and thus the biological activity.
  • the following examples are used to further describe the present invention, but these examples do not limit the scope of the present invention.
  • the experimental methods that do not specify specific conditions in the examples of the present invention usually follow conventional conditions, such as Cold Spring Harbor's antibody technology experimental manual, molecular cloning manual; or according to the conditions recommended by the raw material or commodity manufacturer.
  • the reagents without specific sources are the conventional reagents purchased on the market.
  • TROP-2 (TROP-2-His) protein encoding His tag was synthesized by SinoBiologics (10428-H08H).
  • the indirect ELISA method as described in Example 3 was used for the immunized mouse serum to evaluate the serum titer and the ability to bind to cell surface antigens.
  • the detection of the titer (larger than 100,000 times dilution) determines the start of cell fusion.
  • the immunized mice with strong serum titer, affinity and FACS binding were selected for one final immunization and then sacrificed.
  • the spleen cells and SP2/0 myeloma cells were fused and plated to obtain hybridomas.
  • the target hybridomas were screened by indirect ELISA, and The strain was established as a monoclonal cell strain by the limiting dilution method.
  • the obtained positive antibody strains are further screened using indirect ELISA to select hybridomas that bind to the recombinant protein.
  • the logarithmic growth phase hybridoma cells were collected, and RNA was extracted with Trizol (Invitrogen, 15596-018) and reverse transcribed (PrimeScript TM Reverse Transcriptase, Takara #2680A).
  • the cDNA obtained by reverse transcription was amplified by PCR using a mouse Ig primer set (Novagen, TB326 Rev. B 0503) and then sequenced to finally obtain the sequence of the mouse antibody.
  • the heavy chain and light chain variable region sequences of murine monoclonal antibody M1 are as follows:
  • TROP-2His protein (Sino Biological Inc., cat#10428-H08H) with pH7.4 PBS to a concentration of 1 ⁇ g/ml, add 100 ⁇ l/well to a 96-well high-affinity ELISA plate, and refrigerate at 4°C Incubate overnight (16-20 hours). After washing the plate 4 times with PBST (pH 7.4 PBS containing 0.05% Tween-20), add 150 ⁇ l/well of 3% bovine serum albumin (BSA) blocking solution diluted with PBST, and incubate for 1 hour at room temperature for blocking. After the blocking, the blocking solution was discarded, and the plate was washed 4 times with PBST buffer.
  • BSA bovine serum albumin
  • Collect the cultured TROP-2 high-expressing cells (CHO or 293 cells overexpressing TROP-2 and tumor cells expressing TROP-2, such as HCC-827, MDA-MB-468, etc.), adjust the cell density and spread them 96-well U bottom plate, 1 ⁇ 10 5 to 2 ⁇ 10 5 cells per well. Centrifuge at 1200g for 5min, remove the supernatant, add 100ul of serially diluted antibody solution or mouse immune serum, incubate at 4°C for 60min; centrifuge at 1200g for 5min, remove the supernatant, and wash the cells twice with PBS, add a fluorescently labeled secondary antibody (PE-GAM or PE-GAH) 100ul per well, incubate at 4°C for 60min. Centrifuge at 1200g for 5min to remove the supernatant. After washing the cells twice with PBS, resuspend them in PBS, use a flow cytometer to detect the signal, and make a concentration curve analysis result.
  • the humanization of the murine anti-human TROP-2 monoclonal antibody is carried out according to the methods published in many documents in the field.
  • a human constant domain is used to replace the parental (murine antibody) constant domain, and the human antibody sequence is selected based on the homology of the murine antibody and the human antibody.
  • the present invention humanizes the murine antibody M1.
  • the heavy and light chain variable region sequences are compared with the human antibody germline database to obtain a human germline template with high homology.
  • the CDR region of the murine antibody M1 was transplanted to the selected corresponding humanized template. Then, based on the three-dimensional structure of the murine antibody, the embedded residues, the residues that directly interact with the CDR region, and the residues that have an important impact on the conformation of VL and VH were back-mutated. After expression testing and After comparing the number of back mutations, an antibody designed with a combination of humanized heavy chain variable region HCVR and light chain variable region LCVR sequences was selected. The sequence is as follows:
  • the designed heavy chain and light chain variable region sequences are respectively connected with the IgG heavy chain constant region and human antibody light chain constant region sequences.
  • Exemplary heavy chain constant region and light chain constant region sequences are as follows:
  • the heavy chain and light chain sequences are as follows (where the HU1-HU9 heavy chain is derived from the sequence SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, and SEQ ID NO: 25 are respectively linked to the sequence SEQ ID NO: 49; the HU6DL and HU10 heavy chains are derived from the sequence SEQ ID NO: 19, SEQ ID NO:9 are respectively connected to the sequence SEQ ID NO:48):
  • CDNA fragments were synthesized according to the amino acid sequences of the light and heavy chains of the above humanized antibodies, and inserted into the pcDNA3.1 expression vector (Life Technologies Cat. No. V790-20).
  • the expression vector and the transfection reagent PEI (Polysciences, Inc. Cat. No. 23966) were transfected into HEK293 cells (Life Technologies Cat. No. 11625019) at a ratio of 1:2, and incubated in a CO 2 incubator 4- 5 days.
  • each humanized antibody tested in Example 3 (2) in vitro cell binding experiment was used to test HCC827 tumor cells (non-small cell lung cancer),
  • the affinity (EC 50 ) of MAB-MB-468 tumor cells is shown in the following table:
  • Example 6 Tumor cell killing effect mediated by humanized antibody
  • Humanized antibodies can kill tumor cells from many aspects, one of which is to mediate the killing effect of immune cells on tumor cells.
  • PBMC peripheral blood mononuclear cells
  • HCC827 tumor cells non-small cell lung cancer
  • Collect commercialized human PBMC cells after centrifugal counting, adjust the cell density to 2.2 ⁇ 10 6 cells/mL with complete medium, and spread them in the middle 60 wells of a white 96-well plate with HCC827 cells, 90 ⁇ L per well, the number of cells is 20000. Add 200 ⁇ L of PBS to the remaining side holes, and place the cell plate in a 37°C, 5% CO2 incubator overnight. On the second day of the experiment, the humanized antibody solution was prepared in a 96-well V-bottom plate with PBS, starting at a concentration of 1000 nM, diluted 3 times, and 9 concentrations.
  • SW780 cells were trypsinized, the cells were collected and resuspended in pre-cooled PBS, and the cell concentration was adjusted to 1 ⁇ 10 6 cells/mL .
  • the cell concentration was adjusted to 1 ⁇ 10 6 cells/mL .
  • Take the EP tube add 1mL of cell suspension, centrifuge at 1500rpm for 5 minutes and remove the supernatant, add 1mL of the prepared antibody to be tested to resuspend the cells, the final concentration of the antibody is 20 ⁇ g/ml, and incubate for 1h on a shaker at 4 degrees.
  • All treatment groups were added with 100 ⁇ L of immunostaining fixative, placed at 4°C for more than 30 minutes, and tested with flow cytometer DxFlex on the machine. Take 200 ⁇ l from the tube of the 0min group and add the immunostaining fixative directly. Take 200 ⁇ l from the blank group and add strip buffer and immunostaining fixative directly. On the machine, use the flow cytometer DxFlex for detection.
  • the humanized antibody of the present invention has a very low inhibition rate on the binding of hRS7 antibody and TROP2 protein, suggesting that the humanized antibody of the present invention and hRS7 antibody do not compete for binding to the same epitope.
  • 2a (2g, 17.2mmol) was dissolved in 75mL of acetonitrile, and potassium carbonate (9.27g, 67.2mmol), benzyl bromide (20mL, 167.2mmol) and tetrabutylammonium iodide (620mg, 1.68mmol) were added in sequence.
  • the reaction solution was stirred at room temperature for 48 hours, filtered through celite, the filter cake was rinsed with ethyl acetate (20ml), the combined filtrate was concentrated under reduced pressure, and the resulting residue was purified by silica gel column chromatography with a developing solvent system C to obtain Product 5a (3.2g, yield: 90.1%).
  • reaction solution was filtered with diatomaceous earth, the filter cake was rinsed with ethyl acetate, and the filtrate was concentrated to obtain the crude product 5c 10-cyclopropyl-1-(9H-fluoren-9-yl)-3,6-dioxo- 2,9-dioxa-4,7-diazaundec-11-acid (20mg), the product was directly subjected to the next reaction without purification.
  • 5d (30 mg, 35.7 ⁇ mol) was dissolved in 3 mL of dichloromethane, 1.5 mL of diethylamine was added, and the mixture was stirred at room temperature for 2 hours.
  • the reaction solution was concentrated under reduced pressure, 1.5 mL of toluene was added and concentrated under reduced pressure, repeated twice.
  • the crude product 5e (20mg, 32.3 ⁇ mol) was dissolved in 1mL N,N-dimethylformamide, replaced with argon three times, cooled to 0-5°C in an ice water bath, and 4g (31.8mg, 67.3 ⁇ mol) was added.
  • 4g 31.8mg, 67.3 ⁇ mol
  • 0.5mL N,N-dimethylformamide solution add 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylchloromorpholine salt ( 27.8 mg, 94.3 ⁇ mol)
  • the reaction was stirred in an ice bath for 10 minutes, the ice bath was removed, and the mixture was heated to room temperature and stirred for 1 hour to produce compound 5.
  • reaction solution was purified by high performance liquid chromatography (separation conditions: column: XBridge Prep C18OBD5um 19*250mm; mobile phase: A-water (10mmol NH 4 OAc): B-acetonitrile, gradient elution, flow rate: 18mL/min)
  • the corresponding components were collected and concentrated under reduced pressure to obtain products 5-A and 5-B (3.6 mg, 2.6 mg).
  • the average value y was determined by the ultraviolet method. After placing the cuvette containing sodium succinate buffer in the reference absorption cell and the sample determination absorption cell, after deducting the solvent blank, place the cuvette containing the test solution in the sample determination absorption cell Measure the absorbance at 280nm and 370nm.
  • the average drug load y CDrug/Cmab.
  • the compound MC-MMAF (1.1 mg, 1.2 mol, prepared by the method disclosed in PCT patent WO2005081711) was dissolved in 0.3 mL of acetonitrile, and 2a solution (concentration 6.17 mg/mL, 3.0 mL) was added to 25 After shaking and reacting at °C for 4 hours, the reaction solution was desalted and purified by Sephadex G25 gel column (elution phase: 0.05M PBS solution with pH 6.5), and filtered under sterile conditions with a filter to obtain the product Ab-MC -MMAF antibody-drug conjugate in PBS buffer (3.7 mg/mL, 4.7 mL), refrigerated at 4°C.
  • S-(3-aldehyde propyl) thioacetate (0.7 mg, 5.3 mol) was dissolved in 0.9 mL of acetonitrile solution for later use.
  • Add the pre-prepared acetonitrile solution of S-(3-hydroxypropyl) thioacetate to the acetic acid/sodium acetate buffer (10.35mg/mL, 9.0mL, 0.97mol) of antibody pH 4.3, and then add 1.0mL dropwise
  • An aqueous solution of sodium cyanoborohydride (14.1 mg, 224 mol) was shaken and reacted at 25° C. for 2 hours.
  • the compound MC-VC-PAB-SN-38 (1.3mg, 1.2mol) was dissolved in 0.3ml of acetonitrile, added to the 2h solution (concentration 6.2mg/mL, 3.0mL), and shaken at 25°C After 4 hours of reaction, the reaction solution was desalted and purified with Sephadex G25 gel column (elution phase: 0.05M PBS solution with pH 6.5), and filtered under sterile conditions with a filter to obtain the product Ab-SN-38 antibody -PBS buffer (3.7 mg/mL, 4.7 mL) of the drug conjugate, refrigerated at 4°C.
  • Example 13 The killing effect of antibody-conjugated drugs on tumor cells
  • bladder cancer cell SW780 was used for evaluation. Collect SW780 cells, after centrifugal counting, adjust the cell density to 0.44 ⁇ 10 6 cells/mL with complete medium, and spread them in the middle 60 wells of a white 96-well plate, each with 90 ⁇ L, and the number of cells is 4000. Add 100 ⁇ L PBS to the remaining side holes. The cell plate was placed in a 37°C, 5% CO2 incubator and cultured overnight. On the second day of the experiment, prepare the antibody-drug conjugate solution in a 96-well V-bottom plate with PBS, starting with a concentration of 1000 nM, 3 times dilution, and 9 concentrations.
  • the antibody-drug conjugate was injected intravenously into C57BL/6 mice (dose 10mg/kg). At 1h, 2h, 4h, 8h, 24h, 48h, 96h, 144h and 240h, 20 microliters of blood were drawn, and the concentration of the antibody-drug conjugate in the blood was determined by the ELISA method of Example 3(1) After that, WinNonlin software was used to analyze the pharmacokinetic data to obtain the pharmacokinetic parameters, as shown in Table 12 below.
  • Tumor inhibition rate 100%-(Tumor volume in the administration group on day 28-tumor volume in the administration group on day 0)/(Tumor volume in the control group on day 28-tumor volume in the control group on day 0).
  • the experimental results are shown in Table 13 Shown:
  • the reaction solution was concentrated under reduced pressure, and the obtained crude compound 2-C was purified by high performance liquid chromatography (Separation conditions: Column: XBridge Prep C18 OBD 5um 19*250mm; Mobile phase: A-water (10mmol NH4OAc), B- Acetonitrile, gradient elution, flow rate: 18 mL/min), collect the corresponding components, and concentrate under reduced pressure to obtain the title product (2-A: 1.5 mg, 2-B: 1.5 mg).
  • Example 17 Inhibition test of exenotecan derivatives on tumor cell proliferation in vitro
  • the compounds 2-A and 2-B were tested for their inhibitory activity on the in vitro proliferation of U87MG cells (Cell Bank of Chinese Academy of Sciences, Catalog#TCHu138) and SK-BR-3 tumor cells (human breast cancer cells, ATCC, catalog number HTB-30).
  • the cells were treated in vitro with different concentrations of the compound, and after 6 days of culture, the proliferation of the cells was detected with CTG (Luminescent Cell Viability Assay, Promega, catalog number: G7573) reagent, and the in vitro activity of the compound was evaluated according to the IC50 value.
  • U87MG and SK-BR-3 cells were cultured with 10% FBS in EMEM medium (GE, article number SH30024.01) and McCoy's 5A medium (Gibco, article number 16600-108) containing 10% FBS, respectively.
  • the compound was dissolved in DMSO (dimethyl sulfoxide, Shanghai Titan Technology Co., Ltd.) to prepare a storage solution with an initial concentration of 10 mM.
  • DMSO dimethyl sulfoxide, Shanghai Titan Technology Co., Ltd.
  • the initial concentration of the small molecule compound is 500nM, and the dispensing method is as follows:
  • Adding samples Add 20 ⁇ l of the tested samples of different concentrations to the culture plate, and each sample has two duplicate wells.
  • the culture plate was incubated in an incubator for 6 days (37°C, 5% CO 2 ).
  • Color development Take out the 96-well cell culture plate, add 90 ⁇ l CTG solution to each well, and incubate at room temperature for 10 minutes.
  • Plate reading Take out the 96-well cell culture plate, place it in a microplate reader (BMG labtech, PHERAstar FS), and measure chemiluminescence with the microplate reader.

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Abstract

La présente invention concerne un conjugué anticorps-médicament et son utilisation médicale, en particulier un conjugué anticorps anti-TROP-2-médicament et son utilisation médicale, et en outre, un conjugué anticorps-médicament contenant un anticorps anti-TROP-2 ou un fragment de liaison à l'antigène de celui-ci, ou un sel pharmaceutiquement acceptable ou un composé solvant de celui-ci, et leur utilisation dans la préparation de médicaments pour le traitement de maladies ou de troubles médiés par TROP-2 et leur utilisation dans la détection et le diagnostic de tumeurs.
PCT/CN2021/082294 2020-03-24 2021-03-23 Conjugué anticorps-médicament et son utilisation médicale WO2021190480A1 (fr)

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WO2023241621A1 (fr) * 2022-06-16 2023-12-21 山东博安生物技术股份有限公司 Anticorps anti-liv-1 et conjugué anticorps-médicament
WO2024061173A1 (fr) * 2022-09-19 2024-03-28 上海美雅珂生物技术有限责任公司 Traitement du carcinome nasopharyngé à l'aide d'un conjugué d'anticorps ciblant egfr

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CN105849126A (zh) * 2013-12-25 2016-08-10 第三共株式会社 抗trop2抗体－药物偶联物
WO2016172427A1 (fr) * 2015-04-22 2016-10-27 Immunomedics, Inc. Isolement, détection, diagnostic et/ou caractérisation de cellules cancéreuses trop-2 positives
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WO2023098889A1 (fr) * 2021-12-03 2023-06-08 成都百利多特生物药业有限责任公司 Conjugué anticorps anti-trop2 humain-médicament de camptothécine et son utilisation médicale
WO2023241621A1 (fr) * 2022-06-16 2023-12-21 山东博安生物技术股份有限公司 Anticorps anti-liv-1 et conjugué anticorps-médicament
WO2024061173A1 (fr) * 2022-09-19 2024-03-28 上海美雅珂生物技术有限责任公司 Traitement du carcinome nasopharyngé à l'aide d'un conjugué d'anticorps ciblant egfr

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