WO2016165580A1 - 抗c-Met抗体和抗c-Met抗体-细胞毒性药物偶联物及其医药用途 - Google Patents

抗c-Met抗体和抗c-Met抗体-细胞毒性药物偶联物及其医药用途 Download PDF

Info

Publication number
WO2016165580A1
WO2016165580A1 PCT/CN2016/078699 CN2016078699W WO2016165580A1 WO 2016165580 A1 WO2016165580 A1 WO 2016165580A1 CN 2016078699 W CN2016078699 W CN 2016078699W WO 2016165580 A1 WO2016165580 A1 WO 2016165580A1
Authority
WO
WIPO (PCT)
Prior art keywords
antibody
group
seq
met
antigen
Prior art date
Application number
PCT/CN2016/078699
Other languages
English (en)
French (fr)
Inventor
刘佳建
张连山
陶维康
付雅媛
张玲
马动
崔东冰
王亚里
许建烟
梁金栋
章瑛
蒋贵阳
邱均专
孙自勇
查济平
魏京平
Original Assignee
江苏恒瑞医药股份有限公司
上海恒瑞医药有限公司
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
Priority claimed from CN201510300885.1A external-priority patent/CN106188293A/zh
Priority to MX2017012965A priority Critical patent/MX2017012965A/es
Priority to US15/565,928 priority patent/US10543284B2/en
Priority to BR112017021245-5A priority patent/BR112017021245A2/zh
Priority to EP16779555.8A priority patent/EP3284751A4/en
Priority to AU2016248357A priority patent/AU2016248357A1/en
Application filed by 江苏恒瑞医药股份有限公司, 上海恒瑞医药有限公司 filed Critical 江苏恒瑞医药股份有限公司
Priority to RU2017135257A priority patent/RU2017135257A/ru
Priority to JP2017553320A priority patent/JP2018516539A/ja
Priority to KR1020177031260A priority patent/KR20170138451A/ko
Priority to CA2982777A priority patent/CA2982777A1/en
Priority to CN201680001857.2A priority patent/CN106687480B/zh
Publication of WO2016165580A1 publication Critical patent/WO2016165580A1/zh

Links

Images

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/08—Peptides having 5 to 11 amino acids
    • 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
    • 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
    • A61K31/535—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one oxygen as the ring hetero atoms, e.g. 1,2-oxazines
    • A61K31/537—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one oxygen as the ring hetero atoms, e.g. 1,2-oxazines spiro-condensed or forming part of bridged ring systems
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/06—Tripeptides
    • 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
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • 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
    • 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
    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione

Definitions

  • the present invention discloses an anti-c-Met antibody or antigen-binding fragment thereof, a chimeric antibody, a humanized antibody comprising the anti-c-Met antibody CDR region, and an anti-c-Met antibody-cytotoxic drug conjugate Or a pharmaceutically acceptable salt or solvate thereof, and a pharmaceutical composition comprising a human c-Met antibody or antigen-binding fragment thereof and an antibody-cytotoxic drug conjugate thereof, or a pharmaceutically acceptable salt or solvent compound thereof, and as an anti-antibody
  • cancer drugs used for cancer drugs.
  • a humanized anti-c-Met antibody and an anti-c-Met antibody-cytotoxic drug conjugate or a pharmaceutically acceptable salt or solvate thereof for use in the treatment of a c-Met mediated disease or condition Use of the drug.
  • tyrosine kinases (PTKs)-related cell signal transduction pathways play an extremely important role in the formation and development of tumors, with more than 50% of primary cancers. Both genes and oncogene products have tyrosine kinase activity.
  • the c-Met proto-oncogene belongs to the Ron subfamily of the PTKs family, and its encoded c-Met protein is a high affinity receptor for Hepatocyte Growth Factor/Scatter Factor (HGF/SF).
  • HGF/c-Met signaling pathway is closely related to angiogenesis and tumor growth process. Its continuous activation is an important cause of tissue cell canceration or cancer cell proliferation. Inhibition of this pathway has become a new means of tumor targeted therapy.
  • the c-Met proto-oncogene is located on the long arm of human chromosome 7 (7q31) and is over 120 kb in size. It encodes a c-Met protein precursor with a molecular weight of approximately 150 kD and is locally glycosylated to generate a 170 kD glycoprotein. Shears into the alpha subunit (50 kDa) and the beta subunit (140 kDa), which are linked by disulfide bonds to form a mature c-Met protein receptor.
  • the heterodimer comprises two strands, the beta strand having an extracellular region, a transmembrane region (also referred to as a membrane stretch fragment), and an intracellular region (including an intracellular tyrosine kinase binding site).
  • the alpha chain has only the extracellular portion, but it is highly glycosylated and attached to the beta chain via a disulfide bond.
  • the extracellular region of the two subunits is the recognition site for the corresponding ligand, and the intracellular region has tyrosine kinase activity.
  • c-Met activation The mechanism of c-Met activation is divided into three types: one is the activation mechanism dependent on HGF, the other is independent of HGF activation mechanism, and the third is through other membrane pathways, such as CD44, adhesin, and receptors on the surface of hyaluronic acid membrane. RON signaling pathway and so on. The most common of these is the activation mechanism that relies on HGF.
  • the N-terminus of HGF binds to c-Met, promotes dimerization and autophosphorylation of Tyr1234 and Tyr1235 on the ⁇ chain, and phosphorylation of Tyr1349 and Tyr1356 near the C-terminus produces binding sites for multiple adaptor proteins.
  • c-Met crosslinks with other membrane receptors. (cross-talk), it is now known that this cross-linking can promote tumor formation and metastasis. Since c-Met is the intersection of many pathways leading to tumor formation and metastasis, it is relatively easy to achieve c-Met as a target. For simultaneous interference with many pathways, c-Met is a promising target for anti-tumor production and metastasis.
  • Antibody drug conjugates link monoclonal antibodies or antibody fragments to biologically active cytotoxins via stable chemical linker compounds, making full use of the specificity of antibodies for tumor cell specific or high expression of antigen binding. And the efficiency of cytotoxins to avoid toxic side effects on normal cells. This means that antibody drug conjugates bind precisely to tumor cells and reduce the effects on normal cells compared to traditional chemotherapeutic drugs.
  • the ADC drug consists of three parts: antibody (targeting), linker and toxin. Among them, a good target (antibody part) determines the specificity of the ADC drug, which includes not only specific targeted binding, but also effective endocytosis.
  • c-Met kinase target inhibitors HGF and c-Met bio-antagonists, HGF and c-Met antibodies, and small molecule c-Met inhibitors.
  • HGF and c-Met bio-antagonists HGF and c-Met antibodies
  • small molecule c-Met inhibitors small molecule c-Met inhibitors.
  • ADC drugs for c-Met may be the most effective way to treat tumors at this target.
  • the anti-c-Met antibody ADC drug of the present invention not only retains the antibody-dependent inhibition of cell proliferation of the anti-c-Met antibody of the present invention, but also increases the effect of potential cytotoxic drugs. Moreover, because its toxin is targeted for release in tumor cells, the drug side effects do not increase synchronously with increasing efficacy.
  • the present invention provides a humanized antibody and a chimeric antibody which specifically bind to human c-Met, and the humanized antibody and chimeric antibody are characterized by high affinity, high potency, endocytosis, stability and no c-Met agonistic activity and other characteristics.
  • the present invention also provides an antibody-cytotoxic drug conjugate which specifically binds to human c-Met or a pharmaceutically acceptable salt or solvate thereof, which not only retains the antibody against the c-Met antibody of the present invention Dependent cell proliferation inhibition, while increasing the effects of potential cytotoxic drugs and the broad spectrum of treatment of disease. Moreover, since its toxin is targeted for release in tumor cells (endocytosis of the anti-c-Met antibody of the present invention), the toxic side effects of the drug do not increase synchronously with an increase in therapeutic effect.
  • the present invention provides an antibody or antigen-binding fragment thereof which specifically binds to a c-Met receptor, comprising at least one sequence selected from the following CDR regions or a mutant sequence thereof:
  • Antibody heavy chain variable region HCDR region sequence SEQ ID NO: 6, SEQ ID NO: 7 or SEQ ID NO: 8;
  • Antibody light chain variable region LCDR region sequence SEQ ID NO: 9, SEQ ID NO: 10 or SEQ ID NO: 11.
  • a c-Met antibody or antigen-binding fragment thereof wherein the antibody heavy chain variable region comprises at least one HCDR region sequence selected from the group consisting of Mutant sequence: SEQ ID NO: 6, SEQ ID NO: 7 or SEQ ID NO: 8.
  • a c-Met antibody or antigen-binding fragment thereof wherein the antibody light chain variable region comprises at least one LCDR region sequence selected from the group consisting of Mutant sequence: SEQ ID NO: 9, SEQ ID NO: 10 or SEQ ID NO: 11.
  • a c-Met antibody or antigen-binding fragment thereof as described above wherein the antibody comprises a heavy chain variable region sequence of SEQ ID NO: 6 (HCDR1), SEQ ID NO: 7 (HCDR2) and SEQ ID NO: 8 (HCDR3), or a mutated sequence thereof, and a light chain variable region sequence SEQ ID NO: 9 (LCDR1), SEQ ID NO: 10 (LCDR2) and SEQ ID NO: 11 (LCDR3) or its mutant sequence.
  • the HCDR2 region mutation sequence described therein is preferably SEQ ID NO: 12.
  • a c-Met antibody or antigen-binding fragment thereof as described above, wherein the c-Met antibody or antigen-binding fragment thereof is a murine antibody or a fragment thereof.
  • a c-Met antibody or antigen-binding fragment thereof as described above, wherein the murine antibody heavy chain variable region sequence is: SEQ ID NO: 4.
  • a c-Met antibody or antigen-binding fragment thereof as described above, wherein the murine antibody light chain variable region sequence is: SEQ ID NO: 5.
  • the chain variable region is: SEQ ID NO: 5.
  • a murine antibody or fragment thereof as described above, wherein the antibody heavy chain variable region further comprises a mouse derived IgG1, IgG2, IgG3 or IgG4 or variant thereof Heavy chain FR zone.
  • a murine antibody or fragment thereof as described above, further comprising a heavy chain constant region derived from murine IgGl, IgG2, IgG3 or IgG4, or a variant thereof.
  • a murine antibody or fragment thereof as described above, further comprising a light chain constant region selected from the group consisting of a murine kappa or lambda chain, or variants thereof.
  • a c-Met antibody or antigen-binding fragment thereof as described above, which is a chimeric antibody or a humanized antibody or a fragment thereof.
  • a c-Met antibody or antigen-binding fragment thereof as described above, wherein the humanized antibody heavy chain variable region further comprises human IgG1, IgG2, IgG3, or The heavy chain FR region of IgG4 or a variant thereof.
  • a c-Met antibody or antigen-binding fragment thereof as described above, wherein the heavy chain FR region sequence on the heavy chain variable region of the humanized antibody is derived from a human a germline heavy chain sequence, preferably human germline heavy chain IGHV 3-33*01; a framework sequence comprising the FR1, FR2, FR3 region and FR4 region of human germline heavy chain IGHV 3-33*01 or a mutant sequence thereof, preferably The mutant sequence is a back mutation of 0-10 amino acids.
  • a c-Met antibody or antigen-binding fragment thereof as described above, wherein the humanized antibody comprises a heavy chain variable as set forth in SEQ ID NOs: 13-15 Sequence of regions or variants thereof.
  • a c-Met antibody or antigen-binding fragment thereof wherein the light chain FR region sequence on the light chain variable region of the humanized antibody is selected from the group consisting of a germline light chain sequence, preferably human germline light chain IGKV085 or IGKV 4-1*01, comprising the framework sequences of the FR1, FR2, FR3 and FR4 regions of human germline light chain IGKV085 and IGKV 4-1*01 or A mutant sequence, preferably the mutant sequence is a back mutation of 0-10 amino acids.
  • a c-Met antibody or antigen-binding fragment thereof as described above, wherein the humanized antibody comprises a light chain selected from the group consisting of SEQ ID NOs: 16-18 Variable region sequences or variants thereof.
  • a c-Met antibody or antigen-binding fragment thereof as described above, said humanized antibody comprising a heavy chain variable region selected from the group consisting of SEQ ID NOs: 13-15 And a light chain variable region selected from the group consisting of SEQ ID NOS: 16-18.
  • a c-Met antibody or antigen-binding fragment thereof comprising a heavy chain variable region sequence and a light chain selected from any one of (a) to (c), is provided Combination of variable region sequences:
  • a c-Met antibody or antigen-binding fragment thereof as described above, wherein the heavy chain constant region of the humanized antibody comprises human IgG1 or a variant thereof a constant region of human IgG2 or a variant thereof, human IgG3 or variant thereof or human IgG4 or variant thereof, preferably comprising human IgG1 or a variant thereof or human IgG2 or a variant thereof or human IgG4 or
  • the constant region of its variant is more preferably the constant region of human IgG2 or a variant thereof.
  • a c-Met antibody or antigen-binding fragment thereof comprising a full length selected from or having at least 90% homology to SEQ ID NOs: 23-25 Heavy chain sequence.
  • a c-Met antibody or antigen-binding fragment thereof as described above, wherein the humanized antibody light chain variable region further comprises any one selected from human ⁇ or ⁇ chain Or a light chain FR region of its variant.
  • a c-Met antibody or antigen-binding fragment thereof as described above, wherein the light chain constant region of the humanized antibody comprises a human ⁇ or ⁇ chain or The constant region of its variant.
  • a c-Met antibody or antigen-binding fragment thereof as described above comprising a whole selected from the group consisting of SEQ ID NOs: 26-28 or having at least 90% sequence homology thereto Long light chain sequence.
  • a c-Met antibody or antigen-binding fragment thereof as described above, wherein the humanized antibody comprises a full-length heavy chain selected from the group consisting of SEQ ID NOs: 23-25 Sequence and full length light chain sequences of SEQ ID NOs: 26-28.
  • Ab-9 the heavy chain sequence of SEQ ID NO: 23 and the light chain sequence of SEQ ID NO: 26;
  • Ab-10 the heavy chain sequence of SEQ ID NO: 24 and the light chain sequence of SEQ ID NO: 27;
  • Ab-11 the heavy chain sequence of SEQ ID NO: 25 and the light chain sequence of SEQ ID NO: 28.
  • a c-Met antibody or antigen-binding fragment thereof, as described above, wherein the antigen-binding fragment comprises Fab, Fv, sFv or F(ab') 2 is provided .
  • the invention further provides a DNA molecule encoding an expression precursor product of a c-Met antibody or antigen-binding fragment thereof as described above.
  • the invention further provides an expression vector comprising a DNA molecule as described above.
  • the invention further provides a host cell transformed with an expression vector as described above.
  • a host cell as described above, wherein said host cell is preferably a mammalian cell, more preferably a CHO cell.
  • the invention further provides a pharmaceutical composition
  • a pharmaceutical composition comprising a c-Met antibody or antigen-binding fragment thereof as described above, and one or more pharmaceutically acceptable excipients, diluents or carriers.
  • the present invention further provides a use of a c-Met antibody or antigen-binding fragment thereof as described above, or a pharmaceutical composition comprising the same, for the preparation of a medicament for treating a c-Met mediated disease or condition, wherein
  • the disease or condition described is preferably a cancer; more preferably a cancer expressing c-Met; most preferably gastric cancer, pancreatic cancer, lung cancer, intestinal cancer, renal cancer, melanoma, non-small cell lung cancer; most preferably gastric cancer and non- Small Cell Lung Cancer.
  • the invention further provides a method of treating and preventing a c-Met mediated disease or condition, the method comprising administering to a patient in need thereof a therapeutically effective amount of a c-Met antibody or antigen-binding fragment thereof as described above, or comprising the same a pharmaceutical composition; wherein the disease or condition is preferably cancer; more preferably a cancer expressing c-Met; most preferably gastric cancer, pancreatic cancer, lung cancer, intestinal cancer, renal cancer, melanoma, non-small cell lung cancer; Most preferred are gastric cancer and non-small cell lung cancer.
  • the present invention further provides an antibody-cytotoxic drug conjugate represented by the formula (I) or a pharmaceutically acceptable salt or solvent compound thereof:
  • D is a drug module
  • L 1 , L 2 are joint units
  • t is 0 or 1, preferably 1;
  • y is 1-8, preferably 2-5; y can be a decimal;
  • Ab is an antibody or antigen-binding fragment thereof that specifically binds to a c-Met receptor as described above.
  • X 1 is selected from the group consisting of a hydrogen atom, a halogen, a hydroxyl group, a cyano group, an alkyl group, an alkoxy group, and a cycloalkyl group;
  • X 2 is selected from the group consisting of -alkyl-, -cycloalkyl-, and -heterocyclyl-;
  • n is 0-5, preferably 1-3; S is a sulfur atom.
  • an antibody-cytotoxic drug conjugate represented by the formula (I) or a pharmaceutically acceptable salt or solvate thereof, wherein D is the following formula (D) Compounds shown:
  • R 1 -R 7 are selected from the group consisting of a hydrogen atom, a halogen, a hydroxyl group, a cyano group, an alkyl group, an alkoxy group, and a cycloalkyl group;
  • R 8 -R 11 are optionally selected from the group consisting of a hydrogen atom, a halogen, an alkenyl group, an alkyl group, an alkoxy group, and a cycloalkyl group; and at least one of R 8 to R 11 is preferably selected from the group consisting of a halogen, an alkenyl group, an alkyl group, and a cycloalkyl group.
  • the rest are hydrogen atoms;
  • any two of R 8 -R 11 form a cycloalkyl group, and the remaining two groups are selected from a hydrogen atom, an alkyl group and a cycloalkyl group;
  • R 12 -R 13 is selected from a hydrogen atom, an alkyl group or a halogen
  • R 14 is selected from aryl or heteroaryl, which is optionally further substituted with a substituent selected from the group consisting of a hydrogen atom, a halogen, a hydroxyl group, an alkyl group, an alkoxy group, and a cycloalkyl group;
  • R 15 is selected from the group consisting of halogen, alkenyl, alkyl, cycloalkyl and COO R 17 ;
  • R 16 is selected from the group consisting of a hydrogen atom, a halogen, a hydroxyl group, a cyano group, an alkyl group, an alkoxy group and a cycloalkyl group;
  • R 17 is selected from a hydrogen atom, an alkyl group, and an alkoxy group.
  • an antibody-cytotoxic drug conjugate of the formula (I) or a pharmaceutically acceptable salt or solvate thereof wherein the L 2 is selected from the group consisting of N- Succinimidyl 4-(2-pyridylthio)pentanoate (SPP), N-succinimidyl 4-(N-maleimidomethyl)-cyclohexane-1-carboxylic acid Esters (SMCC), and N-succinimidyl (4-iodo-acetyl)aminobenzoate (SIAB); preferably SPP or SMCC.
  • SPP N- Succinimidyl 4-(2-pyridylthio)pentanoate
  • SMCC N-succinimidyl 4-(N-maleimidomethyl)-cyclohexane-1-carboxylic acid Esters
  • SIAB N-succinimidyl (4-iodo-acetyl)aminobenzoate
  • an antibody-cytotoxic drug of the formula (I) A conjugate or a pharmaceutically acceptable salt or solvate thereof, wherein D is a camptothecin alkaloid; preferably from CPT, 10-hydroxy-CPT, CPT-11 (irinotecan), SN-38 and Pertinol, more preferably SN-38.
  • an antibody-cytotoxic drug conjugate represented by the formula (I) or a pharmaceutically acceptable salt or solvate thereof, which comprises the formula (II) a conjugated drug or a pharmaceutically acceptable salt or solvent compound thereof:
  • R 2 -R 16 are as defined in the formula (D);
  • an antibody-cytotoxic drug conjugate represented by the formula (I) or a pharmaceutically acceptable salt or solvate thereof, which comprises the formula (III) a conjugated drug or a pharmaceutically acceptable salt or solvent compound thereof:
  • R 2 -R 16 are as defined in the formula (D);
  • n 3-6, preferably 5.
  • an antibody-cytotoxic drug of the formula (I) a conjugate or a pharmaceutically acceptable salt or solvate thereof, which comprises a conjugated drug of the formula (IV) or a pharmaceutically acceptable salt or solvate thereof:
  • R 2 -R 16 are as defined in the formula (D);
  • X 1 , X 2 , m are as defined in the formula L2.
  • an antibody-cytotoxic drug conjugate of the formula (I) or a pharmaceutically acceptable salt or solvate thereof which comprises the formula (V) a conjugated drug or a pharmaceutically acceptable salt or solvent compound thereof:
  • X 1 , X 2 , m are as defined in the formula L2.
  • the antibody-cytotoxic drug conjugate of the present invention or a pharmaceutically acceptable salt or solvent compound thereof includes, but is not limited to:
  • Ab-9, Ab-10, Ab-11 are c-Met antibodies as described above, and y is 1-8, preferably 2-5.
  • y ranges from 1 to 8; preferably from 1 to 4.
  • the invention further provides a method of preparing an antibody-cytotoxic drug conjugate of the formula (V), the method comprising:
  • the compound of the formula (Ab-L2) is reacted with a compound of the formula (L1-D) in an organic solvent to obtain a compound of the formula (V);
  • the organic solvent is preferably acetonitrile or ethanol;
  • X 1 is selected from the group consisting of a hydrogen atom, a halogen, a hydroxyl group, a cyano group, an alkyl group, an alkoxy group, and a cycloalkyl group;
  • X 2 is selected from the group consisting of alkyl, cycloalkyl and heterocyclic;
  • X is 0-5, preferably 1-3;
  • n is 0-5, preferably 1-3.
  • the present invention further provides a pharmaceutical composition
  • a pharmaceutical composition comprising the c-Met antibody or antigen-binding fragment thereof as described above, or an antibody-cytotoxic drug conjugate, or a pharmaceutically acceptable salt or solvent compound thereof, and a pharmaceutically acceptable compound Forming agent, diluent or carrier.
  • the present invention further provides a c-Met antibody or antigen-binding fragment thereof, or an antibody-cytotoxic drug conjugate thereof, or a pharmaceutically acceptable salt or solvent compound thereof, or a pharmaceutical composition comprising the same, for use in preparation
  • a medicament for treating a disease or condition mediated by a c-Met wherein the disease or condition is preferably cancer; more preferably a cancer expressing c-Met; most preferably gastric cancer, pancreatic cancer, lung cancer, colon cancer , renal cancer, melanoma, non-small cell lung cancer; most preferred are gastric cancer, pancreatic cancer, non-small cell lung cancer and kidney cancer.
  • the invention further provides a method of treating and preventing a c-Met mediated disease or condition, the method comprising administering to a subject in need thereof a therapeutically effective amount of a c-Met antibody or antigen-binding fragment thereof, or antibody-cell as described above
  • Fig. 1 The anti-c-Met antibody and ADC molecule of the present invention inhibit tumor function, and the results show that the new ADC molecule can achieve complete tumor inhibition by the introduced toxin, and the antibody alone cannot be achieved. The results also showed that the ADC molecule of the present invention did not affect T 1/2 due to the coupling of the toxin, and the ADC drug of the present invention showed no toxicity in the mouse.
  • c-Met or "c-Met polypeptide” or “c-Met receptor” refers to a receptor tyrosine kinase that binds to cell growth factor (HGF).
  • HGF cell growth factor
  • m-c-Met murine c-Met
  • cyno-c-Met monkey c-Met
  • GenBank GenBank
  • NM_000245 or the human protein encoded by the polypeptide sequence provided in GenBank Accession No. NP_000236 or its extracellular domain.
  • the original single-stranded precursor protein is cleaved after translation to produce alpha and beta subunits that are joined by disulfide bonds to form mature receptors.
  • the involvement of the receptor tyrosine kinase c-Met in cellular processes includes, for example, migration, invasion, and morphogenesis processes associated with embryonic tissue regeneration.
  • c-Met-related disorder or condition refers to any unfavorable or lacking expression derived from c-Met, unfavorable regulation or lack of regulation, or deleterious activity or lack of activity, or may be modulated by modulation of c-Met expression or activity.
  • Activation of the HGF/c-Met pathway can be expected, for example, in most cancer patients, or in patients whose disease is indeed driven by changes associated with the c-Met pathway. For example, up-regulation is due to different mechanisms, such as overexpression of HGF and/or c-Met, or constitutive activation by c-Met mutation.
  • C-Met related disorders or conditions include, but are not limited to, for example, proliferative diseases and disorders and inflammatory diseases and disorders.
  • Proliferative diseases include, but are not limited to, for example, cancer, including, for example, gastric cancer, esophageal cancer, renal cancer including papillary renal cell carcinoma, lung cancer, glioma, head and neck cancer, epithelial cancer, skin cancer, leukemia, lymphoma , myeloma, brain cancer, pancreatic cancer, colorectal cancer, gastrointestinal cancer, intestinal cancer, genital cancer, urinary cancer, melanoma, prostate cancer, and other tumors known to those skilled in the art.
  • Inflammatory diseases include, but are not limited to, for example, bacterial infections, including infections caused by Listeria bacteria.
  • the antibody of the present invention refers to an immunoglobulin, which is a tetrapeptide chain structure in which two identical heavy chains and two identical light chains are linked by interchain disulfide bonds.
  • the immunoglobulin heavy chain constant region has different amino acid composition and arrangement order, so its antigenicity is also different. Accordingly, immunoglobulins can be classified into five classes, or isoforms of immunoglobulins, namely IgM, IgD, IgG, IgA and IgE, and the corresponding heavy chains are ⁇ chain, ⁇ chain, ⁇ chain, respectively. , ⁇ chain, ⁇ chain.
  • IgG can be classified into IgG1, IgG2, IgG3, and IgG4.
  • Light chains are classified as either a kappa chain or a lambda chain by the constant region.
  • Each of the five types of Ig may have a kappa chain or a lambda chain.
  • variable region The sequence of about 110 amino acids near the N-terminus of the antibody heavy and light chains varies greatly, being the variable region (V region); the remaining amino acid sequence near the C-terminus is relatively stable and is a constant region (C region).
  • the variable region includes three hypervariable regions (HVR) and four relatively conserved framework regions (FR). The three hypervariable regions determine the specificity of the antibody, also known as the complementarity determining region (CDR).
  • Each of the light chain variable region (LCVR) and the heavy chain variable region (HCVR) consists of three CDR regions and four FR regions, and the order from the amino terminus to the carboxy terminus is: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • the three CDR regions of the light chain refer to LCDR1, LCDR2, and LCDR3; the three CDR regions of the heavy chain refer to HCDR1, HCDR2, and HCDR3.
  • the CDR amino acid residues of the LCVR region and the HCVR region of the antibody or antigen-binding fragment of the invention conform to the known Kabat numbering rules (LCDR1-3, HCDE2-3) in number and position, or conform to the kabat and chothia numbering rules. Then (HCDR1).
  • murine antibody is in the present invention a monoclonal antibody against human c-Met prepared in mice according to the knowledge and skill in the art.
  • the test subject is injected with the c-Met antigen at the time of preparation, and then the hybridoma expressing the antibody having the desired sequence or functional properties is isolated.
  • the murine c-Met antibody or antigen-binding fragment thereof may further comprise a light chain constant region of a murine kappa, lambda chain or variant thereof, or further comprising a murine IgG1 , heavy chain constant region of IgG2, IgG3 or IgG4 or a variant thereof.
  • chimeric antibody is an antibody obtained by fusing a variable region of a murine antibody with a constant region of a human antibody, and can alleviate an immune response induced by a murine antibody.
  • a hybridoma secreting a murine-specific monoclonal antibody is first established, and then the variable region gene is cloned from the mouse hybridoma cell, and then cloned into the constant region gene of the human antibody for recombinant expression.
  • humanized antibody also known as CDR-grafted antibody, refers to the transplantation of mouse CDR sequences into human antibody variable region frameworks, ie different types of An antibody produced in a human germline antibody framework sequence. It is possible to overcome the strong antibody variable antibody response induced by chimeric antibodies by carrying a large amount of mouse protein components.
  • framework sequences can be obtained from public DNA databases including germline antibody gene sequences or published references.
  • the germline DNA sequences of human heavy and light chain variable region genes can be found in the "VBase" human germline sequence database (available on the Internet at www.mrccpe.com.ac.uk/vbase ), as well as in Kabat, EA, etc.
  • the CDR sequence of the c-Met humanized antibody mouse is selected from the group consisting of SEQ ID NO: 6, 7, 8, 9, 10, 11 (please check the #s, in Case just copy from sost draft).
  • the human antibody variable region framework is designed to be selected, wherein the light chain FR region sequence on the antibody light chain variable region is selected from the human germline light chain sequence, preferably the human germline light chain IGKV085 or IGKV 4-1* 01, comprising the FR1, FR2, FR3 region and FR4 region of human germline light chain IGKV085 and IGKV 4-1*01; wherein the heavy chain FR region sequence on the antibody heavy chain variable region is derived from human germline
  • the strand sequence preferably the human germline heavy chain IGHV 3-33*01; comprises the FR1, FR2, FR3 and FR4 regions of the human germline heavy chain IGHV 3-33*01.
  • the human antibody variable region can be subjected to minimal reverse mutation to maintain activity.
  • a humanized antibody can be produced by obtaining an HCVR and LCVR sequence of an anti-c-Met specific antibody (eg, a murine antibody or an antibody produced by a hybridoma), which is grafted to a selected human antibody framework coding sequence.
  • the CDR regions can be optimized by random mutagenesis or mutagenesis at a particular position to replace one or more amino acids in the CDRs with different amino acids prior to grafting the CDR regions into the framework regions.
  • the CDR regions can be optimized after insertion into the human framework regions using methods available to those skilled in the art.
  • a "humanized antibody” has a CDR derived from or derived from a parent antibody (ie, a non-human antibody, preferably a mouse monoclonal antibody), and to the extent that it exists, the framework region and the constant region (or a major portion thereof or The essential portion, ie at least about 90%, 92%, 94%, 95%, 96%, 97%, 98% or 99%) of the sequence is identical to the human germline immunoglobulin region (see, for example, The, International, ImmunoGeneTics, Database, or a recombinant or mutant form thereof, whether or not the antibody is produced in a human cell.
  • a parent antibody ie, a non-human antibody, preferably a mouse monoclonal antibody
  • At least 2, 3, 4, 5 or 6 CDRs of the humanized antibody are optimized from the CDRs of the humanized antibody-derived non-human parent antibody to produce a desired property, such as improved specificity, affinity or And effect, which can be identified by screening assays, such as ELISA assays.
  • the optimized CDRs in the antibodies of the invention comprise at least one amino acid substitution when compared to the CDRs present in the parent antibody.
  • Certain amino acid substitutions in the CDRs of the humanized antibodies of the invention reduce the likelihood of antibody instability (eg, removal of Asn residues in the CDRs) or when compared to the CDRs of the parent antibody Human subjects reduce the immunogenicity of the antibody when administered (eg, as predicted by IMMUNOFILTERTM, Technology).
  • HCVR and LCVR can be expressed as part of the entire anti-sclerostin antibody molecule, ie, a fusion protein expressed as a human constant domain sequence. However, HCVR and LCVR sequences can also be expressed in the absence of a constant sequence to produce a humanized anti-c-Met scFv.
  • the "antigen-binding fragment” as used in the present invention refers to a Fab fragment having antigen-binding activity, a Fab' fragment, an F(ab') 2 fragment, and an Fv fragment scFv fragment which binds to human c-Met;
  • the antibody is selected from one or more of the CDR regions of SEQ ID NO: 3 to SEQ ID NO: 8.
  • the Fv fragment contains the antibody heavy chain variable region and the light chain variable region, but has no constant region and has the smallest antibody fragment of the entire antigen binding site.
  • Fv antibodies also comprise a polypeptide linker between the VH and VL domains and are capable of forming the desired structure for antigen binding.
  • the two antibody variable regions can also be joined by a different linker into a single polypeptide chain, referred to as a single chain antibody or a single chain Fv (scFv).
  • the scFv can also construct a bispecific antibody with other antibodies, such as anti-EGFR antibodies.
  • binding to c-Met in the present invention refers to the ability to interact with human c-Met.
  • antigen binding site as used in the present invention refers to a three-dimensional spatial site that is discrete on an antigen and is recognized by an antibody or antigen-binding fragment of the present invention.
  • the "ADCC” described in the present invention is an antibody-dependent cell-mediated cytotoxicity, which means that a cell expressing an Fc receptor directly kills an antibody by Fc segment of the recognition antibody.
  • Target cells The ADCC effector function of the antibody can be reduced or eliminated by modification of the Fc segment on IgG.
  • the modification refers to mutations in the heavy chain constant region of the antibody, such as N297A, L234A, L235A selected from IgG1; IgG2/4 chimera, F235E of IgG4, or L234A/E235A mutation.
  • the fusion protein described in the present invention is a protein product co-expressed by two genes obtained by DNA recombination.
  • the extracellular region of c-Met refers to a portion of the c-Met protein expressed outside the cell membrane.
  • the engineered antibodies or antigen-binding fragments of the invention can be prepared and purified by conventional methods.
  • the cDNA sequence encoding the heavy chain (SEQ ID NO: 4) and the light chain (SEQ ID NO: 5) can be cloned and recombined into the expression vector pEE6.4 ((Lonza Biologics).
  • the recombinant immunoglobulin expression vector can be Stable transfection of CHO cells.
  • mammalian expression systems result in glycosylation of antibodies, particularly in the highly conserved N-terminus of the FC region. By expression and specificity to human c-Met The bound antibody obtains a stable clone.
  • the positive clone is expanded and cultured in a serum-free medium of the bioreactor to produce an antibody.
  • the antibody-secreted culture solution can be purified by a conventional technique. For example, using A containing an adjusted buffer Or the G Sepharose FF column is passed through the column. The non-specifically bound components are washed away. The bound antibody is eluted by pH gradient method, and the antibody fragment is detected by SDS-PAGE and collected. The antibody can be concentrated by filtration in a conventional manner. Mixtures and multimers can also be removed by conventional methods such as molecular sieves, ion exchange, and the resulting product should be immediately frozen, such as -70 ° C, or lyophilized.
  • the antibody of the present invention refers to a monoclonal antibody.
  • the monoclonal antibody or mAb according to the present invention refers to an antibody obtained from a single clonal cell strain, and the cell strain is not limited to a eukaryotic, prokaryotic or phage clonal cell strain.
  • Monoclonal antibodies or antigen-binding fragments can be obtained recombinantly using, for example, hybridoma technology, recombinant techniques, phage display technology, synthetic techniques (e.g., CDR-grafting), or other prior art techniques.
  • administering when applied to an animal, human, experimental subject, cell, tissue, organ or biological fluid, refers to an exogenous drug, therapeutic agent, diagnostic agent or composition and animal, human, subject Contact of the test subject, cell, tissue, organ or biological fluid.
  • administering can refer to, for example, therapeutic, pharmacokinetic, diagnostic, research, and experimental methods. Treatment of the cells includes contact of the reagents with the cells, and contact of the reagents with the fluid, wherein the fluids are in contact with the cells.
  • Treatment means administering to a patient a therapeutic agent for internal or external use, such as a composition comprising any of the binding compounds of the present invention, the patient having one or more symptoms of the disease, and the therapeutic agent is known to have Therapeutic effect.
  • a therapeutic agent is administered in a subject or population to be treated to effectively alleviate the symptoms of one or more diseases, whether by inducing such symptoms to degenerate or inhibiting the progression of such symptoms to any degree of clinical right measurement.
  • the amount of therapeutic agent also referred to as "therapeutically effective amount” effective to alleviate the symptoms of any particular disease can vary depending on a variety of factors, such as the patient's disease state, age and weight, and the ability of the drug to produce a desired effect in the patient.
  • Whether the symptoms of the disease have been alleviated can be assessed by any clinical test method commonly used by a physician or other professional health care provider to assess the severity or progression of the condition.
  • Embodiments of the invention e.g., methods of treatment or preparations
  • Constantly modified refers to amino acids in other amino acid substitution proteins having similar characteristics (eg, charge, side chain size, hydrophobicity/hydrophilicity, backbone conformation and rigidity, etc.), such that Changes are made without altering the biological activity of the protein. It will be appreciated by those skilled in the art that, in general, a single amino acid substitution in a non-essential region of a polypeptide does not substantially alter biological activity (see, for example, Watson et al. (1987) Molecular Biology of the Gene, The Benjamin/Cummings Pub. Co., Page 224, (4th edition)). In addition, substitution of structurally or functionally similar amino acids is unlikely to disrupt biological activity.
  • a binding compound consisting essentially of the amino acid sequence recited may also include one or more amino acids that do not significantly affect the properties of the binding compound.
  • an "effective amount” includes an amount sufficient to ameliorate or prevent a symptom or condition of a medical condition.
  • An effective amount also means an amount sufficient to allow or facilitate the diagnosis.
  • An effective amount for a particular patient or veterinary subject can vary depending on factors such as the condition to be treated, the overall health of the patient, the route and dosage of the method of administration, and the severity of the side effects.
  • An effective amount can be the maximum dose or dosing regimen that avoids significant side effects or toxic effects.
  • Exogenous refers to a substance that is produced outside of a living organism, cell, or human body depending on the background.
  • Endogenous refers to a substance produced in a cell, organism or human body depending on the background.
  • “Homology” refers to sequence similarity between two polynucleotide sequences or between two polypeptides. When positions in both comparison sequences are occupied by the same base or amino acid monomer subunit, for example if each position of two DNA molecules is occupied by adenine, then the molecule is homologous at that position .
  • the percent homology between the two sequences is a function of the number of matches or homology positions shared by the two sequences divided by the number of positions compared ⁇ 100. For example, in the optimal alignment of sequences, if there are 6 matches or homologs in 10 positions in the two sequences, then the two sequences are 60% homologous. In general, comparisons are made when the maximum sequence of homology is obtained by aligning the two sequences.
  • “Pharmaceutical composition” means a mixture comprising one or more of the compounds described herein, or a physiologically/pharmaceutically acceptable salt or prodrug thereof, and other chemical components, as well as other components such as physiological/pharmaceutically acceptable carriers. And excipients.
  • the purpose of the pharmaceutical composition is to promote the administration of the organism, which facilitates the absorption of the active ingredient and thereby exerts biological activity.
  • carrier refers to a system which changes the manner in which the drug enters the body and the distribution in the body, controls the release rate of the drug, and delivers the drug to the targeted organ.
  • Drug carrier release and targeting systems can reduce drug degradation and loss, reduce side effects, and increase bioavailability.
  • Polymeric surfactants which can be used as carriers, can be self-assembled due to their unique amphiphilic structure to form aggregates of various forms, such as micelles, microemulsions, gels, liquid crystals, vesicles, etc. . These aggregates have the ability to entrap drug molecules while having good permeability to the membrane and can be used as an excellent drug carrier.
  • the term "diluent” is also known as a filler and its primary use is to increase the weight and volume of the tablet. The addition of the diluent not only ensures a certain volume, but also reduces the dose deviation of the main component and improves the compression moldability of the drug. When the drug of the tablet contains an oily component, it is necessary to add an absorbent to absorb the oily substance so as to maintain a "dry" state to facilitate tableting.
  • pharmaceutically acceptable salt refers to a salt of a ligand-cytotoxic drug conjugate of the present invention which is safe and effective for use in a mammal, and which has the desired biological activity, the antibody of the present invention -
  • the antibody drug-conjugated compound contains at least one amino group and thus can form a salt with an acid.
  • solvent compound means that the ligand-drug conjugate compound of the present invention forms a pharmaceutically acceptable solvent compound with one or more solvent molecules.
  • ligand is a macromolecular compound that recognizes and binds to an antigen or receptor associated with a target cell.
  • the role of the ligand is to present the drug to a target cell population that binds to the ligand, including but not limited to protein hormones, lectins, growth factors, antibodies or other molecules capable of binding to cells.
  • a therapeutic agent is a molecule or atom that is administered separately, simultaneously or sequentially with a binding moiety such as an antibody or antibody fragment, or a subfragment thereof, and is useful in the treatment of a disease.
  • therapeutic agents include, but are not limited to, antibodies, antibody fragments, conjugates, drugs, cytotoxic agents, pro-apoptotic agents, toxins, nucleases (including DNase and RNase), hormones, immunomodulators, chelators , boron compounds, photosensitizers or dyes, radioisotopes or radionuclides, oligonucleotides, interfering RNA, peptides, anti-angiogenic agents, chemotherapeutic agents, cytokines, chemokines, prodrugs, enzymes, binding proteins or peptides Or a combination thereof.
  • a conjugate is an antibody component or other targeting moiety that is conjugated to a therapeutic agent as described above.
  • conjugated and “immunoconjugate” are used interchangeably herein.
  • cytotoxic agent refers to a substance that inhibits or prevents the function of a cell and/or causes cell death or destruction.
  • Toxin refers to any substance that is capable of producing a detrimental effect on the growth or proliferation of cells.
  • “Chemotherapeutic agent” refers to a chemical compound that can be used to treat cancer.
  • the definition also includes antihormonal agents that modulate, reduce, block or inhibit the hormonal effects that promote cancer growth, and are often in the form of systemic or systemic therapies. They can be hormones themselves.
  • Auristatin is a fully synthetic drug with a chemical structure that is relatively easy to modify in order to optimize its physical properties and The characteristics of the drug.
  • the auristatin derivatives used for the coupling with the antibody mainly include monomethyl auristatin E (MMAE) and monomethyl auristatin F (MMAF), the former being a natural tubulin polymerase inhibitor tail sea rabbit
  • MMAE monomethyl auristatin E
  • MMAF monomethyl auristatin F
  • a synthetic pentapeptide derived from dolastatin-10 is synthesized by adding a 2-amino-1-phenylpropyl-1-ol at the C-terminus.
  • MMAE has less than one nanomolar inhibitory activity against a variety of human tumor cell lines.
  • MMAF In order to reduce the cytotoxic activity of MMAE, MMAF adds a phenylalanine to the C-terminus of tail rabbit, and because of the structural introduction of a carboxyl group, MMAF has poor cell membrane permeability and thus bioactivity to cells. Significantly decreased, but the inhibitory activity on cells after coupling with antibodies was greatly increased (US7750116).
  • tubulin inhibitor refers to a class of compounds that interfere with the mitotic process of cells by inhibiting the polymerization of tubulin or promoting the polymerization of tubulin, thereby exerting an anti-tumor effect.
  • Non-limiting examples thereof include: maytansinoids, calicheamicin, taxanes, vincristine, colchicine, tail seaweed/aluratin, preferably from maytans or tail sea rabbits Or auristatin; more preferred is a compound represented by the formula D 1 or D M .
  • CPT is an abbreviation for camptothecin, and in the present application CPT is used to denote an analog or derivative of camptothecin itself or camptothecin.
  • the structure of camptothecin and some of its analogs having the number shown and the ring labeled with the letters A-E are provided in the following formula.
  • Intracellular metabolite refers to a compound produced by intracellular reaction to a metabolic process or reaction of an antibody-drug conjugate (ADC).
  • the metabolic process or reaction may be an enzymatic process, such as an egg peptide peptide of an ADC White hydrolysis cleavage, or hydrolysis of functional groups such as hydrazines, esters or amides.
  • Intracellular metabolites include, but are not limited to, antibodies and free drugs that undergo intracellular cleavage upon entry, diffusion, uptake, or transport into cells.
  • intracellular cleavage and “intracellular cleavage” refer to a metabolic process or reaction in a cell to an antibody-drug conjugate (ADC) whereby covalent attachment between the drug moiety (D) and the antibody (Ab) That is, the linker is interrupted, causing the intracellular free drug to dissociate from the antibody.
  • ADC antibody-drug conjugate
  • the module in which the ADC is cleaved is thus an intracellular metabolite.
  • bioavailability refers to the systemic availability (ie, blood/plasma levels) of a given amount of drug administered to a patient. Bioavailability is the absolute term for both the time (rate) and the total amount (degree) of the drug from the administered dosage form to the large cycle.
  • cytotoxic activity refers to the cell killing, cytostatic, or growth inhibiting effect of an intracellular metabolite of an antibody-drug conjugate or antibody-drug conjugate. Cytotoxic activity can be expressed as the IC50 value, ie the concentration per unit volume (molar or mass) at which half of the cells survive.
  • alkyl refers to a saturated aliphatic hydrocarbon group which is a straight or branched chain group containing from 1 to 20 carbon atoms, preferably an alkyl group having from 1 to 12 carbon atoms, more preferably from 1 to 10 carbons.
  • the alkyl group of the atom is most preferably an alkyl group having 1 to 6 carbon atoms.
  • Non-limiting examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1,1-dimethylpropyl, 1 , 2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 2,2-diethyl Hexyl, and various branched isomers thereof.
  • the alkyl group may be substituted or unsubstituted, and when substituted, the substituent may be substituted at any available point of attachment, preferably one or more of the following groups independently selected from the group consisting of an alkane Base, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, fluorenyl, hydroxy, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, naphthenic Oxyl, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, oxo.
  • an alkane Base alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, fluorenyl, hydroxy, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, naphthenic Oxyl, heterocycloal
  • cycloalkyl refers to a saturated or partially unsaturated monocyclic or polycyclic cyclic hydrocarbon substituent containing from 3 to 20 carbon atoms, preferably from 3 to 12 carbon atoms, more preferably from 3 to 10 carbon atoms. One carbon atom, most preferably from 3 to 8 carbon atoms.
  • Non-limiting examples of monocyclic cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cyclohexadienyl, cycloheptyl, cycloheptatriene
  • a polycycloalkyl group includes a spiro ring, a fused ring, and a cycloalkyl group.
  • heterocyclyl refers to a saturated or partially unsaturated monocyclic or polycyclic cyclic hydrocarbon substituent containing from 3 to 20 ring atoms wherein one or more ring atoms are selected from nitrogen, oxygen or S(O).
  • a hetero atom of m (where m is an integer of 0 to 2), but excluding the ring moiety of -OO-, -OS- or -SS-, the remaining ring atoms being carbon. It preferably contains 3 to 12 ring atoms, of which 1 to 4 are hetero atoms; more preferably, the cycloalkyl ring contains 3 to 10 ring atoms.
  • Non-limiting examples of monocyclic heterocyclic groups include pyrrolidinyl, piperidinyl, piperazinyl, morpholinyl, thiomorpholinyl, homopiperazinyl and the like.
  • Polycyclic heterocyclic groups include spiro, fused, and bridged heterocyclic groups.
  • the heterocyclyl ring may be fused to an aryl, heteroaryl or cycloalkyl ring, wherein the ring to which the parent structure is attached is a heterocyclic group, non-limiting examples of which include:
  • the heterocyclic group may be optionally substituted or unsubstituted, and when substituted, the substituent is preferably one or more of the following groups independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkane Thio, alkylamino, halogen, fluorenyl, hydroxy, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio , heterocycloalkylthio, oxo.
  • aryl refers to a 6 to 14 membered all-carbon monocyclic or fused polycyclic ring (ie, a ring that shares a pair of adjacent carbon atoms) having a conjugated ⁇ -electron system, preferably 6 to 10 members, such as benzene.
  • the base and naphthyl are most preferably phenyl.
  • the aryl ring may be fused to a heteroaryl, heterocyclyl or cycloalkyl ring, wherein the ring to which the parent structure is attached is an aryl ring, non-limiting examples of which include:
  • the aryl group may be substituted or unsubstituted, and when substituted, the substituent is preferably one or more of the following groups independently selected from the group consisting of alkyl, alkenyl, alkynyl, alkoxy, alkylthio, Alkylamino, halogen, fluorenyl, hydroxy, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycle Alkylthio group.
  • heteroaryl refers to a heteroaromatic system containing from 1 to 4 heteroatoms, from 5 to 14 ring atoms, wherein the heteroatoms are selected from the group consisting of oxygen, sulfur and nitrogen.
  • the heteroaryl group is preferably 5 to 10 members, more preferably 5 or 6 members, such as furyl, thienyl, pyridyl, pyrrolyl, N-alkylpyrrolyl, pyrimidinyl, pyrazinyl, imidazolyl, tetra Azolyl and the like.
  • the heteroaryl ring may be fused to an aryl, heterocyclic or cycloalkyl ring, wherein the ring to which the parent structure is attached is a heteroaryl ring, non-limiting examples of which include:
  • the heteroaryl group may be optionally substituted or unsubstituted, and when substituted, the substituent is preferably one or more a group independently selected from the group consisting of alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, decyl, hydroxy, nitro, cyano, cycloalkyl, heterocycloalkane Alkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio.
  • alkoxy refers to -O-(alkyl) and -O-(unsubstituted cycloalkyl), wherein alkyl is as defined above.
  • alkoxy groups include: methoxy, ethoxy, propoxy, butoxy, cyclopropoxy, cyclobutoxy, cyclopentyloxy, cyclohexyloxy.
  • the alkoxy group may be optionally substituted or unsubstituted, and when substituted, the substituent is preferably one or more of the following groups independently selected from the group consisting of alkyl, alkenyl, alkynyl, alkoxy, alkane Thio, alkylamino, halogen, fluorenyl, hydroxy, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio , heterocycloalkylthio.
  • bond refers to a covalent bond represented by "-”.
  • hydroxy refers to an -OH group.
  • halogen means fluoro, chloro, bromo or iodo.
  • amino means -NH 2.
  • cyano refers to -CN.
  • nitro refers to -NO 2 .
  • heterocyclic group optionally substituted by an alkyl group means that an alkyl group may be, but not necessarily, present, and the description includes the case where the heterocyclic group is substituted with an alkyl group and the case where the heterocyclic group is not substituted with an alkyl group.
  • Substituted refers to one or more hydrogen atoms in the group, preferably up to 5, more preferably 1 to 3, hydrogen atoms, independently of each other, substituted by a corresponding number of substituents. It goes without saying that the substituents are only in their possible chemical positions, and those skilled in the art will be able to determine (by experiment or theory) substitutions that may or may not be possible without undue effort. For example, an amino group or a hydroxyl group having a free hydrogen may be unstable when combined with a carbon atom having an unsaturated (e.g., olefinic) bond.
  • Linker refers to a chemical moiety comprising a covalent bond or chain of atoms that covalently attaches an antibody to a drug moiety.
  • the linker comprises: a divalent group, such as an alkyldiyl, arylene, heteroarylene, such as -(CR2)nO(CR2)n-, alkoxy repeating unit (eg, poly. Modules such as ethyleneoxy, PEG, polymethyleneoxy, and hydrocarbon amino (eg, polyethylene amino, JeffamineTM); and diesters and amides, including succinate, succinamide, Glycolate, malonate and hexamide.
  • Val-Cit or "vc” valine-citrulline (exemplified dipeptide in a protease cleavable linker)
  • PAB p-aminobenzyloxycarbonyl (exemplified by "self-sacrificing” joint assembly)
  • Me-Val-Cit N-methyl-valine-citrulline (where the linker peptide bond has been modified to prevent its caspase B cleavage)
  • MC(PEG)6-OH Maleimidocaproyl-polyethylene glycol (adhered to antibody cysteine)
  • SPDP N-succinimidyl 3-(2-pyridyldithio)propionate
  • SMCC succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate
  • MMAE monomethyl auristatin E (MW718)
  • MMAF A variant of auristatin E (MMAE) with phenylalanine at the C-terminus of the drug (MW731.5)
  • MMAF-DMAEA MMAF (MW801.5) with DMAEA (dimethylaminoethylamine) linked to C-terminal phenylalanine
  • MMAF-TEG MMAF with tetraethylene glycol esterification to phenylalanine
  • MMAF-NtBu N-tert-butyl as amide attached to the C-terminus of MMAF
  • DM1 N(2')-deacetyl-N(2')-(3-mercapto-1-oxypropyl)-maytansine
  • DM3 N(2')-deacetyl-N2-(4-mercapto-1-oxopentyl)-maytansine
  • DM4 N(2')-deacetyl-N2-(4-mercapto-4-methyl-1-oxopentyl)-maytansine
  • the invention also provides antibody-cytotoxic drug coupling comprising any anti-c-Met antibody of the invention or other endocytogenic c-Met antibody (eg, LY-2875358) conjugated to one or more cytotoxic agents Or a pharmaceutically acceptable salt or solvate thereof (interchangeably referred to as "antibody-drug conjugate” or "ADC"), such as chemotherapeutic agents, drugs, growth inhibitors, toxins (eg, bacteria, An enzymatically active toxin of fungi, plant or animal origin or a fragment thereof) or a radioisotope (ie a radioconjugate).
  • ADC antibody-drug conjugate
  • the antibody-cytotoxic drug conjugate or a pharmaceutically acceptable salt or solvate thereof comprises an anti-c-Met antibody and a chemotherapeutic agent or other toxin.
  • chemotherapeutic agents that can be used to generate antibody-cytotoxic drug conjugates or pharmaceutically acceptable salts or solvate compounds thereof.
  • Enzymatically active toxins and fragments thereof, which are described in the specification, can also be used.
  • the antibody-cytotoxic drug conjugate or a pharmaceutically acceptable salt or solvate thereof comprises an anti-c-Met antibody and one or more small molecule toxins, including but not limited to small molecule drugs, such as hi Alkaloid derivatives, calicheamicin, maytansinoids, dolastatin, auristatin, trichothecene and CC1065 and these drugs A fragment having cytotoxic activity.
  • small molecule drugs such as hi Alkaloid derivatives, calicheamicin, maytansinoids, dolastatin, auristatin, trichothecene and CC1065 and these drugs A fragment having cytotoxic activity.
  • Exemplary linkers L 2 include 6-maleimidocaproyl ("MC”), maleimidopropionyl ("MP”), valine-citrulline (“val-cit”or”Vc”), alanine-phenylalanine (“ala-phe”), p-aminobenzyloxycarbonyl (“PAB”), N-succinimidyl 4-(2-pyridylthio) valerate (“SPP”), N-succinimidyl 4-(N-maleimidomethyl)cyclohexane-1 carboxylate (“SMCC”), and N-succinimidyl (4-iodine) -Acetyl)aminobenzoate (“SIAB”).
  • MC 6-maleimidocaproyl
  • MP maleimidopropionyl
  • Vc valine-citrulline
  • ala-phe alanine-phenylalanine
  • PAB p-aminobenzyloxycarbonyl
  • the linker can be a "cleavable linker” that facilitates release of the drug in the cell.
  • a "cleavable linker” that facilitates release of the drug in the cell.
  • acid labile linkers e.g., hydrazine
  • protease sensitive linkers e.g., peptidase sensitive
  • photolabile linkers e.g., dimethyl linkers, or disulfide containing linkers
  • dimethyl linkers e.g., peptidase sensitive linkers
  • disulfide containing linkers can be used (Chari et al, Cancer Research 52: 127-131). (1992); U.S. Patent No. 5,208,020).
  • the linker member can be a "stretcher unit” that connects the antibody to another linker member or drug module.
  • exemplary extension units are shown below (where the wavy lines indicate sites that are covalently attached to the antibody):
  • the linker unit can be an amino acid unit.
  • the amino acid unit allows the protease to cleave the linker, thereby facilitating the antibody-cytotoxic drug conjugate or a pharmaceutically acceptable salt or solvent compound thereof after exposure to an intracellular protease such as a lysosomal enzyme. Release the drug. See, for example, Doronina et al. (2003) Nat. Biotechnol. 21:778-784.
  • Exemplary amino acid units include, but are not limited to, dipeptides, tripeptides, tetrapeptides, and pentapeptides.
  • Exemplary dipeptides include: valine-citrulline (VC or val-cit); alanine-phenylalanine (AF or ala-phe); phenylalanine-lysine (FK or phe) -lys); or N-methyl- Proline-citrulline (Me-val-cit).
  • Exemplary tripeptides include: glycine-valine-citrulline (gly-val-cit) and glycine-glycine-glycine-glycine-gly-gly-gly.
  • Amino acid units can comprise naturally occurring amino acid residues, as well as minor amino acids and non-naturally occurring amino acid analogs, such as citrulline. Amino acid units can be designed and optimized in terms of their selectivity for enzymatic cleavage of specific enzymes, such as tumor-associated proteases, cathepsins B, C and D, or plasma proteases.
  • the linker member can be a "spacer” unit that attaches the antibody (either directly or through an extender unit and/or an amino acid unit) to the drug module.
  • the spacer unit can be "self-immolative” or “non-self-sacrificing.”
  • a “non-self-sacrificing" spacer unit refers to a spacer unit that remains bound to a drug moiety after enzymatic (proteolytic) cleavage of the ADC, in part or in whole of the spacer unit.
  • non-self-sacrificing spacer units include, but are not limited to, glycine spacer units and glycine-glycine spacer units.
  • peptide spacers susceptible to sequence-specific enzymatic cleavage are also contemplated.
  • enzymatic cleavage of a tumor cell-associated protease to an ADC containing a glycine-glycine spacer unit will result in the release of the glycine-glycine-drug module from the remainder of the ADC.
  • the glycine-glycine-drug module then performs a separate hydrolysis step in the tumor cells such that the glycine-glycine spacer unit is cleaved from the drug moiety.
  • the spacer unit of the linker comprises a p-aminobenzyl unit.
  • p-aminobenzyl alcohol is attached to the amino acid unit via an amide bond and a carbamate, methyl carbamate, or carbonate is formed between the benzyl alcohol and the cytotoxic agent. See, for example, Hamann et al. (2005) Expert Opin. Ther. Patents (2005) 15: 1087-1103.
  • the spacer unit is p-aminobenzyloxycarbonyl (PAB).
  • Exemplary joints in the present invention are as follows:
  • Linkers including extensions, spacers, and amino acid units, can be synthesized by methods known in the art, such as those described in US 2005-0238649 A1.
  • the antibody-cytotoxic drug conjugate, or a pharmaceutically acceptable salt or solvate thereof comprises an antibody of the invention conjugated to one or more maytansinoid molecules.
  • Maytansinoids are mitotic inhibitors that act by inhibiting tubulin multimerization. Maytansine was originally isolated from the East African shrub Maytenus serrata (U.S. Patent No. 3,896,111). It was subsequently discovered that certain microorganisms also produced maytansinoids such as maytansinol and C-3 maytansinol (U.S. Patent No. 4,151,042).
  • Maytansinoid drug modules are attractive drug modules in antibody-drug conjugates because they: (i) are relatively easy to prepare by chemical modification or derivatization of fermentation or fermentation products; (ii) are easy Derivatized with a functional group suitable for coupling to an antibody via a non-disulfide linker; (iii) stable in plasma; (iv) Effective against a variety of tumor cell lines.
  • Maytansine compounds suitable for use as maytansinoid drug modules are well known in the art and can be isolated from natural sources according to known methods or produced using genetic engineering techniques (see Yu et al. (2002) PNAS 99. :7968-7973). Maytanol and maytansinol analogs can also be prepared synthetically according to known methods.
  • Illustrative embodiments of the maytansinoid drug module include: DM1; DM3; and DM4, as disclosed herein.
  • the antibody-cytotoxic drug conjugate or a pharmaceutically acceptable salt or solvate thereof comprises a compound or derivative of dolastatin or dolastatin peptide (eg, auristatin) (eg, auristatin) U.S. Patent No. 5,635,483; 5,780,588) coupled to an antibody of the invention.
  • dolastatin and auristatin have been shown to interfere with microtubule dynamics, GTP hydrolysis, and nuclear and cell division (Woyke et al. (2001) Antimicrob. Agents and Chemother. 45(12): 3580-3584) and have anticancer (U.S. Patent No. 5,663,149) and antifungal activity (Pettit et al.
  • the dolastatin or auristatin drug moiety can be attached to the antibody via the N (amino) terminus or C (carboxyl) terminus of the peptide drug moiety (WO 02/088172).
  • An exemplary auristatin embodiment includes an N-terminally linked monomethyl auristatin drug module DE and DF, as disclosed in Senter et al, Proceedings of the American Association for Cancer Research, Vol. 45, Abstract No. 623, March 2004 On the 28th, the public content of the public content was clearly included in this article as a reference.
  • the peptide drug module can be selected from the following formulas D E and D F :
  • R 2 is selected from the group consisting of H and a C1-C8 hydrocarbon group
  • R 3 is selected from the group consisting of H, C1-C8 hydrocarbyl, C3-C8 carbocyclic, aryl, C1-C8 hydrocarbyl-aryl, C1-C8 hydrocarbyl-(C3-C8 carbocyclic), C3-C8 heterocyclic ring and C1-C8 Hydrocarbyl-(C3-C8 heterocycle);
  • R 4 is selected from the group consisting of H, C1-C8 hydrocarbyl, C3-C8 carbocyclic, aryl, C1-C8 hydrocarbyl-aryl, C1-C8 hydrocarbyl-(C3-C8 carbocyclic), C3-C8 heterocyclic ring and C1-C8 Hydrocarbyl-(C3-C8 heterocycle);
  • R 5 is selected from the group consisting of H and methyl
  • R 4 and R 5 together form a carbocyclic ring and have the formula -(CRaRb)n-, wherein R a and R b are independently selected from the group consisting of H, a C 1 -C 8 hydrocarbon group and a C 3 -C 8 carbon ring, and n is selected from 2 3, 4, 5 and 6;
  • R 6 is selected from the group consisting of H and a C1-C8 hydrocarbon group
  • R 7 is selected from the group consisting of H, C1-C8 hydrocarbyl, C3-C8 carbocyclic, aryl, C1-C8 hydrocarbyl-aryl, C1-C8 hydrocarbyl-(C3-C8 carbocyclic), C3-C8 heterocyclic ring and C1-C8 Hydrocarbyl-(C3-C8 heterocycle);
  • Each R 8 is independently selected from the group consisting of H, OH, a C1-C8 hydrocarbon group, a C3-C8 carbon ring, and an O-(C1-C8 hydrocarbon group);
  • R 9 is selected from the group consisting of H and a C1-C8 hydrocarbon group
  • R 10 is selected from an aryl group or a C3-C8 heterocyclic ring
  • Z is O, S, NH or NR 12 , wherein R 12 is a C1-C8 hydrocarbon group;
  • R 11 is selected from the group consisting of H, C1-C20 hydrocarbyl, aryl, C3-C8 heterocycle, -(R 13 O)mR 14 and -(R 13 O)m-CH(R 15 ) 2 ;
  • n is an integer selected from 1 to 1000;
  • R 13 is a C2-C8 hydrocarbon group
  • R 14 is H or a C1-C8 hydrocarbon group
  • R 15 is independently H, COOH, -(CH 2 )nN(R16) 2 , -(CH 2 )n-SO 3 H or -(CH 2 )n-SO 3 -C1-C8 hydrocarbon group;
  • Each of R 16 is independently H, C1-C8 hydrocarbyl or -(CH 2 ) n-COOH;
  • R 18 is selected from -C(R8) 2 -C(R8) 2 -aryl, -C(R8) 2 -C(R8) 2 -(C3-C8 heterocycle) and -C(R8) 2 -C ( R8) 2 - (C3-C8 carbon ring);
  • n is an integer selected from 0 to 6.
  • An exemplary auristatin of formula D E is MMAE, wherein the wavy line indicates a linker (L) covalently attached to the antibody-drug conjugate:
  • MMAF derivatives selected from the group consisting of wavy lines indicating covalent attachment to Antibody-drug conjugate linker (L):
  • hydrophilic groups may be at the R 11 attached to the drug moiety, the hydrophilic groups include, but are not limited to triethylene glycol (triethylene glycol ester, TEG), as described above. Without being limited to any particular theory, the hydrophilic groups contribute to the internalization and non-agglomeration of the drug moiety.
  • TEG triethylene glycol ester
  • An exemplary embodiment of a Formula I ADC comprising MMAE or MMAF and various linkers has the following structure and abbreviations (wherein “Ab” is an antibody; p is from 1 to about 8; “Val-Cit” is a proline- citramide Acid dipeptide; and “S” is a sulfur atom):
  • peptide-based drug modules can be prepared by forming peptide bonds between two or more amino acids and/or peptide fragments.
  • Such peptide bonds can be prepared, for example, by liquid phase synthesis methods well known in the art of peptide chemistry (see E. Schroder and K. Lübke, "The Peptides", Vol. 1, pp 76-136, 1965, Academic Press).
  • the auristatin/dorlastatin drug module can be prepared according to the methods in the following documents: US2005-0238649A1; U.S. Patent No. 5,356, 548; U.S. Patent No. 5,780,588; Pettit et al. (1989) J. Am. Chem. Soc.
  • the auristatin/dorlastatin drug moiety of the formula DF such as MMAF and its derivatives
  • MMAF auristatin/dorlastatin drug moiety of the formula DF
  • MMAE auristatin/dorlastatin drug moiety of the formula DE
  • MMAE auristatin/dorlastatin drug moiety of the formula DE
  • the drug-linker modules MC-MMAF, MC-MMAE, MC-vc-PAB-MMAF and MC-vc-PAB-MMAE can be conveniently synthesized by conventional methods, for example, Doronina et al. (2003) Nat. Biotech. 21: 778-784 And as described in U.S. Patent Application Publication No. US 2005/0238649 A1, which is then coupled to the antibody of interest.
  • Drug loading is represented by y, which is the average number of drug modules per antibody in the molecule of Formula I.
  • the drug load can range from 1-20 drug modules per antibody (D).
  • An ADC of Formula I includes a collection of antibodies conjugated to a range of (1-20) drug modules.
  • the average number of drug modules per antibody in the ADC preparation from the coupling reaction can be characterized by conventional means such as mass spectrometry, ELISA assays, and HPLC. It is also possible to determine the quantitative distribution of the ADC in terms of y. In some cases, separation, purification, and characterization of a homogenous ADC having a p value from another drug loaded can be accomplished by means such as reverse phase HPLC or electrophoresis.
  • y may be limited by the number of attachment sites on the antibody.
  • the attachment is a cysteine thiol, as in the exemplary embodiments above, the antibody may have only one or several cysteine thiol groups, or there may be only one or several of which are sufficiently reactive.
  • the thiol group can be attached to the linker.
  • a higher drug loading such as y > 5, can cause aggregation, insolubility, toxicity, or loss of cell permeability of certain antibody-drug conjugates.
  • the pharmaceutical loading of the ADC of the invention ranges from 1 to about 8; from about 2 to about 6; from about 3 to about 5; from about 3 to about 4; from about 3.1 to about 3.9; from about 3.2 to about 3.8. From about 3.2 to about 3.7; from about 3.2 to about 3.6; from about 3.3 to about 3.8; or from about 3.3 to about 3.7.
  • the optimal ratio for each antibody drug module has been shown to be less than 8, and may range from about 2 to about 5 for certain ADCs. See US2005-0238649A1 (completely incorporated herein by reference).
  • a drug moiety that is less than a theoretical maximum is coupled to the antibody in a coupling reaction.
  • An antibody can comprise, for example, a lysine residue that does not react with a drug-linker intermediate or a linker reagent, as discussed below. Only the most reactive lysine group can react with the amine reactive linker reagent.
  • antibodies do not contain many free and reactive cysteine thiol groups that can be attached to a drug moiety; in fact, most of the cysteine thiol groups in the antibody exist as disulfide bridges.
  • the antibody can be reduced with a reducing agent such as dithiothreitol (DTT) or tricarbonylethylphosphine (TCEP) under partial or complete reducing conditions to produce a reactive cysteine thiol group.
  • a reducing agent such as dithiothreitol (DTT) or tricarbonylethylphosphine (TCEP) under partial or complete reducing conditions to produce a reactive cysteine thiol group.
  • the antibody is placed under denaturing conditions to expose a reactive nucleophilic group, such as lysine or cysteine.
  • the loading of the ADC can be controlled in different ways, for example by (i) limiting the molar excess of the drug-linker intermediate or linker reagent relative to the antibody, and (ii) limiting the time or temperature of the coupling reaction. , (iii) a cysteine thiol modified moiety or a limiting reducing condition, (iv) engineering the amino acid sequence of the antibody by recombinant techniques such that the number and position of the cysteine residues are in order to control the linker-drug The number and/or position of the attachment is altered (such as thioMab or thioFab prepared as described herein and in WO2006/034488 (completely incorporated herein by reference)).
  • the resulting product is a mixture of ADC compounds having one or more drug modules attached to the antibody.
  • the average number of drugs per antibody can be calculated from the mixture by an antibody-specific and drug-specific dual ELISA antibody assay.
  • Various ADCs in the mixture Molecules can be identified by mass spectrometry and separated by HPLC, such as hydrophobic interaction chromatography. In certain embodiments, a homogenous ADC having a single loading value can be separated from the coupling mixture by electrophoresis or chromatography.
  • the ADC of Formula I can be prepared by several routes using organic chemical reactions, conditions, and reagents known to those skilled in the art, including: (1) The nucleophilic group of the antibody reacts with a bivalent linker reagent to form an Ab by a covalent bond. -L, then reacting with drug moiety D; and (2) the nucleophilic group of the drug moiety is covalently bonded to the bivalent linker reagent to form DL, followed by reaction with the nucleophilic group of the antibody.
  • An exemplary method of preparing an ADC of Formula I via the latter route is described in US 2005-0238649 A1, which is expressly incorporated herein by reference.
  • the nucleophilic group of the antibody includes, but is not limited to, (i) an N-terminal amine group; (ii) a side chain amine group such as lysine; (iii) a side chain thiol group such as cysteine; A glycosylation of a hydroxyl or amino group of a sugar in an antibody.
  • the amine, thiol, and hydroxyl groups are nucleophilic and are capable of reacting with an electrophilic group on the linker module to form a covalent bond
  • the linker reagents include: (i) an active ester such as an NHS ester, a HOBt ester, a halogenated Formates, and acid halides; (ii) hydrocarbyl and benzyl halides, such as haloacetamides; (iii) aldehydes, ketones, carboxyl groups, and maleimide groups.
  • Certain antibodies have a reducible interchain disulfide, a cysteine bridge.
  • the antibody can be completely or partially reduced by treatment with a reducing agent such as DTT (dithiothreitol) or tricarbonylethylphosphine (TCEP) to have reactivity coupled to the linker reagent.
  • a reducing agent such as DTT (dithiothreitol) or tricarbonylethylphosphine (TCEP) to have reactivity coupled to the linker reagent.
  • TCEP tricarbonylethylphosphine
  • TCEP tricarbonylethylphosphine
  • a sulfhydryl group can be introduced into the antibody via modification of a lysine residue, for example by reacting a lysine residue with 2-iminothiolane (Traut's reagent), resulting in the conversion of the amine to a thiol.
  • the antibody-drug conjugates of the invention can also be produced by reaction between an electrophilic group on the antibody, such as an aldehyde or a ketone carbonyl, and a nucleophilic group on a linker reagent or drug.
  • an electrophilic group on the antibody such as an aldehyde or a ketone carbonyl
  • a nucleophilic group on a linker reagent or drug include, but are not limited to, hydrazide, oxime, amino, hydrazine, thiosemicarbazone, hydrazine carboxylate. And arylhydrazide.
  • the sugar of the glycosylated antibody can be oxidized with, for example, a periodate oxidant to form an aldehyde or ketone group that can react with the amine group of the linker reagent or drug moiety.
  • the resulting imine Schiff base can form a stable linkage or can be reduced, for example, with a borohydride reagent to form a stable amine linkage.
  • the reaction of the carbohydrate moiety of the glycosylated antibody with galactose oxidase or sodium metaperiodate can form a carbonyl group (aldehyde group and keto group) in the antibody, which can be combined with a suitable drug group Reaction (Hermanson, Bioconjugate Techniques).
  • an antibody comprising an N-terminal serine or threonine residue can be reacted with sodium metaperiodate, resulting in the formation of an aldehyde at the first amino acid (Geoghegan and Stroh, (1992) Bioconjugate Chem. 3 :138-146; US5362852).
  • aldehydes can react with drug moieties or linker nucleophiles.
  • Nucleophilic groups on the drug moiety include, but are not limited to, amines, thiols, hydroxyl groups, hydrazide, hydrazine, hydrazine, thiosemicarbazone, hydrazine carboxylate, and aryl hydrazide groups, which are capable of interacting with linker modules Electrophilic group Covalent bonds should be formed, and the linker reagents include: (i) active esters such as NHS esters, HOBt esters, haloformates, and acid halides; (ii) hydrocarbyl groups and benzyl halides, such as halogenated Acetamide; (iii) aldehyde, ketone, carboxyl, and maleimide groups.
  • active esters such as NHS esters, HOBt esters, haloformates, and acid halides
  • hydrocarbyl groups and benzyl halides such as halogenated Acetamide
  • the compounds of the present invention expressly include, but are not limited to, ADCs prepared using the following crosslinking reagents: BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, sulfo-EMCS, sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC and sulfo-SMPB, and SVSB (succinimidyl-(4-vinyl sulfone) benzoate) are commercially available (For example, Pierce Biotechnology, Inc., Rockford, IL., USA, see 2003-2004 Applications Handbook and Catalog, pp. 467-498).
  • a plurality of bifunctional protein coupling agents can also be used to prepare antibody-cytotoxic drug conjugates comprising antibodies and cytotoxic agents, or pharmaceutically acceptable salts or solvate compounds thereof, such as N-succinimidyl 3-(2-pyridine Dithio)propionate (SPDP), succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), iminothiolane (IT) , imidate (such as dimethyl adipyl HCl), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), diazide compounds (such as Bis(p-azidobenzoyl)hexanediamine), double nitrogen derivatives (such as bis(p-diazobenzoyl)-ethylenediamine), diisothiocyanates (such as toluene 2,6-) A difunctional derivative of a diisocyanate), and a double active flu
  • a ricin immunotoxin can be prepared as described in Vitetta et al, Science 238: 1098 (1987).
  • Carbon-14-labeled 1-isothiocyanate benzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for coupling radionucleotides to antibodies. See WO94/11026.
  • a fusion protein comprising an antibody and a cytotoxic agent can be prepared by, for example, recombinant techniques or peptide synthesis.
  • the recombinant DNA molecule may comprise regions of the respective antibody and cytotoxic moiety encoding the conjugate, either adjacent to each other or separated by a region encoding a linker peptide that does not disrupt the desired properties of the conjugate.
  • an antibody in yet another embodiment, can be conjugated to a "receptor", such as streptavidin, for use in tumor pretargeting, wherein the antibody-receptor conjugate is administered to the patient, followed by the use of a scavenger The unbound conjugate is cleared in the circulation and then a "ligand” (e.g., avidin) coupled to a cytotoxic agent (e.g., a radionucleotide) is administered.
  • a ligand e.g., avidin
  • cytotoxic agent e.g., a radionucleotide
  • the antibody (light and heavy chain) used in the present invention is constructed by an overlap-extension PCR method well known in the art, and the DNA fragment obtained by overlap extension PCR is inserted into the expression vector pEE6.4 using HindIII/BstBI. Lonza Biologics) was expressed in 293F cells (Invitrogen, Cat# R790-07). The resulting recombinant protein was used for immunization or screening.
  • the c-Met gene template was obtained from origene (Cat. No. RC217003).
  • the cloned DNA sequence was as follows .
  • the experimental procedure was as follows: the antigen (human c-Met-His, Example 1) was diluted to 2 ⁇ g/ml with a coating solution (PBS) (Hyclone, Cat No.: SH30256.01B), and 100 ⁇ l/well was added to the 96-well enzyme label. Plates (Costar 9018, Cat No.: 0313024) were incubated overnight at 4 °C. The next day, the 96-well microtiter plate coated with the antigen was returned to room temperature, and the washing solution (PBS+0.05% Tween20 (Sigma, Cat No.: P1379) was washed three times. Then 200 ⁇ l/well blocking solution (PBS+1% BSA) was added.
  • PBS coating solution
  • a mouse-derived anti-human c-Met monoclonal cell strain is obtained by immunizing a mouse, spleen cell fusion, and hybridoma screening method. This method is well known in the art.
  • the recombinant expression antigen (human c-Met ECD-mFc, human c-Met Sema-flis, see Example 1) was diluted to 1 mg/ml with PBS (Hyclone, Cat No.: SH30256.01B), and Freund's adjuvant.
  • mice with serum titers higher than 1:10 5 after immunization were selected for cell fusion.
  • SIGMA polyethylene glycol
  • RPMI1640 containing hybridoma cell growth factor (Supplier: Roche, Catalogue #1363735001), serum (Supplier: GIBCO, Catalogue #C20270) and HAT (Supplier: Invitrogen, Catalogue #21060-017), per well 10 5 B cells were plated in standard, 100 ⁇ l per well, cultured in a 37 ° C cell culture incubator, and 3 days later, 100 ⁇ l of hybridoma-containing cell growth factor, serum and HT (Supplier: Invitrogen, Catalogue #11067-030) were added per well.
  • RPMI1640 after 2 to 4 days, 150 ⁇ l of RPMI1640 containing hybridoma cell growth factor, serum and HT was added to each well, and positive clones were detected by the next day ELISA (see Example 2). The results are shown in Table 1.
  • the above clones were selected and further cultured to obtain a monoclonal antibody. After the binding activity was confirmed by ELISA, the monoclonal culture supernatant was selected for cell activity detection.
  • the experimental principle is that the anti-human c-Met antibody of the present invention can inhibit the phosphorylation of c-Met on the surface of human gastric cancer cells (MKN45) and inhibit the proliferation of MNK45 cells.
  • Human gastric cancer cells (MKN45, JCRB, JCRB0254, P11) 1 ⁇ 10 5 cells / mL, 50 ⁇ l / well added to 96-well cell culture plates (costar, #3799), medium RPMI 1640medium: (GIBCO, cat#11835 +10% fetal bovine serum (FBS) (GIBCO-10099141). Subsequently, 50 ⁇ l/well of anti-human c-Met antibody to be tested was added, and culture was carried out for 5 days in a 37-degree incubator (manufacturer: SANYO device number TINC035).
  • % cell proliferation rate (1 - experimental group cell reading / no treatment group cell reading) x 100%. The results are shown in Table 2.
  • the single cell strain Ab-5 obtained in Example 4 was subjected to cDNA sequence cloning, and then the monoclonal antibody was recombinantly expressed and each activity was detected.
  • the present invention uses reverse transcription PCR to amplify the heavy and light chain variable regions of an antibody gene, and ligated into a vector to obtain a monoclonal antibody light heavy chain sequence.
  • the total cellular RNA of the single cell strain of the activity of Example 4 was first extracted using an RNA purification kit (Qiagen, Cat. No. 74134, see the specification).
  • the cDNA single strand, the Oligo-dT primers cDNA was then reverse transcribed using Invitrogen's Cat. No. 18080-051 cDNA Synthesis Kit.
  • the antibody light and heavy chain variable region sequences were synthesized by PCR, and the PCR product was cloned into the TA vector pMD-18T, and then sent for sequencing.
  • the obtained antibody light and heavy chain sequences were each cloned into an expression vector (see Example 1), and a recombinant monoclonal antibody was expressed to verify the activity (see Examples 2 and 4), followed by humanization.
  • the sequence of the mouse hybridoma cell monoclonal antibody Ab-5 of the present invention is the sequence of the mouse hybridoma cell monoclonal antibody Ab-5 of the present invention.
  • amino acid residues of the VH/VL CDRs of the anti-human c-Met antibody were determined and annotated by the Kabat numbering system.
  • the CDR sequences of the murine source of the present invention are as described in Table 3:
  • Example 5 After the homology comparison of the murine anti-c-Met monoclonal antibody light heavy chain sequence in the antibody database was carried out in Example 5, a humanized antibody model was established, and the optimal humanized anti-c- was selected according to the model selection of the back mutation. Met monoclonal antibodies are preferred molecules of the invention.
  • the method starts from a published mouse Fab crystal structure model database (such as the PDB database) to find a crystal structure with similar homology to the obtained murine candidate molecule, and picks high resolution (such as The Fab crystal structure was established to establish a mouse Fab model.
  • the sequence of the murine antibody light heavy chain of the present invention is aligned with the sequence in the model, and the sequence in the model which is identical to the murine antibody sequence is retained to obtain a structural model of the murine antibody of the present invention, wherein the inconsistent amino acid is a possible back mutation site.
  • the mouse antibody structural model was run using the Swiss-pdb viewer software to optimize energy (minimization).
  • the different amino acid sites other than the CDRs in the model were back-mutated, and the resulting mutant antibody (humanized) was compared with the antibody before humanization for activity detection. Humanized antibodies with good activity are retained. Thereafter, the CDR regions are optimized to include avoidance of glycosylation, deamidation, oxidation sites, and the like.
  • the CDR regions of the optimized humanized anti-c-Met antibody are shown in Table 4:
  • the light and heavy chain variable regions after humanization are as follows:
  • the light heavy chain and the IgG Fc segment after humanization are recombined to obtain the humanized anti-c-Met monoclonal antibody of the present invention.
  • the Fc sequence used is selected from the following sequences:
  • the binding activity of the anti-c-Met humanized body of the present invention to the c-Met high expression cell line MKN45 was examined by the FACS method.
  • RPMI 1640 medium (GIBCO) with 10% (v/v) fetal bovine serum (FBS) (GIBCO, Cat No.: 10099-141) and penicillin/streptomycin (GIBCO, Cat No.: 15070-063) , Cat No.: 11835-030) medium suspension MKN45 cells (JCRB, Cat No.: JCRB0254) to 10,000,000 cells/mL. 2 mL of resuspended MKN45 cells were added to a 96-well microtiter plate (Corning, Cat No.: 3799) at 150,000 cells/well, and 8 concentrations (20 ⁇ g/ml of 5-fold concentration gradient dilution) of c-Met antibody were added.
  • the final volume was 100 ⁇ l and incubated for 1 hour at 4 degrees.
  • FACS buffer (2.5% (v/v) fetal bovine serum (FBS) in phosphate buffered saline (PBS) (Hyclone, Cat: SH30256.01B), 4 degrees, 1300 rpm, 4 minutes, discard the supernatant, Repeat three times.
  • secondary antibody solution per well (fluorescent labeled goat anti-mouse secondary antibody: 1:200 dilution, Biolegend, Cat#405307; fluorescently labeled anti-human secondary antibody: 1:30 dilution, Biolegend, Cat#409304), at Incubate for 4 hours at 4 degrees.
  • the present invention uses surface plasmon resonance (SPR) to detect the affinity between the c-Met antibody and the c-Met antigen Sema-His.
  • SPR surface plasmon resonance
  • Anti-mouse IgG (GE Life Sciences catalog #BR-1008-38) or anti-human IgG (GE Life Sciences catalog #BR-1008-39) antibody with pH 5.0 sodium acetate solution (GE Healthcare, Cat#BR-1003 -51) Dilution to 30 ⁇ g/ml and 50 ⁇ g/ml, respectively, and immobilization to CM5 chip (GE Life Sciences catalog #BR-1000-12) using an amino coupling kit (GE Life Sciences, Cat#BR100050)
  • the channel, coupling level is set at 15000 RU.
  • Run buffer PBS (Hyclone, Cat#SH30256.01B) + 0.05% P20 (GE Life Sciences, Cat#BR-1000-54)
  • Dilute the c-Met antibody to 1.5 ⁇ g/ml dilute the antigen sema-his to 200 nM in running buffer, and then dilute 1:2 fold to 0.78 nM with the same buffer.
  • the diluted antibody was flowed through the experimental channel for one minute at a flow rate of 30 ⁇ l/min.
  • the antigen was passed through the experimental channel and the comparison channel for 3 minutes at the same flow rate, and the flow rate was adjusted to 10 ⁇ l/min after dissociation for 10 minutes, in the experimental channel and
  • the contrast channel was flowed through the regeneration buffer for 3 minutes.
  • the data was double-deducted and fitted with BiaEvaluation 4.1, and the fitted model was modeled using the 1:1 (Langmuir) model.
  • the above results indicate that the binding activity of the humanized antibody of the present invention and the antigen is 0.13-8 nM, and the detection results are different depending on the detection method.
  • the results indicate that the humanized anti-c-Met antibody retains the binding activity of the antibody prior to humanization.
  • Example 8 In vitro function, cell activity evaluation of anti-c-Met humanized antibody
  • Example 7 In order to examine the function of the antibody of the present invention, the antibody of Example 7 was evaluated by binding experiments blocking c-Met ligand (hepatocyte growth factor HGF) and c-Met, and inhibition of cell proliferation experiments (Example 4). .
  • c-Met ligand hepatocyte growth factor HGF
  • c-Met hepatocyte growth factor HGF
  • Binding of HGF to c-Met results in tyrosine phosphorylation of the c-Met molecule and activation of the c-Met signaling pathway.
  • the anti-c-Met antibody of the present invention was blocked by ELISA to block the activity of HGF binding on the receptor c-Met protein, i.e., IC 50 .
  • c-Met ECD-mFc (Example 1) was diluted in PBS (Hyclone, Cat# SH30256.01B) to a final concentration of 2 ⁇ g/ml, and coated in a 96-well ELISA plate (Costar, cat #2592) at room temperature overnight. Washing was performed 3 times with PBST (PBS + 0.05% Tween 20 (Simga, Cat# P1379)) using a plate washer (Suppler: BioTex; Model: ELX405; S/N: 251504), 300 ⁇ l of blocking solution PBS + 1% BSA (Roche, Cat#738328) was added to a 96-well plate for 60 minutes at 37 °C.
  • PBS Hyclone, Cat# SH30256.01B
  • the humanized antibody of the present invention not only retains the binding activity with the antigen, but also prevents the binding of the antigen and the ligand, and at the same time exhibits inhibition of the growth activity of the tumor cells.
  • Anti-c-Met antibodies block HGF/c-Met binding and may also activate c-Met signaling, i.e., have agonist activity. Anti-c-Met agonist activity is not required for the present invention.
  • three experiments including c-Met phosphorylation, human renal clear cell carcinoma skin metastasis cell (caki-1), and human lung cancer H441 cell migration were evaluated.
  • HGF tyrosine phosphorylation of the c-Met molecule and activates the c-Met signaling pathway. Therefore, HGF activated c-Met as a positive control for agonist experiments, and human lung cancer cell line A459 was used to evaluate the effect of inducing phosphorylation at c-Met tyrosine residue 1349.
  • A549cells were suspended in Ham's F12K + 2 mM glutamine (Invitrogen, #21127-022) + 10% (v/v) fetal bovine serum (FBS) (GIBCO, #10099141), and 0.2 mL of cell suspension was added to 96 wells. Plate (Corning, #3599), cell concentration was 60,000 cells/well. Incubate for 24 hours at 37 ° C, 5% CO 2 . After 24 hours, 96-well medium was discarded, and 100 ⁇ L of low serum medium (Ham's F12K + 2 mM glutamine + 0.5% FBS) was added at 37 ° C under 5% CO 2 for 6 hours.
  • FBS fetal bovine serum
  • the antibody was diluted with the above low serum medium (final concentration 20 ⁇ g/ml).
  • the concentration of the positive control HGF was 200 ng/ml.
  • Cocktail II Sigma #P5726
  • the phosphatase inhibitor cocktail III Sigma #P0044. After cell lysis, c-Met tyrosine phosphorylation was detected by ELISA.
  • the c-Met capture antibody (CST, cat #3148s) was diluted 1:1000 with PBS and added to a 96-well ELISA plate (costar, cat #9018), 100 ⁇ l per well, and incubated overnight at 4 degrees. After washing 3 times with TBS-T, 300 ⁇ l of blocking solution (TBS-T plus 2% (w/v) BSA) was added for 1 hour. After washing 3 times with TBS-T, 75 ⁇ L of cell blocking solution was added, 25 ⁇ l of cell lysate was added, and incubation was carried out overnight at 4 degrees.
  • the cells were washed 3 times with TBS-T, and pY1349c-Met antibody (cell signal, #3133) was diluted 1:1000 with blocking solution, 100 ⁇ l per well. After incubation for 2 hours at room temperature, TBST was washed 4 times, and HRP-labeled goat anti-rabbit polyclonal antibody (cell signaling, cat #7074) was diluted with blocking solution at 1:12000, and 100 ⁇ l per well was incubated for 1 hour at room temperature.
  • TBS-T was washed 5 times, 100 ⁇ L of TMB (ebioscience #TMB, 004201) was added to each well, and 100 ⁇ l of stop solution (2N H 2 SO 4 ) was added, using a 450 nM microplate reader (Supplier: Moleculer Devices; Model: MNR0643) ; Equip ID: TMRP001) reading. Data analysis was performed using SoftMax Pro v5. The results are shown in Table 8.
  • Human renal clear cell carcinoma skin (Caki-1) cells express hepatocyte growth factor receptor (c-Met), and HGF can bind C-Met to stimulate Caki-1 cell proliferation. Therefore, the humanized anti-c-Met antibody of the present invention and HGF are parallelized out of Caki-1 cells, and the agonist activity of the anti-c-Met antibody can be evaluated.
  • Caki-1 Human renal clear cell carcinoma skin
  • HGF hepatocyte growth factor receptor
  • Caki-1 (Shanghai Chinese Academy of Sciences, TCHu135, P12) cells 1000/well were added to 96-well cell culture plates (costar, #3799), medium was McCoy's 5A (invitrogen, #16600) + 10% fetal bovine serum (FBS) (GIBCO-10099141), 37 ° C, 24 hours. The cells were then starved for 24 hours (cell starvation medium was McCoy's 5A + 0.5% fetal bovine serum).
  • Anti-c-Met antibodies can affect the ability of cells to migrate if they have agonist activity.
  • the present invention uses the H441 cell line expressing c-Met to evaluate the ability of the c-Met antibody of the present invention to affect cell migration.
  • RPMI 1640 medium (GIBCO) with 10% (v/v) fetal bovine serum (FBS) (GIBCO, Cat No.: 10099-141) and penicillin/streptomycin (GIBCO, Cat No.: 15070-063) , Cat No.: 11835-030)
  • FBS fetal bovine serum
  • penicillin/streptomycin (GIBCO, Cat No.: 15070-063)
  • GBCO penicillin/streptomycin
  • 11835-030 Medium suspension H441 cells (ATCC, Cat No.: HTB-174) to 500,000 cells/ml.
  • the resuspended H441 cells were added to a 12-well culture plate (Costar, Cat No.: 3513) at 1 ml/well, cultured at 37 ° C, 5% CO 2 for 3 days, and washed twice with phosphate buffered saline (PBS).
  • PBS phosphate buffered saline
  • the cells were added to RPMI 1640 medium containing low concentrations of fetal bovine serum (0.5% FBS), and cultured at 37 ° C, 5% CO 2 for 16 hours. Scratch the bottom of each well with a 5 ml pipette tip, and wash it with low concentration fetal bovine serum medium, add 1 ml of low concentration serum medium RPMI 1640, and randomly select the scratched area under a 4x inverted microscope. The plate was labeled, at this time as time zero, and the cells were treated with 10 ⁇ g/ml c-Met antibody or HGF control (200 ng/ml) (37 ° C, 5% CO 2 ) for 16 hours, then inverted 4 times.
  • the marked scratched area was photographed and stored under a microscope, and this time was recorded as the post-migration time.
  • the percentage of affected cell migration is the migration distance relative to zero divided by the migration distance of the medium group relative to zero multiplied by 100. The results are shown in Table 8.
  • a human gastric cancer MKN45 cell model was subcutaneously transplanted with BALB/c nude mice for detection.
  • MKN45 cells were cultured in monolayer with RPMI-1640 medium (10% fetal calf serum) under the conditions of 37 ° C, 5% CO 2 . Count in the logarithmic growth phase and collect cells. The cells were resuspended in PBS to the appropriate concentration in mice (BALB/c Nude mice, female, 10 weeks, body weight 22-28 g. purchased from Shanghai Slack Laboratory Animal Co., Ltd., animal certificate number: 2007000548777. Feeding environment : SPF grade.) The right wing was subcutaneously inoculated with 0.1 ml of 3 x 10 6 cells. When the average tumor volume reached 114 mm 3 , the body weight was weighed, the tumor volume was measured, grouped, and administration was started.
  • RPMI-1640 medium 10% fetal calf serum
  • the control group was administered with PBS, and the antibody-treated group was given 5 mg/kg of the antibody of the present invention once a week for two times. Tumor volume and body weight were measured twice a week and the trial was terminated on day 25.
  • Tumor size calculation formula: tumor volume (mm 3 ) 0.5 ⁇ (tumor long diameter ⁇ tumor short diameter 2).
  • the tumor inhibition rate calculation formula: tumor inhibition rate (V0-VT) / V0 ⁇ 100%, wherein V 0 , V T are the tumor volume at the beginning of the experiment and at the end of the experiment, respectively.
  • the antibody of the present invention binds to human c-Met, has very good in vitro activity, and inhibits tumor activity in vivo. Furthermore, the antibody has no or very weak agonist activity.
  • human gastric cancer cell MKN45 JCRB, Cat No.: JCRB0254
  • RPMI 1640 medium (GIBCO) with 10% (v/v) fetal bovine serum (FBS) (GIBCO, Cat No.: 10099-141) and penicillin/streptomycin (GIBCO, Cat No.: 15070-063) , Cat No.: 11835-030) medium suspension MKN45 cells to 10,000,000 cells/mL.
  • 2 mL of resuspended MKN45 cells were added to 96-well microtiter plates at 250,000 cells/well, and 10 ⁇ g/ml of c-Met antibody was added to the corresponding wells to a final volume of 100 ⁇ l, and incubated at 4 degrees for 1 hour.
  • FACS buffer phosphate buffer (Hyclone, Cat: SH30256.01B) with 2.5% fetal bovine serum, 4 ° C, 1300 rpm, 4 minutes, discard the supernatant, and repeat three times.
  • FACS buffer phosphate buffer (Hyclone, Cat: SH30256.01B) with 2.5% fetal bovine serum, 4 ° C, 1300 rpm, 4 minutes, discard the supernatant, and repeat three times.
  • secondary antibody solution per well Fluorescently labeled goat anti-mouse secondary antibody: 1:200 dilution, Biolegend, Cat #405307; fluorescently labeled anti-human secondary antibody: 1:30 dilution, Biolegend, Cat #409304
  • FACS buffer The supernatant was centrifuged at 4 ° C, 1300 rpm for 4 minutes, and repeated three times.
  • Percentage of endocytosis of c-Met antibody (fluorescence intensity value at each time point - average fluorescence intensity value at zero point) / average fluorescence intensity value at zero point.
  • the results in Table 9 indicate that the antibody of the present invention has a good endocytosis in addition to agonist activity. After binding to the target cells, the antibody and the receptor are rapidly endocytosed into the target cells, and the endocytosis reaches a maximum within 2-4 hours.
  • Samples were directly LC-MS tested for light weight chain molecular weight analysis for glycosylation. Deamination was analyzed by LC-MS at 4 ° C for a long time (over 3 months), or at 40 ° C under 21 days of accelerated conditions. After the samples were treated under different conditions, the samples were taken out, and the samples were diluted to 2 mg/ml with pH 7.2 Tris-HCl, and added to a final concentration of 10 mM TCEP and 6 M urea (AMRESCO, Cat# 0378) for 3 min at 30 ° C. The final concentration was 20 mM IAA.
  • BiopharmaLynx was used to analyze the presence or absence of deamidation.
  • the obtained mass spectrometry data is obtained by finding the native peptide (native peptide) and the modified product containing the deamidation site, extracting the parent ion to obtain an EIC (Extracted Ion Chromatogram) image, integrating the peak area and calculating the deamidation and oxidation products. proportion.
  • EIC Extra Ion Chromatogram
  • the anti-c-Met antibody of the invention has the properties of receptor binding preventing activity, no agonist activity, endocytic activity of target cells and physical stability, and these characteristics make the antibody of the invention particularly suitable for coupling with toxins into ADC drugs for c -Met expresses cancer treatment.
  • the coupling process is shown in the figure below:
  • 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 Ab-10 monoclonal antibody-propanethiol solution 1c (6.17 mg/) was added. After shaking for 4 hours at 25 ° C in mL, 3.0 mL), the reaction solution was desalted and purified on a Sephadex G25 gel column (elution phase: 0.05 M in PBS containing pH 6.5) under sterile conditions. The title product ADC-1 in PBS buffer (3.7 mg/mL, 4.7 mL) was filtered through a 0.2 ⁇ m filter and stored at 4 ° C.
  • Example 14 anti-c-Met antibody Ab-10 coupled toxin MC-VC-PAB-MMAE
  • the compound MC-VC-PAB-MMAE (1.6 mg, 1.2 ⁇ mol, prepared by the method disclosed in PCT Patent WO2004010957) was dissolved in 0.3 mL of acetonitrile, and Ab-10 monoclonal antibody-propanethiol solution 1c (6.17 mg/) was added. After shaking for 4 hours at 25 ° C in mL, 3.0 mL), the reaction solution was desalted and purified on a Sephadex G25 gel column (elution phase: 0.05 M in PBS containing pH 6.5) under sterile conditions. The title product ADC-2 in PBS buffer (3.6 mg/mL, 4.8 mL) was filtered through a 0.2 ⁇ m filter and stored at 4 ° C.
  • the liquid (10.85 mg/ml, 9.0 mL, 0.976 mmol) was added to the above prepared acetonitrile solution of S-(3-carbonylpropyl)thioacetate, and then 1.0 mL of sodium cyanoborohydride (14.1 mg, 224 ⁇ mol) was added dropwise.
  • the aqueous solution was shaken at 25 ° C for 2 hours.
  • the compound MC-MMAE (1.1 mg, 1.2 ⁇ mol) was dissolved in 0.3 mL of acetonitrile, and added to Ab-9 monoclonal antibody-propanol solution 5c (6.2 mg/mL, 3.0 mL) at 25 ° C. After shaking for 4 hours, the reaction solution was subjected to desalting purification on a Sephadex G25 gel column (elution phase: 0.05 M in PBS containing pH 6.5), and filtered under a sterile condition through a 0.2 ⁇ m filter to obtain the title product ADC- 5 PBS buffer (3.8 mg/mL, 4.6 mL) was stored frozen at 4 °C.
  • the compound MC-VC-PAB-MMAF (1.6 mg, 1.2 ⁇ mol) was dissolved in 0.3 mL of acetonitrile, plus The mixture was shaken into an Ab-9 monoclonal antibody-propanol solution 5c (6.2 mg/mL, 3.0 mL) at 25 ° C for 4 hours, and then the reaction solution was desalted by Sephadex G25 gel column (elution phase: pH).
  • the title product ADC-7 in PBS buffer (3.8 mg/mL, 4.6 mL) was filtered through a 0.2 ⁇ m filter under sterile conditions, and was stored at 4 ° C.
  • the compound MC-VC-PAB-MMAE (1.6 mg, 1.2 ⁇ mol) was dissolved in 0.3 mL of acetonitrile, and added to Ab-9 monoclonal antibody-propanol solution 5c (6.2 mg/mL, 3.0 mL) at 25 ° C. After shaking for 4 hours, the reaction solution was subjected to desalting purification on a Sephadex G25 gel column (elution phase: 0.05 M in PBS containing pH 6.5), and filtered under a sterile condition through a 0.2 ⁇ m filter to obtain the title product ADC. -8 PBS buffer (3.8 mg/mL, 4.6 mL) was stored frozen at 4 °C.
  • the title product 9b solution was obtained as a 6.5 0.05 M PBS solution, and concentrated to about 10 mg/ml (8.3 mg/ml, 11 ml).
  • the compound MC-VC-PAB-SN-38 (1.3 mg, 1.2 ⁇ mol) was dissolved in 0.3 mL of acetonitrile, and added to Ab-9 monoclonal antibody-propanol solution 5c (6.2 mg/mL, 3.0 mL) at 25 After shaking for 4 hours at ° C, the reaction solution was subjected to desalting purification on a Sephadex G25 gel column (elution phase: 0.05 M PBS solution having a pH of 6.5), and filtered under a sterile condition through a 0.2 ⁇ m filter to obtain a title.
  • the product ADC-11 in PBS buffer (3.7 mg/mL, 4.5 mL) was stored frozen at 4 °C.
  • the starting material ((S)-2-amino-3-(2-fluorophenyl)propionic acid 12a (400 mg, 2.18 mmol) was prepared by a known method "Advanced Synthesis & Catalysis, 2012, 354 (17), 3327-3332". It is dissolved in 10 Ml of tert-butyl acetate, added with perchloric acid (300 mg (70%), 3.3 mmol), and stirred at room temperature for 16 hours. After the reaction is completed, 6 Ml of water is added, and the organic phase is saturated with sodium hydrogencarbonate. (5Ml) Washing.
  • reaction mixture was concentrated under reduced pressure, and then diluted with 5 Ml of dichloromethane.
  • the system was layered and the aqueous layer was extracted with dichloromethane (5Ml ⁇ 3).
  • dichloromethane layers were washed with a saturated sodium chloride solution (10 mL) and dried over anhydrous sodium sulfate.
  • N, N', N'-tetramethylurea hexafluorophosphate (84 mg, 0.22 mmol).
  • the reaction system was stirred at room temperature for 1 hour under an argon atmosphere. After the reaction was completed, 10 Ml of water was added and stirred, and the layers were separated. The dichloromethane layer was washed with a saturated sodium chloride solution (10 mL) and dried over anhydrous sodium sulfate. Filtration and concentration of the filtrate under reduced pressure.
  • Toxin intermediates and toxins of the ADC molecules of the invention or other endocytosis c-Met antibody-toxin conjugates eg, LY-2875358-ADC
  • stable toxins in PBS human and monkey plasma sexual evaluation.
  • the toxin intermediates and toxins of the example compounds ADC-1 and ADC-12 were diluted with PBS, human or monkey plasma (Suzhou Xishan Zhongke Pharmaceutical Research and Development Co., Ltd., animal production license number: SCXK (Su) 2012-0009) Incubation to 500 ⁇ g/mL, 7 days at 37 ° C, samples were taken at 0, 3, 7 days to determine the concentration of free toxins and toxin intermediates in the samples.
  • Standard curve analysis method is 50 ⁇ L blank human, monkey plasma or PBS sample, add 50 ⁇ L series working solution, add 20 ⁇ L internal control (camptothecin, 100ng/ml), add 100 ⁇ L acetonitrile, vortex and mix for 3min, 15000rpm, centrifuge for 10min, After 80 ⁇ L of the supernatant was mixed with 80 ⁇ L of 0.2% formic acid, 10 ⁇ L of the injection was carried out.
  • the measured value is the percentage of free toxin contained in the sample. 0.01-0.19% were within the range of detection background values; ND: Not detectable, failed to detect.
  • ND Not detectable, failed to detect.
  • ADC-1, ADC-12 of the present invention was evaluated by FACS (detection and binding of c-Met positive cells) and endocytosis (method see Example 11). The results are shown in Table 13.
  • Test Example 3 anti-c-Met antibody toxin coupling (ADC) molecular cytotoxicity experiment
  • ATP is an indicator of the metabolism of living cells.
  • the detection of ATP can reflect the toxicity of molecules to cells.
  • HepG2 cells (Chinese Academy of Sciences Cell Bank, Cat#TCHu72) were cultured in EMEM complete medium containing 10% FBS, and MKN45 cells were cultured in RPMI1640 complete medium containing 10% FBS, and 2-3 ml trypsin was added to the experiment. 3 min, until the cells were completely digested, the digested cells were eluted by adding 10-15 ml of complete medium, centrifuged at 1000 rpm for 3 min, the supernatant was discarded, and then the cells were resuspended by adding 10-20 ml of complete medium to prepare a single cell suspension. Adjust the cell density to 4 ⁇ 10 4 cells/ml.
  • Example 13 compound and toxin The sample to be tested (Example 13 compound and toxin) was diluted with PBS to different concentration gradients in 2% FBS RPMI1640 medium, 10 ⁇ l per well, and incubated in a 37 ° C 5% CO 2 incubator for 72 hours. use The Luminescent Cell Viability Assay Kit (Promega, Cat# G7571) was tested according to the instructions. Chemiluminescence was detected using a microplate reader (VICTOR 3, PerkinElmer), and data analysis was performed using GraphPad Prism (version 5.0) software. The results will be shown in Table 14.
  • the ADC-1 and ADC-12 of the present invention have no cytotoxic effect on c-Met-negative cells HepG2, indicating that the ADC compound has a specific targeting effect.
  • the cytotoxic effects of the respective toxin fractions on c-Met-negative cells HepG2 were different, and the difference was 82-fold (400.8/4.88).
  • ADC-1 and ADC-12 of the present invention have a specific targeting effect and can inhibit the proliferation of c-Met positive cells, but have no toxic effects on non-specific (normal cells).
  • ADC-1, ADC-12 differ in that the respective free toxins are different in toxicity to both targeted and non-targeted cells.
  • the cytotoxicity of the toxin portion of ADC-12 against c-Met positive cells and negative cells HepG2 was 93, 82 times weaker than that of ADC-1. Therefore, when the molecule reaches the target cell, if the free toxin is released, its non-specific toxic effect is weaker than that of ADC-1. Therefore, the side effects are small and the safety is good.
  • Test Example 4 Inhibition of tumor cell proliferation by anti-c-Met antibody toxin coupled (ADC) molecule
  • ADC-1 (Example 13) is capable of specifically killing tumor target cells expressed by c-Met.
  • a variety of tumor cells were detected using the molecules of the present invention, and the inhibition of cell proliferation by the samples was tested by the CCK method, and the in vitro cell viability of the ADC molecules of the present invention was evaluated according to the IC 50 size.
  • the cells used and the corresponding medium are shown in Table 15 below, and cell proliferation was measured using Cell Counting Kit (Cat# CK04) (operating according to the instructions).
  • the samples were diluted with PBS to different concentration gradients, 10 ⁇ l per well, and incubated for 72 hours in a 37 ° C 5% CO 2 incubator. 10 ⁇ l of CCK8 was added to each well, incubation was continued for 2 hours in the incubator, and OD450 was detected by a microplate reader (VICTOR 3, PerkinElmer), and data analysis was performed using GraphPad Prism (version 5.0) software. The results are shown in Table 16.
  • the results in Table 16 indicate that the anti-c-Met antibody of the present invention has a relatively good activity on the gastric cancer cell line MKN45, SUN, but is weak on other tumor cells having low or no expression of c-Met, such as lung cancer cells, or no activity.
  • the ADC-1 of the present invention has extra toxins, and has no effect on the c-Met-expressing tumor cells, including the gastric cancer cell line MKN45, SUN, and particularly anti-c-Met antibodies, and the lung cancer, pancreatic cancer and It shows good activity on renal cell carcinoma cells.
  • Test Example 5 Evaluation of in vivo efficacy of anti-c-Met antibody toxin coupling (ADC) molecule
  • Example 10 In order to better evaluate the antitumor pharmacodynamic activity of the anti-c-Met antibody and ADC molecule of the present invention, the antibodies Ab-10 and ADC-1 were subjected to parallel comparison experiments by the method of Example 10. In contrast to Example 10, this test example was a single administration, and tumor inhibition was observed until the tendency to resume after the recovery.
  • ADC-1 (2.5mg/kg), stock solution (10mg/ml) was formulated with PBS to a final concentration of 0.25mg/ml;
  • ADC-1 (5mg/kg), stock solution (10mg/ml) was formulated into a final concentration of 0.5mg/ml with PBS;
  • ADC-1 (10mg/kg), stock solution (10mg/ml) was formulated with PBS to a final concentration of 1mg/ml;
  • MKN-45 cells (1 ⁇ 10 6 /piece) were inoculated subcutaneously into the right flank of nude mice, and the tumors were grown to an average volume (150.19+8.44) mm 3 and administered in groups of 8 rats. See Table 17 for specific dosing schedules.
  • the tumor volume was measured twice a week, the body weight was weighed, and data was recorded.
  • V 1/2 ⁇ L length ⁇ L short 2
  • Tumor inhibition rate (V 0 - V T ) / V 0 * 100%
  • V 0 and V T are the tumor volume at the beginning of the experiment and at the end of the experiment, respectively.
  • the antibody and ADC compound of the invention have obvious curative effect on transplanted tumor of MKN-45 nude mice.
  • ADC-1 and ADC-12 were compared in parallel using the same test method described above.
  • Inhibition rate(%) 11 days 15 days 18 days 21 days ADC-1 42.8 44.7 35.4 27.1 ADC-12 44.6 54.5 50.5 50.4
  • ADC-1 and ADC-12 have similar tumor inhibition rates at 11 days, but after 15 days, the efficacy of ADC-1 is weakened (27.1% at 21 days), while the inhibitory effect of ADC-12 remains Level of day 11 (50.4%).
  • Test Example 6 Effect of ADC-12 on human lung cancer NCI-H1993 subcutaneous xenograft in nude mice
  • ADC-12 was dissolved in 20mg/ml solution with water for injection, and stored in a refrigerator at -80 °C. It was diluted with 0.1% BSA physiological saline to the corresponding concentration. The concentration of Ab-10 antibody was 16.3 mg/ml, and it was physiologically treated with 0.1% BSA. After dilution with brine, store in a refrigerator at -80 °C.
  • mice were subcutaneously inoculated with human lung cancer NCI-H1993 cells, and after the tumors were grown to 100-150 mm 3 , the animals were randomly grouped (D0).
  • the dosing dosage and dosing schedule are shown in Table 19.
  • the tumor volume was measured 2-3 times a week, the rats were weighed, and the data were recorded.
  • the tumor volume (V) is calculated as:
  • V 1/2 ⁇ a ⁇ b 2
  • a and b represent length and width, respectively.
  • T/C (%) (TT 0 ) / (CC 0 ) ⁇ 100 where T and C are the tumor volumes at the end of the experiment; T 0 and C 0 are the tumor volumes at the start of the experiment.
  • ADC-12 is an anti-c-Met antibody-toxin conjugate.
  • ADC-12 (1, 3, 10 mg/kg, IV, D0) dose-dependently inhibited the growth of subcutaneous xenografts in nude mice with high expression of c-Met human lung cancer NCI-H1993, with tumor inhibition rates of 45% and 63%, respectively.
  • 124%, 10mg/kg dose group had 7/10 tumor partial regression (D21);
  • Ab-10 antibody stock solution was prepared for naked antibody of ADC-12, Ab-10 antibody stock solution (30mg/kg, IV, twice a week) 6 times)
  • the tumor inhibition rate of NCI-H1993 was 42%; the tumor-bearing mice were well tolerated by the above drugs, and no symptoms such as weight loss occurred.
  • ADC-12 was significantly more effective against NCI-H1993 than Ab-10 antibody stock.
  • ADC-12 (1, 3, 10 mg/kg, IV, D0) dose-dependently inhibited the growth of subcutaneous xenografts in nude mice with high expression of c-Met human lung cancer NCI-H1993, causing partial regression of tumors; Ab-10
  • the antibody stock solution (30 mg/kg, IV, 2 times a week for 6 times) was also effective against NCI-H1993; ADC-12 was significantly more effective against NCI-H1993 than the Ab-10 antibody stock solution. Tumor-bearing mice are well tolerated by the above drugs.

Abstract

提供了抗c-Met的抗体或抗原结合片段，及抗c-Met抗体-细胞毒性药物偶联物，所述抗体或抗原结合片段为嵌合抗体或人源化抗体，还提供了包含人源化抗c-Met抗体、抗原结合片段，抗体-细胞毒性药物偶联物或其可药用盐或溶剂化合物的药物组合物，可用于治疗癌症。

Description

抗c-Met抗体和抗c-Met抗体-细胞毒性药物偶联物及其医药用途 技术领域
本发明公开了一种抗c-Met抗体或其抗原结合片段，包含所述抗c-Met抗体CDR区的嵌合抗体、人源化抗体，及抗c-Met抗体-细胞毒性药物偶联物或其可药用盐或溶剂化合物，以及包含人c-Met抗体或其抗原结合片段及其抗体-细胞毒性药物偶联物或其可药用盐或溶剂化合物的药物组合物，以及其作为抗癌药物的用途。尤其涉及一种人源化的抗c-Met抗体及抗c-Met抗体-细胞毒性药物偶联物或其可药用盐或溶剂化合物，在制备用于治疗c-Met介导的疾病或病症的药物中的用途。
背景技术
近年来分子生物学和肿瘤药理学研究表明，酪氨酸激酶(Protein Tyrosine Kinases,PTKs)相关的细胞信号转导通路在肿瘤的形成和发展中发挥了极其重要的作用，超过50％的原癌基因和癌基因产物都具有酪氨酸激酶活性。c-Met原癌基因属于PTKs家族中Ron亚族，其编码的c-Met蛋白是肝细胞生长因子/离散因子(Hepatocyte Growth Factor/Scatter Factor,HGF/SF)的高亲和性受体。HGF/c-Met信号通路与血管新生和肿瘤生长过程密切相关，其持续激活是组织细胞癌变或癌细胞增殖亢进的重要原因，抑制该通路已成为肿瘤靶向治疗的新手段。
c-Met原癌基因位于人类第7号染色体长臂(7q31)，大小超过120kb，编码分子量约150kD的c-Met蛋白前体，经局部糖基化生成一个170kD的糖蛋白，该糖蛋白进一步剪切为α亚基(50kDa)和β亚基(140kDa)，以二硫键相连，形成成熟的c-Met蛋白受体。该异二聚体包含两条链，β链有胞外区、跨膜区(也称膜伸展片段)和胞内区(包含细胞内酪氨酸激酶结合位点)。α链只有胞外部分，但它是高度糖基化，通过二硫键附着于β链上。两个亚基的胞外区域是相应配体的识别部位，胞内区域具有酪氨酸激酶活性。
c-Met激活的机制分为三种：一是依赖HGF的激活机制，二是不依赖HGF激活机制，三是经过其他膜途径，例如通过透明质酸膜表面受体的CD44、粘附素以及RON信号传导途径等等。其中最常见的是依赖HGF的激活机制。HGF的N末端与c-Met结合，促进β链上Tyr1234和Tyr1235二聚化和自磷酸化，C-末端附近的Tyr1349和Tyr1356磷酸化产生多个接头蛋白的结合位点，这些接头蛋白诱导了P13K/Akt、Ras/Mapk、c-Src和STAT3/5介导的下游信号的激活，引发不同细胞反应，如细胞生存和活动(与P13K/Akt通路密切相关)，肿瘤转移和细胞增殖(主要由Ras/Mapk介导)。此外，c-Met与其它膜受体存在交联 (cross-talk)，现已确知这种交联可促进肿瘤形成及转移，由于c-Met是导致肿瘤形成及转移的许多通路的交叉点，以c-Met为靶标可相对较容易地实现对许多通路的同时干扰，c-Met成为抗肿瘤生成和转移治疗的一个有希望的靶点。
抗体药物偶联物(antibody drug conjugate，ADC)把单克隆抗体或者抗体片段通过稳定的化学接头化合物与具有生物活性的细胞毒素相连，充分利用了抗体对肿瘤细胞特异或高表达抗原结合的特异性和细胞毒素的高效性，避免对正常细胞的毒副作用。这也就意味着，与以往传统的化疗药物相比，抗体药物偶联物能精准地结合肿瘤细胞并降低将对正常细胞的影响。
ADC药物由抗体(靶向)，接头和毒素三部分组成。其中，好的靶点(抗体部分)决定了ADC药物的特异性，这不仅包括特异靶向结合，还包括有效的内吞。
目前针对c-Met激酶靶点抑制剂主要有三类：HGF和c-Met生物拮抗剂、HGF和c-Met抗体，以及小分子c-Met抑制剂。现有的临床结果表明，直接针对HGF,c-Met的抗体，或c-Met小分子抑制疗效不甚理想。针对c-Met的ADC药物可能是该靶点最有效的方法治疗肿瘤。目前，尚没有c-Met ADC药物临床研究。
本发明首创抗c-Met抗体ADC药物，不仅保留了本发明抗c-Met抗体的抗体依赖性的细胞增殖抑制作用，同时又增加了潜在的细胞毒素药物的效应。而且因为其毒素靶向在肿瘤细胞内释放，药物毒副作用并没有随着疗效的增加而同步增大。本发明提供特异性结合人c-Met的人源化抗体和嵌合抗体，并且该人源化抗体和嵌合抗体特征在于具有高亲和力，高药效，有内吞功效，稳定性好和无c-Met激动活性等特点。在这些优良性能基础上，本发明同时还提供特异性结合人c-Met的抗体-细胞毒性药物偶联物或其可药用盐或溶剂化合物，不仅保留了本发明抗c-Met抗体的抗体依赖性的细胞增殖抑制作用，同时又增加了潜在的细胞毒素药物的效应和治疗疾病的广谱性。而且因为其毒素靶向在肿瘤细胞内释放(本发明抗c-Met抗体的内吞作用)，药物毒副作用并没有随着疗效的增加而同步增大。
发明内容
本发明提供一种特异性结合c-Met受体的抗体或其抗原结合片段，其包含至少1个选自以下CDR区的序列或其突变序列：
抗体重链可变区HCDR区序列：SEQ ID NO:6，SEQ ID NO:7或SEQ ID NO:8；和
抗体轻链可变区LCDR区序列：SEQ ID NO:9，SEQ ID NO:10或SEQ ID  NO:11。
在本发明一个优选的实施方案中，提供一种如上所述的c-Met抗体或其抗原结合片段，其中所述的抗体重链可变区包含至少1个选自如下的HCDR区序列或其突变序列：SEQ ID NO:6，SEQ ID NO:7或SEQ ID NO:8。
在本发明一个优选的实施方案中，提供一种如上所述的c-Met抗体或其抗原结合片段，其中所述的抗体轻链可变区包含至少1个选自如下的LCDR区序列或其突变序列：SEQ ID NO:9，SEQ ID NO:10或SEQ ID NO:11。
在本发明一个优选的实施方案中，提供一种如上所述的c-Met抗体或其抗原结合片段，其中所述的抗体包含重链可变区序列SEQ ID NO:6(HCDR1)，SEQ ID NO:7(HCDR2)和SEQ ID NO:8(HCDR3)，或其突变序列，和轻链可变区序列SEQ ID NO:9(LCDR1)，SEQ ID NO:10(LCDR2)和SEQ ID NO:11(LCDR3)或其突变序列。
在本发明一个优选的实施方案中，提供一种如上所述的c-Met抗体或其抗原结合片段，其中所述的CDR区突变序列为CDR区发生1-3个优化抗体活性的氨基酸突变，其中所述的HCDR2区突变序列优选为SEQ ID NO:12。
在本发明一个优选的实施方案中，提供一种如上所述的c-Met抗体或其抗原结合片段，其中所述的c-Met抗体或其抗原结合片段为鼠源抗体或其片段。
在本发明一个优选的实施方案中，提供一种如上所述的c-Met抗体或其抗原结合片段，其中所述的鼠源抗体重链可变区序列为：SEQ ID NO:4。
在本发明一个优选的实施方案中，提供一种如上所述的c-Met抗体或其抗原结合片段，其中所述的鼠源抗体轻链可变区序列为：SEQ ID NO:5。
在本发明一个优选的实施方案中，提供一种如上所述的c-Met抗体或其抗原结合片段，其中所述的鼠源抗体的-重链可变区为：SEQ ID NO:4，轻链可变区为：SEQ ID NO:5。
在本发明一个优选的实施方案中，提供一种如上所述的鼠源抗体或其片段，其抗体重链可变区进一步包含源自鼠源的IgG1、IgG2、IgG3或IgG4或其变体的重链FR区。
在本发明一个优选的实施方案中，提供一种如上所述的鼠源抗体或其片段，其进一步包含源自鼠源IgG1、IgG2、IgG3或IgG4、或其变体的重链恒定区。
在本发明一个优选的实施方案中，提供一种如上所述的鼠源抗体或其片段，其抗体轻链可变区进一步包含选自鼠源κ或λ链、或其变体的轻链FR区。
在本发明一个优选的实施方案中，提供一种如上所述的鼠源抗体或其片段，其进一步包含选自鼠源κ或λ链、或其变体的轻链恒定区。
在本发明一个优选的实施方案中，提供一种如上所述的c-Met抗体或其抗原结合片段，其为嵌合抗体或人源化抗体或其片段。
在本发明一个优选的实施方案中，提供一种如上所述的c-Met抗体或其抗原结合片段，其中所述人源化抗体重链可变区进一步包含人源IgG1、IgG2、IgG3、或IgG4或其变体的重链FR区。
在本发明一个优选的实施方案中，提供一种如上所述的c-Met抗体或其抗原结合片段，其中所述人源化抗体重链可变区上的重链FR区序列，来源于人种系重链序列，优选人种系重链IGHV 3-33*01；包含人种系重链IGHV 3-33*01的FR1，FR2，FR3区和FR4区的框架序列或其突变序列，优选所述突变序列为0-10个氨基酸的回复突变。
在本发明一个优选的实施方案中，提供一种如上所述的c-Met抗体或其抗原结合片段，其中所述的人源化抗体包含SEQ ID NO:13-15所示的重链可变区序列或其变体。
在本发明一个优选的实施方案中，提供一种如上所述的c-Met抗体或其抗原结合片段，其中所述人源化抗体轻链可变区上的轻链FR区序列，选自人种系轻链序列，优选人种系轻链IGKV085或IGKV 4-1*01，包含人种系轻链IGKV085和IGKV 4-1*01的FR1，FR2，FR3区和FR4区的框架序列或其突变序列，优选所述突变序列为0-10个氨基酸的回复突变。
在本发明一个优选的实施方案中，提供一种如上所述的c-Met抗体或其抗原结合片段，其中所述的人源化抗体包含选自SEQ ID NO:16-18所示的轻链可变区序列或其变体。
在本发明一个优选的实施方案中，提供一种如上所述的c-Met抗体或其抗原结合片段，所述的人源化抗体包含选自SEQ ID NO:13-15的重链可变区和选自SEQ ID NO:16-18的轻链可变区。
在本发明一个优选的实施方案中，提供一种如上所述的c-Met抗体或其抗原结合片段，其包含选自(a)至(c)任一的重链可变区序列和轻链可变区序列的组合：
(a)SEQ ID NO:13的重链可变区序列和SEQ ID NO:16的轻链可变区序列；
(b)SEQ ID NO:14的重链可变区序列和SEQ ID NO:17的轻链可变区序列；或
(c)SEQ ID NO:15的重链可变区序列和SEQ ID NO:18的轻链可变区序列。
在本发明一个优选的实施方案中，提供一种如上所述的c-Met抗体或其抗原结合片段，其中所述的人源化抗体的重链恒定区包含源自人源IgG1或其变体、人源IgG2或其变体、人源IgG3或其变体或人源IgG4或其变体的恒定区，优选包含人源IgG1或其变体或人源IgG2或其变体或人源IgG4或其变体的恒定区，更优选人源IgG2或其变体的恒定区。
在本发明一个优选的实施方案中，提供一种如上所述的c-Met抗体或其抗原结合片段，其包含选自SEQ ID NO:23-25或与其具有至少90％同源性的全长重链序列。
在本发明一个优选的实施方案中，提供一种如上所述的c-Met抗体或其抗原结合片段，其中所述人源化抗体轻链可变区进一步包含任选自人源κ或λ链或其变体的轻链FR区。
在本发明一个优选的实施方案中，提供一种如上所述的c-Met抗体或其抗原结合片段，其所述的人源化抗体的轻链恒定区包含选自人源κ或λ链或其变体的恒定区。
在本发明一个优选的实施方案中，提供一种如上所述的c-Met抗体或其抗原结合片段，其包含选自SEQ ID NO:26-28或与其具有至少90％序列同源性的全长轻链序列。
在本发明一个优选的实施方案中，提供一种如上所述的c-Met抗体或其抗原结合片段，其中所述的人源化抗体包含选自SEQ ID NO:23-25的全长重链序列和SEQ ID NO:26-28的全长轻链序列。
在本发明一个优选的实施方案中，提供一种如上所述的c-Met抗体或其抗原结合片段，其中所述的人源化抗体选自(a)至(c)任一的全长轻链序列和全长重链序列的组合：
Ab-9:SEQ ID NO:23的重链序列和SEQ ID NO:26的轻链序列；
Ab-10:SEQ ID NO:24的重链序列和SEQ ID NO:27的轻链序列；或
Ab-11:SEQ ID NO:25的重链序列和SEQ ID NO:28的轻链序列。
在本发明一个优选的实施方案中，提供一种如上所述的c-Met抗体或其抗原结合片段，其中所述的抗原结合片段包含Fab、Fv、sFv或F(ab’)2。
本发明进一步提供一种编码如上所述的c-Met抗体或其抗原结合片段的表达前体产物的DNA分子。
本发明进一步提供一种含有如上所述的DNA分子的表达载体。
本发明进一步提供一种用如上所述的表达载体转化的宿主细胞。
在本发明一个优选的实施方案中，提供一种如上所述的宿主细胞，其中所述的宿主细胞优选为哺乳动物细胞，更优选为CHO细胞。
本发明进一步提供一种药物组合物，其包含含有如上所述的c-Met抗体或其抗原结合片段，和一种或多种可药用的赋形剂、稀释剂或载体。
本发明进一步提供一种如上所述的c-Met抗体或其抗原结合片段、或包含其的药物组合物，在制备用于治疗c-Met介导的疾病或病症的药物中的用途，其中所述的疾病或病症优选为癌症；更优选为表达c-Met的癌症；最优选为胃癌、胰腺癌、肺癌、肠癌、肾癌、黑素瘤、非小细胞肺癌；最优选为胃癌和非小细胞肺癌。
本发明进一步提供一种治疗和预防c-Met介导的疾病或病症的方法，该方法包括给予所需患者治疗有效量的如上所述的c-Met抗体或其抗原结合片段、或包含其的药物组合物；其中所述的疾病或病症优选为癌症；更优选为表达c-Met的癌症；最优选为胃癌、胰腺癌、肺癌、肠癌、肾癌、黑素瘤、非小细胞肺癌；最优选为胃癌和非小细胞肺癌。
本发明进一步提供一种通式(I)所示的抗体-细胞毒性药物偶联物或其药学上可接受的盐或溶剂化合物：
Ab-[(L2)t-L1-D)]y   (I)
其中：
D为药物模块；
L1，L2是接头单元；
t为0或1，优选1；
y为1-8，优选2-5；y可以为小数；
Ab为如上所述的特异性结合c-Met受体的抗体或其抗原结合片段。
在本发明一个优选的实施方案中，提供一种通式(I)所示的抗体-细胞毒性药物偶联物或其药学上可接受的盐或溶剂化合物，其中-L2-为以下通式(-L2-)所示的化合物：
Figure PCTCN2016078699-appb-000001
其中
X1选自自氢原子、卤素、羟基、氰基、烷基、烷氧基和环烷基；
X2选自-烷基-、-环烷基-和-杂环基-；
m为0-5，优选1-3；S为硫原子。
在本发明一个优选的实施方案中，提供一种通式(I)所示的抗体-细胞毒性药物偶联物或其药学上可接受的盐或溶剂化合物，其中所述药物模块D为选自毒素、化疗剂、抗生素、放射性同位素和核溶酶的细胞毒剂。
在本发明一个优选的实施方案中，提供一种通式(I)所示的抗体-细胞毒性药物偶联物或其药学上可接受的盐或溶剂化合物，其中D为以下通式(D)所示的化合物：
Figure PCTCN2016078699-appb-000002
或其互变异构体、内消旋体、外消旋体、对映异构体、非对映异构体、或其混合物形式，或其可药用的盐，其中：
R1-R7选自氢原子、卤素、羟基、氰基、烷基、烷氧基和环烷基；
R8-R11任选自氢原子、卤素、烯基、烷基、烷氧基和环烷基；R8-R11优选至少其中一个选自卤素、烯基、烷基和环烷基，其余为氢原子；
或者R8-R11之中的任意两个形成环烷基，余下的两个基团任选自氢原子、烷基和环烷基；
R12-R13选自氢原子、烷基或卤素；
R14选自芳基或杂芳基，所述的芳基或杂芳基任选进一步被选自氢原子、卤素、羟基、烷基、烷氧基和环烷基的取代基所取代；
R15任选自卤素、烯基、烷基、环烷基和COO R17；
R16选自氢原子、卤素、羟基、氰基、烷基、烷氧基和环烷基；
R17选自氢原子、烷基和烷氧基。
在本发明一个优选的实施方案中，提供一种通式(I)所示的抗体-细胞毒性药物偶联物或其药学上可接受的盐或溶剂化合物，其中L2包含选自Val-Cit，MC，PAB和MC-PAB的接头，优选MC。
在本发明一个优选的实施方案中，提供一种通式(I)所示的抗体-细胞毒性药物偶联物或其药学上可接受的盐或溶剂化合物，其中D是美登木素生物碱；优选自DM1、DM3和DM4，更优选DM1。
在本发明一个优选的实施方案中，提供一种通式(I)所示的抗体-细胞毒性药物偶联物或其药学上可接受的盐或溶剂化合物，其中所述L2选自N-琥珀酰亚氨基4-(2-吡啶基硫代)戊酸酯(SPP)、N-琥珀酰亚氨基4-(N-马来酰亚胺基甲基)-环己烷-1-羧酸酯(SMCC)、和N-琥珀酰亚氨基(4-碘-乙酰基)氨基苯甲酸酯(SIAB)；优选SPP或SMCC。
在本发明一个优选的实施方案中，提供一种通式(I)所示的抗体-细胞毒性药 物偶联物或其药学上可接受的盐或溶剂化合物，其中D是喜树碱类生物碱；优选自CPT、10-羟基-CPT、CPT-11(伊立替康)、SN-38和托泊替康，更优选SN-38。
在本发明一个优选的实施方案中，提供一种通式(I)所示的抗体-细胞毒性药物偶联物或其药学上可接受的盐或溶剂化合物，其中所述接头L2包含任选自Val-Cit，MC，PAB或MC-PAB的结构，优选MC或MC-vc-PAB。
在本发明一个优选的实施方案中，提供一种通式(I)所示的抗体-细胞毒性药物偶联物或其药学上可接受的盐或溶剂化合物，其包括通式(II)所示的偶联药物或其药学上可接受的盐或溶剂化合物：
Figure PCTCN2016078699-appb-000003
其中：
R2-R16如通式(D)中所定义；
Ab，t，y，L1，L2如通式(I)中所定义。
在本发明一个优选的实施方案中，提供一种通式(I)所示的抗体-细胞毒性药物偶联物或其药学上可接受的盐或溶剂化合物，其包括通式(III)所示的偶联药物或其药学上可接受的盐或溶剂化合物：
Figure PCTCN2016078699-appb-000004
其中：
R2-R16如通式(D)中所定义；
Ab，t，y，L1，L2如通式(I)中所定义；
n为3-6，优选5。
在本发明一个优选的实施方案中，提供一种通式(I)所示的抗体-细胞毒性药 物偶联物或其药学上可接受的盐或溶剂化合物，其包括通式(IV)所示的偶联药物或其药学上可接受的盐或溶剂化合物：
Figure PCTCN2016078699-appb-000005
其中：
R2-R16如通式(D)中所定义；
Ab，y如通式(I)中所定义；
n如通式(III)中所定义；
X1，X2，m如通式L2中所定义。
在本发明一个优选的实施方案中，提供一种通式(I)所示的抗体-细胞毒性药物偶联物或其药学上可接受的盐或溶剂化合物，其包括通式(V)所示的偶联药物或其药学上可接受的盐或溶剂化合物：
Figure PCTCN2016078699-appb-000006
其中：
Ab，D，y如通式(I)中所定义；
n如通式(III)中所定义；
X1，X2，m如通式L2中所定义。
本发明所述的抗体-细胞毒性药物偶联物或其可药用盐或溶剂化合物包括但不限于：
Figure PCTCN2016078699-appb-000007
Figure PCTCN2016078699-appb-000008
Figure PCTCN2016078699-appb-000009
Figure PCTCN2016078699-appb-000010
Ab-9，Ab-10，Ab-11为如上所述的c-Met抗体，y为1-8，优选2-5。
其中，y的范围为1-8；优选1-4。
本发明进一步提供一种制备通式(V)所示的抗体-细胞毒性药物偶联物的方法，该方法包括：
Figure PCTCN2016078699-appb-000011
通式(Ab-L2)化合物与通式(L1-D)化合物在有机溶剂中反应，得到通式(V)化合物；所述的有机溶剂优选乙腈或乙醇；
其中：
Ab如前所述的特异性结合c-Met受体的抗体或其抗原结合片段；
X1选自自氢原子、卤素、羟基、氰基、烷基、烷氧基和环烷基；
X2选自烷基、环烷基和杂环基；
X为0-5，优选1-3；
m为0-5，优选1-3。
在本发明一个优选的实施方案中，提供一种如上所述的抗体-细胞毒性药物偶联物或其可药用盐或溶剂化合物，其具有体外或体内细胞杀伤活性。
本发明进一步提供一种药物组合物，其含有如上所述的c-Met抗体或其抗原结合片段、或抗体-细胞毒性药物偶联物或其可药用盐或溶剂化合物和可药用的赋形剂、稀释剂或载体。
本发明进一步提供一种如上所述的c-Met抗体或其抗原结合片段、或抗体-细胞毒性药物偶联物或其可药用盐或溶剂化合物、或包含其的药物组合物，在制备用于治疗c-Met介导的疾病或病症的药物中的用途，其中所述的疾病或病症优选为癌症；更优选为表达c-Met的癌症；最优选为胃癌、胰腺癌、肺癌、肠癌、肾癌、黑素瘤、非小细胞肺癌；最优选为胃癌、胰腺癌、非小细胞肺癌和肾癌。
本发明进一步提供一种治疗和预防c-Met介导的疾病或病症的方法，该方法包括给予所需患者治疗有效量的如上所述的c-Met抗体或其抗原结合片段、或抗体-细胞毒性药物偶联物或其可药用盐或溶剂化合物、或包含其的药物组合物；其中所述的疾病或病症优选为癌症；更优选为表达c-Met的癌症；最优选为胃癌、胰腺癌、肺癌、肠癌、肾癌、黑素瘤、非小细胞肺癌；最优选为胃癌、胰腺癌、非小细胞肺癌和肾癌。
附图说明
图1本发明抗c-Met抗体和ADC分子抑制肿瘤作用，结果表明ADC新分子通过带入的毒素能达到对肿瘤的完全抑制作用，而抗体单独无法达到。结果还表明，本发明ADC分子没有因为毒素的偶联而影响到T1/2，本发明ADC药物在小鼠体内未见毒性。
发明详述
一、术语
为了更容易理解本发明，以下具体定义了某些技术和科学术语。除显而易见在本文件中的它处另有明确定义，否则本文使用的所有其它技术和科学术语都具有本发明所属领域的一般技术人员通常理解的含义。
本发明所用氨基酸三字母代码和单字母代码如J.biol.chem,243,p3558(1968)中所述。
术语“c-Met”或“c-Met多肽”或“c-Met受体”是指结合细胞生长因子(HGF)的受体酪氨酸激酶。本发明中如非特指，比如鼠c-Met(m-c-Met)或猴c-Met(cyno-c-Met)，均指人的c-Met(h-c-Met)。本发明中所用的人、鼠、食蟹猴c-Met均通过GenBank提供的核苷酸序列或多肽序列进行编码，例如GenBank 登录号NM_000245中提供的核苷酸序列编码的人多肽，或由GenBank登录号NP_000236中提供的多肽序列编码的人蛋白质或其细胞外结构域。原始的单链前体蛋白质在翻译后被剪切以产生α和β亚基，其通过二硫键连接以形成成熟受体。受体酪氨酸激酶c-Met参与细胞过程包括，例如伴随胚胎发生的组织再生的迁移、侵入和形态发生的过程。
术语“c-Met相关病症或状况”指任何源自c-Met的不利表达或缺乏表达，不利调控或缺乏调控，或有害活性或缺乏活性，或可以通过调节c-Met表达或活性来进行调节、治疗或治愈的疾病、病症或状况。例如在大部分癌症患者中，或在其疾病确实由与c-Met途径相关的变化驱动的患者中，可以预期HGF/c-Met途径的激活。例如上调归因于不同的机制，像HGF和/或c-Met的过表达，或通过c-Met突变的组成型激活。c-Met相关病症或状况包括但不限于，例如增生性疾病和紊乱和炎性疾病和紊乱。增生性疾病包括但不限于，例如癌症，其包括例如，胃癌、食道癌、肾癌包括乳突状肾细胞癌、肺癌、神经胶质瘤、头颈癌、上皮癌、皮肤癌、白血病、淋巴癌，骨髓瘤、脑癌、胰腺癌，结直肠癌、胃肠癌、肠癌、生殖器癌症、泌尿器癌症、黑色素瘤、前列腺癌以及本领域技术人员已知的其它肿瘤。炎性疾病包括但不限于，例如细菌感染，包括李斯特菌属细菌引起的感染。
本发明所述的抗体指免疫球蛋白，是由两条相同的重链和两条相同的轻链通过链间二硫键连接而成的四肽链结构。免疫球蛋白重链恒定区的氨基酸组成和排列顺序不同，故其抗原性也不同。据此，可将免疫球蛋白分为五类，或称为免疫球蛋白的同种型，即IgM，IgD，IgG，IgA和IgE，其相应的重链分别为μ链，δ链，γ链，α链，ε链。同一类Ig根据其铰链区氨基酸组成和重链二硫键的数目和位置的差别，又可分为不同的亚类，如IgG可分为IgG1，IgG2，IgG3，IgG4。轻链通过恒定区的不同分为κ链或λ链。五类Ig中第每类Ig都可以有κ链或λ链。
抗体重链和轻链靠近N端的约110个氨基酸的序列变化很大，为可变区(V区)；靠近C端的其余氨基酸序列相对稳定，为恒定区(C区)。可变区包括3个高变区(HVR)和4个序列相对保守的骨架区(FR)。3个高变区决定抗体的特异性，又称为互补性决定区(CDR)。每条轻链可变区(LCVR)和重链可变区(HCVR)由3个CDR区4个FR区组成，从氨基端到羧基端依次排列的顺序为：FR1，CDR1，FR2，CDR2，FR3，CDR3，FR4。轻链的3个CDR区指LCDR1，LCDR2，和LCDR3；重链的3个CDR区指HCDR1，HCDR2和HCDR3。发明所述的抗体或抗原结合片段的LCVR区和HCVR区的CDR氨基酸残基在数量和位置符合已知的Kabat编号规则(LCDR1-3，HCDE2-3)，或者符合kabat和chothia的编号规 则(HCDR1)。
术语“鼠源抗体”在本发明中为根据本领域知识和技能用小鼠制备的抗人c-Met的单克隆抗体。制备时用c-Met抗原注射试验对象，然后分离表达具有所需序列或功能特性的抗体的杂交瘤。在本发明一个优选的实施方案中，所述的鼠源c-Met抗体或其抗原结合片段，可进一步包含鼠源κ、λ链或其变体的轻链恒定区，或进一步包含鼠源IgG1,IgG2,IgG3或IgG4或其变体的重链恒定区。
术语“嵌合抗体(chimeric antibody)”，是将鼠源性抗体的可变区与人抗体的恒定区融合而成的抗体，可以减轻鼠源性抗体诱发的免疫应答反应。建立嵌合抗体，要先建立分泌鼠源性特异性单抗的杂交瘤，然后从小鼠杂交瘤细胞中克隆可变区基因，再克隆到人抗体的恒定区基因，进行重组表达。
术语“人源化抗体(humanized antibody)”，也称为CDR移植抗体(CDR-grafted antibody)人源化，是指将小鼠的CDR序列移植到人的抗体可变区框架，即不同类型的人种系抗体构架序列中产生的抗体。可以克服嵌合抗体由于携带大量小鼠蛋白成分，从而诱导的强烈的抗体可变抗体反应。此类构架序列可以从包括种系抗体基因序列的公共DNA数据库或公开的参考文献获得。如人重链和轻链可变区基因的种系DNA序列可以在“VBase”人种系序列数据库(在因特网www.mrccpe.com.ac.uk/vbase可获得)，以及在Kabat，E.A.等人，1991Sequences of Proteins of Immunological Interest，第5版中找到。在本发明一个优选的实施方案中，所述的c-Met人源化抗体小鼠的CDR序列选自SEQ ID NO:6，7，8，9，10，11(please check the#s,in case just copy from sost draft)。人的抗体可变区框架经过设计选择，其中所述抗体轻链可变区上的轻链FR区序列，选自人种系轻链序列，优选人种系轻链IGKV085或IGKV 4-1*01，包含人种系轻链IGKV085和IGKV 4-1*01的FR1，FR2，FR3区和FR4区；其中所述抗体重链可变区上的重链FR区序列，来源于人种系重链序列，优选人种系重链IGHV 3-33*01；包含人种系重链IGHV 3-33*01的FR1，FR2，FR3区和FR4区。为避免免疫原性下降的同时，引起的活性下降，可对所述的人抗体可变区可进行最少反向突变，以保持活性。
本领域中有可获得的多种方法来产生人源化抗体。例如，可通过获得抗c-Met特异抗体(例如，鼠类抗体或由杂交瘤产生的抗体)HCVR和LCVR序列，将其移植到所选的人抗体框架编码序列上来产生人源化抗体。任选地，可通过随机诱变或在特定位置诱变来优化CDR区，以在将CDR区移植到框架区中之前用不同的氨基酸置换CDR中的一个或更多个氨基酸。或者，可使用本领域技术人员可获得的方法在将CDR区插入人框架区后对其进行优化。优选地，“人源化抗体”具有起源于或来自亲本抗体(即非人抗体，优选小鼠单克隆抗体)的CDR，而在其存在的程度上框架区和恒定区(或其主要部分或基本部分，即至少约90％、92％、94％、95％、96％、97％、98％或99％)序列同人类种系免疫球蛋白区(参见，例如 the，International，ImMunoGeneTics，Database)或其重组或突变形式中，无论所述抗体是否在人类细胞中产生。优选地，从人源化抗体来源的非人亲本抗体的CDR优化人源化抗体的至少2、3、4、5或6个CDR，以产生期望的性质，例如改善的特异性、亲和力或中和作用，其可以通过筛选测定，例如ELISA测定来进行鉴定。优选地，本发明抗体中优化的CDR当与亲本抗体中存在的CDR相比时，包含至少一个氨基酸置换。与亲本抗体的CDRs相比，本发明人源化抗体的CDR中某些氨基酸置换(参见本文的实施例6)降低了抗体不稳定性的可能性(例如除去CDR中Asn残基)或当对人受试者施用时降低了抗体的免疫原性(例如，由IMMUNOFILTERTM，Technology预测的)。
在CDR编码序列移植到所选人框架编码序列上之后，然后表达编码人源化可变重链和可变轻链序列的所得DNA序列，以产生结合c-Met的人源化抗体。可将人源化HCVR和LCVR表达为整个抗硬骨素抗体分子的部分，即表达为与人恒定域序列的融合蛋白。然而，HCVR和LCVR序列也可在不存在恒定序列的情况下进行表达，以产生人源化抗c-Met scFv。
进一步描述参与人源化可使用小鼠抗体的方法的文献包括，例如Queen等，Proc.，Natl.Acad.Sci.USA，88，2869，1991和Winter及其同事的方法[Jones等，Nature，321，522(1986)，Riechmann，等，Nature，332，323-327(1988)，Verhoeyen，等，Science，239，1534(1988)]。
本发明中所述的“抗原结合片段”，指具有抗原结合活性的Fab片段，Fab’片段，F(ab’)2片段，以及与人c-Met结合的Fv片段scFv片段；包含本发明所述抗体的选自SEQ ID NO:3至SEQ ID NO:8中的一个或多个CDR区。Fv片段含有抗体重链可变区和轻链可变区，但没有恒定区，并具有全部抗原结合位点的最小抗体片段。一般地，Fv抗体还包含在VH和VL结构域之间的多肽接头，且能够形成抗原结合所需的结构。也可以用不同的连接物将两个抗体可变区连接成一条多肽链，称为单链抗体(single chain antibody)或单链Fv(scFv)。scFv还可以和其它抗体，例如抗EGFR抗体构建双特异抗体(bispecific antibody)本发明的术语“与c-Met结合”，指能与人c-Met相互作用。本发明的术语“抗原结合位点”指抗原上不连续的，由本发明抗体或抗原结合片段识别的三维空间位点。本发明中所述的“ADCC”，即antibody-dependent cell-mediated cytotoxicity，抗体依赖的细胞介导的细胞毒作用，是指表达Fc受体的细胞通过识别抗体的Fc段直接杀伤被抗体包被的靶细胞。可通过对IgG上Fc段的修饰，降低或消除抗体的ADCC效应功能。所述的修饰指在抗体的重链恒定区进行突变，如选自IgG1的N297A，L234A，L235A；IgG2/4chimera，IgG4的F235E,或L234A/E235A突变。
本发明中所述的融合蛋白是一种通过DNA重组，得到的两个基因共表达的蛋白产物。重组c-Met胞外区Fc融合蛋白通过DNA重组，把c-Met胞外区和人 抗体Fc片段共表达的融合蛋白。所述的c-Met胞外区，是指c-Met蛋白表达在细胞膜以外的部分。
本发明工程化的抗体或抗原结合片段可用常规方法制备和纯化。比如，编码重链(SEQ ID NO:4)和轻链(SEQ ID NO:5)的cDNA序列，可以克隆并重组至表达载体pEE6.4((Lonza Biologics)。重组的免疫球蛋白表达载体可以稳定地转染CHO细胞。作为一种更推荐的现有技术，哺乳动物类表达系统会导致抗体的糖基化，特别是在FC区的高度保守N端。通过表达与人c-Met特异性结合的抗体得到稳定的克隆。阳性的克隆在生物反应器的无血清培养基中扩大培养以生产抗体。分泌了抗体的培养液可以用常规技术纯化。比如，用含调整过的缓冲液的A或G Sepharose FF柱进行过柱。洗去非特异性结合的组分。再用PH梯度法洗脱结合的抗体，用SDS-PAGE检测抗体片段，收集。抗体可用常规方法进行过滤浓缩。可溶的混合物和多聚体，也可以用常规方法去除，比如分子筛，离子交换。得到的产物需立即冷冻，如-70℃，或者冻干。
本发明的抗体指单克隆抗体。本发明所述的单克隆抗体或mAb，指由单一的克隆细胞株得到的抗体，所述的细胞株不限于真核的，原核的或噬菌体的克隆细胞株。单克隆抗体或抗原结合片段可以用如杂交瘤技术、重组技术、噬菌体展示技术，合成技术(如CDR-grafting)，或其它现有技术进行重组得到。
“给予”和“处理”当应用于动物、人、实验受试者、细胞、组织、器官或生物流体时，是指外源性药物、治疗剂、诊断剂或组合物与动物、人、受试者、细胞、组织、器官或生物流体的接触。“给予”和“处理”可以指例如治疗、药物代谢动力学、诊断、研究和实验方法。细胞的处理包括试剂与细胞的接触，以及试剂与流体的接触，其中所述流体与细胞接触。
“治疗”意指给予患者内用或外用治疗剂，诸如包含本发明的任一种结合化合物的组合物，所述患者具有一种或多种疾病症状，而已知所述治疗剂对这些症状具有治疗作用。通常，在受治疗患者或群体中以有效缓解一种或多种疾病症状的量给予治疗剂，无论是通过诱导这类症状退化还是抑制这类症状发展到任何临床右测量的程度。有效缓解任何具体疾病症状的治疗剂的量(也称作“治疗有效量”)可根据多种因素变化，例如患者的疾病状态、年龄和体重，以及药物在患者产生需要疗效的能力。通过医生或其它专业卫生保健人士通常用于评价该症状的严重性或进展状况的任何临床检测方法，可评价疾病症状是否已被减轻。尽本发明的实施方案(例如治疗方法或制品)在缓解每个患都有的目标疾病症状方面可能无效，但是根据本领域已知的任何统计学检验方法如Student t检验、卡方检验、依据Mann和Whitney的U检验、Kruskal-Wallis检验(H检验)、Jonckheere-Terpstra检验和Wilcoxon检验确定，其在统计学显著数目的患者中应 当减轻目标疾病症状。
“保守修饰”或“保守置换或取代”是指具有类似特征(例如电荷、侧链大小、疏水性/亲水性、主链构象和刚性等)的其它氨基酸置换蛋白中的氨基酸，使得可频繁进行改变而不改变蛋白的生物学活性。本领域技术人员知晓，一般而言，多肽的非必需区域中的单个氨基酸置换基本上不改变生物学活性(参见例如Watson等(1987)Molecular Biology of the Gene,The Benjamin/Cummings Pub.Co.,第224页，(第4版))。另外，结构或功能类似的氨基酸的置换不大可能破环生物学活性。
整个说明书和权利要求书中使用的术语“基本上由……组成”或其变形表示包括所有所述元件或元件组，并且任选包括与所述元件类似或不同性质的其它元件，所述其它元件非显著改变指定给药方案、方法或组合物的基本性质或新性质。作为非限制性例子，基本上由所提及的氨基酸序列组成的结合化合物还可以包括一种或多种氨基酸，其不显著影响结合化合物的性质。
“有效量”包含足以改善或预防医字病症的症状或病症的量。有效量还意指足以允许或促进诊断的量。用于特定患者或兽医学受试者的有效量可依据以下因素而变化：如待治疗的病症、患者的总体健康情况、给药的方法途径和剂量以及副作用严重性。有效量可以是避免显著副作用或毒性作用的最大剂量或给药方案。
“外源性”指要据背景在生物、细胞或人体外产生的物质。“内源性”指根据背景在细胞、生物或人体内产生的物质。
“同源性”是指两个多核苷酸序列之间或两个多肽之间的序列相似性。当两个比较序列中的位置均被相同碱基或氨基酸单体亚基占据时，例如如果两个DNA分子的每一个位置都被腺嘌呤占据时，那么所述分子在该位置是同源的。两个序列之间的同源怀百分率是两个序列共有的匹配或同源位置数除以比较的位置数×100的函数。例如，在序列最佳比对时，如果两个序列中的10个位置有6个匹配或同源，那么两个序列为60％同源。一般而言，当比对两个序列而得到最大的同源性百分率时进行比较。
“任选”或“任选地”意味着随后所描述地事件或环境可以但不必发生，该说明包括该事件或环境发生或不发生地场合。例如，“任选包含1-3个抗体重链可变区”意味着特定序列的抗体重链可变区可以但不必须存在。
“药物组合物”表示含有一种或多种本文所述化合物或其生理学上/可药用的盐或前体药物与其他化学组分的混合物，以及其他组分例如生理学/可药用的载体和赋形剂。药物组合物的目的是促进对生物体的给药，利于活性成分的吸收进而发挥生物活性。
常规的药物组合物的制备见中国药典。
术语“载体”用于本发明的药物，是指能改变药物进入人体的方式和在体内的分布、控制药物的释放速度并将药物输送到靶向器官的体系。药物载体释放和靶向系统能够减少药物降解及损失，降低副作用，提高生物利用度。如可作为载体的高分子表面活性剂由于其独特的两亲性结构，可以进行自组装，形成各种形式的聚集体，优选的实例如胶束、微乳液、凝胶、液晶、囊泡等。这些聚集体具有包载药物分子的能力，同时又对膜有良好的渗透性，可以作为优良的药物载体。术语“稀释剂”又称填充剂，其主要用途是增加片剂的重量和体积。稀释剂的加入不仅保证一定的体积大小，而且减少主要成分的剂量偏差，改善药物的压缩成型性等。当片剂的药物含有油性组分时，需加入吸收剂吸收油性物，使保持“干燥”状态，以利于制成片剂。
术语“可药用盐”是指本发明配体-细胞毒性药物偶联物的盐，这类盐用于哺乳动物体内时具有安全性和有效性，且具有应有的生物活性，本发明抗体-抗体药物偶联化合物至少含有一个氨基，因此可以与酸形成盐。
术语“溶剂化合物”指本发明的配体-药物偶联化合物与一种或多种溶剂分子形成可药用的溶剂化合物。
术语“配体”是能识别和结合目标细胞相关的抗原或受体的大分子化合物。配体的作用是将药物呈递给与配体结合的目标细胞群，这些配体包括但不限于蛋白类激素、凝集素、生长因子、抗体或其他能与细胞结合的分子。
治疗剂是与结合部分如抗体或抗体片段、或其亚片段分别、同时或相继地给药的分子或原子，并且可用于疾病的治疗。治疗剂的实例包括但不限于抗体、抗体片段、共轭物、药物、细胞毒性剂、促细胞凋亡剂、毒素、核酸酶(包括DNA酶和RNA酶)、激素、免疫调节剂、螯合剂、硼化合物、光敏剂或染料、放射性同位素或放射性核素、寡核苷酸、干扰RNA、肽、抗血管发生剂、化疗剂、细胞因子、趋化因子、前药、酶、结合蛋白或肽、或其组合。
偶联物是与如上所述的治疗剂偶联的抗体组分或其他靶向部分。本文所用的术语“偶联物”和“免疫偶联物”可交换地使用。
术语“细胞毒剂”在用于本文时指抑制或防止细胞的功能和/或引起细胞死亡或破坏的物质。
“毒素”指能够对细胞的生长或增殖产生有害效果的任何物质。
“化疗剂”指可用于治疗癌症的化学化合物。该定义还包括起调节、降低、阻断或抑制可促进癌生长的激素效果作用的抗激素剂，且常常是系统或全身治疗的形式。它们自身可以是激素。
澳瑞他汀是全合成药物，化学结构式相对容易改造，以便优化其物理性质和 成药特性。用于和抗体偶联的澳瑞他汀衍生物主要包括单甲基澳瑞他汀E(MMAE)和单甲基澳瑞他汀F(MMAF)，前者是又天然微管蛋白聚合酶抑制剂尾海兔素-10(dolastatin-10)衍生出的合成五肽，在C-端加上一个2-氨基-1-苯基丙基-1-醇而合成。MMAE对多种人类肿瘤细胞株的抑制活性小于一纳摩尔。为了降低MMAE自身细胞毒活性，MMAF在尾海兔素-10的C-端加上一个苯丙氨酸，因为在结构上引入一个羧基，MMAF的细胞膜通过性较差，因此对细胞的生物活性显著降低，但是和抗体偶联后对细胞的抑制活性大幅度提高(US7750116)。
术语“微管蛋白抑制剂”是指通过抑制微管蛋白的聚合或促进微管蛋白的聚合而干扰细胞的有丝分裂过程，从而发挥抗肿瘤作用的一类化合物。其非限制性实例包括：美登素类、卡利奇霉素、紫杉烷类、长春新碱、秋水仙碱、尾海兔素/澳瑞他汀，优选自美登素类或尾海兔素/澳瑞他汀；更优选自通式D1或DM所示的化合物。
CPT是喜树碱的缩写，并且在本申请中CPT用于表示喜树碱本身或喜树碱的类似物或衍生物。具有所示的编号和用字母A-E标记的环的喜树碱和一些其类似物的结构在以下式提供。
Figure PCTCN2016078699-appb-000012
CPT:R1＝R2＝R3＝H
10-羟基-CPT：R1＝OH；R2＝R3＝H
伊立替康(CPT-11)：
Figure PCTCN2016078699-appb-000013
R2＝乙基；R3＝H
SN-38：R1＝OH；R2＝乙基；R3＝H
托泊替康：R1＝OH；R2＝H；R3＝CH-N(CH3)2
术语“胞内代谢物”指由细胞内对抗体-药物偶联物(ADC)的代谢过程或反应产生的化合物。所述代谢过程或反应可以是酶促过程，诸如ADC的肽接头的蛋 白水解切割、或官能团诸如腙、酯或酰胺的水解。胞内代谢物包括但不限于在进入、扩散、摄取或转运进入细胞后经历胞内切割的抗体和游离药物。
术语“胞内切割的”和“胞内切割”指细胞内对抗体-药物偶联物(ADC)的代谢过程或反应，由此药物模块(D)与抗体(Ab)之间的共价附着，即接头被打断，导致细胞内游离药物与抗体解离。ADC被切割的模块因而是胞内代谢物。
术语“生物利用度”指施用于患者的给定量的药物的系统利用度(即血液/血浆水平)。生物利用度是表明药物从所施用的剂量形式到达大循环的时间(速率)和总量(程度)二者度量的绝对项。
术语“细胞毒活性”指抗体-药物偶联物或抗体-药物偶联物的胞内代谢物的细胞杀伤、细胞抑制、或生长抑制效果。细胞毒活性可以表述为IC50值，即半数细胞存活时每单位体积的浓度(摩尔或质量)。
术语“烷基”指饱和脂肪族烃基团，其为包含1至20个碳原子的直链或支链基团，优选含有1至12个碳原子的烷基，更优选含有1至10个碳原子的烷基，最优选含有1至6个碳原子的烷基。非限制性实例包括甲基、乙基、正丙基、异丙基、正丁基、异丁基、叔丁基、仲丁基、正戊基、1,1-二甲基丙基、1,2-二甲基丙基、2,2-二甲基丙基、1-乙基丙基、2-甲基丁基、3-甲基丁基、正己基、2,2-二乙基己基，及其各种支链异构体等。烷基可以是取代的或非取代的，当被取代时，取代基可以在任何可使用的连接点上被取代，所述取代基优选为一个或多个以下基团，其独立地选自烷基、烯基、炔基、烷氧基、烷硫基、烷基氨基、卤素、巯基、羟基、硝基、氰基、环烷基、杂环烷基、芳基、杂芳基、环烷氧基、杂环烷氧基、环烷硫基、杂环烷硫基、氧代基。
术语“环烷基”指饱和或部分不饱和单环或多环环状烃取代基，环烷基环包含3至20个碳原子，优选包含3至12个碳原子，更优选包含3至10个碳原子，最优选包含3至8个碳原子。单环环烷基的非限制性实例包括环丙基、环丁基、环戊基、环戊烯基、环己基、环己烯基、环己二烯基、环庚基、环庚三烯基、环辛基等；多环环烷基包括螺环、稠环和桥环的环烷基。
术语“杂环基”指饱和或部分不饱和单环或多环环状烃取代基，其包含3至20个环原子，其中一个或多个环原子为选自氮、氧或S(O)m(其中m是整数0至2)的杂原子，但不包括-O-O-、-O-S-或-S-S-的环部分，其余环原子为碳。优选包含3至12个环原子，其中1～4个是杂原子；更优选环烷基环包含3至10个环原子。单环杂环基的非限制性实例包括吡咯烷基、哌啶基、哌嗪基、吗啉基、硫代吗啉基、高哌嗪基等。多环杂环基包括螺环、稠环和桥环的杂环基。
所述杂环基环可以稠合于芳基、杂芳基或环烷基环上，其中与母体结构连接在一起的环为杂环基，其非限制性实例包括：
Figure PCTCN2016078699-appb-000014
等。
杂环基可以是任选取代的或非取代的，当被取代时，取代基优选为一个或多个以下基团，其独立地选自烷基、烯基、炔基、烷氧基、烷硫基、烷基氨基、卤素、巯基、羟基、硝基、氰基、环烷基、杂环烷基、芳基、杂芳基、环烷氧基、杂环烷氧基、环烷硫基、杂环烷硫基、氧代基。
术语“芳基”指具有共轭的π电子体系的6至14元全碳单环或稠合多环(也就是共享毗邻碳原子对的环)基团，优选为6至10元，例如苯基和萘基，最优选苯基。所述芳基环可以稠合于杂芳基、杂环基或环烷基环上，其中与母体结构连接在一起的环为芳基环，其非限制性实例包括：
Figure PCTCN2016078699-appb-000015
芳基可以是取代的或非取代的，当被取代时，取代基优选为一个或多个以下基团，其独立地选自烷基、烯基、炔基、烷氧基、烷硫基、烷基氨基、卤素、巯基、羟基、硝基、氰基、环烷基、杂环烷基、芳基、杂芳基、环烷氧基、杂环烷氧基、环烷硫基、杂环烷硫基。
术语“杂芳基”指包含1至4个杂原子、5至14个环原子的杂芳族体系，其中杂原子选自氧、硫和氮。杂芳基优选为5至10元，更优选为5元或6元，例如呋喃基、噻吩基、吡啶基、吡咯基、N-烷基吡咯基、嘧啶基、吡嗪基、咪唑基、四唑基等。所述杂芳基环可以稠合于芳基、杂环基或环烷基环上，其中与母体结构连接在一起的环为杂芳基环，其非限制性实例包括：
Figure PCTCN2016078699-appb-000016
杂芳基可以是任选取代的或非取代的，当被取代时，取代基优选为一个或多 个以下基团，其独立地选自烷基、烯基、炔基、烷氧基、烷硫基、烷基氨基、卤素、巯基、羟基、硝基、氰基、环烷基、杂环烷基、芳基、杂芳基、环烷氧基、杂环烷氧基、环烷硫基、杂环烷硫基。
术语“烷氧基”指-O-(烷基)和-O-(非取代的环烷基)，其中烷基的定义如上所述。烷氧基的非限制性实例包括：甲氧基、乙氧基、丙氧基、丁氧基、环丙氧基、环丁氧基、环戊氧基、环己氧基。烷氧基可以是任选取代的或非取代的，当被取代时，取代基优选为一个或多个以下基团，其独立地选自烷基、烯基、炔基、烷氧基、烷硫基、烷基氨基、卤素、巯基、羟基、硝基、氰基、环烷基、杂环烷基、芳基、杂芳基、环烷氧基、杂环烷氧基、环烷硫基、杂环烷硫基。
术语“键”指用“—”表示的共价键。
术语“羟基”指-OH基团。
术语“卤素”指氟、氯、溴或碘。
术语“氨基”指-NH2。
术语“氰基”指-CN。
术语“硝基”指-NO2。
术语“氧代基”指＝O。
“任选”或“任选地”意味着随后所描述的事件或环境可以但不必发生，该说明包括该事件或环境发生或不发生地场合。例如，“任选被烷基取代的杂环基团”意味着烷基可以但不必须存在，该说明包括杂环基团被烷基取代的情形和杂环基团不被烷基取代的情形。
“取代的”指基团中的一个或多个氢原子，优选为最多5个，更优选为1～3个氢原子彼此独立地被相应数目的取代基取代。不言而喻，取代基仅处在它们的可能的化学位置，本领域技术人员能够在不付出过多努力的情况下确定(通过实验或理论)可能或不可能的取代。例如，具有游离氢的氨基或羟基与具有不饱和(如烯属)键的碳原子结合时可能是不稳定的。
“接头”指包含使抗体共价附着于药物模块的共价键或原子链的化学模块。在各个实施方案中，接头包括：二价基，诸如亚烃基(alkyldiyl)、亚芳基、亚杂芳基，诸如-(CR2)nO(CR2)n-、烃氧基重复单元(例如聚亚乙基氧基(polyethyleneoxy)、PEG、聚亚甲基氧基(polymethyleneoxy)和烃氨基(例如聚乙烯氨基，JeffamineTM)等模块；及二酸酯和酰胺类，包括琥珀酸酯、琥珀酰胺、二乙醇酸酯、丙二酸酯和己酰胺。
缩写
接头组件
MC＝6-马来酰亚氨基己酰基
Val-Cit或“vc”＝缬氨酸-瓜氨酸(蛋白酶可切割接头中的例示二肽)
瓜氨酸＝2-氨基-5-脲基戊酸
PAB＝对氨基苄氧羰基(“自我牺牲”接头组件的例示)
Me-Val-Cit＝N-甲基-缬氨酸-瓜氨酸(其中接头肽键已经修饰以防止其受到组织蛋白酶B的切割)
MC(PEG)6-OH＝马来酰亚氨基己酰基-聚乙二醇(可附着于抗体半胱氨酸)
SPP＝N-琥珀酰亚氨基4-(2-吡啶基硫代)戊酸酯
SPDP＝N-琥珀酰亚氨基3-(2-吡啶基二硫代)丙酸酯
SMCC＝琥珀酰亚氨基-4-(N-马来酰亚氨基甲基)环己烷-1-羧酸酯
IT＝亚氨基硫烷
细胞毒性药物：
MMAE＝单甲基澳瑞他汀E(MW718)
MMAF＝澳瑞他汀E(MMAE)的变体，其在药物的C-末端处有苯丙氨酸(MW731.5)
MMAF-DMAEA＝有DMAEA(二甲基氨基乙胺)以酰胺连接至C-末端苯丙氨酸的MMAF(MW801.5)
MMAF-TEG＝有四乙二醇酯化至苯丙氨酸的MMAF
MMAF-NtBu＝N-叔丁基作为酰胺附着于MMAF的C-末端
DM1＝N(2’)-脱乙酰基-N(2′)-(3-巯基-1-氧丙基)-美登素
DM3＝N(2’)-脱乙酰基-N2-(4-巯基-1-氧戊基)-美登素
DM4＝N(2’)-脱乙酰基-N2-(4-巯基-4-甲基-1-氧戊基)-美登素
本发明还提供了包含偶联有一种或多种细胞毒剂的本发明任何抗c-Met抗体或其它具有内吞活性的c-Met抗体(如，LY-2875358)的抗体-细胞毒性药物偶联物或其可药用盐或溶剂化合物(可互换的称为“抗体-药物偶联物”或“ADC”)，所述细胞毒剂诸如化疗剂、药物、生长抑制剂、毒素(例如细菌、真菌、植物或动物起源的酶活性毒素或其片段)或放射性同位素(即放射偶联物)。
在某些实施方案中，抗体-细胞毒性药物偶联物或其可药用盐或溶剂化合物包含抗c-Met抗体和化疗剂或其它毒素。本文中(上文))描述了可用于生成抗体-细胞毒性药物偶联物或其可药用盐或溶剂化合物的化疗剂。也可使用酶活性毒素及其片段，这些酶活性毒素及其片段已在说明书中描述。
在某些实施方案中，抗体-细胞毒性药物偶联物或其可药用盐或溶剂化合物包含抗c-Met抗体和一种或多种小分子毒素，包括但不限于小分子药物，诸如喜树碱衍生物、加利车霉素(calicheamicin)、美登木素生物碱(maytansinoids)、多拉司他汀(dolastatin)、澳瑞他汀、单端孢霉素(trichothecene)和CC1065及这些药物 具有细胞毒活性的片段。
例示性的接头L2包括6-马来酰亚氨基己酰基(“MC”)、马来酰亚氨基丙酰基(“MP”)、缬氨酸-瓜氨酸(“val-cit”或“vc”)、丙氨酸-苯丙氨酸(“ala-phe”)、对氨基苄氧羰基(“PAB”)、N-琥珀酰亚氨基4-(2-吡啶基硫代)戊酸酯(“SPP”)、N-琥珀酰亚氨基4-(N-马来酰亚氨基甲基)环己烷-1羧酸酯(“SMCC”)、和N-琥珀酰亚氨基(4-碘-乙酰基)氨基苯甲酸酯(“SIAB”)。本领域知道多种接头，下文也描述了一些。
接头可以是便于在细胞中释放药物的“可切割接头”。例如，可使用酸不稳定接头(例如腙)、蛋白酶敏感(例如肽酶敏感)接头、光不稳定接头、二甲基接头、或含二硫化物接头(Chari等,Cancer Research 52:127-131(1992)；美国专利No.5,208,020)。
在有些实施方案中，接头构件可以是将抗体连接至另一接头构件或药物模块的“延伸物单元”(stretcher unit)。例示性的延伸物单元显示于下文(其中波形线指示共价附着至抗体的位点)：
Figure PCTCN2016078699-appb-000017
在有些实施方案中，接头单元可以是氨基酸单元。在一个这样的实施方案中，氨基酸单元容许蛋白酶切割接头，由此便于在暴露于胞内蛋白酶(诸如溶酶体酶)后从抗体-细胞毒性药物偶联物或其可药用盐或溶剂化合物释放药物。参见例如Doronina等(2003)Nat.Biotechnol.21:778-784。例示性的氨基酸单元包括但不限于二肽、三肽、四肽、和五肽。例示性的二肽包括：缬氨酸-瓜氨酸(VC或val-cit)；丙氨酸-苯丙氨酸(AF或ala-phe)；苯丙氨酸-赖氨酸(FK或phe-lys)；或N-甲基- 缬氨酸-瓜氨酸(Me-val-cit)。例示性的三肽包括：甘氨酸-缬氨酸-瓜氨酸(gly-val-cit)和甘氨酸-甘氨酸-甘氨酸(gly-gly-gly)。氨基酸单元可以包含天然存在的氨基酸残基，以及次要氨基酸和非天然存在氨基酸类似物，诸如瓜氨酸。氨基酸单元可以在它们对特定酶(例如肿瘤相关蛋白酶，组织蛋白酶B、C和D，或血浆蛋白酶)的酶促切割的选择性方面进行设计和优化。
在有些实施方案中，接头构件可以是将抗体连接(或是直接的或是通过延伸物单元和/或氨基酸单元)至药物模块的“间隔物”单元。间隔物单元可以是“自我牺牲的”(self-immolative)或“非自我牺牲的”。“非自我牺牲的”间隔物单元指间隔物单元的部分或整体在ADC的酶促(蛋白水解)切割后保持结合于药物模块的间隔物单元。非自我牺牲的间隔物单元的例子包括但不限于甘氨酸间隔物单元和甘氨酸-甘氨酸间隔物单元。还涵盖对序列特异性酶促切割易感的肽间隔物的其它组合。例如，肿瘤细胞相关蛋白酶对含甘氨酸-甘氨酸间隔物单元的ADC的酶促切割将导致甘氨酸-甘氨酸-药物模块从ADC的剩余部分释放。在一个这样的实施方案中，甘氨酸-甘氨酸-药物模块然后在肿瘤细胞中进行分开的水解步骤，如此从药物模块切割甘氨酸-甘氨酸间隔物单元。
“自我牺牲的”间隔物单元容许释放药物模块而没有分开的水解步骤。在某些实施方案中，接头的间隔物单元包含对氨基苄基单元。在一个这样的实施方案中，将对氨基苯甲醇经酰胺键附着至氨基酸单元，并且在苯甲醇与细胞毒剂之间生成氨基甲酸酯、甲基氨基甲酸酯、或碳酸酯。参见例如Hamann等(2005)Expert Opin.Ther.Patents(2005)15:1087-1103。在一个实施方案中，间隔物单元是对氨基苄氧羰基(PAB)。
本发明中的示例性接头如下：
Figure PCTCN2016078699-appb-000018
接头，包括延伸物、间隔物、和氨基酸单元，可以通过本领域已知方法合成，诸如US2005-0238649A1中所记载的。
例示性的药物模块
美登素和美登木素生物碱
在有些实施方案中，抗体-细胞毒性药物偶联物或其可药用盐或溶剂化合物包含偶联有一个或多个美登木素生物碱分子的本发明抗体。美登木素生物碱是通过抑制微管蛋白多聚化来发挥作用的有丝分裂抑制剂。美登素最初从东非灌木齿叶美登木(Maytenus serrata)分离得到(美国专利No.3,896,111)。随后发现某些微生物也生成美登木素生物碱，诸如美登醇和C-3美登醇酯(美国专利No.4,151,042)。
美登木素生物碱药物模块在抗体-药物偶联物中是有吸引力的药物模块，因为它们：(i)相对易于通过发酵或发酵产物的化学修饰或衍生化来制备；(ii)易于用适于通过非二硫化物接头偶联至抗体的官能团衍生化；(iii)在血浆中稳定；且 (iv)有效针对多种肿瘤细胞系。
适于用作美登木素生物碱药物模块的美登素化合物是本领域公知的，而且可以依照已知方法从天然来源分离，或是使用遗传工程技术生产(参见Yu等(2002)PNAS 99:7968-7973)。美登醇和美登醇类似物也可以依照已知方法合成制备。
美登木素生物碱药物模块的例示性实施方案包括：DM1；DM3；和DM4，正如本文中所公开的。
澳瑞他汀和多拉司他汀
在有些实施方案中，抗体-细胞毒性药物偶联物或其可药用盐或溶剂化合物包含与多拉司他汀(dolastatin)或多拉司他汀肽类似物或衍生物(例如澳瑞他汀)(美国专利No.5,635,483；5,780,588)偶联的本发明抗体。多拉司他汀和澳瑞他汀已经显示出干扰微管动力学、GTP水解、及核和细胞分裂(Woyke等(2001)Antimicrob.Agents and Chemother.45(12):3580-3584)且具有抗癌(美国专利No.5663149)和抗真菌活性(Pettit等(1998)Antimicrob.Agents Chemother.42:2961-2965)。多拉司他汀或澳瑞他汀药物模块可经由肽药物模块的N(氨基)末端或C(羧基)末端附着于抗体(WO02/088172)。
例示性的澳瑞他汀实施方案包括N-末端连接的单甲基澳瑞他汀药物模块DE和DF，披露于Senter等,Proceedings of the American Association for CancerResearch,卷45,摘要号623,2004年3月28日，明确将其公开内容完整收入本文作为参考。肽药物模块可以选自下文通式DE和DF：
Figure PCTCN2016078699-appb-000019
其中DE和DF的波形线指示抗体或抗体-接头的共价附着位点，且每个位置是独立的；
R2选自H和C1-C8烃基；
R3选自H、C1-C8烃基、C3-C8碳环、芳基、C1-C8烃基-芳基、C1-C8烃基-(C3-C8碳环)、C3-C8杂环和C1-C8烃基-(C3-C8杂环)；
R4选自H、C1-C8烃基、C3-C8碳环、芳基、C1-C8烃基-芳基、C1-C8烃 基-(C3-C8碳环)、C3-C8杂环和C1-C8烃基-(C3-C8杂环)；
R5选自H和甲基；
或者R4与R5一起形成碳环且具有通式-(CRaRb)n-，其中Ra和Rb独立地选自H、C1-C8烃基和C3-C8碳环，而n选自2、3、4、5和6；
R6选自H和C1-C8烃基；
R7选自H、C1-C8烃基、C3-C8碳环、芳基、C1-C8烃基-芳基、C1-C8烃基-(C3-C8碳环)、C3-C8杂环和C1-C8烃基-(C3-C8杂环)；
每个R8独立地选自H、OH、C1-C8烃基、C3-C8碳环和O-(C1-C8烃基)；
R9选自H和C1-C8烃基；
R10选自芳基或C3-C8杂环；
Z为O、S、NH或NR12，其中R12为C1-C8烃基；
R11选自H、C1-C20烃基、芳基、C3-C8杂环、-(R13O)m-R14和-(R13O)m-CH(R15)2；
m是选自1-1000的整数；
R13为C2-C8烃基；
R14为H或C1-C8烃基；
R15每次出现独立为H、COOH、-(CH2)n-N(R16)2、-(CH2)n-SO3H或-(CH2)n-SO3-C1-C8烃基；
R16每次出现独立为H、C1-C8烃基或-(CH2)n-COOH；
R18选自-C(R8)2-C(R8)2-芳基、-C(R8)2-C(R8)2-(C3-C8杂环)和-C(R8)2-C(R8)2-(C3-C8碳环)；且
n是为选自0到6的整数。
通式DE的一种例示性澳瑞他汀，实施方案是MMAE，其中波形线指示共价附着至抗体-药物偶联物的接头(L)：
Figure PCTCN2016078699-appb-000020
通式DF的一种例示性澳瑞他汀，实施方案是MMAF，其中波形线指示共价附着至抗体-药物偶联物的接头(L)(参见US2005/0238649及Doronina等(2006)Bioconjugate Chem.17:114-124)：
Figure PCTCN2016078699-appb-000021
其它药物模块包括选自以下的MMAF衍生物，其中波形线指示共价附着至 抗体-药物偶联物的接头(L)：
Figure PCTCN2016078699-appb-000022
Figure PCTCN2016078699-appb-000023
一方面，可以将亲水性基团在R11处附着于药物模块，所述亲水性基团包括但不限于三乙二醇酯(triethylene glycol ester,TEG)，如上所述。不限于任何特定理论，所述亲水性基团有助于药物模块的内在化和不聚集(non-agglomeration)。包含澳瑞他汀/多拉司他汀或其衍生物的通式I ADC的例示性实施方案记载于US2005-0238649A1及Doronina等(2006)Bioconjugate Chem.17:114-124，明确收入本文作为参考。包含MMAE或MMAF及各种接头的通式I ADC的例示性实施方案具有如下结构和缩写(其中“Ab”是抗体；p是1到约8；“Val-Cit”是缬氨酸-瓜氨酸二肽；而“S”是硫原子)：
Figure PCTCN2016078699-appb-000024
                                     Ab-接头1-MC-vc-PAB-MMAF
Figure PCTCN2016078699-appb-000025
                                   Ab-接头1-MC-vc-PAB-MMAE
Figure PCTCN2016078699-appb-000026
                                   Ab-接头1-MC-MMAE
Figure PCTCN2016078699-appb-000027
                                 Ab-接头1-MC-MMAF
典型的是，基于肽的药物模块可通过在两个或更多氨基酸和/或肽片段之间形成肽键来制备。此类肽键可依照例如肽化学领域众所周知的液相合成法来制备(参见E.Schroder和K.Lübke,“The Peptides”,卷1,pp76-136,1965,Academic Press)。澳瑞他汀/多拉司他汀药物模块可依照以下文献中的方法来制备)US2005-0238649A1；美国专利No.5635483；美国专利No.5780588；Pettit等(1989)J.Am.Chem.Soc.111:5463-5465；Pettit等(1998)Anti-Cancer Drug Design 13:243-277；Pettit,G.R.等,Synthesis,1996,719-725；Pettit等(1996)J.Chem.Soc.Perkin Trans.15:859-863；及Doronina(2003)Nat.Biotechnol.21(7):778-784。
具体而言，通式DF的澳瑞他汀/多拉司他汀药物模块诸如MMAF及其衍生物可以使用US2005-0238649A1及Doronina等(2006)Bioconjugate Chem.17:114-124中记载的方法来制备。通式DE的澳瑞他汀/多拉司他汀药物模块诸如MMAE及其衍生物可以使用Doronina等(2003)Nat.Biotech.21:778-784中记载的方法来制备。可以通过常规方法方便地合成药物-接头模块MC-MMAF、MC-MMAE、MC-vc-PAB-MMAF和MC-vc-PAB-MMAE，例如Doronina等(2003)Nat.Biotech.21:778-784及美国专利申请公开号US2005/0238649A1中所记载的，然后将它们偶联至感兴趣的抗体。
药物载荷
药物载荷(loading)由y表示，即通式I的分子中每个抗体的平均药物模块数。 药物载荷的范围可以为每个抗体1-20个药物模块(D)。通式I的ADC包括偶联有一定范围(1-20个)药物模块的抗体的集合。来自偶联反应的ADC制备物中每个抗体的平均药物模块数可以通过常规手段来表征，诸如质谱、ELISA测定法、和HPLC。还可以测定ADC在y方面的定量分布。在有些情况中，将p为某数值的同质ADC从具有其它药物载荷的ADC中分离、纯化、和表征可以通过诸如反相HPLC或电泳的手段来实现。
对于有些抗体-药物偶联物，y可能受到抗体上附着位点数目的限制。例如，若附着是半胱氨酸硫醇，正如上文例示性实施方案中的那样，则抗体可能只有一个或数个半胱氨酸硫醇基，或者可能只有一个或数个有足够反应性的硫醇基，可附着接头。在某些实施方案中，较高的药物载荷，例如y>5，可引起某些抗体-药物偶联物的聚集、不溶性、毒性、或丧失细胞通透性。在某些实施方案中，本发明ADC的药物载荷的范围为1到约8；约2到约6；约3到约5；约3到约4；约3.1到约3.9；约3.2到约3.8；约3.2到约3.7；约3.2到约3.6；约3.3到约3.8；或约3.3到约3.7。事实上，对于某些ADC已经显示了每个抗体药物模块的最佳比率可以为小于8，可以为约2到约5。参见US2005-0238649A1(完整收入本文作为参考)。
在某些实施方案中，在偶联反应中将少于理论最大值的药物模块偶联至抗体。抗体可包含例如赖氨酸残基，其不与药物-接头中间物或接头试剂起反应，如下文所讨论的。只有最具反应性的赖氨酸基团可以与胺反应性接头试剂起反应。一般而言，抗体不包含许多游离的和反应性的半胱氨酸硫醇基，其可连接药物模块；事实上，抗体中的大多数半胱氨酸硫醇基以二硫桥形式存在。在某些实施方案中，可以在部分或完全还原性条件下用还原剂诸如二硫苏糖醇(DTT)或三羰基乙基膦(TCEP)还原抗体以产生反应性半胱氨酸硫醇基。在某些实施方案中，将抗体置于变性条件以暴露反应性亲核基团，诸如赖氨酸或半胱氨酸。
ADC的载荷(药物/抗体比率DAR)可以以不同方式来控制，例如通过：(i)限制药物-接头中间物或接头试剂相对于抗体的摩尔过量，(ii)限制偶联反应的时间或温度，(iii)半胱氨酸硫醇修饰的部分或限制还原性条件，(iv)通过重组技术对抗体的氨基酸序列进行工程改造，使得半胱氨酸残基的数目和位置为了控制接头-药物附着的数目和/或位置而进行改变(诸如如本文中和WO2006/034488(完整收入本文作为参考)中所述而制备的thioMab或thioFab)。
应当理解，若超过一个亲核基团与药物-接头中间物或者与接头试剂和接下来的药物模块试剂起反应，则所得产物是具有一个或多个药物模块附着于抗体之分布的ADC化合物混合物。可以通过对抗体特异性的和对药物特异性的双重ELISA抗体测定法自混合物计算每个抗体的平均药物数。混合物中的各种ADC 分子可以通过质谱来鉴定，并通过HPLC来分离，例如疏水相互作用层析。在某些实施方案中，可以通过电泳或层析从偶联混合物中分离具有单一载荷值的同质ADC。
制备抗体-细胞毒性药物偶联物或其可药用盐或溶剂化合物的某些方法
可以采用本领域技术人员知道的有机化学反应、条件和试剂通过数种路径来制备通式I的ADC，包括：(1)抗体的亲核基团经共价键与二价接头试剂反应形成Ab-L，接着与药物模块D反应；和(2)药物模块的亲核基团经共价键与二价接头试剂反应形成D-L，接着与抗体的亲核基团反应。经后一种路径制备通式I的ADC的例示性方法记载于US2005-0238649A1，明确收入本文作为参考。
抗体的亲核基团包括但不限于：(i)N末端胺基；(ii)侧链胺基，例如赖氨酸；(iii)侧链硫醇基，例如半胱氨酸；和(iv)糖基化抗体中糖的羟基或氨基。胺、硫醇、和羟基是亲核的，能够与接头模块上的亲电子基团反应而形成共价键，而接头试剂包括：(i)活性酯类，诸如NHS酯、HOBt酯、卤代甲酸酯、和酸性卤化物；(ii)烃基和苄基卤化物，诸如卤代乙酰胺；(iii)醛、酮、羧基和马来酰亚胺基团。某些抗体具有可还原的链间二硫化物，即半胱氨酸桥。可通过还原剂诸如DTT(二硫苏糖醇)或三羰基乙基膦(TCEP)处理使抗体完全或部分还原，从而具有与接头试剂偶联的反应性。每个半胱氨酸桥理论上将形成两个反应性硫醇亲核体。或者，可经由赖氨酸残基的修饰将硫氢基引入抗体，例如通过使赖氨酸残基与2-亚氨基硫烷(Traut氏试剂)起反应，导致胺转变为硫醇。
还可通过抗体上的亲电子基团(诸如醛或酮羰基)与接头试剂或药物上的亲核基团之间的反应来生成本发明的抗体-药物偶联物。接头试剂上的有用亲核基团包括但不限于酰肼(hydrazide)、肟(oxime)、氨基(amino)、肼(hydrazine)、缩氨基硫脲(thiosemicarbazone)、肼羧酸酯(hydrazine carboxylate)、和芳基酰肼(arylhydrazide)。在一个实施方案中，可以用例如高碘酸盐氧化剂氧化糖基化抗体的糖，从而形成可与接头试剂或药物模块的胺基团反应的醛或酮基团。所得亚胺Schiff碱基可形成稳定的连接，或者可以用例如硼氢化物试剂还原而形成稳定的胺连接。在一个实施方案中，糖基化抗体的碳水化合物部分与半乳糖氧化酶或偏高碘酸钠的反应可以在抗体中生成羰基(醛基和酮基)，它可与药物上的适宜基团反应(Hermanson,Bioconjugate Techniques)。在另一个实施方案中，包含N-末端丝氨酸或苏氨酸残基的抗体可以与偏高碘酸钠反应，导致在第一个氨基酸处生成醛(Geoghegan和Stroh,(1992)Bioconjugate Chem.3:138-146；US5362852)。这样的醛可与药物模块或接头亲核体反应。
药物模块上的亲核基团包括但不限于：胺、硫醇、羟基、酰肼、肟、肼、缩氨基硫脲、肼羧酸酯、和芳基酰肼基团，它们能够与接头模块上的亲电子基团反 应而形成共价键，而接头试剂包括：(i)活性酯类，诸如NHS酯、HOBt酯、卤代甲酸酯、和酸性卤化物；(ii)烃基和苄基卤化物，诸如卤代乙酰胺；(iii)醛、酮、羧基、和马来酰亚胺基团。
本发明的化合物明确涵盖但不限于用如下交联试剂制备的ADC：BMPS、EMCS、GMBS、HBVS、LC-SMCC、MBS、MPBH、SBAP、SIA、SIAB、SMCC、SMPB、SMPH、sulfo-EMCS、sulfo-GMBS、sulfo-KMUS、sulfo-MBS、sulfo-SIAB、sulfo-SMCC和sulfo-SMPB、及SVSB(琥珀酰亚氨基-(4-乙烯基砜)苯甲酸酯)它们可通过商业途径获得(例如Pierce Biotechnology,Inc.,Rockford,IL.,U.S.A参见2003-2004年度应用手册和产品目录(2003-2004Applications Handbook and Catalog)第467-498页)。
还可使用多种双功能蛋白质偶联剂来制备包含抗体和细胞毒剂的抗体-细胞毒性药物偶联物或其可药用盐或溶剂化合物，诸如N-琥珀酰亚氨基3-(2-吡啶基二硫代)丙酸酯(SPDP)，琥珀酰亚氨基-4-(N-马来酰亚氨基甲基)环己烷-1-羧酸酯(SMCC)，亚氨基硫烷(IT)，亚氨酸酯(诸如盐酸己二酰亚氨酸二甲酯)、活性酯类(诸如辛二酸二琥珀酰亚氨基酯)、醛类(诸如戊二醛)、双叠氮化合物(诸如双(对-叠氮苯甲酰基)己二胺)、双重氮衍生物(诸如双(对-重氮苯甲酰基)-乙二胺)、二异硫氰酸酯(诸如甲苯2,6-二异氰酸酯)、和双活性氟化合物(诸如1,5-二氟-2,4-二硝基苯)的双功能衍生物。例如，可以如Vitetta等,Science 238:1098(1987)中所述制备蓖麻毒蛋白免疫毒素。碳-14标记的1-异硫氰酸苄基-3-甲基二亚乙基三胺五乙酸(MX-DTPA)是用于将放射性核苷酸与抗体偶联的例示性螯合剂。参见WO94/11026。
或者，可通过例如重组技术或肽合成来制备包含抗体和细胞毒剂的融合蛋白。重组DNA分子可以包含各自编码偶联物的抗体和细胞毒部分的区域，彼此或是毗邻或是由编码接头肽的区域分开，该接头肽不破坏偶联物的期望特性。
在又一个实施方案中，可以将抗体与“受体”(诸如链霉亲合素)偶联从而用于肿瘤预先靶向，其中对患者施用抗体-受体偶联物，接着使用清除剂由循环中清除未结合的偶联物，然后施用与细胞毒剂(例如放射性核苷酸)偶联的“配体”(例如亲合素)。提供以下实施例仅为说明性目的，并不旨在限制本发明的范围。
具体实施方式
以下结合实施例用于进一步描述本发明，但这些实施例并非限制本发明的范围。
本发明实施例或测试例中未注明具体条件的实验方法，通常按照常规条件，或按照原料或商品制造厂商所建议的条件。参见Sambrook等，分子克隆，实验 室手册，冷泉港实验室；当代分子生物学方法，Ausubel等著，Greene出版协会，Wiley Interscience，NY。未注明具体来源的试剂，为市场购买的常规试剂。
实施例
实施例1.抗原抗体克隆表达
本发明所用抗体(轻、重链)，抗原用领域周知的重叠延伸PCR方法构建，将重叠延伸PCR得到的DNA片段用HindIII/BstBI这两个酶切位点插入到表达载体pEE6.4((Lonza Biologics)，在293F细胞(Invitrogen,Cat#R790-07)中表达得到。所得重组蛋白用于免疫或筛选。c-Met基因模板来源于origene公司(货号RC217003)。所克隆表达的DNA序列如下。
人c-Met细胞外区域(ECD)和鼠Fc区域融合蛋白(human c-Met ECD-mFc)DNA序列:
Figure PCTCN2016078699-appb-000028
Figure PCTCN2016078699-appb-000029
人c-Met细胞外Sema区域和Flag-His标签(Human c-Met Sema-Flis)DNA序列：
Figure PCTCN2016078699-appb-000030
Figure PCTCN2016078699-appb-000031
人c-Met ECD his标签(Human c-Met ECD-His)重组蛋白DNA序列：
Figure PCTCN2016078699-appb-000032
Figure PCTCN2016078699-appb-000033
实施例2.抗原抗体结合实验(ELISA)
本实验是利用酶联免疫吸附的方法检验抗c-Met抗体(包括杂交瘤上清或重组表达的单克隆抗体等)在体外对c-Met抗原的亲和力。
实验步骤为：用包被液(PBS)(Hyclone,Cat No.:SH30256.01B)稀释抗原(human c-Met-His，实施例1)至2μg/ml，加入100μl/孔于96孔酶标板(Costar9018,Cat No.:03113024)，4℃孵育过夜。次日将包被有抗原的96孔酶标板恢复至室温，洗涤液(PBS+0.05％Tween20(Sigma,Cat No.:P1379)洗涤三次。随后加入200μl/孔封闭液(PBS+1％BSA(Roche,Cat No.:738328)，37℃孵育1小时。洗涤液洗涤三次。加入待测抗c-Met抗体于96孔酶标板，室温孵育1小时。洗涤液洗涤三次。加入封闭液10000倍稀释二抗(Goat anti-Mouse IgG(H+L)(HRP)(Thermo，货号:31432)100μl/孔至96孔酶标板，室温孵育1小时。洗涤液洗涤三次。加入100μl/孔显色液TMB(eBioscience REF:00-4201-56)至96孔酶标板。加入100μl/孔终止液(2N H2SO4)至96孔酶标板。使用读板仪450nm读板。
实施例3.抗人c-Met的小鼠单克隆抗体细胞株产生
本发明通过免疫小鼠，脾细胞融合、杂交瘤筛选方法得到小鼠来源抗人c-Met单克隆细胞株。该方法为本领域熟知。即将重组表达抗原(human c-Met ECD-mFc,human c-Met Sema-flis，见实施例1)用PBS(Hyclone,Cat No.:SH30256.01B)稀释至1mg/ml，与弗氏佐剂(首次免疫用完全弗氏佐剂，加强免疫用不完全弗氏佐剂)乳化后，皮下注射接种Balb/C小鼠(每组5只)，每只小鼠接种100μg抗原，间隔两周加强免疫。从第一次加强免疫开始，每次加强免疫后的7至10天内采集小鼠血清用ELISA测定(方法见实施例2)效价。
选择免疫后血清效价高于1：105的小鼠进行细胞融合。分别无菌制备小鼠B细胞和骨髓瘤细胞(SP2/0，ATCC number：CRL-1581TM)并计数，将两种细胞按照B细胞:SP2/0＝1:4的比例进行混合后离心(1500r/min，7min)，弃上清加入1ml50％聚乙二醇(Supplier:SIGMA,Catalogue#RNBB306)，之后用1ml无血清RPMI1640(Supplier:GIBCO,Catalogue#C22400)终止，离心10min，弃上清， 用含杂交瘤细胞生长因子(Supplier:Roche,Catalogue#1363735001)，血清(Supplier:GIBCO,Catalogue#C20270)和HAT(Supplier:Invitrogen,Catalogue#21060-017)的RPMI1640重悬沉淀，按照每孔105个B细胞的标准铺板，每孔100μl，置37℃细胞培养箱中培养，3天后加入每孔100μl含杂交瘤细胞生长因子，血清和HT(Supplier:Invitrogen,Catalogue#11067-030)的RPMI1640，2至4天后全换液每孔加入150μl含杂交瘤细胞生长因子，血清和HT的RPMI1640，次日ELISA(方法见实施例2)检测阳性克隆。结果见表1.
表1.人c-Met免疫小鼠杂交瘤融合检测
克隆号 检测结果(OD450)
阴性对照 0.07
Ab-1 1.48
Ab-2 1.38
Ab-3 1.29
Ab-4 1.6
Ab-5 1.64
Ab-6 1.75
Ab-7 1.58
Ab-8 1.24
实施例4抗人c-Met单克隆抗体对胃癌细胞MKN45增值的抑制作用
选择上述克隆，进一步培养得到单克隆，ELISA验证了结合活性后，挑选单克隆培养上清进行细胞活性检测。该实验原理是利用本发明抗人c-Met抗体能抑制人胃癌细胞(MKN45)表面c-Met发生磷酸化，而抑制MNK45细胞的增殖。
人胃癌细胞(MKN45，JCRB，JCRB0254，P11)1×105个细胞/mL，50μl/孔加至96孔细胞培养板(costar,#3799)，培养基为RPMI 1640medium:(GIBCO,cat#11835)+10％胎牛血清(FBS)(GIBCO-10099141)。随后加入50μl/孔抗人c-Met待测抗体，37度培养箱(厂商：SANYO设备编号TINC035)中培养5天。使用细胞增殖检测试剂盒(
Figure PCTCN2016078699-appb-000034
Luminescent Cell Viability Assay)(Promega,G7573)，按试剂盒说明书方法检测细胞增殖情况。读板仪(PerkinElmer,TREA001-RDA-IBA100)读板。使用如下公式计算细胞的增值百分率：细胞增殖率％＝(1-实验组细胞读数/不作处理组细胞读数)×100％。结果见表2。
表2.抗人c-Met单克隆抗体细胞活性
克隆号 MKN45抑制率(％)
空白对照 0.01
Ab-1 59.2
Ab-2 58.4
Ab-3 59.6
Ab-4 54.9
Ab-5 77.4
Ab-6 70.8
Ab-7 56.4
Ab-8 53.8
实施例5.抗c-Met抗体序列克隆
将实施例4中得到的活性好的单细胞株Ab-5进行cDNA序列克隆，然后重组表达出单克隆抗体并进行各项活性检测。本发明用逆转录PCR扩增抗体基因的重链和轻链可变区，连接到载体测序得到单克隆抗体轻重链序列。首先采用RNA纯化试剂盒(Qiagen公司，货号74134,步骤见此说明书)提取实施例4中活性好的单细胞株的细胞总RNA。然后使用Invitrogen公司的货号为18080-051cDNA合成试剂盒制备cDNA单链，即Oligo-dT primers cDNA反转录。以此为模版，采用PCR方法合成抗体轻重链可变区序列，PCR产物克隆到TA载体pMD-18T，然后送去测序。将得到的抗体轻重链序列分别克隆到表达载体(见实施例1)，表达重组单克隆抗体，验证活性(见实施例2，4)后，进行人源化工作。
本发明小鼠杂交瘤细胞单克隆抗体Ab-5的序列：
重链可变区：
Figure PCTCN2016078699-appb-000035
轻链可变区：
Figure PCTCN2016078699-appb-000036
抗人c-Met抗体的VH/VL CDR的氨基酸残基由Kabat编号系统确定并注释。
本发明中鼠源的CDR序列如表3所述：
表3.鼠源抗硬骨素抗体的CDR序列
抗体 Ab-5
重链CDR1 NYGVH(SEQ ID NO:6)
重链CDR2 VIWSGGSTNYAAAFVS(SEQ ID NO:7)
重链CDR3 NHDNPYNYAMDY(SEQ ID NO:8)
轻链CDR1 RANKSVSTSTYNYLH(SEQ ID NO:9)
轻链CDR2 LASNLAS(SEQ ID NO:10)
轻链CDR3 QHSRDLPPT(SEQ ID NO:11)
实施例6.抗c-Met抗体人源化
将实施例5得到鼠源抗c-Met单克隆抗体轻重链序列在抗体数据库里进行同源性比较后，建立人源化抗体模型，根据模型选择回复突变筛选最优的人源化抗c-Met单克隆抗体为本发明优选分子。该方法从已经发表的小鼠Fab晶体结构模型数据库(比如PDB数据库)中查找与所得鼠源候选分子同源性相似的晶体结构开始，挑取高分辨率(比如
Figure PCTCN2016078699-appb-000037
)的Fab晶体结构，建立小鼠Fab模型。将本发明鼠源抗体轻重链序列和模型中的序列比对，保留和模型中和鼠源抗体序列一致的序列，得到本发明鼠抗体的结构模型，其中不一致氨基酸为可能的回复突变位点。用Swiss-pdb viewer软件运行鼠抗体结构模型，优化能量(最小化)。将模型中除CDR外的不同氨基酸位点进行回复突变，将所得的突变抗体(人源化)和人源化之前抗体对比进行活性检测。保留活性好的人源化抗体。之后，对CDR区域优化，包括避免糖基化，脱酰胺化，氧化位点等。优化后的人源化抗c-Met抗体的CDR区如表4所示：
表4.优化的抗c-Met抗体的CDR序列
抗体 优化的人源化抗体
重链CDR1 NYGVH(SEQ ID NO:6)
重链CDR2 VIWSGGSTNYAAAFVS(SEQ ID NO:7)
重链CDR3 NHDNPYNYAMDY(SEQ ID NO:8)
轻链CDR1 RADKSVSTSTYNYLH(SEQ ID NO:12)
轻链CDR2 LASNLAS(SEQ ID NO:10)
轻链CDR3 QHSRDLPPT(SEQ ID NO:11)
人源化以后的轻重链可变区如以下所示：
1、重链可变区：
Figure PCTCN2016078699-appb-000038
Figure PCTCN2016078699-appb-000039
2、轻链可变区：
Figure PCTCN2016078699-appb-000040
人源化以后的轻重链和IgG Fc区段重组，得到本发明人源化抗c-Met单克隆抗体。所用的Fc序列任选自以下序列：
重链恒定区：
Figure PCTCN2016078699-appb-000041
Figure PCTCN2016078699-appb-000042
轻链恒定区：
Figure PCTCN2016078699-appb-000043
用基因克隆、重组表达的方法分别克隆、表达、纯化上述抗体，经ELISA(实施例2)和体外结合活性检测(实施例7)，最终选出活性保持最好的人源化抗体Ab-9，Ab-10，Ab-11，序列如下：
Ab-9人源化抗体：
重链：
Figure PCTCN2016078699-appb-000044
Figure PCTCN2016078699-appb-000045
轻链：
Figure PCTCN2016078699-appb-000046
Ab-10人源化抗体：
重链：
Figure PCTCN2016078699-appb-000047
轻链：
Figure PCTCN2016078699-appb-000048
Ab-11人源化抗体：
重链：
Figure PCTCN2016078699-appb-000049
Figure PCTCN2016078699-appb-000050
轻链：
Figure PCTCN2016078699-appb-000051
实施例7.抗c-Met人源化抗体体结合外活性检测
本发明人源化抗体体外活性除了用ELISA(实施例2)检测之外，还检测了其与c-Met高表达细胞株MKN45的结合，及其与c-Met抗原的亲和力(BIACore测定)。结果见表5和表6。
用FACS方法检测本发明抗c-Met人源化体同c-Met高表达细胞株MKN45的结合活性。
在具有10％(v/v)胎牛血清(FBS)(GIBCO，Cat No.:10099-141)和青霉素/链霉素(GIBCO，Cat No.:15070-063)的RPMI 1640培养基(GIBCO，Cat No.:11835-030)中重悬浮MKN45细胞(JCRB，Cat No.:JCRB0254)至10000,000细胞/mL。将2mL重悬的MKN45细胞以150,000细胞/孔加入96孔微滴定板(Corning，Cat No.:3799)中，加入8个浓度(20μg/ml起5倍浓度梯度稀释)的c-Met抗体至对应的孔中，最终体积为100μl，在4度孵育1小时。加入FACS缓冲液(具有2.5％(v/v)胎牛血清(FBS)的磷酸盐缓冲液(PBS)(Hyclone，Cat：SH30256.01B)，4度，1300rpm，4分钟，离心弃上清，重复三次。每孔加入100μl二抗溶液(荧光标记羊抗鼠二抗:1:200稀释，Biolegend，Cat#405307；荧光标记抗人二抗:1:30稀释，Biolegend，Cat#409304)，在4度孵育1小时。加入FACS缓冲液，4度，1300rpm，4分钟，离心弃上清，重复三次。加入200μl FACS缓冲液，重悬细胞，准备好样品，使用流式细胞仪(BD FACS Array)检测。
本发明采用表面等离子共振技术(surface plasmon resonance,SPR)检测c-Met抗体与c-Met抗原Sema-His之间的亲和力。
Anti-mouse IgG(GE Life Sciences catalog#BR-1008-38)或anti-human IgG(GE Life Sciences catalog#BR-1008-39)抗体用pH5.0乙酸钠溶液(GE Healthcare，Cat#BR-1003-51)分别稀释至30μg/ml和50μg/ml，用氨基偶联试剂盒(GE Life Sciences，Cat#BR100050)固定化至CM5芯片(GE Life Sciences catalog#BR-1000-12)的试验及对比通道，偶联水平设定在15000RU。运行缓冲液PBS(Hyclone,Cat#SH30256.01B)+0.05％P20(GE Life Sciences, Cat#BR-1000-54))稀释c-Met抗体至1.5μg/ml，运行缓冲液稀释抗原sema-his至200nM，之后用同样缓冲液1:2倍稀释直至0.78nM。将稀释好的抗体在30μl/min的流速下流过实验通道一分钟，抗原在同样的流速下流经实验通道和对比通道3分钟，解离10分钟后将流速调至10μl/min，在实验通道和对比通道流过再生缓冲液3分钟。数据经双重扣减后用BiaEvaluation 4.1拟合，拟合模型用1：1(Langmuir)模型。
表5.人源化抗c-Met抗体的结合活性
人源化抗体 Ab-9 Ab-10 Ab-11
ELISA检测(EC50,nM) 0.13 0.39 0.2
表6.人源化抗c-Met抗体的MKN45结合活性及抗原亲和力
人源化抗体 MKN45/FCAS结合活性(nM) 和抗原亲和力Biacore(nM)
Ab-9 1.6 4
Ab-10 1.23 8
上述结果表明，本发明人源化抗体和抗原的结合活性在0.13-8nM，因检测方法的不同，检测结果有所不同。结果表明了人源化后的抗c-Met抗体保留了人源化之前抗体的结合活性。
实施例8.抗c-Met人源化抗体体外功能，细胞活性评价
为了检测本发明抗体的功能，用阻断c-Met ligand(肝细胞生长因子HGF)和c-Met的结合实验，以及抑制细胞增值实验(实施例4)对实施例7中的抗体进行了评估。
HGF与c-Met的结合造成c-Met分子的酪氨酸磷酸化以及c-Met信号通路的活化。本实验通过ELISA方法检测本发明抗c-Met抗体阻断HGF结合在受体c-Met蛋白上的活力，即IC50。
c-Met ECD-mFc(实施例子1)稀释在PBS(Hyclone,Cat#SH30256.01B)中，终浓度为2μg/ml,包被在96孔ELISA板(Costar,cat#2592)常温过夜。使用洗板机(Suppler:BioTex；Model:ELX405；S/N:251504)用PBST(PBS+0.05％Tween20(Simga,Cat#P1379))洗涤3次，300μl封闭液PBS+1％BSA(Roche,Cat#738328)加入到96孔板37℃孵育60分钟。用PBST洗涤3次后，封闭液稀释好的抗体50μl加入到96孔中，37℃孵育90分钟。添加封闭液稀释好的终浓度为20ng/ml的人HGF(Sino Biological,#10463-HNAS)50μl到含有c-Met抗体的96孔板中，在室温孵育120分钟。用PBST洗涤三次后，添加100μl封闭液稀释好的终浓度为100ng/ml生物素标记的抗HGF抗体(R&D，Cat#BAF294) 到96孔板中孵育90分钟。PBST洗涤后，添加100μl封闭液稀释好的辣根过氧化物酶(ebioscience,#18-4100-51)到96孔板中孵育30分钟。PBST洗涤后，加入100μl底物(ebioscience,cat#00-4201-56)室温孵育10分钟，加入100μl终止液(2N H2SO4)，用450nM酶标仪(Supplier:Moleculer Devices；Model:MNR0643；Equip ID:TMRP001)读数。使用SoftMax Pro v5进行数据分析。结果见表7。
表7.本发明人源化抗c-Met抗体的体外功能，细胞活性
Figure PCTCN2016078699-appb-000052
上述结果表明，本发明人源化抗体不仅保留了和抗原的结合活性，而且能够阻止抗原和配体(ligand)的结合，同时显示了抑制肿瘤细胞的生长活性。
实施例9本发明抗c-Met人源化抗体的激动剂(agonist)活性评估
抗c-Met抗体阻止HGF/c-Met结合，也有可能激活c-Met信号，即具有激动剂活性。抗c-Met激动剂活性不是本发明所需要的。为了检测本发明抗体是否有激动剂活性，用c-Met磷酸化，人肾透明细胞癌皮肤转移细胞(caki-1)增值，人肺癌H441细胞迁移等三项实验进行检测评估。
HGF与c-Met的结合激活c-Met分子的酪氨酸磷酸化以及活化c-Met信号通路。因此，HGF激活c-Met用于激动剂实验的正对照，用人肺癌细胞株A459来评估诱导c-Met酪氨酸残基1349处磷酸化的作用强弱。
将A549cells悬浮在Ham’s F12K+2mM谷氨酰胺(Invitrogen,#21127-022)+10％(v/v)胎牛血清(FBS)(GIBCO,#10099141)，取0.2mL细胞悬液加入到96孔板(Corning,#3599)，细胞浓度是60,000细胞/孔。37℃，5％CO2条件下孵育24小时。24小时后弃96孔的培养基，加入100μL的低血清培养基(Ham’s F12K+2mM谷氨酰胺+0.5％FBS)37℃，5％CO2条件下饥饿6小时。抗体用上面的低血清培养基进行稀释(终浓度为20μg/ml)。阳性对照HGF的浓度是200ng/ml。37℃孵育15分钟。弃掉培养基后，加入50μl细胞裂解液(10mM Tris,150mM NaCl,2mM EDTA),50mM NaF,1％(v/v)TRITON-X100,蛋白酶抑制剂(Roche cat#05892791001),磷酸酶抑制剂cocktail II(Sigma#P5726)及磷酸酶抑制剂cocktail III(Sigma#P0044)。细胞裂解后用ELISA方法检测c-Met酪氨酸磷酸化。c-Met capture antibody(CST,cat#3148s)用PBS1:1000稀释后加入到96孔ELISA板(costar,cat#9018)，每个孔100μl,在4度孵育过夜。用TBS-T洗涤3次后加入300μl封闭液(TBS-T plus 2％(w/v)BSA)孵 育1小时。用TBS-T洗涤3次后加入75μL细胞封闭液，加入25μl细胞裂解物，4度孵育过夜。用TBS-T洗涤3次，用封闭液1:1000稀释pY1349c-Met抗体(cell signal,#3133)，每个孔100μl。室温孵育2小时后TBST洗涤4次，用封闭液1:12000稀释HRP标记的羊抗兔多抗(cell signaling,cat#7074),每个孔100μl室温孵育1小时。TBS-T洗涤5次，加入100μL of TMB(ebioscience#TMB,004201)到每个孔，再加入100μl终止液(2N H2SO4)，用450nM酶标仪(Supplier:Moleculer Devices；Model:MNR0643；Equip ID:TMRP001)读数。使用SoftMax Pro v5进行数据分析。结果见表8。
人肾透明细胞癌皮肤(Caki-1)细胞表达肝细胞生长因子受体(c-Met)，HGF能结合c-Met刺激Caki-1细胞增殖。因此，本发明的人源化抗c-Met抗体和HGF平行出来Caki-1细胞，可评价抗c-Met抗体的激动剂活性。
Caki-1(上海中科院，TCHu135，P12)细胞1000个/孔加至96孔细胞培养板(costar,#3799)，培养基为McCoy’s 5A(invitrogen,#16600)+10％胎牛血清(FBS)(GIBCO-10099141)，37℃，24小时。随后细胞饥饿24小时(细胞饥饿培养基为McCoy’s 5A+0.5％胎牛血清)。饥饿后，细胞用梯度稀释的抗c-Met抗体(最高浓度为20μg/ml)及阳性对照处理5天后，用细胞增殖检测试剂盒(
Figure PCTCN2016078699-appb-000053
Luminescent Cell Viability Assay(Promega,G7573)检测细胞增殖情况。读板仪(厂商：PerkinElmer设备编号:TREA001-RDA-IBA100)读板。计算细胞的增值百分率：细胞增殖％＝实验组细胞读数/不作处理组细胞读数×100％。结果表明，本发明抗体均对Caki-1细胞没有增值作用。见表8。
抗c-Met抗体如果有激动剂活性，能影响细胞的迁移能力。本发明用表达c-Met的H441细胞株来评估本发明c-Met抗体影响细胞迁移能力。
在具有10％(v/v)胎牛血清(FBS)(GIBCO，Cat No.:10099-141)和青霉素/链霉素(GIBCO，Cat No.:15070-063)的RPMI 1640培养基(GIBCO，Cat No.:11835-030)中重悬浮H441细胞(ATCC，Cat No.:HTB-174)至500,000细胞/ml。将重悬的H441细胞以1ml/孔加入12孔培养板(Costar，Cat No.:3513)中，在37℃，5％CO2下培养3天，用磷酸盐缓冲液(PBS)洗两遍细胞，加入含低浓度胎牛血清(0.5％FBS)的RPMI 1640培养基，37℃，5％CO2下培养16小时。用5ml枪头在每个孔的底部划痕，并用含低浓度胎牛血清培养基清洗一遍，加入1ml低浓度血清培养基RPMI 1640，在4倍倒置显微镜下随机选取划痕区域照相保存并在培养板做好标记，此时的记为时间零点，细胞用10μg/ml的c-Met抗体或HGF对照(200ng/ml)处理(37℃，5％CO2)16小时后，在4倍倒置显微镜下将已标记的划痕区域照相保存，此时的记为迁移后时间。影响细胞迁移百分比为相对于零点时的迁移距离除以培养基组相对于零点时的迁移距离乘以100。结果见 表8。
表8.本发明人源化抗c-Met抗体的激动剂活性评价
人源化抗体 激活c-Met磷酸化(％)* 刺激Caki-1增值所用 H441迁移率(％)#
Ab-9 29.9 无 53
Ab-10 33.7 无 34
*：抗体浓度为20μg/ml,对照HGF(200ng/ml)激活c-Met磷酸化设为100％.#：抗体浓度为20μg/ml,HGF(200ng/ml)影响H441迁移率设为100％。
上述结果表明，本发明人源化抗体激动剂活性比较弱(c-Met磷酸化，H441迁移实验结果)或没有(20μg/ml抗体未能看到刺激Caki-1增值)。
实施例10抗c-Met抗体体内药效评价
为了评价本发明抗体的抗肿瘤活性，用BALB/c裸小鼠皮下移植人源胃癌MKN45细胞模型进行检测。
MKN45细胞用RPMI-1640培养基(10％胎牛血清)单层培养，培养条件为37℃，5％CO2。在对数生长期计数并且收集细胞。将细胞用PBS重悬至合适浓度，在小鼠(BALB/c Nude小鼠，雌性，10周，体重22-28g。购自上海斯莱克实验动物有限公司，动物合格证编号：2007000548777。饲养环境：SPF级。)右翼皮下接种0.1ml3×106细胞。肿瘤平均体积达到114mm3时，称量体重，测量肿瘤体积，分组，开始给药。对照组给PBS,抗体治疗组给本发明抗体5mg/kg,每周一次，给药两次。每周2次，测量肿瘤体积和体重，第25天终止试验。肿瘤大小计算公式：肿瘤体积(mm3)＝0.5×(肿瘤长径×肿瘤短径2)。抑瘤率计算公式：抑瘤率＝(V0-VT)/V0×100％，其中V0、VT分别为实验开始时及实验结束时的肿瘤体积。
结果表明本发明抗体Ab-9，Ab-10，的肿瘤抑制率分别为56％，和64％。小鼠体重在试验过程中无明显变化(22-24g)。该结果表明，本发明人源化抗c-Met抗体在体内有抑制肿瘤生长的活性。
实施例11抗c-Met抗体的内吞作用
本发明抗体结合人c-Met，具有非常好的体外活性，体内抑制肿瘤活性。此外，该抗体没有或有很弱的激动剂活性。为了检测本发明抗体结合人c-Met后，是否能够和人c-Met一起内吞到细胞内，用表达c-Met的人胃癌细胞MKN45(JCRB，Cat No.:JCRB0254)进行了评估。
在具有10％(v/v)胎牛血清(FBS)(GIBCO，Cat No.:10099-141)和青霉素/链 霉素(GIBCO，Cat No.:15070-063)的RPMI 1640培养基(GIBCO，Cat No.:11835-030)中重悬浮MKN45细胞至10000,000细胞/mL。将2mL重悬的MKN45细胞以250,000细胞/孔加入96孔微滴定板中，加入10μg/ml的c-Met抗体至对应的孔中，最终体积为100μl，在4度孵育1小时。加入FACS缓冲液(具有2.5％胎牛血清的磷酸盐缓冲液(Hyclone，Cat：SH30256.01B)，4℃，1300rpm，4分钟，离心弃上清，重复三次。每孔加入100ul二抗溶液(荧光标记羊抗鼠二抗:1:200稀释，Biolegend，Cat#405307；荧光标记抗人二抗:1:30稀释，Biolegend，Cat#409304),在4℃孵育1小时。加入FACS缓冲液，4℃，1300rpm，4分钟，离心弃上清，重复三次。加入细胞完全培养基(具有10％胎牛血清的RPMI 1640培养基)，在37℃，5％CO2下孵育0小时,0.5小时,1小时,2小时,4小时。每孔加入5ul 7-AAD(Biolegend,Cat：420403)至100μl FACS缓冲液中，在4℃孵育30分钟,加入FACS缓冲液，4℃，1300rpm，4分钟，离心弃上清，重复三次。加入200μl Stripping buffer(0.05M甘氨酸，pH3.0；0.1M氯化钠，按照1：1(v/v)混匀)至每孔细胞中，重悬细胞，室温孵育7分钟。室温，1300rpm，4分钟，离心弃上清。加入200μl中和洗液(0.15M三羟甲基氨基甲烷，pH7.4)至每孔细胞中，重悬细胞，室温，1300rpm，4分钟，离心弃上清。加入200μl FACS缓冲液，重悬细胞，准备好样品，使用流式细胞仪(BD FACS Calibur)检测。结果见表9。
c-Met抗体内吞百分比＝(各个时间点荧光强度值-零点时的平均荧光强度值)/零点时的平均荧光强度值。
表9.本发明人源化抗c-Met抗体的细胞内吞性能评价(内吞百分比％)
人源化抗体 0h 0.5h 1h 2h 4h
hIgG(对照)* 0 -0.9 -4.4 -4.9 3.6
Ab-9 0 26 32 32 31
Ab-10 0 24 38 53 59
*:对照组-4.9％-3.6％均为实验误差(背景值)。均视为没有内吞作用
表9结果表明，本发明抗体除了没有激动剂活性外，还有很好的内吞作用。结合靶细胞后，抗体和受体一起迅速内吞到靶细胞内，2-4小时内吞达到最大值。
实施例12抗c-Met抗体生物物理稳定性特性分析
为了评估本发明抗体的生物物理稳定性，比如糖基化，脱氨基化位点是否存在，以及其稳定性等，用质谱(LC-MS)分析方法对本发明抗c-Met抗体进行了综合评价。
样品直接LC-MS检测轻重链分子量分析糖基化。在4℃长时间(3个月以上)，或40℃，21天加速条件下，LC-MS分析脱氨基化。不同条件处理样品后，取出样品，用pH7.2Tris-HCl稀释样品至2mg/ml，加入终浓度为10mM TCEP和6M尿素(AMRESCO,Cat#0378)37℃孵育20min.加入终浓度为20mM IAA(Sigma-Aldrich,Cat#I1149)室温避光孵育15min保护巯基，用pH7.2Tris-HCl稀释样品以调节pH，按照蛋白：酶＝10：1(重量比)的比例添加蛋白酶(Sigma-Aldrich,Cat#T6567)，37℃孵育25min后添加终浓度为0.1％的甲酸(Fluca,Cat#94318)终止反应,离心上LC-MS分析。
用BiopharmaLynx来分析有无脱酰胺基作用。所得到的质谱数据通过找到包含脱酰胺化位点的原生肽(native peptide)和修饰产物，提取母离子得到EIC(Extracted Ion Chromatogram)图，积分得到峰面积并计算脱酰胺化和氧化产物所占比例。结果见表10。
表10.本发明人源化抗c-Met抗体的物理稳定性能评价
Figure PCTCN2016078699-appb-000054
*:重链均有糖基化，分子量均和预期一致。#：脱氨基分子比例(％)。0.66-1.0％在检测背景范围内
上述结果表明，本发明的抗体稳定，物理性能好。
实施例13、抗c-Met抗体Ab-10偶联毒素MC-MMAF
本发明抗c-Met抗体具有受体结合阻止活性、无激动剂活性、具有靶细胞内吞活性和物理稳定性等特性，这些特性使得本发明抗体特别适合和毒素偶联成ADC药物用于c-Met表达癌症治疗。偶联过程见下图：
Figure PCTCN2016078699-appb-000055
Figure PCTCN2016078699-appb-000056
第一步将硫代乙酸S-(3-羰基丙基)酯(0.7mg，5.3μmol)溶解于0.9mL乙腈溶液，备用。向Ab-10单抗pH＝4.3的乙酸/乙酸钠缓冲液(10.35mg/ml，9.0mL，0.97mmol)加入上述预制的硫代乙酸S-(3-羰基丙基)酯的乙腈溶液，然后滴加1.0mL的氰基硼氢化钠(14.1mg,224μmol)的水溶液，于25℃下振荡反应2小时。反应结束后，用Sephadex G25凝胶柱脱盐纯化(洗脱相：pH为6.5的0.05M的PBS溶液)后，得产物1b溶液，浓缩到约10mg/ml后直接进行下一步反应。
第二步，向1b溶液(11.0mL)中加入0.35mL的2.0M盐酸羟胺溶液，于25℃下振荡反应30分钟后，将反应液用Sephadex G25凝胶柱脱盐纯化(洗脱相：pH为6.5的0.05M的PBS溶液)后，得标题产物Ab-10单抗-丙硫醇1c溶液(浓度6.17mg/ml，14.7mL)。
第三步，将化合物MC-MMAF(1.1mg，1.2μmol，采用PCT专利WO2005081711公开的方法制备而得)溶解于0.3mL乙腈中，加入Ab-10单抗-丙硫醇溶液1c(6.17mg/mL，3.0mL)中，于25℃下振荡反应4小时后，将反应液用Sephadex G25凝胶柱脱盐纯化(洗脱相：pH为6.5的含0.05M的PBS溶液)，在无菌条件下通过0.2μm滤器过滤后得标题产物ADC-1的PBS缓冲液(3.7mg/mL，4.7mL)，于4℃冷冻储存。
Q-TOF LC/MS:特征峰：148119.2(MAb+0D)、149278.1(MAb+1D)、150308.1(MAb+2D)、151314.1(MAb+3D)。分析得每个抗体分子连接毒素量(DAR)平均值：y＝1.7。
实施例14、抗c-Met抗体Ab-10偶联毒素MC-VC-PAB-MMAE
Figure PCTCN2016078699-appb-000057
将化合物MC-VC-PAB-MMAE(1.6mg，1.2μmol，采用PCT专利WO2004010957公开的方法制备而得)溶解于0.3mL乙腈中，加入Ab-10单抗-丙硫醇溶液1c(6.17mg/mL，3.0mL)中，于25℃下振荡反应4小时后，将反应液用Sephadex G25凝胶柱脱盐纯化(洗脱相：pH为6.5的含0.05M的PBS溶液)，在无菌条件下通过0.2μm滤器过滤后得标题产物ADC-2的PBS缓冲液(3.6mg/mL，4.8mL)，于4℃冷冻储存。
Q-TOF LC/MS:特征峰：148118.4(MAb+0D)、149509.2(MAb+1D)、150903.1(MAb+2D)、152290.4(MAb+3D)、153680.7(MAb+4D)。分析得每个抗体分子连接毒素量(DAR)平均值：y＝1.8。
实施例15、抗c-Met抗体Ab-10偶联毒素MC-VC-PAB-MMAF
Figure PCTCN2016078699-appb-000058
将化合物MC-VC-PAB-MMAF(1.6mg，1.2μmol，采用PCT专利WO2005081711公开的方法制备而得)溶解于0.3mL乙腈中，加入Ab-10单抗-丙硫醇溶液1c(6.17mg/mL，3.0mL)中，于25℃下振荡反应4小时后，将反应液用Sephadex G25凝胶柱脱盐纯化(洗脱相：pH为6.5的含0.05M的PBS溶液)，在无菌条件下通过0.2μm滤器过滤后得标题产物ADC-3的PBS缓冲液(3.5mg/mL，4.9mL)，于4℃冷冻储存。
Q-TOF LC/MS:特征峰：148119.1(MAb+0D)、149525.3(MAb+1D)、150930.7(MAb+2D)、152335.2(MAb+3D)、153739.8(MAb+4D)。分析得每个抗体分子连接毒素量(DAR)平均值：y＝1.6。
实施例16、抗c-Met抗体Ab-10偶联毒素MC-MMAE
Figure PCTCN2016078699-appb-000059
化合物MC-MMAE(1.2mg，1.2μmol，采用专利申请”US7/750/116B1”公开的方法制备而得)溶解于0.3mL乙腈中，加入Ab-10单抗-丙硫醇溶液1c(6.17mg/mL，3.0mL)中，于25℃下振荡反应4小时后，将反应液用Sephadex G25凝胶柱脱盐纯化(洗脱相：pH为6.5的含0.05M的PBS溶液)，在无菌条件下通过0.2μm滤器过滤后得标题产物ADC-4的PBS缓冲液(3.4mg/mL，5.0mL)，于4℃冷冻储存。
Q-TOF LC/MS:特征峰：148118.6(MAb+0D)、149104.3(MAb+1D)、150090.1(MAb+2D)、151075.8(MAb+3D)。分析得每个抗体分子连接毒素量(DAR)平均值：y＝1.6。
实施例17、抗c-Met抗体Ab-9偶联毒素MC-MMAE
Figure PCTCN2016078699-appb-000060
第一步，将硫代乙酸S-(3-羰基丙基)酯(0.7mg，5.3μmol)，溶解于0.9mL乙腈溶液，备用；向Ab-9单抗pH＝4.3的乙酸/乙酸钠缓冲液(10.85mg/ml，9.0mL，0.976mmol)加入上述预制的硫代乙酸S-(3-羰基丙基)酯的乙腈溶液，然后滴加1.0mL的氰基硼氢化钠(14.1mg,224μmol)的水溶液，于25℃下振荡反应2小时。反应结束后，用Sephadex G25凝胶柱脱盐纯化(洗脱相：pH为6.5的0.05M的PBS溶液)后，得标题产物5b溶液，浓缩到约10mg/ml后直接进行下一步反应。
第二步，向5b溶液(11.0mL)中加入0.35mL的2.0M盐酸羟胺溶液，于25℃下振荡反应30分钟后，将反应液用Sephadex G25凝胶柱脱盐纯化(洗脱相：pH为6.5的0.05M的PBS溶液)后，得标题产物Ab-9单抗-丙硫醇5c溶液(浓度6.2mg/ml，15.0mL)。
第三步，将化合物MC-MMAE(1.1mg，1.2μmol)溶解于0.3mL乙腈中，加入Ab-9单抗-丙硫醇溶液5c(6.2mg/mL，3.0mL)中，于25℃下振荡反应4小时后，将反应液用Sephadex G25凝胶柱脱盐纯化(洗脱相：pH为6.5的含0.05M的PBS溶液)，在无菌条件下通过0.2μm滤器过滤后得标题产物ADC-5的PBS缓冲液(3.8mg/mL，4.6mL)，于4℃冷冻储存。
Q-TOF LC/MS:特征峰：150530.9(MAb+0D)、151915.7(MAb+1D)、153333.6(MAb+2D)、154763.4(MAb+3D)、156271.9(MAb+4D)。分析得每个抗体分子连接毒素量(DAR)平均值：y＝1.5。
实施例18、抗c-Met抗体Ab-9偶联毒素MC-MMAF
Figure PCTCN2016078699-appb-000061
将化合物MC-MMAF(1.1mg，1.2μmol)溶解于0.3mL乙腈中，加入Ab-9单抗-丙硫醇溶液5c(6.2mg/mL，3.0mL)中，于25℃下振荡反应4小时后，将反应液用Sephadex G25凝胶柱脱盐纯化(洗脱相：pH为6.5的含0.05M的PBS溶液)，在无菌条件下通过0.2μm滤器过滤后得标题产物ADC-6的PBS缓冲液(3.8mg/mL，4.6mL)，于4℃冷冻储存。
Q-TOF LC/MS:特征峰：150537.8(MAb+0D)、152087.9(MAb+1D)、153486.5(MAb+2D)、154911.7(MAb+3D)、156499.9(MAb+4D)。分析得每个抗体分子连接毒素量(DAR)平均值：y＝1.7。
实施例19、抗c-Met抗体Ab-9偶联毒素MC-VC-PAB-MMAF
Figure PCTCN2016078699-appb-000062
将化合物MC-VC-PAB-MMAF(1.6mg，1.2μmol)溶解于0.3mL乙腈中，加 入Ab-9单抗-丙硫醇溶液5c(6.2mg/mL，3.0mL)中，于25℃下振荡反应4小时后，将反应液用Sephadex G25凝胶柱脱盐纯化(洗脱相：pH为6.5的含0.05M的PBS溶液)，在无菌条件下通过0.2μm滤器过滤后得标题产物ADC-7的PBS缓冲液(3.8mg/mL，4.6mL)，于4℃冷冻储存。
Q-TOF LC/MS:特征峰：150537.8(MAb+0D)、152087.9(MAb+1D)、153486.5(MAb+2D)、154911.7(MAb+3D)、156499.9(MAb+4D)。分析得每个抗体分子连接毒素量(DAR)平均值：y＝1.8。
实施例20、抗c-Met抗体Ab-9偶联毒素MC-VC-PAB-MMAE
Figure PCTCN2016078699-appb-000063
将化合物MC-VC-PAB-MMAE(1.6mg，1.2μmol)溶解于0.3mL乙腈中，加入Ab-9单抗-丙硫醇溶液5c(6.2mg/mL，3.0mL)中，于25℃下振荡反应4小时后，将反应液用Sephadex G25凝胶柱脱盐纯化(洗脱相：pH为6.5的含0.05M的PBS溶液)，在无菌条件下通过0.2μm滤器过滤后，得标题产物ADC-8的PBS缓冲液(3.8mg/mL，4.6mL)，于4℃冷冻储存。
Q-TOF LC/MS:特征峰：150508.6(MAb+0D)、151903.6(MAb+1D)、153314.5(MAb+2D)、154747.8(MAb+3D)、156039.5MAb+4D)。分析得每个抗体分子连接毒素量(DAR)平均值：y＝1.6。
实施例21、抗c-Met抗体Ab-10偶联毒素SMCC-DM1
Figure PCTCN2016078699-appb-000064
Figure PCTCN2016078699-appb-000065
第一步
将SMCC(4-(N-马来酰亚胺基甲基)环己烷-1-羧酸琥珀酰亚胺酯(1.65mg，4.94μmol，购自上海瀚鸿化工科技有限公司，批号BH-4857-111203)，溶解于0.9mL乙腈溶液，备用；向Ab-10单抗pH＝6.5的PBS缓冲液(10.15mg/ml，9.0mL，0.62μmol)加入上述预制的4-(N-马来酰亚胺基甲基)环己烷-1-羧酸琥珀酰亚胺酯的乙腈溶液，于25℃下振荡反应2小时。反应结束后用Sephadex G25凝胶柱脱盐纯化(洗脱相：pH为6.5的0.05M的PBS溶液)后得标题产物9b溶液，浓缩到约10mg/ml(8.3mg/ml，11ml)后直接进行下一步反应。
第二步
向9b溶液(11.0mL)中加入3.0mg的L-DM1(采用公知的方法文献“Journal of Medicinal Chemistry.2006,49,4392-4408”制备而得)乙醇溶液(3.0mgL-DM1/1.1ml乙醇)，于25℃下振荡反应约4.0小时后，将反应液用Sephadex G25凝胶柱脱盐纯化(洗脱相：pH为6.5的0.05M的PBS溶液)后，得标题产物ADC-9溶液(浓度6.3mg/ml，14mL)，于4℃冷冻储存。Q-TOF LC/MS:特征峰：148119.6(MAb+0D)、149078.1(MAb+1D)、149836.4(MAb+2D)、150593.7(MAb+3D)、151552.5(MAb+4D)。
平均值：y＝2.3。
实施例22、抗c-Met抗体Ab-9偶联毒素SMCC-DM1
Figure PCTCN2016078699-appb-000066
第一步
将SMCC(4-(N-马来酰亚胺基甲基)环己烷-1-羧酸琥珀酰亚胺酯(1.65mg，4.94μmol)，溶解于0.9mL乙腈溶液，备用；向Ab-9单抗pH＝6.5的PBS缓冲液(10.15mg/ml，9.0mL，0.62umol)加入上述预制的4-(N-马来酰亚胺基甲基)环己烷-1-羧酸琥珀酰亚胺酯的乙腈溶液，于25℃下振荡反应2小时。反应结束后用Sephadex G25凝胶柱脱盐纯化(洗脱相：pH为6.5的0.05M的PBS溶液)后，得标题产物10b溶液，浓缩到约10mg/ml(8.3mg/ml，11ml)后直接进行下一步反应。
第二步
向9b溶液(11.0mL)中加入3.0mg的L-DM1(3.0mgL-DM1/1.1ml乙醇)乙醇溶液，于25℃下振荡反应约4.0小时后，将反应液用Sephadex G25凝胶柱脱盐纯化(洗脱相：pH为6.5的0.05M的PBS溶液)后得标题产物ADC-10溶液(浓度6.3mg/ml，14mL)，于4℃冷冻储存。
Q-TOF LC/MS:特征峰：150534.2(MAb+0D)、151492.6(MAb+1D)、152451.7(MAb+2D)、153409.7(MAb+3D)、154368.1(MAb+4D)。
平均值：y＝2.2。
实施例23、抗c-Met抗体Ab-9偶联毒素-SN-38
Figure PCTCN2016078699-appb-000067
将化合物MC-VC-PAB-SN-38(1.3mg，1.2μmol)溶解于0.3mL乙腈中，加入Ab-9单抗-丙硫醇溶液5c(6.2mg/mL，3.0mL)中，于25℃下振荡反应4小时后，将反应液用Sephadex G25凝胶柱脱盐纯化(洗脱相：pH为6.5的含0.05M的PBS溶液)，在无菌条件下通过0.2μm滤器过滤后，得标题产物ADC-11的PBS缓冲液(3.7mg/mL，4.5mL)，于4℃冷冻储存。
Q-TOF LC/MS:特征峰：150537.1(MAb+0D)、151786.6(MAb+1D)、152948.6(MAb+2D)、154161.7(MAb+3D)、155365.9(MAb+4D)、156477.8(MAb+5D)。
平均值：y＝2.6。
实施例24、抗c-Met抗体Ab-10偶联毒素
1、毒素的制备
(S)-2-((2R,3R)-3-((1S,3S,5S)-2-((3R,4S,5S)-4-((S)-N,3-二甲基-2-((S)-3-甲基-2-(甲基氨基)丁酰胺)丁酰胺)-3-甲氧基-5-甲基庚酰基)-2-氮杂双环[3.1.0]己烷-3-基)-3-甲氧基-2-甲基丙酰胺)-3-(2-氟苯基)丙酸
Figure PCTCN2016078699-appb-000068
Figure PCTCN2016078699-appb-000069
第一步
(S)-叔丁酯2-氨基-3-(2-氟苯基)丙酸
将原料((S)-2-氨基-3-(2-氟苯基)丙酸12a(400mg，2.18mmol，采用公知的方法“Advanced Synthesis&Catalysis,2012,354(17),3327-3332”制备而得)溶于10Ml乙酸叔丁酯，加入高氯酸(300mg(70％)，3.3mmol)，于室温下搅拌16小时。反应完毕后加入6Ml水，分液，有机相用饱和碳酸氢钠溶液(5Ml)洗涤。水相用饱和碳酸氢钠溶液调节至Ph＝8，二氯甲烷(5Ml×3)萃取，合并有机相，依次用水(3Ml)，饱和氯化钠溶液(5Ml)洗涤，无水硫酸钠干燥，过滤，滤液减压浓缩得粗品标题产物(S)-叔丁酯2-氨基-3-(2-氟苯基)丙酸12b(390mg，黄色油状物)，产品不经纯化直接进行下一步反应。
第二步
(1S,3S,5S)-叔丁酯3-((1R,2R)-3-(((S)-1-(叔丁氧基)-3-(2-氟苯基)-1-羰基丙基-2-基)
氨基)-1-甲氧基-2-甲基-3-羰基丙基)-2-氮杂双环[3.1.0]己烷-2-羧酸
将原料(2R,3R)-3-((1S,3S,5S)-2-(叔丁氧羰基)-2-氮杂双环[3.1.0]己烷-3-基)-3-甲氧基-2-甲基丙酸12e(100mg，0.334mmol)溶于6Ml二氯甲烷和二甲基甲酰胺(V/V＝5:1)混合溶剂中，加入反应物粗品(S)-叔丁酯2-氨基-3-(2-氟苯基)丙酸12b(80mg，0.334mmol)。再加入N,N-二异丙基乙基胺(0.29Ml，1.67mmol)和2-(7- 偶氮苯并三氮唑)-N,N,N’,N’-四甲基脲六氟磷酸酯(152.3mg，0.40mmol)。反应体系在氩气氛下，于室温搅拌1小时。反应结束后，加10Ml水搅拌，分层，二氯甲烷层用饱和氯化钠溶液(10Ml)洗涤，无水硫酸钠干燥，过滤，滤液减压浓缩。用硅胶柱色谱法以洗脱剂体系B纯化所得残余物，得到标题产物(1S,3S,5S)-叔丁酯3-((1R,2R)-3-(((S)-1-(叔丁氧基)-3-(2-氟苯基)-1-羰基丙基-2-基)氨基)-1-甲氧基-2-甲基-3-羰基丙基)-2-氮杂双环[3.1.0]己烷-2-羧酸12c(173mg，无色液体)，收率99.5％。
MS m/z(ESI):521.2[M+1]
第三步
(S)-叔丁酯2-((2R,3R)-3-((1S,3S,5S)-2-氮杂双环[3.1.0]己烷-3-基)-3-甲氧基-2-甲基丙酰胺)-3-(2-氟苯基)丙酸
将原料(1S,3S,5S)-叔丁酯3-((1R,2R)-3-(((S)-1-(叔丁氧基)-3-(2-氟苯基)-1-羰基丙基-2-基)氨基)-1-甲氧基-2-甲基-3-羰基丙基)-2-氮杂双环[3.1.0]己烷-2-羧酸12c(173mg，0.33mmol)溶于2Ml二氧六环中，加入5.6M的氯化氢二氧六环溶液(0.21Ml，1.16mmol)，氩气氛下，于室温搅拌1小时，置于0℃冰箱内12小时。反应结束后，将反应液减压浓缩，加入5Ml二氯甲烷稀释，加入10Ml饱和碳酸氢钠溶液，搅拌10分钟。体系分层，水层用二氯甲烷萃取(5Ml×3)。合并二氯甲烷层，用饱和氯化钠溶液(10Ml)洗涤，无水硫酸钠干燥。过滤，滤液减压浓缩，得到粗品标题产品(S)-叔丁酯2-((2R,3R)-3-((1S,3S,5S)-2-氮杂双环[3.1.0]己烷-3-基)-3-甲氧基-2-甲基丙酰胺)-3-(2-氟苯基)丙酸12d(77mg，黄色液体)，产品不经纯化直接进行下一步反应。
MS m/z(ESI):421.2[M+1]
第四步
(S)-叔丁酯2-((2R,3R)-3-((1S,3S,5S)-2-((5S,8S,11S,12R)-11-((S)-仲丁基)-1-(9H-芴-9-基)-5,8-二异丙基-12-甲氧基-4,10-二甲基-3,6,9-三羰基-2-氧-4,7,10-三氮杂十四烷基-14-酰基)-2-氮杂双环[3.1.0]己烷-3-基)-3-甲氧基-2-甲基丙酰胺)-3-(2-氟苯基)丙酸
将粗品(S)-叔丁酯2-((2R,3R)-3-((1S,3S,5S)-2-氮杂双环[3.1.0]己烷-3-基)-3-甲氧基-2-甲基丙酰胺)-3-(2-氟苯基)丙酸12d(77mg，0.183mmol)，(5S,8S,11S,12R)-11-((S)-仲丁基)-1-(9H-芴-9-基)-5,8-二异丙基-12-甲氧基-4,10-二甲基-3,6,9-三羰基-2-氧杂-4,7,10-三氮杂十四烷-14-羧酸12i(116.8mg，0.183mmol,采用专利申请“WO 2013072813”公开的方法制备而得)溶于6Ml二氯甲烷和二甲基甲酰胺(V/V＝5:1)混合溶剂中，加入N,N-二异丙基乙基胺(0.16Ml，0.915mmol)和2-(7-偶氮苯并三氮唑)-N,N,N’,N’-四甲基脲六氟磷酸酯(84mg，0.22mmol)。反应体系在氩气氛下，于室温下搅拌1小时。反应结束后，加入10Ml水搅拌，分层。二氯甲烷层用饱和氯化钠溶液(10Ml)洗涤，无水硫酸钠干燥。过滤，滤液减压浓缩。 用硅胶柱色谱法以洗脱剂体系B纯化残留物，得到标题产品(S)-叔丁酯2-((2R,3R)-3-((1S,3S,5S)-2-((5S,8S,11S,12R)-11-((S)-仲丁基)-1-(9H-芴-9-基)-5,8-二异丙基-12-甲氧基-4,10-二甲基-3,6,9-三羰基-2-氧杂-4,7,10-三氮杂十四烷基-14-酰基)-2-氮杂双环[3.1.0]己烷-3-基)-3-甲氧基-2-甲基丙酰胺)-3-(2-氟苯基)丙酸12e(190.5mg，黄色粘稠物)，收率100％。
MS m/z(ESI):1040.6[M+1]
第五步
(S)-叔丁酯2-((2R,3R)-3-((1S,3S,5S)-2-((3R,4S,5S)-4-((S)-N,3-二甲基-2-((S)-3-甲基-2-(甲基氨基)丁酰胺)丁酰胺)-3-甲氧基-5-甲基庚酰基)-2-氮杂双环[3.1.0]己烷-3-基)-3-甲氧基-2-甲基丙酰胺)-3-(2-氟苯基)丙酸
将原料(S)-叔丁酯2-((2R,3R)-3-((1S,3S,5S)-2-((5S,8S,11S,12R)-11-((S)-仲丁基)-1-(9H-芴-9-基)-5,8-二异丙基-12-甲氧基-4,10-二甲基-3,6,9-三羰基-2-氧杂-4,7,10-三氮杂十四烷基-14-酰基)-2-氮杂双环[3.1.0]己烷-3-基)-3-甲氧基-2-甲基丙酰胺)-3-(2-氟苯基)丙酸12e(190.5mg，0.183mmol)溶于1.5Ml二氯甲烷中，加入2Ml二乙胺。反应体系在氩气氛下，于室温搅拌3小时。反应结束后，将反应液减压浓缩，得到粗品标题产品(S)-叔丁酯2-((2R,3R)-3-((1S,3S,5S)-2-((3R,4S,5S)-4-((S)-N,3-二甲基-2-((S)-3-甲基-2-(甲基氨基)丁酰胺)丁酰胺)-3-甲氧基-5-甲基庚酰基)-2-氮杂双环[3.1.0]己烷-3-基)-3-甲氧基-2-甲基丙酰胺)-3-(2-氟苯基)丙酸12f(150mg，黄色粘稠物)，产品不经纯化直接进行下一步反应。
MS m/z(ESI):818.5[M+1]
第六步
(S)-2-((2R,3R)-3-((1S,3S,5S)-2-((3R,4S,5S)-4-((S)-N,3-二甲基-2-((S)-3-甲基-2-(甲基氨基)丁酰胺)丁酰胺)-3-甲氧基-5-甲基庚酰基)-2-氮杂双环[3.1.0]己烷-3-基)-3-甲氧基-2-甲基丙酰胺)-3-(2-氟苯基)丙酸
将粗品(S)-叔丁酯2-((2R,3R)-3-((1S,3S,5S)-2-((3R,4S,5S)-4-((S)-N,3-二甲基-2-((S)-3-甲基-2-(甲基氨基)丁酰胺)丁酰胺)-3-甲氧基-5-甲基庚酰基)-2-氮杂双环[3.1.0]己烷-3-基)-3-甲氧基-2-甲基丙酰胺)-3-(2-氟苯基)丙酸12f(150mg，0.183mmol)溶于1Ml二氧六环中，加入5.6M的氯化氢二氧六环溶液3Ml，氩气氛下，于室温搅拌12小时。反应结束后，将反应液减压浓缩，用乙醚带旋溶剂。所得残余物用高效液相色谱法纯化得标题产品(S)-2-((2R,3R)-3-((1S,3S,5S)-2-((3R,4S,5S)-4-((S)-N,3-二甲基-2-((S)-3-甲基-2-(甲基氨基)丁酰胺)丁酰胺)-3-甲氧基-5-甲基庚酰基)-2-氮杂双环[3.1.0]己烷-3-基)-3-甲氧基-2-甲基丙酰胺)-3-(2-氟苯基)丙酸12g(28mg，白色粉末固体)，收率20％。
MS m/z(ESI):762.7[M+1]
1H NMR(400MHz,CD3OD):δ7.38-7.18(m,2H),7.13-7.01(m,2H),4.80-4.67(m,2H),4.30-4.15(m,1H),4.13-4.01(m,1H),3.96-3.83(m,2H),3.75-3.60(m,2H), 3.42-3.11(m,9H),3.06-2.95(m,1H),2.70-2.58(m,4H),2.28-2.01(m,4H),1.88-1.70(m,3H),1.57-1.25(m,4H),1.22-0.95(m,18H),0.92-0.80(m,4H),0.78-0.65(m,1H).
2、毒素中间体的制备
(S)-2-((2R,3R)-3-((1S,3S,5S)-2-((3R,4S,5S)-4-((S)-2-((S)-2-(6-(2,5-二羰基-2,5-二氢-1H-吡咯-1-基)-N-甲基己酰胺)-3-甲基丁酰胺)-N,3-二甲基丁酰胺)-3-甲氧基-5-甲基庚酰基)-2-氮杂双环[3.1.0]己烷-3-基)-3-甲氧基-2-甲基丙酰胺)-3-(2-氟苯基)丙酸
Figure PCTCN2016078699-appb-000070
将原料(S)-2-((2R,3R)-3-((1S,3S,5S)-2-((3R,4S,5S)-4-((S)-N,3-二甲基-2-((S)-3-甲基-2-(甲基氨基)丁酰胺)丁酰胺)-3-甲氧基-5-甲基庚酰基)-2-氮杂双环[3.1.0]己烷-3-基)-3-甲氧基-2-甲基丙酰胺)-3-(2-氟苯基)丙酸12g(25mg，0.033mmol)溶于3mL二氯甲烷中，加入N,N-二异丙基乙基胺(0.029mL，0.164mmol)，反应体系在氩气氛下，冰浴下滴加预制的6-(2,5-二羰基-2,5-二氢-1H-吡咯-1-基)己酰氯4b(11.3mg，0.049mmol)的二氯甲烷溶液，于室温反应3小时。反应结束后，加入5mL水，搅拌20分钟，分液，有机层用无水硫酸钠干燥，过滤，滤液减压浓缩，残留物用高效液相色谱法纯化得标题产物(S)-2-((2R,3R)-3-((1S,3S,5S)-2-((3R,4S,5S)-4-((S)-2-((S)-2-(6-(2,5-二羰基-2,5-二氢-1H-吡咯-1-基)-N-甲基己酰胺)-3-甲基丁酰胺)-N,3-二甲基丁酰胺)-3-甲氧基-5-甲基庚酰基)-2-氮杂双环[3.1.0]己烷-3-基)-3-甲氧基-2-甲基丙酰胺)-3-(2-氟苯基)丙酸12h(7mg，黄色粘稠物)，收率22.4％。
MS m/z(ESI):955.4[M+1]
1H NMR(400MHz,CD3OD):δ7.36-7.30(m,1H),7.29-7.21(m,1H),7.17-7.02(m,2H),6.83-6.79(m,2H),4.81-4.71(m,2H),4.69-4.55(m,2H),4.25-4.15(m,1H),4.13-4.04(m,1H),3.96-3.85(m,2H),3.70-3.61(m,1H),3.55-3.46(m,3H),3.40-3.21(m,4H),3.18-3.10(m,2H),3.07-2.96(m,4H),2.67-2.56(m,2H),2.54-2.34(m,3H),2.29-2.17(m,2H),2.10-1.99(m,1H),1.89-1.57(m,7H),1.52-1.28(m,6H),1.21-1.11(m,4H),1.07-0.96(m,6H),0.95-0.81(m,12H),0.80-0.69(m,1H).
3、抗体毒素偶联物的制备
Figure PCTCN2016078699-appb-000071
将化合物12h(1.2mg，1.2μmol)溶解于0.3mL乙腈中，加入Ab-10单抗-丙硫醇1c溶液(6.17mg/mL，3.0mL)中，于25℃下振荡反应4小时后将反应液用Sephadex G25凝胶柱脱盐纯化(洗脱相：pH为6.5的含0.05M的PBS溶液)，在无菌条件下通过0.2μm滤器过滤后得标题产物ADC-12的PBS缓冲液(3.3mg/mL，5.0mL)，于4℃冷冻储存。
Q-TOF LC/MS:特征峰：148119.6(MAb+0D)、149150.5(MAb+1D)、150221.1(MAb+2D)、151265.1(MAb+3D)、152314.3(MAb+4D)。
平均值：y＝1.6。
参照实施例13-24，制备实施例25-27的ADC化合物。
Figure PCTCN2016078699-appb-000072
Figure PCTCN2016078699-appb-000073
抗c-Met抗体毒素偶联(ADC)分子测试例
测试例1、抗c-Met抗体毒素偶联(ADC)分子稳定性评价
本发明ADC分子或其它具有内吞作用的c-Met抗体-毒素偶联物(如，LY-2875358-ADC)的毒素中间体和毒素，在PBS、人和猴血浆中进行了游离毒素的稳定性评价。
将实施例化合物ADC-1和ADC-12的毒素中间体和毒素用PBS、人或猴血浆(苏州西山中科药物研究开发有限公司，动物生产许可证号：SCXK(苏)2012-0009)稀释至500μg/mL，37℃孵育7天，于0、3、7天取样测定样品中游离毒素和毒素中间体的浓度。50μL含药人、猴血浆或PBS样品，加入20uL内对照(喜树碱，上海融禾医药科技发展有限公司，批号090107，100ng/ml)，加入150μL乙腈，涡旋混匀3min，15000rpm，离心10min，取上清液80μL与80μL 0.2％甲酸混匀后，10μL进样。标准曲线分析方法为50μL空白人、猴血浆或PBS样品，加入50μL系列工作溶液，加入20μL内对照(喜树碱，100ng/ml)，加入100μL乙腈，涡旋混匀3min，15000rpm，离心10min，取上清液80μL与80μL 0.2％甲酸混匀后，10μL进样。
使用Shimadzu LC-30AD超高效液相色谱系统(日本岛津公司)，UPLC-MS/MS质谱仪为API4000三重四极杆串联质谱仪(美国AB SCIEX公司)，设定色谱条件(色谱柱：Waters XBridgeTM BEH300C18(100mm×4.6mm i.d.,3.5mm)，流动相为0.2％甲酸-乙腈(梯度洗脱)。结果见表11,表12。
表11.本发明ADC-1的毒素中间体和毒素药物血浆稳定性评价
Figure PCTCN2016078699-appb-000074
Figure PCTCN2016078699-appb-000075
*:检测值以样品中含游离毒素的百分比。0.01-0.19％均在检测背景值范围内；ND：Not detectable，未能检测到。
表12.本发明ADC-12的毒素中间体和毒素药物血浆稳定性评价
Figure PCTCN2016078699-appb-000076
ND：Not detectable，未能检测到。
上述结果表明，b本发明ADC-1，ADC-12的毒素中间体和毒素在各种溶剂(PBS,人血浆，猴血浆等)中稳定。37℃孵育0、3和7天均未检测到降解产物游离毒素和毒素中间体(毒素-linker)。
测试例2、抗c-Met抗体毒素偶联(ADC)分子体外活性评价
本发明ADC-1，ADC-12的体外活性用FACS(检测和c-Met阳性细胞的结合力)和内吞作用(方法见实施例11)评价。结果见表13。
表13.本发明ADC分子体外活性
人源化抗体 MKN45/FCAS结合活性(nM) 细胞内吞(％)*
Ab-10 1.01 32.7
ADC-1 1.22 32.9
ADC-12 0.48 31.8
*:数据为1小时内吞比例
上述结果表明，本发明抗体和毒素偶联后，保留了抗体的结合活性和内吞活性。
测试例3、抗c-Met抗体毒素偶联(ADC)分子细胞毒性实验
为了评估本发明ADC分子对细胞的毒性作用，用细胞ATP毒性实验进行了评估。ATP是活细胞新陈代谢的一个指标，检测ATP可以反应分子对细胞的毒性大小。
HepG2细胞(中科院细胞库，Cat#TCHu72)培养在含10％FBS的EMEM完全培养基中，MKN45细胞培养在含10％FBS的RPMI1640完全培养基中，实验时加入2-3ml胰蛋白酶消化2-3min，待细胞消化完全，加入10-15ml完全培养基将经过消化的细胞洗脱下来，1000rpm离心3min，弃上清，接着加入10-20ml完全培养基将细胞重悬，制成单细胞悬液，调整细胞密度为4×104cells/ml。在96孔细胞培养板各孔中加0.1ml上述细胞悬液，37℃5％CO2的培养箱中培养，24小时后去掉培养基，每孔加入90μl含2％FBS的EMEM培养基或含2％FBS的RPMI1640培养基，将待测样品(实施例13化合物和毒素)用PBS稀释成不同浓度梯度，每孔加入10μl，37℃5％CO2的培养箱中孵育72小时。用
Figure PCTCN2016078699-appb-000077
Luminescent Cell Viability Assay试剂盒(Promega，Cat#G7571)按说明书检测。用酶标仪(VICTOR 3，PerkinElmer公司)检测化学发光，GraphPad Prism(version5.0)软件进行数据分析。结果将表14。
表14.本发明ADC分子和相应的游离细胞毒素的细胞毒性作用
测试样品 IC50(nM)in MKN45细胞 IC50(nM)in HepG2细胞
ADC-1 0.51 ND
MMAF 0.85 4.88
ADC-12 0.59 ND
12g 79.4 400.8
ND:没有检测到活性；NA:不适用
讨论：上述结果表明，本发明ADC-1，ADC-12对c-Met阳性细胞MKN45的细胞毒性作用相同(IC50分别为0.51nM,和0.59nM)。但是各自的毒素部分对c-Met阳性细胞MKN45的细胞毒性作用不同，两者相差93倍(79.4/0.85)。
本发明ADC-1，ADC-12对c-Met阴性性细胞HepG2均没有细胞毒性作用，表明ADC化合物具有特异靶向作用。但是各自的毒素部分对c-Met阴性细胞HepG2的细胞毒性作用不同，两者相差82倍(400.8/4.88)。
这些结果表明，本发明ADC-1，ADC-12具有特异靶向作用，能抑制c-Met阳性细胞增值，但对非特异(正常细胞)没有毒性作用。ADC-1，ADC-12不同之处在于，各自的游离毒素对靶向细胞和非靶向细胞的毒性不同。ADC-12的毒素部分对c-Met阳性细胞和阴性细胞HepG2的细胞毒性要比ADC-1的毒素部分弱93，82倍。因此，该分子到达靶细胞过程中，如果有游离的毒素被释放，其非特异的毒性作用要比ADC-1弱。因而，毒副作用要小，安全性好。
测试例4、抗c-Met抗体毒素偶联(ADC)分子对肿瘤细胞的增值抑制作用
上述结果表明，ADC-1(实施例13)能够特异杀死c-Met表达的肿瘤靶细胞。为了检测该毒性作用对肿瘤细胞的增值抑制所用。用本发明分子检测了多种肿瘤细胞，采用CCK法测试样品对细胞增殖的抑制作用，根据IC50大小评价本发明ADC分子体外细胞活性。
所用细胞及相应的培养基见下表15，用Cell Counting Kit(东仁化学科技有限公司，Cat#CK04)检测细胞增值(按说明书进行操作)。
实验时加入2-3ml胰蛋白酶消化2-3min，待细胞消化完全，加入10-15ml完全培养基将经过消化的细胞洗脱下来，1000rpm离心3min，弃上清。接着加入10-20ml培养基将细胞重悬，制成单细胞悬液，调整细胞密度为4×104cells/ml。在96孔细胞培养板各孔中加0.1ml上述细胞悬液，37℃5％CO2的培养箱中培养，24小时后去掉培养基，每孔加入90μl含2％FBS的培养基，将待测样品用PBS稀释成不同浓度梯度，每孔加入10μl，37℃5％CO2的培养箱中孵育72小时。每孔加入10μl CCK8，培养箱中继续孵育2小时，酶标仪(VICTOR 3，PerkinElmer公司)检测OD450，采用GraphPad Prism(version5.0)软件进行数据分析。结果见表16。
表15.本实施例所用细胞的培养基
细胞系 培养基 厂家货号
MKN45 RPMI1640+10％FBS JCRB，JCRB0254
SNU5 IMDM+10％FBS ATCC，Cat#CRL-5973TM
BxPC3 RPMI1640+10％FBS 中科院细胞库(Cat#TCHu 12)
Caki-1 McCOY's 5A+10％FBS 中科院细胞库，Cat#TCHu135
NCI-H1993 RPMI1640+10％FBS ATCC，Cat#CRL-5909TM
PC9 DMEM+10％FBS 上海拜力生物科技有限公司
NCI-H596 DMEM+10％FBS 上海拜力生物科技有限公司
表16.本发明分子对不同癌细胞增殖抑制作用
Figure PCTCN2016078699-appb-000078
Figure PCTCN2016078699-appb-000079
表16结果表明本发明抗c-Met抗体在胃癌细胞系MKN45,SUN上有比较好的活性，而在其它c-Met表达低或不表达的肿瘤细胞，例如肺癌细胞上活性很弱，或者没有。而本发明的ADC-1因为带有额外的毒素，对c-Met表达的肿瘤细胞，包括胃癌细胞系MKN45,SUN，特别抗c-Met抗体没有作用或作用非常微弱的是肺癌、胰腺癌和肾细胞癌细胞上显示出很好的活性。
测试例5、抗c-Met抗体毒素偶联(ADC)分子体内药效评价
1.测试目的
为了更好评价本发明抗c-Met抗体和ADC分子的抗肿瘤药效活性，用实施例10方法对抗体Ab-10和ADC-1进行了平行比较实验。和实施例10不同的是，本测试例是单次给药，观察到肿瘤抑制作用直到有回复后趋势停止试验。
2.待测抗体
Ab-10(5mg/kg)，原液(2.18mg/ml)用PBS配成终浓度0.5mg/ml；
Ab-10(10mg/kg)，原液(2.18mg/ml)用PBS配成终浓度1mg/ml；
Ab-10(30mg/kg)，原液(2.18mg/ml)用PBS配成终浓度3mg/ml；
ADC-1(2.5mg/kg)，原液(10mg/ml)用PBS配成终浓度0.25mg/ml；
ADC-1(5mg/kg)，原液(10mg/ml)用PBS配成终浓度0.5mg/ml；
ADC-1(10mg/kg)，原液(10mg/ml)用PBS配成终浓度1mg/ml；
所有动物给药方式均为尾静脉注射，给药体积0.2ml/只。
3.试验方法
裸小鼠右肋部皮下接种MKN-45细胞(1×106/只)，肿瘤生长至平均体积(150.19+8.44)mm3，随机分组给药，每组8只。具体给药方案见表17。
每周测2次瘤体积，称体重，记录数据。
使用Excel统计软件：平均值以avg计算；SD值以STDEV计算；SEM值以STDEV/SQRT计算；组间差异P值以TTEST计算。
肿瘤体积(V)计算公式为：V＝1/2×L长×L短 2
抑瘤率＝(V0-VT)/V0*100％
其中V0、VT分别为实验开始时及实验结束时的肿瘤体积。
4.试验结果
表17.给药化合物对MKN-45裸小鼠移植瘤的疗效
Figure PCTCN2016078699-appb-000080
Figure PCTCN2016078699-appb-000081
**p<0.01*p<0.05
结论：本发明抗体及ADC化合物对MKN-45裸小鼠移植瘤有明显的疗效。
为了评价本发明ADC-12的体内药效，用上述同样的试验方法，平行比较了ADC-1和ADC-12。同样剂量3mg/kg，单次给药后，结果见表18。
表18.ADC-1和ADC-12对肿瘤的抑制作用
抑制率(％) 11天 15天 18天 21天
ADC-1 42.8 44.7 35.4 27.1
ADC-12 44.6 54.5 50.5 50.4
上述结果表明ADC-1和ADC-12在11天抑瘤率相近，但是从15天之后，ADC-1的药效减弱(21天为27.1％)，而ADC-12的抑瘤作用仍然维持在第11天的水平(50.4％)。
测试例6、ADC-12对人肺癌NCI-H1993裸小鼠皮下移植瘤的疗效
1.实验目的
评价并比较ADC-12、Ab-10抗体原液对人肺癌NCI-H1993裸小鼠皮下移植瘤的疗效。
2.药物配制
ADC-12用注射用水溶解成20mg/ml溶液，分装保存-80℃冰箱，临用时用0.1％BSA生理盐水稀释成相应浓度；Ab-10抗体原液浓度16.3mg/ml,用0.1％BSA生理盐水稀释后，分装保存于-80℃冰箱。
3.实验动物
BALB/cA-nude裸小鼠，6-7周，♀，购自上海灵畅生物科技有限公司。生产许可证号：SCXK(沪)2013－0018；动物合格证号2013001814303。饲养环境：SPF级。
4.实验步骤
裸小鼠皮下接种人肺癌NCI-H1993细胞，待肿瘤生长至100-150mm3后，将动物随机分组(D0)。给药剂量和给药方案见表19。每周测2-3次瘤体积，称鼠重，记录数据。肿瘤体积(V)计算公式为：
V＝1/2×a×b2   其中a、b分别表示长、宽。
T/C(％)＝(T-T0)/(C-C0)×100其中T、C为实验结束时的肿瘤体积；T0、C0为实验开始时的肿瘤体积。
5.结果
ADC-12是抗c-Met抗体-毒素偶联物。ADC-12(1、3、10mg/kg，IV,D0)剂量依赖性地抑制高表达c-Met人肺癌NCI-H1993裸小鼠皮下移植瘤的生长，抑瘤率分别为45％、63％、124％，10mg/kg剂量组有7/10肿瘤部分消退(D21)；Ab-10抗体原液为制备ADC-12的裸抗体，Ab-10抗体原液(30mg/kg,IV,每周2次，共6次)对NCI-H1993的抑瘤率为42％；荷瘤小鼠对以上药物均能很好耐受，没有体重减轻等症状发生。相比较，ADC-12对NCI-H1993的疗效明显强于Ab-10抗体原液。
表19.ADC-12、Ab-10抗体原液对人肺癌NCI-H1993裸小鼠皮下移植瘤的疗效。
Figure PCTCN2016078699-appb-000082
D0:第一次给药时间；P值指与溶剂相比；**P<0.01,与Ab-10抗体原液30mg/kg组比较；均采用Student’s t检验。实验开始时小鼠数目：n＝10。
讨论：ADC-12(1、3、10mg/kg，IV,D0)剂量依赖性地抑制高表达c-Met人肺癌NCI-H1993裸小鼠皮下移植瘤的生长，引起肿瘤部分消退；Ab-10抗体原液(30mg/kg,IV,每周2次，共6次)对NCI-H1993也有效；ADC-12对NCI-H1993的疗效明显强于Ab-10抗体原液。荷瘤小鼠对以上药物均能很好耐受。

Claims (42)

  1. 特异性结合c-Met受体的抗体或其抗原结合片段，其包含至少1个选自以下的CDR区序列或其突变序列：
    抗体重链可变区HCDR区序列：SEQ ID NO:6，SEQ ID NO:7或SEQ ID NO:8；和
    抗体轻链可变区LCDR区序列：SEQ ID NO:9，SEQ ID NO:10或SEQ ID NO:11。
  2. 根据权利要求1所述的特异性结合c-Met受体的抗体或其抗原结合片段，其中所述的抗体重链可变区包含至少1个选自如下的HCDR区序列或其突变序列：SEQ ID NO:6，SEQ ID NO:7或SEQ ID NO:8。
  3. 根据权利要求1所述的特异性结合c-Met受体的抗体或其抗原结合片段，其中所述的抗体轻链可变区包含至少1个选自如下的LCDR区序列或其突变序列：SEQ ID NO:9，SEQ ID NO:10或SEQ ID NO:11。
  4. 根据权利要求1-3任一项所述的特异性结合c-Met受体的抗体或其抗原结合片段，其中所述的抗体包含重链可变区序列SEQ ID NO:6，SEQ ID NO:7和SEQ ID NO:8，或其突变序列，和轻链可变区序列SEQ ID NO:9，SEQ ID NO:10和SEQ ID NO:11或其突变序列。
  5. 根据权利要求1-4任一项所述的特异性结合c-Met受体的抗体或其抗原结合片段，其中所述的突变序列为CDR区发生1-3个优化抗体活性的氨基酸突变，其中HCDR2区突变序列优选为SEQ ID NO:12。
  6. 根据权利要求1-5任一项所述的特异性结合c-Met受体的抗体或其抗原结合片段，其中所述的特异性结合c-Met受体的抗体或其抗原结合片段为鼠源抗体或其片段。
  7. 根据权利要求6所述的特异性结合c-Met受体的抗体或其抗原结合片段，其中所述的鼠源抗体重链可变区序列为：SEQ ID NO:4。
  8. 根据权利要求6所述的特异性结合c-Met受体的抗体或其抗原结合片段，其中所述的鼠源抗体轻链可变区序列为：SEQ ID NO:5。
  9. 根据权利要求6-8任一项所述的特异性结合c-Met受体的抗体或其抗原结合片段，其中所述的鼠源抗体的重链可变区为：SEQ ID NO:4，轻链可变区为：SEQ ID NO:5。
  10. 根据权利要求1-5任一项所述的特异性结合c-Met受体的抗体或其抗原结合片段，其为嵌合抗体或人源化抗体或其片段。
  11. 根据权利要求10所述的特异性结合c-Met受体的抗体或其抗原结合片段，其中所述人源化抗体重链可变区上的重链FR区序列，来源于人种系重链序列，优选人种系重链IGHV 3-33*01；其中所述重链FR区序列包含人种系重链IGHV 3-33*01的FR1，FR2，FR3区和FR4区的框架序列或其突变序列，优选所述突变序列为0-10个氨基酸的回复突变。
  12. 根据权利要求11所述的特异性结合c-Met受体的抗体或其抗原结合片段，其中所述的人源化抗体包含选自SEQ ID NO:13-15所示的重链可变区序列或其变体。
  13. 根据权利要求10所述的特异性结合c-Met受体的抗体或其抗原结合片段，其中所述人源化抗体轻链可变区上的轻链FR区序列，选自人种系轻链序列，优选人种系轻链IGKV085或IGKV 4-1*01，其中所述轻链FR区序列包含人种系轻链IGKV085和IGKV 4-1*01的FR1，FR2，FR3区和FR4区的框架序列或其突变序列，优选所述突变序列为0-10个氨基酸的回复突变。
  14. 根据权利要求13所述的特异性结合c-Met受体的抗体或其抗原结合片段，其中所述的人源化抗体包含选自SEQ ID NO:16-18所示的轻链可变区序列或其变体。
  15. 根据权利要求10-14任一项所述的特异性结合c-Met受体的抗体或其抗原结合片段，所述的人源化抗体包含选自SEQ ID NO:13-15的重链可变区序列和选自SEQ ID NO:16-18的轻链可变区序列。
  16. 根据权利要求1-5和10-15任一项所述的特异性结合c-Met受体的抗体或其抗原结合片段，其包含选自a)至c)任一的重链可变区序列和轻链可变区序列的组合：
    a)SEQ ID NO:13的重链可变区序列和SEQ ID NO:16 的轻链可变区序列；
    b)SEQ ID NO:14的重链可变区序列和SEQ ID NO:17的轻链可变区序列； 或
    c)SEQ ID NO:15的重链可变区序列和SEQ ID NO:18的轻链可变区序列。
  17. 根据权利要求10-16任一项所述的特异性结合c-Met受体的抗体或其抗原结合片段，其中所述的人源化抗体的重链恒定区包含源自人源IgG1或其变体、人源IgG2或其变体、人源IgG3或其变体或人源IgG4或其变体的恒定区，优选包含人源IgG1或其变体或人源IgG2或其变体或人源IgG4或其变体的恒定区，更优选人源IgG2或其变体的恒定区。
  18. 根据权利要求17所述的特异性结合c-Met受体的抗体或其抗原结合片段，其包含选自SEQ ID NO:23-25或与其具有至少90％同源性的全长重链序列。
  19. 根据权利要求10-16任一项所述的特异性结合c-Met受体的抗体或其抗原结合片段，其所述的人源化抗体的轻链恒定区包含选自人源κ或λ链或其变体的恒定区。
  20. 根据权利要求19所述的特异性结合c-Met受体的抗体或其抗原结合片段，其包含选自SEQ ID NO:26-28或与其具有至少90％序列同源性的全长轻链序列。
  21. 根据权利要求10-20任一项所述的特异性结合c-Met受体的抗体或其抗原结合片段，其中所述的人源化抗体包含选自以下全长轻链序列和全长重链序列的组合：
    Ab-9:SEQ ID NO:23的重链序列和SEQ ID NO:26的轻链序列；
    Ab-10:SEQ ID NO:24的重链序列和SEQ ID NO:27的轻链序列；或
    Ab-11:SEQ ID NO:25的重链序列和SEQ ID NO:28的轻链序列。
  22. 一种DNA分子，其编码根据权利要求1-21任一项所述的特异性结合c-Met受体的抗体或其抗原结合片段。
  23. 一种表达载体，其含有根据权利要求22所述的DNA分子。
  24. 一种用根据权利要求23所述的表达载体转化的宿主细胞，其中所述的宿主细胞优选为哺乳动物细胞，更优选为CHO细胞。
  25. 一种药物组合物，其包含权利要求1-21任一项所述的特异性结合c-Met 受体的抗体或其抗原结合片段，和一种或多种可药用的赋形剂、稀释剂或载体。
  26. 一种通式(I)所示的抗体-细胞毒性药物偶联物或其药学上可接受的盐或溶剂化合物:
    Ab-[(L2)t-L1-D)]y   (I)
    其中：
    D为药物模块；
    L1，L2是接头单元；
    t为0或1，优选1；
    y为1-8，优选2-5；
    Ab如权利要求1-21任一项所述的特异性结合c-Met受体的抗体或其抗原结合片段。
  27. 如权利要求26所述的通式(I)所示的抗体-细胞毒性药物偶联物或其药学上可接受的盐或溶剂化合物，其中-L2-为以下通式(-L2-)所示的化合物：
    Figure PCTCN2016078699-appb-100001
    其中
    X1选自自氢原子、卤素、羟基、氰基、烷基、烷氧基和环烷基；
    X2选自-烷基-、-环烷基-和-杂环基-；
    m为0-5，优选1-3；S为硫原子。
  28. 根据权利要求26所述的通式(I)所示的抗体-细胞毒性药物偶联物或其可药用盐或溶剂化合物，其中所述药物模块D为选自毒素、化疗剂、抗生素、放射性同位素和核溶酶的细胞毒剂。
  29. 如权利要求26所述的通式(I)所示的抗体-细胞毒性药物偶联物或其药学上可接受的盐或溶剂化合物，其中D为以下通式(D)所示的化合物：
    Figure PCTCN2016078699-appb-100002
    或其互变异构体、内消旋体、外消旋体、对映异构体、非对映异构体、或其混合物形式，或其可药用的盐，其中：
    R1-R7选自氢原子、卤素、羟基、氰基、烷基、烷氧基和环烷基；
    R8-R11任选自氢原子、卤素、烯基、烷基、烷氧基和环烷基；R8-R11优选至少其中一个选自卤素、烯基、烷基和环烷基，其余为氢原子；
    或者R8-R11之中的任意两个形成环烷基，余下的两个基团任选自氢原子、烷基和环烷基；
    R12-R13选自氢原子、烷基或卤素；
    R14选自芳基或杂芳基，所述的芳基或杂芳基任选进一步被选自氢原子、卤素、羟基、烷基、烷氧基和环烷基的取代基所取代；
    R15任选自卤素、烯基、烷基、环烷基和COO R17；
    R16选自氢原子、卤素、羟基、氰基、烷基、烷氧基和环烷基；
    R17选自氢原子、烷基和烷氧基。
  30. 根据权利要求29所述的通式(I)所示的抗体-细胞毒性药物偶联物或其可药用盐或溶剂化合物，其中L2包含选自Val-Cit，MC，PAB和MC-PAB的接头，优选MC。
  31. 根据权利要求26所述的通式(I)所示的抗体-细胞毒性药物偶联物或其可药用盐或溶剂化合物，其中D是美登木素生物碱；优选DM1、DM3和DM4，更优选DM1。
  32. 根据权利要求31所述的通式(I)所示的抗体-细胞毒性药物偶联物或其可药用盐或溶剂化合物，其中所述L2选自N-琥珀酰亚氨基4-(2-吡啶基硫代)戊酸酯(SPP)、N-琥珀酰亚氨基4-(N-马来酰亚胺基甲基)-环己烷-1-羧酸酯(SMCC)、和N-琥珀酰亚氨基(4-碘-乙酰基)氨基苯甲酸酯(SIAB)；优选SPP或SMCC。
  33. 根据权利要求26所述的通式(I)所示的抗体-细胞毒性药物偶联物或其可药用盐或溶剂化合物，其中D是喜树碱类生物碱；优选自CPT、10-羟基-CPT、CPT-11(伊立替康)、SN-38和托泊替康，更优选SN-38。
  34. 根据权利要求33所述的通式(I)所示的抗体-细胞毒性药物偶联物或其可药用盐或溶剂化合物，其中所述接头L2包含任选自Val-Cit，MC，PAB或MC-PAB的结构，优选MC或MC-vc-PAB。
  35. 如权利要求26所述的通式(I)所示的抗体-细胞毒性药物偶联物或其药学 上可接受的盐或溶剂化合物，其为通式(II)所示的偶联药物或其药学上可接受的盐或溶剂化合物：
    Figure PCTCN2016078699-appb-100003
    其中：
    R2-R16如权利要求27中所定义；
    Ab，t，y，L1，L2如权利要求24中所定义。
  36. 如权利要求26所述的通式(I)所示的抗体-细胞毒性药物偶联物或其药学上可接受的盐或溶剂化合物，其为通式(III)所示的偶联药物或其药学上可接受的盐或溶剂化合物：
    Figure PCTCN2016078699-appb-100004
    其中：
    R2-R16如权利要求27中所定义；
    Ab，t，y，L1，L2如权利要求24中所定义；
    n为3-6，优选5。
  37. 如权利要求26所述的通式(I)所示的抗体-细胞毒性药物偶联物或其药学上可接受的盐或溶剂化合物，其为通式(IV)所示的偶联药物或其药学上可接受的盐或溶剂化合物：
    Figure PCTCN2016078699-appb-100005
    其中：
    R2-R16如权利要求27中所定义；
    Ab，y如权利要求24中所定义；
    n如权利要求34中所定义；
    X1，X2，m如权利要求25中所定义。
  38. 如权利要求26所述的通式(I)所示的抗体-细胞毒性药物偶联物或其药学上可接受的盐或溶剂化合物，其为通式(V)所示的偶联药物或其药学上可接受的盐或溶剂化合物：
    Figure PCTCN2016078699-appb-100006
    其中：
    Ab，D，y如权利要求24中所定义；
    n如权利要求34中所定义；
    X1，X2，m如权利要求25中所定义。
  39. 根据权利要求26-38任一项所述的通式(I)所示的抗体-细胞毒性药物偶联物或其可药用盐或溶剂化合物，其中所述抗体-细胞毒性药物偶联物或其可药用盐或溶剂化合物选自：
    Figure PCTCN2016078699-appb-100007
    Figure PCTCN2016078699-appb-100008
    Figure PCTCN2016078699-appb-100009
    Figure PCTCN2016078699-appb-100010
    Ab-9，Ab-10，Ab-11如权利要求21中所定义，其中，y的范围为1-8；优选2-5。
  40. 一种制备根据权利要求38中所述的通式(V)所示的偶联药物或其药学上可接受的盐或溶剂化合物的方法，该方法包括：
    Figure PCTCN2016078699-appb-100011
    通式(Ab-L2)化合物与通式(L1-D)化合物在有机溶剂中反应，得到通式(V)化合物；所述的有机溶剂优选乙腈或乙醇；
    其中：
    Ab如权利要求1-21任一项所述的特异性结合c-Met受体的抗体或其抗原结合片段；
    X1选自自氢原子、卤素、羟基、氰基、烷基、烷氧基和环烷基；
    X2选自烷基、环烷基和杂环基；
    X为0-5，优选1-3；
    m为0-5，优选1-3。
  41. 一种药物组合物，其含有根据权利要求26-39任一项所述的通式(I)所示的抗体-细胞毒性药物偶联物或其可药用盐或溶剂化合物和可药用的赋形剂、稀释剂或载体。
  42. 根据权利要求1-21任一项所述的特异性结合c-Met受体的抗体或其抗原结合片段、或根据权利要求25所述的药物组合物、或根据权利要求26-39任一项所述的通式(I)所示的抗体-细胞毒性药物偶联物或其可药用盐或溶剂化合 物、或根据权利要求41所述的药物组合物，在制备用于治疗c-Met介导的疾病或病症的药物中的用途，其中所述的疾病或病症优选为癌症；更优选为表达c-Met的癌症；最优选为胃癌、胰腺癌、肺癌、肠癌、肾癌、黑素瘤、非小细胞肺癌；最优选为胃癌、胰腺癌、非小细胞肺癌和肾癌。
PCT/CN2016/078699 2015-04-17 2016-04-07 抗c-Met抗体和抗c-Met抗体-细胞毒性药物偶联物及其医药用途 WO2016165580A1 (zh)

Priority Applications (10)

Application Number Priority Date Filing Date Title
CN201680001857.2A CN106687480B (zh) 2015-04-17 2016-04-07 抗c-Met抗体和抗c-Met抗体-细胞毒性药物偶联物及其医药用途
US15/565,928 US10543284B2 (en) 2015-04-17 2016-04-07 Anti-c-Met antibody and anti-c-Met antibody-cytotoxic drug conjugate and pharmaceutical use thereof
BR112017021245-5A BR112017021245A2 (zh) 2015-04-17 2016-04-07 Anti-c-Met antibody and anti-c-Met antibody - cytotoxic drug conjugates and pharmaceutical use
EP16779555.8A EP3284751A4 (en) 2015-04-17 2016-04-07 Anti-c-met antibody and anti-c-met antibody-cytotoxic drug conjugate and pharmaceutical use thereof
AU2016248357A AU2016248357A1 (en) 2015-04-17 2016-04-07 Anti-c-Met antibody and anti-c-Met antibody-cytotoxic drug conjugate and pharmaceutical use thereof
MX2017012965A MX2017012965A (es) 2015-04-17 2016-04-07 Anticuerpo anti-c-met y conjugado de farmaco citotoxico de anticuerpo anti-c-met y uso farmaceutico del mismo.
RU2017135257A RU2017135257A (ru) 2015-04-17 2016-04-07 Антитело к с-мет и конъюгат антитело к с-мет-цитотоксическое лекарственное средство, и их фармацевтическое применение
JP2017553320A JP2018516539A (ja) 2015-04-17 2016-04-07 抗ｃ−Ｍｅｔ抗体および抗ｃ−Ｍｅｔ抗体−細胞毒性薬物複合体ならびにそれらの医薬用途
KR1020177031260A KR20170138451A (ko) 2015-04-17 2016-04-07 항-ｃ-ｍｅｔ 항체 및 항-ｃ-ｍｅｔ 항체-세포독성 약물 컨쥬게이트 및 이의 약학적 용도
CA2982777A CA2982777A1 (en) 2015-04-17 2016-04-07 Anti-c-met antibody and anti-c-met antibody-cytotoxic drug conjugate and pharmaceutical use thereof

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
CN201510185602 2015-04-17
CN201510185602.3 2015-04-17
CN201510300885.1A CN106188293A (zh) 2015-04-17 2015-06-04 抗c-Met抗体和抗c-Met抗体-细胞毒性药物偶联物及其医药用途
CN201510300885.1 2015-06-04

Publications (1)

Publication Number Publication Date
WO2016165580A1 true WO2016165580A1 (zh) 2016-10-20

Family

ID=57125727

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2016/078699 WO2016165580A1 (zh) 2015-04-17 2016-04-07 抗c-Met抗体和抗c-Met抗体-细胞毒性药物偶联物及其医药用途

Country Status (1)

Country Link
WO (1) WO2016165580A1 (zh)

Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2018068758A1 (zh) * 2016-10-14 2018-04-19 苏州盛迪亚生物医药有限公司 抗c-Met抗体-细胞毒性药物偶联物的医药用途
WO2018223958A1 (zh) 2017-06-06 2018-12-13 江苏恒瑞医药股份有限公司 一种含c-Met抗体药物偶联物的药物组合物及其用途
US10383948B2 (en) * 2016-05-17 2019-08-20 Abbvie Inc. Anti-cMet antibody drug conjugates and methods for their use
WO2020108612A1 (zh) * 2018-11-30 2020-06-04 江苏恒瑞医药股份有限公司 一种c-Met ADC在制备治疗c-Met激酶抑制剂耐药的疾病的药物中的用途
CN111494645A (zh) * 2020-05-20 2020-08-07 中国药科大学 抗人dll4人源化抗体与美登素生物碱dm1的偶联物及其制备方法与应用
WO2021225892A1 (en) * 2020-05-03 2021-11-11 Levena (Suzhou) Biopharma Co., Ltd. Antibody-drug conjugates (adcs) comprising an anti-trop-2 antibody, compositions comprising such adcs, as well as methods of making and using the same
WO2022214517A1 (en) * 2021-04-08 2022-10-13 Byondis B.V. Anti-c-met antibodies and antibody-drug conjugates
CN115297889A (zh) * 2020-09-01 2022-11-04 荣昌生物制药(烟台)股份有限公司 抗c-Met抗体药物偶联物及其应用
WO2023284829A1 (zh) 2021-07-14 2023-01-19 江苏恒瑞医药股份有限公司 特异性结合hgfr和egfr的抗原结合分子及其医药用途

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101035808A (zh) * 2004-08-05 2007-09-12 健泰科生物技术公司 人源化抗c－met拮抗剂
CN103394083A (zh) * 2003-11-06 2013-11-20 西雅图基因公司 能够与配体偶联的单甲基缬氨酸化合物

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103394083A (zh) * 2003-11-06 2013-11-20 西雅图基因公司 能够与配体偶联的单甲基缬氨酸化合物
CN101035808A (zh) * 2004-08-05 2007-09-12 健泰科生物技术公司 人源化抗c－met拮抗剂

Cited By (15)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10383948B2 (en) * 2016-05-17 2019-08-20 Abbvie Inc. Anti-cMet antibody drug conjugates and methods for their use
EP3626273A1 (en) * 2016-05-17 2020-03-25 AbbVie Biotherapeutics Inc. Anti-cmet antibody drug conjugates and methods for their use
US10603389B2 (en) 2016-05-17 2020-03-31 Abbvie Inc. Anti-cMet antibody drug conjugates and methods for their use
EP4233909A3 (en) * 2016-05-17 2023-09-20 AbbVie Biotherapeutics Inc. Anti-cmet antibody drug conjugates and methods for their use
EP3804765A1 (en) * 2016-05-17 2021-04-14 AbbVie Biotherapeutics Inc. Anti-cmet antibody drug conjugates and methods for their use
WO2018068758A1 (zh) * 2016-10-14 2018-04-19 苏州盛迪亚生物医药有限公司 抗c-Met抗体-细胞毒性药物偶联物的医药用途
WO2018223958A1 (zh) 2017-06-06 2018-12-13 江苏恒瑞医药股份有限公司 一种含c-Met抗体药物偶联物的药物组合物及其用途
WO2020108612A1 (zh) * 2018-11-30 2020-06-04 江苏恒瑞医药股份有限公司 一种c-Met ADC在制备治疗c-Met激酶抑制剂耐药的疾病的药物中的用途
CN112996540A (zh) * 2018-11-30 2021-06-18 江苏恒瑞医药股份有限公司 一种c-Met ADC在制备治疗c-Met激酶抑制剂耐药的疾病的药物中的用途
WO2021225892A1 (en) * 2020-05-03 2021-11-11 Levena (Suzhou) Biopharma Co., Ltd. Antibody-drug conjugates (adcs) comprising an anti-trop-2 antibody, compositions comprising such adcs, as well as methods of making and using the same
CN111494645B (zh) * 2020-05-20 2022-09-30 中国药科大学 抗人dll4人源化抗体与美登素生物碱dm1的偶联物及其制备方法与应用
CN111494645A (zh) * 2020-05-20 2020-08-07 中国药科大学 抗人dll4人源化抗体与美登素生物碱dm1的偶联物及其制备方法与应用
CN115297889A (zh) * 2020-09-01 2022-11-04 荣昌生物制药(烟台)股份有限公司 抗c-Met抗体药物偶联物及其应用
WO2022214517A1 (en) * 2021-04-08 2022-10-13 Byondis B.V. Anti-c-met antibodies and antibody-drug conjugates
WO2023284829A1 (zh) 2021-07-14 2023-01-19 江苏恒瑞医药股份有限公司 特异性结合hgfr和egfr的抗原结合分子及其医药用途

Similar Documents

Publication Publication Date Title
TWI731856B (zh) 抗c-Met抗體和抗c-Met抗體-細胞毒性藥物偶聯物及其醫藥用途
JP7064629B2 (ja) 抗ｃｄｈ６抗体及び抗ｃｄｈ６抗体－薬物コンジュゲート
US20210386865A1 (en) Method for selectively manufacturing antibody-drug conjugate
WO2016165580A1 (zh) 抗c-Met抗体和抗c-Met抗体-细胞毒性药物偶联物及其医药用途
KR102508023B1 (ko) 세포-결합제 및 세포독성 약물을 포함하는 콘주게이트
WO2015146132A1 (ja) 抗ｃｄ９８抗体－薬物コンジュゲート
US20160106861A1 (en) Axl antibody-drug conjugate and its use for the treatment of cancer
WO2017161206A1 (en) Conjugates containing conditionally active antibodies or antigen-binding fragments thereof, and methods of use
US11834498B2 (en) Biparatopic FR-alpha antibodies and immunoconjugates
CN115698079A (zh) 抗cd79b抗体药物偶联物、其制备方法及其医药用途
WO2018068758A1 (zh) 抗c-Met抗体-细胞毒性药物偶联物的医药用途
CA3141428A1 (en) Anti-tissue factor antibody-drug conjugates and related methods
CA2930599A1 (en) Compounds to fibroblast growth factor receptor-3 (fgfr3) and therapeutic uses
TW201839001A (zh) 細胞毒性苯并二氮平衍生物及其綴合物
US10159690B2 (en) Chemically modified targeting protein and use thereof

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: 16779555

Country of ref document: EP

Kind code of ref document: A1

ENP Entry into the national phase

Ref document number: 2982777

Country of ref document: CA

ENP Entry into the national phase

Ref document number: 2017553320

Country of ref document: JP

Kind code of ref document: A

WWE Wipo information: entry into national phase

Ref document number: MX/A/2017/012965

Country of ref document: MX

REG Reference to national code

Ref country code: BR

Ref legal event code: B01A

Ref document number: 112017021245

Country of ref document: BR

WWE Wipo information: entry into national phase

Ref document number: 15565928

Country of ref document: US

NENP Non-entry into the national phase

Ref country code: DE

ENP Entry into the national phase

Ref document number: 2016248357

Country of ref document: AU

Date of ref document: 20160407

Kind code of ref document: A

ENP Entry into the national phase

Ref document number: 20177031260

Country of ref document: KR

Kind code of ref document: A

WWE Wipo information: entry into national phase

Ref document number: 2017135257

Country of ref document: RU

ENP Entry into the national phase

Ref document number: 112017021245

Country of ref document: BR

Kind code of ref document: A2

Effective date: 20171003