WO2017088734A1 - 抗ErbB2抗体-药物偶联物及其组合物、制备方法和应用 - Google Patents

抗ErbB2抗体-药物偶联物及其组合物、制备方法和应用 Download PDF

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WO2017088734A1
WO2017088734A1 PCT/CN2016/106802 CN2016106802W WO2017088734A1 WO 2017088734 A1 WO2017088734 A1 WO 2017088734A1 CN 2016106802 W CN2016106802 W CN 2016106802W WO 2017088734 A1 WO2017088734 A1 WO 2017088734A1
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Prior art keywords
antibody
group
drug conjugate
cancer
drug
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PCT/CN2016/106802
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English (en)
French (fr)
Inventor
薛彤彤
苗振伟
汪静
陈刚
卿燕
朱同
肖亮
张宏
杨秋艳
邓大伦
刘立平
曾宏
尹利
石奇峰
宋宏梅
赵曦
王利春
王晶翼
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四川科伦博泰生物医药股份有限公司
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Priority to ES16867957T priority Critical patent/ES2887349T3/es
Priority to CN201811298611.3A priority patent/CN109395090B/zh
Application filed by 四川科伦博泰生物医药股份有限公司 filed Critical 四川科伦博泰生物医药股份有限公司
Priority to EA201792590A priority patent/EA039757B1/ru
Priority to CN201811298547.9A priority patent/CN109395089B/zh
Priority to PL16867957T priority patent/PL3381469T3/pl
Priority to KR1020177036761A priority patent/KR20180080128A/ko
Priority to JP2017566133A priority patent/JP6888871B2/ja
Priority to CN201680036760.5A priority patent/CN107921124B/zh
Priority to DK16867957.9T priority patent/DK3381469T3/da
Priority to EP16867957.9A priority patent/EP3381469B1/en
Priority to CA3000763A priority patent/CA3000763A1/en
Priority to US15/765,685 priority patent/US20190076438A1/en
Priority to AU2016359514A priority patent/AU2016359514A1/en
Publication of WO2017088734A1 publication Critical patent/WO2017088734A1/zh
Priority to HK18103610.4A priority patent/HK1243957B/zh
Priority to US17/169,087 priority patent/US11903948B2/en
Priority to US17/471,653 priority patent/US20220008553A1/en
Priority to US18/324,511 priority patent/US20230293536A1/en

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    • A—HUMAN NECESSITIES
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    • 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
    • A61K31/536—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 ortho- or peri-condensed with carbocyclic ring systems
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    • 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
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
    • A61K39/39558—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against tumor tissues, cells, antigens
    • A—HUMAN NECESSITIES
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    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
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    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
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    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/65—Peptidic linkers, binders or spacers, e.g. peptidic enzyme-labile linkers
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    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A—HUMAN NECESSITIES
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    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6851—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a determinant of a tumour cell
    • A61K47/6869—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a determinant of a tumour cell the tumour determinant being from a cell of the reproductive system: ovaria, uterus, testes, prostate
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/32—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against translation products of oncogenes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered

Definitions

  • the invention belongs to the field of biomedical technology.
  • the invention relates to anti-ErbB2 antibody-drug conjugates, compositions comprising the same, methods for their preparation, and uses.
  • ADC antibody-drug conjugate
  • ADCs typically include three moieties that are joined in a manner: antibodies, linkers, and drugs.
  • Antibodies are good drug targeting carriers.
  • the drug can be linked to the antibody to form a chemical immunoconjugate using a specific functional group such as a hydroxyl group, a thiol group or an amino group in the drug molecule.
  • the targeting performance of the antibody delivers the drug to which it is attached to the target cell "accurately", thereby effectively increasing the concentration of the drug in the local lesion, and greatly reducing the drug concentration of other tissues and organs in the body, thereby achieving synergy and attenuation.
  • Both polyclonal and monoclonal antibodies for these strategies have been reported (Rowland et al., 1986, Cancer Immunol. Immunother., 21: 183-87).
  • PSMA ADC anti-PSMA antibody-MMAE conjugate
  • SGN-75 anti-CD70 antibody-MMAF conjugate
  • T-DM1 curved Humanized antibodies were used in the tocilizumab-DM1 conjugate.
  • FDA approved ADC drugs include (T-DM1) and (brentuximab vedotin).
  • ADCs The drugs used as "warheads" in ADCs are usually cytotoxic drugs, which kill tumor cells mainly by inhibiting cell DNA or protein synthesis, inhibiting cell mitosis. Since cytotoxic drugs are also highly lethal to normal cells, their application and development are greatly limited. Early ADCs used conventional anti-tumor drugs, but the clinical activity of these ADCs was mostly lower than the in vitro activity of chemical monomers.
  • the cytotoxic drugs used in current ADCs mainly include: Maytansinoids (see, for example, EP 0425235, US 5208020, US 5416064, US 7276497, US 7473796, US 7851432, US 2007/0269447, US 2011/0158991, WO 2004/103272, WO 2012/061590), auristatin peptides (see, for example, US 6884869, US 7498298), calicheamicins (see, for example, US 5606040, US 5770710), doxorubicin ( Doxorubicins, see for example Dubowchik et al, 2002, Bioconjugate Chem., 13: 855-869), benzodipyrrole antibiotics (duocarmycins and CC-1065, see for example US 7129261), irinotecan metabolites (see also For example, WO 2015/012904), pyrrolobenzodiazepines (see, for example, Biotechn
  • cytotoxic drugs have strong non-selective toxicity and can cause damage to normal cells, and thus cannot be used as a medicine.
  • the linker in the ADC needs to meet the following requirements: to ensure that the small molecule drug does not detach from the antibody outside the cell; after entering the cell, the cleavable linker cleaves under appropriate conditions, releasing the active small molecule drug, and the non-cleavable linker The active moiety is then formed with the small molecule drug and the amino acid residue from the enzymatic hydrolysis of the antibody.
  • cytotoxic drugs are usually linked to a lysine residue on the surface of the antibody by a linker, or a cysteine residue attached to the hinge region of the antibody (by the interchain disulfide bond moiety) Restore is obtained).
  • a linker or a cysteine residue attached to the hinge region of the antibody (by the interchain disulfide bond moiety) Restore is obtained.
  • the number and location of the coupling are uncertain due to the presence of a large number (more than 80) of lysine residues on the surface of the antibody and the coupling reaction is non-selective. , resulting in the heterogeneity of the generated ADC.
  • T-DM1 has a drug antibody ratio (DAR) distribution of 0-8 and an average DAR value of 3.5 (Lazar et al., 2005, Rapid Commun. Mass Spectrom., 19: 1806-1814).
  • DAR drug antibody ratio
  • the ADC has a DAR value of 2-4.
  • heterogeneity of ADC products is detrimental to their clinical application because of the non-uniformity of pharmacokinetic properties, potency and/or toxicity between components in the product (for example, components with higher DAR values are eliminated in vivo) It is faster and leads to higher toxicity, see for example Boswell et al., 2011, Bioconjugate Chem., 22: 1994-2004), making its stability unsatisfactory.
  • the ErbB receptor tyrosine kinase is an important mediator of cell growth, differentiation and survival.
  • the family includes four members: the epidermal growth factor receptor (EGFR or ErbB1), Her2 (ErbB2), Her3 (ErbB3), and Her4 (ErbB4).
  • the ErbB2-expressing breast cancer is treated clinically with the anti-ErbB2 antibody trastuzumab.
  • 15% of breast cancer patients with immunohistochemistry (IHC) 2+ levels developed a clinical response to trastuzumab with a median response duration of 9.1 months (see, for example, Cobleigh et al., 1996, Journal of Clinical Oncology, 14: 737-744).
  • Trastuzumab (trade name Herceptin) was approved by the US Food and Drug Administration (FDA) on September 25, 1998 for the treatment of breast cancer patients overexpressing ErbB2 protein.
  • trastuzumab rescued some breast cancer patients or prolonged patient survival, it was only effective in patients with high ErbB2 expression, and the clinical response rate was only 15%. Therefore, there is a need for drugs that have better efficacy for more patients.
  • trastuzumab has been coupled with maytansin (DM1) to make trastuzumab emtansine (T-DM1, ), for patients with trastuzumab, other anti-Her2 treatments and common breast cancer treatment for first-line chemotherapy drugs ineffective for taxanes.
  • T-DM1 delivers the drug to the tumor site, which shrinks the tumor, slows disease progression and prolongs survival.
  • the safety and efficacy of T-DM1 was evaluated in clinical trials.
  • T-DM1 is now the fourth FDA-approved drug targeting Her2.
  • T-DM1 has a black box warning at the time of approval to alert patients and health care professionals that the drug can cause liver toxicity, cardiotoxicity and death. This is mainly caused by the DM1 and T-DM1 which are released into the body and released by small molecules containing DM1.
  • T-DM1 was prepared by using the lysine side chain amino group on trastuzumab and sulfosuccinimidyl-4-[N-maleimidomethyl]cyclohexane-1-carboxyl The ester (sulfo-SMCC) is coupled and then coupled to DM1.
  • sulfo-SMCC the ester
  • the average number of DM1 molecules coupled per trastuzumab was 3.5. Since trastuzumab has 88 lysine sites, T-DM1 actually couples trastuzumab with different numbers of DM1 and multiple antibody-drug conjugates with multiple coupling sites. The combination. However, the pharmacodynamic, pharmacokinetic properties and/or toxicity of each antibody-drug conjugate is different.
  • antibody-drug conjugates with high drug loading have stronger in vitro activity, but high drug loading also causes problems such as polymerization due to polymerization between antibody-drug conjugates.
  • Increased body composition decreased stability, increased toxicity, increased immunogenicity, excessive clearance in vivo, short half-life and low actual therapeutic index.
  • the present invention is directed to solving the above problems in the prior art.
  • the invention provides anti-Her2 antibody-drug Conjugates, which inhibit tumor growth in mammals, can be used to treat a variety of cancers.
  • the conjugates have better biological activity, stability and homogeneity, and have reduced toxic side effects.
  • the present invention provides an antibody-drug conjugate of the general formula (I), a pharmaceutically acceptable salt, stereoisomer or metabolite thereof, or a solvate thereof,
  • A is an anti-ErbB2 antibody or an active fragment or variant thereof
  • X and Y are each independently N or CR 1 , and each R 1 is independently H or C 1 -C 10 alkyl;
  • L is a divalent linking group
  • D is a cytotoxic drug group
  • a is an integer selected from 2-10.
  • the invention provides a method of preparing an antibody-drug conjugate of the first aspect of the invention, the method comprising the steps of:
  • G is selected from the group consisting of -F, -Cl, -Br, -I, -N 3 , -OR, -SR, -ONRR', RC(O)O-, -OP(O)RR', RSO 2 -O- with
  • R and R' are each independently a C 1 -C 10 alkyl group, a C 6 -C 14 aryl group, a heterocyclic group having 5 to 10 ring atoms or a phenoxy group, said alkyl group,
  • the aryl, heterocyclic and phenoxy groups are unsubstituted or are each independently selected from one or more selected from the group consisting of halogen, hydroxy, C 1 -C 4 alkyl, C 1 -C 4 alkoxy, C 3 -C a substituent of an 8- cycloalkyl group, a heterocyclic group having 5 to 8 ring atoms, a C 6 -C 10 aryl group or a hetero
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising the antibody-drug conjugate of the first aspect of the invention or a pharmaceutically acceptable salt, stereoisomer or metabolite thereof or a solvate thereof, and A pharmaceutically acceptable carrier.
  • the present invention provides the antibody-drug conjugate of the first aspect of the invention or a pharmaceutically acceptable salt, stereoisomer or metabolite thereof or a solvate thereof for use in the preparation or prevention of cancer Use of the drug.
  • the present invention provides a pharmaceutical preparation comprising the antibody-drug conjugate of the first aspect of the invention or a pharmaceutically acceptable salt, stereoisomer or metabolite thereof or a solvate thereof.
  • the invention provides a method of preventing or treating cancer, the method comprising administering to a patient in need thereof an antibody-drug conjugate of the first aspect of the invention, a pharmaceutically acceptable salt thereof, a stereoisomeric
  • the pharmaceutical composition of the third aspect of the invention or the pharmaceutical preparation of the fifth aspect of the invention is administered to a construct or metabolite or a solvate thereof, or to a patient in need thereof.
  • Figure 1 is a HIC-HPLC spectrum of the crude antibody-drug conjugate I-1.
  • Figure 2 is a HIC-UPLC spectrum of antibody-drug conjugate I-1.
  • Figure 3 is an overlapping reverse phase chromatogram of antibody-drug conjugate I-1 versus trastuzumab naked anti-reduction.
  • Figure 4 is a peptide map obtained by digesting antibody-drug conjugate I-1 and trastuzin naked under the same conditions.
  • Figure 5 is a HIC-HPLC spectrum of the crude antibody-drug conjugate I-2.
  • Figure 6 is a HIC-HPLC spectrum of antibody-drug conjugate I-2.
  • Figure 7 is an overlapping reverse phase chromatogram of antibody-drug conjugate I-2 versus trastuzumab naked anti-reduction.
  • Figure 8 is a diagram showing that the antibody-drug conjugate I-2 and the trastuzin naked antibody were digested under the same conditions. The peptide map obtained.
  • Figure 9 is a HIC-HPLC spectrum of the crude antibody-drug conjugate I-3.
  • Figure 10 is a HIC-HPLC spectrum of antibody-drug conjugate I-3.
  • Figure 11 is an overlapping reverse phase chromatogram of antibody-drug conjugate I-3 versus trastuzumab naked anti-reduction.
  • Figure 12 is a peptide map obtained by digesting antibody-drug conjugate I-3 and trastuzin naked under the same conditions.
  • ranges of values herein are to be understood as encompassing any and all sub-ranges.
  • the range "1 to 10" should be understood to include not only the clearly recited values of 1 to 10, but also any single value in the range of 1 to 10 (eg, 2, 3, 4, 5, 6, 7, 8 and 9) and sub-ranges (eg 1 to 2, 1.5 to 2.5, 1 to 3, 1.5 to 3.5, 2.5 to 4, 3 to 4.5, etc.). This principle also applies to a range where only one value is used as the minimum or maximum value.
  • the present invention provides an antibody-drug conjugate of the general formula (I), a pharmaceutically acceptable salt, stereoisomer or metabolite thereof, or a solvate thereof,
  • A is an anti-ErbB2 antibody or an active fragment or variant thereof
  • X and Y are each independently N or CR 1 (preferably Y is CR 1 , X is N), and each R 1 is independently H or C 1 -C 10 alkyl, preferably H or C 1 -C 6 An alkyl group (eg methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, pentyl or hexyl);
  • L is a divalent linking group
  • D is a cytotoxic drug group
  • a is an integer selected from 2-10, such as 2, 3 or 4, particularly preferably 2.
  • the antibody used in the present invention is an anti-ErbB2 antibody or an active fragment or variant thereof, including a bispecific antibody and an antibody functional derivative.
  • ErbB2 and Her2 are used interchangeably, both of which represent the human HER2 protein of the native sequence (Genebank accession number: X03363, see, eg, Semba et al, 1985, PNAS, 82: 6497-6501; and Yamamoto et al, 1986, Nature, 319: 230-234) and functional derivatives thereof, such as amino acid sequence variants.
  • erbB2 represents the gene encoding human Her2
  • neu represents the gene encoding rat pl85neu.
  • Cancer cells such as breast cancer cells, ovarian cancer cells, gastric cancer cells, endometrial cancer cells, salivary gland cancer cells, lung cancer cells, renal cancer cells, colon cancer cells, thyroid cancer cells, pancreatic cancer cells, bladder cancer cells or liver cancer cells Etc., usually a cell expressing the ErbB2 receptor.
  • the preferred Her2 in the present invention is the native sequence human Her2.
  • anti-Her2 antibodies useful in the present invention include, but are not limited to, MAbs4D5 (ATCC CRL 10463), 2C4 (ATCC HB-12697), 7F3 (ATCC HB-12216), and 7C2 (ATCC HB 12215), which are described in For example, US 5,772,997, WO 98/77797 and US 5,840,525.
  • Humanized anti-Her2 antibodies include huMAb4D5, huMAb4D5-1, huMAb4D5-2, huMAb4D5-3, huMAb4D5-4, huMAb4D5-5, huMAb4D5-6, huMAb4D5-7 and huMAb4D5-8 listed in Table 3 of US 5,821,337; 1370082A 7C2, 7F3, 2C4, 7D3, 3E8, and 2C4 shown in FIG. 1B.
  • Examples of the anti-Her2 antibody which can be used in the present invention may further include: F243L, R292P and Y300L described in WO 2009/123894; F243L, R292P and V305I; F243L, R292P and P396L; and R292P, V305I and P396; WO 2012/079093 MM-111; CN104418952A
  • WO 2010/108127, Dl, Dl. 5, Dl. 5-100, DLI l, DL Ib or DLl If antibody described in Figures 33A and 33B; and humanized 520C9 as described in WO 93/21319.
  • the native sequence Her2 in the present invention can be isolated from nature, or can be prepared by recombinant DNA technology, chemical synthesis, or a combination thereof.
  • the term "functional derivative” includes amino acid sequence variants as well as covalent derivatives of natural polypeptides (eg, derivatives obtained by post-translational modification, pyroglutamate, etc.), provided that they remain comparable or higher than the native polypeptide. Affinity and biological activity.
  • Amino acid sequence variants differ from the natural polypeptide amino acid sequence generally by substitutions, deletions and/or insertions of one or more amino acids in the latter.
  • Deletion variants include fragments of the native polypeptide and N-terminal and/or C-terminal truncated variants. Usually, amino The acid sequence variant should have at least 70% or at least 80% or at least 90% homology to the native polypeptide.
  • Covalent derivatives of the native polypeptide can be derivatives obtained by altering post-translational processing of the antibody, such as changing the number or position of glycosylation sites.
  • homology is used interchangeably with “consistency,” “identity,” or “similarity,” and refers to two nucleic acid sequences to be compared after optimal alignment (optimal alignment). Or the percentage of identical nucleotides or identical amino acid residues between amino acid sequences that are purely statistical and that the differences between the two sequences are randomly distributed and cover the full length thereof. Alignment between two nucleic acid sequences or amino acid sequences is typically performed by comparing the sequences after matching them in an optimal manner, which can be performed by segments or by a "comparison window”. In addition to manual implementation, optimal alignment can also be performed by other methods described in the literature, such as the local homology algorithm described by Smith and Waterman, 1981, Ad. App.
  • antibody is used in its broadest sense and includes intact monoclonal antibodies, polyclonal antibodies, and multispecific antibodies (eg, bispecific antibodies) formed from at least two intact antibodies, as long as they have The biological activity required.
  • the term "monoclonal antibody” refers to an antibody from a population of substantially homogeneous antibodies, ie, the individual antibodies comprising the cluster are identical except for a small number of natural mutations that may be present. Monoclonal antibodies have high specificity for one determinant (epitope) of the antigen, while polyclonal antibodies relative thereto contain different antibodies for different determinants (epitopes). In addition to specificity, monoclonal antibodies have the advantage of being free from contamination by other antibodies during synthesis.
  • the modifier "monoclonal” as used herein means that the antibody is characterized by a substantially homogeneous population of antibodies and is not to be construed as being prepared by a particular method.
  • the monoclonal antibody particularly includes a chimeric antibody, that is, a part of the heavy chain and/or the light chain is identical or homologous to one, a certain class or a subclass of antibodies, and the other part is the other and another One or another subclass of antibodies are identical or homologous as long as they have the desired biological activity (see, for example, US 4,816,567; and Morrison et al., 1984, PNAS, 81:6851-6855).
  • Chimeric antibodies useful in the present invention include primatized antibodies comprising non-human primates (eg, Variable region antigen binding sequences such as ancient monkeys, orangutans, etc. and human constant region sequences.
  • antibody fragment refers to a portion of an antibody, preferably an antigen binding or variable region.
  • antibody fragments include Fab, Fab', F(ab')2 and Fv fragments; diabodies; linear antibodies; and single-chain antibody molecules.
  • bispecific antibody is used interchangeably with "bifunctional antibody conjugate” and refers to a conjugate formed by a first antibody (fragment) and a second antibody (fragment) by a coupling arm. The substance retains the activity of the respective antibody and is therefore bifunctional and bispecific.
  • multispecific antibody includes, for example, trispecific antibodies and tetraspecific antibodies, the former being antibodies having three different antigen binding specificities and the latter being antibodies having four different antigen binding specificities.
  • the term "intact antibody” refers to an antibody comprising an antigen binding variable region and a light chain constant region (CL), a heavy chain constant region (CH1, CH2, and CH3).
  • the constant region can be a native sequence (eg, a human native constant region sequence) or an amino acid sequence variant thereof.
  • An intact antibody is preferably an intact antibody having one or more effector functions.
  • a "humanized" form of a non-human (eg, murine) antibody refers to a chimeric antibody comprising a minimal amount of non-human immunoglobulin sequences. Most humanized antibodies are human receptors. The hypervariable region residues of immunoglobulins are replaced by non-humans (eg, mice, rats, rabbits, or non-human primates) with the desired specificity, affinity, and function. Hypervariable region residues (donor antibodies). In some embodiments, the framework region (FR) residues of the human immunoglobulin are also replaced with non-human residues. Moreover, the humanized antibody may also comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications are intended to further optimize the performance of the antibody.
  • non-human (eg, murine) antibody refers to a chimeric antibody comprising a minimal amount of non-human immunoglobulin sequences. Most humanized antibodies are human receptors. The hypervariable region residues of immunoglobulins are replaced by non-humans (e
  • Humanized antibodies generally comprise at least one, usually two variable regions, wherein all or nearly all of the hypervanable loops correspond to non-human immunoglobulins, while FR is completely or almost entirely human immunoglobulin.
  • a humanized antibody can also comprise at least a portion of an immunoglobulin constant region (Fc, typically a human immunoglobulin Fc).
  • Fc immunoglobulin constant region
  • Intact antibodies can be divided into different "classes” based on the amino acid sequence of the heavy chain constant region.
  • the main five classes are IgA, IgD, IgE, IgG and IgM, several of which can also be divided into different "subclasses” (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2.
  • the heavy chain constant regions of different classes of antibodies are called ⁇ , ⁇ , ⁇ , ⁇ , and ⁇ , respectively.
  • Subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known in the art.
  • one or more D-amino acids can be included in the antibody peptide chain. It is believed that peptides comprising D-amino acids are more stable in the oral, intestinal or plasma than peptides comprising only L-amino acids and are not readily degradable.
  • the monoclonal antibodies used in the present invention can be produced by a number of methods.
  • monoclonal antibodies for use in the present invention can be obtained by hybridoma methods using a variety of species including cells of mice, hamsters, rats, and humans (see, for example, Kohler et al., 1975, Nature, 256:495).
  • Alternatively by recombinant DNA techniques see, for example, US 4,816,567), or isolated from phage antibody libraries (see, for example, Clackson et al, 1991, Nature, 352: 624-628; and Marks et al, 1991, Journal of Molecular Biology , 222: 581-597).
  • the antibody used in the present invention is preferably an anti-human ErbB2 antibody, and preferably, the CDR1, CDR2 and/or CDR3 of the heavy and light chain in the anti-human ErbB2 antibody are trastuzumab heavy and light chain, respectively CDR1, CDR2 and/or CDR3.
  • the anti-human ErbB2 antibody can be a humanized antibody or a fully human antibody.
  • the antibody used in the present invention is more preferably a humanized antibody of the murine anti-human Her2 antibody 4D5 shown in Fig. 1 of US 5,821,337.
  • the antibody used in the present invention is particularly preferably trastuzumab, the sequence of which is disclosed, for example, in CN 103319599A. Lys, the last position of the heavy chain of trastuzumab, is easily deleted, but this deletion does not affect biological activity, see Dick, L. W. et al., Biotechnol. Bioeng., 100: 1132-1143.
  • the above-mentioned trastuzumab and the sequence in which the heavy chain last Lys is deleted or a fragment thereof belong to the trastuzumab of the present invention.
  • the trastuzumab heavy chain sequence is (SEQ ID NO: 1):
  • the trastuzumab light chain sequence is (SEQ ID NO: 2):
  • Antibody fragments can be produced by a variety of methods, such as by proteolysis of intact antibodies (see, for example, Morimoto et al, 1992, Journal of Biochemical and Biophysical Methods, 24: 107-117; and Brennan et al, 1985, Science, 229:81), either directly from recombinant host cells and the antibody phage library discussed above, or directly recover Fab'-SH fragments from E. coli and chemically coupled to form F(ab') fragments (Carter et al., Biotechnology) (N Y), 1992 Feb, 10(2): 163-167.
  • F(ab') fragments can be isolated directly from recombinant host cell culture. Other methods of producing antibody fragments are well known to those skilled in the art.
  • the antibody fragments used in the present invention are single-chain Fv fragments (scFv) (see, for example, WO 93/16185, US 5,571,894, and US 5,587,458).
  • antibody fragments are linear antibody fragments (see, eg, US 5,641,870), which may be monospecific or bispecific.
  • Bispecific antibodies with binding specificities for at least two different epitopes can bind to two different epitopes of the ErbB2 protein.
  • Other bispecific antibodies can combine ErbB2 binding sites with EGFR, ErbB3 and/or ErbB4 binding sites.
  • the anti-ErbB2 arm can be combined with an arm that binds to an elicitor molecule on a leukocyte, such as a T cell receptor molecule (eg, CD2 or CD3), or with an Fc receptor (Fc ⁇ R) such as Fc ⁇ RI (CD64), Fc ⁇ RII (CD32).
  • Bispecific antibodies can also be used to localize cytotoxic agents to cells expressing ErbB2 (see, for example, WO 96/16673, US 5,837,234, WO 98/02463, and US 5,821,337). Methods for purifying bispecific antibodies have been disclosed in the literature (see, for example, WO 93/08829; Traunecker et al, 1991, EMBO J., 10:3655-3659; WO 94/04690; Suresh et al, 1986, Methods in Enzymology, 121:210; US 5,731,168).
  • Leucine zippers can be utilized (see, for example, Kostelny et al., 1992, J. Immunol., 148(5): 1547-1553) and single chain Fv (sFv) dimers (see, for example, Gruber et al., 1994, J. Immunol. 152: 5368) Production of bispecific antibodies.
  • bispecific antibodies from antibody fragments
  • Techniques for producing bispecific antibodies from antibody fragments have been described in the literature, for example, by chemical hydrolysis of F(ab') fragments by proteolytic cleavage of intact antibodies at their positions (Brennan et al., 1985, Science, 229: 81).
  • Fab'-SH fragments can be recovered from E. coli and chemically coupled to form bispecific antibodies (see Shalaby et al., 1992, J. Exp. Med., 175: 217-225).
  • Double antibody The "diabodies” technique provides an alternative method for preparing bispecific antibody fragments (see Hollinger et al, 1993, Proc. Natl. Acad. Sci. USA, 90:6444-6448).
  • Multivalent "octopus"-like antibodies having three or more antigen binding sites and two or more variable regions can be readily produced by recombinant expression of a nucleic acid encoding an antibody polypeptide chain (see, for example, US 2002/0004586 and WO 01/77342).
  • trispecific antibodies can be prepared (see, for example, Tutt et al., 1991, J. Immunol., 147: 60).
  • the CH3 domain of a tri- or tetra-specific antibody in the heavy chain or in the modified heavy chain) can be altered by the "junction-in-hole” technique.
  • the "junction-in-a-well” technique and several examples are described in detail, for example.
  • WO 96/027011 Ridgway, JB et al, 1996, Protein Eng., 9: 617-621; and Merchant, AM et al, 1998, Nat. Biotechnol., 16:677-681.
  • the interaction interfaces of the two CH3 domains are altered to increase heterodimerization of the two heavy chains containing the two CH3 domains.
  • Each of the two CH3 domains (of the two heavy chains) can be a "knot" and the other is a "cavity".
  • Introduction of disulfide bridges further stabilizes heterodimers (see, for example, Merchant, AM et al, 1998, Nature Biotech., 16:677-681; and Atwell, S. et al, 1997, J. Mol. Biol., 270: 26-35) and increase production.
  • nucleic acid molecules encoding antibody amino acid sequence variants can be prepared by a variety of methods known in the art. These methods include, but are not limited to, isolation from natural sources (in the case of naturally occurring amino acid sequence variants) or by oligonucleotide-mediated (or site-directed) mutagenesis, PCR mutagenesis, and changes to the earlier preparation of antibodies. Box mutagenesis by bulk or non-variant patterns. The most interesting substitutional mutagenesis sites include hypervariable regions, but FR changes are also expected.
  • Example 2 of CN103319599A the DNA fragment encoding the heavy chain of trastuzumab heavy chain was subjected to site-directed mutagenesis by conventional molecular biology techniques, and the trastuzumab mutant weight chain K30R was cloned into an antibody heavy chain expression vector, and the enzyme was cloned.
  • the cutting and ligation operations were carried out according to the commercially available kit instructions.
  • the drug used in the present invention is a cytotoxic drug.
  • cytotoxic drug refers to a substance that inhibits or prevents cellular function and/or causes cell destruction.
  • the cytotoxic drug is an auristatin peptide, such as auristatin E (also known as a dolastatin-10 derivative) or a derivative thereof (eg, by ear suppression) An ester of E and keto acid).
  • auristatin E also known as a dolastatin-10 derivative
  • An ester of E and keto acid For example, amphetin E can be reacted with p-acetylbenzoic acid or benzoyl valeric acid to produce AEB (actin EB) and AEVB (5-benzoyl valerate o-statin E ester), respectively.
  • AEB actin EB
  • AEVB 5-benzoyl valerate o-statin E ester
  • Other typical auristatin peptides include AFP (Auroxine F phenylenediamine), MMAF (monomethyl amphetin F), and MMAE (monomethyl amphetin E).
  • the cytotoxic drug is a maytansinoid.
  • Maytansine was first isolated from the East African shrub Maytenus serrata by Kupchan et al. It is more cytotoxic than traditional chemotherapeutic agents such as methotrexate, daunorubicin and vincristine. 100 to 1000 times (see US 3,896,111). Subsequently, some microorganisms were also found to produce maytansinoids such as maytansinol and C-3 esters of maytansinol (see US 4,151,042). Synthetic maytansinol C-3 esters and maytansinol analogs have also been reported (see Kupchan et al, 1978, J. Med.
  • the maytansinol C-3 ester is prepared from maytansinol.
  • examples of maytansinoids include: maytansinol having a modification (e.g., dechlorination) on the aromatic ring, and maytansinol having a modification (e.g., a hydroxylated methyl group) at C-9, C-14. Modified maytansinol at C-15, C-18, C-20 and/or C-4, C-5.
  • the cytotoxic drug is, for example but not limited to, a compound represented by the following structural formula:
  • R, R 1 and R 2 are each independently H, C 1 -C 8 alkyl or C 3 -C 8 cycloalkyl, the alkyl and cycloalkyl optionally being one or more (eg 1, 2, 3, 4 or 5) substituted with a substituent selected from halogen (eg F, Cl, Br or I);
  • X is -S-, -CH 2 -, -CH(OH)-, -CH(OR)-, -CH(ONH 2 )- or -C(O)-;
  • R' is selected from H, -NH 2 , Cl, Br, I, -OS(O) 2 R; and
  • Ar is C 6- C 14 aryl (such as phenyl or naphthyl).
  • the cytotoxic drug group is derived from a compound of the formula (D1) or (D2) or a stereoisomer thereof:
  • R 2 is selected from the group consisting of -CH 2 N 3 , -CONHSO 2 (cyclopropyl), thiazol-2-yl, -CH 3 and -COOH;
  • R 3 is selected from the group consisting of H and -OH;
  • R 4 is selected from the group consisting of H, -NH 2 , Cl, Br, I, -OS(O) 2 R 6 , wherein R 6 is H, C 1 -C 8 alkyl, C 3 -C 8 cycloalkyl or C 6 a -C 14 aryl group, each of which is optionally substituted by one or more (eg 1, 2, 3, 4 or 5) substituents selected from halogen such as F;
  • R 5 is selected from the group consisting of -CH(CH 3 )N(CH 3 )C(O)CH 2 CH 2 SH and -CH(CH 3 )N(CH 3 )C(O)CH 2 C(CH 3 ) 2 SH .
  • the cytotoxic drug group may be a drug of the formula (D1) or (D2) for removing R 4 or R 5 or for removing hydrogen or R 6 of R 4 or R 5 .
  • the resulting group may be a drug of the formula (D1) or (D2) for removing R 4 or R 5 or for removing hydrogen or R 6 of R 4 or R 5 .
  • the cytotoxic drug is selected from the group consisting of:
  • the cytotoxic drug group is linked to the antibody molecule via a divalent linking group.
  • the attachment of the linker to the antibody can be accomplished in a variety of ways, such as via a lysine residue on the surface of the antibody, or reductive coupling to the oxidized hydrocarbyl group of the antibody.
  • the linkage may be a linkage based on a hydrazine, a disulfide bond or a peptide structure. These connections are well known to those skilled in the art.
  • Linkers useful in the present invention include linkable and non-cleavable linkers.
  • a cleavable linker is generally susceptible to cleavage under intracellular conditions.
  • Suitable cleavable linkers include, for example, peptide linkers which can be cleaved by intracellular proteases such as lysosomal proteases or endosomal proteases.
  • the cleavable linker may be a dipeptide linker, such as a valine-citrulline (val-cit) linker, phenylalanine-lysine (phe-lys) Linker or maleimidocaproyl-valine-citrulline-p-aminobenzyloxycarbonyl (mc-Val-Cit-PAB) linker.
  • Suitable cleavable linkers include linkers which are hydrolyzable at a particular pH or pH range (e.g., hydrazone linkages), as well as disulfide linkers.
  • the non-cleavable linker may be a sulfo-SMCC.
  • the coupling of the sulfo-SMCC with the protein occurs via a maleimide, and the sulfo-NHS ester can be reacted with a primary amine such as a lysine ⁇ -amino group in the protein or peptide and an ⁇ -amino group at the N-terminus.
  • a primary amine such as a lysine ⁇ -amino group in the protein or peptide and an ⁇ -amino group at the N-terminus.
  • Another non-cleavable linker is maleimidohexanoyl (MC).
  • the linker can be covalently linked to the antibody, and the covalent linkage should be such that the antibody must be degraded within the cell to release the drug.
  • the linker includes a group for attachment to an antibody, such as an amino group, a hydroxyl group or a carboxyl group.
  • the linker may be derived, for example, from maleimide, haloacetamide (such as iodoacetamide, bromoacetamide or chloroacetamide), a halogenated ester (such as iodoester, bromo ester or chloroester), Halomethyl ketone (such as methyl iodide, bromomethyl ketone or chloromethyl ketone), benzyl halide (such as benzyl iodide, benzyl bromide or benzyl chloride), vinyl sulfone and pyridyl sulphide (pyridylthio) (see for example Wong, 1991, Chemistry of Protein Conjugation and Cross-linking, CRC Press, Inc., Boca Raton).
  • the linker comprises at least Val-Cit, Val-Cit-PAB, Val-Ala-PAB, Val-Lys(Ac)-PAB, Phe-Lys-PAB, Phe-Lys(Ac)- PAB, D-Val-Leu-Lys, Gly-Gly-Arg, Ala-Ala-Asn-PAB, Ala-PAB or PAB.
  • the linker comprises at least a peptide, an oligosaccharide, - (CH 2) n - , - (CH 2 CH 2 O) n -.
  • the linker comprises at least -C(O)-, -NH-C(O)-, -C(O)-O-, -NH-C(O)-NH- or -NH -C(O)-O-.
  • the linker L in formula (I) can be selected from:
  • n is an integer selected from 1-10, preferably 1, 2, 3, 4, 5 or 6.
  • the antibody-drug conjugate of the first aspect of the invention is represented by the general formula (I) as defined above.
  • the most preferred antibody-drug conjugate of formula (I) is selected from the group consisting of I-1, I-2 and I-3:
  • A is trastuzumab.
  • the antibody-drug conjugate of the present invention may be in the form of a pharmaceutically acceptable salt, or in the form of a stereoisomer, or in the form of a metabolite, or in the form of a solvate, and the salt, the stereoisomer
  • the conformation or metabolite may also be in the form of a solvate.
  • pharmaceutically acceptable salt refers to a salt that retains the biological effectiveness and properties of the compound, They are biologically or otherwise desirable for use as a drug.
  • the antibody-drug conjugates of the invention form acid addition salts and/or base addition salts by virtue of the amino and/or carboxyl groups or similar groups present therein.
  • the pharmaceutically acceptable acid addition salt may be a salt formed with an inorganic or organic acid.
  • the inorganic acid includes, for example, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like.
  • the organic acid includes, for example, acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, Methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, and the like.
  • the pharmaceutically acceptable base addition salt may be a salt formed with an inorganic base or an organic base.
  • the salt formed with the inorganic base includes, for example, a sodium salt, a potassium salt, a lithium salt, an ammonium salt, a calcium salt, a magnesium salt, an iron salt, a zinc salt, a copper salt, a manganese salt, and an aluminum salt, and the like, and an ammonium salt is particularly preferable. , potassium salt, sodium salt, calcium salt and magnesium salt.
  • the organic base includes, for example, primary, secondary and tertiary amines, substituted amines (including naturally occurring substituted amines), cyclic amines, basic ion exchange resins and the like. Specific examples of the organic base are isopropylamine, trimethylamine, diethylamine, N-ethylethylamine, tripropylamine and ethanolamine.
  • stereoisomer denotes an isomer formed by at least one asymmetric center. In compounds having one or more asymmetric centers, they can produce racemates, racemic mixtures, single enantiomers, diastereomeric mixtures, and individual diastereomers. Specific individual molecules can also exist as geometric isomers (cis/trans). Unless otherwise stated, when the stereochemistry of the disclosed compounds is not explicitly stated and has one or more asymmetric centers, it is understood that all possible stereoisomers of the compounds are represented.
  • solvate means a solvate formed by the association of one or more solvent molecules with any of the antibody-drug conjugates of Formula I, or a pharmaceutically acceptable salt or isomer thereof.
  • solvate includes hydrates (e.g., hemihydrate, monohydrate, dihydrate, trihydrate, tetrahydrate, and similar hydrates).
  • metabolite means a substance which can be formed by oxidation, reduction, hydrolysis, amidation, deamidation, esterification and/or enzymatic hydrolysis in vivo after administration.
  • the antibody-drug conjugates of the invention can selectively deliver an effective amount of a cytotoxic drug to tumor tissue, thereby achieving better therapeutic selectivity and achieving the desired therapeutic efficacy at lower doses.
  • the invention provides a method of preparing an antibody-drug conjugate of the first aspect of the invention, the method comprising the steps of:
  • G is selected from the group consisting of -F, -Cl, -Br, -I, -N 3 , -OR, -SR, -ONRR', RC(O)O-, -OP(O)RR', RSO 2 -O- with
  • R and R' are each independently a C 1 -C 10 alkyl group, a C 6 -C 14 aryl group, a heterocyclic group having 5 to 10 ring atoms or a phenoxy group, said alkyl group,
  • the aryl, heterocyclic and phenoxy groups are unsubstituted or are each independently selected from one or more selected from the group consisting of halogen, hydroxy, C 1 -C 4 alkyl, C 1 -C 4 alkoxy, C 3 -C a substituent of an 8- cycloalkyl group, a heterocyclic group having 5 to 8 ring atoms, a C 6 -C 10 aryl group or a hetero
  • G in the compound of the formula (I-A-G) in the step (2) is selected from -ONRR' and -OP(O)RR', wherein each of R and R' is independently a phenoxy group.
  • the compound of the formula (I-A-G) in the step (2) is a compound of the formula (I-B) which is formed by reacting a compound of the formula (I-A) with pentafluorophenol:
  • step (4) the purification of step (4) is accomplished by HPLC.
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising the antibody-drug conjugate of the first aspect of the invention or a pharmaceutically acceptable salt, stereoisomer or metabolite thereof or a solvate thereof, and A pharmaceutically acceptable carrier.
  • the pharmaceutical composition further comprises one or more additional anticancer agents, such as chemotherapeutic agents and/or antibodies.
  • pharmaceutical composition refers to a combination of at least one active ingredient and a pharmaceutically acceptable carrier and/or adjuvant that are combined together to achieve a particular purpose.
  • the pharmaceutical composition is in the form of a mixture of components, while in other embodiments, the components of the pharmaceutical composition may be separated in time and/or space, as long as they are capable of acting together To achieve the object of the present invention.
  • the active ingredients may be administered to the individual as a mixture, or may be administered separately to the individual. When administered separately, the active ingredients can be administered simultaneously or sequentially.
  • the choice of pharmaceutically acceptable carrier depends on the dosage form of the pharmaceutical composition, first depending on the route of administration of the dosage form (for example, for oral, nasal, intradermal, subcutaneous, intramuscular or intravenous administration), and secondly on Formulation of the dosage form.
  • the pharmaceutically acceptable carrier may include water (such as water for injection), a buffer, an isotonic saline solution such as PBS (phosphate buffer), dextrose, mannitol, dextrose, lactose, starch, stearin.
  • composition of the present invention may further contain various additives such as a wetting agent, an emulsifier or a buffering agent and the like as needed.
  • the present invention provides the antibody-drug conjugate of the first aspect of the invention or a pharmaceutically acceptable salt, stereoisomer or metabolite thereof or a solvate thereof for use in the preparation or prevention of cancer Use of the drug.
  • the cancer is described below as described in the sixth aspect of the invention, including, but not limited to, breast cancer, gastric cancer, ovarian cancer, non-small cell lung cancer, and liver cancer, particularly breast cancer, such as breast cancer with high expression of ErbB2.
  • the present invention provides a pharmaceutical preparation comprising the antibody-drug conjugate of the first aspect of the invention or a pharmaceutically acceptable salt, stereoisomer or metabolite thereof or a solvate thereof.
  • the pharmaceutical preparation is in the form of a solid preparation, a semisolid preparation, a liquid preparation or a gas preparation.
  • the pharmaceutical preparation is particularly preferably a lyophilized powder injection which has the advantages of less excipients, good stability, and high safety in clinical use.
  • the invention provides a method of preventing or treating cancer, the method comprising administering to a patient in need thereof an antibody-drug conjugate of the first aspect of the invention, a pharmaceutically acceptable salt thereof, a stereoisomeric
  • the pharmaceutical composition of the third aspect of the invention or the pharmaceutical preparation of the fifth aspect of the invention is administered to a construct or metabolite or a solvate thereof, or to a patient in need thereof.
  • the method further comprises administering to the patient one or more additional anticancer agents, such as chemotherapeutic agents and/or antibodies, which can be administered with the antibody-drug conjugate of the invention
  • the pharmaceutical composition or pharmaceutical preparation is carried out simultaneously or sequentially.
  • Such cancers include, but are not limited to, carcinoma, blastoma, sarcoma, leukemia, lymphoma, and other malignant lymphoid diseases. More specific examples of the cancer include: squamous cell carcinoma (e.g., squamous cell carcinoma), lung cancer (e.g., small cell lung cancer, non-small cell lung cancer (NSCLC), lung adenocarcinoma, and lung squamous cell carcinoma), peritoneal cancer , stomach or stomach cancer (such as gastrointestinal cancer and gastrointestinal stromal tumor (GIST)), pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, bladder cancer, breast cancer, colon cancer, rectal cancer, colon Rectal cancer, endometrial cancer or uterine cancer, salivary gland cancer, kidney cancer, prostate cancer, vaginal cancer, thyroid cancer, liver cancer, anal cancer, penile cancer, and head and neck cancer.
  • the cancer is a highly expressed cancer of Erbb2, such as breast cancer, gastric cancer
  • a compound 4 to 6-1 dissolved in DMA was added in an amount of 4 to 6 times.
  • the reaction was carried out for 2 to 6 hours at 2 to 40 ° C with gentle stirring, and the reaction was monitored by HIC-HPLC to obtain a crude product of the antibody-drug conjugate I-1.
  • the HIC-HPLC spectrum is shown in Fig. 1.
  • the antibody-drug conjugate I-1 crude product obtained in the step d was subjected to HIC, and subjected to desalting (i.e., replacement of the buffer) and ultrafiltration to obtain an antibody-drug conjugate I-1.
  • Mobile phase A 1.5M ammonium sulfate aqueous solution, 25m M disodium hydrogen phosphate aqueous solution, pH 7.0
  • Mobile phase B 25 mM aqueous solution of disodium hydrogen phosphate, pH 7.0, 10% isopropanol solution
  • the obtained antibody-drug conjugate I-1 was subjected to DAR analysis by HIC-UPLC, and the obtained spectrum is shown in Fig. 2.
  • the antibody-drug conjugate I-1 was subjected to light heavy chain reduction liquid chromatography to obtain an overlapping reverse phase chromatogram as shown in FIG.
  • the dark curve is the spectrum of trastuzumab naked resistance and the light curve is the spectrum of antibody-drug conjugate I-1.
  • the higher peaks indicate the antibody heavy chain and the lower peaks indicate the antibody light chain.
  • the antibody light chain in the antibody-drug conjugate I-1 is more hydrophobic due to the coupling of the drug molecule than the unconjugated drug trastuzumab naked antibody, so retention time Move back. This indicates that the drug molecules in the antibody-drug conjugate I-1 are all coupled to the antibody light chain.
  • the upper panel is a peptide map of naked anti-214 nm
  • the lower panel is a peptide map of antibody-drug conjugate I-1 at 214 nm.
  • the toxin molecule (D) is all coupled to the lysine of the same peptide of the light chain, thereby realizing Site-directed coupling results in excellent product quality uniformity, quality controllability and reproducibility.
  • the antibody-drug conjugate I-2 crude product obtained in the step c was subjected to HIC (HIC conditions are the same as those listed in the step e of Example 1), and subjected to desalting liquid exchange and ultrafiltration to obtain an antibody-drug conjugate. I-2.
  • the obtained antibody-drug conjugate I-2 was subjected to DAR analysis by HIC-HPLC, and the obtained spectrum is shown in Fig. 6.
  • the product is pure.
  • the antibody-drug conjugate I-2 was subjected to light heavy chain reduction liquid chromatography to obtain an overlapping chromatogram as shown in FIG.
  • the dark curve is the spectrum of trastuzumab naked resistance and the light curve is the spectrum of antibody-drug conjugate I-2.
  • the higher peaks indicate the antibody heavy chain and the lower peaks indicate the antibody light chain.
  • the antibody light chain in the antibody-drug conjugate I-2 is more hydrophobic due to the coupling of the drug molecule than the unconjugated drug trastuzumab naked antibody, so the retention time Move back. This indicates that the drug molecules in the antibody-drug conjugate I-2 are all coupled to the antibody light chain.
  • the upper panel is a peptide map of naked anti-214 nm
  • the lower panel is a peptide map of antibody-drug conjugate I-2 at 214 nm.
  • antibody-drug conjugate I-1 was only increased by one peptide at a retention time of 33.0 min.
  • the toxin molecule (D) is all coupled to the lysine of the same peptide of the light chain, With site-directed coupling, excellent product quality uniformity, quality controllability and reproducibility are achieved.
  • piperidine-4- carboxylic acid (5 eq.) was dissolved in saturated aqueous NaHCO 3 (5 mL), was added the crude compound DA-3 (1 equiv). The reaction mixture was stirred at room temperature for 8 hours. A 10% (w/v) aqueous citric acid solution was added to adjust the pH to 4-5, then extracted with EtOAc (150 mL*2). The organic phase was dried and concentrated to give crude IA-3.
  • the antibody-drug conjugate I-3 crude product obtained in the step c was subjected to HIC (HIC conditions are the same as those listed in the step e of Example 1), and subjected to desalting liquid exchange and ultrafiltration to obtain an antibody-drug conjugate. I-3.
  • the obtained antibody-drug conjugate I-3 was subjected to DAR analysis by HIC-HPLC, and the obtained spectrum is shown in FIG.
  • the product is pure.
  • the antibody-drug conjugate I-3 was subjected to light heavy chain reduction liquid chromatography to obtain an overlapping chromatogram as shown in FIG.
  • the dark curve is the spectrum of trastuzumab naked resistance and the light curve is the spectrum of antibody-drug conjugate I-3.
  • the higher peaks indicate the antibody heavy chain and the lower peaks indicate the antibody light chain.
  • the antibody light chain in the antibody-drug conjugate I-3 is more hydrophobic due to the coupling of the drug molecule than the unconjugated drug trastuzumab naked antibody, so the retention time Move back. This indicates that the drug molecules in the antibody-drug conjugate I-3 are all coupled to the antibody light chain.
  • the upper panel is a peptide map of naked anti-214 nm
  • the lower panel is a peptide map of antibody-drug conjugate I-3 at 214 nm.
  • the antibody-drug conjugate I-1 only one peptide was added at a retention time of 39.0 min.
  • the toxin molecule (D) is all coupled to the lysine of the same peptide of the light chain, thereby realizing Site-directed coupling results in excellent product quality uniformity, quality controllability and reproducibility.
  • the antibody-drug conjugates of Examples 1 to 3 were evaluated for inhibition of tumor growth in mice subcutaneously transplanted with human tumor cells. Specifically, in the present example, the antibody-drug conjugates of Examples 1 to 3 were injected into the human gastric cancer cell line NCI-N87, the breast cancer cell line BT474, and the ovarian cancer cell line SK by single tail vein injection. After the mice of -OV-3, the tumor volume and the body weight change were measured, and the pharmacodynamic effect (antitumor effect) of the antibody-drug conjugate on the tumor-bearing mice was calculated.
  • trastuzumab naked anti-T DM1 (positive control, (ado-trastuzumab emtansine), Roche Pharmaceuticals) or the antibody-drug conjugate of the present invention (I-3, I-1 or I-2, prepared in Examples 1-3), respectively, formulated with sterile ultrapure water A certain concentration of the mother liquor, gently shake, and then stored at -80 ° C. At the time of use, it was diluted with physiological saline according to the dose to obtain a treated solution, and the same concentration of physiological saline was used as a solvent control.
  • a tumor-bearing mouse (model obtained in 1.2) with a tumor volume of 100 to 200 mm 3 (model obtained in 1.2), 7 cells/group, was selected by random grouping (determining the number of cells according to the number of samples).
  • the administration volume was 10 ml/kg.
  • the route of administration is a single tail vein injection.
  • the tumor growth inhibition rate TGI (%) was calculated using the following formula to evaluate the antitumor efficacy of antibody-drug conjugates:
  • TGI (%) [1 - (V T end - V T start ) / (V C end - V C start )] * 100%
  • V T at the beginning mean volume of tumor at the beginning of administration in the treatment group
  • V C mean volume of tumor at the end of the experiment in the solvent control group
  • V C start mean volume of tumor at the start of drug control group administration
  • TGI Group sample Dose (mg/kg) TGI (%) 1 Solvent / / 2 T-DM1 3 54.80% 3 I-1 3 104.75% 4 I-3 3 107.52%
  • the antibody-drug conjugates of the invention also have similar anti-tumor effects on a variety of breast cancer cell lines.
  • TGI Group sample Dose (mg/kg) TGI (%) 1 Solvent / / 2 T-DM1 2 30.136% 3 I-1 2 114.99% 4 I-3 2 87.04% 5 T-DM1 3 65.28% 6 I-3 3 102.72%
  • the antibody-drug conjugates I-1, I-3, and I-2 of the present invention have inhibitory activities against various tumors such as gastric cancer, ovarian cancer, and breast cancer during the evaluation period. They are significantly better than T-DM1, and animal deaths also indicate that they have excellent safety.
  • This example evaluates the stability of the antibody-drug conjugates of Examples 1 to 3 in rats. Specifically, in the present example, after the antibody-drug conjugates of Examples 1 to 3 were intravenously injected into the rat at a dose of 2 mg/kg, the jugular vein was collected periodically, and the blood was measured by ELISA. Antibody-drug conjugate concentration and total antibody concentration, calculating the half-life of antibody-drug conjugates in rats period. The measurement results are shown in Table 5.
  • the antibody-drug conjugates I-1, I-2 and I-3 of the present invention have a longer half-life in rats and are significantly more stable than T-DM1.
  • a lyophilized powder injection of antibody-drug conjugate I-1 was prepared using the starting materials as follows:
  • Antibody-drug conjugate I-1 28g ascorbic acid 20g Lactic acid 10g Polyethylene glycol 4000 63g Water for Injection 2000ml
  • the filtrate is poured into a vial and freeze-dried to obtain a freeze-dried powder needle. After passing the test, the package is packaged.
  • a lyophilized powder injection of antibody-drug conjugate I-2 was prepared using the starting materials as follows:
  • the filtrate is poured into a vial and freeze-dried to obtain a freeze-dried powder needle. After passing the test, the package is packaged.
  • a lyophilized powder injection of antibody-drug conjugate I-3 was prepared using the starting materials as shown in the following table:
  • Antibody-drug conjugate I-3 20g L-histidine 0.32g L-histidine hydrochloride 0.495g Dihydrate trehalose 20g Tween 20 0.09g Water for Injection 1000ml
  • the filtrate is poured into a vial and freeze-dried to obtain a freeze-dried powder needle. After passing the test, the package is packaged.

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Abstract

提供了抗ErbB2抗体-药物偶联物、包含其的组合物、其制备方法和应用。

Description

抗ErbB2抗体-药物偶联物及其组合物、制备方法和应用 发明领域
本发明属于生物医药技术领域。具体而言，本发明涉及抗ErbB2抗体-药物偶联物、包含其的组合物、其制备方法和应用。
发明背景
作为新型的靶向治疗药物，抗体-药物偶联物(antibody-drug conjugate,ADC)开创了肿瘤治疗方法的新纪元。在美国西雅图基因公司(Seattle Genetics,Inc.)和免疫原公司(ImmunoGen,Inc.)的引领下，很多跨国制药企业和初创公司都开展了这一领域的研发。根据Market Research的报道，全球共有45个此类候选药处于临床研究中。
ADC一般包括采用一定方式连接的三个部分：抗体、连接基和药物。
抗体是良好的药物靶向性载体。利用药物分子中的特定官能团如羟基、巯基或氨基，可将药物与抗体连接，组成化学免疫偶联物。抗体的靶向性能将与之相连的药物“精确”地运送到靶细胞，从而有效提高病灶局部的药物浓度，同时极大地降低体内其他组织、器官的药物浓度，从而实现增效减毒。用于这些策略的多克隆抗体和单克隆抗体均已有报导(Rowland等人，1986，Cancer Immunol.Immunother.，21:183-87)。目前临床上所用的ADC中的抗体大多是人源化抗体，例如，PSMA ADC(抗PSMA抗体-MMAE偶联物)、SGN-75(抗CD70抗体-MMAF偶联物)、T-DM1(曲妥珠单抗-DM1偶联物)中所用均为人源化抗体。目前FDA批准的ADC药物包括
Figure PCTCN2016106802-appb-000001
(T-DM1)和
Figure PCTCN2016106802-appb-000002
(brentuximab vedotin)。
ADC中作为“弹头”的药物通常为细胞毒性药物，它们主要通过抑制细胞DNA或蛋白质合成、抑制细胞有丝***等方式来杀伤肿瘤细胞。由于细胞毒性药物对正常细胞同样具有较大杀伤力，因而其应用和发展受到极大限制。早期的ADC利用常规抗肿瘤药物，但这些ADC临床活性大多低于化学药单体的体外活性。目前的ADC所用的细胞毒性药物主要包括：美登素类(Maytansinoids，参见例如EP 0425235、US 5208020、US 5416064、US 7276497、US 7473796、US 7851432、US 2007/0269447、US 2011/0158991、 WO 2004/103272、WO 2012/061590)、耳抑素肽类(Auristatins，参见例如US 6884869、US 7498298)、卡奇霉素类(Calicheamicins，参见例如US 5606040、US 5770710)、阿霉素类(Doxorubicins，参见例如Dubowchik等人，2002，Bioconjugate Chem.，13:855-869)、苯并二吡咯类抗生素(duocarmycins和CC-1065，参见例如US 7129261)、伊立替康代谢产物(Irinotecan metabolites，参见例如WO 2015/012904)、吡咯并苯二氮卓类(Pyrrolobenzodiazepines，参见例如Biotechnol.Healthc.2012Winter，9(4):28-31)以及吡咯并苯二氮卓二聚体类(PBD dimmers，参见例如WO 2005/040170)等。这些细胞毒性药物具有很强的非选择性毒性，会对正常细胞造成伤害，因而本身不能成药。
ADC中的连接基需满足以下要求：在细胞外保证小分子药物不与抗体脱离；进入细胞后，可断裂的连接基在适当条件下断裂，释放出活性小分子药物，而不可断裂的连接基则与小分子药物和来自抗体酶解的氨基酸残基一起形成活性部分。
在目前进入临床试验的ADC中，细胞毒性药物通常籍由连接基连接在抗体表面的赖氨酸残基上，或者连接在抗体铰链区的半胱氨酸残基(由链间二硫键部分还原得到)上。当药物连接在抗体表面的赖氨酸残基上时，由于抗体表面存在大量(超过80个)赖氨酸残基并且偶联反应是非选择性的，因而偶联数目和位点具有不确定性，导致生成的ADC的不均一性。例如，T-DM1的药物/抗体比值(drug antibody ratio，DAR)分布为0-8，平均DAR值为3.5(Lazar等人，2005，Rapid Commun.Mass Spectrom.，19:1806-1814)。通常ADC的DAR值为2-4。当药物连接在抗体铰链区的半胱氨酸残基上时，由于抗体铰链区的链间二硫键只有四对，为了达到平均DAR值为2-4的要求，需要还原部分链间二硫键(Sun等人，2005，Bioconjugate Chem.，16:1282-1290)，然而现有的还原剂如二硫苏糖醇(DTT)和磷酸三氯乙酯(TCEP)等无法选择性地还原链间二硫键，因此所得抗体-药物偶联物也不是均一产物，而是由多种组分组成的混合物，其中主要组分的DAR值为0、2、4、6、8，而且对应每一DAR值的组分又存在由于连接位点不同而形成的异构体。ADC产品的不均一性对于其临床应用是不利的，因为产品中各组分间药代动力学性质、效价和/或毒性不均一(例如，具有较高DAR值的组分在体内被清除得更快，并导致更高的毒性，参见例如Boswell等人，2011，Bioconjugate Chem.，22:1994-2004)，使得其稳定性难以令人满意。
ErbB族受体酪氨酸激酶是细胞生长、分化和存活的重要介质。该家族包括四个成员：上皮生长因子受体(EGFR或ErbB1)、Her2(ErbB2)、Her3(ErbB3)和Her4(ErbB4)。
临床上用抗ErbB2抗体曲妥珠单抗治疗ErbB2高表达的乳腺癌。在临床试验中，15％的具有免疫组织化学(IHC)2+以上水平的乳腺癌患者对曲妥珠单抗产生临床反应，反应持续中值为9.1个月(参见例如Cobleigh等人，1996，临床肿瘤学杂志，14:737-744)。曲妥珠单抗(商品名Herceptin)在1998年9月25日被美国食品与药品管理局(FDA)批准用于治疗过量表达ErbB2蛋白的乳腺癌患者。
曲妥珠单抗虽然挽救了一些乳腺癌患者或延长了患者的生存期，但其仅对ErbB2高表达的患者有效，且临床反应率仅有15％。因此，还需要对更多患者具有更佳疗效的药物。
为了提高治疗指数，已将曲妥珠单抗与美登木素(DM1)偶联，制成trastuzumab emtansine(T-DM1，
Figure PCTCN2016106802-appb-000003
)，用于使用曲妥珠单抗、其它抗Her2治疗药物及常用乳腺癌治疗一线化疗药物紫杉烷类治疗无效的患者。T-DM1将药物输送至肿瘤部位，从而缩小肿瘤，减慢疾病进展并延长生存期。在临床试验中，T-DM1的安全性和有效性得到了评价。T-DM1现已成为FDA批准的第四个以Her2蛋白为靶点的药物。但是，T-DM1在批准时有一项黑框警告，提醒患者与卫生保健专业人员该药物可导致肝毒性、心脏毒性和死亡。这主要是脱落的DM1以及T-DM1进入体内降解释放出的含DM1小分子引起的。
T-DM1的制备是利用曲妥珠单抗上的赖氨酸侧链氨基与磺基琥珀酰亚氨基-4-[N-顺丁烯二酰亚氨基甲基]环己烷-1-羧酸酯(磺基-SMCC)偶联，然后再与DM1偶联。T-DM1中，每分子曲妥珠单抗上偶联的DM1分子平均为3.5个。由于曲妥珠单抗上具有88个赖氨酸位点，因而T-DM1实际为曲妥珠单抗偶联了不同数目DM1和具有多种偶联位点的多种抗体-药物偶联物的组合。然而，每种抗体-药物偶联物的药效、药代动力学性质和/或毒性不同。一般而言，载药量高的抗体-药物偶联物具有更强的体外活性，但高载药量也会带来诸多问题，如因抗体-药物偶联物之间聚合而生成的多聚体成分增加、稳定性下降、毒性增加、免疫原性提高、体内清除速度过快、半衰期过短而实际治疗指数不高。
本发明旨在解决现有技术中的上述问题。本发明提供抗Her2抗体-药物 偶联物，其抑制哺乳动物肿瘤生长，可用于治疗多种癌症。所述偶联物具有更好的生物学活性、稳定性和均一性，并具有降低的毒副作用。
发明概述
在第一方面，本发明提供通式(I)的抗体-药物偶联物，其药学上可接受的盐、立体异构体或代谢物，或者它们的溶剂合物，
Figure PCTCN2016106802-appb-000004
其中：
A为抗ErbB2抗体或者其活性片段或变体；
X和Y各自独立地为N或CR1，且每个R1独立地为H或C1-C10烷基；
L为二价连接基；
D为细胞毒性药物基团；并且
a为选自2-10的整数。
在第二方面，本发明提供本发明第一方面的抗体-药物偶联物的制备方法，所述方法包括以下步骤：
(1).制备通式(I-A)的化合物：
Figure PCTCN2016106802-appb-000005
其中D、L、X和Y如上文关于通式(I)所定义；
(2).活化步骤(1)中得到的通式(I-A)的化合物，得到通式(I-A-G)的化合物
Figure PCTCN2016106802-appb-000006
其中G选自-F、-Cl、-Br、-I、-N3、-OR、-SR、-ONRR’、RC(O)O-、-OP(O)RR’、RSO2-O-和
Figure PCTCN2016106802-appb-000007
其中R和R’每次出现时各自独立地是C1-C10烷基、C6-C14芳基、含5-10个环原子的杂环基或苯氧基，所述烷基、芳基、杂环 基和苯氧基是未取代的或者各自独立地被一个或多个选自卤素、羟基、C1-C4烷基、C1-C4烷氧基、C3-C8环烷基、含5-8个环原子的杂环基、C6-C10芳基或含5-10个环原子的杂芳基的取代基取代；
(3).将步骤(2)中得到的通式(I-A-G)的化合物偶联于所述抗ErbB2抗体或者其活性片段或变体，得到具有不同a值的多种抗体-药物偶联物的混合物；和
(4).经选自离子交换层析、疏水层析、反相层析和亲和层析中的一种或多种层析方法纯化步骤(3)中得到的混合物，得到抗体-药物偶联物。
在第三方面，本发明提供药物组合物，其包含本发明第一方面的抗体-药物偶联物或者其药学上可接受的盐、立体异构体或代谢物或者它们的溶剂合物，以及药学上可接受的载体。
在第四方面，本发明提供本发明第一方面的抗体-药物偶联物或者其药学上可接受的盐、立体异构体或代谢物或者它们的溶剂合物在制备用于预防或治疗癌症的药物中的用途。
在第五方面，本发明提供药物制剂，其包含本发明第一方面的抗体-药物偶联物或者其药学上可接受的盐、立体异构体或代谢物或者它们的溶剂合物。
在第六方面，本发明提供预防或治疗癌症的方法，所述方法包括向有此需要的患者给药本发明第一方面的抗体-药物偶联物，其药学上可接受的盐、立体异构体或代谢物或者它们的溶剂合物，或者向有此需要的患者给药本发明第三方面的药物组合物或本发明第五方面的药物制剂。
附图说明
图1是抗体-药物偶联物I-1粗产物的HIC-HPLC谱图。
图2是抗体-药物偶联物I-1的HIC-UPLC谱图。
图3是抗体-药物偶联物I-1与曲妥珠单抗裸抗还原的重叠反相色谱图。
图4是将抗体-药物偶联物I-1和曲妥珠裸抗在相同条件下进行酶切所得的肽图谱。
图5是抗体-药物偶联物I-2粗产物的HIC-HPLC谱图。
图6是抗体-药物偶联物I-2的HIC-HPLC谱图。
图7是抗体-药物偶联物I-2与曲妥珠单抗裸抗还原的重叠反相色谱图。
图8是将抗体-药物偶联物I-2和曲妥珠裸抗在相同条件下进行酶切所 得的肽图谱。
图9是抗体-药物偶联物I-3粗产物的HIC-HPLC谱图。
图10是抗体-药物偶联物I-3的HIC-HPLC谱图。
图11是抗体-药物偶联物I-3与曲妥珠单抗裸抗还原的重叠反相色谱图。
图12是将抗体-药物偶联物I-3和曲妥珠裸抗在相同条件下进行酶切所得的肽图谱。
发明详述
除非另有定义，本文使用的所有术语具有与本领域普通技术人员通常所理解相同的含义。相关的定义及术语可参见例如Current Protocols in Molecular Biology(Ausubel)。
本文中所述的数值范围应理解为涵盖其中包含的任何和所有子范围。例如，范围“1至10”应理解为不仅包括明确记载的1至10的值，而且还包括1至10范围内的任何单个值(例如2、3、4、5、6、7、8和9)和子范围(例如1至2、1.5至2.5、1至3、1.5至3.5、2.5至4、3至4.5等等)。该原则亦适用于仅用一个数值作为最小值或最大值的范围。
本文中提及的文献均以其整体援引加入本文中。
在第一方面，本发明提供通式(I)的抗体-药物偶联物，其药学上可接受的盐、立体异构体或代谢物，或者它们的溶剂合物，
Figure PCTCN2016106802-appb-000008
其中：
A为抗ErbB2抗体或者其活性片段或变体；
X和Y各自独立地为N或CR1(优选Y为CR1，X为N)，且每个R1独立地为H或C1-C10烷基，优选为H或C1-C6烷基(例如甲基、乙基、正丙基、异丙基、正丁基、异丁基、叔丁基、戊基或己基)；
L为二价连接基；
D为细胞毒性药物基团；并且
a为选自2-10的整数，例如2、3或4，特别优选2。
抗体
本发明中所用的抗体为抗ErbB2抗体或者其活性片段或变体，包括双特异性抗体和抗体功能性衍生物。
在本文中，ErbB2和Her2可互换使用，二者均表示天然序列的人Her2蛋白(Genebank登录号：X03363，参见例如Semba等人，1985，PNAS，82:6497-6501；和Yamamoto等人，1986，Nature，319:230-234)及其功能性衍生物，例如氨基酸序列变体。erbB2表示编码人Her2的基因，neu表示编码大鼠p185neu的基因。癌细胞，例如乳腺癌细胞、卵巢癌细胞、胃癌细胞、子宫内膜癌细胞、唾液腺癌细胞、肺癌细胞、肾癌细胞、结肠癌细胞、甲状腺癌细胞、胰腺癌细胞、膀胱癌细胞或肝癌细胞等，通常为表达ErbB2受体的细胞。
本发明中优选的Her2是天然序列的人Her2。可用于本发明的抗Her2的抗体的实例包括但不限于：MAbs4D5(ATCC CRL 10463)、2C4(ATCC HB-12697)、7F3(ATCC HB-12216)和7C2(ATCC HB 12215)，这些抗体记载于例如US 5,772,997、WO 98/77797和US 5,840,525中。
人源化抗Her2抗体包括US 5,821,337表3中所列的huMAb4D5、huMAb4D5-1、huMAb4D5-2、huMAb4D5-3、huMAb4D5-4、huMAb4D5-5、huMAb4D5-6、huMAb4D5-7和huMAb4D5-8；CN 1370082A图1B中所示的7C2、7F3、2C4、7D3、3E8和2C4。
可用于本发明的抗Her2抗体的实例还可以包括：WO 2009/123894中记载的F243L，R292P和Y300L；F243L，R292P和V305I；F243L，R292P和P396L；以及R292P，V305I和P396；WO 2012/079093中记载的MM-111；CN104418952A权利要求2至10中记载的TPs、TPL、PTs或PTL抗体。WO 2010/108127图33A和图33B中记载的Dl、Dl.5、Dl.5-100、DLI l、DLl Ib或DLl If抗体；以及WO 93/21319中记载的人源化520C9。
本发明中的天然序列的Her2可以从自然界分离得到，也可以通过重组DNA技术、化学合成法或它们的组合制备得到。
术语“功能性衍生物”包括氨基酸序列变体以及天然多肽的共价衍生物(例如通过翻译后修饰、焦谷氨酸化等获得的衍生物)，条件是它们保留与天然多肽相当或更高的亲和力和生物活性。氨基酸序列变体与天然多肽氨基酸序列的差异一般在于后者中的一个或多个氨基酸的取代、缺失和/或***。缺失变体包括天然多肽的片段和N端和/或C端截短变体。通常，氨基 酸序列变体与天然多肽应具有至少70％或至少80％或至少90％的同源性。天然多肽的共价衍生物可以是通过改变抗体的翻译后加工，例如改变糖基化位点的数目或位置获得的衍生物。
术语“同源性”可与“一致性”、“同一性”或“相似性”互换使用，是指在最佳比对(最优比对)后所获得的待比较的两种核酸序列或者氨基酸序列之间相同核苷酸或相同氨基酸残基的百分数，该百分数是纯粹统计学的并且两种序列间的差异随机分布并覆盖其全长。两种核酸序列或者氨基酸序列之间的比对通常是在以最优方式使它们匹配后比较序列而进行，所述比较可通过区段或者通过“比较窗”来实施。除了手工实施外，最优比对还可以通过记载于文献中的其他方法进行，例如Smith和Waterman，1981，Ad.App.Math.，2:482中记载的局部同源性算法，Neddleman和Wunsch，1970，J.MoI.Biol.，48:443中记载的局部同源性算法，Pearson和Lipman，1988，Proc.Natl.Acad.Sci.USA，85:2444中记载的相似性搜索方法，并且可以通过使用这些算法的计算机软件(GAP、BESTFIT、FASTA和TFASTA in the Wisconsin Genetics Software Package，Genetics Computer Group，575Science Dr.，Madison，WI)实施，或者通过BLAST N或BLAST P比较软件实施。
在本文中，术语“抗体”取其最广义的解释，包括完整的单克隆抗体、多克隆抗体以及由至少两个完整抗体形成的多特异性抗体(例如双特异性抗体)，只要它们具有所需的生物学活性。
在本文中，术语“单克隆抗体”指抗体来自一群基本均一的抗体，即构成该集群的各抗体完全相同，除了可能存在的少量天然突变。单克隆抗体具有针对抗原的一个决定簇(表位)的高特异性，而与其相对的多克隆抗体则包含针对不同决定簇(表位)的不同抗体。除了特异性之外，单克隆抗体的优点还在于合成时可以不受其他抗体的污染。此处修饰语“单克隆”表示该抗体的特征在于来自一个基本均一的抗体群，而不应理解成需由特殊方法制得。
在本发明中，单克隆抗体还特别包括嵌合抗体，即重链和/或轻链的一部分与某种、某类或某亚类抗体相同或同源，其余部分则与另一种、另一类或另一亚类抗体相同或同源，只要它们具有所需的生物学活性(参见例如US 4,816,567；和Morrison等人，1984，PNAS，81:6851-6855)。可用于本发明的嵌合抗体包括灵长类化(primatized)抗体，其包含来自非人灵长类(例 如古猴、猩猩等)的可变区抗原结合序列和人恒定区序列。
术语“抗体片段”是指抗体的一部分，优选是抗原结合区或可变区。抗体片段的实例包括Fab、Fab′、F(ab′)2和Fv片段；二抗体(diabody)；线性抗体；和单链抗体分子。
术语“双特异性抗体”与“双功能抗体偶联物”可互换使用，指由第一抗体(片段)和第二抗体(片段)通过偶联臂所形成的偶联物，该偶联物保留了各自抗体的活性，故具有双功能和双特异性。
术语“多特异性抗体”包括例如三特异性抗体和四特异性抗体，前者是具有三种不同抗原结合特异性的抗体，而后者是具有四种不同抗原结合特异性的抗体。
术语“完整抗体”指包含抗原结合可变区和轻链恒定区(CL)、重链恒定区(CH1、CH2和CH3)的抗体。恒定区可以是天然序列(例如人天然恒定区序列)或其氨基酸序列变体。完整抗体优选是具有一种或多种效应功能的完整抗体。
非人(例如鼠)抗体的“人源化”形式指包含最少量非人免疫球蛋白序列的嵌合抗体。大多数人源化抗体是人接受者免疫球蛋白的超变区残基被置换成具有所需特异性、亲和力和功能的非人(例如小鼠、大鼠、兔或非人灵长类)超变区残基(供者抗体)。在一些实施方案中，人免疫球蛋白的框架区(FR)残基也被置换成非人残基。而且，人源化抗体还可以包含受者抗体或供者抗体中没有的残基。这些修饰是为了进一步优化抗体的性能。人源化抗体一般包含至少一个，通常是两个可变区，其中所有或几乎所有超变环(hypervanable loops)与非人免疫球蛋白的相对应，而FR则完全或几乎完全是人免疫球蛋白的序列。人源化抗体还可以包含免疫球蛋白恒定区(Fc，通常是人免疫球蛋白Fc)的至少一部分。有关细节参见例如Jones等人，1986，Nature，321:522-525；Riechmann等人，1988，Nature，332:323-329；和Presta，1992，Curr Op Struct Bwl 2:593-596。
完整抗体可根据重链恒定区的氨基酸序列分为不同的“类”。主要的五类是IgA、IgD、IgE、IgG和IgM，其中几类还可以分为不同的“亚类”(同种型)，例如IgG1、IgG2、IgG3、IgG4、IgA1和IgA2。抗体不同类的重链恒定区分别称为α、β、ε、γ和μ。免疫球蛋白不同类的亚基结构和三维构型是本领域中公知的。
在本发明中，尽管大多数情况下抗体中的氨基酸取代是被L-氨基酸取 代，但也不限于此。在一些实施方案中，抗体肽链中可以包括一个或多个D-氨基酸。认为包含D-氨基酸的肽在口腔、肠道或血浆中比仅包含L-氨基酸的肽更加稳定而不易降解。
本发明所用的单克隆抗体可以由许多方法生产。例如，用于本发明的单克隆抗体可以通过杂交瘤方法，使用许多物种(包括小鼠、仓鼠、大鼠和人的细胞)获得(参见例如Kohler等人，1975，Nature，256:495)，或者通过重组DNA技术制得(参见例如US 4,816,567)，或者从噬菌体抗体库中分离得到(参见例如Clackson等人，1991，Nature，352:624-628；和Marks等人，1991，Journal of Molecular Biology，222:581-597)。
本发明中所用的抗体优选为抗人ErbB2抗体，并且优选地，所述抗人ErbB2抗体中重链和轻链的CDR1、CDR2和/或CDR3分别为曲妥珠单抗重链和轻链的CDR1、CDR2和/或CDR3。所述抗人ErbB2抗体可以为人源化抗体或全人源抗体。
本发明中所用的抗体更优选为US 5,821,337图1中所示的鼠源抗人Her2抗体4D5的人源化抗体。
本发明中所用的抗体特别优选为曲妥珠单抗，其序列已披露于，例如CN 103319599A中。曲妥珠单抗重链末位Lys是容易缺失的，但这种缺失并不影响生物活性，参见Dick,L.W.等人，Biotechnol.Bioeng.，100:1132-1143。上述曲妥珠单抗及其重链末位Lys缺失的序列或其片段均属于本发明所述的曲妥珠单抗。
曲妥珠单抗重链序列为(SEQ ID NO:1)：
Figure PCTCN2016106802-appb-000009
曲妥珠单抗轻链序列为(SEQ ID NO:2)：
Figure PCTCN2016106802-appb-000010
Figure PCTCN2016106802-appb-000011
抗体片段可由多种方法方法生产，例如通过完整抗体的蛋白水解来生产(参见，例如Morimoto等人，1992，Journal of Biochemical and Biophysical Methods，24:107-117；和Brennan等人，1985，Science，229:81)，或者由重组宿主细胞和以上讨论的抗体噬菌体文库直接生产，或者直接从大肠杆菌回收Fab'-SH片段并进行化学偶联以形成F(ab')片段(Carter等人，Biotechnology(N Y)，1992Feb，10(2):163-167。此外，可以直接从重组宿主细胞培养物中分离F(ab')片段。其它生产抗体片段的方法是本领域技术人员公知的。在一些实施方案中，本发明所用的抗体片段是单链Fv片段(scFv)(参见例如WO 93/16185、US 5,571,894以及US 5,587,458)。在一些实施方案中，抗体片段是线性抗体片段(参见例如US 5,641,870)，其可以是单特异性的或双特异性的。
具有针对至少两个不同表位的结合特异性的双特异性抗体(参见例如Millstein等人，1983，Nature，305:537-539)可以结合ErbB2蛋白的两个不同表位。其它双特异性抗体可以将ErbB2结合部位与EGFR、ErbB3和/或ErbB4结合部位组合。或者，抗ErbB2臂可以与结合白细胞上的激发分子如T细胞受体分子(例如CD2或CD3)的臂组合，或着与IgG的Fc受体(FcγR)如FcγRI(CD64)、FcγRII(CD32)和FcγRIII(CD16)的臂组合，以将细胞的防御机制集中于表达ErbB2的细胞。双特异性抗体也可用于将细胞毒性试剂定位于表达ErbB2的细胞(参见例如WO 96/16673、US 5,837,234、WO 98/02463和US 5,821,337)。已有文献披露了双特异性抗体的纯化方法(参见例如WO 93/08829；Traunecker等人，1991，EMBOJ.，10:3655-3659；WO 94/04690；Suresh等人，1986，Methods in Enzymology，121:210；US 5,731,168)。可以利用亮氨酸拉链(参见例如Kostelny等人，1992，J.Immunol.，148(5):1547-1553)和单链Fv(sFv)二聚体(参见例如Gruber等人，1994，J.Immunol.152:5368)生产双特异性抗体。
已有文献描述了从抗体片段生产双特异性抗体的技术，例如利用完整抗体在其位置经蛋白水解裂解产生F(ab')片段的化学键(Brennan等人，1985，Science，229:81)。可以从大肠杆菌回收Fab'-SH片段并化学偶联以形成双特异性抗体(参见Shalaby等人，1992，J.Exp.Med.，175:217-225)。“双抗 体(diabodies)”技术提供了制备双特异性抗体片段的替代方法(参见Hollinger等人，1993，Proc.Natl.Acad.Sci.USA，90:6444-6448)。
可以制备超过二价的抗体。可以通过编码抗体多肽链的核酸的重组表达容易地生产具有三个或更多个抗原结合部位和两个或更多个可变区的多价“章鱼”样抗体(参见例如US 2002/0004586和WO 01/77342)。例如，可以制备三特异性抗体(参见例如Tutt等人，1991，J.Immunol.，147:60)。通过“结-入-穴”技术，可改变三或四特异性抗体(重链中或经修饰的重链中)的CH3域，“结-入-穴”技术及数个实例详细记载于例如WO 96/027011，Ridgway,J.B.等人，1996，Protein Eng.，9:617-621；及Merchant,A.M.等人，1998，Nat.Biotechnol.，16:677-681中。在这种方法中，改变两种CH3域的相互作用界面以提高含有这两种CH3域的两种重链的异二聚化。(两种重链的)两种CH3域的每一个均可以是“结”，而另一是“穴”。引入二硫桥进一步稳定异二聚体(参见例如Merchant,A.M.等人，1998，Nature Biotech.，16:677-681；和Atwell,S.等人，1997，J.Mol.Biol.，270:26-35)及提高产量。
通常通过改变基本的核酸序列来改变氨基酸序列。编码抗体氨基酸序列变体的核酸分子可通过本领域已知的多种方法制备。这些方法包括但不限于从天然来源分离(在天然发生的氨基酸序列变体的情况下)或者通过寡核苷酸介导的(或定点)诱变、PCR诱变以及对抗体的早先制备的变体或非变异型式所进行的盒式诱变。最感兴趣的取代诱变部位包括高变区，但也可预期FR改变。如CN103319599A实施例2利用常规分子生物学技术对全基因合成编码曲妥珠单抗重链的DNA片段进行定点突变，将曲妥珠单抗突变体重链K30R克隆至抗体重链表达载体上，酶切和连接的操作按商业提供的试剂盒说明书进行。
药物
本发明中所用的药物为细胞毒性药物。本文中术语“细胞毒性药物”是指抑制或阻止细胞功能和/或引起细胞破坏的物质。
在一些实施方案中，所述细胞毒性药物是耳抑素肽类，如耳抑素E(auristatin E，亦称为海兔毒素(dolastatin)-10衍生物)或其衍生物(例如由耳抑素E和酮酸形成的酯)。举例来说，耳抑素E可与对乙酰基苯甲酸或苯甲酰基戊酸反应，分别产生AEB(耳抑素EB)及AEVB(5-苯甲酰基戊酸耳抑素E酯)。其它典型耳抑素肽类包括AFP(耳抑素F苯二胺)、MMAF(一甲基耳抑素F)及MMAE(一甲基耳抑素E)。
示例性耳抑素肽类的合成和结构描述于US 6,884,869、US 7,098,308、US 7,256,257、US 7,423,116、US 7,498,298和US 7,745,394中。其他新的耳抑素肽类的合成和结构描述于WO 2013/173393中。这些文献整体援引加入本文。
在一些实施方案中，所述细胞毒性药物是美登素类。美登素(maytansine)首先由Kupchan等人从东非灌木齿叶美登木(Maytenus serrata)中分离得到，它比传统化疗剂如甲氨碟呤、柔红霉素和长春新碱的细胞毒性强100至1000倍(参见US 3,896,111)。随后，发现一些微生物也产生美登素类，如美登醇(maytansinol)和美登醇的C-3酯(参见US 4,151,042)。也已报道了合成的美登醇C-3酯及美登醇类似物(参见Kupchan等人，1978，J.Med.Chem.，21:31-37；Higashide等人，1977，Nature，270:721-722；Kawai等人，1984，Chem.Pharm.Bull.，32:3441-3451)。美登醇C-3酯由美登醇制备得来。美登醇类似物的实例包括：在芳环上有修饰(例如，脱氯)的美登醇，在C-9、C-14处有修饰(例如，羟化的甲基)的美登醇，在C-15、C-18、C-20和/或C-4、C-5处有修饰的美登醇。
在一些实施方案中，所述细胞毒性药物例如是但不限于由以下结构式表示的化合物：
Figure PCTCN2016106802-appb-000012
Figure PCTCN2016106802-appb-000013
Figure PCTCN2016106802-appb-000014
Figure PCTCN2016106802-appb-000015
其中R、R1和R2每次出现时各自独立地是H、C1-C8烷基或C3-C8环烷基，所述烷基和环烷基任选地被一个或多个(如1、2、3、4或5个)选自卤素(如F、Cl、Br或I)的取代基取代；X是-S-、-CH2-、-CH(OH)-、-CH(OR)-、-CH(ONH2)-或-C(O)-；R’选自H、-NH2、Cl、Br、I、-OS(O)2R；且Ar是C6-C14芳基(如苯基或萘基)。
在优选的实施方案中，所述细胞毒性药物基团来自通式(D1)或(D2)的化合物或它们的立体异构体：
Figure PCTCN2016106802-appb-000016
其中：
R2选自-CH2N3、-CONHSO2(环丙基)、噻唑-2-基、-CH3和-COOH；
R3选自H和-OH；并且
R4选自H、-NH2、Cl、Br、I、-OS(O)2R6，其中R6是H、C1-C8烷基、C3-C8环烷基或C6-C14芳基，所述烷基、环烷基和芳基各自任选地被一个或多个(如1、2、3、4或5个)选自卤素的取代基如F取代；
Figure PCTCN2016106802-appb-000017
其中R5选自-CH(CH3)N(CH3)C(O)CH2CH2SH和-CH(CH3)N(CH3)C(O)CH2C(CH3)2SH。
在通式(I)中，所述细胞毒性药物基团可以是通式(D1)或(D2)的药物脱除R4或R5或者脱除R4或R5中的氢或R6而得到的基团。
在优选的实施方案中，所述细胞毒性药物选自：
Figure PCTCN2016106802-appb-000018
Figure PCTCN2016106802-appb-000019
连接基
在本发明的抗体-药物偶联物中，细胞毒性药物基团经二价连接基与抗体分子连接。
所述连接基与抗体的连接可经由多种方式完成，例如经由抗体表面的赖氨酸残基连接，或者还原偶合至抗体的经氧化的烃基。所述连接可以是基于腙、二硫键或肽结构的连接。这些连接方式是本领域技术人员公知的。
本发明可用的连接基包括可断裂及不可断裂的连接基。可断裂的连接基在细胞内的条件下通常易断裂。适当的可断裂的连接基包括例如可被细胞内蛋白酶(如溶酶体蛋白酶(lysosomal protease)或核内体蛋白酶(endosomal protease))切割的肽连接基。在示例性实施方案中，所述可断裂的连接基可为二肽连接基，如缬氨酸-瓜氨酸(val-cit)连接基、苯丙氨酸-赖氨酸(phe-lys)连接基或顺丁烯二酰亚氨基己酰基-缬氨酸-瓜氨酸-对氨基苯甲氧基羰基(mc-Val-Cit-PAB)连接基。其它适当的可断裂的连接基包括可在特定pH或pH范围内被水解的连接基(如腙连接基)，以及二硫化物连接基。不可断裂的连接基可以是磺基-SMCC。磺基-SMCC与蛋白质偶联经由顺丁烯二酰亚氨基发生，其磺基-NHS酯可与伯胺(如蛋白质或肽中的赖氨酸ε-氨基及N端的α-氨基)反应。另一不可断裂的连接基是顺丁烯二酰亚氨基己酰基(MC)。连接基可与抗体共价连接，并且所述共价连接的程度应使所述抗体必须在细胞内被降解才能释放药物。
连接基包括用于与抗体连接的基团，例如氨基、羟基或羧基。连接基可来自例如顺丁烯二酰亚胺、卤代乙酰胺(例如碘乙酰胺、溴乙酰胺或氯乙酰胺)、卤代酯(例如碘代酯、溴代酯或氯代酯)、卤代甲基酮(例如碘甲基酮、溴甲基酮或氯甲基酮)、卤化苄(例如碘化苄、溴化苄或氯化苄)、乙烯砜(vinyl sulfone)及吡啶基硫(pyridylthio)(参见例如Wong，1991，Chemistry of Protein Conjugation and Cross-linking,CRC Press,Inc., Boca Raton)。
在一些实施方案中，所述连接基至少包括Val-Cit、Val-Cit-PAB、Val-Ala-PAB、Val-Lys(Ac)-PAB、Phe-Lys-PAB、Phe-Lys(Ac)-PAB、D-Val-Leu-Lys、Gly-Gly-Arg、Ala-Ala-Asn-PAB、Ala-PAB或PAB。在一些实施方案中，所述连接基至少包括肽、寡糖、-(CH2)n-、-(CH2CH2O)n-。在一些实施方案中，所述连接基至少包括-C(O)-、-NH-C(O)-、-C(O)-O-、-NH-C(O)-NH-或-NH-C(O)-O-。
在一些实施方案中，通式(I)中的连接基L可以选自：
Figure PCTCN2016106802-appb-000020
Figure PCTCN2016106802-appb-000021
Figure PCTCN2016106802-appb-000022
Figure PCTCN2016106802-appb-000023
Figure PCTCN2016106802-appb-000024
Figure PCTCN2016106802-appb-000025
其中m、n每次出现时各自为选自1-10的整数，优选1、2、3、4、5或6。
抗体-药物偶联物
本发明第一方面的抗体-药物偶联物由上文中定义的通式(I)表示。
最优选的通式(I)的抗体-药物偶联物选自I-1、I-2和I-3：
Figure PCTCN2016106802-appb-000026
其中A为曲妥珠单抗。
本发明的抗体-药物偶联物可以为药学可接受的盐的形式，或者为立体异构体的形式，或者为代谢物的形式，或者为溶剂合物的形式，并且所述盐、立体异构体或代谢物也可为溶剂合物的形式。
术语“药学上可接受的盐”是指保留化合物的生物有效性和性质的盐， 它们对于用做药物在生物学上或在其它方面是符合需要的。在许多情况下，本发明的抗体-药物偶联物凭借其中存在的氨基和/或羧基或类似基团来形成酸加成盐和/或碱加成盐。
药学上可接受的酸加成盐可以是与无机酸或有机酸形成的盐。所述无机酸包括，例如，盐酸、氢溴酸、硫酸、硝酸和磷酸等。所述有机酸包括，例如，乙酸、丙酸、羟基乙酸、丙酮酸、草酸、马来酸、丙二酸、琥珀酸、富马酸、酒石酸、柠檬酸、苯甲酸、肉桂酸、扁桃酸、甲磺酸、乙磺酸、对甲苯磺酸和水杨酸等。
药学上可接受的碱加成盐可以是与无机碱或有机碱形成的盐。所述与无机碱形成的盐包括，例如，钠盐、钾盐、锂盐、铵盐、钙盐、镁盐、铁盐、锌盐、铜盐、锰盐和铝盐等，特别优选铵盐、钾盐、钠盐、钙盐和镁盐。所述有机碱包括，例如，伯胺、仲胺和叔胺，取代的胺(包括天然存在的取代的胺)，环胺，碱性离子交换树脂等。有机碱的具体实例为异丙胺、三甲胺、二乙胺、N-乙基乙胺、三丙胺和乙醇胺。
术语“立体异构体”表示由于至少一个不对称中心形成的异构体。在具有一个或多个不对称中心的化合物中，其可产生外消旋体、外消旋混合物、单一对映异构体、非对映异构体混合物和单独的非对映异构体。特定个别分子也可以几何异构体(顺式/反式)存在。除非另外说明，当所公开的化合物的命名或结构中没有明确说明其立体化学并且具有一个或多个不对称中心时，应该理解代表所述化合物的所有可能的立体异构体。
术语“溶剂合物”表示由一个或多个溶剂分子与任一通式I的抗体-药物偶联物或其药学可接受的盐或异构体缔合形成的溶剂合物。术语溶剂合物包括水合物(例如半水合物、一水合物、二水合物、三水合物、四水合物以及类似的水合物)。
术语“代谢物”表示给药后可经体内氧化、还原、水解、酰胺化、脱酰胺化、酯化和/或酶解而形成的物质。
本发明的抗体-药物偶联物可以选择性地将有效量的细胞毒性药物递送到肿瘤组织，由此获得更佳的治疗选择性，并以更低的剂量实现期望的治疗功效。
在第二方面，本发明提供本发明第一方面的抗体-药物偶联物的制备方法，所述方法包括以下步骤：
(1).制备通式(I-A)的化合物：
Figure PCTCN2016106802-appb-000027
其中D、L、X和Y如上文关于通式(I)所定义；
(2).活化步骤(1)中得到的通式(I-A)的化合物，得到通式(I-A-G)的化合物
Figure PCTCN2016106802-appb-000028
其中G选自-F、-Cl、-Br、-I、-N3、-OR、-SR、-ONRR’、RC(O)O-、-OP(O)RR’、RSO2-O-和
Figure PCTCN2016106802-appb-000029
其中R和R’每次出现时各自独立地是C1-C10烷基、C6-C14芳基、含5-10个环原子的杂环基或苯氧基，所述烷基、芳基、杂环基和苯氧基是未取代的或者各自独立地被一个或多个选自卤素、羟基、C1-C4烷基、C1-C4烷氧基、C3-C8环烷基、含5-8个环原子的杂环基、C6-C10芳基或含5-10个环原子的杂芳基的取代基取代；
(3).将步骤(2)中得到的通式(I-A-G)的化合物偶联于所述抗ErbB2抗体或者其活性片段或变体，得到具有不同a值的多种抗体-药物偶联物的混合物；和
(4).经选自离子交换层析、疏水层析、反相层析和亲和层析中的一种或多种层析方法纯化步骤(3)中得到的混合物，得到抗体-药物偶联物。
优选地，步骤(2)中的通式(I-A-G)的化合物中的G选自-ONRR’和-OP(O)RR’，其中R和R’每次出现时各自独立地是苯氧基。
更优选地，步骤(2)中的通式(I-A-G)的化合物为通过使通式(I-A)的化合物与五氟苯酚反应而形成的通式(I-B)的化合物：
Figure PCTCN2016106802-appb-000030
其中D、L、X和Y如上文关于通式(I)所定义，并且所述反应优选使用EDCI、 NHS和/或DCM完成。
优选地，步骤(4)的纯化通过HPLC完成。
在第三方面，本发明提供药物组合物，其包含本发明第一方面的抗体-药物偶联物或者其药学上可接受的盐、立体异构体或代谢物或者它们的溶剂合物，以及药学上可接受的载体。任选地，所述药物组合物进一步包含一种或多种其他抗癌药，如化疗剂和/或抗体。
本文中所用的术语“药物组合物”指组合在一起以实现特定目的的至少一种活性成分及药学上可接受的载体和/或辅料的组合。在一些实施方案中，药物组合物是各组分的混合物的形式，而在另一些实施方案中，药物组合物的各组分可以是在时间和/或空间上分开的，只要其能够共同作用以实现本发明的目的。
当药物组合物中含有两种或更多种活性成分时，所述活性成分可以作为混合物同时施用于个体，也可以分开施用于个体。当分开施用时，各活性成分可以同时或依次施用。
药学上可接受的载体的选择取决于药物组合物的剂型，首先取决于剂型的给药途径(例如用于口服、经鼻、皮内、皮下、肌内或静脉施用的剂型)，其次取决于剂型的配方。例如，所述药学上可接受的载体可以包括水(如注射用水)、缓冲液、等渗盐溶液如PBS(磷酸盐缓冲液)、葡萄糖、甘露醇、右旋葡萄糖、乳糖、淀粉、硬脂酸镁、纤维素、碳酸镁、0.3％甘油、透明质酸、抗坏血酸、乳酸、乙醇、聚亚烷基二醇如聚乙二醇(例如聚乙二醇4000)或聚丙二醇、甘油三酯等。
此外，本发明的药物组合物还可根据需要包含各种添加剂，如润湿剂、乳化剂或缓冲剂等等。
在第四方面，本发明提供本发明第一方面的抗体-药物偶联物或者其药学上可接受的盐、立体异构体或代谢物或者它们的溶剂合物在制备用于预防或治疗癌症的药物中的用途。所述癌症如下文描述本发明的第六方面时所述，例如其包括但不限于乳腺癌、胃癌、卵巢癌、非小细胞肺癌和肝癌，特别是乳腺癌，如ErbB2高表达的乳腺癌。
在第五方面，本发明提供药物制剂，其包含本发明第一方面的抗体-药物偶联物或者其药学上可接受的盐、立体异构体或代谢物或者它们的溶剂合物。
优选地，所述药物制剂为固体制剂、半固体制剂、液体制剂或气体制剂的形式。所述药物制剂特别优选为冻干粉针剂，其具有辅料较少，稳定性好，临床使用安全性较高的优点。
在第六方面，本发明提供预防或治疗癌症的方法，所述方法包括向有此需要的患者给药本发明第一方面的抗体-药物偶联物，其药学上可接受的盐、立体异构体或代谢物或者它们的溶剂合物，或者向有此需要的患者给药本发明第三方面的药物组合物或本发明第五方面的药物制剂。任选地，所述方法进一步包括向所述患者给药一种或多种其他抗癌药，如化疗剂和/或抗体，所述给药可以与给药本发明的抗体-药物偶联物、药物组合物或药物制剂同时或顺序进行。
所述癌症包括但不限于癌(carcinoma)、母细胞瘤、肉瘤、白血病、淋巴瘤及其他恶性淋巴疾病。所述癌症更具体的例子包括：鳞状细胞癌(例如鳞状上皮细胞癌)、肺癌(例如小细胞肺癌、非小细胞肺癌(NSCLC)、肺腺癌和肺鳞状细胞癌)、腹膜癌、、胃或胃部癌症(例如胃肠癌和胃肠基质肿瘤(GIST))、胰腺癌、恶性胶质瘤、***、卵巢癌、膀胱癌、、乳腺癌、结肠癌、直肠癌、结肠直肠癌、子宫内膜癌或子宫癌、唾液腺癌、肾癌、***癌、***癌、甲状腺癌、肝癌、***癌、***癌以及头颈癌。特别地，所述癌症为Erbb2高表达癌症，如乳腺癌、胃癌、子宫内膜癌、唾液腺癌、肺癌、肾癌、结肠癌、甲状腺癌、胰腺癌或膀胱癌。
在以下的实施例中将进一步举例说明本发明。这些实施例仅用于说明本发明，但不以任何方式限制本发明。
实施例
各实施例中使用的缩写的含义如下表所示：
DMF 二甲基甲酰胺
DIC 二异丙基碳二亚胺
HOAt 1-羟基-7-氮杂苯并***
EtOAc 乙酸乙酯
DIEA 二异丙基乙胺
HATU 2-(1H-7-氮杂苯并***-1-基)-1,1,3,3-四甲基脲鎓六氟磷酸盐
PyBOP 1H-苯并***-1-基氧三吡咯烷基六氟磷酸盐
HOBT 1-羟基苯并***
LiOH 氢氧化锂
DCM 二氯甲烷
EDCI 1-乙基-3-(3-二甲基氨基丙基)碳二亚胺
NHS N-羟基琥珀酰亚胺
DMA N,N-二甲基乙酰胺
HIC 疏水作用色谱
HPLC 高效液相色谱
UPLC 超高效液相色谱
THF 四氢呋喃
EtOAc 乙酸乙酯
实施例1.制备抗体-药物偶联物I-1
Figure PCTCN2016106802-appb-000031
步骤a.中间体D-1的制备
Figure PCTCN2016106802-appb-000032
在室温下，将化合物D-0(1mmol，1当量)溶于DMF(50mL)中，依次加入DIC(1.1当量)、HOAt(1.1当量)和4-(3-叠氮-2-氨基丙基)苯胺(1.5当量)。将反应混合物在室温下搅拌8小时，然后加入水(600mL)和EtOAc(200mL*3)，萃取后收集有机相，浓缩并经HPLC纯化，得到中间体D-1。
MS m/z(ESI):773[M+H]+。
步骤b.中间体I-A-1的制备
Figure PCTCN2016106802-appb-000033
在室温下，将化合物S-2(0.1mmol，1当量)溶于DMF(50mL)中，依次加入DIEA(2当量)、HATU(1.05当量)和化合物D-1(2.0当量)。在室温下反应12小时，然后加入水(300mL)和EtOAc(100mL*3)，萃取后收集有机相，浓缩并经HPLC纯化，得到中间体D-A-1。
在室温下，将化合物D-A-1(0.1mmol，1当量)溶于DMF(5mL)中，依次加入DIC(1.1当量)、HOAt(1.1当量)和哌啶4-羧酸(1.2当量)。将反应混合物在室温下搅拌6小时，然后加入水(60mL)和EtOAc(20mL*3)，萃取后收集有机相，浓缩并经HPLC纯化，得到中间体I-A-1。
MS m/z(ESI):1240[M+H]+。
或者
Figure PCTCN2016106802-appb-000034
在冰水浴冷却下，将化合物S-2’(0.1mmol，1.0当量)溶于DMF(10mL)中，依次加入DIEA(2.0当量)、PyBOP(1.0当量)、HOBT(1.0当量)和化合物D-1(0.2mmol，2.0当量)。在室温下反应12小时，然后加入水(30mL)和EtOAc(10mL*3)，萃取后收集有机相，浓缩并经HPLC纯化，得到中间体D-A-1’。
在室温下，将化合物D-A-1’(0.05mmol，1.0当量)溶于THF/H2O(6mL,v:v＝5:1)中，加入LiOH一水合物(3.0当量)。将反应混合物在室温下搅拌16小时，经分离纯化，得到中间体I-A-1。
MS m/z(ESI):1240[M+H]+。
步骤c.中间体I-B-1的制备
Figure PCTCN2016106802-appb-000035
在室温下，将化合物I-A-1(0.1mmol，1当量)溶于DCM(50mL)中，依次加入EDCI(1.5当量)、NHS(1.5当量)和五氟苯酚(2.0当量)。在室温下反应18小时。向反应混合物中依次加入水(30mL)、10％(w/v)柠檬酸水溶液(20mL)和饱和氯化钠水溶液(20mL)进行洗涤，收集有机相，浓缩并经HPLC纯化，得到中间体I-B-1。
MS m/z(ESI):1406[M+H]+。
步骤d.抗体-药物偶联物I-1粗产物的合成
Figure PCTCN2016106802-appb-000036
Figure PCTCN2016106802-appb-000037
I-1粗产物(其中n＝1,2,3,4)
向1ml在pH 7.4的PBS缓冲液中配制的浓度为10～20mg/ml的曲妥珠单抗溶液中加入4～6倍物质的量的溶解在DMA中的化合物I-B-1。在2～40℃和温和搅拌下反应2～6小时，通过HIC-HPLC监测反应，得到抗体-药物偶联物I-1粗产物，HIC-HPLC谱图如图1所示。
HIC-HPLC条件：
色谱柱：   Tosoh TSKgel Butyl-NPR,4.6×100mm
流动相A：  1.5M硫酸铵水溶液
流动相B：  25mM磷酸钠水溶液，pH＝7.0，25％(v/v)异丙醇水溶液
流速：     0.5ml/min
梯度：     0～2min：17％流动相B+83％流动相A
           2～15min：17％～40％流动相B+83％～60％流动相A
           15～15.1min：40％～70％流动相B+60％～30％流动相A
           15.1～17min，70％流动相B+30％流动相A
从图1可以看出，抗体-药物偶联物I-1粗产物为包含I-1-1(图1中的DAR1，n＝1)、I-1-2(图1中的DAR2，n＝2)、I-1-3(图1中的DAR3，n＝3)和I-1-4(图1中的DAR4，n＝4)的混合物。
步骤e.抗体-药物偶联物I-1粗产物的纯化
对步骤d中得到的抗体-药物偶联物I-1粗产物进行HIC，经脱盐换液(即更换缓冲液)和超滤浓缩，获得抗体-药物偶联物I-1。
HIC条件：
填料：      GE填料(Pheynl HP)
流动相A：   1.5M硫酸铵水溶液，25m M磷酸氢二钠水溶液，pH 7.0
流动相B：   25mM磷酸氢二钠水溶液，pH 7.0，10％异丙醇溶液
流速：      1.0ml/min
洗脱条件：  0％～40％流动相B洗脱20CV；40％～100％流动相B洗脱
          30CV分管收集
通过HIC-UPLC对所得抗体-药物偶联物I-1进行DAR分析，所得谱图如图2所示。从图2可以看出，抗体-药物偶联物I-1显示一个单峰，结合图4的肽图谱进行分析，表明抗体药物偶联物I-1的DAR为2，即其中n＝2，其是一分子抗体上偶联两分子药物的抗体-药物偶联物，并且产物纯净。
进一步对抗体-药物偶联物I-1进行轻重链还原液相色谱分析，得到重叠反相色谱图，如图3所示。在图3中，深色曲线是曲妥珠单抗裸抗的谱图，浅色曲线是抗体-药物偶联物I-1的谱图。在两谱图中，较高的峰指示抗体重链，较低的峰指示抗体轻链。从图3可以看出，与未偶联药物的曲妥珠单抗裸抗相比，抗体-药物偶联物I-1中的抗体轻链因偶联药物分子而疏水性增强，因此保留时间后移。这说明，抗体-药物偶联物I-1中药物分子全部偶联在抗体轻链上。
进一步将抗体-药物偶联物I-1和曲妥珠裸抗在相同条件下进行酶切，得到肽图谱，如图4所示。
在图4的肽图谱中，上面的图为裸抗在214nm的肽图谱，下面的图为抗体-药物偶联物I-1在214nm的肽图谱。经比较，抗体-药物偶联物I-1中只在保留时间65.5min处增加了一个肽段。结合图3的轻重链还原色谱图可知，本实施例制备得到的抗体-药物偶联物I-1中，毒素分子(D)全部偶联在轻链同一肽段的赖氨酸上，实现了定点偶联，因而获得优异的产品质量均一性、质量可控性和重现性。
实施例2.制备抗体-药物偶联物I-2
Figure PCTCN2016106802-appb-000038
步骤a.中间体I-A-2的制备
Figure PCTCN2016106802-appb-000039
在室温下，将化合物D-2(购于南京联宁生物制药公司，1mmol，1当量)溶于DMF(50mL)中，加入碳酸钾(2.5当量)和2-氯乙酸甲酯(1.5当量)。将反应混合物升温至40～45℃，反应4小时。反应结束后，过滤除去碳酸钾固体，浓缩滤液。将浓缩所得物质溶于甲醇(30mL)中，加入1M的氢氧化锂水溶液将pH调节至13～14。将反应混合物升温至55℃，搅拌16小时，然后加入10％(w/v)的柠檬酸水溶液，浓缩并经HPLC纯化，得到中间体D-A-2。
在室温下，将化合物D-A-2(0.1mmol，1当量)溶于DMF(5mL)中，依次加入DIC(1.1当量)、HOAt(1.1当量)和哌啶4-羧酸(1.2当量)。将反应混合物在室温下搅拌6小时，然后加入水(60mL)和EtOAc(20mL*3)，萃取后收集有机相，浓缩并经HPLC纯化，得到中间体I-A-2。
MS m/z(ESI):907[M+H]+。
步骤b.中间体I-B-2的制备
Figure PCTCN2016106802-appb-000040
在室温下，将化合物I-A-2(0.1mmol，1当量)溶于DCM(50mL)中，依次加入EDCI(1.5当量)、NHS(1.5当量)和五氟苯酚(2.0当量)。在室温下反应18小时。向反应混合物中依次加入水(30mL)、饱和柠檬酸水溶液(20mL)和饱和氯化钠水溶液(20mL)进行洗涤，萃取后收集有机相，浓缩并经HPLC纯化，得到中间体I-B-2。
MS m/z(ESI):1073[M+H]+。
步骤c.抗体-药物偶联物I-2粗产物的合成
Figure PCTCN2016106802-appb-000041
I-2粗产物(其中n＝1,2,3)
向1ml在pH 7.4的PBS缓冲液中配制的浓度为10mg/ml的曲妥珠单抗溶液中加入8倍物质的量的溶解在DMA中的化合物I-B-2。在室温和温和搅拌下反应16小时，通过HIC-HPLC(HIC-HPLC条件与实施例1步骤d中所列相同)监测反应，得到抗体-药物偶联物I-2粗产物，HIC-HPLC谱图如图5所示。
从图5可以看出，抗体-药物偶联物I-2粗产物为包含I-2-1(图5中的DAR1，n＝1)、I-2-2(图5中的DAR2，n＝2)和I-2-3(图5中的DAR3，n＝ 3)的混合物。
步骤d.抗体-药物偶联物I-2粗产物的纯化
对步骤c中得到的抗体-药物偶联物I-2粗产物进行HIC(HIC条件与实施例1步骤e中所列相同)，经脱盐换液和超滤浓缩，获得抗体-药物偶联物I-2。
通过HIC-HPLC对所得抗体-药物偶联物I-2进行DAR分析，所得谱图如图6所示。从图6可以看出，抗体-药物偶联物I-2的DAR为2，表明该抗体-药物偶联物中n＝2，是一分子抗体上偶联两分子药物的抗体-药物偶联物，并且产物纯净。
进一步对抗体-药物偶联物I-2进行轻重链还原液相色谱分析，得到重叠色谱图，如图7所示。在图7中，深色曲线是曲妥珠单抗裸抗的谱图，浅色曲线是抗体-药物偶联物I-2的谱图。在两谱图中，较高的峰指示抗体重链，较低的峰指示抗体轻链。从图7可以看出，与未偶联药物的曲妥珠单抗裸抗相比，抗体-药物偶联物I-2中的抗体轻链因偶联药物分子而疏水性增强，因此保留时间后移。这说明，抗体-药物偶联物I-2中药物分子全部偶联在抗体轻链上。
进一步将抗体-药物偶联物I-2和曲妥珠裸抗在相同条件下进行酶切，得到肽图谱，如图8所示。
在图8的肽图谱中，上面的图为裸抗在214nm的肽图谱，下面的图为抗体-药物偶联物I-2在214nm的肽图谱。经比较，抗体-药物偶联物I-1中只在保留时间33.0min处增加了一个肽段。结合图7的轻重链还原色谱图可知，本实施例制备得到的抗体-药物偶联物I-2中，毒素分子(D)全部偶联在轻链同一肽段的赖氨酸上，由于实现了定点偶联，可获得优异的产品质量均一性、质量可控性和重现性。
实施例3.制备抗体-药物偶联物I-3
Figure PCTCN2016106802-appb-000042
步骤a.中间体I-A-3的制备
Figure PCTCN2016106802-appb-000043
在室温下，将化合物D-3(根据CN 104662000A第65页实施例V-3合成，1mmol，1当量)溶于THF(60mL)和DMF(30mL)的混合溶液中，依次加入二(对硝基苯基)碳酸酯(3当量)和DIEA(2当量)。将反应混合物在室温下搅拌12小时，然后加入水(600mL)和EtOAc(200mL*3)，萃取后收集有机相，浓缩得到中间体D-A-3粗品，不经纯化而直接用于下一步反应。
在室温下，将哌啶4-羧酸(5当量)溶解在饱和NaHCO3水溶液(5mL)中，加入化合物D-A-3粗品(1当量)。将反应混合物在室温下搅拌8小时。加入10％(w/v)柠檬酸水溶液以将pH调节至4～5，然后用EtOAc(150mL*2)萃取。将有机相干燥并浓缩，得到中间体I-A-3粗品。
MS m/z(ESI):1020[M+H]+。
步骤b.中间体I-B-3的制备
Figure PCTCN2016106802-appb-000044
在室温下，将化合物I-A-3(0.1mmol，1当量)溶于DCM(50mL)中，依次加入EDCI(1.5当量)、NHS(1.5当量)和五氟苯酚(2.0当量)。在室温下反应18小时。向反应混合物中依次加入水(30mL)、10％(w/v)柠檬酸水溶液(20mL)和饱和氯化钠水溶液(20mL)进行洗涤，萃取后收集有机相，浓缩并经HPLC纯化，得到中间体I-B-3。
MS m/z(ESI):1186[M+H]+。
1HNMR(400MHz,DMSO-d6)δ:7.02-7.09(m,4H),4.92(m,1H),4.52(m,1H),3.89(d,1H),3.77(m,1H),3.68-3.55(m,3H),3.46(m,2H),3.24(m,6H),3.05(d,2H),2.92-2.90(m,4H),2.68(m,1H),2.33-2.27(m,11H),1.97-1.93(m,4H),1.73-1.72(m,5H),1.29-1.24(m,5H),1.06-1.01(m,15H),0.96(m,3H),0.74(m,4H),3.34(m,4H)。
步骤c.抗体-药物偶联物I-3粗产物的合成
Figure PCTCN2016106802-appb-000045
I-3粗产物(其中n＝1,2,3)
向1ml在pH 7.8的PBS缓冲液中配制的浓度为10mg/ml的曲妥珠单抗溶液中加入8倍物质的量的溶解在DMA中的化合物I-B-3。在室温和温和搅拌下反应4小时，通过HIC-HPLC(HIC-HPLC条件与实施例1步骤d中所列相同)监测反应，得到抗体-药物偶联物I-3粗产物，HIC-HPLC谱图如图9所示。
从图9可以看出，抗体-药物偶联物I-3粗产物为包含I-3-1(图9中的DAR1，n＝1)、I-3-2(图9中的DAR2，n＝2)和I-3-3(图8中的DAR3，n＝3)的混合物。
步骤d.抗体-药物偶联物I-3粗产物的纯化
对步骤c中得到的抗体-药物偶联物I-3粗产物进行HIC(HIC条件与实施例1步骤e中所列相同)，经脱盐换液和超滤浓缩，获得抗体-药物偶联物I-3。
通过HIC-HPLC对所得抗体-药物偶联物I-3进行DAR分析，所得谱图如图10所示。从图10可以看出，抗体-药物偶联物I-3的DAR为2，表明该抗体-药物偶联物中n＝2，是一分子抗体上偶联两分子药物的抗体-药物偶联物，并且产物纯净。
进一步对抗体-药物偶联物I-3进行轻重链还原液相色谱分析，得到重叠色谱图，如图11所示。在图11中，深色曲线是曲妥珠单抗裸抗的谱图，浅色曲线是抗体-药物偶联物I-3的谱图。在两谱图中，较高的峰指示抗体重链，较低的峰指示抗体轻链。从图11可以看出，与未偶联药物的曲妥珠单抗裸抗相比，抗体-药物偶联物I-3中的抗体轻链因偶联药物分子而疏水性增强，因此保留时间后移。这说明，抗体-药物偶联物I-3中药物分子全部偶联在抗体轻链上。
进一步将抗体-药物偶联物I-3和曲妥珠裸抗在相同条件下进行酶切，得到肽图谱，如图12所示。
在图12的肽图谱中，上面的图为裸抗在214nm的肽图谱，下面的图为抗体-药物偶联物I-3在214nm的肽图谱。经比较，抗体-药物偶联物I-1中只在保留时间39.0min处增加了一个肽段。结合图11的轻重链还原色谱图可知，本实施例制备得到的抗体-药物偶联物I-3中，毒素分子(D)全部偶联在轻链同一肽段的赖氨酸上，实现了定点偶联，因而获得优异的产品质量均一性、质量可控性和重现性。
实施例4.体内活性测试
本实施例评价实施例1～3的抗体-药物偶联物对皮下移植了人肿瘤细胞的小鼠的肿瘤增殖的抑制。具体而言，在本实施例中，将实施例1～3的抗体-药物偶联物单次尾静脉注射给皮下移植人胃癌细胞株NCI-N87、乳腺癌细胞株BT474、卵巢癌细胞株SK-OV-3的小鼠后，测定肿瘤体积和动物体重变化，计算抗体-药物偶联物对荷瘤小鼠的药效(抑瘤疗效)。
称取适量曲妥珠单抗裸抗、T-DM1(阳性对照，
Figure PCTCN2016106802-appb-000046
(ado-trastuzumab emtansine)，罗氏制药)或本发明的抗体-药物偶联物(I-3、I-1或I-2，实施例1～3制备)，分别用无菌超纯水配制成一定浓度的母液，轻柔摇匀后，分装于-80℃保存。使用时用生理盐水根据剂量稀释，得到处理品溶液，并采用相同浓度的生理盐水作为溶剂对照。
选择随机分组(根据样品数量决定分组数)的肿瘤体积在100～200mm3的荷瘤鼠(1.2中得到的模型)，7只/组。给药体积为10ml/kg。给药途径为单次尾静脉注射。给药后观察8周，每周2次用游标卡尺测量肿瘤直径，并按如下计算公式计算肿瘤体积：V＝0.5a2×b，其中a和b分别表示肿瘤的长径和短径。每天观察记录动物死亡情况。
采用以下公式计算肿瘤生长抑制率TGI(％)，用于评价抗体-药物偶联物的抑瘤疗效：
TGI(％)＝[1-(VT末-VT始)/(VC末-VC始)]*100％
其中VT末：处理组实验结束时肿瘤体积均值
VT始：处理组给药开始时肿瘤体积均值
VC末：溶剂对照组实验结束时肿瘤体积均值
VC始：溶剂对照组给药开始时肿瘤体积均值
计算结果见表1～4。
表1.乳腺癌BT-474模型
组别 样品 剂量(mg/kg) TGI(％)
1 溶剂 / /
2 T-DM1 3 54.80％
3 I-1 3 104.75％
4 I-3 3 107.52％
本发明的抗体-药物偶联物对多种乳腺癌细胞系亦具有类似的抑瘤疗效。
表2：胃癌NCI-N87模型
Figure PCTCN2016106802-appb-000047
表3：胃癌NCI-N87模型
组别 样品 剂量(mg/kg) TGI(％)
1 溶剂 / /
2 T-DM1 2 30.136％
3 I-1 2 114.99％
4 I-3 2 87.04％
5 T-DM1 3 65.28％
6 I-3 3 102.72％
表4：卵巢癌SK-OV-3模型
组别 样品 剂量(mg/kg) TGI(％)
1 溶剂 / /
2 T-DM1 6 -11.04％
3 I-2 6 54.72％
4 I-3 6 72.27％
从表1至表4可以看出，本发明的抗体-药物偶联物I-1、I-3和I-2在评价周期内，对多种肿瘤如胃癌、卵巢癌、乳腺癌的抑制活性均明显优于T-DM1，动物死亡情况也表明它们具有优良的安全性。
实施例5.体内稳定性测试
本实施例评价实施例1～3的抗体-药物偶联物在大鼠体内的稳定性。具体而言，在本实施例中，将实施例1～3的抗体-药物偶联物以2mg/kg剂量单次尾静脉注射给大鼠后，定期进行颈静脉采血，用ELISA法测定血中抗体-药物偶联物浓度和总抗体浓度，计算抗体-药物偶联物在大鼠体内的半衰 期。测定结果如表5所示。
表5.抗体-药物偶联物的半衰期
抗体-药物偶联物 T1/2(h)
T-DM1* 114*
I-1 247.6
I-2 211.3
I-3 259.8
*根据Bender等人，The AAPS Journal,Vol.16,No.5,September 2014的报道，T-DM1在大鼠体内的半衰期为114小时。
从表5可以看出，本发明的抗体-药物偶联物I-1、I-2和I-3在大鼠体内具有较长的半衰期，稳定性明显优于T-DM1。
实施例6.制备抗体-药物偶联物I-1的冻干粉针剂
使用如下表所示原料制备抗体-药物偶联物I-1的冻干粉针剂：
抗体-药物偶联物I-1 28g
抗坏血酸 20g
乳酸 10g
聚乙二醇4000 63g
注射用水 2000ml
制备方法：
1、取20g抗坏血酸和10g乳酸，加1000ml注射用水，加热至50～55℃，搅拌使其溶解，取28g抗体-药物偶联物I-1加入溶液中，搅拌溶解后继续搅拌15分钟。
2、取63g聚乙二醇4000，加800ml注射用水，搅拌15分钟。
3、将1、2中所得溶液合并，加注射用水至全量，加0.15％的针用活性炭，搅拌25分钟，过滤脱炭，中间体检查，合格后用0.22μm滤膜过滤除菌。
4、将滤液灌入西林瓶中，进行冷冻干燥，得冻干粉针，检验合格后，包装。
实施例7.制备抗体-药物偶联物I-2的冻干粉针剂
使用如下表所示原料制备抗体-药物偶联物I-2的冻干粉针剂：
抗体-药物偶联物I-2 20g
琥珀酸钠 1.62g
蔗糖 60g
吐温20 0.2g
注射用水 1000ml
制备方法：
1、取16.2g琥珀酸钠、2g吐温20和600g蔗糖，加注射用水定容至10L，搅拌使其溶解，无菌过滤后作为制剂缓冲液备用。取相当于20g抗体-药物偶联物I-2的原液，用制剂缓冲液进行超滤换液，并浓缩至1000ml。
2、将1所述溶液用0.22μm滤膜过滤除菌。
3、将滤液灌入西林瓶中，进行冷冻干燥，得冻干粉针，检验合格后，包装。
实施例8.制备抗体-药物偶联物I-3的冻干粉针剂
使用如下表所示原料制备抗体-药物偶联物I-3的冻干粉针剂：
抗体-药物偶联物I-3 20g
L-组氨酸 0.32g
L-组氨酸盐酸盐 0.495g
二水海藻糖 20g
吐温20 0.09g
注射用水 1000ml
制备方法：
1、取3.2g L-组氨酸、4.95g L-组氨酸盐酸盐、200g二水海藻糖和0.9g吐温20，加注射用水定容至10L，搅拌使其溶解，无菌过滤后作为制剂缓冲液备用。取相当于20g抗体-药物偶联物I-3的原液，用制剂缓冲液进行超滤换液，并浓缩至1000ml。
2、将1所述溶液用0.22μm滤膜过滤除菌。
3、将滤液灌入西林瓶中，进行冷冻干燥，得冻干粉针，检验合格后，包装。
尽管本发明通过之前的具体实施例得到说明，但应当理解，不应将其解释为受此限制。本发明涵盖之前公开的一般方面，并且本领域技术人员可在不背离本发明的精神和范围的情况下进行多种修饰或改变本发明的各 种细节。因此，本说明书仅为说明的目的，而非为限制的目的。

Claims (15)

  1. 通式(I)的抗体-药物偶联物，其药学上可接受的盐、立体异构体或代谢物，或者它们的溶剂合物，
    Figure PCTCN2016106802-appb-100001
    其中：
    A为抗ErbB2抗体或者其活性片段或变体；
    X和Y各自独立地为N或CR1，且每个R1独立地为H或C1-C10烷基；
    L为二价连接基；
    D为细胞毒性药物基团；并且
    a为选自2-10的整数。
  2. 如权利要求1所述的抗体-药物偶联物，其药学上可接受的盐、立体异构体或代谢物，或者它们的溶剂合物，其中所述抗ErbB2抗体为抗人ErbB2抗体，并且优选地，所述抗人ErbB2抗体中重链和轻链的CDR1、CDR2和/或CDR3分别为曲妥珠单抗重链和轻链的CDR1、CDR2和/或CDR3。
  3. 如权利要求2所述的抗体-药物偶联物，其药学上可接受的盐、立体异构体或代谢物，或者它们的溶剂合物，其中所述抗ErbB2抗体为人源化抗体或全人源抗体，优选为曲妥珠单抗。
  4. 如权利要求1-3中任一项所述的抗体-药物偶联物，其药学上可接受的盐、立体异构体或代谢物，或者它们的溶剂合物，其中Y为CR1，X为N。
  5. 如权利要求1-4中任一项所述的抗体-药物偶联物，其药学上可接受的盐、立体异构体或代谢物，或者它们的溶剂合物，其中R1为H。
  6. 如权利要求1-5中任一项所述的抗体-药物偶联物，其药学上可接受的盐、立体异构体或代谢物，或者它们的溶剂合物，其中a为2、3或4。
  7. 如权利要求1-6中任一项所述的抗体-药物偶联物，其药学上可接受的盐、立体异构体或代谢物，或者它们的溶剂合物，其中L选自：
    Figure PCTCN2016106802-appb-100002
    Figure PCTCN2016106802-appb-100003
    Figure PCTCN2016106802-appb-100004
    Figure PCTCN2016106802-appb-100005
    Figure PCTCN2016106802-appb-100006
    Figure PCTCN2016106802-appb-100007
    其中m、n每次出现时各自为选自1-10的整数，优选1、2、3、4、5或6。
  8. 如权利要求1-7中任一项所述的抗体-药物偶联物，其药学上可接受的盐、或代谢物立体异构体，或者它们的溶剂合物，其中所述细胞毒性药 物基团来自通式(D1)或(D2)的化合物或它们的立体异构体：
    Figure PCTCN2016106802-appb-100008
    其中：
    R2选自-CH2N3、-CONHSO2(环丙基)、噻唑-2-基、-CH3和-COOH；
    R3选自H和-OH；并且
    R4选自H、-NH2、Cl、Br、I、-OS(O)2R6，其中R6是H、C1-C8烷基、C3-C8环烷基或C6-C14芳基，所述烷基、环烷基和芳基各自任选地被一个或多个(如1、2、3、4或5个)选自卤素的取代基如F取代；
    Figure PCTCN2016106802-appb-100009
    其中R5选自-C(CH3)N(CH3)C(O)CH2CH2SH和-C(CH3)N(CH3)C(O)CH2C(CH3)2SH。
  9. 如权利要求8所述的抗体-药物偶联物，其药学上可接受的盐、立体异构体或代谢物，或者它们的溶剂合物，其中所述抗体-药物偶联物选自I-1、I-2和I-3：
    Figure PCTCN2016106802-appb-100010
    其中A为曲妥珠单抗。
  10. 权利要求1-9中任一项的抗体-药物偶联物的制备方法，所述方法包括以下步骤：
    (1).制备通式(I-A)的化合物：
    Figure PCTCN2016106802-appb-100011
    其中D、L、X和Y如权利要求1-9中任一项关于通式(I)所定义；
    (2).活化步骤(1)中得到的通式(I-A)的化合物，得到通式(I-A-G)的化合物
    Figure PCTCN2016106802-appb-100012
    其中G选自-F、-Cl、-Br、-I、-N3、-OR、-SR、-ONRR’、RC(O)O-、-OP(O)RR’、RSO2-O-和
    Figure PCTCN2016106802-appb-100013
    其中R和R’每次出现时各自独立地是C1-C10烷基、C6-C14芳基、含5-10个环原子的杂环基或苯氧基，所述烷基、芳基、杂环基和苯氧基是未取代的或者各自独立地被一个或多个选自卤素、羟基、C1-C4烷基、C1-C4烷氧基、C3-C8环烷基、含5-8个环原子的杂环基、C6-C10芳基或含5-10个环原子的杂芳基的取代基取代；优选地，G选自-ONRR’和-OP(O)RR’，其中R和R’每次出现时各自独立地是苯氧基；
    (3).将步骤(2)中得到的通式(I-A-G)的化合物偶联于所述抗ErbB2抗体或者其活性片段或变体，得到具有不同a值的多种抗体-药物偶联物的混合物；和
    (4).经选自离子交换层析、疏水层析、反相层析和亲和层析中的一种或多种层析方法纯化步骤(3)中得到的混合物，得到抗体-药物偶联物。
  11. 权利要求10的制备方法，其中步骤(2)中的通式(I-A-G)的化合物为通过使通式(I-A)的化合物与五氟苯酚反应而形成的通式(I-B)的化合物：
    Figure PCTCN2016106802-appb-100014
    其中D、L、X和Y如权利要求1-9中任一项关于通式(I)所定义，并且所述 反应优选使用1-乙基-3-(3-二甲基氨基丙基)碳二亚胺、N-羟基琥珀酰亚胺和/或二氯甲烷完成。
  12. 药物组合物，其包含权利要求1-9之任一项所述的抗体-药物偶联物或者其药学上可接受的盐、立体异构体或代谢物或者它们的溶剂合物，以及药学上可接受的载体，并且所述药物组合物任选地进一步包含一种或多种其他抗癌药如化疗剂和/或抗体。
  13. 权利要求1-9中任一项所述的抗体-药物偶联物或者其药学上可接受的盐、立体异构体或代谢物或者它们的溶剂合物在制备用于预防或治疗癌症的药物中的用途。
  14. 如权利要求13所述的用途，其中所述癌症选自乳腺癌、胃癌、卵巢癌、非小细胞肺癌、肝癌、子宫内膜癌、唾液腺癌、肾癌、结肠癌、甲状腺癌、胰腺癌或膀胱癌，特别是乳腺癌，如ErbB2高表达的乳腺癌。
  15. 药物制剂，其包含权利要求1-9中任一项的抗体-药物偶联物或者其药学上可接受的盐、立体异构体或代谢物或者它们的溶剂合物，所述制剂优选为固体制剂、半固体制剂、液体制剂或气体制剂的形式，如冻干粉针剂。
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