WO2018036438A1 - 一种抗体-药物偶联物及其制备方法和应用 - Google Patents

一种抗体-药物偶联物及其制备方法和应用 Download PDF

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WO2018036438A1
WO2018036438A1 PCT/CN2017/098140 CN2017098140W WO2018036438A1 WO 2018036438 A1 WO2018036438 A1 WO 2018036438A1 CN 2017098140 W CN2017098140 W CN 2017098140W WO 2018036438 A1 WO2018036438 A1 WO 2018036438A1
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conjugate
cancer
stereoisomer
antibody
group
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PCT/CN2017/098140
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French (fr)
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薛彤彤
汪静
肖亮
宋宏梅
刘登念
张强
袁晓曦
刘立平
杨秋艳
郭大成
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 CN201780004255.7A priority Critical patent/CN108697809A/zh
Publication of WO2018036438A1 publication Critical patent/WO2018036438A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes

Definitions

  • the invention belongs to the field of biomedical technology.
  • the invention relates to an antibody-drug conjugate and methods of making and using same.
  • ADC antibody-drug conjugates
  • 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 using a specific functional group such as a hydroxyl group, a thiol group or an amino group in the drug molecule to constitute an antibody-drug conjugate.
  • 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).
  • the drugs used as "warheads" in ADCs are usually cytotoxic drugs, that is, small molecule toxins, which kill tumor cells mainly by inhibiting cellular 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 Pat. No. 5,416,064, US Pat. No. 7,276,497, US Pat. No. 7,473,796, US Pat. No.
  • 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 must meet the following requirements: sufficient stability outside the cell to ensure that the small molecule drug does not detach from the antibody; after entering the cell, the cleavable linker cleaves under appropriate conditions, releasing the active small molecule drug, The non-cleavable linker forms an active moiety 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).
  • the drug/antibody ratio (DAR) averages 2-4.
  • T-DM1 has a DAR value distribution of 0-8 and an average DAR value of 3.5 (Lazar et al., 2005, Rapid Commun. Mass Spectrom., 19: 1806-1814).
  • the drug is attached to a cysteine residue in the hinge region of the antibody, since there are only four pairs of interchain disulfide bonds in the hinge region of the antibody, in order to achieve an average DAR value of 2-4, partial reduction of interchain disulfide is required.
  • 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.
  • Trop-2 or TROP2 means human trophoblast cell surface antigens 2, also known as TACSTD2, M1S1, GA733-1, EGP-1, which are caused by many human tumors (such as breast cancer, Cell surface receptors expressed in cells of colorectal cancer, lung cancer, pancreatic cancer, ovarian cancer, prostate cancer, and cervical cancer, but Trop-2 has only limited expression in normal human tissues.
  • Trop-2 is a single-transmembrane surface glycoprotein with a molecular weight of 45KD, which is a cell membrane calcium channel-associated protein and is involved in the regulation of intracellular calcium concentration.
  • Trop-2 is involved in cyclin D1 and phosphokinase C and functions to regulate tumor cell growth and promote tumor cell invasion and metastasis.
  • IMMU-132 (Isactuzumab govitecan) is an ADC formed by the cysteine thiol coupling of the humanized antibody Isactuzumab (hRS7) targeting rop-2 and the irinotecan active metabolite SN-38 by antibodies. Because SN-38 has the disadvantage of being toxic and poorly soluble, it cannot be administered directly, while IMMU-132 can improve SN-38. Bioavailability and reduce its toxicity.
  • IMMU-132 an average of about 7 SN-38 molecules are linked per antibody molecule, and the linkage diagram is shown below (see http://www.immunomedics.com/linker-demo.shtml):
  • IMMU-132 has the following shortcomings:
  • IMMU-132 The affinity of IMMU-132 is different from that of hRS7 naked antibiotic. The reason for this change is that the coupling of toxin molecules leads to structural changes of the antibody, and the structural changes of the antibody may cause the hydrophilic region to be exposed. Drug effects cause unpredictable effects;
  • IMMU-132 The DAR value of IMMU-132 is very high and non-uniform, indicating that its drug loading is high. Although the drug with high drug loading may have stronger in vitro activity, high drug loading will bring many problems, such as Polymerization leads to an increase in multimeric components, decreased stability, increased toxicity, increased immunogenicity, rapid elimination in vivo, short half-life and low actual therapeutic index.
  • the present invention provides a conjugate of the formula (I), a pharmaceutically acceptable salt or stereoisomer thereof, or a solvate of the conjugate, salt or stereoisomer,
  • A is an anti-Trop-2 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 1-10.
  • the present invention provides a method of preparing a conjugate of the first aspect of the invention, the method comprising the steps of:
  • R and R' are each, at each occurrence, independently an optionally substituted alkyl, optionally substituted aryl or optionally substituted heterocyclyl; for example, said R and R' each independently occur independently Is a C 1 -C 10 alkyl group, a C 6 -C 14 aryl group, a heterocyclic group having 5 to 10 ring members or a phenoxy group, and the alkyl group, the aryl group, the heterocyclic group and the phenoxy group are not Substituted or independently of one or more selected from the group consisting of halogen, hydroxy, C 1 -C 4 alkyl, C 1 -C 4 alkoxy, C 3 -C 8 cycloalky
  • purifying the mixture obtained in the step (3) to obtain a conjugate preferably, purifying by chromatography; further preferably, the chromatography is selected from ion exchange chromatography, hydrophobic One or more of chromatography, reverse phase chromatography, and affinity chromatography.
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising the conjugate of the first aspect of the invention or a pharmaceutically acceptable salt or stereoisomer thereof or the conjugate, salt or stereoisomer a solvate, and a pharmaceutically acceptable carrier.
  • the present invention provides a pharmaceutical preparation comprising the conjugate of the first aspect of the invention or a pharmaceutical thereof An acceptable salt or stereoisomer or a solvate of the conjugate, salt or stereoisomer.
  • the present invention provides a conjugate of the first aspect of the invention, or a pharmaceutically acceptable salt or stereoisomer thereof, or a solvate of the conjugate, salt or stereoisomer, third Use of the pharmaceutical composition of aspect or the pharmaceutical preparation of the fourth aspect for the preparation of a medicament for preventing or treating cancer.
  • the present invention provides a conjugate of the first aspect of the invention, or a pharmaceutically acceptable salt or stereoisomer thereof, or a solvate of the conjugate, salt or stereoisomer, third
  • the pharmaceutical composition of aspect or the pharmaceutical preparation of the fourth aspect which is for use in the prevention or treatment of cancer.
  • the present invention provides a method of preventing or treating cancer comprising administering to a subject in need thereof an effective amount of the conjugate of the first aspect of the invention or a pharmaceutically acceptable salt thereof Or a stereoisomer or a solvate of the conjugate, a salt or a stereoisomer, a pharmaceutical composition according to the third aspect, or a pharmaceutical preparation according to the fourth aspect.
  • the present invention provides the conjugate of the first aspect of the invention, or a pharmaceutically acceptable salt or stereoisomer thereof, or a solvate of the conjugate, salt or stereoisomer, Use of the pharmaceutical composition of the third aspect or the pharmaceutical preparation of the fourth aspect for the preparation of an agent, wherein the agent is for inhibiting growth, proliferation or migration of cancer cells.
  • the present invention provides the conjugate of the first aspect of the invention, or a pharmaceutically acceptable salt or stereoisomer thereof, or a solvate of the conjugate, salt or stereoisomer,
  • the present invention provides a method of inhibiting growth, proliferation or migration of cancer cells, comprising administering to a cancer cell an effective amount of the conjugate of the first aspect of the invention or a pharmaceutically acceptable salt thereof or A stereoisomer or a solvate of the conjugate, a salt or a stereoisomer, a pharmaceutical composition according to the third aspect, or a pharmaceutical preparation according to the fourth aspect.
  • the present invention provides a kit for inhibiting growth, proliferation or migration of cancer cells, comprising the conjugate of the first aspect of the invention or a pharmaceutically acceptable salt or stereoisomer thereof or The conjugate of the conjugate, the salt or the stereoisomer, the pharmaceutical composition of the third aspect, or the pharmaceutical preparation of the fourth aspect.
  • antibody is used in its broadest sense to include intact monoclonal antibodies, polyclonal antibodies, and bispecific or multispecific antibodies formed from at least two intact antibodies, provided that they have Place 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.
  • a monoclonal antibody also specifically includes a chimeric antibody, ie, a portion of a heavy chain and/or a light chain is identical or homologous to a certain, certain or a subset of antibodies, and the remainder is Another class 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 variable region antigen binding sequences from human non-human primates (e.g., ancient monkeys, orangutans, etc.) and human constant region sequences.
  • bispecific antibody and "bifunctional antibody conjugate” are used interchangeably to mean that a first antibody (fragment) and a second antibody (fragment) are formed by a coupling arm.
  • the conjugate which retains the activity of the respective antibody, is bifunctional and bispecific.
  • bispecific antibodies from antibody fragments
  • Techniques for producing bispecific antibodies from antibody fragments have been described in the literature, for example, using proteolytic cleavage of intact antibodies at their position to produce chemical bonds for F(ab') fragments (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).
  • 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).
  • the preparation of trispecific antibodies can be found, for example, in 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.
  • 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.
  • 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 recovering Fab'-SH fragments from E. coli and chemically coupling to form F(ab') fragments (Carter et al., Biotechnology ( N Y), 1992 Feb, 10(2): 163-167). In addition, F(ab') fragments can be isolated directly from recombinant host cell culture.
  • 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).
  • the antibody fragment is a linear antibody fragment (see, eg, US 5,641,870), which may be monospecific or bispecific.
  • multispecific antibody includes, for example, a trispecific antibody and a tetraspecific antibody, the former being an antibody having three different antigen binding specificities, and the latter having four different antigen binding specificities.
  • Antibodies include, for example, a trispecific antibody and a tetraspecific antibody, the former being an antibody having three different antigen binding specificities, and the latter 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.
  • 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.
  • 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 retain Natural polypeptides have comparable or higher 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.
  • the amino 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.
  • the term “homology” is used interchangeably with “consistency,” “identity,” or “similarity,” and refers to that obtained after optimal alignment (optimal alignment).
  • 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".
  • optimal alignment can also be performed by other methods described in the literature, such as Smith and Waterman. Local homology algorithm described in 1981, Ad. App.
  • 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 certain preferred 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 (
  • a humanized antibody typically comprises at least one, usually two, variable regions in which all or nearly all hypervanable loops correspond to non-human immunoglobulins, while FR is completely or almost entirely human immunoglobulin. the sequence of.
  • 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
  • the amino acid substitution in the antibody is substituted by the L-amino acid in most cases, it is not limited thereto.
  • 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).
  • 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.
  • ranges of values herein are to be understood
  • the range "1 to 10" should be understood to include not only the values of 1 to 10 which are clearly stated, but also the range of 1 to 10 What are the individual values (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 term "pharmaceutically acceptable salt” refers to salts which retain the biological effectiveness and properties of the compounds, which are biologically or otherwise desirable for use as a medicament.
  • the ADCs 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 refers to 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 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, etc. after administration.
  • halogen refers to fluorine (F), chlorine (Cl), bromine (Br), iodine (I), and ruthenium (At).
  • C 1 -C 10 alkyl denotes a straight or branched alkyl group having 1 to 10 carbon atoms, including, for example, “C 1 -C 6 alkyl", “C 1 -C 4 alkyl", “C 1 -C 3 alkyl”, etc., specific examples include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl , tert-butyl, n-pentyl, isopentyl, 2-methylbutyl, neopentyl, 1-ethylpropyl, n-hexyl, isohexyl, 3-methylpentyl, 2-methylpentyl , 1-methylpentyl, 3,3-dimethylbutyl, 2,2-dimethylbutyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 1, 3-
  • C 3 -C 8 cycloalkyl refers to a saturated cyclic alkyl group containing from 3 to 8 carbon atoms, including, for example, “C 3 -C 6 cycloalkyl", “C 4 -C 6 cycloalkyl”, “C 5 -C 7 cycloalkyl” or “C 5 -C 6 cycloalkyl” or the like. Specific examples include, but are not limited to, cyclopropyl, cyclobutane, cyclopentyl, cyclohexane, cycloheptyl, cyclooctyl, and the like.
  • C 6 -C 14 aryl refers to an aromatic monocyclic or polycyclic hydrocarbon group containing from 6 to 14 carbon atoms, such as a C 6 -C 10 aryl group and the like. Specific examples include, but are not limited to, phenyl, naphthyl, anthracenyl, phenanthryl, and the like.
  • heterocyclyl refers to a saturated cyclic group containing at least one heteroatom, wherein the heteroatom is selected from the group consisting of a nitrogen atom, an oxygen atom, and a sulfur atom.
  • heterocyclic group having 5 to 8 ring members “heterocyclic group having 5 to 7 ring members”, “heterocyclic group having 5 to 6 ring members” and the like.
  • Specific examples include, but are not limited to, pyrrolidinyl, tetrahydrofuranyl, piperidinyl, piperazinyl, tetrahydropyranyl, homopiperazinyl and the like.
  • heteroaryl refers to an aromatic cyclic group containing at least one heteroatom, wherein the heteroatom is selected from the group consisting of a nitrogen atom, an oxygen atom, and a sulfur atom.
  • heteroaryl group having 5 to 10 ring members refers to an aromatic cyclic group containing at least one heteroatom, wherein the heteroatom is selected from the group consisting of a nitrogen atom, an oxygen atom, and a sulfur atom.
  • heteroaryl group having 5 to 10 ring members refers to an aromatic cyclic group containing at least one heteroatom, wherein the heteroatom is selected from the group consisting of a nitrogen atom, an oxygen atom, and a sulfur atom.
  • the conjugate represented by the formula (I) has excellent stability, uniformity and/or reduced toxic side effects. Its cell surface receptor Trop-2, which is highly affinity-expressing in tumor cells, exhibits good biological activity.
  • the conjugate inhibits tumor growth in a mammal and is useful for preventing and/or treating a variety of neoplastic diseases, particularly tumors with high expression of Trop-2, such as breast cancer, gastric cancer, pancreatic cancer, ovarian cancer, colorectal Cancer, prostate cancer, cervical cancer, lung cancer (eg non-small cell lung cancer) and liver cancer.
  • the present invention provides a conjugate of the formula (I), a pharmaceutically acceptable salt or stereoisomer thereof, or a solvate of the conjugate, salt or stereoisomer ,
  • A is an anti-Trop-2 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 1-10.
  • Y is CR 1 and X is N.
  • each R 1 is independently H or C 1 -C 6 alkyl (eg, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl) , amyl or hexyl).
  • a is 1, 2, 3 or 4.
  • a is 2.
  • the antibody used in the present invention is an anti-Trop-2 antibody or an active fragment or variant thereof, including a bispecific antibody and an antibody functional derivative.
  • Trop-2 can be used interchangeably with TACSTD2, M1S1, GA733-1, EGP-1, both of which represent the native sequence of human Trop-2 protein (UniProt: P09758, 2) and its functional derivatives, such as amino acids. Sequence variants.
  • anti-Trop-2 antibodies useful in the present invention include, but are not limited to, m7E6, h7E6, h7E6_SVG, h7E6_SVG1, h7E6_SVG2, h7E6_SVG3, h7E6_SVG4, h7E6_SVG5, h7E6_SVG6, h7E6_SVG7, h7E6_SVG8, h7E6_SVG9, h7E6_SVG10, as described in CN104053672A, for example.
  • Anti-Trop-2 antibodies useful in the present invention can also be screened by methods for designing, constructing, and constructing antibody libraries displaying antibodies, as disclosed in CN103476941A, or by Sorrento Medical Corporation (Sorrento Therapeutics, Inc.). The library is screened for.
  • the native sequence of Trop-2 in the present invention can be isolated from nature, or can be produced by recombinant DNA technology, chemical synthesis, or a combination thereof.
  • the antibody used in the present invention is preferably an anti-human Trop-2 antibody.
  • the CDR1, CDR2 and/or CDR3 of the heavy and light chain of the anti-human Trop-2 antibody are CDR1, CDR2 and/or CDR3 of the RS7 mAb heavy and light chain, respectively.
  • the anti-human Trop-2 antibody can be a humanized antibody or a fully human antibody.
  • the antibody is the RS7 antibody of CN 100360567C, the light chain and heavy chain sequences thereof are set forth in SEQ ID NO: 1 and SEQ ID NO: 2, respectively.
  • the underlined region in the above sequence is the CDR region, and the K at the C-terminus of the heavy chain sequence can be taken off.
  • the K at the C-terminus of the heavy chain sequence is detachable, and such deletion does not affect biological activity, such as by deleting the gene encoding K at the C-terminus of the heavy chain sequence.
  • the drug used in the present invention is a cytotoxic drug group.
  • cytotoxic drug refers to a substance that inhibits or prevents cellular function and/or causes cell destruction.
  • the cytotoxic drug group is an auristatin peptide, such as auristatin E (also known as a dolastatin-10 derivative) or a derivative thereof (for example, an ester formed from auristatin E and a keto acid).
  • auristatin E can be reacted with p-acetylbenzoic acid or benzoyl valeric acid to produce AEB (Aesthesia EB) and AEVB (5-benzoylvaleric acid auristatin E ester).
  • AEB esthesia EB
  • AEVB 5-benzoylvaleric acid auristatin E ester
  • Other typical auristatin peptides include AFP (Auroxine F phenylenediamine), MMAF (monomethyl amphetin F), and MMAE (monomethyl amphetin E).
  • the cytotoxic drug group 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, it was found that certain preferred microorganisms also 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 group includes, but is not limited to, the following compounds:
  • R and R' are each independently H, C 1 -C 8 alkyl or C 3 -C 8 cycloalkyl, said alkyl and cycloalkyl being optionally one or more (eg 1, 2, 3, 4 or 5) substituted with a substituent selected from halogen (such as F, Cl, Br or I);
  • Ar is a C 6 -C 14 aryl group (such as phenyl or naphthyl).
  • the cytotoxic drug group is derived from a compound of formula (D1) or (D2) or a stereoisomer thereof:
  • R 2 is selected from -CH 2 N 3 , -CH 3 and -COOH;
  • R 3 is selected from H and -OH;
  • R 5 is selected from the group consisting of H, -OH, -NH 2 , F, Cl, Br, I, OR 7 wherein R 7 is C 1 -C 4 alkyl;
  • R 8 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) from R 4 , R 5 or R 8 or from R 4 , R 5 or R 8 a group derived from hydrogen, R 6 or R 7 .
  • the cytotoxic drug group 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 -.
  • 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 ADC of the present invention is represented by the general formula (I) defined above.
  • ADC of formula (I) is selected from the group consisting of I-1, I-2 and I-3:
  • A is hRS7 monoclonal antibody.
  • the 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 solvate, and the salt or stereoisomer may also be in the form of a solvate. .
  • the conjugates of the invention can selectively deliver an effective amount of a cytotoxic drug group to the tumor tissue, thereby achieving better therapeutic selectivity and achieving the desired therapeutic efficacy at a lower dose.
  • the present invention provides a method of preparing a conjugate of the first aspect of the invention, the method comprising the steps of:
  • R and R' are each independently an optionally substituted alkyl group, an optionally substituted aryl group or an optionally substituted heterocyclic group, for example, each of R and R' is independently C in each occurrence.
  • purifying the mixture obtained in the step (3) to obtain a conjugate preferably, purifying by chromatography; further preferably, the chromatography is selected from ion exchange chromatography, hydrophobic One or more of chromatography, reverse phase chromatography, and affinity chromatography.
  • G in the compound of formula (IAG) in step (2) is -OR, and R is 1, 2, 3, 4 or 5 substituents independently selected from halogen (e.g., fluorine, chlorine, bromine, iodine) substituted C 6 -C 14 aryl.
  • halogen e.g., fluorine, chlorine, bromine, iodine
  • the compound of the formula (I-A-G) in the step (2) is a compound of the formula (I-B) which can be formed, for example, by reacting a compound of the formula (I-A) with pentafluorophenol:
  • D, L, X and Y are as defined above for formula (I) and the reaction is preferably carried out using EDCI, NHS and/or DCM.
  • step (4) the purification of step (4) is accomplished by HPLC.
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising the conjugate of the first aspect of the invention or a pharmaceutically acceptable salt or stereoisomer thereof or the conjugate, salt or stereoisomer a solvate, and a pharmaceutically acceptable carrier.
  • the pharmaceutical composition further comprises one or more anticancer agents, such as a chemotherapeutic agent And / or antibodies.
  • the chemotherapeutic agent is selected from the group consisting of alkylating agents (eg, cyclophosphamide, ifosfamide, etc.), metabolic antagonists (eg, methotrexate, 5-fluorouracil, etc.), Antitumor antibiotics (eg, mitomycin, doxorubicin, etc.), plant-derived antineoplastic agents (eg, vincristine, vindesine, taxol, etc.), cisplatin, carboplatin, etoposide and irinote Kang.
  • the antibody is selected from the group consisting of trastuzumab (particularly when treating breast cancer) and SGN-15 (particularly when treating non-small cell lung cancer).
  • 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 preferred embodiments, the components of the pharmaceutical composition may be separated in time and/or space, as long as It is possible to work together to achieve the objects of the 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 carrier may include water, a buffer, an isotonic saline solution such as PBS (phosphate buffer), dextrose, mannitol, dextrose, lactose, starch, magnesium stearate, fiber. , magnesium carbonate, 0.3% glycerol, hyaluronic acid, ascorbic acid, lactic acid, ethanol, polyalkylene glycol such as polyethylene glycol, polypropylene glycol, triglyceride and the like.
  • PBS phosphate buffer
  • dextrose mannitol
  • dextrose lactose
  • starch magnesium stearate
  • magnesium carbonate 0.3% glycerol, hyaluronic acid, ascorbic acid, lactic acid, ethanol, polyalkylene glycol such as polyethylene glycol, polypropylene glycol, triglyceride and the like.
  • 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 a pharmaceutical preparation comprising the conjugate of the first aspect of the invention or a pharmaceutically acceptable salt, stereoisomer or solvent thereof, or a conjugate of the conjugate, salt or stereoisomer Compound.
  • the pharmaceutical formulation is in the form of a solid formulation, a semi-solid formulation, a liquid formulation, or a gas formulation.
  • 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 present invention provides a conjugate of the first aspect of the invention, or a pharmaceutically acceptable salt or stereoisomer thereof, or a solvate of the conjugate, salt or stereoisomer, Use of the pharmaceutical composition of the third aspect or the pharmaceutical preparation of the fourth aspect for the preparation of a medicament for preventing or treating cancer.
  • the present invention provides the conjugate of the first aspect of the invention, or a pharmaceutically acceptable salt or stereoisomer thereof, or a solvate of the conjugate, salt or stereoisomer,
  • the present invention provides a method of preventing or treating cancer comprising administering to a subject in need thereof an effective amount of the conjugate of the first aspect of the invention or a pharmaceutically acceptable salt thereof Or a stereoisomer or a solvate of the conjugate, a salt or a stereoisomer, a pharmaceutical composition according to the third aspect, or a pharmaceutical preparation according to the fourth aspect.
  • the present invention provides the conjugate of the first aspect of the invention, or a pharmaceutically acceptable salt or stereoisomer thereof, or a solvate of the conjugate, salt or stereoisomer, Use of the pharmaceutical composition of the third aspect or the pharmaceutical preparation of the fourth aspect for the preparation of an agent, wherein the agent is for inhibiting growth, proliferation or migration of cancer cells.
  • the agent is for use in an in vivo method.
  • the agent is for use in an in vitro method.
  • the present invention provides the conjugate of the first aspect of the invention, or a pharmaceutically acceptable salt or stereoisomer thereof, or a solvate of the conjugate, salt or stereoisomer,
  • the conjugate or a pharmaceutically acceptable salt or stereoisomer thereof or a solvate of the conjugate, salt or stereoisomer is used in an in vivo method.
  • the conjugate or a pharmaceutically acceptable salt or stereoisomer thereof or a solvate of the conjugate, salt or stereoisomer is used in an in vitro method.
  • the present invention provides a method of inhibiting growth, proliferation or migration of cancer cells, comprising administering to a cancer cell an effective amount of the conjugate of the first aspect of the invention or a pharmaceutically acceptable salt thereof or A stereoisomer or a solvate of the conjugate, a salt or a stereoisomer, a pharmaceutical composition according to the third aspect, or a pharmaceutical preparation according to the fourth aspect.
  • the method is performed in vivo.
  • the method is performed in vitro.
  • the present invention provides a kit for inhibiting growth, proliferation or migration of cancer cells, comprising the conjugate of the first aspect of the invention or a pharmaceutically acceptable salt or stereoisomer thereof or The conjugate of the conjugate, the salt or the stereoisomer, the pharmaceutical composition of the third aspect, or the pharmaceutical preparation of the fourth aspect.
  • the cancers of the invention 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 (example) Such as squamous cell carcinoma, lung cancer (such as 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 Matrix tumor (GIST), pancreatic cancer, malignant glioma, cervical cancer, ovarian cancer, bladder cancer, breast cancer, colon cancer, rectal cancer, colorectal 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.
  • squamous cell carcinoma such as squamous cell carcinoma
  • lung cancer such as
  • the cancer is a cancer with high expression of Trop-2, such as colorectal cancer, prostate cancer, cervical cancer, breast cancer, gastric cancer, endometrial cancer, salivary gland cancer, lung cancer, kidney cancer, colon Cancer, thyroid cancer, pancreatic cancer or bladder cancer.
  • the cancer is selected from the group consisting of breast cancer, gastric cancer, ovarian cancer, lung cancer (eg, non-small cell lung cancer), and liver cancer, particularly breast cancer, such as Trop-2 high expression breast cancer.
  • the cancer cells of the invention include, but are not limited to, cancer cells, blastoma cells, sarcoma cells, leukemia cells, lymphoma cells. More specific examples of the cancer cells include: squamous cell carcinoma cells (for example, squamous cell carcinoma cells), lung cancer cells (for example, small cell lung cancer cells, non-small cell lung cancer (NSCLC) cells, lung adenocarcinoma cells, and lung scales.
  • squamous cell carcinoma cells for example, squamous cell carcinoma cells
  • lung cancer cells for example, small cell lung cancer cells, non-small cell lung cancer (NSCLC) cells, lung adenocarcinoma cells, and lung scales.
  • Cell carcinoma cells peritoneal cancer cells, gastric or gastric cancer cells (such as gastrointestinal cancer cells and gastrointestinal stromal tumors (GIST) cells), pancreatic cancer cells, glioblastoma cells, cervical cancer cells, ovarian cancer cells , bladder cancer cells, breast cancer cells, colon cancer cells, rectal cancer cells, colorectal cancer cells, endometrial cancer cells or uterine cancer cells, salivary gland cancer cells, renal cancer cells, prostate cancer cells, vaginal cancer cells, thyroid cancer Cells, liver cancer cells, anal cancer cells, penile cancer cells, and head and neck cancer cells.
  • gastric or gastric cancer cells such as gastrointestinal cancer cells and gastrointestinal stromal tumors (GIST) cells
  • pancreatic cancer cells pancreatic cancer cells
  • glioblastoma cells such as gastrointestinal cancer cells and gastrointestinal stromal tumors (GIST) cells
  • pancreatic cancer cells such as gastrointestinal cancer cells and gastrointestinal stromal tumors (GIST) cells
  • pancreatic cancer cells such as gastrointestinal cancer
  • the cancer cell is a cancer cell with high expression of Trop-2, such as colorectal cancer cells, prostate cancer cells, cervical cancer cells, breast cancer cells, gastric cancer cells, endometrial cancer cells, salivary glands Cancer cells, lung cancer cells, renal cancer cells, colon cancer cells, thyroid cancer cells, pancreatic cancer cells or bladder cancer cells.
  • the cancer cell is selected from the group consisting of a breast cancer cell, a gastric cancer cell, an ovarian cancer cell, a lung cancer cell (eg, a non-small cell lung cancer cell), and a liver cancer cell, particularly a breast cancer cell, such as a Trop-2 high. Expression of breast cancer cells.
  • the present invention provides a conjugate of the formula (I), a pharmaceutically acceptable salt or stereoisomer thereof, or a solvate of the conjugate, salt or stereoisomer. It has excellent stability, uniformity and/or reduced toxic side effects.
  • the cell surface receptor Trop-2 which is highly affinity-expressing in tumor cells, exhibits good biological activity.
  • the conjugate inhibits tumor growth in a mammal and is useful for preventing and/or treating a variety of neoplastic diseases, particularly tumors with high expression of Trop-2, such as breast cancer, gastric cancer, pancreatic cancer, ovarian cancer, colorectal Cancer, prostate cancer, cervical cancer, lung cancer (eg non-small cell lung cancer) and liver cancer.
  • Figure 1 shows the results of SDS-PAGE detection of hRS7 antibody, wherein one lane is hRS7 treated under reducing conditions, and the second lane is hRS7 treated under unreduced conditions.
  • Figure 2 is a HIC-HPLC spectrum of the crude product hRS7 and conjugate I-1.
  • Figure 3 is a HIC-HPLC spectrum of the crude product of conjugate I-2.
  • Figure 4 is a HIC-HPLC spectrum of the crude product of conjugate I-3.
  • Figure 5 shows the results of detection of the affinity of antibody hRS7 and conjugate I-1 to tumor cell MDA-MB-468 by flow cytometry.
  • Figure 6 shows the results of detecting the in vitro inhibitory activity of the antibody hRS7 against the tumor cell MDA-MB-468.
  • Figure 7 shows the results of detection of the in vitro inhibitory activity of conjugate I-1 against tumor cell MDA-MB-468.
  • Figure 8 shows the results of detection of the in vitro inhibitory activity of conjugate I-1 on tumor cell BxPC-3.
  • Figure 9 shows the results of detection of the in vitro inhibitory activity of conjugate I-1 against tumor cell NCI-H23.
  • Figure 10 shows the results of detection of the in vitro inhibitory activity of conjugate I-1 against tumor cell NCI-H322M.
  • Figure 11 shows the results of detection of the in vitro inhibitory activity of conjugate I-2 against tumor cell NCI-87.
  • Figure 12 shows the results of detection of the in vitro inhibitory activity of conjugate I-3 against tumor cell HCC827.
  • Figure 13 shows the tumor suppressive effect of conjugate I-1 on mice subcutaneously transplanted into human gastric cancer cell line NCI-N87.
  • the nucleic acid sequence is designed according to the hRS7 antibody light chain sequence and the heavy chain sequence, and then codon optimization and gene synthesis, respectively, to obtain the nucleotide sequence encoding the light chain as shown in SEQ ID NO: 3 and as set forth in SEQ ID NO:
  • the nucleotide sequence encoding the heavy chain is shown, and then the two sequences are inserted into the same expression vector pTT5, and a large number of transfection-grade plasmids are prepared for transfection of CHO-3E7 cells.
  • Nucleotide sequence encoding the heavy chain (SEQ ID NO: 4)
  • CHO-3E7 cells (CNRC #L-11992 Canadian National Research Society) were subcultured and expanded with a serum-free medium added by L-glutamine, FreeStyleTM CHO (Life Technologies, Carlsbad, CA, USA). In a triangular flask, it was carried out on a shaker in a 37 ° C 5% CO 2 incubator. One day before transfection, the cells were inoculated into another flask for culture. On the day of transfection, the recombinant plasmid DNA containing the hRS7 antibody light and heavy chain genes and the transfection reagent (polyetherimide, calcium chloride or DEAE-dextran) were mixed at a ratio of 1:1 to 1:10.
  • the transfection reagent polyetherimide, calcium chloride or DEAE-dextran
  • a shake flask containing 500 mL of CHO-3E7 cell culture medium was added.
  • the shake flask containing the CHO-3E7 cells transfected with the plasmid was placed in an incubator to continue the culture, and the cell culture supernatant was collected on the 6th day after the transfection for purification.
  • the antibody was purified using a Protein A CIP column 5 ml (GenScript, Cat. No. L00433) affinity column.
  • the purified antibody was analyzed for molecular weight, yield and purity by SDS-PAGE, Western blot and SEC-HPLC.
  • the molecular weight analysis results are shown in the SDS-PAGE diagram of Figure 1.
  • Western blot analysis showed that 10.0 mg of hRS7 antibody was obtained.
  • the purity of the antibody was determined by SEC-HPLC to be 99.2%.
  • the binding activity of the antibody to the human Trop-2 antigen was measured by an indirect ELISA method, and it was confirmed that the obtained hRS7 antibody efficiently binds human Trop-2 antigen in a dose-dependent manner, and the binding EC 50 is about 16 ng/ml.
  • the crude product of conjugate I-1 obtained in step d was subjected to HIC detection.
  • 1 mg/ml carboxypeptidase B (CpB, Sigma) was added, and the crude product had a protein content to CpB ratio of 25:1 (w/w), which was digested overnight at 37 °C.
  • the HIC conditions are as follows:
  • Liquid chromatograph Waters Alliance e2695;
  • Liquid chromatography column TOSOH TSKgel Butyl-NPR, 4.6 x 100 mm;
  • Mobile phase A 1.5 M ammonium sulfate
  • Mobile phase B 25 mM Na 2 HPO 4 , pH 7.0, 25% isopropanol;
  • the HIC test results are shown in Figure 2.
  • the hydrophobicity of the conjugate is enhanced compared to the bare resistance, so the peak time is delayed.
  • the crude product of conjugate I-1 showed peaks of two conjugates in HIC detection, and the peak time was later than the peak time of hRS7 naked resistance, indicating that the coupling was successful.
  • the crude product of the conjugate I-1 obtained in the step d was subjected to HIC purification, subjected to desalting liquid exchange (i.e., replacement of the buffer) and concentration by ultrafiltration to obtain a conjugate I-1.
  • the HIC conditions are as follows:
  • Mobile phase A 1.5 M aqueous ammonium sulfate solution, 25 m M aqueous sodium hydrogen phosphate solution, pH 7.0;
  • Mobile phase B 25 mM aqueous solution of sodium hydrogen phosphate, pH 7.0, 10% isopropanol solution;
  • Elution conditions 0% to 40% mobile phase B eluted 20 CV; 40% to 100% mobile phase B eluted 30 CV;
  • the eluted fraction corresponding to the largest main peak collected was desalted by an illustra NAP-5 column (GE Life Science), affinity chromatography was performed with MabPurix (Sai Subtech, 5 ml), and the layer was equilibrated with a 10 mmol/L sodium phosphate aqueous solution of pH 7.4.
  • the column was applied, the sample was loaded, and the column was equilibrated with 5 column volumes of 10 mmol/L sodium phosphate aqueous solution of pH 7.4, and then the column was eluted with 10 mmol/L sodium phosphate and 1 mmol/L NaCl aqueous solution of pH 6.4, and collected and washed.
  • the mixture was deliquored and concentrated by ultrafiltration to obtain a conjugate I-1.
  • the crude product of the conjugate I-2 obtained in the step c was subjected to HIC detection by the method described in the step e of preparing the conjugate I-1, and the results are shown in Fig. 3. It can be seen from Fig. 3 that the crude product of conjugate I-2 showed peaks of two conjugates in HIC detection, and the peak time was later than the peak time of hRS7 naked resistance, indicating that the coupling was successful.
  • the crude product of the conjugate I-2 obtained in the step c is subjected to HIC purification by the method described in the step f of preparing the conjugate I-1, and the eluted portion of the corresponding maximum main peak collected is desalted and exchanged. Affinity chromatography and concentration by ultrafiltration gave conjugate I-2.
  • 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 crude product of the conjugate I-3 obtained in the step c was subjected to HIC detection by the method described in the step e of preparing the conjugate I-1, and the detection results are shown in Fig. 4.
  • the crude product of conjugate I-3 showed multiple peaks in the HIC detection, including peaks of three conjugates, and the peak time was later than the peak time of hRS7 naked resistance, indicating coupling. success.
  • the crude product of the conjugate I-3 obtained in the step c is subjected to HIC purification by the method described in the step f of preparing the conjugate I-1, and the eluted portion corresponding to the second main peak collected is subjected to desalting Liquid, affinity chromatography and concentration by ultrafiltration gave conjugate I-3.
  • the toxin molecules (D) were all coupled to the lysine of the same peptide of the light chain, and the site-directed coupling was achieved to obtain DAR.
  • Uniform antibody-drug conjugates achieve uniformity, controllability and reproducibility of product quality.
  • Example 3 Detection of binding of antibody hRS7 and conjugate I-1 to tumor cell surface antigen Trop-2
  • Table 1 Tumor cells and media components
  • the above tumor cells were treated by a conventional trypsin digestion method, and the number of cells per collection tube was 3 ⁇ 10 5 .
  • a dilution of the antibody hRS7 prepared in Example 1, the conjugate I-1 prepared in Example 2, and the negative control antibody Human IgG1 (purchased from Biolegend) at a concentration of 50 nM was separately prepared with PBS (containing 1% BSA). Each dilution was incubated with each tumor cell for 15 minutes on ice and then washed twice with 400 ⁇ L of PBS. 2 ⁇ L of FITC anti-human IgG Fc antibody (purchased from Biolegend) was added to each tube, incubated on ice for 15 minutes, and then washed twice with 400 ⁇ L of PBS. After resuspending the cells with 400 ⁇ L of PBS in each tube, the fluorescent signal was detected on a CytoFLEX flow cytometer (Beckman Coulter) using a FITC channel.
  • Table 2 Analysis of the positive rate of binding of antibody hRS7 to each tumor cell surface antigen Trop-2 by flow cytometry
  • NCI-H23 As can be seen from Table 2, except for NCI-H23, the other five tumor cells were highly bound to the antibody hRS7, indicating that NCI-H23 is a Trop-2 expression negative cell, while the other five tumor cells are Trop-2 high expressing cells. .
  • the conjugate I-1 of the present invention does not affect the affinity of the antibody hRS7 for the tumor cell surface antigen Trop-2 due to the coupling of the toxin molecule, and thus has an conjugate similar to the existing conjugate such as IMMU-132.
  • Example 4 Inhibition of in vitro activity of tumor cells by antibody hRS7 and conjugates I-1, I-2 and I-3
  • the culture of tumor cells was carried out as described in the first item of Example 3.
  • the antibody hRS7 prepared in Example 1 and the conjugates I-1, I-2 and I-3 prepared in Example 2 were diluted with a medium as shown in Table 1 but with 10% FBS changed to 2% FBS. 5-fold dilution from a starting gradient of 100 ⁇ g/ml gave 100 ⁇ g/ml, 20 ⁇ g/ml, 4 ⁇ g/ml, 0.8 ⁇ g/ml, 0.16 ⁇ g/ml, 0.03 ⁇ g/ml, 0.006 ⁇ g/ml, 0.0012 ⁇ g/ml and A total of 9 concentration points were 0.000024 ⁇ g/ml, and three wells were repeated for each concentration point.
  • the above tumor cells were treated by conventional trypsin digestion, and the cells were resuspended in a medium as shown in Table 1 but changed to 10% FBS to 2% FBS, adjusted to 5*10 4 cells/ml, and added to 100 ⁇ l.
  • 96-well plates containing antibodies at different concentrations The number of cells and co-culture time are shown in Table 3.
  • 20 ⁇ L of CCK8 reagent was added to each well, and reacted to the reaction reading time shown in Table 3, respectively, and then read at 450 nm by SpectraMax M2 microplate reader (Molecular Devices), by detecting mitochondria. Dehydrogenase activity indicates inhibition of cell proliferation.
  • Figure 6 shows that antibody hRS7 has no growth inhibitory effect on tumor cell MDA-MB-468.
  • Figures 7, 8 and 10 show that conjugate I-1 has significant growth inhibitory effects on tumor cells MDA-MB-468, BxPC-3 and NCI-H322M positive for Trop-2 expression, while Figure 9 shows coupling. Compound I-1 had no growth inhibitory effect on the cell line NCI-H23 negative for Trop-2 expression.
  • Figure 11 shows that conjugate I-2 has a significant growth inhibitory effect on tumor cell NCI-N87 positive for Trop-2 expression.
  • Figure 12 shows that conjugate I-3 has a significant growth inhibitory effect on tumor cell HCC827 positive for Trop-2 expression.
  • the conjugates e.g., I-1, I-2, I-3
  • the conjugates are positive for Trop-2 expression, such as MDA-MB-468.
  • BxPC-3, NCI-H322M, NCI-N87 and HCC827 all showed significant growth inhibition, but no growth inhibition effect on the tumor cell NCI-H23 negative for Trop-2 expression.
  • IMMU-132 the in vitro inhibition of BxPC-3 cells by hRS7-SN-38 has an IC 50 of 4.03.
  • the nm/L was converted based on the molecular weight of IMMU-132 (hRS7-SN-38) of about 160 KD, and the IC 50 was converted to 0.6448 ⁇ g/ml.
  • This ratio of the conjugates of the invention I-1 to IC 50 (0.045 ⁇ g / ml) of a high magnitude.
  • IMMU-132 such as Calu-3, Capan-1, SK-MES-1 IC 50 of greater value to other cells, which means that less inhibitory activity.
  • the tumor cell inhibitory activities of the conjugates I-1, I-2 and I-3 of the present invention are significantly superior to those of the prior art IMMU-132, suggesting that the ADC of the present invention is useful for treating gastric cancer, non-small cell lung cancer, Pancreatic cancer and breast cancer have great potential.
  • 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 conjugate of Example I-1 was injected into the human subcutaneously transplanted human gastric cancer cell line NCI-N87 with a single tail vein injection, and the tumor volume and animal body weight change were measured. To calculate the efficacy (antitumor effect) of antibody-drug conjugates in tumor-bearing mice.
  • An appropriate amount of the antibody-drug conjugate I-1 of the present invention is weighed, prepared into a mother liquor of a certain concentration with sterile ultrapure water, gently shaken, and 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 physiological saline was used as a vehicle control.
  • Tumor-bearing mice self-constructed with a tumor volume of 100-250 mm 3 were selected at doses of 0.5 mg/kg and 2 mg/kg.
  • the route of administration is a single tail vein injection.
  • the tumor growth curve is shown in Figure 13.
  • Figure 13 shows that the antibody-drug conjugate I-1 of the present invention has a significant inhibition of NCI-N87 tumor growth and has a good dose-effect relationship, while the vehicle control group has a rapid growth of NCI-N87 tumor. It is shown that the antibody-drug conjugate of the present invention has similar antitumor effects against various breast cancer cell lines BT474.

Abstract

一种抗体-药物偶联物，其药学上可接受的盐或立体异构体，或者所述偶联物、盐或立体异构体的溶剂合物及其制备方法和在预防或治疗癌症中的应用。所述偶联物具有优异的稳定性、均一性和/或降低的毒副作用。所述偶联物能够高度亲和在肿瘤细胞中高表达的细胞表面受体，可用于预防和/或治疗多种肿瘤疾病。

Description

一种抗体-药物偶联物及其制备方法和应用 技术领域
本发明属于生物医药技术领域。具体而言，本发明涉及一种抗体-药物偶联物及其制备方法和应用。
背景技术
作为新型的靶向治疗药物，抗体-药物偶联物(antibody-drug conjugate，下文中简称为“ADC”或“偶联物”)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，组成抗体-药物偶联物。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 PCTCN2017098140-appb-000001
(T-DM1)和
Figure PCTCN2017098140-appb-000002
(brentuximab vedotin)。
ADC中作为“弹头”的药物通常为细胞毒性药物，即小分子毒素，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中的连接基须满足以下要求：在细胞外具有足够的稳定性，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。通常的ADC药物中，药物/抗体比值(drug antibody ratio，DAR)平均为2-4。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DAR值分布为0-8，平均DAR值为3.5(Lazar等人，2005，Rapid Commun.Mass Spectrom.，19：1806-1814)。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，需要部分还原链间二硫键(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。
Trop-2或TROP2含义为人滋养层细胞表面抗原-2(human trophoblast cell-surface antigens 2)，又称为TACSTD2、M1S1、GA733-1、EGP-1，其是由许多人类肿瘤(如乳腺癌、结直肠癌、肺癌、胰腺癌、卵巢癌、***癌、***)细胞表达的细胞表面受体，但Trop-2在正常人体组织只有有限的表达。Trop-2是一种分子量为45KD的单次跨膜表面糖蛋白，为细胞膜钙离子通道相关蛋白，与细胞内钙离子浓度调控有关。Trop-2与细胞周期蛋白D1和磷酸激酶C有关，具有调节肿瘤细胞生长、促进肿瘤细胞侵袭和转移的功能。尽管Trop-2的配体及信号转导通路的具体细节尚未明确，但认为其致瘤性和促侵袭转移特性与其促使细胞内钙离子浓度升高有关。
IMMU-132(Isactuzumab govitecan)是由靶向Trop-2的人源化抗体Isactuzumab(hRS7)和伊立替康活性代谢物SN-38通过抗体的半胱氨酸巯基偶联形成的ADC。由于SN-38具有毒性大和可溶性差的缺点，其无法直接给药，而IMMU-132则可以提高SN-38 的生物利用度并降低其毒性。
在IMMU-132中，平均每个抗体分子连接约7个SN-38分子，其连接示意图如下所示(参见http：//www.immunomedics.com/linker-demo.shtml)：
Figure PCTCN2017098140-appb-000003
然而，IMMU-132存在如下不足：
1、IMMU-132中所用的连接基稳定性较差，导致毒素分子释放过快，不利于药效的发挥；
2、IMMU-132与hRS7裸抗相比，亲和力有所改变，这种改变的原因是毒素分子偶联导致抗体结构改变，而抗体结构的改变可能造成其亲水区域暴露，对于偶联物的药效造成难以预测的影响；
3、IMMU-132的DAR值很高且不均一，表明其载药量较高，尽管载药量高的ADC可能具有更强的体外活性，但高载药量也会带来诸多问题，如因聚合而导致多聚体成分增加、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。
发明内容
在第一方面，本发明提供通式(I)的偶联物，其药学上可接受的盐或立体异构体，或者所述偶联物、盐或立体异构体的溶剂合物，
Figure PCTCN2017098140-appb-000004
其中：
A为抗Trop-2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1-10的整数。
在第二方面，本发明提供本发明第一方面的偶联物的制备方法，所述方法包括以下步骤：
(1).制备通式(I-A)的化合物：
Figure PCTCN2017098140-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 PCTCN2017098140-appb-000006
其通式中G选自-F、-Cl、-Br、-I、-N3、-OR、-SR、-ONRR’、RC(＝O)O-、-OP(＝O)RR’、RSO2-O-和
Figure PCTCN2017098140-appb-000007
其中R和R’每次出现时各自独立地是任选取代的烷基、任选取代的芳基或任选取代的杂环基；例如，所述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个环成员的杂环基或苯氧基，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个环成员的杂环基、C6-C10芳基或含5-10个环成员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Trop-2抗体或者其活性片段或变体，得到具有不同a值的多种偶联物的混合物；和
(4).任选地，纯化步骤(3)中得到的混合物，得到偶联物；优选的，采用层析法进行纯化；进一步优选地，所述层析法选自离子交换层析、疏水层析、反相层析和亲和层析中的一种或多种。
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偶联物或者其药学上可接受的盐或立体异构体或者所述偶联物、盐或立体异构体的溶剂合物，以及药学上可接受的载体。
在第四方面，本发明提供药物制剂，其包含本发明第一方面的偶联物或者其药学上 可接受的盐或立体异构体或者所述偶联物、盐或立体异构体的溶剂合物。
在第五方面，本发明提供本发明第一方面的偶联物或者其药学上可接受的盐或立体异构体或者所述偶联物、盐或立体异构体的溶剂合物、第三方面所述的药物组合物或第四方面所述的药物制剂在制备用于预防或治疗癌症的药物中的用途。
在第六方面，本发明提供本发明第一方面的偶联物或者其药学上可接受的盐或立体异构体或者所述偶联物、盐或立体异构体的溶剂合物、第三方面所述的药物组合物或第四方面所述的药物制剂，其用于预防或治疗癌症。
在第七方面，本发明提供一种预防或治疗癌症的方法，其包括向有此需要的受试者施用有效量的本发明第一方面所述的偶联物或者其药学上可接受的盐或立体异构体或者所述偶联物、盐或立体异构体的溶剂合物、第三方面所述的药物组合物或第四方面所述的药物制剂。
在第八方面，本发明提供本发明第一方面所述的偶联物或者其药学上可接受的盐或立体异构体或者所述偶联物、盐或立体异构体的溶剂合物、第三方面所述的药物组合物或第四方面所述的药物制剂用于制备试剂的用途，其中所述试剂用于抑制癌细胞的生长、增殖或迁移。
在第九方面，本发明提供本发明第一方面所述的偶联物或者其药学上可接受的盐或立体异构体或者所述偶联物、盐或立体异构体的溶剂合物、第三方面所述的药物组合物或第四方面所述的药物制剂，其用于抑制癌细胞的生长、增殖或迁移。
在第十方面，本发明提供一种抑制癌细胞生长、增殖或迁移的方法，其包括给癌细胞施用有效量的本发明第一方面所述的偶联物或者其药学上可接受的盐或立体异构体或者所述偶联物、盐或立体异构体的溶剂合物、第三方面所述的药物组合物或第四方面所述的药物制剂。
在第十一方面，本发明提供一种抑制癌细胞生长、增殖或迁移的试剂盒，其包括本发明第一方面所述的偶联物或者其药学上可接受的盐或立体异构体或者所述偶联物、盐或立体异构体的溶剂合物、第三方面所述的药物组合物或第四方面所述的药物制剂。
发明详述
在本发明中，除非另有说明，否则本文中所用术语具有本领域技术人员通常理解的含义。并且，本文中所用的细胞培养、分子遗传学、核酸化学、免疫学实验室操作步骤均为相应领域内广泛使用的常规步骤。例如可参见Current Protocols in Molecular Biology(Ausubel)。同时，为了更好地理解本发明，下面提供相关术语的定义和解释。
如本文中所使用的，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双特异性抗体或多特异性抗体，只要它们具有所 需的生物学活性。
如本文中所使用的，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。
如本文中所使用的，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。
如本文中所使用的，术语“双特异性抗体”与“双功能抗体偶联物”可以互换使用，是指由第一抗体(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。
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)。
现有技术中公开了制备超过二价的抗体的技术。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，三特异性抗体的制备可参见例如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。
如本文中所使用的，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。
抗体片段可由多种方法生产，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。在某些优选实施方案中，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)。在某些优选实施方案中，抗体片段是线性抗体片段(参见例如US5,641,870)，其可以是单特异性的或双特异性的。
如本文中所使用的，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。
如本文中所使用的，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。
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。
如本文中所使用的，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。
如本文中所使用的，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。
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)。在某些优选实施方案中，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。
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。在某些优选实施方案中，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)。
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。
本文中所述的数值范围应理解为涵盖其中包含的任何数值和所有子范围。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。
如本文中所使用的，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ADC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。
如本文中所使用的，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。
如本文中所使用的，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/或酶解等而形成的物质。
如本文中所使用的，术语“卤素”是指氟(F)、氯(Cl)、溴(Br)、碘(I)和砹(At)。
如本文中所使用的，术语“C1-C10烷基”表示直链或支链的含有1-10个碳原子的烷基，包括例如“C1-C6烷基”、“C1-C4烷基”、“C1-C3烷基”等，具体实例包括但不限于：甲基、乙基、正丙基、异丙基、正丁基、异丁基、仲丁基、叔丁基、正戊基、异戊基、2-甲基丁基、新戊基、1-乙基丙基、正己基、异己基、3-甲基戊基、2-甲基戊基、1-甲基戊基、3，3-二甲基丁基、2，2-二甲基丁基、1，1-二甲基丁基、1，2-二甲基丁基、1，3-二甲基丁基、2，3- 二甲基丁基、2-乙基丁基、1，2-二甲基丙基、正庚基、正辛基、正壬基和正癸基等。
如本文中所使用的，术语“C3-C8环烷基”是指含有3-8个碳原子的饱和环状烷基，包括例如“C3-C6环烷基”、“C4-C6环烷基”、“C5-C7环烷基”或“C5-C6环烷基”等。具体实例包括但不限于：环丙烷基、环丁烷基、环戊烷基、环己烷基、环庚烷基、环辛烷基等。
如本文中所使用的，术语“C6-C14芳基”是指含有6-14个碳原子的具有芳香性的单环或多环烃基，例如C6-C10芳基等。具体的实例包括但不限于苯基、萘基、蒽基、菲基等。
如本文中所使用的，术语“杂环基”是指含有至少一个杂原子的饱和环状基团，其中所述杂原子选自氮原子、氧原子和硫原子。例如“含5-8个环成员的杂环基”、“含5-7个环成员的杂环基”、“含5-6个环成员的杂环基”等。具体的实例包括但不限于吡咯烷基、四氢呋喃基、哌啶基、哌嗪基、四氢吡喃基、高哌嗪基等。
如本文中所使用的，术语“杂芳基”是指含有至少一个杂原子的具有芳香性的环状基团，其中所述杂原子选自氮原子、氧原子和硫原子。例如“含5-10个环成员的杂芳基”、“含5-9个环成员的杂芳基”、“含5-6个环成员的杂芳基”等。具体的实例包括但不限于呋喃基、噻吩基、吡咯基、噻唑基、异噻唑基、噻二唑基、噁唑基、异噁唑基、噁二唑基、咪唑基、吡唑基、1，2，3-***基、1，2，4-***基、1，2，3-噁二唑基、1，2，4-噁二唑基、1，2，5-噁二唑基、1，3，4-噁二唑基、吡啶基、2-吡啶酮基、4-吡啶酮基、嘧啶基、1，4-二氧杂环己二烯基、2H-1，2-噁嗪基、4H-1，2-噁嗪基、6H-1，2-噁嗪基、4H-1，3-噁嗪基、6H-1，3-噁嗪基、4H-1，4-噁嗪基、哒嗪基、吡嗪基、1，2，3-三嗪基、1，3，5-三嗪基、1，2，4，5-四嗪基、氮杂环庚三烯基、1，3-二氮杂环庚三烯基、氮杂环辛四烯基等。
本文中提及的文献均以其整体援引加入本文中。
在本发明中，发明人通过大量的研究发现，如式(I)所示的偶联物具有优异的稳定性、均一性和/或降低的毒副作用。其能高度亲和在肿瘤细胞中高表达的细胞表面受体Trop-2，展现出良好的生物学活性。例如，所述偶联物可抑制哺乳动物肿瘤生长，用于预防和/或治疗多种肿瘤疾病，尤其是Trop-2高表达的肿瘤，如乳腺癌、胃癌、胰腺癌、卵巢癌、结直肠癌、***癌、***、肺癌(例如非小细胞肺癌)和肝癌。
因此，在第一方面，本发明提供通式(I)的偶联物，其药学上可接受的盐或立体异构体，或者所述偶联物、盐或立体异构体的溶剂合物，
Figure PCTCN2017098140-appb-000008
其中：
A为抗Trop-2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1-10的整数。
在某些优选实施方案中，Y为CR1，X为N。
在某些优选实施方案中，R1各自独立地为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)。
在某些优选实施方案中，a为1、2、3或4。
在某些优选实施方案中，a为2。
抗体
本发明中所用的抗体为抗Trop-2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，Trop-2可以与TACSTD2、M1S1、GA733-1、EGP-1互换使用，均表示天然序列的人Trop-2蛋白(UniProt：P09758，2)及其功能性衍生物，例如氨基酸序列变体。
可用于本发明的抗Trop-2的抗体的实例包括但不限于：记载于例如CN104053672A中的m7E6、h7E6、h7E6_SVG、h7E6_SVG1、h7E6_SVG2、h7E6_SVG3、h7E6_SVG4、h7E6_SVG5、h7E6_SVG6、h7E6_SVG7、h7E6_SVG8、h7E6_SVG9、h7E6_SVG10、h7E6_SVG11、h7E6_SVG12、h7E6_SVG13、h7E6_SVG14、h7E6_SVG15、h7E6_SVG16、h7E6_SVG17、h7E6_SVG18、h7E6_SVG19、h7E6_SVG20、h7E6_SVG21、h7E6_SVG22、h7E6_SVG23、h7E6_SVG24、h7E6_SVG25、h7E6_SVG26、h7E6_SVG27、h7E6_SVG28、h7E6_SVG29、h7E6_SVG30、h7E6_SVG31、h7E6_SVG32、h7E6_SVGL、h7E6_SVGL1、h7E6_SVGL2、h7E6_SVGL3、h7E6_SVGL4、h7E6_SVGL5、h7E6_SVGN、m6G11、h6G11、h6G11_FKG_SF；记载于美国专利第7,420,041号中的AR47A6.4.2；记载于美国专利第5,850,854号中的BR110；记载于美国专利第6,653,104号中的RS7；记载于美国专利第7,517,964号中的RS7；以及记载于US2012/0237518中的hRS7。可用于本发明的抗Trop-2抗体还可以通过CN103476941A中公开的载体设计、构建和构建展示抗体的抗体库的方法筛选获得，也可以索伦托医疗公司(Sorrento Therapeutics，Inc.)的
Figure PCTCN2017098140-appb-000009
文 库进行筛选获得。
本发明中的天然序列的Trop-2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。
本发明中所用的抗体优选为抗人Trop-2抗体。
在某些优选实施方案中，所述抗人Trop-2抗体中的重链和轻链的CDR1、CDR2和/或CDR3分别为RS7单抗重链和轻链的CDR1、CDR2和/或CDR3。
在某些优选实施方案中，所述抗人Trop-2抗体可以为人源化抗体或全人源抗体。
在某些优选实施方案中，所述抗体为CN 100360567C所述RS7抗体，其轻链序列及重链序列分别如SEQ ID NO：1和SEQ ID NO：2所示。
轻链序列(SEQ ID NO：1)：
Figure PCTCN2017098140-appb-000010
重链序列(SEQ ID NO：2)：
Figure PCTCN2017098140-appb-000011
以上序列中的下划线区为CDR区，并且重链序列C末端的K可脱掉。在某些优选实施方案中，重链序列C末端的K可脱掉，这种缺失并不影响生物活性，例如通过删除编码重链序列C末端的K的基因去掉K。
药物
本发明中所用的药物即细胞毒性药物基团。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。
在某些优选实施方案中，所述细胞毒性药物基团是耳抑素肽类，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US6,884,869、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WO2013/173393中。这些文献整体援引加入本文。
在某些优选实施方案中，所述细胞毒性药物基团是美登素类。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，发现某些优选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。
在某些优选实施方案中，所述细胞毒性药物基团包括但不限于以下化合物：
Figure PCTCN2017098140-appb-000012
Figure PCTCN2017098140-appb-000013
Figure PCTCN2017098140-appb-000014
其中R和R’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)-；且Ar是C6-C14芳基(如苯基或萘基)。
在某些优选的实施方案中，所述细胞毒性药物基团来自为通式(D1)或(D2)的化合物或它们的立体异构体：
Figure PCTCN2017098140-appb-000015
其中：
R2选自-CH2N3、
Figure PCTCN2017098140-appb-000016
-CH3和-COOH；
R3选自H和-OH；并且，
R4选自H、-OH、-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；
R5选自H、-OH、-NH2、F、Cl、Br、I、OR7，其中R7是C1-C4烷基；
Figure PCTCN2017098140-appb-000017
其中R8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、R5或R8或者脱除R4、R5或R8中的氢、R6或R7而得到的基团。
在某些优选的实施方案中，所述细胞毒性药物基团选自：
Figure PCTCN2017098140-appb-000018
Figure PCTCN2017098140-appb-000019
连接基
在本发明的偶联物中，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)。
在某些优选实施方案中，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。
在某些优选实施方案中，所述连接基至少包括肽、寡糖、-(CH2)n-、-(CH2CH2O)n-。
在某些优选实施方案中，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-。
在某些优选实施方案中，通式(I)中的连接基L可以选自：
Figure PCTCN2017098140-appb-000020
Figure PCTCN2017098140-appb-000021
Figure PCTCN2017098140-appb-000022
Figure PCTCN2017098140-appb-000023
Figure PCTCN2017098140-appb-000024
Figure PCTCN2017098140-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。
ADC
本发明的ADC由上文中定义的通式(I)表示。
最优选的通式(I)的ADC选自I-1、I-2和I-3：
Figure PCTCN2017098140-appb-000026
Figure PCTCN2017098140-appb-000027
其中A为hRS7单抗。
本发明的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，或者为溶剂合物的形式，并且所述盐或立体异构体也可为溶剂合物的形式。
本发明的偶联物可以选择性地将有效量的细胞毒性药物基团递送到肿瘤组织，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 PCTCN2017098140-appb-000028
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 PCTCN2017098140-appb-000029
其中G选自-F、-Cl、-Br、-I、-N3、-OR、SR、-ONRR’、RC(＝O)O-、-OP(＝O)RR’、RSO2-O-和
Figure PCTCN2017098140-appb-000030
其中R和R’每次出现时各自独立地是任选取代的烷基、任选取代的芳基或任选取代的杂环基，例如，R和R’每次出现时各自独立地是C1-C10烷基、C6-C14芳基或者含5-10个环成员的杂环基或苯氧基，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个环成员的杂环基、C6-C10芳基或含5-10个环成员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Trop-2抗体或者其活性片段或变体，得到具有不同a值的多种偶联物的混合物；和
(4).任选地，纯化步骤(3)中得到的混合物，得到偶联物；优选的，采用层析法进行纯化；进一步优选地，所述层析法选自离子交换层析、疏水层析、反相层析和亲和层析中的一种或多种。
在某些优选实施方案中，步骤(2)中的通式(I-A-G)的化合物中的G为-OR，且R是被1、2、3、4或5个独立地选自卤素的取代基(如氟、氯、溴、碘)取代的C6-C14芳基。
在某些优选实施方案中，步骤(2)中的通式(I-A-G)的化合物为通式(I-B)的化合物，其可以通过例如使通式(I-A)的化合物与五氟苯酚反应而形成：
Figure PCTCN2017098140-appb-000031
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。
在某些优选实施方案中，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偶联物或者其药学上可接受的盐或立体异构体或者所述偶联物、盐或立体异构体的溶剂合物，以及药学上可接受的载体。
在某些优选实施方案中，所述药物组合物进一步包含一种或多种抗癌药，如化疗剂 和/或抗体。在某些优选实施方案中，所述化疗剂选自烷基化试剂(例如环磷酰胺，异环磷酰胺(ifosfamide)等)，代谢拮抗剂(例如甲氨喋呤，5-氟尿嘧啶等)，抗肿瘤抗生素(例如，丝裂霉素，阿霉素等)，植物来源的抗肿瘤药(例如长春新碱，长春地辛，泰素等)，顺铂，卡铂，依托泊苷和伊立替康。在某些优选实施方案中，所述抗体选自曲妥单抗(特别是当治疗乳腺癌时)和SGN-15(特别是当治疗非小细胞肺癌时)。
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。在某些优选实施方案中，药物组合物是各组分的混合物的形式，而在另一些优选实施方案中，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，例如用于口服、经鼻、皮内、皮下、肌内或静脉施用的剂型。在某些优选实施方案中，所述载体可以包括水、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、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，本发明提供药物制剂，其包含本发明第一方面的偶联物或者其药学上可接受的盐、立体异构体或者所述偶联物、盐或立体异构体的溶剂合物。
在某些优选实施方案中，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。
在第五方面，本发明提供本发明第一方面的偶联物或者其药学上可接受的盐或立体异构体，或者所述偶联物、盐或立体异构体的溶剂合物、第三方面所述的药物组合物或第四方面所述的药物制剂在制备用于预防或治疗癌症的药物中的用途。
在第六方面，本发明提供本发明第一方面所述的偶联物或者其药学上可接受的盐或立体异构体或者所述偶联物、盐或立体异构体的溶剂合物、第三方面所述的药物组合物或第四方面所述的药物制剂，其用于预防或治疗癌症。
在第七方面，本发明提供一种预防或治疗癌症的方法，其包括向有此需要的受试者施用有效量的本发明第一方面所述的偶联物或者其药学上可接受的盐或立体异构体或者所述偶联物、盐或立体异构体的溶剂合物、第三方面所述的药物组合物或第四方面所述的药物制剂。
在第八方面，本发明提供本发明第一方面所述的偶联物或者其药学上可接受的盐或立体异构体或者所述偶联物、盐或立体异构体的溶剂合物、第三方面所述的药物组合物或第四方面所述的药物制剂用于制备试剂的用途，其中所述试剂用于抑制癌细胞的生长、增殖或迁移。
在某些优选实施方案中，所述试剂用于体内方法中。
在某些优选实施方案中，所述试剂用于体外方法中。
在第九方面，本发明提供本发明第一方面所述的偶联物或者其药学上可接受的盐或立体异构体或者所述偶联物、盐或立体异构体的溶剂合物、第三方面所述的药物组合物或第四方面所述的药物制剂，其用于抑制癌细胞的生长、增殖或迁移。
在某些优选实施方案中，所述偶联物或者其药学上可接受的盐或立体异构体或者所述偶联物、盐或立体异构体的溶剂合物用于体内方法中。
在某些优选实施方案中，所述的偶联物或者其药学上可接受的盐或立体异构体或者所述偶联物、盐或立体异构体的溶剂合物用于体外方法中。
在第十方面，本发明提供一种抑制癌细胞生长、增殖或迁移的方法，其包括给癌细胞施用有效量的本发明第一方面所述的偶联物或者其药学上可接受的盐或立体异构体或者所述偶联物、盐或立体异构体的溶剂合物、第三方面所述的药物组合物或第四方面所述的药物制剂。
在某些优选实施方案中，所述方法在体内进行。
在某些优选实施方案中，所述方法在体外进行。
在第十一方面，本发明提供一种抑制癌细胞生长、增殖或迁移的试剂盒，其包括本发明第一方面所述的偶联物或者其药学上可接受的盐或立体异构体或者所述偶联物、盐或立体异构体的溶剂合物、第三方面所述的药物组合物或第四方面所述的药物制剂。
在某些优选实施方案中，本发明中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。在某些优选实施方案中，所述癌症为Trop-2高表达的癌症，如结直肠癌、***癌、***、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。在某些优选实施方案中，所述癌症选自乳腺癌、胃癌、卵巢癌、肺癌(例如非小细胞肺癌)和肝癌，特别是乳腺癌，如Trop-2高表达的乳腺癌。
在某些优选实施方案中，本发明中所述癌细胞包括但不限于癌(carcinoma)细胞、母细胞瘤细胞、肉瘤细胞、白血病细胞、淋巴瘤细胞。所述癌细胞更具体的例子包括：鳞状细胞癌细胞(例如鳞状上皮细胞癌细胞)、肺癌细胞(例如小细胞肺癌细胞、非小细胞肺癌(NSCLC)细胞、肺腺癌细胞和肺鳞状细胞癌细胞)、腹膜癌细胞、胃或胃部癌症细胞(例如胃肠癌细胞和胃肠基质肿瘤(GIST)细胞)、胰腺癌细胞、恶性胶质瘤细胞、***细胞、卵巢癌细胞、膀胱癌细胞、乳腺癌细胞、结肠癌细胞、直肠癌细胞、结肠直肠癌细胞、子宫内膜癌细胞或子宫癌细胞、唾液腺癌细胞、肾癌细胞、***癌细胞、***癌细胞、甲状腺癌细胞、肝癌细胞、***癌细胞、***癌细胞以及头颈癌细胞。在某些优选实施方案中，所述癌细胞为Trop-2高表达的癌细胞，如结直肠癌细胞、***癌细胞、***细胞、乳腺癌细胞、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、胰腺癌细胞或膀胱癌细胞。在某些优选实施方案中，所述癌细胞选自乳腺癌细胞、胃癌细胞、卵巢癌细胞、肺癌细胞(例如非小细胞肺癌细胞)和肝癌细胞，特别是乳腺癌细胞，如Trop-2高表达的乳腺癌细胞。
发明的有益效果
本发明提供如式(I)所示的偶联物，其药学上可接受的盐或立体异构体，或者所述偶联物、盐或立体异构体的溶剂合物。其具有优异的稳定性、均一性和/或降低的毒副作用。能高度亲和在肿瘤细胞中高表达的细胞表面受体Trop-2，展现出良好的生物学活性。例如，所述偶联物可抑制哺乳动物肿瘤生长，用于预防和/或治疗多种肿瘤疾病，尤其是Trop-2高表达的肿瘤，如乳腺癌、胃癌、胰腺癌、卵巢癌、结直肠癌、***癌、***、肺癌(例如非小细胞肺癌)和肝癌。
具体实施方式
下面将结合实施例对本发明的实施方案进行详细描述，但是本领域技术人员将会理解，下列实施例仅用于说明本发明，而不应视为限定本发明的范围。实施例中未注 明具体条件者，按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者，均为可以通过市购获得的常规产品。
附图说明
图1是hRS7抗体的SDS-PAGE检测结果，其中1道为经还原条件处理的hRS7，2道为未经还原条件处理的hRS7。
图2是抗体hRS7和偶联物I-1粗产物的HIC-HPLC谱图。
图3是偶联物I-2粗产物的HIC-HPLC谱图。
图4是偶联物I-3粗产物的HIC-HPLC谱图。
图5显示用流式细胞仪检测抗体hRS7和偶联物I-1与肿瘤细胞MDA-MB-468的亲和力的检测结果。
图6显示抗体hRS7对肿瘤细胞MDA-MB-468的体外抑制活性的检测结果。
图7显示偶联物I-1对肿瘤细胞MDA-MB-468的体外抑制活性的检测结果。
图8显示偶联物I-1对肿瘤细胞BxPC-3的体外抑制活性的检测结果。
图9显示偶联物I-1对肿瘤细胞NCI-H23的体外抑制活性的检测结果。
图10显示偶联物I-1对肿瘤细胞NCI-H322M的体外抑制活性的检测结果。
图11显示偶联物I-2对肿瘤细胞NCI-87的体外抑制活性的检测结果。
图12显示偶联物I-3对肿瘤细胞HCC827的体外抑制活性的检测结果。
图13显示偶联物I-1对皮下移植人胃癌细胞株NCI-N87的小鼠肿瘤抑制效果。
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
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
THF 四氢呋喃
EtOAc 乙酸乙酯
FITC 异硫氰酸荧光素
实施例1：抗体的制备和检测
1.基因合成与密码子优化、亚克隆和质粒抽提：
按hRS7抗体轻链序列和重链序列设计核酸序列，然后分别进行密码子优化及基因合成，得到如SEQ ID NO：3所示的编码轻链的核苷酸序列和如SEQ ID NO：4所示的编码重链的核苷酸序列，之后将两个序列***到同一表达载体pTT5内，大量抽取制备转染级别质粒，用于转染CHO-3E7细胞。
编码轻链的核苷酸序列(SEQ ID NO：3)
Figure PCTCN2017098140-appb-000032
编码重链的核苷酸序列(SEQ ID NO：4)
Figure PCTCN2017098140-appb-000033
Figure PCTCN2017098140-appb-000034
2.细胞培养和瞬时转染：
将CHO-3E7细胞(CNRC#L-11992加拿大国立研究会)用添加L-谷氨酰胺的无血清培养基FreeStyleTM CHO(Life Technologies，Carlsbad，CA，USA)进行传代扩大培养，所述扩大培养在三角瓶中，于37℃ 5％CO2培养箱中的摇床上进行。转染前一天，将细胞接种到另一三角瓶内进行培养。转染当天，将含有hRS7抗体轻链及重链基因的重组质粒DNA和转染试剂(聚醚酰亚胺、氯化钙或DEAE-葡聚糖)按照1∶1至1∶10的比例混匀孵育后加入装有500mL的CHO-3E7细胞培养液的摇瓶中。将含转染了质粒的CHO-3E7细胞的摇瓶放在培养箱中继续培养，在转染后第6天收集细胞培养上清液，用于纯化。
3.抗体纯化(表达评估)：
采用Protein A CIP column 5ml(GenScript，Cat.No.L00433)亲和柱对抗体进行纯化。采用SDS-PAGE、蛋白质印迹法和SEC-HPLC对纯化后的抗体进行分子量、产量和纯度分析。分子量分析结果如图1的SDS-PAGE图所示。蛋白质印迹法测定结果表明，获得了hRS7抗体10.0mg。SEC-HPLC法测得抗体纯度为99.2％。
4.抗体结合活性检测：
采用间接ELISA方法测定抗体与人Trop-2抗原的结合活性，确认所得hRS7抗体剂量依赖性地有效结合人Trop-2抗原，结合EC50约为16ng/ml。
实施例2：ADC的制备
1.制备偶联物I-1
Figure PCTCN2017098140-appb-000035
步骤a.中间体D-1的制备
Figure PCTCN2017098140-appb-000036
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 PCTCN2017098140-appb-000037
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]+。
步骤c.中间体I-B-1的制备
Figure PCTCN2017098140-appb-000038
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.偶联物I-1粗产物的合成
Figure PCTCN2017098140-appb-000039
向2.5mL在pH 7.2的PBS缓冲液中配制的浓度为10～20mg/ml的hRS7单抗溶液 中加入4～8倍物质的量的溶解在DMA中的化合物I-B-1。在10～40℃和温和搅拌下避光反应3～6小时，得到偶联物I-1粗产物。
步骤e.偶联物I-1粗产物的HIC检测
对步骤d中得到的偶联物I-1粗产物进行HIC检测。向粗产物中加入1mg/ml羧肽酶B(CpB，Sigma)，使所述粗产物中蛋白含量与CpB比例为25∶1(w/w)，37℃酶解过夜。
HIC条件如下：
液相色谱仪：Waters Alliance e2695；
液相色谱柱：TOSOH TSKgel Butyl-NPR，4.6x100mm；
流动相A：1.5M硫酸铵；
流动相B：25mM Na2HPO4，pH 7.0，25％异丙醇；
流速：0.5ml/min；
检测波长：280nm；
柱温：30℃；
样品室温度：8℃；
洗脱条件：
时间(分钟) 0 3 25 30
流动相A(体积％) 70 70 55 15
流动相B(体积％) 30 30 45 85
HIC检测结果如图2所示。当抗体偶联上小分子毒素后，偶联物的疏水性较裸抗有所增强，因此出峰时间会延后。由图2可见，偶联物I-1粗产物在HIC检测中出现两个偶联物的峰，并且出峰时间均迟于hRS7裸抗的出峰时间，表明偶联成功。
f.偶联物I-1粗产物的纯化
对步骤d中得到的偶联物I-1粗产物进行HIC纯化，经脱盐换液(即更换缓冲液)和超滤浓缩，获得偶联物I-1。
HIC条件如下：
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。
将收集所得对应最大主峰的洗脱部分经illustra NAP-5柱(GE生命科学)脱盐，用MabPurix(赛分科技，5ml)进行亲和层析，用pH 7.4的10mmol/L磷酸钠水溶液平衡层 析柱，上样，再用5个柱体积的pH 7.4的10mmol/L磷酸钠水溶液平衡层析柱，然后用pH 6.4的10mmol/L磷酸钠和1mmol/LNaCl水溶液洗脱层析柱，收集洗脱液，超滤浓缩，获得偶联物I-1。
按照文献Methods Mol.Biol.2013，1045：267-73中描述的方法，用紫外分光光度计测定偶联物I-1和hRS7裸抗在280nm和249nm的吸光度值，并按上述文献中的公式计算得到偶联物I-1的DAR值，为约2(具体为2.01)。
2.制备偶联物I-2
Figure PCTCN2017098140-appb-000040
步骤a.中间体I-A-2的制备
Figure PCTCN2017098140-appb-000041
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(30 mL)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 PCTCN2017098140-appb-000042
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.偶联物I-2粗产物的合成
Figure PCTCN2017098140-appb-000043
向1mL在pH 7.4的PBS缓冲液中配制的浓度为10mg/ml的hRS7单抗溶液中加入6倍物质的量的溶解在DMA中的化合物I-B-2。在室温下，温和搅拌反应4-6小时，得到偶联物I-2粗产物。
步骤d.偶联物I-2粗产物的HIC检测
采用制备偶联物I-1的步骤e中所述的方法对步骤c中得到的偶联物I-2粗产物进行HIC检测，检测结果如图3所示。由图3可见，偶联物I-2粗产物在HIC检测中出现两个偶联物的峰，并且出峰时间均迟于hRS7裸抗的出峰时间，表明偶联成功。
步骤e.偶联物I-2粗产物的纯化
采用制备偶联物I-1的步骤f中所述的方法对步骤c中得到的偶联物I-2粗产物进行HIC纯化，并将收集所得对应最大主峰的洗脱部分经脱盐换液，亲和层析，超滤浓缩，获得偶联物I-2。
按照文献Methods Mol.Biol.2013，1045：267-73中描述的方法，用紫外分光光度计测定偶联物I-2和hRS7裸抗在280nm和249nm的吸光度值，并按上述文献中的公式计算得到偶联物I-2的DAR值，为约2(具体为1.96)。
3.制备偶联物I-3
Figure PCTCN2017098140-appb-000044
步骤a.中间体I-A-3的制备
Figure PCTCN2017098140-appb-000045
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 PCTCN2017098140-appb-000046
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]+。
1H NMR(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.偶联物I-3粗产物的合成
Figure PCTCN2017098140-appb-000047
向1mL在pH 7.8的PBS缓冲液中配制的浓度为10mg/ml的hRS7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，得到偶联物I-3粗产物。
步骤d.偶联物I-3粗产物的HIC检测
采用制备偶联物I-1的步骤e中所述的方法对步骤c中得到的偶联物I-3粗产物进行HIC检测，检测结果如图4所示。由图4可见，偶联物I-3粗产物在HIC检测中出现多个峰，其中包括三个偶联物的峰，并且出峰时间均迟于hRS7裸抗的出峰时间，表明偶联成功。
步骤e.偶联物I-3粗产物的纯化
采用制备偶联物I-1的步骤f中所述的方法对步骤c中得到的偶联物I-3粗产物进行HIC纯化，并将收集所得对应第二个主峰的洗脱部分经脱盐换液，亲和层析，超滤浓缩，获得偶联物I-3。
按照文献Methods Mol.Biol.2013，1045：267-73中描述的方法，用紫外分光光度计测定偶联物I-3和hRS7裸抗在280nm和249nm的吸光度值，并按上述文献中的公式计算得到偶联物I-3的DAR值，为约2(具体为2.05)。
本实施例制备得到的偶联物I-1，I-2，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，获得DAR均一的抗体-药物偶联物，实现了产品质量的均一性、可控性和重现性。
实施例3：抗体hRS7及偶联物I-1与肿瘤细胞表面抗原Trop-2结合的检测
1.肿瘤细胞的培养
本实施例中所用的各肿瘤细胞的详细信息以及对应培养基的成份如下表1所示。
表1：肿瘤细胞及培养基成份
细胞名称 肿瘤类型 培养基成份
MDA-MB-468 乳腺癌 DMEM/F12+10％FBS
BxPC-3 胰腺癌 RPMI1640+10％FBS
NCI-H23 非小细胞肺癌 RPMI1640+10％FBS
NCI-H322M 非小细胞肺癌 RPMI1640+10％FBS
NCI-N87 胃癌 RPMI1640+10％FBS
HCC827 非小细胞肺癌 RPMI1640+10％FBS
2.抗体标记和流式细胞仪检测
采用常规胰酶消化方法处理上述肿瘤细胞，使每个收集管细胞数为3×105。用PBS(含 1％BSA)分别配制浓度为50nM的实施例1中制备的抗体hRS7、实施例2中制备的偶联物I-1及阴性对照抗体Human IgG1(购自Biolegend)的稀释液。将各稀释液在冰上与各肿瘤细胞一起孵育15分钟，然后用400μL PBS清洗两遍。每管中加入2μL FITC抗人IgG Fc抗体(购自Biolegend)，在冰上孵育15分钟，然后用400μL PBS清洗两遍。每管中加入400μL PBS使细胞重悬后，在CytoFLEX流式细胞仪(Beckman Coulter)上用FITC通道检测荧光信号。
3.检测结果
抗体hRS7与各肿瘤细胞表面抗原Trop-2结合的检测结果如下表2所示。
表2：流式细胞仪检测抗体hRS7与各肿瘤细胞表面抗原Trop-2结合的阳性率分析
细胞名称 阳性率％
MDA-MB-468 99.51
BxPC-3 96.39
NCI-H23 0.09
NCI-H322M 99.72
NCI-N87 94.61
HCC827 98.97
由表2可以看出，除NCI-H23外，其它五种肿瘤细胞均与抗体hRS7高结合，表明NCI-H23为Trop-2表达阴性细胞，而其它五种肿瘤细胞为Trop-2高表达细胞。
抗体hRS7、偶联物I-1及阴性对照抗体Human IgG1与MDA-MB-468肿瘤细胞表面抗原Trop-2结合的检测结果如图5所示。由图5可以看出，抗体hRS7和偶联物I-1与MDA-MB-468的亲和力没有明显差异。
上述结果表明，本发明的偶联物I-1并未因毒素分子的偶联而影响抗体hRS7对肿瘤细胞表面抗原Trop-2的亲和力，因而与已有偶联物如IMMU-132相比具有优势。
实施例4：抗体hRS7和偶联物I-1、I-2及I-3抑制肿瘤细胞的体外活性检测
1.肿瘤细胞的培养：
如实施例3第1项中所述进行肿瘤细胞的培养。
2.抑制肿瘤细胞的体外活性检测
用如表1中所示但10％FBS改为2％FBS的培养基稀释实施例1中制备的抗体hRS7 和实施例2中制备的偶联物I-1、I-2及I-3，从100μg/ml起始梯度进行5倍稀释，得到100μg/ml、20μg/ml、4μg/ml、0.8μg/ml、0.16μg/ml、0.03μg/ml、0.006μg/ml、0.0012μg/ml和0.000024μg/ml共9个浓度点，每个浓度点重复三个孔。采用常规胰酶消化方法处理上述肿瘤细胞，用如表1中所示但10％FBS改为2％FBS的培养基使细胞重悬，调整到5＊104细胞/ml，并将其加入100μl含不同浓度抗体的96孔板中。细胞数目和共培养时间如表3所示。向每孔中加入20μL CCK8试剂(东仁化学科技有限公司)，分别反应至表3中所示的反应读数时间，然后经SpectraMax M2酶标仪(Molecular Devices)在450nm读数，通过检测线粒体内的脱氢酶活性而指示对细胞增值的抑制作用。
表3：抗体hRS7和偶联物I-1、I-2及I-3的体外活性检测条件及结果
Figure PCTCN2017098140-appb-000048
“-”表示没有出现生长抑制曲线，无法测定IC50。
实验结果进一步在图6-12中示出。
图6显示抗体hRS7对肿瘤细胞MDA-MB-468无生长抑制作用。
图7、8和10显示偶联物I-1对Trop-2表达阳性的肿瘤细胞MDA-MB-468、BxPC-3和NCI-H322M均有明显的生长抑制作用，而图9则显示偶联物I-1对Trop-2表达阴性的细胞NCI-H23无生长抑制作用。
图11显示偶联物I-2对Trop-2表达阳性的肿瘤细胞NCI-N87有明显的生长抑制作用。
图12显示偶联物I-3对Trop-2表达阳性的肿瘤细胞HCC827有明显生长抑制作用。
上述体外实验结果表明，本发明中抗体hRS7偶联毒素分子后所得的偶联物(例如I-1、I-2、I-3)对Trop-2表达阳性的肿瘤细胞例如MDA-MB-468、BxPC-3、NCI-H322M、NCI-N87和HCC827均有明显的生长抑制作用，而对Trop-2表达阴性的肿瘤细胞NCI-H23无生长抑制作用。
根据文献记载(参见Thomas M.Cardillo等人，Clin.Cancer Res.2011，17：3157-3169)，hRS7-SN-38(IMMU-132)对BxPC-3细胞的体外抑制作用的IC50为4.03nm/L，以约160KD的IMMU-132(hRS7-SN-38)分子量为基础进行换算，该IC50可换算为0.6448μg/ml。这比本发明的偶联物I-1的IC50(0.045μg/ml)高了一个数量级。此外IMMU-132对其他细胞如Calu-3、Capan-1、SK-MES-1的IC50值更高，这意味着其更低的抑制活性。因此，本发明的偶联物I-1、I-2及I-3的肿瘤细胞抑制活性明显优于现有技术中的IMMU-132，提示本发明的ADC对于治疗胃癌、非小细胞肺癌、胰腺癌及乳腺癌有很大的潜力。
实施例5：偶联物的体内活性测试
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I-1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)。
称取适量本发明的抗体-药物偶联物I-1，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，并采用生理盐水作为溶媒对照。
选择肿瘤体积在100～250mm3的荷瘤鼠(自行构建)，给药剂量为0.5mg/kg和2mg/kg。给药途径为单次尾静脉注射。给药后观察5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。肿瘤生长曲线如图13。
图13表明本发明的抗体-药物偶联物I-1对NCI-N87肿瘤生长具有显著的抑制，并具有良好的量效关系，而溶媒对照组则NCI-N87肿瘤生长迅速。表明本发明的抗体-药物偶联物对多种乳腺癌细胞系BT474亦具有类似的抑瘤疗效。
尽管本发明的具体实施方式已经得到详细的描述，根据已经公开的所有教导，本领域技术人员可以对本发明技术方案的细节进行各种修改和替换，这些改变均在本发明的保护范围之内。本发明的全部范围由所附权利要求及其任何等同物给出。

Claims (15)

  1. 通式(I)的偶联物，其药学上可接受的盐或立体异构体，或者所述偶联物、盐或立体异构体的溶剂合物，
    Figure PCTCN2017098140-appb-100001
    其中：
    A为抗Trop-2抗体或者其活性片段或变体；
    其中X为N，Y为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1-10的整数。
  2. 如权利要求1所述的偶联物，其药学上可接受的盐或立体异构体，或者所述偶联物、盐或立体异构体的溶剂合物，其中所述抗Trop-2抗体为抗人Trop-2抗体；优选地，所述抗人Trop-2抗体的重链和轻链的CDR1、CDR2和/或CDR3分别为RS7单抗重链和轻链的CDR1、CDR2和/或CDR3。
  3. 如权利要求1或2所述的偶联物，其药学上可接受的盐或立体异构体，或者所述偶联物、盐或立体异构体的溶剂合物，其中所述抗Trop-2抗体为人源化抗体或全人源抗体；优选地，所述抗Trop-2抗体为轻链序列和重链序列分别为SEQ ID NO：1和SEQ ID NO：2的hRS7单抗。
  4. 如权利要求1-3中任一项所述的偶联物，其药学上可接受的盐或立体异构体，或者所述偶联物、盐或立体异构体的溶剂合物，其中R1为H。
  5. 如权利要求1-4中任一项所述的偶联物，其药学上可接受的盐或立体异构体，或者所述偶联物、盐或立体异构体的溶剂合物，其中a为1、2、3或4；优选地，a为2。
  6. 如权利要求1-5中任一项所述的偶联物，其药学上可接受的盐或立体异构体，或 者所述偶联物、盐或立体异构体的溶剂合物，其中L选自：
    Figure PCTCN2017098140-appb-100002
    Figure PCTCN2017098140-appb-100003
    Figure PCTCN2017098140-appb-100004
    Figure PCTCN2017098140-appb-100005
    Figure PCTCN2017098140-appb-100006
    Figure PCTCN2017098140-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。
  7. 如权利要求1-6中任一项所述的偶联物，其药学上可接受的盐或立体异构体，或者所述偶联物、盐或立体异构体的溶剂合物，其中所述细胞毒性药物基团为通式(D1)或(D2)的化合物或它们的立体异构体：
    Figure PCTCN2017098140-appb-100008
    其中：
    R2选自-CH2N3、
    Figure PCTCN2017098140-appb-100009
    -CH3和-COOH；
    R3选自H和-OH；并且，
    R4选自H、-OH、-NH2、Cl、Br、I、-OS(O)2R6，其中R6选自H、C1-C8烷基、C3-C8环烷基或C6-C14芳基，其中所述烷基、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(例如F)的取代基取代；
    R5选自H、-OH、-NH2、F、Cl、Br、I、OR7，其中R7是C1-C4烷基；
    Figure PCTCN2017098140-appb-100010
    其中：
    R8选自-CH(CH3)N(CH3)C(O)CH2CH2SH和-CH(CH3)N(CH3)C(O)CH2C(CH3)2SH。
  8. 如权利要求1-7任一项所述的偶联物，其药学上可接受的盐或立体异构体，或者所述偶联物、盐或立体异构体的溶剂合物，其中所述细胞毒性药物基团选自：
    Figure PCTCN2017098140-appb-100011
  9. 如权利要求7或8所述的偶联物，其药学上可接受的盐或立体异构体，或者所述偶联物、盐或立体异构体的溶剂合物，其中所述偶联物选自I-1、I-2和I-3：
    Figure PCTCN2017098140-appb-100012
    其中A为hRS7单抗。
  10. 权利要求1-9中任一项的偶联物的制备方法，所述方法包括以下步骤：
    (1).制备通式(I-A)的化合物：
    Figure PCTCN2017098140-appb-100013
    其中D、L、X和Y如权利要求1-10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 PCTCN2017098140-appb-100014
    其通式中G选自-F、-Cl、-Br、-I、-N3、-OR、-SR、-ONRR’、RC(O)O-、-OP(O)RR’、RSO2-O-和
    Figure PCTCN2017098140-appb-100015
    其中R和R’每次出现时各自独立地为任选取代的烷基、任选取代的芳基或任选取代的杂环基，例如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为-OR，且R是被1、2、3、4或5个独立地选自卤素的取代基(如氟、氯、溴、碘)取代的C6-C14芳基；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Trop-2抗体或者其活性片段或变体，得到具有不同a值的多种偶联物的混合物；和
    (4).任选地，纯化步骤(3)中得到的混合物，得到偶联物。
  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 PCTCN2017098140-appb-100016
    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中任一项所述的偶联物或者其药学上可接受的 盐或立体异构体或者所述偶联物、盐或立体异构体的溶剂合物，以及药学上可接受的载体。
  13. 药物制剂，其包含权利要求1-9中任一项的偶联物或者其药学上可接受的盐、立体异构体或者所述偶联物、盐或立体异构体的溶剂合物；优选地，其为固体制剂、半固体制剂、液体制剂或气体制剂的形式，如冻干粉针剂。
  14. 权利要求1-9中任一项所述的偶联物或者其药学上可接受的盐或立体异构体或者所述偶联物、盐或立体异构体的溶剂合物、权利要求12的药物组合物或权利要求13的药物制剂在制备用于预防或治疗癌症的药物中的用途。
  15. 如权利要求14所述的用途，其中所述癌症选自癌(carcinoma)、母细胞瘤、肉瘤、白血病、淋巴瘤及其他恶性淋巴疾病；优选地，所述癌症选自鳞状细胞癌(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
PCT/CN2017/098140 2016-08-23 2017-08-18 一种抗体-药物偶联物及其制备方法和应用 WO2018036438A1 (zh)

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