WO2022126593A1 - 一种靶向trop2的抗体药物偶联物、其制备方法及应用 - Google Patents

一种靶向trop2的抗体药物偶联物、其制备方法及应用 Download PDF

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WO2022126593A1
WO2022126593A1 PCT/CN2020/137596 CN2020137596W WO2022126593A1 WO 2022126593 A1 WO2022126593 A1 WO 2022126593A1 CN 2020137596 W CN2020137596 W CN 2020137596W WO 2022126593 A1 WO2022126593 A1 WO 2022126593A1
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alkyl
antibody
residue
trop2
independently
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PCT/CN2020/137596
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English (en)
French (fr)
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郭青松
沈毅珺
杨彤
鲍彬
高贝
吴芳
徐珺
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上海复旦张江生物医药股份有限公司
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Priority to JP2023537436A priority Critical patent/JP2023550532A/ja
Priority to CA3202303A priority patent/CA3202303A1/en
Priority to EP20965609.9A priority patent/EP4265275A4/en
Priority to AU2020482223A priority patent/AU2020482223A1/en
Priority to PCT/CN2020/137596 priority patent/WO2022126593A1/zh
Priority to US18/256,045 priority patent/US20240050584A1/en
Priority to KR1020237024071A priority patent/KR20230121842A/ko
Publication of WO2022126593A1 publication Critical patent/WO2022126593A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6843—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a material from animals or humans
    • 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
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/65—Peptidic linkers, binders or spacers, e.g. peptidic enzyme-labile linkers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A61K47/68037—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a camptothecin [CPT] or derivatives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6851—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a determinant of a tumour cell
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6889—Conjugates wherein the antibody being the modifying agent and wherein the linker, binder or spacer confers particular properties to the conjugates, e.g. peptidic enzyme-labile linkers or acid-labile linkers, providing for an acid-labile immuno conjugate wherein the drug may be released from its antibody conjugated part in an acidic, e.g. tumoural or environment
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/94—Stability, e.g. half-life, pH, temperature or enzyme-resistance

Definitions

  • the invention relates to the field of biomedicine, in particular to an antibody-drug conjugate targeting TROP2, a preparation method and application thereof.
  • Trop2 Tumor-associated calcium signaling protein 2
  • TACSTD2 Tumor-associated calcium signaling protein 2
  • Trop2 expression can activate ERK.
  • Trop2 is mainly expressed in trophoblast cells and play an important role in the development of embryonic organs. It is overexpressed in many human epithelial-derived tumor tissues, including breast cancer, lung cancer, gastric cancer, colorectal cancer, pancreatic cancer, prostate cancer, cervical cancer, ovarian cancer, etc. The expression level is correlated with tumor progression and prognosis. Genomic analysis of breast cancer patients and the results of current clinical studies suggest that Trop2 is a clinically validated therapeutic target.
  • Trop2 has attracted widespread attention due to its high expression on the surface of various tumor cells.
  • the level of Trop2 on the surface of single cells is tens of thousands to hundreds of thousands.
  • Epidemiology has proved that Trop2 is related to the development and prognosis of various tumors, and has become the target of antibody drug development.
  • Trop2 After the antibody binds to the Trop2 protein, its endocytosis efficiency is very high, which is very suitable for the development of antibody conjugated drugs.
  • Antibody-drug conjugate is a new generation of antibody-targeted therapeutic drugs, mainly used in the treatment of cancer tumors.
  • ADC drug is composed of three parts: small molecule cytotoxic drug (Drug), antibody (Antibody) and linker (Linker) connecting antibody and cytotoxic drug.
  • Small molecule cytotoxic drug is bound to antibody protein by chemical coupling method.
  • ADC drugs use antibodies to specifically recognize and guide small-molecule drugs to target cancer cells expressing cancer-specific antigens, and enter cancer cells through endocytosis.
  • the linker part is cleaved under the action of low intracellular pH value or lysosomal protease to release small molecule cytotoxic drugs, thereby achieving the effect of specifically killing cancer cells without damaging normal tissue cells.
  • ADC drugs combine the targeting specificity of antibodies and the high toxicity of small molecule toxins to cancer cells at the same time, which greatly expands the effective therapeutic dose window (Therapeutic window).
  • Clinical studies have proved that ADC drugs have high efficacy and are relatively stable in blood, and can effectively reduce the toxicity of small molecule cytotoxic drugs (chemical drugs) themselves to the circulatory system and healthy tissues. hot spot.
  • the technical problem to be solved by the present invention is to overcome the defect of a single type of the existing antibody-drug conjugates, and provide an antibody-drug conjugate targeting TROP2, its preparation method and application.
  • the antibody-drug conjugate developed in the present invention has good targeting, has good inhibitory effect on tumor cells that positively express TROP2, and has good druggability and high safety.
  • the antibody drug conjugate has the inhibitory effect of TROP2, and also has a good inhibitory effect on at least one of NCI-N87, MDA-MB-468 and BXPC3 cells.
  • the present invention solves the above technical problems through the following technical solutions.
  • the present invention provides an antibody drug conjugate as shown in formula I, a pharmaceutically acceptable salt thereof, a solvate thereof or a solvate of a pharmaceutically acceptable salt thereof;
  • Ab is a TROP2 antibody or a variant of TROP2 antibody
  • amino acid sequence of the light chain in the described TROP2 antibody is shown in SEQ ID NO: 1 in the sequence listing, and the amino acid sequence of the heavy chain is shown in the SEQ ID NO: 2 in the sequence listing;
  • the variant of the TROP2 antibody has at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% homology compared with the TROP2 antibody;
  • n 2 to 8;
  • D is a cytotoxic drug topoisomerase inhibitor
  • R 1 is C 1 -C 6 alkyl substituted with one or more -NR 1-1 R 1-2 , C 1 -C 6 alkane substituted with one or more R 1-3 S(O) 2 - base, C 1 -C 6 alkyl, C 3 -C 10 cycloalkyl, C 6 -C 14 aryl or 5-14-membered heteroaryl; the heteroatom in the 5-14-membered heteroaryl is selected from One or more of N, O and S, the number of heteroatoms is 1, 2, 3 or 4; the R 1-1 , R 1-2 and R 1-3 are independently C 1 ⁇ C 6 alkyl;
  • L1 is independently a phenylalanine residue, alanine residue, glycine residue, glutamic acid residue, aspartic acid residue, cysteine residue, glutamic acid residue, histidine acid residues, isoleucine residues, leucine residues, lysine residues, methionine residues, proline residues, serine residues, threonine residues, tryptophan residues one or more of tyrosine residues, tyrosine residues and valine residues; p is 2-4;
  • n is independently 1 to 12, the c-terminal is connected to L 1 through a carbonyl group, and the f-terminal is connected to the d-terminal of the L 3 ;
  • L3 is Wherein the b end is connected with the Ab, and the d end is connected with the f end of the L 2 .
  • the b-terminal of L3 is preferably connected with the thiol group on the antibody in the form of thioether.
  • the linking form with the cysteine residue in the antibody is
  • the cytotoxic drug topoisomerase inhibitor is preferably R 2 and R 5 are each independently H, C 1 -C 6 alkyl or halogen; R 3 and R 6 are each independently H, C 1 -C 6 alkyl or halogen; R 4 and R 7 are each independently For C 1 -C 6 alkyl.
  • the C 1 -C 6 alkyl group is preferably a C 1 -C 4 alkyl group , more preferably methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl or tert-butyl, and most preferably methyl.
  • said halogen is preferably fluorine, chlorine, bromine or iodine, more preferably fluorine.
  • said C 1 -C 6 alkyl groups are preferably C 1 -C 4 alkyl groups , more preferably methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl or tert-butyl, and most preferably methyl.
  • said halogen is preferably fluorine, chlorine, bromine or iodine, more preferably fluorine.
  • the C 1 -C 6 alkyl group is preferably a C 1 -C 4 alkyl group , more preferably methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl or tert-butyl, most preferably ethyl.
  • R 2 and R 5 are each independently C 1 -C 6 alkyl.
  • R 3 and R 6 are each independently halogen.
  • R 4 and R 7 are ethyl.
  • D is
  • the antibody-drug conjugate when the D is When , the antibody-drug conjugate can be
  • the R 1 is a C 1 -C 6 alkyl group substituted by one or more -NR 1-1 R 1-2
  • the C 1 -C 6 alkane The group is preferably methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl or tert-butyl, most preferably ethyl.
  • the R 1 is a C 1 -C 6 alkyl group substituted by multiple -NR 1-1 R 1-2 , the multiple is two or three .
  • the C 1 -C 6 alkyl group is preferably a methyl group, Ethyl, n-propyl, isopropyl, n-butyl, isobutyl or tert-butyl, most preferably methyl.
  • the -NR 1-1 R 1-2 is preferably -N(CH 3 ) 2 .
  • the C 1 -C 6 alkyl group is preferably a C 1 -C 4 alkyl group, more preferably methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl or tert-butyl, and most preferably ethyl.
  • the R 1 is a C 1 -C 6 alkyl group substituted by multiple R 1-3 S(O) 2 -, the multiple is two or three indivual.
  • the C 1 -C 6 alkyl group is preferably methyl, ethyl, n-propyl, isopropyl propyl, n-butyl, isobutyl or tert-butyl, most preferably methyl.
  • R 1 is a C 1 -C 6 alkyl substituted by an R 1-3 S(O) 2 -
  • the R 1-3 S( O) 2 -substituted C 1 -C 6 alkyl is
  • the C 1 -C 6 alkyl group is preferably methyl, ethyl, n-propyl, isopropyl , n-butyl, isobutyl or tert-butyl, most preferably methyl.
  • the m is an integer (such as 2, 3, 4, 5, 6, 7 or 8) or a non-integer, preferably 3-5, more preferably 3.5-4.5, still further Preferably 3.8-4.2, eg 3.88, 3.90, 3.96, 3.97, 3.98, 4.00, 4.03, 4.05, 4.10, 4.12 or 4.13.
  • the L 1 is preferably a phenylalanine residue, alanine residue, glycine residue, isoleucine residue, leucine residue, proline residue one or more of valine residues, more preferably one or more of phenylalanine residues, alanine residues, glycine residues and valine residues, more preferably For valine residues and/or alanine residues, the multiple is preferably two or three, and the p is preferably 2.
  • the (L 1 ) p is further preferably The g-terminal is connected to the c-terminal of L 2 through a carbonyl group.
  • the n is preferably 8 to 12, such as 8, 9, 10, 11 and 12, for example, 8 or 12.
  • said C 1 -C 6 alkyl is preferably The C 1 -C 4 alkyl group is more preferably methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl or tert-butyl, and most preferably methyl.
  • the R 1 is preferably a C 1 -C 6 alkyl substituted by one or more -NR 1-1 R 1-2 , a C 1-3 S alkyl group substituted by one or more R 1-1 R 1-2 (O) 2 -substituted C 1 -C 6 alkyl, C 1 -C 6 alkyl or C 3 -C 10 cycloalkyl, more preferably substituted by one or more -NR 1-1 R 1-2 C 1 -C 6 alkyl group, C 1 -C 6 alkyl group substituted by one or more R 1-3 S(O) 2 -, or C 1 -C 6 alkyl group , more preferably one or more Multiple -NR 1-1 R 1-2 substituted C 1 -C 6 alkyl, or C 1 -C 6 alkyl substituted by one or more R 1-3 S(O) 2 -, most preferably is C 1 -C 6 alkyl substituted with one or more R 1-3 S(
  • the TROP2 antibody or TROP2 antibody variant is a TROP2 antibody residue (a group formed by replacing a hydrogen on a sulfhydryl group in a TROP2 antibody) group) or a residue of a variant of TROP2 antibody (a group formed by substituting a hydrogen on one sulfhydryl group in a variant of TROP2 antibody).
  • the compound shown in the formula I is any one of the following schemes:
  • Ab is a TROP2 antibody or a variant of a TROP2 antibody
  • D is R 2 and R 5 are each independently H, C 1 -C 6 alkyl or halogen, R 3 and R 6 are each independently H, C 1 -C 6 alkyl or halogen, R 4 and R 7 are each independently is C 1 -C 6 alkyl, D is preferably
  • L1 is independently one of a phenylalanine residue, an alanine residue, a glycine residue, an isoleucine residue, a leucine residue, a proline residue and a valine residue or more;
  • Ab is a TROP2 antibody or a variant of a TROP2 antibody
  • D is Said R 2 and R 5 are independently C 1 -C 6 alkyl; said R 3 and R 6 are each independently halogen, D is preferably
  • R 1 is C 1 -C 6 alkyl substituted with one or more -NR 1-1 R 1-2 , C 1 -C 6 alkane substituted with one or more R 1-3 S(O) 2 - group, or, C 1 -C 6 alkyl;
  • L is independently one or more of a phenylalanine residue, an alanine residue, a glycine residue, and a valine residue;
  • D is Said R 2 and R 5 are independently C 1 -C 6 alkyl; said R 3 and R 6 are each independently halogen, D is preferably
  • R 1 is C 1 -C 6 alkyl substituted with one or more -NR 1-1 R 1-2 , C 1 -C 6 alkane substituted with one or more R 1-3 S(O) 2 - group, or, C 1 -C 6 alkyl;
  • L1 is independently a valine residue and/or an alanine residue
  • R 1 is C 1 -C 6 alkyl substituted with one or more -NR 1-1 R 1-2 , C 1 -C 6 alkane substituted with one or more R 1-3 S(O) 2 - group, or, C 1 -C 6 alkyl;
  • L1 is independently a valine residue and/or an alanine residue
  • R 1 is C 1 -C 6 alkyl substituted with one or more -NR 1-1 R 1-2 , C 1 -C 6 alkane substituted with one or more R 1-3 S(O) 2 - group, or, C 1 -C 6 alkyl;
  • L1 is independently a valine residue and/or an alanine residue.
  • the antibody drug conjugate is preferably
  • the L 2 is preferably
  • the Ab is a TROP2 antibody or a variant of the TROP2 antibody, and the amino acid sequence of the light chain of the TROP2 antibody is preferably as shown in SEQ ID NO: 1 in the sequence table; the The amino acid sequence of its heavy chain of the TROP2 antibody is shown in SEQ ID NO: 2 in the sequence table; D is
  • L 1 is a valine residue and/or an alanine residue, p is 2, and (L 1 )p is preferably R 1 is C 1 -C 6 alkyl substituted with one or more -NR 1-1 R 1-2 , C 1 -C 6 alkane substituted with one or more R 1-3 S(O) 2 - or C 1 -C 6 alkyl, preferably C 1 -C 6 alkyl substituted by one or more -NR 1-1 R 1-2 , or, by one or more R 1-3 S(O ) 2 -substituted C 1 -C 6 alkyl, more preferably C 1 -C 6 alkyl substituted by one or more R 1-3 S(O) 2 -; the R 1-1 and R 1-2 and R 1-3 are independently C 1 -C 4 alkyl, preferably methyl; the C 1 -C 6 alkyl substituted by one or more -NR 1-1 R 1-2 preferably The C 1 -C 6 alkyl substituted by one or
  • the antibody drug conjugate is preferably any of the following compounds:
  • Ab is TROP2 antibody or a variant of TROP2 antibody
  • m is 3.8-4.2, preferably 3.88, 3.90, 3.96, 3.97, 3.98, 4.00, 4.03, 4.05, 4.10, 4.12 or 4.13; the variant of TROP2 antibody
  • the light chain in the body is preferably the amino acid sequence shown in SEQ ID NO: 1 in the sequence listing, and the heavy chain sequence is preferably shown in the SEQ ID NO: 2 in the sequence listing.
  • the antibody drug conjugate is preferably any of the following compounds:
  • Ab is a TROP2 antibody or a variant of a TROP2 antibody, preferably a TROP2 antibody;
  • the light chain in the variant of the TROP2 antibody is preferably the amino acid sequence shown in SEQ ID NO: 1 in the Sequence Listing, and the heavy chain sequence is preferably as shown in the Sequence Listing SEQ ID NO: 2.
  • the antibody drug conjugate is preferably any of the following compounds:
  • Ab is a TROP2 antibody or a variant of a TROP2 antibody; m is preferably 3.90, 4.00 or 4.10; the light chain in the variant of the TROP2 antibody is preferably the amino acid sequence shown in SEQ ID NO: 1 in the sequence table, and the heavy chain The sequence is preferably as shown in SEQ ID NO: 2 of the Sequence Listing.
  • the antibody drug conjugate is preferably any of the following compounds:
  • Ab is a TROP2 antibody or a variant of TROP2 antibody.
  • the antibody drug conjugate is preferably any of the following compounds:
  • Ab is TROP2 antibody, and m is preferably 3.90;
  • Ab is TROP2 antibody, and m is preferably 4.10;
  • Ab is TROP2 antibody, and m is preferably 4.00;
  • Ab is TROP2 antibody, and m is preferably 4.12;
  • Ab is TROP2 antibody, and m is preferably 4.03;
  • Ab is TROP2 antibody, and m is preferably 3.98;
  • Ab is TROP2 antibody, and m is preferably 3.96;
  • Ab is TROP2 antibody, and m is preferably 4.13;
  • Ab is TROP2 antibody, and m is preferably 3.88;
  • Ab is TROP2 antibody, and m is preferably 4.05;
  • Ab is a TROP2 antibody, and m is preferably 4.00.
  • the present invention also provides a method for preparing an antibody-drug conjugate, which comprises the following steps: performing the following coupling reaction with the compound shown in formula II and Ab-hydrogen;
  • L 1 , L 2 , L 3 , R 1 , p and Ab are as described above.
  • the conditions and operations of the coupling reaction can be the conventional conditions and operations of the coupling reaction in the art.
  • the present invention also provides a pharmaceutical composition
  • a pharmaceutical composition comprising substance X and pharmaceutical excipients, wherein the substance X is the above-mentioned antibody-drug conjugate, its pharmaceutically acceptable salt, its solvate or its pharmaceutically acceptable Solvates of the accepted salts.
  • the amount of the aforementioned substance X may be a therapeutically effective amount.
  • the present invention also provides an application of the above-mentioned substance X or the above-mentioned pharmaceutical composition in the preparation of a TROP2 protein inhibitor.
  • the present invention also provides an application of the above-mentioned substance X or the above-mentioned pharmaceutical composition in the preparation of a medicament for treating and/or preventing a tumor, and the tumor is preferably a TROP2 positive tumor.
  • the TROP2-positive tumor is preferably one or more of TROP2-positive gastric cancer, triple-negative breast cancer and human pancreatic cancer.
  • the gastric cancer cells are NCI-N87 cells;
  • the triple-negative breast cancer cells are MDA-MB-468 cells;
  • the pancreatic cancer cells are BXPC3 cells.
  • the pharmaceutical excipients can be widely used in the field of pharmaceutical production. Excipients are mainly used to provide a safe, stable and functional pharmaceutical composition, and can also provide a method to enable the subject to dissolve the active ingredient at a desired rate after administration, or to promote the activity of the subject after the composition is administered. The ingredients are effectively absorbed.
  • the pharmaceutical excipients can be inert fillers, or provide some function, such as stabilizing the overall pH of the composition or preventing degradation of the active ingredients of the composition.
  • the pharmaceutical adjuvants may include one or more of the following adjuvants: buffers, chelating agents, preservatives, cosolvents, stabilizers, excipients and surfactants, colorants, flavors, and sweeteners .
  • the native or native sequence TROP2 can be isolated from nature, or can be prepared by recombinant DNA technology, chemical synthesis, or a combination of the above and similar techniques.
  • Antibodies are here interpreted in the broadest sense, and which bind specifically to targets, such as carbohydrates, polynucleotides, lipids, polypeptides, etc., through at least one antigenic recognition region located in the variable region of the immunoglobulin molecule. Specifically, intact monoclonal antibodies, polyclonal antibodies, bispecific antibodies and antibody fragments are included, so long as they have the desired biological activity. Variants of the antibodies of the present invention refer to amino acid sequence mutants, as well as covalent derivatives of the native polypeptide, provided that biological activity equivalent to the native polypeptide is retained.
  • Amino acid sequence mutants generally differ from the native amino acid sequence by the substitution of one or more amino acids in the native amino acid sequence or the deletion and/or insertion of one or more amino acids in the polypeptide sequence.
  • Deletion mutants include fragments of the native polypeptide and N-terminal and/or C-terminal truncation mutants.
  • amino acid sequence mutants typically have at least 70% (eg, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99%) homology compared to the native sequence.
  • Antibodies of the invention can be prepared using techniques well known in the art, such as hybridoma methods, recombinant DNA techniques, phage display techniques, synthetic techniques, or a combination of these techniques, or other techniques known in the art.
  • DAR drug-antibody ratio
  • m is actually an average between 2 to 8, 3 to 7, or 3 to 5, or m is some integer of 2, 3, 4, 5, 6, 7, or 8; in In some embodiments, m is an average of 3.5-4.5 (eg, 3.88, 3.90, 3.96, 3.97, 3.98, 4.00, 4.03, 4.05, 4.10, 4.12, or 4.13); Average of 4, 5, 6, 7 or 8.
  • a linker refers to a direct or indirect link between an antibody and a drug. Attaching the linker to the mAb can be accomplished via a number of means, such as via surface lysines, reductive coupling to oxidized carbohydrates, and via reduction of cysteine residues released by interchain disulfide bonds.
  • a variety of ADC linkage systems are known in the art, including hydrazone, disulfide, and peptide-based linkages.
  • salts refers to salts of compounds of the present invention prepared with relatively non-toxic, pharmaceutically acceptable acids or bases.
  • base additions can be obtained by contacting neutral forms of such compounds with a sufficient amount of a pharmaceutically acceptable base in neat solution or in a suitable inert solvent.
  • Pharmaceutically acceptable base addition salts include, but are not limited to, lithium, sodium, potassium, calcium, aluminum, magnesium, zinc, bismuth, ammonium, diethanolamine.
  • acids additions can be obtained by contacting the neutral form of such compounds with a sufficient amount of a pharmaceutically acceptable acid in neat solution or in a suitable inert solvent.
  • a salt is not limited to, lithium, sodium, potassium, calcium, aluminum, magnesium, zinc, bismuth, ammonium, diethanolamine.
  • the pharmaceutically acceptable acids include inorganic acids, including but not limited to: hydrochloric acid, hydrobromic acid, hydroiodic acid, nitric acid, carbonic acid, phosphoric acid, phosphorous acid, sulfuric acid, and the like.
  • Described pharmaceutically acceptable acid includes organic acid, described organic acid includes but is not limited to: acetic acid, propionic acid, oxalic acid, isobutyric acid, maleic acid, malonic acid, benzoic acid, succinic acid, suberic acid , fumaric acid, lactic acid, mandelic acid, phthalic acid, benzenesulfonic acid, p-toluenesulfonic acid, citric acid, salicylic acid, tartaric acid, methanesulfonic acid, isonicotinic acid, acid citric acid, oleic acid , tannic acid, pantothenic acid, hydrogen tartrate, ascorbic acid, gentisic acid, fumaric acid, gluc
  • solvate refers to a substance formed by combining a compound of the present invention with a stoichiometric or non-stoichiometric amount of a solvent.
  • Solvent molecules in solvates can exist in ordered or non-ordered arrangements.
  • the solvent includes, but is not limited to, water, methanol, ethanol, and the like.
  • treatment refers to a procedure or process used to reduce or eliminate the number of cancer cells in a patient or to alleviate the symptoms of cancer.
  • Treatment does not necessarily mean that the cancer cells or other disorder will actually be eliminated, the number of cells or disorders will actually be reduced or the symptoms of the cancer or other disorder will actually be alleviated.
  • methods of treating cancer are pursued even with only a low probability of success, but are still considered to induce an overall beneficial course of action, given the patient's medical history and estimated survival expectations.
  • prevention refers to a reduced risk of acquiring or developing a disease or disorder.
  • cycloalkyl refers to a saturated cyclic hydrocarbon radical (eg, C3 - C6 cycloalkyl) having three to twenty carbon atoms, including monocyclic cycloalkyls and polycyclic cycloalkyls.
  • Cycloalkyl groups contain 3 to 20 carbon atoms, preferably 3 to 10 carbon atoms, more preferably 3 to 6 carbon atoms. Examples of cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclononyl, and cyclodecyl.
  • alkyl refers to a straight or branched chain alkyl group having the indicated number of carbon atoms.
  • alkyl groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, isobutyl, sec-butyl, n-pentyl, isopentyl, n-hexyl, n-heptyl, n-Octyl and its analogous alkyl groups.
  • halogen refers to fluorine, chlorine, bromine or iodine.
  • heteroaryl refers to an aryl group (or aromatic ring) containing 1, 2, 3, or 4 heteroatoms independently selected from N, O, and S, which may be a monocyclic, bicyclic, or tricyclic aromatic system , such as furanyl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, thienyl, isoxazolyl, oxazolyl, diazolyl, imidazolyl, pyrrolyl, pyrazolyl, triazolyl, tetrazole base, thiazolyl, isothiazolyl, thiadiazolyl, benzimidazolyl, indolyl, indazolyl, benzothiazolyl, benzisothiazolyl, benzoxazolyl, benzisoxazolyl, quinoline olinyl, isoquinolinyl, etc.
  • aryl refers to any stable monocyclic or bicyclic carbocyclic ring wherein all rings are aromatic. Examples of the above-mentioned aryl unit include phenyl or naphthyl.
  • the room temperature in the present invention refers to 20-30°C.
  • the reagents and raw materials used in the present invention are all commercially available.
  • the antibody-drug conjugate containing TROP2 antibody provided by the present invention has good targeting and has a good inhibitory effect on various tumor cells expressing TROP2 and the like.
  • the antibody-drug conjugate of the present invention has good solubility and good druggability, and no abnormal phenomena such as precipitation occur during the coupling preparation process, which is very beneficial to the preparation of the antibody-drug conjugate.
  • Figure 1 shows the construction of the light and heavy chain expression vectors of FDA018 antibody, wherein Ab-L is the light chain of the antibody, and Ab-H is the heavy chain of the antibody.
  • a monoclonal antibody FDA018 with high affinity and specific targeting of TROP2 is selected, the amino acid sequence of its light chain is shown in SEQ ID NO: 1, and the amino acid sequence of its heavy chain is shown in SEQ ID NO: 2.
  • the light and heavy chain nucleotide sequences of FDA018 were obtained by whole gene synthesis (Suzhou Jinweizhi). It was separately constructed into pV81 vector by double digestion with EcoR I and Hind III (purchased from TAKARA) (as shown in Figure 1), and transformed into Trans 1-T1 competent cells by ligation (purchased from Beijing Quanshijin Bio, Cat.
  • amino acid sequence of the FDA018 light chain is shown below:
  • the amino acid sequence of FDA018 heavy chain is shown below:
  • the intermediate 17 (120 mg, 0.15 mmol) obtained by the synthetic method in the previous step was mixed with the commercially available raw material MC-V (102 mg, 0.33 mmol) in 40 mL of dichloromethane, and the condensing agent 2-ethoxy-1-ethyl was added.
  • Oxycarbonyl-1,2-dihydroquinoline (82 mg, 0.33 mmol) was reacted at room temperature overnight. After the reaction was completed, the solvent was evaporated to dryness under reduced pressure.
  • Intermediate VI can use Fmoc-L-valine-L-alanine as a starting material, with reference to steps a and b in the synthetic method of intermediate 3 in Example 2-1, wherein formazan in step b is used.
  • the amine hydrochloride was prepared by substituting the corresponding commercially available amino compound.
  • Subsequent steps start from intermediate VI, obtain intermediate IX similar to intermediate 9 according to the same method as steps c, d, f and h in Example 2-1, and then follow steps i and j
  • the same procedure was followed to remove the amino protecting group and then condensed with different commercially available maleimides to obtain the final product.
  • the amino compounds and maleimide structures used are shown in Table 1.
  • Exatecan mesylate 0.568 g, 1 mmol
  • 2-(tert-butyldimethylsiloxy)acetic acid CAS: 105459-05-0, 0.38 g, 2 mmol
  • condensing agent HATU 0.76 g, 2 mmol
  • 1 mL of pyridine 1 mL
  • Intermediate V can be prepared by referring to the preparation method of compound 4 in Example 2-1, wherein methylamine hydrochloride in step b is replaced with a corresponding commercially available amino compound.
  • Intermediate V is reacted with DXD-1, followed by 10% trifluoroacetic acid/dichloromethane solution treatment to obtain intermediate X
  • intermediate X refers to the subsequent steps e, g, i and j of compound 5 in Example 2-1
  • Intermediate X obtains amino compound through reduction, the obtained amino compound is condensed with Fmoc-valine hydroxysuccinimide ester, then removes the Fmoc protecting group of the amino group in the obtained product, and the obtained amino product is then combined with 6-(Ma.
  • the reaction of succinimidyl (leimido)hexanoate to give the final product.
  • Compound LE21 yellow solid, ESI-MS m/z: 1141.2 (M+H); compound LE22: yellow solid, ESI-MS m/z: 1106.6 (M+H).
  • the TROP2 antibody FDA018 prepared according to the method in Example 1 was replaced with 50 mM PB/1.0 mM EDTA buffer (pH 7.0) using a G25 desalting column, 5 equivalents of TECP were added, and stirred at 25 ° C for 1 hour to make the antibody.
  • the interchain disulfide bonds were partially opened, and then the pH of the reduced antibody solution was adjusted to 5.0 using citric acid, and the sample was replaced with 20 mM citrate buffer, 1 mM EDTA buffer (pH 5.0) using a G25 desalting column, The water bath temperature was maintained at 25°C for the coupling reaction.
  • the linker-drug conjugates LE12-LE22, LS13 and GGFG-Dxd prepared according to the method of Example 2 above were dissolved in DMSO, respectively, and 4.5 equivalents of the linker-drug conjugate were added dropwise to the reduced antibody.
  • DMSO was added to the final concentration of 5% (v/v), and the reaction was stirred at 25° C. for 0.5 hours. After the reaction was completed, the sample was filtered through a 0.22um membrane. Use a tangential flow ultrafiltration system to purify and remove uncoupled small molecules.
  • the buffer is 25mM His, 6% sucrose solution (pH 6.0), and after purification, it is stored in a -20°C refrigerator.
  • the absorbance values were measured at 280 nm and 370 nm by UV method, and the DAR values were calculated. The results are shown in Table 2 below.
  • the coupling reaction was carried out according to the same operation steps as above in this example, and the samples were prepared according to the highest DAR (ie, excess coupling), and the formation of precipitation when each coupling reaction occurred was observed, and the polymer after each coupling reaction was calculated.
  • the ratio and recovery rate were also shown in Table 2.
  • NCI-N87 (ATCC) cells were selected as the cell line for in vitro activity detection in the experiment, and 2000 cells per well were inoculated in a 96-well cell culture plate and cultured for 20-24 hours.
  • the antibody drug conjugate prepared according to the method of Example 3 was formulated into 11 concentrations of 1000, 166.7, 55.6, 18.6, 6.17, 2.06, 0.69, 0.23, 0.08, 0.008 and 0 nM in L15 cell culture medium containing 10% FBS.
  • This example evaluates the stability of the antibody-drug conjugate prepared according to the method of Example 3 in human plasma. Specifically, in this example, the antibody-drug conjugate of Example 3 was added to human plasma, placed in a 37°C water bath for 1, 3, 7, 14, 21, and 28 days, and the internal standard (ixitecan as the Internal standard substance) and extracted and then detected the release amount of free drug by high performance liquid chromatography, the results are shown in Table 4.
  • the internal standard ixitecan as the Internal standard substance
  • the plasma stability results showed that the ADCs obtained by the new technology were not less stable than FDA018-GGFG-DXD, and some were better. At the same time, the above activity test results also proved that some of the newly obtained ADCs had better activities than FDA018-GGFG-DXD.
  • the linker-drug conjugates (LE14 and GGFG-Dxd) were co-incubated with cathepsin B in three buffers of different pH (5.0, 6.0, 7.0), and samples were taken at different time points into the high performance liquid chromatography-mass spectrometer.
  • the external standard method (with DXD as the external standard) was used to determine the percentage of drug release.
  • the experimental results (as shown in Table 5) show that GGFG-Dxd has a slow enzyme cleavage speed in the used pH range, while the LE14 of the present invention can be rapidly digested in the range of pH 5.0 to pH 7.0.
  • the NCI-N87 cell line was selected as the experimental cell line. After the samples were incubated in cathepsin B system (100 mM sodium acetate-acetate buffer, 4 mM dithiothreitol, pH 5.0) at 37°C for 4 hours, the obtained samples were diluted with culture medium To different concentrations, 8 concentrations (1.5-10 times dilution) were set according to the SN-38 concentration of 70nM-0.003nM, and the changes in the killing (inhibitory) ability of the cell line were observed for 144 hours. Luminescent Cell Viability Assay chemiluminescent staining, and IC50 values were calculated after reading the fluorescence data.
  • the digested samples obtained by incubating in the cathepsin B system at 37°C for 4 hours were subjected to precipitation with an appropriate amount of ethanol to remove proteins, and the released small molecular compounds were detected by high performance liquid chromatography, and the same amount of SN-38 was used as a reference.
  • the release rate was 4 hours, and the results showed that the release rate reached 99%.
  • mice 6-8 week old female Balb/c nude mice were subcutaneously injected with 5 ⁇ 10 6 human gastric cancer cells (NCI-N87) dissolved in 100ul PBS solution on the right back of the neck, and when the tumor grew to an average volume of 150-200 mm 3 , randomly divided into 5 groups according to tumor size and weight of mice, each group of 6 animals, respectively blank control group, and antibody drug conjugate FDA018-GGFG-DXD and FDA018-LE14 two dose groups respectively, specifically, Group 01 was the blank control group; Group 02 was the FDA018-GGFG-DXD group at 4.0 mg/kg; Group 03 was the FDA018-GGFG-DXD group at 2.0 mg/kg; Group 04 was the FDA018-LE14 group at 4.0 mg/kg; The 05 group is the FDA018-LE14 group at 2.0 mg/kg; intraperitoneal administration, once a week.
  • NCI-N87 human gastric cancer cells
  • the body weight and tumor volume of the experimental animals were measured twice a week and the survival status of the animals was observed during the experiment.
  • the results are shown in Table 7.
  • the average tumor volume of the mice in the blank control group was 1388.47 mm 3 after the end of administration.
  • the average tumor volume of the FDA018-GGFG-DXD treatment group with the test drug 2.0 mg/kg was 1235.21 mm 3 on the 14th day after the end of administration, and the FDA018-GGFG-DXD treatment group with 4.0 mg/kg was on the 14th day after the end of the administration.
  • the mean tumor volume was 721.09 mm 3 .
  • the average tumor volume of the FDA018-LE14 treatment group with the test drug 2.0 mg/kg on the 14th day after the end of administration was 1342.31 mm 3
  • the average tumor volume of the FDA018-LE14 treatment group of 4.0 mg/kg on the 14th day after the end of the administration was 435.36mm 3 .
  • the experimental results show that FDA018-LE14 has good anti-tumor activity in vivo, and all experimental mice have no death or weight loss, indicating that FDA018-LE14 has good safety.

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Abstract

一种靶向TROP2的抗体药物偶联物、制备方法及应用。一种如式I所示的抗体药物偶联物，该类化合物具有很好的靶向性，对阳性表达TROP2的肿瘤细胞具有很好的抑制效果，并且，具有很好的成药性，安全性高。该抗体药物偶联物具有TROP2的抑制效果，还对NCI-N87、MDA-MB-468和BXPC3细胞中的至少一种有良好的抑制效果。

Description

一种靶向TROP2的抗体药物偶联物、其制备方法及应用 技术领域
本发明涉及生物医药领域，尤其涉及一种靶向TROP2的抗体药物偶联物、其制备方法及应用。
背景技术
肿瘤相关钙信号传导蛋白2(Trop2，TROP2)是TACSTD2基因编码的一型跨膜糖蛋白，是钙信号传导子与细胞内钙信号调节相关，Trop2表达可以活化ERK，正常情况下Trop2主要表达于滋养层细胞，并在胚胎器官发育过程中起重要作用。在许多人上皮来源肿瘤组织过表达，包括乳腺癌、肺癌、胃癌、结直肠癌、胰腺癌、前列腺癌，子宫颈癌，卵巢癌等，表达水平与肿瘤的恶化及预后相关。乳腺癌患者基因组分析及目前的临床研究结果表明，Trop2是一个经临床确证了的治疗靶点。Trop2因在多种肿瘤细胞表面高表达得到广泛关注，单细胞表面Trop2水平几万至几十万，流行病学证明Trop2与多种肿瘤的发展和预后相关，成为抗体药物研发的靶点，Trop2抗体结合Trop2蛋白后其内吞效率很高，非常适合作为抗体偶联药物的开发。
抗体-小分子药物偶联物(Antibody-drug conjugate，ADC)是新一代抗体靶向治疗药物，主要应用于癌症肿瘤的治疗。ADC药物由小分子细胞毒性药物(Drug)、抗体(Antibody)以及连接抗体和细胞毒性药物的连接子(Linker)三部分组成，小分子细胞毒性药物通过化学偶联的方法结合到抗体蛋白上。ADC药物利用抗体特异地识别并引导小分子药物到达表达癌症特异性抗原的癌细胞靶点，并通过细胞内吞效应进入癌细胞。接头部分在胞内低pH值环境或溶酶体蛋白酶的作用下断裂，释放出小分子细胞毒性药物，从而达到特异杀死癌细胞而不损伤正常组织细胞的作用。因此，ADC药物同时结合了抗体的靶向专一性和小分子毒素对癌细胞的高毒性的特点，大大扩展了药物的有效治疗剂量窗口(Therapeutic window)。临床研究已证明，ADC药物的药效高、在血液中相对稳定，能有效地降低小分子细胞毒性药物(化药)本身对循环系统以及健康组织的毒性，是目前 国际上抗癌药物的研发热点。
发明内容
本发明要解决的技术问题是克服现有的抗体药物偶联物种类单一的缺陷，而提供了一种靶向TROP2的抗体药物偶联物、其制备方法及应用。
本发明开发了的抗体药物偶联物，具有很好的靶向性，对阳性表达TROP2的肿瘤细胞具有很好的抑制效果，并且，具有很好的成药性，安全性高。该抗体药物偶联物具有TROP2的抑制效果，还对NCI-N87、MDA-MB-468和BXPC3细胞中的至少一种有良好的抑制效果。
本发明通过以下技术方案解决上述技术问题。
本发明提供了一种如式I所示的抗体药物偶联物、其药学上可接受的盐、其溶剂合物或其药学上可接受的盐的溶剂合物；
Figure PCTCN2020137596-appb-000001
其中，Ab为TROP2抗体或TROP2抗体的变体；
所述的TROP2抗体中轻链的氨基酸序列如序列表SEQ ID NO:1所示，重链的氨基酸序列如序列表SEQ ID NO:2所示；
所述的TROP2抗体的变体，与TROP2抗体相比，至少具有70％、75％、80％、85％、90％、95％、98％或99％以上的同源性；
m为2～8；
D为细胞毒性药物拓扑异构酶抑制剂；
R 1为被一个或多个-NR 1-1R 1-2取代的C 1～C 6烷基、被一个或多个R 1-3S(O) 2-取代的C 1～C 6烷基、C 1～C 6烷基、C 3～C 10环烷基、C 6～C 14芳基或5～14元杂芳基；所述的5～14元杂芳基中的杂原子选自N、O和S中的一种或多种，杂原子个数为1、2、3或4；所述的R 1-1、R 1-2和R 1-3分别独立地为C 1～C 6烷基；
L 1独立地为苯丙氨酸残基、丙氨酸残基、甘氨酸残基、谷氨酸残基、天冬氨酸残基、半胱氨酸残基、谷氨酸残基、组氨酸残基、异亮氨酸残基、亮氨酸残基、赖氨酸残基、甲硫氨酸残基、脯氨酸残基、丝氨酸残基、苏氨酸残基、色氨酸残基、酪氨酸残基和缬氨酸 残基中的一种或多种；p为2～4；
L 2为
Figure PCTCN2020137596-appb-000002
Figure PCTCN2020137596-appb-000003
其中n独立地为1～12，c端通过羰基与L 1相连，f端与所述的L 3的d端相连；
L 3为
Figure PCTCN2020137596-appb-000004
其中b端和所述的Ab相连，d端与所述的L 2的f端相连。
在本发明一优选实施方案中，上述的抗体偶联药物里，某些基团具有如下定义，未提及的基团的定义如上任一方案所述(本段内容以下简称为“在本发明一优选实施方案中”)：
所述的L 3的b端优选为与所述的抗体上的巯基以硫醚的形式相连。以
Figure PCTCN2020137596-appb-000005
为例，
Figure PCTCN2020137596-appb-000006
与所述的抗体中的半胱氨酸残基的连接形式为
Figure PCTCN2020137596-appb-000007
在本发明一优选实施方案中，当D为细胞毒性药物拓扑异构酶抑制剂时，所述的细胞毒性药物拓扑异构酶抑制剂优选
Figure PCTCN2020137596-appb-000008
R 2和R 5分别独立地为H、C 1-C 6烷基或卤素；R 3和R 6分别独立地为H、C 1-C 6烷基或卤素；R 4和R 7分别独立地为C 1-C 6烷基。
在本发明一优选实施方案中，当所述的R 2和R 5分别独立地为C 1-C 6烷基时，所述的 C 1～C 6烷基优选为C 1～C 4烷基，进一步优选为甲基、乙基、正丙基、异丙基、正丁基、异丁基或叔丁基，最优选为甲基。
在本发明一优选实施方案中，当所述的R 2和R 5分别独立地为卤素时，所述的卤素优选为氟、氯、溴或碘，进一步优选为氟。
在本发明一优选实施方案中，当所述的R 3和R 6分别独立地为C 1-C 6烷基时，所述的C 1～C 6烷基优选为C 1～C 4烷基，进一步优选为甲基、乙基、正丙基、异丙基、正丁基、异丁基或叔丁基，最优选为甲基。
在本发明一优选实施方案中，当所述的R 3和R 6分别独立地为卤素时，所述的卤素优选为氟、氯、溴或碘，进一步优选为氟。
在本发明一优选实施方案中，当所述的R 4和R 7分别独立地为C 1-C 6烷基时，所述的C 1～C 6烷基优选为C 1～C 4烷基，进一步优选为甲基、乙基、正丙基、异丙基、正丁基、异丁基或叔丁基，最优选为乙基。
在本发明一优选实施方案中，R 2和R 5分别独立地为C 1-C 6烷基。
在本发明一优选实施方案中，R 3和R 6分别独立地为卤素。
在本发明一优选实施方案中，R 4和R 7为乙基。
在本发明一优选实施方案中，D为
Figure PCTCN2020137596-appb-000009
在本发明一优选实施方案中，当所述的D为
Figure PCTCN2020137596-appb-000010
时，所述的抗体药物偶联物可为
Figure PCTCN2020137596-appb-000011
在本发明一优选实施方案中，当所述的R 1为被一个或多个-NR 1-1R 1-2取代的C 1～C 6烷基时，所述的C 1～C 6烷基优选为甲基、乙基、正丙基、异丙基、正丁基、异丁基或叔丁基，最优选为乙基。
在本发明一优选实施方案中，当所述的R 1为被多个-NR 1-1R 1-2取代的C 1～C 6烷基时，所述的多个为两个或三个。
在本发明一优选实施方案中，当所述的R 1-1和R 1-2各自独立地为C 1～C 6烷基时，所述的C 1～C 6烷基优选为甲基、乙基、正丙基、异丙基、正丁基、异丁基或叔丁基，最优选为甲基。
在本发明一优选实施方案中，当所述的R 1为被一个或多个-NR 1-1R 1-2取代的C 1～C 6烷基时，所述的-NR 1-1R 1-2优选为-N(CH 3) 2。
在本发明一优选实施方案中，当所述的R 1为被一个-NR 1-1R 1-2取代的C 1～C 6烷基时，所述的被一个-NR 1-1R 1-2取代的C 1～C 6烷基为
Figure PCTCN2020137596-appb-000012
在本发明一优选实施方案中，当所述的R 1为被一个或多个R 1-3S(O) 2-取代的C 1～C 6烷基时，所述的C 1～C 6烷基优选为C 1～C 4烷基，进一步优选为甲基、乙基、正丙基、异丙基、正丁基、异丁基或叔丁基，最优选为乙基。
在本发明一优选实施方案中，当所述的R 1为被多个R 1-3S(O) 2-取代的C 1～C 6烷基时，所述的多个为两个或三个。
在本发明一优选实施方案中，当所述的R 1-3为C 1～C 6烷基时，所述的C 1～C 6烷基优 选为甲基、乙基、正丙基、异丙基、正丁基、异丁基或叔丁基，最优选为甲基。
在本发明一优选实施方案中，当所述的R 1为被一个R 1-3S(O) 2-取代的C 1～C 6烷基时，所述的被一个R 1-3S(O) 2-取代的C 1～C 6烷基为
Figure PCTCN2020137596-appb-000013
在本发明一优选实施方案中，当所述的R 1为C 1～C 6烷基时，所述的C 1～C 6烷基优选为甲基、乙基、正丙基、异丙基、正丁基、异丁基或叔丁基，最优选为甲基。
在本发明一优选实施方案中，所述的m为整数(例如2、3、4、5、6、7或8)或非整数，优选为3-5，进一步优选为3.5-4.5，再进一步优选地为3.8-4.2，例如3.88、3.90、3.96、3.97、3.98、4.00、4.03、4.05、4.10、4.12或4.13。
在本发明一优选实施方案中，所述的L 1优选为苯丙氨酸残基、丙氨酸残基、甘氨酸残基、异亮氨酸残基、亮氨酸残基、脯氨酸残基和缬氨酸残基中的一种或多种，更优选为苯丙氨酸残基、丙氨酸残基、甘氨酸残基和缬氨酸残基中的一种或多种，进一步优选为缬氨酸残基和/或丙氨酸残基，所述的多种优选为两种或三种，所述的p优选为2。
在本发明一优选实施方案中，所述的(L 1) p进一步优选为
Figure PCTCN2020137596-appb-000014
其中g端通过羰基和所述的L 2的c端相连。
在本发明一优选实施方案中，所述的n优选为8～12，例如8、9、10、11和12，再例如8或12。
在本发明一优选实施方案中，当所述的R 1-1、R 1-2和R 1-3独立地为C 1～C 6烷基时，所述的C 1～C 6烷基优选C 1～C 4烷基，进一步优选为甲基、乙基、正丙基、异丙基、正丁基、异丁基或叔丁基，最优选为甲基。
在本发明一优选实施方案中，所述的R 1优选为被一个或多个-NR 1-1R 1-2取代的C 1～C 6烷基、被一个或多个R 1-3S(O) 2-取代的C 1～C 6烷基、C 1～C 6烷基或C 3～C 10环烷基，更优选为被一个或多个-NR 1-1R 1-2取代的C 1～C 6烷基、被一个或多个R 1-3S(O) 2-取代的C 1～C 6烷基、或、C 1～C 6烷基，进一步优选为被一个或多个-NR 1-1R 1-2取代的C 1～C 6烷基、或、被一个或多个R 1-3S(O) 2-取代的C 1～C 6烷基，最优选为被一个或多个R 1-3S(O) 2-取代的C 1～C 6烷基。
在本发明中，当Ab为TROP2抗体或TROP2抗体的变体时，所述的TROP2抗体或TROP2抗体的变体为TROP2抗体的残基(TROP2抗体中的一个巯基上的氢被取代形成的基团)或TROP2抗体的变体的残基(TROP2抗体的变体中的一个巯基上的氢被取代形成的基团)。
在本发明一优选实施方案中，所述的如式I所示的化合物为如下任一方案：
方案一：
Ab为TROP2抗体或TROP2抗体的变体；
D为
Figure PCTCN2020137596-appb-000015
R 2和R 5分别独立地为H、C 1-C 6烷基或卤素，R 3和R 6分别独立地为H、C 1-C 6烷基或卤素，R 4和R 7分别独立地为C 1-C 6烷基，D优选
Figure PCTCN2020137596-appb-000016
L 1独立地为苯丙氨酸残基、丙氨酸残基、甘氨酸残基、异亮氨酸残基、亮氨酸残基、脯氨酸残基和缬氨酸残基中的一种或多种；
方案二：
Ab为TROP2抗体或TROP2抗体的变体；
D为
Figure PCTCN2020137596-appb-000017
所述的R 2和R 5分别独立地为C 1-C 6烷基；所述的R 3和R 6分别独立地为卤素，D优选
Figure PCTCN2020137596-appb-000018
Figure PCTCN2020137596-appb-000019
R 1为被一个或多个-NR 1-1R 1-2取代的C 1～C 6烷基、被一个或多个R 1-3S(O) 2-取代的C 1～C 6烷基、或、C 1～C 6烷基；
L 1独立地为苯丙氨酸残基、丙氨酸残基、甘氨酸残基和缬氨酸残基中的一种或多种；
方案三：
Ab为TROP2抗体；
D为
Figure PCTCN2020137596-appb-000020
所述的R 2和R 5分别独立地为C 1-C 6烷基；所述的R 3和R 6分别独立地为卤素，D优选
Figure PCTCN2020137596-appb-000021
Figure PCTCN2020137596-appb-000022
m为3.5-4.5，
R 1为被一个或多个-NR 1-1R 1-2取代的C 1～C 6烷基、被一个或多个R 1-3S(O) 2-取代的C 1～C 6烷基、或、C 1～C 6烷基；
L 1独立地为缬氨酸残基和/或丙氨酸残基；
方案四：
Ab为TROP2抗体；
D为
Figure PCTCN2020137596-appb-000023
R 1为被一个或多个-NR 1-1R 1-2取代的C 1～C 6烷基、被一个或多个R 1-3S(O) 2-取代的C 1～C 6烷基、或、C 1～C 6烷基；
L 1独立地为缬氨酸残基和/或丙氨酸残基；
方案五：
Ab为TROP2抗体；
D为
Figure PCTCN2020137596-appb-000024
m为3.5-4.5，
R 1为被一个或多个-NR 1-1R 1-2取代的C 1～C 6烷基、被一个或多个R 1-3S(O) 2-取代的C 1～C 6烷基、或、C 1～C 6烷基；
L 1独立地为缬氨酸残基和/或丙氨酸残基。
在本发明一优选实施方案中，所述的抗体药物偶联物优选为
Figure PCTCN2020137596-appb-000025
Figure PCTCN2020137596-appb-000026
在本发明一优选实施方案中，所述的L 2优选为
Figure PCTCN2020137596-appb-000027
在本发明一优选实施方案中，所述的Ab为TROP2抗体或TROP2抗体的变体，所述的TROP2抗体中其轻链的氨基酸序列优选如序列表SEQ ID NO:1所示；所述的TROP2抗体其重链的氨基酸序列如序列表SEQ ID NO:2所示；D为
Figure PCTCN2020137596-appb-000028
L 1为缬氨酸残基和/或丙氨酸残基，p为2，(L 1)p优选为
Figure PCTCN2020137596-appb-000029
R 1为被一个或多个-NR 1-1R 1-2取代的C 1～C 6烷基、被一个或多个R 1-3S(O) 2-取代的C 1～C 6烷基或C 1～C 6烷基，优选为被一个或多个-NR 1-1R 1-2取代的C 1～C 6烷基、或、被一个或多个R 1-3S(O) 2-取代的C 1～C 6烷基，进一步优选为被一个或多个R 1-3S(O) 2-取代的C 1～C 6烷基；所述的R 1-1、R 1-2和R 1-3独立地为C 1～C 4烷基，优选为甲基；所述的被一个或多个-NR 1-1R 1-2取代的C 1～C 6烷基优选为
Figure PCTCN2020137596-appb-000030
所述的被一个或多个R 1- 3S(O) 2-取代的C 1～C 6烷基优选为
Figure PCTCN2020137596-appb-000031
L 2为
Figure PCTCN2020137596-appb-000032
L 3为
Figure PCTCN2020137596-appb-000033
在本发明一优选实施方案中，所述的抗体药物偶联物优选为如下所示的任一化合物：
Figure PCTCN2020137596-appb-000034
Figure PCTCN2020137596-appb-000035
Figure PCTCN2020137596-appb-000036
其中，Ab为TROP2抗体或TROP2抗体的变体，m为3.8-4.2，优选为3.88、3.90、3.96、3.97、3.98、4.00、4.03、4.05、4.10、4.12或4.13；所述的TROP2抗体的变体中轻链优选为如序列表SEQ ID NO:1所示氨基酸序列，重链序列优选如序列表SEQ ID NO：2所示。
在本发明一优选实施方案中，所述的抗体药物偶联物优选为如下所示的任一化合物：
Figure PCTCN2020137596-appb-000037
Figure PCTCN2020137596-appb-000038
Figure PCTCN2020137596-appb-000039
其中，Ab为TROP2抗体或TROP2抗体的变体，优选TROP2抗体；所述的TROP2抗体的变体中轻链优选为如序列表SEQ ID NO:1所示氨基酸序列，重链序列优选如序列表SEQ ID NO：2所示。
在本发明一优选实施方案中，所述的抗体药物偶联物优选为如下所示的任一化合物：
Figure PCTCN2020137596-appb-000040
其中，Ab为TROP2抗体或TROP2抗体的变体；m优选为3.90、4.00或4.10；所述的TROP2抗体的变体中轻链优选为如序列表SEQ ID NO:1所示氨基酸序列，重链序列优选如序列表SEQ ID NO:2所示。
在本发明一优选实施方案中，所述的抗体药物偶联物优选为如下所示的任一化合物：
Figure PCTCN2020137596-appb-000041
Figure PCTCN2020137596-appb-000042
其中，Ab为TROP2抗体或TROP2抗体的变体。
在本发明一优选实施方案中，所述的抗体药物偶联物优选为如下所示的任一化合物：
Figure PCTCN2020137596-appb-000043
Ab为TROP2抗体，m优选为3.90；
Figure PCTCN2020137596-appb-000044
Ab为TROP2抗体，m优选为4.10；
Figure PCTCN2020137596-appb-000045
Ab为TROP2抗体，m优选为4.00；
Figure PCTCN2020137596-appb-000046
Ab为TROP2抗体，m优选为4.12；
Figure PCTCN2020137596-appb-000047
Ab为TROP2抗体，m优选为4.03；
Figure PCTCN2020137596-appb-000048
Ab为TROP2抗体，m优选为3.98；
Figure PCTCN2020137596-appb-000049
Ab为TROP2抗体，m优选为3.96；
Figure PCTCN2020137596-appb-000050
Ab为TROP2抗体，m优选为4.13；
Figure PCTCN2020137596-appb-000051
Ab为TROP2抗体，m优选为3.88；
Figure PCTCN2020137596-appb-000052
Ab为TROP2抗体，m优选为4.05；
Figure PCTCN2020137596-appb-000053
Ab为TROP2抗体，m优选为4.00。
本发明还提供了一种抗体药物偶联物的制备方法，其包括以下步骤：将如式II所示的化合物与Ab-氢进行如下所示的偶联反应即可；
Figure PCTCN2020137596-appb-000054
其中，所述的L 1、L 2、L 3、R 1、p和Ab的定义如上所述。
本发明中，所述的偶联反应的条件和操作可为本领域该偶联反应常规的条件和操作。
本发明还提供了一种药物组合物，其包括物质X和药用辅料，所述的物质X为上述抗体药物偶联物、其药学上可接受的盐、其溶剂合物或其药学上可接受的盐的溶剂合物。
所述的药物组合物中，所述的上述的物质X的用量可为治疗有效量。
本发明还提供了一种上述的物质X或上述的药物组合物在制备TROP2蛋白抑制剂中的应用。本发明还提供了一种上述的物质X或上述的药物组合物在制备用于治疗和/或预防肿瘤的药物中的应用，所述的肿瘤优选TROP2阳性肿瘤。
所述的应用中，所述的TROP2阳性肿瘤优选TROP2阳性胃癌、三阴乳腺癌和人胰腺癌的一种或多种。
在本发明某些实施例中，在所述的胃癌中，胃癌细胞为NCI-N87细胞；
在本发明某些实施例中，在所述的三阴乳腺癌中，三阴乳腺癌细胞为MDA-MB-468细胞；
在本发明某些实施例中，在所述的胰腺癌中，胰腺癌细胞为BXPC3细胞。
除非另有说明，在本发明说明书和权利要求书中出现的以下术语具有下述含义：
所述的药用辅料可为药物生产领域中广泛采用的辅料。辅料主要用于提供一个安全、稳定和功能性的药物组合物，还可以提供方法，使受试者接受给药后活性成分以所期望速率溶出，或促进受试者接受组合物给药后活性成分得到有效吸收。所述的药用辅料可以是惰性填充剂，或者提供某种功能，例如稳定该组合物的整体pH值或防止组合物活性成分的降解。所述的药用辅料可包括下列辅料中的一种或多种：缓冲剂、螯合剂、防腐剂、助溶剂、稳定剂、赋形剂和表面活性剂着色剂、矫味剂和甜味剂。
天然或者天然序列的TROP2可以分离自大自然，也可以用重组DNA技术、化学合成法，或以上及类似技术的联合制得。
抗体在此取其最广义的解释，其可透过位于该免疫球蛋白分子的可变区的至少一个抗原辨认区特异性地与目标结合，诸如碳水化合物、多核苷酸、脂肪、多肽等。具体包括 完整的单克隆抗体，多克隆抗体，双特异抗体以及抗体片段，只要他们具有所需的生物活性。本发明的抗体的变体是指氨基酸序列突变体，以及天然多肽的共价衍生物，条件是保留了与天然多肽相当的生物活性。氨基酸序列突变体与天然氨基酸序列的差异一般在于天然氨基酸序列中的一个活多个氨基酸被取代或在多肽序列中缺失和/或插入一个或多个氨基酸。缺失突变体包括天然多肽的片段和N端和/或C端截短突变体。通常氨基酸序列突变体与天然序列相比至少具有70％(例如70％、75％、80％、85％、90％、95％、98％或99％)的同源性。
本发明的抗体可利用该领域广为周知的技术制备，例如杂交瘤方法、重组DNA技术、噬菌体展示技术、合成技术或该等技术的组合、或该领域己知的其它技术。
关于术语“药物抗体偶联比率”(drug-antibody ratio，即DAR)的说明。L-D是和抗体上的缀合点具反应性的基团，L是连接子，D是在连接L的抗体上进一步缀合的细胞毒剂，在本发明中D为Dxd，每个抗体最终缀合D的DAR数目用m表示或者m也可表示单个抗体缀合D的数量。在一些实施方式中，m实际上为介于2至8、3至7或3至5的平均值，或m为2、3、4、5、6、7或8中的某个整数；在一些实施方式中，m为3.5-4.5的平均值(例如3.88、3.90、3.96、3.97、3.98、4.00、4.03、4.05、4.10、4.12或4.13)；在其它实施方式中，m为2、3、4、5、6、7或8的平均值。
连接子是指抗体与药物间的直接或间接连接。将连接子连接至mAb可经由许多方式完成，诸如经由表面赖氨酸、还原偶合至经氧化的碳水化合物、及经由还原链间二硫键所释放的半胱氨酸残基。多种ADC连接系统是该领域所知，包括以腙、双硫及肽为基底的连接。
术语“药学上可接受的盐”是指本发明化合物与相对无毒的、药学上可接受的酸或碱制备得到的盐。当本发明的化合物中含有相对酸性的功能团时，可以通过在纯的溶液或合适的惰性溶剂中用足够量的药学上可接受的碱与这类化合物的中性形式接触的方式获得碱加成盐。药学上可接受的碱加成盐包括但不限于：锂盐、钠盐、钾盐、钙盐、铝盐、镁盐、锌盐、铋盐、铵盐、二乙醇胺盐。当本发明的化合物中含有相对碱性的官能团时，可以通过在纯的溶液或合适的惰性溶剂中用足够量的药学上可接受的酸与这类化合物的中性形式接触的方式获得酸加成盐。所述的药学上可接受的酸包括无机酸，所述无机酸包括但不限于：盐酸、氢溴酸、氢碘酸、硝酸、碳酸、磷酸、亚磷酸、硫酸等。所述的药学上可接受的酸包括有机酸，所述有机酸包括但不限于：乙酸、丙酸、草酸、异丁酸、马来酸、丙二酸、苯甲酸、琥珀酸、辛二酸、反丁烯二酸、乳酸、扁桃酸、邻苯二甲酸、苯磺酸、对甲苯磺酸、柠檬酸、水杨酸、酒石酸、甲磺酸、异烟酸、酸式柠檬酸、油酸、单 宁酸、泛酸、酒石酸氢、抗坏血酸、龙胆酸、富马酸、葡糖酸、糖酸、甲酸、乙磺酸、双羟萘酸(即4,4’-亚甲基-双(3-羟基-2-萘甲酸))、氨基酸(例如谷氨酸、精氨酸)等。当本发明的化合物中含有相对酸性和相对碱性的官能团时，可以被转换成碱加成盐或酸加成盐。具体可参见Berge et al.,"Pharmaceutical Salts",Journal of Pharmaceutical Science 66:1-19(1977)、或、Handbook of Pharmaceutical Salts:Properties,Selection,and Use(P.Heinrich Stahl and Camille G.Wermuth,ed.,Wiley-VCH,2002)。
术语“溶剂合物”是指本发明化合物与化学计量或非化学计量的溶剂结合形成的物质。溶剂合物中的溶剂分子可以有序或非有序排列的形式存在。所述的溶剂包括但不限于：水、甲醇、乙醇等。
术语“治疗”或它的同等表达当用于例如癌症时，指用来减少或消除患者体内癌细胞数目或减轻癌症的症状的程序或过程。癌症或另外的增生性障碍的“治疗”不一定指癌症细胞或其它障碍会实际上被消除，细胞或障碍的数目会实际上被减少或者癌症或其它障碍的症状会实际上被减轻。通常，即使只具有低的成功可能性也会进行治疗癌症的方法，但是考虑到患者的病史和估计的生存预期，其仍然被认为诱导总体有益的作用过程。
术语“预防”是指获得或发生疾病或障碍的风险降低。
术语“环烷基”是指具有三到二十个碳原子的饱和的环烃原子团(例如C 3-C 6环烷基)，包括单环环烷基和多环环烷基。环烷基包含3至20个碳原子，优选包含3至10个碳原子，更优选包含3至6个碳原子。环烷基的实例包括但不仅限于环丙基、环丁基、环戊基、环己基、环庚基、环辛基、环壬基和环癸基。
术语“烷基”是指具有指定的碳原子数的直链或支链烷基。烷基的实例包括甲基、乙基、正丙基、异丙基、正丁基、叔丁基、异丁基、仲丁基、正戊基、异戊基、正己基、正庚基、正辛基及其类似烷基。
术语“卤素”是指氟、氯、溴或碘。
术语“杂芳基”是指含有1、2、3或4个独立选自N、O和S的杂原子的芳基(或芳环)，其可以是单环、双环或三环的芳香体系，例如呋喃基、吡啶基、哒嗪基、嘧啶基、吡嗪基、噻吩基、异唑基、噁唑基、二唑基、咪唑基、吡咯基、吡唑基、三唑基、四唑基、噻唑基、异噻唑基、噻二唑基、苯并咪唑基、吲哚基、吲唑基、苯并噻唑基、苯并异噻唑基、苯并唑基、苯并异唑基、喹啉基、异喹啉基等。
术语“芳基”是指任何稳定的的单环或者双环碳环，其中所有环均为芳香环。上述芳基单元的实例包括苯基或萘基。
在不违背本领域常识的基础上，上述各优选条件，可任意组合，即得本发明各较佳 实例。
如无特殊说明，本发明中的室温是指20-30℃。
本发明所用试剂和原料均市售可得。
本发明的积极进步效果在于：
1.本发明提供的包含有TROP2抗体的抗体药物偶联物，具有很好的靶向性，对表达TROP2等的多种肿瘤细胞具有很好的抑制效果。
2.体内研究表明，本发明的抗体药物偶联物具有更佳的体外细胞毒性、体内抗肿瘤活性。
3.本发明的抗体药物偶联物溶解性好，具有很好的成药性，偶联制备过程无沉淀等异常现象发生，非常利于抗体药物偶联物的制备。
附图说明
图1为FDA018抗体的轻、重链表达载体构建，其中Ab-L为抗体的轻链，Ab-H为抗体的重链。
具体实施方式
下面通过实施例的方式进一步说明本发明，但并不因此将本发明限制在所述的实施例范围之中。下列实施例中未注明具体条件的实验方法，按照常规方法和条件，或按照商品说明书选择。
缩写说明：
PCR         聚合酶链反应
CHO         中国仓鼠卵巢细胞
HTRF        均相时间分辨荧光
PB          磷酸缓冲液
EDTA        乙二胺四乙酸
TECP        三(2-羧乙基)膦
DMSO        二甲基亚砜
DMF         N,N-二甲基甲酰胺
His         组氨酸
HATU        2-(7-氧化苯并三氮唑)-N,N,N',N'-四甲基脲六氟磷酸盐
v/v         V/V，体积比
UV          紫外可见光
ELISA       酶联免疫吸附试验
BSA         牛血清白蛋白
rpm         转/每分钟
FBS         胎牛血清
实施例1：TROP2抗体的制备
本发明中选取高亲和力并特异性靶向TROP2的单克隆抗体FDA018，其轻链的氨基酸序列如SEQ ID NO:1所示，其重链的氨基酸序列如SEQ ID NO:2所示。FDA018其轻重链核苷酸序列通过全基因合成(苏州金唯智)方式获得。通过EcoR I和Hind III(购于TAKARA)双酶切分别单独构建至pV81载体中(如图1)，通过连接转化至Trans 1-T1感受态细胞(购自北京全式金生物，货号：CD501-03)中，从中挑取克隆进行PCR鉴定并送检、测序确认，培养扩增阳性克隆进行质粒中抽，从而获得抗体轻链真核表达质粒FDA018-L/pV81和抗体重链真核表达质粒FDA018-H/pV81，对这两个质粒使用XbaⅠ(购自Takara，货号：1093S)进行酶切线性化，轻、重链真核表达质粒比例1.5/1，通过电击转化至已适应悬浮生长的CHO细胞中(购于ATCC)，将电击后细胞以2000-5000细胞/孔接至96孔板中，培养3周后使用HTRF法(均相时间分辨荧光)测定表达量，从中挑选表达量前十的细胞池扩增后冻存。复苏一支细胞至125ml摇瓶中(培养体积30ml)，37℃，5.0％CO 2，130rpm震荡培养，3天后扩至1000ml摇瓶中(培养体积300ml)，37℃，5.0％CO 2，130rpm震荡培养，第四天开始隔天补加起始培养体积5-8％的补料培养基，培养至10-12天结束培养，收获液9500rpm离心15min，去除细胞沉淀，收集上清液，再用0.22μm滤膜过滤处理，处理好的样品使用MabSelect亲和层析柱(购于GE公司)进行纯化得到抗体FDA018。
FDA018轻链的氨基酸序列如下所示：
SEQ ID NO：1：
Figure PCTCN2020137596-appb-000055
FDA018重链的氨基酸序列如下所示：
SEQ ID NO：2：
Figure PCTCN2020137596-appb-000056
实施例2：连接基-药物偶联物的合成
实施例2-1：LE12的合成
Figure PCTCN2020137596-appb-000057
中间体2的合成：
将(S)-2-叠氮丙酸(10g，86.9mmol)与4-氨基苄醇(21.40g，173.8mmol)溶于300mL二氯甲烷和甲醇混合溶剂中(体积比2:1)，加入2-乙氧基-1-乙氧碳酰基-1,2-二氢喹啉(21.49g，86.9mmol)，室温反应5小时后，减压蒸干溶剂，然后，所得粗品用硅胶柱层析[二氯甲烷：乙酸乙酯＝1:1(v/v)]纯化得到中间体2(16.3g，收率85％)，ESI-MS m/z：221(M+H)。
中间体3的合成：
将中间体2(15g，68.2mmol)与二(对硝基苯)碳酸酯(22.82g，75.02mmol)混合溶于200mL无水N,N-二甲基甲酰胺中，加入25mL三乙胺，室温反应2小时。通过液质联用色谱监测原料反应完毕后，加入甲胺盐酸盐(6.91g，102.3mmol)，继续室温反应1小时。反应完毕减压蒸馏去除大部分溶剂，然后加入200mL水，200mL乙酸乙酯，分液后收集有机相，有机相经干燥后浓缩，所得粗品经过硅胶柱层析[二氯甲烷：乙酸乙酯＝10:1(v/v)]纯化得到中间体3(18.9g，收率100％)，ESI-MS m/z：278(M+H)。
中间体5的合成：
将中间体3(10g，36.1mmol)与多聚甲醛(1.63g，54.2mmol)混合溶于150mL无水二氯甲烷中，慢慢加入三甲基氯硅烷(6.28g，57.76mmol)，室温反应2小时得到中间体4的粗品溶液，通过取样加甲醇淬灭后液质联用监测反应，待反应完毕将反应液过滤然后向滤液中加入羟基乙酸叔丁酯(9.54g，72.2mmol)和三乙胺(10mL，72.2mmol)，继续室温反应2小时。反应完毕减压蒸馏去除大部分溶剂，所得粗品经过硅胶柱层析[石油醚：乙酸乙酯＝3:1(v/v)]纯化得到中间体5(11.2g，收率74％)，ESI-MS m/z：422(M+H)。
中间体6的合成：
将中间体5(10g，23.8mmol)溶于80mL无水四氢呋喃中，加入80mL水，然后加入三(2-羧乙基膦)盐酸盐(13.6g，47.6mmol)，室温反应4小时。反应完毕减压蒸馏去除四氢呋喃，然后加乙酸乙酯萃取，所得有机相经干燥后减压蒸除溶剂，经硅胶柱层析[二氯甲烷：甲醇＝10:1(v/v)]纯化得到中间体6(8.1g，收率86％)，ESI-MS m/z：396(M+H)。
中间体8的合成：
将的中间体6(5g，12.7mmol)溶于60mL二氯甲烷和甲醇混合溶剂中(v/v＝2：1)中，慢慢加入三氟乙酸3mL，室温反应30分钟。反应完毕加等体积水和乙酸乙酯，有机相干燥后浓缩，所得粗品直接用于下一步。
将上述步所获得的粗品溶于50mL无水N,N-二甲基甲酰胺中，加入Fmoc-缬氨酸羟基琥珀酰亚胺酯(8.3g，19.1mmol)，三乙胺(5mL)，室温反应2小时。反应完毕，减压蒸馏去除大部分溶剂，所得粗品经硅胶柱层析[二氯甲烷：甲醇＝10:1(v/v)]纯化得到中间体8(5.4g，收率64％)，ESI-MS m/z：661(M+H)。
中间体9的合成：
将中间体8(1g，1.5mmol)与Exatecan甲磺酸盐(0.568g，1mmol)混合于30mL无水N,N-二甲基甲酰胺中，加入2-(7-氧化苯并三氮唑)-N,N,N',N'-四甲基脲六氟磷酸盐 (1.14g，3.0mmol)，三乙胺2mL，室温反应2h。反应完毕，减压蒸馏去除溶剂，所得粗品经硅胶柱层析[氯仿：甲醇＝10:1(v/v)]纯化得到中间体9(0.94g，收率87％)，ESI-MS m/z：1078(M+H)。
化合物LE12的合成：
将中间体9(1g，0.929mmol)溶于20mL无水DMF中，加入0.5mL 1,8-二氮杂二环十一碳-7-烯，室温反应1小时。待原料反应完毕，直接加入6-(马来酰亚胺基)己酸琥珀酰亚胺酯(428.5mg，1.39mmol)，室温搅拌1小时。减压馏去溶剂，所得粗品经硅胶柱层析[氯仿：甲醇＝8:1(v/v)]纯化得到标题化合物(0.7g，收率73％)，ESI-MS m/z：1035(M+H)。
实施例2-2：化合物LE13的合成
Figure PCTCN2020137596-appb-000058
中间体14的合成
将市售的中间体12(267mg，0.8mmol)与多聚甲醛(50mg，1.6mmol)混合溶于20mL无水二氯甲烷中，慢慢加入三甲基氯硅烷(0.3mL，3.4mmol)，加料完毕后室温反应2小时。然后通过取样加甲醇淬灭后液质联用监测反应，待反应完毕后将反应液过滤，然后向滤液中加入羟基乙酸叔丁酯(211mg，1.6mmol)和潘必啶0.5mL，继续室温反应约2小时，反应完后减压蒸馏去除大部分溶剂，所得粗品经过硅胶柱层析[二氯甲烷：甲醇＝20:1(v/v)]纯化得到中间体14(260mg，收率68％)，ESI-MS m/z：479(M+H)。
中间体15的合成
将中间体14(238mg,0.50mmol)溶于6mL二氯甲烷和甲醇混合溶剂中(v/v＝2：1)中，慢慢加入三氟乙酸0.3mL，室温反应30分钟。反应完毕加等体积水和乙酸乙酯， 有机相干燥后浓缩，所得粗品直接用于下一步。
中间体16的合成
将上述步骤所获得的粗品与Exatecan甲磺酸盐(170mg，0.30mmol)混合于5mL无水N,N-二甲基甲酰胺中，加入2-(7-氧化苯并三氮唑)-N,N,N',N'-四甲基脲六氟磷酸盐(341mg，0.90mmol)，三乙胺0.60mL，室温反应2h。反应完毕，减压蒸馏去除溶剂，所得粗品经硅胶柱层析[氯仿：甲醇＝10:1(v/v)]纯化得到中间体16(210mg,83％)，ESI-MS m/z：840(M+H)。
中间体17的合成
将中间体16(100mg，0.12mmol)溶于15mL无水四氢呋喃中，加入3mL水，然后加入1摩尔/升的三乙基膦水溶液0.3mL，室温反应4小时。监测反应完毕后将反应液减压蒸馏去除四氢呋喃，向所余水溶液中加碳酸氢钠调节pH至中性，然后加二氯甲烷萃取，所得有机相干燥后减压蒸除溶剂，所得粗品经硅胶柱层析[二氯甲烷：甲醇＝10:1(v/v)]纯化得到中间体17(69mg，收率71％)，ESI-MS m/z：814(M+H)。
化合物LE13的合成
按照上步合成方法得到的中间体17(120mg，0.15mmol)与市售的原料MC-V(102mg，0.33mmol)混合于40mL二氯甲烷中，加入缩合剂2-乙氧基-1-乙氧碳酰基-1,2-二氢喹啉(82mg，0.33mmol)，室温反应过夜，反应完毕减压蒸干溶剂，所得粗品经硅胶柱层析[二氯甲烷：甲醇＝10:1(v/v)]纯化得到化合物LE13(116mg，收率70％)，ESI-MS m/z：1106.5(M+H)。
实施例2-3：化合物LE14的合成
Figure PCTCN2020137596-appb-000059
中间体19的合成
将市售的中间体18(300mg，0.8mmol)与多聚甲醛(50mg，1.6mmol)混合溶于20mL无水二氯甲烷中，慢慢加入三甲基氯硅烷(0.3mL，3.4mmol)，室温反应2小时，通过取样加甲醇淬灭后液质联用监测反应。待反应完毕将反应液过滤，然后向滤液中加入羟基乙酸叔丁酯(211mg，1.6mmol)和三乙胺(0.22m，1.6mmol)，继续室温反应约2小时，反应完后减压蒸馏去除大部分溶剂，所得粗品经过硅胶柱层析[二氯甲烷：甲醇＝20:1(v/v)]纯化得到中间体19(349mg，收率85％)，ESI-MS m/z：514(M+H)， 1H NMR(400MHz,CDCl 3)δ8.13(s,1H),7.56(d,J＝7.5Hz,2H),7.35(s,2H),5.14(s,2H),4.91(s,2H),4.25(q,J＝7.1Hz,1H),3.99(d,J＝42.5Hz,2H),3.85(t,J＝6.2Hz,2H),3.40(dd,J＝18.5,7.6Hz,2H),2.89(d,J＝48.6Hz,3H),1.65(d,J＝6.8Hz,3H),1.46(s,9H)。
中间体20的合成
将中间体19(257mg,0.50mmol)溶于6mL二氯甲烷和甲醇混合溶剂中(v/v＝2：1)中，慢慢加入三氟乙酸0.3mL，室温反应30分钟。反应完毕加等体积水和乙酸乙酯，有机相干燥后浓缩，所得粗品直接用于下一步。
将所得粗品与Exatecan甲磺酸盐(170mg，0.30mmol)混合于5mL无水N,N-二甲基甲酰胺中，加入2-(7-氧化苯并三氮唑)-N,N,N',N'-四甲基脲六氟磷酸盐(341mg，0.90mmol)，三乙胺0.60mL，室温反应2h。反应完毕，减压蒸馏去除溶剂，所得粗品经过硅胶柱层析[二氯甲烷：甲醇＝20:1(v/v)]纯化得到中间体20(212mg，收率81％)， ESI-MS m/z：875(M+H)。 1H NMR(400MHz,CDCl 3)δ8.27(d,J＝34.7Hz,1H),7.63–7.35(m,5H),7.21–7.10(m,1H),5.71–5.48(m,2H),5.24–4.95(m,3H),4.95–4.72(m,4H),4.45(s,1H),4.33–3.97(m,3H),3.75(s,2H),3.39–2.99(m,4H),2.76(d,J＝15.3Hz,3H),2.43–2.15(m,5H),2.04(s,1H),1.94–1.75(m,2H),1.62(d,J＝6.6Hz,3H),1.11–0.89(m,3H)。
中间体21的合成
将中间体20(77mg，0.09mmol)溶于12mL无水四氢呋喃中，加入3mL水，然后加入1摩尔/升的三乙基膦水溶液0.3mL，室温反应4小时。反应完毕减压蒸馏去除四氢呋喃，向所余水溶液中加碳酸氢钠调节pH至中性，然后加二氯甲烷萃取，所得有机相干燥后减压蒸除溶剂，所得粗品经硅胶柱层析[二氯甲烷：甲醇＝10:1(v/v)]纯化得到中间体21(53mg，收率69％)，ESI-MS m/z：849(M+H)。 1H NMR(400MHz,DMSO)δ8.52(s,1H),7.79(d,J＝10.8Hz,1H),7.67–7.55(m,2H),7.47–7.21(m,3H),6.51(s,1H),5.60(s,1H),5.52–5.32(m,2H),5.30–5.11(m,2H),5.11–4.94(m,2H),4.94–4.74(m,2H),4.02(s,2H),3.81–3.66(m,2H),3.60–3.35(m,4H),3.24–3.08(m,2H),2.94(d,J＝30.8Hz,3H),2.39(s,3H),2.28–2.04(m,2H),2.00–1.73(m,2H),1.22(d,J＝6.6Hz,3H),0.96–0.70(m,3H)。
化合物LE14的合成
中间体21(134mg，0.16mmol)与市售的原料MC-V(102mg，0.33mmol)混合于40mL二氯甲烷中，加入缩合剂2-乙氧基-1-乙氧碳酰基-1,2-二氢喹啉(82mg，0.33mmol)，室温反应过夜，反应完毕减压蒸干溶剂，所得粗品经硅胶柱层析[二氯甲烷：甲醇＝10:1(v/v)]纯化得到化合物LE14(137mg，收率75％)，ESI-MS m/z：1141.4(M+H)。 1H NMR(400MHz,DMSO)δ9.97(s,1H),8.52(s,1H),8.27–8.09(m,1H),7.88–7.70(m,2H),7.63–7.51(m,2H),7.28(s,3H),6.99(s,2H),6.51(s,1H),5.59(s,1H),5.50–5.32(m,2H),5.17(s,2H),4.98(s,2H),4.85(d,J＝17.3Hz,2H),4.43–4.33(m,1H),4.21–4.12(m,1H),4.03(s,2H),3.74–3.64(m,2H),3.20–3.03(m,3H),3.02–2.84(m,4H),2.36(s,3H),2.23–2.09(m,4H),2.01–1.90(m,1H),1.90–1.78(m,2H),1.55–1.39(m,4H),1.30(d,J＝6.7Hz,3H),1.23–1.11(m,2H),0.93–0.77(m,9H)。
实施例2-4：化合物LE15-LE20的合成
Figure PCTCN2020137596-appb-000060
中间体VI可以以Fmoc-L-缬氨酸-L-丙氨酸为起始原料，参照实施例2-1中中间体3的合成方法中的步骤a和b，将其中步骤b中的甲胺盐酸盐替换为相应的市售可得的氨基化合物制备而得。后续步骤从中间体VI出发，按照与实施例2-1中的步骤c、d、f和h相同的方法，得到与中间体9相似的中间体IX，然后按照实施例6中的步骤i和j相同的步骤处理，脱除氨基保护基，然后与市售可得的不同马来酰亚胺缩合得到终产物。所用氨基化合物及马来酰亚胺结构见表1。化合物LE15：灰白色固体，ESI-MS m/z：1121.2(M+H)；化合物LE16：淡黄色固体，ESI-MS m/z：1167.1(M+H)；化合物LE17：黄色固体，ESI-MS m/z：1132.3(M+H)；化合物LE18：淡黄色固体，ESI-MS m/z：1305.4(M+H)；化合物LE19：淡黄色固体，ESI-MS m/z：1307.4(M+H)；化合物LE20：淡黄色固体，ESI-MS m/z：1337.6(M+H)。
表1.合成LE15-LE20所用的中间体
Figure PCTCN2020137596-appb-000061
Figure PCTCN2020137596-appb-000062
Figure PCTCN2020137596-appb-000063
实施例2-5：化合物LE21与LE22的合成
Figure PCTCN2020137596-appb-000064
化合物DXD-1的合成
市售的Exatecan甲磺酸盐(0.568g，1mmol)与市售的2-(叔丁基二甲基硅氧)乙酸(CAS：105459-05-0，0.38g，2mmol)混合溶于20mL无水二氯甲烷中，加入缩合剂HATU(0.76g，2mmol)和1mL吡啶，室温搅拌2小时。待反应完毕后，减压蒸干溶剂，所得粗品经柱层析[二氯甲烷：甲醇＝50:1(v/v)]纯化得到标题化合物DXD-1(0.55g，收率90％)，ESI-MS m/z：608.1(M+H)。 1H NMR(400MHz,CDCl 3)δ7.73(d,J＝10.5Hz,1H),7.64(s,1H),7.05(d,J＝9.2Hz,1H),5.80–5.62(m,2H),5.41–5.14(m,4H),4.29–4.15(m,2H),4.08-4.03(m,1H),3.27–3.07(m,2H),2.45(s,3H),2.38–2.28(m,2H),1.96–1.81(m,2H),1.04(t,J＝7.4Hz,3H),0.80(s,9H),0.11(s,3H),0.03(s,3H).
中间体V的制备
中间体V可以参照实施例2-1中化合物4的制备方法，将其中步骤b中的甲胺盐酸盐替换为相应的市售可得的氨基化合物制备而得。
LE21-LE22的合成
中间体V与DXD-1反应，后续经过10％三氟乙酸/二氯甲烷溶液处理得到中间体X，然后中间体X参照实施例2-1中化合物5的后续步骤e、g、i和j进行反应：中间体X经过还原得到氨基化合物，所得氨基化合物和Fmoc-缬氨酸羟基琥珀酰亚胺酯缩合，然后脱去所得产物中氨基的Fmoc保护基，所得氨基产物再和6-(马来酰亚胺基)己酸琥珀酰亚胺酯反应得到最终产物。化合物LE21：黄色固体，ESI-MS m/z：1141.2(M+H)；化合物LE22：黄色固体，ESI-MS m/z：1106.6(M+H)。
Figure PCTCN2020137596-appb-000065
实施例2-6：化合物LS13的合成
参照实施例2-4中LE15的合成方法，SN-38(7-乙基-10-羟基喜树碱)与中间体VII(R 1为甲砜乙基)反应后，经脱保护，缩合等步骤得到化合物LS13： 1H NMR(400MHz,DMSO)δ9.92(d,J＝22.4Hz,1H),8.14(s,1H),8.08(d,J＝9.1Hz,1H),7.81(d,J＝8.0Hz,1H),7.70–7.50(m,3H),7.47(d,J＝7.2Hz,1H),7.34(d,J＝7.2Hz,1H),7.27(s,1H),7.20(s,1H),6.98(s,2H),6.51(s,1H),5.61(s,2H),5.48–5.35(m,2H),5.27(s,2H),5.10(d,J＝20.6Hz,2H),4.36(s,1H),4.21–4.07(m,1H),3.84(s,2H),3.48(s,2H),3.21–2.92(m,6H),2.25–2.04(m,2H),2.04–1.78(m,3H),1.55–1.36(m,4H),1.36–1.10(m,9H),0.95–0.71(m,10H)。
Figure PCTCN2020137596-appb-000066
实施例2-7：化合物GGFG-Dxd的合成
化合物GGFG-Dxd参照WO2015146132A1报道的已知的合成方法制备而得。ESI-MS m/z：1034.5(M+H)， 1H-NMR(400MHz,DMSO-d 6)δ8.61(t,J＝6.4Hz,1H),8.50(d,J＝8.5Hz,1H),8.28(t,J＝5.1Hz,1H),8.11(d,J＝7.5Hz,1H),8.05(t,J＝5.7Hz,1H),7.99(t,J＝5.9Hz,1H),7.77(d,J＝11.0Hz,1H),7.31(s,1H),7.25–7.16(m,5H),6.98(s,2H),6.51(s,1H),5.59(dt,J＝7.4,4.1Hz,1H),5.41(s,2H),5.20(s,2H),4.64(d,J＝6.1Hz,2H),4.53–4.40(m,1H),4.02(s,2H),3.74–3.37(m,8H),3.18–3.00(m,2H),3.04–2.97(m,1H),2.77(dd,J＝13.5,9.4Hz,1H),2.38(s,3H),2.19(dd,J＝14.9,8.5Hz,2H),2.11–2.05(m,2H),1.86(dd,J＝14.0,6.7Hz,2H),1.45(s,4H),1.20–1.14(m,2H),0.87(t,J＝7.1Hz,3H).
Figure PCTCN2020137596-appb-000067
实施例3：抗体药物偶联物的制备
将按照实施例1方法制备得到的TROP2的抗体FDA018使用G25脱盐柱将其置换至50mM PB/1.0mM EDTA缓冲液中(pH 7.0)，加入5当量TECP，于25℃搅拌1小时，以使抗体链间二硫键部分打开，随后使用枸橼酸将还原后的抗体溶液pH调至5.0，使用G25脱盐柱将样品置换至20mM枸橼酸盐缓冲液，1mM EDTA缓冲液中(pH 5.0)，保持水浴温度25℃，以备偶联反应。将按照上述实施例2方法制备得到的连接基-药物偶联物LE12-LE22、LS13和GGFG-Dxd分别用DMSO溶解，从中吸取4.5当量连接基-药物偶联物逐滴加至还原后的抗体溶液中，并补加DMSO至其终浓度为5％(v/v)，25℃搅拌反应0.5个小时，反应 完成后，使用0.22um膜过滤样品。使用切向流超滤系统纯化去除未偶联小分子，缓冲液为25mM His，6％蔗糖溶液(pH 6.0)，纯化后放置于-20℃冰箱中保存。使用UV法分别在280nm和370nm下测定其吸光度值，计算DAR值，结果见下表2。
按照与本实施例以上同样的操作步骤进行偶联反应，样品均按照最高DAR制备(即过量偶联)，观察各偶联反应发生时沉淀的产生情况，并计算各偶联反应完成后的聚体比例和回收率，所得结果同样见表2。
表2 制备不同抗体药物偶联物(ADC)的偶联情况
Figure PCTCN2020137596-appb-000068
“/”表示未计算回收率
在本发明的抗体欧联药物的制备规程中未产生沉淀，且聚体比例在正常范围，表明本发明提供的抗体偶联药物具有很好的理化性质。
效果实施例1：抗体药物偶联物的体外杀伤活性评价
选择NCI-N87(ATCC)细胞作为实验体外活性检测用细胞株，每孔2000个细胞接种于96孔细胞培养板中，培养20-24小时。将按照实施例3方法制备的抗体药物偶联物使用含10％FBS的L15细胞培养基配制成1000、166.7、55.6、18.6、6.17、2.06、0.69、 0.23、0.08、0.008和0nM共11个浓度梯度的供试品溶液，取稀释好的供试品溶液100μL/孔加入到接种细胞的培养板内，置于37℃，5％CO 2培养箱培养144小时后加入CellTiter-
Figure PCTCN2020137596-appb-000069
Luminescent Cell Viability Assay Reagent(50μL/孔)，500rpm室温振荡10分钟混匀，SpectraMaxL酶标仪读取数据(OD570nm，2s间隔读数)后计算IC50结果见表3。
利用上述同样的方法，分别测试各抗体药物偶联物对购自ATCC的MDA-MB-468、和BXPC3肿瘤细胞的细胞毒杀伤活性，结果如表3所示。从表3的结果可以看出，本发明提供的抗体药物偶联物对NCI-N87、MDA-MB-468、和BXPC3等细胞都具有极佳的体外杀伤活性。而对Calu-6阴性细胞不具有杀伤活性，表明所制备的ADC具有特异性的靶向杀伤活性。
表3.抗体药物偶联物的体外杀伤活性
Figure PCTCN2020137596-appb-000070
效果实施例2：体外血浆稳定性试验
本实施例评价按照实施例3方法制得的抗体偶联药物在人血浆中的稳定性。具体而言，在本实施例中，将实施例3的抗体偶联药物加入人血浆中，37℃水浴中放置1、3、7、14、21、28天加内标(依喜替康作为内标物质)并进行萃取然后通过高效液相色谱法检测游离药物的释放量，结果如表4所示。
表4.不同ADC在人血浆中的稳定性评价
Figure PCTCN2020137596-appb-000071
血浆稳定性结果表明采用新技术方案得到的ADC稳定性不劣于FDA018-GGFG-DXD，而且部分更优，同时上述活性测试结果也证明部分新获得的ADC活性优于FDA018-GGFG-DXD。
效果实施例3：连接基-药物偶联物的体外酶切实验
连接基-药物偶联物(LE14和GGFG-Dxd)与组织蛋白酶B在三个不同pH(5.0，6.0，7.0)缓冲液中共孵育，不同时间点取样进入高效液相色谱-质谱联用仪，外标法(以DXD为外标)确定药物的释放百分比。实验结果(如表5所示)表明GGFG-Dxd在所用的pH范围内酶切速度较慢，而本发明的LE14在pH 5.0至pH 7.0的范围内都能快速的酶切。
表5.LE14和GGFG-Dxd体外在不同pH的酶切情况
Figure PCTCN2020137596-appb-000072
效果实施例4：FDA018-LS13的体外酶切实验
选择NCI-N87细胞株作为实验细胞株，样品在组织蛋白酶B体系(100mM醋酸钠 -醋酸缓冲液，4mM二硫苏糖醇，pH 5.0)中37℃孵育4小时后，所得样品用培养基稀释至不同浓度，按照SN-38浓度70nM-0.003nM设定8个浓度(1.5-10倍稀释)，观察144小时对细胞株的杀伤(抑制)能力变化，并通过CellTiter-
Figure PCTCN2020137596-appb-000073
Luminescent Cell Viability Assay化学发光染色，读取荧光数据后计算IC50数值。
将以上在组织蛋白酶B体系中37℃孵育4小时所得的酶切样品经过适量乙醇沉淀去除蛋白，通过高效液相色谱检测释放产生的小分子化合物，并以等量SN-38作为参比，测定4小时释放率，结果显示释放率达到99％。
实验结果(如表6所示)显示，FDA018-LS13经过酶切处理后细胞毒活性与等当量的SN-38几乎相同，也表明FDA018-LS13在组织蛋白酶B作用下几乎完全释放出了SN-38并发挥作用，而FDA018-LS13内吞进入溶酶体则可能发生了不可预知的变化导致SN-38不能有效发挥作用。
表6.FDA018-LS13被组织蛋白酶B体系酶切前后对NCI-N87细胞株的杀伤活性变化
Figure PCTCN2020137596-appb-000074
效果实施例5：测试FDA018-LE14在NCI-N87人胃癌模型中的抗肿瘤活性
选择6-8周大的雌性Balb/c nude小鼠右侧颈背部皮下注射溶于100ul PBS溶液的5×10 6人胃癌细胞(NCI-N87)，待肿瘤生长至平均体积150-200mm 3时，根据肿瘤大小和小鼠体重随机分成5组，每组6只动物，分别为空白对照组、以及抗体药物偶联物FDA018-GGFG-DXD和FDA018-LE14各分别两个剂量组，具体地，01组为空白对照组；02组为4.0mg/kg的FDA018-GGFG-DXD组；03组为2.0mg/kg的FDA018-GGFG-DXD组；04组为4.0mg/kg的FDA018-LE14组；05组为2.0mg/kg的FDA018-LE14组；腹腔给药，每周给药一次。每周两次测量实验动物体重和肿瘤体积并观察实验过程中动物生存状态。结果如表7所示，空白对照组小鼠在结束给药后时平均肿瘤体积为1388.47mm 3。测试药2.0mg/kg的FDA018-GGFG-DXD治疗组在结束给药后第14天平均肿瘤体积为1235.21mm 3，4.0mg/kg的FDA018-GGFG-DXD治疗组在结束给药后第14天平均肿瘤体积为721.09mm 3。测试药2.0mg/kg的FDA018-LE14治疗组在结束给药后第14天平均肿瘤体积为1342.31mm 3，4.0mg/kg的FDA018-LE14治疗组在结束给药后第14天平均肿瘤体积为435.36mm 3。实验结果显示FDA018-LE14具有较好的体内抗肿瘤活性，同时所有 实验小鼠无死亡情况，无体重减轻情况，表明FDA018-LE14具有很好的安全性。
表7.FDA018-LE14在NCI-N87人胃癌模型中模型中的抗肿瘤活性
Figure PCTCN2020137596-appb-000075

Claims (11)

  1. 一种如式I所示的抗体药物偶联物、其药学上可接受的盐、其溶剂合物或其药学上可接受的盐的溶剂合物；
    Figure PCTCN2020137596-appb-100001
    其中，Ab为TROP2抗体或TROP2抗体的变体；
    所述的TROP2抗体中轻链的氨基酸序列如序列表SEQ ID NO:1所示，重链的氨基酸序列如序列表SEQ ID NO:2所示；
    所述的TROP2抗体的变体，与TROP2抗体相比，至少具有70％、75％、80％、85％、90％、95％、98％或99％以上的同源性；
    m为2～8；
    D为细胞毒性药物拓扑异构酶抑制剂；
    R 1为被一个或多个-NR 1-1R 1-2取代的C 1～C 6烷基、被一个或多个R 1-3S(O) 2-取代的C 1～C 6烷基、C 1～C 6烷基、C 3～C 10环烷基、C 6～C 14芳基或5～14元杂芳基；所述的5～14元杂芳基中的杂原子选自N、O和S中的一种或多种，杂原子个数为1、2、3或4；所述的R 1-1、R 1-2和R 1-3分别独立地为C 1～C 6烷基；
    L 1独立地为苯丙氨酸残基、丙氨酸残基、甘氨酸残基、谷氨酸残基、天冬氨酸残基、半胱氨酸残基、谷氨酸残基、组氨酸残基、异亮氨酸残基、亮氨酸残基、赖氨酸残基、甲硫氨酸残基、脯氨酸残基、丝氨酸残基、苏氨酸残基、色氨酸残基、酪氨酸残基和缬氨酸残基中的一种或多种；p为2～4；
    L 2为
    Figure PCTCN2020137596-appb-100002
    Figure PCTCN2020137596-appb-100003
    其中n独立地为1～12，c端通过羰基与L 1相连，f端与所述的L 3的d端相连；
    L 3为
    Figure PCTCN2020137596-appb-100004
    其中b端和所述的Ab相连，d端与所述的L 2的f端相连。
  2. 如权利要求1所述的抗体药物偶联物、其药学上可接受的盐、其溶剂合物或其药学上可接受的盐的溶剂合物，其特征在于：
    所述的L 3的b端与所述的抗体上的巯基以硫醚的形式相连；
    和/或，当D为细胞毒性药物拓扑异构酶抑制剂时，所述的细胞毒性药物拓扑异构酶抑制剂为
    Figure PCTCN2020137596-appb-100005
    R 2和R 5分别独立地为H、C 1-C 6烷基或卤素；R 3和R 6分别独立地为H、C 1-C 6烷基或卤素；R 4和R 7分别独立地为C 1-C 6烷基；
    和/或，当所述的R 1为被一个或多个-NR 1-1R 1-2取代的C 1～C 6烷基时，所述的C 1～C 6烷基为甲基、乙基、正丙基、异丙基、正丁基、异丁基或叔丁基，优选为乙基；
    和/或，当所述的R 1为被多个-NR 1-1R 1-2取代的C 1～C 6烷基时，所述的多个为两个或三个；
    和/或，当所述的R 1-1和R 1-2各自独立地为C 1～C 6烷基时，所述的C 1～C 6烷基为甲基、乙基、正丙基、异丙基、正丁基、异丁基或叔丁基，优选为甲基；
    和/或，当所述的R 1为被一个或多个R 1-3S(O) 2-取代的C 1～C 6烷基时，所述的C 1～C 6烷基为C 1～C 4烷基，优选为甲基、乙基、正丙基、异丙基、正丁基、异丁基或叔丁基，最优选为乙基；
    和/或，当所述的R 1为被多个R 1-3S(O) 2-取代的C 1～C 6烷基时，所述的多个为两个或三个；
    和/或，当所述的R 1-3为C 1～C 6烷基时，所述的C 1～C 6烷基为甲基、乙基、正丙基、异丙基、正丁基、异丁基或叔丁基，优选为甲基；
    和/或，当所述的R 1为C 1～C 6烷基时，所述的C 1～C 6烷基为甲基、乙基、正丙基、异丙基、正丁基、异丁基或叔丁基，优选为甲基；
    和/或，所述的m为整数或非整数，优选为3-5，进一步优选为3.5-4.5；
    和/或，所述的n为8～12；
    和/或，p为2；
    和/或，当所述的R 1-1、R 1-2和R 1-3独立地为C 1～C 6烷基时，所述的C 1～C 6烷基为C 1～C 4烷基，优选为甲基、乙基、正丙基、异丙基、正丁基、异丁基或叔丁基，最优选为甲基。
  3. 如权利要求2所述的抗体药物偶联物、其药学上可接受的盐、其溶剂合物或其药学上可接受的盐的溶剂合物，其特征在于：
    当所述的R 2和R 5分别独立地为C 1-C 6烷基时，所述的C 1～C 6烷基为C 1～C 4烷基，进一步优选为甲基、乙基、正丙基、异丙基、正丁基、异丁基或叔丁基，最优选为甲基；
    和/或，当所述的R 2和R 5分别独立地为卤素时，所述的卤素为氟、氯、溴或碘，优选为氟；
    和/或，当所述的R 3和R 6分别独立地为C 1-C 6烷基时，所述的C 1～C 6烷基为C 1～C 4烷基，进一步优选为甲基、乙基、正丙基、异丙基、正丁基、异丁基或叔丁基，最优选为甲基；
    和/或，当所述的R 3和R 6分别独立地为卤素时，所述的卤素为氟、氯、溴或碘，进一步优选为氟；
    和/或，当所述的R 4和R 7分别独立地为C 1-C 6烷基时，所述的C 1～C 6烷基为C 1～C 4烷基，进一步优选为甲基、乙基、正丙基、异丙基、正丁基、异丁基或叔丁基，最优选为乙基；
    和/或，当所述的R 1为被一个或多个-NR 1-1R 1-2取代的C 1～C 6烷基时，所述的-NR 1-1R 1-2为-N(CH 3) 2；
    和/或，当所述的R 1为被一个-NR 1-1R 1-2取代的C 1～C 6烷基时，所述的被一个-NR 1-1R 1- 2取代的C 1～C 6烷基为
    Figure PCTCN2020137596-appb-100006
    和/或，当所述的R 1为被一个R 1-3S(O) 2-取代的C 1～C 6烷基时，所述的被一个R 1-3S(O) 2-取代的C 1～C 6烷基为
    Figure PCTCN2020137596-appb-100007
    和/或，所述的m为3.8-4.2，例如3.88、3.90、3.96、3.97、3.98、4.00、4.03、4.05、4.10、4.12或4.13；
    和/或，所述的(L 1) p为
    Figure PCTCN2020137596-appb-100008
    其中g端通过羰基和所述的L 2的c端相连；
    和/或，所述的n为8、9、10、11和12。
  4. 如权利要求2所述的抗体药物偶联物、其药学上可接受的盐、其溶剂合物或其药学上可接受的盐的溶剂合物，其特征在于：
    R 2和R 5分别独立地为C 1-C 6烷基；
    和/或，R 3和R 6分别独立地为卤素；
    和/或，R 4和R 7为乙基；
    和/或，所述的L 1为苯丙氨酸残基、丙氨酸残基、甘氨酸残基、异亮氨酸残基、亮氨酸残基、脯氨酸残基和缬氨酸残基中的一种或多种，优选为苯丙氨酸残基、丙氨酸残基、甘氨酸残基和缬氨酸残基中的一种或多种，进一步优选为缬氨酸残基和/或丙氨酸残基，所述的多种优选为两种或三种；
    和/或，所述的R 1为被一个或多个-NR 1-1R 1-2取代的C 1～C 6烷基、被一个或多个R 1-3S(O) 2-取代的C 1～C 6烷基、C 1～C 6烷基或C 3～C 10环烷基，优选为被一个或多个-NR 1-1R 1-2取代的C 1～C 6烷基、被一个或多个R 1-3S(O) 2-取代的C 1～C 6烷基、或、C 1～C 6烷基，进一步优选为被一个或多个-NR 1-1R 1-2取代的C 1～C 6烷基、或、被一个或多个R 1-3S(O) 2-取代的C 1～C 6烷基，最优选为被一个或多个R 1-3S(O) 2-取代的C 1～C 6烷基；
    较佳地，D为
    Figure PCTCN2020137596-appb-100009
  5. 如权利要求1～4中任一项所述的抗体药物偶联物、其药学上可接受的盐、其溶剂合物或其药学上可接受的盐的溶剂合物，其特征在于，所述的抗体偶联药物为如下任一方案：
    方案一：
    Ab为TROP2抗体或TROP2抗体的变体；
    D为
    Figure PCTCN2020137596-appb-100010
    R 2和R 5分别独立地为H、C 1-C 6烷基或卤素，R 3和R 6分别独立地为H、C 1-C 6烷基或卤素，R 4和R 7分别独立地为C 1-C 6烷 基，D优选
    Figure PCTCN2020137596-appb-100011
    L 1独立地为苯丙氨酸残基、丙氨酸残基、甘氨酸残基、异亮氨酸残基、亮氨酸残基、脯氨酸残基和缬氨酸残基中的一种或多种；
    方案二：
    Ab为TROP2抗体或TROP2抗体的变体；
    D为
    Figure PCTCN2020137596-appb-100012
    所述的R 2和R 5分别独立地为C 1-C 6烷基；所述的R 3和R 6分别独立地为卤素，D优选
    Figure PCTCN2020137596-appb-100013
    Figure PCTCN2020137596-appb-100014
    R 1为被一个或多个-NR 1-1R 1-2取代的C 1～C 6烷基、被一个或多个R 1-3S(O) 2-取代的C 1～C 6烷基、或、C 1～C 6烷基；
    L 1独立地为苯丙氨酸残基、丙氨酸残基、甘氨酸残基和缬氨酸残基中的一种或多种；
    方案三：
    Ab为TROP2抗体；
    D为
    Figure PCTCN2020137596-appb-100015
    所述的R 2和R 5分别独立地为C 1-C 6烷基；所述的R 3和R 6分别独立地为卤素，D优选
    Figure PCTCN2020137596-appb-100016
    Figure PCTCN2020137596-appb-100017
    m为3.5-4.5，
    R 1为被一个或多个-NR 1-1R 1-2取代的C 1～C 6烷基、被一个或多个R 1-3S(O) 2-取代的C 1～C 6烷基、或、C 1～C 6烷基；
    L 1独立地为缬氨酸残基和/或丙氨酸残基；
    方案四：
    Ab为TROP2抗体；
    D为
    Figure PCTCN2020137596-appb-100018
    R 1为被一个或多个-NR 1-1R 1-2取代的C 1～C 6烷基、被一个或多个R 1-3S(O) 2-取代的C 1～C 6烷基、或、C 1～C 6烷基；
    L 1独立地为缬氨酸残基和/或丙氨酸残基；
    方案五：
    Ab为TROP2抗体；
    D为
    Figure PCTCN2020137596-appb-100019
    m为3.5-4.5，
    R 1为被一个或多个-NR 1-1R 1-2取代的C 1～C 6烷基、被一个或多个R 1-3S(O) 2-取代的C 1～C 6烷基、或、C 1～C 6烷基；
    L 1独立地为缬氨酸残基和/或丙氨酸残基。
  6. 如权利要求1所述的抗体药物偶联物、其药学上可接受的盐、其溶剂合物或其药学上可接受的盐的溶剂合物，其特征在于，所述的抗体偶联药物为如下所示的任一化合物：
    Figure PCTCN2020137596-appb-100020
    Figure PCTCN2020137596-appb-100021
    Figure PCTCN2020137596-appb-100022
    其中，Ab为TROP2抗体或TROP2抗体的变体，m为3.8-4.2，优选为3.88、3.90、3.96、3.97、3.98、4.00、4.03、4.05、4.10、4.12或4.13；所述的TROP2抗体的变体中轻链优选为如序列表SEQ ID NO:1所示氨基酸序列，重链序列优选如序列表SEQ ID NO：2所示。
  7. 如权利要求1所述的抗体药物偶联物、其药学上可接受的盐、其溶剂合物或其药学上可接受的盐的溶剂合物，其特征在于，所述的抗体偶联药物为如下任一方案：
    方案A：
    Figure PCTCN2020137596-appb-100023
    Figure PCTCN2020137596-appb-100024
    Figure PCTCN2020137596-appb-100025
    其中，Ab为TROP2抗体或TROP2抗体的变体，优选TROP2抗体；所述的TROP2抗体的变体中轻链优选为如序列表SEQ ID NO:1所示氨基酸序列，重链序列优选如序列表SEQ ID NO：2所示；
    方案B：
    Figure PCTCN2020137596-appb-100026
    其中，Ab为TROP2抗体或TROP2抗体的变体；m优选为3.90、4.00或4.10；所述的TROP2抗体的变体中轻链优选为如序列表SEQ ID NO:1所示氨基酸序列，重链序列 优选如序列表SEQ ID NO:2所示；
    方案C：
    Figure PCTCN2020137596-appb-100027
    其中，Ab为TROP2抗体或TROP2抗体的变体；
    方案D：
    Figure PCTCN2020137596-appb-100028
    Ab为TROP2抗体，m优选为3.90；
    Figure PCTCN2020137596-appb-100029
    Ab为TROP2抗体，m优选为4.10；
    Figure PCTCN2020137596-appb-100030
    Ab为TROP2抗体，m优选为4.00；
    Figure PCTCN2020137596-appb-100031
    Ab为TROP2抗体，m优选为4.12；
    Figure PCTCN2020137596-appb-100032
    Ab为TROP2抗体，m优选为4.03；
    Figure PCTCN2020137596-appb-100033
    Ab为TROP2抗体，m优选为3.98；
    Figure PCTCN2020137596-appb-100034
    Ab为TROP2抗体，m优选为3.96；
    Figure PCTCN2020137596-appb-100035
    Ab为TROP2抗体，m优选为4.13；
    Figure PCTCN2020137596-appb-100036
    Ab为TROP2抗体，m优选为3.88；
    Figure PCTCN2020137596-appb-100037
    Ab为TROP2抗体，m优选为4.05；
    Figure PCTCN2020137596-appb-100038
    Ab为TROP2抗体，m优选为4.00。
  8. 一种如权利要求1～7中任一项所述的抗体药物偶联物的制备方法，其特征在于，其包括以下步骤：将如式II所示的化合物与Ab-氢进行如下所示的偶联反应即可；
    Figure PCTCN2020137596-appb-100039
  9. 一种药物组合物，其包括物质X和药用辅料，所述的物质X为如权利要求1～7中任一项所述的抗体药物偶联物、其药学上可接受的盐、其溶剂合物或其药学上可接受的盐的溶剂合物，所述的物质X的量优选为治疗有效量。
  10. 一种物质X或如权利要求9所述的药物组合物在制备TROP2蛋白抑制剂中的应用，所述的物质X为如权利要求1～7中任一项所述的抗体药物偶联物、其药学上可接受的盐、其溶剂合物或其药学上可接受的盐的溶剂合物。
  11. 一种物质X或如权利要求9所述的药物组合物在制备用于治疗和/或预防肿瘤的 药物中的应用，所述的物质X为如权利要求1～7中任一项所述的抗体药物偶联物、其药学上可接受的盐、其溶剂合物或其药学上可接受的盐的溶剂合物；所述的肿瘤优选TROP2阳性肿瘤；所述的TROP2阳性肿瘤优选TROP2阳性胃癌、三阴乳腺癌和人胰腺癌的一种或多种；胃癌细胞优选为NCI-N87细胞；三阴乳腺癌细胞优选为MDA-MB-468细胞；胰腺癌细胞优选为BXPC3细胞。
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