WO2023124963A1 - Antibody-drug conjugate having reduced reversible reaction, and preparation method therefor and application thereof - Google Patents

Antibody-drug conjugate having reduced reversible reaction, and preparation method therefor and application thereof Download PDF

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WO2023124963A1
WO2023124963A1 PCT/CN2022/138621 CN2022138621W WO2023124963A1 WO 2023124963 A1 WO2023124963 A1 WO 2023124963A1 CN 2022138621 W CN2022138621 W CN 2022138621W WO 2023124963 A1 WO2023124963 A1 WO 2023124963A1
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cancer
antibody
stereoisomer
pharmaceutically acceptable
solvate
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PCT/CN2022/138621
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French (fr)
Chinese (zh)
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柯天一
丁会
于海勇
许云雷
劳芳
刘岩
张西东
荣鹏飞
姚德惠
杜旭召
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昆山新蕴达生物科技有限公司
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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
    • 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
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D307/00—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom
    • C07D307/02—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom not condensed with other rings
    • C07D307/04—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom not condensed with other rings having no double bonds between ring members or between ring members and non-ring members
    • C07D307/18—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom not condensed with other rings having no double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D307/20—Oxygen atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants

Definitions

  • the present disclosure relates to the field of biomedicine, in particular, the present disclosure relates to an antibody-drug conjugate with reduced reversible reactions, its preparation method and application, in particular to a new type of linker structure, and a drug-drug conjugate comprising the linker structure.
  • Antibody-drug conjugates have shown unique advantages over simple antibody drugs.
  • the monoclonal antibody is linked to biologically active molecules (such as cytotoxins), thus combining the tumor recognition targeting of the antibody and the efficient killing effect of the cytotoxin, and ingeniously solving the problems caused by the low efficacy of the antibody and the lack of targeting of the toxin.
  • biologically active molecules such as cytotoxins
  • the defect of excessive toxicity This enables ADC to accurately target tumor cells while reducing the impact on normal cells compared with traditional anti-tumor drugs, greatly improving the effectiveness and safety of anti-tumor drugs.
  • ADC generally includes three parts: antibody, linker (Linker) and biologically active molecules.
  • the biologically active molecular part of ADC is a toxin small molecule that plays a killing role, and generally kills tumor cells by inhibiting DNA or protein synthesis, inhibiting cell mitosis, and other methods.
  • Toxins currently used for ADC development mainly include microtubule inhibitors maytansinoids (see EP0425235, US5208020, US5416064, US7276497) and auristatins (MMAE/MMAF, see US2016304621A).
  • cytotoxins also include calicheamicins (Calicheamicin, see US5606040), benzobipyrrole derivatives (duocarmycin, see US7129261), pyrobenzodiazepines (PBDs, see WO2005/040170) and Dendrite derivatives.
  • the camptothecin derivatives include SN-38, DXD, CPT-11, exatecan, 9-nitrocamptothecin, 10-hydroxycamptothecin and the like.
  • the toxin is coupled to the antibody through a linker; the antibody can specifically recognize the target on the surface of the tumor cell, and enrich the ADC on the surface of the tumor cell, so that the ADC enters the tumor cell through the endocytosis effect, releases the toxin, and achieves specific killing role of tumors.
  • ADC joints are divided into two types: cleavable and non-cleavable.
  • the ideal joint should meet the requirements of "good stability and high release efficiency". Unstable joints will cause ADC off-target and increase safety risks, while overly stable joints will Affect the release rate of toxins, thereby affecting the efficacy of drugs. Therefore, the design of the linker is very important, which directly affects the efficacy and safety of ADC.
  • One end of the linker is connected to the antibody, and the other end is connected to the drug.
  • lysine and cysteine are common linking sites between antibodies and linkers. The ⁇ -amino group of the former can react with the activated carboxyl group of the linker to form an amide bond.
  • the antibody has multiple amino groups, which makes the coupling site and DAR value uncertain, and the product is not uniform, which is not conducive to drug stability.
  • ADCs choose cysteine as the coupling site.
  • One of the current methods for coupling linkers to antibody thiols is Michael addition reaction between free thiols on the antibody and maleimide (MC), or a specific substrate and free thiols on the antibody through two passes.
  • the present disclosure relates to a class of linker molecules with a specific structure, and the linker molecule ADC, which is obtained by improving the coupling method of toxins and targeting moieties in ADC or SMDC drugs, and the obtained conjugates have better stability, Higher therapeutic window, better therapeutic effect in colon cancer, pancreatic cancer and other tumor animal models, anti-tumor drug effect is better than traditional ADC with MC as linker.
  • the first aspect of the present disclosure provides an antibody-drug conjugate represented by formula (I), its stereoisomer or a pharmaceutically acceptable salt thereof, or the compound, its stereoisomer or a solvate of a pharmaceutically acceptable salt thereof,
  • Ab is an antibody, such as a monoclonal antibody or an antigen-binding fragment thereof;
  • L 1 is -L 12 -L 13 -L 14 -L 15 -
  • L 12 is selected from 5-6 membered heteroarylene
  • L 13 is selected from direct bonds
  • n is an integer selected from 1-10;
  • L 2 is selected from direct bonds, Preferably, L2 is , the amino terminal is connected to L 1 or L 15 , and the carbonyl terminal is connected to L 3 ;
  • p is an integer selected from 1-10;
  • L3 is an amino acid unit, preferably selected from amino acid residues or peptide residues consisting of 2-10 amino acid residues;
  • L 4 is selected from direct bonds
  • D is a drug linked to L4 through a chemical bond; if L4 is a direct bond, those skilled in the art will understand that D is linked to L3 ;
  • n is any value between 1-20.
  • antibody-drug conjugate represented by the above formula (I) refers to a composition of ADC molecules with the same or different numbers of linked drug molecules.
  • compositions comprising a plurality of ADC molecules.
  • each ADC in the compositions described herein comprises the same number of one or more drug molecules.
  • each ADC in the compositions described herein comprises a different number of one or more drug molecules.
  • each antibody can be coupled with 1, 2, 3, 4, 5, 6, 7, 8 or more drug molecules, such as 1 to 5 (e.g. 1, 2, 3, 4 or 5) drug molecules, 5 to 8 (e.g. 5, 6, 7 or 8) drug molecules or 8 to 12 (e.g. 8, 9, 10, 11 or 12) drug molecules.
  • 1 to 5 e.g. 1, 2, 3, 4 or 5
  • 5 to 8 e.g. 5, 6, 7 or 8
  • 8 to 12 e.g. 8, 9, 10, 11 or 12
  • the Drug Antibody Ratio refers to the number of drug molecules conjugated to the antibody (eg, n in Formula I).
  • the number of drug molecules contained in the ADC described herein is generally an integer, and when the number of drug molecules contained in the ADC described herein (for example, n in formula I) is a fraction, the fraction refers to the number of drug molecules contained in the ADC described herein.
  • L 12 is selected from:
  • L 12 is selected from:
  • L 12 is selected from
  • L 12 is
  • L 13 is selected from direct bonds.
  • m is an integer selected from 1-6.
  • m is selected from 1,2,3.
  • m is 3.
  • p is an integer selected from 1-6.
  • p is selected from 1, 2, 3.
  • p is 2.
  • L is a peptide residue consisting of 2 to 7 (preferably 2 to 4) amino acid residues; preferably, the amino acids are selected from the group consisting of phenylalanine, glycine, valine, lysine amino acid, citrulline, serine, glutamic acid, aspartic acid; more preferably, the amino acid is selected from glycine, phenylalanine, valine, citrulline, alanine.
  • L is selected from the group consisting of valine-citrulline (Val-Cit), alanine-alanine-asparagine (Ala-Ala-Asn), glycine-glycine-lysine ( Gly-Gly-lys), Valine-Lysine (Val-lys), Valine-Alanine (Val-Ala), Valine-Phenylalanine (Val-Phe), or Glycine-Glycine - phenylalanine-glycine (Gly-Gly-Phe-Gly); preferably selected from glycine-glycine-phenylalanine-glycine (Gly-Gly-Phe-Gly), valine-citrulline (Val- Cit), valine-alanine (Val-Ala).
  • L is selected from
  • L3 is glycine-glycine-phenylalanine-glycine (Gly-Gly-Phe-Gly).
  • ADCs comprising Gly-Gly-Phe-Gly exhibit increased stability, reduced off-target cell killing, increased on-target cell killing relative to ADCs comprising other amino acid units or other cleavable moieties , lower aggregation levels, and/or higher drug loading.
  • the amino terminus of L3 is attached to L2 and the carbonyl terminus is attached to L4 .
  • L2 is a direct bond
  • the amino terminal of L3 is connected to L1 or L15 .
  • L 4 is a direct bond
  • the carbonyl end of L 3 is connected to D, for example, to the amino group of eribulin.
  • the linker in the antibody conjugates of the present disclosure comprises at least one spacer unit linking the eribulin derivative D to the cleavable moiety. In some embodiments, the linker comprises a spacer unit attached to D.
  • the spacer unit comprises p-aminobenzyloxycarbonyl (PAB)
  • the spacer unit comprises p-aminobenzoyl
  • L is selected from direct bonds
  • L4 is a direct bond
  • the linker is stable extracellularly such that the ADC remains intact when present in the extracellular environment, but is capable of cleavage when internalized in a cell, eg, a cancer cell.
  • the ADC enters a cell expressing an antigen specific for the antibody portion of the ADC
  • the biologically active molecule portion is cleaved from the antibody portion, and the cleavage releases the unmodified form of the biologically active molecule.
  • the cleavable moiety in the linker is a cleavable peptide moiety.
  • ADCs comprising a cleavable peptide moiety exhibit lower levels of aggregation, improved antibody-to-drug ratios, increased targeted killing of cancer cells, reduced non- Off-target killing of cancer cells, and/or higher drug load (n).
  • addition of a cleavable moiety increases cytotoxicity and/or potency relative to a non-cleavable linker.
  • the increased potency and/or cytotoxicity is in cancers that express moderate levels of the antigen targeted by the antibody portion of the ADC (eg, moderate FRA expression).
  • the cleavable peptide moiety is capable of being cleaved by an enzyme
  • the linker is one that is cleavable by an enzyme.
  • the enzyme is a cathepsin and the linker is a linker that the cathepsin is capable of cleaving.
  • an enzyme-cleavable linker eg, a cathepsin-cleavable linker
  • the linker comprises a cleavable disulfide moiety that is capable of being cleaved under reducing conditions.
  • the linker can be attached via the amino group of D.
  • the linker can be attached via the hydroxyl group of D.
  • D is selected from eribulin or its derivatives (such as eribulin mesylate, etc.), DNA topoisomerase inhibitors, such as topoisomerase I inhibitors (such as camptophyllin Alkaline, Hydroxycamptothecin, 9-Aminocamptothecin, SN-38, Gemitecan, Gematecan, Irinotecan, Topotecan, Exatecan, DXD, Belotecan, Leto tecan, CKD-602, karenitecin, BN-80915, or rubitecan), topoisomerase II inhibitors (eg, actinomycin D, doxorubicin, doxorubicin, duocarmycin, daunorubicin drugs that interfere with DNA synthesis, such as methotrexate, 5-fluorouracil, cytarabine, gemcitabine, mercaptopurine, pentostatin, fludarabine , cladribine or n
  • the drug is selected from eribulin or its derivatives (such as eribulin mesylate, etc.), camptothecin or its derivatives (such as hydroxycamptothecin, 9-aminocamptothecin Alkaline, SN-38, gemitecan, gemotecan, irinotecan, topotecan, exatecan, DXD, belotecan, letotecan, CKD-602, karenitecin, BN-80915 or rubitecan), Monomethyl auristatin E (MMAE), Monomethyl auristatin F (MMAF), MonoMethyl Dolastatin 10 (MMAD).
  • camptothecin or its derivatives such as hydroxycamptothecin, 9-aminocamptothecin Alkaline, SN-38, gemitecan, gemotecan, irinotecan, topotecan, exatecan, DXD, belotecan, letotecan, CKD-60
  • the drug is selected from eribulin or its derivatives (such as eribulin mesylate, etc.), MMAE, DXD, preferably eribulin or its derivatives (such as eribulin mesylate, etc. Riblin, etc.).
  • the drug moiety conjugated to the subject antibody is selected from eribulin or a derivative thereof, eg, the mesylate salt of eribulin.
  • eribulin refers to a synthetic analog of halichondrin B, a macrocyclic compound originally isolated from the sponge Halichondria okadais. Eribulin is a microtubule inhibitor that is thought to bind tubulin and cause cell cycle arrest in G2/M phase by inhibiting mitotic spindle assembly.
  • eribulin mesylate refers to the mesylate salt of eribulin, which is sold under the tradename Halaven TM .
  • the drug is eribulin or its derivatives (such as eribulin mesylate, etc.).
  • D is N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-phenyl
  • n is any value between 1-10.
  • n is any value between 1-5.
  • n is any value between 3.5-5.0, eg, n is 3.9, 4.1 or 4.7.
  • the Ab in the antibody-drug conjugate (ADC) of the present disclosure is an antibody, such as a monoclonal antibody or an antigen-binding fragment thereof, and the antibody or an antigen-binding fragment thereof is selected from anti-Trop-2, CD37, HER2 , CD70, EGFRvIII, Mesothelin, Folate eceoptor1, Mucin 1, CD138, CD20, CD19, CD30, SLTRK6, Nectin 4, Tissue factor, Mucin16, Endothelin receptor, STEAP1, SLC39A6, Guanylylcyclase C, PSMA, CCD79b, CD22, Sodium ph osphate cotransporter2B, GPNMB, Trophoblast glycoprotein, AGS-16, EGFR, CD33, CD66e, CD74, CD56, PD-L1, TACSTD2, DR5, E16, STEAP1, 0772P, MPF, Napi3b, Sema 5b,
  • the monoclonal antibody or antigen-binding fragment thereof comprises Fab, Fab', F(ab') 2 , Fd, Fv, dAb, complementarity determining region fragments, single chain antibody (for example, scFv ), non-human antibody, humanized antibody, chimeric antibody, fully human antibody, pre-antibody (Probody), bispecific antibody or multispecific antibody.
  • the Ab is an anti-Her 2 monoclonal antibody, such as Trastuzumab, Pertuzumab, or an anti-Trop-2 monoclonal antibody, such as Sacituzumab.
  • the antibody in the antibody-drug conjugate is a known antibody, selected from but not limited to Trastuzumab (Trastuzumab), Pertuzumab (Pertuzumab), niger Nimotuzumab, Enoblituzumab, Emibetuzumab, Inotuzumab, Vitin-Pinatuzumab , Brentuximab, Gemtuzumab, Bivatuzumab, Lorvotuzumab, cBR96, and Glematumamab or antigen-binding fragments thereof.
  • the targeting moiety is trastuzumab described in US5821337, pertuzumab described in US7560111 of SEQ ID No.: 16 and SEQ ID No.: 15 ( pertuzumab), the antibodies that can be used in the present disclosure can also be screened by the carrier design, construction and construction of the antibody library displaying antibodies disclosed in CN103476941A, or can be screened with the library of Sorrento Therapeutics, Inc. get.
  • the Lys at the end of the heavy chain of the targeting moiety is easily deleted without affecting the biological activity, see Dick, L.W. et al., Biotechnol. Bioeng., 100: 1132-1143.
  • the targeting moiety is an anti-Trop-2 monoclonal antibody, such as the deletion of Lys at the end of the heavy chain of Sacituzumab, for example, the targeting moiety is an anti-Her 2 monoclonal antibody, such as Trastuzumab, Paragon Deletion of Lys at the end of the heavy chain of Pertuzumab.
  • the Her-2 antibody is the trastuzumab antibody described in US5821337
  • the complementarity determining region (CDR) of the light chain variable region includes CDR1 consisting of the RASQDVNTAVA amino acid sequence; consisting of the SASFLYS amino acid sequence and CDR3 consisting of the QQHYTTPPT amino acid sequence
  • the CDRs of the heavy chain variable region thereof include CDR1 consisting of the DTYIH amino acid sequence; CDR2 consisting of the RIYPTNGYTRY amino acid sequence; and CDR3 consisting of the WGGDGFYAMDY amino acid sequence.
  • the light chain sequence and heavy chain sequence of the trastuzumab antibody are shown in SEQ ID NO: 5 and SEQ ID NO: 6, respectively.
  • Antibodies that retain Her2-binding activity after conservative amino acid substitutions to the above-mentioned antibodies may also be included.
  • trastuzumab antibody Heavy chain amino acid sequence of trastuzumab antibody:
  • ErbB2 and Her2/neu are used interchangeably, both of which refer to the native sequence human Her2 protein (Genebank accession number: X03363, see e.g. Semba et al., 1985, PNAS, 82:6497-6501; and Yamamoto et al., 1986, Nature, 319:230-234) and their functional derivatives, such as amino acid sequence variants.
  • ErbB2 indicates the gene encoding human Her2, and neu indicates the gene encoding rat p185neu.
  • the compounds or conjugates of the present disclosure can inhibit or kill cells expressing ErbB2 receptors, such as breast cancer cells, ovarian cancer cells, gastric cancer cells, endometrial cancer cells, salivary gland cancer cells, lung cancer cells, Kidney, colon, thyroid, pancreatic, bladder, or liver cancer cells.
  • ErbB2 receptors such as breast cancer cells, ovarian cancer cells, gastric cancer cells, endometrial cancer cells, salivary gland cancer cells, lung cancer cells, Kidney, colon, thyroid, pancreatic, bladder, or liver cancer cells.
  • the targeting portion of the anti-Trop-2 antibody is RS7 described in US Patent No. 7,517,964; RS7 described in US Patent No. 6,653,104; and hRS7 described in US2012/0237518.
  • the full name of Trop-2 antibody is human trophoblast cell surface antigen 2 (Trop-2), which is a 40kDa transmembrane glycoprotein encoded by the TACSTD2 gene. Trop-2 was first identified in the human trophoblast choriocarcinoma cell line, and its intracellular tail plays an important role in controlling many signaling pathways that regulate cell functions, such as cell-cell adhesion, cell proliferation, and cell migration.
  • Trop-2 protein in many human tumors such as breast cancer, colorectal cancer, lung cancer, pancreatic cancer, ovarian cancer, prostate cancer, cervical cancer, kidney cancer, urethral cancer, glioma, melanoma, liver cancer, bladder cancer, Gastric cancer, esophageal cancer
  • Trop-2 has the functions of regulating tumor cell growth, promoting tumor cell invasion and metastasis.
  • Trop-2 antibodies that can be used in the present disclosure 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_SVG described, for example, in CN104053672A 8.
  • the anti-Trop-2 antibody that can be used in the present disclosure can also be obtained by screening the carrier design, construction and construction of an antibody library displaying antibodies disclosed in CN103476941A, and can also be obtained from the library of Sorrento Therapeutics, Inc. obtained by screening.
  • the natural sequence Trop-2 in the present disclosure can be isolated from nature, and can also be prepared by recombinant DNA technology, chemical synthesis or a combination thereof.
  • Antibodies used in the present disclosure are preferably anti-human Trop-2 antibodies.
  • the CDR1, CDR2 and/or CDR3 of the heavy chain and light chain of the anti-human Trop-2 antibody are respectively the CDR1, CDR2 and/or CDR3 of the heavy chain and light chain of the RS7 monoclonal antibody.
  • the anti-human Trop-2 antibody can be a humanized antibody or a fully human antibody.
  • the antibody is the RS7 antibody described in CN100360567C, wherein the complementarity determining region (CDR) of the light chain variable region of the humanized or chimeric RS7 antibody includes CDR1 consisting of the KASQDVSIAVA amino acid sequence; CDR2 consisting of the amino acid sequence of SASYRYT; and CDR3 consisting of the amino acid sequence of QQHYITPLT, and wherein the CDR of the heavy chain variable region of the humanized or chimeric RS7 MAb comprises CDR1 consisting of the amino acid sequence of NYGMN; CDR2 consisting of the amino acid sequence of WINTYTGEPTYTDDFKG; and CDR3 consisting of the GGFGSSYWYFDV amino acid sequence.
  • CDR complementarity determining region
  • the light chain sequence and heavy chain sequence of RS7 are respectively shown in SEQ ID NO: 1 and SEQ ID NO: 2;
  • the coding nucleotide sequences of the light chain and heavy chain of the anti-Trop-2 antibody are respectively shown in SEQ Shown in ID NO:3 and SEQ ID NO:4.
  • Antibodies that retain Trop-2 binding activity after conservative amino acid substitutions to the above antibodies may also be included.
  • the antibody drug conjugate is selected from:
  • -S- is not an additional external sulfur atom, but the sulfhydryl group contained in the Ab itself after the disulfide bond is opened. -S- formed after making a ligation.
  • the second aspect of the present disclosure provides a compound represented by formula (II), a pharmaceutically acceptable salt thereof, a stereoisomer thereof, or a solvate thereof,
  • L 1' is L 11 -L 12 -L 13 -L 14 -L 15 -;
  • L 12 , L 13 , L 14 , L 15 are as described above;
  • L 2 , L 3 , L 4 , and D are as described above.
  • the compound represented by formula (II) can be formed by linking the compound represented by the following formula (IV) with a drug, the linking site of the drug is an amino group, and the drug is as described above, Eribulin or its derivatives (such as Eribulin mesylate, etc.) are preferred.
  • provided linkers comprise a cleavable sulfonamide moiety, which linkers are capable of cleavage under reducing conditions.
  • the compound represented by formula (II) is selected from:
  • the third aspect of the present disclosure provides the compound represented by formula (III), its pharmaceutically acceptable salt, its stereoisomer, or its solvate,
  • L 12 , L 13 , L 14 are as described above;
  • the compound represented by formula (III) is N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-N-phenyl
  • the fourth aspect of the present disclosure provides an intermediate compound represented by formula (IV), a pharmaceutically acceptable salt thereof, a stereoisomer thereof, or a solvate thereof,
  • L 1' is L 11 -L 12 -L 13 -L 14 -L 15 -;
  • L 12 , L 13 , L 14 , L 15 are as described above;
  • L 2 and L 3 are as described above.
  • the intermediate compound represented by formula (IV) is selected from
  • the method comprises:
  • the sixth aspect of the present disclosure provides a method for preparing the aforementioned antibody-drug conjugate represented by formula (I), its pharmaceutically acceptable salt, its stereoisomer, or its solvate, which includes:
  • the compound represented by formula (II), its pharmaceutically acceptable salt, its stereoisomer, or its solvate is prepared by the method described in the fifth aspect.
  • the seventh aspect of the present disclosure provides a pharmaceutical composition
  • a pharmaceutical composition comprising the antibody-drug conjugate of the first aspect of the present disclosure, its stereoisomer or a pharmaceutically acceptable salt thereof, or the antibody-drug conjugate
  • the eighth aspect of the present disclosure provides a pharmaceutical preparation, which comprises the antibody-drug conjugate of the first aspect of the present disclosure, its stereoisomer or a pharmaceutically acceptable salt thereof, or the antibody- Drug conjugates, solvates of their stereoisomers or pharmaceutically acceptable salts thereof, or compounds of the second aspect of the disclosure, their stereoisomers, pharmaceutically acceptable salts thereof, or solvates thereof thing.
  • the ninth aspect of the present disclosure provides the antibody-drug conjugate of the first aspect of the present disclosure, its stereoisomer or a pharmaceutically acceptable salt thereof, or the antibody-drug conjugate, its stereo
  • the solvate of the isomer or its pharmaceutically acceptable salt, or the compound of the second aspect of the present disclosure, its stereoisomer, its pharmaceutically acceptable salt, or its solvate, or the seventh aspect of the present disclosure The pharmaceutical composition according to the aspect or the pharmaceutical preparation according to the eighth aspect, which is used for preventing and/or treating tumor or cancer.
  • the tenth aspect of the present disclosure provides a method for preventing or treating cancer, which comprises administering an effective amount of the antibody-drug conjugate described in the first aspect of the present disclosure, its stereoisomer or Its pharmaceutically acceptable salt, or the solvate of the antibody-drug conjugate, its stereoisomer or its pharmaceutically acceptable salt, or the compound of the second aspect of the present disclosure, its stereoisomer , a pharmaceutically acceptable salt thereof, or a solvate thereof, or the pharmaceutical composition described in the seventh aspect or the pharmaceutical preparation described in the eighth aspect of the present disclosure.
  • the eleventh aspect of the present disclosure provides the antibody-drug conjugate of the first aspect of the present disclosure, its stereoisomer or a pharmaceutically acceptable salt thereof, or the antibody-drug conjugate, its A solvate of a stereoisomer or a pharmaceutically acceptable salt thereof, or the compound of the second aspect of the present disclosure, its stereoisomer, a pharmaceutically acceptable salt thereof, or a solvate thereof, or the compound of the second aspect of the present disclosure
  • the twelfth aspect of the present disclosure provides the antibody-drug conjugate described in the first aspect, its stereoisomer or a pharmaceutically acceptable salt thereof, or the antibody-drug conjugate, its stereoisomer or the solvate of the compound or its pharmaceutically acceptable salt, or the compound of the second aspect of the present disclosure, its stereoisomer, its pharmaceutically acceptable salt, or its solvate, or the compound of the seventh aspect of the present disclosure
  • the use of the above-mentioned pharmaceutical composition or the pharmaceutical preparation according to the eighth aspect for preparing a reagent for inhibiting the growth, proliferation or migration of cancer cells.
  • the thirteenth aspect of the present disclosure provides the antibody-drug conjugate described in the first aspect, its stereoisomer or a pharmaceutically acceptable salt thereof, or the antibody-drug conjugate, its stereoisomer or the solvate of the compound or its pharmaceutically acceptable salt, or the compound of the second aspect of the present disclosure, its stereoisomer, its pharmaceutically acceptable salt, or its solvate, or the compound of the seventh aspect of the present disclosure
  • the pharmaceutical composition described above or the pharmaceutical preparation described in the eighth aspect which is used for inhibiting the growth, proliferation or migration of cancer cells.
  • the fourteenth aspect of the present disclosure provides a method for inhibiting the growth, proliferation or migration of cancer cells, which comprises administering to cancer cells an effective amount of the antibody-drug conjugate described in the first aspect of the present disclosure, its stereoisomer or a pharmaceutically acceptable salt thereof, or a solvate of the antibody-drug conjugate, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof, or the compound of the second aspect of the present disclosure, its stereoisomer Construct, its pharmaceutically acceptable salt, or its solvate, or the pharmaceutical composition described in the seventh aspect or the pharmaceutical preparation described in the eighth aspect of the present disclosure.
  • the methods are used for non-therapeutic purposes, such as for scientific research.
  • the fifteenth aspect of the present disclosure provides a kit for inhibiting the growth, proliferation or migration of cancer cells, which includes the antibody-drug conjugate described in the first aspect of the present disclosure, its stereoisomer or its pharmaceutically acceptable Accepted salts, or solvates of the antibody-drug conjugates, stereoisomers thereof, or pharmaceutically acceptable salts thereof, or compounds of the second aspect of the present disclosure, stereoisomers thereof, pharmaceutically acceptable salts thereof, An acceptable salt, or a solvate thereof, or the pharmaceutical composition described in the seventh aspect or the pharmaceutical preparation described in the eighth aspect of the present disclosure.
  • Antibody-drug conjugates of the present disclosure may preferably be administered to mammals, more preferably humans.
  • Substances used in pharmaceutical compositions containing the antibody-drug conjugate of the present disclosure may be appropriately selected from formulation additives or other substances commonly used in this field in consideration of the dosage and concentration to be administered.
  • the antibody-drug conjugate of the present disclosure can be administered in the form of a pharmaceutical composition or pharmaceutical preparation containing one or more pharmaceutically compatible ingredients.
  • the above-mentioned pharmaceutical composition or pharmaceutical preparation may typically contain more than one pharmaceutically acceptable carrier (such as sterile liquid (such as water and oil (including petroleum, animal, vegetable, or synthetic sources) Oil (such as peanut oil, soybean oil, mineral oil, sesame oil, etc.))).
  • sterile liquid such as water and oil (including petroleum, animal, vegetable, or synthetic sources) Oil (such as peanut oil, soybean oil, mineral oil, sesame oil, etc.)
  • Oil such as peanut oil, soybean oil, mineral oil, sesame oil, etc.
  • water is a more representative carrier.
  • saline solution, and aqueous glucose and glycerol solutions are also Can be used as a liquid carrier, especially for injection solutions.
  • Suitable pharmaceutical excipients are known in this field. According to needs, the above-mentioned composition can also contain a small amount of wetting agent or emulsifying agent, or pH buffering Examples of suitable pharmaceutical carriers are described in "Examples of W. Martin Carriers Armaceutical Sciences” by E.W. Martin. The formulation thereof corresponds to the mode of administration.
  • Various delivery systems are known and can be used for administering the antibody-drug conjugates of the present disclosure.
  • the introduction method include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, and subcutaneous routes. Administration may be by infusion or bolus injection, for example.
  • the above-mentioned antibody-drug conjugate is administered by infusion. Parenteral administration is the preferred route of administration.
  • the above-mentioned pharmaceutical composition is formulated into a pharmaceutical composition for intravenous administration to humans, and a prescription is prepared according to conventional procedures.
  • compositions for intravenous administration are solutions in sterile isotonic aqueous buffer.
  • the above-mentioned pharmaceutical composition may also contain a solubilizer and a local anesthetic (such as lidocaine) for relieving pain at the injection site.
  • the above-mentioned ingredients can be supplied by either: as a dry freeze-dried powder or as an anhydrous concentrate in a sealed container, such as an ampoule or sachet, etc., which seals the amount of active agent, respectively.
  • the above-mentioned drug may be supplied mixed together in a unit dosage form.
  • the above-mentioned drug can be put into an infusion bottle containing sterile pharmaceutical-grade water or saline.
  • an ampoule of sterilized water for injection or saline can be provided so that, for example, the above-mentioned components are mixed before administration.
  • the pharmaceutical composition or pharmaceutical preparation of the present disclosure may be a pharmaceutical composition or pharmaceutical preparation containing only the antibody-drug conjugate of the present disclosure, or may contain an antibody-drug conjugate and at least one other cancer therapeutic agent. pharmaceutical composition.
  • the antibody-drug conjugate of the present disclosure can also be administered together with other cancer therapeutic agents, thereby enhancing the anticancer effect.
  • Other anticancer agents used for this purpose may be administered to the individual simultaneously, separately or sequentially with the antibody-drug conjugate, or may be administered with varying intervals of administration.
  • cancer therapeutic agents include nab-paclitaxel, carboplatin, cisplatin, gemcitabine, irinotecan (CPT-11), paclitaxel, pemetrexed, sorafenib, vinblastine, and international patents.
  • Drugs described in pamphlet WO2003/038043, and LH-RH analogs (leuprolide, goserelin, etc.), estramustine phosphate, estrogen antagonists (tamoxifen, ryloxetin, etc.) oxifen, etc.), aromatase inhibitors (anastrozole, letrozole, exemestane), etc., but are not limited as long as they have antitumor activity.
  • Such a pharmaceutical composition can be formulated as a preparation having a selected composition and necessary purity, in the form of a freeze-dried preparation or a liquid preparation.
  • a freeze-dried preparation it may contain appropriate formulation additives available in the art.
  • the preparations can be formed as liquid preparations containing various preparation additives that can be used in this field.
  • composition and concentration of the pharmaceutical composition vary depending on the method of administration, but the affinity of the antibody-drug conjugate contained in the pharmaceutical composition of the present disclosure to the antigen of the antibody-drug conjugate, that is, the affinity for the antigen Considering the dissociation constant (Kd value), the higher the affinity (the lower the Kd value), the more effective the drug can be exhibited even with a small dose. Therefore, when determining the dosage of the antibody-drug conjugate, the dosage can also be set based on the condition of the affinity of the antibody-drug conjugate to the antigen.
  • the antibody-drug conjugate of the present disclosure is administered to humans, for example, it can be administered once at about 0.001 to 100 mg/kg, or administered multiple times at intervals of once every 1 to 180 days.
  • the antibody-drug conjugates, pharmaceutical compositions, and pharmaceutical preparations of the present disclosure can be used to prevent and/or treat tumors or cancers.
  • the tumor or cancer to be prevented and/or treated may be any cancer cell expressing a protein recognizable by the antibody in the antibody-drug conjugate.
  • the tumor or cancer is selected from breast cancer, colorectal cancer, lung cancer, pancreatic cancer, ovarian cancer, prostate cancer, cervical cancer, renal cancer, urethral cancer, glioma, melanoma tumor, liver cancer, bladder cancer, gastric cancer, esophageal cancer; preferably, the cancer is carcinoma in situ or metastatic carcinoma.
  • a prophylactically or therapeutically effective amount of the disclosed antibody-drug conjugate, pharmaceutical composition, or pharmaceutical preparation is administered to a subject in need for inhibiting the growth, proliferation, or migrate.
  • kits for inhibiting growth, proliferation or migration of cancer cells which includes the antibody-drug conjugate, pharmaceutical composition or pharmaceutical preparation of the present disclosure.
  • biologically active molecule refers to a substance that inhibits or prevents cell function and/or causes cell death or destruction.
  • the biologically active substance or bioactive molecule in the conjugate is an anti-tumor active molecule.
  • radioactive isotopes such as those of At 211 , I 131 , I 125 , Y 90 , Re 186 , Re 188 , Sm 153 , Bi 212 , P 32 , Pb 212 and Lu; metal complexes such as metal platinum complexes , metal gold complexes, oxaliplatin, etc.; glycopeptide antibiotics, such as bleomycin, pingyangmycin; DNA topoisomerase inhibitors, such as topoisomerase I inhibitors, camptothecin, hydroxyhippin Tricine, 9-aminocamptothecin, DXD, SN-38, irinotecan, topotecan, belotecan, rubitecan, topoisomerase II inhibitors, actinomycin D, doxorubicin , doxorubicin, docarmycin, daunorubicin, mitoxantrone, podophyllotoxin, etoposide, etc.; drugs
  • toxin refers to a substance capable of having deleterious effects on the growth or proliferation of cells.
  • the biologically active molecule is denoted as D, and immunomodulators, especially TLR8 agonists, can also be selected.
  • linker refers to a chemical structural fragment or bond that is connected to a ligand at one end and a drug at the other end, and can also be connected after other linkers. Then connect with the drug.
  • a joint may comprise one or more joint components.
  • exemplary linker building blocks include 4-(5-methanesulfonyl[1,3,4]-oxadiazole)butanoic acid of the present disclosure, and 6-maleimidocaproyl (MC) of the prior art, Maleimidopropionyl (MP), valine-citrulline (Val-Cit or vc), alanine-phenylalanine (ala-phe), p-aminobenzyloxycarbonyl (PAB), and Those derived from coupling with linker reagents: N-succinimidyl 4-(2-pyridylthio)pentanoate (SPP), N-succinimidyl 4-(N-maleimido Methyl)cyclohexane-1 carboxylate (SMCC, also referred to herein as MCC) and N-succinimidyl (4-iodo-acetyl)aminobenzoate (SIA
  • Linkers can include Stretch units, Spacer units, Amino Acid units and Stretcher units. Can be synthesized by methods known in the art, such as described in US2005-0238649A1.
  • the linker can be a "cleavable linker" that facilitates release of the drug in the cell.
  • acid-labile e.g., hydrazone
  • protease-sensitive e.g., peptidase-sensitive
  • photolabile, dimethyl, or disulfide-containing linkers can be used (Chari et al., Cancer Research 52:127-131 (1992); US Patent No. 5,208,020).
  • stretch unit refers to a chemical structural fragment that is covalently linked to the antibody through a carbon atom at one end and to an amino acid unit, disulfide moiety, sulfonamide moiety, or non-peptidic chemical moiety at the other end.
  • spacer unit is a bifunctional compound structural fragment that can be used to couple amino acid units and cytotoxic drugs to form antibody-drug conjugates. This coupling method can selectively link cytotoxic drugs to amino acid units superior.
  • amino acid refers to an organic compound containing an amino group and a carboxyl group in the molecular structure, and both the amino group and the carboxyl group are directly connected to the -CH- structure.
  • the general formula is H 2 NCHRCOOH, R is H, substituted or unsubstituted alkyl, etc. According to the position of the amino group connected to the carbon atom in the carboxylic acid, it can be divided into ⁇ , ⁇ , ⁇ , ⁇ , ⁇ ...-amino acids.
  • amino acids that make up natural proteins have their specific structural characteristics, that is, their amino groups are directly connected to the ⁇ -carbon atom, that is, ⁇ -amino acids, including glycine (Glycine), alanine (Alanine), valine (Valine), Leucine, Isoleucine, Phenylalanine, Tryptophan, Tyrosine, Aspartic acid, Histidine, Asparagine, Glutamic acid, Lysine, Glutamine, Methionine, Arginine , Serine, Threonine, Cysteine, Proline, etc. Unnatural amino acids such as citrulline.
  • the spacer unit in the present disclosure is PAB, which has a structure such as a p-aminobenzyloxycarbonyl fragment, and is connected to D.
  • adapter components of the present disclosure include, but are not limited to:
  • SM 4-(5-methylsulfonyl[1,3,4]-oxadiazole)butanoic acid
  • MC 6-maleimidocaproyl
  • Val-Cit or "vc” valine-citrulline (an exemplary dipeptide in a protease cleavable linker);
  • Citrulline 2-amino-5-ureidopentanoic acid
  • Me-Val-Cit N-methyl-valine-citrulline (wherein the linker peptide bond has been modified to prevent its cleavage by cathepsin B);
  • MC(PEG) 6 -OH maleimidocaproyl-polyethylene glycol (can be attached to antibody cysteine);
  • SPP N-succinimidyl 4-(2-pyridylthio)pentanoate
  • SPDP N-succinimidyl 3-(2-pyridyldithio)propionate
  • SMCC succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate
  • PBS Phosphate Buffered Saline.
  • antibody-drug conjugate means that a ligand is linked to a biologically active drug through a stable linker unit.
  • antibody drug conjugate refers to linking a monoclonal antibody or antibody fragment with a toxic drug with biological activity through a stable linker unit.
  • drug loading can be expressed as the ratio of the amount of drug to the amount of antibody.
  • the range of drug loading can be 1-20, preferably 1-10 cytotoxic drugs (D) linked to each antibody (Ab).
  • the drug loading is expressed as n, which can be exemplarily 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or the mean of any two values in between.
  • the average amount of drug per ADC molecule after conjugation can be identified using routine methods such as UV/Vis spectroscopy, mass spectrometry, ELISA assay, monoclonal antibody size variant assay (CE-SDS) and HPLC characterization.
  • the loading of antibody-drug conjugates can be controlled by the following non-limiting methods, including:
  • antibody refers to an immunoglobulin, which is a tetrapeptide chain structure composed of two identical heavy chains and two identical light chains linked by interchain disulfide bonds.
  • the amino acid composition and sequence of the constant region of the immunoglobulin heavy chain are different, so their antigenicity is also different.
  • immunoglobulins can be divided into five classes, or isotypes of immunoglobulins, namely IgM, IgD, IgG, IgA, and IgE, and their corresponding heavy chains are ⁇ , ⁇ , and ⁇ chains, respectively. , ⁇ chain, and ⁇ chain.
  • IgG can be divided into different subclasses according to the amino acid composition of its hinge region and the number and position of heavy chain disulfide bonds.
  • IgG can be divided into IgG1, IgG2, IgG3, and IgG4.
  • Light chains are classified as either kappa chains or lambda chains by difference in the constant region.
  • Each of the five Ig classes can have either a kappa chain or a lambda chain.
  • Antibodies of the present disclosure are preferably specific antibodies against cell surface antigens on target cells, non-limiting examples are the following antibodies: anti-HER2 (ErbB2) antibody, anti-EGFR antibody, anti-B7-H3 antibody, anti-c-Met Antibody, anti-HER3 (ErbB3) antibody, anti-HER4 (ErbB4) antibody, anti-CD20 antibody, anti-CD22 antibody, anti-CD30 antibody, anti-CD33 antibody, anti-CD44 antibody, anti-CD56 antibody, anti-CD70 antibody, anti-CD73 antibody, anti-CD105 Antibody, Anti-CEA Antibody, Anti-A33 Antibody, Anti-Cripto Antibody, Anti-EphA2 Antibody, Anti-G250 Antibody, Anti-MUCl Antibody, Anti-Lewis Y Antibody, Anti-VEGFR Antibody, Anti-GPNMB Antibody, Anti-Integrin Antibody, Anti-PSMA Antibody, Anti-Tenascin- C antibody, anti-SLC44A4 antibody, anti-
  • the antibody is selected from Trastuzumab (Trastuzumab, trade name Herceptin), Pertuzumab (Pertuzumab, also known as 2C4, trade name Perjeta), Nimotuzumab (Nimotuzumab, trade name Mingtai Xinsheng), Embolituzumab (Enoblituzumab), Emibetuzumab (Emibetuzumab), Inotuzumab (Inotuzumab), Vitin-Pinatuzumab (Pinatuzumab) Brentuximab, Gemtuzumab, Bivatuzumab, Lorvotuzumab, cBR96, and Glematumamab.
  • Trastuzumab Trastuzumab, trade name Herceptin
  • Pertuzumab Pertuzumab, also known as 2C4, trade name Perjeta
  • Nimotuzumab Nemotuzum
  • variable region The sequence of about 110 amino acids near the N-terminal of the antibody heavy chain and light chain varies greatly, which is the variable region (Fv region); the rest of the amino acid sequence near the C-terminal is relatively stable, which is the constant region.
  • the variable region includes 3 hypervariable regions (HVR) and 4 framework regions (FR) with relatively conserved sequences. Three hypervariable regions determine the specificity of antibodies, also known as complementarity determining regions (CDR).
  • CDR complementarity determining regions
  • Each light chain variable region (LCVR) and heavy chain variable region (HCVR) consists of 3 CDR regions and 4 FR regions, and the sequence from the amino terminal to the carboxyl terminal is: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • the 3 CDR regions of the light chain refer to LCDR1, LCDR2, and LCDR3; the 3 CDR regions of the heavy chain refer to HCDR1, HCDR2, and HCDR3.
  • Antibodies of the present disclosure include murine antibodies, chimeric antibodies, humanized antibodies and fully human antibodies, preferably humanized antibodies and fully human antibodies.
  • murine antibody in this disclosure refers to an antibody prepared using a mouse according to the knowledge and skill in the art. In preparation, test subjects are injected with the specified antigen, and hybridomas expressing antibodies having the desired sequence or functional properties are isolated.
  • chimeric antibody is an antibody formed by fusing the variable region of a murine antibody with the constant region of a human antibody, which can reduce the immune response induced by the murine antibody.
  • To establish a chimeric antibody it is necessary to first establish a hybridoma that secretes a mouse-derived specific monoclonal antibody, then clone the variable region gene from the mouse hybridoma cell, and then clone the constant region gene of the human antibody as required, and then clone the mouse variable region gene It is connected with the human constant region gene to form a chimeric gene and inserted into an expression vector, and finally expresses the chimeric antibody molecule in a eukaryotic system or a prokaryotic system.
  • humanized antibody also known as CDR-grafted antibody (CDR-grafted antibody) refers to the antibody variable region framework grafted with mouse CDR sequences to humans, that is, different types of human germline antibodies Antibodies generated in the framework sequences. It can overcome the heterologous reaction induced by chimeric antibodies due to carrying a large amount of mouse protein components.
  • framework sequences can be obtained from public DNA databases or published references that include germline antibody gene sequences.
  • the germline DNA sequences of the human heavy and light chain variable region genes can be found in the "VBase" human germline sequence database (available on the Internet at www.mrccpe.com.ac.uk/vbase), as well as in Kabat, E.A. et al.
  • the humanized antibody of the present disclosure also includes the humanized antibody after affinity maturation of CDR by phage display. Further descriptions of methods involving the use of mouse antibodies in humanization include, for example, Queen et al., Proc., Natl. 321, 522 (1986), Riechmann, et al., Nature, 332, 323-327 (1988), Verhoeyen, et al., Science, 239, 1534 (1988)].
  • the development of monoclonal antibodies has gone through four stages, namely: murine monoclonal antibodies, chimeric monoclonal antibodies, humanized monoclonal antibodies and fully human monoclonal antibodies.
  • the present disclosure is a fully human monoclonal antibody.
  • the relevant technologies for the preparation of fully human antibodies mainly include: human hybridoma technology, EBV transformed B lymphocyte technology, phage display technology (phage display), transgenic mouse antibody preparation technology (transgenic mouse) and single B cell antibody preparation technology, etc.
  • antigen-binding fragment refers to one or more fragments of an antibody that retain the ability to specifically bind an antigen. It has been shown that fragments of full-length antibodies can be utilized to perform the antigen-binding function of the antibody.
  • binding fragments included in "antigen-binding fragments" include (i) Fab fragments, monovalent fragments consisting of VL, VH, CL and CH1 domains; (ii) F(ab') 2 fragments, comprising (iii) Fd fragment consisting of VH and CH1 domains; (iv) Fv fragment consisting of VH and VL domains of a single arm of an antibody; (v ) a single domain or dAb fragment (Ward et al., (1989) Nature 341:544-546) consisting of a VH domain; and (vi) isolated complementarity determining regions (CDRs) or (vii) optionally via A combination of two or more isolated CDRs joined by a synthetic linker.
  • CDRs complementarity
  • the two domains VL and VH of the Fv fragment are encoded by separate genes, they can be linked by a synthetic linker using recombinant methods, thus making it possible to produce a single protein in which the VL and VH regions pair to form a monovalent molecule. chain (referred to as single-chain Fv (scFv); see, eg, Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci USA 85:5879-5883).
  • single chain antibodies are also intended to be encompassed within the term "antigen-binding fragment" of an antibody.
  • Antigen-binding portions can be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact immunoglobulins.
  • Antibodies can be of different isotypes, eg, IgG (eg, IgGl, IgG2, IgG3, or IgG4 subtype), IgAl, IgA2, IgD, IgE, or IgM antibodies.
  • Fab is an antibody fragment having a molecular weight of about 50,000 and having antigen-binding activity among fragments obtained by treating an IgG antibody molecule with the protease papain (cleaving the amino acid residue at position 224 of the H chain), in which About half and the entire L chain is held together by disulfide bonds.
  • F(ab')2 is an antibody having a molecular weight of about 100,000 and having antigen-binding activity and comprising two Fab regions connected at the hinge position obtained by digesting the lower portion of the two disulfide bonds in the IgG hinge region with the enzyme pepsin fragment.
  • Fab' is an antibody fragment having a molecular weight of about 50,000 and having antigen-binding activity obtained by cleaving the disulfide bond in the hinge region of the above-mentioned F(ab')2.
  • the Fab' fragment can be produced by inserting DNA encoding a Fab' fragment of an antibody into a prokaryote expression vector or a eukaryote expression vector and introducing the vector into a prokaryote or eukaryote to express the Fab'.
  • single-chain antibody single-chain Fv or “scFv” is meant to comprise an antibody heavy chain variable domain (or region; VH) and an antibody light chain variable domain (or region; VL) connected by a linker molecules.
  • Such scFv molecules may have the general structure: NH2 -VL-linker-VH-COOH or NH2 -VH-linker-VL-COOH.
  • Suitable prior art linkers consist of the repeated GGGGS amino acid sequence or variants thereof, for example using 1-4 repeat variants (Holliger et al. (1993), Proc. Natl. Acad. Sci. USA 90:6444-6448) .
  • linkers useful in the present disclosure are described by Alfthan et al. (1995), Protein Eng. 8:725-731, Choi et al. (2001), Eur.J. Immuno 1.31:94-106, Hu et al. (1996) , Cancer Res. 56:3055-3061, described by Kipriyanov et al. (1999), J. Mol. Biol. 293:41-56 and Roovers et al. (2001 ), Cancer Immunol.
  • CDR refers to one of the six hypervariable regions within the variable domain of an antibody that primarily contribute to antigen binding.
  • One of the most commonly used definitions of the six CDRs is provided by Kabat E.A. et al., (1991) Sequences of proteins of immunological interest. NIH Publication 91-3242).
  • the Kabat definition of CDRs applies only to CDR1, CDR2, and CDR3 (CDR L1, CDR L2, CDR L3 or L1, L2, L3) of the light chain variable domain, and to CDR1, CDR L2, and L3 of the heavy chain variable domain.
  • CDR2 and CDR3 CDR H2, CDR H3 or H2, H3).
  • CDR1, HCDR2, HCDR3 there are three CDRs (HCDR1, HCDR2, HCDR3) in each heavy chain variable region and three CDRs (LCDR1, LCDR2, LCDR3) in each light chain variable region.
  • Amino acid sequence boundaries for CDRs can be determined using any of a variety of well-known schemes, including the "Kabat” numbering convention (see Kabat et al.
  • the CDR amino acid residues in the heavy chain variable domain are numbered 31-35 (HCDR1), 50-65 (HCDR2) and 95-102 (HCDR3);
  • the CDR amino acid residues in the chain variable domain (VL) are numbered 24-34 (LCDR1), 50-56 (LCDR2) and 89-97 (LCDR3).
  • the CDR amino acid numbers in VH are 26-32 (HCDR1), 52-56 (HCDR2) and 95-102 (HCDR3); and the amino acid residues in VL are numbered 26-32 (LCDR1), 50- 52 (LCDR2) and 91-96 (LCDR3).
  • the CDRs consist of amino acid residues 26-35 (HCDR1), 50-65 (HCDR2) and 95-102 (HCDR3) in human VH and amino acid residues 24- 34 (LCDR1), 50-56 (LCDR2) and 89-97 (LCDR3).
  • the numbering of CDR amino acid residues in VH is approximately 26-35 (CDR1), 51-57 (CDR2) and 93-102 (CDR3)
  • the numbering of CDR amino acid residues in VL is approximately 27-32 (CDR1 ), 50-52 (CDR2) and 89-97 (CDR3).
  • the CDR regions of antibodies can be determined using the program IMGT/DomainGap Align.
  • antibody framework refers to the portion of a variable domain VL or VH that serves as a scaffold for the antigen-binding loops (CDRs) of the variable domain. Essentially, it is a variable domain without CDRs.
  • epitope refers to the site on an antigen to which an immunoglobulin or antibody specifically binds.
  • An epitope typically comprises at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 contiguous or non-contiguous amino acids in a unique spatial conformation (see, e.g., Epitope Mapping Protocols in Methods in Molecular Biology, Volume 66, G.E. Morris, Ed. (1996)).
  • antibodies bind with an affinity (KD) of less than about 10 "7M , eg, about less than 10 "8M , 10 "9M or 10 " 10M or less.
  • KD affinity
  • nucleic acid molecule refers to DNA molecules and RNA molecules. Nucleic acid molecules can be single-stranded or double-stranded, but are preferably double-stranded DNA. A nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, a promoter or enhancer is operably linked to a coding sequence if the promoter or enhancer affects the transcription of the coding sequence.
  • Antigen-binding fragments can also be prepared by conventional methods.
  • the antibody or antigen-binding fragment of the invention uses genetic engineering methods to add one or more human FR regions to the non-human CDR region.
  • the human FR germline sequence can be obtained from the website http://imgt.cines.fr of ImMunoGeneTics (IMGT) by comparing the IMGT human antibody variable region germline gene database and MOE software, or from Immunoglobulin Journal, 2001ISBN012441351 get.
  • host cell refers to a cell into which an expression vector has been introduced.
  • Host cells can include bacterial, microbial, plant or animal cells.
  • Bacteria that are readily transformed include members of the enterobacteriaceae such as strains of Escherichia coli or Salmonella; the Bacillaceae such as Bacillus subtilis; Pneumococcus; Streptococcus and Haemophilus influenzae.
  • Suitable microorganisms include Saccharomyces cerevisiae and Pichia pastoris.
  • Suitable animal host cell lines include CHO (Chinese Hamster Ovary cell line) and NSO cells.
  • Antibodies or antigen-binding fragments engineered in the present disclosure can be prepared and purified using conventional methods.
  • cDNA sequences encoding heavy and light chains can be cloned and recombined into GS expression vectors.
  • the recombinant immunoglobulin expression vector can stably transfect CHO cells.
  • mammalian expression systems lead to glycosylation of antibodies, especially at the highly conserved N-terminal site of the Fc region. Positive clones are expanded in serum-free medium in bioreactors for antibody production.
  • the culture fluid from which the antibody has been secreted can be purified by conventional techniques. For example, purify with an A or G Sepharose FF column with adjusted buffer.
  • Antibodies can be concentrated by filtration using conventional methods. Soluble mixtures and aggregates can also be removed by conventional methods such as molecular sieves and ion exchange. The obtained product needs to be immediately frozen, such as -70°C, or freeze-dried.
  • 5-6 membered heteroarylene refers to an aromatic 5-membered or 6-membered monocyclic divalent group having at least one heteroatom (O, N or S).
  • Non-limiting examples of the “5-6 membered heteroarylene” are preferred
  • L 1' is L 11 -L 12 -L 13 -L 14 -L 15 -, if L 13 is a direct bond, then L 1' is L 11 -L 12 -L 14 -L 15 -.
  • the compound shown in formula I is Ab-(L 1 -L 2 -L 3 -L 4 -D) n , if L 4 is a direct bond, the compound shown in formula I is Ab-(L 1 - L 2 -L 3 -D) n .
  • Other similar definitions can be understood with reference to the foregoing.
  • pharmaceutically acceptable salt refers to a relatively non-toxic acid addition salt or base addition salt of the compound or conjugate of the present invention.
  • the acid addition salt is a salt formed between the compound or conjugate of the present invention and a suitable inorganic acid or organic acid, and these salts can be prepared by making the compound or conjugate of the present invention and a suitable organic acid or inorganic acid prepared by reacting in a solvent.
  • Representative acid addition salts include hydrobromide, hydrochloride, sulfate, bisulfate, sulfite, acetate, oxalate, valerate, oleate, palmitate, stearate Salt, Luurosilicate, Borate, Benzoate, Lactate, Nitrate, Phosphate, Phosphate, Carbonate, Bicarbonate, Toluate, Citrate, Maleic Acid Salt, fumarate, succinate, malate, ascorbate, tannate, pamoate, alginate, naphthalenesulfonate, tartrate, benzoate, methanesulfonate, p-toluene Sulfonate, Gluconate, Lactobionate and Lauryl Sulfonate etc.
  • the base addition salt is a salt formed between the compound or conjugate of the present invention and a suitable inorganic base or organic base, and these salts can be obtained by making the compound or conjugate of the present invention and a suitable inorganic base or organic base prepared by reacting in a solvent.
  • Representative base addition salts include, for example, salts with alkali metal, alkaline earth metal, quaternary ammonium cations, such as sodium, lithium, potassium, calcium, magnesium, tetramethylquaternary ammonium, tetraethylquaternary ammonium Salts, etc.; amine salts, including salts formed with ammonia (NH 3 ), primary amines, secondary amines or tertiary amines, such as methylamine salts, dimethylamine salts, trimethylamine salts, triethylamine salts, ethylamine salts, etc.
  • quaternary ammonium cations such as sodium, lithium, potassium, calcium, magnesium, tetramethylquaternary ammonium, tetraethylquaternary ammonium Salts, etc.
  • amine salts including salts formed with ammonia (NH 3 ), primary amines, secondary amines or tertiary amines, such as methylamine
  • the compounds or conjugates of the present invention can exist in specific geometric or stereoisomer forms.
  • the chiral center can exist in the drug, in the linker structure, or in the Antibodies and their derivatives.
  • all such compounds including cis and trans isomers, (-)- and (+)-enantiomers, (R)- and (S)-enantiomers, diastereomers isomers, (D)-isomers, (L)-isomers, and racemic and other mixtures thereof, such as enantiomerically or diastereomerically enriched mixtures, are included in the present invention within the range.
  • Optically active (R)- and (S)-isomers as well as D and L-isomers can be prepared by chiral synthesis or chiral reagents or other conventional techniques. If one enantiomer of a compound or conjugate of the invention is desired, it can be prepared by asymmetric synthesis or derivatization with a chiral auxiliary, wherein the resulting diastereomeric mixture is separated and the auxiliary group is cleaved to provide the pure desired enantiomer.
  • a diastereoisomeric salt is formed with an appropriate optically active acid or base, and then a diastereomeric salt is formed by a conventional method known in the art. Diastereomeric resolution is performed and the pure enantiomers are recovered. Furthermore, the separation of enantiomers and diastereomers is usually accomplished by the use of chromatography using chiral stationary phases, optionally in combination with chemical derivatization methods (e.g. amines to amino groups formate).
  • solvates such as hydrates of the compounds, conjugates, pharmaceutically acceptable salts, and stereoisomers of the present invention are also within the scope of the present invention.
  • suitable solvates specifically, solvates formed between compounds or conjugates of the present invention and acetone, 2-butanol, 2-propanol, ethanol, ethyl acetate, tetrahydrofuran, diethyl ether, etc. . Hydrates or ethanolates are also mentioned.
  • treating means that the individual's symptoms are partially or completely relieved, or remain unchanged after treatment.
  • treatment includes prophylaxis, treatment and/or cure.
  • prevention means preventing an underlying disease and/or preventing worsening of symptoms or development of a disease. Treatment or prevention also includes any pharmaceutical use of the provided ADCs and the pharmaceutical compositions, pharmaceutical formulations provided herein.
  • the term “effective dose” refers to the amount of a compound which, when administered, alleviates to some extent one or more symptoms of the condition being treated.
  • the term “effective amount” refers to the amount of a compound which, when administered, inhibits the growth, proliferation or migration of cancer cells to some extent.
  • therapeutic effect means the effect resulting from the treatment of an individual, which alters, usually ameliorates or improves the symptoms of a disease or condition, or cures the disease or condition.
  • “individual” includes human or non-human animal.
  • exemplary human subjects include human subjects suffering from a disease (eg, a disease described herein) (referred to as a patient) or normal subjects.
  • non-human animals include all vertebrates, such as non-mammals (such as birds, amphibians, reptiles) and mammals, such as non-human primates, livestock and/or domesticated animals (such as sheep, dogs, cats, cows, pigs, etc.).
  • the antibody-drug conjugate of the present disclosure has rapid and efficient tumor cell killing activity, and also has good biocompatibility, low immunogenicity, biosafety and stability.
  • a new type of joint structure (such as SM) is designed
  • the present disclosure analyzes the structural characteristics of the toxin molecule eribulin.
  • the ADC molecule in the representative example of the present disclosure saves the commonly used self-breaking structure such as PAB, and directly combines the GGFG unit with Eribulin amino condensation improves the hydrophilicity of ADC, simplifies the complexity of chemical synthesis, reduces the cost of synthesis, and shows excellent efficacy.
  • the ADC prepared by the linker with the novel structure of the present disclosure has better anti-tumor efficacy.
  • Fig. 1 is the SEC-HPLC detection pattern of ADC-1 of the embodiment of the present invention.
  • Fig. 2 is the SEC-HPLC detection pattern of ADC-2 of the embodiment of the present invention.
  • Fig. 3 is the SEC-HPLC detection pattern of ADC-3 of the embodiment of the present invention.
  • Fig. 4 is the SEC-HPLC detection spectrum of the control conjugate 2 (ie ADC-6) of the present invention.
  • Fig. 5 is a graph showing the results of detecting the hydrophilicity and hydrophobicity of each ADC of the present invention by using a protein hydrophobic interaction chromatographic column (HIC).
  • HIC protein hydrophobic interaction chromatographic column
  • Fig. 6 is a graph showing the results of affinity detection between ADCs and TROP proteins of the present invention.
  • Fig. 7 is a statistical graph of the anti-tumor activity of each ADC of the present invention.
  • Fig. 8 is a graph showing body weight changes of experimental animals after treatment with various ADCs of the present invention.
  • Fig. 9 is a photograph taken of tumors taken from experimental animals after each ADC treatment of the present invention.
  • hRS7 antibody was produced in CHO cells.
  • the expression vectors containing the hRS7 antibody gene were constructed by conventional molecular biology methods.
  • the amino acid sequences of the hRS7 antibody light chain and heavy chain are shown in SEQ ID NO: 1 and SEQ ID NO: 2 respectively, and the corresponding nucleotide sequences Shown in SEQ ID NO:3 and SEQ ID NO:4 respectively.
  • Insert the above two sequences into the same expression vector extract a large number of transfection plasmids, and transfect them into CHO-K1 cells (ATCC CCL-61).
  • the specific transfection and antibody preparation processes are as follows:
  • Cell culture CHO-K1 cells were grown in suspension in ActiPro (GE HyClone) medium, and cultured at 37°C, 7% CO 2 , 140 rpm, and 90% relative humidity;
  • the highly expressed cell fluid cultured in shake flasks was collected and purified by protein A affinity purification (GE, Mab Select SuRe) and ion exchange purification (GE, Capto S).
  • SDS-PAGE and SEC-HPLC were used to analyze the molecular weight and purity of the purified antibody.
  • the results of SDS-PAGE showed that the molecular weight of the prepared hRS7 was as expected, and the purity of the antibody measured by SEC-HPLC was 99.1%.
  • Embodiment 2 preparation SM
  • LCMS: [M+1]+ 259.2.
  • Embodiment 3 Preparation of conjugate ADC-1
  • Example 1 The antibody prepared in Example 1 was placed in a 10 mg/mL pH 7.4 PBS solution, using a 25°C water bath, while stirring and mixing, an equal volume of TCEP solution 2.4 times the amount of the substance was added, and the solution was placed in a 25°C water bath for 1.5 Hour. Use a 25°C water bath for the sample, then add 8 times the amount of compound A in DMSO solution (DMSO final concentration 10%) to the antibody solution while stirring and mixing, shake the reaction at 25°C for 90 minutes, and finally stir and mix the sample At the same time, 8 times the amount of cysteine was added, and placed in a 25° C. water bath for 10 minutes to react to obtain the coupling product ADC-1.
  • DMSO solution DMSO final concentration 10%
  • Embodiment 4 Preparation of conjugate ADC-2
  • Example 1 The antibody prepared in Example 1 was placed in a 10 mg/mL pH 7.4 PBS solution, using a 25°C water bath, while stirring and mixing, an equal volume of TCEP solution 2.4 times the amount of the substance was added, and the solution was placed in a 25°C water bath for 1.5 Hour.
  • Use a 25°C water bath for the sample then add 8 times the amount of compound B in DMSO solution (DMSO final concentration 10%) to the antibody solution while stirring and mixing, shake the reaction at 25°C for 90 minutes, and finally stir and mix the sample
  • 8 times the amount of cysteine was added, and placed in a water bath at 25° C. for 10 minutes to react to obtain the coupling product ADC-2.
  • Embodiment 5 Preparation of conjugate ADC-3
  • Example 1 The antibody prepared in Example 1 was placed in a 10 mg/mL pH 7.4 PBS solution, using a 25°C water bath, while stirring and mixing, an equal volume of TCEP solution 2.4 times the amount of the substance was added, and the solution was placed in a 25°C water bath for 1.5 Hour. Use a 25°C water bath for the sample, then add 8 times the amount of compound C in DMSO solution (DMSO final concentration 10%) to the antibody solution while stirring and mixing, shake the reaction at 25°C for 90 minutes, and finally stir and mix the sample At the same time, 8 times the amount of cysteine was added, and placed in a water bath at 25° C. for 10 minutes to react to obtain the coupling product ADC-3.
  • DMSO solution DMSO final concentration 10%
  • Embodiment 6 prepares conjugate ADC-4
  • the coupling with the antibody, the detection of the coupling product, the purification and the determination of the DAR value are the same as in Example 5, and the obtained ADC-4 has a DAR value of 4.7.
  • Example 1 The antibody prepared in Example 1 was placed in an ice-water bath in a 10 mg/mL pH 7.4 PBS solution.
  • an equal volume of TCEP solution 3.5 times the amount of the substance was added while stirring and mixing, and the solution was placed at 25 °C water bath for 1 hour.
  • the temperature drops to 25°C then add 7 times the amount of compound E in 40% DMSO solution to the antibody solution while stirring and mixing, and shake the reaction at 25°C for 90 minutes.
  • cysteine was added in an amount 8 times that of the substance, and placed in a water bath at 25° C. for 10 min to react to obtain the coupling product ADC-5.
  • the detection, purification and DAR value determination methods of the coupling product are the same as in Example 5, and the obtained ADC-5 has a DAR value of 3.8.
  • Example 1 The antibody prepared in Example 1 was placed in a 5 mg/mL pH 7.4 PBS solution, while stirring and mixing, TCEP solution of 7 times the amount of the substance was added, and the solution was placed in a 37°C water bath for 1.5 hours to react. Use a 25°C water bath for the reaction solution. When the temperature drops to 25°C, then add 16 times the amount of compound F in 40% DMSO solution to the antibody solution while stirring and mixing, and shake the reaction at 25°C for 90 minutes. Finally, while the reaction solution was stirred and mixed, cysteine was added in an amount 16 times that of the substance, and placed in a water bath at 25° C. for 10 minutes to react to obtain the coupling product ADC-6.
  • DTT dithiothreitol
  • Embodiment 7 detects the hydrophilicity of each ADC
  • Coating Trop2 protein Dilute Trop2 protein (Beijing Yiqiao Shenzhou Biotechnology Co., Ltd.) with coating solution (0.15M Na 2 CO 3 and 0.35M NaHCO 3 ) to 0.5 ⁇ g/mL, mix well and add to the microtiter plate medium, 100 ⁇ L/well, cover the plate with sealing film, and put it in a refrigerator at 4°C overnight.
  • Washing plate Take out the microplate plate Washing plate: Wash 3 times with 1*PBST.
  • Blocking Take the blocking solution (1% BSA), add 200 ⁇ L/well of the blocking solution, cover the plate with a sealing film, mix at 250 rpm and incubate at 25° C. for 1 h.
  • Plate washing wash 3 times with 1*PBST.
  • Antibody incubation After diluting the ADC prepared in Example 3 and Comparative Example 2 with 1% BSA, and the TROP-2 antibody prepared in Example 1 to 2000 ng/mL, three-fold serial dilutions were made to 12 concentrations, and 100 ⁇ L/well was added to the enzyme Mark the plate, cover the plate with sealing film, mix at 250rpm and incubate at 25°C for 1h.
  • Plate washing Wash the microplate plate 3 times with 1*PBST.
  • Plate washing Wash the microplate plate 3 times with 1*PBST.
  • Color development add TMB (Huzhou Yingchuang) color development substrate: add 100ul per well to the microtiter plate, mix at 250rpm and place at 25°C for 15 minutes.
  • Stop reading add stop solution: add 1M H 2 SO 4 100ul to each well, and read at OD450nm on a microplate reader.
  • BxPC-3 cells human orthotopic pancreatic cancer cells, ATCC CRL-1687
  • the culture conditions were 10% heat-inactivated fetal calf serum and agar in 1640 medium, at 37°C, containing 5% CO 2 in an air incubator. Digested with 0.25% trypsin twice a week for passage. When the cells are in the exponential growth phase, the cells are collected, counted, and inoculated.
  • the P10 generation tumor tissue was used to evaluate the antitumor activity of the test product.
  • the P9 generation tumor grows to 500-800mm 3
  • the tumor-bearing mice are anesthetized with CO 2 and sacrificed, the tumor mass is removed, the surrounding necrotic tissue is removed, and the tumor mass in good condition is cut into small tumors of 20-30mm 3
  • Blocks were inoculated to the right shoulder blade of formal experimental mice, and a total of 65 mice were inoculated.
  • the mice with too small or too large tumor volume were excluded, and the remaining 25 mice were randomly grouped according to tumor volume and started to be administered. See the table below for the dosing regimen.
  • tumor volume (mm 3 ) 1/2 ⁇ (a ⁇ b 2 ) (where a represents the long diameter and b represents the short diameter).
  • the relative tumor proliferation rate, T/C% is the percentage value of the relative tumor volume or tumor weight between the treatment group and the control group at a certain time point.
  • tumors were taken from all mice, weighed and photographed.
  • Figures 7-9 The experimental results are shown in Figures 7-9, Figure 7 is the statistical curve of anti-tumor activity, Figure 8 is the curve of body weight change, and Figure 9 is the photograph taken of the tumor.
  • ADC-1 has the best anti-tumor effect, and it can completely regress the tumor with only one administration, and it is safe.
  • the antitumor effects of ADC-2 and ADC-5 were comparable.

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Abstract

An antibody-drug conjugate having reduced reversible reaction, and a preparation method therefor and an application thereof. Specifically provided are an antibody-drug conjugate represented by formula (I), a pharmaceutically acceptable salt thereof, a stereoisomer thereof, or a solvate thereof. The antibody-drug conjugate has rapid and efficient tumor cell killing activity, and also has good biocompatibility, low immunogenicity, biological safety and stability.

Description

一种可逆反应减少的抗体-药物偶联物，其制备方法及应用Antibody-drug conjugate with reduced reversible reaction, preparation method and application thereof 技术领域technical field
本公开涉及生物医药领域，具体地说，本公开涉及一种可逆反应减少的抗体-药物偶联物，其制备方法及应用，具体涉及一类全新结构的接头结构、包括该接头结构的药物-接头化合物，以及包括所述药物-接头化合物的抗体-药物偶联物、上述药物-接头化合物和抗体-药物偶联物的制备方法及应用。The present disclosure relates to the field of biomedicine, in particular, the present disclosure relates to an antibody-drug conjugate with reduced reversible reactions, its preparation method and application, in particular to a new type of linker structure, and a drug-drug conjugate comprising the linker structure. A linker compound, an antibody-drug conjugate comprising the drug-linker compound, a preparation method and application of the above-mentioned drug-linker compound and antibody-drug conjugate.
背景技术Background technique
抗体-药物偶联物(antibody-drug conjugate，下文简称“ADC”或“偶联物”)相对于单纯的抗体药物而言已经显示出独特的优势，其通过将具有肿瘤细胞表面抗原结合特异性的单克隆抗体与生物活性分子(如细胞毒素)相连，从而将抗体的肿瘤识别靶向性和细胞毒素的高效杀伤作用相结合，巧妙的同时解决了抗体疗效偏低和毒素缺乏靶向性导致毒性过大的缺陷。这使得ADC与传统的抗肿瘤药物相比，在准确的靶向肿瘤细胞的同时，降低对正常细胞的影响，大幅提高了抗肿瘤药物的有效性和安全性。Antibody-drug conjugates (antibody-drug conjugates, hereinafter referred to as "ADC" or "conjugates") have shown unique advantages over simple antibody drugs. The monoclonal antibody is linked to biologically active molecules (such as cytotoxins), thus combining the tumor recognition targeting of the antibody and the efficient killing effect of the cytotoxin, and ingeniously solving the problems caused by the low efficacy of the antibody and the lack of targeting of the toxin. The defect of excessive toxicity. This enables ADC to accurately target tumor cells while reducing the impact on normal cells compared with traditional anti-tumor drugs, greatly improving the effectiveness and safety of anti-tumor drugs.
ADC一般包括三个部分：抗体、接头(Linker)和生物活性分子。ADC的生物活性分子部分为发挥杀伤作用的毒素小分子，一般通过抑制DNA或蛋白合成、抑制细胞有丝分裂等方式来杀伤肿瘤细胞。目前用于ADC开发的毒素主要包括微管抑制剂美登素类(参见EP0425235、US5208020、US5416064、US7276497)和奥瑞他汀(MMAE/MMAF，参见US2016304621A)。其他种类的细胞毒素还包括卡奇霉素类(Calicheamicin，参见US5606040)，苯并二吡咯类衍生物(duocarmycin，参见US7129261)，咯并苯二氮卓类(PBDs，参见WO2005/040170)和喜树碱类衍生物。其中喜树碱类衍生物包括SN-38、DXD、CPT-11、依沙替康、9-硝基喜树碱、10-羟基喜树碱等。毒素通过接头偶联到抗体上；抗体则能够特异性识别肿瘤细胞表面的靶点，将ADC富集到肿瘤细胞表面，使得ADC通过细胞内吞效应进入肿瘤细胞，释放毒素，达到特异性杀灭肿瘤的作用。ADC generally includes three parts: antibody, linker (Linker) and biologically active molecules. The biologically active molecular part of ADC is a toxin small molecule that plays a killing role, and generally kills tumor cells by inhibiting DNA or protein synthesis, inhibiting cell mitosis, and other methods. Toxins currently used for ADC development mainly include microtubule inhibitors maytansinoids (see EP0425235, US5208020, US5416064, US7276497) and auristatins (MMAE/MMAF, see US2016304621A). Other types of cytotoxins also include calicheamicins (Calicheamicin, see US5606040), benzobipyrrole derivatives (duocarmycin, see US7129261), pyrobenzodiazepines (PBDs, see WO2005/040170) and Dendrite derivatives. The camptothecin derivatives include SN-38, DXD, CPT-11, exatecan, 9-nitrocamptothecin, 10-hydroxycamptothecin and the like. The toxin is coupled to the antibody through a linker; the antibody can specifically recognize the target on the surface of the tumor cell, and enrich the ADC on the surface of the tumor cell, so that the ADC enters the tumor cell through the endocytosis effect, releases the toxin, and achieves specific killing role of tumors.
ADC接头分为可裂解和不可裂解两种类型，理想的接头应符合“稳定性好、释放效率高”的要求，不稳定的接头会导致ADC脱靶，增加安全性风险，而过于稳定的接头则影响毒素释放速度，从而影响药效。因此接头的设计至关重要，其直接影响ADC的药效及安全性。接头一端连接抗体，另一端连接药物。目前，抗体与接头的连接位点常见的有赖氨酸和半胱氨酸。前者的ε-氨基可与接头的活化羧基反应形成酰胺键。但此类酰胺键一是在体内酶的作用下容易发生脱落造成毒性，二是抗体自带多个氨基，使得偶联的位点和DAR值不确定，产物不均一，不利于药效稳定。而目前更多的ADC选择半胱氨酸作为偶联位点，在还原条件下使得抗体链间二硫键打开形成巯基后，为接头的偶联提供了相对固定且均一性可控的连接位点。目前将接头与抗体巯基进行偶联的一种方法是抗体上的游离巯基与马来酰亚胺(MC)发生Michael加成反应，也可以是特定的底物与抗体上自由的巯基通过两次Michael加成反应，形成硫桥键，目前所有上市的，以及几乎全部在研的ADC产品均选择马来酰亚胺作为与抗体偶联的接头结构。但已有较多文献报道，通过巯基Michael加成方法得到的ADC，会在体循环中发生逆Michael加成反应，造成毒素过早的在循环中脱落，增加了ADC的毒性。因此，目前仍有待开发与巯基结合更理想的接头结构，以获得安全性更好，稳定性越强，药效越理想的ADC。ADC joints are divided into two types: cleavable and non-cleavable. The ideal joint should meet the requirements of "good stability and high release efficiency". Unstable joints will cause ADC off-target and increase safety risks, while overly stable joints will Affect the release rate of toxins, thereby affecting the efficacy of drugs. Therefore, the design of the linker is very important, which directly affects the efficacy and safety of ADC. One end of the linker is connected to the antibody, and the other end is connected to the drug. Currently, lysine and cysteine are common linking sites between antibodies and linkers. The ε-amino group of the former can react with the activated carboxyl group of the linker to form an amide bond. However, such amide bonds are easy to fall off under the action of enzymes in the body and cause toxicity. Second, the antibody has multiple amino groups, which makes the coupling site and DAR value uncertain, and the product is not uniform, which is not conducive to drug stability. At present, more ADCs choose cysteine as the coupling site. After the disulfide bonds between the antibody chains are opened under reducing conditions to form sulfhydryl groups, it provides a relatively fixed and uniformity-controllable connection site for the coupling of the linker. point. One of the current methods for coupling linkers to antibody thiols is Michael addition reaction between free thiols on the antibody and maleimide (MC), or a specific substrate and free thiols on the antibody through two passes. Michael addition reaction forms a sulfur bridge bond. All currently marketed and almost all ADC products under research choose maleimide as the linker structure for coupling with antibodies. However, there have been many reports in the literature that the ADC obtained by the mercapto Michael addition method will undergo a reverse Michael addition reaction in the systemic circulation, causing the toxin to fall off prematurely in the circulation and increasing the toxicity of the ADC. Therefore, there is still a need to develop a more ideal linker structure for combining with sulfhydryl groups, so as to obtain ADCs with better safety, stronger stability and better drug efficacy.
发明内容Contents of the invention
本公开涉及一类具有特定结构的接头分子，以及包含该接头分子ADC，其通过改进ADC或SMDC药物中毒素与靶向部分的偶联方式获得，获得的偶联物具有更好的稳定性，更高的治疗窗口，在结肠癌、胰腺癌等肿瘤动物模型上体现出更好的治疗效果，抗肿瘤的药效优于传统以MC作为接头的ADC。The present disclosure relates to a class of linker molecules with a specific structure, and the linker molecule ADC, which is obtained by improving the coupling method of toxins and targeting moieties in ADC or SMDC drugs, and the obtained conjugates have better stability, Higher therapeutic window, better therapeutic effect in colon cancer, pancreatic cancer and other tumor animal models, anti-tumor drug effect is better than traditional ADC with MC as linker.
为此，本公开的第一方面，提供一种式(I)表示的抗体-药物偶联物、其立体异构体或其药学上可接受的盐，或所述化合物、其立体异构体或其药学上可接受的盐的溶剂合物，To this end, the first aspect of the present disclosure provides an antibody-drug conjugate represented by formula (I), its stereoisomer or a pharmaceutically acceptable salt thereof, or the compound, its stereoisomer or a solvate of a pharmaceutically acceptable salt thereof,
Ab-(L 1-L 2-L 3-L 4-D) n Ab-(L 1 -L 2 -L 3 -L 4 -D) n
(I)(I)
其中：in:
Ab为抗体，例如单克隆抗体或其抗原结合片段；Ab is an antibody, such as a monoclonal antibody or an antigen-binding fragment thereof;
L 1为-L 12-L 13-L 14-L 15-， L 1 is -L 12 -L 13 -L 14 -L 15 -,
L 12选自5-6元亚杂芳基； L 12 is selected from 5-6 membered heteroarylene;
L 13选自直接键、
Figure PCTCN2022138621-appb-000001
L 13 is selected from direct bonds,
Figure PCTCN2022138621-appb-000001
L 14选自
Figure PCTCN2022138621-appb-000002
L 14 from
Figure PCTCN2022138621-appb-000002
m为选自1-10之间的整数；m is an integer selected from 1-10;
L 15为
Figure PCTCN2022138621-appb-000003
L 15 for
Figure PCTCN2022138621-appb-000003
L 2选自直接键、
Figure PCTCN2022138621-appb-000004
优选地，L 2为
Figure PCTCN2022138621-appb-000005
时，氨基端与L 1或L 15相连，羰基端与L 3相连； L 2 is selected from direct bonds,
Figure PCTCN2022138621-appb-000004
Preferably, L2 is
Figure PCTCN2022138621-appb-000005
, the amino terminal is connected to L 1 or L 15 , and the carbonyl terminal is connected to L 3 ;
p为选自1-10之间的整数；p is an integer selected from 1-10;
L 3为氨基酸单元，优选选自氨基酸残基或由2-10个氨基酸残基组成的肽残基； L3 is an amino acid unit, preferably selected from amino acid residues or peptide residues consisting of 2-10 amino acid residues;
L 4选自直接键、
Figure PCTCN2022138621-appb-000006
L 4 is selected from direct bonds,
Figure PCTCN2022138621-appb-000006
D为与L 4通过化学键相连的药物；若L 4为直接键，则本领域技术人员可以理解，D与L 3相连； D is a drug linked to L4 through a chemical bond; if L4 is a direct bond, those skilled in the art will understand that D is linked to L3 ;
n为1-20之间的任意数值。n is any value between 1-20.
需要说明的是，上述式(I)所示的“抗体药物偶联物”指的是连接的药物分子的个数相同或不同的ADC分子的组合物。It should be noted that the "antibody-drug conjugate" represented by the above formula (I) refers to a composition of ADC molecules with the same or different numbers of linked drug molecules.
具体地，本公开提供了包含多个ADC分子的组合物。在某些情况下，本文所述的组合物中的每个ADC包含相同数目的一个或多个药物分子。在其他情况下，本文所述的组合物中的每个ADC包含不同数目的一个或多个药物分子。In particular, the present disclosure provides compositions comprising a plurality of ADC molecules. In certain instances, each ADC in the compositions described herein comprises the same number of one or more drug molecules. In other cases, each ADC in the compositions described herein comprises a different number of one or more drug molecules.
本文所述的抗体药物偶联物中，每个抗体可以偶联有1个、2个、3个、4个、5个、6个、7个、8个或更多药物分子，例如是1至5个(例如1、2、3、4或5个)药物分子、5至8个(例如5、6、7或8个)药物分子或8至12个 (例如8、9、10、11或12个)药物分子。In the antibody-drug conjugates described herein, each antibody can be coupled with 1, 2, 3, 4, 5, 6, 7, 8 or more drug molecules, such as 1 to 5 (e.g. 1, 2, 3, 4 or 5) drug molecules, 5 to 8 (e.g. 5, 6, 7 or 8) drug molecules or 8 to 12 (e.g. 8, 9, 10, 11 or 12) drug molecules.
药物抗体比(DAR)是指偶联到抗体的药物分子的个数(例如，式I中的n)。本文所述的ADC中包含的药物分子的个数通常是整数，当本文所述的ADC中包含的药物分子的个数(例如，式I中的n)是分数时，该分数指的是包含多个ADC分子的组合物中，每个抗体偶联的药物分子的平均数量。The Drug Antibody Ratio (DAR) refers to the number of drug molecules conjugated to the antibody (eg, n in Formula I). The number of drug molecules contained in the ADC described herein is generally an integer, and when the number of drug molecules contained in the ADC described herein (for example, n in formula I) is a fraction, the fraction refers to the number of drug molecules contained in the ADC described herein. The average number of drug molecules conjugated to each antibody in a composition of multiple ADC molecules.
在一些实施方案中，L 12选自: In some embodiments, L 12 is selected from:
Figure PCTCN2022138621-appb-000007
Figure PCTCN2022138621-appb-000007
Figure PCTCN2022138621-appb-000008
Figure PCTCN2022138621-appb-000008
在一些实施方案中，L 12选自: In some embodiments, L 12 is selected from:
Figure PCTCN2022138621-appb-000009
Figure PCTCN2022138621-appb-000009
在一些实施方案中，L 12选自
Figure PCTCN2022138621-appb-000010
In some embodiments, L 12 is selected from
Figure PCTCN2022138621-appb-000010
在一些实施方案中，L 12为
Figure PCTCN2022138621-appb-000011
In some embodiments, L 12 is
Figure PCTCN2022138621-appb-000011
在一些实施方案中，L 13选自直接键。 In some embodiments, L 13 is selected from direct bonds.
在一些实施方案中，m为选自1-6之间的整数。In some embodiments, m is an integer selected from 1-6.
在一些实施方案中，m选自1、2、3。In some embodiments, m is selected from 1,2,3.
在一些实施方案中，m为3。In some embodiments, m is 3.
在一些实施方案中，p为选自1-6之间的整数。In some embodiments, p is an integer selected from 1-6.
在一些实施方案中，p选自1、2、3。In some embodiments, p is selected from 1, 2, 3.
在一些实施方案中，p为2。In some embodiments, p is 2.
在一些实施方案中，L 3为由2至7个(优选2至4个)氨基酸残基组成的肽残基；优选地，所述氨基酸选自苯丙氨酸、甘氨酸、缬氨酸、赖氨酸、瓜氨酸、丝氨酸、谷氨酸、天冬氨酸；更优选地，所述氨基酸选自甘氨酸、苯丙氨酸、缬氨酸、瓜氨酸、丙氨酸。 In some embodiments, L is a peptide residue consisting of 2 to 7 (preferably 2 to 4) amino acid residues; preferably, the amino acids are selected from the group consisting of phenylalanine, glycine, valine, lysine amino acid, citrulline, serine, glutamic acid, aspartic acid; more preferably, the amino acid is selected from glycine, phenylalanine, valine, citrulline, alanine.
在一些实施方案中，L 3选自缬氨酸-瓜氨酸(Val-Cit)、丙氨酸-丙氨酸-天冬酰胺(Ala-Ala-Asn)、甘氨酸-甘氨酸-赖氨酸(Gly-Gly-lys)、缬氨酸-赖氨酸(Val-lys)、缬氨酸-丙氨酸(Val-Ala)、缬氨酸-苯丙氨酸(Val-Phe)或甘氨酸-甘氨酸-苯丙氨酸-甘氨酸(Gly-Gly-Phe-Gly)；优选选自甘氨酸-甘氨酸-苯丙氨酸-甘氨酸(Gly-Gly-Phe-Gly)、缬氨酸-瓜氨酸(Val-Cit)、缬氨酸-丙氨酸(Val-Ala)。 In some embodiments, L is selected from the group consisting of valine-citrulline (Val-Cit), alanine-alanine-asparagine (Ala-Ala-Asn), glycine-glycine-lysine ( Gly-Gly-lys), Valine-Lysine (Val-lys), Valine-Alanine (Val-Ala), Valine-Phenylalanine (Val-Phe), or Glycine-Glycine - phenylalanine-glycine (Gly-Gly-Phe-Gly); preferably selected from glycine-glycine-phenylalanine-glycine (Gly-Gly-Phe-Gly), valine-citrulline (Val- Cit), valine-alanine (Val-Ala).
在一些实施方案中，L 3选自
Figure PCTCN2022138621-appb-000012
Figure PCTCN2022138621-appb-000013
In some embodiments, L is selected from
Figure PCTCN2022138621-appb-000012
Figure PCTCN2022138621-appb-000013
在一些实施方案中，L 3为甘氨酸-甘氨酸-苯丙氨酸-甘氨酸(Gly-Gly-Phe-Gly)。在一些实施方案中，相对于包含其它氨基酸单元或其它可裂解部分的ADC，包含Gly-Gly-Phe-Gly的ADC显示增加的稳定性，减少的脱靶细胞杀死，增加的靶向细胞杀死，较低的聚集水平，和/或较高的药物负载。 In some embodiments, L3 is glycine-glycine-phenylalanine-glycine (Gly-Gly-Phe-Gly). In some embodiments, ADCs comprising Gly-Gly-Phe-Gly exhibit increased stability, reduced off-target cell killing, increased on-target cell killing relative to ADCs comprising other amino acid units or other cleavable moieties , lower aggregation levels, and/or higher drug loading.
在一些实施方案中，L 3的氨基端与L 2相连，羰基端与L 4相连。本领域技术人员可以理解的是，若L 2为直接键，则L 3的氨基端与L 1或L 15相连。本领 域技术人员同样可以理解的是，若L 4为直接键，则L 3的羰基端与D相连，例如与艾日布林的氨基相连。 In some embodiments, the amino terminus of L3 is attached to L2 and the carbonyl terminus is attached to L4 . Those skilled in the art can understand that if L2 is a direct bond, the amino terminal of L3 is connected to L1 or L15 . Those skilled in the art can also understand that if L 4 is a direct bond, the carbonyl end of L 3 is connected to D, for example, to the amino group of eribulin.
在一些实施方案中，本公开抗体偶联物中连接子包含至少一种将艾日布林衍生物D连接于可裂解部分的间隔单元。在一些实施方案中，所述连接子包含连接于D的间隔单元。In some embodiments, the linker in the antibody conjugates of the present disclosure comprises at least one spacer unit linking the eribulin derivative D to the cleavable moiety. In some embodiments, the linker comprises a spacer unit attached to D.
在一些实施方案中，所述间隔单元包含对氨基苯甲氧基羰基(PAB)In some embodiments, the spacer unit comprises p-aminobenzyloxycarbonyl (PAB)
Figure PCTCN2022138621-appb-000014
Figure PCTCN2022138621-appb-000014
在一些实施方案中，所述间隔单元包含对氨基苯甲酰基In some embodiments, the spacer unit comprises p-aminobenzoyl
Figure PCTCN2022138621-appb-000015
Figure PCTCN2022138621-appb-000015
在一些实施方案中，L 4选自直接键、
Figure PCTCN2022138621-appb-000016
In some embodiments, L is selected from direct bonds,
Figure PCTCN2022138621-appb-000016
在一些实施方案中，L 4为直接键。 In some embodiments, L4 is a direct bond.
在一些实施方案中，连接子在细胞外是稳定的，使得ADC在存在于细胞外环境中时保持完整，但在例如癌细胞的细胞中内化时能够裂解。在一些实施方案中，当ADC进入表达对ADC的抗体部分具有特异性的抗原的细胞时，生物活性分子部分从抗体部分裂解，且裂解释放生物活性分子的未修饰形式。In some embodiments, the linker is stable extracellularly such that the ADC remains intact when present in the extracellular environment, but is capable of cleavage when internalized in a cell, eg, a cancer cell. In some embodiments, when the ADC enters a cell expressing an antigen specific for the antibody portion of the ADC, the biologically active molecule portion is cleaved from the antibody portion, and the cleavage releases the unmodified form of the biologically active molecule.
在一些实施方案中，连接子中的可裂解部分为可裂解肽部分。在一些实施方案中，相对于包含其他可裂解部分的ADC，包含可裂解肽部分的ADC显示较低的聚集水平，改善的抗体与药物比率，增加的癌细胞的靶向杀死，减少的非癌细胞的脱靶杀死，和/或较高的药物负载(n)。在一些实施方案中，相对于不可裂解的连接子，添加可裂解部分增加细胞毒性和/或效力。在一些实施方案中，增加的效力和/或细胞毒性是在表达中等水平的由ADC的抗体部分所靶向的抗原(例如中等FRA表达)的癌症中具有增加的效力和/或细胞毒性。在一些实 施方案中，可裂解肽部分能够由酶裂解，且连接子为酶能够裂解的连接子。在一些实施方案中，酶为组织蛋白酶，且连接子为组织蛋白酶能够裂解的连接子。在某些实施方案中，与其它分裂机制相比，酶能够裂解的连接子(例如组织蛋白酶能够裂解的连接子)显示上述改善特性中的一种或多种。In some embodiments, the cleavable moiety in the linker is a cleavable peptide moiety. In some embodiments, ADCs comprising a cleavable peptide moiety exhibit lower levels of aggregation, improved antibody-to-drug ratios, increased targeted killing of cancer cells, reduced non- Off-target killing of cancer cells, and/or higher drug load (n). In some embodiments, addition of a cleavable moiety increases cytotoxicity and/or potency relative to a non-cleavable linker. In some embodiments, the increased potency and/or cytotoxicity is in cancers that express moderate levels of the antigen targeted by the antibody portion of the ADC (eg, moderate FRA expression). In some embodiments, the cleavable peptide moiety is capable of being cleaved by an enzyme, and the linker is one that is cleavable by an enzyme. In some embodiments, the enzyme is a cathepsin and the linker is a linker that the cathepsin is capable of cleaving. In certain embodiments, an enzyme-cleavable linker (eg, a cathepsin-cleavable linker) exhibits one or more of the improved properties described above compared to other cleavage mechanisms.
在一些实施方案中，所述连接子包含可裂解二硫化物部分，所述连接子在还原条件下能够裂解。In some embodiments, the linker comprises a cleavable disulfide moiety that is capable of being cleaved under reducing conditions.
在一些实施方式中，连接子可以通过与D的氨基连接。In some embodiments, the linker can be attached via the amino group of D.
在一些实施方式中，连接子可以通过与D的羟基连接。In some embodiments, the linker can be attached via the hydroxyl group of D.
在某些实施方式中，D选自艾日布林或其衍生物(如甲磺酸艾日布林等)，DNA拓扑异构酶抑制剂，例如拓扑异构酶I抑制剂(例如喜树碱、羟基喜树碱、9-氨基喜树碱、SN-38、吉咪替康、吉马替康、伊立替康、拓扑替康、依沙替康、DXD、贝洛替康、勒托替康、CKD-602、karenitecin、BN-80915或卢比替康)，拓扑异构酶II抑制剂(例如放线菌素D、阿霉素、多柔比星、多卡米星，柔红霉素、米托蒽醌、鬼臼毒素或依托泊苷)；干扰DNA合成药物，例如甲氨蝶呤、5-氟尿嘧啶、阿糖胞苷、吉西他滨、巯嘌呤、喷司他丁、氟达拉滨、克拉屈滨或奈拉滨；作用于结构蛋白的药物，例如微管蛋白抑制剂如MMAE(Monomethyl auristatin E)、MMAF(Monomethyl auristatin F)、MMAD(MonoMethyl Dolastatin 10)，长春花生物碱类、长春新碱、长春碱、紫杉醇、多西他赛或卡巴他赛；肿瘤信号通路抑制剂，例如丝氨酸/苏氨酸激酶抑制剂、酪氨酸激酶抑制剂、天冬氨酸激酶抑制剂或组氨酸激酶抑制剂；蛋白酶体抑制剂；组蛋白去乙酰化酶抑制剂；肿瘤新生血管生成抑制剂；细胞周期蛋白抑制剂；美登素衍生物，例如Maytansine DM1、Maytansine DM4；卡里奇霉素或其衍生物；奥瑞他汀衍生物；Pyrrolobenzodiazepine dimers(PBD)衍生物；美法仑；丝裂霉素C；苯丁酸氮芥；MGBA(如duocarmycin)；阿霉素(doxorubicin)；蓖麻毒素；白喉毒素；以及其它抑制肿瘤细胞生长、促进肿瘤细胞凋亡或坏死的活性物质。In some embodiments, D is selected from eribulin or its derivatives (such as eribulin mesylate, etc.), DNA topoisomerase inhibitors, such as topoisomerase I inhibitors (such as camptophyllin Alkaline, Hydroxycamptothecin, 9-Aminocamptothecin, SN-38, Gemitecan, Gematecan, Irinotecan, Topotecan, Exatecan, DXD, Belotecan, Leto tecan, CKD-602, karenitecin, BN-80915, or rubitecan), topoisomerase II inhibitors (eg, actinomycin D, doxorubicin, doxorubicin, duocarmycin, daunorubicin drugs that interfere with DNA synthesis, such as methotrexate, 5-fluorouracil, cytarabine, gemcitabine, mercaptopurine, pentostatin, fludarabine , cladribine or nelarabine; drugs that act on structural proteins, such as tubulin inhibitors such as MMAE (Monomethyl auristatin E), MMAF (Monomethyl auristatin F), MMAD (MonoMethyl Dolastatin 10), vinca alkaloids, Vincristine, vinblastine, paclitaxel, docetaxel, or cabazitaxel; tumor signaling pathway inhibitors, such as serine/threonine kinase inhibitors, tyrosine kinase inhibitors, aspartokinase inhibitors, or combinations thereof Amino acid kinase inhibitors; proteasome inhibitors; histone deacetylase inhibitors; tumor angiogenesis inhibitors; cell cyclin inhibitors; maytansine derivatives, such as Maytansine DM1, Maytansine DM4; Pyrrolobenzodiazepine dimers (PBD) derivatives; Melphalan; Mitomycin C; Chlorambucil; MGBA (such as duocarmycin); Doxorubicin; Anesthetic toxin; diphtheria toxin; and other active substances that inhibit tumor cell growth and promote tumor cell apoptosis or necrosis.
在一些实施方案中，所述药物选自艾日布林或其衍生物(如甲磺酸艾日布林等)、喜树碱或其衍生物(如羟基喜树碱、9-氨基喜树碱、SN-38、吉咪替康、吉马替康、伊立替康、拓扑替康、依沙替康、DXD、贝洛替康、勒托替康、CKD-602、karenitecin、BN-80915或卢比替康)、Monomethyl auristatin E(MMAE)、 Monomethyl auristatin F(MMAF)、MonoMethyl Dolastatin 10(MMAD)。In some embodiments, the drug is selected from eribulin or its derivatives (such as eribulin mesylate, etc.), camptothecin or its derivatives (such as hydroxycamptothecin, 9-aminocamptothecin Alkaline, SN-38, gemitecan, gemotecan, irinotecan, topotecan, exatecan, DXD, belotecan, letotecan, CKD-602, karenitecin, BN-80915 or rubitecan), Monomethyl auristatin E (MMAE), Monomethyl auristatin F (MMAF), MonoMethyl Dolastatin 10 (MMAD).
在一些实施方案中，所述药物选自艾日布林或其衍生物(如甲磺酸艾日布林等)、MMAE、DXD，优选艾日布林或其衍生物(如甲磺酸艾日布林等)。In some embodiments, the drug is selected from eribulin or its derivatives (such as eribulin mesylate, etc.), MMAE, DXD, preferably eribulin or its derivatives (such as eribulin mesylate, etc. Riblin, etc.).
在一些实施方案中，缀合于主题抗体的药物部分选自艾日布林或其衍生物，例如艾日布林的甲磺酸盐。如本文所用，术语“艾日布林”是指软海绵素B(最初从海绵冈田软海绵(Halichondria okadais)分离的大环化合物)的合成类似物。艾日布林为微管抑制剂，认为其结合微管蛋白并通过抑制有丝分裂纺锤体组件，在G2/M期引起细胞周期停滞。术语“艾日布林甲磺酸盐”是指艾日布林的甲磺酸盐，其以商品名Halaven TM出售。 In some embodiments, the drug moiety conjugated to the subject antibody is selected from eribulin or a derivative thereof, eg, the mesylate salt of eribulin. As used herein, the term "eribulin" refers to a synthetic analog of halichondrin B, a macrocyclic compound originally isolated from the sponge Halichondria okadais. Eribulin is a microtubule inhibitor that is thought to bind tubulin and cause cell cycle arrest in G2/M phase by inhibiting mitotic spindle assembly. The term "eribulin mesylate" refers to the mesylate salt of eribulin, which is sold under the tradename Halaven ™ .
在某些实施方案中，所述药物为艾日布林或其衍生物(如甲磺酸艾日布林等)。In some embodiments, the drug is eribulin or its derivatives (such as eribulin mesylate, etc.).
在一些实施方案中，D为
Figure PCTCN2022138621-appb-000017
In some embodiments, D is
Figure PCTCN2022138621-appb-000017
在一些实施方案中，n为1-10之间的任意数值。In some embodiments, n is any value between 1-10.
在一些实施方案中，n为1-5之间的任意数值。In some embodiments, n is any value between 1-5.
在一些实施方案中，n为3.5-5.0之间的任意数值，例如，n为3.9、4.1或4.7。In some embodiments, n is any value between 3.5-5.0, eg, n is 3.9, 4.1 or 4.7.
在一些实施方案中，本公开抗体-药物偶联物(ADC)中Ab为抗体，例如单克隆抗体或其抗原结合片段，所述抗体或其抗原结合片段选自抗Trop-2、CD37、HER2、CD70、EGFRvIII、Mesothelin、Folate eceoptor1、Mucin 1、CD138、CD20、CD19、CD30、SLTRK6、Nectin 4、Tissue factor、Mucin16、Endothelinreceoptor、STEAP1、SLC39A6、Guanylylcyclase C、PSMA、CCD79b、CD22、Sodium phosphate cotransporter2B、GPNMB、Trophoblast glycoprotein、AGS-16、EGFR、CD33、CD66e、CD74、CD56、PD-L1、TACSTD2、DR5、E16、STEAP1、0772P、MPF、Napi3b、Sema 5b、PSCA hlg、ETBR、MSG783、STEAP2、TrpM4、CRIPTO、 CD21、CD79b、FcRH2、NCA、MDP、IL20Rα、Brevican、EphB2R、ASLG659、PSCA、GEDA、BAFF-R、CD22、CD79a、CXCR5、HLA-DOB、P2X5、CD72、LY64、FcRH1、IRTA2、TENB2、整合素α5β6，α4β7、FGF2、FGFR2、Her3、CD70、CA6、DLL3、DLL4、P-cadherin、EpCAM、pCAD、CD223、LYPD3、LY6E、EFNA4、ROR1、SLITRK6、5T4、ENPP3、SLC39A6、Claudin18.2、BMPR1B、E16、STEAP1、Tyro7、0772P、MPF、Napi3b、Sema 5b、PSCA hlg、ETBR、MSG783、STEAP2、TrpM4、CRIPTO、CD21、CD79b、FcRH2、NCA、MDP、IL20Rα、Brevican、EphB2R、ASLG659、PSCA、GEDA、CD22、CD79a、CXCR5、HLA-DOB、P2X5、CD72、LY64、FcRH1、IRTA2，c-Met，ApoE、CD1lc、CD40、CD45(PTPRC)、CD49D(ITGA4)、CD80、CSF1R、CTSD、GZMB、Ly86、MS4A7、PIK3AP1、PIK3CD、CCR5、IFNG、IL10RA1、IL-6、ACTA2、COL7A1、LOX、LRRC15、MCPT8、MMP10、NOG、SERPINEl、STAT1、TGFBR1、CTSS、PGF、VEGFA、C1QA、C1QB、ANGPTL4、EGLN、ANGPTL4、EGLN3、BNIP3、AIF1、CCL5、CXCL10、CXCL11、IFI6、PLOD2、KISS1R、STC2、DDIT4、PFKFB3、PGK1、PDK1、AKR1C1、AKR1C2、CADM1、CDH11、COL6A3、CTGF、HMOX1、KRT33A、LUM、WNT5A、IGFBP3、MMP14、CDCP1、PDGFRA、TCF4、TGF、TGFB1、TGFB2、CDl lb、ADGRE1、EMR2、TNFRSF21、UPK1B、TNFSF9、MMP16、MFI2、IGF-1R、RNF43、NaPi2b、BCMA和TENB2。In some embodiments, the Ab in the antibody-drug conjugate (ADC) of the present disclosure is an antibody, such as a monoclonal antibody or an antigen-binding fragment thereof, and the antibody or an antigen-binding fragment thereof is selected from anti-Trop-2, CD37, HER2 , CD70, EGFRvIII, Mesothelin, Folate eceoptor1, Mucin 1, CD138, CD20, CD19, CD30, SLTRK6, Nectin 4, Tissue factor, Mucin16, Endothelin receptor, STEAP1, SLC39A6, Guanylylcyclase C, PSMA, CCD79b, CD22, Sodium ph osphate cotransporter2B, GPNMB, Trophoblast glycoprotein, AGS-16, EGFR, CD33, CD66e, CD74, CD56, PD-L1, TACSTD2, DR5, E16, STEAP1, 0772P, MPF, Napi3b, Sema 5b, PSCA hlg, ETBR, MSG783, STEAP2, TrpM4 , CRIPTO, CD21, CD79b, FcRH2, NCA, MDP, IL20Rα, Brevican, EphB2R, ASLG659, PSCA, GEDA, BAFF-R, CD22, CD79a, CXCR5, HLA-DOB, P2X5, CD72, LY64, FcRH1, IRTA2, TENB2 , Integrin α5β6, α4β7, FGF2, FGFR2, Her3, CD70, CA6, DLL3, DLL4, P-cadherin, EpCAM, pCAD, CD223, LYPD3, LY6E, EFNA4, ROR1, SLITRK6, 5T4, ENPP3, SLC39A6, Claudin18.2 , BMPR1B, E16, STEAP1, Tyro7, 0772P, MPF, Napi3b, Sema 5b, PSCA hlg, ETBR, MSG783, STEAP2, TrpM4, CRIPTO, CD21, CD79b, FcRH2, NCA, MDP, IL20Rα, Brevican, EphB2R, ASLG659, PSCA , GEDA, CD22, CD79a, CXCR5, HLA-DOB, P2X5, CD72, LY64, FcRH1, IRTA2, c-Met, ApoE, CD1lc, CD40, CD45(PTPRC), CD49D(ITGA4), CD80, CSF1R, CTSD, GZMB , Ly86, MS4A7, PIK3AP1, PIK3CD, CCR5, IFNG, IL10RA1, IL-6, ACTA2, COL7A1, LOX, LRRC15, MCPT8, MMP10, NOG, SERPINE1, STAT1, TGFBR1, CTSS, PGF, VEGFA, C1QA, C1QB, ANGPTL4 , EGLN, ANGPTL4, EGLN3, BNIP3, AIF1, CCL5, CXCL10, CXCL11, IFI6, PLOD2, KISS1R, STC2, DDIT4, PFKFB3, PGK1, PDK1, AKR1C1, AKR1C2, CADM1, CDH11, COL6A3, CTGF, HMOX1, KRT33A, LUM , WNT5A, IGFBP3, MMP14, CDCP1, PDGFRA, TCF4, TGF, TGFB1, TGFB2, CD1 lb, ADGRE1, EMR2, TNFRSF21, UPK1B, TNFSF9, MMP16, MFI2, IGF-1R, RNF43, NaPi2b, BCMA, and TENB2.
在一些优选实施方案中，其中，所述单克隆抗体或其抗原结合片段包括Fab、Fab′、F(ab′) 2、Fd、Fv、dAb、互补决定区片段、单链抗体(例如，scFv)、非人抗体、人源化抗体、嵌合抗体、全人抗体、前抗(Probody)、双特异性抗体或多特异性抗体。 In some preferred embodiments, wherein the monoclonal antibody or antigen-binding fragment thereof comprises Fab, Fab', F(ab') 2 , Fd, Fv, dAb, complementarity determining region fragments, single chain antibody (for example, scFv ), non-human antibody, humanized antibody, chimeric antibody, fully human antibody, pre-antibody (Probody), bispecific antibody or multispecific antibody.
在一些优选实施方案中，Ab为抗Her 2的单克隆抗体，例如曲妥珠单抗、帕妥珠单抗(Pertuzumab)或抗Trop-2的单克隆抗体，例如Sacituzumab。In some preferred embodiments, the Ab is an anti-Her 2 monoclonal antibody, such as Trastuzumab, Pertuzumab, or an anti-Trop-2 monoclonal antibody, such as Sacituzumab.
氨基酸在各区域或结构域的分配可遵循Chothia&Lesk(1987)J.Mol.Biol.196：901-917；Chothia等人(1989)Nature 342：878-883的定义。The allocation of amino acids in each region or domain can follow the definition of Chothia & Lesk (1987) J. Mol. Biol. 196: 901-917; Chothia et al. (1989) Nature 342: 878-883.
在一些实施方案中，所述抗体-药物偶联物(ADC)中抗体为已知抗体，选自但不限于选自曲妥珠单抗(Trastuzumab)、帕妥珠单抗(Pertuzumab)、尼妥珠单抗(Nimotuzumab)、恩波妥珠单抗(Enoblituzumab)、依玛妥珠单抗(Emibetuzumab)、奥英妥珠单抗(Inotuzumab)、维汀-匹那妥珠单抗(Pinatuzumab)、维布妥昔单抗(Brentuximab)、吉妥单抗(Gemtuzumab)、比伐珠单抗(Bivatuzumab)、莫洛伐妥单抗(Lorvotuzumab)、cBR96和Glematumamab或其抗原结合片段。In some embodiments, the antibody in the antibody-drug conjugate (ADC) is a known antibody, selected from but not limited to Trastuzumab (Trastuzumab), Pertuzumab (Pertuzumab), niger Nimotuzumab, Enoblituzumab, Emibetuzumab, Inotuzumab, Vitin-Pinatuzumab , Brentuximab, Gemtuzumab, Bivatuzumab, Lorvotuzumab, cBR96, and Glematumamab or antigen-binding fragments thereof.
在本公开的部分实施方案中，靶向部分为记载于US5821337的曲妥珠单抗(trastuzumab)，记载于US7560111的SEQ ID No.：16和SEQ ID No.：15的帕妥珠单抗(pertuzumab)，可用于本公开的抗体还可以通过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.)的文库进行筛选获得。In some embodiments of the present disclosure, the targeting moiety is trastuzumab described in US5821337, pertuzumab described in US7560111 of SEQ ID No.: 16 and SEQ ID No.: 15 ( pertuzumab), the antibodies that can be used in the present disclosure can also be screened by the carrier design, construction and construction of the antibody library displaying antibodies disclosed in CN103476941A, or can be screened with the library of Sorrento Therapeutics, Inc. get.
在本公开的部分实施方案中，所述靶向部分的重链末位Lys是容易缺失的但不影响生物活性，参见Dick，L.W.等人，Biotechnol.Bioeng.，100：1132-1143。例如，所述靶向部分为抗Trop-2的单克隆抗体，例如Sacituzumab重链末位Lys缺失，例如，所述靶向部分为抗Her 2的单克隆抗体，例如曲妥珠单抗、帕妥珠单抗(Pertuzumab)重链末位Lys缺失。In some embodiments of the present disclosure, the Lys at the end of the heavy chain of the targeting moiety is easily deleted without affecting the biological activity, see Dick, L.W. et al., Biotechnol. Bioeng., 100: 1132-1143. For example, the targeting moiety is an anti-Trop-2 monoclonal antibody, such as the deletion of Lys at the end of the heavy chain of Sacituzumab, for example, the targeting moiety is an anti-Her 2 monoclonal antibody, such as Trastuzumab, Paragon Deletion of Lys at the end of the heavy chain of Pertuzumab.
在某些优选实施方案中，所述Her-2抗体为US5821337所述曲妥珠抗体，其轻链可变区的互补决定区(CDR)包括由RASQDVNTAVA氨基酸序列组成的CDR1；由SASFLYS氨基酸序列组成的CDR2；和由QQHYTTPPT氨基酸序列组成的CDR3，并且其重链可变区的CDR包括由DTYIH氨基酸序列组成的CDR1；由RIYPTNGYTRY氨基酸序列组成的CDR2；和由WGGDGFYAMDY氨基酸序列组成的CDR3。曲妥珠抗体的轻链序列及重链序列分别如SEQ ID NO:5和SEQ ID NO:6所示。还可包括那些对上述抗体进行保守氨基酸取代后，保留了Her2结合活性的抗体。In some preferred embodiments, the Her-2 antibody is the trastuzumab antibody described in US5821337, and the complementarity determining region (CDR) of the light chain variable region includes CDR1 consisting of the RASQDVNTAVA amino acid sequence; consisting of the SASFLYS amino acid sequence and CDR3 consisting of the QQHYTTPPT amino acid sequence, and the CDRs of the heavy chain variable region thereof include CDR1 consisting of the DTYIH amino acid sequence; CDR2 consisting of the RIYPTNGYTRY amino acid sequence; and CDR3 consisting of the WGGDGFYAMDY amino acid sequence. The light chain sequence and heavy chain sequence of the trastuzumab antibody are shown in SEQ ID NO: 5 and SEQ ID NO: 6, respectively. Antibodies that retain Her2-binding activity after conservative amino acid substitutions to the above-mentioned antibodies may also be included.
曲妥珠抗体的轻链氨基酸序列：Light chain amino acid sequence of trastuzumab antibody:
diqmtqspss lsasvgdrvt itcrasqdvn tavawyqqkp gkapklliys asflysgvpsdiqmtqspss lsasvgdrvt itcrasqdvn tavawyqqkp gkapklliys asflysgvps
rfsgsrsgtd ftltisslqp edfatyycqq hyttpptfgq gtkveikrtv aapsvfifpprfsgsrsgtd ftltisslqp edfatyycqq hyttpptfgq gtkveikrtv aapsvfifpp
sdeqlksgta svvcllnnfy preakvqwkv dnalqsgasq esvteqdskd styslsstltsdeqlksgta svvcllnnfy preakvqwkv dnalqsgasq esvteqdskd styslsstlt
lskaayekha vyacevthqg lsspvtksfn rgec(SEQ ID NO:5)lskaayekha vyacevthqg lsspvtksfn rgec (SEQ ID NO:5)
曲妥珠抗体的重链氨基酸序列：Heavy chain amino acid sequence of trastuzumab antibody:
evqlvesggg lvqpggslrl scaasgfnik dtyihwvrqa pgkglewvar iyptngytryevqlvesggg lvqpggslrl scaasgfnik dtyihwvrqa pgkglewvar iyptngytry
adsvkgrfti sadtskntay lqmnslraed tavyycsrwg gdgfyamdyw gqgtlvtvssadsvkgrfti sadtskntay lqmnslraed tavyycsrwg gdgfyamdyw gqgtlvtvss
astkgpsvfp lapsskstsg gtaalgclvk dyfpepvtvs wnsgaltsgv htfpavlqssastkgpsvfp lapsskstsg gtaalgclvk dyfpepvtvs wnsgaltsgv htfpavlqss
glyslssvvt vpssslgtqt yicnvnhkps ntkvdkkvep k(SEQ ID NO:6)glyslssvvt vpssslgtqt yiicnvnhkps ntkvdkkvep k (SEQ ID NO:6)
在本公开中，ErbB2和Her2/neu可互换使用，二者均表示天然序列的人Her2蛋白(Genebank登录号：X03363，参见例如Semba等人，1985，PNAS，82：6497-6501；和Yamamoto等人，1986，Nature，319：230-234)及其功能性衍生物，例如氨基酸序列变体。ErbB2表示编码人Her2的基因，neu表示编码大鼠p185neu的基因。在部分实施方案中，本公开的化合物或偶联物能够抑制或杀伤表达ErbB2受体的细胞，例如乳腺癌细胞、卵巢癌细胞、胃癌细胞、子宫内膜癌细胞、唾液腺癌细胞、肺癌细胞、肾癌细胞、结肠癌细胞、甲状腺癌细胞、胰腺癌细胞、膀胱癌细胞或肝癌细胞。In this disclosure, ErbB2 and Her2/neu are used interchangeably, both of which refer to the native sequence human Her2 protein (Genebank accession number: X03363, see e.g. Semba et al., 1985, PNAS, 82:6497-6501; and Yamamoto et al., 1986, Nature, 319:230-234) and their functional derivatives, such as amino acid sequence variants. ErbB2 indicates the gene encoding human Her2, and neu indicates the gene encoding rat p185neu. In some embodiments, the compounds or conjugates of the present disclosure can inhibit or kill cells expressing ErbB2 receptors, such as breast cancer cells, ovarian cancer cells, gastric cancer cells, endometrial cancer cells, salivary gland cancer cells, lung cancer cells, Kidney, colon, thyroid, pancreatic, bladder, or liver cancer cells.
在本公开的部分实施方案中，靶向部分抗Trop-2抗体为记载于美国专利第7,517,964号中的RS7；记载于美国专利第6,653,104号中的RS7；以及记载于US2012/0237518中的hRS7。Trop-2抗体全称为抗体人滋养层细胞表面抗原2(Human trophoblast cell surface antigen 2，Trop-2)，是由TACSTD2基因编码的40kDa跨膜糖蛋白。Trop-2最早鉴定于人滋养层绒毛癌细胞系，其胞内尾巴在控制许多调节细胞功能(如细胞-细胞粘附，细胞增殖和细胞迁移等)的信号通路中发挥着重要的作用。Trop-2蛋白在许多人类肿瘤(如乳腺癌、结直肠癌、肺癌、胰腺癌、卵巢癌、前列腺癌、宫颈癌、肾癌、尿道癌、胶质细胞瘤、黑色素瘤、肝癌、膀胱癌、胃癌、食道癌)细胞表面均有表达，但在正常人体组织只有有限的表达。Trop-2具有调节肿瘤细胞生长、促进肿瘤细胞侵袭和转移的功能。可用于本公开的Trop-2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。可用于本公开的抗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.)的文库进行筛选获得。In some embodiments of the present disclosure, the targeting portion of the anti-Trop-2 antibody is RS7 described in US Patent No. 7,517,964; RS7 described in US Patent No. 6,653,104; and hRS7 described in US2012/0237518. The full name of Trop-2 antibody is human trophoblast cell surface antigen 2 (Trop-2), which is a 40kDa transmembrane glycoprotein encoded by the TACSTD2 gene. Trop-2 was first identified in the human trophoblast choriocarcinoma cell line, and its intracellular tail plays an important role in controlling many signaling pathways that regulate cell functions, such as cell-cell adhesion, cell proliferation, and cell migration. Trop-2 protein in many human tumors (such as breast cancer, colorectal cancer, lung cancer, pancreatic cancer, ovarian cancer, prostate cancer, cervical cancer, kidney cancer, urethral cancer, glioma, melanoma, liver cancer, bladder cancer, Gastric cancer, esophageal cancer) cells are expressed, but only limited expression in normal human tissues. Trop-2 has the functions of regulating tumor cell growth, promoting tumor cell invasion and metastasis. Examples of Trop-2 antibodies that can be used in the present disclosure 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_SVG described, for example, in CN104053672A 8. 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; described in AR47A6.4.2 in US Patent No. 7,420,041. The anti-Trop-2 antibody that can be used in the present disclosure can also be obtained by screening the carrier design, construction and construction of an antibody library displaying antibodies disclosed in CN103476941A, and can also be obtained from the library of Sorrento Therapeutics, Inc. obtained by screening.
本公开中的天然序列的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。The natural sequence Trop-2 in the present disclosure can be isolated from nature, and can also be prepared by recombinant DNA technology, chemical synthesis or a combination thereof.
本公开中所用的抗体优选为抗人Trop-2抗体。Antibodies used in the present disclosure are preferably anti-human Trop-2 antibodies.
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。In certain preferred embodiments, the CDR1, CDR2 and/or CDR3 of the heavy chain and light chain of the anti-human Trop-2 antibody are respectively the CDR1, CDR2 and/or CDR3 of the heavy chain and light chain of the RS7 monoclonal antibody.
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。In some preferred embodiments, the anti-human Trop-2 antibody can be a humanized antibody or a fully human antibody.
在某些优选实施方案中，所述抗体为CN100360567C所述RS7抗体，其中人源化或嵌合RS7抗体的轻链可变区的互补决定区(CDR)包括由KASQDVSIAVA氨基酸序列组成的CDR1；由SASYRYT氨基酸序列组成的CDR2；和由QQHYITPLT氨基酸序列组成的CDR3，并且其中人源化或嵌合RS7MAb的重链可变区的CDR包括由NYGMN氨基酸序列组成的CDR1；由WINTYTGEPTYTDDFKG氨基酸序列组成的CDR2；和由GGFGSSYWYFDV氨基酸序列组成的CDR3。RS7的轻链序列及重链序列分别如SEQ ID NO：1和SEQ ID NO：2所示；优选地，所述抗Trop-2抗体的轻链和重链的编码核苷酸序列分别如SEQ ID NO:3和SEQ ID NO:4所示。还可包括那些对上述抗体进行保守氨基酸取代后，保留了Trop-2结合活性的抗体。In some preferred embodiments, the antibody is the RS7 antibody described in CN100360567C, wherein the complementarity determining region (CDR) of the light chain variable region of the humanized or chimeric RS7 antibody includes CDR1 consisting of the KASQDVSIAVA amino acid sequence; CDR2 consisting of the amino acid sequence of SASYRYT; and CDR3 consisting of the amino acid sequence of QQHYITPLT, and wherein the CDR of the heavy chain variable region of the humanized or chimeric RS7 MAb comprises CDR1 consisting of the amino acid sequence of NYGMN; CDR2 consisting of the amino acid sequence of WINTYTGEPTYTDDFKG; and CDR3 consisting of the GGFGSSYWYFDV amino acid sequence. The light chain sequence and heavy chain sequence of RS7 are respectively shown in SEQ ID NO: 1 and SEQ ID NO: 2; Preferably, the coding nucleotide sequences of the light chain and heavy chain of the anti-Trop-2 antibody are respectively shown in SEQ Shown in ID NO:3 and SEQ ID NO:4. Antibodies that retain Trop-2 binding activity after conservative amino acid substitutions to the above antibodies may also be included.
在一些实施方案中，所述抗体药物偶联物选自：In some embodiments, the antibody drug conjugate is selected from:
Figure PCTCN2022138621-appb-000018
Figure PCTCN2022138621-appb-000018
需要说明的是，以上ADC-1、ADC-2、ADC-3或ADC-4中，-S-并非另外外接的硫原子，而是打开二硫键后的Ab自身所含有巯基与
Figure PCTCN2022138621-appb-000019
进行连接后形成的-S-。 It should be noted that in the above ADC-1, ADC-2, ADC-3 or ADC-4, -S- is not an additional external sulfur atom, but the sulfhydryl group contained in the Ab itself after the disulfide bond is opened.
Figure PCTCN2022138621-appb-000019
-S- formed after making a ligation.
本公开的第二方面，提供式(II)所示化合物、其药学上可接受的盐、其立体异构体、或其溶剂合物，The second aspect of the present disclosure provides a compound represented by formula (II), a pharmaceutically acceptable salt thereof, a stereoisomer thereof, or a solvate thereof,
L 1’-L 2-L 3-L 4-D L 1' -L 2 -L 3 -L 4 -D
(II)(II)
其中：in:
L 1’为L 11-L 12-L 13-L 14-L 15-； L 1' is L 11 -L 12 -L 13 -L 14 -L 15 -;
L 11为
Figure PCTCN2022138621-appb-000020
L 11 is
Figure PCTCN2022138621-appb-000020
L 12、L 13、L 14、L 15如前所述； L 12 , L 13 , L 14 , L 15 are as described above;
L 2、L 3、L 4、D如前所述。 L 2 , L 3 , L 4 , and D are as described above.
在一些实施方案中，式(II)所示的化合物可以将以下式(IV)所示的化合物与药物相连接而形成，所述药物的连接位点为氨基，所述药物如前所述，优选艾日布林或其衍生物(如甲磺酸艾日布林等)。In some embodiments, the compound represented by formula (II) can be formed by linking the compound represented by the following formula (IV) with a drug, the linking site of the drug is an amino group, and the drug is as described above, Eribulin or its derivatives (such as Eribulin mesylate, etc.) are preferred.
在一些实施方案中，提供的连接子包含可裂解磺酰胺部分，所述连接子在还原条件下能够裂解。In some embodiments, provided linkers comprise a cleavable sulfonamide moiety, which linkers are capable of cleavage under reducing conditions.
在一些实施方案中，式(II)所示的化合物选自：In some embodiments, the compound represented by formula (II) is selected from:
Figure PCTCN2022138621-appb-000021
Figure PCTCN2022138621-appb-000021
Figure PCTCN2022138621-appb-000022
Figure PCTCN2022138621-appb-000022
另外，以下两个化合物作为本公开化合物的对照化合物：In addition, the following two compounds are used as reference compounds of the compounds of the present disclosure:
对照化合物1：Control compound 1:
Figure PCTCN2022138621-appb-000023
Figure PCTCN2022138621-appb-000023
对照化合物2：Comparative compound 2:
Figure PCTCN2022138621-appb-000024
Figure PCTCN2022138621-appb-000024
本公开的第三方面，提供式(III)所示化合物、其药学上可接受的盐、其立体异构体、或其溶剂合物，The third aspect of the present disclosure provides the compound represented by formula (III), its pharmaceutically acceptable salt, its stereoisomer, or its solvate,
L 11-L 12-L 13-L 14-L 15’ L 11 -L 12 -L 13 -L 14 -L 15'
(III)(III)
其中：in:
L 11为
Figure PCTCN2022138621-appb-000025
L 11 is
Figure PCTCN2022138621-appb-000025
L 12、L 13、L 14如前所述； L 12 , L 13 , L 14 are as described above;
L 15’为
Figure PCTCN2022138621-appb-000026
L 15' for
Figure PCTCN2022138621-appb-000026
在一些实施方案中，式(III)所示的化合物为In some embodiments, the compound represented by formula (III) is
Figure PCTCN2022138621-appb-000027
Figure PCTCN2022138621-appb-000027
本公开的第四方面，提供式(IV)所示的中间体化合物、其药学上可接受的盐、其立体异构体、或其溶剂合物，The fourth aspect of the present disclosure provides an intermediate compound represented by formula (IV), a pharmaceutically acceptable salt thereof, a stereoisomer thereof, or a solvate thereof,
L 1’-L 2-L 3-OH L 1' -L 2 -L 3 -OH
(IV)(IV)
其中：in:
L 1’为L 11-L 12-L 13-L 14-L 15-； L 1' is L 11 -L 12 -L 13 -L 14 -L 15 -;
L 11为
Figure PCTCN2022138621-appb-000028
L 11 is
Figure PCTCN2022138621-appb-000028
L 12、L 13、L 14、L 15如前所述； L 12 , L 13 , L 14 , L 15 are as described above;
L 2、L 3如前所述。 L 2 and L 3 are as described above.
在一些实施方案中，式(IV)所示的中间体化合物选自In some embodiments, the intermediate compound represented by formula (IV) is selected from
Figure PCTCN2022138621-appb-000029
Figure PCTCN2022138621-appb-000029
Figure PCTCN2022138621-appb-000030
Figure PCTCN2022138621-appb-000030
本公开的第五方面，提供了前述的式(II)所示的化合物、其药学上可接受的盐、其立体异构体、或其溶剂合物的方法，其包括：In a fifth aspect of the present disclosure, there is provided a method for the aforementioned compound represented by formula (II), its pharmaceutically acceptable salt, its stereoisomer, or its solvate, comprising:
将L 1’-L 2-L 3-OH与艾日布林进行酰胺化反应，获得式(II)所示的化合物、其药学上可接受的盐、其立体异构体、或其溶剂合物，其中，L 1’、L 2、L 3如前所述。 Amidation reaction of L 1' -L 2 -L 3 -OH with eribulin to obtain the compound represented by formula (II), its pharmaceutically acceptable salt, its stereoisomer, or its solvate , wherein L 1' , L 2 , and L 3 are as described above.
在一些具体实施方案中，所述方法包括：In some embodiments, the method comprises:
将接头4-(5-甲磺酰基-[1,3,4]噁二唑)-丁酸与H-GGFG-OH或NH2-PEG2-GGFG-OH或H-VA-OH通过(DCC，NHS，有机碱)条件或者(HATU,有机碱)条件进行一步脱水缩合后，再与eribulin通过(HATU,有机碱)条件进行另一步脱水缩合得到目标产物。Pass the linker 4-(5-methylsulfonyl-[1,3,4]oxadiazole)-butyric acid with H-GGFG-OH or NH2-PEG2-GGFG-OH or H-VA-OH through (DCC, NHS , organic base) or (HATU, organic base) conditions for one-step dehydration condensation, and then another step of dehydration condensation with eribulin by (HATU, organic base) conditions to obtain the target product.
本公开的第六方面，提供制备前述的式(I)所示的抗体药物偶联物、其药学上可接受的盐、其立体异构体、或其溶剂合物的方法，其包括：The sixth aspect of the present disclosure provides a method for preparing the aforementioned antibody-drug conjugate represented by formula (I), its pharmaceutically acceptable salt, its stereoisomer, or its solvate, which includes:
将前述的式(II)所示的化合物、其药学上可接受的盐、其立体异构体、或其溶剂合物与前述的Ab反应，获得式(I)所示的抗体药物偶联物、其药学上可接受的盐、其立体异构体、或其溶剂合物。Reacting the compound represented by the aforementioned formula (II), its pharmaceutically acceptable salt, its stereoisomer, or its solvate with the aforementioned Ab to obtain the antibody drug conjugate represented by the formula (I) , a pharmaceutically acceptable salt thereof, a stereoisomer thereof, or a solvate thereof.
在一些实施方案中，所述的式(II)所示的化合物、其药学上可接受的盐、其立体异构体、或其溶剂合物是通过第五方面所述的方法制备得到的。In some embodiments, the compound represented by formula (II), its pharmaceutically acceptable salt, its stereoisomer, or its solvate is prepared by the method described in the fifth aspect.
本公开的第七方面，提供一种药物组合物，其包含本公开第一方面的抗体-药物偶联物、其立体异构体或其药学上可接受的盐，或所述抗体-药物偶联物、其立体异构体或其药学上可接受的盐的溶剂合物，或本公开第二方面的化合物、其立体异构体、其药学上可接受的盐、或其溶剂合物，以及任选的药学上可接受的载体。The seventh aspect of the present disclosure provides a pharmaceutical composition comprising the antibody-drug conjugate of the first aspect of the present disclosure, its stereoisomer or a pharmaceutically acceptable salt thereof, or the antibody-drug conjugate The solvate of the joint product, its stereoisomer or its pharmaceutically acceptable salt, or the compound of the second aspect of the present disclosure, its stereoisomer, its pharmaceutically acceptable salt, or its solvate, and optionally a pharmaceutically acceptable carrier.
本公开的第八方面，提供一种药物制剂，其包含本公开第一方面的所述的抗体-药物偶联物、其立体异构体或其药学上可接受的盐，或所述抗体-药物偶联物、其立体异构体或其药学上可接受的盐的溶剂合物，或本公开第二方面的化合物、其立体异构体、其药学上可接受的盐、或其溶剂合物。The eighth aspect of the present disclosure provides a pharmaceutical preparation, which comprises the antibody-drug conjugate of the first aspect of the present disclosure, its stereoisomer or a pharmaceutically acceptable salt thereof, or the antibody- Drug conjugates, solvates of their stereoisomers or pharmaceutically acceptable salts thereof, or compounds of the second aspect of the disclosure, their stereoisomers, pharmaceutically acceptable salts thereof, or solvates thereof thing.
本公开的第九方面，提供本公开第一方面的所述的抗体-药物偶联物、其立体异构体或其药学上可接受的盐，或所述抗体-药物偶联物、其立体异构体或其药学上可接受的盐的溶剂合物，或本公开第二方面的化合物、其立体异构体、其药学上可接受的盐、或其溶剂合物，或本公开第七方面所述的药物组合物或第八方面所述的药物制剂，其用于预防和/或治疗肿瘤或癌症。The ninth aspect of the present disclosure provides the antibody-drug conjugate of the first aspect of the present disclosure, its stereoisomer or a pharmaceutically acceptable salt thereof, or the antibody-drug conjugate, its stereo The solvate of the isomer or its pharmaceutically acceptable salt, or the compound of the second aspect of the present disclosure, its stereoisomer, its pharmaceutically acceptable salt, or its solvate, or the seventh aspect of the present disclosure The pharmaceutical composition according to the aspect or the pharmaceutical preparation according to the eighth aspect, which is used for preventing and/or treating tumor or cancer.
本公开的第十方面，提供一种预防或治疗癌症的方法，其包括向有此需要的个体施用有效量的本公开第一方面所述的抗体-药物偶联物、其立体异构体或其药学上可接受的盐，或所述抗体-药物偶联物、其立体异构体或其药学上可接受的盐的溶剂合物，或本公开第二方面的化合物、其立体异构体、其药学上可接受的盐、或其溶剂合物，或本公开第七方面所述的药物组合物或第八方面所述的药物制剂。The tenth aspect of the present disclosure provides a method for preventing or treating cancer, which comprises administering an effective amount of the antibody-drug conjugate described in the first aspect of the present disclosure, its stereoisomer or Its pharmaceutically acceptable salt, or the solvate of the antibody-drug conjugate, its stereoisomer or its pharmaceutically acceptable salt, or the compound of the second aspect of the present disclosure, its stereoisomer , a pharmaceutically acceptable salt thereof, or a solvate thereof, or the pharmaceutical composition described in the seventh aspect or the pharmaceutical preparation described in the eighth aspect of the present disclosure.
本公开的第十一方面，提供本公开第一方面的所述的抗体-药物偶联物、其立体异构体或其药学上可接受的盐，或所述抗体-药物偶联物、其立体异构体或其药学上可接受的盐的溶剂合物，或本公开第二方面的化合物、其立体异构体、其药学上可接受的盐、或其溶剂合物，或本公开第七方面所述的药物组合物或第八方面所述的药物制剂在制备用于预防和/或治疗肿瘤或癌症的药物中的用途。The eleventh aspect of the present disclosure provides the antibody-drug conjugate of the first aspect of the present disclosure, its stereoisomer or a pharmaceutically acceptable salt thereof, or the antibody-drug conjugate, its A solvate of a stereoisomer or a pharmaceutically acceptable salt thereof, or the compound of the second aspect of the present disclosure, its stereoisomer, a pharmaceutically acceptable salt thereof, or a solvate thereof, or the compound of the second aspect of the present disclosure Use of the pharmaceutical composition according to the seventh aspect or the pharmaceutical preparation according to the eighth aspect in the preparation of a medicament for preventing and/or treating tumor or cancer.
本公开的第十二方面，提供第一方面所述的抗体-药物偶联物、其立体异构体或其药学上可接受的盐，或所述抗体-药物偶联物、其立体异构体或其药学上可接受的盐的溶剂合物，或本公开第二方面的化合物、其立体异构体、其药学上可接受的盐、或其溶剂合物，或本公开第七方面所述的药物组合物或第八方面所 述的药物制剂用于制备试剂的用途，所述试剂用于抑制癌细胞生长、增殖或迁移。The twelfth aspect of the present disclosure provides the antibody-drug conjugate described in the first aspect, its stereoisomer or a pharmaceutically acceptable salt thereof, or the antibody-drug conjugate, its stereoisomer or the solvate of the compound or its pharmaceutically acceptable salt, or the compound of the second aspect of the present disclosure, its stereoisomer, its pharmaceutically acceptable salt, or its solvate, or the compound of the seventh aspect of the present disclosure The use of the above-mentioned pharmaceutical composition or the pharmaceutical preparation according to the eighth aspect for preparing a reagent for inhibiting the growth, proliferation or migration of cancer cells.
本公开的第十三方面，提供第一方面所述的抗体-药物偶联物、其立体异构体或其药学上可接受的盐，或所述抗体-药物偶联物、其立体异构体或其药学上可接受的盐的溶剂合物，或本公开第二方面的化合物、其立体异构体、其药学上可接受的盐、或其溶剂合物，或本公开第七方面所述的药物组合物或第八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。The thirteenth aspect of the present disclosure provides the antibody-drug conjugate described in the first aspect, its stereoisomer or a pharmaceutically acceptable salt thereof, or the antibody-drug conjugate, its stereoisomer or the solvate of the compound or its pharmaceutically acceptable salt, or the compound of the second aspect of the present disclosure, its stereoisomer, its pharmaceutically acceptable salt, or its solvate, or the compound of the seventh aspect of the present disclosure The pharmaceutical composition described above or the pharmaceutical preparation described in the eighth aspect, which is used for inhibiting the growth, proliferation or migration of cancer cells.
本公开的第十四方面，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公开第一方面所述的抗体-药物偶联物、其立体异构体或其药学上可接受的盐，或所述抗体-药物偶联物、其立体异构体或其药学上可接受的盐的溶剂合物，或本公开第二方面的化合物、其立体异构体、其药学上可接受的盐、或其溶剂合物，或本公开第七方面所述的药物组合物或第八方面所述的药物制剂。在一些实施方案中，所述方法用于非治疗目的，如用于科学研究。The fourteenth aspect of the present disclosure provides a method for inhibiting the growth, proliferation or migration of cancer cells, which comprises administering to cancer cells an effective amount of the antibody-drug conjugate described in the first aspect of the present disclosure, its stereoisomer or a pharmaceutically acceptable salt thereof, or a solvate of the antibody-drug conjugate, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof, or the compound of the second aspect of the present disclosure, its stereoisomer Construct, its pharmaceutically acceptable salt, or its solvate, or the pharmaceutical composition described in the seventh aspect or the pharmaceutical preparation described in the eighth aspect of the present disclosure. In some embodiments, the methods are used for non-therapeutic purposes, such as for scientific research.
本公开的第十五方面，提供一种抑制癌细胞生长、增殖或迁移的试剂盒，其包括本公开第一方面所述的抗体-药物偶联物、其立体异构体或其药学上可接受的盐，或所述抗体-药物偶联物、其立体异构体或其药学上可接受的盐的溶剂合物，或本公开第二方面的化合物、其立体异构体、其药学上可接受的盐、或其溶剂合物，或本公开第七方面所述的药物组合物或第八方面所述的药物制剂。The fifteenth aspect of the present disclosure provides a kit for inhibiting the growth, proliferation or migration of cancer cells, which includes the antibody-drug conjugate described in the first aspect of the present disclosure, its stereoisomer or its pharmaceutically acceptable Accepted salts, or solvates of the antibody-drug conjugates, stereoisomers thereof, or pharmaceutically acceptable salts thereof, or compounds of the second aspect of the present disclosure, stereoisomers thereof, pharmaceutically acceptable salts thereof, An acceptable salt, or a solvate thereof, or the pharmaceutical composition described in the seventh aspect or the pharmaceutical preparation described in the eighth aspect of the present disclosure.
本公开的抗体-药物偶联物可优选向哺乳动物给予，更优选人。Antibody-drug conjugates of the present disclosure may preferably be administered to mammals, more preferably humans.
作为含有本公开的抗体-药物偶联物的药物组合物中使用的物质，可考虑给予量、给予浓度，从本领域中通常使用的制剂添加物或其他物中适当选择使用。Substances used in pharmaceutical compositions containing the antibody-drug conjugate of the present disclosure may be appropriately selected from formulation additives or other substances commonly used in this field in consideration of the dosage and concentration to be administered.
在本公开的某些实施方案中，本公开的抗体-药物偶联物可以以含有1种以上的药学上的适应性成分的药物组合物或药物制剂的形式给予。例如，上述药物组合物或药物制剂中，代表性地，可以含有1种以上的药学上可接受的载体(例如灭菌的液体(例如水及油(包含石油、动物、植物、或合成来源的油(例如花生油、大豆油、矿物油、芝麻油等)))。在静脉内给予上述药物组合物的情况下，水是更具有代表性的载体。另外，食盐水溶液、以及葡萄糖水溶液及甘油水溶液也可作为液体载体，尤其是可用于注射用溶液。适当药学赋形剂在该领域中是已知的。根据需要，上述组合物中还可以含有微量的润湿剂或乳化剂、或pH缓冲化剂。适当的药学载体的例子记载于E.W.Martin的“W.Martin载体的例子 armaceutical Sciences”中。其处方与给予的方式相对应。In some embodiments of the present disclosure, the antibody-drug conjugate of the present disclosure can be administered in the form of a pharmaceutical composition or pharmaceutical preparation containing one or more pharmaceutically compatible ingredients. For example, the above-mentioned pharmaceutical composition or pharmaceutical preparation may typically contain more than one pharmaceutically acceptable carrier (such as sterile liquid (such as water and oil (including petroleum, animal, vegetable, or synthetic sources) Oil (such as peanut oil, soybean oil, mineral oil, sesame oil, etc.))). In the case of intravenous administration of the above-mentioned pharmaceutical composition, water is a more representative carrier. In addition, saline solution, and aqueous glucose and glycerol solutions are also Can be used as a liquid carrier, especially for injection solutions. Suitable pharmaceutical excipients are known in this field. According to needs, the above-mentioned composition can also contain a small amount of wetting agent or emulsifying agent, or pH buffering Examples of suitable pharmaceutical carriers are described in "Examples of W. Martin Carriers Armaceutical Sciences" by E.W. Martin. The formulation thereof corresponds to the mode of administration.
各种输送系统是已知的，可以为了给予本公开的抗体-药物偶联物而使用。作为导入方法，可举出皮内、肌肉内、腹腔内、静脉内、及皮下路径，但不限于这些。例如可以利用输液或快速浓注进行给予。在特定的优选实施方式中，上述抗体-药物偶联物的给予利用输液进行。肠胃外的给予是优选的给予路径。Various delivery systems are known and can be used for administering the antibody-drug conjugates of the present disclosure. Examples of the introduction method include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, and subcutaneous routes. Administration may be by infusion or bolus injection, for example. In a particularly preferred embodiment, the above-mentioned antibody-drug conjugate is administered by infusion. Parenteral administration is the preferred route of administration.
代表性的实施方式中，将上述药物组合物形成向人静脉内给予的药物组合物，按照常规步骤形成处方。代表性地，用于静脉内给予的组合物是灭菌的等张性的水性缓冲液中的溶液。根据需要，上述药物组合物还可以含有增溶剂及用于缓解注射部位的疼痛的局麻剂(例如利多卡因)。通常，上述成分可以通过以下任一方式供给：以经密封的容器(例如将显示活性剂的量的安瓿或药囊等密封)中的干燥冷冻干燥粉末或无水的浓缩物的形式，分别地或在单位剂型中一起混合地供给。预定通过输液来给予上述药物时，例如可将上述药物放入到含有灭菌的制药级的水或食盐水的输液瓶中。当通过注射来给予上述药物时，可提供注射用灭菌水或食盐水的安瓿，以使得例如在给予前混合上述成分。In a typical embodiment, the above-mentioned pharmaceutical composition is formulated into a pharmaceutical composition for intravenous administration to humans, and a prescription is prepared according to conventional procedures. Typically, compositions for intravenous administration are solutions in sterile isotonic aqueous buffer. If necessary, the above-mentioned pharmaceutical composition may also contain a solubilizer and a local anesthetic (such as lidocaine) for relieving pain at the injection site. Generally, the above-mentioned ingredients can be supplied by either: as a dry freeze-dried powder or as an anhydrous concentrate in a sealed container, such as an ampoule or sachet, etc., which seals the amount of active agent, respectively. Or they may be supplied mixed together in a unit dosage form. When the above-mentioned drug is to be administered by infusion, for example, the above-mentioned drug can be put into an infusion bottle containing sterile pharmaceutical-grade water or saline. When the above-mentioned drug is administered by injection, an ampoule of sterilized water for injection or saline can be provided so that, for example, the above-mentioned components are mixed before administration.
本公开的药物组合物或药物制剂，可以是仅含有本公开的抗体-药物偶联物的药物组合物或药物制剂，也可以是含有抗体-药物偶联物及至少一种其他癌治疗剂的药物组合物。也可将本公开的抗体-药物偶联物与其他癌治疗剂一同给予，由此，可增强抗癌效果。出于这种目的使用的其他抗癌剂，可与抗体-药物偶联物同时地、分别地或连续地向个体给予，也可改变给予间隔而进行给予。作为这样的癌治疗剂，可举出白蛋白结合型紫杉醇、卡铂、顺铂、吉西他滨、伊立替康(CPT-11)、紫杉醇、培美曲塞、索拉非尼、长春碱或国际公开第WO2003/038043号小册子中记载的药剂、以及LH-RH类似物(亮丙瑞林、戈舍瑞林等)、磷酸雌二醇氮芥、雌激素拮抗剂(他莫昔芬、雷洛昔芬等)、芳香酶抑制剂(阿那曲唑、来曲唑、依西美坦)等，但只要是具有抗肿瘤活性的药剂，就不受限制。The pharmaceutical composition or pharmaceutical preparation of the present disclosure may be a pharmaceutical composition or pharmaceutical preparation containing only the antibody-drug conjugate of the present disclosure, or may contain an antibody-drug conjugate and at least one other cancer therapeutic agent. pharmaceutical composition. The antibody-drug conjugate of the present disclosure can also be administered together with other cancer therapeutic agents, thereby enhancing the anticancer effect. Other anticancer agents used for this purpose may be administered to the individual simultaneously, separately or sequentially with the antibody-drug conjugate, or may be administered with varying intervals of administration. Examples of such cancer therapeutic agents include nab-paclitaxel, carboplatin, cisplatin, gemcitabine, irinotecan (CPT-11), paclitaxel, pemetrexed, sorafenib, vinblastine, and international patents. Drugs described in pamphlet WO2003/038043, and LH-RH analogs (leuprolide, goserelin, etc.), estramustine phosphate, estrogen antagonists (tamoxifen, ryloxetin, etc.) oxifen, etc.), aromatase inhibitors (anastrozole, letrozole, exemestane), etc., but are not limited as long as they have antitumor activity.
这样的药物组合物可以作为具有选定的组成和必要的纯度的制剂，以冷冻干燥制剂或液状制剂的形式形成制剂。作为冷冻干燥制剂而形成制剂时，可以是含有本领域中可使用的适当的制剂添加物的制剂。另外，在液体制剂的情况也同样，可以作为含有本领域中可使用的各种的制剂添加物的液状制剂而形成制剂。Such a pharmaceutical composition can be formulated as a preparation having a selected composition and necessary purity, in the form of a freeze-dried preparation or a liquid preparation. When the formulation is formed as a freeze-dried formulation, it may contain appropriate formulation additives available in the art. Also in the case of liquid preparations, the preparations can be formed as liquid preparations containing various preparation additives that can be used in this field.
药物组合物的组成及浓度根据给予方法的不同而变化，但从本公开的药物 组合物中包含的抗体-药物偶联物针对抗体-药物偶联物的抗原的亲和性、即针对抗原的解离常数(Kd值)方面考虑，亲和性越高(Kd值越低)时，即使为少量的给予量，也能发挥药效。因此，当确定抗体-药物偶联物的给予量时，也可基于抗体-药物偶联物与抗原的亲和性的状况来设定给予量。当将本公开的抗体-药物偶联物向人给予时，例如，可以以约0.001～100mg/kg给予1次，或以1～180天1次的间隔多次给予。The composition and concentration of the pharmaceutical composition vary depending on the method of administration, but the affinity of the antibody-drug conjugate contained in the pharmaceutical composition of the present disclosure to the antigen of the antibody-drug conjugate, that is, the affinity for the antigen Considering the dissociation constant (Kd value), the higher the affinity (the lower the Kd value), the more effective the drug can be exhibited even with a small dose. Therefore, when determining the dosage of the antibody-drug conjugate, the dosage can also be set based on the condition of the affinity of the antibody-drug conjugate to the antigen. When the antibody-drug conjugate of the present disclosure is administered to humans, for example, it can be administered once at about 0.001 to 100 mg/kg, or administered multiple times at intervals of once every 1 to 180 days.
本公开的抗体-药物偶联物、药物组合物、药物制剂可以用于预防和/或治疗肿瘤或癌症。关于作为预防和/或治疗对象的肿瘤或癌症，只要是表达抗体-药物偶联物中的抗体可识别的蛋白的癌细胞即可。在本公开的某些实施方案中，所述肿瘤或癌症选自乳腺癌、结直肠癌、肺癌、胰腺癌、卵巢癌、前列腺癌、宫颈癌、肾癌、尿道癌、胶质细胞瘤、黑色素瘤、肝癌、膀胱癌、胃癌、食道癌；优选地，所述癌症是原位癌或转移癌。The antibody-drug conjugates, pharmaceutical compositions, and pharmaceutical preparations of the present disclosure can be used to prevent and/or treat tumors or cancers. The tumor or cancer to be prevented and/or treated may be any cancer cell expressing a protein recognizable by the antibody in the antibody-drug conjugate. In certain embodiments of the present disclosure, the tumor or cancer is selected from breast cancer, colorectal cancer, lung cancer, pancreatic cancer, ovarian cancer, prostate cancer, cervical cancer, renal cancer, urethral cancer, glioma, melanoma tumor, liver cancer, bladder cancer, gastric cancer, esophageal cancer; preferably, the cancer is carcinoma in situ or metastatic carcinoma.
在本公开的某些实施方案中，向需要的受试者施用预防或治疗有效量的本公开的抗体-药物偶联物、药物组合物或药物制剂，用于抑制癌细胞的生长、增殖或迁移。In certain embodiments of the present disclosure, a prophylactically or therapeutically effective amount of the disclosed antibody-drug conjugate, pharmaceutical composition, or pharmaceutical preparation is administered to a subject in need for inhibiting the growth, proliferation, or migrate.
在本公开的某些实施方案中，提供一种抑制癌细胞生长、增殖或迁移的试剂盒，其包括本公开的抗体-药物偶联物、药物组合物或药物制剂。In certain embodiments of the present disclosure, a kit for inhibiting growth, proliferation or migration of cancer cells is provided, which includes the antibody-drug conjugate, pharmaceutical composition or pharmaceutical preparation of the present disclosure.
术语the term
除非另有限定，本公开所用的所有技术和科学术语均与本公开所属领域普通技术人员的通常理解一致。虽然也可采用与本公开所述相似或等同的任何方法和材料实施或测试本公开，但本公开描述了优选的方法和材料。描述和要求保护本公开时，依据以下定义使用下列术语。Unless defined otherwise, all technical and scientific terms used in this disclosure are as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein could be used in the practice or testing of the present disclosure, the preferred methods and materials are described herein. In describing and claiming the present disclosure, the following terminology will be used in accordance with the following definitions.
当本公开中使用商品名时，申请人旨在包括该商品名产品的制剂、该商品名产品的非专利药和活性药物部分。When a trade name is used in this disclosure, Applicants intend to include formulations of such trade name products, generics and active drug moieties of such trade name products.
除非有相反陈述，在说明书和权利要求书中使用的术语具有下述含义。Unless stated to the contrary, the terms used in the specification and claims have the following meanings.
术语“生物活性分子”指抑制或防止细胞的功能和/或引起细胞死亡或破坏的物质，在本公开的部分实施方案中，偶联物中的生物活性物或生物活性分子为具有抗肿瘤生物活性的分子。例如：放射性同位素，例如At 211、I 131、I 125、Y 90、Re 186、Re 188、Sm 153、Bi 212、P 32、Pb 212和Lu的放射性同位素；金属配合 物，例如金属铂配合物、金属金配合物，奥沙利铂等；糖肽类抗生素，例如博来霉素、平阳霉素；DNA拓扑异构酶抑制剂，例如拓扑异构酶I抑制剂，喜树碱、羟基喜树碱、9-氨基喜树碱、DXD、SN-38、伊立替康、拓扑替康、贝洛替康、卢比替康，拓扑异构酶II抑制剂，放线菌素D、阿霉素、多柔比星、多卡米星，柔红霉素、米托蒽醌、鬼臼毒素、依托泊苷等；干扰DNA合成药物，例如甲氨蝶呤、5-氟尿嘧啶、阿糖胞苷、吉西他滨、巯嘌呤、喷司他丁、氟达拉滨、克拉屈滨、奈拉滨等；作用于结构蛋白的药物，例如微管蛋白抑制剂，长春花生物碱类、长春新碱、长春碱、紫杉醇、多西他赛、卡巴他赛等；肿瘤信号通路抑制剂，例如丝氨酸/苏氨酸激酶抑制剂、酪氨酸激酶抑制剂、天冬氨酸激酶抑制剂或组氨酸激酶抑制剂等；还包括蛋白酶体抑制剂、组蛋白去乙酰化酶抑制剂、肿瘤新生血管生成抑制剂、细胞周期蛋白抑制剂、美登素衍生物、卡里奇霉素衍生物、奥瑞他汀衍生物、Pyrrolobenzodiazepines(PBD)衍生物、美法仑、丝裂霉素C、苯丁酸氮芥、或其它抑制肿瘤细胞生长、促进肿瘤细胞凋亡和坏死的活性物质；酶及其片段，诸如核溶酶；抗生素；毒素，诸如小分子毒素或者细菌、真菌、植物或动物起源的酶活性毒素，包括其片段和/或变体；生长抑制剂；药物模块。术语“毒素”指能够对细胞的生长或增殖产生有害效果的物质。本公开的一些实施方案中，生物活性分子表示为D，也可以选择免疫调节剂，特别是TLR8激动剂。 The term "biologically active molecule" refers to a substance that inhibits or prevents cell function and/or causes cell death or destruction. In some embodiments of the present disclosure, the biologically active substance or bioactive molecule in the conjugate is an anti-tumor active molecule. For example: radioactive isotopes such as those of At 211 , I 131 , I 125 , Y 90 , Re 186 , Re 188 , Sm 153 , Bi 212 , P 32 , Pb 212 and Lu; metal complexes such as metal platinum complexes , metal gold complexes, oxaliplatin, etc.; glycopeptide antibiotics, such as bleomycin, pingyangmycin; DNA topoisomerase inhibitors, such as topoisomerase I inhibitors, camptothecin, hydroxyhippin Tricine, 9-aminocamptothecin, DXD, SN-38, irinotecan, topotecan, belotecan, rubitecan, topoisomerase II inhibitors, actinomycin D, doxorubicin , doxorubicin, docarmycin, daunorubicin, mitoxantrone, podophyllotoxin, etoposide, etc.; drugs that interfere with DNA synthesis, such as methotrexate, 5-fluorouracil, cytarabine, Gemcitabine, mercaptopurine, pentostatin, fludarabine, cladribine, nelarabine, etc.; drugs acting on structural proteins, such as tubulin inhibitors, vinca alkaloids, vincristine, vinblastine , paclitaxel, docetaxel, cabazitaxel, etc.; tumor signaling pathway inhibitors, such as serine/threonine kinase inhibitors, tyrosine kinase inhibitors, aspartate kinase inhibitors, or histidine kinase inhibitors etc.; also include proteasome inhibitors, histone deacetylase inhibitors, tumor angiogenesis inhibitors, cell cycle protein inhibitors, maytansine derivatives, calicheamicin derivatives, auristatin derivatives , Pyrrolobenzodiazepines (PBD) derivatives, melphalan, mitomycin C, chlorambucil, or other active substances that inhibit tumor cell growth, promote tumor cell apoptosis and necrosis; enzymes and their fragments, such as nucleolytic Enzymes; antibiotics; toxins, such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, including fragments and/or variants thereof; growth inhibitors; drug moieties. The term "toxin" refers to a substance capable of having deleterious effects on the growth or proliferation of cells. In some embodiments of the present disclosure, the biologically active molecule is denoted as D, and immunomodulators, especially TLR8 agonists, can also be selected.
术语“连接子”、“连接单元”、“接头单元”、“接头”或“连接片段”是指一端与配体连接而另一端与药物相连的化学结构片段或键，也可以连接其他接头后再与药物相连。The term "linker", "linker unit", "linker unit", "linker" or "linker segment" refers to a chemical structural fragment or bond that is connected to a ligand at one end and a drug at the other end, and can also be connected after other linkers. Then connect with the drug.
接头可以包含一种或多种接头构件。例示性的接头构件包括本公开的4-(5-甲磺酰基[1,3,4]-噁二唑)丁酸，以及现有技术的6-马来酰亚氨基己酰基(MC)、马来酰亚氨基丙酰基(MP)、缬氨酸-瓜氨酸(Val-Cit或vc)、丙氨酸-苯丙氨酸(ala-phe)、对氨基苄氧羰基(PAB)，及那些源自与接头试剂的偶联的：N-琥珀酰亚氨基4-(2-吡啶基硫代)戊酸酯(SPP)、N-琥珀酰亚氨基4-(N-马来酰亚氨基甲基)环己烷-1羧酸酯(SMCC，在本文中也称作MCC)和N-琥珀酰亚氨基(4-碘-乙酰基)氨基苯甲酸酯(SIAB)。接头可以包括拉伸单元、间隔单元、氨基酸单元和延伸单元。可以通过本领域已知方法合成，诸如US2005-0238649A1中所记 载的。接头可以是便于在细胞中释放药物的“可切割接头”。例如，可使用酸不稳定接头(例如腙)、蛋白酶敏感(例如肽酶敏感)接头、光不稳定接头、二甲基接头、或含二硫化物接头(Chari等,Cancer Research 52:127-131(1992)；美国专利No.5,208,020)。A joint may comprise one or more joint components. Exemplary linker building blocks include 4-(5-methanesulfonyl[1,3,4]-oxadiazole)butanoic acid of the present disclosure, and 6-maleimidocaproyl (MC) of the prior art, Maleimidopropionyl (MP), valine-citrulline (Val-Cit or vc), alanine-phenylalanine (ala-phe), p-aminobenzyloxycarbonyl (PAB), and Those derived from coupling with linker reagents: N-succinimidyl 4-(2-pyridylthio)pentanoate (SPP), N-succinimidyl 4-(N-maleimido Methyl)cyclohexane-1 carboxylate (SMCC, also referred to herein as MCC) and N-succinimidyl (4-iodo-acetyl)aminobenzoate (SIAB). Linkers can include Stretch units, Spacer units, Amino Acid units and Stretcher units. Can be synthesized by methods known in the art, such as described in US2005-0238649A1. The linker can be a "cleavable linker" that facilitates release of the drug in the cell. For example, acid-labile (e.g., hydrazone), protease-sensitive (e.g., peptidase-sensitive) linkers, photolabile, dimethyl, or disulfide-containing linkers can be used (Chari et al., Cancer Research 52:127-131 (1992); US Patent No. 5,208,020).
术语“拉伸单元”指一端通过碳原子与抗体共价连接而另一端与氨基酸单元、二硫化物部分、磺酰胺部分或非肽化学部分相连的化学结构片段。The term "stretch unit" refers to a chemical structural fragment that is covalently linked to the antibody through a carbon atom at one end and to an amino acid unit, disulfide moiety, sulfonamide moiety, or non-peptidic chemical moiety at the other end.
术语“间隔单元”是一种双功能化合结构片段，可用于偶联氨基酸单元和细胞毒性药物最终形成抗体-药物偶联物，这种偶联方式可以将细胞毒性药物选择性的连接到氨基酸单元上。The term "spacer unit" is a bifunctional compound structural fragment that can be used to couple amino acid units and cytotoxic drugs to form antibody-drug conjugates. This coupling method can selectively link cytotoxic drugs to amino acid units superior.
术语“氨基酸”是指分子结构中含有氨基和羧基，并且氨基和羧基都直接连接在-CH-结构上的有机化合物。通式是H 2NCHRCOOH，R为H、取代或未取代烷基等。根据氨基连结在羧酸中碳原子的位置，可分为α、β、γ、δ、ε……-氨基酸。在生物界中，构成天然蛋白质的氨基酸具有其特定的结构特点，即其氨基直接连接在α-碳原子上，即α-氨基酸，包括甘氨酸(Glycine)、丙氨酸(Alanine)、缬氨酸(Valine)、亮氨酸(Leucine)、异亮氨酸(Isoleucine)、苯丙氨酸(Phenylalanine)、色氨酸(Tryptophan)、酪氨酸(Tyrosine)、天冬氨酸(Aspartic acid)、组氨酸(Histidine)、天冬酰胺(Asparagine)、谷氨酸(Glutamic acid)、赖氨酸(Lysine)、谷氨酰胺(Glutamine)、甲硫氨酸(Methionine)、精氨酸(Arginine)、丝氨酸(Serine)、苏氨酸(Threonine)、半胱氨酸(Cysteine)、脯氨酸(Proline)等。非天然氨基酸如瓜氨酸。如本领域技术人员所公知的，非天然氨基酸并不构成天然蛋白质，因此也不参与本公开中抗体的合成。本公开所用氨基酸三字母代码和单字母代码如J.biol.chem,243,p3558(1968)中所述。 The term "amino acid" refers to an organic compound containing an amino group and a carboxyl group in the molecular structure, and both the amino group and the carboxyl group are directly connected to the -CH- structure. The general formula is H 2 NCHRCOOH, R is H, substituted or unsubstituted alkyl, etc. According to the position of the amino group connected to the carbon atom in the carboxylic acid, it can be divided into α, β, γ, δ, ε...-amino acids. In the biological world, the amino acids that make up natural proteins have their specific structural characteristics, that is, their amino groups are directly connected to the α-carbon atom, that is, α-amino acids, including glycine (Glycine), alanine (Alanine), valine (Valine), Leucine, Isoleucine, Phenylalanine, Tryptophan, Tyrosine, Aspartic acid, Histidine, Asparagine, Glutamic acid, Lysine, Glutamine, Methionine, Arginine , Serine, Threonine, Cysteine, Proline, etc. Unnatural amino acids such as citrulline. As is well known to those skilled in the art, unnatural amino acids do not constitute natural proteins and thus do not participate in the synthesis of the antibodies of the present disclosure. The three-letter and one-letter codes for amino acids used in this disclosure are as described in J.biol.chem, 243, p3558 (1968).
在一些实施方案中，本公开中间隔单元为PAB，结构如对氨基苯甲氧羰基片段，连接在D上。In some embodiments, the spacer unit in the present disclosure is PAB, which has a structure such as a p-aminobenzyloxycarbonyl fragment, and is connected to D.
在一些实施方案中，本公开的接头组件包括但不限于：In some embodiments, adapter components of the present disclosure include, but are not limited to:
SM＝4-(5-甲磺酰基[1,3,4]-噁二唑)丁酸；SM=4-(5-methylsulfonyl[1,3,4]-oxadiazole)butanoic acid;
MC＝6-马来酰亚氨基己酰基；MC=6-maleimidocaproyl;
Val-Cit或“vc”＝缬氨酸-瓜氨酸(蛋白酶可切割接头中的例示二肽)；Val-Cit or "vc" = valine-citrulline (an exemplary dipeptide in a protease cleavable linker);
瓜氨酸＝2-氨基-5-脲基戊酸；Citrulline = 2-amino-5-ureidopentanoic acid;
Me-Val-Cit＝N-甲基-缬氨酸-瓜氨酸(其中接头肽键已经修饰以防止其受到组织蛋白酶B的切割)；Me-Val-Cit = N-methyl-valine-citrulline (wherein the linker peptide bond has been modified to prevent its cleavage by cathepsin B);
MC(PEG) 6-OH＝马来酰亚氨基己酰基-聚乙二醇(可附着于抗体半胱氨酸)； MC(PEG) 6 -OH = maleimidocaproyl-polyethylene glycol (can be attached to antibody cysteine);
SPP＝N-琥珀酰亚氨基4-(2-吡啶基硫代)戊酸酯；SPP=N-succinimidyl 4-(2-pyridylthio)pentanoate;
SPDP＝N-琥珀酰亚氨基3-(2-吡啶基二硫代)丙酸酯；SPDP=N-succinimidyl 3-(2-pyridyldithio)propionate;
SMCC＝琥珀酰亚氨基4-(N-马来酰亚氨基甲基)环己烷-1-羧酸酯；SMCC = succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate;
IT＝亚氨基硫烷；IT = iminothiolane;
PBS＝磷酸缓冲盐溶液。PBS = Phosphate Buffered Saline.
术语“抗体-药物偶联物”，指配体通过稳定的连接单元与具有生物活性的药物相连。在本公开中“抗体-药物偶联物”(antibody drug conjugate，ADC)，指把单克隆抗体或者抗体片段通过稳定的连接单元与具有生物活性的毒性药物相连。The term "antibody-drug conjugate" means that a ligand is linked to a biologically active drug through a stable linker unit. In this disclosure, "antibody drug conjugate" (antibody drug conjugate, ADC) refers to linking a monoclonal antibody or antibody fragment with a toxic drug with biological activity through a stable linker unit.
术语“载药量”可以表示为药物量和抗体量的比值。载药量的范围可以是每个抗体(Ab)连接1-20个，优选1-10个细胞毒性药物(D)。在本公开的实施方式中，载药量表示为n，示例性的可以为1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20或任意两数值之间数值的均值。优选1-10，更优选1-8，或2-8，或2-7，或3-8，或3-7，或3-6，或4-7，或4-6，或4-5的均值。可用常规方法如UV/可见光光谱法、质谱、ELISA试验、单抗分子大小变异体测定法(CE-SDS)和HPLC特征鉴定偶联反应后每个ADC分子的药物平均数量。The term "drug loading" can be expressed as the ratio of the amount of drug to the amount of antibody. The range of drug loading can be 1-20, preferably 1-10 cytotoxic drugs (D) linked to each antibody (Ab). In the embodiments of the present disclosure, the drug loading is expressed as n, which can be exemplarily 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or the mean of any two values in between. Preferably 1-10, more preferably 1-8, or 2-8, or 2-7, or 3-8, or 3-7, or 3-6, or 4-7, or 4-6, or 4-5 mean value. The average amount of drug per ADC molecule after conjugation can be identified using routine methods such as UV/Vis spectroscopy, mass spectrometry, ELISA assay, monoclonal antibody size variant assay (CE-SDS) and HPLC characterization.
可以用以下非限制性方法控制抗体-药物偶联物的载量，包括：The loading of antibody-drug conjugates can be controlled by the following non-limiting methods, including:
(1)控制连接试剂和单抗的摩尔比，(1) control the molar ratio of the linking reagent and the monoclonal antibody,
(2)控制反应时间和温度，(2) control reaction time and temperature,
(3)选择不同的反应试剂。(3) Choose different reagents.
术语“抗体”指免疫球蛋白，是由两条相同的重链和两条相同的轻链通过链间二硫键连接而成的四肽链结构。免疫球蛋白重链恒定区的氨基酸组成和排列顺序不同，故其抗原性也不同。据此，可将免疫球蛋白分为五类，或称为免疫 球蛋白的同种型，即IgM、IgD、IgG、IgA和IgE，其相应的重链分别为μ链、δ链、γ链、α链、和ε链。同一类Ig根据其铰链区氨基酸组成和重链二硫键的数目和位置的差别，又可分为不同的亚类，如IgG可分为IgG1、IgG2、IgG3、IgG4。轻链通过恒定区的不同分为κ链或λ链。五类Ig中每类Ig都可以有κ链或λ链。本公开所述的抗体优选为针对靶细胞上细胞表面抗原的特异性抗体，非限制性实施例为以下抗体：抗HER2(ErbB2)抗体、抗EGFR抗体、抗B7-H3抗体、抗c-Met抗体、抗HER3(ErbB3)抗体、抗HER4(ErbB4)抗体、抗CD20抗体、抗CD22抗体、抗CD30抗体、抗CD33抗体、抗CD44抗体、抗CD56抗体、抗CD70抗体、抗CD73抗体、抗CD105抗体、抗CEA抗体、抗A33抗体、抗Cripto抗体、抗EphA2抗体、抗G250抗体、抗MUCl抗体、抗Lewis Y抗体、抗VEGFR抗体、抗GPNMB抗体、抗Integrin抗体、抗PSMA抗体、抗Tenascin-C抗体、抗SLC44A4抗体、抗CD79抗体、抗TROP-2抗体、抗CD79B抗体、抗Mesothelin抗体或其抗原结合片段。在一些实施方案中，抗体选自曲妥珠单抗(Trastuzumab，商品名Herceptin)、帕妥珠单抗(Pertuzumab，也被称作2C4，商品名Perjeta)、尼妥珠单抗(Nimotuzumab，商品名泰欣生)、恩波妥珠单抗(Enoblituzumab)、依玛妥珠单抗(Emibetuzumab)、奥英妥珠单抗(Inotuzumab)、维汀-匹那妥珠单抗(Pinatuzumab)、维布妥昔单抗(Brentuximab)、吉妥单抗(Gemtuzumab)、比伐珠单抗(Bivatuzumab)、莫洛伐妥单抗(Lorvotuzumab)、cBR96和Glematumamab。The term "antibody" refers to an immunoglobulin, which is a tetrapeptide chain structure composed of two identical heavy chains and two identical light chains linked by interchain disulfide bonds. The amino acid composition and sequence of the constant region of the immunoglobulin heavy chain are different, so their antigenicity is also different. Accordingly, immunoglobulins can be divided into five classes, or isotypes of immunoglobulins, namely IgM, IgD, IgG, IgA, and IgE, and their corresponding heavy chains are μ, δ, and γ chains, respectively. , α chain, and ε chain. The same class of Ig can be divided into different subclasses according to the amino acid composition of its hinge region and the number and position of heavy chain disulfide bonds. For example, IgG can be divided into IgG1, IgG2, IgG3, and IgG4. Light chains are classified as either kappa chains or lambda chains by difference in the constant region. Each of the five Ig classes can have either a kappa chain or a lambda chain. Antibodies of the present disclosure are preferably specific antibodies against cell surface antigens on target cells, non-limiting examples are the following antibodies: anti-HER2 (ErbB2) antibody, anti-EGFR antibody, anti-B7-H3 antibody, anti-c-Met Antibody, anti-HER3 (ErbB3) antibody, anti-HER4 (ErbB4) antibody, anti-CD20 antibody, anti-CD22 antibody, anti-CD30 antibody, anti-CD33 antibody, anti-CD44 antibody, anti-CD56 antibody, anti-CD70 antibody, anti-CD73 antibody, anti-CD105 Antibody, Anti-CEA Antibody, Anti-A33 Antibody, Anti-Cripto Antibody, Anti-EphA2 Antibody, Anti-G250 Antibody, Anti-MUCl Antibody, Anti-Lewis Y Antibody, Anti-VEGFR Antibody, Anti-GPNMB Antibody, Anti-Integrin Antibody, Anti-PSMA Antibody, Anti-Tenascin- C antibody, anti-SLC44A4 antibody, anti-CD79 antibody, anti-TROP-2 antibody, anti-CD79B antibody, anti-Mesothelin antibody or an antigen-binding fragment thereof. In some embodiments, the antibody is selected from Trastuzumab (Trastuzumab, trade name Herceptin), Pertuzumab (Pertuzumab, also known as 2C4, trade name Perjeta), Nimotuzumab (Nimotuzumab, trade name Mingtai Xinsheng), Embolituzumab (Enoblituzumab), Emibetuzumab (Emibetuzumab), Inotuzumab (Inotuzumab), Vitin-Pinatuzumab (Pinatuzumab) Brentuximab, Gemtuzumab, Bivatuzumab, Lorvotuzumab, cBR96, and Glematumamab.
抗体重链和轻链靠近N端的约110个氨基酸的序列变化很大，为可变区(Fv区)；靠近C端的其余氨基酸序列相对稳定，为恒定区。可变区包括3个高变区(HVR)和4个序列相对保守的骨架区(FR)。3个高变区决定抗体的特异性，又称为互补性决定区(CDR)。每条轻链可变区(LCVR)和重链可变区(HCVR)由3个CDR区4个FR区组成，从氨基端到羧基端依次排列的顺序为：FR1，CDR1，FR2，CDR2，FR3，CDR3，FR4。轻链的3个CDR区指LCDR1、LCDR2、和LCDR3；重链的3个CDR区指HCDR1、HCDR2和HCDR3。The sequence of about 110 amino acids near the N-terminal of the antibody heavy chain and light chain varies greatly, which is the variable region (Fv region); the rest of the amino acid sequence near the C-terminal is relatively stable, which is the constant region. The variable region includes 3 hypervariable regions (HVR) and 4 framework regions (FR) with relatively conserved sequences. Three hypervariable regions determine the specificity of antibodies, also known as complementarity determining regions (CDR). Each light chain variable region (LCVR) and heavy chain variable region (HCVR) consists of 3 CDR regions and 4 FR regions, and the sequence from the amino terminal to the carboxyl terminal is: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The 3 CDR regions of the light chain refer to LCDR1, LCDR2, and LCDR3; the 3 CDR regions of the heavy chain refer to HCDR1, HCDR2, and HCDR3.
本公开的抗体包括鼠源抗体、嵌合抗体、人源化抗体和全人源抗体，优选人源化抗体和全人源抗体。Antibodies of the present disclosure include murine antibodies, chimeric antibodies, humanized antibodies and fully human antibodies, preferably humanized antibodies and fully human antibodies.
术语“鼠源抗体”在本公开中为根据本领域知识和技能用鼠制备抗体。制备时用特定抗原注射试验对象，然后分离表达具有所需序列或功能特性的抗体的杂交瘤。The term "murine antibody" in this disclosure refers to an antibody prepared using a mouse according to the knowledge and skill in the art. In preparation, test subjects are injected with the specified antigen, and hybridomas expressing antibodies having the desired sequence or functional properties are isolated.
术语“嵌合抗体(chimeric antibody)”，是将鼠源性抗体的可变区与人抗体的恒定区融合而成的抗体，可以减轻鼠源性抗体诱发的免疫应答反应。建立嵌合抗体，要先建立分泌鼠源性特异性单抗的杂交瘤，然后从鼠杂交瘤细胞中克隆可变区基因，再根据需要克隆人抗体的恒定区基因，将鼠可变区基因与人恒定区基因连接成嵌合基因后插入表达载体中，最后在真核系统或原核系统中表达嵌合抗体分子。The term "chimeric antibody" is an antibody formed by fusing the variable region of a murine antibody with the constant region of a human antibody, which can reduce the immune response induced by the murine antibody. To establish a chimeric antibody, it is necessary to first establish a hybridoma that secretes a mouse-derived specific monoclonal antibody, then clone the variable region gene from the mouse hybridoma cell, and then clone the constant region gene of the human antibody as required, and then clone the mouse variable region gene It is connected with the human constant region gene to form a chimeric gene and inserted into an expression vector, and finally expresses the chimeric antibody molecule in a eukaryotic system or a prokaryotic system.
术语“人源化抗体(humanized antibody)”，也称为CDR移植抗体(CDR-grafted antibody)，是指将鼠的CDR序列移植到人的抗体可变区框架，即不同类型的人种系抗体框架序列中产生的抗体。可以克服嵌合抗体由于携带大量鼠蛋白成分，从而诱导的异源性反应。此类构架序列可以从包括种系抗体基因序列的公共DNA数据库或公开的参考文献获得。如人重链和轻链可变区基因的种系DNA序列可以在“VBase”人种系序列数据库(在因特网www.mrccpe.com.ac.uk/vbase可获得)，以及在Kabat，E.A.等人，1991Sequences of Proteins of Immunological Interest，第5版中找到。为避免免疫原性下降的同时，引起的活性下降，可对所述的人抗体可变区框架序列进行最少反向突变或回复突变，以保持活性。本公开的人源化抗体也包括进一步由噬菌体展示对CDR进行亲和力成熟后的人源化抗体。进一步描述参与人源化可使用小鼠抗体的方法的文献包括，例如Queen等，Proc.，Natl.Acad.Sci.USA，88，2869，1991和Winter及其同事的方法[Jones等，Nature，321，522(1986)，Riechmann，等，Nature，332，323-327(1988)，Verhoeyen，等，Science，239，1534(1988)]。The term "humanized antibody", also known as CDR-grafted antibody (CDR-grafted antibody), refers to the antibody variable region framework grafted with mouse CDR sequences to humans, that is, different types of human germline antibodies Antibodies generated in the framework sequences. It can overcome the heterologous reaction induced by chimeric antibodies due to carrying a large amount of mouse protein components. Such framework sequences can be obtained from public DNA databases or published references that include germline antibody gene sequences. For example, the germline DNA sequences of the human heavy and light chain variable region genes can be found in the "VBase" human germline sequence database (available on the Internet at www.mrccpe.com.ac.uk/vbase), as well as in Kabat, E.A. et al. Al, 1991 Sequences of Proteins of Immunological Interest, found in 5th edition. In order to avoid decreased immunogenicity and decreased activity, minimal reverse mutations or back mutations can be performed on the human antibody variable region framework sequence to maintain activity. The humanized antibody of the present disclosure also includes the humanized antibody after affinity maturation of CDR by phage display. Further descriptions of methods involving the use of mouse antibodies in humanization include, for example, Queen et al., Proc., Natl. 321, 522 (1986), Riechmann, et al., Nature, 332, 323-327 (1988), Verhoeyen, et al., Science, 239, 1534 (1988)].
术语“全人源抗体”、“全人抗体”或“完全人源抗体”，也称“全人源单克隆抗体”，其抗体的可变区和恒定区都是人源的，去除免疫原性和毒副作用。单克隆抗体的发展经历了四个阶段，分别为：鼠源性单克隆抗体、嵌合性单克隆抗 体、人源化单克隆抗体和全人源单克隆抗体。本公开为全人源单克隆抗体。全人抗体制备的相关技术主要有：人杂交瘤技术、EBV转化B淋巴细胞技术、噬菌体显示技术(phage display)、转基因小鼠抗体制备技术(transgenic mouse)和单个B细胞抗体制备技术等。The term "fully human antibody", "fully human antibody" or "fully human antibody", also known as "fully human monoclonal antibody", the variable region and constant region of the antibody are all human, and the immunogen sex and side effects. The development of monoclonal antibodies has gone through four stages, namely: murine monoclonal antibodies, chimeric monoclonal antibodies, humanized monoclonal antibodies and fully human monoclonal antibodies. The present disclosure is a fully human monoclonal antibody. The relevant technologies for the preparation of fully human antibodies mainly include: human hybridoma technology, EBV transformed B lymphocyte technology, phage display technology (phage display), transgenic mouse antibody preparation technology (transgenic mouse) and single B cell antibody preparation technology, etc.
术语“抗原结合片段”是指抗体的保持特异性结合抗原的能力的一个或多个片段。已显示可利用全长抗体的片段来进行抗体的抗原结合功能。“抗原结合片段”中包含的结合片段的实例包括(i)Fab片段，由VL、VH、CL和CH1结构域组成的单价片段；(ii)F(ab') 2片段，包含通过铰链区上的二硫桥连接的两个Fab片段的二价片段；(iii)由VH和CH1结构域组成的Fd片段；(iv)由抗体的单臂的VH和VL结构域组成的Fv片段；(v)单结构域或dAb片段(Ward等人,(1989)Nature341：544-546)，其由VH结构域组成；和(vi)分离的互补决定区(CDR)或(vii)可任选地通过合成的接头连接的两个或更多个分离的CDR的组合。此外，虽然Fv片段的两个结构域VL和VH由分开的基因编码，但可使用重组方法，通过合成的接头连接它们，从而使得其能够产生为其中VL和VH区配对形成单价分子的单个蛋白质链(称为单链Fv(scFv)；参见，例如，Bird等人(1988)Science242:423-426；和Huston等人(1988)Proc.Natl.Acad.Sci USA85:5879-5883)。此类单链抗体也意欲包括在术语抗体的“抗原结合片段”中。使用本领域技术人员已知的常规技术获得此类抗体片段，并且以与对于完整抗体的方式相同的方式就功用性筛选片段。可通过重组DNA技术或通过酶促或化学断裂完整免疫球蛋白来产生抗原结合部分。抗体可以是不同同种型的抗体，例如，IgG(例如，IgG1、IgG2、IgG3或IgG4亚型)、IgA1、IgA2、IgD、IgE或IgM抗体。 The term "antigen-binding fragment" refers to one or more fragments of an antibody that retain the ability to specifically bind an antigen. It has been shown that fragments of full-length antibodies can be utilized to perform the antigen-binding function of the antibody. Examples of binding fragments included in "antigen-binding fragments" include (i) Fab fragments, monovalent fragments consisting of VL, VH, CL and CH1 domains; (ii) F(ab') 2 fragments, comprising (iii) Fd fragment consisting of VH and CH1 domains; (iv) Fv fragment consisting of VH and VL domains of a single arm of an antibody; (v ) a single domain or dAb fragment (Ward et al., (1989) Nature 341:544-546) consisting of a VH domain; and (vi) isolated complementarity determining regions (CDRs) or (vii) optionally via A combination of two or more isolated CDRs joined by a synthetic linker. Furthermore, although the two domains VL and VH of the Fv fragment are encoded by separate genes, they can be linked by a synthetic linker using recombinant methods, thus making it possible to produce a single protein in which the VL and VH regions pair to form a monovalent molecule. chain (referred to as single-chain Fv (scFv); see, eg, Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci USA 85:5879-5883). Such single chain antibodies are also intended to be encompassed within the term "antigen-binding fragment" of an antibody. Such antibody fragments are obtained using conventional techniques known to those skilled in the art, and the fragments are screened for functionality in the same manner as for intact antibodies. Antigen-binding portions can be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact immunoglobulins. Antibodies can be of different isotypes, eg, IgG (eg, IgGl, IgG2, IgG3, or IgG4 subtype), IgAl, IgA2, IgD, IgE, or IgM antibodies.
Fab是通过用蛋白酶木瓜蛋白酶(切割H链的224位的氨基酸残基)处理IgG抗体分子所获得的片段中的具有约50,000的分子量并具有抗原结合活性的抗体片段，其中H链N端侧的约一半和整个L链通过二硫键结合在一起。Fab is an antibody fragment having a molecular weight of about 50,000 and having antigen-binding activity among fragments obtained by treating an IgG antibody molecule with the protease papain (cleaving the amino acid residue at position 224 of the H chain), in which About half and the entire L chain is held together by disulfide bonds.
F(ab')2是通过用酶胃蛋白酶消化IgG铰链区中两个二硫键的下方部分而获得的分子量为约100,000并具有抗原结合活性并包含在铰链位置相连的两个Fab区的抗体片段。F(ab')2 is an antibody having a molecular weight of about 100,000 and having antigen-binding activity and comprising two Fab regions connected at the hinge position obtained by digesting the lower portion of the two disulfide bonds in the IgG hinge region with the enzyme pepsin fragment.
Fab'是通过切割上述F(ab')2的铰链区的二硫键而获得的分子量为约50,000并具有抗原结合活性的抗体片段。Fab' is an antibody fragment having a molecular weight of about 50,000 and having antigen-binding activity obtained by cleaving the disulfide bond in the hinge region of the above-mentioned F(ab')2.
此外，可以通过将编码抗体的Fab'片段的DNA插入到原核生物表达载体或真核生物表达载体中并将载体导入到原核生物或真核生物中以表达Fab'来生产所述Fab'。In addition, the Fab' fragment can be produced by inserting DNA encoding a Fab' fragment of an antibody into a prokaryote expression vector or a eukaryote expression vector and introducing the vector into a prokaryote or eukaryote to express the Fab'.
术语“单链抗体”、“单链Fv”或“scFv”意指包含通过接头连接的抗体重链可变结构域(或区域；VH)和抗体轻链可变结构域(或区域；VL)的分子。此类scFv分子可具有一般结构：NH 2-VL-接头-VH-COOH或NH 2-VH-接头-VL-COOH。合适的现有技术接头由重复的GGGGS氨基酸序列或其变体组成，例如使用1-4个重复的变体(Holliger等人(1993)，Proc.Natl.Acad.Sci.USA90:6444-6448)。可用于本公开的其他接头由Alfthan等人(1995)，Protein Eng.8:725-731，Choi等人(2001)，Eur.J.Immuno l.31:94-106，Hu等人(1996)，Cancer Res.56:3055-3061，Kipriyanov等人(1999)，J.Mol.Biol.293:41-56和Roovers等人(2001)，Cancer Immunol.描述。 The term "single-chain antibody", "single-chain Fv" or "scFv" is meant to comprise an antibody heavy chain variable domain (or region; VH) and an antibody light chain variable domain (or region; VL) connected by a linker molecules. Such scFv molecules may have the general structure: NH2 -VL-linker-VH-COOH or NH2 -VH-linker-VL-COOH. Suitable prior art linkers consist of the repeated GGGGS amino acid sequence or variants thereof, for example using 1-4 repeat variants (Holliger et al. (1993), Proc. Natl. Acad. Sci. USA 90:6444-6448) . Other linkers useful in the present disclosure are described by Alfthan et al. (1995), Protein Eng. 8:725-731, Choi et al. (2001), Eur.J. Immuno 1.31:94-106, Hu et al. (1996) , Cancer Res. 56:3055-3061, described by Kipriyanov et al. (1999), J. Mol. Biol. 293:41-56 and Roovers et al. (2001 ), Cancer Immunol.
术语“CDR”是指抗体的可变结构域内主要促成抗原结合的6个高变区之一。所述6个CDR的最常用的定义之一由Kabat E.A.等人，(1991)Sequences of proteins of immunological interest.NIH Publication91-3242)提供。如本文中使用的，CDR的Kabat定义只应用于轻链可变结构域的CDR1、CDR2和CDR3(CDR L1、CDR L2、CDR L3或L1、L2、L3)，以及重链可变结构域的CDR2和CDR3(CDR H2、CDR H3或H2、H3)。通常，每个重链可变区中存在三个CDR(HCDR1、HCDR2、HCDR3)，每个轻链可变区中存在三个CDR(LCDR1、LCDR2、LCDR3)。可以使用各种公知方案中的任何一种来确定CDR的氨基酸序列边界，包括“Kabat”编号规则(参见Kabat等(1991)，“Sequences of Proteins of Immunological Interest”，第5版，Public Health Service，National Institutes of Health，Bethesda，MD)、“Chothia”编号规则(参见Al-Lazikani等人，(1997)JMB 273：927-948)和ImMunoGenTics(IMGT)编号规则(参见Lefranc M.P.，Immunologist，7，132-136(1999)；Lefranc，M.P.等，Dev.Comp.Immunol.，27，55-77(2003))等。例如，对于经典格式，遵循Kabat规则，所述重链可变域(VH)中的CDR氨基酸 残基编号为31-35(HCDR1)、50-65(HCDR2)和95-102(HCDR3)；轻链可变域(VL)中的CDR氨基酸残基编号为24-34(LCDR1)、50-56(LCDR2)和89-97(LCDR3)。遵循Chothia规则，VH中的CDR氨基酸编号为26-32(HCDR1)、52-56(HCDR2)和95-102(HCDR3)；并且VL中的氨基酸残基编号为26-32(LCDR1)、50-52(LCDR2)和91-96(LCDR3)。通过组合Kabat和Chothia两者的CDR定义，CDR由人VH中的氨基酸残基26-35(HCDR1)、50-65(HCDR2)和95-102(HCDR3)和人VL中的氨基酸残基24-34(LCDR1)、50-56(LCDR2)和89-97(LCDR3)构成。遵循IMGT规则，VH中的CDR氨基酸残基编号大致为26-35(CDR1)、51-57(CDR2)和93-102(CDR3)，VL中的CDR氨基酸残基编号大致为27-32(CDR1)、50-52(CDR2)和89-97(CDR3)。遵循IMGT规则，抗体的CDR区可以使用程序IMGT/DomainGap Align确定。The term "CDR" refers to one of the six hypervariable regions within the variable domain of an antibody that primarily contribute to antigen binding. One of the most commonly used definitions of the six CDRs is provided by Kabat E.A. et al., (1991) Sequences of proteins of immunological interest. NIH Publication 91-3242). As used herein, the Kabat definition of CDRs applies only to CDR1, CDR2, and CDR3 (CDR L1, CDR L2, CDR L3 or L1, L2, L3) of the light chain variable domain, and to CDR1, CDR L2, and L3 of the heavy chain variable domain. CDR2 and CDR3 (CDR H2, CDR H3 or H2, H3). Typically, there are three CDRs (HCDR1, HCDR2, HCDR3) in each heavy chain variable region and three CDRs (LCDR1, LCDR2, LCDR3) in each light chain variable region. Amino acid sequence boundaries for CDRs can be determined using any of a variety of well-known schemes, including the "Kabat" numbering convention (see Kabat et al. (1991), "Sequences of Proteins of Immunological Interest", 5th Edition, Public Health Service, National Institutes of Health, Bethesda, MD), the "Chothia" numbering sequence (see Al-Lazikani et al., (1997) JMB 273:927-948) and the ImMunoGenTics (IMGT) numbering sequence (see Lefranc M.P., Immunologist, 7, 132 -136 (1999); Lefranc, M.P. et al., Dev. Comp. Immunol., 27, 55-77 (2003)) et al. For example, following the Kabat rules for the classical format, the CDR amino acid residues in the heavy chain variable domain (VH) are numbered 31-35 (HCDR1), 50-65 (HCDR2) and 95-102 (HCDR3); The CDR amino acid residues in the chain variable domain (VL) are numbered 24-34 (LCDR1), 50-56 (LCDR2) and 89-97 (LCDR3). Following the Chothia rule, the CDR amino acid numbers in VH are 26-32 (HCDR1), 52-56 (HCDR2) and 95-102 (HCDR3); and the amino acid residues in VL are numbered 26-32 (LCDR1), 50- 52 (LCDR2) and 91-96 (LCDR3). Defined by combining the CDRs of both Kabat and Chothia, the CDRs consist of amino acid residues 26-35 (HCDR1), 50-65 (HCDR2) and 95-102 (HCDR3) in human VH and amino acid residues 24- 34 (LCDR1), 50-56 (LCDR2) and 89-97 (LCDR3). Following the IMGT rules, the numbering of CDR amino acid residues in VH is approximately 26-35 (CDR1), 51-57 (CDR2) and 93-102 (CDR3), and the numbering of CDR amino acid residues in VL is approximately 27-32 (CDR1 ), 50-52 (CDR2) and 89-97 (CDR3). Following the IMGT rules, the CDR regions of antibodies can be determined using the program IMGT/DomainGap Align.
术语“抗体框架”，是指可变结构域VL或VH的一部分，其用作该可变结构域的抗原结合环(CDR)的支架。从本质上讲，其是不具有CDR的可变结构域。The term "antibody framework" refers to the portion of a variable domain VL or VH that serves as a scaffold for the antigen-binding loops (CDRs) of the variable domain. Essentially, it is a variable domain without CDRs.
术语“表位”或“抗原决定簇”是指抗原上免疫球蛋白或抗体特异性结合的部位。表位通常以独特的空间构象包括至少3、4、5、6、7、8、9、10、11、12、13、14或15个连续或非连续的氨基酸(参见，例如，Epitope Mapping Protocols in Methods in Molecular B iology，第66卷，G.E.Morris，Ed.(1996))。The term "epitope" or "antigenic determinant" refers to the site on an antigen to which an immunoglobulin or antibody specifically binds. An epitope typically comprises at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 contiguous or non-contiguous amino acids in a unique spatial conformation (see, e.g., Epitope Mapping Protocols in Methods in Molecular Biology, Volume 66, G.E. Morris, Ed. (1996)).
术语“特异性结合”、“选择性结合”、“选择性地结合”和“特异性地结合”是指抗体对预先确定的抗原上的表位的结合。通常，抗体以大约小于10 -7M，例如：大约小于10 -8M、10 -9M或10 -10M或更小的亲和力(KD)结合。 The terms "specifically bind", "selectively bind", "selectively bind" and "specifically bind" refer to the binding of an antibody to a predetermined epitope on an antigen. Typically, antibodies bind with an affinity (KD) of less than about 10 "7M , eg, about less than 10 "8M , 10 "9M or 10 " 10M or less.
术语“核酸分子”是指DNA分子和RNA分子。核酸分子可以是单链或双链的，但优选是双链DNA。当将核酸与另一个核酸序列置于功能关系中时，核酸是“有效连接的”。例如，如果启动子或增强子影响编码序列的转录，那么启动子或增强子有效地连接至所述编码序列。The term "nucleic acid molecule" refers to DNA molecules and RNA molecules. Nucleic acid molecules can be single-stranded or double-stranded, but are preferably double-stranded DNA. A nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, a promoter or enhancer is operably linked to a coding sequence if the promoter or enhancer affects the transcription of the coding sequence.
现有技术中熟知生产和纯化抗体和抗原结合片段的方法，如冷泉港的抗体实验技术指南，5-8章和15章。抗原结合片段同样可以用常规方法制备。发明所述的抗体或抗原结合片段用基因工程方法在非人源的CDR区加上一个或多 个人源FR区。人FR种系序列可以通过比对IMGT人类抗体可变区种系基因数据库和MOE软件，从ImMunoGeneTics(IMGT)的网站http://imgt.cines.fr得到，或者从免疫球蛋白杂志，2001ISBN012441351上获得。Methods for producing and purifying antibodies and antigen-binding fragments are well known in the art, such as Cold Spring Harbor's Antibody Laboratory Technique Guide, Chapters 5-8 and Chapter 15. Antigen-binding fragments can also be prepared by conventional methods. The antibody or antigen-binding fragment of the invention uses genetic engineering methods to add one or more human FR regions to the non-human CDR region. The human FR germline sequence can be obtained from the website http://imgt.cines.fr of ImMunoGeneTics (IMGT) by comparing the IMGT human antibody variable region germline gene database and MOE software, or from Immunoglobulin Journal, 2001ISBN012441351 get.
术语“宿主细胞”是指已向其中引入了表达载体的细胞。宿主细胞可包括细菌、微生物、植物或动物细胞。易于转化的细菌包括肠杆菌科(enterobacteriaceae)的成员，例如大肠杆菌(Escherichia coli)或沙门氏菌(Salmonella)的菌株；芽孢杆菌科(Bacillaceae)例如枯草芽孢杆菌(Bacillus subtilis)；肺炎球菌(Pneumococcus)；链球菌(Streptococcus)和流感嗜血菌(Haemophilus influenzae)。适当的微生物包括酿酒酵母(Saccharomyces cerevisiae)和毕赤酵母(Pichia pastoris)。适当的动物宿主细胞系包括CHO(中国仓鼠卵巢细胞系)和NS0细胞。The term "host cell" refers to a cell into which an expression vector has been introduced. Host cells can include bacterial, microbial, plant or animal cells. Bacteria that are readily transformed include members of the enterobacteriaceae such as strains of Escherichia coli or Salmonella; the Bacillaceae such as Bacillus subtilis; Pneumococcus; Streptococcus and Haemophilus influenzae. Suitable microorganisms include Saccharomyces cerevisiae and Pichia pastoris. Suitable animal host cell lines include CHO (Chinese Hamster Ovary cell line) and NSO cells.
本公开工程化的抗体或抗原结合片段可用常规方法制备和纯化。比如，编码重链和轻链的cDNA序列，可以克隆并重组至GS表达载体。重组的免疫球蛋白表达载体可以稳定地转染CHO细胞。作为一种更推荐的现有技术，哺乳动物类表达系统会导致抗体的糖基化，特别是在Fc区的高度保守N端位点。阳性的克隆在生物反应器的无血清培养基中扩大培养以生产抗体。分泌了抗体的培养液可以用常规技术纯化。比如，用含调整过的缓冲液的A或G Sepharose FF柱进行纯化。洗去非特异性结合的组分。再用PH梯度法洗脱结合的抗体，用SDS-PAGE检测抗体片段，收集。抗体可用常规方法进行过滤浓缩。可溶的混合物和多聚体，也可以用常规方法去除，比如分子筛、离子交换。得到的产物需立即冷冻，如-70℃，或者冻干。Antibodies or antigen-binding fragments engineered in the present disclosure can be prepared and purified using conventional methods. For example, cDNA sequences encoding heavy and light chains can be cloned and recombined into GS expression vectors. The recombinant immunoglobulin expression vector can stably transfect CHO cells. As a more recommended prior art, mammalian expression systems lead to glycosylation of antibodies, especially at the highly conserved N-terminal site of the Fc region. Positive clones are expanded in serum-free medium in bioreactors for antibody production. The culture fluid from which the antibody has been secreted can be purified by conventional techniques. For example, purify with an A or G Sepharose FF column with adjusted buffer. Wash away non-specifically bound components. The bound antibody was then eluted by the pH gradient method, and the antibody fragments were detected by SDS-PAGE and collected. Antibodies can be concentrated by filtration using conventional methods. Soluble mixtures and aggregates can also be removed by conventional methods such as molecular sieves and ion exchange. The obtained product needs to be immediately frozen, such as -70°C, or freeze-dried.
术语“5-6元亚杂芳基”是指具有至少一个杂原子(O、N或S)的芳香族5-元或6-元单环二价基团。所述“5-6元亚杂芳基”的非限制性实例如
Figure PCTCN2022138621-appb-000031
Figure PCTCN2022138621-appb-000032
优选
Figure PCTCN2022138621-appb-000033
The term "5-6 membered heteroarylene" refers to an aromatic 5-membered or 6-membered monocyclic divalent group having at least one heteroatom (O, N or S). Non-limiting examples of the "5-6 membered heteroarylene" are
Figure PCTCN2022138621-appb-000031
Figure PCTCN2022138621-appb-000032
preferred
Figure PCTCN2022138621-appb-000033
术语“直接键”表示其两侧的基团直接相连。例如，L 1’为L 11-L 12-L 13-L 14-L 15-，若L 13为直接键，则L 1’为L 11-L 12-L 14-L 15-。再例如，式I所示的化合物为Ab-(L 1-L 2-L 3-L 4-D) n，若L 4为直接键，则式I所示的化合物为Ab-(L 1-L 2-L 3-D) n。其余类似的定义可以参照前述内容进行理解。 The term "direct bond" means that the groups on both sides are directly connected. For example, L 1' is L 11 -L 12 -L 13 -L 14 -L 15 -, if L 13 is a direct bond, then L 1' is L 11 -L 12 -L 14 -L 15 -. For another example, the compound shown in formula I is Ab-(L 1 -L 2 -L 3 -L 4 -D) n , if L 4 is a direct bond, the compound shown in formula I is Ab-(L 1 - L 2 -L 3 -D) n . Other similar definitions can be understood with reference to the foregoing.
本发明中，“药学上可接受的盐”是指相对无毒的本发明的化合物或偶联物的酸加成盐或碱加成盐。所述酸加成盐为本发明的化合物或偶联物与合适的无机酸或者有机酸形成的盐，这些盐可通过使本发明的化合物或偶联物与适宜的有机酸或无机酸在适当的溶剂中进行反应来制备。代表性酸加成盐包括氢溴酸盐、盐酸盐、硫酸盐、硫酸氢盐、亚硫酸盐、乙酸盐、草酸盐、戊酸盐、油酸盐、棕榈酸盐、硬脂酸盐、月硅酸盐、硼酸盐、苯甲酸盐、乳酸盐、硝酸盐、磷酸盐、磷酸氢盐、碳酸盐、碳酸氢盐、甲苯甲酸盐、柠檬酸盐、马来酸盐、富马酸盐、琥珀酸盐、苹果酸盐、抗坏血酸盐、鞣酸盐、扑酸盐、藻酸盐、萘磺酸盐、酒石酸盐、苯甲酸盐、甲磺酸盐、对甲苯磺酸盐、葡萄糖酸盐、乳糖酸盐和月桂基磺酸盐等。所述碱加成盐为本发明的化合物或偶联物与合适的无机碱或者有机碱形成的盐，这些盐可通过使本发明的化合物或偶联物与适宜的无机碱或者有机碱在适当的溶剂中进行反应来制备。代表性碱加成盐包括例如与碱金属、碱土金属、季铵阳离子形成的盐，例如钠盐、锂盐、钾盐、钙盐、镁盐、四甲基季铵盐、四乙基季铵盐等；胺盐，包括与氨(NH 3)、伯胺、仲胺或叔胺形成的盐，如甲胺盐、二甲胺盐、三甲胺盐、三乙胺盐、乙胺盐等。 In the present invention, "pharmaceutically acceptable salt" refers to a relatively non-toxic acid addition salt or base addition salt of the compound or conjugate of the present invention. The acid addition salt is a salt formed between the compound or conjugate of the present invention and a suitable inorganic acid or organic acid, and these salts can be prepared by making the compound or conjugate of the present invention and a suitable organic acid or inorganic acid prepared by reacting in a solvent. Representative acid addition salts include hydrobromide, hydrochloride, sulfate, bisulfate, sulfite, acetate, oxalate, valerate, oleate, palmitate, stearate Salt, Luurosilicate, Borate, Benzoate, Lactate, Nitrate, Phosphate, Phosphate, Carbonate, Bicarbonate, Toluate, Citrate, Maleic Acid Salt, fumarate, succinate, malate, ascorbate, tannate, pamoate, alginate, naphthalenesulfonate, tartrate, benzoate, methanesulfonate, p-toluene Sulfonate, Gluconate, Lactobionate and Lauryl Sulfonate etc. The base addition salt is a salt formed between the compound or conjugate of the present invention and a suitable inorganic base or organic base, and these salts can be obtained by making the compound or conjugate of the present invention and a suitable inorganic base or organic base prepared by reacting in a solvent. Representative base addition salts include, for example, salts with alkali metal, alkaline earth metal, quaternary ammonium cations, such as sodium, lithium, potassium, calcium, magnesium, tetramethylquaternary ammonium, tetraethylquaternary ammonium Salts, etc.; amine salts, including salts formed with ammonia (NH 3 ), primary amines, secondary amines or tertiary amines, such as methylamine salts, dimethylamine salts, trimethylamine salts, triethylamine salts, ethylamine salts, etc.
本发明的化合物或偶联物可以存在特定的几何或立体异构体形式，本发明的化合物或偶联物中，手性中心可以存在于药物中，可以存在于接头结构中，还可以存在于抗体及其衍生物中。本发明中，所有的这类化合物，包括顺式和反式异构体、(-)-和(+)-对映体、(R)-和(S)-对映体、非对映异构体、(D)-异构体、(L)-异构体，及其外消旋混合物和其他混合物，例如对映异构体或非对映体富集的混合物，均包括在本发明的范围之内。The compounds or conjugates of the present invention can exist in specific geometric or stereoisomer forms. In the compounds or conjugates of the present invention, the chiral center can exist in the drug, in the linker structure, or in the Antibodies and their derivatives. In the present invention, all such compounds, including cis and trans isomers, (-)- and (+)-enantiomers, (R)- and (S)-enantiomers, diastereomers isomers, (D)-isomers, (L)-isomers, and racemic and other mixtures thereof, such as enantiomerically or diastereomerically enriched mixtures, are included in the present invention within the range.
可以通过的手性合成或手性试剂或者其他常规技术制备光学活性的(R)-和(S)-异构体以及D和L异构体。如果想得到本发明的化合物或偶联物的一种对映体，可以通过不对称合成或者具有手性助剂的衍生作用来制备，其中将所得非对映体混合物分离，并且辅助基团裂开以提供纯的所需对映异构体。或者，当分子中含有碱性官能团(如氨基)或酸性官能团(如羧基)时，与适当的光学活性的酸或碱形成非对映异构体的盐，然后通过本领域所公知的常规方法进行非对映异构体拆分，然后回收得到纯的对映体。此外，对映异构体和非对映异构体的分离通常是通过使用色谱法完成的，所述色谱法采用手性固定相，并任选地与化学衍生法 相结合(例如由胺生成氨基甲酸盐)。Optically active (R)- and (S)-isomers as well as D and L-isomers can be prepared by chiral synthesis or chiral reagents or other conventional techniques. If one enantiomer of a compound or conjugate of the invention is desired, it can be prepared by asymmetric synthesis or derivatization with a chiral auxiliary, wherein the resulting diastereomeric mixture is separated and the auxiliary group is cleaved to provide the pure desired enantiomer. Alternatively, when the molecule contains a basic functional group (such as an amino group) or an acidic functional group (such as a carboxyl group), a diastereoisomeric salt is formed with an appropriate optically active acid or base, and then a diastereomeric salt is formed by a conventional method known in the art. Diastereomeric resolution is performed and the pure enantiomers are recovered. Furthermore, the separation of enantiomers and diastereomers is usually accomplished by the use of chromatography using chiral stationary phases, optionally in combination with chemical derivatization methods (e.g. amines to amino groups formate).
本发明中，本发明的化合物、偶联物、药学上可接受的盐、立体异构体的溶剂合物(例如水合物)也在本发明的范围内。作为适当的溶剂合物，具体而言，可以列举本发明的化合物或偶联物与丙酮、2-丁醇、2-丙醇、乙醇、乙酸乙酯、四氢呋喃、二乙醚等形成的溶剂合物。还可以列举出水合物或乙醇化物。In the present invention, solvates (such as hydrates) of the compounds, conjugates, pharmaceutically acceptable salts, and stereoisomers of the present invention are also within the scope of the present invention. As suitable solvates, specifically, solvates formed between compounds or conjugates of the present invention and acetone, 2-butanol, 2-propanol, ethanol, ethyl acetate, tetrahydrofuran, diethyl ether, etc. . Hydrates or ethanolates are also mentioned.
本发明中，“治疗”患有疾病或疾病状况的个体表示所述个体的症状部分或全部缓解，或者在治疗后保持不变。因此，治疗包括预防、治疗和/或治愈。“预防”指防止潜在疾病和/或防止症状恶化或疾病发展。治疗或预防还包括所提供的ADC以及本文所提供的药物组合物、药物制剂的任何药学用途。In the context of the present invention, "treating" an individual suffering from a disease or condition means that the individual's symptoms are partially or completely relieved, or remain unchanged after treatment. Thus, treatment includes prophylaxis, treatment and/or cure. "Prevention" means preventing an underlying disease and/or preventing worsening of symptoms or development of a disease. Treatment or prevention also includes any pharmaceutical use of the provided ADCs and the pharmaceutical compositions, pharmaceutical formulations provided herein.
本公开中，术语“有效剂量”指被给药后会在一定程度上缓解所治疗病症的一种或多种症状的化合物的量。类似地，术语“有效量”指被给药后会在一定程度上抑制癌细胞生长、增殖或迁移的化合物的量。In this disclosure, the term "effective dose" refers to the amount of a compound which, when administered, alleviates to some extent one or more symptoms of the condition being treated. Similarly, the term "effective amount" refers to the amount of a compound which, when administered, inhibits the growth, proliferation or migration of cancer cells to some extent.
本发明中，“疗效”表示由个体的治疗所导致的效果，其改变、通常改良或改善疾病或疾病状况的症状，或者治愈疾病或疾病状况。In the present invention, "therapeutic effect" means the effect resulting from the treatment of an individual, which alters, usually ameliorates or improves the symptoms of a disease or condition, or cures the disease or condition.
本发明中，“个体”包括人或非人动物。示例性人个体包括患有疾病(例如本文所述的疾病)的人个体(称为患者)或正常个体。本发明中，“非人动物”包括所有脊椎动物，例如非哺乳动物(例如鸟类、两栖动物、爬行动物)和哺乳动物，例如非人灵长类、家畜和/或驯化动物(例如绵羊、犬、猫、奶牛、猪等)。In the present invention, "individual" includes human or non-human animal. Exemplary human subjects include human subjects suffering from a disease (eg, a disease described herein) (referred to as a patient) or normal subjects. In the present invention, "non-human animals" include all vertebrates, such as non-mammals (such as birds, amphibians, reptiles) and mammals, such as non-human primates, livestock and/or domesticated animals (such as sheep, dogs, cats, cows, pigs, etc.).
本公开的技术效果：Technical effects of the present disclosure:
本公开的抗体-药物偶联物具有快速高效的肿瘤细胞杀伤活性，同时具有良好的生物相容性、低免疫原性、生物安全性和稳定性。The antibody-drug conjugate of the present disclosure has rapid and efficient tumor cell killing activity, and also has good biocompatibility, low immunogenicity, biosafety and stability.
本公开具有如下的优势：The present disclosure has the following advantages:
(1)设计了一类全新结构的接头(如SM)；(1) A new type of joint structure (such as SM) is designed;
(2)本公开的接头(如SM)与抗体巯基的结合没有逆反应，可以阻止ADC的毒素在血液中因逆反应而脱落，相比传统的MC的接头，增加了ADC的血浆稳定性，降低了毒性；(2) There is no reverse reaction in the combination of the linker (such as SM) of the present disclosure and the sulfhydryl group of the antibody, which can prevent the toxin of the ADC from falling off due to the reverse reaction in the blood. Compared with the traditional MC linker, it increases the plasma stability of the ADC and reduces the toxicity;
(3)在一些实施方案中，本公开通过分析毒素分子艾日布林的结构特点，本公开代表性示例中的ADC分子，省去了常用的自断裂的结构如PAB，将 GGFG单元直接与艾日布林氨基缩合，从而提高了ADC的亲水性，简化了化学合成的复杂性，降低了合成成本，同时显示出优异的药效。(3) In some embodiments, the present disclosure analyzes the structural characteristics of the toxin molecule eribulin. The ADC molecule in the representative example of the present disclosure saves the commonly used self-breaking structure such as PAB, and directly combines the GGFG unit with Eribulin amino condensation improves the hydrophilicity of ADC, simplifies the complexity of chemical synthesis, reduces the cost of synthesis, and shows excellent efficacy.
(4)与MC制备的ADC比，本公开的全新结构的接头制备的ADC的抗肿瘤药效更好。(4) Compared with the ADC prepared by MC, the ADC prepared by the linker with the novel structure of the present disclosure has better anti-tumor efficacy.
附图说明Description of drawings
图1为本发明实施例的ADC-1的SEC-HPLC检测图谱。Fig. 1 is the SEC-HPLC detection pattern of ADC-1 of the embodiment of the present invention.
图2为本发明实施例的ADC-2的SEC-HPLC检测图谱。Fig. 2 is the SEC-HPLC detection pattern of ADC-2 of the embodiment of the present invention.
图3为本发明实施例的ADC-3的SEC-HPLC检测图谱。Fig. 3 is the SEC-HPLC detection pattern of ADC-3 of the embodiment of the present invention.
图4为本发明对照偶联物2(即ADC-6)的SEC-HPLC检测图谱。Fig. 4 is the SEC-HPLC detection spectrum of the control conjugate 2 (ie ADC-6) of the present invention.
图5为采用蛋白疏水作用色谱柱(HIC)检测本发明各ADC的亲疏水性的结果图。Fig. 5 is a graph showing the results of detecting the hydrophilicity and hydrophobicity of each ADC of the present invention by using a protein hydrophobic interaction chromatographic column (HIC).
图6为本发明各ADC与TROP蛋白的亲和力检测结果图。Fig. 6 is a graph showing the results of affinity detection between ADCs and TROP proteins of the present invention.
图7为本发明各ADC的抑瘤活性统计曲线图。Fig. 7 is a statistical graph of the anti-tumor activity of each ADC of the present invention.
图8为本发明各ADC治疗后实验动物体重变化曲线图。Fig. 8 is a graph showing body weight changes of experimental animals after treatment with various ADCs of the present invention.
图9为本发明各ADC治疗后实验动物的取瘤拍照图。Fig. 9 is a photograph taken of tumors taken from experimental animals after each ADC treatment of the present invention.
具体实施方式Detailed ways
下面结合具体实施例，对本公开的方案进行解释。应理解，这些实施例仅用于举例说明本公开而不用于限制本公开的范围。下列实施例中未注明具体技术或条件的，按照本领域内的文献所描述的技术或条件或者按照产品说明书进行。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。The solution of the present disclosure will be explained below in conjunction with specific embodiments. It should be understood that these examples are only for illustrating the present disclosure and are not intended to limit the scope of the present disclosure. If no specific technique or condition is indicated in the following examples, it shall be carried out according to the technique or condition described in the literature in this field or according to the product specification. The reagents or instruments used were not indicated by the manufacturer, and they were all commercially available conventional products.
实施例1：hRS7抗体的制备和检测Example 1: Preparation and detection of hRS7 antibody
1.基因合成、转染和抗体制备1. Gene synthesis, transfection and antibody preparation
hRS7抗体在CHO细胞产生。含hRS7抗体基因的表达载体分别用常规的分子生物学方法构建，hRS7抗体轻链和重链的氨基酸序列分别如SEQ ID NO:1和SEQ ID NO:2所示，其对应的核苷酸序列分别如SEQ ID NO:3和SEQ ID NO:4所示。将上述两序列插入到同一表达载体中，大量提取制备转染质粒，并转染到 CHO-K1细胞(ATCC CCL-61)中，具体转染和抗体制备的过程如下：hRS7 antibody was produced in CHO cells. The expression vectors containing the hRS7 antibody gene were constructed by conventional molecular biology methods. The amino acid sequences of the hRS7 antibody light chain and heavy chain are shown in SEQ ID NO: 1 and SEQ ID NO: 2 respectively, and the corresponding nucleotide sequences Shown in SEQ ID NO:3 and SEQ ID NO:4 respectively. Insert the above two sequences into the same expression vector, extract a large number of transfection plasmids, and transfect them into CHO-K1 cells (ATCC CCL-61). The specific transfection and antibody preparation processes are as follows:
(1)细胞培养：CHO-K1细胞悬浮生长于ActiPro(GE HyClone)培养基，于37℃，7％CO 2，140rpm，90％相对湿度进行培养； (1) Cell culture: CHO-K1 cells were grown in suspension in ActiPro (GE HyClone) medium, and cultured at 37°C, 7% CO 2 , 140 rpm, and 90% relative humidity;
(2)转染：在进入对数生长期后，取细胞离心，重悬于新鲜的ActiPro培养基，计数并调节细胞密度到1.35×10 7个/毫升，转500μl细胞悬液到电击杯中，然后加入40μg构建好的质粒，将细胞与质粒混匀，然后用电转方式导入质粒(Bio-rad电转仪)； (2) Transfection: After entering the logarithmic growth phase, the cells were centrifuged, resuspended in fresh ActiPro medium, counted and adjusted the cell density to 1.35× 107 cells/ml, and transferred 500 μl of the cell suspension to the electric shock cup , then add 40 μg of the constructed plasmid, mix the cells and the plasmid, and then introduce the plasmid by electroporation (Bio-rad electroporator);
(3)亚克隆：电转后的细胞用37℃的ActiPro培养基重悬，每孔100μl分装于96孔板。测定细胞上清以测定抗体的表达水平。将表达水平较高的克隆从96孔板转移到24孔板培养，而后再转入6孔板培养，测定细胞的抗体产量和产率，选择表达量最高的3个克隆进行亚克隆，而后转入摇瓶，放在培养箱中继续培养。(3) Subcloning: The cells after electroporation were resuspended in 37°C ActiPro medium, and 100 μl per well was dispensed into a 96-well plate. Cell supernatants were assayed to determine antibody expression levels. Transfer clones with higher expression levels from 96-well plate to 24-well plate for culture, and then transfer to 6-well plate for culture, measure the antibody production and yield of cells, select the three clones with the highest expression level for subcloning, and then transfer to 6-well plate for culture. into shake flasks and placed in an incubator to continue culturing.
2抗体的纯化2 Antibody purification
收集摇瓶培养的高表达的细胞液，用蛋白A亲和纯化(GE，Mab Select SuRe)和离子交换纯化(GE，Capto S)。采用SDS-PAGE和SEC-HPLC对纯化后的抗体进行分子量和纯度分析。SDS-PAGE测定结果表明制备的hRS7分子量符合预期，用SEC-HPLC法测得抗体纯度为99.1％。The highly expressed cell fluid cultured in shake flasks was collected and purified by protein A affinity purification (GE, Mab Select SuRe) and ion exchange purification (GE, Capto S). SDS-PAGE and SEC-HPLC were used to analyze the molecular weight and purity of the purified antibody. The results of SDS-PAGE showed that the molecular weight of the prepared hRS7 was as expected, and the purity of the antibody measured by SEC-HPLC was 99.1%.
hRS7抗体轻链氨基酸序列hRS7 Antibody Light Chain Amino Acid Sequence
Figure PCTCN2022138621-appb-000034
Figure PCTCN2022138621-appb-000034
hRS7抗体重链氨基酸序列hRS7 Antibody Heavy Chain Amino Acid Sequence
Figure PCTCN2022138621-appb-000035
Figure PCTCN2022138621-appb-000035
Figure PCTCN2022138621-appb-000036
Figure PCTCN2022138621-appb-000036
hRS7抗体轻链核苷酸序列hRS7 Antibody Light Chain Nucleotide Sequence
Figure PCTCN2022138621-appb-000037
Figure PCTCN2022138621-appb-000037
hRS7抗体重链核苷酸序列hRS7 Antibody Heavy Chain Nucleotide Sequence
Figure PCTCN2022138621-appb-000038
Figure PCTCN2022138621-appb-000038
Figure PCTCN2022138621-appb-000039
Figure PCTCN2022138621-appb-000039
实施例2：制备SMEmbodiment 2: preparation SM
Figure PCTCN2022138621-appb-000040
Figure PCTCN2022138621-appb-000040
1.1 SM的合成1.1 Synthesis of SM
Figure PCTCN2022138621-appb-000041
Figure PCTCN2022138621-appb-000041
1.1.1将化合物SM-1(10g,68.43mmol)、DMAP(836mg，6.843mmol)和叔丁醇(100mL,10v/v)加入反应瓶中，氮气保护。在18～22℃缓慢滴加Boc 2O(22.37g,102.64mmol)，期间有微弱升温并产生大量气泡，约一个小时滴完。加毕在18～22℃搅拌2小时。反应液由无色变为红棕色。TLC监测反应结束。向反应液中加冰水(200ml)，用MTBE(100ml*3)萃取，合并有机相，依次用0.5N稀盐酸(200ml)、饱和碳酸氢钠(200ml)和饱和食盐水(200ml)洗，有机相用无水硫酸钠干燥旋干溶剂，得黑色油状物粗品湿法上样过柱，PE/EA＝10/1收集产物得到红棕色油状物SM-2约10g，收率70％。 1.1.1 Add compound SM-1 (10g, 68.43mmol), DMAP (836mg, 6.843mmol) and tert-butanol (100mL, 10v/v) into a reaction flask under nitrogen protection. Boc 2 O (22.37g, 102.64mmol) was slowly added dropwise at 18-22°C, during which there was a slight temperature rise and a large number of bubbles were generated, and the drop was completed in about one hour. After the addition, stir at 18-22°C for 2 hours. The reaction solution changed from colorless to reddish brown. TLC monitored the end of the reaction. Add ice water (200ml) to the reaction solution, extract with MTBE (100ml*3), combine the organic phases, wash with 0.5N dilute hydrochloric acid (200ml), saturated sodium bicarbonate (200ml) and saturated brine (200ml) successively, The organic phase was dried with anhydrous sodium sulfate and the solvent was spin-dried to obtain a crude black oil, which was loaded through the column by wet method, PE/EA=10/1, and the product was collected to obtain about 10 g of reddish-brown oil SM-2, with a yield of 70%.
Figure PCTCN2022138621-appb-000042
Figure PCTCN2022138621-appb-000042
1.1.2将化合物SM-2(5g,24.75mmol)和MeOH(50mL,10v/v)加入反应瓶中，氮气保护,25℃以下滴加80％水合肼(2.32g，37.13mmol),加毕升温到65～70℃反应5小时。LCMS监测反应结束。反应液直接浓缩干，用EA(50ml*2)带旋浓缩得浅黄色油状物粗品SM-3约4g,收率80％。1.1.2 Add compound SM-2 (5g, 24.75mmol) and MeOH (50mL, 10v/v) into the reaction flask, under nitrogen protection, add 80% hydrazine hydrate (2.32g, 37.13mmol) dropwise below 25°C, and complete the addition Raise the temperature to 65-70°C and react for 5 hours. LCMS monitored the completion of the reaction. The reaction solution was directly concentrated to dryness, and concentrated with EA (50ml*2) to give about 4g of crude SM-3 as light yellow oil, with a yield of 80%.
Figure PCTCN2022138621-appb-000043
Figure PCTCN2022138621-appb-000043
1.1.3将化合物SM-3(3.5g,17.3mmol)和THF(70mL,20v/v)加入反应瓶中，氮气保护,加入硫代羰基二咪唑(3.55g，19.9mmol),加毕室温反应1小时。TLC显示原料都转化成中间态。反应液升温到60℃反应6小时，LCMS检测反应终止。向反应液中加入半饱和的氯化钠(100ml),搅拌十分钟后分液。水相用EA(100ml*2)萃取，合并有机相用无水硫酸钠干燥，旋干湿法上样过柱，DCM/MeOH＝40/1收集产品旋干得浅黄色油状物SM-4约2.7g，收率64％。1.1.3 Add compound SM-3 (3.5g, 17.3mmol) and THF (70mL, 20v/v) into the reaction flask, under nitrogen protection, add thiocarbonyldiimidazole (3.55g, 19.9mmol), and complete the reaction at room temperature 1 hour. TLC showed that starting material was all converted to an intermediate state. The temperature of the reaction solution was raised to 60° C. for 6 hours, and the reaction was terminated by LCMS detection. Add half-saturated sodium chloride (100ml) to the reaction solution, stir for ten minutes and then separate the liquids. The aqueous phase was extracted with EA (100ml*2), the combined organic phases were dried with anhydrous sodium sulfate, spin-dried and wet-loaded to the column, DCM/MeOH=40/1, the collected product was spin-dried to obtain light yellow oil SM-4 about 2.7 g, yield 64%.
Figure PCTCN2022138621-appb-000044
Figure PCTCN2022138621-appb-000044
1.1.4将化合物SM-4(2.7g,17.3mmol)和THF(81mL,30v/v)加入反应瓶中，氮气保护,0～10℃时滴加三乙胺(3.35g，33.2mmol),滴毕搅拌5分钟，再滴加CH 3I(3.93g,27.7mmol),滴毕撤去冰浴让其在室温反应16h。向反应液中加入半饱和的氯化钠(100ml),搅拌十分钟后分液。水相用MTBE(50ml*3)萃取，合并有机相用饱和氯化钠(100ml)洗，无水硫酸钠干燥，旋干湿法上样过柱，PE/EA＝5/1收集产品旋干得浅黄色油状物SM-5约2.6g，收率92％。LCMS： [M+1]+＝259.2. 1.1.4 Add compound SM-4 (2.7g, 17.3mmol) and THF (81mL, 30v/v) into the reaction flask, under nitrogen protection, add triethylamine (3.35g, 33.2mmol) dropwise at 0-10°C, Stir for 5 minutes after dropping, then add CH 3 I (3.93g, 27.7mmol) dropwise, remove the ice bath after dropping, let it react at room temperature for 16h. Add half-saturated sodium chloride (100ml) to the reaction solution, stir for ten minutes and then separate the liquids. The aqueous phase was extracted with MTBE (50ml*3), the combined organic phases were washed with saturated sodium chloride (100ml), dried over anhydrous sodium sulfate, spin-dried and loaded through the column by wet method, PE/EA=5/1 to collect the product and spin-dried About 2.6 g of light yellow oil SM-5 was obtained, with a yield of 92%. LCMS: [M+1]+=259.2.
Figure PCTCN2022138621-appb-000045
Figure PCTCN2022138621-appb-000045
1.1.5将化合物SM-5(2.6g,17.3mmol)和DCM(26mL,10v/v)加入反应瓶中，氮气保护,0～10℃时滴加TFA(13ml,5v/v),滴毕撤去冰浴让其在室温反应1h。LCMS监测原料反应完。反应液直接浓缩干，用DCM(50ml*3)带旋，油泵拉干得浅黄色油状物粗品SM-6约3g。LCMS：[M+1]+＝203.1.1.1.5 Add compound SM-5 (2.6g, 17.3mmol) and DCM (26mL, 10v/v) into the reaction flask, under nitrogen protection, add TFA (13ml, 5v/v) dropwise at 0-10°C, dropwise The ice bath was removed and allowed to react at room temperature for 1 h. LCMS monitored the completion of the starting material reaction. The reaction solution was directly concentrated to dryness, swirled with DCM (50ml*3), and pulled dry with an oil pump to obtain about 3g of crude SM-6 as light yellow oil. LCMS: [M+1]+=203.1.
Figure PCTCN2022138621-appb-000046
Figure PCTCN2022138621-appb-000046
1.1.6将化合物SM-6(2g,9.9mmol)和醋酸(20mL,10v/v)加入反应瓶中，氮气保护,0～10℃时分批加入KMnO4(1.88g，11.88mmol),加毕撤去冰浴让其在室温反应2h。LCMS监测原料反应完。将反应液浓缩除去醋酸，残余物用DCM/MeOH＝10/1(50ml)溶解，向其中滴加5％亚硫酸氢钠(100ml)搅拌10分钟后静置分液。水相用DCM/MeOH＝10/1(50ml*3)萃取，合并有机相用无水硫酸钠干燥，旋干得浅黄色固体粗品，溶解后湿法上样过柱，DCM/MeOH＝15/1收集产品得到白色固体SM约520mg，收率22％。LCMS：[M+1]+＝235.1. 1H NMR(400MHz,DMSO-d6)δ12.20(s,1H),3.65(s,3H),3.04(t,J＝4.8Hz,2H),2.41(t,J＝4.8Hz,2H),2.00-1.97(m,2H). 1.1.6 Add compound SM-6 (2g, 9.9mmol) and acetic acid (20mL, 10v/v) into the reaction flask, under nitrogen protection, add KMnO4 (1.88g, 11.88mmol) in batches at 0-10°C, and complete the addition The ice bath was removed and allowed to react at room temperature for 2 h. LCMS monitored the completion of the starting material reaction. The reaction solution was concentrated to remove acetic acid, and the residue was dissolved in DCM/MeOH=10/1 (50ml), and 5% sodium bisulfite (100ml) was added dropwise thereto, stirred for 10 minutes, and then allowed to stand for liquid separation. The aqueous phase was extracted with DCM/MeOH=10/1 (50ml*3), the combined organic phases were dried over anhydrous sodium sulfate, and spin-dried to obtain a light yellow solid crude product, which was dissolved and loaded on the column by wet method, DCM/MeOH=15/ 1. The product was collected to obtain about 520 mg of white solid SM, with a yield of 22%. LCMS: [M+1]+=235.1. 1 H NMR (400MHz, DMSO-d6) δ12.20(s, 1H), 3.65(s, 3H), 3.04(t, J=4.8Hz, 2H), 2.41 (t,J=4.8Hz,2H),2.00-1.97(m,2H).
实施例3：制备偶联物ADC-1Embodiment 3: Preparation of conjugate ADC-1
Figure PCTCN2022138621-appb-000047
Figure PCTCN2022138621-appb-000047
2.1中间体A的制备2.1 Preparation of Intermediate A
Figure PCTCN2022138621-appb-000048
Figure PCTCN2022138621-appb-000048
2.1.1在A1(40mg,0.092mmol)的DCM(2mL)溶液中加入三氟乙酸(1mL)。将反应液在室温条件下反应3小时。将反应液旋干除去溶剂后得粗品目标产物A2大约30mg左右。2.1.1 To a solution of A1 (40 mg, 0.092 mmol) in DCM (2 mL) was added trifluoroacetic acid (1 mL). The reaction solution was reacted at room temperature for 3 hours. After the reaction solution was spin-dried to remove the solvent, about 30 mg of the crude target product A2 was obtained.
Figure PCTCN2022138621-appb-000049
Figure PCTCN2022138621-appb-000049
2.1.2在实施例2制备的SM(20mg,0.085mmol)的NMP(2mL)溶液中加入DIEA(17.3mg,0.17mmol)，HATU(35.71mg,0.093mmol)和A2(28.7mg,0.085mmol)。将反应液在室温条件下搅拌反应3小时。将反应液直接送Pre-HPLC纯化后得目标产物A3大约12mg左右。LCMS：[M+1]+＝553.3.2.1.2 Add DIEA (17.3mg, 0.17mmol), HATU (35.71mg, 0.093mmol) and A2 (28.7mg, 0.085mmol) to the NMP (2mL) solution of SM (20mg, 0.085mmol) prepared in Example 2 . The reaction solution was stirred and reacted at room temperature for 3 hours. The reaction solution was directly sent to Pre-HPLC for purification to obtain about 12 mg of the target product A3. LCMS: [M+1]+=553.3.
Figure PCTCN2022138621-appb-000050
Figure PCTCN2022138621-appb-000050
2.1.3在A3(10mg,0.018mmol)的NMP(2mL)溶液中加入TEA(3.66mg,0.036mmol)，HATU(10.32mg,0.027mmol)和eribulin(13.21mg,0.018mmol)。将反应液于室温条件下搅拌反应3小时。将反应液直接送Pre-HPLC纯化后得目标产物A大约4.5mg左右。LCMS：[M+1]+＝1264.3.2.1.3 Add TEA (3.66mg, 0.036mmol), HATU (10.32mg, 0.027mmol) and eribulin (13.21mg, 0.018mmol) to a solution of A3 (10mg, 0.018mmol) in NMP (2mL). The reaction solution was stirred and reacted at room temperature for 3 hours. The reaction solution was directly sent to Pre-HPLC for purification to obtain about 4.5 mg of the target product A. LCMS: [M+1]+=1264.3.
2.2偶联粗产物ADC-1的合成2.2 Synthesis of coupled crude product ADC-1
Figure PCTCN2022138621-appb-000051
Figure PCTCN2022138621-appb-000051
实施例1制备的抗体在10mg/mL的pH 7.4PBS溶液中，使用25℃水浴锅，搅拌混合的同时加入2.4倍的物质的量的等体积TCEP溶液，溶液置25℃水浴锅静置反应1.5小时。样品使用25℃水浴锅，接着向抗体溶液中搅拌混合的同时加入8倍的物质的量的化合物A的DMSO溶液(DMSO终浓度10％)，25℃摇床摇动反应90分钟，最后样品搅拌混合的同时加入8倍的物质的量的半胱氨酸，置于25℃水浴锅静置反应10min，得到偶联产物ADC-1。The antibody prepared in Example 1 was placed in a 10 mg/mL pH 7.4 PBS solution, using a 25°C water bath, while stirring and mixing, an equal volume of TCEP solution 2.4 times the amount of the substance was added, and the solution was placed in a 25°C water bath for 1.5 Hour. Use a 25°C water bath for the sample, then add 8 times the amount of compound A in DMSO solution (DMSO final concentration 10%) to the antibody solution while stirring and mixing, shake the reaction at 25°C for 90 minutes, and finally stir and mix the sample At the same time, 8 times the amount of cysteine was added, and placed in a 25° C. water bath for 10 minutes to react to obtain the coupling product ADC-1.
2.3偶联粗产物ADC-1的检测2.3 Detection of coupled crude product ADC-1
偶联反应粗产品用SEC检测，The crude product of the coupling reaction is detected by SEC,
SEC色谱条件如下：SEC chromatographic conditions are as follows:
色谱柱型号：TSKgel G4000SWxl 7.8mmI.D.*30cm,8μmColumn model: TSKgel G4000SWxl 7.8mmI.D.*30cm, 8μm
检测器波长：280nm/220nmDetector wavelength: 280nm/220nm
柱温：30℃Column temperature: 30°C
流速：1mL/minFlow rate: 1mL/min
洗脱方式：等度洗脱Elution method: isocratic elution
进样体积：10μLInjection volume: 10μL
运行时间：55minRunning time: 55min
2.4偶联反应产品纯化：2.4 Purification of coupling reaction products:
通过PD-10脱盐柱(填料：sephadex G 25)脱盐纯化后，得偶联物，偶联物再换液到MES 25mM pH5.5海藻糖5％的溶液中，SEC检测，小分子已经完全除掉。纯化后的ADC-1见图1。After being desalted and purified by PD-10 desalting column (filler: sephadex G 25), the conjugate was obtained, and the conjugate was changed into MES 25mM pH5.5 trehalose 5% solution, and the small molecule had been completely removed by SEC detection. Lose. The purified ADC-1 is shown in Figure 1.
2.5 DAR的测定2.5 Determination of DAR
ADC-1和单抗溶液中加入等体积50mM DTT溶液，涡旋混匀，37℃水浴30min。还原后的ADC和抗体用RP-HPLC分析偶联和未偶联的抗体轻链和重链。通过分析其组成确定DAR值为3.9。Add an equal volume of 50mM DTT solution to the ADC-1 and monoclonal antibody solution, vortex and mix well, and bathe in 37°C water for 30min. The reduced ADC and antibody were analyzed by RP-HPLC for conjugated and unconjugated antibody light and heavy chains. The DAR value was determined to be 3.9 by analyzing its composition.
实施例4：制备偶联物ADC-2Embodiment 4: Preparation of conjugate ADC-2
Figure PCTCN2022138621-appb-000052
Figure PCTCN2022138621-appb-000052
3.1中间体B的制备3.1 Preparation of Intermediate B
Figure PCTCN2022138621-appb-000053
Figure PCTCN2022138621-appb-000053
3.1.1将B1(30mg),DCC(23mg)与NHS(13mg)溶解在NMP(1.0mL)中，反应液在室温条件下搅拌过夜，加入B2(GGFG，38mg)，继续反应3小时，HPLC显示有新峰生成，LCMS显示有目标产物生成，Pre-HPLC纯化冻干，得目标化合物B3约44mg。LCMS：[M+1]+＝582.6.3.1.1 Dissolve B1 (30mg), DCC (23mg) and NHS (13mg) in NMP (1.0mL), stir the reaction solution at room temperature overnight, add B2 (GGFG, 38mg), continue to react for 3 hours, HPLC It showed that a new peak was generated, and LCMS showed that the target product was generated. Pre-HPLC purification and lyophilization gave about 44 mg of the target compound B3. LCMS: [M+1]+=582.6.
Figure PCTCN2022138621-appb-000054
Figure PCTCN2022138621-appb-000054
3.1.2将B3(44mg)、HATU(46mg)与Eribulin(50mg)溶解在DMF(2mL)的反应液中，加入DIEA(41微升)，反应液搅拌两小时后，HPLC显示有新峰生成，Pre-HPLC纯化后得目标产物B4约30mg。LCMS：[M+1]+＝1295.0.3.1.2 Dissolve B3 (44mg), HATU (46mg) and Eribulin (50mg) in the reaction solution of DMF (2mL), add DIEA (41 microliters), and stir the reaction solution for two hours, HPLC shows that a new peak is formed , about 30 mg of the target product B4 was obtained after Pre-HPLC purification. LCMS: [M+1]+=1295.0.
Figure PCTCN2022138621-appb-000055
Figure PCTCN2022138621-appb-000055
3.1.3将B4(20mg),溶解在TFA/DCM(0.5mL/1mL)的反应液中，半小时后直接旋转蒸发掉溶液后，直接用于下一步反应。3.1.3 Dissolve B4 (20mg) in the reaction solution of TFA/DCM (0.5mL/1mL). Half an hour later, the solution was directly evaporated by rotary evaporation, and it was directly used in the next reaction.
Figure PCTCN2022138621-appb-000056
Figure PCTCN2022138621-appb-000056
3.1.4在B5旋干的溶液中加入HATU(24mg),实施例2制备的SM(10mg),吡啶(40μL),再加入NMP(0.5mL)，反应4小时后，Pre-HPLC纯化后得目标化合物B约14mg，LCMS：[M+1]+＝1410.0。3.1.4 Add HATU (24 mg), SM (10 mg) prepared in Example 2, pyridine (40 μL) to the spin-dried solution of B5, add NMP (0.5 mL), react for 4 hours, and obtain after Pre-HPLC purification About 14 mg of target compound B, LCMS: [M+1]+=1410.0.
3.2偶联粗产物ADC-2的合成3.2 Synthesis of coupled crude product ADC-2
Figure PCTCN2022138621-appb-000057
Figure PCTCN2022138621-appb-000057
实施例1制备的抗体在10mg/mL的pH 7.4PBS溶液中，使用25℃水浴锅，搅拌混合的同时加入2.4倍的物质的量的等体积TCEP溶液，溶液置25℃水浴锅静置反应1.5小时。样品使用25℃水浴锅，接着向抗体溶液中搅拌混合的同时加入8倍的物质的量的化合物B的DMSO溶液(DMSO终浓度10％)，25℃摇床摇动反应90分钟，最后样品搅拌混合的同时加入8倍的物质的量的半胱氨酸，置于25℃水浴锅静置反应10min，得到偶联产物ADC-2。The antibody prepared in Example 1 was placed in a 10 mg/mL pH 7.4 PBS solution, using a 25°C water bath, while stirring and mixing, an equal volume of TCEP solution 2.4 times the amount of the substance was added, and the solution was placed in a 25°C water bath for 1.5 Hour. Use a 25°C water bath for the sample, then add 8 times the amount of compound B in DMSO solution (DMSO final concentration 10%) to the antibody solution while stirring and mixing, shake the reaction at 25°C for 90 minutes, and finally stir and mix the sample At the same time, 8 times the amount of cysteine was added, and placed in a water bath at 25° C. for 10 minutes to react to obtain the coupling product ADC-2.
3.3偶联粗产物ADC-2的检测3.3 Detection of coupled crude product ADC-2
偶联反应粗产品用SEC检测，The crude product of the coupling reaction is detected by SEC,
SEC色谱条件如下：SEC chromatographic conditions are as follows:
色谱柱型号：TSKgel G4000SWxl 7.8mmI.D.*30cm,8μmColumn model: TSKgel G4000SWxl 7.8mmI.D.*30cm, 8μm
检测器波长：280nm/220nmDetector wavelength: 280nm/220nm
柱温：30℃Column temperature: 30°C
流速：1mL/minFlow rate: 1mL/min
洗脱方式：等度洗脱Elution method: isocratic elution
进样体积：10μLInjection volume: 10μL
运行时间：55minRunning time: 55min
3.4偶联反应产品纯化：3.4 Purification of coupling reaction products:
通过PD-10脱盐柱(填料：sephadex G 25)脱盐纯化后，得偶联物，偶联 物再换液到MES 25mM pH5.5海藻糖5％的溶液中，SEC检测，小分子已经完全除掉。纯化后的ADC-2见图2。After desalting and purifying by PD-10 desalting column (filler: sephadex G 25), the conjugate was obtained, and the conjugate was changed to MES 25mM pH5.5 trehalose 5% solution, and the small molecule was completely removed by SEC detection. Lose. The purified ADC-2 is shown in Figure 2.
3.5 DAR的测定3.5 Determination of DAR
ADC-2和单抗溶液中加入等体积50mM DTT溶液，涡旋混匀，37℃水浴30min。还原后的ADC和抗体用RP-HPLC分析偶联和未偶联的抗体轻链和重链。通过分析其组成确定DAR值为4.1。Add an equal volume of 50mM DTT solution to the ADC-2 and monoclonal antibody solution, vortex and mix well, and bathe in 37°C water bath for 30min. The reduced ADC and antibody were analyzed by RP-HPLC for conjugated and unconjugated antibody light and heavy chains. The DAR value was determined to be 4.1 by analyzing its composition.
实施例5：制备偶联物ADC-3Embodiment 5: Preparation of conjugate ADC-3
Figure PCTCN2022138621-appb-000058
Figure PCTCN2022138621-appb-000058
4.1中间体C的制备4.1 Preparation of Intermediate C
Figure PCTCN2022138621-appb-000059
Figure PCTCN2022138621-appb-000059
4.1.1将实施例2制备的SM(40mg)与C1(32mg)，DCC(35mg),NHS(20mg)溶解在NMP(1mL)的反应液中，反应过夜后有大量固体析出，离心后，固体进行干燥后，直接用于下一步反应，LCMS：[M+1]+＝405.3。4.1.1 Dissolve the SM (40mg) prepared in Example 2, C1 (32mg), DCC (35mg), NHS (20mg) in the reaction solution of NMP (1mL), and a large amount of solids precipitate out after reacting overnight. After centrifugation, After the solid was dried, it was directly used in the next reaction, LCMS: [M+1]+=405.3.
Figure PCTCN2022138621-appb-000060
Figure PCTCN2022138621-appb-000060
4.1.2取干燥后的上一步固体C2(15mg),加入HATU(21mg),eribulin(20 mg),吡啶(40μL),再加入NMP(0.8mL)，反应过夜后，Pre-HPLC纯化后得目标化合物C约8mg，LCMS：[M+1]+＝1116.5。4.1.2 Take the dried solid C2 (15 mg) from the previous step, add HATU (21 mg), eribulin (20 mg), pyridine (40 μL), and then add NMP (0.8 mL), react overnight, and obtain after Pre-HPLC purification About 8 mg of target compound C, LCMS: [M+1]+=1116.5.
4.2偶联粗产物ADC-3的合成4.2 Synthesis of coupled crude product ADC-3
Figure PCTCN2022138621-appb-000061
Figure PCTCN2022138621-appb-000061
实施例1制备的抗体在10mg/mL的pH 7.4PBS溶液中，使用25℃水浴锅，搅拌混合的同时加入2.4倍的物质的量的等体积TCEP溶液，溶液置25℃水浴锅静置反应1.5小时。样品使用25℃水浴锅，接着向抗体溶液中搅拌混合的同时加入8倍的物质的量的化合物C的DMSO溶液(DMSO终浓度10％)，25℃摇床摇动反应90分钟，最后样品搅拌混合的同时加入8倍的物质的量的半胱氨酸，置于25℃水浴锅静置反应10min，得到偶联产物ADC-3。The antibody prepared in Example 1 was placed in a 10 mg/mL pH 7.4 PBS solution, using a 25°C water bath, while stirring and mixing, an equal volume of TCEP solution 2.4 times the amount of the substance was added, and the solution was placed in a 25°C water bath for 1.5 Hour. Use a 25°C water bath for the sample, then add 8 times the amount of compound C in DMSO solution (DMSO final concentration 10%) to the antibody solution while stirring and mixing, shake the reaction at 25°C for 90 minutes, and finally stir and mix the sample At the same time, 8 times the amount of cysteine was added, and placed in a water bath at 25° C. for 10 minutes to react to obtain the coupling product ADC-3.
4.3偶联粗产物ADC-3的检测4.3 Detection of coupled crude product ADC-3
偶联反应粗产品用SEC检测，The crude product of the coupling reaction is detected by SEC,
SEC色谱条件如下：SEC chromatographic conditions are as follows:
色谱柱型号：TSKgel G4000SWxl 7.8mmI.D.*30cm,8μmColumn model: TSKgel G4000SWxl 7.8mmI.D.*30cm, 8μm
检测器波长：280nm/220nmDetector wavelength: 280nm/220nm
柱温：30℃Column temperature: 30°C
流速：1mL/minFlow rate: 1mL/min
洗脱方式：等度洗脱Elution method: isocratic elution
进样体积：10μLInjection volume: 10μL
运行时间：55minRunning time: 55min
4.4偶联反应产品纯化：4.4 Purification of coupling reaction products:
通过PD-10脱盐柱(填料：sephadex G 25)脱盐纯化后，得偶联物，偶联物再换液到MES 25mM pH5.5海藻糖5％的溶液中，SEC检测，小分子已经完全除掉。纯化后的ADC-3见图3。After desalting and purifying by PD-10 desalting column (filler: sephadex G 25), the conjugate was obtained, and the conjugate was changed to MES 25mM pH5.5 trehalose 5% solution, and the small molecule was completely removed by SEC detection. Lose. The purified ADC-3 is shown in Figure 3.
4.5 DAR的测定4.5 Determination of DAR
ADC-3和单抗溶液中加入等体积50mM DTT溶液，涡旋混匀，37℃水浴30min。还原后的ADC和抗体用RP-HPLC分析偶联和未偶联的抗体轻链和重链。通过分析其组成确定DAR值为3.9。Add an equal volume of 50mM DTT solution to the ADC-3 and monoclonal antibody solution, vortex and mix well, and bathe in 37°C water bath for 30min. The reduced ADC and antibody were analyzed by RP-HPLC for conjugated and unconjugated antibody light and heavy chains. The DAR value was determined to be 3.9 by analyzing its composition.
实施例6制备偶联物ADC-4 Embodiment 6 prepares conjugate ADC-4
Figure PCTCN2022138621-appb-000062
Figure PCTCN2022138621-appb-000062
5.中间体D的制备5. Preparation of Intermediate D
Figure PCTCN2022138621-appb-000063
Figure PCTCN2022138621-appb-000063
5.1在D1(34mg,0.091mmol)的DCM(2mL)溶液中加入三氟乙酸(1mL)。将反应液在室温条件下反应3小时。将反应液旋干除去溶剂后得粗品目标产物D2大约24mg左右。5.1 To a solution of D1 (34 mg, 0.091 mmol) in DCM (2 mL) was added trifluoroacetic acid (1 mL). The reaction solution was reacted at room temperature for 3 hours. After the reaction solution was spin-dried to remove the solvent, about 24 mg of the crude target product D2 was obtained.
Figure PCTCN2022138621-appb-000064
Figure PCTCN2022138621-appb-000064
5.2在实施例2制备的SM(20mg,0.085mmol)的NMP(2mL)溶液中加入TEA(17.3mg,0.17mmol)，HATU(35.71mg,0.093mmol)和D2(23.42mg,0.085mmol)。将反应液在室温条件下搅拌反应3小时。将反应液直接送Pre-HPLC纯化后得目标产物D3大约10mg左右。LCMS：[M+1]+＝491.3.5.2 Add TEA (17.3 mg, 0.17 mmol), HATU (35.71 mg, 0.093 mmol) and D2 (23.42 mg, 0.085 mmol) to the NMP (2 mL) solution of SM (20 mg, 0.085 mmol) prepared in Example 2. The reaction solution was stirred and reacted at room temperature for 3 hours. The reaction solution was directly sent to Pre-HPLC for purification to obtain about 10 mg of the target product D3. LCMS: [M+1]+=491.3.
Figure PCTCN2022138621-appb-000065
Figure PCTCN2022138621-appb-000065
在D3(10mg,0.02mmol)的NMP(2mL)溶液中加入TEA(4.13mg,0.04mmol)，HATU(11.63mg,0.03mmol)和eribulin(14.9mg,0.02mmol)。将反应液于室温条件下搅拌反应3小时。将反应液直接送Pre-HPLC纯化后得目标产物D大约4mg左右。LCMS：[M+1]+＝1202.4.To a solution of D3 (10 mg, 0.02 mmol) in NMP (2 mL) was added TEA (4.13 mg, 0.04 mmol), HATU (11.63 mg, 0.03 mmol) and eribulin (14.9 mg, 0.02 mmol). The reaction solution was stirred and reacted at room temperature for 3 hours. The reaction solution was directly sent to Pre-HPLC for purification to obtain about 4 mg of the target product D. LCMS: [M+1]+=1202.4.
与抗体的偶联、偶联产物的检测，纯化以及DAR值的测定同实施例5，获得的ADC-4的DAR值为4.7。The coupling with the antibody, the detection of the coupling product, the purification and the determination of the DAR value are the same as in Example 5, and the obtained ADC-4 has a DAR value of 4.7.
对照例1制备对照化合物1偶联物(ADC-5)Comparative Example 1 Preparation of Comparative Compound 1 Conjugate (ADC-5)
Figure PCTCN2022138621-appb-000066
Figure PCTCN2022138621-appb-000066
6.1中间体E的制备6.1 Preparation of Intermediate E
Figure PCTCN2022138621-appb-000067
Figure PCTCN2022138621-appb-000067
将E1(25mg)与eribulin A2(25mg)溶解在DMF(3mL)中，室温条件下加入DIEA(8.7mg),反应液在室温条件下搅拌2小时，HPLC显示有新峰生成，LCMS显示有目标产物生成，Pre-HPLC纯化冻干，得目标化合物约E 21mg.LCMS：[M+1]+＝1329.4。Dissolve E1 (25 mg) and eribulin A2 (25 mg) in DMF (3 mL), add DIEA (8.7 mg) at room temperature, and stir the reaction solution at room temperature for 2 hours. HPLC shows that a new peak is generated, and LCMS shows that there is a target The product was formed, purified by Pre-HPLC and lyophilized to obtain about E 21 mg of the target compound. LCMS: [M+1]+=1329.4.
6.2偶联粗产物ADC-5的合成6.2 Synthesis of coupled crude product ADC-5
Figure PCTCN2022138621-appb-000068
Figure PCTCN2022138621-appb-000068
实施例1制备的抗体在10mg/mL的pH 7.4PBS溶液中置于冰水浴，当蛋白溶液温度达到4℃时，搅拌混合的同时加入3.5倍的物质的量的等体积TCEP 溶液，溶液置25℃水浴锅静置反应1小时。反应液使用25℃水浴锅，当温度降低至25℃时，接着向抗体溶液中搅拌混合的同时加入7倍的物质的量的化合物E的40％DMSO溶液，25℃摇床摇动反应90分钟，最后反应液搅拌混合的同时加入8倍的物质的量的半胱氨酸，置于25℃水浴锅静置反应10min，得到偶联产物ADC-5。The antibody prepared in Example 1 was placed in an ice-water bath in a 10 mg/mL pH 7.4 PBS solution. When the temperature of the protein solution reached 4°C, an equal volume of TCEP solution 3.5 times the amount of the substance was added while stirring and mixing, and the solution was placed at 25 ℃ water bath for 1 hour. Use a 25°C water bath for the reaction solution. When the temperature drops to 25°C, then add 7 times the amount of compound E in 40% DMSO solution to the antibody solution while stirring and mixing, and shake the reaction at 25°C for 90 minutes. Finally, while the reaction solution was stirred and mixed, cysteine was added in an amount 8 times that of the substance, and placed in a water bath at 25° C. for 10 min to react to obtain the coupling product ADC-5.
偶联产物的检测、纯化和DAR值测定方法同实施例5，获得的ADC-5的DAR值为3.8。The detection, purification and DAR value determination methods of the coupling product are the same as in Example 5, and the obtained ADC-5 has a DAR value of 3.8.
对照例2制备对照化合物2偶联物(ADC-6)Comparative Example 2 Preparation of Comparative Compound 2 Conjugate (ADC-6)
Figure PCTCN2022138621-appb-000069
Figure PCTCN2022138621-appb-000069
7.1中间体F的制备7.1 Preparation of Intermediate F
Figure PCTCN2022138621-appb-000070
Figure PCTCN2022138621-appb-000070
将F1(30mg),eribulin(30mg)与HATU溶解在DMF(0.5mL)中，室温条件下加入DIEA(21微升),反应液在室温条件下搅拌2小时，HPLC显示有新峰生成，LCMS显示有目标产物生成，Pre-HPLC纯化冻干，得目标化合物约F25mg.LCMS：[M+1]+＝1241.6。Dissolve F1 (30 mg), eribulin (30 mg) and HATU in DMF (0.5 mL), add DIEA (21 microliters) at room temperature, and stir the reaction solution at room temperature for 2 hours. HPLC shows that a new peak is generated, and LCMS It was shown that the target product was formed. Pre-HPLC purification and lyophilization gave about F25 mg of the target compound. LCMS: [M+1]+=1241.6.
7.2偶联粗产物ADC-6的合成7.2 Synthesis of coupled crude product ADC-6
Figure PCTCN2022138621-appb-000071
Figure PCTCN2022138621-appb-000071
实施例1制备的抗体在5mg/mL的pH 7.4PBS溶液中，搅拌混合的同时加入7倍的物质的量的TCEP溶液，溶液置37℃水浴锅静置反应1.5小时。反应液使用25℃水浴锅，当温度降低至25℃时，接着向抗体溶液中搅拌混合的同时加入16倍的物质的量的化合物F的40％DMSO溶液，25℃摇床摇动反应90分钟，最后反应液搅拌混合的同时加入16倍的物质的量的半胱氨酸，置于25℃水浴锅静置反应10min，得到偶联产物ADC-6。The antibody prepared in Example 1 was placed in a 5 mg/mL pH 7.4 PBS solution, while stirring and mixing, TCEP solution of 7 times the amount of the substance was added, and the solution was placed in a 37°C water bath for 1.5 hours to react. Use a 25°C water bath for the reaction solution. When the temperature drops to 25°C, then add 16 times the amount of compound F in 40% DMSO solution to the antibody solution while stirring and mixing, and shake the reaction at 25°C for 90 minutes. Finally, while the reaction solution was stirred and mixed, cysteine was added in an amount 16 times that of the substance, and placed in a water bath at 25° C. for 10 minutes to react to obtain the coupling product ADC-6.
7.3偶联粗产物ADC-6的检测7.3 Detection of coupled crude product ADC-6
偶联反应粗产品用SEC检测，The crude product of the coupling reaction is detected by SEC,
SEC色谱条件如下：SEC chromatographic conditions are as follows:
色谱柱型号：TSKgel G4000SWxl 7.8mmI.D.*30cm,8μmColumn model: TSKgel G4000SWxl 7.8mmI.D.*30cm, 8μm
检测器波长：280nm/220nmDetector wavelength: 280nm/220nm
柱温：30℃Column temperature: 30°C
流速：1mL/minFlow rate: 1mL/min
洗脱方式：等度洗脱Elution method: isocratic elution
进样体积：10μLInjection volume: 10μL
运行时间：55minRunning time: 55min
7.4偶联反应产品纯化：7.4 Coupling reaction product purification:
通过PD-10脱盐柱(填料：sephadex G 25)脱盐纯化后，得偶联物，偶联 物再换液到MES 25mM pH5.5海藻糖5％的溶液中，SEC检测，小分子已经完全除掉。纯化后的ADC-6的SEC-HPLC结果见图4。After being desalted and purified by PD-10 desalting column (filler: sephadex G 25), the conjugate was obtained, and the conjugate was changed into MES 25mM pH5.5 trehalose 5% solution, and the small molecule had been completely removed by SEC detection. Lose. The SEC-HPLC results of the purified ADC-6 are shown in FIG. 4 .
7.5 DAR的测定7.5 Determination of DAR
ADC-6和单抗溶液中加入等体积50mM二硫苏糖醇(DTT)溶液，涡旋混匀，37℃水浴30min。还原后的ADC和抗体用RP-HPLC分析偶联和未偶联的抗体轻链和重链。通过分析其组成确定DAR值为7.2。An equal volume of 50 mM dithiothreitol (DTT) solution was added to the ADC-6 and monoclonal antibody solution, vortexed, and placed in a water bath at 37°C for 30 min. The reduced ADC and antibody were analyzed by RP-HPLC for conjugated and unconjugated antibody light and heavy chains. The DAR value was determined to be 7.2 by analyzing its composition.
实施例7检测各ADC的亲水性 Embodiment 7 detects the hydrophilicity of each ADC
采用蛋白疏水作用色谱柱(HIC)检测实施例3-5和对比例1-2中制备的各ADC的亲疏水性，具体实验条件如下：The hydrophilicity and hydrophobicity of each ADC prepared in Example 3-5 and Comparative Example 1-2 were detected by protein hydrophobic interaction chromatographic column (HIC), and the specific experimental conditions were as follows:
取实施例3-5和对比例1-2中制备的各ADC样本30ul进样到HIC(TSKgel Butyl-NPR 4.6mmI.D.×10cm,2.5um)，样品盘温度4℃，柱温40℃，流动相A:1.5M Na 2SO 4&0.025M PB pH7.4&2％IPA(异丙醇)流动相B:0.025M PB pH7.4&11％IPA(异丙醇)，洗脱梯度如下表： Take 30ul of each ADC sample prepared in Example 3-5 and Comparative Example 1-2 and inject it into HIC (TSKgel Butyl-NPR 4.6mmI.D.×10cm, 2.5um), the sample tray temperature is 4°C, and the column temperature is 40°C , mobile phase A: 1.5M Na 2 SO 4 & 0.025M PB pH7.4 & 2% IPA (isopropanol) mobile phase B: 0.025M PB pH7.4 & 11% IPA (isopropanol), the elution gradient is as follows:
Figure PCTCN2022138621-appb-000072
Figure PCTCN2022138621-appb-000072
结果如图5所示，黑色箭头指示的位置为ADC出峰位置，峰位置越靠左说明亲水性越好。比较实施例和对比例的5个ADC的亲水性，能看出亲水性的排序为ADC-6＞ADC-1≈ADC-3＞ADC-5＞ADC-2。因此可见，本公开的SM结构的ADC-1和ADC-3的亲水性能达到与MC结构的ADC-5和ADC-6相近的亲水性。The results are shown in Figure 5. The position indicated by the black arrow is the peak position of the ADC, and the farther to the left the peak position is, the better the hydrophilicity is. Comparing the hydrophilicity of the five ADCs in the example and the comparative example, it can be seen that the ranking of the hydrophilicity is ADC-6>ADC-1≈ADC-3>ADC-5>ADC-2. Therefore, it can be seen that the hydrophilicity of ADC-1 and ADC-3 with SM structure in the present disclosure is close to that of ADC-5 and ADC-6 with MC structure.
实施例8 ADC与TROP蛋白的亲和力实验Example 8 Affinity experiment between ADC and TROP protein
检测实施例3、对比例2制备的ADC与TROP蛋白的亲和力，以比较不同结构的ADC亲和力差别。The affinity between the ADC prepared in Example 3 and Comparative Example 2 and the TROP protein was detected to compare the differences in the affinity of ADCs with different structures.
检测方法：Detection method:
包被Trop2蛋白：将Trop2蛋白(北京义翘神州生物技术有限公司)用包被液(0.15M Na 2CO 3和0.35M NaHCO 3)稀释至0.5μg/mL，混匀后加入到酶标板中，100μL/孔，覆上封板膜，放至4℃冰箱，过夜。 Coating Trop2 protein: Dilute Trop2 protein (Beijing Yiqiao Shenzhou Biotechnology Co., Ltd.) with coating solution (0.15M Na 2 CO 3 and 0.35M NaHCO 3 ) to 0.5 μg/mL, mix well and add to the microtiter plate medium, 100 μL/well, cover the plate with sealing film, and put it in a refrigerator at 4°C overnight.
洗板：拿出酶标板洗板：用1*PBST洗3次。Washing plate: Take out the microplate plate Washing plate: Wash 3 times with 1*PBST.
封闭：取封闭液(1％BSA)，分别加入封闭液200μL/孔，覆上封板膜，250rpm混匀25℃孵育1h。Blocking: Take the blocking solution (1% BSA), add 200 μL/well of the blocking solution, cover the plate with a sealing film, mix at 250 rpm and incubate at 25° C. for 1 h.
洗板：用1*PBST洗3次。Plate washing: wash 3 times with 1*PBST.
孵育抗体：用1％BSA将实施例3、对比例2制备的ADC，和实施例1制备的TROP-2抗体稀释成2000ng/mL后，三倍梯度稀释12个浓度，100μL/孔加至酶标板，覆上封板膜，250rpm混匀25℃孵育1h。Antibody incubation: After diluting the ADC prepared in Example 3 and Comparative Example 2 with 1% BSA, and the TROP-2 antibody prepared in Example 1 to 2000 ng/mL, three-fold serial dilutions were made to 12 concentrations, and 100 μL/well was added to the enzyme Mark the plate, cover the plate with sealing film, mix at 250rpm and incubate at 25°C for 1h.
洗板：将酶标板用1*PBST洗3次。Plate washing: Wash the microplate plate 3 times with 1*PBST.
孵育二抗：将Anti-Human HRP(Boster)用1％BSA稀释成1:10000，100μL/孔加至酶标板，覆上封板膜，250rpm混匀25℃孵育1h。Secondary antibody incubation: Dilute Anti-Human HRP (Boster) with 1% BSA to 1:10000, add 100 μL/well to the microplate, cover the plate with sealing film, mix at 250 rpm and incubate at 25°C for 1 h.
洗板：将酶标板用1*PBST洗3次。Plate washing: Wash the microplate plate 3 times with 1*PBST.
显色：加入TMB(湖州英创)显色底物：每孔100ul加入酶标板，250rpm混匀25℃放置15分钟。Color development: add TMB (Huzhou Yingchuang) color development substrate: add 100ul per well to the microtiter plate, mix at 250rpm and place at 25°C for 15 minutes.
终止读数：加入终止液：每孔加入1M H 2SO 4 100ul，酶标仪OD450nm读数。 Stop reading: add stop solution: add 1M H 2 SO 4 100ul to each well, and read at OD450nm on a microplate reader.
检测结果如图6，圆形代表裸抗的亲和力，三角形代表实施例3制备的ADC-1的亲和力，正方形代表对比例2制备的ADC-6的亲和力。结果表明，ADC-1的亲和力优于对比例2制备的ADC-6，与裸抗更接近。The test results are shown in Figure 6, the circles represent the affinity of the naked antibody, the triangles represent the affinity of ADC-1 prepared in Example 3, and the squares represent the affinity of ADC-6 prepared in Comparative Example 2. The results showed that the affinity of ADC-1 was better than that of ADC-6 prepared in Comparative Example 2, and was closer to the naked antibody.
实施例9 ADC对胰腺癌的抑瘤效果Example 9 Tumor inhibitory effect of ADC on pancreatic cancer
1.实验方法1. Experimental method
1.1细胞培养1.1 Cell culture
BxPC-3细胞(人原位胰腺癌细胞，ATCC CRL-1687)体外单层培养，培养条件为1640培养基中加10％热灭活胎牛血清并加琼脂，于37℃、含5％CO 2空气的培养箱中培养。一周两次用0.25％胰酶进行消化处理传代。当细胞呈指数生长期时，收取细胞，计数，接种。 BxPC-3 cells (human orthotopic pancreatic cancer cells, ATCC CRL-1687) were cultured in a monolayer in vitro. The culture conditions were 10% heat-inactivated fetal calf serum and agar in 1640 medium, at 37°C, containing 5% CO 2 in an air incubator. Digested with 0.25% trypsin twice a week for passage. When the cells are in the exponential growth phase, the cells are collected, counted, and inoculated.
1.2肿瘤细胞接种及瘤块传代1.2 Tumor cell inoculation and passage of tumor mass
将5.0×10 6BxPC-3肿瘤细胞悬浮于0.1ml PBS与Matrigel混合物(1:1)，接种于5只裸鼠右侧肩胛处(P1代)。待肿瘤长至500-800mm 3时，将荷瘤小鼠用CO 2麻醉处死，取出瘤块，去除周围坏死的组织，将瘤块切成20-30mm 3的小瘤块，接种到新的一批裸鼠(P2代)。 5.0×10 6 BxPC-3 tumor cells were suspended in 0.1ml of PBS and Matrigel mixture (1:1), and inoculated on the right scapula of 5 nude mice (P1 generation). When the tumor grows to 500-800 mm 3 , the tumor-bearing mice are anesthetized with CO 2 and killed, the tumor mass is removed, the surrounding necrotic tissue is removed, the tumor mass is cut into small tumor blocks of 20-30 mm 3 , and inoculated into a new tumor mass. Batch nude mice (P2 generation).
1.3瘤块接种及分组给药1.3 Tumor block inoculation and group administration
本试验使用P10代肿瘤组织进行受试品的抗肿瘤活性评价。待P9代肿瘤长至500-800mm 3时，将荷瘤小鼠用CO 2麻醉处死，取出瘤块，去除周围坏死的组织，将状态较好的将瘤块切成20-30mm 3的小瘤块，接种到正式实验用鼠的右侧肩胛处，一共接种65只鼠。瘤块接种14天后肿瘤平均体积达到约200mm 3时，剔除瘤体积过小或过大的小鼠，将剩余的25只小鼠根据瘤体积随机分组并开始给药。给药方案见下表。 In this experiment, the P10 generation tumor tissue was used to evaluate the antitumor activity of the test product. When the P9 generation tumor grows to 500-800mm 3 , the tumor-bearing mice are anesthetized with CO 2 and sacrificed, the tumor mass is removed, the surrounding necrotic tissue is removed, and the tumor mass in good condition is cut into small tumors of 20-30mm 3 Blocks were inoculated to the right shoulder blade of formal experimental mice, and a total of 65 mice were inoculated. When the average tumor volume reached about 200 mm 14 days after tumor block inoculation, the mice with too small or too large tumor volume were excluded, and the remaining 25 mice were randomly grouped according to tumor volume and started to be administered. See the table below for the dosing regimen.
Figure PCTCN2022138621-appb-000073
Figure PCTCN2022138621-appb-000073
1.5实验观察和数据收集1.5 Experimental observation and data collection
肿瘤细胞接种后，除了观察肿瘤生长情况，还对药物治疗对动物行为的影响进行监测：实验动物的活动性，摄食和饮水，体重变化(体重和瘤体积每周测量2次)，眼睛、被毛及其它异常情况。实验过程中观察到的临床症状均记录在原 始数据中。肿瘤体积计算方法为：肿瘤体积(mm 3)＝1/2×(a×b 2)(其中a表示长径，b表示短径)。 After tumor cell inoculation, in addition to observing tumor growth, the effects of drug treatment on animal behavior were also monitored: activity of experimental animals, food intake and drinking, body weight changes (weight and tumor volume were measured twice a week), eyes, Hair and other abnormalities. The clinical symptoms observed during the experiment were recorded in the original data. The calculation method of tumor volume is: tumor volume (mm 3 )=1/2×(a×b 2 ) (where a represents the long diameter and b represents the short diameter).
当单只动物的体重下降超过15％时(BWL>15％)，给予相应单只动物停药处理，体重下降恢复到10％以内，恢复给药。当单只小鼠体重下降>20％，按照动物福利对其实施安乐死。When the body weight of a single animal drops more than 15% (BWL>15%), the corresponding single animal is given drug withdrawal treatment, and the weight loss returns to within 10%, and the drug administration is resumed. When individual mice lost >20% body weight, they were euthanized according to animal welfare guidelines.
1.6疗效评价标准1.6 Curative effect evaluation criteria
相对肿瘤增殖率，T/C％，即在某一时间点，治疗组和对照组的相对肿瘤体积或瘤重的百分比值。计算公式如下：T/C％＝T RTV/C RTV×100％(T RTV：治疗组平均RTV；C RTV：溶媒对照组平均RTV；RTV＝V t/V 0，V 0为分组时该动物的瘤体积，V t为治疗后该动物的瘤体积)；或T/C％＝T TW/C TW×100％(T TW：治疗组实验终结时平均瘤重；C TW：溶媒对照组实验终结时平均瘤重)。 The relative tumor proliferation rate, T/C%, is the percentage value of the relative tumor volume or tumor weight between the treatment group and the control group at a certain time point. The calculation formula is as follows: T/C%=T RTV /C RTV ×100% (T RTV : the average RTV of the treatment group; C RTV : the average RTV of the vehicle control group; RTV=V t /V 0 , where V 0 is the average RTV of the animal in the grouping. V t is the tumor volume of the animal after treatment); or T/C%=T TW /C TW × 100% (T TW : the average tumor weight at the end of the treatment group experiment; C TW : the vehicle control group experiment mean tumor weight at the end).
1.7实验终点1.7 Experimental Endpoint
最后一次给药1周后，所有小鼠取肿瘤，并称重、拍照。One week after the last administration, tumors were taken from all mice, weighed and photographed.
1.8统计分析1.8 Statistical analysis
本实验用one-way ANOVA进行各组间肿瘤均值的比较。方差齐性分析得出F值有显著性差异，在ANOVA分析之后用Dunnet’s T3(方差不齐)法再进行多重比较。用SPSS 17.0进行所有数据分析。p<0.05认为有显著性差异。In this experiment, one-way ANOVA was used to compare the mean values of tumors among the groups. The analysis of the homogeneity of variance shows that there is a significant difference in the F value, and after the ANOVA analysis, multiple comparisons are carried out with Dunnet's T3 (homogeneity of variance) method. All data analyzes were performed with SPSS 17.0. p<0.05 considered significant difference.
2.实验结果2. Experimental results
实验结果如图7-9所示，图7为抑瘤活性统计曲线，图8为体重变化曲线，图9为取瘤拍照图。The experimental results are shown in Figures 7-9, Figure 7 is the statistical curve of anti-tumor activity, Figure 8 is the curve of body weight change, and Figure 9 is the photograph taken of the tumor.
从结果来看，ADC-1的抑瘤效果最好，仅一次用药即可完全让肿瘤消退，且安全性好。ADC-2和ADC-5的抑瘤效果相当。Judging from the results, ADC-1 has the best anti-tumor effect, and it can completely regress the tumor with only one administration, and it is safe. The antitumor effects of ADC-2 and ADC-5 were comparable.

Claims (18)

  1. 式(I)所示的抗体药物偶联物、其药学上可接受的盐、其立体异构体、或其溶剂合物，The antibody-drug conjugate represented by formula (I), its pharmaceutically acceptable salt, its stereoisomer, or its solvate,
    Ab-(L 1-L 2-L 3-L 4-D) n Ab-(L 1 -L 2 -L 3 -L 4 -D) n
    (I)(I)
    其中：in:
    Ab为抗体，例如单克隆抗体或其抗原结合片段；Ab is an antibody, such as a monoclonal antibody or an antigen-binding fragment thereof;
    L 1为-L 12-L 13-L 14-L 15-， L 1 is -L 12 -L 13 -L 14 -L 15 -,
    L 12选自5-6元亚杂芳基； L 12 is selected from 5-6 membered heteroarylene;
    优选地，L 12选自: Preferably, L is selected from:
    Figure PCTCN2022138621-appb-100001
    Figure PCTCN2022138621-appb-100001
    Figure PCTCN2022138621-appb-100002
    Figure PCTCN2022138621-appb-100002
    更优选地，L 12选自: More preferably, L 12 is selected from:
    Figure PCTCN2022138621-appb-100003
    Figure PCTCN2022138621-appb-100003
    Figure PCTCN2022138621-appb-100004
    Figure PCTCN2022138621-appb-100004
    进一步优选地，L 12选自
    Figure PCTCN2022138621-appb-100005
    Further preferably, L 12 is selected from
    Figure PCTCN2022138621-appb-100005
    最优选地，L 12为
    Figure PCTCN2022138621-appb-100006
    Most preferably, L 12 is
    Figure PCTCN2022138621-appb-100006
    L 13选自直接键、
    Figure PCTCN2022138621-appb-100007
    L 13 is selected from direct bonds,
    Figure PCTCN2022138621-appb-100007
    优选地，L 13选自直接键； Preferably, L is selected from direct bonds;
    L 14选自
    Figure PCTCN2022138621-appb-100008
    L 14 from
    Figure PCTCN2022138621-appb-100008
    m为选自1-10之间的整数；m is an integer selected from 1-10;
    优选地，m为选自1-6之间的整数；Preferably, m is an integer selected from 1-6;
    更优选地，m选自1、2、3；More preferably, m is selected from 1, 2, 3;
    最优选地，m为3；Most preferably, m is 3;
    L 15为
    Figure PCTCN2022138621-appb-100009
    L 15 for
    Figure PCTCN2022138621-appb-100009
    L 2选自直接键、
    Figure PCTCN2022138621-appb-100010
    L 2 is selected from direct bonds,
    Figure PCTCN2022138621-appb-100010
    p为选自1-10之间的整数；p is an integer selected from 1-10;
    优选地，p为选自1-6之间的整数；Preferably, p is an integer selected from 1-6;
    更优选地，p选自1、2、3；More preferably, p is selected from 1, 2, 3;
    最优选地，p为2；Most preferably, p is 2;
    L 3为氨基酸单元，优选选自氨基酸残基或由2-10个氨基酸残基组成的肽残基； L3 is an amino acid unit, preferably selected from amino acid residues or peptide residues consisting of 2-10 amino acid residues;
    优选地，L 3为由2-7个(优选2-4个)氨基酸残基组成的肽残基，优选地，所述氨基酸选自苯丙氨酸、甘氨酸、缬氨酸、赖氨酸、瓜氨酸、丝氨酸、谷氨酸、天冬氨酸，更优选地，所述氨基酸选自甘氨酸、苯丙氨酸、缬氨酸、瓜氨酸、丙氨酸； Preferably, L is a peptide residue consisting of 2-7 (preferably 2-4) amino acid residues, preferably, the amino acids are selected from the group consisting of phenylalanine, glycine, valine, lysine, Citrulline, serine, glutamic acid, aspartic acid, more preferably, the amino acid is selected from glycine, phenylalanine, valine, citrulline, alanine;
    更优选地，L 3选自缬氨酸-瓜氨酸(Val-Cit)、丙氨酸-丙氨酸-天冬酰胺(Ala-Ala-Asn)、甘氨酸-甘氨酸-赖氨酸(Gly-Gly-lys)、缬氨酸-赖氨酸(Val-lys)、缬氨酸-丙氨酸(Val-Ala)、缬氨酸-苯丙氨酸(Val-Phe)或甘氨酸-甘氨酸-苯丙氨酸-甘氨酸(Gly-Gly-Phe-Gly)；优选选自甘氨酸-甘氨酸-苯丙氨酸-甘氨酸(Gly-Gly-Phe-Gly)、缬氨酸-瓜氨酸(Val-Cit)、缬氨酸-丙氨酸(Val-Ala)； More preferably, L is selected from valine-citrulline (Val-Cit), alanine-alanine-asparagine (Ala-Ala-Asn), glycine-glycine-lysine (Gly- Gly-lys), Valine-Lysine (Val-lys), Valine-Alanine (Val-Ala), Valine-Phenylalanine (Val-Phe), or Glycine-Glycine-Phenyl Alanine-Glycine (Gly-Gly-Phe-Gly); preferably selected from Glycine-Glycine-Phenylalanine-Glycine (Gly-Gly-Phe-Gly), Valine-Citrulline (Val-Cit) , valine-alanine (Val-Ala);
    最优选地，L 3选自
    Figure PCTCN2022138621-appb-100011
    Figure PCTCN2022138621-appb-100012
    Most preferably, L is selected from
    Figure PCTCN2022138621-appb-100011
    Figure PCTCN2022138621-appb-100012
    L 4选自直接键、
    Figure PCTCN2022138621-appb-100013
    L 4 is selected from direct bonds,
    Figure PCTCN2022138621-appb-100013
    优选地，L 4选自直接键、
    Figure PCTCN2022138621-appb-100014
    Preferably, L is selected from direct bonds,
    Figure PCTCN2022138621-appb-100014
    更优选地，L 4为直接键； More preferably, L 4 is a direct bond;
    D为与L 4通过化学键相连的药物； D is a drug linked to L4 through a chemical bond;
    优选地，D选自艾日布林或其衍生物(如甲磺酸艾日布林等)，DNA拓扑异构酶抑制剂，例如拓扑异构酶I抑制剂(例如喜树碱、羟基喜树碱、9-氨基喜树碱、SN-38、吉咪替康、吉马替康、伊立替康、拓扑替康、依沙替康、DXD、贝洛替康、勒托替康、CKD-602、karenitecin、BN-80915或卢比替康)，拓扑异构酶II抑制剂(例如放线菌素D、阿霉素、多柔比星、多卡米星，柔红霉素、米托蒽醌、鬼臼毒素或依托泊苷)；干扰DNA合成药物，例如甲氨蝶呤、5-氟尿嘧啶、阿糖胞苷、吉西他滨、巯嘌呤、喷司他丁、氟达拉滨、克拉屈滨或奈拉滨；作用 于结构蛋白的药物，例如微管蛋白抑制剂如MMAE(Monomethyl auristatin E)、MMAF(Monomethyl auristatin F)、MMAD(MonoMethyl Dolastatin 10)，长春花生物碱类、长春新碱、长春碱、紫杉醇、多西他赛或卡巴他赛；肿瘤信号通路抑制剂，例如丝氨酸/苏氨酸激酶抑制剂、酪氨酸激酶抑制剂、天冬氨酸激酶抑制剂或组氨酸激酶抑制剂；蛋白酶体抑制剂；组蛋白去乙酰化酶抑制剂；肿瘤新生血管生成抑制剂；细胞周期蛋白抑制剂；美登素衍生物，例如Maytansine DM1、Maytansine DM4；卡里奇霉素或其衍生物；奥瑞他汀衍生物；Pyrrolobenzodiazepine dimers(PBD)衍生物；美法仑；丝裂霉素C；苯丁酸氮芥；MGBA(如duocarmycin)；阿霉素(doxorubicin)；蓖麻毒素；白喉毒素；以及其它抑制肿瘤细胞生长、促进肿瘤细胞凋亡或坏死的活性物质；Preferably, D is selected from eribulin or its derivatives (such as eribulin mesylate, etc.), DNA topoisomerase inhibitors, such as topoisomerase I inhibitors (such as camptothecin, hydroxycycline Tricine, 9-aminocamptothecin, SN-38, gemitecan, gematecan, irinotecan, topotecan, exsatecan, DXD, belotecan, letotecan, CKD -602, karenitecin, BN-80915, or rubitecan), topoisomerase II inhibitors (eg, actinomycin D, doxorubicin, doxorubicin, duocamicin, daunorubicin, mitol anthraquinone, podophyllotoxin, or etoposide); drugs that interfere with DNA synthesis, such as methotrexate, 5-fluorouracil, cytarabine, gemcitabine, mercaptopurine, pentostatin, fludarabine, cladribine or nalarabine; drugs acting on structural proteins, such as tubulin inhibitors such as MMAE (Monomethyl auristatin E), MMAF (Monomethyl auristatin F), MMAD (MonoMethyl Dolastatin 10), vinca alkaloids, vincristine, Vinblastine, paclitaxel, docetaxel, or cabazitaxel; tumor signaling pathway inhibitors, such as serine/threonine kinase inhibitors, tyrosine kinase inhibitors, aspartate kinase inhibitors, or histidine kinase inhibitors proteasome inhibitors; histone deacetylase inhibitors; tumor angiogenesis inhibitors; cell cyclin inhibitors; maytansine derivatives, such as Maytansine DM1, Maytansine DM4; calicheamicin or its derivatives Auristatin derivatives; Pyrrolobenzodiazepine dimers (PBD) derivatives; Melphalan; Mitomycin C; Chlorambucil; MGBA (eg, duocarmycin); Doxorubicin; Ricin; Diphtheria Toxins; and other active substances that inhibit tumor cell growth, promote tumor cell apoptosis or necrosis;
    更优选地，所述药物选自艾日布林或其衍生物(如甲磺酸艾日布林等)、喜树碱或其衍生物(如羟基喜树碱、9-氨基喜树碱、SN-38、吉咪替康、吉马替康、伊立替康、拓扑替康、依沙替康、DXD、贝洛替康、勒托替康、CKD-602、karenitecin、BN-80915或卢比替康)、Monomethyl auristatin E(MMAE)、Monomethyl auristatin F(MMAF)、MonoMethyl Dolastatin 10(MMAD)；More preferably, the drug is selected from eribulin or its derivatives (such as eribulin mesylate, etc.), camptothecin or its derivatives (such as hydroxycamptothecin, 9-aminocamptothecin, SN-38, Gemitecan, Gematecan, Irinotecan, Topotecan, Exatecan, DXD, Belotecan, Letotecan, CKD-602, karenitecin, BN-80915, or Rupees Tecan), Monomethyl auristatin E (MMAE), Monomethyl auristatin F (MMAF), MonoMethyl Dolastatin 10 (MMAD);
    进一步优选地，所述药物选自艾日布林或其衍生物(如甲磺酸艾日布林等)、MMAE、DXD，优选艾日布林或其衍生物(如甲磺酸艾日布林等)；Further preferably, the drug is selected from eribulin or its derivatives (such as eribulin mesylate, etc.), MMAE, DXD, preferably eribulin or its derivatives (such as eribulin mesylate, etc. forest, etc.);
    最优选地，D为
    Figure PCTCN2022138621-appb-100015
    Most preferably, D is
    Figure PCTCN2022138621-appb-100015
    n为1-20之间的任意数值；n is any value between 1-20;
    优选地，n为1-10之间的任意数值；Preferably, n is any value between 1-10;
    更优选地，n为1-5之间的任意数值；More preferably, n is any value between 1-5;
    最优选地，n为3.5-5.0之间的任意数值，例如，n为3.9、4.1或4.7。Most preferably, n is any value between 3.5-5.0, for example, n is 3.9, 4.1 or 4.7.
  2. 权利要求1所述的抗体药物偶联物、其药学上可接受的盐、其立体异构体、或其溶剂合物，其中，所述抗体选自抗Her 2的单克隆抗体，例如曲妥珠单 抗、帕妥珠单抗(Pertuzumab)或抗Trop-2的单克隆抗体，例如Sacituzumab；The antibody-drug conjugate of claim 1, its pharmaceutically acceptable salt, its stereoisomer, or its solvate, wherein the antibody is selected from anti-Her 2 monoclonal antibodies, such as trastole Zhuzumab, Pertuzumab, or anti-Trop-2 monoclonal antibody, such as Sacituzumab;
    优选地，所述抗体为抗TROP-2抗体；Preferably, the antibody is an anti-TROP-2 antibody;
    更优选地，所述抗体为抗TROP-2抗体，且所述抗Trop-2抗体的轻链可变区的互补决定区(CDR)包括由KASQDVSIAVA氨基酸序列组成的CDR1，由SASYRYT氨基酸序列组成的CDR2，和由QQHYITPLT氨基酸序列组成的CDR3；重链可变区的CDR包括由NYGMN氨基酸序列组成的CDR1，由WINTYTGEPTYTDDFKG氨基酸序列组成的CDR2，和由GGFGSSYWYFDV氨基酸序列组成的CDR3；优选地，所述抗Trop-2抗体的轻链及重链的氨基酸序列分别如SEQ ID NO：1和SEQ ID NO：2所示；优选地，所述抗Trop-2抗体的轻链和重链的编码核苷酸序列分别如SEQ ID NO:3和SEQ ID NO:4所示。More preferably, the antibody is an anti-TROP-2 antibody, and the complementarity determining region (CDR) of the light chain variable region of the anti-Trop-2 antibody includes CDR1 consisting of the KASQDVSIAVA amino acid sequence, and CDR1 consisting of the SASYRYT amino acid sequence CDR2, and CDR3 made up of the QQHYITPLT amino acid sequence; the CDR of the heavy chain variable region includes CDR1 made up of the NYGMN amino acid sequence, CDR2 made up of the WINTYTGEPTYTDDFKG amino acid sequence, and CDR3 made up of the GGFGSSYWYFDV amino acid sequence; preferably, the anti The amino acid sequences of the light chain and the heavy chain of the Trop-2 antibody are respectively shown in SEQ ID NO: 1 and SEQ ID NO: 2; preferably, the encoding nucleotides of the light chain and the heavy chain of the anti-Trop-2 antibody The sequences are shown in SEQ ID NO:3 and SEQ ID NO:4 respectively.
  3. 权利要求1-2任一项所述的抗体药物偶联物、其药学上可接受的盐、其立体异构体、或其溶剂合物，其中，所述抗体药物偶联物选自以下：The antibody drug conjugate, its pharmaceutically acceptable salt, its stereoisomer, or its solvate according to any one of claims 1-2, wherein the antibody drug conjugate is selected from the following:
    Figure PCTCN2022138621-appb-100016
    Figure PCTCN2022138621-appb-100016
    Figure PCTCN2022138621-appb-100017
    Figure PCTCN2022138621-appb-100017
  4. 式(II)所示化合物、其药学上可接受的盐、其立体异构体、或其溶剂合物，The compound represented by formula (II), its pharmaceutically acceptable salt, its stereoisomer, or its solvate,
    L 1’-L 2-L 3-L 4-D L 1' -L 2 -L 3 -L 4 -D
    (II)(II)
    其中：in:
    L 1’为L 11-L 12-L 13-L 14-L 15-； L 1' is L 11 -L 12 -L 13 -L 14 -L 15 -;
    L 11为
    Figure PCTCN2022138621-appb-100018
    L 11 is
    Figure PCTCN2022138621-appb-100018
    L 12、L 13、L 14、L 15如权利要求1所限定； L 12 , L 13 , L 14 , L 15 are as defined in claim 1;
    L 2、L 3、L 4、D如权利要求1所限定。 L 2 , L 3 , L 4 , D are as defined in claim 1 .
  5. 权利要求4所述的化合物、其药学上可接受的盐、其立体异构体，或其溶剂合物、其中，所述化合物选自：The compound of claim 4, its pharmaceutically acceptable salt, its stereoisomer, or its solvate, wherein the compound is selected from the group consisting of:
    Figure PCTCN2022138621-appb-100019
    Figure PCTCN2022138621-appb-100019
    Figure PCTCN2022138621-appb-100020
    Figure PCTCN2022138621-appb-100020
  6. 式(III)所示化合物、其药学上可接受的盐、其立体异构体、或其溶剂合物，The compound represented by formula (III), its pharmaceutically acceptable salt, its stereoisomer, or its solvate,
    L 11-L 12-L 13-L 14-L 15’ L 11 -L 12 -L 13 -L 14 -L 15'
    (III)(III)
    其中：in:
    L 11为
    Figure PCTCN2022138621-appb-100021
    L 11 is
    Figure PCTCN2022138621-appb-100021
    L 12、L 13、L 14如权利要求1所限定； L 12 , L 13 , L 14 are as defined in claim 1;
    L 15’为
    Figure PCTCN2022138621-appb-100022
    L 15' for
    Figure PCTCN2022138621-appb-100022
  7. 权利要求6所述的化合物、其药学上可接受的盐、其立体异构体，或其溶剂合物、其中，所述化合物为
    Figure PCTCN2022138621-appb-100023
    The compound according to claim 6, its pharmaceutically acceptable salt, its stereoisomer, or its solvate, wherein the compound is
    Figure PCTCN2022138621-appb-100023
  8. 式(IV)所示的中间体化合物、其药学上可接受的盐、其立体异构体、或其溶剂合物，The intermediate compound represented by formula (IV), its pharmaceutically acceptable salt, its stereoisomer, or its solvate,
    L 1’-L 2-L 3-OH L 1' -L 2 -L 3 -OH
    (IV)(IV)
    其中：in:
    L 1’为L 11-L 12-L 13-L 14-L 15-； L 1' is L 11 -L 12 -L 13 -L 14 -L 15 -;
    L 11为
    Figure PCTCN2022138621-appb-100024
    L 11 is
    Figure PCTCN2022138621-appb-100024
    L 12、L 13、L 14、L 15如权利要求1所限定； L 12 , L 13 , L 14 , L 15 are as defined in claim 1;
    L 2、L 3如权利要求1所限定。 L 2 and L 3 are as defined in claim 1.
  9. 权利要求8所述的中间体化合物、其药学上可接受的盐、其立体异构体，或其溶剂合物、其中，所述中间体化合物选自The intermediate compound, its pharmaceutically acceptable salt, its stereoisomer, or its solvate according to claim 8, wherein the intermediate compound is selected from
    Figure PCTCN2022138621-appb-100025
    Figure PCTCN2022138621-appb-100025
    Figure PCTCN2022138621-appb-100026
    Figure PCTCN2022138621-appb-100026
  10. 制备权利要求4-5任一项所述的式(II)所示的化合物、其药学上可接受的盐、其立体异构体、或其溶剂合物的方法，其包括：The method for preparing the compound represented by formula (II) described in any one of claims 4-5, its pharmaceutically acceptable salt, its stereoisomer, or its solvate, which comprises:
    将L 1’-L 2-L 3-OH与艾日布林进行酰胺化反应，获得式(II)所示的化合物、其药学上可接受的盐、其立体异构体、或其溶剂合物，其中，L 1’、L 2、L 3如权利要求8所限定。 Amidation reaction of L 1' -L 2 -L 3 -OH with eribulin to obtain the compound represented by formula (II), its pharmaceutically acceptable salt, its stereoisomer, or its solvate , wherein L 1' , L 2 , and L 3 are as defined in claim 8.
  11. 制备权利要求1-3任一项所述的式(I)所示的抗体药物偶联物、其药学上可接受的盐、其立体异构体、或其溶剂合物的方法，其包括：The method for preparing the antibody-drug conjugate represented by formula (I), its pharmaceutically acceptable salt, its stereoisomer, or its solvate according to any one of claims 1-3, comprising:
    将权利要求4-5任一项所述的式(II)所示的化合物、其药学上可接受的盐、其立体异构体、或其溶剂合物与权利要求1-2任一项所限定的Ab反应，获得式(I)所示的抗体药物偶联物、其药学上可接受的盐、其立体异构体、或其溶剂合物；The compound represented by formula (II) described in any one of claims 4-5, its pharmaceutically acceptable salt, its stereoisomer, or its solvate and any one of claims 1-2 Defined Ab reaction to obtain the antibody-drug conjugate represented by formula (I), its pharmaceutically acceptable salt, its stereoisomer, or its solvate;
    优选地，所述权利要求4-5任一项所述的式(II)所示的化合物、其药学上可接受的盐、其立体异构体、或其溶剂合物是通过权利要求10所述的方法制备得到的。Preferably, the compound represented by formula (II) according to any one of claims 4-5, its pharmaceutically acceptable salt, its stereoisomer, or its solvate is obtained by claim 10 prepared by the method described above.
  12. 药物组合物，其包含权利要求1-3任一项所述的式(I)所示的抗体药物偶联物、其药学上可接受的盐、其立体异构体、或其溶剂合物，或者包含权利要求4-5任一项所述的式(II)所示的化合物、其药学上可接受的盐、其立体异构体、或其溶剂合物；任选地，还包含一种或多种药用辅料，例如载体和/或赋形 剂。A pharmaceutical composition comprising the antibody-drug conjugate represented by formula (I) according to any one of claims 1-3, its pharmaceutically acceptable salt, its stereoisomer, or its solvate, Or comprise the compound shown in formula (II) described in any one of claim 4-5, its pharmaceutically acceptable salt, its stereoisomer or its solvate; Optionally, also comprise a or various pharmaceutical excipients, such as carriers and/or excipients.
  13. 权利要求1-3任一项所述的式(I)所示的抗体药物偶联物、其药学上可接受的盐、其立体异构体、或其溶剂合物，或者权利要求4-5任一项所述的式(II)所示的化合物、其药学上可接受的盐、其立体异构体、或其溶剂合物，或者权利要求12所述的药物组合物在制备用于预防和/或治疗与细胞活动异常相关的疾病(例如癌症疾病)的药物中的用途；The antibody drug conjugate represented by formula (I) according to any one of claims 1-3, its pharmaceutically acceptable salt, its stereoisomer, or its solvate, or claims 4-5 The compound represented by formula (II) described in any one, its pharmaceutically acceptable salt, its stereoisomer, or its solvate, or the pharmaceutical composition described in claim 12 is used for preventing and/or use in medicines for the treatment of diseases associated with abnormal cell activity (such as cancer diseases);
    优选地，所述癌症选自乳腺癌、结直肠癌、肺癌、胰腺癌、卵巢癌、前列腺癌、宫颈癌、肾癌、尿道癌、胶质细胞瘤、黑色素瘤、肝癌、膀胱癌、胃癌、食道癌；优选地，所述癌症是原位癌或转移癌；Preferably, the cancer is selected from breast cancer, colorectal cancer, lung cancer, pancreatic cancer, ovarian cancer, prostate cancer, cervical cancer, kidney cancer, urethral cancer, glioma, melanoma, liver cancer, bladder cancer, gastric cancer, Esophageal cancer; preferably, said cancer is carcinoma in situ or metastatic carcinoma;
    更优选地，所述癌症选自结肠癌、胰腺癌；More preferably, the cancer is selected from colon cancer, pancreatic cancer;
    最优选地，所述癌症为胰腺癌(如原位胰腺癌)。Most preferably, the cancer is pancreatic cancer (eg pancreatic carcinoma in situ).
  14. 权利要求1-3任一项所述的式(I)所示的抗体药物偶联物、其药学上可接受的盐、其立体异构体、或其溶剂合物，或者权利要求4-5任一项所述的式(II)所示的化合物、其药学上可接受的盐、其立体异构体、或其溶剂合物，或者权利要求12所述的药物组合物，其用于预防和/或治疗与细胞活动异常相关的疾病(例如癌症疾病)；The antibody drug conjugate represented by formula (I) according to any one of claims 1-3, its pharmaceutically acceptable salt, its stereoisomer, or its solvate, or claims 4-5 The compound represented by formula (II) described in any one, its pharmaceutically acceptable salt, its stereoisomer, or its solvate, or the pharmaceutical composition described in claim 12, which is used to prevent and/or to treat diseases associated with abnormal cellular activity (such as cancer diseases);
    优选地，所述癌症选自乳腺癌、结直肠癌、肺癌、胰腺癌、卵巢癌、前列腺癌、宫颈癌、肾癌、尿道癌、胶质细胞瘤、黑色素瘤、肝癌、膀胱癌、胃癌、食道癌；优选地，所述癌症是原位癌或转移癌；Preferably, the cancer is selected from breast cancer, colorectal cancer, lung cancer, pancreatic cancer, ovarian cancer, prostate cancer, cervical cancer, kidney cancer, urethral cancer, glioma, melanoma, liver cancer, bladder cancer, gastric cancer, Esophageal cancer; preferably, said cancer is carcinoma in situ or metastatic carcinoma;
    更优选地，所述癌症选自结肠癌、胰腺癌；More preferably, the cancer is selected from colon cancer, pancreatic cancer;
    最优选地，所述癌症为胰腺癌(如原位胰腺癌)。Most preferably, the cancer is pancreatic cancer (eg pancreatic carcinoma in situ).
  15. 一种预防或治疗与细胞活动异常相关的疾病(例如癌症疾病)的方法，其包括向有此需要的个体施用有效剂量的权利要求1-3任一项所述的式(I)所示的抗体药物偶联物、其药学上可接受的盐、其立体异构体、或其溶剂合物，或者权利要求4-5任一项所述的式(II)所示的化合物、其药学上可接受的盐、其立体异构体、或其溶剂合物，或者权利要求12所述的药物组合物；A method for preventing or treating a disease (such as cancer) associated with abnormal cell activity, comprising administering an effective dose of the formula (I) shown in any one of claims 1-3 to an individual in need thereof Antibody drug conjugate, its pharmaceutically acceptable salt, its stereoisomer, or its solvate, or the compound represented by formula (II) described in any one of claims 4-5, its pharmaceutical Acceptable salts, stereoisomers thereof, or solvates thereof, or the pharmaceutical composition of claim 12;
    优选地，所述癌症选自乳腺癌、结直肠癌、肺癌、胰腺癌、卵巢癌、前列腺癌、宫颈癌、肾癌、尿道癌、胶质细胞瘤、黑色素瘤、肝癌、膀胱癌、胃癌、食道癌；优选地，所述癌症是原位癌或转移癌；Preferably, the cancer is selected from breast cancer, colorectal cancer, lung cancer, pancreatic cancer, ovarian cancer, prostate cancer, cervical cancer, kidney cancer, urethral cancer, glioma, melanoma, liver cancer, bladder cancer, gastric cancer, Esophageal cancer; preferably, said cancer is carcinoma in situ or metastatic carcinoma;
    更优选地，所述癌症选自结肠癌、胰腺癌；More preferably, the cancer is selected from colon cancer, pancreatic cancer;
    最优选地，所述癌症为胰腺癌(如原位胰腺癌)。Most preferably, the cancer is pancreatic cancer (eg pancreatic carcinoma in situ).
  16. 权利要求1-3任一项所述的式(I)所示的抗体药物偶联物、其药学上可接受的盐、其立体异构体、或其溶剂合物，或者权利要求4-5任一项所述的式(II)所示的化合物、其药学上可接受的盐、其立体异构体、或其溶剂合物，或者权利要求12所述的药物组合物在制备用于抑制癌细胞生长、增殖或迁移的试剂中的用途；The antibody drug conjugate represented by formula (I) according to any one of claims 1-3, its pharmaceutically acceptable salt, its stereoisomer, or its solvate, or claims 4-5 The compound represented by formula (II) described in any one, its pharmaceutically acceptable salt, its stereoisomer, or its solvate, or the pharmaceutical composition described in claim 12 is used to inhibit Use in reagents for the growth, proliferation or migration of cancer cells;
    优选地，所述癌症选自乳腺癌、结直肠癌、肺癌、胰腺癌、卵巢癌、前列腺癌、宫颈癌、肾癌、尿道癌、胶质细胞瘤、黑色素瘤、肝癌、膀胱癌、胃癌、食道癌的癌细胞；优选地，所述癌症是原位癌或转移癌的癌细胞；Preferably, the cancer is selected from breast cancer, colorectal cancer, lung cancer, pancreatic cancer, ovarian cancer, prostate cancer, cervical cancer, kidney cancer, urethral cancer, glioma, melanoma, liver cancer, bladder cancer, gastric cancer, Cancer cells of esophageal cancer; preferably, the cancer is cancer cells of carcinoma in situ or metastases;
    更优选地，所述癌细胞选自结肠癌、胰腺癌的癌细胞；More preferably, the cancer cells are selected from cancer cells of colon cancer and pancreatic cancer;
    最优选地，所述癌细胞为胰腺癌癌细胞(如原位胰腺癌细胞)。Most preferably, the cancer cells are pancreatic cancer cells (such as pancreatic cancer cells in situ).
  17. 权利要求1-3任一项所述的式(I)所示的抗体药物偶联物、其药学上可接受的盐、其立体异构体、或其溶剂合物，或者权利要求4-5任一项所述的式(II)所示的化合物、其药学上可接受的盐、其立体异构体、或其溶剂合物，或者权利要求12所述的药物组合物，其用于抑制癌细胞的生长、增殖或迁移；The antibody drug conjugate represented by formula (I) according to any one of claims 1-3, its pharmaceutically acceptable salt, its stereoisomer, or its solvate, or claims 4-5 The compound represented by formula (II) described in any one, its pharmaceutically acceptable salt, its stereoisomer, or its solvate, or the pharmaceutical composition described in claim 12, which is used to inhibit the growth, proliferation, or migration of cancer cells;
    优选地，所述癌症选自乳腺癌、结直肠癌、肺癌、胰腺癌、卵巢癌、前列腺癌、宫颈癌、肾癌、尿道癌、胶质细胞瘤、黑色素瘤、肝癌、膀胱癌、胃癌、食道癌的癌细胞；优选地，所述癌症是原位癌或转移癌的癌细胞；Preferably, the cancer is selected from breast cancer, colorectal cancer, lung cancer, pancreatic cancer, ovarian cancer, prostate cancer, cervical cancer, kidney cancer, urethral cancer, glioma, melanoma, liver cancer, bladder cancer, gastric cancer, Cancer cells of esophageal cancer; preferably, the cancer is cancer cells of carcinoma in situ or metastases;
    更优选地，所述癌细胞选自结肠癌、胰腺癌的癌细胞；More preferably, the cancer cells are selected from cancer cells of colon cancer and pancreatic cancer;
    最优选地，所述癌细胞为胰腺癌癌细胞(如原位胰腺癌细胞)。Most preferably, the cancer cells are pancreatic cancer cells (such as pancreatic cancer cells in situ).
  18. 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权利要求1-3任一项所述的式(I)所示的抗体药物偶联物、其药学上可接受 的盐、其立体异构体、或其溶剂合物，或者权利要求4-5任一项所述的式(II)所示的化合物、其药学上可接受的盐、其立体异构体、或其溶剂合物，或者权利要求12所述的药物组合物；A method for inhibiting cancer cell growth, proliferation or migration, comprising administering to cancer cells an effective amount of the antibody-drug conjugate represented by formula (I) according to any one of claims 1-3, which is pharmaceutically acceptable Acceptable salt, its stereoisomer, or its solvate, or the compound shown in formula (II) described in any one of claim 4-5, its pharmaceutically acceptable salt, its stereoisomer , or a solvate thereof, or the pharmaceutical composition of claim 12;
    优选地，所述癌症选自乳腺癌、结直肠癌、肺癌、胰腺癌、卵巢癌、前列腺癌、宫颈癌、肾癌、尿道癌、胶质细胞瘤、黑色素瘤、肝癌、膀胱癌、胃癌、食道癌的癌细胞；优选地，所述癌症是原位癌或转移癌的癌细胞；Preferably, the cancer is selected from breast cancer, colorectal cancer, lung cancer, pancreatic cancer, ovarian cancer, prostate cancer, cervical cancer, kidney cancer, urethral cancer, glioma, melanoma, liver cancer, bladder cancer, gastric cancer, Cancer cells of esophageal cancer; preferably, the cancer is cancer cells of carcinoma in situ or metastases;
    更优选地，所述癌细胞选自结肠癌、胰腺癌的癌细胞；More preferably, the cancer cells are selected from cancer cells of colon cancer and pancreatic cancer;
    最优选地，所述癌细胞为胰腺癌癌细胞(如原位胰腺癌细胞)。Most preferably, the cancer cells are pancreatic cancer cells (such as pancreatic cancer cells in situ).
PCT/CN2022/138621 2021-12-27 2022-12-13 Antibody-drug conjugate having reduced reversible reaction, and preparation method therefor and application thereof WO2023124963A1 (en)

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