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WO2023143320A1 - Linker for preparing antibody-drug conjugate, compound, and use - Google Patents

Linker for preparing antibody-drug conjugate, compound, and use Download PDF

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Publication number
WO2023143320A1
WO2023143320A1 PCT/CN2023/072958 CN2023072958W WO2023143320A1 WO 2023143320 A1 WO2023143320 A1 WO 2023143320A1 CN 2023072958 W CN2023072958 W CN 2023072958W WO 2023143320 A1 WO2023143320 A1 WO 2023143320A1
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WIPO (PCT)
Prior art keywords
cancer
integer
antibody
linker
tumor
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PCT/CN2023/072958
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French (fr)
Chinese (zh)
Inventor
袁建栋
黄仰青
顾家宁
宋云松
Original Assignee
博瑞生物医药（苏州）股份有限公司
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Priority to CN202380018747.7A priority Critical patent/CN118591544A/en
Publication of WO2023143320A1 publication Critical patent/WO2023143320A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/357—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having two or more oxygen atoms in the same ring, e.g. crown ethers, guanadrel
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
    • A61K31/4015—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil having oxo groups directly attached to the heterocyclic ring, e.g. piracetam, ethosuximide
    • 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
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C237/00—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by amino groups
    • C07C237/02—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by amino groups having the carbon atoms of the carboxamide groups bound to acyclic carbon atoms of the carbon skeleton
    • C07C237/22—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by amino groups having the carbon atoms of the carboxamide groups bound to acyclic carbon atoms of the carbon skeleton having nitrogen atoms of amino groups bound to the carbon skeleton of the acid part, further acylated
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D207/00—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom
    • C07D207/02—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom
    • C07D207/30—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having two double bonds between ring members or between ring members and non-ring members
    • C07D207/34—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having two 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
    • C07D207/36—Oxygen or sulfur atoms
    • C07D207/40—2,5-Pyrrolidine-diones
    • C07D207/416—2,5-Pyrrolidine-diones 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 other ring carbon 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 disclosure belongs to the field of medical technology, specifically, the disclosure relates to a compound shown in formula (I), a linker shown in formula (II), and a compound shown in formula (I), a compound shown in formula (II) ) The use of the linker shown in the preparation of antibody drug conjugates.
  • Antibody-Drug Conjugates are new targeted drugs formed by covalently linking antibodies and cytotoxic drugs through specific linkers. Among them, after the antibody is used as a targeting carrier to transport cytotoxic drugs, that is, "effector molecules" or “warheads” to the target site of action, it specifically binds to the surface antigen of the target cell, enters the interior of the cell through endocytosis, and at the same time brings the small molecule drug into the cell. Internally, the cytotoxic drug is then released to kill the target cell via enzymatic cleavage or self-cleavage of the linker. Compared with chemotherapy and combination therapy, ADC drugs have high drug activity and high selectivity, which can significantly reduce off-target toxicity.
  • ADC drugs such as Brentuximab vedotin (Adcetris, SGN-35) and Trastuzumab emtansine (Kadcyla, T-DM1) have shown good safety and efficacy in clinical citations, and ADC drugs have become the mainstay in the field of disease-targeted therapy. Research hotspots and important development directions.
  • Eribulin is a tubulin polymerization inhibitor developed by Eisai. As a synthetic halichondrin B analogue, Eribulin has unique binding properties.
  • the Eriblin structure is as follows:
  • Eribulin is thought to act by inhibiting the growth phase of microtubule dynamics that prevent cell division.
  • non-clinical studies have shown Eribulin's unique effects in the tumor microenvironment, such as increasing vascular perfusion and permeability in the tumor core area, improving the state of epithelial cells, and reducing the migration ability of breast cancer cells, etc.
  • Eribulin has been approved in more than 70 countries and regions including Europe, America and Asia, and was approved for marketing in China in 2019. Eribulin has high tumor inhibitory and killing activities. Therefore, coupling Eribulin with antibodies is of great significance for realizing specific killing of tumor cells.
  • ADC drugs are composed of three parts: antibody, linker and toxin.
  • Antibodies determine the cell type and target of drug action;
  • cytotoxins can be any compound that can cause cell death, induce cell apoptosis, or reduce cell viability; and linkers are the bridge that organically combines the two, which is the key to ADC drug design.
  • the core part of the drug is the key to achieving targeted drug release.
  • Chemical coupling is the main coupling method currently used, and the coupling is mainly achieved by covalently linking lysine or cysteine residues in antibodies to cytotoxic drugs, for example, through sulfhydryl and maleimide
  • the formed sulfosuccinimide structure realizes the connection of the small molecule drug and the antibody.
  • the structure of sulfosuccinimide is not stable, and it will exchange with other reactive sulfhydryl groups in vivo, resulting in the early release of cytotoxic drugs. On the one hand, it causes toxicity to normal tissues, and on the other hand, it also reduces the drug efficacy of ADC drugs after they reach the target cells.
  • Linkers play a key role in the stability and activity of ADC drugs, and finding more linkers suitable for coupling antibodies and cytotoxic drugs is an important problem that needs to be solved urgently.
  • the present disclosure provides a linker with a novel structure, which is connected with eribulin to form a compound as shown in formula (I).
  • the compound represented by formula (I) can be linked to an antibody to form an antibody-drug conjugate, which can then specifically target target cells such as tumor cells and exert high killing activity on target cells, and has important application prospects in the treatment of diseases such as tumors.
  • the present disclosure provides a compound represented by formula (I) or a pharmaceutically acceptable salt, ester, solvate, optical isomer, tautomer, isotope label or prodrug thereof:
  • L 1 is ( Gly ) p -(Z 1 ) a -, p is any integer of 1-10; a is any integer of 0-20, if present, each Z 1 is independently any Types of amino acid residues;
  • L 3 is wherein m is any integer from 1 to 5, preferably 2; R 2 is -(CH 2 -CH 2 -Y) d -, d is any integer from 1 to 10, and each Y is independently -CH 2 -, -NH-, -O- or -S-;
  • the carboxy-terminal of L1 is connected to the amino-terminus of L2
  • the carboxy-terminus of L2 is connected to the amino-terminus of L3 .
  • a compound according to the present disclosure or a pharmaceutically acceptable salt, ester, solvate, optical isomer, tautomer, isotope label or prodrug thereof, wherein,
  • L 3 is wherein m is any integer from 1 to 5, preferably 2; R 2 is -(CH 2 -CH 2 -Y) d -, d is any integer from 1 to 10, and each Y is independently -CH 2- , -NH-, -O-, or -S-.
  • a compound according to the present disclosure or a pharmaceutically acceptable salt, ester, solvate, optical isomer, tautomer, isotope label or prodrug thereof, wherein,
  • L 1 is ( Gly ) p -, p is any integer of 1-10, preferably any integer of 3-5;
  • L 2 , L 3 are as defined in the first aspect.
  • L 1 , L 2 are as defined in the first aspect.
  • a compound according to the present disclosure or a pharmaceutically acceptable salt, ester, solvate, optical isomer, tautomer, isotope label or prodrug thereof, wherein,
  • L3 is selected from any one of the following:
  • a compound according to the present disclosure or a pharmaceutically acceptable salt, ester, solvate, optical isomer, tautomer, isotope label or prodrug thereof, wherein,
  • R 1 is -(CH 2 -CH 2 -X) b -, b is any integer of 1-10, preferably any integer of 1-5; each X is independently -CH 2 - or - O-; c is any integer of 1-5, preferably any integer of 1-2;
  • L 1 , L 3 are as defined in the first aspect.
  • a compound according to the present disclosure or a pharmaceutically acceptable salt, ester, solvate, optical isomer, tautomer, isotope label or prodrug thereof, wherein,
  • w is any integer of 0-9, preferably any integer of 2-4;
  • c is any integer of 1-5, preferably any integer of 1-2;
  • b is any integer of 1-5, preferably any integer of 2-4, each X is independently -CH 2 - or -O-; and, The structure is:
  • a compound according to the present disclosure or a pharmaceutically acceptable salt, ester, solvate, optical isomer, tautomer, isotope label or prodrug thereof, wherein,
  • w is any integer of 0-9, preferably any integer of 2-4;
  • c is any integer of 1-5, preferably any integer of 1-2;
  • b is any integer of 1-5, preferably any integer of 2-4, each X is independently -CH 2 - or -O-; and, The structure is:
  • the present disclosure provides a compound represented by any one of the following formulas (I-1) to (I-6) or a pharmaceutically acceptable salt, ester, solvate, optical isomer, mutual Variant, isotopically labeled or prodrug:
  • the present disclosure provides a linker for coupling an antibody to a pharmaceutical compound, the linker has a structure as shown in the following formula (II):
  • R 3 is selected from -NH 2 or -OH;
  • L 1 , L 2 , L 3 are as defined in the first aspect.
  • the linker according to the present disclosure wherein the linker has the structure shown in the following formula (II-1) or (II-2):
  • R 3 is selected from -NH 2 or -OH;
  • b is any integer of 1-5, preferably any integer of 2-4, and each X is independently -CH 2 -, -NH-, -O- or -S-.
  • the linker according to the present disclosure wherein the linker has a structure as shown in any one of the following:
  • the present disclosure provides a compound or a pharmaceutically acceptable salt, ester, solvate, optical isomer, tautomer, isotope label or prodrug thereof in the preparation of an antibody according to the first aspect Use in drug conjugates;
  • the antibody drug conjugate according to the compound shown in the first aspect or its pharmaceutically acceptable salt, ester, solvate, optical isomer, tautomer, isotope label or prodrug Conjugate antibody or antigen-binding fragment through L1 ;
  • the antibody drug conjugate is used to prevent and/or treat tumors; optionally, the tumors are tumors related to the abnormal expression of one or more than two proteins in the HER protein family; preferably , the abnormally expressed protein is selected from at least one of HER2 and HER3;
  • the tumor is selected from breast cancer, ovarian cancer, cervical cancer, uterine cancer, prostate cancer, renal cancer, urethral cancer, bladder cancer, liver cancer, gastric cancer, endometrial cancer, salivary gland cancer, esophageal cancer, melanoma , glioma, neuroblastoma, sarcoma, lung cancer, colorectal cancer, leukemia, bone cancer, skin cancer, thyroid cancer, pancreatic cancer, or lymphoma.
  • the present disclosure provides the use of the linker shown in the second aspect in the preparation of an antibody-drug conjugate
  • the L1 group of the linker is connected to the antibody or the antigen-binding fragment, and the L3 group of the linker is connected to the drug compound;
  • the antibody drug conjugate is used to prevent and/or treat tumors; optionally, the tumors are tumors related to the abnormal expression of one or more than two proteins in the HER protein family; preferably , the abnormally expressed protein is selected from at least one of HER2 and HER3;
  • the tumor is selected from breast cancer, ovarian cancer, cervical cancer, uterine cancer, prostate cancer, renal cancer, urethral cancer, bladder cancer, liver cancer, gastric cancer, endometrial cancer, salivary gland cancer, esophageal cancer, melanoma , glioma, neuroblastoma, sarcoma, lung cancer, colorectal cancer, leukemia, bone cancer, skin cancer, thyroid cancer, pancreatic cancer, or lymphoma.
  • the present disclosure provides an antibody-drug conjugate, which has the structure shown in the following formula (III):
  • q is any integer of 2-8, preferably 4;
  • Ab is an antibody or an antigen-binding fragment;
  • Pep is -(X 1 ) e -Leu-Pro-X 2 -Thr-, wherein e is any integer from 0-10, if present, each X 1 is independently Gly or Ala; X 2 is any species amino acid residues;
  • L 1 is -(Gly) p -(Z 1 ) a -, p is any integer of 1-10; a is any integer of 0-20, if present, each Z 1 is independently any kind of amino acid residues;
  • L3 is wherein m is any integer from 1 to 5, preferably 2; R 2 is -(CH 2 -CH 2 -Y) d -, d is any integer from 1 to 10, and each Y is independently -CH 2 -, -NH-, -O- or -S-;
  • the amino terminus of Pep is connected to the carboxyl terminus of the heavy chain or light chain of Ab
  • the carboxyl terminus of Pep is connected to the amino terminus of L1
  • the carboxyl terminus of L1 is connected to the amino terminus of L2
  • the carboxyl terminus of L2 is connected to the carboxy terminus of L3 . amino terminus.
  • Pep is -(Gly-Ala) f -Leu-Pro-X 2 -Thr-, wherein f is any integer of 1-5, preferably 1; X 2 is selected from any kind of amino acid residues, preferably Glu residues ;
  • Pep is -Gly-Ala-Leu-Pro-Glu-Thr-, wherein the amino terminal of Pep is connected to Ab, and the carboxyl terminal of Pep is connected to the amino terminal of L1 .
  • the antibody-drug conjugate according to the present disclosure wherein the carboxyl terminus of the heavy chain of Ab is linked to Pep, and the carboxyl terminus of the light chain of Ab is linked to Pep;
  • the carboxy terminus of the heavy chain of the Ab is linked to -Gly-Ala-Leu-Pro-Glu-Thr-, and the carboxy terminus of the light chain of the Ab is linked to -Gly-Ala-Leu-Pro-Glu-Thr-.
  • the antibody or antigen-binding fragment is specific It specifically binds to at least one protein in the HER protein family; preferably, the antibody or antigen-binding fragment specifically binds to at least one of the following proteins: HER2, HER3.
  • the antibody or antigen-binding fragment comprises a heavy chain variable region, wherein, the heavy chain variable region comprises such as SEQ ID NO: 9, SEQ ID NO: The amino acid sequence shown in at least one of ID NO: 11 and SEQ ID NO: 13; and/or,
  • the antibody or antigen-binding fragment comprises a light chain variable region, wherein the light chain variable region comprises amino acids as shown in at least one of SEQ ID NO: 2, SEQ ID NO: 4 and SEQ ID NO: 6 sequence;
  • the light chain variable region and the heavy chain variable region are coded according to the analysis method of Kabat.
  • the antibody drug conjugate according to the present disclosure wherein the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3;
  • HCDR1 contains the amino acid sequence shown in SEQ ID NO: 9
  • HCDR2 contains the amino acid sequence shown in SEQ ID NO: 11
  • HCDR3 contains the amino acid sequence shown in SEQ ID NO: 13;
  • LCDR1 includes the amino acid sequence shown in SEQ ID NO: 2
  • LCDR2 includes the amino acid sequence shown in SEQ ID NO: 4
  • LCDR3 includes the amino acid sequence shown in SEQ ID NO: 6.
  • amino acid sequence of the heavy chain of the antigen or antigen-binding fragment is shown in SEQ ID NO: 18, and/or,
  • amino acid sequence of the light chain of the antigen or antigen-binding fragment is shown in SEQ ID NO: 17.
  • L 1 , L 2 , and L 3 are as defined in the first aspect.
  • the present disclosure provides an antibody drug conjugate represented by any one of the following formulas (III-1) to (III-6):
  • Ab may be an antibody or antigen-binding fragment that binds to any type of tumor antigen.
  • tumor antigens include but are not limited to PD-1, PD-L1, PDL2, CTLA4, LAG3, TIM3, TIGIT, CD103, CD19, CD20, CD22, CD30, CD33, CD38, CD123, CD138, CD171, AFP, CEA, PSCA, GD2, NKG2D, BCMA, EGFR, HER2, HER3, EGFRvIII, CD171, FAP, IL13Ra2, VEGFR1, VEGFR2, GPC-3, Mesothelin, claudin 18.2, EpCAM, MUC1, MUC16, EPHA2, EPHA3, CD133, PSMA .
  • the antibody or antigen-binding fragment can specifically deliver Eribulin to tumor cells by specifically binding to tumor surface antigens, thereby exerting an efficient and specific tumor cell killing effect.
  • the antibody or antigen-binding fragment specifically binds to at least one protein in the HER protein family; preferably, the antibody or antigen-binding fragment specifically binds to at least one of the following proteins: HER2, HER3.
  • the antibody or antigen-binding fragment comprises a heavy chain variable region, wherein, the heavy chain variable region comprises such as SEQ ID NO: 9, SEQ ID NO: The amino acid sequence shown in at least one of ID NO: 11 and SEQ ID NO: 13; and/or,
  • the antibody or antigen-binding fragment comprises a light chain variable region, wherein the light chain variable region comprises amino acids as shown in at least one of SEQ ID NO: 2, SEQ ID NO: 4 and SEQ ID NO: 6 sequence;
  • the light chain variable region and the heavy chain variable region are coded according to the analysis method of Kabat.
  • the antibody drug conjugate according to the present disclosure wherein the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3;
  • HCDR1 contains the amino acid sequence shown in SEQ ID NO: 9
  • HCDR2 contains the amino acid sequence shown in SEQ ID NO: 11
  • HCDR3 contains the amino acid sequence shown in SEQ ID NO: 13;
  • LCDR1 includes the amino acid sequence shown in SEQ ID NO: 2
  • LCDR2 includes the amino acid sequence shown in SEQ ID NO: 4
  • LCDR3 includes the amino acid sequence shown in SEQ ID NO: 6.
  • the amino acid sequence of the heavy chain of the antigen or antigen-binding fragment is shown in SEQ ID NO: 18, and/or, the antigen or The amino acid sequence of the light chain of the antigen-binding fragment is as SEQ ID NO: 17.
  • the compound shown in the formula (I) provided by the present disclosure is formed by coupling a linker with the cytotoxic drug eribulin; the linker in the compound shown in the formula (I) can be further linked to an antibody to obtain an antibody drug conjugate United things.
  • the antibody-drug conjugate prepared by using the compound represented by formula (I) can specifically target tumor cells; after entering the tumor cell, the linker part of the antibody-drug conjugate can be broken to release the cytotoxic drug Ari Brin has high tumor cell killing activity and has important application prospects in the field of tumor treatment.
  • the compound represented by the formula (I) can be linked with different antibodies against different targets of tumors to achieve efficient and specific killing of different types of tumors.
  • the present disclosure provides a compound shown in any one of formula (I-1) to formula (I-6), and the compound shown in formula (I-1) to formula (I-6) can Through its glycine linker, it is enzymatically coupled with the antibody to obtain a structurally stable antibody-drug conjugate.
  • the antibody-drug conjugate has both specific targeting and high killing activity against tumor cells, and exerts excellent tumor treatment effect.
  • the present disclosure uses compounds represented by formula (I-1) and formula (I-5) to link HER2 antibodies to obtain compounds with formula (III-1), formula (III-5) Antibody drug conjugates showing the structure.
  • the present disclosure finds that the antibody drug conjugates represented by formula (III-1) and formula (III-5) can significantly reduce various types of tumor cells that are HER2 positive (for example, HCC1954, SK-BR-3 and NCI-N87) The in vitro survival rate and high tumor cell killing activity.
  • the antibodies represented by formula (III-1) and formula (III-5) in the present disclosure Compared with the small molecule drug Dxd, Eribulin shows stronger anti-tumor activity; compared with the small molecule drug Eribulin, the antibodies represented by formula (III-1) and formula (III-5) in the present disclosure
  • the drug conjugate has lower IC 50 value and higher tumor cell killing activity.
  • the cytotoxicity of ADC drugs is lower than that of the small molecule drug eribulin; meanwhile, the antibody-drug conjugates shown in formula (III-1) and formula (III-5) are more effective in HER2-positive cells The cytotoxicity was higher than that in HER2-negative cells.
  • the above results indicate that the antibody-drug conjugates in the present disclosure can target and bind tumor cells, and release the cytotoxic drug Eribulin inside the tumor cells to play a targeted and efficient role in killing tumor cells.
  • the linker represented by the formula (II) provided by the present disclosure can respectively connect the antibody and the drug compound to obtain an antibody-drug conjugate.
  • the linker directly affects the stability and drug activity of the antibody-drug conjugate.
  • the L3 group in the linker can break after entering the cell and release cytotoxic drugs; the antibody drug constructed using the linker of the present disclosure
  • the conjugate has high-efficiency and specific tumor cell killing activity, which provides a new choice and conjugation method for the development of antibody-drug conjugates.
  • Figure 1 shows the detection results of the impact of Ab0100-H0037, Ab0100-H0038, BQ3, Eribulin, and Dxd on the survival rate of HCC1954 cells with different drug concentration gradients;
  • Figure 2 shows the detection results of the impact of Ab0100-H0037, Ab0100-H0038, BQ3, Eribulin, and Dxd on the survival rate of SK-BR-3 cells with different drug concentration gradients;
  • Figure 3 shows the detection results of the impact of Ab0100-H0037, Ab0100-H0038, BQ3, Eribulin, and Dxd on the survival rate of NCI-N87 cells with different drug concentration gradients;
  • Figure 4 shows the detection results of the impact of Ab0100-H0037, Ab0100-H0038, BQ3, Eribulin, and Dxd on the survival rate of MDA-MB-468 cells with different drug concentration gradients;
  • Figure 5 shows vehicle (DPBS), Ab0100-H0037 (5mg/kg), Ab0100-H0038 (5mg/kg), and BQ3 (5mg/kg) after administration of tumor-bearing mice (Capan-1 tumor-bearing female BALB /c nude mice) body weight change detection results;
  • Figure 6 shows vehicle (DPBS), Ab0100-H0037 (5mg/kg), Ab0100-H0038 (5mg/kg), and BQ3 (5mg/kg) after administration of tumor-bearing mice (Capan-1 tumor-bearing female BALB /c nude mice) tumor growth curve;
  • Figure 7 shows vehicle (DPBS), Ab0100-H0037 (5mg/kg), Ab0100-H0038 (5mg/kg), and BQ3 (5mg/kg) Tumor growth curve of tumor-bearing mice (NCI-N87 tumor-bearing female BALB/c nude mice) after administration;
  • Figure 8 shows vehicle (DPBS), Ab0100-H0037 (3mg/kg), Ab0100-H0038 (3mg/kg), and BQ3 (5mg/kg) after administration of tumor-bearing mice (HCC1954 tumor-bearing female BALB/c Tumor growth curve of nude mice).
  • any integer from value A to value B covers any type of natural number between value A and value B, and its range covers values A and B including the endpoints.
  • any integer of 1-10 is any integer selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10.
  • amino acid can be common amino acids (amino acids shown in Table 1 in this disclosure), or uncommon amino acids (for example, 2-aminoadipic acid, 2-aminobutyric acid, 2-aminobutyric acid, 2 -aminopimelic acid, 2-carboxamide, etc.).
  • amino acid may be a modified amino acid (for example, acylation, ubiquitination, sugar chain modification, biotinylation, etc.), or an unmodified amino acid.
  • the term "independently" means that at least two groups (or ring systems) present in the structure with the same or similar value range may have the same or different meanings under specific circumstances.
  • the substituent X and the substituent Y are each independently hydrogen, hydroxyl, alkyl or aryl, then when the substituent X is hydrogen, the substituent Y can be either hydrogen or hydroxyl, alkyl or aryl ; Similarly, when the substituent Y is hydrogen, the substituent X can be either hydrogen, or hydroxyl, alkyl or aryl.
  • the number of substituents X is two, and each X is independently hydrogen, hydroxyl, alkyl or aryl; then when one X is hydrogen, the other X can be either hydrogen or hydroxyl, Alkyl or aryl.
  • the number of substituents X is three or more, and each X is independently hydrogen, hydroxyl, alkyl or aryl, which means that any one of X can be selected from hydrogen, hydroxyl, alkyl or aryl .
  • e is any integer of 0-10, and if present, each X 1 is independently Gly or Ala.
  • -(X 1 ) e - may be -Gly-Gly-, -Ala-Ala-, -Ala-Gly- or -Gly-Ala- along the direction from the amino terminal to the carboxyl terminal.
  • linker refers to a chemical structural fragment or bond that is connected to a ligand/antibody at one end and a drug at the other end, and may also be connected to other The connector is then connected to the drug.
  • a linker of the present disclosure is a glycine-terminated amino acid fragment. Amino acid fragments with a glycine terminal can be protected by protecting groups such as Fmoc. As a preferred embodiment, the amino acid fragment at the end can be 3-5 glycines.
  • antibody is used herein in the broadest sense to refer to a protein comprising an antigen binding site, encompassing natural and artificial antibodies of various structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (for example, bispecific antibodies), single chain antibodies, whole antibodies and antibody fragments.
  • antigen-binding fragment is a portion or segment of an intact or complete antibody having fewer amino acid residues than an intact or complete antibody, which is capable of binding antigen or competing with the intact antibody (i.e., the intact antibody from which the antigen-binding fragment is derived) Binding antigen.
  • Antigen-binding fragments can be prepared by recombinant DNA techniques, or by enzymatic or chemical cleavage of intact antibodies.
  • Antigen binding fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab') 2 ; diabodies; linear antibodies; single chain antibodies (e.g. scFv); single domain antibodies; bivalent or bispecific Antibodies or fragments thereof; camelid antibodies (heavy chain antibodies); and bispecific or multispecific antibodies formed from antibody fragments.
  • CDR region is an antibody variable domain that is hypervariable in sequence and forms a structurally defined loop ("hypervariable loop") and/or contains antigen-contacting residues ("antigen contact point").
  • the CDRs are primarily responsible for binding to antigenic epitopes.
  • the CDRs of the heavy and light chains are commonly referred to as CDR1, CDR2 and CDR3, numbered sequentially starting from the N-terminus. located in anti The CDRs within the variable domain of the heavy chain are referred to as HCDR1, HCDR2, and HCDR3, while the CDRs located within the variable domain of the antibody light chain are referred to as LCDR1, LCDR2, and LCDR3.
  • each CDR can be determined using any one or combination of a number of well-known antibody CDR assignment systems, including For example: Chothia based on the three-dimensional structure of antibodies and the topology of the CDR loops (Chothia et al.
  • 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.
  • Such framework sequences can be obtained from public DNA databases or published references that include germline antibody gene sequences.
  • 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.
  • 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) attached to each antibody (Ab); more preferably 2-4.
  • solvation refers to the phenomenon that some solvent molecules are surrounded by solute molecules or ions to form a group.
  • the compounds of the invention may also be provided in the form of salts. These salts can be formed using commonly performed methods. Or, depending on the conditions of the production method of the present invention, the compound (for example, a substance derived from an additive) can be produced in the form of a salt.
  • the compounds of the invention may also be provided in the form of solvates.
  • a solvate with water is mentioned.
  • Such solvates can be formed using commonly performed methods.
  • the compound can be produced in the form of a solvate.
  • salts of the above-mentioned compounds may be produced and provided in the form of solvates.
  • treatment refers to: after suffering from a disease, contacting (for example, administering) a compound of the present disclosure or a pharmaceutically acceptable salt, ester, solvate, optical isomer thereof of a subject , tautomers, isotope labels, prodrugs, or pharmaceutical compositions containing them (hereinafter also referred to as “pharmaceutical compositions of the present disclosure”), thereby reducing the symptoms of the disease compared to when not in contact , does not mean that the symptoms of the disease must be completely suppressed. Suffering from a disease means that the body has symptoms of a disease.
  • prevention used in the context of the present disclosure refers to: prior to suffering from a disease, by making a subject contact (for example, administer) a compound of the present disclosure or a pharmaceutically acceptable salt, ester, solvate, optical isomer isomer, tautomer, isotopic label, prodrug or pharmaceutical composition of the present disclosure, thereby reducing the symptoms after suffering from the disease compared with no contact, it does not mean that the disease must be completely suppressed.
  • mammals including but not Limited to, domesticated animals (e.g., cattle, sheep, cats, dogs, and horses), primates (e.g., humans and nonhuman primates such as monkeys), rabbits, and rodents (e.g., mice and mouse).
  • domesticated animals e.g., cattle, sheep, cats, dogs, and horses
  • primates e.g., humans and nonhuman primates such as monkeys
  • rabbits e.g., mice and mouse.
  • tumor and cancer are used interchangeably herein to encompass both solid and liquid tumors.
  • tumor refers to all neoplastic cell growth and proliferation, whether malignant or benign, and to all pre-cancerous and cancerous cells and tissues.
  • cancer cancerous cells and tissues.
  • cancer cancerous cells and tissues.
  • cancer cancerous cells and tissues.
  • cancer and “cancerous” refer to or describe a physiological disorder in mammals that is often characterized by unregulated cell growth.
  • examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia or lymphoid malignancies.
  • cancers include, but are not limited to, squamous cell carcinoma (e.g., epithelial squamous cell carcinoma), lung cancer (including small cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, and squamous cell carcinoma of the lung), peritoneal carcinoma , hepatocellular carcinoma, gastric cancer (including gastrointestinal and gastrointestinal stromal carcinoma), pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, urethral cancer, hepatoma, breast cancer, colorectal cancer , endometrial or uterine cancer, salivary gland cancer, kidney cancer, prostate cancer, vulvar cancer, thyroid cancer, liver cancer, anal cancer, penile cancer, melanoma, superficial spreading melanoma, lentigo maligna melanoma acral melanoma, nodular melanoma, multiple myeloma and B-cell lymph
  • anti-tumor effect refers to a biological effect that can be exhibited by various means, including but not limited to, for example, reduction in tumor volume, reduction in tumor cell number, reduction in tumor cell proliferation, or reduction in tumor cell survival.
  • an effective amount refers to an amount or dose of a compound or composition of the present disclosure which, when administered to a patient in single or multiple doses, produces the desired effect in a patient in need of treatment or prevention.
  • An effective amount can be readily determined by the attending physician, who is skilled in the art, by considering a variety of factors: the species of the animal, such as a mammal; its size, age and general health; the particular disease involved; the extent or severity of the disease the individual patient's response; the particular antibody administered; the mode of administration; the bioavailability characteristics of the formulation administered; the chosen dosing regimen; and the use of any concomitant therapy.
  • pharmaceutically acceptable salt refers to salts prepared from compounds of the present disclosure with relatively non-toxic acids or bases.
  • base addition salts can be obtained by contacting their free forms with a sufficient amount of base in neat solution or in a suitable inert solvent .
  • pharmaceutically acceptable base addition salts include, but are not limited to, sodium, potassium, ammonium, calcium, magnesium, organic amine, or similar salts.
  • acid addition salts can be obtained by contacting their free forms with a sufficient amount of acid in neat solution or in a suitable inert solvent .
  • pharmaceutically acceptable acid addition salts include, but are not limited to, inorganic acid salts (e.g., hydrochloride, hydrobromide, hydroiodide, nitrate, carbonate, bicarbonate, phosphate , monohydrogen phosphate, dihydrogen phosphate, phosphite, sulfate, bisulfate, etc.), organic acid salts (such as acetate, propionate, isobutyrate, malonate, succinate , suberate, maleate, fumarate, citrate, tartrate, lactate, mandelate, benzoate, phthalate, methanesulfonate, benzenesulfonate salt, p-toluenesulfonate, glucur
  • composition refers to a pharmaceutically acceptable composition comprising one or more compounds or a pharmaceutically acceptable form thereof (e.g. salt, hydrate, solvate, stereoisomeric isomers, tautomers, metabolites, prodrugs, etc.), and other components (such as pharmaceutically acceptable excipients).
  • a pharmaceutically acceptable composition comprising one or more compounds or a pharmaceutically acceptable form thereof (e.g. salt, hydrate, solvate, stereoisomeric isomers, tautomers, metabolites, prodrugs, etc.), and other components (such as pharmaceutically acceptable excipients).
  • pharmaceutically acceptable excipient used in the context of the present disclosure refers to an auxiliary material widely used in the field of pharmaceutical production.
  • the main purpose of using excipients is to provide a pharmaceutical composition that is safe to use, stable in nature and/or has specific functionality, and also to provide a method so that after the drug is administered to the subject, the active ingredient can be used in the desired manner. The rate of dissolution, or the promotion of effective absorption of the active ingredient in the subject to which it is administered.
  • Pharmaceutically acceptable excipients can be inert fillers, or functional ingredients that provide certain functions for the pharmaceutical composition (such as stabilizing the overall pH value of the composition or preventing the degradation of active ingredients in the composition).
  • Non-limiting examples of pharmaceutically acceptable excipients include, but are not limited to, binders, suspending agents, emulsifying agents, diluents (or fillers), granulating agents, adhesives, disintegrants, lubricants, anti-adherents , glidants, wetting agents, gelling agents, absorption delaying agents, dissolution inhibitors, enhancers, adsorbents, buffers, chelating agents, preservatives, coloring agents, flavoring agents, sweeteners, etc.
  • compositions of the present disclosure can be prepared using any method known to those skilled in the art. For example, conventional mixing, dissolving, granulating, emulsifying, milling, encapsulating, entrapping and/or lyophilizing processes.
  • the purpose of using the pharmaceutical composition is to promote the administration to organisms, facilitate the absorption of active ingredients, and then exert biological activity.
  • the pharmaceutical compositions of the present disclosure may be administered by any form, including injection (intra-arterial, intravenous, intramuscular, intraperitoneal, subcutaneous), mucosal, oral (oral solid formulation, oral liquid formulation), rectal, inhalation, implantation , topical (eg ocular) administration, and the like.
  • oral solid formulations include, but are not limited to, powders, capsules, lozenges, granules, tablets, and the like.
  • Non-limiting examples of liquid formulations for oral or mucosal administration include, but are not limited to, suspensions, tinctures, elixirs, solutions, and the like.
  • Non-limiting examples of topical formulations include, but are not limited to, emulsions, gels, ointments, creams, patches, pastes, foams, lotions, drops, or serum formulations.
  • Non-limiting examples of formulations for parenteral administration include, but are not limited to, solutions for injection, dry powder for injection, suspension for injection, emulsion for injection, and the like.
  • the pharmaceutical compositions of the present disclosure can also be formulated as controlled or delayed release dosage forms (eg, liposomes or microspheres).
  • the methods of administration can be varied or adjusted in any suitable manner to meet the needs of the nature of the drug, the convenience of the patient and medical staff, and other relevant factors.
  • Dxd used in the context of this disclosure, also known as Exatecan derivative, is a DNA topoisomerase I inhibitor, which has the structure shown below:
  • the antibody drug conjugates of the present disclosure can be prepared by any method known in the art.
  • the conjugate is prepared by enzyme-catalyzed specific binding of the antibody or antigen-binding fragment to the compound represented by formula (I), wherein the antibody or antigen-binding fragment contains a recognition sequence of the ligase.
  • the ligase is a transpeptidase, which includes but not limited to various natural Sortase enzymes (including Sortase of A, B, C, D, L. plantarum, etc.) and various novel transpeptidases that have been preferably modified.
  • reaction is realized by means of biological enzyme catalysis (refer to WO2015165413A1, which is incorporated herein by reference), the reaction conditions are mild, the physical and chemical damage to the antibody during the coupling process is reduced, the preparation process and process are more optimized, and it is easy to upgrade industrialization. Conducive to the quality control of ADC products.
  • the method includes step A and step B.
  • Step A prepare the compound shown in formula (I)
  • the compound represented by formula (I) is formed by covalently linking a linker with eribulin.
  • the compound represented by formula (I) has a definite structure, definite composition and high purity, so when it is coupled with an antibody, less or no other impurities are introduced. Under the catalysis of ligase, when the compound represented by formula (I) is used to react with the antibody or antigen-binding fragment with the recognition sequence of ligase at a specific site, a highly controllable homogeneous ADC.
  • Step B linking the target molecule with the compound represented by formula (I)
  • the target molecule in the present disclosure can be combined with the compound represented by formula (I) by any method known in the art to obtain the antibody drug conjugate represented by formula (III).
  • the target molecule and the compound represented by the formula (I) are connected to each other through the specific recognition sequence of the ligase of the substrate.
  • the recognition sequence depends on the particular ligase used.
  • the target molecule is an antibody or an antigen-binding fragment, and a recognition-sequence-based terminal modification is introduced at the c-terminus (carboxyl-terminus) of the light chain and/or heavy chain, in wild-type or optimized engineered ligase or Under the catalysis of any combination, the target molecule Combined with the compound represented by formula (I).
  • the ligase is a Sortase enzyme. More specifically, the ligase is Sortase A enzyme.
  • the binding reaction can be represented by the following scheme:
  • the triangle represents a part of the antibody, and the carboxy-terminal of the antibody is connected to the recognition sequence LPXT(G) r of the Sortase A enzyme; wherein, L is leucine, P is proline, X is any type of amino acid, T is threonine, (G) r is glycine in quantity r, and r is any integer of 1-2.
  • the pentagon represents a part of the compound of formula (II), (G) p is glycine with a quantity of p, and p is any integer from 1 to 10. Exemplarily, p is 1, 2, 3, 4, 5, etc. .
  • Sortase A enzyme nucleophilic attack The peptide bond between threonine (T) and glycine (G) in the r- sequence of LPXT(G) in LPXT(G) forms a covalent thio intermediate. This intermediate can capture Glycine linker (G) p in , and re-form an amide bond between threonine (T) and glycine (G), release the Sortase A enzyme, and obtain an antibody drug in which the target molecule is combined with the compound represented by formula (I) Conjugate
  • the laboratory reagents used in the present invention are all from commercial purchases, and the purity is all analytical pure.
  • the abbreviations and full Chinese names of the reagents used in the synthesis process are shown in the table below:
  • Compound BP182a has the structure shown in the following formula (I-5):
  • Step 2 synthesis of BP102c05 and BP102c: for the synthesis of BP102c05 and BP102c, refer to the synthesis method of EP0624377A2, which is incorporated into this application by reference;
  • Step 3 the synthesis of compound BP182a11: add 687mg (1.2eq) BP102c, 565mg (1.0eq) eribulin, 6ml DMF to the flask, stir and dissolve, add 162ul (1.2eq) DIEA, TLC monitors the reaction after the completion of the reaction The solution was poured into TBME, the solid precipitated out, stirred and beaten, filtered, the filter cake was washed with TBME, the filter cake was collected, and vacuum-dried to obtain 1021 mg of off-white solid, namely BP182a11, the sample was directly carried out to the next step reaction without purification.
  • Step 4 the synthesis of compound BP182a: add 402mg (1.0eq) BP182a11, 16ml methanol to the flask, stir to dissolve; weigh 236mg (1.2eq) 1186k, dissolve it with 1ml purified water, and adjust the pH to 5 with sodium bicarbonate solution -6, and then added to the reaction bottle of BP182a11. After 30 minutes, the reaction was monitored by HPLC. The reaction solution was not treated, but was prepared and purified by reverse high performance liquid phase, and the pure product was collected and freeze-dried to obtain 166 mg of white solid, namely BP182a.
  • Compound BP182d has the structure shown in the following formula (I-1):
  • Step 1 the synthesis of BP182d01: synthesized by Fmoc solid-phase synthesis method, using 2ClTrt Resin as a solid-phase carrier, using 20% piperidine/DMF (v/v) solution to remove Fmoc protection, and then using HOBT/DIC as a condensation system, DMF is the reaction solvent, sequentially coupled Fmoc-Gly-OH, Fmoc-Phe-OH, Fmoc-Gly-OH, Fmoc-Gly-OH, Fmoc-AEEA-OH, Fmoc-Gly-OH, Fmoc-Gly-OH, Fmoc-Gly-OH, Fmoc-Gly-OH was then cleaved with 2% TFA/DCM solution, precipitated with methyl tert-butyl ether, centrifuged and dried to obtain BP182d01, which was directly carried out to the next step without purification.
  • DMF is the reaction solvent
  • Step 2 synthesis of BP182d02: add 501mg (1.0eq) BP182d01, 415mg (1.0eq) eribulin, 10ml DCM, 4ml DMF to the flask, stir to dissolve and cool to 5 ⁇ 5°C, add 237ul (2.5eq) DIEA, add 140ul (1.5eq) DEPC, rise to room temperature to react, after TLC monitors the reaction, concentrate the reaction solution in vacuo to remove DCM, then add TBME, stir to make a slurry, filter, wash the filter cake with TBME, collect the filter cake, and vacuum dry to obtain 1.023 g of off-white solid was directly carried out to the next reaction without further purification.
  • Step 3 synthesis of BP182d: add 1000mg BP182d02, 7ml methanol, 7ml THF to the flask, stir to dissolve; weigh 300mg LiOH.H 2 O, dissolve it with 10ml purified water, add it dropwise to the reaction flask, adjust the pH to 12 After the reaction was monitored by TLC, acetic acid was added to quench the reaction, the reaction solution was concentrated in vacuo to remove the organic solvent, and the crude product of BP182d was obtained. The crude product was prepared and purified by forward high performance liquid phase, and the pure product was collected and concentrated to dryness to obtain 195 mg of off-white solid, namely BP182d .
  • Linker Gly-Gly-Gly-PEG4-PA-Cys was synthesized using 2Cl-Trt resin.
  • Coupling and ring-opening reaction The preparation method of the compound of formula I-7 and the compound of formula I-8 is used to prepare the mixture of the compound of I-9 and the compound of formula I-10.
  • Embodiment 5 Preparation of antibody drug conjugate
  • the carboxy-terminal of the heavy chain of antibody Ab0100 is connected with GALPETGG (hereinafter referred to as Ab0100-HCCT L ), and its amino acid sequence is shown in SEQ ID NO: 16; the carboxy-terminal of the light chain of antibody Ab0100 is connected with GALPETGG (hereinafter referred to as Ab0100-LCCT L ), the amino acid sequence of which is shown in SEQ ID NO:15. Therefore, a total of 4 ligase recognition sequences were introduced into antibody Ab0100.
  • the synthesis of antibody Ab0100 can refer to WO1989006692A1, and WO1989006692 is incorporated into the present disclosure by reference.
  • the method of coupling reaction can refer to the method in WO2015165413A1, as follows:
  • Ab0100-H0037 the ADC prepared by enzymatic linking with Sortase A is named Ab0100-H0037.
  • the specific structure of Ab0100-H0037 is shown in the following formula (III-5):
  • Ab0100-H0038 the ADC prepared by enzymatic linking with Sortase a is named Ab0100-H0038.
  • the specific structure of Ab0100-H0038 is shown in the following formula (III-1):
  • the compound LB302-2-4 was coupled to the anti-human ErbB2/Her2 antibody Ab0001, and the obtained ADC information is shown in the table below.
  • the amino acid sequence of the heavy chain of antibody Ab0001 is shown in SEQ ID NO: 19, and the carboxy-terminal of the light chain of antibody Ab0001 is connected with GALPETGG (hereinafter referred to as Ab0001-LCCT L ), and its amino acid sequence is shown in SEQ ID NO: 15 .
  • Example 8 In vitro experiments verify the cytotoxicity of ADC
  • Cell plating 5000 cells/well for NCI-N87 and SK-BR-3, 4000 cells/well for HCC1954 and MDA-MB-468.
  • Sample configuration buffer configuration configure the required amount of sample configuration buffer in the culture medium (10% FBS) used for the administered cells;
  • Sample configuration Dilute the sample according to the ratio of the concentration required for the first concentration, and configure according to the following table in a 24-well plate or a 96-well plate;
  • Detection reagent preparation Equilibrate the CellTiter-Glo Luminescent Cell Viability Assay detection reagent to room temperature in the dark in advance.
  • the gain value is 135. After setting the procedure, remove the cover of the black wall bottom plate and place it in the way specified by the instrument.
  • Figure 1 shows the cytotoxicity detection results of Ab0100-H0038, Ab0100-H0037, BQ3, and small molecule drugs Eribulin and Dxd on human breast cancer cell HCC1954. Among them, the IC50 value of each drug molecule is shown in the table below:
  • Figure 2 shows the cytotoxicity detection results of Ab0100-H0038, Ab0100-H0037, BQ3, small molecule drugs Eribulin and Dxd on human breast cancer cell SK-BR-3.
  • the IC50 value of each drug molecule is shown in the table below:
  • Figure 3 shows the cytotoxicity detection results of Ab0100-H0038, Ab0100-H0037, BQ3, and small molecule drugs Eribulin and Dxd on human gastric cancer cell NCI-N87.
  • the IC50 value of each drug molecule is shown in the table below:
  • Figure 4 shows the cytotoxicity detection results of Ab0100-H0038, Ab0100-H0037, BQ3, and small molecule drugs Eribulin and Dxd on human breast cancer cell line MDA-MB-468. Among them, the IC50 value of each drug molecule is shown in the table below:
  • Ab0100-H0037 and Ab0100-H0038 can significantly reduce the in vitro survival rate of various types of tumor cells that are HER2 positive (for example, HCC1954, SK-BR-3 and NCI-N87), exerting high-efficiency tumor cell
  • the killing activity indicates that the antibody-drug conjugate prepared in the present disclosure has an effective killing effect on HER2-positive tumor cells.
  • the cytotoxicity of Ab0100-H0037 and Ab0100-H0038 was lower than that of the small molecule drug eribulin.
  • the cytotoxicity of Ab0100-H0037 and Ab0100-H0038 to HER2-positive cells HCC1954, SK-BR-3, and NCI-N87 is better than that of eribulin, and significantly better than that of Dxd, indicating that the linker coupling through a special structure in this disclosure
  • the ADC drug obtained from the antibody and the cytotoxic drug eribulin has an improved killing effect on tumor cells.
  • Ab0100-H0037 and Ab0100-H0038 The cytotoxicity in HER2-positive cells is significantly better than that in HER2-negative cells, indicating that Ab0100-H0037 and Ab0100-H0038 can target cells with HER2-positive expression and enter the cells, releasing cytotoxicity in cells Eribulin, a drug, achieves specific and efficient killing of HER2-positive cells, and has good targeting and safety.
  • Example 9 In vivo experiments verify the activity and toxicity of ADC
  • Capan-1 tumor cells were preserved in vitro in IMDM medium supplemented with 20% fetal calf serum and 1% antibiotic-antimycotic at 37°C in an atmosphere of 5% CO2 (ATCC, catalog number HTB-79 ). Tumor cells were routinely subcultured twice a week by trypsin-EDTA treatment. Cells in exponential growth phase were harvested and counted for tumor inoculation.
  • the preparation method of the test sample is shown in the table below:
  • the primary endpoint was to determine whether tumor growth could be delayed or whether the mice could be cured.
  • Figure 5 shows vehicle (DPBS), Ab0100-H0037 (5mg/kg), Ab0100-H0038 (5mg/kg), and BQ3 (5mg/kg) after administration of tumor-bearing mice (Capan-1 tumor-bearing female BALB /c nude mice) body weight change detection results.
  • tumor-bearing mice Capan-1 tumor-bearing female BALB /c nude mice
  • the calculated change based on the body weight of the animals on the first day of administration is shown in percentage change (%) of body weight; the data points represent the percentage group mean change of BW. Error bars represent standard error of the mean (SEM).
  • Figure 5 shows that during the experiment, no significant weight loss, no death and no disease occurred in all groups of the model, and Ab0100-H0037 and Ab0100-H0038 did not show obvious drug toxicity in animal experiments.
  • the mean tumor volume over time in Capan-1 xenograft tumor-bearing female BALB/c nude mice administered Ab0100-H0037, Ab0100-H0038 and BQ3 is shown in FIG. 6 .
  • the entire study was terminated on day 42 under experimental conditions when the mean tumor volume in the vehicle group reached 1,420 mm 3 .
  • T/C % T RTV /C RTV ⁇ 100% (T RTV : RTV of the treatment group; C RTV : RTV of the control group on the same day).
  • RTV V 42 /V 0 , where V 0 is the average tumor volume on the first day of treatment, and V 42 is the average tumor volume on the 42nd day after the start of treatment;
  • TGI(%) [1-(T 42 -T 0 )/(V 42 -V 0 )] ⁇ 100%;
  • Figure 6 shows the tumor growth curves of tumor-bearing mice after administration of vehicle (DPBS), Ab0100-H0037 (5 mg/kg), Ab0100-H0038 (5 mg/kg), and BQ3 (5 mg/kg).
  • vehicle DPBS
  • Ab0100-H0037 5 mg/kg
  • Ab0100-H0038 5 mg/kg
  • BQ3 5 mg/kg
  • the subcutaneous NCI-N87 human gastric cancer xenograft model in female BALB/c nude mice was established as follows: in air containing 5% CO2 at 37°C in RPMI supplemented with 10% fetal bovine serum and 1% antibiotic-antimycotic NCI-N87 tumor cells (ATCC, Manassas, VA, catalog number CRL-5822) were maintained in vitro as monolayer cultures in -1640 medium. Tumor cells were routinely subcultured twice a week by trypsin-EDTA treatment. Cells in exponential growth phase were harvested and counted for tumor inoculation.
  • NCI-N87 tumor cells (10 x 106 ) in 0.2 mL PBS supplemented with Matrigel (1:1) were inoculated subcutaneously on the right side of each mouse for tumor development. Treatment was initiated on day 8 after tumor inoculation when the mean tumor volume reached approximately 188 mm 3 . Animals were allocated into groups using Excel-based randomization software that stratified randomization based on the animals' tumor volume. Each group consists of 6 composed of tumor-bearing mice. Referring to Experimental Example 2, the test article was administered to tumor-bearing mice.
  • Figure 7 shows vehicle (DPBS), Ab0100-H0037 (5mg/kg), Ab0100-H0038 (5mg/kg), and BQ3 (5mg/kg) after administration of tumor-bearing mice (NCI-N87 tumor-bearing female BALB /c tumor growth curve of nude mice).
  • the subcutaneous HCC1954 human breast cancer xenograft model in female BALB/c nude mice was established as follows: Culture in RPMI 1640 supplemented with 10% fetal bovine serum and 1% antibiotic-antimycotic at 37°C and 5% CO2 in the air HCC1954 tumor cells (ATCC, Manassas, VA, catalog number CRL-2338) were preserved in vitro. Tumor cells were routinely subcultured twice a week by trypsin-EDTA treatment. Cells in exponential growth phase were harvested and counted for tumor inoculation.
  • Figure 8 shows vehicle (DPBS), Ab0100-H0037 (3mg/kg), Ab0100-H0038 (3mg/kg), and BQ3 (5mg/kg) after administration of tumor-bearing mice (HCC1954 tumor-bearing female BALB/c Tumor growth curve of nude mice).

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Abstract

The present disclosure relates to the technical field of medicines. Specifically, the present disclosure relates to a compound as shown in formula (I), a linker as shown in formula (II), and a use of the compound as shown in formula (I) and the linker as shown in formula (II) in the preparation of an antibody-drug conjugate. The compound as shown in formula (I) can be linked to an antibody by means of a glycine linker thereof to obtain an antibody-drug conjugate. The antibody-drug conjugate can exert efficient and specific tumor cell killing activity both in vivo and in vitro, significantly inhibits the growth of different types of tumors and does not have obvious cytotoxicity, thus having important application prospects in the field of tumor treatment.

Description

用于制备抗体药物偶联物的连接子、化合物及用途Linkers, compounds and uses for preparing antibody drug conjugates
本公开要求如下专利申请的优先权：于2022年01月28日提交中国专利局、申请号为202210108098.7的中国专利申请，于2022年06月30日提交中国专利局、申请号为202210771649.8的中国专利申请；上述专利申请的全部内容通过引用结合在本公开中。This disclosure claims the priority of the following patent applications: the Chinese patent application with the application number 202210108098.7 submitted to the China Patent Office on January 28, 2022, and the Chinese patent application with the application number 202210771649.8 submitted to the China Patent Office on June 30, 2022 application; the entire content of the aforementioned patent application is incorporated by reference into this disclosure.
技术领域technical field
本公开属于医药技术领域，具体来说，本公开涉及如式(I)所示的化合物、如式(II)所示的连接子，以及如式(I)所示的化合物、如式(II)所示的连接子在制备抗体药物偶联物中的用途。The disclosure belongs to the field of medical technology, specifically, the disclosure relates to a compound shown in formula (I), a linker shown in formula (II), and a compound shown in formula (I), a compound shown in formula (II) ) The use of the linker shown in the preparation of antibody drug conjugates.
背景技术Background technique
抗体药物偶联物(Antibody-Drug Conjugates，ADC)是由抗体与细胞毒性药物通过特定的连接子共价连接而形成的新型靶向药物。其中，抗体作为靶向载体运送细胞毒性药物即“效应分子”或“弹头”至目标作用部位后，与目标细胞表面抗原特异性结合，经内吞进入细胞内部，同时将小分子药物带入细胞内部，然后经酶解或连接子的自身断裂释放细胞毒性药物杀伤目标细胞。与化疗和联合治疗相比，ADC药物的药物活性高，并且具有高的选择性，能显著减少脱靶毒性。近年来，Brentuximab vedotin(Adcetris，SGN-35)和Trastuzumab emtansine(Kadcyla，T-DM1)等ADC药物在临床引用中体现了很好的安全性和有效性，ADC药物已成为疾病靶向治疗领域的研究热点和重要发展方向。Antibody-Drug Conjugates (ADCs) are new targeted drugs formed by covalently linking antibodies and cytotoxic drugs through specific linkers. Among them, after the antibody is used as a targeting carrier to transport cytotoxic drugs, that is, "effector molecules" or "warheads" to the target site of action, it specifically binds to the surface antigen of the target cell, enters the interior of the cell through endocytosis, and at the same time brings the small molecule drug into the cell. Internally, the cytotoxic drug is then released to kill the target cell via enzymatic cleavage or self-cleavage of the linker. Compared with chemotherapy and combination therapy, ADC drugs have high drug activity and high selectivity, which can significantly reduce off-target toxicity. In recent years, ADC drugs such as Brentuximab vedotin (Adcetris, SGN-35) and Trastuzumab emtansine (Kadcyla, T-DM1) have shown good safety and efficacy in clinical citations, and ADC drugs have become the mainstay in the field of disease-targeted therapy. Research hotspots and important development directions.
艾日布林(Eribulin)是由卫材(Eisai)研发的微管蛋白聚合抑制剂，艾日布林作为合成的大田软海绵素(halichondrin B)类似物，具有独特的结合特性。艾日布林结构如下：
Eribulin is a tubulin polymerization inhibitor developed by Eisai. As a synthetic halichondrin B analogue, Eribulin has unique binding properties. The Eriblin structure is as follows:
艾日布林被认为通过抑制阻止细胞分裂的微管动力学的生长期发挥作用。此外，非临床研究显示了艾日布林在肿瘤微环境中的独特作用，如增加肿瘤核心区域的血管灌注和渗透性，改善上皮细胞状态，降低乳腺癌细胞的迁移能力等。目前，艾日布林已在包括欧洲、美洲和亚洲在内的70多个国家和地区获得批准，在中国于2019年获批上市。艾日布林具有高的肿瘤抑制、杀伤活性，因此，将艾日布林与抗体偶联，对于实现肿瘤细胞的特异性杀伤，具有重要意义。Eribulin is thought to act by inhibiting the growth phase of microtubule dynamics that prevent cell division. In addition, non-clinical studies have shown Eribulin's unique effects in the tumor microenvironment, such as increasing vascular perfusion and permeability in the tumor core area, improving the state of epithelial cells, and reducing the migration ability of breast cancer cells, etc. At present, Eribulin has been approved in more than 70 countries and regions including Europe, America and Asia, and was approved for marketing in China in 2019. Eribulin has high tumor inhibitory and killing activities. Therefore, coupling Eribulin with antibodies is of great significance for realizing specific killing of tumor cells.
ADC药物由抗体(antibody)、连接子(linker)和细胞毒性药物(toxin)三部分组成。抗体决定药物作用的细胞类型和靶点；细胞毒素可以是可致细胞死亡、引诱细胞凋亡或减少细胞生存力的任何化合物；而连接子则是将两者有机结合的桥梁，是ADC药物设计的最核心部分，是实现靶向释药的关键。ADC drugs are composed of three parts: antibody, linker and toxin. Antibodies determine the cell type and target of drug action; cytotoxins can be any compound that can cause cell death, induce cell apoptosis, or reduce cell viability; and linkers are the bridge that organically combines the two, which is the key to ADC drug design. The core part of the drug is the key to achieving targeted drug release.
化学偶联是目前主要应用的偶联方法，主要利用抗体中的赖氨酸或半胱氨酸残基与细胞毒性药物共价连接的方式实现偶联，例如，通过巯基与马来酰亚胺形成的硫代琥珀酰亚胺结构实现小分子药物与抗体的连接。然而，硫代琥珀酰亚胺结构并不稳定，在生物体内会与其它反应性巯基发生交换，导致细胞毒性药物的提前释放。一方面对正常组织造成毒性，另一方面也降低了ADC药物达到靶细胞后的药物效能。Chemical coupling is the main coupling method currently used, and the coupling is mainly achieved by covalently linking lysine or cysteine residues in antibodies to cytotoxic drugs, for example, through sulfhydryl and maleimide The formed sulfosuccinimide structure realizes the connection of the small molecule drug and the antibody. However, the structure of sulfosuccinimide is not stable, and it will exchange with other reactive sulfhydryl groups in vivo, resulting in the early release of cytotoxic drugs. On the one hand, it causes toxicity to normal tissues, and on the other hand, it also reduces the drug efficacy of ADC drugs after they reach the target cells.
连接子对于ADC药物的稳定性、药物活性等起到关键作用，发现更多适合偶联抗体与细胞毒性药物的连接子，是目前亟需解决的重要问题。 Linkers play a key role in the stability and activity of ADC drugs, and finding more linkers suitable for coupling antibodies and cytotoxic drugs is an important problem that needs to be solved urgently.
发明内容Contents of the invention
发明要解决的问题The problem to be solved by the invention
鉴于现有技术中存在的问题，例如，需要开发更多适合连接抗体与细胞毒性药物的连接子，以提高ADC药物的稳定性和药物活性。为此，本公开提供了一种结构新颖的连接子，连接子与艾日布林连接形成如式(I)所示的化合物。式(I)所示的化合物能够连接抗体形成抗体药物偶联物，进而特异性靶向肿瘤细胞等目标细胞并发挥高的靶细胞杀伤活性，在肿瘤等疾病的治疗中具有重要的应用前景。In view of the existing problems in the prior art, for example, it is necessary to develop more linkers suitable for linking antibodies and cytotoxic drugs, so as to improve the stability and drug activity of ADC drugs. To this end, the present disclosure provides a linker with a novel structure, which is connected with eribulin to form a compound as shown in formula (I). The compound represented by formula (I) can be linked to an antibody to form an antibody-drug conjugate, which can then specifically target target cells such as tumor cells and exert high killing activity on target cells, and has important application prospects in the treatment of diseases such as tumors.
用于解决问题的方案solutions to problems
第一方面，本公开提供了如式(I)所示的化合物或其药学上可接受的盐、酯、溶剂化物、光学异构体、互变异构体、同位素标记物或前药：
In a first aspect, the present disclosure provides a compound represented by formula (I) or a pharmaceutically acceptable salt, ester, solvate, optical isomer, tautomer, isotope label or prodrug thereof:
其中，L1为(Gly)p-(Z1)a-，p为1-10的任一整数；a为0-20的任一整数，若存在，每一个Z1各自独立地为任意种类的氨基酸残基；Wherein, L 1 is ( Gly ) p -(Z 1 ) a -, p is any integer of 1-10; a is any integer of 0-20, if present, each Z 1 is independently any Types of amino acid residues;
L2为-NH-R1-(CH2)c-C(＝O)-，其中R1为-(CH2-CH2-X)b-，b为1-10的任一整数，每一个X各自独立地为-CH2-、-NH-、-O-或-S-，c为1-5的任一整数；L 2 is -NH-R 1 -(CH 2 ) c -C(=O)-, wherein R 1 is -(CH 2 -CH 2 -X) b -, b is any integer from 1 to 10, each Each X is independently -CH 2 -, -NH-, -O- or -S-, and c is any integer from 1 to 5;
L3为 其中m为1-5的任一整数，优选为2；R2为-(CH2-CH2-Y)d-，d为1-10的任一整数，每一个Y各自独立地为-CH2-、-NH-、-O-或-S-；L 3 is wherein m is any integer from 1 to 5, preferably 2; R 2 is -(CH 2 -CH 2 -Y) d -, d is any integer from 1 to 10, and each Y is independently -CH 2 -, -NH-, -O- or -S-;
L1的羧基端连接L2的氨基端，L2的羧基端连接L3的氨基端。The carboxy-terminal of L1 is connected to the amino-terminus of L2 , and the carboxy-terminus of L2 is connected to the amino-terminus of L3 .
在一些实施方式中，根据本公开所述的化合物或其药学上可接受的盐、酯、溶剂化物、光学异构体、互变异构体、同位素标记物或前药，其中，In some embodiments, a compound according to the present disclosure or a pharmaceutically acceptable salt, ester, solvate, optical isomer, tautomer, isotope label or prodrug thereof, wherein,
L3为 其中m为1-5的任一整数，优选为2；R2为-(CH2-CH2-Y)d-，d为1-10的任一整数，每一个Y各自独立地为-CH2-、-NH-、-O-或-S-。 L 3 is wherein m is any integer from 1 to 5, preferably 2; R 2 is -(CH 2 -CH 2 -Y) d -, d is any integer from 1 to 10, and each Y is independently -CH 2- , -NH-, -O-, or -S-.
在一些实施方式中，根据本公开所述的化合物或其药学上可接受的盐、酯、溶剂化物、光学异构体、互变异构体、同位素标记物或前药，其中，In some embodiments, a compound according to the present disclosure or a pharmaceutically acceptable salt, ester, solvate, optical isomer, tautomer, isotope label or prodrug thereof, wherein,
L1为(Gly)p-，p为1-10的任一整数，优选为3-5的任一整数；L 1 is ( Gly ) p -, p is any integer of 1-10, preferably any integer of 3-5;
L2、L3如第一方面中所定义。L 2 , L 3 are as defined in the first aspect.
在一些实施方式中，根据本公开所述的化合物或其药学上可接受的盐、酯、溶剂化物、光学异构体、互变异构体、同位素标记物或前药，其中，L3选自如下的任意一种：

In some embodiments, the compound according to the present disclosure or a pharmaceutically acceptable salt, ester, solvate, optical isomer, tautomer, isotope label or prodrug thereof, wherein, L is selected from Any of the following:

L1、L2如第一方面中所定义。L 1 , L 2 are as defined in the first aspect.
在一些实施方式中，根据本公开所述的化合物或其药学上可接受的盐、酯、溶剂化物、光学异构体、互变异构体、同位素标记物或前药，其中，In some embodiments, a compound according to the present disclosure or a pharmaceutically acceptable salt, ester, solvate, optical isomer, tautomer, isotope label or prodrug thereof, wherein,
L3选自如下的任意一种：
 L3 is selected from any one of the following:
在一些实施方式中，根据本公开所述的化合物或其药学上可接受的盐、酯、溶剂化物、光学异构体、互变异构体、同位素标记物或前药，其中，In some embodiments, a compound according to the present disclosure or a pharmaceutically acceptable salt, ester, solvate, optical isomer, tautomer, isotope label or prodrug thereof, wherein,
L2为-NH-R1-(CH2)c-C(＝O)-；L 2 is -NH-R 1 -(CH 2 ) c -C(=O)-;
其中R1为-(CH2-CH2-X)b-，b为1-10的任一整数，优选为1-5的任一整数；每一个X各自独立地为-CH2-或-O-；c为1-5的任一整数，优选为1-2的任一整数；Wherein R 1 is -(CH 2 -CH 2 -X) b -, b is any integer of 1-10, preferably any integer of 1-5; each X is independently -CH 2 - or - O-; c is any integer of 1-5, preferably any integer of 1-2;
L1、L3如第一方面中所定义。L 1 , L 3 are as defined in the first aspect.
在一些实施方式中，根据本公开所述的化合物或其药学上可接受的盐、酯、溶剂化物、光学异构体、互变异构体、同位素标记物或前药，其中，In some embodiments, a compound according to the present disclosure or a pharmaceutically acceptable salt, ester, solvate, optical isomer, tautomer, isotope label or prodrug thereof, wherein,
L1-L2-为 L 1 -L 2 -for
其中，w为0-9的任一整数，优选为2-4的任一整数；c为1-5的任一整数，优选为1-2的任一整数；Wherein, w is any integer of 0-9, preferably any integer of 2-4; c is any integer of 1-5, preferably any integer of 1-2;
b为1-5的任一整数，优选为2-4的任一整数，每一个X各自独立地为-CH2-或-O-；并且，
的结构为：
b is any integer of 1-5, preferably any integer of 2-4, each X is independently -CH 2 - or -O-; and,
The structure is:
在一些实施方式中，根据本公开所述的化合物或其药学上可接受的盐、酯、溶剂化物、光学异构体、互变异构体、同位素标记物或前药，其中，In some embodiments, a compound according to the present disclosure or a pharmaceutically acceptable salt, ester, solvate, optical isomer, tautomer, isotope label or prodrug thereof, wherein,
L1-L2-为 L 1 -L 2 -for
其中，w为0-9的任一整数，优选为2-4的任一整数；c为1-5的任一整数，优选为1-2的任一整数；Wherein, w is any integer of 0-9, preferably any integer of 2-4; c is any integer of 1-5, preferably any integer of 1-2;
b为1-5的任一整数，优选为2-4的任一整数，每一个X各自独立地为-CH2-或-O-；并且，
的结构为：
b is any integer of 1-5, preferably any integer of 2-4, each X is independently -CH 2 - or -O-; and,
The structure is:
在一些实施方式中，本公开提供了如下式(I-1)～(I-6)任一项所示的化合物或其药学上可接受的盐、酯、溶剂化物、光学异构体、互变异构体、同位素标记物或前药：


In some embodiments, the present disclosure provides a compound represented by any one of the following formulas (I-1) to (I-6) or a pharmaceutically acceptable salt, ester, solvate, optical isomer, mutual Variant, isotopically labeled or prodrug:


优选如下任一项所示的结构：
A structure as shown in any of the following is preferred:
第二方面，本公开提供了一种用于偶联抗体与药物化合物的连接子，所述连接子具有如下式(II)所示的结构：In a second aspect, the present disclosure provides a linker for coupling an antibody to a pharmaceutical compound, the linker has a structure as shown in the following formula (II):
L1-L2-L3-R3    (II)； L 1 -L 2 -L 3 -R 3 (II);
其中，R3选自-NH2或-OH；Wherein, R 3 is selected from -NH 2 or -OH;
L1、L2、L3如第一方面所定义。L 1 , L 2 , L 3 are as defined in the first aspect.
在一些实施方式中，根据本公开所述的连接子，其中，所述连接子具有如下式(II-1)或(II-2)所示的结构：
In some embodiments, the linker according to the present disclosure, wherein the linker has the structure shown in the following formula (II-1) or (II-2):
其中，R3选自-NH2或-OH；Wherein, R 3 is selected from -NH 2 or -OH;
w为0-9的任一整数，优选为2-4的任一整数；c为1-5的任一整数，优选1-2的任一整数，w is any integer of 0-9, preferably any integer of 2-4; c is any integer of 1-5, preferably any integer of 1-2,
b为1-5的任一整数，优选为2-4的任一整数，每一个X各自独立地为-CH2-、-NH-、-O-或-S-。b is any integer of 1-5, preferably any integer of 2-4, and each X is independently -CH 2 -, -NH-, -O- or -S-.
在一些实施方式中，根据本公开所述的连接子，其中，所述连接子具有如下任一项所示的结构：

In some embodiments, the linker according to the present disclosure, wherein the linker has a structure as shown in any one of the following:

第三方面，本公开提供了根据第一方面所示的化合物或其药学上可接受的盐、酯、溶剂化物、光学异构体、互变异构体、同位素标记物或前药在制备抗体药物偶联物中的用途；In a third aspect, the present disclosure provides a compound or a pharmaceutically acceptable salt, ester, solvate, optical isomer, tautomer, isotope label or prodrug thereof in the preparation of an antibody according to the first aspect Use in drug conjugates;
其中，所述抗体药物偶联物中，根据第一方面所示的化合物或其药学上可接受的盐、酯、溶剂化物、光学异构体、互变异构体、同位素标记物或前药通过L1与抗体或抗原结合片段相偶联；Wherein, in the antibody drug conjugate, according to the compound shown in the first aspect or its pharmaceutically acceptable salt, ester, solvate, optical isomer, tautomer, isotope label or prodrug Conjugate antibody or antigen-binding fragment through L1 ;
可选地，所述抗体药物偶联物用于预防和/或治疗肿瘤；可选地，所述肿瘤为与HER蛋白家族中的一种或两种以上的蛋白异常表达相关的肿瘤；优选地，所述异常表达的蛋白选自HER2、HER3中的至少一种；Optionally, the antibody drug conjugate is used to prevent and/or treat tumors; optionally, the tumors are tumors related to the abnormal expression of one or more than two proteins in the HER protein family; preferably , the abnormally expressed protein is selected from at least one of HER2 and HER3;
可选地，所述肿瘤选自乳腺癌、卵巢癌、宫颈癌、子宫癌、前列腺癌、肾癌、尿道癌、膀胱癌、肝癌、胃癌、子宫内膜癌、唾液腺癌、食道癌、黑色素瘤、神经胶质瘤、神经母细胞瘤、肉瘤、肺癌、结直肠癌、白血病、骨癌、皮肤癌、甲状腺癌、胰腺癌或淋巴瘤。Optionally, the tumor is selected from breast cancer, ovarian cancer, cervical cancer, uterine cancer, prostate cancer, renal cancer, urethral cancer, bladder cancer, liver cancer, gastric cancer, endometrial cancer, salivary gland cancer, esophageal cancer, melanoma , glioma, neuroblastoma, sarcoma, lung cancer, colorectal cancer, leukemia, bone cancer, skin cancer, thyroid cancer, pancreatic cancer, or lymphoma.
第四方面，本公开提供了根据第二方面所示的连接子在制备抗体药物偶联物中的用途；In a fourth aspect, the present disclosure provides the use of the linker shown in the second aspect in the preparation of an antibody-drug conjugate;
其中，所述抗体药物偶联物中，所述连接子的L1基团连接抗体或抗原结合片段，所述连接子的L3基团连接药物化合物；Wherein, in the antibody-drug conjugate, the L1 group of the linker is connected to the antibody or the antigen-binding fragment, and the L3 group of the linker is connected to the drug compound;
可选地，所述抗体药物偶联物用于预防和/或治疗肿瘤；可选地，所述肿瘤为与HER蛋白家族中的一种或两种以上的蛋白异常表达相关的肿瘤；优选地，所述异常表达的蛋白选自HER2、HER3中的至少一种；Optionally, the antibody drug conjugate is used to prevent and/or treat tumors; optionally, the tumors are tumors related to the abnormal expression of one or more than two proteins in the HER protein family; preferably , the abnormally expressed protein is selected from at least one of HER2 and HER3;
可选地，所述肿瘤选自乳腺癌、卵巢癌、宫颈癌、子宫癌、前列腺癌、肾癌、尿道癌、膀胱癌、肝癌、胃癌、子宫内膜癌、唾液腺癌、食道癌、黑色素瘤、神经胶质瘤、神经母细胞瘤、肉瘤、肺癌、结直肠癌、白血病、骨癌、皮肤癌、甲状腺癌、胰腺癌或淋巴瘤。Optionally, the tumor is selected from breast cancer, ovarian cancer, cervical cancer, uterine cancer, prostate cancer, renal cancer, urethral cancer, bladder cancer, liver cancer, gastric cancer, endometrial cancer, salivary gland cancer, esophageal cancer, melanoma , glioma, neuroblastoma, sarcoma, lung cancer, colorectal cancer, leukemia, bone cancer, skin cancer, thyroid cancer, pancreatic cancer, or lymphoma.
第五方面，本公开提供了一种抗体药物偶联物，其具有如下式(III)所示的结构：
In the fifth aspect, the present disclosure provides an antibody-drug conjugate, which has the structure shown in the following formula (III):
其中，q为2-8的任一整数，优选为4；Ab为抗体或抗原结合片段；Wherein, q is any integer of 2-8, preferably 4; Ab is an antibody or an antigen-binding fragment;
Pep为-(X1)e-Leu-Pro-X2-Thr-，其中e为0-10的任一整数，若存在，每一个X1各自独立地为Gly或Ala；X2为任意种类的氨基酸残基；Pep is -(X 1 ) e -Leu-Pro-X 2 -Thr-, wherein e is any integer from 0-10, if present, each X 1 is independently Gly or Ala; X 2 is any species amino acid residues;
L1为-(Gly)p-(Z1)a-，p为1-10的任一整数；a为0-20的任一整数，若存在，每一个Z1各自独立地为任意种类的氨基酸残基；L 1 is -(Gly) p -(Z 1 ) a -, p is any integer of 1-10; a is any integer of 0-20, if present, each Z 1 is independently any kind of amino acid residues;
L2为-NH-R1-(CH2)c-C(＝O)-，其中R1为-(CH2-CH2-X)b-，b为1-10的任一整数，每一个X各自独立地为-CH2-、-NH-、-O-或-S-，c为1-5的任一整数；L 2 is -NH-R 1 -(CH 2 ) c -C(=O)-, wherein R 1 is -(CH 2 -CH 2 -X) b -, b is any integer from 1 to 10, each Each X is independently -CH 2 -, -NH-, -O- or -S-, and c is any integer from 1 to 5;
L3为 L 3 is
或者，L3为其中m为1-5的任一整数，优选为2；R2为-(CH2-CH2-Y)d-，d为1-10的任一整数，每一个Y各自独立地为-CH2-、-NH-、-O-或-S-；Alternatively, L3 is wherein m is any integer from 1 to 5, preferably 2; R 2 is -(CH 2 -CH 2 -Y) d -, d is any integer from 1 to 10, and each Y is independently -CH 2 -, -NH-, -O- or -S-;
并且，Pep的氨基端连接Ab的重链或轻链的羧基端，Pep的羧基端连接L1的氨基端，L1的羧基端连接L2的氨基端，L2的羧基端连接L3的氨基端。And, the amino terminus of Pep is connected to the carboxyl terminus of the heavy chain or light chain of Ab, the carboxyl terminus of Pep is connected to the amino terminus of L1 , the carboxyl terminus of L1 is connected to the amino terminus of L2 , and the carboxyl terminus of L2 is connected to the carboxy terminus of L3 . amino terminus.
在一些实施方式中，根据本公开所述的抗体药物偶联物，其中，In some embodiments, according to the antibody drug conjugate of the present disclosure, wherein,
Pep为-(Gly-Ala)f-Leu-Pro-X2-Thr-，其中f为1-5的任意整数，优选为1；X2选自任意种类的氨基酸残基，优选为Glu残基；Pep is -(Gly-Ala) f -Leu-Pro-X 2 -Thr-, wherein f is any integer of 1-5, preferably 1; X 2 is selected from any kind of amino acid residues, preferably Glu residues ;
优选地，Pep为-Gly-Ala-Leu-Pro-Glu-Thr-，其中Pep的氨基端连接Ab，Pep的羧基端连接L1的氨基端。Preferably, Pep is -Gly-Ala-Leu-Pro-Glu-Thr-, wherein the amino terminal of Pep is connected to Ab, and the carboxyl terminal of Pep is connected to the amino terminal of L1 .
在一些实施方式中，根据本公开所述的抗体药物偶联物，其中，Ab的重链的羧基端连接Pep，且Ab的轻链的羧基端连接Pep；In some embodiments, the antibody-drug conjugate according to the present disclosure, wherein the carboxyl terminus of the heavy chain of Ab is linked to Pep, and the carboxyl terminus of the light chain of Ab is linked to Pep;
优选地，Ab的重链的羧基端连接-Gly-Ala-Leu-Pro-Glu-Thr-，且Ab的轻链的羧基端连接-Gly-Ala-Leu-Pro-Glu-Thr-。Preferably, the carboxy terminus of the heavy chain of the Ab is linked to -Gly-Ala-Leu-Pro-Glu-Thr-, and the carboxy terminus of the light chain of the Ab is linked to -Gly-Ala-Leu-Pro-Glu-Thr-.
在一些实施方式中，根据本公开所述的抗体药物偶联物，其中，所述抗体或抗原结合片段特异 与HER蛋白家族中至少一种蛋白特异性结合；优选地，所述抗体或抗原结合片段与如下的至少一种蛋白特异性结合：HER2、HER3。In some embodiments, according to the antibody drug conjugate of the present disclosure, wherein the antibody or antigen-binding fragment is specific It specifically binds to at least one protein in the HER protein family; preferably, the antibody or antigen-binding fragment specifically binds to at least one of the following proteins: HER2, HER3.
在一些实施方式中，根据本公开所述的抗体药物偶联物，其中，抗体或抗原结合片段包含重链可变区，其中，所述重链可变区包含如SEQ ID NO：9、SEQ ID NO：11和SEQ ID NO：13中至少一项所示的氨基酸序列；和/或，In some embodiments, according to the antibody drug conjugate of the present disclosure, wherein, the antibody or antigen-binding fragment comprises a heavy chain variable region, wherein, the heavy chain variable region comprises such as SEQ ID NO: 9, SEQ ID NO: The amino acid sequence shown in at least one of ID NO: 11 and SEQ ID NO: 13; and/or,
所述抗体或抗原结合片段包含轻链可变区，其中，所述轻链可变区包含如SEQ ID NO：2、SEQ ID NO：4和SEQ ID NO：6中至少一项所示的氨基酸序列；The antibody or antigen-binding fragment comprises a light chain variable region, wherein the light chain variable region comprises amino acids as shown in at least one of SEQ ID NO: 2, SEQ ID NO: 4 and SEQ ID NO: 6 sequence;
所述轻链可变区、所述重链可变区为根据Kabat的分析方法编码。The light chain variable region and the heavy chain variable region are coded according to the analysis method of Kabat.
在一些实施方式中，根据本公开所述的抗体药物偶联物，其中，所述重链可变区包含HCDR1、HCDR2和HCDR3，所述轻链可变区包含LCDR1、LCDR2和LCDR3；In some embodiments, the antibody drug conjugate according to the present disclosure, wherein the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3;
HCDR1包含如SEQ ID NO：9所示的氨基酸序列，HCDR2包含如SEQ ID NO：11所示的氨基酸序列，HCDR3包含如SEQ ID NO：13所示的氨基酸序列；HCDR1 contains the amino acid sequence shown in SEQ ID NO: 9, HCDR2 contains the amino acid sequence shown in SEQ ID NO: 11, and HCDR3 contains the amino acid sequence shown in SEQ ID NO: 13;
LCDR1包含如SEQ ID NO：2所示的氨基酸序列，LCDR2包含如SEQ ID NO：4所示的氨基酸序列，LCDR3包含如SEQ ID NO：6所示的氨基酸序列。LCDR1 includes the amino acid sequence shown in SEQ ID NO: 2, LCDR2 includes the amino acid sequence shown in SEQ ID NO: 4, and LCDR3 includes the amino acid sequence shown in SEQ ID NO: 6.
在一些实施方式中，根据本公开所述的抗体药物偶联物，其中，所述抗原或抗原结合片段的重链的氨基酸序列如SEQ ID NO：18所示，和/或，In some embodiments, according to the antibody drug conjugate of the present disclosure, wherein, the amino acid sequence of the heavy chain of the antigen or antigen-binding fragment is shown in SEQ ID NO: 18, and/or,
所述抗原或抗原结合片段的轻链的氨基酸序列如SEQ ID NO：17所示。The amino acid sequence of the light chain of the antigen or antigen-binding fragment is shown in SEQ ID NO: 17.
在一些实施方式中，根据本公开所述的抗体药物偶联物，其中，L1、L2、L3如第一方面中所定义。In some embodiments, according to the antibody drug conjugate of the present disclosure, L 1 , L 2 , and L 3 are as defined in the first aspect.
在一些实施方式中，本公开提供了如下式(III-1)～(III-6)任一项所示的抗体药物偶联物：

In some embodiments, the present disclosure provides an antibody drug conjugate represented by any one of the following formulas (III-1) to (III-6):

优选如下任一项所示的结构：
A structure as shown in any of the following is preferred:
在本公开中，Ab可以是与任意类型的肿瘤抗原结合的抗体或抗原结合片段。示例性的，肿瘤抗原包括但不限于PD-1、PD-L1、PDL2、CTLA4、LAG3、TIM3、TIGIT、CD103、CD19、CD20、CD22、CD30、CD33、CD38、CD123、CD138、CD171、AFP、CEA、PSCA、GD2、NKG2D、BCMA、EGFR、HER2、HER3、EGFRvIII、CD171、FAP、IL13Ra2、VEGFR1、VEGFR2、GPC-3、Mesothelin、claudin 18.2、EpCAM、MUC1、MUC16、EPHA2、EPHA3、CD133、PSMA。抗体或抗原结合片段通过特异性结合肿瘤表面抗原，从而将艾日布林特异性递送至肿瘤细胞，发挥高效、特异性的肿瘤细胞杀伤作用。In the present disclosure, Ab may be an antibody or antigen-binding fragment that binds to any type of tumor antigen. Exemplary, tumor antigens include but are not limited to PD-1, PD-L1, PDL2, CTLA4, LAG3, TIM3, TIGIT, CD103, CD19, CD20, CD22, CD30, CD33, CD38, CD123, CD138, CD171, AFP, CEA, PSCA, GD2, NKG2D, BCMA, EGFR, HER2, HER3, EGFRvIII, CD171, FAP, IL13Ra2, VEGFR1, VEGFR2, GPC-3, Mesothelin, claudin 18.2, EpCAM, MUC1, MUC16, EPHA2, EPHA3, CD133, PSMA . The antibody or antigen-binding fragment can specifically deliver Eribulin to tumor cells by specifically binding to tumor surface antigens, thereby exerting an efficient and specific tumor cell killing effect.
进一步的，抗体或抗原结合片段与HER蛋白家族中至少一种蛋白特异性结合；优选地，所述抗体或抗原结合片段与如下的至少一种蛋白特异性结合：HER2、HER3。Further, the antibody or antigen-binding fragment specifically binds to at least one protein in the HER protein family; preferably, the antibody or antigen-binding fragment specifically binds to at least one of the following proteins: HER2, HER3.
在一些实施方式中，根据本公开所述的抗体药物偶联物，其中，抗体或抗原结合片段包含重链可变区，其中，所述重链可变区包含如SEQ ID NO：9、SEQ ID NO：11和SEQ ID NO：13中至少一项所示的氨基酸序列；和/或，In some embodiments, according to the antibody drug conjugate of the present disclosure, wherein, the antibody or antigen-binding fragment comprises a heavy chain variable region, wherein, the heavy chain variable region comprises such as SEQ ID NO: 9, SEQ ID NO: The amino acid sequence shown in at least one of ID NO: 11 and SEQ ID NO: 13; and/or,
所述抗体或抗原结合片段包含轻链可变区，其中，所述轻链可变区包含如SEQ ID NO：2、SEQ ID NO：4和SEQ ID NO：6中至少一项所示的氨基酸序列；The antibody or antigen-binding fragment comprises a light chain variable region, wherein the light chain variable region comprises amino acids as shown in at least one of SEQ ID NO: 2, SEQ ID NO: 4 and SEQ ID NO: 6 sequence;
所述轻链可变区、所述重链可变区为根据Kabat的分析方法编码。The light chain variable region and the heavy chain variable region are coded according to the analysis method of Kabat.
在一些实施方式中，根据本公开所述的抗体药物偶联物，其中，所述重链可变区包含HCDR1、HCDR2和HCDR3，所述轻链可变区包含LCDR1、LCDR2和LCDR3；In some embodiments, the antibody drug conjugate according to the present disclosure, wherein the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3;
HCDR1包含如SEQ ID NO：9所示的氨基酸序列，HCDR2包含如SEQ ID NO：11所示的氨基酸序列，HCDR3包含如SEQ ID NO：13所示的氨基酸序列；HCDR1 contains the amino acid sequence shown in SEQ ID NO: 9, HCDR2 contains the amino acid sequence shown in SEQ ID NO: 11, and HCDR3 contains the amino acid sequence shown in SEQ ID NO: 13;
LCDR1包含如SEQ ID NO：2所示的氨基酸序列，LCDR2包含如SEQ ID NO：4所示的氨基酸序列，LCDR3包含如SEQ ID NO：6所示的氨基酸序列。LCDR1 includes the amino acid sequence shown in SEQ ID NO: 2, LCDR2 includes the amino acid sequence shown in SEQ ID NO: 4, and LCDR3 includes the amino acid sequence shown in SEQ ID NO: 6.
在一些实施方式中，根据本公开所述的抗体药物偶联物，其中，所述抗原或抗原结合片段的重链的氨基酸序列如SEQ ID NO：18所示，和/或，所述抗原或抗原结合片段的轻链的氨基酸序列如SEQ  ID NO：17所示。In some embodiments, according to the antibody drug conjugate of the present disclosure, wherein, the amino acid sequence of the heavy chain of the antigen or antigen-binding fragment is shown in SEQ ID NO: 18, and/or, the antigen or The amino acid sequence of the light chain of the antigen-binding fragment is as SEQ ID NO: 17.
发明的效果The effect of the invention
本公开提供的如式(I)所示的化合物，其由连接子与细胞毒性药物艾日布林偶联形成；式(I)所示化合物中的连接子可进一步连接抗体，得到抗体药物偶联物。利用式(I)所示化合物制备的抗体药物偶联物，能够特异性靶向肿瘤细胞；而在进入肿瘤细胞内后，抗体药物偶联物的接头部分可以发生断裂，释放细胞毒性药物艾日布林，具有高的肿瘤细胞杀伤活性，在肿瘤治疗领域具有重要的应用前景。The compound shown in the formula (I) provided by the present disclosure is formed by coupling a linker with the cytotoxic drug eribulin; the linker in the compound shown in the formula (I) can be further linked to an antibody to obtain an antibody drug conjugate United things. The antibody-drug conjugate prepared by using the compound represented by formula (I) can specifically target tumor cells; after entering the tumor cell, the linker part of the antibody-drug conjugate can be broken to release the cytotoxic drug Ari Brin has high tumor cell killing activity and has important application prospects in the field of tumor treatment.
进一步的，如式(I)所示的化合物能够针对肿瘤的不同靶点连接不同的抗体，实现对不同类型肿瘤的高效、特异性杀伤。Furthermore, the compound represented by the formula (I) can be linked with different antibodies against different targets of tumors to achieve efficient and specific killing of different types of tumors.
在一些实施方式中，本公开提供了如式(I-1)～式(I-6)任一项所示的化合物，式(I-1)～式(I-6)所示的化合物能够通过其甘氨酸接头，以酶促偶联的方式与抗体连接得到结构稳定抗体药物偶联物，抗体药物偶联物兼具特异性地靶向作用和对肿瘤细胞的高杀伤活性，发挥优异的肿瘤治疗效果。In some embodiments, the present disclosure provides a compound shown in any one of formula (I-1) to formula (I-6), and the compound shown in formula (I-1) to formula (I-6) can Through its glycine linker, it is enzymatically coupled with the antibody to obtain a structurally stable antibody-drug conjugate. The antibody-drug conjugate has both specific targeting and high killing activity against tumor cells, and exerts excellent tumor treatment effect.
在一些优选的实施方式中，本公开以式(I-1)、式(I-5)所示的化合物连接HER2抗体，得到了具有如式(III-1)、式(III-5)所示结构的抗体药物偶联物。本公开发现，式(III-1)、式(III-5)所示的抗体药物偶联物能够显著降低HER2阳性(例如，HCC1954、SK-BR-3和NCI-N87)的各类肿瘤细胞的体外存活率，发挥高效的肿瘤细胞杀伤活性。与小分子药物Dxd相比，艾日布林显示出更强的抗肿瘤活性；与小分子药物艾日布林相比，本公开中式(III-1)、式(III-5)所示的抗体药物偶联物具有更低的IC50值，和更高的肿瘤细胞杀伤活性。并且，在HER2阴性细胞中，ADC药物的细胞毒性低于小分子药物艾日布林；同时，式(III-1)、式(III-5)所示的抗体药物偶联物在HER2阳性细胞的细胞毒性高于在HER2阴性细胞的细胞毒性。以上结果说明本公开中的抗体药物偶联物能够靶向结合肿瘤细胞，并且在肿瘤细胞内部释放细胞毒性药物艾日布林，发挥靶向、高效杀伤肿瘤细胞的作用。In some preferred embodiments, the present disclosure uses compounds represented by formula (I-1) and formula (I-5) to link HER2 antibodies to obtain compounds with formula (III-1), formula (III-5) Antibody drug conjugates showing the structure. The present disclosure finds that the antibody drug conjugates represented by formula (III-1) and formula (III-5) can significantly reduce various types of tumor cells that are HER2 positive (for example, HCC1954, SK-BR-3 and NCI-N87) The in vitro survival rate and high tumor cell killing activity. Compared with the small molecule drug Dxd, Eribulin shows stronger anti-tumor activity; compared with the small molecule drug Eribulin, the antibodies represented by formula (III-1) and formula (III-5) in the present disclosure The drug conjugate has lower IC 50 value and higher tumor cell killing activity. Moreover, in HER2-negative cells, the cytotoxicity of ADC drugs is lower than that of the small molecule drug eribulin; meanwhile, the antibody-drug conjugates shown in formula (III-1) and formula (III-5) are more effective in HER2-positive cells The cytotoxicity was higher than that in HER2-negative cells. The above results indicate that the antibody-drug conjugates in the present disclosure can target and bind tumor cells, and release the cytotoxic drug Eribulin inside the tumor cells to play a targeted and efficient role in killing tumor cells.
进一步的，在接种Capan-1、NCI-N87和HCC1954不同肿瘤细胞的三种荷瘤小鼠模型中验证式(III-1)、式(III-5)所示抗体药物偶联物的体内肿瘤治疗效果，结果显示，式(III-1)、式(III-5)所示抗体药物偶联物能够显著抑制Capan-1肿瘤、NCI-N87肿瘤和HCC1954肿瘤的生长，高效、特异性杀伤体内肿瘤细胞，并且不具有明显的药物毒性，在肿瘤临床治疗中具有广阔的应用前景。Further, in three tumor-bearing mouse models inoculated with different tumor cells of Capan-1, NCI-N87 and HCC1954, the in vivo tumors of the antibody-drug conjugates represented by formula (III-1) and formula (III-5) were verified. Therapeutic effect, the results show that the antibody-drug conjugates represented by formula (III-1) and formula (III-5) can significantly inhibit the growth of Capan-1 tumors, NCI-N87 tumors and HCC1954 tumors, and efficiently and specifically kill Tumor cells, and does not have obvious drug toxicity, has broad application prospects in the clinical treatment of tumors.
本公开提供的如式(II)所示的连接子，连接子的L1基团和L3基团接头能够分别连接抗体与药物化合物，得到抗体药物偶联物。连接子直接影响抗体药物偶联物的稳定性和药物活性，本公开中连接子中的L3基团在进入细胞内后可发生断裂，释放细胞毒性药物；利用本公开连接子构建的抗体药物偶联物，具有高效、特异性杀伤肿瘤细胞的活性，为抗体药物偶联物的开发提供了新的选择和偶联方式。The linker represented by the formula (II) provided by the present disclosure, the L1 group and the L3 group linker of the linker can respectively connect the antibody and the drug compound to obtain an antibody-drug conjugate. The linker directly affects the stability and drug activity of the antibody-drug conjugate. In the present disclosure, the L3 group in the linker can break after entering the cell and release cytotoxic drugs; the antibody drug constructed using the linker of the present disclosure The conjugate has high-efficiency and specific tumor cell killing activity, which provides a new choice and conjugation method for the development of antibody-drug conjugates.
附图说明Description of drawings
图1示出了不同药物浓度梯度的Ab0100-H0037、Ab0100-H0038、BQ3、艾日布林、Dxd对HCC1954细胞存活率的影响检测结果；Figure 1 shows the detection results of the impact of Ab0100-H0037, Ab0100-H0038, BQ3, Eribulin, and Dxd on the survival rate of HCC1954 cells with different drug concentration gradients;
图2示出了不同药物浓度梯度的Ab0100-H0037、Ab0100-H0038、BQ3、艾日布林、Dxd对SK-BR-3细胞存活率的影响检测结果；Figure 2 shows the detection results of the impact of Ab0100-H0037, Ab0100-H0038, BQ3, Eribulin, and Dxd on the survival rate of SK-BR-3 cells with different drug concentration gradients;
图3示出了不同药物浓度梯度的Ab0100-H0037、Ab0100-H0038、BQ3、艾日布林、Dxd对NCI-N87细胞存活率的影响检测结果；Figure 3 shows the detection results of the impact of Ab0100-H0037, Ab0100-H0038, BQ3, Eribulin, and Dxd on the survival rate of NCI-N87 cells with different drug concentration gradients;
图4示出了不同药物浓度梯度的Ab0100-H0037、Ab0100-H0038、BQ3、艾日布林、Dxd对MDA-MB-468细胞存活率的影响检测结果；Figure 4 shows the detection results of the impact of Ab0100-H0037, Ab0100-H0038, BQ3, Eribulin, and Dxd on the survival rate of MDA-MB-468 cells with different drug concentration gradients;
图5示出了溶媒(DPBS)、Ab0100-H0037(5mg/kg)、Ab0100-H0038(5mg/kg)，以及BQ3(5mg/kg)给药后荷瘤小鼠(Capan-1荷瘤雌性BALB/c裸鼠)的体重变化检测结果；Figure 5 shows vehicle (DPBS), Ab0100-H0037 (5mg/kg), Ab0100-H0038 (5mg/kg), and BQ3 (5mg/kg) after administration of tumor-bearing mice (Capan-1 tumor-bearing female BALB /c nude mice) body weight change detection results;
图6示出了溶媒(DPBS)、Ab0100-H0037(5mg/kg)、Ab0100-H0038(5mg/kg)，以及BQ3(5mg/kg)给药后荷瘤小鼠(Capan-1荷瘤雌性BALB/c裸鼠)的肿瘤生长曲线；Figure 6 shows vehicle (DPBS), Ab0100-H0037 (5mg/kg), Ab0100-H0038 (5mg/kg), and BQ3 (5mg/kg) after administration of tumor-bearing mice (Capan-1 tumor-bearing female BALB /c nude mice) tumor growth curve;
图7示出了溶媒(DPBS)、Ab0100-H0037(5mg/kg)、Ab0100-H0038(5mg/kg)，以及BQ3(5mg/kg) 给药后荷瘤小鼠(NCI-N87荷瘤雌性BALB/c裸鼠)的肿瘤生长曲线；Figure 7 shows vehicle (DPBS), Ab0100-H0037 (5mg/kg), Ab0100-H0038 (5mg/kg), and BQ3 (5mg/kg) Tumor growth curve of tumor-bearing mice (NCI-N87 tumor-bearing female BALB/c nude mice) after administration;
图8示出了溶媒(DPBS)、Ab0100-H0037(3mg/kg)、Ab0100-H0038(3mg/kg)，以及BQ3(5mg/kg)给药后荷瘤小鼠(HCC1954荷瘤雌性BALB/c裸鼠)的肿瘤生长曲线。Figure 8 shows vehicle (DPBS), Ab0100-H0037 (3mg/kg), Ab0100-H0038 (3mg/kg), and BQ3 (5mg/kg) after administration of tumor-bearing mice (HCC1954 tumor-bearing female BALB/c Tumor growth curve of nude mice).
具体实施方式Detailed ways
定义definition
除非有相反陈述，否则在本公开中所使用的术语具有下述含义。Unless stated to the contrary, the terms used in this disclosure have the following meanings.
在本公开的权利要求和/或说明书中，词语“一(a)”或“一(an)”或“一(the)”可以指“一个”，但也可以指“一个或多个”、“至少一个”以及“一个或多于一个”。In the claims and/or specification of the present disclosure, the word "one (a)" or "one (an)" or "one (the)" may mean "one", but may also mean "one or more", "at least one" and "one or more than one".
如在权利要求和说明书中所使用的，词语“包含”、“具有”、“包括”或“含有”是指包括在内的或开放式的，并不排除额外的、未引述的元件或方法步骤。与此同时，“包含”、“具有”、“包括”或“含有”也可以表示封闭式的，排除额外的、未引述的元件或方法步骤。As used in the claims and specification, the words "comprising", "having", "comprising" or "containing" mean inclusive or open-ended and do not exclude additional, non-recited elements or means step. At the same time, "comprising", "having", "including" or "comprising" can also mean enclosing, excluding additional, unrecited elements or method steps.
在整个申请文件中，术语“约”表示：一个值包括测定该值所使用的装置或方法的误差的标准偏差。Throughout this application, the term "about" means that a value includes the standard deviation of error of the apparatus or method used to determine the value.
在本公开中，使用“数值A-数值B的任一整数”覆盖数值A与数值B之间任意类型的自然数，其范围覆盖含端点数值A、B。示例性的，1-10的任一整数为选自1、2、3、4、5、6、7、8、9、10中的任一整数。In this disclosure, the use of "any integer from value A to value B" covers any type of natural number between value A and value B, and its range covers values A and B including the endpoints. Exemplarily, any integer of 1-10 is any integer selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10.
在本公开中，“任意种类的氨基酸”可以是常见氨基酸(本公开中表1所示出的氨基酸)，也可以是非常见氨基酸(例如，2-氨基己二酸，2-氨基丁酸，2-氨基庚二酸，2-羧酸酰胺等等)。进一步的，任意种类的氨基酸可以是修饰的氨基酸(例如，酰基化、泛素化、糖链修饰、生物素化等等)，也可以是未修饰的氨基酸。In this disclosure, "any kind of amino acid" can be common amino acids (amino acids shown in Table 1 in this disclosure), or uncommon amino acids (for example, 2-aminoadipic acid, 2-aminobutyric acid, 2-aminobutyric acid, 2 -aminopimelic acid, 2-carboxamide, etc.). Further, any kind of amino acid may be a modified amino acid (for example, acylation, ubiquitination, sugar chain modification, biotinylation, etc.), or an unmodified amino acid.
术语“各自独立地”是指结构中存在的取值范围相同或相近的至少两个基团(或环系)可以在特定情形下具有相同或不同的含义。例如，取代基X和取代基Y各自独立地为氢、羟基、烷基或芳基，则当取代基X为氢时，取代基Y既可以为氢，也可以为羟基、烷基或芳基；同理，当取代基Y为氢时，取代基X既可以为氢，也可以为羟基、烷基或芳基。或者，取代基X的个数为两个，每个X各自独立地为氢、羟基、烷基或芳基；则当一个X为氢时，另外一个X既可以为氢，也可以为羟基、烷基或芳基。同理，取代基X的个数为三个或以上，每个X各自独立地为氢、羟基、烷基或芳基，意味着其中任意一个X均可以选择氢、羟基、烷基或芳基。The term "independently" means that at least two groups (or ring systems) present in the structure with the same or similar value range may have the same or different meanings under specific circumstances. For example, the substituent X and the substituent Y are each independently hydrogen, hydroxyl, alkyl or aryl, then when the substituent X is hydrogen, the substituent Y can be either hydrogen or hydroxyl, alkyl or aryl ; Similarly, when the substituent Y is hydrogen, the substituent X can be either hydrogen, or hydroxyl, alkyl or aryl. Alternatively, the number of substituents X is two, and each X is independently hydrogen, hydroxyl, alkyl or aryl; then when one X is hydrogen, the other X can be either hydrogen or hydroxyl, Alkyl or aryl. Similarly, the number of substituents X is three or more, and each X is independently hydrogen, hydroxyl, alkyl or aryl, which means that any one of X can be selected from hydrogen, hydroxyl, alkyl or aryl .
示例性地，在-(X1)e-中，e为0-10的任一整数，若存在，每一个X1各自独立地为Gly或Ala。例如，当e为2时，沿氨基端向羧基端的方向，-(X1)e-可以是-Gly-Gly-、-Ala-Ala-，-Ala-Gly-或-Gly-Ala-。Exemplarily, in -(X 1 ) e -, e is any integer of 0-10, and if present, each X 1 is independently Gly or Ala. For example, when e is 2, -(X 1 ) e - may be -Gly-Gly-, -Ala-Ala-, -Ala-Gly- or -Gly-Ala- along the direction from the amino terminal to the carboxyl terminal.
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/抗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。在一些实施方式中，本公开的连接子是末端为甘氨酸的氨基酸片段。末端为甘氨酸的氨基酸片段可以被Fmoc等保护基保护。作为优选的实施例，其末端的氨基酸片段可以是3-5个甘氨酸。The term "linker", "linker unit", "linker unit", "linker" or "linker fragment" refers to a chemical structural fragment or bond that is connected to a ligand/antibody at one end and a drug at the other end, and may also be connected to other The connector is then connected to the drug. In some embodiments, a linker of the present disclosure is a glycine-terminated amino acid fragment. Amino acid fragments with a glycine terminal can be protected by protecting groups such as Fmoc. As a preferred embodiment, the amino acid fragment at the end can be 3-5 glycines.
术语“抗体”在本文中以最广意义使用，指包含抗原结合位点的蛋白质，涵盖各种结构的天然抗体和人工抗体，包括但不限于单克隆抗体、多克隆抗体、多特异性抗体(例如，双特异性抗体)、单链抗体、完整抗体和抗体片段。The term "antibody" is used herein in the broadest sense to refer to a protein comprising an antigen binding site, encompassing natural and artificial antibodies of various structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies ( For example, bispecific antibodies), single chain antibodies, whole antibodies and antibody fragments.
术语“抗原结合片段”是比完整或完全抗体的氨基酸残基数要少的完整或完全抗体的一部分或一段，其能结合抗原或与完整抗体(即与抗原结合片段所来源的完整抗体)竞争结合抗原。可以通过重组DNA技术、或通过酶或化学切割完整的抗体制备抗原结合片段。抗原结合片段包括但不限于Fv，Fab，Fab’，Fab’-SH，F(ab’)2；双抗体；线性抗体；单链抗体(例如scFv)；单域抗体；双价或双特异性抗体或其片段；骆驼科抗体(重链抗体)；和由抗体片段形成的双特异性抗体或多特异性抗体。The term "antigen-binding fragment" is a portion or segment of an intact or complete antibody having fewer amino acid residues than an intact or complete antibody, which is capable of binding antigen or competing with the intact antibody (i.e., the intact antibody from which the antigen-binding fragment is derived) Binding antigen. Antigen-binding fragments can be prepared by recombinant DNA techniques, or by enzymatic or chemical cleavage of intact antibodies. Antigen binding fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab') 2 ; diabodies; linear antibodies; single chain antibodies (e.g. scFv); single domain antibodies; bivalent or bispecific Antibodies or fragments thereof; camelid antibodies (heavy chain antibodies); and bispecific or multispecific antibodies formed from antibody fragments.
术语“互补决定区”或“CDR区”或“CDR”是抗体可变结构域中在序列上高变并且形成在结构上确定的环(“超变环”)和/或含有抗原接触残基(“抗原接触点”)的区域。CDR主要负责与抗原表位结合。重链和轻链的CDR通常被称作CDR1、CDR2和CDR3，从N-端开始顺序编号。位于抗 体重链可变结构域内的CDR被称作HCDR1、HCDR2和HCDR3，而位于抗体轻链可变结构域内的CDR被称作LCDR1、LCDR2和LCDR3。在一个给定的轻链可变区或重链可变区氨基酸序列中，各CDR的精确氨基酸序列边界可以使用许多公知的抗体CDR指派系统的任一种或其组合确定，所述指派系统包括例如：基于抗体的三维结构和CDR环的拓扑学的Chothia(Chothia等人.(1989)Nature 342：877-883，Al-Lazikani等人，“Standard conformations for the canonical structures of immunoglobulins”，Journal of Molecular Biology，273，927-948(1997))，基于抗体序列可变性的Kabat(Kabat等人，Sequences of Proteins of Immunological Interest，第4版，U.S.Department of Health and Human Services，National Institutes of Health(1987))，AbM(University of Bath)，Contact(University College London)，国际ImMunoGeneTics database(IMGT)(在万维网上imgt.cines.fr/上)，以及基于利用大量晶体结构的近邻传播聚类(affinity propagation clustering)的North CDR定义。The term "complementarity determining region" or "CDR region" or "CDR" is an antibody variable domain that is hypervariable in sequence and forms a structurally defined loop ("hypervariable loop") and/or contains antigen-contacting residues ("antigen contact point"). The CDRs are primarily responsible for binding to antigenic epitopes. The CDRs of the heavy and light chains are commonly referred to as CDR1, CDR2 and CDR3, numbered sequentially starting from the N-terminus. located in anti The CDRs within the variable domain of the heavy chain are referred to as HCDR1, HCDR2, and HCDR3, while the CDRs located within the variable domain of the antibody light chain are referred to as LCDR1, LCDR2, and LCDR3. In a given light chain variable region or heavy chain variable region amino acid sequence, the precise amino acid sequence boundaries of each CDR can be determined using any one or combination of a number of well-known antibody CDR assignment systems, including For example: Chothia based on the three-dimensional structure of antibodies and the topology of the CDR loops (Chothia et al. (1989) Nature 342:877-883, Al-Lazikani et al, "Standard conformations for the canonical structures of immunoglobulins", Journal of Molecular Biology, 273, 927-948 (1997)), Kabat based on antibody sequence variability (Kabat et al., Sequences of Proteins of Immunological Interest, 4th ed., USDepartment of Health and Human Services, National Institutes of Health (1987)) , AbM (University of Bath), Contact (University College London), the International ImMunoGeneTics database (IMGT) (on the World Wide Web at imgt.cines.fr/), and affinity propagation clustering based on exploiting a large number of crystal structures The North CDR definition.
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。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.
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“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)；更优先2-4个。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) attached to each antibody (Ab); more preferably 2-4.
在本公开中，“溶剂化”是指：一些溶剂分子被溶质分子或离子包围而成为一个集团的现象。In the present disclosure, "solvation" refers to the phenomenon that some solvent molecules are surrounded by solute molecules or ions to form a group.
本发明的化合物还可以以盐的形式提供。可以利用通常进行的方法形成这些盐。或根据本发明的制造方法的条件，可以以盐的形式生成化合物(例如是源自添加剂的物质)。The compounds of the invention may also be provided in the form of salts. These salts can be formed using commonly performed methods. Or, depending on the conditions of the production method of the present invention, the compound (for example, a substance derived from an additive) can be produced in the form of a salt.
本发明的化合物还可以以溶剂化物的形式提供。例如可列举出与水的溶剂化物。这样的溶剂化物可以利用通常进行的方法来形成。或根据本公开的制造方法的条件，可以以溶剂化物的形式生成化合物。进而，上述化合物的盐可以以溶剂化物的形式生成并提供。The compounds of the invention may also be provided in the form of solvates. For example, a solvate with water is mentioned. Such solvates can be formed using commonly performed methods. Alternatively, depending on the conditions of the production method of the present disclosure, the compound can be produced in the form of a solvate. Furthermore, salts of the above-mentioned compounds may be produced and provided in the form of solvates.
本公开上下文中使用的术语“治疗”是指：在罹患疾病之后，使受试者接触(例如给药)本公开的化合物或其药学上可接受的盐、酯、溶剂化物、光学异构体、互变异构体、同位素标记物、前药或含者有其的药物组合物(以下也称为“本公开的药物组合物”)，从而与不接触时相比使该疾病的症状减轻，并不意味着必需完全抑制疾病的症状。罹患疾病是指：身体出现了疾病症状。The term "treatment" used in the context of the present disclosure refers to: after suffering from a disease, contacting (for example, administering) a compound of the present disclosure or a pharmaceutically acceptable salt, ester, solvate, optical isomer thereof of a subject , tautomers, isotope labels, prodrugs, or pharmaceutical compositions containing them (hereinafter also referred to as "pharmaceutical compositions of the present disclosure"), thereby reducing the symptoms of the disease compared to when not in contact , does not mean that the symptoms of the disease must be completely suppressed. Suffering from a disease means that the body has symptoms of a disease.
本公开上下文中使用的术语“预防”是指：在罹患疾病之前，通过使受试者接触(例如给药)本公开的化合物或其药学上可接受的盐、酯、溶剂化物、光学异构体、互变异构体、同位素标记物、前药或本公开的药物组合物，从而与不接触时相比减轻罹患疾病后的症状，并不意味着必需完全抑制患病。The term "prevention" used in the context of the present disclosure refers to: prior to suffering from a disease, by making a subject contact (for example, administer) a compound of the present disclosure or a pharmaceutically acceptable salt, ester, solvate, optical isomer isomer, tautomer, isotopic label, prodrug or pharmaceutical composition of the present disclosure, thereby reducing the symptoms after suffering from the disease compared with no contact, it does not mean that the disease must be completely suppressed.
本公开上下文中使用的术语“个体”、“患者”或“受试者”包括哺乳动物。哺乳动物包括但不 限于，家养动物(例如，牛，羊，猫，狗和马)，灵长类动物(例如，人和非人灵长类动物如猴)，兔，以及啮齿类动物(例如，小鼠和大鼠)。The term "individual", "patient" or "subject" as used in the context of this disclosure includes mammals. mammals including but not Limited to, domesticated animals (e.g., cattle, sheep, cats, dogs, and horses), primates (e.g., humans and nonhuman primates such as monkeys), rabbits, and rodents (e.g., mice and mouse).
术语“肿瘤”和“癌症”在本文中互换地使用，涵盖实体瘤和液体肿瘤。术语“肿瘤”指所有赘生性(neoplastic)细胞生长和增殖，无论是恶性的还是良性的，及所有癌前(pre-cancerous)和癌性细胞和组织。术语“癌症”、“癌性”和“肿瘤”在本文中提到时并不互相排斥。The terms "tumor" and "cancer" are used interchangeably herein to encompass both solid and liquid tumors. The term "tumor" refers to all neoplastic cell growth and proliferation, whether malignant or benign, and to all pre-cancerous and cancerous cells and tissues. The terms "cancer", "cancerous" and "tumor" are not mutually exclusive when referred to herein.
术语“癌症”和“癌性”指向或描述哺乳动物中特征通常为细胞生长不受调节的生理疾患。癌症的例子包括但不限于癌，淋巴瘤，母细胞瘤，肉瘤和白血病或淋巴样恶性肿瘤。此类癌症的更具体例子包括但不限于鳞状细胞癌(例如上皮鳞状细胞癌)，肺癌(包括小细胞肺癌，非小细胞肺癌，肺的腺癌，和肺的鳞癌)，腹膜癌，肝细胞癌，胃癌(包括胃肠癌和胃肠基质癌)，胰腺癌，成胶质细胞瘤，宫颈癌，卵巢癌，肝癌，膀胱癌，尿道癌，肝瘤，乳腺癌，结肠直肠癌，子宫内膜癌或子宫癌，唾液腺癌，肾癌，前列腺癌，外阴癌，甲状腺癌，肝癌，肛门癌，阴茎癌，黑素瘤，浅表扩散性黑素瘤，恶性雀斑样痣黑素瘤，肢端黑素瘤，结节性黑素瘤，多发性骨髓瘤和B细胞淋巴瘤，慢性淋巴细胞性白血病(CLL)，急性成淋巴细胞性白血病(ALL)，毛细胞性白血病，慢性成髓细胞性白血病，和移植后淋巴增殖性病症(PTLD)，以及与瘢痣病(phakomatoses)，水肿(诸如与脑瘤有关的)和梅格斯氏(Meigs)综合征有关的异常血管增殖，脑瘤和脑癌，以及头颈癌，及相关转移。The terms "cancer" and "cancerous" refer to or describe a physiological disorder in mammals that is often characterized by unregulated cell growth. Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia or lymphoid malignancies. More specific examples of such cancers include, but are not limited to, squamous cell carcinoma (e.g., epithelial squamous cell carcinoma), lung cancer (including small cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, and squamous cell carcinoma of the lung), peritoneal carcinoma , hepatocellular carcinoma, gastric cancer (including gastrointestinal and gastrointestinal stromal carcinoma), pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, urethral cancer, hepatoma, breast cancer, colorectal cancer , endometrial or uterine cancer, salivary gland cancer, kidney cancer, prostate cancer, vulvar cancer, thyroid cancer, liver cancer, anal cancer, penile cancer, melanoma, superficial spreading melanoma, lentigo maligna melanoma acral melanoma, nodular melanoma, multiple myeloma and B-cell lymphoma, chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), hairy cell leukemia, chronic Myeloblastic leukemia, and post-transplantation lymphoproliferative disorder (PTLD), and abnormal vascular proliferation associated with phakomatoses, edema (such as that associated with brain tumors), and Meigs' syndrome , brain tumors and cancers, and head and neck cancers, and related metastases.
术语“抗肿瘤作用”指可以通过多种手段展示的生物学效果，包括但不限于例如，肿瘤体积减少、肿瘤细胞数目减少、肿瘤细胞增殖减少或肿瘤细胞存活减少。The term "anti-tumor effect" refers to a biological effect that can be exhibited by various means, including but not limited to, for example, reduction in tumor volume, reduction in tumor cell number, reduction in tumor cell proliferation, or reduction in tumor cell survival.
术语“有效量”指本公开的化合物或组合物的这样的量或剂量，其以单一或多次剂量施用患者后，在需要治疗或预防的患者中产生预期效果。有效量可以由作为本领域技术人员的主治医师通过考虑以下多种因素来容易地确定：诱如哺乳动物的物种；它的大小、年龄和一般健康；涉及的具体疾病；疾病的程度或严重性；个体患者的应答；施用的具体抗体；施用模式；施用制剂的生物利用率特征；选择的给药方案；和任何伴随疗法的使用。The term "effective amount" refers to an amount or dose of a compound or composition of the present disclosure which, when administered to a patient in single or multiple doses, produces the desired effect in a patient in need of treatment or prevention. An effective amount can be readily determined by the attending physician, who is skilled in the art, by considering a variety of factors: the species of the animal, such as a mammal; its size, age and general health; the particular disease involved; the extent or severity of the disease the individual patient's response; the particular antibody administered; the mode of administration; the bioavailability characteristics of the formulation administered; the chosen dosing regimen; and the use of any concomitant therapy.
本公开上下文中使用的术语“药学上可接受的盐”是指由本公开中的化合物与相对无毒的酸或碱制备得到的盐。当本公开中的化合物含有相对偏酸性的官能团(例如羧基或磺酸基)时，可以通过在纯的溶液或合适的惰性溶剂中用足够量的碱与其游离形式接触的方式获得碱加成盐。药学上可接受的碱加成盐的非限制性实例包括但不限于钠盐、钾盐、铵盐、钙盐、镁盐、有机胺盐或类似的盐。当本公开中的化合物含有相对偏碱性的官能团(例如氨基或胍基)时，可以通过在纯的溶液或合适的惰性溶剂中用足够量的酸与其游离形式接触的方式获得酸加成盐。药学上可接受的酸加成盐的非限制性实例包括但不限于无机酸盐(例如盐酸盐、氢溴酸盐、氢碘酸盐、硝酸盐、碳酸盐、碳酸氢盐、磷酸盐、磷酸一氢盐、磷酸二氢盐、亚磷酸盐、硫酸盐、硫酸氢盐等)、有机酸盐(例如乙酸盐、丙酸盐、异丁酸盐、丙二酸盐、琥珀酸盐、辛二酸盐、马来酸盐、富马酸盐、柠檬酸盐、酒石酸盐、乳酸盐、扁桃酸盐、苯甲酸盐、邻苯二甲酸盐、甲磺酸盐、苯磺酸盐、对甲苯磺酸盐、葡糖醛酸等)以及氨基酸盐(例如精氨酸盐等)。药学上可接受的盐的具体形式还可参见Berge et al.，“Pharmaceutical Salts”，Journal of Pharmaceutical Science，1977，66：1-19)。The term "pharmaceutically acceptable salt" as used in the context of the present disclosure refers to salts prepared from compounds of the present disclosure with relatively non-toxic acids or bases. When the compounds of the present disclosure contain relatively acidic functional groups (such as carboxyl or sulfonic acid groups), base addition salts can be obtained by contacting their free forms with a sufficient amount of base in neat solution or in a suitable inert solvent . Non-limiting examples of pharmaceutically acceptable base addition salts include, but are not limited to, sodium, potassium, ammonium, calcium, magnesium, organic amine, or similar salts. When the compounds of the present disclosure contain relatively basic functional groups (such as amino or guanidino), acid addition salts can be obtained by contacting their free forms with a sufficient amount of acid in neat solution or in a suitable inert solvent . Non-limiting examples of pharmaceutically acceptable acid addition salts include, but are not limited to, inorganic acid salts (e.g., hydrochloride, hydrobromide, hydroiodide, nitrate, carbonate, bicarbonate, phosphate , monohydrogen phosphate, dihydrogen phosphate, phosphite, sulfate, bisulfate, etc.), organic acid salts (such as acetate, propionate, isobutyrate, malonate, succinate , suberate, maleate, fumarate, citrate, tartrate, lactate, mandelate, benzoate, phthalate, methanesulfonate, benzenesulfonate salt, p-toluenesulfonate, glucuronic acid, etc.) and amino acid salts (such as arginine salt, etc.). Specific forms of pharmaceutically acceptable salts can also be found in Berge et al., "Pharmaceutical Salts", Journal of Pharmaceutical Science, 1977, 66: 1-19).
本公开上下文中使用的术语“药物组合物”是指可供药用的组合物，其包含一种或多种化合物或其药学上可接受的形式(例如盐、水合物、溶剂化物、立体异构体、互变异构体、代谢产物、前药等)，以及其他组分(例如药学上可接受的辅料)。The term "pharmaceutical composition" as used in the context of this disclosure refers to a pharmaceutically acceptable composition comprising one or more compounds or a pharmaceutically acceptable form thereof (e.g. salt, hydrate, solvate, stereoisomeric isomers, tautomers, metabolites, prodrugs, etc.), and other components (such as pharmaceutically acceptable excipients).
本公开上下文中使用的术语“药学上可接受的辅料”是指在药物生产领域中广泛采用的辅助物料。使用辅料的主要目的在于提供一种使用安全、性质稳定和/或具有特定功能性的药物组合物，还在于提供一种方法，以便在为受试者施用药物之后，活性成分能够以所期望的速率溶出，或者促进活性成分在接受给药的受试者体内得到有效吸收。药学上可接受的辅料可以是具有惰性的填充剂，也可以是为药用组合物提供某种功能(例如稳定组合物的整体pH值或防止组合物中活性成分的降解)的功效成分。药学上可接受的辅料的非限制性实例包括但不限于粘合剂、助悬剂、乳化剂、稀释剂(或填充剂)、成粒剂、胶粘剂、崩解剂、润滑剂、抗粘着剂、助流剂、润湿剂、胶凝剂、吸收延迟剂、溶解抑制剂、增强剂、吸附剂、缓冲剂、螯合剂、防腐剂、着色剂、矫味剂、甜味剂等。 The term "pharmaceutically acceptable excipient" used in the context of the present disclosure refers to an auxiliary material widely used in the field of pharmaceutical production. The main purpose of using excipients is to provide a pharmaceutical composition that is safe to use, stable in nature and/or has specific functionality, and also to provide a method so that after the drug is administered to the subject, the active ingredient can be used in the desired manner. The rate of dissolution, or the promotion of effective absorption of the active ingredient in the subject to which it is administered. Pharmaceutically acceptable excipients can be inert fillers, or functional ingredients that provide certain functions for the pharmaceutical composition (such as stabilizing the overall pH value of the composition or preventing the degradation of active ingredients in the composition). Non-limiting examples of pharmaceutically acceptable excipients include, but are not limited to, binders, suspending agents, emulsifying agents, diluents (or fillers), granulating agents, adhesives, disintegrants, lubricants, anti-adherents , glidants, wetting agents, gelling agents, absorption delaying agents, dissolution inhibitors, enhancers, adsorbents, buffers, chelating agents, preservatives, coloring agents, flavoring agents, sweeteners, etc.
本公开中的药物组合物可以使用本领域技术人员已知的任何方法来制备。例如，常规混合、溶解、造粒、乳化、磨细、包封、包埋和/或冻干工艺。The pharmaceutical compositions of the present disclosure can be prepared using any method known to those skilled in the art. For example, conventional mixing, dissolving, granulating, emulsifying, milling, encapsulating, entrapping and/or lyophilizing processes.
在本公开中，使用药物组合物的目的在于促进针对生物体的给药，有利于活性成分的吸收，进而发挥生物活性。本公开的药物组合物可以通过任何形式给药，包括注射(动脉内、静脉内、肌肉内、腹膜内、皮下)、粘膜、口服(口服固体制剂、口服液体制剂)、直肠、吸入、植入、局部(例如眼部)给药等。口服固体制剂的非限制性实例包括但不限于散剂、胶囊剂、锭剂、颗粒剂、片剂等。口服或粘膜给药的液体制剂的非限制性实例包括但不限于混悬剂、酊剂、酏剂、溶液剂等。局部给药制剂的非限制性实例包括但不限于乳剂、凝胶剂、软膏剂、乳膏剂、贴剂、糊剂、泡沫剂、洗剂、滴剂或血清制剂。胃肠外给药制剂的非限制性实例包括但不限于注射用溶液剂、注射用干粉剂、注射用悬浮液、注射用乳剂等。本公开的药物组合物还可以制成控制释放或延迟释放剂型(例如脂质体或微球)。In the present disclosure, the purpose of using the pharmaceutical composition is to promote the administration to organisms, facilitate the absorption of active ingredients, and then exert biological activity. The pharmaceutical compositions of the present disclosure may be administered by any form, including injection (intra-arterial, intravenous, intramuscular, intraperitoneal, subcutaneous), mucosal, oral (oral solid formulation, oral liquid formulation), rectal, inhalation, implantation , topical (eg ocular) administration, and the like. Non-limiting examples of oral solid formulations include, but are not limited to, powders, capsules, lozenges, granules, tablets, and the like. Non-limiting examples of liquid formulations for oral or mucosal administration include, but are not limited to, suspensions, tinctures, elixirs, solutions, and the like. Non-limiting examples of topical formulations include, but are not limited to, emulsions, gels, ointments, creams, patches, pastes, foams, lotions, drops, or serum formulations. Non-limiting examples of formulations for parenteral administration include, but are not limited to, solutions for injection, dry powder for injection, suspension for injection, emulsion for injection, and the like. The pharmaceutical compositions of the present disclosure can also be formulated as controlled or delayed release dosage forms (eg, liposomes or microspheres).
在本公开中，施用途经能够以任何适用的方式进行变化或调整，以满足药物的性质、患者和医务人员的便利以及其它相关因素的需求。In this disclosure, the methods of administration can be varied or adjusted in any suitable manner to meet the needs of the nature of the drug, the convenience of the patient and medical staff, and other relevant factors.
本公开上下文中使用的术语“Dxd”又称Exatecan derivative，是一种DNA拓扑异构酶I抑制剂，其具有如下所示结构：
The term "Dxd" used in the context of this disclosure, also known as Exatecan derivative, is a DNA topoisomerase I inhibitor, which has the structure shown below:
在本公开中，当L1位于式(I)所示的化合物，或位于式(II)所示的连接子中，L1为(Gly)p-(Z1)a-，并且，L1中位于氨基末端的甘氨酸残基为NH2-CH2-C(＝O)-。In the present disclosure, when L 1 is located in the compound represented by formula (I), or in the linker represented by formula (II), L 1 is (Gly) p -(Z 1 ) a -, and, L 1 The amino-terminal glycine residue in is NH 2 -CH 2 -C(=O)-.
在本公开中，当L1位于式(III)所示的抗体药物偶联物中，L1为-(Gly)p-(Z1)a-，并且，L1中位于氨基末端的甘氨酸残基为-NH-CH2-C(＝O)-。In the present disclosure, when L 1 is located in the antibody drug conjugate represented by formula (III), L 1 is -(Gly) p -(Z 1 ) a -, and the glycine residue at the amino terminal of L 1 is The group is -NH-CH 2 -C(=O)-.
抗体药物偶联物的制备方法： Preparation method of antibody drug conjugate :
本公开的抗体药物偶联物可以用本领域已知的任何方法制备。在一些实施方式中，偶联物是通过酶催化的抗体或抗原结合片段和式(I)所示的化合物特异性结合制备的，其中，抗体或抗原结合片段中包含连接酶的识别序列。示例性的，连接酶为转肽酶，其包括但不限于各种天然Sortase酶(包括A，B，C，D，L.plantarum的Sortase等)及经过优选改造的各种新型转肽酶。反应通过生物酶催化手段实现(参照WO2015165413A1，其通过引用并入本文)，反应条件温和，降低了偶联过程对抗体的物理、化学损伤，制备工艺与流程更为优化，易于产业化升级，有利于ADC产品的质量控制。The antibody drug conjugates of the present disclosure can be prepared by any method known in the art. In some embodiments, the conjugate is prepared by enzyme-catalyzed specific binding of the antibody or antigen-binding fragment to the compound represented by formula (I), wherein the antibody or antigen-binding fragment contains a recognition sequence of the ligase. Exemplarily, the ligase is a transpeptidase, which includes but not limited to various natural Sortase enzymes (including Sortase of A, B, C, D, L. plantarum, etc.) and various novel transpeptidases that have been preferably modified. The reaction is realized by means of biological enzyme catalysis (refer to WO2015165413A1, which is incorporated herein by reference), the reaction conditions are mild, the physical and chemical damage to the antibody during the coupling process is reduced, the preparation process and process are more optimized, and it is easy to upgrade industrialization. Conducive to the quality control of ADC products.
该方法包括步骤A和步骤B。The method includes step A and step B.
步骤A：制备式(I)所示的化合物Step A: prepare the compound shown in formula (I)
在一些优选的实施方式中，优选实施例中，式(I)所示化合物由连接子与艾日布林共价连接形成。In some preferred embodiments, in a preferred embodiment, the compound represented by formula (I) is formed by covalently linking a linker with eribulin.
式(I)所示的化合物具有明确的结构、确定的组成和高纯度，因此在与抗体进行偶联反应时，引入的杂质较少或不引入其他杂质。在连接酶催化下，当式(I)所示的化合物被用于和带有连接酶识别序列的抗体或抗原结合片段在特定位点发生偶联反应时，可获得质量高度可控的同质ADC。The compound represented by formula (I) has a definite structure, definite composition and high purity, so when it is coupled with an antibody, less or no other impurities are introduced. Under the catalysis of ligase, when the compound represented by formula (I) is used to react with the antibody or antigen-binding fragment with the recognition sequence of ligase at a specific site, a highly controllable homogeneous ADC.
步骤B：将靶分子与式(I)所示化合物连接Step B: linking the target molecule with the compound represented by formula (I)
本公开中的靶分子可以通过本技术已知的任何方法与式(I)所示化合物结合，得到式(III)所示的抗体药物偶联物。The target molecule in the present disclosure can be combined with the compound represented by formula (I) by any method known in the art to obtain the antibody drug conjugate represented by formula (III).
靶分子和式(I)所示化合物通过底物的连接酶特异性识别序列相互连接。识别序列取决于所使用的特定连接酶。在一些实施方式中，靶分子是一种抗体或抗原结合片段，在轻链和/或重链的c端(羧基端)引入基于识别序列的末端修饰，在野生型或优化的工程连接酶或其任何组合的催化下，靶分子 与式(I)所示化合物结合。The target molecule and the compound represented by the formula (I) are connected to each other through the specific recognition sequence of the ligase of the substrate. The recognition sequence depends on the particular ligase used. In some embodiments, the target molecule is an antibody or an antigen-binding fragment, and a recognition-sequence-based terminal modification is introduced at the c-terminus (carboxyl-terminus) of the light chain and/or heavy chain, in wild-type or optimized engineered ligase or Under the catalysis of any combination, the target molecule Combined with the compound represented by formula (I).
在一些具体的实施方式中，连接酶为Sortase酶。更具体的，连接酶为Sortase A酶。In some specific embodiments, the ligase is a Sortase enzyme. More specifically, the ligase is Sortase A enzyme.
示例性的，结合反应可以用以下方案表示：
Exemplary, the binding reaction can be represented by the following scheme:
三角形代表抗体的一部分，抗体的羧基端连接Sortase A酶的识别序列LPXT(G)r；其中，L为亮氨酸，P为脯氨酸，X为任意类型的氨基酸，T为苏氨酸，(G)r是数量为r的甘氨酸，r为1-2的任一整数。五边形表示式(II)化合物的一部分，(G)p是数量为p的甘氨酸，p为1-10的任一整数，示例性的，p为1、2、3、4、5等等。The triangle represents a part of the antibody, and the carboxy-terminal of the antibody is connected to the recognition sequence LPXT(G) r of the Sortase A enzyme; wherein, L is leucine, P is proline, X is any type of amino acid, T is threonine, (G) r is glycine in quantity r, and r is any integer of 1-2. The pentagon represents a part of the compound of formula (II), (G) p is glycine with a quantity of p, and p is any integer from 1 to 10. Exemplarily, p is 1, 2, 3, 4, 5, etc. .
Sortase A酶亲核攻击中LPXT(G)r序列中苏氨酸(T)与甘氨酸(G)之间的肽键，形成共价硫代中间体。该中间体可捕获中的甘氨酸接头(G)p，并在苏氨酸(T)和甘氨酸(G)之间重新形成酰胺键，释放出Sortase A酶，得到靶分子与式(I)所示化合物结合的抗体药物偶联物 Sortase A enzyme nucleophilic attack The peptide bond between threonine (T) and glycine (G) in the r- sequence of LPXT(G) in LPXT(G) forms a covalent thio intermediate. This intermediate can capture Glycine linker (G) p in , and re-form an amide bond between threonine (T) and glycine (G), release the Sortase A enzyme, and obtain an antibody drug in which the target molecule is combined with the compound represented by formula (I) Conjugate
在下面将对本公开进行详细描述。然而，本公开可能具体体现为许多不同的形式，而且它不应该被局限于此处所描述的实施例中，提供这些实施例中的目的是使所披露内容更完整与全面。所用试剂和原料，除了提供制备方法的除外，其余均为市售。除非另有定义，否则本文中所有科技术语具有的含义与权利要求主题所属技术领域人员通常理解的含义相同。The present disclosure will be described in detail below. However, this disclosure may be embodied in many different forms, and it should not be limited to the embodiments described herein, which are provided so that this disclosure will be more complete and comprehensive. All the reagents and raw materials used are commercially available except those provided for the preparation methods. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the claimed subject matter belongs.
本公开中氨基酸及缩写和英文简称如下表所示：Amino acids and their abbreviations and English abbreviations in this disclosure are shown in the following table:
表1
Table 1
本发明所用的实验室试剂均来自于商业化购买，纯度均为分析纯。合成过程中所用的试剂缩写及中文全名如下表所示： The laboratory reagents used in the present invention are all from commercial purchases, and the purity is all analytical pure. The abbreviations and full Chinese names of the reagents used in the synthesis process are shown in the table below:
表2
Table 2
实施例Example
本公开的其他目的、特征和优点将从以下详细描述中变得明显。但是，应当理解的是，详细描述和具体实施例(虽然表示本公开的具体实施方式)仅为解释性目的而给出，因为在阅读该详细说明后，在本公开的精神和范围内所作出的各种改变和修饰，对于本领域技术人员来说将变得显而易见。Other objects, features and advantages of the present disclosure will become apparent from the following detailed description. It should be understood, however, that the detailed description and specific examples, while indicating specific embodiments of the disclosure, are given for illustrative purposes only, since, upon reading this detailed description, all further changes will be made within the spirit and scope of the disclosure. Various changes and modifications will become apparent to those skilled in the art.
除非有特别说明，否则本公开中采用的所有试剂和原料均可以通过商业渠道购买。All reagents and materials used in this disclosure were purchased commercially unless otherwise noted.
实施例1 BP182a的合成The synthesis of embodiment 1 BP182a
化合物BP182a具有如下式(I-5)所示的结构：
Compound BP182a has the structure shown in the following formula (I-5):
化合物BP182a的合成步骤如下所示：
The synthetic steps of compound BP182a are as follows:
步骤1，1186k的合成：1186k以Fmoc固相合成方法合成，以Rink Amide MBHA Resin为固相载体，用20％哌啶/DMF(v/v)溶液脱除Fmoc保护，再以HOBT/DIC为缩合体系，DMF为反应溶剂，依次偶 联Fmoc-Cys(Trt)-OH、Fmoc-NH-PEG4-PA、Fmoc-Gly-OH、Fmoc-Gly-OH、Fmoc-Gly-OH，其中Fmoc-NH-PEG4-PA采用HBTU/DIEA体系缩合，然后经TFA∶TIS∶H2O＝95∶2.5∶2.5裂解，甲基叔丁基醚沉淀，过滤、干燥，得1186k粗品，最后经高效液相色谱纯化、脱盐，然后浓缩、冻干、甲基叔丁基醚打浆、干燥，得1186k纯品。Step 1, synthesis of 1186k: 1186k was synthesized by Fmoc solid-phase synthesis method, using Rink Amide MBHA Resin as a solid-phase carrier, using 20% piperidine/DMF (v/v) solution to remove Fmoc protection, and then using HOBT/DIC as Condensation system, DMF as reaction solvent, followed by coupling Combined Fmoc-Cys(Trt)-OH, Fmoc-NH-PEG4-PA, Fmoc-Gly-OH, Fmoc-Gly-OH, Fmoc-Gly-OH, where Fmoc-NH-PEG4-PA is condensed by HBTU/DIEA system , and then cracked by TFA:TIS:H 2 O=95:2.5:2.5, methyl tert-butyl ether was precipitated, filtered and dried to obtain 1186k crude product, which was finally purified by high performance liquid chromatography, desalted, concentrated, freeze-dried, MTBE was beaten and dried to obtain 1186k pure product.
步骤2，BP102c05、BP102c的合成：BP102c05、BP102c的合成参考EP0624377A2的合成方法，EP0624377A2通过引用并入本申请；Step 2, synthesis of BP102c05 and BP102c: for the synthesis of BP102c05 and BP102c, refer to the synthesis method of EP0624377A2, which is incorporated into this application by reference;
步骤3，化合物BP182a11的合成：向烧瓶中加入687mg(1.2eq)BP102c，565mg(1.0eq)艾日布林，6ml DMF，搅拌溶解，加入162ul(1.2eq)DIEA，TLC监控反应完毕后将反应液倒入至TBME中，固体析出，搅拌打浆后过滤、滤饼用TBME洗涤，收集滤饼，真空干燥得类白色固体1021mg即BP182a11，样品不经纯化，直接进行下一步反应。Step 3, the synthesis of compound BP182a11: add 687mg (1.2eq) BP102c, 565mg (1.0eq) eribulin, 6ml DMF to the flask, stir and dissolve, add 162ul (1.2eq) DIEA, TLC monitors the reaction after the completion of the reaction The solution was poured into TBME, the solid precipitated out, stirred and beaten, filtered, the filter cake was washed with TBME, the filter cake was collected, and vacuum-dried to obtain 1021 mg of off-white solid, namely BP182a11, the sample was directly carried out to the next step reaction without purification.
步骤4，化合物BP182a的合成：向烧瓶中加入402mg(1.0eq)BP182a11，16ml甲醇，搅拌溶清；称取236mg(1.2eq)1186k，用1ml纯化水溶解，并用碳酸氢钠溶液调节pH至5-6，然后加入至BP182a11的反应瓶中，30min后HPLC监控反应完毕，反应液不处理，经反向高效液相制备纯化，收集纯品，冻干得166mg白色固体，即BP182a。LC/MS(m/z)：calcd for C89H135N13O28S 1865.93；found 1867.00[M+H]+、934.10[M+2H]2+、623.30[M+3H]3+。Step 4, the synthesis of compound BP182a: add 402mg (1.0eq) BP182a11, 16ml methanol to the flask, stir to dissolve; weigh 236mg (1.2eq) 1186k, dissolve it with 1ml purified water, and adjust the pH to 5 with sodium bicarbonate solution -6, and then added to the reaction bottle of BP182a11. After 30 minutes, the reaction was monitored by HPLC. The reaction solution was not treated, but was prepared and purified by reverse high performance liquid phase, and the pure product was collected and freeze-dried to obtain 166 mg of white solid, namely BP182a. LC/MS (m/z): calcd for C 89 H 135 N 13 O 28 S 1865.93; found 1867.00 [M+H] + , 934.10 [M+2H] 2+ , 623.30 [M+3H] 3+ .
实施例2 BP182d的合成The synthesis of embodiment 2 BP182d
化合物BP182d具有如下式(I-1)所示的结构：
Compound BP182d has the structure shown in the following formula (I-1):
化合物BP182d的合成步骤如下所示：
The synthetic steps of compound BP182d are as follows:
步骤1，BP182d01的合成：以Fmoc固相合成方法合成，以2ClTrt Resin为固相载体，用20％哌啶/DMF(v/v)溶液脱除Fmoc保护，再以HOBT/DIC为缩合体系，DMF为反应溶剂，依次偶联Fmoc-Gly-OH、Fmoc-Phe-OH、Fmoc-Gly-OH、Fmoc-Gly-OH、Fmoc-AEEA-OH、Fmoc-Gly-OH、Fmoc-Gly-OH、Fmoc-Gly-OH，然后经2％TFA/DCM溶液切割，甲基叔丁基醚沉淀，离心、干燥，得BP182d01，不经纯化，直接进行下一步反应。Step 1, the synthesis of BP182d01: synthesized by Fmoc solid-phase synthesis method, using 2ClTrt Resin as a solid-phase carrier, using 20% piperidine/DMF (v/v) solution to remove Fmoc protection, and then using HOBT/DIC as a condensation system, DMF is the reaction solvent, sequentially coupled Fmoc-Gly-OH, Fmoc-Phe-OH, Fmoc-Gly-OH, Fmoc-Gly-OH, Fmoc-AEEA-OH, Fmoc-Gly-OH, Fmoc-Gly-OH, Fmoc-Gly-OH was then cleaved with 2% TFA/DCM solution, precipitated with methyl tert-butyl ether, centrifuged and dried to obtain BP182d01, which was directly carried out to the next step without purification.
步骤2，BP182d02的合成：向烧瓶中加入501mg(1.0eq)BP182d01，415mg(1.0eq)艾日布林，10mlDCM、4ml DMF，搅拌溶清后降温至5±5℃，加入237ul(2.5eq)DIEA，加入140ul(1.5eq)DEPC，升至室温反应，TLC监控反应完毕后将反应液真空浓缩除去DCM，然后加入TBME，搅拌打浆，过滤、滤饼用TBME洗涤，收集滤饼，真空干燥得类白色固体1.023g，不经纯化，直接进行下一步反应。Step 2, synthesis of BP182d02: add 501mg (1.0eq) BP182d01, 415mg (1.0eq) eribulin, 10ml DCM, 4ml DMF to the flask, stir to dissolve and cool to 5±5°C, add 237ul (2.5eq) DIEA, add 140ul (1.5eq) DEPC, rise to room temperature to react, after TLC monitors the reaction, concentrate the reaction solution in vacuo to remove DCM, then add TBME, stir to make a slurry, filter, wash the filter cake with TBME, collect the filter cake, and vacuum dry to obtain 1.023 g of off-white solid was directly carried out to the next reaction without further purification.
步骤3，BP182d的合成：向烧瓶中加入1000mg BP182d02，7ml甲醇，7ml THF，搅拌溶清；称取300mg LiOH.H2O，用10ml纯化水溶解后滴加至反应瓶中，调节pH至12，TLC监控反应完毕后加入醋酸淬灭反应，将反应液真空浓缩除去有机溶剂，得BP182d粗品，粗品经正向高效液相制备纯化，收集纯品，浓缩至干得195mg类白色固体，即BP182d。LC/MS(m/z)：calcd for C67H97N9O21 1363.68；found 1364.70[M+H]+、682.85[M+2H]2+、1362.75[M-H]-。Step 3, synthesis of BP182d: add 1000mg BP182d02, 7ml methanol, 7ml THF to the flask, stir to dissolve; weigh 300mg LiOH.H 2 O, dissolve it with 10ml purified water, add it dropwise to the reaction flask, adjust the pH to 12 After the reaction was monitored by TLC, acetic acid was added to quench the reaction, the reaction solution was concentrated in vacuo to remove the organic solvent, and the crude product of BP182d was obtained. The crude product was prepared and purified by forward high performance liquid phase, and the pure product was collected and concentrated to dryness to obtain 195 mg of off-white solid, namely BP182d . LC/MS (m/z): calcd for C 67 H 97 N 9 O 21 1363.68; found 1364.70 [M+H] + , 682.85 [M+2H] 2+ , 1362.75 [MH] − .
实施例3 式I-7化合物、式I-8化合物的制备

Embodiment 3 Preparation of formula I-7 compound, formula I-8 compound

在BP182a11与1186k偶联完成后，在反应液中加入LiOH水溶液，发生开环反应，制得式I-7化合物、式I-8化合物的混合物，经HPLC制备纯化后冻干得式I-7化合物、式I-8化合物的混合物。After the coupling of BP182a11 and 1186k is completed, LiOH aqueous solution is added to the reaction solution, and a ring-opening reaction occurs to obtain a mixture of the compound of formula I-7 and the compound of formula I-8, which is purified by HPLC and lyophilized to obtain formula I-7 Compounds, mixtures of compounds of formula I-8.
实施例4 式I-9化合物、式I-10化合物的制备
The preparation of embodiment 4 formula I-9 compound, formula I-10 compound
Linker Gly-Gly-Gly-PEG4-PA-Cys的合成采用2Cl-Trt树脂，合成及纯化方法见1186k的制备方法。偶联及开环反应使用式I-7化合物、式I-8化合物的制备方法制得I-9化合物、式I-10化合物混合物。Linker Gly-Gly-Gly-PEG4-PA-Cys was synthesized using 2Cl-Trt resin. For the synthesis and purification methods, see the preparation method of 1186k. Coupling and ring-opening reaction The preparation method of the compound of formula I-7 and the compound of formula I-8 is used to prepare the mixture of the compound of I-9 and the compound of formula I-10.
实施例5：抗体药物偶联物的制备Embodiment 5: Preparation of antibody drug conjugate
1、抗人ErbB2/Her2抗体的生产、纯化及鉴定：1. Production, purification and identification of anti-human ErbB2/Her2 antibody:
抗体Ab0100重链的羧基端连接有GALPETGG(以下称为Ab0100-HCCTL)，其氨基酸序列如SEQ ID NO：16所示；抗体Ab0100轻链的羧基端连接有GALPETGG(以下称为Ab0100-LCCTL)，其氨基酸序列如SEQ ID NO：15所示。因此，在抗体Ab0100中共引入有4个连接酶的识别序列。抗体Ab0100的合成可参考WO1989006692A1，WO1989006692通过引用并入本公开。 The carboxy-terminal of the heavy chain of antibody Ab0100 is connected with GALPETGG (hereinafter referred to as Ab0100-HCCT L ), and its amino acid sequence is shown in SEQ ID NO: 16; the carboxy-terminal of the light chain of antibody Ab0100 is connected with GALPETGG (hereinafter referred to as Ab0100-LCCT L ), the amino acid sequence of which is shown in SEQ ID NO:15. Therefore, a total of 4 ligase recognition sequences were introduced into antibody Ab0100. The synthesis of antibody Ab0100 can refer to WO1989006692A1, and WO1989006692 is incorporated into the present disclosure by reference.
表3 抗体Ab0100轻链的序列信息
Table 3 Sequence information of antibody Ab0100 light chain
表4 抗体Ab0100重链的序列信息
Table 4 Sequence information of antibody Ab0100 heavy chain
2、偶联抗体与式(I-1)或式(I-5)所示的化合物，偶联反应的方法可已参考WO2015165413A1中的方法，具体如下：2. To couple the antibody with the compound represented by formula (I-1) or formula (I-5), the method of coupling reaction can refer to the method in WO2015165413A1, as follows:
(1)以BP182a所示的化合物为中间体，以上述抗人ErbB2/Her2为抗体，通过Sortase A酶促连接的方法制备得到的ADC，命名为Ab0100-H0037。Ab0100-H0037的具体结构如下式(III-5)所示为：
(1) Using the compound represented by BP182a as an intermediate and the above-mentioned anti-human ErbB2/Her2 as an antibody, the ADC prepared by enzymatic linking with Sortase A is named Ab0100-H0037. The specific structure of Ab0100-H0037 is shown in the following formula (III-5):
(2)以BP182d所示的化合物为中间体，以上述抗人ErbB2/Her2为抗体，通过Sortase a酶促连接的方法制备得到的ADC，命名为Ab0100-H0038。Ab0100-H0038的具体结构如下式(III-1)所示为：
(2) Using the compound represented by BP182d as an intermediate and the above-mentioned anti-human ErbB2/Her2 as an antibody, the ADC prepared by enzymatic linking with Sortase a is named Ab0100-H0038. The specific structure of Ab0100-H0038 is shown in the following formula (III-1):
实施例6：LB302-2-4的合成Example 6: Synthesis of LB302-2-4
本实施例制备如下述结构所示的化合物LB302-2-4：
This example prepares the compound LB302-2-4 shown in the following structure:
化合物LB302-2-4的制备步骤如下所示：The preparation steps of compound LB302-2-4 are as follows:
步骤1，连接剂HX20111的制备：
Step 1, preparation of linker HX20111:
用Rink-amide-MBHA-树脂采用传统固相多肽合成方法合成HX20111。用Fmoc保护连接单元中的氨基酸。偶联试剂从HOBT、HOAt/DIC、DCC、EDCI或HATU中选择。合成后，用三氟乙酸裂解树脂。产品经高效液相色谱纯化，冻干后保存。理论质量：1383.70，测量：[M-H]-＝1382.6。HX20111 was synthesized by traditional solid-phase peptide synthesis method with Rink-amide-MBHA-resin. Amino acids in the linker unit were protected with Fmoc. Coupling reagents are selected from HOBT, HOAt/DIC, DCC, EDCI or HATU. After synthesis, the resin was cleaved with trifluoroacetic acid. The product was purified by high performance liquid chromatography and stored after freeze-drying. Theoretical mass: 1383.70, measured: [MH] - = 1382.6.
步骤2，化合物LB302-2-4的制备：
Step 2, preparation of compound LB302-2-4:
称取HX20111和中间物MC-GGFG-Dxd(摩尔比～1∶2)分别溶于水和DMF中，充分混合得到混合物，在0-40℃下反应0.5-30h。反应完成后，直接加入适量的Tris Base溶液或其他促进开环反应的溶 液，在0-40℃下再进行0.2-20h的反应。反应完成后，用半制备/制备型HPLC对产物进行纯化，并冻干得到连接剂-有效载荷中间体LB302-2-4。理论质量：3486.52，实测：[(M+3H)/3]+＝1163.3。Weigh HX20111 and the intermediate MC-GGFG-Dxd (molar ratio ~1:2) and dissolve them in water and DMF respectively, mix thoroughly to obtain a mixture, and react at 0-40°C for 0.5-30h. After the reaction is completed, directly add an appropriate amount of Tris Base solution or other solutions that promote the ring-opening reaction. solution, and then react at 0-40°C for 0.2-20h. After the reaction was completed, the product was purified by semi-preparative/preparative HPLC and lyophilized to obtain the linker-payload intermediate LB302-2-4. Theoretical mass: 3486.52, measured: [(M+3H)/3] + =1163.3.
实施例7：抗体药物偶联物BQ3的制备Example 7: Preparation of Antibody Drug Conjugate BQ3
采用实施例4中所示的方法，向抗人ErbB2/Her2抗体Ab0001上偶联化合物LB302-2-4，得到的ADC信息如下表所示。其中，抗体Ab0001的重链氨基酸序列如SEQ ID NO：19所示，抗体Ab0001的轻链的羧基端连接有GALPETGG(以下简称为Ab0001-LCCTL)，其氨基酸序列如SEQ ID NO：15所示。Using the method shown in Example 4, the compound LB302-2-4 was coupled to the anti-human ErbB2/Her2 antibody Ab0001, and the obtained ADC information is shown in the table below. Wherein, the amino acid sequence of the heavy chain of antibody Ab0001 is shown in SEQ ID NO: 19, and the carboxy-terminal of the light chain of antibody Ab0001 is connected with GALPETGG (hereinafter referred to as Ab0001-LCCT L ), and its amino acid sequence is shown in SEQ ID NO: 15 .
表5
table 5
表6 抗体Ab0001的重链和轻链的氨基酸信息
Table 6 Amino acid information of heavy chain and light chain of antibody Ab0001
实施例8：体外实验验证ADC的细胞毒性Example 8: In vitro experiments verify the cytotoxicity of ADC
1.1材料与设备1.1 Materials and equipment
本实验例采用的材料与设备如下表所示。The materials and equipment used in this experiment example are shown in the table below.
表7
Table 7
1.2实验步骤1.2 Experimental steps
1.2.1细胞铺板1.2.1 Cell plating
(1)细胞镜检；(1) Microscopic examination of cells;
(2)清洗；(2) cleaning;
(3)消化；(3) digestion;
(3)离心；(3) centrifugal;
(4)细胞计数；(4) cell count;
(5)细胞铺板；其中NCI-N87和SK-BR-3是5000个细胞/孔，HCC1954和MDA-MB-468是4000/孔。(5) Cell plating: 5000 cells/well for NCI-N87 and SK-BR-3, 4000 cells/well for HCC1954 and MDA-MB-468.
1.2.2细胞给药1.2.2 Cell administration
(1)镜检(1) Microscopic examination
(2)药物配制：(2) Drug preparation:
a.样品配置缓冲液配置：将给药细胞所用培养基(10％FBS)配置所需量的样品配置缓冲液；a. Sample configuration buffer configuration: configure the required amount of sample configuration buffer in the culture medium (10% FBS) used for the administered cells;
b.样品配置：按照第一个浓度所需浓度的倍比稀释样品，24孔板或96孔板中按照下表进行配置；b. Sample configuration: Dilute the sample according to the ratio of the concentration required for the first concentration, and configure according to the following table in a 24-well plate or a 96-well plate;
表8
Table 8
(3)加药：从低浓度到高浓度依次加入不同浓度药物，每个浓度设3个复孔，100μl/well，其中puro组为3个复孔，100μl/well。(3) Dosing: Drugs of different concentrations were added sequentially from low concentration to high concentration, with 3 replicate wells for each concentration, 100 μl/well, among which the puro group had 3 replicate wells, 100 μl/well.
(4)药物加完后，将细胞移至培养箱内继续培养120h。培养条件：37度，CO2培养箱中。(4) After adding the drug, the cells were moved to the incubator to continue culturing for 120 h. Culture conditions: 37 degrees, CO 2 incubator.
1.2.3细胞活力检测1.2.3 Cell Viability Detection
(1)检测试剂准备：提前将CellTiter-Glo Luminescent Cell Viability Assay检测试剂避光平衡至室温。(1) Detection reagent preparation: Equilibrate the CellTiter-Glo Luminescent Cell Viability Assay detection reagent to room temperature in the dark in advance.
(2)细胞准备：将待测细胞从培养箱内取出，25℃平衡30min。(2) Cell preparation: take the cells to be tested out of the incubator, and equilibrate at 25° C. for 30 minutes.
(3)ATP检测：弃掉培养基，加入100μl/孔DMEM，50μl/孔CTG加入96孔板中，铝箔纸避光保存，涡旋振荡仪200rpm室温震荡15min(注意：此时可以进行酶标仪程序设置)。(3) ATP detection: Discard the medium, add 100 μl/well DMEM, 50 μl/well CTG into a 96-well plate, store in the dark with aluminum foil, vortex shaker at 200 rpm for 15 minutes at room temperature (note: enzyme labeling can be performed at this time) instrument program settings).
(4)检测程序：增益值为135，设置好程序后，将黑壁底透板去盖，按照仪器指定方式放置。(4) Detection procedure: the gain value is 135. After setting the procedure, remove the cover of the black wall bottom plate and place it in the way specified by the instrument.
(5)数据保存(5) Data storage
1.3实验结果1.3 Experimental results
图1示出了Ab0100-H0038、Ab0100-H0037、BQ3，与小分子药物艾日布林、Dxd对人乳腺癌细胞HCC1954的细胞毒性检测结果。其中，各药物分子的IC50值如下表所示： Figure 1 shows the cytotoxicity detection results of Ab0100-H0038, Ab0100-H0037, BQ3, and small molecule drugs Eribulin and Dxd on human breast cancer cell HCC1954. Among them, the IC50 value of each drug molecule is shown in the table below:
表9
Table 9
图2示出了Ab0100-H0038、Ab0100-H0037、BQ3，与小分子药物艾日布林、Dxd对人乳腺癌细胞SK-BR-3的细胞毒性检测结果。其中，各药物分子的IC50值如下表所示：Figure 2 shows the cytotoxicity detection results of Ab0100-H0038, Ab0100-H0037, BQ3, small molecule drugs Eribulin and Dxd on human breast cancer cell SK-BR-3. Among them, the IC50 value of each drug molecule is shown in the table below:
表10
Table 10
图3示出了Ab0100-H0038、Ab0100-H0037、BQ3，与小分子药物艾日布林、Dxd对人胃癌细胞NCI-N87的细胞毒性检测结果。其中，各药物分子的IC50值如下表所示：Figure 3 shows the cytotoxicity detection results of Ab0100-H0038, Ab0100-H0037, BQ3, and small molecule drugs Eribulin and Dxd on human gastric cancer cell NCI-N87. Among them, the IC50 value of each drug molecule is shown in the table below:
表11
Table 11
图4示出了Ab0100-H0038、Ab0100-H0037、BQ3，与小分子药物艾日布林、Dxd对人乳腺癌细胞MDA-MB-468的细胞毒性检测结果。其中，各药物分子的IC50值如下表所示：Figure 4 shows the cytotoxicity detection results of Ab0100-H0038, Ab0100-H0037, BQ3, and small molecule drugs Eribulin and Dxd on human breast cancer cell line MDA-MB-468. Among them, the IC50 value of each drug molecule is shown in the table below:
表12
Table 12
如图1-3所示，Ab0100-H0037和Ab0100-H0038能够显著降低HER2阳性(例如，HCC1954、SK-BR-3和NCI-N87)的各类肿瘤细胞的体外存活率，发挥高效的肿瘤细胞杀伤活性，说明本公开中制备抗体药物偶联物对HER2阳性的肿瘤细胞具有有效的杀伤作用。如图4所示，在HER2阴性细胞中，Ab0100-H0037和Ab0100-H0038的细胞毒性低于小分子药物艾日布林。As shown in Figures 1-3, Ab0100-H0037 and Ab0100-H0038 can significantly reduce the in vitro survival rate of various types of tumor cells that are HER2 positive (for example, HCC1954, SK-BR-3 and NCI-N87), exerting high-efficiency tumor cell The killing activity indicates that the antibody-drug conjugate prepared in the present disclosure has an effective killing effect on HER2-positive tumor cells. As shown in Figure 4, in HER2-negative cells, the cytotoxicity of Ab0100-H0037 and Ab0100-H0038 was lower than that of the small molecule drug eribulin.
Ab0100-H0037和Ab0100-H0038对HER2阳性细胞HCC1954、SK-BR-3、NCI-N87的细胞毒性优于艾日布林，显著优于Dxd，说明本公开中的通过特殊结构的连接子偶联抗体与细胞毒性药物艾日布林得到的ADC药物，具有提高的肿瘤细胞的杀伤效果。进一步的，Ab0100-H0037和Ab0100-H0038 在HER2阳性细胞中的细胞毒性，明显优于在HER2阴性细胞中的细胞毒性，说明Ab0100-H0037和Ab0100-H0038能够靶向结合HER2阳性表达的细胞并进入细胞内部，通过在细胞内释放细胞毒性药物艾日布林，实现对HER2阳性细胞的特异性、高效杀伤，具有较好的靶向性和安全性。The cytotoxicity of Ab0100-H0037 and Ab0100-H0038 to HER2-positive cells HCC1954, SK-BR-3, and NCI-N87 is better than that of eribulin, and significantly better than that of Dxd, indicating that the linker coupling through a special structure in this disclosure The ADC drug obtained from the antibody and the cytotoxic drug eribulin has an improved killing effect on tumor cells. Further, Ab0100-H0037 and Ab0100-H0038 The cytotoxicity in HER2-positive cells is significantly better than that in HER2-negative cells, indicating that Ab0100-H0037 and Ab0100-H0038 can target cells with HER2-positive expression and enter the cells, releasing cytotoxicity in cells Eribulin, a drug, achieves specific and efficient killing of HER2-positive cells, and has good targeting and safety.
实施例9：体内实验验证ADC的活性和毒性Example 9: In vivo experiments verify the activity and toxicity of ADC
2.1实验动物：2.1 Experimental animals:
商业购买的BALB/c裸鼠，于恒温恒湿条件下饲养。Commercially purchased BALB/c nude mice were raised under constant temperature and humidity conditions.
2.2实验方法和步骤2.2 Experimental methods and steps
2.2.1细胞培养2.2.1 Cell culture
于37℃下和含5％CO2的空气中，在补充有20％胎牛血清和1％抗生素-抗真菌剂的IMDM培养基中体外保存Capan-1肿瘤细胞(ATCC，目录号HTB-79)。通过胰蛋白酶-EDTA处理，每周两次对肿瘤细胞进行常规传代培养。收获处于指数生长期的细胞，并计数用于肿瘤接种。Capan-1 tumor cells were preserved in vitro in IMDM medium supplemented with 20% fetal calf serum and 1% antibiotic-antimycotic at 37°C in an atmosphere of 5% CO2 (ATCC, catalog number HTB-79 ). Tumor cells were routinely subcultured twice a week by trypsin-EDTA treatment. Cells in exponential growth phase were harvested and counted for tumor inoculation.
2.2.2肿瘤接种和动物分组2.2.2 Tumor inoculation and animal grouping
对每只小鼠的右侧皮下接种在0.2mL PBS和Matrigel(PBS∶Matrigel＝1∶1)混合物中的Capan-1肿瘤细胞(5×106)，用于肿瘤发育。在肿瘤接种后第5天，当平均肿瘤体积达到187mm3时，开始治疗。使用基于动物的肿瘤体积对其进行分层随机化的基于Excel的随机化软件将动物分配至各组中。每组由6只荷瘤小鼠组成，对小鼠给予供试品。Capan-1 tumor cells (5×10 6 ) in a mixture of 0.2 mL of PBS and Matrigel (PBS:Matrigel=1:1) were subcutaneously inoculated on the right side of each mouse for tumor development. Treatment was initiated on day 5 after tumor inoculation when the average tumor volume reached 187 mm 3 . Animals were allocated into groups using Excel-based randomization software that stratified randomization based on the animals' tumor volume. Each group consists of 6 tumor-bearing mice, and the mice are given the test substance.
2.2.3供试品制备2.2.3 Preparation of the test article
供试品制备方法如下表所示：The preparation method of the test sample is shown in the table below:
表13
Table 13
注：在给药前现制现配。Note: It is prepared and prepared before administration.
2.2.4肿瘤测量和终点2.2.4 Tumor Measurements and Endpoints
主要终点是确定肿瘤生长是否可被延迟或小鼠是否可被治愈。使用游标卡尺在两个维度上进行肿瘤体积测量，每周两次，并使用如下公式以mm3表示所述体积：V＝0.5a×b2，其中a和b分别是肿瘤的长径和短径。The primary endpoint was to determine whether tumor growth could be delayed or whether the mice could be cured. Tumor volume measurements were performed in two dimensions using vernier calipers, twice a week, and the volume was expressed in mm 3 using the following formula: V=0.5a×b 2 , where a and b are the long and short diameters of the tumor, respectively .
2.3实验结果与讨论2.3 Experimental results and discussion
2.3.1死亡率、患病率和体重增加或减轻2.3.1 Mortality, morbidity and weight gain or loss
作为毒性的间接测量指标，定期监测动物体重。Animal body weights were monitored regularly as an indirect measure of toxicity.
图5示出了溶媒(DPBS)、Ab0100-H0037(5mg/kg)、Ab0100-H0038(5mg/kg)，以及BQ3(5mg/kg)给药后荷瘤小鼠(Capan-1荷瘤雌性BALB/c裸鼠)的体重变化检测结果。其中，图5中基于给药第一天的动物体重计算变化，以体重百分比变化(％)显示；数据点代表BW的百分比组均值变化。误差线表示均值的标准误(SEM)。图5示出了在实验过程中，该模型的所有组中均未见显著体重减轻，未发生死亡和发病，Ab0100-H0037和Ab0100-H0038在动物体内实验中未显示明显药物毒性。Figure 5 shows vehicle (DPBS), Ab0100-H0037 (5mg/kg), Ab0100-H0038 (5mg/kg), and BQ3 (5mg/kg) after administration of tumor-bearing mice (Capan-1 tumor-bearing female BALB /c nude mice) body weight change detection results. Wherein, in Fig. 5, the calculated change based on the body weight of the animals on the first day of administration is shown in percentage change (%) of body weight; the data points represent the percentage group mean change of BW. Error bars represent standard error of the mean (SEM). Figure 5 shows that during the experiment, no significant weight loss, no death and no disease occurred in all groups of the model, and Ab0100-H0037 and Ab0100-H0038 did not show obvious drug toxicity in animal experiments.
2.3.2肿瘤体积 2.3.2 Tumor volume
接受Ab0100-H0037、Ab0100-H0038和BQ3给药的Capan-1异种移植物荷瘤雌性BALB/c裸鼠中平均肿瘤体积随时间的变化显示在图6中。溶媒组的平均肿瘤体积达到1,420mm3时，在试验条件下第42天终止整个研究。The mean tumor volume over time in Capan-1 xenograft tumor-bearing female BALB/c nude mice administered Ab0100-H0037, Ab0100-H0038 and BQ3 is shown in FIG. 6 . The entire study was terminated on day 42 under experimental conditions when the mean tumor volume in the vehicle group reached 1,420 mm 3 .
2.3.3肿瘤生长抑制分析2.3.3 Tumor growth inhibition analysis
基于第42天的肿瘤体积测量值，计算Ab0100-H0037、Ab0100-H0038和BQ3对接种Capan-1荷瘤小鼠的肿瘤生长抑制，结果如下表14所示：Based on the tumor volume measurements on day 42, the tumor growth inhibition of Ab0100-H0037, Ab0100-H0038 and BQ3 on mice inoculated with Capan-1 tumors was calculated, and the results are shown in Table 14 below:
表14
Table 14
注：Note:
1.平均值±SEM；1. Mean ± SEM;
2.使用以下公式计算每组的相对T/C：T/C％＝TRTV/CRTV×100％(TRTV：治疗组RTV；CRTV：同一天的对照组RTV)。RTV＝V42/V0，其中V0为治疗第一天的平均肿瘤体积，V42为治疗开始后第42天的平均肿瘤体积；2. The relative T/C of each group was calculated using the following formula: T/C %=T RTV /C RTV ×100% (T RTV : RTV of the treatment group; C RTV : RTV of the control group on the same day). RTV=V 42 /V 0 , where V 0 is the average tumor volume on the first day of treatment, and V 42 is the average tumor volume on the 42nd day after the start of treatment;
3.TGI(％)＝[1-(T42-T0)/(V42-V0)]×100％；3. TGI(%)=[1-(T 42 -T 0 )/(V 42 -V 0 )]×100%;
4.基于肿瘤体积计算p值。4. Calculation of p-values based on tumor volume.
由表14结果可知，Ab0100-H0037、Ab0100-H0038和BQ3能够对荷瘤小鼠的肿瘤生长产生明显抑制效果，实现对体内肿瘤细胞的高效特异性杀伤。From the results in Table 14, it can be seen that Ab0100-H0037, Ab0100-H0038 and BQ3 can significantly inhibit tumor growth in tumor-bearing mice, and achieve efficient and specific killing of tumor cells in vivo.
2.3.4肿瘤生长曲线2.3.4 Tumor growth curve
图6示出了溶媒(DPBS)、Ab0100-H0037(5mg/kg)、Ab0100-H0038(5mg/kg)，以及BQ3(5mg/kg)给药后荷瘤小鼠的肿瘤生长曲线。Figure 6 shows the tumor growth curves of tumor-bearing mice after administration of vehicle (DPBS), Ab0100-H0037 (5 mg/kg), Ab0100-H0038 (5 mg/kg), and BQ3 (5 mg/kg).
在本实验例中，在皮下接种人胰腺癌细胞Capan-1的荷瘤小鼠模型中评价了Ab0100-H0037、Ab0100-H0038和BQ3的药物毒性及肿瘤治疗效果。结果显示：溶媒对照组的平均肿瘤体积在治疗开始后第42天达到1,420mm3。5mg/kg Ab0100-H0037、Ab0100-H0038和BQ3的所有治疗均显著抑制Capan-1肿瘤生长。在第42天，Ab0100-H0037 5mg/kg(T/C＝10.59％，TGI＝102.98％，p＝0.025)、Ab0100-H00385mg/kg(T/C＝0.75％，TGI＝114.29％，p＝0.018)和BQ35mg/kg(T/C＝2.19％，TGI＝112.62％，p＝0.019)组的平均肿瘤体积分别为150、11和31mm3。Capan-1荷瘤BALB/c裸鼠中，所有供试品，包括Ab0100-H0037、Ab0100-H0038和BQ3均耐受良好。在所有治疗组中均未观察到显著的平均体重减轻。In this experimental example, the drug toxicity and tumor therapeutic effect of Ab0100-H0037, Ab0100-H0038 and BQ3 were evaluated in a tumor-bearing mouse model subcutaneously inoculated with human pancreatic cancer cell Capan-1. The results showed that the average tumor volume of the vehicle control group reached 1,420 mm 3 on the 42nd day after the start of treatment. All treatments with 5 mg/kg Ab0100-H0037, Ab0100-H0038 and BQ3 significantly inhibited Capan-1 tumor growth. On day 42, Ab0100-H0037 5 mg/kg (T/C=10.59%, TGI=102.98%, p=0.025), Ab0100-H0038 5 mg/kg (T/C=0.75%, TGI=114.29%, p=0.018 ) and BQ35mg/kg (T/C=2.19%, TGI=112.62%, p=0.019) groups had mean tumor volumes of 150, 11 and 31 mm 3 , respectively. All tested products, including Ab0100-H0037, Ab0100-H0038 and BQ3, were well tolerated in Capan-1 tumor-bearing BALB/c nude mice. No significant mean body weight loss was observed in any treatment group.
实验例10：体内实验验证ADC的活性和毒性Experimental example 10: In vivo experiment to verify the activity and toxicity of ADC
3.1实验方法与步骤：3.1 Experimental methods and steps:
采用和实验例2相同的实验条件，研究在雌性BALB/c裸鼠皮下NCI-N87人胃癌异种移植模型中Ab0100-H0037、Ab0100-H0038和BQ3抗肿瘤疗效。Using the same experimental conditions as in Experimental Example 2, the antitumor efficacy of Ab0100-H0037, Ab0100-H0038 and BQ3 in the subcutaneous NCI-N87 human gastric cancer xenograft model in female BALB/c nude mice was studied.
雌性BALB/c裸鼠皮下NCI-N87人胃癌异种移植模型建立方法如下：在37℃下含有5％CO2的空气中，在补充有10％胎牛血清和1％抗生素-抗真菌剂的RPMI-1640培养基中，将NCI-N87肿瘤细胞(ATCC，Manassas，VA，目录号CRL-5822)作为单层培养物体外保存。通过胰蛋白酶-EDTA处理，每周两次对肿瘤细胞进行常规传代培养。收获处于指数生长期的细胞，并计数用于肿瘤接种。The subcutaneous NCI-N87 human gastric cancer xenograft model in female BALB/c nude mice was established as follows: in air containing 5% CO2 at 37°C in RPMI supplemented with 10% fetal bovine serum and 1% antibiotic-antimycotic NCI-N87 tumor cells (ATCC, Manassas, VA, catalog number CRL-5822) were maintained in vitro as monolayer cultures in -1640 medium. Tumor cells were routinely subcultured twice a week by trypsin-EDTA treatment. Cells in exponential growth phase were harvested and counted for tumor inoculation.
肿瘤接种和动物分组：Tumor inoculation and animal grouping:
对每只小鼠的右侧皮下接种补充有Matrigel(1∶1)的0.2mL PBS中的NCI-N87肿瘤细胞(10×106)，用于肿瘤发育。在肿瘤接种后第8天，平均肿瘤体积达到大约188mm3时，开始治疗。使用基于动物的肿瘤体积对其进行分层随机化的基于Excel的随机化软件将动物分配至各组中。每组由6 只荷瘤小鼠组成。参照实验例2向荷瘤小鼠给予供试品。NCI-N87 tumor cells (10 x 106 ) in 0.2 mL PBS supplemented with Matrigel (1:1) were inoculated subcutaneously on the right side of each mouse for tumor development. Treatment was initiated on day 8 after tumor inoculation when the mean tumor volume reached approximately 188 mm 3 . Animals were allocated into groups using Excel-based randomization software that stratified randomization based on the animals' tumor volume. Each group consists of 6 composed of tumor-bearing mice. Referring to Experimental Example 2, the test article was administered to tumor-bearing mice.
3.2实验结果：3.2 Experimental results:
3.2.1肿瘤生长曲线3.2.1 Tumor growth curve
图7示出了溶媒(DPBS)、Ab0100-H0037(5mg/kg)、Ab0100-H0038(5mg/kg)，以及BQ3(5mg/kg)给药后荷瘤小鼠(NCI-N87荷瘤雌性BALB/c裸鼠)的肿瘤生长曲线。Figure 7 shows vehicle (DPBS), Ab0100-H0037 (5mg/kg), Ab0100-H0038 (5mg/kg), and BQ3 (5mg/kg) after administration of tumor-bearing mice (NCI-N87 tumor-bearing female BALB /c tumor growth curve of nude mice).
结果显示：在5mg/kg Ab0100-H0037、5mg/kg Ab0100-H0038和5mg/kg BQ3给药的荷瘤小鼠中，NCI-N87肿瘤生长均受到明显抑制；并且，Ab0100-H0038给药的NCI-N87荷瘤BALB/c裸鼠最终具有最小的肿瘤体积。The results showed that: in the tumor-bearing mice administered with 5mg/kg Ab0100-H0037, 5mg/kg Ab0100-H0038 and 5mg/kg BQ3, the growth of NCI-N87 tumors was significantly inhibited; -N87 tumor-bearing BALB/c nude mice finally had the smallest tumor volume.
实验例11：体内实验验证ADC的活性和毒性Experimental Example 11: In vivo experiments to verify the activity and toxicity of ADC
4.1实验方法与步骤：4.1 Experimental method and steps:
采用和试验例2相同的实验条件，研究雌性BALB/c裸鼠皮下HCC1954人乳腺癌异种移植模型中Ab0100-H0037、Ab0100-H0038和BQ3抗肿瘤疗效。Using the same experimental conditions as in Test Example 2, the anti-tumor efficacy of Ab0100-H0037, Ab0100-H0038 and BQ3 in the subcutaneous HCC1954 human breast cancer xenograft model in female BALB/c nude mice was studied.
雌性BALB/c裸鼠皮下HCC1954人乳腺癌异种移植模型建立方法如下：于37℃和5％CO2的空气中，在补充有10％胎牛血清和1％抗生素-抗真菌剂的RPMI 1640培养基中体外保存HCC1954肿瘤细胞(ATCC，Manassas，VA，目录号CRL-2338)。通过胰蛋白酶-EDTA处理，每周两次对肿瘤细胞进行常规传代培养。收获处于指数生长期的细胞，并计数用于肿瘤接种。The subcutaneous HCC1954 human breast cancer xenograft model in female BALB/c nude mice was established as follows: Culture in RPMI 1640 supplemented with 10% fetal bovine serum and 1% antibiotic-antimycotic at 37°C and 5% CO2 in the air HCC1954 tumor cells (ATCC, Manassas, VA, catalog number CRL-2338) were preserved in vitro. Tumor cells were routinely subcultured twice a week by trypsin-EDTA treatment. Cells in exponential growth phase were harvested and counted for tumor inoculation.
肿瘤接种和动物分组：对每只小鼠的右侧皮下接种0.2mLPBS和基质胶(Matrigel)(PBS：基质胶＝1∶1)混合物中的HCC1954肿瘤细胞(5 x 106)，用于肿瘤发育。在肿瘤接种后第13天，平均肿瘤体积达到177mm3时，开始治疗。使用基于动物的肿瘤体积对其进行分层随机化的基于Excel的随机化软件将动物分配至各组中。每组由6只荷瘤小鼠组成。参照实验例2向荷瘤小鼠给予供试品。Tumor inoculation and animal grouping: Subcutaneously inoculate the right side of each mouse with HCC1954 tumor cells (5 x 10 6 ) in a mixture of 0.2 mL of PBS and Matrigel (PBS: Matrigel = 1:1) for tumor development. Treatment was initiated on day 13 after tumor inoculation, when the mean tumor volume reached 177 mm 3 . Animals were allocated into groups using Excel-based randomization software that stratified randomization based on the animals' tumor volume. Each group consisted of 6 tumor-bearing mice. Referring to Experimental Example 2, the test article was administered to tumor-bearing mice.
4.2实验结果：4.2 Experimental results:
图8示出了溶媒(DPBS)、Ab0100-H0037(3mg/kg)、Ab0100-H0038(3mg/kg)，以及BQ3(5mg/kg)给药后荷瘤小鼠(HCC1954荷瘤雌性BALB/c裸鼠)的肿瘤生长曲线。Figure 8 shows vehicle (DPBS), Ab0100-H0037 (3mg/kg), Ab0100-H0038 (3mg/kg), and BQ3 (5mg/kg) after administration of tumor-bearing mice (HCC1954 tumor-bearing female BALB/c Tumor growth curve of nude mice).
结果显示：在3mg/kg Ab0100-H0037、3mg/kg Ab0100-H0038和5mg/kg BQ3给药的荷瘤小鼠中，HCC1954肿瘤生长均受到明显抑制；并且，3mg/kg Ab0100-H0038和5mg/kg BQ3给药的NCI-N87荷瘤BALB/c裸鼠，最终显示完全抑制的肿瘤生长。The results showed that: in the tumor-bearing mice administered with 3mg/kg Ab0100-H0037, 3mg/kg Ab0100-H0038 and 5mg/kg BQ3, the growth of HCC1954 tumors was significantly inhibited; and, 3mg/kg Ab0100-H0038 and 5mg/kg NCI-N87 tumor-bearing BALB/c nude mice administered with kg BQ3 finally showed complete inhibition of tumor growth.
本说明书发明的所有技术特征都可以任何组合方式进行组合。本说明所发明的每个特征也可以被其它具有相同、相等或相似作用的特征所替换。因此，除非特殊说明，所发明的每一特征仅仅是一系列相等或相似特征的实例。All the technical features of the invention in this specification can be combined in any combination. Each feature of the invention described in this description may also be replaced by other features having the same, equivalent or similar effect. Thus, unless stated otherwise, each feature disclosed is only one example of a series of equivalent or similar features.
此外，从上述描述中，本领域技术人员可从本公开中很容易清楚本公开的关键特征，在不脱离本公开的精神及范围的情况下，可对发明进行很多修改以适应各种不同的使用目的及条件，因此这类修改也旨在落入所附权利要求书的范围内。 In addition, from the above description, those skilled in the art can easily understand the key features of the present disclosure from the present disclosure. Without departing from the spirit and scope of the present disclosure, many modifications can be made to the invention to adapt to various purposes and conditions of use, and therefore such modifications are also intended to fall within the scope of the appended claims.

Claims (12)

  1. 如式(I)所示的化合物或其药学上可接受的盐、酯、溶剂化物、光学异构体、互变异构体、同位素标记物或前药：
    Compounds represented by formula (I) or pharmaceutically acceptable salts, esters, solvates, optical isomers, tautomers, isotope labels or prodrugs thereof:
    其中，L1为(Gly)p-(Z1)a-，p为1-10的任一整数；a为0-20的任一整数，若存在，每一个Z1各自独立地为任意种类的氨基酸残基；Wherein, L 1 is (Gly) p -(Z 1 ) a -, p is any integer of 1-10; a is any integer of 0-20, if exists, each Z 1 is independently any kind amino acid residues;
    L2为-NH-R1-(CH2)c-C(＝O)-，其中R1为-(CH2-CH2-X)b-，b为1-10的任一整数，每一个X各自独立地为-CH2-、-NH-、-O-或-S-，c为1-5的任一整数；L 2 is -NH-R 1 -(CH 2 ) c -C(=O)-, wherein R 1 is -(CH 2 -CH 2 -X) b -, b is any integer from 1 to 10, each Each X is independently -CH 2 -, -NH-, -O- or -S-, and c is any integer from 1 to 5;
    L3为
    L 3 is
    L3优选为 L3 is preferably
    其中m为1-5的任一整数，优选为2；R2为-(CH2-CH2-Y)d-，d为1-10的任一整数，每一个Y各自独立地为-CH2-、-NH-、-O-或-S-；wherein m is any integer from 1 to 5, preferably 2; R 2 is -(CH 2 -CH 2 -Y) d -, d is any integer from 1 to 10, and each Y is independently -CH 2 -, -NH-, -O- or -S-;
    L1的羧基端连接L2的氨基端，L2的羧基端连接L3的氨基端。The carboxy-terminal of L1 is connected to the amino-terminus of L2 , and the carboxy-terminus of L2 is connected to the amino-terminus of L3 .
  2. 根据权利要求1所述的化合物或其药学上可接受的盐、酯、溶剂化物、光学异构体、互变异构体、同位素标记物或前药，其特征在于， The compound according to claim 1 or its pharmaceutically acceptable salt, ester, solvate, optical isomer, tautomer, isotope label or prodrug, characterized in that,
    L1为(Gly)p-，p为1-10的任一整数，优选为3-5的任一整数；L 1 is (Gly) p -, p is any integer of 1-10, preferably any integer of 3-5;
    L2、L3如权利要求1中所定义。L 2 , L 3 are as defined in claim 1 .
  3. 根据权利要求1所述的化合物或其药学上可接受的盐、酯、溶剂化物、光学异构体、互变异构体、同位素标记物或前药，其特征在于，L3选自如下的任意一种：

    The compound according to claim 1 or a pharmaceutically acceptable salt, ester, solvate, optical isomer, tautomer, isotope label or prodrug thereof, wherein L is selected from the following Either:

    L3优选自如下的任意一种：
    L 3 is preferably selected from any one of the following:
    L1、L2如权利要求1中所定义。L 1 , L 2 are as defined in claim 1 .
  4. 根据权利要求1所述的化合物或其药学上可接受的盐、酯、溶剂化物、光学异构体、互变异构体、同位素标记物或前药，其特征在于，The compound according to claim 1 or its pharmaceutically acceptable salt, ester, solvate, optical isomer, tautomer, isotope label or prodrug, characterized in that,
    L2为-NH-R1-(CH2)c-C(＝O)-；L 2 is -NH-R 1 -(CH 2 ) c -C(=O)-;
    其中R1为-(CH2-CH2-X)b-，b为1-10的任一整数，优选为1-5的任一整数；每一个X各自独立地为-CH2-或-O-；c为1-5的任一整数，优选为1-2的任一整数；Wherein R 1 is -(CH 2 -CH 2 -X) b -, b is any integer of 1-10, preferably any integer of 1-5; each X is independently -CH 2 - or - O-; c is any integer of 1-5, preferably any integer of 1-2;
    L1、L3如权利要求1中所定义。L 1 , L 3 are as defined in claim 1 .
  5. 根据权利要求1-4任一项所述的化合物或其药学上可接受的盐、酯、溶剂化物、光学异构体、互变异构体、同位素标记物或前药，其特征在于，The compound or its pharmaceutically acceptable salt, ester, solvate, optical isomer, tautomer, isotope label or prodrug according to any one of claims 1-4, characterized in that,
    L1-L2-为 L 1 -L 2 -for
    其中，w为0-9的任一整数，优选为2-4的任一整数；c为1-5的任一整数，优选为1-2的任一整数；Wherein, w is any integer of 0-9, preferably any integer of 2-4; c is any integer of 1-5, preferably any integer of 1-2;
    b为1-5的任一整数，优选为2-4的任一整数，每一个X各自独立地为-CH2-或-O-；并且，
    的结构为：
    b is any integer of 1-5, preferably any integer of 2-4, each X is independently -CH 2 - or -O-; and,
    The structure is:
  6. 根据权利要求1-4任一项所述的化合物或其药学上可接受的盐、酯、溶剂化物、光学异构体、互变异构体、同位素标记物或前药，其特征在于，The compound or its pharmaceutically acceptable salt, ester, solvate, optical isomer, tautomer, isotope label or prodrug according to any one of claims 1-4, characterized in that,
    L1-L2-为 L 1 -L 2 -for
    其中，w为0-9的任一整数，优选为2-4的任一整数；c为1-5的任一整数，优选为1-2的任一整数；Wherein, w is any integer of 0-9, preferably any integer of 2-4; c is any integer of 1-5, preferably any integer of 1-2;
    b为1-5的任一整数，优选为2-4的任一整数，每一个X各自独立地为-CH2-或-O-；并且，
    的结构为：
    b is any integer of 1-5, preferably any integer of 2-4, each X is independently -CH 2 - or -O-; and,
    The structure is:
  7. 如下式(I-1)～(I-10)任一项所示的化合物或其药学上可接受的盐、酯、溶剂化物、光学异构体、互变异构体、同位素标记物或前药：


    Compounds represented by any one of the following formulas (I-1) to (I-10) or pharmaceutically acceptable salts, esters, solvates, optical isomers, tautomers, isotope labels or precursors medicine:


    优选如下任一项所示的结构：
    A structure as shown in any of the following is preferred:
  8. 一种用于偶联抗体与药物化合物的连接子，其特征在于，所述连接子具有如下式(II)所示的结构：A linker for coupling antibodies and pharmaceutical compounds, characterized in that the linker has a structure shown in the following formula (II):
    L1-L2-L3-R3  (II)； L 1 -L 2 -L 3 -R 3 (II);
    其中，R3选自-NH2或-OH；Wherein, R 3 is selected from -NH 2 or -OH;
    L1、L2、L3如权利要求1-4中任一项所定义。L 1 , L 2 , L 3 are as defined in any one of claims 1-4.
  9. 根据权利要求8所述的连接子，其特征在于，所述连接子具有如下式(II-1)或(II-2)所示的结构：
    The linker according to claim 8, wherein the linker has a structure shown in the following formula (II-1) or (II-2):
    其中，R3选自-NH2或-OH；Wherein, R 3 is selected from -NH 2 or -OH;
    w为0-9的任一整数，优选为2-4的任一整数；c为1-5的任一整数，优选1-2的任一整数，w is any integer of 0-9, preferably any integer of 2-4; c is any integer of 1-5, preferably any integer of 1-2,
    b为1-5的任一整数，优选为2-4的任一整数，每一个X各自独立地为-CH2-、-NH-、-O-或-S-。b is any integer of 1-5, preferably any integer of 2-4, and each X is independently -CH 2 -, -NH-, -O- or -S-.
  10. 根据权利要求8或9所述的连接子，其特征在于，所述连接子具有如下任一项所示的结构：

    The linker according to claim 8 or 9, wherein the linker has a structure as shown in any one of the following:

  11. 根据权利要求1-7任一项所示的化合物或其药学上可接受的盐、酯、溶剂化物、光学异构体、互变异构体、同位素标记物或前药在制备抗体药物偶联物中的用途；According to the compound shown in any one of claims 1-7 or its pharmaceutically acceptable salt, ester, solvate, optical isomer, tautomer, isotope label or prodrug in the preparation of antibody drug conjugate use in things;
    其中，所述抗体药物偶联物中，根据权利要求1-7任一项所示的化合物或其药学上可接受的盐、酯、溶剂化物、光学异构体、互变异构体、同位素标记物或前药通过L1与抗体或抗原结合片段相偶联；Wherein, in the antibody drug conjugate, the compound according to any one of claims 1-7 or its pharmaceutically acceptable salt, ester, solvate, optical isomer, tautomer, isotope The label or prodrug is coupled to the antibody or antigen-binding fragment through L1 ;
    可选地，所述抗体药物偶联物用于预防和/或治疗肿瘤；可选地，所述肿瘤为与HER蛋白家族中的一种或两种以上的蛋白异常表达相关的肿瘤；优选地，所述异常表达的蛋白选自HER2、HER3中的至少一种；Optionally, the antibody drug conjugate is used to prevent and/or treat tumors; optionally, the tumors are tumors related to the abnormal expression of one or more than two proteins in the HER protein family; preferably , the abnormally expressed protein is selected from at least one of HER2 and HER3;
    可选地，所述肿瘤选自乳腺癌、卵巢癌、宫颈癌、子宫癌、前列腺癌、肾癌、尿道癌、膀胱癌、肝癌、胃癌、子宫内膜癌、唾液腺癌、食道癌、黑色素瘤、神经胶质瘤、神经母细胞瘤、肉瘤、肺癌、结直肠癌、白血病、骨癌、皮肤癌、甲状腺癌、胰腺癌或淋巴瘤。Optionally, the tumor is selected from breast cancer, ovarian cancer, cervical cancer, uterine cancer, prostate cancer, renal cancer, urethral cancer, bladder cancer, liver cancer, gastric cancer, endometrial cancer, salivary gland cancer, esophageal cancer, melanoma , glioma, neuroblastoma, sarcoma, lung cancer, colorectal cancer, leukemia, bone cancer, skin cancer, thyroid cancer, pancreatic cancer, or lymphoma.
  12. 根据权利要求8-10任一项所示的连接子在制备抗体药物偶联物中的用途；Use of the linker according to any one of claims 8-10 in the preparation of antibody drug conjugates;
    其中，所述抗体药物偶联物中，所述连接子的L1基团连接抗体或抗原结合片段，所述连接子的L3基团连接药物化合物；Wherein, in the antibody-drug conjugate, the L1 group of the linker is connected to the antibody or the antigen-binding fragment, and the L3 group of the linker is connected to the drug compound;
    可选地，所述抗体药物偶联物用于预防和/或治疗肿瘤；可选地，所述肿瘤为与HER蛋白家族中的一种或两种以上的蛋白异常表达相关的肿瘤；优选地，所述异常表达的蛋白选自HER2、HER3中的至少一种；Optionally, the antibody drug conjugate is used to prevent and/or treat tumors; optionally, the tumors are tumors related to the abnormal expression of one or more than two proteins in the HER protein family; preferably , the abnormally expressed protein is selected from at least one of HER2 and HER3;
    可选地，所述肿瘤选自乳腺癌、卵巢癌、宫颈癌、子宫癌、前列腺癌、肾癌、尿道癌、膀胱癌、肝癌、胃癌、子宫内膜癌、唾液腺癌、食道癌、黑色素瘤、神经胶质瘤、神经母细胞瘤、肉瘤、肺癌、结直肠癌、白血病、骨癌、皮肤癌、甲状腺癌、胰腺癌或淋巴瘤。 Optionally, the tumor is selected from breast cancer, ovarian cancer, cervical cancer, uterine cancer, prostate cancer, renal cancer, urethral cancer, bladder cancer, liver cancer, gastric cancer, endometrial cancer, salivary gland cancer, esophageal cancer, melanoma , glioma, neuroblastoma, sarcoma, lung cancer, colorectal cancer, leukemia, bone cancer, skin cancer, thyroid cancer, pancreatic cancer, or lymphoma.
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