WO2023143320A1 - Lieur pour la préparation d'un conjugué anticorps-médicament, composé et utilisation - Google Patents

Lieur pour la préparation d'un conjugué anticorps-médicament, composé et utilisation Download PDF

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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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cancer
integer
antibody
linker
tumor
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PCT/CN2023/072958
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Chinese (zh)
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袁建栋
黄仰青
顾家宁
宋云松
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博瑞生物医药（苏州）股份有限公司
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Publication of WO2023143320A1 publication Critical patent/WO2023143320A1/fr

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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

La présente divulgation se rapporte au domaine technique des médicaments. Plus particulièrement, la présente invention concerne un composé tel que représenté dans la formule (I), un lieur tel que représenté dans la formule (II), et une utilisation du composé tel que représenté dans la formule (I) et du lieur tel que représenté dans la formule (II) dans la préparation d'un conjugué anticorps-médicament. Le composé tel que représenté dans la formule (I) peut être lié à un anticorps au moyen d'un lieur glycine de celui-ci pour obtenir un conjugué anticorps-médicament. Le conjugué anticorps-médicament peut exercer une activité efficace et spécifique de destruction de cellules tumorales à la fois in vivo et in vitro, inhibe de manière significative la croissance de différents types de tumeurs et n'a pas de cytotoxicité évidente, ayant ainsi des perspectives d'application importantes dans le domaine du traitement de tumeurs.
PCT/CN2023/072958 2022-01-28 2023-01-18 Lieur pour la préparation d'un conjugué anticorps-médicament, composé et utilisation WO2023143320A1 (fr)

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