WO2022033578A1 - 抗体药物偶联物 - Google Patents

抗体药物偶联物 Download PDF

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Publication number
WO2022033578A1
WO2022033578A1 PCT/CN2021/112462 CN2021112462W WO2022033578A1 WO 2022033578 A1 WO2022033578 A1 WO 2022033578A1 CN 2021112462 W CN2021112462 W CN 2021112462W WO 2022033578 A1 WO2022033578 A1 WO 2022033578A1
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WIPO (PCT)
Prior art keywords
antibody
heavy chain
light chain
her2
drug conjugate
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/CN2021/112462
Other languages
English (en)
French (fr)
Chinese (zh)
Inventor
张喜全
陈天玺
丰巍伟
张兵
唐小齐
徐同杰
王晓金
盛化策
张正平
王�华
高勇
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Chia Tai Tianqing Pharmaceutical Group Co Ltd
Nanjing Shunxin Pharmaceutical Co Ltd
Original Assignee
Chia Tai Tianqing Pharmaceutical Group Co Ltd
Nanjing Shunxin Pharmaceutical Co Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Chia Tai Tianqing Pharmaceutical Group Co Ltd, Nanjing Shunxin Pharmaceutical Co Ltd filed Critical Chia Tai Tianqing Pharmaceutical Group Co Ltd
Priority to BR112023002417A priority Critical patent/BR112023002417A2/pt
Priority to CN202411649225.XA priority patent/CN119792564A/zh
Priority to CN202180041403.9A priority patent/CN115702008B/zh
Priority to US18/040,498 priority patent/US20230405141A1/en
Priority to EP21855628.0A priority patent/EP4183421A4/en
Priority to CN202411649231.5A priority patent/CN119792566A/zh
Priority to CN202411649220.7A priority patent/CN119792562A/zh
Priority to CN202411649235.3A priority patent/CN119792568A/zh
Priority to CN202411649228.3A priority patent/CN119792565A/zh
Priority to CA3188508A priority patent/CA3188508A1/en
Priority to KR1020237007880A priority patent/KR20230051214A/ko
Priority to MX2023001648A priority patent/MX2023001648A/es
Priority to IL300446A priority patent/IL300446A/en
Priority to AU2021323863A priority patent/AU2021323863A1/en
Priority to CN202411649224.5A priority patent/CN119792563A/zh
Priority to CN202411649232.XA priority patent/CN119792567A/zh
Priority to JP2023508466A priority patent/JP2023537051A/ja
Publication of WO2022033578A1 publication Critical patent/WO2022033578A1/zh
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
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    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/32—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against translation products of oncogenes
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    • A61K47/68037—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a camptothecin [CPT] or derivatives
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    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
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    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
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Definitions

  • the present application relates to antibody drug conjugates comprising linked therapeutic antibody moieties, intermediate linker moieties and cytotoxic drug moieties.
  • the present application also relates to the use of the antibody-drug conjugate in the preparation of a medicament for preventing and treating cancer.
  • Antibody-Drug Conjugate is a class of drugs that combines the high specificity of therapeutic antibodies and the high killing activity of cytotoxic drugs, in which the therapeutic antibody part and the cytotoxic drug part pass through an intermediate linker. Partial connection.
  • ADC drugs on the market worldwide, among which the antibodies of brentuximab vedotin, polatuzumab vedotin and enfortumab vedotin target CD30, CD79b and Nectin-4 respectively, and the antibodies of trastuzumab emtansine and trastuzumab deruxtecan are directed against HER2 targets.
  • the antibody portions of gemtuzumab ozogamicin and inotuzumab ozogamicin were directed against CD33 and CD22 targets, respectively, and the antibody portion of sacituzumab govitecan was directed against TROP2 targets.
  • brentuximab vedotin, polatuzumab vedotin and enfortumab vedotin use auristatins toxin molecules that act on microtubules
  • trastuzumab emtansine uses maytansinoid toxin molecules that act on microtubules
  • gemtuzumab ozogamicin and inotuzumab ozogamicin use calicheamicins toxin molecules that act on DNA
  • the newly launched trastuzumab deruxtecan and sacituzumab govitecan both use camptothecin toxin molecules.
  • Camptothecin (CPT) analogs and derivatives exert antitumor activity by binding to topoisomerase I, and they have shown significant activity against many tumor types.
  • CPT irinotecan hydrochloride
  • CPT-11 irinotecan hydrochloride
  • CPT-11 can only be converted into its active form SN-38 (formula I) after being catalyzed by carboxylesterase in vivo, and the conversion efficiency is extremely low, and SN38 itself is difficult to become a drug due to its poor solubility.
  • Isanotecan (formula II) (English common name: exatecan) is another water-soluble CPT derivative, which was tried to be developed as an anti-tumor drug, but the development was stopped in 2004, and it does not require the use of enzymes. Activated, in addition, the topoisomerase I inhibitory activity of ixatecan was stronger than that of SN-38, which is the pharmacodynamic entity of irinotecan.
  • ADC drugs combine the dual advantages of high efficacy of cytotoxic small molecules and high selectivity of antibodies to specific tumor cells.
  • cytotoxic small molecules and high selectivity of antibodies to specific tumor cells.
  • the application provides antibody drug conjugates containing deuterated modifications or pharmaceutically acceptable salts or solvates thereof, particularly involving deuterated modifications of linkers or cytotoxic drug moieties.
  • the application provides an antibody drug conjugate having the general formula Ab-(LU)n, or a pharmaceutically acceptable salt or solvate thereof, wherein Ab represents an antibody moiety, L represents a linker moiety, and U represents a Cytotoxic drug moiety, n is an integer or decimal selected from 1 to 10.
  • the present application provides an antibody drug conjugate having the general formula Ab-(LU)n or a pharmaceutically acceptable salt or solvate thereof, wherein the Ab (antibody moiety) can specifically bind to a tumor antigen ( Including tumor-specific antigens and tumor-associated antigens), tumor antigens can be selected from any tumor prevention or treatment targets known in the art, such as HER2, EGFR, CD20, CD30, CD33, CD47, CD79b, VEGF, VEGFR, MET, RET, PD-1, PD-L1, etc.
  • tumor antigens can be selected from any tumor prevention or treatment targets known in the art, such as HER2, EGFR, CD20, CD30, CD33, CD47, CD79b, VEGF, VEGFR, MET, RET, PD-1, PD-L1, etc.
  • the application provides an antibody drug conjugate having the general formula Ab-(LU)n, or a pharmaceutically acceptable salt or solvate thereof, wherein the Ab (antibody moiety) is modifiable, For example, one or more amino acid changes, additions or subtractions are included.
  • the application provides an antibody drug conjugate having the general formula Ab-(LU)n or a pharmaceutically acceptable salt or solvate thereof, wherein the antibody moiety Ab is capable of specifically binding HER2 antibody.
  • the antibody moiety Ab of the antibody drug conjugate of the general formula Ab-(LU)n or a pharmaceutically acceptable salt or solvate thereof provided herein is trastuzumab, which has the following The sequences shown in Table S1.
  • the antibody moiety Ab of the antibody drug conjugate of the general formula Ab-(LU)n or a pharmaceutically acceptable salt or solvate thereof provided herein is Pertuzumab, which has the following Sequences shown in Table S2.
  • the application provides an antibody drug conjugate having the general formula Ab-(LU)n, or a pharmaceutically acceptable salt or solvate thereof, wherein the antibody moiety Ab comprises a monovalent and specifically binds A first antigen-binding fragment of the ECD4 epitope of HER2 on a HER2 expressing cell, the first antigen-binding fragment being an scFv comprising a VH and a VL, the VH having a K30 mutation, and/or the VL having an F53 mutation.
  • amino acid 30 in the sequence of the VH is mutated from K to an acidic amino acid, eg, E.
  • the amino acid at position 53 in the sequence of the VL is mutated from F to a neutral or basic amino acid, eg, Y, A, R.
  • the scFv may comprise or be a sequence having a K30 mutation and/or an F53 mutation in the amino acid sequence set forth in SEQ ID NO:1.
  • the application provides an antibody drug conjugate having the general formula Ab-(LU)n, or a pharmaceutically acceptable salt or solvate thereof, wherein the antibody moiety Ab comprises a monovalent and specifically binds HER2 expression
  • the amino acid sequences shown in SEQ ID NOs: 30, 34 and 32 are respectively included; wherein the sequence shown in SEQ ID NO: 27 is GFNIX 2 DTYIH, and X 2 is K or E; the sequence shown in SEQ ID NO
  • the application provides an antibody drug conjugate having the general formula Ab-(LU)n, or a pharmaceutically acceptable salt or solvate thereof, wherein the antibody moiety Ab comprises a monovalent and specifically binds HER2 expression
  • the application provides an antibody drug conjugate having the general formula Ab-(LU)n, or a pharmaceutically acceptable salt or solvate thereof, wherein the antibody moiety Ab comprises a monovalent and specifically binds HER2 expression
  • Ab comprises a monovalent and specifically binds HER2 expression
  • the first antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, and the heavy chain variable region and the light chain variable region respectively comprise the amino acid sequences shown in SEQ ID NOs: 41 and 42; or
  • the first antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, and the heavy chain variable region and the light chain variable region respectively comprise the amino acids shown in SEQ ID NOs: 41 and 42 Amino acid sequences with sequences that are at least 80% identical;
  • sequence shown in SEQ ID NO: 41 is:
  • EVQLVESGGGLVQPGGSLRLSCAASGFNIX 2 DTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSS, X 2 is K or E;
  • sequence shown in SEQ ID NO: 42 is:
  • the application provides an antibody drug conjugate having the general formula Ab-(LU)n, or a pharmaceutically acceptable salt or solvate thereof, wherein the antibody moiety Ab comprises a monovalent and specifically binds HER2 expression
  • the first antigen-binding fragment of the ECD4 epitope of HER2 on the cell is an scFv
  • the first antigen-binding fragment comprises a heavy chain variable region and a light chain variable region
  • the heavy chain can be
  • the variable region and light chain variable region comprise amino acid sequences according to SEQ ID NOs: 35 and 36, respectively.
  • the application provides an antibody drug conjugate having the general formula Ab-(LU)n, or a pharmaceutically acceptable salt or solvate thereof, wherein the antibody moiety Ab comprises a monovalent and specifically binds HER2 expression
  • the application provides an antibody drug conjugate having the general formula Ab-(LU)n or a pharmaceutically acceptable salt or solvate thereof, wherein the antibody moiety Ab further comprises a monovalent and specific binding to HER2 A second antigen-binding fragment of the ECD2 epitope of HER2 on the expressing cell, the second antigen-binding fragment being a Fab.
  • the application provides an antibody drug conjugate having the general formula Ab-(LU)n or a pharmaceutically acceptable salt or solvate thereof, wherein the antibody moiety Ab further comprises a monovalent and specific binding to HER2 Expressing a second antigen-binding fragment of the ECD2 epitope of HER2 on a cell, the second antigen-binding fragment being a Fab and comprising heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2, and light chain CDR3 , the heavy chain CDR1, heavy chain CDR2 and heavy chain CDR3 respectively comprise the amino acid sequences shown in SEQ ID NOs: 45, 46 and 47, and the light chain CDR1, light chain CDR2 and light chain CDR3 respectively comprise SEQ ID NOs: Amino acid sequences shown in 48, 49 and 50.
  • the application provides an antibody drug conjugate having the general formula Ab-(LU)n or a pharmaceutically acceptable salt or solvate thereof, wherein the antibody moiety Ab further comprises a monovalent and specific binding to HER2 A second antigen-binding fragment of the ECD2 epitope of HER2 on the expressing cell, the second antigen-binding fragment being a Fab and comprising a heavy chain variable region and a light chain variable region, which can be The variable regions comprise the amino acid sequences shown in SEQ ID NOs: 37 and 38, respectively.
  • the antibody part Ab of the antibody drug conjugate with the general formula of Ab-(L-U)n or a pharmaceutically acceptable salt or solvate thereof provided by the present application is shown in Table S3.
  • the application provides an antibody drug conjugate having the general formula Ab-(LU)n or a pharmaceutically acceptable salt or solvate thereof, wherein the antibody moiety Ab comprises a first antigen-binding fragment and/or or an immunoglobulin domain to which the second antigen-binding fragment is operably linked, the immunoglobulin domain comprising: i. one or more of CL, CH1, CH2, or CH3, or ii. Fc.
  • the application provides an antibody drug conjugate having the general formula Ab-(LU)n or a pharmaceutically acceptable salt or solvate thereof, wherein the antibody moiety Ab comprises a first antigen-binding fragment and/or or an immunoglobulin domain to which the second antigen-binding fragment is operably linked, the immunoglobulin domain comprising: i. one or more of CL, CH1, CH2, or CH3, or ii. Fc, wherein the Said CL, CH1, CH2, CH3, Fc are derived from CL, CH1, CH2, CH3, Fc of human IgG, respectively.
  • the application provides an antibody drug conjugate having the general formula Ab-(LU)n or a pharmaceutically acceptable salt or solvate thereof, wherein the antibody moiety Ab comprises a first antigen-binding fragment and/or or an immunoglobulin domain to which the second antigen-binding fragment is operably linked, the immunoglobulin domain comprising: i. one or more of CL, CH1, CH2, or CH3, or ii. Fc, wherein the Said CL, CH1, CH2, CH3 or Fc is modified or not; preferably, the modification of said CH3 or Fc is, for example, substitution of amino acids 435 and/or 436 according to the Kabat numbering system.
  • the application provides an antibody drug conjugate having the general formula Ab-(LU)n or a pharmaceutically acceptable salt or solvate thereof, wherein the antibody moiety Ab comprises a first antigen-binding fragment and/or or an immunoglobulin domain to which the second antigen-binding fragment is operably linked, the immunoglobulin domain comprising: i. one or more of CL, CH1, CH2, or CH3, or ii. Fc, wherein the The Fc is a dimeric Fc comprising a first Fc polypeptide and a second Fc polypeptide, the first antigen-binding fragment is operably linked to the first Fc polypeptide, and the second antigen-binding fragment is operably linked to the second Fc polypeptide.
  • the application provides an antibody drug conjugate having the general formula Ab-(LU)n or a pharmaceutically acceptable salt or solvate thereof, wherein the antibody moiety Ab comprises a first antigen-binding fragment and/or or a constant region to which the second antigen-binding fragment is operably linked, which may be an immunoglobulin native sequence constant region or a mutated constant region, such as a native or mutated CL, CH1, CH2, and/or CH3 function
  • the constant region may be derived from the constant region of a human immunoglobulin, such as derived from IgGl, IgG2, IgG3 or IgG4 , in some instances, the constant region may have modifications to improve its ability to mediate effector function.
  • the application provides an antibody drug conjugate having the general formula Ab-(LU)n or a pharmaceutically acceptable salt or solvate thereof, wherein the antibody moiety Ab comprises a first antigen-binding fragment and/or or an immunoglobulin domain or scaffold to which the second antigen-binding fragment is operably linked, e.g.
  • Fc includes native sequence Fc regions and variant Fc regions
  • Fc may be a human Fc such as derived from IgGl, IgG2, IgG3 or IgG4, Fc can have modifications to improve its ability to mediate effector function, e.g., in some embodiments, the backbone has modifications such as knob into hole, H435R, Y436F, defucose, etc.
  • the mutation sites of the knob into hole of the above-mentioned backbone include, for example, Y349C, T366S, L368A, Y407V, S354C and T366W, etc.
  • the application provides an antibody drug conjugate having the general formula Ab-(LU)n or a pharmaceutically acceptable salt or solvate thereof, wherein the antibody moiety Ab comprises a first antigen-binding fragment and/or or a scaffold to which a second antigen-binding fragment is operably linked; in some embodiments, the scaffold is a dimeric Fc comprising a first Fc polypeptide and a second Fc polypeptide; in some embodiments, the dimeric Fc Has modifications; in some embodiments, the dimeric Fc has H435R and/or Y436F, and the modification can be on either the polypeptide chain of the first Fc polypeptide or the second Fc polypeptide; in some specific embodiments, the dimeric Fc has H435R and/or Y436F, the modification occurs only on one Fc polypeptide and not on the other Fc polypeptide; in some embodiments, the dimeric Fc has a knob-into-hole mutation site, eg, Y349C, T
  • the application provides an antibody drug conjugate having the general formula Ab-(LU)n or a pharmaceutically acceptable salt or solvate thereof, wherein the antibody moiety Ab is a bivalent bispecific antibody, which Comprising: a heavy chain comprising the sequence of SEQ ID NO:11, a heavy chain comprising the sequence of SEQ ID NO:13, and a light chain comprising the sequence of SEQ ID NO:15.
  • the antibody Ab portion of the antibody drug conjugate with the general formula of Ab-(L-U)n or a pharmaceutically acceptable salt or solvate thereof provided herein is shown in Table S4.
  • the application provides an antibody drug conjugate of the general formula Ab-(LU)n, or a pharmaceutically acceptable salt or solvate thereof, wherein the cytotoxic drug moiety U is coupled to the antibody moiety Ab through a linker moiety L link.
  • the linker moiety L of the present application can be linked to the antibody moiety by any method known in the art, preferably the linker moiety is linked to the antibody moiety via a sulfhydryl group and/or an amino group. In some more preferred embodiments, the linker moiety of the present application is attached to the antibody moiety through a sulfhydryl group.
  • the application provides an antibody drug conjugate of the general formula Ab-(LU)n, or a pharmaceutically acceptable salt or solvate thereof, wherein the cytotoxic drug moiety U is coupled to the antibody moiety Ab through a linker moiety L ,
  • the linker part can be a cleavable linker or a non-cleavable linker; in some embodiments, the linker part of the present application is a cleavable linker, for example, it can be a low pH-dependent degradation type (including hydrazone bonds, carbonates bond, etc.), proteolytic type (including peptidyl bond) or high glutathione concentration-dependent degradation type (including disulfide bond), etc.; in other embodiments, the linker part of the present application is a non-cleavable linker, for example, it can be It is maleimidocaproyl and so on.
  • the application provides an antibody drug conjugate of the general formula Ab-(LU)n, or a pharmaceutically acceptable salt or solvate thereof, wherein the antibody moiety Ab is combined with one or more cytotoxic drug moieties U coupling
  • cytotoxic drugs can be selected from alkaloids, antimetabolites, antitumor antibiotics, alkylating agents, platinum, etc.
  • the preferred cytotoxic drugs are microtubule inhibitor cytotoxic drugs (including U.S. tansinoids, orlistatins) or cytotoxic drugs acting on DNA (including calicheamicin, duocarmycin, PBD (pyrrolobenzodiazepine), topoisomerase I inhibitors, etc.).
  • the cytotoxic drug moiety U of the antibody drug conjugate of the general formula Ab-(LU)n or a pharmaceutically acceptable salt or solvate thereof provided herein is topoisomerase I inhibition agent.
  • the cytotoxic drug moiety U of the antibody drug conjugate of the general formula Ab-(LU)n or a pharmaceutically acceptable salt or solvate thereof provided herein is a camptothecin-like topoisotropic Construct I inhibitor.
  • the cytotoxic drug moiety U of the antibody drug conjugate of the general formula Ab-(LU)n or a pharmaceutically acceptable salt or solvate thereof provided by the application is selected from SN-38 , SN-38 derivatives, Isanotecan or Isanotecan derivatives.
  • the application provides an antibody drug conjugate having the general formula Ab-(LU)n, or a pharmaceutically acceptable salt or solvate thereof, wherein Ab represents an antibody moiety, L represents a linker moiety, and U represents hi Dendritic topoisomerase I inhibitor, n is an integer or decimal selected from 1 to 10, wherein the L moiety and/or the U moiety has deuterium modification.
  • n is an integer or decimal selected from 2 to 10, such as an integer or decimal from 2 to 9, an integer or decimal from 2 to 8, an integer or decimal from 3 to 9, an integer or decimal from 3 to 8 , an integer or decimal from 4 to 9, an integer or decimal from 4 to 8, an integer or decimal from 5 to 9, or an integer or decimal from 5 to 8.
  • the application provides an antibody drug conjugate having the general formula Ab-(LU)n, or a pharmaceutically acceptable salt or solvate thereof, wherein Ab represents the antibody moiety, L represents the linker moiety, and U represents the hi Dendritic topoisomerase I inhibitor, n is an integer or decimal selected from 1 to 10, wherein the L part and/or the U part is deuterium modified, and the cytotoxic drug part U is selected from SN-38, SN- 38 derivatives, ixatecan or ixatecan derivatives.
  • the antibody drug conjugate of general formula Ab-(L-U)n or a pharmaceutically acceptable salt or solvate thereof provided herein comprises the structure shown in the following formula III:
  • the antibody drug conjugate of general formula Ab-(L-U)n or a pharmaceutically acceptable salt or solvate thereof provided herein comprises the structure shown in the following formula IV:
  • R 1 is selected from hydrogen (H) or deuterium (D).
  • the antibody drug conjugate of general formula Ab-(LU)n or a pharmaceutically acceptable salt or solvate thereof provided herein comprises the structure shown in the following formula IV-1 or IV-2 :
  • the antibody drug conjugate of general formula Ab-(L-U)n or a pharmaceutically acceptable salt or solvate thereof provided herein comprises the structure shown in the following formula V:
  • R 1 and R 2 are independently selected from hydrogen (H) or deuterium (D),
  • the left succinimide terminal of this structure is a linking site to the antibody moiety
  • the right carbonyl terminal is a linking site to the cytotoxic drug moiety.
  • the antibody drug conjugate of general formula Ab-(LU)n or a pharmaceutically acceptable salt or solvate thereof provided herein comprises the following formulae V-1, V-2, V-3 Or the structure shown by V-4, and the structures shown by V-1 to V-4 are respectively connected to the antibody moiety at the left succinimide end, and the right carbonyl end is connected to the cytotoxic drug moiety:
  • the antibody drug conjugate of general formula Ab-(L-U)n or a pharmaceutically acceptable salt or solvate thereof provided herein comprises the structure shown in the following formula VI:
  • R 1 , R 2 are each independently selected from hydrogen (H) or deuterium (D).
  • the antibody drug conjugate of general formula Ab-(LU)n provided herein or a pharmaceutically acceptable salt or solvate thereof comprises the following formulae VI-1, VI-2, VI-3 or the structure shown in VI-4:
  • the application provides an antibody drug conjugate of the structure shown in formula VII or a pharmaceutically acceptable salt or solvate thereof:
  • Ab represents an antibody portion comprising a first antigen-binding fragment and a second antigen-binding fragment, the first antigen-binding fragment being an scFv and comprising heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2 and
  • the light chain CDR3, the heavy chain CDR1, the heavy chain CDR2 and the heavy chain CDR3 respectively comprise the amino acid sequences according to SEQ ID NOs: 43, 28 and 29, the light chain CDR1, the light chain CDR2 and the light chain CDR3 respectively comprise the amino acid sequences according to SEQ ID NOs: 43, 28 and 29 ID NOs: amino acid sequences of 30, 31 and 32;
  • the second antigen-binding fragment is a Fab and comprises heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2 and light chain CDR3, respectively comprising
  • the light chain CDR1, light chain CDR2 and light chain CDR3 comprise the amino acid sequences according to SEQ ID NOs: 48, 49 and 50, respectively,
  • n is an integer or decimal selected from 1 to 10,
  • R 1 , R 2 are each independently selected from hydrogen (H) or deuterium (D).
  • the application provides an antibody drug conjugate or a pharmaceutically acceptable salt or solvate thereof having the structure shown in the following formula VII-1, VII-2, VII-3 or VII-4,
  • Ab represents an antibody portion comprising a first antigen-binding fragment and a second antigen-binding fragment, the first antigen-binding fragment being an scFv and comprising heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2 and
  • the light chain CDR3, the heavy chain CDR1, the heavy chain CDR2 and the heavy chain CDR3 respectively comprise the amino acid sequences according to SEQ ID NOs: 43, 28 and 29, the light chain CDR1, the light chain CDR2 and the light chain CDR3 respectively comprise the amino acid sequences according to SEQ ID NOs: 43, 28 and 29 ID NOs: amino acid sequences of 30, 31 and 32;
  • the second antigen-binding fragment is a Fab and comprises heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2 and light chain CDR3, respectively comprising
  • the light chain CDR1, light chain CDR2 and light chain CDR3 comprise the amino acid sequences according to SEQ ID NOs: 48, 49 and 50, respectively,
  • n is an integer or decimal selected from 1 to 10.
  • the application provides an antibody drug conjugate having a structure shown in formula VII-1 or a pharmaceutically acceptable salt or solvate thereof,
  • Ab represents an antibody portion comprising a first antigen-binding fragment and a second antigen-binding fragment, the first antigen-binding fragment being an scFv and comprising heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2 and
  • the light chain CDR3, the heavy chain CDR1, the heavy chain CDR2 and the heavy chain CDR3 respectively comprise the amino acid sequences according to SEQ ID NOs: 43, 28 and 29, the light chain CDR1, the light chain CDR2 and the light chain CDR3 respectively comprise the amino acid sequences according to SEQ ID NOs: 43, 28 and 29 ID NOs: amino acid sequences of 30, 31 and 32;
  • the second antigen-binding fragment is a Fab and comprises heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2 and light chain CDR3, respectively comprising
  • the light chain CDR1, light chain CDR2 and light chain CDR3 comprise the amino acid sequences according to SEQ ID NOs: 48, 49 and 50, respectively,
  • n is an integer or decimal selected from 1 to 10.
  • the application provides an antibody drug conjugate having a structure shown in formula VII or a pharmaceutically acceptable salt or solvate thereof,
  • n is an integer or decimal selected from 1 to 10,
  • R 1 , R 2 are each independently selected from hydrogen (H) or deuterium (D).
  • the application provides an antibody-drug conjugate having the structure shown in formula VII-1 or a pharmaceutically acceptable salt or solvate thereof,
  • n is an integer or decimal selected from 1 to 10.
  • the application provides a pharmaceutical composition
  • a pharmaceutical composition comprising an antibody drug conjugate according to the application, or a pharmaceutically acceptable salt or solvate thereof, and a pharmaceutically acceptable carrier.
  • the present application provides the use of the antibody drug conjugate of the present application or a pharmaceutically acceptable salt or solvate thereof in the manufacture of a medicament for preventing or treating cancer.
  • the present application provides the use of a pharmaceutical composition comprising the antibody drug conjugate of the present application or a pharmaceutically acceptable salt or solvate thereof and a pharmaceutically acceptable carrier in the preparation of a medicament for preventing or treating cancer .
  • the application provides an antibody drug conjugate or a pharmaceutically acceptable salt or solvate thereof for use in the prevention or treatment of cancer.
  • the present application provides a method of treating or preventing cancer, the method comprising administering to a patient in need thereof a therapeutically effective amount of an antibody drug conjugate of the present application, or a pharmaceutically acceptable salt or solvate thereof, or A pharmaceutical composition comprising the antibody drug conjugate of the present application or a pharmaceutically acceptable salt or solvate thereof and a pharmaceutically acceptable carrier.
  • the antibody drug conjugates of the present application can be used to prevent or treat HER2-positive cancers, HER2-negative cancers (including triple-negative breast cancer), and according to immunohistochemistry
  • the assay detects cancers that show HER2 expression as IHC2+.
  • the application provides a linker-drug intermediate compound having the structure shown in the following formula VI, which is used to obtain an antibody-drug conjugate formed by linking an antibody to the intermediate compound:
  • R 1 , R 2 are each independently selected from hydrogen (H) or deuterium (D).
  • the application provides a linker-drug intermediate compound having the structure of formula VI-1, VI-2, VI-3 or VI-4 below, which is used to obtain a compound formed by linking an antibody to the intermediate compound Antibody Drug Conjugates:
  • the application provides a linker compound having the structure shown in the following formula V, which is used to obtain an antibody-drug conjugate formed by linking a drug and an antibody via the linker:
  • R 1 and R 2 are independently selected from hydrogen (H) or deuterium (D),
  • the left succinimide terminal of this structure is a linking site to the antibody moiety
  • the right carbonyl terminal is a linking site to the cytotoxic drug moiety.
  • the application provides a linker compound having a structure represented by the following formula V-1, V-2, V-3 or V-4, which is used to obtain an antibody drug conjugate formed by linking a drug and an antibody via the linker
  • V-1, V-2, V-3 or V-4 The structures shown in formulas V-1 to V-4 are connected to the antibody moiety at the left succinimide end, and the right carbonyl end is connected to the cytotoxic drug moiety:
  • the application provides compounds having the structure of Formula IV(a) below:
  • R 1 is selected from hydrogen (H) or deuterium (D).
  • the application provides compounds having the following structures of Formula IV(a)-1 or IV(a)-2:
  • the application provides compounds having the structure of Formula III(a) below:
  • the present application provides antibody drug conjugates or pharmaceutically acceptable salts or solvates thereof with improved pharmacokinetic properties. Improvements in pharmacokinetic properties will result in reduced toxicity, improved safety and/or tolerability, improved efficacy, and ultimately improved therapeutic window of the target compound.
  • Figure 1 shows anti-HER2 bispecific antibodies (Expi HER2-1, Expi HER2-2, 23C2 HER2-1 and 23C2 HER2-2) and combined trastuzumab + pertuzumab on BT474 tumor cells kill rate;
  • Figure 2 shows the killing rate of anti-HER2 bispecific antibody, trastuzumab, T-DM1, and combined trastuzumab + pertuzumab on NCI-N87 tumor cells;
  • Figure 3 shows the killing rate of anti-HER2 bispecific antibody, trastuzumab, T-DM1, and combined trastuzumab + pertuzumab on JIMT-1 tumor cells;
  • Figure 4 shows the results of inhibition of the proliferation of BT474 tumor cells by anti-HER2 bispecific antibody, trastuzumab, and combined trastuzumab + pertuzumab;
  • Figure 5 shows the changes in mouse tumor volume of anti-HER2 bispecific antibody, PBS vehicle control, and combined trastuzumab + Pertuzumab (Per + Tra) on gastric cancer N87 mouse xenograft tumor model Impact;
  • Figure 6 shows the effect of anti-HER2 bispecific antibody, PBS vehicle control, and combined trastuzumab + pertuzumab on changes in mouse body weight in the efficacy of gastric cancer N87 mice xenogeneic tumor;
  • Figure 7 is the structure of some exemplary anti-HER2 bispecific antibodies; wherein the dimeric Fc is depicted with one chain shown in black (the first Fc polypeptide) and the other chain shown in grey (the second Fc polypeptide), While one antigen-binding domain (the first antigen-binding fragment) is shown as hatched, the other antigen-binding domain (the second antigen-binding fragment) is shown in white; the first antigen-binding fragment is an scFv, fused to the first Fc polypeptide , and the second antigen-binding fragment is a Fab, fused to the second Fc polypeptide;
  • Figure 8 shows the endocytosis activity of drug conjugates of different antibodies (mono antibody-DDDXD and double antibody-DDDXD) in NCI-N87 tumor cells;
  • Figure 9 shows the endocytosis activities of drug conjugates of different antibodies (mono-DDDXD and double-antibody-DDDXD) in SK-BR-3 tumor cells.
  • substituted means that any one or more hydrogen atoms on a specified atom are replaced by a substituent, so long as the valence of the specified atom is normal and the compound after substitution is stable.
  • Optionally substituted for a group means that the group can be substituted or unsubstituted, for example, an ethyl group is "optionally” substituted with a halogen, meaning that the ethyl group can be unsubstituted ( CH2CH 3 ), monosubstituted (eg CH2CH2F ) , polysubstituted (eg CHFCH2F , CH2CHF2 , etc. ) or fully substituted ( CF2CF3 ) . It will be understood by those skilled in the art that for any group containing one or more substituents, no substitution or substitution pattern is introduced that is sterically impossible and/or cannot be synthesized.
  • Cmn in this context is that the moiety has an integer number of carbon atoms in the given range.
  • C1-6 means that the group may have 1 carbon atom, 2 carbon atoms, 3 carbon atoms, 4 carbon atoms, 5 carbon atoms or 6 carbon atoms.
  • any variable eg, R
  • its definition in each case is independent. So, for example, if a group is substituted with 2 Rs, each R has independent options.
  • linking group When the number of a linking group is 0, such as -(CH 2 ) 0 -, it means that the linking group is a covalent bond.
  • one of the variables is selected from a covalent bond, it means that the two groups connected to it are directly connected.
  • L in A-L-Z represents a covalent bond, it means that the structure is actually A-Z.
  • halo or halogen refers to fluorine, chlorine, bromine and iodine.
  • hydroxy refers to the -OH group.
  • cyano refers to the -CN group.
  • thiol refers to the -SH group.
  • amino refers to the -NH2 group.
  • nitro refers to the -NO 2 group.
  • alkyl refers to a hydrocarbon group of the general formula CnH2n+1 .
  • the alkyl group can be straight or branched.
  • C1-6 alkyl refers to an alkyl group containing 1 to 6 carbon atoms (eg, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, 1-methylbutyl, 2-methylbutyl, 3-methylbutyl, neopentyl, hexyl, 2-methylpentyl, etc.).
  • alkyl portion of alkoxy, alkylamino, dialkylamino, alkylsulfonyl, and alkylthio ie, alkyl
  • alkoxy refers to -O-alkyl
  • alkylamino refers to -NH-alkyl
  • dialkylamino refers to -N(alkyl) 2 .
  • alkylsulfonyl refers to -SO2 -alkyl.
  • alkylthio refers to -S-alkyl.
  • alkenyl refers to a straight or branched chain unsaturated aliphatic hydrocarbon group composed of carbon atoms and hydrogen atoms having at least one double bond.
  • alkenyl groups include, but are not limited to, ethenyl, 1-propenyl, 2-propenyl, 1-butenyl, isobutenyl, 1,3-butadienyl, and the like.
  • alkynyl refers to a straight or branched chain unsaturated aliphatic hydrocarbon group consisting of carbon atoms and hydrogen atoms having at least one triple bond.
  • alkynyl groups include, but are not limited to, ethynyl (-C ⁇ CH), 1-propynyl (-C ⁇ C- CH3 ), 2-propynyl ( -CH2 -C ⁇ CH), 1,3-Butadiynyl (-C ⁇ CC ⁇ CH) and the like.
  • cycloalkyl refers to a carbocyclic ring that is fully saturated and may exist as a monocyclic, bridged or spirocyclic ring. Unless otherwise indicated, the carbocycle is typically a 3- to 10-membered ring.
  • Non-limiting examples of cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, norbornyl (bicyclo[2.2.1]heptyl), bicyclo[2.2.2]octyl, diamond Alkyl etc.
  • cycloalkenyl refers to a non-aromatic carbocyclic ring that is not fully saturated and may exist as a monocyclic, bridged or spirocyclic ring. Unless otherwise indicated, the carbocycle is typically a 5- to 8-membered ring.
  • Non-limiting examples of cycloalkenyl groups include, but are not limited to, cyclopentenyl, cyclopentadienyl, cyclohexenyl, cyclohexadienyl, cycloheptenyl, cycloheptadienyl, and the like.
  • heterocyclyl refers to a non-aromatic ring that is fully saturated or partially unsaturated (but not fully unsaturated heteroaromatic) and may exist as a monocyclic, bridged or spirocyclic ring.
  • the heterocycle is typically a 3- to 7-membered ring containing 1 to 3 heteroatoms (preferably 1 or 2 heteroatoms) independently selected from sulfur, oxygen and/or nitrogen.
  • heterocyclyl groups include, but are not limited to, oxiranyl, tetrahydrofuranyl, dihydrofuranyl, pyrrolidinyl, N-methylpyrrolidinyl, dihydropyrrolyl, piperidinyl, piperazinyl , pyrazolidine, 4H-pyranyl, morpholinyl, thiomorpholinyl, tetrahydrothienyl, etc.
  • heterocycloalkyl refers to a cyclic group that is fully saturated and may exist as a monocyclic, bridged or spirocyclic ring. Unless otherwise indicated, the heterocycle is typically a 3- to 7-membered ring containing 1 to 3 heteroatoms (preferably 1 or 2 heteroatoms) independently selected from sulfur, oxygen and/or nitrogen.
  • 3-membered heterocycloalkyl groups include, but are not limited to, oxiranyl, oxiranyl, ethylene oxide
  • 4-membered heterocycloalkyl groups include, but are not limited to, azetidinyl, oxetine
  • Examples of cyclyl, thibutanyl, 5-membered heterocycloalkyl include, but are not limited to, tetrahydrofuranyl, tetrahydrothienyl, pyrrolidinyl, isoxazolidinyl, oxazolidinyl, isothiazolidinyl, thiazolidine
  • Examples of yl, imidazolidinyl, tetrahydropyrazolyl, 6-membered heterocycloalkyl include, but are not limited to, piperidinyl, tetrahydropyranyl, tetrahydrothiopyranyl, morpholinyl, piperazinyl, 1, Examples of 4-
  • aryl refers to an all-carbon monocyclic or fused polycyclic aromatic ring group having a conjugated pi electron system.
  • an aryl group can have 6-20 carbon atoms, 6-14 carbon atoms, or 6-12 carbon atoms.
  • Non-limiting examples of aryl groups include, but are not limited to, phenyl, naphthyl, anthracenyl, and 1,2,3,4-tetralin, and the like.
  • heteroaryl refers to a monocyclic or fused polycyclic ring system containing at least one ring atom selected from N, O, S, the remaining ring atoms being C, and having at least one aromatic ring.
  • Preferred heteroaryl groups have a single 4 to 8 membered ring, especially 5 to 8 membered ring, or multiple fused rings containing 6 to 14, especially 6 to 10 ring atoms.
  • heteroaryl groups include, but are not limited to, pyrrolyl, furyl, thienyl, imidazolyl, oxazolyl, pyrazolyl, pyridyl, pyrimidinyl, pyrazinyl, quinolinyl, isoquinolinyl , tetrazolyl, triazolyl, triazinyl, benzofuranyl, benzothienyl, indolyl, isoindolyl, etc.
  • Derivative a compound formed by replacing an atom or atomic group in the molecule of the parent compound with another atom or atomic group is called a derivative of the parent compound.
  • Any atom of a compound labeled synthetic in this application may represent any stable isotope of that atom.
  • H ie, hydrogen (H-1)
  • D i.e.
  • deuterium (H-2) that position contains at least 3340 times the isotope (ie, at least 50.1%) greater than the naturally occurring isotope (0.015%)
  • Deuterium isotope when one or more positions in the structure of the labeled synthetic compound are defined as D, namely deuterium (H-2), the content of the compound shown in the structure may be at least 52.5%, at least 60%, At least 67.5%, at least 75%, at least 82.5%, at least 90%, at least 95%, at least 97%, at least 98.5%, at least 99%, at least 99.5%.
  • the deuteration rate of a labeled synthetic compound in this application refers to the ratio of the labeled synthetic isotope content to the naturally occurring isotope content.
  • the deuteration rate of each designated deuterium atom of the compounds labeled and synthesized in this application may be at least 3500 times (52.5%), at least 4000 times (60%), at least 4500 times (67.5%), at least 5000 times (75%), at least 5500 times (82.5%), at least 6000 times (90%), at least 6333.3 times (95%), at least 6466.7 times (97%), at least 6566.7 times (98.5%), At least 6600 times (99%), at least 6633.3 times (99.5%).
  • Isotopologues in this application refer to compounds that differ in chemical structure only in their isotopic composition. Compounds labeled and synthesized in this application have the same chemical structure, with only isotopic variations in the atomic composition of their molecules. Therefore, the deuterium-containing compounds at a specific position marked and synthesized in this application will also contain very little hydrogen isotope at this position, and the amount of hydrogen isotope at a certain position in the compounds marked and synthesized in this application depends on many factors. , including the deuterium isotope purity of the deuterated reagents ( D2O , D2, NaBD4 , LiAlD4 , etc. ) and the effectiveness of introducing deuterium isotope synthesis methods.
  • the total amount of hydrogen isotopologues at such a location will be less than 49.9% as previously described.
  • the total amount of hydrogen isotope at a position in a compound labeled synthetic in this application will be less than 47.5%, 40%, 32.5%, 25%, 17.5%, 10%, 5%, 3%, 1%, or 0.5% %.
  • any atom not designated as deuterium is present in its natural isotopic abundance.
  • treating means administering a compound or formulation described herein to prevent, ameliorate or eliminate a disease or one or more symptoms associated with said disease, and includes: (i) prevention of a disease or disease state during breastfeeding occurs in animals, especially when such mammals are susceptible to the disease state but have not been diagnosed with the disease state; (ii) inhibit the disease or disease state, i.e. arrest its development; (iii) alleviate the disease or disease state, even if the disease or disease state resolves.
  • terapéuticaally effective amount means (i) treating or preventing a particular disease, condition or disorder, (ii) alleviating, ameliorating or eliminating one or more symptoms of a particular disease, condition or disorder, or (iii) preventing or delaying The amount of a compound of the present application for the onset of one or more symptoms of a particular disease, condition or disorder described herein.
  • the amount of a compound of the present application that constitutes a “therapeutically effective amount” will vary depending on the compound, the disease state and its severity, the mode of administration, and the age of the mammal to be treated, but can be routinely determined by those skilled in the art according to its own knowledge and the present disclosure.
  • pharmaceutically acceptable refers to those compounds, materials, compositions and/or dosage forms that, within the scope of sound medical judgment, are suitable for use in contact with human and animal tissue without more toxicity, irritation, allergic reactions or other problems or complications, commensurate with a reasonable benefit/risk ratio.
  • salts for example, metal salts, ammonium salts, salts with organic bases, salts with inorganic acids, salts with organic acids, salts with basic or acidic amino acids and the like can be mentioned .
  • solvate refers to a substance formed by the association of a compound with a solvent molecule.
  • composition refers to a mixture of one or more compounds of the present application or salts thereof and pharmaceutically acceptable excipients.
  • the purpose of a pharmaceutical composition is to facilitate administration of a compound of the present application to an organism.
  • pharmaceutically acceptable excipients refers to those excipients which are not significantly irritating to the organism and which do not impair the biological activity and properties of the active compound. Suitable excipients are well known to those skilled in the art, such as carbohydrates, waxes, water-soluble and/or water-swellable polymers, hydrophilic or hydrophobic materials, gelatin, oils, solvents, water, and the like.
  • tautomer or "tautomeric form” refers to structural isomers of different energies that are interconvertible via a low energy barrier.
  • proton tautomers also known as proton tautomers
  • proton tautomers include interconversions via migration of protons, such as keto-enol and imine-enamine isomerizations.
  • a specific example of a proton tautomer is an imidazole moiety in which a proton can move between two ring nitrogens.
  • Valence tautomers include interconversions through recombination of some of the bonding electrons.
  • antibody used in its broadest sense, specifically covering whole monoclonal antibodies, polyclonal antibodies, multispecific antibodies (eg bispecific antibodies) formed from at least two whole antibodies, multifunctional antibodies and antibody fragments , as long as they have the desired biological activity.
  • humanized antibody refers to an antibody comprising CDR regions derived from a non-human antibody and the remainder of the antibody molecule derived from one or more human antibodies.
  • mutant is used to refer to a peptide comprising an amino acid sequence derived from the amino acid sequence of the peptide by substitution of one or two or more amino acids with amino acids different from the original peptide, deletion of one or two or more amino acids Wild type amino acids, insertion of one or two or more amino acids not present in wild type, and/or addition of amino acids not present in wild type to the amino terminus (N terminus) and/or carboxy terminus of wild type (C-terminal) (hereinafter, collectively referred to as “mutation"). In this application, “insertion” may also be included in “addition”.
  • CDR complementarity determining region
  • hypervariable region refers to each region of an antibody variable domain that is hypervariable in sequence and/or forms structurally defined loops.
  • Native tetrabodies typically contain six CDRs, three in the heavy chain variable region and three in the light chain variable region.
  • variable region An antibody building block consists of two pairs of polypeptide chains, each pair having one heavy and one light chain, the N-terminal domain of each chain defining a major amino acid of about 100 to 110 or more amino acids The region responsible for antigen recognition is the variable region.
  • Fab refers to containing the constant domain (CL) of the light chain and the first constant domain (CH1) of the heavy chain together with the variable domain VL (light chain variable region) on the light and heavy chains, respectively and VH (variable heavy chain region).
  • the variable domains contain complementarity determining regions (CDRs) that are involved in antigen binding.
  • scFv includes the VH and VL domains of antibodies, wherein these domains are present in a single polypeptide chain.
  • the scFv further comprises a polypeptide linker between the VH and VL domains that enables the scFv to form the structure required for antigen binding.
  • ECD is the extracellular domain.
  • HER receptors are receptor protein tyrosine kinases that belong to the human epidermal growth factor receptor (HER) family and include EGFR, HER2, HER3 and HER4 receptors, HER2 receptors typically contain an extracellular domain that binds HER ligands , a lipophilic transmembrane domain, a conserved intracellular tyrosine kinase domain, and a carboxy-terminal signaling domain with several tyrosine residues that can be phosphorylated, the extracellular domain of HER2 includes four structures domains, ECD1, ECD2, ECD3, and ECD4, respectively.
  • antibody moiety refers to an antibody moiety in an antibody drug conjugate which, in certain specific embodiments, is linked to an intermediate linker moiety through a specific functional group, which antibody moiety can specifically bind to an antigen.
  • linker moiety refers to the part of the antibody drug conjugate that connects the antibody moiety to the cytotoxic drug moiety, which may be cleavable or non-cleavable. Release cytotoxic drugs.
  • cytotoxic drug moiety refers to the cytotoxic drug moiety in the antibody drug conjugate, in some specific schemes, it is linked to the intermediate linker moiety through a functional group, and in tumor cells, the cytotoxic drug will be freed molecules, thereby exerting anti-tumor effect.
  • trastuzumab generic name Trastuzumab, is a recombinant humanized monoclonal antibody that selectively acts on the extracellular site of human epidermal growth factor receptor-4 (HER4) and can be used for therapy HER2-positive cancer, an example of which is the trade name Marketed therapeutic monoclonal antibody products.
  • HER4 human epidermal growth factor receptor-4
  • Pertuzumab generic name Pertuzumab, is a recombinant humanized monoclonal antibody that selectively acts on the extracellular site of human epidermal growth factor receptor-2 (HER2) and can be used for therapy HER2-positive cancer.
  • HER2 human epidermal growth factor receptor-2
  • HER2 is the second member of the EGFR family with tyrosine kinase activity, wherein the expression level of HER2 can be detected according to immunohistochemical detection methods, HER2 positive refers to IHC3+, HER2 negative refers to IHC1+/0, for IHC2+ The situation should be further confirmed by ISH testing.
  • cancer refers to the physiological condition in mammals that is often characterized by unregulated cell growth.
  • triple negative breast cancer refers to breast cancer that is negative for estrogen receptor, progesterone receptor and human epidermal growth factor receptor 2 expression.
  • the percent identity (degree of homology) between sequences can be determined, for example, by using freely available computer programs on the World Wide Web (eg www.ncbi.nlm.nih.gov) commonly used for this purpose (eg with default Set BLASTp or BLASTn) to compare the two sequences to determine.
  • Trastuzumab and Pertuzumab used in the examples of this application are both prepared according to conventional methods for antibodies. First, construct a vector, and then purify and express it after transfecting eukaryotic cells. The sequence of Touzumab is referred to WHO DRUG INFORMATION INN RL78, and the sequence of Pertuzumab is referred to the Examples section of WO0100245.
  • DS-8201 is the active ingredient of the commercial preparation Enhertu of Daiichi Sankyo Company, and its structure is the same as that of Trastuzumab-DXD prepared in this application (see Example 15 for the structure).
  • the Fc part adopts human IgG1, and the variable region sequence of the anti-Her2 arm is based on The sequence of the monoclonal antibody, through the designed linker 1 (ie, (GGGGS) 3 ) will The light and heavy chain variable regions of the monoclonal antibody are connected in series to form an anti-Her2 scFv-Fc (SEQ ID NO: 1), and its amino acid sequence is as follows:
  • anti-Her2-scFv-VL-F53Y-Fc (SEQ ID NO: 3): derived from wild-type anti-Her2 scFv-Fc, with F53Y mutation in the VL region; its amino acid sequence is as follows:
  • anti-Her2-scFv-VL-F53A-Fc (SEQ ID NO: 5): derived from wild-type anti-Her2 scFv-Fc, the VL region has the F53A mutation; its amino acid sequence is as follows:
  • anti-Her2-scFv-VL-F53R-Fc (SEQ ID NO: 7): derived from wild-type anti-Her2 scFv-Fc, the VL region has the F53R mutation; its amino acid sequence is as follows:
  • anti-Her2-scFv-VH-K30E-Fc SEQ ID NO: 9: derived from wild-type anti-Her2 scFv-Fc, the VH region has K30E mutation; its amino acid sequence is as follows:
  • the DNA sequences of anti-Her2 scFv-Fc and its variants were synthesized and cloned into the pcDNA3.1 expression vector, respectively.
  • the expression vector for anti-Her2 scFv-Fc or its variants was co-transfected into ExpiCHO cells (CHO-S, Thermo Corporation) by using the ExpiCHO TM Expression Kit (Thermo Fisher, Cat. No. A29133). Cells were grown in ExpiCHO Expression Medium at 37°C in a humidified atmosphere containing 8% CO2 incubator on an orbital shaker platform rotating at 130 rpm. The culture supernatant was collected for protein purification using protein A magnetic beads (Genscript, cat. no. L00273). Protein concentration was measured by UV-Vis spectrophotometer (NanoDrop lite, Thermo Scientific).
  • Table 1 Sequence information of anti-Her2 scFv-Fc and its variants
  • Example 2 Aggregate verification of anti-Her2 scFv-Fc and its variants and detection of binding to human Her2 antigen
  • Biacore T200 was used for real-time detection. Reaction signals to obtain association and dissociation curves.
  • the buffer used in the experiment was Biacore general buffer (137mM NaCl, 2.7mM KCl, 10mM Na2HPO4 ⁇ 12H2O , 1.8mM KH2PO4 , 0.05% surfactant P20 (GE, catalog number BR- 1000-54), pH 7.4).
  • Anti-hIgG (captured with human antibody capture kit, GE, catalog number 29-2346-00) was coupled to the surface of the CM5 chip and the reaction value reached about 9000RU, and the reaction value after capturing anti-Her2 scFv-Fc or its variants was about 200RU, and then detect the signal value of the interaction between different concentrations of human HER2 protein (100nM, 50nM, 25nM, 12.5nM, 6.25nM, 3.125nM) and anti-Her2 scFv-Fc or its variants.
  • the flow rate of the flow cell was 50 ⁇ l/min, the binding time was 240 s, the dissociation time was 1400 s, the regeneration was performed with 3M MgCl 2 (GE) for 60 s, and the baseline was stable.
  • the results are obtained according to the 1:1 combination of affinity and kinetics in the biacore evaluation software.
  • the affinity of anti-Her2 scFv-Fc or variants to antigenic human Her2 protein is shown in Table 2.
  • has a similar binding affinity to the antigenic human Her2 protein as anti-Her2 scFv-Fc (KD 0.77nM)
  • the variant anti-Her2-scFv- KD 0.54 nM for binding of VH-K30E-Fc to antigenic human Her2 protein. It can be seen that the introduction of F53Y and K30E mutations will not reduce the binding affinity to the antigen human Her2 protein.
  • the components of anti-Her2 scFv-Fc or its variants were separated by gel chromatography, and the components were eluted in descending order of molecular weight.
  • the column is an ACQUITY UPLC Protein BEH SEC Column 1.7 ⁇ m, 4.6*300mm size gel chromatography column, column temperature 25°C.
  • the mobile phase is 50mmol/L phosphate buffer solution-200mmol/L sodium chloride, pH 7.0 (we weigh 2.33g of sodium dihydrogen phosphate dihydrate, 12.53g of sodium hydrogen phosphate dodecahydrate, 11.69g of sodium chloride, add about 800ml ultrapure water, stir until fully dissolved, add ultrapure water to 1000ml, mix well and filter through a 0.22 ⁇ m filter).
  • Table 2 shows the main peak and aggregate content ratio of anti-Her2 scFv-Fc or its variants.
  • the variants anti-Her2-scFv-VL-F53Y-Fc and anti-Her2-scFv-VH-K30E-Fc significantly reduced aggregates from 6.31% of anti-Her2 scFv-Fc to 4.94% and 3.39%, respectively .
  • Table 2 Aggregate content and binding affinity to antigen Her2 of anti-Her2 scFv-Fc and its variants
  • Anti-Her2 bispecific antibodies were produced as human IgGl with Knobs-into-holes (Ridgway, et al., 1996) Fc engineering.
  • the Fc region sequence of one of the heavy chains is designed with H435R and Y436F mutations (Jendeberg et al., 1997) to reduce the affinity of Fc for protein A, which is beneficial to the removal of isoforms formed during the assembly of bispecific antibodies in protein A affinity purification.
  • Source dimer (patent US5945311A).
  • anti-Her2-scFv-VL-F53Y-Fc mutation and anti-Her2-scFv-VH-K30E-Fc mutation can significantly reduce anti-Her2-scFv aggregates without reducing the affinity for Her2 antigen
  • the antigen binding domain of an anti-Her2 arm of the anti-Her2 bispecific antibody is in the form of scFv (VH-linker-VL structure), and the variable region sequence contains the mutation K30E (b-anti-Her2-scFv - VH-K30E-Fc, SEQ ID NO: 21), mutant F53Y (b-anti-Her2-scFv-VL-F53Y-Fc, SEQ ID NO: 23) or both point mutations anti-Her2-scFv - VH-K30E-VL-F53Y-Fc (SEQ ID NO: 11).
  • the antigen-binding domain of the other anti-Her2 arm of the anti-Her2 bispecific antibody is in Fab form, including anti-Her2-domain2-HC-Fc (SEQ ID NO: 13) and anti-Her2-domain2 -LC (SEQ ID NO: 15); additionally an anti-Her2 bispecific antibody was constructed with scFv (VH-linker-VL structure or VL-linker-VH structure) without mutation as a control.
  • the DNA sequences of the anti-Her2 bispecific antibodies (SEQ ID NOs: 12, 14 and 16) were synthesized and cloned into the pcDNA3.1 expression vector, respectively. by using The high-yield expression system (Cat. No.: MIR 6270) combines anti-Her2-scFv-VH-K30E-VL-F53Y-Fc (SEQ ID NO:11), anti-Her2-domain2-HC-Fc (SEQ ID NO:13) and The expression vector of anti-Her2-domain2-LC (SEQ ID NO: 15) was co-transfected into ExpiCHO cells at a transfection ratio of 1:1:1.5. The transfection density was 6 ⁇ 10 6 cells/ml.
  • the medium is Expression medium (Cat. No.
  • fucose knockout anti-Her2 bispecific antibodies were prepared using FUT8- knockout CHO-S cells (designated CHO FUT8-/- cells).
  • the DNA sequences of the anti-Her2 bispecific antibodies (SEQ ID NOs: 12, 14 and 16) were synthesized and cloned into the pcDNA3.1 expression vector, respectively. by using The high-yield expression system (Cat.
  • MIR 6270 uses the expression vectors of anti-Her2-scFv-VH-K30E-VL-F53Y-Fc, anti-Her2-domain2-HC-Fc and anti-Her2-domain2-LC according to the transfection ratio 1:1:1.5 co-transfection into FUT8-knockout CHO-S cells.
  • the transfection density was 6 ⁇ 10 6 cells/ml.
  • the medium is Expression medium (Cat. No. MIR 6200, manufacturer Mirus). Cell culture supernatants were collected by centrifugation on the 10th day after transfection. Protein purification was performed using Protein A magnetic beads (Genscript, cat. no. L00273). Protein concentration was measured by UV-Vis spectrophotometer (NanoDrop lite, Thermo Scientific).
  • This sample was named 23C2 Her2-2.
  • the sequences of Expi Her2-1, Expi Her2-3, Expi Her2-4 and Expi Her2-5 were expressed in CHO FUT8-/- cells, correspondingly to obtain a fucose knockout anti-Her2 bispecific antibody 23C2 Her2-1, 23C2 Her2-3, 23C2 Her2-4 and 23C2 Her2-5.
  • Anti-Her2 bispecific antibody samples expressed in CHO FUT8-/- cells and CHO-S cells were processed using the GlycoWorks RapiFluor-MS N-glycan kit (Waters, Milford, Mass., USA) to convert N-glycans from The N-sugar is released on the protein, and then the N-sugar is labeled, separated by a chromatographic column, and analyzed by an FLR detector (Waters, Milford, Mass., USA), and the structure and content of the N-sugar can be obtained. The content ratio of glycoforms is shown in the table. 4.
  • the proportion of normal anti-Her2 bispecific antibody Expi HER2-2 without fucose is 21.85%, and the anti-Her2 bispecific antibody 23C2 HER2-2 expressed in FUT8- knockout CHO-S cells does not contain rock
  • the algalose ratio is 99.40%.
  • This example relates to the aggregation verification of anti-her2 bispecific antibodies 23C2 Her2-1, 23C2 Her2-2, 23C2 Her2-3, 23C2 Her2-4, 23C2 Her2-5.
  • the anti-her2 bispecific antibody was separated by a gel chromatography column to verify the aggregate content, and each component was washed out in order of molecular weight from large to small.
  • the column is an ACQUITY UPLC Protein BEH SEC Column 1.7 ⁇ m, 4.6*300mm size gel chromatography column, column temperature 25°C.
  • the mobile phase is 50mmol/L phosphate buffer solution-200mmol/L sodium chloride, pH 7.0 (we weigh 2.33g of sodium dihydrogen phosphate dihydrate, 12.53g of sodium hydrogen phosphate dodecahydrate, 11.69g of sodium chloride, add about 800ml ultrapure water, stir until fully dissolved, add ultrapure water to 1000ml, mix well and filter through a 0.22 ⁇ m filter).
  • sample name Aggregates monomer low molecular weight 23C2 Her2-2 8.51% 91.02% 0.47% 23C2 Her2-1 19.55% 80.05% 0.40%
  • the expressed and purified anti-Her2 bispecific antibody was tested by Biacore T200 (GE Company) to determine the affinity of the tested molecule and HER2 protein.
  • the experimental process is described as follows:
  • a certain amount of anti-Her2 bispecific antibody was captured by a chip coupled with Anti-hIgG, and then flowed through human Her2 (Sino Biological, catalog number 10004-H08H) on the surface of the chip, and the reaction signal was detected in real time by Biacore T200, thereby obtaining Binding and dissociation curves.
  • the buffer used in the experiment was Biacore universal buffer (137 mM NaCl, 2.7 mM KCl, 10 mM Na 2 HPO 4 ⁇ 12H 2 O, 1.8 mM KH 2 PO 4 , 0.05% surfactant P20, pH 7.4).
  • Anti-hIgG (captured with human antibody capture kit, GE, catalog number 29-2346-00) was coupled to the surface of the CM5 chip and the reaction value reached about 9000RU, and the reaction value after capturing the anti-Her2 bispecific antibody was about 200RU, and then The signal values of the interaction between different concentrations of Her2 protein (100nM, 50nM, 25nM, 12.5nM, 6.25nM, 3.125nM) and anti-Her2 bispecific antibodies were detected respectively.
  • the flow cell flow rate was 50 ⁇ l/min
  • the binding time was 240 s
  • the dissociation time was 1400 s
  • the baseline was stable for 60 s regeneration with 3M MgCl 2 (GE).
  • Anti-Her2 bispecific antibody 23C2 Her2-2 and control trastuzumab and pertuzumab binding affinities to antigen Her2 are shown in Table 6, KD of anti-Her2 bispecific antibody 23C2 Her2-2 binding to antigen Her2 is 6.11E-10M, the KD of trastuzumab binding to antigen Her2 is 1.22E-09M, the KD of pertuzumab binding to antigen Her2 is 2.35E-09M, anti-Her2 bispecific antibody 23C2 Her2- 2 has a higher affinity for the antigen Her2 than trastuzumab and pertuzumab.
  • Example 6 Referring to the experimental process of Example 6, the expression and purification of anti-Her2 bispecific antibodies 23C2Her2-1, 23C2 Her2-2, 23C2 Her2-3, 23C2 Her2-4, 23C2 Her2-5 and HER2 protein affinity.
  • Human PBMC peripheral blood mononuclear cells
  • NK cells peripheral blood mononuclear cells
  • target cells BT474Her2+++, source: Cell Bank of the Chinese Academy of Sciences Type Culture Collection
  • anti-Her2 antibody was evaluated according to the EC50.
  • In vitro activity of Her2 bispecific antibodies were evaluated according to the EC50.
  • BT474 cells were adjusted to 3 ⁇ 10 5 cells/ml in 1640 experimental medium containing 2% FBS (fetal bovine serum), and 50 ⁇ l per well were inoculated into 96-well cell culture plates (eppendorf, catalog number: 0030730199 ).
  • FBS fetal bovine serum
  • Target cell + effector cell + antibody target cell group
  • target cell group BT474 cell
  • effector cell group human PBMC
  • target cell + effector cell group blank control group (medium) and lysate control group
  • the effect-to-target ratio was 10:1.
  • 20 ⁇ l/well of lysate Promega, Cat. No. G182A
  • Nonradioactive cytotoxicity assay kit cytotox 96 nonradioactive cytotoxicity assay, Promega, G1780 was used to measure the cell lysis rate.
  • Lysis rate (%) (OD administration group- OD target cell+effector cell group )/(OD target cell maximum release group- OD target cell group ) ⁇ 100%
  • FIG. 1 shows the killing rate of anti-Her2 bispecific antibodies on BT474Her2+++ tumor cells.
  • ADCC-enhanced anti-Her2 bispecific antibodies (denoted as 23C2 HER2-1 and 23C2 HER2-2 in Figure 1) were more effective in killing BT474 tumor cells than the combined trastuzumab + pertuzumab, Anti-Her2 bispecific antibodies expressed in CHO-S (Expi HER2-1 and Expi HER2-2); the EC50 of the combination of Trastuzumab + Pertuzumab (1:1) is 8.627ng/ ml, the EC50 of Expi HER2-1 is 38.05ng/ml, Expi HER2-2 is better than Expi HER2-1, its EC50 is 35.17ng/ml; the EC50 of ADCC-enhanced 23C2 HER2-1 is 4.728ng/ml, ADCC-enhanced The 23C2 HER2-2 was superior to the ADCC-enhance
  • NCI-N87Her2++ source: Cell Bank of the Chinese Academy of Sciences Type Culture Collection
  • NK cells from human PBMC (peripheral blood mononuclear cells), and evaluated according to the EC50 size In vitro activity of anti-Her2 bispecific antibodies.
  • NCI-N87 cells were adjusted to 3 ⁇ 10 5 cells/ml in 1640 experimental medium containing 2% FBS (fetal bovine serum), and 50 ⁇ l per well were inoculated into 96-well cell culture plates (eppendorf, Catalog No. : 0030730199).
  • Different concentrations 8.1nM, 2.7nM, 0.9nM, 0.3nM, 0.1nM, 0.03nM, 0.01nM, 0.003nM, 0.001nM, 0.0004nM
  • Different concentrations of antibody or control drug were added to the above 96-well cell culture plate, 50 ⁇ l per well.
  • Human PBMC were adjusted to a cell density of 1.5 ⁇ 10 6 cells/ml using experimental medium, 100 ⁇ l per well.
  • Set up administration group target cells + effector cells + antibody or control drug
  • target cell group NCI-N87 cells
  • effector cell group human PBMC
  • target cell + effector cell group blank control group (medium)
  • the lysate control group and the target cell maximum release group had an effect-to-target ratio of 10:1. 45 min before detection, 20 ⁇ l/well of lysate (Promega, Cat. No. G182A) was added to the target cell maximum release group and the lysate control group.
  • Nonradioactive cytotoxicity assay kit cytotox 96 nonradioactive cytotoxicity assay, Promega, G1780
  • trastuzumab T-DM1 (trastuzumab-Maytansine conjugate, trade name )
  • trastuzumab + Pertuzumab (1:1), Expi HER2-1 as control drugs.
  • Lysis rate (%) (OD administration group- OD target cell+effector cell group )/(OD target cell maximum release group- OD target cell group ) ⁇ 100%
  • FIG. 2 shows the killing rate of anti-Her2 bispecific antibodies against NCI-N87 tumor cells.
  • ADCC-enhanced anti-Her2 bispecific antibody 23C2 HER2-2 kills NCI-N87 tumor cells better than combined trastuzumab + pertuzumab, trastuzumab, T-DM1 and Expi HER2-1;
  • the EC50 of ADCC-enhanced 23C2 HER2-2 is 0.02447nM
  • the EC50 of trastuzumab + pertuzumab combination is 0.08267nM
  • the EC50 of Expi HER2-1 is 0.1048nM
  • the EC50 of T-DM1 The EC50 was 0.07392 nM and the EC50 of trastuzumab was 0.07468 nM.
  • JIMT-1 source: AddexBio, Catalog#: C0006005
  • NK cells peripheral blood mononuclear cells
  • anti-Her2 was evaluated according to the EC50 size In vitro activity of bispecific antibodies.
  • JIMT-1 cells were adjusted to 3 ⁇ 10 5 cells/ml using 1640 experimental medium containing 2% FBS (fetal bovine serum), and 50 ⁇ l per well were inoculated into 96-well cell culture plates (eppendorf, Catalog No. : 0030730199).
  • Different concentrations 88.1nM, 2.7nM, 0.9nM, 0.3nM, 0.1nM, 0.03nM, 0.01nM, 0.003nM, 0.001nM, 0.0004nM
  • Different concentrations of antibody or control drug were added to the above 96-well cell culture plate, 50 ⁇ l per well.
  • Human PBMC were adjusted to a cell density of 1.5 ⁇ 10 6 cells/ml using experimental medium, 100 ⁇ l per well.
  • Set up administration group target cells + effector cells + antibody or control drug
  • target cell group BT474 cells
  • effector cell group human PBMC
  • target cell + effector cell group blank control group (medium) and lysate
  • the effect-to-target ratio was 20:1. 45 min before detection, 20 ⁇ l/well of lysate (Promega, Cat. No. G182A) was added to the target cell maximum release group and the lysate control group.
  • Nonradioactive cytotoxicity assay kit (cytotox 96 nonradioactive cytotoxicity assay, Promega, G1780) was used to measure the cell lysis rate.
  • Lysis rate (%) (OD administration group- OD target cell+effector cell group )/(OD target cell maximum release group- OD target cell group ) ⁇ 100%
  • FIG. 3 shows the killing rate of JIMT-1 tumor cells by anti-Her2 bispecific antibodies.
  • ADCC-enhanced anti-Her2 bispecific antibody 23C2 HER2-2 kills JIMT-1 tumor cells better than combined trastuzumab + pertuzumab, trastuzumab, T-DM1, Expi HER2-1; where ADCC-enhanced 23C2 HER2-2 has an EC50 of 0.01006nM, trastuzumab + pertuzumab combination has an EC50 of 0.06727nM, Expi HER2-1 has an EC50 of 0.08066nM, T-DM1 The EC50 is 0.08357nM for trastuzumab and 0.07443nM for trastuzumab, in addition, the anti-Her2 bispecific antibody 23C2 HER2-2 has a higher cell lysis rate.
  • NCI-N87 Her2++ gastric cancer cells Cell Bank of the Type Culture Collection, Chinese Academy of Sciences. Prepare NCI-N87 Her2++ gastric cancer cells at a concentration of 5 ⁇ 10 7 cells/ml*0.1ml/cell, inoculated into nude mice (from Changzhou Cavens Laboratory Animal Co., Ltd., nude mice, 14-17g) under sterile conditions , male, rearing environment: SPF grade) under the right armpit.
  • the diameter of the transplanted tumor in nude mice was measured with a vernier caliper. After the tumor grew to 100-250 mm, the animals were divided into 5 groups:
  • Group 5 Per+Tra (trastuzumab+pertuzumab), 5mg/kg+5mg/kg
  • Each administration group was administered intravenously according to the corresponding dose, twice a week, for about 3 weeks (6 times). Except group 5, the administration volume of the two drugs in the combination group was 5ml/kg, and the administration volume of the other groups was 10ml/kg.
  • Group 1 was simultaneously administered i.v. 10 ml/kg PBS (Hyclone; Cat. No.: sh30256.01). When the combination group was administered, trastuzumab was administered at least 30 minutes after the administration of Pertuzumab.
  • the anti-tumor effect of the test substance was dynamically observed, the tumor volume was measured 2-3 times a week, the mice were weighed at the same time, and the data were recorded; the general performance of the mice was observed daily.
  • Tumor volume (TV)
  • Step 4 D5([( ⁇ N-[(9H-fluoren-9-ylmethoxy)carbonyl]glycyl ⁇ amino)methoxy]acetyl-N-[(2- ⁇ [(1S,9S) -9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de ]pyrano[3',4':6'7']indolizino[1,2-b]quinolin-1-yl]amino ⁇ -2-oxoethoxy)methyl]glycinamide )Synthesis
  • Step 5 ( ⁇ [(glycyl)amino]methoxy ⁇ acetyl-N-[(2- ⁇ [(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl Base-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6'7']indium Synthesis of azino[1,2-b]quinolin-1-yl]amino ⁇ -2-oxoethoxy)methyl]glycinamide)
  • DXD was prepared with reference to the method disclosed in Example 76 in the specification of patent WO2014057687.
  • Solution A pH7.4 PBS buffer
  • Solution B 10 mM TCEP (tris(2-carboxyethyl)phosphine hydrochloride) in water
  • Solution D Histidine buffer (containing 0.89mg/ml L-histidine and 4.04mg/ml L-histidine hydrochloride monohydrate)
  • Solution E 700 mg/ml sucrose solution (prepared with solution D)
  • Solution F 20 mg/ml Tween 80 (prepared with Solution D)
  • Antibodies Trastuzumab, 23C2 Her2-2
  • linker-payload (linker-cytotoxic drug part): MC-GGFG-DXD, MC-GGFG-DDDXD
  • the DAR value was determined by LC-MS method. Take 50 ⁇ g of the prepared ADC sample, add 1 ⁇ l of glycosidase PNGase F (Shui On Bio, China), and incubate at 37°C for 20 hours.
  • the mass spectrometer used in the experiment was a high-resolution Xevo G2-XS (Waters, USA).
  • the sample concentration was adjusted to 5 ⁇ M, and mass spectrometry data were collected in positive ion mode using the direct injection method.
  • the acquired nondenaturing mass spectrometry data were analyzed and processed using the software UNIFI1.8.2.169 (Waters, USA).
  • the protein concentration was detected by the lowry method.
  • Trastuzumab, 23C2 Her2-2 was used as a standard. Measure the absorbance values of the standard and the prepared ADC samples at the OD650 wavelength with a microplate reader, fit the standard curve, bring the sample absorbance values into the standard curve, and calculate the protein concentration.
  • Negative control 14 ⁇ l ultrapure water+2 ⁇ l 10 ⁇ DNA Topoismerasel Buffer+2 ⁇ l 0.1%BSA+1 ⁇ l pBR322DNA+1 ⁇ l topoisomerase I working solution;
  • anti-Her2 double antibody had stronger antigen binding activity than trastuzumab
  • anti-Her2 double antibody ADC had stronger antigen binding activity than trastuzumab ADC.
  • DS-8201, and control IgG1 a non-HER2 target-specific IgG1; Yiqiao Shenzhou, product number HG1K
  • DDDXD conjugate IgG1-DDDXD were used as controls.
  • the serially diluted sample solution and the labeled endocytosis reagent (sartorius, 90564) were added to the cell plate, 20 ⁇ l per well, and incubated at 37° C. for 15 min.
  • the 96-well cell culture plate was taken out, the above two kinds of cells were added at 20 ⁇ l/well, the cell culture plate was placed at 37° C., and incubated for 2 h.
  • the 96-well cell culture plate was taken out and placed in a flow cytometer to measure the signal intensity.
  • DXD and DDDXD were pre-diluted to 140000ng/ml in culture medium, marked as S1, and then subjected to five-fold gradient dilution to obtain their corresponding reference samples S1-S9.
  • the final drug concentration range is 35000ng/ml ⁇ 0.0896ng/ml , a total of 9 concentrations.
  • DDDXD showed stronger tumor cell proliferation inhibitory activity than DXD.
  • the antibody to be tested and the antibody-drug conjugate prepared in Example 15 were pre-diluted to 20 ⁇ g/ml in culture medium, marked as S1, and then subjected to five-fold gradient dilution to obtain corresponding samples S1 to S9.
  • the final drug concentration range is 5000ng/ml ⁇ 0.0128ng/ml, with 9 concentrations in total.
  • the anti-Her2 dual-antibody ADC has better cell killing ability than the trastuzumab ADC.
  • Each incubation system contains phosphate buffered saline (PBS, pH 7.4), liver microsomal protein, substrate (acetonitrile solution of the sample to be tested) and NADPH, and incubated in a 37°C water bath, respectively, at reaction 0, 5, 15, After 30 and 60 min, the same volume of ice-cold acetonitrile was added to terminate the reaction. Negative controls were incubated with heat-inactivated liver microsomes of the corresponding species. The remaining content of the original substrate was detected by LC/MS/MS method.
  • PBS phosphate buffered saline
  • substrate acetonitrile solution of the sample to be tested
  • NADPH acetonitrile solution of the sample to be tested
  • Negative controls were incubated with heat-inactivated liver microsomes of the corresponding species. The remaining content of the original substrate was detected by LC/MS/MS method.
  • model group vehicle (including: L-histidine 0.89 mg/ml, L-histidine hydrochloride 4.04 mg/ml , polysorbate 80 0.3mg/ml, sucrose 90mg/ml), 6 rats; 23C2 Her2-2-DXD group: 1.68 mg/kg, qw, iv 6 rats; 23C2 Her2-2-DDDXD group: 1.59 mg/kg, qw, iv6 only.
  • the tumor volume was measured 2-3 times a week, the mice were weighed at the same time, and the data were recorded; the animal performance was observed daily.
  • Wt 0 is the animal body weight at the time of administration in separate cages (ie d0), and Wt is the animal body weight at each measurement.
  • TGI tumor growth inhibition
  • TW is the tumor weight in the administration group
  • TW 0 is the tumor weight in the model group.

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