WO2023280092A1 - 抗体药物偶联物及其应用 - Google Patents

抗体药物偶联物及其应用 Download PDF

Info

Publication number
WO2023280092A1
WO2023280092A1 PCT/CN2022/103592 CN2022103592W WO2023280092A1 WO 2023280092 A1 WO2023280092 A1 WO 2023280092A1 CN 2022103592 W CN2022103592 W CN 2022103592W WO 2023280092 A1 WO2023280092 A1 WO 2023280092A1
Authority
WO
WIPO (PCT)
Prior art keywords
antibody
drug conjugate
heavy chain
conjugate according
group
Prior art date
Application number
PCT/CN2022/103592
Other languages
English (en)
French (fr)
Other versions
WO2023280092A9 (zh
Inventor
徐霆
王媲琳
郭康平
赵永浩
彭建建
Original Assignee
江苏康宁杰瑞生物制药有限公司
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 江苏康宁杰瑞生物制药有限公司 filed Critical 江苏康宁杰瑞生物制药有限公司
Priority to CN202280047851.4A priority Critical patent/CN117616052A/zh
Priority to US18/575,293 priority patent/US20240307551A1/en
Priority to EP22836843.7A priority patent/EP4386006A1/en
Priority to JP2024500541A priority patent/JP2024525624A/ja
Publication of WO2023280092A1 publication Critical patent/WO2023280092A1/zh
Publication of WO2023280092A9 publication Critical patent/WO2023280092A9/zh

Links

Images

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6849—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a receptor, a cell surface antigen or a cell surface determinant
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A61K47/68037—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a camptothecin [CPT] or derivatives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6851—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a determinant of a tumour cell
    • A61K47/6855—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a determinant of a tumour cell the tumour determinant being from breast cancer cell
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6873—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting an immunoglobulin; the antibody being an anti-idiotypic antibody
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6875—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody being a hybrid immunoglobulin
    • A61K47/6879—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody being a hybrid immunoglobulin the immunoglobulin having two or more different antigen-binding sites, e.g. bispecific or multispecific immunoglobulin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6889—Conjugates wherein the antibody being the modifying agent and wherein the linker, binder or spacer confers particular properties to the conjugates, e.g. peptidic enzyme-labile linkers or acid-labile linkers, providing for an acid-labile immuno conjugate wherein the drug may be released from its antibody conjugated part in an acidic, e.g. tumoural or environment
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/32—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against translation products of oncogenes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/40—Immunoglobulins specific features characterized by post-translational modification
    • C07K2317/41—Glycosylation, sialylation, or fucosylation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C07K2317/732—Antibody-dependent cellular cytotoxicity [ADCC]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

  • This application relates to the field of biomedicine, in particular to an antibody-drug conjugate and its application.
  • HER receptor tyrosine kinase family are important mediators of cell growth, differentiation and survival.
  • This family of receptors includes four distinct members, including epidermal growth factor receptor (EGFR, ErbB1, or HER1), HER2 (ErbB2 or p185neu), HER3 (ErbB3) and HER4 (ErbB4 or tyro2).
  • EGFR epidermal growth factor receptor
  • HER2 ErbB2 or p185neu
  • HER3 ErbB3
  • HER4 ErbB4 or tyro2
  • Bispecific antibodies are immunoglobulin molecules that contain two different ligand binding sites. It replaces the same sequence of the two arms of the classic antibody Fab, but uses two different Fab sequences, so the Y-shaped arms can bind different epitopes.
  • the use of bispecific antibodies in cancer therapy has been reviewed in several literatures (Carter 2001; Chames and Baty 2009; Chames and Baty 2009).
  • Antibody-drug conjugates obtained by linking a cytotoxic drug to an antibody that binds to an antigen expressed on the surface of cancer cells and can be internalized into cells can selectively deliver drugs to cancer cells, and it is expected that The drug accumulates in the cancer cells and kills the cancer cells.
  • the present application provides an antibody-drug conjugate, including a bispecific antibody targeting HER2 or an antigen-binding fragment thereof.
  • the antibody drug conjugate can specifically bind at least one (eg, at least 2) epitopes in human HER2.
  • the antibody-drug conjugates described in this application can have the following properties: (1) effectively kill tumor cells, wherein the antibody part and drug part can play a synergistic function of killing tumors; (2) specifically bind tumor cells; ( 3) good stability in serum; (4) stronger bystander killing effect on tumor cells; (5) good ADCC effect; and/or (6) better endocytic efficiency.
  • the present application also provides a preparation method of the antibody-drug conjugate, a composition comprising the antibody-drug conjugate, and applications of the antibody-drug conjugate and the composition.
  • the present application provides an antibody drug conjugate comprising a bispecific antibody targeting HER2 or an antigen-binding fragment thereof.
  • the HER2-targeting bispecific antibody or antigen-binding fragment thereof specifically binds at least one epitope of human HER2.
  • the HER2-targeting bispecific antibody or antigen-binding fragment thereof specifically binds the extracellular domain II of human HER2 and/or the extracellular domain IV of human HER2.
  • the HER2-targeting bispecific antibody or antigen-binding fragment thereof comprises a first light chain and a second light chain.
  • the first light chain comprises a first LCDR1-3, wherein the first LCDR1 comprises the amino acid sequence shown in SEQ ID NO.4.
  • the first LCDR2 comprises the amino acid sequence shown in SEQ ID NO.5.
  • the first LCDR3 comprises the amino acid sequence shown in SEQ ID NO.6.
  • the first light chain is capable of binding to the heavy chain of Pertuzumab.
  • the second light chain comprises a second LCDR1-3, wherein the second LCDR1 comprises the amino acid sequence shown in SEQ ID NO.1.
  • the second LCDR2 comprises the amino acid sequence shown in SEQ ID NO.2.
  • the second LCDR3 comprises the amino acid sequence shown in SEQ ID NO.3.
  • the second light chain is capable of binding to the heavy chain of trastuzumab.
  • variable regions of the first light chain and the second light chain comprise the amino acid sequence shown in any one of SEQ ID NOs. 7-12.
  • variable regions of the first light chain and the second light chain comprise the amino acid sequence shown in SEQ ID NO.7.
  • said first light chain and said second light chain comprise the amino acid sequence set forth in any one of SEQ ID NOs. 13-18.
  • the first light chain is selected from the group consisting of a light chain of Pertuzumab or a mutant thereof and a light chain of Trastuzumab or a mutant thereof; and/or, the The second light chain is selected from the group consisting of the light chain of Pertuzumab or a mutant thereof and the light chain of Trastuzumab or a mutant thereof.
  • the first light chain and the second light chain have the same amino acid sequence.
  • said first light chain and said second light chain comprise the amino acid sequence set forth in SEQ ID NO.13.
  • the HER2-targeting bispecific antibody or antigen-binding fragment thereof comprises a first heavy chain and a second heavy chain, wherein the first heavy chain can be expressed under physiological conditions or in vitro. correctly bind to the first light chain.
  • the second heavy chain can correctly combine with the second light chain under physiological conditions or protein expression in vitro.
  • the first heavy chain comprises a first heavy chain variable region that is the heavy chain variable region of Pertuzumab.
  • the second heavy chain comprises a second heavy chain variable region that is the heavy chain variable region of trastuzumab.
  • said first heavy chain and said second heavy chain comprise a heavy chain constant region, wherein said heavy chain constant region is derived from a human IgG constant region.
  • the Fc fragment of the first heavy chain and the second heavy chain comprises the amino acid sequence of any one of SEQ ID NOs. 25-57.
  • the first heavy chain comprises the amino acid sequence shown in 21 or 23.
  • the second heavy chain comprises the amino acid sequence set forth in 22 or 24.
  • the antibody drug conjugate comprises the structure shown in Formula 1:
  • M (L1) a -(L2) b -D (Formula 1), wherein M represents the HER2-targeting bispecific antibody or its antigen-binding fragment described in this application; L1 represents the linker connected to M, and L2 Represents the linker connected to D, a, b are each independently selected from 0-10, and D represents the drug.
  • said L1 and/or L2 are selected from the group consisting of cleavable linkers, non-cleavable linkers, hydrophilic linkers, hydrophobic linkers, charged linkers, Charged linkers and dicarboxylic acid-based linkers.
  • the L1 is connected to the M through a sulfhydryl group, an azido group or an amide group on the M.
  • said M comprises a first heavy chain and a second heavy chain, said first heavy chain and/or said second heavy chain comprising an attachment site capable of attachment to said L1.
  • the linking site comprises a group capable of linking to the L1 after deglycosylation modification.
  • the group is located at the side group of amino acid Q at position 297 of the first heavy chain; and/or at the side group of amino acid Q at position 298 of the second heavy chain.
  • the groups include amido groups.
  • the linking site comprises a group capable of linking to the L1 after glycosylation modification.
  • the group is located at the side group of amino acid N at position 299 of the first heavy chain; and/or at the side group of amino acid N at position 300 of the second heavy chain.
  • the group comprises -N 3 .
  • said glycosylated modification comprises: said M has been contacted with UDP-GalNAz, ⁇ -1,4-galactosyltransferase, or a variant thereof.
  • the L1 is capable of participating in a SPAAC reaction.
  • the L1 is selected from the group consisting of maleimide, succinimid-3-yl-N and DBCO.
  • the L1 is DBCO-(PEG) n1 , wherein n1 is an integer of 0-10, or, the L1 is maleimide.
  • the L2 is selected from the group consisting of polypeptides, VC-PAB, N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), N-succinimidyl Imidyl 4-(2-pyridyldithio)pentanoate (SPP), N-succinimidyl 4-(2-pyridyldithio)butyrate (SPDB), N-succinimidyl Imido-4-(2-pyridyldithio)-2-sulfobutyrate (sulfo-SPDB), N-succinimidyl iodoacetate (SIA), N-succinyl Amino (4-iodoacetyl) aminobenzoate (SIAB), maleimide PEG NHS, N-4-(maleimidomethyl) cyclohexyl carboxylate succinamido ester (SMCC ), N-sulfo-(
  • the L2 is GGFG.
  • the drug has the ability to kill tumor cells and/or inhibit the growth of tumor cells.
  • the drug comprises a small molecule drug.
  • the drug is selected from the group consisting of V-ATPase inhibitors, Bcl2 inhibitors, MCL1 inhibitors, HSP90 inhibitors, IAP inhibitors, mTor inhibitors, microtubule stabilizers, microtubule destabilizing agents agent, auristatin, dolastatin, maytansinoid, MetAP (methionine aminopeptidase), nuclear export inhibitor of protein CRM1, DPPIV inhibitor, proteasome inhibitor, phosphotransfer reaction inhibitor in mitochondria, protein synthesis inhibitor CDK2 inhibitors, CDK9 inhibitors, kinesin inhibitors, HDAC inhibitors, DNA disruptors, DNA alkylating agents, DNA intercalators, DNA minor groove binders, DHFR inhibitors, nucleoside analogs, HDAC inhibitors agents, anthracyclines, NAMPT inhibitors, SN-38 glucuronic acid, etoposide phosphate, nitrogen mustard, proteosome inhibitors, cytokines, and Toll-like receptor agonist
  • the drug is selected from the group consisting of DM1, exatecan, DXd, MMAE, SN-38, Calicheamicin, Anthracyclin-5G, DM4, microtubule inhibitor SHR153024, PNU-159682, Duo5 toxin, SN38 derived substances or their derivatives.
  • the antibody drug conjugate has a structure selected from the group consisting of:
  • the antibody drug conjugate has a drug/antibody ratio of about 2-6.
  • the application provides a compound for preparing the antibody-drug conjugate described in the application, which has the structure shown in Formula 2: M-(L1) a (Formula 2), wherein M represents the compound described in the application.
  • M represents the compound described in the application.
  • L1 represents a linker connected to M;
  • a is selected from 0-10.
  • the L1 is selected from the group consisting of cleavable linkers, non-cleavable linkers, hydrophilic linkers, hydrophobic linkers, charged linkers, uncharged linkers and dicarboxylic acid-based linkers.
  • the L1 is connected to the M through a sulfhydryl group, an azido group or an amide group on the M.
  • said M comprises a first heavy chain and a second heavy chain, said first heavy chain and/or said second heavy chain comprising an attachment site capable of attachment to said L1.
  • the linking site comprises a group capable of linking to the L1 after deglycosylation modification.
  • the group is located at the side group of amino acid Q at position 297 of the first heavy chain; and/or at the side group of amino acid Q at position 298 of the second heavy chain.
  • the groups include amido groups.
  • the linking site comprises a group capable of linking to the L1 after glycosylation modification.
  • the group is located at the side group of amino acid N at position 299 of the first heavy chain; and/or at the side group of amino acid N at position 300 of the second heavy chain.
  • the group comprises -N 3 .
  • said glycosylated modification comprises: said M has been contacted with UDP-GalNAz, ⁇ -1,4-galactosyltransferase, or a variant thereof.
  • the L1 is capable of participating in a SPAAC reaction.
  • the L1 is selected from the group consisting of maleimide, succinimid-3-yl-N and DBCO.
  • the L1 is DBCO-(PEG) n1 , wherein n1 is an integer of 0-10, or, the L1 is maleimide.
  • the compound comprises a structure selected from the group consisting of:
  • the present application provides a method for preparing the antibody-drug conjugate described in the present application, which includes the following steps: contacting the compound described in the present application with the drug described in the present application.
  • the present application provides a pharmaceutical composition, which comprises the antibody-drug conjugate described in the present application, or a pharmaceutically acceptable carrier.
  • the present application provides a method for regulating the tumor microenvironment of a subject, which includes the following steps: administering the antibody-drug conjugate described in the present application, or the pharmaceutical composition described in the present application to the subject.
  • the present application provides a method for regulating the immune response of a subject, which includes the following steps: administering the antibody-drug conjugate described in the present application, or the pharmaceutical composition described in the present application to the subject.
  • the present application provides the application of the antibody-drug conjugate described in the present application, or the pharmaceutical composition described in the present application in the preparation of medicines, wherein the medicines can prevent and/or treat tumors.
  • the tumor comprises solid tumors and/or non-solid tumors.
  • Figure 1 shows the glycosylation modification reaction of the HER2-targeting bispecific antibody described in this application.
  • Fig. 2 shows the HPLC profile of the glycosylation-modified HER2-targeting bispecific antibody described in the present application.
  • Figure 3 shows the reaction steps for obtaining the antibody-drug conjugates described in this application.
  • FIG. 4 shows the HPLC profile of the antibody drug conjugate described in this application.
  • Figure 5 shows the reaction steps for obtaining the antibody drug conjugates described in this application.
  • Figure 6 shows the Waters Xevo G2-QTOF mass spectrometry analysis results of the antibody-drug conjugate described in this application.
  • Figure 7 shows the Waters Xevo G2-QTOF mass spectrometry analysis results of the antibody-drug conjugate described in this application.
  • Figure 8 shows the results of Waters Xevo G2-QTOF mass spectrometry analysis of the light chain fraction in DS-8201.
  • Figure 9 shows the results of Waters Xevo G2-QTOF mass spectrometry analysis of the heavy chain portion of DS-8201.
  • Figure 10 shows the binding affinity of the HER2-targeting bispecific antibody described in this application to Fc ⁇ RIIIa.
  • Figure 11 shows the binding affinity of the antibody drug conjugates described in this application to Fc ⁇ RIIIa.
  • Figure 12 shows the binding affinity of the HER2-targeting bispecific antibody described in this application to Fc ⁇ RI.
  • Figure 13 shows the binding affinity of the antibody drug conjugates described in this application to Fc ⁇ RI.
  • Figure 14 shows the binding ability of the antibody drug conjugates described in this application to tumor cells.
  • Figure 15 shows the killing ability of the antibody drug conjugates described in this application on tumor cells treated for 3 days.
  • Figure 16 shows the killing ability of the antibody-drug conjugate described in the present application on tumor cells treated for 5 days.
  • Figure 17 shows the killing ability of the antibody drug conjugates described in this application on tumor cells treated for 3 days.
  • Fig. 18 shows the results of inhibition of proliferation of SK-BR-3 cells in each experimental group.
  • Figure 19 shows the results of inhibition of the proliferation of MDA-MB-468 cells in each experimental group.
  • Figure 20 shows the analysis results of the ADCC activity of the antibody drug conjugates described in this application.
  • Figure 21 shows the steps of measuring the ADCC activity of the antibody-drug conjugate described in this application using the PBMC system.
  • Figures 22-23 show the results of measuring the ADCC activity of the antibody-drug conjugate described in this application using the PBMC system.
  • FIGS 24-26 show the endocytosis of the antibody drug conjugates described in this application in tumor cells.
  • Figure 27 shows the endocytosis of the antibody drug conjugates described in this application in tumor cells.
  • Figure 28 shows the pharmacokinetic study results of the antibody-drug conjugates described in this application.
  • Figure 29 shows the results of the ratio of NCI-N87 positive cells after endocytosis induced by the antibody drug conjugates described in this application.
  • Figure 30 shows the growth curves of the tumor volumes of mice in each group in the PDX tumor model of human gastric cancer.
  • Figure 31 shows the growth curve of the tumor volume of the human gastric cancer HER2 IHC-PDX tumor model mice.
  • antibody drug conjugate generally refers to an ADC, ie, a binding protein (eg, an antibody or antigen-binding fragment thereof) linked to one or more chemical drugs.
  • the chemical drug can be any therapeutic and/or cytotoxic agent.
  • the antibody drug conjugate can have any number from 1-8 of drugs conjugated to the antibody, for example can include 2, 4, 6 or 8 drug loaded species.
  • the drugs may include mitotic inhibitors, anti-tumor antibiotics, immunomodulators, vectors for gene therapy, alkylating agents, anti-angiogenic agents, antimetabolites, boron-containing agents, chemoprotective agents, Hormones, antihormones, corticosteroids, phototherapeutics, oligonucleotides, radionuclide agents, topoisomerase inhibitors, tyrosine kinase inhibitors, and/or radiosensitizers.
  • the term “drug/antibody ratio” or “DAR” generally refers to the number of drug linked to the antibody of the ADC.
  • the DAR of an ADC can range from 1 to 8, or higher loadings (eg, 10), and the range of DAR can depend on the number of attachment sites on the antibody.
  • the DAR can be the number of drugs loaded on a single antibody.
  • the DAR can also be the average or average DAR of a group of ADCs.
  • HER2 generally refers to human epidermal growth factor receptor 2 (SwissProt P04626).
  • the HER2 can also be called rbB-2, NEU, HER-2 or CD340.
  • the HER2 may include any variant, isoform, and species homologue of HER2 that is naturally expressed by cells, including tumor cells, or expressed by cells transfected with the HER2 gene or cDNA.
  • extracellular domain generally refers to the extracellular domain of human HER2 (SwissProt P04626), which may include four extracellular domains I, II, III and IV.
  • antibody generally refers to an immunoglobulin molecule composed of two identical pairs of polypeptide chains, each pair having a "light” (L) chain and a “heavy” (H) chain.
  • Antibody light chains can be classified as kappa and lambda light chains.
  • Heavy chains can be classified as mu, delta, gamma, alpha, or epsilon, and define the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively.
  • the variable and constant regions are joined by a "J" region of about 12 or more amino acids, with the heavy chain also comprising a "D" region of about 3 or more amino acids.
  • Each heavy chain is composed of a heavy chain variable region (VH) and a heavy chain constant region (CH).
  • the heavy chain constant region consists of 3 domains (CH1, CH2 and CH3).
  • Each light chain is composed of a light chain variable region (VL) and a light chain constant region (CL).
  • the light chain constant region consists of one domain, CL.
  • the constant regions of the antibodies mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (eg, effector cells) and the first component (Clq) of the classical complement system.
  • the VH and VL regions can also be subdivided into regions of high variability called complementarity determining regions (CDRs) interspersed with more conserved regions called framework regions (FRs).
  • CDRs complementarity determining regions
  • Each VH and VL consists of 3 CDRs and 4 FRs arranged in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4, from amino-terminus to carboxy-terminus.
  • the variable regions (VH and VL) of each heavy chain/light chain pair form the antibody binding site, respectively. Assignment of amino acids to regions or domains follows the Kabat Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)), or Chothia & Lesk (1987) J.Mol.Biol.196:901-917 ; Definition by Chothia et al. (1989) Nature 342:878-883.
  • antibody is not limited to any particular method of producing antibodies. For example, it includes, inter alia, recombinant antibodies, monoclonal antibodies and polyclonal antibodies. Antibodies can be of different isotypes, eg, IgG (eg, IgGl, IgG2, IgG3, or IgG4 subtype), IgAl, IgA2, IgD, IgE, or IgM antibodies.
  • IgG eg, IgGl, IgG2, IgG3, or IgG4 subtype
  • IgAl IgA2, IgD, IgE, or IgM antibodies.
  • antigen-binding portion refers to one or more portions of a full-length antibody that retain the ability to bind to the same antigen (e.g., HER2) to which the antibody binds, competing with the intact antibody for antigen specificity. sexual union. See generally, Fundamental Immunology, Ch.7 (Paul, W., ed., 2nd ed., Raven Press, N.Y. (1989), which is incorporated herein by reference in its entirety for all purposes. or by enzymatic or chemical cleavage of intact antibodies to produce antigen-binding portions.
  • antigen e.g., HER2
  • antigen-binding portions include Fab, Fab', F(ab')2, Fd, Fv, dAb, and complementarity determining region (CDR) fragments, Single-chain antibodies (for example, scFv), chimeric antibodies, diabodies (diabodies) and such polypeptides, which comprise at least a portion of an antibody sufficient to confer polypeptide-specific antigen-binding ability.
  • CDR complementarity determining region
  • an antigen-binding portion of an antibody e.g., the antibody fragments described above
  • a given antibody e.g., monoclonal antibody 2E12
  • the method is to screen the antigen-binding portion of the antibody for specificity.
  • Fd fragment means an antibody fragment consisting of VH and CH1 domains
  • Fv fragment means an antibody fragment consisting of VL and VH domains of a single arm of an antibody
  • dAb fragment means an antibody fragment consisting of VH domains (Ward et al., Nature 341:544 546 (1989))
  • Fab fragment means an antibody fragment consisting of VL, VH, CL and CH1 domains
  • F(ab')2 fragment means an antibody fragment comprising two Fab fragments connected by a disulfide bridge at the hinge region.
  • antibody Fc generally refers to the constant region of a human immunoglobulin chain, defined based on papain cleavage of antibodies.
  • the Fc may be the carboxy-terminal or a part of the constant region of an immunoglobulin heavy chain.
  • an immunoglobulin Fc region may comprise two or more domains of heavy chains CH2, CH3, CH4 in combination with an immunoglobulin hinge region.
  • immunoglobulins can be divided into different classes, there are five main classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, some of which are further divided into subclasses (isotypes) , such as IgG-1, IgG-2, IgG-3, IgG-4, IgA-1 and IgA-2.
  • subclasses such as IgG-1, IgG-2, IgG-3, IgG-4, IgA-1 and IgA-2.
  • the selection of particular immunoglobulin Fc regions from particular immunoglobulin classes and subclasses is within the purview of those skilled in the art.
  • the Fc may comprise at least one immunoglobulin hinge region, a CH2 domain and a CH3 domain, such as human IgG1 Fc.
  • the term "bispecific antibody” generally refers to an antibody capable of binding to two antigens or epitopes respectively.
  • the bispecific antibody may comprise the light chain and heavy chain of an antibody capable of specifically binding a first antigen or antigenic epitope, and the light chain and heavy chain of an antibody capable of specifically binding a second antigen or antigenic epitope.
  • the light chain of the antibody capable of specifically binding the first antigen or antigenic epitope and the light chain of the antibody capable of specifically binding the second antigen or antigenic epitope in the bispecific antibody have the same sequence.
  • the heavy chain of the antibody capable of specifically binding the first antigen or antigenic epitope and the heavy chain of the antibody capable of specifically binding the second antigen or antigenic epitope have different sequences.
  • epitope or “antigenic epitope” generally refers to the site on an antigen that is specifically bound by an immunoglobulin or an antibody.
  • An “epitope” is also referred to in the art as an "antigenic determinant”.
  • Epitopes or antigenic determinants usually consist of chemically active surface groups of molecules such as amino acids or carbohydrate or sugar side chains and usually have specific three-dimensional structural characteristics as well as specific charge characteristics.
  • an epitope typically includes at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 consecutive or non-contiguous amino acids in a unique spatial conformation, which may be "linear of" or “conformational”.
  • the term "specific binding” generally refers to binding as opposed to non-specific adsorption.
  • a dissociation constant for example, "KD”
  • KD dissociation constant
  • the KD value of the bispecific antibody or antigen-binding fragment thereof for HER2 protein is less than 1 ⁇ 10 -5 M, less than 5 ⁇ 10 -6 M, less than 2 ⁇ 10 -6 M, or 1 ⁇ 10 -6 M or less; 5 ⁇ 10 -9 M or less, 2 ⁇ 10 -9 M or less, or 1 ⁇ 10 -9 M or less.
  • the binding can be measured by known methods such as surface plasmon resonance (Surface Plasmon Resonance) method, ELISA method, RIA method and the like.
  • CDR in this specification refers to a complementarity determining region (CDR: Complementarity deterring region). It is known that the heavy chain and light chain of an antibody molecule each have three CDRs. CDR is also called hypervariable domain (hypervariable domain). On the primary structure, they are separated in 3 places, respectively.
  • CDRH1, CDRH2, and CDRH3 from the amino-terminal side of the heavy chain amino acid sequence for the CDRs of the heavy chain, and are described as CDRH1, CDRH2, and CDRH3 from the amino-terminal side of the light chain amino acid sequence for the CDRs of the light chain.
  • Pertuzumab generally refers to Pertuzumab, also known as 2C4, trade name Perjeta.
  • the amino acid sequences of the light chain and heavy chain variable regions of Pertuzumab can be found in Figure 2 of US20090285837A1.
  • the pertuzumab is a recombinant humanized monoclonal antibody that can specifically bind to the extracellular dimerization domain (subdomain II) of epidermal growth factor receptor 2 (HER2).
  • HER2 epidermal growth factor receptor 2
  • the pertuzumab can block the heterodimerization of HER2 and other HER receptors by binding to HER2, thereby slowing down the growth of tumors.
  • the pertuzumab can be used to treat HER2-positive metastatic breast cancer, and can be used to treat early breast cancer.
  • trastuzumab generally refers to Trastuzumab, trade name
  • the amino acid sequences of the light chain and heavy chain of trastuzumab can be found in Figure 16 of US20090285837A1.
  • the trastuzumab is a humanized monoclonal antibody derived from recombinant DNA, which can be produced by mammalian cells (Chinese hamster ovary cells CHO) cultured in suspension in sterile medium.
  • the trastuzumab can specifically bind to the extracellular domain of HER2, and can also stimulate the body's own immune cells to destroy tumor cells.
  • the trastuzumab can be used to treat HER2-positive metastatic breast cancer, early breast cancer, and HER2-positive metastatic gastric adenocarcinoma or gastroesophageal junction adenocarcinoma.
  • Exatecan generally refers to Exatecan (DX-8951), which is a DNA topoisomerase I inhibitor with CAS number 171335-80-1.
  • DXd is a derivative of Exitecan (DX-8951) with a CAS number of 1599440-33-1.
  • exitecan and DXd belong to moderately toxic drugs, and the application of these drugs in ADC can reduce the toxic and side effects caused by toxin shedding.
  • linker generally refers to a chemical moiety that is bifunctional or multifunctional and is used to link the bispecific antibody or antigen-binding fragment thereof described herein to the drug described herein.
  • the linker may comprise one coupling component, or may comprise multiple coupling components.
  • tumor generally refers to the physiological condition in mammals that is often characterized by unregulated cell growth.
  • the tumor includes one or more cancerous cells.
  • the tumors may include solid tumors and/or non-solid tumors.
  • tumor microenvironment generally refers to the environment in which a tumor exists, which is the acellular area within the tumor and the area immediately outside the tumor tissue but not belonging to the intracellular compartment of the cancer cells themselves. Tumors are closely related to the tumor microenvironment and can continuously interact with each other. For example, tumors can alter the tumor microenvironment, which can affect tumor growth and spread.
  • the tumor microenvironment has a low pH in the range of 5.8 to 7.0.
  • the tumor microenvironment may have lower concentrations of glucose and other nutrients, but higher concentrations of lactic acid.
  • the temperature of the tumor microenvironment can be 0.3 to 1 °C higher than the normal physiological temperature.
  • the tumor microenvironment has been described in "MRI of the Tumor Microenvironment” by Gillies et al., Journal of Magnetic Resonance Imaging, Vol. 16, pp. 430-450, 2002.
  • treatment generally refers to both therapeutic treatment and prophylactic or preventive measures, where the goal is to prevent or slow down (lessen) an unwanted physiological change or condition, such as hyperproliferative conditions such as cancer To grow, form or spread.
  • beneficial or desired clinical outcomes include, but are not limited to: relief of symptoms, diminishment of extent of disease, stabilization of disease state (i.e. not worsening), delay or slowing of disease progression, amelioration or palliation of disease state, and recovery (whether partial or complete), whether detectable or undetectable.
  • Treatment or “treating” can also refer to prolonging survival as compared to expected survival if not receiving treatment.
  • Those in need of treatment include those already with the disease or disorder as well as those prone to develop the disease or disorder or those in which the condition or disorder is to be prevented.
  • the present application provides an antibody drug conjugate comprising a bispecific antibody targeting HER2 or an antigen-binding fragment thereof.
  • the antibody drug conjugate may comprise the structure shown in Formula 1: M-(L1) a -(L2) b -D (Formula 1), wherein M represents the A bispecific antibody targeting HER2 or an antigen-binding fragment thereof; L1 represents a linker connected to M, L2 represents a linker connected to D, a and b are each independently selected from 0-10, and D represents a drug.
  • the HER2-targeting bispecific antibody or antigen-binding fragment thereof can specifically bind at least one epitope of human HER2.
  • two different epitopes of human HER2 can be specifically bound.
  • the at least two epitopes may be located in the same domain of the human HER2 protein, for example, may also be located in at least two different domains of the human HER2 protein.
  • the HER2-targeting bispecific antibody or antigen-binding fragment thereof can specifically bind to the extracellular domain II of human HER2 and/or the extracellular domain IV of human HER2.
  • the HER2-targeting bispecific antibody or antigen-binding fragment thereof can specifically bind to extracellular domain II of human HER2 and extracellular domain IV of human HER2.
  • the HER2-targeting bispecific antibody or antigen-binding fragment thereof may comprise a first light chain and a second light chain.
  • the first light chain may comprise the first LCDR1-3, wherein the first LCDR1 may comprise the amino acid sequence shown in SEQ ID NO.4.
  • the first LCDR2 may comprise the amino acid sequence shown in SEQ ID NO.5.
  • the first LCDR3 may comprise the amino acid sequence shown in SEQ ID NO.6.
  • the first LCDR1 may comprise the amino acid sequence shown in SEQ ID NO.4; the first LCDR2 may comprise the amino acid sequence shown in SEQ ID NO.5; and the first LCDR2 may comprise the amino acid sequence shown in SEQ ID NO.5;
  • An LCDR3 may comprise the amino acid sequence shown in SEQ ID NO.6.
  • the first light chain is capable of binding to the heavy chain of Pertuzumab.
  • the second light chain may comprise a second LCDR1-3, wherein the second LCDR1 may comprise the amino acid sequence shown in SEQ ID NO.1.
  • the second LCDR2 may comprise the amino acid sequence shown in SEQ ID NO.2.
  • the second LCDR3 may comprise the amino acid sequence shown in SEQ ID NO.3.
  • said second light chain is capable of binding to the heavy chain of trastuzumab.
  • the first light chain and/or the second light chain can be engineered from two original monoclonal antibodies (eg, known monoclonal antibodies).
  • the two original monoclonal antibodies may target HER2.
  • the two original monoclonal antibodies can specifically bind at least one epitope of human HER2.
  • the two original monoclonal antibodies can respectively specifically bind to two different epitopes of human HER2.
  • the two original monoclonal antibodies can specifically bind to the extracellular domain II of human HER2 and the extracellular domain IV of human HER2, respectively.
  • the two original monoclonal antibodies may be trastuzumab and pertuzumab.
  • the first light chain and/or the second light chain may differ in amino acid sequence from the light chains of either of the two original monoclonal antibodies.
  • the first light chain and/or the second light chain may have the same amino acid sequence as the light chain of any one of the two original monoclonal antibodies, or, the first light chain and/or the second light chain may Or the amino acid sequence of the second light chain can be obtained through modification on the basis of the amino acid sequence of the light chain of any one of the two original monoclonal antibodies.
  • the modification may include amino acid sequence modification.
  • the purpose of the modification may be to maintain as much as possible the affinity with the antigen or epitope corresponding to the two original monoclonal antibodies.
  • the modification may include mutation, deletion or addition of amino acids, for example, mutation, deletion or addition of no more than 3 amino acids, no more than 2 amino acids, or no more than 1 amino acid.
  • amino acid sequence of the variable region of the first light chain may be identical to the amino acid sequence of the variable region of the second light chain.
  • variable regions of the first light chain and the second light chain may comprise the amino acid sequence shown in any one of SEQ ID NO.7-12.
  • variable regions of the first light chain and the second light chain may comprise the amino acid sequence shown in SEQ ID NO.7.
  • the first light chain and the second light chain may comprise a light chain constant region.
  • the light chain constant region may be of the kappa or lambda type.
  • the ⁇ -type light chain constant region includes various allotypes, such as Km1, Km1,2, Km3;
  • the lambda-type light chain constant region includes various allotypes, such as CL1, CL2, CL3, CL6 and CL7.
  • the first light chain and the second light chain may comprise the amino acid sequence described in any one of SEQ ID NO.13-18.
  • the first light chain may be selected from the group consisting of the light chain of Pertuzumab or a mutant thereof and the light chain of Trastuzumab or a mutant thereof.
  • the second light chain may be selected from the group consisting of the light chain of Pertuzumab or a mutant thereof and the light chain of Trastuzumab or a mutant thereof.
  • the first light chain may be the light chain of Pertuzumab or a mutant thereof.
  • the second light chain may be the light chain of trastuzumab or a mutant thereof.
  • amino acid sequences of the first light chain and the second light chain may be the same.
  • the first light chain and the second light chain may comprise the amino acid sequence described in SEQ ID NO.13.
  • the HER2-targeting bispecific antibody or antigen-binding fragment thereof may comprise a first heavy chain and a second heavy chain, wherein the first heavy chain can be expressed under physiological conditions or in vitro. Properly associated with the first light chain.
  • the second heavy chain can correctly combine with the second light chain under physiological conditions or protein expression state in vitro.
  • the first heavy chain may comprise a first heavy chain variable region, and the first heavy chain variable region may be the heavy chain variable region of Pertuzumab.
  • the second heavy chain may comprise a second heavy chain variable region, and the second heavy chain variable region may be the heavy chain variable region of trastuzumab.
  • the first heavy chain and the second heavy chain may comprise a heavy chain constant region, wherein the heavy chain constant region may be derived from a constant region of human IgG.
  • the heavy chain constant region of the first heavy chain, and/or the heavy chain constant region of the second heavy chain may comprise an Fc region.
  • the Fc region can be modified, for example, the modification can increase the ratio of the first heavy chain and the second heavy chain to form a heterodimer.
  • modified technology can be well known to those skilled in the art, for example, refer to Ridgway, Presta et al. 1996; Carter 2001, patent CN 102558355A, patent CN 103388013A.
  • the heavy chain type of the heavy chain constant region of the first heavy chain and the heavy chain constant region of the second heavy chain may be the same or different.
  • the heavy chain constant region of the first heavy chain and the heavy chain constant region of the second heavy chain may differ in amino acid sequence from the heavy chain constant regions of the two original monoclonal antibodies.
  • the amino acid sequence of the heavy chain constant region of the first heavy chain may be identical to the amino acid sequence of the heavy chain constant region of one of the original monoclonal antibodies; and/or, the heavy chain of the second heavy chain
  • the amino acid sequence of the chain constant region may be identical to the amino acid sequence of the heavy chain constant region of another strain of the original monoclonal antibody.
  • the Fc fragments of the first heavy chain and the second heavy chain may comprise the amino acid sequence described in any one of SEQ ID NO.24-57.
  • the first heavy chain may comprise the amino acid sequence shown in any one of SEQ ID NO.21-24.
  • the first heavy chain may comprise the amino acid sequence shown in SEQ ID NO.21 or 23.
  • the second heavy chain may comprise the amino acid sequence shown in any one of SEQ ID NO.21-24.
  • the second heavy chain may comprise the amino acid sequence shown in SEQ ID NO. 22 or 24.
  • the first heavy chain may comprise the amino acid sequence shown in SEQ ID NO.21 or 23, and the second heavy chain may comprise the amino acid sequence shown in SEQ ID NO.22 or 24.
  • the first heavy chain may comprise the amino acid sequence shown in SEQ ID NO.21
  • the second heavy chain may comprise the amino acid sequence shown in SEQ ID NO.22.
  • the first heavy chain may comprise the amino acid sequence shown in SEQ ID NO.23
  • the second heavy chain may comprise the amino acid sequence shown in SEQ ID NO.24.
  • the HER2-targeting bispecific antibody or antigen-binding fragment thereof can be obtained according to conventional technical means in the art.
  • it can be purified from host cells.
  • the purification methods may include chromatographic techniques such as size exclusion, ion exchange, affinity chromatography and ultrafiltration.
  • the antibody drug conjugate may comprise the structure shown in Formula 1: M-(L1) a -(L2) b -D (Formula 1), wherein M represents the HER2-targeting drug described in this application
  • M represents the HER2-targeting drug described in this application
  • a compound for preparing the antibody drug conjugate described in this application has a structure shown in Formula 2: M-(L1) a (Formula 2), wherein M represents the target drug conjugate described in this application A bispecific antibody to HER2 or an antigen-binding fragment thereof; L1 represents a linker connected to M; a is selected from 0-10.
  • the L1 and the M may be connected through the thiol, azido or amide group on the M.
  • the M may comprise a first heavy chain and a second heavy chain, and the first heavy chain and/or the second heavy chain may comprise a linking site capable of linking with the L1.
  • the linking site may include a group capable of linking with the L1 after deglycosylation modification.
  • the group may be located at the side group of amino acid Q at position 297 of the first heavy chain; and/or may be located at the side group of amino acid Q at position 298 of the second heavy chain.
  • the group may include an amide group.
  • transglutaminases TGs
  • TGs transglutaminases
  • the linking site may include a group capable of linking with the L1 after glycosylation modification.
  • the group may be located at the side group of the 299th amino acid N of the first heavy chain; and/or may be located at the side group of the 300th amino acid N of the second heavy chain.
  • N299 or N300 may have a conserved glycosylation site. The glycosylation site at the N can be utilized for site-directed coupling.
  • the numbering of the amino acid position can be calculated from the amino acid at the N-terminal of the first heavy chain and/or the second heavy chain.
  • glycosylation modification can be performed on the glycosylation modification site.
  • said glycosylation form can be accomplished, for example, by expressing the protein in a cell with an altered glycosylation structure.
  • Cells with altered glycosylation structures have been described in the art and can be used to express therein proteins with such glycosylation modifications (e.g., HER2-targeting bispecific antibodies or antigens thereof described herein) combined fragments).
  • Glycosylation of a protein may depend on the amino acid sequence of the protein (eg, the HER2-targeting bispecific antibody or antigen-binding fragment thereof described herein) and the host cell in which the protein is expressed. Different organisms can produce different glycosylases (eg, glycosyltransferases and glycosidases) and have different available substrates (nucleotide sugars). Due to these factors, protein glycosylation patterns and the composition of residues at glycosylation modification sites may vary depending on the host system in which a particular protein is expressed. Glycosyl residues useful herein may include glucose, galactose, mannose, fucose, n-acetylglucosamine, and/or sialic acid. For example, the glycosylation modification may include a human-adapted glycosylation modification pattern.
  • the glycosylation modification can change the properties of the protein (eg, the HER2-targeting bispecific antibody or antigen-binding fragment thereof described in the present application).
  • Different protein glycosylation can affect and/or result in different protein properties (e.g. in terms of expression, half-life in vivo, protein folding, solubility, susceptibility to proteases, transport, transport, compartmentalization, secretion, other protein or factors such as recognition, antigenicity or allergenicity changes).
  • the specific structure and preparation method of the protein glycosylation can be adjusted according to different glycosylation purposes, and those skilled in the art can make adjustments according to the existing technology in the field.
  • the glycosylation modification can be adjusted according to human and/or animal species specificity.
  • the glycosylation modification can be achieved with the aid of a glycosylase (e.g., a heterologous glycosylase derived from the host cell).
  • the glycosylation modification can obtain a protein exhibiting glycosylation characteristics of a human protein (eg, the HER2-targeting bispecific antibody or antigen-binding fragment thereof described herein).
  • the glycosylase may be of natural or non-natural origin.
  • the glycosylated modification may include: the M has been contacted with UDP-GalNAz, ⁇ -1,4-galactosyltransferase or a variant thereof.
  • the ⁇ -1,4-galactosyltransferase or variant thereof e.g., ⁇ -1,4-Gal-T1-Y289
  • GalNAc N-acetylgalactosamine
  • It may be linked to a terminal N-acetylglucosamine (GlcNAc) residue on a glycan of a protein (such as the HER2-targeting bispecific antibody or antigen-binding fragment thereof).
  • the M may comprise the group described in the present application.
  • the side group of amino acid N at position N299 of said first heavy chain of said M; and/or the side group of amino acid N at position N300 of said second heavy chain of said M can obtain -N 3 base modification, become glycosylation modified M.
  • the Fc region of the M can be linked to the L1 through the group.
  • the M in the antibody drug conjugates described herein and/or the compounds described herein, can be linked to the L1 via the group.
  • the glycosylated modified M can undergo an addition reaction with the L1.
  • the a may be an integer selected from 0-10, for example, may be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.
  • b may be an integer selected from 0-10, for example, may be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.
  • the L1 and/or L2 may be selected from the group consisting of cleavable linkers, non-cleavable linkers, hydrophilic linkers, hydrophobic linkers, charged linkers, uncharged linkers, linkers and dicarboxylic acid-based linkers.
  • both the L1 and the L2 can be regarded as linkers.
  • the linker may be a part that extends the drug linkage to avoid, for example, shielding the active site of the antibody or increasing the solubility of the ADC.
  • the linker may comprise stretcher units and/or amino acid units.
  • the linker can be used to couple the antibody (for example, the HER2-targeting bispecific antibody or antigen-binding fragment thereof described in the present application) to the drug (for example, by covalent connect).
  • the conjugation can be via the antibody's cysteine sulfhydryl or amine (eg N-terminal or amino acid side chain such as lysine) forming a bond with the functional group of the linker.
  • cysteine sulfhydryl or amine eg N-terminal or amino acid side chain such as lysine
  • the linker may have a functional group capable of reacting with a free cysteine present on the antibody to form a bond (e.g., a covalent bond).
  • the functional groups may include: maleimide, haloacetamides, ⁇ -haloacetyl, active esters such as succinimide ester, 4-nitrophenyl ester, pentafluorophenyl ester, Tetrafluorophenyl esters, acid anhydrides, acid chlorides, sulfuryl chlorides, isocyanates and/or isothiocyanates.
  • the linker may include a functional group capable of reacting with an electrophilic group present on the antibody.
  • the functional groups may include: aldehyde, ketone carbonyl, hydrazine, oxime, amino, hydrazine, thiosemicarbazone, hydrazine carboxylate, and/or aryl hydrazide.
  • the linker may include a cleavable linker and a non-cleavable linker.
  • the linker eg, L2
  • the linker can be a cleavable (eg, self immolative) linker, which can facilitate release of the drug.
  • the linker eg, L1
  • the linker can be a non-cleavable linker that can be attached to the antibody and is non-cleavable.
  • the cleavable linker can be cleaved under intracellular conditions.
  • the cleavable linkers can include: acid labile linkers (e.g., comprising hydrazones), protease sensitive linkers (e.g., sensitive to peptidases), photolabile linkers and/or comprising disulfide
  • the linker of the thing may comprise a peptide linker capable of being cleaved by intracellular proteases (eg, lysosomal proteases) and/or endosomal proteases.
  • the linker is cleavable under the intracellular environment. For example, cleavage of the linker can allow the drug in the antibody drug conjugate to exert an effective therapeutic effect.
  • the structure and/or properties of the linker are stable extracellularly.
  • the antibody drug conjugates described herein may be structurally stabilized prior to being transported or delivered into cells.
  • the HER2-targeting bispecific antibody or its antigen-binding fragment remains coupled to the drug.
  • the linker in the antibody drug conjugate can, for example, maintain the specific binding properties of the HER2-targeting bispecific antibody or antigen-binding fragment thereof; participate in the antibody drug Delivery of the conjugate, and/or, maintaining the therapeutic effect (eg, cytotoxic effect) of the drug.
  • the linker may include a hydrophilic linker (eg, PEG4Mal and sulfo-SPDB) and a hydrophobic linker.
  • a hydrophilic linker eg, PEG4Mal and sulfo-SPDB
  • the hydrophilic linker can reduce the extent to which the antibody drug conjugate can be pumped out of resistant cancer cells by MDR (multidrug resistance) or functionally similar transporters.
  • the linker may also function to directly or indirectly inhibit cell growth and/or cell proliferation.
  • the linker may function as an intercalator in the cleavage.
  • the linker may act indirectly to inhibit macromolecule biosynthesis.
  • the linker can facilitate the entry of the antibody drug conjugate into cells (for example, can promote "internalization”).
  • the linker can also be designed to improve the stability of the antibody drug conjugate.
  • said L1 is capable of participating in the SPAAC reaction.
  • the SPAAC reaction is an azide-alkynyl cycloaddition reaction.
  • the SPAAC reaction can be used as one of the addition reactions in the preparation process of the antibody-drug conjugate.
  • the L1 may be selected from the group consisting of maleimide, succinimid-3-yl-N and DBCO.
  • the L1 may be DBCO-(PEG) n1 , wherein n1 is an integer of 0-10, or the L1 is maleimide.
  • the L1 can be DBCO-(PEG) 4 , DBCO-(PEG) 3 .
  • the PEG molecule can be used to increase the hydrophilicity of the antibody drug conjugate.
  • the PEG molecule can be used to meet the requirement of spatial distance for L2 (eg GGFG) cleavage (eg enzyme cleavage).
  • said L2 can be selected from the group consisting of polypeptide, VC-PAB, N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), N-succinyl Imino 4-(2-pyridyldithio)pentanoate (SPP), N-succinimidyl 4-(2-pyridyldithio)butyrate (SPDB), N-succinyl Imino-4-(2-pyridyldithio)-2-sulfobutyrate (sulfo-SPDB), N-succinimidyl iodoacetate (SIA), N-succinimide Succinyl (4-iodoacetyl) aminobenzoate (SIAB), maleimide PEG NHS, N-4-(maleimidomethyl) cyclohexyl carboxylate succinamido ester (SMCC) , N-sulfo
  • SPDP
  • the L2 can be GGFG, LP, VK, VC, GFG, GGFGG, GGFGS, GGFGGG, GGFGGE, GGFGGGFG, DGGF, DGGFG, D d GGFG, DG Me GFG, DGGFS, DDGGFG, KDGGFG, KGGFG, EGGFG or SGGFG.
  • the L2 can be GGFG.
  • Cathepsin B cleavable tetrapeptide Gly-Gly-Phe-Gly (GGFG) is a highly hydrophilic (eg stronger than Gly-Phe-Leu-Gly) linker.
  • the drug may have the ability to kill tumor cells and/or inhibit the growth of tumor cells.
  • the drugs may include small molecule drugs.
  • the drug may be selected from the group consisting of V-ATPase inhibitors, Bcl2 inhibitors, MCL1 inhibitors, HSP90 inhibitors, IAP inhibitors, mTor inhibitors, microtubule stabilizers, microtubule destabilizers , auristatin, dolastatin, maytansinoid, MetAP (methionine aminopeptidase), nuclear export inhibitor of protein CRM1, DPPIV inhibitor, proteasome inhibitor, phosphotransfer reaction inhibitor in mitochondria, protein synthesis inhibitor , CDK2 inhibitors, CDK9 inhibitors, kinesin inhibitors, HDAC inhibitors, DNA disruptors, DNA alkylating agents, DNA intercalators, DNA minor groove binders, DHFR inhibitors, nucleoside analogs, HD AC inhibitors , anthracyclines, NAMPT inhibitors, SN-38 glucuronic acid, etoposide phosphate, nitrogen mustard, proteosome inhibitors, cytokines, and Toll-like
  • the drug may be selected from the group consisting of DM1, exatecan, DXd, MMAE, SN-38, Calicheamicin, Anthracyclin-5G, DM4, microtubule inhibitor SHR153024, PNU-159682, Duo5 toxin, SN38 derivatives or their derivatives.
  • the drug may be camptothecin or its derivatives.
  • the drug may be a DNA topoisomerase I inhibitor.
  • the drug can be Exatecan (DX-8951), DXd, Ducamycin, Mitomycin C, Talithromycin, Maytansine, TLR7 agonist, TLR8 agonist , a TLR7/8 agonist or a TLR9 agonist.
  • the drug can have the following structure:
  • the antibody drug conjugate may have a structure selected from the following group:
  • the drug/antibody ratio of the antibody drug conjugate may be about 2-6.
  • the drug/antibody ratio can be about 1, about 2, about 3, about 4, about 5, or about 6.
  • the drug/antibody ratio can be four.
  • the drug/antibody ratio can be three.
  • the application provides a compound for the antibody drug conjugate described in the application, which has the structure shown in formula 2: M-(L1) a (formula 2), wherein M The bispecific antibody targeting HER2 or its antigen-binding fragment; L1 represents the linker connected to M; a is selected from 0-10.
  • the compound may include a structure selected from the group consisting of:
  • the present application provides a method for preparing the antibody-drug conjugate described in the present application, which includes the following steps: contacting the compound described in the present application with the drug described in the present application.
  • the preparation method of the antibody-drug conjugate may include the following steps: (1) contacting the L1 described in this application, the L2 and the drug; (2) obtaining Compound (L1) a -(L2) b -D; (3) contacting M described in this application with said compound (L1) a -(L2) b -D to obtain the antibody drug conjugate described in this application things. For example, M in step (3) undergoes the glycosylation modification.
  • the present application provides a pharmaceutical composition, which comprises the antibody-drug conjugate described in the present application, or a pharmaceutically acceptable carrier.
  • the pharmaceutical composition may include a "therapeutically effective dose” or “prophylactically effective dose” of the antibody-drug conjugate described in the present application.
  • the “therapeutically effective dose” may be an amount effective, at dosages and for durations necessary, to achieve the desired therapeutic result.
  • the therapeutically effective dose can be determined by those skilled in the art, for example, according to the state of the disease, the age, sex, body weight of the subject, the ability of the antibody-drug conjugate to elicit a desired response in the subject, etc. Factors change.
  • the “prophylactically effective dose” may be an amount effective to achieve the desired prophylactic result at the necessary dosage and duration. For example, the prophylactically effective dose may be lower than the therapeutically effective dose.
  • the pharmaceutical composition may be formulated in a form suitable for administration (eg, suitable for parenteral, intradermal, intramuscular, intraperitoneal, intravenous and subcutaneous, intratumoral and/or mucosal administration).
  • the pharmaceutical composition may include other pharmaceutically active ingredients.
  • the pharmaceutically acceptable carrier may include any or all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, etc. that are physiologically compatible.
  • the pharmaceutically acceptable carrier may include one or more of water, saline, phosphate-buffered saline, dextrose, glycerol, ethanol, etc., and combinations thereof.
  • the pharmaceutically acceptable carrier may also include isotonic agents, wetting agents, emulsifying agents, preservatives and/or buffering agents.
  • the present application provides a method for regulating the tumor microenvironment of a subject, which includes the following steps: administering the antibody-drug conjugate described in the present application, or the pharmaceutical composition described in the present application to the subject.
  • the present application provides a method for regulating the immune response of a subject, which includes the following steps: administering the antibody-drug conjugate described in the present application, or the pharmaceutical composition described in the present application to the subject.
  • the present application provides the application of the antibody drug conjugate described in the present application, or the pharmaceutical composition described in the present application in the preparation of medicines, wherein the medicines can regulate the tumor microenvironment of the subject and/or regulate the subject immune response.
  • the present application provides the antibody-drug conjugate described in the present application, or the pharmaceutical composition described in the present application, which is used for regulating the tumor microenvironment of the subject and/or regulating the immune response of the subject.
  • the present application provides the application of the antibody-drug conjugate described in the present application, or the pharmaceutical composition described in the present application in the preparation of medicines, wherein the medicines can prevent and/or treat tumors.
  • the present application provides a method for preventing and/or treating tumors, which includes the following steps: administering the antibody-drug conjugate described in the present application or the pharmaceutical composition described in the present application to prepare a drug.
  • the present application provides the antibody drug conjugate or the pharmaceutical composition described in the present application, which is used for preventing and/or treating tumors.
  • the tumor may include solid tumors and/or non-solid tumors.
  • the tumor may comprise gastric cancer, breast cancer (eg breast ductal carcinoma), and/or pancreatic cancer.
  • the tumors of the present application may include HER2 weakly positive tumors and/or HER2 negative tumors, for example, the technical solution of the present application may have a bystander killing effect.
  • antibody A A bispecific antibody targeting HER2 (hereinafter referred to as antibody A), wherein antibody A comprises a first light chain, a second light chain, a first heavy chain and a second heavy chain, wherein the first light chain and the amino acid sequence of the second light chain is shown in SEQ ID NO.13, wherein the amino acid sequence of the heavy chain variable region of the first heavy chain is shown in SEQ ID NO.19; the second heavy chain The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.20. Wherein the amino acid sequence of the first heavy chain is shown in SEQ ID NO.21; the amino acid sequence of the second heavy chain is shown in SEQ ID NO.22.
  • antibody A was artificially synthesized and isolated and purified.
  • DS-8201 (also known as: T-DXd, trade name ENHERTU) is manufactured by Daiichi Sankyo and AstraZeneca. DS-8201 is composed of anti-HER2 IgG1 monoclonal antibody coupled with topoisomerase I inhibitor DXd through a linker.
  • U3-1402 was manufactured by Daiichi Sankyo. U3-1402 is formed by covalently coupling HER3 antibody Patritumab and topoisomerase inhibitor DXd.
  • DXd that is, exatecan DX-8951 derivatives, purchased from Lianning Biology
  • compound 1 comprising a linker and a toxin: DBCO-PEG4-GGFG-DXd, which has the following structural formula:
  • Antibody A was contacted with ⁇ -1,4-galactosyltransferase ( ⁇ -1,4-al-T1) (purchased from Shanghai Baosen Biology) in the presence of UDP-GalNAz.
  • ⁇ -1,4-galactosyltransferase ⁇ -1,4-al-T1
  • UDP-GalNAz the structural formula of UDP-GalNAz is
  • the obtained glycosylation-modified antibody A was detected by HPLC, and the detection results are shown in Figure 2.
  • the results in Figure 2 indicate that the glycosylation-modified antibody A has its unique characteristic peak and its molecular weight is 148796Da.
  • step (1) Contact DBCO-PEG4-GGFG-DXd in step (1) with the glycosylated antibody A prepared in step (2) and react at 25-35°C for 10-15 hours, wherein DBCO and glycosyl SPAAC reaction occurs at the -N3 group in Antibody A modified by B to obtain Antibody A-ADC1 molecule.
  • the reaction to obtain the antibody A-ADC1 molecule is shown in Figure 3.
  • Antibody A-ADC1 was detected by HPLC, and the detection results are shown in Figure 4.
  • the results in Figure 4 indicate that the antibody A-ADC1 molecule has its unique characteristic peak, and its molecular weight is 154299.
  • Herceptin was glycosylated according to the method described in Example 1, and reacted with DBCO-PEG4-GGFG-DXd to obtain Herceptin-ADC1 molecule, wherein its DAR was 8.
  • Antibody A is reacted with TECP, so that antibody A has sulfhydryl groups (for example, 8 -SH), and modified antibody A is obtained.
  • TECP sulfhydryl groups
  • the modified antibody A prepared in the step (2) is contacted with the compound 2 prepared in the step (1) comprising a linker and a toxin to obtain an antibody A-ADC2 molecule.
  • the stability of the antibody A-ADC1 prepared in Example 1, the antibody A-ADC2 prepared in Example 2, and DS-8201 in human serum were investigated respectively.
  • the result is shown in Figure 6.
  • the results in Figure 6 show that the molecular weight of antibody A-ADC1 (about 154299) after incubation in human serum for 0 days, 3 days, and 7 days basically did not change, and the DAR value remained at 4 after 3 days and 7 days in serum.
  • step (1) the stability of antibody A-ADC2 in serum was detected.
  • the results of Waters Xevo G2-QTOF mass spectrometry analysis are shown in Figure 7.
  • the results in Figure 7 illustrate that when the antibody A-ADC2 was incubated in human serum for 0 day, 3 days, and 7 days, respectively. After 3 days of incubation, the maleimide on the light chain can exist stably in human serum due to ring-opening hydrolysis; while the DAR value of the heavy chain decreased from 6 to about 2 after 3 days of incubation, and the DAR of the heavy chain after 7 days of incubation The value is almost 0, indicating that the heavy chain of antibody A-ADC2 will be eliminated in serum after 7 days.
  • step (1) test the stability of DS-8201 in serum.
  • Biofilm interferometry (BLI) (Fortebio Macromolecular Interaction Instrument of Danaher Company) was used to measure the affinity of antibody A, antibody A-ADC1 prepared in Example 1, and Fc ⁇ RI, Fc ⁇ RIIIa, etc. respectively.
  • the curing concentration is 10 ⁇ g/ml, and the curing height is 2nm.
  • the curing concentration is 10 ⁇ g/ml, and the curing height is 2nm.
  • Example 4 The binding ability of ADC molecules comprising antibody A to tumor cells
  • antibody A-ADC1 prepared in Example 1
  • antibody A-ADC2 prepared in Example 2
  • antibody A to NCI-N87 cells were investigated respectively.
  • the EC50 of antibody A-ADC1 is consistent with that of parent antibody A, both are about 1.1 ⁇ g/ml, indicating that antibody A-ADC1 is compatible with parent antibody A.
  • the binding activity of molecular antibody A to NCI-N87 cells with high expression of HER2 is consistent; the peak value of antibody A-ADC1 is greater than that of antibody A-ADC2, indicating that the binding activity of antibody A-ADC1 to NCI-N87 cells with high expression of HER2 is slightly stronger than that of antibody A -ADC2.
  • Example 5 The ability of ADC molecules comprising antibody A to kill tumor cells
  • test results of BT474 cells treated for 3 days are shown in FIG. 17 .
  • the NCI-N87, BT474 tumor cell killing test shows that the antibody A-ADC1 and the antibody A-ADC2 have good killing ability to the HER2 high expression positive tumor cells (NCI-N87, BT474) cells; and the antibody A-ADC1 and the The killing ability of antibody A-ADC2 on HER2 high expression positive tumor cells (NCI-N87, BT474 cells) is better than that of DS-8201.
  • ADC drugs are usually combined with antigens on the cell membrane first, and then the antibody-antigen complex enters the cell through endocytosis and forms inclusion bodies, and then the inclusion bodies further mature and fuse into lysosomes.
  • the cytotoxic drug is released (through the corresponding specific protease to decompose the linker, such as cathepsin B (cathepsin B), or the entire ADC drug is decomposed in the lysosome), and the resulting cytotoxic drug penetrates the lysosome Membrane, binding to DNA or microtubules leads to apoptosis.
  • These drugs can also be pumped out to the tumor microenvironment through the protein transport on the cell membrane, enter the adjacent tumor cells, and have a killing effect on them, which can lead to the "bystander effect".
  • Antibody A-ADC1 is a sugar site-directed conjugated drug targeting a biepitope antibody targeting HER2, using a cleavable linker.
  • SK with antibodies A-ADC1 and DS-8201 respectively -B-R-3 (HER2 high expression) single cell system, MDA-MB-468 (HER2 no expression) single cell system, SK-BR-3 and MDA-MB-468 (1:1) mixed cell system, after treatment for 3 days , Count the cells in all sample wells, and stain the mixed cell system with APC anti-CD340Antibody fluorescent antibody, and detect it by flow cytometry.
  • SK-BR-3 and MDA-MB-468 cells in the logarithmic growth phase in T75 culture flasks discard the medium, wash twice with PBS and drain the liquid; add 2ml of trypsin for digestion, and place in a 37°C incubator for 4 minutes ;Transfer the cell digestion solution to a 15ml centrifuge tube, add 8ml growth medium to stop the digestion and pipette the cells to a single cell suspension, centrifuge at 1000rpm for 5min; discard the supernatant, add 3ml medium (RPMI-1640+1%FBS) Resuspend cells, pipette 20 ⁇ l cell suspension and 20 ⁇ l 0.2% trypan blue to mix.
  • 3ml medium RPMI-1640+1%FBS
  • Counting Mono-culture: Both cell densities were adjusted to 1*10 5 cells/ml with culture medium (RPMI-1640+1% FBS). Plate 96-well plates at 100 ⁇ l/well, the number of cells per well is 1*10 4 , and culture overnight at 37°C in a 5% CO2 incubator; mixed cell plating (co-culture): medium (RPMI-1640+1 %FBS) both cell densities were adjusted to 2*10 5 cells/ml, and 50 ⁇ l of SK-BR-3 cells and 50 ⁇ l of MDA-MB-468 cells were added to each well. The total number of cells per well is 2*10 4 , cultured overnight at 37°C in a 5% CO 2 incubator;
  • Antibody drug configuration Dilute the test sample with RPMI-1640 medium containing 1% FBS, from the highest working concentration of 50nM, downward 5-fold gradient dilution, a total of 4 concentrations; set the control group at the same time, that is, no drug group ( Negative Control); add 100 ⁇ l of the above-mentioned diluted test sample to each well, and directly add 100 ⁇ l of RPMI-1640+1% FBS medium to the control group (Negative Control); culture at 37°C, 5% CO2 for 72 hours; suck into the 96-well plate Supernatant, wash once with 200 ⁇ l 1XPBS, add 20 ⁇ l trypsin to each well for digestion, place in 37°C incubator for 4min; add 100 ⁇ l medium (RPMI-1640+10% FBS) to stop digestion and pipette cells to single cell suspension, 1000rpm Centrifuge for 5 min; discard the supernatant, add 40 ⁇ l of 1XPBS to each well to resuspend the cells, draw 20
  • the inhibition rate of antibody A-ADC1 on the proliferation of MDA-MB-468 cells in the mixed cell system is: 68.65%, 46.87%, 31.61%, -4.95%; ENHERTU
  • the proliferation inhibition rates of MDA-MB-468 cells in the mixed cell system were: 45.02%, 25.4%, -20.02%, -22.08%.
  • the bystander killing ability of antibody A-ADC1 on HER2-negative cells is about 1.5 times that of ENHERTU; when the concentration is 10nM, the bystander killing ability of antibody A-ADC1 on HER2-negative cells is about 1.8 times that of ENHERTU; At a concentration of 2nM, antibody A-ADC1 still has 31.61% bystander killing ability, and ENHERTU has no bystander killing ability; therefore, antibody A-ADC1 has stronger bystander killing ability than ENHERTU at a concentration of 2-50nM.
  • the following table shows the results of the ratio of HER2-positive and HER2-negative cells in the mixed cell group
  • the bystander killing ability of antibody A-ADC1 for HER2-negative cells is about 1.5 times that of ENHERTU (DS-8201); when the concentration is 10nM, the bystander killing ability of antibody A-ADC1 for HER2-negative cells is about It is 1.8 times that of ENHERTU; when the concentration is 2nM, antibody A-ADC1 still has bystander killing ability of 31.61%, and ENHERTU has no bystander killing ability; therefore, antibody A-ADC1 has stronger killing ability than ENHERTU at the concentration of 2-50nM Bystander lethality.
  • the target cell NCI-N87, the effector cell Jurkat-Fc ⁇ RIIIa-V158-NFAT and the antibody A-ADC1 prepared in Example 1 were co-cultured for 6 hours, and the ADCC activity was measured by detecting the fluorescent RUL value.
  • reaction medium 2% FBS+1640;
  • step (2) Use the reaction medium prepared in step (1) to prepare antibody A-ADC1, antibody A-ADC2 and antibody A at different concentrations. 9 concentrations;
  • step (2) Add 25 ⁇ L of antibody A prepared in step (2) or ADC containing antibody A, 25 ⁇ L of N8 cells and 25 ⁇ L of Jurkat-Fc ⁇ RIIIa-V158-NFAT cells into the 96-well plate, and place in the incubator for 6 hours;
  • the results are shown in Figure 20, and the results of Figure 20 are illustrated, wherein 1-3 show the results of Antibody A, Antibody A-ADC1 and Antibody A-ADC2, respectively.
  • the antibody A-ADC1 molecule prepared in Example 1 still retains good ADCC activity in HER2 positive/highly expressed cells (NCI-N87 cells), and its ADCC activity is 87.7% of the parent molecule antibody A (antibody A EC50/ADC1 EC50); and the antibody A-ADC2 prepared in Example 2 lost most of its ADCC activity, and its activity was only 37.2% of the parent molecule antibody A (antibody A EC50/ADC2 EC50). It shows that the coupling method has a great influence on the ADCC activity of ADC.
  • the OD value is detected at 490nm.
  • the OD value is detected at 490nm.
  • the ADCC activity of antibody A-ADC1, antibody A-ADC2, DS8201 and antibody A in the cell system with high expression of HER2 were respectively measured, and the target cells were cells with high expression of HER2: BT747 and NCI-N87 , the effector cells were PBMCs (PBMC-qc and PBMC-z) in the peripheral blood of two donors.
  • the detection process is shown in Figure 21, and the detection results are shown in Figure 22-23.
  • NCI-N87 cells purchased from Shanghai Chinese Academy of Sciences
  • NCI-N87 cells were digested and counted, 4 ⁇ 10 4 cells/50 ⁇ L/well were added to each drug-containing well, and placed in the incubator for 2, 5 and 23 hours;
  • BT474 cells were digested and counted, and 4 ⁇ 10 4 cells/50 ⁇ L/well were added to each drug-containing well, and placed in an incubator for 24 hours;
  • antibody-endocytosis reagent mixtures such as antibody A-ADC1, antibody A-ADC2, DS-8201, and antibody A were incubated with the tumor cell system respectively, and the endocytosis of each molecule by the cells was detected by cell flow cytometry efficiency.
  • the antibody A-ADC1 retains the tumor cell endocytosis effect equivalent to that of the parent molecule, and is far superior to DS-8201, indicating that the ADC described in this application can quickly bind to the tumor cell surface HER2 achieves endocytosis, blocks HER2 signaling pathway, and plays a tumor-killing role.
  • the measurement results show that the endocytosis efficiency of antibody A-ADC1 and antibody A-ADC2 is equivalent, and the endocytosis efficiency reaches more than 70% at 40nM. It can be seen that the endocytosis efficiency of ADC containing antibody A described in this application is DS-8201 under the same conditions (30%) 2.33 times, and the targeting of the former to BT474 cells is much stronger than that of DS-8201. It shows that the antibody A-ADC1 has a stronger ability to inhibit the signal pathway mediated by the target, and its tumor lethality is much stronger than that of DS-8201, and its tumor suppression ability is stronger.
  • Group administration test plan BxPC-3 cells were subcutaneously injected into the underarm of nude mice to establish the model, and the inoculation concentration was 1 ⁇ 10 6 /0.1mL/mouse. When the tumor size grew to an average of about 190mm 3 , they were randomly divided into groups according to tumor size and body weight, with 4 females in each group. The day of randomization was recorded as day 0. The test drug was administered intraperitoneally on the 0th day, and the test substance was administered intraperitoneally according to the dose every 3 days thereafter, for a total of 2 administrations. Animals not selected were euthanized with CO 2 .
  • the experimental animals were divided into three groups: A, B and C.
  • Group A was administered with PBS
  • the specific grouping dosing regimen is shown in Table 4.
  • NCI-N87 tumor model mouse antibody A-ADC1 After a single intravenous injection of NCI-N87 tumor model mouse antibody A-ADC1, the Cmax of the total antibody and ADC were 45422.727 ⁇ g/L and 70056.81675 ⁇ g/L, and the AUC (0-t) were 1598292.175 ⁇ g/L* h, 2098916.987 ⁇ g/L*h, T max were 0.033h, 0.033h, T 1/2 were 100.976h, 95.32h, respectively.
  • the C max of the total antibody and ADC were 12343.966 ⁇ g/L and 19604.51225 ⁇ g/L, respectively, and the AUC (0-t) were 421103.345 ⁇ g/L*h, 586509.313 ⁇ g/L*h, T max are 0.033h, 0.033h, T 1/2 are 86.744h, 65.384h respectively.
  • the ADC (Antibody A-ADC1) of this application is an ADC drug that is coupled with a small molecule DXd on the basis of a biepitope antibody targeting HER2, such as Antibody A, by selecting the human gastric cancer cell line NCI-N87 cells overexpressing Her2 , to compare the endocytosis effects of Antibody A-ADC1, Antibody A and the marketed similar target drug DS8201.
  • NC hole 25ul Buffer+25ul PHrodo+50ul cell (1E5);
  • BLANK well 50ul Buffer+50ul cell (1E5) with a volume of 100ul per well and pipetting and mixing;
  • Antibody A, antibody A-ADC1 and DS8201 can all induce HER2 endocytosis on the surface of NCI-N87 cells, and the endocytosis effect of antibody A-ADC1 is comparable to that of antibody A.
  • the endocytosis effect of antibody A-ADC1 was significantly stronger than that of DS8201 at three concentration points. Under the conditions of 50nM, 10nM, and 2nM concentrations, the endocytosis ratio of antibody A was 92.25%, 91.82%, and 65.71%, respectively.
  • Antibody A The endocytosis ratios of ADC1 were 85.48%, 84.65%, 58.95%, and the endocytosis ratios of DS8201 were 34.79%, 28.36%, 21.26%.
  • Table 6 shows the proportion of positive NCI-N87 cells after endocytosis of Antibody A, Antibody A-ADC1 and DS8201
  • Figure 29 shows the proportion of NCI-N87 positive cells after endocytosis induced by Antibody A, Antibody A-ADC1 and DS8201.
  • Example 12 Inhibitory Effect of ADC Molecules Containing Antibody A on HER2 Weakly Positive Gastric Cancer PDX Subcutaneous Xenograft NOD/SCID Mouse Model
  • the following table shows the sample information of human gastric cancer PDX xenograft model
  • mice For each PDX model, 30 4-5 week-old NOD/SCID female mice were subcutaneously inoculated with gastric cancer PDX tumor masses with a diameter of 2-3 mm in the right front scapula. When the average volume of the tumor was 213.40 mm 3 The mice were randomly grouped by body weight (see the table below), and the day of the grouping was defined as the first day, and the administration was started immediately after the grouping, and the day of the administration was defined as the first day after the administration.
  • the average tumor volume of the PBS control group (CTL) mice was 725.39 ⁇ 190.25mm 3 on the 27th day after the first administration of the group, and the relative tumor volume was 3.58 ⁇ 1.11; On the 27th day after administration, the average tumor volume was 112.89 ⁇ 61.65mm 3 , the relative tumor volume was 0.50 ⁇ 0.16, and the relative tumor inhibition rate TGI (%) was 86.05%, which was statistically significantly different from the PBS control group (p ⁇ 0.0001); the positive control drug ENHERTU (DS-8201) (10mg/kg) had an average tumor volume of 106.41 ⁇ 17.77mm 3 and a relative tumor volume of 0.50 ⁇ 0.11 on the 27th day after the first administration of the group, and the relative tumor inhibition rate TGI (%) was 86.08%, which was statistically significantly different from the PBS control group (p ⁇ 0.0001).
  • the antibody A-ADC1 has a significant tumor inhibitory effect on the human HER2 IHC 2+ gastric cancer PDX tumor model at a dose of 10 mg/kg, and the antibody A-ADC1 with a DAR of 4 and the positive control drug ENHERTU with a DAR of 8 The antitumor efficacy is equivalent.
  • Figure 30 shows the growth curves of the tumor volumes of mice in each group in the PDX tumor model of human gastric cancer.
  • the following table shows the drug efficacy analysis table of each group in the Case 168 human gastric cancer PDX tumor model
  • the following table shows the sample information of human gastric cancer HER2 IHC 2+PDX xenograft model
  • mice For each PDX model, 30 4-5-week-old female NOD/SCID mice were subcutaneously inoculated with a gastric cancer PDX tumor mass with a diameter of 2-3 mm in the right anterior shoulder blade. When the average tumor volume was 166.53 mm 3 , the mice were randomly divided into groups according to tumor size and body weight. The day of grouping was defined as the first day, and the administration was started immediately after grouping, and the day of administration was defined as the first day after administration.
  • the average tumor volume of mice in the PBS control group was 629.24 ⁇ 146.59mm 3 on the 17th day after the first administration of the group, and the relative tumor volume was 4.13 ⁇ 0.97; On day 17, the average tumor volume was 238.32 ⁇ 84.57mm 3 , the relative tumor volume was 1.68 ⁇ 1.01, and the relative tumor inhibition rate TGI (%) was 59.41%, which was statistically significantly different from the PBS control group (p ⁇ 0.0001)
  • the positive control drug ENHERTU (ie DS-8201) (10mg/kg) was 174.06 ⁇ 71.09mm 3 on the 17th day after the first administration of the group, and the relative tumor volume was 1.02 ⁇ 0.34, and the relative tumor inhibition rate TGI (% ) was 75.19%, which was statistically significantly different from the PBS control group (p ⁇ 0.0001).
  • the average tumor volume of the test drug antibody A-ADC1 treatment group was slightly larger than that of the positive control drug ENHERTU treatment group on the 17th day after the first administration of the group, but there was no statistically significant difference.
  • the antibody A-ADC1 has a significant tumor inhibitory effect on the human HER2-gastric cancer PDX tumor model at a dose of 10 mg/kg, and the anti-tumor effect of the antibody A-ADC1 with a DAR of 4 and the positive control drug ENHERTU with a DAR of 8 The efficacy is equivalent.
  • Figure 31 shows the growth curve of the tumor volume of the human gastric cancer HER2 IHC-PDX tumor model mice (drug treatment was given only once on day 1, tail vein injection, 10mg/kg).
  • the following table shows the drug efficacy analysis table of each group in the Case 111 human gastric cancer PDX tumor model

Landscapes

  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Immunology (AREA)
  • Epidemiology (AREA)
  • Organic Chemistry (AREA)
  • Cell Biology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • General Chemical & Material Sciences (AREA)
  • Oncology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Molecular Biology (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Preparation (AREA)
  • Peptides Or Proteins (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

一种抗体药物偶联物，其包含靶向HER2的双特异性抗体或其抗原结合片段。该抗体药物偶联物的制备方法以及用途，以及包括所述抗体药物偶联物的药物组合物。所述抗体药物偶联物可以有效地杀伤肿瘤。

Description

抗体药物偶联物及其应用 技术领域
本申请涉及生物医药领域，具体的涉及一种抗体药物偶联物及其应用。
背景技术
HER受体酪氨酸激酶家族的成员是细胞生长、分化和存活的重要介导物。该受体家族包括四种独特的成员，包括表皮生长因子受体(EGFR、ErbB1、或HER1)、HER2(ErbB2或p185neu)、HER3(ErbB3)和HER4(ErbB4或tyro2)。该受体家族的成员已牵连于多种类型的人恶性肿瘤。
双特异性抗体(Bispecific antibody,BsAbs)是含有两个不同配体结合位点的免疫球蛋白分子。它取代了经典的抗体Fab两臂相同的序列，而是用两个不同的Fab序列，因此Y型两臂可以结合不同的抗原表位。双特异性抗体在癌症治疗中的应用已经被多篇文献所综述(Carter 2001；Chames and Baty 2009；Chames and Baty 2009)。
向与在癌细胞表面表达、并且能向细胞内化的抗原结合的抗体上连接具有细胞毒性的药物得到的抗体-药物偶联物能够选择性地向癌细胞输送药物，从而可预计其能使药物在癌细胞内蓄积，并杀死癌细胞。
发明内容
本申请提供了一种抗体药物偶联物，包括靶向HER2的双特异性抗体或其抗原结合片段。所述抗体药物偶联物可以特异性地结合人HER2中的至少一个(例如至少2个)表位。本申请所述的抗体药物偶联物可以有以下的性质：(1)有效地杀伤肿瘤细胞，其中抗体部分和药物部分可以发挥协同杀伤肿瘤的功能；(2)特异性地结合肿瘤细胞；(3)在血清中有良好的稳定性；(4)对肿瘤细胞有更强的旁观者杀伤效应；(5)具有良好的ADCC效应；和/或(6)具有较好的内吞效率。本申请还提供了所述抗体药物偶联物的制备方法，包含所述抗体药物偶联物的组合物，以及所述抗体药物偶联物和组合物的应用。
一方面，本申请提供了一种抗体药物偶联物，其包含靶向HER2的双特异性抗体或其抗原结合片段。
在某些实施方式中，所述靶向HER2的双特异性抗体或其抗原结合片段特异性结合人 HER2的至少一个表位。
在某些实施方式中，所述靶向HER2的双特异性抗体或其抗原结合片段特异性结合人HER2的胞外结构域II和/或人HER2的胞外结构域IV。
在某些实施方式中，所述靶向HER2的双特异性抗体或其抗原结合片段包含第一轻链和第二轻链。
在某些实施方式中，所述第一轻链包含第一LCDR1-3，其中所述第一LCDR1包含SEQ ID NO.4所示的氨基酸序列。
在某些实施方式中，所述第一LCDR2包含SEQ ID NO.5所示的氨基酸序列。
在某些实施方式中，所述第一LCDR3包含SEQ ID NO.6所示的氨基酸序列。
在某些实施方式中，所述第一轻链能够与帕妥珠单抗的重链结合。
在某些实施方式中，所述第二轻链包含第二LCDR1-3，其中所述第二LCDR1包含SEQ ID NO.1所示的氨基酸序列。
在某些实施方式中，所述第二LCDR2包含SEQ ID NO.2所示的氨基酸序列。
在某些实施方式中，所述第二LCDR3包含SEQ ID NO.3所示的氨基酸序列。
在某些实施方式中，所述第二轻链能够与曲妥珠单抗的重链结合。
在某些实施方式中，所述第一轻链和所述第二轻链的可变区包含SEQ ID NO.7-12中任一项所示的氨基酸序列。
在某些实施方式中，所述第一轻链和所述第二轻链的可变区包含SEQ ID NO.7所示的氨基酸序列。
在某些实施方式中，所述第一轻链和所述第二轻链包含SEQ ID NO.13-18中任一项所述的氨基酸序列。
在某些实施方式中，所述第一轻链选自下组：帕妥珠单抗的轻链或其突变体和曲妥珠单抗的轻链或其突变体；和/或，所述第二轻链选自下组：帕妥珠单抗的轻链或其突变体和曲妥珠单抗的轻链或其突变体。
在某些实施方式中，所述第一轻链和所述第二轻链的氨基酸序列相同。
在某些实施方式中，所述第一轻链和所述第二轻链包含SEQ ID NO.13所述的氨基酸序列。
在某些实施方式中，所述靶向HER2的双特异性抗体或其抗原结合片段包含第一重链和第二重链，其中所述第一重链能够在生理条件或体外的蛋白表达状态下与所述第一轻链正确结合。
在某些实施方式中，所述第二重链能够在生理条件或体外的蛋白表达状态下与所述第二轻链正确结合。
在某些实施方式中，所述第一重链包含第一重链可变区，所述第一重链可变区为帕妥珠单抗的重链可变区。
在某些实施方式中，所述第二重链包含第二重链可变区，所述第二重链可变区为曲妥珠单抗的重链可变区。
在某些实施方式中，所述第一重链和所述第二重链包含重链恒定区，其中所述重链恒定区源自人IgG的恒定区。
在某些实施方式中，所述第一重链和所述第二重链的Fc片段包含SEQ ID NO.25-57中任一项所述的氨基酸序列。
在某些实施方式中，所述第一重链包含21或23所示的氨基酸序列。
在某些实施方式中，所述第二重链包含22或24所示的氨基酸序列。
在某些实施方式中，所述抗体药物偶联物，包含式1所示的结构：
M-(L1) a-(L2) b-D(式1)，其中M表示本申请所述的靶向HER2的双特异性抗体或其抗原结合片段；L1表示与M连接的连接子，L2表示与D连接的连接子，a，b各自独立地选自0-10，D表示药物。
在某些实施方式中，所述L1和/或L2选自下组：可裂解的连接子、不可裂解的连接子、亲水的连接子、疏水的连接子、带电荷的连接子、不带电荷的连接子和基于二羧酸的连接子。
在某些实施方式中，所述L1与所述M通过所述M上的巯基、叠氮基或酰胺基连接。
在某些实施方式中，所述M包含第一重链和第二重链，所述第一重链和/或所述第二重链包含能够与所述L1连接的连接位点。
在某些实施方式中，所述连接位点包含去糖基化修饰后能够与所述L1连接的基团。
在某些实施方式中，所述基团位于所述第一重链的第297位氨基酸Q的侧基；和/或，位于所述第二重链的第298位氨基酸Q的侧基。
在某些实施方式中，所述基团包括酰胺基。
在某些实施方式中，所述连接位点包含经糖基化修饰后能够与所述L1连接的基团。
在某些实施方式中，所述基团位于所述第一重链的第299位氨基酸N的侧基；和/或，位于所述第二重链的第300位氨基酸N的侧基。
在某些实施方式中，所述基团包含-N 3。
在某些实施方式中，所述经糖基化修饰包括：所述M已经与UDP-GalNAz、β-1,4-半乳糖基转移酶或其变体接触。
在某些实施方式中，所述L1能够参与SPAAC反应。
在某些实施方式中，所述L1选自下组：马来酰亚胺、琥珀酰亚胺-3-基-N和DBCO。
在某些实施方式中，所述L1为DBCO-(PEG) n1，其中 n1为0-10的整数，或者，所述L1为马来酰亚胺。
在某些实施方式中，所述L2选自下组：多肽、VC-PAB、N-琥珀酰亚胺基-3-(2-吡啶基二硫代)丙酸酯(SPDP)、N-琥珀酰亚胺基4-(2-吡啶基二硫代)戊酸酯(SPP)、N-琥珀酰亚胺基4-(2-吡啶基二硫代)丁酸酯(SPDB)、N-琥珀酰亚胺基-4-(2-吡啶基二硫基)-2-磺基丁酸酯(sulfo-SPDB)、N-琥珀酰亚胺基碘乙酸酯(SIA)、N-琥珀酰亚胺基(4-碘乙酰基)氨基苯甲酸酯(SIAB)、马来酰亚胺PEG NHS、N-4-(马来酰亚胺基甲基)环己基羧酸琥珀酰胺基酯(SMCC)、N-磺基(4-亚马来酰亚胺甲基)环己基羧酸磺基琥珀酯(磺基-SMCC)和2,5-二氧吡咯烷基-1-基17-(2,5-二氧代-2,5-二氢-1H-吡咯-1-基)-5,8,11,14-四氧-4,7,10,13-四氮杂十八烷-1-酸酯(CX1-1)。
在某些实施方式中，所述L2为GGFG。
在某些实施方式中，所述药物具有杀伤肿瘤细胞，和/或抑制肿瘤细胞生长的能力。
在某些实施方式中，所述药物包括小分子药物。
在某些实施方式中，所述药物选自下组：V-ATPase抑制剂、Bcl2抑制剂、MCL1抑制剂、HSP90抑制剂、IAP抑制剂、mTor抑制剂、微管稳定剂、微管去稳定剂、auristatin、dolastatin、美登木素生物碱、MetAP(蛋氨酸氨基肽酶)、蛋白质CRM1的核输出抑制剂、DPPIV抑制剂、蛋白酶体抑制剂、线粒体中的磷酸转移反应抑制剂、蛋白质合成抑制剂、CDK2抑制剂、CDK9抑制剂、驱动蛋白抑制剂、HDAC抑制剂、DNA破坏剂、DNA烷化剂、DNA嵌入剂、DNA小沟结合剂、DHFR抑制剂、核苷类似物、HD AC抑制剂、蒽环类、NAMPT抑制剂、SN-38葡糖醛酸、依托泊苷磷酸酯、氮芥末、蛋白体抑制剂、细胞因子和Toll样受体激动剂。
在某些实施方式中，所述药物选自下组：DM1、exatecan、DXd、MMAE、SN-38、Calicheamicin、Anthracyclin-5G、DM4、微管抑制剂SHR153024、PNU-159682、Duo5毒素、SN38衍生物或他们的衍生物。
在某些实施方式中，所述的抗体药物偶联物具有选自下组的结构：
Figure PCTCN2022103592-appb-000001
在某些实施方式中，所述抗体药物偶联物的药物/抗体比率约2-6。
另一方面，本申请提供一种制备本申请所述的抗体药物偶联物的化合物，其具有式2所示的结构：M-(L1) a(式2)，其中M表示本申请所述靶向HER2的双特异性抗体或其抗原结合片段；L1表示与M连接的连接子；a选自0-10。
在某些实施方式中，所述L1选自下组：可裂解的连接子、不可裂解的连接子、亲水的连接子、疏水的连接子、带电荷的连接子、不带电荷的连接子和基于二羧酸的连接子。
在某些实施方式中，所述L1与所述M通过所述M上的巯基、叠氮基或酰胺基连接。
在某些实施方式中，所述M包含第一重链和第二重链，所述第一重链和/或所述第二重链包含能够与所述L1连接的连接位点。
在某些实施方式中，所述连接位点包含去糖基化修饰后能够与所述L1连接的基团。
在某些实施方式中，所述基团位于所述第一重链的第297位氨基酸Q的侧基；和/或，位于所述第二重链的第298位氨基酸Q的侧基。
在某些实施方式中，所述基团包括酰胺基。
在某些实施方式中，所述连接位点包含经糖基化修饰后能够与所述L1连接的基团。
在某些实施方式中，所述基团位于所述第一重链的第299位氨基酸N的侧基；和/或，位于所述第二重链的第300位氨基酸N的侧基。
在某些实施方式中，所述基团包含-N 3。
在某些实施方式中，所述经糖基化修饰包括：所述M已经与UDP-GalNAz、β-1,4-半乳糖基转移酶或其变体接触。
在某些实施方式中，所述L1能够参与SPAAC反应。
在某些实施方式中，所述L1选自下组：马来酰亚胺、琥珀酰亚胺-3-基-N和DBCO。
在某些实施方式中，所述L1为DBCO-(PEG) n1，其中n1为0-10的整数，或者，所述L1为马来酰亚胺。
在某些实施方式中，所述化合物包括选自下组的结构：
Figure PCTCN2022103592-appb-000002
另一方面，本申请提供了制备本申请所述的抗体药物偶联物的方法，其包括以下步骤：将本申请所述的化合物与本申请所述的药物接触。
另一方面，本申请提供了药物组合物，其包含本申请所述的抗体药物偶联物，或药学上可接受的载体。
另一方面，本申请提供了调节受试者的肿瘤微环境的方法，其包括以下步骤：向受试者施用本申请所述抗体药物偶联物、或者本申请所述的药物组合物。
另一方面，本申请提供了调节受试者的免疫反应的方法，其包括以下步骤：向受试者施用本申请所述的抗体药物偶联物、或者本申请所述的药物组合物。
另一方面，本申请提供了本申请所述的抗体药物偶联物、或者本申请所述的药物组合物制备药物中的应用，其中所述药物可以预防和/或治疗肿瘤。
在某些实施方式中，所述肿瘤包括实体瘤和/或非实体瘤。
本领域技术人员能够从下文的详细描述中容易地洞察到本申请的其它方面和优势。下文的详细描述中仅显示和描述了本申请的示例性实施方式。如本领域技术人员将认识到的，本申请的内容使得本领域技术人员能够对所公开的具体实施方式进行改动而不脱离本申请所涉及发明的精神和范围。相应地，本申请的附图和说明书中的描述仅仅是示例性的，而非为限 制性的。
附图说明
本申请所涉及的发明的具体特征如所附权利要求书所显示。通过参考下文中详细描述的示例性实施方式和附图能够更好地理解本申请所涉及发明的特点和优势。对附图简要说明书如下：
图1显示的是本申请所述靶向HER2的双特异性抗体进行糖基化修饰的反应。
图2显示的是糖基化修饰的本申请所述靶向HER2特异性的双特异性抗体的HPLC图谱。
图3显示的是获得本申请所述抗体药物偶联物的反应步骤。
图4显示的是本申请所述抗体药物偶联物的HPLC图谱。
图5显示的是获得本申请所述抗体药物偶联物的反应步骤。
图6显示的是本申请所述抗体药物偶联物的Waters Xevo G2-QTOF质谱分析结果。
图7显示的是本申请所述抗体药物偶联物的Waters Xevo G2-QTOF质谱分析结果。
图8显示的是DS-8201中的轻链部分的Waters Xevo G2-QTOF质谱分析结果。
图9显示的是DS-8201中的重链部分的Waters Xevo G2-QTOF质谱分析结果。
图10显示的是本申请所述靶向HER2的双特异性抗体与FcγRIIIa的结合亲和力情况。
图11显示的是本申请所述抗体药物偶联物与FcγRIIIa的结合亲和力情况。
图12显示的是本申请所述靶向HER2的双特异性抗体与FcγRI的结合亲和力情况。
图13显示的是本申请所述抗体药物偶联物与FcγRI的结合亲和力情况。
图14显示的是本申请所述抗体药物偶联物对肿瘤细胞的结合能力。
图15显示的是本申请所述抗体药物偶联物处理3天对肿瘤细胞的杀伤能力。
图16显示的是本申请所述抗体药物偶联物处理5天对肿瘤细胞的杀伤能力。
图17显示的是本申请所述抗体药物偶联物处理3天对肿瘤细胞的杀伤能力。
图18显示的是各个实验组对于SK-BR-3细胞的增殖抑制结果。
图19显示的是各个实验组对于MDA-MB-468细胞的增殖抑制结果。
图20显示的是本申请所述抗体药物偶联物ADCC活性的分析结果。
图21显示的是利用PBMC体系测定本申请所述抗体药物偶联物ADCC活性的步骤。
图22-23显示的是利用PBMC体系测定本申请所述抗体药物偶联物ADCC活性的结果。
图24-26显示的是本申请所述抗体药物偶联物在肿瘤细胞中的内吞作用。
图27显示的是本申请所述抗体药物偶联物在肿瘤细胞中的内吞作用。
图28显示的是本申请所述抗体药物偶联物的药代动力学研究结果。
图29显示的是本申请所述抗体药物偶联物的诱导内吞后NCI-N87阳性细胞比例结果。
图30显示的是人源胃癌PDX肿瘤模型中各组小鼠肿瘤体积的生长曲线。
图31显示的是人源胃癌HER2IHC-PDX肿瘤模型小鼠肿瘤体积的生长曲线。
具体实施方式
以下由特定的具体实施例说明本申请发明的实施方式，本领域技术人员可由本说明书所公开的内容容易地了解本申请发明的其他优点及效果。
术语定义
在本申请中，术语“抗体药物偶联物”通常是指ADC，即与一个或多个化学药物连接的结合蛋白(例如抗体或其抗原结合片段)。所述化学药物可以为任意的治疗剂和/或细胞毒性及。所述抗体药物偶联物可以具有从1-8任何数目的与抗体偶联的药物，例如可以包括2、4、6或8的载药种类(drug loaded species)。在本申请中，所述药物可以包括有丝分裂抑制剂、抗肿瘤抗生素、免疫调节剂、用于基因疗法的载体、烷化剂、抗血管生成剂、抗代谢物、含硼剂、化疗保护剂、激素、抗激素剂、皮质类固醇、光敏治疗剂、寡核苷酸、放射性核素剂、拓扑异构酶抑制剂、酪氨酸激酶抑制剂和/或放射致敏剂。
在本申请中，术语“药物/抗体比率”或“DAR”通常是指与ADC的抗体连接的药物的数目。ADC的DAR范围可以为1-8，也可以为更高载量(例如10)，DAR的范围可以取决于抗体上连接位点的数目。在本申请中，所述DAR可以为负载到单个抗体上的药物的数目。所述DAR也可以为一组ADC的平均或均值DAR。
在本申请中，术语“HER2”通常是指人表皮生长因子受体2(SwissProt P04626)。在本申请中，所述HER2也可以称为rbB-2、NEU、HER-2或CD340。所述HER2可以包括HER2的任何变体、同工型、和物种同源物，其由细胞-包括肿瘤细胞-天然地表达，或由以HER2基因或cDNA转染的细胞表达。
在本申请中，术语“胞外结构域”通常是指人HER2(SwissProt P04626)的胞外结构域，其可以包括I、II、III和IV四个胞外结构域。
在本申请中，术语“抗体”通常是指由两对相同的多肽链(每对具有一条“轻”(L)链和一条“重”(H)链)组成的免疫球蛋白分子。抗体轻链可分类为κ和λ轻链。重链可分类为μ、δ、γ、α或ε，并且分别将抗体的同种型定义为IgM、IgD、IgG、IgA和IgE。在轻链和重链内，可变区和恒定区通过大约12或更多个氨基酸的“J”区连接，重链还包含大约3个或更多 个氨基酸的“D”区。各重链由重链可变区(VH)和重链恒定区(CH)组成。重链恒定区由3个结构域(CH1、CH2和CH3)组成。各轻链由轻链可变区(VL)和轻链恒定区(CL)组成。轻链恒定区由一个结构域CL组成。抗体的恒定区可介导免疫球蛋白与宿主组织或因子，包括免疫系统的各种细胞(例如，效应细胞)和经典补体系统的第一组分(C1q)的结合。VH和VL区还可被细分为具有高变性的区域(称为互补决定区(CDR))，其间散布有较保守的称为构架区(FR)的区域。各VH和VL由按下列顺序：FR1、CDR1、FR2、CDR2、FR3、CDR3、FR4从氨基末端至羧基末端排列的3个CDR和4个FR组成。各重链/轻链对的可变区(VH和VL)分别形成抗体结合部位。氨基酸至各区域或结构域的分配遵循Kabat Sequences of Proteins of Immunological Interest(National Institutes of Health,Bethesda,Md.(1987 and 1991))，或Chothia&Lesk(1987)J.Mol.Biol.196:901-917；Chothia等人(1989)Nature 342:878-883的定义。术语“抗体”不受任何特定的产生抗体的方法限制。例如，其包括，特别地，重组抗体、单克隆抗体和多克隆抗体。抗体可以是不同同种型的抗体，例如，IgG(例如，IgG1，IgG2，IgG3或IgG4亚型)，IgA1，IgA2，IgD，IgE或IgM抗体。
在本申请中，术语“抗原结合部分”是指全长抗体的一个或多个部分，所述部分保持结合抗体所结合的相同抗原(例如，HER2)的能力，与完整抗体竞争对抗原的特异性结合。通常参见，Fundamental Immunology,Ch.7(Paul,W.,ed.,第2版，Raven Press,N.Y.(1989)，其以其全文通过引用合并入本文，用于所有目的。可通过重组DNA技术或通过完整抗体的酶促或化学断裂产生抗原结合部分。在一些情况下，抗原结合部分包括Fab、Fab'、F(ab')2、Fd、Fv、dAb和互补决定区(CDR)片段、单链抗体(例如，scFv)、嵌合抗体、双抗体(diabody)和这样的多肽，其包含足以赋予多肽特异性抗原结合能力的抗体的至少一部分。可使用本领域技术人员已知的常规技术(例如，重组DNA技术或酶促或化学断裂法)从给定的抗体(例如单克隆抗体2E12)获得抗体的抗原结合部分(例如，上述抗体片段)，并且以与对于完整抗体的方式相同的方式就特异性筛选抗体的抗原结合部分。
在本申请中，术语“Fd片段”意指由VH和CH1结构域组成的抗体片段；术语“Fv片段”意指由抗体的单臂的VL和VH结构域组成的抗体片段；术语“dAb片段”意指由VH结构域组成的抗体片段(Ward等人,Nature 341:544 546(1989))；术语“Fab片段”意指由VL、VH、CL和CH1结构域组成的抗体片段；术语“F(ab')2片段”意指包含通过铰链区上的二硫桥连接的两个Fab片段的抗体片段。
在本申请中，术语“抗体Fc”通常是指基于抗体的木瓜蛋白酶裂解而定义的，人免疫球蛋白链恒定区。所述Fc可以是免疫球蛋白重链恒定区的羧基端或其中的一部分。例如，免疫球 蛋白Fc区可包括重链CH2、CH3、CH4的两个或更多结构域与免疫球蛋白铰链区的组合。根据重链恒定区的氨基酸序列，免疫球蛋白可以分为不同的种类，主要有5类免疫球蛋白：IgA，IgD，IgE，IgG和IgM，其中一些还可进一步分成亚类(同种型)，如IgG-l，IgG-2，IgG-3，IgG-4，IgA-l和IgA-2。从特定的免疫球蛋白类别和亚类中选择特定的免疫球蛋白Fc区在本领域技术人员所掌握的范围之内。例如，所述Fc可以包括至少一个免疫球蛋白绞链区，一个CH2结构域和一个CH3结构域，例如为人IgG1 Fc。
在本申请中，术语“双特异性抗体”通常是指能够分别和两种抗原或抗原表位结合的抗体。所述双特异性抗体可以包括能够特异性结合第一抗原或抗原表位的抗体的轻链和重链，以及能够特异性结合第二抗原或抗原表位的抗体的轻链和重链。在本申请的一个实施方案中，所述双特异性抗体中能够特异性结合第一抗原或抗原表位的抗体的轻链和能够特异性结合第二抗原或抗原表位的抗体的轻链具有相同的序列。在本申请的一个实施方案中，所述双特异性抗体中能够特异性结合第一抗原或抗原表位的抗体的重链和能够特异性结合第二抗原或抗原表位的抗体的重链具有不同的序列。
在本申请中，术语“表位”或“抗原表位”通常是指，抗原上被免疫球蛋白或抗体特异性结合的部位。“表位”在本领域内也称为“抗原决定簇”。表位或抗原决定簇通常由分子的化学活性表面基团例如氨基酸或碳水化合物或糖侧链组成并且通常具有特定的三维结构特征以及特定的电荷特征。例如，表位通常以独特的空间构象包括至少3，4，5，6，7，8，9，10，11，12，13，14或15个连续或非连续的氨基酸，其可以是“线性的”或“构象的”。参见，例如，Epitope Mapping Protocols in Methods in Molecular Biology，第66卷，G.E.Morris，Ed.(1996)。在线性表位中，蛋白质与相互作用分子(例如抗体)之间的所有相互作用的点沿着蛋白质的一级氨基酸序列线性存在。在构象表位中，相互作用的点跨越彼此分开的蛋白质氨基酸残基而存在。
在本申请中，术语“特异性结合”通常是指相对于非特异的吸附的结合。作为结合是否特异的判定基准，例如，可举出解离常数(例如，“KD”)。例如，所述双特异性抗体或其抗原结合片段对HER2蛋白的KD值为1×10 -5M以下、5×10 -6M以下、2×10 -6M以下、或1×10 -6M以下；为5×10 -9M以下、2×10 -9M以下、或1×10 -9M以下。所述结合可利用表面等离子共振(Surface Plasmon Resonance)法、ELISA法、RIA法等已知的方法测定。
本说明书中的“CDR”是指，互补性决定区(CDR：Complementarity deterring region)。已知抗体分子的重链及轻链中分别具有3个CDR。CDR也被称为高度可变区(hypervariable domain)，其为在抗体的重链及轻链的可变区域内一级结构的变异性特别高的部位，在重链及 轻链的多肽链的一级结构上，分别地，分离在3处。本说明书中，关于抗体的CDR，针对重链的CDR，从重链氨基酸序列的氨基末端侧开始记载为CDRH1、CDRH2、CDRH3，针对轻链的CDR，从轻链氨基酸序列的氨基末端侧开始，记载为CDRL1、CDRL2、CDRL3。这些部位在立体结构上相互接近，决定针对结合的抗原的特异性。
在本申请中，20种常规氨基酸和其缩写遵从常规用法。参见Immunology A Synthesis(第2版,E.S.Golub和D.R.Gren,Eds.,Sinauer Associates,Sunderland,Mass.(1991)),其通过引用合并入本文。
在本申请中，术语“帕妥珠单抗”通常是指Pertuzumab，也被称作2C4，商品名Perjeta。所述帕妥珠单抗的轻链和重链可变区的氨基酸序列可以参见US20090285837A1的图2。所述帕妥珠单抗是重组人源化单克隆抗体，可以与表皮生长因子受体2(HER2)的细胞外二聚化结构域(亚结构域II)发生特异性结合。所述帕妥珠单抗可以通过结合HER2，阻滞了HER2与其它HER受体的异二聚化，从而可以减缓了肿瘤的生长。所述帕妥珠单抗可以用于治疗HER2阳性的转移性乳腺癌，可以用于治疗早期乳腺癌。
在本申请中，术语“曲妥珠单抗”通常是指Trastuzumab，商品名
Figure PCTCN2022103592-appb-000003
所述曲妥珠单抗的轻链和重链的氨基酸序列可以参见US20090285837A1的图16。所述曲妥珠单抗是一种重组DNA衍生的人源化单克隆抗体，其可以是由悬浮培养于无菌培养基中的哺乳动物细胞(中国仓鼠卵巢细胞CHO)生产的。所述曲妥珠单抗可以特异性结合HER2的胞外结构域，也可以以刺激身体自身的免疫细胞去摧毁肿瘤细胞。所述曲妥珠单抗可以用于治疗HER2阳性的转移性乳腺癌、早期乳腺癌和HER2阳性的转移性胃腺癌或胃食管交界腺癌。
在本申请中，术语“Exatecan”通常是指依喜替康(DX-8951)，其是一种DNA拓扑异构酶I抑制剂，CAS号为171335-80-1。DXd是依喜替康(DX-8951)衍生物，CAS号为1599440-33-1。作为一种药物喜树碱类衍生物，依喜替康和DXd属于中等毒性类药物，此类药物应用于ADC中可降低因毒素脱落而带来的毒副作用。
在本申请中，术语“连接子”通常是指为双功能或多功能的且用于将本申请所述双特异性抗体或其抗原结合片段连接于本申请所述药物的化学部分。所述连接子可以包括一个偶联成分，或者可以包括多个偶联成分。
在本申请中，术语“肿瘤”通常是指哺乳动物中通常特征是不受调节的细胞生长的生理状况。所述肿瘤包括一个或多个癌性细胞。所述肿瘤可以包括实体瘤和/或非实体瘤。
在本申请中，术语“肿瘤微环境”通常是指肿瘤存在其中的环境，该环境是肿瘤内的非细胞区域和直接在肿瘤组织外侧但不属于癌细胞本身的细胞内区室的区域。肿瘤与肿瘤微环境 密切相关，并可以不断相互作用。例如，肿瘤可以改变所述肿瘤微环境，所述肿瘤微环境可以影响肿瘤的生长和扩散。通常，所述肿瘤微环境具有5.8至7.0范围内的低pH。所述肿瘤微环境可以具有较低浓度的葡萄糖和其他营养物，但具有较高浓度的乳酸。此外，所述肿瘤微环境的温度可以比正常生理温度高0.3至1℃。所述肿瘤微环境已经描述于Gillies等的“MRI of the Tumor Microenvironment”，Journal of Magnetic Resonance Imaging，第16卷，第430-450页，2002。
在本申请中，术语“治疗”通常是指治疗性处理及预防性或防范性措施二者，其中目标是预防或减缓(减轻)不想要的生理学变化或病症，诸如高增殖性状况诸如癌症的生长，形成或传播。为了本发明，有利或期望的临床结果包括但不限于：缓解症状，削弱疾病的程度，疾病状态稳定(即不恶化)，延迟或减缓疾病发展，改善或减轻疾病状态，及康复(无论是部分的还是完全的)，无论是可检测的还是不可检测的。“治疗”或“处理”还可以指与不接受治疗的预期存活相比延长存活。需要治疗的受试者包括早就患有疾病或病症的受试者以及倾向于患上疾病或病症的受试者或者要预防状况或病症的受试者。
发明详述
一方面，本申请提供一种抗体药物偶联物，其包含靶向HER2的双特异性抗体或其抗原结合片段。
在某些实施方式中，所述抗体药物偶联物，可以包含式1所示的结构：M-(L1) a-(L2) b-D(式1)，其中M表示本申请所述的靶向HER2的双特异性抗体或其抗原结合片段；L1表示与M连接的连接子，L2表示与D连接的连接子，a，b各自独立地选自0-10，D表示药物。
靶向HER2的双特异性抗体或其抗原结合部分
在本申请中，所述靶向HER2的双特异性抗体或其抗原结合片段可以特异性结合人HER2的至少一个表位。例如，可以特异性结合人HER2的2个不同的表位。在本申请中，所述至少两个表位可以位于人HER2蛋白中的同一个结构域，例如，也可以位于人HER2蛋白中的至少两个不同的结构域。
例如，所述靶向HER2的双特异性抗体或其抗原结合片段可以特异性结合人HER2的胞外结构域II和/或人HER2的胞外结构域IV。例如，所述靶向HER2的双特异性抗体或其抗原结合片段可以特异性结合人HER2的胞外结构域II和人HER2的胞外结构域IV。
在本申请中，所述靶向HER2的双特异性抗体或其抗原结合片段可以包含第一轻链和第二轻链。
在本申请中，所述第一轻链可以包含第一LCDR1-3，其中所述第一LCDR1可以包含SEQ  ID NO.4所示的氨基酸序列。
在本申请中，所述第一LCDR2可以包含SEQ ID NO.5所示的氨基酸序列。
在本申请中，所述第一LCDR3可以包含SEQ ID NO.6所示的氨基酸序列。
例如，所述第一轻链中，所述第一LCDR1可以包含SEQ ID NO.4所示的氨基酸序列；所述第一LCDR2可以包含SEQ ID NO.5所示的氨基酸序列；且所述第一LCDR3可以包含SEQ ID NO.6所示的氨基酸序列。
在本申请中，所述第一轻链能够与帕妥珠单抗的重链结合。
在本申请中，所述第二轻链可以包含第二LCDR1-3，其中所述第二LCDR1包可以含SEQ ID NO.1所示的氨基酸序列。
在本申请中，所述第二LCDR2可以包含SEQ ID NO.2所示的氨基酸序列。
在本申请中，所述第二LCDR3可以包含SEQ ID NO.3所示的氨基酸序列。
例如，所述第二轻链中，所述第二LCDR1包可以含SEQ ID NO.1所示的氨基酸序列；所述第二LCDR2可以包含SEQ ID NO.2所示的氨基酸序列；且所述第二LCDR3可以包含SEQ ID NO.3所示的氨基酸序列。
在本申请中，所述第二轻链能够与曲妥珠单抗的重链结合。
在本申请中，所述第一轻链和/或所述第二轻链可以从两株原始单克隆抗体(例如，可以为已知单克隆抗体)改造获得。在本申请中，所述两株原始单克隆抗体可以靶向HER2。例如，所述两株原始单克隆抗体可以特异性结合人HER2的至少一个表位。例如，所述两株原始单克隆抗体可以分别特异性结合人HER2的2个不同的表位。例如，所述两株原始单克隆抗体可以分别特异性结合人HER2的胞外结构域II和人HER2的胞外结构域IV。例如，所述两株原始单克隆抗体可以为曲妥珠单抗和帕妥珠单抗。
例如，所述第一轻链和/或所述第二轻链可以与所述两株原始单克隆抗体中任意一株的轻链的氨基酸序列不同。又例如，所述第一轻链和/或所述第二轻链可以与所述两株原始单克隆抗体中任意一株的轻链的氨基酸序列相同，或者，所述第一轻链和/或所述第二轻链的氨基酸序列，可以是在所述两株原始单克隆抗体中任意一株的轻链的氨基酸序列的基础上经过改造获得的。例如，所述改造可以包括氨基酸序列改造。例如，所述改造的目的可以为尽可能保持与所述两株原始单克隆抗体所对应的抗原或抗原表位的亲和力。
在本申请中，所述改造可以包括氨基酸的突变、缺失或添加，例如突变、缺失或添加不超过3个氨基酸，不超过2个氨基酸，或者不超过1个氨基酸。
在本申请中，所述第一轻链的可变区的氨基酸序列可以与所述第二轻链的可变区的氨基酸序列相同。
在本申请中，所述第一轻链和所述第二轻链的可变区可以包含SEQ ID NO.7-12中任一项所示的氨基酸序列。
在本申请中，所述第一轻链和所述第二轻链的可变区可以包含SEQ ID NO.7所示的氨基酸序列。
在本申请中，所述第一轻链和所述第二轻链可以包括轻链恒定区。所述轻链恒定区可以为κ型或λ型。例如，所述κ型轻链恒定区包括各种同种异型，如Km1、Km1,2、Km3；所述λ型轻链恒定区包括各种同种异型，如CL1、CL2、CL3、CL6和CL7。
在本申请中，所述第一轻链和所述第二轻链可以包含SEQ ID NO.13-18中任一项所述的氨基酸序列。
在本申请中，所述第一轻链可以选自下组：帕妥珠单抗的轻链或其突变体和曲妥珠单抗的轻链或其突变体。在本申请中，所述第二轻链可以选自下组：帕妥珠单抗的轻链或其突变体和曲妥珠单抗的轻链或其突变体。
在本申请中，所述第一轻链可以为帕妥珠单抗的轻链或其突变体。
在本申请中，所述第二轻链可以为曲妥珠单抗的轻链或其突变体。
在本申请中，所述第一轻链和所述第二轻链的氨基酸序列可以相同。
在本申请中，所述第一轻链和所述第二轻链可以包含SEQ ID NO.13所述的氨基酸序列。
在本申请中，所述靶向HER2的双特异性抗体或其抗原结合片段可以包含第一重链和第二重链，其中所述第一重链能够在生理条件或体外的蛋白表达状态下与所述第一轻链正确结合。
在本申请中，所述第二重链能够在生理条件或体外的蛋白表达状态下与所述第二轻链正确结合。
在本申请中，所述第一重链可以包含第一重链可变区，所述第一重链可变区可以为帕妥珠单抗的重链可变区。
在本申请中，所述第二重链可以包含第二重链可变区，所述第二重链可变区可以为曲妥珠单抗的重链可变区。
在本申请中，所述第一重链和所述第二重链可以包含重链恒定区，其中所述重链恒定区可以源自人IgG的恒定区。
在某些情况下，所述第一重链的重链恒定区，和/或所述第二重链的重链恒定区可以包含Fc区域。所述Fc区域可以经过改造，例如，所述改造可以提高所述第一重链和所述第二重链形成异二聚体的比例。
在本申请中，所述改造的技术可以是本领域技术人员所熟知的，例如可以参见参考 Ridgway,Presta et al.1996；Carter 2001，专利CN 102558355A，专利CN 103388013A。
在本申请中，所述第一重链的重链恒定区和所述第二重链的重链恒定区的重链类型可以相同，也可以不同。例如，所述第一重链的重链恒定区和所述第二重链的重链恒定区可以与两株原始单克隆抗体的重链恒定区的氨基酸序列不同。又例如，所述第一重链的重链恒定区的氨基酸序列可以与其中一株所述原始单克隆抗体的重链恒定区的氨基酸序列相同；和/或，所述第二重链的重链恒定区的氨基酸序列可以与其中另一株所述原始单克隆抗体的重链恒定区的氨基酸序列相同。
在本申请中，所述第一重链和所述第二重链的Fc片段可以包含SEQ ID NO.24-57中任一项所述的氨基酸序列。
在本申请中，所述第一重链可以包含SEQ ID NO.21-24中任一项所示的氨基酸序列。例如，所述第一重链可以包含SEQ ID NO.21或23所示的氨基酸序列。
在本申请中，所述第二重链可以包含SEQ ID NO.21-24中任一项所示的氨基酸序列。例如，所述第二重链可以包含SEQ ID NO.22或24所示的氨基酸序列。
在本申请中，所述第一重链可以包含SEQ ID NO.21或23所示的氨基酸序列，且所述第二重链可以包含SEQ ID NO.22或24所示的氨基酸序列。例如，在本申请中，所述第一重链可以包含SEQ ID NO.21所示的氨基酸序列，且所述第二重链可以包含SEQ ID NO.22所示的氨基酸序列。例如，在本申请中，所述第一重链可以包含SEQ ID NO.23所示的氨基酸序列，且所述第二重链可以包含SEQ ID NO.24所示的氨基酸序列。
在本申请中，所述靶向HER2的双特异性抗体或其抗原结合片段可以根据本领域常规的技术手段获得。例如，可以从宿主细胞中纯化获得。所述纯化方法可以包括色谱技术如体积排阻、离子交换、亲和色谱法及超滤法。
药物偶联物及化合物
在本申请中，所述抗体药物偶联物可以包含式1所示的结构：M-(L1) a-(L2) b-D(式1)，其中M表示本申请所述的靶向HER2的双特异性抗体或其抗原结合片段；L1表示与M连接的连接子，L2表示与D连接的连接子，a，b各自独立地选自0-10，D表示药物。
在本申请中，一种制备本申请所述的抗体药物偶联物的化合物，其具有式2所示的结构：M-(L1) a(式2)，其中M表示本申请所述靶向HER2的双特异性抗体或其抗原结合片段；L1表示与M连接的连接子；a选自0-10。
M的修饰与L1的连接
在本申请中，所述L1与所述M可以通过所述M上的巯基、叠氮基或酰胺基连接。
在本申请中，所述M可以包含第一重链和第二重链，所述第一重链和/或所述第二重链可以包含能够与所述L1连接的连接位点。
在本申请中，所述连接位点可以包含去糖基化修饰后能够与所述L1连接的基团。
在本申请中，所述基团可以位于所述第一重链的第297位氨基酸Q的侧基；和/或，可以位于所述第二重链的第298位氨基酸Q的侧基。例如，所述基团可以包括酰胺基。例如，可以使用谷氨酰胺转移酶(TGs)将所述氨基酸Q作为所述连接位点，形成DAR等于约2的所述抗体药物偶联物。
在本申请中，所述连接位点可以包含经糖基化修饰后能够与所述L1连接的基团。
在本申请中，所述基团可以位于所述第一重链的第299位氨基酸N的侧基；和/或，可以位于所述第二重链的第300位氨基酸N的侧基。在重链的CH2域内，N299或N300可以具有一个保守的糖基化位点。可以利用所述N处的糖基化位点进行定点偶联。
其中所述氨基酸的位置的编号可以自所述第一重链和/或所述第二重链的N端的氨基酸起始计算。
在本申请中，所述糖基化修饰位点上可以进行糖基化修饰。在本申请中，所述糖基化形式可以通过，例如，在具有改变的糖基化结构的细胞中表达蛋白质而完成。具有改变的糖基化结构的细胞已经在本领域中描述且可以用作在其中表达具有所述糖基化修饰的蛋白质(例如，本申请所述的靶向HER2的双特异性抗体或其抗原结合片段)。
蛋白质的糖基化可以取决于蛋白质(例如，本申请所述的靶向HER2的双特异性抗体或其抗原结合片段)的氨基酸序列以及蛋白质在其中表达的宿主细胞。不同生物体可以产生不同的糖基化酶(例如，糖基转移酶和糖苷酶)，且具有不同的可用底物(核苷酸糖)。由于这些因素，蛋白质糖基化模式和糖基化修饰位点的残基的组成可能根据在其中表达特定蛋白质的宿主系统而不同。可用于本申请中的糖基残基可以包括葡萄糖、半乳糖、甘露糖、岩藻糖、n-乙酰萄糖胺和/或唾液酸。例如，所述糖基化修饰可以包括适应于人的糖基化修饰的模式。
在本申请中，所述糖基化修饰可以使蛋白质(例如，本申请所述的靶向HER2的双特异性抗体或其抗原结合片段)的性质产生变化。
不同的蛋白质糖基化可以影响和/或导致不同的蛋白质特性(例如在表达量、体内半衰期、蛋白质折叠、溶解性、对蛋白酶的易感性、运输、转运、区室化、分泌、其它蛋白质或因子的识别、抗原性或变应原性等特性的变化)。
在本申请中，所述蛋白质糖基化的具体结构和制备方式可以根据不同的糖基化目的而进行调整，本领域技术人员可以根据本领域现有技术来进行调整。在某些情况下，所述糖基化修饰可以根据人和/或动物的物种特异性来进行调整。在某些情况下，所述糖基化修饰可以通 过借助糖基化酶(例如，来源于宿主细胞的异源糖基化酶)来得以实现。例如，所述糖基化修饰可以获得表现出人蛋白糖基化特征的蛋白质(例如，本申请所述的靶向HER2的双特异性抗体或其抗原结合片段)。所述糖基化酶可以是天然来源的，也可以是非天然来源的。
在本申请中，所述经糖基化修饰可以包括：所述M已经与UDP-GalNAz、β-1,4-半乳糖基转移酶或其变体接触。例如，所述β-1,4-半乳糖基转移酶或其变体(例如β-1,4-Gal-T1-Y289)可以具有整合修饰的N-乙酰半乳糖胺(GalNAc)的能力，可以与蛋白质(例如所述靶向HER2的双特异性抗体或其抗原结合片段)聚糖上的末端N-乙酰氨基葡萄糖(GlcNAc)残基相连。
在本申请中，所述M与UDP-GalNAz、β-1,4-半乳糖基转移酶或其变体接触后，所述M可以包含本申请所述的基团。例如，所述M的所述 第一重链的N299位点上氨基酸N的侧基；和/或所述M的所述第二重链的N300位点上氨基酸N的侧基可以获得-N 3基修饰，成为糖基化修饰的M。
例如，在本申请所述的抗体药物偶联物和/或本申请所述的化合物中，通过所述基团，所述M的Fc区域可以与所述L1连接。例如，在本申请所述的抗体药物偶联物和/或本申请所述的化合物中，通过所述基团，所述M可以与所述L1连接。例如，所述糖基化修饰的M可以与所述L1进行加成反应。
连接子
在本申请中，所述a可以为选自0-10的整数，例如，可以为0、1、2、3、4、5、6、7、8、9或10。
在本申请中，所述b可以为选自0-10的整数，例如，可以为0、1、2、3、4、5、6、7、8、9或10。
在本申请中，所述L1和/或L2可以选自下组：可裂解的连接子、不可裂解的连接子、亲水的连接子、疏水的连接子、带电荷的连接子、不带电荷的连接子和基于二羧酸的连接子。
在本申请中，所述L1和所述L2都可以被认为属于连接子。
在本申请中，所述连接子可以为延伸药物连接以避免例如屏蔽抗体的活性位点或提高ADC的溶解性的部分。所述连接子可以包括延伸体(stretcher)单元和/或氨基酸单元。
在本申请中，所述连接子可以用于将抗体(例如，本申请所述的靶向HER2的双特异性抗体或其抗原结合片段)与所述药物进行偶联(例如，可以通过共价连接)。
例如，所述偶联可以通过抗体的半胱氨酸巯基或胺(例如N-末端或氨基酸侧链如赖氨酸)与所述连接子的官能团形成键。
例如，所述连接子可以具有能够与所述抗体上存在的游离的半胱氨酸反应以形成键(例 如共价键)的官能团。例如，所述官能团可以包括：马来酰亚胺、卤代乙酰胺类、α-卤代乙酰基、活性酯如琥珀酰亚胺酯、4-硝基苯基酯、五氟苯基酯、四氟苯基酯、酸酐、酰基氯、磺酰氯、异氰酸酯和/或异硫氰酸酯。
在某些情况下，所述连接子可以包括能够与抗体上存在的亲电子基团反应的官能团。例如，所述官能团可以包括：醛、酮羰基、肼、肟、氨基、肼、缩氨基硫脲、肼羧酸酯和/或芳基酰肼。
在本申请中，所述连接子可以包括可裂解的连接子和不可裂解的连接子。例如，所述连接子(例如L2)可以为可裂解(例如，可以自切割的(self immolative))的连接子，其可以利于所述药物的释放。例如，所述连接子(例如L1)可以为不可裂解的连接子，其可以与所述抗体连接且是不可切割的。在本申请中，所述可裂解的连接子可以在细胞内的条件下发生裂解。例如，所述可裂解的连接子可以包括：酸不稳定的连接子(例如，包含腙)蛋白酶敏感的连接子(例如，对肽酶敏感)、光不稳定的连接子和/或包含二硫化物的连接子。例如，所述可裂解的连接子可以包括能够被细胞内蛋白酶(例如溶酶体蛋白酶)和/或胞内体蛋白酶切割的肽连接子。在某些情况下，所述连接子在细胞内环境下是可裂解的。例如，所述连接子的裂解可以使得所述抗体药物偶联物中的药物发挥有效的治疗作用。
在本申请中，所述连接子的结构和/或性质在细胞外是稳定的。本申请所述的抗体药物偶联物在被转运或递送进入细胞之前，其结构可以是稳定的。例如，所述抗体药物偶联物中，所述靶向HER2的双特异性抗体或其抗原结合片段和所述药物保持偶联的状态。
在某些情况下，如果所述抗体药物偶联物中的所述连接子能够例如，保持所述靶向HER2的双特异性抗体或其抗原结合片段的特异性结合性质；参与所述抗体药物偶联物的递送，和/或，保持所述药物的治疗效果(例如，细胞毒性效应)。
在本申请中，所述连接子可以包括亲水的连接子(例如，可以为PEG4Mal和磺基-SPDB)和疏水的连接子。例如，所述亲水的连接子可以降低所述抗体药物偶联物可通过MDR(多药耐药性)或功能上类似的转运蛋白泵送到抗性癌细胞外的程度。
在本申请中，所述连接子也可以发挥直接或间接地抑制细胞生长和/或细胞增殖的功能。例如，所述连接子可以在所述裂解中发挥嵌入剂的功能。例如，所述连接子可以发挥间接的作用以抑制大分子生物合成。
在本申请中，所述连接子可以促进所述抗体药物偶联物进入细胞(例如可以促进“内化”作用)。在本申请中，所述连接子还可以被设计以提高所述抗体药物偶联物的稳定性。
在本申请中，所述L1能够参与SPAAC反应。所述SPAAC反应为叠氮-炔基环加成反应。SPAAC反应可以作为加成反应中的一种，应用于所述抗体药物偶联物的制备过程中。
在本申请中，所述L1可以选自下组：马来酰亚胺、琥珀酰亚胺-3-基-N和DBCO。
在本申请中，所述L1可以为DBCO-(PEG) n1，其中 n1为0-10的整数，或者，所述L1为马来酰亚胺。例如，所述L1可以为DBCO-(PEG) 4、DBCO-(PEG) 3。在本申请中，所述PEG分子可以用来增加所述抗体药物偶联物的亲水性。所述PEG分子可以用来满足L2(例如GGFG)裂解(例如酶切时)对空间距离的要求。
在本申请中，所述可以L2选自下组：多肽、VC-PAB、N-琥珀酰亚胺基-3-(2-吡啶基二硫代)丙酸酯(SPDP)、N-琥珀酰亚胺基4-(2-吡啶基二硫代)戊酸酯(SPP)、N-琥珀酰亚胺基4-(2-吡啶基二硫代)丁酸酯(SPDB)、N-琥珀酰亚胺基-4-(2-吡啶基二硫基)-2-磺基丁酸酯(sulfo-SPDB)、N-琥珀酰亚胺基碘乙酸酯(SIA)、N-琥珀酰亚胺基(4-碘乙酰基)氨基苯甲酸酯(SIAB)、马来酰亚胺PEG NHS、N-4-(马来酰亚胺基甲基)环己基羧酸琥珀酰胺基酯(SMCC)、N-磺基(4-亚马来酰亚胺甲基)环己基羧酸磺基琥珀酯(磺基-SMCC)和2,5-二氧吡咯烷基-1-基17-(2,5-二氧代-2,5-二氢-1H-吡咯-1-基)-5,8,11,14-四氧-4,7,10,13-四氮杂十八烷-1-酸酯(CX1-1)。
在本申请中，所述L2可以为GGFG、LP、VK、VC、GFG、GGFGG、GGFGS、GGFGGG、GGFGGE、GGFGGGFG、DGGF、DGGFG、D d GGFG、DG  Me GFG、DGGFS、DDGGFG、KDGGFG、KGGFG、EGGFG或者SGGFG。例如，所述L2可以为GGFG。组织蛋白酶B可裂解的四肽Gly-Gly-Phe-Gly(GGFG)是一种亲水性强(例如强于Gly-Phe-Leu-Gly)的连接子。
药物
在本申请中，所述药物可以具有杀伤肿瘤细胞，和/或抑制肿瘤细胞生长的能力。
在本申请中，所述药物可以包括小分子药物。
在本申请中，所述药物可以选自下组：V-ATPase抑制剂、Bcl2抑制剂、MCL1抑制剂、HSP90抑制剂、IAP抑制剂、mTor抑制剂、微管稳定剂、微管去稳定剂、auristatin、dolastatin、美登木素生物碱、MetAP(蛋氨酸氨基肽酶)、蛋白质CRM1的核输出抑制剂、DPPIV抑制剂、蛋白酶体抑制剂、线粒体中的磷酸转移反应抑制剂、蛋白质合成抑制剂、CDK2抑制剂、CDK9抑制剂、驱动蛋白抑制剂、HDAC抑制剂、DNA破坏剂、DNA烷化剂、DNA嵌入剂、DNA小沟结合剂、DHFR抑制剂、核苷类似物、HD AC抑制剂、蒽环类、NAMPT抑制剂、SN-38葡糖醛酸、依托泊苷磷酸酯、氮芥末、蛋白体抑制剂、细胞因子和Toll样受体激动剂。
在本申请中，所述药物可以选自下组：DM1、exatecan、DXd、MMAE、SN-38、Calicheamicin、Anthracyclin-5G、DM4、微管抑制剂SHR153024、PNU-159682、Duo5毒素、SN38衍生物或他们的衍生物。
在本申请中，所述药物可以为喜树碱或其衍生物。在本申请中，所述药物可以为DNA拓扑异构酶I抑制剂。
在本申请中，所述药物可以为依喜替康Exatecan(DX-8951)、DXd、杜卡霉素、丝裂霉素C、塔利霉素、美登素、TLR7激动剂、TLR8激动剂、TLR7/8激动剂或TLR9激动剂。
例如，所述药物可以具有以下的结构：
Figure PCTCN2022103592-appb-000004
在本申请中，所述的抗体药物偶联物可以具有选自下组的结构：
Figure PCTCN2022103592-appb-000005
在本申请中，所述抗体药物偶联物的药物/抗体比率可以为约2-6。例如，所述药物/抗体比率可以为约1、约2、约3、约4、约5或约6。例如，所述药物/抗体比率可以为4。例如，所述药物/抗体比率可以为3。
化合物和制备方法
另一方面，本申请提供了一种用以本申请所述的抗体药物偶联物的化合物，其具有式2所示的结构：M-(L1) a(式2)，其中M本申请所述靶向HER2的双特异性抗体或其抗原结合片段；L1表示与M连接的连接子；a选自0-10。
在本申请中，所述化合物可以包括选自下组的结构：
Figure PCTCN2022103592-appb-000006
另一方面，本申请提供了制备本申请所述的抗体药物偶联物的方法，其包括以下步骤：将本申请所述的化合物与本申请所述的药物接触。
在某些情况下，所述的抗体药物偶联物的制备方法可以包括以下的步骤：(1)将本申请所述的L1、所述的L2和所述的药物的接触；(2)获得化合物(L1) a-(L2) b-D；(3)将本申请所述的M与所述化合物(L1) a-(L2) b-D接触，得到本申请所述的抗体药物偶联物。例如，步骤(3)中的M进行了所述糖基化修饰。
药物组合物和应用
另一方面，本申请提供了药物组合物，其包含本申请所述的抗体药物偶联物，或药学上可接受的载体。
在本申请中，所述药物组合物可以包括“治疗有效剂量”或“预防有效剂量”的本申请所述的抗体药物偶联物。所述“治疗有效剂量”可以为在必要的剂量和持续时间下有效地实现所需治疗结果的量。所述治疗有效剂量可以由本领域技术人员确定，例如，可以根据疾病的状态、受试者的年龄、性别、体重、所述抗体药物偶联物在受试者体内引起说所需反应的能力等因素变化。所述“预防有效剂量”可以为在必要的剂量和持续时间下，有效地实现所需预防结果的量。例如，所述预防有效剂量可以低于所述治疗有效剂量。
在本申请中，所述药物组合物可以被配置为适用于施用的形式(例如，适用于肠胃外、皮内、肌肉内、腹膜内、静脉内和皮下、肿瘤内和/或粘膜施用)。在本申请中，所述药物组合物可以包括其他的药物活性成分。
在本申请中，所述药学上可接受的载体可以包括生理上相容的任何或所有溶剂、分散介质、包衣、抗细菌剂和抗真菌剂、等渗剂和吸收延迟剂等。所述药学上可接受的载体可以包括水、盐水、磷酸盐缓冲盐水、右旋糖、甘油、乙醇等等以及其组合中的一种或多种。所述 药学上可接受的载体还可以包括等渗即、润湿剂、乳化剂、防腐剂和/或缓冲剂。
另一方面，本申请提供了调节受试者的肿瘤微环境的方法，其包括以下步骤：向受试者施用本申请所述抗体药物偶联物、或者本申请所述的药物组合物。
另一方面，本申请提供了调节受试者的免疫反应的方法，其包括以下步骤：向受试者施用本申请所述的抗体药物偶联物、或者本申请所述的药物组合物。
本申请提供了本申请所述的抗体药物偶联物、或者本申请所述的药物组合物制备药物中的应用，其中所述药物可以调节受试者的肿瘤微环境和/或调节受试者的免疫反应。
本申请提供了所述的抗体药物偶联物、或者本申请所述的药物组合物，其用于调节受试者的肿瘤微环境和/或调节受试者的免疫反应。
另一方面，本申请提供了本申请所述的抗体药物偶联物、或者本申请所述的药物组合物制备药物中的应用，其中所述药物可以预防和/或治疗肿瘤。
本申请提供了一种预防和/或治疗肿瘤的方法，其包括以下步骤：向受试者施用本申请所述的抗体药物偶联物或者本申请所述的药物组合物制备药物。
本申请提供了所述的抗体药物偶联物、或者本申请所述的药物组合物，其用于预防和/或治疗肿瘤。
在本申请中，所述肿瘤可以包括实体瘤和/或非实体瘤。
例如，所述肿瘤可以包括胃癌、乳腺癌(例如乳腺导管癌)和/或胰腺癌。例如，本申请的肿瘤可以包含HER2弱阳性肿瘤和/或HER2阴性肿瘤，例如本申请的技术方案可以具有旁观者杀伤效应。
不欲被任何理论所限，下文中的实施例仅是为了阐释本申请发明的各个技术方案，而不用于限制本申请发明的范围。
实施例
靶向HER2的双特异性抗体(以下简称抗体A)，其中抗体A包含一个第一轻链，一个第二轻链，一个第一重链和一个第二重链，其中所述第一轻链和所述第二轻链的氨基酸序列如SEQ ID NO.13所示，其中所述第一重链的重链可变区的氨基酸序列如SEQ ID NO.19所示；所述第二重链的重链可变区的氨基酸序列如SEQ ID NO.20所示。其中所述第一重链的氨基酸序列如SEQ ID NO.21所示；所述第二重链的氨基酸序列如SEQ ID NO.22所示。
根据抗体A的氨基酸序列，人工合成并分离纯化抗体A。
DS-8201(又称：T-DXd，商品名ENHERTU)由第一三共、阿斯利康制备。DS-8201由抗HER2的IgG1单抗通过连接体与拓扑异构酶Ⅰ抑制剂DXd偶联而成。
U3-1402由第一三共制备。U3-1402由HER3抗体Patritumab与拓扑异构酶抑制剂DXd通过共价键偶联而成。
实施例1制备抗体A-ADC1分子
(1)制备包含连接子和毒素的化合物
获得DXd(即exatecan DX-8951衍生物，购自联宁生物)，并包含连接子和毒素的化合物1：DBCO-PEG4-GGFG-DXd，其具有以下的结构式：
Figure PCTCN2022103592-appb-000007
(2)制备糖基化的抗体A
将抗体A与β-1,4-半乳糖基转移酶(β-1,4-al-T1)(购自上海保森生物)在存在UDP-GalNAz的情况下接触。其中UDP-GalNAz的结构式为
Figure PCTCN2022103592-appb-000008
经接触后，抗体A上第一重链的N299和第二重链的N300位点上获得了-N 3基修饰，成为糖基化修饰的抗体A。抗体A糖基化修饰的反应如图1所示。
对所得的糖基化修饰的抗体A进行HPLC检测，检测结果如图2所示，图2的结果说明，糖基化修饰的抗体A具有其独特的特征峰，且分子量为148796Da。
(3)制备抗体A-ADC1分子
将步骤(1)中的DBCO-PEG4-GGFG-DXd与步骤(2)制得的糖基化修饰的抗体A在25-35℃条件下接触并反应10-15个小时，其中DBCO与糖基化修饰的抗体A中的-N 3基发生SPAAC反应，获得抗体A-ADC1分子。
获得抗体A-ADC1分子的反应如图3所示。对抗体A-ADC1进行HPLC检测，检测结果如图4所示，图4的结果说明，抗体A-ADC1分子具有其独特的特征峰，且分子量为154299。
(4)制备Herceptin-ADC1分子
将Herceptin按照实施例1记载的方法进行糖基化修饰，并与DBCO-PEG4-GGFG-DXd 反应，获得Herceptin-ADC1分子，其中其DAR为8。
实施例2制备抗体A-ADC2分子
(1)制备包含连接子和毒素的化合物
将马来酰亚胺与GGFG-DXd反应，获得包含连接子和毒素的化合物2，其结构式为：
Figure PCTCN2022103592-appb-000009
(2)制备修饰的抗体A
将抗体A与TECP反应，使得抗体A存在巯基(例如8个-SH)，获得修饰的抗体A。
(3)制备抗体A-ADC2分子
将步骤(2)制得的修饰的抗体A与步骤(1)制得的包含连接子和毒素的化合物2接触，获得抗体A-ADC2分子。
获得抗体A-ADC2分子的反应如图5所示。
实施例3包含抗体A的ADC分子的稳定性分析
分别考察实施例1制备的抗体A-ADC1、实施例2制备的抗体A-ADC2、以及DS-8201在人血清中的稳定性。
(1)抗体A-ADC1在血清中的稳定性
利用Protein L把加入血清里的抗体A-ADC1纯化出来，发现3天、7天后没有明显硫醇交换发生。
具体实验步骤：分别取处理后的人血清100μL加入到1.5mL的离心管中，然后加入抗体A-ADC1,使抗体A-ADC1的终浓度为0.1mg/mL，放在37℃培养箱分别孵育3、7天。
取20μL的Protein L磁珠悬浮液加入到1.5mL离心管中，放在磁力架上分离除去保存液。加入200μL的DPBS混匀磁珠，放在磁力架上分离除去上清液(该步骤重复2次)。
将孵育3天或7天的抗体A-ADC1加入到装有磁转的1.5ml离心管中，混匀，室温下置于混匀仪中1h，然后放在磁力架上除去上清液。接着加入200μL的DPBS，混匀，放在磁力架上分离除去上清液(该步骤重复3次)。最后加入20μL的洗脱液(100mM Glycine,PH＝2.8)，放在磁力架上室温混匀，收集上清液，然后向收集的上清液中加入20μL中和缓冲液 (200mM tris,PH＝8.0))，测其浓度，通过Waters Xevo G2-QTOF质谱分析纯化后的抗体A-ADC1。
结果如图6所示。图6的结果说明，在人血清中孵育0天、3天、7天后的抗体A-ADC1的分子量(154299左右)基本没有变化，在血清中3天、7天后DAR值仍然保持在4，偶联药物分子在血清中孵育3天、7天后没有明显硫醇交换发生，说明抗体A-ADC1在血清中的稳定性良好，不易发生在体内运送过程中将毒素释放到血液中的情况，更利于将偶联的小分子毒素药物运送到肿瘤靶点，可降低毒素分子因脱靶而对正常机体组织产生毒副作用风险。
(2)抗体A-ADC2在血清中的稳定性
按照步骤(1)涉及的方法，检测抗体A-ADC2在血清中的稳定性。
Waters Xevo G2-QTOF质谱分析的结果如图7所示。图7的结果说明，当抗体A-ADC2在人血清中分别孵育0天，3天、7天。孵育3天后，轻链上的马来酰亚胺因发生开环水解，所以能在人血清中稳定存在；而重链孵育3天后DAR值由6减少到2左右，孵育7天后重链的DAR值几乎为0，说明7天后抗体A-ADC2的重链会在血清中发生消除。
(3)DS-8201在血清中的稳定性
按照步骤(1)涉及的方法，检测DS-8201在血清中的稳定性。
Waters Xevo G2-QTOF质谱分析DS-8201重链和轻链在血清中孵育的结果分别如图8和图9所示。图8和图9的结果说明，当DS-8201在人血清中分别孵育0天、3天、7天。孵育3天后轻链上的马来酰亚胺因发生开环水解，所以能在人血清中稳定存在；而7天后重链的DAR值由6减少到2左右，DS-8201的重链在血清中发生了明显的消除，重链上的小分子毒素脱靶严重。
实施例3包含抗体A的ADC分子与FcγR的结合亲和力测试
采用生物膜干涉法(BLI)(丹纳赫公司的Fortebio大分子相互作用仪)分别测定抗体A、实施例1制备的抗体A-ADC1与FcγRI、FcγRIIIa等的亲和力。
亲和力测试的具体步骤：
固定抗体A-WS-161018(母分子抗体A)或者抗体A-ADC1到FAB2G biosensor，固化浓度为10μg/ml，固化高度2nm。
将FcγRIIIa稀释至1000nM、500nM、250nM、125nM、62.5nM，基线60s，结合30s，解离150s。稀释液为动力学缓冲液，再生液为甘氨酸-HCl(pH1.7)，中和液为稀释液。拟合时解离时间按10s拟合，实验结果如图10-图11和表1所示。图10和图11分别显示了抗体A和抗体A-ADC1与FcγRIIIa的结合亲和力情况。
表1
样品 KD(M) Kon(1/Ms) Kdis(1/s)
抗体A 1.65E-07 5.60E+05 9.21E-02
抗体A-ADC1 1.50E-07 5.76E+05 8.61E-02
固定抗体A-WS-161018(母分子抗体A)或者抗体A-ADC1到FAB2G biosensor，固化浓度为10μg/ml，固化高度2nm。
将FcγRI稀释至200nM、100nM、250nM、50nM、25nM，基线60s，结合30s，解离150s。稀释液为0.02％PBST20，再生液为甘氨酸-HCl(pH1.7)，中和液为稀释液。拟合时解离时间按10s拟合，实验结果如图12-图13和表2所示。图12和图13分别显示了抗体A和抗体A-ADC1与FcγRI的结合亲和力情况。
表2
样品 KD(M) Kon(1/Ms) Kdis(1/s)
抗体A 2.89E-10 8.54E+05 2.47E-04
抗体A-ADC1 6.05E-10 9.23E+05 5.58E-04
以上的结果说明，抗体A-ADC1与FcγRIIIa亲和力与母分子抗体A一致；抗体A-ADC1与FcγRI具有较强的结合亲和力，其KD值达到了6.05×10 -10M，与母分子抗体A的亲和力类似。
FcγR结合实验说明，抗体A-ADC1涉及的定点偶联不影响ADC中抗体部分的Fc功能，抗体A-ADC1仍然保留了母分子抗体A的Fc功能，抗体A-ADC1中的抗体部分可与毒素协同作用共同杀伤肿瘤细胞。
实施例4包含抗体A的ADC分子对肿瘤细胞的结合能力
分别考察实施例1制备的抗体A-ADC1、实施例2制备的抗体A-ADC2以及抗体A对NCI-N87细胞的结合活性。
具体试验步骤：
(1)1％BSA/PBS配置药物，配制最高浓度120μg/ml(工作浓度60μg/ml)，向下3倍稀释，共10个浓度；
(2)收集NCI-N87细胞(购自上海中科院)，离心，计数，5％BSA/PBS封闭15min，1.5mL离心管每管2×10 5个细胞/50μL，每管加入50μL抗体药物，4℃放置1小时；
(3)2000rpm/5min，离心洗涤2次，每次洗涤加入1000μL 1％BSA/PBS，最后一次吸干， 每孔加入90μL 1％BSA/PBS稀释的APC-IgG(1:100稀释)，4℃放置30min，避光。
(4)2000rpm/5min，离心洗涤2次，每次洗涤加入1000μL 1％BSA/PBS，最后一次吸干；最后每管加入300μL 1％BSA/PBS，避光，进行FACS检测。
结果如图14和表3所示。
表3
  抗体A 抗体A-ADC1 抗体A-ADC2
谷值 675.3 485.8 660.8
峰值 40787 36596 30780
EC50 1.103 1.154 0.9369
图14和表3的结果显示，高浓度抗体A的结合活性最高,抗体A-ADC1略低于抗体A，二者平均荧光强度(MFI)分别达到峰值后趋于稳定；而抗体A-ADC2活性较低。说明抗体A-ADC1及其母分子抗体A与NCI-N87细胞的结合更稳定。
从EC50值同样可以看出，在与HER2高表达的NCI-N87细胞结合性方面，抗体A-ADC1与母分子抗体A的EC50一致，都为1.1μg/ml左右，说明抗体A-ADC1与母分子抗体A与HER2高表达的NCI-N87细胞的结合活性一致；抗体A-ADC1的峰值大于抗体A-ADC2，说明抗体A-ADC1与HER2高表达的NCI-N87细胞结合活性稍强于抗体A-ADC2。
实施例5包含抗体A的ADC分子对肿瘤细胞的杀伤能力
分别考察实施例1制备的抗体A-ADC1、实施例2制备的抗体A-ADC2、抗体A以及DS-8201对NCI-N87细胞和BT474细胞的杀伤能力。
具体试验步骤：
(a)针对5天处理时间的NCI-N87细胞
(1)配制生长培养基：RPMI-1640+10％FBS+1％P/S，胰酶消化NCI-N87细胞，按10000个细胞/孔/150μL接种96孔板，37℃培养过夜；
(2)实验培养基配制：RPMI-1640+0.1％FBS+1％P/S，将抗体A、抗体A-ADC1、抗体A-ADC2和DS-8201分别配制成15μg/mL的最高工作浓度，3倍稀释，共9个浓度点；
(3)每孔150μL各浓度药物稀释液，设置阴性对照孔，37℃培养72±3小时或者；
(4)每孔吸取70μL上清，弃去。补入70μL新配置的15μg/mL抗体A、抗体A-ADC1、抗体A-ADC2或DS-8201，37℃继续培养48±2小时；
(5)96孔板每孔加入15μL CCK-8，轻拍板子摇匀，37℃放置2小时左右；
(6)以650nm为参比波长，在450nm下读取吸光值。
(b)针对3天处理时间NCI-N87、BT474细胞
(1)配制生长培养基：RPMI-1640+10％FBS+1％P/S，胰酶消化NCI-N87细胞或BT474细胞，按10000个细胞/孔/150μL接种96孔板，37℃培养过夜；
(2)实验培养基配制：RPMI-1640+0.1％FBS+1％P/S，将抗体A、抗体A-ADC1、抗体A-ADC2和DS-8201分别配制成30μg/mL的最高工作浓度，8倍稀释，共5个浓度点；
(3)每孔150μL各浓度药物稀释液，设置阴性对照孔，37℃培养72±3小时或者；
(4)96孔板每孔加入15μL CCK-8，轻拍板子摇匀，37℃放置2小时左右；
(6)以650nm为参比波长，在450nm下读取吸光值。
试验结果：
(1)NCI-N87肿瘤细胞杀伤试验
对NCI-N87细胞处理3天、处理5天的试验结果分别如图15、图16所示。
图15-图16的结果显示，按照上述方法步骤，分别处理NCI-N87细胞3天，抗体A-ADC1对NCI-N87的抑制率达到80％以上，抗体A仅次之，DS8201(Herceptin-ADC1)最低仅达60％。处理NCI-N87细胞5天，抗体A-ADC1的抑制率仍然保持最高，略高于其母分子以及DS-8201(Herceptin-ADC1)。由此可见抗体A-ADC1对NCI-N87细胞的杀伤效果显著高于DS-8201(Herceptin-ADC1)。
(2)BT474肿瘤细胞杀伤试验
对BT474细胞处理3天的试验结果分别如图17所示。
图17的结果显示，按照上述方法步骤，分别处理BT474细胞3天，抗体A-ADC1对BT474细胞的抑制率达到了55％，抗体A-ADC2达36％，而DS-8201则仅为23％，抗体A-ADC1对BT474细胞的抑制率远优于抗体A-ADC2、DS-8201，杀伤力是DS-8201的2.4倍。
对NCI-N87、BT474肿瘤细胞杀伤试验说明，抗体A-ADC1和抗体A-ADC2对HER2高表达阳性肿瘤细胞(NCI-N87、BT474)细胞均有较好的杀伤能力；并且抗体A-ADC1和抗体A-ADC2对HER2高表达阳性肿瘤细胞(NCI-N87、BT474细胞)的杀伤能力优于DS-8201。
实施例6包含抗体A的ADC分子对HER2阴性细胞的旁观者杀伤效应效果
ADC药物通常先与细胞膜上的抗原相结合，然后抗体抗原复合物通过细胞内吞作用进入细胞内，并形成内含体，随后内含体进一步成熟并融合成溶酶体，在溶酶体内，细胞毒药物被释放(通过与之相对应的特异性蛋白酶分解连接子，如组织蛋白酶B(cathepsin B)，或者 整个ADC药物在溶酶体被分解)，产生的细胞毒药物穿透溶酶体膜，绑定至DNA或者微管导致细胞凋亡。这些药物也可以通过细胞膜上的蛋白转运泵出至肿瘤微环境，进入到相邻的肿瘤细胞上，对其产生杀伤作用，这可以导致“旁观者效应”(Bystander Effect)。
抗体A-ADC1一种靶向HER2的双表位抗体的糖定点偶联药物，使用可裂解linker，为了考察抗体A-ADC1的旁观者效应，我们分别用抗体A-ADC1、DS-8201处理SK-B-R-3(HER2高表达)单细胞体系、MDA-MB-468(HER2不表达)单细胞体系、SK-BR-3与MDA-MB-468(1:1)混合细胞体系，处理3天后，对所有样本孔细胞进行计数，并且用APC anti-CD340Antibody荧光抗体对混合细胞体系染色，经流式仪检测。
实验方法
T75培养瓶中对数生长期的SK-BR-3，MDA-MB-468细胞，弃去培养基，PBS洗涤两次后吸干液体；加入胰酶2ml进行消化，37℃培养箱放置4分钟；转移细胞消化液至15ml离心管中，加入8ml生长培养基终止消化并吹打细胞至单细胞悬液，1000rpm离心5min；弃去上清液，加入3ml培养基(RPMI-1640+1％FBS)重悬细胞，吸取20μl细胞悬液和20μl 0.2％台盼蓝混合。
计数：单个细胞铺板(mono-culture)：用培养基(RPMI-1640+1％FBS)将两种细胞密度都调整为1*10 5cells/ml。各自按100μl/well铺板96孔板，每孔细胞个数为1*10 4，37℃，5％CO2培养箱培养过夜；混合细胞铺板(co-culture)：用培养基(RPMI-1640+1％FBS)将两种细胞密度都调整为2*10 5cells/ml，每孔加入50μl SK-BR-3细胞，以及50μl MDA-MB-468细胞。每孔细胞总数为2*10 4，37℃，5％CO 2培养箱培养过夜；
抗体药物配置：用含1％FBS的RPMI-1640培养基稀释受试样品，从最高工作浓度50nM，向下5倍梯度稀释，共4个浓度；同时设置对照组，即不加药物组(Negative Control)；每孔加入100μl上述稀释的受试样品,对照组(Negative Control)直接加入RPMI-1640+1％FBS培养基100μl；37℃，5％CO2培养72h；吸去96孔板中上清，200μl 1XPBS洗涤一次后，每孔加入20μl胰酶进行消化，37℃培养箱放置4min；加入100μl培养基(RPMI-1640+10％FBS)终止消化并吹打细胞至单细胞悬液，1000rpm离心5min；弃去上清液，每孔加入40μl 1XPBS重悬细胞，吸取20μl细胞悬液和20μl 0.2％台盼蓝混合计数；计数后，将混合细胞铺板组中每孔加入100μl(1：100倍1XPBS稀释的APC anti-human CD340 Antibody)荧光抗体，混匀后，2～8℃，避光孵育15min；1000rpm离心5min，弃去上清，每孔加入200μl 1XPBS，1000rpm离心5min洗涤一次，吸干上清，加入100μl 1XPBS重悬，流式上机检测。
如图18显示的是，各个实验组对于SK-BR-3细胞的增殖抑制结果；如图19显示的是，各个实验组对于MDA-MB-468细胞的增殖抑制结果。结果显示：(a)抗体A-ADC1和 ENHERTU(即DS-8201)对SK-BR-3单细胞体系、SK-BR-3与MDA-MB-468混合体系中的SK-BR-3细胞都有旁观者杀伤效果；(b)抗体A-ADC1与ENHERTU对HER2阴性MDA-MB-468单细胞体系的杀伤效果不明显，而对混合体系中的MDA-MB-468细胞具有明显的杀伤作用。样品浓度为50nM，10nM，2nM,0.4nM时，抗体A-ADC1对混合细胞体系中的MDA-MB-468细胞的增殖抑制率分别为：68.65％，46.87％，31.61％，-4.95％；ENHERTU对混合细胞体系中的MDA-MB-468细胞的增殖抑制率分别为：45.02％，25.4％，-20.02％，-22.08％。浓度为50nM时，抗体A-ADC1对于HER2阴性细胞的旁观者杀伤能力约为ENHERTU的1.5倍；浓度为10nM时，抗体A-ADC1对于HER2阴性细胞的旁观者杀伤能力约为ENHERTU的1.8倍；浓度为2nM时，抗体A-ADC1仍然具有旁观者杀伤能力31.61％，ENHERTU无旁观者杀伤能力；因此，抗体A-ADC1在2-50nmM浓度下，比ENHERTU有更强的旁观者杀伤能力。
下表显示了CountStar细胞计数的结果
Figure PCTCN2022103592-appb-000010
下表显示了混合细胞组HER2阳性，HER2阴性细胞比例的结果
Figure PCTCN2022103592-appb-000011
下表显示了SK-BR-3细胞增殖抑制率的结果
Figure PCTCN2022103592-appb-000012
Figure PCTCN2022103592-appb-000013
下表显示了MDA-MB-468细胞增殖抑制率的结果
Figure PCTCN2022103592-appb-000014
结果与结论
浓度为50nM时，抗体A-ADC1对于HER2阴性细胞的旁观者杀伤能力约为ENHERTU(即DS-8201)的1.5倍；浓度为10nM时，抗体A-ADC1对于HER2阴性细胞的旁观者杀伤能力约为ENHERTU的1.8倍；浓度为2nM时，抗体A-ADC1仍然具有旁观者杀伤能力31.61％，ENHERTU无旁观者杀伤能力；因此，抗体A-ADC1在2-50nmM浓度下，比ENHERTU有更强的旁观者杀伤能力。
实施例7包含抗体A的ADC分子的ADCC活性分析
(a)利用报告基因体系测定抗体A-ADC1的ADCC活性
将靶细胞NCI-N87、效应细胞Jurkat-FcγRIIIa-V158-NFAT与实施例1制备的抗体A-ADC1共培养6小时，通过检测荧光RUL值测定ADCC活性。
具体实验步骤：
(1)配制反应培养基：2％FBS+1640；
(2)利用步骤(1)制备的反应培养基制备不同浓度的抗体A-ADC1、抗体A-ADC2和抗体A，其中浓度最高为15000ng/ml(终浓度：10000ng/ml)，4倍稀释至9个浓度；
(3)收集NCI-N87细胞，利用步骤(1)制备的反应培养基调整细胞数至1×10 6个细胞/mL；收集Jurkat-FcγRIIIa-V158-NFAT细胞，利用步骤(1)制备的反应培养基调整细胞数至4×10 6个细胞/mL；
(4)96孔板中加入25μL步骤(2)配制的抗体A或包含抗体A的ADC、25μLN8细胞和25μL Jurkat-FcγRIIIa-V158-NFAT细胞，培养箱放置6小时；
(5)每孔加入10μL Bio-Glo TMluciferase substrate，立即于荧光酶标仪上检测每孔的RLU 值。
结果如图20所示，图20的结果说明，其中1-3分别显示抗体A、抗体A-ADC1和抗体A-ADC2的结果。实施例1制备的抗体A-ADC1分子在HER2阳性/高表达的细胞中(NCI-N87细胞)仍然保留很好的ADCC活性，其ADCC活性是母分子抗体A的87.7％(抗体A EC50/ADC1 EC50)；而实施例2制备的抗体A-ADC2丧失了大部分的ADCC活性，其活性仅为母分子抗体A的37.2％(抗体A EC50/ADC2 EC50)。说明偶联方式对ADC的ADCC活性影响较大。
(b)利用PBMC体系测定包含抗体A的ADC的ADCC活性
具体试验步骤(BT474细胞)：
(1)收集300IU/ml利用IL-2活化24小时的PBMC(活化用培养基：10％FBS+1640+300IU/ml)，利用10％FBS+1640+150IU/ml调整细胞数至4.5×10 6个细胞/mL；
(2)利用活化用培养基配制不同浓度抗体A-ADC1、抗体A-ADC2、DS8201和人IgG1，其中浓度最高为3μg/mL(终浓度为1μg/mL)，10倍稀释，3个浓度；
3、收集BT474细胞，利用活化用培养基调整细胞数至3×10 5个细胞/mL；
4、96孔板中加入50μL步骤(2)配制的包含抗体A的ADC、50μL BT474，50μL PBMC，培养箱放置4小时；
5、检测前30min，在TMR和VCC孔中加入10μL细胞裂解液；
6、1500rpm/5min，取上清50μL至新的96孔板中，每孔加入50μL LDH检测底物，室温放置30min；
7、490nm处检测OD值。
具体试验步骤(NCI-N87细胞)：
(1)收集300IU/ml利用IL-2活化24小时的PBMC(活化用培养基：10％FBS+1640+300IU/ml)，利用10％FBS+1640+150IU/ml调整细胞数至4.5×10 6个细胞/mL；
(2)利用活化用培养基配制不同浓度抗体A-ADC1、抗体A-ADC2、DS8201和人IgG1，其中浓度最高为3000ng/ml(终浓度为1000ng/ml)，6倍稀释，5个浓度；
3、收集NCI-N87细胞，利用活化用培养基调整细胞数至3×10 5个细胞/mL；
4、96孔板中加入50μL步骤(2)配制的包含抗体A的ADC、50μL BT474，50μL PBMC，培养箱放置4小时；
5、检测前30min，在TMR和VCC孔中加入10μL细胞裂解液；
6、1500rpm/5min，取上清50μL至新的96孔板中，每孔加入50μL LDH检测底物，室温 放置30min；
7、490nm处检测OD值。
按照上述方法步骤，分别测定抗体A-ADC1、抗体A-ADC2、DS8201和抗体A等在HER2高表达的细胞体系中的ADCC活性，靶细胞选用的是HER2高表达的细胞:BT747和NCI-N87，效应细胞是两个捐赠者外周血中的PBMC(PBMC-qc和PBMC-z)。检测流程如图21所示，检测结果如图22-23所示。
结果显示，抗体A-ADC1在PBMC-qc:BT474(效靶比15:1)细胞体系、PBMC-z:NCI-N87(效靶比15:1)细胞体系中的活性均强于抗体A-ADC2、DS-8201，说明抗体A-ADC1在HER2高表达的细胞体系中较DS-8201显示更高的ADCC活性，可见本申请所述的ADC可以保留偶联母分子抗体A的ADCC活性。
实施例8检测包含抗体A的ADC分子在肿瘤细胞中的内吞效率
具体实验步骤(NCI-N87细胞，购自上海中科院)：
(1)使用含有10％FBS的1640培养基配制各浓度的抗体A-ADC1、抗体A-ADC2、DS8201和抗体A(与内吞试剂混合)：96孔板中加入50μL终浓度分别为：50nM、10nM、2nM的抗体A-ADC1、抗体A-ADC2、DS8201或抗体A；
(2)NCI-N87细胞消化，计数，每含药孔中加入4×10 4个细胞/50μL/孔，培养箱中放置2、5和23小时；
(3)96孔板细胞弃上清，每孔加入50μL胰酶消化5min，
(4)每孔加入200μL含有10％FBS的1640培养基重悬，并吹打成单细胞悬液；
(5)转移至1.5ml EP管中，FACS检测。
具体实验步骤(BT474细胞)：
(1)使用含有10％FBS的1640培养基配制各浓度的抗体A-ADC1、抗体A-ADC2、DS8201和抗体A(与内吞试剂混合)：96孔板中加入50μL终浓度分别为：：40nM、8nM、1.6nM的抗体A-ADC1、抗体A-ADC2、DS8201或抗体A；
(2)BT474细胞消化，计数，每含药孔中加入4×10 4个细胞/50μL/孔，培养箱中放置24小时；
(3)96孔板细胞弃上清，每孔加入50μL胰酶消化5min，
(4)每孔加入200μL含有10％FBS的1640培养基重悬，并吹打成单细胞悬液；
(5)转移至1.5ml EP管中，FACS检测。
按照上述方法步骤，将抗体A-ADC1、抗体A-ADC2、DS-8201、抗体A等抗体-内吞试 剂混合物与肿瘤细胞体系中分别孵育后，通过细胞流式检测细胞对各分子的内吞效率。
(1)在NCI-N87肿瘤细胞中的内吞作用
当抗体A-ADC1、抗体A-ADC2、DS-8201、抗体A各分子在NCI-N87细胞体系中分别孵育2h、5h、23h后，通过细胞流式检测NCI-N87细胞对各分子的内吞作用。孵育2h、5h、23h后的结果分别如图24、25、26所示。
(a)当孵育达到2h时，细胞对各分子的内吞效率为：抗体A>抗体A-ADC1>抗体A-ADC2>DS-8201；孵育5h时，内吞效率为：抗体A＝抗体A-ADC1>>抗体A-ADC2>>DS-8201，此时抗体A-ADC1与母分子抗体A的内吞效率相当，且远远高于DS-8201、抗体A-ADC2。说明经过短时间的孵育(5h)，抗体A-ADC1保留了与母分子相当的肿瘤细胞内吞效果，且远远优于DS-8201，说明本申请所述的ADC能够快速结合肿瘤细胞表面的HER2实现内吞，阻断HER2信号通路，发挥肿瘤杀伤作用。
(b)当孵育达到23h时，抗体A-ADC1、抗体A-ADC2、抗体A等的内吞效率相当，50nM时内吞效率均达到55％以上，该内吞效率优于DS-8201，说明施用本申请所述ADC 23h后有55％药物(50nM)都达到了NCI-N87肿瘤靶细胞内，肿瘤靶向性较DS-8201略强。
(2)在BT474肿瘤细胞中的内吞作用
当抗体A-ADC1、抗体A-ADC2、DS-8201在BT474细胞体系中孵育达到24小时后通过细胞流式检测。孵育24小时的结果如图27所示。
测定结果显示抗体A-ADC1与抗体A-ADC2的内吞效率相当，40nM时内吞效率达到70％以上，可见本申请所述包含抗体A的ADC的内吞效率在同等条件下是DS-8201(30％)的2.33倍，同时前者对BT474细胞的靶向性远强于DS-8201。说明抗体A-ADC1对靶点介导的信号通路抑制能力更强，肿瘤杀伤力远强于DS-8201，肿瘤抑制能力更强。
实施例9包含抗体A的ADC分子在动物模型中抑制肿瘤生长
分组给药试验方案：于裸鼠腋下皮下注射BxPC-3细胞建立模型，接种浓度为1×10 6/0.1mL/只。待肿瘤大小长到平均约190mm 3时，按肿瘤大小和体重随机分组，每组4只，雌性。随机分组当天记为0天。于第0天腹腔给予受试药物，以后每3天1次按给药剂量腹腔给予受试物，共2次给药。未选入的动物CO 2安乐处死。
将试验动物分为A、B、C三组，A组施用PBS，B组施用抗体A-ADC1(DAR＝4)，C组施用DS-8201(DAR＝8)。具体的分组给药方案如表4所示。
表4
Figure PCTCN2022103592-appb-000015
Figure PCTCN2022103592-appb-000016
按照上述试验方案，对A、B、C三组试验动物小鼠分别施用三种受试物24天，(A组PBS有2只动物因肿瘤生长过大，施用受试物20天后被安乐死)。得到三种受试物对BxPC-3肿瘤模型的抑瘤曲线。
抑瘤曲线的结果说明，对BxPC-3肿瘤模型动物施用抗体A-ADC1(DAR＝4)与DS-8201(DAR＝8)，对肿瘤很大程度的抑制，随着时间的推进肿瘤抑制效果明显，且抗体A-ADC1与DS-8201的抑瘤效果类似。
实施例10包含抗体A的ADC分子在Balb/c-Nu裸鼠NCI-N87肿瘤模型中药代动力学研究
通过在Balb/c-Nu裸鼠皮下接种人胃癌细胞NCI-N87，采用NCI-N87肿瘤模型，单次静脉注射(iv)给予抗体A-ADC1 1mg/kg、ENHERTU(即DS-8201)1mg/kg，于不同时间点采集血样，Elisa法检测动物血清样本中总抗体及ADC含量(方法一：ELISA检测总抗体含量，包含ADC和裸抗体；方法二：ELISA检测ADC含量(包括含有1-4个药物的ADC)，不包括释放毒素后的裸抗体)，使用DAS(3.2.8)软件非房室模型计算计算药代动力学参数。
表5
Figure PCTCN2022103592-appb-000017
单次静脉注射给予NCI-N87肿瘤模型小鼠抗体A-ADC1后，总抗体和ADC的C max分别为45422.727μg/L、70056.81675μg/L，AUC (0-t)分别为1598292.175μg/L*h、2098916.987μg/L*h，T max均为0.033h、0.033h，T 1/2分别为100.976h、95.32h。
单次静脉注射给药NCI-N87肿瘤模型小鼠ENHERTU(即DS-8201)后，总抗体和ADC的C max分别为12343.966μg/L、19604.51225μg/L，AUC (0-t)分别为421103.345μg/L*h、586509.313μg/L*h，T max均为0.033h、0.033h，T 1/2分别为86.744h、65.384h。
综上，单次静脉注射给予NCI-N87肿瘤模型小鼠抗体A-ADC1、ENHERTU，Elisa方法一(总抗体浓度)结果显示T 1/2分别为100.976h、86.744h，抗体A-ADC1T 1/2是ENHERTU的1.164倍，Cmax是ENHERTU的3.680倍，AUC (0-t)是ENHERTU的3.795倍。方法二(抗体药物偶联物含量)结果显示抗体A-ADC1和ENHERTU T 1/2分别为95.32h、65.384h，抗体A-ADC1T 1/2是ENHERTU的1.460倍，Cmax是ENHERTU的3.574倍，AUC (0-t)是ENHERTU的3.580倍。结果可以参见图28。
实施例11包含抗体抗体A的ADC分子对NCI-N87细胞表面HER2内吞的影响
靶向Her2的单克隆抗体可促进Her2内在化降解，来进一步抑制Her2的下游信号转导，从而抑制细胞增殖。本申请ADC(抗体A-ADC1)是在一种靶向HER2双表位抗体，例如抗体A的基础上偶联小分子DXd的ADC药物，通过选用Her2过表达的人胃癌细胞系NCI-N87细胞，比较抗体A-ADC1，抗体A以及上市的同类靶点药物DS8201的内吞效果。
实验步骤：
1)配置实验Buffer:1640+10％FBS培养基；
2)将N87细胞按350g，5min离心后，去掉上清，用实验buffer调整密度为1*106cells/ml；
3)将实验所需抗体用实验buffer稀释为首浓度200nM将pHrodo用实验buffer稀释成浓度为600nM；
4)稀释好的200nM抗体与600nM pHrodo体积1：1混合孵育15min后，用实验Buffer将抗体与pHrodo混合物5倍稀释共3个浓度点，孵育5min后加入50ul细胞；
5)样本孔中加入抗体和pHrodo混合物50ul与50ul细胞；
6)NC孔：25ul Buffer+25ul PHrodo+50ul cell(1E5)；
7)BLANK孔：50ul Buffer+50ul cell(1E5)每孔体积为100ul吹打混匀；
8)37℃细胞培养箱孵育5h；
9)孵育结束350xg离心5min后，去掉上清，每孔加胰酶消化细胞培养基重悬为单细胞悬液350xg离心5min按200μl/孔中加入1xPBS重悬；
10)350xg离心5min后，按150μl/孔中加入1xPBS重悬细胞,细胞移入新96孔板流式上机；
实验结果：
抗体A，抗体A-ADC1及DS8201都可以诱导NCI-N87细胞表面的HER2内吞作用，其中抗体A-ADC1与抗体A的内吞效果相当。另外，抗体A-ADC1的内吞效果在三个浓度点都明显强于DS8201，在50nM，10nM,2nM浓度条件下，抗体A的内吞比例分别是92.25％，91.82％，65.71％，抗体A-ADC1的内吞比例分别是85.48％，84.65％，58.95％，DS8201的内吞比例分别是34.79％，28.36％，21.26％。抗体A-ADC1对NCI-N87的内吞效果与抗体A相当，并显著强于DS8201。
表6
Figure PCTCN2022103592-appb-000018
表6显示了抗体A，抗体A-ADC1和DS8201内吞后阳性NCI-N87细胞比例
图29显示的是，抗体A、抗体A-ADC1和DS8201诱导内吞后NCI-N87阳性细胞比例。
实施例12包含抗体A的ADC分子对HER2弱阳性胃癌PDX皮下异种移植NOD/SCID小鼠模型的抑制效果
评价本申请ADC(抗体A-ADC1)在人源HER2弱阳性(2+)胃癌PDX皮下异种移植NOD/SCID小鼠动物模型中的抗肿瘤作用，和已上市阳性对照药物ENHERTU(即DS-8201)进行对比。
模型信息
下表显示的是人源胃癌PDX异种移植模型样本信息
模型ID 癌症类型 种族 特性
Case168 胃腺癌 亚洲 HER2(2+)P6
实验过程
每例PDX模型选取30只4-5周龄性NOD/SCID雌性小鼠右前肩胛处皮下接种直径为2-3mm的胃癌PDX肿瘤瘤块,待肿瘤平均体积为213.40mm 3时，根据肿瘤大小和小鼠体重随机分组(见下表)，分组当天定义为第1天，分组后立即开始给药，给药当天定义为给药后第1天。
下表显示的是测试药物在人源胃癌PDX肿瘤模型中的抗肿瘤作用实验设计
Figure PCTCN2022103592-appb-000019
实验结果：
对照组和测试药组在首次给药第27天时(此时对照组肿瘤长径最大径已达小鼠实验伦理上限1.5cm)，各组内动物全部存活，因此选用此时肿瘤体积数据用于分析评估测试药物本申请ADC(抗体A-ADC1)抗肿瘤作用。PBS对照组(CTL)小鼠在分组首次给药后第27天时平均肿瘤体积为725.39±190.25mm 3，相对肿瘤体积为3.58±1.11；测试药抗体A-ADC1(10mg/kg)在分组首次给药后第27天时平均肿瘤体积为112.89±61.65mm 3，相对肿瘤体积为0.50±0.16，相对肿瘤抑制率TGI(％)为86.05％，相对于PBS对照组在统计学上有显著性差异(p＜0.0001)；阳性对照药ENHERTU(即DS-8201)(10mg/kg)在分组首次给药后第27天时平均肿瘤体积为106.41±17.77mm 3，相对肿瘤体积为0.50±0.11，相对肿瘤抑制率TGI(％)为86.08％，相对于PBS对照组在统计学上有显著性差异(p＜0.0001)。此外，在10mg/kg剂量水平下，分组首次给药后第27天时测试药物抗体A-ADC1治疗组中的平均肿瘤体积与ENHERTU治疗组平均肿瘤体积大小相近，且在统计学上无显著性差异(抗体A-ADC1 VS ENHERTU,P＝0.8362＞0.05，ns.)。总体来说，抗体A-ADC1在10mg/kg剂量下对人源HER2IHC 2+胃癌PDX肿瘤模型具有显著的的肿瘤抑制作用，DAR为4的抗体A-ADC1与DAR为8的阳性对照药物ENHERTU的抗肿瘤药效相当。图30显示的是人源胃癌PDX肿瘤模型中各组小鼠肿瘤体积的生长曲线。
下表显示的是在Case 168人源胃癌PDX肿瘤模型中各组药效分析表
Figure PCTCN2022103592-appb-000020
Figure PCTCN2022103592-appb-000021
注：1.数据以“平均值
Figure PCTCN2022103592-appb-000022
±标准误差(S)”表示；
2.T/C％＝T RTV/C RTV×100％；TGI％＝(1-T RTV/C RTV)×100％。
3.CTL VS本申请ADC(抗体A-ADC1),P＜0.0001；CTL VS ENHERTU(即DS-8201),P＜0.0001；抗体A-ADC1 VS ENHERTU,P＝0.8362＞0.05.
实施例13包含抗体A的ADC分子对HER2阴性胃癌PDX皮下异种移植NOD/SCID小鼠模型的抑制效果
评价本申请ADC(抗体A-ADC1)在HER2表达阴性的人源胃癌PDX肿瘤皮下异种移植NOD/SCID小鼠动物模型中的抗肿瘤作用，和已上市阳性对照药物ENHERTU(即DS-8201)进行对比
实验过程：
模型信息
下表显示的是人源胃癌HER2 IHC 2+PDX异种移植模型样本信息
模型ID 癌症类型 种族 特性
Case 111 胃腺癌 亚洲 HER2(-)P6
实验过程
每例PDX模型选取30只4-5周龄雌性NOD/SCID小鼠小鼠右前肩胛处皮下接种直径为2-3mm的胃癌PDX肿瘤瘤块。待肿瘤平均体积为166.53mm 3时，根据肿瘤大小和小鼠体重随机分组，分组当天定义为第1天，分组后立即开始给药，给药当天定义为给药后第1天。
下表显示的是测试药物在人源胃癌PDX肿瘤模型中的抗肿瘤作用实验设计
Figure PCTCN2022103592-appb-000023
实验结果：
对照组和测试药组在分组首次给药后第17天时(此时对照组肿瘤长径最大径已达小鼠实验伦理上限1.5cm)，各组内动物全部存活，因此选用此时的肿瘤体积数据用于评估测试药物本申请ADC(抗体A-ADC1)抗肿瘤作用。PBS对照组小鼠在分组首次给药后第17天时平均肿瘤体积为629.24±146.59mm 3，相对肿瘤体积为4.13±0.97；测试药抗体A-ADC1(10mg/kg)在分组首次给药后第17天时平均肿瘤体积为238.32±84.57mm 3，相对肿瘤体积为1.68±1.01，相对肿瘤抑制率TGI(％)为59.41％，相对于PBS对照组在统计学上有显著性差异(p＜0.0001)；阳性对照药ENHERTU(即DS-8201)(10mg/kg)在分组首次给药后第17天时平均肿瘤体积为174.06±71.09mm 3，相对肿瘤体积为1.02±0.34，相对肿瘤抑制率TGI(％)为75.19％，相对于PBS对照组在统计学上有显著性差异(p＜0.0001)。此外，在10mg/kg剂量水平下，分组首次给药后第17天时测试药物抗体A-ADC1治疗组的平均肿瘤体积要稍大于阳性对照药物ENHERTU治疗组的平均肿瘤体积，但是在统计学上无显著性差异(抗体A-ADC1 VS ENHERTU,P＝0.526＞0.05，ns.)。总体来说抗体A-ADC1在10mg/kg剂量下对人源HER2-胃癌PDX肿瘤模型具有显著的的肿瘤抑制作用，DAR为4的抗体A-ADC1与DAR为8的阳性对照药物ENHERTU的抗肿瘤药效相当。图31显示的是人源胃癌HER2 IHC-PDX肿瘤模型小鼠肿瘤体积的生长曲线(仅于day1给一次药物处理，尾静脉注射，10mg/kg)。
下表显示的是在Case 111人源胃癌PDX肿瘤模型中各组药效分析表
Figure PCTCN2022103592-appb-000024
注：1.数据以“平均值
Figure PCTCN2022103592-appb-000025
±标准误差(S)”表示；
2.T/C％＝T RTV/C RTV×100％；TGI％＝(1-T RTV/C RTV)×100％。
3.CTL VS本申请ADC(抗体A-ADC1),P＜0.0001；CTL VS ENHERTU(即DS-8201),P＜0.0001；抗体A-ADC1 VS ENHERTU,P＝0.526＞0.05，ns.。
前述详细说明是以解释和举例的方式提供的，并非要限制所附权利要求的范围。目前本 申请所列举的实施方式的多种变化对本领域普通技术人员来说是显而易见的，且保留在所附的权利要求和其等同方式的范围内。

Claims (67)

  1. 抗体药物偶联物，其包含靶向HER2的双特异性抗体或其抗原结合片段。
  2. 根据权利要求1所述的抗体药物偶联物，其中所述靶向HER2的双特异性抗体或其抗原结合片段特异性结合人HER2的至少一个表位。
  3. 根据权利要求1-2中任一项所述的抗体药物偶联物，其中所述靶向HER2的双特异性抗体或其抗原结合片段特异性结合人HER2的胞外结构域II和/或人HER2的胞外结构域IV。
  4. 根据权利要求1-3中任一项所述的抗体药物偶联物，其中所述靶向HER2的双特异性抗体或其抗原结合片段包含第一轻链和第二轻链。
  5. 根据权利要求4所述的抗体药物偶联物，其中所述第一轻链包含第一LCDR1-3，其中所述第一LCDR1包含SEQ ID NO.4所示的氨基酸序列。
  6. 根据权利要求5所述的抗体药物偶联物，其中所述第一LCDR2包含SEQ ID NO.5所示的氨基酸序列。
  7. 根据权利要求5-6中任一项所述的抗体药物偶联物，其中所述第一LCDR3包含SEQ ID NO.6所示的氨基酸序列。
  8. 根据权利要求4-7中任一项所述的抗体药物偶联物，其中所述第一轻链能够与帕妥珠单抗的重链结合。
  9. 根据权利要求4-8中任一项所述的抗体药物偶联物，其中所述第二轻链包含第二LCDR1-3，其中所述第二LCDR1包含SEQ ID NO.1所示的氨基酸序列。
  10. 根据权利要求9所述的抗体药物偶联物，其中所述第二LCDR2包含SEQ ID NO.2所示的氨基酸序列。
  11. 根据权利要求9-10中任一项所述的抗体药物偶联物，其中所述第二LCDR3包含SEQ ID NO.3所示的氨基酸序列。
  12. 根据权利要求4-11中任一项所述的抗体药物偶联物，其中所述第二轻链能够与曲妥珠单抗的重链结合。
  13. 根据权利要求4-12中任一项所述的抗体药物偶联物，其中所述第一轻链和所述第二轻链的可变区包含SEQ ID NO.7-12中任一项所示的氨基酸序列。
  14. 根据权利要求4-13中任一项所述的抗体药物偶联物，其中所述第一轻链和所述第二轻链的可变区包含SEQ ID NO.7所示的氨基酸序列。
  15. 根据权利要求4-14中任一项所述的抗体药物偶联物，其中所述第一轻链和所述第二轻链包含SEQ ID NO.13-18中任一项所述的氨基酸序列。
  16. 根据权利要求4-15中任一项所述的抗体药物偶联物，其中所述第一轻链选自下组：帕妥 珠单抗的轻链或其突变体和曲妥珠单抗的轻链或其突变体；和/或，所述第二轻链选自下组：帕妥珠单抗的轻链或其突变体和曲妥珠单抗的轻链或其突变体。
  17. 根据权利要求4-16中任一项所述的抗体药物偶联物，其中所述第一轻链和所述第二轻链的氨基酸序列相同。
  18. 根据权利要求4-17中任一项所述的抗体药物偶联物，其中所述第一轻链和所述第二轻链包含SEQ ID NO.13所述的氨基酸序列。
  19. 根据权利要求1-18中任一项所述的抗体药物偶联物，其中所述靶向HER2的双特异性抗体或其抗原结合片段包含第一重链和第二重链，其中所述第一重链能够在生理条件或体外的蛋白表达状态下与所述第一轻链正确结合。
  20. 根据权利要求19所述的抗体药物偶联物，其中所述第二重链能够在生理条件或体外的蛋白表达状态下与所述第二轻链正确结合。
  21. 根据权利要求19-20中任一项所述的抗体药物偶联物，其中所述第一重链包含第一重链可变区，所述第一重链可变区为帕妥珠单抗的重链可变区。
  22. 根据权利要求19-21中任一项所述的抗体药物偶联物，其中所述第二重链包含第二重链可变区，所述第二重链可变区为曲妥珠单抗的重链可变区。
  23. 根据权利要求19-22中任一项所述的抗体药物偶联物，其中所述第一重链和所述第二重链包含重链恒定区，其中所述重链恒定区源自人IgG的恒定区。
  24. 根据权利要求19-23中任一项所述的抗体药物偶联物，其中所述第一重链和所述第二重链的Fc片段包含SEQ ID NO.25-57中任一项所述的氨基酸序列。
  25. 根据权利要求19-24中任一项所述的抗体药物偶联物，其中所述第一重链包含SEQ ID NO.21或23所示的氨基酸序列。
  26. 根据权利要求19-25中任一项所述的抗体药物偶联物，其中所述第二重链包含SEQ ID NO.22或24所示的氨基酸序列。
  27. 根据权利要求1-26中任一项所述的抗体药物偶联物，其包含式1所示的结构：
    M-(L1) a-(L2) b-D(式1)，
    其中M表示权利要求1-26中任一项所述的靶向HER2的双特异性抗体或其抗原结合片段；
    L1表示与M连接的连接子，L2表示与D连接的连接子，
    a，b各自独立地选自0-10，
    D表示药物。
  28. 根据权利要求27所述的抗体药物偶联物，其中所述L1和/或L2选自下组：可裂解的连接 子、不可裂解的连接子、亲水的连接子、疏水的连接子、带电荷的连接子、不带电荷的连接子和基于二羧酸的连接子。
  29. 根据权利要求27-28中任一项所述的抗体药物偶联物，其中所述L1与所述M通过所述M上的巯基、叠氮基或酰胺基连接。
  30. 根据权利要求27-29中任一项所述的抗体药物偶联物，其中所述M包含第一重链和第二重链，所述第一重链和/或所述第二重链包含能够与所述L1连接的连接位点。
  31. 根据权利要求30所述的抗体药物偶联物，其中所述连接位点包含去糖基化修饰后能够与所述L1连接的基团。
  32. 根据权利要求31所述的抗体药物偶联物，其中，所述基团位于所述第一重链的第297位氨基酸Q的侧基；和/或，位于所述第二重链的第298位氨基酸Q的侧基。
  33. 根据权利要求30所述的抗体药物偶联物，其中所述连接位点包含经糖基化修饰后能够与所述L1连接的基团。
  34. 根据权利要求33所述的抗体药物偶联物，其中所述基团位于所述第一重链的第299位氨基酸N的侧基；和/或，位于所述第二重链的第300位氨基酸N的侧基。
  35. 根据权利要求33-34中任一项所述的抗体药物偶联物，其中所述基团包含-N 3。
  36. 根据权利要求33-35中任一项所述的抗体药物偶联物，其中所述经糖基化修饰包括：所述M已经与UDP-GalNAz、β-1,4-半乳糖基转移酶或其变体接触。
  37. 根据权利要求27-36中任一项所述的抗体药物偶联物，其中所述L1能够参与SPAAC反应。
  38. 根据权利要求27-37中任一项所述的抗体药物偶联物，其中所述L1选自下组：马来酰亚胺、琥珀酰亚胺-3-基-N和DBCO。
  39. 根据权利要求27-38中任一项所述的抗体药物偶联物，其中所述L1为DBCO-(PEG) n1，其中 n1为0-10的整数，或者，所述L1为马来酰亚胺。
  40. 根据权利要求27-39中任一项所述的抗体药物偶联物，其中所述L2选自下组：多肽、VC-PAB、N-琥珀酰亚胺基-3-(2-吡啶基二硫代)丙酸酯(SPDP)、N-琥珀酰亚胺基4-(2-吡啶基二硫代)戊酸酯(SPP)、N-琥珀酰亚胺基4-(2-吡啶基二硫代)丁酸酯(SPDB)、N-琥珀酰亚胺基-4-(2-吡啶基二硫基)-2-磺基丁酸酯(sulfo-SPDB)、N-琥珀酰亚胺基碘乙酸酯(SIA)、N-琥珀酰亚胺基(4-碘乙酰基)氨基苯甲酸酯(SIAB)、马来酰亚胺PEG NHS、N-4-(马来酰亚胺基甲基)环己基羧酸琥珀酰胺基酯(SMCC)、N-磺基(4-亚马来酰亚胺甲基)环己基羧酸磺基琥珀酯(磺基-SMCC)和2,5-二氧吡咯烷基-1-基17-(2,5-二氧代-2,5-二氢-1H-吡咯-1-基)-5,8,11,14-四氧-4,7,10,13-四氮杂十八烷-1-酸酯(CX1-1)。
  41. 根据权利要求27-40中任一项所述的抗体药物偶联物，其中所述L2为GGFG。
  42. 根据权利要求27-41中任一项所述的抗体药物偶联物，其中所述药物具有杀伤肿瘤细胞，和/或抑制肿瘤细胞生长的能力。
  43. 根据权利要求27-42中任一项所述的抗体药物偶联物，其中所述药物包括小分子药物。
  44. 根据权利要求27-43中任一项所述的抗体药物偶联物，其中所述药物选自下组：V-ATPase抑制剂、Bcl2抑制剂、MCL1抑制剂、HSP90抑制剂、IAP抑制剂、mTor抑制剂、微管稳定剂、微管去稳定剂、auristatin、dolastatin、美登木素生物碱、MetAP(蛋氨酸氨基肽酶)、蛋白质CRM1的核输出抑制剂、DPPIV抑制剂、蛋白酶体抑制剂、线粒体中的磷酸转移反应抑制剂、蛋白质合成抑制剂、CDK2抑制剂、CDK9抑制剂、驱动蛋白抑制剂、HDAC抑制剂、DNA破坏剂、DNA烷化剂、DNA嵌入剂、DNA小沟结合剂、DHFR抑制剂、核苷类似物、HD AC抑制剂、蒽环类、NAMPT抑制剂、SN-38葡糖醛酸、依托泊苷磷酸酯、氮芥末、蛋白体抑制剂、细胞因子和Toll样受体激动剂。
  45. 根据权利要求27-44中任一项所述的抗体药物偶联物，其中所述药物选自下组：DM1、exatecan、DXd、MMAE、SN-38、Calicheamicin、Anthracyclin-5G、DM4、微管抑制剂SHR153024、PNU-159682、Duo5毒素、SN38衍生物或他们的衍生物。
  46. 根据权利要求27-45中任一项所述的抗体药物偶联物，其具有选自下组的结构：
    Figure PCTCN2022103592-appb-100001
    Figure PCTCN2022103592-appb-100002
  47. 根据权利要求1-46中任一项所述的抗体药物偶联物，其药物/抗体比率为约1-6。
  48. 制备权利要求1-47中任一项所述的抗体药物偶联物的化合物，其具有式2所示的结构：
    M-(L1) a(式2)，其中M表示权利要求1-26中任一项所述靶向HER2的双特异性抗体或其抗原结合片段；
    L1表示与M连接的连接子；
    a选自0-10。
  49. 根据权利要求48所述的化合物，其中所述L1选自下组：可裂解的连接子、不可裂解的连接子、亲水的连接子、疏水的连接子、带电荷的连接子、不带电荷的连接子和基于二羧酸的连接子。
  50. 根据权利要求48-49中任一项所述的化合物，其中所述L1与所述M通过所述M上的巯基、叠氮基或酰胺基连接。
  51. 根据权利要求48-50中任一项所述的化合物，其中所述M包含第一重链和第二重链，所述第一重链和/或所述第二重链包含能够与所述L1连接的连接位点。
  52. 根据权利要求51所述的化合物，其中所述连接位点包含去糖基化修饰后能够与所述L1连接的基团。
  53. 根据权利要求51所述的化合物，其中，所述基团位于所述第一重链的第297位氨基酸Q的侧基；和/或，位于所述第二重链的第298位氨基酸Q的侧基。
  54. 根据权利要求51所述的化合物，其中所述连接位点包含经糖基化修饰后能够与所述L1连接的基团。
  55. 根据权利要求54所述的化合物，其中所述基团位于所述第一重链的第299位氨基酸N的侧基；和/或，位于所述第二重链的第300位氨基酸N的侧基。
  56. 根据权利要求54-55中任一项所述的化合物，其中所述基团包含-N 3。
  57. 根据权利要求53-56中任一项所述的化合物，其中所述经糖基化修饰包括：所述M已经与UDP-GalNAz、β-1,4-半乳糖基转移酶或其变体接触。
  58. 根据权利要求48-57中任一项所述的化合物，其中所述L1能够参与SPAAC反应。
  59. 根据权利要求48-58中任一项所述的化合物，其中所述L1选自下组：马来酰亚胺、琥珀酰亚胺-3-基-N和DBCO。
  60. 根据权利要求48-59中任一项所述的化合物，其中所述L1为DBCO-(PEG) n1，其中 n1为0-10的整数，或者，所述L1为马来酰亚胺。
  61. 根据权利要求48-60中任一项所述的化合物，其包括选自下组的结构：
    Figure PCTCN2022103592-appb-100003
  62. 制备权利要求1-47中任一项所述的抗体药物偶联物的方法，其包括以下步骤：
    将权利要求48-61中任一项所述的化合物与权利要求1-47中任一项所述的药物接触。
  63. 药物组合物，其包含权利要求1-47中任一项所述的抗体药物偶联物，任选地，包含药学上可接受的载体。
  64. 调节受试者的肿瘤微环境的方法，其包括以下步骤：向受试者施用权利要求1-47中任一项所述的抗体药物偶联物、或者权利要求63所述的药物组合物。
  65. 调节受试者的免疫反应的方法，其包括以下步骤：向受试者施用权利要求1-47中任一项所述的抗体药物偶联物、或者权利要求63所述的药物组合物。
  66. 权利要求1-47中任一项所述的抗体药物偶联物、或者权利要求63所述的药物组合物制备药物中的应用，其中所述药物可以预防和/或治疗肿瘤。
  67. 根据权利要求66所述的应用，其中所述肿瘤包括实体瘤和/或非实体瘤。
PCT/CN2022/103592 2021-07-05 2022-07-04 抗体药物偶联物及其应用 WO2023280092A1 (zh)

Priority Applications (4)

Application Number Priority Date Filing Date Title
CN202280047851.4A CN117616052A (zh) 2021-07-05 2022-07-04 抗体药物偶联物及其应用
US18/575,293 US20240307551A1 (en) 2021-07-05 2022-07-04 Antibody-drug conjugate and application thereof
EP22836843.7A EP4386006A1 (en) 2021-07-05 2022-07-04 Antibody-drug conjugate and application thereof
JP2024500541A JP2024525624A (ja) 2021-07-05 2022-07-04 抗体薬物コンジュゲート及びその使用

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202110757803.1 2021-07-05
CN202110757803 2021-07-05

Publications (2)

Publication Number Publication Date
WO2023280092A1 true WO2023280092A1 (zh) 2023-01-12
WO2023280092A9 WO2023280092A9 (zh) 2023-02-09

Family

ID=84801059

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2022/103592 WO2023280092A1 (zh) 2021-07-05 2022-07-04 抗体药物偶联物及其应用

Country Status (5)

Country Link
US (1) US20240307551A1 (zh)
EP (1) EP4386006A1 (zh)
JP (1) JP2024525624A (zh)
CN (1) CN117616052A (zh)
WO (1) WO2023280092A1 (zh)

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116751319A (zh) * 2023-07-19 2023-09-15 首都医科大学 一种壳聚糖六肽衍生物及其制备方法和应用
WO2024222870A1 (zh) * 2023-04-27 2024-10-31 石药集团巨石生物制药有限公司 一种抗体药物偶联物
WO2025103474A1 (zh) * 2023-11-17 2025-05-22 成都康弘生物科技有限公司 双毒素抗体药物偶联物及其用途

Citations (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20090285837A1 (en) 2004-07-22 2009-11-19 Genentech, Inc. Her2 antibody composition
CN102558355A (zh) 2011-12-31 2012-07-11 苏州康宁杰瑞生物科技有限公司 基于电荷网络的异二聚体fc改造方法及异二聚体蛋白的制备方法
CN103388013A (zh) 2012-07-25 2013-11-13 苏州康宁杰瑞生物科技有限公司 利用电荷排斥作用制备同二聚体蛋白混合物的方法
CN105820251A (zh) * 2015-01-08 2016-08-03 苏州康宁杰瑞生物科技有限公司 具有共同轻链的双特异性抗体或抗体混合物
CN105829347A (zh) * 2013-12-20 2016-08-03 豪夫迈·罗氏有限公司 双特异性her2抗体及使用方法
CN105980409A (zh) * 2013-11-27 2016-09-28 酵活有限公司 靶向her2的双特异性抗原结合构建体
CN106232139A (zh) * 2014-04-11 2016-12-14 免疫医疗有限责任公司 双特异性her2抗体
CN107446045A (zh) * 2016-07-22 2017-12-08 北京天广实生物技术股份有限公司 一种抗her2的抗体、其药物组合物及用途
CN111518213A (zh) * 2019-02-03 2020-08-11 苏州康聚生物科技有限公司 抗her2的双特异性抗体及其应用
WO2021110107A1 (en) * 2019-12-04 2021-06-10 Jiangsu Alphamab Biopharmaceuticals Co., Ltd. COMBINATION OF BISPECIFIC FUSION PROTEIN AND ANTI-Her2 ANTIBODY FOR TUMOR TREATMENT
WO2021174113A1 (en) * 2020-02-28 2021-09-02 Regeneron Pharmaceuticals, Inc. Bispecific antigen binding molecules that bind her2, and methods of use thereof
WO2021209007A1 (en) * 2020-04-15 2021-10-21 Shenzhen Enduring Biotech, Ltd. Antibody-drug conjugate

Patent Citations (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20090285837A1 (en) 2004-07-22 2009-11-19 Genentech, Inc. Her2 antibody composition
CN102558355A (zh) 2011-12-31 2012-07-11 苏州康宁杰瑞生物科技有限公司 基于电荷网络的异二聚体fc改造方法及异二聚体蛋白的制备方法
CN103388013A (zh) 2012-07-25 2013-11-13 苏州康宁杰瑞生物科技有限公司 利用电荷排斥作用制备同二聚体蛋白混合物的方法
CN105980409A (zh) * 2013-11-27 2016-09-28 酵活有限公司 靶向her2的双特异性抗原结合构建体
CN105829347A (zh) * 2013-12-20 2016-08-03 豪夫迈·罗氏有限公司 双特异性her2抗体及使用方法
CN106232139A (zh) * 2014-04-11 2016-12-14 免疫医疗有限责任公司 双特异性her2抗体
CN105820251A (zh) * 2015-01-08 2016-08-03 苏州康宁杰瑞生物科技有限公司 具有共同轻链的双特异性抗体或抗体混合物
CN107446045A (zh) * 2016-07-22 2017-12-08 北京天广实生物技术股份有限公司 一种抗her2的抗体、其药物组合物及用途
CN111518213A (zh) * 2019-02-03 2020-08-11 苏州康聚生物科技有限公司 抗her2的双特异性抗体及其应用
WO2021110107A1 (en) * 2019-12-04 2021-06-10 Jiangsu Alphamab Biopharmaceuticals Co., Ltd. COMBINATION OF BISPECIFIC FUSION PROTEIN AND ANTI-Her2 ANTIBODY FOR TUMOR TREATMENT
WO2021174113A1 (en) * 2020-02-28 2021-09-02 Regeneron Pharmaceuticals, Inc. Bispecific antigen binding molecules that bind her2, and methods of use thereof
WO2021209007A1 (en) * 2020-04-15 2021-10-21 Shenzhen Enduring Biotech, Ltd. Antibody-drug conjugate

Non-Patent Citations (13)

* Cited by examiner, † Cited by third party
Title
"Epitope Mapping Protocols in Methods in Molecular Biology", vol. 66, 1996
"Fundamental Immunology", 1989, RAVEN PRESS
"Immunology A Synthesis", 1991, SINAUER ASSOCIATES
"Innovations for Next- Generation Antibody-Drug Conjugates", 31 December 2018, SPRINGER INTERNATIONAL PUBLISHING , Cham , ISBN: 978-3-319-78154-9, article KEVIN J HAMBLETT: "HER2-Targeted ADCs: At the Forefront of ADC Technology Development", pages: 163 - 185, XP009542492, DOI: 10.1007/978-3-319-78154-9_7 *
CHOTHIA ET AL., NATURE, vol. 341, 1989, pages 544 546 - 883
CHOTHIALESK, J. MOL. BIOL., vol. 196, 1987, pages 901 - 917
GILLIES ET AL.: "MRI of the Tumor Microenvironment", JOURNAL OF MAGNETIC RESONANCE IMAGING, vol. 16, no. 1599440-33-1, 2002, pages 430 - 450, XP055045243, DOI: 10.1002/jmri.10181
KEVIN J. HAMBLETT, PHIL W. HAMMOND, STUART D. BARNSCHER, VINCENT K. FUNG, RUPERT H. DAVIES, GRANT R. WICKMAN, ANDREA HERNANDEZ, TO: "Abstract 3914: ZW49, a HER2-targeted biparatopic antibody-drug conjugate for the treatment of HER2-expressing cancers", CANCER RESEARCH, vol. 78, no. 13, 31 July 2018 (2018-07-31), US, pages 1 - 4, XP009523638, ISSN: 0008-5472, DOI: 10.1158/1538-7445.AM2018-3914 *
KJ HAMBLETT; SD BARNSCHER; RH DAVIES; PW HAMMOND; A HERNANDEZ; GR WICKMAN; VK FUNG; T DING; G GARNETT; AS GALEY; P ZWIERZCHOWSKI; : "Abstract P 6-17 -13: ZW 49, a HER2 targeted biparatopic antibody drug conjugate for the treatment of HER2 expressing cancers", CANCER RESEARCH, vol. 79, no. 4_suppl., 15 February 2019 (2019-02-15), US, pages 1 - 4, XP009542493, ISSN: 0008-5472, DOI: 10.1158/1538-7445.SABCS18-P6-17-13 *
LI JOHN Y; PERRY SAMUEL R; MUNIZ-MEDINA VANESSA; WANG XINZHONG; WETZEL LESLIE K; REBELATTO MARLON C; HINRICHS MARY JANE MASSON; BE: "A Biparatopic HER2-Targeting Antibody-Drug Conjugate Induces Tumor Regression in Primary Models Refractory to or Ineligible for HER2-Targeted Therapy", CANCER CELL, vol. 29, no. 1, 11 January 2016 (2016-01-11), US , pages 117 - 129, XP029383988, ISSN: 1535-6108, DOI: 10.1016/j.ccell.2015.12.008 *
M. PEGRAM, E. HAMILTON ,A.R. TAN ,A.M. STORNIOLO ,N. ELGEIOUSHI ,S. MARSHALL ,S. ABDULLAH ,M. PATEL: "Phase 1 study of bispecific HER2 antibody-drug conjugate MEDI4276 in patients with advanced HER2-positive breast or gastric cancer", ANNALS OF ONCOLOGY, vol. 29, no. Supplement 3, 31 March 2018 (2018-03-31), pages iii8 - iii8, XP093023064, DOI: 10.1093/annonc/mdy048.005 *
PEGRAM MARK D., HAMILTON ERIKA P., TAN ANTOINETTE R., STORNIOLO ANNA MARIA, BALIC KEMAL, ROSENBAUM ANTON I., LIANG MEINA, HE PENG,: "First-in-Human, Phase 1 Dose-Escalation Study of Biparatopic Anti-HER2 Antibody–Drug Conjugate MEDI4276 in Patients with HER2-positive Advanced Breast or Gastric Cancer", MOLECULAR CANCER THERAPEUTICS, vol. 20, no. 8, 1 August 2021 (2021-08-01), US , pages 1442 - 1453, XP093023048, ISSN: 1535-7163, DOI: 10.1158/1535-7163.MCT-20-0014 *
PERNAS SONIA, SARA M. TOLANEY: "HER2-positive breast cancer: new therapeutic frontiers and overcoming resistance", THERAPEUTIC ADVANCES IN MEDICAL ONCOLOGY, 31 December 2019 (2019-12-31), pages 1 - 16, XP093023061, DOI: 10.1177/1758835919833519 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2024222870A1 (zh) * 2023-04-27 2024-10-31 石药集团巨石生物制药有限公司 一种抗体药物偶联物
CN116751319A (zh) * 2023-07-19 2023-09-15 首都医科大学 一种壳聚糖六肽衍生物及其制备方法和应用
WO2025103474A1 (zh) * 2023-11-17 2025-05-22 成都康弘生物科技有限公司 双毒素抗体药物偶联物及其用途

Also Published As

Publication number Publication date
US20240307551A1 (en) 2024-09-19
JP2024525624A (ja) 2024-07-12
EP4386006A1 (en) 2024-06-19
WO2023280092A9 (zh) 2023-02-09
CN117616052A (zh) 2024-02-27

Similar Documents

Publication Publication Date Title
US11077202B2 (en) Anti-CDH6 antibody and anti-CDH6 antibody-drug conjugate
WO2023280092A1 (zh) 抗体药物偶联物及其应用
KR102084806B1 (ko) 인테그린 αvβ6에 대한 항체 및 암을 치료하기 위한 그의 용도
US20240409658A1 (en) Anti-cd228 antibodies and antibody-drug conjugates
TW201738271A (zh) 對表皮生長因子受體變異體ｉｉｉ具特異性之抗體類及彼等之用途
CN114269389A (zh) 靶向紧密连接蛋白18.2的抗体药物偶联物
US20230310629A1 (en) Bispecific antibody-drug conjugates targeting egfr and muc1 and uses thereof
CN115884793A (zh) 具有含肽接头的位点特异性抗体-药物缀合物
US20240390512A1 (en) Methods of treating chemotherapy-resistant cancer with an antibody-drug conjugate
TW202245844A (zh) 抗dll3抗體-藥物結合物
EP4494658A1 (en) Antibody-drug conjugate and use thereof
US20250228766A1 (en) Polynucleotides encoding antibodies which bind the ec3 domain of cadherin-6 (cdh6) and possess internalization ability
WO2024208354A1 (en) Antibody drug conjugates
TW202432186A (zh) 配體-細胞毒藥物綴合物及其醫藥用途
TW202444760A (zh) 抗ｃｄ２５抗體及抗ｃｄ２５抗體－藥物複合體
WO2025140548A1 (zh) 一种免疫缀合物及其用途
TW202417054A (zh) 配體－細胞毒性藥物偶聯物及其藥物用途
HK40084709A (zh) 具有含肽接頭的位點特異性抗體-藥物綴合物
HK40072804B (zh) 靶向緊密連接蛋白18.2的抗體藥物偶聯物
HK40072804A (zh) 靶向緊密連接蛋白18.2的抗體藥物偶聯物
HK40027154A (zh) 抗cdh6抗體和抗cdh6抗體-藥物綴合物
HK40027154B (zh) 抗cdh6抗體和抗cdh6抗體-藥物綴合物

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 22836843

Country of ref document: EP

Kind code of ref document: A1

ENP Entry into the national phase

Ref document number: 2024500541

Country of ref document: JP

Kind code of ref document: A

WWE Wipo information: entry into national phase

Ref document number: 202280047851.4

Country of ref document: CN

WWE Wipo information: entry into national phase

Ref document number: 2022836843

Country of ref document: EP

NENP Non-entry into the national phase

Ref country code: DE

ENP Entry into the national phase

Ref document number: 2022836843

Country of ref document: EP

Effective date: 20240205