WO2022228422A1 - 一种双特异抗体偶联物 - Google Patents

一种双特异抗体偶联物 Download PDF

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WO2022228422A1
WO2022228422A1 PCT/CN2022/089229 CN2022089229W WO2022228422A1 WO 2022228422 A1 WO2022228422 A1 WO 2022228422A1 CN 2022089229 W CN2022089229 W CN 2022089229W WO 2022228422 A1 WO2022228422 A1 WO 2022228422A1
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bispecific antibody
chain variable
heavy chain
variable domain
cancer
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PCT/CN2022/089229
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English (en)
French (fr)
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朱晓东
叶金统
王金凤
何伟
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轩竹生物科技股份有限公司
北京轩竹康明生物科技有限公司
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Application filed by 轩竹生物科技股份有限公司, 北京轩竹康明生物科技有限公司 filed Critical 轩竹生物科技股份有限公司
Priority to JP2023565593A priority Critical patent/JP2024519523A/ja
Priority to EP22794892.4A priority patent/EP4332120A1/en
Priority to KR1020237040340A priority patent/KR20240000575A/ko
Priority to CN202280031170.9A priority patent/CN117321086A/zh
Publication of WO2022228422A1 publication Critical patent/WO2022228422A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • 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
    • A61K47/6801—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
    • A61K47/68031—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being an auristatin
    • 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
    • A61K47/6889—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
    • 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
    • A61K2039/545—Medicinal preparations containing antigens or antibodies characterised by the dose, timing or administration schedule
    • 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
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/52—Constant or Fc region; Isotype
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/64—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising a combination of variable region and constant region components

Definitions

  • the invention belongs to the technical field of antibody drugs, and in particular, relates to a bispecific antibody conjugate; and a pharmaceutical composition comprising the bispecific antibody conjugate, and the preparation of the bispecific antibody conjugate and the pharmaceutical composition thereof Use in medicaments for the prevention and/or treatment of cancer.
  • Human epidermal growth factor receptor 2 (HER2, also known as ErbB2) is a member of the ErbB family of tyrosine protein kinase receptors. It is a type I membrane protein with a single channel transmembrane domain, an extracellular domain and a cytoplasmic kinase domain.
  • the HER2 gene is amplified in approximately 20% of breast cancer patients (HER2 + ). Targeting HER2 with monoclonal antibodies has been shown to be very effective in the treatment of patients with HER2-amplified breast cancer.
  • Trastuzumab (mAb 4D5, Herceptin ) is a humanized anti-HER2 monoclonal antibody that binds to domain IV of HER2 ECD and was approved by the FDA in 1998 for the treatment of HER2 + breast cancer.
  • Pertuzumab (rhuMab 2C4, ) is another humanized anti-HER2 monoclonal antibody, but binds to domain II of HER2 ECD, which is a separate epitope from that of trastuzumab on HER2. Since domain II of HER2 ECD is involved in dimerization, the binding of Pertuzumab to HER2 prevents HER2 from dimerizing with another receptor such as EGFR, HER3 or HER4.
  • pertuzumab and trastuzumab have shown superior efficacy compared to trastuzumab or pertuzumab alone and has been approved by the FDA for HER2+ metastatic breast cancer (2012 ), and one year later for neoadjuvant treatment of HER2+ breast cancer (2013).
  • Antibody editing refers to the targeted and precise modification and transformation of antibodies, including knockout, substitution and addition of sugars in antibody sugar chains, site-directed or non-site-directed coupling of antibody payloads, and precise improvement of antibody Fab and Fc engineering. .
  • Antibody editing emphasizes precise modification and transformation, making full use of and regulating each functional area and different modules of antibodies.
  • Antibody editing of natural or engineered antibody molecules can construct multifunctional novel antibodies with diverse molecular structures such as glycoengineered antibodies and ADCs.
  • ADCs Antibody-drug conjugates
  • biological missiles conjugated antibodies with targeting properties and strong cytotoxic drugs.
  • ADCs can specifically bind to antigen-positive tumor cell membranes and exert pharmacological effects; they can also form endosomes into cells through tumor cell endocytosis, and enter endosomes and lysosomes after entering the cytoplasm. It binds to the body, dissociates the coupled small molecule toxin and restores its intrinsic properties, thereby killing tumor cells.
  • ADCs can be regarded as a kind of prodrug, which can target small molecule toxins into tumor cells, increase the exposure of small molecule toxins in tumor cells, reduce the exposure to normal tissues, and expand the exposure of such small molecule toxins.
  • Safety window therapeutic window
  • ADCs are mainly composed of three parts, namely, targeting antibodies, conjugated toxins, and linkers between antibodies and toxins, and their targets are usually related antigens or specific receptors on the surface of tumor cells.
  • the mAb in the ADC must have the following characteristics: 1. It can maintain its own characteristics after coupling with small molecule toxins; 2. It has good targeting to the antigen; 3. It only binds to the antigen of the target cell; 4. Feedback; 5 The non-specific binding to other cells is rare.
  • TDM1 Trastuzumab-emtansine conjugate
  • Kadcyla was approved by the FDA for the treatment of HER2-positive advanced metastatic breast cancer.
  • TDM1 became the first ADC drug to enter China.
  • ADCs Bispecific antibody conjugates
  • the present invention provides a bispecific antibody conjugate (ADC), a pharmaceutically acceptable salt, solvate or deuterated product thereof, comprising a bispecific antibody, a toxin and a linker;
  • ADC bispecific antibody conjugate
  • the bispecific antibody comprises a first paratope capable of recognizing and/or binding domain II of HER2, and a second paratope capable of recognizing and/or binding domain IV of HER2;
  • the toxin is selected from maytansinoids, hamitrines, amanitamines, auristatins, calicheamicins or duocarmycins;
  • the linker is selected from a cleavable linker and a non-cleavable linker
  • one or more hydrogen atoms in the toxin and/or linker are optionally deuterated.
  • the deuterated species means that one or more hydrogen atoms in the toxin and/or linker in the bispecific antibody conjugate (ADC) are optionally deuterated.
  • the bispecific antibody comprises a first paratope capable of recognizing and/or binding domain II of HER2, and a second paratope capable of recognizing and/or binding domain IV of HER2.
  • the bispecific antibody comprises a pair of homologous light chains and a pair of heterologous heavy chains comprising two different heavy chain variable domains.
  • the pair of homologous light chains comprises a modified trastuzumab light chain variable domain
  • the modified trastuzumab light chain variable domain is based on the unmodified trastuzumab light chain variable domain, and subjected to sequence modification, and the sequence modification includes:
  • the modified trastuzumab light chain variable domain is based on the unmodified trastuzumab light chain variable domain with the following sequence modifications: N30S, S56Y and T94W any one, two or three of these three modifications;
  • the sequence of the unmodified trastuzumab light chain variable domain is shown in SEQ ID NO:5.
  • one heavy chain variable domain is selected from the unmodified Pertuzumab heavy chain variable domain or the modified Pertuzumab heavy chain variable domain, the unmodified Pertuzumab heavy chain variable domain
  • the sequence of the monoclonal antibody heavy chain variable domain is shown in SEQ ID NO: 3;
  • the modified Pertuzumab heavy chain variable domain is based on the unmodified Pertuzumab heavy chain variable domain, and undergoes sequence modification, and the sequence modification includes:
  • the modified Pertuzumab heavy chain variable domain is based on the unmodified Pertuzumab heavy chain variable domain, with sequence modifications comprising : T30A and/or G56A;
  • the other heavy chain variable domain is selected from the unmodified trastuzumab heavy chain variable domain or the modified trastuzumab heavy chain variable domain; the unmodified trastuzumab
  • the sequence of the variable domain of the heavy chain of benzumab is shown in SEQ ID NO:7;
  • the modified trastuzumab heavy chain variable domain is based on the unmodified trastuzumab heavy chain variable domain, and is subjected to sequence modification, and the sequence modification includes:
  • the modified trastuzumab heavy chain variable domain is based on the unmodified trastuzumab heavy chain variable domain, with sequence modifications comprising : N54T and/or D98S.
  • the bispecific antibody comprises a pair of homologous light chains and a pair of heterologous heavy chains comprising two different heavy chain variable domains;
  • the pair of homologous light chains comprises a modified trastuzumab light chain variable domain
  • the modified trastuzumab light chain variable domain is based on the unmodified trastuzumab light chain variable domain with the following sequence modifications: N30S, S56Y and T94W among the three modifications. any one, two or three;
  • the sequence of the unmodified trastuzumab light chain variable domain is shown in SEQ ID NO: 5;
  • one heavy chain variable domain is selected from the unmodified Pertuzumab heavy chain variable domain or the modified Pertuzumab heavy chain variable domain, the unmodified Pertuzumab heavy chain variable domain
  • the sequence of the monoclonal antibody heavy chain variable domain is shown in SEQ ID NO: 3;
  • the modified Pertuzumab heavy chain variable domain is based on the unmodified Pertuzumab heavy chain variable domain, and the sequence modification is carried out, and the sequence modification comprises: T30A and/or G56A ;
  • the other heavy chain variable domain is selected from the unmodified trastuzumab heavy chain variable domain or the modified trastuzumab heavy chain variable domain; the unmodified trastuzumab
  • the sequence of the variable domain of the heavy chain of benzumab is shown in SEQ ID NO:7;
  • the modified trastuzumab heavy chain variable domain is based on the unmodified trastuzumab heavy chain variable domain, and is subjected to sequence modification, and the sequence modification comprises: N54T and/or D98S .
  • the bispecific antibody is in the form of a Fab-Ig, Ig-Fab, or a heterodimeric Ig.
  • the bispecific antibody is in the form of a heterodimeric Ig.
  • the Fc region in the bispecific antibody is a knob-into-holes structure comprising two complementary Fc regions, the two complementary Fc regions being the Fc regions of the knob portion and the Fc region of the hole part.
  • the two Fc regions of the bispecific antibody are modified Fc regions comprising a substitution consisting of M428L.
  • the Fc of the bispecific antibody comprises:
  • ADCP antibody-dependent cellular phagocytosis
  • the bispecific antibody is a defucosylated antibody.
  • the defucosylated antibody is obtained by being produced by a host cell deficient in fucosylation.
  • the fucosylation defect is a knockout of the FUT8 gene.
  • the defucosylated antibody is obtained by having S239D, I332E, A330L substitutions (Kabat numbering) or any or all combinations of these variations in the Fc domain of the antibody.
  • the bispecific antibody is in the form of a heterodimeric Ig in which the Fc regions of the two heterologous heavy chains are connected by knobs-into-holes;
  • the bispecific antibody is in the form of a heterodimeric Ig in which the Fc regions of the two heterologous heavy chains are connected by a knob-hole structure, and the Fc region of the knob portion is unmodified
  • the modified Pertuzumab heavy chain Fc region either includes the following modifications: T366W; the Fc region of the hole portion is an unmodified trastuzumab heavy chain Fc region or includes any of the following modifications Combination: T366S, L368A and Y407V;
  • the bispecific antibody is in the form of a heterodimeric Ig, the Fc regions of the two heterologous heavy chains are connected by a knob-hole structure, and the Fc region of the knob part is in the form of a knob.
  • the unmodified Pertuzumab heavy chain Fc is based on sequence modifications, which include the following modifications: T366W and M428L; the Fc region of the hole portion is based on unmodified trast
  • the sequence modification was carried out based on the Fc region of the heavy chain of benzumab, and the sequence modification included any combination of the following modifications: T366S, L368A and Y407V; and the Fc region of the hole part of the bispecific antibody also included Modified as follows: M428L.
  • the bispecific antibody comprises a pair of homologous light chains and a pair of heterologous heavy chains comprising two different heavy chain variable domains;
  • the pair of homologous light chains comprises a modified trastuzumab light chain variable domain
  • the modified trastuzumab light chain variable domain is based on the unmodified trastuzumab light chain variable domain with the following sequence modifications: N30S, S56Y and T94W among the three modifications. any one, two or three;
  • the sequence of the unmodified trastuzumab light chain variable domain is shown in SEQ ID NO: 5;
  • one heavy chain variable domain is selected from the unmodified Pertuzumab heavy chain variable domain or the modified Pertuzumab heavy chain variable domain, the unmodified Pertuzumab heavy chain variable domain
  • the sequence of the monoclonal antibody heavy chain variable domain is shown in SEQ ID NO: 3;
  • the modified Pertuzumab heavy chain variable domain is based on the unmodified Pertuzumab heavy chain variable domain, and the sequence modification is carried out, and the sequence modification comprises: T30A and/or G56A ;
  • the other heavy chain variable domain is selected from the unmodified trastuzumab heavy chain variable domain or the modified trastuzumab heavy chain variable domain; the unmodified trastuzumab
  • the sequence of the variable domain of the heavy chain of benzumab is shown in SEQ ID NO:7;
  • the modified trastuzumab heavy chain variable domain is based on the unmodified trastuzumab heavy chain variable domain, and is subjected to sequence modification, and the sequence modification comprises: N54T and/or D98S ;
  • the bispecific antibody is in the form of a heterodimer Ig, the Fc regions of the two heterologous heavy chains are connected by a knob-hole structure, and the Fc region of the knob part is an unmodified Pertuzumab Sequence modifications were made based on the anti-heavy chain Fc, and the sequence modifications included the following modifications: T366W and M428L; the Fc region of the hole part was the unmodified trastuzumab heavy chain Fc region Based on the sequence modification, the sequence modification includes any combination of the following modifications: T366S, L368A and Y407V; and the bispecific antibody also includes the following modification in the Fc region of the hole part: M428L.
  • the bispecific antibody comprises a pair of homologous light chains and a pair of heterologous heavy chains comprising two different heavy chain variable domains;
  • the pair of homologous light chains comprises a modified trastuzumab light chain variable domain
  • the modified trastuzumab light chain variable domain is based on the unmodified trastuzumab light chain variable domain with the following sequence modifications: N30S, S56Y and T94W among the three modifications. any one, two or three;
  • the sequence of the unmodified trastuzumab light chain variable domain is shown in SEQ ID NO: 5;
  • one heavy chain variable domain is selected from the unmodified Pertuzumab heavy chain variable domain or the modified Pertuzumab heavy chain variable domain, the unmodified Pertuzumab heavy chain variable domain
  • the sequence of the monoclonal antibody heavy chain variable domain is shown in SEQ ID NO: 3;
  • the modified Pertuzumab heavy chain variable domain is based on the unmodified Pertuzumab heavy chain variable domain, and the sequence modification is carried out, and the sequence modification comprises: T30A and/or G56A ;
  • the other heavy chain variable domain is selected from the unmodified trastuzumab heavy chain variable domain or the modified trastuzumab heavy chain variable domain; the unmodified trastuzumab
  • the sequence of the variable domain of the heavy chain of benzumab is shown in SEQ ID NO:7;
  • the modified trastuzumab heavy chain variable domain is based on the unmodified trastuzumab heavy chain variable domain, and is subjected to sequence modification, and the sequence modification comprises: N54T and/or D98S ;
  • the bispecific antibody is in the form of a heterodimer Ig, the Fc regions of the two heterologous heavy chains are connected by a knob-hole structure, and the Fc region of the knob part is an unmodified Pertuzumab Sequence modifications were made based on the anti-heavy chain Fc, and the sequence modifications included the following modifications: T366W and M428L; the Fc region of the hole part was the unmodified trastuzumab heavy chain Fc region Based on the sequence modification, the sequence modification includes any combination of the following modifications: T366S, L368A and Y407V; and the bispecific antibody also includes the following modification in the Fc region of the hole part: M428L;
  • the bispecific antibody is a defucosylated antibody.
  • the bispecific antibody is selected from the group consisting of T51, T52, T53, T54, T55, T56, T57 and T58.
  • the bispecific antibody is selected from the group consisting of defucosylated T51 (T51-AF), defucosylated T52 (T52-AF), defucosylated T53 ( T53-AF), Defucosylated T54 (T54-AF), Defucosylated T55 (T55-AF), Defucosylated T56 (T56-AF), Defucosylated T56 (T56-AF) Glycosylated T57 (T57-AF) and defucosylated T58 (T58-AF).
  • Said toxins are selected from maytansinoids, hamitrins, amanitamines, auristatins, calicheamicins or duocarmycins, and their deuterated counterparts.
  • the toxin is selected from the group consisting of DM1, DM4, E7974, HTI-286, ⁇ -Amanita, ⁇ -Amanita, ⁇ -Amanita, ⁇ -Amanita, a Hydroxy amanita amide, trihydroxy amanita toxin, trihydroxy amanita toxin amide, ⁇ -trihydroxy amanita toxin, monomethyl auristatin F, monomethyl auristatin E, auristatin EB, Auristatin EVB, Auristatin F phenylenediamine, calicheamicin gamma, duocarmycin A, adozelesin and CC-1065, and their deuterated counterparts.
  • DM1, DM4, E7974, HTI-286 ⁇ -Amanita, ⁇ -Amanita, ⁇ -Amanita, ⁇ -Amanita, a Hydroxy amanita amide, trihydroxy
  • the linker is selected from a cleavable linker and a non-cleavable linker, and one or more hydrogen atoms in the linker are optionally deuterated;
  • the cleavable linker includes but is not limited to: chemically cleavable linking and/or an enzymatically cleavable linker, such as a linker containing a peptide component.
  • the enzymatically cleavable linker is selected from valine-citrulline, or phenylalanine-lysine, and their deuterated counterparts.
  • the present invention provides a bispecific antibody conjugate (ADC), a pharmaceutically acceptable salt, solvate or deuterated thereof, comprising a bispecific antibody, a toxin and a linker;
  • ADC bispecific antibody conjugate
  • the bispecific antibody comprises a pair of homologous light chains and a pair of heterologous heavy chains comprising two different heavy chain variable domains;
  • the pair of homologous light chains comprises a modified trastuzumab light chain variable domain
  • the modified trastuzumab light chain variable domain is based on the unmodified trastuzumab light chain variable domain with the following sequence modifications: N30S, S56Y and T94W among the three modifications. any one, two or three;
  • the sequence of the unmodified trastuzumab light chain variable domain is shown in SEQ ID NO: 5;
  • one heavy chain variable domain is selected from the unmodified Pertuzumab heavy chain variable domain or the modified Pertuzumab heavy chain variable domain, the unmodified Pertuzumab heavy chain variable domain
  • the sequence of the monoclonal antibody heavy chain variable domain is shown in SEQ ID NO: 3;
  • the modified Pertuzumab heavy chain variable domain is based on the unmodified Pertuzumab heavy chain variable domain, and the sequence modification is carried out, and the sequence modification comprises: T30A and/or G56A ;
  • the other heavy chain variable domain is selected from the unmodified trastuzumab heavy chain variable domain or the modified trastuzumab heavy chain variable domain; the unmodified trastuzumab
  • the sequence of the variable domain of the heavy chain of benzumab is shown in SEQ ID NO:7;
  • the modified trastuzumab heavy chain variable domain is based on the unmodified trastuzumab heavy chain variable domain, and is subjected to sequence modification, and the sequence modification comprises: N54T and/or D98S ;
  • the bispecific antibody is in the form of a heterodimer Ig, the Fc regions of the two heterologous heavy chains are connected by a knob-hole structure, and the Fc region of the knob part is an unmodified Pertuzumab Sequence modifications were made based on the anti-heavy chain Fc, and the sequence modifications included the following modifications: T366W and M428L; the Fc region of the hole part was the unmodified trastuzumab heavy chain Fc region Based on the sequence modification, the sequence modification includes any combination of the following modifications: T366S, L368A and Y407V; and the bispecific antibody also includes the following modification in the Fc region of the hole part: M428L;
  • the bispecific antibody is a defucosylated antibody
  • the toxin is selected from the group consisting of DM1, DM4, E7974, HTI-286, monomethyl auristatin F, monomethyl auristatin E, auristatin EB, auristatin EVB, auristatin F phenylenediamine, and their deuterated products;
  • the linker is selected from valine-citrulline, or phenylalanine-lysine, and their deuterated counterparts.
  • the present invention provides a bispecific antibody conjugate (ADC), a pharmaceutically acceptable salt, solvate or deuterated thereof, comprising a bispecific antibody, a toxin and a linker;
  • ADC bispecific antibody conjugate
  • the bispecific antibody comprises a pair of homologous light chains and a pair of heterologous heavy chains comprising two different heavy chain variable domains;
  • the pair of homologous light chains comprises a modified trastuzumab light chain variable domain
  • the modified trastuzumab light chain variable domain is based on the unmodified trastuzumab light chain variable domain with the following sequence modifications: N30S, S56Y and T94W among the three modifications. any one, two or three;
  • the sequence of the unmodified trastuzumab light chain variable domain is shown in SEQ ID NO: 5;
  • one heavy chain variable domain is selected from the unmodified Pertuzumab heavy chain variable domain or the modified Pertuzumab heavy chain variable domain, the unmodified Pertuzumab heavy chain variable domain
  • the sequence of the monoclonal antibody heavy chain variable domain is shown in SEQ ID NO: 3;
  • the modified Pertuzumab heavy chain variable domain is based on the unmodified Pertuzumab heavy chain variable domain, and the sequence modification is carried out, and the sequence modification comprises: T30A and/or G56A ;
  • the other heavy chain variable domain is selected from the unmodified trastuzumab heavy chain variable domain or the modified trastuzumab heavy chain variable domain; the unmodified trastuzumab
  • the sequence of the variable domain of the heavy chain of benzumab is shown in SEQ ID NO:7;
  • the modified trastuzumab heavy chain variable domain is based on the unmodified trastuzumab heavy chain variable domain, and is subjected to sequence modification, and the sequence modification comprises: N54T and/or D98S ;
  • the bispecific antibody is in the form of a heterodimer Ig, the Fc regions of the two heterologous heavy chains are connected by a knob-hole structure, and the Fc region of the knob part is an unmodified Pertuzumab Sequence modifications were made based on the anti-heavy chain Fc, and the sequence modifications included the following modifications: T366W and M428L; the Fc region of the hole part was the unmodified trastuzumab heavy chain Fc region Based on the sequence modification, the sequence modification includes any combination of the following modifications: T366S, L368A and Y407V; and the bispecific antibody also includes the following modification in the Fc region of the hole part: M428L;
  • the bispecific antibody is a defucosylated antibody
  • the toxin is selected from monomethyl auristatin E and its deuterated products;
  • the linker is selected from valine-citrulline and its deuterated counterparts.
  • the present invention provides a bispecific antibody conjugate (ADC), a pharmaceutically acceptable salt, solvate or deuterated thereof, comprising a bispecific antibody, a toxin and a linker;
  • ADC bispecific antibody conjugate
  • the bispecific antibody is selected from T51, T52, T53, T54, T55, T56, T57 and T58; in some embodiments, the bispecific antibody is defucosylated antibody T51-AF, T52-AF, T53-AF, T54-AF, T55-AF, T56-AF, T57-AF and T58-AF;
  • the toxin is selected from the group consisting of DM1, DM4, E7974, HTI-286, monomethyl auristatin F, monomethyl auristatin E, auristatin EB, auristatin EVB, auristatin F benzodiazepine Amines, and their deuterated products; in some embodiments, the toxin is selected from monomethyl auristatin E and its deuterated products;
  • the linker is selected from valine-citrulline, or phenylalanine-lysine, and their deuterated products; in some embodiments, the linker is valine-citrulline Acids and their deuterated products.
  • the present invention provides a bispecific antibody conjugate (ADC), a pharmaceutically acceptable salt, solvate or deuterated thereof, comprising a bispecific antibody, a toxin and a linker;
  • ADC bispecific antibody conjugate
  • the bispecific antibody is selected from T51, T52, T53, T54, T55, T56, T57 and T58, and defucosylated antibodies T51-AF, T52-AF, T53-AF, T54-AF , T55-AF, T56-AF, T57-AF and T58-AF;
  • the toxin is selected from the group consisting of DM1, DM4, E7974, HTI-286, monomethyl auristatin F, monomethyl auristatin E, auristatin EB, auristatin EVB, auristatin F benzodiazepine Amines, and their deuterated products;
  • the linker is selected from valine-citrulline, or phenylalanine-lysine, and their deuterated products.
  • the present invention provides a bispecific antibody conjugate (ADC), a pharmaceutically acceptable salt, solvate or deuterated thereof, comprising a bispecific antibody, a toxin and a linker;
  • ADC bispecific antibody conjugate
  • the bispecific antibody is selected from T51, T52, T53, T54, T55, T56, T57 and T58, and defucosylated antibodies T51-AF, T52-AF, T53-AF, T54-AF , T55-AF, T56-AF, T57-AF and T58-AF;
  • the toxin is selected from monomethyl auristatin E and its deuterated products
  • the linker is selected from valine-citrulline and its deuterated products.
  • the valine-citrulline linker is linked to the bispecific antibody via 6-maleimidohexanoic acid and to monomethyl auristatin E toxin via p-aminobenzyl alcohol .
  • the bispecific antibody conjugate has an average drug:antibody ratio (DAR) of about 1-6, eg, about 1, 2, 3, 4, 5, 6, eg, about 2.5, 2.6 , 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.
  • DAR average drug:antibody ratio
  • the structure of the bispecific antibody conjugate is shown in the following formula (I):
  • Vc valine-citrulline
  • the linker is connected to the bispecific antibody through 6-maleimidohexanoic acid , linked to the rightmost Drug (toxin moiety) through p-aminobenzyl alcohol.
  • the DAR value (n) is further specified in the structure of the bispecific antibody conjugate, as shown in the following formula (I'):
  • the middle part is the linker valine-citrulline (Val-Cit, referred to as Vc) part
  • the linker is connected to the bispecific antibody through 6-maleimidohexanoic acid , connected to the rightmost Drug (toxin moiety) through p-aminobenzyl alcohol
  • the toxin is selected from DM1, DM4, E7974, HTI-286, monomethyl auristatin F, monomethyl auristatin E, Ristatin EB, Auristatin EVB, Auristatin F phenylenediamine, and their deuterated products;
  • n represents the average drug:antibody ratio (DAR)
  • DAR drug:antibody ratio
  • n is selected from 1-6, such as about 1, 2, 3, 4, 5, 6, such as about 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.
  • the structure of the bispecific antibody conjugate is shown in the following formula (I'):
  • the bispecific antibody is connected to the rightmost Drug (toxin moiety) through p-aminobenzyl alcohol, and the toxin is selected from DM1, DM4, E7974, HTI-286, monomethyl auristatin F, monomethyl Australian Auristatin E, Auristatin EB, Auristatin EVB, Auristatin F phenylenediamine, and their deuterated products;
  • n represents the average drug:antibody ratio (DAR), n is selected from 3-4, eg, about 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.
  • DAR drug:antibody ratio
  • the structure of the bispecific antibody conjugate is shown in the following formula (II):
  • the structure in [ ] is the linker-toxin moiety Vc-MMAE, Vc is linked to the bispecific antibody through 6-maleimidohexanoic acid, and Vc is linked to the bispecific antibody through p-aminobenzyl alcohol.
  • the toxin monomethyl auristatin E (MMAE) is linked.
  • n is the DAR value
  • n is 1-6, such as about 1, 2, 3, 4, 5, 6, such as 3-4, such as about 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.65, 3.7, 3.75, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.
  • the bispecific antibody conjugate has an average drug:antibody ratio (DAR) of 3-4, eg, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.65, 3.7, 3.75, 3.8 , 3.9, 4.
  • DAR drug:antibody ratio
  • the structure of the bispecific antibody conjugate is shown in the following formula (II):
  • n is the DAR value, and n is selected from 3-4, for example, about 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, and 4.
  • the structure of the bispecific antibody conjugate is shown in the following formula (II):
  • n is the DAR value, and n is selected from 3-4, for example, about 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, and 4.
  • the bispecific antibody is selected from the group consisting of T51, T52, T53, T54, T55, T56, T57 and T58.
  • the bispecific antibody is selected from the group consisting of defucosylated T51 (T51-AF), defucosylated T52 (T52-AF), defucosylated T53 ( T53-AF), Defucosylated T54 (T54-AF), Defucosylated T55 (T55-AF), Defucosylated T56 (T56-AF), Defucosylated T56 (T56-AF) Glycosylated T57 (T57-AF) and defucosylated T58 (T58-AF).
  • the bispecific antibody is selected from the group consisting of T54, T58, defucosylated T54 (T54-AF) and defucosylated T58 (T58-AF).
  • the bispecific antibody is selected from defucosylated T54 (T54-AF) and defucosylated T58 (T58-AF).
  • the bispecific antibody conjugate of the invention is T51-MMAE, T52-MMAE, T53-MMAE, T54-MMAE, T55-MMAE, T56-MMAE, T57-MMAE, T58-MMAE, and corresponding
  • the defucosylated bispecific antibody conjugates T51-AF-MMAE, T52-AF-MMAE, T53-AF-MMAE, T54-AF-MMAE, T55-AF-MMAE, T56-AF-MMAE, T57 -AF-MMAE and T58-AF-MMAE.
  • the bispecific antibody conjugates of the invention are T54-MMAE and T58-MMAE, and the corresponding defucosylated bispecific antibody conjugates T54-AF-MMAE and T58-AF-MMAE .
  • a pharmaceutical composition comprising the above-mentioned bispecific antibody conjugate, a pharmaceutically acceptable salt, solvate or deuterated compound thereof, and a pharmaceutically acceptable carrier or excipients.
  • a bispecific antibody conjugate as described above, a pharmaceutically acceptable salt thereof, a solvate or a deuterated product thereof, or a pharmaceutical composition as described above in preparation for prophylaxis and/or use or in the use of drugs for the treatment of cancer.
  • the cancer is a HER2-expressing cancer.
  • the cancer is a cancer that expresses low levels of HER2.
  • the cancer includes breast cancer, ovarian cancer, endometrial cancer, cervical cancer, gastric cancer, esophageal cancer, lung cancer, head and neck cancer, bladder cancer, bile duct cancer, and colorectal cancer.
  • the present invention also relates to the application of the bispecific antibody conjugates, their pharmaceutically acceptable salts, solvates or their deuterated products or pharmaceutical compositions in the preparation of anti-tumor combined preparations. Also included are the optional following tumor-treating active ingredients:
  • a method for preventing and/or treating cancer comprising administering to a patient in need thereof a therapeutically effective amount of the bispecific antibody conjugate as described above, pharmaceutically acceptable The salt, solvate or deuterated form thereof or the pharmaceutical composition as described above.
  • the cancer is a HER2-expressing cancer.
  • the cancer is a cancer that expresses low levels of HER2.
  • the cancer includes breast cancer, ovarian cancer, endometrial cancer, cervical cancer, gastric cancer, esophageal cancer, lung cancer, head and neck cancer, bladder cancer, bile duct cancer, and colorectal cancer.
  • the present invention also relates to the application of the bispecific antibody conjugate, its pharmaceutically acceptable salt, solvate or its deuterated product or pharmaceutical composition in the treatment of cancer, which is characterized in that it is combined with the following drugs for the treatment of cancer or in combination with treatment regimens;
  • the bispecific antibody conjugates of the present invention have excellent antitumor activity, and at specific doses, in multiple different types of mouse tumor models Its anti-tumor activity is better than TDM1 (or DS8201), and at the same dose, it can better inhibit tumor growth in a shorter period of time than TDM1 (or DS8201).
  • the bispecific antibody conjugate according to the present invention has good tolerance and low toxicity.
  • FIG. 1 Schematic representation of the Fab-Ig format of bispecific antibodies with homologous light chains designed from Antibody A and Antibody B.
  • FIG. 1 Schematic representation of the Ig-Fab format of bispecific antibodies with homologous light chains designed from Antibody A and Antibody B.
  • FIG. 3 Schematic representation of the heterodimeric IgG format of bispecific antibodies with homologous light chains designed from Antibody A and Antibody B, the heterodimers being linked by a knob-hole structure.
  • FIG 4 Schematic representation of the heterodimeric IgG format of bispecific antibodies with homologous light chains designed from Antibody A and Antibody B, the heterodimers are linked by an electrostatic steering mechanism.
  • Figure 5 shows the SEC purity graphs of antibodies T54 (5A) and T54-AF (5B), wherein the abscissa represents time (min) and the ordinate represents absorbance value (mAU).
  • Fig. 6 SEC purity graph of antibody conjugates T54-MMAE (6A) and T54-AF-MMAE (6B), wherein the abscissa represents time (min) and the ordinate represents absorbance value (mAU).
  • FIG. 7 shows the HIC-HPLC results of antibody T54, wherein the abscissa represents time (min) and the ordinate represents absorbance value (mAU).
  • Fig. 8 HIC-HPLC results of antibody conjugates T54-MMAE (8A) and T54-AF-MMAE (8B), wherein the abscissa represents time (min) and the ordinate represents absorbance value (mAU).
  • Figure 10 The growth inhibition curve of antibodies herceptin, T54 and their corresponding conjugates TDM1, T54-MMAE on breast cancer cells SKBR3, wherein the abscissa represents the logarithm of the concentration, and the ordinate represents the cell survival of breast cancer cells SKBR3 Rate.
  • Figure 11 Growth inhibition curve of antibodies herceptin, T54 and their corresponding conjugates TDM1, T54-MMAE on gastric cancer cell N87, wherein the abscissa represents the logarithm of the concentration, and the ordinate represents the cell survival rate of gastric cancer cell N87.
  • Figure 12 Detects the inhibitory effect of T54-AF-MMAE, DS8201, T54-AF, herceptin, perjeta, Isotype (isotype control, as negative antibody) on various cell lines, wherein (A) BT474 cell line, (B) NCI- H2170 cell line (C) MDA-MB-175 VII cell line (D) SKOV-3 cell line (E) MCF-7 cell line (F) MDA-MB-435 cell line (G) NCI-H446 cell line.
  • Figure 13 shows the growth curve of human xenograft breast cancer model BT474 after the initiation of treatment with antibody conjugates TDM1 and T54-MMAE, wherein the abscissa represents days after tumor transplantation, and the ordinate represents tumor volume (mm 3 ).
  • Figure 14 is a graph of the growth curve of human xenograft breast cancer model BT474 after the initiation of treatment with positive control ADC (DS8201) and defucosylated bispecific antibody conjugate T54-AF-MMAE (T54-AF-ADC), wherein , the abscissa represents the days after tumor transplantation, and the ordinate represents the tumor volume (mm 3 ).
  • FIG. 15 Growth curve of human xenograft gastric model N87 after treatment with positive control ADC (DS8201) and defucosylated bispecific antibody conjugate T54-AF-MMAE (T54-AF-ADC), wherein, The abscissa represents days after tumor transplantation, and the ordinate represents tumor volume (mm 3 ).
  • the present invention discloses a bispecific antibody conjugate, its pharmaceutically acceptable salt, solvate or its deuterated product, including bispecific antibody, toxin and linker; the bispecific antibody recognizes and/or binds to HER2 Domain II, and domain IV that recognizes and/or binds HER2; the toxin is selected from maytansinoids, hamitrines, amanitamines, auristatins, calicheamicins or Oncomycins; the linker is selected from a cleavable linker and a non-cleavable linker; one or more hydrogen atoms in the toxin and linker are optionally deuterated.
  • the term "antibody” refers to a molecule having the general structure of a naturally occurring mammalian immunoglobulin (Ig) of which IgG is an example. That is, two identical light chains comprising one variable domain and one constant domain and two identical heavy chains comprising one variable domain and three constant domains. The light and heavy chains associate with each other through their variable domains, and the constant domain of the light chain and the first constant domain of the heavy chain. The two heavy chains are associated with each other through the second and third constant domains.
  • the antigen binding site of an antibody is formed by two variable domains, and in particular by three complementarity determining regions (CDRs) of each variable domain.
  • an antibody is said to bind to a molecule (antigen) if it is able to specifically interact with and adsorb to a molecule. Antibody binding does not include nonspecific or low affinity interactions. While “antibody” is meant to refer to the structure of a naturally occurring immunoglobulin, it also includes designed molecules that retain this general structure, such as chimeric, CDR-grafted, and humanized antibodies.
  • epitopope refers to the portion of an antigen that mediates the specific binding of an antigen to an antibody (or antibody construct) through contact with the antigen binding site of the antibody (or antibody construct).
  • paratope refers to the portion of an antibody (or antibody construct) that mediates the specific binding of an antibody (or antibody construct) to an antigen through contact with an epitope of the antigen.
  • substitution refers to engineered changes in amino acid sequence. Unless the context dictates otherwise, a mutation refers to a sequence change caused by an artificially assisted biological process. Conventionally, substitutions are represented by the three-part one-letter code of the amino acid in the reference sequence, the position in the reference sequence, and the one-letter code of the amino acid in the resulting sequence. For example, A26S means that alanine (A) at position 26 in the reference sequence was changed to serine (S) in the resulting sequence.
  • homologous refers to two or more peptide chains that are identical in amino acid sequence.
  • heterologous refers to two or more peptide chains that differ, are not identical in amino acid sequence.
  • deuterated refers to a structure in which one or more hydrogen atoms (H-1) in the structure of an antibody conjugate are replaced by a deuterium atom (H-2).
  • any atom of the compounds of the present invention may represent any stable isotope of that atom.
  • H ie, hydrogen (H-1)
  • H-1 hydrogen
  • D i.e. deuterium (H-2)
  • H-2 deuterium
  • Deuterium isotope when one or more positions in the structure of the compound of the present application is defined as D, namely deuterium (H-2), the content of the compound shown in the structure can be at least 52.5%, at least 60%, at least 67.5%, at least 75%, at least 82.5%, at least 90%, at least 95%, at least 97%, at least 98.5%, at least 99%, at least 99.5%.
  • the deuteration rate of the compounds of the present application refers to the ratio of the isotope content of label synthesis to the amount of naturally occurring isotopes.
  • the deuteration rate of each designated deuterium atom of the compounds of the present application may be at least 3500 times (52.5%), at least 4000 times (60%), at least 4500 times (67.5%), at least 5000 times (75%), at least 5000 times (75%), At least 5500 times (82.5%), at least 6000 times (90%), at least 6333.3 times (95%), at least 6466.7 times (97%), at least 6566.7 times (98.5%), at least 6600 times ( 99%), at least 6633.3 times (99.5%).
  • Isotopologues in this application refer to compounds that differ in chemical structure only in their isotopic composition.
  • the deuterium-containing compounds of the present application at a particular position will also contain very little hydrogen isotopologues at that position, and the amount of hydrogen isotopologues at the deuterated positions in the deuterated compounds of the present application depends on many factors, including the deuterated reagent ( D2O, D2, NaBD4, L1AID4, etc.) and the effectiveness of introducing deuterium isotope synthesis methods.
  • the total amount of hydrogen isotopologues at such deuterated positions will be less than 49.9%.
  • the total amount of hydrogen isotopes at the deuterated positions in the deuterated compounds of the present application will be less than 47.5%, 40%, 32.5%, 25%, 17.5%, 10%, 5%, 3%, 1% or 0.5%.
  • any atom not designated as deuterium is present in its natural isotopic abundance.
  • the numbering of the amino acid sequence of the light chain variable domain and the amino acid sequence of the heavy chain variable domain are based on Vajdos et al., J. Mol. Biol. 320:415 (2002), using Kabat No. (Kabat et al., NIH publication no. 91-3242, pp 662, 680, 689 (1991).
  • the bispecific antibodies of the present invention can take on a number of different general structures.
  • the bispecific antibodies of the invention are specific for two epitopes in the same antigen or in different antigens.
  • the bispecific antibodies of the invention have two paratopes.
  • variable domains may be added to each heavy chain such that the heavy chain includes, for example, 3, 4, 5 or more variable domains that facilitate the formation of the same number of paratopes.
  • the third paratope may confer specificity for the third epitope, making the antibody trispecific.
  • the third paratope may be specific for one of the epitopes recognized by either of the first two paratopes, making the antibody still bispecific, but with increased sensitivity to one of the epitopes. price.
  • the fourth paratope may be specific for the fourth epitope, making the antibody construct tetraspecific, or it may be specific for one of the epitopes recognized by any of the first three paratopes are specific so that the antibody construct is trispecific or bispecific.
  • the bispecific antibody comprises a first paratope capable of recognizing and/or binding domain II of HER2, and a second paratope capable of recognizing and/or binding domain IV of HER2.
  • the bispecific antibody comprises a pair of homologous light chains and a pair of heterologous heavy chains comprising two different heavy chain variable domains.
  • the pair of homologous light chains comprise a modified trastuzumab light chain variable domain
  • the modified trastuzumab light chain variable domain is based on the unmodified trastuzumab light chain variable domain, and subjected to sequence modification, and the sequence modification includes:
  • the modified trastuzumab light chain variable domain is based on the unmodified trastuzumab light chain variable domain with the following sequence modifications: N30S, S56Y and T94W any one, two or three of these three modifications;
  • the sequence of the unmodified trastuzumab light chain variable domain is shown in SEQ ID NO:5.
  • one heavy chain variable domain is selected from the unmodified Pertuzumab heavy chain variable domain or the modified Pertuzumab heavy chain variable domain, the unmodified Pertuzumab heavy chain variable domain
  • the sequence of the monoclonal antibody heavy chain variable domain is shown in SEQ ID NO: 3;
  • the modified Pertuzumab heavy chain variable domain is based on the unmodified Pertuzumab heavy chain variable domain, and undergoes sequence modification, and the sequence modification includes:
  • the modified Pertuzumab heavy chain variable domain is based on the unmodified Pertuzumab heavy chain variable domain, with sequence modifications comprising : T30A and/or G56A;
  • the other heavy chain variable domain is selected from the unmodified trastuzumab heavy chain variable domain or the modified trastuzumab heavy chain variable domain; the unmodified trastuzumab
  • the sequence of the variable domain of the heavy chain of benzumab is shown in SEQ ID NO:7;
  • the modified trastuzumab heavy chain variable domain is based on the unmodified trastuzumab heavy chain variable domain, and is subjected to sequence modification, and the sequence modification includes:
  • the modified trastuzumab heavy chain variable domain is based on the unmodified trastuzumab heavy chain variable domain, with sequence modifications comprising : N54T and/or D98S.
  • the bispecific antibody is in the form of a Fab-Ig, Ig-Fab, or a heterodimeric Ig.
  • the bispecific antibody is in the form of a heterodimeric Ig.
  • Fc region refers to the C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region.
  • the term includes native sequence Fc regions and variant Fc regions.
  • the numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, also known as the EU index, as in Kabat et al., Sequences of Proteins of Immunological Interest, 5th edition, Public Health Service , described in National Institutes of Health, Bethesda, MD (1991).
  • the constant domains are human immunoglobulins, such as IgG, IgM, IgA, IgD; or IgG and its subtypes IgG1, IgG2, IgG3, IgG4; or recombinant from these types and subtypes A combination of CH1 , CH2 and CH3 domains.
  • the Fc of the bispecific antibody comprises an Fc with increased antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), or complement-dependent cytotoxicity (CDC) activity due to Caused by increased or decreased binding affinity of Fc receptors such as CD16a, CD16b, CD32a, CD16b, CD64 and C1q proteins.
  • ADCC antibody-dependent cellular cytotoxicity
  • ADCP antibody-dependent cellular phagocytosis
  • CDC complement-dependent cytotoxicity
  • the bispecific antibody comprises a mutation in the Fc domain to reduce ADCC activity or CDC activity.
  • mutations at N297 in the Fc domain such as, but not limited to, N297A, N297G; (2) mutations at L234, such as L234A, L234G, and/or L235, such as L235A or L235G; (3) a mutation of P329, such as P329G; or (4) a mutation of D265, such as D265A; or a combination of substitutions at any or all of these positions.
  • Fc mutations include mutations that increase serum half-life.
  • the Fc has the following substitutions: T250Q , or M428L, or T250Q/M428L double mutation in CH3 (Hinton et al., J Biol Chem. 279(8):6213-6, 2004).
  • the Fc of the bispecific antibody has the M252Y/S254T/T256E triple mutation (Dall'Acqua WF et al., J Immunol 169(9):5171-80, 2002).
  • the Fc of the bispecific antibody has the N434A mutation (Petkova SB et al., International Immunology 18(12):1759-1769, 2006.) or the M428L/N434S double mutation, or the M428L/N434A double mutation ( Zalevsky J et al., Nat Biotechnol. 28(2):157–159, 2010).
  • the Fc region of the diabody has been modified to increase its serum half-life.
  • the modification that increases serum half-life is M428L.
  • Bispecific antibodies of the invention may be in homodimeric form (comprising two identical heavy chains), eg Fab-Ig (as shown in Figure 1) or Ig-Fab (as shown in Figure 2).
  • Linkers consist of 0 to 100 amino acids of any composition.
  • the adaptor is, for example, ( G3S ) nG3 , where n is any number between 1 and 20 (as shown in Table 8).
  • the bispecific antibodies of the invention may also be in the form of heterodimeric Ig.
  • Two different heavy chains from two different antibodies can be formed into heterodimers using techniques described in the art, including but not limited to knobs-into-holes (shown in Figure 3), electrostatic steering mechanisms (as shown in Figure 4) and so on.
  • the bispecific antibodies of the invention are selected from those disclosed in International Patent Publication WO2018/191188A1.
  • the bispecific antibody of the invention is selected from the group consisting of T51, T52, T53, T54, T55, T56, T57 and T58.
  • T51 to T58, T54 and T58 are in the form of heterodimeric Ig and adopt a knobs-into-holes structure, and the remaining 6 are in the form of homodimers, among which T53 and T57 In Ig-Fab format, T51, T52, T55 and T56 in Fab-Ig format.
  • the heavy chain codes and amino acid sequences corresponding to the eight bispecific antibodies T51 to T58 are shown in Tables 8 and 9.
  • the homologous light chains from T51 to T58 start with the unmodified trastuzumab light chain variable domain and are obtained by site mutation.
  • the mutation method can be: N30 is mutated to S (N30S), S56 is mutated to Y(S56Y) or T94 is mutated to W(T94W), or any combination of two or three of the above mutations, such as N30S/S56Y, N30S/T94W, S56Y/T94W, N30S/S56Y/T94W, etc.
  • the light chain code numbers corresponding to T51 to T58 are shown in Table 8, and the sequence of the light chain variable domain of the initial trastuzumab is shown in SEQ ID NO: 5.
  • the bispecific antibodies disclosed herein can be produced by recombinant methods. Methods for recombinant production are well known in the art and include protein expression in prokaryotic and eukaryotic cells, and subsequent isolation of bispecific antibodies, and typically purification to pharmaceutically acceptable purity.
  • the nucleic acid encoding the antibody sequence is inserted into an expression vector by standard methods. Expression is performed in appropriate prokaryotic or eukaryotic host cells, such as CHO cells, NSO cells, SP2/0 cells, HEK293 cells, COS cells, PER.C6 cells, yeast or E. coli cells, and the antibodies are derived from Recovered from cells (lysed cells or supernatant).
  • certain embodiments disclosed herein include methods for making bispecific antibodies comprising the steps of: a) transforming a host cell with at least one expression vector comprising a nucleic acid molecule encoding the antibody; b) in a process allowing synthesis of the antibody molecule culturing the host cells under conditions; c) recovering the antibody from the culture.
  • Antibodies are suitably isolated from the culture medium by conventional immunoglobulin purification procedures, such as protein A-sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis or affinity chromatography.
  • cell As used herein, the expressions "cell”, “cell line” and “cell culture” are used interchangeably and all such designations include progeny.
  • the terms “transformants” and “transformed cells” include primary test cells and cultures derived therefrom, regardless of the number of passages. It should also be understood that all progeny may not be identical in DNA content due to intentional or unintentional mutations. Variant progeny that are screened for the same function or biological activity in the original transformed cell are included. Where a different name is intended, it will be clear from the context as “cell”, “cell line” or “cell culture”.
  • transfection refers to the process of transforming a vector/nucleic acid into a host cell. If cells without a strong cell wall barrier are used as host cells, transfection can be carried out, for example, by calcium phosphate precipitation. However, other methods for introducing DNA into cells, such as by nuclear injection or by protoplast fusion, can also be used. If prokaryotic cells or cells containing solid cell wall structures are used, for example one method of transfection is calcium treatment with calcium chloride.
  • expression refers to the process of transcription of nucleic acid into mRNA and/or the subsequent translation of post-transcribed mRNA (also referred to as transcript) into peptides, polypeptides or proteins.
  • Transcripts and encoded polypeptides may be collectively referred to as "gene products.” If the polynucleotide includes sequences derived from genomic DNA, expression in eukaryotic cells may include splicing of mRNA.
  • a "vector” is a nucleic acid molecule, particularly an autonomously replicating nucleic acid molecule, that transfers an inserted nucleic acid molecule into and/or between host cells.
  • the term includes vectors used primarily for the insertion of DNA or RNA into cells (eg, chromosomal integration), replication vectors used primarily for DNA or RNA replication, and expression vectors for transcription and/or translation of DNA or RNA. Also included are vectors that provide more than one of the above functions.
  • an "expression vector” is a polynucleotide that, when introduced into an appropriate host cell, can be transcribed and translated into a polypeptide.
  • An “expression system” generally refers to a suitable host cell that includes an expression vector that can be used to produce the desired expression product.
  • host cell refers to any type of cellular system that can be engineered to produce the antibodies disclosed herein.
  • HEK293 cells and CHO cells are used as host cells.
  • Control sequences suitable for prokaryotes include, for example, promoters, optionally operator sequences, and ribosome binding sites.
  • Eukaryotic cells are known to utilize promoters, enhancers and polyadenylation signals.
  • a nucleic acid is "operably linked" when it is in a functional relationship with another nucleic acid sequence.
  • the DNA of a leader sequence or secreted leader is operably linked to the DNA of a polypeptide if it is expressed as a preprotein involved in the secretion of the polypeptide;
  • a promoter or enhancer is operably linked to the coding sequence if it affects the transcription of the sequence or, if the ribosome binding site is positioned to facilitate translation, it is operably linked to the coding sequence.
  • "operably linked” means that the DNA sequences being linked are contiguous, and in the case of a secretory leader, contiguous and in reading frame. However, enhancers need not be contiguous.
  • introns may be present between operably linked nucleic acid sequences. Ligation is accomplished by ligation at convenient restriction sites. If no such site exists, synthetic oligonucleotide adaptors or linkers are used according to conventional practice.
  • Antibody-encoding nucleic acid sequences can be readily obtained from the literature or by back translation with reference to the preferred codons of the intended host cell. Encoding nucleic acids may be assembled from chemically synthesized polynucleotides and/or previously cloned antibody-encoding DNA, possibly by means of site-directed mutagenesis.
  • the nucleic acid encoding the antibody can be isolated and inserted into a replicable vector for further cloning (DNA amplification) or expression.
  • Antibody-encoding DNA can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes capable of binding specifically to genes encoding the heavy and light chains of the antibody).
  • Vector components generally include, but are not limited to, one or more of the following: signal sequences, origins of replication, one or more marker genes, enhancer elements, promoters, and transcription termination sequences, such as those described in US 5,534,615, which relate to protein expression The entire disclosure of is specifically incorporated herein by reference.
  • Suitable host cells for cloning or expressing DNA in vectors herein are prokaryotic cells, yeast or higher eukaryotic cells as described above.
  • Suitable prokaryotes for this purpose include eubacteria, such as Gram-negative and Gram-positive organisms, eg, Enterobacteriaceae, such as Escherichia, eg E. coli , Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella such as S. typhimurium, Serratia such as S. marcescans and Shigella, and Bacilli such as B. subtilis and Bacillus licheniformis ( B. licheniformis), Pseudomonas such as P.
  • eubacteria such as Gram-negative and Gram-positive organisms, eg, Enterobacteriaceae, such as Escherichia, eg E. coli , Enterobacter, Erwinia, Klebsiella, Proteus
  • E. coli cloning host is E. coli 294 (ATCC 31,446), but other strains such as E. coli B, E. coli X1776 (ATCC 31,537) and E. coli W3110 (ATCC 27,325) are also suitable. These examples are illustrative, not restrictive.
  • eukaryotic microorganisms such as filamentous fungi or yeast are suitable cloning or expression hosts for antibody-encoding vectors.
  • Saccharomyces cerevisiae is a common baker's yeast and the most commonly used yeast in lower eukaryotic host microorganisms.
  • many other genera, species and strains are also common and useful herein, such as Schizosaccharomyces pombe; Kluyveromyces hosts such as, for example, K.
  • Suitable host cells for expression of glycosylated antibodies are derived from multicellular organisms, including invertebrate cells such as plant and insect cells. Numerous baculovirus strains and variants and corresponding permissive insect host cells have been identified, such as Spodoptera frugiperda (caterpillar), Aedes aegypti (mosquito), Aedes albopictus albopictus) (mosquito), Drosophila melanogaster (fruit fly) and silkworm (Bombyx mori). Various viral strains for transfection are publicly available, for example, the L-1 variant of Autographa californica NPV and the Bm-5 strain of B.
  • the virus herein according to the invention is in particular used for the transfection of Spodoptera frugiperda cells.
  • Plant cell cultures of cotton, corn, potato, soybean, petunia, tomato and tobacco may also serve as hosts.
  • vertebrate cells are of greatest interest, and propagation of vertebrate cells in culture (tissue culture) has become a routine procedure.
  • useful mammalian host cell lines are the monkey kidney CV1 line (COS-7, ATCC CRL 1651) transformed by SV40; the human embryonic kidney line (293 or subclone 293 cells for growth in suspension culture); Baby hamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovary cells/-DHFR (CHO); mouse Sertoli cells (TM4); monkey kidney cells (CV1ATCC CCL 70); African green monkey kidney cells ( VERO-76, ATCC CRL-1587); human cervical cancer cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); Buffalo rat hepatocytes (BRL 3A, ATCC CRL 1442); human Lung cells (W138, ATCC CCL 75); Human hepatoma cells (Hep G2, HB 8065); Mouse mammary tumor (MMT 060562, ATCC
  • Host cells are transformed with the above expression vectors for antibody production and cultured in a modified conventional nutrient medium suitable for inducing promoters, selecting transformants, or amplifying genes encoding the desired sequences.
  • Host cells for antibody production can be cultured in a variety of media.
  • Commercially available media such as Ham's F10, Minimum Essential Medium (MEM), RPMI-1640 and Dulbecco's Modified Eagle's Medium (DMEM) are suitable for culturing host cells.
  • antibodies can be produced intracellularly, in the periplasmic space, or secreted directly into the culture medium. If the antibody is produced intracellularly, as a first step, particulate debris, either host cells or lysed fragments, can be removed, for example, by centrifugation or ultrafiltration.
  • Antibody compositions prepared from cells can be purified using, for example, hydroxyapatite chromatography, gel electrophoresis, dialysis, and affinity chromatography, with affinity chromatography being the preferred purification technique.
  • affinity chromatography is the preferred purification technique.
  • the suitability of Protein A as an affinity ligand depends on the species and isotype of any immunoglobulin Fc domain present in the antibody. Protein A can be used to purify antibodies based on human ⁇ 1, ⁇ 2 or ⁇ 4 heavy chains. Protein G is recommended for all mouse isotypes and for human ⁇ 3.
  • the matrix to which the affinity ligand is attached is most often agarose, but other matrices are also available.
  • Mechanically stable matrices such as controlled pore glass or poly(styrenedivinyl)benzene allow for faster flow rates and shorter processing times than can be achieved with agarose.
  • Bakerbond ABX TM resin can be used for purification when the antibody includes a CH3 domain.
  • Other protein purification techniques such as fractionation on ion exchange columns, ethanol precipitation, reverse phase HPLC, silica chromatography, chromatography on heparin SEPHAROSE TM , chromatography on anion or cation exchange resins (such as polyaspartic acid) column), chromatographic focusing, SDS-PAGE and ammonium sulfate precipitation are also available, depending on the antibody to be recovered.
  • the mixture comprising the antibody of interest and contaminants can be subjected to low pH hydrophobic interaction chromatography using an elution buffer with a pH between about 2.5-4.5, preferably in low salt concentration (eg, from about 0-0.25M salt).
  • the antibody can be dissolved in an aqueous solvent including any pharmaceutically acceptable buffers, salts or other excipients. Solubilized antibodies can be refrigerated or frozen until use. Alternatively, it can be lyophilized and reconstituted shortly before use.
  • Toxins used for coupling with antibodies generally need to meet the following three conditions: (1) the mechanism of action is clear, such as blocking mitosis and causing DNA damage; (2) high activity, generally requiring EC 90 to be less than 1 nmol ⁇ L -1 ; (3) The chemical method can be used to couple and release the highly active toxin itself or its highly active derivatives in tumor cells.
  • the toxin employed in the present invention can be any toxin commonly used in the art, such as microtubule inhibitors, DNA damaging agents, and other toxin molecules.
  • the toxins of the present invention are selected from, for example, maytansines, hemiasterlins, amanitins, auristatins, californica calicheamicins or Duocarmycins, etc., and their deuterated products.
  • Maytansinoids are a class of cytotoxins that are widely used in ADCs. By blocking the polymerization of tubulin, cells are blocked in the G2/M phase of the cell cycle, thereby inhibiting the progress of cell mitosis and causing cells apoptosis.
  • the maytansine derivatives can be, for example, DM1, DM4, and their deuterated derivatives.
  • Hamitrine is a class of tripeptides modified from the original natural product Hamitrine, which is an inhibitor of tubulin polymerization and can bind to the vinblastine site of tubulin non-competitively. point.
  • Hamitrin derivatives may be, for example, E7974 (Eisai), HTI-286 (Wyeth), and their deuterated products.
  • Amanita muscarines are a class of bicyclic octapeptides extracted from poisonous mushrooms that can efficiently bind to mammalian RNA polymerase II and kill cells by terminating DNA transcription. These molecules have high hydrophilicity and high selectivity. toxicity.
  • Amanitas can be, for example, alpha-amanita, beta-amanita, gamma- amanita, epsilon-amanita, monohydroxyamanita amide or monohydroxyamanita Dideoxy variants of carboxylic acids, or mono-deoxy variants of trihydroxy amanita, trihydroxy amanita amide, gamma-trihydroxy amanita or gamma-trihydroxy amanita amide, etc., and their deuterated counterparts.
  • Auristatins are synthetic derivatives of dolastatin 10, which achieve potent mitotic inhibition by inhibiting tubulin polymerization.
  • Auristatins can be, for example, monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), auristatin EB (AEB), auristatin EVB (AEVB) and auristatin F benzene Diamines (AFP), and their deuterated derivatives.
  • MMAE monomethyl auristatin E
  • MMAF monomethyl auristatin F
  • AEB auristatin EB
  • AEVB auristatin EVB
  • AFP auristatin F benzene Diamines
  • calicheamicin There are 7 main derivatives of calicheamicin, among which calicheamicin ⁇ has the highest activity and is most used clinically.
  • the calicheamicin contains three moieties, oligosaccharide, californone, and methyltrisulfide, in which methyltrisulfide acts as an initiating device, and upon reduction, initiates the Bergman rearrangement reaction and leads to DNA cleavage.
  • Duocarmycins can specifically recognize the minor groove of DNA and effectively alkylate the adenine at the N3 position of DNA bases, and have high anticancer activity.
  • Duocarmycin toxoids can be, for example, duocarmycin A, adozelesin, CC-1065, and their deuterated counterparts.
  • Toxins used in the present invention may also be, for example, docarmycins, doxorubicins (eg morpholino-doxorubicin and cyanomorpholino-doxorubicin), dolastatin, dolestatin -10, combretastatin, calicheamicin, tubulysins, disorazole, epothilone, paclitaxel, docetaxel, SN-38, topotecan, rhizomycin, echinomycin, colchicine, Vinblastine, Vindesine, Estrogen, Cimadotine, eleutherobin, Methotrexate, Methylfolate, Dichloromethotrexate, 5-Fluorouracil, 6-Mercaptopurine, Mercaptopurine, Melphalan, Vinca rosin, leurosideine, actinomycin, daunorubicin and daunorubicin conjugates, mitomycin C, mitomycin A, carcino
  • the toxins of the present invention are selected from maytansinoids and their deuterated counterparts, eg, DM1, DM4, and their deuterated counterparts.
  • the toxins of the present invention are selected from the group consisting of Hamitrines and their deuterated products, eg, E7974, HTI-286, and their deuterated products.
  • the toxins of the present invention are selected from the group consisting of amanitas and their deuterated products, such as alpha-amotamine, beta- amanita, gamma- amanita, epsilon-amotamine etc., and their deuterated counterparts.
  • the toxins of the present invention are selected from the group of auristatins and their deuterated derivatives, including but not limited to monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), auristatin EB (AEB), Auristatin EVB (AEVB) and Auristatin F phenylenediamine (AFP), and their deuterated counterparts.
  • auristatins and their deuterated derivatives including but not limited to monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), auristatin EB (AEB), Auristatin EVB (AEVB) and Auristatin F phenylenediamine (AFP), and their deuterated counterparts.
  • MMAE monomethyl auristatin E
  • MMAF monomethyl auristatin F
  • AEB auristatin EB
  • AEVB Auristatin EVB
  • AFP Auristatin F phenylenediamine
  • the toxin of the present invention is monomethyl auristatin F (MMAF) and its deuterated products. In some embodiments, the toxin of the present invention is monomethyl auristatin E (MMAE) and deuterated products thereof.
  • MMAF monomethyl auristatin F
  • MMAE monomethyl auristatin E
  • the toxins of the present invention are selected from the class of calicheamicins and their deuterated products, eg, calicheamicin gamma and their deuterated products.
  • the toxins of the present invention are selected from the group consisting of duocarmycins and their deuterated counterparts, eg, duocarmycin A, adozelesin, CC-1065, and their deuterated counterparts.
  • the toxins of the present invention are selected from, eg, maytansinoids (eg, DM1, DM4), hamitrines (eg, E7974, HTI-286), amanitamines (eg, alpha-amanita Alkaline, ⁇ -Amantine, ⁇ -Amantine, ⁇ -Amanita), Auristatins (e.g. MMAE, MMAF, AEB, AEVB and AFP), calicheamicins (e.g. gamma), duocarmycins (eg duocarmycin A, adozelesin, CC-1065), etc., and deuterated products of these toxins.
  • maytansinoids eg, DM1, DM4
  • hamitrines eg, E7974, HTI-286
  • amanitamines eg, alpha-amanita Alkaline, ⁇ -Amantine, ⁇ -Amantine, ⁇ -Aman
  • Linkers of the present invention may be monofunctional, attaching a single toxin molecule to a single site on the antibody; or polyfunctional, attaching two/more toxin molecules to a single site on the antibody, or One toxin molecule is attached to two/more sites on the antibody.
  • the linker used as an antibody conjugate needs to meet at least the following two conditions: (1) It is stable enough in the body and will not fall off in the blood circulation to avoid toxicity due to toxin shedding; (2) Effectively release the toxin at the target site .
  • Linkers can be classified into cleavable linkers and non-cleavable linkers in which one or more hydrogen atoms are optionally deuterated.
  • the cleavable linker includes chemically cleavable linker and enzymatic cleavable linker.
  • the linker of the present invention is a cleavable linker or a deuterated product thereof.
  • Cleavable linkers are generally susceptible to cleavage under intracellular conditions, such as by lysosomal processes.
  • the linkers of the present invention are chemically cleavable linkers or deuterated products thereof.
  • the linker of the present invention is an enzymatically cleavable linker or a deuterated form thereof.
  • the enzymatically cleavable linker may, for example, be a linker comprising a peptide component comprising two or more amino acids and cleavable by intracellular proteases such as lysosomal or endosomal proteases.
  • the peptide component may comprise natural amino acid residues and/or minor amino acids and/or non-naturally occurring amino acid analogs, such as citrulline.
  • Peptide components can be designed and optimized for enzymatic cleavage by specific enzymes, eg, tumor-associated proteases, cathepsins B, C or D, or plasmin proteases.
  • the enzymatically cleavable linker may be a dipeptide-containing linker, such as a valine-citrulline (Val-Cit) or phenylalanine-lysine (Phe-Lys)-containing linker.
  • a dipeptide-containing linker such as a valine-citrulline (Val-Cit) or phenylalanine-lysine (Phe-Lys)-containing linker.
  • Suitable dipeptides for inclusion in linkers include Val-Lys, Ala-Lys, Me-Val-Cit, Phe-homoLys, Phe-Cit, Leu-Cit, Ile-Cit, Trp-Cit, Phe-Arg , Ala-Phe, Val-Ala, Met-Lys, Asn-Lys, Ile-Pro, Ile-Val, Asp-Val, His-Val, Met-(D)Lys, Asn-(D)Lys, Val-( D)Asp, NorVal-(D)Asp, Ala-(D)Asp, Me3Lys-Pro, Phenyl Gly-(D)Lys, Met-(D)Lys, Asn-(D)Lys, Pro-(D) Lys and Met-(D)Lys.
  • Cleavable linkers can also include longer peptide components, such as tripeptides, tetrapeptides, or pentapeptides. Examples include, but are not limited to, the tripeptides Met-Cit-Val, Gly-Cit-Val, (D)Phe-Phe-Lys, and (D)Ala-Phe-Lys, and the tetrapeptides Gly-Phe-Leu-Gly and Ala- Leu-Ala-Leu.
  • the linkers of the invention are selected from valine-citrulline (Val-Cit) or phenylalanine-lysine (Phe-Lys), and their deuterated counterparts.
  • the linker of the present invention is selected from valine-citrulline (Val-Cit, Vc) or its deuterated products.
  • the linker of the present invention is valine-citrulline (Val-Cit, Vc) and the toxin is MMAE, ie, the linker-toxin of the present invention is Vc-MMAE.
  • Vc-MMAE The structure of Vc-MMAE (mc-vc-PAB-MMAE, Maleimidocaproyl-valine-citrulline-p-aminobenzyloxycarbonyl monomethylauristatin E, Vedotin) is shown below.
  • mc in the present invention refers to 6-maleimidohexanoic acid, or a group formed by linking it with a bispecific antibody and/or a linker through a chemical bond.
  • PAB in the present invention refers to p-aminobenzyl alcohol, or a group formed by linking it with a linker and/or a toxin through a chemical bond.
  • PAB p-aminobenzyl alcohol
  • the antibody can be any of the above-mentioned bispecific antibodies;
  • the linker can be any of the above-mentioned linkers, such as a dipeptide-containing linker, such as a valine-citrulline-containing linker amino acid (Val-Cit) or phenylalanine-lysine (Phe-Lys) linker;
  • the toxin can be any of the toxins described above, eg DM1, DM4, MMAE, MMAF, etc.
  • the bispecific antibody conjugate has an average drug:antibody ratio (DAR) of about 1-6. In some embodiments, the average drug:antibody ratio (DAR) of the bispecific antibody conjugate is, for example, about 1, 2, 3, 4, 5, 6. In some embodiments, the DAR of the bispecific antibody conjugate is about 2, 3, 4, 5. In some embodiments, the DAR of the bispecific antibody conjugate is 3-4. In some embodiments, the DAR of the bispecific antibody conjugate is about 3,4.
  • DAR average drug:antibody ratio
  • the DAR of the bispecific antibody conjugate is about 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, etc.
  • the middle part is the linker valine-citrulline (Val-Cit, Vc) part
  • the rightmost Drug represents the toxin part.
  • the free sulfhydryl group on the reduced antibody can interact with a maleamide group (eg, 6-maleimidohexanoic acid (mc)) to achieve the linkage between the antibody and the linker (Val-Cit, Vc).
  • a maleamide group eg, 6-maleimidohexanoic acid (mc)
  • the linker is attached to the rightmost Drug (toxin moiety) via p-aminobenzyl alcohol.
  • Bispecific antibodies of formula (I) are bispecific antibodies as described above.
  • the DAR value (n) is further specified in the structure of the bispecific antibody conjugate, as shown in the following formula (I'):
  • n represents the average drug:antibody ratio (DAR)
  • DAR drug:antibody ratio
  • the structure of the bispecific antibody conjugate can be shown in the following formula (II).
  • n ie the average drug:antibody ratio DAR value
  • Vc-MMAE linker-toxin moiety
  • n ie the average drug:antibody ratio DAR value
  • n can be, for example, about 1-6, such as 1, 2, 3, 4, 5, 6, such as 3-4, such as 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, etc.
  • the bispecific antibody conjugate has an average drug:antibody ratio (DAR) of 3-4, eg, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.65, 3.7, 3.75, 3.8 , 3.9, 4.
  • DAR drug:antibody ratio
  • the free sulfhydryl group on the reduced antibody can interact with a maleamide group (eg, 6-maleimidohexanoic acid (mc)) to achieve the linkage between the antibody and the linker (Val-Cit, Vc).
  • a maleamide group eg, 6-maleimidohexanoic acid (mc)
  • the linker is attached to the rightmost toxin monomethyl auristatin E (MMAE) via p-aminobenzyl alcohol.
  • MMAE monomethyl auristatin E
  • Bispecific antibodies of formula (II) are bispecific antibodies as described above.
  • the bispecific antibody conjugate of the invention may be T51-MMAE, T52-MMAE, T53-MMAE, T54-MMAE, T55-MMAE, T56-MMAE, T57-MMAE, T58-MMAE.
  • Bispecific antibodies T51, T52, T53, T54, T55, T56, T57 and T58 in these bispecific antibody conjugates may be defucosylated (ie T51-AF, T52-AF, T53-AF, T54-AF, T55-AF, T56-AF, T57-AF and T58-AF), the corresponding bispecific antibody conjugates are T51-AF-MMAE, T52-AF-MMAE, T53-AF-MMAE, T54- AF-MMAE, T55-AF-MMAE, T56-AF-MMAE, T57-AF-MMAE and T58-AF-MMAE.
  • T54-MMAE in the present invention refers to a bispecific antibody conjugate with a specific structure of T54-(mc-vc-PAB-MMAE)n, where n is the average drug:antibody ratio (DAR); T54, mc , vc, PAB and MMAE are as defined above.
  • T54-AF-MMAE in the present invention refers to a bispecific antibody conjugate with a specific structure of T54-AF-(mc-vc-PAB-MMAE)n, where n is the average drug:antibody ratio (DAR) ; T54-AF, mc, vc, PAB and MMAE are as defined above.
  • T51-MMAE T52-MMAE, T53-MMAE, T55-MMAE, T56-MMAE, T57-MMAE, T58-MMAE, make a similar understanding to the definition of "T54-MMAE".
  • T51-AF-MMAE T52-AF-MMAE, T53-AF-MMAE, T55-AF-MMAE, T56-AF-MMAE, T57-AF-MMAE, T58-AF-MMAE, match "T54-AF-MMAE”
  • T54-AF-MMAE The definition of MMAE" is similarly understood.
  • ADCs of the present disclosure can be prepared by one of several routes known in the art using organic chemical reactions, conditions and reagents known to those skilled in the art (see, e.g., Bioconjugate Techniques (G.T. Hermanson, 2013, Academic Press, and examples provided herein).
  • conjugation can be accomplished by (1) reacting a nucleophilic or electrophilic group of an antibody with a bifunctional linker to form an antibody-linker intermediate via a covalent bond , and then react with the activated toxin (such as DM1, DM4, MMAE, MMAF, etc.); or (2) the nucleophilic group or electrophilic group of the toxin reacts with the linker to form a linker-toxin via a covalent bond, and then Reacts with nucleophilic or electrophilic groups of antibodies.
  • the activated toxin such as DM1, DM4, MMAE, MMAF, etc.
  • toxins can be coupled to various groups on the antibody via appropriate linkers to provide ADCs.
  • conjugation can be performed via antibody surface lysines, or via oxidized carbohydrates or via cysteine residues that have been released by reduction of one or more interchain disulfide bonds.
  • antibodies can be modified to include other cysteine residues or unnatural amino acids that provide reactive handles, such as selenomethionine, p-acetylphenylalanine, formylglycine, or p-azidomethyl -L-Phenylalanine.
  • cysteine residues or unnatural amino acids that provide reactive handles, such as selenomethionine, p-acetylphenylalanine, formylglycine, or p-azidomethyl -L-Phenylalanine.
  • modifications are well known in the art (see, eg, US Pat. Nos.
  • the ADCs of the present disclosure comprise an auristatin conjugated via an appropriate linker to a cysteine residue on the bispecific antibody that has been released by reduction of one or more interchain disulfide bonds Toxins (eg MMAE, MMAF).
  • an auristatin conjugated via an appropriate linker to a cysteine residue on the bispecific antibody that has been released by reduction of one or more interchain disulfide bonds Toxins eg MMAE, MMAF.
  • Suitable reducing agents include, for example, dithiothreitol (DTT), tris(2-carboxyethyl)phosphine (TCEP), 2-mercaptoethanol, cysteamine, and many water-soluble phosphines.
  • DTT dithiothreitol
  • TCEP tris(2-carboxyethyl)phosphine
  • 2-mercaptoethanol 2-mercaptoethanol
  • cysteamine and many water-soluble phosphines.
  • the average DAR of an ADC can be determined by standard techniques such as UV/VIS spectroscopic analysis, ELISA-based techniques, chromatographic techniques such as hydrophobic interaction chromatography (HIC), UV-MALDI mass spectrometry (MS) and MALDI-TOF MS . Additionally, the distribution of drug-linked forms (eg, percentages of DAR0, DAR1, DAR2, etc.
  • MS with or without accompanying chromatographic separation steps
  • Hydrophobic interaction chromatography with or without accompanying chromatographic separation steps
  • HPLC reversed-phase HPLC
  • IEF isoelectric focusing gel electrophoresis
  • the average DAR of the ADC is determined by hydrophobic interaction chromatography (HIC) techniques.
  • HIC hydrophobic interaction chromatography
  • the ADC can be purified and separated from unconjugated reactants and/or any conjugate aggregates by purification methods known in the art. Such methods include, but are not limited to, size exclusion chromatography (SEC), hydrophobic interaction chromatography (HIC), ion exchange chromatography, chromatographic focusing, ultrafiltration, centrifugal ultrafiltration, and combinations thereof.
  • SEC size exclusion chromatography
  • HIC hydrophobic interaction chromatography
  • ion exchange chromatography chromatographic focusing
  • ultrafiltration centrifugal ultrafiltration, and combinations thereof.
  • the present invention also relates to pharmaceutical compositions comprising the bispecific antibody conjugates described above.
  • treating includes: (1) preventing or delaying the development of a subject who may have or are prone to have the state, disorder or disorder but has not yet experienced or exhibits clinical or subclinical symptoms of the state, disorder or disorder The occurrence of at least one clinical or subclinical symptom of the state, disorder or disorder; or (2) inhibiting the state, disorder or disorder, i.e. preventing, reducing or delaying the progression of the disease or its recurrence (in the case of maintenance therapy) or at least one clinical or subclinical symptom thereof; or (3) alleviating disease, ie, causing a reduction in the state, disorder or condition or at least one clinical or subclinical symptom thereof.
  • the benefit achieved by the treated subject is statistically significant, or at least perceptible to the patient or physician.
  • the term "therapeutically effective” or “effective” as applied to a dose or amount means, when administered to a subject for the treatment (eg, prevention or amelioration) of a condition, disorder or condition, sufficient to effect such treatment or prevention The amount of a compound or pharmaceutical composition that produces an effective result.
  • a “therapeutically effective amount” will vary depending on the compound or bacteria or analog being administered, as well as the disease and its severity, and the age, weight, physical condition and responsiveness of the mammal being treated.
  • compositions of the present disclosure means that the molecular entities and other components of such compositions are physiologically tolerable, and when administered to mammals (eg, humans) ) usually does not produce adverse reactions.
  • pharmaceutical formulation or “pharmaceutical composition” refers to a form that allows for biological activity of the active ingredients contained therein to be effective and does not contain additional components that would be unacceptably toxic to the subject to which the formulation is to be administered. formulation or composition.
  • carrier refers to a diluent, adjuvant, excipient, or vehicle for administering a compound.
  • Such pharmaceutical carriers can be sterile liquids such as water and oils, including those of petroleum, animal, vegetable or synthetic materials, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Particularly for injectable solutions, aqueous or aqueous saline solutions and aqueous dextrose and glycerol solutions are employed as carriers.
  • the carrier can be a solid dosage form carrier including, but not limited to, one or more binders (for compressed pellets), glidants, encapsulating agents, flavoring agents, and coloring agents. Suitable pharmaceutical carriers are described in Remington's Pharmaceutical Sciences by E.W. Martin.
  • a bispecific antibody conjugate according to the present disclosure or a pharmaceutical composition comprising the same for the prevention and/or treatment of cancer.
  • a bispecific antibody conjugate in the manufacture of a medicament for the prevention and/or treatment of cancer.
  • the use is for the treatment of cancer.
  • Other similar embodiments are methods of preventing and/or treating cancer comprising administering to a patient in need thereof a therapeutically effective amount of a bispecific antibody conjugate.
  • the subject is a mammal. In some embodiments, the subject is a human.
  • Cancers described herein are, for example, HER2-expressing cancers, such as cancers that express low levels of HER2, such as, but not limited to, HER2 + breast cancer, ovarian cancer, endometrial cancer, cervical cancer, gastric cancer, esophageal cancer, lung cancer, head and neck cancer cancer, bladder cancer, bile duct cancer and/or colorectal cancer.
  • HER2 + breast cancer, ovarian cancer, endometrial cancer, cervical cancer, gastric cancer, esophageal cancer, lung cancer, head and neck cancer cancer, bladder cancer, bile duct cancer and/or colorectal cancer such as, but not limited to, HER2 + breast cancer, ovarian cancer, endometrial cancer, cervical cancer, gastric cancer, esophageal cancer, lung cancer, head and neck cancer cancer, bladder cancer, bile duct cancer and/or colorectal cancer.
  • the bispecific antibody conjugates of the present invention can be administered alone or in combination with other agents suitable for the treatment of cancer.
  • the bispecific antibody conjugate can be used in combination with other therapies, such as for chemotherapy or targeted therapy, or for immunotherapy.
  • they can be used in conjunction with radiation therapy or surgery.
  • Bispecific antibody conjugates can be delivered by any suitable route.
  • the conjugate is delivered by injection or infusion, by a skin patch, by a depot/pump device, or by inhalation.
  • the conjugate is administered intravenously, intraperitoneally, subcutaneously, or intramuscularly.
  • the conjugate of some embodiments may be administered once a day or less frequently. For example, in some embodiments, once every two days, once every three days, once every four days, once every five days, once every six days, once a week, once every two weeks, once every three weeks, once every four weeks , monthly, every two months, every three months, or every six months to administer the bispecific antibody conjugate.
  • the optimal dosage level of the effective molecule will depend on a variety of factors, including the patient's age, weight, medical condition, possible combinations with other drugs, and the severity of the condition.
  • the specific dosage can be determined by those skilled in the art according to the specific situation.
  • kits, unit dosage forms, and articles of manufacture comprising the conjugates described herein or compositions thereof.
  • Kits, unit dosage forms, and articles of manufacture can include, for example, vials (such as sealed vials) prefilled syringes and autoinjectors (pen type) of the conjugates described herein.
  • Amino acids are referred to herein by their common 3-letter symbols or 1-letter (single-letter) symbols for amino acids according to the IUPAC-IUB Biochemical Nomenclature Committee.
  • CHO-K1 cells were co-transfected with bispecific antibody-containing heavy chain plasmid and light chain plasmid.
  • Cells were resuspended in fresh opti medium (without glutamine, obtained commercially) and seeded in 96-well plates, cultured and recovered in a 37°C, 5% CO 2 incubator for 24 h. After 24 hours of recovery, resuspend in fresh opti medium of 25 ⁇ M MSX, replace with fresh medium every 3 days, and continue to culture under 25 ⁇ M MSX pressure for 2-4 weeks, and pick out the surviving mixed cell clones.
  • the mixed cells were seeded in a 96-well plate with fresh opti medium at a density of 0.5 cells/well by limiting dilution method, and cultured at 37°C in a 5% CO 2 incubator for about 2 weeks.
  • the clones capable of expressing antibodies were detected by ELISA. After two rounds of limiting dilution screening, a monoclonal capable of expressing antibody T54 was finally obtained.
  • CHO-K1 cells were subjected to TALEN knockout FUT8 gene and LCA pressurization to construct fucose knockout cell line D57, using D57 cell line, similar to Example 1 to prepare T54 method to prepare the defucosylated bispecific antibody T54-AF.
  • bispecific antibodies T54 and T54-AF After culturing the stable line of bispecific antibodies for several days, the supernatant was purified by protein A affinity and cation exchange to obtain bispecific antibodies T54 and T54-AF.
  • CHO-K1 cells were co-transfected with the corresponding bispecific antibody heavy chain plasmid and light chain plasmid, respectively, to prepare antibodies T51, T51-AF, T52, T52-AF, T53, T53 -AF, T55, T55-AF, T56, T56-AF, T57, T57-AF, T58, T58-AF.
  • the bispecific antibody T54 obtained in Example 1 was dialyzed into a coupling buffer (25mM Na 2 B 4 O 7 /25mM NaCl, 1mM DTPA, pH 7.4), concentrated to a concentration of 5mg/ml or more by ultrafiltration, and adjusted to a concentration of 5mg/ml. ml.
  • the reducing agent TCEP was added, the water bath was 25°C, and the reaction was carried out for 2h.
  • mc block refers to 6-maleimidohexanoic acid, and the structural formula of 6-maleimidohexanoic acid is:
  • PAB refers to p-aminobenzyl alcohol, and the structural formula of p-aminobenzyl alcohol (PAB) is:
  • Vc-MMAE mc-vc-pab-MMAE
  • mc-vc-pab-MMAE was dissolved in DMSO to 10 mM.
  • the mc-vc-pab-MMAE was mixed with the antibody in a molar ratio of 10:1, the water bath was 25°C, and the reaction was carried out for 2h for coupling.
  • the conjugated product was dialyzed into dialysis buffer (20 mM His-acetate, pH 5.5) and filtered through a 0.22 ⁇ m membrane to obtain the bispecific antibody conjugate T54-MMAE.
  • Mobile phase A 50 mM Na3PO4, 1.5M ( NH4 ) 2SO4 , 5 % isopropanol, pH 6.95
  • Mobile phase B 50 mM Na 3 PO 4 , 5% isopropanol, pH 6.95
  • the antibody T54 and its conjugate T54-MMAE were subjected to SEC purity analysis respectively, and the results were shown in Figure 5A and Figure 6A, respectively. It can be seen from the figure that the polymer contents of T54 and T54-MMAE are 1.95% and 0.24%, respectively, and the monomer contents are 97.17% and 96.75%, respectively. The monomer contents of the two are not much different.
  • the antibody T54 and its conjugate T54-MMAE were analyzed by HIC-HPLC, and the results were shown in Figure 7 and Figure 8A, respectively. It can be seen from the figure that the antibody conjugate T54-MMAE is conjugated with different numbers of toxins, of which the ratio of 4 toxins is 73.31%, and the average DAR value is 3.70. The specific data are shown in Table 1-1.
  • T54-AF and its conjugate T54-AF-MMAE were subjected to SEC purity analysis, respectively, and the results were shown in Figure 5B and Figure 6B, respectively. It can be seen from the figure that the polymer contents of T54-AF and T54-AF-MMAE are 0.72% and 0.17%, respectively, and the monomer contents are 99.28% and 99.83%, respectively. The monomer contents of the two are not much different.
  • the antibody conjugate T54-AF-MMAE was subjected to HIC-HPLC to analyze the number of conjugated toxins, and the results are shown in Figure 8B. It can be seen from the figure that the antibody conjugate T54-AF-MMAE is conjugated with different numbers of toxins, of which the ratio of 4 toxins is 66.61%, and the average DAR value is 3.46. The specific data are shown in Table 1-2. Show.
  • Positive control ADC DS8201 (Trastuzumab Deruxtecan, an antibody-drug conjugate targeting Her2, derived from humanized anti-Her2 antibody trastuzumab via a tetrapeptide (GGFG) linker coupled to the topoisomerase-I inhibitor camptothecin (DX-8951 derivative DXd) was obtained).
  • GGFG tetrapeptide
  • the binding activity of antibodies, antibody conjugates and antigen HER2 was detected by ELISA. Coated with antigen HER2 (2 ⁇ g/ml), 100 ⁇ l per well, overnight at 4°C. Add 300 ⁇ l of PBS and wash 4 times repeatedly, add 300 ⁇ l of 3% BSA in PBS, block at 37° C. for 2 h, and repeat washing 4 times with PBS. Add 100 ⁇ l of the antibody to be detected, and incubate at 37°C for 1 h. Repeat washing 4 times with PBS, add 100 ⁇ l of the diluted secondary antibody, incubate at 37°C for 1 h, discard the secondary antibody, and repeat washing 4 times with PBS. Add 100 ⁇ l of TMB chromogenic solution and incubate at 37°C for 5-10 min. The reaction was terminated by adding 50 ⁇ l of dilute sulfuric acid stop solution. OD 450 was detected by a microplate reader.
  • Table 2 EC 50 values of antibody/antibody conjugate binding to HER2 antigen
  • HER2 expression-positive tumor cells (SKBR3, N87) in logarithmic growth phase were washed with PBS, digested with trypsin, neutralized with 10% FBS medium (DMEM, RPMI1640) and trypsinized, centrifuged, and washed with the corresponding 10%
  • the target cells were resuspended in FBS medium (DMEM, RPMI1640), the cell density was adjusted to 2E4, added to a 96-well plate, and 100 ⁇ l was added to each well. After culturing for 24 hours, different concentrations of drugs diluted with the medium were added. , 100 ⁇ l per well (initial concentration 300 ⁇ g/ml, 8-fold dilution, 8 concentrations). After 72 h, the cell supernatant was aspirated, 100 ⁇ l of medium supplemented with cck8 was added, and incubated for 4 h, and the OD 450 was detected.
  • the data were analyzed with the software GraphPad 5.0.
  • the inhibition curve of each antibody and antibody conjugate on breast cancer cell SKBR3 is shown in Figure 10, and the IC 50 value is shown in Table 3.
  • the results showed that T54, T54-MMAE, TDM1, and Herceptin all inhibited the growth of SKBR3 tumor cells, while the antibody conjugates TDM1 and T54-MMAE had more obvious inhibition on tumor cells, and the maximum inhibition rate could be as high as 80%.
  • the effect is second, the maximum inhibition rate can reach 52.51%, Herceptin is the weakest, and the maximum inhibition rate is only 33.54%.
  • the data were analyzed with the software GraphPad 5.0.
  • the inhibition curve of each antibody and antibody conjugate on gastric cancer cell N87 is shown in Figure 11, and the IC 50 value is shown in Table 4.
  • the results show that T54, T54-MMAE and TDM1 can inhibit the growth of N87 tumor cells, while the antibody conjugates TDM1 and T54-MMAE have more obvious inhibition on tumor cells, and the maximum inhibition rate can be as high as 70%, followed by T54. , the maximum inhibition rate can reach 55.6%.
  • Herceptin had no obvious inhibitory effect on N87.
  • the tumor cells in the logarithmic growth phase were washed with PBS, digested with trypsin, neutralized with the corresponding medium (DMEM, RPMI1640, MEM, etc.) containing 10% FBS, after the trypsin was neutralized, centrifuged, and the corresponding 10% FBS medium was used.
  • DMEM, RPMI1640, MEM, etc. resuspend the target cells, adjust the cell density to 4x10 4 /ml, add to a 96-well plate, add 100 microliters to each well, and after culturing for 24 hours, add different cells diluted with culture medium.
  • T54-AF-MMAE proliferation inhibitory activity of T54-AF-MMAE, DS8201, T54-AF, herceptin, perjeta, and Isotype (isotype control, as a negative antibody) on tumor cells with high, medium and low expression levels of HER2 was detected respectively, and the data were analyzed. The results are shown in Figure 12.
  • the growth inhibitory effect of T54-AF-MMAE on HER2-positive tumor cells was basically better than that of DS8201 and T54-AF, herceptin and perjeta mAbs.
  • BT474 tumor cells were purchased from ATCC under the designation HTB-20 TM .
  • Tumor cells were cultured with inactivated 10% fetal bovine serum, 1 mM Napyr, 1X MEM-NEAA, 1X MEM VITAMIN, 10 ⁇ g/mL human insulin, and RPMI 1640 medium in an incubator at 37 °C, 5% CO , each After 3 to 4 days, the cells were subcultured, and the tumor cells in the logarithmic growth phase were used for inoculation of tumors in vivo.
  • BT474 tumor cells were resuspended with PBS+Matrigel (1:1) at a concentration of 1 x 10 8 /mL, and inoculated into the right flank of experimental animals subcutaneously, 100 ⁇ L / animal, when the tumor grew to an average volume of about 111 mm 3
  • Group administration a total of 3 groups, 5 animals in each group, the specific dosing schedule is shown in Table 5-1.
  • Dosing frequency 1 5 solvent control — i.p. qw x 2 2 5 TDM1 2 i.p. qw x 2 3 5 T54-MMAE 2 i.p. qw x 2
  • qw x 2 means: once a week for a total of 2 weeks
  • volume 0.5 ⁇ long diameter ⁇ short diameter 2 , twice a week.
  • volume 0.5 ⁇ long diameter ⁇ short diameter 2 , twice a week.
  • T/C value was calculated according to the tumor volume, where T was the mean relative tumor volume (RTV) of each test substance-treated group, and C was the mean relative tumor volume (RTV) of the solvent control group.
  • RTV is the ratio of tumor volume after administration to that before administration.
  • Tumor growth inhibition rate (%) (1-T/C) ⁇ 100%, tumor growth inhibition rate ⁇ 60%, and statistical processing p ⁇ 0.05 is effective.
  • mice in each group were euthanized, the tumor tissue was removed, and the tumor tissue was weighed and placed neatly for pictures.
  • mice in each group are shown in Figure 13 and Table 6-1.
  • Both TDM1 (2mg/kg) and T54-MMAE (2mg/kg) showed strong antitumor effects in human xenograft breast cancer model BT474, effectively inhibiting tumor growth.
  • the tumor suppressor effect of T54-MMAE was better than that of TDM1.
  • the tumor-bearing mice did not lose weight, and showed good tolerance to the test substances.
  • BT474 cells were cultured in RPMI-1640 medium containing inactivated 10% fetal bovine serum, 100 U/mL of penicillin and 100 ⁇ g/mL of streptomycin, and 2 mM glutamine in an incubator at 37 °C, 5% CO , after the cells were fully grown every 3 to 4 days, the cells were subcultured, and the tumor cells in the logarithmic growth phase were used for inoculation of tumors in vivo.
  • Tumor cells were washed twice with PBS, then resuspended with PBS:Matrigel (mixed at a volume ratio of 1:1), adjusted to a cell concentration of 1 ⁇ 10 8 /mL, and inoculated subcutaneously in the right anterior flank of experimental animals, 100 ⁇ L/small Rat, i.e. 1 ⁇ 10 7 /mouse.
  • the experimental animals were subcutaneously injected with estradiol benzoate injection (2 mL: 4 mg, Ningbo No. 2 Hormone Factory), 40 ⁇ g per animal, once a week from the day before the inoculation.
  • estradiol benzoate injection (2 mL: 4 mg, Ningbo No. 2 Hormone Factory
  • qw x 4 means: once a week for a total of 4 weeks
  • volume 0.5 ⁇ long diameter ⁇ short diameter 2 , twice a week.
  • volume 0.5 ⁇ long diameter ⁇ short diameter 2 , twice a week.
  • T/C value was calculated according to the tumor volume, where T was the mean relative tumor volume (RTV) of each test substance-treated group, and C was the mean relative tumor volume (RTV) of the solvent control group.
  • RTV is the ratio of tumor volume after administration to that before administration.
  • Tumor growth inhibition rate (%) (1-T/C) ⁇ 100%, tumor growth inhibition rate ⁇ 60%, and statistical processing p ⁇ 0.05 is effective.
  • mice in each group were euthanized, the tumor tissue was removed, and the tumor tissue was weighed and placed neatly for pictures.
  • T54-AF-MMAE 4mpk started to show a significant tumor inhibitory effect at D26; T54-AF-MMAE1mpk started to show a significant tumor inhibitory effect at D33 (7 days after the second administration). ;
  • T54-AF-MMAE high (4mpk) showed a significant improvement in D26 (7 days after the first dose) and D33 (7 days after the second dose). effectiveness.
  • the tumor-bearing mice showed good tolerance to the high-dose, medium-dose and low-dose dual-antibody-ADC test drugs.
  • Tumor cells were cultured in RPMI-1640 medium containing inactivated 10% fetal bovine serum, 100 U/mL of penicillin and 100 ⁇ g/mL of streptomycin, and 2 mM glutamine in an incubator at 37 °C, 5% CO , after the cells were fully grown every 3 to 4 days, the cells were subcultured, and the tumor cells in the logarithmic growth phase were used for inoculation of tumors in vivo.
  • the tumor cells were washed twice with PBS, adjusted to a cell concentration of 1 ⁇ 10 8 /mL, and inoculated into the right anterior flank of experimental animals subcutaneously, 100 ⁇ L/mouse, ie, 1 ⁇ 10 7 /mouse.
  • the tumor grows to an average tumor volume of 80-120 mm 3
  • the patients are administered into groups according to the tumor volume. There are 8 groups in total, with 8 animals in each group.
  • the specific dosing schedule is shown in Table 7-1 below.
  • Dosing volume is 10 ⁇ L/g according to animal body weight; i.p. is intraperitoneal injection; qw x 4 means: once a week for a total of 4 weeks.
  • volume 0.5 ⁇ long diameter ⁇ short diameter 2 , twice a week.
  • volume 0.5 ⁇ long diameter ⁇ short diameter 2 , twice a week.
  • T/C value was calculated according to the tumor volume, where T was the mean relative tumor volume (RTV) of each test substance-treated group, and C was the mean relative tumor volume (RTV) of the solvent control group.
  • RTV is the ratio of tumor volume after administration to that before administration.
  • Tumor growth inhibition rate (%) (1-T/C) ⁇ 100%, tumor growth inhibition rate ⁇ 60%, and statistical processing p ⁇ 0.05 is effective.
  • mice in each group were euthanized, the tumor tissue was removed, and the tumor tissue was weighed and placed neatly for pictures.
  • DS8201 1mpk, T54-AF-MMAE 0.25mpk, 1mpk and 4mpk can significantly inhibit the growth of NCI-N87 tumor.
  • the tumor-bearing mice showed good tolerance to the high-dose, medium-dose and low-dose dual-antibody-ADC test drugs.
  • diabodies T51 to T58 and their corresponding defucosylated diabodies T51-AF to T58-AF contain the M428L substitution in the Fc region to increase the serum half-life of the diabodies .
  • VH-Pert Pertuzumab heavy chain variable region
  • VH-Tra trastuzumab heavy chain variable region
  • Tra-LC Trastuzumab light chain.
  • **The signal sequence is used to aid in the secretion of the antibody from the host cell and subsequent cleavage by the enzyme.

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Abstract

提供一种双特异抗体偶联物，其包括双特异抗体、毒素和连接子；以及包含该双特异抗体偶联物的药物组合物、以及该双特异抗体偶联物及其药物组合物在制备用于预防和/或治疗癌症的药物中的应用。所述双特异抗体包含能够识别和/或结合HER2的结构域II的第一互补位，和能够识别和/或结合HER2的结构域IV的第二互补位；所述毒素选自美登素类、哈米特林类、鹅膏蕈碱类、澳瑞他汀类、刺孢霉素类或倍癌霉素类；所述连接子选自可裂解连接子和不可裂解连接子；可选的，所述毒素和/或连接子中的一个或多个氢原子任选地被氘代。

Description

一种双特异抗体偶联物 技术领域
本发明属于抗体药物技术领域，具体的，涉及一种双特异抗体偶联物；以及包含该双特异抗体偶联物的药物组合物、以及该双特异抗体偶联物及其药物组合物在制备用于预防和/或治疗癌症的药物中的应用。
背景技术
人表皮生长因子受体2(HER2，又称ErbB2)是酪氨酸蛋白激酶受体ErbB家族的成员。它是I型膜蛋白，具有单通道跨膜结构域、胞外结构域和胞质激酶结构域。HER2基因在大约20％的乳腺癌患者中被扩增到(HER2 +)。已证明用单克隆抗体靶向HER2在治疗HER2扩增性乳腺癌患者中是非常有效的。
曲妥珠单抗(mAb 4D5，赫赛汀
Figure PCTCN2022089229-appb-000001
)是一种人源化抗HER2单克隆抗体，与HER2ECD的结构域IV结合，已在1998年获FDA批准用于治疗HER2 +乳腺癌。
帕妥珠单抗(rhuMab 2C4，
Figure PCTCN2022089229-appb-000002
)是另一种人源化抗HER2单克隆抗体，但与HER2ECD的结构域II结合，结构域II是与HER2上与曲妥珠单抗的表位分开的表位。由于HER2ECD的结构域II参与二聚作用，帕妥珠单抗与HER2的结合阻止了HER2与另一受体(诸如EGFR、HER3或HER4)二聚化。
帕妥珠单抗与曲妥珠单抗的组合显示出相比于单独的曲妥珠单抗或帕妥珠单抗更卓越的功效，并已被FDA批准用于HER2+转移性乳腺癌(2012)，且一年后用于HER2+乳腺癌新辅助治疗(2013)。
国际专利公开文本WO2018191188A1公开了一种含有共同轻链的双特异抗体，其同时靶向HER2ECD的结构域II和HER2ECD的结构域IV。
抗体编辑(Mab Edit)是指对抗体定向精准的修饰与改造，包括抗体糖链中糖的敲除、取代与增加，抗体payload的定点或非定点偶联，抗体Fab与Fc工程精准改进等方式。抗体编辑强调精准修饰与改造，充分利用和调节抗体各个功能区和不同模块。对天然或工程化抗体分子进行抗体编辑，可以构建如糖工程抗体、ADCs等分子结构多样的多功能新型抗体。
抗体偶联物(antibody-drug conjugates，ADCs)是将具有靶向性质的抗体和强细胞毒性的药物偶联而成的一种新型抗肿瘤药物，俗称“生物导弹”。ADCs通过抗体的靶向作用，可特异地结合到抗原阳性的肿瘤细胞膜，发挥药理作用；也可通过肿瘤细胞内吞作用形成内吞体进入细胞内，进入胞浆后的内吞体与溶酶体结合，使偶联的小分子毒素解离并恢复其固有性质，从而杀灭肿瘤细胞。
ADCs可以看作一种前药，可靶向性地将小分子毒素导入肿瘤细胞，增加了小分子毒素在肿瘤细胞中的暴露，减少了对正常组织的暴露，扩大了此类小分子毒素的安全窗(治疗窗)。
ADCs主要由三部分组成，即靶向性的抗体、偶联的毒素、抗体与毒素之间的连接子(linker)，其作用的靶点通常为肿瘤细胞表面的相关抗原或特定受体。ADC中的mAb须具有以下特点：①偶联小分子毒素后还能维持自身特性；②对抗原的靶向性好；③只与目标靶细胞的抗原结合；④与抗原结合后不对mAb产生负反馈；⑤与其它细胞的非特异性结合很少。
曲妥珠单抗-emtansine偶联物(TDM1)是将小分子毒素DM1和曲妥珠单抗连接起来得到的一种抗体偶联物。2013年2月，TDM1(Kadcyla)被FDA批准用于治疗HER2阳性晚期转移性乳腺癌。2019年，TDM1成为第一个进入中国的ADC药物。
双特异抗体偶联物(ADC)能够同时靶向两个不同的靶点、或者同一靶点的两个不同结构域，其治疗效果值得期待。然而，对于采用双特异抗体的ADC仍有很大的未被满足的临床需求。
发明内容
本发明提供了一种双特异抗体偶联物(ADC)、其药学上可接受的盐、溶剂化物或其氘代物，其包括双特异抗体、毒素和连接子；
所述双特异抗体包含能够识别和/或结合HER2的结构域II的第一互补位，和能够识别和/或结合HER2的结构域IV的第二互补位；
所述毒素选自美登素类、哈米特林类、鹅膏蕈碱类、澳瑞他汀类、刺孢霉素类或倍癌霉素类；
所述连接子选自可裂解连接子和不可裂解连接子；
可选的，所述的毒素和/或连接子中的一个或多个氢原子任选地被氘代。
优选的，所述氘代物指所述双特异抗体偶联物(ADC)中的毒素和/或连接子中的一个或多个氢原子任选地被氘代。
在一些实施方式中，所述双特异抗体包含能够识别和/或结合HER2的结构域II的第一互补位，和能够识别和/或结合HER2的结构域IV的第二互补位。
在一些实施方式中，所述双特异抗体包括一对同源的轻链和一对包含两个不同的重链可变结构域的异源重链。
所述一对同源的轻链包括修改后的曲妥珠单抗轻链可变结构域；
所述修改后的曲妥珠单抗轻链可变结构域以未修改的曲妥珠单抗轻链可变结构域为基础，进行序列修改，所述的序列修改包含：
a)S56Y/T/A中的任意一种；
b)N30S/A中的任意一种；
c)T94W/F/Y中的任意一种；
d)H91Y/F/W中的任意一种；
e)P96Y/F/W中的任意一种；或
f)a)至e)的这五类修改方式的任意组合；
在一些实施方式中，所述修改后的曲妥珠单抗轻链可变结构域以未修改的曲妥珠单抗轻链可变结构域为基础，进行如下序列修改：N30S、S56Y和T94W这三种修改中的任意一种、两种或三种；
所述未修改的曲妥珠单抗轻链可变结构域的序列如SEQ ID NO:5所示。
所述的两个不同的重链可变结构域中，
(1)一个重链可变结构域选自未修改的帕妥珠单抗重链可变结构域或修改后的帕妥珠单抗重链可变结构域，所述未修改的帕妥珠单抗重链可变结构域的序列如SEQ ID NO:3所示；
所述修改后的帕妥珠单抗重链可变结构域以未修改的帕妥珠单抗重链可变结构域为基础，进行序列修改，所述的序列修改包含：
i)T30A/S/N/D中的任意一种；
ii)G56A/S/T中的任意一种；或
iii)i)和ii)这两类修改方式的任意组合；
在一些实施方案中，所述修改后的帕妥珠单抗重链可变结构域以未修改的帕妥珠单抗重链可变结构域为基础，进行序列修改，所述的序列修改包含：T30A和/或G56A；
(2)另一个重链可变结构域选自未修改的曲妥珠单抗重链可变结构域或修改后的曲妥珠单抗重链可变结构域；所述未修改的曲妥珠单抗重链可变结构域的序列如SEQ ID NO:7所示；
所述修改后的曲妥珠单抗重链可变结构域以未修改的曲妥珠单抗重链可变结构域为基础，进行序列修改，所述的序列修改包含：
i)N54T/S/A中的任意一种；
ii)D98S/W/T/R中的任意一种；或
iii)i)和ii)这两类修改方式的任意组合；
在一些实施方式中，所述修改后的曲妥珠单抗重链可变结构域以未修改的曲妥珠单抗重链可变结构域为基础，进行序列修改，所述的序列修改包含：N54T和/或D98S。
在一些实施方式中，所述双特异抗体包括一对同源的轻链和一对包含两个不同的重链可变结构域的异源重链；
所述一对同源的轻链包括修改后的曲妥珠单抗轻链可变结构域；
所述修改后的曲妥珠单抗轻链可变结构域以未修改的曲妥珠单抗轻链可变结构域为基础，进行如下序列修改：N30S、S56Y和T94W这三种修改中的任意一种、两种或三种；
所述未修改的曲妥珠单抗轻链可变结构域的序列如SEQ ID NO:5所示；
所述的两个不同的重链可变结构域中，
(1)一个重链可变结构域选自未修改的帕妥珠单抗重链可变结构域或修改后的帕妥珠单抗重链可 变结构域，所述未修改的帕妥珠单抗重链可变结构域的序列如SEQ ID NO:3所示；
所述修改后的帕妥珠单抗重链可变结构域以未修改的帕妥珠单抗重链可变结构域为基础，进行序列修改，所述的序列修改包含：T30A和/或G56A；
(2)另一个重链可变结构域选自未修改的曲妥珠单抗重链可变结构域或修改后的曲妥珠单抗重链可变结构域；所述未修改的曲妥珠单抗重链可变结构域的序列如SEQ ID NO:7所示；
所述修改后的曲妥珠单抗重链可变结构域以未修改的曲妥珠单抗重链可变结构域为基础，进行序列修改，所述的序列修改包含：N54T和/或D98S。
在一些实施方式中，所述双特异抗体为Fab-Ig、Ig-Fab或异二聚体Ig形式。
在一些实施方式中，所述双特异抗体为异二聚体Ig形式。
在一些实施方式中，所述的双特异抗体中的Fc区为杵臼结构(knobs-into-holes)，其包含两个互补的Fc区，所述两个互补的Fc区为knob部分的Fc区和hole部分的Fc区。
在一些实施方式中，所述的双特异抗体的两个Fc为包括由M428L组成的替换的修改后的Fc区。
在一些实施方式中，所述双特异抗体的Fc包含：
(1)增加抗体依赖性细胞毒性(ADCC)效应的Fc，或
(2)增加抗体依赖性细胞吞噬作用(ADCP)的Fc，或
(3)增加补体依赖性细胞毒性(CDC)活性的Fc。
在一些实施方式中，所述双特异抗体为去岩藻糖基化抗体。
在一些实施方式中，所述去岩藻糖基化抗体的获得方法为：由具有岩藻糖基化缺陷的宿主细胞产生。
在一些实施方式中，所述岩藻糖基化缺陷为敲除FUT8基因。
在一些实施方式中，所述去岩藻糖基化抗体的获得方法为：在抗体的Fc结构域中具有S239D、I332E、A330L替换(Kabat编号)或这些变异的任何或所有的组合。
在一些实施方式中，所述的双特异抗体为异二聚体Ig形式，其两条异源重链的Fc区通过杵臼结构(knobs-into-holes)连接；
在一些实施方式中，所述的双特异抗体为异二聚体Ig形式，其两条异源重链的Fc区通过杵臼结构连接，并且，所述的knob(杵)部分的Fc区是未修改的帕妥珠单抗重链Fc区或者包括了以下修改：T366W；所述的hole(臼)部分的Fc区是未修改的曲妥珠单抗重链Fc区或者包括了以下修改的任意组合：T366S、L368A和Y407V；
在一些实施方式中，所述的双特异抗体为异二聚体Ig形式，其两条异源重链的Fc区通过杵臼结构连接，并且，所述的knob(杵)部分的Fc区是以未修改的帕妥珠单抗重链Fc为基础进行了序列修改，所述的序列修改包括了以下修改：T366W和M428L；所述的hole(臼)部分的Fc区是以未修改的曲妥珠单抗重链Fc区为基础进行了序列修改，并且所述的序列修改包括了以下修改的任意组合：T366S、 L368A和Y407V；并且所述的双特异抗体在hole部分的Fc区还包括了如下修改：M428L。
在一些实施方式中，所述双特异抗体包括一对同源的轻链和一对包含两个不同的重链可变结构域的异源重链；
所述一对同源的轻链包括修改后的曲妥珠单抗轻链可变结构域；
所述修改后的曲妥珠单抗轻链可变结构域以未修改的曲妥珠单抗轻链可变结构域为基础，进行如下序列修改：N30S、S56Y和T94W这三种修改中的任意一种、两种或三种；
所述未修改的曲妥珠单抗轻链可变结构域的序列如SEQ ID NO:5所示；
所述的两个不同的重链可变结构域中，
(1)一个重链可变结构域选自未修改的帕妥珠单抗重链可变结构域或修改后的帕妥珠单抗重链可变结构域，所述未修改的帕妥珠单抗重链可变结构域的序列如SEQ ID NO:3所示；
所述修改后的帕妥珠单抗重链可变结构域以未修改的帕妥珠单抗重链可变结构域为基础，进行序列修改，所述的序列修改包含：T30A和/或G56A；
(2)另一个重链可变结构域选自未修改的曲妥珠单抗重链可变结构域或修改后的曲妥珠单抗重链可变结构域；所述未修改的曲妥珠单抗重链可变结构域的序列如SEQ ID NO:7所示；
所述修改后的曲妥珠单抗重链可变结构域以未修改的曲妥珠单抗重链可变结构域为基础，进行序列修改，所述的序列修改包含：N54T和/或D98S；
所述双特异抗体为异二聚体Ig形式，其两条异源重链的Fc区通过杵臼结构连接，并且，所述的knob(杵)部分的Fc区是以未修改的帕妥珠单抗重链Fc为基础进行了序列修改，所述的序列修改包括了以下修改：T366W和M428L；所述的hole(臼)部分的Fc区是以未修改的曲妥珠单抗重链Fc区为基础进行了序列修改，并且所述的序列修改包括了以下修改的任意组合：T366S、L368A和Y407V；并且所述的双特异抗体在hole部分的Fc区还包括了如下修改：M428L。
在一些实施方式中，所述双特异抗体包括一对同源的轻链和一对包含两个不同的重链可变结构域的异源重链；
所述一对同源的轻链包括修改后的曲妥珠单抗轻链可变结构域；
所述修改后的曲妥珠单抗轻链可变结构域以未修改的曲妥珠单抗轻链可变结构域为基础，进行如下序列修改：N30S、S56Y和T94W这三种修改中的任意一种、两种或三种；
所述未修改的曲妥珠单抗轻链可变结构域的序列如SEQ ID NO:5所示；
所述的两个不同的重链可变结构域中，
(1)一个重链可变结构域选自未修改的帕妥珠单抗重链可变结构域或修改后的帕妥珠单抗重链可变结构域，所述未修改的帕妥珠单抗重链可变结构域的序列如SEQ ID NO:3所示；
所述修改后的帕妥珠单抗重链可变结构域以未修改的帕妥珠单抗重链可变结构域为基础，进行序列 修改，所述的序列修改包含：T30A和/或G56A；
(2)另一个重链可变结构域选自未修改的曲妥珠单抗重链可变结构域或修改后的曲妥珠单抗重链可变结构域；所述未修改的曲妥珠单抗重链可变结构域的序列如SEQ ID NO:7所示；
所述修改后的曲妥珠单抗重链可变结构域以未修改的曲妥珠单抗重链可变结构域为基础，进行序列修改，所述的序列修改包含：N54T和/或D98S；
所述双特异抗体为异二聚体Ig形式，其两条异源重链的Fc区通过杵臼结构连接，并且，所述的knob(杵)部分的Fc区是以未修改的帕妥珠单抗重链Fc为基础进行了序列修改，所述的序列修改包括了以下修改：T366W和M428L；所述的hole(臼)部分的Fc区是以未修改的曲妥珠单抗重链Fc区为基础进行了序列修改，并且所述的序列修改包括了以下修改的任意组合：T366S、L368A和Y407V；并且所述的双特异抗体在hole部分的Fc区还包括了如下修改：M428L；
并且，所述双特异抗体为去岩藻糖基化抗体。
在一些实施方式中，所述双特异抗体选自T51、T52、T53、T54、T55、T56、T57和T58。
在一些实施方式中，所述双特异抗体选自去岩藻糖基化的T51(T51-AF)、去岩藻糖基化的T52(T52-AF)、去岩藻糖基化的T53(T53-AF)、去岩藻糖基化的T54(T54-AF)、去岩藻糖基化的T55(T55-AF)、去岩藻糖基化的T56(T56-AF)、去岩藻糖基化的T57(T57-AF)和去岩藻糖基化的T58(T58-AF)。
所述毒素选自美登素类、哈米特林类、鹅膏蕈碱类、澳瑞他汀类、刺孢霉素类或倍癌霉素类，以及它们的氘代物。
在一些实施方式中，所述毒素选自DM1、DM4、E7974、HTI-286、α-鹅膏蕈碱、β-鹅膏蕈碱、γ-鹅膏蕈碱、ε-鹅膏蕈碱、一羟鹅膏毒肽酰胺、三羟鹅膏毒肽、三羟鹅膏毒肽酰胺、γ-三羟鹅膏毒肽、单甲基澳瑞他汀F、单甲基澳瑞他汀E、澳瑞他汀EB、澳瑞他汀EVB、澳瑞他汀F苯二胺、刺孢霉素γ、倍癌霉素A、adozelesin和CC-1065，以及它们的氘代物。
所述连接子选自可裂解连接子和不可裂解连接子，所述连接子中的一个或多个氢原子任选地被氘代；所述可裂解连接子包括但不限于：化学裂解性连接子和/或酶催化裂解性连接子，所述酶催化裂解性连接子例如是含肽组分的连接子。
在一些实施方式中，所述酶催化裂解性连接子选自缬氨酸-瓜氨酸、或苯丙氨酸-赖氨酸，以及它们的氘代物。
在一些实施方式中，本发明提供了一种双特异抗体偶联物(ADC)、其药学上可接受的盐、溶剂化物或其氘代物，其包括双特异抗体、毒素和连接子；
所述双特异抗体包括一对同源的轻链和一对包含两个不同的重链可变结构域的异源重链；
所述一对同源的轻链包括修改后的曲妥珠单抗轻链可变结构域；
所述修改后的曲妥珠单抗轻链可变结构域以未修改的曲妥珠单抗轻链可变结构域为基础，进行如下序列修改：N30S、S56Y和T94W这三种修改中的任意一种、两种或三种；
所述未修改的曲妥珠单抗轻链可变结构域的序列如SEQ ID NO:5所示；
所述的两个不同的重链可变结构域中，
(1)一个重链可变结构域选自未修改的帕妥珠单抗重链可变结构域或修改后的帕妥珠单抗重链可变结构域，所述未修改的帕妥珠单抗重链可变结构域的序列如SEQ ID NO:3所示；
所述修改后的帕妥珠单抗重链可变结构域以未修改的帕妥珠单抗重链可变结构域为基础，进行序列修改，所述的序列修改包含：T30A和/或G56A；
(2)另一个重链可变结构域选自未修改的曲妥珠单抗重链可变结构域或修改后的曲妥珠单抗重链可变结构域；所述未修改的曲妥珠单抗重链可变结构域的序列如SEQ ID NO:7所示；
所述修改后的曲妥珠单抗重链可变结构域以未修改的曲妥珠单抗重链可变结构域为基础，进行序列修改，所述的序列修改包含：N54T和/或D98S；
所述双特异抗体为异二聚体Ig形式，其两条异源重链的Fc区通过杵臼结构连接，并且，所述的knob(杵)部分的Fc区是以未修改的帕妥珠单抗重链Fc为基础进行了序列修改，所述的序列修改包括了以下修改：T366W和M428L；所述的hole(臼)部分的Fc区是以未修改的曲妥珠单抗重链Fc区为基础进行了序列修改，并且所述的序列修改包括了以下修改的任意组合：T366S、L368A和Y407V；并且所述的双特异抗体在hole部分的Fc区还包括了如下修改：M428L；
所述双特异抗体为去岩藻糖基化抗体；
所述毒素选自DM1、DM4、E7974、HTI-286、单甲基澳瑞他汀F、单甲基澳瑞他汀E、澳瑞他汀EB、澳瑞他汀EVB、澳瑞他汀F苯二胺，以及它们的氘代物；
所述连接子选自缬氨酸-瓜氨酸、或苯丙氨酸-赖氨酸，以及它们的氘代物。
在一些实施方式中，本发明提供了一种双特异抗体偶联物(ADC)、其药学上可接受的盐、溶剂化物或其氘代物，其包括双特异抗体、毒素和连接子；
所述双特异抗体包括一对同源的轻链和一对包含两个不同的重链可变结构域的异源重链；
所述一对同源的轻链包括修改后的曲妥珠单抗轻链可变结构域；
所述修改后的曲妥珠单抗轻链可变结构域以未修改的曲妥珠单抗轻链可变结构域为基础，进行如下序列修改：N30S、S56Y和T94W这三种修改中的任意一种、两种或三种；
所述未修改的曲妥珠单抗轻链可变结构域的序列如SEQ ID NO:5所示；
所述的两个不同的重链可变结构域中，
(1)一个重链可变结构域选自未修改的帕妥珠单抗重链可变结构域或修改后的帕妥珠单抗重链可变结构域，所述未修改的帕妥珠单抗重链可变结构域的序列如SEQ ID NO:3所示；
所述修改后的帕妥珠单抗重链可变结构域以未修改的帕妥珠单抗重链可变结构域为基础，进行序列修改，所述的序列修改包含：T30A和/或G56A；
(2)另一个重链可变结构域选自未修改的曲妥珠单抗重链可变结构域或修改后的曲妥珠单抗重链可变结构域；所述未修改的曲妥珠单抗重链可变结构域的序列如SEQ ID NO:7所示；
所述修改后的曲妥珠单抗重链可变结构域以未修改的曲妥珠单抗重链可变结构域为基础，进行序列修改，所述的序列修改包含：N54T和/或D98S；
所述双特异抗体为异二聚体Ig形式，其两条异源重链的Fc区通过杵臼结构连接，并且，所述的knob(杵)部分的Fc区是以未修改的帕妥珠单抗重链Fc为基础进行了序列修改，所述的序列修改包括了以下修改：T366W和M428L；所述的hole(臼)部分的Fc区是以未修改的曲妥珠单抗重链Fc区为基础进行了序列修改，并且所述的序列修改包括了以下修改的任意组合：T366S、L368A和Y407V；并且所述的双特异抗体在hole部分的Fc区还包括了如下修改：M428L；
所述双特异抗体为去岩藻糖基化抗体；
所述毒素选自单甲基澳瑞他汀E及其氘代物；
所述连接子选自缬氨酸-瓜氨酸及其氘代物。
在一些实施方式中，本发明提供了一种双特异抗体偶联物(ADC)、其药学上可接受的盐、溶剂化物或其氘代物，其包括双特异抗体、毒素和连接子；
(1)所述双特异抗体选自T51、T52、T53、T54、T55、T56、T57和T58；在一些实施方式中，所述双特异抗体为去岩藻糖基化的抗体T51-AF、T52-AF、T53-AF、T54-AF、T55-AF、T56-AF、T57-AF和T58-AF；
(2)所述毒素选自DM1、DM4、E7974、HTI-286、单甲基澳瑞他汀F、单甲基澳瑞他汀E、澳瑞他汀EB、澳瑞他汀EVB、澳瑞他汀F苯二胺，以及它们的氘代物；在一些实施方式中，所述毒素选自单甲基澳瑞他汀E及其氘代物；
(3)所述连接子选自缬氨酸-瓜氨酸、或苯丙氨酸-赖氨酸，以及它们的氘代物；在一些实施方式中，所述连接子为缬氨酸-瓜氨酸及其氘代物。
在一些实施方式中，本发明提供了一种双特异抗体偶联物(ADC)、其药学上可接受的盐、溶剂化物或其氘代物，其包括双特异抗体、毒素和连接子；
(1)所述双特异抗体选自T51、T52、T53、T54、T55、T56、T57和T58，以及去岩藻糖基化的 抗体T51-AF、T52-AF、T53-AF、T54-AF、T55-AF、T56-AF、T57-AF和T58-AF；
(2)所述毒素选自DM1、DM4、E7974、HTI-286、单甲基澳瑞他汀F、单甲基澳瑞他汀E、澳瑞他汀EB、澳瑞他汀EVB、澳瑞他汀F苯二胺，以及它们的氘代物；
(3)所述连接子选自缬氨酸-瓜氨酸、或苯丙氨酸-赖氨酸，以及它们的氘代物。
在一些实施方式中，本发明提供了一种双特异抗体偶联物(ADC)、其药学上可接受的盐、溶剂化物或其氘代物，其包括双特异抗体、毒素和连接子；
(1)所述双特异抗体选自T51、T52、T53、T54、T55、T56、T57和T58，以及去岩藻糖基化的抗体T51-AF、T52-AF、T53-AF、T54-AF、T55-AF、T56-AF、T57-AF和T58-AF；
(2)所述毒素选自单甲基澳瑞他汀E及其氘代物；
(3)所述连接子选自缬氨酸-瓜氨酸及其氘代物。
在一些实施方式中，所述缬氨酸-瓜氨酸连接子通过6-马来酰亚胺己酸与所述双特异抗体连接，通过对氨基苄醇与单甲基澳瑞他汀E毒素连接。
在一些实施方式中，所述双特异抗体偶联物的平均药物：抗体比(DAR)约为1-6，例如约为1、2、3、4、5、6，例如约为2.5、2.6、2.7、2.8、2.9、3、3.1、3.2、3.3、3.4、3.5、3.6、3.7、3.8、3.9、4、4.1、4.2、4.3、4.4、4.5、4.6、4.7、4.8、4.9、5。
在一些实施方式中，所述双特异抗体偶联物的结构如下式(I)所示：
Figure PCTCN2022089229-appb-000003
其中，
Figure PCTCN2022089229-appb-000004
表示如上所述的双特异抗体，中间部分为连接子缬氨酸-瓜氨酸(Val-Cit，简称Vc)部分，连接子通过6-马来酰亚胺己酸与所述双特异抗体连接，通过对氨基苄醇与最右侧的Drug(毒素部分)连接。
在一些实施方式中，所述双特异抗体偶联物的结构中进一步明确了DAR值(n)，如下式(I’)所示：
Figure PCTCN2022089229-appb-000005
其中，
Figure PCTCN2022089229-appb-000006
表示如上所述的双特异抗体，中间部分为连接子缬氨酸-瓜氨酸(Val-Cit，简称Vc)部分，连接子通过6-马来酰亚胺己酸与所述双特异抗体连接，通过对氨基苄醇与最右侧的Drug(毒素部分)连接，所述毒素选自DM1、DM4、E7974、HTI-286、单甲基澳瑞他汀F、单甲基澳瑞他汀E、澳瑞他汀EB、澳瑞他汀EVB、澳瑞他汀F苯二胺，以及它们的氘代物；
n表示平均药物：抗体比(DAR)，n选自1-6，例如约为1、2、3、4、5、6，例如约为2.5、2.6、2.7、2.8、2.9、3、3.1、3.2、3.3、3.4、3.5、3.6、3.7、3.8、3.9、4、4.1、4.2、4.3、4.4、4.5、4.6、4.7、4.8、4.9、5。
在一些实施方式中，所述双特异抗体偶联物的结构如下式(I’)所示：
Figure PCTCN2022089229-appb-000007
其中，
Figure PCTCN2022089229-appb-000008
表示双特异抗体，选自T51、T52、T53、T54、T55、T56、T57和T58，以及去岩藻糖基化的抗体T51-AF、T52-AF、T53-AF、T54-AF、T55-AF、T56-AF、T57-AF和T58-AF；中间部分为连接子缬氨酸-瓜氨酸(Val-Cit，简称Vc)部分，连接子通过6-马来酰亚胺己酸与所述双特异抗体连接，通过对氨基苄醇与最右侧的Drug(毒素部分)连接，所述毒素选自DM1、DM4、E7974、HTI-286、单甲基澳瑞他汀F、单甲基澳瑞他汀E、澳瑞他汀EB、澳瑞他汀EVB、澳瑞他汀F苯二胺，以及它们的氘代物；
n表示平均药物：抗体比(DAR)，n选自3-4，例如约为3、3.1、3.2、3.3、3.4、3.5、3.6、3.7、 3.8、3.9、4。
在一些实施方式中，所述双特异抗体偶联物的结构如下式(II)所示：
Figure PCTCN2022089229-appb-000009
其中，
Figure PCTCN2022089229-appb-000010
表示如上所述的双特异抗体，[]中的结构为连接子-毒素部分Vc-MMAE，Vc通过6-马来酰亚胺己酸与所述双特异抗体连接，Vc通过对氨基苄醇与毒素单甲基澳瑞他汀E(MMAE)连接。式中，n即为DAR值，n为1-6，例如约1、2、3、4、5、6，例如3-4，例如约2.5、2.6、2.7、2.8、2.9、3、3.1、3.2、3.3、3.4、3.5、3.6、3.65、3.7、3.75、3.8、3.9、4、4.1、4.2、4.3、4.4、4.5、4.6、4.7、4.8、4.9、5。
在一些实施方案中，所述双特异抗体偶联物的平均药物：抗体比(DAR)为3-4，例如3、3.1、3.2、3.3、3.4、3.5、3.6、3.65、3.7、3.75、3.8、3.9、4。
在一些实施方式中，所述双特异抗体偶联物的结构如下式(II)所示：
Figure PCTCN2022089229-appb-000011
其中，
Figure PCTCN2022089229-appb-000012
表示双特异抗体，选自T51、T52、T53、T54、T55、T56、T57和T58，以及去岩藻糖基化的抗体T51-AF、T52-AF、T53-AF、T54-AF、T55-AF、T56-AF、T57-AF和T58-AF；[]中的结构为连接子-毒素部分Vc-MMAE，Vc通过6-马来酰亚胺己酸与所述双特异抗体连接，Vc通过对氨基苄醇与毒素单甲基澳瑞他汀E(MMAE)连接。式中，n即为DAR值，n选自3-4，例如约为3、3.1、3.2、3.3、3.4、3.5、3.6、3.7、3.8、3.9、4。
在一些实施方式中，所述双特异抗体偶联物的结构如下式(II)所示：
Figure PCTCN2022089229-appb-000013
其中，
Figure PCTCN2022089229-appb-000014
表示双特异抗体，选自T54、T58，以及去岩藻糖基化的抗体T54-AF和T58-AF；[]中的结构为连接子-毒素部分Vc-MMAE，Vc通过6-马来酰亚胺己酸与所述双特异抗体连接，Vc通过对氨基苄醇与毒素单甲基澳瑞他汀E(MMAE)连接。式中，n即为DAR值，n选自3-4，例如约为3、3.1、3.2、3.3、3.4、3.5、3.6、3.7、3.8、3.9、4。
在一些实施方式中，所述双特异抗体选自T51、T52、T53、T54、T55、T56、T57和T58。
在一些实施方式中，所述双特异抗体选自去岩藻糖基化的T51(T51-AF)、去岩藻糖基化的T52(T52-AF)、去岩藻糖基化的T53(T53-AF)、去岩藻糖基化的T54(T54-AF)、去岩藻糖基化的T55(T55-AF)、去岩藻糖基化的T56(T56-AF)、去岩藻糖基化的T57(T57-AF)和去岩藻糖基化的T58(T58-AF)。
在一些实施方式中，所述双特异抗体选自T54、T58、去岩藻糖基化的T54(T54-AF)和去岩藻糖基化的T58(T58-AF)。
在一些实施方式中，所述双特异抗体选自去岩藻糖基化的T54(T54-AF)和去岩藻糖基化的T58(T58-AF)。
在一些实施方式中，本发明的双特异抗体偶联物是T51-MMAE、T52-MMAE、T53-MMAE、T54-MMAE、T55-MMAE、T56-MMAE、T57-MMAE、T58-MMAE，以及对应的去岩藻糖基化双特异抗体偶联物T51-AF-MMAE、T52-AF-MMAE、T53-AF-MMAE、T54-AF-MMAE、T55-AF-MMAE、T56-AF-MMAE、T57-AF-MMAE和T58-AF-MMAE。
在一些实施方式中，本发明的双特异抗体偶联物是T54-MMAE和T58-MMAE，以及对应的去岩藻糖基化双特异抗体偶联物T54-AF-MMAE和T58-AF-MMAE。
根据本发明的另一方面，提供了一种药物组合物，包括如上所述的双特异抗体偶联物、其药学上可接受的盐、溶剂化物或其氘代物，以及药学上可接受的载体或赋形剂。
根据本发明的另一方面，提供了如上所述的双特异抗体偶联物、其药学上可接受的盐、溶剂化物或 其氘代物或如上所述的药物组合物在制备用于预防和/或治疗癌症的药物中的应用。
在一些实施方式中，所述癌症为表达HER2的癌症。
在一些实施方式中，所述癌症为HER2中低水平表达的癌症。
在一些实施方式中，所述癌症包括乳腺癌、卵巢癌、子宫内膜癌、子宫颈癌、胃癌、食管癌、肺癌、头颈癌、膀胱癌、胆管癌和结直肠癌。
本发明还涉及所述的双特异抗体偶联物、其药学上可接受的盐、溶剂化物或其氘代物或药物组合物在制备抗肿瘤联用制剂中的应用，所述的联用制剂中还包括任选的如下治疗肿瘤的活性成分：
(1)靶向HER2以外的其他靶点的药物；
(2)细胞治疗类药物；
(3)免疫治疗类药物。
根据本发明的另一方面，提供了一种用于预防和/或治疗癌症的方法，包括向有需要的患者施用治疗有效量的如上所述的双特异抗体偶联物、其药学上可接受的盐、溶剂化物或其氘代物或如上所述的药物组合物。
在一些实施方式中，所述癌症为表达HER2的癌症。
在一些实施方式中，所述癌症为HER2中低水平表达的癌症。
在一些实施方式中，所述癌症包括乳腺癌、卵巢癌、子宫内膜癌、子宫颈癌、胃癌、食管癌、肺癌、头颈癌、膀胱癌、胆管癌和结直肠癌。
本发明还涉及所述的双特异抗体偶联物、其药学上可接受的盐、溶剂化物或其氘代物或药物组合物在治疗癌症中的应用，其特征在于，与下述治疗癌症的药物或治疗方案联用；
(1)靶向HER2以外的其他靶点的药物；
(2)细胞治疗类药物；
(3)免疫治疗类药物；
(4)手术；
(5)物理疗法如放疗。
本发明具有以下有益效果：
(1)根据本发明的双特异抗体偶联物、其药学上可接受的盐、溶剂化物或其氘代物具有优异的抗肿瘤活性，特定剂量下，在多个不同类型的小鼠肿瘤模型中的抗肿瘤活性优于TDM1(或DS8201)，相比于TDM1(或DS8201)，在相同的剂量下，能够在更短的时间内更好地抑制肿瘤的增长。
(2)根据本发明的双特异抗体偶联物、其药学上可接受的盐、溶剂化物或其氘代物具有良好的耐 受性和较低的毒性。
附图说明
图1由抗体A和抗体B设计的具有同源轻链的双特异抗体的Fab-Ig形式的示意图。
图2由抗体A和抗体B设计的具有同源轻链的双特异抗体的Ig-Fab形式的示意图。
图3由抗体A和抗体B设计的具有同源轻链的双特异抗体的异二聚体IgG形式的示意图，异二聚体通过杵臼结构连接。
图4由抗体A和抗体B设计的具有同源轻链的双特异抗体的异二聚体IgG形式的示意图，异二聚体通过静电转向机制连接。
图5抗体T54(5A)和T54-AF(5B)的SEC纯度图，其中，横坐标表示时间(min)，纵坐标表示吸光度值(mAU)。
图6抗体偶联物T54-MMAE(6A)和T54-AF-MMAE(6B)的SEC纯度图，其中，横坐标表示时间(min)，纵坐标表示吸光度值(mAU)。
图7抗体T54的HIC-HPLC结果图，其中，横坐标表示时间(min)，纵坐标表示吸光度值(mAU)。图8抗体偶联物T54-MMAE(8A)和T54-AF-MMAE(8B)的HIC-HPLC结果图，其中，横坐标表示时间(min)，纵坐标表示吸光度值(mAU)。
图9阳性对照ADC(DS8201)、T54-AF及T54-AF-MMAE(T54-AF-ADC)与HER2抗原的结合曲线图，其中，横坐标表示浓度的对数，纵坐标表示OD 450值。
图10抗体herceptin、T54及其各自对应的偶联物TDM1、T54-MMAE对乳腺癌细胞SKBR3的生长抑制曲线图，其中，横坐标表示浓度的对数，纵坐标表示乳腺癌细胞SKBR3的细胞存活率。
图11抗体herceptin、T54及其各自对应的偶联物TDM1、T54-MMAE对胃癌细胞N87的生长抑制曲线图，其中，横坐标表示浓度的对数，纵坐标表示胃癌细胞N87的细胞存活率。
图12检测T54-AF-MMAE、DS8201、T54-AF、herceptin、perjeta、Isotype(同型对照，作为阴性抗体)对各个不同细胞株的抑制效果，其中(A)BT474细胞株、(B)NCI-H2170细胞株(C)MDA-MB-175Ⅶ细胞株(D)SKOV-3细胞株(E)MCF-7细胞株(F)MDA-MB-435细胞株(G)NCI-H446细胞株。
图13抗体偶联物TDM1和T54-MMAE治疗开始后人源异种移植乳腺癌模型BT474的生长曲线图，其中，横坐标表示移植肿瘤后的天数，纵坐标表示肿瘤体积(mm 3)。
图14阳性对照ADC(DS8201)和去岩藻糖基化双特异抗体偶联物T54-AF-MMAE(T54-AF-ADC)治疗开始后人源异种移植乳腺癌模型BT474的生长曲线图，其中，横坐标表示移植肿瘤后的天数，纵坐标表示肿瘤体积(mm 3)。
图15阳性对照ADC(DS8201)和去岩藻糖基化双特异抗体偶联物T54-AF-MMAE(T54-AF-ADC)治 疗开始后人源异种移植胃模型N87的生长曲线图，其中，横坐标表示移植肿瘤后的天数，纵坐标表示肿瘤体积(mm 3)。
具体实施方式
本发明公开了一种双特异抗体偶联物、其药学上可接受的盐、溶剂化物或其氘代物，包括双特异抗体、毒素和连接子；所述双特异抗体识别和/或结合HER2的结构域II，并且识别和/或结合HER2的结构域IV；所述毒素选自美登素类、哈米特林类、鹅膏蕈碱类、澳瑞他汀类、刺孢霉素类或倍癌霉素类；所述连接子选自可裂解连接子和不可裂解连接子；所述毒素和连接子中的一个或多个氢原子任选地被氘代。
如本文所用，术语“抗体”是指具有天然存在的哺乳动物免疫球蛋白(Ig)(IgG是其范例)的一般结构的分子。即包括一个可变结构域和一个恒定结构域的两条相同的轻链以及包括一个可变结构域和三个恒定结构域的两条相同的重链。轻链和重链通过它们的可变结构域、以及轻链的恒定结构域与重链的第一恒定结构域相互联合。这两条重链通过第2和第3恒定结构域相互联合。抗体的抗原结合位点由两个可变结构域形成，且特别地由每个可变结构域的三个互补决定区(CDR)形成。如果抗体能够特异性地与分子相互作用并吸附到分子上，则称抗体与分子(抗原)结合。抗体结合不包括非特异性或低亲和力的相互作用。虽然“抗体”的本意是指天然存在的免疫球蛋白的结构，但它也包括保留这种一般结构的设计的分子，诸如嵌合、CDR移植和人源化抗体。
如本文所用，术语“表位”是指抗原上通过与抗体(或抗体构建体)的抗原结合位点进行接触而介导抗原与抗体(或抗体构建体)特异性结合的部分。
如本文所用，术语“互补位”是指抗体(或抗体构建体)上通过与抗原的表位进行接触而介导抗体(或抗体构建体)与抗原特异性结合的部分。
如本文所用，术语“修改”、“突变”和“替换”(及其语法形式)是指氨基酸序列中工程化的变化。除非上下文另有说明，否则突变是指由人工辅助的生物过程引起的序列变化。常规地，替换用参考序列中的氨基酸的单字母码、在参考序列中位置、和产生的序列中的氨基酸的单字母码三部分来表示。例如，A26S表示在参考序列中第26位处的丙氨酸(A)在产生的序列中被改变为丝氨酸(S)。
如本文所用，术语“同源”指的是两条或多条肽链的氨基酸序列完全相同。
如本文所用，术语“异源”指的是两条或多条肽链的氨基酸序列有差异、不完全相同。
如本文所用，术语“氘代物”、“氘代”指的是抗体偶联物结构中的一个或多个氢原子(H-1)被氘原子(H-2)取代所得到的结构。
本发明中的化合物的任何原子若没有特别指定，可代表该原子的任何一种稳定的同位素。除非特别说明，当结构中某一位置被定义为H即氢(H-l)时，该位置仅含天然存在的同位素。同样，除非特别 说明，当结构中某一位置被定义为D即氘(Η-2)时，该位置含同位素量至少比天然存在的同位素量(0.015％)大3340倍(即至少含50.1％氘同位素)，当本申请化合物的结构中某一个或多个位置被定义为D即氘(Η-2)时，该结构所示的化合物的含量可至少为52.5％、至少为60％、至少为67.5％、至少为75％、至少为82.5％、至少为90％、至少为95％、至少为97％、至少为98.5％、至少为99％、至少为99.5％。
本申请化合物的氘代率是指标记合成的同位素含量与天然存在的同位素量的比值。本申请化合物的每个指定氘原子的氘代率可至少为3500倍(52.5％)、至少为4000倍(60％)、至少为4500倍(67.5％)、至少为5000倍(75％)、至少为5500倍(82.5％)、至少为6000倍(90％)、至少为6333.3倍(95％)、至少为6466.7倍(97％)、至少为6566.7倍(98.5％)、至少为6600倍(99％)、至少为6633.3倍(99.5％)。
本申请中的同位素体(isotopologues)是指在化学结构方面仅有同位素组成上不同的化合物。本申请中的在特定位置含氘化合物也会含非常少的该位置的氢同位素体，本申请氘代化合物中的氘代位置的氢同位素体的量取决于许多因素，其中包括氘代试剂(D2O、D2、NaBD4、L1AID4等)的氘同位素纯度以及引入氘同位素合成方法的有效性。然而，如前所述，这种氘代位置的氢同位素体的量总数将少于49.9％。本申请氘代化合物中的氘代位置的氢同位素体的量总数将少于47.5％、40％、32.5％、25％、17.5％、10％、5％、3％、1％或0.5％。
本申请中，任何未指定为氘的各原子以其天然同位素丰度存在。
本申请中，对于轻链可变结构域的氨基酸序列、以及重链可变结构域的氨基酸序列的编号，均基于Vajdos et al.,J.Mol.Biol.320:415(2002)，使用Kabat编号(Kabat et al.,NIH publication no.91-3242,pp 662,680,689(1991)。
双特异抗体
本发明的双特异抗体可采用许多不同的总体结构。在一些实施方式中，本发明的双特异抗体对同一抗原或不同抗原中的两个表位具有特异性。在一些实施方式中，本发明的双特异抗体具有两个互补位。
应该理解的是，可以向每个重链添加额外的可变结构域，使得重链包括例如3、4、5或更多的可变结构域，其促进形成相同数量的互补位。在这些多互补位抗体中，第三个互补位可能赋予对第三个表位的特异性，使抗体具有三特异性。可替代地，第三个互补位可能对被前两个互补位中任一个所识别的表位之一有特异性，使得抗体仍然是双特异性的，但是增加了对表位中的一个的价数。类似地，第四个互补位可能对第四个表位具有特异性，使得抗体构建体是四特异性的，或者它可能对被前三个互补位中的任一个所识别的表位之一有特异性，使得抗体构建体是三特异性的或双特异性的。
在一些实施方式中，双特异抗体包含能够识别和/或结合HER2的结构域II的第一互补位，和能够识别和/或结合HER2的结构域IV的第二互补位。
在一些实施方式中，所述双特异抗体包括一对同源的轻链和一对包含两个不同的重链可变结构域的 异源重链。
在一些实施方式中，所述一对同源的轻链包括修改后的曲妥珠单抗轻链可变结构域；
所述修改后的曲妥珠单抗轻链可变结构域以未修改的曲妥珠单抗轻链可变结构域为基础，进行序列修改，所述的序列修改包含：
a)S56Y/T/A中的任意一种；
b)N30S/A中的任意一种；
c)T94W/F/Y中的任意一种；
d)H91Y/F/W中的任意一种；
e)P96Y/F/W中的任意一种；或
f)a)至e)的这五类修改方式的任意组合；
在一些实施方式中，所述修改后的曲妥珠单抗轻链可变结构域以未修改的曲妥珠单抗轻链可变结构域为基础，进行如下序列修改：N30S、S56Y和T94W这三种修改中的任意一种、两种或三种；
所述未修改的曲妥珠单抗轻链可变结构域的序列如SEQ ID NO:5所示。
所述的两个不同的重链可变结构域中，
(1)一个重链可变结构域选自未修改的帕妥珠单抗重链可变结构域或修改后的帕妥珠单抗重链可变结构域，所述未修改的帕妥珠单抗重链可变结构域的序列如SEQ ID NO:3所示；
所述修改后的帕妥珠单抗重链可变结构域以未修改的帕妥珠单抗重链可变结构域为基础，进行序列修改，所述的序列修改包含：
i)T30A/S/N/D；
ii)G56A/S/T；或
iii)i)和ii)这两类修改方式的任意组合；
在一些实施方案中，所述修改后的帕妥珠单抗重链可变结构域以未修改的帕妥珠单抗重链可变结构域为基础，进行序列修改，所述的序列修改包含：T30A和/或G56A；
(2)另一个重链可变结构域选自未修改的曲妥珠单抗重链可变结构域或修改后的曲妥珠单抗重链可变结构域；所述未修改的曲妥珠单抗重链可变结构域的序列如SEQ ID NO:7所示；
所述修改后的曲妥珠单抗重链可变结构域以未修改的曲妥珠单抗重链可变结构域为基础，进行序列修改，所述的序列修改包含：
i)N54T/S/A；
ii)D98S/W/T/R；或
iii)i)和ii)这两类修改方式的任意组合；
在一些实施方式中，所述修改后的曲妥珠单抗重链可变结构域以未修改的曲妥珠单抗重链可变结构域为基础，进行序列修改，所述的序列修改包含：N54T和/或D98S。
在一些实施方式中，所述双特异抗体为Fab-Ig、Ig-Fab或异二聚体Ig形式。
在一些实施方式中，所述双特异抗体为异二聚体Ig形式。
如本文所用的术语“Fc区”、“Fc”或“Fc结构域”是指含有恒定区的至少一部分的免疫球蛋白重链C端区域。该术语包括天然序列Fc区和变体Fc区。除非本文另有说明，否则Fc区或恒定区中的氨基酸残基的编号是根据EU编号系统，也称为EU索引，如Kabat等人,Sequences of Proteins of Immunological Interest,第5版,Public Health Service,National Institutes of Health,Bethesda,MD(1991)中所述。
恒定结构域(C H1、C H2和C H3)为人免疫球蛋白，诸如IgG、IgM、IgA、IgD；或IgG及其亚型IgG1、IgG2、IgG3、IgG4；或者从这些类型和亚型中重组的C H1、C H2和C H3结构域的组合。
在一些实施方式中，双特异抗体的Fc包含具有增加的抗体依赖性细胞毒性(ADCC)、抗体依赖性细胞吞噬作用(ADCP)或补体依赖性细胞毒性(CDC)活性的Fc，其是由于与Fc受体(诸如CD16a、CD16b、CD32a、CD16b、CD64和C1q蛋白)的结合亲和力增强或减弱所致。这包括但不限于ADCC增强的去岩藻糖基化抗体，获得方法为(1)通过在具有岩藻糖基化缺陷(诸如敲除FUT8基因)的宿主细胞中产生双特异抗体(Yamane-Ohnuki N et al.,Biotechnol Bioeng.87(5):614-22,2004)；(2)在抗体的Fc结构域中具有S239D、I332E、A330L替换(Kabat编号)或这些变异的任何或所有的组合(Lazar GA et al.,Proc Natl Acad Sci U S A.103(11):4005–4010,2006)；或(3)如中国专利申请CN201610194325.7中所示。
在一些实施方式中，双特异抗体在Fc结构域中包含突变，以减小ADCC活性或CDC活性。这些可包括但不限于在Fc结构域中(1)N297的突变，诸如但不限于N297A、N297G；(2)L234的突变，诸如L234A、L234G，和/或L235的突变，诸如L235A或L235G；(3)P329的突变，诸如P329G；或(4)D265的突变，诸如D265A；或者在任何或所有这些位置处的替换的组合。
在一些实施方式中，Fc突变(所有编号均为Kabat编号系统的Eu索引)包括增加血清半衰期的突变。在一种实施方式中，Fc具有以下替换：在C H3中的T250Q、或M428L、或T250Q/M428L双突变(Hinton et al.,J Biol Chem.279(8):6213-6,2004)。在另一实施方式中，双特异抗体的Fc具有M252Y/S254T/T256E三重突变(Dall’Acqua WF et al.,J Immunol169(9):5171-80,2002)。在另一实施方式中，双特异抗体的Fc具有N434A突变(Petkova SB et al.,International Immunology 18(12):1759–1769,2006.)或M428L/N434S双突变、或M428L/N434A双突变(Zalevsky J et al.,Nat Biotechnol.28(2):157–159,2010)。
在一些实施方式中，双特异抗体的Fc区已经被修改以增加其血清半衰期。在一些实施方式中，增加血清半衰期的修改为M428L。
本发明的双特异抗体可以为同二聚体形式(包括两个相同的重链)，例如Fab-Ig(如图1所示)或Ig-Fab(如图2所示)。接头由任何组成的0至100个氨基酸组成。在一些实施方式中，接头铰链(adapter)是例如(G 3S) nG 3，其中n是1至20之间的任意数字(如表8所示)。
本发明的双特异抗体也可以为异二聚体Ig形式。来自两种不同抗体的两种不同的重链可以利用本领域中描述的技术来形成异二聚体，包括但不限于杵臼结构(knobs-into-holes，如图3所示)、静电转向机制(如图4所示)等。
在一些实施方式中，本发明的双特异抗体选自国际专利公开文本WO2018/191188A1中公开的那些。
在一些实施方式中，本发明的双特异抗体选自T51、T52、T53、T54、T55、T56、T57和T58。
T51至T58这8个双特异抗体中，T54和T58为异二聚体Ig形式，且采取了杵臼结构(knobs-into-holes)，其余6个均为同二聚体形式，其中T53和T57采取Ig-Fab形式，T51、T52、T55和T56采取Fab-Ig形式。
T51至T58这8个双特异抗体所对应的重链代号及其氨基酸序列如表8和表9所示。
T51至T58的同源轻链以未修改的曲妥珠单抗轻链可变结构域作为起始，经位点突变得到，突变方式例如可以是：N30突变为S(N30S)、S56突变为Y(S56Y)或T94突变为W(T94W)，或者上述突变中的两种或三种的任意组合，例如N30S/S56Y、N30S/T94W、S56Y/T94W、N30S/S56Y/T94W等。T51至T58所对应的轻链代号如表8所示，作为起始的曲妥珠单抗轻链可变结构域的序列如SEQ ID NO:5所示。
本文公开的双特异抗体可通过重组方法来产生。用于重组生产的方法在本领域中是众所周知的，且包括：在原核细胞和真核细胞中的蛋白表达，和随后分离双特异抗体，以及通常纯化至药学上可接受的纯度。对于在宿主细胞中的表达上述抗体，通过标准方法将编码抗体序列的核酸插入表达载体中。表达在适当的原核或真核宿主细胞中进行，如CHO细胞、NSO细胞、SP2/0细胞、HEK293细胞、COS细胞、PER.C6细胞、酵母或大肠杆菌(E.coli)细胞，且抗体从细胞(溶解后的细胞或上清液)中回收。
因此，本文公开的某些实施方式包括用于制备双特异抗体的方法，包括以下步骤：a)用包括编码抗体的核酸分子的至少一种表达载体转化宿主细胞；b)在允许合成抗体分子的条件下培养宿主细胞；c)从培养物中回收抗体。
通过常规免疫球蛋白纯化程序，诸如蛋白A-琼脂糖凝胶、羟基磷灰石层析、凝胶电泳、透析或亲和层析，从培养基中适当地分离抗体。
如本文所用，表达“细胞”、“细胞系”和“细胞培养物”可以互换使用，且所有这样的名称都包括子代。词语“转化株”和“转化细胞”包括初代受试细胞和由此衍生的培养物，而不考虑传代次数。还应理解，由于有意或无意的突变，所有后代的DNA含量可能并不完全相同。包括与在原转化细胞中筛 选的功能或生物活性相同的变异子代。在意为不同名称的地方，将从上下文中清楚理解其为“细胞”、“细胞系”或“细胞培养物”。
如本文所用，术语“转化”是指将载体/核酸转化到宿主细胞中的过程。如果使用没有强大细胞壁屏障的细胞作为宿主细胞，则可以例如通过磷酸钙沉淀法进行转染。然而，也可以使用用于将DNA引入细胞的其他方法，诸如通过核注射或通过原生质体融合。如果使用原核细胞或包含坚实细胞壁结构的细胞，则例如一种转染方法是使用氯化钙进行钙处理。
如本文所用，“表达”是指核酸转录成mRNA的过程和/或转录后的mRNA(也称为转录物)随后翻译成肽类、多肽或蛋白质的过程。转录物和编码的多肽可以统称为“基因产物”。如果多核苷酸包括源自基因组DNA的序列，则真核细胞中的表达可包括mRNA的剪接。
“载体”是核酸分子，特别是自主复制的核酸分子，它将插入的核酸分子转移到宿主细胞内和/或在宿主细胞之间转移。该术语包括主要用于将DNA或RNA插入细胞的载体(例如染色体整合)，主要用于DNA或RNA复制的复制载体，以及用于DNA或RNA的转录和/或翻译的表达载体。还包括提供多于一种上述功能的载体。
“表达载体”是当被引入适当的宿主细胞时，可以被转录并翻译成多肽的多核苷酸。“表达系统”通常是指包括可用于产生所需表达产物的表达载体的合适的宿主细胞。
如本文所用，术语“宿主细胞”是指可被工程化为产生本文公开的抗体的任何类型的细胞系统。在一些实施方式中，HEK293细胞和CHO细胞被用作宿主细胞。
适合于原核生物的控制序列例如包括启动子、任选地操作子序列和核糖体结合位点。已知真核细胞利用启动子、增强子和多聚腺苷酸化信号。
当核酸与另一核酸序列处于功能关系时，它是“可操作地连接”的。例如，如果前导序列或分泌型前导物的DNA表达为参与多肽分泌的前蛋白，则其与多肽的DNA可操作地连接；如果启动子或增强子影响序列的转录，则其与编码序列可操作地连接；或者，如果核糖体结合位点的定位是为了便于翻译，则其与编码序列可操作地连接。通常，“可操作地连接”意为被连接的DNA序列是连续的，且在分泌型前导物的情况下是连续且在阅读框中的。然而，增强子不必是连续的。类似地，在一些情况下，内含子可存在于可操作地连接的核酸序列之间。通过在方便的限制性位点的连接反应来实现连接。如果不存在这样的位点，则按照常规实践使用合成寡核苷酸衔接子或接头。
编码抗体的核酸序列可以很容易地从文献中获得，或者参照预期宿主细胞的优选的密码子通过反向翻译获得。编码核酸可以由化学合成的多核苷酸和/或之前克隆的抗体编码DNA组装而成，可能地借助于定点突变。
对于抗体的重组生产，可以将编码该抗体的核酸分离并插入可复制载体中用于进一步克隆(DNA扩增)或表达。使用常规程序(例如，通过使用能够与编码抗体的重链和轻链的基因特异性结合的寡核 苷酸探针)可以很容易地对编码抗体的DNA进行分离和测序。许多载体是可用的。载体成分一般包括但不限于以下一种或多种：信号序列、复制起点、一个或多个标记基因、增强子元件、启动子和转录终止序列，例如US5,534,615中所述，其关于蛋白表达的全部公开通过引用具体并入本文。
本文用于在载体中克隆或表达DNA的合适的宿主细胞是上文所述的原核细胞、酵母或高等真核细胞。为此目的的合适的原核生物包括真细菌，诸如革兰氏阴性和革兰氏阳性生物，例如，肠杆菌科(Enterobacteriaceae)，诸如埃希氏菌属(Escherichia)例如大肠杆菌(E.coli)、肠杆菌属(Enterobacter)、欧文氏菌属(Erwinia)、克雷伯氏菌属(Klebsiella)、变形杆菌属(Proteus)、沙门氏菌属(Salmonella)例如鼠伤寒沙门氏杆菌(S.typhimurium)、沙雷氏菌属(Serratia)例如粘质沙雷氏菌(S.marcescans)和志贺氏菌属(Shigella)，以及芽孢杆菌属(Bacilli)诸如枯草芽孢杆菌(B.subtilis)和地衣芽孢杆菌(B.licheniformis)、假单胞菌属(Pseudomonas)诸如铜绿假单胞菌(P.aeruginosa)等假单胞菌和链霉菌属(Streptomyces)。一种示例性大肠杆菌克隆宿主是大肠杆菌294(ATCC 31,446)，但诸如大肠杆菌B、大肠杆菌X1776(ATCC 31,537)和大肠杆菌W3110(ATCC 27,325)的其他菌株也是合适的。这些实例是说明性的，而非限制性的。
除原核生物外，真核微生物诸如丝状真菌或酵母也是用于抗体编码载体的合适的克隆或表达宿主。酿酒酵母菌(Saccharomyces cerevisiae)是常见的面包酵母，是低等真核宿主微生物中最常用的酵母。然而，许多其他属、物种和菌株在本文中也是常见的且可用的，诸如粟酒裂殖酵母(Schizosaccharomyces pombe)；克鲁维酵母(Kluyveromyces)宿主，诸如例如乳酸克鲁维酵母(K.lactis)、脆壁克鲁维酵母(K.fragilis)(ATCC 12,424)、保加利亚克鲁维酵母(K.bulgaricus)(ATCC 16,045)、K.wickeramii(ATCC24,178)、K.Waltii(ATCC 56,500)、果蝇克鲁维酵母(K.drosophilarum)(ATCC 36,906)、耐热克鲁维酵母(K.thermotolerans)和马克斯克鲁维酵母(K.marxianus)；耶氏酵母属(Yarrowia)(EP 402,226)；毕赤酵母(Pichia pastoris)(EP 183,070)；假丝酵母(Candida)：里氏木霉(Trichoderma reesia)(EP244,234)；粗糙脉孢菌(Neurospora crassa)；雪旺酵母属(Schwanniomyces)，诸如西方雪旺酵母(S.occidentalis)，以及丝状真菌，诸如例如脉孢菌属(Neurospora)、青霉菌属(Penicillium)、弯颈霉属(Tolypocladium)，以及曲霉属(Aspergillus)宿主，诸如构巢曲霉(A.nidulans)和黑曲霉(A.niger)。
用于糖基化抗体的表达的合适的宿主细胞源自多细胞生物，包括无脊椎动物细胞，诸如植物和昆虫细胞。已经鉴定出许多杆状病毒株和变异体以及相应的允许的昆虫宿主细胞，诸如草地贪夜蛾(Spodoptera frugiperda)(毛虫)、埃及伊蚊(Aedes aegypti)(蚊子)、白纹伊蚊(Aedes albopictus)(蚊子)、黑腹果蝇(Drosophila melanogaster)(果蝇)和家蚕(Bombyx mori)。各种用于转染的病毒株均可公开获得，例如，苜蓿尺蠖(Autographa californica)NPV的L-1变体和家蚕(B.mori)NPV的Bm-5株，且这样的病毒可以用作根据本发明的本文的病毒，特别是用于草地贪夜蛾细胞的转染。棉花、玉米、马铃薯、大豆、矮牵牛、番茄和烟草的植物细胞培养物也可作为宿主。
然而，对脊椎动物细胞的兴趣最大，且在培养物(组织培养物)中繁殖脊椎动物细胞已成为常规程序。可用的哺乳动物宿主细胞系的实例有通过SV40转化的猴肾CV1系(COS-7，ATCC CRL 1651)；人胚胎肾系(293或用于在悬浮培养物中生长的亚克隆293细胞)；幼仓鼠肾细胞(BHK，ATCC CCL 10)；中国仓鼠卵巢细胞/-DHFR(CHO)；小鼠支持细胞(sertoli cells)(TM4)；猴肾细胞(CV1ATCC CCL 70)；非洲绿猴肾细胞(VERO-76，ATCC CRL-1587)；人宫颈癌细胞(HELA，ATCC CCL 2)；犬肾细胞(MDCK，ATCC CCL 34)；布法罗大鼠肝细胞(BRL 3A，ATCC CRL 1442)；人肺细胞(W138，ATCC CCL 75)；人肝癌细胞(Hep G2,HB 8065)；小鼠乳腺肿瘤(MMT 060562，ATCC CCL51)；TRI细胞；MRC 5细胞；FS4细胞。
用上述表达载体转化宿主细胞用于抗体生产，并在适合用于诱导启动子、选择转化子或扩增编码所需序列的基因的改良的常规营养培养基中培养。
用于产生抗体的宿主细胞可以在多种培养基中培养。可商购的培养基诸如Ham's F10、最小必需培养基(MEM)、RPMI-1640和Dulbecco改良Eagle培养基(DMEM)适合于培养宿主细胞。此外，US4,767,704；US4,657,866；US4,927,762；US4,560,655；或US5,122,469；WO 90/03430；WO 87/00195；或US Re.30,985可以用作宿主细胞的培养基。任何这些培养基均可按需要补充激素和/或其他生长因子(诸如胰岛素、转铁蛋白或表皮生长因子)、盐(诸如氯化钠、钙、镁和磷酸盐)、缓冲液(诸如HEPES)、核苷酸(诸如腺甘和胸苷)、抗生素(诸如庆大霉素 TM)、微量元素(定义为通常在微摩尔的范围内存在于最终浓度的无机化合物)以及葡萄糖或等效的能量来源。任何其他必需的补充剂也可以以适当的浓度包括在内，其是本领域技术人员已知的。培养条件，诸如温度、pH等，是之前被选择用于表达的宿主细胞所使用的那些培养条件，且对于普通技术人员是显而易见的。
当使用重组技术时，抗体可以在细胞内、周质空间中产生，或直接分泌到培养基中。如果抗体是在细胞内产生的，则作为第一步，颗粒碎片，无论是宿主细胞还是溶解片段，都可以通过例如离心或超滤来去除。
可以使用例如羟基磷灰石层析、凝胶电泳、透析和亲和层析来纯化由细胞制备的抗体组合物，亲和层析是优选的纯化技术。蛋白A作为亲和配体的适合性取决于抗体中存在的任何免疫球蛋白Fc结构域的种类和同种型。蛋白A可用于纯化基于人γ1、γ2或γ4重链的抗体。推荐蛋白G用于所有小鼠同种型和用于人γ3。亲和配体所附连的基质最经常是琼脂糖，但也有其他基质。机械稳定的基质诸如可控孔玻璃或聚(苯乙烯二乙烯基)苯允许比用琼脂糖可获得的更快的流速和更短的处理时间。当抗体包括C H3结构域时，Bakerbond ABX TM树脂可用于纯化。其他蛋白质纯化技术，诸如在离子交换柱上的分级、乙醇沉淀、反相HPLC、二氧化硅色谱法、肝素SEPHAROSE TM的色谱法、阴离子或阳离子交换树脂上的色谱法(诸如聚天冬氨酸柱)、层析聚焦、SDS-PAGE和硫酸铵沉淀也是可用的，取决于待回收的抗体。
在任何初步纯化步骤(一步或多步)之后，包括感兴趣的抗体和污染物的混合物可使用pH在约2.5-4.5之间的洗脱缓冲液进行低pH疏水作用色谱，优选地在低盐浓度(例如，从约0-0.25M盐)下进行。
一旦纯化之后，抗体可以溶解于包括任何药学上可接受的缓冲液、盐或其他赋形剂的水性溶剂中。溶解的抗体在使用之前可以冷藏或冷冻。可替代地，可以冻干并在使用前不久重构。
毒素
用于和抗体偶联的毒素一般需要满足以下三个条件：(1)作用机制清楚,如阻断有丝分裂和造成DNA损伤等；(2)高活性,一般要求EC 90小于1nmol·L -1；(3)可以采用化学方法偶联，并在肿瘤细胞内释放高活性的毒素本身或其高活性衍生物。
本发明所采用的毒素可以是本领域通常使用的任何毒素，例如可以是微管抑制剂、DNA损伤剂以及其他毒素分子。
在一些实施方式中，本发明的毒素例如选自美登素类(maytansine)、哈米特林(hemiasterlin)类、鹅膏蕈碱(amanitin)类、澳瑞他汀(auristatin)类、刺孢霉素(calicheamicins)类或倍癌霉素(Duocarmycins)类等，以及它们的氘代物。
美登素类是在ADC中使用较为广泛的一类细胞毒素，通过阻断微管蛋白的聚合作用，将细胞阻滞于细胞周期的G2/M期，从而抑制了细胞有丝分裂的进行，导致细胞凋亡。
美登素类衍生物例如可以是DM1、DM4，以及它们的氘代物。
Figure PCTCN2022089229-appb-000015
哈米特林类是由所述原始天然产品哈米特林修饰而来的一类三肽，是一种微管蛋白聚合的抑制剂，可以非竞争地结合在微管蛋白的长春花碱位点。
哈米特林类衍生物例如可以是E7974(卫材)、HTI-286(惠氏)，以及它们的氘代物。
Figure PCTCN2022089229-appb-000016
鹅膏蕈碱类是一类从毒蘑菇提取的、可高效结合哺乳动物RNA聚合酶II的双环八肽，通过终止DNA转录而杀死细胞，这类分子具有高的亲水性和高选择性毒性。
鹅膏蕈碱类例如可以是α-鹅膏蕈碱、β-鹅膏蕈碱、γ-鹅膏蕈碱、ε-鹅膏蕈碱、一羟鹅膏毒肽酰胺或一羟鹅膏毒肽羧酸的二脱氧变体，或者三羟鹅膏毒肽、三羟鹅膏毒肽酰胺、γ-三羟鹅膏毒肽或γ-三羟鹅膏毒肽酰胺的单脱氧变体等，以及它们的氘代物。
澳瑞他汀类是海兔毒素10的合成衍生物，通过抑制微管蛋白聚合来达到有效的有丝分裂抑制作用。澳瑞他汀类例如可以是单甲基澳瑞他汀E(MMAE)、单甲基澳瑞他汀F(MMAF)、澳瑞他汀EB(AEB)、澳瑞他汀EVB(AEVB)和澳瑞他汀F苯二胺(AFP)，以及它们的氘代物。
Figure PCTCN2022089229-appb-000017
刺孢霉素有7个主要衍生物，其中刺孢霉素γ活性最高，在临床上运用最多。刺孢霉素含有寡糖、刺孢酮和甲基三硫三个部分，其中甲基三硫充当引发装置，还原后启动Bergman重排反应并导致DNA裂解。
倍癌霉素类能特异性地识别DNA小沟,并有效地烷基化DNA碱基N3位的腺嘌呤,具有很高的抗癌活性。倍癌霉素类毒素例如可以是倍癌霉素A、adozelesin、CC-1065，以及它们的氘代物。
Figure PCTCN2022089229-appb-000018
用于本发明的毒素还可以例如是多卡米星类、多柔比星类(例如吗啉代-多柔比星和氰基吗啉代-多柔比星)、多拉司他汀、dolestatin-10、考布他汀、加利车霉素、tubulysins、disorazole、埃坡霉素、紫杉醇、多西他赛、SN-38、托泊替康、根霉素、棘霉素、秋水仙碱、长春碱、长春地辛、雌氮芥、西马多丁、eleutherobin、甲氨蝶呤、甲基叶酸、二氯甲氨喋呤、5-氟尿嘧啶、6-巯嘌呤、巯嘌呤、美法仑、长春罗新、leurosideine、放线菌素、柔红霉素和柔红霉素轭合物、丝裂霉素C、丝裂霉素A、洋红霉素、氨基蝶呤、他利霉素、鬼臼毒素类(例如依托泊苷或磷酸依托泊苷)、长春新碱、喜树碱等，以及它们的氘代物。
在一些实施方式中，本发明的毒素选自美登素类及其氘代物，例如DM1、DM4，以及它们的氘代物。
在一些实施方式中，本发明的毒素选自哈米特林类及其氘代物，例如E7974、HTI-286，以及它们的氘代物。
在一些实施方式中，本发明的毒素选自鹅膏蕈碱类及其氘代物，例如α-鹅膏蕈碱、β-鹅膏蕈碱、γ-鹅膏蕈碱、ε-鹅膏蕈碱等，以及它们的氘代物。
在一些实施方式中，本发明的毒素选自澳瑞他汀类及其氘代物，包括但不限于单甲基澳瑞他汀E(MMAE)、单甲基澳瑞他汀F(MMAF)、澳瑞他汀EB(AEB)、澳瑞他汀EVB(AEVB)和澳瑞他汀F苯二胺(AFP)，以及它们的氘代物。
在一些实施方式中，本发明的毒素为单甲基澳瑞他汀F(MMAF)及其氘代物。在一些实施方式中，本发明的毒素为单甲基澳瑞他汀E(MMAE)及其氘代物。
在一些实施方式中，本发明的毒素选自刺孢霉素类及其氘代物，例如刺孢霉素γ及其氘代物。
在一些实施方式中，本发明的毒素选自倍癌霉素类及其氘代物，例如倍癌霉素A、adozelesin、CC-1065，以及它们的氘代物。
在一些实施方式中，本发明的毒素选自例如美登素类(例如DM1、DM4)、哈米特林类(例如E7974、HTI-286)、鹅膏蕈碱类(例如α-鹅膏蕈碱、β-鹅膏蕈碱、γ-鹅膏蕈碱、ε-鹅膏蕈碱)、澳瑞他汀类(例如MMAE、MMAF、AEB、AEVB和AFP)、刺孢霉素类(例如刺孢霉素γ)、倍癌霉素类(例如倍癌霉素A、adozelesin、CC-1065)等，以及上述这些毒素的氘代物。
连接子
本发明的双特异抗体偶联物中，通过连接子实现双特异抗体与毒素的连接。本发明的连接子可以是单官能的，将单个毒素分子连接至抗体上的单个位点；也可以是多官能的，将两个/多个毒素分子连接至抗体上的单个位点、或者将一个毒素分子连接至抗体上的两个/多个位点。
作为抗体偶联物的连接子至少需要满足以下两个条件：(1)在体内足够稳定，不会在血液循环中脱落，避免因毒素脱落而产生毒性；(2)在靶点有效地释放毒素。
连接子可以分为可裂解连接子和不可裂解连接子，所述连接子中的一个或多个氢原子任选地被氘代。其中，可裂解连接子又包括化学裂解性连接子和酶催化裂解性连接子两种。
在一些实施方式中，本发明的连接子为可裂解连接子或其氘代物。可裂解连接子通常易于在细胞内条件下裂解，例如通过溶酶体过程裂解。
在一些实施方式中，本发明的连接子为化学裂解性连接子或其氘代物。
在一些实施方式中，本发明的连接子为酶催化裂解性连接子或其氘代物。
酶催化裂解性连接子例如可以是包含肽组分的连接子，所述肽组分包括两个或更多个氨基酸并且可被细胞内蛋白酶诸如溶酶体蛋白酶或内体蛋白酶裂解。肽组分可以包含天然氨基酸残基和/或次要氨基酸和/或非天然存在的氨基酸类似物，诸如瓜氨酸。肽组分可以设计和优化以供特定酶进行酶促裂解，所述特定酶例如，肿瘤相关的蛋白酶、组织蛋白酶B、C或D或纤维蛋白溶酶蛋白酶。
酶催化裂解性连接子可以是含二肽的连接子，例如含有缬氨酸-瓜氨酸(Val-Cit)或苯丙氨酸-赖氨酸(Phe-Lys)的连接子。包含在连接子中的合适二肽的其它实例包括Val-Lys、Ala-Lys、Me-Val-Cit、Phe-homoLys、Phe-Cit、Leu-Cit、Ile-Cit、Trp-Cit、Phe-Arg、Ala-Phe、Val-Ala、Met-Lys、Asn-Lys、Ile-Pro、 Ile-Val、Asp-Val、His-Val、Met-(D)Lys、Asn-(D)Lys、Val-(D)Asp、NorVal-(D)Asp、Ala-(D)Asp、Me3Lys-Pro、苯基Gly-(D)Lys、Met-(D)Lys、Asn-(D)Lys、Pro-(D)Lys和Met-(D)Lys。可裂解连接子也可以包括更长的肽组分，诸如三肽、四肽或五肽。实例包括但不限于三肽Met-Cit-Val、Gly-Cit-Val、(D)Phe-Phe-Lys和(D)Ala-Phe-Lys，以及四肽Gly-Phe-Leu-Gly和Ala-Leu-Ala-Leu。
在一些实施方式中，本发明的连接子选自缬氨酸-瓜氨酸(Val-Cit)或苯丙氨酸-赖氨酸(Phe-Lys)，以及它们的氘代物。在一些实施方式中，本发明的连接子选自缬氨酸-瓜氨酸(Val-Cit,Vc)或其氘代物。
在一些实施方式中，本发明的连接子为缬氨酸-瓜氨酸(Val-Cit，Vc)且毒素为MMAE，即，本发明的连接子-毒素为Vc-MMAE。Vc-MMAE(mc-vc-PAB-MMAE,Maleimidocaproyl-valine-citrulline-p-aminobenzyloxycarbonyl monomethylauristatin E，Vedotin)的结构如下所示。
Figure PCTCN2022089229-appb-000019
本发明的Vc-MMAE(mc-vc-PAB-MMAE)的各个连接子嵌段和毒素MMAE都可以通过商购的方式获得。
本发明所述的“mc”是指6-马来酰亚胺己酸，或者其与双特异抗体和/或连接子通过化学键连接后形成的基团。
6-马来酰亚胺己酸(mc)的结构式为：
Figure PCTCN2022089229-appb-000020
本发明所述的“PAB”是指对氨基苄醇，或者其与连接子和/或毒素通过化学键连接后形成的基团。
对氨基苄醇(PAB)的结构式为：
Figure PCTCN2022089229-appb-000021
双特异抗体偶联物
本发明的双特异抗体偶联物中，抗体可以是如上所述的任意双特异抗体；连接子可以是如上所述的任意连接子，例如含二肽的连接子，例如含有缬氨酸-瓜氨酸(Val-Cit)或苯丙氨酸-赖氨酸(Phe-Lys)的连接子；毒素可以是如上所述的任意毒素，例如DM1、DM4、MMAE、MMAF等。
在一些实施方式中，双特异抗体偶联物的平均药物：抗体比(DAR)约为1-6。在一些实施方式中，双特异抗体偶联物的平均药物：抗体比(DAR)例如约为1、2、3、4、5、6。在一些实施方式中，双特异抗体偶联物的DAR约为2、3、4、5。在一些实施方案中，所述双特异抗体偶联物的DAR为3-4。在一些实施方式中，双特异抗体偶联物的DAR约为3、4。在一些实施方式中，双特异抗体偶联物的DAR约为2.5、2.6、2.7、2.8、2.9、3、3.1、3.2、3.3、3.4、3.5、3.6、3.7、3.8、3.9、4、4.1、4.2、4.3、4.4、4.5、4.6、4.7、4.8、4.9、5等。
双特异抗体偶联物的结构可以如下式(I)所示：
Figure PCTCN2022089229-appb-000022
其中，
Figure PCTCN2022089229-appb-000023
表示双特异抗体，中间部分为连接子缬氨酸-瓜氨酸(Val-Cit,Vc)部分，最右侧的Drug表示毒素部分。
还原型抗体上的游离巯基可以与马来酰胺基团(例如，6-马来酰亚胺己酸(mc))作用，从而实现抗体与连接子(Val-Cit,Vc)之间的连接。
连接子通过对氨基苄醇与最右侧的Drug(毒素部分)连接。
式(I)的双特异抗体
Figure PCTCN2022089229-appb-000024
为如上文所述的双特异抗体。
在一些实施方式中，所述双特异抗体偶联物的结构中进一步明确了DAR值(n)，如下式(I’)所示：
Figure PCTCN2022089229-appb-000025
其中，
Figure PCTCN2022089229-appb-000026
表示如上所述的双特异抗体，中间部分为连接子缬氨酸-瓜氨酸(Val-Cit，简称Vc)部分，连接子通过6-马来酰亚胺己酸与所述双特异抗体连接，通过对氨基苄醇与最右侧的Drug(毒素部分)连接，n表示平均药物：抗体比(DAR)，n选自1-6，例如约为1、2、3、4、5、6，例如约为2.5、2.6、2.7、2.8、2.9、3、3.1、3.2、3.3、3.4、3.5、3.6、3.7、3.8、3.9、4、4.1、4.2、4.3、4.4、4.5、4.6、4.7、4.8、4.9、5。
双特异抗体偶联物的结构可以如下式(II)所示。
Figure PCTCN2022089229-appb-000027
其中，
Figure PCTCN2022089229-appb-000028
表示双特异抗体，[]中的结构为连接子-毒素部分Vc-MMAE，n(即平均药物：抗体比DAR值)可以例如约为1-6，例如1、2、3、4、5、6，例如3-4，例如2.5、2.6、2.7、2.8、2.9、3、3.1、3.2、3.3、3.4、3.5、3.6、3.7、3.8、3.9、4、4.1、4.2、4.3、4.4、4.5、4.6、4.7、4.8、4.9、5等。
在一些实施方案中，所述双特异抗体偶联物的平均药物：抗体比(DAR)为3-4，例如3、3.1、3.2、3.3、3.4、3.5、3.6、3.65、3.7、3.75、3.8、3.9、4。
还原型抗体上的游离巯基可以与马来酰胺基团(例如，6-马来酰亚胺己酸(mc))作用，从而实现抗体与连接子(Val-Cit,Vc)之间的连接。
连接子通过对氨基苄醇与最右侧的毒素单甲基澳瑞他汀E(MMAE)连接。
式(II)的双特异抗体
Figure PCTCN2022089229-appb-000029
为如上文所述的双特异抗体。
在一些实施方式中，本发明的双特异抗体偶联物可以是T51-MMAE、T52-MMAE、T53-MMAE、T54-MMAE、T55-MMAE、T56-MMAE、T57-MMAE、T58-MMAE。这些双特异抗体偶联物中的双特异抗体T51、T52、T53、T54、T55、T56、T57和T58可以是去岩藻糖基化的(即T51-AF、T52-AF、T53-AF、T54-AF、T55-AF、T56-AF、T57-AF和T58-AF)，对应的双特异抗体偶联物为T51-AF-MMAE、T52-AF-MMAE、T53-AF-MMAE、T54-AF-MMAE、T55-AF-MMAE、T56-AF-MMAE、T57-AF-MMAE和T58-AF-MMAE。
本发明所述的“T54-MMAE”是指双特异抗体偶联物，具体结构为T54-(mc-vc-PAB-MMAE)n，其中n 为平均药物：抗体比(DAR)；T54、mc、vc、PAB和MMAE的定义如上文所述。
本发明所述的“T54-AF-MMAE”是指双特异抗体偶联物，具体结构为T54-AF-(mc-vc-PAB-MMAE)n，其中n为平均药物：抗体比(DAR)；T54-AF、mc、vc、PAB和MMAE的定义如上文所述。
对于T51-MMAE、T52-MMAE、T53-MMAE、T55-MMAE、T56-MMAE、T57-MMAE、T58-MMAE，作与“T54-MMAE”的定义相类似的理解。
对于T51-AF-MMAE、T52-AF-MMAE、T53-AF-MMAE、T55-AF-MMAE、T56-AF-MMAE、T57-AF-MMAE、T58-AF-MMAE，作与“T54-AF-MMAE”的定义相类似的理解。
双特异抗体偶联物的制备
可以通过本领域已知的几种途径之一，采用本领域技术人员已知的有机化学反应、条件和试剂来制备本公开的ADC(参见，例如，Bioconjugate Techniques(G.T.Hermanson,2013,Academic Press，以及本文提供的实施例)。例如，可以通过以下方式实现偶联：(1)抗体的亲核基团或亲电子基团与双官能连接子反应，经由共价键形成抗体-连接子中间体，然后与活化的毒素(例如DM1、DM4、MMAE、MMAF等)反应；或(2)毒素的亲核基团或亲电子基团与连接子反应，经由共价键形成连接子-毒素，然后与抗体的亲核基团或亲电子基团反应。
如上所述，毒素可以经由适当的连接子与抗体上的各个基团偶联以提供ADC。例如，偶联可以通过抗体表面赖氨酸，或通过氧化的碳水化合物或通过已经通过还原一个或多个链间二硫键而释放的半胱氨酸残基来进行。可选地，可以将抗体修饰为包括其它半胱氨酸残基或提供反应性把手的非天然氨基酸，诸如硒代蛋氨酸、对乙酰基苯丙氨酸、甲酰甘氨酸或对叠氮基甲基-L-苯丙氨酸。此类修饰是本领域熟知的(参见，例如，美国专利号7,521,541；8,455,622和9,000,130；Hofer等人，Biochemistry,48:12047-12057(2009)；Axup等人，PNAS,109:16101-16106(2012)；Wu等人，PNAS,106:3000-3005(2009)；Zimmerman等人，Bioconj.Chem.,25:351-361(2014))。
在某些实施方式中，本公开的ADC包含经由适当的连接子偶联至双特异抗体上已通过还原一个或多个链间二硫键而释放的半胱氨酸残基的澳瑞他汀类毒素(例如MMAE、MMAF)。
为了与半胱氨酸残基偶联，可以进行抗体链间二硫键的部分还原，然后与连接子-毒素偶联。合适的还原剂是本领域已知的，并且包括例如二硫苏糖醇(DTT)、三(2-羧乙基)膦(TCEP)、2-巯基乙醇、半胱胺和许多水溶性膦。
ADC的平均DAR可以通过标准技术，诸如UV/VIS光谱分析、基于ELISA的技术、色谱技术(诸如疏水相互作用色谱法(HIC))、UV-MALDI质谱法(MS)和MALDI-TOF MS来测定。另外，还可以通过本领域已知的各种技术来分析药物连接形式的分布(例如，DAR0、DAR1、DAR2等种类的百分率)，所述技术包括MS(有或无伴随的色谱分离步骤)、疏水相互作用色谱法、反相HPLC或等电聚焦凝胶电泳(IEF)(参见，例如，Sun等人,Bioconj Chem.,28:1371-81(2017)；Wakankar等人,mAbs,3:161-172(2011))。
在某些实施方案中，通过疏水相互作用色谱法(HIC)技术测定ADC的平均DAR。
偶联后，可通过本领域已知的纯化方法将ADC纯化并与未偶联的反应物和/或任何偶联物聚集体分离。此类方法包括但不限于尺寸排阻色谱法(SEC)、疏水相互作用色谱法(HIC)、离子交换色谱法、色谱聚焦、超滤、离心超滤及其组合。
本发明还涉及包括如上所述的双特异抗体偶联物的药物组合物。
术语“治疗”包括：(1)预防或延迟在可能患有或有倾向患该状态、疾患或病症但还未经历或显示该状态、疾患或病症的临床或亚临床症状的受试者中发展的该状态、疾患或病症的至少一种临床或亚临床症状出现；或(2)抑制该状态、疾患或病症，即阻止、减少或延缓疾病的发展或其复发(在维持治疗的情况下)或者其至少一种临床症状或者亚临床症状；或(3)减轻疾病，即，导致该状态、疾患或病症或至少一种其临床或亚临床症状的减退。接受治疗的受试者所获得的益处在统计学上是显著的，或者至少对患者或医师来说是可察觉的。
如本文所用，术语“治疗有效”或“有效”应用于剂量或量是指当向受试者给药用于治疗(例如预防或改善)状态、疾患或病症时，足以使这样的治疗或预防产生有效结果的化合物或药物组合物的量。“治疗有效量”将根据所给药的化合物或细菌或类似物，以及疾病和其严重程度，和接受治疗的哺乳动物的年龄、体重、身体状况和反应情况而变化。
如与本公开的组合物一起使用的短语“药学上可接受的”，是指这样的组合物的分子实体和其他成分在生理上是可耐受的，并且当给药至哺乳动物(例如人类)时通常不会产生不良反应。
术语“药物制剂”或“药物组合物”是指处于允许其中包含的活性成分的生物活性有效的形式且不包含对将被给药制剂的受试者有不可接受的毒性的额外的组分的制剂或组合物。
术语“载体”是指用于给药化合物的稀释剂、佐剂、赋形剂或媒介物。这样的药物载体可以是无菌液体，诸如水和油，包括石油、动物、植物或合成原料的无菌液体，诸如花生油、大豆油、矿物油、芝麻油等。特别是对于可注射溶液，使用水或水溶液盐水溶液和水性葡萄糖和甘油溶液作为载体。可替代地，载体可以是固体剂型载体，包括但不限于一种或多种粘结剂(用于压缩丸)、助流剂、包封剂、调味剂和着色剂。在E.W.Martin的《雷明顿药学(Remington’s Pharmaceutical Sciences)》中描述了合适的药物载体。
其他实施方式涉及如本公开的双特异抗体偶联物或包含该双特异抗体偶联物的药物组合物用于预防和/或治疗癌症的用途。在另一实施方式中，提供了双特异抗体偶联物在制备用于预防和/或治疗癌症的药物中的用途。在一些实施方式中，所述用途是用于治疗癌症。其他类似的实施方式为预防和/或治疗癌症的方法，包括向有需要的患者施用治疗有效量的双特异抗体偶联物。在一些实施方式中，受试者是哺乳动物。在一些实施方式中，受试者是人类。
本文描述的癌症例如为表达HER2的癌症，例如为HER2中低水平表达的癌症，例如但不限于HER2 + 乳腺癌、卵巢癌、子宫内膜癌、子宫颈癌、胃癌、食管癌、肺癌、头颈癌、膀胱癌、胆管癌和/或结直肠癌。
本发明的双特异抗体偶联物可以单独给药，或者与其他适合治疗癌症的药剂联合给药。例如，该双特异抗体偶联物可以与其他疗法联合使用，诸如用于化疗或靶向疗法，或用于免疫疗法。类似地，它们可以与放射疗法或外科手术联合使用。
双特异抗体偶联物可以通过任何合适的途径递送。在一些实施方式中，通过注射或输注、通过皮肤贴片、通过库/泵装置或通过吸入来递送该偶联物。在这些实施方式的各个方面，通过静脉内、腹腔内、皮下或肌肉内给药该偶联物。
可以一天一次或更少的频率施用一些实施方式中的偶联物。例如，在一些实施方式中，每两天一次、每三天一次、每四天一次、每五天一次、每六天一次、每周一次、每两周一次、每三周一次、每四周一次、每月、每两个月一次、每三个月一次或每六个月一次施用双特异抗体偶联物。
有效分子的最佳剂量水平将取决于多种因素，包括患者的年龄、体重、身体状况、与其他药物的可能组合以及病情的严重程度。具体剂量可由本领域技术人员根据具体情况来进行确定。
试剂盒、单位剂型和制造物品
本公开还提供了包括本文所述偶联物或其组合物的试剂盒、单位剂型和制造物品。试剂盒、单位剂型、和制造物品可包括，例如，本文所述偶联物的小瓶(诸如密封小瓶)预充注射器以及自注射器(笔型)。
本文通过根据IUPAC-IUB生物化学命名委员会的氨基酸常见的3字母符号或1字母(单字母)符号来指代氨基酸。
实施例
下面将结合具体实施例对本发明技术方案进行描述，对本发明的上述内容作进一步的详细说明，但不应将此理解为本发明上述主题的范围仅限于以下实施例。凡基于本发明上述内容所实现的技术均属于本发明的范围。
实施例1双特异抗体的制备
利用电转染技术，将含双特异抗体重链质粒和轻链质粒分别共转染CHO-K1细胞。用新鲜的opti培养基(无谷氨酰胺，通过商购获得)重悬细胞并接种于96孔板中，37℃，5％CO 2培养箱培养恢复24h。恢复24h后，25μM MSX的新鲜opti培养基重悬，每3天更换新鲜培养基，于25μM MSX压力下持续培养2-4周，挑出存活的混合细胞克隆。通过有限稀释法将混合细胞用新鲜的opti培养基以0.5个细胞/孔密度接种于96孔板，37℃，5％CO 2培养箱培养2周左右，ELISA方法检测能够表达抗体的克隆，经过两轮的有限稀释法筛选，最终得到能够表达抗体T54的单克隆。
参照中国专利申请CN201610194325.7描述的方法，将CHO-K1细胞经过TALEN敲除FUT8基因和 LCA加压，构建岩藻糖敲除细胞株D57，使用D57细胞株，采用与实施例1制备T54类似的方法来制备去岩藻糖基化的双特异抗体T54-AF。
将双特异抗体的稳定系培养数天后，上清液经protein A亲和纯化和阳离子交换纯化，得到双特异抗体T54、T54-AF。
采用与以上实施例1类似的方法，利用相应的双特异抗体重链质粒和轻链质粒分别共转染CHO-K1细胞，制备得到抗体T51、T51-AF、T52、T52-AF、T53、T53-AF、T55、T55-AF、T56、T56-AF、T57、T57-AF、T58、T58-AF。
实施例2双特异抗体偶联物的制备
将实施例1得到的双特异抗体T54透析到偶联缓冲液(25mM Na 2B 4O 7/25mM NaCl,1mM DTPA,pH 7.4)，超滤浓缩至浓度5mg/ml以上，调整浓度至5mg/ml。加入还原剂TCEP，水浴25℃，反应2h。
Vc-MMAE(mc-vc-PAB-MMAE)的各个连接子嵌段和毒素MMAE都可以通过商购的方式获得。
其中，“mc嵌段”是指6-马来酰亚胺己酸，6-马来酰亚胺己酸的结构式为：
Figure PCTCN2022089229-appb-000030
“PAB”是指对氨基苄醇，对氨基苄醇(PAB)的结构式为：
Figure PCTCN2022089229-appb-000031
Vc-MMAE(mc-vc-pab-MMAE)的化学结构式如下：
Figure PCTCN2022089229-appb-000032
各个嵌段的化学偶联都使用本领域常规的酯化或脱水反应完成即可。
将mc-vc-pab-MMAE溶解于DMSO至10mM。将mc-vc-pab-MMAE与抗体按摩尔比10：1混合，水浴25℃，反应2h偶联。
将偶联产物透析到透析缓冲液(20mM His-acetate,pH 5.5)，并经0.22μm膜过滤，得到双特异抗体偶联物T54-MMAE。
采用与以上实施例2类似的方法，制备得到双特异抗体偶联物T54-AF-MMAE、T51-MMAE、 T51-AF-MMAE、T52-MMAE、T52-AF-MMAE、T53-MMAE、T53-AF-MMAE、T55-MMAE、T55-AF-MMAE、T56-MMAE、T56-AF-MMAE、T57-MMAE、T57-AF-MMAE、T58-MMAE、T58-AF-MMAE。
实施例3双特异抗体T54、T54-AF和双特异抗体偶联物T54-MMAE、T54-AF-MMAE的表征
对双特异抗体T54、T54-AF和双特异抗体偶联物T54-MMAE、T54-AF-MMAE进行SEC纯度分析和HIC-HPLC分析。
SEC纯度分析所用的仪器和条件如下：
仪器:Thermo Ultimate 3000
柱子:Waters,XBridge
Figure PCTCN2022089229-appb-000033
SEC 3.5μm(7.8×300mm).
流动相:15％异丙醇的PBS,pH 7.4
流速:0.5ml/min,30min
上样量:40μg。
HIC-HPLC分析所用的仪器和条件如下：
柱子:Thermo MAbPac HIC-Butyl
流动相:
流动相A:50mM Na 3PO 4，1.5M(NH 4) 2SO 4，5％异丙醇，pH6.95
流动相B:50mM Na 3PO 4，5％异丙醇，pH6.95
流速:0.5ml/min
上样量:100μg。
1、对双特异抗体及其偶联物的检测结果
将抗体T54及其偶联物T54-MMAE分别进行SEC纯度分析，结果分别如图5A、图6A所示。由图中可以看出，T54及T54-MMAE的聚体含量分别为1.95％、0.24％，单体含量分别为97.17％、96.75％，二者的单体含量相差不大。
将抗体T54及其偶联物T54-MMAE分别进行HIC-HPLC分析，结果分别如图7、图8A所示。由图中可以看出，抗体偶联物T54-MMAE偶联了不同数量的毒素，其中偶联4个毒素的比例为73.31％，平均DAR值为3.70，具体数据如表1-1所示。
表1-1不同DAR值的偶联物T54-MMAE的比例
  DAR 0(％) DAR 2(％) DAR 4(％) DAR 6(％) DAR 8(％) 平均DAR
T54-MMAE 1.29 20.29 73.31 2.58 2.53 3.70
2、对去岩藻糖基化的双特异抗体及其偶联物的检测结果
将抗体T54-AF及其偶联物T54-AF-MMAE分别进行SEC纯度分析，结果分别如图5B、图6B所 示。由图中可以看出，T54-AF及T54-AF-MMAE的聚体含量分别为0.72％、0.17％，单体含量分别为99.28％、99.83％，二者的单体含量相差不大。
将抗体偶联物T54-AF-MMAE进行HIC-HPLC分析偶联毒素个数，结果如图8B所示。由图中可以看出，抗体偶联物T54-AF-MMAE偶联了不同数量的毒素，其中偶联4个毒素的比例为66.61％，平均DAR值为3.46，具体数据如表1-2所示。
表1-2不同DAR值的偶联物T54-AF-MMAE的比例
  DAR 0(％) DAR 2(％) DAR 4(％) DAR 6(％) DAR 8(％) 平均DAR
T54-AF-MMAE 5.81 22.34 66.61 3.39 1.85 3.46
实施例4与HER2的结合活性测试
分析对象：抗体T54-AF；
抗体偶联物T54-AF-MMAE；
阳性对照ADC DS8201(Trastuzumab Deruxtecan，一款靶向Her2的抗体偶联药物，由人源化抗Her2抗体trastuzumab通过一种四肽(GGFG)linker偶联拓扑异构酶-I抑制剂喜树碱衍生物(DX-8951衍生物DXd)得到)。
以ELISA的方法检测抗体、抗体偶联物和抗原HER2的结合活性。包被抗原HER2(2μg/ml)，每孔100μl，4℃过夜。加入300μl PBS反复洗4次，加入300μl 3％BSA的PBS，37℃封闭2h，PBS重复洗4次。加入100μl待检测的抗体，37℃孵育1h。PBS重复洗4次，加入100μl稀释好的二抗，37℃孵育1h，弃去二抗，PBS重复洗4次。加入100μl TMB显色液，37℃孵育5-10min。加入50μl稀硫酸终止液终止反应。酶标仪检测OD 450。
分析数据，抗体T54-AF及抗体偶联物T54-AF-MMAE、DS8201与HER2抗原的结合活性曲线如图9所示，EC 50值如表2所示。
表2抗体/抗体偶联物与HER2抗原结合的EC 50值
  T54-AF T54-AF-MMAE DS8201
EC 50(ng/ml) 71.04 430.7 130.4
实施例5对HER2表达阳性的肿瘤细胞(SKBR3、N87)的增殖抑制试验
将对数生长期的HER2表达阳性的肿瘤细胞(SKBR3、N87)用PBS清洗，加入胰酶消化，用10％FBS培养基(DMEM、RPMI1640)中和胰酶后，离心，用相应的10％FBS培养基(DMEM、RPMI1640)将靶细胞重悬，将细胞密度调整到2E4，加入到96孔板中，每孔加100微升，培养24h后，加入用培养基稀释好的不同浓度的药物，每孔100μl(起始浓度300μg/ml，8倍稀释，8个浓度)。72h后，将细胞上清吸出，加入添加有cck8的培养基100μl，孵育4h，检测OD 450。
1、对双特异抗体及其偶联物的检测结果
(1)乳腺癌细胞SKBR3的生长抑制
用软件GraphPad 5.0分析数据，各抗体、抗体偶联物对乳腺癌细胞SKBR3的抑制曲线如图10所示，IC 50值如表3所示。结果显示，T54、T54-MMAE、TDM1、Herceptin都有抑制SKBR3肿瘤细胞生长作用，而抗体偶联物TDM1、T54-MMAE对肿瘤细胞的抑制更明显，最大抑制率可高达80％以上，T54抑制效果次之，最大抑制率可达52.51％，Herceptin最弱，最大抑制率仅为33.54％。
表3抗体/抗体偶联物对乳腺癌细胞SKBR3的生长抑制IC 50值及最大抑制率
  T54 T54-MMAE Herceptin TDM1
IC 50(ng/ml) 90.57 18.63 36.7 14.73
最大抑制率(％) 52.51％ 85.32％ 33.54％ 86.06％
(2)胃癌细胞N87的生长抑制
用软件GraphPad 5.0分析数据，各抗体、抗体偶联物对胃癌细胞N87的抑制曲线如图11所示，IC 50值如表4所示。结果显示，T54、T54-MMAE、TDM1有抑制N87肿瘤细胞生长作用，而抗体偶联物TDM1、T54-MMAE对肿瘤细胞的抑制更明显，最大抑制率可高达70％以上，T54抑制效果次之，最大抑制率可达55.6％。Herceptin对N87没有明显的抑制效果。
表4抗体/抗体偶联物对胃癌细胞N87的生长抑制IC 50值及最大抑制率
  T54 T54-MMAE Herceptin TDM1
IC 50(ng/ml) 118.1 59.74 / 24.49
最大抑制率(％) 55.6％ 82.73％ / 74..86％
2、对去岩藻糖基化的双特异抗体及其偶联物的检测结果
将对数生长期的肿瘤细胞用PBS清洗，加入胰酶消化，用含10％FBS相应的培养基(DMEM、RPMI1640、MEM等)中和胰酶后，离心，用相应的10％FBS培养基(DMEM、RPMI1640、MEM等)将靶细胞重悬，将细胞密度调整到4x10 4/ml，加入到96孔板中，每孔加100微升，培养24h后，加入用培养基稀释好的不同浓度的药物，每孔100μl(起始浓度100μg/ml，6倍稀释，8个浓度)。72h后，将细胞上清吸出，加入添加有cck8的培养基100μl，孵育4h，检测OD450。
分别检测T54-AF-MMAE、DS8201、T54-AF、herceptin、perjeta、Isotype(同型对照，作为阴性抗体)对HER2高中低表达水平的肿瘤细胞的增殖抑制活性，分析数据，结果如图12，显示T54-AF-MMAE对HER2表达阳性的不同肿瘤细胞的抑制生长效果基本上均优于DS8201及T54-AF、herceptin、perjeta单抗。
实施例6对小鼠体内BT474乳腺癌肿瘤细胞的增殖抑制试验
1、对双特异抗体偶联物的检测结果
(1)细胞培养
BT474肿瘤细胞购自ATCC，编号为HTB-20 TM。用含有灭活的10％胎牛血清，1mM Napyr，1X MEM-NEAA，1X MEM VITAMIN，10μg/mL人胰岛素以及RPMI 1640培养基在37℃、5％CO 2的培养箱中培养肿瘤细胞，每隔3至4天待细胞长满后分瓶传代，将处于对数生长期的肿瘤细胞用于体内肿瘤的接种。
(2)肿瘤细胞接种与分组
用PBS+Matrigel(1:1)重悬BT474肿瘤细胞，浓度为1x 10 8/mL，接种于实验动物的右侧胁肋部皮下，100μL/只，在肿瘤生长至平均体积为111mm 3左右时分组给药，共3组，每组5只，具体给药方案见表5-1。
表5-1给药方案表
组别 动物数(只) 治疗 剂量(mg/kg) 给药途径 给药频率
1 5 溶剂对照 — i.p. qw x 2
2 5 TDM1 2 i.p. qw x 2
3 5 T54-MMAE 2 i.p. qw x 2
qw x 2表示：每周给药一次，共给药2周
(3)小鼠体重的测量及实验指标
使用游标卡尺测量肿瘤的长短径，按照肿瘤体积计算公式：体积＝0.5×长径×短径 2计算肿瘤体积，每周2次。在进行肿瘤体积测量的同时，称量小鼠体重。记录小鼠体重的变化与给药时间的关系。同时观察小鼠的存活情况和健康状况，如给药期间动物活动、进食等一般状态。
根据肿瘤体积计算T/C值，其中T为各受试物处理组相对肿瘤体积(RTV)的平均值，C为溶剂对照组相对肿瘤体积(RTV)的平均值。RTV为给药后与给药前的肿瘤体积比值。肿瘤生长抑制率(％)＝(1－T/C)×100％，肿瘤生长抑制率≥60％，并经统计学处理p<0.05为有效。
(4)样品取材与处理
实验结束后，各组小鼠安乐死，剥离肿瘤组织，肿瘤组织称重后摆放整齐拍照。
各组小鼠肿瘤生长曲线及肿瘤生长抑制率见图13、表6-1。TDM1(2mg/kg)、T54-MMAE(2mg/kg)在人源异种移植乳腺癌模型BT474中均表现出很强的抗肿瘤作用，有效地抑制了肿瘤生长。T54-MMAE的抑瘤效果优于TDM1。给药前后，荷瘤鼠的体重均没有减轻，对测试物均表现出良好的耐受性。
表6-1双特异抗体偶联物对人源异种移植乳腺癌模型BT474的抑瘤作用(肿瘤体积)
Figure PCTCN2022089229-appb-000034
Figure PCTCN2022089229-appb-000035
2、对去岩藻糖基化的双特异抗体偶联物的检测结果
(1)细胞培养
用含有灭活的10％胎牛血清，100U/mL的青霉素和100μg/mL的链霉素以及2mM谷氨酰胺的RPMI-1640培养基在37℃、5％CO 2的培养箱中培养BT474细胞，每隔3至4天待细胞长满后分瓶传代，将处于对数生长期的肿瘤细胞用于体内肿瘤的接种。
(2)肿瘤细胞接种与分组
肿瘤细胞用PBS洗涤两次，然后用PBS：Matrigel(体积比1：1混合)重悬，调整细胞浓度为1×10 8/mL，接种于实验动物的右侧前肋部皮下，100μL/小鼠，即1×10 7/小鼠。实验动物在接种前一天起皮下注射苯甲酸雌二醇注射液(2mL：4mg，宁波第二激素厂)，40μg/只，每周一次。待肿瘤生长至平均肿瘤体积达到113mm 3时，根据肿瘤体积分组给药，共7组，每组5只动物，具体给药方案见表5-2。
表5-2给药方案表
组别 动物数 受试物 剂量(mg/kg，mpk) 给药途径 给药频率
G1 5 Vehicle - i.p. qw×4
G2 5 T54-AF-MMAE 4 i.p. qw×4
G3 5 T54-AF-MMAE 1 i.p. qw×4
G4 5 T54-AF-MMAE 0.25 i.p. qw×4
G5 5 DS8201 4 i.p. qw×4
G6 5 DS8201 1 i.p. qw×4
G7 5 DS8201 0.25 i.p. qw×4
qw x 4表示：每周给药一次，共给药4周
(3)小鼠体重的测量及实验指标
使用游标卡尺测量肿瘤的长短径，按照肿瘤体积计算公式：体积＝0.5×长径×短径 2计算肿瘤体积，每周2次。在进行肿瘤体积测量的同时，称量小鼠体重。记录小鼠体重的变化与给药时间的关系。同时观察小鼠的存活情况和健康状况，如给药期间动物活动、进食等一般状态。
根据肿瘤体积计算T/C值，其中T为各受试物处理组相对肿瘤体积(RTV)的平均值，C为溶剂对照组相对肿瘤体积(RTV)的平均值。RTV为给药后与给药前的肿瘤体积比值。肿瘤生长抑制率(％)＝(1－T/C)×100％，肿瘤生长抑制率≥60％，并经统计学处理p<0.05为有效。
(4)样品取材与处理
实验结束后，各组小鼠安乐死，剥离肿瘤组织，肿瘤组织称重后摆放整齐拍照。
(5)实验结果
各组小鼠肿瘤生长曲线及给药后的肿瘤生长抑制情况见图14、表6-2、续表6-2-1和续表6-2-2。结果显示，在人源异种移植乳腺癌模型BT474中，
1)相比空白对照(Vehicle)，T54-AF-MMAE 4mpk在D26开始呈现显著的抑瘤作用；T54-AF-MMAE1mpk在D33(第二次给药后7天)开始呈现显著的抑瘤作用；
2)相比相同剂量的对照药(DS8201)，T54-AF-MMAE高(4mpk)在D26(第一次给药后7天)和D33(第二次给药后7天)呈现显著的优效性。
治疗期间，荷瘤鼠对高、中、低剂量双抗-ADC测试药物均表现出很好的耐受性，各组小鼠体重正常，无异常表现，一般状态良好。
表6-2去岩藻糖基化的双特异抗体偶联物对人源异种移植乳腺癌模型BT474的抑瘤作用(肿瘤体积)
Figure PCTCN2022089229-appb-000036
续表6-2-1去岩藻糖基化的双特异抗体偶联物对人源异种移植乳腺癌模型BT474的抑瘤作用
Figure PCTCN2022089229-appb-000037
Figure PCTCN2022089229-appb-000038
续表6-2-2去岩藻糖基化的双特异抗体偶联物对人源异种移植乳腺癌模型BT474的抑瘤作用
Figure PCTCN2022089229-appb-000039
注：a.均数±标准误差；b.与Vehicle组比较；c.与对照药相同剂量比较；*为<0.001，**为<0.05。
实施例7对小鼠体内N87胃癌肿瘤细胞的增殖抑制试验
(1)细胞培养
用含有灭活的10％胎牛血清、100U/mL的青霉素和100μg/mL的链霉素以及2mM谷氨酰胺的RPMI-1640培养基在37℃、5％CO 2的培养箱中培养肿瘤细胞，每隔3至4天待细胞长满后分瓶传代，将处于对数生长期的肿瘤细胞用于体内肿瘤的接种。
(2)肿瘤细胞接种与分组
肿瘤细胞用PBS洗涤两次，调整细胞浓度为1×10 8/mL，接种于实验动物的右侧前肋部皮下，100μL/小鼠，即1×10 7/小鼠。待肿瘤生长至平均肿瘤体积达到80-120mm 3时，根据肿瘤体积分组给药，共8组，每组8只动物，具体给药方案见下表7-1。
表7-1给药方案表
组别 动物数 受试物 剂量(mg/kg，mpk) 给药途径 给药频率
G1 8 Vehicle - i.p. qw×4
G2 8 DS8201 0.1 i.p. qw×4
G3 8 DS8201 0.25 i.p. qw×4
G4 8 DS8201 1 i.p. qw×4
G5 8 T54-AF-MMAE 0.1 i.p. qw×4
G6 8 T54-AF-MMAE 0.25 i.p. qw×4
G7 8 T54-AF-MMAE 1 i.p. qw×4
G8 8 T54-AF-MMAE 4 i.p. qw×4
注：给药容积依动物体重按10μL/g；i.p.为腹腔注射；qw x 4表示：每周给药一次，共给药4周。
(3)小鼠体重的测量及实验指标
使用游标卡尺测量肿瘤的长短径，按照肿瘤体积计算公式：体积＝0.5×长径×短径 2计算肿瘤体积，每周2次。在进行肿瘤体积测量的同时，称量小鼠体重。记录小鼠体重的变化与给药时间的关系。同时观察小鼠的存活情况和健康状况，如给药期间动物活动、进食等一般状态。
根据肿瘤体积计算T/C值，其中T为各受试物处理组相对肿瘤体积(RTV)的平均值，C为溶剂对照组相对肿瘤体积(RTV)的平均值。RTV为给药后与给药前的肿瘤体积比值。肿瘤生长抑制率(％)＝(1－T/C)×100％，肿瘤生长抑制率≥60％，并经统计学处理p<0.05为有效。
(4)样品取材与处理
实验结束后，各组小鼠安乐死，剥离肿瘤组织，肿瘤组织称重后摆放整齐拍照。
(5)实验结果
各组小鼠肿瘤生长曲线及给药后的肿瘤生长抑制情况如图15和表7-2所示。结果显示，在人源异种移植胃癌模型N87中，
1)DS8201 1mpk，T54-AF-MMAE 0.25mpk、1mpk和4mpk能够显著抑制NCI-N87肿瘤的生长。
2)相同剂量下，T54-AF-MMAE(1mpk)的抑瘤效果显著优于DS8201(1mpk)，p＝0.005(Student's t test)。
治疗期间，荷瘤鼠对高、中、低剂量双抗-ADC测试药物均表现出很好的耐受性，各组小鼠体重正常，无异常表现，一般状态良好。
表7-2去岩藻糖基化的双特异抗体偶联物对人源异种移植胃癌模型N87的抑瘤作用
Figure PCTCN2022089229-appb-000040
注：a.均数±标准误差；
实施例8、本申请各双特异抗体的结构及序列
本申请涉及的双特异抗体T51-T58的结构和序列分别如表8、9所示。
尽管在表8中没有显示，但双特异抗体T51至T58及其对应的去岩藻糖基化双特异抗体T51-AF至T58-AF在Fc区中包含M428L替换以提高双特异抗体的血清半衰期。
表8各双特异抗体的结构
Figure PCTCN2022089229-appb-000041
Figure PCTCN2022089229-appb-000042
*除Fc区中的替换使用Eu编号外，本表使用Kabat编号；
V H-Pert：帕妥珠单抗重链可变区；
V H-Tra：曲妥珠单抗重链可变区；
Tra-LC：曲妥珠单抗轻链。
表8中“重链#1”这一列的具体氨基酸序列如表9所示。
表9抗体的相关描述及其氨基酸序列
Figure PCTCN2022089229-appb-000043
Figure PCTCN2022089229-appb-000044
Figure PCTCN2022089229-appb-000045
Figure PCTCN2022089229-appb-000046
*V-区替换用Kabat编号表示；Fc区替换用Eu编号表示。
**信号序列用于帮助抗体从宿主细胞中分泌，随后被酶切除。
其他说明
本申请包含文件名为“序列表-一种双特异抗体偶联物”的序列表，该序列表的全部内容通过引用并入本文。
除非另有说明，否则在说明书和权利要求书中使用的表示成分的数量、诸如分子量的性质、反应条件等的所有数字都应理解为在所有情况下都被术语“约”修饰。如本文所用，术语“约”和“大约”意为10至15％之内，优选地5至10％之内。因此，除非指示相反情况，否则说明书和所附权利要求书中提出的数值参数是可以根据本发明所寻求获得的所需性质而改变的近似值。至少并且不是为了限制本权利要求范围的等效方案的应用，每个数值参数应当至少是根据所给出的有效数字并采用通常的四舍五入方法来构成的。尽管描述本发明的宽范围的数字范围和参数是近似值，但是特定实施例中给出的数值是尽可能精确的。然而，任何数值固有地包含一定的误差，这些误差必然地由各自测试量度中所存在的标准偏差引起。
除非本文另有说明或明显违背上下文，否则在描述本发明的上下文中(特别是在所附权利要求的上下文中)所使用的术语“一个”、“一种”、“所述/该”以及类似指示词应被解释为既包括单数也包括复数。对本文数值范围的叙述仅旨在用作为引用落入该范围的每个单独的值的简写方法(shorthand method)。除非本文另有说明，每个单独的值都被并入说明书，就像其被单独地并入本文一样。除非本文另有说明或上下文明显矛盾，否则，本文所描述的所有方法都可按任何适宜的顺序执行。本文提供的任何和所有的实施例或示例性语言(例如“诸如”)的使用，仅仅旨在更好地阐明本发明并且不是对本发明的范围进行限制，除非另有说明。说明书中的所有语言都不应解释为指示对本发明的实施所必需的任何非要求的元素。
本文公开的本发明的可替代的元素或实施方式的分组不应被解释为限制。各个组成员可以单独地或者以与所述组的其他成员或者本文内的其他要素任意组合被指代且要求保护。可以预料的是组中的一个或多个成员可因便利性和/或专利性原因包含在组中或从组中删除。当任何这类包括或删除发生时，本申请文件被认为含有经修改的组，以满足对附加的权利要求书中所用的所有马库什组的书面描述。
本文描述了本发明的某些实施方案，包括发明人已知的实施本发明的最佳模式。当然，在阅读上述描述之后，这些所描述的实施方式的变体对于本领域普通技术人员将是显而易见的。本发明人预期普通的技术人员能适当地采用这类变体，且本发明人旨在使得本发明以与本文特定描述不同的方式应用。因此，如适用的法律允许，本发明包括在此附上的权利要求书中述及的主题的所有修改和等同物。此外，除非本文另有说明或上下文另有明确相反指示，否则本发明涵盖上述元素以所有可能的变体的任何组合。
本文公开的特定实施方式可以通过使用由……组成或基本上由……组成的语言进一步在权利要求 中得到限制。当用于权利要求时，无论是提交还是按照修改添加，过渡术语“由……组成”排除未在权利要求中指明的任何元素、步骤或成分。过渡术语“基本上由……组成”将权利要求的范围限制在特定材料或步骤以及那些不会实质上影响基本和新颖的特性(一个或多个)的材料或步骤。所要求保护的本发明的实施方式在本文内在地或明确地描述并实现。
另外，在整个本说明书中引用了大量专利和印刷出版物作为参考文献。上述各参考文献和印刷出版物的全部内容通过引用各自并入本文。
最后，应理解本文公开的本发明的实施方式仅用于阐明本发明的原理。可以采用的其他修改形式也在本发明的范围内。因此，通过举例而非限制的方式，可根据本文的教导利用本发明的可替代的配置。因此，本发明不局限于精确显示和描述的内容。

Claims (20)

  1. 一种双特异抗体偶联物(ADC)、其药学上可接受的盐、溶剂化物或其氘代物，其包括双特异抗体、毒素和连接子；其特征在于，
    所述双特异抗体包含能够识别和/或结合HER2的结构域II的第一互补位，和能够识别和/或结合HER2的结构域IV的第二互补位；所述双特异抗体为Fab-Ig、Ig-Fab或异二聚体Ig形式，优选的，所述双特异抗体为异二聚体Ig形式；
    所述毒素选自美登素类、哈米特林类、鹅膏蕈碱类、澳瑞他汀类、刺孢霉素类或倍癌霉素类；
    所述连接子选自可裂解连接子和不可裂解连接子，所述可裂解连接子包括但不限于化学裂解性连接子和酶催化裂解性连接子，所述酶催化裂解性连接子包括含肽组分的连接子；
    可选的，所述毒素和/或连接子中的一个或多个氢原子任选地被氘代。
  2. 根据权利要求1所述的双特异抗体偶联物、其药学上可接受的盐、溶剂化物或其氘代物，其特征在于，所述双特异抗体包括一对同源的轻链和一对包含两个不同的重链可变结构域的异源重链，其中：
    (1)所述一对同源的轻链包括修改后的曲妥珠单抗轻链可变结构域；
    所述修改后的曲妥珠单抗轻链可变结构域以未修改的曲妥珠单抗轻链可变结构域为基础，进行序列修改，所述的序列修改包含：
    a)S56Y/T/A的任意一种；
    b)N30S/A的任意一种；
    c)T94W/F/Y的任意一种；
    d)H91Y/F/W的任意一种；
    e)P96Y/F/W的任意一种；或
    f)a)至e)的这五类修改方式的任意组合；
    优选的，所述修改后的曲妥珠单抗轻链可变结构域以未修改的曲妥珠单抗轻链可变结构域为基础，进行如下序列修改：N30S、S56Y和T94W这三种修改中的任意一种、两种或三种；
    所述未修改的曲妥珠单抗轻链可变结构域的序列如SEQ ID NO:5所示；
    (2)所述的两个不同的重链可变结构域中，
    1)一个重链可变结构域，其选自未修改的帕妥珠单抗重链可变结构域或修改后的帕妥珠单抗重链可变结构域，所述未修改的帕妥珠单抗重链可变结构域的序列如SEQ ID NO:3所示；
    所述修改后的帕妥珠单抗重链可变结构域以未修改的帕妥珠单抗重链可变结构域为基础，进行序列修改，所述的序列修改包含：
    i)T30A/S/N/D中的任意一种；
    ii)G56A/S/T中的任意一种；或
    iii)i)和ii)这两类修改方式的任意组合；
    优选的，所述修改后的帕妥珠单抗重链可变结构域以未修改的帕妥珠单抗重链可变结构域为基础，进行序列修改，所述的序列修改包含：T30A和/或G56A；
    2)另一个重链可变结构域，其选自未修改的曲妥珠单抗重链可变结构域或修改后的曲妥珠单抗重链可变结构域；所述未修改的曲妥珠单抗重链可变结构域的序列如SEQ ID NO:7所示；
    所述修改后的曲妥珠单抗重链可变结构域以未修改的曲妥珠单抗重链可变结构域为基础，进行序列修改，所述的序列修改包含：
    i)N54T/S/A中的任意一种；
    ii)D98S/W/T/R中的任意一种；或
    iii)i)和ii)这两类修改方式的任意组合；
    优选的，所述修改后的曲妥珠单抗重链可变结构域以未修改的曲妥珠单抗重链可变结构域为基础，进行序列修改，所述的序列修改包含：N54T和/或D98S。
  3. 根据权利要求1或2所述的双特异抗体偶联物、其药学上可接受的盐、溶剂化物或其氘代物，其特征在于，所述的双特异抗体为异二聚体Ig形式，其两条异源重链的Fc区通过杵臼结构(knobs-into-holes)连接。
  4. 根据权利要求1-3任一所述的双特异抗体偶联物、其药学上可接受的盐、溶剂化物或其氘代物，其特征在于，所述的双特异抗体的两个Fc区为包括由M428L组成的替换的修改后的Fc区。
  5. 根据权利要求1-4任一所述的双特异抗体偶联物、其药学上可接受的盐、溶剂化物或其氘代物，其特征在于，所述双特异抗体的Fc包含：
    (1)增加抗体依赖性细胞毒性(ADCC)效应的Fc，或
    (2)增加抗体依赖性细胞吞噬作用(ADCP)的Fc，或
    (3)增加补体依赖性细胞毒性(CDC)活性的Fc。
  6. 根据权利要求1-5任一所述的双特异抗体偶联物、其药学上可接受的盐、溶剂化物或其氘代物，其特征在于，所述双特异抗体为去岩藻糖基化抗体；优选的，所述去岩藻糖基化抗体的获得方法为：由具有岩藻糖基化缺陷的宿主细胞产生；所述岩藻糖基化缺陷为敲除FUT8基因。
  7. 根据权利要求1-6任一所述的双特异抗体偶联物、其药学上可接受的盐、溶剂化物或其氘代物，其特征在于，
    所述的双特异抗体为异二聚体Ig形式，其两条异源重链的Fc区通过杵臼结构(knobs-into-holes)连接；
    优选的，所述的knob部分的Fc区是未修改的帕妥珠单抗重链Fc区或者包括了以下修改：T366W；所述的hole部分的Fc区是未修改的曲妥珠单抗重链Fc区或者包括了以下修改的任意组合：T366S、L368A和Y407V；
    最优选的，所述的knob部分的Fc区是以未修改的帕妥珠单抗重链Fc为基础进行了序列修改，所述的序列修改包括了以下修改：T366W和M428L；所述的hole部分的Fc区是以未修改的曲妥珠单抗重链Fc区为基础进行了序列修改，并且所述的序列修改包括了以下修改：T366S、L368A和Y407V；并且所述的双特异抗体在hole部分的Fc区还包括了如下修改：M428L。
  8. 根据权利要求1-5任一所述的双特异抗体偶联物、其药学上可接受的盐、溶剂化物或其氘代物，其特征在于，
    所述双特异抗体选自T51、T52、T53、T54、T55、T56、T57和T58；或者，
    所述双特异抗体选自去岩藻糖基化的T51(T51-AF)、去岩藻糖基化的T52(T52-AF)、去岩藻糖基化的T53(T53-AF)、去岩藻糖基化的T54(T54-AF)、去岩藻糖基化的T55(T55-AF)、去岩藻糖基化的T56(T56-AF)、去岩藻糖基化的T57(T57-AF)和去岩藻糖基化的T58(T58-AF)。
  9. 根据权利要求1-8任一所述的双特异抗体偶联物、其药学上可接受的盐、溶剂化物或其氘代物，其特征在于，
    所述毒素选自DM1、DM4、E7974、HTI-286、α-鹅膏蕈碱、β-鹅膏蕈碱、γ-鹅膏蕈碱、ε-鹅膏蕈碱、一羟鹅膏毒肽酰胺、三羟鹅膏毒肽、三羟鹅膏毒肽酰胺、γ-三羟鹅膏毒肽、单甲基澳瑞他汀F、单甲基澳瑞他汀E、澳瑞他汀EB、澳瑞他汀EVB、澳瑞他汀F苯二胺、刺孢霉素γ、倍癌霉素A、adozelesin和CC-1065，以及它们的氘代物；优选的，所述毒素选自单甲基澳瑞他汀E和单甲基澳瑞他汀F，以及它们的氘代物；
    所述酶催化裂解性连接子选自缬氨酸-瓜氨酸、或苯丙氨酸-赖氨酸，以及它们的氘代物；优选的，所述酶催化裂解性连接子选自缬氨酸-瓜氨酸及其氘代物。
  10. 一种双特异抗体偶联物(ADC)、其药学上可接受的盐、溶剂化物或其氘代物，其包括双特异 抗体、毒素和连接子；其特征在于，
    (1)所述双特异抗体选自T51、T52、T53、T54、T55、T56、T57和T58；优选的，所述双特异抗体为去岩藻糖基化的抗体T51-AF、T52-AF、T53-AF、T54-AF、T55-AF、T56-AF、T57-AF和T58-AF；
    (2)所述毒素选自DM1、DM4、E7974、HTI-286、单甲基澳瑞他汀F、单甲基澳瑞他汀E、澳瑞他汀EB、澳瑞他汀EVB、澳瑞他汀F苯二胺，以及它们的氘代物；优选的，所述毒素选自单甲基澳瑞他汀E和单甲基澳瑞他汀F，以及它们的氘代物；
    (3)所述连接子选自缬氨酸-瓜氨酸、或苯丙氨酸-赖氨酸，以及它们的氘代物；优选的，所述连接子选自缬氨酸-瓜氨酸及其氘代物。
  11. 根据权利要求10所述的双特异抗体偶联物、其药学上可接受的盐、溶剂化物或其氘代物，其特征在于，
    所述双特异抗体偶联物的结构如下式(I’)所示：
    Figure PCTCN2022089229-appb-100001
    其中：
    (1)
    Figure PCTCN2022089229-appb-100002
    表示如权利要求10所述的双特异抗体；
    (2)中间部分为连接子缬氨酸-瓜氨酸(Val-Cit，简称Vc)部分，连接子通过6-马来酰亚胺己酸与所述双特异抗体连接，通过对氨基苄醇与最右侧的Drug(毒素部分)连接；
    所述毒素选自DM1、DM4、E7974、HTI-286、单甲基澳瑞他汀F、单甲基澳瑞他汀E、澳瑞他汀EB、澳瑞他汀EVB、澳瑞他汀F苯二胺，以及它们的氘代物；
    (3)n表示所述双特异抗体偶联物的平均药物：抗体比(DAR)，选自1-6。
  12. 根据权利要求11所述的双特异抗体偶联物、其药学上可接受的盐、溶剂化物或其氘代物，其特征在于，
    所述双特异抗体偶联物的结构如下式(II)所示：
    Figure PCTCN2022089229-appb-100003
    其中，
    (1)
    Figure PCTCN2022089229-appb-100004
    表示如权利要求10所述的双特异抗体；
    (2)[]中的结构为连接子-毒素部分Vc-MMAE，Vc通过6-马来酰亚胺己酸与所述双特异抗体连 接，Vc通过对氨基苄醇与毒素单甲基澳瑞他汀E(MMAE)连接；
    (3)n为DAR值，选自1-6。
  13. 根据权利要求10-12任一所述的双特异抗体偶联物、其药学上可接受的盐、溶剂化物或其氘代物，其特征在于，所述双特异抗体选自T54、T58、去岩藻糖基化的T54(T54-AF)或去岩藻糖基化的T58(T58-AF)。
  14. 一种药物组合物，包括权利要求1-13任一所述的双特异抗体偶联物、其药学上可接受的盐、溶剂化物或其氘代物，以及药学上可接受的载体或赋形剂。
  15. 权利要求1-13任一所述的双特异抗体偶联物、其药学上可接受的盐、溶剂化物或其氘代物或权利要求14所述的药物组合物在制备用于预防和/或治疗癌症的药物中的应用。
  16. 根据权利要求15所述的应用，其特征在于，所述癌症为表达HER2的癌症，优选的，所述癌症为HER2中低水平表达的癌症；优选的，所述癌症包括乳腺癌、卵巢癌、子宫内膜癌、子宫颈癌、胃癌、食管癌、肺癌、头颈癌、膀胱癌、胆管癌和结直肠癌。
  17. 权利要求1-13任一所述的双特异抗体偶联物、其药学上可接受的盐、溶剂化物或其氘代物或权利要求14所述的药物组合物在制备抗肿瘤联用制剂中的应用，所述的联用制剂中还包括任选的如下治疗肿瘤的活性成分：
    (1)靶向HER2以外的其他靶点的药物；
    (2)细胞治疗类药物；
    (3)免疫治疗类药物。
  18. 一种用于预防和/或治疗癌症的方法，包括向有需要的患者施用治疗有效量的如权利要求1-13任一所述的双特异抗体偶联物、其药学上可接受的盐、溶剂化物或其氘代物或权利要求14所述的药物组合物。
  19. 根据权利要求18所述的方法，其特征在于，所述癌症为表达HER2的癌症，优选的，所述癌症为HER2中低水平表达的癌症；优选的，
    所述癌症包括乳腺癌、卵巢癌、子宫内膜癌、子宫颈癌、胃癌、食管癌、肺癌、头颈癌、膀胱癌、胆管癌和结直肠癌。
  20. 如权利要求1-13任一所述的双特异抗体偶联物、其药学上可接受的盐、溶剂化物或其氘代物或权利要求14所述的药物组合物在治疗癌症中的应用，其特征在于，与下述治疗癌症的药物或治疗方案联用；
    (1)靶向HER2以外的其他靶点的药物；
    (2)细胞治疗类药物；
    (3)免疫治疗类药物；
    (4)手术；
    (5)物理疗法如放疗。
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