WO2023087255A1 - 双特异性抗体及其应用 - Google Patents

双特异性抗体及其应用 Download PDF

Info

Publication number
WO2023087255A1
WO2023087255A1 PCT/CN2021/131804 CN2021131804W WO2023087255A1 WO 2023087255 A1 WO2023087255 A1 WO 2023087255A1 CN 2021131804 W CN2021131804 W CN 2021131804W WO 2023087255 A1 WO2023087255 A1 WO 2023087255A1
Authority
WO
WIPO (PCT)
Prior art keywords
seq
cancer
sequence
heavy chain
light chain
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/CN2021/131804
Other languages
English (en)
French (fr)
Inventor
方丽娟
张敬
华珊
周鹏飞
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Wuhan YZY Biopharma Co Ltd
Original Assignee
Wuhan YZY Biopharma Co Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Wuhan YZY Biopharma Co Ltd filed Critical Wuhan YZY Biopharma Co Ltd
Priority to CN202180104172.1A priority Critical patent/CN118201960B/zh
Priority to CN202510748238.0A priority patent/CN120818062A/zh
Priority to JP2024529262A priority patent/JP2024540461A/ja
Priority to PCT/CN2021/131804 priority patent/WO2023087255A1/zh
Priority to MX2024006079A priority patent/MX2024006079A/es
Priority to US18/711,094 priority patent/US20250002605A1/en
Priority to AU2021474386A priority patent/AU2021474386A1/en
Priority to EP21964409.3A priority patent/EP4435006A4/en
Priority to CA3238406A priority patent/CA3238406A1/en
Priority to KR1020247020170A priority patent/KR20240100453A/ko
Priority to CN202510748232.3A priority patent/CN120818061A/zh
Publication of WO2023087255A1 publication Critical patent/WO2023087255A1/zh
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6849—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a receptor, a cell surface antigen or a cell surface determinant
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/10—Antioedematous agents; Diuretics
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2809—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against the T-cell receptor (TcR)-CD3 complex
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0681—Cells of the genital tract; Non-germinal cells from gonads
    • C12N5/0682—Cells of the female genital tract, e.g. endometrium; Non-germinal cells from ovaries, e.g. ovarian follicle cells
    • 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
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A61K2039/507—Comprising a combination of two or more separate antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/51—Complete heavy chain or Fd fragment, i.e. VH + CH1
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/515—Complete light chain, i.e. VL + CL
    • 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
    • C07K2317/522—CH1 domain
    • 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
    • C07K2317/524—CH2 domain
    • 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
    • C07K2317/526—CH3 domain
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/55—Fab or Fab'
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • 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
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C07K2317/732—Antibody-dependent cellular cytotoxicity [ADCC]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/94—Stability, e.g. half-life, pH, temperature or enzyme-resistance
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2510/00—Genetically modified cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2800/00—Nucleic acids vectors
    • C12N2800/10—Plasmid DNA
    • C12N2800/106—Plasmid DNA for vertebrates
    • C12N2800/107—Plasmid DNA for vertebrates for mammalian

Definitions

  • the present invention relates to the technical field of immunology. Specifically, it relates to bispecific antibodies against EpCAM and CD3 and applications thereof.
  • Bi-specific antibodies (bi-specific antibodies, BsAbs), also known as dual-targeting antibodies, can simultaneously recognize and bind two different antigens or epitopes, and block two different signaling pathways to exert their effects.
  • BsAbs monoclonal antibody
  • mAb monoclonal Ab
  • double antibodies have many advantages: 1 It can redirect specific immune effector cells to adjacent tumor cells to enhance tumor killing, which is achieved by combining mAb therapy.
  • the present invention develops a novel bispecific antibody characterized by comprising an antigen-binding domain specifically binding to EpCAM and an antigen-binding domain specifically binding to CD3; and applications thereof.
  • the present invention relates to the following aspects:
  • a bispecific antibody characterized in that it comprises an antigen-binding domain specifically binding to EpCAM and an antigen-binding domain specifically binding to CD3,
  • antigen-binding domain specifically binding to EpCAM is selected from the group consisting of:
  • an antigen-binding domain specifically binding to EpCAM comprising the following CDRs or variants thereof:
  • CDRL1, CDRL2 and CDRL3 contained in the light chain variable region shown in SEQ ID NO: 13,
  • the sequence of CDRL1 is as shown in SEQ ID NO:32
  • the sequence of CDRL2 is as shown in SEQ ID NO:33
  • the sequence of CDRL3 is as shown in SEQ ID NO:34
  • the sequence of CDRH1 is as shown in SEQ ID NO:34
  • the sequence of CDRH2 is shown in SEQ ID NO:36
  • the sequence of CDRH3 is shown in SEQ ID NO:37; or
  • an antigen-binding domain specifically binding to EpCAM comprising the following CDRs or variants thereof:
  • CDRL1, CDRL2 and CDRL3 contained in the light chain variable region shown in SEQ ID NO: 15,
  • the sequence of CDRL1 is as shown in SEQ ID NO:38
  • the sequence of CDRL2 is as shown in SEQ ID NO:39
  • the sequence of CDRL3 is as shown in SEQ ID NO:40
  • CDRH1 The sequence of CDRH2 is shown in SEQ ID NO:41
  • the sequence of CDRH2 is shown in SEQ ID NO:42
  • the sequence of CDRH3 is shown in SEQ ID NO:43;
  • the antigen-binding domain specifically binding to CD3 is selected from the group consisting of:
  • an antigen-binding domain specifically binding to CD3 comprising the following CDRs or variants thereof:
  • CDRH1, CDRH2 and CDRH3 contained in the heavy chain variable region shown in SEQ ID NO:50, and CDRL1, CDRL2 and CDRL3 contained in the light chain variable region shown in SEQ ID NO:51,
  • the sequence of CDRH1 is shown in SEQ ID NO:44
  • the sequence of CDRH2 is shown in SEQ ID NO:45
  • the sequence of CDRH3 is shown in SEQ ID NO:46
  • the sequence of CDRL1 is shown in SEQ ID As shown in NO:47
  • the sequence of CDRL2 is shown in SEQ ID NO:48
  • the sequence of CDRL3 is shown in SEQ ID NO:49; or
  • an antigen-binding domain specifically binding to CD3 comprising the following CDRs or variants thereof:
  • CDRH1, CDRH2 and CDRH3 contained in the heavy chain variable region shown in SEQ ID NO:58, and CDRL1, CDRL2 and CDRL3 contained in the light chain variable region shown in SEQ ID NO:59,
  • the sequence of CDRH1 is shown in SEQ ID NO:52
  • the sequence of CDRH2 is shown in SEQ ID NO:53
  • the sequence of CDRH3 is shown in SEQ ID NO:54
  • the sequence of CDRL1 is shown in SEQ ID As shown in NO:55
  • the sequence of CDRL2 is shown in SEQ ID NO:56
  • the sequence of CDRL3 is shown in SEQ ID NO:57; wherein the variants of the CDRs and the corresponding CDRs have 3, 2 or 1 amino acid respectively Difference or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity, respectively.
  • bispecific antibody of item 1, wherein the antigen-binding domain specifically binding to EpCAM comprises the following heavy chain variable region and light chain variable region (or variants thereof):
  • antigen-binding domain specifically binding to CD3 comprises the following heavy chain variable region and light chain variable region (or variants thereof):
  • the antigen-binding domain specifically binding to EpCAM is in the form of a Fab fragment
  • the antigen-binding domain specifically binding to CD3 is in the form of ScFv
  • the antigen-binding domain specifically binding to EpCAM comprises the following heavy chain variable region and light chain variable region (or variants thereof):
  • said antigen binding domain that specifically binds CD3 is selected from the group consisting of:
  • said variant has 3, 2 or 1 amino acid difference or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, respectively, from said corresponding variable region or ScFv, respectively. 95%, 96%, 97%, 98%, 99% identity.
  • a light chain-heavy chain pair specifically binding to EpCAM said light chain-heavy chain pair comprising a light chain and a heavy chain, or consisting of it; wherein said light chain comprises a light chain variable region and a light chain constant region (preferred sequence as shown in any of SEQ ID NO:1, 60-65), the heavy chain comprises a heavy chain variable region, CH1 (preferred sequence as shown in SEQ ID NO:2) and the first Fc fragment;
  • the first Fc fragment comprises a hinge region (preferred sequence as shown in SEQ ID NO: 3), CH2 (preferred sequence as shown in any of SEQ ID NO: 6, 7, 66-71) and CH3a;
  • the fusion peptide comprising or consisting of a ScFv specifically binding to CD3 and a second Fc fragment; preferably, the ScFv comprises a heavy chain sequentially from the N-terminus to the C-terminus Variable region, connecting peptide (preferred sequence as shown in SEQ ID NO: 4) and light chain variable region, the second Fc fragment comprises a hinge region in sequence from the N-terminal to the C-terminal direction (preferred sequence is as shown in SEQ ID NO: 3), CH2 (preferred sequence as shown in any of SEQ ID NO: 6, 7, 66-71) and CH3b, preferably the hinge between the C-terminus of the light chain variable region and the second Fc fragment
  • the regions are connected by a linker peptide (preferred sequence as shown in SEQ ID NO:5);
  • the first Fc fragment and the second Fc fragment are human or humanized Fc fragments, such as human IgG Fc fragments, such as IgG1, IgG2, IgG3, IgG4, IgG5 Fc fragments;
  • the first Fc fragment and/or the second Fc fragment comprises one or more substitutions that form a knob-hole pairing between the heavy chain and the fusion peptide, such as one T366 on the CH3 domain is replaced by a relatively large amino acid residue, such as tyrosine (Y) or tryptophan (W), and Y407 on the other CH3 domain is replaced by a relatively small amino acid residue, such as threonine (T), alanine (A) or valine (V), for example comprising one or more substitutions in Table 6;
  • the first Fc fragment and/or the second Fc fragment comprises one or more substitutions, 1) the substitution forms a salt bridge pairing between the heavy chain and the fusion peptide, for example, a CH3 domain contains a one or more substitutions by amino acid residues that are positively charged under physiological conditions, and the other CH3 domain contains one or more substitutions by one or more amino acid residues that are negatively charged under physiological conditions , for example the positively charged amino acid residue is arginine (R), histidine (H) or lysine (K), for example the negatively charged amino acid residue can be aspartic acid (D) or glutamic acid (E), for example, the replaced amino acid residues include one or more of D356, L368, K392, D399 and K409, for example, one or more replacements in Table 7, 2) the replacement is at this heavy A disulfide bond is formed between the chain and the fusion peptide, such as the substitution in Table 8, and/or 3) the substitution leads to a significant reduction in the binding ability between Fc and protein
  • CH3b of the fusion peptide and CH3a of the heavy chain have substitution pairs forming a knob-hole structure
  • CH3b of the fusion peptide and CH3a of the heavy chain have a substitution pair that forms an ionic bond
  • CH3b of the fusion peptide and CH3a of the heavy chain have a substitution pair that forms a disulfide bond
  • CH3b of the fusion peptide and CH3a of the heavy chain have substitutions that lead to decreased binding ability to protein A;
  • CH1 comprises the sequence of SEQ ID No: 2; and/or CL comprises a sequence selected from any one of SEQ ID Nos: 1, 60-65;
  • the first Fc fragment and/or the second Fc fragment comprises CH2 selected from any one of SEQ ID Nos:6, 7, 66-71 and/or SEQ ID Nos:8, 9, 11, 12. CH3 of the sequence of any one of 72-76;
  • sequences of CH3a and CH3b are selected from the group consisting of:
  • said bispecific antibody is selected from the group consisting of:
  • (1) It comprises or consists of a fusion peptide, a heavy chain and a light chain; wherein the fusion peptide comprises SEQ ID NO:18, SEQ ID NO:5, SEQ ID NO:3, SEQ ID NO:6 and SEQ ID NO :11, or consisting of it; the heavy chain comprising or consisting of SEQ ID NO:14, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:6 and SEQ ID NO:8; the light chain comprising SEQ ID NO:1 ID NO:13 and SEQ ID NO:1, or consisting of it;
  • the fusion peptide comprises SEQ ID NO:19, SEQ ID NO:5, SEQ ID NO:3, SEQ ID NO:6 and SEQ ID NO :11, or consisting of it; the heavy chain comprising or consisting of SEQ ID NO:14, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:6 and SEQ ID NO:8; the light chain comprising SEQ ID NO:1 ID NO:13 and SEQ ID NO:1, or consisting of it;
  • fusion peptide comprises SEQ ID NO: 18, SEQ ID NO: 5, SEQ ID NO: 3, SEQ ID NO: 6 and SEQ ID NO :11, or consisting of it; the heavy chain comprising or consisting of SEQ ID NO:16, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:6 and SEQ ID NO:8; the light chain comprising SEQ ID NO: ID NO:15 and SEQ ID NO:1, or consisting of it;
  • fusion peptide comprises SEQ ID NO:19, SEQ ID NO:5, SEQ ID NO:3, SEQ ID NO:6 and SEQ ID NO :11, or consisting of it;
  • the heavy chain comprising or consisting of SEQ ID NO:16, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:6 and SEQ ID NO:8;
  • the light chain comprising SEQ ID NO: ID NO:15 and SEQ ID NO:1, or consisting of it;
  • fusion peptide comprises SEQ ID NO: 18, SEQ ID NO: 5, SEQ ID NO: 3, SEQ ID NO: 7 and SEQ ID NO :12, or consisting of it;
  • the heavy chain comprising or consisting of SEQ ID NO:14, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:7 and SEQ ID NO:9;
  • the light chain comprising SEQ ID NO: ID NO:13 and SEQ ID NO:1, or consisting of it;
  • fusion peptide comprises SEQ ID NO:19, SEQ ID NO:5, SEQ ID NO:3, SEQ ID NO:7 and SEQ ID NO :12, or consisting of it; the heavy chain comprising or consisting of SEQ ID NO:14, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:7 and SEQ ID NO:9; the light chain comprising SEQ ID NO: ID NO:13 and SEQ ID NO:1, or consisting of it;
  • fusion peptide comprises SEQ ID NO: 18, SEQ ID NO: 5, SEQ ID NO: 3, SEQ ID NO: 6 and SEQ ID NO :8, or consisting of it;
  • the heavy chain comprising or consisting of SEQ ID NO:14, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:6 and SEQ ID NO:11;
  • the light chain comprising SEQ ID NO:11; ID NO:13 and SEQ ID NO:1, or consisting of it;
  • fusion peptide comprises SEQ ID NO:19, SEQ ID NO:5, SEQ ID NO:3, SEQ ID NO:6 and SEQ ID NO :8, or consisting of it; the heavy chain comprising or consisting of SEQ ID NO:14, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:6 and SEQ ID NO:11; the light chain comprising SEQ ID NO:11; ID NO:13 and SEQ ID NO:1, or consisting of it;
  • (9) It comprises or consists of a fusion peptide, a heavy chain and a light chain; wherein the fusion peptide comprises SEQ ID NO: 18, SEQ ID NO: 5, SEQ ID NO: 3, SEQ ID NO: 6 and SEQ ID NO :8, or consisting of it; the heavy chain comprising or consisting of SEQ ID NO:16, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:6 and SEQ ID NO:11; the light chain comprising SEQ ID NO:11; ID NO:15 and SEQ ID NO:1, or consisting of it;
  • (10) It comprises or consists of a fusion peptide, a heavy chain and a light chain; wherein the fusion peptide comprises SEQ ID NO:19, SEQ ID NO:5, SEQ ID NO:3, SEQ ID NO:6 and SEQ ID NO :8, or consisting of it; the heavy chain comprising or consisting of SEQ ID NO:16, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:6 and SEQ ID NO:11; the light chain comprising SEQ ID NO:11; ID NO:15 and SEQ ID NO:1, or consisting of it;
  • (11) It comprises or consists of a fusion peptide, a heavy chain and a light chain; wherein the fusion peptide comprises SEQ ID NO:18, SEQ ID NO:5, SEQ ID NO:3, SEQ ID NO:7 and SEQ ID NO :9, or consisting of it; the heavy chain comprising or consisting of SEQ ID NO:14, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:7 and SEQ ID NO:12; the light chain comprising SEQ ID NO:12; ID NO:13 and SEQ ID NO:1, or consisting of it;
  • fusion peptide comprises SEQ ID NO:19, SEQ ID NO:5, SEQ ID NO:3, SEQ ID NO:7 and SEQ ID NO :9, or consisting of it; the heavy chain comprising or consisting of SEQ ID NO:14, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:7 and SEQ ID NO:12; the light chain comprising SEQ ID NO:12; ID NO:13 and SEQ ID NO:1, or consist thereof.
  • a nucleic acid composition comprising: a nucleic acid sequence encoding the bispecific antibody described in any one of items 1-3, preferably,
  • the nucleic acid composition comprises:
  • A) a first expression vector comprising a first nucleic acid encoding an antigen-binding domain or a light chain-heavy chain pair that specifically binds EpCAM as defined in any one of items 1-3;
  • a second expression vector comprising a second nucleic acid encoding a CD3-specific antigen-binding domain or fusion peptide as defined in any one of items 1-3.
  • An expression vector comprising the nucleic acid composition according to item 4.
  • a host cell comprising the expression vector of item 5.
  • a pharmaceutical composition comprising the bispecific antibody described in any one of items 1-3, and a pharmaceutically acceptable carrier, and optionally, treating cancer (EpCAM-positive tumors, such as colorectal cancer, gastric cancer, breast cancer , ovarian cancer, lung cancer (such as non-small cell lung cancer), prostate cancer, pancreatic cancer, liver cancer, retinoblastoma, esophageal cancer, kidney cancer, renal clear cell tumor, squamous cell carcinoma of the skin, basal cell carcinoma of the skin, sarcoma, nasal nerve Glioma, craniopharyngioma, thyroid cancer, cholangiocytoma, bladder cancer, head and neck cancer, cervical cancer, or oral cancer, etc.) and/or drugs for malignant ascites, malignant effusion, malignant pleural effusion, etc.
  • cancer EpCAM-positive tumors, such as colorectal cancer, gastric cancer, breast cancer , ovarian cancer, lung cancer (such as non-small cell lung cancer), prostate cancer
  • the dosage form of the pharmaceutical composition includes gastrointestinal administration dosage form or parenteral administration dosage form; more preferably, the dosage form of the pharmaceutical composition is injection, including intravenous Injection, intravenous drip, subcutaneous injection, local injection, intramuscular injection, intratumoral injection, intraperitoneal injection, intracranial injection, or intracavitary injection.
  • a conjugate or fusion protein comprising the bispecific antibody according to any one of items 1-3, preferably comprising a substance A conjugated or fused to said bispecific antibody, said substance A being selected from Therapeutic agents, prodrugs, proteins (e.g., enzymes), viruses, lipids, biological response modifiers (e.g., immunomodulators), PEGs, hormones, oligonucleotides, diagnostic agents, cytotoxic agents, which may be drugs or Toxins, ultrasound enhancers, nonradioactive labels, detectable labels, such as chemiluminescent labeling compounds (such as luminol, isoluminol, thermal acridinium esters, imidazoles, acridinium salts, and oxalate ), or fluorescent metals (such as 152Eu, or lanthanide labels).
  • Therapeutic agents e.g., prodrugs, proteins (e.g., enzymes), viruses, lipids, biological response modifiers (e.g., immunomodulators), PEG
  • a kit comprising the bispecific antibody described in any one of items 1-3, and optionally, treating cancer (EpCAM-positive tumors, such as colorectal cancer, gastric cancer, breast cancer, ovarian cancer, lung cancer ( Such as non-small cell lung cancer), prostate cancer, pancreatic cancer, liver cancer, retinoblastoma, esophageal cancer, renal cancer, renal clear cell tumor, squamous cell carcinoma of the skin, basal cell carcinoma of the skin, sarcoma, nasal glioma, craniopharynx tumor, thyroid cancer, cholangiocytoma, bladder cancer, head and neck cancer, cervical cancer, or oral cancer, etc.) and/or drugs for malignant ascites, malignant effusion, malignant pleural effusion, etc. drug).
  • cancer EpCAM-positive tumors, such as colorectal cancer, gastric cancer, breast cancer, ovarian cancer, lung cancer ( Such as non-small cell lung cancer), prostate cancer, pancreatic cancer, liver cancer, retin
  • the above-mentioned cancers such as EpCAM-positive tumors, such as colorectal cancer, gastric cancer, breast cancer, ovarian cancer, lung cancer (such as non-small cell lung cancer), prostate cancer, pancreatic cancer, liver cancer, retinoblastoma, esophageal cancer, renal cancer, kidney cancer Clear cell tumor, squamous cell carcinoma of the skin, basal cell carcinoma of the skin, sarcoma, nasal glioma, craniopharyngioma, thyroid cancer, cholangiocytoma, bladder cancer, head and neck tumor, cervical cancer, or oral cavity cancer.
  • EpCAM-positive tumors such as colorectal cancer, gastric cancer, breast cancer, ovarian cancer, lung cancer (such as non-small cell lung cancer), prostate cancer
  • a method for treating cancer and/or malignant ascites, malignant effusion, and malignant pleural effusion comprising administering to a subject a therapeutically effective amount of the bispecific antibody described in any one of items 1-3, the cancer EpCAM-positive Tumors, such as colorectal cancer, gastric cancer, breast cancer, ovarian cancer, lung cancer (such as non-small cell lung cancer), prostate cancer, pancreatic cancer, liver cancer, retinoblastoma, esophageal cancer, kidney cancer, clear cell tumor of the kidney, squamous cell tumor of the skin Carcinoma, basal cell carcinoma of the skin, sarcoma, nasal glioma, craniopharyngioma, thyroid cancer, cholangiocytoma, bladder cancer, head and neck cancer, cervical cancer, or oral cavity cancer.
  • the cancer EpCAM-positive Tumors such as colorectal cancer, gastric cancer, breast cancer, ovarian cancer, lung cancer (such as non-small cell lung cancer), prostate cancer, pan
  • the CDR regions of an antibody are responsible for the binding specificity of the antibody to an antigen.
  • the known antibody heavy and light chain variable region sequences there are currently several methods for determining antibody CDR regions, including the Kabat, IMGT, Chothia, and AbM numbering systems.
  • each application of the definition to the CDRs of an antibody or variant thereof will be within the scope of the terms as defined and used herein.
  • the variable region amino acid sequence of the antibody one skilled in the art can generally determine the specific CDRs without reliance on any experimental data other than the sequence itself.
  • Antibody or “antigen-binding fragment” as used herein refers to a polypeptide or polypeptide complex that specifically recognizes and binds to an antigen.
  • the term “antibody” is used in a broad sense and includes immunoglobulin or antibody molecules, including monoclonal or polyclonal human, humanized, composite and chimeric antibodies and antibody fragments.
  • the term “antibody” thus includes any protein or peptide comprising a molecule comprising at least a portion of an immunoglobulin molecule that has the biological activity of binding to an antigen.
  • antibodies include murine, chimeric, humanized or fully human antibodies prepared by techniques well known to those skilled in the art.
  • Recombinant antibodies such as chimeric and humanized monoclonal antibodies, including human and non-human portions, can be prepared using recombinant DNA techniques well known in the art.
  • the immunoglobulin molecule or antibody molecule of the present application may be of any type (e.g.
  • IgG, IgE, IgM, IgD, IgA and IgY any class of immunoglobulin molecule (e.g. IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclasses.
  • antibody fragment or "antigen-binding fragment” includes, but is not limited to, F(ab')2, F(ab)2, Fab', Fab, Fv, Fd, dAb, Fab/c, complementarity determining regions (CDRs) Fragment, Single-chain Fvs (ScFv), Disulfide-stabilized Fv fragment (dsFv), (dsFv)2, Bispecific dsFv (dsFv-dsFv'), Diabody, Disulfide Bond-stabilized diabodies (ds-Diabody), ScFv multimers (such as ScFv dimers, ScFv trimers), multispecific antibodies formed from a part of an antibody comprising one or more CDRs, Nanobodies, A single domain antibody (sdab), domain antibody, bivalent domain antibody, or any other antibody fragment that binds to an antigen but does not contain a complete antibody structure.
  • CDRs complementarity determining regions
  • an antigen-binding fragment includes any polypeptide or polypeptide complex capable of binding to the same antigen as the parent antibody or parent antibody fragment.
  • the term “antibody fragment” includes aptamers, aptamer aptamer aptamer aptamer aptamer aptamer aptamer aptamer aptamer aptamer aptamer aptamer aptamer aptamer aptamer aptamer aptamer aptamer aptamer aptamer aptamer aptamer aptamer aptamer aptamer aptamer aptamer aptamer aptamer aptamer aptamer aptamer aptamer aptamer aptamer aptamer aptamer aptamer aptamer aptamer aptamer aptamer aptamer aptamer aptamer aptamer aptamer aptamer aptamer aptamer aptamer
  • Single-chain variable fragment refers to a fusion protein of the variable regions of the heavy (VH) and light (VL) chains of an immunoglobulin. In certain aspects, these regions are linked with short linker peptides of 10 to about 25 amino acids.
  • the linker can be rich in glycine for flexibility, serine or threonine for solubility, and can link the N-terminus of VH to the C-terminus of VL and vice versa.
  • the protein retains the properties of the original immunoglobulin, except that the constant regions have been removed and linkers have been introduced.
  • ScFv molecules are known in the art, such as those described in US Patent 5,892,019.
  • the antigen-binding domain binding EpCAM and CD3 is Fab, or ScFv, or non-covalent pairing (Fv) between heavy chain variable region (VH)-light chain variable region (VL).
  • Any of the above-mentioned antibodies or polypeptides may also include additional polypeptides, eg, a signal peptide at the N-terminus of the antibody to direct secretion, or other heterologous polypeptides as described herein, such as a 6 ⁇ His tag for purification.
  • the present invention includes not only complete antibodies, but also antibody fragments with immunological activity or fusion proteins formed by antibodies and other sequences. The invention also provides other proteins or fusion expression products having the antibodies of the invention.
  • the present invention includes any protein or protein conjugates and fusion expression products (ie immunoconjugates and fusion expression products) having heavy chains and light chains containing variable regions, as long as the variable regions are compatible with the antibody of the present invention
  • the variable regions of the heavy and light chains are identical or at least 90% homologous, preferably at least 95% homologous, most preferably 96%, 97%, 98% or more than 99% homologous. Therefore, the present invention includes those molecules having the light and heavy chain variable regions of monoclonal antibodies with CDRs, as long as their CDRs have more than 90% (preferably more than 95%, most preferably 96%, 97% of the CDRs of the present invention). %, 98% or more than 99%) of homology.
  • the invention also includes fragments, variants, derivatives and analogs of the antibodies.
  • Antibodies, antigen-binding fragments of the present application, their variants or derivatives including but not limited to, polyclonal antibodies, monoclonal antibodies, multispecific antibodies (such as bispecific antibodies, trispecific antibodies, etc.), human antibodies , animal-derived antibody, humanized antibody, primatized (primatized) antibody, or chimeric antibody, CDR-grafted and/or modified antibody, single-chain antibody (for example, ScFv), double-chain antibody, antigenic expression
  • Site-binding fragments e.g., Fab, Fab' and F(ab')2, Fd, Fv, single chain Fv (ScFv), scFv, disulfide-linked Fv (dsFv), VL domain- or VH-containing Fragments of domains, fragments produced from Fab expression libraries, and anti-idiotypic (anti-Id) antibodies.
  • the antibody fragment, antigen-binding fragment, derivative or analog of the present invention may be (i) a polypeptide having one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) substituted, and such substitution
  • the amino acid residues may or may not be encoded by the genetic code, or (ii) a polypeptide with a substituent group in one or more amino acid residues, or (iii) a mature polypeptide combined with another compound (such as to extend the half-life of the polypeptide (e.g., polyethylene glycol), or (iv) an additional amino acid sequence fused to the polypeptide sequence (such as a leader sequence or secretory sequence or a sequence or proprotein used to purify the polypeptide sequence, or a fusion protein with a 6 ⁇ His tag).
  • Such fragments, derivatives and analogs are within the purview of those skilled in the art in light of the teachings herein.
  • the antibody of the present invention refers to a polypeptide that has human EpCAM and CD3 binding activity and includes the above-mentioned CDR region.
  • the term also includes variant forms of polypeptides comprising the above CDR regions that have the same function as the antibodies of the present invention. These variations include (but are not limited to): one or more (usually 1-50, preferably 1-30, more preferably 1-20, and most preferably 1-10) amino acid deletions , insertion and/or substitution, and addition of one or several (usually within 20, preferably within 10, more preferably within 5) amino acids at the C-terminal and/or N-terminal. For example, in the art, substitutions with amino acids with similar or similar properties generally do not change the function of the protein.
  • adding one or several amino acids at the C-terminus and/or N-terminus usually does not change the function of the protein.
  • the term also includes active fragments and active derivatives of the antibodies of the invention.
  • Variants of the polypeptide include: homologous sequences, conservative variants, allelic variants, natural mutants, induced mutants, encoded by DNA that can hybridize with the DNA encoding the antibody of the present invention under high or low stringency conditions The protein, and the polypeptide or protein obtained by using the antiserum against the antibody of the present invention.
  • Antibodies of the invention may be (i) polypeptides having one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) substituted, or (ii) having substitutions in one or more amino acid residues or (iii) a mature polypeptide fused to another compound (such as a compound that extends the half-life of the polypeptide, such as polyethylene glycol), or (iv) an additional amino acid sequence fused to the polypeptide sequence to form The polypeptide (such as a leader sequence or a secretory sequence or a sequence or protein sequence used to purify the polypeptide, or a fusion protein formed with a 6His tag). Such fragments, derivatives and analogs are within the purview of those skilled in the art in light of the teachings herein.
  • a “conservative amino acid substitution” is one in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues with similar side chains have been defined in the art and include basic side chains (e.g. lysine, arginine, histidine), acidic side chains (e.g.
  • aspartic acid glutamic acid
  • uncharged polar side chains e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine
  • nonpolar side chains e.g., alanine , valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan
  • side chains of ⁇ -branches for example, threonine, valine, isoleucine amino acids
  • aromatic side chains e.g. tyrosine, phenylalanine, tryptophan, histidine
  • nonessential amino acid residues of an immunoglobulin polypeptide are preferably replaced with other amino acid residues from the same side chain family.
  • a string of amino acids may be replaced by a structurally similar string of amino acids that differ in sequence and/or composition of side chain families.
  • Non-limiting examples of conservative amino acid substitutions are provided in the table below, where a similarity score of 0 or higher indicates a conservative substitution between these two amino acids.
  • the conservative substitution is preferably a substitution wherein one amino acid residue within the following groups (a)-(e) is replaced by another amino acid residue within the same group: (a) small Aliphatic, non-polar or weakly polar residues: Ala, Ser, Thr, Pro and Gly; (b) polar, negatively charged residues and their (uncharged) amides: Asp, Asn, Glu and Gln; (c) polar, positively charged residues: His, Arg, and Lys; (d) large aliphatic, nonpolar residues: Met, Leu, Ile, Val, and Cys; and (e ) Aromatic residues: Phe, Tyr and Trp.
  • Particularly preferred conservative substitutions are as follows: Ala by Gly or by Ser; Arg by Lys; Asn by Gln or His; Asp by Glu; Cys by Ser; Gln by Asn; Glu by Asp; Gly replaced by Ala or replaced by Pro; His replaced by Asn or replaced by Gln; Ile replaced by Leu or replaced by Val; Leu replaced by Ile or replaced by Val; Lys replaced by Arg, replaced by Gln or replaced by Glu; Met Substitution of Leu, substitution of Tyr or substitution of Ile; substitution of Phe by Met, substitution of Leu or substitution of Tyr; substitution of Ser by Thr; substitution of Thr by Ser; substitution of Trp by Tyr; substitution of Tyr by Trp; and/or substitution of Phe To Val, to Ile or to Leu.
  • Fc amino acid numbering follows Kabat numbering. "Kabat numbering” refers to the numbering system described by Kabat et al., as described in the US Department of Health and Human Services, "Sequence of Proteins of Immunological Interest” (1983). See the table below for the specific numbers:
  • the 221-227th amino acid is the hinge (hinge) domain
  • the 228th-340th amino acid is the CH2 domain of the second constant region of the heavy chain
  • Amino acids 341-447 are the CH3 domain of the third constant region of the heavy chain.
  • Antibodies can be modified to increase heterodimer pairing efficiency.
  • the Fc fragment of the heavy chain of the monovalent unit and/or the Fc fragment of the fusion peptide may comprise one or more substitutions that form a knob-hole structure as compared to the wild-type antibody fragment right.
  • Knob-and-hole configurations are known in the art. See, eg, Ridgway et al., "'Knob-into-holes' engineering of antibody CH3 domains for heavy chain heterodimerization," Protein Engineering 9(7):617-21 (1996).
  • T366 on one CH3 domain is replaced by a relatively large amino acid residue, such as tyrosine (Y) or tryptophan (W).
  • Y407 on the other CH3 domain can be replaced by relatively smaller amino acid residues, such as threonine (T), alanine (A) or valine (V).
  • one of the CH3 domains contains one or more substitutions by amino acid residues that are positively charged under physiological conditions and the other CH3 domain contains one or more substitutions by one or more Substitution of amino acid residues that have a negative charge under physiological conditions.
  • the positively charged amino acid residue can be arginine (R), histidine (H) or lysine (K).
  • the negatively charged amino acid residue can be aspartic acid (D) or glutamic acid (E).
  • Amino acid residues that may be substituted include, but are not limited to, D356, L368, K392, D399, and K409.
  • H435 and Y436 on one CH3 domain were replaced by arginine and phenylalanine, respectively, which resulted in a significant decrease in the binding ability between Fc and protein A, thus making heterodimer and homodimer There are different protein A binding activities between the two entities, and the two can be easily separated in the process of affinity chromatography.
  • the CH3 amino acid sequence of Fc forming a heterodimer is shown in the following table:
  • heterodimeric antibody comprising two different antigen-binding polypeptide units.
  • the heterodimer differs in size from its corresponding homodimer, and the size difference can be exploited to facilitate separation of the heterodimer and homodimer.
  • one of the two antigen-binding polypeptide units comprises a light chain-heavy chain pair similar to a wild-type antibody. Throughout this application, this unit is also referred to as "monovalent unit”.
  • the other antigen binding polypeptide unit comprises a single chain variable fragment (ScFv). Such ScFv can be fused to the N-terminus of the constant fragment (Fc) of an antibody, called a fusion peptide. This fusion peptide is also referred to as a "single-chain unit" throughout this application.
  • any of the above-mentioned antibodies or polypeptides may also include additional polypeptides, eg, encoded polypeptides as described herein, signal peptides of antibody constant regions used to direct secretion, or other heterologous polypeptides as described herein.
  • the antibodies described herein may be modified such that their amino acid sequence differs from the naturally occurring binding polypeptides from which they are derived.
  • a polypeptide or amino acid sequence from a given protein may be similar to the starting sequence, e.g., have a certain percentage identity to the starting sequence, e.g., it may be 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% identity.
  • nucleotide or amino acid substitutions, deletions, or insertions may also be made to make conservative substitutions or changes in "non-essential" amino acid regions.
  • the polypeptide or amino acid sequence from a given protein can be identical to the starting sequence, except for one or more independent amino acid substitutions, insertions, or deletions, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20 or more independent amino acid substitutions, insertions, or deletions.
  • the polypeptide or amino acid sequence from the specified protein has 1 to 5, 1 to 10, 1 to 15, or 1 to 20 independent amino acid substitutions, insertions, or missing.
  • the term "detectable label” refers to a directly or indirectly detectable compound or composition that is directly or indirectly bound to a composition to be detected (e.g., a polynucleotide or protein, such as Antibodies) to obtain "tagged” compositions.
  • a composition to be detected e.g., a polynucleotide or protein, such as Antibodies
  • the term also includes sequences bound to the polynucleotide that provide a signal through the expression of an intervening sequence, such as green fluorescent protein (GFP) and the like.
  • GFP green fluorescent protein
  • the tag itself can be detected (eg a radioisotope tag or a fluorescent tag) or, in the case of an enzymatic tag, can catalyze a chemical change in the substrate compound or composition which can be detected. This tag can be used for small-scale assays or more suitable for high-throughput screening.
  • suitable labels include, but are not limited to, radioisotopes, fluorescent dyes, chemiluminescent compounds, dyes, and proteins (including enzymes).
  • the label can be detected only, or it can be quantified. Merely detectable responses generally include those whose presence can only be confirmed, where quantifiable responses generally include those that have quantifiable (eg, numerically reportable) values such as intensity, polarization, and/or other properties.
  • the detectable reaction can be directly using a luminophore or fluorophore associated with the assay component that actually involves binding, or indirectly using a luminophore linked to another (e.g. reporter or indicator) component or fluorophores.
  • the antibodies of the invention can bind to a therapeutic agent (such as a chemotherapeutic drug, such as cisplatin, carboplatin), a prodrug, a peptide, a protein, an enzyme, a virus, a lipid, a biological response modifier, a pharmaceutical agent, or PEG.
  • a therapeutic agent such as a chemotherapeutic drug, such as cisplatin, carboplatin
  • a prodrug such as a chemotherapeutic drug, such as cisplatin, carboplatin
  • a prodrug such as a chemotherapeutic drug, such as cisplatin, carboplatin
  • a prodrug such as a chemotherapeutic drug, such as cisplatin, carboplatin
  • a prodrug such as a chemotherapeutic drug, such as cisplatin, carboplatin
  • a prodrug such as a chemotherapeutic drug, such as cisp
  • an antigen binding polypeptide comprises an amino acid sequence or one or more groups that are not normally bound by an antibody.
  • a single chain Fv antibody fragment of the present application may comprise a flexible linker sequence, or may be modified to add functional groups (eg, polyethylene glycol (PEG), drug, toxin, or label).
  • PEG polyethylene glycol
  • Antibodies of the present application, variants or derivatives thereof include modified derivatives, ie, any type of molecule is covalently attached to the antibody and the covalent attachment does not prevent the antibody from binding to the antigenic epitope.
  • the antibody may comprise one or more non-canonical amino acids.
  • treatment refers to both therapeutic treatment and prophylactic or preventive measures, wherein prevention or slowing (lessening) of undesirable physiological changes or disease, such as the development of cancer, is performed in a subject.
  • beneficial or desired clinical outcomes include, but are not limited to, alleviation of symptoms, reduction of disease severity, stabilization (e.g., non-exacerbation) of disease state, delay or slowing of disease progression, amelioration or palliation of disease state, and remission (whether part or all), whether detectable or not.
  • Treatment can also mean prolonging survival as compared to expected survival if not receiving treatment. Those conditions requiring treatment include those already with the disorder or symptom as well as those prone to have the disorder or symptom or those in which the disorder or symptom is to be prevented.
  • subject or “individual” or “animal” or “patient” or “mammal” is meant any subject, especially a mammalian subject, for whom diagnosis, prognosis or treatment is desired.
  • Mammalian subjects include humans, domestic animals, farm animals, zoos, playgrounds, or pets such as dogs, cats, guinea pigs, rabbits, rats, mice, mice, horses, cows, cows, primates (e.g., Humans, monkeys such as cynomolgus monkeys, macaques, baboons, and chimpanzees, etc.), etc.
  • the antigen-binding polypeptides, variants or derivatives of the present application can be used in certain therapeutic and diagnostic methods related to cancer or infectious diseases.
  • the present application also relates to antibody-based therapy comprising administering the bispecific antibodies of the present application to patients, such as animals, mammals and humans, for the treatment of one or more of the diseases or conditions described herein.
  • Therapeutic drugs of the present application include, but are not limited to, the antibodies of the present application (including their variants and derivatives as described herein) and nucleic acids or polynucleotides encoding the antibodies of the present application (including their variants as described herein) variants and derivatives).
  • the antibodies of the present application can also be used to treat, inhibit or prevent a disease, disorder or condition, including a malignant disease, disorder, or a condition associated with such a disease or disorder, such as an immune response-related disease.
  • the antibodies of the invention can be used as immunosuppressants.
  • the antibodies of the invention can be used to treat autoimmune diseases.
  • the antigen binding polypeptides of the present application, variants or derivatives thereof are useful for inhibiting the growth, development and/or metastasis of cancers, especially those listed above or in the following paragraphs.
  • Antibodies of the present application or variants or derivatives thereof may be used to treat, prevent, diagnose and/or predict other diseases or conditions associated with increased cell survival, including but not limited to cancer or tumors, including the development of malignancies and/or Metastasis, and related diseases (such as malignant ascites, malignant pleural effusion, nausea and effusion), such as EpCAM-positive tumors.
  • Methods of administering antibodies, variants or derivatives thereof include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural and oral routes.
  • the antibody or composition can be administered by any convenient route, such as by infusion or bolus injection, absorbed through epithelial or mucosal and skin linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.), and can interact with other biological Active agents are administered together.
  • the antibody-containing pharmaceutical composition of the present application can be administered orally, rectally, parenterally, intracisternally, intravaginally, intraperitoneally, topically (such as via powder, ointment, drops, or transdermal patch), buccal administration, or as an oral or nasal spray.
  • parenteral refers to modes of administration including intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous and intraarticular injection and infusion. Administration can be systemic or local.
  • an antigen-binding polypeptide or composition of the present application may also be desirable to administer an antigen-binding polypeptide or composition of the present application locally to the area in need of treatment, such as by, but not limited to, local infusion during surgery, topical application, for example in conjunction with a post-surgical wound dressing , by injection, by catheter, by suppository, or by an implant of porous, non-porous, or gel-like material, including membranes or fibers.
  • care must be taken when administering the proteins of the present application, including antibodies, to use materials that do not absorb the protein.
  • Figure 1 Schematic diagram of the YBODY antibody structure.
  • FIG. 1 The negative control flow diagram of HCT116+Jurkat without antibody, where Q1 is the CFSE-stained Jurkat cells, Q2 is the co-combined Jurkat and HCT116 cells, Q3 is the PKH26-stained HCT116 cells, and Q4 is the unstained cells;
  • B Flow chromatogram of HCT116+Jurkat+M701A 10 ⁇ g/ml experimental group, the meaning of each quadrant is the same as before;
  • C The concentration gradient curve of the binding of HCT116 and Jurkat cells mediated by different antibodies.
  • FIG. 4 Detection of bispecific antibody-mediated killing in vitro.
  • FIG. 1 In vivo efficacy of bispecific antibodies in HCT116 human colon cancer model.
  • Figure 6 In vivo efficacy of bispecific antibodies in the OVCAR-3 human ovarian cancer model.
  • Embodiment 1 the expression vector construction of bispecific antibody
  • the bispecific antibody structure targeting EpCAM and CD3 includes an anti-EpCAM binding region and an anti-CD3 binding region.
  • the monovalent unit is the pairing formed by the anti-EpCAM heavy chain and light chain
  • the single-chain unit is the ScFv-Fc form of anti-CD3. It is defined as a YBODY structure (as shown in Figure 1), in which the anti-CD3 VL is connected to the hinge region and CH2 through a linker.
  • the heavy chain Fc of the monovalent unit and the Fc of the single chain unit undergo amino acid mutation modification, so that they are not easy to form homodimers (homodimers), but easy to form heterodimers (heterodimers).
  • each chain corresponding to the bispecific antibody was amplified by PCR and overlapping PCR, and each antibody chain was cloned into the pcDNA3.1 vector ( Invitrogen Corporation).
  • the specific sequence information of each chain of the antibody is shown in Table 1 and the sequence listing.
  • Plasmids were extracted according to conventional plasmid extraction methods, and used for chemical transfection of CHO-S cells (from Gibco).
  • the transfected cells were cultured with suspension and shaking in a shaker at 37° C. and 5% CO 2 for 7-10 days.
  • the supernatant was harvested by centrifugation at 3000 x g and filtered through a 0.22 ⁇ m filter.
  • the preliminarily purified bispecific antibody was obtained by protein A affinity chromatography, the concentration of the purified protein was determined by the UV absorbance at 280nm and the corresponding extinction coefficient, and the purity of the antibody was tested by high performance size exclusion chromatography (HPLC-SEC) , and calculate the corresponding expression level of each protein.
  • HPLC-SEC high performance size exclusion chromatography
  • bispecific antibody The molecular expression of bispecific antibody is between 40 mg/L and 91 mg/L, and the initial purity is between 45% and 81%. The expression and The initial strength is significantly better than that of M701.
  • affinity sample was purified by cation exchange chromatography, and bispecific antibodies with HPLC-SEC purity >95% were finally obtained.
  • the purification recovery rates of each bispecific antibody are shown in Table 2.
  • M701 47.2mg/L 45.91% 14% M701A 90.8mg/L 81.12% 57% M701B 84.1mg/L 78.53% 53% M701C 54.4mg/L 64.54% 32% M701D 65.8mg/L 58.63% twenty three% M701E 49mg/L 55.76% twenty two% M701F 52.4mg/L 66.32% 30% M701G 41mg/L 70.76% 25% M701H 73.3mg/L 72.03% 43% M701I 80.6mg/L 73.21% 45% M701J 87.7mg/L 78.21% 47% M701K 81.3mg/L 74.50% 41%
  • n.d. means not detected.
  • human colon cancer cells HCT116 (Shanghai Institute of Life Sciences, Chinese Academy of Sciences) were used as positive cells expressing human EpCAM on the cell membrane surface, and the affinity of the antibody to human EpCAM on the cell surface was detected.
  • HCT116 cells were collected by centrifugation, resuspended in buffer (PBS+1% FBS), and added to 96-well plate at 2 ⁇ 10 5 cells/well, 50 ⁇ L per well. After centrifugation at 350 ⁇ g for 5 min, the supernatant was removed.
  • buffer PBS+1% FBS
  • the human CD3 antigen (SB, Cat:CT038-H2508H) was immobilized on the CM5 chip by the amino coupling method, and the antigen coupling amount was 1500RU.
  • the sample was diluted with 1 ⁇ HBS-EP+buffer to The initial concentration, then 2-fold gradient dilution to 4 concentrations, was tested from low concentration to high concentration on the machine, the binding flow rate was 30 ⁇ L/min, the binding time was 120 s, and the dissociation time was 300 s; the chip was regenerated with pH 1.5 Glycine solution, and the regeneration flow rate 10 ⁇ L/min, regeneration time 30s.
  • the software Biacore T200Evaluation Software was used to fit the data of the resulting spectrum in a 1:1 Binding fitting method to obtain the dissociation equilibrium constant (KD).
  • KD dissociation equilibrium constant
  • the series of bispecific antibodies all have a binding effect on human CD3 antigen.
  • the anti-EpCAM antibody variable region sequences of M701A and M701B are the same (SEQ ID NO:13 and SEQ ID NO:14), and the anti-CD3 antibody variable region sequences are respectively SEQ ID NO:18 and SEQ ID NO:19, corresponding to The affinities were 21.15 nM and 28.27 nM, respectively.
  • the anti-EpCAM antibody variable region sequence becomes another (SEQ ID NO:15 and SEQ ID NO:16), the anti-CD3 antibody variable region sequence is still SEQ ID NO:18 and SEQ ID NO:19, the corresponding double
  • the affinities of specific antibodies M701H and M701I were 95.26nM and 40.03nM, respectively. This shows that the combination of different anti-EpCAM antibodies and different anti-CD3 antibodies forms bispecific antibodies, and the expression of affinity is not regular.
  • EpCAM-positive cell line HCT116 was stained with PKH26, and CD3-positive cell line Jurkat (Shanghai Institute of Biology , Chinese Academy of Sciences) was stained with CFSE. cells) and mixed with the antibody to be tested in a gradient dilution for co-incubation, wherein M700 is the anti-EpCAM monoclonal antibody control (light chain SEQ ID NO: 25 and heavy chain SEQ ID NO: 26), and M100 is the anti-CD3 monoclonal antibody control (light chain SEQ ID NO: 27 and heavy chain SEQ ID NO: 28), Mco101 is an anti-luciferase and anti-CD3 bispecific antibody, as the CD3 terminal isotype control of bispecific antibody (light chain SEQ ID NO: 29, heavy chain Chain SEQ ID NO: 30 and single chain SEQ ID NO: 31, the structure is the same as Fig.
  • the biological activity of the bispecific antibody was detected using Jurkat-CD3-NFAT-RE-Luc cells (Promega).
  • the pLV-puro (Inovogen Tech.Co., cat.No.VL3001) vector containing DNA encoding human EpCAM gene (NCBI sequence number: NM_002354.3) was transfected into CHO-K1 cells to obtain cells stably expressing human EpCAM Strain CHO-K1-huEpCAM, collect CHO-K1-huEpCAM as target cells, and resuspend in buffer (PBS+1%FBS), add 4 ⁇ 104 cells/well to all white 96-well culture plate, and Place in a 37°C, 5% CO 2 incubator for overnight culture for 18-24 hours, remove the medium in the plate, and add 40 ⁇ L of antibody diluent to each well.
  • buffer PBS+1%FBS
  • the isolated PBMCs were used as effector cells, and EpCAM expressing cells were used as target cells to detect the killing effect mediated by bispecific antibodies in vitro.
  • the pLV-puro (Inovogen Tech.Co., cat.No.VL3001) vector containing DNA encoding human EpCAM gene (NCBI sequence number: NM_002354.3) was transfected into mouse melanoma cell B16 cells (Shanghai Biological Research Institute, Chinese Academy of Sciences) Hospital), the cell line B16-EpCAM stably expressing human EpCAM was obtained, wherein B16 was used as a negative cell not expressing EpCAM.
  • EpCAM target cells expressing EpCAM
  • human colon cancer cell HCT116 (Shanghai Institute of Life Sciences, Chinese Academy of Sciences), human ovarian cancer cell OVCAR-3 (CCTCC, China Center for Type Culture Collection) and CHO-K1-huEpCAM.
  • the cells were digested with trypsin to form a single-cell suspension, collected by centrifugation at 300 g for 5 min, and stained with 5 ⁇ M hydroxyfluorescein diacetate, succinimidyl ester (CFSE) (37°C , 15min), washed with complete medium twice, counted on a Vi-cell cell counter, and then added to a 96-well plate according to the experimental design, 2 ⁇ 10 4 cells/100 ⁇ L per well.
  • CFSE succinimidyl ester
  • Antibodies at corresponding concentrations were added at 50 ⁇ L/well, and hPBMC were counted on a Cellometer and placed in a 96-well plate (2 ⁇ 10 5 cells/50 ⁇ L per well, and the effect-to-target ratio was 10:1). Place the cell culture plate in a cell culture incubator for 72 h, digest the cells into a single cell suspension, add propidium bromide (PI) solution with a final concentration of 1 ⁇ g/mL, incubate for 10 min, and use a flow cytometer (BD Accuri TM C6) was tested on the machine, and the percentage of CFSE+PI+ double positive cells accounting for CFSE+ positive cells was analyzed.
  • PI propidium bromide
  • M701A only has a killing effect on B16-EpCAM cells with EpCAM expression, but has no killing effect on B16 cells without EpCAM expression, while the control antibody Mco101 has no killing effect on any cells, indicating bispecificity Antibodies are targeted.
  • M701A had a significant killing effect on both HCT116 and OVCAR-3 cells, and it was stronger than the control Mco101.
  • bispecific antibodies M701A, M701B, M701H, and M701I all had significant killing effects on CHO-K1-huEpCAM cells, and the effects of M701A and M701B were significantly stronger than those of M701H and M701I.
  • both bispecific antibodies M701J and M701K had significant killing effects on HCT116 cells, with EC50 of 7.814-25.43ng/ml, and the killing level of M701J was not significantly different from that of M701A.
  • the killing activity of the above-mentioned bispecific antibodies was significantly stronger than that of M701 (the EC50 of M701 was 69.17 ng/ml).
  • Example 9 In vivo efficacy of bispecific antibody in HCT116 human colon cancer heterotopic xenograft tumor model
  • HCT116 cells and effector cells CIK cytokine-induced killer, cytokine-induced killer cells
  • CIK cytokine-induced killer, cytokine-induced killer cells
  • Plasma cells Since this kind of cells express both CD3+ and CD56+ membrane protein molecules, they are also called NK cell-like T lymphocytes), the cells were collected and counted.
  • the inoculation volume is 0.1ml/mouse, to establish human colon cancer HCT116 xenografts Tumor model.
  • the treatment was started 1 hour after inoculation.
  • the experiment was divided into test drug M701A 2mg/kg group, M701A 1mg/kg group, M701 1mg/kg group, monoclonal antibody control M700 2mg/kg group and vehicle control group (normal saline), with 8 mice in each group.
  • Tail vein injection was administered on the 0th, 2nd, and 4th days after inoculation, respectively, and administered three times in total. Efficacy was evaluated based on relative tumor inhibition rate and complete tumor regression rate, and safety was evaluated based on animal body weight changes and death.
  • TGI Relative tumor inhibition rate
  • Complete regression rate of tumor (complete regression): defined as tumor volume less than 63 mm 3 during or after treatment.
  • Complete tumor regression rate (%) number of animals in a group that achieved complete regression/total number of animals in this group ⁇ 100%.
  • the test drug M701A (2mg/kg, 1mg/kg) showed a significant tumor inhibitory effect on the 30th day after drug withdrawal in the treatment group (that is, the 33rd day after inoculation), and the relative tumor inhibition rate TGI (%) were 100% and 93.82%, respectively.
  • the vehicle control group there were statistically significant differences (all p ⁇ 0.001), and all tumors in the M701A (2mg/kg) group reached the standard of complete regression in the two groups.
  • M701B, M701J and M701K all showed similar tumor inhibitory effects to M701A at the same dose, and there was no decrease in body weight.
  • Example 10 In vivo efficacy of bispecific antibody in OVCAR-3 human ovarian cancer heterotopic xenograft tumor model
  • OVCAR-3 cells and effector cells CIK were cultured according to the culture conditions, and the cells were collected and counted. Inoculate pre-mixed OVCAR-3 cells (1 ⁇ 10 7 cells/mouse) and CIK (1 ⁇ 10 7 cells/mouse) on the right back of each mouse, the inoculation volume is 0.2ml/mouse, and the content of Matrigel glue is 50%. (0.1ml/mouse), to establish human ovarian cancer OVCAR-3 heterotopic transplantation tumor model. The treatment was administered 1 hour after inoculation.
  • mice The experiment was divided into the test drug M701A (5 mg/kg), the CD3 end isotype control Mco101 (5 mg/kg), the monoclonal antibody control M700 (5 mg/kg) group and the vehicle control group (normal saline). Group 8 mice. Tail vein injection was administered on the 0th, 2nd, and 4th days after inoculation, respectively, and administered three times in total. Efficacy was evaluated based on relative tumor inhibition rate (TGI) and complete tumor regression rate, and safety was evaluated based on animal body weight changes and death.
  • TGI tumor inhibition rate
  • the test drug M701A (5mg/kg) showed a significant tumor inhibitory effect on the 44th day after drug withdrawal (that is, the 48th day after inoculation) in the treatment group, and the relative tumor inhibition rate TGI (%) was 98.97%.
  • TGI tumor inhibition rate
  • TGI 68.87%)
  • M701B, M701J and M701K all showed similar tumor inhibitory effects to M701A at the same dose, and there was no decrease in body weight.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Immunology (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Genetics & Genomics (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Molecular Biology (AREA)
  • Engineering & Computer Science (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Cell Biology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Biomedical Technology (AREA)
  • Biotechnology (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • General Engineering & Computer Science (AREA)
  • Microbiology (AREA)
  • Reproductive Health (AREA)
  • Physics & Mathematics (AREA)
  • Diabetes (AREA)
  • Epidemiology (AREA)
  • Plant Pathology (AREA)
  • Hematology (AREA)
  • Peptides Or Proteins (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Medicinal Preparation (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

本发明提供了双特异性抗体及其应用，所述双特异性抗体包含特异性结合EpCAM的抗原结合结构域和特异性结合CD3的抗原结合结构域。

Description

双特异性抗体及其应用 技术领域
本发明涉及免疫学的技术领域。具体地说，涉及抗EpCAM和CD3的双特异性抗体及应用。
背景技术
双特异性抗体(bi-specific antibody，BsAb)又称双靶向抗体，可同时识别和结合两种不同的抗原或表位，并阻断两种不同的信号通路以发挥其作用。与识别单一抗原的单克隆抗体(monoclonal Ab，mAb)相比，双抗体有诸多优势：①可将特异性免疫效应细胞重定向到邻近的肿瘤细胞，以增强肿瘤杀伤，这是通过组合mAb治疗策略无法实现的；②通过两个不同的细胞表面抗原的相互作用增加结合特异性；③与组合疗法中单一抗体的药物开发相比，可降低开发成本、临床试验、法规审查的预算；④与联合疗法中单一抗体的药物相比，能够同时阻断在发病机理中发挥独特或重叠功能的两种不同途径。
癌症和其他疾病均是由多因素导致的，在病原学上有许多信号通路，单一靶点的免疫治疗不能有效杀死靶向细胞。接受mAb治疗的患者可能会产生抗药性或对治疗无应答。因此，双抗体已成为癌症、炎症、病毒感染及自身免疫病等许多疾病治疗的主要选择。然而，双抗体在自然环境下不存在，需通过重组DNA、细胞融合或化学结合的技术来实现，其中重组DNA技术是目前制备BsAb使用最多的技术，但是仍然存在着BsAb表达困难、产量低、纯化难、稳定性差等诸多障碍，因此，构建新的双特异性抗体，使其能克服上述障碍，并建立相应的免疫杀伤动物模型，是非常有必要的。本发明提供了一种新型双特性抗体，并描述了对其药效学的研究方法及结果。
发明内容
本发明开发了一种新的双特异性抗体，其特征在于包含特异性结合EpCAM的抗原结合结构域和特异性结合CD3的抗原结合结构域；以及其应用。
具体地，本发明涉及以下几个方面：
1.双特异性抗体，其特征在于包含特异性结合EpCAM的抗原结合结构域和特异性结合CD3的抗原结合结构域，
其中所述特异性结合EpCAM的抗原结合结构域，选自以下各项组成的组：
1)包含以下CDRs或其变体的特异性结合EpCAM的抗原结合结构域：
(i)SEQ ID NO:14所示的重链可变区中包含的CDRH1，CDRH2和CDRH3，和
(ii)SEQ ID NO:13所示的轻链可变区中包含的CDRL1，CDRL2和CDRL3，
优选地，按照Kabat序列编号系统，CDRL1的序列如SEQ ID NO:32所示，CDRL2的序列如SEQ ID NO:33所示，CDRL3的序列如SEQ ID NO:34所示，CDRH1的序列如SEQ ID NO:35所示，CDRH2的序列如SEQ ID NO:36所示，CDRH3的序列如SEQ ID NO:37所示；或
2)包含以下CDRs或其变体的特异性结合EpCAM的抗原结合结构域：
(i)SEQ ID NO:16所示的重链可变区中包含的CDRH1，CDRH2和CDRH3，和
(ii)SEQ ID NO:15所示的轻链可变区中包含的CDRL1，CDRL2和CDRL3，
优选地，按照Kabat序列编号系统和CDR定义系统，CDRL1的序列如SEQ ID NO:38所示，CDRL2的序列如SEQ ID NO:39所示，CDRL3的序列如SEQ ID NO:40所示，CDRH1的序列如SEQ ID NO:41所示，CDRH2的序列如SEQ ID NO:42所示，和CDRH3的序列如SEQ ID NO:43；
所述特异性结合CD3的抗原结合结构域，选自以下各项组成的组：
1)包含以下CDRs或其变体的特异性结合CD3的抗原结合结构域：
SEQ ID NO:50所示的重链可变区中包含的CDRH1，CDRH2和CDRH3，以及SEQ ID NO:51所示的轻链可变区中包含的CDRL1，CDRL2和CDRL3，
优选地，按照Kabat序列编号系统，CDRH1的序列如SEQ ID NO:44所示，CDRH2的序列如SEQ ID NO:45所示，和CDRH3的序列如SEQ ID NO:46，CDRL1的序列如SEQ ID NO:47所示，CDRL2的序列如SEQ ID NO:48所示，CDRL3的序列如SEQ ID NO:49所示；或
2)包含以下CDRs或其变体的特异性结合CD3的抗原结合结构域：
SEQ ID NO:58所示的重链可变区中包含的CDRH1，CDRH2和CDRH3，以及SEQ ID NO:59所示的轻链可变区中包含的CDRL1，CDRL2和CDRL3，
优选地，按照Kabat序列编号系统，CDRH1的序列如SEQ ID NO:52所示，CDRH2的序列如SEQ ID NO:53所示，和CDRH3的序列如SEQ ID NO:54，CDRL1的序列如SEQ ID NO:55所示，CDRL2的序列如SEQ ID NO:56所示，CDRL3的序列如SEQ ID NO:57所示；其中所述CDRs的变体与对应的CDRs分别具有3，2或1个氨基酸差异或分别具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％同一性。
2.项目1所述的双特异性抗体，其中所述特异性结合EpCAM的抗原结合结构域包含以下重链可变区和轻链可变区(或其变体)：
(i)SEQ ID NO:14所示的重链可变区，SEQ ID NO:13所示的轻链可变区；或者
(ii)SEQ ID NO:16所示的重链可变区，SEQ ID NO:15所示的轻链可变区；和
其中所述特异性结合CD3的抗原结合结构域包含以下重链可变区和轻链可变区(或其变体)：
(1)SEQ ID NO:50所示的重链可变区和SEQ ID NO:51所示的轻链可变区，或
(2)SEQ ID NO:58所示的重链可变区和SEQ ID NO:59所示的轻链可变区；
优选地，其中特异性结合EpCAM的抗原结合结构域为Fab片段形式，特异性结合CD3的抗原结合结构域为ScFv形式，
优选地，其中所述特异性结合EpCAM的抗原结合结构域包含以下重链可变区和轻链可变区(或其变体)：
(i)SEQ ID NO:14所示的重链可变区，SEQ ID NO:13所示的轻链可变区；和
其中所述特异性结合CD3的抗原结合结构域选自以下各项组成的组：
(1)SEQ ID NO:18所示的ScFv或其变体，
(2)SEQ ID NO:19所示的ScFv或其变体，
其中所述变体与所述对应的可变区或ScFv分别具有3，2或1个氨基酸差异或分别具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％同一性。
3.项目1或2所述的双特异性抗体，其中所述双特异性抗体包含
(1)特异性结合EpCAM的轻链-重链对，所述轻链-重链对包含轻链和重链，或由其组成；其中所述轻链包含轻链可变区和轻链恒定区(优选序列如SEQ ID NO:1、60-65任一所示)，所述重链包含重链可变区、CH1(优选序列如SEQ ID NO:2所示)和第一Fc片段；优选地，所述第一Fc片段包含铰链区(优选序列如SEQ ID NO:3所示)，CH2(优选序列如SEQ ID NO:6、7、66-71任一所示)和CH3a；
(2)特异性结合CD3的融合肽，所述融合肽包含特异性结合CD3的ScFv和第二Fc片段，或由其组成；优选地，所述ScFv从N端至C端方向依次包含重链可变区，连接肽(优选序列如SEQ ID NO:4所示)和轻链可变区，所述第二Fc片段从N端至C端方向依次包含铰链区(优选序列如SEQ ID NO:3所示)，CH2(优选序列如SEQ ID NO:6、7、66-71任一所示)和CH3b，优选地所述轻链可变区的C端与所述第二Fc片段的铰链区通过连接肽(优选序列如SEQ ID NO:5所示)连接；
优选地，所述第一Fc片段和第二Fc片段为人或者人源化的Fc片段，例如人IgG Fc片段，例如IgG1、IgG2、IgG3、IgG4、IgG5Fc片段；
优选地，与野生型抗体比，所述第一Fc片段和/或第二Fc片段包含一处或多处替换，该替换在该重链和融合肽之间形成杵-臼结构配对，例如一个CH3结构域上的T366被相对较大的氨基酸残基替换，如酪氨酸(Y)或色氨酸(W)替换，另一CH3结构域上的Y407被相对较小的氨基酸残基替换，如苏氨酸(T)，丙氨酸(A)或缬氨酸(V)，例如包含表6的一处或多处替换；
优选地，所述第一Fc片段和/或第二Fc片段包含一处或多处替换，1)该替换在该重链和融合肽之间形成盐桥配对，例如一个CH3结构域含有一处或多处替换，经在生理条件下有正电荷的氨基酸残基替换，而另一CH3结构域包含一处或多处替换，经一个或多个在生理条件下具有负电荷的氨基酸残基替换，例如该带正电的氨基酸残基为精氨酸(R)，组氨酸(H)或赖氨酸(K)，例如该带负电荷的氨基酸残基可为天冬氨酸(D)或谷氨酸(E)，例如被替换的氨基酸残基包括D356、L368、K392、D399和K409中的一个或多个，例如表7的一处或多处替换，2)该替换在该重链和融合肽之间形成二硫键，例如表8的替换，和/或3)该替换导致Fc与蛋白A之间的结合能力显著降低，例如一个CH3结构域上的H435和Y436分别被替换为精氨酸和苯丙氨酸，如表9所示；
优选地，其中：
a)融合肽的CH3b和重链的CH3a具有形成杵-臼结构的替换对；
b)融合肽的CH3b和重链的CH3a具有形成离子键的替换对；
c)融合肽的CH3b和重链的CH3a具有形成二硫键的替换对；和/或
d)融合肽的CH3b和重链的CH3a具有导致与蛋白A的结合能力下降的替换；
优选地，CH1包括SEQ ID No:2的序列；和/或CL包括选自SEQ ID Nos:1、60-65任一项的序列；
优选地，所述第一Fc片段和/或第二Fc片段包含选自SEQ ID Nos:6、7、66-71任一项的序列的CH2和/或SEQ ID Nos:8、9、11、12、72-76任一项的序列的CH3；
优选地，CH3a和CH3b的序列选自以下各项组成的组：
(1)其中一个序列如SEQ ID NO:8所示，另一个序列如SEQ ID NO:11所示；
(2)其中一个序列如SEQ ID NO:9所示，另一个序列如SEQ ID NO:12所示；
(3)其中一个序列如SEQ ID NO:72所示，另一个序列如SEQ ID NO:74所示；
(4)其中一个序列如SEQ ID NO:9所示，另一个序列如SEQ ID NO:75所示；
(5)其中一个序列如SEQ ID NO:73所示，另一个序列如SEQ ID NO:76所示；
优选地，所述双特异性抗体选自以下各项组成的组：
(1)其包含融合肽，重链和轻链，或由其组成；其中融合肽包含SEQ ID NO:18，SEQ ID NO:5，SEQ ID NO:3，SEQ ID NO:6和SEQ ID NO:11，或由其组成；重链包含SEQ ID NO:14，SEQ ID NO:2，SEQ ID NO:3，SEQ ID NO:6和SEQ ID NO:8，或由其组成；轻链包含SEQ ID NO:13和SEQ ID NO:1，或由其组成；
(2)其包含融合肽，重链和轻链，或由其组成；其中融合肽包含SEQ ID NO:19，SEQ ID NO:5，SEQ ID NO:3，SEQ ID NO:6和SEQ ID NO:11，或由其组成；重链包含SEQ ID NO:14，SEQ ID NO:2，SEQ ID NO:3，SEQ ID NO:6和SEQ ID NO:8，或由其组成；轻链包含SEQ ID NO:13和SEQ ID NO:1，或由其组成；
(3)其包含融合肽，重链和轻链，或由其组成；其中融合肽包含SEQ ID NO:18，SEQ ID NO:5，SEQ ID NO:3，SEQ ID NO:6和SEQ ID NO:11，或由其组成；重链包含SEQ ID NO:16，SEQ ID NO:2，SEQ ID NO:3，SEQ ID NO:6和SEQ ID NO:8，或由其组成；轻链包含SEQ ID NO:15和SEQ ID NO:1，或由其组成；
(4)其包含融合肽，重链和轻链，或由其组成；其中融合肽包含SEQ ID NO:19，SEQ ID NO:5，SEQ ID NO:3，SEQ ID NO:6和SEQ ID NO:11，或由其组成；重链包含SEQ ID NO:16，SEQ ID NO:2，SEQ ID NO:3，SEQ ID NO:6和SEQ ID NO:8，或由其组成；轻链包含SEQ ID NO:15和SEQ ID NO:1，或由其组成；
(5)其包含融合肽，重链和轻链，或由其组成；其中融合肽包含SEQ ID NO:18，SEQ ID NO:5，SEQ ID NO:3，SEQ ID NO:7和SEQ ID NO:12，或由其组成；重链包含SEQ ID NO:14，SEQ ID NO:2，SEQ ID NO:3，SEQ ID NO:7和SEQ ID NO:9，或由其组成；轻链包含SEQ ID NO:13和SEQ ID NO:1，或由其组成；
(6)其包含融合肽，重链和轻链，或由其组成；其中融合肽包含SEQ ID NO:19，SEQ ID NO:5，SEQ ID NO:3，SEQ ID NO:7和SEQ ID NO:12，或由其组成；重链包含SEQ ID NO:14，SEQ ID NO:2，SEQ ID NO:3，SEQ ID NO:7和SEQ ID NO:9，或由其组成；轻链包含SEQ ID NO:13和SEQ ID NO:1，或由其组成；
(7)其包含融合肽，重链和轻链，或由其组成；其中融合肽包含SEQ ID NO:18，SEQ ID NO:5，SEQ ID NO:3，SEQ ID NO:6和SEQ ID NO:8，或由其组成；重链包含SEQ ID NO:14，SEQ ID NO:2，SEQ ID NO:3，SEQ ID NO:6和SEQ ID NO:11，或由其组成；轻链包含SEQ ID NO:13和SEQ ID NO:1，或由其组成；
(8)其包含融合肽，重链和轻链，或由其组成；其中融合肽包含SEQ ID NO:19，SEQ ID NO:5，SEQ ID NO:3，SEQ ID NO:6和SEQ ID NO:8，或由其组成；重链包含 SEQ ID NO:14，SEQ ID NO:2，SEQ ID NO:3，SEQ ID NO:6和SEQ ID NO:11，或由其组成；轻链包含SEQ ID NO:13和SEQ ID NO:1，或由其组成；
(9)其包含融合肽，重链和轻链，或由其组成；其中融合肽包含SEQ ID NO:18，SEQ ID NO:5，SEQ ID NO:3，SEQ ID NO:6和SEQ ID NO:8，或由其组成；重链包含SEQ ID NO:16，SEQ ID NO:2，SEQ ID NO:3，SEQ ID NO:6和SEQ ID NO:11，或由其组成；轻链包含SEQ ID NO:15和SEQ ID NO:1，或由其组成；
(10)其包含融合肽，重链和轻链，或由其组成；其中融合肽包含SEQ ID NO:19，SEQ ID NO:5，SEQ ID NO:3，SEQ ID NO:6和SEQ ID NO:8，或由其组成；重链包含SEQ ID NO:16，SEQ ID NO:2，SEQ ID NO:3，SEQ ID NO:6和SEQ ID NO:11，或由其组成；轻链包含SEQ ID NO:15和SEQ ID NO:1，或由其组成；
(11)其包含融合肽，重链和轻链，或由其组成；其中融合肽包含SEQ ID NO:18，SEQ ID NO:5，SEQ ID NO:3，SEQ ID NO:7和SEQ ID NO:9，或由其组成；重链包含SEQ ID NO:14，SEQ ID NO:2，SEQ ID NO:3，SEQ ID NO:7和SEQ ID NO:12，或由其组成；轻链包含SEQ ID NO:13和SEQ ID NO:1，或由其组成；
(12)其包含融合肽，重链和轻链，或由其组成；其中融合肽包含SEQ ID NO:19，SEQ ID NO:5，SEQ ID NO:3，SEQ ID NO:7和SEQ ID NO:9，或由其组成；重链包含SEQ ID NO:14，SEQ ID NO:2，SEQ ID NO:3，SEQ ID NO:7和SEQ ID NO:12，或由其组成；轻链包含SEQ ID NO:13和SEQ ID NO:1，或由其组成。
4.核酸组合物，其包含:编码项目1-3任一项所述的双特异性抗体的核酸序列，优选地，
核酸组合物包含：
a)第一表达载体，所述第一表达载体包含编码项目1-3任一项中定义的特异性结合EpCAM的抗原结合结构域或轻链-重链对的第一核酸；
b)第二表达载体，所述第二表达载体包含编码项目1-3任一项中定义的特异性结合CD3的抗原结合结构域或融合肽的第二核酸。
5.表达载体，其包含项目4的核酸组合物。
6.宿主细胞，其包含项目5的表达载体。
7.药物组合物，其包含项目1-3任一项所述的双特异性抗体，和药用载体，和任选地，治疗癌症(EpCAM阳性的肿瘤，例如结直肠癌，胃癌，乳腺癌，卵巢癌，肺癌(如非小细胞肺癌)，前列腺癌，胰腺癌，肝癌，视网膜母细胞瘤，食管癌，肾癌，肾透明细胞瘤，皮肤鳞癌，皮肤基底细胞癌，肉瘤，鼻腔神经胶质瘤，颅咽管瘤，甲状腺癌，胆管细胞瘤，膀胱癌，头颈部肿瘤，子宫颈癌，或口腔癌等，)和/或恶性腹水、恶性积液、恶性胸水等的药物(如小分子药物或大分子药物)，优选地，所述药物组合物的剂型包括胃肠给药剂型或胃肠外给药剂型；更优选地，所述药物组合物的剂型为注射剂，包括静脉注射、静脉滴注、皮下注射、局部注射、肌肉注射、瘤内注射、腹腔内注射、颅内注射、或腔内注射。
8.缀合物或融合蛋白，其包含项目1-3任一项的双特异性抗体，优选地，其包含与所述双特异性抗体缀合或融合的物质A，所述物质A选自治疗剂、药物前体、蛋白(例如酶)、病毒、脂质、生物反应调节剂(如免疫调节剂)、PEG、激素、寡核苷酸、诊断 剂、细胞毒性剂，其可为药物或毒素，超声增强剂，非放射性标记物，可检测标记物，如化学发光物标记化合物(如鲁米诺、异鲁米诺、热性吖啶鎓酯、咪唑、吖啶鎓盐和草酸酯)，或荧光发光金属(如152Eu，或镧系标记)。
9.试剂盒，其包含项目1-3任一项所述的双特异性抗体，和任选地，治疗癌症(EpCAM阳性的肿瘤，例如结直肠癌，胃癌，乳腺癌，卵巢癌，肺癌(如非小细胞肺癌)，前列腺癌，胰腺癌，肝癌，视网膜母细胞瘤，食管癌，肾癌，肾透明细胞瘤，皮肤鳞癌，皮肤基底细胞癌，肉瘤，鼻腔神经胶质瘤，颅咽管瘤，甲状腺癌，胆管细胞瘤，膀胱癌，头颈部肿瘤，子宫颈癌，或口腔癌等)和/或恶性腹水、恶性积液、恶性胸水等的药物(如小分子药物或大分子药物)。
10.项目1-3任一项所述的双特异性抗体，用于治疗癌症，或在制备治疗癌症和/或恶性腹水、恶性积液、恶性胸水等的药物或试剂盒中的应用，所述癌症例如EpCAM阳性的肿瘤，例如结直肠癌，胃癌，乳腺癌，卵巢癌，肺癌(如非小细胞肺癌)，前列腺癌，胰腺癌，肝癌，视网膜母细胞瘤，食管癌，肾癌，肾透明细胞瘤，皮肤鳞癌，皮肤基底细胞癌，肉瘤，鼻腔神经胶质瘤，颅咽管瘤，甲状腺癌，胆管细胞瘤，膀胱癌，头颈部肿瘤，子宫颈癌，或口腔癌。
11.治疗癌症和/或恶性腹水、恶性积液、恶性胸水的方法，包含对受试者施用治疗有效量的项目1-3任一项所述的双特异性抗体，所述癌症EpCAM阳性的肿瘤，例如结直肠癌，胃癌，乳腺癌，卵巢癌，肺癌(如非小细胞肺癌)，前列腺癌，胰腺癌，肝癌，视网膜母细胞瘤，食管癌，肾癌，肾透明细胞瘤，皮肤鳞癌，皮肤基底细胞癌，肉瘤，鼻腔神经胶质瘤，颅咽管瘤，甲状腺癌，胆管细胞瘤，膀胱癌，头颈部肿瘤，子宫颈癌，或口腔癌。
应理解，在本发明范围内，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
在本发明中涉及的术语具备本领域技术人员理解的常规含义。在本技术领域内使用和/或可接受的情况下，一个术语有两个或两个以上定义时，本文使用的术语的定义用于包括所有的含义。
本领域普通技术人员可以理解，抗体的CDR区负责抗体对抗原的结合特异性。在已知抗体重链和轻链可变区序列的情况下，目前有几种确定抗体CDR区的方法，包括Kabat，IMGT，Chothia和AbM编号系统。然而，每种关于抗体或其变体的CDR的定义的应用都将在本文定义和使用的术语的范围内。如果给定该抗体的可变区氨基酸序列，则本领域技术人员通常可确定特定CDR，而不依赖于该序列自身之外的任何实验数据。
本文使用的“抗体”或者“抗原结合片段”指特定识别并结合抗原的多肽或多肽复合体。术语“抗体”以广义使用，并且包括免疫球蛋白或抗体分子，所述抗体分子包括单克隆或多克隆的人、人源化、复合和嵌合抗体以及抗体片段。因此术语“抗体”包括含有特定分子的任何蛋白或肽，该特定分子含有至少一部分的免疫球蛋白分子，该免疫球蛋白分子具有结合至抗原的生物活性。此种情况的实例包括但不限于，重链或轻链或其配体结合部分的互补决定区(CDR)，重链或轻链可变区，重链或轻链恒定区，框架(FR) 区或其任何部分，或结合蛋白的至少一部分。在本发明中，抗体包括用本领域技术人员熟知技术所制备的鼠的、嵌合的、人源化的或者全人的抗体。重组抗体，例如嵌合的和人源化的单克隆抗体，包括人的和非人的部分，可以采用本领域熟知的DNA重组技术制备。本申请的免疫球蛋白分子或抗体分子可为任何类型(例如IgG、IgE、IgM、IgD、IgA和IgY)、免疫球蛋白分子的任何类(例如IgG1、IgG2、IgG3、IgG4、IgA1和IgA2)或亚类。
术语“抗体片段”或“抗原结合片段”包括但不限于，F(ab')2、F(ab)2、Fab'、Fab、Fv、Fd、dAb、Fab/c、互补决定区(CDR)片段、单链Fvs(ScFv)、二硫键稳定性Fv(Disulfide-stabilized Fv fragment,dsFv)、(dsFv)2、双特异性dsFv(dsFv-dsFv')、双链抗体(Diabody)、二硫键稳定的双链抗体(ds-Diabody)、ScFv多聚体(如ScFv二聚体、ScFv三聚体)、由包含一个或多个CDR的抗体的一部分形成的多特异性抗体、纳米抗体、单域抗体(sdab)、结构域抗体、二价结构域抗体，或任何其他与抗原结合但不包含完整的抗体结构的抗体片段。不管结构如何，抗原结合片段包括任何能够与亲本抗体或亲本抗体片段结合的相同抗原结合的多肽或多肽复合体。术语“抗体片段”包括适配体、适配体对映体(spiegelmers)和双体(diabodies)。术语“抗体片段”也包括任何合成的或基因改造的蛋白，它们与抗体一样可结合至特定的抗原以形成复合体。通常，抗体片段具有本发明抗体的至少约50个连续氨基酸，较佳地至少约50个连续氨基酸，更佳地至少约80个连续氨基酸，最佳地至少约100个连续氨基酸。
“单链可变片段”或“ScFv”指免疫球蛋白的重链(VH)和轻链(VL)的可变区域的融合蛋白。在某些方面，这些区域用10至约25个氨基酸的短接头肽连接。该接头可富含甘氨酸以具有柔性，也含有丝氨酸或苏氨酸以具有可溶性，且能将VH的N-末端连接至VL的C末端，反过来也一样。该蛋白质保留了原始的免疫球蛋白的特性，只是去除了恒定区并引入了接头。ScFv分子为本领域已知的，例如美国专利5,892,019中描述的那些。
结合EpCAM和CD3的抗原结合结构域为Fab，或ScFv，或重链可变区(VH)-轻链可变区(VL)之间非共价配对(Fv)。任何上述的抗体或多肽还可包括额外的多肽，例如，抗体N端的信号肽，该信号肽用于指导分泌，或如本文所述的其他异源多肽，如用于纯化的6×His标签。本发明不仅包括完整的抗体，还包括具有免疫活性的抗体片段或抗体与其他序列形成的融合蛋白。本发明还提供了具有本发明抗体的其他蛋白质或融合表达产物。具体地，本发明包括具有含可变区的重链和轻链的任何蛋白质或蛋白质偶联物及融合表达产物(即免疫偶联物及融合表达产物)，只要该可变区与本发明抗体的重链和轻链的可变区相同或至少90％同源性，较佳地至少95％同源性，最佳地96％、97％、98％或99％以上同源性。因此，本发明包括那些具有带CDR的单克隆抗体轻链和重链可变区的分子，只要其CDR与本发明CDR具有90％以上(较佳地95％以上，最佳地96％、97％、98％或99％以上)的同源性。
本发明还包括所述抗体的片段、变体、衍生物和类似物。本申请的抗体、抗原结合片段、它们的变体或衍生物，包括但不限于，多克隆抗体、单克隆抗体、多特异性抗体(如双特异性抗体、三特异性抗体等)、人抗体、动物源抗体、人源化的抗体、灵长类化的(primatized)抗体、或嵌合抗体、CDR嫁接和/或修饰的抗体、单链抗体(例如，ScFv)、 双链抗体、抗原表位结合片段，例如，Fab、Fab'和F(ab')2、Fd、Fv、单链Fv(ScFv)、单链抗体、二硫键连接的Fv(dsFv)、包含VL结构域或VH结构域的片段、由Fab表达库产生的片段、和抗独特型(idiotypic)(抗-Id)抗体。本发明的抗体片段、抗原结合片段、衍生物或类似物可以是(i)有一个或多个保守或非保守性氨基酸残基(优选保守性氨基酸残基)被取代的多肽，而这样的取代的氨基酸残基可以是也可以不是由遗传密码编码的，或(ii)在一个或多个氨基酸残基中具有取代基团的多肽，或(iii)成熟多肽与另一个化合物(比如延长多肽半衰期的化合物，例如聚乙二醇)融合所形成的多肽，或(iv)附加的氨基酸序列融合到此多肽序列而形成的多肽(如前导序列或分泌序列或用来纯化此多肽的序列或蛋白原序列，或与6×His标签形成的融合蛋白)。根据本文的教导，这些片段、衍生物和类似物属于本领域熟练技术人员公知的范围。
本发明抗体指具有人EpCAM和CD3结合活性的、包括上述CDR区的多肽。该术语还包括具有与本发明抗体相同功能的、包含上述CDR区的多肽的变异形式。这些变异形式包括(但并不限于)：一个或多个(通常为1-50个，较佳地1-30个，更佳地1-20个，最佳地1-10个)氨基酸的缺失、插入和/或取代，以及在C末端和/或N末端添加一个或数个(通常为20个以内，较佳地为10个以内，更佳地为5个以内)氨基酸。例如，在本领域中，用性能相近或相似的氨基酸进行取代时，通常不会改变蛋白质的功能。又比如，在C末端和/或N末端添加一个或数个氨基酸通常也不会改变蛋白质的功能。该术语还包括本发明抗体的活性片段和活性衍生物。该多肽的变异形式包括：同源序列、保守性变异体、等位变异体、天然突变体、诱导突变体、在高或低的严紧条件下能与本发明抗体的编码DNA杂交的DNA所编码的蛋白、以及利用抗本发明抗体的抗血清获得的多肽或蛋白。
本发明的抗体可以是(i)有一个或多个保守或非保守性氨基酸残基(优选保守性氨基酸残基)被取代的多肽，或(ii)在一个或多个氨基酸残基中具有取代基团的多肽，或(iii)成熟多肽与另一个化合物(比如延长多肽半衰期的化合物，例如聚乙二醇)融合所形成的多肽，或(iv)附加的氨基酸序列融合到此多肽序列而形成的多肽(如前导序列或分泌序列或用来纯化此多肽的序列或蛋白原序列，或与6His标签形成的融合蛋白)。根据本文的教导，这些片段、衍生物和类似物属于本领域熟练技术人员公知的范围。
“保守氨基酸替换”是其中氨基酸残基被具有类似侧链的氨基酸残基替换。具有类似侧链的氨基酸残基家族已在本领域中定义，其包括碱性侧链(例如赖氨酸、精氨酸、组氨酸)，酸性侧链(例如天冬氨酸，谷氨酸)，不带电荷的极性侧链(例如，甘氨酸、天冬酰胺、谷氨酰胺、丝氨酸、苏氨酸、酪氨酸、半胱氨酸)，非极性侧链(例如，丙氨酸、缬氨酸、亮氨酸、异亮氨酸、脯氨酸、苯丙氨酸、蛋氨酸、色氨酸)，β-支链的侧链(例如，苏氨酸、缬氨酸、异亮氨酸)和芳族侧链(例如酪氨酸、苯丙氨酸、色氨酸、组氨酸)。因此，免疫球蛋白多肽的非必需氨基酸残基优选被来自相同侧链家族的其他氨基酸残基替换。在另一些实施方案中，一串氨基酸可被结构上类似的氨基酸串替换，后者在顺序上和/或侧链家族的组成上不同。
在下表中提供了保守性氨基酸替换的非限制性实例，其中相似性得分为0或更高表示在这两个氨基酸之间有保守替换。
  C G P S A T D E N Q H K R V M I L F Y W
W -8 -7 -6 -2 -6 -5 -7 -7 -4 -5 -3 -3 2 -6 -4 -5 -2 0 0 17
Y 0 -5 -5 -3 -3 -3 -4 -4 -2 -4 0 -4 -5 -2 -2 -1 -1 7 10  
F -4 -5 -5 -3 -4 -3 -6 -5 -4 -5 -2 -5 -4 -1 0 1 2 9    
L -6 -4 -3 -3 -2 -2 -4 -3 -3 -2 -2 -3 -3 2 4 2 6      
I -2 -3 -2 -1 -1 0 -2 -2 -2 -2 -2 -2 -2 4 2 5        
M -5 -3 -2 -2 -1 -1 -3 -2 0 -1 -2 0 0 2 6          
V -2 -1 -1 -1 0 0 -2 -2 -2 -2 -2 -2 -2 4            
R -4 -3 0 0 -2 -1 -1 -1 0 1 2 3 6              
K -5 -2 -1 0 -1 0 0 0 1 1 0 5                
H -3 -2 0 -1 -1 -1 1 1 2 3 6                  
Q -5 -1 0 -1 0 -1 2 2 1 4                    
N -4 0 -1 1 0 0 2 1 2                      
E -5 0 -1 0 0 0 3 4                        
D -5 1 -1 0 0 0 4                          
T -2 0 0 1 1 3                            
A -2 1 1 1 2                              
S 0 1 1 1                                
P -3 -1 6                                  
G -3 5                                    
C 12                                      
在一些实施方案中，所述保守替换优选地是这样的替换，即，其中下列组(a)–(e)内的一个氨基酸被同组内的另一氨基酸残基替换：(a)小的脂肪族、非极性或弱极性的残基：Ala,Ser,Thr,Pro和Gly；(b)极性、带负电荷的残基及其(不带电荷的)酰胺：Asp,Asn,Glu和Gln；(c)极性、带正电荷的残基:His,Arg和Lys；(d)大的脂肪族、非极性残基:Met,Leu,Ile,Val和Cys；以及(e)芳族残基:Phe,Tyr和Trp。
特别优选的保守替换如下：Ala替换成Gly或替换成Ser；Arg替换成Lys；Asn替换成Gln或替换成His；Asp替换成Glu；Cys替换成Ser；Gln替换成Asn；Glu替换成Asp；Gly替换成Ala或替换成Pro；His替换成Asn或替换成Gln；Ile替换成Leu或替换成Val；Leu替换成Ile或替换成Val；Lys替换成Arg,替换成Gln或替换成Glu；Met替换成Leu,替换成Tyr或替换成Ile；Phe替换成Met,替换成Leu或替换成Tyr；Ser替换成Thr；Thr替换成Ser；Trp替换成Tyr；Tyr替换成Trp；和/或Phe替换成Val,替换成Ile或替换成Leu。
Fc氨基酸编号遵从Kabat编号。“Kabat编号”是指Kabat等描述的编号系统，其内容记载于美国卫生和公共服务部，“Sequence of Proteins of Immunological Interest”(1983)。具体的编号见下表：
基于Kabat编号系统的Fc氨基酸编号
Figure PCTCN2021131804-appb-000001
其中，
第221-227位氨基酸为铰链(hinge)结构域，
第228-340位氨基酸为重链第二恒定区CH2结构域，
第341-447位氨基酸为重链第三恒定区CH3结构域。
可对抗体进行修改以提高异二聚体配对效率。比如，在某些方面，与野生型的抗体片段比较，该单价单元重链的Fc片段和/或该融合肽的Fc片段可包含一处或多处替换，这些替换之间形成杵-臼结构对。杵-臼构型在本领域中是已知的。参见，例如Ridgway等的“‘Knob-into-holes’engineering of antibody CH3domains for heavy chain heterodimerization,”Protein Engineering 9(7):617-21(1996)。
一方面，一个CH3结构域上的T366被相对较大的氨基酸残基替换，如酪氨酸(Y)或色氨酸(W)替换。然后，另一CH3结构域上的Y407可被相对较小的氨基酸残基替换，如苏氨酸(T)，丙氨酸(A)或缬氨酸(V)。
表6.Fc氨基酸替换的组合在单价单元和单链单元之间形成杵-臼结构对提高异二聚体配对效率
组合序号 一个CH3上的替换 另一CH3上的替换
1 T366W Y407A
2 T366W Y407V
3 T366Y Y407A
4 T366Y Y407V
5 T366W T366S，L368A，Y407V
一方面，该CH3结构域之一含有一处或多处替换，经在生理条件下有正电荷的氨基酸残基替换，而另一CH3结构域包含一处或多处替换，经一个或多个在生理条件下具有负电荷的氨基酸残基替换。一方面，该带正电的氨基酸残基可为精氨酸(R)，组氨酸(H)或赖氨酸(K)。另一方面，该带负电荷的氨基酸残基可为天冬氨酸(D)或谷氨酸(E)。可被替换的氨基酸残基包括，但不限于，D356、L368、K392、D399和K409。
表7.CH3氨基酸替换的组合在单价单元和单链单元之间形成离子键提高异二聚体配对效率
组合序号 一个CH3上的替换 另一CH3上的替换
1 D356K D399K K392D K409D
2 L368R D399K K392D K409D
3 L368K D399K K392D K409D
4 L368R D399K K409D
5 L368K D399K K409D
6 L368R K409D
7 L368K K409D
一方面，一个CH3结构域上的S354被替换为半胱氨酸，另一CH3结构域上的Y349也被替换为半胱氨酸，两个被替换位置的残基形成了二硫键。
表8.CH3氨基酸替换的组合在单价单元和单链单元之间形成二硫键提高异二聚体配对效率
组合序号 一个CH3上的替换 另一CH3上的替换
1 S354C Y349C
一方面，一个CH3结构域上的H435和Y436分别被替换为精氨酸和苯丙氨酸，该替换导致Fc与蛋白A之间的结合能力显著降低，从而使异二聚体和同二聚体之间具有不同的蛋白A结合活性，在亲和层析过程中易于将两者分离。
表9.一个CH3的氨基酸替换导致与蛋白A的结合能力下降
组合序号 CH3上的替换
1 H435R，Y436F
在本发明的一个优选例中，形成异二聚体的Fc的CH3氨基酸序列如下表所示：
Figure PCTCN2021131804-appb-000002
本申请的一个实施例提供了一种异二聚体抗体，该抗体包含两个不同的抗原结合多肽单元。在某些方面，该异二聚体与其对应的同二聚体大小不同，可利用大小上的区别来促进分离异二聚体和同二聚体。
在某些方面，如图1中，这两个抗原结合多肽单元之一包含类似于野生型抗体的轻 链-重链对。在整个本申请中，该单元也称为“单价单元”。在某些方面，如图1中，其他抗原结合多肽单元包含单链可变片段(ScFv)。这样的ScFv可融合至抗体的恒定片段(Fc)的N端，称为融合肽。在本申请全文中此融合肽也被称为“单链单元”。
任何上述的抗体或多肽还可包括额外的多肽，例如，如本文所述的编码的多肽，抗体恒定区的信号肽，该信号肽用于指导分泌，或如本文所述的其他异源多肽。本文所述的抗体可会被修改，以使得它们的氨基酸序列与天然存在的结合多肽不同，它们得自这些天然存在的结合多肽。例如，来自于指定的蛋白的多肽或氨基酸序列可与起始序列类似，例如，与起始序列具有一定百分比的同一性，例如，其可与起始序列有60％、70％、75％、80％、85％、90％、95％、98％或99％的同一性。此外，也可进行核苷酸或氨基酸替换，缺失，或插入以在“非必需”氨基酸区域进行保守替换或改变。例如，来自指定的蛋白质的多肽或氨基酸序列可与启动顺序相同，除了一个或多个独立的氨基酸的替换，插入，或缺失，例如，1个、2个、3个、4个、5个、6个、7个、8个、9个、10个、15个、20个或更多独立的氨基酸替换，插入，或缺失。在某些实施例中，来自指定的蛋白质的多肽或氨基酸序列相对于起始序列有1至5个、1至10个、1至15个或1至20个独立的氨基酸的替换，插入，或缺失。
本文使用的术语“可检测的标签”指可直接或间接检测的化合物或组合物，该化合物或组合物直接或间接地结合至待检测的组合物(例如，多核苷酸或蛋白质，该蛋白如抗体)以获得“有标签的”组合物。该术语还包括，结合至该多核苷酸的序列，其通过插入序列的表达提供信号，如绿色荧光蛋白(GFP)等。该标签自身可被检测(例如放射性同位素标签或荧光标签)或者，在酶标签情况下，可催化底物化合物或组合物的化学改变，该改变可被检测。该标签可用于小规模检测或更适于高通量筛选。同样地，合适的标签包括但不限于放射性同位素、荧光染料、化学发光化合物、染料、和蛋白(包括酶)。该标签可仅被检测，也可被定量。仅被检测的反应通常包括仅能证实其存在的反应，其中可被定量的反应通常包括具有可定量的(例如可通过数字报告的)值例如强度、极化和/或其他性质的反应。在发光或荧光分析中，可检测的反应可直接使用与分析成分相关的实际上涉及结合的发光体或荧光基团，或间接使用与另一(例如报告分子或指示剂)成分连接的发光体或荧光基团。
在一些实施方案中，本发明所述抗体可以结合治疗剂(如化疗药，如顺铂、卡铂)、药物前体、肽、蛋白、酶、病毒、脂质、生物反应调节剂、药剂或PEG。本发明抗体可以连接至或融合至治疗剂上，该治疗剂可包括可检测标记物，如放射性标记物、免疫调节剂、激素、酶、寡核苷酸、光活性治疗剂或诊断剂、细胞毒性剂，其可为药物或毒素，超声增强剂，非放射性标记物，它们的组合和其他这类本领域已知的成分。
在某些实施例中，抗原结合多肽包含通常不与抗体结合的氨基酸序列或一个或多个基团。例如，本申请的单链Fv抗体片段可包含柔性接头序列，或可以被修改以添加的官能基团(例如，聚乙二醇(PEG)、药物、毒素或标记物)。本申请的抗体，其变体或衍生物包括被修改的衍生物，即，将任何类型的分子共价连接至抗体且该共价连接不会阻止抗体结合至抗原表位。此外，该抗体可以包含一种或多种非经典氨基酸酸。
应注意非明确数量的实体限定应指一或多个(种)该实体；例如，“多功能抗体”应理解为表示一或多个(种)多功能抗体。同样地，非明确数量限定的、术语“一个或多个” 和“至少一个”本文中可互换使用。
本文使用的术语“治疗”是指治疗性治疗和预防或防治措施，其中对于受试者进行防止或减慢(减轻)不良的生理变化或疾病，如癌症的发展。有益的或所需的临床结果包括但不限于，减轻症状，降低疾病的程度、稳定(例如使其不恶化)疾病的状态，延迟或减缓疾病发展，改善或缓和疾病状态，并缓解(无论是部分或全部)，无论可否被检测到。“治疗”也可指与不接受治疗时的预计生存期相比能延长生存期。那些需要治疗的状况包括那些已经具有病症或症状以及那些容易具有病症或症状或那些将对病症或症状进行预防的情况。
所谓“受试者”或“个体”或“动物”或“患者”或“哺乳动物”是指任何受试者，特别是哺乳动物受试者，它们需要进行诊断，预后或治疗。哺乳动物受试者包括人类，家养动物，农场动物，动物园，运动场，或宠物，如狗，猫，豚鼠，兔，大鼠、小鼠，老鼠，马，牛，奶牛，灵长类(例如，人、猴如食蟹猴，猕猴，狒狒，和黑猩猩等)等。
本文所述的，本申请的抗原结合多肽、变体或衍生物可用于与癌症或传染病相关的某些治疗和诊断方法中。本申请还涉及基于抗体的治疗，此种治疗包括将本申请的双特异性抗体给予至患者，例如动物、哺乳动物和人，用于治疗本文描述的一种或多种疾病或状况。本申请的治疗药物包括但不限于，本申请的抗体(包括如本文所述的它们的变体和衍生物)和核酸或编码本申请的抗体的多核苷酸(包括如本文所述的它们的变体和衍生物)。本申请的抗体也可以用于治疗，抑制或预防疾病，病症或状况，包括恶性的疾病，病症，或与这样的疾病或病症相关的状况，如免疫应答相关的疾病。在一些实施方案中，本发明的抗体可以用作免疫抑制剂。在一些实施方案中，本发明的抗体可以用于治疗自体免疫疾病。本申请的抗原结合多肽，其变体或衍生物用于抑制癌症的生长，发展和/或转移，特别是上面列出的或下面段落中列出的那些。
可以用本申请的抗体或其变体或衍生物治疗，预防，诊断和/或预测的与增加细胞存活相关的其他疾病或状况，包括但不限于癌症或肿瘤，包括恶性肿瘤的发展和/或转移，以及相关疾病(如恶性腹水、恶性胸水、恶心积液)，如EpCAM阳性的肿瘤。
给予抗体、其变体或衍生物的方法包括但不限于皮内、肌内、腹膜内、静脉内、皮下、鼻内、硬膜外和口服途径。该抗体或组合物可以通过任何方便的途径给药，例如通过输注或大剂量注射，通过上皮或粘膜与皮肤内层(例如，口腔粘膜，直肠和肠粘膜等)吸收，并且可以与其他生物活性剂一起给药。因此，本申请的含抗体的药物组合物可被口服给药、直肠给药、非肠道给药、脑池内给药、阴道内给药、腹膜内给药、局部给药(如经粉剂、软膏、滴剂或透皮贴剂)、含服给药或作为口腔或鼻腔喷剂。本文使用的术语“非肠道”是指包括静脉内、肌内、腹膜内、胸骨内、皮下和关节内注射和输注的给药模式。给药可以是全身或局部给药。还可能需要的是将本申请的抗原结合多肽或组合物局部给药至需要治疗的区域，这可通过，例如但不限于，在手术过程中局部灌注，局部应用，例如与手术后伤口敷料结合，通过注射、通过导管、通过栓剂、或通过植入物实现，所述植入物为多孔的、无孔的、或凝胶状材料、包括膜或纤维。优选地，当给予本申请的蛋白质(包括抗体)时必须小心使用不吸收蛋白质的材料。
附图说明
图1.YBODY抗体结构示意图。
图2.双特异性抗体介导的HCT116和Jurkat细胞结合作用检测。A：HCT116+Jurkat不加抗体的阴性对照流式图，其中Q1为CFSE染色的Jurkat细胞，Q2为共结合的Jurkat和HCT116细胞，Q3为PKH26染色的HCT116细胞，Q4为无染色细胞；B：HCT116+Jurkat+M701A 10μg/ml实验组流式图，各象限表征意义同前；C：不同抗体介导的HCT116和Jurkat细胞结合的浓度梯度曲线。
图3.双特异性抗体的生物学活性检测(报告基因系统)。
图4.双特异性抗体介导的体外杀伤作用检测。A：M701A对B16-EpCAM的体外杀伤；B：M701A对B16的体外杀伤；C：M701A对HCT116的体外杀伤；D：M701A对OVCAR-3的体外杀伤；E：不同双特异性抗体对CHO-K1-huEpCAM的体外杀伤；F：不同双特异性抗体对HCT116的体外杀伤。
图5.双特异性抗体在HCT116人源结肠癌模型中的体内药效。A：小鼠肿瘤体积的生长变化情况；B：小鼠体重变化情况。
图6.双特异性抗体在OVCAR-3人源卵巢癌模型中的体内药效。A：小鼠肿瘤体积的生长变化情况；B：小鼠体重变化情况。
具体实施方式
以下结合附图对本发明的方法和应用进行描述，所举实例只用于解释本发明，并非用于限定本发明的范围。对于本发明所属技术领域的普通技术人员来说，在不脱离本发明构思的前提下，还可以做出若干简单推演或替换，都应当视为属于本发明的保护范围。
实施例1:双特异性抗体的表达载体构建
以EpCAM和CD3为靶点的双特异性抗体结构包括抗EpCAM结合区域和抗CD3结合区域，单价单元为抗EpCAM重链与轻链形成的配对，单链单元为抗CD3的ScFv-Fc形式，定义为YBODY结构(如图1)，其中抗CD3的VL通过接头与绞链区和CH2连接。其中单价单元的重链Fc和单链单元(以人IgG重链Fc为骨架)的Fc进行氨基酸突变改造，使其各自不易形成同二聚体(homodimer)，而易于形成异二聚体(heterodimer)。以已有质粒或合成基因片段为模版，通过PCR以及重叠PCR扩增双特异性抗体对应的各条链，并通过酶切连接或重组的方法，将各条抗体链克隆至pcDNA3.1载体(Invitrogen公司)中。抗体各条链的具体序列信息如表1和序列表所示。
表1.各抗体分子对应的氨基酸序列信息
Figure PCTCN2021131804-appb-000003
Figure PCTCN2021131804-appb-000004
实施例2：双特异性抗体的表达与纯化
按常规的质粒提取方法进行质粒的提取，并用于化学转染CHO-S细胞(来源于Gibco)。转染后的细胞在37℃、5％CO 2摇床中悬浮震荡培养7-10天。通过3000×g离心收获上清并用0.22μm滤膜过滤。通过蛋白A亲和层析获得初步纯化的双特异性抗体，通过在280nm处的UV吸光度以及相应的消光系数测定纯化的蛋白浓度，通过高性能尺寸排阻色谱法(HPLC-SEC)测试抗体纯度，并计算各蛋白对应的表达量。双特异性抗体分子表达量在40mg/L～91mg/L，初始纯度在45％-81％，M701A、M701B、M701C、M701D、M701E、M701F、M701G、M701H、M701I、M701J和M701K的表达量和起始初度均明显优于M701。接下来，通过阳离子交换层析对亲和样品进行精纯，最终获得HPLC-SEC纯度>95％的双特异性抗体，各双特异性抗体的纯化回收率如表2。
表2.双特异性抗体的表达量和起始HPLC-SEC纯度
抗体编号 表达量 HPLC-SEC 纯化回收率
M701 47.2mg/L 45.91％ 14％
M701A 90.8mg/L 81.12％ 57％
M701B 84.1mg/L 78.53％ 53％
M701C 54.4mg/L 64.54％ 32％
M701D 65.8mg/L 58.63％ 23％
M701E 49mg/L 55.76％ 22％
M701F 52.4mg/L 66.32％ 30％
M701G 41mg/L 70.76％ 25％
M701H 73.3mg/L 72.03％ 43％
M701I 80.6mg/L 73.21％ 45％
M701J 87.7mg/L 78.21％ 47％
M701K 81.3mg/L 74.50％ 41％
实施例3：双特异性抗体的的热稳定性检测
将各双特异性抗体的精纯样品用缓冲液(25mM柠檬酸+50mM NaCl，pH6.0)稀释至0.5mg/ml，以100μL/管分装到1.5mL EP管中，放入40℃水浴中进行14天的热加速实验，并检测纯度和亲和力的变化情况。放入40℃水浴当天计为D0，第14天记为D14。
采用氨基偶联方法分别将人EpCAM(SB，Cat:10694-H08H)和人CD3抗原(SB，Cat:CT038-H2508H)固定于CM5芯片上，抗原偶联量为1500RU，检测抗原端结合活性时，采用1×HBS-EP+buffer稀释样品至起始浓度，再2倍梯度稀释4个浓度，上机从低浓度至高浓度进行检测，结合流速30μL/min，结合时间120s，解离时间300s； 采用pH1.5的Glycine溶液再生芯片，再生流速10μL/min，再生时间30s。检测结束后，采用软件Biacore T200Evaluation Software以1:1Binding拟合方式对结果图谱进行数据拟合，得到解离平衡常数(KD)。
结果如表3所示，M701A、M701B、M701H、M701I、M701J、M701K热加速第14天纯度下降小于5％，其中M701A、M701B、M701J、M701K纯度变化小于2％，且两端亲和力基本未变，说明M701A、M701B、M701J、M701K具有良好的热稳定性。
表3.双特异性抗体热加速样品的纯度和亲和力检测
Figure PCTCN2021131804-appb-000005
说明：n.d.表示未检测。
实施例4：双特异性抗体EpCAM端细胞亲和力检测
利用FACS方法，以人结肠癌细胞HCT116(中国科学院上海生命研究院)作为细胞膜表面表达人EpCAM的阳性细胞，检测抗体与细胞表面人EpCAM的亲和力。
离心收集HCT116细胞，并重悬于缓冲液中(PBS+1％FBS)，按照2×10 5个细胞/孔加入96孔板中，每孔50μL。后按照350×g离心5min后去除上清。将双抗用缓冲液稀释至1000nM，并倍比梯度稀释，而后按照50μL/孔加入96孔板中，重悬后避光孵育1h，离心后去上清，用缓冲液洗两次后再重悬于稀释好的PE标记抗人IgG Fc抗体(Biolegend,409304)中，避光孵育30min，用缓冲液洗两次后再重悬于100μL缓冲液中，通过流式细胞仪(BD Accuri TM C6)上机检测。
各双特异性抗体均与HCT116细胞有明显结合作用，其中M700为抗EpCAM端单抗对照(轻链SEQ ID NO：25和重链SEQ ID NO：26)，具体EC50值如表4所示。
表4.双特异性抗体与HCT116细胞的结合能力
抗体编号 M700 M701 M701A M701B M701H M701I M701J M701K
EC50(nM) 1.782 10.501 3.565 3.719 7.151 7.086 3.779 5.652
实施例5：双特异性抗体CD3端亲和力检测(Biacore)
采用氨基偶联方法将人CD3抗原(SB，Cat:CT038-H2508H)固定于CM5芯片上，抗原偶联量为1500RU，检测CD3抗原端结合活性时，采用1×HBS-EP+buffer稀释样品至起始浓度，再2倍梯度稀释4个浓度，上机从低浓度至高浓度进行检测，结合流 速30μL/min，结合时间120s，解离时间300s；采用pH1.5的Glycine溶液再生芯片，再生流速10μL/min，再生时间30s。检测结束后，采用软件Biacore T200Evaluation Software以1:1Binding拟合方式对结果图谱进行数据拟合，得到解离平衡常数(KD)。如表5所示，系列双特异性抗体均与人CD3抗原有结合作用。其中，M701A和M701B的抗EpCAM抗体可变区序列相同(SEQ ID NO:13和SEQ ID NO:14)，抗CD3抗体可变区序列分别为SEQ ID NO:18和SEQ ID NO:19，对应亲和力分别为21.15nM和28.27nM。但抗EpCAM抗体可变区序列变成另一种(SEQ ID NO:15和SEQ ID NO:16)，抗CD3抗体可变区序列还是SEQ ID NO:18和SEQ ID NO:19，对应的双特异性抗体M701H和M701I的亲和力分别为95.26nM和40.03nM。这说明不同抗EpCAM抗体和不同抗CD3抗体组合形成双特异性抗体，亲和力的表现不具有规律。
表5.Biacore检测各双特异性抗体与人CD3抗原结合能力
抗体编号 M701 M701A M701B M701H M701I M701J M701K
Kd(nM) 25.44 21.15 28.27 95.26 40.03 23.45 28.78
实施例6：双特异性抗体介导的细胞桥接作用
EpCAM阳性细胞株HCT116用PKH26染色，CD3阳性细胞株Jurkat(中国科学院上海生命研究院)用CFSE染色，染色后的细胞以1:1比例(1×10 5个HCT116细胞:1×10 5个Jurkat细胞)并加入梯度稀释的待测抗体混合共孵育，其中M700为抗EpCAM单抗对照(轻链SEQ ID NO：25和重链SEQ ID NO：26)，M100为抗CD3单抗对照(轻链SEQ ID NO：27和重链SEQ ID NO：28)，Mco101为抗荧光素酶和抗CD3的双特异性抗体，作为双特异性抗体的CD3端同型对照(轻链SEQ ID NO：29、重链SEQ ID NO：30和单链SEQ ID NO：31，结构同图1)。共孵育1h，洗涤重悬后，流式细胞仪(BD Accuri TM C6)上机检测，CFSE和PKH26双阳性细胞的即为被抗体桥接的HCT116和Jurkat细胞。
结果如图2所示，1:1的比例混合Jurkat和HCT116细胞，在加入hIgG、M700、M100、Mco101作用1h后，HCT116和Jurkat细胞没有被桥接；而加入M701A和M701B后，桥接的细胞占到PKH26阳性细胞总数的40％左右，M701A与M701B作用活性相当，双特异性抗体介导的细胞相互作用与抗体浓度呈现正的量效关系。
实施例7：双特异性抗体的生物学活性检测(报告基因法)
利用Jurkat-CD3-NFAT-RE-Luc细胞(Promega)检测双特异性抗体的生物学活性。将含有编码人EpCAM基因(NCBI序列号：NM_002354.3)DNA的pLV-puro(Inovogen Tech.Co.,cat.No.VL3001)载体转染到CHO-K1细胞中，获得稳定表达人EpCAM的细胞株CHO-K1-huEpCAM，收集CHO-K1-huEpCAM作为靶细胞，并重悬于缓冲液中(PBS+1％FBS)，按照4×10 4个细胞/孔加入全白96孔培养板中，并置于37℃，5％CO 2培养箱中过夜培养18～24小时，移除板中的培养基，每孔加入40μL抗体稀释液。取出Jurkat-CD3-NFAT-RE-Luc细胞即效应细胞，将细胞吹散，制备成单细胞悬液，按照效靶比E:T＝1.5:1，在全白96孔培养板内，每孔加入40μL，即6×10 4个/孔铺板，将全 白96孔培养板置于37℃，5％CO 2培养箱中培养6小时，每孔加入80μL Bio-Glo荧光素酶检测液，避光室温孵育15分钟，置于多功能读数仪中，采用化学发光读取发光值。
结果如图3所示，在该报告基因评估体系中，双特异性抗体M701、M701A、M701B、M701H、M701I、M701J、M701K均表现出生物学活性，M701A、M701J、M701K活性强于M701。
实施例8：双特异性抗体介导的体外杀伤作用检测
用分离获得的PBMC作为效应细胞，以EpCAM表达细胞作为靶细胞检测双特异性抗体介导的体外杀伤作用。将含有编码人EpCAM基因(NCBI序列号：NM_002354.3)DNA的pLV-puro(Inovogen Tech.Co.,cat.No.VL3001)载体转染到小鼠黑色素瘤细胞B16细胞(中国科学院上海生命研究院)中，获得稳定表达人EpCAM的细胞株B16-EpCAM，其中B16作为不表达EpCAM的阴性细胞。其它EpCAM表达的靶细胞还包括人结肠癌细胞HCT116(中国科学院上海生命研究院)、人卵巢癌细胞OVCAR-3(CCTCC，中国典型培养物保藏中心)以及CHO-K1-huEpCAM。用胰酶将细胞消化成为单细胞悬液，300g离心5min收集细胞并用5μM羟基荧光素二醋酸盐琥珀酰亚胺脂(5，6-carboxyfluorescein diacetate，succinimidyl ester，CFSE)将细胞染色(37℃，15min)，完全培养基洗两次后于Vi-cell细胞计数仪上计数，后按照实验设计加入96孔板中，每孔2×10 4个细胞/100μL。按照50μL/孔加入相应浓度的抗体，将hPBMC于Cellometer细胞计数仪上计数后入96孔板中(每孔2×10 5个细胞/50μL，效靶比为10:1)。将细胞培养板置于细胞培养箱中培养72h，将细胞消化成单细胞悬液后加入终浓度为1μg/mL的溴化丙锭(Propidium iodide，PI)溶液，孵育10min后用流式细胞仪(BD Accuri TM C6)上机检测，并分析CFSE+PI+双阳性细胞占CFSE+阳性细胞百分比。
如图4A和4B所示，M701A只对有EpCAM表达的B16-EpCAM细胞有杀伤作用，对无EpCAM表达的B16细胞无杀伤作用，而对照抗体Mco101对任何细胞都无杀伤作用，说明双特异性抗体作用具有靶向性。如图4C和4D所示，M701A对HCT116和OVCAR-3细胞均有明显杀伤作用，且强于对照Mco101。如图4E所示，双特异性抗体M701A、M701B、M701H、M701I均对CHO-K1-huEpCAM细胞均有明显杀伤作用，且M701A和M701B作用明显强于M701H和M701I。如图4F所示，双特异性抗体M701J、M701K均对HCT116细胞均有明显杀伤作用，杀伤的EC50均在7.814-25.43ng/ml，且M701J杀伤水平与M701A无明显差异。上述双特异性抗体的杀伤活性均明显强于M701(M701的EC50为69.17ng/ml)。
实施例9：双特异性抗体在HCT116人源结肠癌异位异种移植瘤模型中的体内药效
按照培养条件培养足够量的HCT116细胞和效应细胞CIK(cytokine-induced killer，细胞因子诱导的杀伤细胞，是将人外周血单个核细胞在体外用多种细胞因子共同培养一段时间后获得的一群异质细胞。由于该种细胞同时表达CD3+和CD56+两种膜蛋白分子，故又被称为NK细胞样T淋巴细胞)，收集细胞并计数。每只小鼠右侧背部接种预先混合好的HCT116细胞(2×10 6细胞/只)和CIK(2×10 6细胞/只)，接种体积0.1ml/ 只，建立人源结肠癌HCT116异种移植肿瘤模型。接种1h后开始给药治疗。实验分为测试药M701A 2mg/kg组、M701A 1mg/kg组、M701 1mg/kg组、单抗对照M700 2mg/kg组及溶媒对照组(生理盐水)，每组8只小鼠。尾静脉注射给药，分别于接种后第0，2，4天给药，共给药三次。根据相对肿瘤抑制率及肿瘤完全消退率进行疗效评价，根据动物体重变化和死亡情况进行安全性评价。
肿瘤大小计算公式：肿瘤体积(mm 3)＝0.5×(肿瘤长径×肿瘤短径 2)。
相对肿瘤抑制率TGI(％)：TGI＝1-T/C(％)。T和C分别为治疗组和对照组在某一特定时间点的肿瘤体积(TV)。计算公式如下：T/C％＝T TV/C TV×100％(T TV：治疗组平均肿瘤体积；C TV：溶媒对照组平均肿瘤体积)。
肿瘤完全消退率(complete regression)：定义为肿瘤在治疗过程中或治疗后肿瘤体积低于63mm 3。肿瘤完全消退率(％)＝一组中达到完全消退的动物数/该组动物总数×100％。
如图5A所示，测试药M701A(2mg/kg、1mg/kg)在治疗组中停药后第30天(即接种后第33天)时均表现出显著的抑瘤作用，相对肿瘤抑制率TGI(％)分别为100％和93.82％。相对溶媒对照组统计学上均有显著性差异(p值均<0.001)，两组中M701A(2mg/kg)组所有肿瘤达到完全消退标准。M701A在这两个剂量下药效均显著优于2mg/kg的M700(p值<0.001)，相同剂量下M701A的药效明显优于M701(均为1mg/kg)。如图5B所示，治疗过程中均无动物体重下降，未见药物毒性表现。
在相同的肿瘤模型中，M701B，M701J和M701K在相同剂量下均表现出跟M701A类似的抑瘤效果，且体重均无下降。
实施例10：双特异性抗体在OVCAR-3人源卵巢癌异位异种移植瘤模型中的体内药效
按照培养条件培养足够量的OVCAR-3细胞和效应细胞CIK，收集细胞并计数。每只小鼠右侧背部接种预先混合好的OVCAR-3细胞(1×10 7细胞/只)和CIK(1×10 7细胞/只)，接种体积0.2ml/只，Matrigel胶含量为50％(0.1ml/鼠)，建立人源卵巢癌OVCAR-3异位移植肿瘤模型。接种后1h给药治疗，实验分为测试药M701A(5mg/kg)、CD3端同型对照Mco101(5mg/kg)，单抗对照M700(5mg/kg)组及溶媒对照组(生理盐水)，每组8只小鼠。尾静脉注射给药，分别于接种后第0，2，4天给药，共给药三次。根据相对肿瘤抑制率(TGI)及肿瘤完全消退率进行疗效评价，根据动物体重变化和死亡情况进行安全性评价。
如图6A所示，测试药M701A(5mg/kg)在治疗组中停药后第44天(即接种后第48天)时表现出显著的抑瘤作用，相对肿瘤抑制率TGI(％)为98.97％。相对溶媒对照组统计学上均有显著性差异(p值均<0.001)，M701A(5mg/kg)组肿瘤完全消退率为87.5％，该组药效显著优于5mg/kg的M700(TGI＝70.42％)(p值<0.001)和5mg/kg的Mco101(TGI＝68.87％)(p值<0.001)。如图6B所示，治疗过程中均无动物体重下降，未见药物毒性表现。
在相同的肿瘤模型中，M701B，M701J和M701K在相同剂量下均表现出跟M701A类似的抑瘤效果，且体重均无下降。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独 引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。
序列表
Figure PCTCN2021131804-appb-000006
Figure PCTCN2021131804-appb-000007
Figure PCTCN2021131804-appb-000008
Figure PCTCN2021131804-appb-000009
Figure PCTCN2021131804-appb-000010

Claims (11)

  1. 双特异性抗体，其特征在于包含特异性结合EpCAM的抗原结合结构域和特异性结合CD3的抗原结合结构域，
    其中所述特异性结合EpCAM的抗原结合结构域，选自以下各项组成的组：
    1)包含以下CDRs或其变体的特异性结合EpCAM的抗原结合结构域：
    (i)SEQ ID NO:14所示的重链可变区中包含的CDRH1，CDRH2和CDRH3，和
    (ii)SEQ ID NO:13所示的轻链可变区中包含的CDRL1，CDRL2和CDRL3，
    优选地，按照Kabat序列编号系统，CDRL1的序列如SEQ ID NO:32所示，CDRL2的序列如SEQ ID NO:33所示，CDRL3的序列如SEQ ID NO:34所示，CDRH1的序列如SEQ ID NO:35所示，CDRH2的序列如SEQ ID NO:36所示，CDRH3的序列如SEQ ID NO:37所示；或
    2)包含以下CDRs或其变体的特异性结合EpCAM的抗原结合结构域：
    (i)SEQ ID NO:16所示的重链可变区中包含的CDRH1，CDRH2和CDRH3，和
    (ii)SEQ ID NO:15所示的轻链可变区中包含的CDRL1，CDRL2和CDRL3，
    优选地，按照Kabat序列编号系统和CDR定义系统，CDRL1的序列如SEQ ID NO:38所示，CDRL2的序列如SEQ ID NO:39所示，CDRL3的序列如SEQ ID NO:40所示，CDRH1的序列如SEQ ID NO:41所示，CDRH2的序列如SEQ ID NO:42所示，和CDRH3的序列如SEQ ID NO:43；
    所述特异性结合CD3的抗原结合结构域选自以下各项组成的组：
    2)包含以下CDRs或其变体的特异性结合CD3的抗原结合结构域：
    SEQ ID NO:50所示的重链可变区中包含的CDRH1，CDRH2和CDRH3，以及SEQ ID NO:51所示的轻链可变区中包含的CDRL1，CDRL2和CDRL3，
    优选地，按照Kabat序列编号系统，CDRH1的序列如SEQ ID NO:44所示，CDRH2的序列如SEQ ID NO:45所示，和CDRH3的序列如SEQ ID NO:46，CDRL1的序列如SEQ ID NO:47所示，CDRL2的序列如SEQ ID NO:48所示，CDRL3的序列如SEQ ID NO:49所示；或
    2)包含以下CDRs或其变体的特异性结合CD3的抗原结合结构域：
    SEQ ID NO:58所示的重链可变区中包含的CDRH1，CDRH2和CDRH3，以及SEQ ID NO:59所示的轻链可变区中包含的CDRL1，CDRL2和CDRL3，
    优选地，按照Kabat序列编号系统，CDRH1的序列如SEQ ID NO:52所示，CDRH2的序列如SEQ ID NO:53所示，和CDRH3的序列如SEQ ID NO:54，CDRL1的序列如SEQ ID NO:55所示，CDRL2的序列如SEQ ID NO:56所示，CDRL3的序列如SEQ ID NO:57所示；其中所述CDRs的变体与对应的CDRs分别具有3，2或1个氨基酸差异或分别具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％同一性，
    优选地，其中特异性结合EpCAM的抗原结合结构域为Fab片段形式，特异性结合CD3的抗原结合结构域为ScFv形式。
  2. 权利要求1所述的双特异性抗体，其中所述特异性结合EpCAM的抗原结合结构域包含以下重链可变区和轻链可变区(或其变体)：
    (i)SEQ ID NO:14所示的重链可变区，SEQ ID NO:13所示的轻链可变区；或者
    (ii)SEQ ID NO:16所示的重链可变区，SEQ ID NO:15所示的轻链可变区；和
    其中所述特异性结合CD3的抗原结合结构域包含以下重链可变区和轻链可变区(或其变体)：
    (1)SEQ ID NO:50所示的重链可变区和SEQ ID NO:51所示的轻链可变区，或
    (2)SEQ ID NO:58所示的重链可变区和SEQ ID NO:59所示的轻链可变区；
    优选地，
    其中所述特异性结合EpCAM的抗原结合结构域包含以下重链可变区和轻链可变区(或其变体)：
    (i)SEQ ID NO:14所示的重链可变区，SEQ ID NO:13所示的轻链可变区；和
    其中所述特异性结合CD3的抗原结合结构域选自以下各项组成的组：
    (1)SEQ ID NO:18所示的ScFv或其变体，
    (2)SEQ ID NO:19所示的ScFv或其变体，
    其中所述变体与所述对应的可变区或ScFv分别具有3，2或1个氨基酸差异或分别具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％同一性。
  3. 权利要求1或2所述的双特异性抗体，其中所述双特异性抗体包含
    (1)特异性结合EpCAM的轻链-重链对，所述轻链-重链对包含轻链和重链，或由其组成；其中所述轻链包含轻链可变区和轻链恒定区(优选序列如SEQ ID NO:1、60-65任一所示)，所述重链包含重链可变区、CH1(优选序列如SEQ ID NO:2所示)和第一Fc片段；优选地，所述第一Fc片段包含铰链区(优选序列如SEQ ID NO:3所示)，CH2(优选序列如SEQ ID NO:6、7、66-71任一所示)和CH3a；
    (2)特异性结合CD3的融合肽，所述融合肽包含特异性结合CD3的ScFv和第二Fc片段，或由其组成；优选地，所述ScFv从N端至C端方向依次包含重链可变区，连接肽(优选序列如SEQ ID NO:4所示)和轻链可变区，所述第二Fc片段从N端至C端方向依次包含铰链区(优选序列如SEQ ID NO:3所示)，CH2(优选序列如SEQ ID NO:6、7、66-71任一所示)和CH3b，优选地所述轻链可变区的C端与所述第二Fc片段的铰链区通过连接肽(优选序列如SEQ ID NO:5所示)连接；
    优选地，所述第一Fc片段和第二Fc片段为人或者人源化的Fc片段，例如人IgG Fc片段，例如IgG1、IgG2、IgG3、IgG4、IgG5 Fc片段；
    优选地，与野生型抗体比，所述第一Fc片段和/或第二Fc片段包含一处或多处替换，该替换在该重链和融合肽之间形成杵-臼结构配对，例如一个CH3结构域上的T366被相对较大的氨基酸残基替换，如酪氨酸(Y)或色氨酸(W)替换，另一CH3结构域上的Y407被相对较小的氨基酸残基替换，如苏氨酸(T)，丙氨酸(A)或缬氨酸(V)，例如包含表6的一处或多处替换；
    优选地，所述第一Fc片段和/或第二Fc片段包含一处或多处替换，1)该替换在该重链和融合肽之间形成盐桥配对，例如一个CH3结构域含有一处或多处替换，经在 生理条件下有正电荷的氨基酸残基替换，而另一CH3结构域包含一处或多处替换，经一个或多个在生理条件下具有负电荷的氨基酸残基替换，例如该带正电的氨基酸残基为精氨酸(R)，组氨酸(H)或赖氨酸(K)，例如该带负电荷的氨基酸残基可为天冬氨酸(D)或谷氨酸(E)，例如被替换的氨基酸残基包括D356、L368、K392、D399和K409中的一个或多个，例如表7的一处或多处替换，2)该替换在该重链和融合肽之间形成二硫键，例如表8的替换，和/或3)该替换导致Fc与蛋白A之间的结合能力显著降低，例如一个CH3结构域上的H435和Y436分别被替换为精氨酸和苯丙氨酸，如表9所示；
    优选地，其中：
    a)融合肽的CH3b和重链的CH3a具有形成杵-臼结构的替换对；
    b)融合肽的CH3b和重链的CH3a具有形成离子键的替换对；
    c)融合肽的CH3b和重链的CH3a具有形成二硫键的替换对；和/或
    d)融合肽的CH3b和重链的CH3a具有导致与蛋白A的结合能力下降的替换；
    优选地，CH1包括SEQ ID No:2的序列；和/或CL包括选自SEQ ID Nos:1、60-65任一项的序列；
    优选地，所述第一Fc片段和/或第二Fc片段包含选自SEQ ID Nos:6、7、66-71任一项的序列的CH2和/或SEQ ID Nos:8、9、11、12、72-76任一项的序列的CH3；
    优选地，CH3a和CH3b的序列选自以下各项组成的组：
    (1)其中一个序列如SEQ ID NO:8所示，另一个序列如SEQ ID NO:11所示；
    (2)其中一个序列如SEQ ID NO:9所示，另一个序列如SEQ ID NO:12所示；
    (3)其中一个序列如SEQ ID NO:72所示，另一个序列如SEQ ID NO:74所示；
    (4)其中一个序列如SEQ ID NO:9所示，另一个序列如SEQ ID NO:75所示；
    (5)其中一个序列如SEQ ID NO:73所示，另一个序列如SEQ ID NO:76所示；
    优选地，所述双特异性抗体选自以下各项组成的组：
    (1)其包含融合肽，重链和轻链，或由其组成；其中融合肽包含SEQ ID NO:18，SEQ ID NO:5，SEQ ID NO:3，SEQ ID NO:6和SEQ ID NO:11，或由其组成；重链包含SEQ ID NO:14，SEQ ID NO:2，SEQ ID NO:3，SEQ ID NO:6和SEQ ID NO:8，或由其组成；轻链包含SEQ ID NO:13和SEQ ID NO:1，或由其组成；
    (2)其包含融合肽，重链和轻链，或由其组成；其中融合肽包含SEQ ID NO:19，SEQ ID NO:5，SEQ ID NO:3，SEQ ID NO:6和SEQ ID NO:11，或由其组成；重链包含SEQ ID NO:14，SEQ ID NO:2，SEQ ID NO:3，SEQ ID NO:6和SEQ ID NO:8，或由其组成；轻链包含SEQ ID NO:13和SEQ ID NO:1，或由其组成；
    (3)其包含融合肽，重链和轻链，或由其组成；其中融合肽包含SEQ ID NO:18，SEQ ID NO:5，SEQ ID NO:3，SEQ ID NO:6和SEQ ID NO:11，或由其组成；重链包含SEQ ID NO:16，SEQ ID NO:2，SEQ ID NO:3，SEQ ID NO:6和SEQ ID NO:8，或由其组成；轻链包含SEQ ID NO:15和SEQ ID NO:1，或由其组成；
    (4)其包含融合肽，重链和轻链，或由其组成；其中融合肽包含SEQ ID NO:19，SEQ ID NO:5，SEQ ID NO:3，SEQ ID NO:6和SEQ ID NO:11，或由其组成；重链包含SEQ ID NO:16，SEQ ID NO:2，SEQ ID NO:3，SEQ ID NO:6和SEQ ID NO:8，或由其组成；轻链包含SEQ ID NO:15和SEQ ID NO:1，或由其组成；
    (5)其包含融合肽，重链和轻链，或由其组成；其中融合肽包含SEQ ID NO:18，SEQ ID NO:5，SEQ ID NO:3，SEQ ID NO:7和SEQ ID NO:12，或由其组成；重链包含SEQ ID NO:14，SEQ ID NO:2，SEQ ID NO:3，SEQ ID NO:7和SEQ ID NO:9，或由其组成；轻链包含SEQ ID NO:13和SEQ ID NO:1，或由其组成；
    (6)其包含融合肽，重链和轻链，或由其组成；其中融合肽包含SEQ ID NO:19，SEQ ID NO:5，SEQ ID NO:3，SEQ ID NO:7和SEQ ID NO:12，或由其组成；重链包含SEQ ID NO:14，SEQ ID NO:2，SEQ ID NO:3，SEQ ID NO:7和SEQ ID NO:9，或由其组成；轻链包含SEQ ID NO:13和SEQ ID NO:1，或由其组成；
    (7)其包含融合肽，重链和轻链，或由其组成；其中融合肽包含SEQ ID NO:18，SEQ ID NO:5，SEQ ID NO:3，SEQ ID NO:6和SEQ ID NO:8，或由其组成；重链包含SEQ ID NO:14，SEQ ID NO:2，SEQ ID NO:3，SEQ ID NO:6和SEQ ID NO:11，或由其组成；轻链包含SEQ ID NO:13和SEQ ID NO:1，或由其组成；
    (8)其包含融合肽，重链和轻链，或由其组成；其中融合肽包含SEQ ID NO:19，SEQ ID NO:5，SEQ ID NO:3，SEQ ID NO:6和SEQ ID NO:8，或由其组成；重链包含SEQ ID NO:14，SEQ ID NO:2，SEQ ID NO:3，SEQ ID NO:6和SEQ ID NO:11，或由其组成；轻链包含SEQ ID NO:13和SEQ ID NO:1，或由其组成；
    (9)其包含融合肽，重链和轻链，或由其组成；其中融合肽包含SEQ ID NO:18，SEQ ID NO:5，SEQ ID NO:3，SEQ ID NO:6和SEQ ID NO:8，或由其组成；重链包含SEQ ID NO:16，SEQ ID NO:2，SEQ ID NO:3，SEQ ID NO:6和SEQ ID NO:11，或由其组成；轻链包含SEQ ID NO:15和SEQ ID NO:1，或由其组成；
    (10)其包含融合肽，重链和轻链，或由其组成；其中融合肽包含SEQ ID NO:19，SEQ ID NO:5，SEQ ID NO:3，SEQ ID NO:6和SEQ ID NO:8，或由其组成；重链包含SEQ ID NO:16，SEQ ID NO:2，SEQ ID NO:3，SEQ ID NO:6和SEQ ID NO:11，或由其组成；轻链包含SEQ ID NO:15和SEQ ID NO:1，或由其组成；
    (11)其包含融合肽，重链和轻链，或由其组成；其中融合肽包含SEQ ID NO:18，SEQ ID NO:5，SEQ ID NO:3，SEQ ID NO:7和SEQ ID NO:9，或由其组成；重链包含SEQ ID NO:14，SEQ ID NO:2，SEQ ID NO:3，SEQ ID NO:7和SEQ ID NO:12，或由其组成；轻链包含SEQ ID NO:13和SEQ ID NO:1，或由其组成；
    (12)其包含融合肽，重链和轻链，或由其组成；其中融合肽包含SEQ ID NO:19，SEQ ID NO:5，SEQ ID NO:3，SEQ ID NO:7和SEQ ID NO:9，或由其组成；重链包含SEQ ID NO:14，SEQ ID NO:2，SEQ ID NO:3，SEQ ID NO:7和SEQ ID NO:12，或由其组成；轻链包含SEQ ID NO:13和SEQ ID NO:1，或由其组成。
  4. 核酸组合物，其包含:编码权利要求1-3任一项所述的双特异性抗体的核酸序列，优选地，
    核酸组合物包含：
    a)第一表达载体，所述第一表达载体包含编码权利要求1-3任一项中定义的特异性结合EpCAM的抗原结合结构域或轻链-重链对的第一核酸；
    b)第二表达载体，所述第二表达载体包含编码权利要求1-3任一项中定义的特异性结合CD3的抗原结合结构域或融合肽的第二核酸。
  5. 表达载体，其包含权利要求4的核酸组合物。
  6. 宿主细胞，其包含权利要求5的表达载体。
  7. 药物组合物，其包含权利要求1-3任一项所述的双特异性抗体，和药用载体，和任选地，治疗癌症(EpCAM阳性的肿瘤，例如结直肠癌，胃癌，乳腺癌，卵巢癌，肺癌(如非小细胞肺癌)，前列腺癌，胰腺癌，肝癌，视网膜母细胞瘤，食管癌，肾癌，肾透明细胞瘤，皮肤鳞癌，皮肤基底细胞癌，肉瘤，鼻腔神经胶质瘤，颅咽管瘤，甲状腺癌，胆管细胞瘤，膀胱癌，头颈部肿瘤，子宫颈癌，或口腔癌等，)和/或恶性腹水、恶性积液、恶性胸水等的药物(如小分子药物或大分子药物)，优选地，所述药物组合物的剂型包括胃肠给药剂型或胃肠外给药剂型；更优选地，所述药物组合物的剂型为注射剂，包括静脉注射、静脉滴注、皮下注射、局部注射、肌肉注射、瘤内注射、腹腔内注射、颅内注射、或腔内注射。
  8. 缀合物或融合蛋白，其包含权利要求1-3任一项的双特异性抗体，优选地，其包含与所述双特异性抗体缀合或融合的物质A，所述物质A选自治疗剂、药物前体、蛋白(例如酶)、病毒、脂质、生物反应调节剂(如免疫调节剂)、PEG、激素、寡核苷酸、诊断剂、细胞毒性剂，其可为药物或毒素，超声增强剂，非放射性标记物，可检测标记物，如化学发光物标记化合物(如鲁米诺、异鲁米诺、热性吖啶鎓酯、咪唑、吖啶鎓盐和草酸酯)，或荧光发光金属(如152Eu，或镧系标记)。
  9. 试剂盒，其包含权利要求1-3任一项所述的双特异性抗体，和任选地，治疗癌症(EpCAM阳性的肿瘤，例如结直肠癌，胃癌，乳腺癌，卵巢癌，肺癌(如非小细胞肺癌)，前列腺癌，胰腺癌，肝癌，视网膜母细胞瘤，食管癌，肾癌，肾透明细胞瘤，皮肤鳞癌，皮肤基底细胞癌，肉瘤，鼻腔神经胶质瘤，颅咽管瘤，甲状腺癌，胆管细胞瘤，膀胱癌，头颈部肿瘤，子宫颈癌，或口腔癌等)和/或恶性腹水、恶性积液、恶性胸水等的药物(如小分子药物或大分子药物)。
  10. 权利要求1-3任一项所述的双特异性抗体，用于治疗癌症，或在制备治疗癌症和/或恶性腹水、恶性积液、恶性胸水等的药物或试剂盒中的应用，所述癌症例如EpCAM阳性的肿瘤，例如结直肠癌，胃癌，乳腺癌，卵巢癌，肺癌(如非小细胞肺癌)，前列腺癌，胰腺癌，肝癌，视网膜母细胞瘤，食管癌，肾癌，肾透明细胞瘤，皮肤鳞癌，皮肤基底细胞癌，肉瘤，鼻腔神经胶质瘤，颅咽管瘤，甲状腺癌，胆管细胞瘤，膀胱癌，头颈部肿瘤，子宫颈癌，或口腔癌。
  11. 治疗癌症和/或恶性腹水、恶性积液、恶性胸水的方法，包含对受试者施用治疗有效量的权利要求1-3任一项所述的双特异性抗体，所述癌症EpCAM阳性的肿瘤，例如结直肠癌，胃癌，乳腺癌，卵巢癌，肺癌(如非小细胞肺癌)，前列腺癌，胰腺癌，肝癌，视网膜母细胞瘤，食管癌，肾癌，肾透明细胞瘤，皮肤鳞癌，皮肤基底细胞癌，肉瘤，鼻腔神经胶质瘤，颅咽管瘤，甲状腺癌，胆管细胞瘤，膀胱癌，头颈部肿瘤，子宫颈癌，或口腔癌。
PCT/CN2021/131804 2021-11-19 2021-11-19 双特异性抗体及其应用 Ceased WO2023087255A1 (zh)

Priority Applications (11)

Application Number Priority Date Filing Date Title
CN202180104172.1A CN118201960B (zh) 2021-11-19 2021-11-19 双特异性抗体及其应用
CN202510748238.0A CN120818062A (zh) 2021-11-19 2021-11-19 双特异性抗体及其应用
JP2024529262A JP2024540461A (ja) 2021-11-19 2021-11-19 二重特異性抗体及びその用途
PCT/CN2021/131804 WO2023087255A1 (zh) 2021-11-19 2021-11-19 双特异性抗体及其应用
MX2024006079A MX2024006079A (es) 2021-11-19 2021-11-19 Anticuerpo biespecifico y uso del mismo.
US18/711,094 US20250002605A1 (en) 2021-11-19 2021-11-19 Bispecific antibody and use thereof
AU2021474386A AU2021474386A1 (en) 2021-11-19 2021-11-19 Bispecific antibody and use thereof
EP21964409.3A EP4435006A4 (en) 2021-11-19 2021-11-19 Bispecific Antibodies and Their Use
CA3238406A CA3238406A1 (en) 2021-11-19 2021-11-19 Bispecific antibody and use thereof
KR1020247020170A KR20240100453A (ko) 2021-11-19 2021-11-19 이중 특이적 항체 및 이의 응용
CN202510748232.3A CN120818061A (zh) 2021-11-19 2021-11-19 双特异性抗体及其应用

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/CN2021/131804 WO2023087255A1 (zh) 2021-11-19 2021-11-19 双特异性抗体及其应用

Publications (1)

Publication Number Publication Date
WO2023087255A1 true WO2023087255A1 (zh) 2023-05-25

Family

ID=86395992

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2021/131804 Ceased WO2023087255A1 (zh) 2021-11-19 2021-11-19 双特异性抗体及其应用

Country Status (9)

Country Link
US (1) US20250002605A1 (zh)
EP (1) EP4435006A4 (zh)
JP (1) JP2024540461A (zh)
KR (1) KR20240100453A (zh)
CN (3) CN120818062A (zh)
AU (1) AU2021474386A1 (zh)
CA (1) CA3238406A1 (zh)
MX (1) MX2024006079A (zh)
WO (1) WO2023087255A1 (zh)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN120346170A (zh) * 2025-03-13 2025-07-22 武汉友芝友生物制药股份有限公司 抗EpCAM和CD3双特异性抗体药物组合物
WO2025190337A1 (zh) * 2024-03-13 2025-09-18 武汉友芝友生物制药股份有限公司 使用抗EpCAM×CD3双特异性抗体改善或治疗恶性腹水的方法

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5892019A (en) 1987-07-15 1999-04-06 The United States Of America, As Represented By The Department Of Health And Human Services Production of a single-gene-encoded immunoglobulin
WO2004106383A1 (en) * 2003-05-31 2004-12-09 Micromet Ag Pharmaceutical composition comprising a bispecific antibody for epcam
CN1867586A (zh) * 2003-10-16 2006-11-22 麦克罗梅特股份公司 多特异性的去免疫化cd3结合物
WO2018028449A1 (en) * 2016-08-08 2018-02-15 Beigene, Ltd. Method for predicting efficacy of immune checkpoint inhibitors in cancer patients
CN110914296A (zh) * 2019-02-22 2020-03-24 武汉友芝友生物制药有限公司 改造的Fc片段，包含其的抗体及其应用
WO2021047558A1 (zh) * 2019-09-09 2021-03-18 武汉友芝友生物制药有限公司 断裂型内含肽、使用其的重组多肽的制备方法

Family Cites Families (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DK1629011T3 (da) * 2003-05-31 2010-05-03 Micromet Ag Humane anti-humane-DC3-bindingsmolekyler
EP2370467B1 (en) * 2008-10-01 2016-09-07 Amgen Research (Munich) GmbH Cross-species-specific pscaxcd3, cd19xcd3, c-metxcd3, endosialinxcd3, epcamxc d3, igf-1rxcd3 or fapalpha xcd3 bispecific single chain antibody
SG10201405434VA (en) * 2009-09-18 2014-10-30 Amgen Res Munich Gmbh Dosage regimen for administering an epcamxcd3 bispecific antibody
PT3434767T (pt) * 2010-11-30 2026-01-23 Chugai Pharmaceutical Co Ltd Agente terapêutico indutor de citotoxicidade
US9382329B2 (en) * 2012-08-14 2016-07-05 Ibc Pharmaceuticals, Inc. Disease therapy by inducing immune response to Trop-2 expressing cells
EA035419B9 (ru) * 2014-05-29 2020-08-07 Мэкроудженикс, Инк. Триспецифичные связывающие молекулы и способы их применения
CN104592392B (zh) * 2015-01-21 2018-08-28 武汉友芝友生物制药有限公司 一种双特异性抗体EpCAM×CD3的构建及应用
CN104592391B (zh) * 2015-01-21 2020-08-21 武汉友芝友生物制药有限公司 一种双特异性抗体EpCAM×CD3的构建及应用
IL303187A (en) * 2016-08-29 2023-07-01 Akamis Bio Ltd Adenovirus with bispecific T cell activator

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5892019A (en) 1987-07-15 1999-04-06 The United States Of America, As Represented By The Department Of Health And Human Services Production of a single-gene-encoded immunoglobulin
WO2004106383A1 (en) * 2003-05-31 2004-12-09 Micromet Ag Pharmaceutical composition comprising a bispecific antibody for epcam
CN1867586A (zh) * 2003-10-16 2006-11-22 麦克罗梅特股份公司 多特异性的去免疫化cd3结合物
WO2018028449A1 (en) * 2016-08-08 2018-02-15 Beigene, Ltd. Method for predicting efficacy of immune checkpoint inhibitors in cancer patients
CN110914296A (zh) * 2019-02-22 2020-03-24 武汉友芝友生物制药有限公司 改造的Fc片段，包含其的抗体及其应用
WO2021047558A1 (zh) * 2019-09-09 2021-03-18 武汉友芝友生物制药有限公司 断裂型内含肽、使用其的重组多肽的制备方法

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
"Sequence of Proteins of Immunological Interest", 1983, UNITED STATES DEPARTMENT OF HEALTH AND HUMAN SERVICES
RIDGWAY ET AL.: "Knob-into-holes' engineering of antibody CH3 domains for heavy chain heterodimerization", PROTEIN ENGINEERING, vol. 9, no. 7, 1996, pages 617 - 21
See also references of EP4435006A4

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2025190337A1 (zh) * 2024-03-13 2025-09-18 武汉友芝友生物制药股份有限公司 使用抗EpCAM×CD3双特异性抗体改善或治疗恶性腹水的方法
CN120346170A (zh) * 2025-03-13 2025-07-22 武汉友芝友生物制药股份有限公司 抗EpCAM和CD3双特异性抗体药物组合物

Also Published As

Publication number Publication date
CN118201960A (zh) 2024-06-14
US20250002605A1 (en) 2025-01-02
JP2024540461A (ja) 2024-10-31
AU2021474386A1 (en) 2024-06-27
CN118201960B (zh) 2025-05-16
MX2024006079A (es) 2024-07-29
EP4435006A4 (en) 2026-04-29
EP4435006A1 (en) 2024-09-25
CA3238406A1 (en) 2023-05-25
CN120818061A (zh) 2025-10-21
KR20240100453A (ko) 2024-07-01
CN120818062A (zh) 2025-10-21

Similar Documents

Publication Publication Date Title
US20200055932A1 (en) Antibody Constructs for CLDN18.2 and CD3
TWI564306B (zh) 雙特異性抗體
JP7851303B2 (ja) ヒトｃｄ３イプシロンに結合する新規なヒト抗体
WO2021197359A1 (zh) 一种构建多特异性抗体的平台
JP2022546002A (ja) ４価の対称型二重特異性抗体
EP4242232A1 (en) Bispecific antibody and use thereof
WO2022068810A1 (zh) 抗Claudin18.2和CD3的双特异性抗体以及其用途
WO2021047386A1 (zh) 靶向caix抗原的纳米抗体及其应用
WO2018176992A1 (zh) 新型双特异性抗体及其用途
KR20250027562A (ko) Dll3 및 cd3에 특이적으로 결합하는 항원 결합 분자 및 이의 의약적 용도
CN118201960B (zh) 双特异性抗体及其应用
WO2024082269A1 (zh) 双特异性抗体在免疫细胞治疗方面的应用
US20230340119A1 (en) Composition of triax antibodies and method of making and using thereof
US20230406887A1 (en) Antigen binding domain with reduced clipping rate
WO2024109657A1 (zh) 抗ccr8抗体及其用途
RU2839153C2 (ru) Биспецифическое антитело и его применение
WO2022117003A1 (zh) 抗PD-L1/TGF-β双功能抗体及其用途
HK40117294A (zh) 双特异性抗体及其应用
WO2025157244A1 (zh) 抗b7h3抗体及其应用
TW202544031A (zh) 抗tigit/抗pd-l1雙特異性抗體及其用途
CN120858119A (zh) 抗muc17、cd3和cd28三特异性抗体
WO2025162454A1 (zh) 双特异性抗体和细胞因子融合蛋白及其应用
WO2025002251A1 (zh) 抗gprc5d抗体及其医药用途
WO2023011650A1 (zh) 一种多特异性抗体及其用途
CN121591898A (zh) 靶向cd228的抗体和抗体-药物偶联物

Legal Events

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

Ref document number: 21964409

Country of ref document: EP

Kind code of ref document: A1

WWE Wipo information: entry into national phase

Ref document number: 202180104172.1

Country of ref document: CN

ENP Entry into the national phase

Ref document number: 2024529262

Country of ref document: JP

Kind code of ref document: A

ENP Entry into the national phase

Ref document number: 3238406

Country of ref document: CA

WWE Wipo information: entry into national phase

Ref document number: 18711094

Country of ref document: US

WWE Wipo information: entry into national phase

Ref document number: 2021474386

Country of ref document: AU

Ref document number: AU2021474386

Country of ref document: AU

WWE Wipo information: entry into national phase

Ref document number: 1020247020170

Country of ref document: KR

WWE Wipo information: entry into national phase

Ref document number: 2024116487

Country of ref document: RU

Ref document number: 2021964409

Country of ref document: EP

NENP Non-entry into the national phase

Ref country code: DE

ENP Entry into the national phase

Ref document number: 2021474386

Country of ref document: AU

Date of ref document: 20211119

Kind code of ref document: A

ENP Entry into the national phase

Ref document number: 2021964409

Country of ref document: EP

Effective date: 20240619

WWG Wipo information: grant in national office

Ref document number: 2024116487

Country of ref document: RU

WWG Wipo information: grant in national office

Ref document number: 202180104172.1

Country of ref document: CN