WO2020135804A1 - 异源二聚体融合蛋白 - Google Patents

异源二聚体融合蛋白 Download PDF

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WO2020135804A1
WO2020135804A1 PCT/CN2019/129591 CN2019129591W WO2020135804A1 WO 2020135804 A1 WO2020135804 A1 WO 2020135804A1 CN 2019129591 W CN2019129591 W CN 2019129591W WO 2020135804 A1 WO2020135804 A1 WO 2020135804A1
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seq
fusion protein
antigen
binding domain
polypeptide chain
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PCT/CN2019/129591
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French (fr)
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王峰
郑花鸯
张雨菡
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上海一宸医药科技有限公司
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Priority to CN201980086672.XA priority Critical patent/CN113490690A/zh
Priority to US17/419,314 priority patent/US20220089722A1/en
Publication of WO2020135804A1 publication Critical patent/WO2020135804A1/zh

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    • 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
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], 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 [IGs], 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
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], 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
    • C07K16/2833—Immunoglobulins [IGs], 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 MHC-molecules, e.g. HLA-molecules
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2851—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the lectin superfamily, e.g. CD23, CD72
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2863—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for growth factors, growth regulators
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2878—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—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
    • C07K2317/60—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
    • 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
    • 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
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/33—Fusion polypeptide fusions for targeting to specific cell types, e.g. tissue specific targeting, targeting of a bacterial subspecies

Definitions

  • the invention belongs to the field of biotechnology pharmaceuticals, and is specifically based on a heterodimer fusion protein of immunoglobulin fragments and its preparation and application.
  • bispecific antibodies that can simultaneously bind two different epitopes of the same antigen or different antigens has long been an important area of research and development of new structural antibodies. So far, three bispecific antibodies have been approved for marketing. The earliest double antibody was Catumaxomab produced by the hybridization and fusion of mouse and rat hybridoma cells by Frenesius and Trion. (anti-EpCAM ⁇ anti-CD3), approved by EMA (European Medicines Agency) in 2009 for the treatment of malignant ascites caused by EpCAM (Epithelial cell adhesion molecule) positive tumors.
  • EMA European Medicines Agency
  • bispecific antibodies that are currently undergoing preclinical or clinical research can be divided into two major categories: immunoglobulin-like (IgG-like)-based double antibodies and non-immune globules based on antibody fragments Non-IgG-like double antibody.
  • the non-IgG-like dual antibody represented by the BiTE technology platform has a small molecular weight and good tissue penetration, but due to its lack of Fc fragments, the half-life in vivo is very short.
  • Blincyto which has been approved for marketing, has an official published half-life in vivo of only 2 hours. It requires continuous injection in the clinic, and the patient's compliance is poor.
  • IgG-like double Anti-Fc fragments containing IgG molecules retain Fc-mediated antibody-dependent cell-mediated cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), Antibody-dependent cellular phagocytosis (ADCP) and other effect functions; in addition, Fc can be used for the purification of IgG-like double antibodies, which helps to improve solubility and greatly improve the stability of such double antibodies Sex; due to the large molecular weight and FcRn-mediated recycling mechanism, Ig-like double antibodies usually have a longer plasma half-life.
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • CDC complement-dependent cytotoxicity
  • ADCP Antibody-dependent cellular phagocytosis
  • Fc can be used for the purification of IgG-like double antibodies, which helps to improve solubility and greatly improve the stability of such double antibodies Sex; due to the large molecular weight and FcRn-mediated recycling mechanism, Ig-like double antibodies usually have a longer plasma half-life.
  • the Ig-like double antibody represented by Roche's CrossMab technology is effective by swapping the VH of one of the antibody fragments with VL, CH1-CL or VH/CH1-VL/CL and knobs-into-holes on the Fc backbone It solves the problems of light chain-light chain pairing, heavy chain-heavy chain pairing, and heavy chain light chain mismatch of two antibody fragments in the molecule.
  • BsAbs with multiple antibodies in clinical trials have adopted this technology.
  • one of the antibody fragments becomes an unconventional Fab type due to the exchange of domains, the affinity of the antibody fragment to the antigen is affected to some extent, and the pharmacokinetic properties are not particularly good.
  • the homologous 2+2 type dual antibody represented by DVD-Ig the two arms of Fc can bind two antigens, which greatly improves the affinity of BsAb, but because it is a homodimer, it is easy to activate T cells Therefore, this type of BsAbs is not suitable for recruiting T cells to kill tumor cells. Therefore, the preparation of BsAbs with excellent pharmacokinetic properties, relatively simple process, and maintaining the natural binding activity of the two antibodies in the molecule is crucial for clinical drug development.
  • the present invention provides a heterodimeric fusion protein in which the Fab heavy chain (FabH) and Fab light chain (FabL) capable of constituting the first antigen-binding domain Fab are fused (directly or through a linker) in two The N-terminus of a single-chain Fc, and the resulting structure has an antibody-like stable protein fold.
  • the present invention also provides a heterodimer fusion protein in which scfv, Fab2, Fv, Nanobody or physiologically active peptide constituting the second antigen-binding domain and the Fab heavy chain and/or Or the N-terminus of the light chain or the C-terminus of any of the single-chain Fc fusions.
  • the first antigen-binding domain and the second antigen-binding domain in the molecule of the heterodimeric fusion protein thus obtained can form and maintain their functional conformations, respectively, so that the two antigen-binding structures can be played well
  • the activity and pharmaceutical properties of the domain itself; at least one of the antigen-binding domains can monovalently bind antigen.
  • the heterodimer fusion protein of the present invention has a longer half-life.
  • the present invention provides a heterodimer fusion protein based on immunoglobulin fragments, the heterodimer fusion protein including:
  • a second polypeptide chain comprising a Fab light chain and a second single-chain Fc, the Fab light chain being fused to the N-terminus of the second Fc single chain directly or through a linker;
  • the Fab heavy chain of the first polypeptide chain and the Fab light chain of the second polypeptide chain form a first antigen-binding domain Fab
  • the first single-chain Fc and the second single-chain Fc form Fc dimerization domain ( Figure 1A).
  • the linker is GGSGAKLAALKAKLAALKGGGGS. In some embodiments, the linker is GGGGSELAALEAELAALEAGGSG. In some embodiments, the Fab heavy chain of the first polypeptide chain is fused to the N-terminus of the first Fc single chain through the linker GGSGAKLAALKAKLAALKGGGGS, and the Fab light chain of the second polypeptide chain is linked to the GGGGSELAALEAELAALEAGGSG with the linker The N-terminal fusion of the second Fc single chain.
  • the Fab heavy chain of the first polypeptide chain is fused to the N-terminus of the first Fc single chain through GGGGSELAALEAELAALEAGGSG, and the Fab light chain of the second polypeptide chain is linked to the Fab through the linker GGSGAKLAALKAKLAALKGGGGS The N-terminal fusion of the second Fc single chain.
  • the first single-chain Fc and the second single-chain Fc are preferably derived from the same antibody isotypes. But it can also come from different antibody isotypes, as long as the two can pair to form a dimer.
  • both the first single-chain Fc and the second single-chain Fc are derived from IgG, more specifically, both are derived from IgG1.
  • the first single-chain Fc and the second single-chain Fc pair through an interchain disulfide bond and intermolecular interaction to form a dimer.
  • the first and second single-chain Fc are wild-type Fc.
  • the wild-type Fc has the amino acid sequence shown in SEQ ID NO. 147.
  • the first and second single-chain Fc are Fc variants.
  • the Fc variant is free of glycosylation modification sites.
  • the Fc variant comprises N297 deglycosylated modified amino acid substitutions.
  • the Fc variant comprising N294 deglycosylated modified amino acid modification has the amino acid sequence shown in SEQ ID NO. 143.
  • the Fc variant comprises one or more amino acid substitutions that reduce Fc binding to Fc receptors and/or effector functions.
  • the amino acid substitution in the Fc variant comprises one or more of E233P, L234V, L235A, delG236, A327G, A330S, and A331S.
  • the Fc variant comprising one or more amino acid substitutions that reduce Fc binding to Fc receptors and/or effector function has the amino acid sequence shown in SEQ ID. 144.
  • one of the first Fc variant and the second Fc variant further comprises the amino acid substitution S354C, T366W, and the other of the first Fc and the second Fc further comprises the amino acid substitution Y349C , T366S, L368A and Y407V.
  • the first Fc variant has the amino acid sequence shown in SEQ ID NO. 145
  • the second Fc variant has the amino acid sequence shown in SEQ ID NO. 146.
  • the first Fc variant has the amino acid sequence shown in SEQ ID NO.
  • the second Fc variant has the amino acid sequence shown in SEQ ID NO. 145.
  • the first Fc variant has the amino acid sequence shown in SEQ ID NO. 147
  • the second Fc variant has the amino acid sequence shown in SEQ ID NO. 148.
  • the first Fc variant has the amino acid sequence shown in SEQ ID NO. 148
  • the second Fc variant has the amino acid sequence shown in SEQ ID NO. 147.
  • the heterodimeric fusion protein further comprises a second antigen binding domain.
  • the second antigen-binding domain of the heterodimeric fusion protein is a single chain Fv (scfv).
  • the scfv constituting the second antigen binding domain is fused to the N-terminus of the first polypeptide chain directly or through a linker (FIG. 1B).
  • the first antigen-binding domain Fab of the heterodimeric fusion protein binds CD3.
  • the first antigen-binding domain Fab that binds CD3 has a Fab heavy chain shown in SEQ ID NO. 125 and a Fab light chain shown in SEQ ID NO. 126.
  • the second antigen binding domain scfv of the heterodimeric fusion protein binds EGFR.
  • the second antigen binding domain scfv that binds EGFR has the amino acid sequence shown in SEQ ID NO. 142.
  • the heterodimeric fusion protein binds CD3 and EGFR.
  • the heterodimer fusion protein that binds CD3 and EGFR has a first antigen-binding domain having a Fab heavy chain as shown in SEQ ID NO. 125 and a Fab heavy chain as shown in SEQ ID NO. 126 In the Fab light chain, the second antigen-binding domain has the amino acid sequence shown in SEQ ID NO. 142.
  • the heterodimer fusion protein that binds CD3 and EGFR has a first polypeptide chain that includes the amino acid sequence shown in SEQ ID NO. 18 and an amino acid sequence that includes the amino acid sequence shown in SEQ ID NO. 12. Second polypeptide chain.
  • scfv constituting the second antigen binding domain is fused to the N-terminus of the second polypeptide chain directly or through a linker (FIG. 1C).
  • the first antigen-binding domain Fab of the heterodimeric fusion protein binds CD3.
  • the CD3-binding first antigen-binding domain Fab has FabH shown in SEQ ID NO. 125 and FabL shown in SEQ ID NO. 126.
  • the second antigen binding domain scfv of the heterodimeric fusion protein binds EGFR.
  • the second antigen binding domain scfv that binds EGFR has the amino acid sequence shown in SEQ ID NO. 142.
  • the heterodimeric fusion protein binds CD3 and EGFR.
  • the first antigen-binding domain of the heterodimeric fusion protein that binds CD3 and EGFR has the Fab heavy chain shown in SEQ ID NO. 125 and the Fab shown in SEQ ID NO. 126
  • the second antigen-binding domain of the light chain has the amino acid sequence shown in SEQ ID NO. 142.
  • the heterodimer fusion protein that binds CD3 and EGFR has a first polypeptide chain having the amino acid sequence shown in SEQ ID NO. 30 and a protein having the amino acid sequence shown in SEQ ID NO. 58 The second polypeptide chain.
  • scfv constituting the second antigen binding domain is fused to the C-terminus of the first polypeptide chain directly or through a linker (FIG. ID).
  • the first antigen-binding domain Fab of the heterodimeric fusion protein binds CD3.
  • the first antigen-binding domain Fab that binds CD3 has a Fab heavy chain shown in SEQ ID NO. 125 and a Fab light chain shown in SEQ ID NO. 126.
  • the first antigen-binding domain Fab that binds CD3 has a Fab heavy chain shown in SEQ ID NO. 129 and a Fab light chain shown in SEQ ID NO. 130.
  • the first antigen-binding domain Fab that binds CD3 has a Fab heavy chain shown in SEQ ID NO. 131 and a Fab light chain shown in SEQ ID NO. 132.
  • the scfv that constitutes the second antigen-binding domain of the heterodimeric fusion protein binds CD19. In some embodiments, the scfv constituting the second antigen-binding domain that binds to CD19 has the amino acid sequence shown in SEQ ID NO.139. In some embodiments, the scfv that constitutes the second antigen-binding domain of the heterodimeric fusion protein binds EGFR. In some embodiments, the scfv constituting the second antigen-binding domain that binds EGFR has the amino acid sequence shown in SEQ ID NO. 142. In some embodiments, the heterodimeric fusion protein binds CD3 and CD19.
  • the first antigen-binding domain of the heterodimeric fusion protein that binds CD3 and CD19 has the Fab heavy chain shown in SEQ ID NO. 125 and the Fab shown in SEQ ID NO. 126
  • the second antigen-binding domain of the light chain has the amino acid sequence shown in SEQ ID NO.139.
  • the heterodimer fusion protein that binds CD3 and CD19 has a first polypeptide chain having the amino acid sequence shown in SEQ ID NO. 2 and a protein having the amino acid sequence shown in SEQ ID NO. 4. The second polypeptide chain.
  • the heterodimer fusion protein that binds CD3 and CD19 has a first antigen-binding domain having a Fab heavy chain shown in SEQ ID NO. 129 and a Fab shown in SEQ ID NO. 130
  • the second antigen-binding domain of the light chain has the amino acid sequence shown in SEQ ID NO.139.
  • the heterodimer fusion protein that binds CD3 and CD19 has a first polypeptide chain having the amino acid sequence shown in SEQ ID NO. 6 and a protein having the amino acid sequence shown in SEQ ID NO. 8. The second polypeptide chain.
  • the heterodimeric fusion protein binds CD3 and EGFR.
  • the heterodimer fusion protein that binds CD3 and EGFR has a first antigen-binding domain having a Fab heavy chain shown in SEQ ID NO. 131 and a Fab shown in SEQ ID NO. 132
  • the second antigen-binding domain of the light chain has the amino acid sequence shown in SEQ ID NO. 142.
  • the heterodimeric fusion protein that binds CD3 and EGFR has a first polypeptide chain having the amino acid sequence shown in SEQ ID NO. 14 and a protein having the amino acid sequence shown in SEQ ID NO. 16 The second polypeptide chain.
  • scfv constituting the second antigen-binding domain is fused to the C-terminus of the second polypeptide chain directly or through a linker (FIG. 1E).
  • the first antigen-binding domain Fab of the heterodimeric fusion protein binds CD3.
  • the first antigen-binding domain Fab that binds CD3 has a Fab heavy chain shown in SEQ ID NO. 125 and a Fab light chain shown in SEQ ID NO. 126.
  • the first antigen-binding domain Fab that binds CD3 has a Fab heavy chain shown in SEQ ID NO. 129 and a Fab light chain shown in SEQ ID NO. 130.
  • the first antigen-binding domain Fab that binds CD3 has a Fab heavy chain shown in SEQ ID NO. 131 and a Fab light chain shown in SEQ ID NO. 132.
  • the scfv that constitutes the second antigen-binding domain of the heterodimeric fusion protein binds CD19. In some embodiments, the scfv constituting the second antigen-binding domain that binds to CD19 has the amino acid sequence shown in SEQ ID NO.139. In some embodiments, the scfv that constitutes the second antigen binding domain of the heterodimeric fusion protein binds BCMA. In some embodiments, the scfv constituting the second antigen-binding domain that binds to BCMA has the amino acid sequence shown in SEQ ID NO. 140. In some embodiments, the second antigen binding domain scfv of the heterodimeric fusion protein binds CLL-1.
  • the scfv that constitutes the second antigen-binding domain that binds CLL-1 has the amino acid sequence shown in SEQ ID NO. 141. In some embodiments, the scfv that constitutes the second antigen-binding domain of the heterodimeric fusion protein binds EGFR. In some embodiments, the scfv constituting the second antigen-binding domain that binds EGFR has the amino acid sequence shown in SEQ ID NO. 142. In some embodiments, the heterodimeric fusion protein binds CD3 and CD19. In some embodiments, the first antigen-binding domain of the heterodimeric fusion protein that binds CD3 and CD19 has the Fab heavy chain shown in SEQ ID NO.
  • the second antigen-binding domain of the light chain has the amino acid sequence shown in SEQ ID NO.139.
  • the heterodimer fusion protein that binds CD3 and CD19 has a first antigen-binding domain having a Fab heavy chain shown in SEQ ID NO. 129 and a Fab shown in SEQ ID NO. 130
  • the second antigen-binding domain of the light chain has the amino acid sequence shown in SEQ ID NO.139.
  • the heterodimeric fusion protein that binds CD3 and CD19 has a first polypeptide chain having the amino acid sequence shown in SEQ ID NO. 20 and a protein having the amino acid sequence shown in SEQ ID NO.
  • the heterodimer fusion protein that binds CD3 and CD19 has a first polypeptide chain that includes the amino acid sequence shown in SEQ ID NO. 24 and includes an amino acid sequence shown in SEQ ID NO. 26 Second polypeptide chain.
  • the heterodimeric fusion protein binds CD3 and BCMA.
  • the first antigen-binding domain of the heterodimeric fusion protein that binds CD3 and BCMA has the Fab heavy chain shown in SEQ ID NO. 125 and the Fab shown in SEQ ID NO. 126
  • the second antigen-binding domain of the light chain has the amino acid sequence shown in SEQ ID NO.140.
  • the heterodimer fusion protein that binds CD3 and BCMA has a first polypeptide chain that includes the amino acid sequence shown in SEQ ID NO. 30 and contains an amino acid sequence shown in SEQ ID NO. 40 Second polypeptide chain.
  • the heterodimeric fusion protein binds CD3 and CLL-1.
  • the first antigen-binding domain of the heterodimeric fusion protein that binds CD3 and CLL-1 has the Fab heavy chain shown in SEQ ID NO. 125 and SEQ ID NO. 126 Fab light chain
  • the second antigen-binding domain has the amino acid sequence shown in SEQ ID NO.141.
  • the heterodimer fusion protein that binds CD3 and CLL-1 has a first polypeptide chain that includes the amino acid sequence shown in SEQ ID NO. 30 and contains a SEQ ID NO. 42 The second polypeptide chain of amino acid sequence.
  • the heterodimeric fusion protein binds CD3 and EGFR.
  • the first antigen-binding domain of the heterodimeric fusion protein that binds CD3 and EGFR has the Fab heavy chain shown in SEQ ID NO. 125 and the Fab shown in SEQ ID NO. 126
  • the second antigen-binding domain of the light chain has the amino acid sequence shown in SEQ ID NO. 142.
  • the heterodimeric fusion protein that binds CD3 and EGFR has a first polypeptide chain that includes the amino acid sequence shown in SEQ ID NO. 24 and an amino acid sequence that includes the amino acid sequence shown in SEQ ID NO. 28 Second polypeptide chain.
  • the heterodimer fusion protein that binds CD3 and EGFR has a first polypeptide chain that includes the amino acid sequence shown in SEQ ID NO. 30 and contains an amino acid sequence shown in SEQ ID NO. 32 Second polypeptide chain.
  • the heterodimeric fusion protein that binds CD3 and EGFR has a first polypeptide chain that includes the amino acid sequence shown in SEQ ID NO.
  • the heterodimer fusion protein that binds CD3 and EGFR has a first polypeptide chain that includes the amino acid sequence shown in SEQ ID NO. 44 and an amino acid sequence that includes the amino acid sequence shown in SEQ ID NO. 46 Second polypeptide chain.
  • the heterodimer fusion protein that binds CD3 and EGFR has a first polypeptide chain that includes the amino acid sequence shown in SEQ ID NO. 44 and an amino acid sequence that includes the amino acid sequence shown in SEQ ID NO. 52 Second polypeptide chain.
  • the heterodimeric fusion protein that binds CD3 and EGFR has a first polypeptide chain that includes the amino acid sequence shown in SEQ ID NO. 48 and an amino acid sequence that includes the amino acid sequence shown in SEQ ID NO. 50 Second polypeptide chain.
  • the heterodimer fusion protein that binds CD3 and EGFR has a first antigen-binding domain having a Fab heavy chain shown in SEQ ID NO. 131 and a Fab shown in SEQ ID NO. 132
  • the second antigen-binding domain of the light chain has the amino acid sequence shown in SEQ ID NO. 142.
  • the heterodimer fusion protein that binds CD3 and EGFR has a first polypeptide chain that includes the amino acid sequence shown in SEQ ID NO. 36 and an amino acid sequence that includes the amino acid sequence shown in SEQ ID NO. 38 Second polypeptide chain.
  • the second antigen-binding domain of the heterodimeric fusion protein is a physiologically active peptide.
  • the physiologically active peptide is fused to the N-terminus of the first polypeptide chain directly or through a linker (FIG. 1B).
  • the physiologically active peptide is fused to the N-terminus of the second polypeptide chain directly or through a linker ( Figure 1C).
  • the physiologically active peptide is fused to the C-terminus of the first polypeptide chain directly or through a linker ( Figure ID).
  • the physiologically active peptide is EGF4. In some embodiments, the physiologically active peptide EGF4 has the amino acid sequence shown in SEQ ID NO. 150. In some embodiments, the first antigen-binding domain Fab of the heterodimeric fusion protein binds CD3. In some embodiments, the first antigen-binding domain Fab that binds CD3 has a Fab heavy chain shown in SEQ ID NO. 125 and a Fab light chain shown in SEQ ID NO. 126. In some embodiments, the heterodimeric fusion protein binds CD3 and EGFR.
  • the first antigen-binding domain of the heterodimeric fusion protein that binds CD3 and EGFR has the Fab heavy chain shown in SEQ ID NO. 125 and the Fab shown in SEQ ID NO. 126
  • the second antigen-binding domain physiologically active peptide has the amino acid sequence shown in SEQ ID NO. 150.
  • the heterodimer fusion protein that binds CD3 and EGFR has a first polypeptide chain that includes the amino acid sequence shown in SEQ ID NO. 10 and a protein sequence that includes the amino acid sequence shown in SEQ ID NO. 12. The second polypeptide chain.
  • the physiologically active peptide is fused to the C-terminus of the second polypeptide chain directly or through a linker (FIG. 1E).
  • the physiologically active peptide is EGF4. In some embodiments, the physiologically active peptide EGF4 has the amino acid sequence shown in SEQ ID NO. 150.
  • the first antigen-binding domain Fab of the heterodimeric fusion protein binds CD3. In some embodiments, the first antigen-binding domain Fab that binds CD3 has a Fab heavy chain shown in SEQ ID NO. 125 and a Fab light chain shown in SEQ ID NO. 126. In some embodiments, the heterodimeric fusion protein has a first antigen-binding domain having a Fab heavy chain as shown in SEQ ID NO. 125 and a Fab light chain as shown in SEQ ID NO. 126.
  • the second antigen binding domain has the amino acid sequence shown in SEQ ID NO. 150.
  • the heterodimeric fusion protein has a first polypeptide chain comprising the amino acid sequence shown in SEQ ID NO. 30 and a second polypeptide chain comprising the amino acid sequence shown in SEQ ID NO. 34 .
  • the second antigen-binding domain of the heterodimeric fusion protein consists of first and second physiologically active peptides.
  • the first and second physiologically active peptides are fused to the N-terminus of the first and second polypeptide chains directly or through a linker, respectively.
  • first and second physiologically active peptides are fused to the C-terminus of the first and second polypeptide chains directly or through a linker (FIG. 1F).
  • first and second physiologically active peptides are different. In some embodiments, the first and the second physiologically active peptide are the same.
  • the first and second physiologically active peptides are NKG2D.
  • the physiologically active peptide NKG2D has the amino acid sequence shown in SEQ ID NO. 151.
  • the first antigen-binding domain Fab of the heterodimer binds CD3.
  • the CD3-binding first antigen-binding domain has a Fab heavy chain shown in SEQ ID NO. 125 and a Fab light chain shown in SEQ ID NO. 126.
  • the heterodimeric fusion protein binds CD3 and MIC-A.
  • the first antigen-binding domain of the heterodimeric fusion protein that binds CD3 and MIC-A has the Fab heavy chain shown in SEQ ID NO. 125 and SEQ ID NO. 126 Fab light chain
  • the second antigen-binding domain has the amino acid sequence shown in SEQ ID NO.151.
  • the heterodimeric fusion protein that binds CD3 and MIC-A has a first polypeptide chain that includes the amino acid sequence shown in SEQ ID NO. 122 and contains the amino acid shown in SEQ ID NO. 124 The second polypeptide chain of the sequence.
  • the second antigen-binding domain of the heterodimeric fusion protein is Fv.
  • the heavy chain variable region of the Fv constituting the second antigen-binding domain is fused directly or through a linker to the C-terminus of the first polypeptide chain to constitute the second antigen-binding domain
  • the light chain variable region of Fv is fused directly or via a linker to the C-terminus of the second polypeptide chain ( Figure 1G).
  • the first antigen-binding domain Fab of the heterodimeric fusion protein binds CD3.
  • the CD3-binding first antigen-binding domain has a Fab heavy chain shown in SEQ ID NO. 125 and a Fab light chain shown in SEQ ID NO. 126.
  • the Fv that constitutes the second binding domain of the heterodimeric fusion protein binds EGFR.
  • the Fv constituting the second antigen-binding domain that binds to EGFR has a heavy chain variable region shown in SEQ ID NO. 135 and a light chain variable region shown in SEQ ID NO. 136.
  • the heterodimeric fusion protein binds CD3 and EGFR.
  • the first antigen-binding domain of the heterodimeric fusion protein that binds CD3 and EGFR has the Fab heavy chain shown in SEQ ID NO. 125 and the Fab shown in SEQ ID NO. 126
  • the second antigen-binding domain of the light chain has a heavy chain variable region shown in SEQ ID NO. 135 and a light chain variable region shown in SEQ ID NO. 136.
  • the heterodimer fusion protein that binds CD3 and EGFR has a first polypeptide chain that includes the amino acid sequence shown in SEQ ID NO. 60 and a protein sequence that includes the amino acid sequence shown in SEQ ID NO.
  • the heterodimer fusion protein that binds CD3 and EGFR has a first polypeptide chain that includes the amino acid sequence shown in SEQ ID NO. 64 and a protein sequence that includes the amino acid sequence shown in SEQ ID NO. 66 The second polypeptide chain.
  • the heterodimeric fusion protein that binds CD3 and EGFR has a first polypeptide chain that includes the amino acid sequence shown in SEQ ID NO. 68 and a protein sequence that includes the amino acid sequence shown in SEQ ID NO. 70 The second polypeptide chain.
  • the heavy chain variable region of the second antigen-binding domain Fv is fused directly or through a linker to the C-terminus of the second polypeptide chain, the lightness of the second antigen-binding domain Fv
  • the chain variable region is fused to the C-terminus of the first polypeptide chain directly or through a linker ( Figure 1H).
  • the first antigen-binding domain Fab of the heterodimeric fusion protein binds CD3.
  • the CD3-binding first antigen-binding domain has a Fab heavy chain shown in SEQ ID NO. 125 and a Fab light chain shown in SEQ ID NO. 126.
  • the second antigen-binding domain of the heterodimeric fusion protein binds EGFR.
  • the Fv constituting the second antigen-binding domain that binds to EGFR has a heavy chain variable region shown in SEQ ID NO. 135 and a light chain variable region shown in SEQ ID NO. 136.
  • the heterodimeric fusion protein binds CD3 and EGFR.
  • the first antigen-binding domain of the heterodimeric fusion protein that binds CD3 and EGFR has the Fab heavy chain shown in SEQ ID NO. 125 and the Fab shown in SEQ ID NO. 126
  • the second antigen-binding domain of the light chain has a heavy chain variable region shown in SEQ ID NO. 135 and a light chain variable region shown in SEQ ID NO. 136.
  • the heterodimeric fusion protein that binds CD3 and EGFR has a first polypeptide chain that includes the amino acid sequence shown in SEQ ID NO. 72 and a first polypeptide chain that includes the amino acid sequence shown in SEQ ID NO. 74 The second polypeptide chain.
  • the heterodimeric fusion protein that binds CD3 and EGFR has a first polypeptide chain that includes the amino acid sequence shown in SEQ ID NO. 76 and a first polypeptide chain that includes the amino acid sequence shown in SEQ ID NO. 78 The second polypeptide chain.
  • the heterodimeric fusion protein that binds CD3 and EGFR has a first polypeptide chain that includes the amino acid sequence shown in SEQ ID NO. 80 and a protein sequence that includes the amino acid sequence shown in SEQ ID NO. 82 The second polypeptide chain.
  • the heavy chain variable region of the Fv constituting the second antigen-binding domain is fused directly or through a linker to the N-terminus of the second polypeptide chain to constitute the second antigen-binding domain
  • the light chain variable region of Fv is fused to the N-terminus of the first polypeptide chain directly or through a linker ( Figure 1J).
  • the heavy chain variable region of the Fv that constitutes the second antigen-binding domain is fused to the N-terminus of the first polypeptide chain directly or through a linker, and the second antigen-binding domain Fv The light chain variable region is fused to the N-terminus of the second polypeptide chain directly or through a linker ( Figure 11).
  • the first antigen-binding domain of the heterodimeric fusion protein binds EGFR. In some embodiments, the first antigen-binding domain that binds EGFR has a Fab heavy chain shown in SEQ ID NO. 127 and a Fab light chain shown in SEQ ID NO. 128. In some embodiments, the second antigen-binding domain of the heterodimeric fusion protein binds CD3. In some embodiments, the Fv constituting the second antigen-binding domain that binds to CD3 has a heavy chain variable region shown in SEQ ID NO. 133 and a light chain variable region shown in SEQ ID NO. 134. In some embodiments, the heterodimeric fusion protein binds CD3 and EGFR.
  • the first antigen-binding domain of the heterodimeric fusion protein that binds CD3 and EGFR has the Fab heavy chain shown in SEQ ID NO. 127 and the Fab shown in SEQ ID NO. 128
  • the second antigen-binding domain of the light chain has a heavy chain variable region shown in SEQ ID NO. 133 and a light chain variable region shown in SEQ ID NO. 134.
  • the heterodimer fusion protein that binds CD3 and EGFR has a first polypeptide chain that includes the amino acid sequence shown in SEQ ID NO. 104 and an amino acid sequence that includes the amino acid sequence shown in SEQ ID NO. 106 Second polypeptide chain.
  • the first antigen-binding domain Fab of the heterodimeric fusion protein binds CD3.
  • the first antigen-binding domain Fab that binds CD3 has a Fab heavy chain shown in SEQ ID NO. 125 and a Fab light chain shown in SEQ ID NO. 125.
  • the Fv that constitutes the second antigen binding domain of the heterodimeric fusion protein binds EGFR.
  • the second antigen-binding domain that binds EGFR has a heavy chain variable region shown in SEQ ID NO. 135 and a light chain variable region shown in SEQ ID NO. 136.
  • the heterodimeric fusion protein binds CD3 and EGFR.
  • the first antigen-binding domain Fab of the heterodimer that binds CD3 and EGFR has a Fab heavy chain shown in SEQ ID NO. 125 and a Fab light shown in SEQ ID NO. 125
  • the second antigen binding domain Fv of the chain has a heavy chain variable region shown in SEQ ID NO. 135 and a light chain variable region shown in SEQ ID NO. 136.
  • the heterodimer fusion protein that binds CD3 and EGFR has a first polypeptide chain that includes the amino acid sequence shown in SEQ ID NO. 108 and an amino acid sequence that includes the amino acid sequence shown in SEQ ID NO. 110 Second polypeptide chain.
  • the second antigen-binding domain of the heterodimeric fusion protein is Fab2.
  • the heavy chain of the second antigen-binding domain Fab2 is fused directly or through a linker to the C-terminus of the first polypeptide chain, and the light chain of the Fab2 is directly or through a linker to the The C-terminal fusion of the second polypeptide chain (Figure 1K).
  • the first antigen-binding domain Fab of the heterodimer binds CD3.
  • the first antigen-binding domain Fab that binds CD3 has a Fab heavy chain shown in SEQ ID NO. 125 and a Fab light chain shown in SEQ ID NO. 126.
  • Fab2 constituting the second antigen binding domain of the heterodimer binds EGFR.
  • Fab2 constituting the second antigen-binding domain that binds to EGFR has a Fab heavy chain shown in SEQ ID NO. 127 and a Fab light chain shown in SEQ ID NO. 128.
  • the heterodimeric fusion protein binds CD3 and EGFR.
  • the first antigen-binding domain Fab of the heterodimeric fusion protein that binds CD3 and EGFR has a Fab heavy chain as shown in SEQ ID NO. 125 and as shown in SEQ ID NO. 126
  • the second antigen-binding domain Fab2 has the Fab heavy chain shown in SEQ ID NO. 127 and the Fab light chain shown in SEQ ID NO. 128.
  • the heterodimeric fusion protein that binds CD3 and EGFR has a first polypeptide chain that includes the amino acid sequence shown in SEQ ID NO. 84 and a protein sequence that includes the amino acid sequence shown in SEQ ID NO. 86 The second polypeptide chain.
  • the heavy chain of the second antigen-binding domain Fab2 is fused directly or through a linker to the C-terminus of the second polypeptide chain, and the light chain of the Fab2 is directly or through a linker to the The C-terminal fusion of the first polypeptide chain ( Figure 1N).
  • the first antigen-binding domain of the heterodimer binds CD3.
  • the first antigen-binding domain Fab that binds CD3 has a Fab heavy chain shown in SEQ ID NO. 125 and a Fab light chain shown in SEQ ID NO. 126.
  • the second antigen-binding domain Fab2 of the heterodimer binds EGFR.
  • the second antigen-binding domain Fab2 that binds EGFR has a Fab heavy chain as shown in SEQ ID NO. 127 and a Fab light chain as shown in SEQ ID NO. 128.
  • the heterodimeric fusion protein binds CD3 and EGFR.
  • the first antigen-binding domain Fab of the heterodimeric fusion protein that binds CD3 and EGFR has a Fab heavy chain as shown in SEQ ID NO. 125 and as shown in SEQ ID NO. 126
  • the second antigen-binding domain Fab2 has the Fab heavy chain shown in SEQ ID NO. 127 and the Fab light chain shown in SEQ ID NO. 128.
  • the heterodimer fusion protein that binds CD3 and EGFR has a first polypeptide chain that includes the amino acid sequence shown in SEQ ID NO. 88 and a protein sequence that includes the amino acid sequence shown in SEQ ID NO. 90 The second polypeptide chain.
  • the heavy chain of the second antigen-binding domain Fab2 is fused directly or via a linker to the N-terminus of the second polypeptide chain, and the light chain of the Fab2 is directly or via a linker to the The N-terminal fusion of the first polypeptide chain (Figure 1M).
  • the heavy chain of the second antigen-binding domain Fab2 is fused directly or through a linker to the N-terminus of the first polypeptide chain, and the light chain of the Fab2 is directly or through a linker to the The N-terminal fusion of the second polypeptide chain ( Figure 1L).
  • the first antigen-binding domain Fab of the heterodimeric fusion protein binds EGFR.
  • the EGFR-binding first antigen-binding domain Fab has a Fab heavy chain as shown in SEQ ID NO. 127 and a Fab light chain as shown in SEQ ID NO. 128.
  • the second antigen-binding domain Fab2 of the heterodimeric fusion protein binds CD3.
  • the second antigen-binding domain Fab2 that binds CD3 has a Fab heavy chain shown in SEQ ID NO. 125 and a Fab light chain shown in SEQ ID NO. 126.
  • the heterodimer binds EGFR and CD3.
  • the first antigen-binding domain Fab of the heterodimeric fusion protein that binds EGFR and CD3 has a Fab heavy chain as shown in SEQ ID NO. 127 and as shown in SEQ ID NO. 128 In the Fab light chain, the second antigen-binding domain Fab2 has the Fab heavy chain shown in SEQ ID NO. 125 and the Fab light chain shown in SEQ ID NO. 126.
  • the heterodimeric fusion protein that binds EGFR and CD3 has a first polypeptide chain that includes the amino acid sequence shown in SEQ ID NO. 112 and an amino acid sequence that includes the amino acid sequence shown in SEQ ID NO. 114 Second polypeptide chain.
  • the first antigen-binding domain Fab of the heterodimeric fusion protein binds CD3.
  • the CD3-binding first antigen-binding domain has a Fab heavy chain shown in SEQ ID NO. 125 and a Fab light chain shown in SEQ ID NO. 126.
  • the second antigen-binding domain Fab2 of the heterodimeric fusion protein binds EGFR.
  • the second antigen-binding domain Fab2 that binds EGFR has a Fab heavy chain as shown in SEQ ID NO. 127 and a Fab light chain as shown in SEQ ID NO. 128.
  • the heterodimeric fusion protein binds CD3 and EGFR.
  • the first antigen-binding domain Fab of the heterodimeric fusion protein that binds CD3 and EGFR has a Fab heavy chain as shown in SEQ ID NO. 125 and as shown in SEQ ID NO. 126
  • the second antigen-binding domain Fab2 has the Fab heavy chain shown in SEQ ID NO. 127 and the Fab light chain shown in SEQ ID NO. 128.
  • the heterodimer fusion protein that binds CD3 and EGFR has a first polypeptide chain that includes the amino acid sequence shown in SEQ ID NO. 116 and a protein sequence that includes the amino acid sequence shown in SEQ ID NO. 118 The second polypeptide chain.
  • the second antigen-binding domain of the heterodimeric fusion protein is a Nanobody.
  • the second antigen-binding domain Nanobody is fused to the N-terminus of the first polypeptide chain directly or through a linker. In some embodiments, the second antigen-binding domain Nanobody is fused to the N-terminus of the second polypeptide chain directly or through a linker.
  • the second antigen-binding domain Nanobody is fused to the C-terminus of the first polypeptide chain directly or through a linker. In some embodiments, the second antigen-binding domain Nanobody is fused to the C-terminus of the second polypeptide chain directly or through a linker.
  • the present invention provides a heterodimer fusion protein based on immunoglobulin fragments, the heterodimer comprising:
  • the first polypeptide chain which in order from N-terminal to C-terminal includes: Fc, (L1)n, CH1, L2, VH,
  • the second polypeptide chain which comprises N, C and C in order from Fc to (L3)n, CL, L4 and VL;
  • n is 0 or 1
  • L1, L2, L3, and L4 are linkers, and VH and VL form a first antigen-binding domain Fv.
  • the heterodimeric fusion protein further includes a second antigen binding domain.
  • the second antigen-binding domain is a physiologically active peptide.
  • the physiologically active peptide is fused to the N-terminus of the first or second polypeptide chain directly or through a linker (Figure 1P and Figure 1S).
  • the physiologically active peptide is fused to the C-terminus of the first or second polypeptide chain directly or through a linker (Figure 1T and Figure 1U).
  • the second antigen binding domain is Fab2.
  • the heavy chain of the second antigen-binding domain Fab2 is fused directly or through a linker to the N-terminus of the first polypeptide chain, and the light chain of the Fab2 is directly or through a linker to the The N-terminal fusion of the second polypeptide chain.
  • the heavy chain of the second antigen-binding domain Fab2 is fused directly or via a linker to the N-terminus of the second polypeptide chain, and the light chain of the Fab2 is directly or via a linker to the The N-terminal fusion of the second polypeptide chain.
  • the second antigen-binding domain of the heterodimeric fusion protein is Fv.
  • the heavy chain variable region of the second antigen-binding domain Fv is fused to the N-terminus of the first polypeptide chain directly or through a linker, and the lightness of the second antigen-binding domain Fv The chain variable region is fused to the N-terminus of the second polypeptide chain directly or through a linker.
  • the heavy chain variable region of the second antigen-binding domain Fv is fused to the N-terminus of the second polypeptide chain directly or through a linker, the lightness of the second antigen-binding domain Fv
  • the chain variable region is fused to the N-terminus of the first polypeptide chain directly or through a linker.
  • the second antigen binding domain is a Nanobody.
  • the second antigen-binding domain Nanobody is fused to the N-terminus of the first polypeptide chain directly or through a linker. In some embodiments, the second antigen-binding domain Nanobody is fused to the N-terminus of the second polypeptide chain directly or through a linker.
  • the second antigen binding domain is scfv. In some embodiments, the second antigen-binding domain scfv is fused to the C-terminus of the first or second polypeptide chain directly or through a linker (FIG. 1T and FIG. 1U).
  • the second antigen binding domain scfv is fused to the N-terminus of the second polypeptide chain directly or through a linker (FIG. 1S).
  • the second antigen binding domain scfv is fused to the N-terminus of the first polypeptide chain directly or through a linker (FIG. 1P).
  • the first antigen binding domain Fv of the heterodimer binds CD3.
  • the first antigen-binding domain Fv that binds CD3 has a VH as shown in SEQ ID NO. 133 and a VL as shown in SEQ ID NO. 134.
  • the first antigen-binding domain Fv that binds CD3 has a VH shown in SEQ ID NO. 137 and a VL shown in SEQ ID NO. 138.
  • the second antigen binding domain scfv binds to CD19.
  • the second antigen binding domain scfv that binds CD19 has the amino acid sequence shown in SEQ ID NO.139.
  • the heterodimeric fusion protein binds CD3 and CD19.
  • the heterodimeric fusion protein that binds CD3 and CD19 has a first antigen-binding domain Fv having a VH shown in SEQ ID NO. 133 and a VL shown in SEQ ID NO. 134, Its second antigen binding domain scfv has the amino acid sequence shown in SEQ ID NO.139.
  • the heterodimer fusion protein that binds CD3 and EGFR has a first polypeptide chain of the amino acid sequence shown in SEQ ID NO. 96 and a second amino acid sequence shown in SEQ ID NO. 98 Polypeptide chain.
  • the first antigen-binding domain Fv of the heterodimeric fusion protein that binds CD3 and CD19 has a VH as shown in SEQ ID NO. 137 and a VL as shown in SEQ ID NO. 138, Its second antigen binding domain scfv has the amino acid sequence shown in SEQ ID NO.139.
  • the heterodimer fusion protein that binds CD3 and EGFR has a first polypeptide chain that includes the amino acid sequence shown in SEQ ID NO. 92 and an amino acid sequence that includes the amino acid sequence shown in SEQ ID NO. 94 Second polypeptide chain.
  • the present invention provides a heterodimer fusion protein based on immunoglobulin fragments, the heterodimer comprising:
  • the first polypeptide chain which in order from N-terminal to C-terminal includes: Fc, (L1)n, CL, L2, VH,
  • n is 0 or 1
  • L1, L2, L3, and L4 are linkers, and VH and VL form a first antigen-binding domain Fv.
  • the heterodimeric fusion protein further includes a second antigen binding domain.
  • the second antigen-binding domain is a physiologically active peptide.
  • the physiologically active peptide is fused to the N-terminus of the first or second polypeptide chain directly or through a linker (Figure 1Q and Figure 1R).
  • the physiologically active peptide is fused to the C-terminus of the first or second polypeptide chain directly or through a linker (FIG. 1V and FIG. 1W).
  • the second antigen binding domain is Fab2.
  • the heavy chain of the second antigen-binding domain Fab2 is fused directly or through a linker to the N-terminus of the first polypeptide chain, and the light chain of the Fab2 is directly or through a linker to the The N-terminal fusion of the second polypeptide chain.
  • the heavy chain of the second antigen-binding domain Fab2 is fused directly or via a linker to the N-terminus of the second polypeptide chain, and the light chain of the Fab2 is directly or via a linker to the The N-terminal fusion of the second polypeptide chain.
  • the second antigen-binding domain of the heterodimeric fusion protein is Fv.
  • the heavy chain variable region of the second antigen-binding domain Fv is fused to the N-terminus of the first polypeptide chain directly or through a linker, and the lightness of the second antigen-binding domain Fv The chain variable region is fused to the N-terminus of the second polypeptide chain directly or through a linker.
  • the heavy chain variable region of the second antigen-binding domain Fv is fused to the N-terminus of the second polypeptide chain directly or through a linker, the lightness of the second antigen-binding domain Fv
  • the chain variable region is fused to the N-terminus of the first polypeptide chain directly or through a linker.
  • the second antigen binding domain is a Nanobody.
  • the second antigen-binding domain Nanobody is fused to the N-terminus of the first polypeptide chain directly or through a linker. In some embodiments, the second antigen-binding domain Nanobody is fused to the N-terminus of the second polypeptide chain directly or through a linker.
  • the second antigen binding domain is scfv. In some embodiments, the second antigen-binding domain scfv is fused to the C-terminus of the first or second polypeptide chain directly or through a linker (FIG. 1V and FIG. 1W).
  • the second antigen binding domain scfv is fused to the N-terminus of the second polypeptide chain directly or through a linker (FIG. 1Q).
  • the second antigen binding domain scfv is fused to the N-terminus of the first polypeptide chain directly or through a linker (FIG. 1R).
  • the first antigen-binding domain of the heterodimer binds CD3.
  • the first antigen-binding domain Fv that binds CD3 has a VH as shown in SEQ ID NO. 133 and a VL as shown in SEQ ID NO. 134.
  • the second antigen binding domain scfv binds to CD19.
  • the second antigen binding domain scfv that binds CD19 has the amino acid sequence shown in SEQ ID NO.139.
  • the heterodimeric fusion protein binds CD3 and CD19.
  • the heterodimeric fusion protein that binds CD3 and CD19 has a first antigen-binding domain Fv having a VH shown in SEQ ID NO. 133 and a VL shown in SEQ ID NO. 134, Its second antigen binding domain scfv has the amino acid sequence shown in SEQ ID NO.139.
  • the heterodimer fusion protein that binds CD3 and CD19 has a first polypeptide chain that includes the amino acid sequence shown in SEQ ID NO. 100 and contains an amino acid sequence shown in SEQ ID NO. 102 Second polypeptide chain.
  • the heavy chain and light chain of the first antigen-binding domain Fab are fused (directly or through a linker) to the N-terminus of the two Fcs.
  • the second antigen-binding domains scfv, Fab2, Fv, Nanobody or physiologically active peptide is fused to the N-terminus of Fab or C-terminus of Fc, effectively solving the heavy chain and heavy chain, light chain and light chain, heavy chain and light chain between the two antigen binding domains Mismatch problem.
  • the heterodimer fusion protein provided by the present invention comprises at least one linker.
  • the linker is optionally selected from the following linkers: GGGGSGGGGSGGGGS, SGGGGSGGGGSGGGGS, GGSGGSGGGGSGGGG, GGSGGSGGGGSGGGGS, GGGSGAKLAALKAKLAALKGGGGS, GGGGSELAALEAELAALEAGGSG, APATSLQSGQLGFQCGELCSAG, GAGAP, GTKAP, TGGGP, TVA
  • the linkers are the same. In some embodiments, the linker is different.
  • the invention also provides a polynucleotide encoding the heterodimeric fusion protein of the invention.
  • the invention also relates to an expression vector comprising the polynucleotide of the invention.
  • the invention also relates to a host cell comprising the expression vector of the invention.
  • the invention also relates to a pharmaceutical composition comprising the heterodimer fusion protein of the invention.
  • the invention also relates to a method of treating cancer and autoimmune diseases in a subject in need.
  • the method comprises administering to the subject an effective amount of the heterodimer fusion protein provided herein, or a pharmaceutical combination of the heterodimer fusion protein provided herein and a pharmaceutically acceptable carrier Thing.
  • the present invention provides a method for treating B cell blood cancer in a subject in need thereof, the method comprising administering to the subject the heterodimeric fusion protein of the present invention, wherein The heterodimer fusion protein can bind CD3 and CD19.
  • the heterodimeric fusion protein comprises SEQ ID No: 2 and SEQ ID No: 4; SEQ ID No: 6 and SEQ ID No: 8; SEQ ID No: 20 and SEQ ID No: 22; SEQ ID No: 24 and SEQ ID No: 26; SEQ ID No: 92 and SEQ ID No: 94; SEQ ID No: 96 and SEQ ID No: 98; SEQ ID No: 100 and SEQ ID No : Amino acid sequence of 102.
  • the B-cell blood cancer is selected from the group consisting of: Hodgkin's lymphoma, non-Hodgkin's lymphoma (NHL), precursor B-cell lymphoblastic leukemia/lymphoma, mature B-cell neoplasms, B-cell chronic lymphocytic leukemia/small lymphocytic lymphoma, B-cell prolymphocytic leukemia, lymphoplasmacytic lymphoma, mantle cell lymphoma, follicular lymphoma, cutaneous follicular center lymphoma, Marginal zone B-cell lymphoma, hairy cell leukemia, diffuse large B-cell lymphoma, Burkitt's lymphoma, plasmacytoma, plasma cell myeloma, post-transplant lymphoproliferative disorders, shifted Valdence Tren's macroglobulinemia (Waldenstrom's macroglobulinemia) and anaplastic large cell lymphoma.
  • NHL non-
  • the present invention provides a method for treating cancer in a subject in need
  • the cancer is selected from the group consisting of: melanoma (eg, metastatic malignant melanoma) Tumor), kidney cancer (eg, clear cell carcinoma), prostate cancer (eg, hormone-refractory prostate adenocarcinoma), pancreatic cancer, breast cancer, colon cancer, lung cancer (eg, non-small cell lung cancer), esophageal cancer, head and neck Squamous cell carcinoma, liver cancer, ovarian cancer, cervical cancer, thyroid cancer, glioblastoma, glioma, leukemia, lymphoma and other neoplastic malignant diseases.
  • melanoma eg, metastatic malignant melanoma
  • Tumor eg, metastatic malignant melanoma
  • kidney cancer eg, clear cell carcinoma
  • prostate cancer eg, hormone-refractory prostate adenocarcinoma
  • pancreatic cancer breast cancer
  • colon cancer
  • the present invention provides a method for treating lung cancer, colon cancer and the like with high expression of EGFR in a subject in need thereof, the method comprising administering the heterodimer of the present invention to the subject Fusion protein, wherein the heterodimer fusion protein is capable of binding CD3 and EGFR.
  • the heterodimeric fusion protein comprises the following amino acid sequences: SEQ ID No: 10 and SEQ ID No: 12; SEQ ID No: 14 and SEQ ID No: 16; SEQ ID No: 18 and SEQ ID No: 12; SEQ ID No: 24 and SEQ ID No: 28; SEQ ID No: 30 and SEQ ID No: 32; SEQ ID No: 30 and SEQ ID No: 34; SEQ ID No: 36 and SEQ ID No: 38; SEQ ID No: 44 and SEQ ID No: 46; SEQ ID No: 48 and SEQ ID No: 50; SEQ ID No: 44 and SEQ ID No: 52; SEQ ID No: 54 and SEQ ID No: 56, SEQ ID No: 30 and SEQ ID No: 58; SEQ ID No: 60 and SEQ ID No: 62; SEQ ID No: 64 and SEQ ID No: 66; SEQ ID No: 68 and SEQ ID No: 70; SEQ ID No: 72 and SEQ ID No: 74; SEQ ID No:
  • the present invention provides a method for treating multiple myeloma in a subject in need thereof, the method comprising administering to the subject the heterodimeric fusion protein of the present invention, wherein The heterodimer fusion protein is capable of binding CD3 and BCMA.
  • the heterodimeric fusion protein has SEQ ID No: 30 and SEQ ID No: 40 amino acid sequences.
  • the present invention provides a method for treating acute myeloid leukemia in a subject in need thereof, the method comprising administering to the subject the heterodimeric fusion protein of the present invention, wherein The heterodimer fusion protein is capable of binding CD3 and CLL-1.
  • the heterodimer fusion protein has the amino acid sequence set forth in SEQ ID No: 30 and SEQ ID No: 42.
  • the present invention provides a method for treating a viral infection in a subject in need, the method comprising administering to the subject the heterodimeric fusion protein of the present invention, wherein the The source dimer fusion protein can bind CD3 and MICA.
  • the heterodimeric fusion protein has the amino acid sequence set forth in SEQ ID No: 122 and SEQ ID No: 124.
  • FIG. 1A is a schematic diagram of the basic skeleton of a heterodimer fusion protein, where the chain where FabH is located is the first polypeptide chain, and the chain where FabL is located is the second polypeptide chain.
  • 1B-1F are schematic diagrams of heterodimer structures in which the first antigen-binding domain is Fab and the second antigen-binding domain is scfv or active peptide.
  • the heterodimer fusion protein in which the second antigen-binding domain is fused at the N-terminus of the first polypeptide chain is shown in FIG.
  • the heterodimer in the second antigen-binding domain is fused at the N-terminus of the second polypeptide chain
  • the fusion protein is shown in Figure 1C; the heterodimer fusion protein with the second antigen-binding domain fused at the C-terminus of the first polypeptide chain is shown in Figure 1D; the second antigen-binding domain is fused to the second polypeptide chain C
  • the terminal heterodimer fusion protein is shown in FIG. 1E; the second antigen-binding domain is fused to the first polypeptide chain and the second polypeptide chain C-terminal heterodimer fusion protein as shown in FIG. 1F.
  • FIG. 1G-1J are schematic diagrams of the structure of a heterodimeric fusion protein in which the first antigen-binding domain is Fab and the second antigen-binding domain is Fv.
  • the heterodimeric fusion protein of the VH and VL of the second antigen binding domain Fv fused to the C-terminus of the first polypeptide chain and the second polypeptide chain is shown in FIG. 1G; the second antigen binding domain Fv
  • the heterodimeric fusion protein fused to the C-terminus of the first polypeptide chain and the second polypeptide chain by VL and VH, respectively, is shown in FIG.
  • 1K-1N are schematic diagrams of the structure of a heterodimer fusion protein in which the first antigen-binding domain is Fab and the second antigen-binding domain is Fab2.
  • the heterodimer fusion protein in which the Fab2H and Fab2L of the second antigen binding domain Fab2 are fused to the C-terminus of the first polypeptide chain and the second polypeptide chain is shown in FIG. 1K; the Fab2 of the second antigen binding domain
  • the heterodimeric fusion protein with Fab2L and Fab2H fused to the C-terminus of the first and second polypeptide chains is shown in FIG.
  • FIG. 10 is a schematic diagram of the exchange positions of the VH and VL of the Fab in the first antigen binding domain and the second antigen binding domain on the two polypeptide chains.
  • FIGS. 1P-1X are schematic diagrams of the structure of a heterodimeric fusion protein in which the first antigen-binding domain is Fv and the second antigen-binding domain is scfv or active peptide.
  • the heterodimer fusion protein in which the scfv or physiologically active peptide of the second antigen-binding domain is fused to the N-terminus of the first polypeptide is shown in FIGS. 1P and 1Q; the scfv or physiological of the second antigen-binding domain
  • FIGS. 1P and 1Q the heterodimeric fusion protein in which the active peptide is fused to the N-terminus of the second polypeptide is shown in FIGS.
  • FIGS. 1T and 1U The heterodimer fusion protein is shown in FIGS. 1T and 1U; the heterodimer fusion protein in which the scfv or physiologically active peptide of the second antigen-binding domain is fused to the C-terminus of the second polypeptide is shown in FIGS. 1V and 1W;
  • FIG. 1X is a heterodimer fusion protein in which the physiologically active peptide is fused to the N-terminus of the first polypeptide and the second polypeptide chain, respectively.
  • Figure 2 shows an SDS-PAGE gel image of an exemplary heterodimeric fusion protein purified with ProteinA or CH1 resin.
  • M means protein marker;
  • - means to load without adding beta-mercaptoethanol;
  • + means to load after adding beta-mercaptoethanol; 1:IgFD-24; 2:IgFD-11; 3:IgFD -25; 4: IgFD-26; 5: IgFD-31; 6: IgFD-27; 7: IgFD-30; 7 (CH1): IgFD-30 (CH1 resin purification); 8: IgFD-29; 8 (CH1 ): IgFD-29 (CH1 resin purification); 9: IgFD-28; 9 (CH1): IgFD-28 (CH1 resin purification).
  • Figure 3-1 shows an exemplary heterodimer fusion protein gel exclusion chromatography chromatogram.
  • Figure 3-2 is an exemplary heterodimer antibody ion exchange chromatography chromatogram.
  • Figure 4 shows the use of flow cytometry to detect the binding of different concentrations of anti-CD3/CD19 heterodimer fusion proteins IgFD-6 and IgFD-7 to the surface of NALM-6 cells.
  • Figure 5 shows the use of flow cytometry to detect the anti-CD3/anti-CD19 heterodimer fusion proteins IgFD-6 and IgFD-7 to promote the killing effect of PMBC on Nalm-6 cells.
  • Figure 6 shows the ELISA detection of the binding strength of different anti-CD3/anti-EGFR heterodimer fusion proteins to human EGFR antigen.
  • Figure 7 shows the use of flow cytometry to detect the binding of different anti-CD3/anti-EGFR heterodimer fusion proteins to the surface of F98-EGFR cells.
  • ProA represents the fusion protein purified by proteinA Resin;
  • CH1 represents the fusion protein purified by CH1 resin.
  • Figure 8 shows the binding of anti-CD3/anti-EGFR heterodimer fusion proteins IgFD-8, IgFD-18 and IgFD-19 to PBMC-T cells using flow cytometry.
  • Figure 9 shows the use of flow cytometry to detect the binding of different anti-CD3/anti-EGFR heterodimer fusion proteins to the surface of Jurkat T cells, where ProA represents the fusion protein purified by proteinA Resin; CH1 represents the purification by CH1 resin Fusion protein.
  • Figure 10 shows the use of LDH method to detect different anti-CD3/anti-EGFR heterodimer fusion proteins to promote the killing effect of PBMC on F98-EGFR cells. Size represents the protein after gel chromatography; Monos represents the protein after ion exchange chromatography.
  • Figure 11 shows the detection of anti-CD3/anti-BCMA heterodimer fusion protein IgFD-22 using LDH assay (a) and flow cytometry (b) to promote the killing effect of PBMC on MM1.R cells.
  • Figure 12 shows the binding strength of the heterodimeric fusion protein IgFD-37 targeting CD3 and MICA to human MICA antigen by ELISA, of which IgFD-36 is a control.
  • Figure 13 shows the binding strength of the heterodimeric fusion protein IgFD37 targeting CD3 and MICA at different concentrations and the MICA on the cell surface using flow cytometry;
  • A PANC-1 cells
  • B BXPC-3 Cells
  • C is K562 cells.
  • Figure 14 shows the use of LDH assay to detect CD3 and MICA heterodimer fusion protein IgFD-37 to promote the killing effect of PBMC on K562 cells (A) and PANC-1 cells (B).
  • Figure 15 shows the use of flow cytometry to detect the anti-CD3/anti-CLL-1 heterodimer fusion protein IgFD-23 to promote the killing effect of PBMC on HL-60 cells.
  • Figure 16 is the pharmacokinetic curves of IgFD-25 and IgFD-33 after intraperitoneal administration in rats.
  • Figure 17 is the tumor growth inhibition curve of IgFD-33 in A431 lung cancer mouse model.
  • heterodimeric fusion protein means an antibody or antibody-based fusion protein consisting of two different polypeptide chains each containing an Fc, wherein the Fc of one polypeptide chain forms the Fc with the Fc of the other polypeptide chain Dimer, two polypeptide chains form at least one antigen binding domain.
  • antigen binding domain means a portion of an antigen binding molecule that specifically binds an antigenic determinant. More specifically, the term “antigen binding domain” refers to a portion of an antibody that includes a region that specifically binds to and complements a part or all of an antigen. In the case of a large antigen, the antigen-binding molecule may only bind to a specific part of the antigen, which is called an epitope.
  • the antigen binding domain may be provided by, for example, one or more variable domains (also referred to as variable regions).
  • the antigen binding domain may be derived from any animal species, such as rodents (e.g. rabbits, rats or hamsters) and humans.
  • Non-limiting examples of antigen-binding domains include: single-chain antibodies, Fab, F(ab')2, Fd fragments, Fv, single-chain Fv (scFv) molecules, dAb fragments, and amino acid residues that mimic the hypervariable regions of antibodies The smallest unit of recognition.
  • the antigen binding domain is Fab.
  • the antigen binding domain is Fv.
  • the antigen binding domain is scfv.
  • the antigen-binding domain is a physiologically active peptide.
  • antigen is synonymous with “antigenic determinant” and “epitope”, meaning a site on a polypeptide macromolecule that binds to an antigen-binding domain, thereby forming an antigen-binding domain-antigen complex (e.g. A stretch of consecutive amino acids or a conformational configuration consisting of different regions of non-contiguous amino acids).
  • Antigens can be found, for example, on the surface of tumor cells, on the surface of virus-infected cells, on the surface of other diseased cells, on the surface of immune cells, free matter in serum, and/or in extracellular matrix (ECM).
  • ECM extracellular matrix
  • the protein referred to herein as an antigen may be from any vertebrate source, including mammals such as primates (eg, humans) and rodents (eg, mice and rats) and other step-in animals.
  • mammals such as primates (eg, humans) and rodents (eg, mice and rats) and other step-in animals.
  • the term encompasses "full-length", unprocessed protein, and any form of protein produced by intracellular processing.
  • the term also covers naturally occurring protein variants, such as splice variants or allelic variants.
  • the antigen is a human protein.
  • Exemplary human proteins that can be used as antigens include, but are not limited to: BCMA, CLL-1, EpCAM, CD19, CCR5, EGFR, HER2, HER3, HER4, EGF4, PSMA, CEA, MUC-1 (Mucin), MUC-2 , MUC-3, MUC-4, MUC-5 AC , MUC-5 B , MUC7, ⁇ hCG, Lewis-Y, CD20, CD33, CD30, CD16A, B7-H3, CD123, gpA33, P-Cadherin, GPC3, CLEC12A , CD32B, TROP-2, ganglioside GD3, 9-O-Acetyl-GD3, GM2, Globo H, fucosyl GM1, Poly SA, GD2, Carboanhydrase IX (MN/CA IX), CD44v6, Sonic Hedgehog (Shh), Wue- 1.
  • Plasma Cell Antigen (membrane-bound) IgE, Melanoma Chondroitin Sulfate Proteoglycan (MCSP), CCR8, TNF-alpha precursor, STEAP, mesothelin, A33, Prostate Stem Cell Antigen (PSCA), Ly-6, desmoglein 4, E -cadherin neoepitope, Fetal Acetylcholine Receptor, CD25, CA19-9marker, CA-125marker and Muellerian Inhibitory Substance (MIS) Receptor type II, sTn (sialylated Tn antigen; TAG-72), FAP (fibroblast activation antigen), endosialin, EGFRvIII, LG, SAS, CD63, 2B4 (CD244), ⁇ 4 ⁇ 1 integrin, ⁇ 2 integrin (e.g.
  • Fc is used to define a C-terminal domain in an immunoglobulin heavy chain that contains at least a portion of a constant region. It means a polypeptide comprising a constant region of an antibody (excluding the first constant region immunoglobulin domain) and in some cases a hinge [Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature 332: 323-329 (1988); and Presta, Curr. Op. Struct. BIOL. 2:529-596 (1992)].
  • Fc refers to the last two constant region immunoglobulin domains of human IgA, IgD, and IgG, the last three constant region immunoglobulin domains of IgE and IgM, and the flexible hinge N-terminus of these domains.
  • Fc may include the J chain.
  • the Fc domain contains the immunoglobulin domains C ⁇ 2 and C ⁇ 3 (C ⁇ 2 and C ⁇ 3) and the lower hinge region between C ⁇ 1 (C ⁇ 1) and C ⁇ 2 (C ⁇ 2).
  • Fc variant means an Fc sequence that differs from a wild Fc sequence due to at least one amino acid modification, such as a substitution, deletion, or insertion, but still retains the ability to pair with the corresponding Fc single chain to form an Fc dimer .
  • the amino acid modification of the "Fc variant” changes the effector functional activity relative to the parent Fc region activity.
  • the variant Fc region may have altered (ie, increased or decreased) antibody-dependent cytotoxicity (ADCC), complement-mediated cytotoxicity (CDC), phagocytosis, opsonization Role or cell binding.
  • ADCC antibody-dependent cytotoxicity
  • CDC complement-mediated cytotoxicity
  • phagocytosis opsonization Role or cell binding.
  • the Fc amino acid modification can change (ie, increase or decrease) the affinity of the variant Fc region for Fc[gamma]R relative to wild Fc.
  • the variant Fc can change the affinity for FcyRI, FcyRII, FcyRIII.
  • the variant Fc has E233P, L234V, del235L, G236A, A327G, A330S, A331S, E356D, M358L amino acid modifications.
  • the "variant Fc” is a deglycosylated modified Fc, that is, an Fc containing an N297A amino acid modification.
  • the variant Fc further comprises S354C, T366W amino acid modifications, or S354C, T366W, Y349C, T366S, L368A, Y407V amino acid modifications.
  • Wild type or “WT” means herein an amino acid sequence or nucleotide sequence found in nature, including allelic variation.
  • the WT protein has an amino acid sequence or nucleotide sequence that has not been intentionally modified.
  • variant polypeptide sequence As used herein, “variant” means a polypeptide sequence that differs from the parent polypeptide sequence due to at least one amino acid modification.
  • a variant polypeptide may refer to the polypeptide itself, the composition comprising the polypeptide, or the amino sequence encoding it.
  • the variant polypeptide has at least one amino acid modification compared to the parent polypeptide, such as about 1 to about 10 amino acid modifications compared to the parent polypeptide, and preferably about 1 to about 5 amino acid modifications.
  • the variant polypeptide sequence herein preferably has at least about 80% homology with the parent polypeptide sequence, most preferably at least about 90% homology, and more preferably at least about 95% homology.
  • amino acid modification herein means amino acid substitutions, insertions and/or deletions in the polypeptide sequence. For clarity, unless otherwise stated, amino acid modifications are usually directed to amino acids encoded by DNA, such as the 20 amino acids with codons in DNA and RNA.
  • amino acid substitution or “substitution” means herein that the amino acid at a specific position in the parent polypeptide sequence is replaced with a different amino acid. Specifically, in some embodiments, the substitutions are for non-naturally occurring amino acids at specific positions that are not naturally occurring in the organism or in any organism.
  • substitution E272Y refers to a variant polypeptide in which glutamic acid at position 272 is replaced with tyrosine, in this case an Fc variant.
  • the protein is engineered to change the nucleic acid coding sequence but not the starting amino acid (eg CGG (encoding arginine) to CGA (still encoding arginine) to increase the expression level of the host organism) is not " "Amino acid substitution”; that is, although a new gene encoding the same protein is produced, if the protein has the same amino acid at a specific position at the beginning, it is not an amino acid substitution.
  • the variant Fc of the present invention has conservative substitutions relative to the parent or natural Fc, ie substitutions with amino acid residues having amino acid side chain groups of the same or similar properties.
  • the variant Fc of the present invention has no more than 40, 30, 20, 10, or 5 conservative substitutions relative to the parent or native Fc, as long as it still retains the ability to pair with the corresponding Fc single chain to form an Fc dimer That's it.
  • Effective function refers to those biological activities attributable to the Fc region of an antibody, which vary with the antibody isotype.
  • Examples of antibody effector functions include: C1q binding and complement-dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); down-regulation of cell surface receptors (eg, B cell receptors) ; And B cell activation.
  • ADCC antibody-dependent cytotoxicity
  • NK cells non-specific cytotoxic cells expressing FcR (such as natural killer (NK) cells, neutrophils, and macrophages) recognize bound on target cells The antibody then causes lysis of the target cells.
  • the main cells (NK cells) used to mediate ADCC express FcyRIII only, while monocytes express FcyRI, FcyRII and FcyRIII.
  • FcR expression on hematopoietic cells is summarized in Table 3 on pages 464 of Ravetch and Kinet, Annu. Rev. Immunol 9 (1991) 457-492.
  • “Complement-dependent cytotoxicity (CDC)” refers to a mechanism that induces cell death, in which the Fc effector molecular domain(s) of the target-bound antibody activates a series of enzymatic reactions, causing pores to form in the target cell membrane.
  • an antigen-antibody complex such as an antigen-antibody complex on an antibody-coated target cell, binds and activates the complement component C1q, which in turn activates the complement cascade, resulting in the death of the target cell.
  • Activation of complement can also lead to the deposition of complement components on the surface of target cells, which facilitates ADCC by binding to complement receptors on leukocytes (eg CR3).
  • Fab heavy chain or “FabH” means a polypeptide comprising immunoglobulin domains of VH and CH1;
  • Fab light chain or “FabL” means a polypeptide comprising immunoglobulin domains of VL and CL, That is the immunoglobulin light chain.
  • CH1 and CL in the Fab can exchange positions, that is, FabH includes VH and CL, and FabL includes VL-CH1.
  • Fab means a polypeptide comprising VH, CH1, VL, and CL immunoglobulin domains.
  • Fab can refer to an isolated domain, or this region in the case of a full-length antibody, antibody fragment, or Fab fusion protein.
  • Fab is generally composed of two chains, such as Fab heavy chain and Fab light chain.
  • CH1 and CL in the Fab can exchange positions with each other. Therefore, Fabs in which CH1 and CL exchange positions with each other are also included in the present invention.
  • Fv means a non-fused dimer comprising one VL and one VH.
  • Single-chain Fv or “scfv” means a polypeptide comprising the VL and VH domains of a single antibody, and the VL and VH are on the same polypeptide.
  • linker includes one or more amino acids of any reasonable sequence that provides flexibility/rigidity.
  • the linker is selected from, but not limited to, the group consisting of: GGGGSGGGGSGGGGS, SGGGGSGGGGSGGGGS, GGSGGSGGGGSGGGG, GGSGGSGGGGSGGGGS, GGGSGAKLAALKAKLAALKGGGGS, GGGGSELAALEAELAALEAGGSG, APATSLQSGQLGFQCGELCSGS, GTKAP, GTKAPPP, TAGGP, TVA GSG, SGG, GGG, GGGS, SGGG, GGGGSGS, GGGGSGS, GGGGSGGS, GGGGSGGGGS, AKTTPKLEEGEFSEAR, AKTTPKLEEGEFSEARV, AKTTPKLGG, SAKTTPKLGG, AKTTPKLEEGEFSEARV, SAKTTP, SAKTTPKLGG, RAGAPGS, RAGAPGS, RAGAPGS, RAGAPGS, RAG
  • the linker may also be a peptide linker cleavable in vivo, a protease (such as MMP) sensitive linker, a disulfide bond-based linker that can be cleaved by reduction as described previously, etc. (FusionProtein Technologies for Biopharmaceuticals : Applications and Challenges, edited by Stefan R. Schmidt) or any cleavable linker known in the art.
  • physiologically active peptide of the present invention includes not only proteins that exhibit physiological functions in vivo after binding to an antigen binding domain, but also polypeptides that only participate in antigen binding but have no physiological functions.
  • physiologically active peptides include receptors, ligand proteins, hormones, cytokines, interleukins, interleukin-binding proteins, enzymes, growth factors, transcriptional regulators, coagulation factors, vaccines, structural proteins, and cell surfaces Antigens, receptor antagonists and their derivatives.
  • PCR amplifies the above gene fragments respectively, and overlap PCR will obtain the anti-CD3 VH-1 and anti-CD3 VL-1, anti-EGFR VH and anti-EGFR VL, anti-CD3 VL-3 and anti-CD3 VH -3, anti-CD19 VH and anti-CD19 VL, anti-BCMA VH and anti-BCMA VL, anti-CLL-1 VH and anti-CLL-1 VL are connected through linker to get scfv1 (anti-CD3 scfv-1) , Scfv2 (anti-EGFR scfv), scfv3 (anti-CD3 scfv-3), scfv5 (anti-CD19 scfv), scfv6 (anti-BCMA scfv), scfv7 (anti-CLL-1 scfv) fragments, sequence verification.
  • PCR amplifies the 1.1 synthetic gene fragments, and connects the amplified PCR products anti-CD3, VL-1, anti-EGFR, VL, anti-CD3, VL-3, and anti-CD3, and VL-4 to CL ⁇ to obtain anti-CD3 CD3 VL-1-CL (Fab1L), anti-EGFR VL-CL (Fab2L), anti-CD3 VL-3-CL (Fab3L), anti-CD3 VL-4-CL (Fab4L), sequencing verification.
  • Fab1L anti-EGFR VL-CL
  • Fab3L anti-CD3 VL-3-CL
  • Fab4L anti-CD3 VL-4-CL
  • PCR amplifies the 1.1 synthesized gene fragments respectively, and connects the amplified anti-CD3 VH-1, anti-EGFR VH, anti-CD3 VH-3, anti-CD3 VH-4 with CH1 to obtain anti-CD3 CD3 VH-1-CH1 (Fab1H), anti-EGFR VH-CH1 (Fab2H), anti-CD3 VH-3-CH1 (Fab3H), anti-CD3 VH-4-CH1 (Fab4H), sequencing verification.
  • Fab1H anti-EGFR VH-CH1
  • Fab3H anti-CD3 VH-3-CH1
  • Fab4H anti-CD3 VH-4-CH1
  • the FabH and FabL verified by the above sequencing were further cloned into the pFuse-hIgG1-Fc2 vector (InvivoGen, CA) by in-frame connection.
  • the Fc fragment on the vector had the following mutations: N297A or E233P, L234V, L235A, delG236, A327G, A330S, A331S.
  • the Fc further comprises S354C, T366W mutation or Y349C, T366S, L368A, Y407V mutation. If necessary, either scfv or active peptide or VH or VL or Fab is connected to the above vector through a linker, and all the constructed sequences are verified by sequencing.
  • the nucleotide and amino acid sequences of each construct are shown in the sequence table: Seq No. 1-Seq. No. 142.
  • the cell culture supernatants were collected 48h and 96h after transfection, respectively, and the heterodimer fusion was purified using CH1 Select resin (Thermo Fisher Scientific, IL), ProteinG and/or Protein A Resin (Genscript) according to the manufacturer's instructions protein.
  • CH1 Select resin Thermo Fisher Scientific, IL
  • ProteinG ProteinG
  • Protein A Resin Genscript
  • the composition and purity of the purified heterodimeric fusion protein were analyzed by SDS-PAGE under reducing and non-reducing conditions. And the concentration was determined by A280 and BCA (Pierce, Rockford, IL).
  • the obtained CH1 resin, ProteinG and/or Protein purified heterodimer fusion protein was analyzed by GE's AKTA chromatography column.
  • the column used was: Superdex 200Increase 10/300GL gel exclusion layer Separation column and/or Mono 5/50GL ion exchange chromatography column.
  • IgFD-6 10.7 IgFD-21 3.75 IgFD-22 4.58 IgFD-23 8.3 IgFD-9 30 IgFD-10 25.3 IgFD-33 18
  • Anti-CD3/anti-CD19 heterodimer antibody promotes the specific killing of NALM-6 by PBMC
  • PBMCs peripheral blood mononuclear cells
  • FBS FBS complete medium
  • NALM6 cells After culturing NALM6 cells (RPMI1640 medium contains 10% FBS), after labeling with Green fluorescent cell linker mini kit (Sigma), 10 4 green fluorescent labeled NALM6 cells are taken, and the T cells activated with the above stimulation are 1:5 (NALM6 cells are 10 4 , T cells are in the ratio of 5*10 4 ), add gel-exclusion chromatography purified gradient diluted IgFD-6 or IgFD-7, after incubation at 37 °C for 24h, add 1% 7-AAD, upflow Analyze with a cytometer. Green fluorescence positive/7-AAD negative cells are viable NALM6 cells.
  • hEGFR-6-his (SinoBiological) (100 ng/well) in a 96-well plate and incubated overnight at 4°C; PBST (0.5% Tween-20 in PBS) containing 2% skimmed milk powder was blocked at room temperature for 1 hour, and each was added with a gradient dilution Heterodimeric antibodies IgFD-11, IgFD-24, IgFD-25, IgFD-26, IgFD-31, anti-EGFR were incubated at room temperature for 2h.
  • F98-EGFR cells were cultured (DMEM medium containing 10% FBS, 200 ⁇ g/ml G418). And gel exclusion chromatography were taken after 2x10 5 cells were washed three times with cold PBS, 2% FBS (dissolved in PBS) of purified closing IgFD-8, IgFD-9, IgFD-10, IgFD-11, IgFD -18, IgFD-19, IgFD-25, IgFD-26 or IgFD-31 (laboratory expressed aEGFR as control) Incubate at 4°C for 2h (mix gently during incubation), wash with 2% FBS (dissolved in PBS) The unbound antibody was removed, and stained with FITC anti-human IgG Fc (KPL, Inc., MD) for 1 h at 4°C, and eluted with 2% FBS (dissolved in PBS) for FACS analysis. As shown in Figure 7 and Table 4, different fusion forms of anti-CD3/anti-EGFR heterodimers can bind to EGFR on the surface
  • PBMCs peripheral blood mononuclear cells
  • FBS FBS complete medium
  • the cells were incubated with FITC anti-human IgG Fc (KPL, Inc., MD) at 4°C for 1 h (mixed gently during the incubation), 2% FBS (dissolved in PBS) washes away unbound antibody, and then FACS analysis was performed.
  • FITC anti-human IgG Fc KPL, Inc., MD
  • Anti-CD3/anti-EGFR heterodimer promotes the detection of cell-specific killing LDH of F98-EGFR by PBMC
  • PBMCs peripheral blood mononuclear cells
  • FBS FBS complete medium
  • %Cytotoxicity (Experimental--Effector Spontaneous--Target Spontaneous)/(Target Maximum--Target Spontaneous) x100
  • Target Maximum is the LDH content in the supernatant after F98-EGFR cell lysis
  • Target Spontaneous is the LDH content in the supernatant of F98-EGFR cells only
  • Effector Spontaneous is the LDH content in the supernatant of only effector cells (T cells).
  • Anti-CD3/BCMA heterodimer promotes the killing effect of PBMC on MM1.R cells
  • PBMCs peripheral blood mononuclear cells
  • FBS FBS complete medium
  • MM1.R cells Cultivate MM1.R cells (RPMI1640 medium contains 10% FBS), take 10 4 and incubate with the above stimulated T cells at a ratio of 1:5 (MM1.R cells are 10 4 , T cells are 5*10 4 ), IgFD-22 purified by gradient exclusion chromatography was added and incubated at 37°C for 24h.
  • Cytotox-96 nonradioactive cytotoxicity assay kit (Promega) was used to detect the LDH content in the culture supernatant. SpectraMax 250 reads the OD value at 490nm. The cytotoxicity (in %) is calculated as follows:
  • %Cytotoxicity (experimental–Effector Spontaneous–Target Spontaneous)/(Target Maximum–Target Spontaneous)x100
  • Target Maximum is the LDH content in the supernatant after lysis of only MM1.R cells
  • Target Spontaneous is the LDH content in the supernatant of only MM1.R cells.
  • Effector Spontaneous is the LDH content in the supernatant of only effector cells (T cells).
  • MM1.R cells RPMI1640 medium containing 10% FBS
  • Green fluorescent cell linker mini kit Sigma
  • 10 4 green fluorescently labeled MM1.R cells and the T cells activated by the above stimulation at 1: 5 (MM1.R cells are 10 4 , T cells are 5*10 4 ) incubate, add gel exclusion chromatography purified gradient diluted IgFD-22 37 °C incubation 24h, after adding 1% 7-AAD, Upflow cytometry for analysis. Green fluorescence positive/7-AAD negative cells are viable MM1.R cells.
  • PANC-1 DMEM medium containing 10% FBS
  • BXPC-3 RPMI1640 medium containing 10% FBS
  • K562 cells RPMI1640 medium containing 10% FBS
  • PBMCs peripheral blood mononuclear cells
  • FBS FBS complete medium
  • K562 and PANC-1 cells take 10 4 and the above activated T cells at a ratio of 1:5 (K562 or PANC-1 cells are 10 4 , T cells are 5*10 4 ), and add a gradient dilution of IgFD- 36 and IgFD-37 were incubated at 37°C for 24h.
  • Cytotox-96 nonradioactive cytotoxicity assay kit (Promega) was used to detect the content of LDH in the culture supernatant.
  • SpectraMax 250 reads the OD value at 490nm.
  • the cytotoxicity (in %) is calculated as follows:
  • %Cytotoxicity (Experimental--Effector Spontaneous--Target Spontaneous)/(Target Maximum--Target Spontaneous) x100
  • Target Maximum is the LDH content in the supernatant of only K562 or PANC-1 cells after lysis
  • Target Spontaneous is the LDH content in the supernatant of only K562 or PANC-1 cells.
  • Effector Spontaneous is the LDH content in the supernatant of only effector cells (T cells).
  • Anti-CD3/anti-CLL-1 heterodimer promotes the killing effect of PBMC on HL-60 cells
  • PBMCs peripheral blood mononuclear cells
  • FBS FBS complete medium
  • IgFD-23 can effectively call T cells in PBMC, and then have a specific killing effect on HL-60.
  • the samples IgFD-6 and IgFD-7 were mixed with freshly prepared thermal shift dye and shift buffer (Protein Thermal Shift TM Dye Kit, ThermoFisher Scientific, Cat. 4461146) at the ratio recommended by the manufacturer, using ViiA TM 7 Real-Time PCR System Thermal scanning was performed at a heating rate of 0.05°C/s at 25-99°C.
  • the Tm value was calculated using the "Area under curve (AUC)" analysis model of GraphPad Prism7 software. Each set of data is repeated 2 times to ensure the repeatability of the results.
  • IgFD-33 SD male rats (3) were intraperitoneally injected (I.P.) with IgFD-33. Heparin anticoagulated blood was collected from the tail vein, and the blood collection time was as follows: 2h, 4h, 8h, 24h, 36h, 4d, 7d, 11d, and 14d. After centrifugation, the plasma was taken and stored at -80°C until use. The content of IgFD-33 in plasma was measured according to Example 4.2(1). The results are shown in Figure 16. The half-life of IgFD-33 in rats can reach 2.5 days.

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Abstract

本发明提供了异源二聚体融合蛋白及其制备与应用。本发明将第一抗原结合结构域Fab的重链和轻链分别融合在两条Fc的N端，并在此基础上将第二抗原结合结构域scfv、Fab2、Fv、纳米抗体或生理活性肽与Fab的N端或Fc的C端融合，由此获得的异源二聚体融合蛋白由于其分子内的每个抗原结合结构域均为其本身的结构，很好地保留了抗原结合结构域本身的活性和药学性质；其中至少一个抗原结合结构域可以单价结合抗原；由于保留了Fc片段，本发明的异源二聚体融合蛋白具有较长的半衰期。

Description

异源二聚体融合蛋白 技术领域
本发明属于生物技术制药领域，具体基于免疫球蛋白片段的异源二聚体融合蛋白及其制备与应用。
背景技术
自1986年美国FDA批准第一个治疗性抗体muromonab-CD3用于治疗器官移植相关的急性排斥反应以来，截止至2018年5月，FDA批准的治疗性单抗已经超过80个，平均每年2-3个单抗获批。近几年来，治疗性单抗的获批呈井喷之势。2015年批准的治疗性单抗10个、2016年10个，2017年FDA批准的治疗性单抗破纪录地达到了17个。据估计，2020年治疗性单抗的市场份额将超过1250亿美元(Expert Opin Ther Pat.2018；28(3):251-276)。尽管单抗市场获得了巨大的成功，但是，单抗治疗的缺陷却不容忽视。传统抗体仅结合单一靶点的单一表位，其疗效受到一定限制。药理学研究揭示，多数复杂疾病都涉及多种与疾病相关的信号通路，例如肿瘤坏死因子TNF、白介素6等多种促炎症细胞因子同时介导免疫炎性疾病，而肿瘤细胞的增殖往往是由多个生长因子受体的异常上调造成的。单一信号通路的阻断通常疗效有限，而且容易形成耐药性。
因此，开发能够同时结合同一抗原或不同抗原的两个不同表位的双特异性抗体，长期以来成为新结构抗体研发的重要领域。截止至目前，已经有三个双特异性抗体被批准上市。最早的双抗是Frenesius和Trion公司利用小鼠和大鼠杂交瘤细胞杂交融合产生的Catumaxomab
Figure PCTCN2019129591-appb-000001
(anti-EpCAM×anti-CD3)，于2009年被EMA(欧洲药品管理局)批准用于治疗EpCAM(Epithelial cell adhesion molecule)阳性肿瘤所引起的恶性腹水。2014年12月，Amgen公司研发的双特异性靶向(anti-CD3×anti-CD19)抗体药物Blinatumomab
Figure PCTCN2019129591-appb-000002
获得FDA的批准，用于治疗费城染色体阴性(Ph-)的复发性或难治性B细胞急性淋巴细胞白血病(acute lymphoblastic leukemia，ALL)。Blinatumomab享受了FDA所有的审批优惠，包括优先评审、加速批准、突破性药物认定和孤儿药。2017年，Chugai和Roche的Emicizumab
Figure PCTCN2019129591-appb-000003
(anti-factor X×anti-factor IX)被FDA批准用于A型血友病的治疗。据统计，目前有超过60种双特异性抗体处于临床前研究阶 段，超过30种处于不同的临床试验阶段。根据2014年的双特异性抗体治疗市场估计(Bispecifc antibody therapeutics market(3 rd edition),2017–2030)，至2024年双特异性抗体每年的市场份额将达到58亿美元，双特异性抗体市场巨大。
根据其分子结构，目前正在进行临床前或临床研究的双特异性抗体可以分为两大类：以免疫球蛋白样(IgG-like)为基础的双抗和以抗体片段为基础的非免疫球蛋白样(non-IgG-like)的双抗。以BiTE技术平台为代表的non-IgG-like双抗分子量小，组织穿透性好，但是由于其缺少Fc片段，体内半衰期很短。例如，已被批准上市的Blincyto其官方公布的体内半衰期仅2h，临床上需连续注射给药，患者的依从性差；此外，其稳定性相对较差，容易产生无定形聚集；IgG-like的双抗含有IgG分子中的Fc片段，保留了Fc介导的如抗体依赖细胞介导的细胞毒性(antibody-dependent cell-mediated cytotoxicity，ADCC)、补体依赖的细胞毒性(complement-dependent cytotoxicity，CDC)、抗体依赖的细胞吞噬作用(antibody-dependent cellular phagocytosis，ADCP)等效应功能；除此之外，Fc可用于IgG-like双抗的纯化，有助于改善溶解性，大大提高这类双抗的稳定性；由于分子量大且可通过FcRn介导的再循环机制，Ig-like的双抗通常具有较长的血浆半衰期。以Roche的CrossMab技术为代表的Ig-like双抗通过将其中一个抗体片段的VH与VL、CH1-CL或VH/CH1-VL/CL互换以及Fc骨架上的knobs-into-holes，有效地解决了分子内两个抗体片段各自的轻链-轻链配对、重链-重链配对以及重链轻链错配问题，目前已有多个抗体处于临床试验阶段的BsAbs采用了该技术。然而，其中一个抗体片段由于结构域互换而变为非常规的Fab类型，该抗体片段对抗原的亲和力受到一定的影响，药代动力学特性也不是特别佳。而以DVD-Ig为代表的同源2+2型双抗，其Fc的两臂分别可以结合两个抗原，大大提高了BsAb的亲和力，但是由于其为同源二聚体，容易激活T细胞，因此该类BsAbs不适合用于召集T细胞杀伤肿瘤细胞。因此，制备具有优良药代动力学特性、工艺相对简单而又保持分子内两个抗体的天然结合活性的BsAbs对于临床药物开发至关重要。
本发明简述:
本发明提供这样的异源二聚体融合蛋白，其中，将能够构成第一抗原结合结构域Fab的Fab重链(FabH)和Fab轻链(FabL)分别融合(直接或通过连接子)在两条单链Fc的N端，由此形成的结构具有类似抗体的稳定蛋白折叠。在此基础上，本发明还提供这样的异源二聚体融合蛋白，其中进一步将构成第二抗原结合结构域的scfv、Fab2、Fv、纳米抗体或生理活性肽与所述Fab重链和/或轻链的N端或任一所述单链Fc的C端融合。由此获得的异源二聚体融合蛋白的分子内的第一抗原结合结构域和第二抗原结合结构域分别可以 形成并保持其功能性的构象，从而可以很好地发挥两个抗原结合结构域本身的活性和药学性质；其中至少一个抗原结合结构域可以单价结合抗原。此外，由于保留了Fc片段，本发明的异源二聚体融合蛋白具有较长的半衰期。
据此，在一方面，本发明提供了基于免疫球蛋白片段的异源二聚体融合蛋白，所述异源二聚体融合蛋白包括：
a)第一多肽链，其包含Fab重链和第一单链Fc，所述Fab重链直接或通过连接子与所述第一Fc单链的N端融合；
b)第二多肽链，其包含Fab轻链和第二单链Fc，所述Fab轻链直接或通过连接子与所述第二Fc单链的N端融合；
其中，所述第一多肽链的Fab重链和所述第二多肽链的Fab轻链形成第一抗原结合结构域Fab，所述第一单链Fc和所述第二单链Fc形成Fc二聚结构域(图1A)。
在一些实施方案中，所述连接子为GGSGAKLAALKAKLAALKGGGGS。在一些实施方案中，所述连接子为GGGGSELAALEAELAALEAGGSG。在一些实施方案中，所述第一多肽链的Fab重链通过连接子GGSGAKLAALKAKLAALKGGGGS与所述第一Fc单链的N端融合，所述第二多肽链的Fab轻链通过连接子GGGGSELAALEAELAALEAGGSG与所述第二Fc单链的N端融合。在一些实施方案中，所述第一多肽链的Fab重链通过GGGGSELAALEAELAALEAGGSG与所述第一Fc单链的N端融合，所述第二多肽链的Fab轻链通过连接子GGSGAKLAALKAKLAALKGGGGS与所述第二Fc单链的N端融合。
所述第一单链Fc和第二单链Fc优选源自相同的抗体同种型(isotypes)。但也可以来自不同的抗体同种型，只要二者能够配对形成二聚体即可。在一些实施方案中，第一单链Fc和第二单链Fc均源自IgG，更具体地，均源自IgG1。第一单链Fc和第二单链Fc通过链间二硫键以及分子间相互作用配对形成二聚体。在一些实施方案中，所述第一和第二单链Fc为野生型Fc。在一些实施方案中，所述野生型Fc具有如SEQ ID NO.147所示的氨基酸序列。在一些实施方案中，所述第一和第二单链Fc为Fc变体。在一些实施方案中，所述Fc变体不含糖基化修饰位点。在一些实施方案中，所述Fc变体包含N297去糖基化修饰的氨基酸置换。在一些实施方案中，所述包含N294去糖基化修饰氨基酸修饰的Fc变体具有如SEQ ID NO.143所示的氨基酸序列。在一些实施方案中，所述Fc变体包含一处或多处降低Fc对Fc受体的结合和/或效应功能的氨基酸置换。在一些实施方案中，所述Fc变体中的氨基酸置换包含E233P、L234V、L235A、delG236、A327G、A330S及 A331S中的一种或多种。在一些实施方案中，所述包含一处或多处降低Fc对Fc受体的结合和/或效应功能的氨基酸置换的Fc变体具有如SEQ ID.144所示的氨基酸序列。在一些实施方案中，所述第一Fc变体和第二Fc变体之一进一步包含氨基酸置换S354C、T366W，且所述第一Fc和所述第二Fc中的另一个进一步包含氨基酸置换Y349C、T366S、L368A和Y407V。在一些实施方案中，所述第一Fc变体具有如SEQ ID NO.145所示的氨基酸序列，所述第二Fc变体具有如SEQ ID NO.146所示的氨基酸序列。在一些实施方案中，所述第一Fc变体具有如SEQ ID NO.146所示的氨基酸序列，所述第二Fc变体具有如SEQ ID NO.145所示的氨基酸序列。在一些实施方案中，所述第一Fc变体具有如SEQ ID NO.147所示的氨基酸序列，所述第二Fc变体具有如SEQ ID NO.148所示的氨基酸序列。在一些实施方案中，所述第一Fc变体具有如SEQ ID NO.148所示的氨基酸序列，所述第二Fc变体具有如SEQ ID NO.147所示的氨基酸序列。
在一些实施方案中，所述异源二聚体融合蛋白进一步包含第二抗原结合结构域。
在一些实施方案中，所述异源二聚体融合蛋白的第二抗原结合结构域为单链Fv(scfv)。
在一些实施方案中，构成所述第二抗原结合结构域的scfv直接或通过连接子与所述第一多肽链的N端融合(图1B)。
在一些实施方案中，所述异源二聚体融合蛋白的第一抗原结合结构域Fab结合CD3。在一些实施方案中，所述结合CD3的第一抗原结合结构域Fab具有如SEQ ID NO.125所示的Fab重链和SEQ ID NO.126所示的Fab轻链。在一些实施方中，所述异源二聚体融合蛋白的第二抗原结合结构域scfv结合EGFR。在一些实施方案中，所述结合EGFR的第二抗原结合结构域scfv具有如SEQ ID NO.142所示的氨基酸序列。在一些实施方案中，所述异源二聚体融合蛋白结合CD3和EGFR。在一些实施方中，所述结合CD3和EGFR的异源二聚体融合蛋白其第一抗原结合结构域具有如SEQ ID NO.125所示的Fab重链和如SEQ ID NO.126所示的Fab轻链，其第二抗原结合结构域具有如SEQ ID NO.142所示的氨基酸序列。在一些实施方案中，所述结合CD3和EGFR的异源二聚体融合蛋白具有包含如SEQ ID NO.18所示氨基酸序列的第一多肽链和包含如SEQ ID NO.12所示氨基酸序列的第二多肽链。
在一些实施方案中，构成所述第二抗原结合结构域的scfv直接或通过连接子与所述第二多肽链的N端融合(图1C)。
在一些实施方案中，所述异源二聚体融合蛋白的第一抗原结合结构域Fab结合CD3。在一些实施方案中，所述结合CD3的第一抗原结合结构域Fab具有如SEQ ID NO.125所示的FabH和SEQ ID NO.126所示的FabL。在一些实施方案中，所述异源二聚体融合蛋白的第二抗原结合结构域scfv结合EGFR。在一些实施方案中，所述结合EGFR的第二抗原结合结构域scfv具有如SEQ ID NO.142所示的氨基酸序列。在一些实施方案中，所述异源二聚体融合蛋白结合CD3和EGFR。在一些实施方中，所述结合CD3和EGFR的异源二聚体融合蛋白的第一抗原结合结构域具有如SEQ ID NO.125所示的Fab重链和SEQ ID NO.126所示的Fab轻链，其第二抗原结合结构域具有如SEQ ID NO.142所示的氨基酸序列。在一些实施方案中，所述结合CD3和EGFR的异源二聚体融合蛋白具有如SEQ ID NO.30所示氨基酸序列的第一多肽链和具有如SEQ ID NO.58所示氨基酸序列的第二多肽链。
在一些实施方案中，构成所述第二抗原结合结构域的scfv直接或通过连接子与所述第一多肽链的C端融合(图1D)。
在一些实施方案中，所述异源二聚体融合蛋白的第一抗原结合结构域Fab结合CD3。在一些实施方案中，所述结合CD3的第一抗原结合结构域Fab具有如SEQ ID NO.125所示的Fab重链和SEQ ID NO.126所示的Fab轻链。在一些实施方案中，所述结合CD3的第一抗原结合结构域Fab具有如SEQ ID NO.129所示的Fab重链和SEQ ID NO.130所示的Fab轻链。在一些实施方案中，所述结合CD3的第一抗原结合结构域Fab具有如SEQ ID NO.131所示的Fab重链和SEQ ID NO.132所示的Fab轻链。在一些实施方案中，构成所述异源二聚体融合蛋白的第二抗原结合结构域的scfv结合CD19。在一些实施方案中，构成所述结合CD19的第二抗原结合结构域的scfv具有如SEQ ID NO.139所示的氨基酸序列。在一些实施方案中，构成所述异源二聚体融合蛋白的第二抗原结合结构域的scfv结合EGFR。在一些实施方案中，构成所述结合EGFR的第二抗原结合结构域的scfv具有如SEQ ID NO.142所示的氨基酸序列。在一些实施方案中，所述异源二聚体融合蛋白结合CD3和CD19。在一些实施方案中，所述结合CD3和CD19的异源二聚体融合蛋白的第一抗原结合结构域具有如SEQ ID NO.125所示的Fab重链和SEQ ID NO.126所示的Fab轻链，其第二抗原结合结构域具有如SEQ ID NO.139所示的氨基酸序列。在一些实施方案中，所述结合CD3和CD19的异源二聚体融合蛋白具有如SEQ ID NO.2所示氨基酸序列的第一多肽链和具有如SEQ ID NO.4所示氨基酸序列的第二多肽链。在一些实施方案中，所述结合CD3和CD19的异源二聚体融合蛋白其第一抗原结合结构域具有如SEQ ID NO.129所示的Fab重链和SEQ ID NO.130所示的Fab轻链，其第二抗原结合结构域具有如SEQ ID NO.139所示的氨基酸序列。在一些实施方案中，所述结合CD3和CD19的异源二聚体融 合蛋白具有如SEQ ID NO.6所示氨基酸序列的第一多肽链和具有如SEQ ID NO.8所示氨基酸序列的第二多肽链。在一些实施方案中，所述异源二聚体融合蛋白结合CD3和EGFR。在一些实施方案中，所述结合CD3和EGFR的异源二聚体融合蛋白其第一抗原结合结构域具有如SEQ ID NO.131所示的Fab重链和SEQ ID NO.132所示的Fab轻链，其第二抗原结合结构域具有如SEQ ID NO.142所示的氨基酸序列。在一些实施方案中，所述结合CD3和EGFR的异源二聚体融合蛋白具有如SEQ ID NO.14所示氨基酸序列的第一多肽链和具有如SEQ ID NO.16所示氨基酸序列的第二多肽链。
在一些实施方案中，构成所述第二抗原结合结构域的scfv直接或通过连接子与所述第二多肽链的C端融合(图1E)。
在一些实施方案中，所述异源二聚体融合蛋白的第一抗原结合结构域Fab结合CD3。在一些实施方案中，所述结合CD3的第一抗原结合结构域Fab具有如SEQ ID NO.125所示的Fab重链和SEQ ID NO.126所示的Fab轻链。在一些实施方案中，所述结合CD3的第一抗原结合结构域Fab具有如SEQ ID NO.129所示的Fab重链和SEQ ID NO.130所示的Fab轻链。在一些实施方案中，所述结合CD3的第一抗原结合结构域Fab具有如SEQ ID NO.131所示的Fab重链和SEQ ID NO.132所示的Fab轻链。在一些实施方案中，构成所述异源二聚体融合蛋白的第二抗原结合结构域的scfv结合CD19。在一些实施方案中，构成所述结合CD19的第二抗原结合结构域的scfv具有如SEQ ID NO.139所示的氨基酸序列。在一些实施方案中，构成所述异源二聚体融合蛋白的第二抗原结合结构域的scfv结合BCMA。在一些实施方案中，构成所述结合BCMA的第二抗原结合结构域的scfv具有如SEQ ID NO.140所示的氨基酸序列。在一些实施方案中，所述异源二聚体融合蛋白的第二抗原结合结构域scfv结合CLL-1。在一些实施方案中，构成所述结合CLL-1的第二抗原结合结构域的scfv具有如SEQ ID NO.141所示的氨基酸序列。在一些实施方案中，构成所述异源二聚体融合蛋白的第二抗原结合结构域的scfv结合EGFR。在一些实施方案中，构成所述结合EGFR的第二抗原结合结构域的scfv具有如SEQ ID NO.142所示的氨基酸序列。在一些实施方案中，所述异源二聚体融合蛋白结合CD3和CD19。在一些实施方案中，所述结合CD3和CD19的异源二聚体融合蛋白的第一抗原结合结构域具有如SEQ ID NO.125所示的Fab重链和SEQ ID NO.126所示的Fab轻链，其第二抗原结合结构域具有如SEQ ID NO.139所示的氨基酸序列。在一些实施方案中，所述结合CD3和CD19的异源二聚体融合蛋白其第一抗原结合结构域具有如SEQ ID NO.129所示的Fab重链和SEQ ID NO.130所示的Fab轻链，其第二抗原结合结构域具有如SEQ ID NO.139所示的氨基酸序列。在一些实施方案中，所述结合CD3和CD19的异源二聚体融合蛋白具有如SEQ ID NO.20所示氨 基酸序列的第一多肽链和具有如SEQ ID NO.22所示氨基酸序列的第二多肽链。在一些实施方案中，所述结合CD3和CD19的异源二聚体融合蛋白具有包含如SEQ ID NO.24所示氨基酸序列的第一多肽链和包含如SEQ ID NO.26所示氨基酸序列的第二多肽链。在一些实施方案中，所述异源二聚体融合蛋白结合CD3和BCMA。在一些实施方案中，所述结合CD3和BCMA的异源二聚体融合蛋白的第一抗原结合结构域具有如SEQ ID NO.125所示的Fab重链和SEQ ID NO.126所示的Fab轻链，其第二抗原结合结构域具有如SEQ ID NO.140所示的氨基酸序列。在一些实施方案中，所述结合CD3和BCMA的异源二聚体融合蛋白具有包含如SEQ ID NO.30所示氨基酸序列的第一多肽链和包含如SEQ ID NO.40所示氨基酸序列的第二多肽链。在一些实施方案中，所述异源二聚体融合蛋白结合CD3和CLL-1。在一些实施方案中，所述结合CD3和CLL-1的异源二聚体融合蛋白的第一抗原结合结构域具有如SEQ ID NO.125所示的Fab重链和SEQ ID NO.126所示的Fab轻链，其第二抗原结合结构域具有如SEQ ID NO.141所示的氨基酸序列。在一些实施方案中，所述结合CD3和CLL-1的异源二聚体融合蛋白具有包含如SEQ ID NO.30所示氨基酸序列的第一多肽链和包含如SEQ ID NO.42所示氨基酸序列的第二多肽链。在一些实施方案中，所述异源二聚体融合蛋白结合CD3和EGFR。在一些实施方案中，所述结合CD3和EGFR的异源二聚体融合蛋白的第一抗原结合结构域具有如SEQ ID NO.125所示的Fab重链和SEQ ID NO.126所示的Fab轻链，其第二抗原结合结构域具有如SEQ ID NO.142所示的氨基酸序列。在一些实施方案中，所述结合CD3和EGFR的异源二聚体融合蛋白具有包含如SEQ ID NO.24所示氨基酸序列的第一多肽链和包含如SEQ ID NO.28所示氨基酸序列的第二多肽链。在一些实施方案中，所述结合CD3和EGFR的异源二聚体融合蛋白具有包含如SEQ ID NO.30所示氨基酸序列的第一多肽链和包含如SEQ ID NO.32所示氨基酸序列的第二多肽链。在一些实施方案中，所述结合CD3和EGFR的异源二聚体融合蛋白具有包含如SEQ ID NO.54所示氨基酸序列的第一多肽链和包含如SEQ ID NO.56所示氨基酸序列的第二多肽链。在一些实施方案中，所述结合CD3和EGFR的异源二聚体融合蛋白具有包含如SEQ ID NO.44所示氨基酸序列的第一多肽链和包含如SEQ ID NO.46所示氨基酸序列的第二多肽链。在一些实施方案中，所述结合CD3和EGFR的异源二聚体融合蛋白具有包含如SEQ ID NO.44所示氨基酸序列的第一多肽链和包含如SEQ ID NO.52所示氨基酸序列的第二多肽链。在一些实施方案中，所述结合CD3和EGFR的异源二聚体融合蛋白具有包含如SEQ ID NO.48所示氨基酸序列的第一多肽链和包含如SEQ ID NO.50所示氨基酸序列的第二多肽链。在一些实施方案中，所述结合CD3和EGFR的异源二聚体融合蛋白其第一抗原结合结构域具有如SEQ ID NO.131所示的Fab重链和 SEQ ID NO.132所示的Fab轻链，其第二抗原结合结构域具有如SEQ ID NO.142所示的氨基酸序列。在一些实施方案中，所述结合CD3和EGFR的异源二聚体融合蛋白具有包含如SEQ ID NO.36所示氨基酸序列的第一多肽链和包含如SEQ ID NO.38所示氨基酸序列的第二多肽链。
在一些实施方案中，所述异源二聚体融合蛋白的第二抗原结合结构域为生理活性肽。
在一些实施方案中，所述生理活性肽直接或通过连接子与所述第一多肽链的N端融合(图1B)。
在一些实施方案中，所述生理活性肽直接或通过连接子与所述第二多肽链的N端融合(图1C)。
在一些实施方案中，所述生理活性肽直接或通过连接子与所述第一多肽链的C端融合(图1D)。
在一些实施方案中，所述生理活性肽为EGF4。在一些实施方案中，所述生理活性肽EGF4具有如SEQ ID NO.150所示的氨基酸序列。在一些实施方案中，所述异源二聚体融合蛋白的第一抗原结合结构域Fab结合CD3。在一些实施方案中，所述结合CD3的第一抗原结合结构域Fab具有如SEQ ID NO.125所示的Fab重链和SEQ ID NO.126所示的Fab轻链。在一些实施方案中，所述异源二聚体融合蛋白其结合CD3和EGFR。在一些实施方案中，所述结合CD3和EGFR的异源二聚体融合蛋白的第一抗原结合结构域具有如SEQ ID NO.125所示的Fab重链和SEQ ID NO.126所示的Fab轻链，其第二抗原结合结构域生理活性肽具有如SEQ ID NO.150所示的氨基酸序列。在一些实施方案中，所述结合CD3和EGFR的异源二聚体融合蛋白具有包含如SEQ ID NO.10所示氨基酸序列的第一多肽链和包含SEQ ID NO.12所示氨基酸序列的第二多肽链。
在一些实施方案中，所述生理活性肽直接或通过连接子与所述第二多肽链的C端融合(图1E)。
在一些实施方案中，所述生理活性肽为EGF4。在一些实施方案中，所述生理活性肽EGF4具有如SEQ ID NO.150所示的氨基酸序列。在一些实施方案中，所述异源二聚体融合蛋白的第一抗原结合结构域Fab结合CD3。在一些实施方案中，所述结合CD3的第一抗原结合结构域Fab具有如SEQ ID NO.125所示的Fab重链和SEQ ID NO.126所示的Fab轻链。在一些实施方案中，所述异源二聚体融合蛋白其第一抗原结合结构域具有如SEQ ID NO.125所示的Fab重链和SEQ ID NO.126所示的Fab轻链，其第二抗原结合结构域具有如SEQ ID NO.150所示的氨基酸序列。在一些实施方案中，所述异源二聚体融合蛋白具有 包含如SEQ ID NO.30所示氨基酸序列的第一多肽链和包含SEQ ID NO.34所示氨基酸序列的第二多肽链。在一些实施方案中，所述异源二聚体融合蛋白的第二抗原结合结构域由第一和第二生理活性肽组成。
在一些实施方案中，所述第一和第二生理活性肽分别与所述第一多肽链和第二多肽链的N端直接或通过连接子融合。
在一些实施方案中，所述第一和第二生理活性肽分别与所述第一多肽链和第二多肽链的C端直接或通过连接子融合(图1F)。
在一些实施方案中，所述第一和第二生理活性肽不同。在一些实施方案中，所述第一和所述第二生理活性肽相同。
在一些实施方案中，所述第一和第二生理活性肽为NKG2D。在一些实施方案中，所述生理活性肽NKG2D具有如SEQ ID NO.151所示的氨基酸序列。在一些实施方案中，所述异源二聚体的第一抗原结合结构域Fab结合CD3。在一些实施方案中，所述结合CD3的第一抗原结合结构域具有如SEQ ID NO.125所示的Fab重链和SEQ ID NO.126所示的Fab轻链。在一些实施方案中，所述异源二聚体融合蛋白结合CD3和MIC-A。在一些实施方案中，所述结合CD3和MIC-A的异源二聚体融合蛋白的第一抗原结合结构域具有如SEQ ID NO.125所示的Fab重链和SEQ ID NO.126所示的Fab轻链，其第二抗原结合结构域具有如SEQ ID NO.151所示的氨基酸序列。在一些实施方案中，所述结合CD3和MIC-A的异源二聚体融合蛋白具有包含如SEQ ID NO.122所示氨基酸序列的第一多肽链和包含SEQ ID NO.124所示氨基酸序列的第二多肽链。
在一些实施方案中，所述异源二聚体融合蛋白的第二抗原结合结构域为Fv。
在一些实施方案中，构成所述第二抗原结合结构域的Fv的重链可变区直接或通过连接子与所述第一多肽链的C端融合，构成所述第二抗原结合结构域的Fv的轻链可变区直接或通过连接子与所述第二多肽链的C端融合(图1G)。
在一些实施方案中，所述异源二聚体融合蛋白的第一抗原结合结构域Fab结合CD3。在一些实施方案中，所述结合CD3的第一抗原结合结构域具有如SEQ ID NO.125所示的Fab重链和SEQ ID NO.126所示的Fab轻链。在一些实施方案中，构成所述异源二聚体融合蛋白的第二结合结构域的Fv结合EGFR。在一些实施方案中，构成所述结合EGFR的第二抗原结合结构域的Fv具有如SEQ ID NO.135所示的重链可变区和SEQ ID NO.136所示的轻链可变区。在一些实施方案中，所述异源二聚体融合蛋白结合CD3和EGFR。在一些实施方案中，所述结合CD3和EGFR的异源二聚体融合蛋白的第一抗原结合结构域具有如SEQ ID NO.125所示的Fab重链和SEQ ID NO.126所示的Fab轻链，其第二抗原结合结 构域具有如SEQ ID NO.135所示的重链可变区和SEQ ID NO.136所示的轻链可变区。在一些实施方案中，所述结合CD3和EGFR的异源二聚体融合蛋白具有包含如SEQ ID NO.60所示氨基酸序列的第一多肽链和包含SEQ ID NO.62所示氨基酸序列的第二多肽链。在一些实施方案中，所述结合CD3和EGFR的异源二聚体融合蛋白具有包含如SEQ ID NO.64所示氨基酸序列的第一多肽链和包含SEQ ID NO.66所示氨基酸序列的第二多肽链。在一些实施方案中，所述结合CD3和EGFR的异源二聚体融合蛋白具有包含如SEQ ID NO.68所示氨基酸序列的第一多肽链和包含SEQ ID NO.70所示氨基酸序列的第二多肽链。
在一些实施方案中，所述第二抗原结合结构域Fv的重链可变区直接或通过连接子与所述第二多肽链的C端融合，所述第二抗原结合结构域Fv的轻链可变区直接或通过连接子与所述第一多肽链的C端融合(图1H)。
在一些实施方案中，所述异源二聚体融合蛋白的第一抗原结合结构域Fab结合CD3。在一些实施方案中，所述结合CD3的第一抗原结合结构域具有如SEQ ID NO.125所示的Fab重链和SEQ ID NO.126所示的Fab轻链。在一些实施方案中，所述异源二聚体融合蛋白的第二抗原结合结构域结合EGFR。在一些实施方案中，构成所述结合EGFR的第二抗原结合结构域的Fv具有如SEQ ID NO.135所示的重链可变区和SEQ ID NO.136所示的轻链可变区。在一些实施方案中，所述异源二聚体融合蛋白结合CD3和EGFR。在一些实施方案中，所述结合CD3和EGFR的异源二聚体融合蛋白的第一抗原结合结构域具有如SEQ ID NO.125所示的Fab重链和SEQ ID NO.126所示的Fab轻链，其第二抗原结合结构域具有如SEQ ID NO.135所示的重链可变区和SEQ ID NO.136所示的轻链可变区。在一些实施方案中，所述结合CD3和EGFR的异源二聚体融合蛋白具有包含如SEQ ID NO.72所示氨基酸序列的第一多肽链和包含SEQ ID NO.74所示氨基酸序列的第二多肽链。在一些实施方案中，所述结合CD3和EGFR的异源二聚体融合蛋白具有包含如SEQ ID NO.76所示氨基酸序列的第一多肽链和包含SEQ ID NO.78所示氨基酸序列的第二多肽链。在一些实施方案中，所述结合CD3和EGFR的异源二聚体融合蛋白具有包含如SEQ ID NO.80所示氨基酸序列的第一多肽链和包含SEQ ID NO.82所示氨基酸序列的第二多肽链。
在一些实施方案中，构成所述第二抗原结合结构域的Fv的重链可变区直接或通过连接子与所述第二多肽链的N端融合，构成所述第二抗原结合结构域的Fv的轻链可变区直接或通过连接子与所述第一多肽链的N端融合(图1J)。
在一些实施方案中，构成所述第二抗原结合结构域的Fv的重链可变区直接或通过连接子与所述第一多肽链的N端融合，所述第二抗原结合结构域Fv的轻链可变区直接或通过连接子与所述第二多肽链的N端融合(图1I)。
在一些实施方案中，所述异源二聚体融合蛋白的第一抗原结合结构域结合EGFR。在一些实施方案中，所述结合EGFR的第一抗原结合结构域具有如SEQ ID NO.127所示的Fab重链和SEQ ID NO.128所示的Fab轻链。在一些实施方案中，所述异源二聚体融合蛋白的第二抗原结合结构域结合CD3。在一些实施方案中，构成所述结合CD3的第二抗原结合结构域的Fv具有如SEQ ID NO.133所示的重链可变区和SEQ ID NO.134所示的轻链可变区。在一些实施方案中，所述异源二聚体融合蛋白结合CD3和EGFR。在一些实施方案中，所述结合CD3和EGFR的异源二聚体融合蛋白的第一抗原结合结构域具有如SEQ ID NO.127所示的Fab重链和SEQ ID NO.128所示的Fab轻链，其第二抗原结合结构域具有如SEQ ID NO.133所示的重链可变区和SEQ ID NO.134所示的轻链可变区。在一些实施方案中，所述结合CD3和EGFR的异源二聚体融合蛋白具有包含如SEQ ID NO.104所示氨基酸序列的第一多肽链和包含如SEQ ID NO.106所示氨基酸序列的第二多肽链。
在一些实施方案中，所述异源二聚体融合蛋白的第一抗原结合结构域Fab结合CD3。在一些实施方案中，所述结合CD3的第一抗原结合结构域Fab具有如SEQ ID NO.125所示的Fab重链和SEQ ID NO.125所示的Fab轻链。在一些实施方案中，构成所述异源二聚体融合蛋白的第二抗原结合结构域的Fv结合EGFR。在一些实施方案中，所述结合EGFR的第二抗原结合结构域具有如SEQ ID NO.135所示的重链可变区和SEQ ID NO.136所示的轻链可变区。在一些实施方案中，所述异源二聚体融合蛋白结合CD3和EGFR。在一些实施方案中，所述结合CD3和EGFR的异源二聚体的第一抗原结合结构域Fab具有如SEQ ID NO.125所示的Fab重链和SEQ ID NO.125所示的Fab轻链，其第二抗原结合结构域Fv具有如SEQ ID NO.135所示的重链可变区和SEQ ID NO.136所示的轻链可变区。在一些实施方案中，所述结合CD3和EGFR的异源二聚体融合蛋白具有包含如SEQ ID NO.108所示氨基酸序列的第一多肽链和包含如SEQ ID NO.110所示氨基酸序列的第二多肽链。
在一些实施方案中，所述异源二聚体融合蛋白的第二抗原结合结构域为Fab2。
在一些实施方案中，所述第二抗原结合结构域Fab2的重链直接或通过连接子与所述第一多肽链的C端融合，所述Fab2的轻链直接或通过连接子与所述第二多肽链的C端融合(图1K)。
在一些实施方案中，所述异源二聚体的第一抗原结合结构域Fab结合CD3。在一些实施方案中，所述结合CD3的第一抗原结合结构域Fab具有如SEQ ID NO.125所示的Fab重链和SEQ ID NO.126所示的Fab轻链。在一些实施方案中，构成所述异源二聚体的第二抗原结合结构域的Fab2结合EGFR。在一些实施方案中，构成所述结合EGFR的第二抗原结合结构域的Fab2具有如SEQ ID NO.127所示的Fab重链和SEQ ID NO.128所示的Fab轻链。在一些实施方案中，所述异源二聚体融合蛋白结合CD3和EGFR。在一些实施方案中，所述结合CD3和EGFR的异源二聚体融合蛋白的第一抗原结合结构域Fab具有如SEQ ID NO.125所示的Fab重链和SEQ ID NO.126所示的Fab轻链，其第二抗原结合结构域Fab2具有如SEQ ID NO.127所示的Fab重链和SEQ ID NO.128所示的Fab轻链。在一些实施方案中，所述结合CD3和EGFR的异源二聚体融合蛋白具有包含如SEQ ID NO.84所示氨基酸序列的第一多肽链和包含SEQ ID NO.86所示氨基酸序列的第二多肽链。
在一些实施方案中，所述第二抗原结合结构域Fab2的重链直接或通过连接子与所述第二多肽链的C端融合，所述Fab2的轻链直接或通过连接子与所述第一多肽链的C端融合(图1N)。
在一些实施方案中，所述异源二聚体的第一抗原结合结构域结合CD3。在一些实施方案中，所述结合CD3的第一抗原结合结构域Fab具有如SEQ ID NO.125所示的Fab重链和SEQ ID NO.126所示的Fab轻链。在一些实施方案中，所述异源二聚体的第二抗原结合结构域Fab2结合EGFR。在一些实施方案中，所述结合EGFR的第二抗原结合结构域Fab2具有如SEQ ID NO.127所示的Fab重链和SEQ ID NO.128所示的Fab轻链。在一些实施方案中，所述异源二聚体融合蛋白结合CD3和EGFR。在一些实施方案中，所述结合CD3和EGFR的异源二聚体融合蛋白的第一抗原结合结构域Fab具有如SEQ ID NO.125所示的Fab重链和SEQ ID NO.126所示的Fab轻链，其第二抗原结合结构域Fab2具有如SEQ ID NO.127所示的Fab重链和SEQ ID NO.128所示的Fab轻链。在一些实施方案中，所述结合CD3和EGFR的异源二聚体融合蛋白具有包含如SEQ ID NO.88所示氨基酸序列的第一多肽链和包含SEQ ID NO.90所示氨基酸序列的第二多肽链。
在一些实施方案中，所述第二抗原结合结构域Fab2的重链直接或通过连接子与所述第二多肽链的N端融合，所述Fab2的轻链直接或通过连接子与所述第一多肽链的N端融合(图1M)。
在一些实施方案中，所述第二抗原结合结构域Fab2的重链直接或通过连接子与所述第一多肽链的N端融合，所述Fab2的轻链直接或通过连接子与所述第二多肽链的N端融合(图1L)。
在一些实施方案中，所述异源二聚体融合蛋白的第一抗原结合结构域Fab结合EGFR。在一些实施方案中，所述结合EGFR的第一抗原结合结构域Fab具有如SEQ ID NO.127所示的Fab重链和SEQ ID NO.128所示的Fab轻链。在一些实施方案中，所述异源二聚体融合蛋白的第二抗原结合结构域Fab2结合CD3。在一些实施方案中，所述结合CD3的第二抗原结合结构域Fab2具有如SEQ ID NO.125所示的Fab重链和SEQ ID NO.126所示的Fab轻链。在一些实施方案中，所述异源二聚体结合EGFR和CD3。在一些实施方案中，所述结合EGFR和CD3的异源二聚体融合蛋白的第一抗原结合结构域Fab具有如SEQ ID NO.127所示的Fab重链和SEQ ID NO.128所示的Fab轻链，其第二抗原结合结构域Fab2具有如SEQ ID NO.125所示的Fab重链和SEQ ID NO.126所示的Fab轻链。在一些实施方案中，所述结合EGFR和CD3的异源二聚体融合蛋白具有包含如SEQ ID NO.112所示氨基酸序列的第一多肽链和包含如SEQ ID NO.114所示氨基酸序列的第二多肽链。
在一些实施方案中，所述异源二聚体融合蛋白的第一抗原结合结构域Fab结合CD3。在一些实施方案中，所述结合CD3的第一抗原结合结构域具有如SEQ ID NO.125所示的Fab重链和SEQ ID NO.126所示的Fab轻链。在一些实施方案中，所述异源二聚体融合蛋白的第二抗原结合结构域Fab2结合EGFR。在一些实施方案中，所述结合EGFR的第二抗原结合结构域Fab2具有如SEQ ID NO.127所示的Fab重链和SEQ ID NO.128所示的Fab轻链。在一些实施方案中，所述异源二聚体融合蛋白结合CD3和EGFR。在一些实施方案中，所述结合CD3和EGFR的异源二聚体融合蛋白的第一抗原结合结构域Fab具有如SEQ ID NO.125所示的Fab重链和SEQ ID NO.126所示的Fab轻链，其第二抗原结合结构域Fab2具有如SEQ ID NO.127所示的Fab重链和SEQ ID NO.128所示的Fab轻链。在一些实施方案中，所述结合CD3和EGFR的异源二聚体融合蛋白具有包含如SEQ ID NO.116所示氨基酸序列的第一多肽链和包含SEQ ID NO.118所示氨基酸序列的第二多肽链。
在一些实施方案中，所述异源二聚体融合蛋白的第二抗原结合结构域为纳米抗体。
在一些实施方案中，所述第二抗原结合结构域纳米抗体直接或通过连接子与所述第一多肽链的N端融合。在一些实施方案中，所述第二抗原结合结构域纳米抗体直接或通过连接子与所述第二多肽链的N端融合。
在一些实施方案中，所述第二抗原结合结构域纳米抗体直接或通过连接子与所述第一多肽链的C端融合。在一些实施方案中，所述第二抗原结合结构域纳米抗体直接或通过连接子与所述第二多肽链的C端融合。
另一方面，本发明提供了基于免疫球蛋白片段的异源二聚体融合蛋白，所述异源二聚体包括：
a)第一多肽链，其按从N端到C端的顺序依次包含：Fc、(L1)n、CH1、L2、VH，
b)第二多肽链，其按从N端到C端的顺序依次包含：Fc、(L3)n、CL、L4、VL；
其中，n为0或1，L1、L2、L3和L4为连接子，VH和VL形成第一抗原结合结构域Fv。
在一些实施方案中，所述异源二聚体融合蛋白进一步包括第二抗原结合结构域。
在一些实施方案中，所述第二抗原结合结构域为生理活性肽。
在一些实施方案中，所述生理活性肽直接或通过连接子与所述第一或第二多肽链的N端融合(图1P和图1S)。
在一些实施方案中，所述生理活性肽直接或通过连接子与所述第一或第二多肽链的C端融合(图1T和图1U)。
在一些实施方案中，所述第二抗原结合结构域为Fab2。
在一些实施方案中，所述第二抗原结合结构域Fab2的重链直接或通过连接子与所述第一多肽链的N端融合，所述Fab2的轻链直接或通过连接子与所述第二多肽链的N端融合。
在一些实施方案中，所述第二抗原结合结构域Fab2的重链直接或通过连接子与所述第二多肽链的N端融合，所述Fab2的轻链直接或通过连接子与所述第二多肽链的N端融合。
在一些实施方案中，所述异源二聚体融合蛋白的第二抗原结合结构域为Fv。
在一些实施方案中，所述第二抗原结合结构域Fv的重链可变区直接或通过连接子与所述第一多肽链的N端融合，所述第二抗原结合结构域Fv的轻链可变区直接或通过连接子与所述第二多肽链的N端融合。
在一些实施方案中，所述第二抗原结合结构域Fv的重链可变区直接或通过连接子与所述第二多肽链的N端融合，所述第二抗原结合结构域Fv的轻链可变区直接或通过连接子与所述第一多肽链的N端融合。
在一些实施方案中，所述第二抗原结合结构域为纳米抗体。
在一些实施方案中，所述第二抗原结合结构域纳米抗体直接或通过连接子与所述第一多肽链的N端融合。在一些实施方案中，所述第二抗原结合结构域纳米抗体直接或通过连接子与所述第二多肽链的N端融合。
在一些实施方案中，所述第二抗原结合结构域为scfv。在一些实施方案中，所述第二抗原结合结构域scfv直接或通过连接子与所述第一或第二多肽链的C端融合(图1T和图1U)。
在一些实施方案中，所述第二抗原结合结构域scfv直接或通过连接子与所述第二多肽链的N端融合(图1S)。
在一些实施方案中，所述第二抗原结合结构域scfv直接或通过连接子与第一多肽链的N端融合(图1P)。
在一些实施方案中，所述异源二聚体的第一抗原结合结构域Fv结合CD3。在一些实施方案中，所述结合CD3的第一抗原结合结构域Fv具有如SEQ ID NO.133所示的VH和SEQ ID NO.134所示的VL。在一些实施方案中，所述结合CD3的第一抗原结合结构域Fv具有如SEQ ID NO.137所示的VH和SEQ ID NO.138所示的VL。在一些实施方案中，所述第二抗原结合结构域scfv结合CD19。在一些实施方案中，所述结合CD19的第二抗原结合结构域scfv具有如SEQ ID NO.139所示的氨基酸序列。在一些实施方案中，所述异源二聚体融合蛋白结合CD3和CD19。在一些实施方案中，所述结合CD3和CD19的异源二聚体融合蛋白其第一抗原结合结构域Fv具有如SEQ ID NO.133所示的VH和SEQ ID NO.134所示的VL，其第二抗原结合结构域scfv具有如SEQ ID NO.139所示的氨基酸序列。在一些实施方案中，所述结合CD3和EGFR的异源二聚体融合蛋白具有如SEQ ID NO.96所示氨基酸序列的第一多肽链和SEQ ID NO.98所示氨基酸序列的第二多肽链。在一些实施方案中，所述结合CD3和CD19的异源二聚体融合蛋白的第一抗原结合结构域Fv具有如SEQ ID NO.137所示的VH和SEQ ID NO.138所示的VL，其第二抗原结合结构域scfv具有如SEQ ID NO.139所示的氨基酸序列。在一些实施方案中，所述结合CD3和EGFR的异源二聚体融合蛋白具有包含如SEQ ID NO.92所示氨基酸序列的第一多肽链和包含如SEQ ID NO.94所示氨基酸序列的第二多肽链。
一方面，本发明提供了基于免疫球蛋白片段的异源二聚体融合蛋白，所述异源二聚体包括：
a)第一多肽链，其按从N端到C端的顺序依次包含：Fc、(L1)n、CL、L2、VH，
b)第二多肽链，其按从N端到C端的顺序依次包含：Fc、(L3)n、CH1、L2、VL，
其中，n为0或1，L1、L2、L3和L4为连接子，VH和VL形成第一抗原结合结构域Fv。
在一些实施方案中，所述异源二聚体融合蛋白进一步包括第二抗原结合结构域。
在一些实施方案中，所述第二抗原结合结构域为生理活性肽。
在一些实施方案中，所述生理活性肽直接或通过连接子与所述第一或第二多肽链的N端融合(图1Q和图1R)。
在一些实施方案中，所述生理活性肽直接或通过连接子与所述第一或第二多肽链的C端融合(图1V和图1W)。
在一些实施方案中，所述第二抗原结合结构域为Fab2。
在一些实施方案中，所述第二抗原结合结构域Fab2的重链直接或通过连接子与所述第一多肽链的N端融合，所述Fab2的轻链直接或通过连接子与所述第二多肽链的N端融合。
在一些实施方案中，所述第二抗原结合结构域Fab2的重链直接或通过连接子与所述第二多肽链的N端融合，所述Fab2的轻链直接或通过连接子与所述第二多肽链的N端融合。
在一些实施方案中，所述异源二聚体融合蛋白的第二抗原结合结构域为Fv。
在一些实施方案中，所述第二抗原结合结构域Fv的重链可变区直接或通过连接子与所述第一多肽链的N端融合，所述第二抗原结合结构域Fv的轻链可变区直接或通过连接子与所述第二多肽链的N端融合。
在一些实施方案中，所述第二抗原结合结构域Fv的重链可变区直接或通过连接子与所述第二多肽链的N端融合，所述第二抗原结合结构域Fv的轻链可变区直接或通过连接子与所述第一多肽链的N端融合。
在一些实施方案中，所述第二抗原结合结构域为纳米抗体。
在一些实施方案中，所述第二抗原结合结构域纳米抗体直接或通过连接子与所述第一多肽链的N端融合。在一些实施方案中，所述第二抗原结合结构域纳米抗体直接或通过连接子与所述第二多肽链的N端融合。
在一些实施方案中，所述第二抗原结合结构域为scfv。在一些实施方案中，所述第二抗原结合结构域scfv直接或通过连接子与所述第一或第二多肽链的C端融合(图1V和图1W)。
在一些实施方案中，所述第二抗原结合结构域scfv直接或通过连接子与所述第二多肽链的N端融合(图1Q)。
在一些实施方案中，所述第二抗原结合结构域scfv直接或通过连接子与第一多肽链的N端融合(图1R)。
在一些实施方案中，所述异源二聚体的第一抗原结合结构域结合CD3。在一些实施方案中，所述结合CD3的第一抗原结合结构域Fv具有如SEQ ID NO.133所示的VH和SEQ ID NO.134所示的VL。在一些实施方案中，所述第二抗原结合结构域scfv结合CD19。在一些实施方案中，所述结合CD19的第二抗原结合结构域scfv具有如SEQ ID NO.139所示的氨基酸序列。在一些实施方案中，所述异源二聚体融合蛋白结合CD3和CD19。在一些 实施方案中，所述结合CD3和CD19的异源二聚体融合蛋白其第一抗原结合结构域Fv具有如SEQ ID NO.133所示的VH和SEQ ID NO.134所示的VL，其第二抗原结合结构域scfv具有如SEQ ID NO.139所示的氨基酸序列。在一些实施方案中，所述结合CD3和CD19的异源二聚体融合蛋白具有包含如SEQ ID NO.100所示氨基酸序列的第一多肽链和包含如SEQ ID NO.102所示氨基酸序列的第二多肽链。
本发明将第一抗原结合结构域Fab的重链和轻链分别融合(直接或通过连接子)在两条Fc的N端，在此基础上，进一步将第二抗原结合结构域scfv、Fab2、Fv、纳米抗体或生理活性肽与Fab的N端或Fc的C端融合，有效地解决了两个抗原结合结构域之间的重链与重链、轻链与轻链、重链与轻链的错配问题。
本发明提供的异源二聚体融合蛋白包含至少一个连接子。在一些实施例中，所述连接子任选自以下的连接子：GGGGSGGGGSGGGGS、SGGGGSGGGGSGGGGS、GGSGGSGGGGSGGGG、GGSGGSGGGGSGGGGS、GGSGAKLAALKAKLAALKGGGGS、GGGGSELAALEAELAALEAGGSG、APATSLQSGQLGFQCGELCSASA、ASTKGP、TVAAPSVFIFPP、PNLLGGP、GGGGS、GGGEAAAKEAAAKEAAAKAGG。在一些实施方案中，所述连接子相同。在一些实施方案中，所述连接子不同。
一方面，本发明还提供编码本发明所述的异源二聚体融合蛋白的多核苷酸。
一方面、本发明还涉及一种表达载体，其包含本发明的多核苷酸。
一方面，本发明还涉及一种宿主细胞，其包含本发明的表达载体。
一方面，本发明还涉及一种药物组合物，其包含本发明的异源二聚体融合蛋白。
另一方面，本发明还涉及治疗有需要的受试者的癌症和自身免疫性疾病的方法。在一些实施例中，所述方法包括向受试者施用有效量的本文提供的异源二聚体融合蛋白，或者本文提供的异源二聚体融合蛋白和药学上可接受的载体的药物组合物。
在一些实施例中，本发明提供了用于治疗有需要的受试者的B细胞血癌的方法，所述方法包括向受试者施用本发明所述的异源二聚体融合蛋白，其中所述异源二聚体融合蛋白能够结合CD3和CD19。在一些实施例中，所述异源二聚体融合蛋白包含选自：SEQ ID No:2和SEQ ID No:4；SEQ ID No:6和SEQ ID No:8；SEQ ID No:20和SEQ ID No:22；SEQ ID No:24和SEQ ID No:26；SEQ ID No:92和SEQ ID No:94；SEQ ID No:96和SEQ ID No:98；SEQ ID No:100和SEQ ID No:102的氨基酸序列。在一些实施例中，所述B细胞血癌选自：霍奇金氏淋巴瘤(Hodgkin's lymphoma)、非霍奇金氏淋巴瘤(NHL)、前体B细胞淋巴母细胞性白血病/淋巴瘤、成熟B细胞赘瘤、B细胞慢性淋巴细胞白血病/小淋巴细 胞性淋巴瘤、B细胞幼淋巴细胞白血病、淋巴浆细胞淋巴瘤、套细胞淋巴瘤、滤泡性淋巴瘤、皮肤滤泡中心淋巴瘤、边缘区B细胞淋巴瘤、毛细胞白血病、弥漫性大B细胞淋巴瘤、伯基特氏淋巴瘤(Burkitt's lymphoma)、浆细胞瘤、浆细胞性骨髓瘤、移植后淋巴增生病症、移瓦尔登斯特伦氏巨球蛋白血症(Waldenstrom's macroglobulinemia)及间变性大细胞淋巴瘤。
在一些实施例中，本发明提供了用于治疗有需要的受试者的癌症的方法，在一些实施例中，所述癌症选自以下组成的组：黑素瘤(例如，转移性恶性黑素瘤)、肾癌(例如，透明细胞癌)、前列腺癌(例如，激素难治性前列腺腺癌)、胰腺癌、乳癌、结肠癌、肺癌(例如，非小细胞肺癌)、食道癌、头颈鳞状细胞癌、肝癌、卵巢癌、宫颈癌、甲状腺癌、成胶质细胞瘤、神经胶质瘤、白血病、淋巴瘤及其它赘生性恶性疾病。
在一些实施例中，本发明提供了用于治疗有需要的受试者体内EGFR高表达的肺癌、结肠癌等方法，所述方法包括向受试者施用本发明所述的异源二聚体融合蛋白，其中所述异源二聚体融合蛋白能够结合CD3和EGFR。在一些实施例中，所述异源二聚体融合蛋白包含如下氨基酸序列：SEQ ID No:10和SEQ ID No:12；SEQ ID No:14和SEQ ID No:16；SEQ ID No:18和SEQ ID No:12；SEQ ID No:24和SEQ ID No:28；SEQ ID No:30和SEQ ID No:32；SEQ ID No:30和SEQ ID No:34；SEQ ID No:36和SEQ ID No:38；SEQ ID No:44和SEQ ID No:46；SEQ ID No:48和SEQ ID No:50；SEQ ID No:44和SEQ ID No:52；SEQ ID No:54和SEQ ID No:56、SEQ ID No:30和SEQ ID No:58；SEQ ID No:60和SEQ ID No:62；SEQ ID No:64和SEQ ID No:66；SEQ ID No:68和SEQ ID No:70；SEQ ID No:72和SEQ ID No:74；SEQ ID No:76和SEQ ID No:78；SEQ ID No:80和SEQ ID No:82；SEQ ID No:84和SEQ ID No:86、SEQ ID No:88和SEQ ID No:90；SEQ ID No:104和SEQ ID No:106；SEQ ID No:108和SEQ ID No:110；SEQ ID No:112和SEQ ID No:114；SEQ ID No:116和SEQ ID No:118。
在一些实施例中，本发明提供了用于治疗有需要的受试者的多发性骨髓瘤的方法，所述方法包括向受试者施用本发明所述的异源二聚体融合蛋白，其中所述异源二聚体融合蛋白能够结合CD3和BCMA。在一些实施例中，所述异源二聚体融合蛋白具有SEQ ID No:30和SEQ ID No:40氨基酸序列。
在一些实施例中，本发明提供了用于治疗有需要的受试者的急性髓系白血病的方法，所述方法包括向受试者施用本发明所述的异源二聚体融合蛋白，其中所述异源二聚 体融合蛋白能够结合CD3和CLL-1。在一些实施例中，所述异源二聚体融合蛋白具有SEQ ID No:30和SEQ ID No:42所述的氨基酸序列。
在一些实施例中，本发明提供了用于治疗有需要的受试者病毒感染的方法，所述方法包括向受试者施用本发明所述的异源二聚体融合蛋白，其中所述异源二聚体融合蛋白能够结合CD3和MICA。在一些实施例中，所述异源二聚体融合蛋白具有SEQ ID No:122和SEQ ID No:124所述的氨基酸序列。
附图说明：
图1A为异源二聚体融合蛋白的基本骨架示意图，其中FabH所在链为第一多肽链，FabL所在链为第二多肽链。图1B-图1F为第一抗原结合结构域为Fab，第二抗原结合结构域为scfv或活性肽的异源二聚体结构示意图。其中，第二抗原结合结构域融合在第一多肽链N端的异源二聚体融合蛋白如图1B所示；第二抗原结合结构域融合在第二多肽链N端的异源二聚体融合蛋白如图1C所示；第二抗原结合结构域融合在第一多肽链C端的异源二聚体融合蛋白如图1D所示；第二抗原结合结构域融合在第二多肽链C端的异源二聚体融合蛋白如图1E所示；第二抗原结合结构域分别融合在第一多肽链和第二多肽链C端的异源二聚体融合蛋白如图1F所示。图1G-图1J为第一抗原结合结构域为Fab，第二抗原结合结构域为Fv的异源二聚体融合蛋白结构示意图。其中，第二抗原结合结构域Fv的VH和VL分别融合至第一多肽链和第二多肽链C端的异源二聚体融合蛋白如图1G所示；第二抗原结合结构域Fv的VL和VH分别融合至第一多肽链和第二多肽链C端的异源二聚体融合蛋白如图1H所示；第二抗原结合结构域Fv的VH和VL分别融合至第一多肽链和第二多肽链N端的异源二聚体融合蛋白如图1I所示；第二抗原结合结构域Fv的VL和VH分别融合至第一多肽链和第二多肽链N端的异源二聚体融合蛋白如图1J所示。
图1K-图1N为第一抗原结合结构域为Fab，第二抗原结合结构域为Fab2的异源二聚体融合蛋白结构示意图。其中，第二抗原结合结构域Fab2的Fab2H和Fab2L分别融合至第一多肽链和第二多肽链C端的异源二聚体融合蛋白如图1K所示；第二抗原结合结构域Fab2的Fab2L和Fab2H分别融合至第一多肽链和第二多肽链C端的异源二聚体融合蛋白如图1L所示；第二抗原结合结构域Fab2的Fab2H和Fab2L分别融合至第一多肽链和第二多肽链N端的异源二聚体融合蛋白如图1M所示；第二抗原结合结构域Fab2的Fab2L和Fab2H分别融合至第一多肽链和第二多肽链N端的异源二聚体融合蛋白如图1N所示。图1O为第一抗 原结合结构域和第二抗原结合结构域中Fab的VH和VL在两条多肽链上互相交换位置的示意图。图1P-图1X为第一抗原结合结构域为Fv，第二抗原结合结构域为scfv或活性肽的异源二聚体融合蛋白结构示意图。其中，第二抗原结合结构域的scfv或生理活性肽与第一多肽的N端融合的异源二聚体融合蛋白如图1P和图1Q所示；第二抗原结合结构域的scfv或生理活性肽与第二多肽的N端融合的异源二聚体融合蛋白如图1R和图1S所示；第二抗原结合结构域的scfv或生理活性肽与第一多肽的C端融合的异源二聚体融合蛋白如图1T和图1U所示；第二抗原结合结构域的scfv或生理活性肽与第二多肽的C端融合的异源二聚体融合蛋白如图1V和图1W所示；图1X为生理活性肽分别与第一多肽和第二多肽链的N端融合的异源二聚体融合蛋白。
图2展示了示例性异源二聚体融合蛋白用ProteinA或CH1 resin纯化后的SDS-PAGE凝胶图像。其中，“M”表示蛋白marker；“-”表示不加beta-巯基乙醇上样；“+”表示加beta-巯基乙醇后上样；1:IgFD-24；2:IgFD-11；3:IgFD-25；4:IgFD-26；5:IgFD-31；6:IgFD-27；7:IgFD-30；7(CH1):IgFD-30(CH1 resin纯化)；8:IgFD-29；8(CH1):IgFD-29(CH1 resin纯化)；9:IgFD-28；9(CH1):IgFD-28(CH1 resin纯化)。
图3-1展示了示例性异源二聚体融合蛋白凝胶排阻层析色谱图。图3-2为示例性异源二聚体抗体离子交换层析色谱图。
图4显示了使用流式细胞术检测不同浓度anti-CD3/CD19异源二聚体融合蛋白IgFD-6、IgFD-7与NALM-6细胞表面的结合。
图5显示了使用流式细胞术检测anti-CD3/anti-CD19异源二聚体融合蛋白IgFD-6、IgFD-7促进PMBC对Nalm-6细胞的杀伤作用。
图6显示了ELISA检测不同anti-CD3/anti-EGFR异源二聚体融合蛋白与人EGFR抗原的结合强度。
图7显示了使用流式细胞术检测不同anti-CD3/anti-EGFR异源二聚体融合蛋白与F98-EGFR细胞表面的结合。ProA表示通过proteinA Resin纯化的融合蛋白；CH1表示通过CH1 resin纯化的融合蛋白。
图8显示了使用流式细胞术检测anti-CD3/anti-EGFR异源二聚体融合蛋白IgFD-8、IgFD-18和IgFD-19与PBMC-T细胞的结合。
图9显示了使用流式细胞术检测不同anti-CD3/anti-EGFR异源二聚体融合蛋白与Jurkat T细胞表面的结合，其中ProA表示通过proteinA Resin纯化的融合蛋白；CH1表示通过CH1 resin纯化的融合蛋白。
图10显示了使用LDH方法检测不同anti-CD3/anti-EGFR异源二聚体融合蛋白促进PBMC对F98-EGFR细胞的杀伤作用。Size表示凝胶层析后的蛋白；Monos表示离子交换层析后的蛋白。
图11显示了使用LDH assay(a)和流式细胞术(b)检测anti-CD3/anti-BCMA异源二聚体融合蛋白IgFD-22促进PBMC对MM1.R细胞的杀伤作用。
图12显示了用ELISA检测靶向CD3和MICA的异源二聚体融合蛋白IgFD-37与人MICA抗原的结合强度，其中IgFD-36为对照。
图13显示了使用流式细胞数检测不同浓度的靶向CD3和MICA的异源二聚体融合蛋白IgFD37与细胞表面MICA的结合强度；(A)PANC-1细胞，(B)为BXPC-3细胞，(C)为K562细胞。
图14显示了使用LDH assay检测靶向CD3和MICA的异源二聚体融合蛋白IgFD-37促进PBMC对K562细胞(A)和对PANC-1细胞(B)的杀伤作用。
图15显示了使用流式细胞术检测anti-CD3/anti-CLL-1异源二聚体融合蛋白IgFD-23促进PBMC对HL-60细胞的杀伤作用。
图16为IgFD-25和IgFD-33在大鼠中腹腔给药后的药代动力学曲线。
图17为IgFD-33在A431肺癌小鼠模型中的肿瘤生长抑制曲线。
本发明详述
为了能更全面地了解本申请，下文阐述若干定义。此类定义旨在涵盖语法等效物。所有在本文中提及的专利和公开文献的内容，包括在这些专利和公开中披露的所有序列，明确地通过提述并入本文。
如本文所用，“异源二聚体融合蛋白”意指由两条各自包含Fc的不同多肽链组成的抗体或基于抗体的融合蛋白，其中一条多肽链的Fc与另一条多肽链的Fc形成Fc二聚体，两条多肽链形成至少一个抗原结合结构域。
如本文所用，“抗原结合结构域”意指抗原结合分子中特异性结合抗原决定簇的部分。更具体地，术语“抗原结合结构域”是指抗体的一部分，所述部分包含与抗原的一部分或全部特异性结合并互补的区域。在抗原很大的情况下，抗原结合分子可以仅结合抗原的特定部分，该部分称为表位。抗原结合结构域可以由例如一个或多个可变结构域(也称为可变区)提供。抗原结合结构域可源自任何动物物种，如啮齿类(例如兔、大鼠或 仓鼠)和人。抗原结合结构域的非限制性实例包括：单链抗体、Fab、F(ab’)2、Fd片段、Fv、单链Fv(scFv)分子、dAb片段和由模拟抗体高变区的氨基酸残基组成的最小识别单元。在本发明的一些具体实施例中，抗原结合结构域为Fab。在一些具体的实施例中，抗原结合结构域为Fv。在一些具体的实施例中，抗原结合结构域为scfv。在一些具体的实施例中，抗原结合结构域为生理活性肽。
如本文所用，术语“抗原”与“抗原决定簇”和“表位”同义，意指多肽大分子上与抗原结合结构域结合，从而形成抗原结合结构域—抗原复合物的位点(例如一段连续的氨基酸或由非连续氨基酸的不同区域组成的构象构型)。“抗原”可以在例如肿瘤细胞表面上、病毒感染细胞的表面上、其他患病细胞的表面上、免疫细胞的表面上、血清中的游离物和/或在胞外基质(ECM)中找到。除非另有说明，否则本文中称为抗原的蛋白质可以是来自任何脊椎动物来源，包括哺乳动物如灵长类(例如人)和啮齿类(例如小鼠和大鼠)等步入动物。当提及本文中的特定蛋白质时，该术语涵盖“全长”、未加工的蛋白质，以及由细胞内加工而产生的任何形式的蛋白质。该术语还涵盖天然存在的蛋白质变体，例如剪接变体或等位基因变体。在一些实施例中，抗原是人蛋白。可用作抗原的示例性人蛋白包括但不限于：BCMA、CLL-1、EpCAM、CD19、CCR5、EGFR、HER2、HER3、HER4、EGF4、PSMA、CEA、MUC-1(Mucin)、MUC-2、MUC-3、MUC-4、MUC-5 AC、MUC-5 B、MUC7、βhCG、Lewis-Y、CD20、CD33、CD30、CD16A、B7-H3、CD123、gpA33、P-Cadherin、GPC3、CLEC12A、CD32B、TROP-2、ganglioside GD3、9-O-Acetyl-GD3,GM2,Globo H、fucosyl GM1、Poly SA、GD2、Carboanhydrase IX(MN/CA IX)、CD44v6、Sonic Hedgehog(Shh)、Wue-1、Plasma Cell Antigen、(membrane-bound)IgE、Melanoma Chondroitin Sulfate Proteoglycan(MCSP)、CCR8、TNF-alpha precursor、STEAP、mesothelin、A33、Prostate Stem Cell Antigen(PSCA)、Ly-6、desmoglein 4、E-cadherin neoepitope、Fetal Acetylcholine Receptor、CD25、CA19-9marker、CA-125marker and Muellerian Inhibitory Substance(MIS)Receptor type II、sTn(sialylated Tn antigen；TAG-72)、FAP(fibroblast activation antigen)、endosialin、EGFRvIII、LG、SAS、CD63、2B4(CD244)、α4β1整联蛋白、β2整联蛋白(例如CD11a-CD18、CD11b-CD18、CD11c-CD18)、CD2(LFA2、OX34)、CD16、CD27(TNFRSF7)、CD38、CD96、CD100、CD160、CD137、CEACAM1(CD66)、CRTAM、CS1(CD319)、DNAM-1(CD226)、GITR(TNFRSF18)、KIR的活化形式(例如KIR2DS1、KIR2DS4、KIR-S)、NKG2C、NKG2D、NKG2E、天然细胞毒性受体(例如NKp30、NKp44、NKp46、NKp80)、NTB-A和PEN-5、CD2(LFA2、OX34)、CD3、CD5、CD27(TNFRSF7)、CD28、CD30(TNFRSF8)、CD40L、CD84(SLAMF5)、 CD137(4-1BB)、CD226、CD229(Ly9、SLAMF3)、CD244(2B4、SLAMF4)、CD319(CRACC、BLAME)、CD352(Ly108、NTBA、SLAMF6)、CRTAM(CD355)、DR3(TNFRSF25)、GITR(CD357)、HVEM(CD270)、ICOS、LIGHT、LTβR(TNFRSF3)、OX40(CD134)、SLAM(CD150、SLAMF1)、TCRα、TCRβ、TCRδγ和TIM1(HAVCR、KIM1)等。美国专利号7235641提供的肿瘤细胞表面抗原实例、在Miller，Hematology,2013,2013(1):247-253；Mentlik等人,Frontiers in Immunology,2013,4:481(1-12)；Stein等人,Antibodies,2012,1:88-123；Pegram等人,Immunology and Cell Biology,2011,89:216-224；和Vivier等人,Nature Immunology,2008,9:503-510中提供的NK细胞表面抗原更多信息、以及在Stein等人，Antibodies,2012,1:88-123；Chen和Flies,Nature Reviews Immunology,2013,13:227-242；和Pardoll,NatureReviews Cancer,2012,12:252-264中提供的T细胞表面抗原的更多信息，上述文献所述全部内容通过引用并入本文。
如本文所用，“Fc”用于定义免疫球蛋白重链中至少含有恒定区的一部分的C端结构域。意指包含抗体的恒定区(不包含第一恒定区免疫球蛋白域)且在一些情况下包含铰链的多肽[Jones等.,Nature 321:522-525(1986)；Riechmann等.,Nature 332:323-329(1988)；和Presta,Curr.Op.Struct.BIOL.2:529-596(1992)]。因此，Fc是指人IgA、IgD和IgG的最后两个恒定区免疫球蛋白域、IgE和IgM的最后三个恒定区免疫球蛋白域，以及这些域的柔性铰链N末端。对于IgA和IgM来说，Fc可以包括J链。对于IgG来说，Fc域包含免疫球蛋白域Cγ2和Cγ3(Cγ2和Cγ3)以及位于Cγ1(Cγ1)与Cγ2(Cγ2)之间的较低铰链区。
如本文所用，“Fc变体”意指由于至少一个氨基酸修饰，如取代、缺失或插入而不同于野生Fc序列，但仍保留与相应的Fc单链配对形成Fc二聚体的能力的Fc序列。在一些实施方案中，相对于亲本Fc区活性，“Fc变体”的氨基酸修饰改变了效应功能活性。举例来说，在一个实施方案中，变体Fc区可以具有改变的(即，增加的或降低的)抗体依赖性细胞毒性(ADCC)、补体介导的细胞毒性(CDC)、吞噬作用、调理作用或细胞结合。在另外的实施方案中，相对于野生Fc，Fc氨基酸修饰可以改变(即，增加或降低)变体Fc区对FcγR的亲和力。举例来说，变体Fc可以改变对FcγRI、FcγRII、FcγRIII的亲和力。在一些实施方案中，变体Fc具有E233P、L234V、del235L、G236A、A327G、A330S、A331S、E356D、M358L氨基酸修饰。在一些实施方案中，“变体Fc”为去糖基化修饰的Fc，也即含有N297A氨基酸修饰的Fc。在另一些实施方案中，变体Fc进一步包含S354C,T366W氨基酸修饰，或者S354C、T366W、Y349C、T366S、L368A、Y407V氨基酸修饰。
“野生型”或“WT”在本文中意指在自然界中见到的氨基酸序列或核苷酸序列，包括等位基因变异。WT蛋白具有未被有意修饰的氨基酸序列或核苷酸序列。
本文中使用的“变体”意指由于至少一个氨基酸修饰而不同于亲本多肽序列的多肽序列。变体多肽可指多肽自身、包含该多肽的组合物或编码其的氨基序列。优选地，与亲本多肽相比该变体多肽具有至少一个氨基酸修饰，如与亲本多肽相比约1到约10个氨基酸修饰，以及优选约1到约5个氨基酸修饰。本文中该变体多肽序列与亲本多肽序列优选具有至少约80％的同源性，最优选至少约90％的同源性，更优选至少约95％的同源性。
“氨基酸修饰”在本文中意指多肽序列中的氨基酸取代、插入和/或缺失。为清楚起见，除非另外说明，否则氨基酸修饰通常是针对DNA编码的氨基酸，例如在DNA和RNA中具有密码子的20种氨基酸。
“氨基酸取代”或“取代”在本文中意指亲本多肽序列中特定位置处的氨基酸被不同氨基酸置换。确切地说，在一些实施例中，取代是针对特定位置处非天然存在的氨基酸，这些氨基酸不是生物体内天然存在的或是任何生物体中的。举例来说，取代E272Y是指272位的谷氨酸被酪氨酸置换的变异体多肽，在此情形中是Fc变体。为清楚起见，蛋白质被工程改造成改变核酸编码序列但不改变起始氨基酸(例如CGG(编码精氨酸)变为CGA(仍编码精氨酸)，用以增加宿主生物体表达水平)不是“氨基酸取代”；也就是说，尽管产生了编码同一蛋白质的新基因，但如果该蛋白质在其起始的特定位置处具有相同氨基酸，就不是氨基酸取代。优选地，本发明的变体Fc相对于亲本或天然Fc具有保守取代，即以具有性质相同或相近的氨基酸侧链基团的氨基酸残基进行的取代。优选地，本发明的变体Fc相对于亲本或天然Fc具有不超过40，30，20，10，或5个保守取代，只要其仍保留与相应的Fc单链配对形成Fc二聚体的能力即可。
“效应功能”指可归因于抗体的Fc区的那些生物学活性，其随抗体同种型而不同。抗体效应功能的实例包括：C1q结合和依赖补体的细胞毒性(CDC)；Fc受体结合；依赖抗体的细胞介导的细胞毒性(ADCC)；细胞表面受体(例如B细胞受体)的下调；及B细胞活化。
“抗体依赖性细胞毒性(ADCC)”指细胞介导的反应，其中表达FcR的非特异性细胞毒性细胞(例如天然杀伤(NK)细胞、中性粒细胞和巨噬细胞)识别靶细胞上结合的抗体，随后引起靶细胞的裂解。用于介导ADCC的主要细胞(NK细胞)仅表达FcγRIII，而单核细胞表达FcγRI、FcγRII和FcγRIII。造血细胞上的FcR表达总结在Ravetch和Kinet，Annu.Rev.Immunol9(1991)457-492的464页上表3中。
“依赖补体的细胞毒性(CDC)”指诱导细胞死亡的机制，其中结合靶标的抗体的Fc效应分子结构域(一个或多个)活化一系列酶促反应，造成孔洞在靶细胞膜中形成。典型地，抗原-抗体复合物，诸如抗体包被的靶细胞上的抗原-抗体复合物，结合并活化 补体成分C1q，其转而活化补体级联，从而导致靶细胞死亡。补体的活化还可以导致补体成分在靶细胞表面上的沉积，其通过结合白细胞上的补体受体(例如CR3)利于ADCC。
如本文所用，“Fab重链”或“FabH”意指包含VH和CH1的免疫球蛋白域的多肽；“Fab轻链”或“FabL”意指包含VL和CL的免疫球蛋白域的多肽，也即免疫球蛋白轻链。在本发明的一些实施方案中，Fab中的CH1和CL可以交换位置，也即FabH包括VH和CL，FabL包括VL-CH1。
如本文所用，“Fab”意指包含VH、CH1、VL和CL免疫球蛋白结构域的多肽。Fab可以指孤立的结构域，或在全长抗体、抗体片段或Fab融合蛋白的情况下的此区。如本领域中的技术人员将了解，Fab通常由Fab重链和Fab轻链等两条链构成。在本发明的一些实施方案中，Fab中的CH1和CL可以互相交换位置，因此，CH1和CL互相交换位置的Fab也包括在本发明中。
如本文所用，“Fv”意指包含一个VL和一个VH的非融合的二聚体。“单链Fv”或“scfv”意指包含单一抗体的VL和VH结构域的多肽，且VL和VH在同一多肽上。
本文中使用的“连接子”包含了提供柔性/刚性的任何合理序列的一个或多个氨基酸。优选地，所述连接子选自但不限于由以下组成的组：GGGGSGGGGSGGGGS、SGGGGSGGGGSGGGGS、GGSGGSGGGGSGGGG、GGSGGSGGGGSGGGGS、GGSGAKLAALKAKLAALKGGGGS、GGGGSELAALEAELAALEAGGSG、APATSLQSGQLGFQCGELCSASA、ASTKGP、TVAAPSVFIFPP、PNLLGGP、GGGGS、GGGEAAAKEAAAKEAAAKAGG、G、GS、SG、GGS、GSG、SGG、GGG、GGGS、SGGG、GGGGSGS、GGGGSGS、GGGGSGGS、GGGGSGGGGS、AKTTPKLEEGEFSEAR、AKTTPKLEEGEFSEARV、AKTTPKLGG、SAKTTPKLGG、AKTTPKLEEGEFSEARV、SAKTTP、SAKTTPKLGG、RADAAP、RADAAPTVS、RADAAAAGGPGS、RADAAAA(G4S)4、SAKTTP、SAKTTPKLGG、SAKTTPKLEEGEFSEARV、ADAAP、ADAAPTVSIFPP、TVAAP、QPKAAP、QPKAAPSVTLFPP、AKTTPP、AKTTPPSVTPLAP、AKTTAP、AKTTAPSVYPLAP、ASTKGPSVFPLAP、GENKVEYAPALMALS、GPAKELTPLKEAKVS及GHEAAAVMQVQYPAS。所述连接子也可以是在体内可裂解的肽连接子、蛋白酶(如MMP)敏感性连接子、如先前所描述的可以通过还原进行裂解的基于二硫键的连接子等(FusionProtein Technologies for Biopharmaceuticals:Applications and Challenges,由Stefan R.Schmidt编辑)或本领域中已知的任何可裂解的连接子。
本发明的术语“生理活性肽”不仅包括与抗原结合结构域结合后呈现出体内生理学功能的蛋白，而且还包括仅仅参与抗原结合，但没有生理功能的多肽。可应用于本发明 的生理活性肽举例来说有受体、配体蛋白质、激素、细胞因子、白介素、白介素结合蛋白、酶、生长因子、转录调控因子、凝血因子、疫苗、结构蛋白质、细胞表面抗原、受体拮抗剂及它们的衍生物等。
实施例
尽管处于清楚理解的目的，下面通过举例说明和实施例相当详细地描述了前述发明，但是根据本发明的教义，本领域的普通技术人员将显而易见的是，在不背离所附权利要求的精神和范围情况下，可以对本发明进行某些改变和修改。以下实施例仅以说明方式提供，而并不起限制作用。本领域的技术人员将易于认识到多种非关键性参数，这些参数可以改变或修改得到基本上类似的结果。
对于在本发明中讨论的所有恒定区位置，编号规则依照如Kabat中的EU索引(Kabat等人，1991，Sequences of proteins of Immunological Interest,5 th Ed.,United Stetes Public Health Servic,National Institutes of Health,Bethesda,通过引用整体地并入)。抗体领域中的技术人员会理解由在免疫球蛋白序列的特定区域中的非顺序性编号构成的这种惯例能够标准化地参照免疫球蛋白家族中的保守位置。因此，如有EU索引定义的任何给定的免疫球蛋白的位置不一定对应于其顺序性序列。
实施例1异源二聚体融合蛋白真核表达载体的构建
1.1基因合成
合成anti-CD19 VH、anti-CD19 VL、anti-CD3 VH-1、anti-CD3 VL-1、anti-CD3 VH-3、anti-CD3 VL-3、anti-EGFR VH、anti-EGFR VL、活性肽EGF4、活性肽NKG2D、anti-CD3 VH-4、anti-CD3 VL-4、anti-BCMA VH、anti-BCMA VL、anti-CLL-1 VH、anti-CLL-1 VL、抗体轻链CL及抗体重链CH1基因(IDT公司合成)。
1.2 scfv的构建
PCR分别扩增上述基因片段，overlap PCR将扩增获得的anti-CD3 VH-1与anti-CD3 VL-1、anti-EGFR VH与anti-EGFR VL、anti-CD3 VL-3与anti-CD3 VH-3、anti-CD19 VH与anti-CD19 VL、anti-BCMA VH与anti-BCMA VL、anti-CLL-1 VH与anti-CLL-1 VL分别通过linker连接获得scfv1(anti-CD3 scfv-1)、scfv2(anti-EGFR scfv)、scfv3(anti-CD3 scfv-3)、scfv5(anti-CD19 scfv)、scfv6(anti-BCMA scfv)、scfv7(anti-CLL-1 scfv)片段，测序验证。
1.3 Fab片段的构建
PCR分别扩增1.1合成的基因片段，将扩增获得的PCR产物anti-CD3 VL-1、anti-EGFR VL、anti-CD3 VL-3和anti-CD3 VL-4分别与CL`连接获得anti-CD3 VL-1-CL(Fab1L)、anti-EGFR VL-CL(Fab2L)、anti-CD3 VL-3-CL(Fab3L)、anti-CD3 VL-4-CL(Fab4L)，测序验证。
PCR分别扩增1.1合成的基因片段，通过overlap PCR将扩增获得的anti-CD3 VH-1、anti-EGFR VH、anti-CD3 VH-3、anti-CD3 VH-4分别与CH1连接获得anti-CD3 VH-1-CH1(Fab1H)、anti-EGFR VH-CH1(Fab2H)、anti-CD3 VH-3-CH1(Fab3H)、anti-CD3 VH-4-CH1(Fab4H)，测序验证。
将上述测序验证的FabH和FabL，进一步通过in-frame连接分别克隆至pFuse-hIgG1-Fc2载体上(InvivoGen,CA)，载体上的Fc片段具有如下突变：N297A或E233P,L234V,L235A,delG236,A327G,A330S,A331S。在某些实施例中，Fc进一步包含S354C,T366W突变或Y349C,T366S,L368A,Y407V突变。根据需要，将或scfv或活性肽或VH或VL或Fab通过连接子与上述载体连接，所有构建的序列均测序验证。每个构建体的核苷酸及氨基酸序列见序列表：Seq No.1-Seq.No.142。
表1构建体及序列编号
Figure PCTCN2019129591-appb-000004
Figure PCTCN2019129591-appb-000005
Figure PCTCN2019129591-appb-000006
Figure PCTCN2019129591-appb-000007
实施例2示例性异源二聚体融合蛋白的表达、纯化和凝胶排阻层析
将实施例1构建好的异源二聚体融合蛋白真核表达载体的两条链瞬时共转染FreeStyle HEK293细胞(ThermoFisher)：将28ml FreeStyle HEK 293(3×10 7细胞/ml)接 种至125ml细胞培养瓶，质粒用1ml Opti-MEM(Invitrogen)稀释后加至1ml含60μl 293 Fectin(Invitrogen,Inc)的Opti-MEM中，室温静置30min，将质粒-293fectin mixture加至细胞培养液中125rpm，37℃，5％CO2培养。分别于转染后48h和96h收集细胞培养上清，并根据制造商的说明书，使用CH1 Select resin(Thermo Fisher Scientific,IL)、ProteinG和/或Protein A Resin(Genscript)纯化异源二聚体融合蛋白。通过SDS-PAGE，在还原条件和非还原条件下分析纯化的异源二聚体融合蛋白的组成和纯度。并通过A280和BCA(Pierce，Rockford，IL)测定其浓度。
将获得的CH1 resin、ProteinG和/或Protein A resin纯化后的异源二聚体融合蛋白用GE的AKTA chromatography过柱分析，所用的层析柱为：Superdex 200 Increase 10/300GL凝胶排阻层析柱和/或Mono S 5/50GL离子交换层析柱。凝胶排阻层析所用的溶液为PBS缓冲液(0.010M phosphate buffer,0.0027M KCl,0.14M NaCl,pH7.4)，离子交换层析所用的溶液为Buffer A:20mM NaOAc,pH＝5和Buffer B:20mM NaOAc,1M NaCl,pH＝5。从图2的SDS胶图、图3的色谱图及表2的蛋白表达结果看，不同的异源二聚体融合蛋白表达具有相当的纯度，而且其表达水平与常规mAb的表达水平相当，表明这些异源二聚体抗体可以在哺乳动物细胞中高效地表达。
表2蛋白表达分析
蛋白 表达水平(mg/L)
IgFD-6 10.7
IgFD-21 3.75
IgFD-22 4.58
IgFD-23 8.3
IgFD-9 30
IgFD-10 25.3
IgFD-33 18
实施例3异源二聚体抗体质谱分析
将凝胶排阻层析纯化的样品与PNGase F(NEB)37℃孵育8h后，加10mM DTT处理后，将样品注射进HPLC-Q-TOF-MS(Agilent,USA)的300SB-C8,2.1x 50mm柱，进行MS分析。结果见表3，不同异源二聚体融合蛋白的两条链的分子量理论预测值与利用质谱检测得到的分子量基本一致。
表3蛋白质谱分析
Figure PCTCN2019129591-appb-000008
Figure PCTCN2019129591-appb-000009
实施例4体外活性研究
4.1 anti-CD3/CD19异源二聚体融合蛋白体外活性检测
(1)流式细胞术检测anti-CD3/CD19异源二聚体融合蛋白与NALM-6细胞的结合
培养NALM6细胞(RPMI1640培养基含10％FBS)。取2x10 5细胞用预冷的PBS清洗3次，2％FBS(溶于PBS)封闭后分别与分子筛纯化的不同浓度(200nM，40nM，8nM，1.6nM)IgFD-6或IgFD-7 4℃孵育2h(孵育过程中轻轻混匀，2％FBS(溶于PBS)洗去未结合的抗体，用FITC anti-human IgG Fc(KPL,Inc.,MD)4℃染色1h，2％FBS(溶于PBS)洗脱后进行FACS分析.
结果见图4。与对照FITC anti-human IgG Fc相比，不同浓度的IgFD-6和IgFD-7与CD19+NALM6细胞均具有较强的结合，浓度越高，结合强度越大。
(2)anti-CD3/anti-CD19异源二聚体抗体促进PBMC对NALM-6的细胞特异性杀伤
收集健康志愿者外周血，Ficoll-Hypaque(GE Healthcare)梯度离心分离外周血单核细胞(PBMCs)，RPMI 1640/10％FBS完全培养基重悬。将PBMCs与固相结合的anti-CD3(Clone OKT3,eBiosciences)、2μg/mL anti-CD28(Clone CD28.2,eBiosciences)37℃孵育，48h后加入20U/ml IL2(R&D Systems)刺激活化的T细胞扩增10天。
培养NALM6细胞(RPMI1640培养基含10％FBS)，Green fluorescent cell linker mini kit(Sigma)标记后，取10 4绿色荧光标记的NALM6细胞，与上述刺激活化的T细胞以1:5(NALM6细胞为10 4，T细胞为5*10 4)的比例孵育，加入凝胶排阻层析纯化的梯度稀释的IgFD-6或IgFD-7，37℃孵育24h后，加入1％7-AAD，上流式细胞仪进行分析。绿色荧光阳性/7-AAD阴性的细胞即为活的NALM6细胞。
结果如图5所示。IgFD-6和IgFD-7都能够有效促进PBMC中的T细胞对Nalm6细胞的杀伤作用。
4.2 anti-CD3/EGFR异源二聚体体外活性检测
(1)anti-CD3/EGFR异源二聚体结合人EGFR ELISA检测
包被hEGFR-6-his(SinoBiological)(100ng/孔)于96孔板，4℃孵育过夜；含2％脱脂奶粉的PBST(0.5％Tween-20in PBS)室温封闭1小时，分别加入梯度稀释的异源二聚体抗体IgFD-11、IgFD-24、IgFD-25、IgFD-26、IgFD-31、anti-EGFR室温孵育2h，含2％脱脂奶粉的PBST洗4-5次后，加入Anti-Human IgG(FC)Antibody-HRP(KPL)二抗室温孵育1h，含2％脱脂奶粉的PBST洗4-5次后，TMB显色试剂(BioLegend,Cat.421101)显色后于650nm处读数。如图6显示，不同融合形式的anti-CD3/anti-EGFR异源二聚体与人EGFR抗原均有较强的结合能力。
(2)流式细胞术检测anti-CD3/anti-EGFR异源二聚体与F98-EGFR细胞结合
培养F98-EGFR细胞(DMEM培养基含10％FBS,200μg/ml G418)。取2x10 5细胞用预冷的PBS清洗3次，2％FBS(溶于PBS)封闭后分别与凝胶排阻层析纯化的IgFD-8、IgFD-9、IgFD-10、IgFD-11、IgFD-18、IgFD-19、IgFD-25、IgFD-26或IgFD-31(实验室表达的aEGFR为对照)4℃孵育2h(孵育过程中轻轻混匀)，2％FBS(溶于PBS)洗去未结合的抗体，用FITC anti-human IgG Fc(KPL,Inc.,MD)4℃染色1h，2％FBS(溶于PBS)洗脱后进行FACS分析。如图7和表4所示，不同融合形式的anti-CD3/anti-EGFR异源二聚体可与F98-EGFR细胞表面的EGFR结合，不同异源二聚体与F98-EGFR的结合力无显著差异。
(3)流式细胞术检测anti-CD3/anti-EGFR异源二聚体与PBMC-T细胞结合
收集健康志愿者外周血，Ficoll-Hypaque(GE Healthcare)梯度离心分离外周血单核细胞(PBMCs)，RPMI 1640/10％FBS完全培养基重悬。将PBMCs与固相结合的anti-CD3 (Clone OKT3,eBiosciences)、2μg/mL anti-CD28(Clone CD28.2,eBiosciences)37℃孵育，48h后加入20U/ml IL2(R&D Systems)刺激活化的T细胞扩增。
取2x10 5细胞，用预冷的PBS洗3次，2％FBS(溶于PBS)封闭后，与凝胶排阻层析纯化的不同浓度(25nM,7.5nM,2.5nM,0.75nM or 0.25nM)IgFD-8、IgFD-18或IgFD-19 4℃孵育1h(孵育过程中轻轻混匀)，2％FBS(溶于PBS)洗去未结合的抗体。细胞与FITC anti-human IgG Fc(KPL,Inc.,MD)4℃孵育1h(孵育过程中轻轻混匀)，2％FBS(溶于PBS)洗去未结合的抗体后进行FACS分析。
结果见图8。不同融合形式的异源二聚体与PBMC-T细胞结合的强弱依次是IgFD-8>IgFD-19>IgFD-18，与F98-EGFR细胞结合的强弱为IgFD-18>IgFD-19>IgFD-8(图6)，提示scfv(anti-EGFR scfv和anti-CD3 scfv)与抗原的亲和力比Fab(anti-EGFR Fab和anti-CD3Fab)与抗原的结合力强。
(4)流式细胞术检测anti-CD3/anti-EGFR异源二聚体与Jurkat细胞结合
培养Jurkat细胞(DMEM培养基含10％FBS)。取2x10 5细胞用预冷的PBS清洗3次，2％FBS(溶于PBS)封闭后分别与凝胶排阻层析纯化的IgFD-11、IgFD-8、IgFD-25、IgFD-26、IgFD-31或IgFD-36(实验室表达的aEGFR为对照)4℃孵育2h(孵育过程中轻轻混匀)，2％FBS(溶于PBS)洗去未结合的抗体，用FITC anti-human IgG Fc(KPL,Inc.,MD)4℃染色1h，2％FBS(溶于PBS)洗脱后进行FACS分析。
结果见图9。不同融合形式的anti-CD3/anti-EGFR异源二聚体均能与Jurkat T细胞很好第结合
(5)anti-CD3/anti-EGFR异源二聚体促进PBMC对F98-EGFR的细胞特异性杀伤LDH检测
收集健康志愿者外周血，Ficoll-Hypaque(GE Healthcare)梯度离心分离外周血单核细胞(PBMCs)，RPMI 1640/10％FBS完全培养基重悬。将PBMCs与固相结合的anti-CD3(Clone OKT3,eBiosciences)、2μg/mL anti-CD28(Clone CD28.2,eBiosciences)37℃孵育，48h后加入20U/ml IL2(R&D Systems)刺激活化的T细胞扩增10天。
培养F98-EGFR细胞(DMEM培养基含10％FBS,200μg/ml G418)，取10 4与上述刺激活化的T细胞以1:5(F98-EGFR细胞为10 4，T细胞为5*10 4)的比例孵育，加入梯度稀释的IgFD-8、IgFD-18、IgFD-19、IgFD-21、IgFD-20、IgFD-25、IgFD-26、IgFD-28、IgFD-29或IgFD-30 37℃孵育24h。Cytotox-96nonradioactive cytotoxicity assay kit(Promega)检测培养上清中LDH含量。SpectraMax 250读取490nm处OD值。细胞毒性(％表示)计算如下：
％Cytotoxicity＝(Experimental–Effector Spontaneous–Target Spontaneous)/(Target Maximum–Target Spontaneous)x100
其中，Target Maximum为仅有F98-EGFR细胞裂解后的上清中LDH含量
Target Spontaneous为仅有F98-EGFR细胞的上清中LDH含量
Effector Spontaneous为仅有效应细胞(T细胞)的上清中LDH含量。
结果见图10。不同融合形式的anti-EGFR&anti-CD3均能有效召集PBMC中的T细胞对F98-EGFR细胞产生相应的杀伤作用。
4.3 anti-CD3/BCMA异源二聚体促进PBMC对MM1.R细胞的杀伤作用
收集健康志愿者外周血，Ficoll-Hypaque(GE Healthcare)梯度离心分离外周血单核细胞(PBMCs)，RPMI 1640/10％FBS完全培养基重悬。将PBMCs与固相结合的anti-CD3(Clone OKT3,eBiosciences)、2μg/mL anti-CD28(Clone CD28.2,eBiosciences)37℃孵育，48h后加入20U/ml IL2(R&D Systems)刺激活化的T细胞扩增10天。
(1)LDH释放实验检测：
培养MM1.R细胞(RPMI1640培养基含10％FBS)，取10 4与上述刺激活化的T细胞以1:5(MM1.R细胞为10 4，T细胞为5*10 4)的比例孵育，加入梯度稀释的凝胶排阻层析纯化的IgFD-22 37℃孵育24h。
用Cytotox-96 nonradioactive cytotoxicity assay kit(Promega)检测上述培养上清中LDH含量。SpectraMax 250读取490nm处OD值。细胞毒性(％表示)计算如下：
％Cytotoxicity＝(experimental–Effector Spontaneous–Target Spontaneous)/(Target Maximum–Target Spontaneous)x100
其中，Target Maximum为仅有MM1.R细胞裂解后的上清中LDH含量
Target Spontaneous为仅有MM1.R细胞的上清中LDH含量。
Effector Spontaneous为仅有效应细胞(T细胞)的上清中LDH含量。
(2)FACS检测
培养MM1.R细胞(RPMI1640培养基含10％FBS)，用Green fluorescent cell linker mini kit(Sigma)标记后，取10 4绿色荧光标记的MM1.R细胞，与上述刺激活化的T细胞以1:5(MM1.R细胞为10 4，T细胞为5*10 4)的比例孵育，加入凝胶排阻层析纯化的梯度稀释的IgFD-22 37℃孵育24h，加入1％7-AAD后，上流式细胞仪进行分析。绿色荧光阳性/7-AAD阴性的细胞即为活的MM1.R细胞。
LDH释放和FACS检测结果见图11(A)和图10(B)。IgFD-22能有效促进PMBC对MM1.R细胞的杀伤作用。
4.4靶向CD3和MICA异源二聚体体外活性检测
(1)靶向CD3和MICA的异源二聚体结合人MICA ELISA检测
包被MICA(北京义翘神州)(100ng/孔)于96孔板，4℃孵育过夜；含2％脱脂奶粉的PBST(0.5％Tween-20in PBS)室温封闭1小时，分别加入梯度稀释的异源二聚体抗体IgFD-36、IgFD-37于37℃孵育1h，含2％脱脂奶粉的PBST洗3次后，加入anti-human Fc-HRP二抗(KPL 5200-0279,1:1000)室温孵育1h，含2％脱脂奶粉的PBST洗5次后，TMB显色试剂(BioLegend,Cat.421101)显色后于652nm处读数。如图12显示，IgFD-37异源二聚体与人MICA抗原有较强的结合能力。
(2)流式细胞术检测靶向CD3和MICA异源多聚体抗体与PANC-1、BXPC-3、K562细胞结合
分别培养PANC-1(DMEM培养基含10％FBS)、BXPC-3(RPMI1640培养基含10％FBS)、K562细胞(RPMI1640培养基含10％FBS)。取2x10 5细胞用预冷的PBS清洗3次，2％FBS(溶于PBS)封闭后分别与凝胶排阻层析纯化的IgFD-36、IgFD-37 4℃孵育2h(孵育过程中轻轻混匀)，2％FBS(溶于PBS)洗去未结合的抗体，用anti-human kappa light chain-FITC(Biolegend，316506)4℃染色1h，2％FBS(溶于PBS)洗脱后进行FACS分析。如图13和表4所示，靶向CD3和MICA的异源二聚体可与PANC-1、BXPC-3或K562细胞表面的NKG2DL亚基MICA结合。
(3)靶向CD3和MICA的异源二聚体促进PBMC对表达MICA的细胞特异性杀伤
收集健康志愿者外周血，Ficoll-Hypaque(GE Healthcare)梯度离心分离外周血单核细胞(PBMCs)，RPMI 1640/10％FBS完全培养基重悬。将PBMCs与固相结合的anti-CD3(Clone OKT3,eBiosciences)、2μg/mL anti-CD28(Clone CD28.2,eBiosciences)37℃孵育，48h后加入20U/ml IL2(R&D Systems)刺激活化的T细胞扩增10天。
培养K562、PANC-1细胞，取10 4与上述刺激活化的T细胞以1:5(K562或PANC-1细胞为10 4，T细胞为5*10 4)的比例，加入梯度稀释的IgFD-36和IgFD-37于37℃孵育24h。Cytotox-96 nonradioactive cytotoxicity assay kit(Promega)检测培养上清中LDH含量。SpectraMax 250读取490nm处OD值。细胞毒性(％表示)计算如下：
％Cytotoxicity＝(Experimental–Effector Spontaneous–Target Spontaneous)/(Target Maximum–Target Spontaneous)x100
其中，Target Maximum为仅有K562或PANC-1细胞裂解后的上清中LDH含量
Target Spontaneous为仅有K562或PANC-1细胞的上清中LDH含量。
Effector Spontaneous为仅有效应细胞(T细胞)的上清中LDH含量。
结果见图14。靶向CD3和MICA的异源二聚体能有效召集PBMC中的T细胞对MICA阳性的细胞产生相应的杀伤作用。
4.5 anti-CD3/anti-CLL-1异源二聚体促进PBMC对HL-60细胞的杀伤作用
收集健康志愿者外周血，Ficoll-Hypaque(GE Healthcare)梯度离心分离外周血单核细胞(PBMCs)，RPMI 1640/10％FBS完全培养基重悬。将PBMCs与固相结合的anti-CD3(Clone OKT3,eBiosciences)、2μg/mL anti-CD28(Clone CD28.2,eBiosciences)37℃孵育，48h后加入20U/ml IL2(R&D Systems)刺激活化的T细胞扩增10天。
培养HL-60细胞(RPMI1640培养基含10％FBS)，用Green fluorescent cell linker mini kit(Sigma)标记后，取10 4绿色荧光标记的HL-60细胞，与上述刺激活化的T细胞以1:5(HL-60细胞为10 4，T细胞为5*10 4)的比例孵育，加入凝胶排阻层析纯化的梯度稀释的IgFD-23 37℃孵育24h，加入1％7-AAD后，上流式细胞仪进行分析。绿色荧光阳性/7-AAD阴性的细胞即为活的NALM6细胞。
结果见图15。IgFD-23能够有效召集PBMC中的T细胞，进而对HL-60产生特异性杀伤作用。
表4不同异源二聚体与细胞的结合及促进PBMC对靶细胞的特异杀伤
Figure PCTCN2019129591-appb-000010
Figure PCTCN2019129591-appb-000011
实施例5，热力学稳定性测试
将样品IgFD-6、IgFD-7与新鲜配制的thermal shift dye、shift buffer(Protein Thermal Shift TM Dye Kit,ThermoFisher Scientific,Cat.4461146)按厂家推荐的比例混合，利用ViiA TM 7 Real-Time PCR System以0.05℃/s的加热速率在25-99℃进行热扫描。用GraphPad  Prism7软件的“Area under curve(AUC)”分析模型计算Tm值。每组数据均重复2次试验以保证结果的重复性。
结果如表5所示，IgFD-6和IgFD-7具有类似的Tm值。
表5蛋白Tm值
Figure PCTCN2019129591-appb-000012
实施例6异源二聚体大鼠体内PK研究
将IgFD-33腹腔注射(I.P.)SD雄性大鼠(3只)。尾静脉采集肝素抗凝血，采血时间如下：2h、4h、8h、24h、36h、4d、7d、11d和14d。离心后取血浆，-80℃保存备用。血浆中IgFD-33的含量检测参照实施例4.2(1)进行。结果如图16所示，IgFD-33在大鼠体内半衰期可达2.5天。
实施例7异源二聚体小鼠体内活性研究
IgFD-33抑制荷瘤小鼠肿瘤块能力的检测在6-8周雌性NCG小鼠上进行。将5×10 6A431细胞和2.5×10 6新鲜的PBMC重悬于200ul无血清培养基后皮下注射小鼠右胁腹(Day0)，用卡钳测量实验小鼠肿瘤块大小。按下式计算肿瘤体积：肿瘤体积＝宽度*宽度*长度/2。待肿瘤大小为50-100mm3时(Day7)，分别于Day8和Day11注射1.0×10 7体外活化的T细胞(用固相结合的anti-CD3 antibody(clone OKT3,eBioscience)、2μg/mL anti-CD28抗体(clone CD28.2,eBioscience)、50IU/mL重组人IL-2(R&D Systems)体外刺激PBMCs)，同时于Day7-Day13每天注射异源二聚体抗体IgFD-33(其中空白组：A431 without PBMC without activate T cells+生理盐水；对照组：A431&新鲜的PBMC+活化的T细胞+生理盐水)，每隔一天称量小鼠体重并用卡钳测量肿瘤块大小。
结果如图17所示，与空白组或对照组相比，A431接种的大鼠其肿瘤块在IgFD-33给药后持续消退，在A431接种后的35天未见肿瘤复发，提示IgFD-33具有较高的体内活性。

Claims (46)

  1. 异源二聚体融合蛋白，其包括：
    a)第一多肽链，其包含Fab重链和第一单链Fc，所述Fab重链直接或通过连接子与所述第一Fc单链的N端融合；
    b)第二多肽链，其包含Fab轻链和第二单链Fc，所述Fab轻链直接或通过连接子与所述第二Fc单链的N端融合；
    其中，所述第一多肽链的Fab重链和所述第二多肽链的Fab轻链形成第一抗原结合结构域Fab，所述第一Fc单链和所述第二Fc单链形成Fc二聚结构域。
  2. 权利要求1的异源二聚体融合蛋白，其进一步包含第二抗原结合结构域。
  3. 权利要求2的异源二聚体融合蛋白，其中，所述第二抗原结合结构域由scfv或生理活性肽构成。
  4. 权利要求3的异源二聚体融合蛋白，其中，所述构成第二抗原结合结构域的scfv或生理活性肽直接或通过连接子与所述第一多肽链或第二多肽链的N端融合。
  5. 权利要求3的异源二聚体融合蛋白，其中，所述构成第二抗原结合结构域的scfv或生理活性肽直接或通过连接子与所述第一多肽链或第二多肽链的C端融合。
  6. 权利要求3的异源二聚体融合蛋白，其中，所述第二抗原结合结构域由第一和第二生理活性肽构成，所述第一和第二生理活性肽分别与所述第一多肽链和第二多肽链的N端直接或通过连接子融合。
  7. 权利要求3的异源二聚体融合蛋白，其中所述第二抗原结合结构域由第一和第二生理活性肽构成，所述第一和第二生理活性肽分别与所述第一多肽链和第二多肽链的C端直接或通过连接子融合。
  8. 权利要求2的异源二聚体融合蛋白，其中所述第二抗原结合结构域由Fv构成。
  9. 权利要求8的异源二聚体融合蛋白，其中所述Fv的重链可变区直接或通过连接子与所述第一多肽链或第二多肽链的C端融合，相应地，所述Fv的轻链可变区直接或通过连接子与所述第二多肽链或第一多肽链的C端融合。
  10. 权利要求9的异源二聚体融合蛋白，其中所述Fv的重链可变区直接或通过连接子与所述第一多肽链或第二多肽链的N端融合，相应地，所述Fv的轻链可变区直接或通过连接子与所述第二多肽链或第一多肽链的N端融合。
  11. 权利要求2的异源二聚体融合蛋白，其中所述第二抗原结合结构域由Fab2构成。
  12. 权利要求11的异源二聚体融合蛋白，其中所述Fab2的重链直接或通过连接子与所述第一多肽链或第二多肽链的C端融合，相应地，所述Fab2的轻链直接或通过连接子与所述第二多肽链或第一多肽链的C端融合。
  13. 权利要求11的异源二聚体融合蛋白，其中所述Fab2的重链直接或者通过连接子与所述第一多肽链或第二多肽链的N端融合，相应地，所述Fab2的轻链直接或者通过连接子与所述第二多肽链或第一多肽链的N端融合。
  14. 权利要求1至13任一项的异源二聚体融合蛋白，其中进一步地，其分子内Fab的恒定结构域CH1和CL在两条链上互相交换位置。
  15. 权利要求1至14任一项的异源二聚体融合蛋白，其中所述连接子独立地选自但不限于以下氨基酸序列组成的组：GGSGAKLAALKAKLAALKGGGGS、GGGGSELAALEAELAALEAGGSG、GGGGSGGGGSGGGGS、SGGGGSGGGGSGGGGS、GGSGGSGGGGSGGGG、GGSGGSGGGGSGGGGS、GGSGAKLAALKAKLAALKGGGGS、GGGGSELAALEAELAALEAGGSG、APATSLQSGQLGFQCGELCSASA、ASTKGP、TVAAPSVFIFPP、PNLLGGP、GGGGS、GGGEAAAKEAAAKEAAAKAGG。
  16. 异源二聚体融合蛋白，其包含：
    a)第一多肽链，其按从N端到C端的顺序依次包含：Fc、(L1)n、CH1、L2、VH，
    b)第二多肽链，其按从N端到C端的顺序依次包含：Fc、(L3)n、CL、L4、VL；或者
    a)第一多肽链，其按从N端到C端的顺序依次包含：Fc、(L1)n、CL、L2、VH，
    b)第二多肽链，其按从N端到C端的顺序依次包含：Fc、(L3)n、CH1、L4、VL，其中，n为0或1，L1、L2、L3和L4为连接子，VH和VL形成第一抗原结合结构域。
  17. 权利要求16的异源二聚体融合蛋白，其进一步包括第二抗原结合结构域。
  18. 权利要求17的异源二聚体融合蛋白，其中，所述第二抗原结合结构域由scfv或生理活性肽构成。
  19. 权利要求18的异源二聚体融合蛋白，其中，所述构成第二抗原结合结构域的scfv或生理活性肽直接或通过连接子与第一多肽链或第二多肽链的N端融合。
  20. 权利要求18的异源二聚体融合蛋白，其中，所述第二抗原结合结构域由第一和第二生理活性肽构成，所述第一和第二生理活性肽分别与所述第一多肽链和第二多肽链的N端直接或通过连接子融合。
  21. 权利要求16-20中任一项的异源二聚体融合蛋白，其中，L1、L2、L3和L4连接子独立地选自：G、GS、SG、SS、GGS、GSG、SGG、GGG、GGGS、SGGG、GGGGS、GGGGSGS、GGGGSGGS、GGGGSGGGGS、GGGGSGGGGSGGGGS、AKTTPKLEEGEFSEAR、AKTTPKLEEGEFSEARV、AKTTPKLGG、SAKTTPKLGG、AKTTPKLEEGEFSEARV、SAKTTP、SAKTTPKLGG、RADAAP、RADAAPTVS、RADAAAAGGPGS、RADAAAA(G4S)4、SAKTTP、SAKTTPKLGG、SAKTTPKLEEGEFSEARV、ADAAP、ADAAPTVSIFPP、TVAAP、TVAAPSVFIFPP、QPKAAP、QPKAAPSVTLFPP、AKTTPP、AKTTPPSVTPLAP、AKTTAP、AKTTAPSVYPLAP、ASTKGP、ASTKGPSVFPLAP、GENKVEYAPALMALS、GPAKELTPLKEAKVS及GHEAAAVMQVQYPAS；其中L1、L2、L3和L4可以相同，也可以不同。
  22. 权利要求1至21任一项的异源二聚体融合蛋白，其中所述Fc为人IgG1的Fc。
  23. 权利要求22的异源二聚体融合蛋白，其中所述Fc为Fc变体。
  24. 权利要求23的异源二聚体融合蛋白，其中所述Fc变体不含糖基化修饰。
  25. 权利要求24的异源二聚体融合蛋白，其中所述Fc变体包含N297去糖基化修饰的氨基酸置换。
  26. 权利要求22的异源二聚体融合蛋白，其中所述Fc变体包含一处或多处降低Fc对Fc受体的结合和/或效应功能的氨基酸置换。
  27. 权利要求26异源二聚体融合蛋白，其中所述Fc变体中的氨基酸置换包含E233P、L234V、L235A、delG236、A327G、A330S及A331S中的一种或多种。
  28. 权利要求22-27的异源二聚体融合蛋白，其中所述第一Fc和所述第二Fc之一进一步包含氨基酸置换S354C、T366W，且所述第一Fc和所述第二Fc中的另一个进一步包含氨基酸置换Y349C、T366S、L368A和Y407V。
  29. 前述权利要求中任一项的异源二聚体融合蛋白，其可结合抗原CD3、CD16、CD2、CD28、CD25、NKG2D、NKp46、BCMA、CLL-1、EpCAM、CD19、CCR5、EGFR、HER2、HER3、HER4、EGF4、PSMA、CEA、MUC-1(Mucin)、MUC-2、MUC-3、MUC-4、MUC-5 AC、MUC-5 B、MUC7、βhCG、Lewis-Y、CD20、CD33、CD30、CD16A、 B7-H3、CD123、gpA33、P-Cadherin、GPC3、CLEC12A、CD32B、TROP-2、ganglioside GD3、9-O-Acetyl-GD3,GM2,Globo H、fucosyl GM1、Poly SA、GD2、Carboanhydrase IX(MN/CA IX)、CD44v6、Sonic Hedgehog(Shh)、Wue-1、Plasma Cell Antigen、(membrane-bound)IgE、Melanoma Chondroitin Sulfate Proteoglycan(MCSP)、CCR8、TNF-alpha precursor、STEAP、mesothelin、A33Antigen、Prostate Stem Cell Antigen(PSCA)、Ly-6、desmoglein 4、E-cadherin neoepitope、Fetal Acetylcholine Receptor、CD25、CA19-9marker、CA-125marker and Muellerian Inhibitory Substance(MIS)Receptor type II、sTn(sialylated Tn antigen；TAG-72)、FAP(fibroblast activation antigen)、endosialin、EGFRvIII、LG、SAS、CD63中的一种或其组合。
  30. 前述权利要求中任一项的异源二聚体融合蛋白，其可结合由下列抗原对组成的抗原：CD3和CD19；CD3和CD20；CD3和BCMA；CD3和CLL-1；CD3和EGFR；CD3和HER2；CD3和MIC-A；CD3和CEA；CD3和PSMA；CD3和EpCAM。
  31. 权利要求30的异源二聚体融合蛋白，其可同时结合CD3和CD19。
  32. 权利要求31的异源二聚体融合蛋白，其包括可结合CD3和CD19的两条多肽链组成的组：SEQ ID No:2和SEQ ID No:4；SEQ ID No:6和SEQ ID No:8；SEQ ID No:20和SEQ ID No:22；SEQ ID No:24和SEQ ID No:26；SEQ ID No:92和SEQ ID No:94；SEQ ID No:96和SEQ ID No:98；SEQ ID No:100和SEQ ID No:102。
  33. 权利要求30的异源二聚体融合蛋白，其可同时结合CD3和EGFR。
  34. 权利要求33的异源二聚体融合蛋白，其包括可结合CD3和EGFR的两条多肽链组成的组：SEQ ID No:10和SEQ ID No:12；SEQ ID No:14和SEQ ID No:16；SEQ ID No:18和SEQ ID No:12；SEQ ID No:24和SEQ ID No:28；SEQ ID No:30和SEQ ID No:32；SEQ ID No:30和SEQ ID No:34；SEQ ID No:36和SEQ ID No:38；SEQ ID No:44和SEQ ID No:46；SEQ ID No:48和SEQ ID No:50；SEQ ID No:44和SEQ ID No:52；SEQ ID No:54和SEQ ID No:56、SEQ ID No:30和SEQ ID No:58；SEQ ID No:60和SEQ ID No:62；SEQ ID No:64和SEQ ID No:66；SEQ ID No:68和SEQ ID No:70；SEQ ID No:72和SEQ ID No:74；SEQ ID No:76和SEQ ID No:78；SEQ ID No:80和SEQ ID No:82；SEQ ID No:84和SEQ ID No:86、SEQ ID No:88和SEQ ID No:90；SEQ ID No:104和SEQ ID No:106；SEQ ID No:108和SEQ ID No:110；SEQ ID No:112和SEQ ID No:114；SEQ ID No:116和SEQ ID No:118。
  35. 权利要求30的异源二聚体融合蛋白，其可同时结合CD3和BCMA。
  36. 权利要求35的异源二聚体融合蛋白，其包括可结合CD3和BCMA的两条多肽链组成的组：SEQ ID No:30和SEQ ID No:40。
  37. 权利要求30的异源二聚体融合蛋白，其可同时结合CD3和CLL-1。
  38. 权利要求37的异源二聚体融合蛋白，其包括可结合CD3和CLL-1的两条多肽链组成的组：SEQ ID No:30和SEQ ID No:42。
  39. 权利要求30的异源二聚体融合蛋白，其可同时结合CD3和MIC-A。
  40. 权利要求39的异源二聚体融合蛋白，其包括可结合CD3和MIC-A的两条多肽链组成的组：SEQ ID No:122和SEQ ID No:124。
  41. 一种多核苷酸，其编码如权利要求1-40任一项所述的异源二聚体融合蛋白。
  42. 一种载体，特别是表达载体，其包括权利要求41的多核苷酸。
  43. 一种宿主细胞，其包括：
    ---表达载体，其包括编码所述第一多肽链的多核苷酸，和
    ---表达载体，其包括编码所述第二多肽链的多核苷酸。
  44. 制备权利要求1-40中任一项的异源二聚体融合蛋白，其包括下列步骤：
    1)用以下各项瞬时转染哺乳动物宿主细胞：
    ---表达载体，其包括编码所述第一多肽链的多核苷酸，和
    ---表达载体，其包括编码所述第二多肽链的多核苷酸；
    在容许所述异源二聚体融合蛋白表达的条件下培养所述哺乳动物宿主细胞；和
    从培养上清中收集分泌的异源二聚体融合蛋白。
  45. 一种药物组合物，其包含权利要求1-40的异源二聚体融合蛋白。
  46. 一种治疗有需要的受试者的癌症、自身免疫性疾病或病毒感染的方法，所述方法包括对所述受试者施用治疗有效量的组合物，所述组合物包含药学可接受形式的权利要求1-40任一项的异源二聚体融合蛋白。
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