WO2023016568A1 - 一种双特异性重组蛋白及其用途 - Google Patents

一种双特异性重组蛋白及其用途 Download PDF

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WO2023016568A1
WO2023016568A1 PCT/CN2022/112300 CN2022112300W WO2023016568A1 WO 2023016568 A1 WO2023016568 A1 WO 2023016568A1 CN 2022112300 W CN2022112300 W CN 2022112300W WO 2023016568 A1 WO2023016568 A1 WO 2023016568A1
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amino acid
acid sequence
region
chain
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French (fr)
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何柯
宋利平
范艺
陈英娇
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上海才致药成生物科技有限公司
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Priority to AU2022326107A priority Critical patent/AU2022326107A1/en
Priority to CA3228311A priority patent/CA3228311A1/en
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/21—Interferons [IFN]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • 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
    • A61P35/00—Antineoplastic agents
    • 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
    • A61P37/02—Immunomodulators
    • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/46—Hybrid immunoglobulins
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material

Definitions

  • the invention belongs to the field of biomedicine, and specifically relates to a bispecific recombinant protein and its application.
  • Interferons are a group of active proteins with multiple functions. They are mainly cytokines produced by monocytes and lymphocytes. They are divided into three types: type I, type II and type III. IFN ⁇ subtypes may also be included, such as IFN ⁇ 2, IFN ⁇ 4, etc. Type II and Type III interferons may include IFN ⁇ , IFN ⁇ 1 (IL-29), IFN ⁇ 2 (IL-28a) and IFN ⁇ 3 (IL-28b). Interferon has broad-spectrum anti-virus, affects cell growth and differentiation, regulates immune function and many other biological activities. Among them, interferon ⁇ (IFN ⁇ ) has a wide range of antiviral and antitumor effects.
  • IFN ⁇ interferon ⁇
  • liver diseases such as chronic hepatitis B, hepatitis C, etc.
  • antiviral such as viral respiratory diseases, skin diseases, blood diseases, gynecological diseases, etc.
  • specific tumors such as chronic myelogenous leukemia, melanoma, lymphoma, kidney cancer, etc.
  • IFN ⁇ receptors are widely expressed in various tissues and organs of the human body, they are often accompanied by toxic side effects such as fever, fatigue, myalgia, liver toxicity, bone marrow suppression, and neurotoxicity during clinical use.
  • a large number of clinical research data show that PEG-IFN ⁇ (6 ⁇ g/kg/week treatment for 8 weeks; 3 ⁇ g/kg/week maintenance) will cause more than 60% of patients to have grade 3 or higher serious adverse events during the adjuvant treatment of melanoma ( SAE) (Practical Guidelines for the Management of Interferon-a-2b Side Effects in Patients Receiving Adjuvant Treatment for Melanoma.
  • PEG-IFN ⁇ treatment of chronic myeloid leukemia CML clinical maximum tolerated dose is 7.5-9 ⁇ g/kg
  • MTD chronic myeloid leukemia CML clinical maximum tolerated dose
  • the optimal tumor inhibitory dose should be at least 100 times higher than the clinically tolerated dose
  • the freeze-thaw stability is generally poor.
  • the recombinant human albumin interferon- ⁇ 2b fusion protein rHSA-IFN- ⁇ 2b
  • the body content reaches 17.91%, and the recombinant human albumin interferon- ⁇ 2b fusion protein should not be frozen and thawed (Xia Yi et al., Stability research of recombinant human albumin interferon ⁇ -2b fusion protein, Journal of Biology, 2008,25(006) :38-40), so that there are more restrictions on production, transportation and use.
  • bispecific recombinant protein a high-efficiency and low-toxicity antibody cytokine fusion protein (i.e., bispecific recombinant protein), which can significantly increase the therapeutic dose of IFN ⁇ on the premise of human tolerance, and improve the effects of anti-tumor, anti-virus and liver disease treatment.
  • the bispecific recombinant protein has better freeze-thaw stability than interferon.
  • the technical problem to be solved by the present invention is to provide a bispecific recombinant protein and its use in order to overcome the poor efficacy of monoclonal antibodies and the deficiencies in the safety and freeze-thaw stability of interferon alpha in the prior art.
  • the bispecific recombinant protein of the present invention has the function of directional regulation of immunity, can significantly improve the target targeting of the recombinant protein, and can also significantly reduce the generation of non-target proteins in the preparation process that contain non-target proteins that target non-target organs, tissues, and cells. caused toxic side effects.
  • the present invention solves the above-mentioned technical problems through the following technical solutions.
  • the first aspect of the present invention provides a bispecific recombinant protein, which includes a first functional binding region, a second functional binding region and an Fc region; the first functional binding region includes a first chain and In the second chain, the C-terminus of the first chain and the C-terminus of the second function-binding region are connected to the N-terminus of the Fc region directly or through a linker sequence; the C-terminus of the second chain is combined with the second function The N-terminus of the region is connected directly or through a linker sequence;
  • the first functional binding region includes a heavy chain variable region (VH), a light chain variable region (VL), a heavy chain constant region 1 (CH1) and a light chain constant region (CL);
  • the second functional binding region comprises interferon, its truncation or its mutant.
  • the C-terminus of the CL or CH1 is directly connected to the N-terminus of the Fc region or through a linker sequence; the C-terminus of the corresponding CH1 or CL is directly connected to the N-terminus of the second functional binding region or Linked by a linker sequence.
  • the first chain includes VH and CH1, VH and CL, VL and CH1, or VL and CL; the second chain includes VH and CH1, VH and CL, VL and CH1, or VL and CL.
  • the first chain is different from the second chain, and the combination of the first chain and the second chain includes four elements of VH, CH1, VL and CL.
  • the first chain includes VH and CH1
  • the second chain includes VL and CL
  • the first chain includes VH and CL
  • the second chain includes VL and CH1.
  • the interferon is type I interferon, such as IFN ⁇ .
  • the IFN ⁇ is selected from IFN- ⁇ 1a, IFN- ⁇ 1b, IFN- ⁇ 2a, IFN- ⁇ 2b, IFN- ⁇ 4a, IFN- ⁇ 4b, IFN- ⁇ 5, IFN- ⁇ 6, IFN- ⁇ 7, One or more of IFN- ⁇ 8, IFN- ⁇ 10, IFN- ⁇ 14, IFN- ⁇ 16, IFN- ⁇ 17, and IFN- ⁇ 21.
  • the interferon is IFN- ⁇ 2b.
  • the second functional binding region comprises an amino acid sequence selected from any one of the following a1)-a2): a1) SEQ ID NO: 17; a2) the amino acid sequence shown in SEQ ID NO: 17 Amino acid sequence obtained by adding, deleting, modifying and/or substituting at least one amino acid residue, its monomer has binding affinity to IFN ⁇ receptor and the binding affinity is not higher than the binding affinity of a1) monomer to IFN ⁇ receptor .
  • the amino acid sequence of the monomer is an amino acid sequence obtained by substitution at the corresponding position of the amino acid sequence shown in SEQ ID NO: 17; the substitution at the corresponding position is selected from L26A, One or more of L30A, A145G, R149A and S152A.
  • the a2) monomer is a single-site replacement, and the replacement sites are respectively L26A, L30A, A145G, R149A or S152A, and the corresponding amino acid sequences are shown in SEQ ID NO: 18-22. Show.
  • the VH is directly connected to CH1 or CL or connected through a linker sequence; the VL is directly connected to CL or CH1 or connected through a linker sequence.
  • VH when VL is connected to CL, VH is connected to CH1; when VL is connected to CH1, VH is connected to CL.
  • the first functional binding region targets tumor cells or immune cells.
  • the first functional binding region targets any one or more of the following targets: GPC3, BCMA, PD-L1, Trop-2, 5T4, AGS-16, ALK1, ANG-2, B7-H3 , B7-H4, c-fms, c-Met, CA6, CD123, CD19, CD20, CD22, CD24, EpCAM, CD30, CD32b, CD37, CD38, CD40, CD52, CD70, CD74, CD79b, CD98, CEA, CEACAM5 , CLDN18.2, CLDN6, CS1, CXCR4, DLL-4, EGFR, EGFRvIII, EGP-1, ENPP3, EphA3, ETBR, FGFR2, FN, FR- ⁇ , GCC, GD2, GPNMB, HER2, HER3, HLA-DR , ICAM-1, IGF-1R, IL-3R, LIV-1, MSLN, MUC16, MUC1, NaPi2b, Internexin-4, Notch 2, Notch 1,
  • the first functional binding region targets GPC3; the first functional binding region comprises an antigen-binding fragment of a specific anti-GPC3 monoclonal antibody.
  • the heavy chain variable region (VH) sequence of the specific anti-GPC3 monoclonal antibody is as shown in SEQ ID NO: 1
  • the light chain variable region (VL) sequence is as shown in SEQ ID NO: 2.
  • the first functional binding region targets PD-L1; the first functional binding region comprises an antigen-binding fragment of a specific anti-PD-L1 monoclonal antibody.
  • the heavy chain variable region (VH) sequence of the specific anti-PD-L1 monoclonal antibody is shown in SEQ ID NO: 5
  • the light chain variable region (VL) sequence is shown in SEQ ID NO: Shown in 6.
  • the first functional binding region targets BCMA; the first functional binding region comprises an antigen-binding fragment of a specific anti-BCMA monoclonal antibody.
  • the heavy chain variable region (VH) sequence of the specific anti-BCMA monoclonal antibody is shown in SEQ ID NO: 7 or SEQ ID NO: 9, and the corresponding light chain variable region ( VL) sequence as shown in SEQ ID NO:8 or SEQ ID NO:10.
  • the first functional binding region targets Trop-2; the first functional binding region comprises an antigen-binding fragment of a specific anti-Trop-2 monoclonal antibody.
  • the heavy chain variable region (VH) sequence of the specific anti-Trop-2 monoclonal antibody is as shown in SEQ ID NO: 11
  • the light chain variable region (VL) sequence is as shown in SEQ ID NO:12 shown.
  • the length of the linker sequence is no more than 20 amino acid residues, and the linker sequence is selected from (GGGGS)n, (GGGS)n, (GGS)n, (G)n, (GS) n, (EAAAK)n, or (XP)n, n is a natural number; the amino acid sequence of the linker sequence is, for example, shown in SEQ ID NO: 13-16.
  • the bispecific recombinant protein is composed of A chain and B chain; wherein, the structure of A chain is VH-CH1-FcA region, and the structure of B chain is VL-CL-second functional binding region - FcB region.
  • the structure of the A chain is VH-CL-FcA region
  • the structure of the B chain is VL-CH1-second functional binding region-FcB region.
  • the structure of the A chain is VL-CH1-FcA region
  • the structure of the B chain is VH-CL-second functional binding region-FcB region.
  • the structure of the A chain is VL-CL-FcA region
  • the structure of the B chain is VH-CH1-second functional binding region-FcB region.
  • the A chain and the B chain are combined by one or more selected from intermolecular force, covalent bond and salt bond.
  • a second aspect of the present invention provides a bispecific recombinant protein, which includes a first functional binding region, a second functional binding region and an Fc region; the first functional binding region includes a heavy chain variable region (VH), light chain variable region (VL), heavy chain constant region 1 (CH1) and light chain constant region (CL); the second functional binding region comprises interferon, its truncation or its mutant
  • the bispecific recombinant protein is composed of a first arm and a second arm, and the first arm is composed of a chain 1 and a chain 2; wherein, the structure of the chain 1 is a VH-CH1-FcA region, and the chain The structure of 2 is VL-CL; or, the structure of chain 1 is VH-CL-FcA region, and the structure of chain 2 is VL-CH1; or, the structure of chain 1 is VL-CH1-FcA region , the structure of the chain 2 is VH-CL; or, the structure of the chain 1 is VL-CL-F
  • the first functional binding region comprises an antigen-binding fragment of a specific anti-GPC3 monoclonal antibody; when the first functional binding region targets PD-L1, the The first functional binding region comprises an antigen-binding fragment of a specific anti-PD-L1 monoclonal antibody; when the first functional binding region targets BCMA, the first functional binding region comprises an antigen of a specific anti-BCMA monoclonal antibody Binding fragment; when the first functional binding region targets Trop-2, the first functional binding region comprises an antigen-binding fragment of a specific anti-Trop-2 monoclonal antibody.
  • the amino acid sequence of VH comprises SEQ ID NO:1
  • the amino acid sequence of CH1 comprises SEQ ID NO:31
  • VL The amino acid sequence of CL comprises SEQ ID NO:2
  • the amino acid sequence of CL comprises SEQ ID NO:32.
  • the amino acid sequence of VH comprises SEQ ID NO:5
  • the amino acid sequence of CH1 comprises SEQ ID NO:31
  • the amino acid sequence of VL comprises SEQ ID NO:6
  • the amino acid sequence of CL comprises SEQ ID NO:32.
  • the amino acid sequence of VH comprises SEQ ID NO: 7 or 9, and the amino acid sequence of CH1 comprises SEQ ID NO: 31
  • the amino acid sequence of VL comprises SEQ ID NO: 8 or 10
  • the amino acid sequence of CL comprises 32.
  • the amino acid sequence of VH comprises SEQ ID NO: 11
  • the amino acid sequence of CH1 comprises SEQ ID NO: 31
  • the amino acid sequence of VL comprises SEQ ID NO:12
  • the amino acid sequence of CL comprises SEQ ID NO:32.
  • the Fc region of the bispecific recombinant protein is an Fc region from IgG1, IgG2, IgG3 or IgG4; the Fc region comprises a natural sequence or a non-natural sequence.
  • the Fc region is an Fc region from human IgG1, IgG2, IgG3 or IgG4.
  • the FcA region and the FcB region are combined through knobs-into-holes.
  • the amino acid sequence of the FcA region comprises SEQ ID NO:25
  • the amino acid sequence of the FcB region comprises SEQ ID NO:25
  • the amino acid sequence of the FcA region comprises SEQ ID NO:26
  • the amino acid sequence of the FcB region comprises SEQ ID NO:27.
  • the amino acid sequence of the FcA region comprises SEQ ID NO:27
  • the amino acid sequence of the FcB region comprises SEQ ID NO:26.
  • the amino acid sequence of the FcA region comprises SEQ ID NO:28
  • the amino acid sequence of the FcB region comprises SEQ ID NO:28
  • the amino acid sequence of the FcA region comprises SEQ ID NO:29
  • the amino acid sequence of the FcB region comprises SEQ ID NO:30.
  • the amino acid sequence of the FcA region comprises SEQ ID NO:30
  • the amino acid sequence of the FcB region comprises SEQ ID NO:29.
  • the first functional region targets GPC3; in the A chain, the amino acid sequence of VH includes SEQ ID NO:1, the amino acid sequence of VL includes SEQ ID NO:2, and the amino acid sequence of CH1 includes SEQ ID NO:31, the amino acid sequence of CL comprises SEQ ID NO:32, the amino acid sequence of the FcA region comprises SEQ ID NO:26 or SEQ ID NO:27;
  • the amino acid sequence of VH comprises SEQ ID NO:1
  • the amino acid sequence of VL comprises SEQ ID NO:2
  • the amino acid sequence of CH1 comprises SEQ ID NO:31
  • the amino acid sequence of CL comprises SEQ ID NO:32
  • the amino acid sequence of the second functional binding region comprises SEQ ID NO: 17, 18, 19, 21 or 22, and the amino acid sequence of the FcB region comprises SEQ ID NO: 27 or SEQ ID NO: 26.
  • the C-terminal of SEQ ID NO:2 or SEQ ID NO:1 is directly connected to the N-terminal of SEQ ID NO:32, and the C-terminal of SEQ ID NO:32 Connected to the N-terminus of SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 21 or SEQ ID NO: 22 through a linker sequence, the linker sequence is selected from SEQ ID NO: 13- Any of 16.
  • amino acid sequence of the A chain is composed of SEQ ID NO: 1, 31 and FcA region sequentially connected
  • the amino acid sequence of the B chain is composed of SEQ ID NO: 2, 32, 13, 17 and FcB region sequentially connected;
  • amino acid sequence of the B chain is composed of SEQ ID NO: 2, 32, 13, 18 and FcB region sequentially connected;
  • amino acid sequence of the B chain is composed of SEQ ID NO: 2, 32, 13, 19 and FcB region sequentially connected;
  • amino acid sequence of the B chain is composed of SEQ ID NO: 2, 32, 13, 20 and FcB region sequentially connected;
  • amino acid sequence of the B chain is composed of SEQ ID NO: 2, 32, 13, 21 and FcB region connected in sequence;
  • amino acid sequence of the B chain is composed of SEQ ID NO: 2, 32, 13, 22 and FcB region connected sequentially;
  • amino acid sequence of the B chain is composed of SEQ ID NO: 2, 32, 14, 17 and FcB region sequentially connected;
  • amino acid sequence of the B chain is composed of SEQ ID NO: 2, 32, 14, 18 and FcB region sequentially connected;
  • amino acid sequence of the B chain is composed of SEQ ID NO: 2, 32, 14, 19 and FcB region sequentially connected;
  • amino acid sequence of the B chain is composed of SEQ ID NO: 2, 32, 14, 20 and FcB region sequentially connected;
  • amino acid sequence of the B chain is composed of SEQ ID NO: 2, 32, 14, 21 and FcB region connected sequentially;
  • amino acid sequence of the B chain is composed of SEQ ID NO: 2, 32, 14, 22 and FcB region connected in sequence;
  • amino acid sequence of the B chain is composed of SEQ ID NO: 2, 32, 15, 17 and FcB region sequentially connected;
  • amino acid sequence of the B chain is composed of SEQ ID NO: 2, 32, 15, 18 and FcB region sequentially connected;
  • amino acid sequence of the B chain is composed of SEQ ID NO: 2, 32, 15, 19 and FcB region sequentially connected;
  • amino acid sequence of the B chain is composed of SEQ ID NO: 2, 32, 15, 20 and FcB region connected in sequence;
  • amino acid sequence of the B chain is composed of SEQ ID NO: 2, 32, 15, 21 and FcB region sequentially connected;
  • amino acid sequence of the B chain is composed of SEQ ID NO: 2, 32, 15, 22 and FcB region connected in sequence;
  • amino acid sequence of the B chain is composed of SEQ ID NO: 2, 32, 16, 17 and FcB region sequentially connected;
  • amino acid sequence of the B chain is composed of SEQ ID NO: 2, 32, 16, 18 and FcB region sequentially connected;
  • amino acid sequence of the B chain is composed of SEQ ID NO: 2, 32, 16, 19 and FcB region sequentially connected;
  • amino acid sequence of the B chain is composed of SEQ ID NO: 2, 32, 16, 21 and FcB region sequentially connected;
  • amino acid sequence of the B chain is composed of SEQ ID NO: 2, 32, 16, 22 and FcB region sequentially connected.
  • the amino acid sequence of the FcA region is SEQ ID NO:26
  • the amino acid sequence of the FcB region is SEQ ID NO:27.
  • the amino acid sequence of the FcB region is SEQ ID NO:26.
  • the first functional binding region targets PD-L1; in the A chain, the amino acid sequence of VH includes SEQ ID NO: 5, the amino acid sequence of VL includes SEQ ID NO: 6, and the amino acid sequence of CH1
  • the amino acid sequence comprises SEQ ID NO:31
  • the amino acid sequence of CL comprises SEQ ID NO:32
  • the amino acid sequence of the FcA region comprises SEQ ID NO:26 or SEQ ID NO:27 or SEQ ID NO:29 or SEQ ID NO:30;
  • the amino acid sequence of VH comprises SEQ ID NO:5
  • the amino acid sequence of VL comprises SEQ ID NO:6
  • the amino acid sequence of CH1 comprises SEQ ID NO:31
  • the amino acid sequence of CL comprises SEQ ID NO:32
  • the amino acid sequence of the second functional binding region comprises any one of SEQ ID NO:17-22
  • the amino acid sequence of the FcB region comprises SEQ ID NO:27 or SEQ ID NO:26 or SEQ ID NO:30 or SEQ ID NO:29 .
  • the C-terminal of SEQ ID NO: 6 or SEQ ID NO: 5 is directly connected to the N-terminal of SEQ ID NO: 32, and the C-terminal of SEQ ID NO: 32 It is connected with the N-terminus of any one of SEQ ID NOs: 17-22 through a linker sequence, and the linker sequence is selected from any one of SEQ ID NOs: 13-16.
  • amino acid sequence of the A chain is composed of SEQ ID NO: 5, 31, and FcA region sequentially connected
  • the amino acid sequence of the B chain is composed of SEQ ID NO: 6, 32, 13, 17 and FcB region connected in sequence;
  • amino acid sequence of the B chain is composed of SEQ ID NO: 6, 32, 13, 18 and FcB region connected sequentially;
  • amino acid sequence of the B chain is composed of SEQ ID NO: 6, 32, 13, 19 and FcB region sequentially connected;
  • amino acid sequence of the B chain is composed of SEQ ID NO: 6, 32, 13, 20 and FcB region sequentially connected;
  • amino acid sequence of the B chain is composed of SEQ ID NO: 6, 32, 13, 21 and FcB region sequentially connected;
  • amino acid sequence of the B chain is composed of SEQ ID NO: 6, 32, 13, 22 and FcB region connected sequentially;
  • amino acid sequence of the B chain is composed of SEQ ID NO: 6, 32, 14, 17 and FcB region sequentially connected;
  • amino acid sequence of the B chain is composed of SEQ ID NO: 6, 32, 14, 18 and FcB region sequentially connected;
  • amino acid sequence of the B chain is composed of SEQ ID NO: 6, 32, 14, 19 and FcB region sequentially connected;
  • amino acid sequence of the B chain is composed of SEQ ID NO: 6, 32, 14, 20 and FcB region sequentially connected;
  • amino acid sequence of the B chain is composed of SEQ ID NO: 6, 32, 14, 21 and FcB region sequentially connected;
  • amino acid sequence of the B chain is composed of SEQ ID NO: 6, 32, 14, 22 and FcB region connected sequentially;
  • amino acid sequence of the B chain is composed of SEQ ID NO: 6, 32, 15, 17 and FcB region connected in sequence;
  • amino acid sequence of the B chain is composed of SEQ ID NO: 6, 32, 15, 18 and FcB region sequentially connected;
  • amino acid sequence of the B chain is composed of SEQ ID NO: 6, 32, 15, 19 and FcB region sequentially connected;
  • amino acid sequence of the B chain is composed of SEQ ID NO: 6, 32, 15, 20 and FcB region sequentially connected;
  • amino acid sequence of the B chain is composed of SEQ ID NO: 6, 32, 15, 21 and FcB region sequentially connected;
  • amino acid sequence of the B chain is composed of SEQ ID NO: 6, 32, 15, 22 and FcB region connected sequentially;
  • amino acid sequence of the B chain is composed of SEQ ID NO: 6, 32, 16, 17 and FcB region connected in sequence;
  • amino acid sequence of the B chain is composed of SEQ ID NO: 6, 32, 16, 18 and FcB region sequentially connected;
  • amino acid sequence of the B chain is composed of SEQ ID NO: 6, 32, 16, 19 and FcB region sequentially connected;
  • amino acid sequence of the B chain is composed of SEQ ID NO: 6, 32, 16, 20 and FcB region sequentially connected;
  • amino acid sequence of the B chain is composed of SEQ ID NO: 6, 32, 16, 21 and FcB region sequentially connected;
  • amino acid sequence of the B chain is composed of SEQ ID NO: 6, 32, 16, 22 and FcB region connected in sequence.
  • the amino acid sequence of the FcA region is SEQ ID NO:26
  • the amino acid sequence of the FcB region is SEQ ID NO:27
  • the amino acid sequence of the FcB region is SEQ ID NO:26
  • the amino acid sequence of the FcA region is SEQ ID NO:29
  • the amino acid sequence of the FcB region is SEQ ID NO:30.
  • the amino acid sequence of the FcA region is SEQ ID NO:30
  • the amino acid sequence of the FcB region is SEQ ID NO:29.
  • the first functional binding region targets BCMA.
  • the amino acid sequence of VH comprises SEQ ID NO: 7 or 9
  • the amino acid sequence of VL comprises SEQ ID NO: 8 or 10
  • the amino acid sequence of CH1 comprises SEQ ID NO: 31.
  • the amino acid sequence of CL comprises SEQ ID NO:32
  • the amino acid sequence of the FcA region comprises SEQ ID NO:26 or SEQ ID NO:27;
  • the amino acid sequence of VH includes SEQ ID NO: 7 or 9
  • the amino acid sequence of VL includes SEQ ID NO: 8 or 10
  • the amino acid sequence of CH1 includes SEQ ID NO: 31
  • the amino acid sequence of CL includes SEQ ID NO:32
  • the amino acid sequence of the second functional binding region comprises any one of SEQ ID NO:17-22
  • the amino acid sequence of the FcB region comprises SEQ ID NO:27 or SEQ ID NO:26.
  • the C-terminal of SEQ ID NO: 7, 8, 9 or 10 in the amino acid sequence of the B chain is directly connected to the N-terminal of SEQ ID NO: 32, and the C-terminal of SEQ ID NO: 32 It is connected with the N-terminus of any one of SEQ ID NOs: 17-22 through a linker sequence, and the linker sequence is selected from any one of SEQ ID NOs: 13-16.
  • amino acid sequence of the A chain is composed of SEQ ID NO: 7, 31, and FcA region sequentially connected
  • the amino acid sequence of the B chain is composed of SEQ ID NO: 8, 32, 13, 17 and FcB region sequentially connected;
  • amino acid sequence of the B chain is composed of SEQ ID NO: 8, 32, 13, 18 and FcB region connected in sequence;
  • amino acid sequence of the B chain is composed of SEQ ID NO: 8, 32, 13, 19 and FcB region sequentially connected;
  • amino acid sequence of the B chain is composed of SEQ ID NO: 8, 32, 13, 20 and FcB region connected in sequence;
  • amino acid sequence of the B chain is composed of SEQ ID NO: 8, 32, 13, 21 and FcB region sequentially connected;
  • amino acid sequence of the B chain is composed of SEQ ID NO: 8, 32, 13, 22 and FcB region connected in sequence;
  • amino acid sequence of the B chain is composed of SEQ ID NO: 8, 32, 14, 17 and FcB region sequentially connected;
  • amino acid sequence of the B chain is composed of SEQ ID NO: 8, 32, 14, 18 and FcB region sequentially connected;
  • amino acid sequence of the B chain is composed of SEQ ID NO: 8, 32, 14, 19 and FcB region sequentially connected;
  • amino acid sequence of the B chain is composed of SEQ ID NO: 8, 32, 14, 20 and FcB region sequentially connected;
  • amino acid sequence of the B chain is composed of SEQ ID NO: 8, 32, 14, 21 and FcB region connected sequentially;
  • amino acid sequence of the B chain is composed of SEQ ID NO: 8, 32, 14, 22 and FcB region connected in sequence;
  • amino acid sequence of the B chain is composed of SEQ ID NO: 8, 32, 15, 17 and FcB region sequentially connected;
  • amino acid sequence of the B chain is composed of SEQ ID NO: 8, 32, 15, 18 and FcB region sequentially connected;
  • amino acid sequence of the B chain is composed of SEQ ID NO: 8, 32, 15, 19 and FcB region connected in sequence;
  • amino acid sequence of the B chain is composed of SEQ ID NO: 8, 32, 15, 20 and FcB region connected in sequence;
  • amino acid sequence of the B chain is composed of SEQ ID NO: 8, 32, 15, 21 and FcB region connected sequentially;
  • amino acid sequence of the B chain is composed of SEQ ID NO: 8, 32, 15, 22 and FcB region connected sequentially;
  • amino acid sequence of the B chain is composed of SEQ ID NO: 8, 32, 16, 17 and FcB region sequentially connected;
  • amino acid sequence of the B chain is composed of SEQ ID NO: 8, 32, 16, 18 and FcB region sequentially connected;
  • amino acid sequence of the B chain is composed of SEQ ID NO: 8, 32, 16, 19 and FcB region sequentially connected;
  • amino acid sequence of the B chain is composed of SEQ ID NO: 8, 32, 16, 20 and FcB region sequentially connected;
  • amino acid sequence of the B chain is composed of SEQ ID NO: 8, 32, 16, 21 and FcB region sequentially connected;
  • amino acid sequence of the B chain is composed of SEQ ID NO: 8, 32, 16, 22 and FcB region connected in sequence.
  • amino acid sequence of the A chain is composed of SEQ ID NO: 9, 31, and FcA region sequentially connected
  • the amino acid sequence of the B chain is composed of SEQ ID NO: 10, 32, 13, 17 and FcB region sequentially connected;
  • amino acid sequence of the B chain is composed of SEQ ID NO: 10, 32, 13, 18 and FcB region sequentially connected;
  • amino acid sequence of the B chain is composed of SEQ ID NO: 10, 32, 13, 19 and FcB region sequentially connected;
  • amino acid sequence of the B chain is composed of SEQ ID NO: 10, 32, 13, 20 and FcB region sequentially connected;
  • amino acid sequence of the B chain is composed of SEQ ID NO: 10, 32, 13, 21 and FcB region sequentially connected;
  • amino acid sequence of the B chain is composed of SEQ ID NO: 10, 32, 13, 22 and FcB region sequentially connected;
  • amino acid sequence of the B chain is composed of SEQ ID NO: 10, 32, 14, 17 and FcB region sequentially connected;
  • amino acid sequence of the B chain is composed of SEQ ID NO: 10, 32, 14, 18 and FcB region sequentially connected;
  • amino acid sequence of the B chain is composed of SEQ ID NO: 10, 32, 14, 19 and FcB region sequentially connected;
  • amino acid sequence of the B chain is composed of SEQ ID NO: 10, 32, 14, 20 and FcB region sequentially connected;
  • amino acid sequence of the B chain is composed of SEQ ID NO: 10, 32, 14, 21 and FcB region sequentially connected;
  • amino acid sequence of the B chain is composed of SEQ ID NO: 10, 32, 14, 22 and FcB region sequentially connected;
  • amino acid sequence of the B chain is composed of SEQ ID NO: 10, 32, 15, 17 and FcB region sequentially connected;
  • amino acid sequence of the B chain is composed of SEQ ID NO: 10, 32, 15, 18 and FcB region sequentially connected;
  • amino acid sequence of the B chain is composed of SEQ ID NO: 10, 32, 15, 19 and FcB region sequentially connected;
  • amino acid sequence of the B chain is composed of SEQ ID NO: 10, 32, 15, 20 and FcB region sequentially connected;
  • amino acid sequence of the B chain is composed of SEQ ID NO: 10, 32, 15, 21 and FcB region sequentially connected;
  • amino acid sequence of the B chain is composed of SEQ ID NO: 10, 32, 15, 22 and FcB region sequentially connected;
  • amino acid sequence of the B chain is composed of SEQ ID NO: 10, 32, 16, 17 and FcB region sequentially connected;
  • amino acid sequence of the B chain is composed of SEQ ID NO: 10, 32, 16, 18 and FcB region sequentially connected;
  • amino acid sequence of the B chain is composed of SEQ ID NO: 10, 32, 16, 19 and FcB region sequentially connected;
  • amino acid sequence of the B chain is composed of SEQ ID NO: 10, 32, 16, 20 and FcB region sequentially connected;
  • amino acid sequence of the B chain is composed of SEQ ID NO: 10, 32, 16, 21 and FcB region sequentially connected;
  • amino acid sequence of the B chain is composed of SEQ ID NO: 10, 32, 16, 22 and FcB region sequentially connected.
  • the amino acid sequence of the FcA region is SEQ ID NO:26
  • the amino acid sequence of the FcB region is SEQ ID NO:27.
  • the amino acid sequence of the FcB region is SEQ ID NO:26.
  • the first functional binding region targets Trop-2; in the A chain, the amino acid sequence of VH comprises SEQ ID NO: 11, the amino acid sequence of VL comprises SEQ ID NO: 12, and the amino acid sequence of CH1
  • the amino acid sequence comprises SEQ ID NO:31
  • the amino acid sequence of CL comprises SEQ ID NO:32
  • the amino acid sequence of the FcA region comprises SEQ ID NO:26 or SEQ ID NO:27;
  • the amino acid sequence of VH comprises SEQ ID NO:11
  • the amino acid sequence of VL comprises SEQ ID NO:12
  • the amino acid sequence of CH1 comprises SEQ ID NO:31
  • the amino acid sequence of CL comprises SEQ ID NO:32
  • the amino acid sequence of the second functional binding region comprises any one of SEQ ID NO:17-22
  • the amino acid sequence of the FcB region comprises SEQ ID NO:27 or SEQ ID NO:26.
  • the C-terminal of SEQ ID NO: 12 or SEQ ID NO: 11 is directly connected to the N-terminal of SEQ ID NO: 32, and the C-terminal of SEQ ID NO: 32 It is connected with the N-terminus of any one of SEQ ID NOs: 17-22 through a linker sequence, and the linker sequence is selected from any one of SEQ ID NOs: 13-16.
  • amino acid sequence of the A chain is composed of SEQ ID NO: 11, 31, and FcA region sequentially connected
  • the amino acid sequence of the B chain is composed of SEQ ID NO: 12, 32, 13, 17 and FcB region sequentially connected;
  • amino acid sequence of the B chain is composed of SEQ ID NO: 12, 32, 13, 18 and FcB region sequentially connected;
  • amino acid sequence of the B chain is composed of SEQ ID NO: 12, 32, 13, 19 and FcB region sequentially connected;
  • amino acid sequence of the B chain is composed of SEQ ID NO: 12, 32, 13, 20 and FcB region sequentially connected;
  • amino acid sequence of the B chain is composed of SEQ ID NO: 12, 32, 13, 21 and FcB region sequentially connected;
  • amino acid sequence of the B chain is composed of SEQ ID NO: 12, 32, 13, 22 and FcB region sequentially connected;
  • amino acid sequence of the B chain is composed of SEQ ID NO: 12, 32, 14, 17 and FcB region sequentially connected;
  • amino acid sequence of the B chain is composed of SEQ ID NO: 12, 32, 14, 18 and FcB region sequentially connected;
  • amino acid sequence of the B chain is composed of SEQ ID NO: 12, 32, 14, 19 and FcB region sequentially connected;
  • amino acid sequence of the B chain is composed of SEQ ID NO: 12, 32, 14, 20 and FcB region sequentially connected;
  • amino acid sequence of the B chain is composed of SEQ ID NO: 12, 32, 14, 21 and FcB region sequentially connected;
  • amino acid sequence of the B chain is composed of SEQ ID NO: 12, 32, 14, 22 and FcB region connected in sequence;
  • amino acid sequence of the B chain is composed of SEQ ID NO: 12, 32, 15, 17 and FcB region sequentially connected;
  • amino acid sequence of the B chain is composed of SEQ ID NO: 12, 32, 15, 18 and FcB region sequentially connected;
  • amino acid sequence of the B chain is composed of SEQ ID NO: 12, 32, 15, 19 and FcB region sequentially connected;
  • amino acid sequence of the B chain is composed of SEQ ID NO: 12, 32, 15, 20 and FcB region sequentially connected;
  • amino acid sequence of the B chain is composed of SEQ ID NO: 12, 32, 15, 21 and FcB region sequentially connected;
  • amino acid sequence of the B chain is composed of SEQ ID NO: 12, 32, 15, 22 and FcB region sequentially connected;
  • amino acid sequence of the B chain is composed of SEQ ID NO: 12, 32, 16, 17 and FcB region sequentially connected;
  • amino acid sequence of the B chain is composed of SEQ ID NO: 12, 32, 16, 18 and FcB region sequentially connected;
  • amino acid sequence of the B chain is composed of SEQ ID NO: 12, 32, 16, 19 and FcB region sequentially connected;
  • amino acid sequence of the B chain is composed of SEQ ID NO: 12, 32, 16, 20 and FcB region sequentially connected;
  • amino acid sequence of the B chain is composed of SEQ ID NO: 12, 32, 16, 21 and FcB region sequentially connected;
  • amino acid sequence of the B chain is composed of SEQ ID NO: 12, 32, 16, 22 and FcB region sequentially connected.
  • the amino acid sequence of the FcA region is SEQ ID NO:26
  • the amino acid sequence of the FcB region is SEQ ID NO:27.
  • the amino acid sequence of the FcB region is SEQ ID NO:26.
  • the first functional binding region targets GPC3
  • the amino acid sequence of chain 1 in the first arm is composed of SEQ ID NO: 1, 31 and FcA region sequentially connected
  • the second The amino acid sequence of chain 2 in one arm is composed of the sequential connection of SEQ ID NO: 2 and 32
  • the amino acid sequence of the second arm is composed of any one of SEQ ID NO: 17-22 and the sequence of the FcB region; when the When the amino acid sequence of the FcA region is SEQ ID NO: 26, the amino acid sequence of the FcB region is SEQ ID NO: 27; or, when the amino acid sequence of the FcA region is SEQ ID NO: 27, the amino acid sequence of the FcB region The amino acid sequence is SEQ ID NO:26.
  • the amino acid sequence of chain 1 in the first arm is composed of SEQ ID NO:5, 31 and FcA region sequentially connected
  • the amino acid sequence of chain 2 in the first arm is composed of SEQ ID NO: 6 and 32 sequentially connected
  • the amino acid sequence of the second arm is composed of any one of SEQ ID NO: 17-22 and the FcB region sequentially connected
  • the amino acid sequence of the FcA region is SEQ ID NO:26
  • the amino acid sequence of the FcB region is SEQ ID NO:27
  • the amino acid sequence of the FcA region is SEQ ID NO:27
  • the amino acid sequence of the FcB region is SEQ ID NO: 26; or, when the amino acid sequence of the FcA region is SEQ ID NO: 29, the amino acid sequence of the FcB region is SEQ ID NO: 30; or, when the FcA When the amino acid sequence of the FcB region is SEQ ID NO:30, the amino acid sequence
  • the first functional binding region targets BCMA
  • the amino acid sequence of chain 1 in the first arm is composed of SEQ ID NO: 7, 31 and FcA region sequentially connected
  • the The amino acid sequence of chain 2 in the first arm is composed of SEQ ID NO: 8, 32 in sequence; or, when the amino acid sequence of chain 1 in the first arm is composed of SEQ ID NO: 9, 31 and FcA region in sequence,
  • the amino acid sequence of chain 2 in the first arm is composed of SEQ ID NO: 10 and 32
  • the amino acid sequence of the second arm is composed of any one of SEQ ID NO: 17-22 and the FcB region;
  • the amino acid sequence of the FcA region is SEQ ID NO:26
  • the amino acid sequence of the FcB region is SEQ ID NO:27
  • the amino acid sequence of the FcA region is SEQ ID NO:27
  • the The amino acid sequence of the FcB region is SEQ ID NO: 26.
  • the amino acid sequence of chain 1 in the first arm is composed of SEQ ID NO: 11, 31 and FcA region sequentially connected
  • the amino acid sequence of chain 2 in the first arm is composed of SEQ ID NO: 12 and 32 sequentially connected
  • the amino acid sequence of the second arm is composed of any one of SEQ ID NO: 17-22 and the FcB region sequentially connected
  • the amino acid sequence of the FcA region is SEQ ID NO:26
  • the amino acid sequence of the FcB region is SEQ ID NO:27; or, when the amino acid sequence of the FcA region is SEQ ID NO:27, the The amino acid sequence of the FcB region is SEQ ID NO: 26.
  • the third aspect of the present invention provides a nucleic acid molecule encoding the bispecific recombinant protein described in the first aspect or the second aspect.
  • the fourth aspect of the present invention provides a recombinant expression vector comprising the nucleic acid molecule as described in the third aspect.
  • the fifth aspect of the present invention provides a recombinant cell obtained by transforming a host cell with the expression vector described in the fourth aspect.
  • the sixth aspect of the present invention provides a method for preparing the bispecific recombinant protein as described in the first aspect or the second aspect, the method comprising using the recombinant cell as described in the fifth aspect to express the bispecific protein Steps for recombinant protein.
  • the seventh aspect of the present invention provides a drug or pharmaceutical composition, which comprises the bispecific recombinant protein as described in the first aspect or the second aspect.
  • the pharmaceutical composition further includes a pharmaceutically acceptable carrier.
  • the eighth aspect of the present invention provides the bispecific recombinant protein described in the first aspect or the second aspect, or the medicine or pharmaceutical composition described in the seventh aspect in the preparation of medicines for treating, adjuvantly treating or preventing diseases use.
  • the disease is selected from tumors, liver diseases and viral infection diseases.
  • the disease is GPC3, BCMA, PD-L1, Trop-2, 5T4, AGS-16, ALK1, ANG-2, B7-H3, B7-H4, c-fms, c- Met, CA6, CD123, CD19, CD20, CD22, CD24, EpCAM, CD30, CD32b, CD37, CD38, CD40, CD52, CD70, CD74, CD79b, CD98, CEA, CEACAM5, CLDN18.2, CLDN6, CS1, CXCR4, DLL-4, EGFR, EGFRvIII, EGP-1, ENPP3, EphA3, ETBR, FGFR2, FN, FR- ⁇ , GCC, GD2, GPNMB, HER2, HER3, HLA-DR, ICAM-1, IGF-1R, IL- 3R, LIV-1, MSLN, MUC16, MUC1, NaPi2b, Connexin-4, Notch 2, Notch 1, PD-L2, PDGFR-
  • the tumor is selected from breast cancer, intestinal cancer, pancreatic cancer, esophageal cancer, ovarian cancer, gastric cancer, prostate cancer, kidney cancer, cervical cancer, myeloma, lymphoma, leukemia, thyroid cancer, uterine cancer cancer, bladder cancer, neuroendocrine cancer, head and neck cancer, liver cancer, nasopharyngeal cancer, testicular cancer, lung cancer, melanoma, skin cancer, sarcoma, glioma, mesothelioma, and myelodysplastic syndrome.
  • the bowel cancer includes colorectal cancer
  • the ovarian cancer includes yolk sac tumor
  • the neuroendocrine cancer includes Merkel cell carcinoma
  • the lung cancer includes small cell lung cancer and non-small cell lung cancer
  • the skin cancer includes basal cell skin cancer and squamous cell skin cancer
  • the sarcoma includes fibrosarcoma protuberance
  • the glioma includes glioblastoma
  • the uterine cancer includes endometrial cancer and Uterine sarcoma.
  • the term "recombinant protein” refers to an artificially designed/constructed protein rather than a naturally occurring protein.
  • the "recombinant” in the “recombinant protein” of the present invention does not represent its production method, it is only used to indicate that the "recombinant protein” does not exist naturally.
  • the recombinant protein of the present invention may be an expressed protein or an assembled protein.
  • antibody generally refers to a protein comprising one or more polypeptides substantially encoded by immunoglobulin genes or fragments of immunoglobulin genes.
  • Immunoglobulin genes can include kappa, lambda, alpha, gamma, delta, epsilon, and mu constant region genes, as well as a myriad of immunoglobulin variable region genes.
  • light chains can be classified as either kappa or lambda.
  • Heavy chains can be classified as gamma, mu, alpha, delta, or epsilon, which in turn define the immunoglobulin classes: IgG, IgM, IgA, IgD, and IgE, respectively.
  • Antibodies used in the present application may have structural units comprising tetramers.
  • Each tetramer can be composed of two identical pairs of polypeptide chains, each pair having one "light” chain (about 25 kD) and one "heavy” chain (about 50-70 kD).
  • the N-terminus of each member may define a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition.
  • the terms light chain variable region (VL) and heavy chain variable region (VH) generally refer to these regions of the light chain and heavy chain, respectively.
  • Antibodies can exist as intact immunoglobulins or as a number of well-characterized fragments produced by digestion with various peptidases or de novo expression (for a more detailed description of other antibody fragments, see Fundamental Immunology, edited by W.E. Paul, Raven Press, N.Y. (1993)).
  • first functional antigen or “antigen of interest” refers to the antigen that binds to the first functional binding region.
  • second functional antigen refers to an antigen that binds to a second functional binding region.
  • fragment crystallizable (Fc) fragment crystallizable, Fc consists of IgG constant region CH2 and CH3 domains and a hinge region.
  • the term "antigen-binding fragment” or “Fab fragment” or “Fab” consists of the variable region (VL) of the light chain, the constant region (CL) of the light chain, the variable region (VH) of the heavy chain, the The constant region 1 (CH1) domain of the chain can bind to the antigen.
  • buttons (knobs) and/or buttons (holes) can be made on one heavy chain, and correspondingly, multiple buttons (holes) and/or buttons (knobs) can be made on another heavy chain. ).
  • antigen-specific refers to a specific antigen or epitope thereof that is selectively recognized by an antigen-binding molecule.
  • substitution or “substitution” or “mutation” when applied to an amino acid residue refers to the substitution of one or more naturally occurring or introduced amino acids with another in a peptide, polypeptide or protein, forming Novel peptides, polypeptides or proteins (as in the term “mutant” or “mutant” herein). Substitutions in a polypeptide or protein can result in reduced or unchanged function of the polypeptide or protein.
  • substitutions can also be "conservative substitutions", which, when referring to amino acid sequences, refer to the substitution of one amino acid residue with another with a different amino acid residue in the side chain with similar physicochemical properties, or for those that are not critical to the activity of the polypeptide Amino acid substitutions.
  • nonpolar side chain amino acid residues such as Met, Ala, Val, Leu and Ile, Pro, Phe, Trp
  • uncharged polar side chain residues such as Cys, Ser, Thr, Asn, Gly, and Gln
  • acidic side chain residues such as Asp, Glu
  • basic side chain amino acids such as His, Lys, and Arg
  • ⁇ -branched side chain amino acids such as Thr, Val, and Ile
  • substitutions, deletions or additions may also be considered “conservative substitutions.”
  • the number of amino acids inserted or deleted may range from about 1 to 5. Conservative substitutions generally do not cause significant changes in the conformational structure of the protein, and thus maintain the biological activity of the protein.
  • double positive expression cell or “target cell” or “target target cell” refers to a cell that can interact with the first functional binding domain and the second functional binding domain at the same time.
  • second functional antigen-specific cell or “non-target cell” or “non-target target cell” refers to a cell that does not interact with the first functional binding domain, but only interacts with the second functional binding domain.
  • interferon generally refers to a signaling protein produced and released by host cells in response to the presence of pathogens, such as viruses, bacteria, parasites or tumor cells.
  • pathogens such as viruses, bacteria, parasites or tumor cells.
  • type I interferon can include IFN ⁇ and IFN ⁇ , and IFN ⁇ can also include IFN ⁇ subtypes, such as IFN ⁇ 2, IFN ⁇ 4, and the like.
  • Type I interferons can inhibit viral replication, have antiparasitic activity, inhibit cell proliferation, stimulate the cytotoxic activity of immune cells, participate in immune regulation, and exhibit antitumor effects.
  • Type II and Type III interferons may include IFN ⁇ , IFN ⁇ 1 (IL-29), IFN ⁇ 2 (IL-28a) and IFN ⁇ 3 (IL-28b).
  • interferon may include full-length interferon or a fragment (e.g., a truncated form) or variant thereof that binds to the corresponding interferon receptor and retains its cell proliferation inhibited biological activity.
  • an interferon can be from any mammalian species. In some embodiments, the interferon is from a species selected from the group consisting of human, equine, bovine, murine, porcine, rabbit, cat, dog, rat, goat, sheep, and non-human primate.
  • interferon alpha includes all natural or recombinant interferon alpha, particularly preferably human interferon alpha, such as recombinant human interferon alpha, including but not limited to IFN- ⁇ 2b (available for example from Schering Corporation, Kenilworth, NJ interferon or Interferon), IFN- ⁇ 2a (such as available from Hoffmann-La Roche, Nutley, NJ interferon); for example a mixture of natural alpha-type interferons including but not limited to IFN-alpha-n1 (for example available from Sumitomo, Japan or Glaxo-Wellcome Ltd., London, Great Britain Interferon ⁇ -n1) or IFN- ⁇ -n3 (such as Alferon® available from Interferon Sciences interferon).
  • IFN- ⁇ 2b available for example from Schering Corporation, Kenilworth, NJ interferon or Interferon
  • IFN- ⁇ 2a such as available from Hoffmann-La Roche, Nutley, NJ interferon
  • IFN- ⁇ 2a such
  • the term “IFN ⁇ ” or “interferon- ⁇ ” also includes any substance having biological activity of IFN ⁇ , such as mutated or modified IFN ⁇ , such as PEG derivative of IFN ⁇ (PEG-IFN ⁇ ).
  • IFN ⁇ or " ⁇ -type interferon” is not limited by any specific source, and can be obtained from commercially available sources or produced by conventional techniques known to those skilled in the art. The production methods include But not limited to biological source extraction method and genetic engineering extraction method, which are described in detail in eg "Pestka S. Arch Biochem Biophys. 1983 Feb 15; 221(1): 1-37".
  • the IFN ⁇ is from a species selected from the group consisting of human, equine, bovine, murine, porcine, rabbit, cat, dog, rat, goat, sheep, and non-human primate.
  • IFN ⁇ 2b or “IFN- ⁇ 2b” or “IFN- ⁇ 2b” or “interferon- ⁇ 2b” is a subtype of IFN- ⁇ , both expressions of interferon- ⁇ 2b.
  • the amino acid sequence of human wild-type interferon- ⁇ 2b containing signal peptide is shown in SEQ ID NO:15.
  • low affinity mutant refers to an IFN ⁇ mutant whose proliferation inhibition or antiviral function activity is unchanged or decreased compared to wild-type IFN ⁇ .
  • linker sequence refers to connecting different functional binding fragments (such as the first functional binding region and the second functional binding region, the first functional binding region or the second functional binding region and the Fc region), Or link the amino acid sequences of different structural domains in the same functional binding fragment.
  • GC33 Codrituzumab
  • Anti-GPC3 mAb are used interchangeably in the present invention to denote the anti-GPC3 antibody Codrituzumab.
  • atezolizumab As used herein, the terms “atezolizumab”, “atezolizumab”, “Anti-PD-L1 mAb” are used interchangeably in the present invention to denote the anti-PD-L1 antibody Atezolizumab.
  • the term "host cell” generally includes a single cell, cell line or cell culture that can be or has been the recipient of a subject plasmid or vector, comprising a polynucleotide disclosed herein, or expressing the present The protein heterodimers of the application (eg, heterodimeric proteins).
  • a host cell can include progeny of a single host cell. The progeny may not necessarily be identical (morphologically or in the total DNA complement of the genome) to the original parent cell due to natural, accidental or deliberate mutations.
  • Host cells may include cells transfected in vitro with the vectors disclosed herein.
  • the host cell may be a bacterial cell such as Escherichia coli (E.
  • the host cell is a mammalian cell.
  • the mammalian cells are CHO cells.
  • vector generally refers to a nucleic acid molecule capable of self-replication in a suitable host, which transfers an inserted nucleic acid molecule into and/or between host cells.
  • the term can include vectors used primarily for the insertion of DNA or RNA into cells, vectors used primarily for the replication of DNA or RNA, and expression vectors used for the transcription and/or translation of DNA or RNA. Also included are vectors that provide more than one of the above functions.
  • An "expression vector” is a polynucleotide that can be transcribed and translated into a polypeptide when introduced into a suitable host cell.
  • “Expression system” generally means a suitable host cell comprising an expression vector capable of producing the desired yield of expression.
  • an effective amount refers to an amount of a composition (eg, a bispecific recombinant protein described herein) sufficient to achieve an intended application, including but not limited to disease treatment.
  • a therapeutically effective amount may vary depending on the intended application (e.g., in vitro or in vivo) or the subject and condition being treated, such as the subject's weight and age, severity of the condition, mode of administration, etc., which may readily to be determined by one of ordinary skill in the art.
  • the term also applies to doses that induce a particular response (eg, target gene induction, proliferation and/or apoptosis) in target cells.
  • the particular dosage will vary depending on the particular compound chosen, the dosing regimen followed, whether it is administered in combination with other compounds, the timing of administration, the tissue to which it is applied, and the physical delivery system in which it is administered.
  • therapeutic benefit generally refers to eradication or lessening of the severity of the underlying condition being treated. Additionally, by eradicating, lessening the severity, or reducing the incidence of one or more physiological symptoms associated with the underlying condition such that improvement is observed in the subject (although the subject may still be afflicted by the underlying condition) Therapeutic benefit.
  • compositions may be administered to subjects who are at risk of developing a particular disease, or who report one or more physical symptoms of a disease, even though a diagnosis of the disease may not have been made.
  • the term "therapeutic effect" generally includes therapeutic and/or prophylactic benefits as described above.
  • Prophylaxis includes delaying or eliminating the onset of a disease or condition, delaying or eliminating the onset of symptoms of a disease or condition, slowing, stopping or reversing the progression of a disease or condition, or any combination thereof.
  • co-administration generally include the administration of two or more agents directed to an animal such that the agents and/or their metabolites are simultaneously present in the subject.
  • Co-administration includes simultaneous administration in separate compositions, administration at different times in separate compositions, or administration in a composition in which both agents are present.
  • cell proliferation generally refers to the phenomenon in which the number of cells changes due to division. For example, cell proliferation can result in an increase in cell number.
  • the term also includes cell growth by which the cell morphology has changed (eg, increased in size), consistent with a proliferative signal.
  • the term “inhibition of proliferation” or “inhibition of cell proliferation” generally refers to a reduction in the growth rate and/or proliferation rate of cancer cells. For example, this can include the death of cancer cells (eg, by apoptosis). In some embodiments, the term may also refer to inhibiting the growth and/or proliferation of a solid tumor and/or inducing a reduction in size or elimination of a tumor.
  • freeze-thaw stability generally refers to the stability of an emulsion system when subjected to alternating changes of freezing and thawing.
  • the term "subject” or “individual” or “animal” or “patient” refers to a human or non-human animal, including a mammal or primates. Mammalian subjects include humans, farm animals, farm animals, and zoo, sporting or pet animals such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, pigs, cows, bears, and the like.
  • in vivo generally refers to an event that occurs within the body of a subject.
  • an in vitro assay generally refers to events that occur outside the body of a subject.
  • an in vitro assay includes any assay performed outside of a subject.
  • In vitro assays include cell-based assays in which dead or living cells are used.
  • In vitro assays also include cell-free assays in which intact cells are not used.
  • administering refers to delivering a therapeutically effective amount of a pharmaceutical composition comprising a recombinant protein or fusion protein of the present invention to a subject.
  • Administration can be systemic or local.
  • Administration can be via an administration device, such as a syringe.
  • the administration methods include but are not limited to embedding, snorting, spraying, injection and the like.
  • Routes of administration include inhalation, intranasal, oral, intravenous, subcutaneous or intramuscular administration, and the like.
  • the present invention has following beneficial effect:
  • novel bispecific recombinant protein comprising interferon, its truncated body or mutant provided by the present invention has two types of structures, and has high safety and freeze-thaw stability while having high activity.
  • the bispecific recombinant protein of the present invention has significantly stronger binding activity, ADCC activity, and proliferation inhibitory activity to target cells that are positive for the target antigen than IFN ⁇ at an equimolar concentration, and it is not effective for non-target cells that are negative for the target antigen.
  • the binding activity and anti-proliferation activity of cells are not obviously weaker than that of IFN ⁇ at equimolar concentrations, and even the anti-proliferation activity of some experimental bispecific recombinant proteins on non-target cells is negative. That is, when the bispecific recombinant protein of the present invention does not have the targeting ability of the target antigen, the activity of IFN ⁇ in the second functional binding region on cells expressing IFN ⁇ receptors is significantly reduced or even no related activity is seen.
  • the C-terminus of the CH1 domain or the C-terminus of the CL domain and the N-terminus of the second functional binding region are connected by different linker sequences to the purpose of the bispecific recombinant protein of the first type of structure of the present invention
  • Antigen-positive target cells have strong anti-proliferation activity, and the anti-proliferation activity (IC50) is higher than that of interferon control; the activity of bispecific recombinant protein with shorter linker sequence is relatively weak, so it can be passed through
  • the length of the linker sequence is adjusted to modulate the activity of the second functional binding domain on cells expressing IFN[alpha] receptors.
  • the bispecific recombinant protein of the present invention containing an IFN- ⁇ 2b low-affinity mutant has a proliferation inhibitory activity on non-target target cells that are negative for the target antigen compared with the wild-type bispecific recombinant protein containing IFN- ⁇ 2b on the target antigen.
  • the decrease degree of the anti-proliferation activity of the negative non-target target cells is equivalent or more significant than that of the target antigen-positive target cells containing the IFN- ⁇ 2b low-affinity mutant bispecific recombinant protein.
  • the potential risk impurities (homodimers of the B chain or the second arm) of the bispecific recombinant protein of the present invention have very weak inhibitory activity on the proliferation of non-target target cells negative for the target antigen, wherein the first type of structural double Compared with the potential risk impurities of the second type of structural bispecific recombinant protein, the specific recombinant protein has weaker proliferation inhibitory activity on non-target target cells negative for the target antigen. Therefore, the potential risk impurities of the bispecific recombinant protein of the present invention have The potential safety risks or potential side effects are extremely low, and the potential safety risks or potential side effects brought by the potential risk impurities of the bispecific recombinant protein with the first type of structure are even lower.
  • the bispecific recombinant protein of the present invention has good freeze-thaw stability. After repeated freeze-thaw 5 times, the purity is above 95%, the appearance is clear, and the freeze-thaw stability is significantly better than that of IFN- ⁇ 2b monomer or PEGylation IFN- ⁇ 2b.
  • the structure of the bispecific recombinant protein of the present invention is highly expandable, and its first functional binding region can be used to construct the antigen-binding fragment of a specific antibody targeting the target antigen according to the needs of clinical indications, which significantly reduces the time required for conventional antibody drug screening. Time-consuming, improve drug screening efficiency, reduce screening costs.
  • the bispecific recombinant protein involved in the present invention can significantly improve the killing effect on target cells, and at the same time significantly reduce the toxic and side effects caused by binding to non-target cells, and has better safety, and Has good freeze-thaw stability.
  • Fig. 1 is a schematic diagram of the structure of the bispecific recombinant protein of the first type of structure in the present invention.
  • Fig. 2 is a schematic diagram of the structure of the bispecific recombinant protein of the second type of structure in the present invention.
  • Fig. 3 is a non-reducing SDS-PAGE electrophoresis image of the bispecific recombinant protein with the first structure of the present invention after affinity capture.
  • Fig. 4 is a reduced SDS-PAGE electrophoresis image of the bispecific recombinant protein with the first structure of the present invention after affinity capture.
  • Fig. 5 is a non-reducing SDS-PAGE electrophoresis diagram of the purified bispecific recombinant protein of the first structure of the present invention.
  • Fig. 6 is a non-reducing SDS-PAGE electrophoresis image of the bispecific recombinant protein of the second structure of the present invention after Kappa purification.
  • Fig. 7 is a graph showing the binding activity of the bispecific recombinant protein (the first type of structure) of the present invention and the control sample in the target target cell HepG2 liver cancer cells determined by flow cytometry.
  • Fig. 8 is a histogram showing the binding activity of the bispecific recombinant protein of the present invention (the first type of structure) and the control sample in the target target cell HuH-7 determined by flow cytometry.
  • Fig. 9 is a graph showing the binding activity of the bispecific recombinant protein (the second type of structure) of the present invention and the control sample in the target cell HepG2 determined by flow cytometry.
  • Fig. 10 is a graph showing the ADCC activity of the bispecific recombinant protein (the first type of structure) of the present invention measured by the LDH method in the target cell HepG2.
  • Fig. 11 is a graph showing the ADCC activity curves of the bispecific recombinant protein of the present invention (the second type of structure) and the control sample in the target cell HepG2 determined by LDH method.
  • Fig. 12 is a graph showing the inhibitory activity of bispecific recombinant proteins (first type of structure) with different linker sequences of the present invention on the proliferation of the target target cell HuH-7.
  • Fig. 13 is a graph showing the growth inhibitory activity of the bispecific recombinant protein (the first type of structure) with the GPC3 antigen targeting function and the control sample on the GPC3-positive target cell HuH-7.
  • Fig. 14 is a graph showing the anti-proliferation activity of the bispecific recombinant protein (the first type of structure) of the present invention on the PD-L1-positive target cell MDA-MB-231.
  • Figure 15 is a graph showing the proliferation inhibitory activity of MDA-MB-231 blocked by the anti-PD-L1 antibody of the bispecific recombinant protein (the first type of structure) of the present invention.
  • Fig. 16 is a graph showing the anti-proliferation activity of the bispecific recombinant protein (the first type of structure) containing different IFN- ⁇ 2b low-affinity mutants against GPC3-positive target cell HuH-7 of the present invention.
  • Fig. 17 is a graph showing the anti-proliferation activity of the bispecific recombinant protein (the first type of structure) containing different IFN- ⁇ 2b low-affinity mutants against GPC3-negative non-target target cell SW480 of the present invention.
  • Fig. 18 is a graph showing the anti-proliferation activity of the bispecific recombinant protein (the first type of structure) containing different IFN- ⁇ 2b low-affinity mutants against GPC3-negative non-target target cell U266 of the present invention.
  • Fig. 19 is a graph showing the anti-proliferation activity of the bispecific recombinant protein (first type of structure) containing different IFN- ⁇ 2b low-affinity mutants against Trop-2-positive target cell OVCAR3 of the present invention.
  • Fig. 20 is a graph showing the anti-proliferation activity of the bispecific recombinant protein (the first type of structure) containing different IFN- ⁇ 2b low-affinity mutants against the Trop-2-positive target cell MDA-MB-231 of the present invention.
  • Fig. 21 is a graph showing the anti-proliferation activity of the bispecific recombinant protein (first type of structure) containing different IFN- ⁇ 2b low-affinity mutants of the present invention on the Trop-2 negative non-target target cell NCI-H929.
  • Fig. 22 is a graph showing the anti-proliferation activity of the bispecific recombinant protein (first type of structure) containing different IFN- ⁇ 2b low-affinity mutants against BCMA-positive target cell NCI-H929 of the present invention.
  • Fig. 23 is a graph showing the anti-proliferation activity of the bispecific recombinant protein (the first type of structure) containing different IFN- ⁇ 2b low-affinity mutants against BCMA-negative non-target target cell Huh-7 of the present invention.
  • Fig. 24 is a graph showing the anti-proliferation activity of the bispecific recombinant protein (first type of structure) containing different IFN- ⁇ 2b low-affinity mutants against BCMA-negative non-target target cell MDA-MB-231 of the present invention.
  • Figure 25 is a graph showing the anti-proliferation activity of the bispecific recombinant protein (first type of structure) containing different IFN- ⁇ 2b low-affinity mutants of the present invention on the PD-L1-positive target cell MDA-MB-231.
  • Fig. 26 is a graph showing the anti-proliferation activity of the bispecific recombinant protein (first type of structure) containing different IFN- ⁇ 2b low-affinity mutants against PD-L1-negative target cell OVCAR-3 of the present invention.
  • Fig. 27 is a graph showing the anti-proliferation activity of the bispecific recombinant protein (the second type of structure) containing different IFN- ⁇ 2b low-affinity mutants against GPC3-positive target cell Huh-7 of the present invention.
  • Fig. 28 is a graph showing the anti-proliferation activity of the bispecific recombinant protein (the second type of structure) containing different IFN- ⁇ 2b low-affinity mutants against GPC3-negative non-target target cell SW480 of the present invention.
  • Fig. 29 is a graph showing the anti-proliferation activity of the bispecific recombinant protein (second structure) containing different IFN- ⁇ 2b low-affinity mutants against GPC3-negative non-target target cell MDA-MB-231 of the present invention.
  • Fig. 30 is a graph showing the anti-proliferation activity of the bispecific recombinant protein (second type of structure) containing different IFN- ⁇ 2b low-affinity mutants against GPC3-negative non-target target cell U266 of the present invention.
  • Fig. 31 is a graph showing the growth inhibitory activity of the potential risk impurities of the bispecific recombinant protein of the present invention on the GPC3-negative non-target target cell MDA-MB-231.
  • Fig. 32 is a graph showing the antitumor efficacy curve of the bispecific recombinant protein of the present invention on the HuH-7 subcutaneous tumor model of BALB/c nude mice.
  • Figure 33 is a histogram of the antitumor effect of the bispecific recombinant protein of the present invention on the HuH-7 subcutaneous tumor model of BALB/c nude mice.
  • Fig. 34 is a histogram of the antitumor effect of the bispecific recombinant protein of the present invention on the PBMC humanized M-NSG mouse MDA-MB-231 model.
  • the structure of the bispecific recombinant protein of the present invention is shown in Figure 1 and Figure 2.
  • the bispecific recombinant protein contains the first functional binding region targeting the target antigen, composed of IFN ⁇ or its low-affinity mutant or retaining the function of IFN ⁇
  • the second functional binding region and the Fc region constituted by the truncation body or mutant thereof.
  • the C-terminal of the second functional binding region is directly connected to the Fc region or connected through a linker sequence
  • the C-terminal of the first functional binding region is directly connected to the Fc region or connected through a linker sequence
  • the variable region of the first functional binding region ( The V region) and the constant region (C region) are connected directly or through a linker sequence
  • the Fc region can be a conventional IgG structure, or knobs-into-holes technology can be used
  • the connection between the first functional binding region and the second functional binding region Relationships have two structures, namely:
  • the N-terminal of the second functional binding region is directly connected to the C-terminal of CL or CH1 that is not connected to the Fc region in the first functional binding region or connected through a linker sequence (as shown in Figure 1);
  • the N-terminal of the second functional binding region is separated from the first functional binding region, that is, the N-terminal of the second functional binding region is not connected to other functional fragments (except signal peptide) (as shown in Figure 2).
  • This example illustrates the design of the bispecific recombinant protein of the present invention by taking the second functional binding region selected from human IFN- ⁇ 2b (Genebank: AAP20099.1) as an example.
  • IFN- ⁇ 2b Human IFN- ⁇ 2b
  • the other subtypes of IFN ⁇ can achieve the same technical effect (British Journal of Pharmacology (2013) 168 1048-1058), and the present invention will not repeat them here.
  • the bispecific recombinant proteins with the first type of structure are shown in Table 1
  • the bispecific recombinant proteins with the second type of structure are shown in Table 2
  • the control samples are shown in Table 3.
  • Table 1 Exemplary molecular structure of bispecific recombinant protein of the first type of structure
  • (H) refers to the heavy chain VH and CH1 structural domains
  • (L) refers to the light chain VL and CL structural domains
  • GC33 represents anti-glypican (Glypican3, GPC3) single Anti-codrituzumab
  • Fc means wild type Fc region
  • Fc1 means Fc region with hole or holes mutation
  • Fc2 means Fc region with knob or knobs mutation.
  • the amino acid sequence of IFN- ⁇ 2b-Fc is composed of SEQ ID NO:17 and SEQ ID NO:25 connected sequentially, and the corresponding signal peptide sequence is as SEQ ID NO:24; the amino acid sequence of IFN- ⁇ 2b is as SEQ ID NO: 17; the amino acid sequence of Codrituzumab is quoted from patent US7919086, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:1, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:2.
  • the amino acid sequence of atezolizumab is quoted from patent US20100203056, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:5, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:6.
  • the amino acid sequence of Palivizumab is quoted from patent WO1994017105, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:3, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:4.
  • the heavy and light chain variable region sequence of Belantamab is quoted from the patent US20190194338, the heavy chain variable region sequence is shown in SEQ ID NO:9, and the light chain variable region sequence is shown in SEQ ID NO:10.
  • the heavy and light chain variable region sequence of Elranatamab is quoted from the patent US20160297885, the heavy chain variable region sequence is shown in SEQ ID NO:7, and the light chain variable region sequence is shown in SEQ ID NO:8.
  • the heavy and light chain variable region sequence of hRS7 is quoted from the patent US7517964, the heavy chain variable region sequence is shown in SEQ ID NO:11, and the light chain variable region sequence is shown in SEQ ID NO:12.
  • Human IgG1 isotype control (B117901) was purchased from Baiying Biotechnology.
  • Recombinantly expressed IFN- ⁇ 2b protein Z03003 was purchased from Nanjing GenScript Biotechnology Co., Ltd.
  • the bispecific recombinant protein expression plasmid was synthesized by GENEWIZ Company after codon optimization according to the protein sequence, connected into the pCDNA3.1 plasmid, and the sequence was verified by sequencing.
  • Sample loading use a sample pump to load the sample, the retention time is 5min, and the loading capacity is ⁇ 30mg/ml;
  • TTM101-LC01 takes TTM101-LC01 as an example to describe the sample purification process of the bispecific recombinant protein with the first type of structure.
  • the expression supernatant of the bispecific recombinant protein TTM101-LC01 uses SULFATE 650F filler (TOSOH) to remove aggregates and other impurities in the sample.
  • TOSOH SULFATE 650F filler
  • Sample loading use a sample pump to load the sample, the retention time is 5min, and the loading capacity is ⁇ 50mg/ml;
  • TTM101-01 This example takes TTM101-01 as an example to describe the sample purification process of the bispecific recombinant protein with the second type of structure.
  • Sample loading use a sample pump to load the sample, the retention time is 5min, and the loading capacity is ⁇ 30mg/ml;
  • Elution use the eluent (0.1M Gly-HCl, pH2.7) to elute the target protein, adjust the pH of the eluent to 5.5 with 1M Tris buffer, and detect the protein purity by SDS-PAGE.
  • eluent 0.1M Gly-HCl, pH2.7
  • the purification method of the control samples Codrituzumab, IFN- ⁇ 2b-Fc and Atezolizumab in Table 3 is the same as 2.1.
  • the theoretical molecular weight of the bispecific recombinant protein shown in Table 1 and Table 2 is about 120kD
  • the molecular weight of the control antibody Codrituzumab shown in Table 3 is 150KD
  • the molecular weight of IFN ⁇ 2b-Fc is 88KD
  • the molecular weight of IFN ⁇ 2b monomer is 19.2KD.
  • the binding affinity of the GPC3-targeting bispecific recombinant protein to the target GPC3-positive liver cancer cell line was determined by flow cytometry.
  • the following method takes TTM101-LC01, TTM101-LC02, TTM101-LC03, and TTM101-01 as examples, and is suitable for double-labeled drugs whose first functional binding domain binds to GPC3 antigen and whose second functional binding domain is IFN- ⁇ 2b or its low-affinity mutants. Detection of specific recombinant proteins.
  • the detected cells are HepG2 cells (Nanjing Kebai Biotechnology Co., Ltd.) and HuH-7 (Shanghai Cell Bank of Chinese Academy of Sciences).
  • the well-grown cells were collected and counted, centrifuged and resuspended with FACS buffer (PBS+2% FBS) to 1 ⁇ 10 6 cells/ml concentration.
  • the cells were added to a 96-well U-plate (product number: 3799, Corning) at 100 ⁇ l/well, and 7 dilutions of bispecific recombinant protein or control protein were added (starting from 100 nM, 3-fold serial dilution, a total of 7 concentrations), incubated at 4°C for 1 hour; after washing with FACS buffer, add goat anti-human IgG (Alexa Fluor488 goat anti-human IgG (H+L), Invitrogen) and incubate at 4°C for 1 hour; wash and resuspend with FACS buffer Afterwards, the fluorescence value was detected by flow cytometry (Attune Nxt, invitrogen).
  • the experimental results are shown in Figures 7 and 8.
  • the bispecific recombinant proteins TTM101-LC01, TTM101-LC02, and TTM101-LC03 with the first type of structure can all be combined with the target cells HepG2 and HuH that simultaneously express GPC3 and IFN ⁇ receptors.
  • -7 cells have a certain binding activity, and the binding of the bispecific recombinant protein to HepG2 and HuH-7 cells has a higher maximum average fluorescence intensity than that of anti-GPC3 mAb (anti-GPC3 mAb, the control sample Codrituzumab).
  • the length of the linker sequence has a weak influence on the binding activity of the bispecific recombinant protein to the target cell, and the binding activity (EC50) of the recombinant protein is relatively lowest when the linker sequence is one GGGGS.
  • the bispecific recombinant protein (TTM101-01) with the second type of structure has a certain binding activity to the target cell HepG2 cells expressing GPC3 and IFN receptors at the same time, and the EC50 is comparable to that of the anti-GPC3 monoclonal antibody (anti -GPC3mAb, namely the control sample Codrituzumab), and compared with the anti-GPC3 monoclonal antibody (anti-GPC3mAb, namely the control sample Codrituzumab), the bispecific recombinant protein (TTM101-01) has a higher maximum mean fluorescence intensity.
  • the ADCC activity of the bispecific recombinant protein against the target GPC3-positive liver cancer cell line was determined by the lactate dehydrogenase (LDH) method.
  • LDH lactate dehydrogenase
  • the following method takes TTM101-LC01, TTM101-LC02, TTM101-LC03, and TTM101-01 as examples, and is suitable for double-labeled drugs whose first functional binding domain binds to GPC3 antigen and whose second functional binding domain is IFN- ⁇ 2b or its low-affinity mutants. Detection of specific recombinant protein ADCC activity.
  • PBMC Human peripheral blood mononuclear cells
  • target cells were the GPC3-positive HepG2 liver cancer cell line
  • LDH detection kit was (CytoTox-ONETM-Homogeneous Membrance Integrity Assay, Promega, G7892).
  • Target cells and effector cells were plated at a ratio of 1:20, and bispecific recombinant protein or control protein (starting at 150nM, 5-fold serial dilution, a total of 8 concentrations) was added, and incubated at 37°C for 4 hours.
  • bispecific recombinant proteins with the first type of structure such as TTM101-LC01, TTM101-LC02, TTM101-LC03
  • bispecific recombinant proteins with the second type of structure such as TTM101- 01
  • the ADCC activity of the bispecific recombinant protein is equivalent to that of anti-GPC3 mAb (anti-GPC3 mAb, ie the control sample Codrituzumab) (see Figure 11).
  • the proliferation inhibitory activity of bispecific recombinant proteins targeting GPC3 on different tumor cell lines was determined by cell titer glo kit (Promega, Cat: G7558).
  • the following method takes TTM101-LC01, TTM101-LC02, and TTM101-LC03 as examples, which are suitable for the detection of bispecific recombinant proteins whose first functional binding domain binds GPC3 antigen and whose second functional binding domain is IFN ⁇ 2b or its low-affinity mutants .
  • GPC3-positive cell line HuH-7 or GPC3-negative tumor cell line U266 purchasedd from Nanjing Kebai Biotechnology Co., Ltd.
  • GPC3-negative tumor cell line SW480 purchased from Nanjing Kebai Biotechnology Co., Ltd.
  • add bispecific recombinant protein or control protein 52nM starting, 10-fold serial dilution, a total of 6 points; or 10nM starting, 5-fold serial dilution, a total of 8 points
  • place CO 2 Incubate at 37°C in an incubator for 3 days add Cell titer glo, and detect signal value with Multimode Plate Reader (PerkinElmer, Envision2105).
  • the bispecific recombinant proteins (such as TTM101-LC01, TTM101-LC02, TTM101-LC03) had higher inhibitory activity (IC50) on the proliferation of GPC3-positive (GPC3+) target target cell HuH-7 than the control IFN- ⁇ 2b (see FIG. 12 ), which is also higher than that of anti-GPC3 monoclonal antibody (anti-GPC3 mAb, that is, the control sample Codrituzumab, which has no inhibitory effect on the proliferation of HuH-7).
  • anti-GPC3 mAb that is, the control sample Codrituzumab, which has no inhibitory effect on the proliferation of HuH-7.
  • the proliferation inhibitory activity of the bispecific recombinant protein TTM101-LC04 lacking the GPC3 targeting function and the Fc fusion protein of IFN- ⁇ 2b on HuH-7 was significantly lower than that of the GPC3-positive target cell.
  • TTM101-LC02 a bispecific recombinant protein with targeting function
  • TTM101-LC02 shows that the binding of bispecific recombinant protein with GPC3 targeting function to GPC3 on the target cell can significantly enhance the proliferation inhibitory activity of IFN- ⁇ 2b without targeting
  • the anti-proliferation activity of IFN- ⁇ 2b of the bispecific recombinant protein targeting target cells is relatively low, revealing that the bispecific recombinant protein of the present invention only has a strong anti-proliferation effect on the target target cells with the target antigen.
  • the effect on non-target target cells that do not have the target antigen is weak or does not bind, indicating that the safety of the bispecific recombinant protein of the present invention is relatively high.
  • the bispecific recombinant proteins of the first type of structure with different linker sequences are effective against GPC3-positive (GPC3+) target cells (taken as HuH-7 as an example) all have stronger anti-proliferation activity than IFN- ⁇ 2b, and bispecific recombinant proteins with different linker sequences have slightly different anti-proliferation activities.
  • Bispecific recombinant proteins containing one GGGGS linker sequence i.e.
  • the proliferation inhibitory activity of bispecific recombinant proteins was significantly lower than that of IFN- ⁇ 2b (that is, the relative activity of the bispecific recombinant protein ⁇ 1), this result shows that in cells that do not express GPC3 (that is, non-target cells that do not express the target antigen), the proliferation inhibitory activity of the bispecific recombinant protein is extremely low, suggesting that Its security is higher.
  • the proliferation inhibitory activity of the bispecific recombinant protein on target cells is at least 700 times that of the bispecific recombinant protein on non-target cells.
  • the proliferation inhibitory activity of bispecific recombinant proteins targeting PD-L1 on different tumor cell lines was determined by cell titer glo kit (Promega, Cat: G7558).
  • the following methods take TTM101-LC05, TTM101-LC06, and TTM101-LC07 as examples, which are suitable for bispecifics whose first functional binding domain binds to PD-L1 antigen and whose second functional binding domain is IFN- ⁇ 2b or its low-affinity mutants Recombinant protein.
  • the PD-L1 positive cell line MDA-MB-231 (purchased from Nanjing Kebai Biotechnology Co., Ltd.) (human breast cancer cells) was plated in a 96-well black bottom transparent plate (Corning, 3904), and bispecific recombinant protein or The control protein (starting at 52nM, 10-fold serial dilution, 6 concentration points in total), was placed in a CO 2 incubator and cultured at 37°C for 3 days, Cell titer glo was added, and the signal value was detected by Multimode Plate Reader (PerkinElmer, Envision2105).
  • bispecific recombinant proteins targeting PD-L1 (TTM101-LC05, TTM101-LC06 and TTM101-LC07) Both have proliferation inhibitory activity, and the activity is significantly higher than that of the control bispecific recombinant protein TTM101-LC04 and IFN- ⁇ 2b without PD-L1 targeting function, indicating that targeting PD-L1 can significantly enhance the proliferation inhibitory activity of IFN- ⁇ 2b .
  • the bispecific recombinant protein with PD-L1 targeting function can bind to PD-L1 on the target target cell MDA-MB-231, which can significantly enhance the proliferation inhibitory activity of IFN- ⁇ 2b without target cell
  • the anti-proliferation activity of IFN- ⁇ 2b of the bispecific recombinant protein that acts on it is relatively low, revealing that the bispecific recombinant protein of the present invention only has a strong anti-proliferation effect on the target cells that have the target antigen, but does not express The non-target target cells of the target antigen have a weak or no binding effect, indicating that the safety of the bispecific recombinant protein of the present invention is relatively high.
  • PD-L1-positive target cells MDA-MB-231 were plated in a 96-well black bottom transparent plate (Corning, 3904), and PD-L1 antibody Atezolizumab or isotype control 200nM was added, and incubated at 37°C for 30 minutes.
  • Add bispecific recombinant protein or control protein initial concentration 20nM, 6-fold dilution, a total of 6 concentration points), place in a CO2 box at 37°C for 3 days, add Cell titer glo, Multimode Plate Reader (PerkinElmer, Envision2105) detection signal value.
  • the proliferation inhibitory activity of TTM101-LC07 was significantly reduced.
  • the proliferation inhibitory activity of the tested cells was significantly decreased, demonstrating from another perspective that the bispecific recombinant protein of the present invention has a weak or non-binding effect on non-target target cells that do not have the ability to bind the target antigen, indicating that the bispecific recombinant protein of the present invention higher security.
  • the present invention also designed A series of bispecific recombinant proteins containing low-affinity mutants of IFN- ⁇ 2b.
  • the mutation design based on TTM101-LC02 is taken as an example to design a bispecific recombinant protein containing IFN- ⁇ 2b low-affinity mutant, and the mutant based on the mutation site of wild-type IFN- ⁇ 2b and IFNAR2 (IFN ⁇ receptor 2)
  • the relative affinity (data refer to Cell.2011 August 19; 146(4):621–632.) is shown in Table 6, and the proliferation inhibitory activity of bispecific recombinant proteins containing different IFN- ⁇ 2b low-affinity mutants was detected, and the experimental method With embodiment 5.
  • IFN- ⁇ 2b mutation site IFNAR2 relative affinity L26A 0.22 L30A 0.0013 A145G 0.03 R149A 0.01 S152A 0.1
  • the results showed that the bispecific recombinant protein possessing IFN- ⁇ 2b low-affinity mutants was effective in GPC3-positive target cells (HuH-7) or GPC3-negative non-target cells (U266, SW480 ), the proliferation inhibitory activities of TTM101-LC02-M2, TTM101-LC02-M3 and TTM101-LC02-M4 were all decreased relative to TTM101-LC02.
  • the IFN- ⁇ 2b-containing low-affinity mutant has the same activity as IFN- ⁇ 2b in the target antigen-positive target cell line of the GPC3-targeting bispecific recombinant protein with the first type of structure, but it does not express the target antigen. Reduced activity in non-target cell lines of interest. It shows that the A145G or R149A mutation has good targeting and high safety in targeting GPC3 or PD-L1 bispecific recombinant protein with the first type of structure.
  • TTM101-LC10, TTM101-LC10-M3 and TTM101-LC10-M4 had better or equivalent proliferation inhibitory activity compared with IFN- ⁇ 2b
  • Trop-2-negative target cells NCI-H929
  • the proliferation inhibitory activities of TTM101-LC10, TTM101-LC10-M3 and TTM101-LC10-M4 were significantly reduced compared with IFN- ⁇ 2b, especially TTM101-LC10-M3 and Compared with IFN- ⁇ 2b, the activity of TTM101-LC10-M4 is more than 20 times lower.
  • the above results show that the A145G or R149A mutation has good targeting and high safety in the Trop-2-targeting bispecific recombinant protein with the first type of structure.
  • TTM101-LC08-M3, TTM101 -LC09-M3 and TTM101-LC08-M4 have comparable proliferation inhibitory activity to IFN- ⁇ 2b, and TTM101-LC09-M4 is about 7 times weaker than IFN- ⁇ 2b.
  • TTM101-LC08-M3, TTM101-LC09-M3, TTM101-LC08-M4, and TTM101-LC09-M4 had significantly lower proliferation inhibitory activity on BCMA-negative off-target target cells (HuH-7 and MDA-MB-231) in IFN- ⁇ 2b. It shows that the A145G or R149A mutation has good targeting and high safety in the BCMA-targeting bispecific recombinant protein with the first type of structure.
  • TTM101-LC07 and TTM101-LC07-M3’s proliferation inhibitory activity increased about 5 times, and TTM101-LC07-M4’s activity was equivalent to IFN- ⁇ 2b’s, but
  • the anti-proliferation activity of OVCAR3 on PD-L1-negative cells was significantly weaker than that of IFN- ⁇ 2b, indicating that A145G or R149A mutations have good targeting and high safety in PD-L1-targeting bispecific recombinant proteins with the first type of structure.
  • the inventors took the A145G and R149A mutations as an example to construct a GPC3-targeted bispecific recombinant protein and a PD-L1-targeted bispecific recombinant protein with the second type of structure.
  • TTM101-01-M3 and TTM101-01-M4 as examples to illustrate the GPC3-targeted bispecific recombinant proteins containing IFN- ⁇ 2b low-affinity mutants with the second type of structure on the GPC3-positive targets.
  • Cells HuH-7 and GPC3-negative non-target target cells SW480, MDA-MB-231 and U266 were tested for proliferation inhibitory activity.
  • the experimental method was the same as in Example 5.
  • the protein to be tested and the control protein were diluted 5 times from 100 nM. 9 gradients.
  • TTM101-01-M3 and TTM101-01-M4 in GPC3-positive target cell HuH-7 proliferation inhibitory activity IC50 and IFN- ⁇ 2b are basically equivalent ( Figure 27); )
  • TTM101-01-M3 and TTM101-01-M4 had more than a hundred times lower inhibitory activity than IFN- ⁇ 2b (Fig. 28-30), especially for U266 and MDA-MB-231, which were lower than IFN- ⁇ 2b More than tens of thousands of times.
  • IFN- ⁇ 2b low-affinity mutants with A145G or R149A mutations were as active as IFN- ⁇ 2b in cell lines targeting GPC3 or PD-L1 with the second type of structure in cell lines positive for the target antigen. Comparable, but significantly less active in off-target cell lines that do not express the antigen of interest, and the R149A mutation was relatively less active. It shows that the A145G or R149A mutation has good targeting and high safety in targeting GPC3 or PD-L1 bispecific recombinant protein with the second type of structure.
  • the bispecific recombinant protein of the present invention containing the IFN- ⁇ 2b low-affinity mutant has an inhibitory activity on the proliferation of non-target target cells negative for the target antigen compared with the wild-type bispecific recombinant protein containing IFN- ⁇ 2b on the target cell.
  • the decrease of the anti-proliferation activity of antigen-negative non-target target cells is equivalent or more significant than that of the target antigen-positive target cells containing the IFN- ⁇ 2b low-affinity mutant bispecific recombinant protein.
  • the present invention takes TTM101-LC02 and TTM101-01 as examples to analyze the impact of potential risk impurities (B chain or second arm homodimer) on Proliferation inhibition of non-target cells of interest.
  • TTM101-01 second arm homodimer inhibited the proliferation of MDA-MB-231 (GPC3-negative cells, non-target cells) by 66.8% (down by about 24.5% compared with IFN- ⁇ 2b), and the homodimer of the B chain of TTM101-LC02 on MDA -
  • the proliferation inhibition rate of MB-231 was only 16.2% (about 75.1% lower than IFN- ⁇ 2b, about 50% lower than IFN- ⁇ 2b-Fc).
  • potential risk impurities have only a very weak effect on non-target target cells, that is, the potential safety risks or potential toxic side effects are extremely low.
  • Example 9 In vivo detection of anti-tumor pharmacodynamic activity of bispecific recombinant protein containing IFN- ⁇ 2b low-affinity mutant
  • a ⁇ 30 mm 3 Huh-7 tumor mass was inoculated on the right side of the neck and back of BALB/c nude mice, and the experimental animals were marked with ear tags at the same time as the only confirmation mark for subsequent experiments. Wait for the tumor to grow, and when the average volume of the tumor reaches 150 mm 3 , start random grouping of mice, with 6 mice in each group. All were administered by intravenous injection, and the administration volume was 10mL/kg. The administration was continued for 3 weeks, twice a week, and a total of 6 administrations were administered.
  • the experimental index is to investigate whether tumor growth is inhibited, delayed or cured.
  • Tumor diameters were measured twice a week with vernier calipers.
  • Statistical analysis including mean and standard error (SEM) of tumor volume at each time point for each group. Comparisons between two groups were analyzed using a one-tailed T test, and GraphPad Prism was used for all data analysis. p ⁇ 0.05 considered significant difference.
  • M-NSG (6-8w, female) was subcutaneously inoculated with MDA-MB-231 cells (number of inoculated cells: 1E7/mouse + 30% Matrigel) as day 0, and the survival status and subcutaneous tumor formation of the mice were observed the next day.
  • mice Seven days after inoculation, PBMC cells (0205C) were injected into the tail vein (the number of inoculated cells: 5E6/monkey), and on the seventh day after PBMC inoculation, the whole blood of the mice was collected to detect hCD45%, according to the average tumor volume ( ⁇ 200mm 3 ) and Ratio of h CD45% The mice were randomly grouped, and the day of the grouping was D0, all of which were administered by intravenous injection, and the administration volume was 10mL/kg, administered continuously for 3 weeks, administered twice a week, and administered 6 times in total .
  • the experimental index is to investigate whether tumor growth is inhibited, delayed or cured.
  • Tumor diameters were measured twice a week with vernier calipers.
  • Statistical analysis including mean and standard error (SEM) of tumor volume at each time point for each group. Comparisons between two groups were analyzed using a one-tailed T test, and GraphPad Prism was used for all data analysis. p ⁇ 0.05 considered significant difference.
  • TTM101-LC07-M3(N297G) inhibited tumor growth in a dose-dependent manner, and the tumor inhibitory effect was better than atezolizumab at the same dose.
  • Dose PEG-IFN- ⁇ 2b dose higher than clinically tolerated dose.
  • TTM101-LC07-M4(N297G) has the same efficacy as 1mg/kg Atezolizumab at the dose level of 0.2mg/kg.
  • the above results indicate that PD-L1 targeting A145G and R149A attenuating mutant IFN- ⁇ 2b can effectively inhibit tumor growth.
  • the existing recombinant human albumin interferon- ⁇ 2b fusion protein has poor freeze-thaw stability and is not suitable for repeated freeze-thaw.
  • pegylated long-acting interferon cannot be frozen and shaken, and has high requirements for transportation and storage conditions.
  • repeated freeze-thaw stability tests were performed on the bispecific recombinant protein with the first type of structure.
  • the samples before and after freeze-thaw were analyzed for purity (size exclusion chromatography, SEC) and appearance.
  • TTM101-LC03 and its mutants have good freeze-thaw stability, and the purity is above 95%. After 5 repeated freeze-thaw cycles, the appearance is clear, indicating that the IFN- ⁇ 2b protein part of the bispecific recombinant protein of the present invention The freeze-thaw stability is significantly better than that of IFN- ⁇ 2b monomer or PEGylated IFN- ⁇ 2b.
  • Targets for example 5T4, AGS-16, ALK1, ANG-2, B7-H3, B7-H4, c-fms, c-Met, CA6, CD123, CD19, CD20, CD22, CD24, EpCAM, CD30, CD32b, CD37, CD38, CD40, CD52, CD70, CD74, CD79b, CD98, CEA, CEACAM5, CLDN18.2, CLDN6, CS1, CXCR4, DLL-4, EGFR, EGFRvIII, EGP- 1.
  • targets for example 5T4, AGS-16, ALK1, ANG-2, B7-H3, B7-H4, c-fms, c-Met, CA6, CD123, CD19, CD20, CD22, CD24, EpCAM, CD30, CD32b, CD37, CD38, CD40, CD52, CD70, CD74, CD79b, CD98, CEA, CEACAM5, CLDN18.2, CLDN6, CS1, CXCR4, DLL-4, EGFR

Abstract

本发明公开了一种双特异性重组蛋白及其用途。所述双特异性重组蛋白包括第一功能结合区、第二功能结合区和Fc区；所述第一功能结合区包括第一链和第二链，所述第一链的C端和所述第二功能结合区的C端分别与Fc区的N端直接或通过接头序列连接；所述第二链的C端与第二功能结合区的N端直接或通过接头序列连接。本发明还公开了该重组蛋白在抗肿瘤、抗病毒和治疗肝病等方面的应用。重组蛋白可显著提高对目标靶细胞的杀伤功效，同时显著降低因对非目标靶细胞结合所导致的毒副作用，具有更佳的安全性，且具有良好的冻融稳定性。

Description

一种双特异性重组蛋白及其用途
本申请要求申请日为2021/8/12的中国专利申请2021109267431的优先权。本申请引用上述中国专利申请的全文。
技术领域
本发明属于生物医药领域，具体涉及一种双特异性重组蛋白及其用途。
背景技术
干扰素是一组具有多种功能的活性蛋白质，主要由单核细胞和淋巴细胞产生的细胞因子，分为I型、II型和III型三类，其中I型干扰素包括IFNα和IFNβ，IFNα还可包含IFNα亚型，例如IFNα2、IFNα4等，II型和III型干扰素可包括IFNγ、IFNλ1(IL-29)、IFNλ2(IL-28a)和IFNλ3(IL-28b)。干扰素具有广谱的抗病毒、影响细胞生长和分化、调节免疫功能等多种生物活性。其中，干扰素α(IFNα)具有广泛的抗病毒、抗肿瘤作用，通过与IFNα受体的结合激活效应细胞，提高自然杀伤细胞等免疫细胞的活性，抑制病毒及肿瘤细胞增殖，诱导肿瘤细胞凋亡等，因此是目前临床上广泛应用的生物制品之一，主要治疗肝病(例如慢性乙型肝炎、丙型肝炎等)、抗病毒(如病毒性呼吸道疾病、皮肤病、血液疾病、妇科疾病等)和特定的肿瘤(如慢性粒细胞白血病、黑色素瘤、淋巴瘤、肾癌等)，以及作为肿瘤和恶性血液病的辅助治疗。
但是，由于IFNα受体广泛表达于人体各组织器官，在临床使用过程中常伴有发热、疲劳、肌痛、肝毒性、骨髓抑制以及神经毒性等毒副作用。大量临床研究数据显示，PEG-IFNα(6μg/kg/week治疗8周；3μg/kg/week维持)在黑色素瘤辅助治疗过程中会导致超过60％以上的患者产生3级以上的严重不良事件(SAE)(Practical Guidelines for the Management of Interferon-a-2b Side Effects in Patients Receiving Adjuvant Treatment for Melanoma.Cancer 2008；112:982–94)，PEG-IFNα治疗慢性髓细胞白血病CML临床最大耐受剂量(MTD)为7.5-9μg/kg(Long-Term Results of the Randomized Phase III Trial EORTC 18991 of Adjuvant Therapy With Pegylated Interferon Alfa-2b Versus Observation in Resected Stage III Melanoma.JOURNAL OF CLINICAL ONCOLOGY,VOLUME 30,NUMBER 31,NOVEMBER 1 2012)，而根据临床前体内外药效实验数据，较佳的抑瘤剂量相较于临床耐受剂量还需至少提高100倍(Treating Cancer with PEG Intron Pharmacokinetic Profile and Dosing Guidelines for an Improved Interferon-Alpha-2b Formulation.TALPAZ et al BLOOD,15SEPTEMBER 2001.VOLUME 98,NUMBER 6)。
此外，干扰素因其产品特性，冻融稳定性一般较差，如重组人白蛋白干扰素-α2b融合蛋白(rHSA-IFN-α2b)反复冻融后聚合体逐渐增加，冻融4次后聚合体含量达到17.91％，重组人白蛋白干扰素-α2b融合蛋白不宜冻融(夏毅等，重组人白蛋白干扰素α-2b融合蛋白的稳定性研究，生物学杂志，2008,25(006):38-40)，以至于对生产、运输及使用的限制较多。
因此急需发明一种高效低毒的抗体细胞因子融合蛋白(即双特异性重组蛋白)，在人体耐受的前提上，显著提升IFNα的治疗剂量，提高抗肿瘤、抗病毒和肝病治疗等效果。同时，该双特异性重组蛋白 具备相较于干扰素更佳的冻融稳定性。
发明内容
本发明所要解决的技术问题是为了克服现有技术中单抗药效不佳以及干扰素α安全性和冻融稳定性上的不足，提供一种双特异性重组蛋白及其用途。本发明的双特异性重组蛋白具有定向调节免疫的作用，可显著提高重组蛋白的目标靶向性，还能显著降低制备过程中生成的含有非目标蛋白靶向非目标脏器、组织、细胞而引起的毒副作用。
本发明通过以下技术方案解决上述技术问题。
本发明的第一方面提供一种双特异性重组蛋白，所述双特异性重组蛋白包括第一功能结合区、第二功能结合区和Fc区；所述第一功能结合区包括第一链和第二链，所述第一链的C端和所述第二功能结合区的C端分别与Fc区的N端直接或通过接头序列连接；所述第二链的C端与第二功能结合区的N端直接或通过接头序列连接；
所述第一功能结合区包括重链可变区(VH)、轻链可变区(VL)、重链恒定区1(CH1)和轻链恒定区(CL)；
所述第二功能结合区包含干扰素、其截短体或其突变体。
本发明一些实施方案中，所述CL或CH1的C末端与所述Fc区的N端直接或通过接头序列连接；相应的CH1或CL的C末端与第二功能结合区的N端直接连接或通过接头序列连接。
本发明一些实施方案中，所述第一链包括VH和CH1、VH和CL、VL和CH1，或VL和CL；所述第二链包括VH和CH1、VH和CL、VL和CH1，或VL和CL。所述第一链与第二链不相同，且第一链与第二链的组合包括VH、CH1、VL和CL四个元件。例如，当所述第一链包括VH和CH1时，第二链包括VL和CL；当所述第一链包括VH和CL时，第二链包括VL和CH1。
本发明一些实施方案中，所述干扰素为I型干扰素，例如为IFNα。
本发明一些较佳实施方案中，所述IFNα选自IFN-α1a、IFN-α1b、IFN-α2a、IFN-α2b、IFN-α4a、IFN-α4b、IFN-α5、IFN-α6、IFN-α7、IFN-α8、IFN-α10、IFN-α14、IFN-α16、IFN-α17和IFN-α21中的一种或多种。
本发明一些具体实施方案中，所述干扰素为IFN-α2b。
本发明一些实施方案中，所述第二功能结合区包含选自如下a1)-a2)任意一种的氨基酸序列：a1)SEQ ID NO:17；a2)SEQ ID NO:17所示的氨基酸序列经过添加、缺失、修饰和/或置换至少1个氨基酸残基获得的氨基酸序列，其单体具有对IFNα受体的结合亲和力并且该结合亲和力不高于a1)单体对IFNα受体的结合亲和力。
本发明一些较佳实施方案中，a2)单体的氨基酸序列为在如SEQ ID NO:17所示的氨基酸序列的相应位点置换得到的氨基酸序列；所述相应位点的置换选自L26A、L30A、A145G、R149A和S152A中的一种或者多种。
本发明一些具体实施方案中，所述a2)单体是单一位点的置换，置换位点分别为L26A、L30A、A145G、R149A或S152A，相应的氨基酸序列分别如SEQ ID NO:18～22所示。
本发明一些实施方案中，所述VH与CH1或CL直接连接或通过接头序列连接；所述VL与CL或CH1直接连接或通过接头序列连接。
本发明一些具体实施方案中，当VL与CL连接时，VH与CH1连接；当VL与CH1连接时，VH与CL连接。
本发明一些实施方案中，所述第一功能结合区靶向肿瘤细胞或免疫细胞。
具体地，所述第一功能结合区靶向下列靶标中的任意一种或多种：GPC3、BCMA、PD-L1、Trop-2、5T4、AGS-16、ALK1、ANG-2、B7-H3、B7-H4、c-fms、c-Met、CA6、CD123、CD19、CD20、CD22、CD24、EpCAM、CD30、CD32b、CD37、CD38、CD40、CD52、CD70、CD74、CD79b、CD98、CEA、CEACAM5、CLDN18.2、CLDN6、CS1、CXCR4、DLL-4、EGFR、EGFRvIII、EGP-1、ENPP3、EphA3、ETBR、FGFR2、FN、FR-α、GCC、GD2、GPNMB、HER2、HER3、HLA-DR、ICAM-1、IGF-1R、IL-3R、LIV-1、MSLN、MUC16、MUC1、NaPi2b、结合素-4、Notch 2、Notch 1、PD-L2、PDGFR-α、PS、PSMA、SLTRK6、STEAP1、TEM1、VEGFR、CD25、CD27L、DKK-1、CSF-1R、MSB0010718C、CD138、Siglec15和CD155。
本发明一些实施方案中，所述第一功能结合区靶向GPC3；所述第一功能结合区包含特异性抗GPC3单克隆抗体的抗原结合片段。
本发明一些具体实施方案中，所述特异性抗GPC3单克隆抗体的重链可变区(VH)序列如SEQ ID NO:1所示，轻链可变区(VL)序列如SEQ ID NO:2所示。
本发明另一些实施方案中，所述第一功能结合区靶向PD-L1；所述第一功能结合区包含特异性抗PD-L1单克隆抗体的抗原结合片段。
本发明一些具体实施方案中，所述特异性抗PD-L1单克隆抗体的重链可变区(VH)序列如SEQ ID NO:5所示，轻链可变区(VL)序列如SEQ ID NO:6所示。
本发明另一些实施方案中，所述第一功能结合区靶向BCMA；所述第一功能结合区包含特异性抗BCMA单克隆抗体的抗原结合片段。
本发明一些具体实施方案中，所述特异性抗BCMA单克隆抗体的重链可变区(VH)序列如SEQ ID NO:7或SEQ ID NO:9所示，相应的轻链可变区(VL)序列如SEQ ID NO:8或SEQ ID NO:10所示。
本发明另一些实施方案中，所述第一功能结合区靶向Trop-2；所述第一功能结合区包含特异性抗Trop-2单克隆抗体的抗原结合片段。
本发明一些具体实施方案中，所述特异性抗Trop-2单克隆抗体的重链可变区(VH)序列如SEQ ID NO:11所示，轻链可变区(VL)序列如SEQ ID NO:12所示。
本发明一些实施方案中，所述接头序列的长度不超过20个氨基酸残基，所述接头序列选自(GGGGS)n、(GGGS)n、(GGS)n、(G)n、(GS)n、(EAAAK)n、或(XP)n，n为自然数；所述接头序列的氨基酸序列例如如SEQ ID NO:13～16所示。
本发明一些实施方案中，所述双特异性重组蛋白由A链和B链组成；其中，A链的结构为VH-CH1-FcA区，B链的结构为VL-CL-第二功能结合区-FcB区。
本发明另一些实施方案中，A链的结构为VH-CL-FcA区，B链的结构为VL-CH1-第二功能结合区-FcB区。
本发明另一些实施方案中，A链的结构为VL-CH1-FcA区，B链的结构为VH-CL-第二功能结合区-FcB区。
本发明另一些实施方案中，A链的结构为VL-CL-FcA区，B链的结构为VH-CH1-第二功能结合区-FcB区。
本发明中，所述A链与B链通过选自分子间作用力、共价键和盐键中一种或多种结合。
本发明的第二方面提供一种双特异性重组蛋白，所述双特异性重组蛋白包括第一功能结合区、第二功能结合区和Fc区；所述第一功能结合区包括重链可变区(VH)、轻链可变区(VL)、重链恒定区1(CH1)和轻链恒定区(CL)；所述第二功能结合区包含干扰素、其截短体或其突变体；所述双特异性重组蛋白由第一臂和第二臂组成，所述第一臂由链1和链2组成；其中，所述链1的结构为VH-CH1-FcA区，所述链2的结构为VL-CL；或者，所述链1的结构为VH-CL-FcA区，所述链2的结构为VL-CH1；或者，所述链1的结构为VL-CH1-FcA区，所述链2的结构为VH-CL；或者，所述链1的结构为VL-CL-FcA区，所述链2的结构为VH-CH1；所述第二臂的结构为第二功能结合区-FcB区，所述FcA区和FcB区组成所述双特异性重组蛋白的Fc区；所述第二臂中，第二功能结合区的氨基酸序列包含SEQ ID NO:17～22中任一种；
当所述第一功能结合区靶向GPC3时，所述第一功能结合区包含特异性抗GPC3单克隆抗体的抗原结合片段；当所述第一功能结合区靶向PD-L1时，所述第一功能结合区包含特异性抗PD-L1单克隆抗体的抗原结合片段；当所述第一功能结合区靶向BCMA时，所述第一功能结合区包含特异性抗BCMA单克隆抗体的抗原结合片段；当所述第一功能结合区靶向Trop-2时，所述第一功能结合区包含特异性抗Trop-2单克隆抗体的抗原结合片段。
本发明一些实施方案中，当所述第一功能结合区靶向GPC3时，所述第一臂中，VH的氨基酸序列包含SEQ ID NO:1，CH1的氨基酸序列包含SEQ ID NO:31，VL的氨基酸序列包含SEQ ID NO:2，CL的氨基酸序列包含SEQ ID NO:32。
本发明一些实施方案中，当所述第一功能结合区靶向PD-L1时，所述第一臂中，VH的氨基酸序列包含SEQ ID NO:5，CH1的氨基酸序列包含SEQ ID NO:31，VL的氨基酸序列包含SEQ ID NO:6，CL的氨基酸序列包含SEQ ID NO:32。
本发明一些实施方案中，当所述第一功能结合区靶向BCMA时，所述第一臂中，VH的氨基酸序列包含SEQ ID NO:7或9，CH1的氨基酸序列包含SEQ ID NO:31，VL的氨基酸序列包含SEQ ID NO:8或10，CL的氨基酸序列包含32。
本发明一些实施方案中，当所述第一功能结合区靶向Trop-2时，所述第一臂中，VH的氨基酸序列包含SEQ ID NO:11，CH1的氨基酸序列包含SEQ ID NO:31，VL的氨基酸序列包含SEQ ID NO:12，CL的氨基酸序列包含SEQ ID NO:32。
本发明中，所述双特异性重组蛋白的Fc区为来自IgG1、IgG2、IgG3或IgG4的Fc区；所述Fc区包含天然序列或非天然序列。
本发明一些实施方案中，所述Fc区为来自人IgG1、IgG2、IgG3或IgG4的Fc区。
本发明一些较佳实施方案中，所述FcA区和FcB区通过knobs-into-holes结合。
本发明一些具体实施方案中，当所述FcA区的氨基酸序列包含SEQ ID NO:25时，所述FcB区的氨基酸序列包含SEQ ID NO:25。
本发明另一些具体实施方案中，当所述FcA区的氨基酸序列包含SEQ ID NO:26时，所述FcB区的氨基酸序列包含SEQ ID NO:27。
本发明另一些具体实施方案中，当所述FcA区的氨基酸序列包含SEQ ID NO:27时，所述FcB区的氨基酸序列包含SEQ ID NO:26。
本发明另一些具体实施方案中，当所述FcA区的氨基酸序列包含SEQ ID NO:28时，所述FcB区的氨基酸序列包含SEQ ID NO:28。
本发明另一些具体实施方案中，当所述FcA区的氨基酸序列包含SEQ ID NO:29时，所述FcB区的氨基酸序列包含SEQ ID NO:30。
本发明另一些具体实施方案中，当所述FcA区的氨基酸序列包含SEQ ID NO:30时，所述FcB区的氨基酸序列包含SEQ ID NO:29。
本发明一些实施方案中，所述第一功能区靶向GPC3；所述A链中，VH的氨基酸序列包含SEQ ID NO:1，VL的氨基酸序列包含SEQ ID NO:2，CH1的氨基酸序列包含SEQ ID NO:31，CL的氨基酸序列包含SEQ ID NO:32，FcA区的氨基酸序列包含SEQ ID NO:26或SEQ ID NO:27；
所述B链中，VH的氨基酸序列包含SEQ ID NO:1，VL的氨基酸序列包含SEQ ID NO:2，CH1的氨基酸序列包含SEQ ID NO:31，CL的氨基酸序列包含SEQ ID NO:32，第二功能结合区的氨基酸序列包含SEQ ID NO:17、18、19、21或22，FcB区的氨基酸序列包含SEQ ID NO:27或SEQ ID NO:26。
本发明一些具体实施方案中，所述B链的氨基酸序列中SEQ ID NO:2或SEQ ID NO:1的C端与SEQ ID NO:32的N端直接连接，SEQ ID NO:32的C端与SEQ ID NO:17、SEQ ID NO:18、SEQ ID NO:19、SEQ ID NO:21或SEQ ID NO:22的N端通过接头序列连接，所述接头序列选自SEQ ID NO:13-16中的任一种。
本发明一些实施方案中，当所述A链的氨基酸序列由SEQ ID NO:1、31和FcA区顺序连接组成时，
所述B链的氨基酸序列由SEQ ID NO:2、32、13、17和FcB区顺序连接组成；
或者，所述B链的氨基酸序列由SEQ ID NO:2、32、13、18和FcB区顺序连接组成；
或者，所述B链的氨基酸序列由SEQ ID NO:2、32、13、19和FcB区顺序连接组成；
或者，所述B链的氨基酸序列由SEQ ID NO:2、32、13、20和FcB区顺序连接组成；
或者，所述B链的氨基酸序列由SEQ ID NO:2、32、13、21和FcB区顺序连接组成；
或者，所述B链的氨基酸序列由SEQ ID NO:2、32、13、22和FcB区顺序连接组成；
或者，所述B链的氨基酸序列由SEQ ID NO:2、32、14、17和FcB区顺序连接组成；
或者，所述B链的氨基酸序列由SEQ ID NO:2、32、14、18和FcB区顺序连接组成；
或者，所述B链的氨基酸序列由SEQ ID NO:2、32、14、19和FcB区顺序连接组成；
或者，所述B链的氨基酸序列由SEQ ID NO:2、32、14、20和FcB区顺序连接组成；
或者，所述B链的氨基酸序列由SEQ ID NO:2、32、14、21和FcB区顺序连接组成；
或者，所述B链的氨基酸序列由SEQ ID NO:2、32、14、22和FcB区顺序连接组成；
或者，所述B链的氨基酸序列由SEQ ID NO:2、32、15、17和FcB区顺序连接组成；
或者，所述B链的氨基酸序列由SEQ ID NO:2、32、15、18和FcB区顺序连接组成；
或者，所述B链的氨基酸序列由SEQ ID NO:2、32、15、19和FcB区顺序连接组成；
或者，所述B链的氨基酸序列由SEQ ID NO:2、32、15、20和FcB区顺序连接组成；
或者，所述B链的氨基酸序列由SEQ ID NO:2、32、15、21和FcB区顺序连接组成；
或者，所述B链的氨基酸序列由SEQ ID NO:2、32、15、22和FcB区顺序连接组成；
或者，所述B链的氨基酸序列由SEQ ID NO:2、32、16、17和FcB区顺序连接组成；
或者，所述B链的氨基酸序列由SEQ ID NO:2、32、16、18和FcB区顺序连接组成；
或者，所述B链的氨基酸序列由SEQ ID NO:2、32、16、19和FcB区顺序连接组成；
或者，所述B链的氨基酸序列由SEQ ID NO:2、32、16、21和FcB区顺序连接组成；
或者，所述B链的氨基酸序列由SEQ ID NO:2、32、16、22和FcB区顺序连接组成。
上述实施方案中，当所述FcA区的氨基酸序列为SEQ ID NO:26时，所述FcB区的氨基酸序列为SEQ ID NO:27。当所述FcA区的氨基酸序列为SEQ ID NO:27时，所述FcB区的氨基酸序列为SEQ ID NO:26。
本发明一些实施方案中，所述第一功能结合区靶向PD-L1；所述A链中，VH的氨基酸序列包含SEQ ID NO:5，VL的氨基酸序列包含SEQ ID NO:6，CH1的氨基酸序列包含SEQ ID NO:31，CL的氨基酸序列包含SEQ ID NO:32，FcA区的氨基酸序列包含SEQ ID NO:26或SEQ ID NO:27或SEQ ID NO:29或SEQ ID NO:30；
所述B链中，VH的氨基酸序列包含SEQ ID NO:5，VL的氨基酸序列包含SEQ ID NO:6，CH1的氨基酸序列包含SEQ ID NO:31，CL的氨基酸序列包含SEQ ID NO:32，第二功能结合区的氨基酸序列包含SEQ ID NO:17-22中任一种，FcB区的氨基酸序列包含SEQ ID NO:27或SEQ ID NO:26或SEQ ID NO:30或SEQ ID NO:29。
本发明一些具体实施方案中，所述B链的氨基酸序列中SEQ ID NO:6或SEQ ID NO:5的C端与SEQ ID NO:32的N端直接连接，SEQ ID NO:32的C端与SEQ ID NO:17-22中任一种的N端通过接头序列连接，所述接头序列选自SEQ ID NO:13-16中的任一种。
本发明一些实施方案中，当所述A链的氨基酸序列由SEQ ID NO:5、31、FcA区顺序连接组成时，
所述B链的氨基酸序列由SEQ ID NO:6、32、13、17和FcB区顺序连接组成；
或者，所述B链的氨基酸序列由SEQ ID NO:6、32、13、18和FcB区顺序连接组成；
或者，所述B链的氨基酸序列由SEQ ID NO:6、32、13、19和FcB区顺序连接组成；
或者，所述B链的氨基酸序列由SEQ ID NO:6、32、13、20和FcB区顺序连接组成；
或者，所述B链的氨基酸序列由SEQ ID NO:6、32、13、21和FcB区顺序连接组成；
或者，所述B链的氨基酸序列由SEQ ID NO:6、32、13、22和FcB区顺序连接组成；
或者，所述B链的氨基酸序列由SEQ ID NO:6、32、14、17和FcB区顺序连接组成；
或者，所述B链的氨基酸序列由SEQ ID NO:6、32、14、18和FcB区顺序连接组成；
或者，所述B链的氨基酸序列由SEQ ID NO:6、32、14、19和FcB区顺序连接组成；
或者，所述B链的氨基酸序列由SEQ ID NO:6、32、14、20和FcB区顺序连接组成；
或者，所述B链的氨基酸序列由SEQ ID NO:6、32、14、21和FcB区顺序连接组成；
或者，所述B链的氨基酸序列由SEQ ID NO:6、32、14、22和FcB区顺序连接组成；
或者，所述B链的氨基酸序列由SEQ ID NO:6、32、15、17和FcB区顺序连接组成；
或者，所述B链的氨基酸序列由SEQ ID NO:6、32、15、18和FcB区顺序连接组成；
或者，所述B链的氨基酸序列由SEQ ID NO:6、32、15、19和FcB区顺序连接组成；
或者，所述B链的氨基酸序列由SEQ ID NO:6、32、15、20和FcB区顺序连接组成；
或者，所述B链的氨基酸序列由SEQ ID NO:6、32、15、21和FcB区顺序连接组成；
或者，所述B链的氨基酸序列由SEQ ID NO:6、32、15、22和FcB区顺序连接组成；
或者，所述B链的氨基酸序列由SEQ ID NO:6、32、16、17和FcB区顺序连接组成；
或者，所述B链的氨基酸序列由SEQ ID NO:6、32、16、18和FcB区顺序连接组成；
或者，所述B链的氨基酸序列由SEQ ID NO:6、32、16、19和FcB区顺序连接组成；
或者，所述B链的氨基酸序列由SEQ ID NO:6、32、16、20和FcB区顺序连接组成；
或者，所述B链的氨基酸序列由SEQ ID NO:6、32、16、21和FcB区顺序连接组成；
或者，所述B链的氨基酸序列由SEQ ID NO:6、32、16、22和FcB区顺序连接组成。
上述实施方案中，当所述FcA区的氨基酸序列为SEQ ID NO:26时，所述FcB区的氨基酸序列为SEQ ID NO:27。当所述FcA区的氨基酸序列为SEQ ID NO:27时，所述FcB区的氨基酸序列为SEQ ID NO:26。当所述FcA区的氨基酸序列为SEQ ID NO:29时，所述FcB区的氨基酸序列为SEQ ID NO:30。当所述FcA区的氨基酸序列为SEQ ID NO:30时，所述FcB区的氨基酸序列为SEQ ID NO:29。
本发明一些实施方案中，所述第一功能结合区靶向BCMA。
本发明一些较佳实施方案中，所述A链中，VH的氨基酸序列包含SEQ ID NO:7或9，VL的氨基酸序列包含SEQ ID NO:8或10，CH1的氨基酸序列包含SEQ ID NO:31，CL的氨基酸序列包含SEQ ID NO:32，FcA区的氨基酸序列包含SEQ ID NO:26或SEQ ID NO:27；
所述B链中，VH的氨基酸序列包含SEQ ID NO:7或9，VL的氨基酸序列包含SEQ ID NO:8或10，CH1的氨基酸序列包含SEQ ID NO:31，CL的氨基酸序列包含SEQ ID NO:32，第二功能结合区的氨基酸序列包含SEQ ID NO:17-22中任一种，FcB区的氨基酸序列包含SEQ ID NO:27或SEQ ID NO:26。
本发明一些具体实施方案中，所述B链的氨基酸序列中SEQ ID NO:7、8、9或10的C端与SEQ ID NO:32的N端直接连接，SEQ ID NO:32的C端与SEQ ID NO:17-22中任一种的N端通过接头序列连接，所述接头序列选自SEQ ID NO:13-16中的任一种。
本发明一些实施方案中，当所述A链的氨基酸序列由SEQ ID NO:7、31、FcA区顺序连接组成时，
所述B链的氨基酸序列由SEQ ID NO:8、32、13、17和FcB区顺序连接组成；
或者，所述B链的氨基酸序列由SEQ ID NO:8、32、13、18和FcB区顺序连接组成；
或者，所述B链的氨基酸序列由SEQ ID NO:8、32、13、19和FcB区顺序连接组成；
或者，所述B链的氨基酸序列由SEQ ID NO:8、32、13、20和FcB区顺序连接组成；
或者，所述B链的氨基酸序列由SEQ ID NO:8、32、13、21和FcB区顺序连接组成；
或者，所述B链的氨基酸序列由SEQ ID NO:8、32、13、22和FcB区顺序连接组成；
或者，所述B链的氨基酸序列由SEQ ID NO:8、32、14、17和FcB区顺序连接组成；
或者，所述B链的氨基酸序列由SEQ ID NO:8、32、14、18和FcB区顺序连接组成；
或者，所述B链的氨基酸序列由SEQ ID NO:8、32、14、19和FcB区顺序连接组成；
或者，所述B链的氨基酸序列由SEQ ID NO:8、32、14、20和FcB区顺序连接组成；
或者，所述B链的氨基酸序列由SEQ ID NO:8、32、14、21和FcB区顺序连接组成；
或者，所述B链的氨基酸序列由SEQ ID NO:8、32、14、22和FcB区顺序连接组成；
或者，所述B链的氨基酸序列由SEQ ID NO:8、32、15、17和FcB区顺序连接组成；
或者，所述B链的氨基酸序列由SEQ ID NO:8、32、15、18和FcB区顺序连接组成；
或者，所述B链的氨基酸序列由SEQ ID NO:8、32、15、19和FcB区顺序连接组成；
或者，所述B链的氨基酸序列由SEQ ID NO:8、32、15、20和FcB区顺序连接组成；
或者，所述B链的氨基酸序列由SEQ ID NO:8、32、15、21和FcB区顺序连接组成；
或者，所述B链的氨基酸序列由SEQ ID NO:8、32、15、22和FcB区顺序连接组成；
或者，所述B链的氨基酸序列由SEQ ID NO:8、32、16、17和FcB区顺序连接组成；
或者，所述B链的氨基酸序列由SEQ ID NO:8、32、16、18和FcB区顺序连接组成；
或者，所述B链的氨基酸序列由SEQ ID NO:8、32、16、19和FcB区顺序连接组成；
或者，所述B链的氨基酸序列由SEQ ID NO:8、32、16、20和FcB区顺序连接组成；
或者，所述B链的氨基酸序列由SEQ ID NO:8、32、16、21和FcB区顺序连接组成；
或者，所述B链的氨基酸序列由SEQ ID NO:8、32、16、22和FcB区顺序连接组成。
本发明另一些实施方案中，当所述A链的氨基酸序列由SEQ ID NO:9、31、FcA区顺序连接组成时，
所述B链的氨基酸序列由SEQ ID NO:10、32、13、17和FcB区顺序连接组成；
或者，所述B链的氨基酸序列由SEQ ID NO:10、32、13、18和FcB区顺序连接组成；
或者，所述B链的氨基酸序列由SEQ ID NO:10、32、13、19和FcB区顺序连接组成；
或者，所述B链的氨基酸序列由SEQ ID NO:10、32、13、20和FcB区顺序连接组成；
或者，所述B链的氨基酸序列由SEQ ID NO:10、32、13、21和FcB区顺序连接组成；
或者，所述B链的氨基酸序列由SEQ ID NO:10、32、13、22和FcB区顺序连接组成；
或者，所述B链的氨基酸序列由SEQ ID NO:10、32、14、17和FcB区顺序连接组成；
或者，所述B链的氨基酸序列由SEQ ID NO:10、32、14、18和FcB区顺序连接组成；
或者，所述B链的氨基酸序列由SEQ ID NO:10、32、14、19和FcB区顺序连接组成；
或者，所述B链的氨基酸序列由SEQ ID NO:10、32、14、20和FcB区顺序连接组成；
或者，所述B链的氨基酸序列由SEQ ID NO:10、32、14、21和FcB区顺序连接组成；
或者，所述B链的氨基酸序列由SEQ ID NO:10、32、14、22和FcB区顺序连接组成；
或者，所述B链的氨基酸序列由SEQ ID NO:10、32、15、17和FcB区顺序连接组成；
或者，所述B链的氨基酸序列由SEQ ID NO:10、32、15、18和FcB区顺序连接组成；
或者，所述B链的氨基酸序列由SEQ ID NO:10、32、15、19和FcB区顺序连接组成；
或者，所述B链的氨基酸序列由SEQ ID NO:10、32、15、20和FcB区顺序连接组成；
或者，所述B链的氨基酸序列由SEQ ID NO:10、32、15、21和FcB区顺序连接组成；
或者，所述B链的氨基酸序列由SEQ ID NO:10、32、15、22和FcB区顺序连接组成；
或者，所述B链的氨基酸序列由SEQ ID NO:10、32、16、17和FcB区顺序连接组成；
或者，所述B链的氨基酸序列由SEQ ID NO:10、32、16、18和FcB区顺序连接组成；
或者，所述B链的氨基酸序列由SEQ ID NO:10、32、16、19和FcB区顺序连接组成；
或者，所述B链的氨基酸序列由SEQ ID NO:10、32、16、20和FcB区顺序连接组成；
或者，所述B链的氨基酸序列由SEQ ID NO:10、32、16、21和FcB区顺序连接组成；
或者，所述B链的氨基酸序列由SEQ ID NO:10、32、16、22和FcB区顺序连接组成。
上述实施方案中，当所述FcA区的氨基酸序列为SEQ ID NO:26时，所述FcB区的氨基酸序列为SEQ ID NO:27。当所述FcA区的氨基酸序列为SEQ ID NO:27时，所述FcB区的氨基酸序列为SEQ ID NO:26。
本发明一些实施方案中，所述第一功能结合区靶向Trop-2；所述A链中，VH的氨基酸序列包含SEQ ID NO:11，VL的氨基酸序列包含SEQ ID NO:12，CH1的氨基酸序列包含SEQ ID NO:31，CL的氨基酸序列包含SEQ ID NO:32，FcA区的氨基酸序列包含SEQ ID NO:26或SEQ ID NO:27；
所述B链中，VH的氨基酸序列包含SEQ ID NO:11，VL的氨基酸序列包含SEQ ID NO:12，CH1的氨基酸序列包含SEQ ID NO:31，CL的氨基酸序列包含SEQ ID NO:32，第二功能结合区的氨基酸序列包含SEQ ID NO:17-22中任一种，FcB区的氨基酸序列包含SEQ ID NO:27或SEQ ID NO:26。
本发明一些具体实施方案中，所述B链的氨基酸序列中SEQ ID NO:12或SEQ ID NO:11的C端与SEQ ID NO:32的N端直接连接，SEQ ID NO:32的C端与SEQ ID NO:17-22中任一种的N端通过接头序列连接，所述接头序列选自SEQ ID NO:13-16中的任一种。
本发明一些实施方案中，当所述A链的氨基酸序列由SEQ ID NO:11、31、FcA区顺序连接组成时，
所述B链的氨基酸序列由SEQ ID NO:12、32、13、17和FcB区顺序连接组成；
或者，所述B链的氨基酸序列由SEQ ID NO:12、32、13、18和FcB区顺序连接组成；
或者，所述B链的氨基酸序列由SEQ ID NO:12、32、13、19和FcB区顺序连接组成；
或者，所述B链的氨基酸序列由SEQ ID NO:12、32、13、20和FcB区顺序连接组成；
或者，所述B链的氨基酸序列由SEQ ID NO:12、32、13、21和FcB区顺序连接组成；
或者，所述B链的氨基酸序列由SEQ ID NO:12、32、13、22和FcB区顺序连接组成；
或者，所述B链的氨基酸序列由SEQ ID NO:12、32、14、17和FcB区顺序连接组成；
或者，所述B链的氨基酸序列由SEQ ID NO:12、32、14、18和FcB区顺序连接组成；
或者，所述B链的氨基酸序列由SEQ ID NO:12、32、14、19和FcB区顺序连接组成；
或者，所述B链的氨基酸序列由SEQ ID NO:12、32、14、20和FcB区顺序连接组成；
或者，所述B链的氨基酸序列由SEQ ID NO:12、32、14、21和FcB区顺序连接组成；
或者，所述B链的氨基酸序列由SEQ ID NO:12、32、14、22和FcB区顺序连接组成；
或者，所述B链的氨基酸序列由SEQ ID NO:12、32、15、17和FcB区顺序连接组成；
或者，所述B链的氨基酸序列由SEQ ID NO:12、32、15、18和FcB区顺序连接组成；
或者，所述B链的氨基酸序列由SEQ ID NO:12、32、15、19和FcB区顺序连接组成；
或者，所述B链的氨基酸序列由SEQ ID NO:12、32、15、20和FcB区顺序连接组成；
或者，所述B链的氨基酸序列由SEQ ID NO:12、32、15、21和FcB区顺序连接组成；
或者，所述B链的氨基酸序列由SEQ ID NO:12、32、15、22和FcB区顺序连接组成；
或者，所述B链的氨基酸序列由SEQ ID NO:12、32、16、17和FcB区顺序连接组成；
或者，所述B链的氨基酸序列由SEQ ID NO:12、32、16、18和FcB区顺序连接组成；
或者，所述B链的氨基酸序列由SEQ ID NO:12、32、16、19和FcB区顺序连接组成；
或者，所述B链的氨基酸序列由SEQ ID NO:12、32、16、20和FcB区顺序连接组成；
或者，所述B链的氨基酸序列由SEQ ID NO:12、32、16、21和FcB区顺序连接组成；
或者，所述B链的氨基酸序列由SEQ ID NO:12、32、16、22和FcB区顺序连接组成。
上述实施方案中，当所述FcA区的氨基酸序列为SEQ ID NO:26时，所述FcB区的氨基酸序列为SEQ ID NO:27。当所述FcA区的氨基酸序列为SEQ ID NO:27时，所述FcB区的氨基酸序列为SEQ ID NO:26。
本发明一些实施方案中，当所述第一功能结合区靶向GPC3时，所述第一臂中链1的氨基酸序列由SEQ ID NO:1、31和FcA区顺序连接组成时，所述第一臂中链2的氨基酸序列由SEQ ID NO:2、32顺序连接组成，所述第二臂的氨基酸序列由SEQ ID NO:17-22中任一种和FcB区顺序连接组成；当所述FcA区的氨基酸序列为SEQ ID NO:26时，所述FcB区的氨基酸序列为SEQ ID NO:27；或者，当所述FcA区的氨基酸序列为SEQ ID NO:27时，所述FcB区的氨基酸序列为SEQ ID NO:26。
本发明另一些实施方案中，当所述第一功能结合区靶向PD-L1时，所述第一臂中链1的氨基酸序列由SEQ ID NO:5、31和FcA区顺序连接组成时，所述第一臂中链2的氨基酸序列由SEQ ID NO:6、32顺序连接组成，所述第二臂的氨基酸序列由SEQ ID NO:17-22中任一种和FcB区顺序连接组成；当所述FcA区的氨基酸序列为SEQ ID NO:26时，所述FcB区的氨基酸序列为SEQ ID NO:27；或者，当所述FcA区的氨基酸序列为SEQ ID NO:27时，所述FcB区的氨基酸序列为SEQ ID NO:26；或者，当所述FcA区的氨基酸序列为SEQ ID NO:29时，所述FcB区的氨基酸序列为SEQ ID NO:30；或者，当所述FcA区的氨基酸序列为SEQ ID NO:30时，所述FcB区的氨基酸序列为SEQ ID NO:29。
本发明另一些实施方案中，当所述第一功能结合区靶向BCMA时，所述第一臂中链1的氨基酸序列由SEQ ID NO:7、31和FcA区顺序连接组成时，所述第一臂中链2的氨基酸序列由SEQ ID NO:8、32顺序连接组成；或，所述第一臂中链1的氨基酸序列由SEQ ID NO:9、31和FcA区顺序连接组成时，所述第一臂中链2的氨基酸序列由SEQ ID NO:10、32顺序连接组成；所述第二臂的氨基酸序列由SEQ  ID NO:17-22中任一种和FcB区顺序连接组成；当所述FcA区的氨基酸序列为SEQ ID NO:26时，所述FcB区的氨基酸序列为SEQ ID NO:27；或者，当所述FcA区的氨基酸序列为SEQ ID NO:27时，所述FcB区的氨基酸序列为SEQ ID NO:26。
本发明另一些实施方案中，当所述第一功能结合区靶向Trop-2时，所述第一臂中链1的氨基酸序列由SEQ ID NO:11、31和FcA区顺序连接组成时，所述第一臂中链2的氨基酸序列由SEQ ID NO:12、32顺序连接组成，所述第二臂的氨基酸序列由SEQ ID NO:17-22中任一种和FcB区顺序连接组成，当所述FcA区的氨基酸序列为SEQ ID NO:26时，所述FcB区的氨基酸序列为SEQ ID NO:27；或者，当所述FcA区的氨基酸序列为SEQ ID NO:27时，所述FcB区的氨基酸序列为SEQ ID NO:26。
本发明的第三方面提供一种编码如第一方面或第二方面所述的双特异性重组蛋白的核酸分子。
本发明的第四方面提供一种包含如第三方面所述的核酸分子的重组表达载体。
本发明的第五方面提供一种由第四方面所述的表达载体转化宿主细胞得到的重组细胞。
本发明的第六方面提供一种如第一方面或第二方面所述的双特异性重组蛋白的制备方法，所述方法包括使用如第五方面所述的重组细胞表达得到所述双特异性重组蛋白的步骤。
本发明的第七方面提供一种药物或药物组合物，其包含如第一方面或第二方面所述的双特异性重组蛋白。
本发明一些实施方案中，所述药物组合物还包括药学上可接受的载体。
本发明的第八方面提供一种第一方面或第二方面所述的双特异性重组蛋白或如第七方面所述的药物或药物组合物在制备治疗、辅助治疗或预防疾病的药物中的用途。
本发明一些实施方案中，所述疾病选自肿瘤、肝病和病毒感染疾病。
本发明一些具体实施方案中，所述疾病为GPC3、BCMA、PD-L1、Trop-2、5T4、AGS-16、ALK1、ANG-2、B7-H3、B7-H4、c-fms、c-Met、CA6、CD123、CD19、CD20、CD22、CD24、EpCAM、CD30、CD32b、CD37、CD38、CD40、CD52、CD70、CD74、CD79b、CD98、CEA、CEACAM5、CLDN18.2、CLDN6、CS1、CXCR4、DLL-4、EGFR、EGFRvIII、EGP-1、ENPP3、EphA3、ETBR、FGFR2、FN、FR-α、GCC、GD2、GPNMB、HER2、HER3、HLA-DR、ICAM-1、IGF-1R、IL-3R、LIV-1、MSLN、MUC16、MUC1、NaPi2b、结合素-4、Notch 2、Notch 1、PD-L2、PDGFR-α、PS、PSMA、SLTRK6、STEAP1、TEM1、VEGFR、CD25、CD27L、DKK-1、CSF-1R、MSB0010718C、CD138、Siglec15或CD155阳性的相关疾病。
本发明一些实施方案中，所述肿瘤选自乳腺癌、肠癌、胰腺癌、食管癌、卵巢癌、胃癌、前列腺癌、肾癌、宫颈癌、骨髓瘤、淋巴瘤、白血病、甲状腺癌、子宫癌、膀胱癌、神经内分泌癌、头颈癌、肝癌、鼻咽癌、睾丸癌、肺癌、黑色素瘤、皮肤癌、肉瘤、神经胶质瘤、间皮瘤和骨髓发育不良综合征。
本发明一些具体实施方案中，所述肠癌包括结肠直肠癌，所述卵巢癌包括卵黄囊瘤，所述神经内分泌癌包括梅克尔细胞癌，所述肺癌包括小细胞肺癌和非小细胞肺癌，所述皮肤癌包括基底细胞皮肤癌和鳞状细胞皮肤癌，所述肉瘤包括隆突性纤维肉瘤，所述神经胶质瘤包括胶质母细胞瘤，所述子宫癌包括子宫内膜癌和子宫肉瘤。
如本文所用，术语“重组蛋白”是指人工设计/构建的蛋白，而不是天然存在的蛋白质。本发明的“重 组蛋白”中的“重组”不代表其生产方式，其仅用于表示“重组蛋白”并不天然存在。本发明的重组蛋白可以是表达的蛋白，可以是组装的蛋白。
如本文中所用，术语“抗体”通常是指包含一个或多个基本上由免疫球蛋白基因或免疫球蛋白基因片段编码的多肽的蛋白质。免疫球蛋白基因可以包括κ、λ、α、γ、δ、ε和μ恒定区基因，以及无数的免疫球蛋白可变区基因。如本文所用，轻链可被分类为κ或λ。重链可被分类为γ、μ、α、δ或ε，其依次分别定义免疫球蛋白类别：IgG、IgM、IgA、IgD和IgE。本申请中使用的抗体可具有包含四聚体的结构单元。每个四聚体可由两对相同的多肽链组成，每对具有一条“轻”链(约25kD)和一条“重链”(约50-70kD)。每个成员的N末端可以界定约100至110个或更多个氨基酸的可变区，其主要负责抗原识别。如本文中所用，术语轻链可变区(VL)和重链可变区(VH)通常分别指轻链和重链的这些区域。抗体可作为完整免疫球蛋白存在或作为通过用各种肽酶消化或从头表达产生的许多充分表征的片段存在(关于其它抗体片段的更详细描述，参见Fundamental Immunology，W.E.Paul编辑，Raven Press,N.Y.(1993))。
如本文所用，术语“第一功能抗原”或“目的抗原”指与第一功能结合区结合的抗原。
如本文所用，术语“第二功能抗原”指与第二功能结合区结合的抗原。
如本文所用，术语“Fc区”(fragment crystallizable，Fc)由IgG恒定区CH2和CH3结构域及铰链区组成。
如本文所用，术语“抗原结合片段”或“Fab段”或“Fab”由轻链的可变区(VL)、轻链的恒定区(CL)、重链的可变区(VH)、重链的恒定区1(CH1)结构域组成，可与抗原结合。
如本文所用，术语“Knob(s)-into-Hole(s)技术”或“杵臼”技术或“隆突-入-空穴”技术或“纽扣”技术，是利用基因工程技术，在重链两个CH3结构域引入不同的突变来促使重链发生异源二聚化，一条重链上做一个钮(knob)，在另一条重链上做一个扣(hole)，然后两者优先咬合在一起形成不对称抗体(Ridgway JB,et al.'Knobs-into-holes'engineering ofantibody CH3domains for heavy chain heterodimerization.Protein Engineering,1996,9(7):617-621)。如本领域技术人员所知，一条重链上可以做多个钮(knob)和/或扣(hole)，对应的，另一条重链上可以做多个扣(hole)和/或钮(knob)。
术语“抗原特异性”是指被抗原结合分子选择性地识别的特定抗原或其表位。
如本文所用，术语“取代”或“置换”或“突变”在用于氨基酸残基时是指在肽、多肽或蛋白中将天然存在的或引入的一个或多个氨基酸用另一个取代，形成新的肽、多肽或蛋白(如本文术语“突变体”或“突变型”)。多肽或蛋白中的取代可导致多肽或蛋白功能的减弱或不变。取代还可以是“保守取代”，其针对氨基酸序列时是指将一个氨基酸残基用另一个具有相似理化性质的侧链的不同氨基酸残基取代，或者对那些对多肽的活性并非至关重要的氨基酸的取代。例如，可以在非极性侧链氨基酸残基间(例如Met、Ala、Val、Leu和Ile、Pro、Phe、Trp)、不带电的极性侧链残基间(例如Cys、Ser、Thr、Asn、Gly和Gln)、酸性侧链残基间(例如Asp、Glu)、碱性侧链氨基酸间(例如His、Lys和Arg)、β分支侧链氨基酸间(例如Thr、Val和Ile)、含硫侧链氨基酸间(例如Cys和Met)或芳香侧链残基间(例如Trp、Tyr、His和Phe)进行保守取代。在某些实施方式中，取代、删除或添加也可以认为是“保守取代”。插入或删除的氨基酸的数量的范围可以为约1至5。保守取代通常不引起蛋白构象结构上的显著变化，并且因此能保持蛋白的生物学活性。
如本文所用，术语“双阳表达细胞”或“目标细胞”或“目标靶细胞”指可以同时与第一功能结合区和第二功能结合区作用的细胞。
如本文所用，术语“第二功能抗原单阳细胞”或“非目标细胞”或“非目标靶细胞”指不与第一功能结合区作用、只与第二功能结合区作用的细胞。
如本文中所用，术语“干扰素”(IFN)通常是指响应于病原体(诸如，病毒、细菌、寄生虫或肿瘤细胞)的存在由宿主细胞产生和释放的信号蛋白。有三种主要类型的干扰素(即I型、II型和III型)，其中I型干扰素可包括IFNα和IFNβ，IFNα还可包含IFNα亚型，例如IFNα2、IFNα4等。I型干扰素可抑制病毒复制，具有抗寄生虫活性，抑制细胞增殖，刺激免疫细胞的细胞毒性活性，参与免疫调节，并表现出抗肿瘤效果。II型和III型干扰素可包括IFNγ、IFNλ1(IL-29)、IFNλ2(IL-28a)和IFNλ3(IL-28b)。如本文中所用，术语“干扰素”可包括全长干扰素或者其片段(例如，截短形式)或变体，所述其片段或变体与相应干扰素受体可以结合并保留其细胞增殖抑制的生物活性。如本文中所用，干扰素可来自任何哺乳动物物种。在一些实施方案中，干扰素来自选自人、马、牛、鼠、猪、兔、猫、狗、大鼠、山羊、绵羊和非人灵长类动物的物种。
如本文所用，术语“IFNα”或“α型干扰素”包括所有天然或重组的α型干扰素，特别优选地为人α型干扰素，例如重组人α型干扰素，包括但不限于IFN-α2b(例如可获得自Schering Corporation,Kenilworth,N.J.的
Figure PCTCN2022112300-appb-000001
干扰素或
Figure PCTCN2022112300-appb-000002
干扰素)、IFN-α2a(例如可获得自Hoffmann-La Roche,Nutley,N.J.的
Figure PCTCN2022112300-appb-000003
干扰素)；例如天然α型干扰素的混合物，包括但不限于IFN-α-n1(例如可获得自Sumitomo,Japan的或Glaxo-Wellcome Ltd.,London,Great Britain的
Figure PCTCN2022112300-appb-000004
干扰素α-n1)或IFN-α-n3(例如可获得自Interferon Sciences的Alferon
Figure PCTCN2022112300-appb-000005
干扰素)。在本发明中，术语“IFNα”或“α型干扰素”还包括任何具有IFNα生物学活性的物质，例如突变或修饰过的IFNα，例如IFNα的PEG衍生物(PEG-IFNα)。在本发明中，术语“IFNα”或“α型干扰素”不受任何特定的获得来源的限制，可通过市售来源获得或通过本领域技术人员已知的常规技术产生，所述生产方法包括但不限于生物来源提取法和基因工程提取法，其详细描述于例如“Pestka S.Arch Biochem Biophys.1983 Feb 15；221(1):1-37”。在一些实施方案中，IFNα来自选自人、马、牛、鼠、猪、兔、猫、狗、大鼠、山羊、绵羊和非人灵长类动物的物种。
如本文所用，术语“IFNα2b”或“IFN-α2b”或“IFN-α2b”或“干扰素-α2b”是IFN-α的一种亚型，均是对干扰素-α2b的表述。人源野生型含信号肽的干扰素-α2b氨基酸序列如SEQ ID NO:15所示。
如本文所用，术语“低亲和突变体”指相较野生型IFNα，其增殖抑制或抗病毒功能活性不变或下降的IFNα突变体。
如本文所用，术语“接头序列”或“Linker”是指连接不同功能结合片段(如第一功能结合区和第二功能结合区、第一功能结合区或第二功能结合区和Fc区)，或连接同一功能结合片段内不同结构域的氨基酸序列。
如本文所用，术语“GC33”、“Codrituzumab”、“Anti-GPC3mAb”在本发明中可以互换使用，表示抗GPC3抗体Codrituzumab。
如本文所用，术语“阿特珠单抗”、“Atezolizumab”、“Anti-PD-L1mAb”在本发明中可以互换使用， 表示抗PD-L1抗体Atezolizumab。
如本文所使用的术语“宿主细胞”通常包括可以是或已经是受试者质粒或载体的接受者的单个细胞、细胞系或细胞培养物，其包含本申请公开的多核苷酸，或表达本申请的蛋白质异二聚体(例如，异二聚体蛋白)。宿主细胞可以包括单个宿主细胞的后代。由于天然、偶然或有意的突变，后代可以不一定与原始亲本细胞完全相同(在形态上或在基因组总DNA互补体上)。宿主细胞可包括用本申请公开的载体在体外转染的细胞。宿主细胞可以是细菌细胞(例如大肠杆菌(E.coli))、酵母细胞或其它真核细胞，例如HEK293细胞、COS细胞、中国仓鼠卵巢(CHO)细胞、HeLa细胞或骨髓瘤细胞。在一些实施方案中，宿主细胞是哺乳动物细胞。在一些实施方案中，所述哺乳动物细胞是CHO细胞。
如在本文中所用，术语“载体”通常是指能够在合适的宿主中自我复制的核酸分子，其将插入的核酸分子转移至宿主细胞中和/或在宿主细胞之间转移。该术语可包括主要用于将DNA或RNA插入细胞的载体，主要用于DNA或RNA的复制的载体，以及用于DNA或RNA的转录和/或翻译的表达载体。还包括提供不止一种上述功能的载体。“表达载体”是当被引入合适的宿主细胞时可被转录并翻译成多肽的多核苷酸。“表达系统”通常意味着合适的宿主细胞，其包含能够产生期望的表达产量的表达载体。
术语“有效量”或“治疗有效量”是指足以实现预期应用(包括但不限于疾病治疗)的组合物(例如，本文所述的双特异性重组蛋白)的量。治疗有效量可根据预期的应用(例如，体外或体内)或所治疗的受试者和疾病状况，例如受试者的体重和年龄、疾病状况的严重性、施用方式等而变化，其可以容易地由本领域普通技术人员来确定。该术语还可应用于在靶细胞中诱导特定应答(例如，靶基因诱导、增殖和/或凋亡)的剂量。具体剂量将根据所选择的具体化合物、所遵循的给药方案、其是否与其它化合物组合施用、施用的时间安排、其所施用至的组织以及其所处的物理递送系统而变化。
术语“治疗”或“医治”或“缓解”或“改善”在本文中可互换使用，并且是指获得有益或所需的结果(包括但不限于治疗益处和/或预防益处)的方法。如本文中所用，治疗益处通常是指根除或减轻所治疗的潜在病症的严重性。此外，通过根除、减轻严重性或减少与潜在病症相关的一种或多种生理症状的发生率，以使得在受试者中观察到改善(尽管受试者仍然可能受到潜在病症折磨)来实现治疗益处。对于预防益处，可向处于发展特定疾病的风险中的受试者，或报告疾病的一种或多种生理症状的受试者施用组合物，即使可能尚未进行该疾病的诊断。
如本文中所用，术语“治疗作用”通常包括如上所述的治疗益处和/或预防益处。预防作用包括延迟或消除疾病或病况的出现，延迟或消除疾病或病况的症状的发作，减缓、停止或逆转疾病或病况的进展，或其任何组合。
如本文中所用，术语“共施用”、“与……组合施用”及其语法上等同物通常包括指向动物施用两种或更多种试剂，以使得试剂和/或其代谢物同时存在于受试者中。共施用包括在单独的组合物中同时施用，在单独的组合物中在不同时间施用，或在其中两种试剂均存在的组合物中施用。
如本文中所用，术语“细胞增殖”通常是指细胞数目由于分裂而改变的现象。例如，细胞增殖可导致细胞数目的增加。该术语还包括细胞形态通过其已发生改变(例如，尺寸增加)的细胞生长，其与增殖信号一致。
如本文中所用，术语“增殖抑制”或“抑制细胞增殖”通常是指癌细胞的生长速率和/或增殖速率的降 低。例如，这可包括癌细胞的死亡(例如通过凋亡)。在一些实施方案中，该术语还可指抑制实体瘤的生长和/或增殖和/或诱导肿瘤的尺寸减小或消除。
如本文中所用，术语“冻融稳定性”通常是指乳液体系经受冻结和融化交替变化时的稳定性。
本申请使用的术语“受试者”或“个体”或“动物”或“患者”是指需要诊断、预后、缓解、预防和/或治疗疾病或病症的人或非人动物，包含哺乳动物或灵长类。哺乳类受试者包含人、畜养动物、农场动物，以及动物园、体育或宠物动物，如狗、猫、豚鼠、兔、大鼠、小鼠、马、猪、牛、熊等等。
如本文中所用，术语“体内”通常是指在受试者体内发生的事件。
如本文中所用，术语“体外”通常是指发生在受试者体外的事件。例如，体外测定包括在受试者外进行的任何测定。体外测定包括其中使用死细胞或活细胞的基于细胞的测定。体外测定还包括其中不使用完整细胞的无细胞测定。
如本文所用，术语“施用”是指将治疗有效量的包含本发明的重组蛋白或融合蛋白的药物组合物递送至受试者。施用可以全身施用也可以局部施用。施用可以通过施用装置进行，例如注射器。施用方式包括但不限于包埋、鼻吸、喷雾、注射等。施用途径包括吸入、鼻内、口服、静脉内、皮下或肌内施用等。
与现有技术相比，本发明具有如下有益效果:
本发明提供的两类结构的新的包含干扰素、其截短体或突变体的双特异性重组蛋白，具有高活性的同时，具有高的安全性及冻融稳定性。
对于表达IFNα受体的细胞，其有益效果表现在：
1、本发明的双特异性重组蛋白其对目的抗原阳性的目标靶细胞的结合活性、ADCC活性、增殖抑制活性等皆显著强于等摩尔浓度的IFNα，且其对目的抗原阴性的非目标靶细胞(如正常细胞)的结合活性、增殖抑制活性等均非显而易见性地弱于等摩尔浓度的IFNα，甚至部分实验双特异性重组蛋白对非目标靶细胞的增殖抑制活性检测结果为阴性。即当本发明双特异性重组蛋白不具备目的抗原的靶向能力时，第二功能结合区中的IFNα对表达IFNα受体的细胞的活性显著降低甚至未见相关活性。
2、抗原结合片段中CH1结构域的C末端或CL结构域的C末端与第二功能结合区的N端之间不同的接头序列连接的本发明第一类结构的双特异性重组蛋白对目的抗原阳性的目标靶细胞均具有较强的增殖抑制活性，且增殖抑制活性(IC50)均高于干扰素对照；具有较短接头序列的双特异性重组蛋白，其活性相对较弱，因此可以通过调整接头序列长度来调节第二功能结合区对表达IFNα受体的细胞的活性。
3、含IFN-α2b低亲和突变体的本发明双特异性重组蛋白，其对目的抗原阴性的非目标靶细胞的增殖抑制活性相较含IFN-α2b野生型双特异性重组蛋白对目的抗原阴性的非目标靶细胞的增殖抑制活性的下降程度，相较含IFN-α2b低亲和突变体双特异性重组蛋白在目的抗原阳性的目标靶细胞的下降程度相当或更显著。
4、本发明的双特异性重组蛋白其潜在风险杂质(B链或第二臂的同源二聚体)对目的抗原阴性的非目标靶细胞的增殖抑制活性极弱，其中第一类结构双特异性重组蛋白相较于第二类结构双特异性重组蛋白的潜在风险杂质对目的抗原阴性的非目标靶细胞的增殖抑制活性更弱，因此本发明双特异性重 组蛋白，其潜在风险杂质带来的潜在安全性风险或潜在毒副作用极低，第一类结构的双特异性重组蛋白其潜在风险杂质带来的潜在安全性风险或潜在毒副作用更低。
5、本发明的双特异性重组蛋白具有良好的冻融稳定性，反复冻融5次后，纯度均在95％以上，外观澄清，冻融稳定性明显优于IFN-α2b单体或者PEG化的IFN-α2b。
6、本发明的双特异性重组蛋白结构可拓展性强，其第一功能结合区可根据临床适应症的需要构建靶向目的抗原的特异性抗体的抗原结合片段，显著降低常规抗体药物筛选所耗费的时间，提高药物筛选效率，降低筛选成本。
综上所述，本发明所涉及的双特异性重组蛋白可显著提高对目标靶细胞的杀伤功效，同时显著降低因对非目标靶细胞结合所导致的毒副作用，具有更佳的安全性，且具有良好的冻融稳定性。以下将结合附图对本发明的构思、具体结构及产生的技术效果作进一步说明，以充分地了解本发明的目的、特征和效果。
附图说明
图1是本发明第一类结构的双特异性重组蛋白的结构示意图。
图2是本发明第二类结构的双特异性重组蛋白的结构示意图。
图3是本发明第一类结构的双特异性重组蛋白经亲和捕获后的非还原SDS-PAGE电泳图。
图4是本发明第一类结构的双特异性重组蛋白经亲和捕获后的还原SDS-PAGE电泳图。
图5是本发明第一类结构的双特异性重组蛋白经精纯后的非还原SDS-PAGE电泳图。
图6是本发明第二类结构的双特异性重组蛋白经Kappa纯化后的非还原SDS-PAGE电泳图。
图7是流式细胞术测定本发明的双特异性重组蛋白(第一类结构)与对照样品在目标靶细胞HepG2肝癌细胞的结合活性曲线图。
图8是流式细胞术测定本发明的双特异性重组蛋白(第一类结构)与对照样品在目标靶细胞HuH-7的结合活性柱状图。
图9是流式细胞术测定本发明的双特异性重组蛋白(第二类结构)与对照样品在目标靶细胞HepG2的结合活性曲线图。
图10是LDH方法测定本发明的双特异性重组蛋白(第一类结构)在目标靶细胞HepG2的ADCC活性曲线图。
图11是LDH方法测定本发明的双特异性重组蛋白(第二类结构)与对照样品在目标靶细胞HepG2的ADCC活性曲线图。
图12是本发明不同接头序列的双特异性重组蛋白(第一类结构)对目标靶细胞HuH-7增殖抑制活性曲线图。
图13是具有GPC3抗原靶向功能的双特异性重组蛋白(第一类结构)与对照样品对GPC3阳性的目标靶细胞HuH-7增殖抑制活性曲线图。
图14是本发明的双特异性重组蛋白(第一类结构)对PD-L1阳性的目标靶细胞MDA-MB-231的增殖抑制活性曲线图。
图15是本发明的双特异性重组蛋白(第一类结构)对抗PD-L1抗体封闭后的MDA-MB-231的增殖抑制活性曲线图。
图16是本发明含不同IFN-α2b低亲和突变体的双特异性重组蛋白(第一类结构)对GPC3阳性的目标靶细胞HuH-7增殖抑制活性曲线图。
图17是本发明含不同IFN-α2b低亲和突变体的双特异性重组蛋白(第一类结构)对GPC3阴性的非目标靶细胞SW480增殖抑制活性曲线图。
图18是本发明含不同IFN-α2b低亲和突变体的双特异性重组蛋白(第一类结构)对GPC3阴性的非目标靶细胞U266增殖抑制活性曲线图。
图19是本发明含不同IFN-α2b低亲和突变体的双特异性重组蛋白(第一类结构)对Trop-2阳性的目标靶细胞OVCAR3增殖抑制活性曲线图。
图20是本发明含不同IFN-α2b低亲和突变体的双特异性重组蛋白(第一类结构)对Trop-2阳性的目标靶细胞MDA-MB-231增殖抑制活性曲线图。
图21是本发明含不同IFN-α2b低亲和突变体的双特异性重组蛋白(第一类结构)对Trop-2阴性的非目标靶细胞NCI-H929增殖抑制活性曲线图。
图22是本发明含不同IFN-α2b低亲和突变体的双特异性重组蛋白(第一类结构)对BCMA阳性的目标靶细胞NCI-H929增殖抑制活性曲线图。
图23是本发明含不同IFN-α2b低亲和突变体的双特异性重组蛋白(第一类结构)对BCMA阴性的非目标靶细胞Huh-7增殖抑制活性曲线图。
图24是本发明含不同IFN-α2b低亲和突变体的双特异性重组蛋白(第一类结构)对BCMA阴性的非目标靶细胞MDA-MB-231增殖抑制活性曲线图。
图25是本发明含不同IFN-α2b低亲和突变体的双特异性重组蛋白(第一类结构)对PD-L1阳性的目标靶细胞MDA-MB-231增殖抑制活性曲线图。
图26是本发明含不同IFN-α2b低亲和突变体的双特异性重组蛋白(第一类结构)对PD-L1阴性的目标靶细胞OVCAR-3增殖抑制活性曲线图。
图27是本发明含不同IFN-α2b低亲和突变体的双特异性重组蛋白(第二类结构)对GPC3阳性的目标靶细胞Huh-7增殖抑制活性曲线图。
图28是本发明含不同IFN-α2b低亲和突变体的双特异性重组蛋白(第二类结构)对GPC3阴性的非目标靶细胞SW480增殖抑制活性曲线图。
图29是本发明含不同IFN-α2b低亲和突变体的双特异性重组蛋白(第二类结构)对GPC3阴性的非目标靶细胞MDA-MB-231增殖抑制活性曲线图。
图30是本发明含不同IFN-α2b低亲和突变体的双特异性重组蛋白(第二类结构)对GPC3阴性的非目标靶细胞U266增殖抑制活性曲线图。
图31是本发明的双特异性重组蛋白潜在风险杂质对GPC3阴性的非目标靶细胞MDA-MB-231增殖抑制活性曲线图。
图32是本发明的双特异性重组蛋白在BALB/c nude小鼠HuH-7皮下瘤模型上的抑瘤药效曲线图。
图33是本发明的双特异性重组蛋白在BALB/c nude小鼠HuH-7皮下瘤模型上的抑瘤药效柱状图。
图34是本发明的双特异性重组蛋白在PBMC人源化M-NSG小鼠MDA-MB-231模型上的抑瘤药效柱状图。
具体实施方式
为了使发明实现的技术手段、创造特征、达成目的和功效易于明白了解，下结合具体图示，进一步阐述本发明。但本发明不仅限于以下实施的案例。
实施例1双特异性重组蛋白的设计
本发明双特异性重组蛋白的结构如图1和图2所示，所述双特异性重组蛋白包含靶向目的抗原的第一功能结合区、由IFNα或其低亲和突变体或保留IFNα功能的截短体或其突变体构成的第二功能结合区、以及Fc区。其中，第二功能结合区的C端与Fc区直接连接或通过接头序列连接，第一功能结合区的C端与Fc区直接连接或通过接头序列连接；第一功能结合区的可变区(V区)和恒定区(C区)直接连接或通过接头序列连接；Fc区可以是常规的IgG结构，也可以采用knobs-into-holes技术；第一功能结合区与第二功能结合区的连接关系有两种结构，分别为：
第一类结构，第二功能结合区的N端与第一功能结合区中未与Fc区连接的CL或CH1的C末端直接连接或通过接头序列连接(如图1所示)；
第二类结构，第二功能结合区的N端与第一功能结合区分离，即第二功能结合区的N端未连接其他功能片段(信号肽除外)(如图2所示)。
本实施例以第二功能结合区选自人IFN-α2b(Genebank：AAP20099.1)为例阐述本发明双特异性重组蛋白的设计。IFNα家族共15个亚型，不同亚型结构相似，序列高度同源(80-99％)，且所结合的IFN受体一致，都具有抗病毒、增殖抑制、抗肿瘤和免疫调节的功能。对于IFN-α2b可以得到的技术效果，IFNα其余亚型可以达到一致的技术效果(British Journal of Pharmacology(2013)168 1048–1058)，本发明在这里不再赘述。
具有第一类结构的双特异性重组蛋白见表1，具有第二类结构的双特异性重组蛋白见表2，对照样品见表3。
表1第一类结构的双特异性重组蛋白示例性分子结构
Figure PCTCN2022112300-appb-000006
Figure PCTCN2022112300-appb-000007
Figure PCTCN2022112300-appb-000008
表2第二类结构双特异性重组蛋白示例性分子结构
Figure PCTCN2022112300-appb-000009
表3对照样品示例性分子结构
Figure PCTCN2022112300-appb-000010
以上表1和表2中，(H)指重链VH和CH1组成结构域，(L)指轻链VL和CL组成结构域；GC33表示抗磷脂酰肌醇蛋白聚糖(Glypican3，GPC3)单抗codrituzumab；Fc表示野生型Fc区、Fc1表示具有hole或holes突变的Fc区、Fc2表示具有knob或knobs突变的Fc区。
表1和表2所述序列名称对应序列号见表4-1和表4-2。其中，具有第一类结构的A链和B链信号肽以及具有第二类结构序列第一臂(H+L)信号肽如SEQ ID NO:23所示；具有第二类结构的第二臂信号肽如SEQ ID NO:24所示，为IFNα2b天然信号肽。表3中，IFN-α2b-Fc的氨基酸序列由SEQ ID NO:17和SEQ ID NO:25顺序连接组成，对应信号肽序列如SEQ ID NO:24；IFN-α2b的氨基酸序列如SEQ ID NO:17所示；Codrituzumab的氨基酸序列引用自专利US7919086，重链可变区氨基酸序列如SEQ ID NO:1所示，轻链可变区氨基酸序列如SEQ ID NO:2所示。Atezolizumab的氨基酸序列引用自专利US20100203056，重链可变区氨基酸序列如SEQ ID NO:5所示，轻链可变区氨基酸序列如SEQ ID NO:6所示。Palivizumab的氨基酸序列引用自专利WO1994017105，重链可变区氨基酸序列如SEQ ID NO:3所示，轻链可变区氨基酸序列如SEQ ID NO:4所示。Belantamab的重轻链可变区序列引用自专利US20190194338，重链可变区序列如SEQ ID NO:9所示，轻链可变区序列如SEQ ID NO:10所示。Elranatamab重轻链可变区序列引用自专利US20160297885，重链可变区序列如SEQ ID NO:7所示，轻链可变区序列如SEQ ID NO:8所示。hRS7的重轻链可变区序列引用自专利US7517964，重链可变区序列如SEQ ID NO:11所示，轻链可变区序列如SEQ ID NO:12所示。人IgG1同型对照(B117901)购买自百英生物。重组表达IFN-α2b蛋白(Z03003)购买自南京金斯瑞生物科技有限公司。
表4-1部分序列名称与对应序列编号组成
Figure PCTCN2022112300-appb-000011
Figure PCTCN2022112300-appb-000012
Figure PCTCN2022112300-appb-000013
表4-2其他序列名称与对应序列编号组成
Figure PCTCN2022112300-appb-000014
Figure PCTCN2022112300-appb-000015
Figure PCTCN2022112300-appb-000016
Figure PCTCN2022112300-appb-000017
实施例2双特异性重组蛋白质粒构建、表达和纯化
1、质粒构建、细胞转染和蛋白表达
双特异性重组蛋白表达质粒由GENEWIZ公司根据蛋白序列进行密码子优化后合成，连接入 pCDNA3.1质粒中，经测序验证序列。
将所得表达质粒用0.22μm滤膜过滤后，吸取50μg质粒(其中，具有第一类结构的双特异性重组蛋白，其A链和B链表达质粒的质量比例为2:1或者3:1；具有第二类结构的双特异性重组蛋白，其轻链、重链和第二臂表达质粒的质量比例为3:2:2)加入到2mL OptiPRO SFM Medium(GIBCO)中混匀。吸取160μL转染试剂ExpiFectamine CHO Reagent加入到2mL OptiPRO SFM Medium中混匀。将得到的转染试剂混合溶液加入到含有质粒的混合溶液中混匀。将质粒和转染试剂的混合物缓慢均匀加入体积为50mL，细胞密度为6×10 6viable cells/mL的宿主细胞ExpiCHO-S(Thermo Fisher)悬液中，置于37℃、8％CO 2培养箱中培养。第1天(18～22小时后)加入300μL ExpiCHO Enhancer和8mL ExpiCHO Feed，将培养温度降低至32℃；第5天进行第二次补料，补加8mL ExpiCHO Feed，培养12天后收获。将细胞悬液8000rpm离心15分钟，将离心所得上清即细胞培养收获液用于目的蛋白纯化。
2、蛋白纯化
2.1、样品捕获
双特异性重组蛋白表达上清使用Mabselect Sure(即蛋白A，购自思拓凡)亲和填料捕获目的蛋白，实验过程如下：
a)平衡：使用平衡液(50mM Tris-HCl，150mM NaCl，pH7.2)平衡层析柱，直至紫外检测线平稳；
b)上样：用样品泵上样，保留时间5min，载量≤30mg/ml；
c)再平衡：使用平衡液(50mM Tris-HCl，150mM NaCl，pH7.2)冲洗层析柱5个柱体积；
d)洗脱：使用洗脱液(50mM NaAC-HAC，Ph3.5)洗脱目的蛋白，并用1M Tris缓冲液将洗脱液pH调制5.5，SDS-PAGE检测蛋白纯度。
2.2、样品精纯
2.2.1本实施例以TTM101-LC01为例描述具有第一类结构的双特异性重组蛋白样品精纯过程。
双特异性重组蛋白TTM101-LC01表达上清使用SULFATE 650F填料(TOSOH)除去样品中的聚体及其它杂质，实验过程如下：
a)平衡：使用平衡液(50mM NaAC-HAC，pH5.5)平衡层析柱，直至紫外检测线平稳；
b)上样：用样品泵上样，保留时间5min，载量≤50mg/ml；
c)再平衡：使用平衡液(50mM NaAC-HAC，pH5.5)冲洗层析柱5个柱体积；
d)洗脱：使用洗脱液(50mM NaAC-HAC，250Mm，pH5.5)洗脱目的蛋白，SDS-PAGE检测蛋白纯度。
2.2.2本实施例以TTM101-01为例描述具有第二类结构的双特异性重组蛋白样品精纯过程。
双特异性重组蛋白TTM101-01表达上清使用Kappa select亲和填料(思拓凡)除去样品中的杂质，实验过程如下：
a)平衡：使用平衡液(50mM Tris-HCl，150mM NaCl，pH7.2)平衡层析柱，直至紫外检测线平稳；
b)上样：用样品泵上样，保留时间5min，载量≤30mg/ml；
c)再平衡：使用平衡液(50mM Tris-HCl，150mM NaCl，pH7.2)冲洗层析柱5个柱体积；
d)洗脱：使用洗脱液(0.1M Gly-HCl，pH2.7)洗脱目的蛋白，并用1MTris缓冲液将洗脱液pH调制 5.5，SDS-PAGE检测蛋白纯度。
表3中对照样品Codrituzumab、IFN-α2b-Fc和Atezolizumab的纯化方式同2.1。
表1和表2所示双特异性重组蛋白的理论分子量均为120kD左右，表3所示对照抗体Codrituzumab分子量为150KD，IFNα2b-Fc的分子量为88KD，IFNα2b单体的分子量为19.2KD。具有第一类结构的双特异性重组蛋白(见图1)亲和捕获(即经蛋白A纯化后)的样品非还原SDS-PAGE蛋白电泳检测结果如图3所示，还原SDS-PAGE蛋白电泳检测结果如图4所示，电泳结果显示该结构表达抗体聚体含量较多，精纯后(即经SULFATE 650F纯化后)的非还原SDS-PAGE蛋白电泳检测结果如图5所示，可见通过阳离子交换层析填料可去除大部分聚体。具有第二类结构的双特异性重组蛋白(见图2)精纯后(即经Kappa纯化后)的非还原SDS-PAGE蛋白电泳检测结果如图6所示，通过蛋白A亲和纯化和Kappa亲和纯化，获得纯度较高双特异性重组蛋白。
实施例3靶向GPC3双特异性重组蛋白对GPC3阳性肝癌细胞系(双阳表达细胞，目标靶细胞)结合活性检测
靶向GPC3双特异性重组蛋白对靶标GPC3阳性肝癌细胞系的结合亲和力通过流式细胞术进行测定。以下方法以TTM101-LC01、TTM101-LC02、TTM101-LC03、TTM101-01为例，适用于第一功能结合区结合GPC3抗原、第二功能结合区为IFN-α2b或其低亲和突变体的双特异性重组蛋白的检测。
检测细胞为HepG2细胞(南京科佰生物科技有限公司)和HuH-7(上海中科院细胞库)，收集生长良好的细胞并计数，离心并用FACS缓冲液(PBS+2％FBS)重悬细胞至1×10 6个细胞/ml的浓度。将细胞以100μl/孔加入到96孔板U型板(货号：3799，Corning)，分别加入7个稀释度的双特异性重组蛋白或对照蛋白(从100nM起始，3倍梯度稀释，共7个浓度)，4℃孵育1小时；FACS缓冲液清洗后，加入山羊抗人IgG(Alexa Fluor488 goat anti-human IgG(H+L),Invitrogen)4℃孵育1小时；用FACS缓冲液清洗重悬后，通过流式细胞仪(Attune Nxt，invitrogen)检测荧光值。
实验结果如图7和图8所示，具有第一类结构的双特异性重组蛋白TTM101-LC01、TTM101-LC02、TTM101-LC03均可与同时表达GPC3和IFNα受体的目标靶细胞HepG2和HuH-7细胞具有一定的结合活性，双特异性重组蛋白与HepG2和HuH-7细胞的结合相较于抗GPC3单抗(anti-GPC3mAb，即对照样品Codrituzumab)具有更高的最大平均荧光强度。同时，如图7和图8所示，接头序列的长短对双特异性重组蛋白与目标靶细胞的结合活性影响较弱，当接头序列为1个GGGGS的重组蛋白结合活性(EC50)相对最低。
如图9所示，具有第二类结构的双特异性重组蛋白(TTM101-01)与同时表达GPC3和IFN受体的目标靶细胞HepG2细胞具有一定的结合活性，EC50与抗GPC3单抗(anti-GPC3mAb，即对照样品Codrituzumab)相当，且相较于抗GPC3单抗(anti-GPC3mAb，即对照样品Codrituzumab)，双特异性重组蛋白(TTM101-01)具有更高的最大平均荧光强度。
实施例4靶向GPC3双特异性重组蛋白的抗体依赖细胞介导的细胞毒作用(antibody-dependent cell-mediated cytotoxicity，ADCC)活性检测
双特异性重组蛋白对靶标GPC3阳性肝癌细胞系的ADCC活性通过乳酸脱氢酶(LDH)方法进行测定。以下方法以TTM101-LC01、TTM101-LC02、TTM101-LC03、TTM101-01为例，适用于第一功能结合区结合GPC3抗原、第二功能结合区为IFN-α2b或其低亲和突变体的双特异性重组蛋白ADCC活性检测。
人外周血单核细胞(Peripheral Blood Mononuclear Cell，PBMC)作为效应细胞,靶细胞为GPC3阳性的HepG2肝癌细胞系，LDH检测试剂盒为(CytoTox-ONETM-Homogeneous Membrance Integrity Assay,Promega,G7892)。靶细胞和效应细胞以1:20的条件铺板，加入双特异性重组蛋白或对照蛋白(从150nM起始，5倍梯度稀释，共8个浓度)，37℃共孵育4小时。37℃孵育3.5小时后向对照组加入裂解试剂，显微镜下观察细胞状态，待细胞裂解完全后10000转离心5分钟，取上清转至96孔黑底透明板内(Corning,3904)，与反应底物于37℃孵育30分钟,加入终止液，避光振荡3到5分钟，酶标仪(SpectraMax M2)检测信号值，详细步骤参考LDH检测试剂盒说明书。
如图10和图11所示，具有第一类结构的双特异性重组蛋白(如TTM101-LC01、TTM101-LC02、TTM101-LC03)和具有第二类结构的双特异性重组蛋白(如TTM101-01)均保留有ADCC活性，且双特异性重组蛋白的ADCC活性与抗GPC3单抗(anti-GPC3mAb，即对照样品Codrituzumab)相当(见图11)。
实施例5靶向GPC3双特异性重组蛋白增殖抑制活性检测
靶向GPC3的双特异性重组蛋白对不同肿瘤细胞系的增殖抑制活性通过cell titer glo试剂盒(Promega,Cat:G7558)进行测定。以下方法以TTM101-LC01、TTM101-LC02、TTM101-LC03为例，适用于第一功能结合区结合GPC3抗原、第二功能结合区为IFNα2b或其低亲和突变体的双特异性重组蛋白的检测。
GPC3阳性细胞系HuH-7或者GPC3阴性肿瘤细胞系U266(购自南京科佰生物科技有限公司)和GPC3阴性肿瘤细胞系SW480(购自南京科佰生物科技有限公司)铺板于96孔黑底透明板内(Corning,3904)，加入双特异性重组蛋白或对照蛋白(52nM起始，10倍梯度稀释，共6个点；或10nM起始，5倍梯度稀释，共8个点)，放置CO 2培养箱37℃培养3天，加入Cell titer glo，Multomode Plate Reader(PerkinElmer,Envision2105)检测信号值。
结果显示，双特异性重组蛋白(如TTM101-LC01、TTM101-LC02、TTM101-LC03)对GPC3阳性(GPC3+)的目标靶细胞HuH-7增殖抑制活性(IC50)均高于对照IFN-α2b(见图12)，也高于抗GPC3单抗(anti-GPC3mAb，即对照样品Codrituzumab，该抗体对HuH-7未见增殖抑制作用)。如图13所示，缺乏GPC3靶向功能的双特异性重组蛋白TTM101-LC04以及IFN-α2b的Fc融合蛋白对HuH-7(GPC3阳性的目标靶细胞)的增殖抑制活性均显著低于拥有GPC3靶向功能的双特异性重组蛋白TTM101-LC02，表明拥有GPC3靶向功能的双特异性重组蛋白其与目标靶细胞上GPC3的结合，可以显著地增强IFN-α2b增殖抑制活性，而不具有目标靶细胞靶向作用的双特异性重组蛋白其IFN-α2b所起到的增殖抑制活性较低，揭示本发明双特异性重组蛋白仅对具有目的抗原的目标靶细胞有较强的增殖抑制作用，而对于不具有目的抗原的非目标靶细胞则作用较弱或不结合，说明本发明双特异性重组蛋 白的安全性较高。
另，如表5结果显示，不同接头序列的第一类结构的双特异性重组蛋白(以TTM101-LC01、TTM101-LC02、TTM101-LC03为例)对GPC3阳性(GPC3+)的目标靶细胞(以HuH-7为例)均具有相较IFN-α2b更强的增殖抑制活性，不同接头序列的双特异性重组蛋白其增殖抑制活性略有差异，含有1个GGGGS作为接头序列的双特异性重组蛋白(即以较短长度接头序列连接的双特异性重组蛋白，以TTM101-LC01为例)活性相对较低(图12、表5)，该结果与图8所示不同接头序列的重组蛋白结合活性结果相一致。且在GPC3阴性(GPC3-)的非目标靶细胞U266和SW480细胞系中，双特异性重组蛋白(以TTM101-LC01、TTM101-LC02、TTM101-LC03为例)的增殖抑制活性显著低于IFN-α2b(即双特异重组蛋白的相对活性<<1)，该结果表明在不表达GPC3的细胞(即不表达目的抗原的非目标靶细胞)中，双特异性重组蛋白增殖抑制活性极低，提示其安全性较高。同时，如表5所示，双特异性重组蛋白对目标靶细胞的增殖抑制活性是该双特异性重组蛋白对非目标靶细胞的至少700倍。
表5不同接头序列双特异性重组蛋白增殖抑制相对活性
Figure PCTCN2022112300-appb-000018
实施例6靶向PD-L1双特异性重组蛋白增殖抑制活性检测
靶向PD-L1的双特异性重组蛋白对不同肿瘤细胞系的增殖抑制活性通过cell titer glo试剂盒(Promega,Cat:G7558)进行测定。以下方法以TTM101-LC05、TTM101-LC06、TTM101-LC07为例，适用于第一功能结合区结合PD-L1抗原、第二功能结合区为IFN-α2b或其低亲和突变体的双特异性重组蛋白。
PD-L1阳性细胞系MDA-MB-231(购自南京科佰生物科技有限公司)(人乳腺癌细胞)铺板于96孔黑底透明板内(Corning,3904)，加入双特异性重组蛋白或对照蛋白(52nM起始，10倍梯度稀释，共6个浓度点)，放置CO 2培养箱37℃培养3天，加入Cell titer glo，Multomode Plate Reader(PerkinElmer,Envision2105)检测信号值。
如图14所示，对于PD-L1阳性(PD-L1+)的目标靶细胞MDA-MB-231，靶向PD-L1的双特异性重组蛋白(TTM101-LC05、TTM101-LC06和TTM101-LC07)都具有增殖抑制活性，并且活性显著高于不具有PD-L1靶向功能的对照双特异性重组蛋白TTM101-LC04以及IFN-α2b，表明靶向PD-L1可以显著地增强IFN-α2b增殖抑制活性。表明拥有PD-L1靶向功能的双特异性重组蛋白其与目标靶细胞MDA-MB-231上PD-L1结合，可以显著地增强IFN-α2b的增殖抑制活性，而不具有目标靶细胞靶向作用的双特异性重组蛋白其IFN-α2b所起到的增殖抑制活性较低，揭示本发明双特异性重组蛋白仅对具 有目的抗原的目标靶细胞有较强的增殖抑制作用，而对于不表达目的抗原的非目标靶细胞则作用较弱或不结合，说明本发明双特异性重组蛋白的安全性较高。
PD-L1阳性的目标靶细胞MDA-MB-231铺板于96孔黑底透明板内(Corning，3904)，加入PD-L1抗体Atezolizumab或者同型对照200nM，37℃培养30分钟。加入双特异性重组蛋白或对照蛋白(起始浓度20nM，6倍稀释，共6个浓度点)，放置CO 2箱37℃培养3天，加入Cell titer glo，Multomode Plate Reader(PerkinElmer，Envision2105)检测信号值。
如图15所示，加入Atezolizumab封闭PD-L1抗原与抗体的结合位点后，TTM101-LC07增殖抑制活性显著降低，该结果证明封闭待测细胞上目的抗原的结合功能，双特异性重组蛋白对待测细胞的增殖抑制活性显著下降，从另一个角度论证了本发明双特异性重组蛋白对于不具有目的抗原结合能力的非目标靶细胞则作用较弱或不结合，说明本发明双特异性重组蛋白的安全性较高。
实施例7含IFN-α2b低亲和突变体双特异性重组蛋白增殖抑制活性检测
考虑到IFN-α2b的人体耐受剂量与常规抗体起效剂量间存在差异，为更好匹配目的抗原靶向的半抗体与IFN-α2b的效果，实现高效低毒的效果，本发明还设计了一系列包含IFN-α2b低亲和突变体的双特异性重组蛋白。本实施例以基于TTM101-LC02的突变设计为例设计含IFN-α2b低亲和突变体双特异性重组蛋白，基于野生型IFN-α2b的突变位点的突变体与IFNAR2(IFNα受体2)的相对亲和力(数据参考Cell.2011 August 19；146(4):621–632.)见表6，检测含不同IFN-α2b低亲和突变体的双特异性重组蛋白的增殖抑制活性，实验方法同实施例5。
表6 IFN-α2b突变位点及IFNAR2相对亲和力
IFN-α2b突变位点 IFNAR2相对亲和力
L26A 0.22
L30A 0.0013
A145G 0.03
R149A 0.01
S152A 0.1
如图16-18所示，结果表明，拥有IFN-α2b低亲和突变体的双特异性重组蛋白在GPC3阳性的目标靶细胞(HuH-7)或GPC3阴性的非目标靶细胞(U266、SW480)，TTM101-LC02-M2、TTM101-LC02-M3和TTM101-LC02-M4增殖抑制活性相对于TTM101-LC02均有降低。例如，TTM101-LC02-M3在HuH-7(GPC3阳性细胞系，目标靶细胞)的增殖抑制活性IC50与IFN-α2b相当(IC50 IFN-α2b＝0.1793，IC50 TTM101-LC02-M3＝0.179)，但TTM101-LC02-M3在GPC3阴性的非目标靶细胞(U266)的增殖抑制相对活性(IC50IFN-α2b/双特异性重组蛋白)更弱，例如，TTM101-LC02-M3在U266上的增殖抑制相对活性(IC50IFN-α2b/双特异性重组蛋白)为0.000615，显著弱于IFN-α2b，弱于TTM101-LC02，TTM101-LC02-M2、TTM101-LC02-M4、TTM101-LC03-M3和TTM101-LC03-M4也有类似的结果。
以上结果表明含IFN-α2b低亲和突变体在具有第一类结构的靶向GPC3双特异性重组蛋白在目的抗原阳性的目标靶细胞系中活性与IFN-α2b相当，但在不表达目的抗原的非目标靶细胞系中活性降低。 表明A145G或R149A突变在具有第一类结构的靶向GPC3或PD-L1双特异性重组蛋白的靶向性好且安全性高。
为了进一步验证第一类结构的靶向性，分别检测靶向Trop-2、BCMA和PD-L1的双特异性重组蛋白在靶点阴性和阳性的肿瘤细胞上活性差异。实验方法同实施例5和6。
如图19-21所示，检测拥有IFN-α2b低亲和突变体的双特异性重组蛋白在Trop-2阳性的目标靶细胞(OVCAR3和MDA-MB-231)和Trop-2阴性的目标靶细胞(NCI-H929)上的活性。结果表明，TTM101-LC10-M3和TTM101-LC10-M4增殖抑制活性在Trop-2阴性和阳性的细胞上增殖抑制活性与TTM101-LC10相比均有降低。但是，在Trop-2阳性的目标靶细胞(OVCAR3和MDA-MB-231)上，TTM101-LC10、TTM101-LC10-M3和TTM101-LC10-M4增殖抑制活性与IFN-α2b比较更优或相当，在Trop-2阴性的目标靶细胞(NCI-H929)上，TTM101-LC10、TTM101-LC10-M3和TTM101-LC10-M4增殖抑制活性与IFN-α2b比较明显降低，尤其是TTM101-LC10-M3和TTM101-LC10-M4，活性与IFN-α2b比较降低20倍以上。以上结果表明A145G或R149A突变在具有第一类结构的靶向Trop-2双特异性重组蛋白的靶向性好且安全性高。
如图22-24所示，检测拥有IFN-α2b低亲和突变体的双特异性重组蛋白在BCMA阳性的目标靶细胞(NCI-H929)上的活性，结果表明，TTM101-LC08-M3、TTM101-LC09-M3和TTM101-LC08-M4增殖抑制活性与IFN-α2b相当，TTM101-LC09-M4比IFN-α2b活性减弱7倍左右。在BCMA阴性的非目标靶细胞(HuH-7和MDA-MB-231)上，TTM101-LC08-M3、TTM101-LC09-M3、TTM101-LC08-M4和TTM101-LC09-M4增殖抑制活性均显著低于IFN-α2b。表明A145G或R149A突变在具有第一类结构的靶向BCMA双特异性重组蛋白的靶向性好且安全性高。
如图25和图26所示，检测拥有IFN-α2b低亲和突变体的双特异性重组蛋白在PD-L1阳性的目标靶细胞MDA-MB-231和PD-L1阴性的目标靶细胞OVCAR3上的活性。结果表明，TTM101-LC07-M3和TTM101-LC07-M4增殖抑制活性在PD-L1阳性的细胞上增殖抑制活性与TTM101-LC07相比均有降低。TTM101-LC07、TTM101-LC07-M3增殖抑制活性与IFN-α2b比较提高5倍左右，TTM101-LC07-M4与IFN-α2b比较活性相当，但是TTM101-LC07-M3和TTM101-LC07-M4增殖抑制活性在PD-L1阴性细胞OVCAR3上增殖抑制活性显著弱于IFN-α2b，表明A145G或R149A突变在具有第一类结构的靶向PD-L1双特异性重组蛋白的靶向性好且安全性高。
同时，发明人以A145G和R149A突变为例构建具有第二类结构的靶向GPC3双特异性重组蛋白和靶向PD-L1双特异性重组蛋白。
本实施例以TTM101-01-M3、TTM101-01-M4为例阐述具有第二类结构的靶向GPC3的含IFN-α2b低亲和突变体的双特异性重组蛋白分别在GPC3阳性的目标靶细胞HuH-7和GPC3阴性的非目标靶细胞SW480、MDA-MB-231和U266上检测增殖抑制活性，实验方法同实施例5，待测蛋白和对照蛋白从100nM开始进行5倍梯度稀释，共9个梯度。
结果表明，在GPC3阳性的目标细胞(HuH-7，目标靶细胞)、GPC3阴性的非目标细胞(U266、MDA-MB-231或SW480，非目标靶细胞)中，TTM101-01-M3、TTM101-01-M4增殖抑制活性相较于TTM101-01均有降低，但对目标靶细胞和非目标靶细胞的降低程度有显著区别。其中TTM101-01-M3和TTM101- 01-M4在GPC3阳性的目标靶细胞HuH-7的增殖抑制活性IC50与IFN-α2b基本相当(图27)；而在GPC3阴性细胞系(即非目标靶细胞)中，TTM101-01-M3和TTM101-01-M4增殖抑制活性比IFN-α2b低百倍以上(图28-30)，尤其是对U266、MDA-MB-231的增殖抑制活性比IFN-α2b低上万倍以上。
以上结果表明，具有A145G或R149A突变的IFN-α2b低亲和突变体在具有第二类结构的靶向GPC3或PD-L1双特异性重组蛋白在目的抗原阳性的细胞系中活性与IFN-α2b相当，但是在不表达目的抗原的非目标靶细胞系中的活性显著降低，并且R149A突变的活性相对更低。表明A145G或R149A突变在具有第二类结构的靶向GPC3或PD-L1双特异性重组蛋白的靶向性好且安全性高。
综上，含IFN-α2b低亲和突变体的本发明双特异性重组蛋白，其对目的抗原阴性的非目标靶细胞的增殖抑制活性相较含IFN-α2b野生型双特异性重组蛋白对目的抗原阴性的非目标靶细胞的增殖抑制活性的下降程度，相较含IFN-α2b低亲和突变体双特异性重组蛋白在目的抗原阳性的目标靶细胞的下降程度相当或更显著。
实施例8双特异性重组蛋白潜在风险杂质对非目标靶细胞的增殖抑制活性检测
为降低双特异性重组蛋白未来制备工艺过程中潜在的杂质安全性风险，本发明以TTM101-LC02和TTM101-01为例，分析其潜在风险杂质(B链或第二臂同二聚体)对非目标靶细胞的增殖抑制情况。
在TTM101-LC02精纯过程(如实施例2所述)中，分离TTM101-LC02的B链同二聚体(图3，同二聚体分子量约140kD)，检测其与IFN-α2b-Fc1(TTM101-01第二臂同二聚体)在非目标靶细胞(GPC3阴性细胞)MDA-MB-231中的增殖抑制活性。结果如图31显示，在16.7nM浓度下，IFN-α2b对MDA-MB-231(GPC3阴性细胞，非目标靶细胞)增殖抑制率为91.3％，TTM101-01第二臂同二聚体(IFN-α2b-Fc1)对MDA-MB-231(GPC3阴性细胞，非目标靶细胞)增殖抑制率为66.8％(较IFN-α2b下降约24.5％)，TTM101-LC02的B链同二聚体对MDA-MB-231(GPC3阴性细胞，非目标靶细胞)增殖抑制率仅为16.2％(较IFN-α2b降低约75.1％，较IFN-α2b-Fc降低约50％)。综上，潜在风险杂质对非目标靶细胞仅有极弱的作用，即潜在的安全性风险或带来的潜在毒副作用极低。
实施例9含IFN-α2b低亲和突变体双特异性重组蛋白体内抑瘤药效活性检测
靶向GPC3双特异性重组蛋白在BALB/c nude小鼠HuH-7皮下瘤模型上的抑瘤药效
在BALB/c nude小鼠的右侧颈背部接种～30mm 3的Huh-7肿瘤块，接种同时将实验动物进行耳标号标记，作为后续实验的唯一确认标志。等待肿瘤生长，肿瘤平均体积达到150mm 3时，开始进行随机分组给药，每组6只小鼠。均采用静脉注射给药，给药体积均为10mL/kg，连续给药3周，每周给药2次，共给药6次。
实验指标是考察肿瘤生长是否被抑制、延缓或治愈。每周两次用游标卡尺测量肿瘤直径。肿瘤体积的计算公式为：V＝0.5a×b 2，a和b分别表示肿瘤的长径和短径。统计分析，包括每个组的每个时间点的肿瘤体积的平均值和标准误差(SEM)。两组间比较用one–tailed T test进行分析，使用GraphPad Prism进行所有数据分析。p<0.05认为有显著性差异。
结果如图32和图33所示，TTM101-LC03-M3剂量依赖抑制肿瘤生长，并且抑瘤效果优于等剂量下GC33药效，同时也优于高剂量PEG-IFN-α2b(剂量高于临床耐受剂量)。以上结果表明GPC3靶向A145G减弱突变IFN-α2b可有效抑制肿瘤生长。
靶向PD-L1双特异性重组蛋白在PBMC人源化M-NSG小鼠MDA-MB-231模型上的抑瘤药效
M-NSG(6-8w，雌性)皮下接种MDA-MB-231细胞(接种细胞数：1E7/只+30％Matrigel)记为第0天，隔天观察小鼠生存状态及皮下成瘤情况。接种7天后，尾静脉注射PBMC细胞(0205C)(接种细胞数：5E6/只)，PBMC接种后第7天，采集小鼠全血流式检测hCD45％，根据平均肿瘤体积(～200mm 3)和h CD45％比例将小鼠随机分组，分组当天即为D0，均采用静脉注射给药，给药体积均为10mL/kg，连续给药3周，每周给药2次，共给药6次。
实验指标是考察肿瘤生长是否被抑制、延缓或治愈。每周两次用游标卡尺测量肿瘤直径。肿瘤体积的计算公式为：V＝0.5a×b 2，a和b分别表示肿瘤的长径和短径。统计分析，包括每个组的每个时间点的肿瘤体积的平均值和标准误差(SEM)。两组间比较用one–tailed T test进行分析，使用GraphPad Prism进行所有数据分析。p<0.05认为有显著性差异。
结果如图34所示，TTM101-LC07-M3(N297G)剂量依赖抑制肿瘤生长，并且抑瘤效果优于等剂量下Atezolizumab药效，4mg/kg TTM101-LC07-M3(N297G)药效优于高剂量PEG-IFN-α2b(剂量高于临床耐受剂量)。TTM101-LC07-M4(N297G)在0.2mg/kg剂量水平下，药效与1mg/kg Atezolizumab相当。以上结果表明PD-L1靶向A145G和R149A减弱突变IFN-α2b均可有效抑制肿瘤生长。
实施例10双特异性重组蛋白冻融稳定性考察
现有的重组人白蛋白干扰素-α2b融合蛋白冻融稳定性较差，不宜反复冻融，如PEG化的长效干扰素不可冷冻和震荡，对运输和保存条件要求较高。为了研究本发明双特异性重组蛋白的冻融稳定性，对具有第一类结构的双特异性重组蛋白进行反复冻融稳定性测试。蛋白放置于20mM NaAc(PH＝5)缓冲液，在-40℃条件下进行5次的反复冻融。对冻融前后的样品进行纯度(尺寸排阻层析，SEC)和外观分析。结果如表7所示，TTM101-LC03及其突变体冻融稳定性良好，纯度均在95％以上，5次反复冻融后外观澄清，表明本发明双特异性重组蛋白其IFN-α2b蛋白部分冻融稳定性明显优于IFN-α2b单体或者PEG化的IFN-α2b。
表7具有第一类结构的双特异性重组蛋白冻融稳定性
Figure PCTCN2022112300-appb-000019
对于本发明第一功能结合区靶向其他靶标(例如5T4、AGS-16、ALK1、ANG-2、B7-H3、B7-H4、c-fms、c-Met、CA6、CD123、CD19、CD20、CD22、CD24、EpCAM、CD30、CD32b、CD37、CD38、CD40、CD52、CD70、CD74、CD79b、CD98、CEA、CEACAM5、CLDN18.2、CLDN6、CS1、CXCR4、 DLL-4、EGFR、EGFRvIII、EGP-1、ENPP3、EphA3、ETBR、FGFR2、FN、FR-α、GCC、GD2、GPNMB、HER2、HER3、HLA-DR、ICAM-1、IGF-1R、IL-3R、LIV-1、MSLN、MUC16、MUC1、NaPi2b、结合素-4、Notch 2、Notch 1、PD-L2、PDGFR-α、PS、PSMA、SLTRK6、STEAP1、TEM1、VEGFR、CD25、CD27L、DKK-1、CSF-1R、MSB0010718C、CD138、Siglec15和CD155)的双特异性重组蛋白，也能够达到与上述靶标类似的效果。
本文提供的任何和所有实施例或示例性语言(例如，“诸如”)的使用仅旨在更好地说明本发明，而不对本发明的范围构成限制，除非另有要求。说明书中的语言不应被解释为指示任何未要求保护的元件对于实施本发明是必要的。
本说明书中引用的所有出版物和专利申请通过引用并入本文，如同每个单独的出版物或专利申请被具体地和单独地指明通过引用并入。此外，本文所述的任何理论、机制、证明或发现旨在进一步增强对本发明的理解，并且不意图以任何方式将本发明限制到这样的理论、机制、证明或发现。尽管已经在附图和前面的描述中详细地示出和描述了本发明，但是本发明应当被认为是说明性的而不是限制性的。

Claims (23)

  1. 一种双特异性重组蛋白，其特征在于，所述双特异性重组蛋白包括第一功能结合区、第二功能结合区和Fc区；所述第一功能结合区包括第一链和第二链，所述第一链的C端和所述第二功能结合区的C端分别与Fc区的N端直接或通过接头序列连接；所述第二链的C端与第二功能结合区的N端直接或通过接头序列连接；
    所述第一功能结合区包括重链可变区(VH)、轻链可变区(VL)、重链恒定区1(CH1)和轻链恒定区(CL)；
    所述第二功能结合区包含干扰素、其截短体或其突变体；
    较佳地，所述CL或CH1的C末端与所述Fc区的N端直接或通过接头序列连接；相应的CH1或CL的C末端与第二功能结合区的N端直接连接或通过接头序列连接。
  2. 如权利要求1所述的双特异性重组蛋白，其特征在于，所述干扰素为I型干扰素；较佳地，所述I型干扰素为IFNα；更佳地，所述IFNα选自IFN-α1a、IFN-α1b、IFN-α2a、IFN-α2b、IFN-α4a、IFN-α4b、IFN-α5、IFN-α6、IFN-α7、IFN-α8、IFN-α10、IFN-α14、IFN-α16、IFN-α17和IFN-α21中的一种或多种。
  3. 如权利要求2所述的双特异性重组蛋白，其特征在于，所述干扰素为IFN-α2b；
    较佳地，所述第二功能结合区包含选自如下a1)-a2)任意一种的氨基酸序列：a1)SEQ ID NO:17；a2)SEQ ID NO:17所示的氨基酸序列经过添加、缺失、修饰和/或置换至少1个氨基酸残基获得的氨基酸序列，其单体具有对IFNα受体的结合亲和力并且该结合亲和力不高于a1)单体对IFNα受体的结合亲和力。
  4. 如权利要求3所述的双特异性重组蛋白，其特征在于，a2)单体的氨基酸序列为在如SEQ ID NO:17所示的氨基酸序列的相应位点置换得到的氨基酸序列；所述相应位点的置换选自L26A、L30A、A145G、R149A和S152A中的一种或者多种；
    较佳地，所述a2)单体是单一位点的置换，置换位点分别为L26A、L30A、A145G、R149A或S152A，相应的氨基酸序列分别如SEQ ID NO:18～22所示。
  5. 如权利要求1所述的双特异性重组蛋白，其特征在于，所述VH与CH1或CL直接连接或通过接头序列连接；所述VL与CL或CH1直接连接或通过接头序列连接；
    较佳地，当VL与CL连接时，VH与CH1连接；当VL与CH1连接时，VH与CL连接。
  6. 如权利要求1所述的双特异性重组蛋白，其特征在于，所述第一功能结合区靶向肿瘤细胞或免疫细胞；较佳地，所述第一功能结合区靶向下列靶标中的任意一种或多种：GPC3、BCMA、PD-L1、Trop-2、5T4、AGS-16、ALK1、ANG-2、B7-H3、B7-H4、c-fms、c-Met、CA6、CD123、CD19、CD20、CD22、CD24、EpCAM、CD30、CD32b、CD37、CD38、CD40、CD52、CD70、CD74、CD79b、CD98、CEA、CEACAM5、CLDN18.2、CLDN6、CS1、CXCR4、DLL-4、EGFR、EGFRvIII、EGP-1、ENPP3、EphA3、ETBR、FGFR2、FN、FR-α、GCC、GD2、GPNMB、HER2、HER3、HLA-DR、ICAM-1、IGF-1R、IL-3R、LIV-1、MSLN、MUC16、MUC1、NaPi2b、结合素-4、Notch 2、Notch 1、PD-L2、PDGFR-α、PS、 PSMA、SLTRK6、STEAP1、TEM1、VEGFR、CD25、CD27L、DKK-1、CSF-1R、MSB0010718C、CD138、Siglec15和CD155。
  7. 如权利要求6所述的双特异性重组蛋白，其特征在于，所述第一功能结合区靶向GPC3；所述第一功能结合区包含特异性抗GPC3单克隆抗体的抗原结合片段；优选地，所述特异性抗GPC3单克隆抗体的重链可变区(VH)序列如SEQ ID NO:1所示，轻链可变区(VL)序列如SEQ ID NO:2所示；
    或者，所述第一功能结合区靶向PD-L1；所述第一功能结合区包含特异性抗PD-L1单克隆抗体的抗原结合片段；优选地，所述特异性抗PD-L1单克隆抗体的重链可变区(VH)序列如SEQ ID NO:5所示，轻链可变区(VL)序列如SEQ ID NO:6所示；
    或者，所述第一功能结合区靶向BCMA；所述第一功能结合区包含特异性抗BCMA单克隆抗体的抗原结合片段；优选地，所述特异性抗BCMA单克隆抗体的重链可变区(VH)序列如SEQ ID NO:7或SEQ ID NO:9所示，相应的轻链可变区(VL)序列如SEQ ID NO:8或SEQ ID NO:10所示；
    或者，所述第一功能结合区靶向Trop-2；所述第一功能结合区包含特异性抗Trop-2单克隆抗体的抗原结合片段；优选地，所述特异性抗Trop-2单克隆抗体的重链可变区(VH)序列如SEQ ID NO:11所示，轻链可变区(VL)序列如SEQ ID NO:12所示。
  8. 如权利要求1～7任一项所述的双特异性重组蛋白，其特征在于，所述接头序列的长度不超过20个氨基酸残基，所述接头序列选自(GGGGS)n、(GGGS)n、(GGS)n、(G)n、(GS)n、(EAAAK)n、或(XP)n，n为自然数；
    较佳地，所述接头序列的氨基酸序列如SEQ ID NO:13～16所示。
  9. 如权利要求8所述的双特异性重组蛋白，其特征在于，所述双特异性重组蛋白由A链和B链组成；其中，A链的结构为VH-CH1-FcA区，B链的结构为VL-CL-第二功能结合区-FcB区；或者A链的结构为VH-CL-FcA区，B链的结构为VL-CH1-第二功能结合区-FcB区；或者，A链的结构为VL-CH1-FcA区，B链的结构为VH-CL-第二功能结合区-FcB区；或者A链的结构为VL-CL-FcA区，B链的结构为VH-CH1-第二功能结合区-FcB区；所述A链与B链通过选自分子间作用力、共价键和盐键中一种或多种结合。
  10. 一种双特异性重组蛋白，其特征在于，所述双特异性重组蛋白包括第一功能结合区、第二功能结合区和Fc区；所述第一功能结合区包括重链可变区(VH)、轻链可变区(VL)、重链恒定区1(CH1)和轻链恒定区(CL)；所述第二功能结合区包含干扰素、其截短体或其突变体；所述双特异性重组蛋白由第一臂和第二臂组成，所述第一臂由链1和链2组成；其中，所述链1的结构为VH-CH1-FcA区，所述链2的结构为VL-CL；或者，所述链1的结构为VH-CL-FcA区，所述链2的结构为VL-CH1；或者，所述链1的结构为VL-CH1-FcA区，所述链2的结构为VH-CL；或者，所述链1的结构为VL-CL-FcA区，所述链2的结构为VH-CH1；所述第二臂的结构为第二功能结合区-FcB区，所述FcA区和FcB区组成所述双特异性重组蛋白的Fc区；所述第二臂中，第二功能结合区的氨基酸序列包含SEQ ID NO:17～22中任一种；
    当所述第一功能结合区靶向GPC3时，所述第一功能结合区包含特异性抗GPC3单克隆抗体的抗原结合片段；优选地，所述第一臂中，VH的氨基酸序列包含SEQ ID NO:1，CH1的氨基酸序列包含 SEQ ID NO:31，VL的氨基酸序列包含SEQ ID NO:2，CL的氨基酸序列包含SEQ ID NO:32；
    当所述第一功能结合区靶向PD-L1时，所述第一功能结合区包含特异性抗PD-L1单克隆抗体的抗原结合片段；优选地，所述第一臂中，VH的氨基酸序列包含SEQ ID NO:5，CH1的氨基酸序列包含SEQ ID NO:31，VL的氨基酸序列包含SEQ ID NO:6，CL的氨基酸序列包含SEQ ID NO:32；
    当所述第一功能结合区靶向BCMA时，所述第一功能结合区包含特异性抗BCMA单克隆抗体的抗原结合片段；优选地，所述第一臂中，VH的氨基酸序列包含SEQ ID NO:7或9，CH1的氨基酸序列包含SEQ ID NO:31，VL的氨基酸序列包含SEQ ID NO:8或10，CL的氨基酸序列包含32；
    当所述第一功能结合区靶向Trop-2时，所述第一功能结合区包含特异性抗Trop-2单克隆抗体的抗原结合片段；优选地，所述第一臂中，VH的氨基酸序列包含SEQ ID NO:11，CH1的氨基酸序列包含SEQ ID NO:31，VL的氨基酸序列包含SEQ ID NO:12，CL的氨基酸序列包含SEQ ID NO:32。
  11. 如权利要求9或10所述的双特异性重组蛋白，其特征在于，所述双特异性重组蛋白的Fc区为来自IgG1、IgG2、IgG3或IgG4的Fc区；所述Fc区包含天然序列或非天然序列；
    较佳地，所述Fc区为来自人IgG1、IgG2、IgG3或IgG4的Fc区；
    更佳地，所述FcA区和FcB区通过knobs-into-holes结合；
    进一步更佳地，当所述FcA区的氨基酸序列包含SEQ ID NO:25时，所述FcB区的氨基酸序列包含SEQ ID NO:25；或者，当所述FcA区的氨基酸序列包含SEQ ID NO:26时，所述FcB区的氨基酸序列包含SEQ ID NO:27；当所述FcA区的氨基酸序列包含SEQ ID NO:27时，所述FcB区的氨基酸序列包含SEQ ID NO:26；或者，当所述FcA区的氨基酸序列包含SEQ ID NO:28时，所述FcB区的氨基酸序列包含SEQ ID NO:28；或者，当所述FcA区的氨基酸序列包含SEQ ID NO:29时，所述FcB区的氨基酸序列包含SEQ ID NO:30；或者，当所述FcA区的氨基酸序列包含SEQ ID NO:30时，所述FcB区的氨基酸序列包含SEQ ID NO:29。
  12. 如权利要求11所述的双特异性重组蛋白，其特征在于，所述第一功能区靶向GPC3；所述A链中，VH的氨基酸序列包含SEQ ID NO:1，VL的氨基酸序列包含SEQ ID NO:2，CH1的氨基酸序列包含SEQ ID NO:31，CL的氨基酸序列包含SEQ ID NO:32，FcA区的氨基酸序列包含SEQ ID NO:26或SEQ ID NO:27；
    所述B链中，VH的氨基酸序列包含SEQ ID NO:1，VL的氨基酸序列包含SEQ ID NO:2，CH1的氨基酸序列包含SEQ ID NO:31，CL的氨基酸序列包含SEQ ID NO:32，第二功能结合区的氨基酸序列包含SEQ ID NO:17、18、19、21或22，FcB区的氨基酸序列包含SEQ ID NO:27或SEQ ID NO:26；
    较佳地，所述B链的氨基酸序列中SEQ ID NO:2或SEQ ID NO:1的C端与SEQ ID NO:32的N端直接连接，SEQ ID NO:32的C端与SEQ ID NO:17、SEQ ID NO:18、SEQ ID NO:19、SEQ ID NO:21或SEQ ID NO:22的N端通过接头序列连接，所述接头序列选自SEQ ID NO:13-16中的任一种；
    更佳地，当所述A链的氨基酸序列由SEQ ID NO:1、31和FcA区顺序连接组成时，
    所述B链的氨基酸序列由SEQ ID NO:2、32、13、17和FcB区顺序连接组成；
    或者，所述B链的氨基酸序列由SEQ ID NO:2、32、13、18和FcB区顺序连接组成；
    或者，所述B链的氨基酸序列由SEQ ID NO:2、32、13、19和FcB区顺序连接组成；
    或者，所述B链的氨基酸序列由SEQ ID NO:2、32、13、20和FcB区顺序连接组成；
    或者，所述B链的氨基酸序列由SEQ ID NO:2、32、13、21和FcB区顺序连接组成；
    或者，所述B链的氨基酸序列由SEQ ID NO:2、32、13、22和FcB区顺序连接组成；
    或者，所述B链的氨基酸序列由SEQ ID NO:2、32、14、17和FcB区顺序连接组成；
    或者，所述B链的氨基酸序列由SEQ ID NO:2、32、14、18和FcB区顺序连接组成；
    或者，所述B链的氨基酸序列由SEQ ID NO:2、32、14、19和FcB区顺序连接组成；
    或者，所述B链的氨基酸序列由SEQ ID NO:2、32、14、20和FcB区顺序连接组成；
    或者，所述B链的氨基酸序列由SEQ ID NO:2、32、14、21和FcB区顺序连接组成；
    或者，所述B链的氨基酸序列由SEQ ID NO:2、32、14、22和FcB区顺序连接组成；
    或者，所述B链的氨基酸序列由SEQ ID NO:2、32、15、17和FcB区顺序连接组成；
    或者，所述B链的氨基酸序列由SEQ ID NO:2、32、15、18和FcB区顺序连接组成；
    或者，所述B链的氨基酸序列由SEQ ID NO:2、32、15、19和FcB区顺序连接组成；
    或者，所述B链的氨基酸序列由SEQ ID NO:2、32、15、20和FcB区顺序连接组成；
    或者，所述B链的氨基酸序列由SEQ ID NO:2、32、15、21和FcB区顺序连接组成；
    或者，所述B链的氨基酸序列由SEQ ID NO:2、32、15、22和FcB区顺序连接组成；
    或者，所述B链的氨基酸序列由SEQ ID NO:2、32、16、17和FcB区顺序连接组成；
    或者，所述B链的氨基酸序列由SEQ ID NO:2、32、16、18和FcB区顺序连接组成；
    或者，所述B链的氨基酸序列由SEQ ID NO:2、32、16、19和FcB区顺序连接组成；
    或者，所述B链的氨基酸序列由SEQ ID NO:2、32、16、21和FcB区顺序连接组成；
    或者，所述B链的氨基酸序列由SEQ ID NO:2、32、16、22和FcB区顺序连接组成；
    当所述FcA区的氨基酸序列为SEQ ID NO:26时，所述FcB区的氨基酸序列为SEQ ID NO:27；或，当所述FcA区的氨基酸序列为SEQ ID NO:27时，所述FcB区的氨基酸序列为SEQ ID NO:26。
  13. 如权利要求11所述的双特异性重组蛋白，其特征在于，所述第一功能结合区靶向PD-L1；所述A链中，VH的氨基酸序列包含SEQ ID NO:5，VL的氨基酸序列包含SEQ ID NO:6，CH1的氨基酸序列包含SEQ ID NO:31，CL的氨基酸序列包含SEQ ID NO:32，FcA区的氨基酸序列包含SEQ ID NO:26或SEQ ID NO:27或SEQ ID NO:29或SEQ ID NO:30；
    所述B链中，VH的氨基酸序列包含SEQ ID NO:5，VL的氨基酸序列包含SEQ ID NO:6，CH1的氨基酸序列包含SEQ ID NO:31，CL的氨基酸序列包含SEQ ID NO:32，第二功能结合区的氨基酸序列包含SEQ ID NO:17-22中任一种，FcB区的氨基酸序列包含SEQ ID NO:27或SEQ ID NO:26或SEQ ID NO:30或SEQ ID NO:29；
    较佳地，所述B链的氨基酸序列中SEQ ID NO:6或SEQ ID NO:5的C端与SEQ ID NO:32的N端直接连接，SEQ ID NO:32的C端与SEQ ID NO:17-22中任一种的N端通过接头序列连接，所述接头序列选自SEQ ID NO:13-16中的任一种；
    更佳地，
    当所述A链的氨基酸序列由SEQ ID NO:5、31、FcA区顺序连接组成时，
    所述B链的氨基酸序列由SEQ ID NO:6、32、13、17和FcB区顺序连接组成；
    或者，所述B链的氨基酸序列由SEQ ID NO:6、32、13、18和FcB区顺序连接组成；
    或者，所述B链的氨基酸序列由SEQ ID NO:6、32、13、19和FcB区顺序连接组成；
    或者，所述B链的氨基酸序列由SEQ ID NO:6、32、13、20和FcB区顺序连接组成；
    或者，所述B链的氨基酸序列由SEQ ID NO:6、32、13、21和FcB区顺序连接组成；
    或者，所述B链的氨基酸序列由SEQ ID NO:6、32、13、22和FcB区顺序连接组成；
    或者，所述B链的氨基酸序列由SEQ ID NO:6、32、14、17和FcB区顺序连接组成；
    或者，所述B链的氨基酸序列由SEQ ID NO:6、32、14、18和FcB区顺序连接组成；
    或者，所述B链的氨基酸序列由SEQ ID NO:6、32、14、19和FcB区顺序连接组成；
    或者，所述B链的氨基酸序列由SEQ ID NO:6、32、14、20和FcB区顺序连接组成；
    或者，所述B链的氨基酸序列由SEQ ID NO:6、32、14、21和FcB区顺序连接组成；
    或者，所述B链的氨基酸序列由SEQ ID NO:6、32、14、22和FcB区顺序连接组成；
    或者，所述B链的氨基酸序列由SEQ ID NO:6、32、15、17和FcB区顺序连接组成；
    或者，所述B链的氨基酸序列由SEQ ID NO:6、32、15、18和FcB区顺序连接组成；
    或者，所述B链的氨基酸序列由SEQ ID NO:6、32、15、19和FcB区顺序连接组成；
    或者，所述B链的氨基酸序列由SEQ ID NO:6、32、15、20和FcB区顺序连接组成；
    或者，所述B链的氨基酸序列由SEQ ID NO:6、32、15、21和FcB区顺序连接组成；
    或者，所述B链的氨基酸序列由SEQ ID NO:6、32、15、22和FcB区顺序连接组成；
    或者，所述B链的氨基酸序列由SEQ ID NO:6、32、16、17和FcB区顺序连接组成；
    或者，所述B链的氨基酸序列由SEQ ID NO:6、32、16、18和FcB区顺序连接组成；
    或者，所述B链的氨基酸序列由SEQ ID NO:6、32、16、19和FcB区顺序连接组成；
    或者，所述B链的氨基酸序列由SEQ ID NO:6、32、16、20和FcB区顺序连接组成；
    或者，所述B链的氨基酸序列由SEQ ID NO:6、32、16、21和FcB区顺序连接组成；
    或者，所述B链的氨基酸序列由SEQ ID NO:6、32、16、22和FcB区顺序连接组成；
    当所述FcA区的氨基酸序列为SEQ ID NO:26时，所述FcB区的氨基酸序列为SEQ ID NO:27；或者，当所述FcA区的氨基酸序列为SEQ ID NO:27时，所述FcB区的氨基酸序列为SEQ ID NO:26；或者，当所述FcA区的氨基酸序列为SEQ ID NO:29时，所述FcB区的氨基酸序列为SEQ ID NO:30；或者，当所述FcA区的氨基酸序列为SEQ ID NO:30时，所述FcB区的氨基酸序列为SEQ ID NO:29。
  14. 如权利要求11所述的双特异性重组蛋白，其特征在于，所述第一功能结合区靶向BCMA；
    较佳地，所述A链中，VH的氨基酸序列包含SEQ ID NO:7或9，VL的氨基酸序列包含SEQ ID NO:8或10，CH1的氨基酸序列包含SEQ ID NO:31，CL的氨基酸序列包含SEQ ID NO:32，FcA区的氨基酸序列包含SEQ ID NO:26或SEQ ID NO:27；
    所述B链中，VH的氨基酸序列包含SEQ ID NO:7或9，VL的氨基酸序列包含SEQ ID NO:8或10，CH1的氨基酸序列包含SEQ ID NO:31，CL的氨基酸序列包含SEQ ID NO:32，第二功能结合区的氨基酸序列包含SEQ ID NO:17-22中任一种，FcB区的氨基酸序列包含SEQ ID NO:27或SEQ ID NO:26；
    更佳地，所述B链的氨基酸序列中SEQ ID NO:7、8、9或10的C端与SEQ ID NO:32的N端直接连接，SEQ ID NO:32的C端与SEQ ID NO:17-22中任一种的N端通过接头序列连接，所述接头序列选自SEQ ID NO:13-16中的任一种；
    进一步更佳地，
    当所述A链的氨基酸序列由SEQ ID NO:7、31、FcA区顺序连接组成时，
    所述B链的氨基酸序列由SEQ ID NO:8、32、13、17和FcB区顺序连接组成；
    或者，所述B链的氨基酸序列由SEQ ID NO:8、32、13、18和FcB区顺序连接组成；
    或者，所述B链的氨基酸序列由SEQ ID NO:8、32、13、19和FcB区顺序连接组成；
    或者，所述B链的氨基酸序列由SEQ ID NO:8、32、13、20和FcB区顺序连接组成；
    或者，所述B链的氨基酸序列由SEQ ID NO:8、32、13、21和FcB区顺序连接组成；
    或者，所述B链的氨基酸序列由SEQ ID NO:8、32、13、22和FcB区顺序连接组成；
    或者，所述B链的氨基酸序列由SEQ ID NO:8、32、14、17和FcB区顺序连接组成；
    或者，所述B链的氨基酸序列由SEQ ID NO:8、32、14、18和FcB区顺序连接组成；
    或者，所述B链的氨基酸序列由SEQ ID NO:8、32、14、19和FcB区顺序连接组成；
    或者，所述B链的氨基酸序列由SEQ ID NO:8、32、14、20和FcB区顺序连接组成；
    或者，所述B链的氨基酸序列由SEQ ID NO:8、32、14、21和FcB区顺序连接组成；
    或者，所述B链的氨基酸序列由SEQ ID NO:8、32、14、22和FcB区顺序连接组成；
    或者，所述B链的氨基酸序列由SEQ ID NO:8、32、15、17和FcB区顺序连接组成；
    或者，所述B链的氨基酸序列由SEQ ID NO:8、32、15、18和FcB区顺序连接组成；
    或者，所述B链的氨基酸序列由SEQ ID NO:8、32、15、19和FcB区顺序连接组成；
    或者，所述B链的氨基酸序列由SEQ ID NO:8、32、15、20和FcB区顺序连接组成；
    或者，所述B链的氨基酸序列由SEQ ID NO:8、32、15、21和FcB区顺序连接组成；
    或者，所述B链的氨基酸序列由SEQ ID NO:8、32、15、22和FcB区顺序连接组成；
    或者，所述B链的氨基酸序列由SEQ ID NO:8、32、16、17和FcB区顺序连接组成；
    或者，所述B链的氨基酸序列由SEQ ID NO:8、32、16、18和FcB区顺序连接组成；
    或者，所述B链的氨基酸序列由SEQ ID NO:8、32、16、19和FcB区顺序连接组成；
    或者，所述B链的氨基酸序列由SEQ ID NO:8、32、16、20和FcB区顺序连接组成；
    或者，所述B链的氨基酸序列由SEQ ID NO:8、32、16、21和FcB区顺序连接组成；
    或者，所述B链的氨基酸序列由SEQ ID NO:8、32、16、22和FcB区顺序连接组成；
    当所述A链的氨基酸序列由SEQ ID NO:9、31、FcA区顺序连接组成时，
    所述B链的氨基酸序列由SEQ ID NO:10、32、13、17和FcB区顺序连接组成；
    或者，所述B链的氨基酸序列由SEQ ID NO:10、32、13、18和FcB区顺序连接组成；
    或者，所述B链的氨基酸序列由SEQ ID NO:10、32、13、19和FcB区顺序连接组成；
    或者，所述B链的氨基酸序列由SEQ ID NO:10、32、13、20和FcB区顺序连接组成；
    或者，所述B链的氨基酸序列由SEQ ID NO:10、32、13、21和FcB区顺序连接组成；
    或者，所述B链的氨基酸序列由SEQ ID NO:10、32、13、22和FcB区顺序连接组成；
    或者，所述B链的氨基酸序列由SEQ ID NO:10、32、14、17和FcB区顺序连接组成；
    或者，所述B链的氨基酸序列由SEQ ID NO:10、32、14、18和FcB区顺序连接组成；
    或者，所述B链的氨基酸序列由SEQ ID NO:10、32、14、19和FcB区顺序连接组成；
    或者，所述B链的氨基酸序列由SEQ ID NO:10、32、14、20和FcB区顺序连接组成；
    或者，所述B链的氨基酸序列由SEQ ID NO:10、32、14、21和FcB区顺序连接组成；
    或者，所述B链的氨基酸序列由SEQ ID NO:10、32、14、22和FcB区顺序连接组成；
    或者，所述B链的氨基酸序列由SEQ ID NO:10、32、15、17和FcB区顺序连接组成；
    或者，所述B链的氨基酸序列由SEQ ID NO:10、32、15、18和FcB区顺序连接组成；
    或者，所述B链的氨基酸序列由SEQ ID NO:10、32、15、19和FcB区顺序连接组成；
    或者，所述B链的氨基酸序列由SEQ ID NO:10、32、15、20和FcB区顺序连接组成；
    或者，所述B链的氨基酸序列由SEQ ID NO:10、32、15、21和FcB区顺序连接组成；
    或者，所述B链的氨基酸序列由SEQ ID NO:10、32、15、22和FcB区顺序连接组成；
    或者，所述B链的氨基酸序列由SEQ ID NO:10、32、16、17和FcB区顺序连接组成；
    或者，所述B链的氨基酸序列由SEQ ID NO:10、32、16、18和FcB区顺序连接组成；
    或者，所述B链的氨基酸序列由SEQ ID NO:10、32、16、19和FcB区顺序连接组成；
    或者，所述B链的氨基酸序列由SEQ ID NO:10、32、16、20和FcB区顺序连接组成；
    或者，所述B链的氨基酸序列由SEQ ID NO:10、32、16、21和FcB区顺序连接组成；
    或者，所述B链的氨基酸序列由SEQ ID NO:10、32、16、22和FcB区顺序连接组成；
    当所述FcA区的氨基酸序列为SEQ ID NO:26时，所述FcB区的氨基酸序列为SEQ ID NO:27；当所述FcA区的氨基酸序列为SEQ ID NO:27时，所述FcB区的氨基酸序列为SEQ ID NO:26。
  15. 如权利要求11所述的双特异性重组蛋白，其特征在于，所述第一功能结合区靶向Trop-2；
    所述A链中，VH的氨基酸序列包含SEQ ID NO:11，VL的氨基酸序列包含SEQ ID NO:12，CH1的氨基酸序列包含SEQ ID NO:31，CL的氨基酸序列包含SEQ ID NO:32，FcA区的氨基酸序列包含SEQ ID NO:26或SEQ ID NO:27；
    所述B链中，VH的氨基酸序列包含SEQ ID NO:11，VL的氨基酸序列包含SEQ ID NO:12，CH1的氨基酸序列包含SEQ ID NO:31，CL的氨基酸序列包含SEQ ID NO:32，第二功能结合区的氨基酸序列包含SEQ ID NO:17-22中任一种，FcB区的氨基酸序列包含SEQ ID NO:27或SEQ ID NO:26；
    较佳地，所述B链的氨基酸序列中SEQ ID NO:12或SEQ ID NO:11的C端与SEQ ID NO:32的N端直接连接，SEQ ID NO:32的C端与SEQ ID NO:17-22中任一种的N端通过接头序列连接，所述接头序列选自SEQ ID NO:13-16中的任一种；
    更佳地，
    当所述A链的氨基酸序列由SEQ ID NO:11、31、FcA区顺序连接组成时，
    所述B链的氨基酸序列由SEQ ID NO:12、32、13、17和FcB区顺序连接组成；
    或者，所述B链的氨基酸序列由SEQ ID NO:12、32、13、18和FcB区顺序连接组成；
    或者，所述B链的氨基酸序列由SEQ ID NO:12、32、13、19和FcB区顺序连接组成；
    或者，所述B链的氨基酸序列由SEQ ID NO:12、32、13、20和FcB区顺序连接组成；
    或者，所述B链的氨基酸序列由SEQ ID NO:12、32、13、21和FcB区顺序连接组成；
    或者，所述B链的氨基酸序列由SEQ ID NO:12、32、13、22和FcB区顺序连接组成；
    或者，所述B链的氨基酸序列由SEQ ID NO:12、32、14、17和FcB区顺序连接组成；
    或者，所述B链的氨基酸序列由SEQ ID NO:12、32、14、18和FcB区顺序连接组成；
    或者，所述B链的氨基酸序列由SEQ ID NO:12、32、14、19和FcB区顺序连接组成；
    或者，所述B链的氨基酸序列由SEQ ID NO:12、32、14、20和FcB区顺序连接组成；
    或者，所述B链的氨基酸序列由SEQ ID NO:12、32、14、21和FcB区顺序连接组成；
    或者，所述B链的氨基酸序列由SEQ ID NO:12、32、14、22和FcB区顺序连接组成；
    或者，所述B链的氨基酸序列由SEQ ID NO:12、32、15、17和FcB区顺序连接组成；
    或者，所述B链的氨基酸序列由SEQ ID NO:12、32、15、18和FcB区顺序连接组成；
    或者，所述B链的氨基酸序列由SEQ ID NO:12、32、15、19和FcB区顺序连接组成；
    或者，所述B链的氨基酸序列由SEQ ID NO:12、32、15、20和FcB区顺序连接组成；
    或者，所述B链的氨基酸序列由SEQ ID NO:12、32、15、21和FcB区顺序连接组成；
    或者，所述B链的氨基酸序列由SEQ ID NO:12、32、15、22和FcB区顺序连接组成；
    或者，所述B链的氨基酸序列由SEQ ID NO:12、32、16、17和FcB区顺序连接组成；
    或者，所述B链的氨基酸序列由SEQ ID NO:12、32、16、18和FcB区顺序连接组成；
    或者，所述B链的氨基酸序列由SEQ ID NO:12、32、16、19和FcB区顺序连接组成；
    或者，所述B链的氨基酸序列由SEQ ID NO:12、32、16、20和FcB区顺序连接组成；
    或者，所述B链的氨基酸序列由SEQ ID NO:12、32、16、21和FcB区顺序连接组成；
    或者，所述B链的氨基酸序列由SEQ ID NO:12、32、16、22和FcB区顺序连接组成；
    当所述FcA区的氨基酸序列为SEQ ID NO:26时，所述FcB区的氨基酸序列为SEQ ID NO:27；当所述FcA区的氨基酸序列为SEQ ID NO:27时，所述FcB区的氨基酸序列为SEQ ID NO:26。
  16. 如权利要求10所述的双特异性重组蛋白，其特征在于，
    当所述第一功能结合区靶向GPC3时，所述第一臂中链1的氨基酸序列由SEQ ID NO:1、31和FcA区顺序连接组成时，链2的氨基酸序列由SEQ ID NO:2、32顺序连接组成，所述第二臂的氨基酸序列由SEQ ID NO:17-22中任一种和FcB区顺序连接组成；当所述FcA区的氨基酸序列为SEQ ID NO:26时，所述FcB区的氨基酸序列为SEQ ID NO:27；或者，当所述FcA区的氨基酸序列为SEQ ID NO:27时，所述FcB区的氨基酸序列为SEQ ID NO:26；
    当所述第一功能结合区靶向PD-L1时，所述第一臂中链1的氨基酸序列由SEQ ID NO:5、31和FcA区顺序连接组成时，链2的氨基酸序列由SEQ ID NO:6、32顺序连接组成，所述第二臂的氨基酸序列由SEQ ID NO:17-22中任一种和FcB区顺序连接组成；当所述FcA区的氨基酸序列为SEQ ID NO:26时，所述FcB区的氨基酸序列为SEQ ID NO:27；或者，当所述FcA区的氨基酸序列为SEQ ID NO:27时，所述FcB区的氨基酸序列为SEQ ID NO:26；或者，当所述FcA区的氨基酸序列为SEQ ID NO:29 时，所述FcB区的氨基酸序列为SEQ ID NO:30；或者，当所述FcA区的氨基酸序列为SEQ ID NO:30时，所述FcB区的氨基酸序列为SEQ ID NO:29；
    当所述第一功能结合区靶向BCMA时，所述第一臂中链1的氨基酸序列由SEQ ID NO:7、31和FcA区顺序连接组成时，链2的氨基酸序列由SEQ ID NO:8、32顺序连接组成；或，所述第一臂中链1的氨基酸序列由SEQ ID NO:9、31和FcA区顺序连接组成时，所述第一臂中链2的氨基酸序列由SEQ ID NO:10、32顺序连接组成；所述第二臂的氨基酸序列由SEQ ID NO:17-22中任一种和FcB区顺序连接组成；当所述FcA区的氨基酸序列为SEQ ID NO:26时，所述FcB区的氨基酸序列为SEQ ID NO:27；或者，当所述FcA区的氨基酸序列为SEQ ID NO:27时，所述FcB区的氨基酸序列为SEQ ID NO:26；
    当所述第一功能结合区靶向Trop-2时，所述第一臂中链1的氨基酸序列由SEQ ID NO:11、31和FcA区顺序连接组成时，链2的氨基酸序列由SEQ ID NO:12、32顺序连接组成，所述第二臂的氨基酸序列由SEQ ID NO:17-22中任一种和FcB区顺序连接组成，当所述FcA区的氨基酸序列为SEQ ID NO:26时，所述FcB区的氨基酸序列为SEQ ID NO:27；或者，当所述FcA区的氨基酸序列为SEQ ID NO:27时，所述FcB区的氨基酸序列为SEQ ID NO:26。
  17. 一种编码如权利要求1-16任一项所述的双特异性重组蛋白的核酸分子。
  18. 一种包含如权利要求17所述的核酸分子的重组表达载体。
  19. 一种由如权利要求18所述的表达载体转化宿主细胞得到的重组细胞。
  20. 一种如权利要求1-16任一项所述的双特异性重组蛋白的制备方法，其特征在于，所述方法包括使用如权利要求19所述的重组细胞表达得到所述双特异性重组蛋白的步骤。
  21. 一种药物或药物组合物，其包含如权利要求1-16任一项所述的双特异性重组蛋白；
    较佳地，所述药物组合物还包括药学上可接受的载体。
  22. 一种如权利要求1-16任一项所述的双特异性重组蛋白或如权利要求21所述的药物或药物组合物在制备治疗、辅助治疗或预防疾病的药物中的用途；
    较佳地，所述疾病选自肿瘤、肝病和病毒感染疾病；
    更佳地，所述疾病为GPC3、BCMA、PD-L1、Trop-2、5T4、AGS-16、ALK1、ANG-2、B7-H3、B7-H4、c-fms、c-Met、CA6、CD123、CD19、CD20、CD22、CD24、EpCAM、CD30、CD32b、CD37、CD38、CD40、CD52、CD70、CD74、CD79b、CD98、CEA、CEACAM5、CLDN18.2、CLDN6、CS1、CXCR4、DLL-4、EGFR、EGFRvIII、EGP-1、ENPP3、EphA3、ETBR、FGFR2、FN、FR-α、GCC、GD2、GPNMB、HER2、HER3、HLA-DR、ICAM-1、IGF-1R、IL-3R、LIV-1、MSLN、MUC16、MUC1、NaPi2b、结合素-4、Notch 2、Notch 1、PD-L2、PDGFR-α、PS、PSMA、SLTRK6、STEAP1、TEM1、VEGFR、CD25、CD27L、DKK-1、CSF-1R、MSB0010718C、CD138、Siglec15或CD155阳性的相关疾病。
  23. 如权利要求22所述的应用，其特征在于，所述肿瘤选自乳腺癌、肠癌、胰腺癌、食管癌、卵巢癌、胃癌、前列腺癌、肾癌、宫颈癌、骨髓瘤、淋巴瘤、白血病、甲状腺癌、子宫癌、膀胱癌、神经内分泌癌、头颈癌、肝癌、鼻咽癌、睾丸癌、肺癌、黑色素瘤、皮肤癌、肉瘤、神经胶质瘤、间皮瘤和 骨髓发育不良综合征；
    较佳地，所述肠癌包括结肠直肠癌，所述卵巢癌包括卵黄囊瘤，所述神经内分泌癌包括梅克尔细胞癌，所述肺癌包括小细胞肺癌和非小细胞肺癌，所述皮肤癌包括基底细胞皮肤癌和鳞状细胞皮肤癌，所述肉瘤包括隆突性纤维肉瘤，所述神经胶质瘤包括胶质母细胞瘤，所述子宫癌包括子宫内膜癌和子宫肉瘤。
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