WO2023078245A1 - Il10单体融合蛋白及其应用 - Google Patents

Il10单体融合蛋白及其应用 Download PDF

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WO2023078245A1
WO2023078245A1 PCT/CN2022/128998 CN2022128998W WO2023078245A1 WO 2023078245 A1 WO2023078245 A1 WO 2023078245A1 CN 2022128998 W CN2022128998 W CN 2022128998W WO 2023078245 A1 WO2023078245 A1 WO 2023078245A1
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antibody
terminal
monomer
fusion protein
connecting peptide
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French (fr)
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芦迪
霍永庭
张轶博
闫加庆
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广东菲鹏制药股份有限公司
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/54—Interleukins [IL]
    • C07K14/5428—IL-10
    • 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
    • A61K38/20—Interleukins [IL]
    • 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
    • A61K38/20—Interleukins [IL]
    • A61K38/2066—IL-10
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • 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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6849—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a receptor, a cell surface antigen or a cell surface determinant
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/54—Interleukins [IL]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/54—Interleukins [IL]
    • C07K14/55—IL-2
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • 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
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention relates to the field of biomedicine, specifically, fusion protein and its application, more specifically, a fusion protein, nucleic acid, expression vector, recombinant cell, the fusion protein or nucleic acid, expression vector or recombinant cell in the preparation of drugs Uses and pharmaceutical compositions.
  • Interleukin 10 Interleukin 10, IL-10, or IL10
  • CCF human cytokine synthesis inhibitory factor
  • 10 protein has safety problems. After a small amount of natural dimerized IL-10 molecule binds to its receptor IL-10R ⁇ with high affinity, it further binds to IL-10R ⁇ to form a hexameric complex and activate downstream signals, causing biological functional response.
  • the current clinical IL-10 drug molecules lack targeting, and the drug molecules are easy to off-target and cause side effects; and most of the IL-10 antibody fusion proteins under development are designed to use antibody targeting to introduce IL-10 into function in the local environment.
  • These fusion protein molecules often have a normal IL-10 dimer structure, but due to the high affinity binding of IL-10 dimer to IL-10R ⁇ , it may affect the targeting effect of the antibody end, which leads to the failure of the design idea, and if The activity of the antibody side requires a larger dose, and the side effects of the IL-10 side are still a thorny issue.
  • the drug molecules are easy to miss the target. Not only the drug effect is not easy to exert, but also easy to produce side effects.
  • the inventors explored the connection mode of IL-10 monomer molecules and antibody molecules, and prepared a A fusion protein comprising an IL-10 monomer molecule and an antibody molecule, the IL-10 molecule in the fusion protein is in the form of a monomer before the antibody binds to the target cell, cannot bind to its receptor, and will not interfere with the interaction of the antibody molecule with the target cell
  • Antigen binding on target cells after the antibody binds to the antigen, a large number of IL-10 monomer molecules aggregate, so that two IL-10 monomers that are close to each other function as a dimer and bind to the IL-10 receptor, thereby causing
  • the normal biological response makes the targeting and safety of IL-10 drugs significantly improved, and the efficacy of the drug is fully exerted.
  • the present invention proposes a fusion protein.
  • it includes an antibody and an IL-10 monomer, wherein the antibody includes two identical light chains and two identical heavy chains, and one IL-10 monomer is connected to the antibody heavy chain or light chain , the N-terminal of the IL-10 monomer is not linked to the C-terminal of the antibody heavy chain.
  • the natural IL-10 molecule is in the form of a dimer
  • the fusion protein according to the embodiment of the present invention is in the form of a monomer before the antibody binds to the target cell, and cannot bind to its receptor, and will not interfere with the binding of the antibody molecule to the target cell .
  • IL-10 monomer fusion protein After the antibody binds to the target cells, a large number of IL-10 monomer molecules aggregate, so that the two IL-10 monomers that are close to each other function as a dimer and bind to the IL-10 receptor, thereby causing a normal biological response. And because IL-10 monomer is difficult to trigger biological function, so IL-10 monomer fusion protein will not produce natural dimer IL-10 molecules or the dose limit of antibody fusion protein, making IL-10 monomer fusion The dosage of protein administration has been significantly increased.
  • the fusion protein can be used in combination with more antibody molecules, so that the targeting and safety of the fusion protein can be significantly improved, the drug effect can be fully exerted, and various tumors or inflammatory diseases can be treated or prevented.
  • the above-mentioned fusion protein may further include at least one of the following additional technical features:
  • each antibody light chain includes a VL region and a CL region
  • each antibody heavy chain includes a VH region, a CH1 region, a CH2 region, and a CH3 region
  • the C-terminal of the chain is connected, or the C-terminal of the IL-10 monomer is connected with the N-terminal of the light chain of the antibody, or the N-terminal of the IL-10 monomer is connected with the C-terminal of the VL region of the hydrogen chain, and the IL-10
  • the C-terminal of the monomer is connected to the N-terminal of the CL region of the hydrogen chain, or the C-terminal of the IL-10 monomer is connected to the N-terminal of the antibody heavy chain, or the N-terminal of the IL-10 monomer is connected to the heavy chain VH
  • the C-terminal of the IL-10 monomer is connected to the N-terminal of the CH1 region of the heavy chain, or the N-terminal of the IL-10 monomer is connected to the C-
  • the IL-10 monomer includes natural IL-10 monomer.
  • the IL-10 monomer is a modified natural IL-10 monomer, and the modification includes at least one of the following (i) and (ii): (i) removing natural IL-10 The first 2 amino acids at the N-terminal of the monomer; (ii) inserting a first connecting peptide between the 116th and 117th amino acids, and the first connecting peptide is a flexible connecting peptide.
  • the stability of the IL-10 monomer was improved by removing the first 2 amino acids at the N-terminus of the native IL-10 monomer, while inserting a first linkage between amino acids 116 and 117 of the IL-10 monomer
  • the IL-10 monomer molecule in the fusion protein is closed to become a closed-loop IL-10 monomer molecule (the two IL10 chains of the natural IL10 dimer are interlaced to form, and after being inserted into the linker, the IL10 Monomers can independently form structural domains (that is, closed-loop monomer molecules), and two IL-10 monomers in the same fusion protein will not interfere with each other such as connection.
  • the amino acid sequence of the first connecting peptide is shown in SEQ ID NO: 21.
  • the IL-10 monomer has the amino acid sequence shown in SEQ ID NO:2.
  • the IL-10 monomer is connected to the antibody heavy chain or light chain through the second linker peptide or/and the third linker peptide.
  • the IL10 monomer is connected to the heavy chain or light chain of the antibody by using the second linking peptide or/and the third linking peptide, after the antibody part in the fusion protein binds to the corresponding target cell, the 2 close to each other
  • the IL-10 monomer of each fusion protein is more likely to form a dimer to play a role and bind to the IL-10 receptor.
  • the second connecting peptide and/or the third connecting peptide may be a flexible connecting peptide or a rigid connecting peptide.
  • the second connecting peptide has the amino acid sequence shown in SEQ ID NO:12
  • the third connecting peptide has the amino acid sequence shown in SEQ ID NO:22.
  • the IL-10 monomer is connected to the heavy chain end or light chain end of the antibody through the second linking peptide.
  • the N-terminus of the second connecting peptide is connected to the C-terminus of the light chain of the antibody, and the C-terminus of the second connecting peptide is connected to the N-terminus of the IL-10 monomer; or
  • the N-terminal of the second connecting peptide is connected to the C-terminal of the IL-10 monomer, and the C-terminal of the second connecting peptide is connected to the N-terminal of the light chain of the antibody; or the second connecting peptide
  • the N-terminus is connected to the C-terminus of the IL-10 monomer, and the C-terminus of the second connecting peptide is connected to the N-terminus of the heavy chain of the antibody.
  • the IL-10 monomer is connected to the end of the heavy chain or the end of the light chain of the antibody through the third connecting peptide;
  • the N-terminus of the third connecting peptide is connected to the C-terminus of the light chain of the antibody, and the C-terminus of the third connecting peptide is connected to the N-terminus of the IL-10 monomer; or
  • the N-terminal of the third connecting peptide is connected to the C-terminal of the IL-10 monomer, and the C-terminal of the third connecting peptide is connected to the N-terminal of the antibody light chain; or the third connecting peptide
  • the N-terminal is connected to the C-terminal of the IL-10 monomer, and the C-terminal of the third connecting peptide is connected to the N-terminal of the heavy chain of the antibody.
  • the N-terminal of the third connecting peptide is connected to the C-terminal of the VL region of the light chain, and the C-terminal of the third connecting peptide is connected to the N-terminal of the IL-10 monomer; Or the N-terminal of the third connecting peptide is connected to the C-terminal of the IL-10 monomer, and the C-terminal of the third connecting peptide is connected to the N-terminal of the light chain CL region; or the third connecting The N-terminal of the peptide is connected to the C-terminal of the heavy chain VH region, the C-terminal of the third connecting peptide is connected to the N-terminal of the IL-10 monomer; or the N-terminal of the third connecting peptide is connected to the The C-terminal of the IL-10 monomer is connected, the C-terminal of the third connecting peptide is connected with the N-terminal of the CH1 region of the heavy chain; or the N-terminal of the third connecting
  • the N-terminal of the IL-10 monomer is connected to the C-terminal of the VL region of the light chain of the antibody through the second connecting peptide, and the C-terminal of the IL-10 monomer is connected through
  • the second connecting peptide is connected to the N-terminal of the light chain CL region of the antibody; or the N-terminal of the IL-10 monomer is connected to the C-terminal of the heavy chain VH region of the antibody through the second connecting peptide connected, the C-terminal of the IL-10 monomer is connected to the N-terminal of the heavy chain CH1 region of the antibody through the second connecting peptide; or the N-terminal of the IL-10 monomer is connected through the second
  • the peptide is connected to the C-terminal of the heavy chain CH2 region of the antibody, and the C-terminal of the IL-10 monomer is connected to the N-terminal of the heavy chain CH3 region of the antibody through the second connecting peptide.
  • the antibody is selected from immune cell antibodies and tumor cell antibodies.
  • the antibody is anti-PD-1 antibody, anti-PD-L1 antibody, anti-Her2 antibody, anti-CCR8 antibody, anti-VEGFR2 antibody, anti-claudin18.2 antibody, anti-CD39 antibody, anti-SMA4D antibody, Anti-GUCY2C antibody, anti-CTLA-4 antibody, anti-TIM3 antibody, anti-TIGIT antibody, anti-CD47 antibody, or anti-TNF ⁇ antibody.
  • the fusion protein has the amino acid sequence described in SEQ ID NO:3 and SEQ ID NO:9; or the fusion protein has the amino acid sequence described in SEQ ID NO:3 and SEQ ID NO:5 sequence; or the fusion protein has the amino acid sequence described in SEQ ID NO:4 and SEQ ID NO:6; or the fusion protein has the amino acid sequence described in SEQ ID NO:7 and SEQ ID NO:6; or the The fusion protein has the amino acid sequence described in SEQ ID NO:3 and SEQ ID NO:8; or the fusion protein has the amino acid sequence described in SEQ ID NO:10 and SEQ ID NO:6.
  • the invention proposes a nucleic acid.
  • the nucleic acid encodes the fusion protein described in the first aspect.
  • the IL-10 molecule in the fusion protein encoded by the nucleic acid according to the embodiment of the present invention is in the form of a monomer before the antibody part binds to the target cell, cannot bind to its receptor, and will not interfere with the binding of the antibody molecule to the target cell; the antibody binds to the target cell Afterwards, a large number of IL-10 monomer molecules aggregate, so that two IL-10 monomers that are close to each other function as a dimer and bind to IL-10 receptors, thereby causing normal biological responses; and because IL-10 10 monomer is difficult to trigger biological function, so IL-10 monomer fusion protein will not produce natural dimeric IL-10 molecule or its antibody fusion protein. Dose was significantly increased.
  • the fusion protein can be used in combination with more antibody molecules, so that the targeting and safety of the fusion
  • the nucleic acid molecule is DNA or RNA.
  • the present invention provides an expression vector.
  • the expression vector comprises the nucleic acid described in the second aspect.
  • the expression vector according to the embodiment of the present invention is introduced into a suitable recipient cell, under the mediation of the regulatory system, the expression of the aforementioned fusion protein can be effectively realized, and the IL-10 molecule in the obtained fusion protein is bound to the antibody part
  • the target cells are in the form of monomers before, unable to bind to their receptors, and will not interfere with the binding of antibody molecules to target cells; after the antibody binds to target cells, a large number of IL-10 monomer molecules aggregate, making the two IL-10 monomers close to each other
  • the IL-10 monomer functions as a dimer and binds to the IL-10 receptor, thereby causing normal biological responses; and because IL-10 monomers are difficult to trigger biological functions, IL-10 monomer fusion proteins will not produce natural
  • the dosage limitation of the dimer IL-10 molecule or its antibody fusion protein makes the dosage of the
  • the present invention provides a recombinant cell.
  • the recombinant cell carries the nucleic acid described in the second aspect, the expression vector described in the third aspect, or expresses the fusion protein described in the first aspect.
  • the recombinant cells according to the embodiments of the present invention can be used for the expression and large-scale acquisition of the aforementioned fusion protein.
  • the IL-10 molecule of the obtained fusion protein is in the form of a monomer, and cannot bind to its receptor.
  • IL-10 monomer fusion proteins Interfering with the binding of antibody molecules to target cells; after the antibody binds to target cells, a large number of IL-10 monomer molecules aggregate, making the two IL-10 monomers close to each other function as dimers and bind to IL-10 receptors , so as to cause normal biological responses; and because IL-10 monomers are difficult to trigger biological functions, IL-10 monomer fusion proteins will not produce natural dimer IL-10 molecules or their antibody fusion protein doses Restriction makes the dosage of IL-10 monomer fusion protein significantly increased.
  • the fusion protein can be used in combination with more antibody molecules, so that the targeting and safety of the fusion protein can be significantly improved, the drug effect can be fully exerted, and various tumors or inflammatory diseases can be treated or prevented.
  • the present invention proposes the fusion protein described in the first aspect, the nucleic acid described in the second aspect, the expression vector described in the third aspect or the recombinant cell described in the fourth aspect in the preparation of drugs the use of.
  • the medicament is used for treating or preventing tumors or inflammatory diseases.
  • the drug can effectively treat or alleviate tumors and inflammatory diseases, and has high safety.
  • the present invention provides a pharmaceutical composition. According to an embodiment of the present invention, it comprises the fusion protein described in the first aspect, the nucleic acid described in the second aspect, the expression vector described in the third aspect or the recombinant cell described in the fourth aspect.
  • the medicine according to the specific embodiment of the present invention can effectively treat or alleviate tumors and inflammatory diseases, and has high safety.
  • the above pharmaceutical composition may further include at least one of the following additional technical features (i) and (ii):
  • the pharmaceutical composition is used for treating or preventing tumors or inflammatory diseases.
  • the pharmaceutical composition may include: pharmaceutically acceptable adjuvants, which include stabilizers, wetting agents, emulsifiers, binders, etc. at least one of osmotic agents; the pharmaceutical composition is at least one of tablet, granule, powder, capsule, solution, suspension, freeze-dried preparation.
  • pharmaceutically acceptable adjuvants which include stabilizers, wetting agents, emulsifiers, binders, etc. at least one of osmotic agents
  • the pharmaceutical composition is at least one of tablet, granule, powder, capsule, solution, suspension, freeze-dried preparation.
  • the present invention proposes a method for diagnosing, treating, preventing or alleviating a disease, disorder or condition associated with a tumor or an inflammatory disease in a subject, which comprises administering to the above-mentioned subject A therapeutically effective amount of the aforementioned fusion protein and/or the aforementioned pharmaceutical composition.
  • Figure 1 is a schematic diagram of the binding mode of natural IL-10 molecules and IL-10 monomers and IL-10 receptors according to an embodiment of the present invention, wherein, A represents the schematic diagram of the binding mode of natural IL-10 molecules and IL-10 receptors , R1 represents IL-10 receptor (IL-10R ⁇ ), R2 represents IL-10 receptor (IL-10R ⁇ ); B represents the schematic diagram of the binding mode of IL-10 monomer and IL-10 receptor, R1 represents IL-10 Receptor (IL-10R ⁇ ), IL-10M1 means IL-10 monomer;
  • FIG. 2 is a schematic diagram of the binding of the antibody to the antigen on the target cell and the binding of the IL-10 monomer to the IL-10 receptor in the fusion protein according to an embodiment of the present invention.
  • the affinity of the IL-10 monomer is weak and the targeting effect is relatively weak. Poor, therefore, the antibody end can normally play a targeting role.
  • the local enrichment of the antibody can gather IL-10 monomers together and become a dimer IL-10(M)*2 to play a role.
  • Function where IL-10R means IL10R ⁇ R ⁇ complex, Her2 means human epidermal growth factor receptor, which is an example of antibody targeting antigen, T-cell means T cell, and Tumor means tumor;
  • FIG. 3 is a schematic diagram of the positional structure of IL-10 monomers inserted into antibody molecules according to an embodiment of the present invention, wherein, A represents the position where IL-10 monomers can be inserted on the antibody (position 1-position 7), and B represents the Schematic diagram of the structure of an IL-10 monomer inserted between the VL and CL of each light chain of the antibody (that is, position 1), and connected using linker2, the resulting fusion protein molecule is named R0987; C represents the VH, Schematic diagram of the structure of an IL-10 monomer inserted between CH1 (ie, position 2) and connected using linker2, the resulting fusion protein molecule is named R0988; D is between CH2 and CH3 of the antibody heavy chain (ie, position 3) Schematic diagram of the structure of an IL-10 monomer inserted and linked using linker2, and the resulting fusion protein molecule was named R0989; E Schematic diagram of the structure of an IL-10 monomer inserted at the N-terminal of the antibody light chain (that is
  • Fig. 4 is a graph of SDS-PAGE detection results of fusion proteins R0987, R0988, R0989, R0990, R0991, R0992, and R0993 according to an embodiment of the present invention
  • Fig. 5 is a result diagram of the fusion protein according to an embodiment of the present invention using flow cytometry to detect the binding activity of the antibody end, wherein the abscissa (Ab conc. Log (nM)) represents the antibody concentration (nM), and the ordinate MFI PE represents the average The fluorescence intensity;
  • Fig. 6 is a result diagram of the fusion protein according to an embodiment of the present invention using flow cytometry to detect the binding activity of the IL-10 monomer end, wherein the abscissa (Ab conc. Log (nM)) represents the antibody concentration (nM), and the ordinate MFI PE means mean fluorescence intensity;
  • Fig. 7 is the result figure that the fusion protein according to the embodiment of the present invention uses reporter gene method to detect IL-10 monomer terminal binding activity, wherein, abscissa (Ab conc.Log ( ⁇ g/mL)) represents antibody concentration ( ⁇ g/mL ), the ordinate (Lum) represents the Luminescence fluorescence intensity;
  • Figure 8 is a graph showing the results of statistical analysis of tumor volume in mice after the fusion protein treats MC38 tumor model mice according to an embodiment of the present invention, wherein the abscissa (Days post engraftment) indicates the number of days after the transplantation of the mouse, and the ordinate ( Tumor volume) represents the tumor volume;
  • Figure 9 is a graph showing the results of statistical analysis of the tumor volume in mice after the fusion protein treats CT26 tumor model mice according to an embodiment of the present invention, wherein the abscissa (Days post engraftment) indicates the number of days after the transplantation of the mouse, and the ordinate ( Tumor volume) represents the tumor volume.
  • first and second are used for descriptive purposes only, and cannot be interpreted as indicating or implying relative importance or implicitly specifying the quantity of indicated technical features.
  • the features defined as “first” and “second” may explicitly or implicitly include at least one of these features.
  • “plurality” means at least two, such as two, three, etc., unless otherwise specifically defined.
  • the term "antibody” is an immunoglobulin molecule capable of specifically binding to an antigen. It includes two light chains with lighter molecular weight and two heavy chains with heavier molecular weight. The heavy chain (H chain) and light chain (L chain) are connected by disulfide bonds to form a tetrapeptide chain molecule. Among them, the amino-terminal (N-terminal) amino acid sequence of the peptide chain varies greatly, which is called the variable region (V region), and the carboxy-terminal (C-terminal) is relatively stable, with little change, called the constant region (C region). The V regions of the L chain and H chain are called VL and VH, respectively, the C region of the L chain is CL, and the C region of the H chain includes CH1, CH2 and CH3 regions.
  • the natural IL-10 molecule has a dimer structure (including two natural IL-10 monomers), the binding mode of the natural IL-10 molecule and the IL-10 receptor (IL-10R) is shown in Figure 1-A, and IL-10
  • the activity of -10 monomer on IL-10R ⁇ is 10 times weaker than that of natural IL-10 dimer (specific experimental data reference: J Biol Chem.2000 May 5; 275(18):13552-7.doi:10.1074/jbc .275.18.13552.), and can hardly mediate further binding with IL-10R ⁇ , so it is difficult to activate downstream signals to cause biological functional responses.
  • the binding mode of IL-10 monomer and IL-10 receptor is shown in Figure 1-B shown.
  • the present invention links IL-10 monomers to different parts of the antibody molecule (which may be symmetrical or asymmetrical structures) to construct the fusion protein (IL-10 monomer fusion protein), through which the antibody itself The steric hindrance of the structure isolates the IL-10 monomer.
  • the IL-10 monomer fusion protein is in a free state, even if the IL-10 monomer combines with IL-10R ⁇ , it cannot cause a functional response.
  • the fusion protein obtained in the present invention A wider space is provided for the fused antibody end to function, so that the IL-10 monomer fusion protein can be used in conjunction with more antibody molecules.
  • the present invention provides a fusion protein comprising an antibody and an IL-10 monomer, wherein the antibody comprises two identical light chains and two identical heavy chains, one IL-10 monomer It is connected with the heavy chain or light chain of the antibody, and the N-terminal of the IL-10 monomer is not connected with the C-terminal of the heavy chain of the antibody.
  • the natural IL-10 molecule is in the form of a dimer
  • the fusion protein according to some specific embodiments of the present invention is in the form of a monomer before the antibody binds to the target cell, and cannot bind to its receptor, and will not interfere with the binding of the antibody molecule to the target cell.
  • each antibody light chain includes a VL region and a CL region
  • each antibody heavy chain includes a VH region, a CH1 region, a CH2 region, and a CH3 region
  • the N-terminus of the IL-10 monomer is connected to The C-terminal of the antibody light chain is connected, or the C-terminal of the IL-10 monomer is connected with the N-terminal of the antibody light chain, or the N-terminal of the IL-10 monomer is connected with the C-terminal of the VL region of the hydrogen chain
  • the IL-10 The C-terminal of the -10 monomer is connected to the N-terminal of the CL region of the hydrogen chain, or the C-terminal of the IL-10 monomer is connected to the N-terminal of the antibody heavy chain, or the N-terminal of the IL-10 monomer is connected to the heavy chain
  • the C-terminal of the VH region of the chain is connected, the C-terminal of the IL-10 monomer is connected with the
  • the IL-10 monomer comprises a natural IL-10 monomer, and the natural IL-10 monomer has the amino acid sequence shown in SEQ ID NO:1.
  • the IL-10 monomer includes a modified natural IL-10 monomer, and the modification includes at least one of the following (i) and (ii): (i) removing natural IL - the first 2 amino acids at the N-terminus of the 10 monomer; (ii) inserting a first connecting peptide between the 116th and 117th amino acids, the first connecting peptide being a flexible connecting peptide.
  • the stability of the IL-10 monomer was improved by removing the first 2 amino acids at the N-terminus of the native IL-10 monomer, while inserting a first linkage between amino acids 116 and 117 of the IL-10 monomer
  • the IL-10 monomer molecule in the fusion protein becomes a closed-loop monomer molecule (the two IL10 chains of the natural IL10 dimer are interlaced to form, and after being inserted into the linker, the IL10 monomer can be formed independently domain, that is, a closed-loop monomer molecule), the two IL-10 monomers in the same fusion protein will not interfere with each other such as connection.
  • the first connecting peptide has the amino acid sequence shown in SEQ ID NO:21.
  • the IL-10 monomer has the amino acid sequence shown in SEQ ID NO:2.
  • the IL-10 monomer is connected to the antibody heavy chain or light chain through the second linker peptide or/and the third linker peptide.
  • the IL10 monomer is connected to the heavy chain or light chain of the antibody by using the second linking peptide or/and the third linking peptide, after the antibody part in the fusion protein binds to the corresponding target cell, the 2 close to each other
  • the IL-10 monomer of each fusion protein is more likely to form a dimer to play a role and bind to the IL-10 receptor.
  • the second connecting peptide and/or the third connecting peptide are not particularly limited, either a flexible connecting peptide or a rigid connecting peptide.
  • the second connecting peptide has the amino acid sequence shown in SEQ ID NO:12
  • the third connecting peptide has the amino acid sequence shown in SEQ ID NO:22.
  • the IL-10 monomer is connected to the heavy chain end or light chain end of the antibody through the second linking peptide.
  • the N-terminus of the second connecting peptide is connected to the C-terminus of the light chain of the antibody, and the C-terminus of the second connecting peptide is connected to the N-terminus of the IL-10 monomer; or
  • the N-terminal of the second connecting peptide is connected to the C-terminal of the IL-10 monomer, and the C-terminal of the second connecting peptide is connected to the N-terminal of the light chain of the antibody; or the second connecting peptide
  • the N-terminus is connected to the C-terminus of the IL-10 monomer, and the C-terminus of the second connecting peptide is connected to the N-terminus of the heavy chain of the antibody.
  • the IL-10 monomer is connected to the end of the heavy chain or the end of the light chain of the antibody through the third connecting peptide;
  • the N-terminus of the third connecting peptide is connected to the C-terminus of the light chain of the antibody, and the C-terminus of the third connecting peptide is connected to the N-terminus of the IL-10 monomer; or
  • the N-terminal of the third connecting peptide is connected to the C-terminal of the IL-10 monomer, and the C-terminal of the third connecting peptide is connected to the N-terminal of the antibody light chain; or the third connecting peptide
  • the N-terminal is connected to the C-terminal of the IL-10 monomer, and the C-terminal of the third connecting peptide is connected to the N-terminal of the heavy chain of the antibody.
  • the N-terminal of the third connecting peptide is connected to the C-terminal of the VL region of the light chain, and the C-terminal of the third connecting peptide is connected to the N-terminal of the IL-10 monomer; Or the N-terminal of the third connecting peptide is connected to the C-terminal of the IL-10 monomer, and the C-terminal of the third connecting peptide is connected to the N-terminal of the light chain CL region; or the third connecting The N-terminal of the peptide is connected to the C-terminal of the heavy chain VH region, the C-terminal of the third connecting peptide is connected to the N-terminal of the IL-10 monomer; or the N-terminal of the third connecting peptide is connected to the The C-terminal of the IL-10 monomer is connected, the C-terminal of the third connecting peptide is connected with the N-terminal of the CH1 region of the heavy chain; or the N-terminal of the third connecting
  • the N-terminal of the IL-10 monomer is connected to the C-terminal of the VL region of the light chain of the antibody through the second connecting peptide, and the C-terminal of the IL-10 monomer is connected through
  • the second connecting peptide is connected to the N-terminal of the light chain CL region of the antibody; or the N-terminal of the IL-10 monomer is connected to the C-terminal of the heavy chain VH region of the antibody through the second connecting peptide connected, the C-terminal of the IL-10 monomer is connected to the N-terminal of the heavy chain CH1 region of the antibody through the second connecting peptide; or the N-terminal of the IL-10 monomer is connected through the second
  • the peptide is connected to the C-terminal of the heavy chain CH2 region of the antibody, and the C-terminal of the IL-10 monomer is connected to the N-terminal of the heavy chain CH3 region of the antibody through the second connecting peptide.
  • the antibody is selected from immune cell antibodies and tumor cell antibodies.
  • the antibody is anti-PD-1 antibody, anti-PD-L1 antibody, anti-Her2 antibody, anti-CCR8 antibody, anti-VEGFR2 antibody, anti-claudin18.2 antibody, anti-CD39 antibody, anti-SMA4D Antibody, anti-GUCY2C antibody, anti-CTLA-4 antibody, anti-TIM3 antibody, anti-TIGIT antibody, anti-CD47 antibody, or anti-TNF ⁇ antibody.
  • the fusion protein has the amino acid sequence described in SEQ ID NO:3 and SEQ ID NO:9; or the fusion protein has the amino acid sequence described in SEQ ID NO:3 and SEQ ID NO:5 or the fusion protein has the amino acid sequence described in SEQ ID NO:4 and SEQ ID NO:6; or the fusion protein has the amino acid sequence described in SEQ ID NO:7 and SEQ ID NO:6; Or the fusion protein has the amino acid sequence described in SEQ ID NO:3 and SEQ ID NO:8; or the fusion protein has the amino acid sequence described in SEQ ID NO:10 and SEQ ID NO:6.
  • the present invention provides an isolated nucleic acid encoding the fusion protein of the first aspect.
  • the fusion protein encoded by the nucleic acid is in the form of a monomer before the antibody part binds to the target cell, and cannot bind to its receptor, and will not interfere with the binding of the antibody molecule to the target cell; antibody binding After target cells, a large number of IL-10 monomer molecules aggregate, so that two IL-10 monomers close to each other function as a dimer and bind to IL-10 receptors, thereby causing normal biological responses; and because IL-10 monomer is difficult to trigger biological function, so IL-10 monomer fusion protein will not produce natural dimer IL-10 molecule or its antibody fusion protein dose limit, making the IL-10 monomer fusion protein The dosage was significantly increased.
  • the fusion protein can be used in combination with more antibody molecules, so that the targeting and safety of the fusion protein can be significantly improved, the drug effect can be fully exerted, and various tumors or
  • the nucleic acid molecule is DNA or RNA.
  • the present invention provides an expression vector comprising the nucleic acid of the second aspect.
  • the nucleic acid molecule can be directly or indirectly linked to the control elements on the carrier, as long as these control elements can control the translation and expression of the nucleic acid molecule.
  • these control elements can come directly from the vector itself, or they can be exogenous, that is, not from the vector itself.
  • it is sufficient that the nucleic acid molecule is operably linked to the control element.
  • “Operably linked” herein refers to linking the exogenous gene to the carrier, so that the control elements in the carrier, such as transcription control sequences and translation control sequences, can exert their intended functions of regulating the transcription and translation of the exogenous gene. Function.
  • the expression vector after the expression vector is introduced into a suitable recipient cell, under the mediation of the regulatory system, the expression of the aforementioned fusion protein can be effectively realized, and the obtained fusion protein can be obtained before the antibody part binds to the target cell.
  • IL-10 molecules are in the form of monomers, which cannot bind to their receptors, and will not interfere with the binding of antibody molecules to target cells; after the antibody binds to target cells, a large number of IL-10 monomer molecules aggregate, making the two IL-10 molecules that are close to each other
  • the monomer functions as a dimer and binds to the IL-10 receptor, thereby causing normal biological responses; and because IL-10 monomers are difficult to trigger biological functions, IL-10 monomer fusion proteins will not produce
  • the dosage limitation of natural dimer IL-10 molecule or its antibody fusion protein makes the dosage of IL-10 monomer fusion protein significantly increased.
  • the fusion protein can be used in combination with more antibody molecules, so that the targeting and safety of the fusion protein can be significantly improved, the drug effect can be fully exerted, and various tumors or inflammatory diseases can be treated or prevented.
  • the expression vector is a eukaryotic expression vector.
  • the present invention provides a recombinant cell carrying the nucleic acid of the second aspect, the expression vector of the third aspect, or expressing the fusion protein of the first aspect.
  • Recombinant cells according to specific embodiments of the present invention can be used for the expression and large-scale acquisition of the aforementioned fusion protein.
  • the IL-10 molecule of the obtained fusion protein is in the form of a monomer and cannot bind to its receptor. It will interfere with the combination of antibody molecules and target cells; after the antibody binds to target cells, a large number of IL-10 monomer molecules aggregate, so that two IL-10 monomers that are close to each other act as a dimer and interact with IL-10 receptors.
  • IL-10 monomer fusion proteins will not produce natural dimeric IL-10 molecules or administration of their antibody fusion proteins Dose limitation makes the dosage of IL-10 monomer fusion protein significantly increased.
  • the fusion protein can be used in combination with more antibody molecules, so that the targeting and safety of the fusion protein can be significantly improved, the drug effect can be fully exerted, and various tumors or inflammatory diseases can be treated or prevented.
  • the recombinant cells are mammalian cells, such as human, monkey, rabbit, dog, bovine, etc.; mammalian cells such as human HEK-293F cells or CHO-K1 cells.
  • the recombinant cells do not include animal germ cells, fertilized eggs or embryonic stem cells.
  • the present invention proposes the use of the aforementioned fusion protein, nucleic acid, expression vector or recombinant cell in the preparation of medicines for treating or preventing tumors or inflammatory diseases.
  • the drug can effectively treat or alleviate tumors and inflammatory diseases, and has high safety.
  • the present invention provides a pharmaceutical composition comprising the aforementioned fusion protein, nucleic acid, expression vector or recombinant cell.
  • the pharmaceutical composition can accurately bind to the target cells that the antibody contained in the pharmaceutical composition can bind to, and before the antibody binds to the target cell, the IL-10 monomer molecules have low binding activity to IL-10 receptors, therefore, the IL-10 monomers will not interfere with the targeting of the antibodies contained in the pharmaceutical composition, and in the drug composition After the antibody contained in the composition is combined with the target cells, it can fully exert its medicinal effect, effectively treat or alleviate tumors and inflammatory diseases, and has high safety.
  • the pharmaceutical composition is used for treating or preventing tumors or inflammatory diseases.
  • the fusion protein provided by the present invention can be incorporated into a pharmaceutical composition suitable for administration to a subject.
  • these pharmaceutical compositions include fusion proteins provided by the invention.
  • the pharmaceutical composition further includes a pharmaceutically acceptable carrier, including any solvent, solid excipient, diluent, binder, disintegrant, or other liquid excipient, Dispersing agents, flavoring agents or suspending agents, surfactants, isotonic agents, thickeners, emulsifiers, preservatives, solid binders, glidants or lubricants, etc., are suitable for the specific target dosage form.
  • a pharmaceutically acceptable carrier including any solvent, solid excipient, diluent, binder, disintegrant, or other liquid excipient, Dispersing agents, flavoring agents or suspending agents, surfactants, isotonic agents, thickeners, emulsifiers, preservatives, solid binders, glidants or lubricants, etc.
  • fusion proteins of the invention may be incorporated into pharmaceutical compositions suitable for parenteral administration (eg, intravenous, subcutaneous, intraperitoneal, intramuscular).
  • parenteral administration eg, intravenous, subcutaneous, intraperitoneal, intramuscular
  • These pharmaceutical compositions can be prepared in various forms.
  • liquid, semi-solid and solid dosage forms and the like including but not limited to liquid solutions (eg, injection solutions and infusion solutions), dispersions or suspensions, tablets, pills, powders, liposomes and suppositories.
  • the fusion protein can be administered by intravenous infusion or intramuscular or subcutaneous injection.
  • treat and prevention and words derived therefrom as used herein do not necessarily imply 100% or complete treatment or prevention. Rather, there are varying degrees of treatment or prophylaxis that would be considered by one of ordinary skill in the art to be of potential benefit or therapeutic effect.
  • the treatment or prevention provided herein may include the treatment or prevention of one or more conditions or symptoms of the disease being treated or prevented, such as cancer. Additionally, for purposes herein, "prevention” may encompass delaying the onset of a disease or a symptom or condition thereof.
  • the present invention provides a method for diagnosing, treating, preventing or alleviating a disease, disorder or condition associated with a tumor or an inflammatory disease in a subject, which comprises administering to the above-mentioned subject A therapeutically effective amount of the aforementioned fusion protein and/or the aforementioned pharmaceutical composition is administered.
  • IL-10 monomers are inserted into different positions of antibody molecules (anti-PD-1 antibodies are used as an example in this example) to obtain fusion proteins.
  • the specific experimental operations for the construction of IL-10 monomer molecules are mainly Refer to "Molecular Cloning Laboratory Guide”.
  • the natural IL-10 monomer molecular sequence is shown in SEQ ID NO:1, and the peptide of the amino acid sequence shown in SEQ ID NO:21 is inserted between the 116N and 117K positions of the natural IL-10 monomer molecular sequence Segment (that is, the first connecting peptide, linker1), while removing the first 2 amino acids of the N-terminal of the natural IL-10 monomer, thereby forming the IL-10 monomer of the embodiment of the present invention, its sequence is as SEQ ID NO:2 shown; the heavy chain of the antibody is shown in SEQ ID NO:3, and the light chain of the antibody is shown in SEQ ID NO:6.
  • R0987 Insert the IL-10 monomer between the VL and CL of the antibody light chain (position 1), and use linker2 (GSGSGSGS) to link, the resulting fusion protein has such as SEQ ID NO: 3 (heavy chain) and SEQ ID The amino acid sequence shown in NO:5 (light chain), as shown in Figure 3B;
  • R0988 Insert the IL-10 monomer between the VH and CH1 of the antibody heavy chain (position 2), and use linker2 (GSGSGSGS) to link, the resulting fusion protein has such as SEQ ID NO: 4 (heavy chain) and SEQ ID The amino acid sequence shown in NO:6 (light chain), as shown in Figure 3C;
  • R0989 The IL-10 monomer is inserted between CH2 and CH3 of the antibody heavy chain (position 3), and linked using linker2 (GSGSGSGS), the resulting fusion protein has such as SEQ ID NO: 7 (heavy chain) and SEQ ID The amino acid sequence shown in NO:6 (light chain), as shown in Figure 3D;
  • R0990 Connect the IL-10 monomer to the N-terminal (position 4) of the light chain of the antibody through a linker, and use linker3 (GGGGSGGGGSGGGGSGGGGS) to link, the resulting fusion protein has such as SEQ ID NO: 3 (heavy chain) and SEQ ID NO : the amino acid sequence shown in 8 (light chain), as shown in Figure 3E;
  • R0991 Connect the IL-10 monomer to the C-terminal of the antibody light chain (position 5) through a linker, and use linker3 (GGGGSGGGGSGGGGSGGGGS) to link, the resulting fusion protein has such as SEQ ID NO: 3 (heavy chain) and SEQ ID NO : the amino acid sequence shown in 9 (light chain), as shown in Figure 3F;
  • R0992 Link the IL-10 monomer to the N-terminal (position 6) of the heavy chain of the antibody through a linker, and use linker3 (GGGGSGGGGSGGGGSGGGGS) to link, the resulting fusion protein has such as SEQ ID NO: 10 (heavy chain) and SEQ ID NO : the amino acid sequence shown in 6 (light chain), as shown in Figure 3G;
  • R0993 Link the IL-10 monomer to the C-terminal (position 7) of the heavy chain of the antibody through a linker and use linker3 (GGGGSGGGGSGGGGSGGGGS) to link, the resulting fusion protein has such as SEQ ID NO: 11 (heavy chain) and SEQ ID NO: The amino acid sequence shown in 6 (light chain), as shown in Figure 3H;
  • the second connecting peptide (linker2) and the third connecting peptide (linker3) are flexible or rigid connecting peptides, wherein the second connecting peptide has the amino acid sequence shown in SEQ ID NO: 12, and the third connecting peptide The peptide has the amino acid sequence shown in SEQ ID NO: 22;
  • the amino acid sequences of the heavy chain and light chain of the above fusion protein are shown in Table 1 below, wherein the sequence GSGSGSGS (SEQ ID NO: 12) marked in bold italics is the amino acid sequence of the second linker peptide (linker2), only with the addition of
  • the roughly marked sequence GGGGSGGGGSGGGGSGGGGSG (SEQ ID NO: 22) is the amino acid sequence of the third connecting peptide (linker3), and only the underlined sequence GQGTQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTL RLRLRRCHRFLPCENGGGSGGKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRN (SEQ ID NO: 2) is the amino acid sequence of the modified IL-10 monomer .
  • Embodiment 2 Preparation of fusion protein
  • a plasmid containing the gene of interest (encoding the fusion protein in Example 1) was constructed and prepared by conventional methods.
  • the plasmid containing the target gene is introduced into the host cell Expi293 after forming a cationic complex with the transfection reagent PEI, and the foreign gene on the plasmid is transcribed and translated in the cell while the plasmid is in the cell, thereby obtaining the fusion protein,
  • the specific experimental operation is as follows:
  • Expi293 was cultured at 37°C, 8% carbon dioxide, and 130rpm, and the cells were counted before transfection.
  • the 2E6 cells were inoculated into 1L shake flasks, and the culture system was about 300mL.
  • the transient cell expression solution was centrifuged at 9000rpm/20min, the supernatant was collected, and then sterilized and filtered through a 0.22 ⁇ m filter membrane. Purification using ProA affinity chromatography. The process is as follows: Use AKTA york 150 chromatographic equipment, equilibrate the chromatographic column (such as MabSelectSuRe LX, GE) with at least 5CV equilibration buffer (10mM PBS), load the sample to the chromatographic column, and make the target protein adsorb on the chromatographic column and Other impurities are separated by breakthrough.
  • chromatographic column such as MabSelectSuRe LX, GE
  • the expression data of IL-10 monomer fusion protein is shown in Table 2. Except for the expression of R0987 which was 97.731 mg/mL, the transient expression of other fusion proteins were all greater than 100 mg/L, and the expression of R0990 even reached 194.409 mg/mL. L. After the one-step affinity purification, the samples were tested by SEC-HPLC, and the target purity was above 85%, and the purity of R0989 was even greater than 95%. The sample was detected by SDS-PAGE, which showed the correct distribution of light and heavy chains, and the electrophoresis results are shown in Figure 4. It was concluded that IL-10 monomer fusion proteins with different insertion positions all exhibited good production properties.
  • Example 3 The in vitro activity of the fusion protein
  • FACS Flow cytometry fluorescence sorting technique
  • All fusion protein molecules R0428 (a positive control antibody without IL-10, whose heavy chain amino acid sequence and light chain amino acid sequence are shown in SEQ ID NO: 13, 14) or R0594 ( IL-10-Fc fusion protein control, its amino acid sequence is shown in SEQ ID NO: 15, wherein IL-10 has removed the first 2 amino acids of natural IL-10, and the first connecting peptide has not been inserted)
  • the protein molecule is diluted to the initial concentration 400nM, volume 180 ⁇ l, 3-fold serial dilution (60 ⁇ l sample + 120 ⁇ l dilution buffer), a total of 11 concentration gradient points were obtained.
  • R0594 contains two identical peptide chains, the amino acid sequence of one chain:
  • the detection results of the binding activity of the antibody end showed that the activity of the antibody end of the fusion proteins R0987, R0988, R0989, and R0991 was relatively strong, and that of R0990 was the weakest. It shows that the IL-10 monomer is inserted between VL and CL of the light chain, or between VH and CH1 of the heavy chain, and between CH2 and CH3 of the heavy chain, and the C-terminus of the light chain will not affect the binding activity of the antibody.
  • the detection results of the binding activity of IL-10 monomer end to IL-10R ⁇ are shown in Figure 6.
  • the binding activity of IL-10 monomer end of fusion proteins R0989 and R0993 to IL-10R ⁇ is obviously weak, which may be related to IL-10R ⁇ .
  • the insertion positions of the 10 monomers are all close to the C-terminus of the antibody, and the binding activity of the other fusion proteins to IL-10R ⁇ is relatively close, which is consistent with the positive control molecule R0594. It shows that the IL-10 monomer inserted into the antibody domain can still form the expected spatial structure after recombinant expression, and normally bind IL-10R ⁇ .
  • the reporter gene method was used to detect the activity of the IL-10 monomer end of the IL-10 monomer fusion protein.
  • Cell preparation Take out the cultured HEK293-hIL-10-Report gene cell (Cat#GM-C07927, Genomeditech) and observe it under a microscope. Cells with normal cell adhesion, transparent granules, and moderate density can be used as effector cells for experiments ;Digest the above cells with TE, centrifuge at 300g for 4min after the digestion is terminated, remove the supernatant, resuspend with 1% FBS-PBS and wash again; discard the supernatant, and finally use the medium to resuspend the cells, and count the cells Adjust the cell density to 5 ⁇ 10 5 cells/mL.
  • the test results are shown in Figure 7.
  • the results of the reporter gene are similar to the Binding results.
  • the binding activity of the IL-10 monomer end of the fusion proteins R0989 and R0993 is low, while the activity of the positive control R0579 is much stronger than that of the fusion protein tested this time.
  • the test results of the reporter gene method showed that the binding activity of the modified IL-10 monomer was lower than that of the dimer IL-10 molecule.
  • mice The inventor carried out experiments in mice on the basis of the detection in Example 3, and the specific experimental operations were as follows:
  • MC38 tumor cells (source: National Experimental Cell Resource Sharing Platform, resource number: 3111C0001CCC000523) were cultured in the addition of 10% FBS (inactivated fetal bovine serum, supplier: Gibco, product number: 10091148), 100 U/mL penicillin and 100 ⁇ g /mL streptomycin in DMEM (supplier: Gibco, product number: 11965084) medium.
  • FBS inactivated fetal bovine serum
  • supplier Gibco, product number: 10091148
  • penicillin 100 ⁇ g /mL streptomycin
  • DMEM supply: Gibco, product number: 11965084
  • the temperature of the incubator was controlled at 37°C, supplemented with 5% carbon dioxide.
  • Tumor cells were passaged twice a week and digested with trypsin-EDTA.
  • Inoculation collect MC38 tumor cells in logarithmic growth phase, centrifuge at 250g for 10 minutes after digestion. After washing twice with ice-cold PBS, the cells were counted, and the PBS was adjusted to a cell concentration of 3 ⁇ 10 6 cells/mL. The right abdomen of the mice was shaved in advance to prepare the skin, and each mouse was inoculated with 0.1 mL of cell suspension, which was 3 ⁇ 10 5 tumor cells per mouse.
  • mice On days 6-8 after inoculation, tumors of all animals were measured and weighed. To avoid the impact of initial tumor volume on treatment effectiveness, mice were randomly assigned into groups based on tumor volume, ensuring that all groups had comparable mean tumor volumes, making treatment effects comparable at baseline.
  • fusion proteins R0987, R0988, R0989, R0991, R0992, R0993 and R0862 (i.e. Isotype, negative control antibody; its heavy chain amino acid sequence and light chain amino acid sequence are shown in SEQ ID NO: 16, 17), R0428, R0676 (IL-10-Fc fusion protein control, its amino acid sequence is shown in SEQ ID NO: 18, wherein the first 2 amino acids of natural IL-10 are removed from the IL-10, and the first connecting peptide is not inserted) ), R0579 protein molecules were injected intraperitoneally into mice at doses of 0.61, 0.61, 0.61, 0.61, 0.61, 0.61, 0.5, 0.5, 0.3, and 0.6 mg/kg, respectively, Q3D ⁇ 3 (once every 3 days, a total of Administer 3 times), measure tumor volume 3 times a week, examine and record animal tumor growth and the effect of treatment on normal
  • R0676 contains two identical peptide chains, the amino acid sequence of one chain:
  • Endpoints The primary endpoint of the experiment is whether the tumor growth is delayed or whether the mice are cured.
  • TGI Tumor Growth Inhibition
  • Calculation method independent sample T test can be used for comparison between two groups. More than 3 groups were compared using One-WayANOVA. If the F value shows a significant difference, post hoc analysis among multiple groups can be performed. The data were processed using GraphPad Prism, when p ⁇ 0.05 indicated a significant statistical difference.
  • Tumor volume V 0.5a ⁇ b2, where a and b are the long and short diameters of the tumor, respectively.
  • TGI (%) [1-(Ti-T0)/(Vi-V0)] ⁇ 100
  • Ti is the average tumor volume of the treatment group on day i
  • T0 is the average tumor volume of the treatment group at the beginning of treatment
  • Vi is the average tumor volume of the vehicle control group on day i
  • V0 is the average tumor volume of the vehicle control group S at the beginning of treatment.
  • TGI was used to express the effect of drugs on tumor growth inhibition.
  • the inventor further verified the fusion protein on the basis of the experimental results obtained in Example 4, and the specific experimental operations were as follows:
  • CT26 tumor cells (supplier: Cell Bank of Chinese Academy of Sciences, article number: NA) were cultured in 10% FBS (inactivated fetal bovine serum, supplier: Gibco, article number: 10091148), 100 U/mL penicillin and 100 ⁇ g/mL Streptomycin RPMI-1640 (supplier: Gibco, product number: 11875085) medium.
  • FBS inactivated fetal bovine serum
  • penicillin 100 U/mL
  • Streptomycin RPMI-1640 (supplier: Gibco, product number: 11875085) medium.
  • the temperature of the incubator was controlled at 37°C, supplemented with 5% carbon dioxide.
  • Tumor cells were passaged twice a week and digested with trypsin-EDTA.
  • Inoculation collect the cells in the logarithmic growth phase, centrifuge at 250g for 10 minutes after digestion. After washing twice with ice-cold PBS, the cells were counted, and the cell concentration in PBS was adjusted to 1 ⁇ 10 6 /mL. The right abdomen of the mice was shaved in advance to prepare the skin, and each mouse was inoculated with 0.1 mL of cell suspension, which was 1 ⁇ 10 5 tumor cells per mouse.
  • mice On days 6-8 after inoculation, tumors of all animals were measured and weighed. To avoid the impact of initial tumor volume on treatment effectiveness, mice were randomly assigned into groups based on tumor volume, ensuring that all groups had comparable mean tumor volumes, making treatment effects comparable at baseline.
  • the protein molecules were injected intraperitoneally into mice at doses of 10, 10, 10, 10, 5, and 7.9 mg/kg, respectively, Q3D ⁇ 3 (administrated once every 3 days, total administration 3 times), the tumor volume was measured 2 times a week, and the tumor growth of the animals was examined and recorded, as well as the effects of treatment on normal behavior, such as mobility, food and water consumption, weight gain/loss, eyes/hair/skin, etc.
  • Endpoints The primary endpoint of the experiment is whether the tumor growth is delayed or whether the mice are cured.
  • TGI Tumor Growth Inhibition
  • Calculation method independent sample T test was used for comparison between two groups. More than 3 groups were compared using One-WayANOVA. If the F value shows a significant difference, post hoc analysis among multiple groups can be performed. The data were processed using GraphPad Prism, when p ⁇ 0.05 indicated a significant statistical difference.
  • Tumor volume V 0.5a ⁇ b2, where a and b are the long and short diameters of the tumor, respectively.
  • TGI (%) [1-(Ti-T0)/(Vi-V0)] ⁇ 100
  • Ti is the average tumor volume of the treatment group on day i
  • T0 is the average tumor volume of the treatment group at the beginning of treatment
  • Vi is the average tumor volume of the vehicle control group on day i
  • V0 is the average tumor volume of the vehicle control group S at the beginning of treatment.
  • TGI was used to express the effect of drugs on tumor growth inhibition. According to the specific experiment, the survival curve is also used as one of the standards to measure the efficacy of the drug.

Abstract

提供了一种IL10单体融合蛋白及其应用，涉及生物医药领域，所述融合蛋白包括抗体和IL-10单体。本发明所述的融合蛋白具有较强的靶向性，安全性得到显著提高，且可有效治疗或预防多种肿瘤或炎性疾病。

Description

IL10单体融合蛋白及其应用
优先权声明
本申请要求申请号为202111286063.4，申请日为2021年11月2日，发明名称为IL10单体融合蛋白及其应用的中国发明专利申请的优先权，其全部内容通过引用并入本文中。
技术领域
本发明涉及生物医药领域，具体地，涉及融合蛋白及其应用，更具体地，涉及一种融合蛋白、核酸、表达载体、重组细胞、所述融合蛋白或核酸或表达载体或重组细胞在制备药物中的用途以及药物组合物。
背景技术
白介素10(Interleukin 10，IL-10，或IL10)也被称为人类细胞因子合成抑制因子(CSIF)，是一种抗炎细胞因子，属于同源二聚体分泌物，目前临床上的IL-10蛋白存在安全性问题，少量的天然二聚化的IL-10分子与其受体IL-10Rα高亲和力结合后，进一步与IL-10Rβ结合，形成六聚体复合物并激活下游信号，引起生物学功能反应。
目前的临床IL-10药物分子缺乏靶向性，药物分子容易脱靶，并产生副作用；而大部分在开发的IL-10抗体融合蛋白，其设计思路是利用抗体靶向作用将IL-10引到局部环境中发挥作用。这些融合蛋白分子往往都具有正常的IL-10二聚体结构，但是由于IL-10二聚体与IL-10Rα高亲和力结合，可能会影响抗体端的靶向作用，从而导致设计思路失败，并且如果抗体端的活性需求剂量较大，则IL-10端的副作用仍然是一个棘手的问题。
因此，仍需积极探索靶向性强、副作用小的IL-10药物，该研究方向对预防或治疗肿瘤或炎性疾病具有重要意义。
发明内容
本申请是基于发明人对以下问题的发现和认识作出的：
目前临床IL-10药物存在缺乏靶向性，药物分子容易脱靶，不仅药效不易发挥且容易产生副作用，发明人通过对IL-10单体分子和抗体分子的连接方式进行探索，制备了一种包含IL-10单体分子和抗体分子的融合蛋白，所述融合蛋白中的IL-10分子在抗体结合靶细胞前IL-10呈单体形式，无法与其受体结合，不会干扰抗体分子与靶细胞上抗原的结合，抗体结合抗原后，大量IL-10单体分子聚集，使得相互靠近的2个IL-10单体作为二聚体发挥作用，与IL-10受体进行结合，从而引起正常的生物学反应，使得IL-10药物的靶向性和安全性得到显著提高，药效得到充分发挥。
在本发明的第一方面，本发明提出了一种融合蛋白。根据本发明的实施例，包括抗体和IL-10单体，其中，所述抗体包括两条相同的轻链和两条相同的重链，一条IL-10单体与抗体重链或轻链连接，所述IL-10单体的N末端不与抗体重链的C末端连接。天然IL-10分子为二聚体形式，根据本发明实施例的融合蛋白在抗体结合靶细胞前IL-10分子呈单体形式，无法与其受体结合，不会干扰抗体分子与靶细胞的结合。抗体结合靶细胞后，大量IL-10单体分子聚集，使得相互靠近的2个IL-10单体作为二聚体发挥作用，与IL-10受体进行结合，从而引起正常的生物学反应，且由于IL-10单体很难引发生物功能，所以IL-10单体融合蛋白不会产生天然二聚体IL-10分子或其抗体融合蛋白的给药剂量限制，使得IL-10单体融合蛋白的给药剂量得到显著提高。所述融合蛋白能够与更多的抗体分子搭配使用，使得融合蛋白的靶向性和安全性得到显著提高，药效得到充分发挥，且可治疗或预防多种肿瘤或炎性疾病。
根据本发明的实施例，上述融合蛋白还可以进一步包括如下附加技术特征至少之一：
根据本发明的实施例，每条抗体轻链包括VL区、CL区，每条抗体重链包括VH区、CH1区、CH2区、CH3区；所述IL-10单体的N末端与抗体轻链C末端相连，或所述IL-10单体的C末端与抗体轻链N末端相连，或所述IL-10单体的N末端与氢链VL区的C末端相连，所述IL-10单体的C末端与氢链CL区的N末端相连，或所述IL-10单体的C末端与抗体重链的N末端相连，或所述IL-10单体的N末端与重链VH区的C末端相连，所述IL-10单体的C末端与重链CH1区的N末端相连，或所述IL-10单体的N末端与重链CH2区的C末端相连，所述IL-10单体的C末端与重链CH3区的N末端相连。
根据本发明的实施例，所述IL-10单体包括天然IL-10单体。
根据本发明的实施例，所述IL-10单体为修饰后的天然IL-10单体，所述修饰包括下列(i)和(ii)的至少之一：(i)去除天然IL-10单体的N末端的前2个氨基酸；(ii)在第116位和117位氨基酸之间插入第一连接肽，所述第一连接肽为柔性连接肽。
将天然IL-10单体的N末端的前2个氨基酸去除后，IL-10单体的稳定性得到提升，同时在IL-10单体的第116位和117位氨基酸之间插入第一连接肽后，所述融合蛋白中的IL-10单体分子封闭，成为闭环IL-10单体分子(天然IL10二聚体的两条IL10链是交错在一起形成的，经过插入linker改造后，IL10单体可以独立形成结构域，即闭环单体分子)，同一融合蛋白中的两个IL-10单体不会出现相互连接等干扰。
根据本发明的实施例，所述第一连接肽的氨基酸序列如SEQ ID NO:21所示。
Figure PCTCN2022128998-appb-000001
根据本发明的实施例，所述IL-10单体具有SEQ ID NO:2所示的氨基酸序列。
Figure PCTCN2022128998-appb-000002
根据本发明的实施例，所述IL-10单体与抗体重链或轻链通过第二连接肽或/和第三连接肽连接。利 用第二连接肽或/和第三连接肽将所述IL10单体与所述抗体重链或轻链相连后，使得所述融合蛋白中的抗体部分与相应靶细胞结合后，相互靠近的2个融合蛋白的IL-10单体更易形成二聚体发挥作用，与IL-10受体进行结合。
根据本发明的实施例，所述第二连接肽或/和第三连接肽可以是柔性连接肽或刚性连接肽。
根据本发明的实施例，所述第二连接肽具有SEQ ID NO:12所示的氨基酸序列，所述第三连接肽具有SEQ ID NO:22所示的氨基酸序列。
Figure PCTCN2022128998-appb-000003
根据本发明的实施例，所述IL-10单体与所述抗体的重链末端或轻链末端通过所述第二连接肽连接。
根据本发明的实施例，所述第二连接肽的N末端与所述抗体轻链的C末端相连，所述第二连接肽的C末端与所述IL-10单体的N末端相连；或所述第二连接肽的N末端与所述IL-10单体的C末端相连，所述第二连接肽的C末端与所述抗体轻链的N末端相连；或所述第二连接肽的N末端与所述IL-10单体的C末端相连，所述第二连接肽的C末端与所述抗体重链的N末端相连。
根据本发明的实施例，所述IL-10单体与所述抗体的重链末端或轻链末端通过所述第三连接肽连接；
根据本发明的实施例，所述第三连接肽的N末端与所述抗体轻链的C末端相连，所述第三连接肽的C末端与所述IL-10单体的N末端相连；或所述第三连接肽的N末端与所述IL-10单体的C末端相连，所述第三连接肽的C末端与所述抗体轻链的N末端相连；或所述第三连接肽的N末端与所述IL-10单体的C末端相连，所述第三连接肽的C末端与所述抗体重链的N末端相连。
根据本发明的实施例，所述第三连接肽的N末端与所述轻链VL区的C末端相连，所述第三连接肽的C末端与所述IL-10单体的N末端相连；或所述第三连接肽的N末端与所述IL-10单体的C末端相连，所述第三连接肽的C末端与所述轻链CL区的N末端相连；或所述第三连接肽的N末端与所述重链VH区的C末端相连，所述第三连接肽的C末端与所述IL-10单体的N末端相连；或所述第三连接肽的N末端与所述IL-10单体的C末端相连，所述第三连接肽的C末端与所述重链CH1区的N末端相连；或所述第三连接肽的N末端与所述重链CH2区的C末端相连，所述第三连接肽的C末端与所述IL-10单体的N末端相连；或所述第三连接肽的N末端与所述IL-10单体的C末端相连，所述第三连接肽的C末端与所述重链CH3区的N末端相连。
根据本发明的实施例，所述IL-10单体的N末端通过所述第二连接肽与所述抗体的轻链的VL区的C末端相连，所述IL-10单体的C末端通过所述第二连接肽与所述抗体的轻链CL区的N末端连接；或所述IL-10单体的N末端通过所述第二连接肽与所述抗体的重链VH区的C末端相连，所述IL-10单体的C末端通过所述第二连接肽与所述抗体的重链CH1区的N末端相连；或所述IL-10单体的N末端通过所述第二连接肽与所述抗体的重链CH2区的C末端相连，所述IL-10单体的C末端通过所述第二连接肽与所述抗体的重链CH3区的N末端相连。
根据本发明的实施例，所述抗体选自免疫细胞类抗体和肿瘤细胞类抗体。
根据本发明的实施例，所述抗体为抗PD-1抗体、抗PD-L1抗体、抗-Her2抗体、抗CCR8抗体、抗VEGFR2抗体、抗claudin18.2抗体、抗CD39抗体、抗SMA4D抗体、抗GUCY2C抗体、抗CTLA-4抗体、抗TIM3抗体、抗TIGIT抗体、抗CD47抗体或抗TNFα抗体。
根据本发明的实施例，所述融合蛋白具有SEQ ID NO:3和SEQ ID NO:9所述的氨基酸序列；或所述融合蛋白具有SEQ ID NO:3和SEQ ID NO:5所述的氨基酸序列；或所述融合蛋白具有SEQ ID NO:4和SEQ ID NO:6所述的氨基酸序列；或所述融合蛋白具有SEQ ID NO:7和SEQ ID NO:6所述的氨基酸序列；或所述融合蛋白具有SEQ ID NO:3和SEQ ID NO:8所述的氨基酸序列；或所述融合蛋白具有SEQ ID NO:10和SEQ ID NO:6所述的氨基酸序列。
Figure PCTCN2022128998-appb-000004
Figure PCTCN2022128998-appb-000005
Figure PCTCN2022128998-appb-000006
在本发明的第二方面，本发明提出了一种核酸。根据本发明的实施例，所述核酸编码第一方面所述的融合蛋白。根据本发明实施例的核酸编码的融合蛋白中的IL-10分子在抗体部分结合靶细胞前呈单体形式，无法与其受体结合，不会干扰抗体分子与靶细胞的结合；抗体结合靶细胞后，大量IL-10单体分子聚集，使得相互靠近的2个IL-10单体作为二聚体发挥作用，与IL-10受体进行结合，从而引起正常的生物学反应；且由于IL-10单体很难引发生物功能，所以IL-10单体融合蛋白不会产生天然二聚体IL-10分子或其抗体融合蛋白的给药剂量限制，使得IL-10单体融合蛋白的给药剂量得到显著提高。所述融合蛋白能够与更多的抗体分子搭配使用，使得融合蛋白的靶向性和安全性得到显著提高，药效得到充分发挥，且可治疗或预防多种肿瘤或炎性疾病。
根据本发明的实施例，所述核酸分子为DNA或RNA。
在本发明的第三方面，本发明提出了一种表达载体。根据本发明的实施例，所述表达载体包含第二方面所述的核酸。根据本发明实施例的表达载体导入合适的受体细胞后，可在调控系统的介导下，有效实现前面所述的融合蛋白的表达，获得的融合蛋白中的IL-10分子在抗体部分结合靶细胞前呈单体形式，无法与其受体结合，不会干扰抗体分子与靶细胞的结合；抗体结合靶细胞后，大量IL-10单体分子聚集，使得相互靠近的2个IL-10单体作为二聚体发挥作用，与IL-10受体进行结合，从而引起正常的生物学反应；且由于IL-10单体很难引发生物功能，所以IL-10单体融合蛋白不会产生天然二聚体IL-10分子或其抗体融合蛋白的给药剂量限制，使得IL-10单体融合蛋白的给药剂量得到显著提高。所述融合蛋白能够与更多的抗体分子搭配使用，使得融合蛋白的靶向性和安全性得到显著提高，药效得到充分发挥，且可治疗或预防多种肿瘤或炎性疾病。
在本发明的第四方面，本发明提出了一种重组细胞。根据本发明的实施例，所述重组细胞携带第二方面所述的核酸、第三方面所述的表达载体或表达第一方面所述的融合蛋白。根据本发明实施例的重组细胞可用于前面所述的融合蛋白的表达和大量获得，获得的融合蛋白在抗体部分结合靶细胞前IL-10分子呈单体形式，无法与其受体结合，不会干扰抗体分子与靶细胞的结合；抗体结合靶细胞后，大量IL-10单体分子聚集，使得相互靠近的2个IL-10单体作为二聚体发挥作用，与IL-10受体进行结合，从而引起正常的生物学反应；且由于IL-10单体很难引发生物功能，所以IL-10单体融合蛋白不会产生天然二聚体IL-10分子或其抗体融合蛋白的给药剂量限制，使得IL-10单体融合蛋白的给药剂量得到显著提高。 所述融合蛋白能够与更多的抗体分子搭配使用，使得融合蛋白的靶向性和安全性得到显著提高，药效得到充分发挥，且可治疗或预防多种肿瘤或炎性疾病。
在本发明的第五方面，本发明提出了第一方面所述的融合蛋白、第二方面所述的核酸、第三方面所述的表达载体或第四方面所述的重组细胞在制备药物中的用途。根据本发明的实施例，所述药物用于治疗或预防肿瘤或炎性疾病。根据本发明的具体实施例，所述药物可有效治疗或缓解肿瘤以及炎性疾病，且安全性较高。
在本发明的第六方面，本发明提出了一种药物组合物。根据本发明的实施例，包含第一方面所述的融合蛋白、第二方面所述的核酸、第三方面所述的表达载体或第四方面所述的重组细胞。根据本发明的具体实施例的药物可有效治疗或缓解肿瘤以及炎性疾病，且具有较高的安全性。
根据本发明的实施例，上述药物组合物还可以进一步包括如下附加技术特征(i)和(ii)中的至少之一：
(i)根据本发明的实施例，所述药物组合物用于治疗或预防肿瘤或炎性疾病。
(ii)根据本发明的实施例，所述药物组合物可包括：药学上可接受的辅剂，所述药学上可接受的辅剂包括稳定剂、湿润剂、乳化剂、粘合剂、等渗剂的至少之一；所述药物组合物呈片剂、颗粒剂、散剂、胶囊剂、溶液剂、悬浮剂、冻干制剂的至少一种。
在本发明的第七方面，本发明提出了一种在受试者中诊断、治疗、预防或减轻与肿瘤或炎性疾病相关的疾病、病症或状况的方法，其包括向上述受试者施用治疗有效量的前述融合蛋白和/或前述药物组合物。
本发明的附加方面和优点将在下面的描述中部分给出，部分将从下面的描述中变得明显，或通过本发明的实践了解到。
附图说明
本发明的上述和/或附加的方面和优点结合下面附图对实施例的描述将变得明显和容易理解，其中：
图1是根据本发明实施例的天然IL-10分子及IL-10单体与IL-10受体结合方式的示意图，其中，A表示天然IL-10分子与IL-10受体结合方式的示意图，R1表示IL-10受体(IL-10Rα)，R2表示IL-10受体(IL-10Rβ)；B表示IL-10单体与IL-10受体结合方式的示意图，R1表示IL-10受体(IL-10Rα)，IL-10M1表示IL-10单体；
图2是根据本发明实施例的融合蛋白中所述抗体与靶细胞上的抗原结合、IL-10单体与IL-10受体结合的示意图，IL-10单体亲和力较弱，靶向作用差，因此，抗体端可以正常发挥靶向作用，靶向肿瘤表面后，抗体的局部富集，可以将IL-10单体聚集在一起，变成二聚体IL-10(M)*2发挥作用，其中，IL-10R表示IL10RαRβ复合物，Her2表示人类表皮生长因子受体，是抗体靶向抗原的举例，T-cell表示T细胞，Tumor表示肿瘤；
图3是根据本发明实施例的IL-10单体插入抗体分子中的位置结构示意图，其中，A表示IL-10单体在抗体上可插入的位置(位置1-位置7)，B表示在抗体每条轻链的VL、CL之间(即位置1)插入一条IL-10单体的结构示意图，并使用linker2连接，得到的融合蛋白分子命名为R0987；C表在抗体重链的VH、CH1之间(即位置2)插入一条IL-10单体的结构示意图，并使用linker2连接，得到的融合蛋白分子命名为R0988；D在到抗体重链的CH2、CH3之间(即位置3)插入一条IL-10单体的结构示意图，并使用linker2连接，得到的融合蛋白分子命名为R0989；E在抗体轻链的N端(即位置4)插入一条IL-10单体的结构示意图，并使用linker2连接，得到的融合蛋白分子命名为R0990；F在抗体轻链的C端(即位置5)插入一条IL-10单体的结构示意图，并使用linker3连接，得到的融合蛋白分子命名为R0991；G表示在抗体重链的N端(即位置6)插入一条IL-10单体的结构示意图，并使用linker3连接，得到的融合蛋白分子命名为R0992；H表示在抗体重链的C端(即位置7)插入一条IL-10单体的结构示意图，并使用linker3连接，得到的融合蛋白分子命名为R0993；I是本发明实施例3-5中的对照分子(R0428、R0594、R0862、R0676、R0579、R1049)的结构图；
图4是根据本发明实施例的融合蛋白R0987、R0988、R0989、R0990、R0991、R0992、R0993的SDS-PAGE检测结果图；
图5是根据本发明实施例的融合蛋白采用流式法检测抗体端结合活性的结果图，其中，横坐标(Ab conc.Log(nM))表示抗体浓度(nM)，纵坐标MFI PE表示平均荧光强度；
图6是根据本发明实施例的融合蛋白采用流式法检测IL-10单体端结合活性的结果图，其中，横坐标(Ab conc.Log(nM))表示抗体浓度(nM)，纵坐标MFI PE表示平均荧光强度；
图7是根据本发明实施例的融合蛋白采用报告基因法检测IL-10单体端结合活性的结果图，其中，横坐标(Ab conc.Log(μg/mL))表示抗体浓度(μg/mL)，纵坐标(Lum)表示Luminescence荧光强度；
图8是根据本发明实施例的融合蛋白治疗MC38肿瘤模型小鼠后的小鼠体内肿瘤体积的统计分析结果图，其中，横坐标(Days post engraftment)表示小鼠移植后的天数，纵坐标(Tumor volume)表示肿瘤体积；
图9是根据本发明实施例的融合蛋白治疗CT26肿瘤模型小鼠后的小鼠体内肿瘤体积的统计分析结果图，其中，横坐标(Days post engraftment)表示小鼠移植后的天数，纵坐标(Tumor volume)表示肿瘤体积。
具体实施方式
下面详细描述本发明的实施例，所述实施例的示例在附图中示出。下面通过参考附图描述的实施例是示例性的，旨在用于解释本发明，而不能理解为对本发明的限制。
此外，术语“第一”、“第二”仅用于描述目的，而不能理解为指示或暗示相对重要性或者隐含指明所指示的技术特征的数量。由此，限定有“第一”、“第二”的特征可以明示或者隐含地包括至少一个该特征。在本发明的描述中，“多个”的含义是至少两个，例如两个，三个等，除非另有明确具体的限定。
本文中，术语“抗体”是能够与抗原特异性结合的免疫球蛋白分子。包括两条分子量较轻的轻链和两条分子量较重的重链，重链(H链)和轻链(L链)由二硫键连接形成一个四肽链分子。其中，肽链的氨基端(N端)氨基酸序列变化很大，称为可变区(V区)，羧基端(C端)相对稳定，变化很小，称为恒定区(C区)。L链和H链的V区分别称为VL和VH，L链的C区为CL，H链的C区包含CH1区、CH2区和CH3区。
天然IL-10分子为二聚体结构(包括两条天然IL-10单体)，天然IL-10分子与IL-10受体(IL-10R)结合方式如图1-A所示，而IL-10单体对IL-10Rα的活性比天然IL-10二聚体弱10倍(具体实验数据参考文献：J Biol Chem.2000 May 5；275(18):13552-7.doi:10.1074/jbc.275.18.13552.)，且几乎无法介导与IL-10Rβ的进一步结合，因此很难激活下游信号引起生物学功能反应，IL-10单体与IL-10受体结合方式如图1-B所示。
因此，本发明将IL-10单体连接在所述抗体分子的不同部位(可以是对称的或非对称的结构)构建所述融合蛋白(IL-10单体融合蛋白)，通过所述抗体自身结构的空间位阻使IL-10单体被隔离开，当IL-10单体融合蛋白处于游离状态时，即使IL-10单体与IL-10Rα结合也无法引起功能反应。只有当抗体端结合到靶细胞后，才能通过聚集效应，将邻近IL-10单体-IL-10Rα复合物聚拢，并进一步与IL-10Rβ结合形成复合物产生下游信号并引发生物功能，具体结合方式如图2所示，这样可以避免药物分子在没有到达靶部位前IL-10分子对所述抗体的靶向性产生干扰，不会产生临床副作用；且由于IL-10单体很难引发生物功能，所以IL-10单体融合蛋白不会产生天然二聚体IL-10分子或其抗体融合蛋白的给药剂量限制，使得IL-10单体融合蛋白的给药剂量得到显著提高。同时，由于不同抗体的临床使用剂量具有差异，当IL-10分子的使用剂量较低，会严重限制IL-10单体融合蛋白的中抗体靶标的选择，因此，本发明获得的所述融合蛋白为融合的抗体端发挥功能提供了更广泛的空间，从而使所述IL-10单体融合蛋白能够与更多的抗体分子搭配使用。
在一些实施方案中，本发明提出了一种融合蛋白，包括抗体和IL-10单体，其中，所述抗体包括两条相同的轻链和两条相同的重链，一条IL-10单体与抗体重链或轻链连接，所述IL-10单体的N末端不与抗体重链的C末端连接。天然IL-10分子为二聚体形式，根据本发明的一些具体实施例的融合蛋白在抗体结合靶细胞前IL-10分子呈单体形式，无法与其受体结合，不会干扰抗体分子与靶细胞的结合，抗体结合靶细胞后，大量IL-10单体分子聚集，使得相互靠近的2个IL-10单体作为二聚体发挥作用，与IL-10受体进行结合，从而引起正常的生物学反应，使得融合蛋白的靶向性和安全性得到显著提高，药效得到充分发挥。
根据本发明的一些具体实施方案，每条抗体轻链包括VL区、CL区，每条抗体重链包括VH区、 CH1区、CH2区、CH3区；所述IL-10单体的N末端与抗体轻链C末端相连，或所述IL-10单体的C末端与抗体轻链N末端相连，或所述IL-10单体的N末端与氢链VL区的C末端相连，所述IL-10单体的C末端与氢链CL区的N末端相连，或所述IL-10单体的C末端与抗体重链的N末端相连，或所述IL-10单体的N末端与重链VH区的C末端相连，所述IL-10单体的C末端与重链CH1区的N末端相连，或所述IL-10单体的N末端与重链CH2区的C末端相连，所述IL-10单体的C末端与重链CH3区的N末端相连。
根据本发明的一些具体实施方案，所述IL-10单体包括天然IL-10单体，所述天然IL-10单体具有SEQ ID NO:1所示的氨基酸序列。
Figure PCTCN2022128998-appb-000007
根据本发明的一些具体实施方案，所述IL-10单体包括修饰后的天然IL-10单体，所述修饰包括下列(i)和(ii)的至少之一：(i)去除天然IL-10单体的N末端的前2个氨基酸；(ii)在第116位和117位氨基酸之间插入第一连接肽，所述第一连接肽为柔性连接肽。
将天然IL-10单体的N末端的前2个氨基酸去除后，IL-10单体的稳定性得到提升，同时在IL-10单体的第116位和117位氨基酸之间插入第一连接肽后，所述融合蛋白中的IL-10单体分子成为闭环单体分子(天然IL10二聚体的两条IL10链是交错在一起形成的，经过插入linker改造后，IL10单体可以独立形成结构域，即闭环单体分子)，同一融合蛋白中的两个IL-10单体不会出现相互连接等干扰。
根据本发明的一些具体实施方案，所述第一连接肽具有SEQ ID NO:21所示的氨基酸序列。
GGGSGG(SEQ ID NO:21)
根据本发明的一些具体实施方案，所述IL-10单体具有SEQ ID NO:2所示的氨基酸序列。
Figure PCTCN2022128998-appb-000008
根据本发明的实施例，所述IL-10单体与抗体重链或轻链通过第二连接肽或/和第三连接肽连接。利用第二连接肽或/和第三连接肽将所述IL10单体与所述抗体重链或轻链相连后，使得所述融合蛋白中的抗体部分与相应靶细胞结合后，相互靠近的2个融合蛋白的IL-10单体更易形成二聚体发挥作用，与IL-10受体进行结合。
根据本发明的实施例，所述第二连接肽或/和第三连接肽不受特别限制，柔性连接肽或刚性连接肽均可。
根据本发明的实施例，所述第二连接肽具有SEQ ID NO:12所示的氨基酸序列，所述第三连接肽具有SEQ ID NO:22所示的氨基酸序列。
Figure PCTCN2022128998-appb-000009
根据本发明的实施例，所述IL-10单体与所述抗体的重链末端或轻链末端通过所述第二连接肽连接。
根据本发明的实施例，所述第二连接肽的N末端与所述抗体轻链的C末端相连，所述第二连接肽的C末端与所述IL-10单体的N末端相连；或所述第二连接肽的N末端与所述IL-10单体的C末端相连，所述第二连接肽的C末端与所述抗体轻链的N末端相连；或所述第二连接肽的N末端与所述IL-10单体的C末端相连，所述第二连接肽的C末端与所述抗体重链的N末端相连。
根据本发明的实施例，所述IL-10单体与所述抗体的重链末端或轻链末端通过所述第三连接肽连接；
根据本发明的实施例，所述第三连接肽的N末端与所述抗体轻链的C末端相连，所述第三连接肽的C末端与所述IL-10单体的N末端相连；或所述第三连接肽的N末端与所述IL-10单体的C末端相连，所述第三连接肽的C末端与所述抗体轻链的N末端相连；或所述第三连接肽的N末端与所述IL-10单体的C末端相连，所述第三连接肽的C末端与所述抗体重链的N末端相连。
根据本发明的实施例，所述第三连接肽的N末端与所述轻链VL区的C末端相连，所述第三连接肽的C末端与所述IL-10单体的N末端相连；或所述第三连接肽的N末端与所述IL-10单体的C末端相连，所述第三连接肽的C末端与所述轻链CL区的N末端相连；或所述第三连接肽的N末端与所述重链VH区的C末端相连，所述第三连接肽的C末端与所述IL-10单体的N末端相连；或所述第三连接肽的N末端与所述IL-10单体的C末端相连，所述第三连接肽的C末端与所述重链CH1区的N末端相连；或所述第三连接肽的N末端与所述重链CH2区的C末端相连，所述第三连接肽的C末端与所述IL-10单体的N末端相连；或所述第三连接肽的N末端与所述IL-10单体的C末端相连，所述第三连接肽的C末端与所述重链CH3区的N末端相连。
根据本发明的实施例，所述IL-10单体的N末端通过所述第二连接肽与所述抗体的轻链的VL区的C末端相连，所述IL-10单体的C末端通过所述第二连接肽与所述抗体的轻链CL区的N末端连接；或所述IL-10单体的N末端通过所述第二连接肽与所述抗体的重链VH区的C末端相连，所述IL-10单体的C末端通过所述第二连接肽与所述抗体的重链CH1区的N末端相连；或所述IL-10单体的N末端通过所述第二连接肽与所述抗体的重链CH2区的C末端相连，所述IL-10单体的C末端通过所述第二连接肽与所述抗体的重链CH3区的N末端相连。
根据本发明的一些具体实施方案，所述抗体选自免疫细胞类抗体和肿瘤细胞类抗体。
根据本发明的一些具体实施方案，所述抗体为抗PD-1抗体、抗PD-L1抗体、抗-Her2抗体、抗CCR8抗体、抗VEGFR2抗体、抗claudin18.2抗体、抗CD39抗体、抗SMA4D抗体、抗GUCY2C抗体、抗CTLA-4抗体、抗TIM3抗体、抗TIGIT抗体、抗CD47抗体或抗TNFα抗体。
根据本发明的一些具体实施方案，所述融合蛋白具有SEQ ID NO:3和SEQ ID NO:9所述的氨基酸序列；或所述融合蛋白具有SEQ ID NO:3和SEQ ID NO:5所述的氨基酸序列；或所述融合蛋白具有SEQ  ID NO:4和SEQ ID NO:6所述的氨基酸序列；或所述融合蛋白具有SEQ ID NO:7和SEQ ID NO:6所述的氨基酸序列；或所述融合蛋白具有SEQ ID NO:3和SEQ ID NO:8所述的氨基酸序列；或所述融合蛋白具有SEQ ID NO:10和SEQ ID NO:6所述的氨基酸序列。
Figure PCTCN2022128998-appb-000010
Figure PCTCN2022128998-appb-000011
Figure PCTCN2022128998-appb-000012
在一些实施方案中，本发明提出了一种分离的核酸，所述核酸编码第一方面所述的融合蛋白。根据本发明的一些具体实施方案中的核酸编码的融合蛋白在抗体部分结合靶细胞前IL-10分子呈单体形式，无法与其受体结合，不会干扰抗体分子与靶细胞的结合；抗体结合靶细胞后，大量IL-10单体分子聚集，使得相互靠近的2个IL-10单体作为二聚体发挥作用，与IL-10受体进行结合，从而引起正常的生物学反应；且由于IL-10单体很难引发生物功能，所以IL-10单体融合蛋白不会产生天然二聚体IL-10分子或其抗体融合蛋白的给药剂量限制，使得IL-10单体融合蛋白的给药剂量得到显著提高。所述融合蛋白能够与更多的抗体分子搭配使用，使得融合蛋白的靶向性和安全性得到显著提高，药效得到充分发挥，且可治疗或预防多种肿瘤或炎性疾病。
根据本发明的一些具体实施方案，所述核酸分子为DNA或RNA。
在一些实施方案中，本发明提出了一种表达载体，所述表达载体包含第二方面所述的核酸。在将上述核酸分子连接到载体上时，可以将核酸分子与载体上的控制元件直接或者间接相连，只要这些控制元件能够控制核酸分子的翻译和表达等即可。当然这些控制元件可以直接来自于载体本身，也可以是外源性的，即并非来自于载体本身。当然，核酸分子与控制元件进行可操作地连接即可。本文中“可操作地连接”是指将外源基因连接到载体上，使得载体内的控制元件，例如转录控制序列和翻译控制序列等，能够发挥其预期的调节外源基因的转录和翻译的功能。根据本发明一些具体实施方案中的表达载体导入合适的受体细胞后，可在调控系统的介导下，有效实现前面所述的融合蛋白的表达，获得的融合蛋白在抗体部分结合靶细胞前IL-10分子呈单体形式，无法与其受体结合，不会干扰抗体分子与靶细胞的结合；抗体结合靶细胞后，大量IL-10单体分子聚集，使得相互靠近的2个IL-10单体作为二聚体发挥作用，与IL-10受体进行结合，从而引起正常的生物学反应；且由于IL-10单体很难引发生物功能，所以IL-10单体融合蛋白不会产生天然二聚体IL-10分子或其抗体融合蛋白的给药剂量限制，使得IL-10单体融合蛋白的给药剂量得到显著提高。所述融合蛋白能够与更多的抗体分子搭配使用，使得融合蛋白的靶向性和安全性得到显著提高，药效得到充分发挥，且可治疗或预防多种肿瘤或炎性疾病。
根据本发明的一些具体的实施方案，所述表达载体为真核表达载体。
在一些实施方案中，本发明提出了一种重组细胞，所述重组细胞携带第二方面所述的核酸、第三方面所述的表达载体或表达第一方面所述的融合蛋白。根据本发明具体实施例的重组细胞可用于前面所述的融合蛋白的表达和大量获得，获得的融合蛋白在抗体部分结合靶细胞前IL-10分子呈单体形式，无法与其受体结合，不会干扰抗体分子与靶细胞的结合；抗体结合靶细胞后，大量IL-10单体分子聚集，使得相互靠近的2个IL-10单体作为二聚体发挥作用，与IL-10受体进行结合，从而引起正常的生物学反应；且由于IL-10单体很难引发生物功能，所以IL-10单体融合蛋白不会产生天然二聚体IL-10分子或其抗体融合蛋白的给药剂量限制，使得IL-10单体融合蛋白的给药剂量得到显著提高。所述融合蛋白能够与更多的抗体分子搭配使用，使得融合蛋白的靶向性和安全性得到显著提高，药效得到充分发挥，且 可治疗或预防多种肿瘤或炎性疾病。
根据本发明的一些具体的实施方案，所述重组细胞为哺乳动物细胞，哺乳动物例如：人、猴、兔、犬、牛等；哺乳动物细胞如：人HEK-293F细胞或CHO-K1细胞。
根据本发明的一些具体实施方案，所述重组细胞不包括动物生殖细胞、受精卵或胚胎干细胞。
在一些实施方案中，本发明提出了前面所述的融合蛋白、核酸、表达载体或重组细胞在制备药物中的用途，所述药物用于治疗或预防肿瘤或炎性疾病。根据本发明的一些具体实施方案，所述药物可有效治疗或缓解肿瘤以及炎性疾病，且安全性较高。
在一些实施方案中，本发明提出了一种药物组合物，包含前面所述的融合蛋白、核酸、表达载体或重组细胞。根据本发明的一些具体实施方案的药物组合物可准确与所述药物组合物中包含的所述抗体可以结合的靶细胞进行结合，在所述抗体和靶细胞结合前所述药物组合物中的IL-10单体分子与IL-10受体结合活性较低，因此，所述IL-10单体不会干扰所述药物组合物中包含的所述抗体的靶向性，并在所述药物组合物包含的所述抗体与靶细胞结合后充分的发挥药效，有效治疗或缓解肿瘤以及炎性疾病，且具有较高的安全性。
根据本发明的一些具体实施方案，所述药物组合物用于治疗或预防肿瘤或炎性疾病。
根据本发明的一些具体实施方案，本发明提供的融合蛋白可以掺入适合受试者施用的药物组合物中。通常，这些药物组合物包括本发明提供的融合蛋白。
根据本发明的一些具体实施方案，所述药物组合物进一步包括药学上可接受的载体，包括任何溶剂、固体赋形剂、稀释剂、粘合剂、崩解剂、或其他液体赋形剂、分散剂、矫味剂或悬浮剂、表面活性剂、等渗剂、增稠剂、乳化剂、防腐剂、固体粘合剂、助流剂或润滑剂，等等，适合于特有的目标剂型。除了任何常规的辅料与本发明的融合蛋白不相容的范围，例如所产生的任何不良的生物效应或与药学上可接受的组合物的任何其他组分以有害的方式产生的相互作用，它们的用途也是本发明所考虑的范围。
例如，本发明的融合蛋白可掺入适用于胃肠外施用(例如静脉内、皮下、腹膜内、肌肉内)的药物组合物中。这些药物组合物可以被制备成各种形式。例如液体、半固体和固体剂型等，包括但不限于液体溶液(例如，注射溶液和输注溶液)、分散剂或悬浮剂、片剂、丸剂、粉末、脂质体和栓剂。所述融合蛋白可通过静脉输注射或肌肉内或皮下注射来施用。
本文使用的术语“治疗”和“预防”以及源自于此的词不必暗示100％或完全治疗或预防。相反，存在不同程度的治疗或预防，本领域普通技术人员认为所述治疗或预防具有潜在的益处或治疗效果。而且，本发明提供的治疗或预防可包括正在治疗或预防的疾病，如癌症的一种或多种病患或症状的治疗或预防。另外，为了本文的目的，“预防”可涵盖延缓疾病或其症状或病患的发作。
根据本发明的一些具体实施方案，本发明提供了一种在受试者中诊断、治疗、预防或减轻与肿瘤或炎性疾病相关的疾病、病症或状况的方法，其包括向上述受试者施用治疗有效量的前述融合蛋白和/或前述药物组合物。
下面参考具体实施例，对本发明进行描述，需要说明的是，这些实施例仅仅是描述性的，而不以任何方式限制本发明。
实施例中未注明具体技术或条件的，按照本领域内的文献所描述的技术或条件或者按照产品说明书进行。所用试剂或仪器未注明生产厂商者，均为可以通过市购获得的常规产品。
实施例1 融合蛋白的构建
本实施例将IL-10单体插入到抗体分子(本实施例以抗PD-1抗体为例)的不同位置中以获得融合蛋白，其中，IL-10单体分子的构建的具体实验操作主要参考《分子克隆实验指南》。天然IL-10单体分子序列如SEQ ID NO:1所示，在天然IL-10单体分子序列的116N、117K两个位点之间插入如SEQ ID NO:21所示的氨基酸序列的肽段(即，第一连接肽，linker1)，同时去除天然IL-10单体的N末端的前2个氨基酸，从而形成本发明实施例的IL-10单体，其序列如SEQ ID NO:2所示；所述抗体重链如SEQ ID NO:3所示，抗体轻链如SEQ ID NO:6所示。
Figure PCTCN2022128998-appb-000013
本实施例共构建7种融合蛋白，融合蛋白的具体结构如图3所示(图中展示了IL-10单体的插入位置)；所述融合蛋白中IL-10单体与抗体分子的连接方式描述如下：
R0987：将IL-10单体插入到抗体轻链的VL、CL之间(位置1)，并使用linker2(GSGSGSGS)进行连接，所得融合蛋白具有如SEQ ID NO:3(重链)和SEQ ID NO:5(轻链)所示的氨基酸序列，如图3B所示；
R0988：将IL-10单体插入到抗体重链的VH、CH1之间(位置2)，并使用linker2(GSGSGSGS)进行连接，所得融合蛋白具有如SEQ ID NO:4(重链)和SEQ ID NO:6(轻链)所示的氨基酸序列,如图3C所示；
R0989：将IL-10单体插入到抗体重链的CH2、CH3之间(位置3)，并使用linker2(GSGSGSGS)进行连接，所得融合蛋白具有如SEQ ID NO:7(重链)和SEQ ID NO:6(轻链)所示的氨基酸序列，如图3D所示；
R0990：将IL-10单体通过linker连接到抗体轻链的N端(位置4)，并使用linker3(GGGGSGGGGSGGGGSGGGGS)进行连接，所得融合蛋白具有如SEQ ID NO:3(重链)和SEQ ID NO:8(轻链)所示的氨基酸序列，如图3E所示；
R0991：将IL-10单体通过linker连接到抗体轻链的C端(位置5)，并使用linker3(GGGGSGGGGSGGGGSGGGGS)进行连接，所得融合蛋白具有如SEQ ID NO:3(重链)和SEQ ID NO:9(轻链)所示的氨基酸序列，如图3F所示；
R0992：将IL-10单体通过linker连接到抗体重链的N端(位置6)，并使用linker3(GGGGSGGGGSGGGGSGGGGS)进行连接，所得融合蛋白具有如SEQ ID NO:10(重链)和SEQ ID NO:6(轻链)所示的氨基酸序列，如图3G所示；
R0993：将IL-10单体通过linker连接到抗体重链的C端(位置7)并使用linker3(GGGGSGGGGSGGGGSGGGGS)进行连接，所得融合蛋白具有如SEQ ID NO:11(重链)和SEQ ID NO:6(轻链)所示的氨基酸序列，如图3H所示；
本发明实施例3-5中的对照分子(R0428、R0594、R0862、R0676、R0579、R1049)的结构图如图3I所示。
所述第二连接肽(linker2)和第三连接肽(linker3)为柔性或刚性连接肽，其中，所述第二连接肽具有如SEQ ID NO:12所示的氨基酸序列，所述第三连接肽具有如SEQ ID NO:22所示的氨基酸序列；
上述融合蛋白的重链和轻链的氨基酸序列如下表1所示,其中，用加粗斜体标注的序列GSGSGSGS(SEQ ID NO:12)是第二连接肽(linker2)的氨基酸序列，仅用加粗标注的序列GGGGSGGGGSGGGGSGGGGSG(SEQ ID NO:22)是第三连接肽(linker3)的氨基酸序列，仅用下划线标注的序列GQGTQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENGGGSGGKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRN(SEQ ID NO:2)是改造后的IL-10单体的氨基酸序列。
表1
Figure PCTCN2022128998-appb-000014
Figure PCTCN2022128998-appb-000015
Figure PCTCN2022128998-appb-000016
Figure PCTCN2022128998-appb-000017
Figure PCTCN2022128998-appb-000018
Figure PCTCN2022128998-appb-000019
Figure PCTCN2022128998-appb-000020
Figure PCTCN2022128998-appb-000021
实施例2 融合蛋白的制备
通过常规方法构建、制备含目的基因(编码实施例1中的融合蛋白)的质粒。将含有目的基因的质粒通过与转染试剂PEI形成阳离子复合物后，导入到宿主细胞Expi293，质粒在细胞内期间，质粒上的外源基因在细胞内发生转录翻译，从而得到所述融合蛋白，具体实验操作如下：
Expi293在37℃、8％二氧化碳、130rpm条件下培养，并在转染前通过细胞计数，将2E6的细胞接种至1L摇瓶中，培养体系约为300mL。配制转染复合物准备转染：首先将750μg目标质粒加入到含有15mLOpti-MEM试剂的50mL离心管中，轻轻混匀，标记为A管；将1.5mg转染试剂PEI加入到含有15mLOpti-MEM试剂的50mL离心管中，轻轻混匀后，室温孵育5min，标记为B管；将B管PEI稀释液逐滴加入到A管DNA稀释液中，轻轻混匀后，室温孵育15min，孵育结束后，将PEI-目标质粒复合物加入到Expi293细胞，置于37℃摇床中继续培养，培养至D5-D10后收样。
瞬转细胞表达液经过9000rpm/20min离心，收集上清，再经过0.22μm滤膜除菌过滤。纯化采用ProA亲和层析。过程如下：使用AKTA avant 150层析设备，用至少5CV平衡缓冲液(10mM PBS)平衡层析柱(如MabSelectSuRe LX，GE)，加载样品至层析柱，使目标蛋白吸附在层析柱上而其他杂质穿透分离。完成上样后使用至少5CV平衡缓冲液(10mM PBS)再次冲洗层析柱，随后使用洗脱缓冲液(20mM NaAc，pH＝3.4)洗脱目标蛋白，收集管中预先加入中和缓冲液(1M Tris，pH＝8.0)，中和缓冲液的加入体积根据洗脱样品的预估含量而定，一般加入10％洗脱体积量。
IL-10单体融合蛋白表达数据如表2所示，除R0987表达量为97.731mg/mL外，其余融合蛋白的瞬转表达量均都大于100mg/L，R0990的表达量甚至达到194.409mg/L。一步亲和纯化后样品经SEC-HPLC检测，目标纯度都在85％以上，R0989的纯度甚至大于95％。样品经SDS-PAGE检测，显示正确的轻重链分布，电泳结果如图4所示。得出结论，不同插入位置的IL-10单体融合蛋白均展现出良好的生产性质。
表2
Figure PCTCN2022128998-appb-000022
实施例3 融合蛋白的体外活性
3.1流式细胞荧光分选技术(FACS)检测融合蛋白抗体端、IL-10单体端的结合活性
用含3％BSA的PBS buffer将所有融合蛋白分子、R0428(不含IL-10的阳性对照抗体，其重链氨基酸序列、轻链氨基酸序列如SEQ ID NO:13、14所示)或R0594(IL-10-Fc融合蛋白对照，其氨基酸序列如SEQ ID NO:15所示，其中IL-10去除了天然IL-10的前2个氨基酸，未插入第一连接肽)蛋白分子稀释成初始浓度400nM，体积180μl，3倍梯度稀释(60μl样品+120μl稀释Buffer)，共得到11个浓度梯度点。将CHO-mPD1或者CHO-hIL-10R细胞于250g条件下离心5min后弃去上清，用3％BSA的PBS buffer调整细胞密度为2E+06，按100μL/管均分到96孔V型板中；将上述稀释好的融合蛋白分子加入到细胞中，100μL/孔，2-8℃孵育0.5h；取出96孔板，250g离心5min，小心去上清后，加入3％BSA的PBS buffer200μl/孔，再次250g离心5min，小心去上清；用含3％BSA的PBS buffer配制PE荧光二抗(1:500稀释)，按100μl/孔的体积添加至对应的96孔板中，重悬细胞，2-8℃孵育30min；取出96孔板，250g离心5min，小心去上清后，加入含3％BSA的PBS buffer200μl/孔，再次250g离心5min，小心去上清；用1xPBS按照100μL/孔的体积重悬，重悬后进行FACS检测。
R0428的重链序列：
Figure PCTCN2022128998-appb-000023
R0428的轻链序列：
Figure PCTCN2022128998-appb-000024
R0594含两条相同的肽链，其中一条链的氨基酸序列：
Figure PCTCN2022128998-appb-000025
抗体端结合活性检测结果如图5所示，融合蛋白R0987、R0988、R0989、R0991抗体端的活性比较强，R0990最弱。说明IL-10单体插入在轻链VL、CL之间，或者重链VH、CH1之间，重链CH2、CH3之间，轻链的C端并不会影响抗体的结合活性。
IL-10单体端对IL-10Rα的结合活性检测结果如图6所示，其中，融合蛋白R0989、R0993的IL-10单体端对IL-10Rα的结合活性明显较弱，可能与IL-10单体插入位置都靠近抗体的C端部位有关，其余融合蛋白对IL-10Rα的结合活性比较接近，与阳性对照分子R0594一致。说明插入到抗体结构域中的IL-10单体，经过重组表达后仍然可以形成预期的空间结构，并且正常结合IL-10Rα。
3.2报告基因法检测IL-10单体融合蛋白的IL-10单体端的活性。
用1640+10％FBS将所有融合蛋白分子及对照蛋白分子R0579(IL-10-阳性抗体融合蛋白对照，其重链氨基酸序列如SEQ ID NO:23所示，其轻链氨基酸序列如SEQ ID NO:14所示，其中所述IL-10去除了天然IL-10的前2个氨基酸，未插入第一连接肽)稀释成初始浓度20μg/mL，体积450μl，3倍梯度稀释(150μl+300μl)，共获得9个浓度。细胞准备：取出正在培养的HEK293-hIL-10-Report gene cell(Cat#GM-C07927，Genomeditech)在显微镜下观察，细胞贴壁正常、颗粒透亮，且密度适中的细胞可以作为实验所用的效应细胞；将上述细胞用TE消化，终止消化后在300g条件下离心4min后去除上清，用1％FBS-PBS重悬后再清洗一遍；弃上清，最终使用培养基重悬细胞，细胞计数后调整细胞密度为5×10 5个/mL。按照上面的实验设计先将稀释好的药物以50μL/孔的体积添加到对应的孔板中，再将上述重悬后的细胞也以50μL/孔的体积加入，在Medium only的孔里补加100μL的培养基，Cell only的孔中补加50μL的培养基，最终所有孔的终体积为100μL。将96孔板继续在培养箱中培养16h。提前解冻Bright-LumiTM萤火虫荧光素酶检测试剂，平衡至室温。细胞培养16h后取出细胞培养板平衡至室温10min(不宜超过30min)。每孔加入100μLBright-LumiTM萤火虫荧光素酶检测试剂，室温孵育5-10min。使用多功能酶标仪中的化学发光模式检测信号。
检测结果如图7所示，报告基因的结果与Binding结果类似，融合蛋白R0989、R0993的IL-10单体端的结合活性较低，而阳性对照R0579的活性远强于本次检测的融合蛋白。报告基因法的检测结果表明改造后的IL-10单体结合活性是低于二聚体形式的IL-10分子的。
实施例4 融合蛋白对MC38肿瘤模型小鼠的影响
发明人在实施例3的检测基础上进行小鼠体内实验，具体实验操作如下：
细胞培养：MC38肿瘤细胞(来源：国家实验细胞资源共享平台，资源编号：3111C0001CCC000523)培养在添加了10％FBS(inactivated fetal bovine serum，供应商：Gibco，货号:10091148)、100U/mL盘尼西林和100μg/mL链霉素的DMEM(供应商：Gibco，货号:11965084)培养基中。培养箱温度控制在37℃，补充5％二氧化碳。肿瘤细胞一周传代两次，以胰酶-EDTA消化。
接种：收集对数增长期的MC38肿瘤细胞，消化后250g条件下离心10分钟。以冰冷的PBS洗涤 两次后进行细胞计数，调节PBS至细胞浓度为3×10 6个/mL。小鼠右侧腹部提前剃毛备皮，每只鼠接种0.1mL细胞悬液，接种量即每只3×10 5个肿瘤细胞。
分组：接种后第6-8天，测量所有动物的肿瘤并称重。为避免初始肿瘤体积对治疗有效性的影响，将小鼠根据肿瘤体积随机分配成组，确保所有组的平均瘤体积相当，使治疗效果在基线上具有可比性。
观察：接种后，每天检查动物的发病率和死亡率。治疗开始后，将融合蛋白R0987、R0988、R0989、R0991、R0992、R0993以及R0862(即Isotype，阴性对照抗体；其重链氨基酸序列、轻链氨基酸序列如SEQ ID NO:16、17所示)、R0428、R0676(IL-10-Fc融合蛋白对照，其氨基酸序列如SEQ ID NO:18所示，其中所述IL-10去除了天然IL-10的前2个氨基酸，未插入第一连接肽))、R0579蛋白分子分别以0.61、0.61、0.61、0.61、0.61、0.61、0.5、0.5、0.3、0.6mg/kg的剂量对小鼠进行腹腔注射，Q3D×3(每3天给药一次，一共给药3次)，每周3次测量肿瘤体积，检查并记录动物肿瘤生长以及治疗对正常行为的影响，如行动能力、食物和水消耗、体重增加/损失、眼睛/毛发/皮肤等的影响。
R0862的重链序列：
Figure PCTCN2022128998-appb-000026
R0862的轻链序列：
Figure PCTCN2022128998-appb-000027
R0676含两条相同的肽链，其中一条链的氨基酸序列：
Figure PCTCN2022128998-appb-000028
Figure PCTCN2022128998-appb-000029
R0579的重链序列：
Figure PCTCN2022128998-appb-000030
R0579的轻链序列：
Figure PCTCN2022128998-appb-000031
实验终点(Endpoints)：实验的主要终点是肿瘤生长是否被延缓或小鼠是否被治愈。根据每组肿瘤体积计算TGI(Tumor Growth Inhibition)。计算方法：两组之间比较可用独立样本T检验。3组以上比较应用One-WayANOVA。如果F值显示有显著性差异，可进行多组间的事后分析。数据使用GraphPad Prism处理，当p<0.05表示具有显著性统计学差异。肿瘤体积V＝0.5a×b2，a和b分别为肿瘤的长、短径。肿瘤生长抑制TGI(％)＝[1-(Ti-T0)/(Vi-V0)]×100，Ti为第i天治疗组的平均瘤体积，T0为治疗开始时治疗组的平均瘤体积，Vi为第i天溶剂对照组的平均瘤体积，V0为治疗开始时溶剂对照组S的平均瘤体积。以TGI来表示药物对肿瘤生长抑制的效果。
实验结果如图8所示，经融合蛋白R0987、R0991、R0993治疗后的小鼠体内肿瘤生长速度降低，肿瘤体积较小；相比于单抗或IL-10融合蛋白，融合蛋白R0987、R0991、R0993具有更好的抑瘤效果。
实施例5 融合蛋白对CT26肿瘤模型小鼠的影响
发明人在实施例4所得实验结果的基础上对所述融合蛋白进行进一步验证，具体实验操作如下：
细胞培养：CT26肿瘤细胞(供应商：中国科学院细胞库，货号：NA)培养在添加了10％FBS(inactivated fetal bovine serum，供应商：Gibco，货号:10091148)、100U/mL盘尼西林和100μg/mL链霉素的 RPMI-1640(供应商：Gibco，货号:11875085)培养基中。培养箱温度控制在37℃，补充5％二氧化碳。肿瘤细胞一周传代两次，以胰酶-EDTA消化。
接种：收集对数增长期的细胞，消化后250g离心10分钟。以冰冷的PBS洗涤两次后计数，调节PBS中细胞浓度为1×10 6/mL。小鼠右侧腹部提前剃毛备皮，每只鼠接种0.1mL细胞悬液，接种量即每只1×10 5个肿瘤细胞。
分组：接种后第6-8天，测量所有动物的肿瘤并称重。为避免初始肿瘤体积对治疗有效性的影响，将小鼠根据肿瘤体积随机分配成组，确保所有组的平均瘤体积相当，使治疗效果在基线上具有可比性。
观察：本研究方案中所涉及的动物使用和护理，在进行之前由广东菲鹏制药股份有限公司动物伦理委员会审查和批准。在研究期间，根据SPF动物房标准操作规程对动物进行护理和使用。接种后，每天检查动物的发病率和死亡率。治疗开始后，将融合蛋白R0987、R0991、R0993以及R0862、R0579、R0676、R1049(抗PD-1的单抗对照，其重链氨基酸序列如SEQ ID NO:19所示，轻链氨基酸序列如SEQ ID NO:20所示)蛋白分子分别以10、10、10、10、10、5、7.9mg/kg的剂量对小鼠进行腹腔注射，Q3D×3(每3天给药一次，一共给药3次)，每周2次测量肿瘤体积，检查并记录动物肿瘤生长以及治疗对正常行为的影响，如行动能力、食物和水消耗、体重增加/损失、眼睛/毛发/皮肤等的影响。
R1049重链序列：
Figure PCTCN2022128998-appb-000032
R1049轻链序列：
Figure PCTCN2022128998-appb-000033
实验终点(Endpoints)：实验的主要终点是肿瘤生长是否被延缓或小鼠是否被治愈。根据每组肿瘤体积计算TGI(Tumor Growth Inhibition)，计算方法：两组之间比较可用独立样本T检验。3组以上比较应用One-WayANOVA。如果F值显示有显著性差异，可进行多组间的事后分析。数据使用GraphPad Prism处理，当p<0.05表示具有显著性统计学差异。肿瘤体积V＝0.5a×b2，a和b分别为肿瘤的长、短径。肿瘤生长抑制TGI(％)＝[1-(Ti-T0)/(Vi-V0)]×100，Ti为第i天治疗组的平均瘤体积，T0为治疗开始时治 疗组的平均瘤体积，Vi为第i天溶剂对照组的平均瘤体积，V0为治疗开始时溶剂对照组S的平均瘤体积。以TGI来表示药物对肿瘤生长抑制的效果。根据具体实验，生存曲线也作为衡量药效的标准之一。
实验结果如图9所示，相对于融合蛋白R0987、R0993，经融合蛋白R0991治疗后小鼠体内肿瘤生长速度显著降低，肿瘤体积较小，具有更好的抑瘤效果。
在本说明书的描述中，参考术语“一个实施例”、“一些实施例”、“示例”、“具体示例”、或“一些示例”等的描述意指结合该实施例或示例描述的具体特征、结构、材料或者特点包含于本发明的至少一个实施例或示例中。在本说明书中，对上述术语的示意性表述不必须针对的是相同的实施例或示例。而且，描述的具体特征、结构、材料或者特点可以在任一个或多个实施例或示例中以合适的方式结合。此外，在不相互矛盾的情况下，本领域的技术人员可以将本说明书中描述的不同实施例或示例以及不同实施例或示例的特征进行结合和组合。
尽管上面已经示出和描述了本发明的实施例，可以理解的是，上述实施例是示例性的，不能理解为对本发明的限制，本领域的普通技术人员在本发明的范围内可以对上述实施例进行变化、修改、替换和变型。

Claims (17)

  1. 一种融合蛋白，其特征在于，包括抗体和IL-10单体，其中，所述抗体包括两条相同的轻链和两条相同的重链，一条IL-10单体与所述抗体的重链或轻链连接，所述IL-10单体的N末端不与所述抗体的重链的C末端连接。
  2. 根据权利要求1所述的融合蛋白，其特征在于，所述抗体的每条轻链包括VL区、和CL区，所述抗体的每条重链包括VH区、CH1区、CH2区、和CH3区；所述抗体的每条轻链或每条重链连接有一条所述IL-10单体；
    任选地，所述IL-10单体的N末端与所述抗体的轻链C末端相连；或
    所述IL-10单体的C末端与所述抗体的轻链N末端相连；或
    所述IL-10单体的N末端与所述抗体的轻链的VL区的C末端相连，所述IL-10单体的C末端与所述抗体的轻链的CL区的N末端相连；或
    所述IL-10单体的C末端与所述抗体的重链的N末端相连；或
    所述IL-10单体的N末端与所述抗体的重链VH区的C末端相连，所述IL-10单体的C末端与所述抗体的重链CH1区的N末端相连；或
    所述IL-10单体的N末端与所述抗体的重链CH2区的C末端相连，所述IL-10单体的C末端与所述抗体的重链CH3区的N末端相连。
  3. 根据权利要求1-2任一项所述的融合蛋白，其特征在于，所述IL-10单体包括天然IL-10单体。
  4. 根据权利要求1-3任一项所述的融合蛋白，其特征在于，所述IL-10单体包括修饰后的天然IL-10单体，所述修饰包括在第116位和117位氨基酸之间插入第一连接肽，所述第一连接肽为柔性连接肽；
    任选地，所述第一连接肽具有SEQ ID NO:21所示的氨基酸序列；
    任选地，所述IL-10单体具有SEQ ID NO:2所示的氨基酸序列。
  5. 根据权利要求1-4任一项所述的融合蛋白，其特征在于，所述IL-10单体与所述抗体的重链或轻链通过第二连接肽和/或第三连接肽连接；
    任选地，所述第二连接肽具有SEQ ID NO:12所示的氨基酸序列，所述第三连接肽具有SEQ ID NO:22所示的氨基酸序列。
  6. 根据权利要求5所述的融合蛋白，其特征在于，所述IL-10单体与所述抗体的重链末端或轻链末端通过所述第二连接肽连接；任选地，
    所述第二连接肽的N末端与所述抗体的轻链的C末端相连，所述第二连接肽的C末端与所述IL-10单体的N末端相连；或
    所述第二连接肽的N末端与所述IL-10单体的C末端相连，所述第二连接肽的C末端与所述抗体的轻链的N末端相连；或
    所述第二连接肽的N末端与所述IL-10单体的C末端相连，所述第二连接肽的C末端与所述抗体的重链的N末端相连。
  7. 根据权利要求5所述的融合蛋白，其特征在于，所述IL-10单体与所述抗体的重链末端或轻链末端通过所述第三连接肽连接；
    所述第三连接肽的N末端与所述抗体的轻链的C末端相连，所述第三连接肽的C末端与所述IL-10单体的N末端相连；或
    所述第三连接肽的N末端与所述IL-10单体的C末端相连，所述第三连接肽的C末端与所述抗体的轻链的N末端相连；或
    所述第三连接肽的N末端与所述IL-10单体的C末端相连，所述第三连接肽的C末端与所述抗体的重链的N末端相连。
  8. 根据权利要求5所述的融合蛋白，其特征在于，所述第三连接肽的N末端与所述轻链VL区的C末端相连，所述第三连接肽的C末端与所述IL-10单体的N末端相连；或
    所述第三连接肽的N末端与所述IL-10单体的C末端相连，所述第三连接肽的C末端与所述轻链CL区的N末端相连；或
    所述第三连接肽的N末端与所述重链VH区的C末端相连，所述第三连接肽的C末端与所述IL-10单体的N末端相连；或
    所述第三连接肽的N末端与所述IL-10单体的C末端相连，所述第三连接肽的C末端与所述重链CH1区的N末端相连；或
    所述第三连接肽的N末端与所述重链CH2区的C末端相连，所述第三连接肽的C末端与所述IL-10单体的N末端相连；或
    所述第三连接肽的N末端与所述IL-10单体的C末端相连，所述第三连接肽的C末端与所述重链CH3区的N末端相连。
  9. 根据权利要求5所述的的融合蛋白，其特征在于，所述IL-10单体的N末端通过所述第二连接肽与所述抗体的轻链的VL区的C末端相连，所述IL-10单体的C末端通过所述第二连接肽与所述抗体的轻链CL区的N末端连接；或
    所述IL-10单体的N末端通过所述第二连接肽与所述抗体的重链VH区的C末端相连，所述IL-10单体的C末端通过所述第二连接肽与所述抗体的重链CH1区的N末端相连；或
    所述IL-10单体的N末端通过所述第二连接肽与所述抗体的重链CH2区的C末端相连，所述IL-10单体的C末端通过所述第二连接肽与所述抗体的重链CH3区的N末端相连。
  10. 根据权利要求1-9任一项所述的融合蛋白，其特征在于，所述抗体选自免疫细胞类抗体和肿瘤细胞类抗体；
    任选地，所述抗体为抗PD-1抗体、抗PD-L1抗体、抗Her2抗体、抗CCR8抗体、抗VEGFR2抗 体、抗claudin18.2抗体、抗CD39抗体、抗SMA4D抗体、抗GUCY2C抗体、抗CTLA-4抗体、抗TIM3抗体、抗TIGIT抗体、抗CD47抗体或抗TNFα抗体；
    任选地，所述抗体为抗PD-1抗体。
  11. 根据权利要求1-10任一项所述的融合蛋白，其特征在于，所述融合蛋白具有SEQ ID NO:3和SEQ ID NO:9所述的氨基酸序列；或
    所述融合蛋白具有SEQ ID NO:3和SEQ ID NO:5所述的氨基酸序列；或
    所述融合蛋白具有SEQ ID NO:4和SEQ ID NO:6所述的氨基酸序列；或
    所述融合蛋白具有SEQ ID NO:7和SEQ ID NO:6所述的氨基酸序列；或
    所述融合蛋白具有SEQ ID NO:3和SEQ ID NO:8所述的氨基酸序列；或
    所述融合蛋白具有SEQ ID NO:10和SEQ ID NO:6所述的氨基酸序列。
  12. 一种核酸，其特征在于，所述核酸编码权利要求1-11任一项所述的融合蛋白。
  13. 一种表达载体，其特征在于，所述表达载体包含权利要求12所述的核酸。
  14. 一种重组细胞，其特征在于，所述重组细胞携带权利要求12所述的核酸、权利要求13所述的表达载体或表达权利要求1～11任一项所述的融合蛋白。
  15. 权利要求1-11任一项所述的融合蛋白、权利要求12所述的核酸、权利要求13所述的表达载体或权利要求14所述的重组细胞在制备药物中的用途，所述药物用于治疗或预防肿瘤或炎性疾病。
  16. 一种药物组合物，其特征在于，包含权利要求1-11任一项所述的融合蛋白、权利要求12所述的核酸、权利要求13所述的表达载体或权利要求14所述的重组细胞。
  17. 根据权利要求16所述的药物组合物，其特征在于，所述药物组合物用于治疗或预防肿瘤或炎性疾病。
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