WO2021143733A1 - 一种融合蛋白及其制法和用途 - Google Patents

一种融合蛋白及其制法和用途 Download PDF

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WO2021143733A1
WO2021143733A1 PCT/CN2021/071544 CN2021071544W WO2021143733A1 WO 2021143733 A1 WO2021143733 A1 WO 2021143733A1 CN 2021071544 W CN2021071544 W CN 2021071544W WO 2021143733 A1 WO2021143733 A1 WO 2021143733A1
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fusion protein
opg
tnfr2
cells
another preferred
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PCT/CN2021/071544
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English (en)
French (fr)
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蔡则玲
陈羿
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丁邦
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Priority to CN202180001300.XA priority Critical patent/CN113396163A/zh
Priority to US17/792,700 priority patent/US20230203204A1/en
Publication of WO2021143733A1 publication Critical patent/WO2021143733A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • 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
    • 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
    • A61K38/191—Tumor necrosis factors [TNF], e.g. lymphotoxin [LT], i.e. TNF-beta
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
    • A61P19/10—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease for osteoporosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • 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
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70575—NGF/TNF-superfamily, e.g. CD70, CD95L, CD153, CD154
    • 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
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70578—NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95
    • 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
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/715—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons
    • 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
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/715—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons
    • C07K14/7151—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons for tumor necrosis factor [TNF], for lymphotoxin [LT]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/0006—Modification of the membrane of cells, e.g. cell decoration

Definitions

  • the present invention relates to the fields of biology and medicine, and more specifically to a fusion protein and its preparation method and use.
  • RA Rheumatoid arthritis
  • TNF ⁇ TNF ⁇
  • TNFa blockers drugs that inhibit the function of TNFa
  • RANKL receptor activator NF- ⁇ B ligand
  • OPG Osteoprotegrin
  • the purpose of the present invention is to provide drugs that can not only improve inflammation in RA patients, but also prevent focal bone erosion and systemic bone loss caused by inflammation.
  • a fusion protein comprising the following elements fused together: (a) TNF receptor or active fragment thereof; (b) OPG or active fragment thereof; and optionally (C) Fc fragment.
  • the fusion protein retains the biological activities of the aforementioned elements (a) and (b).
  • the TNF receptor is selected from the following group: TNFR2, TNFR1, or a combination thereof.
  • the TNF receptor is derived from human or non-human mammals, more preferably from rodents (such as mice, rats), primates and humans.
  • the TNF receptor includes wild type and mutant type.
  • the TNF receptor includes a full-length, mature form of TNF receptor, or an active fragment thereof.
  • the TNF receptor also includes a derivative of TNF receptor.
  • the derivative of the TNF receptor includes a modified TNF receptor, a protein molecule whose amino acid sequence is homologous to the natural TNF receptor and has natural TNF receptor activity, and a dimer of TNF receptor Or a multimer, a fusion protein containing the amino acid sequence of the TNF receptor.
  • the modified TNF receptor is a PEGylated TNF receptor.
  • the "protein molecule with an amino acid sequence homologous to the natural TNF receptor and having natural TNF receptor activity” means that its amino acid sequence has ⁇ 85% homology with the TNF receptor, Preferably ⁇ 90% homology, more preferably ⁇ 95% homology, and most preferably ⁇ 98% homology; and protein molecules with TNF receptor activity.
  • the TNF receptor includes a first domain, a second domain, a third domain, and/or a fourth domain.
  • first domain, the second domain, the third domain, and/or the fourth domain are each independently a cysteine-rich domain (CRD).
  • CCD cysteine-rich domain
  • the TNF receptor is the second and third domains of the extracellular region of the TNF receptor, or the extracellular region of the TNF receptor containing the second and third domains.
  • the TNF receptor contains or has the amino acid sequence of TNFR2 (SEQ ID NO.: 4) at positions 1-257, or 23-257, or 77-162, or No. 205-257.
  • the OPG is derived from human or non-human mammals, more preferably from rodents (such as mice, rats), primates and humans.
  • the OPG includes wild type and mutant type.
  • the OPG includes a full-length, mature form of OPG, or an active fragment thereof.
  • the OPG also includes OPG derivatives.
  • the OPG derivatives include modified OPG, protein molecules whose amino acid sequence is homologous to natural OPG and have natural OPG activity, dimers or multimers of OPG, and those containing OPG amino acid sequence. Fusion protein.
  • the modified OPG is PEGylated OPG.
  • the "protein molecule whose amino acid sequence is homologous to natural OPG and has natural OPG activity” means that its amino acid sequence has ⁇ 85% homology with OPG, preferably ⁇ 90% The homology, preferably ⁇ 95% homology, and best ⁇ 98% homology; and protein molecules with OPG activity.
  • the OPG includes a first domain, a second domain, a third domain, and/or a fourth domain.
  • first domain, the second domain, the third domain, and/or the fourth domain are each independently a cysteine-rich domain (CRD).
  • CCD cysteine-rich domain
  • the OPG is the second domain, the third domain, and the fourth domain of the extracellular region of OPG, or the OPG cell containing the second domain, the third domain, and the fourth domain. Outer zone.
  • the OPG contains or has positions 1 to 194, or 22 to 194, or 22 to 194 of the OPG amino acid sequence (SEQ ID NO.: 5).
  • the Fc fragment is derived from humans or non-human mammals, more preferably from rodents (such as mice, rats), primates and humans.
  • the Fc fragment is the Fc fragment of immunoglobulin IgG, preferably the Fc portion of IgG1.
  • the Fc fragment includes natural Fc fragments and Fc mutants.
  • the Fc fragment contains or has positions 99 (Glu)-330 (Lys) of the human IgG1 amino acid sequence (accession number UniProtKB-P01857).
  • the Fc fragment contains or has positions 487-718 of SEQ ID NO.:1.
  • the Fc fragment contains or has positions 430-661 of SEQ ID NO.:2.
  • amino acid sequence of the Fc fragment is shown in SEQ ID NO.:3.
  • the fusion protein has the structure shown in the following formula I or II:
  • X is TNF receptor or its active fragment
  • Y is OPG or its active fragment
  • Z is an optional Fc fragment
  • any two of the X, Y, and Z are connected in a head-to-head, head-to-tail, tail-to-head or tail-to-tail manner.
  • the "head” refers to the N-terminus of the polypeptide or fragments thereof, especially the N-terminus of the wild-type polypeptide or fragments thereof.
  • the "tail” refers to the C-terminus of the polypeptide or fragments thereof, especially the C-terminus of the wild-type polypeptide or fragments thereof.
  • the length of the peptide linker is 0-20 amino acids, preferably, 0-10 amino acids.
  • the fusion protein is a homodimer.
  • the fusion protein is selected from the following group:
  • (B) It has ⁇ 80% homology with the amino acid sequence shown in SEQ ID NO:1 or 2 (preferably ⁇ 90% homology; etc. preferably ⁇ 95% homology; most preferably, ⁇ 97% homology, such as 98% or more, 99% or more) polypeptide, and the polypeptide has (a) inflammation inhibitory activity or (b) osteoporosis and/or bone loss inhibitory activity;
  • the amino acid sequence shown in SEQ ID NO: 1 or 2 is formed by substitution, deletion or addition of 1-5 amino acid residues, and retains (a) inflammation inhibitory activity or (b) osteoporosis and/ Or a derivative polypeptide with bone loss inhibitory activity.
  • amino acid sequence of the fusion protein is shown in SEQ ID NO.: 1 or 2.
  • the second aspect of the present invention provides an isolated polynucleotide which encodes the fusion protein of the first aspect of the present invention.
  • the polynucleotide additionally contains an auxiliary element selected from the group consisting of signal peptide, secretory peptide, tag sequence (such as 6His), or flank of the ORF of the mutein or fusion protein. Its combination.
  • the polynucleotide is selected from the following group: DNA sequence, RNA sequence, or a combination thereof.
  • the third aspect of the present invention provides a vector containing the polynucleotide according to the second aspect of the present invention.
  • the vector includes one or more promoters, which are operably linked to the nucleic acid sequence, enhancer, transcription termination signal, polyadenylation sequence, origin of replication, and selectable marker. , Nucleic acid restriction sites, and/or homologous recombination sites.
  • the vectors include plasmids and viral vectors.
  • the viral vector is selected from the group consisting of adeno-associated virus (AAV), adenovirus, lentivirus, retrovirus, herpes virus, SV40, poxvirus, or a combination thereof.
  • AAV adeno-associated virus
  • adenovirus adenovirus
  • lentivirus lentivirus
  • retrovirus lentivirus
  • herpes virus SV40
  • poxvirus poxvirus
  • the vector includes an expression vector, a shuttle vector, and an integration vector.
  • the fourth aspect of the present invention provides a host cell, which contains the vector according to the third aspect of the present invention, or its genome integrates the polynucleotide according to the second aspect of the present invention.
  • the host cell is a eukaryotic cell, such as a yeast cell, a plant cell or a mammalian cell (including human and non-human mammals).
  • the host cell is a prokaryotic cell, such as Escherichia coli.
  • the yeast cells are selected from one or more sources of yeast from the following group: Pichia pastoris, Kluyveromyces, or a combination thereof; preferably, the yeast cells include:
  • the yeast Luveyi is more preferably Kluyveromyces marxianus, and/or Kluyveromyces lactis.
  • the host cell is selected from the group consisting of E. coli, wheat germ cells, insect cells, SF9, SP2/0, Hela, HEK293, CHO (such as CHOKS), yeast cells, or a combination thereof.
  • the fifth aspect of the present invention provides a method for producing the fusion protein of the first aspect of the present invention, and the method includes the steps:
  • the fusion protein is isolated or purified.
  • the sixth aspect of the present invention provides a pharmaceutical composition, which contains the fusion protein described in the first aspect of the present invention and a pharmaceutically acceptable carrier thereof.
  • the pharmaceutical composition further includes other drugs for inhibiting inflammatory activity.
  • other drugs for inhibiting inflammatory activity are selected from the following group: hormone drugs, non-steroidal anti-inflammatory drugs, immunosuppressive drugs, small molecule targeted drugs, biological agents, or combinations thereof.
  • the hormone drug is selected from the group consisting of hydrocortisone, prednisone, prednisolone, dexamethasone, or a combination thereof.
  • the non-steroidal anti-inflammatory drug is selected from the group consisting of aspirin, indomethacin, naproxen, ibuprofen, diclofenac, loxoprofen, meloxicam, celecoxime Coxib, etoricoxib, parecoxib, or a combination thereof.
  • the immunosuppressive drug is selected from the group consisting of methotrexate, cyclophosphamide, azathioprine, cyclosporine, mycophenolate mofetil, tacrolimus, sirolimus, and Flumet, or a combination thereof.
  • the small molecule targeted drug is selected from the group consisting of tofacitib, baritinib, or a combination thereof.
  • the biological agent is selected from the group consisting of etanercept, certuzumab, adalimumab, golimumab, infliximab, tocilizumab, sucralose Kimumab, ustekinumab, kanasimab, anakinra, linalicept, abatacept, rituximab, belimumab, or a combination thereof.
  • the seventh aspect of the present invention provides a fusion protein according to the first aspect of the present invention, a polynucleotide according to the second aspect of the present invention, a vector according to the third aspect of the present invention, and a vector according to the fourth aspect of the present invention.
  • the infectious disease includes septic shock (such as LPS-induced septic shock).
  • the autoimmune disease is selected from the group consisting of rheumatoid arthritis, ankylosing spondylitis, or a combination thereof.
  • osteoporosis or loss is selected from the group consisting of: osteoporosis or/and loss caused by rheumatoid arthritis, osteoporosis in women after menopause, or a combination thereof.
  • the tumor-related disease is selected from the group consisting of multiple myeloma, bone-related bone metastasis solid tumor, or a combination thereof.
  • composition or preparation is also used for one or more purposes selected from the following group:
  • the cell is selected from the group consisting of fibroblasts, mononuclear macrophages, vascular endothelial cells, or a combination thereof.
  • the composition is a pharmaceutical composition.
  • the eighth aspect of the present invention provides a method for inhibiting cell apoptosis, including the steps:
  • cells are cultured, thereby inhibiting cell apoptosis.
  • the cells are selected from the group consisting of fibroblasts, hematological tumor cells, solid tumor cells, or a combination thereof.
  • the hematological tumor cells are selected from the group consisting of B-cell malignant tumor cells, acute myeloid leukemia cells, Hodgkin’s lymphoma cells, T-cell malignant tumor cells, multiple myeloma cells, or Its combination.
  • the solid tumor cells are selected from the group consisting of breast cancer cells, non-small cell lung cancer cells, liver cancer cells, colon cancer cells, gastric cancer cells, or a combination thereof.
  • the cell is a cell cultured in vitro.
  • the method is non-therapeutic and non-diagnostic.
  • the apoptosis includes TNFa-induced apoptosis; and/or TRAIL-induced apoptosis.
  • the ninth aspect of the present invention provides a method for inhibiting the differentiation of mononuclear macrophages, including the steps:
  • monocyte macrophages are cultured, thereby inhibiting the differentiation of monocyte macrophages.
  • the mononuclear macrophages are cells cultured in vitro.
  • the method is non-therapeutic and non-diagnostic.
  • the differentiation of mononuclear macrophages is RANKL-induced differentiation of mononuclear macrophages.
  • the tenth aspect of the present invention provides a method for preventing and/or treating diseases, including the step of administering the fusion protein according to the first aspect of the present invention to a subject in need.
  • the fusion protein is administered in the form of monomer and/or dimer.
  • the subject is a human.
  • the disease is selected from the following group: infectious disease, autoimmune disease, osteoporosis or loss, tumor-related disease, or a combination thereof.
  • FIG. 1 is a schematic diagram of the structure of the fusion proteins OPG-TNFR2-Fc and TNFR2-OPG-Fc.
  • OPG-TNFR2-Fc contains (from N-terminal to C-terminal): OPG amino acid sequence 22-194 includes 4 CRDs, TNFR2 amino acid sequence 23-257 includes 4 CRDs, and IgG1 Fc.
  • TNFR2-OPG-Fc contains (from N-terminal to C-terminal): TNFR2 amino acid sequence 23-257 includes 4 CRDs, OPG amino acid sequence 22-194 includes 4 CRDs, TNFR2 amino acid sequence 203-257, and IgG1 Fc.
  • Figure 2 shows the SDS-PAGE analysis of the fusion protein with reduced and non-reduced denatured protein gels.
  • the fusion protein purified by the Protein A affinity chromatography column was analyzed and identified by 6% non-reducing denaturing SDS-PAGE (A) and 10% reducing denaturing SDS-PAGE (B).
  • A Human IgG1
  • 2 OPG-TNFR2-Fc
  • 3 TNFR2-OPG-Fc. Each sample is loaded with 5 mg.
  • FIG. 3 shows the HPLC-SEC analysis of the fusion protein.
  • the fusion protein was analyzed by HPLC-SEC (TSKgel G3000SWXL) chromatography column.
  • the red curve represents OPG-TNFR2-Fc; the green curve represents TNFR2-OPG-Fc.
  • the molecular weight is represented by the blue curve.
  • Figure 4 shows the results of an in vitro binding to recombinant human TNFa ELISA study.
  • Figure 5 shows the results of an ELISA study on in vitro binding to recombinant human RANKL.
  • Figure 6 shows the results of an ELISA study on in vitro binding to recombinant human TRAIL.
  • Figure 7 shows the results of studies on inhibiting TNFa-induced apoptosis of L929 cells.
  • Figure 8 shows the results of studies on inhibiting Trail-induced apoptosis of L929 cells.
  • Figure 9 shows the results of inhibition of RANKL-induced cell differentiation of RAW264.7 cells.
  • Figure 10 shows the results of a study to inhibit the death of mice from septic shock induced by LPS.
  • Figure 11 shows (A): reducing the incidence of CIA-induced inflammation in mice; (B) reducing the degree of inflammation in CIA-induced mice.
  • the inventors unexpectedly discovered that by fusing (a) TNF receptor or its active fragment; (b) OPG or its active fragment; and optionally (c) Fc fragment, the resulting fusion protein has Very excellent biological activity, and can significantly (a) inhibit TNFa-induced apoptosis; and/or (b) inhibit TRAIL-induced apoptosis; and/or (c) inhibit RANKL-induced mononuclear macrophages Cell differentiation.
  • the fusion protein has good stability and long half-life, so it helps (i) prevent and/or treat infectious diseases; and/or (ii) prevent and/or treat autoimmune diseases; and/or (iii ) Prevention and/or treatment of tumor-related diseases.
  • the present invention has been completed on this basis.
  • Fc refers to the Fc fragment of human immunoglobulin.
  • immunoglobulin Fc region refers to the constant region of an immunoglobulin chain, particularly the carboxyl end or a part of the constant region of an immunoglobulin heavy chain.
  • the immunoglobulin Fc region may include two heavy chains CH1, CH2, and CH3. A combination of one or more domains and an immunoglobulin hinge region.
  • the Fc region of the immunoglobulin used includes at least one immunoglobulin hinge region, one CH2 domain and one CH3 domain, preferably lacking CH1 domain.
  • Human immunoglobulins are known to have multiple classes, such as IgA, IgD, IgE, IgM and IgG (including four subclasses of IgG1, IgG2, IgG3, and IgG4). Choose a specific immune globulin from specific immunoglobulin classes and subclasses
  • the globulin Fc region is within the scope of those skilled in the art.
  • the immunoglobulin Fc region can optionally include the coding sequence of the human immunoglobulin IgG4 subclass Fc region, in which one immune system is deleted.
  • the globulin heavy chain 1 domain (CH1) but includes the hinge region and the coding sequence of CH2, CH3, and the two domains.
  • containing includes “comprising”, “consisting mainly of”, “consisting essentially of”, and “consisting of”; Mainly composed of”, “basically composed of” and “consisted of” belong to the subordinate concepts of "containing", “having” or “including”.
  • the fusion protein is an isolated protein that is not related to other proteins, polypeptides or molecules, and is a purified product of recombinant host cell culture or as a purified extract.
  • TNF receptor or its active fragment
  • TNFR1 and 2 belong to the TNF receptor superfamily and are type I transmembrane proteins.
  • the molecular weights of TNFR1 and TNFR2 are 55 and 75kDa, respectively, and their extracellular regions each contain 4 cysteine-rich regions, and the amino acid sequence is 23% identical.
  • TNFR1 is widely expressed in various types of cells, while TNFR2 is mainly expressed in hematopoietic cells, such as T cells and natural killer cells, as well as endothelial cells, nerve cells, thymocytes and mesenchymal stem cells.
  • TNFR1 mainly binds to soluble TNFa
  • TNFR2 binds to soluble TNFa and can also bind to TNFa in the form of membrane protein.
  • TNF receptors include, but are not limited to, TNFR2 and TNFR1.
  • TNF-R2, TNFR II, and hTNFR II are used interchangeably, and all refer to the human tumor necrosis factor II receptor.
  • OPG osteoprotegrin
  • TNFR tumor necrosis factor receptor superfamily
  • the mature OPG contains 4 cysteine-rich regions, 2 death domains and 1 heparin binding domain.
  • the cysteine-rich region is essential for the interaction between OPG and its ligand, and the C-terminal cystein mediates the formation of homodimers.
  • OPG is widely and continuously expressed in mesenchymal stem cells, fibroblasts and endothelial cells.
  • OPG is also known as the decoy receptor for TNF superfamily ligands, and binds to RANKL and TRAIL.
  • TRAIL reduces the release of OPG from expressing cells, while OPG inhibits TRAIL-induced apoptosis.
  • OPG can inhibit RANKL to promote osteoclast formation and promote osteoclastogenesis.
  • the lack of OPG in the human body causes Paget disease in adolescents. When there is not enough OPG to balance the functions of RANKL and RANK, it will lead to osteoporosis and vascular calcification.
  • fusion protein of the present invention or “polypeptide” refers to the fusion protein described in the first aspect of the present invention.
  • the fusion protein of the present invention comprises the following elements: (a) TNF receptor or its active fragment, (b) OPG or its active fragment, and optionally (c) Fc fragment.
  • the various elements such as between element a and element b, element b or element c
  • the linking sequence is usually a sequence that does not affect the two proteins.
  • the structure of the fusion protein is as shown in XYZ(I) or YXZ(II), where X is TNF receptor or its active fragment; Y is OPG or its active fragment; Z is optional Fc fragment.
  • the fusion protein has an amino acid sequence as shown in SEQ ID NO.: 1 or 2.
  • fusion protein also includes variant forms of the fusion protein (such as the sequence shown in SEQ ID NO.: 1 or 2) having the above-mentioned activities. These variant forms include (but are not limited to): 1-3 (usually 1-2, more preferably 1) amino acid deletion, insertion and/or substitution, and addition or addition at the C-terminus and/or N-terminus One or several (usually 3 or less, preferably 2 or less, more preferably 1 or less) amino acids are deleted. For example, in the field, when amino acids with similar or similar properties are substituted, the function of the protein is usually not changed. For another example, adding or deleting one or several amino acids at the C-terminus and/or N-terminus usually does not change the structure and function of the protein. In addition, the term also includes the polypeptide of the present invention in monomeric and multimeric forms. The term also includes linear and non-linear polypeptides (such as cyclic peptides).
  • the present invention also includes active fragments, derivatives and analogs of the above-mentioned fusion protein.
  • fragment refers to a polypeptide that substantially retains the function or activity of the fusion protein of the present invention.
  • polypeptide fragments, derivatives or analogues of the present invention can be (i) one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) are substituted, or (ii) in one or more A polypeptide with substitution groups in three amino acid residues, or (iii) a polypeptide formed by fusing an antigenic peptide with another compound (such as a compound that prolongs the half-life of the polypeptide, such as polyethylene glycol), or (iv) an additional amino acid sequence A polypeptide fused to this polypeptide sequence (a fusion protein fused with a leader sequence, a secretory sequence, or a tag sequence such as 6 ⁇ His). According to the teachings herein, these fragments, derivatives and analogs belong to the scope well known to those skilled in the art.
  • a preferred type of active derivative means that compared with the amino acid sequence of Formula I or Formula II, there are at most 3, preferably at most 2, and more preferably at most 1 amino acid replaced by an amino acid with similar or similar properties. Peptides. These conservative variant polypeptides are best produced according to Table A by performing amino acid substitutions.
  • substitutions Ala(A) Val; Leu; Ile Val Arg(R) Lys; Gln; Asn Lys Asn(N) Gln; His; Lys; Arg Gln Asp(D) Glu Glu Cys(C) Ser Ser Gln(Q) Asn Asn Glu(E) Asp Asp Gly(G) Pro; Ala Ala His(H) Asn; Gln; Lys; Arg Arg Ile(I) Leu; Val; Met; Ala; Phe Leu Leu(L) Ile; Val; Met; Ala; Phe Ile Lys(K) Arg; Gln; Asn Arg Met(M) Leu; Phe; Ile Leu Phe(F) Leu; Val; Ile; Ala; Tyr Leu Pro(P) Ala Ala Ser(S) Thr Thr Thr(T) Ser Ser Trp(W) Tyr; Phe Tyr Tyr(Y) Trp; Phe; Thr; Ser Preferred substitution Ala(
  • the present invention also provides analogs of the fusion protein of the present invention.
  • the difference between these analogues and the polypeptide shown in SEQ ID NO.: 1 or SEQ ID NO.: 2 may be a difference in amino acid sequence, a difference in modified form that does not affect the sequence, or both.
  • Analogs also include analogs having residues different from natural L-amino acids (such as D-amino acids), and analogs having non-naturally occurring or synthetic amino acids (such as ⁇ , ⁇ -amino acids). It should be understood that the polypeptide of the present invention is not limited to the representative polypeptides exemplified above.
  • Modified (usually not changing the primary structure) forms include: chemically derived forms of polypeptides in vivo or in vitro, such as acetylation or carboxylation. Modifications also include glycosylation, such as those polypeptides produced by glycosylation modifications during the synthesis and processing of the polypeptide or during further processing steps. This modification can be accomplished by exposing the polypeptide to an enzyme that performs glycosylation (such as a mammalian glycosylase or deglycosylase). Modified forms also include sequences with phosphorylated amino acid residues (such as phosphotyrosine, phosphoserine, phosphothreonine). It also includes polypeptides that have been modified to improve their anti-proteolytic properties or optimize their solubility properties.
  • the present invention also relates to a vector containing the polynucleotide of the present invention, a host cell produced by genetic engineering using the vector of the present invention or the fusion protein coding sequence of the present invention, and a method for producing the polypeptide of the present invention through recombinant technology.
  • the polynucleotide sequence of the present invention can be used to express or produce a recombinant fusion protein. Generally speaking, there are the following steps:
  • polynucleotide (or variant) of the present invention encoding the fusion protein of the present invention, or use a recombinant expression vector containing the polynucleotide to transform or transduce a suitable host cell;
  • the polynucleotide sequence encoding the fusion protein can be inserted into the recombinant expression vector.
  • recombinant expression vector refers to bacterial plasmids, bacteriophages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenovirus, retrovirus or other vectors well known in the art. Any plasmid and vector can be used as long as it can be replicated and stabilized in the host.
  • An important feature of an expression vector is that it usually contains an origin of replication, a promoter, a marker gene, and translation control elements.
  • Methods well known to those skilled in the art can be used to construct an expression vector containing the DNA sequence encoding the fusion protein of the present invention and appropriate transcription/translation control signals. These methods include in vitro recombinant DNA technology, DNA synthesis technology, and in vivo recombination technology.
  • the DNA sequence can be effectively linked to an appropriate promoter in the expression vector to guide mRNA synthesis.
  • promoters are: Escherichia coli lac or trp promoter; lambda phage PL promoter; eukaryotic promoters include CMV immediate early promoter, HSV thymidine kinase promoter, early and late SV40 promoter, anti Transcriptional virus LTRs and some other known promoters that can control gene expression in prokaryotic or eukaryotic cells or viruses.
  • the expression vector also includes a ribosome binding site for translation initiation and a transcription terminator.
  • the expression vector preferably contains one or more selectable marker genes to provide phenotypic traits for selecting transformed host cells, such as dihydrofolate reductase for eukaryotic cell culture, neomycin resistance, and green Fluorescent protein (GFP), or tetracycline or ampicillin resistance for E. coli.
  • selectable marker genes to provide phenotypic traits for selecting transformed host cells, such as dihydrofolate reductase for eukaryotic cell culture, neomycin resistance, and green Fluorescent protein (GFP), or tetracycline or ampicillin resistance for E. coli.
  • a vector containing the above-mentioned appropriate DNA sequence and an appropriate promoter or control sequence can be used to transform an appropriate host cell so that it can express the protein.
  • the host cell can be a prokaryotic cell (such as Escherichia coli), or a lower eukaryotic cell, or a higher eukaryotic cell, such as yeast cells, plant cells or mammalian cells (including human and non-human mammals).
  • a prokaryotic cell such as Escherichia coli
  • yeast cells such as Pichia pastoris, Kluyveromyces, or a combination thereof
  • yeast cells include: Kluyveromyces, more preferably Maxsk Luwei and/or Kluyveromyces lactis) are host cells.
  • Enhancers are cis-acting factors of DNA, usually about 10 to 300 base pairs, acting on promoters to enhance gene transcription. Examples include the 100 to 270 base pair SV40 enhancer on the late side of the replication initiation point, the polyoma enhancer on the late side of the replication initiation point, and adenovirus enhancers.
  • Transformation of host cells with recombinant DNA can be performed by conventional techniques well known to those skilled in the art.
  • the host is a prokaryotic organism such as Escherichia coli
  • competent cells that can absorb DNA can be harvested after the exponential growth phase and treated with the CaCl 2 method.
  • the steps used are well known in the art.
  • Another method is to use MgCl 2 .
  • the transformation can also be carried out by electroporation.
  • the host is a eukaryote
  • the following DNA transfection methods can be selected: calcium phosphate co-precipitation method, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.
  • the obtained transformants can be cultured by conventional methods to express the polypeptide encoded by the gene of the present invention.
  • the medium used in the culture can be selected from various conventional mediums.
  • the culture is carried out under conditions suitable for the growth of the host cell. After the host cell has grown to a suitable cell density, the selected promoter is induced by a suitable method (such as temperature conversion or chemical induction), and the cell is cultured for a period of time.
  • the recombinant polypeptide in the above method can be expressed in the cell or on the cell membrane, or secreted out of the cell. If necessary, the physical, chemical, and other characteristics can be used to separate and purify the recombinant protein through various separation methods. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to: conventional renaturation treatment, treatment with a protein precipitation agent (salting out method), centrifugation, osmotic sterilization, ultra-treatment, ultra-centrifugation, molecular sieve chromatography (gel filtration), adsorption layer Analysis, ion exchange chromatography, high performance liquid chromatography (HPLC) and various other liquid chromatography techniques and combinations of these methods.
  • the present invention provides a fusion protein, which may optionally contain a peptide linker.
  • the size and complexity of the peptide linker may affect the activity of the protein.
  • the peptide linker should have sufficient length and flexibility to ensure that the two proteins connected have enough freedom in space to perform their functions. At the same time, the influence of the formation of ⁇ helix or ⁇ sheet in the peptide linker on the stability of fusion protein is avoided.
  • the length of the connecting peptide is generally 0-20 amino acids, preferably 0-10 amino acids.
  • the composition is a pharmaceutical composition, which contains the above-mentioned fusion protein, and a pharmaceutically acceptable carrier, diluent, stabilizer and/or thickener, and can be prepared as a lyophilized powder , Tablets, capsules, syrups, solutions or suspensions.
  • “Pharmaceutically acceptable carrier or excipient (excipient)” refers to: one or more compatible solid or liquid fillers or gel substances, which are suitable for human use, and must have sufficient purity and sufficient Low toxicity. "Compatibility” here means that the components in the composition can be blended with the active ingredients of the present invention and between them without significantly reducing the efficacy of the active ingredients.
  • the composition may be liquid or solid, such as powder, gel or paste.
  • the composition is a liquid, preferably an injectable liquid. Suitable excipients will be known to those skilled in the art.
  • Examples of pharmaceutically acceptable carriers include cellulose and its derivatives (such as sodium carboxymethyl cellulose, sodium ethyl cellulose, cellulose acetate, etc.), gelatin, talc, and solid lubricants (such as stearic acid). , Magnesium stearate), calcium sulfate, vegetable oils (such as soybean oil, sesame oil, peanut oil, olive oil, etc.), polyols (such as propylene glycol, glycerin, mannitol, sorbitol, etc.), emulsifiers (such as Tween) ), wetting agents (such as sodium lauryl sulfate), coloring agents, flavoring agents, stabilizers, antioxidants, preservatives, pyrogen-free water, etc.
  • cellulose and its derivatives such as sodium carboxymethyl cellulose, sodium ethyl cellulose, cellulose acetate, etc.
  • gelatin such as sodium carboxymethyl cellulose, sodium ethyl cellulose, cellulose
  • the composition may comprise physiologically acceptable sterile aqueous or non-aqueous solutions, dispersions, suspensions or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions.
  • Suitable aqueous and non-aqueous carriers, diluents, solvents or excipients include water, ethanol, polyols and suitable mixtures thereof.
  • these substances can be formulated in a non-toxic, inert and pharmaceutically acceptable aqueous carrier medium, where the pH is usually about 5-8, preferably about 6-8, although the pH can be The nature of the formulated substance and the condition to be treated vary.
  • the formulated pharmaceutical composition can be administered by conventional routes, including (but not limited to): intraperitoneal, intravenous, or topical administration.
  • the pharmaceutical composition is used for (i) preventing and/or treating infectious diseases; and/or (ii) preventing and/or treating autoimmune diseases; and/or (iii) preventing and/or treating osteoporosis or Loss; and/or (iv) prevention and/or treatment of tumor-related diseases; and/or (v) inhibition of TNFa-induced apoptosis; and/or (vi) inhibition of TRAIL-induced apoptosis; and/or (vii ) Inhibit RANKL-induced differentiation of mononuclear macrophages; and/or (viii) inhibit TNFa-induced immune activation; and/or (ix) inhibit RANKL-induced osteoclast formation.
  • the fusion protein of the present invention has a long half-life.
  • the fusion protein of the present invention can not only improve inflammation in RA patients, but also prevent focal bone erosion and systemic bone loss caused by inflammation.
  • the present invention found for the first time that the fusion protein of the present invention can significantly (i) prevent and/or treat infectious diseases; and/or (ii) prevent and/or treat autoimmune diseases; and/or (iii) inhibit TNFa-induced apoptosis; and/or (iv) inhibition of TRAIL-induced apoptosis; and/or (v) inhibition of RANKL-induced differentiation of monocytes and macrophages; and/or (vi) inhibition of autoimmune diseases Caused osteoporosis and loss; and/or (vii) inhibition of bone metastases.
  • TNFR2-OPG-Fc fusion protein hereinafter referred to as OTFc
  • the human OPG gene sequence is derived from the human OPG full-length cDNA (Open Biosystems, MHS1011-7509022), and the human TNFR2 and human IgG Fc sequences are derived from the human TNFR2(1-257)-Fc fusion protein gene. Has been built.
  • the polymerase chain reaction (PCR) method was used to synthesize the genes encoding OPG amino acids 1-194 (5' and 3'end primers) using human OPG full-length cDNA as a template.
  • primer-1 and primer-2 they are primer-1 and primer-2, the primer sequences are shown in Table 1, the same below) and the human TNFR2(1-257)-Fc fusion protein gene is used as a template to synthesize the gene encoding human TNFR2-Fc fusion protein (without protein signal)
  • Set coding sequence 1-22, 5'and 3'primers are primer-3 and primer-4 respectively), and then use overlap extension PCR method to connect the above two PCR fragments to synthesize the encoding OPG(1-194)-TNFR2(23 -257)
  • the complete gene of the Fc fusion protein (5' and 3'end primers are primer-1 and primer-4, respectively).
  • the first 23 nucleotide sequences of primer-2 are complementary to the nucleotide sequence of primer-3, so that the two PCR fragments can be joined during the second step of overlap extension PCR.
  • Primer-1 contains the restriction endonuclease Not I sequence
  • primer-4 contains the restriction endonuclease Xba I sequence, which is used to insert the protein expression vector.
  • the synthesized PCR fragments were separated by agarose gel electrophoresis, and purified by DNA gel purification kit (QIAGEN).
  • the OPG-TNFR2-Fc fusion protein gene synthesized by overlap extension PCR was first cloned into pCR2.1 vector (Invitrogen) with the T/A vector cloning kit, and then the fusion protein was cut from this vector with Not I and Xba I restriction enzymes The gene is inserted into a mammalian expression plasmid that is also digested with Not I and Xba I, such as pcDNA3.1 (Invitrogen).
  • the DNA sequence of OPG(1-194)-TNFR2(23-257)-Fc inserted in pCR2.1 was confirmed by DNA sequencing.
  • TNFR2(1-257)-OPG(22-194)-TNFR2(202-257)-Fc hereinafter referred to as TOFc
  • TNFR2-OPG-Fc polymerase chain reaction
  • the synthesized fusion protein gene was first cloned into the pCR-Blunt II-TOPO vector (Invitrogen) with the T/A vector cloning kit, and then the fusion protein gene was cut from this vector with Not I and Xba I restriction enzymes, and inserted into the same In mammalian expression plasmids digested with Not I and Xba I, such as pcDNA3.1 (Invitrogen).
  • the DNA sequence of TNFR2(1-257)-OPG(22-194)-TNFR2(202-257)-Fc inserted in the pCR-Blunt II-TOPO vector was confirmed by DNA sequencing.
  • the host cell used to construct cells stably expressing the fusion protein is Chinese hamster ovary cell CHO-KS.
  • CHO-KS is a CHO-K1 cell grown in a medium containing fetal bovine serum (FBS) after gradually reducing the FBS content in the medium until it is cultured in a FBS-free medium, and finally acclimatized to the OptiCHO medium without FBS ( Cells grown in suspension in Invitrogen).
  • FBS fetal bovine serum
  • the anti-neomycin gene in the pcDNA3.1 vector containing the fusion protein gene was replaced with the rat glutamine synthetase gene, and the fusion protein expression vector was transfected by electrotransfection (Bio-Rad, Gene Pulser Xcell) Into CHO-KS cells, the transfected cells were cultured for 24 hours, and then the transfected cells were screened and cultured on a 96-well culture plate by the limiting dilution method.
  • the selection medium is OptiCHO, 5 ⁇ g/ml recombinant human insulin and 10 ⁇ M aminosulfoxide methionine (MSX). Culture the cells in an incubator at 37°C and 8% CO 2.
  • the cell culture medium of each well with cell population was analyzed by ELISA method (alkaline phosphatase-conjugated goat anti-human IgG Fc antibody, Jackson ImmunoResearch Lab), and the cells with positive expression of the fusion protein were analyzed.
  • the population is further amplified, then detected by ELISA, and then amplified, and finally a population of stable cells expressing fusion protein is obtained.
  • the fusion protein cell line constructed in Example 2 was cultured and expanded to 2 liters. The culture supernatant was harvested, and the fusion protein was purified with a Protein-A affinity chromatography column (POROS MabCapture A, Life Tech).
  • the purified fusion protein was analyzed by reducing and non-reducing SDS-PAGE electrophoresis and HPLC-SEC (high pressure liquid phase-molecular sieve) (TSKgel G3000SWXL, TOSOH Bioscience).
  • OPG-TNFR2-Fc fusion protein is a homodimer with a theoretical molecular weight of about 142kDa.
  • the TNFR2-OPG-Fc fusion protein is a homodimer with a theoretical molecular weight of about 153kDa.
  • the non-reducing SDS-PAGE electrophoresis gel (Figure 2A) showed that the molecular weights of these two homodimer fusion proteins were larger than human immunoglobulin IgG1, which were similar to their theoretical values.
  • Figure 2B shows the results of the reduced SDS-PAGE gel electrophoresis of the single chain of the fusion protein.
  • Figure 3 shows the HPLC-SEC (molecular sieve chromatography column) analysis results of OPG-TNFR2-Fc fusion protein and TNFR2-OPG-Fc fusion protein.
  • the result shows that the fusion protein peak is located near 600kDa.
  • OPG and TNFR2 each have 4 cysteine-rich regions (CRD), 8 CRDs are connected together, so that the fusion protein molecules form a rod-shaped structure, and the molecular sieve chromatography column analysis shows that the molecular weight of the standard protein is larger than the compact structure. .
  • rhTNFa Recombinant human TNFa (rhTNFa, SinoBiological) was dissolved in PBS (pH 7.0) solution, added to a 96-well ELISA plate, and placed in a refrigerator at 4°C overnight. On the second day, free rhTNFa was washed away with PBST (PBS containing 0.05% Tween-20), and PBST blocking solution containing 3% BSA was added.
  • PBS pH 7.0
  • the blocked ELISA plate is added with serially diluted fusion proteins of different concentrations, and the fusion protein that binds to TNFa is detected by alkaline phosphatase-conjugated goat anti-human IgG Fc antibody (Jackson ImmunoResearch Lab), and the substrate (PNPP) is added for color development. Read the plate with a microplate reader at a wavelength of 405nm/655nm.
  • fusion protein bound to RANKL was detected with alkaline phosphatase-conjugated goat anti-human IgG Fc antibody (Jackson ImmunoResearch Lab), and the substrate (PNPP) was added for color development, and the plate was read with a microplate reader at dual wavelengths of 405nm/490nm.
  • the in vitro specific binding activity of the fusion protein and TRAIL was studied by ELISA method. Dilute OPG-TNFR2-Fc, TNFR2-OPG-Fc and OPG-Fc (Sino Biological Inc.) fusion proteins with PBS to 1 ⁇ g/mL respectively, add 50 ⁇ L to each well of 96-well ELISA plate, and place in refrigerator at 4°C overnight . On the second day, wash with PBST (PBS containing 0.05% Tween-20) three times, and add 200 ⁇ L of PBST blocking solution containing 3% BSA to each well.
  • PBST PBS containing 0.05% Tween-20
  • the biological activity of TNFR2 of the fusion protein adopts an in vitro biological activity detection method that neutralizes TNFa.
  • the biological activity of TNFa was tested with mouse fibroblast L929 cytotoxicity.
  • RhTNFa and serial dilutions of fusion proteins of different concentrations, OPG-TNFR2-Fc, TNFR2-OPG-Fc and TNFR2 were mixed to L929 cells, cultured in a cell incubator for 20 hours and then used The crystal seed staining method was used to detect the viability of L929 cells.
  • OPG-TNFR2-Fc fusion protein and TNFR2-OPG-Fc fusion protein can inhibit rhTNFa-induced apoptosis of L929 cells (Figure 7).
  • Recombinant human TRAIL (R&D Systems Inc.) was mixed with serially diluted fusion proteins of different concentrations and added to L929 cells. After 20 hours of culture in a cell incubator, the viability of L929 cells was detected by crystal staining.
  • Recombinant human OPG-Fc fusion protein Novo Protein
  • TNFR2-OPG-Fc fusion protein The ability of TNFR2-OPG-Fc fusion protein to inhibit rhTRAIL-induced apoptosis of L929 cells was significantly stronger than that of OPG-Fc and OPG-TNFR2-Fc ( Figure 8).
  • the IC50 of TNFR2-OPG-Fc fusion protein to inhibit rhTRAIL-induced apoptosis of L929 cells was 0.02nM, and the inhibitory activities of OPG-Fc and OPG-TNFR2-Fc were equivalent, with IC50 of 0.27nM and 0.39nM, respectively.
  • RANKL The activity of RANKL to induce the differentiation of mouse monocyte macrophages RAW264.7 and the activity of OPG-TNFR2-Fc to inhibit the differentiation of RANKL-induced cells were tested by the anti-tartrate acid phosphate staining method (TRAP).
  • Raw264.7 cells (cell bank of the Type Culture Collection Committee of the Chinese Academy of Sciences) were cultured in DMEM+10% FBS medium. One day before the experiment, 100 ⁇ L of each well of a 96-well cell culture plate was added containing 5 x 10 3 cells.
  • mice To detect the effect of the fusion protein on the death of mice from septic shock induced by LPS to study the in vivo biological activity of the fusion protein TNFR2. Twenty-four Balb/c mice aged 8-10 weeks were divided into 4 groups, each with 6 mice (male and female). Each mouse was intraperitoneally injected with 1 mg LPS, and then intravenously injected with different doses of TNFR2-OPG-Fc fusion protein and Etanercept (Pfize). The state of the mouse was observed in the following 2 days and the time of death of the mouse was recorded.
  • mice in the control group died within 26 hours.
  • Two groups of mice were injected with TNFR2-OPG-Fc fusion protein 100 ⁇ g/mouse and 150 ⁇ g/mouse, each of which had 2 and 2 survived , And 1 and 2 each survived at 46 hours.
  • the mice injected with TNFR2-Fc (Etanercept) 100 ⁇ g/mouse (a dose equivalent to TNFR2-OPG-Fc 150 ⁇ g/mouse) also died within 26 hours ( Figure 10).
  • CIA mouse arthritis model establishment methods refer to Feige et al., Cell Mol Life Sci, 57: 1457-1470, 2000 and Schett et al., Arthritis Rheum. 52: 1604-1611, 2005.
  • CIA model mice were randomly divided into 4 groups, namely the negative control group (group 1, no immunization, no administration), positive control group (group 2, immunization but no administration), and Etanercept group (group 3, 25mg/kg)
  • the OGP-TNFR2-Fc fusion protein group group 4, 30 mg/kg, molar content of Etanercept equivalent to 25 mg/kg contained 8 mice in each group. Mice in groups 1-3 were immunized twice on D1 and D22, respectively, and then mice in groups 3 and 4 were intraperitoneally injected with the corresponding fusion protein 3 times a week for a total of 11 administrations.
  • mice The clinical symptoms of mice are as follows:
  • clinical joint scoring was started, and the clinical scoring was performed 3 times a week.
  • the clinical scoring criteria are as follows:
  • the degree of infiltration of inflammatory cells in the ankles and vertebrae of each group of mice was evaluated. Serum was collected on D35 and 24 hours after the last dose. At the end point, the right hind paw ankle joint was collected, fixed with 4% paraformaldehyde, and sliced after decalcification for HE staining for histopathological score. The parameters of cell infiltration, bone erosion and cartilage damage were graded separately.
  • Bone mineral density (BMD) evaluation At the end of the experiment, the left posterior bone and vertebrae were soaked and dehydrated with 70% ethanol, and the bone density was measured by micro-CT.
  • TRAP-5b anti-tartrate acid phosphatase-5b
  • Table 2 shows the study of reducing the concentration of TRAP-5b, a marker of bone resorption in the serum of cynomolgus monkeys.

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Abstract

提供了一种融合蛋白及其制法和用途，该蛋白可以： (a)抑制TNFa诱导的细胞凋亡；和/或(b)抑制TRAIL诱导的细胞凋亡；和/或(c)抑制RANKL诱导的单核巨噬细胞的分化；能够(i)预防和/或治疗感染性疾病；和/或(ii)预防和/或治疗自身免疫性疾病；和/或(iii)预防和/或治疗骨质疏松或丢失；和/或(iv)预防和/或***相关疾病。

Description

一种融合蛋白及其制法和用途 技术领域
本发明涉及生物和药物领域，更具体地涉及一种融合蛋白及其制法和用途。
背景技术
自身免疫性疾病是一种免疫介导的疾病，是免疫***对宿主自身抗原发生正性应答、造成组织或器官的病理性损伤、影响其生理功能、并最终导致各种临床症状的状态。类风湿性关节炎(RA)是自身免疫性疾病中最常见、发病率最高的一种，它对滑膜、关节软骨和骨骼造成伤害。RA是由一系列促炎细胞因子通过相互作用而引起并维持的，其中，TNFα是炎症性关节炎患者关键的炎症介导细胞因子。目前有多种抑制TNFa功能的药物(TNFa阻断剂)在临床上治疗各种自身免疫性疾病。
关节炎是慢性软组织炎症，最后通常伴随着关节的破坏。免疫介导的关节炎通常会造成炎症关节内的局灶性骨糜烂，这主要是由于受体激活剂NF-κB配体(RANKL)介导的破骨细胞活性过多而引起。RANKL是骨吸收的重要介质，骨保护素osteoprotegrin(OPG)是RANKL的诱饵受体、能特异性结合RANKL的可溶性蛋白，是RANKL的生理抑制剂。
然而，目前的药物虽然能改善RA患者的炎症，但对有炎症关节的局灶性骨侵蚀和全身性骨丢失没有或很有限的预防和治疗作用。
因此，本领域迫切需要开发既能改善RA患者的炎症，也能防止炎症造成的局灶性骨糜烂和全身性骨丢失的药物。
发明内容
本发明的目的就是提供既能改善RA患者的炎症，也能防止炎症造成的局灶性骨糜烂和全身性骨丢失的药物。
在本发明的第一方面，提供了一种融合蛋白，所述融合蛋白包括融合在一起的以下元件：(a)TNF受体或其活性片段；(b)OPG或其活性片段；和任选的(c)Fc 片段。
在另一优选例中，所述的融合蛋白保留了上述元件(a)和(b)的生物活性。
在另一优选例中，所述TNF受体选自下组：TNFR2、TNFR1、或其组合。
在另一优选例中，所述TNF受体来源于人或非人哺乳动物，更佳地来源于啮齿动物(如小鼠、大鼠)、灵长动物和人。
在另一优选例中，所述TNF受体包括野生型和突变型。
在另一优选例中，TNF受体包括全长的、成熟形式的TNF受体，或其活性片段。
在另一优选例中，所述TNF受体还包括TNF受体的衍生物。
在另一优选例中，所述TNF受体的衍生物包括经修饰的TNF受体、氨基酸序列与天然TNF受体同源且具有天然TNF受体活性的蛋白分子、TNF受体的二聚体或多聚体、含有TNF受体氨基酸序列的融合蛋白。
在另一优选例中，所述经修饰的TNF受体是PEG化的TNF受体。
在另一优选例中，所述“氨基酸序列与天然TNF受体同源且具有天然TNF受体活性的蛋白分子”是指其氨基酸序列与TNF受体相比具有≥85％的同源性，较佳地≥90％的同源性，更佳地≥95％的同源性，最佳地≥98％同源性；并且具有TNF受体活性的蛋白分子。
在另一优选例中，所述TNF受体包括第一结构域、第二结构域、第三结构域、和/或第四结构域。
在另一优选例中，所述第一结构域、第二结构域、第三结构域、和/或第四结构域各自独立地为半胱氨酸富集区(CRD)。
在另一优选例中，所述TNF受体为TNF受体胞外区的第二结构域和第三结构域、或含第二结构域、第三结构域的TNF受体胞外区。
在另一优选例中，所述的TNF受体为含有或具有TNFR2氨基酸序列(SEQ ID NO.:4)的第1－257位，或第23－257位，或第77－162位，或第205－257位。
在另一优选例中，所述OPG来源于人或非人哺乳动物，更佳地来源于啮齿动物(如小鼠、大鼠)、灵长动物和人。
在另一优选例中，所述OPG包括野生型和突变型。
在另一优选例中，所述OPG包括全长的、成熟形式的OPG，或其活性片段。
在另一优选例中，所述OPG还包括OPG的衍生物。
在另一优选例中，所述OPG的衍生物包括经修饰的OPG、氨基酸序列与天然OPG同源且具有天然OPG活性的蛋白分子、OPG的二聚体或多聚体、含有OPG氨基酸序列的融合蛋白。
在另一优选例中，所述经修饰的OPG是PEG化的OPG。
在另一优选例中，所述“氨基酸序列与天然OPG同源且具有天然OPG活性的蛋白分子”是指其氨基酸序列与OPG相比具有≥85％的同源性，较佳地≥90％的同源性，更佳地≥95％的同源性，最佳地≥98％同源性；并且具有OPG活性的蛋白分子。
在另一优选例中，所述OPG包括第一结构域、第二结构域、第三结构域、和/或第四结构域。
在另一优选例中，所述第一结构域、第二结构域、第三结构域、和/或第四结构域各自独立地为半胱氨酸富集区(CRD)。
在另一优选例中，所述OPG为OPG胞外区的第二结构域、第三结构域和第四结构域、或含第二结构域、第三结构域、第四结构域的OPG胞外区。
在另一优选例中，所述OPG含有或具有OPG氨基酸序列(SEQ ID NO.:5)的第1－194位，或第22－194位，或第22－194位。
在另一优选例中，所述Fc片段来源于人或非人哺乳动物，更佳地来源于啮齿动物(如小鼠、大鼠)、灵长动物和人。
在另一优选例中，所述Fc片段为免疫球蛋白IgG的Fc片段，较佳地为IgG1的Fc部分。
在另一优选例中，所述Fc片段包括天然的Fc片段及Fc突变体。
在另一优选例中，所述的Fc片段含有或具有人IgG1氨基酸序列(登录号为UniProtKB-P01857)的第99(Glu)－330(Lys)位。
在另一优选例中，所述Fc片段含有或具有SEQ ID NO.:1的第487-718位。
在另一优选例中，所述Fc片段含有或具有SEQ ID NO.:2的第430-661位。
在另一优选例中，所述Fc片段的氨基酸序列如SEQ ID NO.:3所示。
在另一优选例中，所述的融合蛋白具有下式I或II所示的结构：
X-Y-Z  (I)
Y-X-Z  (II)；
式中，
X为TNF受体或其活性片段；
Y为OPG或其活性片段；
Z为任选的Fc片段；
“-”表示连接上述元件的肽键或肽接头。
在另一优选例中，所述的X、Y、Z中的任何两者以头-头、头-尾、尾-头或尾-尾方式相连。
在另一优选例中，所述的“头部”指多肽或其片段的N端，尤其是野生型多肽的或其片段的N端。
在另一优选例中，所述的“尾部”指多肽或其片段的C端，尤其是野生型多肽的或其片段的C端。
在另一优选例中，所述的肽接头的长度为0-20个氨基酸，较佳地，0-10个氨基酸。
在另一优选例中，所述融合蛋白为同源二聚体。
在另一优选例中，所述融合蛋白选自下组：
(A)具有SEQ ID NO:1或2所示氨基酸序列的多肽；
(B)具有与SEQ ID NO:1或2所示氨基酸序列≥80％同源性(优选地，≥90％的同源性；等优选地≥95％的同源性；最优选地，≥97％的同源性，如98％以上，99％以上)的多肽，且所述多肽具有(a)炎症抑制活性或(b)骨质疏松和/或骨丢失抑制活性；
(C)将SEQ ID NO:1或2所示氨基酸序列经过1-5个氨基酸残基的取代、缺失或添加而形成的，且保留(a)炎症抑制活性或(b)骨质疏松和/或骨丢失抑制活性的衍生多肽。
在另一优选例中，所述融合蛋白的氨基酸序列如SEQ ID NO.:1或2所示。
本发明第二方面提供了一种分离的多核苷酸，所述的多核苷酸编码本发明第一方面所述的融合蛋白。
在另一优选例中，所述的多核苷酸在所述突变蛋白或融合蛋白的ORF的侧翼还额外含有选自下组的辅助元件：信号肽、分泌肽、标签序列(如6His)、或其组合。
在另一优选例中，所述的多核苷酸选自下组：DNA序列、RNA序列、或其组合。
本发明第三方面提供了一种载体，它含有本发明第二方面所述的多核苷酸。
在另一优选例中，所述载体包含一个或多个启动子，所述启动子可操作地与所述核酸序列、增强子、转录终止信号、多腺苷酸化序列、复制起点、选择性标记、核酸限制性位点、和/或同源重组位点连接。
在另一优选例中，所述载体包括质粒、病毒载体。
在另一优选例中，所述的病毒载体选自下组：腺相关病毒(AAV)、腺病毒、慢病毒、逆转录病毒、疱疹病毒、SV40、痘病毒、或其组合。
在另一优选例中，所述载体包括表达载体、穿梭载体、整合载体。
本发明第四方面提供了一种宿主细胞，所示所述宿主细胞含有本发明第三方面所述的载体，或其基因组中整合有本发明第二方面所述的多核苷酸。
在另一优选例中，所述的宿主细胞为真核细胞，如酵母细胞、植物细胞或哺乳动物细胞(包括人和非人哺乳动物)。
在另一优选例中，所述的宿主细胞为原核细胞，如大肠杆菌。
在另一优选例中，所述酵母细胞选自下组的一种或多种来源的酵母：毕氏酵母、克鲁维酵母、或其组合；较佳地，所述的酵母细胞包括：克鲁维酵母，更佳地为马克斯克鲁维酵母、和/或乳酸克鲁维酵母。
在另一优选例中，所述宿主细胞选自下组：大肠杆菌、麦胚细胞，昆虫细胞，SF9、SP2/0、Hela、HEK293、CHO(比如CHOKS)、酵母细胞、或其组合。
本发明第五方面提供了一种产生本发明第一方面所述的融合蛋白的方法，所述的方法包括步骤：
在适合表达的条件下，培养本发明第四方面所述的宿主细胞，从而表达融合蛋白；和/或
分离或纯化所述融合蛋白。
本发明第六方面提供了一种药物组合物，所述的药物组合物含有本发明第一方面所述的融合蛋白及其药物学上可接受的载体。
在另一优选例中，所述药物组合物还包括其他用于抑制炎症活性的药物。
在另一优选例中，其他用于抑制炎症活性的药物选自下组：激素类药物、非甾体类抗炎药、免疫抑制药物、小分子靶向药物、生物制剂、或其组合。
在另一优选例中，所述激素类药物选自下组：氢化可的松、强的松、波尼松龙、***、或其组合。
在另一优选例中，所述非甾体类抗炎药选自下组：阿司匹林、吲哚美辛、萘普生、布洛芬、双氯芬酸、洛索洛芬、美洛昔康、塞来昔布、依托考昔、帕瑞昔布、或其组合。
在另一优选例中，所述免疫抑制药物选自下组：甲氨蝶呤、环磷酰胺、咪唑硫嘌呤、环孢素、霉酚酸酯、他克莫司、西罗莫司、来氟米特、或其组合。
在另一优选例中，所述小分子靶向药物选自下组：托法替布、巴瑞替尼、或其组合。
在另一优选例中，所述生物制剂选自下组：依那西普、赛妥珠单抗、阿达木单抗、戈利木单抗、英夫利昔单抗、托珠单抗、苏金单抗、优特克单抗、卡纳单抗、阿那白滞素、利纳西普、阿巴西普、利妥昔单抗、贝利单抗、或其组合。
本发明第七方面提供了一种本发明第一方面所述的融合蛋白、本发明第二方面所述的多核苷酸、本发明第三方面所述的载体、本发明第四方面所述的宿主细胞的用途，用于制备组合物或制剂，所述组合物或制剂用于(i)预防和/或治疗感染性疾病；和/或(ii)预防和/或治疗自身免疫性疾病；和/或(iii)预防和/或治疗骨质疏松或丢失；和/或(iv)预防和/或***相关疾病。
在另一优选例中，所述感染性疾病包括感染性休克(比如LPS诱导的感染性休克)。
在另一优选例中，所述自身免疫性疾病选自下组：类风湿性关节炎、强直性脊柱炎、或其组合。
在另一优选例中，骨质疏松或丢失选自下组：类风湿性关节炎引起的骨质疏松或/和丢失、妇女绝经后的骨质疏松、或其组合。
在另一优选例中，所述肿瘤相关疾病选自下组：多发性骨髓瘤、骨相关的骨转移实体瘤、或其组合。
在另一优选例中，所述组合物或制剂还用于选自下组的一种或多种用途：
(a)抑制TNFa诱导的细胞凋亡；
(b)抑制TNFa诱导的免疫活化；
(c)抑制TRAIL诱导的细胞凋亡；
(d)抑制RANKL诱导的单核巨噬细胞的分化；
(e)抑制RANKL诱导的破骨细胞形成。
在另一优选例中，所述细胞选自下组：成纤维细胞、单核巨噬细胞、血管内皮细胞、或其组合。
在另一优选例中，所述的组合物为药物组合物。
本发明第八方面提供了一种抑制细胞凋亡的方法，包括步骤：
在本发明第一方面所述的融合蛋白存在下，培养细胞，从而抑制细胞凋亡。
在另一优选例中，所述细胞选自下组：成纤维细胞、血液肿瘤细胞、实体肿 瘤细胞、或其组合。
在另一优选例中，所述血液肿瘤细胞选自下组：B细胞恶性肿瘤细胞、急性髓细胞性白血病细胞、霍奇金淋巴瘤细胞、T细胞恶性肿瘤细胞、多发性骨髓瘤细胞、或其组合。
在另一优选例中，所述实体肿瘤细胞选自下组：乳腺癌细胞、非小细胞肺癌细胞、肝癌细胞、结肠癌细胞、胃癌细胞、或其组合。
在另一优选例中，所述细胞为体外培养的细胞。
在另一优选例中，所述方法为非治疗性和非诊断性的。
在另一优选例中，所述细胞凋亡包括TNFa诱导的细胞凋亡；和/或TRAIL诱导的细胞凋亡。
本发明第九方面提供了一种抑制单核巨噬细胞的分化的方法，包括步骤：
在本发明第一方面所述的融合蛋白存在下，培养单核巨噬细胞，从而抑制单核巨噬细胞的分化。
在另一优选例中，所述单核巨噬细胞为体外培养的细胞。
在另一优选例中，所述方法为非治疗性和非诊断性的。
在另一优选例中，所述单核巨噬细胞的分化为RANKL诱导的单核巨噬细胞的分化。
本发明第十方面提供了一种预防和/或治疗疾病的方法，包括步骤：给需要的对象施用本发明第一方面所述的融合蛋白。
在另一优选例中，所述的融合蛋白以单体和/或二聚体形式施用。
在另一优选例中，所述的对象是人。
在另一优选例中，所述疾病选自下组：感染性疾病、自身免疫性疾病、骨质疏松或丢失、肿瘤相关疾病、或其组合。
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
附图说明
图1是融合蛋白OPG-TNFR2-Fc和TNFR2-OPG-Fc结构示意图。OPG-TNFR2-Fc含有(从N端到C端)：OPG氨基酸序列22-194包括4个CRDs，TNFR2氨基酸 序列23-257包括4个CRDs，以及IgG1 Fc。TNFR2-OPG-Fc含有(从N端到C端)：TNFR2氨基酸序列23-257包括4个CRDs，OPG氨基酸序列22-194包括4个CRDs，TNFR2氨基酸序列203-257，以及IgG1 Fc。
图2显示了还原和非还原变性蛋白胶SDS-PAGE分析融合蛋白。Protein A亲和层析柱纯化的融合蛋白用6％非还原变性SDS-PAGE(A)和10％还原变性SDS-PAGE(B)分析鉴定。1：人IgG1；2：OPG-TNFR2-Fc；3：TNFR2-OPG-Fc。每个样品上样5mg。
图3显示了HPLC-SEC分析融合蛋白。融合蛋白用HPLC-SEC(TSKgel G3000SWXL)层析柱分析。红色曲线代表OPG-TNFR2-Fc；绿色曲线代表TNFR2-OPG-Fc。分子量用蓝色曲线表示。
图4显示了体外结合重组人TNFa的ELISA研究结果。
图5显示了体外结合重组人RANKL的ELISA研究结果。
图6显示了体外结合重组人TRAIL的ELISA研究结果。
图7显示了抑制TNFa诱导的L929细胞凋亡研究结果。
图8显示了抑制Trail诱导的L929细胞凋亡研究结果。
图9显示了抑制RANKL诱导的RAW264.7细胞细胞分化研究结果。
图10显示了抑制LPS诱导的小鼠感染性休克死亡研究结果。
图11显示了(A)：降低CIA诱导小鼠炎症的发生率；(B)降低CIA诱导小鼠发生炎症的程度。
具体实施方式
本发明人经过深入的研究，意外地发现，将(a)TNF受体或其活性片段；(b)OPG或其活性片段；和任选的(c)Fc片段相融合，获得的融合蛋白具有极其优异的生物活性，并且可以非常显著地(a)抑制TNFa诱导的细胞凋亡；和/或(b)抑制TRAIL诱导的细胞凋亡；和/或(c)抑制RANKL诱导的单核巨噬细胞的分化。此外，该融合蛋白且稳定性好、半衰期长，因此有助于(i)预防和/或治疗感染性疾病；和/或(ii)预防和/或治疗自身免疫性疾病；和/或(iii)预防和/或***相关疾病。在此基础上完成了本发明。
如本文所用，除非另外说明，Fc是指人免疫球蛋白的Fc片段。术语“免疫球蛋白Fc区”指免疫球蛋白链恒定区，特别是免疫球蛋白重链恒定区的羧 基端或其中的一部分，例如免疫球蛋白Fc区可包括重链CH1、CH2、CH3的两个或更多结构域与免疫球蛋白铰链区的组合，在优选例中，所用的免疫球蛋白的Fc区包括至少一个免疫球蛋白绞链区，一个CH2结构域和一个CH3结构域，优选缺少CH1结构域。
已知人免疫球蛋白有多种类别，如IgA、IgD、IgE、IgM及IgG(包括IgG1、IgG2、IgG3、IgG4四个亚类)，从特定的免疫球蛋白类别和亚类中选择特定的免疫球蛋白Fc区是在本领域技术人员所掌握的范围之内的，在一个优选的实例中，免疫球蛋白Fc区可选择包含有人免疫球蛋白IgG4亚类Fc区的编码序列，其中缺失一个免疫球蛋白重链1结构域(CH1)，但包括了铰链区以及CH2、CH3、二个结构域的编码序列。
如本文所用，所述的“含有”，“具有”或“包括”包括了“包含”、“主要由……构成”、“基本上由……构成”、和“由……构成”；“主要由……构成”、“基本上由……构成”和“由……构成”属于“含有”、“具有”或“包括”的下位概念。
如本文所用，除非另外说明，所述的融合蛋白是一种分离的蛋白，与其它蛋白、多肽或分子无联系，是重组宿主细胞培养的纯化产物或作为一种纯化的提取物。
TNF受体或其活性片段
TNFR1和2属于TNF受体超级家族成员，是I型跨膜蛋白。TNFR1和TNFR2的分子量分别为55和75kDa，它们的胞外区各自都含有4个半胱氨酸富集区，氨基酸序列有23％一致性。TNFR1广泛表达在各类细胞中，而TNFR2主要表达在造血系细胞中，比如T细胞和自然杀伤细胞，以及内皮细胞、神经细胞、胸腺细胞和间质干细胞等。TNFR1主要结合可溶性的TNFa，而TNFR2结合可溶性的TNFa，也能结合膜蛋白形式的TNFa。
在本发明中，TNF受体包括但并不限于，TNFR2、TNFR1。如本文所用，除非另外说明，TNF-R2，TNFR II，hTNFR II可互换使用，都指人肿瘤坏死因子II型受体。
OPG
OPG(osteoprotegrin)是分泌蛋白，分子量约55-60kDa，具有抑制破骨 细胞生成、调节骨密度功能。OPG属于肿瘤坏死因子受体超级家族(TNFR)成员，成熟的OPG含有4个半胱氨酸富集区、2个死亡结构域和1个heparin结合域。半胱氨酸富集区对OPG与其配体的相互作用至关重要，C末端的cystein介导蛋白形成同源二聚体。OPG广泛和持续表达在间质干细胞、成纤维细胞和内皮细胞。OPG也被称为TNF超级家族配体的诱惑受体，与RANKL和TRAIL结合。TRAIL降低OPG从表达的细胞中释放，而OPG抑制TRAIL诱导的细胞凋亡。OPG能抑制RANKL的促进破骨细胞形成、促进破骨发生功能。人体缺乏OPG造成青少年Paget疾病，当没有足够的OPG平衡RANKL和RANK功能时，会导致骨质疏松和血管钙化。
融合蛋白
如本文所用，“本发明的融合蛋白”、或“多肽”均指本发明第一方面所述的融合蛋白。
在一优选实施方式中，本发明的融合蛋白包含以下元件：(a)TNF受体或其活性片段、(b)OPG或其活性片段、和任选的(c)Fc片段。本发明所述的融合蛋白中，所述的各元件之间(如元件a与元件b、元件b或元件c之间)，可以含有或不含有连接序列。所述的连接序列通常是对两个蛋白不产生影响作用的序列。
在另一优选例中，所述融合蛋白的结构如X-Y-Z(I)或Y-X-Z(II)所示，式中X为TNF受体或其活性片段；Y为OPG或其活性片段；Z为任选的Fc片段。
在另一优选例中，所述融合蛋白具有如SEQ ID NO.:1或2所示的氨基酸序列。
如本文所用，术语“融合蛋白”还包括具有上述活性的融合蛋白(如SEQ ID NO.:1或2所示的序列)的变异形式。这些变异形式包括(但并不限于)：1-3个(通常为1-2个，更佳地1个)氨基酸的缺失、***和/或取代，以及在C末端和/或N末端添加或缺失一个或数个(通常为3个以内，较佳地为2个以内，更佳地为1个以内)氨基酸。例如，在本领域中，用性能相近或相似的氨基酸进行取代时，通常不会改变蛋白质的功能。又比如，在C末端和/或N末端添加或缺失一个或数个氨基酸通常也不会改变蛋白质的结构和功能。此外，所述术语还包括单体和多聚体形式的本发明多肽。该术语还包括线性以及非线性的多肽(如环肽)。
本发明还包括上述融合蛋白的活性片段、衍生物和类似物。如本文所用，术语“片段”、“衍生物”和“类似物”是指基本上保持本发明融合蛋白的功能或活性 的多肽。本发明的多肽片段、衍生物或类似物可以是(i)有一个或几个保守或非保守性氨基酸残基(优选保守性氨基酸残基)被取代的多肽，或(ii)在一个或多个氨基酸残基中具有取代基团的多肽，或(iii)抗原肽与另一个化合物(比如延长多肽半衰期的化合物，例如聚乙二醇)融合所形成的多肽，或(iv)附加的氨基酸序列融合于此多肽序列而形成的多肽(与前导序列、分泌序列或6×His等标签序列融合而形成的融合蛋白)。根据本文的教导，这些片段、衍生物和类似物属于本领域熟练技术人员公知的范围。
一类优选的活性衍生物指与式I或式II的氨基酸序列相比，有至多3个，较佳地至多2个，更佳地至多1个氨基酸被性质相似或相近的氨基酸所替换而形成多肽。这些保守性变异多肽最好根据表A进行氨基酸替换而产生。
表A
最初的残基 代表性的取代 优选的取代
Ala(A) Val；Leu；Ile Val
Arg(R) Lys；Gln；Asn Lys
Asn(N) Gln；His；Lys；Arg Gln
Asp(D) Glu Glu
Cys(C) Ser Ser
Gln(Q) Asn Asn
Glu(E) Asp Asp
Gly(G) Pro；Ala Ala
His(H) Asn；Gln；Lys；Arg Arg
Ile(I) Leu；Val；Met；Ala；Phe Leu
Leu(L) Ile；Val；Met；Ala；Phe Ile
Lys(K) Arg；Gln；Asn Arg
Met(M) Leu；Phe；Ile Leu
Phe(F) Leu；Val；Ile；Ala；Tyr Leu
Pro(P) Ala Ala
Ser(S) Thr Thr
Thr(T) Ser Ser
Trp(W) Tyr；Phe Tyr
Tyr(Y) Trp；Phe；Thr；Ser Phe
Val(V) Ile；Leu；Met；Phe；Ala Leu
本发明还提供本发明融合蛋白的类似物。这些类似物与SEQ ID NO.:1或SEQ ID NO.:2所示的多肽的差别可以是氨基酸序列上的差异，也可以是不影响序列的修饰形式上的差异，或者兼而有之。类似物还包括具有不同于天然L-氨基酸的残基(如D-氨基酸)的类似物，以及具有非天然存在的或合成的氨基酸(如β、γ-氨基酸) 的类似物。应理解，本发明的多肽并不限于上述例举的代表性的多肽。
修饰(通常不改变一级结构)形式包括：体内或体外的多肽的化学衍生形式如乙酰化或羧基化。修饰还包括糖基化，如那些在多肽的合成和加工中或进一步加工步骤中进行糖基化修饰而产生的多肽。这种修饰可以通过将多肽暴露于进行糖基化的酶(如哺乳动物的糖基化酶或去糖基化酶)而完成。修饰形式还包括具有磷酸化氨基酸残基(如磷酸酪氨酸，磷酸丝氨酸，磷酸苏氨酸)的序列。还包括被修饰从而提高了其抗蛋白水解性能或优化了溶解性能的多肽。
表达载体和宿主细胞
本发明也涉及包含本发明的多核苷酸的载体，以及用本发明的载体或本发明融合蛋白编码序列经基因工程产生的宿主细胞，以及经重组技术产生本发明所述多肽的方法。
通过常规的重组DNA技术，可利用本发明的多聚核苷酸序列可用来表达或生产重组的融合蛋白。一般来说有以下步骤：
(1).用本发明的编码本发明融合蛋白的多核苷酸(或变异体)，或用含有该多核苷酸的重组表达载体转化或转导合适的宿主细胞；
(2).在合适的培养基中培养的宿主细胞；
(3).从培养基或细胞中分离、纯化蛋白质。
本发明中，编码融合蛋白的多核苷酸序列可***到重组表达载体中。术语“重组表达载体”指本领域熟知的细菌质粒、噬菌体、酵母质粒、植物细胞病毒、哺乳动物细胞病毒如腺病毒、逆转录病毒或其他载体。只要能在宿主体内复制和稳定，任何质粒和载体都可以用。表达载体的一个重要特征是通常含有复制起点、启动子、标记基因和翻译控制元件。
本领域的技术人员熟知的方法能用于构建含本发明融合蛋白编码DNA序列和合适的转录/翻译控制信号的表达载体。这些方法包括体外重组DNA技术、DNA合成技术、体内重组技术等。所述的DNA序列可有效连接到表达载体中的适当启动子上，以指导mRNA合成。这些启动子的代表性例子有：大肠杆菌的lac或trp启动子；λ噬菌体PL启动子；真核启动子包括CMV立即早期启动子、HSV胸苷激酶启动子、早期和晚期SV40启动子、反转录病毒的LTRs和其他一些已知的可控制基因在原核或真核细胞或其病毒中表达的启动子。表达载体还包括翻译起始用的核糖体结合位点和转录终止子。
此外，表达载体优选地包含一个或多个选择性标记基因，以提供用于选择转化的宿主细胞的表型性状，如真核细胞培养用的二氢叶酸还原酶、新霉素抗性以及绿色荧光蛋白(GFP)，或用于大肠杆菌的四环素或氨苄青霉素抗性。
包含上述的适当DNA序列以及适当启动子或者控制序列的载体，可以用于转化适当的宿主细胞，以使其能够表达蛋白质。
宿主细胞可以是原核细胞(如大肠杆菌)，或是低等真核细胞，或是高等真核细胞，如酵母细胞、植物细胞或哺乳动物细胞(包括人和非人哺乳动物)。代表性例子有：大肠杆菌、麦胚细胞，昆虫细胞，SF9、Hela、HEK293、CHO、酵母细胞等。在本发明的一个优选实施方式中，选择酵母细胞(如毕氏酵母、克鲁维酵母、或其组合；较佳地，所述的酵母细胞包括：克鲁维酵母，更佳地为马克斯克鲁维酵母、和/或乳酸克鲁维酵母)为宿主细胞。
本发明的多核苷酸在高等真核细胞中表达时，如果在载体中***增强子序列时将会使转录得到增强。增强子是DNA的顺式作用因子，通常大约有10到300个碱基对，作用于启动子以增强基因的转录。可举的例子包括在复制起始点晚期一侧的100到270个碱基对的SV40增强子、在复制起始点晚期一侧的多瘤增强子以及腺病毒增强子等。
本领域一般技术人员都清楚如何选择适当的载体、启动子、增强子和宿主细胞。
用重组DNA转化宿主细胞可用本领域技术人员熟知的常规技术进行。当宿主为原核生物如大肠杆菌时，能吸收DNA的感受态细胞可在指数生长期后收获，用CaCl 2法处理，所用的步骤在本领域众所周知。另一种方法是使用MgCl 2。如果需要，转化也可用电穿孔的方法进行。当宿主是真核生物，可选用如下的DNA转染方法：磷酸钙共沉淀法，常规机械方法如显微注射、电穿孔、脂质体包装等。
获得的转化子可以用常规方法培养，表达本发明的基因所编码的多肽。根据所用的宿主细胞，培养中所用的培养基可选自各种常规培养基。在适于宿主细胞生长的条件下进行培养。当宿主细胞生长到适当的细胞密度后，用合适的方法(如温度转换或化学诱导)诱导选择的启动子，将细胞再培养一段时间。
在上面的方法中的重组多肽可在细胞内、或在细胞膜上表达、或分泌到细胞外。如果需要，可利用其物理的、化学的和其它特性通过各种分离方法分离和纯化重组的蛋白。这些方法是本领域技术人员所熟知的。这些方法的例子包 括但并不限于：常规的复性处理、用蛋白沉淀剂处理(盐析方法)、离心、渗透破菌、超处理、超离心、分子筛层析(凝胶过滤)、吸附层析、离子交换层析、高效液相层析(HPLC)和其它各种液相层析技术及这些方法的结合。
肽接头
本发明提供了一种融合蛋白，它可任选地含有肽接头。肽接头大小和复杂性可能会影响蛋白的活性。通常，肽接头应当具有足够的长度和柔韧性，以保证连接的两个蛋白在空间上有足够的自由度以发挥其功能。同时避免肽接头中形成α螺旋或β折叠等对融合蛋白的稳定性的影响。
连接肽的长度一般为0-20个氨基酸，较佳地0-10个氨基酸。
药物组合物
本发明还提供了一种药物组合物。在优选例中，所述的组合物是药物组合物，它含有上述的融合蛋白，以及药学上可接受的载体、稀释剂、稳定剂和/或增稠剂，并可制备成如冻干粉、片剂、胶囊、糖浆、溶液或悬浮液的药剂类型。
“药学上可接受的载体或赋形剂(excipient)”指的是：一种或多种相容性固体或液体填料或凝胶物质，它们适合于人使用，而且必须有足够的纯度和足够低的毒性。“相容性”在此指的是组合物中各组份能和本发明的活性成分以及它们之间相互掺和，而不明显降低活性成分的药效。
组合物可以是液体或固体，例如粉末、凝胶或糊剂。优选地，组合物是液体，优选地可注射液体。合适的赋形剂将是本领域技术人员己知的。
药学上可以接受的载体部分例子有纤维素及其衍生物(如羧甲基纤维素钠、乙基纤维素钠、纤维素乙酸酯等)、明胶、滑石、固体润滑剂(如硬脂酸、硬脂酸镁)、硫酸钙、植物油(如豆油、芝麻油、花生油、橄榄油等)、多元醇(如丙二醇、甘油、甘露醇、山梨醇等)、乳化剂(如吐温
Figure PCTCN2021071544-appb-000001
)、润湿剂(如十二烷基硫酸钠)、着色剂、调味剂、稳定剂、抗氧化剂、防腐剂、无热原水等。
组合物可包含生理上可接受的无菌含水或无水溶液、分散液、悬浮液或乳液，和用于重新溶解成无菌的可注射溶液或分散液的无菌粉末。适宜的含水和非水载体、稀释剂、溶剂或赋形剂包括水、乙醇、多元醇及其适宜的混合物。
通常，可将这些物质配制于无毒的、惰性的和药学上可接受的水性载体介质中，其中pH通常约为5-8，较佳地pH约为6-8，尽管pH值可随被配制物质的性质 以及待治疗的病症而有所变化。配制好的药物组合物可以通过常规途径进行给药，其中包括(但并不限于)：腹膜内、静脉内、或局部给药。所述药物组合物用于(i)预防和/或治疗感染性疾病；和/或(ii)预防和/或治疗自身免疫性疾病；和/或(iii)预防和/或治疗骨质疏松或丢失；和/或(iv)预防和/或***相关疾病；和/或(v)抑制TNFa诱导的细胞凋亡；和/或(vi)抑制TRAIL诱导的细胞凋亡；和/或(vii)抑制RANKL诱导的单核巨噬细胞的分化；和/或(viii)抑制TNFa诱导的免疫活化；和/或(ix)抑制RANKL诱导的破骨细胞形成。
本发明的主要优点包括：
(a)本发明融合蛋白的半衰期长。
(b)本发明的融合蛋白既能改善RA患者的炎症，也能防止炎症造成的局灶性骨糜烂和全身性骨丢失。
(c)本发明首次发现，本发明的融合蛋白可显著(i)预防和/或治疗感染性疾病；和/或(ii)预防和/或治疗自身免疫性疾病；和/或(iii)抑制TNFa诱导的细胞凋亡；和/或(iv)抑制TRAIL诱导的细胞凋亡；和/或(v)抑制RANKL诱导的单核巨噬细胞的分化；和/或(vi)抑制自身免疫性疾病引起的骨质疏松和丢失；和/或(vii)抑制骨转移瘤。
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件，例如Sambrook等人，分子克隆：实验室手册(New York:Cold Spring Harbor Laboratory Press,1989)中所述的条件，或按照制造厂商所建议的条件。除非另外说明，否则百分比和份数是重量百分比和重量份数。
除非有特别说明，否则本发明实施例中的试剂和材料均为市售产品。
实施例1 TNFR2和OPG的IgG Fc融合蛋白表达质粒的构建
1.TNFR2-OPG-Fc融合蛋白(以下简称OTFc)表达质粒的构建
人OPG基因序列来自人OPG全长cDNA(Open Biosystems，MHS1011-7509022)，人TNFR2和人IgG Fc序列来自人TNFR2(1-257)-Fc融合蛋白基因。已经构建。
为构建OPG-TNFR2-Fc融合蛋白表达载体，先用聚合酶链式反应技术(PCR)方法分别以人OPG全长cDNA为模板合成编码OPG氨基酸1-194的基因(5’和 3’端引物分别为引物-1和引物-2，引物序列见表1，下同)和以人TNFR2(1-257)-Fc融合蛋白基因为模板合成编码人TNFR2-Fc融合蛋白的基因(不含蛋白信号台编码序列1-22，5’和3’引物分别为引物-3和引物-4)，然后用重叠延伸PCR方法把上述2个PCR片段连接，合成编码OPG(1-194)-TNFR2(23-257)-Fc融合蛋白的完整基因(5’和3’端引物分别为引物-1和引物-4)。引物-2的头23个核苷酸序列与引物-3的核苷酸序列互补，这样在第二步的重叠延伸PCR过程中，可以把这2个PCR片段连接起来。引物-1含有限制性内切酶Not I序列，引物-4含有限制性内切酶Xba I序列，用于***蛋白表达载体。合成的PCR片段用琼脂糖凝胶电泳分离，用DNA胶纯化试剂盒(QIAGEN)纯化。重叠延伸PCR合成的OPG-TNFR2-Fc融合蛋白基因首先用T/A载体克隆试剂盒克隆到pCR2.1载体(Invitrogen)，然后用Not I和Xba I限制性内切酶从此载体切下融合蛋白基因，***到同样被用Not I和Xba I酶切的哺乳细胞表达质粒中，比如pcDNA3.1(Invitrogen)。***在pCR2.1里的OPG(1-194)-TNFR2(23-257)-Fc DNA序列用DNA测序确认。
2.TNFR2(1-257)-OPG(22-194)-TNFR2(202-257)-Fc(以下简称TOFc)融合蛋白表达质粒的构建
为构建TNFR2(1-257)-OPG(22-194)-TNFR2(202-257)-Fc(以下简称TNFR2-OPG-Fc)融合蛋白表达载体，先用聚合酶链式反应技术(PCR)方法分别合成编码TNFR2氨基酸1-257基因(5’和3’端引物分别为引物-5和引物-6)、编码OPG氨基酸22-194的基因(不含编码蛋白信号肽1-21基因，5’和3’端引物分别为引物-7和引物-8)和编码Fc基因(含TNFR2氨基酸202-257，5’和3’端引物分别为引物-9和引物-4)，然后用重叠延伸PCR方法分别把上述第1和第2个PCR片段连接(5’和3’端引物分别为引物-5和引物-8)，第2和第3个PCR片段连接(5’和3’端引物分别为引物-7和引物-4)，再用重叠延伸PCR方法把这2个PCR片段连接(5’和3’端引物分别为引物-5和引物-4)，合成编码TNFR2(1-257)-OPG(22-194)-TNFR2(202-257)-Fc融合蛋白OPG-TNFR2-Fc融合蛋白的全长基因。合成的融合蛋白基因首先用T/A载体克隆试剂盒克隆到pCR-Blunt II-TOPO载体(Invitrogen)，然后用Not I和Xba I限制性内切酶从此载体切下融合蛋白基因，***到同样被用Not I和Xba I酶切的哺乳细胞表达质粒中，比如pcDNA3.1(Invitrogen)。***在pCR-Blunt II-TOPO载体里的TNFR2(1-257)-OPG(22-194)-TNFR2(202-257) -Fc DNA序列用DNA测序确认。
上述构建的2个融合蛋白的结构示意图如图1所示。
表1
Figure PCTCN2021071544-appb-000002
实施例2构建融合蛋白稳定表达细胞株
用于构建稳定表达融合蛋白细胞的宿主细胞为中国仓鼠卵巢细胞CHO-KS。CHO-KS是生长在含胎牛血清(FBS)培养基里的CHO-K1细胞经过逐渐降低培养基中FBS含量的培养直至无FBS培养基培养，最终驯化成在不含FBS的OptiCHO培养基(Invitrogen)中悬浮生长的细胞。含有融合蛋白基因的pcDNA3.1载体中的抗新霉素基因用大鼠谷氨酰胺合成酶基因取代，采用电转染(Bio-Rad，Gene Pulser Xcell)的方法把此融合蛋白表达载体转染进CHO-KS细胞，转染 的细胞在培养24个小时后，用有限稀释法在96孔培养板上对转染的细胞进行筛选培养。筛选培养基是OptiCHO，5μg/ml重组人胰岛素和10μM氨基亚砜蛋氨酸(MSX)。在37℃，8％CO 2的培养箱里培养细胞。3个星期后，用ELISA方法(碱性磷酸酶偶联的羊抗人IgG Fc抗体，Jackson ImmunoResearch Lab)对每个长有细胞群的孔的细胞培养液进行分析，把融合蛋白表达阳性的细胞群进一步扩增，再ELISA检测，再扩增，最后得到融合蛋白表达稳定细胞群。
实施例3融合蛋白的表达和纯化
将实施例2中构建的融合蛋白细胞株，培养扩增至2升。收获培养液上清，用Protein-A亲和层析柱(POROS MabCapture A，Life Tech)纯化融合蛋白。
纯化的融合蛋白用还原和非还原SDS-PAGE电泳分析和HPLC-SEC(高压液相-分子筛)(TSKgel G3000SWXL，TOSOH Bioscience)分析。
结果与分析
OPG-TNFR2-Fc融合蛋白是同源二聚体，理论分子量约为142kDa。TNFR2-OPG-Fc融合蛋白为同源二聚体，理论分子量约为153kDa。非还原SDS-PAGE电泳胶(图2A)显示这2个同源二聚体融合蛋白的分子量大于人免疫球蛋白IgG1，与各自的理论值相近。图2B显示了融合蛋白单链的还原SDS-PAGE电泳胶结果。
OPG-TNFR2-Fc融合蛋白和TNFR2-OPG-Fc融合蛋白的HPLC-SEC(分子筛层析柱)分析结果见图3。该结果显示融合蛋白峰位于600kDa附近。OPG和TNFR2各自有4个半胱氨酸富集区域(CRD)，8个CRDs连在一起，使融合蛋白分子形成棒状结构，在分子筛层析柱分析中表现出大于紧密结构的标准蛋白的分子量。
实施例4与TNFa体外结合研究
融合蛋白与TNFa的体外专一性结合活性用ELISA方法研究。将重组人TNFa(rhTNFa，SinoBiological)溶解在PBS(pH7.0)溶液里，加入96-孔ELISA板，于4℃冰箱里过夜。第二天，用PBST(PBS含0.05％Tween-20)洗去游离的rhTNFa，加入含3％BSA的PBST封闭溶液。封闭的ELISA板加入系列稀释的不同浓度的融合蛋白，与TNFa结合的融合蛋白用碱性磷酸酶偶联的羊抗人IgG Fc抗体(Jackson ImmunoResearch Lab)检测，加入底物(PNPP)显色， 用酶标仪在波长405nm/655nm下读板。
结果与分析
提供了OPG-TNFR2-Fc融合蛋白和TNFR2-OPG-Fc融合蛋白与rhTNFa的体外结合ELISA研究结果见图4。结果显示，这2个融合蛋白均能与rhTNFa特异性结合，EC 50分别为33ng/ml和68ng/ml。
实施例5与RANKL体外结合
融合蛋白与RANKL的体外专一性结合活性用ELISA方法研究。将重组人RANKL-鼠源Fc融合蛋白(Sino Biological Inc.)溶解在PBS(pH7.0)溶液里至1μg/mL，往96-孔ELISA板里每孔加入50μL，于4℃冰箱里过夜。第二天，用PBST(PBS含0.05％Tween-20)清洗3次，每孔加入200μL，加入含3％BSA的PBST封闭溶液。倒空封闭溶液，ELISA板中加入系列稀释的以下3种融合蛋白，OPG-TNFR2-Fc、TNFR2-OPG-Fc和OPG-Fc(Sino Biological Inc.，作为对照样品)。与RANKL结合的融合蛋白用碱性磷酸酶偶联的羊抗人IgG Fc抗体(Jackson ImmunoResearch Lab)检测，加入底物(PNPP)显色，用酶标仪在双波长405nm/490nm下读板。
结果与分析
实验结果显示(图5)，这3个融合蛋白均能与人RANKL专一性结合，但OPG-TNFR2-Fc和TNFR2-OPG-Fc结合RANKL的活性强于OPG-Fc，它们的结合活性EC 50分别为9.9ng/mL、9.4ng/mL和40ng/mL。OPG-TNFR2-Fc和TNFR2-OPG-Fc与RANKL的结合活性没有显著差别。
实施例6与TRAIL体外结合
融合蛋白与TRAIL的体外专一性结合活性用ELISA方法研究。将OPG-TNFR2-Fc、TNFR2-OPG-Fc和OPG-Fc(Sino Biological Inc.)融合蛋白分别用PBS稀释至1μg/mL，在96-孔ELISA板每孔加入50μL，于4℃冰箱里过夜。第二天，用PBST(PBS含0.05％Tween-20)清洗3次，每孔加入200μL含3％BSA的PBST封闭溶液。倒空封闭溶液，往封闭的ELISA板中加入系列稀释的rhTRAIL(Histidine tag，R&D Systems)，与融合蛋白结合的rhTRAIL用辣根过氧化物酶(HRP)偶联的鼠抗Histidine抗体(Sino Biological Inc.)检测， 加入底物(TMB)显色，用酶标仪在双波长405nm/490nm下读板。
结果与分析
实验结果显示(图6)，rhTRAIL均能与3个融合蛋白专一性结合，虽然OPG-TNFR2-Fc和TNFR2-OPG-Fc结合TRAIL的活性弱于OPG-Fc，它们的结合活性EC 50分别为43ng/mL、78ng/mL和14ng/mL。OPG-TNFR2-Fc结合TRAIL的活性稍微强于TNFR2-OPG-Fc。
实施例7 TNFR2体外生物活性研究
融合蛋白的TNFR2生物学活性采用在体外中和TNFa的生物活性检测方法。TNFa生物活性用小鼠成纤维细胞L929细胞毒性检测。rhTNFa与系列稀释不同浓度的融合蛋白，OPG-TNFR2-Fc、TNFR2-OPG-Fc和TNFR2(Etanercept，Biogen Inc，作为对照样品)，混合后到L929细胞，在细胞培养箱里培养20小时后用结晶子染色方法检测L929细胞活率。
结果与分析
OPG-TNFR2-Fc融合蛋白和TNFR2-OPG-Fc融合蛋白均能抑制rhTNFa诱导的L929细胞凋亡(图7)。OPG-TNFR2-Fc抑制TNFa诱导的L929细胞凋亡活性(EC 50＝0.11nM)强于Etanercept(EC 50＝0.19nM),也强于TNFR2-OPG-Fc(EC 50＝0.35nM)。
实施例8 OPG体外生物活性研究
1.抑制TRAIL诱导的L929细胞凋亡
重组人TRAIL(R&D Systems Inc.)与系列稀释不同浓度的融合蛋白混合加入到L929细胞，在细胞培养箱里培养20小时后用结晶子染色方法检测L929细胞活率。重组人OPG-Fc融合蛋白(Novo Protein)
结果与分析
TNFR2-OPG-Fc融合蛋白抑制rhTRAIL诱导L929细胞凋亡的能力显著强于OPG-Fc和OPG-TNFR2-Fc(图8)。TNFR2-OPG-Fc融合蛋白抑制rhTRAIL诱导L929细胞凋亡的活性IC50为0.02nM，OPG-Fc和OPG-TNFR2-Fc的抑制活性相当，IC50分别为0.27nM和0.39nM。
2.抑制RANKL诱导小鼠单核巨噬细胞RAW264.7分化
RANKL诱导小鼠单核巨噬细胞RAW264.7分化的活性和OPG-TNFR2-Fc抑制RANKL诱导的细胞分化活性采用抗酒石酸酸性磷酸染色方法(TRAP)检测。Raw264.7细胞(中国科学院典型培养物保藏委员会细胞库)培养在DMEM+10％FBS培养基里，实验前一天，在96-孔细胞培养板里每孔加入100μL含有5 x 10 3个细胞。培养细胞24小时后倒空上清，每孔加入200μL-MEM培养基+10％FBS含有20ng/mL RANKL和不同浓度的OPG-TNFR2-Fc融合蛋白。培养细胞2天后倒空上清，加入新鲜配制的同样浓度的RANKL和相应浓度的OPG-TNFR2-Fc，在继续培养细胞3天。倒空上清，用PBS溶液洗细胞2次，然后每孔加入100μL的1％triton X-100溶液(PBS稀释)裂解细胞，高速离心后取上清，用TRAP检测试剂盒(碧云天生物技术有限公司)检测细胞裂解液中TRAP的含量。
结果与分析
实验结果显示(图9)，随着OPG-TNFR2-Fc融合蛋白的浓度增加，RANKL诱导的RAW264.7细胞产生的酸性磷酸酶的含量随着降低，表明OPG-TNFR2-Fc融合蛋白能抑制RANKL诱导的小鼠单核巨噬细胞RAW264.7分化的功能。
实施例9小鼠模型研究
1.抑制LPS诱导的小鼠感染性休克死亡研究
检测融合蛋白对LPS诱导的小鼠感染性休克死亡的影响研究融合蛋白TNFR2的体内生物活性。24只8-10周龄Balb/c小鼠分成4组，每组6只(雌雄各半)。每只小鼠腹腔注射1mg LPS，随即静脉注射不同剂量的TNFR2-OPG-Fc融合蛋白和Etanercept(Pfize)，在随后的2天内观察小鼠状态，记录小鼠死亡的时间。
结果与分析
在注射致死剂量1mg LPS/只后，对照组小鼠在26小时内全部死亡，注射TNFR2-OPG-Fc融合蛋白100μg/只和150μg/只的2组小鼠，各有2只和2只存活，在46小时仍各有1只和2只存活。而注射TNFR2-Fc(Etanercept)100μg/只(剂量相当于TNFR2-OPG-Fc 150μg/只)的小鼠在26小时内也全部死亡(图10)。
2.CIA诱导的小鼠关节炎模型
CIA小鼠关节炎模型建立方法分别参考文献Feige et al.,Cell Mol Life Sci,57:1457-1470,2000和Schett et al.,Arthritis Rheum.52:1604-1611,2005。CIA模型小鼠随机分成4组，分别是阴性对照组(组1，不免疫、不给药)、阳性对照组(组2，免疫但不给药)、Etanercept组(组3，25mg/kg)和OGP-TNFR2-Fc融合蛋白组(组4，30mg/kg，摩尔含量相当于25mg/kg的Etanercept)每组含8只小鼠。组1-3的小鼠在D1和D22分别免疫2次，随后组3和组4的小鼠腹腔注射相应的融合蛋白，每周3次，共给药11次。
小鼠临床症状检测如下：
在D22动物第2次免疫结束，开始进行临床关节评分，临床评分每周3次。临床评分标准如下：
Figure PCTCN2021071544-appb-000003
评估各组小鼠踝和椎骨被炎症细胞浸润程度。分别在D35和最后1次给药后24小时采集血清。在终点采集右后爪踝关节，4％多聚甲醛固定，脱钙后切片进行HE染色，进行组织病理学评分。对于细胞浸润、骨侵蚀和软骨损伤的参数，分别分级。
骨密度(BMD)评价。在实验结束时，取左后骨和椎骨用70％乙醇浸泡脱水，用micro-CT检测骨密度。
结果与分析
本实验结果显示，在实验结束时，阳性对照组所有小鼠的爪子都表现出关节炎症状，而OPG-TNFR2-Fc和etanercept组小鼠的炎症发生率在50-60％之间(图11A)。对小鼠爪炎症的临床评分结果显示，OPG-TNFR2-Fc组小鼠爪的平均炎症程度显著低于阳性对照组的小鼠，与etanercept组小鼠表现出的炎症程度相当(图 11B)。本实验结果证明，OPG-TNFR2-Fc融合蛋白有较好的保护和降低CIA诱导的小鼠关节炎程度的功能。
实施例10 OPG-TNFR2-Fc融合蛋白对食蟹猴骨质吸收的影响研究
通过研究OPG-TNFR2-Fc融合蛋白对食蟹猴血清中抗酒石酸酸性磷酸酶-5b(TRAP-5b)浓度的影响来研究融合蛋白对食蟹猴骨质吸收的影响。TRAP-5b是骨质吸收过程中的代谢产物，被作为体现骨质吸收的生物标志物。6只雄食蟹猴(3～5周岁)分成3组，其中1组静脉给予15mg/kg的OPG-TNFR2-Fc融合蛋白，另2组分别皮下注射5mg/kg和15mg/kg的OPG-TNFR2-Fc融合蛋白。在以下时间取血保留血清，给药前、给药后24小时、48小时和168小时。用TRAP-5b检测试剂盒(上海蓝基生物科技有限公司)检测血清中TRAP-5b的浓度
结果与分析
表2显示了降低食蟹猴血清中骨质吸收标志物TRAP-5b浓度的研究。
表2
Figure PCTCN2021071544-appb-000004
研究结果显示(表2)，在给予OPG-TNFR2-Fc融合蛋白后24小时，所有食蟹猴血清中TRAP-5b的浓度均降低，降低程度从11％到40％不等。在皮下给药组里，TRAP-5b浓度的降低程度与给药剂量成正比。在给药后24、48和168小时后，所有食蟹猴血清TRAP-5b的平均浓度分别下降24％、11％和8％。本研究表明，OPG-TNFR2-Fc融合蛋白能抑制食蟹猴骨质的吸收。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (10)

  1. 一种融合蛋白，其特征在于，所述融合蛋白包括融合在一起的以下元件：(a)TNF受体或其活性片段；(b)OPG或其活性片段；和任选的(c)Fc片段。
  2. 如权利要求1所述的融合蛋白，其特征在于，所述TNF受体选自下组：TNFR2、TNFR1、或其组合。
  3. 一种分离的多核苷酸，其特征在于，所述的多核苷酸编码权利要求1所述的融合蛋白。
  4. 一种载体，其特征在于，它含有权利要求3所述的多核苷酸。
  5. 一种宿主细胞，其特征在于，所示所述宿主细胞含有权利要求4所述的载体，或其基因组中整合有权利要求3所述的多核苷酸。
  6. 一种产生权利要求1所述的融合蛋白的方法，其特征在于，所述的方法包括步骤：
    在适合表达的条件下，培养权利要求5所述的宿主细胞，从而表达融合蛋白；和/或
    分离或纯化所述融合蛋白。
  7. 一种药物组合物，其特征在于，所述的药物组合物含有权利要求1所述的融合蛋白及其药物学上可接受的载体。
  8. 一种权利要求1所述的融合蛋白、权利要求3所述的多核苷酸、权利要求4所述的载体、权利要求5所述的宿主细胞的用途，其特征在于，用于制备组合物或制剂，所述组合物或制剂用于(i)预防和/或治疗感染性疾病；和/或(ii)预防和/或治疗自身免疫性疾病；和/或(iii)预防和/或治疗骨质疏松或丢失；和/或(iv)预防和/或***相关疾病。
  9. 一种抑制细胞凋亡的方法，其特征在于，包括步骤：
    在权利要求1所述的融合蛋白存在下，培养细胞，从而抑制细胞凋亡。
  10. 一种抑制单核巨噬细胞的分化的方法，其特征在于，包括步骤：
    在权利要求1所述的融合蛋白存在下，培养单核巨噬细胞，从而抑制单核巨噬细胞的分化。
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