WO2015113494A1 - 双功能融合蛋白及其制备方法和用途 - Google Patents

双功能融合蛋白及其制备方法和用途 Download PDF

Info

Publication number
WO2015113494A1
WO2015113494A1 PCT/CN2015/071718 CN2015071718W WO2015113494A1 WO 2015113494 A1 WO2015113494 A1 WO 2015113494A1 CN 2015071718 W CN2015071718 W CN 2015071718W WO 2015113494 A1 WO2015113494 A1 WO 2015113494A1
Authority
WO
WIPO (PCT)
Prior art keywords
seq
fusion protein
bifunctional fusion
amino acid
sequence
Prior art date
Application number
PCT/CN2015/071718
Other languages
English (en)
French (fr)
Inventor
陈国强
刘家望
宋楠萌
杨亚平
车美英
Original Assignee
北京韩美药品有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 北京韩美药品有限公司 filed Critical 北京韩美药品有限公司
Priority to EP15742538.0A priority Critical patent/EP3101035B1/en
Priority to KR1020167023486A priority patent/KR20160113268A/ko
Priority to US15/114,006 priority patent/US20160340422A1/en
Priority to JP2016548092A priority patent/JP2017508446A/ja
Priority to CN201580004917.1A priority patent/CN105916883B/zh
Publication of WO2015113494A1 publication Critical patent/WO2015113494A1/zh

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/24—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against cytokines, lymphokines or interferons
    • C07K16/244—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
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/7051—T-cell receptor (TcR)-CD3 complex
    • 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
    • A61K38/177—Receptors; Cell surface antigens; Cell surface determinants
    • A61K38/1774—Immunoglobulin superfamily (e.g. CD2, CD4, CD8, ICAM molecules, B7 molecules, Fc-receptors, MHC-molecules)
    • 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
    • A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
    • A61K39/3955—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against proteinaceous materials, e.g. enzymes, hormones, lymphokines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/04—Drugs for disorders of the alimentary tract or the digestive system for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • A61P13/12—Drugs for disorders of the urinary system of the kidneys
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/06—Antipsoriatics
    • 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
    • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
    • 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
    • A61P19/06—Antigout agents, e.g. antihyperuricemic or uricosuric agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • 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
    • A61P3/00—Drugs for disorders of the metabolism
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/04—Antihaemorrhagics; Procoagulants; Haemostatic agents; Antifibrinolytic 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
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/70521—CD28, CD152
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/24—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against cytokines, lymphokines or interferons
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • the present invention relates to a bifunctional fusion protein, a preparation method thereof and use thereof.
  • the present invention relates to a bifunctional recombinant fusion protein capable of simultaneously blocking the B7/CD28 and IL-17A/IL-17RA signaling pathways, a gene encoding the bifunctional fusion protein, a vector containing the gene, and a host containing the vector Cells and pharmaceutical compositions containing the bifunctional fusion protein.
  • Cytotoxic T lymphocyte-associated antigen 4 also known as CD152, is a leukocyte differentiation antigen.
  • the ligands of CTLA4 and CD28 are B7 molecules, and CTLA4 binds to B7 molecule to induce T cell non-reactivity and participate in the negative regulation of immune response.
  • Abatacept (trade name OrenciaTM, Bristol-Myers Squibb ) , which was marketed in 2005 for the treatment of rheumatoid arthritis, is a monofunctional fusion protein consisting of two extracellular regions of CTLA4 , ECD) binds to the Fc segment of human IgG1 and inhibits T cell proliferation and activation by competitively blocking the binding of B7 to CD28 (Genovese MC, Becker JC, Schiff M, et al. N. Engl. J .Med., 2005, 353: 1114-1123).
  • Interleukin-17A is a pro-inflammatory cytokine produced primarily from Th17 cells and is the most representative member of the IL-17 family (Miossec P, Kolls JK. Nat. Rev .Drug.Discov., 2012, 11: 763-776).
  • IL-17A binds to its receptor (IL-17RA)
  • IL-17RA receptor
  • IL-17RA IL-17RA
  • IL-17RA IL-17A
  • IL-17A binds to its receptor
  • IL-17RA When IL-17A binds to its receptor (IL-17RA), it induces the expression of inflammatory cytokines and chemokines in a variety of cells (such as fibroblasts, epithelial cells, and endothelial cells) and plays an important role in immune defense.
  • Lin Y, Ritchea S, Logar A. Immunity, 2009, 31: 799-810) can cause inflammatory diseases.
  • IL-17A neutralizing antibodies Chobaud M, Durand JM, Buchs N
  • Animal models of many autoimmune diseases have demonstrated that the use of antibodies to neutralize IL-17A can effectively inhibit the pathological development of inflammation (Lubberts E, Koenders MI, Oppers-Walgreen B, et al. Arthritis Rheum., 2004, 50: 650- 659).
  • IL-17A related antibody drugs have been approved for marketing, but three drugs are in clinical research, namely Secukinumab (IL-17A). Targeting antibodies), Ixekizumab (IL-17A targeting antibody) and Brodarumab (IL-17RA targeting antibody).
  • IL-17A targeting antibody Ixekizumab
  • IL-17RA targeting antibody IL-17RA targeting antibody
  • clinical studies of drugs have shown that these drugs do not exhibit the desired therapeutic effect for certain chronic inflammatory diseases such as rheumatoid arthritis (Kellner H, Ther Adv Musculoskelet Dis., 2013, 5: 141-152). Therefore, treatment with co-administration may increase the clinical benefit of certain chronic inflammations such as rheumatoid arthritis.
  • Combination administration requires sequential injection of two or more antibodies, or the same dosage form of the antibody.
  • sequential injection of antibodies reduces the patient's treatment adherence and increases pain.
  • due to differences in the physicochemical properties of different antibodies it is difficult or almost impossible to make different antibodies into the same dosage form.
  • the present invention provides a bifunctional fusion protein that simultaneously blocks the B7/CD28 and IL-17A/IL-17RA signaling pathways, a gene encoding the bifunctional fusion protein, a vector containing the gene, a host cell containing the vector, and the like A pharmaceutical composition of the bifunctional fusion protein.
  • a first aspect of the invention relates to a bifunctional fusion protein comprising a CTLA4 molecule extracellular domain and a functional fragment that neutralizes IL-17 activity, in one embodiment, said IL-17 is IL-17A.
  • the bifunctional fusion protein further comprises a linker peptide, wherein the extracellular region of the CTLA4 molecule is linked to the N-terminus or C-terminus of a functional fragment that neutralizes IL-17 activity via a linker peptide, in one implementation
  • the amino acid sequence of the linker peptide is 1-25 amino acids in length, for example, the amino acid sequence of the linker peptide is 20, 21, 22, 23, 24 or 25 amino acids in length, for example, the linker peptide is The amino acid sequence shown in SEQ ID NO: 3.
  • the functional fragment that neutralizes IL-17 activity is an anti-IL-17 antibody or a functional fragment thereof, a chimeric antibody, a humanized antibody, a fully human antibody, a single-chain antibody, a bispecific antibody,
  • the extracellular region of the CTLA4 molecule is linked to the N-terminus of the heavy or light chain of the anti-IL-17 antibody by a linker peptide.
  • the anti-IL-17 antibody is an IgG, IgA, IgD, IgE, IgM antibody or a hybrid thereof, and in certain embodiments, the anti-IL-17 antibody is an IgG antibody
  • the extracellular region of the CTLA4 molecule comprises the amino acid sequence set forth in SEQ ID NO: 2 or a functional fragment thereof, or the sequence as described above is replaced, deleted or added with one or more amino acid residues.
  • An equivalent functional amino acid sequence such as 2, 3, 4, 5, 10, 15, 20, 30, 50 amino acid residues, or an amino acid sequence having at least 70% identity and equivalent functionality thereto, such as at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6% identity.
  • the heavy chain sequence of the anti-IL-17 antibody comprises the amino acid sequence set forth in SEQ ID NO: 28, or the sequence as described above is replaced, deleted or added with one or more amino acid residues.
  • An equivalent functional amino acid sequence such as 2, 3, 4, 5, 10, 15, 20, 30, 50 amino acid residues, or an amino acid sequence having at least 70% identity and equivalent functionality thereto, such as at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4% 99.5%, 99.6% identity
  • the light chain sequence of the anti-IL-17 antibody comprises the amino acid sequence set forth in SEQ ID NO: 27, or the sequence shown above is substituted, deleted or added with one or more amino acids
  • An equivalent functional amino acid sequence formed by a residue such as 2, 3, 4, 5, 10, 15, 20, 30, 50 amino acid residues, or an amino acid sequence having at least 70% identity and equivalent function thereto, Such as at least about 75%, 80%,
  • the extracellular region of the CTLA4 molecule set forth in SEQ ID NO: 2 is linked to the heavy chain of the anti-IL-17 antibody set forth in SEQ ID NO: 4 or 12 by the linker peptide set forth in SEQ ID NO: The N-terminus of the sequence or the N-terminus of the light chain sequence of the anti-IL-17 antibody shown in SEQ ID NO: 5 or 13.
  • the extracellular region of the CTLA4 molecule set forth in SEQ ID NO: 2 is linked to the heavy chain of the anti-IL-17 antibody set forth in SEQ ID NO: 4 or 12 by the linker peptide set forth in SEQ ID NO: The C-terminus of the sequence or the C-terminus of the light chain sequence of the anti-IL-17 antibody shown in SEQ ID NO: 5 or 13.
  • the amino acid sequence of the bifunctional fusion protein is selected from the group consisting of SEQ ID NOs: 5 and 6, SEQ ID NOs: 13 and 14, SEQ ID NOs: 4 and 19, and SEQ ID NO: 24. And 25, SEQ ID NOS: 26 and 27, or an amino acid sequence of the same function formed by substitution, deletion or addition of one or more amino acid residues, such as 2, 3, 4, 5, 10 , 15, 20, 30, 50 amino acid residues, or an amino acid sequence having at least 70% identity and equivalent functionality thereto, such as at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6% identity.
  • a second aspect of the invention relates to a gene encoding a bifunctional fusion protein as described above.
  • the coding gene comprises the nucleotide sequences set forth in SEQ ID NOs: 7 and 8 or SEQ ID NOs: 15 and 16.
  • nucleic acid sequences encoding the light and heavy chains in the vector are present in different expression cassettes.
  • a third aspect of the invention relates to a recombinant vector comprising a coding gene as described above operably linked thereto.
  • the vector is a eukaryotic expression vector, and in certain embodiments, the vector is a vector X0GC engineered to have two expression cassettes.
  • a fourth aspect of the invention relates to a host cell comprising the recombinant vector as described above.
  • a fifth aspect of the invention relates to a method of producing a bifunctional fusion protein as described above, comprising the steps of:
  • the eukaryotic expression vector is X0GC, and in one embodiment, the host cell is HEK293-T or CHO cells.
  • a sixth aspect of the invention relates to a method for producing a bifunctional fusion protein as described above, which comprises culturing a host cell as described above under suitable expression conditions, and isolating and purifying the expressed protein.
  • a seventh aspect of the invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising a bifunctional fusion protein as described above or a bifunctional fusion protein produced by the method as described above.
  • the bifunctional fusion protein as described above or the pharmaceutical composition as described above is for use in preventing, treating or ameliorating rheumatoid arthritis, psoriasis, psoriatic arthritis, ankylosing spondylitis, gram Ron's disease, systemic lupus erythematosus, lupus nephritis, multiple sclerosis, autoimmune encephalomyelitis, glomerulonephritis, idiopathic thrombocytopenic purpura, primary Sjogren's syndrome, gout, or organ transplantation .
  • the bifunctional fusion protein as described above or the pharmaceutical composition as described above is for use in the prevention, treatment or amelioration of rheumatoid arthritis, psoriasis, psoriatic arthritis, ankylosing spondylitis Crohn's disease.
  • An eighth aspect of the invention relates to the use of a bifunctional fusion protein as described above or a bifunctional fusion protein produced by the above method or a pharmaceutical composition as described above for the preparation of a medicament for preventing, treating or ameliorating a disease.
  • the disease is rheumatoid arthritis, psoriasis, psoriatic arthritis, ankylosing spondylitis, Crohn's disease, systemic lupus erythematosus, lupus nephritis, multiple sclerosis, self Immune encephalomyelitis, glomerulonephritis, idiopathic thrombocytopenic purpura, primary Sjogren's syndrome, gout, or organ transplantation.
  • a ninth aspect of the invention relates to a method of preventing, treating or ameliorating a disease, the method comprising administering to a subject in need thereof a therapeutically effective amount of a bifunctional fusion protein as described above, produced as described above A step of a bifunctional fusion protein or a pharmaceutical composition as described above.
  • the disease is rheumatoid arthritis, psoriasis, psoriatic arthritis, ankylosing spondylitis, Crohn's disease, systemic lupus erythematosus, lupus nephritis, multiple sclerosis, self Immune encephalomyelitis, kidney Small ball nephritis, idiopathic thrombocytopenic purpura, primary Sjogren's syndrome, gout, or organ transplantation.
  • a tenth aspect of the invention relates to a kit comprising a bifunctional fusion protein as described above, a nucleic acid molecule as described above, a vector as described above or a host cell as described above.
  • the experimental results of the present invention indicate that the bifunctional fusion protein of the present invention has a high expression level and strongly inhibits the secretion of inflammatory cytokines in an in vitro experiment, and can effectively inhibit the development of joint inflammation in an animal model. It is known to those skilled in the art that the combined use or fusion of several different mechanisms of action may result in different effects such as antagonism, irrelevance, accumulation or synergy, and the mutual use of two or more drugs in combination or fusion. The role can only be determined through specific experimental research and a lot of labor.
  • the enhanced therapeutic effect of rheumatoid arthritis achieved by the bifunctional fusion protein of the present invention provides a drug candidate that may be more excellent in the treatment of patients with rheumatoid arthritis, especially in patients with moderate to severe rheumatoid arthritis.
  • the bifunctional fusion protein of the invention simultaneously acts on two different targets for treating rheumatoid arthritis, and reduces the probability of failure or poor effect of single target treatment, and has important economic and social benefits.
  • the present invention can reduce the production cost, reduce the volume and frequency of clinical administration, improve subject compliance, and have a huge prevention and treatment of immune diseases. Application prospects.
  • Figures 1.A and B show the purification of the bifunctional fusion proteins A and B in embodiments of the invention by Protein A affinity chromatography, respectively.
  • Figures 2.A and B show the purification of the bifunctional fusion proteins A and B in an embodiment of the invention by ion exchange chromatography, respectively.
  • Figures 3.A and B show the purity of the monomers of the bifunctional fusion proteins A and B in the embodiments of the present invention, respectively, by high performance liquid chromatography.
  • Bifunctional fusion protein A inhibits IL-17A-induced secretion of GRO ⁇ by HT-29 cells.
  • Figure 7 Mixed lymphocyte reaction assay for immunosuppressive activity of bifunctional fusion protein B.
  • Figure 8 Reduced effect of bifunctional fusion protein A humanized analog on rat CIA joint inflammation index.
  • the extracellular domain of the CTLA4 molecule is operably linked to functional fragments that neutralize IL-17 activity, maintaining their respective spatial structures and exerting their respective physiological activities.
  • the extracellular region of the CTLA4 molecule and the functional fragments that neutralize IL-17 activity can be directly fused together without affecting their respective functions, and the extracellular region of the CTLA4 molecule is linked to a functional fragment that neutralizes IL-17 activity.
  • N-terminal or C-terminal, other spacer sequences, such as a linker peptide, may also be added therebetween.
  • bifunctional fusion protein it does not mean that any other sequence not related to its function can be added to the bifunctional fusion protein. .
  • a fusion protein of a complex composition such as the bifunctional fusion protein
  • one skilled in the art may add one or more additional amino acid residues between the respective components or both ends of the fusion protein as needed in preparing the fusion protein.
  • the bifunctional fusion protein is defined by a closed expression, these conditions will not be truly covered.
  • CTLA4 molecular extracellular region refers to the extracellular region of cytotoxic T lymphocyte-associated antigen 4 (CTLA4).
  • CCD extracellular region
  • SEQ ID NO: 2 is amino acid sequence 37-161 of the human CTLA4 protein (Genbank Accession No. NM_005214.4).
  • the term "functional fragment that neutralizes IL-17 activity" as used herein refers to an immunoglobulin that targets IL-17 or a modification, functional equivalent, functional fragment or variant thereof.
  • the functional fragment that neutralizes IL-17 activity is an IgG antibody that targets IL-17.
  • the IgG is a chimeric, humanized or fully human IgG.
  • the modifications may be chemical modifications, such as acylation, alkylation, PEGylation products, so long as these modifications retain the ability to target IL-17.
  • the functional equivalent refers to other polypeptide fragments that are capable of effecting the ability of the immunoglobulin to target IL-17 binding.
  • the functional fragment refers to a protein fragment that retains the ability to target IL-17, such as a single domain antibody, a single chain antibody, a single chain variable fragment (scFv), a Fab fragment, or F ( Ab') 2 fragment.
  • a variant refers to a polypeptide derived from a parent protein by one or more alterations at one or more (several) positions, ie, substitutions, insertions, and/or deletions.
  • the linker is 5-30 amino acids in length, and in certain embodiments, the linker is 25 amino acids in length, and in certain embodiments, the sequence of the linker is GGGGSGGGGSGGGGSGGGGSGGGGS.
  • the ligation fragment used in the present invention is not particularly limited as long as it functions as a spacer for the two components of the fusion protein, so that each component can correctly form its respective spatial structure, retain its biological activity, and express the cell expression level.
  • the amino acid sequence of the bifunctional fusion protein is selected from the group consisting of SEQ ID NOs: 5 and 6, SEQ ID NOs: 13 and 14, SEQ ID NOs: 4 and 19, and SEQ ID NO: 24 and 25, SEQ ID NOS: 26 and 27, or an amino acid sequence of the same function formed by substitution, deletion or addition of one or more amino acid residues, such as 1, 2, 3, 4, 5, 10, 15, 20, 30, 50 amino acid residues, or an amino acid sequence having at least 70% identity and equivalent functionality thereto, such as at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6% identity.
  • conserveed amino acids of amino acids are well known in the art.
  • identity has the meaning commonly known in the art, and those skilled in the art are also familiar with the rules and criteria for determining identity between different sequences. Sequences defined by the present invention with varying degrees of identity must also have the extracellular domain of the CTLA4 molecule or neutralize the activity of IL-17. Methods and means for screening variant sequences using the above activities are well known to those skilled in the art. Such variant sequences can be readily obtained by those skilled in the art in light of the teachings of the present disclosure.
  • the invention features a gene encoding a nucleotide sequence encoding a bifunctional fusion protein as described above.
  • one or more codons in the coding gene sequence can be replaced equidistantly, such as one or several codons, such as 1, 2, 3, without altering the encoded amino acid. , 4, 5, 6, 8, 9, 10, 15, 20, 30, 40, 50 codons. Codon usage tables are well known in the art.
  • the invention features a recombinant vector comprising a coding gene as described above operably linked thereto.
  • the recombinant vector is a recombinant expression vector, which may be a prokaryotic expression vector or a eukaryotic expression vector, but is preferably a eukaryotic expression vector, more preferably a recombinant expression vector for eukaryotic expression in mammals.
  • operably linked refers to a manner in which the coding gene is placed in a suitable position in the vector such that the coding gene is correctly and smoothly replicated, transcribed or expressed.
  • the invention relates to a host cell transformed or transfected with a recombinant vector as described above, the host cell comprising a mammalian cell, a bacterial cell, a yeast cell, an insect cell, and a plant cell.
  • the host cell comprises CHO cells, HEK293 cells, NSO cells, and SP 2/0 cells.
  • the invention relates to a method of producing the bifunctional fusion protein comprising culturing the host cell described above under suitable polypeptide expression conditions and isolating and purifying the expressed polypeptide from the cell culture medium.
  • the purified polypeptide has a purity of greater than 50%, more preferably greater than 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97% 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8% or 99.9%.
  • the invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising a bifunctional fusion protein as described above and a pharmaceutically acceptable carrier.
  • the bifunctional fusion protein may be the sole active ingredient of the pharmaceutical composition, or may be one of the active ingredients of the pharmaceutical composition, and other active ingredients are other therapeutic agents that can be used in combination with the bifunctional fusion protein.
  • compositions of the present invention comprise a single administration dosage form, a topically administered dosage form, and a systemic administration dosage form.
  • the invention relates to said bifunctional fusion protein or pharmaceutical composition as described above for use in the prevention, treatment or amelioration of rheumatoid arthritis, psoriasis, psoriatic arthritis, ankylosing spondylitis Crohn's disease, systemic lupus erythematosus, lupus nephritis, multiple sclerosis, autoimmune encephalomyelitis, glomerulonephritis, idiopathic thrombocytopenic purpura, primary Sjogren's syndrome, gout, or Organ transplantation.
  • the invention relates to a method of preventing, treating or ameliorating a disease, the method comprising administering to a subject in need thereof a therapeutically effective amount of a bifunctional fusion protein as described above, produced as described above The step of a bifunctional fusion protein or a pharmaceutical composition as described above.
  • the disease is rheumatoid arthritis, psoriasis, psoriatic arthritis, ankylosing spondylitis, Crohn's disease, systemic lupus erythematosus, lupus nephritis, multiple sclerosis, self Immune encephalomyelitis, glomerulonephritis, idiopathic thrombocytopenic purpura, primary Sjogren's syndrome, gout, or organ transplantation.
  • the term "therapeutically effective amount” refers to a dose which, when administered, can exert a pharmacological effect in a subject.
  • the “therapeutically effective amount” can be easily determined by a person skilled in the art according to the condition of the patient such as age, body weight, disease state, and the like.
  • the invention relates to the use of a bifunctional fusion protein as described above or a bifunctional fusion protein produced by the above method or a pharmaceutical composition as described above for the preparation of a medicament for the prevention, treatment or amelioration of a disease .
  • the disease is rheumatoid arthritis, psoriasis, psoriatic arthritis, ankylosing spondylitis, Crohn's disease, systemic lupus erythematosus, lupus nephritis, multiple sclerosis, self Immune encephalomyelitis, glomerulonephritis, idiopathic thrombocytopenic purpura, primary Sjogren's syndrome, gout, or organ transplantation.
  • the conditions in which the bifunctional fusion protein of the present invention can prevent, treat or ameliorate are not limited.
  • the specific conditions listed above, any condition that can achieve prophylactic, therapeutic or ameliorating benefits by simultaneously blocking the B7/CD28 and IL-17A/IL-17RA signaling pathways are all within the scope of the present invention.
  • the present invention has the following beneficial effects:
  • the extracellular domain of human CTLA4 protein is linked to an anti-IL-17 antibody, and the target protein forms a tetrameric form, which satisfies the requirement that the extracellular domain of CTLA4 needs to form a dimeric form, and retains anti-IL.
  • the titer of the -17A antibody is still bivalent, and therefore, the total titer of the protein of interest is trivalent.
  • the screening and comparison by ligation and in vitro binding experiments also showed that the effective ligation method in the present invention does not bind the extracellular region of CTLA4 molecule to its ligand B7, and the anti-IL-17 antibody and IL-17. influences;
  • the bifunctional protein of the present invention after further modification, can form a chimeric or humanized analog, further reducing immunogenicity while retaining its binding activity.
  • Example 1 Construction of expression vector for bifunctional fusion protein A
  • the signal peptide sequence for expression of the bifunctional fusion protein A is shown in SEQ ID NO: 1; the extracellular region (ECD) sequence of the human CTLA4 protein is shown in SEQ ID NO: 2, which is amino acids 37-161 of the human CTLA4 protein. sequence (Genbank Accession No.
  • connecting peptide is (G 4 S) 5, a sequence such as SEQ ID NO: 3 as shown; bifunctional fusion protein a in the variable anti IL-17A antibody (control antibody a),
  • the region sequence is from patent US7846443 (rat 1D10), the constant region sequence is the constant region sequence of mouse IgG2a, ie the heavy chain (hc) sequence of the anti-IL-17A antibody is shown in SEQ ID NO: 4, light chain (lc)
  • the sequence is shown in SEQ ID NO: 5; the amino acid sequence of human CTLA4 ECD-linker peptide-anti-IL-17A mAb hc-A is set forth in SEQ ID NO: 6.
  • the nucleotide sequence encoding the amino acid sequence set forth in SEQ ID NO: 6 is optimized for mammalian cell codon preference and the sequence is set forth in SEQ ID NO: 7.
  • the nucleotide sequence encoding the amino acid sequence set forth in SEQ ID NO: 5 is optimized for mammalian cell codon preference and the sequence is set forth in SEQ ID NO: 8.
  • SEQ ID NO: 7 and SEQ ID NO: 8 were synthesized by Nanjing Kingsray Biotechnology Co., Ltd., and cloned into pUC57 vector by TA cloning and the obtained sequence-verified positive clones were named pUC57-CTLA4-anti-IL- 17A mAb hc-A and pUC57-anti-IL-17A mAb lc-A.
  • the pUC57-CTLA4-anti-IL-17A mAb hc-A plasmid (Nanjing Kingsray Biotechnology Co., Ltd.) was used as a template to amplify the signal peptide-human CTLA4ECD-linker peptide-anti-IL-17A mAb hc- by conventional PCR.
  • the coding sequence for A, the upstream primer used carries the Hind IIII restriction site, and the sequence CCCAAGCTTGCCACCATGGGGGTCCTG (SEQ ID NO: 9).
  • the downstream primer carries the EcoR I restriction site and the sequence is CCGGAATTCTCATTTGCCTGGGGTTCT (SEQ ID NO: 10).
  • the coding sequence of anti-IL-17A mAb lc-A was amplified by conventional PCR using pUC57-anti-IL-17A mAb lc-A plasmid (Nanjing Kingsray Biotechnology Co., Ltd.) as a template.
  • the upstream primer used was Hind.
  • the III cleavage site is SEQ ID NO: 9.
  • the downstream primer carries the EcoR I restriction site, and the sequence is CCGGAATTCTCAGCACTCATTCCG (SEQ ID NO: 11).
  • the amplified signal peptide-human CTLA4 ECD-linked peptide-anti-IL-17A mAb hc-A and anti-IL-17A mAb lc-A coding sequences (lengths are 1881 bp and 717 bp, respectively), 1% agar
  • the corresponding fragment was recovered after glycophore electrophoresis.
  • the recovered gene fragment and the company's eukaryotic expression vector X0GC (Patent US20100120089) were digested with Hind III and EcoR I to obtain recombinant plasmids X0GC-CTLA4-anti-IL-17A mAb hc-A and X0GC-anti-.
  • IL-17A mAb lc-A was transformed into E.
  • coli DH5 ⁇ coli DH5 ⁇ , respectively, to obtain recombinant DH5 ⁇ /X0GC-CTLA4-anti-IL-17A mAb hc-A and DH5 ⁇ /X0GC-anti-IL-17A mAb lc-A. Positive clones were screened by PCR and DNA sequencing was performed to verify that the recombinant plasmid was constructed correctly.
  • DH5 ⁇ /X0GC-CTLA4-anti-IL-17A mAb hc-A and DH5 ⁇ /X0GC-anti-IL-17A mAb lc-A were inoculated separately into 1 L of LB/Amp liquid medium (composed of 1% peptone (BD) Company), 0.5% yeast extract (BD company), 1% NaCl (Nippon Pharmaceutical Group Chemical Reagent Co., Ltd.), shake culture at 37 ° C, 180 rpm overnight. The next day, the plasmid was extracted using the endotoxin-free plasmid (Qiagen, 12381) for transfection of HEK293-T (Cell Resource Center, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences).
  • the signal peptide sequence for expression of the bifunctional fusion protein B is shown in SEQ ID NO: 1; the ECD sequence of the human CTLA4 protein is shown in SEQ ID NO: 2, which is the amino acid sequence of amino acids 37-161 of the human CTLA4 protein (Genbank accession number) NM_005214.4); the linker peptide is (G 4 S) 5 , the sequence is shown in SEQ ID NO: 3; the sequence of the anti-IL-17A antibody in the bifunctional fusion protein B is from US Pat. No. 7,784,443 (humanized 16C10).
  • hc Its heavy chain (hc) sequence is set forth in SEQ ID NO: 12, and the light chain (lc) sequence is set forth in SEQ ID NO: 13; the amino acid sequence of human CTLA4 ECD-linker peptide-anti-IL-17A mAb hc-B is as SEQ ID NO:14.
  • the nucleotide sequence encoding the amino acid sequence set forth in SEQ ID NO: 14 is optimized for mammalian cell codon preference and the sequence is set forth in SEQ ID NO: 15.
  • the nucleotide sequence encoding the amino acid sequence set forth in SEQ ID NO: 13 is optimized for mammalian cell codon preference and the sequence is set forth in SEQ ID NO: 16.
  • SEQ ID NO: 15 and SEQ ID NO: 16 were synthesized by Nanjing Kingsray Biotechnology Co., Ltd., and cloned into pUC57 vector by TA cloning and the obtained sequence-verified positive clones were named pUC57-CTLA4-anti-IL-. 17A mAb hc-B and pUC57-anti-IL-17A mAb lc-B.
  • the coding sequence of anti-IL-17A mAb lc-B was amplified by conventional PCR using pUC57-anti-IL-17A mAb lc-B plasmid (Nanjing Kingsray Biotechnology Co., Ltd.) as a template.
  • the upstream primer used was Hind. III cleavage site, the sequence is SEQ ID NO: 9.
  • the downstream primer carries the EcoR I restriction site and the sequence is CCGGAATTCTCAGCACTCGCCCCGGTTG (SEQ ID NO: 18).
  • the amplified signal peptide-human CTLA4 ECD-linker peptide-anti-IL-17A mAb hc-B and anti-IL-17A mAb lc-B coding sequence (length 1887 bp and 735 bp, respectively), 1% agar
  • the corresponding fragment was recovered after glycophore electrophoresis.
  • the recovered gene fragment and the company's eukaryotic expression vector X0GC (Patent US20100120089) were digested with Hind III and EcoR I to obtain recombinant plasmids X0GC-CTLA4-anti-IL-17A mAb hc-B and X0GC-anti-.
  • IL-17A mAb lc-B which was transformed into E.
  • DH5 ⁇ /X0GC-CTLA4-anti-IL-17A mAb hc-B and DH5 ⁇ /X0GC-anti-IL-17A mAb lc-B were inoculated separately into 1 L of LB/Amp liquid medium (composed of 1% peptone (BD) Company), 0.5% yeast extract (BD company), 1% NaCl (Nippon Pharmaceutical Group Chemical Reagent Co., Ltd.), shake culture at 37 ° C, 180 rpm overnight.
  • the plasmid was extracted for HEK293-T transfection using the endotoxin-free plasmid (Qiagen, 12381) the next day.
  • HEK293-T cells with a good growth state and viability (live cell ratio, also can be written as "live rate") of 95% or more were inoculated into a ten-layer cell factory at an inoculation amount of 1.8 ⁇ 10 7 cells (NUNC, product number) :140400), cultured in DMEM medium (Corning) containing 10% fetal bovine serum (Gibco).
  • the cell factory was repeatedly inverted and mixed, and then cultured in a 37 ° C, 5% CO 2 incubator for 48 hours. The wall is complete and the density reaches 80%, which can be used for transient transfection.
  • Plasmids X0GC-CTLA4-anti-IL-17A mAb hc-A and X0GC-anti-IL-17A mAb lc-A were simultaneously transfected into cells for expression of bifunctional fusion protein A.
  • the plasmid X0GC-CTLA4-anti-IL-17A mAb hc-B and X0GC-anti-IL-17A mAb lc-B were simultaneously transfected into cells for expression of the bifunctional fusion protein B. After filtration through a 0.22 ⁇ m filter, 1330 ⁇ g of each was aspirated and added to 66 ml of serum-free DMEM medium.
  • the supernatant from the cell factory was filtered through a 0.2 ⁇ m filter and concentrated by membrane filtration ultrafiltration (nominal molecular weight cutoff 10 kDa), and the solution was replaced with a 20 mM phosphate solution (pH 7.4) containing 150 mM NaCl.
  • the precipitate was removed by filtration through a 0.2 ⁇ m filter before applying column chromatography. This step was carried out at 4 °C.
  • Purification was carried out at 4 °C using an AKTA explorer 100 protein purification system (GE Healthcare) and an affinity chromatography column rProtein A Sepharose Fast Flow (16 mm I.D., 22 ml, GE Healthcare).
  • the column was first equilibrated with mobile phase A (20 mM sodium phosphate buffer, 150 mM sodium chloride, pH 7.4), and the supernatant of the above treated cells was loaded after the baseline was stabilized at a flow rate of 5 ml/min. After the sample, rinse with mobile phase A.
  • Example 5 Construction of a stable cell line of a bifunctional fusion protein
  • CHO/DG44 (dhfr-) cells are passed through 2-3 passages after resuscitation and can be used in transfection experiments when their survival rate reaches 95%.
  • Transfection of CHO/DG44 cells was performed using Lipofectamine 2000 transfection reagent (Invitrogen, 11668019) according to the instructions. Specifically, 5 ⁇ g of the plasmid and 3 mL of ⁇ -MEM medium (SAFC, 51451 C) were mixed in one centrifuge tube while 20 ⁇ L of the transfection agent and 3 mL of ⁇ -MEM medium were mixed in another centrifuge tube. After standing at room temperature for 5 minutes in a clean bench, the diluted plasmid and the transfection agent were mixed and allowed to stand for 30 minutes.
  • the cell suspension was diluted with ⁇ -MEM medium supplemented with 10% dialyzed fetal bovine serum and 1 mg/mL G418 to a cell concentration of 5 cells/mL, and the cell suspension was seeded in a 96-well plate.
  • the cells were cultured in a cell culture incubator (37 ° C, 5% CO 2 ) until the single cells grew into separate colonies.
  • the colony-derived colonies were selected by trypsinization and passaged into 24-well plates. The cells were then passaged sequentially into 12-well plates and 6-well plates.
  • Example 6 Small-scale fermentation of bifunctional fusion proteins
  • the clone No. 103 was selected for production in a 5 L bioreactor (Sartorius, Bplus twin 5L).
  • the cells were inoculated to Hyclone SFM4 CHO medium (4 g/L glucose, 0.3 g/L glutamine ammonia) at a cell density of 5 x 10 5 .
  • the working volume of the bioreactor was 2.4 L, and the initial process conditions were: 37 ° C, pH 7.1, dissolved oxygen 40% - 60%, and stirring speed 40 rpm. Thereafter, the stirring speed was increased by 5 rpm per day, and after 5 days, the temperature was lowered at a rate of 0.5 ° C / hour, the temperature was stopped at 34 ° C, and 0.3 mM sodium butyrate was added. Two weeks later, the reactor fermentation broth was harvested, centrifuged at 7000 rpm for 20 minutes, and the supernatant was harvested and stored. The concentration was 240 mg/L as determined by ELISA.
  • Example 7 Binding affinity of bifunctional fusion proteins A and C to human and mouse IL-17A
  • a bifunctional fusion protein C similar to the bifunctional fusion protein A was constructed.
  • the amino acid sequence of the bifunctional fusion protein C is as shown in SEQ ID NO: 19 (CTLA4 ECD-linker peptide-anti-IL-17A mAb lc) and SEQ ID NO: 4, ie its CTLA4 ECD is linked to anti-IL-17A via a linker peptide Light mAb
  • the strand (ie, linked to SEQ ID NO: 5), the CTLA4 ECD different from the bifunctional fusion protein A is linked to the heavy chain of the anti-IL-17A mAb by a linker peptide (ie, ligated to SEQ ID NO: 4).
  • control antibody A serves as a control antibody for bifunctional fusion proteins A and C, the amino acid sequences of which are shown in SEQ ID NO: 4 (the heavy chain of control antibody A) and SEQ ID NO: 5 (light chain of control antibody A) That is, the control antibody A corresponds to a portion of the anti-IL-17A antibody in the bifunctional fusion proteins A and C.
  • the specific implementation process of the method is as follows. Human IL-17A (Beijing Yiqiao Shenzhou, Cat. No. 12047-HNAE) or mouse IL-17A (Beijing Yiqiao Shenzhou, Cat. No. 51065-MNAE) was coated with sodium carbonate solution (15 mM sodium carbonate, 35 mM sodium bicarbonate, pH 9.6). On a 96-well high adsorption ELISA plate (Corning, 2592), the coating concentration was 1 ⁇ g/mL, 100 ⁇ L per well, and overnight coating at 4 °C. The plate was washed 5 times with PBST (Sigma, Cat. No. P-3563).
  • PBST Sigma, Cat. No. P-3563
  • the plate was blocked with PBST containing 1% BSA and incubated at 300 °L per well for at least 1 hour at 25 °C.
  • the plate was washed 5 times with PBST.
  • a specific concentration of the test sample diluted in PBST containing 1% BSA was added, 100 ⁇ L per well, and incubated at 25 ° C for 1 hour.
  • the plate was washed 5 times with PBST.
  • horseradish peroxidase-labeled anti-mouse IgG antibody (Abeam, Cat. No. Ab7068) diluted 1:10000 in PBST containing 1% BSA was added, and 100 ⁇ L per well was incubated at 25 ° C for 1 hour.
  • the plate was washed 5 times with PBST.
  • a colorimetric substrate TMB (BD OptEIA, Cat. No. 555214) was added, 100 ⁇ L per well, and color developed for 10 minutes at room temperature. Color development was stopped by adding 1 M H 2 SO 4 , 100 ⁇ L per well. The absorbance at 450 nm was read on a microplate reader.
  • the bifunctional fusion protein A and the bifunctional protein C of the present invention have high affinity for binding to their antigens human IL-17A (Fig. 4) and mouse IL-17A (Fig. 5), and Affinity is similar to control antibody A.
  • CTLA4 ECD linked to the N-terminus of the heavy chain or light chain of the control antibody A by the linker peptide does not significantly affect the affinity of the control antibody A itself to the antigen.
  • Example 8 Effect of different ligation forms of CTLA4 on the affinity of bifunctional fusion protein to B7
  • Bifunctional fusion protein C the sequence is as described above, ie SEQ ID NO: 19 (CTLA4 ECD-linker peptide-anti-IL-17A mAb lc) and SEQ ID NO: 4, ie its CTLA4 ECD is linked by a linker peptide
  • the N-terminus of the anti-IL-17A mAb light chain ie, SEQ ID NO: 5
  • the CTLA4 ECD different from the bifunctional fusion protein A is linked to the N-terminus of the anti-IL-17A mAb heavy chain by a linker peptide (ie, ligation) To SEQ ID NO: 4).
  • Bifunctional fusion protein D the signal peptide sequence used is shown in SEQ ID NO: 1; the extracellular region (ECD) sequence of human CTLA4 protein is shown in SEQ ID NO: 2; the linker peptide is (G 4 S) 5 , the sequence is shown in SEQ ID NO: 3, the heavy chain (hc) sequence of the anti-IL-17A antibody is shown in SEQ ID NO: 4, and the light chain (lc) sequence is shown in SEQ ID NO: 5;
  • the amino acid sequence of IL-17A mAb hc-A-linker peptide-CTLA4 ECD is set forth in SEQ ID NO: 20, that is, CTLA4 of the bifunctional fusion protein D is linked to the C-terminus of the heavy chain of the control antibody A via a linker peptide.
  • Bifunctional fusion protein E the signal peptide sequence used is shown in SEQ ID NO: 1; the extracellular region (ECD) sequence of human CTLA4 protein is shown in SEQ ID NO: 2; the linker peptide is (G4S) 5, The sequence is as shown in SEQ ID NO: 3, the heavy chain (hc) sequence of the anti-IL-17A antibody is shown in SEQ ID NO: 4, the light chain (lc) sequence is shown in SEQ ID NO: 5; anti-IL- The amino acid sequence of the 17A mAb lc-A-linker peptide-CTLA4ECD is set forth in SEQ ID NO: 21, that is, the CTLA4 of the bifunctional fusion protein E is linked to the C-terminus of the light chain of the control antibody A via a linker peptide.
  • CTLA4ECD binds to its ligand B7 molecule.
  • B7 binding affinity assay the binding ability of the bifunctional fusion proteins A and C and D and E to the B7 molecule was determined by flow cytometry on Raji cells highly expressing B7 molecules.
  • the specific implementation process of the method is as follows. Raji cells (ATCC, Cat. No. CCL-86) were collected by centrifugation at 1000 rpm for 5 minutes. Wash once with PBS (PromoCell, Cat. No. C-40232) containing 2% FBS (GIBCO, Cat. No. 10099141).
  • Raji cells were resuspended in EP tubes, 1 x 10 6 cells per tube, resuspended in 200 ⁇ L of cold PBS containing 2% FBS and containing a final sample concentration of 0.5 nM.
  • the EP tube was incubated on ice for 30 minutes. Wash twice with PBS containing 2% FBS. It was then resuspended in 200 ⁇ L of cold PBS containing 2% FBS and containing FITC-labeled anti-mouse IgG antibody (1:50 dilution). Incubate on ice for 30 minutes in the dark. Wash twice with PBS containing 2% FBS. The cells were resuspended in 500 ⁇ L of cold PBS containing 2% FBS, and the cell suspension was assayed on a flow cytometer.
  • the bifunctional fusion protein A has the ability to bind to Raji cells highly expressing its ligand B7 molecule, and the binding ability is significantly greater than that of the bifunctional fusion protein C, the bifunctional fusion protein D, and the bifunctional fusion protein E. .
  • the above results indicate that the different ligation forms of CTLA4ECD and anti-IL-17A antibodies can significantly affect their ability to bind to B7.
  • Example 9 Bifunctional fusion protein A inhibits IL-17A-induced secretion of GRO ⁇ by HT-29 cells
  • Human colorectal cancer cell line HT-29 is from ATCC.
  • HT-29 cells were cultured in RPMI-1640 medium (Gibco, Cat. No.: 22400) containing 10% fetal bovine serum (Gibco, Cat. No. 10099), placed in a 37 ° C, 5% CO 2 cell incubator, weekly. Passage twice.
  • the specific procedure for inhibiting IL-17A-induced GRO ⁇ secretion assay is as follows.
  • the sample to be tested was diluted in RPMI 1640 complete medium containing 10% fetal calf serum, and 50 ⁇ L per well was added to a cell culture plate (Coring, Cat. No. 3599).
  • Human IL-17A at a concentration of 240 ng/mL diluted to the same complete medium was added to the cell culture plate at 50 ⁇ L per well. Incubate for 1 hour at 37 ° C in a 5% CO 2 incubator.
  • the HT-29 cells were then resuspended in complete medium and plated into 96-well cell culture plates at 100 ⁇ L per well, approximately 20,000 cells.
  • the cells were incubated for 48 hours at 37 ° C in a 5% CO 2 incubator.
  • the cell culture plate was centrifuged at 1000 rpm for 5 minutes, and the culture supernatant was taken out.
  • GRO ⁇ levels were determined using a GRO ⁇ ELISA kit (R&D, Cat. No. DY275) according to the instructions.
  • both the control antibody A and the bifunctional fusion protein A inhibited IL-17A-induced secretion of GRO ⁇ by HT-29 cells with IC 50 of 532 pM and 911 pM, respectively.
  • Example 10 Bifunctional fusion protein B inhibits IL-2 secretion by human peripheral blood mononuclear cells
  • the immunosuppressive activity of the bifunctional fusion protein B of the present invention is determined by a mixed lymphocyte reaction. Briefly, two human peripheral blood mononuclear cells (PBMC) from two different individuals were taken, one of which was inactivated as a stimulating cell and the other PBMC as a reactive cell, which mixed the two PBMCs and stimulated the cells. It can activate the immune response of the reaction cells and secrete a large amount of the cytokine IL-2. Samples were incubated with this mixed PBMC system to determine their effect on IL-2 secretion.
  • PBMC peripheral blood mononuclear cells
  • the specific implementation method of the lymphocyte mixing reaction is as follows. Two different batches of PBMC (Lonza, Cat. No. CC-2702) from different individuals were resuspended and resuspended in RPMI-1640 (Gibco, Cat. No. 22400) complete medium containing 10% fetal calf serum (Gibco, Cat. No. 10099). One PBMC acts as a stimulating cell and one PBMC acts as a reactive cell. Stimulated cells were incubated with 50 ⁇ g/ml mitomycin C (Wako, Cat. No. 50-07-7) for 45 minutes at 37 ° C, then washed twice with DPBS (PromoCell, Cat. No.
  • IL-2 levels were determined using an IL-2 ELISA kit (Raybiotech, Cat. ELH-IL2-001) according to the instructions.
  • Example 11 Chimera and humanized analogs of bifunctional fusion protein A
  • Example 12 Pharmacodynamic study of a bifunctional fusion protein A humanized analog in a type II collagen induced rat arthritis (CIA) model
  • the rat CIA model is an animal model widely used in the pathogenesis of rheumatoid arthritis and in the screening of therapeutic drugs.
  • 7-week-old female Wistar rats purchased from Beijing Huakang Biotechnology Co., Ltd.
  • 5 rats were randomly selected as normal control rats, and the remaining rats were used to establish rat CIA. model.
  • Rats were given a CIA model by primary immunization and booster immunization. The primary immunization was mixed with 200 ⁇ g of type II bovine collagen (Chondrex, Cat. No. 20022) and Freund's incomplete adjuvant (Sigma-Aldrich, Cat. No.
  • the experimental results are shown in Fig. 8.
  • the rats in the normal control group did not produce an inflammatory reaction, and the CIA model rats in the vehicle group produced a significant inflammatory reaction.
  • the CIA model of the bifunctional fusion protein A humanized analog administration group was subjected to arthritis. The index was significantly inhibited, showing a better inflammatory remission effect than the abatacept group.

Abstract

本发明提供了包含CTLA4胞外区和抗IL-17抗体的双功能融合蛋白、编码该蛋白的基因、含有该基因的载体、含有该载体的宿主细胞以及含有该蛋白的药物组合物。

Description

双功能融合蛋白及其制备方法和用途 技术领域
本发明涉及双功能融合蛋白及其制备方法和用途。特别的，本发明涉及可同时阻断B7/CD28和IL-17A/IL-17RA信号通路的双功能重组融合蛋白、编码该双功能融合蛋白的基因、含有该基因的载体、含有该载体的宿主细胞以及含有该双功能融合蛋白的药物组合物。
背景技术
调节和抑制T细胞功能被视为一种治疗慢性炎症的有效方式。细胞毒T淋巴细胞相关抗原4(cytotoxic T lymphocyte-associated antigen 4,CTLA4)，又名CD152，是一种白细胞分化抗原。CTLA4和CD28的配体均为B7分子，而CTLA4与B7分子结合后诱导T细胞无反应性，参与免疫反应的负调节。2005年上市的用于类风湿性关节炎治疗的阿巴西普(abatacept，商品名OrenciaTM，百时美施贵宝公司)是一种单功能融合蛋白，由2个CTLA4分子的胞外区(extracellular domain,ECD)与人IgG1的Fc段结合而成，通过竞争性阻断B7与CD28分子的结合，从而抑制T细胞增殖和激活(Genovese MC,Becker JC,Schiff M,et al.N.Engl.J.Med.,2005,353:1114-1123)。
白细胞介素17A(IL-17或IL-17A)是一种促炎性细胞因子，主要产生自Th17细胞，是IL-17家族中最具代表性的成员(Miossec P,Kolls JK.Nat.Rev.Drug.Discov.,2012,11:763-776)。IL-17A与其受体(IL-17RA)结合后，可诱导多种细胞(如成纤维细胞、上皮细胞和内皮细胞)表达炎症性细胞因子以及趋化因子，在机体免疫防御中发挥重要作用(Lin Y,Ritchea S,Logar A.Immunity,2009,31:799-810)。然而，过高表达的IL-17A可引起炎症疾病。类风湿关节炎患者的滑膜组织高表达IL-17A，并导致重要炎症因子——白细胞介素6的产生，但该作用可被IL-17A中和抗体抑制(Chabaud M,Durand JM,Buchs N,et al.Arthritis Rheum.1999,42:963-70)。很多自身免疫疾病的动物模型实验证明，使用抗体中和IL-17A，可有效抑制炎症的病理发展(Lubberts E,Koenders MI,Oppers-Walgreen B,et al.Arthritis Rheum.,2004,50:650-659)。目前，尚无IL-17A相关抗体药物被批准上市，但有3个药物正处于临床研究阶段，分别是Secukinumab(IL-17A 靶向抗体)、Ixekizumab(IL-17A靶向抗体)和Brodalumab(IL-17RA靶向抗体)。然而，药物临床研究结果表明，对于某些慢性炎症疾病如类风湿性关节炎，这些药物并未表现出预期的治疗效果(Kellner H,Ther Adv Musculoskelet Dis.,2013,5:141-152)。因此，采用联合给药的治疗模式，可能会增加某些慢性炎症如类风湿关节炎患者的临床获益。
联合给药需要依次注射两种或多种抗体，或将抗体做成同一种剂型。然而，一方面，依次注射抗体会降低患者的治疗依从性，并增加疼痛。另一方面，由于不同抗体的物化性质存在差异，很难或几乎不太可能将不同抗体做成同一种剂型。
鉴于此，仍然有必要研究一种同时阻断B7/CD28和IL-17A/IL-17RA信号通路的新型治疗药物。
发明内容
本发明提供一种同时阻断B7/CD28和IL-17A/IL-17RA信号通路的双功能融合蛋白、编码该双功能融合蛋白的基因、含有该基因的载体、含有该载体的宿主细胞以及含有该双功能融合蛋白的药物组合物。
本发明的第一方面涉及一种双功能融合蛋白，其包含CTLA4分子胞外区和中和IL-17活性的功能片段，在一个实施方案中，所述IL-17是IL-17A。
在一个实施方案中，所述的双功能融合蛋白还包含连接肽，其中所述CTLA4分子胞外区通过连接肽连接于中和IL-17活性的功能片段的N端或C端，在一个实施方案中，所述连接肽的氨基酸序列长度为1-25个氨基酸，例如，所述连接肽的氨基酸序列长度为20、21、22、23、24或25个氨基酸，例如，所述连接肽是SEQ ID NO：3所示的氨基酸序列。
在一个实施方案中，中和IL-17活性的功能片段是抗IL-17抗体或其功能片段、嵌合抗体、人源化抗体、完全人源抗体、单链抗体、双特异性抗体，在一个实施方案中，CTLA4分子胞外区通过连接肽连接于抗IL-17抗体重链或轻链的N端。在某些实施方案中，抗IL-17抗体为IgG、IgA、IgD、IgE、IgM抗体或者它们的杂合体，在某些实施方案中，抗IL-17抗体为IgG抗体
在一个实施方案中，所述CTLA4分子胞外区包含SEQ ID NO：2所示的氨基酸序列或其功能片段，或如上述所示的序列经替换、缺失或添加一个或多个氨基酸残基形成的具有同等功能的氨基酸序列，如2、3、4、5、10、15、20、30、50个氨基酸残基，或与其具有至少70％同一性且具有同等功能的氨基酸序列，如至少约75％、80％、85％、90％、 91％、92％、93％、94％、95％、96％、97％、98％、99％、99.1％、99.2％、99.3％、99.4％、99.5％、99.6％同一性。
在一个实施方案中，所述抗IL-17抗体的重链序列包含SEQ ID NO：28所示的氨基酸序列，或如上述所示的序列经替换、缺失或添加一个或多个氨基酸残基形成的具有同等功能的氨基酸序列，如2、3、4、5、10、15、20、30、50个氨基酸残基，或与其具有至少70％同一性且具有同等功能的氨基酸序列，如至少约75％、80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、99.1％、99.2％、99.3％、99.4％、99.5％、99.6％同一性；所述抗IL-17抗体的轻链序列包含SEQ ID NO：27所示的氨基酸序列，或如上述所示的序列经替换、缺失或添加一个或多个氨基酸残基形成的具有同等功能的氨基酸序列，如2、3、4、5、10、15、20、30、50个氨基酸残基，或与其具有至少70％同一性且具有同等功能的氨基酸序列，如至少约75％、80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、99.1％、99.2％、99.3％、99.4％、99.5％、99.6％同一性。
在一个实施方案中，SEQ ID NO：2所示的CTLA4分子胞外区通过SEQ ID NO：3所示的连接肽连接至SEQ ID NO：4或12所示的抗IL-17抗体的重链序列的N端或连接至SEQ ID NO：5或13所示的抗IL-17抗体的轻链序列的N端。
在一个实施方案中，SEQ ID NO：2所示的CTLA4分子胞外区通过SEQ ID NO：3所示的连接肽连接至SEQ ID NO：4或12所示的抗IL-17抗体的重链序列的C端或连接至SEQ ID NO：5或13所示的抗IL-17抗体的轻链序列的C端。
在一个实施方案中，所述双功能融合蛋白的氨基酸序列选自下述组合：SEQ ID NO：5和6、SEQ ID NO：13和14、SEQ ID NO：4和19、SEQ ID NO：24和25、SEQ ID NO：26和27、或如上述所示的序列经替换、缺失或添加一个或多个氨基酸残基形成的具有同等功能的氨基酸序列，如2、3、4、5、10、15、20、30、50个氨基酸残基，或与其具有至少70％同一性且具有同等功能的氨基酸序列，如至少约75％、80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、99.1％、99.2％、99.3％、99.4％、99.5％、99.6％同一性。
本发明的第二方面涉及一种如上所述的双功能融合蛋白的编码基因。
在一个实施方案中，所述的编码基因包含SEQ ID NO：7和8或SEQ ID NO：15和16所示的核苷酸序列。
在某些实施方案中，所述载体中编码轻链和重链的核酸序列存在于不同的表达框中。
本发明的第三方面涉及一种重组载体，其包含有效连接其中的如上所述的编码基因。
在某些实施方案中，所述的载体为真核细胞表达载体，在某些实施方案中，所述的载体为经改造而具有两个表达框的载体X0GC。
本发明的第四方面涉及一种宿主细胞，其含有如上所述的重组载体。
本发明的第五方面涉及一种制备如上所述的双功能融合蛋白的方法，其包括步骤：
(1)将如上所述编码基因有效连接至真核表达载体中并转染至宿主细胞进行表达或将如上所述的重组载体转染至宿主细胞进行表达；和
(2)纯化所述双功能融合蛋白，
在一个实施方案中，所述真核表达载体为X0GC，在一个实施方案中，所述宿主细胞为HEK293-T或CHO细胞。
本发明的第六方面涉及一种生产如上所述的双功能融合蛋白的方法，其包括在适宜表达条件下培养如上所述的宿主细胞，并分离、纯化表达的蛋白。
本发明的第七方面涉及一种药物组合物，其包含如上所述的双功能融合蛋白或如上所述的方法产生的双功能融合蛋白。
在一个实施方案中，如上所述的双功能融合蛋白或如上所述的药物组合物用于预防、治疗或改善类风湿关节炎、银屑病、银屑病关节炎、强直性脊柱炎、克罗恩氏病、系统性红斑狼疮、狼疮肾炎、多发性硬化症、自身免疫性脑脊髓炎、肾小球肾炎、特发性血小板减少性紫癜、原发性干燥综合征、痛风、或器官移植。
在另一个实施方案中，如上所述的双功能融合蛋白或如上所述的药物组合物用于预防、治疗或改善类风湿性关节炎、银屑病、银屑病关节炎、强直性脊柱炎、克罗恩氏病。
本发明的第八方面涉及如上所述的双功能融合蛋白或者由如上方法产生的双功能融合蛋白或者如上所述的药物组合物在制备用于预防、治疗或改善疾病的药物中的应用。
在一个实施方案中，所述疾病为类风湿关节炎、银屑病、银屑病关节炎、强直性脊柱炎、克罗恩氏病、系统性红斑狼疮、狼疮肾炎、多发性硬化症、自身免疫性脑脊髓炎、肾小球肾炎、特发性血小板减少性紫癜、原发性干燥综合征、痛风、或器官移植。
本发明的第九方面涉及一种预防、治疗或改善疾病的方法，所述方法包括向有此需要的受试者施用治疗有效量的如上所述的双功能融合蛋白、如上所述方法生产的双功能融合蛋白或如上所述的药物组合物的步骤。
在一个实施方案中，所述疾病为类风湿关节炎、银屑病、银屑病关节炎、强直性脊柱炎、克罗恩氏病、系统性红斑狼疮、狼疮肾炎、多发性硬化症、自身免疫性脑脊髓炎、肾 小球肾炎、特发性血小板减少性紫癜、原发性干燥综合征、痛风、或器官移植。
本发明的第十方面涉及一种试剂盒，其包含如上所述的双功能融合蛋白、如上所述的核酸分子、如上所述的载体或如上所述的宿主细胞。
本发明具有以下几方面优点：
本发明的实验结果表明，本发明的双功能融合蛋白表达量高，体外实验中强烈抑制炎症细胞因子的分泌，动物模型中能有效地抑制关节炎症的发展。本领域技术人员知晓，几个不同作用机理的药物联合使用或融合使用可能会出现拮抗、无关、累加或协同等不同的效果，两种或更多种药物联合使用或融合使用到底呈现何种相互作用只能通过具体的实验研究、付出大量劳动的情况下才能确定。虽然现有技术中有可能会组合使用CTLA4分子和抗IL-17抗体治疗了类风湿关节炎，但由于人类机体的复杂性，尤其是对类风湿关节炎这样的发病机理极其复杂、发病机理尚未完全洞悉的全身性、免疫性疾病，药物即使联合使用或融合使用也罕有实现各个不同作用机理药物所能实现的治疗效果的完全累加或协同的效果，很多都呈现组合无关甚或拮抗的效果。本发明的双功能融合蛋白实现的对类风湿关节炎的增强的治疗效果为类风湿关节炎患者、尤其是中重度类风湿关节炎患者的治疗提供了一种治疗效果可能更加优异的候选药物。同时，本发明的双功能融合蛋白同时作用于治疗类风湿关节炎的两个不同的靶点，降低了单一靶点治疗失败或效果不佳的机率，具有重要的经济意义和社会效益。与使用单功能的蛋白(例如CTLA4-Fc)情况相比，本发明可降低生产成本，并减少临床给药体积和频率，提高受试者顺从性，在免疫性疾病的预防和治疗上具有巨大的应用前景。
附图说明
图1.A和B分别显示了Protein A亲和色谱法纯化本发明实施方式中的双功能融合蛋白A和B。
图2.A和B分别显示了离子交换色谱法纯化本发明实施方式中的双功能融合蛋白A和B。
图3.A和B分别显示了高效液相排阻色谱法分析本发明实施方式中的双功能融合蛋白A和B的单体纯度。
图4.双功能融合蛋白A和C与人IL-17A的结合力。
图5.双功能融合蛋白A和C与小鼠IL-17A的结合力。
图6.双功能融合蛋白A抑制IL-17A诱导的HT-29细胞分泌GROα。
图7.混合淋巴细胞反应测定双功能融合蛋白B的免疫抑制活性。
图8.双功能融合蛋白A人源化类似物对大鼠CIA关节炎症指数的降低作用。
具体实施方式
本发明的一个目的在于提供一种双功能融合蛋白，其包含CTLA4分子胞外区和中和IL-17活性的功能片段。CTLA4分子胞外区与中和IL-17活性的功能片段有效连接，保持其各自的空间结构并发挥其各自的生理活性。所述CTLA4分子胞外区与中和IL-17活性的功能片段可以在不影响其各自功能的情况下直接融合在一起，且CTLA4分子胞外区连接在中和IL-17活性的功能片段的N端或C端，也可以在其间加入其它间隔序列，如连接肽。
本领域技术人员知晓，虽然本发明在限定所述双功能融合蛋白时所用限定语为“包括”，但其并不意味着可以在所述双功能融合蛋白中任意加入与其功能不相关的其他序列。在制备复杂组成的融合蛋白如所述双功能融合蛋白时，为了保证融合蛋白各个组成成分的空间结构、生物活性、细胞表达水平，以及为了将所述各种组分适当的融合在一起，或为了增强所述融合蛋白的抗水解能力，本领域技术人员在制备所述融合蛋白时，会根据需要在各个组分之间或所述融合蛋白的两端加入一个或多个额外的氨基酸残基，因此，如果用封闭式的表述来限定所述双功能融合蛋白将不能真实地覆盖这些情形。
本发明所用的术语“CTLA4分子胞外区”是指细胞毒T淋巴细胞相关抗原4(cytotoxic T lymphocyte-associated antigen 4,CTLA4)的胞外区。在某个实施方案中，人CTLA4蛋白的胞外区(ECD)序列如SEQ ID NO：2所示，其为人CTLA4蛋白的第37-161位氨基酸序列(Genbank登录号NM_005214.4)。
在某些实施方案中，本发明所用的术语“中和IL-17活性的功能片段”是指靶向IL-17的免疫球蛋白或其修饰物、功能等同物、功能片段或变体。在某些实施方案中，中和IL-17活性的功能片段为靶向IL-17的IgG抗体。在某些实施方案中，所述IgG为嵌合的、人源化的或全人的IgG。在某些实施方案中，所述修饰物可以是化学修饰物，如酰基化、烷基化、PEG化产物，只要这些修饰物保留了靶向IL-17的能力即可。在某些实施方案中，所述功能等同物是指能够实现所述免疫球蛋白靶向结合IL-17能力的其他多肽片段。在某些实施方案中，所述功能片段是指保留了靶向IL-17的能力的蛋白质片段，如单一结构域抗体、单链抗体、单链可变片段(scFv)、Fab片段或F(ab’)2片段。在某些实施方案中，所 述变体是指通过在一个或多个(几个)位置的一个或多个改变，即取代、插入和/或缺失而从亲本蛋白衍生的多肽。
在某些实施方案中，所述连接片段长度为5-30个氨基酸，在某些实施方案中，所述连接片段长度为25个氨基酸，在某些实施方案中，所述连接片段的序列为GGGGSGGGGSGGGGSGGGGSGGGGS。本发明所用的连接片段并无特殊限制，只要其起到间隔融合蛋白的两个组分，使各个组分能正确形成其各自的空间结构、保留其生物活性、细胞表达水平即可。
在某些实施方案中，所述双功能融合蛋白的氨基酸序列选自下述组合：SEQ ID NO：5和6、SEQ ID NO：13和14、SEQ ID NO：4和19、SEQ ID NO：24和25、SEQ ID NO：26和27、或如上述所示的序列经替换、缺失或添加一个或多个氨基酸残基形成的具有同等功能的氨基酸序列，如1、2、3、4、5、10、15、20、30、50个氨基酸残基，或与其具有至少70％同一性且具有同等功能的氨基酸序列，如至少约75％、80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、99.1％、99.2％、99.3％、99.4％、99.5％、99.6％同一性。氨基酸的保守氨基酸是本领域公知的。
本发明使用的术语“同一性”具有本领域通常已知的含义，本领域技术人员也熟知测定不同序列间同一性的规则、标准。本发明用不同程度同一性限定的序列还必须要同时具有CTLA4分子胞外区或中和IL-17的活性。本领域技术人员公知如何利用上述活性筛选变体序列的方法和手段。本领域技术人员可以在本申请公开内容的教导下容易地获得这样的变体序列。
在一个实施方案中，本发明涉及一种编码基因，其包含编码如上所述的双功能融合蛋白的核苷酸序列。
本领域技术人员知晓，虽然本发明在限定所述编码基因时所用限定语为“包括”，但其并不意味着可以在所述编码基因两端任意加入与其功能不相关的其他序列。本领域技术人员知晓，为了满足重组操作的要求，需要在所述编码基因的两端添加合适的限制性内切酶的酶切位点，或者额外增加启动密码子、终止密码子等，因此，如果用封闭式的表述来限定所述编码基因将不能真实地覆盖这些情形。
本领域技术人员公知，在不改变所编码的氨基酸的情况下，所述编码基因序列中的一个或多个密码子可以进行等义替换，如一个或几个密码子，如1、2、3、4、5、6、8、9、10、15、20、30、40、50个密码子。密码子使用表是本领域公知的。
在一个实施方案中，本发明涉及一种重组载体，其包含有效连接其中的如上所述的编码基因。所述重组载体为重组表达载体，可以是原核表达载体也可以是真核表达载体，但优选真核表达载体，更优选用于哺乳动物真核表达的重组表达载体。
本发明所用的术语“有效连接”是指这样的连接方式，其中所述编码基因置于载体的适当位置，使得所述编码基因正确地、顺利地复制、转录或表达。
在一个实施方案中，本发明涉及一种宿主细胞，其转化或转染有如上所述的重组载体，所述宿主细胞包括哺乳动物细胞、细菌细胞、酵母菌细胞、昆虫细胞和植物细胞。
在一个优选的实施方案中，所述的宿主细胞包含CHO细胞、HEK293细胞、NSO细胞和SP 2/0细胞。
在一个实施方案中，本发明涉及一种生产所述双功能融合蛋白的方法，其包括在适宜的多肽表达条件下培养上述宿主细胞，并从细胞培养基中分离、纯化表达的多肽。优选地，所述纯化后的多肽的纯度为大于50％，更优选地，大于60％、65％、70％、75％、80％、85％、90％、95％、96％、97％、98％、99％、99.5％、99.6％、99.7％、99.8％或99.9％。
在一个实施方案中，本发明涉及一种药物组合物，其包含如上所述的双功能融合蛋白和一种药学上可接受的载体。所述双功能融合蛋白可以是所述药物组合物的唯一活性成分，也可以是所述药物组合物活性成分之一，其他活性成分为可以与所述双功能融合蛋白组合使用的其他治疗剂。
在一个优选的实施方案中，本发明的药物组合物包含单次给药的剂型、局部给药的剂型和全身给药剂型。
在一个实施方案中，本发明涉及所述的双功能融合蛋白或如上所述的药物组合物用于预防、治疗或改善类风湿关节炎、银屑病、银屑病关节炎、强直性脊柱炎、克罗恩氏病、系统性红斑狼疮、狼疮肾炎、多发性硬化症、自身免疫性脑脊髓炎、肾小球肾炎、特发性血小板减少性紫癜、原发性干燥综合征、痛风、或器官移植。在某些实施方案中，如上所述的双功能融合蛋白或如上所述的药物组合物用于预防、治疗或改善类风湿性关节炎、银屑病、银屑病关节炎、强直性脊柱炎、克罗恩氏病。
在某些实施方案中，本发明涉及预防、治疗或改善疾病的方法，所述方法包括向有此需要的受试者施用治疗有效量的如上所述的双功能融合蛋白、如上所述方法生产的双功能融合蛋白或如上所述的药物组合物的步骤。
在一个实施方案中，所述疾病为类风湿关节炎、银屑病、银屑病关节炎、强直性脊柱炎、克罗恩氏病、系统性红斑狼疮、狼疮肾炎、多发性硬化症、自身免疫性脑脊髓炎、肾小球肾炎、特发性血小板减少性紫癜、原发性干燥综合征、痛风、或器官移植。
本发明所用的术语“治疗有效量”是指在给药时，可以在受试者体内发挥药理作用的剂量。“治疗有效量”可以由本领域技术人员根据患者的情况如年龄、体重、疾病状态等容易地确定。
在一个实施方案中，本发明涉及如上所述的双功能融合蛋白或者由如上方法产生的双功能融合蛋白或者如上所述的药物组合物在制备用于预防、治疗或改善疾病的药物中的应用。
在一个实施方案中，所述疾病为类风湿关节炎、银屑病、银屑病关节炎、强直性脊柱炎、克罗恩氏病、系统性红斑狼疮、狼疮肾炎、多发性硬化症、自身免疫性脑脊髓炎、肾小球肾炎、特发性血小板减少性紫癜、原发性干燥综合征、痛风、或器官移植。
本领域技术人员知晓，虽然本发明上述内容中列出了本发明所述的双功能融合蛋白所能预防、治疗或改善的病状，但本发明的双功能融合蛋白所能处理的病状并不仅限于上述列出的具体病状，任何可以通过同时阻断B7/CD28和IL-17A/IL-17RA信号通路而获得预防、治疗或改善益处的病状都包括在本发明的保护范围之内。
根据本发明的上述技术方案，本发明具有如下有益效果：
1)将B7和IL-17A两条信号通路的阻断分子(人CTLA4的胞外区和抗IL-17抗体)融合入一个分子进行表达和生产，与分别生产CTLA4蛋白的胞外区和抗IL-17抗体相比，极大地降低了操作方法和生产成本；
2)将人CTLA4蛋白的胞外区和和抗IL-17抗体连接，目的蛋白形成四聚体形式，既满足了CTLA4胞外区发挥活性需要形成二聚体形式的要求，又保留了抗IL-17A抗体的效价仍为二价，因此，目的蛋白的总效价是三价。并且，经过连接方式的筛选对比以及体外结合实验也显示，本发明中的有效连接方式，对CTLA4分子的胞外区与其配体B7的结合，以及对抗IL-17抗体和IL-17的结合没有影响；
3)本发明的双功能蛋白经过进一步改造后，可形成嵌合体或人源化类似物，在保留其结合活性的情况下进一步减小了免疫原性。
4)疾病模型实验结果显示，双功能融合蛋白能有效地缓解疾病动物模型的炎症反应。
下面将通过下述非限制性实施例进一步说明本发明，本领域技术人员公知，在不背离本发明精神的情况下，可以对本发明做出许多修改，这样的修改也落入本发明的范围。
下述实验方法如无特别说明，均为常规方法，所使用的实验材料如无特别说明，均可容易地从商业公司获取。
实施例
实施例1：双功能融合蛋白A的表达载体的构建
(1)双功能融合蛋白A的氨基酸和编码核苷酸序列
表达双功能融合蛋白A所用信号肽序列如SEQ ID NO：1所示；人CTLA4蛋白的胞外区(ECD)序列如SEQ ID NO：2所示，其为人CTLA4蛋白的第37-161位氨基酸序列(Genbank登录号NM_005214.4)；连接肽为(G4S)5，序列如SEQ ID NO：3所示；双功能融合蛋白A中的抗IL-17A抗体(对照抗体A)的可变区序列来自专利US7846443(大鼠1D10)，恒定区序列为小鼠IgG2a的恒定区序列，即抗IL-17A抗体的重链(hc)序列如SEQ ID NO：4所示，轻链(lc)序列如SEQ ID NO：5所示；人CTLA4ECD-连接肽-anti-IL-17A mAb hc-A的氨基酸序列如SEQ ID NO：6所示。
编码SEQ ID NO：6所示氨基酸序列的核苷酸序列经过哺乳动物细胞密码子偏好性的优化，序列如SEQ ID NO：7所示。编码SEQ ID NO：5所示氨基酸序列的核苷酸序列经过哺乳动物细胞密码子偏好性的优化，序列如SEQ ID NO：8所示。SEQ ID NO：7和SEQ ID NO：8由南京金斯瑞生物科技有限公司合成，利用TA克隆进入pUC57载体并将所获得的经过测序验证的阳性克隆分别命名为pUC57-CTLA4-anti-IL-17A mAb hc-A和pUC57-anti-IL-17A mAb lc-A。
(2)双功能融合蛋白A表达载体的构建和转染质粒的准备
用pUC57-CTLA4-anti-IL-17A mAb hc-A质粒(南京金斯瑞生物科技有限公司)为模板，通过常规PCR扩增信号肽-人CTLA4ECD-连接肽-anti-IL-17A mAb hc-A的编码序列，所用上游引物带有Hind IIII酶切位点，序列CCCAAGCTTGCCACCATGGGGGTCCTG(SEQ ID NO：9)。下游引物带有EcoR I酶切位点，序列为CCGGAATTCTCATTTGCCTGGGGTTCT(SEQ ID NO：10)。
用pUC57-anti-IL-17A mAb lc-A质粒(南京金斯瑞生物科技有限公司)为模板，通过常规PCR扩增anti-IL-17A mAb lc-A的编码序列，所用上游引物带有Hind III酶切位点，为SEQ ID NO：9。下游引物带有EcoR I酶切位点，序列为 CCGGAATTCTCAGCACTCATTCCG(SEQ ID NO：11)。
将扩增得到的信号肽-人CTLA4ECD-连接肽-anti-IL-17A mAb hc-A和anti-IL-17A mAb lc-A编码序列(长度分别是1881bp和717bp)，经1％浓度的琼脂糖凝胶电泳后回收相应片段。将回收获得的基因片段和本公司真核表达载体X0GC(专利US20100120089)用Hind III和EcoR I酶切后连接，获得重组质粒X0GC-CTLA4-anti-IL-17A mAb hc-A和X0GC-anti-IL-17A mAb lc-A，分别将其转化大肠杆菌DH5α，获得重组菌DH5α/X0GC-CTLA4-anti-IL-17A mAb hc-A和DH5α/X0GC-anti-IL-17A mAb lc-A。PCR筛选阳性克隆并进行DNA测序，验证重组质粒构建正确。
将阳性DH5α/X0GC-CTLA4-anti-IL-17A mAb hc-A和DH5α/X0GC-anti-IL-17A mAb lc-A分别接种至1L的LB/Amp液体培养基(组成为1％蛋白胨(BD公司)、0.5％酵母提取物(BD公司)、1％NaCl(国药集团化学试剂有限公司))，于37℃、180rpm条件下振荡培养过夜。第二天使用无内毒素质粒大提试剂盒(Qiagen，12381)提取质粒用于HEK293-T(中国科学院上海生命科学研究院细胞资源中心)转染。
实施例2：双功能融合蛋白B的表达载体的构建
(1)双功能融合蛋白B的氨基酸和编码核苷酸序列
表达双功能融合蛋白B所用信号肽序列如SEQ ID NO：1所示；人CTLA4蛋白的ECD序列如SEQ ID NO：2所示，其为人CTLA4蛋白的第37-161位氨基酸序列(Genbank登录号NM_005214.4)；连接肽为(G4S)5，序列如SEQ ID NO：3所示；双功能融合蛋白B中的抗IL-17A抗体的序列来自专利US7846443(人源化的16C10)，其重链(hc)序列如SEQ ID NO：12所示，轻链(lc)序列如SEQ ID NO：13所示；人CTLA4ECD-连接肽-anti-IL-17A mAb hc-B的氨基酸序列如SEQ ID NO：14所示。
编码SEQ ID NO：14所示氨基酸序列的核苷酸序列经过哺乳动物细胞密码子偏好性的优化，序列如SEQ ID NO：15所示。编码SEQ ID NO：13所示氨基酸序列的核苷酸序列经过哺乳动物细胞密码子偏好性的优化，序列如SEQ ID NO：16所示。SEQ ID NO：15和SEQ ID NO：16由南京金斯瑞生物科技有限公司合成，利用TA克隆进入pUC57载体并将所获得的经过测序验证的阳性克隆分别命名为pUC57-CTLA4-anti-IL-17A mAb hc-B和pUC57-anti-IL-17A mAb lc-B。
(2)双功能融合蛋白B表达载体的构建和转染质粒的准备
用pUC57-CTLA4-anti-IL-17A mAb hc-B质粒(南京金斯瑞生物科技有限公司)为模 板，通过常规PCR扩增信号肽-人CTLA4ECD-连接肽-anti-IL-17A mAb hc-B的编码序列，所用上游引物带有Hind III酶切位点，序列为SEQ ID NO：9。下游引物带有EcoR I酶切位点，序列为CCGGAATTCTCACTTTCCTGGTGACAGACTCA(SEQ ID NO：17)。
用pUC57-anti-IL-17A mAb lc-B质粒(南京金斯瑞生物科技有限公司)为模板，通过常规PCR扩增anti-IL-17A mAb lc-B的编码序列，所用上游引物带有Hind III酶切位点，序列为SEQ ID NO：9。下游引物带有EcoR I酶切位点，序列为CCGGAATTCTCAGCACTCGCCCCGGTTG(SEQ ID NO：18)。
将扩增得到的信号肽-人CTLA4ECD-连接肽-anti-IL-17A mAb hc-B和anti-IL-17A mAb lc-B编码序列(长度分别为1887bp和735bp)，经1％浓度的琼脂糖凝胶电泳后回收相应片段。将回收获得的基因片段和本公司真核表达载体X0GC(专利US20100120089)用Hind III和EcoR I酶切后连接，获得重组质粒X0GC-CTLA4-anti-IL-17A mAb hc-B和X0GC-anti-IL-17A mAb lc-B，分别将其转化大肠杆菌DH5α，获得重组菌DH5α/X0GC-CTLA4-anti-IL-17A mAb hc-B和DH5α/X0GC-anti-IL-17A mAb lc-B。PCR筛选阳性克隆并进行DNA测序，验证重组质粒构建正确。
将阳性DH5α/X0GC-CTLA4-anti-IL-17A mAb hc-B和DH5α/X0GC-anti-IL-17A mAb lc-B分别接种至1L的LB/Amp液体培养基(组成为1％蛋白胨(BD公司)、0.5％酵母提取物(BD公司)、1％NaCl(国药集团化学试剂有限公司))，于37℃、180rpm条件下振荡培养过夜。第二天使用无内毒素质粒大提试剂盒(Qiagen,12381)提取质粒用于HEK293-T转染。
实施例3：双功能融合蛋白A和B的表达
将生长状态良好、活力(活细胞比率，也可以写成“活率”)达到95％以上的HEK293-T细胞以1.8×107个细胞的接种量接种于十层细胞工厂(NUNC公司，产品号：140400)，用含10％胎牛血清(Gibco公司)的DMEM培养基(Corning公司)培养，细胞工厂反复颠倒混匀后置于37℃、5％CO2培养箱内培养48小时，细胞贴壁完全，密度达到80％，即可用于瞬时转染。
将质粒X0GC-CTLA4-anti-IL-17A mAb hc-A和X0GC-anti-IL-17A mAb lc-A同时转染细胞，用于双功能融合蛋白A的表达。将质粒X0GC-CTLA4-anti-IL-17A mAb hc-B和X0GC-anti-IL-17A mAb lc-B同时转染细胞，用于双功能融合蛋白B的表达。用0.22μm滤膜过滤后，各吸取1330μg，加入到66ml无血清DMEM培养基中。吸取2660μg转 染试剂PEI(Sigma公司)加入到66ml无血清DMEM培养基中，分别混匀后，将获得的PEI混合液倒入含有质粒的DMEM中，混匀。室温静置15分钟后，将含有质粒与PEI的混合物加入到1.3L的无血清DMEM培养基内，充分混匀后缓慢加入到细胞工厂内。将细胞工厂置于37℃、5％CO2培养箱内培养。4小时后，加入266ml Cell Boost 5(Thermo Fisher公司)，混匀后继续培养6天，7000rpm条件下离心20min，收集上清液用于蛋白纯化。上清液经ELISA浓度测定，双功能融合蛋白A和B各批次蛋白表达量为4-8mg/L。
实施例4：双功能融合蛋白A和B的纯化
来自细胞工厂的上清液经0.2μm滤膜过滤后，再通过膜包超滤浓缩(标称截留分子量10kDa)，将溶液置换为含150mM NaCl的20mM磷酸盐溶液(pH 7.4)。在应用层析柱纯化之前，以0.2μm滤膜过滤以去除沉淀物。此步骤在4℃下进行。
采用AKTA explorer 100型蛋白纯化系统(GE Healthcare)以及亲和色谱柱rProtein A Sepharose Fast Flow(16mm I.D.，22ml，GE Healthcare)于4℃下进行纯化。首先以流动相A(20mM磷酸钠缓冲液，150mM氯化钠，pH 7.4)平衡色谱柱，在基线稳定后将经过上述处理的细胞上清液进行上样，流速为5ml/min，并在上样后以流动相A进行冲洗。之后，首先以流动相B1(含有0.5M精氨酸的流动相A)冲洗5个柱体积；然后以流动相B2(100mM柠檬酸，pH 3.0)洗脱5个柱体积，收集洗脱峰即为目的蛋白峰；以上洗脱步骤流速都为5ml/min。色谱图如图1A和B所示。收集标示的洗脱峰(图1A和B的灰色区域)，并通过滴加1M醋酸钠溶液将pH调至5.0。
采用AKTA explorer 100型蛋白纯化系统(GE Healthcare)以及强阴离子交换色谱柱HiTrap Q Sepharose FF(5ml，GE Healthcare)于4℃下进行纯化。首先以流动相A(20mM醋酸钠，pH 5.0)平衡色谱柱，在基线稳定后将上一步实验中收集并调整pH后的洗脱液进行上样，流速为5ml/min，收集流穿峰即为目的蛋白峰。上样完毕后，应用流动相B(20mM醋酸钠，1M氯化钠，pH 5.0)进行等度洗脱，流速为5ml/min。色谱图如图2A和B所示。收集标示组分(图2A和B的灰色区域)，用超滤管(标称截留分子量30kDa)浓缩，在超净工作台用0.2μm滤膜无菌过滤，然后用紫外分光光度计(280nm)进行定量，并用高效液相排阻色谱(SE-HPLC)进行纯度分析。结果如图3A和B所示，双功能融合蛋白A和B的单体纯度分别为96.6％和97.4％。
实施例5：双功能融合蛋白的稳定细胞系构建
CHO/DG44(dhfr-)细胞复苏之后传2-3代，当其存活率达到95％时，可以用于转染实验。利用Lipofectamine 2000转染试剂(Invitrogen，11668019)按照说明书进行CHO/DG44细胞的转染。具体地，将5μg质粒和3mLα-MEM培养基(SAFC,51451C)在一个离心管中混合，同时在另一个离心管里混合20μL转染剂和3mLα-MEM培养基。在洁净工作台里室温静置5分钟后，将上述稀释的质粒和转染剂混合，继续静置30分钟。然后将6mL混合物缓慢加入75cm2的细胞培养方瓶(含汇合度为70～80％的CHO/DG44细胞)中，然后将方瓶置于细胞培养箱(37℃、5％CO2)。培养过夜后，移去转染试剂混合物，加入新鲜α-MEM培养基，并添加10％透析胎牛血清(Gibco,30067-334)。然后将方瓶置于细胞培养箱(37℃、5％CO2)，培养1～2天。之后在新鲜培养基中加入1mg/mL G418(Cellgro,61-234RG)，以此培养基对细胞进行培养，持续2周时间。
为得到单克隆细胞株，需要进行有限稀释操作实验。具体地，用添加有10％透析胎牛血清和1mg/mL G418的α-MEM培养基，稀释细胞悬液，使细胞浓度为5个细胞/mL，将细胞悬液接种于96孔板中。将细胞置于细胞培养箱(37℃、5％CO2)里培养，直到单细胞长成单独的群落。通过胰酶消化的方法，挑选由单细胞长成的群落，传代到24孔板中。然后把细胞依次传代到12孔板、6孔板中。当6孔板中的单克隆细胞株汇合度达到90％时，使用ELISA方法测定目的蛋白的表达水平，并鉴定得到了表达水平较高的克隆：克隆52号(14mg/L)、克隆85号(56mg/L)、克隆103号(59mg/L)。
实施例6：双功能融合蛋白的小规模发酵
选择克隆103号在5L生物反应器(Sartorius，Bplus twin 5L)中进行生产。以5×105细胞密度接种至Hyclone SFM4CHO培养基(4g/L葡萄糖，0.3g/L谷氨酰氨)。生物反应器工作体积为2.4L，起始工艺条件为：37℃、pH 7.1、溶氧40％-60％、搅拌速度40rpm。之后，搅拌速度每天提高5rpm，并于5天后，以0.5℃/小时的速度降温，至34℃时停止降温，并加入0.3mM丁酸钠。两周后，收获反应器发酵液，7000rpm离心20分钟，收获上清液并保存。经ELISA测定，浓度为240mg/L。
实施例7：双功能融合蛋白A和C与人和小鼠IL-17A的结合亲和力
在结合亲和力实验中，构建一种类似于双功能融合蛋白A的双功能融合蛋白C。双功能融合蛋白C的氨基酸序列如SEQ ID NO：19(CTLA4ECD-连接肽-anti-IL-17A mAb lc)和SEQ ID NO：4所示，即其CTLA4ECD通过连接肽连接至anti-IL-17A mAb的轻 链(即连接至SEQ ID NO：5)，不同于双功能融合蛋白A的CTLA4ECD通过连接肽连接至anti-IL-17A mAb的重链(即连接至SEQ ID NO：4)。此外，对照抗体A作为双功能融合蛋白A和C的对照抗体，其氨基酸序列如SEQ ID NO:4(对照抗体A的重链)和SEQ ID NO:5(对照抗体A的轻链)所示，即对照抗体A对应于双功能融合蛋白A和C中的抗IL-17A抗体的部分。
该方法具体实施过程如下。用碳酸钠溶液(15mM碳酸钠，35mM碳酸氢钠，pH 9.6)包被人IL-17A(北京义翘神州，货号12047-HNAE)或者小鼠IL-17A(北京义翘神州，货号51065-MNAE)于96孔高吸附酶标板(Corning，2592)上，包被浓度为1μg/mL，100μL每孔，4℃包被过夜。PBST(Sigma,货号P-3563)洗涤该板5次。用含1％BSA的PBST封闭该板，300μL每孔，25℃孵育至少1小时。PBST洗涤该板5次。加入稀释在含1％BSA的PBST里的特定浓度的待测样品，100μL每孔，25℃孵育1小时。PBST洗涤该板5次。然后加入1:10000稀释在含1％BSA的PBST里的辣根过氧化物酶标记的抗小鼠IgG抗体(Abcam，货号Ab7068)，100μL每孔，25℃孵育1小时。PBST洗涤该板5次。加入比色底物TMB(BD OptEIA，货号555214)，100μL每孔，室温显色10分钟。加入1M H2SO4，100μL每孔，终止显色。在酶标仪上读取450nm处的吸光度。
结果如图4和5所示，本发明中的双功能融合蛋白A和双功能蛋白C具有与其抗原人IL-17A(图4)和小鼠IL-17A(图5)结合的高亲和力，且亲和力与对照抗体A相似。以上结果表明，通过连接肽连接于对照抗体A重链或轻链N端的CTLA4ECD，不会显著影响对照抗体A本身与抗原的亲合力。
此外，需要说明的是，本发明中的双功能融合蛋白B不能与小鼠IL-17A结合，但具有与人IL-17A结合的高亲和力(本发明利用ELISA方法测得的数值是EC50＝103pM)，与专利(US7846443)中的数值接近。
实施例8：CTLA4的不同连接形式对双功能融合蛋白与B7亲和力的影响
为了研究比较CTLA4的不同连接形式对双功能融合蛋白与B7亲合力的影响，我们构建了具有不同CTLA4连接形式的双功能融合蛋白类似物：
(1)双功能融合蛋白C：序列如前所述，即SEQ ID NO：19(CTLA4ECD-连接肽-anti-IL-17A mAb lc)和SEQ ID NO：4，即其CTLA4ECD通过连接肽连接至anti-IL-17A mAb轻链的N端(即连接至SEQ ID NO：5)，不同于双功能融合蛋白A的CTLA4ECD通过连接肽连接至anti-IL-17A mAb重链的N端(即连接至SEQ ID NO：4)。
(2)双功能融合蛋白D：所用信号肽序列如SEQ ID NO：1所示；人CTLA4蛋白的胞外区(ECD)序列如SEQ ID NO：2所示；连接肽为(G4S)5，序列如SEQ ID NO：3所示，抗IL-17A抗体的重链(hc)序列如SEQ ID NO：4所示，轻链(lc)序列如SEQ ID NO：5所示；anti-IL-17A mAb hc-A-连接肽-CTLA4ECD的氨基酸序列如SEQ ID NO：20所示，即双功能融合蛋白D的CTLA4通过连接肽连接于对照抗体A重链的C端。
(3)双功能融合蛋白E：所用信号肽序列如SEQ ID NO：1所示；人CTLA4蛋白的胞外区(ECD)序列如SEQ ID NO：2所示；连接肽为(G4S)5，序列如SEQ ID NO：3所示，抗IL-17A抗体的重链(hc)序列如SEQ ID NO：4所示，轻链(lc)序列如SEQ ID NO：5所示；anti-IL-17A mAb lc-A-连接肽-CTLA4ECD的氨基酸序列如SEQ ID NO：21所示，即双功能融合蛋白E的CTLA4通过连接肽连接于对照抗体A轻链的C端。
CTLA4ECD可与其配体B7分子结合。在B7结合亲和力测定实验中，用流式细胞术在高表达B7分子的Raji细胞上测定双功能融合蛋白A和C以及D和E与B7分子的结合能力。该方法具体实施过程如下。1000rpm离心5分钟收集Raji细胞(ATCC，货号CCL-86)。用含2％FBS(GIBCO，货号10099141)的PBS(PromoCell，货号C-40232)洗涤一次。将Raji细胞在EP管中重悬，每管1×106个细胞，重悬于200μL含2％FBS且含有终浓度为0.5nM的待测样品的冷PBS中。EP管于冰上孵育30分钟。用含2％FBS的PBS洗涤两次。然后再重悬于200μL含2％FBS且含有FITC标记的抗小鼠IgG抗体(1:50稀释)的冷PBS中。冰上避光孵育30分钟。用含2％FBS的PBS洗涤两次。再重悬于500μL含2％FBS的冷PBS中，该细胞悬液于流式细胞仪上进行检测分析。
结果如表1所示，双功能融合蛋白A具有与高表达其配体B7分子的Raji细胞结合的能力，且结合力显著大于双功能融合蛋白C、双功能融合蛋白D和双功能融合蛋白E。以上结果表明，CTLA4ECD与抗IL-17A抗体的不同连接形式可显著影响其与B7的结合能力。
表1.双功能融合蛋白与高表达B7分子的Raji细胞的结合能力
Figure PCTCN2015071718-appb-000001
Figure PCTCN2015071718-appb-000002
实施例9：双功能融合蛋白A抑制IL-17A诱导的HT-29细胞分泌GROα
人结直肠癌细胞株HT-29来自ATCC。HT-29细胞在含有10％胎牛血清(Gibco,货号:10099)的RPMI-1640培养基(Gibco,货号:22400)中培养，置于37℃、5％CO2细胞孵箱中，每周传代两次。
抑制IL-17A诱导的GROα分泌实验具体操作如下。将待测样品梯度稀释于含10％胎牛血清的RPMI 1640完全培养基中，每孔50μL加入细胞培养板(Coring，货号3599)中。再向细胞培养板中加入稀释于相同完全培养基的浓度为240ng/mL的人IL-17A，每孔50μL。置于37℃、5％CO2培养箱中孵育1小时。然后将HT-29细胞重悬在完全培养基中，接种到96孔细胞培养板内，每孔100μL，约20000个细胞。细胞在37℃、5％CO2培养箱中孵育培养48小时。将细胞培养板1000rpm离心5分钟，取出培养上清。按照说明书，用GROαELISA试剂盒(R&D，货号DY275)测定GROα水平。
结果如图6所示，对照抗体A和双功能融合蛋白A均可抑制IL-17A诱导的HT-29细胞分泌GROα，IC50分别是532pM和911pM。
实施例10：双功能融合蛋白B抑制人外周血单个核细胞分泌IL-2
通过混合淋巴细胞反应测定本发明的双功能融合蛋白B的免疫抑制活性，即抑制T淋巴细胞活化的活性。简要的说，取来自两位不同个体的两份人外周血单个核细胞(PBMC)，其中一份PBMC经灭活作为刺激细胞，另一份PBMC作为反应细胞，混合这两个PBMC，刺激细胞即可活化反应细胞的免疫反应，并大量分泌细胞因子IL-2。将样品与这个混合PBMC体系共孵育，检测其对IL-2分泌的影响。
淋巴细胞混合反应的具体实施方法如下。将两支来自不同个体的不同批号PBMC(Lonza，货号CC-2702)复苏重悬于含10％胎牛血清(Gibco,货号10099)的RPMI-1640(Gibco,货号22400)完全培养基中。一支PBMC作为刺激细胞，一支PBMC作为反应细胞。刺激细胞与50μg/ml的丝裂霉素C(Wako,货号50-07-7)在37℃条件下共孵育45分钟，然后用DPBS(PromoCell,货号C-40232)洗涤细胞两次，然后细胞再重悬于RPMI1640完全培养基中。刺激细胞和反应细胞均进行计数(Innovatis Cedex)和细胞活率测定(台盼蓝染料，Invitrogen公司，货号15250-061)。然后将反应细胞接种于96孔细胞培养板(Corning，货号3799)，每孔50μL，每孔1×105个细胞，再每孔加入100μL 的设定浓度的双功能融合蛋白B、已上市的阳性对照药物阿巴西普(OrenciaTM，百时美施贵宝公司)或依那西普(益赛普，上海中信国健药业股份有限公司)，每个样品三个复孔。每孔加入50μL刺激细胞，每孔1×105个细胞。将细胞培养板置于37℃、5％CO2细胞孵箱中孵育。72小时之后，每孔取出100μL培养上清，保存于-70℃。按照说明书，用IL-2ELISA试剂盒(Raybiotech，货号ELH-IL2-001)测定IL-2水平。
结果如图7所示，在混合人PBMC反应中，双功能融合蛋白B、阿巴西普和依那西普抑制IL-2分泌的EC50分别是3nM、12nM和21nM，双功能融合蛋白B显示出了比已上市的阳性对照药物阿巴西普和依那西普更强的IL-2分泌抑制活性。额外的试验也表明，双功能融合蛋白A也表现出了较强的IL-2分泌抑制活性，活性接近于阿巴西普。
实施例11：双功能融合蛋白A的嵌合体与人源化类似物
我们将双功能融合蛋白A的恒定区氨基酸序列，即对照抗体A的恒定区氨基酸序列，替换为人IgG1恒定区序列。人IgG1重链恒定区序列如SEQ ID NO：22所示，轻链恒定区序列如SEQ ID NO：23所示。由此得到了双功能融合蛋白A嵌合体类似物，其氨基酸序列如SEQ ID NO：24和SEQ ID NO：25所示。该嵌合体类似物可减少鼠源性成分，从而有效减少蛋白的免疫原性。
此外，我们进一步对SEQ ID NO：24和SEQ ID NO：25进行部分序列改造，将其可变区域内的骨架区氨基酸进行替换和突变，以进一步减少鼠源性成分。由此得到的双功能融合蛋白A人源化类似物的氨基酸序列如SEQ ID NO：26和SEQ ID NO：27所示。
我们将双功能融合蛋白A、嵌合体及其人源化类似物进行了抗原亲合力测定。结果如表2所示，嵌合体和人源化类似物较好的保留了母体的抗原结合活性。
表2.双功能融合蛋白A、嵌合体和人源化类似物与人IL-17A和B7的亲合力
Figure PCTCN2015071718-appb-000003
实施例12：双功能融合蛋白A人源化类似物在Ⅱ型胶原诱导的大鼠关节炎(CIA)模型中的药效研究
大鼠CIA模型是广泛应用于类风湿性关节炎发病机制研究和治疗药物筛选的动物模型。实验动物选用7周龄雌性Wistar大鼠(购自北京华阜康生物科技股份有限公司)，适应环境一周后，随机选取5只大鼠作为正常对照大鼠，其余大鼠用于建立大鼠CIA模型。大鼠经初次免疫和加强免疫得到CIA模型。初次免疫使用200μgⅡ型牛胶原(Chondrex,货号20022)与弗氏不完全佐剂(Sigma-Aldrich,货号F5506)混合形成乳液，于大鼠尾根部皮内注射。一周以后，进行加强免疫。加强免疫使用100μgⅡ型牛胶原与弗氏不完全佐剂混合形成乳液，于大鼠尾根部皮内注射。在加强免疫后观察到大鼠四肢足爪红肿等临床性关节炎症状之后，随机进行分组，每组6只。
对CIA模型大鼠各组分别给药。分别给予药物溶媒(PBS)、阿巴西普(272nmol/kg，分别在第1、5、8、11天进行给药)、双功能融合蛋白A人源化类似物(33nmol/kg，每两天给药一次，腹腔注射，共给药7次)。给药后，观察前后四肢足爪病变情况，并进行关节炎指数评分：0＝无红肿，1＝踝关节或者跗骨关节有红斑、轻微肿胀，2＝从踝关节到跗骨关节均有红斑、轻微肿胀，3＝从踝关节到跖骨关节均有红斑、中等程度肿胀，4＝踝关节、足爪包括趾骨关节均有红斑、严重程度肿胀或者四肢关节僵硬，大鼠四肢都给予炎症评分，每只鼠的最大评分为16分。
实验结果如图8所示，正常对照组大鼠没有产生炎症反应，溶媒组的CIA模型大鼠产生明显炎症反应，双功能融合蛋白A人源化类似物给药组的CIA模型大鼠关节炎指数得到了明显的抑制，显示出了比阿巴西普给药组更好的炎症缓解效果。

Claims (10)

  1. 一种双功能融合蛋白，其包含CTLA4分子胞外区和抗IL-17抗体，优选地，所述IL-17是IL-17A。
  2. 权利要求1所述的双功能融合蛋白，其氨基酸序列选自以下序列的组合：
    a)如SEQ ID NO：5和6、SEQ ID NO：13和14、SEQ ID NO：4和19、SEQ ID NO：24和25、SEQ ID NO：26和27所示的序列；
    b)如SEQ ID NO：5和6、SEQ ID NO：13和14、SEQ ID NO：4和19、SEQ ID NO：24和25、SEQ ID NO：26和27所示的序列经替换、缺失或添加一个或多个氨基酸残基形成的具有同时阻断B7和IL-17A信号通路活性的氨基酸序列；或
    c)与SEQ ID NO：5和6、SEQ ID NO：13和14、SEQ ID NO：4和19、SEQ ID NO：24和25、SEQ ID NO：26和27所示的序列具有至少70％同一性并具有同时阻断B7和IL-17A信号通路活性的氨基酸序列。
  3. 一种能够编码权利要求1-2任一项所述的双功能融合蛋白的编码基因。
  4. 一种重组载体，其包含有效连接其中的权利要求3所述的编码基因。
  5. 一种宿主细胞，其含有权利要求4所述的重组载体。
  6. 一种制备权利要求1至2任一项所述的双功能融合蛋白的方法，其包括步骤：
    (1)将权利要求3所述编码基因有效连接至真核表达载体中并转染至宿主细胞进行表达或将权利要求4所述的重组载体转染至宿主细胞进行表达；和
    (2)纯化所述双功能融合蛋白，
    优选地，所述真核表达载体为X0GC，优选地，所述宿主细胞为HEK293-T或CHO细胞。
  7. 一种药物组合物，其包含权利要求1至2任一项所述的双功能融合蛋白或权利要求6所述的方法产生的双功能融合蛋白。
  8. 权利要求1至2所述的蛋白或者由权利要求6产生的蛋白或者权利要求7所述的药物组合物在制备用于预防、治疗或改善疾病的药物中的应用。
  9. 如权利要求8所述的应用，所述疾病为类风湿关节炎、银屑病、银屑病关节炎、强直性脊柱炎、克罗恩氏病、系统性红斑狼疮、狼疮肾炎、多发性硬化症、自身免疫性脑脊髓炎、肾小球肾炎、特发性血小板减少性紫癜、原发性干燥综合征、痛风、或器官移植。
  10. 一种试剂盒，其包含如权利要求1至2所述的蛋白、如权利要求3所述的编码基因、如权利要求4所述的重组载体或如权利要求5所述的宿主细胞。
PCT/CN2015/071718 2014-01-28 2015-01-28 双功能融合蛋白及其制备方法和用途 WO2015113494A1 (zh)

Priority Applications (5)

Application Number Priority Date Filing Date Title
EP15742538.0A EP3101035B1 (en) 2014-01-28 2015-01-28 Bifunctional fusion protein, preparation method therefor, and use thereof
KR1020167023486A KR20160113268A (ko) 2014-01-28 2015-01-28 이기능 융합단백질,이의 제조방법 및 용도
US15/114,006 US20160340422A1 (en) 2014-01-28 2015-01-28 Bifunctional fusion protein, preparation method therefor, and use thereof
JP2016548092A JP2017508446A (ja) 2014-01-28 2015-01-28 二機能性融合タンパク質及びその製造方法並びに使用
CN201580004917.1A CN105916883B (zh) 2014-01-28 2015-01-28 双功能融合蛋白及其制备方法和用途

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201410042808.6 2014-01-28
CN201410042808 2014-01-28

Publications (1)

Publication Number Publication Date
WO2015113494A1 true WO2015113494A1 (zh) 2015-08-06

Family

ID=53756234

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2015/071718 WO2015113494A1 (zh) 2014-01-28 2015-01-28 双功能融合蛋白及其制备方法和用途

Country Status (6)

Country Link
US (1) US20160340422A1 (zh)
EP (1) EP3101035B1 (zh)
JP (1) JP2017508446A (zh)
KR (1) KR20160113268A (zh)
CN (1) CN105916883B (zh)
WO (1) WO2015113494A1 (zh)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11319359B2 (en) 2015-04-17 2022-05-03 Alpine Immune Sciences, Inc. Immunomodulatory proteins with tunable affinities
US11753458B2 (en) 2017-10-10 2023-09-12 Alpine Immune Sciences, Inc. CTLA-4 variant immunomodulatory proteins and uses thereof

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2019079520A2 (en) 2017-10-18 2019-04-25 Alpine Immune Sciences, Inc. ICOS VARIANT LIGAND IMMUNOMODULATORY IMMUNOMODULATORY PROTEINS, COMPOSITIONS AND METHODS THEREOF
EP4308608A1 (en) * 2021-03-16 2024-01-24 JN Biosciences LLC Bifunctional molecules for treatment of immune disorders

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101001645A (zh) * 2004-08-05 2007-07-18 诺瓦提斯公司 Il－17拮抗抗体
WO2009147362A1 (en) * 2008-06-03 2009-12-10 Imperial Innovations Limited Method of rheumatoid arthritis treatment
US20100120089A1 (en) 2006-10-16 2010-05-13 Hanmi Pharmaceutical Co., Ltd. A novel vector and expression cell line for mass production of recombinant protein and a process of producing recombinant protein using same
US7846443B2 (en) 2006-08-11 2010-12-07 Schering Corporation Antibodies to IL-17A
WO2013041029A1 (en) * 2011-09-23 2013-03-28 Igenimed Pharmaceuticals Inc. Novel soluble ctla4 variants

Family Cites Families (14)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101370525B (zh) * 2005-08-19 2013-09-18 Abbvie公司 双重可变结构域免疫球蛋白及其用途
GB0620934D0 (en) * 2006-10-20 2006-11-29 Cambridge Antibody Tech Protein variants
EP2331575B1 (en) * 2008-09-29 2014-05-28 Roche Glycart AG Antibodies against human il 17 and uses thereof
TWI496582B (zh) * 2008-11-24 2015-08-21 必治妥美雅史谷比公司 雙重專一性之ｅｇｆｒ／ｉｇｆｉｒ結合分子
EP2435477A1 (en) * 2009-05-28 2012-04-04 Glaxo Group Limited Antigen-binding proteins
US20120100166A1 (en) * 2010-07-15 2012-04-26 Zyngenia, Inc. Ang-2 Binding Complexes and Uses Thereof
KR20180129991A (ko) * 2010-11-05 2018-12-05 노파르티스 아게 Ｉｌ-１７ 길항제를 사용한 류마티스성 관절염의 치료 방법
SG11201406238UA (en) * 2012-04-05 2014-10-30 Hoffmann La Roche Bispecific antibodies against human tweak and human il17 and uses thereof
AU2013260172B2 (en) * 2012-05-11 2017-11-23 Medimmune Limited CTLA-4 variants
US20140219913A1 (en) * 2012-12-28 2014-08-07 Abbvie, Inc. Dual Specific Binding Proteins Having a Receptor Sequence
CN103232542B (zh) * 2013-02-01 2015-05-13 殷勇 一种双靶标嵌合蛋白
WO2014149067A1 (en) * 2013-03-15 2014-09-25 Momenta Pharmaceuticals, Inc. Methods related to ctla4-fc fusion proteins
BR112015023752B1 (pt) * 2013-03-15 2023-11-14 Zyngenia, Inc. Domínio de reconhecimento modular (mrd), complexo compreendendo mrd e cetuximabe, usos do complexo para inibir a angiogênese e tratar câncer e composição farmacêutica compreendendo o dito complexo
WO2015126548A1 (en) * 2014-02-21 2015-08-27 Ibc Pharmaceuticals, Inc. Disease therapy by inducing immune response to trop-2 expressing cells

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101001645A (zh) * 2004-08-05 2007-07-18 诺瓦提斯公司 Il－17拮抗抗体
US7846443B2 (en) 2006-08-11 2010-12-07 Schering Corporation Antibodies to IL-17A
US20100120089A1 (en) 2006-10-16 2010-05-13 Hanmi Pharmaceutical Co., Ltd. A novel vector and expression cell line for mass production of recombinant protein and a process of producing recombinant protein using same
WO2009147362A1 (en) * 2008-06-03 2009-12-10 Imperial Innovations Limited Method of rheumatoid arthritis treatment
WO2013041029A1 (en) * 2011-09-23 2013-03-28 Igenimed Pharmaceuticals Inc. Novel soluble ctla4 variants

Non-Patent Citations (7)

* Cited by examiner, † Cited by third party
Title
CHABAUD M; DURAND JM; BUCHS N ET AL., ARTHRITIS RHEUM., vol. 42, 1999, pages 963 - 70
GENOVESE MC; BECKER JC; SCHIFFM ET AL., N. ENGL. J. MED., vol. 353, 2005, pages 1114 - 1123
KELLNER H, THER ADV MUSCULOSKELET DIS., vol. 5, 2013, pages 141 - 152
LIN Y; RITCHEA S; LOGAR A., IMMUNITY, vol. 31, 2009, pages 799 - 810
LUBBERTS E; KOENDERS MI; OPPERS-WALGREEN B ET AL., ARTHRITIS RHEUM., vol. 50, 2004, pages 650 - 659
MIOSSEC P; KOLLS JK, NAT. REV. DRUG. DISCOV., vol. 11, 2012, pages 763 - 776
See also references of EP3101035A4

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11319359B2 (en) 2015-04-17 2022-05-03 Alpine Immune Sciences, Inc. Immunomodulatory proteins with tunable affinities
US11753458B2 (en) 2017-10-10 2023-09-12 Alpine Immune Sciences, Inc. CTLA-4 variant immunomodulatory proteins and uses thereof

Also Published As

Publication number Publication date
CN105916883A (zh) 2016-08-31
EP3101035A1 (en) 2016-12-07
EP3101035B1 (en) 2021-10-06
KR20160113268A (ko) 2016-09-28
CN105916883B (zh) 2019-12-03
JP2017508446A (ja) 2017-03-30
EP3101035A4 (en) 2017-07-19
US20160340422A1 (en) 2016-11-24

Similar Documents

Publication Publication Date Title
CN107001468B (zh) Cd3结合结构域
JP6152433B2 (ja) 抗ｔｎｆ−抗ｉｌ−１７二重特異性抗体
WO2020186974A1 (zh) 一种双特异性抗体及其制备方法与应用
TWI701259B (zh) ４﹘１ｂｂ抗體及其製備方法和應用
CA3004830A1 (en) Composition and methods for anti-tnfr2 antibodies
KR20070020048A (ko) 항체
CN101983207A (zh) 抗mif抗体
CA2976623C (en) Fc fusion high affinity ige receptor .alpha.-chain
WO2017107885A1 (zh) 抗人程序性死亡受体1抗体及其制备方法和用途
TW201726731A (zh) 對TNF-α、IL-17A及IL-17F具特異性之多重特異性抗體分子
CN106336459B (zh) 抗人白细胞介素-17a单克隆抗体、其制备方法和应用
WO2015113494A1 (zh) 双功能融合蛋白及其制备方法和用途
KR20240046224A (ko) 이중특이성 항체 및 그 용도
CA3144567A1 (en) Polypeptides
US11667704B2 (en) Anti-IL-17 antibody/TNFR ECD fusion protein and use thereof
CN104231086B (zh) 双功能融合蛋白及其制备方法和用途
WO2019192493A1 (zh) 抗人lag-3单克隆抗体及其应用
CN109641958B (zh) huTNFR1相互作用的单价抑制剂
TWI825214B (zh) 抗TNFα/抗IL-17A天然抗體結構樣異源二聚體形式雙特異抗體及其製備
CN116323671A (zh) 具有增加的选择性的多靶向性双特异性抗原结合分子
CN107074976B (zh) 同时阻断b7/cd28和il6/il6r/gp130信号通路的重组融合蛋白
WO2022141378A1 (zh) 一种抗pd-1的单域抗体
WO2024046301A1 (zh) 包含taci多肽的融合蛋白及其用途
WO2023186113A9 (zh) 靶向pd-l1和cd40的抗原结合蛋白及其制备和应用
CA3194384A1 (en) Caninized rat antibodies to canine interleukin-31 receptor alpha

Legal Events

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

Ref document number: 15742538

Country of ref document: EP

Kind code of ref document: A1

ENP Entry into the national phase

Ref document number: 2016548092

Country of ref document: JP

Kind code of ref document: A

REEP Request for entry into the european phase

Ref document number: 2015742538

Country of ref document: EP

WWE Wipo information: entry into national phase

Ref document number: 2015742538

Country of ref document: EP

WWE Wipo information: entry into national phase

Ref document number: 15114006

Country of ref document: US

NENP Non-entry into the national phase

Ref country code: DE

ENP Entry into the national phase

Ref document number: 20167023486

Country of ref document: KR

Kind code of ref document: A