WO2018032638A1 - 用于构建融合蛋白的连接肽 - Google Patents

用于构建融合蛋白的连接肽 Download PDF

Info

Publication number
WO2018032638A1
WO2018032638A1 PCT/CN2016/106011 CN2016106011W WO2018032638A1 WO 2018032638 A1 WO2018032638 A1 WO 2018032638A1 CN 2016106011 W CN2016106011 W CN 2016106011W WO 2018032638 A1 WO2018032638 A1 WO 2018032638A1
Authority
WO
WIPO (PCT)
Prior art keywords
fusion protein
linker peptide
peptide
amino acid
family
Prior art date
Application number
PCT/CN2016/106011
Other languages
English (en)
French (fr)
Inventor
李强
李媛丽
陈思
王著
董炤
李子瑞
马心鲁
杨璐
高永娟
郑云程
孙乃超
Original Assignee
安源医药科技（上海）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
Priority claimed from CN201610694914.1A external-priority patent/CN106317226B/zh
Priority claimed from CN201610692679.4A external-priority patent/CN106117370B/zh
Application filed by 安源医药科技（上海）有限公司 filed Critical 安源医药科技（上海）有限公司
Priority to EP16913393.1A priority Critical patent/EP3502143A4/en
Priority to US16/326,412 priority patent/US11123438B2/en
Publication of WO2018032638A1 publication Critical patent/WO2018032638A1/zh
Priority to US17/391,535 priority patent/US11833212B2/en
Priority to US18/492,958 priority patent/US20240108743A1/en

Links

Images

Classifications

    • 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
    • A61P3/04—Anorexiants; Antiobesity agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/65—Peptidic linkers, binders or spacers, e.g. peptidic enzyme-labile linkers
    • 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
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • 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
    • C07K14/475—Growth factors; Growth regulators
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/54—Interleukins [IL]
    • C07K14/5418—IL-7
    • 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
    • C07K14/745—Blood coagulation or fibrinolysis factors
    • C07K14/755—Factors VIII, e.g. factor VIII C (AHF), factor VIII Ag (VWF)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/06—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies from serum
    • C07K16/065—Purification, fragmentation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2809—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against the T-cell receptor (TcR)-CD3 complex
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2887—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against CD20
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/46—Hybrid immunoglobulins
    • C07K16/468—Immunoglobulins having two or more different antigen binding sites, e.g. multifunctional antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells

Definitions

  • the present invention relates to the field of fusion proteins and, more particularly, to a linker peptide for use in the construction of a fusion protein.
  • linkers has several advantages over other fusion strategies.
  • the amino acids that make up the linker peptide are diverse (20 common amino acids), and the length of the linker peptide is also an important tunable parameter, which can lead to a rich diversity of linker peptides (20 n-th power, n is a link
  • the number of amino acid residues of the peptide is favorable for engineering;
  • the linker peptide can provide a certain spatial interval for the two proteins to be fusion, so as to tend to fold correctly without mutual interference;
  • the linker peptide It also provides the possibility of more interactions between the two proteins to be fused, promoting synergy between them.
  • linker peptides There are currently two commonly used linker peptides, a rigid linker peptide in the form of a helix (such as A(EAAAK) n A) and a flexible linker peptide composed of a low hydrophobicity, low charge effect amino acid.
  • the rigid linker peptide in the form of a helix can effectively separate the different functional regions of the fusion protein.
  • flexible linker peptides include the Huston designed (GGGGS) 3 sequence.
  • GGGGS Huston designed
  • CTP carboxy terminal peptide of the human chorionic gonadotropin
  • CTP carboxy terminal peptide of the human chorionic gonadotropin
  • CTP acts as a linker between the beta subunit and the alpha subunit of follicle stimulating hormone; among the fusion proteins disclosed in WO2005058953A2, CTP is used as a linker for The glycoprotein hormone beta subunit and alpha subunit are linked.
  • CTP has the effect of prolonging the half-life of certain proteins in vivo, it is mainly disclosed in more patent documents as an extended half-life part of the fusion protein, which can optionally use immunoglobulin Fc, CTP or other prolonged half-life. Fusion ligand.
  • Luo et al found the dengue virus NS2B protein (the N-terminal serine protease, the C-terminal RNA helicase, the linker peptide is a polypeptide of 11 amino acid residues), by inserting glycine between the original 173 and 174 sites, or The substitution of proline at position 174 to glycine resulted in a significant decrease in the activity of the protein at both ends, indicating that the length and rigidity of the naturally linked peptide are the result of long-term evolution and are important for the function of the natural fusion protein ( Luo D et al, J Biol Chem., 2010, 285(24): 18818-18827).
  • the linker peptide should have the following characteristics: 1) enable the linked protein to be effectively folded, in a proper configuration, without causing molecular dynamics changes, and preferably selecting a non-immunogenic natural amino acid; ) should have anti-protease attack ability; 3) should avoid the mutual interference of the amino acid sequences at both ends of the fusion protein.
  • linker peptides In the absence of clear guidelines for the design and construction of linker peptides, the inventors have been inspired by their long-term research experience, especially in the study of Fc fusion proteins, and developed new linker peptides for the construction of fusion proteins. Surprisingly, it is more conducive to maintaining the biological activity of the protein or polypeptide, and has wide applicability and portability.
  • a first aspect of the invention provides a linker peptide consisting of a flexible peptide comprising one or more flexible units, and a rigid peptide comprising one or more rigid units, wherein
  • the flexible unit comprises two or more amino acid residues selected from the group consisting of Gly, Ser, Ala and Thr; and the rigid unit comprises a carboxy terminal peptide of human chorionic gonadotropin beta subunit (referred to herein as CTP) ).
  • the linker peptide is glycosylated. More preferably, a glycosylation site is located on the CTP. More preferably, the glycosylation process is carried out in mammalian cells (e.g., Chinese hamster ovary cells) or other expression vectors having a suitable glycosylation modification system.
  • mammalian cells e.g., Chinese hamster ovary cells
  • other expression vectors having a suitable glycosylation modification system.
  • the rigid peptide constituting the linker peptide of the present invention is located at the N-terminus or C-terminus of the flexible peptide; preferably, the rigid peptide is located at the C-terminus of the flexible peptide.
  • the structural formula of the linker peptide of the present invention may be represented by F-R or R-F, wherein F and R represent a flexible peptide and a rigid peptide, respectively.
  • the flexible peptide preferably comprises 1, 2, 3, 4 or 5 flexible units
  • the rigid peptide preferably comprises 1, 2, 3, 4 or 5 rigid units.
  • the flexible unit comprises 2 or more G and S residues. More preferably, the flexible unit has the amino acid sequence of (GS) a (GGS) b (GGGS) c (GGGGS) d , wherein a, b, c and d respectively represent a structure composed of G and S residues The number of cells, and all are integers greater than or equal to 0, and a+b+c+d ⁇ 1.
  • the rigid unit is selected from the full length sequence consisting of amino acids 113 to 145 of the carboxy terminus of human chorionic gonadotropin ⁇ subunit or a fragment thereof (hereinafter referred to as CTP), in particular, the rigid unit comprises SEQ ID NO: 1 or its truncated sequence.
  • the CTP comprises at least 2 glycosylation sites; for example, a preferred embodiment of the invention Wherein the CTP comprises two glycosylation sites, exemplarily, the CTP comprises 10 amino acids of the N-terminus of SEQ ID NO: 1 , ie SSSS*KAPPPS*, or the CTP comprises 14 of the SEQ ID NO: 1 C-terminus An amino acid, ie, S*RLPGPS*DTPILPQ; as another example, the CTP comprises three glycosylation sites, exemplarily, the CTP comprises 16 amino acids of the N-terminus of SEQ ID NO: 1 SSSS*KAPPPS*LPSPS*R; as in other embodiments, the CTP comprises four glycosylation sites, exemplarily, the CTP comprises 28, 29, 30, 31, 32 or 33 amino acids It begins at position 113, 114, 115, 116, 117 or 118 of the human chorionic gonadotropin beta subunit and ends at position 145.
  • the CTP comprises 28 amino acids of the N-terminus of SEQ ID NO: 1, namely SSSS*KAPPPS*LPSPS*RLPGPS*DTPILPQ.
  • * represents a glycosylation site.
  • the rigid units provided herein are at least 70% homologous to the native CTP amino acid sequence; in other embodiments, the rigid units provided herein are at least 80% homologous to the native CTP amino acid sequence; In some embodiments, the rigid units provided herein are at least 90% homologous to the native CTP amino acid sequence; in other embodiments, the rigid units provided herein are at least 95% homologous to the native CTP amino acid sequence.
  • the rigid peptide of the present invention may further comprise two or three of the above CTP rigid units.
  • the rigid peptide comprises two R3 rigid units: SSSSKAPPPSSSSSKAPPPS (denoted as R3R3);
  • the rigid peptide comprises three R4 rigid units: SRLPGPSDTPILPQSRLPGPSDTPILPQSRLPGPSDTPILPQ (denoted as R4R4R4).
  • the linker peptide has the amino acid sequence set forth in SEQ ID NO: 2 (GSGGGGSGGGGSGGGGSGGGGSGGGGGGSSSSSKAPPPSLPSPSRLPGPSDTPILPQ) (denoted as F2-R1).
  • the linker peptide has the amino acid sequence (denoted as F1-R2) represented by SEQ ID NO: 3 (GSGGGSGGGGSGGGGSPRFQDSSSSKAPPPSLPSPSRLPGPSDTPILPQ).
  • the linker peptide has the amino acid sequence (denoted as F4-R1) represented by SEQ ID NO: 4 (GSGGGGSGGGGSGGGGSSSSSKAPPPSLPSPSRLPGPSDTPILPQ).
  • the linker peptide has the amino acid sequence (expressed as F5-R5) represented by SEQ ID NO: 5 (GGGSGGGSGGGSGGGSGGGSSSSSKAPPPSLPSPSR).
  • the linker peptide has the amino acid sequence (expressed as F4-R3R3) represented by SEQ ID NO: 6 (GSGGGGSGGGGSGGGGSSSSSKAPPPSSSSSKAPPPS).
  • the linker peptide has the amino acid sequence (expressed as F6-R4R4R4) represented by SEQ ID NO: 7 (GGSGGSGGSGGSSRLPGPSDTPILPQSRLPGPSDTPILPQSRLPGPSDTPILPQSRLPGPSDTPILPQ).
  • a fusion protein comprising the linker peptide.
  • the fusion protein comprises two biologically active molecules and a linker peptide linking the two active molecules.
  • the structural formula of the fusion protein is represented by: K1-L-K2 or K2-L-K1, wherein K1 is the first biologically active molecule; L is the above-described linking peptide; K2 is the second biologically active molecule, and is composed of The portions of the fusion protein are sequentially linked from the N-terminus to the C-terminus.
  • the active molecule may be selected from a protein or protein domain, a polypeptide, an antibody or an antibody fragment, preferably a protein or protein domain, an antibody or an antibody fragment.
  • the active molecule K1 of the fusion protein comprises a biologically functional protein or protein domain, a polypeptide, in particular a soluble or membrane signaling molecule, a cytokine, a growth factor, a hormone, a costimulatory molecule, an enzyme, a receptor And a protein or polypeptide having a ligand function for the receptor; and the active molecule K2 is a serum protein or protein domain that prolongs the circulating half-life, for example, human serum albumin (HSA), transferrin (TF) or antibody/ Immunoglobulin Fc fragment and the like.
  • HSA human serum albumin
  • TF transferrin
  • the active molecule K1 of the fusion protein comprises a toxin, an enzyme, a cytokine, a membrane protein or an immunomodulatory cytokine; and the active molecule K2 comprises an antibody or antibody fragment, and K1 and K2 are linked by the linker peptide
  • An antibody fusion protein is formed.
  • K2 is an antibody Fv segments (V L or V H); another example, K2 is a single chain antibody (ScFv).
  • the biologically active molecule K1 comprises, but is not limited to, adenosine A1 receptor, angiotensin converting enzyme ACE, Activin family, ADAM family, ALK family, ⁇ -1-antitrypsin, family of apoptosis-related proteins.
  • EGF epidermal growth factor EGF and receptor family
  • coagulation factor IIa factor VII, factor VIII, factor IX
  • ferritin fibroblast growth factor FGF and receptor family
  • FZD follicle stimulating hormone
  • HGF glucagon
  • cardiac myosin growth hormone, Ig, IgA receptor, IgE, insulin-like growth factor IGF and binding protein family, interleukin IL superfamily and its receptor superfamily, interference INF family, iNOS, integrin family, kallikrein family, laminin, L-selectin, luteinizing hormone, MMP family, mucin family, cadherin superfamily , platelet-derived growth factor PDGF and receptor family, par
  • the active molecule K1 is human factor VII (FVII), human factor VIII (FVIII), GLP-1 analogue Exendin-4 (Ex4), human interleukin 7 (IL-7) or Human growth hormone (hGH);
  • the linker L of the fusion protein is shown as SEQ ID NO: 2, 3, 4, 5, 6 or 7;
  • the active molecule K2 is selected from the group consisting of human immunoglobulin IgG, IgM, IgA Fc fragment; more preferably an Fc fragment from human IgG1, IgG2, IgG3 or IgG4; further, the Fc may be a wild type or a mutant comprising at least one of the wild type human immunoglobulin Fc Amino acid modifications, and variants have reduced effector function (ADCC effect or CDC effect) and/or enhanced binding affinity to the neonatal receptor FcRn.
  • ADCC effect or CDC effect human interleukin 7
  • hGH Human growth hormone
  • the Fc mutant is preferably selected from the group consisting of: (i) vFc ⁇ 1 : human IgG1 hinge region, CH2 and CH3 regions containing Leu234Val, Leu235Ala and Pro331Ser mutations (eg SEQ ID NO (8) amino acid sequence); (ii) vFc ⁇ 2-1 : human IgG2 hinge region, CH2 and CH3 region containing the Pro331Ser mutation (such as the amino acid sequence shown in SEQ ID NO: 9); (iii) vFc ⁇ 2-2 : human IgG2 hinge region, CH2 and CH3 regions containing the Thr250Gln and Met428Leu mutations (such as the amino acid sequence shown in SEQ ID NO: 10); (iv) vFc ⁇ 2-3 : human IgG2 hinge containing the Pro331Ser, Thr250Gln and Met428Leu mutations Region, CH2 and CH3 regions (such as the amino acid sequence
  • the FVII-Fc fusion protein comprises, in order from the N-terminus to the C-terminus, FVII (having the amino acid sequence shown in SEQ ID NO: 13) and a linker peptide (having the SEQ ID NO: the amino acid sequence shown by 2) and human IgG Fc (having the amino acid sequence shown in SEQ ID NO: 11).
  • the FVIII-Fc fusion protein comprises, in order from the N-terminus to the C-terminus, FVIII (having the amino acid sequence set forth in SEQ ID NO: 14), a linker peptide (having the SEQ ID NO: the amino acid sequence shown by 2) and human IgG Fc (having the amino acid sequence shown in SEQ ID NO: 11).
  • the Exendin-4-Fc fusion protein contains Exendin-4 (having the amino acid sequence shown in SEQ ID NO: 15) and a linker peptide in this order from the N-terminus to the C-terminus. Having the amino acid sequence set forth in SEQ ID NO: 2, 3, 5 or 7 and human IgG Fc (having the amino acid sequence set forth in SEQ ID NO: 11).
  • the IL-7-Fc fusion protein contains IL-7 (having the amino acid sequence shown in SEQ ID NO: 16) and a linker peptide in this order from the N-terminus to the C-terminus.
  • IL-7 having the amino acid sequence shown in SEQ ID NO: 16
  • linker peptide in this order from the N-terminus to the C-terminus.
  • the hGH-Fc fusion protein comprises, in order from the N-terminus to the C-terminus, hGH (having the amino acid sequence shown in SEQ ID NO: 17) and a linker peptide (having as SEQ ID NO: the amino acid sequence shown by 2) and human IgG Fc (having the amino acid sequence shown in SEQ ID NO: 11).
  • the fusion protein is an antibody heavy chain variable region (V H); and K2 is an antibody light chain variable region (V L). And K1 and K2 are joined by the linker peptide to form a single chain antibody (ScFv).
  • the active molecule K1 of the fusion protein comprises a first antibody, or an antibody fragment; and the active molecule K2 comprises a second antibody, or an antibody fragment, and K1 and K2 are joined by the linker peptide to form a bispecific antibody.
  • K1 is a complete double-stranded antibody of Anti-CD20
  • K2 is a single-chain antibody of Anti-CD3
  • K1 and K2 are joined by the linker peptide to form a bispecific antibody.
  • the heavy chain of the Anti-CD20 diabody comprising the bispecific antibody has the amino acid sequence set forth in SEQ ID NO: 18, and the light chain has the amino acid set forth in SEQ ID NO: a sequence; and the anti-CD3 single-chain antibody comprising the bispecific antibody has the amino acid sequence set forth in SEQ ID NO: 20; and the linker peptide has the amino acid sequence set forth in SEQ ID NO: 4 or 6; And the heavy chain of the Anti-CD20 diabody is linked to the Anti-CD3 single chain antibody by the linker peptide.
  • the present invention provides a method for preparing a fusion protein, wherein the structural expression of the fusion protein is represented by: K1-L-K2 or K2-L-K1, wherein K1 is the first Bioactive molecule; L is the linker peptide; K2 is a second bioactive molecule, and the parts constituting the fusion protein are sequentially linked from the N-terminus to the C-terminus.
  • the active molecule may be selected from a protein or protein domain, a polypeptide, an antibody or an antibody fragment, preferably a protein or protein domain, an antibody or an antibody fragment.
  • the preparation method comprises the step of connecting K1 and K2 through the L phase; in a preferred embodiment of the invention, the method comprises the steps of:
  • step (b) introducing the fusion gene obtained in step (a) into a eukaryotic or prokaryotic expression host;
  • step (c) culturing step (b) screening the high-yield expression host to express the fusion protein;
  • the linker peptide is used to link active protein/polypeptide and long-circulating half-life serum proteins, such as antibody/immunoglobulin Fc fragments, human serum albumin (HSA), transferrin (TF), and the like.
  • serum proteins such as antibody/immunoglobulin Fc fragments, human serum albumin (HSA), transferrin (TF), and the like.
  • the method for producing a FVII-Fc fusion protein comprises the step of linking the active molecule FVII and the human IgG Fc through the linker peptide (SEQ ID NO: 2); in a preferred embodiment of the present invention, The method for producing a FVIII-Fc fusion protein comprises the step of linking the active molecule FVIII and the human IgG Fc through the linker peptide (SEQ ID NO: 2); in another preferred embodiment of the invention, the preparation of the Exendin4-Fc fusion protein The method comprises the step of linking the active molecule Exendin4 to the human IgG Fc via the linker peptide (SEQ ID NO: 2, 3, 5 or 7); in another preferred embodiment of the invention, the IL-7-Fc fusion protein The method of preparation comprises ligating the active molecule IL-7 and human IgG Fc via the linker peptide (SEQ ID NO: 2) In another preferred embodiment of the present invention, the method for producing a h
  • the linker peptide is used in the construction of a bispecific antibody.
  • the method for producing a bispecific antibody of Anti-CD3XCD20 comprises passing a double-chain antibody of Anti-CD20 and a single-chain antibody of Anti-CD3 through the linker peptide (SEQ ID NO: 4 or 6) The steps to connect.
  • the CTP of the connecting peptide of the present invention which comprises a plurality of O-glycosyl side chains, can form a relatively stable stereoconfiguration, and thus has a rigid structure, which can more effectively isolate the two parts of the fusion protein and eliminate The steric hindrance effect between each other.
  • a series of fusion proteins consisting of active protein and Fc, such as FVII-Fc fusion protein, and the addition of a CTP rigid unit in the linker peptide ensures that the N-terminally fused active protein does not affect the binding site of the Fc variant and FcRn, thereby affecting The half-life; in addition, the Fc's ProteinA binding site is important for the purification step in the preparation process.
  • the CTP rigid unit is ligated to ensure that the N-terminally fused active protein does not "cover" its binding site to protein A.
  • the addition of a CTP rigid unit also allows the Fc fragment of about 25 kD size to not interfere with the correct folding of the N-terminally fused active protein, resulting in a decrease or loss of its biological activity/function. It has been shown in various embodiments of the invention that the addition of a CTP rigid unit results in a significant increase in the biological activity of the fusion protein.
  • This may be interpreted as a CTP rigid polypeptide with multiple glycosyl side chains, which can form a stable stereoconfiguration relative to the random coiling of a flexible linker peptide such as (GGGGS)n, which effectively pulls the fusion protein apart.
  • the spatial distance between the two semi-molecules causes the active protein and the Fc segment to fold independently to form the correct three-dimensional conformation without affecting their respective biological activities, thereby reducing the activity loss or even loss or formation due to misfolding or conformational changes of the active protein.
  • the linker peptide of the present invention has wide applicability and portability, and the combined sequence of the rigid unit and the flexible unit can impart a conformation between the complete rigidity and the complete flexibility of the linker peptide, and the specific rigidity of the polypeptide (or The degree of flexibility varies depending on the ratio and arrangement between the two sequences.
  • the rigidity of the linker peptide can be finely regulated to meet the different requirements for the rigidity of the linker peptide in the construction of the fusion protein.
  • CTP contains glycosylation sites, negatively charged, highly sialylated CTP can resist the removal of the kidney, further extend the half-life of the fusion protein; improve pharmacokinetic parameters, such as reducing clearance, reducing the apparent volume of distribution Increasing the AUC (0-t) increases the bioavailability of the fusion protein, and it is expected that the clinical dose will also decrease.
  • the protective effect of the glycosyl side chain attached to the CTP can reduce the sensitivity of the linker to the protease, making the fusion protein less susceptible to degradation in the junction region.
  • Antibody fragment means an antigen-binding fragment of an antibody and an antibody analog, which typically comprises at least a portion of an antigen binding or variable region (eg, one or more CDRs) of a parental antibody.
  • the "Fc" region contains two heavy chain fragments comprising the CH2 and CH3 domains of the antibody.
  • the two heavy chain fragments are held together by two or more disulfide bonds and by the hydrophobic action of the CH3 domain.
  • the "Fv region” contains variable regions from both heavy and light chains, but lacks a constant region.
  • single-chain Fv antibody refers to an antibody fragment comprising the VH and VL domains of an antibody, wherein these domains are present in a single polypeptide chain.
  • Fv polypeptides additionally comprise a polypeptide linker between the VH and VL domains that enables the ScFv to form the desired structure for antigen binding.
  • Bispecific antibody refers to a region comprising two variable domains or ScFv such that the resulting antibody recognizes two different antigenic determinants.
  • Antibody fusion protein refers to a product obtained by fusing an antibody fragment with other biologically active proteins using genetic engineering techniques. Due to the difference in fusion proteins, this antibody fusion protein has a variety of biological functions.
  • an antibody fusion protein containing a Fv segment splicing a Fab or Fv with certain toxins, enzymes, and cytokine genes, and directing the biologically active molecules to specific parts of the target cells by the guidance of these antibodies, so-called “bio-missile missiles" ";
  • a chimeric receptor a fusion protein formed by fusing ScFv with certain cell membrane protein molecules, which can be expressed on the cell surface, called a chimeric receptor, confers the ability of a particular cell to bind to an antigen;
  • an Fc-containing antibody fusion protein an antibody IgG constant region Fc segment is fused to a biologically active molecule to form an Fc fusion protein.
  • the Fc fusion protein not only exerts the biological activity of the active molecule, but also imparts similar antibody-like properties, including prolonging plasma half-life and a series of effector functions specific to the Fc fragment.
  • the Fc segment plays an important role in eradicating the immune defense of pathogens. effect.
  • Fc-mediated IgG The effector function of Fc-mediated IgG is exerted through two mechanisms: (1) binding to cell surface Fc receptors (Fc ⁇ Rs), digestion of pathogens by phagocytosis or cleavage or killer cells via antibody-dependent cellular cytotoxicity (ADCC) pathway , Or (2) binding to C1q of the first complement component C1, eliciting a complement-dependent cytotoxicity (CDC) pathway, thereby lysing the pathogen.
  • ADCC antibody-dependent cellular cytotoxicity
  • CDC complement-dependent cytotoxicity
  • FcRn is active in adult epithelial tissues and is expressed in epithelial cells of the intestinal lumen, pulmonary trachea, nasal cavity, vagina, colon and rectum.
  • the fusion protein consisting of the Fc segment can efficiently shuttle the epithelial barrier by FcRn-mediated transcytosis.
  • hCG-beta carboxy terminal peptide is a short peptide derived from the carboxy terminus of the ⁇ -subunit of human chorionic gonadotropin (hCG).
  • reproductive-related peptide hormones FSH
  • FSH follicle stimulating hormone
  • LH luteinizing hormone
  • TSH thyrotropin
  • hCG chorionic gonadotropin
  • CTP contains 37 amino acid residues with four O-glycosylation sites and a sugar side chain terminal that is a sialic acid residue. Negatively charged, highly sialylated CTP is resistant to the clearance of the kidneys, thereby prolonging the half-life of the protein in vivo (Fares FA et al, Proc Natl Acad Sci USA, 1992, 89: 4304-4308).
  • Figure 1 Comparison of the duration of bleeding after administration of FP-A1 and Novo for 1 h and 2 h. Compared with HA-N-1h group, * P ⁇ 0.05, *** P ⁇ 0.01; compared with C57-NS group, # P ⁇ 0.05, ### P ⁇ 0.01.
  • Figure 4 Effect of FP-C1 in different dose groups on HbA1c (%) in db/db diabetic mice; statistical difference labeling annotation: FP-C1 group compared with model group, * P ⁇ 0.05, ** P ⁇ 0.01.
  • Figure 10 Effect of FP-C1 on the cross-sectional area of fat-fed cells fed with high fat diet. Note: A: normal group; B: high fat group; C: FP-C1 group.
  • Figure 11 The ability of the IL7-Fc fusion proteins FP-D1 and FP-D2 to stimulate mouse mononuclear cell proliferation.
  • Figure 12 The ability of hGH-Fc fusion proteins FP-E1 and FP-E2 to stimulate Nb2 cell proliferation.
  • Figure 13 Curves of blood concentration of hGH-Fc fusion proteins FP-E1 and FP-E2 as a function of time.
  • Figure 14 Growth curves of each group after administration of the hGH-Fc fusion protein FP-E1.
  • Anti-CD3 x CD20 bispecific antibody FP-F1 results in the concentration-dependent activation of human PBMC.
  • the inventors constructed a series of fusion proteins containing linker peptides K1-L-K2.
  • the composition of each fusion protein is shown in Table 1.
  • a DNA sequence encoding the fusion protein active molecule K1 and the active molecule K2 is linked by a DNA sequence linked to the peptide L to form a fusion gene sequence.
  • they are all manually optimized CHO cell preferred codons. It is preferably obtained using a chemical synthesis method.
  • a restriction enzyme site, SpeI and EcoRI was inserted at each of the 5' and 3' ends of the synthesized fragment.
  • the fusion protein gene verified by sequencing was digested with the corresponding restriction endonuclease, and then inserted into the corresponding restriction sites of the expression plasmid PXY1A1 which was modified with PCDNA3.1 as a template to obtain a fusion protein high expression plasmid.
  • the PXY1A1 plasmid includes, but is not limited to, the following important expression components: 1) human cytomegalovirus early promoter and mammalian cells with high exogenous expression of the desired enhancer; 2) dual screening marker with kanamycin resistance in bacteria sexual, feeding G418 resistance in milk animal cells; 3) Mouse dihydrofolate reductase (DHFR) gene expression cassette, when the host cell is DHFR gene-deficient, methotrexate (MTX) can amplify the fusion gene and DHFR gene (See U.S. Patent 4,399,216).
  • DHFR Mouse dihydrofolate reductase
  • the fusion protein expression plasmid is transfected into a mammalian host cell line, and in order to obtain stable high levels of expression, the preferred host cell line is a DHFR enzyme deficient CHO cell (see U.S. Patent 4,818,679).
  • the medium was changed to a screening medium containing 0.6 mg/mL G418, and the cells were seeded in a 96-well culture plate at a concentration (5000-10000 viable cells/well) for 10-14 days until large. Discrete cell clones appear. Transfectants resistant to the selected drug were screened by ELISA assay. Subclones were generated to produce high levels of fusion protein wells by limiting dilution of 96-well plates.
  • the fusion protein with good multi-party validation in order to achieve higher level expression, it is preferred to co-amplify with the DHFR gene inhibited by MTX drugs.
  • the transfected fusion protein gene was co-amplified with the DHFR gene in growth medium containing increasing concentrations of MTX.
  • the obtained highly expressed monoclonal cell strain was first fed in a shake flask or a 5 liter fermentor, and the fusion protein was purified by Protein A affinity chromatography column and other ion exchange chromatography columns.
  • the inventors constructed a series of fusion proteins containing the flexible-rigid unit linker peptides of the present invention, and also constructed a plurality of fusion proteins containing only flexible linker peptides of different lengths as controls.
  • FVII-Fc fusion protein CTP-containing FP-A1; CTP-free FP-A2 and FP-A3
  • FVIII-Fc fusion protein CTP-containing FP-B1; CTP-free FP-B2 and FP) -B3
  • Exendin-4 and its analog Fc fusion protein CTP-containing FP-C1, FP-C2, FP-C3, FP-C4; CTP-free FP-C5
  • IL7-Fc fusion protein including CTP FP-D1; CTP-free FP-D2
  • hGH-Fc fusion protein CTP-containing FP-E1; CTP-free FP-E2
  • the inventors also constructed an Anti-CD20 ⁇ CD3 bispecific antibody (CTP-containing FP-F1 and FP-F2).
  • the composition of each fusion protein is shown in Table 1, and the amino acid composition of each fusion protein component is shown in the sequence listing.
  • the FP-A1, FP-A2 and FP-A3CHO stably expressing cell lines obtained in Example 1 were cultured in a shake flask for 10-14 days, subjected to Protein A affinity chromatography, multi-dimensional mode chromatography, anion exchange layer.
  • the purification and molecular sieve chromatography were carried out in four steps for purification, and then the fusion protein was activated by a solution incubation self-activation method.
  • SDS-PAGE protein electrophoresis showed that under the reducing conditions, the unactivated FP-A2 had two distinct bands near 70-85kDa and 40kDa, indicating the presence of degraded fragments, accounting for about 20-30%.
  • the purified protein migrated to approximately 130 kDa with a band >200 kDa, indicating partial polymerization of the fusion protein.
  • the unactivated FP-A3 has a single-stranded molecule close to 100 kDa and no impurity band.
  • the partially purified protein migrates to >200 kDa, indicating that FP-A3 is a multimer.
  • the unactivated FP-A1 has a single-stranded molecule of 100-110 kDa, and no obvious hybrid band.
  • the purified protein migrates to 150 kDa.
  • the activated FP-A1 showed two clear bands under reducing conditions, namely 74.3KDa HC-L-CTP-Fc and about 24.0KDa LC, no other bands, and the purified protein migrated to 150kDa under non-reducing conditions. It indicates that the fusion protein FP-A1 has not undergone significant degradation and no obvious polymerization phenomenon, and has higher thermodynamic stability and stronger resistance to protease hydrolysis. This example shows that the linker peptide contains a CTP rigid unit, which increases the stability of the fusion protein, is not easily degraded, and reduces the formation of the polymer.
  • Determination of the biological activity of FVIIa by coagulation is obtained by correcting the ability of FVIIa-factor-deficient plasma to cause prolonged clotting time.
  • the detection method firstly mixes diluted human lyophilized plasma of known FVII activity (Unicalibrator, Cat. No. 00625) with VII matrix plasma, determines prothrombin time (PT), establishes a standard curve, and then tests The plasma was moderately diluted and mixed with spent FVII matrix plasma for PT assay.
  • the logarithmic equation of the activity percentage C (%) and the PT time t(s) fitted by the standard curve can be used to measure the activity of the sample FVIIa, and the result is expressed as a percentage of normal plasma (%).
  • the results showed that under the optimal experimental conditions, the highest activities of FP-A1, FP-A2 and FP-A3 were about 20,000 IU/mg, 4000 IU/mg and 7000 IU/mg, respectively.
  • wild-type male C57BL/6J mice purchased from Shanghai Southern Model Biological Research Center
  • an equal volume dose of physiological saline was administered by tail vein injection.
  • the two groups of HA mice given FP-A1 were subjected to a tail-tailing test at 1 h and 2 h after administration; the HA mice given Novo were tail-tested 1 h after administration; C57BL/6J normal control group (C57-NS) Group) Mice were subjected to a tail-break test 2 h after injection. All data in mean ⁇ standard error T-test analysis was used for comparison between the experimental groups.
  • FP-A1 2.5 mg/kg warfarin (Orion Corporation, Finland, batch number: 1569755), and intravenous administration of 10,000 IU/kg of FP-A1 or Noci after 24 hours. Novo Nordisk)).
  • the FP-A1 group was collected at 0.05, 0.5, 1, 2, 3, 5, 8, and 12 h after administration; the Noqi group was collected at 0.05, 0.5, 1, 2, 3, and 5 h after administration.
  • the blood sample was treated with sodium citrate at a final concentration of 0.013 M as an anticoagulant, and the supernatant was taken by centrifugation at 3000 rpm for 10 min. The activity of the sample was measured and the activity half-life was calculated according to the method in 2.2.
  • the activity half-life of FP-A1 was calculated to be 3.03 ⁇ 0.35 h; the active half-life of Novo was 1.01 ⁇ 0.16 h.
  • the activity half-life of FP-A1 was about 3 times longer in rats, and the blood coagulation activity was about 40% after a single injection of FP-A1 for 3 hours, and the activity of the same active Noki group was 3 hours later. It has been reduced to 3%; while the clotting activity of FP-A1 remains above 7% after 12 hours of administration.
  • the test results of 2.3 and 2.4 indicate that the fusion protein consisting of the linker peptide (comprising flexible unit and CTP rigid unit) of the present invention has a significantly prolonged functional half-life, indicating that the linker peptide eliminates the active protein FVII against Fc and its receptor.
  • the Shielding Effect of the FcRn Binding Site A Significantly Extended Half-Life was Obtained, and it was also demonstrated that FVII was not affected by the Fc steric hindrance effect of C-terminal fusion by the addition of the linker peptide of the present invention, forming a correct three-dimensional conformation and maintaining Higher biological activity.
  • Example 3 production of blood coagulation factor FVIII-Fc fusion protein, determination of biological activity
  • the FP-B1, FP-B2 and FP-B3CHO stably expressing cell lines obtained in Example 1 were subjected to 7-12 days of fed culture or semi-continuous tank culture, and the supernatant was harvested immediately for Protein A and/or VIII. -select (GE) affinity chromatography purification. Under optimal culture conditions, the supernatant harvested from FP-B2 was purified by Protein A and VIII-select (GE) two-step affinity chromatography and still contained various components. SDS-PAGE protein electrophoresis showed that under the reducing condition, the main band of 180kDa and multiple fragments of 40-100kDa size were observed; under non-reducing conditions, most of the purified protein migrated to >300kDa.
  • FP-B2 product is mostly in the form of a polymer, which is unstable and easily degradable.
  • the supernatant of FP-B1 harvested was purified by two-step affinity chromatography using Protein A and VIII-select (GE). Under reducing conditions, clear three bands appeared, which were single-chain FVIII- Fc (190 kDa), light chain-Fc (105 KDa) and heavy chain (90 KDa), no bands; under non-reducing conditions, FP-B1 and FP-B3 purified proteins migrated to >200 kDa, while most of FP-B3 The protein is located in the concentrated gel site, indicating that FP-B3 is also present in the form of a polymer.
  • the coagulation method for determining the biological activity of FVIII is obtained by correcting the ability of FVIII factor-deficient plasma to cause prolonged clotting time.
  • the invention adopts the factor VIII (FVIII) measuring kit produced by the French STAGO company (coagulation method) - Deficient FVIII (Cat. No. 00725).
  • the detection method is to measure the activated partial thromboplastin time (APTT) of normal human freeze-dried plasma (Unicalibrator (Cat No. 00625)) with known factor VIII activity, and the test instrument is STAGO.
  • a series of blood coagulation analyzers first establish a standard curve, and then mix the FVIII fusion protein with the matrix plasma of the VIII factor, determine the APTT value, the percentage of activity C (%) and the APTT time t(s) fitted by the standard curve.
  • the difference in activity between FP-B1 and FP-B2 is significant, indicating that the linker peptide contains a CTP rigid unit, which can reduce the steric hindrance effect of the Fc ligand and increase the activity of the FVIII-Fc fusion protein.
  • Example 4 Exendin4-Fc fusion protein production, biological activity and in vivo half-life determination
  • the CHO stably expressing cell lines of FP-C1, FP-C2, FP-C3, FP-C4 and FP-C5 obtained in Example 1 were cultured in shake flasks for 12-14 days, and the Protein A affinity layer was used. Column purification, the purity of both fusion proteins is above 95%, the molecular size is also in line with expectations, and then used for activity analysis.
  • the in vitro activity assay method is described in the literature (Zlokarnik G et al, Science, 1998, 279 (5347): 84-88).
  • the expression plasmid PGL-4.29 (Luc2P/CRE/Hygro) (Promega) carrying the human GLP-1R expression plasmid and the CRE-Luc reporter gene was co-transfected into CHO-K1 cells and screened by antibiotic pressure. Stable cell lines co-expressing both plasmids were obtained.
  • the fusion proteins FP-C1, FP-C2, FP-C3, FP-C4 and FP-C5 were diluted with DMEM medium containing 10% FBS, and 10 ⁇ l/well was added, and the concentration gradient was set to 0.010, 0.020, 0.039, 0.078, 0.156, 0.313, 0.625, 1.25, and 2.5 nM were simultaneously set to an equal concentration of duraglutide positive control (manufactured by Eli Lilly Company, Cat. No. 9301897).
  • the supernatant was aspirated, the cells were washed slowly by adding 300 ⁇ l PBS/well, then PBS was aspirated, 40 ⁇ l of lysate was added, shaken for 15 min, and then 40 ⁇ l of fluorescence was added per well.
  • the enzyme substrate (Luciferase reporter gene detection kit, article number: GM-040501B, product of Jiman Biotechnology Co., Ltd.), reaction for 2 minutes, using a multi-function microplate reader (SpectraMax M5system, Molecular Device)
  • the fluorescence value was measured at a wavelength of 560 nm, and a dose response curve was plotted based on the fluorescence value, and the EC 50 value was calculated.
  • Table 2 The results are shown in Table 2.
  • EC 50 values of FP-C1, FP-C2, FP-C3, FP-C4 and FP-C5 were approximately 0.03086 nM, 0.03156 nM, 0.03684 nM, 0.04012 nM and 0.03586 nM, and the duraglutide EC 50 value was approximately 0.02987.
  • CTP-containing FP-C1, FP-C2, FP-C3, FP-C4 and CTP-free FP-C5 have comparable in vitro biological activities.
  • mice Female diabetic db/db mice (purchased from Shanghai Slack Laboratory Animals Co., Ltd.), 8 weeks old, weighing 42 ⁇ 2 g, were randomly divided into 3 groups according to body weight, with 6 rats in each group.
  • the test group was injected subcutaneously with FP-C1 and FP-C5 at a dose of 3 mg/kg, and the positive group was injected with 3 mg/kg of duraglutide (produced by Eli Lilly and Company, Cat. No. 9301897), and the model group was injected with an equal volume (10 mL). /kg) of PBS buffer.
  • Blood samples were collected from the tail veins before administration (0h), 1h, 2h, 4h, 6h, 24h, 48h, 72h, 96h, 120h, 144h, 168h, 192h and 216h.
  • the blood glucose meter is used to measure random blood glucose (RBG) and record the data.
  • Blood glucose data were expressed as mean ⁇ standard deviation (means ⁇ SD), and data were analyzed using SPSS 18.0 statistical software.
  • both FP-C1 and FP-C5 and the positive control drug duraglutide had significant hypoglycemic effects, as shown in Figure 3, as can be seen from the curve of mouse RBG values within 9 days after administration.
  • the blood glucose lowering effect of duraglutide was only maintained until the fourth day (P>0.05).
  • the blood glucose level was not statistically different from the model group; while FP-C1 could be maintained.
  • the blood glucose level of the mice at 168 hours after the administration, that is, the 7th day, was still statistically different (P ⁇ 0.05); FP-C5 was maintained until 144 hours after the administration, that is, the 6th day.
  • the blood glucose levels of the rats were still statistically different compared to the model group.
  • FP-C1 has a longer-term hypoglycemic effect in a diabetic mouse model relative to FP-C5, indicating that the linker CTP rigid unit can further extend the in vivo functional half-life of Exendin-4.
  • SPF female db/db mice purchased from Shanghai Slack Laboratory Animal Co., Ltd.
  • 24 db/db mice were random blood glucose (RBG)
  • RBG blood glucose
  • the model group and FP-C1 were administered at low (0.75 mg/kg), medium (1.5 mg/kg), and high (3 mg/kg) doses.
  • Each drug-administered group was given a corresponding dose of the drug solution by subcutaneous injection, and the model group was subcutaneously injected with PBS buffer at a dose of 10 ml/kg.
  • Each group of animals was administered once a week for 10 weeks, and the RBG values of the mice in each group at different time points after drug administration were measured by a blood glucose meter (Anzheng blood glucose meter, Changsha Sannuo Biosen Co., Ltd. product). And record the data. Blood collection time points were set: before the first administration (0d), after the administration, on the 7th, 14d, 21d, 28d, 35d, 42d, 49d, 56d, 63d and 70d.
  • HbA1c Glycated hemoglobin
  • HbA1c is a product of the binding of blood glucose to hemoglobin of red blood cells, which is directly proportional to the level of glucose in the blood. Since the life span of red blood cells in the blood circulation is about 120 days, glycated hemoglobin can reflect the total level of blood glucose 4-12 weeks before blood collection, which makes up for the deficiency of fasting blood glucose. Therefore, HbA1c is the most important indicator for long-term control of blood glucose, and it is also an important basis for clinical decision whether to replace the treatment plan. In the present example, the HbA1c test results can stably and reliably reflect the blood glucose control of the mice 2 to 3 months before the blood collection.
  • SPF male Kunming mice purchased from Shanghai Slack Laboratory Animal Co., Ltd.
  • the Kunming mice which were kept for 1 week, were fasted for 18 hours and weighed.
  • An equal volume of citric acid-sodium citrate buffer (purchased from Sinopharm Chemical Reagent Co., Ltd.) was administered intraperitoneally.
  • RBG blood glucose
  • RBG ⁇ 16.7mmol/L was a successfully modeled diabetic mouse.
  • 32 STZ-induced diabetic mice were randomly divided into 4 groups, 8 rats in each group, respectively, given subcutaneous injection of 3 mg/kg FP-C1. And 3 mg/kg of duraglutide.
  • An equal volume (10 ml/kg) of PBS buffer was administered to the model group and the normal group, respectively.
  • Blood samples were collected from the tail vein before administration (0h), 1h, 2h, 4h, 6h, 24h, 48h, 72h, 96h, 120h, 144h, 168h, 192h, 216h and 240h.
  • Peripheral blood glucose meter The random blood glucose level RBG of each group of animals was measured, and data was recorded. Data in mean ⁇ standard deviation Formal representation, using SPSS 18.0 statistical software to analyze the data. Normal distribution, one-way ANOVA was used for the mean difference between groups, LSD test was used for variance homogeneity, Dunnet T3 test for variance variance; non-parametric test for non-normal distribution, P ⁇ 0.05 for significant statistics difference.
  • FP-C1 As shown in Fig. 5, the 0-240 hour random blood glucose level curve of STZ-induced diabetic mice after single injection of FP-C1 showed that FP-C1 can effectively reduce the random blood glucose level of STZ-induced diabetic mice. FP-C1 had the lowest blood glucose level at the 24th hour after administration, and then slowly recovered. At the 96th hour, the random blood glucose level was significantly different from that of the model group (P ⁇ 0.05).
  • C57BL/6J male mice at 7 weeks of age purchased from Shanghai Slack Laboratory Animal Co., Ltd., animal production license number SCXK (Shanghai): 2012-0002). Breeding environment: temperature 22-25 ° C, relative humidity 45-65%, lighting time 12h / d.
  • C57BL/6J mice were randomly divided into 3 groups according to body weight: normal group (NFD), high fat group (HFD), and FP-C1 group (HFD+FP-C1 0.3 mg/kg).
  • the high fat group and the FP-C1 group were fed with high fat diet (D12492 high fat diet, product of Research Diets, USA), and the normal group mice were fed with normal fat diet (NFD).
  • the FP-C1 group was subcutaneously injected with the corresponding drug solution every 6 days at a dose of 0.3 mg/kg.
  • the normal group and the model group were subcutaneously injected with PBS buffer at a dose of 10 ml/kg.
  • each group of mice was fasted for 16 hours, weighed and tested for fasting blood glucose, blood was taken from the eyelids, centrifuged at 400 ⁇ g for 15 min, and serum was isolated. After the blood was taken, the mice were sacrificed by cervical dislocation, and the length of the nose from the tip of the mouse to the length of the anus (body length) was measured, and the Lee's index was calculated.
  • the adipose tissue around the epididymis was separated and weighed. The same site of epididymal adipose tissue was stored in 10% formalin solution for pathological examination.
  • mice were weighed once every 6 days, and the mouse weight gain curve was plotted and the weight gain of the mice was calculated.
  • Mouse weight gain body weight at the time of final weighing in mice - body weight at time of grouping in mice.
  • the degree of obesity in mice was evaluated by Lee's index.
  • each group of mice was fasted for 16h (17:00am-9:00pm), and the fasting blood glucose level of each group was detected by blood glucose meter (Anzheng blood glucose meter, Changsha Sannuo Biosen Co., Ltd.).
  • FBG blood glucose meter
  • Oral (i.g) was given 2g/kg glucose solution (analytically pure anhydrous glucose, product of Sinopharm Chemical Reagent Co., Ltd.), and blood glucose of each group was detected 30 min, 60 min, 90 min and 120 min after gavage. Value, plot the glucose tolerance curve, and calculate the area under the corrected blood glucose curve (iAUC) by the trapezoidal method.
  • TG triglyceride detection kit, Ningbo Meike Biotechnology Co., Ltd.
  • serum serum was detected by automatic biochemical analyzer (Ou Ba XL-200 automatic biochemical analyzer, German Ou Pa company) and supporting kits.
  • automatic biochemical analyzer Ou Ba XL-200 automatic biochemical analyzer, German Ou Pa company
  • the content of TC total cholesterol test kit, Ningbo Meike Biotechnology Co., Ltd.), the specific operation according to the instrument manual.
  • mice insulin resistance index was calculated.
  • HE staining hematoxylin-eosin staining
  • iAUC was significantly higher in the high-fat group than in the normal group (P ⁇ 0.05).
  • the iAUC of the FP-C1 group was significantly lower than that of the high fat group (P ⁇ 0.05).
  • HE staining showed that the cross-sectional area of epididymal fat cells in the high-fat group was significantly increased compared with the normal group. Compared with the high-fat group, the cross-sectional area of epididymal adipocytes in the FP-C1 group was significantly reduced. The results are shown in Fig. 10.
  • Example 5 IL-7-Fc fusion protein production, biological activity and in vivo half-life determination
  • the FP-D1 and FP-D2CHO stably expressing cell lines obtained in Example 1 were purified by Protein A affinity chromatography after 12-14 days of shake flask feeding, and the purity of both fusion proteins was above 95%. The molecular size was also as expected and then used for activity analysis.
  • the in vitro biological activity analysis method of IL-7 and its fusion protein is as follows: After the mouse spleen-derived mononuclear cells are activated by concanavalin A (ConA), 100 ⁇ L of the cells are seeded into a 96-well plate, followed by a series of concentrations.
  • ConA concanavalin A
  • Figure 11 shows the ability of hIL-7, FP-D1 and FP-D2 fusion proteins to stimulate mouse mononuclear cell proliferation.
  • the dose response curves plotted obtained FP-D1 and FP-D2 fusion protein EC50 values (EC 50) 0.039 0.048nM.
  • the recombinant fusion proteins exhibit superior biological activity in vitro expression E.coli hIL-7 (EC 50 value of 0.08nM).
  • FP-D1 adds the rigid structure of CTP to FP-D2, which reduces the clearance rate, reduces the apparent volume of distribution, and increases the AUC (0-t) . Therefore, the efficacy of FP-D1 may be better than that of FP-D2, and the bioavailability is higher, and it is expected that the clinical dose will also be lowered. Thus, FP-D1 exhibits excellent performance in terms of biological activity and pharmacokinetics. The results of this experiment indicate that the negatively charged, highly sialylated CTP in the ligation peptide can resist the scavenging effect of the kidney, further prolong the half-life of the fusion protein, and increase the bioavailability of the fusion protein.
  • Example 6 hGH-Fc fusion protein production, biological activity and in vivo half-life determination
  • the CHO stably expressing cell lines of FP-E1 and FP-E2 obtained in Example 1 were purified by Protein A affinity chromatography after 12-14 days of shake flask feeding, and the purity of all three fusion proteins was above 95%. The molecular size is also in line with expectations and is then used for activity analysis.
  • In vitro biological activity can be determined by transfectants or purified proteins to stimulate the proliferation of rat lymphoma cell Nb2 cells. Since the proliferation of Nb2 cells is stimulated by the stimulation of hGH on the lactating receptors on the cells, the Nb2 cell proliferation ability assay can be used to evaluate the biological activity of growth hormone (see, for example, Uchida et al., J Mol Endocrinol., 1999). , 23:347-353).
  • Mouse lymphoma cell Nb2-11 (US ATCC cell bank) was normally cultured in Fischer's medium containing 10% fetal bovine serum. Thereafter, the fusion protein was diluted with serum-free medium, diluted 3 to 1000 gradients from 1000 ng/ml, 100 ⁇ l per well, and added to a 96-well plate, and the last column was negative for the medium. The cells grown in the log phase were washed with serum-free medium, and then adjusted to a density of 3 ⁇ 10 6 cells per ml, and 100 ⁇ l per well was added to the above 96-well plate.
  • the culture was continued for 48 hours at 37 ° C in a 5% CO 2 incubator, and cell proliferation was measured using a CCK-8 kit (Cell Counting Kit, purchased from Shanghai Shengsheng Biotechnology Co., Ltd., Cat. No. 40203ES80).
  • the absorbance at 450 nm was measured with a microplate reader and the OD reading was plotted against the concentration of the fusion protein.
  • the biological activity of the fusion protein can be determined from the resulting dose response curve.
  • Figure 12 shows the ability of hGH fusion protein to stimulate proliferation of Nb2 cells.
  • Table 9 for different values of EC 50 fusion protein. Since the amino acid at the C-terminus of growth hormone is closely related to its function, Fc directly linked to the C segment of hGH affects its biological activity. After the ligation peptide is added between hGH and Fc, the activity of the hGH fusion protein is increased. As can be seen from the results, the activity of FP-E1 was nearly doubled compared with FP-E2. This may be due to the fact that CTP exerts its own function on the one hand, and on the other hand, the CTP rigid structure is coupled with a flexible peptide linker as a linker sequence linking the Fc to the target protein. This novel peptide linker facilitates folding of the fusion protein into a better three-dimensional structure. Thus the biological activity of hGH is increased.
  • mice Male SPF SD rats (purchased from Shanghai Bikai Experimental Animal Co., Ltd.), after one week of pre-feeding, weighed about 290 g, randomized by body weight, 3 in each group, and 0.176 mg/kg FP-E1 in a single intravenous injection. And FP-E2, to examine the changes in plasma concentration and time.
  • the control group and the drug-administered group were collected by eyelids at 0, 0.5, 1, 2, 3, 4, 5, 8, 10, 24, 48, and 72 h after administration.
  • the blood was placed at room temperature for 30 min and then centrifuged at 5000 r/min for 10 min.
  • the serum was separated and stored at -20 °C. Serum hGH levels were determined at various time points using an ELISA specific for hGH.
  • the main pharmacokinetic parameters of each group were calculated by the software PKSOLVER. The results of the pharmacokinetic parameters of each group are shown in Table 10.
  • FP-E1 adds CTP rigid structure to FP-E2, reduces clearance rate and increases AUC (0-t) , further reduces apparent volume of distribution, making it less distributed in tissues, blood drugs
  • AUC AUC (0-t)
  • FP-E1 has a higher blood concentration after modification, it means that its bioavailability is higher, so FP-E1 is better than FP-E2, and its clinical dose can be expected to decrease. Thus, FP-E1 exhibits superior performance in terms of biological activity and pharmacokinetics.
  • the pituitary was removed under the condition of cleansing. The recovery period was 2 weeks after the pituitary surgery.
  • the qualified healthy animals whose body weight change was less than ⁇ 10% before the operation were selected.
  • the pituitary rats were randomly divided according to their body weight. For 6 groups, 5 groups/group.
  • Short-acting rhGH (trade name norditropin, manufactured by Novo Nordisk A/S) was used as the positive reference drug 1 (Y1) of this experiment, and the specific activity was 3 IU/mg; positive by PEG-rhGH (Changchun Jinsai Pharmaceutical Co., Ltd.) Reference drug 2 (Y2), specific activity 6IU / mg; FP-E1 low, medium and high dose settings were 5mg / kg / 14d, 15mg / kg / 14d and 45mg / kg / 14d.
  • the medium dose of FP-E1, 15 mg/kg/14d was comparable to the positive reference drugs Y1 and Y2, depending on the molecular weight and the number of moles.
  • the administration method was subcutaneous injection in the neck, different doses of FP-E1 fusion protein and Y2, and each dose was administered once every day on the first day and the eighth day; Y1 was administered once a day for 14 consecutive days.
  • ⁇ bw of each of the administration groups was significantly increased on the 8th day after administration, but the difference in ⁇ bw values between the administration groups was not significant compared with the model group; and on the 15th day after administration, FP -E1 has a very significant effect on the body weight gain of rats.
  • the high dose FP-E1 induced pituitary resection rats weight gain ( ⁇ bw value) is about 1.5 times that of rhGH (Y1) and PEG-rhGH (Y2) group;
  • the weight gain induced by the medium dose FP-E1 was slightly better than Y1, which was basically consistent with Y2.
  • the CHO stably expressing cell lines of FP-F1 and FP-F2 obtained in Example 1 were cultured in a shake flask for 10-14 days, and purified by Protein A affinity chromatography. Both fusion proteins were more than 95% pure, and the molecular size was also as expected, followed by activity analysis.
  • PBMCs were prepared from human fresh blood by density gradient centrifugation and resuspended at 5 x 10 6 cells/ml with 10% heat-inactivated FBS medium, ready for use.
  • the antibody (Anti-CD3 monoclonal antibody OKT3 or bispecific antibody FP-F1) was diluted to 2 ⁇ g/ml using complete medium, and diluted to 8 gradients at 1:5 magnification, and added to a 96-well plate at 100 ⁇ l per well. , set three holes.
  • the medium was used as a negative control and ConA was used as a positive control.
  • the PBMC cell suspension prepared above was added to a 96-well plate at 100 ⁇ l/well, and cultured at 37 ° C, 5% CO 2 for 72 hours.
  • the 96-well plate culture supernatant was carefully aspirated, 100 ⁇ l per well, and the IFN- ⁇ content in the supernatant was measured by an IFN- ⁇ ELISA kit (purchased from BD) (Fig. 15).
  • 10 ⁇ l of CCK-8 reagent was added to each well of a 96-well plate and incubation was continued for 4 h.
  • a four-parameter S-curve was fitted and the EC 50 value was calculated (Fig. 16).
  • Another Anti-CD3 ⁇ CD20 bispecific antibody FP-F2 constructed in accordance with the present invention also activates T cells in a concentration-dependent manner in an activated human PBMC cell assay, and produces IFN- ⁇ , and FP-F2 activates human PBMC.
  • the EC 50 value was 0.214 nM, which is comparable to FP-F1.
  • Anti-CD3 ⁇ CD20 bispecific antibody FP-F1 kills subcutaneous xenografts
  • the control group was: (1) negative control group: FP-F1 solvent PBS; (2) positive control group: (Anti-CD20 antibody, Genentech), administered at a dose of 10 mg/kg, twice a week intravenously.
  • the body weight and tumor volume of the mice were measured twice a week, and the volume was calculated as 1/2 x length x width x width (mm 3 ).
  • FP-F1 different doses of FP-F1 showed a good tumor growth inhibiting effect, in which 2 mice in the 10 mg/kg administration group completely disappeared, and 1 mouse in the 1 mg/kg administration group had complete tumors. Disappeared, the 0.1 mg/kg dose of the treatment group also showed a certain effect of inhibiting tumor growth.
  • the tumor growth of the negative control group was normal, reaching 400 mm 3 at 14 days.
  • the results showed that the FP-F1 1 mg/kg dose group was equivalent to the Rituxan 10 mg/kg dose group, indicating that the in vivo inhibitory effect of the bispecific antibody FP-F1 constructed in the present invention on tumor growth was only Anti-CD20 monoclonal antibody. At 1/10, it can be expected that the clinically administered dose will also be greatly reduced.

Abstract

一种用于构建融合蛋白的连接肽，包含柔性肽和刚性肽两部分，所述柔性肽由1个或多个柔性单元组成，所述刚性肽由1个或多个刚性单元组成，其中，所述柔性单元包含2个或更多个选自Gly，Ser，Ala和Thr的氨基酸残基；和所述刚性单元包含多个糖基化位点的人绒毛膜促性腺激素β亚基的羧基末端肽(CTP)。所述连接肽能更有效的消除两个融合分子间彼此的空间位阻效应，减少因活性蛋白错误折叠或构象变化而导致的聚合或活性降低/丧失；而且，另一方面，带负电、高度唾液酸化的CTP能抵抗肾脏对其的清除作用，进一步延长融合分子的半衰期，提高融合蛋白的生物利用度；再一方面，CTP糖基侧链的保护作用可以降低连接肽对蛋白酶的敏感性，使融合蛋白不易在连接区被降解。

Description

用于构建融合蛋白的连接肽 技术领域
本发明涉及融合蛋白领域，更具体地，涉及一种用于构建融合蛋白的连接肽。
背景技术
近二十年，蛋白融合技术已广泛应用于构建双功能抗体、双功能酶和双功能蛋白等。然而，在构建融合蛋白过程中遇到了各种问题，例如在单独表达时能正确折叠的蛋白在融合蛋白中不能正确折叠；融合的两个蛋白因空间距离靠近从而导致活性位点被屏蔽；融合蛋白分子因不能正确折叠或其构象变化原因而容易被蛋白酶降解；原本具有一定柔性的蛋白催化域在融合之后失去了原有的功能等。这些问题的出现，往往导致融合蛋白活性降低甚至完全丧失。一般认为，构建融合蛋白后，原蛋白分子的活性会有一定程度的下降；一个有意义的融合蛋白是其各部分的活性保持在原蛋白分子的50％以上。为了解决以上问题，人们对融合蛋白的设计和构建进行了许多研究和探索，以期提高融合蛋白的活性。如通过改变融合蛋白的首尾顺序，变换不同的融合位点，采用不同的融合配体，或采用连接肽等方法。
相比于其它融合策略，使用连接肽(linker)具有多种优势。第一、组成连接肽的氨基酸具有多样性(20种常见氨基酸)，而且连接肽长度也是重要可调参数，由此可带来连接肽丰富的多样性(20的n次方种，n是连接肽的氨基酸残基数)，有利于对其进行工程改造；第二、连接肽能为两个待融合蛋白提供一定的空间间隔，以达到趋向于正确折叠而不相互干扰；第三、连接肽还能为两个待融合蛋白提供更多相互作用的可能性，促进相互间协同作用。
目前常用的连接肽有两种，螺旋形式的刚性连接肽(如A(EAAAK)nA)和由低疏水性、低电荷效应氨基酸组成的柔性连接肽。螺旋形式的刚性连接肽能有效分开融合蛋白的不同功能区。柔性连接肽的例子包括Huston设计的(GGGGS)3序列。另外，人绒毛膜促性腺激素(hCG)β链的羧基末端肽(以下称其为CTP)也有报道可以单独作为连接肽，主要用于连接同一个蛋白的不同亚基。例如，中国专利CN103539860A、CN103539861A、CN103539868A和CN103539869A公开的融合蛋白中，CTP作为接头，位于促卵泡激素的beta亚基和alpha亚基之间； 专利WO2005058953A2公开的融合蛋白中，CTP作为接头，用于连接糖蛋白激素beta亚基和alpha亚基。但是，CTP因为具有延长某些蛋白体内半衰期的作用，因而在更多的专利文献中主要被公开作为融合蛋白中的延长半衰期部分，该部分可以选择使用免疫球蛋白Fc、CTP或其他能延长半衰期的融合配体。
有不少文献报道了连接肽序列对于融合蛋白构建和表达的影响。例如，在构建单链抗体1F7研究中，发现采用一个常规Genex212连接肽(GSTSGSGKSSEGKG)来连接其轻链和重链时，不能获得蛋白原有的催化活性，且蛋白不稳定；通过构建具有随机序列的连接肽(18个氨基酸残基)库进行筛选，才获得具有催化活性的单链抗体(Tang Y et al.，J Biol Chem.，1996，271(26)：15682-15686)。Arai等在研究螺旋形式连接肽时，发现随着连接肽长度增加，从EBFP到EGFP的荧光共振能量转移减少，预示连接肽长度增加能有效分开两个功能区(Arai R等，Protein Engineering，2001，14(8)：529-532)。另外，Luo等发现登革热病毒NS2B蛋白(N端为丝氨酸蛋白酶，C端为RNA解旋酶，连接肽为11个氨基酸残基的多肽)，通过在原173和174位点之间插入甘氨酸，或将174位的脯氨酸替换成甘氨酸，均导致两端蛋白活性的显著下降，表明其天然连接肽所具有的长度和刚柔性是长期进化的结果，对该天然融合蛋白的功能发挥具有重要意义(Luo D等，J Biol Chem.，2010，285(24)：18817-18827)。
本发明人发现，连接肽长度及其氨基酸组成、是否含有糖基化位点、连接肽与两个活性分子间的适配性等多个方面都会影响融合蛋白的功能及稳定性。本发明人认为，连接肽应该具备以下特性：1)能使所连接蛋白实现有效折叠，使其处于适当构型，不引起分子动力学变化，且最好选择非免疫原性的天然氨基酸；2)应具备防蛋白酶攻击能力；3)应尽量避免融合蛋白两端氨基酸序列的互相干扰。
在对设计和构建连接肽缺乏明确指导原则的情况下，本发明人根据自身的长期研究经验，尤其是在对Fc融合蛋白的研究中受到启发，开发了新的用于构建融合蛋白的连接肽，令人意外的是，其更利于保持蛋白或多肽的生物活性，而且具有广泛适用性和可移植性。
发明内容
本发明的目的在于提供一种用于构建融合蛋白的新型连接肽。
本发明的第一个方面提供一种连接肽，由柔性肽和刚性肽组成，所述柔性肽包括1个或多个柔性单元，和所述刚性肽包括1个或多个刚性单元；其中，所述柔性单元包含2个或更多个选自Gly、Ser、Ala和Thr的氨基酸残基；和所述刚性单元包含人绒毛膜促性腺激素β亚基的羧基末端肽(本文中简称为CTP)。
优选地，所述连接肽是糖基化的。较优选地，糖基化位点位于所述CTP上。更优选地，所述糖基化过程通过在哺乳动物细胞(如，中国仓鼠卵巢细胞)或其他具有合适的糖基化修饰系统的表达载体中完成。
进一步地，组成本发明所述连接肽的刚性肽位于所述柔性肽的N末端或C末端；优选地，所述刚性肽位于柔性肽的C末端。具体地，本发明所述连接肽的结构通式可表示为F-R或R-F，其中，F和R分别代表柔性肽和刚性肽。进一步地，所述柔性肽优选地包含1，2，3，4或5个柔性单元，和所述刚性肽优选地包含1，2，3，4或5个刚性单元。
较佳地，所述柔性单元包含2个或更多个G和S残基。更优地，所述柔性单元的氨基酸序列通式为(GS)a(GGS)b(GGGS)c(GGGGS)d，其中a，b，c和d分别代表由G和S残基所组成结构单元的数目，且都是大于或等于0的整数，且a+b+c+d≥1。
示例性地，以Fi表示各柔性单元，且i＝1，2，3，4，5......n，在本发明一些实施例中，所述柔性肽优选地包含但不限于如下柔性单元：
Figure PCTCN2016106011-appb-000001
其中，所述刚性单元选自由人绒毛膜促性腺激素β亚基羧基末端第113至145位氨基酸所组成的全长序列或其片段(以下简称CTP)，具体地，所述刚性单元包含SEQ ID NO：1或其截短的序列。
优选地，所述CTP包含至少2个糖基化位点；例如，本发明的一优选实施例 中，所述CTP包含2个糖基化位点，示例性地，所述CTP包含SEQ ID NO：1N端的10个氨基酸，即SSSS*KAPPPS*，或所述CTP包含SEQ ID NO：1C端的14个氨基酸，即S*RLPGPS*DTPILPQ；又如，另一实施例中，所述CTP包含3个糖基化位点，示例性地，所述CTP包含SEQ ID NO：1N端的16个氨基酸，即SSSS*KAPPPS*LPSPS*R；再如，另一些实施例中，所述CTP包含4个糖基化位点，示例性地，所述CTP包含28、29、30、31、32或33个氨基酸并开始于人绒毛膜促性腺激素β亚基的第113、114、115、116、117或118位，终止于第145位。具体地，所述CTP包含SEQ ID NO：1N端的28个氨基酸，即SSSS*KAPPPS*LPSPS*RLPGPS*DTPILPQ。在本文中，*代表糖基化位点。每种可能性都代表本发明的独立实施方式。
在另一些实施例中，本发明提供的刚性单元与天然CTP氨基酸序列至少70％同源；在另一些实施例中，本发明提供的刚性单元与天然CTP氨基酸序列至少80％同源；在另一些实施例中，本发明提供的刚性单元与天然CTP氨基酸序列至少90％同源；在另一些实施例中，本发明提供的刚性单元与天然CTP氨基酸序列至少95％同源。
示例性地，以Ri表示各刚性单元，且i＝1，2，3，4，5......n，本发明一些实施例中所述刚性肽可优选地包含但不限于如下CTP刚性单元：
Figure PCTCN2016106011-appb-000002
本发明所述刚性肽还可包含2个或3个上述CTP刚性单元，如本发明的一实施例中，所述刚性肽包含2个R3刚性单元：SSSSKAPPPSSSSSKAPPPS(表示为R3R3)；本发明的另一实施例中，所述刚性肽包含3个R4刚性单元：SRLPGPSDTPILPQSRLPGPSDTPILPQSRLPGPSDTPILPQ(表示为R4R4R4)。
本发明一些优选实施例中，所述连接肽具有SEQ ID NO：2(GSGGGGSGGGGSGGGGSGGGGSGGGGSSSSSKAPPPSLPSPSRLPGPSDTPILPQ)所示的氨基酸序列(表示为F2-R1)。
本发明另一优选实施例中，所述连接肽具有SEQ ID NO：3(GSGGGSGGGGSGGGGSPRFQDSSSSKAPPPSLPSPSRLPGPSDTPILPQ)所示的氨基酸序列(表示为F1-R2)。
本发明另一优选实施例中，所述连接肽具有SEQ ID NO：4(GSGGGGSGGGGSGGGGSSSSSKAPPPSLPSPSRLPGPSDTPILPQ)所示的氨基酸序列(表示为F4-R1)。
本发明另一优选实施例中，所述连接肽具有SEQ ID NO：5(GGGSGGGSGGGSGGGSGGGSSSSSKAPPPSLPSPSR)所示的氨基酸序列(表示为F5-R5)。
本发明另一优选实施例中，所述连接肽具有SEQ ID NO：6(GSGGGGSGGGGSGGGGSSSSSKAPPPSSSSSKAPPPS)所示的氨基酸序列(表示为F4-R3R3)。
本发明另一优选实施例中，所述连接肽具有SEQ ID NO：7(GGSGGSGGSGGSSRLPGPSDTPILPQSRLPGPSDTPILPQSRLPGPSDTPILPQ)所示的氨基酸序列(表示为F6-R4R4R4)。
本发明另一方面，还提供一种包含所述连接肽的融合蛋白。所述融合蛋白包含两种生物活性分子及连接两种活性分子的连接肽。所述融合蛋白的结构通式表示为：K1-L-K2或K2-L-K1，其中K1是第一种生物活性分子；L是上述连接肽；K2是第二种生物活性分子，且组成所述融合蛋白的各部分自N端至C端首尾依次相连。进一步地，所述活性分子可选自蛋白或蛋白结构域、多肽、抗体或抗体片段，优选为蛋白或蛋白结构域、抗体或抗体片段。
示例性地，所述融合蛋白的活性分子K1包含具有生物学功能的蛋白或蛋白结构域、多肽，特别是可溶性或者膜信号分子、细胞因子、生长因子、激素、共刺激分子、酶、受体以及对于受体具有配体功能的蛋白或者多肽；和所述活性分子K2为延长循环半衰期的血清蛋白或蛋白结构域，例如，人血白蛋白(HSA)、转铁蛋白(TF)或抗体/免疫球蛋白Fc片段等。
示例性地，所述融合蛋白的活性分子K1包含毒素、酶、细胞因子、膜蛋白或免疫调节细胞因子；和所述活性分子K2包含抗体或抗体片段，且K1和K2通过所述连接肽连接形成抗体融合蛋白。例如，K2为抗体Fv段(VL或VH)； 又如，K2为单链抗体(ScFv)。
进一步地，所述生物活性分子K1包含但不限于腺苷A1受体、血管紧张素转换酶ACE、Activin家族、ADAM家族、ALK家族、α-1-抗胰蛋白酶、细胞程序性死亡相关蛋白家族、神经生长因子及受体家族、骨形态发生蛋白BMP及受体家族、补体因子、降钙素、癌症相关抗原、组织蛋白酶家族、CCL趋化因子及受体家族、CD超家族、CFTR、CXCL趋化因子及受体家族、EGF、表皮生长因子EGF及受体家族、凝血因子IIa、因子VII、因子VIII、因子IX、铁蛋白、成纤维细胞生长因子FGF及受体家族、促卵泡激素、FZD家族、HGF、胰高血糖素、心肌肌球蛋白、生长激素、Ig、IgA受体、IgE、胰岛素样生长因子IGF及结合蛋白家族、白细胞介素IL超家族及其受体超家族、干扰素INF家族、iNOS、整联蛋白家族、激肽释放酶家族、层粘连蛋白、L-选择蛋白、黄体生成素、MMP家族、粘蛋白家族、钙粘蛋白超家族、血小板衍生生长因子PDGF及受体家族、甲状旁腺激素、血清白蛋白、T-细胞相关受体超家族、TGF-α、转化生长因子TGFβ超家族、促甲状腺激素、促甲状旁腺激素、肿瘤坏死因子TNF超家族及其受体TNFRSF超家族、尿激酶、WNT信号通路蛋白家族、胸腺肽α1、胸腺肽β4、VEGF、血管内皮生长因子VEGF及其受体家族。
本发明的优选实施例中，所述活性分子K1为人凝血因子VII(FVII)、人凝血因子VIII(FVIII)、GLP-1类似物Exendin-4(Ex4)、人白介素7(IL-7)或人生长激素(hGH)；所述融合蛋白的连接肽L如SEQ ID NO：2，3，4，5，6或7所示；所述活性分子K2选自人免疫球蛋白IgG、IgM、IgA的Fc片段；更优选自人IgG1、IgG2、IgG3或IgG4的Fc片段；进一步地，Fc可以是野生型或突变体，所述Fc突变体包含位于野生型人免疫球蛋白Fc中的至少一种氨基酸修饰，且变体具有降低的效应子功能(ADCC效应或CDC效应)和/或与新生儿受体FcRn的结合亲和力增强。进一步地，本发明的优选实施方式中，所述Fc突变体优选自下组：(i)vFcγ1：含有Leu234Val、Leu235Ala和Pro331Ser突变的人IgG1绞链区、CH2和CH3区域(如SEQ ID NO：8所示氨基酸序列)；(ii)vFcγ2-1：含有Pro331Ser突变的人IgG2绞链区、CH2和CH3区域(如SEQ ID NO：9所示氨基酸序列)；(iii)vFcγ2-2：含有Thr250Gln和Met428Leu突变的人IgG2绞链区、CH2和CH3区域(如SEQ ID NO：10所示氨基酸序列)；(iv)vFcγ2-3：含有 Pro331Ser、Thr250Gln和Met428Leu突变的人IgG2绞链区、CH2和CH3区域(如SEQ ID NO：11所示氨基酸序列)；(v)vFcγ4：含有Ser228Pro和Leu235Ala突变的人IgG4绞链区、CH2和CH3区域(如SEQ ID NO：12所示氨基酸序列)。
更优选地，本发明的一实施例中，所述FVII-Fc融合蛋白从N端至C端依次含有FVII(具有如SEQ ID NO：13所示的氨基酸序列)、连接肽(具有如SEQ ID NO：2所示的氨基酸序列)和人IgG Fc(具有如SEQ ID NO：11所示的氨基酸序列)。
更优选地，本发明的一实施例中，所述FVIII-Fc融合蛋白从N端至C端依次含有FVIII(具有如SEQ ID NO：14所示的氨基酸序列)、连接肽(具有如SEQ ID NO：2所示的氨基酸序列)和人IgG Fc(具有如SEQ ID NO：11所示的氨基酸序列)。
更优选地，本发明的一实施例中，所述Exendin-4-Fc融合蛋白从N端至C端依次含有Exendin-4(具有如SEQ ID NO：15所示的氨基酸序列)、连接肽(具有如SEQ ID NO：2，3，5或7所示的氨基酸序列)和人IgG Fc(具有如SEQ ID NO：11所示的氨基酸序列)。
更优选地，本发明的一实施例中，所述IL-7-Fc融合蛋白从N端至C端依次含有IL-7(具有如SEQ ID NO：16所示的氨基酸序列)、连接肽(具有如SEQ ID NO：2所示的氨基酸序列)和人IgG Fc(具有如SEQ ID NO：11所示的氨基酸序列)。
更优选地，本发明的一实施例中，所述hGH-Fc融合蛋白从N端至C端依次含有hGH(具有如SEQ ID NO：17所示的氨基酸序列)、连接肽(具有如SEQ ID NO：2所示的氨基酸序列)和人IgG Fc(具有如SEQ ID NO：11所示的氨基酸序列)。
本发明的另一些优选实施例中，所述融合蛋白的活性分子K1为抗体的重链可变区(VH)；和K2为抗体的轻链可变区(VL)。且K1和K2通过所述连接肽连接形成单链抗体(ScFv)。
本发明的另一些优选实施例中，所述融合蛋白的活性分子K1包含第一种抗体、或者抗体片段；和所述活性分子K2包含第二种抗体、或者抗体片段，且 K1和K2通过所述连接肽连接形成双特异性抗体。
优选地，本发明的一实施例中，K1为Anti-CD20的完整双链抗体，K2为Anti-CD3的单链抗体，K1和K2通过所述连接肽连接形成双特异性抗体。更优选地，所述双特异性抗体包含的Anti-CD20双链抗体的重链具有如SEQ ID NO：18所示的氨基酸序列，且所述轻链具有如SEQ ID NO：19所示的氨基酸序列；和所述双特异性抗体包含的Anti-CD3单链抗体具有如SEQ ID NO：20所示的氨基酸序列；和所述连接肽具有如SEQ ID NO：4或6所示的氨基酸序列；且所述Anti-CD20双链抗体的重链通过所述连接肽与所述Anti-CD3单链抗体连接。
另一方面，本发明还提供了所述连接肽用于制备融合蛋白的方法，所述融合蛋白的结构通式表示为：K1-L-K2或K2-L-K1，其中K1是第一种生物活性分子；L是所述连接肽；K2是第二种生物活性分子，且组成所述融合蛋白的各部分自N端至C端首尾依次相连。其中，所述活性分子可选自蛋白或蛋白结构域、多肽、抗体或抗体片段，优选为蛋白或蛋白结构域、抗体或抗体片段。所述制备方法包括可以使K1和K2通过L相连接的步骤；本发明的优选实施例中，所述方法包括如下步骤：
(a)将编码所述融合蛋白的第一种活性分子K1和第二种活性分子K2的DNA序列通过连接肽L的DNA序列连接组成融合基因；
(b)将步骤(a)中获得的融合基因引入真核或原核表达宿主；
(c)培养步骤(b)筛选到的高产量表达宿主，表达融合蛋白；
(d)收获步骤(c)得到的发酵液，并分离纯化融合蛋白。
示例性地，所述连接肽用于连接活性蛋白/多肽和长循环半衰期的血清蛋白，例如抗体/免疫球蛋白Fc片段、人血白蛋白(HSA)、转铁蛋白(TF)等。本发明一优选实施例中，FVII-Fc融合蛋白的制备方法中包含使活性分子FVII和人IgG Fc通过所述连接肽(SEQ ID NO：2)连接的步骤；本发明一优选实施例中，FVIII-Fc融合蛋白的制备方法中包含使活性分子FVIII和人IgG Fc通过所述连接肽(SEQ ID NO：2)连接的步骤；本发明另一优选实施例中，Exendin4-Fc融合蛋白的制备方法中包含使活性分子Exendin4与人IgG Fc通过所述连接肽(SEQ ID NO：2、3、5或7)连接的步骤；本发明另一优选实施例中，IL-7-Fc融合蛋白的制备方法中包含使活性分子IL-7和人IgG Fc通过所述连接肽(SEQ ID NO：2)连接的 步骤；本发明另一优选实施例中，hGH-Fc融合蛋白的制备方法中包含使活性分子hGH和人IgG Fc通过所述连接肽(SEQ ID NO：2)连接的步骤。
示例性地，所述连接肽用于双特异性抗体的构建。本发明优选实施例中，Anti-CD3XCD20的双特异性抗体的制备方法中包含使Anti-CD20的双链抗体和Anti-CD3的单链抗体通过所述连接肽(SEQ ID NO：4或6)连接的步骤。
本发明人发现，本发明技术效果可以概况如下：
1、本发明组成所述连接肽的CTP，它含有多个O-糖基侧链，能形成相对稳定的立体构象，因而具有刚性结构，能更有效地将融合蛋白的两部分隔离开，消除彼此间的空间位阻效应。由活性蛋白与Fc组成的一系列融合蛋白，例如FVII-Fc融合蛋白，连接肽中增加CTP刚性单元保证了N-端融合的活性蛋白不会影响Fc变体与FcRn的结合位点，从而影响半衰期；另外Fc的ProteinA结合位点对于制备工艺中纯化步骤很重要，连接CTP刚性单元保证N-端融合的活性蛋白也不会“罩住”它与protein A的结合位点。另一方面，CTP刚性单元的添加也使得约25KD大小的Fc片段不会干扰N-端融合的活性蛋白的正确折叠，造成其生物学活性/功能的下降或丧失。本发明多个实施例中都表明CTP刚性单元的加入使得融合蛋白的生物学活性显著提高。这可能解释为具有多个糖基侧链的CTP刚性多肽，相对于(GGGGS)n这类柔性连接肽的无规则卷曲，它可以形成稳定的立体构象，这种构象能有效地拉开融合蛋白两个半分子间的空间距离，促使活性蛋白和Fc段独立折叠形成正确的三维构象而互不影响各自的生物活性，从而减少因活性蛋白错误折叠或构象变化而导致的活性降低甚至丧失或形成聚合体，以提高融合蛋白的生物活性。
2、本发明所述连接肽具有广泛适用性和可移植性，刚性单元和柔性单元的组合序列，则能赋予连接肽具有介于完全刚性和完全柔性之间的构象，多肽具体的刚性(或柔性)程度因两种序列之间的比例、排列而异。通过设计不同比例和排列的刚性单元和柔性单元的组合序列，能对连接肽的刚性进行细微调控，以满足融合蛋白构建中对连接肽刚性的不同要求。
3、CTP含有糖基化位点，带负电、高度唾液酸化的CTP能够抵抗肾脏对其清除作用，进一步延长融合蛋白的半衰期；改善药代动力学参数，如降低清除率、 降低表观分布容积、增加AUC(0-t)，使融合蛋白的生物利用度提高，可以预期其临床给药剂量也将降低。
4、CTP上连接的糖基侧链的保护作用可以降低连接肽对蛋白酶的敏感性，使融合蛋白不易在连接区被降解。
术语解释
“抗体片段”意即抗体的抗原结合片段及抗体类似物，其通常包含至少部分母体抗体(parental antibody)的抗原结合区或可变区(例如一个或多个CDR)。
“Fc”区含有包含抗体的CH2和CH3结构域的两个重链片段。两个重链片段由两个或多个二硫键并通过CH3结构域的疏水作用保持在一起。
“Fv区”包含来自重链和轻链二者的可变区，但缺少恒定区。
“单链Fv抗体”(或“ScFv抗体”)是指包含抗体的VH和VL结构域的抗体片段，其中这些结构域存在于单个多肽链中。一般而言，Fv多肽另外在VH和VL结构域之间包含多肽接头，该接头使得ScFv能形成用于抗原结合的所需结构。
“双特异性抗体”是指包含两个可变结构域或ScFv单位使得所得抗体识别两种不同抗原决定簇。
“抗体融合蛋白”是指利用基因工程技术将抗体片段与其他生物活性蛋白融合所得的产物。由于融合蛋白的不同，这种抗体融合蛋白具有多种生物学功能。
例如，含Fv段的抗体融合蛋白：将Fab或Fv与某些毒素、酶、细胞因子基因拼接，通过这些抗体的引导，可将其生物活性分子导向靶细胞特定部位，即所谓的“生物导弹”；
例如，嵌合受体：将ScFv与某些细胞膜蛋白分子融合，形成的融合蛋白，可表达于细胞表面，称为嵌合受体，赋予特定细胞结合某种抗原的能力；
例如，含Fc段的抗体融合蛋白：将抗体IgG恒定区Fc段与生物活性分子融合，形成Fc融合蛋白。Fc融合蛋白不仅可发挥活性分子的生物学活性，还赋予其类似抗体的特性，包括延长血浆半衰期以及Fc片段特有的一系列效应功能，例如，一方面Fc段在消灭病原体的免疫防御中起重要作用。Fc介导的IgG的效应子功能发挥通过两种机制：(1)与细胞表面Fc受体(FcγRs)结合，由吞噬作用或裂解作用或杀伤细胞通过抗体依赖性细胞毒性(ADCC)途径消化病原体， 或(2)与第一补体成分C1的C1q结合，引发补体依赖性细胞毒性(CDC)途径，从而裂解病原体。另一方面，抗体Fc段与FcRn受体结合可以避免抗体进入溶酶体而被降解。包含Fc段的融合蛋白被表达FcRn的细胞内吞和保护。这些融合蛋白不被标记为降解，而是再次进入循环系统，因而增加了这些蛋白的体内半衰期。再一方面，FcRn在成年上皮组织中有活性，并且在肠腔、肺气管、鼻腔、阴道、结肠和直肠的上皮细胞中表达。由Fc段组成的融合蛋白可通过FcRn介导的胞转作用，有效地穿梭上皮屏障。
“hCG-β羧基末端肽(CTP)”是一段来自人绒毛膜促性腺激素(hCG)的β-亚基羧基末端的短肽。四种与生殖相关的多肽类激素促卵泡激素(FSH)、黄体生成素(LH)、促甲状腺素(TSH)和绒毛膜促性腺激素(hCG)含有相同的α-亚基和各自特异的β-亚基。与其它三种激素相比，hCG体内半衰期明显延长，这主要来源于其β-亚基上特有的羧基末端肽(CTP)。CTP含有37个氨基酸残基，它具有4个O-糖基化位点，糖侧链终端是唾液酸残基。带负电、高度唾液酸化的CTP能够抵抗肾脏对其的清除作用，从而延长蛋白在体内的半衰期(Fares FA等，Proc Natl Acad Sci USA，1992，89：4304-4308)。
附图说明
图1、HA小鼠等活性给予FP-A1和诺其1h和2h后出血时长比较。与HA-N-1h组相比，*P＜0.05，***P＜0.01；与C57-NS组相比，#P＜0.05，###P＜0.01。
图2、FP-A1及诺其在华法林大鼠上的活性半衰期。
图3、db/db糖尿病小鼠单次注射FP-C1和FP-C5在0～216h间RBG值变化曲线。
图4、不同剂量组FP-C1对db/db糖尿病小鼠HbA1c(％)的影响；统计学差异标记注释：FP-C1组与模型组相比，*P＜0.05，**P＜0.01。
图5、STZ诱导糖尿病小鼠单次注射FP-C1在0～240h间RBG值变化曲线；统计学差异标记注释：FP-C1组与模型组相比，*P＜0.05，**P＜0.01；杜拉鲁肽组与模型组相比，△P＜0.05，△△P＜0.01。
图6、FP-C1对高脂饲料喂养小鼠体重增长的影响。
图7、FP-C1对高脂饲料喂养小鼠糖耐量的影响(means±SD，n＝8)。注：与 正常组相比，#P＜0.05，##P＜0.01；与高脂组相比，*P＜0.05，**P＜0.01。
图8、FP-C1对高脂饲料喂养小鼠血清胰岛素含量的影响(means±SD，n＝8)。注：与正常组相比，#P＜0.05，##P＜0.01；与高脂组相比，*P＜0.05，**P＜0.01。
图9、FP-C1对高脂饲料喂养小鼠胰岛素耐受指数的影响(means±SD，n＝8)。注：与正常组相比，#P＜0.05，##P＜0.01；与高脂组相比，*P＜0.05，**P＜0.01。
图10、FP-C1对高脂饲料喂养脂肪细胞横截面积的影响。注：A：正常组；B：高脂组；C：FP-C1组。
图11、IL7-Fc融合蛋白FP-D1和FP-D2刺激小鼠单个核细胞增殖的能力。
图12、hGH-Fc融合蛋白FP-E1和FP-E2刺激Nb2细胞增殖的能力。
图13、hGH-Fc融合蛋白FP-E1和FP-E2血药浓度随时间变化曲线。
图14、hGH-Fc融合蛋白FP-E1给药后各组的生长曲线。
图15、Anti-CD3×CD20双特异性抗体FP-F1浓度依赖地活化人PBMC细胞分泌IFN-γ的检测结果。
图16、Anti-CD3×CD20双特异性抗体FP-F1浓度依赖地活化人PBMC结果。
图17、Anti-CD3×CD20双特异性抗体FP-F1杀伤皮下移植瘤的药效检测结果。
具体实施方式
实施例1、连接肽用于融合蛋白的构建
本发明人构建了一系列含连接肽的融合蛋白K1-L-K2。各融合蛋白的组成如表1所示。将编码所述融合蛋白活性分子K1和活性分子K2的DNA序列通过连接肽L的DNA序列相连组成融合基因序列。优选地，它们都是经人工优化过的CHO细胞偏爱密码子。优选地使用化学合成方法获得。为了便于将上述获得的融合基因目的片段插入表达载体的特定位点，在所合成片段5’和3’端各插入一个限制性酶切位点，分别为SpeI和EcoRI。经测序验证后的融合蛋白基因以相应的限制性内切酶酶切，然后插入到以PCDNA3.1为模板并改造后的表达质粒PXY1A1的相应酶切位点间，得到融合蛋白高表达质粒。PXY1A1质粒包含但不限于以下重要表达元器件：1)人巨细胞病毒早期启动子和哺乳动物细胞高外源表达所需增强子；2)双重筛选标记物，在细菌中具有卡那霉素抗性，在哺 乳动物细胞中具有G418抗性；3)鼠二氢叶酸还原酶(DHFR)基因表达框，当宿主细胞为DHFR基因缺陷型时，氨甲蝶呤(MTX)能共扩增融合基因和DHFR基因(参见美国专利US 4,399,216)。将融合蛋白表达质粒转染入哺乳动物宿主细胞系，为了获得稳定高水平的表达，优选的宿主细胞系是DHFR酶缺陷型CHO细胞(参见美国专利US 4,818,679)。转染两天后，将培养基换成含0.6mg/mL G418的筛选培养基，细胞以一定浓度(5000-10000个活细胞/孔)接种在96孔培养板里，培养10-14天直至大的离散细胞克隆出现。用ELISA分析方法，筛选对选择用药具有抗性的转染子。通过极限稀释96孔培养板，亚克隆产生高水平融合蛋白的孔。对于多方验证效果良好的融合蛋白，为了实现更高水平表达，宜用受MTX药物抑制的DHFR基因进行共扩增。在含有递增浓度MTX的生长培养基中，用DHFR基因共扩增转染的融合蛋白基因。获得的高表达单克隆细胞株先在摇瓶或5升发酵罐中进行补料培养，再以Protein A亲和层析柱和其它离子交换层析柱纯化融合蛋白。
发明人构建了一系列含本发明所述柔性-刚性单元连接肽的融合蛋白，同时还构建了多种仅含不同长度柔性连接肽的融合蛋白作为对照。例如，FVII-Fc融合蛋白(含CTP的FP-A1；不含CTP的FP-A2和FP-A3)、FVIII-Fc融合蛋白(含CTP的FP-B1；不含CTP的FP-B2和FP-B3)、Exendin-4及其类似物Fc融合蛋白(含CTP的FP-C1、FP-C2、FP-C3、FP-C4；不含CTP的FP-C5)、IL7-Fc融合蛋白(含CTP的FP-D1；不含CTP的FP-D2)、hGH-Fc融合蛋白(含CTP的FP-E1；不含CTP的FP-E2)。此外，发明人还构建了Anti-CD20×CD3双特异性抗体(含CTP的FP-F1和FP-F2)。各融合蛋白组成如表1所示，各融合蛋白组成部分的氨基酸组成见序列表。
表1、各种融合蛋白组成(从N端至C端)
Figure PCTCN2016106011-appb-000003
Figure PCTCN2016106011-appb-000004
实施例2、凝血因子FVII-Fc融合蛋白制备、生物学活性和体内活性半衰期测定
2.1凝血因子FVII-Fc融合蛋白制备和鉴定
实施例1中获得的FP-A1、FP-A2和FP-A3CHO稳定表达细胞株，经过10-14天的摇瓶补料培养，经Protein A亲和层析、多维模式层析、阴离子交换层析和分子筛层析四个步骤进行纯化，然后再以溶液孵育自激活法活化融合蛋白。SDS-PAGE蛋白电泳检测显示，未活化的FP-A2在还原条件下，单链分子分别在70-85kDa和40kDa附近有两条明显条带，预示降解片段的存在，大约占20-30％；在非还原条件下，纯化蛋白迁移至约130kDa，并伴有部分＞200kDa的条带，表明部分融合蛋白发生聚合。未活化的FP-A3在还原条件下，单链分子接近100kDa，无杂带，在非还原条件下，部分纯化蛋白迁移至＞200kDa，表明FP-A3都是多聚体。未活化的FP-A1在还原条件下，单链分子100-110kDa，无明显杂带，在非还原条件下，纯化蛋白迁移至150kDa。活化的FP-A1在还原条件下呈现两条清晰条带，分别为74.3KDa的HC-L-CTP-Fc和约24.0KDa的LC，无其它杂带，在非还原条件下，纯化蛋白迁移至150kDa，表明融合蛋白FP-A1未发生明显降解且未出现明显的聚合现象，具有更高的热力学稳定性和更强的抗蛋白酶水解能力。该实施例表明，连接肽中含有CTP刚性单元，能够增加融合蛋白的稳定性，不易降解，并减少聚合体的形成。
2.2凝血法直接测定融合蛋白的生物学活性
凝血法测定FVIIa生物学活性是通过纠正FVIIa因子缺乏血浆所导致凝固时间延长的能力而获得的。采用法国STAGO公司生产的试剂盒
Figure PCTCN2016106011-appb-000005
-Deficient FVII(Cat.No.00743)。检测方法首先是将稀释的已知FVII活性的正常人冻干血浆(Unicalibrator，Cat.No.00625)与乏VII基质血浆混合，测定凝血酶原时间(PT)，建立标准曲线，再将待测血浆适度稀释后与乏FVII基质血浆混合，进行PT测定。通过标准曲线所拟合的活性百分比C(％)与PT时间t(s)的对数方程，可测得待测样本FVIIa的活性多少，其结果用正常血浆的百分比来表示(％)，试剂盒中所提供的标准品百分比活性(％)与国际酶活单位IU的对应关系为100％＝1IU，据此可求算出待测样品FVII的酶比活性大小，单位为IU/mg。结果显示，在最佳实验条件下，FP-A1、FP-A2和FP-A3的最高活性分别约20000IU/mg、4000IU/mg和7000IU/mg。如上实验结果表明，连接肽的类型和长度对于FVII-Fc的活性和稳定性影响都很大，含有刚性单元连接肽的融合蛋白FP-A1体外生物学活性远高于不含刚性单元的融合蛋白FP-A2和FP-A3，且表明仅靠延长柔性连接肽的长度，并不能有效的提高融合蛋白的活性。融合分子间的空间位阻效应影响融合蛋白的正确构象的形成，因而稳定性降低，易形成聚合体。该实施例表明连接肽中含有CTP刚性单元，能够降低Fc配体的空间位阻效应，提高融合蛋白的活性及稳定性。
2.3测定FVII-Fc融合蛋白的体内抑制出血作用
以FVIII因子基因剔除纯合子甲型血友病小鼠(hemophilia A，HA，购自上海南方模式生物研究中心)的断尾出血模型(tail vein transection(TVT)bleeding model)评估FVII-Fc融合蛋白的对血友病小鼠的止血作用。16-20周龄雄性HA小鼠适应性饲养一周后，随机分为3组，每组6只。其中两组给予300,000IU/kg的FP-A1，另一组给予300,000IU/kg的诺其(
Figure PCTCN2016106011-appb-000006
Novo Nordisk公司)。同时以16-20周龄，野生型雄性C57BL/6J小鼠(购自上海南方模式生物研究中心)作为正常对照组(n＝6)，尾静脉注射给予等体积剂量的生理盐水。给予FP-A1的两组HA小鼠分别于给药后1h和2h进行断尾试验；给予诺其的HA小鼠于给药后1h进行断尾试验；C57BL/6J正常对照组(C57-NS组)小鼠于注射后2h进 行断尾试验。所有数据以均数±标准误
Figure PCTCN2016106011-appb-000007
表示，各实验组间比较采用t-test检验分析，分析软件采用Graphpad Prism 5.0，p＜0.05认为有统计学意义。
如图1所示，诺其组给药1h后，小鼠出血时间为30分钟，说明它已无促凝止血效果(HA-N-1h组)，而给予FP-A1 1h(HA-F-1h组)和2h后(HA-F-2h组)仍然能有效止血，出血时间均比诺其组显著缩短(P＜0.05)。这说明FP-A1相对于诺其具有显著延长的活性半衰期。
2.4.测定FVII-Fc融合蛋白的体内活性半衰期
本实验为考察FP-A1在华法林致大鼠凝血障碍模型上的活性半衰期。据文献报道的方法(Joe Salas等，Thrombosis Research，2015，135(5)：970-976或Gerhard Dickneite等，Thrombosis Research，2007，119：643-651)进行实验，选取8-12周龄200-220g SD大鼠16只(购于北京维通利华实验动物技术有限公司)，将大鼠随机分为2组，每组8只。以2.5mg/kg华法林(Orion Corporation，Finland，批号：1569755)灌胃，24h后分别静脉给予10,000IU/kg的FP-A1或诺其(
Figure PCTCN2016106011-appb-000008
Novo Nordisk公司)。FP-A1组于给药后0.05、0.5、1、2、3、5、8、12h采血；诺其组于给药后0.05、0.5、1、2、3、5h采血。血样以终浓度0.013M的柠檬酸钠为抗凝剂，3000rpm离心10min取上清，按2.2中的方法进行样品活性测定并计算活性半衰期。
如图2所示，计算得出FP-A1的活性半衰期为3.03±0.35h；诺其的活性半衰期为1.01±0.16h。相比等活性的诺其，FP-A1在大鼠体内活性半衰期延长了约3倍，单次注射FP-A1 3h后血浆凝血活性约40％，而等活性给药的诺其组3h后活性已降至3％；而FP-A1给药12h后血浆凝血活性仍保持在7％以上。
2.3和2.4的试验结果表明，由本发明所述连接肽(包含柔性单元和CTP刚性单元)所组成的融合蛋白，具有显著延长的功能半衰期，表明该连接肽消除了活性蛋白FVII对Fc与其受体FcRn结合位点的屏蔽效应，获得了显著延长的半衰期，同时也证明FVII因添加本发明所述连接肽而未受到C端融合的Fc位阻效应的影响，形成了正确的三维构象，保持了较高的生物学活性。
实施例3、凝血因子FVIII-Fc融合蛋白生产、生物学活性测定
3.1凝血因子FVIII-Fc融合蛋白生产和鉴定
实施例1中获得的FP-B1、FP-B2和FP-B3CHO稳定表达细胞株，经过7-12天的补料培养或半连续罐流培养，收获上清液立即进行Protein A和/或VIII-select(GE)亲和层析纯化。在最佳培养条件下，FP-B2收获的上清液经过Protein A和VIII-select(GE)两步亲和层析纯化，仍然含有多种组分。SDS-PAGE蛋白电泳检测显示，在还原条件下，显现180kDa的主带和多条40-100kDa大小的片段；在非还原条件下，绝大部分纯化蛋白迁移至＞300kDa。表明FP-B2产物多以聚合体形式存在，且不稳定，易降解。在同样培养条件下，FP-B1收获的上清液经过Protein A和VIII-select(GE公司)两步亲和层析纯化，在还原条件下，显现清晰的三条带，分别为单链FVIII-Fc(190kDa)、轻链-Fc(105KDa)和重链(90KDa)，无杂带；在非还原条件下，FP-B1和FP-B3纯化蛋白迁移至＞200kDa，而FP-B3中大部分蛋白位于浓缩胶部位，预示着FP-B3也多以聚合体形式存在。
据报道FVIII 2303-2332区域的脂质结合区(Lipid binding region)对其功能至关重要，该区域极微小的构象变化即会引起蛋白的聚合，导致其活性丧失(Gilbert GE等，Biochemistry，1993，32(37)：9577-9585)。该试验结果表明，含有CTP刚性单元的连接肽可以消除C端Fc对FVIII脂质结合区的位阻效应，使其空间构象几乎不受影响，从而减少蛋白的聚合，增强其稳定性，大大提高了FVIII-Fc融合蛋白的生物活性。
3.2凝血法直接测定FVIII-Fc融合蛋白的生物学活性
凝血法测定FVIII生物学活性是通过纠正FVIII因子缺乏血浆所导致凝固时间延长的能力而获得的。本发明采用法国STAGO公司生产的VIII因子(FVIII)测定试剂盒(凝固法)
Figure PCTCN2016106011-appb-000009
-Deficient FVIII(Cat.No.00725)。检测方法是将稀释的已知凝血因子VIII活性的正常人冻干血浆(Unicalibrator(Cat No.00625))做活化部分凝血活酶时间(APTT)测定，测试仪器为STAGO公司
Figure PCTCN2016106011-appb-000010
系列血凝仪，先建立标准曲线，再将FVIII融合蛋白与乏VIII因子的基质血浆混合，测定其APTT值，通过标准曲线所拟合的活性百分比C(％)与APTT时间t(s)的对数方程，可测得待测样本FVIII的活性大小，其结果用正常血浆的活性百分比表示(％)，试剂盒中所提供的标准品百分比活性(％)与国际酶活单位IU的对 应关系为100％＝1IU，据此可求算出待测样品FVIII的酶比活性大小，表示为IU/mg。结果显示，在最佳实验条件下，FP-B1、FP-B2和FP-B3的最高活性分别为10000IU/mg、150IU/mg和1300IU/mg。考虑到FP-B2蛋白分子中大部分为无活性的多聚体或降解片段，所以FP-B2和FP-B3中实际具有活性的分子比活并不一定差别很大，这预示着延长柔性连接肽长度对提高目的蛋白FVIII的活性作用有限。FP-B1与FP-B2的活性差异显著，充分表明连接肽中含有CTP刚性单元，能够降低Fc配体的空间位阻效应，提高FVIII-Fc融合蛋白的活性。
实施例4、Exendin4-Fc融合蛋白生产，生物学活性和体内半衰期测定
4.1体外生物学活性
实施例1中获得的FP-C1、FP-C2、FP-C3、FP-C4和FP-C5的CHO稳定表达细胞株，经过12-14天的摇瓶补料培养，以Protein A亲和层析柱纯化，两种融合蛋白纯度都在95％以上，分子大小也符合预期，然后用于活性分析。体外活性测定方法参照文献(Zlokarnik G等，Science，1998，279(5347)：84-88)。方法简述如下：首先将带有人GLP-1R表达质粒和CRE-Luc报告基因的表达质粒PGL-4.29(Luc2P/CRE/Hygro)(Promega公司)共转染CHO-K1细胞，通过抗生素加压筛选获得共表达两种质粒的稳定细胞株。体外活性分析时，以16000个细胞/孔，每孔200μl接种到96孔细胞培养板中，用含10％FBS的DMEM培养基培养16～24小时，待细胞生长至覆盖板底90％以上面积时，用含10％FBS的DMEM培养基梯度稀释融合蛋白FP-C1、FP-C2、FP-C3、FP-C4和FP-C5，10μl/孔加入，浓度梯度设置为0.010、0.020、0.039、0.078、0.156、0.313、0.625、1.25和2.5nM，同时设置等浓度的杜拉鲁肽阳性对照组(Eli Lilly Company生产，货号：9301897)。在37℃，5％CO2条件下孵育5-6小时后，吸去上清，缓慢加入300μl PBS/孔洗涤细胞，随后吸去PBS，加入40μl裂解液，震荡15min，随后每孔加入40μl荧光素酶底物(荧光素酶(Luciferase)报告基因检测试剂盒，货号：GM-040501B，吉满生物有限公司产品)，反应2分钟，以多功能酶标仪(SpectraMax M5system，Molecular Device公司)于波长560nm下测定荧光值，并根据荧光值绘制剂量反应曲线，计算出EC50值，结果见表2。FP-C1、FP-C2、FP-C3、FP-C4和FP-C5的EC50值约为0.03086nM、0.03156nM、0.03684nM、0.04012nM和 0.03586nM，杜拉鲁肽EC50值约为0.02987nM，含CTP的FP-C1、FP-C2、FP-C3、FP-C4与不含CTP的FP-C5的体外生物学活性相当。发明人理解的原因可能是对于Exendin-4这类结构简单的小分子多肽，它与融合配体Fc相互间的空间位阻作用极小，因此CTP对消除位阻效应，提高融合蛋白活性方面的作用不显著。
表2、各融合蛋白的体外活性EC50值比较
Figure PCTCN2016106011-appb-000011
4.2db/db糖尿病小鼠单次注射FP-C1和FP-C5的血糖变化情况
雌性糖尿病db/db小鼠(购自上海斯莱克实验动物有限责任公司)，8周龄，体重42±2g，按体重随机分为3组，每组6只。受试药组按3mg/kg剂量皮下注射FP-C1和FP-C5，阳性组注射3mg/kg的杜拉鲁肽(Eli Lilly and Company生产，货号：9301897)，模型组注射等体积剂量(10mL/kg)的PBS缓冲液。各组动物分别于给药前(0h)、给药后1h、2h、4h、6h、24h、48h、72h、96h、120h、144h、168h、192h和216h尾静脉采血，用血糖仪(三诺安准血糖仪)测定随机血糖值(Random blood glucose，RBG)，并记录数据。血糖数据以均数±标准差(means±SD)形式表示，采用SPSS18.0统计软件分析数据。
在相同剂量下，FP-C1和FP-C5与阳性对照药杜拉鲁肽均有明显降血糖作用，如图3所示，从给药后9天内小鼠RBG值变化曲线图中可以看出，杜拉鲁肽降血糖作用仅能维持至第4天(P＞0.05)，在给药后第120小时，其血糖值与模型组相比已不具有统计学差异；而FP-C1能维持至给药后168小时，即第7天小鼠的血糖水平与模型组相比，仍具有统计学差异(P＜0.05)；FP-C5能维持至给药后144小时，即第6天小鼠的血糖水平与模型组相比，仍具有统计学差异。因而，可知FP-C1相对于FP-C5，在糖尿病小鼠模型中具有更长效的降血糖作用，表明连接肽CTP刚性单元可进一步延长Exendin-4的体内功能半衰期。
4.3Exendin4-Fc融合蛋白的在大鼠体内的药代动力学特征
雄性SPF级SD大鼠(购自上海必凯实验动物有限公司)，预饲一周后单次 皮下注射(sc)0.5mg/kg的FP-C1和FP-C5，每组4只，分别于给药前0h，给药后2h、8h、24h、32h、48h、56h、72h、96h、120h和144h眼眶取血，每次约0.3ml左右，分别记作：T0、T2、T8、T24、T32、T48、T56、T72、T96、T120和T144。取血后静置，再以5000rpm离心10min分离血清，于-70℃冷冻保存后合并检验。用双抗夹心ELISA测定时，以自制或市售的抗Exendin-4或GLP-1的NH2末端单克隆抗体(如Santa Cruz公司生产，货号SC-65389)包被、以自制或市售的辣根过氧化物酶标记的鼠抗人IgG Fc单抗(如北京义翘神州生物技术有限公司，货号：10702-MM01E-50)进行检测。后将数据输入分析软件PKSOLVER，得出待测药物在血液中T1/2，Cmax和AUC(0～t)等药代动力学参数。
如表3中结果显示，0.5mg/kg的FP-C5在大鼠体内的循环半衰期T1/2为14.9±1.29小时，而0.5mg/kg的FP-C1在大鼠体内的T1/2分别为21.4±2.51小时。FP-C1的最大血药浓度Cmax值显著高于FP-C5。另外，通过比较表3中不同采血时间点测得的AUC0～t(t＝2h、5h、8h、24h、28h、32h或48h)可得知，在给药剂量相同的情况下，FP-C1的药物暴露量显著高于FP-C5，即FP-C1在大鼠体内的绝对生物利用度较不含CTP的FP-C5更高，可以预期其临床给药剂量也将降低。
表3、雄性SD大鼠单次皮下注射0.5mg/kg的FP-C1、FP-C5的药代动力学参数
Figure PCTCN2016106011-appb-000012
4.4、db/db糖尿病小鼠连续10周给予FP-C1的随机血糖及HbA1c含量变化
SPF级雌性db/db小鼠(购自上海斯莱克实验动物有限公司)，8周龄，适应性饲养1周后，将24只db/db小鼠按随机血糖值(random blood glucose，RBG)随机分为4组(n＝6)：模型组、FP-C1按低(0.75mg/kg)、中(1.5mg/kg)、高(3mg/kg)剂量给药。各给药组皮下注射给予相应剂量的药物溶液，模型组皮下注射PBS缓冲液，给药体积均为10ml/kg。各组动物每周给药一次，连续给药10周，分别用血糖仪(安准血糖仪，长沙三诺生物传感股份有限公司产品)检测给药后不同采血时间点各组小鼠RBG值，并记录数据。采血时间点设定：第一次给药前(0d)、给药后第7d、14d、21d、28d、35d、42d、49d、56d、63d和70d。第70d，各组 小鼠禁食14h后眼眶取血，然后立即用糖化血红蛋白测定试剂盒(免疫比浊法)(深圳希莱恒医用电子有限公司产品，产品注册号：粤食药监械(准)字2013第2400025号)及其配套仪器H700特定蛋白分析仪(深圳希莱恒医用电子有限公司产品)检测全血中糖化血红蛋白(HbA1c)含量，结果以HbA1c占总血红蛋白的百分比(％)表示。
数据以均数±标准差(means±SD)形式表示，采用SPSS18.0统计软件分析数据。正态分布，多组间均数差异采用单因素方差分析，方差齐性采用Dunnett t-检验，方差不齐采用Dunnett’T3检验；非正态性分布采用非参数检验，P＜0.05表示具有显著性统计学差异。
从表4中各组小鼠连续给药10周随机血糖值的变化趋势可知，FP-C1高、中、低剂量组的小鼠血糖值相对模型组都有一定程度的降低，并且其降血糖活性呈现剂量依赖性。提示FP-C1能有效、持续地控制db/db糖尿病小鼠的血糖水平。而且，首次给药和末次给药FP-C1的降糖效果相似，说明未出现由于受体的快速抗药性反应，引发对FP-C1的耐受作用。
糖化血红蛋白(HbA1c)是血中葡萄糖与红细胞的血红蛋白相结合的产物，它与血中的葡萄糖水平呈正比的关系。由于红细胞在血循环中的寿命约为120天，因此糖化血红蛋白可反映取血前4-12周血糖的总水平，弥补了空腹血糖只反映瞬时血糖的不足。因而，HbA1c是长期控制血糖最重要的评估指标，也是临床决定是否要更换治疗方案的重要依据。本实施例中HbA1c检查结果可以稳定、可靠的反映出取血前2～3个月小鼠的血糖控制情况。连续给药10周后各组小鼠HbA1c含量测定结果如图4所示，显示高、中、低剂量组FP-C1糖化HbA1c含量较模型组均发生显著性降低(P＜0.01)，且呈现剂量依赖性，其中高剂量组HbA1c(％)降低最显著(6.38±1.63)，但仍高于正常C57BL/6J小鼠的HbA1c水平(正常值参考范围：2.5％-3.5％)，说明即使3mg/kg的FP-C1也不会引起长期低血糖反应的发生；以上结果提示FP-C1能够长期、有效、平稳地控制小鼠血糖，且不增加引起低血糖的风险，这与表4中血糖变化趋势相符。
表4.FP-C1对db/db小鼠随机血糖的影响(means±SD，n＝6)
Figure PCTCN2016106011-appb-000013
Figure PCTCN2016106011-appb-000014
注：各剂量组与模型组相比*P＜0.05；**P＜0.01。
续表4.FP-C1对db/db小鼠随机血糖的影响(means±SD，n＝6)
Figure PCTCN2016106011-appb-000015
注：各剂量组与模型组相比*P＜0.05；**P＜0.01。
4.5、STZ诱导糖尿病小鼠单次注射FP-C1的药效学研究
选取SPF级雄性昆明小鼠(购自上海斯莱克实验动物有限公司)，体重25±2g，按体重随机分为糖尿病组和正常组。适应饲养1周的昆明小鼠，禁食18h，称重，糖尿病组按150mg/kg腹腔注射给予1％STZ溶液，pH＝4.4(购自Sigma公司，货号S0130)；正常组小鼠(n＝8)腹腔注射给予等体积的柠檬酸-柠檬酸钠缓冲液(购自国药集团化学试剂有限公司)。注射后第10天检测糖尿病组小鼠随机血糖值(Random blood glucose，RBG)，其中RBG≥16.7mmol/L为造模成功的糖尿病小鼠。为观察受试药物对STZ诱导的糖尿病小鼠的降血糖作用，选取STZ-诱导糖尿病小鼠32只，随机分为4组，每组8只，分别于皮下注射给予3mg/kg的FP-C1和3mg/kg的杜拉鲁肽。模型组和正常组分别给予等体积剂量(10ml/kg)的PBS缓冲液。分别于给药前(0h)、给药后1h、2h、4h、6h、24h、48h、72h、96h、120h、144h、168h、192h、216h和240h尾静脉采血，用血糖仪(三诺安准血糖仪)测定各组动物的随机血糖值RBG，并记录数据。数据以均数±标差
Figure PCTCN2016106011-appb-000016
形式表示，采用SPSS 18.0统计软件分析数据。正态分布，多组间均数差异采用单因素方差分析，方差齐性采用LSD检验，方差不齐采用Dunnet T3检验；非正态性分布采用非参数检验，P＜0.05表示具有显著性统计学差异。
如图5所示，STZ诱导的糖尿病小鼠单次注射FP-C1后0-240小时随机血糖值变化曲线图显示，FP-C1能有效地降低STZ诱导的糖尿病小鼠的随机血糖值。FP-C1在给药后第24小时血糖水平降至最低，其后缓慢回升，在第96小时其随机血糖值与模型组相比，仍具有显著性差异(P＜0.05)。
4.5、FP-C1对高脂饲料诱导的肥胖小鼠预防减肥作用的实验研究
一、模型建立与分组给药
7周龄C57BL/6J雄性小鼠24只(购买自上海斯莱克实验动物有限公司，动物生产许可证号SCXK(沪)：2012-0002)。饲养环境：温度22-25℃，相对湿度45-65％，照明时间12h/d。C57BL/6J小鼠适应性饲养1周后，按体重随机分为3组：正常组(NFD)、高脂组(HFD)、FP-C1组(HFD+FP-C1 0.3mg/kg)。高脂组、FP-C1组给予高脂饲料(D12492高脂饲料，美国Research Diets公司产品)饲喂，正常组小鼠给予标准饲料(normal fat diet，NFD)喂养。FP-C1组按0.3mg/kg剂量每6天皮下注射相应药物溶液一次，正常组和模型组皮下注射PBS缓冲液，给药体积10ml/kg。96天后，各组小鼠禁食16小时，称重并检测空腹血糖值，眼眶取血，400×g离心15min，分离得血清。取血后，脱颈椎处死小鼠，测定小鼠鼻尖到肛门长度(体长)，计算Lee’s指数。分离双侧附睾周围脂肪组织，称湿重。取同一部位附睾脂肪组织保存于10％福尔马林溶液中，用于病理形态学检测。
二、指标检测
2.1、体重及肥胖程度
每6天小鼠称重一次，绘制小鼠体重增长曲线，并计算小鼠体重增长量。小鼠体重增长量＝小鼠末次称量时体重-小鼠分组时体重。以Lee’s指数评价小鼠肥胖程度。
2.2、脂肪重量及指数
以分析天平(BSA223S电子天平，赛多利斯科学仪器(北京)有限公司产品)称取小鼠两侧附睾周围脂肪组织，称湿重。计算附睾脂肪质量分数(mg/g)：附睾脂肪质量分数＝附睾脂肪质量(mg)/空腹体重(g)。
2.3、口服糖耐量实验
试验开展84天后，各组小鼠禁食16h(17:00am-9:00pm)，用血糖仪(安准血糖仪，长沙三诺生物传感股份有限公司产品)检测各组小鼠空腹血糖值(FBG)，称重。口服(i.g)给予2g/kg葡萄糖溶液(分析纯无水葡萄糖，国药集团化学试剂有限公司产品)，检测灌胃后30min，60min，90min和120min各组小鼠血糖 值，绘制糖耐量曲线，梯形法计算修正血糖曲线下面积值(iAUC)。
2.4、血清生化检测
用全自动生化分析仪(欧霸XL-200全自动生化分析仪，德国欧霸公司产品)及配套试剂盒检测血清中TG(甘油三酯检测试剂盒，宁波美康生物科技有限公司产品)和TC(总胆固醇检测试剂盒，宁波美康生物科技有限公司产品)的含量，具体操作按照仪器说明书进行。
2.5、胰岛素及胰岛素耐受指数
用ELISA法(小鼠胰岛素ELISA检测试剂盒，美国ALPCO公司产品)检测小鼠血清胰岛素含量，并计算胰岛素耐受指数。
2.6、脂肪组织病理检测
取同一侧附睾脂肪组织，以苏木精-伊红染色法(简称HE染色)观察脂肪细胞组织形态学。
三、统计与分析
数据以均数±标准差(means±SD)形式表示，采用SPSS18.0统计软件分析数据。正态分布，多组间均数差异采用单因素方差分析，方差齐性采用LSD检验，方差不齐采用Dunnet T3检验；非正态性分布采用非参数检验，P＜0.05表示具有显著性统计学差异。
四、结果
4.1、FP-C1对高脂饲料喂养小鼠体重及肥胖程度的的影响
与正常组相比，高脂组小鼠体重、体重增长量及Lee’s指数均显著升高(P＜0.01)。FP-C1能显著降低高脂饲料饲喂小鼠的体重，体重增长量及Lee’s指数(P＜0.01)，结果见表5和图6。
表5.FP-C1对高脂饲料喂养小鼠体重和Lee’s指数的影响(means±SD，n＝8)
Figure PCTCN2016106011-appb-000017
注：与正常组相比，#P＜0.05，##P＜0.01；与高脂组相比，*P＜0.05，**P＜0.01.
4.2、FP-C1对附睾脂肪质量和质量分数的影响
如表6所示，与正常组相比，高脂组小鼠附睾脂肪质量及质量分数均显著升 高(P＜0.01)。与高脂组相比，FP-C1组小鼠附睾脂肪质量及质量分数均显著降低(P＜0.05)。
表6.FP-C1对附睾脂肪质量和质量分数的影响(means±SD，n＝8)
Figure PCTCN2016106011-appb-000018
注：与正常组相比，#P＜0.05，##P＜0.01；与高脂组相比，*P＜0.05，**P＜0.01.
4.3、FP-C1对小鼠血清TG和TC含量的影响
与正常组相比，高脂组小鼠血清TC和TG含量均显著升高(P＜0.01)。与高脂组相比，FP-C1组小鼠血清TC含量显著降低(P＜0.01)，TG含量也发生显著降低(P＜0.01)。结果见表7。
表7.FP-C1对小鼠血清TG和TC含量的影响(means±SD，n＝8)
Figure PCTCN2016106011-appb-000019
注：与正常组相比，#P＜0.05，##P＜0.01；与高脂组相比，*P＜0.05，**P＜0.01.
4.4、FP-C1对高脂饲料喂养小鼠糖耐量的影响
如图7所示，高脂组iAUC显著高于正常组(P＜0.05)。与高脂组相比，FP-C1组的iAUC显著降低(P＜0.05)。
4.5、FP-C1对高脂饲料喂养小鼠血清胰岛素和胰岛素耐受指数的影响
与正常组相比，高脂组小鼠的血清胰岛素浓度(P＜0.01)及胰岛素耐受指数(P＜0.05)均显著性升高，即小鼠已发生明显的胰岛素抵抗现象，因而会代偿性的分泌过多胰岛素产生高胰岛素血症。与高脂组相比，FP-C1能显著降低小鼠血清胰岛素(INS)含量(P＜0.05)，改善小鼠胰岛素耐受指数(HOMA-IR)(P＜0.05)。结果分别见图8和图9。
4.6、病理形态学检查
HE染色结果显示，与正常组相比，高脂组小鼠附睾脂肪细胞横截面积显著增加。与高脂组相比，FP-C1组小鼠附睾脂肪细胞横截面积显著缩小，结果见图10。
综合以上研究结果，证实FP-C1可以有效地控制高脂饲料诱导的肥胖小鼠的 体重，具有减肥作用。
实施例5、IL-7-Fc融合蛋白生产、生物学活性和体内半衰期测定
5.1体外生物学活性
实施例1中获得的FP-D1和FP-D2CHO稳定表达细胞株，经过12-14天的摇瓶补料培养，以Protein A亲和层析纯化，两种融合蛋白纯度都在95％以上，分子大小也符合预期，然后用于活性分析。IL-7及其融合蛋白的体外生物活性分析方法如下：小鼠脾脏来源的单个核细胞通过刀豆蛋白A(ConA)活化后，将100μL的细胞接种到96孔板中，之后加入一系列浓度梯度的FP-D1和FP-D2，37℃，5％CO2培养72小时后，加入20μL MTT试剂，继续培养4小时，吸出培养基后每孔加入100μL二甲基亚砜(DMSO)，检测在492nm的吸光度，以此测定细胞的增殖情况。每个处理设三个重复，每个重复测定两次，以重组IL-7(hIL-7，购自北京义翘神州生物技术有限公司)作为阳性对照，以培养基作为阴性对照。图11显示了hIL-7、FP-D1和FP-D2融合蛋白刺激小鼠单个核细胞增殖的能力。根据绘制的剂量反应曲线，可获得FP-D1和FP-D2融合蛋白的半数有效浓度值(EC50)分别为0.039和0.048nM。按摩尔浓度计，重组融合蛋白表现出的体外生物活性优于E.coli表达的hIL-7(EC50值为0.08nM)。
5.2IL-7-Fc融合蛋白的药代动力学测定
雄性SPF级SD大鼠(购自上海必凯实验动物有限公司)，预饲一周后分别单次静脉注射(iv)和皮下注射(sc)2mg/kg的FP-D1和FP-D2，每组3只，考察血药浓度随时间的变化规律。在注射后0、3、6、24、48、72、96、120、126、144以及172小时，通过眼眶采血，每次约0.3ml。取血后静置，之后5000转/分钟离心10分钟，取血清待测。采用对人IL-7特异的ELISA方法，测定每个时间点的融合蛋白浓度。通过软件PKSOLVER将得到的数据进行分析，计算T1/2，AUC(0-t)等药代动力学参数，结果见表8。
表8、融合蛋白的药代动力学参数
Figure PCTCN2016106011-appb-000020
Figure PCTCN2016106011-appb-000021
数据显示，FP-D2的消除相半衰期时间分别为12.86(iv)和13.81(sc)小时，二者接近，符合线性代谢动力学特征。FP-D1的消除相半衰期分别为18(iv)和19(sc)小时，稍长于FP-D2。静脉给药方式的总体清除率(C1)FP-D1比FP-D2要低，说明FP-D1在FP-D2的基础上经过改造后，体内清除速率变慢了。除此之外，我们还发现由于引入了CTP结构，FP-D1的表观分布容积也降低，使其在组织中分布更少，血液中药物含量更高，导致体内暴露量更高。FP-D1在FP-D2的基础上增加了CTP的刚性结构，降低清除率、降低表观分布容积、增加AUC(0-t)。所以FP-D1的药效上可能优于FP-D2，生物利用度更高，可以预期其临床给药剂量也将降低。由此可见，FP-D1在生物学活性和药代动力学方面表现出优异的性能。该试验结果表明，连接肽中含有带负电、高度唾液酸化的CTP能够抵抗肾脏对其清除作用，进一步延长融合蛋白的半衰期，且提高融合蛋白的生物利用度。
实施例6、hGH-Fc融合蛋白生产、生物学活性和体内半衰期测定
6.1MTT法测定融合蛋白的体外生物学活性
实施例1中获得的FP-E1和FP-E2的CHO稳定表达细胞株，经过12-14天的摇瓶补料培养，以ProteinA亲和层析纯化，三种融合蛋白纯度都在95％以上，分子大小也符合预期，然后用于活性分析。体外生物活性可以用转染子或纯化蛋白刺激大鼠淋巴瘤细胞Nb2细胞的增殖能力来测定。因Nb2细胞的增殖是受到hGH对细胞上的泌乳受体刺激而起反应，因而，Nb2细胞增殖能力测定可以用来评价生长激素的生物活性(参见，例如内田等人，J Mol Endocrinol.，1999，23：347-353)。
在含有10％胎牛血清的Fischer′s培养基中正常培养小鼠淋巴瘤细胞Nb2-11(美国ATCC细胞库)。之后，使用无血清培养基稀释融合蛋白，从1000ng/ml 3倍稀释至8个梯度，每孔100μl，添加到96孔板中，最后一列以培养基为阴性对 照。取对数期生长的细胞，用无血清培养基洗涤细胞，然后调整密度为每毫升3×106个细胞，每孔100μl加入到上述96孔板中。在37℃、5％CO2培养箱中继续培养48小时，使用CCK-8试剂盒(Cell Counting Kit，购自上海翊圣生物科技有限公司，货号：40203ES80)检测细胞增殖情况。用酶标仪测定在450nm处的吸光度，将OD读数相对于融合蛋白的浓度作图。由所得剂量反应曲线可测定融合蛋白的生物活性。
图12显示了hGH融合蛋白刺激Nb2细胞增殖的能力。表9为不同融合蛋白的EC50值。由于生长激素C端的氨基酸与其功能密切相关，Fc直接与hGH的C段连接会影响其生物活性。在hGH与Fc之间加入连接肽后，hGH融合蛋白的活性提高。由结果可以看出，FP-E1的活性与FP-E2相比，活性提高了近1倍。这可能由于CTP一方面发挥自身的功能，另一方面CTP刚性结构与柔性肽接头偶联作为连接Fc和目的蛋白的接头序列，这种新型肽接头有利于融合蛋白折叠成更良好的三维结构，因而提高了hGH的生物活性。
表9、hGH融合蛋白的EC50值
Figure PCTCN2016106011-appb-000022
6.2体内循环半衰期
雄性SPF级SD大鼠(购自上海必凯实验动物有限公司)，预饲一周后，体重约290g，按体重随机分组，每组3只，单次静脉注射给予0.176mg/kg的FP-E1和FP-E2，考察血药浓度和时间的变化规律。对照组和给药组分别在给药后0、0.5、1、2、3、4、5、8、10、24、48、72h经眼眶采血，血液在室温放置30min后5000r/min离心10min，分离出血清，-20℃保存。用针对hGH特异的ELISA方法测定各时间点血清中hGH含量。通过软件PKSOLVER，计算各组主要药代动力学参数。各组药代动力学参数结果如表10。
表10、hGH融合蛋白的药代动力学参数
Figure PCTCN2016106011-appb-000023
Figure PCTCN2016106011-appb-000024
由结果看出，FP-E1和FP-E2融合蛋白的体内半衰期分别为2.6和2.4小时，半衰期基本一致，这一点也可以从MRT参数中看出来，二者在体内停留的时间也接近。从图13的药时曲线上发现FP-E1的血药浓度一直比FP-E2高，推测可能是FP-E1结构的改变导致了其药代动力学的性质发生了变化。FP-E1的总体清除率只有FP-E2的一半，表明FP-E1的体内清除相对慢，而FP-E2的表观分布容积是FP-E1的两倍，说明FP-E2进入体内后迅速的分布到组织中，导致了其血药浓度低于FP-E1。FP-E1在FP-E2的基础上增加了CTP刚性结构，降低清除率并使AUC(0-t)增加，近一步降低了表观分布容积，使其在组织中分布更少，血液中药物含量更高，导致体内暴露量更高，所以FP-E1的结构优势不仅体现在优越的药代参数上，而且其药效可能也优于FP-E2。因FP-E1经过改造后血药浓度更高，意味着其生物利用度更高，所以FP-E1的药效好于FP-E2，可以预期其临床给药剂量也将降低。由此可见，FP-E1在生物学活性和药代动力学方面表现出更优异的性能。
6.3融合蛋白的体内生物学活性测定
选取4周龄SPF级雄性SD大鼠，体重60-80g，由中国食品药品检定研究院实验动物中心提供。
试验前2周清洁条件下手术摘除垂体，去垂体手术后2周为恢复期，给药前选择大鼠体重变化小于手术前体重±10％的合格健康动物，去垂体大鼠按体重均匀随机分为6组，5只/组。以短效rhGH(商品名norditropin，Novo Nordisk A/S生产)作为本实验的阳性参照药1(Y1)，比活3IU/mg；以PEG-rhGH(长春金赛药业有限责任公司)作为阳性参照药2(Y2)，比活6IU/mg；FP-E1低、中、高剂量设定分别为5mg/kg/14d、15mg/kg/14d和45mg/kg/14d。根据分子量大小和摩尔数，FP-E1的中剂量15mg/kg/14d与阳性参照药Y1和Y2相当。给药方式为颈部皮下注射，不同剂量FP-E1融合蛋白和Y2，每鼠于第1天和第8天各给药1次；Y1每天给药1次，连续14天。
给药后每只大鼠每天称重，第15天用二氧化碳窒息处死全部大鼠，称重。 每只动物第i天(di)体重(bwi)与给药前即第1天(d1)体重(bw1)之差即增加的体重(必要时，实验结束后可进行尸检，切开蝶鞍区，肉眼检查有无垂体残留，剔除有垂体残存的动物)。体重增加计算公式：Δbw＝bwi-bw1，其中：Δbw为体重增重；bw1为给药前d1体重；bwi为给药后第i天体重。计量资料以均数标准差
Figure PCTCN2016106011-appb-000025
表示，结果见表11。图14显示了给药后各组的生长曲线。
表11、hGH融合蛋白给药前后大鼠体重变化(
Figure PCTCN2016106011-appb-000026
n＝7)
Figure PCTCN2016106011-appb-000027
从结果可以看出，与模型组相比，在给药后第8天各给药组Δbw均显著增加，但各给药组间Δbw值差异不显著；而在给药后第15天，FP-E1对大鼠的体重增长具有非常显著的促进作用，高剂量FP-E1诱导垂体切除大鼠体重增加(Δbw值)是rhGH(Y1)和PEG-rhGH(Y2)组的约1.5倍；而中剂量FP-E1诱导的体重增加略优于Y1，与Y2基本一致。我们发现，FP-E1高剂量组在第2次给药后对大鼠体重增加，与PEG-rhGH组相比表现出更显著的促进作用，而在第1次给药后一周内，两组的Δbw值却不具有统计学差异。从这种差异性结果分析推测出FP-E1相比PEG-rhGH应具有更长的体内活性半衰期，因此重复给药后，这种体内蓄积效应使得FP-E1组在第2次给药后生长曲线的上升趋势更陡峭。
实施例7、Anti-CD3×CD20双特异性抗体的生产和生物学活性分析
7.1Anti-CD3×CD20双特异性抗体的制备和鉴定
实施例1中获得的FP-F1和FP-F2的CHO稳定表达细胞株，经过10-14天的摇瓶补料培养，以ProteinA亲和层析纯化。两种融合蛋白纯度都在95％以上，分子大小也符合预期，然后进行活性分析。
7.2Anti-CD3×CD20双特异性抗体的体外活性测定
采用密度梯度离心法从人新鲜血液制备PBMC，用含10％热灭活的FBS培养基重悬为5×106个/ml，备用。使用完全培养基将抗体(Anti-CD3单抗OKT3或双特异性抗体FP-F1)稀释到2μg/ml，再按1∶5倍率稀释至8个梯度，以每孔100μl加入到96孔板中，设三复孔。使用培养基作为阴性对照，并以ConA为阳性对照。上述制备的PBMC细胞悬液，以100μl/孔加入到96孔板中，37℃，5％CO2培养72h。孵育后，小心吸取96孔板培养上清，每孔100μl，并以IFN-γELISAkit(购自BD公司)检测上清中IFN-γ的含量(图15)。96孔板中各孔加入10μl CCK-8试剂，继续孵育4h。以抗体浓度作为X轴，492nm的吸光度值作为Y轴，拟合四参数S曲线，计算EC50值(图16)。结果显示，FP-F1活化T细胞产生IFN-γ的量与抗体浓度呈正相关，并且略低于对照抗体OKT3产生的量，表明组成双特异性抗体的Anti-CD3单链抗体功能良好；对照抗体OKT3活化人PBMC的EC50值为0.015nM，而FP-F1为0.233nM，活化能力下降约两个数量级，这有助于降低双特异性抗体的临床毒性。
本发明构建的另一个Anti-CD3×CD20双特异性抗体FP-F2在活化人PBMC细胞试验中，也同样呈浓度依赖性地活化T细胞，并产生IFN-γ，并且FP-F2活化人PBMC的EC50值为0.214nM，与FP-F1相当。
7.3Anti-CD3×CD20双特异性抗体FP-F1杀伤皮下移植瘤的药效试验
人Burkitt’s淋巴瘤细胞Raji(购自中国科学院典型培养物保藏委员会细胞库)与Matrigel(购自BD公司，货号354234)以1∶1的比例共同接种于雌性SCID Beige小鼠(购自上海灵畅生物科技有限公司)皮下，接种密度为8×106个细胞。生长6天后，根据体重和肿瘤体积进行分组，每组7只，将培养好的LAK细胞以1×106细胞/50μL的量注射到肿瘤组织中，同时在当天进行给药，剂量分别为10、1和0.1mg/kg的FP-F1，一周两次尾静脉注射。对照组为：(1)阴性对照组：FP-F1溶剂PBS；(2)阳性对照组：
Figure PCTCN2016106011-appb-000028
(Anti-CD20抗体，Genentech公司)，给药剂量为10mg/kg，一周两次尾静脉注射。每周测量小鼠体重和肿瘤体积两次，体积计算公式为1/2×长×宽×宽(mm3)。
在图17中，不同剂量的FP-F1显示出良好的抑制肿瘤生长的效果，其中 10mg/kg给药组有2只小鼠肿瘤完全消失，1mg/kg给药组有1只小鼠肿瘤完全消失，0.1mg/kg给药剂量治疗组也显示出一定的抑制肿瘤生长的作用。阴性对照组肿瘤生长情况正常，在14天时达到400mm3。结果显示，FP-F1 1mg/kg剂量组与Rituxan 10mg/kg剂量组疗效相当，表明本发明构建的双特异性抗体FP-F1的体内抑制肿瘤生长的起效剂量仅为Anti-CD20单抗
Figure PCTCN2016106011-appb-000029
的1/10，可以预期其临床给药剂量也将大大降低。
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 (30)

  1. 一种用于构建融合蛋白的连接肽，由柔性肽和刚性肽组成，所述柔性肽包含1个或多个柔性单元，和所述刚性肽包含1个或多个刚性单元；其中，所述柔性单元包含2个或更多个选自Gly、Ser、Ala和Thr的氨基酸残基；和所述刚性单元包含人绒毛膜促性腺激素β亚基的羧基末端肽。
  2. 如权利要求1所述的连接肽，其特征在于，刚性肽位于柔性肽的C末端。
  3. 如权利要求1所述的连接肽，其特征在于，所述柔性单元包含2个或更多个选自Gly和Ser的氨基酸残基。
  4. 如权利要求3所述的连接肽，其特征在于，所述柔性单元氨基酸组成的结构通式为(GS)a(GGS)b(GGGS)c(GGGGS)d，其中a，b，c和d是大于或等于0的整数，且a+b+c+d≥1。
  5. 如权利要求4所述的连接肽，其特征在于，所述柔性单元选自如下序列：
    (i)GSGGGSGGGGSGGGGS；
    (ii)GSGGGGSGGGGSGGGGSGGGGSGGGGS；
    (iii)GSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS；
    (iv)GSGGGGSGGGGSGGGGS；
    (v)GGGSGGGSGGGSGGGSGGGS；
    (vi)GGSGGSGGSGGS。
  6. 如权利要求1所述的连接肽，其特征在于，所述刚性单元包含如SEQ ID NO：1所示氨基酸序列或其片段，其中，所述片段包含至少2个糖基化位点。
  7. 如权利要求6所述的连接肽，其特征在于，所述刚性单元选自如下序列：
    (i)SSSSKAPPPSLPSPSRLPGPSDTPILPQ；
    (ii)PRFQDSSSSKAPPPSLPSPSRLPGPSDTPILPQ；
    (iii)SSSSKAPPPS；
    (iv)SRLPGPSDTPILPQ；
    (v)SSSSKAPPPSLPSPSR。
  8. 如权利要求1所述的连接肽，其特征在于，所述人绒毛膜促性腺激素β亚基的羧基末端肽与权利要求6或7所述连接肽中刚性单元的氨基酸序列至少具有70％，80％，90％或95％的同一性。
  9. 如权利要求1所述的连接肽，其特征在于，所述刚性肽包含1，2，3，4 或5个刚性单元。
  10. 如权利要求1所述的连接肽，其特征在于，所述连接肽具有如SEQ ID NO：2、3、4、5、6或7所示的氨基酸序列。
  11. 一种融合蛋白，其结构通式表示为K1-L-K2，其中K1是第一种生物活性分子；L是如权利要求1-10任一项所述连接肽；K2是第二种生物活性分子；且所述活性分子K1或K2选自蛋白或蛋白结构域、多肽、抗体或抗体片段。
  12. 如权利要求11所述的融合蛋白，其特征在于，所述活性分子K1包含可溶性或者膜信号分子、细胞因子、生长因子、激素、共刺激分子、酶、受体或对于受体具有配体功能的蛋白或者多肽；和所述活性分子K2为能够延长活性分子K1循环半衰期的蛋白或蛋白结构域。
  13. 如权利要求12所述的融合蛋白，其特征在于，所述活性分子K2选自人血白蛋白、转铁蛋白和免疫球蛋白Fc片段。
  14. 如权利要求11所述的融合蛋白，其特征在于，所述活性分子K1包含毒素、酶、细胞因子、膜蛋白或免疫调节细胞因子；和所述活性分子K2包含抗体或抗体片段，且K1和K2通过所述连接肽L连接形成抗体融合蛋白。
  15. 如权利要求11所述的融合蛋白，其特征在于，所述生物活性分子K1包含腺苷A1受体、血管紧张素转换酶ACE、Activin家族、ADAM家族、ALK家族、α-1-抗胰蛋白酶、细胞程序性死亡相关蛋白家族、神经生长因子及受体家族、骨形态发生蛋白BMP及受体家族、补体因子、降钙素、癌症相关抗原、组织蛋白酶家族、CCL趋化因子及受体家族、CD超家族、CFTR、CXCL趋化因子及受体家族、EGF、表皮生长因子EGF及受体家族、凝血因子IIa、因子VII、因子VIII、因子IX、铁蛋白、成纤维细胞生长因子FGF及受体家族、促卵泡激素、FZD家族、HGF、胰高血糖素、心肌肌球蛋白、生长激素、Ig、IgA受体、IgE、胰岛素样生长因子IGF及结合蛋白家族、白细胞介素IL超家族及其受体超家族、干扰素INF家族、iNOS、整联蛋白家族、激肽释放酶家族、层粘连蛋白、L-选择蛋白、黄体生成素、MMP家族、粘蛋白家族、钙粘蛋白超家族、血小板衍生生长因子PDGF及受体家族、甲状旁腺激素、血清白蛋白、T-细胞相关受体超家族、TGF-α、转化生长因子TGFβ超家族、促甲状腺激素、促甲状旁腺激素、肿瘤坏死因子TNF超家族及其受体TNFRSF超家族、尿激酶、WNT信号通路蛋 白家族、胸腺肽α1、胸腺肽β4、VEGF、血管内皮生长因子VEGF及其受体家族。
  16. 如权利要求15所述的融合蛋白，其特征在于，所述活性分子K1为人凝血因子VII、人凝血因子VIII或人生长激素；和所述融合蛋白的连接肽L如SEQ ID NO：2，3，4，5，6或7所示；和所述活性分子K2为人IgG Fc；其中，Fc可以是野生型或突变体，所述Fc突变体包含位于野生型人免疫球蛋白Fc中的至少一种氨基酸修饰，且变体具有降低的效应子功能和/或与新生儿受体FcRn的结合亲和力增强。
  17. 如权利要求15所述的融合蛋白，其特征在于，所述融合蛋白从N端至C端依次含有Exendin-4及其突变体、连接肽和人免疫球蛋白Fc。
  18. 如权利要求17所述的融合蛋白，其特征在于，所述连接肽氨基酸序列如SEQ ID NO：2，3，4，5，6或7所示；和所述Exendin-4具有如SEQ ID NO：15所示的氨基酸序列；和所述人免疫球蛋白Fc如SEQ ID NO：8，9，10，11或12所示。
  19. 如权利要求18所述的融合蛋白在制备用于治疗非胰岛素依赖性的II型糖尿病药物中的用途。
  20. 如权利要求18所述的融合蛋白在制备治疗或预防肥胖症的药物中的用途。
  21. 如权利要求15所述的融合蛋白，其特征在于，所述融合蛋白从N端至C端依次含有IL-7、连接肽和人免疫球蛋白Fc。
  22. 如权利要求21所述的融合蛋白，其特征在于，所述IL-7具有如SEQ ID NO：16所示的氨基酸序列；和所述连接肽具有如SEQ ID NO：2所示的氨基酸序列；和所述人免疫球蛋白Fc具有如SEQ ID NO：11所示的氨基酸序列。
  23. 如权利要求11所述的融合蛋白，其特征在于，所述活性分子K1包含第一种抗体、或者抗体片段；和所述活性分子K2包含第二种抗体、或者抗体片段，且K1和K2通过所述连接肽L连接形成双特异性抗体。
  24. 如权利要求23所述的融合蛋白，其特征在于，所述活性分子K1为Anti-CD20的双链抗体，K2为Anti-CD3的单链抗体，K1和K2通过所述连接肽连接形成双特异性抗体，
  25. 如权利要求24所述的融合蛋白，其特征在于，所述双特异性抗体包含的Anti-CD20双链抗体的重链具有如SEQ ID NO：18所示的氨基酸序列，且所述轻链具有如SEQ ID NO：19所示的氨基酸序列；和所述双特异性抗体包含的Anti-CD3单链抗体具有如SEQ ID NO：20所示的氨基酸序列；和所述连接肽具有如SEQ ID NO：4或6所示的氨基酸序列；且所述Anti-CD20双链抗体的重链通过所述连接肽与所述Anti-CD3单链抗体连接。
  26. 如权利要求11所述的融合蛋白，其特征在于，所述融合蛋白是糖基化的。
  27. 如权利要求26所述的融合蛋白，其特征在于，所述融合蛋白是通过在哺乳动物细胞中表达而糖基化的。
  28. 如权利要求27所述的融合蛋白，其特征在于，所述融合蛋白是通过在中国仓鼠卵巢细胞中表达而糖基化的。
  29. 制备或生产如权利要求11-28任一项所述的融合蛋白的方法，所述制备方法包括可以使K1和K2通过L相连接的步骤。
  30. 如权利要求29所述的方法，其特征在于，所述方法包括如下步骤：
    (a)将编码所述融合蛋白的第一种活性分子K1和第二种活性分子K2的DNA序列通过连接肽L的DNA序列连接组成融合基因；
    (b)将步骤(a)中获得的融合基因引入真核或原核表达宿主；
    (c)培养步骤(b)筛选到的高产量表达宿主，表达融合蛋白；
    (d)收获步骤(c)得到的发酵液，并分离纯化融合蛋白。
PCT/CN2016/106011 2016-08-19 2016-11-16 用于构建融合蛋白的连接肽 WO2018032638A1 (zh)

Priority Applications (4)

Application Number Priority Date Filing Date Title
EP16913393.1A EP3502143A4 (en) 2016-08-19 2016-11-16 BINDING PEPTIDE FOR THE CONSTRUCTION OF A FUSION PROTEIN
US16/326,412 US11123438B2 (en) 2016-08-19 2016-11-16 Linker peptide for constructing fusion protein
US17/391,535 US11833212B2 (en) 2016-08-19 2021-08-02 Linker peptide for constructing fusion protein
US18/492,958 US20240108743A1 (en) 2016-08-19 2023-10-24 Linker peptide for constructing fusion protein

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
CN201610692679.4 2016-08-19
CN201610694914.1 2016-08-19
CN201610694914.1A CN106317226B (zh) 2016-08-19 2016-08-19 用于构建融合蛋白的连接肽
CN201610692679.4A CN106117370B (zh) 2016-08-19 2016-08-19 高糖基化Exendin‑4及其类似物的融合蛋白、其制备方法和用途

Related Child Applications (2)

Application Number Title Priority Date Filing Date
US16/326,412 A-371-Of-International US11123438B2 (en) 2016-08-19 2016-11-16 Linker peptide for constructing fusion protein
US17/391,535 Continuation US11833212B2 (en) 2016-08-19 2021-08-02 Linker peptide for constructing fusion protein

Publications (1)

Publication Number Publication Date
WO2018032638A1 true WO2018032638A1 (zh) 2018-02-22

Family

ID=61196273

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2016/106011 WO2018032638A1 (zh) 2016-08-19 2016-11-16 用于构建融合蛋白的连接肽

Country Status (3)

Country Link
US (3) US11123438B2 (zh)
EP (1) EP3502143A4 (zh)
WO (1) WO2018032638A1 (zh)

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112661862A (zh) * 2020-12-25 2021-04-16 深圳大学 一种融合蛋白及其制备方法和应用
US11472863B2 (en) 2016-08-19 2022-10-18 Ampsource Biopharma Shanghai Inc. Human coagulation factor IX (FIX) fusion protein, preparation method therefor, and use thereof
US11471513B2 (en) 2016-08-19 2022-10-18 Ampsource Biopharma Shanghai Inc. Highly glycosylated human blood-clotting factor VIII fusion protein, and manufacturing method and application of same

Families Citing this family (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11123438B2 (en) 2016-08-19 2021-09-21 Ampsource Biopharma Shanghai Inc. Linker peptide for constructing fusion protein
JP7137696B2 (ja) 2019-05-14 2022-09-14 プロヴェンション・バイオ・インコーポレイテッド １型糖尿病を予防するための方法および組成物
US11601276B2 (en) 2021-04-30 2023-03-07 Mobeus Industries, Inc. Integrating and detecting visual data security token in displayed data via graphics processing circuitry using a frame buffer
US11682101B2 (en) 2021-04-30 2023-06-20 Mobeus Industries, Inc. Overlaying displayed digital content transmitted over a communication network via graphics processing circuitry using a frame buffer
US11586835B2 (en) 2021-04-30 2023-02-21 Mobeus Industries, Inc. Integrating overlaid textual digital content into displayed data via graphics processing circuitry using a frame buffer
US11483156B1 (en) 2021-04-30 2022-10-25 Mobeus Industries, Inc. Integrating digital content into displayed data on an application layer via processing circuitry of a server
US11562153B1 (en) 2021-07-16 2023-01-24 Mobeus Industries, Inc. Systems and methods for recognizability of objects in a multi-layer display
KR20230018189A (ko) * 2021-07-29 2023-02-07 주식회사 유비프로틴 이중 기능성 화장료 조성물 및 이의 제조방법
WO2023102156A1 (en) 2021-12-03 2023-06-08 Wisconsin Alumni Research Foundation Mutant ace2 proteins and methods of using same

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1290301A (zh) * 1997-11-17 2001-04-04 华盛顿大学 单链双功能的糖蛋白激素
CN104427994A (zh) * 2012-02-14 2015-03-18 奥普科生物制品有限公司 长效凝血因子和生产它们的方法
CN104519897A (zh) * 2012-06-08 2015-04-15 比奥根艾迪克Ma公司 促凝血化合物
CN104903352A (zh) * 2012-12-28 2015-09-09 艾伯维公司 多价结合蛋白组合物
CN105753945A (zh) * 2014-12-15 2016-07-13 清华大学无锡应用技术研究院 一种连接肽及其应用

Family Cites Families (74)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4399216A (en) 1980-02-25 1983-08-16 The Trustees Of Columbia University Processes for inserting DNA into eucaryotic cells and for producing proteinaceous materials
US4818679A (en) 1985-02-19 1989-04-04 The Trustees Of Columbia University In The City Of New York Method for recovering mutant cells
US6225449B1 (en) 1991-10-04 2001-05-01 Washington University Hormone analogs with multiple CTP extensions
US6660843B1 (en) 1998-10-23 2003-12-09 Amgen Inc. Modified peptides as therapeutic agents
WO2001032678A1 (en) 1999-11-05 2001-05-10 Smithkline Beecham Corporation sbgFGF-19a
AU2002322394A1 (en) 2001-07-30 2003-02-17 Eli Lilly And Company Method for treating diabetes and obesity
EP1329227A1 (en) 2002-01-22 2003-07-23 Deutsches Krebsforschungszentrum Stiftung des öffentlichen Rechts Diagnostic conjugate useful for intercellular imaging and for differentiating between tumor- and non-tumor cells
US7081446B2 (en) 2002-01-31 2006-07-25 The Trustees Of Columbia University In The City Of New York Long-acting follicle stimulating hormone analogues and uses thereof
US20050176108A1 (en) 2003-03-13 2005-08-11 Young-Min Kim Physiologically active polypeptide conjugate having prolonged in vivo half-life
DE602004031390D1 (de) 2003-05-06 2011-03-24 Syntonix Pharmaceuticals Inc Gerinnungsfaktor VII-Fc chimäre Proteine zur Behandlung von hämostatischen Krankheiten
KR100758755B1 (ko) 2003-06-12 2007-09-14 일라이 릴리 앤드 캄파니 Ｇｌｐ-1 유사체 융합 단백질
EP1641483B1 (en) 2003-06-12 2008-02-13 Eli Lilly And Company Fusion proteins
ZA200706129B (en) 2003-12-03 2009-07-29 Novo Nordisk As Glycopegylated Factor IX
US20050250185A1 (en) 2003-12-12 2005-11-10 Murphy Andrew J OGH fusion polypeptides and therapeutic uses thereof
WO2005058953A2 (en) 2003-12-12 2005-06-30 Regeneron Pharmaceuticals, Inc. Ogh fusion polypeptides and therapeutic uses thereof
CA2557782A1 (en) 2004-03-17 2005-10-06 Eli Lilly And Company Glycol linked fgf-21 compounds
DK1751184T3 (da) 2004-05-13 2009-11-23 Lilly Co Eli FGF-21 fusionsproteiner
BRPI0514790A (pt) 2004-09-02 2008-06-24 Lilly Co Eli muteìna de fgf-21 de humano, ou um seu peptìdeo biologicamente ativo, método para produzir a muteìna, composição farmacêutica, e, uso da muteìna de fgf-21 de humano
WO2006028714A1 (en) 2004-09-02 2006-03-16 Eli Lilly And Company Muteins of fibroblast growth factor 21
US20090043080A1 (en) 2004-09-29 2009-02-12 Novo Nordisk Healthcare A/G Purification of a Bulk of a Factor VII Polypeptide by Fractionated Elution from an Anion-Exchange Material
JP5948627B2 (ja) 2004-10-29 2016-07-20 レイショファーム ゲーエムベーハー 線維芽細胞成長因子（ｆｇｆ）のリモデリングと糖質ペグ化
BRPI0517837A (pt) 2004-11-12 2008-10-21 Xencor Inc variantes fc com ligação alterada a fcrn
JP2008522617A (ja) 2004-12-14 2008-07-03 イーライ リリー アンド カンパニー 線維芽細胞成長因子２１の突然変異タンパク質
JP5461008B2 (ja) 2005-04-13 2014-04-02 アストラゼネカ アクチボラグ ベクター
US20070129298A1 (en) 2005-07-22 2007-06-07 Maxygen Holdings, Ltd. In-solution activation of factor vii
US8048849B2 (en) 2006-02-03 2011-11-01 Modigene, Inc. Long-acting polypeptides and methods of producing same
US8304386B2 (en) 2006-02-03 2012-11-06 Prolor Biotech, Inc. Long-acting growth hormone and methods of producing same
EP1816201A1 (en) 2006-02-06 2007-08-08 CSL Behring GmbH Modified coagulation factor VIIa with extended half-life
AU2008232937B2 (en) 2007-03-30 2012-09-27 Ambrx, Inc. Modified FGF-21 polypeptides and their uses
EP4098661A1 (en) 2007-12-26 2022-12-07 Xencor, Inc. Fc variants with altered binding to fcrn
US8420088B2 (en) 2008-01-28 2013-04-16 Novartis Ag Methods and compositions using FGF23 fusion polypeptides
JOP20190083A1 (ar) 2008-06-04 2017-06-16 Amgen Inc بولي ببتيدات اندماجية طافرة لـfgf21 واستخداماتها
WO2010042747A2 (en) 2008-10-10 2010-04-15 Amgen Inc. Fgf21 mutants and uses thereof
KR20110117666A (ko) 2009-01-23 2011-10-27 노보 노르디스크 에이/에스 알부민 바인더 ａ-ｂ-ｃ-ｄ-ｅ-를 갖는 ｆｇｆ21 유도체 및 그것의 사용
WO2010129600A2 (en) 2009-05-05 2010-11-11 Amgen Inc. Fgf21 mutants and uses thereof
SG10201707763PA (en) 2009-05-05 2017-11-29 Amgen Inc FGF21 Mutants And Uses Thereof
JP2012529463A (ja) 2009-06-11 2012-11-22 ノヴォ ノルディスク アー／エス ２型糖尿病を治療するための、ｇｌｐ−１とｆｇｆ２１との組合せ
UA109888C2 (uk) 2009-12-07 2015-10-26 ІЗОЛЬОВАНЕ АНТИТІЛО АБО ЙОГО ФРАГМЕНТ, ЩО ЗВ'ЯЗУЄТЬСЯ З β-КЛОТО, РЕЦЕПТОРАМИ FGF І ЇХНІМИ КОМПЛЕКСАМИ
EP2977055A1 (en) 2010-02-16 2016-01-27 Novo Nordisk A/S Factor viii fusion protein
JP2013523184A (ja) 2010-04-15 2013-06-17 アムジエン・インコーポレーテツド ヒトＦＧＦ受容体およびβ−ＫＬＯＴＨＯ結合性タンパク質
SG10201505218YA (en) 2010-07-09 2015-08-28 Biogen Hemophilia Inc Factor ix polypeptides and methods of use thereof
CN103415300B (zh) 2010-07-20 2018-02-23 诺沃—诺迪斯克有限公司 N‑末端修饰的fgf21化合物
US9023791B2 (en) 2010-11-19 2015-05-05 Novartis Ag Fibroblast growth factor 21 mutations
PL2710035T3 (pl) 2011-05-16 2017-09-29 F.Hoffmann-La Roche Ag Agoniści fgfr1 i sposoby stosowania
US9574002B2 (en) 2011-06-06 2017-02-21 Amgen Inc. Human antigen binding proteins that bind to a complex comprising β-Klotho and an FGF receptor
EP2537864B1 (en) 2011-06-24 2019-08-07 Laboratoire Français du Fractionnement et des Biotechnologies Fc variants with reduced effector functions
SG10201806648TA (en) 2011-07-01 2018-09-27 Ngm Biopharmaceuticals Inc Compositions, uses and methods for treatment of metabolic disorders and diseases
EA029045B1 (ru) 2011-07-08 2018-02-28 Байоджен Хемофилия Инк. Химерные и гибридные полипептиды фактора viii и способы их применения
TW201315742A (zh) 2011-09-26 2013-04-16 Novartis Ag 治療代謝病症之雙功能蛋白質
UY34346A (es) 2011-09-26 2013-04-30 Novartis Ag Proteínas de fusión para tratar trastornos metabólicos
US9573987B2 (en) 2011-12-20 2017-02-21 Indiana University Research And Technology Corporation CTP-based insulin analogs for treatment of diabetes
KR102041412B1 (ko) 2011-12-30 2019-11-11 한미사이언스 주식회사 면역글로불린 Ｆｃ 단편 유도체
WO2013152351A2 (en) 2012-04-06 2013-10-10 The Trustees Of Columbia University In The City Of New York Fusion polypeptides and methods of use thereof
AU2013270683A1 (en) 2012-06-08 2014-12-11 Biogen Ma Inc. Chimeric clotting factors
TWI513705B (zh) 2012-06-11 2015-12-21 Lilly Co Eli 纖維母細胞生長因子２１蛋白質
WO2014026954A1 (en) 2012-08-13 2014-02-20 Novo Nordisk A/S Liquid factor viii formulations
KR20150043505A (ko) 2012-09-07 2015-04-22 사노피 대사 증후군의 치료용 융합 단백질
EP2900328A4 (en) 2012-09-25 2016-05-11 Biogen Ma Inc PROCESS FOR USE OF FIXED POLYPEPTIDES
TW201536811A (zh) 2013-05-31 2015-10-01 Biogen Idec Inc 嵌合ｆｖｉｉ－ｘｔｅｎ分子及其用途
CN104774269B (zh) 2013-08-16 2016-02-24 安源生物科技(上海)有限公司 改良型人凝血因子FVII-Fc融合蛋白及其制备方法与用途
CN103539869B (zh) 2013-10-11 2015-11-18 浙江凯胜畜产品加工有限公司 一种提高粗肝素钠提取工艺酶解效率的方法
CN103539860B (zh) 2013-11-01 2014-12-03 广州优联康医药科技有限公司 一种长效重组人促卵泡激素融合蛋白
CN103539868B (zh) 2013-11-01 2016-05-04 上海春露生物化学有限公司 一种制备羧甲基壳聚糖的方法
CN103539861B (zh) 2013-11-01 2015-02-18 广州联康生物科技有限公司 长效重组人促卵泡激素融合蛋白
CN103897064B (zh) 2013-11-29 2016-05-11 广州优联康医药科技有限公司 长效重组人绒促性素融合蛋白
CN104693270B (zh) 2013-12-10 2018-10-16 清华大学 一种用于融合蛋白的连接肽
KR20160088656A (ko) 2015-01-16 2016-07-26 주식회사유한양행 지속형 fgf21 융합 단백질 및 이를 포함하는 약학적 조성물
KR20170049320A (ko) 2015-10-28 2017-05-10 주식회사유한양행 이중 작용 단백질 및 이를 포함하는 약학적 조성물
CN106279437B (zh) 2016-08-19 2017-10-31 安源医药科技（上海）有限公司 高糖基化人凝血因子viii融合蛋白及其制备方法与用途
CN106256835A (zh) 2016-08-19 2016-12-28 安源医药科技（上海）有限公司 高糖基化人生长激素融合蛋白及其制备方法与用途
CN107759694B (zh) 2016-08-19 2023-01-13 安源医药科技（上海）有限公司 双特异性抗体及其制备方法与用途
US11123438B2 (en) 2016-08-19 2021-09-21 Ampsource Biopharma Shanghai Inc. Linker peptide for constructing fusion protein
CN106279436B (zh) 2016-08-19 2017-10-31 安源医药科技（上海）有限公司 活化的人凝血因子vii融合蛋白及其制备方法与用途
CN110028587B (zh) 2018-01-11 2021-10-08 安源医药科技（上海）有限公司 用于调节血糖和脂质的增效型双功能蛋白

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1290301A (zh) * 1997-11-17 2001-04-04 华盛顿大学 单链双功能的糖蛋白激素
CN104427994A (zh) * 2012-02-14 2015-03-18 奥普科生物制品有限公司 长效凝血因子和生产它们的方法
CN104519897A (zh) * 2012-06-08 2015-04-15 比奥根艾迪克Ma公司 促凝血化合物
CN104903352A (zh) * 2012-12-28 2015-09-09 艾伯维公司 多价结合蛋白组合物
CN105753945A (zh) * 2014-12-15 2016-07-13 清华大学无锡应用技术研究院 一种连接肽及其应用

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
LI JIANFANG ET AL: "Design of Linker Peptides and Its Application in Fusion Protein", JOURNAL OF WUXI UNIVERSITY OF LIGHT INDUSTRY, vol. 34, no. 11, 30 November 2015 (2015-11-30), pages 1121 - 1127, XP009512737 *
See also references of EP3502143A4 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11472863B2 (en) 2016-08-19 2022-10-18 Ampsource Biopharma Shanghai Inc. Human coagulation factor IX (FIX) fusion protein, preparation method therefor, and use thereof
US11471513B2 (en) 2016-08-19 2022-10-18 Ampsource Biopharma Shanghai Inc. Highly glycosylated human blood-clotting factor VIII fusion protein, and manufacturing method and application of same
CN112661862A (zh) * 2020-12-25 2021-04-16 深圳大学 一种融合蛋白及其制备方法和应用

Also Published As

Publication number Publication date
US20220105193A1 (en) 2022-04-07
US11833212B2 (en) 2023-12-05
US20190184026A1 (en) 2019-06-20
EP3502143A1 (en) 2019-06-26
US20240108743A1 (en) 2024-04-04
US11123438B2 (en) 2021-09-21
EP3502143A4 (en) 2020-07-15

Similar Documents

Publication Publication Date Title
US11833212B2 (en) Linker peptide for constructing fusion protein
CN107759694B (zh) 双特异性抗体及其制备方法与用途
JP6722175B2 (ja) 代謝障害を処置するための組成物及びその使用方法
CN103649127B (zh) 用于代谢病症和疾病治疗的组合物、应用和方法
CN104394882B (zh) 成纤维细胞生长因子21蛋白
RU2741345C2 (ru) Белки с двойной функцией и содержащая их фармацевтическая композиция
CN101321870B (zh) 在体内具有延长的半衰期和增强的红细胞生成活性的重组人EPO-Fc融合蛋白
KR102202255B1 (ko) 생식샘자극 호르몬 카복시 말단 펩타이드들과 부착하여 폴리펩타이드들의 유체역학적 부피를 증가시키는 방법
JP6356115B2 (ja) ラクトフェリン融合タンパク質及びその製造方法
US11858975B2 (en) Multi-domain active protein for treating metabolic diseases
US11911443B2 (en) Fusion proteins with extended serum half life
WO2018032787A1 (zh) 高糖基化人生长激素融合蛋白及其制备方法与用途
TW201427993A (zh) 用於治療肥胖之升糖素與ｇｌｐ－１共促效劑
KR20170049319A (ko) 지속형 fgf21 융합 단백질 및 이를 포함하는 약학적 조성물
US10023624B2 (en) Long-acting recombinant human follicle-stimulating hormone-Fc fusion protein
EP3738977A1 (en) Extracellular domain of alpha subunit of ige fc receptor, pharmaceutical composition comprising same and method for producing same
KR20180052545A (ko) 융합 단백질을 포함하는 간염증, 간섬유화 및 간경화 예방 또는 치료용 약학적 조성물
WO2017159540A1 (ja) 血清アルブミン－２０ｋ成長ホルモン融合タンパク質
EP4119571A1 (en) Novel bispecific protein and use thereof
KR20190076126A (ko) Glp-1 및 유전자 전달체를 포함하는 경구용 항당뇨 조성물
NZ624338A (en) Method for preparing physiologically active polypeptide complex
NZ624338B2 (en) Method for preparing physiologically active polypeptide complex

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: 16913393

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

ENP Entry into the national phase

Ref document number: 2016913393

Country of ref document: EP

Effective date: 20190319