WO2014101287A1 - Protéine hybride d'un polypeptide thérapeutique présentant un profil pharmacocinétique amélioré, et son utilisation - Google Patents

Protéine hybride d'un polypeptide thérapeutique présentant un profil pharmacocinétique amélioré, et son utilisation Download PDF

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WO2014101287A1
WO2014101287A1 PCT/CN2013/001602 CN2013001602W WO2014101287A1 WO 2014101287 A1 WO2014101287 A1 WO 2014101287A1 CN 2013001602 W CN2013001602 W CN 2013001602W WO 2014101287 A1 WO2014101287 A1 WO 2014101287A1
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pcloud
fusion protein
glp
protein
polypeptide
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PCT/CN2013/001602
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English (en)
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Xinguo QIAN
Wei Hong
Xiaoyu Ma
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Beijing Anxinhuaide Biotech. Co., Ltd
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Application filed by Beijing Anxinhuaide Biotech. Co., Ltd filed Critical Beijing Anxinhuaide Biotech. Co., Ltd
Priority to US14/655,282 priority Critical patent/US10246503B2/en
Priority to EP13869513.5A priority patent/EP2935338A4/fr
Priority to CN201380067873.8A priority patent/CN104870478B/zh
Publication of WO2014101287A1 publication Critical patent/WO2014101287A1/fr
Priority to PCT/CN2014/094429 priority patent/WO2015090234A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C07K14/605—Glucagons
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/22—Hormones
    • A61K38/26—Glucagons
    • 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
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
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    • 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
    • C07K14/575—Hormones
    • C07K14/635—Parathyroid hormone, i.e. parathormone; Parathyroid hormone-related peptides
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70578—NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95
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    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/71—Receptors; Cell surface antigens; Cell surface determinants for growth factors; for growth regulators
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    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/715—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons
    • C07K14/7151—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons for tumor necrosis factor [TNF], for lymphotoxin [LT]
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    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/715—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons
    • C07K14/7156—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons for interferons [IFN]
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    • 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
    • C07K14/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
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    • A61K38/177—Receptors; Cell surface antigens; Cell surface determinants
    • A61K38/179—Receptors; Cell surface antigens; Cell surface determinants for growth factors; for growth regulators
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/18—Growth factors; Growth regulators
    • A61K38/1816—Erythropoietin [EPO]
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/193—Colony stimulating factors [CSF]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/21—Interferons [IFN]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/22—Hormones
    • A61K38/27—Growth hormone [GH], i.e. somatotropin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/22—Hormones
    • A61K38/29—Parathyroid hormone, i.e. parathormone; Parathyroid hormone-related peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/31—Fusion polypeptide fusions, other than Fc, for prolonged plasma life, e.g. albumin

Definitions

  • the present invention relates generally to a fusion protein with therapeutic efficacy.
  • the present invention relates to a method of improving the half life of a therapeutic polypeptide by fusing with one or more flexible un-structured polypeptide sequences and a trimeric scaffold protein and the use thereof.
  • therapeutic polypeptides suffer from short terminal in vivo half life and poor thermal stability when injected into a subject. Short plasma half life is commonly due to fast renal clearance as well as to enzymatic degradation occurring during systemic circulation. The long half life time is usually required for a therapeutic polypeptide to achieve its optimal efficacy. Increasing the in vivo residence times of the therapeutic polypeptides could decrease their dosing frequencies and make them more convenient for the patients to use.
  • PEGylation has been widely utilized to extend the half life of a therapeutic polypeptide (see review paper [1 -4], patents 1-9).
  • PEGylation changes the physical and chemical properties of the biomedical molecule, such as its conformation, electrostatic binding, and hydrophobicity, and results in an improvement in the pharmacokinetic behavior of the drug.
  • PEGylation improves drug solubility and decreases immunogenicity.
  • PEGylation also increases drug stability and the retention time of the conjugates in blood.
  • PEGylation has severe consequences for the biological activities of the protein.
  • the activity of the PEGylated protein usually reduces by 20-50 fold [2, 5](patents 1 -9).
  • PEG protein styrene glycosyl
  • the site for PEGylation needs to be carefully decided to avoid interfering with the active site of the therapeutic polypeptide.
  • some short peptides such as GLP-1 , PTH and Calcitonin, it would be difficult to choose the proper site for PEGylation without disturbing the biological activity of the peptides.
  • PEG is a heterogeneous mixture of related polymers, its conjugation to a therapeutic polypeptide results in numerous distinct species with similar molecular sizes and chemical properties. This complicates the purification and increases the production costs of the PEGylated products.
  • fusion of a therapeutic polypeptide with human IgG Fc fragment or human serum albumin may significantly increase the half life of the therapeutic polypeptide [6-9] (patents 10, 1 1 , 12).
  • recombinant fusion protein with IgG Fc fragment or HSA needs to be produced from eukaryotic systems such as mammalian cell lines or yeast cells, which significantly increases the cost of the recombinant protein.
  • the present invention is directed to enhance the pharmaceutical properties, stability, solubility and safety of the therapeutic polypeptides.
  • the present invention is particularly useful for improving the pharmacokinetic properties, such as in vivo terminal half-life, of a therapeutic polypeptide.
  • the present invention provides a fusion protein comprising a therapeutic polypeptide fused to a scaffold protein which forms a homo-trimer in solution.
  • the fusion protein further comprises one or more flexible un-structured polypeptide sequences.
  • the fusion protein further comprises a proteinous connecting moiety (PCM) of human origin.
  • PCM proteinous connecting moiety
  • the proteinous connecting moiety is a proteinous sequence having an elongated shape, such as a human Fibronectin type III domain.
  • the flexible un-structured polypeptide sequence contains 1 to 3000 amino acid residues, wherein the sum of G, S, E, A, P and T constitutes more than 90% of the flexible un-structured polypeptide sequence; and the flexible un-structured polypeptide sequence has greater than 90% unstructured random coil formation as determined by GOR algorithm[10].
  • Fusing the therapeutic polypeptide with one or more flexible unstructured polypeptide sequences and the trimeric scaffold protein can significantly increase the apparent molecular weight of the fusion protein and improve the in vivo half life of the therapeutic polypeptide.
  • this method renders the therapeutic polypeptide with tri-valency, which may greatly enhance the potency and efficacy of the therapeutic polypeptide (reviewed in [1 1] ).
  • This novel method provided by the invention can efficiently increase the hydrodynamic radius and/or the radius of gyration (Rg) of the polypeptide molecule to extend its half life in vivo.
  • the flexible unstructured polypeptide sequences and the proteinous connecting moiety (PCM) within the fusion protein contribute significantly to increasing the apparent molecular weight of the fusion proteins.
  • the therapeutic polypeptide may be fused with one or more flexible unstructured polypeptides and the trimeric scaffold protein in a number of ways.
  • the fusion protein of the present invention may be configured, from N-terminus to C-terminus, using the following formula:
  • Linker is the flexible un-structured polypeptide linker above;
  • TP is a therapeutic polypeptide
  • the fusion protein may further contain a proteinous connecting moiety of human origin.
  • the therapeutic polypeptide is connected with the flexible un-structured polypeptide sequence via the proteinous connecting moiety of human origin.
  • the fusion protein exhibits an improved pharmacokinetic profile when administered to a subject compared with the therapeutic polypeptide by itself.
  • the fusion protein may be configured, from N-terminus to C-terminus, according to the following formula:
  • Linker is the flexible un-structured polypeptide linker characterized above;
  • TP is the therapeutic polypeptide
  • PCM is the proteinous connecting moiety of human origin
  • Loop is a flexible loop which refers to the protein sequence which has the variable lengths from 0 to 100 residues. These flexible loops are rich in glycine (G) and serine (S). These flexible loops may also contain glutamate(E), alanine (A), proline (P) and threonine(T). These flexible loops have greater than 95% unstructured random coil formation as determined by GOR algorithm.
  • the flexible un-structured linker is exhibited as one or more flexible un-structured pCloud polypeptides.
  • the pCloud sequence is characterized in: (a) the total pCloud amino acid residues is at least 100 to about 3000 amino acid residues; (b) the pCloud polypeptide is generated by use of some or all of the fragments derived from human fibrinogen alpha chain (table 1). In pCloud sequence, the fibrinogen fragments are flanked by flexible loops with various lengths. Therefore the pCloud polypeptide is primarily human originated and has low immunogenicity. (c) the pCloud polypeptide is rich in glycine (G), serine (S) and Glutamate (E). The pCloud polypeptide also contains alanine (A), proline (P), arginine (R) and threonine (T).
  • the sum of G, S, E, A, P and T constitutes more than 90% of the pCloud sequence, (d) The pCloud sequence has greater than 90% unstructured random coil formation as determined by GOR algorithm [10]; and (e) the pCloud sequence does not contain any T-cell epitopes as predicted by TEPITOPE algorithm [ 12].
  • the pCloud polypeptide represents a flexible unstructured polypeptide originated from human fibrinogen alpha chain.
  • the fragments derived from human fibrinogen alpha chain (listed in table 1) can be utilized as the building blocks to constitute the pCloud polypeptide.
  • the human fibrinogen alpha chain fragments are flanked by flexible loops with variable lengths from 0 to 100 residues. Attaching one or more pCloud polypeptide to a therapeutic polypeptide may significantly increase the apparent molecular weight of the therapeutic polypeptide and improve the in vivo half life of the therapeutic polypeptide.
  • the in vivo half life of the therapeutic polypeptide connected with the pCloud sequence can be adjusted by varying the length of the pCloud sequence. More importantly, the pCloud sequence is generated based on the human fibrinogen alpha chain, therefore, the pCloud polypeptide may not stimulate the immune responses from human patients when administrated.
  • the scaffold protein of the invention is selected from the group consisting of human collagen noncollagenous (NC) domains which form stable homo-trimers in solution, proteins which form homo-trimers in solution with Clq-like molecular structures, proteins which form homo-trimers in solution with TNF-like molecular structures, and proteins with C-type lectin-like domains (CTLD) which form homo-trimers in solution.
  • NC human collagen noncollagenous domains
  • CTL C-type lectin-like domains
  • the scaffold protein is selected from the group consisting of the NC I domain within Multiplexin type of human Collagen, NC2 domain within FACIT type of collagen, human Cl q A chain, Cl q B chain, C l q C chain, cbln family members, human EMILIN- 1 , multimerin, AC P30/adiponectin, adipolin, resistin, resistin-like molecule (RELM) hormone family members, human TNFalpha, TNFbeta, TRAIL, RANK ligand, Fas ligand, CD 30 ligand, CD40 ligand, CD27 ligand, OX40L, CD 137, mannan-binding lectin (MBL), surfactant protein A (SP-A), surfactant protein D (SP-D), collectin liver 1 (CL-L1), collectin placenta 1 (CL-P1), conglutinin, collectin of 43 kDa (CL-43) and collectin of 46 kDa (CL
  • the therapeutic polypeptide is selected from the group consisting of human glucagon-like peptide- 1 (GLP-1), Exenatide, GLP-2, C-peptide, Calcitonin, human Parathyroid hormone (PTH), glucagon, G-CSF, GM-CSF, Interferon, interleukin factors, VEGF receptors, TNF alpha receptors, RANK, Growth hormone, Erythropoietin, blood-coagulation factors, single-chain Fv, single domain antibodies and functional variants thereof.
  • GLP-1 human glucagon-like peptide- 1
  • Exenatide GLP-2
  • C-peptide Calcitonin
  • PTH human Parathyroid hormone
  • glucagon G-CSF
  • GM-CSF GM-CSF
  • Interferon Interleukin factors
  • VEGF receptors vascular endothelial growth factor
  • TNF alpha receptors fibroblast growth factor
  • RANK Growth hormone
  • the therapeutic polypeptide is connected with the pCloud sequence via a proteinous connecting moiety of human origin.
  • the proteinous connecting moiety is a proteinous sequence having an elongated shape, such as human Fibronectin type III domain.
  • the fusion protein of the invention comprises, from N-terminus to C-terminus, a therapeutic polypeptide selected from the group consisting of GLP- 1 , GLP- 1 (A8G/G22E), GLP- 1 (A8G/G22E/R36S) and GLP-1 (A8G/G22E/R36G); a flexible loop; a proteinous connecting moiety selected from the group consisting of Fn7, Fn8 and TNCfn3; a pCloud sequence; and a scaffold protein selected from the group consisting of COL18NC1 , COL15NC 1 , COL19NC2, and ACRP30 C l q-like domain.
  • a therapeutic polypeptide selected from the group consisting of GLP- 1 , GLP- 1 (A8G/G22E), GLP- 1 (A8G/G22E/R36S) and GLP-1 (A8G/G22E/R36G); a flexible loop; a proteinous connecting moiety selected from the group
  • the fusion protein of the invention comprises, from N-terminus to C-terminus, GLP- 1 (A8G/G22E/R36G), a flexible loop, Fn8, a pCloud sequence, and COL18NC1.
  • the fusion protein of the invention comprises, from N-terminus to C-terminus, a first pCloud sequence, human growth hormone, a second pCloud sequence and COL18NC 1.
  • the present invention also provides a polynucleotide sequence encoding the fusion protein, a pharmaceutical composition comprising the fusion protein and a pharmaceutically acceptable carrier, and an expression vector comprising the polynucleotide sequence and expression control elements.
  • the present invention provides a method of improving the pharmacokinetic property of a therapeutic polypeptide, comprising the steps of fusing the therapeutic polypeptide to one or more pCloud polypeptide and a trimeric scaffold protein.
  • the therapeutic polypeptide is connected with the pCloud sequence via a proteinous connecting moiety of human origin.
  • the fusion protein of the present invention achieves a property characterized in that (a) the terminal half-life of the therapeutic polypeptide linked to the scaffold protein and one or more pCloud sequence is significantly longer as compared to the terminal half- life of the therapeutic polypeptide by itself; (b) stability and solubility under physiologic conditions of the therapeutic polypeptide linked to the scaffold protein and one or more pCloud sequence are improved as compared to the stability and solubility of the therapeutic polypeptide by itself.
  • Fig. 1 shows the schematic drawings illustrating some mechanisms of the present invention.
  • the therapeutic polypeptide is shown as a red star in Fig. la and Fig. l c and as a red helix in Fig. lb.
  • a) The therapeutic polypeptide is connected directly to the pCloud polypeptide and the scaffold protein
  • b) The therapeutic polypeptide is fused with the pCloud polypeptide and the scaffold protein via a proteinous connecting moiety of human origin, preferably a proteinous sequence with an elongated shape
  • the therapeutic polypeptide and the pCloud polypeptide can be fused at the opposite terminus of the scaffold protein.
  • Fig. 2 The amino acid sequence of the mature human fibrinogen alpha chain.
  • the amino acid residue numbers of the fibrinogen alpha chain are listed.
  • the 12 unstructured fragments containing primarily the residues glycine (G), serine (S) and Glutamate (E), proline (P), arginine (R) and threonine (T) are underlined.
  • mutations Y277S, V379A and D396E were introduced.
  • the residues Y277, V379 and D396 are in bold.
  • Fig. 3 shows the gel filtration chromatography profiles for purified GLP- 1 (A8G/G22E/R36S)-Fn8-COL 18NC 1 ,
  • GLP- 1 (A8G/G22E/R36S)-Fn8-COL 18NC 1 -20
  • GLP-l (A8G/G22E/R36S)-Fn8-COL18NC l-30, and GLP- l (A8G/G22E/R36S)-Fn8-COL18NCl -54 using the analytical column Superdex200 (GE Healthcare).
  • the profiles of these proteins are labeled as NC I , NCI -20, NCI -30 and NCI -54.
  • the elution time for the molecular marker proteins (158Kd, 67Kd and 44Kd) are shown by arrows.
  • the X-axis refers to elution time and the Y-axis refers to UV280 absorbance intensity.
  • Fig. 4 shows the pharmacokinetics profiles of the GLP-1 containing proteins (GLP- 1 (A8G/G22E/R36S)-Fn8-COLl 8NC 1 ,
  • GLP- 1 (A8G/G22E/R36S)-Fn8-COL 18NC 1 -20
  • GLP- l (A8G/G22E/R36S)-Fn8-COL18NC l-30 and GLP- 1 (A8G/G22E/R36S)-Fn8-COL 18NC 1 -54) in Sprague Dawley rats measured by use of the sandwich ELISA method.
  • the profiles of these proteins are labeled as NCI , NC20, NC30 and NC54, respectively.
  • the vertical axis indicates the percentage of the measured protein concentration by use of sandwich ELISA method compared with C max .
  • Fig. 5 shows the gel filtration chromatography profiles for purified
  • GLP- 1 -Fn8 GLP- 1 -Fn8-COL 18NC 1 ,
  • GLP- l (A8G/G22E/R36G)-Fn8-p246-COL18NCl by use of the analytical column Superdex200 (GE Healthcare).
  • the elution time for the molecular marker proteins are shown by arrows.
  • the X-axis refers to elution time and the Y-axis refers to UV280 absorbance intensity, respectively.
  • Fig. 6 shows the results of cAMP assays for GLP-1 (7-37) peptide, GLP-1 -Fn8-C0L18NC1 and GLPl (A8G/G22E/R36G)-Fn8-p246-COL18NCl .
  • This assay is based on competitive binding technique.
  • a monoclonal antibody specific for cAMP becomes bound to the goat anti-mouse antibody coated onto the microplate.
  • cAMP present in a sample competes with a fixed amount of horseradish peroxidase (HRP)-labeled cAMP for sites on the monoclonal antibody. This is followed by another wash to remove excess conjugate and unbound sample.
  • HRP horseradish peroxidase
  • a substrate solution is added to the wells to determine the bound enzyme activity.
  • the color development is stopped and the absorbance is read at 450nm.
  • the intensity of the color is inversely proportional to the concentration of cAMP in the sample.
  • the Y-axis refers to the OD 450 obtained by the plate reader and the X-axis refers to the concentration of GLP-1 (7-37) peptide, GLP-1-Fn8-C0L18NC1 and GLP 1 (A8G/G22E/R36G)-Fn8-p246-COL 18NC 1.
  • Fig. 7 shows the pharmacokinetics profiles of the fusion proteins GLP- 1 -Fn8-C0L18NC 1 , GLP-l (A8G/G22E/R36G)-Fn8-p246-COL18NCl , and GLP-l (A8G/G22E/R36G)-Fn8-p285-COL18NCl in Sprague Dawley rats.
  • the protein concentration in the blood samples were measured by use of the sandwich ELISA method.
  • the profiles of these proteins are labeled as NC I , p246 and p285, respectively.
  • the errors bars calculated from a group of six rats are labeled.
  • Fig. 8 shows the pharmacokinetics profiles of the fusion proteins GLP-l(A8G/G22E/R36G)-Fn8-p246-COL18NCl in three cynomolgus monkeys.
  • the protein concentration in the serum samples were measured by use of the sandwich ELISA method.
  • the three cynomolgus monekys were labeled as #1 , 2 and 3, respectively.
  • Fig. 9 shows the Intraperitoneal glucose tolerance test (IPGTT) results of GLPl (A8G/G22E/R36G)-Fn8-p246-COL18NC l in SD rats.
  • GLPl (A8G/G22E/R36G)-Fn8-p246-COL18NC l is labeled as p246.
  • the rats were injected with saline, GLPl (A8G/G22E/R36G)-Fn8-p246-COL18NC l at the dose of l Onmol/kg and 20nmol/kg, respectively. 8 hours after the administrations of the fusion protein (and control), the IPGTT experiments were conducted.
  • the three curves indicated the glucose levels in the IPGTT for rats that received negative control (saline), GLPl (A8G/G22E/R36G)-Fn8-p246-COL18NCl at the doses of lOnmol/kg and 20nmol/kg, respectively.
  • the rats were injected with saline, GLPl (A8G/G22E/R36G)-Fn8-p246-COL18NCl at the dose of 20nmol/kg. 32 hours and 102 hours after the administrations of the fusion protein (and the control), the IPGTT experiments were conducted.
  • the three curves indicated the glucose levels in the IPGTT for rats that 32 hours and 102 hours after administration of negative control (saline),
  • GLP l (A8G/G22E/R36G)-Fn8-p246-COL18NCl respectively.
  • polypeptide polypeptide
  • peptide protein
  • polymers of amino acids of any length may be linear or branched, it may comprise modified amino acids, and it may be interrupted by non-amino acids.
  • the terms also encompass an amino acid polymer that has been modified, for example, by disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation, such as conjugation with a labeling component.
  • flexible unstructured polypeptide flexible unstructured polypeptide sequence
  • flexible unstructured linker flexible unstructured polypeptide linker
  • the flexible un-structured polypeptide sequence contains 1 to 3000 amino acid residues, wherein the sum of G, S, E, A, P and T constitutes more than 90% of the flexible un-structured polypeptide sequence; and the flexible un-structured polypeptide sequence has greater than 90% unstructured random coil formation as determined by GOR algorithm[10].
  • pCloud polypeptide is characterized in: (a) the total pCloud amino acid residues is at least 100 to about 3000 amino acid residues; (b) the pCloud polypeptide sequence is generated by use of some or all of the fragments derived from human fibrinogen alpha chain. In pCloud sequence, the fibrinogen fragments are flanked by flexible loops with various lengths. Therefore pCloud is primarily human originated and has low immunogenicity when administered to human, (c) the pCloud sequence is rich in glycine (G), serine (S) and Glutamate (E). The pCloud also contains alanine (A), proline (P), arginine (R) and threonine (T).
  • the sum of G, S, E, A, P and T constitutes more than 90% of the pCloud sequence, (d)
  • the pCloud sequence has greater than 90% unstructured random coil formation as determined by GOR algorithm; and (e) the pCloud sequence does not contain any T-cell epitopes as predicted by TEPITOPE algorithm.
  • the human fibrinogen fragments are flanked by flexible loops with variable lengths from 0 to 100 residues.
  • flexible loop in this invention refers to the protein sequence which has the variable lengths from 0 to 100 residues. These flexible loops are rich in glycine (G) and serine (S). These flexible loops may also contain glutamate(E), alanine (A), proline (P) and threonine(T). These flexible loops have greater than 95% unstructured random coil formation as determined by GOR algorithm.
  • the flexible loops are generally the flexible unstructured polypeptide linkers with shorter lengths and more flexibility. A skilled artisan will appreciate that the flexible loop may be utilized in the fusion protein as a spacer to provide flexibility.
  • a “fragment” is a truncated form of a native protein.
  • variant or “functional variant” of a protein refers to a modified version of the native protein which comprises substitutions, deletions and/or additions of one or several amino acids, and which substantially retains the biological activity of the native protein.
  • a variant protein may share at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity with the reference protein.
  • conservative substitutions of amino acids are preferred which are well known to a skilled artisan.
  • Deletions are preferably deletions of amino acids from regions not involved in the biological function of the protein.
  • GLP- 1 (A8G/G22E/R36G) is a functional variant of wild type GLP- 1 , which contains three substitutions of amino acids and which substantially retains or increases its biological activity as shown by the cAMP assay.
  • Conjugated "linked,” “connected”, “fused,” and “fusion” are used interchangeably herein. These terms refer to the joining together of two more chemical elements or components, by whatever means including chemical conjugation or recombinant means.
  • two distinct proteins can be connected together by "in- frame fusion", which refers to the joining of two or more open reading frames (ORFs) to form a continuous longer ORF, in a manner that maintains the correct reading frame of the original ORFs.
  • ORFs open reading frames
  • the resulting recombinant fusion protein is a single protein containing two or more segments that correspond to polypeptides encoded by the original ORFs (which segments are not normally so joined in nature).
  • the two proteins can also be linked together by use of a chemical crosslinker, which results in a protein conjugate that contains two individual polypeptides connected by a crosslinker.
  • sequence is an order of amino acids in a polypeptide in an amino to carboxyl terminus direction in which residues that neighbor each other in the sequence are contiguous in the primary structure of the polypeptide.
  • DNA refers to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof.
  • Polynucleotides may have any three-dimensional structure, and may perform any function, known or unknown.
  • polynucleotides coding or non-coding regions of a gene or gene fragment, loci (locus) defined from linkage analysis, exons, introns, messenger RNA (mPvNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers.
  • a polynucleotide may comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs.
  • modifications to the nucleotide structure may be imparted before or after assembly of the polymer.
  • the sequence of nucleotides may be interrupted by non-nucleotide components.
  • a polynucleotide may be further modified after polymerization, such as by conjugation with a labeling component.
  • the term "functional variant" of a protein refers to a modified version of the native protein which comprises substitutions, deletions and/or additions of one or several amino acids, e.g., less than 15 amino acids, or preferably less than 10 or 5 amino acids, and which substantially retains the biological activity of the native protein. Typically, conservative substitutions of amino acids are preferred which are well known to a skilled artisan. Deletions are preferably deletions of amino acids from regions not involved in the biological function of the protein.
  • GLP- 1 (A8G/G22E) and GLP- 1 (A8G/G22E/R36G) are the functional variants of wild type GLP- 1 , which contains two or three substitutions of amino acids and which substantially retains its biological activity such as increasing cAMP level.
  • GLP- 1 A8G/G22E and GLP- 1 (A8G/G22E/R36G) are the functional variants of wild type GLP- 1 , which contains two or three substitutions of amino acids and which substantially retains its biological activity such as increasing cAMP level.
  • a method to increase the half life of a therapeutic polypeptide by fusing the therapeutic polypeptide to one or more flexible un-structured polypeptide sequences and a scaffold protein.
  • the scaffold protein can form a stable homo-trimer in solution.
  • This method can efficiently increase the hydrodynamic radius and/or the radius of gyration (Rg) of the polypeptide molecule to extend its half life in vivo.
  • Rg radius of gyration
  • changing the length of the flexible unstructured polypeptide linker within the fusion protein can adjust the in vivo half life of the fusion protein in a tunable manner.
  • the fusion protein of the invention may further comprise a proteinous connecting moiety of human origin, preferably a proteinous sequence with an elongated shape.
  • the proteinous connecting moiety can be connected to the therapeutic polypeptide via a flexible loop.
  • the proteinous connecting moiety can be linked to the scaffold protein via a flexible, un-structured linker whose length is adjustable.
  • the flexible un-structured linker is exhibited as one or more un-structured pCloud polypeptides.
  • the therapeutic polypeptide, the pCloud polypeptides and the scaffold protein can be arranged in a number of manners.
  • the therapeutic polypeptide is connected directly to the pCloud polypeptide and the scaffold protein (Fig. la).
  • the therapeutic polypeptide is fused with the pCloud polypeptide and the scaffold protein via a proteinous connecting moiety of human origin, preferably a proteinous sequence with an elongated shape (Fig. lb).
  • the therapeutic polypeptide and the pCloud polypeptide can be fused at the opposite terminus of the scaffold protein (Fig. lc).
  • the method of the invention may have several major advantages over the traditional PEGylation method or Fc/HSA fusion method.
  • PEGylation on the polypeptide molecule is not essential, therefore the biological activity of the therapeutic polypeptide is fully retained.
  • the scaffold protein forms a homo-trimer, the fusion protein of the therapeutic polypeptide with the scaffold protein may greatly increase the apparent size of the fusion protein to slow down renal filtration. Moreover, the trimer formation also renders the fusion protein tri-valency. This may greatly increase the activity of the therapeutic protein. 3.
  • the length of the flexible unstructured polypeptide linker within the fusion protein plays an important role in determining the in vivo half life of the fusion protein.
  • the method of the invention provides a platform to fine tune the in vivo half life of a therapeutic polypeptide by varying the length of the flexible unstructured polypeptide linker within the fusion protein.
  • the scaffold protein and pCloud polypeptide is preferably generated from human proteins, usually from human extracellular proteins, therefore, no foreign protein sequences are introduced into the fusion protein.
  • the immunogenicity of the fusion proteins generated using the method is low.
  • the recombinant fusion protein of the present invention can be generated using E.coli expression system, which eliminates the need of the expensive chemical synthesis process for some therapeutic polypeptides or the need of using the eukaryotic expression systems.
  • the flexible unstructured polypeptide sequences within the fusion protein play critical roles in extending the half-life of the therapeutic polypeptide.
  • the lengths of the flexible unstructured polypeptide sequences play important role in determining the hydrodynamic radius and/or the radius of gyration of the fusion protein.
  • the primary sequences of the flexible unstructured polypeptide heavily affect the stability and solubility of the fusion protein.
  • flexible un-structured polypeptide refers to an amino acid sequence which is flexible in movement and which does not form any regular stable secondary and tertiary protein structures.
  • the flexible un-structured polypeptide sequence contains 1 to 3000 amino acid residues, wherein the sum of G, S, E, A, P and T constitutes more than 90% of the flexible un-structured polypeptide sequence; and the flexible un-structured polypeptide sequence has greater than 90% unstructured random coil formation as determined by GOR algorithm[10].
  • the therapeutic polypeptide is a relatively large protein (such as Interferon, Growth hormone, Erythropoietin, G-CSF, or TNFR2, usually a protein with more than 100 amino acid residues), it may be directly fused to the scaffold protein through a flexible, un-structured polypeptide linker.
  • the therapeutic polypeptide might be a short peptide (such as GLP-1 , Exenatide, GLP-2, C-peptide, Calcitonin or PTH, usually a peptide not more than 100 residues).
  • the fusion protein may further contain a proteinous connecting moiety of human origin, preferably a proteinous sequence with an elongated shape, such as the human Fibronectin type III domain.
  • the therapeutic polypeptide and flexible unstructured polypeptide linker is connected by use of the proteinous connecting moiety.
  • the proteinous connecting moiety can further increase the hydrodynamic radius and/or the radius of gyration (Rg) of the fusion protein.
  • the proteinous connecting moiety can stabilize the therapeutic polypeptide.
  • the proteinous connecting moiety may comprise a whole protein, a truncated version of a protein, a protein domian or domains in tandem, or protein fragments.
  • the proteinous connecting moiety may comprise some non-proteinous modifications which are not formed by amino acids, such as PEG.
  • the length of the flexible, unstructured linker may play an important role in determining the hydrodynamic radius and/or the radius of gyration (Rg) and the in vivo half life of the fusion protein.
  • the flexible, unstructured polypeptide linker may contain sequences such as (G5S)n, (G4S)n, (G3S)n, (GS)n, (G2S2)n, (G3S3)n, (GS3)n where n is an integer, or other sequences that are rich in G, S, A, T or P.
  • the length of the flexible linker may vary from 1 to 3000 amino acid residues, and particularly within the range of 5 to 500 amino acid residues.
  • the fusion protein may consist of one or more flexible, un-structured polypeptide linkers. It will be appreciated by a skilled artisan that these flexible, un-structured linkers within the fusion protein may be the same or different.
  • compositions comprising the "pCloud" polypeptide.
  • the flexible un-structured linker is exhibited as one or more un-structured pCloud polypeptides.
  • pCloud polypeptides are generally extended polypeptides that have low degree or no secondary or tertiary structures under physiologic conditions.
  • the pCloud polypeptide is characterized in: (a) the total pCloud amino acid residues is at least 100 to about 3000 amino acid residues; (b) the pCloud polypeptide sequence is generated by use of some or all of the fragments derived from human fibrinogen alpha chain. In pCloud sequence, the fibrinogen fragments are flanked by flexible loops with various lengths. Therefore pCloud is primarily human originated and has low immunogenicity when administered to human, (c) the pCloud sequence is rich in glycine (G), serine (S) and Glutamate (E). The pCloud also contains alanine (A), proline (P), arginine (R) and threonine (T).
  • A alanine
  • P proline
  • R arginine
  • T threonine
  • the sum of G, S, E, A, P and T constitutes more than 90% of the pCloud sequence, (d) The pCloud sequence has greater than 90% unstructured random coil formation as determined by GOR algorithm; and (e) the pCloud sequence does not contain any T-cell epitopes as predicted by TEPITOPE algorithm.
  • XTEN technology it has been reported that fusing an unstructured polypeptide to the therapeutic polypeptide can significantly extend the in vivo half life of the therapeutic polypeptide (XTEN technology)[13].
  • the unstructured polypeptide is generated using artificial peptide fragments and it is not fused with a trimeric scaffold protein.
  • These artificial peptides in XTEN technology represent foreign peptides to human body and it is likely that these foreign peptides may elicit immune responses within the patients when administrated. Because many therapeutic polypeptide, such as human Growth hormone and GLP-1 analogues, needs to be applied to the patients for an extended period, the foreign peptides introduced by the XTEN technology may represent a potential threat for the patients.
  • pCloud polypeptide generated by use of human fibrinogen alpha chain sequences was demonstrated to efficiently extend the in vivo half life of therapeutic polypeptides.
  • the pCloud polypeptide is further fused with a trimeric scaffold protein, which further enhances the pharmacokinetic profile of the therapeutic polypeptide.
  • Human Fibrinogen (factor ⁇ ) is a soluble, 340 kDa plasma glycoprotein, that is converted by thrombin into fibrin during blood clot formation [14-16]. Fibrinogen is synthesized in the liver by the hepatocytes. The normal concentration of fibrinogen in the human blood plasma is quite high (1.5-3 mg/ml), which strongly suggests that the human fibrinogen sequence may exhibit very low immunogenicity. Human fibrinogen is a hetero-hexamer that contains two sets of three different chains ( ⁇ , ⁇ , and ⁇ ), linked to each other by disulfide bonds.
  • an intrinsic unstructured region (residues 262-455) is present (Fig. 2).
  • This unstructured region of fibrinogen contains minimum secondary structures as determined by GOR algorithm [10, 17].
  • 12 fragments within this fibrinogen unstructured region (residues 262-455) have been identified to contain primarily the residues glycine (G), serine (S) and Glutamate (E), proline (P), arginine (R) and threonine (T).
  • mutations Y277S, V379A and D396E were introduced in fragment 1 , 9 and 1 1 , respectively (Fig. 2).
  • the resultant 12 fragments derived from human fibrinogen alpha chain sequence are utilized as the building blocks to generate the pCloud polypeptides (Table. 1).
  • the variants of these fragments that share at least 70%, 75%, 80%, 85%o or 90% amino acid sequence identity with the fragments listed in table 1 may be utilized as the building blocks for pCloud polypeptides.
  • Table 1. the protein sequences of the 12 fragments derived from human fibrinogen alpha chain
  • the fragments listed in table 1 derived from human fibrinogen alpha chain are flanked by flexible loops with variable lengths from 0 to 100 residues.
  • These flexible loops are rich in glycine (G) and serine (S). These loops also contain glutamate(E), alanine (A), proline (P) and threonine(T).
  • the flexible loops have greater than 95% unstructured random coil formation as determined by GOR algorithm.
  • the flexible loop sequences can be selected, but not limited, from the table 2.
  • the flexible loops are generally the flexible unstructured polypeptide linkers with shorter lengths and more flexibility.
  • the flexible loops are utilized to connect the human fibrinogen alpha chain fragments to constitute the pCloud polypeptide.
  • the flexible loops are also utilized to connect therapeutic polypeptide and the proteinous connecting moiety of human origin in the fusion protein.
  • the flexible loops may also be utilized to link the therapeutic polypeptide with the scaffold protein, the scaffold protein with the flexible unstructured polypeptide (or the pCloud polypeptide), and the proteinous connecting moiety with the flexible unstructured polypeptide (or the pCloud polypeptide).
  • the flexible loop may be utilized in the fusion protein as a spacer to provide flexibility.
  • Table 2 the protein sequences of the flexible loops utilized in the pCloud sequence to connect the fibrinogen alpha chain fragments.
  • n is an integer that can be adjusted based on needs.
  • the fragments listed in table 1 are utilized to generate the pCloud sequence, or alternatively at least 10 fragments listed in table 1 , or alternatively at least 8 fragments in table 1 , or alternatively at least 6 fragments in table 1 , or alternatively at least 5 fragments, or alternatively at least 3 fragments in table 1 , or at least one fragment in table 1 are utilized to generate the pCloud sequence.
  • the fragments listed in table 1 can be connected in the order as they appear in the human fibrinogen alpha chain sequence to constitute the pCloud sequence.
  • the fragments listed in table 1 can be connected in the order that is distinct from they appear in the human fibrinogen alpha chain sequence to generate the pCloud sequence.
  • the pCloud polypeptide contains 100 to 3000 amino acid residues generated by use of the human fibrinogen derived fragments listed in table 1.
  • the pCloud sequence has greater than 90% unstructured random coil formation as determined by GOR algorithm.
  • a pCloud sequence may comprise charged residues separated by other residues such as serine or glycine, which may lead to better expression or purification behavior.
  • the charged residues such as D, E, K and R, may also prevent the aggregations of the pCloud polypeptide.
  • the pCloud polypeptide may carry a net negative charge under physiologic conditions that may contribute to the unstructured conformation and reduced binding of the pCloud polypeptide component to the mammalian proteins and tissues. Based on the net charge, pCloud polypeptide may have an isoelectric point (pi) of 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, or even 6.5. In preferred embodiments, the pCloud polypeptide will have an isoelectric point between 2.0 and 5.0.
  • the content of the hydrophobic amino acids (I, L, V, M, F, W, Y) in the pCloud polypeptide will typically be less than 5%, or less than 2%, or less than 1 % of the total amino acid residues.
  • the invention provides compositions in which the pCloud sequences have a low degree of immunogenicity or are essentially non- immunogenic.
  • Several facts can contribute to the low immunogenicity of pCloud, such as the unstructured conformation, the high degree of solubility, the low degree or lack of residues with large side chains, the low degree or lack of self-aggregation, the low degree or lack of proteolytic sites within the sequence, the low degree or lack of hydrophobic residues, and the lack of epitopes in the pCloud sequence.
  • the pCloud polypeptides are generated by use of human fibrinogen alpha chain fragment sequences, therefore, the pCloud polypeptides may not stimulate any immune responses from human body. Moreover, the pCloud polypeptide primarily consists of unstructured sequence and contains low degree of secondary structures, which will prevent the pCloud polypeptide to activate the B-cells. To be efficiently recognized by the host humoral immune system, the foreign polypeptide needs to form a stable conformation. The precise folding of the polypeptide may allow it to form the epitope that can be recognized as "foreign" by the host humoral immune system, resulting in the production of antibodies to the polypeptide or triggering a cell-mediated immune response.
  • the pCloud polypeptides lack a predicted T-cell epitope when analyzed by TEPITOPE algorithm [12], wherein the TEPITOPE algorithm prediction for epitopes within the pCloud sequence is based on a score of -7 or greater, or -8 or greater, or -9 or greater.
  • the data strongly suggest that the pCloud polypeptide may not be recognized by MHC molecules and T-cell receptors to trigger the T-cell activations which may lead to the release of cytokines further activating other lymphocytes such as B cells to produce antibodies or activating T killer cells as a full cellular immune response.
  • the therapeutic polypeptide is connected to one or more pCloud sequences and the scaffold protein to extend the in vivo half life of the therapeutic polypeptide.
  • the pCloud sequence can be placed at either or both of the N-terminal and the C-terminal end of the therapeutic polypeptide.
  • the pCloud sequences can also be placed at either or both of the N-terminal and the C-terminal end of the scaffold protein.
  • the pCloud sequences within the fusion protein could be the same or be different from each other.
  • the fusion protein of the present invention is configured using the following formula:
  • pCloud is the pCloud polypeptide characterized above, they could be different from each other.
  • TP is a therapeutic polypeptide selected, but not limited from the group consisting of human glucagon-like peptide- 1 (GLP-1), Exenatide, GLP-2, C-peptide, Calcitonin, human Parathyroid hormone (PTH), glucagon, G-CSF, GM-CSF, Interferon, interleukin factors, VEGF receptors, TNF alpha receptors, RANK, Growth hormone, Erythropoietin, blood-coagulation factors, single-chain Fv, single domain antibodies and functional variants thereof.
  • GLP-1 human glucagon-like peptide- 1
  • Exenatide GLP-2
  • C-peptide Calcitonin
  • PTH human Parathyroid hormone
  • glucagon G-CSF
  • GM-CSF GM-CSF
  • Interferon Interleukin factors
  • VEGF receptors vascular endothelial growth factor
  • TNF alpha receptors RANK
  • Growth hormone Ery
  • the therapeutic polypeptide might be a short peptide (such as
  • the therapeutic polypeptide is connected with the pCloud sequence via a proteinous connecting moiety of human origin.
  • a flexible loop may be utilized to fuse the therapeutic polypeptide and the proteinous connecting moiety.
  • the flexible loop has been characterized above.
  • the proteinous connecting moiety can stabilize the therapeutic polypeptide and further increase the hydrodynamic radius and/or the Rg of the fusion protein.
  • the proteinous connecting moiety may comprise a whole protein, a truncated version of a protein, a protein domain or domains in tandem, or protein fragments.
  • the proteinous connecting moiety may comprise some non-proteinous modifications which are not formed by amino acids, such as PEG.
  • the fusion protein containing the therapeutic polypeptide, the proteinous connecting moiety, the pCloud polypeptides, and the scaffold protein of the present invention is configured according to the following formula: (pCloud) m -TP-Loop-PCM-(pCloud) n -Scaffold- (pCloud) k or
  • pCloud is the pCloud polypeptide characterized above;
  • TP is the therapeutic polypeptide selected, but not limited from the group consisting of human glucagon-like peptide- 1 (GLP-1), Exenatide, GLP-2, C-peptide, Calcitonin, human Parathyroid hormone (PTH), glucagon, G-CSF, GM-CSF, Interferon, interleukin factors, VEGF receptors, TNF alpha receptors, RANK, Growth hormone, Erythropoietin, blood-coagulation factors, single-chain Fv, single domain antibodies and functional variants thereof;
  • Loop is a flexible loop characterized above.
  • PCM is the proteinous connecting moiety of human origin.
  • the proteinous connecting moiety within the fusion protein is a proteinous sequence having an elongated shape of human origin.
  • the proteinous connecting moiety within the fusion protein is a human Fibronectin type III domain.
  • the proteinous connecting moiety within the fusion protein contains a human Fibronectin type III domain 8 (Fn8).
  • Fn8 human Fibronectin type III domain 8
  • Fibronectin(Fn) is a high-molecular weight ( ⁇ 440kDa) glycoprotein of the extracellular matrix that binds to a number of proteins including integrins, collagen, fibrin and heparan sulfate proteoglycans (e.g. syndecans) [18] .
  • Fibronectin exists as a protein dimer, consisting of two nearly identical polypeptide chains linked by a pair of C-terminal disulfide bonds.
  • Each fibronectin monomer has a molecular weight of 230-250 kDa and contains three types of domains: type I, II, and III.
  • Type I and type II are stabilized by intra-chain disulfide bonds, while fibronectin type III domains do not contain any disulfide bonds [19] .
  • the Fibronectin type III domain is an evolutionary conserved protein domain that is widely found in animal proteins. The human fibronectin protein in which this domain was first identified contains 16 copies of this domain (Fnl to Fnl6).
  • the fibronectin type III domain family (pfam ID: PF00041) member contains about 95 amino acids long and possesses a beta sandwich structure.
  • Fibronection type III domain forms a very stable domain structure with the melting temperature of -70 °C as measured by DSC [20 ' 21] .
  • Fibronectin type III domains are found in a wide variety of extracellular proteins. In human genome, fibronection type III domain exists in many proteins including Tenascin, Usherin, Titin, tripartite motif (TRIM) family members, tissue factor, TIE1 , TIE2, SPEG, SORL1 , SDK1 , ROBOl , ROB02, SDK2, Receptor-type tyrosine-protein phosphatase, prolactin receptor, LI CAM, NCAM1 , NCAM2, myomesin 1 , myomesin 2, Myosin-binding protein C, LIFR, Leptin receptor, Integrin, Insulin receptor, Contactin, Collagen, Cytokine receptor-like factor, Inteferon receptor, Growth hormone receptor, fibronectin, leucine rich transmembrane protein (FLRT
  • Fibronectin type III domain as the proteinous connecting moiety in our method may have low immunogenicity.
  • the Fibronectin type III domains can also be used in tandem fashion in the proteinous connecting moiety.
  • Fibronectin type III domain can be expressed in recombinant form using a number of expression systems including E. coli, using Fibronectin type III domain as the proteinous connecting moiety in the method of the invention may greatly increase the expression yield of the fusion protein.
  • Collagens are a diverse family of proteins that constitute the major structural component of the extracellular matrix [22-24]. Collagen is composed of a triple helix, which generally consists of two identical chains (oil) and an additional chain that differs slightly in its chemical composition (a2). Classification according to supramolecular structure assigns collagens to fibril, fibril-associated containing interrupted triple helicies (FACIT), beaded filament, anchoring fibril, network-forming, transmembrane or multiple triple helicies with interruptions (Multiplexin) families[25]. To date, 43 unique -chains that belong to 28 types of collagens (types I-XXVIII) have been discovered in vertebrates.
  • FACIT interrupted triple helicies
  • Multiplexin multiple triple helicies with interruptions
  • the alpha chains of collagens consist of at least one triple helical collagenous domain of varying length and two noncollagenous (NC) domains of variable sequence, size, and shape that are positioned at the N and C terminus.
  • the collagenous domains contain the G-X-Y repeats and form the typical triple helix within the collagen molecule while some of the NC domains form homo-trimers to stabilize the collagen triple helix.
  • NC I carboxy-terminal NC
  • the Multiplexin family members also utilize NC I domains for trimerization.
  • Fibril associated-collagens have recently been shown to trimerize via their NC2 domains (the second NC domain from the carboxy-terminal end)[27, 28].
  • About 130 a. a. residues are present in each monomer.
  • the NCI domains from the multiplexin family members such as Collagen XV and XVIII formed the much smaller homo-trimer with the length of about 55 residues in each monomer[26, 32].
  • Both types of the collagen NC domains form very stable homo-trimers in solution (Tm>60°C) and are suitable as the scaffold protein as described in the invention.
  • the Clq family is characterized by a C-terminal conserved globular Cl q domain (pfam ID: PF00386), which can form a stable homo-trimer [33-35].
  • the C l q-like protein family includes, but not limited to, human C l q A chain, C l q B chain, C lq C chain, cbln family members, human EMILI -1 , multimerin, ACRP30/adiponectin, adipolin, resistin and resistin-like molecule (RELM) hormone family members.
  • TNF Tumor necrosis factor
  • TNF family members include, but not limited, human TNFalpha, TNFbeta, TRAIL, RANK ligand, Fas ligand, CD 30 ligand, CD40 ligand, CD27 ligand, OX40L and CD137.
  • TNF family members form homo-trimers in solution and demonstrated the similar molecular structure as the C l q family members. Therefore, these two family members are also named as Cl q/TNF-related proteins (CTRP)[34].
  • CRP Cl q/TNF-related proteins
  • CTLDs C-type lectin-like domains
  • PF00059 C-type lectin-like domains
  • a number of CTLD proteins contain a neck and a C-terminal C-type carbohydrate-recognition domain (CRD) and they form homo-trimer in solution.
  • CCD carbohydrate-recognition domain
  • CTLD includes mannan-binding lectin (MBL), surfactant protein A (SP-A), surfactant protein D (SP-D), collectin liver 1 (CL-L1), collectin placenta 1 (CL-P1), conglutinin, collectin of 43 kDa (CL-43) and collectin of 46 kDa (CL-46), Langerin and Tetranectin [39-42].
  • MBL mannan-binding lectin
  • SP-A surfactant protein A
  • SP-D surfactant protein D
  • collectin liver 1 CL-L1
  • CL-P1 collectin placenta 1
  • conglutinin collectin of 43 kDa
  • CL-46 collectin of 46 kDa
  • Langerin and Tetranectin [39-42].
  • the CTRP family members including the Clq-like domains and TNF family members, can be utilized to fuse with the therapeutic polypeptide to extend the in vivo half life of the fusion protein.
  • the CTLD family members can be employed as the scaffold protein to drive the trimerization of the therapeutic polypeptides.
  • the NCI domain within Multiplexin type of human Collagen (such as collagen XV and XVIII) and NC2 domain within FACIT type of collagen (such as collagen IX, XII, XIV, XVI, XIX, XX, XXI, and XXII) may serve as the scaffold proteins in the method of this invention.
  • the therapeutic polypeptide and the pCloud polypeptides can be fused to either the N-terminus or the C-terminus of the scaffold protein.
  • the trimer formation of the fusion protein can efficiently increase the hydrodynamic radius of the protein molecule.
  • the fusion protein may demonstrate a much larger apparent size than a compact molecule with the same molecular weight. Therefore the fusion protein will show a much reduced clearing rate by renal filtration and will exhibit an extended half life in vivo.
  • the NCI domain within Multiplexin type of human Collagen (such as collagen XV and XVIII) were selected as the scaffold protein in the present invention.
  • NC I domain does not utilize the G-X-Y repeats and does not form the typical triple helix.
  • no disulfide bridges between the NC I domains are required to form the stable homo-trimer.
  • NCI domain contains only about 55 amino acid residues and represents a small protein, which makes it less likely to interfere with the proper functions of the therapeutic polypeptide in the fusion protein. All the features of NC 1 domain within Multiplexin type of human Collagen (such as collagen XV and XVIII) make it an ideal scaffold protein in the present invention.
  • Using NCI domain as the scaffold protein is preferred in the present invention, which is quite different from using other collagen domains as the scaffold protein as described elsewhere (patent 14, 15).
  • scaffold protein does not necessarily mean an entire wild type protein; a domain or functional variant thereof which can form a stable homo-trimer in solution and can therefore serve the purpose of the invention may also be used in our method.
  • the NCI domain within Multiplexin type of human Collagen such as collagen XV and XVIII was used as a scaffold protein.
  • the therapeutic polypeptide may be selected from, but not limited to, human glucagon-like peptide- 1 (GLP-1), Exenatide, GLP-2, C-peptide, Calcitonin, human Parathyroid hormone (PTH), glucagon, G-CSF, GM-CSF, Interferon, interleukin factors, VEGF receptors, TNF alpha receptors, RANK, Growth hormone, Erythropoietin, blood-coagulation factors, single-chain Fv, single domain antibodies and functional variants thereof.
  • GLP-1 human glucagon-like peptide- 1
  • Exenatide GLP-2
  • C-peptide Calcitonin
  • PTH human Parathyroid hormone
  • glucagon G-CSF
  • GM-CSF GM-CSF
  • Interferon Interleukin factors
  • VEGF receptors vascular endothelial growth factor
  • TNF alpha receptors fibroblast growth factor
  • RANK Growth hormone
  • GLP- 1 in a preferred embodiment of this invention, we use GLP- 1 as one of the examples to illustrate how the method of the invention can significantly improve the pharmacokinetics property of GLP- 1 and its mutants while retaining its biological activity.
  • the natural incretin hormone glucagon-like peptide- 1 (GLP- 1) supports glucose homeostasis by enhancing glucose-dependent insulin secretion from ?-cells and suppressing inappropriately elevated postprandial glucagon secretion from ct-cells.
  • GLP- 1 has been demonstrated to reduce appetite and food intake and inhibit gastric emptying, which may facilitate weight management [43, 44]. Therefore, GLP-1 remains to be a very promising therapeutic polypeptide for type 2 diabetes and weight loss.
  • GLP-1 is a 30 residue polypeptide with a very short half life in vivo, which severely limits its applications. In the present invention, we demonstrated data to show that the half life of GLP-1 can be significantly extended by use of the method of the invention.
  • pCloud polypeptides and the scaffold protein with the human growth hormone, human Interferon alpha-2b and human G-CSF.
  • These therapeutic polypeptides suffer significantly from their short in vivo half life. Fusing with pCloud polypeptide and the scaffold protein may greatly improve the pharmacokinetic profiles of the therapeutic polypeptides in vivo.
  • Antibody IgG is a Y-shaped molecule with bi-valency and utilizes two identical variable domains to interact with its ligand.
  • the fusion protein generated using the method of the invention has tri-valency and therefore might behave better than the traditional human monoclonal antibody IgG in interacting with the ligand. For example, TNF alpha forms a homo-trimer in solution and interact with three TNF receptors simultaneously.
  • TNF receptor 2 TNF receptor 2
  • TNFR2, p75 TNF receptor 2
  • Etanercept Etanercept
  • one Enbrel molecule can only block two out of three possible binding sites located on TNF alpha homo-trimer.
  • our fusion protein of TNFR2 and collagen XVIII NC 1 domain generated can form a homo-trimer and block all three binding sites of TNFalpha while retaining a long half life in vivo.
  • the scaffold protein utilized in this invention can form homo-trimers by simultaneous self assembly.
  • the NCI domain within Multiplexin type of human Collagen such as collagen XV and XVIII
  • NC 1 domain no inter-chain disulfide bonds are needed to drive the trimerization, which makes it more convenient for protein expression.
  • Many expression systems such as E. coli, yeast, insect cell and mammalian cell systems can be utilized to express the fusion proteins generated by the invention. In the sharp contrast, therapeutic monoclonal antibodies rely purely on the mammalian systems for mass productions. Summary for Trident technology
  • Improving the pharmacokinetic property of a therapeutic polypeptide may have major impacts on its clinical application.
  • GLP-1 extending its in vivo half life transformed it into a practical drug with great efficacy and broad markets.
  • increasing the apparent molecular weight (hydrodynamic radius and/or radius of gyration) of a therapeutic polypeptide can result in an improvement in the pharmacokinetic behavior of the therapeutic polypeptide possibly due to the slower renal clearance.
  • the apparent molecular weight (the hydrodynamic radius and/or Rg) of a protein is determined by its molecular weight as well as by its structure, including shape and compactness.
  • the flexible unstructured polypeptides (in preferred embodiments, pCloud polypeptides) can adopt unstructured conformations due to electrostatic repulsion between individual charges of the polypeptide and/or the inherent flexibility imparted by the particular amino acids in the sequence that lack potential to confer secondary structures.
  • the extended and unstructured conformation of the flexible unstructured polypeptides may have a greater proportional hydrodynamic radius and/or Rg compared to polypeptides of a comparable sequence length and/or molecular weight that have tight secondary and/or tertiary structures, such as typical globular proteins.
  • Methods for determining the hydrodynamic radius and/or Rg are well known in the art, such as by the use of size exclusion chromatography (SEC), as described in U.S. Patent Nos. 6,406,632 and 7,294,513.
  • the present invention provides a novel technique termed as
  • trimer formation may greatly increase the hydrodynamic radius of the fusion molecule and improve the in vivo half life of the fusion protein.
  • the flexible unstructured polypeptides in preferred embodiments, pCloud polypeptides
  • fusing the therapeutic polypeptide with flexible unstructured polypeptides (in preferred embodiments, pCloud polypeptides) and the trimeric scaffold protein may render the fusion protein a much larger apparent molecular size compared to a compactly folded globular protein with the same molecular weight.
  • This will greatly improve the pharmacokinetic profile of the therapeutic polypeptide.
  • fragments derived from human fibrinogen alpha chain sequence were utilized as the building blocks to generate the pCloud polypeptides which rendered low immunogenicity when administered to human.
  • the method of the invention can provide the therapeutic polypeptide with tri-valency, which may greatly increase the affinity and avidity of the fusion protein toward the ligand.
  • the fusion protein can be further modified by PEGylation.
  • the PEG moiety may have a molecular weight of between 2 kDa and 100 kDa.
  • the Cys residue may need to be generated in the fusion protein using site-directed mutagenesis.
  • the fusion proteins of the present invention can be produced through the application of recombinant DNA technology.
  • Recombinant polynucleotide constructs encoding a fusion polypeptide of the present invention typically include an expression control sequence operably-linked to the coding sequences of the fusion polypeptide, including naturally-associated or heterologous promoter regions.
  • another aspect of the invention includes vectors containing one or more nucleic acid sequences encoding a fusion polypeptide of the present invention.
  • the nucleic acid containing all or a portion of the nucleotide sequence encoding the fusion polypeptide is inserted into an appropriate cloning vector, or an expression vector (i.e.
  • expression vectors useful in recombinant DNA techniques are often in the form of plasmids.
  • "plasmid” and “vector” can be used interchangeably as plasmid is the most commonly used form of vector.
  • the invention is intended to include such other forms of expression vectors that are not technically plasmids, such as viral vectors (e.g. , replication defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions.
  • the expression control sequences are eukaryotic promoter systems in vectors capable of transforming or transfecting eukaryotic host cells.
  • expression vectors are typically replicable in the host organisms either as episomes or as an integral part of the host chromosomal DNA.
  • expression vectors contain selection markers, e.g. , ampicillin-resistance or kanamycin-resistance, to permit detection of those cells transformed with the desired DNA sequences.
  • Vectors can also encode a signal peptide, e.g. , pectate lyase, useful to direct the secretion of extracellular antibody fragments. See U.S. Pat. No. 5,576, 195.
  • the recombinant expression vectors of the invention may comprise a nucleic acid encoding a fusion polypeptide in a form suitable for expression of the nucleic acid in a host cell, which means that the recombinant expression vectors may include one or more regulatory sequences selected on the basis of the host cells to be used for expression that are operatively-linked to the nucleic acid sequence to be expressed.
  • "operably-linked" is intended to mean that the nucleotide sequence of interest is linked to the regulatory sequence(s) in a manner that allows for expression of the nucleotide sequence (e.g. , in an in vitro transcription/translation system or in a host cell when the vector is introduced into the host cell).
  • regulatory sequence is intended to include promoters, enhancers and other expression control elements (e.g. , polyadenylation signals). Such regulatory sequences are described, e.g. , in Goeddel, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990). Regulatory sequences include those that direct constitutive expression of a nucleotide sequence in many types of host cell and those that direct expression of the nucleotide sequence only in certain host cells (e.g. , tissue-specific regulatory sequences). It will be appreciated by those skilled in the art that the design of the expression vector can depend on such factors as the choice of the host cell to be transformed, the level of expression of polypeptide desired, etc.
  • fusion polypeptide-expressing host cells which contain a nucleic acid encoding one or more fusion polypeptides.
  • the recombinant expression vectors of the invention can be designed for expression of a fusion polypeptide in prokaryotic or eukaryotic cells.
  • a fusion polypeptide can be expressed in bacterial cells such as Escherichia coli, insect cells, fungal cells, e.g. , yeast, or mammalian cells. Suitable host cells are discussed further in Goeddel, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. ( 1990).
  • the recombinant expression vector can be transcribed and translated in vitro, e.g. using T7 promoter regulatory sequences and T7 polymerase.
  • polypeptides in prokaryotes are most often carried out in E. coli with vectors containing constitutive or inducible promoters directing the expression of the recombinant polypeptides.
  • the vectors may add a number of amino acids to a polypeptide encoded therein, usually to the amino terminus of the recombinant polypeptide.
  • Such vectors with extra amino acid residues typically serve three purposes: (i) to increase expression of recombinant polypeptide; (ii) to direct the recombinant protein to periplasmic space; and (iii) to aid in the purification of the recombinant polypeptide by acting as a ligand in affinity purification.
  • Typical expression vectors serving this purpose include pGEX (GE Healthcare), pMAL (New England Biolabs), pET20b(Novagen), pET43b (Novagen), pET32b(Novagen) and pRIT5 (GE Healthcare).
  • E. coli expression vectors examples include pTrc vectors (Invitrogen), pQE (Qiagen) and pET vectors (Novagen).
  • One strategy to maximize recombinant polypeptide expression is to alter the nucleic acid sequence of the nucleic acid to be inserted into an expression vector so that the individual codons for each amino acid are those preferentially utilized in the expression host, e.g. , E. coli 48 .
  • Such alteration of nucleic acid sequences of the invention can be carried out by standard DNA synthesis techniques.
  • the fusion polypeptide expression vector is a yeast expression vector.
  • yeast Saccharomyces cerivisae examples include pYES2 (Invitrogen), pMFa 49 and pJRY88 50 .
  • the fusion protein may also be expressed in Pichia system using the vectors pPICZ pGAPZ and pPIC9(InVitrogen).
  • a fusion polypeptide can be expressed in insect cells using baculovirus expression vectors or using the stable insect cell lines. Baculovirus systems available for expression of polypeptides in cultured insect cells ⁇ e.g.
  • SF9 cells include the BaculoGold system (BD Biosciences), BaculoDirect system (Invitrogen) and BacVector system (Novagen).
  • the stable insect expression systems include, but not limited to, DES system (Invitrogen) and InsectDirect (Novagen).
  • a nucleic acid encoding a fusion polypeptide of the invention is expressed in mammalian cells using a mammalian expression vector.
  • mammalian expression vectors include, e.g., but are not limited to, pcDNA3.1 (Invitrogen), pSecTag (invitrogen), and pTriEx series vectors (Novagen).
  • the expression vector's control functions are often provided by viral regulatory elements.
  • commonly used promoters are derived from polyoma, adenovirus 2, cytomegalovirus, and simian virus 40.
  • the recombinant fusion protein can be expressed in the cytoplasm.
  • the fusion protein can be secreted into the medium by adding an N-terminal secretion signal.
  • host cell and "recombinant host cell” are used interchangeably herein. It is understood that such terms refer not only to the particular subject cell but also to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein.
  • a host cell can be any prokaryotic or eukaryotic cell.
  • a fusion polypeptide can be expressed in bacterial cells such as E. coli, insect cells, yeast or mammalian cells. Mammalian cells are a preferred host for expressing nucleotide segments encoding immunoglobulins or fragments thereof. See Winnacker, From Genes To Clones, (VCH Publishers, NY, 1987).
  • a number of suitable host cell lines capable of secreting intact heterologous proteins have been developed in the art, and include Chinese hamster ovary (CHO) cell lines, 293 cells, various COS cell lines, HeLa cells, L cells and myeloma cell lines. Preferably, the cells are nonhuman.
  • Expression vectors for these cells can include expression control sequences, such as an origin of replication, a promoter, an enhancer, and necessary processing information sites, such as ribosome binding sites, RNA splice sites, polyadenylation sites, and transcriptional terminator sequences.
  • Preferred expression control sequences are promoters derived from endogenous genes, cytomegalovirus, SV40, adenovirus, bovine papillomavirus, and the like.
  • Other suitable host cells are known to those skilled in the art.
  • a gene that encodes a selectable marker (e.g. , resistance to antibiotics) is generally introduced into the host cells along with the gene of interest.
  • selectable markers include those that confer resistance to drugs, such as G418, hygromycin and methotrexate.
  • Nucleic acid encoding a selectable marker can be introduced into a host cell on the same vector as that encoding the fusion polypeptide or can be introduced on a separate vector. Cells stably transfected with the introduced nucleic acid can be identified by drug selection (e.g., cells that have incorporated the selectable marker gene will survive, while the other cells die).
  • the fusion polypeptides are purified from culture media and/or host cells. Purification of recombinant polypeptides is well known in the art and includes ammonium sulfate precipitation, affinity chromatography purification technique, column chromatography, ion exchange purification technique, gel filtration and the like (see generally Scopes, Protein Purification (Springer- Verlag, N.Y., 1982).
  • the present invention envisions treating a disease, for example, type II diabetes, in a mammal by the administration of the fusion protein compositions of the present invention.
  • Administration of the fusion protein in accordance with the present invention may be continuous or intermittent, depending, for example, upon the recipient's physiological condition, whether the purpose of the administration is therapeutic or prophylactic, and other factors known to skilled practitioners.
  • the administration of the vaccines of the invention may be essentially continuous over a preselected period of time or may be in a series of spaced doses. Both local and systemic administration is contemplated.
  • the pharmaceutical composition of the present invention may be delivered via various routes and to various sites in a mammal body to achieve a particular effect.
  • routes e.g., a particular route can provide a more immediate and more effective reaction than another route.
  • Local or systemic delivery can be accomplished by administration comprising application or instillation of the formulation into body cavities, inhalation or insufflation of an aerosol, or by parenteral introduction, comprising intramuscular, intravenous, peritoneal, subcutaneous, intradermal, as well as topical administration.
  • the amount of the administered fusion protein of the present invention will vary depending on various factors including, but not limited to, the particular disease, the weight, the physical condition, and the age of the mammal, and whether prevention or treatment is to be achieved. Such factors can be readily determined by the clinician employing animal models or other test systems which are well known to the art.
  • the amount of the fusion protein of the present invention to be administered to a mammal subject may vary in the range of lng/kg to l OOmg/kg of the subject body weight. In an embodiment of the invention, the amount of administration was from lug/kg to l .Omg/kg.
  • the fusion proteins of the invention are prepared for administration, they are preferably combined with a pharmaceutically acceptable carrier to form a pharmaceutical formulation, or unit dosage form.
  • a pharmaceutically acceptable carrier commonly known to a skilled artisan in the field of pharmacy.
  • the total active ingredients in such formulations include from 0.1 to 99.9% by weight of the formulation.
  • the active ingredient for administration may be present as a powder or as granules; as a solution, a suspension or an emulsion.
  • the therapeutic agent may be formulated for parenteral administration (e.g., by injection, for example, bolus injection or continuous infusion) and may be presented in unit dosage form in ampules, pre-filled syringes, small volume infusion containers or in multi-dose containers with an added preservative.
  • the active ingredients may take such forms as suspensions, solutions, or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
  • the active ingredients may be in powder form, obtained by aseptic isolation of sterile solid or by lyophilization from solution, for re-constitution with a suitable vehicle, e.g. , sterile, pyrogen-free water, before use.
  • water, suitable oil, saline, aqueous dextrose (glucose), and related sugar solutions and glycols such as propylene glycol or polyethylene glycols are suitable carriers for parenteral solutions.
  • Solutions for parenteral administration contain the active ingredient, suitable stabilizing agents and, if necessary, buffer substances.
  • Antioxidizing agents such as sodium bisulfate, sodium sulfite or ascorbic acid, either alone or combined, are suitable stabilizing agents.
  • parenteral solutions can contain preservatives such as benzalkonium chloride, methyl- or propyl-paraben and chlorobutanol.
  • Suitable pharmaceutical carriers are described in Remington's Pharmaceutical Sciences, a standard reference text in this field.
  • control release preparations can include appropriate macromolecules, for example polymers, polyesters, polyamino acids, polyvinyl, pyrolidone, ethylenevinylacetate, methyl cellulose, carboxymethyl cellulose or protamine sulfate.
  • concentration of macromolecules as well as the methods of incorporation can be adjusted in order to control release.
  • the agent can be incorporated into particles of polymeric materials such as polyesters, polyamino acids, hydrogels, poly (lactic acid) or ethylenevinylacetate copolymers. In addition to being incorporated, these agents can also be used to trap the compound in microcapsules.
  • compositions, carriers, and reagents are used interchangeably and represent that the materials are capable of administration to or upon a subject without the production of undesirable physiological effects to a degree that would prohibit administration of the composition.
  • Such carriers include, but are not limited to, water, saline, Ringer's solutions, and dextrose solution. Liposomes and non-aqueous vehicles such as fixed oils may also be used.
  • the use of such media and compounds for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or compound is incompatible with the fusion polypeptides, use thereof in the compositions is contemplated. Supplementary active compounds can also be incorporated into the compositions.
  • a pharmaceutical composition of the invention is formulated to be compatible with its intended route of administration.
  • the fusion polypeptides compositions of the present invention can be administered by parenteral, topical, intravenous, oral, subcutaneous, intraarterial, intradermal, transdermal, rectal, intracranial, intraperitoneal, intranasal; intramuscular route or as inhalants.
  • the fusion polypeptides can optionally be administered in combination with other agents that are at least partly effective in treating various diseases including various actin- or microfilament-related diseases.
  • Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial compounds such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating compounds such as ethylenediaminetetraacetic acid (EDTA); buffers such as acetates, citrates or phosphates, and compounds for the adjustment of tonicity such as sodium chloride or dextrose.
  • the pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide.
  • the parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.
  • compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion.
  • suitable carriers include physiological saline, bacteriostatic water, Cremophor ELTM (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS).
  • the composition must be sterile and should be fluid to the extent that easy syringeability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi.
  • the carrier can be a solvent or dispersion medium containing, e.g., water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof.
  • the proper fluidity can be maintained, e.g. , by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal compounds, e.g. , parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like.
  • isotonic compounds e.g. , sugars, polyalcohols such as manitol, sorbitol, sodium chloride in the composition.
  • Prolonged absorption of the injectable compositions can be brought about by including in the composition a compound which delays absorption, e.g. , aluminum monostearate and gelatin.
  • Sterile injectable solutions can be prepared by incorporating the fusion polypeptides in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization.
  • dispersions are prepared by incorporating the binding agent into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above.
  • methods of preparation are vacuum drying and freeze-drying that yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
  • the agents of this invention can be administered in the form of a depot injection or implant preparation which can be formulated in such a manner as to permit a sustained or pulsatile release of the active ingredient.
  • Oral compositions generally include an inert diluent or an edible carrier. They can be enclosed in gelatin capsules or compressed into tablets.
  • the binding agent can be incorporated with excipients and used in the form of tablets, troches, or capsules.
  • Oral compositions can also be prepared using a fluid carrier for use as a mouthwash, wherein the compound in the fluid carrier is applied orally and swished and expectorated or swallowed.
  • Pharmaceutically compatible binding compounds, and/or adjuvant materials can be included as part of the composition.
  • the tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating compound such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening compound such as sucrose or saccharin; or a flavoring compound such as peppermint, methyl salicylate, or orange flavoring.
  • a binder such as microcrystalline cellulose, gum tragacanth or gelatin
  • an excipient such as starch or lactose, a disintegrating compound such as alginic acid, Primogel, or corn starch
  • a lubricant such as magnesium stearate or Sterotes
  • a glidant such as colloidal silicon dioxide
  • the fusion polypeptides are delivered in the form of an aerosol spray from pressured container or dispenser which contains a suitable propellant, e.g. , a gas such as carbon dioxide, or a nebulizer.
  • a suitable propellant e.g. , a gas such as carbon dioxide, or a nebulizer.
  • Systemic administration can also be by transmucosal or transdermal means.
  • penetrants appropriate to the barrier to be permeated are used in the formulation.
  • penetrants are generally known in the art, and include, e.g., for transmucosal administration, detergents, bile salts, and fusidic acid derivatives.
  • Transmucosal administration can be accomplished through the use of nasal sprays or suppositories.
  • the fusion polypeptides are formulated into ointments, salves, gels, or creams as generally known in the art.
  • the fusion polypeptides can also be prepared as pharmaceutical compositions in the form of suppositories (e.g. , with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.
  • suppositories e.g. , with conventional suppository bases such as cocoa butter and other glycerides
  • retention enemas for rectal delivery.
  • Example 1 Construction of an expression vector of GLP- 1 fused with human Fibronectin type III domain 7 (Fn7) and human collagen XVIII NC I domain (COL 18NC 1 )
  • GLP- 1 polypeptide was fused to the N-terminus of the human collagen XVIII NC I domain (COL 18NC 1 ) which forms a stable homo-trimer.
  • the human Fibronectin type III domain 7 was utilized as the proteinous connecting moiety.
  • Fn7 was connected to the GLP- 1 polypeptide and COL 18NC 1 through a flexible loop (GGGSGGGG) and a flexible, unstructured linker (GGGSGG).
  • the pET29b vector Novagen was used to construct a recombinant plasmid containing the GLP- 1 -Fn7-C0L 18NC 1 fusion gene.
  • human COL 18NC 1 was cloned into pET29b by BamUl and Xhol to result in the pET29b-COL18NCl vector.
  • PCR reaction was carried out using human collagen XVIII cDNA as the template by using the following primers:
  • the PCR product was digested by restrictive enzyme Ndel and BamUl (Fermentas) and ligated into the digested pET29b-COL18NCl vector.
  • the optimized DNA sequence of human GLP- 1 (7-37) was included in the primer named as Glp 1 -Fn7-Forward.
  • the cloned GLP-l-Fn7-COL18NCl fusion gene was confirmed by DNA sequencing.
  • the protein sequence of GLP- 1 -Fn7-C0L18NC 1 was listed as SEQ ID NO: 1.
  • GLP-l-Fn7-COL18NC l protein sequence Fn7 is connected to GLP- 1 and COL18NC 1 by use of a flexible loop and a flexible unstructured linker.
  • the flexible loop between GLP-1 and Fn7 (GGGSGGGG) is underlined.
  • the flexible, unstructured linker (GGGSGG) between Fn7 and COL18NC 1 is also underlined.
  • GLP- 1 sequence is in italic.
  • Fibronectin type III domains may also act as the proteinous connecting moiety between the therapeutic polypeptide and the scaffold protein in our method.
  • Fibronectin type III domain 8 Fn8
  • the gene encoding GLP- 1 and human Fibronectin type III domain 8 (Fn8) was amplified by PCR using the following primers and human Fibronectin cDNA as the template:
  • the PCR product was digested by Ndel and BamR ⁇ .
  • the digested insert was ligated into the digested vector pET29b-COL 18NC l (generated in example 1 ).
  • the resulted vector was named as pET29b-GLP- l -Fn8-COL18NC l .
  • the protein sequence of the fusion protein GLP- 1 -Fn8-C0L 18NC 1 was listed as SEQ ID NO: 2.
  • GLP- 1-Fn8-C0L18NC1 protein sequence GLP-1 sequence is in italic.
  • Fn8 is connected to GLP-1 and COL18NC 1 by use of a flexible loop and a flexible unstructured linker.
  • the flexible loop between GLP- 1 and Fn8 (GGGSGGGGS) is underlined.
  • the flexible unstructured linker (GGGSGG) between Fn8 and COL18NC 1 is also underlined.
  • the constructed expression vector pET29b-Glp l -Fn8-COL18NC l was used to transform Escherichia coli BL21 (DE3) for protein expression (for detailed protocols of the transformation, see Molecular Coining: A Laboratory Manual).
  • a single colony was selected from the culture dish, and placed into a 10 ml LB liquid medium with kanamycin (final concentration, 50 iglm ⁇ ), then shaken at 37 °C at 220 rpm overnight. 1 L LB culture was inoculated and allowed to grown until OD 60 o reached 0.4- 1.0. Isopropyl thiogalactoside (IPTG) was added to a final concentration of 0.2mM.
  • IPTG Isopropyl thiogalactoside
  • A8G/G22E/R36S and A8G/G22E/R36G within the GLP- 1 sequence may increase its resistance to protease digestion, reduce immunogenicity and boost its biological activity [6, 48].
  • the PCR reaction was carried out using vector pET29b-GLP l -Fn8-COL 18NC l prepared in example 2 as the template with the following primers: Glp- l (A8G/G22E)-Fn8-forward:
  • the PCR product was digested by Ndel and Xhol (Fermentas).
  • the digested insert was ligated into the digested vector of pET29b.
  • the resulted vector was named as pET29b-GLP l (A8G/G22E)-Fn8-COL18NC l .
  • the expression and purification protocol of the fusion protein of GLP- l (A8G/G22E)-Fn8-COL 18NCl was the same as described in example 3.
  • GLP- 1 Other mutations within the GLP- 1 , such as A8V/G22E, A8S/G22E, A8G/G22E/R36S and A8G/G22E/R36G, can be generated using the Quikchange II site-directed mutagenesis kit (Agilent) using the GLP- 1 (A8G/G22E)-Fn8-C0L18NC 1 gene as the template.
  • the protein sequences of these GLP 1 mutants fused with Fn8-C0L 18NC 1 were listed as SEQ ID NO: 3-7.
  • the expression and purification protocol of these fusion proteins can be carried out using similar protocols described in example 3.
  • GLP-1 (A8G/G22E)-Fn8-C0L18NC 1 protein sequence, the GLP- 1 mutation sites (A8G/G22E) are underlined.
  • GLP- 1 (A8S/G22E)-Fn8-COL18NC l protein sequence SEQ ID NO: 5
  • GLP- 1 mutation sites (A8S/G22E) are underlined.
  • HGEGTFTSDVSSYLEEQAA EFIAWLVKGSGGGGSGGGGSAVPPPTDLRFTNIGPDTMRVTWAPPPSIDLTNFLVRYSPV
  • GLP- 1 (A8G/G22E/R36G)-Fn8-COL 18NC l protein sequence, the GLP-1 mutation sites
  • Example 5 Between the therapeutic polypeptides and the scaffold proteins, various lengths of the flexible un-structured linkers can be used to generate the fusion proteins with the desired hydrodynamic radius and/or radius of gyration (Rg)
  • the flexible unstructured linker between Fn8 and COL18NC 1 contains six residues (GGGSGG).
  • GGGSGG GGGSGG
  • GLP- 1 (A8G/G22E/R36S)-Fn8-COLl 8NC 1 -30
  • the length of the flexible unstructured polypeptide linker between Fn8 and COL18NC1 contained 20, 30, 54 and 60 residues, respectively.
  • GLP- 1 (A8G/G22E/R36S)-Fn8-COLl 8NC 1 -20
  • GLP- 1 (A8G/G22E/R36S)-Fn8-COLl 8NC 1 -30
  • GLP- 1 (A8G/G22E/R36S)-Fn8-COLl 8NC 1 -54 and
  • GLP-l(A8G/G22E/R36S)-Fn8-COL18NCl-60 are listed as SEQ ID NO: 8-1 1.
  • GLP-l(A8G/G22E/R36S)-Fn8-COL18NC l-54 and GLP-l(A8G/G22E/R36S)-Fn8-COL18NCl-60 were grafted to the pET29b by Ndel and Xhol for protein expressions.
  • the expression and purification of these fusion proteins were carried out using similar protocols described in example 3. To estimate the hydrodynamic radius and/or the Rg of the fusion proteins, the purified proteins were loaded on an analytical gel filtration column Superdex200 (GE Healthcare).
  • GLP-l(A8G/G22E/R36S)-Fn8-COL18NC l-20, GLP-l(A8G/G22E/R36S)-Fn8-COL18NCl-30 exhibited larger apparent molecular weight than their genuine molecular weight as well.
  • our method can provide the therapeutic polypeptide with a larger hydrodynamic Radius and/or Rg which exhibited increased apparent molecular size on gel filtration profile.
  • the flexible unstructured linker between the therapeutic polypeptide and the scaffold protein may adjust the hydrodynamic radius and/or the Rg of the fusion molecule in a tunable manner.
  • GLP-l A8G/G22E/R36S
  • Fn8-COL18NC l-54 protein sequence of GLP-l (A8G/G22E/R36S)-Fn8-COL18NC l-54, the flexible unstructured linker (54 residues) between Fn8 and COL 18NC1 is underlined.
  • the flexible loop between GLP-1 and Fn8 (GGGSGG) is also underlined.
  • HGEGTFTSDVSSYLEEOAAKEFIAWLVKGSGGGGSGGAVPPPTDLRFTNIGPDTMRVTWAPPPSIDLTNFLVRYSPVKN EEDVAELSISPSDNAVVLTNLLPGTEYVVSVSSVYEQHESTPLRGRQ TG GGGGSGGGGSTASSASTGGPSGGGGSGGGGSAPSSGSTSGGTAAGGGGSGGGGS GASSGVRLWATRQAMLGQVHEVPEGWLIFVAEQEELYVRVQNGFRKVQLEARTPLPRG
  • SEQ ID NO: 1 1 protein sequence of GLP-l (A8G/G22E/R36S)-Fn8-COL18NC l-60, the flexible unstructured linker (60 residues) between Fn8 and COL18NC1 is underlined.
  • the flexible loop between GLP- 1 and Fn8 (GGGSGG) is also underlined.
  • HGEGTFTSDVSSYLEEQAA £FIAWLV GSGGGGSGGAVPPPTDLRFTNIGPDTMRVTWAPPPSIDLTNFLVRYSPVKN EEDVAELSISPSDNAVVLTNLLPGTEYVVSVSSVYEQHESTPLRGRQKTG GGGSGGGSGGGSTASSASTKGPSGGGSGGGSGGGSAPSS STSGGTAAGGGSGGGSGGGS GASSGVRLWATRQAMLGQVHEVPEGWLIFVAEQEELYVRVQNGFR VQLEARTPLPRG
  • Example 6 The pharmacokinetics studies for GLP-l (A8G/G22E/R36S)-Fn8-COL18NCl ,
  • GLP-l (A8G/G22E/R36S)-Fn8-COL18NCl-30
  • GLP- 1 A8G/G22E/R36S)-Fn8-COL 18NC 1-54.
  • GLP- 1 (A8G/G22E/R36S)-Fn8-COLl 8NC 1 -30
  • GLP- 1 (A8G/G22E/R36S)-Fn8-COLl 8NC 1 -54 in PBS buffer, pH 7.2.
  • SD Sprague-Dawley
  • Blood samples were taken at various time points after injections such as 0-min, 30-min, 1-hour, 2-hour, 4-hour, 8-hour, 24-hour, 48-hour, 3-day, 4-day, 5-day. 7-day, 10-day.
  • the serum samples were centrifuged and kept at -80°C freezer.
  • the GLP-1 concentrations within the serum samples were examined by use of the sandwich ELISA method.
  • the rabbit polyclonal antibody against human Fibronectin at the concentration of 3ug/ml (Ab299, Abeam company) was coated on ELISA plate for 1 hour at room temperature. Then the plate was washed by PBST buffer three times and the wells were blocked by PBS with 10% FBS for 1 hour at room temperature. The plate was washed three times before the serum samples containing GLP-1 fusion proteins were added. The serum samples could be diluted to 20-10000 folds before use. The ELISA plate was incubated with the serum samples at room temperature for 1 hour and then washed by PBST buffer five times.
  • mouse monoclonal against human GLP-1 peptide antibody (sc57510, Santa Cruz Biotich) at the concentration of lug/ml in PBST buffer was added to the wells.
  • the plate was washed extensively after incubation of 1 hour at room temperature.
  • the secondary antibody Goat anti-rabbit IgG HRP conjugated antibody (Beijing ZSGB-Bio company, ZB 5301), was added into the wells and the color was developed using TMB (3,3 ',5,5 '-tetramethylbenzidine, BD biosciences, Cat 555214 ).
  • the plate reader Bio-Rad microplate reader Model 680 was utilized to obtain the OD450 readings. This method has been calibrated using purified proteins first.
  • Fig. 4 showed the pharmacokinetics profiles of the GLP- 1 containing proteins by use of the sandwich ELISA method described above.
  • the pharmacokinetics parameters were obtained by using the WinNonlin software (Table 3).
  • the data clearly showed that the GLP- 1 containing fusion proteins generated by use of the method of the invention exhibited much extended in vivo half life possibly due to their enlarged Rg.
  • the data also showed that the flexible, unstructured linker between the therapeutic polypeptide and the scaffold protein may adjust the in vivo half life of the fusion molecules in a tunable manner.
  • GLP- 1 (A8G/G22E/R36S)-Fn8-COL 18NC 1 -30
  • GLP-l(A8G/G22E/R36S)-Fn8-COL18NCl-54 were shown in abbreviation as NCI, NCI -20, NCI -30, and NCI -54 respectively.
  • Example 7 construction of the fusion protein containing GLP-1 mutants, Fn8, pCloud sequence and COL18NC1
  • the pCloud sequence in this example (p246) comprises all the 12 fibrinogen fragments listed in table 1.
  • the 12 fragments listed in table 1 were placed in the order as they appear in the human fibrinogen alpha chain sequence.
  • the flexible loops that were utilized to connect these fibrinogen-derived fragments in p246 sequence are GSGSESGSG, GGGSGGGS and GGSGGGSGG.
  • the optimized gene encoding p246 andCOL18NCl was synthesized, digested by BamHI and Xhol and ligated into the digested vector of pET29b. The resulted vector was named as pET29b-p246-COL18NCl.
  • the optimized gene encoding GLP-1(A8G/G22E) and Fn8 was synthesized, digested by Ndel and BamHI and ligated into the digested pET29b-p246-COL18NCl.
  • the resulted vector was named as pET29b-GLP-l(A8G/G22E)-Fn8-p246-COL18NCl.
  • the protein sequence of the fusion protein GLP-l(A8G/G22E)-Fn8-p246-COL18NCl was listed as SEQ ID NO: 12.
  • GLP- l (A8G/G22E)-Fn8-p246-COL 18NCl contains GLP- 1 (A8G/G22E), a flexible loop, Fn8, pCloud sequence p246 and the scaffold protein COL18NC 1 .
  • GLP- 1 Other mutations within the GLP- 1 , such as A8V/G22E, A8S/G22E,
  • A8G/G22E/R36S and A8G/G22E/R36G can be generated using the Quikchange II site-directed mutagenesis kit (Agilent) using the GLP- l (A8G/G22E)-Fn8-p246-COL 18NCl gene as the template.
  • the resulted vectors of the fusion proteins were named as P ET29b-GLP- l (A8V/G22E)-Fn8-p246-COL 18NC l ,
  • P ET29b-GLP- l (A8G/G22E/R36S)-Fn8-p246-COL 18NC l and pET29b-GLP- 1 ( A8G/G22E/R36G)-Fn8 -p246-COL 18NC 1.
  • the protein sequences of these fusion proteins were listed as SEQ ID NO: 13- 16.
  • the expression and purification protocol of these fusion proteins can be carried out using similar protocols described in example 3.
  • GLP-l(A8G/G22E)-Fn8-p246-COL18NCl protein sequence, the GLP-1 mutation sites (A8G/G22E) are in Italic and bold.
  • the flexible loop between GLP1(A8G/G22E) and Fn8 is in bold.
  • the pCloud sequence p246 is underlined.
  • the pCloud sequence p246 is underlined.
  • the pCloud sequence p246 is underlined.
  • GLP-l(A8G/G22E/R36S)-Fn8-p246-COL18NCl protein sequence the GLP-1 mutation sites (A8G/G22E/R36S) are in Italic and bold.
  • the pCloud sequence p246 is underlined.
  • GLP-l(A8G/G22E/R36G)-Fn8-p246-COL18NCl protein sequence the GLP-1 mutation sites (A8G/G22E/R36G) are in Italic and bold.
  • the flexible loop between GLP1(A8G/G22E/R36G) and Fn8 is in bold.
  • the pCloud sequence p246 is underlined.
  • Example 8 Cloning and expression of GLP- 1 (A8G/G22E/R36G) fused with Tenascin C fibronectin type III domain 3 (TNCfn3), pCloud sequence p246 and human collagen XVIII NC 1 (COL 18NC 1 )
  • the fibronectin type III domain that can be used as the proteinous connecting moiety for our method is not limited within human fibronectin.
  • Other suitable fibronectin type III domain may alternatively be utilized as the proteinous connecting moiety in the method of the invention.
  • a fibronectin type III domain from human Tenascin C can be utilized to connect the therapeutic polypeptide and the pCloud sequence as well.
  • the human Tenascin C fibronectin type III domain 3 (TNCfn3) was connected to the GLP- 1 (A8G/G22E/R36G) and the pCloud sequence as the proteinous connecting moiety.
  • the gene encoding GLP-1 (A8G/G22E/R36G), the flexible loop and TNCfn3 was synthesized, digested by Ndel and BamHI and ligated into the digested pET29b-GLP-l(A8G/G22E/R36G)-p246-COL 18NCl vector.
  • the resulted vector was named as pET29b-GLP l (A8G/G22E/R36G)-TNCfn3-p246-COL18NC l .
  • the expression and purification protocol of the fusion protein GLP l (A8G/G22E/R36G)-TNCfn3-p246-COL 18NC l (SEQ ID NO: 17) was the same as described in example 3.
  • SEQ ID NO: 17 SEQ ID NO: 17, GLP-1 (A8G/G22E/R36G)- TNCfh3-p246-COL18NCl protein sequence,
  • the flexible unstructured linker between GLP1(A8G/G22E/R36G) and TNCfh3 is in bold.
  • the TNCfn3 sequence is in italic.
  • the pCloud sequence p246 is underlined.
  • NLKPD TE YEVSLISRR GDMS SNPAKE TF TG GGGSGGGSGSGSESGSGGGSTSSGTGSETESPGSG
  • Example 9 Cloning and expression of GLP- 1 (A8G/G22E/R36G) fused with Fn8, pCloud sequence p246 and human collagen XV NC I (COL 15NC 1 )
  • the gene encoding pCloud sequence p246 and COL15NC 1 domain was synthesized, digested by BamHI and Xhol and ligated into the digested pET29b-GLP- l (A8G/G22E/R36G)-Fn8-p246-COL 18NC l vector generated before.
  • the resulted vector was named as pET29b-GLP l (A8G/G22E/R36G)-Fn8-p246-COL15NC l and the sequence of the vector was confirmed by DNA sequencing.
  • the protein sequence of GLP 1 (A8G/G22E/R36G)-Fn8-C0L15NC 1 was listed as SEQ ID NO: 18.
  • the expression and purification protocol of the fusion protein GLP 1 (A8G/G22E/R36G)-Fn8-C0L 15NC 1 was the same as described in example 3.
  • the GLP-1 mutation sites (A8G/G22E/R36G) are in Italic.
  • Example 10 Cloning and expression of GLP- 1 (A8G/G22E/R36G) fused with Fn8, pCloud sequence and human collagen XIX NC2 domain (COL19NC2)
  • NC2 domain from collagen XIX can be utilized as the scaffold protein in our method. It has been shown that human collagen XIX NC2 domain (COL 19NC2) forms a highly stable homo-trimer[28].
  • the gene encoding pCloud sequence p246 and COL19NC2 domain was synthesized, digested by BamHI and Xhol and ligated into the digested pET29b-GLP- 1 (A8G/G22E/R36G)-Fn8 -p246-COL 15NC 1 vector generated before.
  • the resulted vector was named as pET29b-GLP l (A8G/G22E/R36G)-Fn8-p246-COL19NC2 and the sequence of the vector was confirmed by DNA sequencing.
  • the protein sequence of GLP l (A8G/G22E/R36G)-Fn8-p246-COL19NC2 was listed as SEQ ID NO: 19.
  • the expression and purification protocol of the fusion protein GLP l (A8G/G22E/R36G)-Fn8-p246-COL19NC2 was the same as described in example 3.
  • GLP-l(A8G/G22E/R36G)-Fn8-p246-COL19NC2 protein sequence the sequence of COL19NC2 is underlined.
  • the GLP-1 mutation sites (A8G/G22E/R36G) are in Italic.
  • Example 1 1 Cloning and expression of GLP- 1 (A8G/G22E/R36G) fused with Fn8, pCloud sequence and human ACRP30 C-terminal Cl q-like domain
  • ACRP30 (also referred to as Adiponectin, GBP-28, apMl and AdipoQ) is a protein hormone that modulates a number of metabolic processes, including glucose regulation and fatty acid catabolism[49].
  • ACRP30 contains a C-terminal globular domain that forms a homo-trimer with typical Clq-like structure [50].
  • the C l q-like domain such as the ACRP30 C l q-like domain, can be utilized as the scaffold protein in our method.
  • the gene encoding pCloud sequence p246 and ACRP30 C l q-like domain was synthesized, digested by BamHI and Xhol and ligated into the digested pET29b-GLP- l (A8G/G22E/R36G)-Fn8-p246-COL15NC l vector generated before.
  • the resulted vector was named as pET29b-GLP l (A8G/G22E/R36G)-Fn8-p246-ACRP30 and the sequence of the vector was confirmed by DNA sequencing.
  • the protein sequence of GLPl (A8G/G22E/R36G)-Fn8-p246-ACRP30 was listed as SEQ ID NO:20.
  • SEQ ID NO: 20 SEQ ID NO: 20
  • the GLP-1 mutation sites (A8G/G22E/R36G) are in Italic.
  • Example 12 Constructions of the fusion proteins of GLP- 1 (A8G/G22E/R36G), Fn8, pCloud sequences with various lengths and COL18NC l
  • Numerous pCloud sequences can be generated by use of the human Fribrinogen fragments listed in Table 1 and the flexible loops listed in table 2.
  • the fibrinogen fragments are flanked by flexible loops.
  • the 12 human fibrinogen alpha chain fragments listed in table 1 were connected in the order as they appear in the human fibrinogen alpha chain sequence to constitute the pCloud sequences.
  • the fragments listed in table 1 were connected in the order that is distinct from they appear in the human fibrinogen alpha chain sequence to generate the pCloud sequence.
  • p285 some fibrinogen alpha chain fragments were utilized more than once.
  • the flexible loops GGGSGGGSGS, GSGSESTSG, GSTSESGSG, GSTSGSESG, GSESGSTSG, GSESTSGSG, GSGSTSESG and GGSGGGSGG listed in table 2 were utilized to connect the human fribrinogen alpha chain fragments.
  • the flexible loops GGGSGGGS, GGSGGGSGG and GGSGSESGSGG were utilized to connect the human fibrinogen alpha chain fragments.
  • the flexible loops GGGSGGGS, GGSGGGSGG and GSGSESGSG were utilized to connect the fibrinogen alpha chain fragments.
  • the genes encoding pCloud sequence (p245, p271 and p285, respectively) and the scaffold protein COL18NC 1 was synthesized, digested by BamHI and Xhol and ligated into the digested vector pET29b-GLP- 1 (A8G/G22E/R36G)-Fn8 -p246-COL 18NC 1 generated before.
  • the resulted vector were named as pET29b-GLP- l (A8G/G22E/R36G)-Fn8-p245-COL18NC l ,
  • GLP- l (A8G/G22E/R36G)-Fn8-p245-COL18NC l ,
  • GLP- 1 A8 G/G22E/R36G-Fn8 -p271 -COL 18NC 1
  • GLP- l A8G/G22E/R36G-Fn8-p285-COL18NC l , respectively.
  • the protein sequences of these fusion proteins were listed as SEQ ID NO:21 -23. These fusion proteins were expressed and purified using the protocols described in example 3.
  • SEQ ID NO: 21 GLP-l (A8G/G22E/R36G)-Fn8-p245-COL18NCl protein sequence, the GLP-1 mutation sites (A8G/G22E/R36G) are in Italic and bold.
  • the flexible loop between GLP1(A8G/G22E/R36G) and Fn8 is in bold.
  • the pCloud sequence p245 is underlined.
  • GLP- l(A8G/G22E/R36G)-Fn8-p271 -COL18NCl protein sequence the GLP-1 mutation sites (A8G/G22E/R36G) are in Italic and bold.
  • the flexible loop between GLP1 (A8G/G22E/R36G) and Fn8 is in bold.
  • the pCloud sequence p271 is underlined.
  • GLP-1 (A8G/G22E/R36G)-Fn8-p285-COL18NCl protein sequence the GLP- 1 mutation sites (A8G/G22E/R36G) are in Italic and bold.
  • the flexible loop between GLP1(A8G/G22E/R36G) and Fn8 is in bold.
  • the pCloud sequence p285 is underlined.
  • Example 13 The therapeutic polypeptide fused with pCloud polypeptides and the scaffold protein exhibits a much larger hydrodynamic radius and/or Rg for its molecular weight
  • GLP-1 -Fn8 is a fusion protein of GLP- 1 and Fn8 and contains the first 144 amino acid residues of the fusion protein GLP- 1 -Fn8-C0L18NC 1 (SEQ ID NO:2).
  • GLP- 1 -Fn8 does not contain the scaffold protein COL 18NC 1 , so it forms a monomer in solution.
  • Fig. 5 showed the chromatography profiles for these fusion proteins.
  • the apparent molecular weights for GLP-1 -Fn8, GLP- 1 -Fn8-C0L18NC 1 , GLP- 1 (A8G/G22E/R36G)-Fn8-p246-COL18NCl showed by gel filtration analysis were ⁇ 20Kd, ⁇ 100Kd and ⁇ 550Kd, respectively.
  • GLP-l (A8G/G22E/R36G)-Fn8-p246-COL18NCl trimer were ⁇ 15Kd, 65Kd and 123Kd, respectively.
  • GLP-1 receptor GLP-1 receptor
  • GLP-1 R Chinese Hamster Ovary (CHO) cells stably transfected with human GLP-1 receptor (GLP-1 R) were generated and named as S-CHO cells.
  • S-CHO cells were propagated in DMEM medium with 10% FCS containing 0.05mg/ml G418. Before analysis, SCHO cells were grown to 70-80% confluence in 6-well plates at 37°C. The cells were treated 0.2mM 3-isobutyl-l -methylxanthine (IBMX).
  • IBMX 3-isobutyl-l -methylxanthine
  • GLP- 1 fusion proteins were incubated with GLP- 1 fusion proteins at various concentrations of InM, 3nM, l OnM, 33nM, lOOnM for 15 min at 37°C. The cells were then lysed by use of cold lysis buffer. The supernatants of the cell extracts were used for cAMP level determinations. The Parameter cAMP ELISA kit from R&D Systems was utilized to measure the cAMP concentrations in the cell lysates. The EC50 values of the GLP- 1 fusion proteins were generated by using the software Origin. The GLP- 1 (7-37) peptide (Anaspec) and BSA were used as positive and negative controls. Fig.
  • Example 15 The pharmacokinetics studies for GLP- 1 -Fn8-COL 18NC 1 ,
  • GLP l (A8G/G22E/R36G)-Fn8-p246-COL18NCl and GLP l (A8G/G22E/R36G)-Fn8-p285-COL 18NC l
  • Blood samples were taken at various time points after injections such as 0-min, 30-min, 1 -hour, 2-hour, 4-hour,6-hour, 8-hour, 24-hour, 48-hour, 3-day, 4-day, -day, 6-day and 7-day.
  • the serum samples were centrifuged and kept at -80°C freezer.
  • the GLP- 1 concentrations within the samples were examined by use of the sandwich ELISA method.
  • the rabbit polyclonal antibody against human Fibronectin at the concentration of 3ug/ml (Ab299, Abeam company) was coated on ELISA plate for 1 hour at room temperature. Then the plate was washed by PBST buffer three times and the wells were blocked by PBS with 10% FBS for 1 hour at room temperature. The plate was washed three times before the serum samples containing GLP- 1 fusion proteins were added. The serum samples could be diluted to 20-10000 folds before use. The ELISA plate was incubated with the serum samples at room temperature for 1 hour and then washed by PBST buffer five times.
  • mouse monoclonal against human GLP- 1 peptide antibody (sc57510, Santa Cruz Biotich) at the concentration of lug/ml in PBST buffer was added to the wells.
  • the plate was washed extensively after incubation of 1 hour at room temperature.
  • the secondary antibody Goat anti-rabbit IgG HRP conjugated antibody (Beijing ZSGB-Bio company, ZB 5301), was added into the wells and the color was developed using TMB (3,3',5,5 '-tetramethylbenzidine, BD biosciences, Cat 555214 ).
  • the plate reader Bio-Rad microplate reader Model 680 was utilized to obtain the OD450 readings. This method has been calibrated using purified proteins first.
  • Fig. 7 showed the pharmacokinetics profiles of the GLP-1 containing proteins in SD rats by use of the sandwich ELISA method described above.
  • the pharmacokinetics parameters were obtained by using the WinNonlin software (Table 5).
  • the data clearly showed that the GLP- 1 containing fusion proteins generated by use of the method of the invention exhibited much extended in vivo half life possibly due to their enlarged hydrodynamic radius and/or Rg.
  • Particularly, including the pCloud polypeptide into the fusion protein dramatically improved the pharmacokinetic profile of fusion protein.
  • the half life of GLP- 1 -Fn8-C0L 18NC 1 reached 5.3 hours compared with the half life of a couple of minutes for GLP- 1 peptide.
  • GLP 1 A8G/G22E/R36G-Fn8-p246-COLl 8NC 1
  • GLP l A8G/G22E/R36G-Fn8-p285-COL 18NC l were further extended to 30.8 and 31.2 hours, respectively, due to the applications of the flexible unstructured pCloud sequences within the fusion proteins.
  • the fusion protein GLP l (A8G/G22E/R36G)-Fn8-p246-COL18NC l was administrated into the cynomolgus monkeys by subcutaneous injection.
  • the fusion protein was administered on three cynomolgus monkeys at the dose of 5 nmol/kg.
  • Blood samples were taken at various time points after injections such as 0-min, , 2-hour, 4-hour, 6-hour, 8-hour, 24-hour, 48-hour, and daily until the 15th day.
  • the serum samples were centrifuged and kept at -80°C freezer.
  • the concentrations of the fusion protein within the serum samples were examined by using the sandwich ELISA method described above.
  • the pharmacokinetics profile of the fusion protein GLPl (A8G/G22E/R36G)-Fn8-p246-COL18NCl in cynomolgus monkeys was shown in Fig. 8.
  • the pharmacokinetics parameters were obtained by using the WinNonlin software (Table 5).
  • the half life of the fusion protein GLPl (A8G/G22E/R36G)-Fn8-p246-COL18NCl in in cynomolgus monkeys was estimated to be -67 hours, which is significantly longer than the half life of GLP- 1 -Fc fusion protein (half life -51 hours) in monkeys [6].
  • the pharmacokinetics data in rats and monkeys strongly suggested that a weekly dose or even once every ten day of GLP l (A8G/G22E/R36G)-Fn8-p246-COL18NCl in human may be effective.
  • the serum samples of the cynomolgus monkeys receiving the fusion protein GLP l (A8G/G22E/R36G)-Fn8-p246-COL18NC l were withdrawn one month after dosing to examine whether specific antibody against GLPl (A8G/G22E/R36G)-Fn8-p246-COL 18NC l has been generated.
  • the analysis of the serum samples of the three monkeys receiving the fusion protein by use of ELISA method indicated that no specific antibody was induced against GLP l (A8G/G22E/R36G)-Fn8-p246-COL18NC l in any of the cynomolgus monkeys.
  • GLP 1 A8 G/G22E/R36G-Fn8 -p246-COL 18NC 1
  • GLP l A8G/G22E/R36G-Fn8-p285-COL18NCl were shown in abbreviations as NC I , p246-NC l and p285-NC l .
  • Example 16 the Intraperitoneal glucose tolerance test (IPGTT) of GLPl (A8G/G22E/R36G)-Fn8-p246-COL18NC l in SD rats.
  • IPGTT Intraperitoneal glucose tolerance test
  • GLPl A8G/G22E/R36G-Fn8-p246-COL 18NC l to reduce the glucose level in animal models.
  • IPGTT Intraperitoneal glucose tolerance test
  • the fusion protein GLPl (A8G/G22E/R36G)-Fn8-p246-COL18NC l was administered on SD(Sprague-Dawley) rats by intraperitoneal injections at the dose of 10 nmol/kg and 20 nmol/kg for animals, respectively.
  • Glucose was injected into the animals with the dose of 2g/kg at 8hours, 32 hours and 100 hours after the injection of the fusion protein
  • GLPl (A8G/G22E/R36G)-Fn8-p246-COL18NC l Blood samples were taken at various time points after injections such as 0-min, 10-min, 20-min,30-min, 60-min and 120-min. The glucose levels within the blood samples were measured using a Accu-Chek Performa blood glucose meter (Roche) immediately.
  • Fig. 9a the rats were injected with GLPl (A8G/G22E/R36G)-Fn8-p246-COL18NC l at the dose of l Onmol/kg and 20nmol/kg about 8 hours before the IPGTT experiments were conducted. The data showed in Fig.
  • Fig. 9a indicated that the fusion protein GLP l (A8G/G22E/R36G)-Fn8-p246-COL18NC l at the dose of l Onmol/kg and 20nmol/kg can efficiently reduce the glucose level.
  • Fig. 9b about 32 hours and 100 hours after the rats were injected with GLPl (A8G/G22E/R36G)-Fn8-p246-COL18NC l at the dose of 20nmol/kg , the IPGTT experiments were conducted. The data in Fig.
  • Example 17 Construction of the fusion protein of Interferon, pCloud polypeptide and the scaffold protein COL 18NC 1
  • the resultant fusion protein was named as IFN-p246-COL18NC l and IFN-p271 -COL 18NC l , respectively.
  • the protein sequences of these fusion proteins were listed as SEQ ID NO: 24 and 25.
  • the Interferon containing fusion proteins were expressed by use of the Pichia expression system (Life Technologies).
  • the genes encoding IFN-p246-COL18NCl and IFN-p271 -COL 18NC l were amplified by PCR and cloned into the expression vector pPICZalphaA(Life Technologies) by use of Xhol and Notl.
  • the protein expression was carried out by following the protocols from Life Technologies.
  • the secreted recombinant proteins were purified using the similar protocol described in example 3.
  • the fusion protein IFN-p246-COL18NC l and IFN-p271 -COL 18NC l can be expressed by use of the E.coli expression system as described in example 3.
  • the Cignal ISRE Luciferase Reporter Assay Kit (Qiagen) was utilized. Hela cells were transfected with vector that contains the interferon stimulated response element (ISRE) reporter. After 16 hours of transfection, medium was changed to assay medium (Opti-MEM + 10% heat inactivated FBS + 0.1 mM NEAA + ImM Sodium pyruvate + 100 U/ml penicillin + 100 ⁇ g/ml streptomycin).
  • SEQ ID NO: 24 protein sequence of IFN-p246-COL18NCl , The pCloud sequence p246 is underlined.
  • Example 18 Construction of the fusion protein of TNFR2, COL 18NC1 and pCloud polypeptide
  • TNF Receptor (TNFR2, or p75) has been fused to IgGl Fc fragment to constitute a fusion protein Etanercept (Enbrel). Etanercept has been successfully utilized to treat severe active rheumatoid arthritis by blocking the TNF alpha functions[5 1 ] .
  • Etanercept has been successfully utilized to treat severe active rheumatoid arthritis by blocking the TNF alpha functions[5 1 ] .
  • TNFR2 we applied the method of the present invention on TNFR2 to generate the fusion protein of TNFR2, COL18NC1 and the pCloud polypeptide.
  • the TNFR2 has been placed at the N-terminus of the scaffold protein COL18NC 1 while the pCloud polypeptide has been positioned at the C-terminus of the scaffold protein COL18NC 1 .
  • the rationale for this design is to make sure that three TNFR2 can properly interact with and block the function of the TNF alpha trimer simultaneously.
  • the resultant TNFR2-COL 18NC 1 -pCloud fusion protein is tri-valent and can block all three binding sites of TNFalpha while retaining a long half life in vivo.
  • one Etanercept molecule can only block two out of three possible binding sites located on TNF alpha homo-trimer.
  • the optimized gene encoding TNFR2 was synthesized and cloned into the pPICZalphaA(Life Technologies) by Xhol and BamHI.
  • the resultant vector is named as pPICZalphaA-TNFR2.
  • the optimized gene encoding COL18NC 1 and p246 was synthesized and cloned into the vector pPICZalphaA-TNFR2 by use of BamHI and Notl.
  • the protein sequence of the resultant fusion protein TNFR2-COL 18NC l -p246 is listed as SEQ ID NO: 26.
  • the optimized gene encoding COL 18NC 1 and p271 was synthesized and cloned into the vector pPICZalphaA-TNFR2 by use of BamHI and Notl.
  • the protein sequence of the resultant fusion protein TNFR2-COL 18NCl -p271 is listed as SEQ ID NO: 27.
  • SEQ ID NO: 26 TNFR2-COL18NC l -p246 protein sequence.
  • the COL18NC1 protein sequence is in italic and bold.
  • the pCloud sequence p246 is underlined.
  • SEQ ID NO: 27 TNFR2-COL18NCl -p271 protein sequence.
  • the COL18NC1 protein sequence is in italic and bold.
  • the pCloud sequence p271 is underlined.
  • the protein expressions of TNFR2-COL18NC l -p246 and TNFR2-COL 18NC l -p271 were carried out by use of the pichia system as described before.
  • the secreted recombinant proteins were purified using the similar protocol described in example 3.
  • the purified protein was kept in 20mM Hepes buffer (pH 7.5), NaCl 150mM.
  • the purity of the fusion protein was examined by SDS-PAGE electrophoresis (purity >95%).
  • the biological activity of TNFR2-COL18NC l -p246 and TNFR2-COL 18NC l -p271 was measured by its ability to block the TNF-alpha signaling.
  • the positive control TNFR2-Fc fusion protein (R&D systems) can efficiently block the apoptosis of L-929 mouse fibroblast cells induced by TNF-alpha (0.25 ng/mL) in the presence of actinomycin D.
  • the ED50 of TNFR2-Fc fusion protein was shown to be ⁇ 5ng/ml using the abovementioned assay.
  • Our data indicated that TNFR2-COL 18NC l -p246 and TNFR2-COL 18NC l -p271 can inhibit the cell killing activity of TNF alpha for L929 cells with the ED50 of l -5ng/ml using the same assay. This suggested that TNFR2-COL18NC l -p246 and TNFR2-COL18NC l -p271 can function as efficiently as TNFR2-IgGl Fc fusion protein in the in vitro studies.
  • Example 19 Construction of the fusion protein containing VEGFRl R2, COL18NC 1 and pCloud sequences
  • VEGF Vascular endothelial growth factor
  • VEGFRl R2 vascular endothelial growth factor receptor 1
  • VEGFR2 vascular endothelial growth factor receptor 2
  • the synthetic gene that encodes the human VEGFRl R2 was grafted into the digested vectors pPICZalphaA-TNFR2-COL 18NC l -p246 and pPICZalphaA-TNFR2-COL18NC l -p271 generated in example 14 by use of Xhol and BamHI.
  • the resultant fusion proteins were named as VEGFRl R2-COL18NC l -p246 and VEGFRlR2-COL18NC l -p271 and their protein sequences were listed as SEQ ID NO: 28 and 29.
  • VEGFRlR2-COL18NCl-p246 and VEGFRlR2-COL18NCl-p271 were carried out by use of the pichia system as described in example 13.
  • the secreted recombinant proteins were purified using the similar protocol described in example 3.
  • the purified protein was kept in 20mM Hepes buffer (pH 7.5), NaCl 150mM.
  • the purity of the fusion protein was examined by SDS-PAGE electrophoresis (purity >95%).
  • the biological activities of VEGFRlR2-COL18NCl-p246 and VEGFRlR2-COL18NCl-p271 were shown by its ability to interact with VEGF.
  • VEGFRlR2-COL18NCl-p246 and VEGFRlR2-COL18NCl-p271 fusion proteins can bind VEGF with the similar affinity as the VEGFRlR2-IgG Fc fusion protein.
  • SEQ ID NO: 28 VEGFRlR2-COL18NCl-p246 fusion protein sequence.
  • the COL18NC1 protein sequence is in italic and bold.
  • the pCloud sequence p271 is underlined.
  • Example 20 Cloning and expression of Exenatide (EX) fused with Fn8, pCloud sequence and human collagen XVIII NC I (COL 18NC 1 )
  • Exenatide (INN, marketed as Byetta, Bydureon) is a glucagon-like peptide- 1 agonist (GLP-1 agonist) medication, belonging to the group of incretin mimetics, approved in April 2005 for the treatment of type II diabetes mellitus.
  • GLP-1 agonist glucagon-like peptide- 1 agonist
  • Exenatide and Fn8 was synthesized, digested by Ndel and BamHI and ligated into the digested vectors pET29b-GLP- 1 (A8G/G22E/R36G)-Fn8-p246-COL 18NC 1 and pET29b-GLP- 1 (A8G/G22E/R36G)-Fn8-p271 -COL 18NC 1 generated in examples 4 and 9.
  • the resulted fusion proteins were named as EX-Fn8-p246-COL18NC l and EX-Fn8-p271 -COL18NCl and their protein sequences were listed as SEQ ID NO: 30 and 31.
  • EX-Fn8-p246-COL 18NC l and EX-Fn8-p271 -COL 18NC l were carried out as described in example 3.
  • the biological activities of the fusion proteins could be measured by use of the cAMP assay described in example 1 1.
  • EX-Fn8-p246-COL18NCl protein sequence the Exenatide sequence is in Italic.
  • the flexible loop between EX and Fn8 is in bold.
  • the pCloud sequence p246 is underlined.
  • SEQ ID NO: 31 EX-Fn8-p271-COL18NCl protein sequence, the Exenatide sequence is in Italic.
  • the flexible unstructured linker between EX and Fn8 is in bold.
  • the pCloud sequence p271 is underlined.
  • NCI domain trimer Structure, 2002.10(2): p. 165-73.

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Abstract

La présente invention concerne une protéine hybride comprenant un polypeptide thérapeutique hybridé avec un ou plusieurs polypeptides non-structurés flexibles et une protéine trimère de structure. La séquence polypeptidique non-structurée flexible au sein de la protéine hybride est présentée comme une ou plusieurs des séquences pCloud dérivées de la chaîne alpha d'un fibrinogène humain, et peut être entourée d'une fraction de liaison protéique d'origine humaine. L'invention concerne également des compositions pharmaceutiques comprenant la protéine hybride, des molécules d'acides nucléiques codant pour la protéine hybride, des vecteurs contenant les acides nucléiques, des cellules hôtes transformées avec les vecteurs, et des procédés de fabrication des protéines hybrides selon l'invention, et leur utilisation.
PCT/CN2013/001602 2012-12-24 2013-12-19 Protéine hybride d'un polypeptide thérapeutique présentant un profil pharmacocinétique amélioré, et son utilisation WO2014101287A1 (fr)

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US14/655,282 US10246503B2 (en) 2012-12-24 2013-12-19 Method of improving the pharmacokinetic profile of a therapeutic polypeptide and the use thereof
EP13869513.5A EP2935338A4 (fr) 2012-12-24 2013-12-19 Protéine hybride d'un polypeptide thérapeutique présentant un profil pharmacocinétique amélioré, et son utilisation
CN201380067873.8A CN104870478B (zh) 2012-12-24 2013-12-19 具有改善的药代动力学性质的治疗性多肽融合蛋白及其应用
PCT/CN2014/094429 WO2015090234A1 (fr) 2013-12-19 2014-12-19 Amélioration du profile pharmacocinétique d'un polypeptide inhibant l'angiopoïetine-2 ou la thymalfasine

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Cited By (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2015090234A1 (fr) * 2013-12-19 2015-06-25 Beijing Anxinhuaide Biotech. Co., Ltd Amélioration du profile pharmacocinétique d'un polypeptide inhibant l'angiopoïetine-2 ou la thymalfasine
US9376672B2 (en) 2009-08-24 2016-06-28 Amunix Operating Inc. Coagulation factor IX compositions and methods of making and using same
US10370430B2 (en) 2012-02-15 2019-08-06 Bioverativ Therapeutics Inc. Recombinant factor VIII proteins
US10421798B2 (en) 2012-02-15 2019-09-24 Bioverativ Therapeutics Inc. Factor VIII compositions and methods of making and using same
US10548953B2 (en) 2013-08-14 2020-02-04 Bioverativ Therapeutics Inc. Factor VIII-XTEN fusions and uses thereof
CN111153965A (zh) * 2018-11-07 2020-05-15 浙江道尔生物科技有限公司 用于改善活性蛋白或多肽性能的人工重组蛋白及其应用
US10676733B2 (en) 2014-10-28 2020-06-09 Merck Patent Gmbh Methods for non-covalent Fc-domain-containing protein display on the surface of cells and methods of screening thereof
US10745680B2 (en) 2015-08-03 2020-08-18 Bioverativ Therapeutics Inc. Factor IX fusion proteins and methods of making and using same
US11066465B2 (en) 2015-12-30 2021-07-20 Kodiak Sciences Inc. Antibodies and conjugates thereof
US11155610B2 (en) 2014-06-28 2021-10-26 Kodiak Sciences Inc. Dual PDGF/VEGF antagonists
US11912784B2 (en) 2019-10-10 2024-02-27 Kodiak Sciences Inc. Methods of treating an eye disorder

Families Citing this family (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110092835A (zh) * 2018-01-30 2019-08-06 上海惠盾生物技术有限公司 一种glp-1类似物-col3a1融合蛋白
CA3179246A1 (fr) * 2020-05-19 2021-11-25 Yves Durocher Utilisation de resistine en tant que partenaire de trimerisation pour l'expression de proteines trimeriques
CN117794908A (zh) 2021-08-02 2024-03-29 巴斯夫欧洲公司 (3-喹啉基)-喹唑啉
CN114644717B (zh) * 2022-04-15 2024-04-26 江苏农牧科技职业学院 一种重组人胰高糖素样肽-1及其构建方法和应用

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5961973A (en) * 1992-03-06 1999-10-05 Crea; Roberto Pathogen-targeted biocatalysts
EP2441776A1 (fr) * 2010-10-15 2012-04-18 Leadartis, S.L. Génération de complexes polyvalents et multifonctionnels avec un domaine de trimérisation XVIII du collagène

Family Cites Families (15)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1017829A2 (fr) * 1997-08-26 2000-07-12 Ariad Gene Therapeutics, Inc. Proteines de fusion a domaine de dimerisation, de trimerisation ou de tetramerisation, et a domaine additionnel d'activation de transcription heterologue, d'inhibition de transcription, de liaison d'adn ou de liaison de ligand
US7090976B2 (en) * 1999-11-10 2006-08-15 Rigel Pharmaceuticals, Inc. Methods and compositions comprising Renilla GFP
DE10122140A1 (de) * 2001-05-08 2002-11-28 Apotech Res & Dev Ltd Rekombinante Fusionsproteine und deren Trimere
US7846445B2 (en) * 2005-09-27 2010-12-07 Amunix Operating, Inc. Methods for production of unstructured recombinant polymers and uses thereof
US7855279B2 (en) * 2005-09-27 2010-12-21 Amunix Operating, Inc. Unstructured recombinant polymers and uses thereof
US10183986B2 (en) * 2005-12-15 2019-01-22 Industrial Technology Research Institute Trimeric collagen scaffold antibodies
EP1894940A1 (fr) * 2006-08-28 2008-03-05 Apogenix GmbH Protéines de fusion de la superfamille TNF
JP2010536341A (ja) * 2007-08-15 2010-12-02 アムニクス， インコーポレイテッド 生物学的に活性なポリペプチドの特性を改変するための組成物および方法
DK2393828T3 (en) * 2009-02-03 2017-01-23 Amunix Operating Inc Extended recombinant polypeptides and compositions comprising same
US20120263701A1 (en) * 2009-08-24 2012-10-18 Volker Schellenberger Coagulation factor vii compositions and methods of making and using same
WO2011123830A2 (fr) * 2010-04-02 2011-10-06 Amunix Operating Inc. Compositions d'alpha 1-antitrypsine, leurs procédés de préparation et d'utilisation
TW201138808A (en) * 2010-05-03 2011-11-16 Bristol Myers Squibb Co Serum albumin binding molecules
EP2420253A1 (fr) * 2010-08-20 2012-02-22 Leadartis, S.L. Synthèse de molécules polyvalentes et multifonctionnelles avec un domaine de trimérisation de collagène XV
CN102153652A (zh) * 2010-12-10 2011-08-17 浙江大学 一种融合蛋白的表达方法及用途
EP3406347A3 (fr) * 2012-02-27 2019-02-13 Amunix Operating Inc. Compositions de conjugués xten et leurs procédés de fabrication

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5961973A (en) * 1992-03-06 1999-10-05 Crea; Roberto Pathogen-targeted biocatalysts
EP2441776A1 (fr) * 2010-10-15 2012-04-18 Leadartis, S.L. Génération de complexes polyvalents et multifonctionnels avec un domaine de trimérisation XVIII du collagène

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
LIU W. ET AL.: "The mechanical properties of single fibrin fibers.", JOURNAL OF THROMBOSIS AND HAEMOSTASIS., vol. 8, no. 5, May 2010 (2010-05-01), pages 1030 - 1036, XP055135666 *
OHASHI T. ET AL.: "An experimental study of GFP-based FRET, with the application to intrinsically unstructured proteins.", PROTEIN SCIENCE, vol. 16, no. 7, July 2007 (2007-07-01), pages 1429 - 1438, XP055270976 *
See also references of EP2935338A4 *

Cited By (13)

* Cited by examiner, † Cited by third party
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US9376672B2 (en) 2009-08-24 2016-06-28 Amunix Operating Inc. Coagulation factor IX compositions and methods of making and using same
US9758776B2 (en) 2009-08-24 2017-09-12 Amunix Operating Inc. Coagulation factor IX compositions and methods of making and using same
US10370430B2 (en) 2012-02-15 2019-08-06 Bioverativ Therapeutics Inc. Recombinant factor VIII proteins
US10421798B2 (en) 2012-02-15 2019-09-24 Bioverativ Therapeutics Inc. Factor VIII compositions and methods of making and using same
US11685771B2 (en) 2012-02-15 2023-06-27 Bioverativ Therapeutics Inc. Recombinant factor VIII proteins
US10548953B2 (en) 2013-08-14 2020-02-04 Bioverativ Therapeutics Inc. Factor VIII-XTEN fusions and uses thereof
WO2015090234A1 (fr) * 2013-12-19 2015-06-25 Beijing Anxinhuaide Biotech. Co., Ltd Amélioration du profile pharmacocinétique d'un polypeptide inhibant l'angiopoïetine-2 ou la thymalfasine
US11155610B2 (en) 2014-06-28 2021-10-26 Kodiak Sciences Inc. Dual PDGF/VEGF antagonists
US10676733B2 (en) 2014-10-28 2020-06-09 Merck Patent Gmbh Methods for non-covalent Fc-domain-containing protein display on the surface of cells and methods of screening thereof
US10745680B2 (en) 2015-08-03 2020-08-18 Bioverativ Therapeutics Inc. Factor IX fusion proteins and methods of making and using same
US11066465B2 (en) 2015-12-30 2021-07-20 Kodiak Sciences Inc. Antibodies and conjugates thereof
CN111153965A (zh) * 2018-11-07 2020-05-15 浙江道尔生物科技有限公司 用于改善活性蛋白或多肽性能的人工重组蛋白及其应用
US11912784B2 (en) 2019-10-10 2024-02-27 Kodiak Sciences Inc. Methods of treating an eye disorder

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