WO2015090234A1 - Amélioration du profile pharmacocinétique d'un polypeptide inhibant l'angiopoïetine-2 ou la thymalfasine - Google Patents

Amélioration du profile pharmacocinétique d'un polypeptide inhibant l'angiopoïetine-2 ou la thymalfasine Download PDF

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WO2015090234A1
WO2015090234A1 PCT/CN2014/094429 CN2014094429W WO2015090234A1 WO 2015090234 A1 WO2015090234 A1 WO 2015090234A1 CN 2014094429 W CN2014094429 W CN 2014094429W WO 2015090234 A1 WO2015090234 A1 WO 2015090234A1
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fusion protein
thymalfasin
pcloud
protein
scaffold
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PCT/CN2014/094429
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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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Priority claimed from PCT/CN2013/001602 external-priority patent/WO2014101287A1/fr
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Publication of WO2015090234A1 publication Critical patent/WO2015090234A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C07K14/57581—Thymosin; Related peptides
    • 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
    • C07K14/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • the present invention relates to a fusion protein containing an angiopoietin-2 inhibiting polypeptide or Thymalfasin with improved therapeutic efficacy.
  • the present invention relates to a fusion protein comprising an angiopoietin-2 inhibiting polypeptide or Thymalfasin fused to a proteinous connecting moiety of human origin, a pCloud sequence and a trimeric scaffold protein.
  • the recombinant fusion proteins of the present invention can be expressed in soluble form by use of prokaryotic system and exhibits the increased plasma half-life and stability compared to the angiopoietin-2 inhibiting polypeptide or Thymalfasin by itself.
  • Angiopoietins form a family of vascular growth factors that play a role in embryonic and postnatal angiogenesis. Angiopoietin signaling most directly corresponds with angiogenesis through the cell surface receptors Tie1 and Tie2 [1, 2] .
  • Angiopoietin-2 (Ang2) are ligands of the endothelial cell receptor Tie2 and have crucial roles in the tumour angiogenic switch [1] . Increased expression of Ang2 in tumors correlates with poor prognosis [3] .
  • Thymalfasin (Thymosin alpha 1) is a 28-amino acid polypeptide and the synthetic Thymalfasin has been approved for clinical uses to boost immune responses.
  • Thymalfasin can influence T-cell production and maturation, stimulate production of Th1 cytokines such as interferon-gamma and interleukin-2, and activate natural killer cell-mediated cytotoxicity [5, 6] .
  • the half life of Thymalfasin in human is about one and a half hours, which requires Thymalfasin to be injected every two days. Improving the pharmacokinetic profile of Thymalfasin can make it more convenient for the patients.
  • fusion of a therapeutic polypeptide with human IgG Fc fragment or human serum albumin may significantly increase the half life of the therapeutic polypeptide [7] .
  • recombinant fusion protein with IgG Fc fragment or HSA usually 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 IgG Fc or HSA fusion protein can be generated in the inclusion body from the E. coli expression system. Time-consuming refolding procedure has to be carried out for the fusion protein to gain biological activity.
  • fusion protein comprising an angiopoietin-2 inhibiting polypeptide or Thymalfasin fused to a proteinous connecting moiety of human origin, a pCloud sequence and a trimeric scaffold protein.
  • the recombinant fusion proteins of the present invention can be expressed in soluble form to large quantities by use of prokaryotic system. These fusion proteins exhibit increased plasma half-life and stability compared to the angiopoietin-2 inhibiting polypeptide or Thymalfasin by itself.
  • a method termed as “Trident technology” has been described in PCT/CN2013/001602 for improving the pharmacokinetic properties of a therapeutic polypeptide, such as in vivo terminal half-life.
  • the method has been applied to the angiopoietin-2 inhibiting polypeptide or Thymalfasin to extend the in vivo half life for these two peptides.
  • the recombinant fusion proteins of the present invention can be expressed in soluble form to large quantities by use of prokaryotic system, which renders the lower manufacturing cost and more convenient purification process compared with other types of fusion protein such as Fc fusion and BSA fusion.
  • the present invention provides a fusion protein comprising an angiopoietin-2 inhibiting polypeptide or Thymalfasin fused to a proteinous connecting moiety (PCM) of human origin, an un-structured pCloud sequence and a trimeric scaffold protein.
  • the proteinous connecting moiety is a proteinous sequence having an elongated shape, such as a human Fibronectin type III domain.
  • the therapeutic polypeptide is connected with a proteinous connecting moiety (PCM) of human origin, an un-structured pCloud sequence and a trimeric scaffold protein.
  • PCM proteinous connecting moiety
  • This method 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 angiopoietin-2 inhibiting polypeptide or Thymalfasin with tri-valency, which may greatly enhance the potency and efficacy of the therapeutic polypeptide (reviewed in [8] ) .
  • the fusion protein of the invention comprises, from N-terminus to C-terminus, the angiopoietin-2 inhibiting polypeptide or Thymalfasin; a flexible loop; a proteinous connecting moiety selected from the group consisting of human fibronectin domain 7 (Fn7) , human fibronectin domain 8 (Fn8) , and human Tenascin fibronectin type III domain 3 (TNCfn3) ; a pCloud sequence; and a scaffold protein selected from the group consisting of COL18NC1, COL15NC1, COL19NC2, and ACRP30 C1q-like domain.
  • the fusion protein of the invention comprises, from N-terminus to C-terminus, the angiopoietin-2 inhibiting polypeptide or Thymalfasin, a flexible loop, Fn8, a pCloud sequence, and COL18NC1.
  • 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.
  • Fig. 1 shows the schematic drawing illustrating the fusion proteins of the present invention.
  • the therapeutic polypeptide (the angiopoietin-2 inhibiting polypeptide or Thymalfasin) was shown as a red helix.
  • the therapeutic polypeptide was 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. 2 shows the expression profiles of L17-Fn8-p246-COL18NC1 and Thymalfasin-Fn8-p246-COL18NC1 by 12%SDS-PAGE analysis.
  • lane 1 indicated the molecular weight markers and lane 2 denoted the supernatant portion of L17-Fn8-p246-COL18NC1 expressed in E. coli strain BL21 (DE3) .
  • the soluble L17-Fn8-p246-COL18NC1 accounted for ⁇ 40%of the total soluble proteins.
  • Fig. 2 shows the expression profiles of L17-Fn8-p246-COL18NC1 and Thymalfasin-Fn8-p246-COL18NC1 by 12%SDS-PAGE analysis.
  • lane 1 indicated the molecular weight markers
  • lane 2 denoted the supernatant portion of L17-Fn8-p246-COL18NC1 expressed in E. coli strain BL21
  • lane 1 indicated the molecular weight markers and lane 2 showed the supernatant of Thymalfasin-Fn8-p246-COL18NC1 expressed in E. coli strain BL21 (DE3) .
  • the soluble Thymalfasin-Fn8-p246-COL18NC1 accounted for ⁇ 20%of the total soluble proteins.
  • Fig. 3 shows that L17-Fn8-p246-COL18NC1 can neutralize the interactions between Ang2 and Tie2.0.1ug/ml Ang2 was used to coat the 96 plate for 1hour, then blocked by 1%BSA for 1 hour. 1nM Tie2-Fc was generated in PBS buffer, 0.1%BSA. Various concentrations of L17-Fn8-p246-COL18NC1 (final concentrations of 0pM, 0.1pM, 1pM, 10pM, 30pM, 100pM, 1nM, and 3nM) were added in the Tie2-Fc solution and incubated for 30min. Then 100ul of the mixture was added into the well and incubated for 1 hour.
  • mouse anti-human Fc Antibody-HRP conjugate was used to detect the Tie2-Fc.
  • the assay indicated that the fusion protein L17-Fn8-p246-COL18NC1 could efficiently inhibit the binding between Ang2 and Tie2.
  • Vertical axis indicated the OD450 readings and the horizontal axis showed the concentrations of L17-Fn8-p246-COL18NC1 added in the well.
  • Fig. 4 shows the inhibitory effects of the fusion protein L17-Fn8-p246-COL18NC1 on the growth of Colo205 tumor xenografts in nude mice.
  • Vertical axis indicated the averaged tumor volume (mm 3 ) and the horizontal axis showed the days after the injection of tumor cells into mice.
  • Treatment of L17-Fn8-p246-COL18NC1 at the dose of 2mg/kg was initiated from 3 days post-injection of tumor cells Colo205 into the mice (red curve) .
  • PBS was injected as the control (blue curve) .
  • the treatments were given to the animals once every three days. Tumor volumes were measured every three days after treatments.
  • Fig. 5 shows the pharmacokinetics profile of the purified L17-Fn8-p246-COL18NC1 in Sprague Dawley rats measured by use of the sandwich ELISA method.
  • the vertical axis indicated the measured protein concentration (ug/ml) by use of sandwich ELISA method.
  • the horizontal axis indicated the hours after the injection of the fusion protein.
  • Fig. 6 shows the inhibitory effects of purified Thymalfasin-Fn8-p246-COL18NC1 on human leukemia cell line HL-60 in vitro.
  • Cultured cells (5 ⁇ 10 3 cells/well) were exposed to various concentrations of Thymalfasin-Fn8-p246-COL18NC1 and Thymalfasin as the control (5uM, 10uM, 20uM, 40uM, 80uM, 160uM and 320uM) for 48 hrs.
  • Cell proliferation was determined by the MTT viability assay. The assays were repeated in triplicate.
  • Red curve showedthe data for Thymalfasin-Fn8-p246-COL18NC1 and the blue curve indicated that for Thymalfasin.
  • the horizontal axis showed the concentrations of Thymalfasin-Fn8-p246-COL18NC1 and Thymalfasin.
  • the vertical axis indicated the live cell percentage measured by MTT assay.
  • Fig. 7 shows the pharmacokinetics profile of the purified Thymalfasin-Fn8-p246-COL18NC1 in Sprague Dawley rats measured by use of the sandwich ELISA method.
  • the vertical axis indicated the measured protein concentration (ug/ml) by use of sandwich ELISA method.
  • the horizontal axis indicated the hours after the injection of the fusion protein.
  • polypeptide polypeptide
  • peptide protein
  • proteins polymers of amino acids of any length.
  • 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 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.
  • 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.
  • 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.
  • 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.
  • angiopoietin-2 inhibiting polypeptide by fusing the angiopoietin-2 inhibiting polypeptide to a proteinous connecting moiety of human origin, a pCloud sequence and a trimeric scaffold protein.
  • a method to increase the half life of Thymalfasin by fusing Thymalfasin to a proteinous connecting moiety of human origin, a pCloud sequence and a trimeric scaffold protein.
  • compositions comprising the unstructured “pCloud” polypeptide.
  • the pCloud polypeptide is characterized in: (a) the total pCloud amino acid residues are 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.
  • the pCloud sequence has greater than 90%unstructured random coil formation as determined by GOR algorithm [9] ; and (e) the pCloud sequence does not contain any T-cell epitopes as predicted by TEPITOPE algorithm [10] .
  • the angiopoietin-2 inhibiting polypeptide or Thymalfasin is fused with the proteinous connecting moiety, the pCloud polypeptide and the scaffold protein.
  • a flexible loop may be utilized to fuse the therapeutic polypeptide and the proteinous connecting moiety.
  • the presence of the proteinous connecting moiety in the fusion protein is important because it can significantly enhance the biological activity of the therapeutic polypeptide, e.g. Thymalfasin, as shown in the examples. This is possibly due to the stabilization of the Thymalfasin helix formation.
  • the fusion protein containing the angiopoietin-2 inhibiting polypeptide, the proteinous connecting moiety, the pCloud polypeptides, and the scaffold protein of the present invention is configured, from N-terminus to C-terminus, according to the following formula:
  • AIP is the angiopoietin-2 inhibiting polypeptide
  • pCloud is the pCloud polypeptide defined in PCT/CN2013/001602;
  • Loop is a flexible loop defined above.
  • PCM is the proteinous connecting moiety of human origin.
  • the angiopoietin-2 inhibiting polypeptides have been identified by use of phage display library screening [4, cited patent 2] . These Ang2 inhibiting polypeptides contain ⁇ 25 residues and bind Ang2 with high affinities to block the interactions between Ang2 and Tie2. In the present invention, these Ang2 inhibiting polypeptides have been utilized to construct the fusion proteins as potential cancer therapy.
  • the fusion protein containing Thymalfasin, the proteinous connecting moiety, the pCloud polypeptides, and the scaffold protein of the present invention is configured, from N-terminus to C-terminus, according to the following formula:
  • Thymalfasin is a polypeptide which can be used to boost the immune response in the treatment of bacterial and viral infections and cancer; it has the primary sequence of SDAAVDTSSEITTKDLKEKKEVVEEAEN;
  • pCloud is the pCloud polypeptide defined in PCT/CN2013/001602;
  • Loop is a flexible loop defined above.
  • PCM is the proteinous connecting moiety 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) .
  • the NC1 domain within Multiplexin type of human Collagen (such as collagen XV and XVIII) were selected as the scaffold protein in the present invention.
  • 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 this invention has tri-valency and therefore might behave better than the traditional human monoclonal antibody IgG in interacting with the ligand.
  • the scaffold protein utilized in this invention can form homo-trimers by simultaneous self assembly.
  • the NC1 domain within Multiplexin type of human Collagen such as collagen XV and XVIII
  • the scaffold protein was selected as the scaffold protein.
  • no inter-chain disulfide bonds are needed to drive the trimerization, which makes it more convenient for protein expression.
  • therapeutic monoclonal antibodies of Fc fusion protein rely heavily on the mammalian systems for mass productions or time consuming refolding protocols.
  • 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., a vector that contains the necessary elements for the transcription and translation of the inserted polypeptide coding sequence) by recombinant DNA techniques well known in the art and as detailed below. Methods for producing diverse populations of vectors have been described by Lerner et al., U.S. Pat. No. 6,291,160; 6,680,192.
  • 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.
  • 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 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 active ingredient for administration may be present as a powder or as granules; as a solution, a suspension or an emulsion.
  • a typical daily dose may range from 0.01 mg/kg to 100 mg/kg of the therapeutic agent.
  • Example 1 Construction of an expression vector of Ang2 inhibiting polypeptide L17 fused with human Fibronectin type III domain 8 (Fn8) , pCloud and human collagen XVIII NC1 domain (COL18NC1)
  • an Ang2 inhibiting polypeptide L17 was fused to the human Fibronectin type III domain 8 (Fn8) , pCloud and human collagen XVIII NC1 domain (COL18NC1) to improve the pharmacokinetics profile of L17 (Fig. 1 for schematic drawing) .
  • Fn8 human Fibronectin type III domain 8
  • pCloud human collagen XVIII NC1 domain
  • COL18NC1 human collagen XVIII NC1 domain
  • the resultant vector was named as pET29b-L17-Fn8-p246-COL18NC1.
  • the protein sequence of the fusion protein L17-Fn8-p246-COL18NC1 was listed as SEQ ID NO: 1. From N-terminus to C-terminus, L17-Fn8-p246-COL18NC1 contains L17, a flexible loop, Fn8, pCloud sequence p246 and the scaffold protein COL18NC1.
  • the constructed expression vector pET29b-L17-Fn8-p246-COL18NC1 was used to transform Escherichia coli BL21 (DE3) for protein expression.
  • a single colony was selected from the culture dish, and placed into a 10 ml LB liquid medium with kanamycin (final concentration, 50 ⁇ g/ml) , then shaken at 37 °Cat 220 rpm overnight.
  • 1L LB culture was inoculated and allowed to grown until OD 600 reached 0.4-1.0.
  • Isopropyl thiogalactoside (IPTG) was added to a final concentration of 0.2mM. After a successive culture at 30°Cfor overnight, cells were collected by centrifugation.
  • the cells were diluted 1: 20 with 20mM Tris, NaCl 50mM, 2mM EDTA, pH 8.0, and, after a thorough mix, disrupted by sonication. Insoluble precipitates were removed by centrifugation at 13,000 RCF for 30 min. The proteins of interest were present in the supernatant, with the expressed product comprising 40%of soluble proteins. 50ml of the supernatant was loaded on a HiTrap Q column (5ml) (GE healthcare) . The fusion protein of L17-Fn8-p246-COL18NC1 was eluted with about 0.2 M NaCl in the buffer. The eluted protein was further purified by use of a gel filtration column S-200 (GE Healthcare) , and the buffer was replaced with PBS (pH 7.5) . The final product was confirmed by SDS-PAGE electrophoresis.
  • L17-Fn8-p246-COL18NC1 can be expressed in soluble form to large quantities by use of E. coli expression.
  • the soluble L17-Fn8-p246-COL18NC1 portion accounted for about 40%of the total E. coli protein (Fig. 2a) .
  • the presence of large amount of soluble L17-Fn8-p246-COL18NC1 made the purification process easy and efficient. This is to sharp contrast with the Ang-2 inhibiting peptide fused to Fc, which required the time consuming refolding protocol [4] . Refolding process also greatly reduced the yield for the purified recombinant fusion protein.
  • Example 3 Constructions of the fusion proteins L17-Fn8-p271-COL18NC1 and L17-Fn8-p285-COL18NC1
  • the pCloud sequences with different lengths were introduced into the fusion protein.
  • the synthetic gene encoding the Ang2 inhibiting polypeptide L17 and Fn8 was ligated into the digested vectors pET29b-p271-COL18NC1 and pET29b-p285-COL18NC1.
  • These vectors contain pCloud sequences with different lengths and have been described in PCT/CN2013/001602.
  • the sequences of the resultant fusion proteins L17-Fn8-p271-COL18NC1 and L17-Fn8-p285-COL18NC1 are listed as SEQ ID NO: 2 and 3.
  • the protein expressions and purifications of L17-Fn8-p271-COL18NC1 and L17-Fn8-p285-COL18NC1 were carried out using the similar protocols described in example 2.
  • Example 4 The fusion proteins L17-Fn8-p246-COL18NC1, L17-Fn8-p271-COL18NC1 and L17-Fn8-p285-COL18NC1 can efficiently neutralize the interactions between Ang2 and Tie2
  • Table 1 The EC50 values for the fusion protein to inhibit the binding between Ang2 and Tie2
  • Example 5 The fusion protein L17-Fn8-p246-COL18NC1 can significantly suppress the tumor growth in vivo
  • Fig. 4 the colon cancer cell line Colo205 (Fig. 4) .
  • Nude mice were injected subcutaneously with 0.2 ml of tumor cell suspension in RPMI1640 medium, containing 2 ⁇ 10 6 Colo205 cells.
  • Treatment with fusion protein and PBS as the negative control was performed from day 3 to day 27 post-injection of cells.
  • the fusion protein L17-Fn8-p246-COL18NC1 was administered subcutaneously at the dose of 2mg/kg.
  • the injection was performed once every three days. Tumor volumes were recorded at the interval of 3 days. Tumor volume was calculated as length ⁇ width ⁇ height/2 in mm 3 .
  • Example 6 The pharmacokinetics studies for the fusion protein L17-Fn8-p246-COL18NC1, L17-Fn8-p271-COL18NC1 and L17-Fn8-p285-COL18NC1 in SD rats
  • the fusion protein L17-Fn8-p246-COL18NC1 was administered on Sprague-Dawley (SD) rats by intraperitoneal injections at the doses of 25nmol/kg. Blood samples were taken at various time points after injections. The serum samples were centrifuged and kept at -80°C freezer.
  • the concentration of L17-Fn8-p246-COL18NC1 in the serum samples was examined by use of the sandwich ELISA method.
  • the rabbit polyclonal antibody against human Fibronectin at the concentration of 2ug/ml (Ab299, Abcam company) was coated on ELISA plate for 1.5hour at 37 degree. Then the plate was washed by PBST buffer three times and the wells were blocked by PBST with 5%BSA for 1.5 hour at 37 degree. The plate was washed three times before the serum samples containing the fusion protein were added.
  • the ELISA plate was incubated with serially diluted serum samples and stood at room temperature for 1 hour and then washed by PBST buffer five times.
  • Fig. 5 showed the pharmacokinetics profiles of L17-Fn8-p246-COL18NC1 by use of the sandwich ELISA method described above. Similar PK data have also been generated for the fusion proteins L17-Fn8-p271-COL18NC1 and L17-Fn8-p285-COL18NC1. The PK parameters are listed in Table 2 for the fusion proteins. The data clearly showed that the fusion proteins generated by use of the present invention exhibited much extended in vivo half life and now are suitable for clinical applications.
  • Example 7 Constructions and expressions of the fusion proteins Thymalfasin-Fn8-p246-COL18NC1, Thymalfasin-Fn8-p271-COL18NC1 and Thymalfasin-Fn8-p285-COL18NC1
  • the synthetic gene encoding the Thymalfasin and Fn8 was ligated into the digested vectors pET29b-p246-COL18NC1, pET29b-p271-COL18NC1 and pET29b-p285-COL18NC1.
  • the sequences of the resultant fusion proteins Thymalfasin-Fn8-p246-COL18NC1, Thymalfasin-Fn8-p271-COL18NC1 and Thymalfasin-Fn8-p285-COL18NC1 are listed as SEQ ID NO: 4, 5 and 6.
  • Thymalfasin-Fn8-p246-COL18NC1 The protein expressions and purifications of Thymalfasin-Fn8-p246-COL18NC1, Thymalfasin -Fn8-p271-COL18NC1 and Thymalfasin-Fn8-p285-COL18NC1 were carried out using the similar protocols described in example 2.
  • These fusion proteins can be expressed in soluble forms by use of the E. coli expression system.
  • Fig. 2b showed that the soluble recombinant Thymalfasin-Fn8-p246-COL18NC1 accounted for ⁇ 20%of the total E. coli proteins.
  • Example 8 The fusion proteins Thymalfasin-Fn8-p246-COL18NC1, Thymalfasin-Fn8-p271-COL18NC1 and Thymalfasin-Fn8-p285-COL18NC1 are biologically active to inhibit tumor cell growth in vitro
  • Thymalfasin can inhibit a number of tumor cell growth in vitro [6] .
  • a proliferation assay to test how the purified Thymalfasin-Fn8-p246-COL18NC1 affect the growth for human leukemia cell HL-60 (Fig. 6) .
  • Cultured cells (5 ⁇ 10 3 cells/well) were exposed to various concentrations of Thymalfasin-Fn8-p246-COL18NC1 and Thymalfasin as the control (5uM, 10uM, 20uM, 40uM, 80uM, 160uM and 320uM) for 48 hrs.
  • Cell proliferation was determined by the MTT viability assay.
  • Thymalfasin-Fn8-p246-COL18NC1 can effectively inhibit the tumor cell growth.
  • Similar data have been obtained for the fusion proteins Thymalfasin-Fn8-p271-COL18NC1 and Thymalfasin-Fn8-p285-COL18NC1.
  • the fusion proteins were more potent in suppressing the tumor cell growth than Thymalfasin by itself as shown by the EC50 values in table 3. This is possibly due to the fact that Thymalfasin needs to form a helix to be biologically active. Fusing Thymalfasin with the proteinous connecting moiety Fn8 may stabilize the helix formation and increase the biological activity of the fusion protein.
  • Table 3 The EC50 values for Thymalfasin and Thymalfasin-containing fusion proteins to inhibit the tumor cell HL-60 growth.
  • Thymalfasin-Fn8-p246-COL18NC1 Thymalfasin-Fn8-p271-COL18NC1
  • Thymalfasin-Fn8-p285-COL18NC1 are shown in abbreviations of Tp246, Tp271 and Tp285, respectively.
  • Thymalfasin is shown in TA.
  • Example 9 The pharmacokinetics studies for Thymalfasin-Fn8-p246-COL18NC1, Thymalfasin-Fn8-p271-COL18NC1 and Thymalfasin-Fn8-p285-COL18NC1 in SD rats
  • Thymalfasin-containing fusion proteins generated in the present invention we purified the recombinant fusion proteins of Thymalfasin-Fn8-p246-COL18NC1 in PBS buffer, pH 7.2.
  • the fusion protein was administered into SD (Sprague-Dawley) rats by intraperitoneal injections at the dose of 25 nmol/kg. Blood samples were taken at various time points after injections. The serum samples were centrifuged and kept at -80°C freezer.
  • the fusion protein 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, Abcam 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 PBST with 5%FBS for 1 hour at room temperature. The plate was washed three times before the serum samples were added. The ELISA plate was incubated with the serially diluted serum samples at room temperature for 1 hour and then washed by PBST buffer five times.
  • Fig. 7 showed the pharmacokinetics profile of Thymalfasin-Fn8-p246-COL18NC1 in SD rats by use of the sandwich ELISA method described above.
  • the pharmacokinetics profiles of Thymalfasin-Fn8-p271-COL18NC1 and Thymalfasin-Fn8-p285-COL18NC1 were also obtained.
  • the pharmacokinetics parameters were obtained by using the WinNonlin software (Table 4) .
  • Thymalfasin-Fn8-p246-COL18NC1 Main pharmacokinetic parameters for Thymalfasin-Fn8-p246-COL18NC1, Thymalfasin-Fn8-p271-COL18NC1 and Thymalfasin-Fn8-p285-COL18NC1.
  • Thymalfasin-Fn8-p246-COL18NC1, Thymalfasin-Fn8-p271-COL18NC1 and Thymalfasin-Fn8-p285-COL18NC1 are shown in abbreviations of Tp246, Tp271 and Tp285, respectively.
  • Thymalfasin is shown in TA.
  • the half life T1/2 for Thymalfasin in SD rats is estimated from the T1/2 of Thymalfasin in human (*) .
  • Thymalfasin containing fusion proteins generated in the present invention exhibited much extended in vivo half life compared to Thymalfasin by itself.
  • Thymalfasin containing fusion proteins showed stronger biological activities compared to Thymalfasin (Table 3) .
  • these fusion proteins may represent better alternatives for Thymalfasin for boosting immune responses to treat bacterial and viral infections.

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Abstract

La présente invention concerne une protéine de fusion comprenant un polypeptide inhibant l'angiopoïétine-2 ou de la thymalfasine fusionnée à une fraction de connexion protéique d'origine humaine, une séquence pCloud et une protéine d'échafaudage trimérique. Ce procédé augmente grandement la demi-vie plasmatique et la stabilité du polypeptide inhibant l'angiopoïétine-2 ou la thymalfasine. La protéine de fusion recombinante peut être exprimée sous forme soluble et biologiquement active à l'aide d'un système d'expression procaryote, qui rend le coût de fabrication plus faible et le procédé de purification plus pratique comparé à d'autres types de protéine de fusion tels que la fusion Fc et la fusion BSA.
PCT/CN2014/094429 2013-12-19 2014-12-19 Amélioration du profile pharmacocinétique d'un polypeptide inhibant l'angiopoïetine-2 ou la thymalfasine WO2015090234A1 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107098978A (zh) * 2017-05-05 2017-08-29 中国药科大学 一种抗肿瘤和免疫增强双重功效融合蛋白
WO2021258650A1 (fr) * 2020-06-24 2021-12-30 广州暨南大学医药生物技术研究开发中心有限公司 Procédé de préparation d'une protéine structurale de matrice extracellulaire humaine recombinante

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US5961973A (en) * 1992-03-06 1999-10-05 Crea; Roberto Pathogen-targeted biocatalysts
CN1840682A (zh) * 2005-04-01 2006-10-04 北京诺思兰德生物技术有限责任公司 用基因工程方法大量生产高活性的胸腺素a1
CN101787072A (zh) * 2001-10-11 2010-07-28 安姆根有限公司 人血管生成素-2的特异结合剂
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
WO2014101287A1 (fr) * 2012-12-24 2014-07-03 Beijing Anxinhuaide Biotech. Co., Ltd Protéine hybride d'un polypeptide thérapeutique présentant un profil pharmacocinétique amélioré, et son utilisation

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Publication number Priority date Publication date Assignee Title
US5961973A (en) * 1992-03-06 1999-10-05 Crea; Roberto Pathogen-targeted biocatalysts
CN101787072A (zh) * 2001-10-11 2010-07-28 安姆根有限公司 人血管生成素-2的特异结合剂
CN1840682A (zh) * 2005-04-01 2006-10-04 北京诺思兰德生物技术有限责任公司 用基因工程方法大量生产高活性的胸腺素a1
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
WO2014101287A1 (fr) * 2012-12-24 2014-07-03 Beijing Anxinhuaide Biotech. Co., Ltd Protéine hybride d'un polypeptide thérapeutique présentant un profil pharmacocinétique amélioré, et son utilisation
WO2014100913A1 (fr) * 2012-12-24 2014-07-03 Beijing Anxinhuaide Biotech. Co., Ltd Amélioration de la demi-vie d'un polypeptide thérapeutique par fusion avec une protéine d'échafaudage trimère via un espaceur

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107098978A (zh) * 2017-05-05 2017-08-29 中国药科大学 一种抗肿瘤和免疫增强双重功效融合蛋白
WO2021258650A1 (fr) * 2020-06-24 2021-12-30 广州暨南大学医药生物技术研究开发中心有限公司 Procédé de préparation d'une protéine structurale de matrice extracellulaire humaine recombinante

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