Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Patent
1994-09-29
1997-07-15
Draper, Garnette D.
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
435 694, 4352523, 4353201, 536 235, 530350, 530395, 530399, C12P 2106, C12N 120, C07K 100
Patent
active
056482337
DESCRIPTION:
BRIEF SUMMARY
FIELD OF THE INVENTION
This invention is related to genetically engineered modified TCFs which contain different number of N-linked oligosaccharide chains compared with wild type TCF, and have new amino-acid sequences. These modified TCFs obtained in this invention have longer serum half-lives, have growth stimulating activities for hepatocytes and cytotoxic activities against tumor cells. Therefore, the modified TCFs are useful as therapeutic agents against liver diseases and as anti-cancer drugs.
BACKGROUND OF THE INVENTION
TCF-II, tumor cytotoxic factor derived from human fibroblast, is a novel anti-tumor protein which is different from any other proteins so far reported. The inventors succeeded in cDNA cloning of this protein, deduced its entire amino-acid sequence and confirmed the usefulness. This novel anti-tumor protein and its cDNA were disclosed in W090/10651. They were designated as TCF-II. In this invention, the glycoprotein which has the amino-acid sequence disclosed in W090/10651 is called TCF. TCF is a substance which has been called TCF-II.
TCF has both strong cytotoxic activity against tumor cells and growth stimulating activity for normal cells. And it has been confirmed that TCF is member of a family including HGF, a growth factor for hepatocytes. The. molecular weight of TCF is 78000.+-.2000 daltons and/or 74000.+-.2000 daltons on SDS polyacrylamide gel electrophoresis. Under reducing conditions, it showed a polypeptide band called A chain with a molecular mass of 52000.+-.2000 daltons and two polypeptide bands called B chain and/or C chain with molecular masses of 30000.+-.2000 daltons and/or 26000.+-.2000 daltons, respectively.
Because TCF is a growth factor for hepatocytes, application to liver regeneration after hepatectomy has been examined. Since biological half-life of TCF is very short, attempts have been made to obtain more effective modified TCFs with prolonged biological half-lives.
Relationship between oligosaccharide chains and serum half-lives has been investigated in some glycoproteins including erythropoietin. The investigations have demonstrated that glycoproteins with slightly different structure in oligosaccharide chains and with different biological activities can be synthesized from the same gene. It was known that glycosylated erythropoietin is different in the biological activities from non-glycosylated one. However, about the relationship between oligosaccharide chains and biological activities of TCF little was known.
DISCLOSURE OF THE INVENTION
The present inventors took notice of the usefulness of TCF, and investigated application of TCF to the treatment of tumors or liver diseases and utilization as diagnostic markers of diseases. TCF has a very short half-life of approximately 2 minutes. To obtain modified proteins with prolonged biological half-lives, the inventors constructed several genetically engineered TCFs with mutations in the polypeptide moiety and analyzed them. However, most of the modified proteins appeared to lose the biological activities. Then, to prolong the biological half-life without loss of the biological activity; the inventors paid attention to the four N-linked oligosaccharide chains attached to TCF and attempted to construct the new modified TCFs which have a deletion of one or more oligosaccharide chains.
The theme of this invention is to present the new modified TCFs which have different amino-acid sequences compared to the wild-type TCF, and decreased number of N-linked oligosaccharide chains and longer biological half-lives than the wild-type TCF. These modified TCFs can be obtained by altering the nucleotide sequence of TCF cDNA encoding the amino acid residues responsible for N-glycosylation and by expressing the genetically mutagenized TCF cDNAs. Modified TCFs presented by the invention have one or more deletion(s) of the four N-linked oligosaccharide chains present in the wild-type TCF. These are the first modified TCFs with prolonged biological half lives without any loss of the biological activities, obtained by alter
REFERENCES:
Goto, M. et al.; "Production of Recombinant Human Erythropoietin in Mammalian Cells: Host-Cell Dependency of the Biological Activity of the Cloned Glycoprotein". Bio/Technology, vol. 6, pp. 67-71, Jan. 1988.
Hofmann, R. et al., "Scatter Factor is a Glycoprotein but Glycosylation is not Required for its Activity", Biochimica et Biophysica Acta, 1120, 343-350 (1992).
Yamaguchi et al, 1991 JBC vol. 266 No. 30 pp. 20434-20439.
Dube et al, 1988 vol. 263 No. 33 JBC pp. 17516-17521.
Shima et al 1991 vol. 180 No. 2 Biochem & Biophy Res Comm pp. 1151-1158.
Weidner et al 1991 vol. 88 PNAS USA pp. 7001-7005.
Rubin et al 1991 vol. 88 pp. 415-419 PNAS.
Goto Masaaki
Higashio Kanji
Masunaga Hiroaki
Murakami Akihiko
Oogaki Fumiko
Draper Garnette D.
Snow Brand Milk Products Co. Ltd.
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