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Type II TGF-beta receptor/immunoglobulin constant region fusion proteins

A technology of immunoglobulin and fusion protein, which is applied in the field of type II TGF-β receptor/immunoglobulin constant region fusion protein, and can solve problems such as fibrous tissue accumulation

Inactive Publication Date: 2000-06-21
BIOGEN INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Therefore, TGF-β can lead to the accumulation of fibrous tissue

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0194] The invention is illustrated by the following non-limiting examples. Example 1: Construction and expression of soluble rabbit TGFβR: Fc fusion protein

[0195] A full-length clone encoding the rabbit type II TGF-beta (TGFβ) receptor (MIS -3fl1). The extracellular domain comprising the rabbit type II receptor (nucleotides 1312-1995 of SEQ ID NO.: 2) fused to the Fc portion of human IgG1 (nucleotides 1312-1995 of SEQ ID NO.: 2) was constructed as follows 832-1311) recombinant rabbit TGFβRH: Fc fusion gene:

[0196] Fragment 1) A 479bp fragment, containing the extracellular domain of the rabbit type II TGFβ receptor, was extracted from Clone MIS-3f11 was amplified. These oligonucleotides flank the resulting PCR product with Not1 and Sal1 restriction enzyme sites, which are then digested by these two restriction enzymes.

[0197]Fragment 2) pSAB132 (Miller et al., J. Exp. Med. 178:211 [1993]) was digested with the restriction enzyme Notl and the terminal phosphate grou...

Embodiment 2

[0202] Recombinant rabbit TGFβR:Fc fusion gene (SEQ ID NO.: 2 contained in SEQ ID NO.: 1) was transfected into COS cells by electroporation at 280 volts. Transfected cells were grown on DMEM supplemented with 10% fetal bovine serum. After 5 days, the cells were removed from the medium by centrifugation, and the supernatant was detected by SDS / polyacrylamide gel electrophoresis (SDS / PAGE) for the expression of recombinant rabbit TGFβRII:Fc. The ability of TGF[beta]RII:Fc present in cell culture supernatants to bind TGF[beta] was tested. [ 125 I]-TGFβ (NEN Life Science Products) was incubated with conditioned cell culture medium containing rabbit TGFβR:Fc, or control IgG, and protein A-agarose bound to the Fc portion of IgG for 2.5 hours. The resulting protein A:Fc complexes were collected by centrifugation, washed with phosphate-buffered saline, and analyzed by SDS / PAGE and autoradiography. TGFβRII:Fc, but not control IgG was able to immunoprecipitate[ 125 I]-TGFβ. Example...

Embodiment 3

[0202] Recombinant rabbit TGFβR:Fc fusion gene (SEQ ID NO.: 2 contained in SEQ ID NO.: 1) was transfected into COS cells by electroporation at 280 volts. Transfected cells were grown on DMEM supplemented with 10% fetal bovine serum. After 5 days, the cells were removed from the medium by centrifugation, and the supernatant was detected by SDS / polyacrylamide gel electrophoresis (SDS / PAGE) for the expression of recombinant rabbit TGFβRII:Fc. The ability of TGF[beta]RII:Fc present in cell culture supernatants to bind TGF[beta] was tested. [ 125 I]-TGFβ (NEN Life Science Products) was incubated with conditioned cell culture medium containing rabbit TGFβR:Fc, or control IgG, and protein A-agarose bound to the Fc portion of IgG for 2.5 hours. The resulting protein A:Fc complexes were collected by centrifugation, washed with phosphate-buffered saline, and analyzed by SDS / PAGE and autoradiography. TGFβRII:Fc, but not control IgG was able to immunoprecipitate[ 125 I]-TGFβ. Example...

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Abstract

Fusion proteins comprising the TGF-beta Type II receptor linked to a portion of an immunoglobulin constant chain are described. Also described are methods of using the fusion proteins of the invention to treat a variety of fibroproliferative disorders.

Description

Background of the invention [0001] The present invention relates to a soluble TGF-beta receptor fusion protein and its use in the treatment of diseases characterized by excessive production of TGF-beta, including fibroplasia. [0002] background [0003] Transforming growth factor-β (TGF-β) represents a family of polypeptides of which three, TGF-β1, TGF-β2 and TGF-β3, are present in mammals. These factors have overall effects on cell growth and differentiation (Roberts and Sporn (1990) Handbk. Exp. Pharm. 95:419-58). Accumulating evidence indicates that TGF-β also regulates immune processes (Wahl et al. (1989) Immunol. Today 10:258-61). In addition to stimulating the accumulation of immune cells at the site of injury, TGF-β also provides a strong positive feedback for its continuous synthesis (Kim et al. (1990) Molecular Cell Biology (Mol.Cell.Biol.) 10: 1492- 1497). [0004] TGF-β may be a major factor in the etiology of fibroproliferative disorders. TGF-beta is known to...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/09A61K38/00A61P29/00A61P43/00C07K14/71C07K19/00C12N1/15C12N1/19C12N1/21C12N5/10C12P21/02
CPCC07K14/71C07K2319/00A61K38/00A61P13/12A61P17/00A61P19/02A61P21/00A61P25/00A61P27/02A61P29/00A61P43/00C12N15/62C12N15/11
Inventor P·果特瓦斯V·克特里安斯基R·卡特M·萨尼克拉-纳德尔
Owner BIOGEN INC
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