Chimeric protein comprising a fibrinogen fragment and a laminin fragment and use thereof

Patent metadata

Field	Value
Publication number	US-2023203131-A1
Application number	US-202117923323-A
Country	US
Kind code	A1
Filing date	May 7, 2021
Priority date	May 8, 2020
Publication date	Jun 29, 2023
Grant date	—

Abstract

The present invention provides a gel formed of fibrin and a molecule generated by thrombin treatment of a chimeric protein that comprises a fibrinogen fragment capable of binding to fibrinogen upon thrombin treatment and a laminin fragment having integrin-binding activity, and optionally further comprises a protein having growth factor-binding activity. The gel of the present invention is suitable as a gel substrate that has properties of the basement membrane and can be used in medical applications.

First claim

1 . A chimeric protein comprising a fibrinogen fragment capable of binding to fibrinogen upon thrombin treatment and a laminin fragment having integrin-binding activity, wherein the laminin fragment is in the form of a heterotrimer comprising C-terminal regions of laminin α, β, and γ chains, wherein the fibrinogen fragment is in the form of a heterotrimer comprising N-terminal regions of fibrinogen Aα, Bβ, and γ chains, and wherein each of the Aα, Bβ, and γ chains of the fibrinogen fragment is fused to any one of the α, β, and γ chains of the laminin fragment in any one of the following combinations (1) to (3):
(1) the fibrinogen Aα chain is fused to the laminin β chain, the fibrinogen Bβ chain is fused to the laminin α chain, and the fibrinogen γ chain is fused to the laminin γ chain;
(2) the fibrinogen Aα chain is fused to the laminin α chain, the fibrinogen Bβ chain is fused to the laminin γ chain, and the fibrinogen γ chain is fused to the laminin β chain; and

(3) the fibrinogen Aα chain is fused to the laminin γ chain, the fibrinogen Bβ chain is fused to the laminin β chain, and the fibrinogen γ chain is fused to the laminin α chain.

2 - 10 . (canceled)

11 . The chimeric protein according to claim 1 , wherein the fibrinogen fragment comprises two fibrinogen fragments, and wherein the two fibrinogen fragments are each in the form of a heterotrimer and associate with each other through their N-terminal regions to form a hexamer.

12 . The chimeric protein according to claim 1 , further comprising a protein having growth factor-binding activity.

13 . The chimeric protein according to claim 12 , wherein the protein having growth factor-binding activity is a heparan sulfate proteoglycan.

14 . A gel formed of fibrin and a molecule generated by thrombin treatment of the chimeric protein according to claim 1 .

15 . A method for producing a gel, comprising the step of preparing a mixture of the chimeric protein according to claim 1 , fibrinogen, and thrombin.

16 . The method according to claim 15 , wherein the molar ratio of the chimeric protein to the fibrinogen in the mixture is 1:5 to 1:20,000.

17 . A composition for gel preparation comprising the chimeric protein according to claim 1 and fibrinogen.

18 . A kit for preparing a gel, the kit comprising the chimeric protein according to claim 1 , fibrinogen, and thrombin.

19 . A method for three-dimensional culture of cells or tissue fragments comprising culturing cells or tissue fragments using the gel according to claim 14 .

20 . A method for producing cells or organoids for transplantation medicine comprising culturing cells or tissue fragments according to claim 19 under conditions producing cells or organoids for transplantation.

Assignees

• Univ Osaka
• Matrixome Inc

Inventors

• Sekiguchi Kiyotoshi
• Takizawa Mamoru
• Taniguchi Yukimasa

Classifications

• C07K2319/00

  Fusion polypeptide · CPC title

• C07K14/78

  Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG] · CPC title

• C12N5/0062

  General methods for three-dimensional culture · CPC title

• C12N2513/00

  3D culture · CPC title

• C12N5/0068 Primary

  General culture methods using substrates (for specific animal cell type C12N5/06) · CPC title