Expression of IL-12 family heterodimers

Patent metadata

Field	Value
Publication number	US-9546202-B2
Application number	US-201414244768-A
Country	US
Kind code	B2
Filing date	Apr 3, 2014
Priority date	May 11, 2008
Publication date	Jan 17, 2017
Grant date	Jan 17, 2017

Abstract

The present invention provides methods of improving the levels and stability of expression of interleukin-12 family cytokine polypeptides by expressing the alpha and beta subunits of the polypeptides at their determined relative molar ratios that increase the levels and stability of expression of the heterodimer, e.g., in comparison to heterodimer expressed at an equimolar ratio.

First claim

What is claimed is:

1. A dual expression vector for expressing a first subunit and a second subunit of an IL-12 family cytokine protein, comprising a first expression cassette for expressing the first subunit and a second expression cassette for expressing the second subunit, wherein the first subunit and the second subunit are expressed from the dual expression vector at a relative ratio other than 1:1, and further, wherein the IL-12 family cytokine protein is IL 23 or IL-27.

2. The dual expression vector of claim 1 , wherein the first subunit and the second subunit are expressed at a ratio in the range of about 3:1 to about 15:1.

3. The dual expression vector of claim 1 , wherein the first subunit is expressed under the control of a human CMV promoter and the second subunit is expressed under the control of a simian CMV promoter.

4. The dual expression vector of claim 2 , wherein the IL-12 family cytokine is IL-23, wherein the first subunit is IL-23 p40 and the second subunit is IL-23 p19.

5. The dual expression vector of claim 4 , wherein the p19 subunit is encoded by a nucleic acid sequence that shares at least 95% nucleic acid sequence identity with SEQ ID NO:26 and the p40 subunit is encoded by a nucleic acid sequence hat shares at least 95% nucleic acid sequence identity with SEQ ID NO:33.

6. The dual expression vector of claim 1 , wherein the IL-12 family cytokine is IL-27.

7. The dual expression vector of claim 1 , wherein the IL-12 family cytokine is IL-27 and further, wherein the p28 subunit is encoded by a nucleic acid sequence that shares at least 95% sequence identity with SEQ ID NO:29 and the EBI3 subunit is encoded by a nucleic acid sequence that shares at least 95% sequence identity with SEQ ID NO:30.

8. A mammalian cell comprising the dual promoter expression vector of claim 1 .

9. A composition comprising the dual expression vector of claim 1 and a pharmaceutically acceptable excipient.

10. A method of promoting the stability and secretion of an IL-27 heterodimer comprised of a p28 subunit and an EBI3 subunit, comprising expressing the p28 and the EBI3 subunit in a relative ratio that is not equimolar.

11. The method of claim 10 , wherein the p28 subunit is expressed from a nucleic acid sequence that shares at least 95% sequence identity with SEQ ID NO:29 and the EBI3 subunit is expressed from a nucleic acid sequence that shares at least 95% sequence identity with SEQ ID NO:30.

12. A method of increasing the levels of expression of an IL-12 family cytokine protein, wherein the IL-12 family cytokine protein comprises an alpha subunit and a beta subunit, the method comprising:
a) determining the ratio of the alpha subunit and the beta subunit that produces an increased level of expression; and
b) expressing the alpha subunit and the beta subunit from a cell at the determined ratio; wherein:
(i) the IL-12 family cytokine is cytokine is IL-12, and the alpha subunit (p35) and the beta subunit (p40) are expressed at a ratio in the range of 1:3 to about 1:15;
(ii) the IL-12 family cytokine is IL-23, and the alpha subunit (p19) and the beta subunit (p40) are expressed at a ratio in the range of about 1:3 to about 1:15; or
(iii) the IL-12 family cytokine is IL-27, and the alpha subunit (p28) and the beta subunit (EBI3) are expressed in non-equimolar amounts.

13. The method of claim 12 , wherein the IL-12 family cytokine is IL-12, and the alpha subunit (p35) and the beta subunit (p40) are expressed at a ratio in the range of 1:3 to about 1:15.

14. The method of claim 12 , wherein the IL-12 family cytokine is IL-23, and the alpha subunit (p19) and the beta subunit (p40) are expressed at a ratio in the range of about 1:3 to about 1:15.

15. The method of claim 12 , wherein the IL-12 family cytokine is IL-27, and the alpha subunit (p28) and the beta subunit (EBI3) are expressed in non-equimolar amounts.

16. The method of claim 12 , wherein the level of expression of the IL-12 family cytokine protein is increased at least 3-fold in comparison to expressing the alpha subunit and the beta subunit at a 1:1 ratio.

Assignees

• Us Health

Inventors

• Felber Barbara K
• Pavlakis George N

Classifications

• C07K14/54

  Interleukins [IL] · CPC title

• C07K14/5434 Primary

  IL-12 · CPC title