Stable Form Of Signal Converting Protein Fusion Proteins, And Methods Of Use And Preparation Thereof

• US 20150376260A1
• Filed: 12/31/2013
• Published: 12/31/2015
• Est. Priority Date: 01/01/2013
• Status: Active Grant

• Current Assignee: KAHR Medical (2005) Ltd.
• Original Assignee: KAHR Medical (2005) Ltd.
• Inventors: ELHALEL, Michal Dranitzki, SHANI, Noam
• Granted Patent: US 10,040,841 B2
• Time in Patent Office: Days
• US Class Current: 1/1
• CPC Class Codes: C07K 14/70521 CD28, CD152
• CPC Class Codes: C07K 14/70575 NGF/TNF-superfamily, e.g. CD70, CD95L, CD153, CD154 NGF C07K14/48, TNF C07K14/525
• CPC Class Codes: C07K 14/70578 NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95 NGF-receptor C07K14/71, TNF-receptor C07K14/7151
• CPC Class Codes: C07K 2319/00 Fusion polypeptide

Abstract

A stable fusion protein, wherein in solution, a majority of the fusion proteins are in the homo-hexamer form, which may be prepared for example as a CTLA4-FasL fusion protein.

Claims

1. Stable CTLA4-FasL fusion protein, comprising a majority of CTLA4-FasL fusion protein as a homo-hexamer.

16. The protein of claim 1, wherein the homo-hexamer form is the majority form before purification.

17. The protein of claim 1, wherein the homo-hexamer form is the majority form after purification.

30. The method of claim 27 wherein said CTLA4-FasL fusion protein is provided according to any of the above claims.

2. Stable CTLA4-FasL fusion protein, comprising a majority of CTLA4-FasL fusion protein as a multimer of approximately 250 kD in molecular weight.

11. The protein of claim 2, wherein the homo-hexamer form is at least 51% of the total fusion protein.

12. The protein of claim 11, wherein the homo-hexamer form is at least 60% of the total fusion protein.

13. The protein of claim 12, wherein the homo-hexamer form is at least 70% of the total fusion protein.

14. The protein of claim 13, wherein the homo-hexamer form is at least 80% of the total fusion protein.

15. The protein of claim 14, wherein the homo-hexamer form is at least 90% of the total fusion protein.

18. The protein of claim 2, wherein the dodecamer form is no more than 5% of the total fusion protein.

19. The protein of claim 18, wherein the dodecamer form is no more than 4% of the total fusion protein.

20. The protein of claim 19, wherein the dodecamer form is no more than 3% of the total fusion protein.

21. The protein of claim 20, wherein the dodecamer form is no more than 2% of the total fusion protein.

22. The protein of claim 21, wherein the dodecamer form is no more than 1% of the total fusion protein.

23. The protein of claim 2, wherein the dodecamer form is the majority form during purification.

25. A pharmaceutical composition, comprising the CTLA4-FasL fusion protein of claim 2 and a pharmaceutically acceptable carrier.

27. A method of treating cancer in a subject, comprising:

determining whether the cancer expresses B7 receptors; and

if the cancer expresses B7 receptors, administering CTLA4-FasL fusion protein according to claim 2 to the subject.

28. The method of claim 27, wherein said determining whether the cancer expresses FasR and B7 receptors, such that and if the cancer expresses B7 receptors and FasR, administering CTLA4-FasL fusion protein to the subject.

29. The method of claim 28, wherein said administering CTLA4-FasL fusion protein comprises administering a majority of CTLA4-FasL fusion protein as a homo-hexamer to the subject.

3. Stable CTLA4-FasL fusion protein, comprising no more than 5% of CTLA4-FasL fusion protein as a dodecamer.

4. Stable CTLA4-FasL fusion protein, comprising no more than 5% of CTLA4-FasL fusion protein as a multimer of approximately 500 kD in molecular weight.

5. (canceled)

6. A stable CTLA4-FasL fusion protein, wherein in solution, a majority of the fusion proteins are a multimer of approximate 250 kD in molecular weight.

7. (canceled)

8. A method for purifying a CTLA4-FasL fusion protein after expression in cells in cell media, comprising separating the cell media from the cells, the cell media comprising a majority of CTLA4-FasL fusion protein as a multimer of approximate 250 kD in molecular weight.

9. The method of claim 8, further comprising applying the cell media to a Size-Exclusion-Chromatography (SEC) column.

10. The method of claim 9, further comprising applying the cell media to a Concavalin-A (Con A) column before said applying to said SEC column.

24. (canceled)

26. (canceled)

31. (canceled)

32. (canceled)

33. A stable homo-hexamer SCP fusion protein, comprising a Component 1 protein and a Component 2 protein, wherein the Component 1 protein is selected from the group consisting of BTN3A1, CD27, CD80, CD86, ENG, NLGN4X, CD84, TIGIT, CD40, IL-8, IL-10, CD164, LY6G6F, CD28, CTLA4, TYROBP, ICOS, VEGFA, CSF1, VEGFB, BMP2, BMP3, GDNF, PDGFC, PDGFD, TGFB1, LY96, CD96 and GFER;

and wherein the Component 2 protein comprises any TNF-superfamily ligand.

34. The protein of claim 33, wherein the component 2 protein is preferably selected from the group consisting of FasL, TRAIL, TNF-alpha, TNF-beta, OX40L, CD40L, CD27L, CD30L, 4-1BBL, RANKL, TWEAK, APRIL, BAFF, LIGHT, VEGI, GITRL, EDA1/2, Lymphotoxin alpha and Lymphotoxin beta.

35. The protein of claim 34, wherein CTLA4, LY6G6F, CD28, LY96, CD96 and CD247 form disulfide links to form the homo-hexamer.

36. The protein of claim 35, comprising an extracellular domain of the component 1 protein and an extracellular domain of the component 2 protein.

37. The protein of claim 36, having an N-terminal side which is the extracellular domain of the component 1 protein and a C-terminal side which is the extracellular domain of the component 2 protein.

38. The protein of claim 35, wherein the homo-hexamer is formed through the formation of covalent bonds, non-covalent bonds or a combination thereof between portions of the fusion protein.