Ligation of stapled polypeptides

• US 9,458,189 B2
• Filed: 06/24/2015
• Issued: 10/04/2016
• Est. Priority Date: 07/23/2008
• Status: Active Grant

• Current Assignee: President and Fellows of Harvard College (Harvard Corporation)
• Original Assignee: President and Fellows of Harvard College (Harvard Corporation)
• Inventors: Verdine, Gregory L., Kennedy, Eileen Jeanne
• Primary Examiner(s): Bradley, Christina
• Application Number:
  US14/748,287
• Publication Number:
  US 20150376227A1
• Time in Patent Office: 468 Days
• Field of Search: None
• US Class Current: 1/1
• CPC Class Codes: A61K 38/00 Medicinal preparations containing peptides peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00
• CPC Class Codes: A61K 38/1709 from mammals
• CPC Class Codes: A61K 38/2086 IL-13 to IL-16
• CPC Class Codes: C07K 1/107 by chemical modification of precursor peptides
• CPC Class Codes: C07K 14/4702 Regulators; Modulating activity
• CPC Class Codes: C07K 14/5437 IL-13

Abstract

The present invention provides technology for making large (e.g., greater than 50 amino acids), semi-synthetic, stapled or stitched proteins. The method essentially involves ligating a synthetically produced stapled or stitched peptide to a larger protein. Modified version of IL-13 and MYC are provided as illustrative examples.

Claims

1. A method of preparing a stabilized, non-immunogenic, and folded protein comprising a stitched α

-helical peptide segment, the method comprising steps of;

providing a stitched α

-helical peptide segment, wherein the stitched α

-helical peptide segment is of formula;

2. The method of claim 1, wherein the stitched α

-helical peptide segment is less than 30 amino acids in length.

3. The method of claim 1, wherein the stitched peptide is of formula:

4. The method of claim 1, wherein the stitched peptide is of formula:

5. The method of claim 1, wherein the step of providing a stitched α

-helical peptide segment comprises steps of;

synthetically producing an unstitched peptide segment, wherein the peptide segment comprises unnatural amino acid residues suitable for stitching; and

stitching the unstitched peptide.

6. The method of claim 1, wherein the step of providing a stitched α

-helical peptide segment comprises steps of;

providing a peptide with at least two α

-methyl,α

-alkenylglycine or α

-hydro,α

-alkenylglycine residues and at least one dialkenylglycine residue; and

stitching the peptide using a Grubbs catalyst to form at least two macrocyclic hydrocarbon staples.

7. The method of claim 6, wherein the two α

-methyl,α

-alkenylglycine or α

-hydro,α

-alkenylglycine residues are at positions i and i+3, at positions i and i+4, and/or at positions i and i+7.

8. The method of claim 6, wherein the α

-methyl,α

-alkenylglycine or α

-hydro, α

-alkenylglycine residue is of the formula;

9. The method of claim 1, wherein the second protein is prepared recombinantly.

10. The method of claim 1, wherein the second protein is treated with a protease to yield an N-terminal cysteine.

11. The method of claim 10, wherein the protease is Factor Xa, Tobacco Etch Virus, enterokinase, or ubiquitin C-terminal hydrolase.

12. The method of claim 1, wherein the second protein is produced by having cysteine as the second residue in an expression construct following an N-terminal methionine;

wherein the methionine is processed during protein expression to yield an N-terminal cysteine.

13. The method of claim 1, wherein the step of ligating comprises ligating using Expressed Protein Ligation (EPL).

14. The method of claim 1, wherein the step of ligating produces a scarless protein.

15. The method of claim 1, wherein stitched protein comprises a stitched version of IL-13.

16. The method of claim 15, wherein Helix A of IL-13 is stitched.

17. The method of claim 15, wherein Helix D of IL-13 is stitched.

18. The method of claim 1, wherein the stitched protein comprises a stitched version of c-myc, a stitched version of insulin, a stitched version of a transcription factor, or a stitched version of a cytokine.

19. The method of claim 1, wherein the stitched α

-helical peptide segment is less than 25 amino acids in length.

20. The method of claim 1, wherein the stitched α

-helical peptide segment is less than 20 amino acids in length.

21. The method of claim 1, wherein the stitched α

-helical peptide is a bifunctional peptide.

22. A stitched protein made by the method of claim 1.