Compositions and methods for selective protein degradation

Patent metadata

Field	Value
Publication number	US-11999802-B2
Application number	US-201816757026-A
Country	US
Kind code	B2
Filing date	Oct 18, 2018
Priority date	Oct 18, 2017
Publication date	Jun 4, 2024
Grant date	Jun 4, 2024

Abstract

The invention provides compositions including a fusion polypeptide and methods for making a fusion polypeptide that includes a COF1/CRBN-binding polypeptide, COF2/CRBN-binding polypeptide, or COF3/CRBN-binding polypeptide and a heterologous polypeptide of interest.

First claim

What is claimed is:

1. A fusion polypeptide comprising:
(i) a CRBN (cereblon)-binding polypeptide, and
(ii) a chimeric antigen receptor (CAR) that comprises, in a N-terminal to C-terminal direction, an antigen binding domain, a transmembrane domain, and one or more intracellular signaling domains, wherein the CRBN-binding polypeptide comprises the amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 1.

2. The fusion polypeptide of claim 1 , wherein the CRBN-binding polypeptide is fused to the CAR.

3. The fusion polypeptide of claim 1 , wherein the association of CRBN with the CRBN-binding polypeptide or the fusion polypeptide in the absence of lenalidomide or pomalidomide, or a pharmaceutically acceptable salt thereof, is no more than 20% of the association of CRBN with the CRBN-binding polypeptide or the fusion polypeptide in the presence of lenalidomide or pomalidomide, or a pharmaceutically acceptable salt thereof.

4. The fusion polypeptide of claim 1 , wherein the degradation or ubiquitination of the fusion polypeptide in the absence of lenalidomide or pomalidomide, or a pharmaceutically acceptable salt thereof, is no more than 70% of the degradation or ubiquitination of the fusion polypeptide in the presence of lenalidomide or pomalidomide, or a pharmaceutically acceptable salt thereof.

5. The fusion polypeptide of claim 1 , wherein the fusion polypeptide further comprises a degradation domain, wherein the degradation domain is separated from the CRBN-binding polypeptide and the CAR by a heterologous protease cleavage site.

6. The fusion polypeptide of claim 5 , wherein the fusion polypeptide comprises, from N-terminus to C-terminus:
i) the degradation domain, the heterologous protease cleavage site, the CAR, and the CRBN-binding polypeptide;
ii) the degradation domain, the heterologous protease cleavage site, the CRBN-binding polypeptide, and the CAR;
iii) the CRBN-binding polypeptide, the CAR, the heterologous protease cleavage site, and the degradation domain; or
iv) the CAR, the CRBN-binding polypeptide, the heterologous protease cleavage site, and the degradation domain.

7. A composition comprising the fusion polypeptide of claim 1 , and a pharmaceutically acceptable carrier, excipient or stabilizer.

8. The fusion polypeptide of claim 1 , wherein the association of CRBN with the CRBN-binding polypeptide or the fusion polypeptide in the absence of lenalidomide or pomalidomide, or a pharmaceutically acceptable salt thereof, is no more than 10% of the association of CRBN with the CRBN-binding polypeptide or the fusion polypeptide in the presence of lenalidomide or pomalidomide, or a pharmaceutically acceptable salt thereof.

9. The fusion polypeptide of claim 1 , wherein the association of CRBN with the CRBN-binding polypeptide or the fusion polypeptide in the absence of lenalidomide or pomalidomide, or a pharmaceutically acceptable salt thereof, is no more than 5% of the association of CRBN with the CRBN-binding polypeptide or the fusion polypeptide in the presence of lenalidomide or pomalidomide, or a pharmaceutically acceptable salt thereof.

10. The fusion polypeptide of claim 1 , wherein the association of CRBN with the CRBN-binding polypeptide or the fusion polypeptide in the absence of lenalidomide or pomalidomide, or a pharmaceutically acceptable salt thereof, is no more than 1% of the association of CRBN with the CRBN-binding polypeptide or the fusion polypeptide in the presence of lenalidomide or pomalidomide, or a pharmaceutically acceptable salt thereof.

11. The fusion polypeptide of claim 1 , wherein the degradation or ubiquitination of the fusion polypeptide in the absence of lenalidomide or pomalidomide, or a pharmaceutically acceptable salt thereof, is no more than 50% of the degradation or ubiquitination of the fusion polypeptide in the presence of lenalidomide or pomalidomide, or a pharmaceutically acceptable salt thereof.

12. The fusion polypeptide of claim 1 , wherein the degradation or ubiquitination of the fusion polypeptide in the absence of lenalidomide or pomalidomide, or a pharmaceutically acceptable salt thereof, is no more than 10% of the degradation or ubiquitination of the fusion polypeptide in the presence of lenalidomide or pomalidomide, or a pharmaceutically acceptable salt thereof.

13. The fusion polypeptide of claim 1 , wherein the degradation or ubiquitination of the fusion polypeptide in the absence of lenalidomide or pomalidomide, or a pharmaceutically acceptable salt thereof, is no more than 1% of the degradation or ubiquitination of the fusion polypeptide in the presence of lenalidomide or pomalidomide, or a pharmaceutically acceptable salt thereof.

14. The fusion polypeptide of claim 1 , wherein the CRBN-binding polypeptide comprises the amino acid sequence of SEQ ID NO: 1.

15. The fusion polypeptide of claim 1 , wherein the CRBN-binding polypeptide consists of the amino acid sequence of SEQ ID NO: 1 or 3.

16. The fusion polypeptide of claim 5 , wherein the degradation domain is an estrogen receptor (ER) domain.

17. The fusion polypeptide of claim 5 , wherein
the degradation domain is an ER domain and comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 46 or 48.

18. The fusion polypeptide of claim 1 , wherein the CRBN-binding polypeptide is linked to the CAR by a peptide bond.

19. The fusion polypeptide of claim 1 , wherein the CRBN-binding polypeptide is linked to the CAR by a bond other than a peptide bond.

20. The fusion polypeptide of claim 1 , wherein the CAR is linked directly to the CRBN-binding polypeptide.

21. The fusion polypeptide of claim 1 , wherein the CAR is linked indirectly to the CRBN-binding polypeptide.

22. The fusion polypeptide of claim 1 , wherein the CRBN-binding polypeptide is operatively linked to the CAR via a linker, a glycine serine linker, or a linker comprising the amino acid sequence of SEQ ID NO: 28.

23. The fusion polypeptide of claim 5 , wherein the heterologous protease cleavage site is cleaved by a protease selected from the group consisting of furin, PCSK1, PCSK5, PCSK6, PCSK7, cathepsin B, Granzyme B, Factor XA, Enterokinase, genenase, sortase, precission protease, thrombin, TEV protease, and elastase 1.

24. The fusion polypeptide of claim 5 , wherein the heterologous protease cleavage site comprises a sequence having a cleavage motif selected from the group consisting of Arg-X-Lys/Arg-Arg consensus motif (X can be any amino acid; SEQ ID NO: 52), Arg-X-X-X-Lys/Arg-Arg consensus motif (X can be any amino acid; SEQ ID NO: 53), Arg-Arg-X consensus motif (SEQ ID NO: 54), Ile-Glu-Pro-Asp-X consensus motif (SEQ ID NO: 55), Ile-Glu/Asp-Gly-Arg (SEQ ID NO: 56), Asp-Asp-Asp-Asp-Lys (SEQ ID NO: 57), Pro-Gly-Ala-Ala-His-Tyr (SEQ ID NO: 58), Leu-Pro-X-Thr-Gly/Ala consensus motif (SEQ ID NO: 59), Leu-Glu-Val-Phe-Gln-Gly-Pro (SEQ ID NO: 60), Leu-Val-Pro-Arg-Gly-Ser (SEQ ID NO: 61), Glu-Asn-Leu-Tyr-Phe-Gln-Gly (SEQ ID NO: 62), and [Ala-Gly-Ser-Val]X (X can be any amino acid; SEQ ID NO: 63).

25. The fusion polypeptide of claim 5 , wherein the heterologous protease cleavage site is cleaved by furin.

26. The fusion polypeptide of claim 25 , wherein the heterologous protease cleavage site comprises a furin cleavage site selected from the group consisting of RTKR (SEQ ID NO: 123); GTGAEDPRPSRKRRSLGDVG (SEQ ID NO: 125); GTGAEDPRPSRKRR (SEQ ID NO: 127); LQWLEQQVAKRRTKR (SEQ ID NO: 129); GTGAEDPRPSRKRRSLGG (SEQ ID NO: 1

Assignees

• Novartis Ag

Inventors

• Bradner James E
• Golosov Andrei
• Goold Carleton Proctor
• Guimaraes Carla Patricia Pinto
• Hild Marc Horst Peter
• Motz Gregory
• Ross Nathan Thomas
• Solomon Jonathan M
• Beckwith Rohan Eric John
• Carbonneau Seth

Classifications

• C12N2740/15043

  viral genome or elements thereof as genetic vector · CPC title

• C07K2319/81

  containing a Zn-finger domain for DNA binding · CPC title

• C07K2319/20

  containing a tag with affinity for a non-protein ligand · CPC title

• C07K19/00 Primary

  Hybrid peptides {, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes} · CPC title

• C07K16/2863

  against receptors for growth factors, growth regulators · CPC title