Force sensor cleavage domain containing chimeric polypeptides and methods of use thereof

Patent metadata

Field	Value
Publication number	US-12065479-B2
Application number	US-201816763524-A
Country	US
Kind code	B2
Filing date	Nov 15, 2018
Priority date	Nov 16, 2017
Publication date	Aug 20, 2024
Grant date	Aug 20, 2024

Abstract

Provided are chimeric polypeptides which modulate various cellular processes following cleavage of a force sensor cleavage domain, including non-Notch force sensor cleavage domains, induced upon binding of a specific binding member of the chimeric polypeptide with its binding partner. Methods of using force sensor cleavage domain-containing chimeric polypeptides to modulate cellular functions, including e.g., modulation (including induction or repression) of gene expression, are also provided. Nucleic acids encoding the subject chimeric polypeptides and associated expression cassettes and vectors as well as cells that contain such nucleic acids and/or expression cassettes and vectors are provided. Also provided, are methods of monitoring cell-cell signaling and method of treating a subject using the described components, as well as kits for practicing the subject methods.

First claim

What is claimed is:

1. A chimeric polypeptide comprising, from N terminus to C terminus:
a) an extracellular domain comprising a first member of a binding pair;
b) a non-Notch force sensor cleavage domain comprising a proteolytic cleavage site;
c) a cleavable transmembrane domain; and
d) an intracellular domain that is not a Notch intracellular signaling domain and does not induce expression of Notch target genes,
wherein binding of the first member of the specific binding pair to the second member of the specific binding pair, present on a cell or other solid support, induces cleavage at the proteolytic cleavage site thereby releasing the intracellular domain, and
wherein the non-Notch force sensor cleavage domain is a von Willebrand Factor (vWF) A2 domain.

2. The chimeric polypeptide according to claim 1 , wherein the first member of the binding pair comprises at least a portion of a receptor that binds a ligand and the second member of the binding pair comprises at least a portion of the ligand.

3. The chimeric polypeptide according to claim 1 , wherein the first member of the binding pair comprises at least a portion of a ligand that binds a receptor and the second member of the binding pair comprises at least a portion of the receptor.

4. The chimeric polypeptide according to claim 1 , wherein the first member of the binding pair comprises an antibody.

5. The chimeric polypeptide according to claim 4 , wherein the antibody is a nanobody, a diabody, a triabody, or a minibody, a F(ab′) 2 fragment, a Fab fragment, a single chain variable fragment (scFv) or a single domain antibody (sdAb).

6. The chimeric polypeptide according to claim 1 , wherein the intracellular domain comprises a transcriptional activator or repressor.

7. A nucleic acid encoding the chimeric polypeptide according to claim 1 .

8. The nucleic acid according to claim 1 , wherein the nucleic acid further comprises a transcriptional control element responsive to the released intracellular domain operably linked to a nucleic acid sequence encoding a polypeptide of interest (POI).

9. The nucleic acid according to claim 8 , wherein the POI is a heterologous polypeptide selected from the group consisting of: a reporter protein, an immunoactivator, an immune suppression factor, a transcription factor, a site-specific nuclease, a recombinase, a chimeric antigen receptor (CAR), an antibody, a chimeric bispecific binding member, an engineered T cell receptor (TCR) an innate-immune response inducer.

10. A recombinant expression vector comprising the nucleic acid according to claim 1 .

11. A method of modulating expression of a heterologous polypeptide in a cell, the method comprising:
contacting a cell with a second member of a binding pair, wherein the cell expresses a chimeric polypeptide and comprises a sequence encoding the heterologous polypeptide operably linked to a transcriptional control element responsive to the intracellular domain of the chimeric polypeptide, thereby releasing the intracellular domain of the chimeric polypeptide and modulating expression of the heterologous polypeptide, wherein the chimeric polypeptide comprises, from N terminus to C terminus:
a) an extracellular domain comprising a first member of a binding pair;
b) a non-Notch force sensor cleavage domain comprising a proteolytic cleavage site;
c) a cleavable transmembrane domain; and
d) an intracellular domain that is not a Notch intracellular signaling domain and does not induce expression of Notch target genes,

wherein binding of the first member of the specific binding pair to the second member of the specific binding pair, present on a cell or other solid support, induces cleavage at the proteolytic cleavage site thereby releasing the intracellular domain, and
wherein the non-Notch force sensor cleavage domain is a von Willebrand Factor (vWF) A2 domain.

12. The method according to claim 11 , wherein the heterologous polypeptide is selected from the group consisting of: a reporter protein, an immunoactivator, an immune suppression factor, a transcription factor, a site-specific nuclease, a recombinase, a chimeric antigen receptor (CAR), an antibody, a chimeric bispecific binding member, an engineered T cell receptor (TCR) an innate-immune response inducer.

13. A method of modulating an activity of a cell that expresses a chimeric polypeptide, the method comprising:
contacting the cell with a second member of the specific binding pair, wherein binding of the first member of the specific binding pair to the second member of the specific binding pair induces cleavage of the chimeric polypeptide at the proteolytic cleavage site, thereby releasing the intracellular domain, wherein release of the intracellular domain modulates the activity of the cell, wherein the chimeric polypeptide comprises, from N terminus to C terminus:
a) an extracellular domain comprising a first member of a binding pair;
b) a non-Notch force sensor cleavage domain comprising a proteolytic cleavage site;
c) a cleavable transmembrane domain; and
d) an intracellular domain that is not a Notch intracellular signaling domain and does not induce expression of Notch target genes,

wherein binding of the first member of the specific binding pair to the second member of the specific binding pair, present on a cell or other solid support, induces cleavage at the proteolytic cleavage site thereby releasing the intracellular domain, and

wherein the non-Notch force sensor cleavage domain is a von Willebrand Factor (vWF) A2 domain.

14. The method according to claim 13 , wherein release of the intracellular domain induces cell death by a mechanism other than apoptosis.

15. The method according to claim 13 , wherein release of the intracellular domain modulates gene expression in the cell through transcriptional regulation, chromatin regulation, translation, trafficking or post-translational processing.

16. The method according to claim 13 , wherein release of the intracellular domain induces de novo expression or modulates expression of a gene product in the cell.

17. The method according to claim 16 , wherein the gene product is a transcriptional activator, a transcriptional repressor, a chimeric antigen receptor, a second chimeric Notch receptor polypeptide, a translation regulator, a cytokine, a hormone, a chemokine, or an antibody.

18. A host cell comprising:
a) a nucleic acid encoding a chimeric polypeptide according to claim 1 ; and
b) a transcriptional control element responsive to the intracellular domain of the chimeric polypeptide operably linked to a nucleic acid encoding a polypeptide of interest (POI).

19. The host cell according to claim 18 , wherein the heterologous polypeptide is selected from the group consisting of: a reporter protein, an immunoactivator, an immune suppression factor, a transcription factor, a site-specific nuclease, a recombinase, a chimeric antigen receptor (CAR), an antibody, a chimeric bispecific binding member, an engineered T cell receptor (TCR) an innate-immune response inducer.

20. The host cell according to claim 18 , wherein the cell is a T cell, a B cell, a monocyte, a natural killer cell, a dendritic cell, a macrophage, a regulatory T cell, a helper T cell, or a cytotoxic T cell.

21. The method according to claim 11 , wherein the vWF A2 domain comprises a sequence having at least 77% identity to SEQ ID NO: 69.

22. The method according to claim 13 , wherein the vWF A2 domain comprises a sequence having at l

Assignees

• Univ California
• Univ Columbia

Inventors

• Choe Joseph H
• Langridge Paul
• Lim Wendell A
• Roybal Kole T
• Struhl Gary

Classifications

• A61K40/4211

  CD19 or B4 · CPC title

• A61K40/31

  Chimeric antigen receptors [CAR] · CPC title

• A61K40/11

  T-cells, e.g. tumour infiltrating lymphocytes [TIL] or regulatory T [Treg] cells; Lymphokine-activated killer [LAK] cells · CPC title

• G01N33/6803

  General methods of protein analysis not limited to specific proteins or families of proteins · CPC title

• G01N33/5005

  involving human or animal cells (immunoassay G01N33/56966; immunoassays of protozoa G01N33/56905; protozoa in screening assays C12Q1/025) · CPC title