Binding-triggered transcriptional switches and methods of use thereof

Patent metadata

Field	Value
Publication number	US-2016264665-A1
Application number	US-201615096971-A
Country	US
Kind code	A1
Filing date	Apr 12, 2016
Priority date	Feb 24, 2015
Publication date	Sep 15, 2016
Grant date	—

Abstract

The present disclosure provides binding-triggered transcriptional switch polypeptides, nucleic acids comprising nucleotide sequences encoding the binding-triggered transcriptional switch polypeptides, and host cells genetically modified with the nucleic acids. The present disclosure also provides chimeric Notch receptor polypeptides, nucleic acids comprising nucleotide sequences encoding the chimeric Notch receptor polypeptides, and host cells transduced and/or genetically modified with the nucleic acids. The present disclosure provides transgenic organisms comprising a nucleic acid encoding a binding triggered transcriptional switch polypeptide and/or a chimeric Notch receptor polypeptide of the present disclosure. Binding triggered transcriptional switch polypeptides and chimeric Notch receptor polypeptides of the present disclosure are useful in a variety of applications, which are also provided.

First claim

1 .- 143 . (canceled)

144 . A nucleic acid comprising a nucleotide sequence encoding a chimeric polypeptide comprising, from N-terminal to C-terminal and in covalent linkage:
a) an extracellular domain comprising a single-chain Fv (scFv) or a nanobody that specifically binds to an antigen; b) a Notch receptor polypeptide comprising one or more proteolytic cleavage sites and having at least 85% amino acid sequence identity to any one of SEQ ID NOs:131, 132 and 135-137; and c) an intracellular domain comprising a transcriptional activator, wherein binding of the scFv or the nanobody to the antigen induces cleavage of the Notch receptor polypeptide at the one or more proteolytic cleavage sites, thereby releasing the intracellular domain.

145 . The nucleic acid of claim 144 , wherein release of the intracellular domain causes the transcriptional activator to induce expression of an endogenous gene product in a cell comprising the nucleic acid.

146 . The nucleic acid of claim 144 , wherein release of the intracellular domain causes the transcriptional activator to induce expression of a heterologous gene product in a cell comprising the nucleic acid.

147 . The nucleic acid of claim 144 , wherein the nucleic acid further comprises a transcriptional control element, responsive to the transcriptional activator, operably linked to a nucleotide sequence encoding a chimeric antigen receptor (CAR).

148 . The nucleic acid of claim 144 , wherein the nucleic acid further comprises a transcriptional control element, responsive to the transcriptional activator, operably linked to a nucleotide sequence encoding a therapeutic antibody for the treatment of cancer.

149 . The nucleic acid of claim 148 , wherein the therapeutic antibody for the treatment of cancer is selected from pembrolizumab and tremelimumab.

150 . The nucleic acid of claim 144 , wherein the Notch receptor polypeptide has at least 85% amino acid sequence identity to SEQ ID NO:131 or SEQ ID NO:132.

151 . The nucleic acid of claim 144 , wherein the Notch receptor polypeptide comprises, at its N-terminus, one or more epidermal growth factor (EGF) repeats.

152 . The nucleic acid of claim 151 , wherein the Notch receptor polypeptide comprises, at its N-terminus, 2 to 11 EGF repeats.

153 . The nucleic acid of claim 144 , wherein the Notch receptor polypeptide comprises a synthetic linker.

154 . The nucleic acid of claim 151 , wherein the Notch receptor polypeptide comprises a synthetic linker between the one or more EGF repeats and the one or more proteolytic cleavage sites.

155 . The nucleic acid of claim 144 , wherein the Notch receptor polypeptide has a length from 50 amino acids to 1000 amino acids.

156 . The nucleic acid of claim 155 , wherein the Notch receptor polypeptide has a length from 300 amino acids to 400 amino acids.

157 . The nucleic acid of claim 144 , wherein the antigen is a cancer-associated antigen.

158 . The nucleic acid of claim 144 , wherein the one or more proteolytic cleavage sites comprises an S2 proteolytic cleavage site, an S3 proteolytic cleavage site or a combination thereof.

159 . The nucleic acid of claim 158 , wherein the one or more proteolytic cleavage sites comprises an S2 proteolytic cleavage site that is an ADAM-17-type protease cleavage site comprising an Ala-Val dipeptide sequence.

160 . The nucleic acid of claim 158 , wherein the one or more proteolytic cleavage sites comprises an S3 proteolytic cleavage site that is a gamma-secretase (γ-secretase) cleavage site comprising a Gly-Val dipeptide sequence.

161 . The nucleic acid of claim 158 , wherein the one or more proteolytic cleavage sites further comprises an S1 proteolytic cleavage site.

162 . The nucleic acid of claim 161 , wherein the S1 proteolytic cleavage site is a furin-like protease cleavage site comprising the amino acid sequence Arg-X-(Arg/Lys)-Arg, where X is any amino acid.

163 . The nucleic acid of claim 158 , wherein the Notch receptor polypeptide lacks an S1 proteolytic cleavage site.

164 . A recombinant expression vector comprising the nucleic acid of claim 144 .

165 . The recombinant expression vector of claim 164 , wherein the recombinant expression vector further comprises a transcriptional control element, responsive to the transcriptional activator, operably linked to a nucleotide sequence encoding a chimeric antigen receptor (CAR).

166 . The recombinant expression vector of claim 164 , wherein the recombinant expression vector further comprises a transcriptional control element, responsive to the transcriptional activator, operably linked to a nucleotide sequence encoding a therapeutic antibody for the treatment of cancer.

167 . A cell genetically modified to produce a chimeric polypeptide, wherein the chimeric polypeptide comprises:
a) an extracellular domain comprising a single-chain Fv (scFv) or nanobody that specifically binds to an antigen; b) a Notch receptor polypeptide comprising one or more proteolytic cleavage sites and having at least 85% amino acid sequence identity to any one of SEQ ID NOs:131, 132 and 135-137; and c) an intracellular domain comprising a transcriptional activator, wherein binding of the scFv or the nanobody to the antigen induces cleavage of the Notch receptor polypeptide at the one or more proteolytic cleavage sites, thereby releasing the intracellular domain.

168 . The cell of claim 167 , wherein the cell is an immune cell, a neuron, an epithelial cell, an endothelial cell, or a stem cell.

169 . The cell of claim 168 , wherein the immune 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.

170 . The cell of claim 169 , wherein the immune cell is a T cell.

171 . A method of treating cancer in an individual, the method comprising administering to the individual the cell of claim 167 , wherein the antigen is a cancer-associated antigen.

172 . The method of claim 171 , wherein release of the intracellular domain causes the transcriptional activator to induce expression of an endogenous gene product in the cell.

173 . The method of claim 171 , wherein release of the intracellular domain causes the transcriptional activator to induce expression of a heterologous gene product in the cell.

Assignees

• Univ California

Inventors

• Lim Wendell A
• Morsut Leonardo
• Roybal Kole T

Classifications

• C07K2319/74

  containing a fusion for binding to a cell surface receptor · CPC title

• C07K2317/622

  Single chain antibody (scFv) · CPC title

• G01N33/53

  Immunoassay; Biospecific binding assay; Materials therefor · CPC title

• C07K19/00

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

• C07K16/28 Primary

  against receptors, cell surface antigens or cell surface determinants · CPC title