Binding-triggered transcriptional switches and methods of use thereof

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.

1 - 143 . (canceled) 
     
     
         144 . A nucleic acid encoding a chimeric polypeptide comprising:
 (a) an extracellular domain that comprises an antigen binding region of an antibody;   (b) transmembrane region and one or more protease cleavage sites; and   (c) an intracellular domain that comprises a DNA binding domain:
 i. wherein the intracellular domain does not comprise an immunoreceptor activation domain or a co-stimulatory domain; and 
 ii. when the chimeric polypeptide is expressed in a mammalian cell, binding of the antigen binding region of the antibody to a corresponding antigen induces proteolytic cleavage of the chimeric polypeptide at one or more of the protease cleavage sites to release the intracellular domain. 
   
     
     
         145 . The nucleic acid of  claim 144 , wherein the antigen binding region is a scFv or a nanobody. 
     
     
         146 . The nucleic acid of  claim 144 , wherein the antigen is a cancer-associated antigen. 
     
     
         147 . The nucleic acid of  claim 144 , wherein the chimeric polypeptide comprises a Notch regulatory region between the antigen binding region of (a) and the DNA binding domain of (c). 
     
     
         148 . The nucleic acid of  claim 147 , wherein the Notch regulatory region comprises a Lin 12-Notch repeat, a heterodimerization domain comprising an S2 proteolytic cleavage site and a transmembrane domain comprising an S3 proteolytic cleavage site. 
     
     
         149 . The nucleic acid of  claim 147 , wherein the Notch regulatory region comprises, at its N-terminus, one or more epidermal growth factor (EGF) repeats. 
     
     
         150 . The nucleic acid of  claim 149 , wherein the Notch regulatory region comprises, at its N-terminus, 2 to 11 EGF repeats. 
     
     
         151 . The nucleic acid of  claim 148 , wherein the S2 proteolytic cleavage site is an ADAM-17-type protease cleavage site comprising an Ala-Val dipeptide sequence. 
     
     
         152 . The nucleic acid of  claim 148 , wherein the S3 proteolytic cleavage site is a gamma-secretase (γ-secretase) cleavage site comprising a Gly-Val dipeptide sequence. 
     
     
         153 . The nucleic acid of  claim 144 , wherein the chimeric polypeptide further comprises an S1 proteolytic cleavage site. 
     
     
         154 . The nucleic acid of  claim 144 , wherein the chimeric polypeptide lacks an S1 proteolytic cleavage site. 
     
     
         155 . The nucleic acid of  claim 153 , 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. 
     
     
         156 . The nucleic acid of  claim 147 , wherein the Notch regulatory region has at least 85% amino acid sequence identity to any one of the Notch regulatory regions of SEQ ID NO:131, 132 and 135-137. 
     
     
         157 . The nucleic acid of  claim 144 , wherein the intracellular domain comprises a transcription factor or a first half of a split transcription factor. 
     
     
         158 . A genetically modified cell comprising the nucleic acid of  claim 144 , wherein the nucleic acid is operably linked to a promoter and the chimeric polypeptide is expressed in the cell. 
     
     
         159 . The genetically modified cell of  claim 158 , wherein the cell further comprises
 a second nucleic acid comprising a transcriptional control element that is operably linked to a coding sequence for a therapeutic agent, wherein binding of the antigen binding region of the chimeric polypeptide to an antigen releases the intracellular domain and activates expression of the therapeutic agent.   
     
     
         160 . The genetically modified cell of  claim 159 , wherein the therapeutic agent is immunosuppressive. 
     
     
         161 . The genetically modified cell of  claim 159 , wherein the therapeutic agent stimulates immune cells. 
     
     
         162 . The genetically modified cell of  claim 159 , wherein the therapeutic agent is selected from the group consisting of: an antibody, a chemokine, a chemokine receptor, a cytokine, a cytokine receptor, a chimeric antigen receptor (CAR), a T cell receptor (TCR), an activating immunoreceptor, an inhibiting immunoreceptor, an immunoactivator, an immunoinhibitor, an apoptosis inducer, an apoptosis inhibitor, a toxin derived protein and a site-specific nuclease. 
     
     
         163 . The genetically modified cell of  claim 159 , wherein the therapeutic agent is a chimeric antigen receptor (CAR). 
     
     
         164 . The genetically modified cell of  claim 158 , wherein the cell is an immune cell, a neuron, an epithelial cell, an endothelial cell, or a stem cell. 
     
     
         165 . The genetically modified cell of  claim 158 , 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. 
     
     
         166 . The genetically modified cell of  claim 158 , wherein the cell is a T cell.