Hook fusion protein for regulating the cellular trafficking of a target protein

Abstract

The present invention relates to a hook fusion protein comprising a hook domain and at least one cytoplasmic carboxy terminal endoplasmic reticulum (ER) retention signal and/or at least one cytoplasmic amino terminal endoplasmic reticulum (ER) retention signal; wherein the hook fusion protein is a soluble protein that localizes in the cytoplasm. The present invention also relates to a nucleic acid system for intracellular targeting control comprising a nucleic acid encoding a hook fusion protein as herein disclosed, and a nucleic acid encoding a target fusion protein comprising a hook-binding domain; wherein said target fusion protein in a membrane protein; and wherein the hook fusion protein localizes in the ER when bound to the target fusion protein. The invention also encompasses a vector system, viral particle system, host cell and kit comprising said nucleic acids. The invention also includes the vector system, viral particle system, host cell or kit for use as a medicament, in particular for immunotherapy.

1 . A hook fusion protein comprising
 a hook core   at least one cytoplasmic carboxy terminal endoplasmic reticulum (ER) retention signal and/or at least one cytoplasmic amino terminal endoplasmic reticulum (ER) retention signal;   wherein the hook fusion protein is a soluble protein that localizes in the cytoplasm.   
     
     
         2 . A hook fusion protein according to  claim 1 , wherein the hook core is a streptavidin sequence. 
     
     
         3 . A hook fusion protein according to  claim 1  wherein the carboxy terminal endoplasmic reticulum (ER) retention signal is K(X)KXX, with X being any amino acids and/or the amino terminal endoplasmic reticulum (ER) retention signal is a fragment of the isoform of the human invariant chain of the major histocompatibility complex protein Ii, optionally wherein the fragment of the isoform of the human invariant chain of the major histocompatibility complex protein Ii has an amino acid sequence selected from SEQ ID NO: 5 or 14. 
     
     
         4 . A hook fusion protein according to  claim 1  further comprising an endocytosis signal, preferably consisting of YXXI with X being any amino acids. 
     
     
         5 . A nucleic acid comprising a nucleic acid sequence encoding the hook fusion protein according to  claim 1 . 
     
     
         6 . The nucleic acid according to  claim 5  further comprising a nucleic acid sequence encoding a target fusion protein comprising a hook-binding domain,
 wherein said target fusion protein is a chimeric antigen receptor comprising:
 a binding domain; 
 a hook-binding domain, and 
 at least one activation domain; 
 
 or alternatively comprising:
 the full NKG2D or a functional variant thereof, 
 at least one activation domain, and 
 a hook-binding domain. 
 
 
     
     
         7 . A nucleic acid system for intracellular targeting control comprising
 (a) a nucleic acid encoding a hook fusion protein according to  claim 1 , and   (b) a nucleic acid encoding a target fusion protein comprising a hook-binding domain;
 wherein said target fusion protein is a membrane protein; and 
 wherein the hook fusion protein localizes in the ER when bound to the target fusion protein; optionally wherein the hook fusion protein comprises a streptavidin domain and the target fusion protein comprises a streptavidin-binding domain, optionally 
 wherein the target fusion protein is a chimeric antigen receptor as defined in  claim 6  comprising:
 a binding domain; 
 a hook-binding domain, and 
 at least one activation domain; 
 
 or alternatively comprising:
 the full NKG2D or a functional variant thereof, 
 at least one activation domain, and 
 a hook-binding domain. 
 
   
     
     
         8 . A vector system comprising one or more vectors comprising
 (a) the nucleic acid sequence of  claim 6 , and optionally   (b) a nucleic acid encoding a target fusion protein comprising a hook-binding domain;
 wherein the nucleic acids (a) and (b) are located on the same or on different vectors; optionally 
 wherein the hook fusion protein comprises a streptavidin domain and the target fusion protein comprises a streptavidin-binding domain. 
   
     
     
         9 . A vector system according to  claim 8 , wherein the nucleic acids (a) and (b) are located on the same vector and wherein the nucleic acid (a) is inserted upstream of an IRES sequence and the nucleic acid (b) is inserted downstream of said IRES sequence. 
     
     
         10 . A vector system according to  claim 8 , wherein the nucleic acids (a) and (b) are located on the same vector and wherein:
 i) the nucleic acid (a) comprises an Ii retention signal in its N terminal sequence and is inserted upstream of a 2A peptide sequence, or   ii) the nucleic acid (a) comprises a K(X)KXX retention signal in its C terminal sequence and is inserted downstream of a 2A peptide sequence.   
     
     
         11 . A vector system according to  claim 10  further comprising the nucleic acid sequence (b)
 wherein said nucleic acid sequence (b) comprises a streptavidin-binding domain, and 
 wherein said nucleic acid sequence (b) is inserted downstream of the 2A peptide in the i) configuration or upstream of the 2A peptide in the ii) configuration. 
 
     
     
         12 . A vector system according to  claim 8 , wherein the target fusion protein encoded by the nucleic acid (b) is a chimeric antigen receptor comprising:
 a binding domain;   a hook-binding domain, and   at least one activation domain;   
       or alternatively comprising:
 the full NKG2D or a functional variant thereof, 
 at least one activation domain, and 
 a hook-binding domain. 
 
     
     
         13 . A viral particle system comprising a vector system according to  claim 8 ; optionally wherein the viral particle system is a lentiviral particle. 
     
     
         14 . An isolated cell comprising a vector system as defined in  claim 8 . 
     
     
         15 . An in vitro method for regulating the intracellular trafficking in a host cell of a target protein;
 wherein said target protein is a fusion protein comprising a hook binding domain; and   wherein the method comprises expressing in said host cell a vector system according to  claim 8 ;   wherein the hook fusion protein and the target fusion protein are capable of conditional interaction in the absence of a ligand for the hook core domain, optionally wherein the hook core domain is streptavidin, the hook-binding domain is a streptavidin-binding domain and the ligand is biotin.   
     
     
         16 . (canceled) 
     
     
         17 . (canceled) 
     
     
         18 . (canceled)