Engineered cytolytic immunecell

Abstract

The present invention relates to engineered a cytolytic immune cell comprising: i) a releasable protein which comprises a polypeptide of interest (POI) and a first interaction domain; and ii) a retention protein which is retained within an intracellular compartment of the cell and comprises a second interaction domain which binds to the first protein interaction domain, wherein binding between the first protein interaction domain and second protein interaction domain is disrupted by the presence of an agent, such that in the absence of the agent, the first protein interaction domain and second protein interaction domain bind and result in retention of the POI within an intracellular compartment; whereas in the presence of the agent, the first protein interaction domain and second protein interaction do not bind and the POI is released from the intracellular compartment and expressed at the cell surface or secreted by the cell.

1 . An engineered cytolytic immune cell comprising:
 i) a releasable protein which comprises a polypeptide of interest (POI) and a first interaction domain; and   ii) a retention protein which is retained within an intracellular compartment of the cell and comprises a second interaction domain which binds to the first protein interaction domain,   wherein binding between the first protein interaction domain and second protein interaction domain is disrupted by the presence of an agent, such that in the absence of the agent, the first protein interaction domain and second protein interaction domain bind and result in retention of the POI within an intracellular compartment; whereas in the presence of the agent, the first protein interaction domain and second protein interaction do not bind and the POI is released from the intracellular compartment and expressed at the cell surface or secreted by the cell.   
     
     
         2 . An engineered cytolytic immune cell according to  claim 1 , wherein in the presence of the agent, the POI is exported to and expressed at the cell surface. 
     
     
         3 . An engineered cytolytic immune cell according to  claim 1 , wherein in the presence of the agent, the POI is secreted by the cell. 
     
     
         4 - 11 . (canceled) 
     
     
         12 . An engineered cytolytic immune cell according to  claim 1 , wherein the intracellular retention domain is a golgi retention domain. 
     
     
         13 . An engineered cytolytic immune cell according to  claim 1 , wherein the intracellular retention domain is an endoplasmic reticulum retention domain. 
     
     
         14 . An engineered cytolytic immune cell according to  claim 1 , wherein the intracellular retention domain is located at the C-terminus or the N-terminus of the retention protein. 
     
     
         15 - 20 . (canceled) 
     
     
         21 . An engineered cytolytic immune cell according to  claim 1 , wherein the POI is a secreted protein. 
     
     
         22 . An engineered cytolytic immune cell according to  claim 1 , wherein the POI is a cell surface membrane protein. 
     
     
         23 . An engineered cytolytic immune cell according to  claim 1 , wherein the releasable protein comprises two or more POIs. 
     
     
         24 . (canceled) 
     
     
         25 . An engineered cytolytic immune cell according to  claim 1 , wherein the cell further comprises a chimeric antigen receptor (CAR) or transgenic T cell receptor (TCR). 
     
     
         26 . A nucleic acid construct which comprises:
 (i) a first nucleic acid sequence encoding a releasable protein which comprises a polypeptide of interest (POI and a first interaction domain;   (ii) a retention protein;   (iii) a second nucleic acid sequence encoding a retention protein which is retained within an intracellular compartment of the cell in which it is expressed and comprises a second interaction domain which binds to the first protein interaction domain; and   (iv) a third nucleic acid sequence which encodes a CAR or transgenic TCR.   
     
     
         27 . (canceled) 
     
     
         28 . A vector which comprises a nucleic acid construct according to  claim 26 . 
     
     
         29 . (canceled) 
     
     
         30 . A method for making an engineered cytolytic immune cell, which comprises the step of introducing to a cytolytic immune cell a nucleic acid construct according to  claim 26 . 
     
     
         31 . (canceled) 
     
     
         32 . A method for controlling the secretion of a POI from a cell or the cell-surface expression of a POI on a cell, which comprises the step of administering an agent which disrupts binding between the first protein interaction domain and second protein interaction domain to the engineered cytolytic immune cell according to  claim 1 . 
     
     
         33 . (canceled) 
     
     
         34 . A pharmaceutical composition which comprises a plurality of engineered cytolytic immune cells according to  claim 1 . 
     
     
         35 .- 36 . (canceled) 
     
     
         37 . A method for treating a disease in a subject, which comprises the step of administering a pharmaceutical composition according to  claim 34  to a subject in need thereof. 
     
     
         38 . A method for treating a disease, which comprises the step of administering an agent to a subject to which an engineered cytolytic immune cell has been administered,
 wherein the engineered cytolytic immune cell comprises:
 i) a releasable protein which comprises a polypeptide of interest (POI) and a first interaction domain; and 
 ii) a retention protein which is retained within an intracellular compartment of the cell and comprises a second interaction domain which binds to the first protein interaction domain, 
 wherein binding between the first protein interaction domain and second protein interaction domain is disrupted by the presence of the agent, such that in the absence of the agent, the first protein interaction domain and second protein interaction domain bind and result in retention of the POI within an intracellular compartment; whereas in the presence of the agent, the first protein interaction domain and second protein interaction do not bind and the POI is released from the intracellular compartment and expressed at the cell surface or secreted by the cell. 
   
     
     
         39 - 42 . (canceled) 
     
     
         43 . A method according to  claim 37  for treating cancer, which comprises the step of monitoring toxic activity and/or anti-tumour activity in the subject and further comprises the step of: stopping or reducing the administration of an agent which disrupts binding between the first protein interaction domain and second protein interaction domain to the subject in order to reduce adverse toxic effects; and/or increasing the administration of such an agent to the subject in order to increase anti-tumour effects. 
     
     
         44 - 46 . (canceled) 
     
     
         47 . A method according to  claim 38  for treating cancer, which comprises the step of monitoring toxic activity and/or anti-tumour activity in the subject and further comprises the step of: stopping or reducing the administration of the agent to the subject in order to reduce adverse toxic effects; and/or increasing the administration of the agent to the subject in order to increase anti-tumour effects.