Genetically Modified Immune Cells Targeting NY-ESO-1 and Methods of Use Thereof

Abstract

The present disclosure provides modified immune cell (e.g., modified T cell) comprising an exogenous T cell receptor (TCR) having specificity for NY-ESO-1. The present disclosure provides modified immune cells or precursors thereof (e.g., modified T cells) comprising an exogenous TCR and a switch receptor. Gene edited modified cells are also provided, such that the expression of one or more of an endogenous T-cell receptor gene (e.g., TRAC, TRBC) or an endogenous immune checkpoint gene (e.g. PD-1 or TIM-3) is downregulated.

1 .- 24 . (canceled) 
     
     
         25 . A method for generating a modified T cell comprising:
 a) introducing into a T cell a first nucleic acid comprising a nucleic acid sequence encoding an exogenous T cell receptor (TCR) having affinity for NY-ESO-1 on a target cell; and   b) introducing into the T cell one or more nucleic acids capable of downregulating gene expression of one or more endogenous genes selected from the group consisting of TCR alpha chain, and TCR beta chain.   
     
     
         26 . The method of  claim 25 , wherein the one or more nucleic acids capable of downregulating gene expression comprises a CRISPR system comprising a Cas9/gRNA ribonucleoprotein (RNP) complex. 
     
     
         27 . The method of  claim 25 , wherein the first nucleic acid further comprises a nucleic acid sequence encoding a switch receptor. 
     
     
         28 . The method of  claim 27 , wherein the nucleic acid sequence encoding the switch receptor comprises the nucleic acid sequence set forth in SEQ ID NOs: 15, 135, 137, or 139. 
     
     
         29 . The method of  25 , further comprising:
 c) introducing into the T cell a nucleic acid capable of downregulating gene expression of an endogenous PD1 coding sequence.   
     
     
         30 . The method of  claim 29 , wherein the one or more nucleic acids capable of downregulating gene expression comprises a CRISPR system comprising a Cas9/gRNA ribonucleoprotein (RNP) complex. 
     
     
         31 . The method of  claim 25 , wherein the exogenous TCR comprises:
 (i) a TCR alpha chain comprising the amino acid sequence set forth in SEQ ID NO:5; and/or   (ii) a TCR beta chain comprising the amino acid sequence set forth in SEQ ID NO: 12.   
     
     
         32 . The method of  claim 25 , wherein:
 (i) the endogenous TCR alpha chain gene comprises the nucleic acid sequence set forth in SEQ ID NO:128; and/or   (ii) the endogenous TCR beta chain gene comprises the nucleic acid sequence set forth in SEQ ID NO:129.   
     
     
         33 . The method of  claim 29 , wherein the endogenous PD1 coding sequence comprising the nucleic acid sequence set forth in SEQ ID NO:130. 
     
     
         34 . The method of  claim 27 , wherein the switch receptor comprises:
 a first domain, wherein the first domain is derived from a first polypeptide that is associated with a negative signal;   a second domain comprising a switch receptor transmembrane domain; and   a third domain, wherein the third domain is derived from a second polypeptide that is associated with a positive signal.   
     
     
         35 . The method of  claim 34 , wherein the first polypeptide that is associated with a negative signal is PD-1. 
     
     
         36 . The method of  claim 34 , wherein the first polypeptide that is associated with a negative signal is a variant of PD-1 having an alanine-to-leucine substitution at amino acid position 132 relative to the wild-type PD-1 amino acid sequence. 
     
     
         37 . The method of  claim 34 , wherein the third polypeptide that is associated with a positive signal is CD28 or 4-1BB. 
     
     
         38 . The method of  claim 34 , wherein the switch receptor comprises:
 a first domain comprising at least a portion of the extracellular domain of PD1;   a second domain comprising a switch receptor transmembrane domain comprising at least a portion of the transmembrane domain of CD28; and   a third domain comprising at least a portion of the intracellular domain of CD28.   
     
     
         39 . The method of  claim 34 , wherein the switch receptor comprises:
 a first domain comprising at least a portion of the extracellular domain of PD-1;   a second domain comprising a switch receptor transmembrane domain comprising at least a portion of the transmembrane domain of CD8alpha; and   a third domain comprising at least a portion of the intracellular domain of 4-1BB.   
     
     
         40 . The method of  claim 34 , wherein the switch receptor comprises:
 a first domain comprising at least a portion of the extracellular domain of PD-1, wherein the PD-1 is a variant having an alanine-to-leucine substitution at amino acid position 132 relative to the wild-type PD-1 amino acid sequence;   a second domain comprising a switch receptor transmembrane domain comprising at least a portion of the transmembrane domain of CD28; and   a third domain comprising at least a portion of the intracellular domain of CD28.   
     
     
         41 . The method of  claim 34 , wherein the switch receptor comprises:
 a first domain comprising at least a portion of the extracellular domain of PD-1, wherein the PD-1 is a variant having an alanine-to-leucine substitution at amino acid position 132 relative to the wild-type PD-1 amino acid sequence;   a second domain comprising a switch receptor transmembrane domain comprising at least a portion of the transmembrane domain of CD8alpha; and   a third domain comprising at least a portion of the intracellular domain of 4-1BB.   
     
     
         42 . The method of  claim 34 , wherein the switch receptor comprises the amino acid sequence set forth in any one of SEQ ID NOs: 14, 134, 136, or 138. 
     
     
         43 . The method of  claim 25 , wherein T cell is an autologous T cell derived from a human.