Reducing fratricide of immune cells expressing nkg2d-based receptors

Abstract

The present application relates to the field of immunotherapy, more particularly to the manufacture of cells for adoptive cell therapy. Provided herein are methods to prevent and/or reduce fratricide during manufacturing of such cells, particularly of cells expressing a chimeric NKG2D receptor. Also provided are cells and compositions comprising cells in which fratricide is prevented and/or reduced.

1 . A method of reducing and/or preventing fratricide during manufacturing of immune cells expressing a chimeric NKG2D receptor, comprising functional inhibition of NKG2D signaling during the manufacturing process of the cells. 
     
     
         2 . The method of  claim 1 , wherein functional inhibition of NKG2D signaling is achieved by one or more of:
 permanent or transient inhibition of one or more of the NKG2D ligands of the immune cells;   transient inhibition of the chimeric NKG2D receptor;   transient inhibition of downstream signaling of the chimeric NKG2D receptor.   
     
     
         3 . (canceled) 
     
     
         4 . (canceled) 
     
     
         5 . (canceled) 
     
     
         6 . The method of  claim 1 , wherein functional inhibition is achieved using shRNA or an antibody against the NKG2D receptor or against one or more of its ligands. 
     
     
         7 . The method of  claim 1 , wherein the immune cells are cells for adoptive cell transfer such as T cells, NK cells, NKT cells, stem cells or iPSCs. 
     
     
         8 . An engineered immune cell comprising a nucleic acid molecule encoding a chimeric NKG2D receptor according to  claim 1  and at least one of:
 one or more endogenous genes encoding a NKG2D ligand that have been engineered to be inactivated; 
 one or more shRNAs directed against the chimeric NKG2D receptor and/or one or more NKG2D ligands. 
 
     
     
         9 . The engineered immune cell of  claim 8 , wherein the NKG2D ligands are selected from: MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5 and ULBP6. 
     
     
         10 . A composition of immune cells comprising a nucleic acid molecule encoding a chimeric NKG2D receptor according to  claim 1 , the cells further comprising
 one or more endogenous genes encoding a NKG2D ligand that have been engineered to be inactivated;   One or more shRNAs directed against the chimeric NKG2D receptor and/or one or more NKG2D ligands;   
       and/or the composition further comprising
 one or more antibodies directed against the chimeric NKG2D receptor and/or one or more NKG2D ligands; and 
 an inhibitor of downstream signaling of the chimeric NKG2D receptor, 
 optionally a PI3K inhibitor. 
 
     
     
         11 . (canceled) 
     
     
         12 . (canceled) 
     
     
         13 . A method of treating cancer, comprising administering an effective amount of the engineered immune cell of  claim 8  to a subject in need thereof. 
     
     
         14 . A method of treating cancer, comprising administering the composition of  claim 10  to a subject in need thereof. 
     
     
         15 . A composition comprising immune cells expressing a chimeric NKG2D receptor, wherein said chimeric NKG2D receptor comprises (a) a NKG2D receptor polypeptide that binds to at least one NKG2D ligand selected from MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5 and ULBP6 and (b) a signaling moiety that transduces a signal in immune cells that express the chimeric NKG2D receptor, wherein said composition comprising said cells comprises reduced or no susceptibility to fratricide during the manufacturing of said immune cells expressing said chimeric NKG2D receptor, and comprises one or more of the following:
 (i) the composition which comprises said immune cells expressing the chimeric NKG2D receptor and further comprises an antibody or small molecule that specifically binds to a Phosphoinositide 3-kinase (PI3K) polypeptide thereby inhibiting PI3K signaling;   (ii) the immune cells expressing the chimeric NKG2D receptor in the composition comprise at least one nucleic acid that specifically binds to a Phosphoinositide 3-kinase (PI3K) nucleic acid which inhibits PI3K signaling;   (iii) the composition which comprises said immune cells expressing the chimeric NKG2D receptor further comprises at least one antibody specific to the NKG2D receptor polypeptide comprised in the chimeric NKG2D receptor and/or at least one antibody specific to a NKG2D ligand selected from MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5 and ULBP6; and/or   (iv) the immune cells expressing the chimeric NKG2D receptor in the composition comprise at least one nucleic acid that specifically binds to the nucleic acid in the chimeric NKG2D receptor nucleic acid which encodes for said NKG2D receptor polypeptide and/or at least one nucleic acid that specifically binds to a nucleic acid encoding a NKG2D ligand selected from MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5 and ULBP6, wherein the binding of said nucleic acid which specifically binds to said NKG2D receptor nucleic acid or to said NKG2D ligand nucleic acid downregulates the expression of the NKG2D receptor or said NKG2D ligand.   
     
     
         16 . The composition of  claim 15  (i), wherein the small molecule or antibody is LY294002 or idelalisib. 
     
     
         17 . The composition of  claim 15  (ii), wherein the at least one nucleic acid that specifically binds to a Phosphoinositide 3-kinase (PI3K) nucleic acid which inhibits PI3K signaling comprises one or more shRNAs or siRNAs that specifically bind to the PI3K nucleic acid. 
     
     
         18 . The composition of  claim 15  (iii) which further comprises at least one antibody specific to a NKG2D ligand selected from MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5 and ULBP6. 
     
     
         19 . The composition of  claim 15  (iv), wherein the at least one nucleic acid comprises one or more shRNAs or siRNAs that specifically bind to the nucleic acid in the chimeric NKG2D receptor nucleic acid which encodes for said NKG2D receptor polypeptide. 
     
     
         20 . The composition of  claim 15  (iv), wherein the at least one nucleic acid that specifically binds to a nucleic acid encoding a NKG2D ligand selected from MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5 and ULBP6 comprises one or more shRNAs or siRNAs that specifically bind to said nucleic acid encoding a NKG2D ligand. 
     
     
         21 . The composition of  claim 15  (i), (ii), (iii) or (iv), wherein the immune cells comprise one or more of T cells, NK cells, NKT cells, stem cells and iPSCs. 
     
     
         22 . A method of treatment in a subject comprising a disease characterized by NKG2D expressing cells which are involved in the disease pathology comprising administering an effective amount of immune cells expressing a chimeric NKG2D receptor, wherein said chimeric NKG2D receptor comprises (a) a NKG2D receptor polypeptide that binds to at least one NKG2D ligand selected from MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5 and ULBP6 and (b) a signaling moiety that transduces a signal in immune cells that express the chimeric NKG2D receptor, wherein said immune cells are derived from a composition according to  claim 15 .