Methods for engineering allogeneic and highly active t cell for immunotherapy

Abstract

The present invention relates to methods for developing engineered T-cells for immunotherapy that are non-alloreactive. The present invention relates to methods for modifying T-cells by inactivating both genes encoding T-cell receptor and an immune checkpoint gene to unleash the potential of the immune response. This method involves the use of specific rare cutting endonucleases, in particular TALE-nucleases (TAL effector endonuclease) and polynucleotides encoding such polypeptides, to precisely target a selection of key genes in T-cells, which are available from donors or from culture of primary cells. The invention opens the way to standard and affordable adoptive immunotherapy strategies for treating cancer and viral infections.

1 . A method of preparing T-cells for immunotherapy comprising:
 (a) modifying T-cells by inactivating at least:
 a first gene encoding a immune checkpoint protein, and 
 a second gene encoding a component of the T-cell receptor (TCR); and 
   (b) expanding said cells.   
     
     
         2 . The method of  claim 1  comprising modifying T-cells by:
 (a) introducing into said T-cell rare-cutting endonucleases able to selectively inactivate by DNA cleavage respectively:
 said gene encoding a immune checkpoint protein, and 
 at least one gene encoding one component of the T-cell receptor (TCR); and 
 
 (b) expanding said cells. 
 
     
     
         3 . The method according to  claim 1 , wherein said immune checkpoint gene is selected from the group consisting of: PD1, CTLA-4, LAG3, Tim3, BTLA, BY55, TIGIT, B7H5, LAIR1, SIGLEC10, and 2B4. 
     
     
         4 . The method according to  claim 1 , wherein said transformed T-cells are expanded in a patient's blood. 
     
     
         5 . The method according to  claim 1 , wherein said transformed T-cells are expanded in-vivo. 
     
     
         6 . The method according to  claim 1 , wherein said gene is PD1 or CTLA-4. 
     
     
         7 . The method according to  claim 1 , wherein the at least two inactivated genes are selected from the group consisting of PD1 and TCR alpha, PD1 and TCR beta, CTLA-4 and TCR alpha, CTLA-4 and TCR beta, LAG3 and TCR alpha, LAG3 and TCR beta, Tim3 and TCR alpha, Tim3 and TCR beta, BTLA and TCR alpha, BTLA and TCR beta, BY55 and TCR alpha, BY55 and TCR beta, TIGIT and TCR alpha, TIGIT and TCR beta, B7H5 and TCR alpha, B7H5 and TCR beta, LAIR1 and TCR alpha, LAIR1 and TCR beta, SIGLEC10 and TCR alpha, SIGLEC10 and TCR beta, 2B4 and TCR alpha, and 2B4 and TCR beta. 
     
     
         8 . The method according to  claim 2 , wherein said rare-cutting endonucleases are encoded by mRNA. 
     
     
         9 . The method according to  claim 2  that comprises introducing one or more rare-cutting endonucleases into said cell in step (a) by way of RNA electroporation. 
     
     
         10 . The method according to  claim 2 , wherein said rare-cutting endonucleases are TALE-nucleases. 
     
     
         11 . The method according to  claim 10 , wherein at least one of these TALE-nuclease is directed against one of the gene target sequences of TCRalpha selected from SEQ ID NO: 37, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59 and SEQ ID NO: 60. 
     
     
         12 . The method according to  claim 10 , wherein at least one of these TALE-nuclease is directed against one of the gene target sequences of TCRbeta selected from the group consisting of SEQ ID NO: 38 and SEQ ID NO: 39. 
     
     
         13 . The method according to  claim 10 , wherein at least one of these TALE-nucleases is directed against one of the gene target sequences of PD1 selected from the group consisting of SEQ ID NO: 77 and SEQ ID NO: 78. 
     
     
         14 . The method according to  claim 10 , wherein at least one of these TALE-nucleases is directed against one of the gene target sequences of CTLA-4 selected from the group consisting of SEQ ID NO: 74 and SEQ ID NO: 76. 
     
     
         15 . The method according to  claim 1 , further comprising introducing into said T-cells a chimeric antigen receptor (CAR). 
     
     
         16 . The method according to  claim 1 , wherein said T-cells in step a) are derived from inflammatory T-lymphocytes, cytotoxic T-lymphocytes, regulatory T-lymphocytes or helper T-lymphocytes. 
     
     
         17 . The method according to  claim 1 , wherein said T-cells in step a) are derived from CD4+ T-lymphocytes and/or CD8+ T-lymphocytes. 
     
     
         18 . An isolated T-cell or cell line obtainable by the method of  claim 1 . 
     
     
         19 . An isolated T-cell, in which at least two genes selected from the group consisting of: PD1 and TCR alpha, PD1 and TCR beta, CTLA-4 and TCR alpha, CTLA-4 and TCR beta, LAG3 and TCR alpha, LAG3 and TCR beta, Tim3 and TCR alpha, Tim3 and TCR beta, BTLA and TCR alpha, BTLA and TCR beta, BY55 and TCR alpha, BY55 and TCR beta, TIGIT and TCR alpha, TIGIT and TCR beta, B7H5 and TCR alpha, B7H5 and TCR beta, LAIR1 and TCR alpha, LAIR1 and TCR beta, SIGLEC10 and TCR alpha, SIGLEC10 and TCR beta, 2B4 and TCR alpha, and 2B4 and TCR beta, have been inactivated. 
     
     
         20 . An isolated T-cell according to  claim 19 , further comprising an exogenous polynucleotide sequence encoding a Chimeric Antigen Receptor. 
     
     
         21 . An isolated T-cell of  claim 20  wherein said Chimeric Antigen Receptor is a multi-chain Chimeric Antigen Receptor. 
     
     
         22 . An isolated T-cell of  claim 19 , further comprising an exogenous nucleic acid comprising at least a fragment of pTalpha transgene to support CD3 surface expression. 
     
     
         23 . An isolated T-cell of  claim 19  for its use as a medicament. 
     
     
         24 . An isolated T-cell of  claim 19  for treating a cancer or a viral infection. 
     
     
         25 . An isolated T-cell of  claim 19  for treating lymphoma. 
     
     
         26 . A pharmaceutical composition comprising at least one isolated T-cell of  claim 19 . 
     
     
         27 . A method for treating a patient comprising:
 (a) preparing a population of modified T-cells according to the method of  claim 1 ; and   (b) administrating said transformed T-cells to said patient.   
     
     
         28 . The method according to  claim 27 , wherein said patient is diagnosed with cancer, a viral infection, an autoimmune disorder or Graft versus Host Disease (GvHD).