Method for generating t cells progenitors

Abstract

The invention relates to an in vitro method to generate T cell progenitors, comprising the step of culturing CD34+ cells in a medium containing TNF-alpha and/or an antagonist of the Aryl hydrocarbon/Dioxin receptor, in particular StemRegenin 1 (SR1), in presence of a Notch ligand and optionally a fibronectin fragment.

1 .- 20 . (canceled) 
     
     
         20 . A CD7+ T cell progenitors population comprising T cells that are CD7+CD34−. 
     
     
         21 . The CD7+ T cell progenitors population according to  claim 20 , wherein more than 80% of the CD7+ cells are CD34−CD5−, CD34−CD1a−, or CD34−CD1a−CD5−. 
     
     
         22 . The CD7+ T cell progenitors population according to  claim 20 , comprising a transgene coding for a Chimeric Antigen Receptor (CAR). 
     
     
         23 . An in vitro method for generating a T cell progenitors population comprising T cells that are CD7+CD34−, comprising the step of culturing CD34+ cells in a medium comprising TNF-alpha and/or an antagonist of the Aryl hydrocarbon/Dioxin receptor, in the presence of an immobilized Notch ligand. 
     
     
         24 . The in vitro method according to  claim 23 , wherein the antagonist of the Aryl hydrocarbon/Dioxin receptor is StemRegenin 1 (SR1). 
     
     
         25 . The in vitro method according to  claim 23 , wherein the culture medium contains both TNF-alpha and the antagonist of the Aryl hydrocarbon/Dioxin receptor. 
     
     
         26 . The in vitro method according to  claim 23 , wherein TNF-alpha and/or the antagonist of the Aryl hydrocarbon/Dioxin receptor is/are present, in the culture medium, from day 0 of the culture. 
     
     
         27 . The in vitro method according to  claim 23 , wherein the CD34+ cells have been isolated from an adult donor. 
     
     
         28 . The in vitro method according to  claim 23 , wherein the cells are cultured in presence of TNF-alpha and/or SR1 for at most 10 days. 
     
     
         29 . The in vitro method according to  claim 23 , wherein the cells are cultured in presence of TNF-alpha and/or the antagonist of the Aryl hydrocarbon/Dioxin receptor between 3 and 7 days. 
     
     
         30 . The in vitro method according to  claim 23 , wherein the TNF-alpha and/or the antagonist of the Aryl hydrocarbon/Dioxin receptor is/are added to the medium culture at a concentration higher or equal to 3 ng/ml. 
     
     
         31 . The in vitro method according to  claim 23 , wherein the Notch ligand is the soluble domain of the Delta-like-4 ligand, fused to a Fc region of an IgG protein. 
     
     
         32 . The in vitro method according to  claim 23 , wherein the cells are also exposed to a fibronectin fragment, wherein said fragment comprises the RGDS and CS-1 patterns as well as a heparin-binding domain. 
     
     
         33 . The in vitro method according to  claim 32 , wherein the fibronectin fragment is Retronectin®. 
     
     
         34 . The in vitro method according to  claim 23 , wherein the culture medium contains at least three cytokines or growth factors chosen from the group consisting of Interleukin 7 (IL-7), Stem Cells Factor (SCF), thrombopoietin (TPO), and Flt3 ligand (FLT3L). 
     
     
         35 . The in vitro method according to  claim 23 , further comprising the steps of
 a. purifying the generated T cells progenitors   b. optionally conditioning the T cells progenitors in a pouch for injection to a patient.   
     
     
         36 . The in vitro method according to  claim 23 , further comprising a step of exposing the cells to a vector intended for transfection or transduction of CD34+ cells. 
     
     
         37 . The in vitro method according to  claim 23 , further comprising a step of exposing the cells to a vector or nucleic acid sequences containing the element appropriate for gene editing. 
     
     
         38 . A kit for performing the method according to  claim 23 , comprising:
 (i) a coating medium containing a ligand of Notch, and optionally a fibronectin fragment   (ii) a medium adapted for culturing CD34+ cells and T cells   (iii) a progenitor expansion medium containing TNF-alpha and/or an antagonist of the Aryl hydrocarbon/Dioxin receptor and three or four cytokines selected from the group consisting of SCF, TPO, Flt3L, and IL-7.