METHOD FOR GENERATING T CELLS PROGENITORS
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.