METHOD FOR NATURAL KILLER CELL EXPANSION
The present invention provides a method for in-vitro culturing and expanding natural killer (NK) cells in a cell culture medium comprising a population of NK cells, the method comprising a) adding an effective concentration of interleukin-21 (IL-21) at the beginning of the culturing process to said medium, b) adding repeatedly an effective concentration of interleukin-2 (IL-2) and/or interleukin-15 (IL-15) to said medium, and c) adding repeatedly feeder cells or membrane particles thereof to said medium, wherein said feeder cells are B cell derived which are EBV immortalized; and wherein said expansion of NK cells in said cell culture medium is maintained for at least 3 weeks.
1 ) A method for in-vitro culturing and expanding natural killer (NK) cells in a cell culture medium comprising a population of NK cells, the method comprising
a) adding an effective concentration of interleukin-21 (IL-21) at the beginning of the culturing process to said medium
b) adding repeatedly an effective concentration of interleukin-2 (IL-2) and/or interleukin-15 (IL-15) to said medium
c) adding repeatedly feeder cells or membrane particles thereof to said medium, wherein said feeder cells are B cell derived which are EBV immortalized; and
wherein said expansion of NK cells in said cell culture medium is maintained for at least 3 weeks.
2 ) The method according to claim 1 , wherein said effective concentration of IL-21 in said cell culture medium is between 0.1 and 1000 ng/mL.
3 ) The method according to claim 1 or 2 , wherein said effective concentration of IL-2 in said cell culture system is between 1 U/mL and 5000 U/mL and/or said effective concentration of IL-15 in said cell culture system is between 0.1 and 1000 ng/mL.
4 ) The method according to any one of claims 1 to 3 , wherein said adding repeatedly feeder cells to said medium is performed between day 5 and day 16 after the previous feeder cell addition.
5 ) The method according to any one of claims 1 to 4 , wherein said expansion of NK cells in said cell culture medium is maintained for at least 5 weeks.
6 ) The method according to any one of claims 1 to 5 , wherein said NK cells in a cell culture medium comprising a population of NK cells are purified NK cells.
7 ) The method of any one of claims 1 to 6 , wherein the starting concentration of said NK cells in a cell culture medium comprising a population of NK cells is between 20 cells/mL and 2×10 7 cells/mL.
8 ) The method of any one of claims 1 to 7 , wherein the starting concentration of said NK cells in a cell culture medium comprising a population of NK cells is between 20 cells/mL and 2.5×10 4 cells/mL.
9 ) The method of any one of claims 1 to 8 , wherein said feeder cells are genetically non-modified cells with exception of the integration of the EBV genome.
10 ) The method of any one of claims 1 to 9 , wherein said NK cells in a cell culture medium comprising a population of NK cells are genetically modified.
11 ) The method according to claim 10 , wherein said genetically modified NK cells express a chimeric antigen receptor (CAR).
12 ) The method according to any one of claims 1 to 11 , wherein said method is performed in an automated process.
13 ) The method according to claim 12 , wherein said process is performed in a closed system.
14 ) A pharmaceutical composition comprising a population of NK cells produced according to the method of claims 1 - 13 .