Car nk cells

A method for the manufacturing of a population of NK-cells, genetically modified with a Chimeric Antigen Receptor (CAR) comprising:

• (i) A first phase of production of an intermediate cellular population containing CD34+ cells carrying at least one polynucleotide coding for a CAR according to the following steps:
• a) obtaining a biological starting sample selected from the group consisting of bone marrow, peripheral blood, placenta or umbilical cord blood,
• b) isolating hematopoietic stem cells from such biological sample,
• c) culturing the isolated hematopoietic stem cells in the presence of a culture medium I,
• d) transducing cultured stem cells by incubating such cells with a lentiviral vector carrying at least a polynucleotide coding for a CAR, thus obtaining an intermediate cellular population containing CAR-CD34+ stem cells, and
• e) optionally, culturing the intermediate cellular population containing CAR-CD34+ stem cells in culture medium I for at least one day, wherein
• culture medium I is a basic culture medium, comprising a collection of cytokines, wherein said collection of cytokines comprises Interleukin-7 and one or more of stem cell factor (SCF), flt-3Ligand (FLT-3L), thrombopoietin (TPO), and two or more of granulocyte-macrophage-colony-stimulating factor (GM-CSF), granulocyte-colony-stimulating factor (G-CSF), and interleukin-6 (IL-6),
• (ii) a second phase comprising:
• an optional, preliminary step of further expanding the intermediate cellular population containing CAR-CD34+ stem cells from the first phase in culture medium II, thereby obtaining a cellular population containing CAR-CD34+ stem cells and CAR-NK progenitor cells, wherein culture medium II is a basic culture medium comprising a collection of cytokines, wherein said collection of cytokines comprises two or more of SCF, FLT-3L interleukin-15 (IL-15) and IL-7 and two or more of GM-CSF, G-CSF, and IL6, and
• a step of expanding and differentiating the intermediate cellular population containing CAR-CD34+ stem cells from phase I or the cellular population containing CAR-CD34+ stem cells and CAR-NK progenitor cells from the optional preliminary step into a cellular population containing CAR-NK cells, the step comprising culturing the intermediate cellular population from phase I containing CAR-CD34+ stem cells or the cellular population from the optional preliminary step containing CAR-CD34+ stem cells and CAR-NK progenitor cells in culture medium III, thereby obtaining a cellular population containing NK-cells wherein the culture medium III is a basic culture medium comprising a collection of cytokines, wherein said collection of cytokines comprises two or more of SCF, IL-7, IL-15 and interleukin-2 (IL-2) and two or more of GM-CSF, G-CSF, and IL-6.