CAR NK cells

A method for the manufacturing of a population of NK-cells, genetically modified with a Chimeric Antigen Receptor (CAR), the method 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 material, or umbilical cord blood,
• b) isolating hematopoietic stem cells from such biological sample,
• c) obtaining a culture medium I by adding to a first basic culture medium a first collection of cytokines, wherein said first collection of cytokines comprises Interleukin-7 (IL-7), stem cell factor (SCF), flt-3Ligand (FLT-3L), thrombopoietin (TPO), granulocyte-macrophage-colony-stimulating factor (GM-CSF), granulocyte-colony-stimulating factor (G-CSF), and Interleukin-6 (IL-6),
• d) culturing the isolated hematopoietic stem cells in the presence of the culture medium I,
• e) 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
• f) culturing the intermediate cellular population containing CAR-CD34+ stem cells in the culture medium I for at least one day,
• (ii) a second phase comprising:
• g) obtaining a culture medium II by adding to a second basic culture medium a second collection of cytokines, wherein said second collection of cytokines comprises SCF, FLT-3L, Interleukin-15 (IL-15), IL-7, GM-CSF, G-CSF, and IL6,
• h) obtaining a culture medium III by adding to a third basic culture medium a third collection of cytokines, wherein said third collection of cytokines comprises SCF, IL-7, IL-15, Interleukin-2 (IL-2), GM-CSF, G-CSF, and IL-6,
• i) a step of further expanding the intermediate cellular population containing CAR-CD34+ stem cells from the first phase in the culture medium II, thereby obtaining a cellular population containing CAR-CD34+ stem cells and CAR-NK progenitor cells, and
• j) a step of expanding and differentiating the cellular population containing CAR-CD34+ stem cells and CAR-NK progenitor cells from step i) 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 the culture medium III, thereby obtaining a cellular population containing NK-cells.