Enhanced expansion and cytotoxicity of engineered natural killer cells and uses thereof

A population of engineered natural killer cells comprising,

• an engineered chimeric receptor configured to bind a marker on a target cancer cell and upon binding, induce the NK cell to exert a cytotoxic effect against the target cancer cell,
• wherein the NK cell was expanded by co-culturing for a first time, in a culture media comprising interleukin 2 (IL2), interleukin 12 (IL12), and interleukin 18 (IL18), a starting population of natural killer (NK) cells with a first population of feeder cells,
• wherein the first population of feeder cells comprises cells engineered to express 4-1 BBL and membrane-bound interleukin-15 (mbIL15),
• wherein the starting population of NK cells comprises fewer cells than the first population of feeder cells,
• wherein the first co-culturing results in an intermediate expanded population of NK cells;
• separating, after the first co-culturing, at least a portion of the intermediate expanded population of NK cells from the feeder cells,
• co-culturing for at least as second time, in fresh culture media, at least a portion of the intermediate expanded population of NK cells with a second population of the feeder cells,
• wherein the portion of the population of NK cells co-cultured with the second population of feeder cells comprises fewer cellsthan the second population of feeder cells,
• wherein the at least a second co-culturing results in a further expanded population of NK cells.

A method for enhancing the expansion of natural killer cells for use in immunotherapy, comprising:

• co-culturing, in a culture media, a population of natural killer (NK) cells with a first population of feeder cells for a first period of time,
• wherein the first feeder cell population comprises cells engineered to express 4-1 BBL and membrane-bound interleukin-15 (mbIL15),
• wherein the population of NK cells comprises fewer cells than the population of feeder cells,
• wherein the culture media comprises interleukin 2 (IL2), interleukin 12 (IL12), and interleukin 18 (IL18),
• wherein the IL2 is present in the media in a concentration between about 0.5 ng/mL and about 6.0 ng/mL, wherein the IL12 is present in the media at a concentration between about 10 ng/mL and about 100 ng/mL, and wherein the IL18 is present in the media at a concentration between about 0.01 ng/mL and about 30 ng/mL,
• wherein the co-culturing for the first period of time results in an expanded population of NK cells;
• separating, after the first period of time, at least a portion of the expanded population of NK cells from the feeder cells,
• co-culturing, in fresh culture media, the at least a portion of the expanded population of NK cells with a second population of the feeder cells for a second period of time,
• wherein the population of NK cells comprises fewer cells than the population of feeder cells,
• wherein the culture media comprises interleukin 2 (IL2), interleukin 12 (IL12), and interleukin 18 (IL18),
• wherein the co-culturing for the second period of time results in a further expanded population of NK cells;
• and,
• optionally repeating the separating and co-culturing steps at least one additional time using fresh culture media comprising IL2, IL12, and IL18, thereby resulting in additional expansion of the further expanded population of NK cells.