Generation of Highly Cytotoxic Natural Killer Cells for Treatment of Acute Myelogenous Leukemia Using a Feeder-Free, Particle-Based Approach

• NK cell immunotherapy is promising, but consistent NK expansion remains challenging.
• We developed novel particle-based method for selective and efficient NK expansion.
• This method yields 250 fold NK cell expansion in 14 days from unselected PBMCs, and maximal expansion of 1,800 fold in 17-21 days with NK cell content increasing to 86%.
• Expanded NK cells are cytotoxic against AML/CML cell lines and also against patient AML blasts.
• Analysis of markers is consistent with a cytotoxic phenotype of expanded NK cells.

Natural killer (NK) cell immunotherapy as a cancer treatment shows promise, but expanding NK cells consistently from a small fraction (∼5%) of peripheral blood mononuclear cells (PBMCs) to therapeutic amounts remains challenging. Most current ex vivo expansion methods use co-culture with feeder cells (FC), but their use poses challenges for wide clinical application. We developed a particle-based NK cell expansion technology that uses plasma membrane particles (PM-particles) derived from K562-mbIL15-41BBL FCs. These PM-particles induce selective expansion of NK cells from unsorted PBMCs, with NK cells increasing 250-fold (median, 35; 10 donors; range, 94 to 1492) after 14 days of culture and up to 1265-fold (n = 14; range, 280 to 4426) typically after 17 days. The rate and efficiency of NK cell expansions with PM-particles and live FCs are comparable and far better than stimulation with soluble 41BBL, IL-15, and IL-2. Furthermore, NK cells expand selectively with PM-particles to 86% (median, 35; range, 71% to 99%) of total cells after 14 days. The extent of NK cell expansion and cell content was PM-particle concentration dependent. These NK cells were highly cytotoxic against several leukemic cell lines and also against patient acute myelogenous leukemia blasts. Phenotype analysis of these PM-particle–expanded NK cells was consistent with an activated cytotoxic phenotype. This novel NK cell expansion methodology has promising clinical therapeutic implications.