In Vitro Preactivation of PBMCs with PM-mb21-41BBL Particles Stimulates in Vivo Expansion of NK Cells Under Low IL-2 in NSG Mice

NK cell immunotherapy as a cancer treatment shows promise, but methods for consistent expansion of NK cells from a small fraction (~5%) of peripheral blood mononuclear cells (PBMCs) to therapeutically effective amounts are not widely accessible. Strategies that promote in vivo expansion rely on high-dose IL-2 in combination with lymphodepletion and suffer from severe treatment related toxicities as well as limited expansion. On the other hand, in vitro methods are available for robust NK cell expansion, but rely on prolonged in vitro co-culture with feeder cells that is costly and complex and requires high dose IL-2 (25,000-35,000 U) for persistence after injection into mice. An optimal NK cell expansion method would rely on “off the shelf” reagents, would not require long-term complex cultures, and would induce a rapid, sustained in vivo expansion of NK cells from unsorted PBMCs under low concentration of IL-2. We have developed a method to prepare particles from K562 cells engineered to express specific cytokines, and such particles stimulate NK cell expansion. Particles prepared using K562 cells expressing IL-21 and 41BBL (PM21 particles) cause highly specific expansion of cytotoxic NK cells from unsorted PBMCs that achieves 95% NK cells in ~14 days and ~10,000 fold expansion of NK in about 21 days. NK cell expansion is consistent using different preparations of PM-particles or leukocyte sources, and the PM-particles retain expansion efficacy with storage. The PM-mb21-41BBL particles were also used to stimulate in vivo NK cell expansion in NSG mice under ultralow IL-2 (1,000 units per injection, 3 injections per week, per mouse). Unsorted human PBMCs, either shortly pre-activated in culture for two days or not pre-activated, were injected (2 x 10⁶ PBMCs per mice by i.p.) and human CD56⁺CD3^- NK cells and other relevant hCD45⁺ lymphocytes were monitored in the peripheral blood over time and in tissues and fluids at the time of euthanization. In vivo NK cell expansion was observed in mice injected with PBMCs that were pre-activated with PM21 particles. The extent of NK cell expansion observed in the peripheral blood was in levels that would be relevant for clinical cancer treatment (>50,000 NK cells/mL of mouse blood 14 days post injection). In vivo NK cell expansion was further confirmed with analogous experiments using PBMCs that were stained with CellTrace Violet to monitor proliferation. Notably, NK cells were also found in spleen, bone marrow, lung, liver and brain. These results taken together provide proof of principle that PM-particle technology is effective for direct in vivo NK cell expansion from unsorted PBMCs under low dose IL-2.