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

Abstract

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.

PD-L1 blockade enhances anti-tumor efficacy of NK cells

“…NK cells were expanded using PM21-particles as previously described. 24,25 Briefly, whole PBMCs or T-cell depleted PBMCs (EasySep CD3 positive selection kit; StemCell Technologies) were cultured for 14 days with 100 U/mL IL2 (Peprotech) and 200 µg/mL PM21 particles in SCGM media (CellGenix) supplemented with 10% FBS, 2 mM Glutamax. For mouse experiments, expanded NK cells were further T-cell depleted on day 14 if T cell content was above 3% and used fresh or frozen for future use (see Supplemental Table 1 for exact information about T cell content and procedure used).…”

“…15 Recently, the K562-mb21 cell based method was modified to a cell-free, PM21-particle based method for both ex vivo and in vivo specific expansion of NK cells which can eliminate some logistical and safety concerns while also retaining the benefits of the feeder-cell based expansion. 24,25 These significant breakthroughs made in regards to generating large doses of NK cells allow for their potential use as a viable and attractive therapeutic option for cancer treatment.…”

Natural killer cell immunosenescence in acute myeloid leukaemia patients: new targets for immunotherapeutic strategies?

“…Recently, a particle-based NK cell expansion technology has been developed using plasma membrane particles derived from the K562 cell line engineered to express IL-15 and CD137. This technology induces the selective expansion of large numbers of NK cells with high cytotoxic capacity against leukaemic cell lines and leukaemia blasts, facilitating the expansion of NK cells for clinical use [59]. The development of methods for the expansion of large numbers of NK cells from blood or pluripotent progenitors may contribute to the feasibility of adoptive transfer clinical trials.…”

NK cell therapy after hematopoietic stem cell transplantation: can we improve anti-tumor effect?

“…Although the culture conditions have been standardized according to good manufacturing practices, the use of cancer cell lines for NK cell production and clinical applications is still controversial. Therefore, new feeder cell-free methods are under investigation and one very successful method, among others, is the use of particles prepared from plasma membrane of K562-MB21-41BBL cells that are derived from the previously described K562 cell line expressing 4-1BB and membrane-bound IL-21 (PM21 particles) [39,40], which yields high numbers of cytotoxic NK cells that home to different organ sites after infusion in NSG mice.…”