An Improved Method to Produce Clinical-Scale Natural Killer Cells from Human Pluripotent Stem Cells.

Human natural killer (NK) cell-based adoptive anticancer immunotherapy has gained intense interest with many clinical trials actively recruiting patients to treat a variety of both hematological malignancies and solid tumors. Most of these trials use primary NK cells isolated either from peripheral blood (PB-NK cells) or umbilical cord blood (UCB-NK cells), though these sources require NK cell collection for each patient leading to donor variability and heterogeneity in the NK cell populations. In contrast, NK cells derived human embryonic stem cells (hESC-NK cells) or induced pluripotent stem cells (hiPSC-NK cells) provide more homogeneous cell populations that can be grown at clinical scale, and genetically engineered if needed. These characteristics make hESC-/iPSC-derived NK cells an ideal cell population for developing standardized, "off-the-shelf" immunotherapy products. Additionally, production of NK cells from undifferentiated human pluripotent stem cells enables studies to better define pathways that regulate human NK cell development and function. Our group previously has established a stromal-free, two-stage culture system to derive NK cells from hESC/hiPSC in vitro followed by clinical-scale expansion of these cells using interleukin (IL)-21 expressing artificial antigen-presenting cells. However, prior to differentiation, this method requires single-cell adaptation of hESCs/hiPSCs which takes months. Recently we optimized this method by adapting the mouse embryonic fibroblast-dependent hESC/hiPSC to feeder-free culture conditions. These feeder-free hESCs/hiPSCs are directly used to form embryoid body (EB) to generate hemato-endothelial precursor cells. This new method produces mature, functional NK cells with higher efficiency to enable rapid production of an essentially unlimited number of homogenous NK cells that can be used for standardized, targeted immunotherapy for the treatment of refractory cancers and infectious diseases.