An improved method to produce clinical scale natural killer cells from human pluripotent stem cells

Abstract

Human natural killer (NK) cell-based adoptive anti-cancer 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 desired. 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 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-21 expressing artificial antigen-presenting cells. However, prior to differentiation, this method requires single cell adaption 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 hESC/hiPSCs are directly used to generate hematoendothelial 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.

Recent Advances in CAR-Based Solid Tumor Immunotherapy

“…CAR-NK cells can be generated from various sources, including peripheral and cord blood, induced pluripotent stem cells (iPSCs), and cell lines [99][100][101][102][103]. The tumor-killing activity of CAR-NK cells can be regulated by CAR-dependent and -independent ADCC mechanisms [99].…”

“…Can be generated from different sources (peripheral blood, cord blood, iPSCs, and cell lines)[99][100][101][102][103]]• Anti-tumor activity is derived from both CAR-dependent and -independent ADCC mechanisms [99] • Low risk of GvHD, CRS, and ICANS [99] • "off-the-shelf" allogeneic CAR-NK cell therapy [99] NK cells to express chemokine receptors [41] -CAR-NK cells designed to target Tregs, mesenchymal stromal cells, and cancer-associated fibroblasts [104] • Low CAR transduction efficiency -Non-viral electroporation (mRNA, transposon) [105,106] -Small molecular compounds to enhance the viral transduction of NK cells (e.g., retronectin, vectofusin-1, and PEG2) [106] • Limited ex vivo expansion -Engineered feeder cells to express immunostimulatory signaling molecules (e.g., IL-21, IL-15, and 4-1BB) [109,110] -Engineered CAR-NK cells to express IL-12, IL-15, and IL-18 [104,111,112] • Limited persistence in the immunosuppressive TME -CAR-NK cells expressing immune cell-stimulating cytokines (e.g., IL-12, IL-18, and IL-15) [41,106] -CAR-NK cells in combination with immune checkpoints inhibitors[41,104] …”