Differentiation of Human Natural Killer Cells From Pluripotent Stem Cells In a Feeder Free System Amenable to Clinical Translation

Abstract 107

Human natural killer (NK) cells are an attractive source of lymphocytes for adoptive immunotherapy. Although our understanding of natural killer cell biology continually grows, translating these concepts to the clinic has fallen behind. Currently, NK cell adoptive immunotherapy is only beneficial to a limited number of patients, particularly those with acute myelogenous leukemia (AML). In order to generate NK cells that are effective against a broader range of malignancies, our lab has focused on the generation of NK cells from human pluripotent stem cells. We have previously demonstrated the potency of NK cells derived from human embryonic stem cells (hESCs) both in vitro and in vivo. These previous studies utilized a stromal cell co-culture system to derive hematopoietic progenitor cells (CD34⁺CD45⁺ cells) from hESCs that can then produce CD45⁺CD56⁺ NK cells in a secondary culture system. More recently, we used a similar co-culture system to generate fully functional NK cells from several human induced pluripotent stem cell (iPSC) lines, which could provide NK cells on a patient-specific basis. Both hESC and iPSC-derived NK cells consist of a mature, homogenous population of cells expressing CD56, CD94, killer immunoglobulin-like receptors (KIRs), CD16, and the apoptosis-inducing ligands FasL and TRAIL. We now aim to convert this system into a completely defined stromal cell-free system. These studies will make this system more amenable to clinical scale up and allow us to better define elements essential for hematopoietic and NK cell development from human pluripotent stem cells. For example, hESC-derived NK cells would require minimal cell processing compared peripheral blood NK (PB-NK) cells that typically requires depletion of CD3⁺ and CD20⁺ cells to prevent GVH and passenger lymphocyte syndrome, respectively. Neither T cells nor B cells are present in our cultures, preventing these complications. Here, we have now used a spin embryoid body (“spin-EB”) approach potentially suitable for clinical scale-up. In these spin-EB cultures, defined numbers of undifferentiated hESCs or iPSCs are first aggregated in 96 well plates by centrifugation (3000 cells per well) in serum-free media containing only the cytokines SCF, BMP4, and VEGF. Under these stage 1 conditions, hematopoietic progenitor cells that express CD34, CD45, CD43, and CD31 develop and expand over 6–11 days. After this time, EBs are directly transferred (without dissociation or sorting) to Stage 2 cultures with EL08 stromal cells or stroma-free conditions in serum-free media containing NK cell-initiating cytokines (IL15, SCF, FLT3L, IL7, and IL3). We find that these Stage 2 cells acquire all the typical markers of mature NK cells (CD56, CD94, KIRs, etc). They also kill the CML target K562 cells at similar effector-to-target ratios as stromal-derived NK cells and PB-NK cells. Most notably, spin-EB-derived NK cells generated in entirely feeder free conditions also exhibit a typical NK cell phenotype and kill K562 targets. However, the NK cells derived in feeder-free Stage 2 conditions are less proliferative than those cultured in Stage 2 conditions on EL08 cells. Specifically, 12,000 undifferentiated hESCs or iPSCs give rise to 0.8–1 × 10⁶ NK cells using EL08 in Stage 2 cultures compared to 0.4– 1 × 10⁵ cells using the feeder-free Stage 2 conditions. Additionally, the efficiency of NK cell development from spin-EB cultures is not uniform: 58% of EBs produce NK cells in Stage 2 using EL08 stromal cells, and 25% of EBs produce NK cells in the completely feeder-free system. Based on these calculations, given 100% efficiency of this spin-EB NK cell developmental system, we estimate that 240,000 starting hESCs would provide enough NK cells to treat a single patient with a dose of 20 × 10⁶ NK cells. Even at the lower efficiencies demonstrated here, one plate of undifferentiated hESCs or iPSCs (typically with 5–10 × 10⁶ undifferentiated cells) could treat several patients. Ongoing work is focused on enhancing the percentage of hematopoietic progenitors obtained from the spin EB approach and expanding feeder-free NK cells with artificial antigen presenting cells in Stage 2 conditions.