Engineering Human Induced Pluripotent Stem Cells with Novel Chimeric Antigen Receptors to Generate Natural Killer (NK) Cell Cancer Immunotherapies with Targeted Anti-Tumor Activity

Chimeric antigen receptors (CARs) provide a powerful strategy to direct and enhance anti-tumor activity of immune effector cells. While most studies have evaluated CAR-expression in T cells, here we evaluate and optimize CAR constructs that are specifically designed with natural killer (NK) cell transmembrane and signaling domains. Since NK cell-mediated cytotoxicity does not require self-HLA expression, derivation of NK cells from human induced pluripotent stem cells (iPSCs), combined with the use of NK cell-specific CARs, enables production of a standardized, targeted allogeneic effector cell population. After screening 10 combinations of CARs using an anti-mesothelin (meso) scFv to target ovarian cancer cells, we identified a CAR construct containing the transmembrane domain of NKG2D, the 2B4 co-stimulatory domain, and the CD3ζ signaling domain that mediates a strong increase in intracellular NK cell signaling. iPSC-derived NK cells that express this novel CAR (NKCAR-iPSC-NK cells) have a surface phenotype similar to NK cells isolated from peripheral blood (PB-NK) and unmodified iPSC-derived NK cells. Interestingly, the NKCAR-iPSC-NK cells mediate improved killing of target-expressing targets more than iPSC-derived NK cells expressing a 3^rd generation CAR with CD28, CD137, and ζ signaling elements optimized for T cell-mediated activity (TCAR-iPSC-NK cells). We examined intracellular signaling pathways stimulated by our NKG2D-2B4-ζ CAR construct. In NKCAR-iPSC-NK cells, antigen (meso)-expressing targets promoted phosphorylation of Syk and Erk. Site-directed mutagenesis studies demonstrate the requirement for the NKG2D and 2B4 domains to mediate release of cytotoxic granules (CD107a), cytokine (IFN-γ) release and robust tumor cell lysis. Using an ovarian cancer xenograft model, a single dose of NKCAR-iPSC-NK cells markedly inhibited tumor growth, and mediated significantly enhanced survival (84 days) compared to NK cells isolated from peripheral blood (PB-NK cells) (70 day survival), non-CAR-expressing iPSC-NK cells (57 days), and TCAR-iPSC-NK cells (63 days) (p < 0.002 for NKCAR-iPSC-NK cells compared to each of the other 3 NK cell populations). As an additional strategy, we have engineered iPSCs both with this novel CAR using an anti-CD19 scFv and with a high-affinity non-cleavable CD16 receptor (hnCD16 with F158V (high-affinity) and S197P (non-cleavable) substitutions). To prevent antigen loss variants that promote resistance of B cell malignancies to anti-CD19-CAR-T cell-based therapies, this approach now allows us to combine the use of anti-CD19-CAR with anti-CD20 antibody to target B cell malignancies using engineered iPSC-NK cells. This combinatorial targeting of Raji cells (CD19+CD20+ B cell lymphoma) demonstrates enhanced upregulation of CD107a and TNFα with improved elimination of CD19-negative target cells. Together, these strategies provide a comprehensive approach to utilize NKCAR-iPSC-NK cells as a novel strategy to produce "off-the-shelf", targeted, allogeneic lymphocytes suitable to treat refractory solid tumor and hematologic malignancies.