CAR19 iPSC-Derived NK Cells Utilize the Innate Functional Potential Mediated through NKG2A-Driven Education and Override the HLA-E Check Point to Effectively Target B Cell Lymphoma

Induced pluripotent stem cell (iPSC)-derived natural killer (iNK) cells offer a promising platform for off-the-shelf immunotherapy against cancer. A unique benefit of iPSC-derived immune effector cells is the possibility to perform multiple precision editing steps at the single cell level to achieve a homogenous effector cell population tailored to target a desired cancer type and equipped with selected functional properties. These functional edits are superimposed on the innate reactivity of NK cells to stress ligands and MHC downregulation (missing self). The ability of NK cells to sense missing self is based on a functional calibration to self MHC during a process termed NK cell education, the latter being critically dependent on signaling through inhibitory receptors, including CD94/NKG2A and killer cell immunoglobulin-like receptors (KIR). Whereas the process of NK cell differentiation into mature effector cells from iPSCs has been well characterized, the role of natural variation in inhibitory receptor expression and NK cell education remains poorly defined in iNK cells.

We used mass cytometry to map the receptor repertoire in series of iNK cell lines and genetic edits thereof during differentiation and in vitro expansion (Figure 1A and B). Similar to peripheral blood NK cells, the receptor repertoire was diversified but genetically hardwired showing consistent patterns within each iNK cell line but with slight variation between genetically distinct lines. NKG2A was the dominantly expressed inhibitory receptor ranging from 13% to 87% with the highest expression in multi-edited iNK cell lines engineered to express a chimeric antigen receptor against CD19, a high affinity, non-cleavable FcγRIIIa receptor (CD16) and a recombinant IL15 signaling complex (CAR19-iNK cells). KIR expression was generally low in all tested iNK cell lines but increased gradually during culture and was further increased by genetic silencing of NKG2A receptors. Interestingly, silencing of NKG2A lead to increased levels of the activating receptor NKG2C.

We monitored degranulation by iNK cell variants against K562 engineered to express varying levels of HLA-E as well as CD19⁺ Nalm-6 cells. Genetic silencing of ß2microglobulin (ß2m), associated with reduced levels of HLA-class I and HLA-E, led to dampened global functional responses in iNK cells, suggesting a positive impact of education during iNK cell differentiation and expansion (Figure 1C). Subset stratification revealed that NKG2A⁺ iNK cells showed superior functionality compared to NKG2A^- iNK cells across all iNK cell lines tested, albeit less striking in CAR19-iNK cells that showed the highest overall natural cytotoxicity (Figure 1D). Knockdown of NKG2A led to a general reduction in functional capacity of NK92 cells (Figure 1E-F) and CAR19-iNK cells (Figure 1H), supporting a critical role for NKG2A-driven education in iNK cells. Given the superior functionality of NKG2A⁺ iNK cells, we next addressed whether this advantage was countered by expression of the check point ligand HLA-E during target cell interactions. Although we noted a slight inhibitory impact on natural cytotoxicity in NK cells isolated and expanded from peripheral blood (PB-NK) against K562 cells expressing physiological levels of HLA-E, this effect was completely overridden in iNK cells and did not interfere with NKG2A⁺ CAR-iNK cell recognition of HLA-E expressing CD19⁺ target cells (Figure 1G-H). Indeed, NKG2A⁺ CAR19-iNK showed superior degranulation against HLA-E expressing CD19⁺ Nalm-6 targets compared to CRISPR-edited NKG2A^-/- CAR19-iNK cells (Figure 1I).

Our results shed light on the regulatory gene circuits and cellular programs that determine functional potential in iPSC-derived NK cells products. Specifically, our results point to a crucial role for NKG2A-driven acquisition of a mature effector cell phenotype in combination with functional education through cognate ligands. Importantly, iNK cell education is operational during iNK cell differentiation and expansion without interfering with recognition of tumor targets expressing HLA-E.

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