Engineering iPSC-derived NK cells with autocrine feeder signaling to enhance cytotoxic function

Natural killer (NK) cells are key effectors in cancer immunotherapy, but their clinical translation is hindered by limited cell expansion and persistence. Feeder-based expansion systems using tumor-derived K562 cells achieve robust cell proliferation but raise safety concerns. To address these challenges, we generated human induced pluripotent stem cell (iPSC)-derived NK cells endowed with self-feeder properties and autocrine signaling that promote cell expansion and functional activation while minimizing oncogenic risk. Specifically, iPSCs were differentiated into hematopoietic progenitors (iHPCs) under defined conditions and subsequently matured into NK (iNK) cells. The self-supporting iNK cells were then established by viral transduction to express CD86, 4-1BB ligand (4-1BBL), and interleukin-21 (IL-21) bispecific antibodies engineered in a single-chain variable fragment (scFv) format. We showed that the optimized hybrid differentiation protocol yielded highly pure (92.1% CD56⁺) NK cells that expressed early activation markers (CD161⁺) but remained phenotypically immature (CD94⁺ 2.2%, CD16⁺ 17.7%), consistent with an early-stage but lineage-committed NK phenotype. The engineered self-supporting iNK cells exhibited robust expression of CD86, 4-1BBL, and IL-21 bispecific scFv. Co-culturing the self-supporting iNK cells with primary NK cells isolated from peripheral blood prolonged the viability of the primary NK cells beyond the typical two-week lifespan, indicating self-feeder supportive function. Importantly, the self-supporting iNK cells exhibited enhanced cytotoxicity against K562 cells and retained their cytolytic function even after repeated re-challenge with K562 targets. In conclusion, the engineered iNK cells demonstrated dual functionality, acting both as feeder cells and as cytotoxic effectors against target cancer cells. These self-supporting iNK cells with autocrine feeder signaling hold potential to advance NK cell-based cancer immunotherapy.