Combination of cytokines, PM21-particle stimulation, and TGF-beta conditioning results in Natural Killer cells with enhanced cytotoxicity and infiltration of solid tumors

In this study, feeder cell-free PM21 particle activation of Natural Killer (NK) cells was combined with cytokine activation and TGF-beta conditioning to produce highly proliferative and cytotoxic NK cells that can infiltrate solid tumors. Development of NK cell therapeutics is a promising anti-cancer therapy. For clinical applications, NK cells are typically either ex vivo activated with cytokines or expanded with feeder cells or by feeder cell-free methods. One such method for feeder cell-free expansion of highly cytotoxic NK cells uses plasma membrane particles containing surface IL-21 and 41BBL (PM21). Additionally, NK cells expanded with IL-12, IL-15, and IL-18 in combination with PM21 stimulation (CAP-NK cells) exhibit robust proliferation, potent cytotoxicity, and memory-like features. To further improve their activity against solid tumors-a setting often resistant to NK cells-we introduced TGF-beta conditioning. Previous studies have shown that ex vivo TGF-beta conditioning induces IFNgamma hypersecretion. To assess whether this could enhance anti-tumor function, PM21- and CAP-NK cells were expanded in the presence of TGF-beta. The CAP-based expansion method still resulted in enhanced NK-cell expansion, even with TGF-beta conditioning. CAP-based expansion remained highly effective even with TGF-beta conditioning, achieving an average 4000±700-fold expansion by day 14 compared to 1800±60-fold for PM21-NK cells. TGF-beta conditioned NK cells retained high viability after cryopreservation (>80% immediately and at 16 h post-thaw) and 99±1% expressed CD25 by day 7. These cells produced significantly more IFNgamma upon stimulation than unconditioned CAP-NK cells. Moreover, TGF-beta conditioned CAP-NK cells acquired a tissue-resident-like phenotype (CD103+, CD49a+, CD300a−) not observed in PM21- or CAP-NK cells, which would be expected to enhance their ability to infiltrate tumors. To test this, labeled TGF-beta conditioned CAP-NK and PM21-NK cells were co-cultured with large lung tumor spheroids and monitored via live-cell imaging. TGF-beta conditioned NK cells penetrated tumors faster and deeper than PM21-NK cells, which remained mostly at the periphery, resulting in greater spheroid killing-even in tumors overexpressing TGF-beta.TGF-beta conditioning also enhanced cytotoxicity against multiple solid tumor cell line spheroids, including lung, pancreatic, and neuroblastoma. Overall, these findings support TGF-beta conditioned CAP-NK cells as a potent cellular therapy candidate for solid tumors.