Sequential Exposure to Interleukin-21 and Interleukin-15 During Human Natural Killer Cell Expansion Optimizes Yield and Function

NK cells are frequently expanded for the clinic using irradiated engineered K562 feeder cells expressing a core transgene set of membrane-bound mb IL-15 and/or mbIL-21 together with 41BBL. Prior comparisons of mbIL-15 to mbIL-21 for NK expansion lack comparisons of key attributes of the resulting NK cells including their high dimensional phenotype, polyfunctionality, the breadth and potency of cytotoxicity, cellular metabolism, and activity in xenograft tumor models. Moreover, despite multiple rounds of K562 stimulation, studies of sequential use of mbIL-15- and mbIL-21-based feeder cells are absent. We addressed these gaps and found that using mbIL-15 versus mbIL-21-based feeder cells drove distinct phenotypic and functional profiles. Feeder cells expressing mbIL-15 alone drove superior functionality by nearly all measures, while those expressing mbIL-21 alone drove superior yield. In combination, most attributes resembled those imparted by mbIL-21, whereas in sequence NK yield approximated that imparted by the first cytokine, and the phenotype, transcriptome, and function resembled that driven by the second cytokine highlighting the plasticity of NK cell differentiation. The sequence mbIL-21 followed by mbIL-15 was especially advantageous in achieving significant yields of highly functional NK cells that demonstrated equivalent in vivo activity to those expanded by mbIL-15 alone in 2 of 3 xenograft models. Our findings define the impact of mbIL-15 versus mbIL-21 during NK expansion and reveal a previously under appreciated tradeoff between NK yield and function for which sequential use of mbIL-21- followed by mbIL-15-based feeder cells may be the optimal approach in many settings. To characterize NK cells expanded with different K562 feeders expressing mbIL15 or/and mbIL21 and optimize the NK expansion process for producing sufficient functional NK cells, We co-cultured pure NK cells with irradiated K562-41BBL-CD40-mbIL15 or K562-41BBL-CD40-mbIL21 or K562-41BBL-CD40-mbIL15-mbIL21 or sequential K562-41BBL-CD40-mbIL15 and K562-41BBL-CD40-mbIL21. On day 20 of the expansion, NK cell yield, phenotype, functionality, metabolism, and transcriptional expression were assessed.