382 Generation of immune-evasive allogeneic adaptive NK cell therapies

<h3>Background</h3> Differences in the persistence and anti-tumor responses of various NK cell subsets represent key hurdles to the implementation of NK cell-based cancer immunotherapies. Our lab has recently developed a protocol for the specific expansion of the adaptive NK cell subpopulation.¹ Due to their unique KIR repertoire with dominant expression of one single KIR, adaptive NK cells deliver a strong, predictable missing-self response in an allogeneic, HLA-C/KIR mismatch setting. However, allorejection responses may significantly curtail their efficacy. In the present study, we explore a novel strategy for engineering immune-evasive allogeneic NK cells based on unidirectional manipulation of the immune synapse. <h3>Methods</h3> CD3/CD19-depleted PBMCs from adaptive donors were co-cultured with HLA-E-expressing, irradiated K562 cells in IL-2 supplemented media. On day 10 of the expansion, ICAM-1 and/or CD58 were knocked out using CRISPR/Cas9. On day 17, residual CD54+ or CD58+ NK cells were MACS depleted using FITC-conjugated anti-CD54 or -CD58 antibodies and anti-FITC MicroBeads. Adaptive NK cell potency in HLA-C/KIR matched and mismatched settings was explored in an Incucyte killing assay. <h3>Results</h3> We have previously shown that knockout of the adhesion ligands ICAM-1 and CD58 protect B2M-knockout iPSC-derived NK cells from host NK cell-mediated rejection.² Here, we develop a protocol for ICAM-1 and CD58 knockout in <i>ex vivo</i>-expanded adaptive NK cells to potentiate their use in an allogeneic setting. We achieved robust expansion (median 470-fold) of single self-KIR+NKG2C+ adaptive NK cells from donors with our adaptive NK cell expansion protocol. Combining this expansion protocol with multiplexed CRISPR/Cas9 editing at day 10 resulted in knockout efficiencies of over 80% for ICAM-1 and 50% for CD58 in difficult-to-edit, adaptive NK cells. Additionally, we demonstrate that our expanded, multiplex-edited adaptive NK cells retain their potent targeting of allogeneic HLA-C/KIR mismatched targets. <h3>Conclusions</h3> This protocol makes progress towards the development of highly potent, immune-evasive adaptive NK cells for use as off-the-shelf cancer immunotherapies. Future work will test the potency of these cells in models of allorejection. <h3>References</h3> Haroun-Izquierdo A,<i> et al</i>. Adaptive single-KIR+NKG2C+ NK cells expanded from select superdonors show potent missing-self reactivity and efficiently control HLA-mismatched acute myeloid leukemia. <i>J Immunother Cancer.</i> 2022;<b>10</b>(11):e005577. Hammer Q, Perica K, <i>et al</i>. Genetic ablation of adhesion ligands averts rejection of allogeneic immune cells.<i> Cell Stem Cell</i>. 2024; In press.