CRISPR/Cas9-mediated CISH deletion and CD16a upregulation potentiates ADCC in NK cells against relapsed/refractory multiple myeloma

Abstract Background Relapsed/refractory multiple myeloma (RRMM) remains incurable for many patients despite recent advances in proteasome inhibitors and monoclonal antibodies. Although intrinsic checkpoint regulators like CISH and the restricted expression of FCGR3A (CD16a), the crucial receptor for ADCC, limit the activity of natural killer (NK) cells, which are becoming increasingly potent mediators of antibody-dependent cellular cytotoxicity (ADCC). Methods To increase CD16a expression, Cas9 ribonucleoprotein complexes that target the CISH locus were nucleofected into primary human NK cells along with a homology-directed repair template. To verify robust proliferation (> 20-fold expansion over 14 days), a two-fold increase in CD16a surface density, and editing efficiency (> 80% by flow cytometry), gene-edited cells were expanded ex vivo. IFN-γ and TNF-α secretion were measured by ELISA, and specific lysis was measured by chromium release in in vitro cytotoxicity tests against MM.1S and U266 myeloma lines with and without daratumumab (10 µg/mL). NSG mice with established MM.1S xenografts were given a single adoptive transfer of 1 × 10^7 engineered or control NK cells in conjunction with daratumumab for in vivo validation. Tumor volumes and overall survival were tracked for 60 days. Results In the presence of daratumumab, CRISPR-edited NK cells achieved 65 ± 5% specific lysis of RRMM targets compared to 28 ± 4% by unedited controls (p < 0.001). Engineered cells secreted 3.2-fold more IFN-γ and 2.8-fold more TNF-α (p < 0.01 for both cytokines). In vivo, treated mice exhibited a prolonged tumor doubling time (28 vs. 14 days; p = 0.002) and extended median survival (57 vs. 39 days; p = 0.005), without evidence of body-weight loss or cytokine release syndrome. Conclusion In both preclinical in vitro and in vivo models, CRISPR/Cas9 deletion of CISH in conjunction with CD16a upregulation significantly increases NK-cell ADCC against RRMM. For patients who are not responding to current treatments, this dual-engineering strategy offers a promising next-generation cellular immunotherapy.