Efficacy of Human iPSC-Derived CAR-NK Cells Targeting Multiple Myeloma Cells

NK cells armed with chimeric antigen receptors (CAR) enable NK cells specifically to target cancer cells by recognizing tumor associated antigens. Previous studies have proven that human iPSC-derived CAR-NK cells have enhanced anti-tumor activity. Targeted B-cell maturation antigen (BCMA) therapy for relapsed refractory MM (rrMM) has been widely used with antibody-drug conjugate (ADC), bispecific antibodies, and CART cells, which has produced a rapid response, but relapse is common. Recent work has revealed GPRC5D as an alternative target in MM for immunotherapy and patients who have previously received anti-BCMA therapy have responded to GPRC5D CART cells. To assess target GPRC5D therapy with CAR-NK cells, which may in future serve as allogeneic immunotherapy, we transduced anti-GPRC5D CAR into an iPSC line derived from a healthy donor using a piggybac transposon system. More than 97% of CAR inserted-iPSCs (CAR-iPSCs) expressed anti-GPRC5D scFV with a similar expression level identified in CAR-iPSC-derived-NK cells (CAR-iNK, 92.7%). The anti-GPRC5D CAR-iNK demonstrated high purity (>99.9% for CD45 ⁺ and CD56 ⁺) and expressed a high level of CD16 (63.6%), the NK cell activating receptor NKG2D (96.3%) and NKp30 (98.7%), and the co-stimulatory receptors CD244 (99.6%) and CD226 (97.8%). Nonetheless expression of TCR⍺β and TCRƳ- was relatively low (<2%). Cytotoxicity assay revealed that anti-GPRC5D CAR-iNK had similar cytotoxicity against K562 cells (No GPRC5D antigen) to cord blood-derived NK cells (CB-NK), wide type (WT) iNK and anti-BCMA CAR-iNK. Cytotoxicity was dose-dependent indicating that these NK cells had similar innate anti-cancer cytotoxicity. When incubated at a ratio of 4:1 with NCI-H929 cells (MM) for 4 hours, which express a high level of both BCMA and GPRC5D, anti-GPRC5D CAR-iNK demonstrated an overwhelming advantage (approximately 90% killing rate) over the CB-NK (no killing) and WT iNK (around 10% killing rate). Furthermore, the anti-GPRC5D CAR-iNK also showed superior cytotoxicity to anti-BCMA CAR-iNK (Figure 1), indicating that GPRC5D may be a more efficient target for CAR-NK-based immunotherapy in MM. We further confirmed that anti-GPRC5D CAR-NK have acceptable cytotoxicity to NCI-H929 and OPM-2 cells, even when transported over a long distance (more than 2000 km) and following cryopreservation. The lower expression by OPM-2 cells of BCMA and GPRC5D compared with NCI-H929 indicates that both fresh and cryopreserved anti-GPRC5D CAR-iNK can specifically target MM with a higher and lower expression of GPRC5D. In addition, cryopreserved anti-GPRC5D CAR-iNK cells maintain comparable antigen-specific cytotoxicity in vitro, and demonstrate the ability to reduce tumor burden in an antigen-specific manner in an OPM-2 xenograft model. Our results demonstrate that genome-engineered human iPSCs can provide an unlimited source for manufacturing scalable, off-the-shelf, and cost-effective CAR-NK cells, and GPRC5D is a potent immunotherapeutic target in multiple myeloma.