CIB315: An Allogeneic, Off-the-Shelf Anti-GPRC5D iPSC-Derived CAR-NK Product Targeting Multiple Myeloma

Human induced-Pluripotent Stem Cells (hiPSCs)-derived NK cells have been widely used to develop anti-tumor therapy. iPSCs can self-renew and are easy to engineer with designer nucleases. They provide a robust platform for developing anti-tumor iPSC-derived NK therapies with enhanced functions that include antigen-specific cytotoxicity, long term persistence and enhanced antibody-dependent cell-mediated cytotoxicity (ADCC). GPRC5D has been identified as a novel target for the treatment of multiple myeloma (MM), and recent data from clinical trials show that MM patients treated with anti-GPRC5D bispecific antibodies or CAR-T cells achieved a good response. Anti-GPRC5D therapy is also effective in patients with previous anti-BCMA therapy. In this context, we developed an anti-GPRC5D therapy using human iPSC-derived CAR-NK cells, termed CIB315. CIB315 contains multiple gene edits, aimed at killing GPCR5D⁺ cells in an antigen-specific manner with an anti-GPRC5D CAR, as well as enhanced persistence through metabolic reprogramming by ATOD4 knockout, and enhanced ADCC by expression of CD16. In addition, CIB315 also has an edit of CD38 knockout to avoid daratumumab-induced fratricide. We conducted a multiplexed gene editing simultaneously at the iPSC stage. CIB315 was manufactured from a genome-edited iPSC and further expanded using a serum-free and aAPC-free protocol. With this protocol, CIB315 can be generated in 30 days with 99.4% of cells expressing anti-GPRC5D CAR and CD16. Compared with WT iNK, CIB315 demonstrated antigen-specific cytotoxicity (50.4% ± 16.7% vs. 17.2% ± 6.3%; incubated for 4 hours at E:T 4:1) when incubated with MM.1S-Luc, which was derived from an MM patient, and expressed GPRC5D. After adding 0.2 ug/mL daratumumab, CIB315 showed a robust increase in cytotoxicity (96.9% ± 0.2%) to MM.1S-Luc, demonstrating an enhanced ADCC effect. As well as daratumumab, CIB315 could effectively deplete U87 MG in a tumor spheroid model by Cetuximab (anti-EGFR)-mediated ADCC. In addition, there was no evidence of CIB315 fratricide when the concentration of daratumumab was increased from 2 ug/mL or 10 ug/mL. Interestingly, CIB315 also supported killing of MM.1S-Luc for multiple rounds. Our data also revealed that CD38 knockout was helpful to these multiple rounds of killing target cells. Cryopreserved CIB315 cells that were subsequently thawed and rested demonstrated strong viability (>90%) and maintained their expression of activating receptors when compared with fresh cells with no significant decrease in cytotoxicity to K562 cells or MM tumor cell lines. In vivo study revealed that both low dose and high dose CIB315 was safe with no associated severe toxic reactions in each group of animals. A qRT-PCR assay was used to measure the in vivopersistence of CIB315 according to the number of anti-GPRC5D CAR copies per microgram of genomic DNA. Our results showed that CIB315 could persist for at least two weeks in NOG mice, and IL-2 could better support NK cell proliferation in vivo. Our results demonstrate that CIB315 can serve as a potent allogeneic, off-the-shelf anti-GPRC5D therapy for multiple myeloma.