246 A GPRC5D and BCMA bispecific CAR-T product demonstrated dual antigen targeting capability and efficacy against multiple myeloma cells bearing BCMA mutations associated with treatment resistance

Background

Although B-cell Maturation Antigen (BCMA)-targeting immunotherapies greatly improve survival in multiple myeloma (MM) patients, antigen escape remains a significant challenge preventing long-term tumor control.^{1 2}. Post-BCMA treatment and relapsed patients often exhibit mutations, biallelic loss, and downregulation of BCMA gene expression.³ We previously reported the development of a G Protein-Coupled Receptor Class C Group 5 Member D (GPRC5D)/BCMA bispecific CAR that demonstrated robust efficacy against MM tumor lines both in vitro and in vivo.⁴ Here, we present a dual-targeting CAR that successfully addresses antigen escape challenges, providing an alternative treatment option for refractory/relapsed (R/R) MM.

Methods

The GPRC5D/BCMA bispecific CAR was developed with novel binders with high binding affinity and specificity. These CARs were optimized for binder combination, binder order, and CAR backbone structure. To assess binder affinity and killing efficacy, flow cytometry and in vitro long-term cytotoxicity assays were utilized against tumor cells expressing wild-type and mutant BCMA antigens.

Results

The GPRC5D/BCMA bispecific CAR was designed to address antigen escape resulting from loss or downregulation of antigens, or reduced binding of BCMA-targeting therapy to the mutated BCMA. To confirm the dual antigen cytotoxicity of the CAR, we expressed GPRC5D, BCMA, or both in HEK293 cells for in vitro killing assay. Notably, no significant cytotoxicity was observed in parental HEK293 cells, which lack expression of either antigen. Importantly, the GPRC5D or BCMA mono-targeting control CARs selectively controlled HEK293-GPRC5D and HEK293-BCMA cells, respectively. In comparison, the dual-targeting CAR demonstrated remarkable efficacy against HEK293 cells expressing either single or dual antigens (table 1). Recently, alterations in BCMA (R27P, P33S, S30del, and P34del) have been reported in MM patients relapsed after BCMA-targeting T cell engagers (TCEs) treatment. The BCMA binder in the bispecific CAR exhibited nanomolar-level EC50s, substantially lower than TCE controls, for all four mutant proteins (table 1). Additionally, in vitro cytotoxicity assay demonstrated that the bispecific CAR effectively suppressed the growth of cells expressing BCMA mutant proteins (table 1).

Conclusions

In our current investigation, we elucidated the dual-targeting capacity of the previously reported GPRC5D/BCMA bispecific CAR. Moreover, the BCMA binder within the bispecific CAR exhibits a high binding affinity to BCMA mutants associated with resistance to BCMA TCEs. Notably, the bispecific CAR also demonstrated robust cytotoxicity against cells expressing these BCMA mutants. These compelling findings strongly suggest that the GPRC5D/BCMA bispecific CAR holds promise in addressing antigen escape, meeting the unmet medical needs of R/R MM patients, and potentially improving survival rate.

References

Paula Rodriguez-Otero, et al. Ide-cel or standard regimens in relapsed and refractory multiple myeloma. N Eng J Med 2023;388:1002–1014. Jesús San-Miguel, et al. Cilta-cel or standard care in lenalidomide-refractory multiple myeloma. N Engl J Med. 2023;389(4):335–347. Holly Lee, et al. Mechanisms of antigen escape from BCMA- or GPRC5D-targeted immunotherapies in multiple myeloma. Nature Medicine. 2023;29:2295–2306. Chia-Wei Chang, et al. G Protein-Coupled receptor class C group 5 member D (Gprc5d) and B-Cell Maturation Antigen (BCMA) Bi-specific dual chimeric antigen receptors (CARS) Effectively address antigen escape and tumor heterogeneity challenge in multiple myeloma (MM). ASGCT 27th Annual Meeting. Molecular Therapy 2024;32:4S1:205–206.