Identification of LPAR1/LPAR5 As Novel GPCR Partners of GPRC5D for the Efficient CAR-T Therapy of Multiple Myeloma

Introduction: The emergence of chimeric antigen receptor T-cell (CAR T) therapy has brought hope to the treatment of refractory or relapsed multiple myeloma (RRMM). GPRC5D is an orphan G protein-coupled receptor (GPCR), which has a seven-transmembrane structure, and really high expression in multiple myeloma cells. So that CAR-T cells that target GPRC5D and this has been validated in a phase I/II clinical trials with encouraging clinical efficacy and manageable safety profiles of anti-GPRC5D CAR T-cell therapy for RRMM. It is of great clinical significance to find ways to further improve the efficacy of anti-GPRC5D CAR T-cell therapy.

Methods: Our previous study based on a single-cell transcriptome analysis revealed a strong co-expression correlation of GPRC5D and lysophosphatidic acid receptor 1/5 (LPAR1/5). To further confirm the interaction with GPRC5D, LPAR1 and LPAR5 were tested by co-immunoprecipitation (co-IP) screening, bimolecular fluorescence complementation (BiFC), multiple pharmacological ELISA assays, and flow cytometry (FACS) experiments. The MM cell lines (RPMI 8226, L363 and KMS11) were treated with activator or antagonists of LPAR1 or LPAR5, and then co-cultured with anti-GPRC5D CAR T-cells. The cytotoxicity and cytokine analysis were determined by luciferase-base killing assay and FACS.

Results: The results showed the obvious interactions between LPAR1/LPAR5 and GPRC5D from co-IP and BiFC assays, strongly supported the prediction from the bioinformatic analysis. The heterodimers of GPRC5D and LPAR1/LPAR5 influenced membrane expression of GPRC5D to different degrees based on ELISA assay. The HEK293T surface expression of GPRC5D was reduced in the presence of LPA receptors activator (1-Oleoyl lysophosphatidic acid, 1-Oleoyl-LPA), which indicated that both LPAR1 and LPAR5 had trafficking effect on GPRC5D. To further confirm these effects, the MM cell lines, RPMI 8226, L363 and KMS11, were cultured with 1-Oleoyl-LPA or combining antagonists of LPAR1 (AM966) or LPAR5 (TC LPA5 4). The cell-surface change of GPRC5D expression had no statistical difference when MM cells were only treated with 1-Oleoyl-LPA. However, the surface expression of GPRC5D of MM cell lines was increased with the combination of LPAR1/LPAR5 activator and antagonists by FACS detection

To further investigate the above effects could promote the efficacy of anti-GPRC5D CAR T-cell therapy, MM cell lines and anti-GPRC5D CAR T-cells were co-cultured, with or without the treatment of activator and antagonists. The results showed that the CAR T-cell killing capacity to MM cell lines was reduced after the treatment of 1-Oleoyl-LPA, while its killing capacity was significantly recovered after performing a combination of 1-Oleoyl-LPA and AM966/TC LPA5 4. Notably, the levels of IL-2 and TNF-α in 1-Oleoyl-LPA groups were lower than the mock groups, and increased after the combination of AM966/TC LPA5 4. But, IFN-γ did not display significant difference. These data indicated that the regulation of LPAR1 and LPAR5 function might have enhance the efficacity in anti-GPRC5D CAR T-cell therapy.

Conclusions: Our study provides hint evidence that LPAR1 and LPAR5 increase the MM cell-surface expression of GPRC5D and improve the efficacy of anti-GPRC5D CAR T-cell therapy, which might be as novel GPCR partners of GPRC5D for the efficient CAR-T therapy of multiple myeloma.