Talquetamab alters T cell transcriptome in presence of GPRC5D on MM cells

Abstract Background: G protein-coupled receptor class C group 5 member D (GPRC5D) has emerged as an important immune target in multiple myeloma (MM) however, its biological function still remains to be fully understood. Talquetamab, a GPRC5D-directed bispecific antibody, has shown an extraordinary efficacy of 70% in triple class refractory MM patients and received a regulatory approval for the treatment of RRMM patients. Despite this encouraging response patients continue to relapse and genomic loss and epigenetic regulation have been identified as a mechanism to downregulate the receptor. In order to better understand the interplay of talquetamab on GPRC5D loss models and T cells within the immune microenvironment, we performed single cell sequencing on a coculture of GPRC5D loss models and healthy T cells in presence and absence of talquetamab. Methods: GPRC5D mono- and biallelic knockout models were generated using CRISPR-Cas9 technology. Cocultures were established using healthy T cells with or without talquetamab for 24 hours. We performed CITE-seq to decipher the impact of talquetamab on MM cells and T cells in presence of GPRC5D. Cell hashing with TotalSeq-C antibodies enabled multiplexing. Pooled cells underwent 10x Genomics 5' GEM-X workflow and were sequenced on Illumina NovaSeq X Plus. Gene expression and ADT data were normalized, scaled, and analyzed via weighted nearest neighbor (WNN) analyses, followed by uniform manifold approximation and projection (UMAP) and unsupervised clustering using the Louvain algorithm. T and MM cell clusters were identified by canonical marker genes. Differential gene expression was assessed using the Wilcoxon rank-sum test, and interferon response scores were calculated using AddModuleScore. Results: Using CITE-seq we observed that both mono- and bi-allelic GPRC5D loss induced pronounced transcriptomic changes in MM cells. Among T cell clusters, identified based on expression of canonical markers (IL2RA, IRF4, TBX21, TNFRSF4, TNFRSF18, TNF, IFNG), CD4 and CD8 activated T cells were only observed in presence of talquetamab and GPRC5D+ cells and completely disappeared when stimulated with GPRC5DDel/Del models. To assess the impact of talquetamab on T cell transcriptomics across subpopulations, we performed pairwise differential gene expression analysis and observed a significant induction of interferon stimulated genes (GBP2, GBP4, GBP5, IFI44, IFIT2, ISG15) in presence of talquetamab with GPRC5D on MM cells (p-value < 0.001, absolute log2FC > 2). Interferon signaling genes (STAT1, IRF1) were found to be upregulated in T cells by talquetamab alone even in absence of GPRC5D+ cells in coculture. Talquetamab induced an increase in interferon response across T cell subpopulations (p<0,0001) and this response was exaggerated based on the presence of GPRC5D expressing MM cells (p<0,0001). Additionally, we also identified genes only upregulated in presence of GPRC5D and talquetamab, including SOCS3, CD69, JUNB, BATF, CISH, and PRDM1, indicating a unique activation status of these T cells. This upregulation was not observed in presence of GPRC5DDel/Del cells in coculture. To understand the impact of gene alterations on cell signaling pathways in different T cell subpopulations, we performed Gene set enrichment analysis. An enrichment of IFN-γ and IFNA-signaling (NES>2.0, FDR 0,0) was observed in CD4, CD8, Tcm and Treg populations. Additionally, IL2-STAT5 signaling and inflammatory response (NES>2.0, FDR 0.0) pathways were found to be enriched only in presence of talquetamab and GPRC5D+ cells. Of note, in the absence of talquetamab, T cells transcriptomic profile did not change in response to GPRC5D presence or absence on MM cells. Conclusion Our findings demonstrate that talquetamab even in the absence of GPRC5D upregulates the expression of interferon signaling genes in T cells and this response is enhanced when GPRC5D is present on MM cells. Moreover, talquetmab alters the activation profile across T cells subsets and both abundance and distribution of different T cells subpopulations is changed by GPRC5D status of the MM cells in coculture.