Bi-Allelic GPRC5D Alterations Provide Proliferative Advantage in Presence of Talquetamab
Authors: Munawar Umair , Han Seungbin , Nerreter Silvia , Vogt Cornelia , Kurian Shilpa , Eiring Patrick , Weingart Julia , Zhou Xiang , Sauer Markus , Einsele Hermann , Rasche Leo , Waldschmidt Johannes , Kortüm K. Martin 
Published: December 2024

Background:
G protein-coupled receptor class C group 5 member D (GPRC5D) has recently emerged as a promising immunotherapy target in Multiple Myeloma (MM) although its function remains unknown. Mono-allelic GPRC5D alterations are found in 15% patients with newly diagnosed MM and these numbers are likely to increase in relapsed / refractory MM where most patients have been exposed to the anti-GPRC5D-CD3 bispecific antibody talquetamab. The functional impact of such baseline mono-allelic and acquired bi-allelic alterations of the GPRC5D gene remains to be elucidated. In this study, we modeled various GPRC5D genetic alterations and studied their impact on the biology of the disease and treatment responsiveness to anti-MM immunotherapies.
Methods:
Mono- and bi-allelic GPRC5D knock-out (KO) cell models were created using the MM cell line OPM-2 via CRISPR-Cas9 technology. GPRC5D allelic status in the cells was confirmed using digital droplet PCR (ddPCR) and Sanger sequencing. Direct stochastic optical reconstruction microscopy (dSTORM) was utilized for estimation of receptor distribution and epitope quantification of common immunotherapeutic targets such as GPRC5D, CD38, BCMA and SLAMF7. Functional assays were conducted using healthy effector T cells and treatment efficacy was tested for various immunotherapies and conventional anti-MMdrugs.
Results:
Gene expression profiling with RT-PCR confirmed that GPRC5DWt/Del cells had a 50% reduction in expression of GPRC5D mRNA (2 folds decrease in 2-ΔΔct) compared to WT cells. Whereas, GPRC5DDel/Del cells had 98% reduction in GPRC5D expression at mRNA level. These transcriptomic results were confirmed by ultra-high resolution microscopy (dSTORM). There was a 2 folds reduction in GPRC5D surface expression on GPRC5DWt/Del cells (0.1624±0.0222 clusters/µm2) compared to WT cells (0.3237±0.1024 clusters/µm2). GPRC5DDel/Del had barely detectable GPRC5D expression with 0.0322±0.007 clusters/µm2. These results were also confirmed at protein level with Western blots. When exposed to the anti-GPRC5D-CD3 bispecific antibody talquetamab, GPRC5DWt/Del cells depicted a significant resistance which was further enhanced in GPRC5DDel/Del cells. Surprisingly, in the presence of talquetamab GPRC5D deficient models showed a proliferation gain (0.5 fold, p=0.016) compared to the WT parental cells . Notably, this was most pronounced in the biallelelicly inactivated cells (2 folds increase, p&lt;0.0001). In absence of talquetamab, the baseline cell proliferation rate did not differ between the WT cells and our models. No proliferation changes were seen in cell models treated with talquetamab in absence of effector T cells. High content Cyto/Chemokine profiling on the GPRC5D coculture experiments was perfomed using the scioCyto microarray platform which revealed significantly altered cyto/chemokine profiles, including growth and proliferation promoting cytokines such as CCL28, IL34, HGF and CXCL9. In addition, differential gene expression analysis from Bulk RNA sequencing of the GPRC5D WT and KO cells confirmed upregulation of MAPK and PI3K pathways involved in growth proliferation and downregulation of the RAP1 signaling pathway involved in cell adhesion. Unspecific or alternate binding of talquetamab to the GPRC5DDel/Del cell surface was not detectable using flow cytometry, to decipher potential mechanism of growth induction via non-specific binding of talquetamab, pulldown assays combined with proteomic based analysis are ongoing.
Conclusion
Here we provide first data that monoallelic GPRC5D inactivation impairs talquetamab efficiency. Our work supports an anti-proliferative function of GPRC5D and suggests that treatment over progression with talquetamab may favor tumor growth in GPRC5D deficient cells.