Introduction: Bispecific T cell engagers (TCEs) and chimeric antigen receptor T cells (CAR T) directed against GPRC5D have demonstrated efficacy in relapsed multiple myeloma (MM). However, patients invariably relapse with the emergence of mutant GPRC5D clones. Whether these identified GPRC5D mutants, in particular non-truncating SNVs involving non-extracellular domain residues, confer resistance to anti-GPRC5D therapies remain to be defined.
Methods: Serially collected bone marrow aspirates pre- and post- GPRC5D TCE therapy were subjected to magnetic beads CD138+ sorting followed by whole genome sequencing (100x), scCNV, and scRNA. Identified GPRC5D SNVs were cloned and stably transduced in K562 cells for functional characterization.
Results: Study cohort included 12 patients who received talquetamab. 11/12 had ≥VGPR and mPFS of 13.5 months. Among 5/12 with progressive disease, all had newly detected GPRC5D mutations. Patient MM-19 had clonal GPRC5D biallelic deletion, while MM-18 had a major subclone (90%) harboring biallelic GPRC5D deletion and a minor subclone (5%) with SNV leading to a frameshift mutation (p. Leu174Trpfs180ter). MM-03 progressed with GPRC5D monoallelic loss coupled with clonal p.Asp239Asn (p.D239N). MM-31 at progression harbored 4 subclones with either GPRC5D biallelic deletion (12%), p.Arg233ter (45%), p.Tyr257Ser (28%), or rearrangement between chr 2 and GPRC5D locus on chr 12 (15%). Similarly, MM-32 demonstrated convergent evolution of mutant subclones harboring biallelic deletion 12p (7%) and another subclone with monoallelic loss coupled with GPRC5D p.Glu146ter (35%). Among the GPRC5D SNVs, 3 mapped to its extracellular domain and 2 to its transmembrane domain. Notably, all GPRC5D SNVs clustered within conserved motifs across GPCR family of proteins known to be implicated in protein trafficking across golgi/ endoplasmic reticulum (ER) and cell membrane. Indeed, confocal microscopy and flow cytometry assays of K562 cells stably expressing WT or mutant GPRC5D demonstrated that the identified GPRC5D mutants, except p.D239N, were retained in the ER (co-localizing with calnexin), abolishing GPRC5D surface expression. Meanwhile, GPRC5D p.D239N was detectable on the cell surface with its expression level comparable to WT GPRC5D. We evaluated the binding of talquetamab (monovalent GPRC5D binding) and forimtamig (bivalent) on p.D239N expressing clones. While forimtamig demonstrated dose dependent linear binding to D239N, talquetamab binding to p.D239N was only detectable at high concentrations ≥ 10 nM. Consistent with their binding, forimtamig demonstrated enhanced cytolytic activity against GPRC5D p.D239N.
Conclusions: GPRC5D antigen escape predominantly involve convergence of multiple clones harboring monoallelic chr.12p deletions coupled with GPRC5D mutations. GPRC5D mutations cluster within GPCR conserved motifs involved in protein trafficking and hence prevent its membrane localization.