Multimodal antigenic escape to GPRC5D-targeted T-cell engagers in multiple myeloma [scATAC-seq]

Tumor intrinsic adaptations with emerging resistant clones following T cell targeted immunotherapies pose a major barrier to durable remissions in multiple myeloma (MM). Through integrated genomic, transcriptomic, and epigenomic interrogation of clonal plasma cells, we observed antigenic drift in 68.4% of relapsed cases following anti-GPRC5D T cell engager (TCE) therapy. These escape events were driven by three distinct mutational mechanisms involving i) focal to large biallelic deletions at the GPRC5D gene locus, ii) monoallelic deletion coupled with GPRC5D single nucleotide variants (SNVs) or insertions/deletions (indels) on the remaining allele, as well as iii) epigenetic GPRC5D promoter/enhancer silencing. Beyond biallelic deletions resulting in complete antigenic loss, we demonstrate that GPRC5D SNVs and indels mutate anti-GPRC5D TCE binding epitopes or more commonly affect G-protein couple receptor family conserved motifs critical for protein membrane trafficking resulting in endoplasmic reticulum (ER) GPRC5D trapping. Multiple subclones bearing distinct genomic alterations at GPRC5D locus co-emerged within individual cases, depicting their convergent evolutionary trajectories. Importantly, anti-GPRC5D TCEs with varying epitope specificity, affinity, and valency differentially targeted mutant subclones, underscoring their non-redundant functional roles in overcoming resistance Overall design: Experiments were conducted on primary patient bone marrow samples, after informed consent, in accordance with the Declaration of Helsinki and following the approval by the Medical Center Institutional Review Board. Sorted CD138+ cells were collected from patients' bone marrow aspirates pre-and post-treatment with anti-GPRC5D T cell engagers, counted and viably frozen. Frozen cells were then thawed at 37°C, resuspended in RPMI 1640 medium (Gibco) and washed twice by centrifugation at 2000 rpm for 5 min. The desired number of nuclei [2000-8000] was targeted and processed according to the Nuclei Isolation for Single Cell ATAC sequencing (CG000169, 10x Genomics, Pleasanton, CA, USA). Nuclei isolation was performed as indicated in the nuclei isolation protocol for Single Cell ATAC Sequencing (10x Genomics). Based on the starting number of cells and desired final nuclei concentration, primary MM cells were lysed and resuspended in an appropriate volume of chilled Diluted Nuclei Buffer. The resulting nuclei were immediately used to generate scATAC-seq libraries using the Chromium Single Cell ATAC reagent kit (CG000168, 10x Genomics) according to the manufacturer's protocol.