scCNVseq Datasets Supporting Tumor Intrinsic Mechanisms of Antigen Escape to Anti-BCMA and Anti-GPRC5D Targeted Immunotherapies in Multiple Myeloma
Multiple myeloma (MM) immune escape resulting from B cell maturation antigen (BCMA) target loss is considered to be a rare event that mediates MM resistance to anti-BCMA chimeric antigen receptor T cell (CAR T) or bispecific T cell engager (TCE) therapies. Emerging clinical data also suggests that downregulation of G protein coupled receptor family C group 5 member D (GPRC5D), another promising target antigen expressed on MM cells, is observed in patients at relapse post anti-GPRC5D CAR T, but the genomic mechanisms that underlie GPRC5D loss has not been described. In order to examine the tumor intrinsic factors that promote MM antigen escape, we performed combined bulk whole genome sequencing and single cell copy number variation analysis on CD138+ cells from bone marrow aspirates of patients before and after relapse from anti-BCMA or -GPRC5D CAR T/ TCE. We describe five cases with distinct biallelic events on TNFRSF17 at MM relapse after CAR T/ TCE. In addition to focal biallelic deletions at the TNFRSF17 locus acquired at relapse, BCMA negative clones can emerge from the selective expansion of subclones with homozygous TNFRSF17 loss that exist prior to any anti-BCMA therapy exposure. Furthermore, we corroborate with functional data to demonstrate that three different non-truncating mutations in the extracellular domain of BCMA negates the efficacies of BCMA directed TCEs and mediate disease relapse in patients. With respect to GPRC5D, we report four cases of MM relapse with biallelic loss of GPRC5D following anti-GPRC5DxCD3e. Our data support that immunoselection of BCMA negative or mutant clones post anti-BCMA therapies may be more frequent than currently accepted in the field, and that an all or none screening approach for BCMA expression is inadequate to detect pertinent mutations that affect patient response to targeted therapies. We also highlight the importance of developing immunotherapies targeting novel and non-redundant epitopes or antigens in MM Overall design: Experiments were conducted on primary multiple myeloma patients bone marrow samples provided after informed consent, in accordance with the Declaration of Helsinki and following the approval by the Medical Center Institutional Review Board. Unbiased mRNA and DNA profiling were conducted by using scRNA-seq and sc-CNV-seq from the GemCode system (10x Genomics) accordingly to the manufacturer's protocols. For single-cell DNA library generation for CNV single-cell suspensions of primary MM cells were processed according to 10X Genomics Reagent Kits User Guide (CG000153). Single cells were partitioned in a hydrogel matrix by combining with a CB polymer to form Cell beads (CBs) using a microfluidic chip. Post a first encapsulation, CBs were treated to lyse the encapsulated cells and denature the genomic DNA (gDNA). The denatured gDNAs in the CB were then accessible to amplification and barcoding. A second microfluidic encapsulation step was then required to partition the CB with 10x barcode Gel Beads (GBs) to generate GEMs. Immediately after barcoding and amplification, 10x barcoded fragments were pooled and attached to standard Illumina adaptors. For all the single cell methods, quality control and quantification was performed using a Kapa Library Quantification qPCR kit (Kapa Biosystems) on a BioRad qPCR instrument prior to preparing a single pool containing equal amounts of each library. This pool was then subjected to on-board cluster formation and sequencing on an Illumina NextSeq 500 sequencer with a high-output v2.5 150 sequencing kit for RNA-seq and  300 cycle sequencing kit for CNV-seq as per the standard Illumina protocols. After sequencing, the bcl data was converted to fastq data files using the Illumina BCL2FASTQ utility. Samples were processed with CellRanger suite v3.0 and downstream analyses were realized with Seurat, Loupe and custom R packages.