A single-cell multi-omics assay for simultaneous measurement of vector copy number and protein expression in CAR T cells

Chimeric antigen receptor T cell (CAR T) immunotherapies have transformed cancer treatment. These therapies typically involve lentiviral vector-mediated CAR gene integration, followed by reinfusion into patients, necessitating rigorous quality control. Critical parameters, including transduction efficiency and vector copy number (VCN), must be accurately quantified. However, conventional gene transfer assays lack the resolution to capture cellular heterogeneity, relying on bulk population averages or labor-intensive clonal outgrowth methods. In order to address these limitations, we have developed an end-to-end solution from panel design to data analysis pipeline for targeted single-cell interrogation of transgene copy number. We applied this single-cell protein + DNA multi-omics VCN workflow to analyze a bi-cistronic CD19xCD22 CAR T product. Our VCN analysis exhibited high sensitivity, specificity, and average VCN linearity, as validated by an orthogonal method. Additionally, it uniquely revealed the unprecedented single-cell resolution of the VCN distribution profile. Furthermore, we quantitatively measured surface protein expression for lineage assignment, which revealed differential transduction percentages and VCN distribution patterns across cell lineages (e.g., CD4+ and CD8+ T cells), providing insights into factors potentially influencing treatment outcomes. This single-cell genomic/proteomic workflow could be a powerful research tool with potential clinical applications to enhance our ability to deliver optimal CAR T products to patients.