Multi-omics profiling with InTraSeq™ uncovers novel single-cell RNA and Proteomics events in CARTs 2265562

Abstract Introduction CAR T-cell therapy has transformed treatment of blood cancers but faces persistent barriers in solid tumors. A major limitation is the absence of single-cell approaches that integrate gene expression with protein and post-translational modification (PTM) data to capture dynamic CAR signaling. Conventional scRNA-seq provides only transcriptional snapshots. We applied InTraSeq™, a multimodal single-cell platform enabling simultaneous quantification of mRNA, surface, cytoplasmic, and nuclear proteins–including PTMs–to comprehensively profile CAR activity. Methods Jurkat and anti-CD20 CAR-Jurkat cells were analyzed under four conditions: monocultures or co-cultures with CD20+ Raji cells. Each was assessed at 2 and 24 hours (eight total samples). Samples were labeled with unique Histone H3 InTraSeq™ barcodes and pooled for processing with Genomics 3’ kits. An anti-Whitlow/218 linker antibody InTraSeq™ conjugate distinguished CAR-Jurkat cells. Combined transcriptomic and proteomic data identified activation- and signaling-related changes. Results All samples were accurately demultiplexed using Histone H3 barcodes. The anti-Whitlow/218 probe confirmed CAR expression and enabled detection of engineered Jurkats. In CAR-Jurkat co-cultures, unique RNA clusters and protein signaling states emerged at both timepoints, highlighting CAR activation. Apoptosis scoring revealed elevated apoptotic protein signals specifically in Raji cells after 24-hour co-culture with CAR-Jurkats, validating targeted cytotoxic function. Conclusion InTraSeq™ provides high-resolution, multi-omic insight into CAR-T activation, integration of RNA and protein data, and effector-target interactions. This approach delivers a unified view of CAR signaling and function, offering a valuable framework for refining CAR-T therapy design and optimization. Funding Source n/a Topic Categories Technological Innovations in Immunology (TECH)