IMMU-11. Spike-in probe-enhanced single-cell RNA-sequencing for precise tracking and profiling of “prime-and-kill” synNotch-CAR-T cells

Abstract To enhance the safety and efficacy of chimeric antigen receptor (CAR)-T cell therapy for glioblastoma, the synthetic Notch (synNotch)-CAR-T cell system integrates multi-antigen “prime-and-kill” recognition circuits. We developed synNotch-CAR-T cells that express anti-IL13ɑ2/anti-EphA2 dual “killing” receptors (becoming “primed”) exclusively upon encountering tumor/brain-specific “priming” antigens, such as EGFRvIII (currently in a phase I clinical trial [NCT06186401]) and Brevican (BCAN). However, precise monitoring of infused cells at single-cell resolution remains technically challenging and underdeveloped. Here, we present a novel single-cell RNA-sequencing (scRNA-seq)-based method for synNotch-CAR tracking and profiling. Using the 10X Genomics Flex Gene Expression platform, we incorporated 20 custom-designed “spike-in” probes targeting various regions of the synNotch-CAR transcript, enabling simultaneous assessment of priming status. In vitro (n = 5 samples with 53,933 cells in total), individual spike-in probes demonstrated a mean sensitivity of 34.0% (range: 15.6–57.3) and specificity of 99.7% (98.5–100), substantially outperforming our previous 3’ Gene Expression workflow (detection < 2%). We further developed machine-learning prediction models using random forest and multivariate logistic regression, achieving 60.1–78.9% sensitivity and 99.1–99.2% specificity for synNotch-positive cell identification, and 99–99.1% specificity for detecting CAR-expressing primed cells. In mouse xenograft-derived samples (n = 11 samples with 4,366 cells), these prediction models determined that 72.2%, 51.3%, and 46.4% of brain-, spleen-, and lung-derived human T cells were synNotch positive. Among these cells, primed cells were significantly enriched in the brain compared to the spleen and lung (28.4%, 19.6%, and 14.6%, respectively; Fisher’s exact test P = 5.1×10-11). Transcriptomic profiling revealed that primed synNotch-CAR-T cells were strongly associated with antigen experience, cytotoxicity, metabolic activity, and cell proliferation signatures. In summary, our spike-in probe-enhanced scRNA-seq approach provides unprecedented resolution of synNotch-CAR-T cell in vivo dynamics, offering deeper biological insights. Additionally, this approach is expected to contribute to improved patient monitoring and be broadly applicable to other engineered cell therapies.