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bioRxiv · 10.64898/2026.03.26.713760

Spike-in probe-enhanced single-cell RNA-seq reveals post-infusion transcriptomic remodeling of "prime-and-kill" synNotch-CAR-T cells

Nejo, T.·Watchmaker, P. B.·Simic, M. S.·Yamamichi, A.·Lakshmanachetty, S.·Zhao, A.·Lu, J.·Gallus, M.·Benway, H. L.·Zhu, R.·Almeida, R.·Lim, W. A.·Okada, H.
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Abstract

We previously developed synthetic Notch (synNotch)-chimeric antigen receptor (CAR)-T cells to improve the safety and efficacy of CAR-T therapy for glioblastoma. In this system, an anti-EphA2/IL13R2-dual-CAR is expressed only upon recognition of tumor- or brain-specific "priming" antigens, EGFRvIII (termed E-SYNC cells) or brevican (B-SYNC), respectively, with E-SYNC currently under phase I clinical evaluation (NCT06186401). However, tracking and profiling these engineered cells in vivo remain challenging, limiting our understanding of their activity and therapeutic potential. To address this gap, we developed a single-cell RNA-sequencing (scRNA-seq) workflow with custom spike-in probes for synNotch-CAR transcripts, enabling simultaneous detection of engineered cells and transcriptomic profiling. In vitro, integration of multiple probes using machine-learning-assisted classifiers detected 78.2% of E-SYNC cells and 60.0% of B-SYNC cells with 98.0% specificity. In a xenograft model, synNotch-positive cells were detected across the spleen, lung, and brain, with the highest frequency and most robust priming and activation observed in the brain. Single-cell transcriptomic analyses revealed tissue-specific differentiation programs, including cytotoxicity, proliferation, metabolic activity, and acquisition of tissue-resident memory phenotypes, shaped by both environmental cues and synNotch-mediated antigen recognition. In summary, this spike-in probe-enhanced scRNA-seq workflow enables robust detection and high-resolution characterization of synNotch-CAR-T cell dynamics and provides a broadly applicable platform for monitoring engineered immune cells in diverse clinical contexts. One Sentence SummaryOur spike-in probe-enhanced single-cell RNA-sequencing method enables analysis of tissue-dependent activation and transcriptional states of synNotch-CAR-T cells, providing a robust and scalable platform for in vivo tracking and transcriptomic profiling of engineered cell therapies.

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Nejo, T., Watchmaker, P. B., Simic, M. S., Yamamichi, A., Lakshmanachetty, S., Zhao, A., Lu, J., Gallus, M., Benway, H. L., Zhu, R., Almeida, R., Lim, W. A., Okada, H.. 2026-03-29. Spike-in probe-enhanced single-cell RNA-seq reveals post-infusion transcriptomic remodeling of "prime-and-kill" synNotch-CAR-T cells. https://doi.org/10.64898/2026.03.26.713760

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