Development of a DNA Aptamer‐Based Approach to Noninvasively Image CAR‐T Cells In Vivo and Traceless Enrichment In Vitro

Abstract Chimeric antigen receptor (CAR) T cells offered a potential cure for malignancies, however, their outcomes and dynamics across different anatomical sites remained inadequately characterized. Monitoring the bio‐distribution and tumor‐homing of CAR‐T cells in vivo is crucial, as it provides patient‐specific data that might inform on treatment success, potential failure, and off‐target toxicities. Herein, an Aptamer A3 by Cell‐SELEX (systematic evolution of ligands by exponential enrichment) is generated, which can bind with CAR‐T cells with nanomolar affinity. After CAR‐T cells are injected into Nalm6 xenograft tumor model mice through tail vein, Cy5‐labeled A3 is injected into mice for fluorescence time‐delay imaging in vivo. The fluorescence signal produced by the Cy5‐labeled A3 is accumulated in the tumor area and reached its maximum at day 14. Moreover, A3 could enrich CAR‐T cells in mixed cell populations in a traceless way. A3 is screened for CAR‐T cells imaging and CAR‐T cells enrichment, which may provide assistance for the evaluation of CAR‐T cells efficacy and the manufacture of CAR‐T cells. Overall, this research shows that A3 enabled repeated, sensitive, and specific assessment of the infused CAR‐T cells in vivo. The screened aptamer will have broad applications for tracking CAR‐T cells in patients, providing insights into treatment success, potential failure, and off‐target toxicities.