Validation of a High sensitivity assay for detection of CAR T cell vectors using low partition digital PCR technology.

Although in-vivo engraftment, expansion, and persistence of CAR T cells are pivotal components of treatment efficacy, quantitative monitoring has not been implemented in routine clinical practice. We describe the development and analytical validation of a digital-PCR (dPCR) assay for ultra-sensitive detection of CAR constructs post-treatment, circumventing known technical limitations of low partitioning platforms. Primers and probes, designed for detection of axicabtagene, brexucabtagene and MSK CAR constructs, were employed to validate testing on the Bio-Rad dPCR low-partitioning platform; results were compared to Raindrop, a high-partitioning system, as reference method. Bio-Rad protocols were modified to enable testing of DNA inputs as high as 500ng. Using dual input reactions (20 and 500ng) and a combined analysis approach, the assay demonstrated consistent target detection around 1x10-5 (0.001%) with excellent specificity and reproducibility and 100% accuracy compared to the reference method. Dedicated analysis of 53 clinical samples received during validation/implementation phases, showed the assay effectively enabled monitoring across multiple time points of early expansion (day 6-28) and long-term persistence (up to 479 days). CAR vectors were detected at levels ranging from 0.005-74% (vector vs. reference gene copies). The highest levels observed in our cohort correlated strongly with the temporal diagnosis of grade 2 and 3 cytokine release syndrome diagnosis (p<0.005). Only 3 patients with undetectable constructs had disease progression at the time of sampling.