Real Time, Point-of-Need Measurement of Blood CD19 CAR-T Vector Load Is Concordant with Standard Ddpcr Method

Introduction CD19-directed chimeric antigen receptor T cell (CAR-T) therapy has revolutionized treatment of non-Hodgkin lymphoma; however, significant toxicities have been associated with CAR-T therapies. Peripheral blood quantification of CAR-T vector load may have prognostic value and can aid in toxicity management for large B cell lymphoma (LBCL) patients undergoing axicabtagene ciloleucel (axi-cel) treatment. We assessed the concordance of the IdyllaTM platform, a point-of-need (PON), cartridge-based system for real-time assessment of CAR-T vector load from peripheral blood, with standard droplet digital polymerase chain reaction (ddPCR) and flow cytometry (FACS) methods. Methods Thirty-nine consecutively enrolled subjects who received axi-cel CAR-T therapy for LBCL were consented on an IRB-approved protocol. Blood samples for ddPCR and Idylla vector load measurement were collected at pre-lymphodepletion and days 1, 3, 5, 7, 14, and 28 after axi-cel infusion. Blood samples for FACS were collected on days 5, 7, 14 and 28 after axi-cel infusion. ddPCR was used to detect and quantify T cells carrying the anti-CD19 CAR transgene from PBMCs. The Idylla CD19 CAR-T Prototype is a fully automated, qPCR based-assay that quantitatively detects CD19 CAR transgene starting from 500μL EDTA peripheral blood. CD19 CAR-T expansion was measured by FACS with anti-idiotype-FMC63 conjugated to Dylight 65013. The concordance between Idylla, ddPCR, and FACS analysis was evaluated using Z-score standardization and Bland-Altman analysis. Results 246 samples were collected across 39 participants. CAR-T vector load was measured by Idylla (copies per uL) and ddPCR (copies per cell) (Fig. 1). A Bland-Altman analysis was performed on the standardized CD19 transgene values of all samples collected between days 1-28. The concordance between Idylla and ddPCR measurements was within the acceptable range, with 95% of all samples within ± 2 SD of the mean difference (Fig. 2). Restricting analysis to day 7, Bland-Altman analysis also found concordance between standardized Idylla and log transformed FACS (Fig. 3a) and ddPCR values (Fig. 3b) for samples collected at the time of peak CAR-T expansion. Discussion Previous studies of CAR-T expansion have used reverse transcriptase PCR and/or FACS approaches, which require technical laboratory skills and have multi-day turnaround times. The Idylla platform enables fully automated detection of CAR-T vector load, with 2 minutes of hands-on time. Results are reported in ∼90 minutes. This PON device may ultimately enable standardization of CAR-T vector load measurements across clinical sites, development of predictive algorithms for toxicity, and management of patients showing primary or secondary treatment failure. The peak blood CAR-T vector load and its association to toxicity and efficacy outcomes will be reported in future studies.