(T1330-01-08) Multiplexed Quantification of CAR-T Subtypes by PCR

Purpose: The rapid advancement of chimeric antigen receptor T-cell (CAR-T) therapies over the past decade has had a transformative effect on disease management and patient health. Although much success has been achieved with single-target CAR-T modalities in hematologic cancers, acquired antigen loss can result in relapse, while in solid tumors, multiple, co-stimulatory antigen targets are frequently required to avoid off-target effects (Shah 2019, Newick 2017). In both environments, accurate quantification of CAR-T levels post-infusion is critical for elucidation of pharmacokinetic parameters. Herein, we describe a system for use with complex, multi-target and/or secondary-line CAR-T therapies that permits quantitative detection of up to three (3) unique transgenes within a single digital PCR reaction from limited patient material.

Methods: Three (3) validated qPCR assays for CAR-T targets were selected for combination into a multiplexed dPCR reaction. A specific region of gDNA isolated from the peripheral blood of healthy donor served as the endogenous control gene. A suite of primers and probes (P/P) were designed for the engineered targets and regions of the housekeeping gene. Control plasmids were generated for each target of interest, and titrated into PB/BM at high, medium, and low transgene levels. Assay performance was assessed by comparing the measured limit of detection (LOD), limits of quantification (LOQ), accuracy, and precision of the multiplexed reaction to the validated qPCR assays, as well as droplet digital PCR (ddPCR) duplex reactions.

Results: Overall, performance of the multiplexed dPCR was comparable to the established qPCR and duplex ddPCR assays. For all platforms, measured LOD levels fell below 10 cp/rxn, with LLOQ values aligned at 20 cp/rxn. The percent difference in measured transgene levels was ≤20% between dPCR and ddPCR platforms, and transgene levels were highly correlated (R² >0.95, Spearman’s Rho >0.98) across all methodologies. Inter and Intra-precision results for the multiplexed reaction exhibited < 20% CV across replicates. Maximum DNA input was slightly reduced for the multiplexed reaction (~400 ng) compared to the established qPCR limits ( >1 µg), and the optimal input for dPCR was identified as 100 ng per reaction versus the requisite 200 ng per reaction, per assay, required for the duplex qPCR tests.

Conclusion: Excellent concordance was observed across platforms between the measured transgene levels when targets were multiplexed and when assessed independently. Although dynamic range was slightly reduced for dPCR systems compared to qPCR, increased resolution at lower target copies and direct quantification by dPCR provides analytical and logistical advantages. Critically, the multiplexed dPCR format significantly reduces the required total assays and DNA input, minimizing sample collection burden for patients, while ensuring efficient quantification of all desired targets for clinical researchers. As CAR-T therapies continue to evolve beyond single-transgenes, multiplexed vector copy number testing will be an indispensable tool.

References: Newick, K. et.al. "CAR T Cell Therapy for Solid Tumors." Annu. Rev. Med. 2017.68:139-152.
Shah, N, Fry, T. "Mechanisms of Resistance to CAR T Cell Therapy." Nat Rev Clin Oncol. 2019 June ; 16(6): 372–385.

Acknowledgements: All authors are employed and receive funding from Navigate BioPharma Services.