Validation of a Digital Polymerase Chain Reaction Assay to Quantify Chimeric Antigen Receptor-Positive T-Cells Relative to Total T-Cells

Chimeric antigen receptor T-cells (CAR-T) have become a widely-utilized therapy for B-cell malignancies in pediatric and adult patients, and are under investigation in early-phase clinical trials for a host of other hematologic and solid malignancies. Whereas monitoring of CAR-T persistence in B-cell malignancies has largely relied on assessment of B-cell aplasia, similar measures of CAR-T persistence are not applicable in the context of other malignancies. Digital PCR (dPCR) has gained favor as a sensitive and user-friendly method for CAR-T persistence monitoring in patients after infusion and can be adapted to any CAR-T construct. Historically, CAR-T quantitation has been expressed in copies per microliter or as a percentage of total nucleated cells, both of which fail to provide information on the broader immunologic context for the patient. Total T-cell counts can be determined by flow cytometry, but this method requires a high level of expertise given the low sensitivity and high background of many CAR detection reagents. Additionally, employing both dPCR and flow cytometry increases demand for both personnel hours and source sample quantity, either of which may at times prohibit the application of 2 investigative modalities.

We developed a T-cell specific dPCR assay that can be multiplexed with CAR-T and control gene dPCR assays to provide quantitation of CAR-T cells, total T-cells, and total nucleated cells. This assay eliminates the need for redundant quantitation of T-cells by flow cytometry and in combination with ultra-sensitive CAR-T dPCR assays can allow a greater depth of CAR-T quantitation relative to total T cells with minimal source sample needs. The assay is built around the T-cell receptor excision circle (TREC) locus within the T-cell receptor alpha (TCRa) gene, such that the dPCR amplicon is only present in TCRa germline or non-T cells. The percentage of T-cells can then be calculated relative to the total nucleated cells in the sample.

This TCRa dPCR assay (VIC fluorophore) was developed to be multiplexed with customized CAR-specific dPCR assays (FAM fluorophore) and with an albumin reference gene (Cy5 fluorophore) on a Qiagen Qiacuity dPCR instrument. We validated the assay using the T-cell line Jurkat and the AML cell line MOLM13 in serial dilutions, as well as in bulk mononuclear cells and T-cell enriched peripheral blood samples from healthy donors (Pearson correlation coefficients, r, ranging 0.86-0.99). We then confirmed strong correlation between flow cytometry assessments of T-cell quantity and TCRa assay-based T-cell quantitation in banked peripheral blood samples from patients treated with the CAR-T construct UCD19 (r=0.95)(NCT05535855); these latter experiments also confirmed multiplexing capability with the UCD19 CAR-specific assay (r=0.99 between our analysis and the trial dPCR analysis).

In summary, the TCRa dPCR assay allows quantitation of CAR+ T-cells relative to total T-cells and total nucleated cells in patient samples in a single multiplexed assay without requiring additional clinical sample usage.