Characterization of a multiplex digital PCR assay to quantify total T cells relative to chimeric antigen receptor-positive T cells

Chimeric antigen receptor-T cells (CAR-T) have become a widely utilized therapy for B cell malignancies and are under investigation in early-phase clinical trials for a host of other hematologic and solid malignancies. Monitoring of CAR-T persistence has largely relied on quantitative PCR, flow cytometry, or a combination of these methodologies. Digital PCR (dPCR) has gained favor as a sensitive and user-friendly method for monitoring CAR-T persistence in patients after infusion and can be adapted to any CAR-T construct. Historically, CAR-T quantitation has been expressed in copies per microliter (copies/μL) or as a percentage of total nucleated cells, both of which fail to provide information on the broader immunologic context for the patient. We have developed a T cell-specific dPCR assay that can be multiplexed with CAR-T and control gene assays to provide quantitation of total T cells as well as CAR-T 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 assays can allow a greater depth of CAR-T quantitation relative to total T cells with minimal source sample needs.