Comparison of FACS and PCR for Detection of BCMA-CAR-T Cells

Abstract

Chimeric-antigen-receptor (CAR)-T-cell therapy is already widely used to treat patients who are relapsed or refractory to chemotherapy, antibodies, or stem-cell transplantation. Multiple myeloma still constitutes an incurable disease. CAR-T-cell therapy that targets BCMA (B-cell maturation antigen) is currently revolutionizing the treatment of those patients. To monitor and improve treatment outcomes, methods to detect CAR-T cells in human peripheral blood are highly desirable. In this study, three different detection reagents for staining BCMA-CAR-T cells by flow cytometry were compared. Moreover, a quantitative polymerase chain reaction (qPCR) to detect BCMA-CAR-T cells was established. By applying a cell-titration experiment of BCMA-CAR-T cells, both methods were compared head-to-head. In flow-cytometric analysis, the detection reagents used in this study could all detect BCMA-CAR-T cells at a similar level. The results of false-positive background staining differed as follows (standard deviation): the BCMA-detection reagent used on the control revealed a background staining of 0.04% (±0.02%), for the PE-labeled human BCMA peptide it was 0.25% (±0.06%) and for the polyclonal anti-human IgG antibody it was 7.2% (±9.2%). The ability to detect BCMA-CAR-T cells down to a concentration of 0.4% was similar for qPCR and flow cytometry. The qPCR could detect even lower concentrations (0.02–0.01%). In summary, BCMA-CAR-T-cell monitoring can be reliably performed by both flow cytometry and qPCR. In flow cytometry, reagents with low background staining should be preferred.

An IQ Consortium Perspective on Best Practices for Bioanalytical and Immunogenicity Assessment Aspects of CAR‐T and TCR‐T Cellular Therapies Development

“…Reagents used to prepare the standard curve and quality controls for PCR should be evaluated for both sensitivity and specificity that meet the assay needs as well as efficiency and linearity. Efficiency and linearity of the curve, extraction recovery efficiency, and specificity are evaluated when using a qPCR method 15 . An appropriate lower limit of quantitation and limit of detection (as needed) for the qPCR assay should be defined to support validation.…”

“…Efficiency and linearity of the curve, extraction recovery efficiency, and specificity are evaluated when using a qPCR method. 15 An appropriate lower limit of quantitation and limit of detection (as needed) for the qPCR assay should be defined to support validation. The use of dPCR, which applies absolute quantitation, therefore eliminats the need for a standard curve, is an alternative approach to quantify vector copy numbers of CAR-Ts.…”

“…Difference of 20 to 40-fold. 15 Multiple cell type identification on a per cell basis Potential to simultaneously assess expression of other proteins on CAR-Ts on a per-cell basis. This allows, for example, the separate enumeration of CD4+ and CD8+ subsets of CAR-T cells, and the determination of memory phenotype of circulating CAR-Ts.…”

Bioanalytical Methods for Characterization of CAR‐T Cellular Kinetics: Comparison of PCR Assays and Matrices

“…The conventional ligand binding‐based approach utilized for biologics for pharmacokinetic (PK) assessments is not feasible for CAR‐T‐cell therapies. Currently, characterizing the cellular kinetics (PK analysis) of CAR‐T‐cell therapies involves enumerating CAR expressing T‐cells by flow cytometry and/or determination of vector copy number of the CAR‐T‐cell gene construct by polymerase chain reaction (PCR) 8–10 . Each approach has advantages and limitations that need to be considered carefully.…”

“…Currently, characterizing the cellular kinetics (PK analysis) of CAR-T-cell therapies involves enumerating CAR expressing T-cells by flow cytometry and/or determination of vector copy number of the CAR-T-cell gene construct by polymerase chain reaction (PCR). [8][9][10] Each approach has advantages and limitations that need to be considered carefully. Flow cytometric analysis provides a direct assessment of CAR-expressing T-cell numbers in fresh or cryopreserved peripheral blood mononuclear cells (PBMCs).…”