Sensitivity and Specificity of CD19.CAR-T Cell Detection by Flow Cytometry and PCR

Abstract

Chimeric-antigen-receptor-T (CAR-T) cells are currently revolutionizing the field of cancer immunotherapy. Therefore, there is an urgent need for CAR-T cell monitoring by clinicians to assess cell expansion and persistence in patients. CAR-T cell manufacturers and researchers need to evaluate transduction efficiency and vector copy number for quality control. Here, CAR expression was analyzed in peripheral blood samples from patients and healthy donors by flow cytometry with four commercially available detection reagents and on the gene level by quantitative polymerase chain reaction (qPCR). Flow cytometric analysis of CAR expression showed higher mean CAR expression values for CD19 CAR detection reagent and the F(ab’)2 antibody than Protein L and CD19 Protein. In addition, the CD19 CAR detection reagent showed a significantly lower median background staining of 0.02% (range 0.007–0.06%) when compared to the F(ab’)2 antibody, CD19 protein and Protein L with 0.80% (range 0.47–1.58%), 0.65% (range 0.25–1.35%) and 0.73% (range 0.44–1.23%). Furthermore, flow cytometry-based CAR-T cell frequencies by CD19 CAR detection reagent showed a good correlation with qPCR results. In conclusion, quality control of CAR-T cell products can be performed by FACS and qPCR. For the monitoring of CAR-T cell frequencies by FACS in patients, CAR detection reagents with a low background staining are preferable.

Comparison of single copy gene‑based duplex quantitative PCR and digital droplet PCR for monitoring of expansion of CD19‑directed CAR T cells in treated patients

“…Therefore, the survival of transgenic cells not expressing the CAR is unlikely. Accordingly, high correlation levels of CAR quantification data obtained by PCR-based approaches determining the transgene at the genomic level and FC detecting the CAR protein on the CAR T cell surface have been reported by our group ( 18 , 32 ) and others ( 10 ).…”

A new story for an old challenge: Would flow cytometry beat molecular biology in monitoring chimeric antigen receptor T cell pharmacokinetics?

“…Jiali Cheng et al [1] answered positively this question, which has also been confirmed in previous studies [13, 14]. CAR T cell were efficiently detected by flow cytometry, especially using an anti‐idiotype antibody, with a LOD at 0.03678% of lymphocytes (mean + 3SD in healthy whole blood) with reasonable coefficient of variance below 11%. The next question constitutes the main objective of the study: Would flow cytometry compete with droplet digital PCR (dd‐RT PCR) efficiency in detecting rare CAR T cells? …”

“…This needs to be adapted to monitor new CAR programs using other cell types like NK cells [9,10] and other tumor types like solid tumors, for which, TAAs are highly variable [11,12]. Jiali Cheng et al [1] answered positively this question, which has also been confirmed in previous studies [13,14]. CAR T cell were efficiently detected by flow cytometry, especially using an anti-idiotype antibody, with a LOD at 0.03678% of lymphocytes (mean + 3SD in healthy whole blood) with reasonable coefficient of variance below 11%.…”

Current state of CAR-T therapy for T-cell malignancies

“…36 Moreover, given that the transduction efficiency and vector copy number of CAR-T-cells can be detected and evaluated by flow cytometry and qPCR, improvements in these technologies may be helpful to this problem. 122 Other solutions to enhance CAR-T efficacy For the CAR-T therapy for B-cell malignancies, investigators have explored a variety of methods to enhance the efficacy, such as combination strategies and dual-target CAR-T-cell therapy. 123,124 A preclinical study showed that the expression of PD-1 and PD-L1 was upregulated after CAR-T infusion, which will inhibit the effector function of CAR-T-cells.…”

“… 36 Moreover, given that the transduction efficiency and vector copy number of CAR-T-cells can be detected and evaluated by flow cytometry and qPCR, improvements in these technologies may be helpful to this problem. 122 …”