CD19.CAR-T Cell Analysis Using Flow Cytometry and PCR

Introduction: After approval of CD19.CAR-T cell therapy by both FDA and EMA, chimeric antigen receptor T (CAR-T) cell therapy has been established as a new and innovative therapy method for patients with relapsed/refractory acute lymphoblastic leukemia (ALL) and non-Hodgkin lymphoma (NHL). Because of this, there is great need for reliable methods for CAR-T cell monitoring by clinicians to further assess cell expansion, distribution and persistence in patients. This is both necessary for CAR-T cell manufacturing sites and researchers to assess transduction efficiency and vector copy numbers per cell. Therefore, we analyzed CAR expression of CD19.CAR-T cells using flow cytometry as well as quantitative polymerase chain reaction (qPCR).

Methods: In this study, four different commercially available CD19.CAR-T cell staining reagents (Protein L, CD19 protein, F(ab`)2 antibody, and CD19.CAR-T cell detection reagent) were used to evaluate CAR-T cell products and peripheral blood samples of both patients and healthy donors by flow cytometry. Furthermore, qPCR was performed with data comparison using flow cytometry results.

Results: 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. Moreove, the CD19 CAR detection reagent showed a significantly lower median background staining of 0.02% (range 0.007-0.06%) when compared to F(ab')2 antibody, CD19 protein and Protein L with 0.80% (range 0.47-1.58%.), 0.68% (range 0.30-1.38%) 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.

Conclusion: CAR-T staining was successfully performed with all tested commercially available CAR detection reagents. Evaluation of manufactured CAR-T cells as well as quality control was comparably done using FACS and qPCR. Because of lower frequencies of CAR-T cells in the patient samples, CAR-T cell staining reagents with a low background staining should be preferably used.