CD19.CAR-T Cell Analysis Using Flow Cytometry and PCR
Authors: Schanda Nicola , Kunz Alexander , Sauer Tim , Schubert Maria-Luisa , Korell Felix , Hückelhoven-Krauss Angela , Neuber Brigitte , Hinkelbein Mandy , Müller-Tidow Carsten , Schmitt Michael , Schmitt Anita 
Published: November 2021

Abstract
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.

Disclosures
Sauer: Abbvie: Consultancy, Speakers Bureau; Pfizer: Consultancy, Speakers Bureau; Matterhorn Biosciences AG: Consultancy, Other: DSMB/SAB Member; Takeda: Consultancy, Other: DSMB/SAB Member. Schubert: Gilead: Consultancy. Müller-Tidow: Janssen: Consultancy, Research Funding; Bioline: Research Funding; Pfizer: Research Funding. Schmitt: TolerogenixX: Current holder of individual stocks in a privately-held company; Novartis: Other: Travel grants, Research Funding; MSD: Membership on an entity's Board of Directors or advisory committees; Kite Gilead: Other: Travel grants; Apogenix: Research Funding; Hexal: Other: Travel grants, Research Funding; Bluebird Bio: Other: Travel grants. Schmitt: Hexal: Other: Travel grant; Therakos/Mallinckrodt: Research Funding; Jazz Pharmaceuticals: Other: Travel grant; TolerogenixX Ltd: Current Employment.