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. 2021 Nov 17;10(11):3208.
doi: 10.3390/cells10113208.

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

Nicola Schanda  1 , Tim Sauer  1 , Alexander Kunz  1 , Angela Hückelhoven-Krauss  1 , Brigitte Neuber  1 , Lei Wang  1 , Mandy Hinkelbein  1 , David Sedloev  1 , Bailin He  1 , Maria-Luisa Schubert  1 , Carsten Müller-Tidow  1 , Michael Schmitt  1 , Anita Schmitt  1
Affiliations

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

Nicola Schanda et al. Cells. .

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.

Keywords: CD19.CAR-T cells; detection reagent; flow cytometry (FACS); polymerase chain reaction (PCR).

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Conflict of interest statement

M.S. received funding for collaborative research from Apogenix, Hexal and Novartis, travel grants from Hexal and Kite, he received financial support for educational activities and conferences from bluebird bio, Kite and Novartis, he is a board member for MSD and (co-)PI of clinical trials of MSD, GSK, Kite and BMS, as well as co-Founder and shareholder of TolerogenixX Ltd. A.S. received travel grants from Hexal and Jazz Pharmaceuticals, research grant from Therakos/Mallinckrodt and is co-founder of TolerogenixX Ltd. A.S. is part-time employee of TolerogenixX Ltd. M.-L.S.: consultancy for Kite/Gilead, Takeda. Advisory board Kite/Gilead, Janssen. C.M.-T.: Bayer AG (research support). Pfizer, Janssen-Cilag GmbH (advisory board member). Pfizer, Daiichi Sankyo, BiolineRx (grants and/or provision of investigational medicinal products). The other authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Binding mechanisms of the different CD19.CAR detection reagents. Universal detection reagents, Protein L (1) and F(ab’)2 fragment (2) are binding to the immunoglobulin light chain and to the Fab portion of the immunoglobulin. Antigen-specific detection reagents (3 and 4) are binding to the CD19 binding site of the scFv. The reagents are either directly conjugated to a fluorochrome as recombinant CD19 protein (4) and F(ab’)2 fragment (2) or are conjugated to biotin binding to an anti-biotin antibody or fluorochrome conjugated streptavidin in a second staining step as CD19.CAR detection reagent (3) and Protein L (1). The CD19.CAR consists of a CD3ζ cytoplasmatic domain fused to the CD28 and 4-1BB costimulatory domains. The light and the heavy chain variable domains (VL and VH) separated by a linker are building the single chain variable fragment (scFv), which is linked via a hinge-region to the transmembrane domain.
Figure 2
Figure 2
Comparison of four different antibodies to detect CD19-specific CAR-T cells. Both graphs show the different percentages for CAR-T cell detection when using the displayed staining reagents. (A) depicts the percentage of CAR-T cells produced from five different HD samples. (B) shows the percentage of CAR-T cells produced from five different patient samples (Pat). (C) shows the contour plots for CAR-T cells of one HD stained with all four different antibodies. The gate for the non-transduced cells is the same as in the respective CD19.CAR-T cell group. Comparison for HDs and patient samples were evaluated in three independent experiments respectively. (*) p < 0.05; (**) p < 0.01 by one-way ANOVA.
Figure 3
Figure 3
Sensitivity of the detection reagents to detect CD19-specific CAR-T cells in PBMCs. Graph (A) shows CD19-specific CAR-T cells that were serially diluted in PBMCs of the same HD at six different dilutions (1:1 to 1:1000). The graph displays the mean values ± standard error of mean of CAR-T cells to PBMCs from four HDs. (B) The dot plots display representative data obtained from one out of four different HDs. Data are representative of four different HDs acquired in one experiment.
Figure 4
Figure 4
Specificity of the different detection reagents. PBMCs were stained with the respective CAR-detecting reagents to assess background staining. (A) shows the percentage of CD19.CAR-T cells in PBMCs only, for eight different donors. (B) displays results from one donor stained with different detection reagents. Data are representative of eight different HDs acquired in one experiment. (**) p < 0.01; (***) p < 0.001 by one-way ANOVA.
Figure 5
Figure 5
Comparison of CAR-T cell detection using flow cytometry and qPCR. CD19.CAR-T cells were diluted at six different dilutions (1:1 to 1:1000) and the percentage was compared with either flow cytometry using the CD19 CAR detection reagent or qPCR. Experiments were performed using three different HDs and yielded similar results. The graph displays the mean values and standard deviation.
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