Detection of CAR‐T19 cells in peripheral blood and cerebrospinal fluid: An assay applicable to routine diagnostic laboratories

Abstract

Background: Chimeric antigen receptor-modified T-cells targeting CD19 (CAR-T19) are licensed for treating relapsed/refractory diffuse large B-cell lymphoma and B-acute lymphoblastic leukemia. Predicting treatment responses and toxicity (e.g., cytokine release syndrome and neurotoxicity) remains a big challenge. CAR-T19 monitoring could increase our understanding of treatment responses and be of relevance to patient management. A robust method for accurate CAR-T19 detection is therefore extremely desirable.Methods: An assay that uses fluorochrome-conjugated human recombinant soluble CD19 was tested against two commercially available CAR-T19 therapies and a CAR-T19 cell line developed in-house. Precision, concordance, and analyte stability were tested using peripheral blood obtained from CAR-T19-treated patients and controls. Results:The assay showed good accuracy, and had a limit of blank for whole blood samples of 0.13%. Reproducibility and inter-operator concordance were satisfactory (CVs <15%). The assay distinguished CAR-T19 from reactive T-cells in cerebrospinal fluid (CSF) from patients with suspected immune effector cell-associated neurotoxicity syndrome (ICANS), and was adapted to study memory T-cell compartments in treated patients. Conclusion:The assay enabled routine monitoring of CAR-T19 in blood and CSF samples. Despite profound cytopenia in many lymphoma patients, results were obtained regularly from only 4 ml of blood. The assay can be adapted easily to characterize the memory and exhaustion status of CAR-T19 and native T-cells. Importantly, it does not rely on CAR construct specificity; thus, it can be used to detect any CD19-targeted CAR cell. Finally, our validation process can serve as a blueprint for other fluorochrome proteins used to detect CAR cells.

Optimization of a flow cytometry test for routine monitoring of B cell maturation antigen targeted CAR in peripheral blood

“…The gating strategy for reporting the proportion of lymphocytes that are CAR+ T cells was based upon that described in Johansson et al, 2021. T cells, NK cells and B cells were identified within an enlarged lymphocyte gate from which CD14+ monocytes were excluded.…”

Optimization and validation of in vivo flow cytometry chimeric antigen receptor T cell detection method using CD19his indirect staining

“…To detect and quantify CAR-T cell, current approaches are represented by the quantification of the CAR transgene sequence using qPCR [20] or flow cytometry [17,21]. Different efforts were made to propose flow cytometry assays for CAR-T cell detection, not only for the anti-CD19 CAR but also for the anti-BCMA CAR [22][23][24][25][26][27].…”

Molecular monitoring of T-cell kinetics and migration in severe neurotoxicity after real-world CD19-specific chimeric antigen receptor T cell therapy

“…10,11,16 However, to date systematic data on CAR-T cell kinetics within the cerebrospinal fluid (CSF) are missing, which partly reflects the lack of broadly available diagnostic CAR-specific monitoring tools to easily examine the frequently scanty CSF specimen in the non-trial setting. 17,18 In our prior work, we developed and validated digital-PCR (dPCR) assays for CD19-CAR-T cell tracking. [19][20][21][22] Here, we applied these assays to assess the CD19-CAR-T cell kinetics in diagnostic CSF samples and corresponding peripheral blood specimen from patients experiencing severe grade ≥3 neurotoxicity of CD19-CAR-T cell therapy.…”