Optimization and validation of in vivo flow cytometry chimeric antigen receptor T cell detection method using CD19his indirect staining.
Cytometry A
; 105(2): 112-123, 2024 02.
Article
em En
| MEDLINE
| ID: mdl-37707318
ABSTRACT
CD19-targeted chimeric antigen receptor T (CAR-T) cell therapy has shown unprecedented results in patients with B cell relapsed/refractory acute lymphoblastic leukemia (R/R-ALL) and B cell non-Hodgkin lymphomas where no other curative options are available. In vivo monitoring of CAR-T cell kinetics is fundamental to understand the correlation between CAR-T cells expansion and persistence with treatment response and toxicity development. The aim of this study was to define a robust, sensitive, and universal method for CAR-T cell detection using flow cytometry. We set up and compared with each other three assays for CD19 CAR-T cell detection, all based on commercially available reagents. All methods used a recombinant human CD19 protein fragment recognized by the single-chain variable fragment of the CAR construct. The two indirect staining assays (CD19his + APC-conjugated antihistidine antibody and CD19bio + APC-conjugated antibiotin antibody) showed better sensitivity and specificity compared with the direct staining with CD19-FITC, and CD19his had a better cost-effective profile. We validated CAR detection with CD19his with parallel quantitative real-time polymerase chain reaction data and we could demonstrate a strong positive correlation. We also showed that CD19his staining can be easily included in a multicolor flow cytometry panel to achieve additional information about the cell phenotype of CAR-T cell positive subpopulations. Finally, this method can be used for different anti-CD19 CAR-T cell products and for different sample sources. These data demonstrate that detection of CAR-T cells by CD19his flow cytometry staining is a reliable, robust, and broadly applicable tool for in vivo monitoring of CAR-T cells.
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Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Receptores de Antígenos Quiméricos
Tipo de estudo:
Diagnostic_studies
Limite:
Humans
Idioma:
En
Revista:
Cytometry A
Ano de publicação:
2024
Tipo de documento:
Article
País de afiliação:
Itália