Monitoring of tisagenlecleucel transgene DNA using a quantitative polymerase chain reaction assay
| Autor: | Lisa Davis, Irene L. Ch’en, Shabnam Tangri, Reinhold Pollner, Creton Kalfoglou, Nancy Valencia, Karen Thudium Mueller, Nathan Riccitelli, Jennifer Brogdon |
|---|---|
| Rok vydání: | 2020 |
| Předmět: |
0301 basic medicine
CAR-T cell therapy lcsh:QH426-470 Transgene Biology 03 medical and health sciences chemistry.chemical_compound 0302 clinical medicine expansion In vivo cellular kinetics Genetics medicine Digital polymerase chain reaction lcsh:QH573-671 Molecular Biology lcsh:Cytology persistence Molecular biology lcsh:Genetics genomic DNA qPCR 030104 developmental biology Real-time polymerase chain reaction medicine.anatomical_structure chemistry 030220 oncology & carcinogenesis Cancer cell Molecular Medicine Original Article Bone marrow CAR transgene DNA |
| Zdroj: | Molecular Therapy. Methods & Clinical Development Molecular Therapy: Methods & Clinical Development, Vol 20, Iss, Pp 535-541 (2021) |
| ISSN: | 2329-0501 |
| Popis: | Chimeric antigen receptor (CAR)-T cell therapies reprogram T cells to engage and eliminate cancer cells. Patients’ T cells are transduced in vitro using lentiviral or retroviral vectors containing a CAR transgene. Following infusion, CAR-T cells expand in vivo and may persist in the peripheral blood and bone marrow for years. Therefore, monitoring in vivo copies of the CAR transgene requires highly sensitive, validated analytical methods. Herein, we describe the validation of a qPCR assay to detect tisagenlecleucel transgene in patient samples. The limit of detection and lower limit of quantitation were 3.1 and 10 copies/200 ng genomic DNA, respectively, equivalent to ∼50 copies/μg genomic DNA and in alignment with US Food and Drug Administration guidance on bioanalytical method validation. The assay allowed quantitation of the tisagenlecleucel transgene over a wide dynamic range with a high degree of linearity, that is, 101–106 copies/200 ng genomic DNA (R2 ≥ 0.9988). Coefficients of variation of measured transgene copies ranged from 0.2% to 12.8%. A droplet digital PCR assay was performed as a method of validation and showed a strong correlation with the qPCR assay (R2 = 0.9980, p < 0.0001). This qPCR assay is being utilized to monitor tisagenlecleucel expansion and persistence in clinical trials. Graphical Abstract Highly specific, sensitive, and quantitative analytical assays are needed to monitor the in vivo cellular kinetics of CAR-T cells in the patient after infusion. In this study, Davis and colleagues describe the validation results of a novel qPCR assay designed to specifically identify the tisagenlecleucel CAR transgene in patient samples. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|