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Monitoring of tisagenlecleucel transgene DNA using a quantitative polymerase chain reaction assay.

ABSTRACT: 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/?g 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, 10^1-10⁶ 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.

SUBMITTER: Davis L

PROVIDER: S-EPMC7878179 | biostudies-literature | 2021 Mar

REPOSITORIES: biostudies-literature

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Monitoring of tisagenlecleucel transgene DNA using a quantitative polymerase chain reaction assay.

Davis Lisa L Riccitelli Nathan N Valencia Nancy N Ch'en Irene L IL Tangri Shabnam S Brogdon Jennifer L JL Kalfoglou Creton C Mueller Karen Thudium KT Pollner Reinhold R

Molecular therapy. Methods & clinical development 20201210

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 o ...[more]

PMID: 33614827

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Project description:Background and objectivesThe variable response of hepatitis C virus (HCV) genotypes towards anti-viral treatment requires prior information on the genotype status before planning a therapeutic strategy. Although assays for typing or subtyping of HCV are available, however, a fast and reliable assay system is still needed. The present study was planned to develop a single-step multiplex quantitative real time polymerase chain reaction (qPCR) assay to determine HCV genotypes in patients' sera.MethodsThe conserved sequences from 5' UTR, core and NS5b regions of HCV genome were used to design primers and hydrolysis probes labeled with fluorophores. Starting with the standardization of singleplex (qPCR) for each individual HCV-genotype, the experimental conditions were finally optimized for the development of multiplex assay. The sensitivity and specificity were assessed both for singleplex and multiplex assays. Using the template concentration of 102 copies per microliter, the value of quantification cycle (Cq) and the limit of detection (LOD) were also compared for both singleplex and multiplex assays. Similarly, the merit of multiplex assay was also compared with sequence analysis and restriction fragment length polymorphism (RFLP) techniques used for HCV genotyping. In order to find the application of multiplex qPCR assay, it was used for genotyping in a panel of 98 sera positive for HCV RNA after screening a total number of 239 patients with various liver diseases.ResultsThe results demonstrated the presence of genotype 1 in 26 of 98 (26.53%) sera, genotype 3 in 65 (66.32%) and genotype 4 in 2 (2.04%) sera samples, respectively. One sample showed mixed infection of genotype 1 and 3. Five samples could not show the presence of any genotype. Genotypes 2, 5 and 6 could not be detected in these sera samples. The analysis of sera by singleplex and RFLP indicated the results of multiplex to be comparable with singleplex and with clear merit of multiplex over RFLP. In addition, the results of multiplex assay were also found to be comparable with those from sequence analysis. The sensitivity, specificity, Cq values and LOD values were compared and found to be closely associated both for singleplex and multiplex assays.ConclusionThe multiplex qPCR assay was found to be a fast, specific and sensitive method that can be used as a technique of choice for HCV genotyping in all routine laboratories.

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Monitoring of tisagenlecleucel transgene DNA using a quantitative polymerase chain reaction assay.

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Monitoring of tisagenlecleucel transgene DNA using a quantitative polymerase chain reaction assay.

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