Considerations in the Development and Validation of Real-Time Quantitative Polymerase Chain Reaction and its Application in Regulated Bioanalysis to Characterize the Cellular Kinetics of Car-T Products in Clinical Studies

Yang, Tong‐Yuan; Doddareddy, Rajitha

doi:10.4155/bio-2020-0221

Cited by 13 publications

(16 citation statements)

References 23 publications

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“…These criteria were in line with the recommendations from the Global CRO Council in Bioanalysis (GCC) (29). Although some studies have proposed different criteria (30)(31)(32)(33)(34), there is no one criterion for the spike-in method similar to our method. The discussion should be continued to provide recommendations or regulatory guidance/guidelines on assay qualification/validation using the spike-in method for the quantification of CTPs using ddPCR.…”

Section: Discussionsupporting

confidence: 71%

Novel Cell Quantification Method Using a Single Surrogate Calibration Curve Across Various Biological Samples

Nakayama

Yamamoto

Hirabayashi

2023

AAPS J

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Quantitative polymerase chain reaction (qPCR) is generally used to quantify transplanted cell therapy products in biological samples. As the matrix effects on PCR amplification and variability in DNA recovery from biological samples are well-known limitations that hinder the assay’s performance, a calibration curve is conventionally established for each matrix. Droplet digital PCR (ddPCR) is based on the endpoint assay and advantageous in avoiding matrix effects. Moreover, the use of an external control gene may correct assay fluctuations to minimize the effects caused by inconsistent DNA recovery. In this study, we aimed to establish a novel and robust ddPCR method capable of quantifying human cells across various mouse biological samples using a single surrogate calibration curve in combination with an external control gene and DNA recovery normalization. Acceptable accuracy and precision were observed for quality control samples from different tissues, indicating the excellent quantitative and versatile potential of the developed method. Furthermore, the established method enabled the evaluation of human CD8+ T cell biodistribution in immunodeficient mice. Our findings provide new insights into the use of ddPCR-based quantification methods in biodistribution studies of cell therapy products. Graphical Abstract

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Section: Discussionsupporting

confidence: 71%

Novel Cell Quantification Method Using a Single Surrogate Calibration Curve Across Various Biological Samples

Nakayama

Yamamoto

Hirabayashi

2023

AAPS J

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“…Persistence of CAR-CLDN18.2 cells in peripheral blood was determined at the central laboratory of CARsgen Therapeutics by quantification of the woodchuck hepatitis post-transcriptional regulatory element region of the lentiviral transgene by qPCR (Supplementary Table 9 ) 42 . Patient samples of 10 ml of peripheral blood were collected in K2EDTA tubes at baseline and after infusion.…”

Section: Methodsmentioning

confidence: 99%

Claudin18.2-specific CAR T cells in gastrointestinal cancers: phase 1 trial interim results

Gong

et al. 2022

Nat Med

312

155

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Despite success in hematologic malignancies, the treatment landscape of chimeric antigen receptor (CAR) T cell therapy for solid tumors remains limited. Claudin18.2 (CLDN18.2)-redirected CAR T cells showed promising efficacy against gastric cancer (GC) in a preclinical study. Here we report the interim analysis results of an ongoing, open-label, single-arm, phase 1 clinical trial of CLDN18.2-targeted CAR T cells (CT041) in patients with previously treated, CLDN18.2-positive digestive system cancers (NCT03874897). The primary objective was safety after CT041 infusion; secondary objectives included CT041 efficacy, pharmacokinetics and immunogenicity. We treated 37 patients with one of three CT041 doses: 2.5 × 108, 3.75 × 108 or 5.0 × 108 cells. All patients experienced a grade 3 or higher hematologic toxicity. Grade 1 or 2 cytokine release syndrome (CRS) occurred in 94.6% of patients. No grade 3 or higher CRS or neurotoxicities, treatment-related deaths or dose-limiting toxicities were reported. The overall response rate (ORR) and disease control rate (DCR) reached 48.6% and 73.0%, respectively. The 6-month duration of response rate was 44.8%. In patients with GC, the ORR and DCR reached 57.1% and 75.0%, respectively, and the 6-month overall survival rate was 81.2%. These initial results suggest that CT041 has promising efficacy with an acceptable safety profile in patients with heavily pretreated, CLDN18.2-positive digestive system cancers, particularly in those with GC.

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“…The qPCR is prone to quantitation bias and errors that includes (i) dependence on accurate quantification of calibrators to generate standard curve, with an assumption of no loss of calibrator molecules (ii) potential for errors during initial preparation and quantification of the standards, resuspensions, serial dilutions etc., and (iii) during PCR amplification itself 47 . In addition, the qPCR reaction efficiency is vulnerable to the presence of inhibitors (from blood sample, reagents, salts, anti‐coagulants, etc 29–31 . On the contrary, the absolute quantification of gene copy numbers by ddPCR is independent of the use of standard curve and the partition of the reaction in individual droplets largely overcomes the limitations mentioned above for qPCR.…”

Section: Discussionmentioning

confidence: 99%

“…Since then, several other studies have been reported where qPCR was used to measure transgene levels 19–26 . Although the results from flow cytometry and qPCR have shown to be correlative, 27,28 the differences in these techniques and samples may not always produce concordant data sets attributed to various factors 17,29 . QPCR has been traditionally used for estimating the copy number of CAR transgenes 29 .…”

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confidence: 99%

Bioanalytical Methods for Characterization of CAR‐T Cellular Kinetics: Comparison of PCR Assays and Matrices

Masilamani

Jawa

Dai

et al. 2023

Clin Pharma and Therapeutics

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Recently, multiple chimeric antigen receptor T‐cell (CAR‐T)‐based therapies have been approved for treating hematological malignancies, targeting CD19 and B‐cell maturation antigen. Unlike protein or antibody therapies, CAR‐T therapies are “living cell” therapies whose pharmacokinetics are characterized by expansion, distribution, contraction, and persistence. Therefore, this unique modality requires a different approach for quantitation compared with conventional ligand binding assays implemented for most biologics. Cellular (flow cytometry) or molecular assays (polymerase chain reaction (PCR)) can be deployed with each having unique advantages and disadvantages. In this article, we describe the molecular assays utilized: quantitative PCR (qPCR), which was the initial platform used to estimate transgene copy numbers and more recently droplet digital PCR (ddPCR) which quantitates the absolute copy numbers of CAR transgene. The comparability of the two methods in patient samples and of each method across different matrices (isolated CD3+ T‐cells or whole blood) was also performed. The results show a good correlation between qPCR and ddPCR for the amplification of same gene in clinical samples from a CAR‐T therapy trial. In addition, our studies show that the qPCR‐based amplification of transgene levels was well‐correlated, independent of DNA sources (either CD3+ T‐cells or whole blood). Our results also highlight that ddPCR can be a better platform for monitoring samples at the early phase of CAR‐T dosing prior to expansion and during long‐term monitoring as they can detect samples with very low copy numbers with high sensitivity, in addition to easier implementation and sample logistics.

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Considerations in the Development and Validation of Real-Time Quantitative Polymerase Chain Reaction and its Application in Regulated Bioanalysis to Characterize the Cellular Kinetics of Car-T Products in Clinical Studies

Cited by 13 publications

References 23 publications

Novel Cell Quantification Method Using a Single Surrogate Calibration Curve Across Various Biological Samples

Novel Cell Quantification Method Using a Single Surrogate Calibration Curve Across Various Biological Samples

Claudin18.2-specific CAR T cells in gastrointestinal cancers: phase 1 trial interim results

Bioanalytical Methods for Characterization of CAR‐T Cellular Kinetics: Comparison of PCR Assays and Matrices

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