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Detection and quantification of integrated vector copy number by multiplex droplet digital PCR in dual-transduced CAR
Wei Wang1, Muhammad Al-Hajj1, Alireza S Alavi1
1Autolus Therapeutics, The MediaWorks, 191 Wood Lane, W12 7FP London, UK.
Abstract:
The success of chimeric antigen receptor (CAR) T cell therapies in refractory hematologic malignancies has prompted investigation of their efficacy in solid tumors. AUTO6NG is a dual-transduced GD2-targeting CAR that encodes distinct modules designed to enhance T cell activity in relapsed/refractory neuroblastoma. The ability to detect and precisely quantify vector copy number (VCN) for each integrated vector is essential for assessing the effect of each module on T cell tumor infiltration, persistence, and clinical activity. Droplet digital PCR (ddPCR) enables accurate, sensitive, and absolute quantification of specific nucleic acid sequences. Compared to standard detection of two targets, multiplex ddPCR assays allow simultaneous detection of up to four targets by selective modulation of signal amplitude while retaining the ability to quantify the target. We have developed a multiplex assay based on the two-channel system for simultaneous detection and quantification of three targets in AUTO6NG CAR T cells. The assay was highly specific, sensitive, accurate, and reproducible across time and samples. No differences were observed in measuring VCN between standard duplex and multiplex assays. Our results demonstrate that ddPCR is an accurate and cost-effective method for simultaneous detection of multiple targets in genomic DNA derived from engineered CAR T cells.
Insights
Multiplex droplet digital PCR (ddPCR) accurately quantifies multiple targets in engineered CAR T-cells. This method is essential for assessing chimeric antigen receptor (CAR) T-cell therapies in neuroblastoma treatment.
Area of Science:
- Biotechnology
- Molecular Biology
- Immunotherapy
Background:
- Chimeric antigen receptor (CAR) T-cell therapies show success in hematologic malignancies.
- Investigation into CAR T-cell efficacy in solid tumors, like neuroblastoma, is ongoing.
- AUTO6NG is a dual-transduced GD2-targeting CAR designed to enhance T-cell activity.
Purpose of the Study:
- To develop and validate a multiplex ddPCR assay for quantifying vector copy number (VCN) in AUTO6NG CAR T-cells.
- To assess the accuracy, sensitivity, and reproducibility of the multiplex ddPCR assay.
- To enable precise quantification of integrated vectors for evaluating CAR T-cell therapy efficacy.
Main Methods:
- Development of a multiplex ddPCR assay using a two-channel system.
- Simultaneous detection and quantification of three targets in genomic DNA from AUTO6NG CAR T-cells.
- Validation of the assay for specificity, sensitivity, accuracy, and reproducibility.
Main Results:
- The developed multiplex ddPCR assay demonstrated high specificity, sensitivity, accuracy, and reproducibility.
- No significant differences were observed in VCN measurements between standard duplex and multiplex ddPCR assays.
- The assay effectively enables simultaneous detection and quantification of multiple targets.
Conclusions:
- Multiplex ddPCR is an accurate and cost-effective method for simultaneous target quantification in engineered CAR T-cells.
- This validated assay is crucial for assessing the performance of novel CAR T-cell therapies like AUTO6NG.
- The findings support the use of ddPCR for robust quality control and clinical monitoring of CAR T-cell products.
