Abstract
BackgroundGenetically engineered T cells have become an important therapy for B-cell malignancies. Measuring the efficiency of vector integration into the T cell genome is important for assessing the potency and safety of these cancer immunotherapies.MethodsA digital droplet polymerase chain reaction (ddPCR) assay was developed and evaluated for assessing the average number of lenti- and retroviral vectors integrated into Chimeric Antigen Receptor (CAR) and T Cell Receptor (TCR)-engineered T cells.ResultsThe ddPCR assay consistently measured the concentration of an empty vector in solution and the average number of CAR and TCR vectors integrated into T cell populations. There was a linear relationship between the average vector copy number per cell measured by ddPCR and the proportion of cells transduced as measured by flow cytometry. Similar vector copy number measurements were obtained by different staff using the ddPCR assay, highlighting the assays reproducibility among technicians. Analysis of fresh and cryopreserved CAR T and TCR engineered T cells yielded similar results.ConclusionsddPCR is a robust tool for accurate quantitation of average vector copy number in CAR and TCR engineered T cells. The assay is also applicable to other types of genetically engineered cells including Natural Killer cells and hematopoietic stem cells.
Highlights
Engineered T cells have become an important therapy for B-cell malignancies
The cells were transduced with a γ-retroviral vector that encoded a Chimeric antigen receptor (CAR) and 7 days after initiation of the cultures the CAR T cells were collected for vector copy number analysis
We showed that there was no significant difference in copy number at multiple multiplicity of infections (MOI) when comparing results from targeting either of these two regions (Figure A)
Summary
Engineered T cells have become an important therapy for B-cell malignancies. Chimeric antigen receptor (CAR) T cells are a novel cellular therapy wherein autologous T cells are harvested from a patient and genetically modified to express chimeric antigen receptors. These receptors are composed of an extracellular scFv designed to bind to a target with high specificity, and an intracellular portion consisting of a costimulatory structure and the T. T cells genetically engineered to express TCRs specific for cancer antigens are being used for cancer immunotherapy. T cells engineered to express TCRs directed to the cancer/testis antigen NY-ESO-1 are being used to treat melanoma and synovial cell sarcoma [7].
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