An adjusted droplet digital PCR assay for quantification of vector copy number in CAR-T cell and TCR-T cell products.

BACKGROUND: Genetically engineered T-cell therapy holds immense promise in cancer immunotherapy. These T-cell products are typically engineered by vectors that permanently integrate into the T-cell genome, thus raising concerns about potential risks of insertional mutagenesis. Therefore, it becomes imperative to assess the integrated vector copy number (VCN) as a critical safety parameter for gene-engineered cell products. MATERIALS AND METHODS: In this study, we developed a robust assay for assessing the VCN of chimeric antigen receptor-T cell and T-cell receptor T-cell products, based on the droplet digital polymerase chain reaction (ddPCR) method. To provide accurate representation of the VCN in gene-engineered cells, we implemented a calculation that factors in the putative transduction efficiency based on Poisson distribution statistics. The adjusted VCN value (VCN(adj)) was also compared with VCN value from sorted transgene-positive cell populations, to validate its accuracy. RESULTS: This assay consistently and accurately determines the average VCN for cell products. By comparing the VCN in sorted transgene-positive cell populations, we validated the refinement calculation provides a closer approximation to the actual VCN within transduced cells, offering a more realistic representation of the VCN for engineered cell products. CONCLUSION: In summary, we present a reliable and robust ddPCR-based assay for quantification of VCN in gene-engineered cell products.