Vector copy number quality control testing for CAR T-cells: critical validation parameters

Background & Aim Background Retroviral and lentiviral vectors have been broadly used in Chimeric Antigen Receptor (CAR) T-cell therapy clinical trials. These vectors have the capacity to integrate permanently into host cell DNA. There is an increased risk of oncogenesis if the vector copy number (VCN) per cell is high. The Food and Drug Administration (FDA) recommends that the VCN shall be <5 copies per genome. Accurate and rapid measurement of VCN is an important quality control step required for release of CAR T-cell products for patient infusion. Aims Establish a reliable test for detection of VCN in patient samples. Evaluate the linearity of the vector copy number qPCR and primer multiplexing. Methods, Results & Conclusion Methods CD3+ T-cells from three individuals with no tumour burden were transduced using a retroviral vector to generate CAR T-cells. CAR T-cells and the Un-transduced T-cells (controls) were expanded in the same culturing conditions for 14 days. Genomic DNA was extracted from the six samples using Qiagen DNA extraction kit. Human albumin (huALB) and the retroviral vector backbone copy numbers were detected using qPCR with TaqMan probes against a standard curve. Results The Vector Copy Number of the CAR-T cell products were 5, 3, and 2 copies for Donor 1, 2 and 3, respectively. Multiplexing of huALB and vector probes did not impact the accuracy of VCN detection. The linear correlation coefficient was r2 = 0.992 ± 0.003. Conclusion qPCR is a sensitive and reliable quality control test for detection of VCN in CAR T-cells. A number of critical process parameters and key variables need to be considered when preparing VCN validation protocols. Retroviral and lentiviral vectors have been broadly used in Chimeric Antigen Receptor (CAR) T-cell therapy clinical trials. These vectors have the capacity to integrate permanently into host cell DNA. There is an increased risk of oncogenesis if the vector copy number (VCN) per cell is high. The Food and Drug Administration (FDA) recommends that the VCN shall be <5 copies per genome. Accurate and rapid measurement of VCN is an important quality control step required for release of CAR T-cell products for patient infusion. Establish a reliable test for detection of VCN in patient samples. Evaluate the linearity of the vector copy number qPCR and primer multiplexing. CD3+ T-cells from three individuals with no tumour burden were transduced using a retroviral vector to generate CAR T-cells. CAR T-cells and the Un-transduced T-cells (controls) were expanded in the same culturing conditions for 14 days. Genomic DNA was extracted from the six samples using Qiagen DNA extraction kit. Human albumin (huALB) and the retroviral vector backbone copy numbers were detected using qPCR with TaqMan probes against a standard curve.