Table 1.
List of samples used for assay development and validation.
Fig 1.
(A) JCAR014 CAR-T cell plasmid standard curves using data points from three different PCR runs, with reactions run in triplicate [R2 (0.9842–0.9972); P < 0.0001 for each triplicate]. JCAR014 CAR-T cell standard curve runs 1 and 2 were done on the same day on two different machines (the ABI QuantStudioTM Real-Time PCR system and Bio-Rad CFX 384 Real-Time PCR Detection System, respectively). Run 3 was performed six days later on the same machine as run 1. (B) MAGEA1 TCR-T cell plasmid standard curves using data points from three different PCR runs, with reactions run in triplicate, on three different days [R2 (0.9640–0.9908); P < 0.0001 for each triplicate]. MAGEA1 TCR-T cell standard curve runs 1 and 3 were performed using the Bio-Rad CFX 384 Real-Time PCR Detection System, while run 2 was performed using the ABI QuantStudioTM Real-Time PCR system.
Fig 2.
Gel electrophoresis imaging of traditional PCR products using WPRE- targeted primers.
Left: 10-fold dilution series of fresh HA-1 TCR-T cell product DNA in a background of fresh PBMC DNA. Right: 10-fold dilution series of fresh JCAR014 CAR-T cell product DNA in a background of fresh PBMC DNA, with 100% PBMC and molecular biology grade water as negative controls. Ladders of 50 bp and 100 bp were used as size markers on 2% agarose gels shown.
Table 2.
Quantification cycles (Cq) and melt temperatures (Tm) for serial dilutions of DNA from JCAR014 CAR-T or HA-1 TCR-T cell DNA into DNA from normal healthy donor PBMCs are shown. Sample results were considered positive if Tm value was between 81.00 and 84.50°C.
Table 3.
qPCR results from FFPE samples using SYBR green with WPRE-specific primers.
Table 4.
List of real-time PCR results from patient FFPE samples using SYBR green qPCR with WPRE-specific primers.