GMP validation of the SOP for early transduction of TriVSTs. (A) Diagram of the finalized SOP. Day 0, PBMC were stimulated with irradiated, Ad5f35-transduced autologous LCLs and then transduced on day 3. On day 9, triVSTs were harvested and restimulated in a G-Rex100 gas permeable flask. Cryopreservation was performed after the second stimulation on days 16–18 of culture. (B) Transgene expression after the 1st and 2nd stimulation was determined by flow cytometry in 4 donors. (C) Trivirus specificity was determined by γ-IFN ELIspot assay after the 1st and 2nd stimulations. 1x105 responder cells per well were pulsed with overlapping peptide libraries for hexon, penton (to test for adenovirus specificity) and pp65 (to test for CMV specificity) and LCLs (to test for EBV specificity). One representative donor of three is shown. (D) Three clinical products of CAR-transduced triVSTs were cryopreserved in 10% DMSO, 12.5% HSA (Flexbumin, Baxter) and 1xHBSS in a Cryomed controlled-rate freezer. Viability of fresh and thawed cells was evaluated using trypan blue exclusion method. (E) INF-γ ELISPOT assay results for fresh and thawed CAR-transduced tri-VSTs. Adenovirus-specific responses were evaluated using pepmix libraries spanning hexon and penton, CMV with a pepmix library for pp65 and EBV-specific responses were analyzed using autologous LCLs.