Rapid Expansion Protocol (REP) With Viral Transduction

This process describes the standard rapid expansion protocol of TIL cells for clinical use, just in a smaller scale (9, 10). TIL were thawed in CM with 3,000 IU/ml IL-2 and allowed to rest for a period of 2 days at a concentration of 1.0×10⁶/ml in a 24-well plate. A mini-scale REP was initiated by stimulating TIL with 30 ng/ml OKT3, 3,000 IU/ml IL-2 and irradiated PBMC from non-related donors as feeder cells (5000 rad, TIL to feeder cells ratio = 1:100) in 50% CM and 50% AIM-V medium in T25 flasks. On the day of viral transduction (REP day 7), TIL were harvested, counted and adjusted to a concentration of 0.5×10⁶/ml in CM with 3,000 IU/ml IL-2. Four ml of the cell suspension was distributed per well of a 6-well plate layered with viral vector as follows: Plate coating and viral transduction were performed as described above for the SCP. The viral supernatant was then aspirated and 2 × 10⁶ TIL in Stim medium with 3,000 IU/ml IL-2 were added to each well, centrifuged for 10 min at 1,000g and incubated at 37°C overnight. The next day the cells were transferred to T75 culture flasks and maintained at a concentration of 0.5–2.0 × 10⁶ cells/ml in Stim medium with 3,000 IU/ml IL-2 until day 14 (potential day of TIL infusion).