Human serum

Healthy donor-derived enriched CD4+/8+ T cells were isolated from leukocyte cones using SepMate, RosetteSep reagent (Stemcell Technologies) and Ficoll Paque density grade centrifugation. Excess cryopreserved leukapheresis from B-ALL patients on the ALLCAR19 trial (NCT02935257) were thawed, rested overnight in TexMACS (Miltenyi Biotec) supplemented with 3% human serum (Sigma Aldrich, Life Science Production) and enriched for CD4+/8+T cells using a microbead-based pan T-cell isolation kit (Miltenyi Biotec). Activation/transduction at small-scale was designed to mimic the cGMP Miltenyi CliniMACS Prodigy-based CAR-T manufacture process on ALLCAR19.20 (link) Selected T-cells were maintained in TexMACS supplemented with 3% human serum and 10 ng/mL IL7/IL15 (cTexMACS). Cells were activated with TransAct (Miltenyi Biotec) and transduced with concentrated lentivirus on retronectin-coated plates at a multiplicity of infection of 5, cells were maintained for a further 4 days for a total manufacturing time of 8 days. AKT inhibitor VIII (Merck Millipore/Ardena) was added at T-cell activation at a concentration of 1–5 µM and maintained throughout the culture period, with a media change every 48 hours.

The manufacturing procedure was performed in cleanrooms according to GMP EU Annex 1. TILs were expanded in vitro according to the “Young TIL” method.^{25 (link),26 (link)} Within hours from surgical removal of the tumor, the tumor specimen was cut into small fragments and placed in individual wells with culture medium containing RPMI 1640 (Thermo Fisher Scientific), 10% human serum (Sigma-Aldrich), IL-2 6000 IU/mL (Novartis), and Penicillin, Streptomycin and Fungizone (Thermo Fisher Scientific). During expansion, TIL cultures were pooled together to generate a bulk TIL product.
Following initial expansion, TILs were further expanded according to the Rapid Expansion Protocol (REP) for fourteen days.^{27 (link)} The REP was initiated in static culture containers with 2 × 10^{7 (link)} TILs in REP medium containing RPMI 1640/AIM-V (Thermo Fisher Scientific), 10% human serum, IL-2, anti-CD3-antibody (OKT3) (Miltenyi Biotec) and irradiated allogeneic feeder cells in a 1:200 ratio. Between day 6 and 9, depending on cell growth, the cell culture was transferred to a dynamic Xuri W25 bioreactor (Cytiva). On day 14, the cell culture was harvested, centrifuged, and washed.