Vectofusin

On day 7 of culture, 0.25 × 10⁶ NK cells were seeded in Opti‐MEM + 0.1% heat‐inactivated FCS + 20 ng mL⁻¹ IL‐15 + 10 μg mL⁻¹ vectofusin (Miltenyi Biotec) in a 48 well plate with or without FMC63‐CD28 (hinge)‐CD28 (TM)‐CD28‐CD3z‐MSCV‐pRRL‐BaEV at a MOI of 5. The plate was centrifuged at 1200 g for 1 h at 32°C. After overnight incubation, the cells were harvested, washed and reseeded in GMP‐SCGM + 10% human serum + GlutaMax + 5 ng mL⁻¹ IL‐15 in a G‐Rex 48 well plate. For cord blood‐derived NK cells, 250 000 freshly thawed irradiated iKE2‐F9 feeder cells were added, with 330 000 iKE2‐F9 feeder cells being added to PB‐derived NK cells. Four days after transduction, cells were phenotyped to determine transduction efficiency.

CAR T cell production was performed on the Prodigy device (Miltenyi). Pheresis was washed in DPBS, stimulated with transact (Miltenyi) and cultured in IL-7 and IL-15. Transduction was performed on d 3 after stimulation initially in retronectin-coated bags, but subsequently was performed in suspension within the Miltenyi Prodigy bioreactor facilitated by vectofusin (Miltenyi). T cells were expanded in the Prodigy device until the dose was reached. Total production time ranged from 7 to 14 d. Products were cryopreserved in DMSO containing cryoprotectant.

Target cells were seeded at 5000 or 10,000 cells per well of a 96-well plate and directly incubated with viral particles at the indicated multiplicities of infection (MOI) in their cognate media (in the presence of FBS). Twenty-four hours later, the media was exchanged with fresh media, and cells were incubated for at least another 72 h before analysis. For transduction enhancer tests, 8 µg/ml of Polybrene (Santa Cruz Biotechnology Cat. # sc-134220) or 10 µg/ml of vectofusin (Miltenyi Biotec, Cat. # 130-111-163) were added. Spinoculations were performed at 1500 × g for 90 min at 33 °C. For DIRECTED tropism, the indicated antibody amounts were used unless otherwise stated. For the in vitro transduction of Kasumi-1 cells, lentiviral particles were co-incubated with αCD117 or αCD20 antibody for 30 min at room temperature, before excess antibody was removed using Amicon Ultra-0.5 Centrifugal Filter Units (Millipore Sigma, Cat. # UFC510096) by washing three times with excess PBS.