CAR-encoding vector and lentivirus production

The second generation HER2 CAR and mock lentiviral constructs have been described previously (Hammill et al., 2015; Helsen et al., 2018). The lentiviral vector encoding the anti-HER2 CAR consists of a human DARPin28z, a CD8 hinge region, a transmembrane and cytoplasmic domain of CD28, and the cytoplasmic region of CD3zeta (pCCL-Darpin-hCD8a NGFR) under the control of the human EF-1α promoter and truncated NGFR under the control of a human cytomegalovirus (hCMV) promoter as a transduction marker. The CAR negative control vector only includes the truncated NGFR gene under the hCMV promoter (pCCL-CMV-NGFR).

Third generation, self-inactivating and non-replicative lentivirus was produced as previously described (Hammill et al., 2016; Helsen et al., 2018). Briefly, 9 × 10⁶ HEK 293T cells were first cultured on 15 cm diameter tissue culture-treated plates (NUNC; Thermo Fisher) until 70% confluency. The HEK 293T cells were then transfected with the packaging plasmids pRSV-REV (6.25 μg), pMD2.G (9 μg), pMDLg-pRRE (12.5 μg), and the transfer plasmid pCCL containing the transgene (32 μg) using Opti-MEM (Thermo Fisher) and Lipofectamine 2000 (Thermo Fisher). Twelve to sixteen hours after transfection, media was removed and supplemented with fresh HEK 293T media containing sodium butyrate (1mM; Sigma-Aldrich Canada Co.). After 36–48 hr post media replenishment, media containing lentivirus particles was collected, filtered with 0.45 μm filters and concentrated by ultracentrifugation (4°C, 1 hr 40min, 1.3 × 10 rcf). Viral pellet was resuspended in PBS (phosphate buffered saline) and stocks were stored at −80°C. Viral titer in TU/mL was determined by serial dilution and transduction of HEK 293T cells with thawed virus aliquots. Titer was determined by measuring the %NGFR positive population by flow cytometry using a VioBrightFITC-conjugated anti-NGFR antibody (Miltenyi Biotec).