Lentivirus vector design and lentivirus production
Lentivirus for target cell modification: A number of third-generation self-inactivating lentiviral plasmids expressing two transgenes separated by the sequence for the Thosea asigna virus 2A (T2A) peptide were constructed using pGreenPuro (SBI System Biosciences, Mountain View, CA). The plasmids are denoted pBMN(TurboRFP-Luc2), pBMN(copGFP-PSCA) and pBMN(copGFP-TARP), where TurboRFP encodes turbo red fluorescent protein, Luc2 encodes codon-optimized firefly luciferase, copGFP encodes copepod green fluorescent protein, PSCA encodes the human prostate stem cell antigen and TARP encodes human T cell receptor γ-chain alternate reading frame protein.
Lentivirus for T cell engineering: An anti-PSCA CAR-expressing lentiviral plasmid, pBMN(PSCA-CAR), was generated by fusing a PSCA-recognizing single chain antibody fragment, obtained through reversed genetics
 with the signaling moieties of CD28, OX-40 and CD3 ζ chain, from a plasmid obtained from M Brenner, Baylor College of Medicine, Houston, TX
Lentiviruses were produced in HEK-293 T cells using polyethyleneimine (Sigma-Aldrich, St Louis, MO) transfection. The pBMN-based lentiviral plasmid and the packaging plasmids pLP1, pLP2 and pVSV-G (Invitrogen) were used at a ratio of 2:1:1:1. The supernatant was harvested 48 and 72 hours post-transfection, concentrated through ultracentrifugation at 75,000 × g for 90 minutes and stored at -80°C. Mock lentivirus was produced using an empty pRRL lentiviral plasmid (Addgene, Cambridge, MA).
Target cell lines
The mel526 cell line was obtained from T Boon, Ludwig Institute for Cancer Research, Brussels, Belgium and cultured in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) (Invitrogen, Carlsbad, CA). Mel526-based target cells were produced through lentiviral transduction followed by sorting using a FACS Aria III sorter (BD Biosciences, Franklin Lakes, NJ). Mel526 cells co-expressing TARP, copGFP, Luc2 and turboRFP will be referred to in the text as mel526(TARP), and mel526 cells co-expressing PSCA, copGFP, Luc2 and turboRFP will be referred to as mel526(PSCA).
T cells from activated and lentivirus transducted of PBMCs
Peripheral blood mononuclear cells (PBMCs) were isolated from buffy coats from healthy donors using Ficoll-Paque (GE Healthcare, Uppsala, Sweden) and cultured in RPMI-1640 supplemented with 10% human AB serum (our own production), 2 mM L-glutamine, 10 mM HEPES, 20 μM β-mercaptoethanol and 1% penicillin/streptomycin. The PBMCs were activated with 100 ng/ml OKT-3 (Nordic Biosite, Täby, Sweden) and 100 IU/ml IL-2 (Proleukin, Novartis, Basel, Switzerland) for 2 days to selectively stimulate T cells. Activated cells were transduced with 50 μl concentrated PSCA-CAR-encoding lentivirus or Mock lentivirus for 4 hours at 37°C in the presence of 10 μg/ml protamine sulphate and 100 IU IL-2 (Sigma-Aldrich). Transduction was repeated 24 hours later and the cells were cultured and expanded for 2-4 weeks before analysis. For analysis of PSCA-CAR expression, cells were stained with biotinylated protein-L (Genscript, Piscataway, NJ)
, washed 3 times with PBS containing 4% BSA, followed by labeling with phycoerythrin (PE)-conjugated streptavidin (BD Biosciences) or stained with Alexa Fluor® 647 F(ab')2 Fragment of Goat Anti-Mouse IgG (H + L) (Invitrogen) and stained with an allophycocyanin (APC)-conjugated anti-CD3 or fluorescein isothiocyanate (FITC)-conjugated anti-CD3 antibody (Nordic Biosite). Flow cytometry analysis was performed using FACSCanto II or LSRII (BD Biosciences).
IFN-γ and IL-2 ELISA
Activated and PSCA-CAR-transduced or Mock-transduced T cells (105 cells) were co-cultured with mel526(PSCA) or mel526(TARP) cells at a 1:1 ratio in 96-well plates. Supernatants were collected after 16 hours. ELISA (Mabtech, Nacka Strand, Sweden) was used to detect IFN-γ and IL-2 secretion.
Activated and PSCA-CAR-transduced or Mock-transduced T cells (105 cells) were labeled for 20 minutes at 37°C with 5 μM Cell Trace Violet (Invitrogen) in PBS and then washed with cold cell culture medium containing 10% human serum to stop the labeling reaction. Labeled PBMCs were co-cultured with irradiated (50 Gy) mel526(PSCA) or mel526(TARP) cells at a 1:1 ratio in 96-well plates for 5 days. The T cells received a low dose of IL-2 (10 IU/ml) on day 1. The labeled cells were then collected and stained with an APC-conjugated anti-CD3 antibody followed by flow cytometry analysis.
CD107a degranulation flow cytometry analysis
Activated and PSCA-CAR-transduced or Mock-transduced T cells (105 cells) were co-cultured with mel526(PSCA) or mel526(TARP) cells at a 1:1 ratio in 96-well plates for 16 hours. Cells were stained with a FITC-conjugated anti-CD107a antibody and an APC-conjugated anti-CD3 antibody followed by flow cytometry analysis.
Bioluminescence in vitro killing assay
Activated and PSCA-CAR-transduced or Mock-transduced T cells were co-cultured with luciferase-expressing mel526(PSCA) or mel526(TARP) (15000 cells) in various effector to target cell ratios (0.4:1–50:1) in flat-bottomed 96-well plates. Co-cultures were harvested 48 hours later and analyzed for luciferase expression using Steady-Glo® Luciferase Assay System (Promega Corporation, Madison, WI), according to the manufacturer’s instruction, and the luminescence was measured in a luminometer (Wallac Victor 2 Multi-label Counter, Perkin Elmer, Waltham, MA). Luciferase activity from target cells not exposed to T cells was set as 100% cell viability (survival).
Animal model for T cell treatment
Nude NMRI mice (Harlan, Netherlands) were injected subcutaneously (hind flank) with 3 × 106 mel526(PSCA) cells. One, seven and fourteen days later the mice received intravenous injection of 1 × 107 PSCA-CAR-transduced T cells or Mock-transduced T cells. Twelve mice per group were used. The tumors were measured by caliper and tumor volume was calculated using the equation (length × width2)/2. Animals were sacrificed, when tumors reached over 1000 mm3. The Uppsala Animal Ethics Committee has approved the animal studies (ID numbers C319/9 and C195/11).
Statistics were performed using GraphPad prism software version 5.04 (La Jolla, CA, USA). Statistical analysis for IFN-γ and IL-2 secretion, cell proliferation and CD107a degranulation were performed using paired Student’s t-test. Log-rank test was used to compare survival curves created by the Kaplan-Meier method. Values of p < 0.05 were considered statistically significant.