Developing retroviruses encoding an EpCAM-specific CAR
The EpCAM-specific CAR construct is similar to the FMC63-28z CAR (Genebank identifier HM852952.1), except the anti-CD19, single-chain variable fragment sequence is replaced with an anti-EpCAM fragment (sequence corresponds to Genebank identifier AJ564232.1). The construct was synthesized and inserted into a pLNCX retroviral vector. Retroviruses encoding the EpCAM-specific CAR or an empty pLNCX vector for controls were generated using the retrovirus packaging kit, Ampho (Takara), and a 293 T packaging cell line, following the manufacturer’s protocol.
PBL preparation and retrovirus transduction
For PBL preparation, donor blood was obtained from healthy volunteers with consent from the Institutional Review Board of the Cancer Institute, Chinese Academy of Medical Sciences, and written informed consent for participation in the study was obtained from participants. After centrifugation on Ficoll-Hypaque density gradients (Sigma-Aldrich), PBMCs were plated at 2 × 106 cells/mL in cell culture for 2 h and the non-adherent cells were collected. The cells were then stimulated for 2 d on a non-tissue-culture-treated 24-well plate coated with 1 μg/mL OKT3 (Biolegend) at 1 × 106 cells/mL and in the presence of 1 μg/mL of anti-human CD28 antibody (Biolegend). For retrovirus transduction, a 24-well plate was coated with RetroNectin (Takara) at 4°C overnight, according to the manufacturer’s protocol, and then blocked with 2% BSA at room temperature for 30 min. The plate was then loaded with retrovirus supernatants at 300 μL/well and incubated at 37°C for 6 h. Next, 1 × 106 stimulated PBLs in 1 mL of medium were added to 1 mL of retrovirus supernatants before being transferred to the pre-coated wells and cultured at 37°C for 2 d. The cells were then transferred to a tissue-culture-treated plate at 1 × 106 cells/mL and cultured in the presence of 100 U/mL of recombinant human IL-2.
PC3, PC3M, Hela, and 293 T cells were obtained from ATCC and were maintained in culture with DMEM medium (Gibco) supplemented with 10% FBS. PBLs were cultured in RPMI (Gibco) supplemented with 10% FBS, 1× nonessential amino acid, L-glutamine, sodium pyruvate, penicillin-streptomycin, and 0.1% β-mercaptoethanol. To establish PC3-luc, PC3M-luc, and Hela-luc stable cell lines, the luciferase gene was cloned from pGL4.17-luc/Neo and inserted into pLNCX retroviral vectors; the retroviruses were prepared as described above. To transduce the tumor cell lines, retrovirus supernatants were mixed with cell culture medium at a ratio of 1:1, which was added to the tumor cells at 60–70% confluence with 15 μg/mL polybrene. Twenty-four hours after transduction, the cells were split into ten plates and 400 μg/mL of G418 was added to the culture. Culture medium was changed every 2–3 d, and 10–14 d later selected cells were passaged and maintained in culture medium supplemented with 200 μg/mL of G418.
Male NOD/SCID mice, 5–8 weeks of age, were purchased from Vital River Laboratories, and used in compliance with institutional animal healthcare regulations. For the PC3M in vivo model, 5 × 105 PC3M-luc cells were intraperitoneally injected into mice and 5 d later 1 × 107 PBLs transduced with the CAR or control vector were injected. For the PC3 metastasis model, PC3-luc cells were injected intravenously at 5 × 106 cells/mouse and 6 h later 5 × 106 PBLs transduced with the CAR or control vector were injected intravenously. Live animal imaging was performed as described previously , briefly, the mice were intraperitoneally injected with 15 μg/μL of luciferin (Promega) in 200 μL and 10 min later luminescence imaging was conducted with an IVIS system (Xenogen/Caliper Life Sciences). For the in vivo experiments, five mice were used per group and each experiment was repeated at least twice.
Sorted or unsorted PC3 cells in 100 μL of medium were seeded in a 96-well plate at 2,500 cells/well; control wells received 100 μL of medium only. Ten microliters of CCK-8 solution (Dojindo) was added to each well and after 4 h of incubation at 37°C, the cell number was determined by measuring the absorbance at 450 nm using a microplate reader. Cells were cultured for 24, 48, and 72 h and a CCK-8 assay was performed at each time point. The absorbance was subtracted with that of the control well and the resulting OD450 at each time point was divided by the starting value to calculate the relative proliferation ratio.
Flow cytometry and cell sorting
PBLs were stained with FITC, PE, or Percp-Cy5.5 conjugated CD3, CD4, or CD8 antibodies (eBioscience). Fluorescence was measured using a FACS Calibur flow cytometer and was analyzed using Flowjo software. To detect CAR transduced cells, PBLs were stained with an optimal concentration of biotinylated protein L (GeneScript), followed by staining with PE conjugated streptavidin (eBioscience). A PE-conjugated anti-human EpCAM antibody (eBioscience) was used to stain the tumor cells PC3 and PC3M and a FACSAria II cell sorter was used to sort EpCAM+ and EpCAM− cells.
Luciferase-expressing tumor cells were seeded in a 96-well plate at 1 × 105 cells/well and PBLs transduced with retroviruses were added at different E:T ratios. After incubation at 37°C for 24 h, luciferin (Promega) was added at a final concentration of 0.3 mg/mL and cytotoxicity was determined by luminescence imaging.
CFSE proliferation assay
Retrovirus-transduced PBLs were labeled with CFSE (Invitrogen) according to the manufacturer’s protocol and incubated with tumor cells at an E:T ratio of 2:1. Three days later, cells were collected and stained with Percp-Cy5.5 conjugated CD8 antibodies (eBioscience) and were analyzed by flow cytometry. The analysis was carried out on the CD8+ population.
Data are presented as means ± standard error of the mean. To determine the significance of differences between samples or groups, a student’s t-test or two-way analysis of variance was used as indicated in the figure legends.