Human NSLCL cell lines H520, H1975 and H1299, SCLC cell lines H446 and H69, pancreatic cancer cell line BxPC3, breast cancer cell line MDA-DB-468, ovarian cancer cell line OVCAR3, CHO and HEK-293 T cells were purchased from American Type Culture Collection (ATCC) and maintained in DMEM medium (Thermo Fisher Scientific) supplemented with 10% heat-inactivated fetal bovine serum (FBS), 2 mM glutamine and 1% penicillin/streptomycin (all from Thermo Fisher Scientific) and all cell lines were cultured at 37 °C in a humidified chamber with 5% CO2. Stably transfected PTK7-CHO cell line was constructed by infecting parental CHO cells with lentiviral supernatants containing PTK7 gene (#HG19399-UT, Sino Biological) and sorting for PTK7 expression by using MoFloTM XDP cell sorting system (Beckmam Coulter). These cell lines were also infected with the lentiviral supernatants containing Luciferase-IRES-GFP (GL) and were then sorted for GFP expression to obtain GL-expressing cell lines. Human primary normal epithelial cell lines (Mammary, Small Airway and Renal Epithelial Cells) and human umbilical vein endothelial cells (HUVECs) were obtained from PriCells (Wuhan, China) and cultured according to the supplier’s instructions.
Sequences of 3 humanized mouse anti-human PTK7 antibodies (Hu23, Hu24 and Hu58) were obtained from an US patent (US20150315293A1). The variable region sequences of heavy (VH) and light chain (VL) of these antibodies were used to design scFv with the sequence of VH-G4S Linker-VL. PTK7-CARs containing scFv from Hu23, Hu24 and Hu58 were designated as PTK7-CAR1, PTK7-CAR2, and PTK7-CAR3 respectively. From the 5′-end to 3′-end, each CAR is comprised of the human CD8α signal peptide sequence, PTK7-scFv, hinge and TM regions of the human CD8α molecule, 4-1BB intracellular domain sequence (BB-ICD), and CD3ζ intracellular domain sequence (CD3ζ-ICD) as previously described . Following CAR, a truncated tEGFR sequence is included via T2A ribosomal skipping sequence in the construct to allow for potential enrichment, tracking and depletion if needed of transduced T cells . DNA encoding the CARs was codon-optimized and synthesized by General Biosystems (Anhui, China) with appropriate restriction sites. The CAR sequences were then cloned into third generation self-inactivated lentiviral vector pLVEF derived from pRRLSIN.cPPT.PGK-GFP.WPRE vector (Plasmid #12252, Addgene) with replacing its original human PGK promoter with human EF1α promoter from pWPXLd vector (Plasmid #12258, Addgene). As a negative control, lentiviral vector encoding truncated tEGFR was constructed.
High-titer replication-incompetent lentiviruses were produced and concentrated as described previously . Briefly, HEK-293 T cells were transfected with pVSV-G (VSV glycoprotein expression plasmid), pRSV-Rev (Rev expression plasmid), pMDLg/p.RRE (Gag/Pol expression plasmid), and pLVEF transfer plasmid using polyethylenimine (PEI, Sigma). The viral supernatant was harvested at 24 and 48 hours after transfection and concentrated by using Lenti-X Concentrator (Clontech) in accordance with manufacturer’s instructions.
CAR T-cell production
Human PBMCs were obtained from healthy donors under protocols approved by the Institutional Review Board of Harbin Medical University and isolated by density gradient centrifugation over Ficoll-Paque (GE Healthcare). Freshly isolated PBMCs were then activated with anti-human CD3/CD28 Dynabeads (Thermo Fisher Scientific) at a 3:1 ratio for 48 hours followed by two sequential transduction with lentiviruses on RetroNectin-coated non-tissue treated plates and maintained in culture in RPMI-1640 (Thermo Fisher Scientific) supplemented with 10% FBS (Thermo Fisher Scientific) and recombinant human IL-2 (300 U/mL). Fresh media containing cytokine were replenished every other day to maintain T-cell concentration at 0.5×106 cells/mL. Five days after transduction, the CD3/CD28 Dynabeads were removed from the culture by magnetic separation, and CAR T cells were propagated for 14 days in total before using for functional assays. To track T cell numbers over time, viable cells were counted using trypan blue.
PTK7 expression on tumor cells was detected by mouse monoclonal anti-PTK7 antibody (clone OTI2E7, Invitrogen) and goat-anti-mouse IgG-phycoerythrin (PE)-conjugated antibody (Jackson ImmunoResearch). CAR expression on 293T cells was detected by APC-conjugated rabbit monoclonal anti-EGFR antibody (Sino Biological) and biotin-conjugated goat-anti-human IgG F(ab′)2 (Jackson ImmunoResearch) and streptavidin-PE (Biolegend). CAR expression on T cells was detected by FITC-conjugated CD4 (clone OKT4), PE-conjugated CD8 (clone SK1, all from Biolegend), and APC-conjugated anti-EGFR antibody. The phenotype and effector molecule expression on T cells were detected with a panel of monoclonal anti-human antibodies as follow: BV510-conjugated CD3 (clone UCHT1), BV421-conjugated CD4 (clone OKT4), APC-Cy7-conjugated CD8 (clone SK1), APC-conjugated CD45RO (clone UCHL1), PE-conjugated CCR7 (clone G043H7), PE-conjugated TIM-3 (clone F38-2E2), APC-conjugated PD-1 (clone EH12.2H7), and PE-conjugated Granzyme B (clone GB11, all from Biolegend). CAR T cells in spleens from tumor-bearing tumor was detected by BV510-conjugated CD3 and APC-conjugated rabbit anti-EGFR antibody. In most assays, cells were stained with Zombie Aqua™ Fixable Viability Kit (BioLegend) to exclude dead cells from analysis. Flow cytometry data were acquired with a FACSCantoTM system (BD Biosciences) using DIVA software according to the manufacturers’ instructions.
Cytokine release assays
Control or PTK7-CAR T cells (1×105 cells/100 μl media) were co-cultured with an equal number of target cells for 24 hours, after which cell-free supernatants were harvested for testing IL-2 and IFN-γ secretion by ELISA kits (R&D Systems) according to the manufacturer’s instructions.
Control or CAR T cells were first labeled with 5 μM fluorescent dye carboxyfluorescein diacetate succinimidyl ester (CFSE; Invitrogen) according to the manufacturer’s instructions, and then co-cultured with tumor cells at an effector-to-target ratio of 1:1. CFSE dilution was measured on gated T cells on day 3 using flow cytometry.
In vitro killing assays
For tumor cell killing assays, GL-expressing target cells (1x104 cells/100 μl media) were co-cultured with control or PTK7-CAR T cells at the varying effector-to-target ratios in triplicate wells of white 96-well plates. In some assays, it was conducted in the presence of soluble PTK7 protein (OriGene). Target cell viability was monitored 18 h later by using Bright-Glo™ Luciferase Assay System (Promega) according to the manufacturer’s instructions. The percent lysis (%) was calculated by using the following equation: 1-[bioluminescence value in sample well (target cells + CAR T cells)/maximum bioluminescence value (target cells alone)].
For human primary normal cell killing assays, target cells were first labeled with 5 μM fluorescent dye CFSE according to the manufacturer’s instructions, and then co-cultured with control or PTK7-CAR T cells at the indicated effector-to-target ratios in triplicates. After 18-hour incubation at 37°C, mixed cells were harvested and stained with 7-AAD and then subjected to flow cytometric analysis to quantify remaining live (7-AAD negative) target cells. The cytotoxicity was calculated as 100% - the percentage of alive target cells/alive target cells in control wells without effectors.
In vitro recursive cytotoxicity assays
GL-expressing tumor cells (1x105 cells/500 μl media) were seeded in 12-well tissue culture plates, after overnight plating, 2.5 × 104 (effector-to-target ratio of 1:4) CAR T cells were added to the monolayer of tumor cells. Three days later when tumor cells had been completely eradicated (round 1), all cells in the well were collected and washed with PBS, resuspended in fresh medium and added to a new plate seeded with tumor cells for 3 days (round 2). This procedure was repeated one more time, if applicable (round 3). At the end of each round, a duplicate well was harvested for counting of residual tumor cells (GFP+) and CAR T cells (CD3+) and other phenotypic analysis (granzyme B, PD-1, TIM-3) of CAR T cells by flow cytometry.
In vivo tumor models
All animal experiments were conducted under a protocol approved by the Institutional Animal Care and Use Committee of the Harbin Medical University. Six- to 8-week-old B-NSG mice (NOD-Prkdcscid Il2rgtm1/Bcgen) were obtained from Biocytogen Co.,Ltd (Beijing, China) and maintained on a 12-h light–dark cycle in a temperature-controlled high barrier facility with free access to food and water and treated under specific pathogen-free conditions at the Animal Centre of the Harbin Medical University. The tumor xenograft model was established by subcutaneous (s.c.) inoculation with 3 x 105 H520 or H69 tumor cells suspended in 100µl PBS. After 7 days when the tumor was consistently palpable (50-100 mm3), mice were randomized into 3 groups (3-5 mice per group) and intravenously (i.v.) injected with control or PTK7-CAR T cells suspended in 100µl PBS and repeated once one week later. Mice were weekly monitored for tumor growth by using a calliper for 60 days, and then euthanized by cervical dislocation with blood and tumor harvested for analysis when they seemed moribund or their tumors reached 15 mm in diameter. Tumor volume (V) was calculated according to the following formula: V (mm3) = length x width2/2.
Tumor tissues were fixed with formalin and embedded in paraffin. Then, 4-mm-thick sections were deparaffinized with xylene and rehydrated in decreasing concentrations of ethanol. After heat-induced antigen retrieval, slides were then blocked by 3% BSA and stained with rabbit monoclonal anti-human CD3ε antibody (clone SP162, Abcam) or rabbit polyclonal anti-PTK7 antibody (Invitrogen) in the blocking solution overnight at 4℃. Slides were then rinsed with Tris-HCl/0.05% Tween-20 buffer and visualized with a horseradish peroxidase (HRP)-conjugated anti-rabbit EnVision+ Kit (Dako). PBS substituted for the primary antibody was used as the negative control.
Statistical analyses were performed with GraphPad Prism software (version 7.0). Differences in groups were determined by two-way ANOVA with Tukey’s multiple comparison test with P<0.05 considered to be a statistically significant. The survival curves were constructed using the Kaplan-Meier method and analyzed by using a log-rank test. All values and error bars represent the mean ± SEM. In the figures, significance of findings was defined as follows: p > 0.05; *p < 0.05; **p < 0.01; ***p < 0.001, or ****p < 0.0001.