Construction of parental plasmid vectors and production of mcDNA
Based on previous reports, we designed a third-generation GPC3-CAR [23] structure and a second-generation CD133-CAR structure [24, 25]. The DNA sequences of GPC3 scFv and CD133 scFv were derived from monoclonal antibodies (mAbs) described by Nakano et al. [26] and Swaminathan et al. [27]. The GPC3-CAR was composed of the GPC3 scFv, human CD8α hinge and transmembrane domain (nucleotides 412-609, GenBank NM 001768.6), human CD28 molecule (nucleotides 538-660, GenBank NM 006139.3), human CD137 molecule (nucleotides 640-765, GenBank NM 001561.5) and human CD3ζ molecule (nucleotides 154-492, GenBank NM 198253.2). The CD133-CAR, containing the CD133 scFv, was linked to the intracellular domains from the human CD137 and CD3ζ molecules via the human CD8α hinge and CD8α transmembrane regions. NcoI and EcoRI sites were incorporated at both ends. We humanized the 2 CAR gene sequences and then synthesized (Detai Biologics, Nanjing, China) and confirmed them by genetic sequencing (Sango Biotech, Shanghai, China). We cloned these 2 CAR structures into pUC57 vectors and then into the parental minicircle plasmid pMC.CMV-EasyTM (System Biosciences, CA, USA). The pMC.CMV-Easy-GFP-CD133-CAR (8513 bp) parental minicircle plasmid contained the CD133-CAR (1455 bp) and a GFP cassette (758 bp), and pMC.CMV-Easy-GPC3-CAR (7923 bp) contained the GPC3-CAR (1608 bp) without a GFP cassette (to clearly distinguish the constructs in subsequent experiments). We transformed the parental minicircle plasmids into E. coli strain ZYCY10P3S2T (System Biosciences), and then added the inducer L- (+)-arabinose (Sigma Chemical, MO, USA) into the bacterial growth medium to mediate recombination between attB and attP. The recombinase φC31 was produced after the recombination and separated the parental minicircle plasmid into mcDNA and the parental bacterial backbone. We extracted the CD133-CAR mcDNA and GPC3-CAR mcDNA with an Endo-Free Plasmid DNA Maxi Kit (Omega Bio-tek, GA, USA) and confirmed them via restriction analysis.
Generation and proliferation of CoG133-CAR T cells
Peripheral blood mononuclear cells (PBMCs) derived from healthy donors were obtained from the Hebei Blood Center. All donors gave informed consent to use their samples for research purposes. All procedures were performed in accordance with the guidelines approved by Hebei Medical University. PBMCs were isolated with lymphocyte separation medium (Tonbo Biosciences, CA, USA). Primary human CD3+ T cells were negatively selected from PBMCs with MACS CD3 MicroBeads (Miltenyi Biotec, Bergish Gladbach, Germany) and cultured in RPMI-1640 medium (Thermo Fisher Scientific, MA, USA) supplemented with 10% heat-inactivated fetal bovine serum (FBS, Thermo Fisher Scientific) at 37°C in 5% CO2. Primary T cells were activated with 1000 U/L IFN-γ (Peprotech, NJ, USA), cultured with 1 μg/ml anti-CD3 and anti-CD28 antibodies (Miltenyi Biotec) for 1 day, and then expanded in the presence of 500 U/ml recombinant human interleukin-2 (IL-2, Peprotech) and 10 U/ml recombinant human interleukin-15 (IL-15, Peprotech) for 2-5 days. We transfected 5×106 T cells via electroporation with a 4D-NucleofectorTM system (Lonza, Cologne, Germany); 3μg of mcDNA control plasmid (System Biosciences), CD133-CAR plasmid or GPC3-CAR plasmid, and 100μl of P3 Primary Cell Buffer (Lonza) was added according to the manufacturer’s instructions. The EO-115 program was used. CoG133-CAR T cells were generated by simultaneously electroporating 1.5μg of CD133-CAR plasmid and 1.5μg of GPC3-CAR plasmid into T cells. The transfected T cells were cultured in fresh medium supplemented with 500U/ml IL-2. Fresh medium was added every two days to maintain a concentration of 8 × 105cells/ml.
Cell lines and culture conditions
The human HCC cell lines HepG2 and PLC8024 were obtained from the American Type Culture Collection (ATCC, VA, USA) and cultured in minimal essential medium (MEM, Thermo Fisher Scientific). Huh7 and SK-HEP-1 cells were obtained from the Shanghai Cell Bank (Shanghai, China) and cultured in Dulbecco’s modified Eagle’s medium (DMEM, Thermo Fisher Scientific). All cell lines were cultured in medium supplemented with 10% FBS (Thermo Fisher Scientific) and 1% penicillin-streptomycin (Thermo Fisher Scientific) at 37°C in 5% CO2. For bioluminescence assays, we generated a firefly luciferase expressed Huh7 cell line.
Flow cytometry
All cell samples were analyzed with a BD FACSCanto™ flow cytometry system (BD Bioscience, CA, USA), and statistical analysis was conducted in FlowJo software (FlowJo, OR, USA). The phenotype of T cells was assessed with fluorescently labeled antibodies specific for human CD3-PC5, CD4-fluorescein isothiocyanate (FITC) and CD8-FITC, which were obtained from BD Bioscience. Tumor surface antigen expression was detected with antibodies against human CD133- phycoerythrin (PE) (BioLegend, CA, USA) and GPC3-PE (Abcam, MA, USA); isotype control groups were stained with IgG1-PE (Abcam). The expression of GFP in T cells was evaluated in the FITC channel to demonstrate the expression of CD133-CAR. The expression of GPC3-CAR was assessed by recombinant biotinylated protein L (Thermo Fisher Scientific) binding PE-conjugated streptavidin (PE-SA, BD Bioscience). All FACS-related cell samples were handled on ice and washed three times with 1× PBS (Thermo Fisher Scientific) containing 1% FBS before staining the corresponding antibodies.
In vitro cytotoxicity assays
Effector cells were cocultured with target cells at increasing effector: target ratios of 1:5, 1:1, 5:1 and 10:1 in flat-bottom 96-well plates (Corning, NY, USA) containing 100 μl of T cell culture medium at 37°C in 5% CO2 for 18 hours. Then, we measured the absorbance at 450nm according to the Cell Counting Kit-8 instructions (Dojindo Molecular Technologies, Kumamoto, Japan) using an Epoch microplate spectrophotometer (BioTek, VT, USA). We calculated the cytotoxicity of the effector cells with the following formula: specific lysis (%) = [1– (mixture cell experiment–medium control)/ (target cell spontaneous–medium control)] ×100.
Cytokine secretion assays
Effector cells were cocultured with target cells in 96-well plates at an effector: target ratio of 5:1 for 24 hours. Supernatants were collected to measure the levels of cytokines, including IL-2, IFN-γ and TNF-α, according to the protocols of the enzyme-linked immunosorbent assay (ELISA) kit (Thermo Fisher Scientific). Additionally, 5×106 effector cells were collected for in vitro experiments, and 100μl of peripheral blood was collected from treated xenograft mice for in vivo experiments.
Western blot analysis
T cells and tumor tissues were lysed with Radioimmunoprecipitation (RIPA) Lysis and Extraction Buffer (Thermo Fisher Scientific) and quantified with a BCA Protein Assay Kit (Thermo Fisher Scientific). Protein lysates were separated on a 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel and transferred to a polyvinylidene fluoride (PVDF) membrane (Thermo Fisher Scientific). The PVDF membrane was blocked in AquaBlock Blocking Buffer (EastCoast Bio, ME, USA) for 2 hours, followed by overnight incubation at 4°C with the following primary antibodies: anti-CD133 (1:1000, Abcam), anti-GPC3 (1:400, Abcam), anti-β-actin (1:5000, Abcam) and anti-CD3ζ (1:5000, Abcam). Unbound antibodies were washed away with Tris-HCl buffer containing Tween 20, and the PVDF membrane was then incubated with a horseradish peroxidase (HRP)-conjugated secondary antibody (Abcam) for 50 minutes at room temperature. Blots were detected using SuperSignalTM West Pico PLUS Chemiluminescent Substrate (Thermo Fisher Scientific) and visualized with a ChemiDocTM Touch Imaging System (BIO-RAD, CA, USA).
Xenograft mouse models
All animal experiments were conducted in the Clinical Research Center of Hebei General Hospital (HBGH), and all animal procedures were approved by the Animal Care and Management Committee of HBGH. All animal protocols were approved by the Hebei Medical University Animal Care and Use Committee, Hebei, China. Six- to eight-week-old female nonobese diabetic/severe combined immuno-deficiency (NOD/SCID) mice were purchased from Vital River, Beijing, China and were raised in specific pathogen-free (SPF)-grade cages and provided autoclaved food and water.
For the subcutaneous HCC models, mice were inoculated subcutaneously with 5×106 SK-HEP-1, HepG2, PLC8024 or Huh7 cells on day 0, and the volumes of tumors derived from these cells were 100mm3 on day 14, day 12, day 17 and day 15, respectively. Then, the xenograft mice received two intravenous injections of 1×107 effector cells on the 3rd and 10th days after the tumor volume reached 100 mm3. For the bioluminescent Huh7 models, mice received 5×106 luciferase-labeled Huh7 cells subcutaneously and were then divided randomly into 5 groups (n=5) and injected intravenously with two doses of 1×107 effector cells at the abovementioned time points. We measured the tumor volumes and mouse body weights three times weekly, and tumor volumes were calculated with the following formula: V =1/2 (length ×width2). Tumor weights were measured after the mice were sacrificed.
Histopathological, immunohistochemical (IHC) and immunofluorescence analyses
After sacrifice, the Huh7 xenograft mice were perfused with saline and paraformaldehyde at the apex of the heart, and the heart, liver, brain, lung, pancreas, spleen, and intestine were placed in a paraformaldehyde fixative for more than 24 hours. All tissues were embedded in paraffin and sliced. Paraffin sections were first dewaxed and stained with hematoxylin. Then, the sections were dehydrated in an alcohol gradient and stained with eosin. Finally, the sections were sealed in neutral gum after dehydration. Three sections were randomly selected from each mouse and photographed under an optical microscope (NIKON, Tokyo, Japan). Paraffin sections of mouse tumors were subjected to the HE staining method described above. After dewaxing, the tumor tissue sections were placed in a repair kit filled with EDTA antigen retrieval buffer (pH 8.0) for repair. A tissue pen was used to outline the tissue, and an autofluorescence quencher was added. Bovine serum albumin (BSA) was added dropwise in the circle for 30 minutes. For immunohistochemical staining, sections were incubated with anti-CD133 (1:1000, Abcam) and anti-GPC3 (1:200, Abcam) antibodies at 4°C overnight and were then washed and incubated with the corresponding secondary antibody at room temperature for 50 minutes. Color development was carried out with 3,3’-diaminobenzidine (DAB), and nuclei were counterstained with hematoxylin. Finally, sections were observed and images were acquired under a microscope. For immunofluorescence staining, sections were incubated with anti-CD133 (1:1000, Abcam), anti-GPC3 (1:200, Abcam) and anti-CD3ζ (1:200, Abcam) antibodies overnight at 4°C and then with the corresponding secondary antibody for 50 minutes at room temperature after washing. After incubation with 4’,6-diamidino-2-phenylindole (DAPI) for 10 minutes at room temperature, images were acquired under a fluorescence microscope (NIKON, Tokyo, Japan).
Bioluminescence assays
Cultured Huh7 cells were inoculated bilaterally into the backs of mice to observe tumor growth, and tumors were imaged in vivo when the average volume reached 100 mm3. Ten minutes after subcutaneous injection of 100 mg/kg D-fluorescein (Solarbio, Beijing, China), mice were anesthetized with isoflurane and were then imaged with a cooled charge-coupled device (CCD) camera system (IVIS Lumina LT Series Ⅲ, Perkin Elmer, Waltham, MA, USA). The results were analyzed quantitatively in Living Image software.
Statistical analysis
Data are presented as the means ± SDs and were analyzed using Prism 8.0 (GraphPad Software, San Diego, CA). Statistical analysis was carried out using Student's t-test (two-group comparisons), one-way ANOVA with Tukey’s post hoc test, and two-way repeated-measures ANOVA followed by Bonferroni's post hoc test. Comparison of survival curves was performed using the log-rank (Mantel-Cox) test. P <0.05 was considered statistically significant.