Construction of the HER2-, MSLN-, and EpCAM-targeting CAR vectors
HER2-specific, MSLN-specific, and EpCAM-specific single-chain antibody fragments (scFvs) with a CD8 leading sequence, a CD8 hinge, and transmembrane sequence, as well as the intracellular signaling domain of 4-1BB, CD28, and CD3ζ in tandem, were engineered into third generation HER2-, MSLN-, and EpCAM-CAR T cells. The sequences, except for scFv, have been previously reported (19). The full-length nucleotide sequence was synthesized by Sangon Biotech (Shanghai, China), and was subsequently inserted into a CAR lentiviral expression vector [pCDH-EF1-MCS-EF1-puro] at two specific restriction enzyme sites (EcoR1 and Not1).
Establishment of HER2-targeted CAR-T cells
The pCDH-CMV-MCS-EF1-puro lentivirus system was used to generate a virus against HER2-targeted CAR. HEK293T cells were co-transfected with a HER2-targeting CAR vector, or control vector, with PLP1, PLP2, and PLP-VSVG, at a ratio of 23.1:16.5:16.5:9.9, polyethyleneimine (Polysciences, Warrington, PA, USA). Six hours after transfection, cells were re-fed with DMEM containing 20% FBS. After transfection, cell supernatant was collected at 48 h and 72 h, centrifuged at 4000 rpm for 5 min to remove cell debris, then filtered through a 0.45 µm filter. To concentrate the virus, 1: 4 PEG8000 (Sigma, Merk, Shanghai, China) was added and mixed every 30 minutes four times. After that, the virus was placed overnight at 4°C, then centrifuged at 4000 g for 30 min. The supernatant was removed, and lentivirus particles were resuspended in PBS and stored at -80°C.
Human peripheral blood mononuclear cells (PBMCs) were isolated by density gradient centrifugation with a Ficoll kit (GE, Shanghai, China) and subsequently activated with anti-human CD3 (100 ng/mL; T&L Biotechnology) and anti-human CD28 (100 ng/mL; T&L Biotechnology). Recombinant human IL-2 (30 ng/mL; Novoprotein) and 1% penicillin–streptomycin (Gibco, Life Technologies, Shanghai, China) were added to X-VIVO 15 medium (Lonza, USA) for T cell proliferation. After activation for 24 h, T cells were transduced with HER2-targeted CAR lentiviral particles, selected with puromycin, and collected for subsequent in vivo and in vitro experiments after 12–14 days. Non-transduced T cells were used as a control group.
Cell lines and culture conditions
HOS, U118MG, U251, U87MG, HepG2, MKN-45, and SK-OV-3 cells were obtained from the American Type Culture Collection (ATCC, USA). HOS and SK-OV-3 cells were maintained in RPMI 1640 medium (Gibco, USA), and 293T, U118MG, U251, U87MG, HepG2, and MKN-45 cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM, Gibco, USA). All cells were cultured in a medium supplemented with 10% FBS (BI, China), penicillin (60 µg/mL), and streptomycin (100 µg/mL) (Sangon, China) and maintained in an incubator with 5% CO2 at 37°C.
Immunohistochemistry
Tissues of human glioblastoma were obtained from the Second Hospital of Dalian Medical University. Surgical glioblastoma tissues were fixed in 4% paraformaldehyde and embedded into paraffin. 4 µm thickness sections were deparaffinized in xylene and rehydrated with 100, 90, 80, and 70% ethanol to PBS. HER2 antibody (Cell Signaling Technology, USA) was utilized for immunostaining at room temperature (RT) for 2 hrs. After the slides were incubated with the HRP-labeled secondary Abs at RT for 1 h, 3,3' -diaminobenzidine (DAB) was added for coloration. The intensity of staining was analyzed by integrated optical density using Image-Pro R Plus software (version 6.0; Media Cybernetics, USA).
Flow cytometry and antibodies
Cells were incubated with antiCD16/CD32 (2.4G2) mAb to block Fcγ receptors. Recombinant anti-HER2 FITC-conjugated antibody and recombinant anti-EpCAM FITC-conjugated antibody (Sino Biological Inc., Beijing, China) were used to detect the expression of HER2 and EpCAM protein. Recombinant anti-mesothelin FITC antibody (Abcam, Cambridge, UK) was used to detect the expression of mesothelin. Cells were stained with HER2, EpCAM, or mesothelin antibody for 1h on ice. The expression of CAR on CAR-T cells was detected using biotinylated human HER2/MSLN/EpCAM (Acro, Beijing, China), followed by staining with allophycocyanin (APC) streptavidin (BioLegend, CA, USA).
Anti-mouse CD3 (17A2, BioLegend, USA) was used to detect the presence of CD3+ T cells in mouse peripheral blood. A FACS-Calibur (Becton Dickinson, USA) was used to perform flow cytometry according to prior guidelines (20), and FlowJo software (Tree Star) was utilized to analyze the data.
Cytotoxicity assays
Tumor cells were regarded as target cells (T) and suspended at a density (2 × 105 cells/ mL). Then, 0.1 mL cell suspension was transferred into a 96-well E-plate (ACEA Biosciences, Menlo Park, CA, USA) and cultured for 20 h. After that, HER2-targeting CAR-T cells (HER2-CAR-T) and untransfected T cells (NC-T) were regarded as effector cells (E) and added into each well separately at different E: T ratios (E: T of 5:1, or 2.5:1). The co-cultures were further cultured for the indicated times. RTCA software (xCELLigence RTCASP, ACEA, Los Angeles, CA, USA) was used to measure the viability of target cells in real time.
Measurement of cytokine secretion
HER2-CAR-T cells and NC-T cells were co-cultured with tumor cells for 24 h in a 96-well plate without any cytokines added. Enzyme-linked immunosorbent assay (ELISA) kits (eBioscience, Grand Island, NY, USA) for specific cytokines (IFN-γ, TNF-α, GM-CSF, IL-6, and IL-8) were used to detect the level of cytokine production in the supernatant and assess the cell-killing efficacy.
Tumor models and treatment
6- to 8-week-old NODPrkdcem26IL2rgem26/Nju (NCG) mice were obtained from NBRI (Nanjing Biomedical Research Institute of Nanjing University and Nanjing Galaxy Biopharma, Nanjing, China). Mice were maintained at 24 ± 1°C with free water and food intake, and illuminated for 12 hrs (08:00 to 20:00) in the specific pathogen-free (SPF) laboratory animal facility of Dalian Medical University (Dalian, China).
For CDX (cell-derived xenograft, CDX) mouse models, 1 × 106 U118MG tumor cells in 100 µL of PBS were injected subcutaneously (s.c.) into the axilla of NCG mice. Tumor size was measured every 4 days. Mice were divided into 3 groups with 6 mice per group until the tumor volume reached 50–100 mm3. Tumor volumes were calculated according to the following formula: tumor volume = (length) × (width)2 × 0.5, in which the length represented the longer dimension and tumor weights were recorded. Mice were monitored according to the Institutional Animal Care and Use Committee (IACUC) animal facilities rules and regulations. Situations when the experiment needed to be paused immediately were listed, as previously reported (21).
For in vivo tumor killing, HER2-CAR-Ts were administered by two methods, peritumoral injection (p.v.) and intravenous injection (i.v.). Each U118MG-CDX mouse model was injected with 5 × 106 HER2-CAR-T cells in 200 µL PBS. Non-injected mice (non-transduced-T cells, named NC-T) were regarded as control groups.
Statistical analysis
Data are presented as mean ± standard deviation (SD) from at least three experiments. Two-tailed Student’s t-tests were performed to compare the statistical differences between groups, using GraphPad Prism software version 9. A p < 0.05 was considered to indicate statistical significance. *p < 0.05, **p < 0.01, ***p < 0.001.