Article Figures & Data
Figures
- Figure 1.
FACS analysis demonstrated the binding of scFv-M27-Fc, scFv-806-Fc, and scFv-C225-Fc to the indicated target cells. To determine the binding of scFv-Fc proteins to untransfected or transfected U87MG, U251 glioblastoma cells, and three primary keratinocytes, 1 × 106 cells were isolated and incubated with 5 μg/mL of scFv-M27-Fc, scFv-806-Fc, and scFv-C225-Fc antibodies for 45 minutes at 4°C. After wash with FACS buffer, the cells were stained with an FITC-conjugated goat ant human IgG (H+L) for 45 minutes at 4°C. A, Fluorescence was assessed by using a BD FACSCelesta flow cytometer. B, The mean fluorescence intensity (MFI) of scFv proteins binding to cells was determined by flow-cytometric analysis.
- Figure 2.
Construction and characterization of EGFR/EGFRvIII bitargeted CAR T cells. A, Schematic representation of CARs. M27-28Z CAR consists of an scFv, a human CD8α hinge and a transmembrane (TM) region derived from human CD28, and an intracellular signaling domain from human CD28 and human CD3ζ. M27-28BBZ or 806-28BBZ CAR comprises an scFv, a human CD8α hinge, a CD28 transmembrane domain, and an intracellular signaling domain derived from human CD28, CD137, and CD3ζ. B, Expression of EGFR-specific CARs on the lentivirus-transduced human T cells was analyzed using flow cytometry. Transduction efficiency is shown. C and D, In vitro cytotoxic activities of EGFR/EGFRvIII-targeted CAR T cells (M27-28Z or M27-28BBZ). Primary human T cells transduced with the indicated lentiviral vectors were incubated with the untransfected and transfected glioblastoma cells at the various E:T ratios for 18 hours. Cell lysis was determined using a standard nonradioactive cytotoxic assay. Data are representative of three independent experiments. Each data point reflects the mean ± SEM of triplicates (*, P < 0.05; **, P < 0.01; ***, P < 0.001; two-tailed Student t test).
- Figure 3.
In vitro cytotoxic activity of and cytokine production by M27-28BBZ T cells in the presence of tumor cells. Primary human T cells transduced with the indicated lentiviral vectors were incubated with cell lines at different E:T ratios for 18 hours. Cell lysis was determined using a standard nonradioactive cytotoxic assay. Data are representative of three independent experiments and shown as the mean ± SEM of triplicates. A, The cytotoxic activities of M27-28BBZ and 806-28BBZ to U87MG, U87MG-EGFR, and U87MG-EGFRvIII cells. B, The cytotoxic assay of M27-28BBZ and 806-28BBZ using U251, U251-EGFR, and U251-EGFRvIII cells. C, The specific lysis of M27-28BBZ and 806-28BBZ of primary keratinocytes from three different human skin specimens. D, The specific lysis of M27-28BBZ and 806-28BBZ to A431 and CAL27 cells. Data are representative of three independent experiments and shown as the mean ± SEM of triplicates (*, P < 0.05; **, P< 0.01; ***, P< 0.001; two-tail Student t test). E, Human T cells transduced with lentiviral vectors were cocultured with indicated cell lines at a 3:1 E:T ratio. Culture supernatants harvested at 24 hours after coculture were assayed for TNFα, IL2, and IFNγ by ELISA. Data are representative of three independent experiments and presented as the mean ± SEM of triplicates.
- Figure 4.
In vivo antitumor activities of M27-28BBZ CAR T cells on established subcutaneous EGFR- or EGFRvIII-overexpressing glioblastoma xenografts. A, NOD/SCID mice were subcutaneously inoculated with 2 × 106 U251-EGFRvIII cells on day 0. Two doses of 1 × 107 M27-28BBZ CAR T cells or mock T cells were intravenously administered on days 12 and 15. Data are presented as the mean tumor volume ± SEM. Statistically significant differences between M27-28BBZ CAR T cells and mock T cells are marked with asterisks (***, P < 0.001; two-way ANOVA with Bonferroni posttest). B, On day 42 after tumor cell inoculation, the mice were euthanized. Tumor weight was measured. Endpoint was defined by the tumors reaching a mean volume of 2,000 mm3, more than 20% body weight loss in mice, tumor ulceration, or inability of the mice to ambulate. Efficacy was evaluated by measuring the reduction of the mean tumor weight relative to mock T cells (***, P < 0.001; two-tailed Student t test). C, NOD/SCID mice were subcutaneously inoculated with 3 × 106 U251-EGFR cells. When the tumor volume was approximately 100–120 mm3, mice bearing established subcutaneous tumors were treated with two doses of 1 × 107 M27-28BBZ CAR T cells or mock T cells on days 14 and 17. Data are presented as the mean tumor volume ± SEM (*, P < 0.05; two-way ANOVA with Bonferroni posttest). D, On day 49 after tumor cell inoculation, the mice were euthanized. Tumor weight was measured. Efficacy was evaluated by measuring the reduction of the mean tumor weight relative to mock control (*, P < 0.05; two-tailed Student t test).
- Figure 5.
Antitumor activities of M27-28BBZ T cells on established intracranial glioblastoma xenografts in vivo. For an orthotopic model of glioblastoma, 1 × 106 U251-luci-EGFR cells were implanted intracranially into NOD/SCID mice (n = 8 mice per group). For U251-luci-EGFR/U251-luci-EGFRvIII models, 7.5 × 105 U251-luci-EGFR and 2.5 × 105 U251-luci-EGFRvIII cells were implanted intracranially into mice (n = 7 mice per group). For U251-luci-EGFRvIII models, 5 × 105 U251-luci-EGFRvIII cells were implanted intracranially into mice on day 0 (n = 6 mice per group). On day 7, mice were injected intravenously with 1 × 107 M27 CAR T cells or mock T cells. A, Representative IVIS images depicting 6–7 of the mice from each of the groups treated with the indicated CAR T cells. Mice were imaged weekly. Endpoint was defined by mice inability to ambulate. B, Tumor burden was quantified as total flux in units of photons/second. Bars indicate means ± SE (*, P < 0.05; **, P < 0.01; ***, P < 0.001; two-way ANOVA with Bonferroni posttest). C, Survival was plotted using a Kaplan–Meier curve; statistically significant differences between the experimental groups were determined using log-rank Mantel–Cox test (*, P < 0.05).
- Figure 6.
The persistence of human T cells from mice treated with the indicated genetically modified T cells in orthotopic glioblastoma xenografts models. A, The quantification of CD4+ and CD8+ T cells in murine peripheral blood of intracranial tumor-bearing mice on day 15. Fifty microliters of murine blood was drawn at 8 days after indicated T-cell infusion to determine T-cell counts. T cells were analyzed by flow cytometry using anti-CD3-PerCP/anti-CD4-FITC/anti-CD8-PE and were enumerated using the count beads. Data indicate means ± SEM number of T cells per microliter of blood as measured by flow cytometry. Statistically significant differences were calculated by the two-tailed Student t test (*, P < 0.05). B–D, The representative immunostaining images of CD3+ T-cell infiltration in tumor-bearing mice brains. The brain tissues were harvested from intracranial tumor-bearing mice on day 15. Formalin-fixed, paraffin-embedded mice brain tissue sections were consecutively cut and stained for human CD3 (brown). [The images were taken with the microscope (BX41, Olympus) and camera (DP70) under × 200 magnifications. Scale bar, 100 μm]. E, The quantification of T-cell infiltration in brain tissues of intracranial tumor-bearing mice treated with M27-28BBZ and mock T cells. Data are expressed as mean ± SEM (*, P < 0.05; **, P < 0.01; (**); ***, P < 0.001; two-tailed Student t test).
Additional Files
Supplementary Data
- Supplementary Materials and Methods - Supplementary Materials and Methods
- Supplementary Figures - Figure S1-S6
- Supplementary Table - Table S1
Volume 6, Issue 11
