Fig 1.
G3-EGFRvIII CAR expression, cell surface trafficking, and artificial antigen presenting cells.
(A)Schematic representation of G3-EGFRvIII CAR along with G1-EGFRvIII CAR, and delζ-EGFRvIII CAR (partially deleted TCR). The MR1-scFv was coupled with a CD8 hinge and the CD8 transmembrane TM domain. (B)CD28 and OX40 signaling domains were incorporated between CD8 TM domain and TCR signaling domains. A 9 a.a. c-myc epitope was incorporated between scFv and CD8 hinge and CD8 TM regions. (C)A deletion mutant confers lower signaling capacity in the delζ mutant. (D)Verification of G3-EGFRvIII CAR (MR1-CD8TM-CD20-OX40-CD3) by RT-PCR amplification. (E)Flow cytometric detection of cell surface G1-EGFRvIII CAR (G1) or G3-EGFRvIII CAR (G3) on PBMC transfectants 9 days post nucleofection.
Fig 2.
Artificial antigen presenting cells (aAPCs).
(A)aAPC based on the K562 cell line encoding a non-signaling EGFRvIII mutant. (B)aAPC based on the K562 cell line encoding a non-signaling EGFRvIII mutant in addition to CD32, CD80, and CD137L. (C)G1-EGFRvIII CAR (D)G3-EGFRvIII CAR (E)Overall transduction and stimulation protocol for (F)three week and (G)six week (under selection) of CAR T-cells.
Fig 3.
CAR T-cell expansion with and without selective conditions.
(A)Short-term culture. G3 CAR T-cells were co-cultured with three different aAPCs as shown. (B)G1 vs G3 CAR T-cells grown for 6 restimulation cycles under hygromycin selection.
Fig 4.
G1 vs G3 CAR and durability of cytoxicity in culture.
(A)Short term biophotonic cytolysis assay. Seven days after nucleofection of PBMCs with G1-EGFRvIII CAR (G1), G3-EGFRvIII CAR (G3), or delζ-EGFRvIII CAR [2] plasmids, cells were added to U87vIIIffluc cells at a 25:1 ratio. 5 hours after incubation, T-cell activity was measured by bioluminescence release, and percentage of specific release was calculated for each group. Mean cytolysis values ± SEM is shown for n = 3 for all groups. (B)Flow cytometric detection of G1-EGFRvIII, G3-EGFRvIII, or delζ-EGFRvIII CAR cell surface expression on PBMC transfectants 7 days post nucleofection. Expression of c-myc was typically detected as low magnitude population shift of mean fluorescence of 0.5 log units. (C)Post-culturing cytolysis in PBMCs that were nucleofected with G3-MR1 plasmids and cocultured with irradiated CD32-80-137L-EGFRVIIIΔ654 for 6 restimulation cycles (7-day cycles). Cells were added to U87vIIIfluc target cells at 5:1 or 10:1 ratios. Mean cytolysis values ± SEM is shown for n = 3 for all groups. (D)Flow cytometric detection of GFP in control PMG-GFP-T cells following six-week expansion. GFP expression was observed as a low fluorescence intensity shift of the entire transduced population versus control cells.
Fig 5.
Comparison of CAR T-cell cytokine expression by CAR T cells cocultured with different feeder cell lines.
G1 (1x106), G3 (1x106), or delζ (8x104)-T-cells were stimulated in 7-day cycles with irradiated CD32-80-137L (A. left panel), CD32-80-137L-EGFRVIIIΔ654 (B. middle panel) (both preloaded with anti-CD3, and anti-CD28), or EGFRVIIIΔ654aAPCs (C. right panel) at a 2:1 ratio. CAR T-cells were then exposed to U87-EGFRvIII tumor cells and supernatants were collected 24 hours thereafter. IFN-γ (solid black bar), and IL-2 (grey bar) expression was measured by ELISA. Significant difference in IFN-γ expression was observed in G3 CAR T-cells when cocultured with CD32-80-137L aAPC compared to delζ CAR T-cells (F2,2 = 8717, p = .0002), as well as in G1 CAR-T cells compared to delζ CAR T-cells [(F2,2 = 8157, p = .0002), A]. When cocultured with EGFRVIII654 aAPC, G3 CAR T-cells demonstrated significantly robust IFN-γ expression compared to G1 (F2,2 = 91.47, p = .0216) and delζ CAR T-cells [(F2,2 = 98.59, p = .0201), B]. Significant IFN-γ production was observed when G3 CAR-T cells were cocultured with highly modified CD32-80-137L-EGFRVIII654 aAPC compared to G1 CAR-T cells [(F2,2 = 1194, p = .0017), C]. No exogenous IL-2 was utilized during the preceding restimulation cycles or during the co-incubation with tumor cells. Mean cytokine expression values ±SEM is shown for n = 3 for all groups. Asterisks indicate significant differences (***, P ≤ 0.0001; **, P ≤ 0.001, *, P ≤ 0.01, see Results).
Fig 6.
In vivo activity of G3-T-cells in an intracranial glioblastoma model.
(A-C)25,000 firefly luciferase expressing U87vIIIffluc human glioblastoma cells were mixed with 25,000 G1, G3, or GFP-T-cells (1:1) immediately before intracranial implantation into the forebrain of NOD-scid mice. Starting form day 1, bioluminescence was measured every 3-5 days for 121 days using Xenogen living image system (IVIS®, Xenogen). Prior to imaging, animals were anesthetized and luciferin substrate was administered. Living image®software (Xenogen) was used for imagind and signal quantification. (D)Kaplan-Meier survival curve for single, low-dose treatment cohort.
Fig 7.
Histological analysis of T-cell persistence.
(A)Representative hematoxylin and eosin sections are presented for G3 mouse prior to natural death at day 90 (A, G-I). (B-F)Representative immunofluorescence anti-human CD3 brain histology images of tumor bed of G3 (D-F), G1 (B), or GFP-T (C) cell treated mouse groups prior to natural death at day 90 (G3), or 35 days (G1 and GFP) after initial tumor and T-cell grafting. G3 tumor tissue was pictured at 200x (D) and 400x of the same area (E), and at 400x of a different tumor area of the same tissue (F). Pictures are shown the demolished tumor tissue disconnected to tumor bed at 20x magnification (D). Same tumor tissue was pictured at 100x (G), 200x (H) and 400x (I) magnification, demonstrating demolished tumor tissue (dT) within the tumor bed (T).