The oncolytic virus Delta-24-RGD elicits an antitumor effect in pediatric glioma and DIPG mouse models

Abstract

Pediatric high-grade glioma (pHGG) and diffuse intrinsic pontine gliomas (DIPGs) are aggressive pediatric brain tumors in desperate need of a curative treatment. Oncolytic virotherapy is emerging as a solid therapeutic approach. Delta-24-RGD is a replication competent adenovirus engineered to replicate in tumor cells with an aberrant RB pathway. This virus has proven to be safe and effective in adult gliomas. Here we report that the administration of Delta-24-RGD is safe in mice and results in a significant increase in survival in immunodeficient and immunocompetent models of pHGG and DIPGs. Our results show that the Delta-24-RGD antiglioma effect is mediated by the oncolytic effect and the immune response elicited against the tumor. Altogether, our data highlight the potential of this virus as treatment for patients with these tumors. Of clinical significance, these data have led to the start of a phase I/II clinical trial at our institution for newly diagnosed DIPG (NCT03178032).

Conflict of interest statement

H.J., C.G.-M. and J.F. report ownership interest (including patents) in and are consultants for DNATrix. M.M.A., S.T.-S. and R.D.-V. report DNAtrix sponsored research not related with this article. The remaining authors do not have potential conflicts of interest to disclose.

Figures

Fig. 1

Delta-24-RGD determinants of replication and infection in pHGG and DIPG. a Assessment of integrin and CAR expression in pHGG and DIPGs. b Characterization of amplifications and deletions of RB pathway genes in pHGG and DIPGs. Source data are provided as a Source Data file

Fig. 2

Delta-24-RGD exerts a potent oncolytic effect in DIPG and pHGG cell lines. a Flow cytometry analyses of CAR and integrin expression. DIPG and pHGG cell lines were incubated with fluorescent antibodies against ανβ3 and ανβ5 integrins and CAR. The data are shown as the relative percentage (mean ± SD) of positive expression scored among 10,000 cells. b Assessment of infectivity in DIPG and pHGG cell lines. The indicated cell lines were infected with a replication-deficient construct expressing a modified fiber knob (AdGFP-RGD). The data are shown as the relative percentage (mean ± SD) of GFP-positive cells scored among 10,000 cells per treatment group. c Assessment of viral protein expression in pHGG and DIPG cell lines infected with Delta-24-RGD by western blotting. One representative blot is shown of three independent experiments. d Quantification of Delta-24-RGD replication in the indicated cell lines. Viral titers were determined three days after infection at an MOI of 10 (106 pfu/ml) by an anti-hexon staining-based method in 293 cells and expressed as plaque-forming units (pfu) per milliliter. The dashed line indicates the input virus. The data are shown as the mean ± SD of three independent experiments. e Cell proliferation analyses of Delta-24-RGD-infected DIPG and pHGG cell lines. Cell viability was assessed using MTS assays 5 days after infection. The data are shown as the percentage (mean ± SD of three independent experiments) of cells alive after infection with Delta-24-RGD at the indicated multiplicities of infection (MOIs) relative to the non-infected cells (control, equal to 100%). Source data are provided as a Source Data file

Fig. 3

Delta-24-RGD increases overall survival in DIPG and pHGG models. Tumors were developed by orthotopic intracranial injection of 500,000 cells in female nude mice. a Kaplan–Meier survival curves of Delta-24-RGD (108 pfu)- and control (PBS)- treated athymic mice bearing a TP80 (Control N = 8, Delta-24-RGD-treated N = 9), b TP54 (Control N = 8, Delta-24-RGD-treated N = 9), c CHLA-03-AA (Control N = 9, Delta-24-RGD-treated N = 11), and d PBT-24 (Control N = 9, Delta-24-RGD-treated N = 10). Animals were treated 3 days post-tumor cell injection. The shaded area represents a 50-day interval from the time of cell implantation. Log-rank test was used as statistical analyses. e Representative images of the histopathological analyses of TP80 and TP54 (H&E) of longitudinal slides of control (left images) and Delta-24-RGD-treated tumors (right image). f Representative images of the histopathological analyses of CHLA-03-AA and PBT-24 (H&E) (magnification × 1) of control (left image) and Delta-24-RGD-treated (right images). Mice were treated with either PBS (control) or Delta-24-RGD 3 days post injection. For comparison studies, analyzed brains are derived from mice that died at a similar time point in both groups: CHLA-03-AA = 50 ± 10 days; PBT-24 = 60 ± 5 days; TP80 = 200 ± 5 days; TP54 = 90 ± 5 days. g Representative images of H&E, hexon, and E1A immunostaining of TP80 or CHLA-03-AA tumors, non-treated, or treated with Delta-24-RGD (scale bars, 100 μm). Images correspond to the brains shown in e, f. h, i Kaplan–Meier survival curves of established tumors h CHLA-03-AA (Control N = 10, Delta-24-RGD-treated N = 10) and i PBT-24 (Control N = 8, Delta-24-RGD-treated N = 9) treated with Delta-24-RGD 15 days post cell implantation. The shaded area represents a 50-day interval from the time of cell implantation. Source data are provided as a Source Data file

Fig. 4

Delta-24-RGD exerts a potent oncolytic effect in DIPG murine cell lines. a Flow cytometry analyses of infectivity in DIPG murine cell lines. The indicated cell lines were infected at 0.1, 1, and 10 MOIs with a replication-deficient construct expressing a modified fiber knob (AdGFP-RGD). The data are shown as the relative percentage (mean ± SD) of GFP-positive cells scored among 10,000 cells per treatment group. b Western blot analysis of late protein fiber expressed in murine cell lines 42 h after infection at different viral doses and a human positive control (C+). MK = Marker. A representative blot is shown of three independent experiments. c Quantification of Delta-24-RGD replication in the indicated cell lines. Viral titers were determined 3 days after infection at MOIs of 50, 100, and 300 and control (C) that corresponds to infected cells with 300 MOIs recollected at 16 h by an anti-hexon staining-based method in 293 cells and expressed as plaque-forming units (pfu) per milliliter. The data are shown as the mean ± SD of three independent experiments, two-tailed Student t test analysis was used for comparison. d Representative electronic microscopy images showing viral progeny 72 h after viral infection of the NP53 (300 MOIs) or XFM (200 MOIs). e Cell proliferation analyses of Delta-24-RGD-infected DIPG murine cell lines. Cell viability was assessed using an MTS assay 5 days after infection. The data are shown as the percentage (mean ± SD of three independent experiments) of cells alive after infection with Delta-24-RGD at the indicated multiplicities of infection (MOIs) relative to the non-infected cells (control, equal to 100%). Source data are provided as a Source Data file

Fig. 5

Delta-24-RGD effect DIPG immunocompetent models. Brain tumors were developed by intracranial injection of the NP53 cell line, and PBS or Delta-24-RGD were administered intratumorally 3 days after cell implantation and animals were killed at day15 after cell implantation (a–h). a Representative images (scale bar, 100 µm) of CD3, CD4, CD8, and FoxP3 immunostaining of DIPG tumors from control mice or Delta-24-RGD-treated mice. b Quantification of positive CD3+, c CD4+, d CD8+, and e FoxP3/CD4+ cell infiltration per mm2 of DIPG tumors. Graph showing the quantification of positive cells infiltrating 15 days after cell implantation per mm2 of PBS or Delta-24-RGD-treated tumors (n = 3). P values were calculated using two-tailed Student t test. f Quantification of IFN gamma (mean fold change 193.7), CD8a (mean fold change 29.1), and CD4 (mean fold change 2.9) mRNA expression. The data shown represent the mRNA expression in tumors treated with Delta-24-RGD normalized to PBS-tumor mRNA expression (N = 3). Two-tailed Student t test was used for comparison between control and treated mice. g ELISA quantification of IFN gamma production in splenocytes from control and Delta-24-RGD-treated animals co-cultured with tumor cells. P values were calculated using two-tailed Student t test. h NP53 cells (5 × 106) were implanted subcutaneously in mice flank. Seven days later subcutaneous tumors were visible and ranged between 40 and 80 mm3. Mice were randomized in two groups and treated with three administrations of Delta-24-RGD. Tumor volumes were measured every 2–3 days until the end of the experiment (day 25). Tumor volume (N = 8) was calculated by the equation V (mm3) = π/6 × W2 × L; W is tumor width and L is tumor length. Graph showing percentage of tumor growth was calculated as (V−V0/V0 × 100) where V0 is the tumor volume present when treatment starts, comparison between groups were performed with two-tailed Student t test. i Brain tumors were developed by intracranial injection of the NP53 cell line, and PBS or Delta-24-RGD were administered intratumorally 3 days after cell implantation. Kaplan–Meier survival curves of Delta-24-RGD (107 pfu)- and control (PBS)- treated immunocompetent mice (N = 10, both groups) bearing intracranial NP53 tumors. P value was calculated with the log-rank test. j Rechallenge experiment of the long-term survivor from i. The long-term survivor from the Delta-24-RGD-treated group was subjected to a rechallenge with NP53 cells and compared with a control untreated mice. k Kaplan–Meier survival curves of Delta-24-RGD (107 pfu)- and control (PBS)-treated immunodeficient (athymic nude) mice (N = 11; both groups) bearing intracranial NP53 tumors. P value was calculated with the log-rank test. Source data are provided as a Source Data file