Targeting the CD40 costimulatory receptor to improve virotherapy efficacy in diffuse midline gliomas

Abstract

Diffuse midline glioma (DMG) is a devastating pediatric brain tumor. The oncolytic adenovirus Delta-24-RGD has shown promising efficacy and safety in DMG patients but is not yet curative. Thus, we hypothesized that activating dendritic cells (DCs) through the CD40 costimulatory receptor could increase antigen presentation and enhance the anti-tumor effect of the virus, resulting in long-term responses. This study shows that the intratumoral co-administration of Delta-24-RGD and a CD40 agonistic antibody is well tolerated and induces long-term anti-tumor immunity, including complete responses (up to 40%) in DMG preclinical models. Mechanistic studies revealed that this therapy increased tumor-proliferating T lymphocytes and proinflammatory myeloid cells, including mature DCs with superior tumor antigen uptake capacity. Moreover, the lack of cross-presenting DCs and the prevention of DC recruitment into the tumor abolish the Delta-24-RGD+anti-CD40 anti-DMG effect. This approach shows potential for combining virotherapy with activating antigen-presenting cells in these challenging tumors.

Keywords: CD40; DIPG; DMG; Delta-24-RGD; dendritic cells; diffuse intrinsic pontine glioma; diffuse midline glioma; oncolytic adenovirus.

Graphical abstract

Figure 1

The combination of Delta-24-RGD with the anti-CD40 is safe and produces complete responses in DMG-bearing mice (A) Experimental schedule combining the treatment with anti-CD40 (24 μg; clone FGK4.5 intratumoral [i.t.]) and the oncolytic virus Delta-24-RGD (10⁷ plaque-forming unit [PFU], i.t.) in the XFM tumor model. (B) Survival of XFM-bearing mice treated 4 days after tumor injection. (C) Experimental schedule combining the treatment with anti-CD40 (24 μg; clone FGK4.5 i.t.) the same day or 3 days after virus injection (10⁷ PFU of Delta-24-RGD i.t.). (D) Survival curves of XFM-bearing mice treated with the indicated schedules. (E) Images of bioluminescence were obtained from XFM-bearing mice treated with IgG control or the combination at the indicated time points. (F) Measurement of tumor growth over time by bioluminescence. (G) Tumor growth change at day 10 compared to day 7 post-treatment in mice from (E). (H) Survival of UC-BL6-C7-bearing mice treated 7 days after tumor injection. (I) Percentage of body weight change of DMG-bearing mice on day 3 compared to the day before mice were treated with the indicated agents. BW, body weight. Error bars represent mean ± SEM. The log rank test was used for statistical analysis comparing the indicated groups in the survival experiments. n = 8–20 mice per group. One-way ANOVA and the corresponding non-parametric test were used to statistically analyze body weight. n = 7–10 mice per group. The Mann-Whitney test was used for tumor growth rate analysis (n = 4 mice per group) ∗p < 0.05.

Figure 2

Delta-24-RGD and anti-CD40 therapy promotes a durable anti-tumor immune response (A and B) Survival curves of Rag2Il2Rγ^−/− immunodeficient mice bearing XFM and UC-BL6-C7 tumors; treated as indicated in the experimental schedule. (C) Survival curves of long-term survivors subjected to rechallenge with XFM cells as shown in the experimental illustration. (D) Survival curves of Delta-24-RGD+anti-CD40-long-term survivors subjected to FTY720 and rechallenged with XFM cells in the pons. Log rank test was used for statistical analysis comparing the indicated group with untreated mice in the survival experiments. n = 4–9 mice per group.

Figure 3

The combination increases proliferating tumor-infiltrating lymphocytes (A) Schedule of the experimental procedures performed in the tumor microenvironment study in XFM tumors 6 days after the indicated treatments. (B–E) Numbers of the total immune-infiltrating populations (CD45 high expressing cells), CD8 and CD4 T lymphocytes, and regulatory T cells (Tregs) per mg of tumor assessed by flow cytometry. (F) Number of proliferating CD8 and CD4 T cells (measured by ki67-expressing cells) upon the indicated conditions. (G and H) Percentage of CD4 and CD8 T cells in the tumor expressing the indicated activation molecules. (I) Numbers of natural killer (NK) cells per tumor mg at day 6 post-treatment. Error bars represent mean ± SEM. One-way ANOVA and the Kruskal-Wallis test were used for statistical analyses. n = 5–10 samples per group. (∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001).

Figure 4

The combination enhances a proinflammatory phenotype of the tumor myeloid compartment, with cDC1 playing a pivotal role in the anti-tumor response (A) Numbers of microglia, macrophages, and monocytes present per mg of tumor and assessed by flow cytometry in XFM tumors 6 days after being treated with the indicated conditions. (B and C) Representative images and analysis of the number of CD11c-positive cells (dendritic cells) assessed by immunofluorescence staining of XFM tumors at day six post-treatment. Scale bar: 100 μm. (D and E) Number and percentage of dendritic cells (DCs) from the total immune tumor infiltrate. (F) The concentration of the indicated proinflammatory chemokines and cytokines in the tumor microenvironment 6 days after treatment was assessed by LEGENDplex. (G) Volcano plot showing the differentially expressed genes (assessed by RNA-seq) in XFM tumors treated with the combination compared to IgG control at day 6. (H) Gene set enrichment analysis of pathways involved in myeloid cell activation and differentiation present in Delta-24-RGD+anti-CD40-treated tumors compared to IgG control and anti-CD40 alone. (I) Mean of fluorescence intensity (MFI) of MHC-II on microglia and tumor macrophages was measured by flow cytometry 6 days after treatment. (J) Number of DCs expressing high levels of MHC-II molecules per tumor mg. (K) Number of tumor DCs expressing CD40 and CD86 per tumor mg. (L) MFI of PDL1 present on the membrane of tumor DCs at day 6 post-treatment. (M) Representative example of flow cytometry plots showing the mCherry signal in the indicated populations infiltrating the NP53-mCherry orthotopic DMG model. (N) Quantification of the mCherry-positive cells from (E) in the tumor niche 24 h after the indicated treatments. (O) Survival curve of Batf3ko mice bearing UC-BL6-C7 tumor and treated with either the combination or an IgG control. (P) Representative H&E images of two brains treated with the IgG control and the combination that were collected at the endpoint. Error bars represent mean ± SEM. One-way ANOVA and the Kruskal-Wallis test were used for statistical analyses from (A)–(H) data. n = 3–10 mice per group. Log rank test was used for statistical analysis comparing the combination with IgG control-treated Batf3ko mice in the survival experiments. n = 4 mice per group. (∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001).

Figure 5

The inhibition of CSF1R avoids the generation of complete responses upon the combination therapy and the recruitment of DCs into the tumor and draining lymph nodes (A) Experimental overview of the syngeneic model of DMG. 1,000 XFM cells were injected into the pons of BALB/c mice. At day 3, mice were treated via oral gavage with the CSF1R inhibitor (PLX3397, 50 μg/g). On day 5, mice were treated intratumorally with the combination therapy. (B) Survival curves of XFM-bearing mice treated with the combination in the presence or absence of the CSF1R inhibitor. (C) Analysis of DC infiltration in XFM tumors, cervical deep and superficial lymph nodes, and spleen 6 days post-treatment. (D and E) Numbers of cDC1 and cDC2 present in the tumor and deep LN upon RGD+anti-CD40 with or without PLX3397. (F) Concentration of the indicated proinflammatory chemokines and cytokines in the tumor microenvironment 6 days post-treatment with or without CSF1R inhibition. LN, lymph nodes. cDC1 and cDC2, conventional dendritic cell type 1 and type 2. Log rank test was used for statistical analysis comparing the combination with IgG control-treatedmice in the survival experiments in (B). The fraction of complete responses per treatment is indicated in brackets. n = 12 mice per group. One-way ANOVA and the Kruskal-Wallis test were used for statistical analyses of (C)–(F) data. n = 6–7 samples per group. Error bars represent mean ± SEM. (∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001).