Oncolytic herpes virus G47Δ works synergistically with CTLA-4 inhibition via dynamic intratumoral immune modulation

Abstract

Oncolytic virus therapy can increase the immunogenicity of tumors and remodel the immunosuppressive tumor microenvironment, leading to an increased antitumor response to immune-checkpoint inhibitors. Here, we investigated the therapeutic potential of G47Δ, a third-generation oncolytic herpes simplex virus type 1, in combination with immune-checkpoint inhibitors using various syngeneic murine subcutaneous tumor models. Intratumoral inoculations with G47Δ and systemic anti-cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) antibody administration caused an enhanced antitumor activity when combined and worked synergistically. Conversely, the efficacy of G47Δ in combination with anti-programmed cell death protein-1 (PD-1) antibody was equivalent to that of the anti-PD-1 antibody alone in all murine models examined. The combination of intratumoral G47Δ and systemic anti-CTLA-4 antibody was shown to recruit effector T cells into the tumor efficiently while decreasing regulatory T cells. Furthermore, a wide range of gene signatures related to inflammation, lymphoid lineage, and T cell activation was highly upregulated with the combination therapy, suggesting the conversion of immune-insusceptible tumors to immune susceptible. The therapeutic effect proved tumor specific and long lasting. Immune cell subset depletion studies demonstrated that CD4⁺ T cells were required for synergistic curative activity. The results depict the dynamics of immune modulation of the tumor microenvironment and provide a clinical rationale for using G47Δ with immune checkpoint inhibitors.

Keywords: CTLA-4; G47Δ; PD-1; antitumor immunity; esophageal carcinoma; herpes simplex virus type 1; immune-checkpoint inhibitors; oncolytic virus therapy; regulatory T cells; tumor microenvironment.

Graphical abstract

Figure 1

In vitro and in vivo effects of G47Δ in murine carcinomas (A) Cytopathic effects of G47Δ in vitro. Murine cancer cells were infected with G47Δ (AKR, HNM007, MOI of 0.1 [●] or 1 [○]; SCCVII, MOI of 0.01 [▲] or 0.1 [●]) or mock. Cell viability was expressed as a percentage of the mock-infected controls. G47Δ exhibited a good cytopathic effect in AKR and HNM007 cell lines at an MOI of 1.0 and in SCCVII at an MOI of 0.1. Data are presented as the mean of triplicates ± SD. (B) Virus yields of G47Δ in vitro. Murine cancer cells were infected with G47Δ at an MOI of 0.1, and recovered virus yields were determined at 24 and 48 h after infection. All murine cancer cell lines tested supported the replication of G47Δ to a certain extent. The results are presented as the mean of triplicates ± SD. (C) Four syngeneic murine subcutaneous tumor models (AKR [upper left], HNM007 [upper right], SCCVII [lower left], and B16-F10 [lower right]) were used. Established tumors, 5–6 mm in diameter, were inoculated with G47Δ (2 × 10⁵ for SCCVII and 5 × 10⁶ PFUs for others) or mock on days 0 and 3. The G47Δ treatment significantly inhibited the growth of subcutaneous tumors compared with the mock treatment in all models. The results are presented as the mean ± SEM (n = 7). One-way ANOVA followed by Dunnett’s test was used to determine statistical significance (∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001).

Figure 2

Efficacy of G47Δ in combination with CTLA-4 or PD-1 inhibition in a murine subcutaneous AKR tumor model (A−D) Effects of G47Δ or CTLA-4 inhibition, either alone or in combination, on tumor growth in the murine subcutaneous AKR tumor model. (A) Experimental design. C57BL/6 mice harboring unilateral subcutaneous AKR tumors were given intratumoral injections with G47Δ (5 × 10⁶ PFUs on days 0 and 3) or mock in combination with intraperitoneal injections with the anti-CTLA-4 antibody (25 μg on days 0, 3, and 6) as indicated. (B) Delayed tumor growth was observed with either G47Δ (p < 0.05) or CTLA-4 inhibition (p < 0.01) alone, but the combination treatment was associated with a significant decrease in tumor growth compared with each monotherapy (versus G47Δ, p < 0.001; versus αCTLA-4, p < 0.01). The results are presented as the mean ± SEM (n = 8 per group). (C) Individual tumor growth curves of AKR tumors. The combination therapy achieved a cure in 5/8 animals. (D) Kaplan-Meier survival curves. The combination therapy resulted in significantly prolonged survival (p < 0.001 versus control; G47Δ, p < 0.01 versus αCTLA-4). (E−H) Efficacies of G47Δ and PD-1 inhibition, either alone or in combination, in the murine subcutaneous AKR tumor model. (E) Experimental design. C57BL/6 mice harboring subcutaneous AKR tumors were treated with G47Δ (5 × 10⁶ PFUs) or mock together with intraperitoneal injections with the anti-PD-1 antibody (100 μg) as indicated. (F) Tumor growth was significantly inhibited by the anti-PD-1 antibody alone (versus control, p < 0.001). The efficacy of G47Δ combined with systemic PD-1 inhibition was equivalent to that of PD-1 inhibition alone. The results are presented as the mean ± SEM (n = 7 per group). (G) Individual tumor growth curves. The combination therapy did not yield a cure. (H) Kaplan-Meier survival curves. One-way ANOVA followed by Dunnett’s test was used for the comparisons of tumor growth. For survival analysis, the log-rank test followed by Holm’s sequential Bonferroni corrections was used to determine statistical significance (∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; ns, not significant).

Figure 3

Tumor-infiltrating lymphocytes in subcutaneous AKR tumors treated with the combination of G47Δ and CTLA-4 inhibition C57BL/6 mice harboring subcutaneous AKR tumors were treated according to the schedule shown in Figure 2A. Tumor-infiltrating immune cells were analyzed by flow cytometry 7 days after the initial treatments. Comparisons of absolute numbers of (A) CD3 cells, (B) CD4⁺ T cells, (C) CD8⁺ T cells, and (D) Tregs (CD4⁺Foxp3⁺) per gram of tumor tissue. (E) The percentage of Tregs, gated on CD4⁺ T cells. (F) The ratio of CD8⁺ T cells to Tregs. (G) The percentage of CD3, CD8⁺, CD4⁺, and Tregs gated on CD45 cells. Results are representative of two independent experiments with 7 animals per group, and bars represent the SEM. One-way ANOVA followed by Dunnett’s test was used to determine statistical significance (∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; ns, not significant).

Figure 4

Intratumoral immune-related gene-expression changes in subcutaneous AKR tumors 7 days after the initial treatments Gene expression analyses from AKR tumors, focusing on selected mRNAs related to inflammation, lymphocytes, activation markers, exhaustion signatures, and apoptosis. C57BL/6 mice harboring subcutaneous AKR tumors were treated with intratumoral inoculations with G47Δ (5 × 10⁶ PFUs) and intraperitoneal injections with an anti-CTLA-4 antibody (25 μg). The tumor tissues were harvested 7 days after the initial treatments, total RNA was extracted, cDNA was reverse transcribed, and gene expression analysis was performed using qPCR analysis. The details of the gene symbols are presented in Table S1. The fold change in expression of the indicated genes (A) with G47Δ treatment over control, (B) with CTLA-4 inhibition over control, (C) with the combination therapy over control, and (D) the combination therapy over CTLA-4 inhibition. The bar represents mean fold change + SEM (n = 6). The yellow bars represent mRNAs that were significantly upregulated (p < 0.05, fold change ≥ 2) as compared with the reference group. The blue bars show mRNAs that were significantly downregulated (p < 0.05, fold change < 0.5) as compared with the reference group. The expression data were normalized to the geometric mean of three housekeeping genes (Actb, Gapdh, and Hprt1). One-way ANOVA followed by Dunnett’s test was used to determine statistical significance. All experiments were performed twice, with six samples for each group.

Figure 5

Immune-related gene expressions in AKR tumors in early and late phases of the combination therapy Expressions of the 12 genes in subcutaneous AKR tumors in an early phase (day 3) and a late phase (day 7) of the combination therapy. Comparisons of gene expressions related to (A−C) inflammation (Ccl5, Il1a, and Il1b), (D−F) lymphocytes (Cd4, Cd8a, and Foxp3), (G and H) Th1 response (Cxcl10 and Cxcr3), (I) Th2 response (Il2), (J) exhaustion marker (Cd274), and (K and L) T cell activation (Gzmb and Prf1). Top and bottom lines represent data on day 3 and day 7, respectively. The bar represents mean fold change + SEM (n = 6). One-way ANOVA followed by Dunnett’s test was used to determine statistical significance (∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; ns, not significant).

Figure 6

The combination therapy enhanced specific antitumor immune responses (A−C) An IFN-γ ELISpot assay of splenocytes from each treatment group. (A) The number of IFN-γ spots that responded to AKR cells was significantly higher in the combination group than in the other groups (p < 0.001). (B) A representative well from each group. (C) The number of IFN-γ-secreting spots stimulated by AKR cells, but not by Hepa1-6 cells, was significantly increased in the combination therapy group. The results are presented as the mean ± SEM (n = 7). (D and E) Rechallenge study. Mice (n = 5) in which subcutaneous AKR tumors were cured by the combination therapy were re-challenged on day 90 with one-fifth the number of AKR cells implanted in the contralateral hemisphere. As control, age-matched (3 months) naive mice were given the same challenge, i.e., the same dose of tumor cells (n = 5). (D) Growth of rechallenged AKR tumors in cured mice and naive mice. The results are presented as the mean ± SEM (n = 5). (E) Kaplan-Meier survival analysis. One-way ANOVA followed by Dunnett’s test (A), Student’s t test (C and D), and log-rank analysis (E) were used to determine the statistical significance of differences (∗∗p < 0.01; ns, not significant).

Figure 7

Depletion of CD4⁺ T cells abrogated the enhanced efficacy of the combination therapy The experimental designs are presented in Figure S4. (A and B) Tumor growth (A) and survival analysis (B) of CD8⁺ T cell depletion assay (n = 7). A CD8⁺ T cell depletion decreased but did not abolish the enhanced efficacy of the combination therapy (0/7 cures). (C and D) Tumor growth (C) and survival analysis (D) of CD4⁺ T cell depletion assay (n = 7). The efficacy of the combination therapy was completely abrogated by a depletion of CD4⁺ T cells. (E and F) Tumor growth (E) and survival analysis (F) of the NK cell depletion assay (n = 7). The synergistic therapeutic effect and the survival benefit by the combination therapy were retained under a depletion of NK cells (1/7 cure). The results are presented as the mean ± SEM. One-way ANOVA followed by Dunnett’s test was used for comparisons of tumor growth. For the survival analysis, the log-rank test followed by Holm’s sequential Bonferroni corrections was used to determine statistical significance (∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; ns, not significant).