Treatment of HPV-Related Uterine Cervical Cancer with a Third-Generation Oncolytic Herpes Simplex Virus in Combination with an Immune Checkpoint Inhibitor

Abstract

Cervical cancer is one of the most common cancers in women. The development of new therapies with immune checkpoint inhibitors (ICIs) is being investigated for cervical cancer; however, their efficacy is not currently sufficient. Oncolytic virus therapy can increase tumor immunogenicity and enhance the antitumor effect of ICIs. In this report, the therapeutic potential of a triple-mutated oncolytic herpes virus (T-01) with an ICI for human papillomavirus (HPV)-related cervical cancer was evaluated using a bilateral syngeneic murine model. The efficacy of intratumoral (i.t.) administration with T-01 and subcutaneous (s.c.) administration of anti-programmed cell death ligand 1 (PD-L1) antibody (Ab) was equivalent to that of anti-PD-L1 Ab alone on the T-01-injected side. Moreover, combination therapy had no significant antitumor effect compared to monotherapy on the T-01-non-injected side. Combination therapy significantly increased the number of tumor specific T cells in the tumor. While T-01 could not be isolated from tumors receiving combination therapy, it could be isolated following T-01 monotherapy. Furthermore, T-01 had a cytotoxic effect on stimulated T cells. These results suggest that T-01 and anti-PD-L1 Ab partially counteract and therefore concomitant administration should be considered with caution.

Keywords: cervical cancer; herpes simplex virus; immune checkpoint inhibitor; oncolytic viral therapy; oncolytic virus.

Figure 1

Experimental design of T-01 treatment in combination with PD-L1 inhibition in a murine bilateral subcutaneous TC-1 tumor mode.

Figure 2

Efficacy of T-01 in combination with PD-L1 inhibition in a murine bilateral subcutaneous TC-1 tumor model. (a): Individual tumor growth curves of T-01 tumors. (b): Growth of TC-1 tumors in the T-01-injected side in animals. The results are presented as the mean ± SD (n = 9–18 per group) (** p < 0.01, *** p < 0.001, **** p < 0.0001 n.s.: not significant). (c): Growth of TC-1 tumors in the non-injected side in animals. These results are presented as the mean ± SD (n = 9–18 per group) (** p < 0.01, *** p < 0.001, **** p < 0.0001 n.s.: not significant).

Figure 3

Tumor-specific and HSV-specific CTLs in bilateral subcutaneous TC-1 tumors treated with the combination of T-01 and anti-PD-L1 Ab. Thirteen days after tumor implantation, tumors were harvested and characterized for the presence of E7-specific CD8+ T-cells and HSV- specific CD8+ T-cells using E7-specific tetramer and HSV gB tetramer staining followed by flow cytometry analysis. (a): Tumor-infiltrating E7-specific CD8+ T-cells were analyzed by flow cytometry. (b): Tumor-infiltrating HSV-specific CD8+ T-cells were analyzed by flow cytometry. The results are presented as boxplots, which display the dataset based on the five-number summary: minimum, maximum, sample median, and the first and third quartiles (n = 8 per group). A two-way ANOVA followed by Sidak’s multiple comparisons test was used to determine statistical significance (* p < 0.05, n.s.: not significant).

Figure 4

Analysis of viral titers in TC-1 tumor. Thirteen and eighteen days after tumor implantation, quantification of T-01 in the tumors of the T-01 and combination groups was performed. Day 13 is 2 days after the second virus administration and 2 days after the anti-PD-L1 antibody administration. Day 18 is 2 days after the third virus administration and 7 days after the anti-PD-L1 antibody administration. These results are presented as the mean + SD (n = 3 per group). The Mann–Whitney test was used to determine statistical significance (* p < 0.05, n.s.: not significant).

Figure 5

Cytotoxicity of T-01 against activated lymphocytes. Lymphocytes were isolated from the spleens of naïve mice, cultured in the presence or absence of PHA, and then infected with T-01. Cell survival was assayed 24 h after infection. The results are presented as boxplots, which display the dataset based on the five-number summary: minimum, maximum, sample median, and the first and third quartiles. The Student’s t-test was used to determine statistical significance (* p < 0.05). The experiments were performed four times. These data are the mean of 4 experiments.