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. 2016 Oct 27;9(1):70.
doi: 10.1186/s13048-016-0282-3.

IL-12 Expressing oncolytic herpes simplex virus promotes anti-tumor activity and immunologic control of metastatic ovarian cancer in mice

Eric D Thomas  1 , Selene Meza-Perez  2 , Kerri S Bevis  3 , Troy D Randall  2 , G Yancey Gillespie  4 , Catherine Langford  4 , Ronald D Alvarez  3
Affiliations

IL-12 Expressing oncolytic herpes simplex virus promotes anti-tumor activity and immunologic control of metastatic ovarian cancer in mice

Eric D Thomas et al. J Ovarian Res. .

Abstract

Background: Despite advances in surgical aggressiveness and conventional chemotherapy, ovarian cancer remains the most lethal cause of gynecologic cancer mortality; consequently there is a need for new therapeutic agents and innovative treatment paradigms for the treatment of ovarian cancer. Several studies have demonstrated that ovarian cancer is an immunogenic disease and immunotherapy represents a promising and novel approach that has not been completely evaluated in ovarian cancer. Our objective was to evaluate the anti-tumor activity of an oncolytic herpes simplex virus "armed" with murine interleukin-12 and its ability to elicit tumor-specific immune responses. We evaluated the ability of interleukin-12-expressing and control oncolytic herpes simplex virus to kill murine and human ovarian cancer cell lines in vitro. We also administered interleukin-12-expressing oncolytic herpes simplex virus to the peritoneal cavity of mice that had developed spontaneous, metastatic ovarian cancer and determined overall survival and tumor burden at 95 days. We used flow cytometry to quantify the tumor antigen-specific CD8+ T cell response in the omentum and peritoneal cavity.

Results: All ovarian cancer cell lines demonstrated susceptibility to oncolytic herpes simplex virus in vitro. Compared to controls, mice treated with interleukin-12-expressing oncolytic herpes simplex virus demonstrated a more robust tumor antigen-specific CD8+ T-cell immune response in the omentum (471.6 cells vs 33.1 cells; p = 0.02) and peritoneal cavity (962.3 cells vs 179.5 cells; p = 0.05). Compared to controls, mice treated with interleukin-12-expressing oncolytic herpes simplex virus were more likely to control ovarian cancer metastases (81.2 % vs 18.2 %; p = 0.008) and had a significantly longer overall survival (p = 0.02). Finally, five of 6 mice treated with interleukin-12-expressing oHSV had no evidence of metastatic tumor when euthanized at 6 months, compared to two of 4 mice treated with sterile phosphate buffer solution.

Conclusion: Our pilot study demonstrates that an interleukin-12-expressing oncolytic herpes simplex virus effectively kills both murine and human ovarian cancer cell lines and promotes tumor antigen-specific CD8+ T-cell responses in the peritoneal cavity and omentum, leading to reduced peritoneal metastasis and improved survival in a mouse model.

Keywords: Immunotherapy for treatment of ovarian cancer; Oncolytic herpes simplex virus.

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Figures

Fig. 1
Fig. 1
In vitro 4 h cytotoxicity of oHSV towards murine ovarian cancer cell lines. 48 h after M002 was added to the incubating murine cell lines, Alamar Blue dye was added to each well. 4 and 8 h after the Alamar Blue dye was added to the wells, the mean optical density for each well was calculated and used to construct a dose response plot and the number of viable cells was calculated utilizing regression analysis. A representative figure at 4 h demonstrates the cytotoxicity of M002 towards the syngeneic murine cell lines (a), and the mean number of PFU/TD50 at 4 and 8 h is demonstrated (b)
Fig. 2
Fig. 2
In vitro 4 h cytotoxicity of oHSV towards human ovarian cancer cell lines. 48 h after M032 was added to the incubating murine cell lines, Alamar Blue dye was added to each well. 4 and 8 h after the Alamar Blue dye was added to the wells, the mean optical density for each well was calculated and used to construct a dose response plot and the number of viable cells was calculated utilizing regression analysis. A representative figure at 4 h demonstrates the cytotoxicity of M032 towards the human ovarian cancer cell lines (a), and the mean number of PFU/TD50 at 4 and 8 h is demonstrated (b)
Fig. 3
Fig. 3
Anti-tumor effect in MISIIR-TAg mice treated with IP M002 or PBS in vivo MISIIR mice were injected with IP M002 or PBS weekly for a total of three doses. They were followed until death at which time a necropsy was performed to determine the presence and extent of tumor burden. The weight of each mouse’s tumor is depicted. Each circle represents a mouse treated with IP PBS, whereas each square represents a mouse treated with IP M002. Two (18 %) mice treated with PBS were without evidence of tumor compared to 9 (81 %) mice treated with M002 (p = 0.055)
Fig. 4
Fig. 4
Overall survival in MISIIR-TAg mice treated with IP M002 or PBS. MISIIR mice were observed following 3 weekly treatments with IP M002 or PBS. Necropsy was performed at time of death evaluating for the presence of metastatic ovarian carcinoma. If the mice were still alive at 6 months, a planned euthanasia date to evaluate for metastatic disease was predetermined prior to mouse randomization. Mice treated with M002 had a significantly improved overall survival compared to those treated with placebo (p = 0.02)
Fig. 5
Fig. 5
Day 7 tumor antigen-specific CD8+ T cell immune response. MISIIR mice were treated with either IP M002 or PBS weekly for a total of 3 weeks. Mice were subsequently euthanized on day 7 or day 10 following the third injection. A representative day 7 flow cytometry gate demonstrating the number of tumor antigen-specific CD8+ T-cells for the Mesothelin tetramer in the omentum is illustrated for mice treated with M002 (a) and PBS (b). The day 7 tumor antigen-specific CD8+ T-cell immune response for the SV40-TAg and Mesothelin tetramer from the omentum, peritoneal fluid, draining lymph nodes, and spleen is demonstrated. The number of antigen-specific CD8+ T-cells was significantly larger to the SV40-TAg in the omentum (p = 0.04) and to Mesothelin in the omentum (p = 0.002) and peritoneal cavity (p = 0.02) (c)
Fig. 6
Fig. 6
Day 10 tumor antigen-specific CD8+ T cell immune response. MISIIR mice were treated with either IP M002 or PBS weekly for a total of 3 weeks. Mice were subsequently euthanized on day 7 or day 10 following the third injection. A representative day 10 flow cytometry gate demonstrating the numbers of tumor antigen-specific CD8+ T-cells for the Mesothelin tetramer in the omentum is illustrated for mice treated with M002 (a) and PBS (b). The day 10 tumor antigen-specific CD8+ T-cell immune response for the SV40-TAg and Mesothelin tetramer from the omentum, peritoneal fluid, draining lymph nodes, and spleen is demonstrated. The number of antigen-specific CD8+ T-cells was significantly larger to Mesothelin in the peritoneal cavity (p = 0.03) (c)
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