Antitumor‐specific T‐cell responses induced by oncolytic adenovirus ONCOS‐102 (AdV5/3‐D24‐GM‐CSF) in peritoneal mesothelioma mouse model

Oncolytic adenoviral immunotherapy activates the innate immune system with subsequent induction of adaptive tumor‐specific immune responses to fight cancer. Hence, oncolytic viruses do not only eradicate cancer cells by direct lysis, but also generate antitumor immune response, allowing for long‐lasting cancer control and tumor reduction. Their therapeutic effect can be further enhanced by arming the oncolytic adenovirus with costimulatory transgenes and/or coadministration with other antitumor therapies. ONCOS‐102 has already been found to be well tolerated and efficacious against some types of treatment‐refractory tumors, including mesothelin‐positive ovarian cancer (NCT01598129). It induced local and systemic CD8+ T‐cell immunity and upregulated programmed death ligand 1. These results strongly advocate the use of ONCOS‐102 in combination with other therapeutic strategies in advanced and refractory tumors, especially those expressing the mesothelin antigen. The in vivo work presented herein describes the ability of the oncolytic adenovirus ONCOS‐102 to induce mesothelin‐specific T‐cells after the administration of the virus in bagg albino (BALB/c) mice with mesothelin‐positive tumors. We also demonstrate the effectiveness of the interferon‐γ the enzyme‐linked immunospot (ELISPOT) assay to detect the induction of T‐cells recognizing mesothelin, hexon, and E1A antigens in ONCOS‐102‐treated mesothelioma‐bearing BALB/c mice. Thus, the ELISPOT assay could be useful to monitor the progress of therapy with ONCOS‐102.

mesothelioma, ovarian, and lung adenocarcinoma. Therefore, it could potentially be used as a tumor marker or as an antigenic target of vaccines. 4 No effective therapeutic modalities exist for malignant mesothelioma apart from surgical resection in 10% to 15% of the patients. In the advanced disease, chemotherapy has a marginal effect and the prognosis is extremely poor. 5 Hence, there is an urgent need for new and more effective therapies. Oncolytic adenoviruses are promising immunotherapeutic agents for advanced and treatment-refractory cancer patients. Their antitumor activity is based on the direct lysis of cancer cells and the induction of systemic antitumor immunity. [6][7][8][9] Oncolysis leads to the release of tumor epitopes that can be processed by antigen-presenting cells [10][11][12][13][14][15][16][17][18] to activate antigen-specific CD4+ and CD8+ T-cell responses. Immunogenic cell death leads to changes in cell surface structure, such as exposure of calreticulin in the outer plasma membrane and subsequent release of high-mobility group box 1 protein and adenosine triphosphate. 19 Activated CD8+ T-cells can expand into cytotoxic effector cells and infiltrate tumors where they mediate antitumor immunity after antigen recognition.
Granulocyte-macrophage colony-stimulating factor (GM-CSF) mediates antitumor effects by mobilizing and maturing dendritic cells as well as increasing the activity of cytotoxic T-cells. [15][16][17] Systemic use of recombinant GM-CSF is associated with well-
Splenocytes were isolated to determine counts of T-cells responding to mesothelin, human adenovirus 5 E1A, and hexon peptides by secretion of IFN-γ. Harvested splenocytes were stimulated with peptide pools of the complete murine mesothelin protein sequence, human adenovirus 5 E1A, and hexon proteins. IFN-γ production by T-cells was evaluated by using IFN-γ ELISPOT (Abcam, Cambridge, UK). A single-cell suspension of 2.5 × 10 5 splenocytes/well was plated in RPMI medium including 200 ng of peptide. After incubating overnight at 37°C and 5% CO 2 , plates were washed and stained with biotinylated anti-mouse IFN-γ and incubated for 2 hours, followed by streptavidin conjugate enzyme. The spots were counted using the ELISpot Reader (AID, Strasberg, Germany).

| Statistical analysis
Statistical significance was analyzed by using the Mann-Whitney test.
All statistical analysis, calculations and tests were performed using GraphPad Prism 7 (GraphPad Software, San Diego, CA). Results are presented as mean ± standard deviation. All P values were 2 sided and considered statistically significant when ≤.05. Oncolytic adenoviruses are immunotherapeutic agents with the ability to prime and boost immune responses, leading to the development of anticancer immunity. 15 ONCOS-102 is an engineered adenovirus (Ad5/3) that codes for GM-CSF. Its chimeric 5/3 capsid contains a fiber with a c-terminal knob derived from serotype 3, which binds to tumor-associated desmoglein 2 receptor instead of the coxsackieadenovirus receptor, which is found to be downregulated in advanced tumors. 7 The 24-bp deletion in the Rb binding site of the E1A gene causes the virus to replicate selectively in cells with p16-Rb pathway defects, which includes most cancers. 20 ONCOS-102 causes immunogenic cancer cell death 19  T-cell epitopes that could be used to prime antigen-specific T-cells and challenge adoptively transferred T-cells in vivo. These epitopes span conserved regions of the hexon protein and would be useful to monitor immune response before and after immunotherapy. 24 As the capsid of ONCOS-102 contains hexon protein, which plays an important role in virus entry into cells, and E1A protein, which binds to pRb/p300 family of histone acetyltransferases and induces p53-dependent apoptosis in cancer cells 5 ; they were used to stimulate the splenocytes in ELISPOT.
Mice treated with the virus generated specific T-cells against hexon and E1A antigen, as can be seen in Figure 1, in which the signal was detected from only ONCOS-102-treated mice. This is not surprising as viral capsid components such as hexon, 22