Background: ONCOS-102 is an oncolytic adenovirus expressing granulocyte-macrophage colony stimulating factor (GM-CSF; Ad5/3-D24-GMCSF) that stimulates immune responses and modulates the tumor microenvironment, both as a monotherapy and in combination with programmed cell death protein 1 (PD-1) blockade. We present granular analyses of individual immune-related gene expression in response to ONCOS-102 in a randomized phase I/II study in MPM. Methods: Following a safety run-in (n = 6), 25 pts were randomized to receive ONCOS-102 (3 x 10¹¹ virus particles in 2.5 mL) intratumorally under CT or US guidance on Days 1, 4, 8, 36, 78 and 120, plus six cycles of P/C starting on Day 22 (ONCOS+P/C), or six cycles of P/C only (control). Both treatment-naïve (1L) and previously treated patients were enrolled. Survival was followed up to 30 months. Tumor biopsies were collected at baseline and Day 36, and immunofluorescence for immune cell subsets, RNASeq and qPCR were performed. Results: In the pooled population (n = 31), median overall survival (OS) was 16.6 vs 18.3 months for ONCOS-102 + P/C vs control arms (P> 0.05). In 1L patients (n = 17) median OS was 20.3 vs 13.5 months (P> 0.05), and 30-month OS rate was 34.1% vs 0. A significant increase in tumour-infiltrating immune cells was observed (baseline to Day 36) with ONCOS-102 + P/C (n = 20) but not control (n = 11) using immunofluorescence and transcriptome analysis. CD8+, GranzymeB+ CD8+ cells, and CD8+:regulatory T-cell and M1:M2 macrophage ratios increased in ONCOS-102-treated pts (P≤0.016). A difference in tumor immune-infiltration was observed in ONCOS-102 + P/C pts stratified by survival at Month 18: while deregulation of complement pathways and inflammatory response was seen at baseline, at Day 36 adaptive immune response and T-cell activation were observed in pts who were alive but not in those deceased. Additionally, GM-CSF protein, likely derived from ONCOS-102, was transiently present in higher concentration at Days 4 and 8 in 18-month survivors vs deceased pts. Based on a previously described predictive inflammatory gene signature for treatment with checkpoint inhibitors in mesothelioma, a binomial model assessing baseline transcriptome levels demonstrated 89% accuracy in predicting 18-month survival. Conclusions: ONCOS-102 caused increased T-cell infiltration and upregulated immune response-related gene expression in tumor lesions. A trend for prolonged OS was observed in the 1L setting, only. Consistent with observations in PD-1 resistant melanoma, ONCOS-102-induced immune response in the tumor is hallmarked by a high prevalence of cytotoxic T-cells and depolarization of macrophages, which may support addition of checkpoint inhibitors to the combination of ONCOS-102 plus P/C in MPM. Clinical trial information: NCT02879669.