Abstract:
Tumor-infiltrating lymphocytes (TILs) strongly influence clinical outcomes in many solid cancers, yet ovarian tumors frequently retain an immunologically “cold” phenotype that prevents effective immune entry and sustains immune evasion. Although oncolytic adenoviruses (OAds) can initiate immune awakening by driving tumor-restricted lysis and potent immunogenic cell death (ICD), this newly inflamed tumor microenvironment (TME) often triggers compensatory PD-L1 upregulation, re-establishing a barrier to durable antitumor immunity. To overcome this self-limiting loop, we developed a next-generation Ad5/35 oncolytic platform engineered to secrete carboxylesterase-2 (CES2) and exploit the broadly expressed CD46 receptor for efficient infection of ovarian cancer cells. When paired with systemic irinotecan (CPT-11), intratumoral CES2 locally converts the prodrug into the potent chemotherapeutic SN-38, enabling high intratumoral exposure with reduced systemic toxicity. Across 3D ovarian tumor spheroid models, CES2-mediated SN-38 production profoundly amplified tumor destruction beyond viral oncolysis alone. More importantly, SN38 acted as a powerful TME remodeller. It suppressed the PD-L1 surge typically induced during OAd-driven inflammation, preventing premature T-cell dysfunction and preserving cytotoxic T-cell activity within the tumor milieu. Simultaneously, SN-38 elevated surface MICA/B—key ligands for NKG2D receptors—thereby enhancing recognition and engagement by NK cells and CD8? T cells. This stress-ligand upregulation strengthened immunosurveillance at a stage when tumors typically resist immune infiltration.SN-38 also increased MHC-I expression on cancer cells, improving antigen presentation and enabling more efficient tumor recognition by TILs. The combined enhancement of MICA/B and MHC-I established a more immunogenic tumor phenotype, converting the formerly resistant TME into one that actively supports immune engagement. Additionally, because CES2 is secreted, SN-38 diffused throughout the tumor mass, generating a substantial bystander effect that eliminated non-infected neighbouring cells—an essential advantage in heterogeneous and poorly penetrated ovarian tumors. In vivo, we evaluated antitumor efficacy using bilateral flank xenografts in BALB/c nude mice. The CES2-armed OAd was injected directly into the right-flank tumor, while CPT-11 was administered intraperitoneally to enable systemic prodrug availability. As expected, significant tumor regression occurred in the virus-injected right tumor, confirming efficient local conversion of CPT-11 into SN-38. Remarkably, the untreated left-flank tumors also exhibited substantial volume reduction, demonstrating a systemic bystander effect mediated by secreted CES2 and diffusing SN-38. These findings validate the therapeutic synergy between OAd-mediated intratumoral CES2 delivery and CPT-11 activation, even in the absence of adaptive immunity. Overall, our study introduces a dual-function therapeutic strategy in which an Ad5/35 CES2-secreting oncolytic adenovirus couples direct tumor oncolysis with localized SN-38 generation and robust bystander killing. By simultaneously restraining PD-L1 induction and enhancing MICA/B and MHC-I expression, this approach reshapes the ovarian TME into a state more conducive to immune infiltration and tumor clearance. These results position CES2-armed OAds as a promising platform for combination viro-chemotherapy in difficult-to-treat ovarian cancers.
