An ICOSL-armed oncolytic adenovirus activates CD4+ T cell to potentiate antitumor immunity and synergizes with anti-PD-1 or CAR-T cell therapy in colorectal cancer

The efficacy of oncolytic adenoviruses (ADVs) in colorectal cancer (CRC) is limited by their inability to effectively expand CD4 + T cells within the tumor microenvironment (TME). This study aimed to elucidate the underlying mechanism and overcome this bottleneck through viral engineering. We constructed an oncolytic adenovirus expressing inducible T cell co-stimulator ligand (ICOSL), designated ADV-ICOSL, based on the backbone virus ADV-NC. Its antitumor efficacy, safety, and ability to induce immunological memory were systematically evaluated in MC38 and CT26 murine colorectal cancer (CRC) models. The mechanism of action was investigated using flow cytometry, co-culture assays, RNA sequencing, and specific pathway inhibitors. The synergistic potential with anti-PD-1 or chimeric antigen receptor T cell (CAR-T) therapy was assessed. Furthermore, a humanized ICOSL-expressing virus (ADV-hICOSL) was developed and validated in cell line-derived xenograft (CDX) and patient-derived xenograft (PDX) models. We initially discovered that ADV-NC treatment led to decreased ICOSL levels in the TME, resulting in insufficient co-stimulation for CD4 + T cells. In contrast, ADV-ICOSL significantly elevated ICOSL expression and demonstrated superior antitumor efficacy and the capacity to induce long-term immune memory. Mechanistic studies revealed that ADV-ICOSL, via elevating ICOSL, activated the PI3K/Akt-NF-κB signaling axis in CD4 + T cells, promoting their secretion of IL-2. IL-2, in a non-contact-dependent manner, potently enhanced the proliferation and cytotoxicity of CD8 + T cells. This process was strictly dependent on CD4 + T cells. Furthermore, ADV-ICOSL exhibited significant synergistic effects when combined with either anti-PD-1 or CAR-T therapy. Importantly, ADV-hICOSL effectively increased the infiltration of CD4 + T and GZMB + CD8 + T cells in CDX/PDX models, exerting potent antitumor effects. Our study not only reveals that the suboptimal efficacy of conventional oncolytic adenovirus is associated with ICOSL downregulation and impaired CD4 + T cell help, but also successfully develops ADV-ICOSL to reverse this limitation. This study provides a novel combinatorial strategy and a solid experimental foundation for oncolytic virotherapy in CRC.