Enhancing oncolytic virotherapy with a tri-specific T-cell engager targeting CD3ε, EpCaM, and 4-1BB: preclinical evaluation and implications for cancer immunotherapy

BACKGROUND: Cytotoxic T-cell-mediated tumor lysis is a key mechanism of oncolytic virotherapy. While oncolytic viruses expressing bispecific T-cell engagers (BiTE) enhance tumor targeting, they lack costimulatory signals, leading to T-cell exhaustion. We developed an oncolytic adenovirus expressing a trispecific T-cell engager (TriTE) to improve antitumor responses in colorectal carcinoma models. METHODS: An oncolytic adenovirus (Ad5-TriTE) was engineered to express a TriTE molecule targeting EpCAM (tumor antigen), CD3ε (T-cell activation), and 4-1BB (co-stimulation). For comparison, a BiTE-expressing virus (Ad5-BiTE) lacking 4-1BB was used. BiTE (αCD3ε-αEpCAM) facilitates T-cell redirection, whereas TriTE (αCD3ε-α4-1BBL-αEpCAM) adds co-stimulation for enhanced T-cell activation. Antitumor efficacy was evaluated in syngeneic and humanized colorectal carcinoma mouse models. RESULTS: Ad5-TriTE demonstrated efficient tumor infection and TriTE secretion, leading to superior tumor control compared to Ad5-BiTE. Enhanced CD8 + T-cell infiltration and activation correlated with improved antitumor effects in both subcutaneous and peritoneal metastasis models. The humanized version, Ad5-hTriTE, exhibited potent activity in a humanized colon cancer model. CONCLUSIONS: Oncolytic adenovirus armed with TriTE enhances antitumor immunity by integrating costimulatory signaling. Incorporating costimulatory molecules into oncolytic virotherapy may offer more effective cancer immunotherapy strategies.