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

This study reports the development of an oncolytic adenovirus engineered to express a trispecific T‑cell engager (TriTE) that simultaneously targets EpCAM on tumor cells, CD3ε on T cells, and 4‑1BB to provide costimulation.

Key points:

• Rationale: Conventional oncolytic viruses expressing BiTEs redirect T cells to tumors but lack costimulatory signaling, contributing to T‑cell exhaustion and suboptimal antitumor activity. The TriTE design aims to add a 4‑1BB costimulatory signal to enhance and sustain T‑cell function.

• Design:

• Ad5-TriTE encodes a TriTE molecule (αCD3ε–α4‑1BBL–αEpCAM).
• The comparator virus, Ad5-BiTE, encodes a BiTE lacking the 4‑1BB component (αCD3ε–αEpCAM).
• Efficacy was tested in syngeneic and humanized mouse models of colorectal carcinoma, including subcutaneous tumors and peritoneal metastases.

• Main findings:

• Ad5-TriTE efficiently infected tumors and secreted functional TriTE.
• Compared with Ad5-BiTE, Ad5-TriTE achieved superior tumor control in multiple colorectal cancer models.
• This was associated with greater CD8+ T‑cell infiltration and activation within tumors.
• A human-adapted construct (Ad5-hTriTE) showed strong antitumor activity in a humanized colon cancer model.

• Conclusion: Incorporating a 4‑1BB costimulatory element into T‑cell engagers delivered by oncolytic adenovirus augments antitumor immunity beyond BiTE-based approaches. This strategy may represent a more effective form of oncolytic viro-immunotherapy for epithelial tumors such as colorectal cancer.