Hangiu, OanaRubio Pérez, LauraÁlvarez-Vallina, Luis2023-04-042023-04-0420222589-0042https://hdl.handle.net/10641/3333Costimulation of tumor-infiltrating T lymphocytes by anti-4-1BB monoclonal antibodies (mAbs) has shown anti-tumor activity in human trials, but can be associated with significant off-tumor toxicities involving FcgR interactions. Here, we introduce albumin-fused mouse and human bispecific antibodies with clinically favorable pharmacokinetics designed to confine 4-1BB costimulation to the tumor microenvironment. These Fc-free 4-1BB agonists consist of an EGFR-specific VHH antibody, a 4-1BB-specific scFv, and a human albumin sequence engineered for high FcRn binding connected in tandem (LiTCo-Albu). We demonstrate in vitro cognate target engagement, EGFR-specific costimulatory activity, and FcRn-driven cellular recycling similar to non-fused FcRn high-binding albumin. The mouse LiTCo-Albu exhibited a prolonged circulatory half-life and in vivo tumor inhibition,with no indication of 4-1BBmAb-associated toxicity. Furthermore, we show a greater therapeutic effect when used in combination with PD-1-blocking mAbs. These findings demonstrate the feasibility of tumor-specific LiTCo- Albu antibodies for safe and effective costimulatory strategies in cancer immunotherapy.engAtribución-NoComercial-SinDerivadas 3.0 Españahttp://creativecommons.org/licenses/by-nc-nd/3.0/es/journal articleopen access10.1016/j.isci.2022.104958