Hangiu et al. generated small (47kDa) mouse and human bispecific Fc-free agonist antibodies comprising an EGFR-specific single-domain VHH antibody linked to a 4-1BB-specific scFv and a human albumin sequence (LiTCo-Albu). In vitro, LiTCo-Albu bound specifically to its cognate targets and mediated EGFR-targeted 4-1BB costimulatory activity and its own cellular recycling via FcRn. In mice, mouse LiTCo-Albu had an extended circulatory half-life, and delayed established EGFR+ CRC growth; therapeutic activity was enhanced by a blocking anti-PD-1 mAb. LiTCo-Albu did not display Fc-dependent 4-1BB mAb-associated toxicities.
Contributed by Paula Hochman
ABSTRACT: Costimulation 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 Fc_R 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 V(HH) 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 4-1BB mAb-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.