An Fc-free EGFR-specific 4-1BB-agonistic trimerbody displays broad anti-tumor activity in humanized murine cancer models without toxicity.

Purpose: The induction of 4-1BB signaling by agonistic antibodies can drive the activation and proliferation of effector T cells and thereby enhance a T cell-mediated anti-tumor response. Systemic administration of anti-4-1BB-agonistic IgGs, althougheffective preclinically, has not advanced in clinical development due to their severe hepatotoxicity. Experimental Design: Here, we generated a humanized EGFR-specific 4-1BB-agonistic trimerbody, which replaces the IgG Fc region with a human collagen homotrimerization domain. It was characterized by structural analysis and in vitro functional studies. We also assessed pharmacokinetics, anti-tumor efficacy, and toxicity in vivo. Results: In the presence of a T cell receptor signal, the trimerbody provided potent T cell costimulation that was strictly dependent on 4-1BB hyperclustering at the point of contact with a tumor antigen-displaying cell surface. It exhibits significant anti-tumor activity in vivo, without hepatotoxicity, in a wide range of human tumors including colorectal and breast cancer cell-derived xenografts, and non-small-cell lung cancer patient-derived xenografts associated with increased tumor-infiltrating CD8+ T cells. The combination of the trimerbody with a PD-L1-blocker led to increased IFNg secretion in vitro and resulted in tumor regression in humanized mice bearing aggressive triple-negative breast cancer. Conclusions: These results demonstrate the non-toxic broad anti-tumor activity of humanized Fc-free tumor-specific 4-1BB-agonistic trimerbodies and their synergy with checkpoint blockers, which may provide a way to elicit responses in most cancer patients while avoiding Fc-mediated adverse reactions.