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The costimulation of immune cells using first-generation anti-4-1BB monoclonal antibodies (mAbs) has demonstrated anti-tumor activity in human trials. Further clinical development, however, is restricted by significant off-tumor toxicities associated with FcγR interactions. Here, we have designed an Fc-free tumor-targeted 4-1BB-agonistic trimerbody, 1D8N/CEGa1, consisting of three anti-4-1BB single-chain variable fragments and three anti-EGFR single-domain antibodies positioned in an extended hexagonal conformation around the collagen XVIII homotrimerization domain. The1D8N/CEGa1 trimerbody demonstrated high-avidity binding to 4-1BB and EGFR and a potent in vitro costimulatory capacity in the presence of EGFR. The trimerbody rapidly accumulates in EGFR-positive tumors and exhibits anti-tumor activity similar to IgG-based 4-1BB-agonistic mAbs. Importantly, treatment with 1D8N/CEGa1 does not induce systemic inflammatory cytokine production or hepatotoxicity associated with IgG-based 4-1BB agonists. These results implicate FcγR interactions in the 4-1BB-agonist-associated immune abnormalities, and promote the use of the non-canonical antibody presented in this work for safe and effective costimulatory strategies in cancer immunotherapy.

Original publication




Journal article


Nature communications

Publication Date





Department of Antibody Engineering, Leadartis SL, 28008, Madrid, Spain.


CD8-Positive T-Lymphocytes, Lymphocytes, Tumor-Infiltrating, Cell Line, Tumor, Animals, Mice, Inbred BALB C, Humans, Mice, Mice, Nude, Skin Neoplasms, Immunoglobulin G, Epitopes, Epitopes, B-Lymphocyte, Xenograft Model Antitumor Assays, Epitope Mapping, Cytotoxicity, Immunologic, Female, Adaptive Immunity, Single-Chain Antibodies, ErbB Receptors, Tumor Necrosis Factor Receptor Superfamily, Member 9