An Fc-free EGFR-specific 4-1BB-agonistic Trimerbody Displays Broad Antitumor Activity in Humanized Murine Cancer Models without Toxicity

Clin Cancer Res. 2021 Jun 1;27(11):3167-3177. doi: 10.1158/1078-0432.CCR-20-4625. Epub 2021 Mar 30.


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 antitumor response. Systemic administration of anti-4-1BB-agonistic IgGs, although effective 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, antitumor 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 antitumor 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 IFNγ secretion in vitro and resulted in tumor regression in humanized mice bearing aggressive triple-negative breast cancer.

Conclusions: These results demonstrate the nontoxic broad antitumor 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 patients with cancer while avoiding Fc-mediated adverse reactions.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Breast Neoplasms / immunology
  • Breast Neoplasms / pathology*
  • Breast Neoplasms / therapy*
  • Carcinoma, Non-Small-Cell Lung / immunology
  • Carcinoma, Non-Small-Cell Lung / pathology*
  • Carcinoma, Non-Small-Cell Lung / therapy*
  • Cell Line
  • Disease Models, Animal
  • ErbB Receptors*
  • Female
  • Immunotherapy / methods*
  • Lung Neoplasms / immunology
  • Lung Neoplasms / pathology*
  • Lung Neoplasms / therapy*
  • Lymphocyte Activation / genetics
  • Lymphocyte Activation / physiology
  • Mice, Transgenic
  • T-Lymphocytes / immunology
  • Tumor Necrosis Factor Receptor Superfamily, Member 9 / immunology
  • Tumor Necrosis Factor Receptor Superfamily, Member 9 / metabolism
  • Tumor Necrosis Factor Receptor Superfamily, Member 9 / therapeutic use*


  • Tumor Necrosis Factor Receptor Superfamily, Member 9
  • EGFR protein, human
  • EGFR protein, mouse
  • ErbB Receptors