Abstract

Activation of TNF-related apoptosis-inducing ligand receptor 2 (TRAILR2) can induce apoptosis in a variety of human cancer cell lines and xenografts, while lacking toxicity in normal cells. The natural ligand and agonistic antibodies show antitumor activity in preclinical models of cancer, and this had led to significant excitement in the clinical potential of these agents. Unfortunately, this optimism has been tempered by trial data that, thus far, are not showing clear signs of efficacy in cancer patients. The reasons for discrepant preclinical and clinical observations are not understood, but one possibility is that the current TRAILR2 agonists lack sufficient potency to achieve a meaningful response in patients. Toward addressing that possibility, we have developed multivalent forms of a new binding scaffold (Tn3) that are superagonists of TRAILR2 and can induce apoptosis in tumor cell lines at subpicomolar concentrations. The monomer Tn3 unit was a fibronectin type III domain engineered for high-affinity TRAILR2 binding. Multivalent presentation of this basic unit induced cell death in TRAILR2-expressing cell lines. Optimization of binding affinity, molecular format, and valency contributed to cumulative enhancements of agonistic activity. An optimized multivalent agonist consisting of 8 tandem Tn3 repeats was highly potent in triggering cell death in TRAIL-sensitive cell lines and was 1 to 2 orders of magnitude more potent than TRAIL. Enhanced potency was also observed in vivo in a tumor xenograft setting. The TRAILR2 superagonists described here have the potential for superior clinical activity in settings insensitive to the current therapeutic agonists that target this pathway.

Highlights

  • Cells can undergo apoptosis via 2 different pathways: the intrinsic and extrinsic

  • The extrinsic pathway is triggered by activation of cell surface death receptors, which belong to the tumor necrosis factor (TNF) receptor superfamily

  • We describe the isolation of monomeric TNF-related apoptosis-inducing ligand receptor 2 (TRAILR2)-binding scaffold proteins based on a fibronectin type III scaffold, and engineering of these into multivalent proteins that induced cell death in TRAIL-sensitive and TRAIL-resistant cell lines

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Summary

Introduction

Cells can undergo apoptosis via 2 different pathways: the intrinsic and extrinsic. The intrinsic pathway is preferentially triggered by intracellular proteins such as p53 in response to many different damaging influences including DNA damage, a defective cell-cycle or loss of cell survival factors. Triggered apoptosis is mainly regulated by proteins of the Bcl-2 family that control the release of proapoptotic factors from the mitochondrial intermembrane space. The extrinsic pathway is triggered by activation of cell surface death receptors, which belong to the tumor necrosis factor (TNF) receptor superfamily. Ligand-induced crosslinking of these receptors initiates a p53-independent intracellular signal transduction cascade that leads to cellular apoptosis. Authors' Affiliations: Departments of 1Antibody Discovery and Protein Engineering, and 2Oncology, MedImmune, LLC, Gaithersburg, Maryland

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