Multivalent Scaffold Proteins as Superagonists of TRAIL Receptor 2–Induced Apoptosis

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.

Genetic and Pharmacological Screens Converge in Identifying FLIP, BCL2, and IAP Proteins as Key Regulators of Sensitivity to the TRAIL-Inducing Anticancer Agent ONC201/TIC10

“…These findings suggest that other therapies that synergize with TRAIL, such as quinacrine, are worth investigating for combinatorial activity with ONC201 (13). By a similar notion, these findings also suggest that sorafenib may sensitize other TRAIL-based agents such as TRAIL-receptor agonists that have been developed including antibody- and protein scaffold-based approaches (14–16). …”

Multivalent Conjugates of Sonic Hedgehog Accelerate Diabetic Wound Healing

“…28 Other recent studies have also demonstrated that multivalent presentation of peptide and protein ligands is sufficient to induce enhanced cellular bioactivity relative to an unconjugated monovalent control. [63][64][65] In a particularly noteworthy study, multivalent conjugates of ephrin-B2-HyA, synthesized using the conjugation protocol described here, demonstrated enhanced differentiation of neural progenitor cells in vitro and in vivo. 66 In addition, the authors reported that clustering of ephrin-B2 receptors on the cell surface and the downstream pathway activity were correlated directly with ephrin-B2 valency, thereby providing further evidence that the multivalent presentation of protein ligands was sufficient to enhance the activation of their cellular targets.…”

RG7386, a Novel Tetravalent FAP-DR5 Antibody, Effectively Triggers FAP-Dependent, Avidity-Driven DR5 Hyperclustering and Tumor Cell Apoptosis

“…In addition to full-length antibodies, different scaffolds such as atrimers (31) or multivalent monobodies (32,33) are under evaluation to target DRs. However, the lack of clinical efficacy observed with untargeted DR5 agonistic antibodies illustrates that the activity of these molecules in vitro may not necessarily translate into clinical efficacy.…”