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.

Non-antibody scaffolds are increasingly used to generate novel binding proteins for both research and therapeutic applications. Our group has developed the tenth fibronectin type III domain of human tenascin-C (TNfn3) as one such scaffold. As a scaffold, TNfn3 must tolerate extensive mutation to introduce novel binding sites. However, TNfn3's marginal stability (T(m) ∼ 59°C, ΔG(unfolding) = 5.7 kcal/mol) stands as a potential obstacle to this process. To address this issue, we sought to engineer highly stable TNfn3 variants. We used two parallel strategies. Using insights gained from structural analysis of other FN3 family members, we (1) rationally designed stabilizing point mutations or (2) introduced novel stabilizing disulfide bonds. Both strategies yielded highly stable TNfn3 variants with T(m) values as high as 83°C and ΔG(unfolding) values as high as 9.4 kcal/mol. Notably, only three or four mutations were required to achieve this level of stability with either approach. These results validate our rational design strategies and illustrate that substantial stability increases can be achieved with minimal mutation. One TNfn3 variant reported here has now been successfully used as a scaffold to develop two promising therapeutic molecules. We anticipate that other variants described will exhibit similar utility.

TRAILR2 (DR5, TNFRSF10B) is a member of the TNF-receptor superfamily that mediates programmed cell death following stimulation with its ligand. Binding of homotrimeric ligand TRAIL to TRAILR2 induces oligomerization of the receptors and triggers the apoptotic pathway through induction of caspases. Although TRAILR2 is found to be expressed in a number of normal cell types it is routinely found at high levels in many cancer types. Since TRAIL has been shown to preferentially induce apoptosis in cancer cells versus normal cells, several TRAIL receptor agonists including monoclonal antibodies as well as the ligand itself, are currently in clinical development. Here, we describe a novel protein scaffold (Tn3) based on a fibronectin type-III domain from the protein Tenascin that was engineered to specifically agonize TRAILR2. Connecting TRAILR2-binding Tn3 domains together significantly improved the in vitro potency of the molecule in several cancer cell models. For example, a hexameric TRAILR2-binding Tn3 construct has an EC50 of 0.5 pM in a 72 hour cell viability assay with Colo-205 cells. This is approximately 2 logs better than TRAIL. Furthermore, a subset of TRAIL-resistant cancer cell lines derived from multiple tissues of origin is sensitive to the multimeric Tn3. The improved potency over TRAIL was demonstrated in colorectal cancer xenograft models. In addition, tumor regression was also observed at doses as low as 0.3 mg/kg. These results suggest that a multimeric Tn3 protein scaffold targeting TRAILR2 has more potent anti-tumor activity in vitro and in vivo compared than the natural ligand. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 239. doi:1538-7445.AM2012-239