Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) induces apoptotic cell death in a variety of tumor cells without significant cytotoxicity on normal cells. However, many cancer cells with apoptotic defects are resistant to treatment with TRAIL alone, limiting its potential as an anticancer therapeutic. Here, we report on the tumoricidal activity of a human single-chain fragment variable, HW1, which specifically binds to TRAIL receptor 2 (TR2) without competing with TRAIL for the binding. HW1 treatment as a single agent induces autophagic cell death in a variety of both TRAIL-sensitive and TRAIL-resistant cancer cells, but exhibits much less cytotoxicity on normal cells. The HW1-induced autophagic cell death was inhibited by an autophagy inhibitor, 3-methyladenine, or by RNA interference knockdown of Beclin-1 and Atg7. We also show that the HW1-mediated autophagic cell death occurs predominantly via the c-Jun NH 2 -terminal kinase pathway in a caspase-independent manner. Analysis of the death-inducing signaling complex induced by HW1 binding to TR2 exhibits the recruitment of TNF receptor-associated death domain and TNF receptorassociated factor 2, but not Fas-associated death domain, caspase-8, or receptor-interacting protein, which is distinct from that induced by TRAIL. Our results reveal a novel TR2-mediated signaling pathway triggering autophagic cell death and provides a new strategy for the elimination of cancer cells, including TRAIL-resistant tumors, through nonapoptotic cell death. [Cancer Res 2007;67(15):7327-34]

Here, we report the development of target-specific binding proteins based on the kringle domain (KD) (∼80 residues), a ubiquitous modular structural unit occurring across eukaryotic species. By exploiting the highly conserved backbone folding by core residues, but using extensive sequence variations in the seven loop regions of naturally occurring human KDs, we generated a synthetic KD library on the yeast cell surface by randomizing 45 residues in the loops of a human KD template. We isolated KD variants that specifically bind to anticancer target proteins, such as human death receptor 4 (DR4) and/or DR5, and that function as agonists to induce apoptotic cell death in several cancer cell lines in vitro and inhibit tumor progression in mouse models. Combined treatments with KD variants possessing different recognition sites on the same target protein exerted synergisitic tumoricidal activities, compared to treatment with individual variants. In addition to the agonists, we isolated an antagonistic KD variant that binds human tumor necrosis factor-α (TNFα) and efficiently neutralizes TNFα-induced cytotoxicity in vitro and in vivo. The KD scaffold with seven flexible loops protruding from the central core was strongly sequence-tolerant to mutations in the loop regions, offering a potential advantage of distinct binding sites for target recognition on the single domain. Our results suggest that the KD scaffold can be used to develop target-specific binding proteins that function as agonists or antagonists toward given target molecules, indicative of their potential use as biotherapeutics.

Background:We report the cell death mechanism of KD548-Fc, an Fc-fused DR4/DR5 dual-specific Kringle domain variant. Results: KD548-Fc-stimulated DR4/DR5 activate Nox1 NADPH oxidase to generate superoxide anion, leading to reactive oxygen species-mediated apoptotic cell death. Conclusion: DR4/DR5 have the capability to activate Nox1. Significance: Our results provide a new signaling pathway of DR4/DR5, distinct from the conventional apoptosis pathway.

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