earticle

영어

Here, we report the development of novel 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 7 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 anti-cancer 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 protruding 7 loops from the central core was strongly sequence-tolerant to mutations in the loop regions, offering favorable characteristics, such as the extensive randomizable sequence space and potentially multiple binding sites on the single domain for target recognition. 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.