earticle

영어

Here we report a novel non-antibody protein scaffold developed based on the fold of kringle domain (KD) derived from plasminogen. Based on the sequence analysis of 39 unique KD in 31 human proteins (1), we designed KD libraries with a rationale to conserve the critical residues for the KD fold, but to introduce mutations which are exposed to the surface in the flexible loops, using synthetic shuffling technique (2). For target proteins as a proof-of-concept, we used death receptor 4 (DR4), DR5 and TNFα. Isolated KD mutants against the individual target proteins, using yeast surface display techniques combined with FACS, were solubly expressed well and
specifically bind to the targeted proteins with affinity of 10-6-10-8 M, without significant cross-reactivity, assessed by ELISA and SPR. Some KD mutants selected against DR4 or DR5 induced apoptosis in several carcinoma cell lines as a single agent. Also, some KD mutants isolated against TNFα efficiently protected TNFα-induced cell death by neutralizing the cytotoxicity of TNFα. Our results suggest that a novel non-antibody scaffold based on KD fold can be developed as a specific binder to given targets to modulate the biological activity.