Aptide-based fusion proteins for biomedical applications

Abstract

Very recently, we have developed a β-hairpin scaffold based high affinity peptide ligand, termed ‘aptide’, presenting a comparable affinity and specificity to antibodies. Encouraged by it smaller size (3 kDa), stability, easy of genetic fusion, low immunogenic property, and specially less patent competition, aptide based potential applications can cover broad range of biomedical application, from applied research to therapeutics. This thesis has demonstrated that the application of aptide-based fusion proteins to targeted cancer therapy and the development of new antibody purification methods can broaden and enhance the applicability of aptide.

In chapter II, I described the fusion protein of TNFαand EDB-specific aptide for targeted cancer therapy. Although tumor necrosis factor (TNF)-α has shown potent antitumor effects, severe side effects at less than therapeutic doses have limited its systemic administration. As a new strategy for targeted delivery of TNF to tumors, I designed a fusion protein (mTNFα-$APT_{EDB}$) consisting of mouse TNFα and an aptide specific to extra domain B of fibronectin (EDB). mTNFα-$APT_{EDB}$ presented high selectivity and affinity (~6 nM) against EDB. When tested against the EDB-positive, TNFα-sensitive mouse fibrosarcoma cell line, WEHI-164, mTNFα-$APT_{EDB}$ exhibited a modest four-fold decrease in anticancer activity compared with unconjugated mTNFα. However, in EDB-overexpressing fibrosarcoma-bearing mice, systemically delivered mTNFα-$APT_{EDB}$ showed more uptake in tumor tissue and much greater tumor growth inhibition than mTNFα alone without substantial body weight loss. Moreover, the in vivo antitumor efficacy of mTNFα-$APT_{EDB}$ was further significantly enhanced by combination treatment with the chemotherapeutic drug, melphalan, suggesting that TNFα-mediated enhanced vascular permeability increase drug uptake in tumor and produce a synergistic effect. Taken together, these results demonstrate that a fusion protein of mTNFα with a cancer-targeting aptide could be a new anticancer therapeutic option for ensuring potent antitumor efficacy with low toxicity after systemic administration.

In chapter III, I described $Ca^{2+}$ ion induced affinity precipitation using a calsequestrin (CSQ) and Z-domain fusion proteins for antibody purification. In addition, the screening and identification of human Fc fragment (hFc)-specific aptide ($APT_{hFc}$) was studied for the construction of CSQ-$APT_{hFc}$ fusion protein. Affinity precipitation is an alternative strategy to conventional chromatographic methods for therapeutic antibody purification due to operational benefits of stimuli sensitive precipitation. CSQ is the calcium binding protein and its polymerization by $Ca^{2+}$ ion induce insoluble precipitation. In this study, the features of CSQ fused Z-domain (ZCSQ) for antibody purification were characterized. With 5 mM $Ca^{2+}$ ion concentration, ZCSQ efficiently precipitated the ZCSQ/IgG complex followed by released IgG with high purity in acidic buffer (pH 3.5 sodium citrate) and ensured at least 4 times regeneration by EDTA chelation of $Ca^{2+}$ ion. In CHO cell culture media, ZCSQ fusion protein clearly purify IgG and effectively removed host cell DNA contamination than protein A chromatography. Notably, the $Ca^{2+}$ ion induced ZCSQ/IgG precipitation neither affect the antigen binding ability nor cause the critical aggregation of the purified IgG. At the same time, I confirmed that $APT_{hFc}$ specifically bind to hFc protein with ~72 nM affinity and expected that $APT_{hFc}$ be a potential candidate for next-generation non-immunogenic affinity ligand of CSQ fusion protein for antibody purification. Taken together, these results demonstrate that $Ca^{2+}$ ion and CSQ based affinity precipitation is a proof of concept method for antibody purification