The intracellular delivery of proteins with high efficiency in a receptor-specific manner is of great significance in molecular medicine and biotechnology, but remains a challenge. Herein, we present the development of a highly efficient and receptor-specific delivery platform for protein cargos by combining the receptor binding domain of Escherichia coli Shiga-like toxin and the translocation domain of Pseudomonas aeruginosa exotoxin A. We demonstrated the utility and efficiency of the delivery platform by showing a cytosolic delivery of diverse proteins both in vitro and in vivo in a receptor-specific manner. In particular, the delivery system was shown to be effective for targeting an intracellular protein and consequently suppressing the tumor growth in xenograft mice. The present platform can be widely used for intracellular delivery of diverse functional macromolecules with high efficiency in a receptor-specific manner. Biotechnol. Bioeng. 2016;113: 1639-1646. © 2016 Wiley Periodicals, Inc.

Age-related macular degeneration (AMD) is the leading cause of vision loss and blindness among people over the age of 60. Vascular endothelial growth factor (VEGF) plays a major role in pathological angiogenesis in AMD. Herein, we present the development of an anti- human VEGF repebody, which is a small-sized protein binder consisting of leucine-rich repeat (LRR) modules. The anti-VEGF repebody selected through a phage-display was shown to have a high affinity and specificity for human VEGF. We demonstrate that this repebody effectively inhibits in vitro angiogenic cellular processes, such as proliferation and migration, by blocking the VEGF-mediated signaling pathway. The repebody was also shown to have a strong suppression effect on choroidal neovascularization (CNV) and vascular leakage in vivo. Our results indicate that the anti-VEGF repebody has a therapeutic potential for treating neovascular AMD as well as other VEGF-involved diseases including diabetic retinopathy and metastatic cancers.

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With the high efficacy of protein-based therapeutics and plenty of intracellular drug targets, cytosolic protein delivery in a cell-specific manner has attracted considerable attention in the field of precision medicine. Herein, we present an intracellular protein delivery system based on a target-specific repebody and the translocation domain of Pseudomonas aeruginosa exotoxin A. The delivery platform was constructed by genetically fusing an EGFR-specific repebody as a targeting moiety to the translocation domain, while a protein cargo was fused to the C-terminal end of the delivery platform. The delivery platform was revealed to efficiently translocate a protein cargo to the cytosol in a target-specific manner. We demonstrate the utility and potential of the delivery platform by showing a remarkable tumor regression with negligible toxicity in a xenograft mice model when gelonin was used as the cytotoxic protein cargo. The present platform can find wide applications to the cell-selective cytosolic delivery of diverse proteins in many areas.

RATIONALE Idiopathic Pulmonary Fibrosis (IPF) is a progressive, irreversible and fatal lung disease. Its risk factors include age, genetic and environmental/occupational factors. Currently available therapies only slow down disease progression. Autotaxin (ATX) is an extracellular enzyme involved in the hydrolysis of lysophosphatidylcholine (LPC) to form lysophosphatidic acid (LPA). The ATX-LPA-LPA receptor (LPAR) axis has been suggested to play a pivotal role in the pathogenesis and the progression of IPF. Genetic deletion of ATX, LPAR1 and LPAR2 significantly improved the severity of bleomycin-induced pulmonary fibrosis in mouse. Pharmacological inhibition of ATX and LPAR1 reduced lung fibrosis parameters resulted from the bleomycin treatment in mouse. Positive efficacy data were obtained from clinical trials with drugs targeting the ATX-LPAR pathway. METHODS The inhibitory potency of BBT-877, an orally available small molecule inhibitor of ATX, was measured by FS-3 (in vitro) and plasma LPC (ex vivo) assays. Transwell cell migration assay was used to quantify in vitro anti-migratory efficacy of BBT-877. In mouse disease model, bleomycin was intranasally administrated to induce pulmonary fibrosis at day 0, and BBT-877 was given orally twice a day from day 7 to 21. RESULTS In vitro and ex vivo IC 50 of BBT-877 were determined to be 2.4 nM and 6.89 nM (LPA 18:2), respectively. LPA-induced cell migration was effectively inhibited by BBT-877. In vivo anti-fibrotic efficacy of BBT-877 was shown in the mouse model of bleomycin-induced pulmonary fibrosis. BBT-877 did not significantly impair the viability of various cell types even when treated at high concentrations (CC 50 : >100 M). IND-enabling toxicology studies have been completed in the rat and monkey models, with demonstrated good safety profiles and no remarkable findings up to 1000 and 300 mg/kg/day, respectively. CONCLUSION Results of comprehensive in vitro and in vivo studies with BBT-877 demonstrate this compound is a very potent, selective, potentially best-in-class ATX inhibitor with a very favorable safety profile and support further investigation in clinical testing for the treatment of IPF. Phase 1 clinical studies are currently ongoing and will be completed by Aug 2019.

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The modulation of a cell signaling process using a molecular binder followed by an analysis of the cellular response is crucial for understanding its role in the cellular function and developing pharmaceuticals. Herein, we present the modulation of the ERK2-mediated signaling pathway through the cytosolic delivery of a native regulatory protein for ERK2, that is, PEA-15 (phosphoprotein enriched in astrocytes, 15 kDa), and its engineered variants using a bacterial toxin-based delivery system. Based on biochemical and structural analyses, PEA-15 variants with different phosphorylation sites and a high affinity for ERK2 were designed. Semi-rational approach led to about an 830-fold increase in the binding affinity of PEA-15, resulting in more effective modulation of the ERK2-mediated signaling. Our approach enabled an understanding of the cellular function of the ERK2-mediated signaling process and the effect of PEA-15 phosphorylation on its action as an ERK2 blocker. We demonstrated the utility and potential of our approach by showing an efficient cytosolic delivery of these PEA-15 variants and the effective suppression of cell proliferation through the inhibition of the ERK2 function. The present approach can be used broadly for modulating the cell signaling processes and understanding their roles in cellular function, as well as for the development of therapeutics.

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