Modulation of cellular signaling by intracellular delivery of protein binders

Abstract

Over decades, many types of therapeutics for cancer have been developed, and they mostly targeted receptors on cell surface or kinases by chemical drugs with low specificities. However, there are numerous intracellular proteins that are connected in cellular signaling processes, and the proteins are fundamentally related with cellular functions such as cell proliferation. Based on this fact, signaling proteins have been targeted for therapeutic effects, but despite intense efforts in medical industry and academia, a therapeutic potential on intracellular signaling proteins has remained elusive due to the difficulty in efficient intracellular accessibility. In particular, proteins do not enter mammalian cells directly, which severely limits many disease-relevant targets. In this regard, we developed protein binders and delivered the binders for modulation of signaling pathways. Bruton’s tyrosine kinase (Btk) is one of intracellular protein kinases, and it is an important regulator in B cell receptor (BCR) signaling pathway, in which plays a key role in processes such as cell proliferation. We previously developed a Btk-specific protein binder, rF10, which was designed to target the SH2 domain of human Btk (hBtk). We present an approach to deliver proteins to B-cell lymphoma in a highly efficient and versatile way. The hBtk-specific protein binder, rF10, was genetically fused with a cell-penetrating peptide and intracellularly delivered. We observed that the repebody effectively inhibits the activity of hBtk in B-cell lymphoma, leading a down-regulation of BCR-mediated signaling pathway, showing its potent strategy for therapeutic proteins. We demonstrate the modulation of the ERK2-mediated signaling pathway through the cytosolic delivery of a native regulatory protein for ERK2, that is, PEA-15 and its engineered variants, PEA-16, using a bacterial toxin-based delivery system. Based on biochemical and structural analyses, a high-affinity variant for ERK2 was 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. Also, we demonstrated the utility and potential of our approach by showing an efficient cytosolic delivery of PEA-16 and the effective suppression of cell proliferation through the inhibition of the ERK2 function. We present the development of an anti-Bcl-2 protein binder as a potent anti-cancer agent and delivered for targeted cancer therapy. We developed anti-Bcl-2 repebody based on modular approach, and it was demonstrated that the repebody showed high specificity. Also, the repebody was applied to the EGFR-specific repebody and the bacterial toxin-based delivery system for high-efficiency targeted delivery, and the repebody showed effective inhibitory effects in cell proliferation, suggesting the general utility of the delivery approach in targeted cancer therapy. In this study, we demonstrated the utility and potential of protein binders and intracellular delivery system for modulation of cellular signaling pathways. 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.