Development of Protein-Based Biotherapeutics That Penetrates Cell-Membrane and Induces Anti-Hepatocellular Carcinoma Effect - Improved Cell-Permeable Suppressor of Cytokine Signaling (iCP-SOCS3) Proteins, Polynucleotides Encoding the Same, and Anti-Hepatocellular Carcinoma Compositions Comprising the Same

摘要

Protein transduction exploits the ability of some cell-penetrating peptide (CPP) sequences to enhance the uptake of proteins and other macromolecules by mammalian cells. Previously developed hydrophobic CPPs, named membrane translocating sequence (MTS), membrane translocating motif (MTM) and macromolecule transduction domain (MTD), are able to deliver biologically active proteins into a variety of cells and tissues. Various cargo proteins fused to these CPPs have been used to test the functional and/or therapeutic efficacy of protein transduction. For example, recombinant proteins consisting of suppressor of cytokine signaling 3 protein (CP-SOCS3) fused to the fibroblast growth factor (FGF) 4-derived MTM were developed to inhibit inflammation and apoptosis. However, CP-SOCS3 fusion proteins expressed in bacteria were hard to purify in soluble form. To address these critical limitations, CPP sequences called advanced MTDs (aMTD) have been developed in this art. This is accomplished by (i) analyzing previous developed hydrophobic CPP sequences to identify specific critical factors (CFs) that affect intracellular delivery potential and (ii) constructing artificial aMTD sequences satisfied for each critical factor. In addition, solubilization domains (SDs) have been incorporated into the aMTD-fused SOCS3 recombinant proteins to enhance solubility with corresponding increases in protein yield and cell-/tissue-permeability. These recombinant SOCS3 proteins fused to aMTD/SD having much higher solubility/yield and cell-/tissue-permeability have been named as improved cell-permeable SOCS3 (iCP-SOCS3) proteins. Previously developed CP-SOCS3 proteins fused to MTM were only tested or used as anti-inflammatory agents to treat acute liver injury. In the present art, iCP-SOCS3 proteins have been tested for use as anti-cancer agents in the treatment of hepatocellular carcinoma. Since SOCS3 is frequently deleted in and loss of SOCS3 in hepatocytes promotes resistance to apoptosis and proliferation, we reasoned that iCP-SOCS3 could be used as a protein-based intracellular replacement therapy for the treatment of hepatocellular carcinoma. The results support this reasoning: treatment of hepatocellular carcinoma cells with iCP-SOCS3 results in reduced cancer cell viability, enhanced apoptosis and loss of cell migration/invasion potential. Furthermore, iCP-SOCS3 inhibits the growth of hepatocellular carcinoma in a subcutaneous xenografts model. In the present invention with iCP-SOCS3 fused to an empirically determined combination of newly developed aMTD and customized SD, macromolecule intracellular transduction technology (MITT) enabled by the advanced MTD may provide novel protein therapy against hepatocellular carcinoma.