| 摘要 |
In principle, protein-based biotherapeutics offers a way to control biochemical processes in living cells under non-steady state conditions and with fewer off-target effects than conventional small molecule therapeutics. However, systemic protein delivery in vivo has been proven difficult due to poor tissue penetration and rapid clearance. 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. Previously, recombinant proteins consisting of suppressor of cytokine signaling 3 (CP-SOCS3) protein 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 cells were hard to be purified in soluble form. To address these critical limitations, CPP sequences called advanced MTDs (aMTDs) have been developed in this art. The development of this art has been 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 that satisfy 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 neoplasia in lung. Since SOCS3 is frequently deleted in cancer cells and loss of SOCS3 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 lung cancer. The results demonstrated in this art support this reasoning: treatment of human non-small cell lung carcinoma cells with iCP-SOCS3 results in reduced cancer cell viability, enhanced apoptosis. Furthermore, iCP-SOCS3 inhibited migration/invasion of lung cancer cells. In the present invention with iCP-SOCS3, where SOCS3 is fused to an empirically determined combination of newly developed aMTD and customized SD, macromolecule intracellular transduction technology (MITT) enabled by the advanced MTDs may provide novel protein therapy against lung cancer. |
