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 (SOSC3) fused to the
fibroblast growth factor (FGF) 4-derived MTM were developed to inhibit
inflammation and
apoptosis. However, this 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 each critical factor. Furthermore, 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 SOCS3 recombinant 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-angiogenic agents. Since SOCS3 is known to be an endogenous inhibitor of
pathological angiogenesis, we reasoned that iCP-SOCS3 could be used as a protein-based
intracellular replacement therapy for inhibiting
angiogenesis in
tumor cells. The results demonstrated in this art support this following reasoning:
Cancer treatment with iCP-SOCS3 results in reduced endothelial cell viability, loss of
cell migration potential and suppressed vascular
sprouting potentials. 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
cancer cell-mediated
angiogenesis.