Development of a Protein-Based Biotherapeutic Agent That Penetrates Cell-Membrane and Induces Anti-Tumor Effect in Solid Tumors - Improved Cell-Permeable Suppressor of Cytokine Signaling (iCP-SOCS3) Proteins, Polynucleotides Encoding the Same, and Anti-Tumor Compositions Comprising the Same

Abstract

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. The 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. 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 various cancers likes gastric, colorectal and breast cancer, and glioblastoma. Since SOCS3 is frequently deleted in and loss of SOCS3 in tumors 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 various cancers. The results demonstrated in this art support the reasoning: treatment of cancer cells with iCP-SOCS3 results in reduced cancer cell viability, enhanced apoptosis of solid tumors including gastric, colorectal and breast cancer, and glioblastoma and loss of cell migration / invasion potential. Furthermore, iCP-SOCS3 inhibits the growth of gastric and colorectal tumors in a subcutaneous xenografts model. 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 various tumors such as gastric cancer, colorectal cancer, glioblastoma, and breast cancer.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS[0001]This application claims the benefit of the filing date of U.S. Provisional Application No. 62 / 042,493, filed on Aug. 27, 2014, in the United States Patent and Trademark Office, the disclosure of which is incorporated herein in its entirety by reference.TECHNICAL FIELD[0002]The present invention pertains to (i) improved cell-permeable SOCS3 (iCP-SOCS3) proteins as protein-based biotherapeutics, which are well-enhanced in their ability to transport biologically active SOCS3 proteins across the plasma membrane, to increase in its solubility and manufacturing yield, and to induce anti-tumor effect in solid tumors; (ii) polynucleotides that encode the same, and (iii) anti-solid tumor compositions that comprise the same.BACKGROUND ART[0003]The Janus kinase signal transducers and activators of transcription signaling (JAK / STAT) plays important roles in immune responses, including oncogenesis. So many investigations demonstrated that STAT-3, an i...

AI Technical Summary

Benefits of technology

The patent describes a new way to make proteins that can better penetrate membranes in the body, which can improve their effectiveness as medications. This is done by adding special domains to the protein that help it cross membranes more easily. The protein also becomes more soluble and can be produced in larger quantities, which makes it easier to develop as a drug. Overall, this invention creates a better way to make proteins that can be used as medicines.

Problems solved by technology

However, effective treatment regimens for gastric cancers, especially advanced gastric cancer, are still lacking; therefore, the prognosis of patients with this disease remains poor.

Colorectal cancer is one of the most fatal neoplastic diseases worldwide and a serious global health problem, with over one million new cases and half million mortalities worldwide each year.

Gliomas are the most common type of primary brain tumors are highly malignant and are associated with a very poor prognosis.

Glioblastoma is a very aggressive subtype of glioma with very short life expectancy and limited treatment options.

However, the SOCS3 proteins fused to FGF4-derived MTM displayed extremely low solubility, poor yields and relatively low cell- and tissue-permeability.

Therefore, the MTM-fused SOCS3 proteins were not suitable for further clinical development as therapeutic agents.

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