Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

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

a technology of protein-based biotherapeutics and cell membranes, applied in the direction of peptide/protein ingredients, osteogenic factors, peptide sources, etc., can solve the problems of poor yield, low cell- and tissue-permeability, and low solubility of socs3 proteins fused to fgf4-derived mtm, so as to improve cell-/tissue-permeability, anti-angiogenic effect, and so on

Inactive Publication Date: 2016-03-03
CELLIVERY THERAPEUTICS
View PDF1 Cites 19 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The patent describes a way to improve the ability of recombinant proteins to penetrate membranes and be active inside cells. This is achieved by fusing the proteins with special domains that help them pass through membranes. The method also makes the proteins soluble and increases their yield during manufacturing. This results in improved cell-permeable versions of the proteins, with potential uses in drug delivery and protein therapy.

Problems solved by technology

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.

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Development of Protein-Based Biotherapeutics That Penetrates Cell-Membrane and Induces Anti-Angiogenic Effect - Improved Cell-Permeable Suppressor of Cytokine Signaling (iCP-SOCS3) Proteins, Polynucleotides Encoding the Same, and Anti-Angiogenic Compositions Comprising the Same
  • Development of Protein-Based Biotherapeutics That Penetrates Cell-Membrane and Induces Anti-Angiogenic Effect - Improved Cell-Permeable Suppressor of Cytokine Signaling (iCP-SOCS3) Proteins, Polynucleotides Encoding the Same, and Anti-Angiogenic Compositions Comprising the Same
  • Development of Protein-Based Biotherapeutics That Penetrates Cell-Membrane and Induces Anti-Angiogenic Effect - Improved Cell-Permeable Suppressor of Cytokine Signaling (iCP-SOCS3) Proteins, Polynucleotides Encoding the Same, and Anti-Angiogenic Compositions Comprising the Same

Examples

Experimental program
Comparison scheme
Effect test

example 1

Development of Novel Advanced Macromolecule Transduction Domain (aMTD)

[0081]H-regions of signal sequences (HRSP)-derived CPPs (MTM, MTS and MTD) do not have a common sequence, a sequence motif, and / or a common structural homologous feature. In this invention, the aim is to develop improved hydrophobic CPPs formatted in the common sequence and structural motif that satisfy newly determined ‘critical factors’ to have a ‘common function’, to facilitate protein translocation across the membrane with similar mechanism to the analyzed CPPs. 6 critical factors have been selected to artificially develop novel hydrophobic CPP, namely advanced macromolecule transduction domain (aMTD). These 6 critical factors include the followings: amino acid length of the peptides (ranging from 9 to 13 amino acids), bending potentials (dependent with the presence and location of proline in the middle of sequence (at 5′, 6′, 7′ or 8′ amino acid) and at the end of peptide (at 12′)), instability index (II) for...

example 2

Construction of Expression Vectors for Recombinant SOCS3 Proteins

[0082]Histidine-tagged human SOCS3 proteins were constructed by amplifying the SOCS3 cDNA (225 amino acids) for aMTD fused to SOCS3 cargo. The PCR reactions (100 ng genomic DNA, 10 pmol each primer, each 0.2 mM dNTP mixture, 1X reaction buffer and 2.5 U Pfu(+) DNA polymerase (Doctor protein, Korea)) were digested on the restriction enzyme site between Nde I (5′) and Sal I (3′) involving 35 cycles of denaturing (95° C.), annealing (62° C.), and extending (72° C.) for 45 sec each. For the last extension cycle, the PCR reactions remained for 10 min at 72° C. The PCR products were subcloned into 6x His expression vector, pET-28a(+) (Novagen). Coding sequence for SDA or SDB fused to C terminus of his-tagged aMTD-SOCS3 was cloned at BamHl (5′) and Sall (3′) in pET-28a(+) from PCR-amplified DNA segments and confirmed by DNA sequence analysis of the resulting plasmids.

example 3

Inducible Expression, Purification, and Preparation of Recombinant Proteins

[0083]The recombinant proteins were purified from E. coli BL21-CodonPlus (DE3) cells grown to an A600 of 0.6 and induced for 3 hrs with 0.6 mM IPTG. Denatured recombinant proteins were purified by Ni2+ affinity chromatography as directed by the supplier (Qiagen, Hilden, Germany). After purification, they were dialyzed against a refolding buffer (0.55 M guanidine HCl, 0.44 M L-arginine, 50 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, 100 mM NDSB, 2 mM reduced glutathione, and 0.2 mM oxidized glutathione) and changed to a physiological buffer such as DMEM medium.

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

PropertyMeasurementUnit
temperatureaaaaaaaaaa
hydrophobicaaaaaaaaaa
solubilityaaaaaaaaaa
Login to View More

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. 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.

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 have (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-angiogenic effect; (ii) polynucleotides that encode the same, and (iii) anti-angiogenesis compositions that comprise the same.BACKGROUND ART[0003]Tumor cells have the ability to spread to adjacent or distant organs, penetrate blood or lymphatic vessels, circulate through the intravascular stream, and then proliferate at another site: metastasis. For the met...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
Patent Type & Authority Applications(United States)
IPC IPC(8): C07K14/47C07K7/08
CPCC07K14/4703C07K2319/10A61K38/00C07K7/08C07K14/51C07K2319/21A61K38/08A61K38/10A61K38/1709A61K38/1761C07K7/06C07K14/4702C07K2319/01C07K2319/00C07K2319/40C07K14/47
Inventor JO, DAEWOONGCHOI, YOUNG, SILNA, WON, HEUMOH, KI, JOON
Owner CELLIVERY THERAPEUTICS
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com