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US20160060313A1 - 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 - Google Patents

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 Download PDF

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US20160060313A1
US20160060313A1 US14/838,295 US201514838295A US2016060313A1 US 20160060313 A1 US20160060313 A1 US 20160060313A1 US 201514838295 A US201514838295 A US 201514838295A US 2016060313 A1 US2016060313 A1 US 2016060313A1
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socs3
proteins
cell
protein
fused
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Daewoong Jo
Young Sil CHOI
Won Heum NA
Ki Joon OH
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Cellivery Therapeutics Inc
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Cellivery Therapeutics Inc
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Assigned to JO, DAEWOONG, CELLIVERY THERAPEUTICS, INC. reassignment JO, DAEWOONG ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: CHOI, YOUNG SIL, JO, DAEWOONG, KI JOON, OH, NA, WON HEUM
Publication of US20160060313A1 publication Critical patent/US20160060313A1/en
Priority to PCT/KR2016/009456 priority patent/WO2017034349A1/fr
Priority to EP16839637.2A priority patent/EP3341396B1/fr
Assigned to CELLIVERY THERAPEUTICS, INC. reassignment CELLIVERY THERAPEUTICS, INC. ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: JO, DAEWOONG
Priority to US15/631,982 priority patent/US10787492B2/en
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    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/475—Growth factors; Growth regulators
    • C07K14/51—Bone morphogenetic factor; Osteogenins; Osteogenic factor; Bone-inducing factor
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    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
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    • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/08—Peptides having 5 to 11 amino acids
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    • A61K38/00—Medicinal preparations containing peptides
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    • A61K38/10—Peptides having 12 to 20 amino acids
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    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
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    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
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    • C07K2319/10—Fusion polypeptide containing a localisation/targetting motif containing a tag for extracellular membrane crossing, e.g. TAT or VP22
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    • C07—ORGANIC CHEMISTRY
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    • C07K2319/20—Fusion polypeptide containing a tag with affinity for a non-protein ligand
    • C07K2319/21—Fusion polypeptide containing a tag with affinity for a non-protein ligand containing a His-tag
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    • C—CHEMISTRY; METALLURGY
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    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
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    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids

Definitions

  • 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.
  • iCP-SOCS3 cell-permeable SOCS3
  • 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.
  • growth of the vascular network is important.
  • the processes whereby new blood and lymphatic vessels form are called angiogenesis, resulting in the excessive proliferation of cancer cells through the formation of a new vascular network to supply nutrients, oxygen and immune cells and also to remove waste products.
  • Cytokines including IL-6 and interferon-gamma (IFN- ⁇ ) activate the Janus kinase (JAK)/signal transducers and activators of transcription (STAT) signaling pathway, a vital role promoting the inflammation, carcinogenesis and angiogenesis.
  • Cytokine signaling is strictly regulated by the SOCS family proteins induced by different classes of agonists, including cytokines, hormones and infectious agents.
  • SOCS1 and SOCS3 are relatively specific to STAT1 and STAT3, respectively.
  • SOCS1 inhibits JAK activation through its N-terminal kinase inhibitory region (KIR) by the direct binding to the activation loop of JAKs, while SOCS3 binds to janus kinases (JAKs)-proximal sites on the receptor through its SH2 domain and inhibits JAK activity that blocks recruitment of STAT3.
  • KIR N-terminal kinase inhibitory region
  • SOCS3 binds to janus kinases (JAKs)-proximal sites on the receptor through its SH2 domain and inhibits JAK activity that blocks recruitment of STAT3.
  • Both SOCS1 and SOCS3 promote anti-inflammaory effects due to the suppression of inflammation-inducing cytokine signaling.
  • the SOCS box another domain in SOCS proteins, interacts with E3 ubiquitin ligases and/or couples the SH2 domain-binding proteins to the ubiquitin—proteasome pathway. Therefore, SOCSs inhibit cytokine signaling by suppressing JAK
  • SOCS3 In connection with SOCSs and angiogenesis, the SOCS3 gene has been implicated as an angiogenesis inhibitor in the cancer development. Previous studies have reported that SOCS3 is transiently induced by inflammatory mediators and inhibits cytoplasmic effectors such as the JAK/STAT kinases. In addition, SOCS3 deactivates tyrosine kinase receptor signaling, including the IGF-1 receptor, resulting in the suppression of apoptosis and vascular sprouting of the endothelial cells (ECs). This means that SOCS3 plays a important role in the negative regulation of the JAK/STAT pathway and contributes to the suppression of angiogenesis by regulating the angiogenic potentials of endothelial cells.
  • 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.
  • improved SOCS3 recombinant proteins iCP-SOCS3 fused to the combination of novel hydrophobic CPPs, namely advanced macromolecule transduction domains (aMTDs), to greatly improve the efficiency of membrane penetrating ability in vitro and in vivo with solubilization domains to increase their solubility and manufacturing yield when expressed and purified from bacteria cells.
  • aMTD/SD-fused iCP-SOCS3 recombinant proteins have much improved physicochemical characteristics (solubility & yield) and functional activity (cell-/tissue-permeability) compared to the protein fused only to FGF-4-derived MTM.
  • the newly developed iCP-SOCS3 proteins have now been demonstrated to have therapeutic application in treating angiogenesis, exploiting the ability of SOCS3 to suppress JAK/STAT signaling.
  • the present invention represents that macromolecule intracellular transduction technology (MITT) enabled by the new hydrophobic CPPs that are aMTDs may provide novel protein therapy through SOCS3-intracellular protein replacement against the angiogenesis in tumor.
  • MITT macromolecule intracellular transduction technology
  • An aspect of the present invention relates to improved cell-permeable SOCS3 (iCP-SOCS3) recombinant proteins capable of mediating the transduction of biologically active macromolecules into live cells.
  • iCP-SOCS3 cell-permeable SOCS3
  • aMTDs advanced macromolecule transduction domains
  • iCP-SOCS3 recombinant proteins fused to solubilization domains greatly increase in their solubility and manufacturing yield when they are expressed and purified in the bacteria system.
  • An aspect of the present invention also relates to its therapeutic application for delivery of a biologically active molecule to a cell involving a cell-permeable SOCS3 recombinant protein, where the aMTD is attached to a biologically active cargo molecule.
  • aspects of the present invention relate to an efficient use of aMTD sequences for drug delivery, protein therapy, intracellular protein therapy, protein replacement therapy and peptide therapy.
  • the present invention provides improved cell-permeable SOCS3 as a biotherapeutics having improved solubility/yield, cell-/tissue-permeability and anti-angiogenic effects. Therefore, this would allow their practically effective applications in drug delivery and protein therapy, including intracellular protein therapy and protein replacement therapy.
  • FIG. 1 shows the structure of SOCS3 recombinant proteins.
  • a schematic diagram of the His-tagged SOCS3 recombinant protein is illustrated and constructed according to the present invention.
  • the his-tag for affinity purification (white), aMTD165 (black), SOCS3 (gray) and solubilization domain A and B (SDA & SDB, hatched) are shown.
  • FIG. 2 shows the construction of expression for SOCS3 recombinant proteins
  • FIG. 3 shows the inducible expression and purification of SOCS3 recombinant proteins.
  • Expression of SOCS3 recombinant proteins in E.coli before ( ⁇ ) and after (+) induction with IPTG and purification by Ni2+ affinity chromatography (P) were monitored by SDS-PAGE, and stained with Coomassie blue.
  • FIG. 4 shows the improvement of solubility/yield with aMTD/SD-fusion.
  • the solubility, yield and recovery (in percent) of soluble form from denatured form are indicated (left). Relative yield of recombinant proteins is normalized to the yield of HS3 protein (Right).
  • FIG. 5 shows aMTD-mediated cell-permeability of SOCS3 recombinant proteins.
  • RAW264.7 cells were exposed to FITC-labeled SOCS3 recombinant proteins (10 ⁇ M) for 1 hr, treated with proteinase K to remove cell-associated but non-internalized proteins and analyzed by flow cytometry. Untreated cells (gray) and equimolar concentration of unconjugated FITC (FITC only, green)-treated cells were served as control.
  • FIG. 6 shows aMTD-mediated intracellular delivery and localization of SOCS3 recombinant proteins.
  • Each of NIH3T3 cells was incubated for 1 hour at 37° C. with 10 ⁇ M FITC-labeled SOCS3 protein.
  • Cell-permeability of SOCS3 recombinant proteins was visualized by utilizing confocal microscopy LSM700 version.
  • FIG. 7 shows the systemic delivery of aMTD/SD-fused SOCS3 recombinant proteins in vivo.
  • Cryosections of saline-perfused organs were prepared from mice 1 hr after intraperitoneal injection of FITC only or 600 ⁇ g FITC-conjugated recombinant SOCS3 proteins, and were analyzed by fluorescence microscopy.
  • FIG. 8 shows the structure of SDB-fused SOCS3 recombinant protein.
  • a schematic diagram of the SOCS3 recombinant protein is illustrated and constructed according to the present invention.
  • the his-tag for affinity purification (white), SOCS3 (gray) and solubilization domain B (SDB, hatched) are shown.
  • FIG. 9 shows the expression, purification and determination of solubility/yield of SD-fused SOCS3 protein.
  • Expression of SOCS3 recombinant proteins in E.coli before ( ⁇ ) and after (+) induction with IPTG and purification by Ni2+ affinity chromatography (P) were monitored by SDS-PAGE, and stained with Coomassie blue (Left, top). The solubility, yield and recovery (in percent) of soluble form from denatured form are indicated (Left, bottom). Relative yield of recombinant proteins is normalized to the yield of HS3 protein (Right).
  • FIG. 10 shows the mechanism of aMTD-mediated SOCS3 protein uptake into cells.
  • A-D RAW264.7 cells were treated with 100 mM EDTA for 3 hrs (A), 5 mg/ml Proteinase K for 10 mins (B), 20 mM taxol for 30 mins (C), or 10 ⁇ M antimycin for 2 hrs either without or with 1 mM supplemental ATP for 3 hrs.
  • Cells were exposed for 1 hr to 10 ⁇ M FITC-labeled HS3 (black), -HS3B (blue) or -HM165S3B (red), treated with proteinase K for 20 mins, and analyzed by flow cytometry.
  • Untreated cells (gray) and equimolar concentration of unconjugated FITC (FITC only, green)-treated cells were served as control.
  • E RAW264.7 cells were exposed for the indicated times to 10 ⁇ M FITC-labeled HS3 (black), -HS3B (blue) or -HM165S3B (red), treated with proteinase K, and analyzed by flow cytometry.
  • FIG. 11 shows aMTD-mediated cell-to-cell delivery.
  • the top (right) panel shows a mixture of double negative cells (cells exposed to FITC-HS3B that did not incorporate the protein) and single positive Cy5.5 labeled cells; whereas, second panel from the left contains FITC-Cy5.5 double-positive cells generated by the transfer of FITC-HM 165 S3B to Cy5.5 labeled cells and the remaining FITC and Cy5.5 single-positive cells.
  • the bottom panels show FITC fluorescence profiles of cell populations before mixing (coded as before) and 1 hr after the same cells were mixed with Cy5.5-labeled cells.
  • FIG. 12 shows the inhibition of STAT Phosphorylation Induced by IFN-y. Inhibition of STAT1 phosphorylation detected by immunoblotting analysis. The levels of phosphorylated STAT1 and STAT3 untreated and treated with IFN-y were compared to the levels in IFN- ⁇ -treated RAW 264.7 cells that were pulsed with 10 ⁇ M of indicated proteins.
  • FIG. 13 shows the inhibition of Cytokines Secretion Induced by LPS. Inhibition of TNF- ⁇ and IL-6 expression by recombinant SOCS3 proteins in primary macrophages isolated from peritoneal exudates of C3H/HeJ mice. Error bars indicate+s.d. of the mean value derived from each assay done in triplicate.
  • FIG. 14 shows the inhibition of proliferation in endothelial cells with iCP-SOCS3.
  • HUVECs were seeded in 96 well plates and then treated with DMEM (vehicle) or SOCS3 recombinant proteins. Cell viability was evaluated with the CellTiter-Glo Cell Viability Assay. *, p ⁇ 0.01
  • FIG. 15 shows the inhibition of migration in endothelial cells with iCP-SOCS3. HUVECs were treated with SOCS3 recombinant proteins for 2 hrs, and cell migration were measured by Transwell assay. *, p ⁇ 0.01
  • FIG. 16 shows the inhibition of tube formation in endothelial cells with iCP-SOCS3.
  • HUVECs were treated with 10 ⁇ M of SOCS3 recombinant protein for 8 hrs and then the trypsinized cells (5 ⁇ 10 4 /well) were seeded on the surface of the Matrigel. Tube formation is monitored after 24 hrs and the images were analyzed using a service provided by Wimasis. The data shown are representative of three independent experiments. *, p ⁇ 0.01.
  • Average length, molecular weight and pl value of the peptides analyzed were 10.8 ⁇ 2.4, 1,011 ⁇ 189.6 and 5.6 ⁇ 0.1, respectively.
  • Bending potential was determined based on the fact whether proline (P) exists and/or where the amino acid(s) providing bending potential to the peptide in recombinant protein is/are located.
  • Proline differs from the other common amino acids in that its side chain is bonded to the backbone nitrogen atom as well as the alpha-carbon atom.
  • the resulting cyclic structure markedly influences protein architecture which is often found in the bends of folded peptide/protein chain. Eleven out of 17 were determined as ‘Bending’ peptide which means that proline should be present in the middle of sequence for peptide bending and/or located at the end of the peptide for protein bending.
  • peptide sequences could penetrate the plasma membrane in a “bent” configuration. Therefore, bending or no-bending potential is considered as one of the critical factors for the improvement of current hydrophobic CPPs.
  • instability index (II) of the sequence was determined.
  • the index value representing rigidity/flexibility of the peptide was extremely varied (8.9-79.1), but average value was 40.1 ⁇ 21.9 which suggested that the peptide should be somehow flexible, but not too rigid or flexible.
  • Alanine (V), valine (V), leucine (L) and isoleucine (I) contain aliphatic side chain and are hydrophobic—that is, they have an aversion to water and like to cluster. These amino acids having hydrophobicity and aliphatic residue enable them to pack together to form compact structure with few holes. Analyzed peptide sequence showed that all composing amino acids were hydrophobic (A, V, L and I) except glycine (G) in only one out of 17 and aliphatic (A, V, L,
  • the CPP sequences may be supposed to penetrate the plasma membrane directly after inserting into the membranes in a “bent” configuration with hydrophobic sequences adopting an a-helical conformation.
  • our analysis strongly indicated that bending potential was crucial. Therefore, structural analysis of the peptides conducted to determine whether the sequence was to form helix or not.
  • Nine peptides were helix and 8 were not. It seems to suggest that helix structure may not be required.
  • Critical Factors for the development of new hydrophobic CPPs—advanced MTDs: i) amino acid length, ii) bending potential (proline presence and location), iii) rigidity/flexibility (instability index: II), iv) structural feature (aliphatic index: AI), v) hydropathy (GRAVY) and vi) amino acid composition/residue structure (hydrophobic and aliphatic A/a) .
  • Amino Acid Composition Hydrophobic and Aliphatic amino acids—A, V, L, I and P
  • All 240 aMTD sequences have been designed and developed based on six critical factors (TABLES 2-1 to 2-6).
  • the aMTD amino sequences are SEQ ID NOS: 1 to 240, and the aMTD nucleotide sequences are SEQ ID NOS: 241 to 480.
  • All 240 aMTDs hydrophobic, flexible, bending, aliphatic and helical 12 a/a-length peptides
  • To determine the cell-permeability of aMTDs and random peptides which do not satisfy one or more critical factors have also been designed and tested.
  • Amino Acid Composition Hydrophobic and Aliphatic amino acids—A, V, L, I and P
  • aMTDs advanced macromolecule transduction domains
  • cell-permeable SOCS3 recombinant proteins have been developed by adopting aMTD165 (TABLE 4) that satisfied all 6 critical factors (TABLE 5).
  • recombinant cargo (SOCS3) proteins fused to hydrophobic CPP could be expressed in bacteria system and purified with single-step affinity chromatography; however, protein dissolved in physiological buffers (e.q. PBS, DMEM or RPMI1640 etc.) was highly insoluble and had extremely low. Therefore, an additional non-functional protein domain (solubilization domain: SD; TABLE 6) has been fused to the recombinant proteins at their C terminus to improve low solubility/yield and to enhance relative cell-/tissue-permeability.
  • physiological buffers e.q. PBS, DMEM or RPMI1640 etc.
  • solubilization domain A SDA
  • SDB solubilization domain B
  • NTD N-terminal domain
  • Histidine-tagged human SOCS3 proteins were designed ( FIG. 1 ) and constructed by amplifying the SOCS3 cDNA (225 amino acids) from nt 4 to 678 using primers [TABLE 7] for SOCS3 cargo fused to aMTD.
  • the PCR products were subcloned with NdeI (5′) and BamHl (3′) into pET-28a(+). Coding sequences for SDA or SDB were fused to the C terminus of his-tagged aMTD-fused SOCS3 and cloned at between the BamHl (5′) and Sall (3′) sites in pET-28a(+) ( FIG. 2 ).
  • PCR primers for SOCS3 and SDA and/or SDB fused to SOCS3 are summarized in TABLES 7, 8 and 9, respectively.
  • the cDNA and amino acid sequences of histidine tag are provided in SEQ ID NO: 481 and 482, and cDNA and amino acid sequences of aMTDs are indicated in SEQ ID NO: 483 and 484, respectively.
  • the cDNA and amino acid sequences are displayed in SEQ ID NO: 485 and 486 (SOC53); SEQ ID NO: 487 and 488 (SDA); and SEQ ID NO: 489 and 450 (SDB), respectively.
  • the SOCS3 recombinant proteins were expressed in E. coli BL21-CodonPlus (DE3) cells, grown to an OD 600 of 0.6 and induced for 3 hrs with 0.6 mM isopropyl- ⁇ -D-thiogalactopyranoside (IPTG).
  • IPTG isopropyl- ⁇ -D-thiogalactopyranoside
  • the proteins were purified by Ni2 + affinity chromatography and dissolved in a physiological buffer such as DMEM medium.
  • the histidine-tagged SOCS3 proteins were expressed, purified, and prepared in soluble form ( FIG. 3 ).
  • the yield of each soluble SOCS3 recombinant proteins was determined by measuring absorbance (A450).
  • SOCS3 recombinant proteins containing aMTD165 and solubilization domain had little tendency to precipitate whereas recombinant SOCS3 proteins lacking a solubilization domain (HS3 and HM 165 S3) were largely insoluble. Solubility of aMTD/SD-fused SOCS3 proteins was scored on a 5 point scale compared with that of SOCS3 proteins lacking the solubilization domain ( FIG. 4 ).
  • Yields per L of E.coli for each recombinant protein ranged from 1 to 47 mg/L ( FIG. 4 ). Yields of SOCS3 proteins containing an aMTD and SDB (HM 165 S3B) were 50% higher than his-tagged SOCS3 protein (HS3).
  • aMTD/SD-Fused SOCS3 Recombinant Proteins Significantly Increase Cell- and Tissue-Permeability 3-1.
  • aMTD/SD-Fused SOC53 Recombinant Proteins Are Cell-Permeable
  • SOCS3 recombinant proteins were conjugated to 5/6-fluorescein isothiocyanate (FITC).
  • RAW 264.7 FIG. 5
  • NIH3T3 cells FIG. 6
  • the cells were washed three times with ice-cold PBS and treated with proteinase K to remove surface-bound proteins, and internalized proteins were measured by flow cytometry ( FIG. 5 ) and visualized by confocal laser scanning microscopy ( FIG. 6 ).
  • SOCS3 proteins containing aMTD165 HM 165 S3, HM 165 S3A and HM 165 S3B
  • SOCS3 recombinant proteins were monitored following intraperitoneal (IP) injections in mice. Tissue distributions of fluorescence-labeled-SOCS3 proteins in different organs was analyzed by fluorescence microscopy ( FIG. 7 ).
  • SOCS3 recombinant proteins fused to aMTD165 HM 165 S3, HM 165 S3A and HM165S3B
  • HM 165 S3, HM 165 S3A and HM165S3B were distributed to a variety of tissues (liver, kidney, spleen, lung, heart and, to a lesser extent, brain).
  • liver showed highest levels of fluorescent cell-permeable SOCS3 since intraperitoneal administration favors the delivery of proteins to this organ via the portal circulation.
  • SOCS3 containing aMTD165 was detectable to a lesser degree in lung, spleen and heart.
  • aMTD/SDB-fused SOCS3 recombinant protein (HM 165 S3B) showed the highest systemic delivery of SOCS3 protein to the tissues comparable to the SOCS3 containing only aMTD (HM 165 S3) or aMTD/SDA (HM 165 S3A) proteins.
  • SOCS3 protein containing both of aMTD165 and SDB leads to higher cell-/tissue-permeability due to the increase in solubility and stability of the protein, and it displayed a dramatic synergic effect on cell-/tissue-permeability.
  • SOCS3 recombinant proteins lacking SD were less soluble, produced lower yields, and showed tendency to precipitate when they were expressed and purified in E. coli. Therefore, we additionally designed ( FIG. 8 ) and constructed SOCS3 recombinant protein containing only SDB (without aMTD165: HS3B) as a negative control. As expected, its solubility and yield increased compared to that of SOCS3 proteins lacking SDB (HS3; FIG. 9 ). Therefore, HS3B proteins were used as a control protein.
  • aMTD/SD-Fused SOCS3 Protein Efficiently Inhibits Cellular Processes 4-1.
  • aMTD/SD-Fused SOCS3 Protein Inhibits the Activation of STATs Induced by INF- ⁇
  • the ultimate test of cell-penetrating efficiency is a determination of intracellular activity of SOCS3 proteins transported by aMTD. Since endogenous SOCS3 are known to block phosphorylation of STAT1 and STAT3 by IFN- ⁇ -mediated JAK1 and JAK2 activation, we demonstrated whether cell-permeable SOCS3 inhibits the phosphorylation of STATs. All SOCS3 recombinant proteins containing aMTD (HM 165 S3, HM 165 S3A and HM 165 S3B), suppressed IFN- ⁇ -induced phosphorylation of STAT1 and STAT3 ( FIG. 12 ). In contrast, STAT phosphorylation was readily detected in cells exposed to HS3, which lacks the aMTD motif required for membrane penetration ( FIG. 12 ), indicating that HS3, which lacks an MTD sequence and did not enter the cells, has no biological activity.
  • SOCS3 recombinant protein containing aMTD and SDB (HM165S3B) is a prototype of a new generation of improved cell-permeable SOCS3 (iCP-SOCS3), and will be selected for further evaluation as a potential anti-angiogenic agent.
  • iCP-SOCS3 Suppresses Pro-Angiogenic Functions in Endothelial Cells 5-1.
  • iCP-SOC53 Inhibits Cell Viability in Endothelial Cells
  • SOCS3 recombinant proteins containing aMTD165 and solubilization domain significantly suppressed cell proliferation—over 60% in 10 ⁇ M treatment (p ⁇ 0.01)—than other proteins, including HS3B or vehicle alone (i.e. exposure of cells to culture media without recombinant proteins) in human umbilical vein endothelial cells (HUVECs).
  • HM 165 S3B solubilization domain
  • HUVECs were treated with 10 ⁇ M SOCS3 recombinant proteins for 2 hrs, and cell migration was measure by Transwell migration assay ( FIG. 15 ).
  • Cell migration was markedly suppressed in HUVECs treated with HM 165 S3B (iCP-SOCS3) protein although SOCS3 protein lacking aMTD165 (HS3B) had no effect on the cell migration.
  • iCP-SOCS3 Protein Inhibits Vasculogenic Activity of Endothelial Cells
  • iCP-SOCS3 To determine whether the morphogenesis of the endothelial cells into capillary tube structures was regulated by iCP-SOCS3 through proteins transduction, endothelial cell tube-forming assays were performed in 24-well Matrigel coated plates. HUVECs were treated with 5 ⁇ M of SOCS3 recombinant protein for 8 hrs and tube-formation was monitored after 24 hrs. As a result, HUVECs treated with HM 165 S3B proteins significantly suppressed the tube formation compared with negative controls including HS3B or vehicle ( FIG. 16 ). Taken together, these results suggest that iCP-SOCS3 directly plays an important role in the inhibition of angiogenesis and may provide the way for the development of novel therapies aimed at reducing the angiogenesis.
  • 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.
  • 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).
  • 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 rigidity/flexibility II: 40-60
  • GRAVY grand average of hydropathy
  • Al aliphatic index
  • new hydrophobic peptide sequences namely advanced macromolecule transduction domain peptides (aMTDs)
  • aMTDs advanced macromolecule transduction domain peptides
  • 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.
  • 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.
  • 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).
  • 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
  • a physiological buffer such as DMEM medium.
  • recombinant SOCS3 proteins were conjugated to 5/6-fluorescein isothiocyanate (FITC) according to the manufacturer's instructions (Sigma-Aldrich, St. Louis, Mo.).
  • FITC 5/6-fluorescein isothiocyanate
  • RAW 264.7 cells were treated with 10 ⁇ M FITC-labeled recombinant proteins for 1 hr at 37° C., washed three times with cold PBS, and treated with proteinase K (10 ⁇ g/mL) for 20 min at 37° C. to remove cell-surface bound proteins.
  • Cell-permeability of these recombinant proteins was analyzed by flow cytometry (Guava, Millipore, Darmstadt, Germany) using the Flowio cytometric analysis software.
  • NIH3T3 cells were cultured on coverslips in 24-well plates and with 10 ⁇ M FITC-conjugated recombinant proteins for 1 hr at 37° C. These cells on coverslips were washed with PBS, fixed with 4% formaldehyde for 10 min, and washed three times with PBS at room temperature. Coverslips were mounted with VECTASHIELD Mounting Medium (Vector laboratories, Burlingame, Calif.) with DAPI (4′,6-diamidino-2-phenylindole) for nuclear staining. Intracellular localization of fluorescent signal was determined by confocal laser scanning microscopy (LM700, Zeiss, Germany).
  • ICR mice (6-week-old, female) were injected intraperitoneally (600 ⁇ g/head) with either FITC only or FITC-conjugated SOCS3 recombinant proteins. After 2 hrs, the liver, kidney, spleen, lung, heart, and brain were isolated, washed with an O.C.T. compound (Sakura), and frozen on dry ice. Cryosections (20 ⁇ m) were analyzed by fluorescence microscopy (Carl Zeiss, Gottingen, Germany).
  • RAW264.7 cells were pretreated with different agents to assess the effect of various conditions on protein uptake: (i) 5 ⁇ g/ml proteinase K for 10 min, (ii) 20 ⁇ M Taxol for 30 min, (iii) 10 ⁇ M antimycin in the presence or absence of 1 mM ATP for 2 hrs, (iv) incubation on ice (or maintained at 37 ° C.) for 60 min, and (v) 100 mM EDTA for 3 hrs. These agents were used at concentrations known to be active in other applications.
  • the cells were then incubated with 10 ⁇ M FITC-labeled proteins for 1 hr at 37° C., washed three times with ice-cold phosphate-buffered saline, treated with proteinase K (10 ⁇ g/ml for 5 min at 37° C.) to remove cell-surface bound proteins, and analyzed by flow cytometry.
  • FITC-labeled proteins 10 ⁇ M FITC-labeled proteins for 1 hr at 37° C.
  • proteinase K 10 ⁇ g/ml for 5 min at 37° C.
  • PANC-1 cells (Korean Cell Line Bank, Seoul, Korea) were cultured in modified Eagle's medium (DMEM; Welgene, Daege, Korea) supplemented with 10% (v/v) FBS, penicillin (100 units/ml), and streptomycin (10 ⁇ g/ml, Gibco BRL) and pretreated with 10 ⁇ M of SOCS3 recombinant proteins for 2 hrs followed by exposing the cells to agonists (100 ng/ml IFN- ⁇ ) for 15 min.
  • DMEM modified Eagle's medium
  • FBS penicillin
  • streptomycin 10 ⁇ M of SOCS3 recombinant proteins
  • RIPA lysis buffer 50 mM Tris pH 8.0, 150 mM NaCl, 1% Nonidet P-40, 0.1% SDS, 0.5% sodium deoxycholate, 10 mM NaF, and 2 mM Na3VO4
  • RIPA lysis buffer 50 mM Tris pH 8.0, 150 mM NaCl, 1% Nonidet P-40, 0.1% SDS, 0.5% sodium deoxycholate, 10 mM NaF, and 2 mM Na3VO4
  • Equal amounts of lysates were resolved by SDS-PAGE, transferred onto PVDF membranes, and probed with phospho (pY701)-specific STAT1 (Cell Signaling, Danvers, Mass.).
  • Cytokine Measurement Cytometric Bead Array (CBA) Assay
  • Peritoneal macrophages were obtained from C3H/HeJ mice. Peritoneal macrophages were incubated with 10 ⁇ M recombinant proteins (1:HS3, 2:HM 165 S3, 3:HM 165 S3A and 4:HM 165 S3B, respectively) for 1 hr at 37° C. and then stimulated them with LPS (500 ng/ml) and/or IFN- ⁇ (100 U/ml) without removing iCP-SOCS3 proteins for 3, 6, or 9 hrs. The culture media were collected, and the extracellular levels of cytokine were measured by a cytometric bead array (BD Biosciences, Pharmingen) according to the manufacturer's instructions.
  • a cytometric bead array BD Biosciences, Pharmingen
  • HUVECs were purchased (ATCC, Manassas, Va.) and maintained as recommended by the supplier. These cells (3 ⁇ 10 3 /well) were seeded in 96 well plates. The next day, cells were treated with DMEM (vehicle) or recombinant SOCS3 proteins for 96 hrs in the presence of serum (2%). Proteins were replaced daily. Cell growth and survival were evaluated with the CellTiter-Glo Cell Viability Assay (Promega, Madison, Wis.). Measurements using a Luminometer (Turner Designs, Sunnyvale, Calif.) were conducted following the manufacturer's protocol.
  • DMEM vehicle
  • 10 ⁇ M SOCS3 recombinant proteins lysed in RIPA lysis buffer containing proteinase inhibitor cocktail, incubated for 15 min at 4° C., and centrifuged at 13,000 rpm for 10 min at 4° C.
  • Equal amounts of lysates were separated on 15% SDS-PAGE gels and transferred to a nitrocellulose membrane.
  • the membranes were blocked using 5% skim milk or 5% Albumin in TBST and incubated with the following antibodies: anti-Bcl-2 (Santa Cruz biotechnology) and anti-Cleaved Caspase 3 (Cell Signaling Technology), then HRP conjugated anti-mouse or anti-rabbit secondary antibody.
  • Transwell inserts (Costar) was coated with gelatin (10 ⁇ g/ml), and the membranes were allowed to dry for 1 hr at room temperature.
  • the Transwell inserts were assembled into a 24-well plate, and the lower chamber was filled with growth media containing 10% FBS and FGF2 (10 ⁇ g/ml).
  • Cells (5 ⁇ 10 5 ) were added to each upper chamber, and the plate was incubated at 37° C. in a 5% CO2 incubator for 24 hrs. Migrated cells were stained with 0.6% hematoxylin and 0.5% eosin and counted.
  • Endothelial cell tube-forming assays are performed in 24-well Matrigel (BD Bioscience) coated plates.
  • Human umbilical-vein endothelial cells (HUVECs) are treated with 10 ⁇ M of recombinant SOCS3 protein for 8 hrs and then the trypsinized cells were seeded on the surface of the Matrigel (5 ⁇ 10 4 /well). Tube formation is monitored after 24 hrs and the images were analyzed using a service provided by Wimasis.

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US20180051060A1 (en) 2018-02-22
US20170190754A1 (en) 2017-07-06
US20180237485A1 (en) 2018-08-23
US20160060310A1 (en) 2016-03-03
EP3341396A4 (fr) 2019-03-06
US20160060312A1 (en) 2016-03-03
WO2017034349A1 (fr) 2017-03-02
US10385103B2 (en) 2019-08-20
US10961292B2 (en) 2021-03-30
US10774123B2 (en) 2020-09-15
US20170226168A1 (en) 2017-08-10
US10975132B2 (en) 2021-04-13
US20160060311A1 (en) 2016-03-03
EP3341394B1 (fr) 2021-07-28
EP3341395A4 (fr) 2018-08-08
US10781241B2 (en) 2020-09-22
WO2017034330A1 (fr) 2017-03-02
US20170137482A1 (en) 2017-05-18
US20160060314A1 (en) 2016-03-03
WO2017034335A1 (fr) 2017-03-02
US20160060319A1 (en) 2016-03-03
EP3341394A4 (fr) 2019-01-09
EP3341396A1 (fr) 2018-07-04
US10787492B2 (en) 2020-09-29
US20200299348A1 (en) 2020-09-24
EP3341396B1 (fr) 2021-04-07
EP3341400A1 (fr) 2018-07-04
WO2017034344A1 (fr) 2017-03-02
WO2017034347A1 (fr) 2017-03-02
US20170198019A1 (en) 2017-07-13
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EP3341395B1 (fr) 2023-11-29
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EP3341395A1 (fr) 2018-07-04
US11279743B2 (en) 2022-03-22
US20190359669A1 (en) 2019-11-28
EP3341394A1 (fr) 2018-07-04

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