US20160060314A1 - 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 - Google Patents

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 Download PDF

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US20160060314A1
US20160060314A1 US14/838,304 US201514838304A US2016060314A1 US 20160060314 A1 US20160060314 A1 US 20160060314A1 US 201514838304 A US201514838304 A US 201514838304A US 2016060314 A1 US2016060314 A1 US 2016060314A1
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socs3
aliphatic
proteins
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Daewoong Jo
Young Sil CHOI
Seul Mee SHIN
Ju Hyun Nam
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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, NAM, JU HYUN, SHIN, SEUL MEE
Publication of US20160060314A1 publication Critical patent/US20160060314A1/en
Priority to PCT/KR2016/009414 priority patent/WO2017034333A1/fr
Priority to EP16839621.6A priority patent/EP3341394B1/fr
Priority to US15/361,701 priority patent/US20170137482A1/en
Priority to US16/426,751 priority patent/US10961292B2/en
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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 (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.
  • iCP-SOCS3 cell-permeable SOCS3
  • the Janus kinase signal transducers and activators of transcription signaling plays important roles in immune responses, including oncogenesis. So many investigations demonstrated that STAT-3, an important member of STAT proteins, was considered as a protooncogene in various types of disorder. STAT-3 is phosphorylated and dimerizes by the Janus kinase (JAK), and its overexpression and constitutive activation can significantly induce cell proliferation, tumor angiogenesis, invasion. Meanwhile, inhibition of JAK-STAT signaling led to suppress the cancer cell growth and induce apoptosis.
  • SOCS3 cytokine signaling-3
  • Gastric cancer remains the second leading cause of cancer-related death in the world. Advances in early detection and decreased chronic Helicobacter pylori infection rates have led to a substantial reduction in gastric cancer rates worldwide. However, effective treatment regimens for gastric cancers, especially advanced gastric cancer, are still lacking; therefore, the prognosis of patients with this disease remains poor.
  • SOCS3 mRNA levels are higher in adjacent normal mucosal tissues, however, gastric cancer patients with high simultaneous expression of SOCS3 have a better overall survival than those with low simultaneous expression. Based on this, SOCS3 may represent new therapeutic target to treat gastric cancer.
  • 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. It has been reported as being relevant to some inflammatory bowel diseases, such as Crohn's disease and ulcerative colitis.
  • the pathogenesis of colorectal carcinoma is complex, with the involvement of multiple cellular transduction pathways including IL-6/STAT3 signaling.
  • Reduced or silenced SOCS3 has been found in many human types of cancer including colorectal cancer, and restoring SOCS3 expression in the cancer cells inhibits IL-6-mediated STAT3 activation, induces tumor cell apoptosis and decreases cell proliferation. Therefore, suppression of the IL-6/STAT3 pathway via modulation of SOCS3 has been a promising strategy for anti-colon/colorectal cancer therapy.
  • Glioblastoma the most common neoplasm among diffuse infiltrating astrocytomas, is notorious for its ability to evade immune-surveillance as well as for its invasive and angiogenic properties. 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. A hallmark of this lethal disorder is the presence of activated STAT3. Because SOCS3 is a negative regulator of STAT-3 activation, it hypothesized that SOCS3 may function as a tumor suppressor in glioblastoma tissues.
  • Breast cancer is a disease that arises from the accumulation of alterations in the genome of cells that make up the mammary gland.
  • Breast cancer is the most common type of cancer among women, with an estimated 1.38 million new cases of cancer diagnosed in 2008 (23% of all cancers), and the second most common type of cancer overall (10.9% of all cancers).
  • Expression of SOCS3 protein is significantly down-regulated in breast cancer specimens and replacing of SOCS3 protein may directly influence the treatment of breast cancer.
  • 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. Both promote anti-inflammaory effects due to the suppression of inflammation-inducing cytokine signaling.
  • KIR N-terminal kinase inhibitory region
  • JAKs janus kinases
  • 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 kinase activity and degrading the activated cytokine receptor complex.
  • the SOCS1 gene has been implicated as an anti-oncogene in the tumor development.
  • Previous studies have reported that aberrant methylation in the CpG island of SOCS1 induces its transcriptional silencing in cancer cell lines, and SOCS1 heterozygous mice are hypersensitive to various cancers.
  • abnormalities of SOCS3 are also associated with the solid tumors. Hypermethylation of CpG islands in the SOCS3 promoter is correlated with its transcriptional silencing in tumors cell lines.
  • SOCS3 overexpression down-regulates active STAT3, induces apoptosis, and suppresses growth in cancer 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 in their solubility and manufacturing yield when expressed and purified from bacteria cells.
  • aMTD/SD-fused iCP-SOCS3 proteins iCP-SOCS3 proteins
  • iCP-SOCS3 proteins much improved physicochemical characteristics (solubility & yield) and functional activity (cell-/tissue-permeability) compared with 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 the tumors, 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 aMTD may provide novel protein therapy through SOCS3-intracellular protein replacement against the various cancer cells.
  • MITT macromolecule intracellular transduction technology
  • An aspect of the present invention relates to improved cell-permeable SOCS3 (iCP-SOCS3) capable of mediating the transduction of biologically active macromolecules into live cells.
  • aMTDs advanced macromolecule transduction domains
  • iCP-SOCS3 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-tumor effect in solid tumors. 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 -HM 165 S3B (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 -HM 165 S3B (red), treated with proteinase K, and analyzed by flow cytometry.
  • FIG. 11 shows the 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- ⁇ . Inhibition of STAT1 phosphorylation detected by immunoblotting analysis. The levels of phosphorylated STAT1 and STAT3 untreated and treated with IFN- ⁇ 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 cell-permeability of iCP-SOCS3 (HM 165 S3B) in various cancer cells.
  • RAW264.7 cells were exposed to FITC-labeled SOCS3 recombinant proteins (10 ⁇ M) for 1 hr, treated with proteinase K to remove cell-associated proteins 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.
  • FIG. 15 shows the tissue distribution of iCP-SOCS3 (HM 165 S3B) into various tissues.
  • 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. 16 shows the inhibition of proliferation in gastric cancer cells with iCP-SOCS3.
  • Gastric cancer cells AGS, MKN75, MKN45, NCI-N87
  • DMEM fetal calf serum
  • HM 165 S3A HM 165 S3A
  • HM 165 S3B recombinant proteins for 96 h in the presence of serum (2%).
  • Cell viability was evaluated with the CellTiter-Glo Cell Viability Assay.
  • FIG. 17 shows the inhibition of proliferation in colorectal cancer cells with iCP-SOCS3.
  • HCT 116 cell was seeded in 96 well plates. Next day, cells were treated with DMEM (V), HS3 (1), HM 165 S3 (2), HM 165 S3A (3) or HM 165 S3B (4) recombinant proteins for 96 h in the presence of serum (2%). Cell viability was evaluated with the CellTiter-Glo Cell Viability Assay.
  • FIG. 18 shows the inhibition of proliferation in glioblastoma cells with iCP-SOCS3.
  • U-87 MG cell was seeded in 96 well plates. Next day, cells were treated with DMEM (V), HS3 (1), HM 165 S3 (2), HM 165 S3A (3) or HM 165 S3B (4) recombinant proteins for 96 h in the presence of serum (2%). Cell viability was evaluated with the CellTiter-Glo Cell Viability Assay.
  • FIG. 19 shows the inhibition of proliferation in breast cancer cells with iCP-SOCS3.
  • MDA-MB-231 cell was seeded in 96 well plates. Next day, cells were treated with DMEM (V), HS3 (1), HM 165 S3 (2), HM 165 S3A (3) or HM 165 S3B (4) recombinant proteins for 96 h in the presence of serum (2%). Cell viability was evaluated with the CellTiter-Glo Cell Viability Assay.
  • FIG. 20 shows the induction of apoptosis in colorectal cancer cells with iCP-SOCS3.
  • HCT116 cells were treated for 24 hr with 10 ⁇ M HS3B or HM 165 S3B proteins and apoptotic cells were visualized by TUNEL staining.
  • FIG. 21 shows the stimulation of apoptosis in gastric cancer cells with iCP-SOCS3.
  • AGS cells were treated for 24 hr with 10 ⁇ M HS3B or HM 165 S3B proteins and analyzed by flow cytometry of cells stained with annexin-V and 7-AAD.
  • FIG. 22 shows the stimulation of apoptosis in colorectal cancer cells with iCP-SOCS3.
  • HCT116 cells were treated for 24 hr with 10 ⁇ M HS3B or HM 165 S3B proteins and analyzed by flow cytometry of cells stained with annexin-V and 7-AAD.
  • FIG. 23 shows the stimulation of apoptosis in glioblastoma cells with iCP-SOCS3.
  • U-87 MG cells were treated for 24 hr with 10 ⁇ M HS3B or HM 165 S3B proteins and analyzed by flow cytometry of cells stained with annexin-V and 7-AAD.
  • FIG. 24 shows the stimulation of apoptosis in breast cancer cells with iCP-SOCS3.
  • MDA-MB-231 cells were treated for 24 hr with 10 M HS3B or HM 165 S3B proteins and analyzed by flow cytometry of cells stained with annexin-V and 7-AAD.
  • FIG. 25 shows the expression of biomarker genes associated to apoptosis in breast cancer cells with iCP-SOCS3.
  • MDA-MB-231 cells were treated for 24 hr with 10 ⁇ M HS3B or HM 165 S3B proteins and lysed. The expression of each protein was determined by immunoblotting with indicated antibodies. An antibody against ⁇ -actin was used as a loading control.
  • FIG. 26 shows the inhibition of migration in gastric cancer cells with iCP-SOCS3.
  • AGS cells were grown to 100% confluence and these procedures were performed on wound-healing assays The wound areas were examined and photographed at 0 and 24 hrs post-wounding.
  • FIG. 27 shows the inhibition of migration in colorectal cancer cells with iCP-SOCS3.
  • HCT116 cells were grown to 100% confluence and these procedures were performed on wound-healing assays The wound areas were examined and photographed at 0 and 48 hrs post-wounding.
  • FIG. 28 shows the inhibition of migration in glioblastoma cells with iCP-SOCS3.
  • U-87 MG cells were grown to 100% confluence and these procedures were performed on wound-healing assays The wound areas were examined and photographed at 0 and 72 hrs post-wounding.
  • FIG. 29 shows the inhibition of migration in breast cancer cells with iCP-SOCS3.
  • MDA-MB-231 cells were grown to 100% confluence and these procedures were performed on wound-healing assays The wound areas were examined and photographed at 0 and 24 hrs post-wounding.
  • FIG. 30 shows the inhibition of migration/invasion in gastric cancer cells with iCP-SOCS3.
  • AGS cells were treated with SOCS3 recombinant proteins for 24 hrs, and migration/invasion were measured by Transwell assay. The data shown are representative of three independent experiments. **, p ⁇ 0.01.
  • FIG. 31 shows the external appearance of gastric tumor bearing mice.
  • Female Balb/c nu/nu mice were subcutaneously implanted with NCI-N87 tumor block (1 mm 3 ) into the left side of the back. After tumors reached a size of 50-80 mm 3 (start), the mice were injected daily (I.V.) for 3 w with diluent alone (black) or with HS3B (blue) or HM 165 S3B (iCP-SOCS3, red) and observed for 2 w following the termination of the treatment. Representative mice treated with diluent alone or with SOCS3 proteins were photographed on day 0 and 35 after starting protein therapy.
  • FIG. 32 shows the suppression of subcutaneously implanted gastric cancer with iCP-SOCS3.
  • Female Balb/c nu/nu mice were subcutaneously implanted with NCI-N87 tumor block (1 mm 3 ) into the left side of the back. After tumors reached a size of 50-80 mm 3 (start), the mice were injected daily (I.V.) for 3 w with diluent alone (black) or with HS3B (blue) or HM 165 S3B (iCP-SOCS3, red) and observed for 2 w following the termination of the treatment. Tumor weight (left) and volume (right) were measured in the indicated day.
  • FIG. 33 shows the differential expression of biomarkers in gastric cancer with iCP-SOCS3.
  • the expression of each protein was determined by immunoblotting with anti-p21, Bax, cleaved caspase-3, and CD31 antibodies in protein-treated tumors at day 35.
  • An antibody against ⁇ -actin was used as a loading control.
  • Tumor tissues from mice treated daily for 3 w with indicated proteins and observed for 2 w following the termination of the treatment were sectioned and immunostained with antibodies against p21, Bax, cleaved caspased-3, and VEGF.
  • FIG. 34 shows the external appearance of colorectal cancer bearing mice.
  • Female Balb/c nu/nu mice were subcutaneously implanted with HCT116 tumor block (1 mm 3 ) into the left side of the back. After tumors reached a size of 50-80 mm 3 (start), the mice were injected daily (I.V.) for 3 w with diluent alone (black) or with HS3B (blue) or HM 165 S3B (iCP-SOCS3, red) and observed for 2 w following the termination of the treatment. Representative mice treated with diluent alone or with SOCS3 proteins were photographed on day 0 and 35 after starting protein therapy.
  • FIG. 35 shows the suppression of subcutaneously implanted colorectal cancer with iCP-SOCS3.
  • Female Balb/c nu/nu mice were subcutaneously implanted with HCT116 tumor block (1 mm 3 ) into the left side of the back. After tumors reached a size of 50-80 mm 3 (start), the mice were injected daily (I.V.) for 3 w with diluent alone (black) or with HS3B (blue) or HM 165 S3B (iCP-SOCS3, red) and observed for 2 w following the termination of the treatment. Tumor weight (left) and volume (right) were measured in the indicated day.
  • FIG. 36 shows the differential expression of biomarkers in colorectal cancer with iCP-SOCS3.
  • the expression of each protein was determined by immunoblotting with anti-p21 and CD31 antibodies in protein-treated tumors at day 35.
  • An antibody against ⁇ -actin was used as a loading control.
  • Tumor tissues from mice treated daily for 3 w with indicated proteins and observed for 2 w following the termination of the treatment were sectioned and immunostained with antibodies against p21, Bax, and cleaved caspased-3.
  • FIG. 37 shows the suppression of subcutaneously implanted glioblastoma with iCP-SOCS3.
  • Female Balb/c nu/nu mice were subcutaneously implanted with U-87 MG tumor block (1 mm 3 ) into the left side of the back. After tumors reached a size of 50-80 mm 3 (start), the mice were injected daily (I.V.) for 3 w with diluent alone (black) or with HS3B (blue) or HM 165 S3B (iCP-SOCS3, red) and observed for 2 w following the termination of the treatment. Tumor weight (left) and volume (right) were measured in the indicated day.
  • Average length, molecular weight and pI 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, I, and P). Their hydropathic index (Grand Average of Hydropathy: GRAVY) and aliphatic index (AI) were 2.5 ⁇ 0.4 and 217.9 ⁇ 43.6, respectively.
  • 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 ⁇ -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).
  • 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.
  • 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 BamHI (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 BamHI (5′) and SalI (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 NOs: 483 and 484, respectively.
  • the cDNA and amino acid sequences are displayed in SEQ ID NOs: 485 and 486 (SOCS3); SEQ ID NOs: 487 and 488 (SDA); and SEQ ID NOs: 489 and 490 (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 SOCS3 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 HM 165 S3B
  • HM 165 S3, HM 165 S3A and HM 165 S3B 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.
  • aMTD165-mediated intracellular delivery was occurred.
  • the aMTD-mediated intracellular delivery of SOCS3 protein did not require protease-sensitive protein domains displayed on the cell surface ( FIG. 10B ), microtubule function ( FIG. 10C ), or ATP utilization ( FIG. 10D ), since aMTD165-dependent uptake [compare to HS3 (black) and HS3B (blue)] was essentially unaffected by treating cells with proteinase K, taxol, or the ATP depleting agent, antimycin.
  • aMTD165-fused SOCS3 proteins uptake was blocked by treatment with EDTA and low temperature ( FIGS. 10A and E), indicating the importance of membrane integrity and fluidity for aMTD-mediated protein transduction.
  • 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 Janus kinases (JAK) 1 and 2 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.
  • HM 165 S3, HM 165 S3A and HM 165 S3B suppressed
  • SOCS3 recombinant protein containing aMTD and SDB (HM 165 S3B) 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-tumor agent.
  • iCP-SOCS3 Suppresses Pro-Tumorigenic Functions in Solid Cancer Cells 5-1. iCP-SOCS3 Enhances the Cellular Uptake into Various Cancer Cells and Systemic Delivery to Various Tissues
  • solid tumor is one of the most cancers with a high mortality rate, there are few drugs for treating this lethal disorder. Since constitutive activation of STAT3 is found in various types of tumors and SOCS3 is closely related to the development of various solid tumors including gastric, colorectal and breast cancer, and glioblastoma, we first chose the various tumors as a primary indication of the iCP-SOCS3 as an anti-cancer agent.
  • FITC-HM 165 S3B recombinant protein FITC-HM 165 S3B recombinant protein (iCP-SOCS3) promoted the transduction into cultured cancer cells ( FIG. 14 ).
  • iCP-SOCS3 proteins enhanced the systemic delivery to liver after intraperitoneal injection ( FIG. 15 ). Therefore, these data indicate that iCP-SOCS3 protein could be intracellularly delivered and distributed to the various cells and tissue, contributing for beneficial biotherapeutic effects.
  • SOCS3 Since the endogenous level of SOCS3 protein is reduced in solid tumor—gastric, colorectal and breast cancer, and glioblastoma—patients, and SOCS3 negatively regulates cell growth and motility in cultured tumor cells, we investigated whether iCP-SOCS3 inhibits cell viability through SOCS3 intracellular delivery in solid tumor cells. As shown in FIG. 16-19 , SOCS3 recombinant proteins containing aMTD165 significantly suppressed gastric, colorectal and breast cancer, and glioblastoma cell proliferation. HM 165 S3B (iCP-SOCS3) protein was the most cytotoxic to various solid tumor cells—over 80% in 10 ⁇ M treatment (p ⁇ 0.01)—especially compared to vehicle alone (i.e.
  • HM 165 S3B protein (iCP-SOCS3) was a considerably efficient inducer of apoptosis in HCT116 cells, as assessed either by a fluorescent terminal dUTP nick-end labeling (TUNEL) assay ( FIG. 20 ) and Annexin V staining ( FIGS. 21-24 ). Consistently, no changes in TUNEL and Annexin V staining were observed in colorectal cancer cells, HCT116, treated with HS3B compared to untreated cell (Vehicle).
  • TUNEL fluorescent terminal dUTP nick-end labeling
  • iCP-SOCS3 HM 165 S3B protein
  • Bcl-2 B-cell lymphoma 2
  • caspase-3 cleaved cysteine-aspartic acid protease
  • iCP-SOCS3 Inhibits Migration/Invasion of Gastric, Colorectal and Breast Cancer, and Glioblastoma
  • iCP-SOCS3 iCP-SOCS3 to influence cell migration to various cancer cells, such as gastric (AGS), colorectal (HCT116) and breast cancer (MDA-MB-231), and glioblastoma (U-87 MG) cells.
  • AGS gastric
  • HCT116 colorectal
  • MDA-MB-231 breast cancer
  • U-87 MG glioblastoma
  • cancer cells treated with HM 165 S3B recombinant protein also showed significant inhibitory effect on their Transwell migration compared with untreated cells (Vehicle) and non-permeable SOCS3 protein-treated cells (HS3B; FIG. 30 ).
  • gastric cancer cells, AGS treated with HM 165 S3B recombinant protein (iCP-SOCS3) caused remarkable decrease in invasion compared with the control proteins (HS3B; FIG. 30 ).
  • iCP-SOCS3 Suppresses Pro-Tumorigenic Functions in Various Cancer Cells 6-1.
  • iCP-SOCS3 Suppresses the Gastric and Colorectal Cancer, and Glioblastoma Xenograft
  • mice were subcutaneously implanted with tumor block (1 mm 3 ) of tumor cells into the left side of the back.
  • Tumor-bearing mice were intravenously administered HM 165 S3B or control proteins (HS3B; 600 ⁇ g/head, respectively) for 21 days and observed for 2 weeks following the termination of the treatment ( FIGS. 31 , 34 and 37 ).
  • HM 165 S3B protein significantly suppressed the tumor growth (p ⁇ 0.05) during the treatment and the effect persisted for at least 2 weeks after the treatment was terminated (65% inhibition in the gastric cancer xenograft, 79% inhibition in the colorectal cancer xenograft at day 35, 78% inhibition in the glioblastoma xenograft at day 42, respectively).
  • the growth of HS3B-treated tumors increased, matching the rates observed in control mice (Vehicle; FIGS. 31 , 32 , 34 , 35 and 37 ).
  • iCP-SOCS3 Regulates the Expression of Tumor-Associated Markers in Human Tumor Xenograft
  • HM 165 S3B The anti-tumor activity of HM 165 S3B at day 35 was accompanied by changes in the expression of biomarkers linked to SOCS3 signaling, including p21, Bax, cleaved caspase-3, CD31, and VEGF ( FIGS. 33 and 36 ).
  • Expression of tumor suppressors (p21, Bax, and cleaved caspase-3) was dramatically enhanced in tumor tissues treated with HM 165 S3B recombinant protein ( FIGS. 33 and 36 ), suggesting that iCP-SOCS3 inhibits tumor growth by regulating tumor-specific protein expression in vivo.
  • VEGF vascular endothelial growth factor
  • CD31 a pro-angiogenic factor
  • iCP-SOCS3 targets tumor cells directly and may be developed for use as novel therapy against various solid tumors including gastric, colorectal and breast cancer, and glioblastoma.
  • 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
  • AI 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, 1 ⁇ 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 6 ⁇ His expression vector, pET-28a(+) (Novagen). Coding sequence for SDA or SDB fused to C terminus of his-tagged aMTD-SOCS3 was cloned at BamHI (5′) and SalI (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
  • Apoptotic cells were analyzed using terminal dUTP nick-end labeling (TUNEL) assay with In Situ Cell Death Detection kit TMR red (Roche, 4056 Basel, Switzerland).
  • Cells were treated with either 10 ⁇ M SOCS3 recombinant protein or buffer alone for 16 hrs with 2% fetal bovine serum.
  • Treated cells were washed with cold PBS two times, fixed in 4% paraformaldehyde (PFA, Junsei, Tokyo, Japan) for 1 hr at room temperature, and incubated in 0.1% Triton X-100 for 2 min on the ice.
  • Cells were washed with cold PBS twice, and treated TUNEL reaction mixture for 1 hr at 37° C. in dark, washed cold PBS three times and observed by fluorescence microscopy (Nikon, Tokyo, Japan).
  • Annexin V/7-Aminoactinomycin D (7-AAD) staining was performed using flow cytometry according to the manufacturer's guidelines. Briefly, 1 ⁇ 10 6 cells were washed three times with ice-cold PBS. The cells were then resuspended in 100 ⁇ l of binding buffer and incubated with 1 ⁇ l of 7-AAD and 1 ⁇ l of annexin V-PE for 30 min in the dark at 37° C. Flow cytometric analysis was immediately performed using a guava easyCyteTM 8 Instrument (Merck Millipore).
  • 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.
  • Cells were seeded into 12-well plates, grown to 90% confluence, and incubated with 10 ⁇ M HS3, HM 165 S3A, HM 165 S3A or HM 165 S3B in serum-free medium for 2 hrs prior to changing the growth medium.
  • the cells were washed twice with PBS, and the monolayer at the center of the well was “wounded” by scraping with a pipette tip.
  • Cells were cultured for an additional 72 hrs and cell migration was observed by phase contrast microscopy. The migration is quantified by counting the number of cells that migrated from the wound edge into the clear area.
  • 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.
  • Transwell inserts The lower surface of Transwell inserts (Costar) was coated with gelatin (10 ⁇ g/ml), the upper surface of Transwell inserts was coated with matrigel (40 ⁇ g per well; BD Biosciences), 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.
  • mice Female Balb/c nu/nu mice were subcutaneously implanted with NCI-N87, HCT116 or U-87 MG tumor block (1 mm 3 ) into the left back side of the mouse. Tumor-bearing mice were intravenously administered with iCP-SOCS3 or the control proteins (600 ⁇ g/head) for 21 days and observed for 2 weeks following the termination of the treatment. Tumor size was monitored by measuring the longest (length) and shortest dimensions (width) once a day with a dial caliper, and tumor volume was calculated as width 2 ⁇ length ⁇ 0.5.
  • mice After protein treatment, mice were killed, and six organs (brain, heart, lung, liver, kidney, and spleen) from each were collected and kept in a suitable fixation solution until the next step.
  • organs brain, heart, lung, liver, kidney, and spleen
  • Tissue samples were fixed in 4% Paraformaldehyde (Duksan) for 3 days, dehydrated, cleared with xylene and embedded in Paraplast. Sections (6 ⁇ m thick) of tumor were placed onto poly-L-lysine coated slides. To block endogenous peroxidase activity, sections were incubated for 15 min with 3% H 2 O 2 in methanol. After washing three times with PBS, slides were incubated for 30 min with blocking solution (5% fetal bovine serum in PBS).
  • blocking solution 5% fetal bovine serum in PBS
  • Rabbit anti-p21 antibody (sc-397, SantaCruz), mouse anti-Bax antibody (sc-7480, SantaCruz) and rabbit anti-VEGF (ab46154, abcam) were diluted 1:1000 (to protein concentration 0.1 ⁇ g/ml) in blocking solution, applied to sections, and incubated at 4° C. for 24 hrs. After washing three times with PBS, sections were incubated with biotinylated mouse and rabbit IgG (Vector Laboratories) at a 1:1000 dilution for 1 hr at room temperature, then incubated with avidin-biotinylated peroxidase complex using a Vectorstain ABC Kit (Vector Laboratories) for 30 min at room temperature.

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  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
US14/838,304 2014-08-27 2015-08-27 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 Abandoned US20160060314A1 (en)

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US14/838,304 US20160060314A1 (en) 2014-08-27 2015-08-27 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
PCT/KR2016/009414 WO2017034333A1 (fr) 2014-08-27 2016-08-25 Protéine recombinée socs3 à perméabilité cellulaire améliorée (icp) et ses utilisations
EP16839621.6A EP3341394B1 (fr) 2014-08-27 2016-08-25 Protéine recombinée socs3 à perméabilité cellulaire améliorée (icp) et ses utilisations
US15/361,701 US20170137482A1 (en) 2014-08-27 2016-11-28 Cell-permeable (icp)-socs3 recombinant protein and uses thereof
US16/426,751 US10961292B2 (en) 2014-08-27 2019-05-30 Cell-permeable (ICP)-SOCS3 recombinant protein and uses thereof

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US14/838,304 Abandoned US20160060314A1 (en) 2014-08-27 2015-08-27 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
US14/838,288 Abandoned US20160060312A1 (en) 2014-08-27 2015-08-27 Development of Protein-Based Biotherapeutics That Penetrates Cell-Membrane and Induces Anti-Pancreatic Cancer Effect - Improved Cell-Permeable Suppressor of Cytokine Signaling (iCP-SOCS3) Proteins, Polynucleotides Encoding the Same, and Anti-Pancreatic Cancer Compositions Comprising the Same
US14/838,318 Abandoned US20160060319A1 (en) 2014-08-27 2015-08-27 Development of Protein-Based Biotherapeutics That Induced Osteogenesis for Bone Healing Therapy: Cell-Permeable BMP2 and BMP7 Recombinant Proteins (CP-BMP2 & CP-BMP7), Polynucleotides Encoding the Same and Pro-osteogenic Compositions Comprising the Same
US14/838,260 Abandoned US20160060310A1 (en) 2014-08-27 2015-08-27 Development of Protein-Based Biotherapeutics That Penetrates Cell-Membrane and Induces Anti-Hepatocellular Carcinoma Effect - Improved Cell-Permeable Suppressor of Cytokine Signaling (iCP-SOCS3) Proteins, Polynucleotides Encoding the Same, and Anti-Hepatocellular Carcinoma Compositions Comprising the Same
US14/838,280 Abandoned US20160060311A1 (en) 2014-08-27 2015-08-27 Development of Protein-Based Biotherapeutics That Penetrates Cell-Membrane and Induces Anti-Lung Cancer Effect - Improved Cell-Permeable Suppressor of Cytokine Signaling (iCP-SOCS3) Proteins, Polynucleotides Encoding the Same, and Anti-Lung Cancer Compositions Comprising the Same
US14/838,295 Abandoned US20160060313A1 (en) 2014-08-27 2015-08-27 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
US15/361,701 Abandoned US20170137482A1 (en) 2014-08-27 2016-11-28 Cell-permeable (icp)-socs3 recombinant protein and uses thereof
US15/408,123 Active US10781241B2 (en) 2014-08-27 2017-01-17 Cell-permeable (iCP)-SOCS3 recombinant protein and uses thereof
US15/408,230 Abandoned US20170198019A1 (en) 2014-08-27 2017-01-17 Cell-permeable (icp)-socs3 recombinant protein and uses thereof
US15/432,662 Active US10385103B2 (en) 2014-08-27 2017-02-14 Cell-permeable (ICP)-SOCS3 recombinant protein and uses thereof
US15/631,982 Active US10787492B2 (en) 2014-08-27 2017-06-23 Cell-permeable (iCP)-SOCS3 recombinant protein and uses thereof
US15/884,884 Active US10774123B2 (en) 2014-08-27 2018-01-31 Cell-permeable bone morphogenetic protein (CP-BMP) recombinant protein and use thereof
US16/426,751 Active US10961292B2 (en) 2014-08-27 2019-05-30 Cell-permeable (ICP)-SOCS3 recombinant protein and uses thereof
US16/426,864 Active US10975132B2 (en) 2014-08-27 2019-05-30 Cell-permeable (ICP)-SOCS3 recombinant protein and uses thereof
US16/831,520 Active 2036-01-18 US11279743B2 (en) 2014-08-27 2020-03-26 Cell-permeable bone morphogenetic protein (CPBMP) recombinant protein and use thereof

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US14/838,288 Abandoned US20160060312A1 (en) 2014-08-27 2015-08-27 Development of Protein-Based Biotherapeutics That Penetrates Cell-Membrane and Induces Anti-Pancreatic Cancer Effect - Improved Cell-Permeable Suppressor of Cytokine Signaling (iCP-SOCS3) Proteins, Polynucleotides Encoding the Same, and Anti-Pancreatic Cancer Compositions Comprising the Same
US14/838,318 Abandoned US20160060319A1 (en) 2014-08-27 2015-08-27 Development of Protein-Based Biotherapeutics That Induced Osteogenesis for Bone Healing Therapy: Cell-Permeable BMP2 and BMP7 Recombinant Proteins (CP-BMP2 & CP-BMP7), Polynucleotides Encoding the Same and Pro-osteogenic Compositions Comprising the Same
US14/838,260 Abandoned US20160060310A1 (en) 2014-08-27 2015-08-27 Development of Protein-Based Biotherapeutics That Penetrates Cell-Membrane and Induces Anti-Hepatocellular Carcinoma Effect - Improved Cell-Permeable Suppressor of Cytokine Signaling (iCP-SOCS3) Proteins, Polynucleotides Encoding the Same, and Anti-Hepatocellular Carcinoma Compositions Comprising the Same
US14/838,280 Abandoned US20160060311A1 (en) 2014-08-27 2015-08-27 Development of Protein-Based Biotherapeutics That Penetrates Cell-Membrane and Induces Anti-Lung Cancer Effect - Improved Cell-Permeable Suppressor of Cytokine Signaling (iCP-SOCS3) Proteins, Polynucleotides Encoding the Same, and Anti-Lung Cancer Compositions Comprising the Same
US14/838,295 Abandoned US20160060313A1 (en) 2014-08-27 2015-08-27 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
US15/361,701 Abandoned US20170137482A1 (en) 2014-08-27 2016-11-28 Cell-permeable (icp)-socs3 recombinant protein and uses thereof
US15/408,123 Active US10781241B2 (en) 2014-08-27 2017-01-17 Cell-permeable (iCP)-SOCS3 recombinant protein and uses thereof
US15/408,230 Abandoned US20170198019A1 (en) 2014-08-27 2017-01-17 Cell-permeable (icp)-socs3 recombinant protein and uses thereof
US15/432,662 Active US10385103B2 (en) 2014-08-27 2017-02-14 Cell-permeable (ICP)-SOCS3 recombinant protein and uses thereof
US15/631,982 Active US10787492B2 (en) 2014-08-27 2017-06-23 Cell-permeable (iCP)-SOCS3 recombinant protein and uses thereof
US15/884,884 Active US10774123B2 (en) 2014-08-27 2018-01-31 Cell-permeable bone morphogenetic protein (CP-BMP) recombinant protein and use thereof
US16/426,751 Active US10961292B2 (en) 2014-08-27 2019-05-30 Cell-permeable (ICP)-SOCS3 recombinant protein and uses thereof
US16/426,864 Active US10975132B2 (en) 2014-08-27 2019-05-30 Cell-permeable (ICP)-SOCS3 recombinant protein and uses thereof
US16/831,520 Active 2036-01-18 US11279743B2 (en) 2014-08-27 2020-03-26 Cell-permeable bone morphogenetic protein (CPBMP) recombinant protein and use thereof

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US20160060312A1 (en) * 2014-08-27 2016-03-03 Cellivery Therapeutics, Inc. Development of Protein-Based Biotherapeutics That Penetrates Cell-Membrane and Induces Anti-Pancreatic Cancer Effect - Improved Cell-Permeable Suppressor of Cytokine Signaling (iCP-SOCS3) Proteins, Polynucleotides Encoding the Same, and Anti-Pancreatic Cancer Compositions Comprising the Same
US11279743B2 (en) 2014-08-27 2022-03-22 Cellivery Therapeutics, Inc. Cell-permeable bone morphogenetic protein (CPBMP) recombinant protein and use thereof
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US20160060311A1 (en) * 2014-08-27 2016-03-03 Daewoong Jo Development of Protein-Based Biotherapeutics That Penetrates Cell-Membrane and Induces Anti-Lung Cancer Effect - Improved Cell-Permeable Suppressor of Cytokine Signaling (iCP-SOCS3) Proteins, Polynucleotides Encoding the Same, and Anti-Lung Cancer Compositions Comprising the Same
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US10385103B2 (en) 2014-08-27 2019-08-20 Cellivery Therapeutics, Inc. Cell-permeable (ICP)-SOCS3 recombinant protein and uses thereof
US10975132B2 (en) 2014-08-27 2021-04-13 Cellivery Therapeutics, Inc. Cell-permeable (ICP)-SOCS3 recombinant protein and uses thereof
US10961292B2 (en) 2014-08-27 2021-03-30 Cellivery Therapeutics, Inc. Cell-permeable (ICP)-SOCS3 recombinant protein and uses thereof
US10787492B2 (en) * 2014-08-27 2020-09-29 Cellivery Therapeutics, Inc. Cell-permeable (iCP)-SOCS3 recombinant protein and uses thereof
US10781241B2 (en) 2014-08-27 2020-09-22 Cellivery Therapeutics, Inc. Cell-permeable (iCP)-SOCS3 recombinant protein and uses thereof
US20180195047A1 (en) * 2015-08-10 2018-07-12 Cellivery Therapeutics, Inc. Cell-permeable reprogramming factor (icp-rf) recombinant protein and use thereof
US10508265B2 (en) * 2015-08-10 2019-12-17 Cellivery Therapeutics, Inc. Cell-permeable reprogramming factor (iCP-RF) recombinant protein and use thereof
WO2017180587A2 (fr) 2016-04-11 2017-10-19 Obsidian Therapeutics, Inc. Systèmes de biocircuits régulés
WO2019241315A1 (fr) 2018-06-12 2019-12-19 Obsidian Therapeutics, Inc. Constructions régulatrices dérivées de pde5 et procédés d'utilisation en immunothérapie
WO2020086742A1 (fr) 2018-10-24 2020-04-30 Obsidian Therapeutics, Inc. Régulation de protéine accordable par er
WO2020150584A1 (fr) * 2019-01-18 2020-07-23 Children's Medical Center Corporation Compositions et méthodes pour induire ou renforcer un socs3 afin de bloquer la croissance tumorale et la rétinopathie proliférante
WO2021046451A1 (fr) 2019-09-06 2021-03-11 Obsidian Therapeutics, Inc. Compositions et méthodes de régulation de protéine accordable dhfr

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EP3341400B1 (fr) 2020-10-21
US11279743B2 (en) 2022-03-22
EP3341400A1 (fr) 2018-07-04
US10385103B2 (en) 2019-08-20
US10961292B2 (en) 2021-03-30
EP3341396A4 (fr) 2019-03-06
WO2017034349A1 (fr) 2017-03-02
EP3341396B1 (fr) 2021-04-07
US20180237485A1 (en) 2018-08-23
EP3341395A1 (fr) 2018-07-04
US10781241B2 (en) 2020-09-22
WO2017034330A1 (fr) 2017-03-02
WO2017034333A1 (fr) 2017-03-02
WO2017034347A1 (fr) 2017-03-02
US20170190754A1 (en) 2017-07-06
EP3341394A1 (fr) 2018-07-04
EP3341395B1 (fr) 2023-11-29
EP3341400A4 (fr) 2018-09-12
US20200299348A1 (en) 2020-09-24
EP3341394B1 (fr) 2021-07-28
US20170198019A1 (en) 2017-07-13
US20170226168A1 (en) 2017-08-10
US20190338000A1 (en) 2019-11-07
US20160060313A1 (en) 2016-03-03
US20160060319A1 (en) 2016-03-03
US20190359669A1 (en) 2019-11-28
EP3341396A1 (fr) 2018-07-04
EP3341394A4 (fr) 2019-01-09
US20180051060A1 (en) 2018-02-22
US20170137482A1 (en) 2017-05-18
US20160060312A1 (en) 2016-03-03
US10975132B2 (en) 2021-04-13
WO2017034344A1 (fr) 2017-03-02
US10787492B2 (en) 2020-09-29
EP3341395A4 (fr) 2018-08-08
US20160060311A1 (en) 2016-03-03
US20160060310A1 (en) 2016-03-03
WO2017034335A1 (fr) 2017-03-02

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