US20160060311A1 - 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 - Google Patents

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 Download PDF

Info

Publication number
US20160060311A1
US20160060311A1 US14/838,280 US201514838280A US2016060311A1 US 20160060311 A1 US20160060311 A1 US 20160060311A1 US 201514838280 A US201514838280 A US 201514838280A US 2016060311 A1 US2016060311 A1 US 2016060311A1
Authority
US
United States
Prior art keywords
socs3
aliphatic
proteins
protein
cell
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Abandoned
Application number
US14/838,280
Other languages
English (en)
Inventor
Daewoong Jo
Young Sil CHOI
Kuy Sook LEE
Min Seok Jang
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Cellivery Therapeutics Inc
Original Assignee
Cellivery Therapeutics Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Cellivery Therapeutics Inc filed Critical Cellivery Therapeutics Inc
Priority to US14/838,280 priority Critical patent/US20160060311A1/en
Assigned to CELLIVERY THERAPEUTICS, INC., JO, DAEWOONG reassignment CELLIVERY THERAPEUTICS, INC. ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: CHOI, YOUNG SIL, JANG, MIN SEOK, JO, DAEWOONG, LEE, KYU SOOK
Publication of US20160060311A1 publication Critical patent/US20160060311A1/en
Priority to PCT/KR2016/009441 priority patent/WO2017034344A1/fr
Priority to US15/408,230 priority patent/US20170198019A1/en
Assigned to CELLIVERY THERAPEUTICS, INC. reassignment CELLIVERY THERAPEUTICS, INC. ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: JO, DAEWOONG
Priority to US16/426,864 priority patent/US10975132B2/en
Abandoned legal-status Critical Current

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • 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
    • A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A61K38/1761—Apoptosis related proteins, e.g. Apoptotic protease-activating factor-1 (APAF-1), Bax, Bax-inhibitory protein(s)(BI; bax-I), Myeloid cell leukemia associated protein (MCL-1), Inhibitor of apoptosis [IAP] or Bcl-2
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • 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
    • C07K14/4702—Regulators; Modulating activity
    • C07K14/4703—Inhibitors; Suppressors
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • 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
    • A—HUMAN NECESSITIES
    • 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
    • A61K38/10—Peptides having 12 to 20 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • 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
    • A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • 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
    • C07K14/4702—Regulators; Modulating activity
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/10—Fusion polypeptide containing a localisation/targetting motif containing a tag for extracellular membrane crossing, e.g. TAT or VP22
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/40—Fusion polypeptide containing a tag for immunodetection, or an epitope for immunisation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • 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-non-small cell lung carcinoma effect; (ii) polynucleotides that encode the same, and (iii) anti-lung cancer compositions that comprise the same.
  • iCP-SOCS3 cell-permeable SOCS3
  • NSCLC non-small cell lung cancer
  • SCLC small cell lung cancer
  • Squamous cell carcinoma, adenocarcinoma, and large cell carcinoma are all subtypes of non-small cell lung cancer.
  • 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 metastasis in the lung.
  • IFN- interferon-gamma
  • JNK Janus kinase
  • STAT3 which functions as an oncogene downstream of IL-6/gp130, is hyper-activated in lung cancer cells contributes to increase cell proliferation and inhibits apoptosis.
  • 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-inflammatory 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.
  • SOCS3 may significantly inhibit the proliferation of lung cancer cells in vitro and indicated that SOCS3 may act as an anti-oncogene involved in the development of tumors. Furthermore, SOCS3 may regulate the movement and migration of tumor cells. Methylation-mediated silencing of SOCS3 has been reported in non-small lung cancer (NSCLC) and other human cancers. In addition to the effect of SOCS3 in inflammation, abnormalities of the JAK/STAT pathway are also associated with cancer. It has been reported that methylationin of CpG islands in the functional SOCS3 promoter is correlated with its transcription silencing in the lung cancer cell lines.
  • 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 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 the lung cancer, 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 lung cancer.
  • 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.
  • An aspect of the present invention provides improved cell-permeable SOCS3 as a biotherapeutics having improved solubility/yield, cell-/tissue-permeability and anti-lung cancer 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 This figure shows the agarose gel electrophoresis analysis showing plasmid DNA fragments encoding SOCS3, aMTDs fused SOCS3 and SD cloned into the pET28 (+) vector according to the present invention.
  • FIG. 3 shows 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 recombinant 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-HM165S3B 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- .
  • 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 (HM165S3B) in lung cancer cells.
  • A549 lung cancer 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 (HM165S3B) into lung tissue.
  • 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 lung cancer cells with iCP-SOCS3.
  • A549 lung cancer cells were seeded in 96 well plates. Next day, cells were treated with DMEM (V), HS3 (1), HM165S3 (2), HM165S3A (3) or HM165S3B (4) recombinant proteins for 96 hrs in the presence of serum (2%). Cell viability was evaluated with the CellTiter-Glo Cell Viability Assay.
  • FIG. 17 shows the induction of apoptosis in lung cancer cells with iCP-SOCS3.
  • A549 lung cancer cells were treated for 24 hrs with 10 ⁇ M HS3B or HM165S3B proteins and apoptotic cells were visualized by TUNEL staining.
  • FIG. 18 shows the stimulation of apoptosis in lung cancer cells with iCP-SOCS3.
  • A549 lung cancer cells were treated for 24 hr with 10 ⁇ M HS3B or HM165S3B proteins and analyzed by flow cytometry of cells stained with annexin-V and 7-AAD.
  • FIG. 19 shows the inhibition of migration in lung cancer cells with iCP-SOCS3.
  • A549 lung cancer 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. 20 shows the inhibition of migration/invasion in lung cancer cells with iCP-SOCS3.
  • A549 lung cancer 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.
  • 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, 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).
  • 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) were practically confirmed by their quantitative and visual cell-permeability. 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. Relative cell-permeability of 240 aMTDs to the negative control (random peptide, hydrophilic & non-aliphatic 12A/a length peptide) was significantly increased by up to 164 fold, with average increase of 19.6 ⁇ 1.6.
  • novel 240 aMTDs averaged of 13 ⁇ 1.1 (maximum 109.9) and 6.6 ⁇ 0.5 (maximum 55.5) fold higher cell-permeability, respectively.
  • the empirically optimized critical factors are provided below.
  • 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 NO: 483 and 484, respectively.
  • the cDNA and amino acid sequences are displayed in SEQ ID NO: 485 and 486 (S0053); SEQ ID NO: 487 and 488 (SDA); and SEQ ID NO: 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 50053 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 (HM165S3, HM165S3A and HM165S3B) efficiently entered the cells ( FIGS.
  • 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 (HM165S3A) 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 Lung Cancer Cells 5-1.
  • iCP-50053 Enhances the Cellular Uptake into Lung Cancer Cells and the Systemic Delivery to the Lung Tissue
  • lung cancer is one of the most common cancers with a high mortality rate, there are few drugs for treating this lethal disorder. Since constitutive activation of STAT3 is found in various cancers and SOCS3 is closely related to the development of lung cancer, we first chose lung cancer 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 A549 lung cancer cells ( FIG. 14 ).
  • iCP-SOCS3 proteins enhanced the systemic delivery to lung tissue after intraperitoneal injection ( FIG. 15 ). Therefore, these data indicate that iCP-SOCS3 protein could be intracellularly delivered and distributed to the lung cells and tissue, contributing for beneficial biotherapeutic effects.
  • iCP-SOCS3 inhibits cell viability through SOCS3 intracellular delivery in lung cancer cells.
  • SOCS3 recombinant proteins containing aMTD165 significantly suppressed cancer cell proliferation.
  • HM 165 S3B (iCP-SOCS3) protein was the most cytotoxic to A549 lung cancer cells—over 80% in 10 ⁇ M treatment (p ⁇ 0.01)—especially compared to vehicle alone (i.e. exposure of cells to culture media without recombinant proteins; FIG. 16 , left).
  • HM 165 S3B protein (iCP-SOCS3) was a considerably efficient inducer of apoptosis in A549 cells, as assessed either by a fluorescent terminal dUTP nick-end labeling (TUNEL) assay ( FIG. 17 ) and Annexin V staining ( FIG. 18 ). Consistently, no changes in TUNEL and Annexin V staining were observed in A549 cells treated with HS3B compared to untreated cells (Vehicle).
  • TUNEL fluorescent terminal dUTP nick-end labeling
  • iCP-SOCS3 A549 cells treated with HM 165 S3B recombinant protein (iCP-SOCS3) caused remarkable decrease in invasion compared with the control proteins (HS3B; FIG. 21 ). Taken together, these data indicate that iCP-SOCS3 contributes to inhibit tumorigenic activities of lung cancer cells.
  • 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, lx 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 FlowJo 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 g/ml, Gibco BRL
  • 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:HM165S3, 3:HM165S3A and 4:HM165S3B, 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
  • Cells originated from lung cancer and mouse fibroblast (NIH3T3) were purchased (ATCC, Manassas, Va.) and maintained as recommended by the supplier. These cells (3 ⁇ 103/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.
  • 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 ⁇ 106 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).
  • Cells were seeded into 12-well plates, grown to 90% confluence, and incubated with 10 ⁇ M HS3, HM16553, HM165S3A or HM165S3B 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 48 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 ⁇ 105) 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 ⁇ 105) 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.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • General Health & Medical Sciences (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Genetics & Genomics (AREA)
  • Biophysics (AREA)
  • Biochemistry (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Zoology (AREA)
  • Toxicology (AREA)
  • Orthopedic Medicine & Surgery (AREA)
  • Animal Behavior & Ethology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Engineering & Computer Science (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Epidemiology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Immunology (AREA)
  • Marine Sciences & Fisheries (AREA)
  • Oncology (AREA)
  • Hematology (AREA)
  • Peptides Or Proteins (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
US14/838,280 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 Abandoned US20160060311A1 (en)

Priority Applications (4)

Application Number Priority Date Filing Date Title
US14/838,280 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
PCT/KR2016/009441 WO2017034344A1 (fr) 2014-08-27 2016-08-25 Protéine recombinante (icp)-socs3 de pénétration cellulaire améliorée et ses utilisations
US15/408,230 US20170198019A1 (en) 2014-08-27 2017-01-17 Cell-permeable (icp)-socs3 recombinant protein and uses thereof
US16/426,864 US10975132B2 (en) 2014-08-27 2019-05-30 Cell-permeable (ICP)-SOCS3 recombinant protein and uses thereof

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US201462042493P 2014-08-27 2014-08-27
US14/838,280 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

Related Child Applications (1)

Application Number Title Priority Date Filing Date
US15/408,230 Continuation-In-Part US20170198019A1 (en) 2014-08-27 2017-01-17 Cell-permeable (icp)-socs3 recombinant protein and uses thereof

Publications (1)

Publication Number Publication Date
US20160060311A1 true US20160060311A1 (en) 2016-03-03

Family

ID=55401724

Family Applications (15)

Application Number Title Priority Date Filing Date
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
US14/838,260 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
US14/838,295 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
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
US14/838,304 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
US14/838,318 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
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
US16/426,864 Active US10975132B2 (en) 2014-08-27 2019-05-30 Cell-permeable (ICP)-SOCS3 recombinant protein and uses thereof
US16/426,751 Active US10961292B2 (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

Family Applications Before (3)

Application Number Title Priority Date Filing Date
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
US14/838,260 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
US14/838,295 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

Family Applications After (11)

Application Number Title Priority Date Filing Date
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
US14/838,318 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
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
US16/426,864 Active US10975132B2 (en) 2014-08-27 2019-05-30 Cell-permeable (ICP)-SOCS3 recombinant protein and uses thereof
US16/426,751 Active US10961292B2 (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

Country Status (3)

Country Link
US (15) US20160060312A1 (fr)
EP (4) EP3341396B1 (fr)
WO (6) WO2017034347A1 (fr)

Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
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
WO2017180587A2 (fr) 2016-04-11 2017-10-19 Obsidian Therapeutics, Inc. Systèmes de biocircuits régulés
WO2018017747A3 (fr) * 2016-07-19 2018-04-12 University Of Pittsburgh - Of The Commonwealth System Of Higher Education Virus oncolytiques ciblant stat3
CN108727469A (zh) * 2017-04-17 2018-11-02 北京翼方生物科技有限责任公司 一种介导药物递送的新型穿膜肽及其应用
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
WO2021046451A1 (fr) 2019-09-06 2021-03-11 Obsidian Therapeutics, Inc. Compositions et méthodes de régulation de protéine accordable dhfr
US11963990B2 (en) 2021-04-30 2024-04-23 Kalivir Immunotherapeutics, Inc. Oncolytic viruses for modified MHC expression
US12036257B2 (en) 2017-10-31 2024-07-16 Kalivir Immunotherapeutics, Inc. Platform oncolytic vector for systemic delivery

Families Citing this family (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20170029798A1 (en) * 2015-07-27 2017-02-02 Cellivery Therapeutics, Inc. Development of Improved Cell-Permeable (iCP) Parkin Recombinant Protein as a Protein-Based Anti-Neurodegenerative Agent for the Treatment of Parkinson's Disease-Associated Phenotypes by Utilizing BBB-Penetrating Protein Delivery System MITT, Enabled by Advanced Macromolecule Transduction Domain (aMTD)
WO2017026776A1 (fr) * 2015-08-10 2017-02-16 Cellivery Therapeutics, Inc. Protéine recombinante de facteur de reprogrammation à perméabilité cellulaire améliorée (icp-rf), et utilisation de ladite protéine
EP3337815B1 (fr) * 2015-08-18 2020-12-16 Cellivery Therapeutics, Inc. Protéine recombinante perméable à la cellule (cp)- socs3 et utilisations de ladite protéine
US11319546B2 (en) * 2016-09-28 2022-05-03 Cellivery Therapeutics, Inc. Cell-permeable (CP)-Cas9 recombinant protein and uses thereof
KR101944517B1 (ko) 2016-12-27 2019-02-01 서울대학교산학협력단 세포 투과능 및 골조직 재생능을 가지고 있는 이중 기능성 신규 펩타이드 및 이의 용도
EP3645557B1 (fr) * 2017-06-28 2024-10-23 The Cleveland Clinic Foundation Traitement d'une lésion du système nerveux et de troubles neurodégénératifs et d'affections associés
CN109411465B (zh) * 2017-08-17 2022-04-15 联华电子股份有限公司 半导体结构及虚拟图案布局的设计方法
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
BR112022011744A2 (pt) * 2020-02-18 2022-09-20 Cellivery Therapeutics Inc Peptídeo sintético inibidor de importação nuclear permeável à célula melhorado para inibição de tempestade de citocina ou doença inflamatória e uso do mesmo

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20090209458A1 (en) * 2004-03-04 2009-08-20 Vanderbilt University Cell-penetrating socs polypeptides that inhibit cytokine-induced signaling
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

Family Cites Families (32)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999023220A1 (fr) 1997-11-03 1999-05-14 Incyte Pharmaceuticals, Inc. Suppresseur de la signalisation cytokinaire
US7892532B2 (en) * 1999-04-30 2011-02-22 Warsaw Orthopedic, In Emory University Intracellular delivery of osteoinductive proteins and peptides
US20030104622A1 (en) 1999-09-01 2003-06-05 Robbins Paul D. Identification of peptides that facilitate uptake and cytoplasmic and/or nuclear transport of proteins, DNA and viruses
JP4799790B2 (ja) 1999-09-27 2011-10-26 メリオン リサーチ スリー リミテッド 膜転位ペプチド、同ペプチドを含有する組成物、同ペプチドを含有する医薬製剤及び同ペプチドの製剤の調製における使用
WO2003066801A2 (fr) 2002-02-05 2003-08-14 Indian Institute Of Technology Procede d'integration specifique d'un gene d'arn polymerase t7 dans le chromosome d'une corynebacterie et systeme de vecteur navette base sur le promoteur de corynebacterie t7 resultant
EP1495045B1 (fr) 2002-03-29 2009-09-02 Creagene, Inc. Peptides de transduction cytoplasmiques et leurs utilisations
US6835810B2 (en) 2002-05-13 2004-12-28 Geneshuttle Biopharma, Inc. Fusion protein for use as vector
US7897394B2 (en) * 2006-09-21 2011-03-01 Intrexon Corporation Endoplasmic reticulum localization signals
KR20080044710A (ko) * 2006-11-17 2008-05-21 김정문 향상된 세포 투과능 및 조직재생 기능을 가지는 비활성폴리펩티드, 및 그 용도
WO2008093982A1 (fr) * 2007-01-29 2008-08-07 Procell Therapeutics Inc. Nouveaux domaines de transduction de macromolécules et procédés d'identification et utilisations correspondantes
JP2010537632A (ja) 2007-08-29 2010-12-09 タフツ ユニバーシティー 組織及び細胞への核酸、タンパク質、薬物、及びアデノウイルスの送達を向上させるための細胞透過性ペプチドの製造及び使用方法、組成物並びにキット
KR20110016867A (ko) 2008-05-16 2011-02-18 주식회사 프로셀제약 세포투과성 ｐ２７ 재조합 단백질, 이를 코딩하는 폴리뉴클레오티드 및 이를 유효성분으로 함유하는 항암 조성물
US20110229525A1 (en) * 2010-03-12 2011-09-22 Vanderbilt University Modulation of cytokine signaling
WO2012050402A2 (fr) * 2010-10-14 2012-04-19 주식회사 프로셀제약 Protéine parkin recombinante à perméation cellulaire et composition pharmaceutique de traitement des maladies dégénératives du cerveau l'incluant
EA025152B1 (ru) 2010-12-02 2016-11-30 Бионор Иммуно Ас Конструкция пептидного каркаса
EP2714971A4 (fr) 2011-05-23 2015-01-21 Phylogica Ltd Procédé de détermination, d'identification ou d'isolement de peptides pénétrant dans des cellules
CN104039811B (zh) 2011-11-23 2018-02-23 株式会社普罗赛尔制药 具有改善的细胞渗透性的新的大分子转导结构域的研发及其使用方法
KR101258279B1 (ko) 2011-11-23 2013-04-25 주식회사 프로셀제약 세포 투과능을 개선한 개량형 신규 거대 분자 전달 도메인 개발 및 이의 이용방법
EP2931923A1 (fr) * 2012-12-13 2015-10-21 Baylor Research Institute Signatures de transcription sanguine de la tuberculose et de la sarcoïdose pulmonaires actives
CN105247067A (zh) 2013-05-24 2016-01-13 诺和诺德股份有限公司 融合蛋白酶
CA3236835A1 (fr) * 2013-11-22 2015-05-28 Mina Therapeutics Limited Compositions d'arn a activation courte de c/ebp alpha et methodes d'utilisation
DK3096775T5 (da) * 2014-01-24 2024-10-14 Univ Florida SOCS-mimetika til behandling af sygdomme
KR20160009456A (ko) * 2014-07-16 2016-01-26 계명대학교 산학협력단 차량시트의 유아보호장치
KR101643718B1 (ko) * 2014-07-16 2016-07-28 한국항공우주연구원 지주형 무인비행체 격납충전장치 및 이를 이용한 무인비행체의 격납 및 충전방법
KR101694161B1 (ko) * 2014-07-16 2017-01-09 엘지전자 주식회사 조명 디바이스 및 조명 디바이스를 포함하는 조명 시스템
US10323063B2 (en) * 2014-08-17 2019-06-18 Cellivery Therapeutics, Inc. Advanced macromolecule transduction domain (aMTD) sequences for improvement of cell-permeability, polynucleotides encoding the same, method to identify the unique features of aMTDs comprising the same, method to develop the aMTD sequences comprising the same
US20160068825A1 (en) * 2014-09-04 2016-03-10 Daewoong Jo Development of Protein-Based Biotherapeutics That Penetrate Cell-Membrane and Induce Anti-Cancer Effect- Cell-Permeable Glutathione Peroxidase7 (CP-GPX7) in Gastrointestinal Track (GIT), Polynucleotides Encoding the Same, and Anti-Cancer Compositions Comprising the Same
US20160083441A1 (en) * 2014-09-24 2016-03-24 Daewoong Jo Development of Protein-Based Biotherapeutics That Penetrate Cell-Membrane and Induce Anti-Cancer Effect - Cell-Permeable Trefoil Factor 1 (CP-TFF1) in Gastrointestinal Track (GIT) Cancer, Polynucleotides Encoding The Same, and Anti-Cancer Compositions Comprising The Same
US20170029798A1 (en) * 2015-07-27 2017-02-02 Cellivery Therapeutics, Inc. Development of Improved Cell-Permeable (iCP) Parkin Recombinant Protein as a Protein-Based Anti-Neurodegenerative Agent for the Treatment of Parkinson's Disease-Associated Phenotypes by Utilizing BBB-Penetrating Protein Delivery System MITT, Enabled by Advanced Macromolecule Transduction Domain (aMTD)
WO2017026779A1 (fr) * 2015-08-10 2017-02-16 Cellivery Therapeutics, Inc. Protéine recombinée cre à perméabilité cellulaire améliorée (icp-cre) et son utilisation
WO2017026776A1 (fr) * 2015-08-10 2017-02-16 Cellivery Therapeutics, Inc. Protéine recombinante de facteur de reprogrammation à perméabilité cellulaire améliorée (icp-rf), et utilisation de ladite protéine
EP3337815B1 (fr) * 2015-08-18 2020-12-16 Cellivery Therapeutics, Inc. Protéine recombinante perméable à la cellule (cp)- socs3 et utilisations de ladite protéine

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20090209458A1 (en) * 2004-03-04 2009-08-20 Vanderbilt University Cell-penetrating socs polypeptides that inhibit cytokine-induced signaling
US8420096B2 (en) * 2004-03-04 2013-04-16 Vanderbilt University Cell-penetrating SOCS polypeptides that inhibit cytokine-induced signaling
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
US20160060314A1 (en) * 2014-08-27 2016-03-03 Daewoong Jo 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
US20160060313A1 (en) * 2014-08-27 2016-03-03 Daewoong Jo 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
US20160060310A1 (en) * 2014-08-27 2016-03-03 Daewoong Jo 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

Non-Patent Citations (9)

* Cited by examiner, † Cited by third party
Title
Bechara, Cherine and Sagan, Sandrine, "Cell penetrating peptides: 20 years later, where do we stand." FEBS Let. (2013) 587 p1693-1702 *
Forli, Stephano, "Charting a path to success in virtual screening." Molecules (2015) 20(10) p18732-18758 *
He, Biao et al, "Activity of the suppressor of cytokine signaling-3 promotor in human non small cell lung cancer." Clin. Lung. Canc. (2004) 5(6) 366-370 *
Kato, Atsushi et al, "Mutational analysis of protein solubility enhancement using short peptide tags." Biopolymers (2006) 85(1) p12-18 *
the web page Wine Turtle, http://www.wineturtle.com/fining-wine-clarification-agents/, downloaded 4 Aug, 2016 *
the webpage for Croda health care's arlasolveTM, http://www.crodahealthcare.com/home.aspx?view=dtl&d=content&s=149&r=346&p=2204&productname=Arlasolve&chemicaldescription=&chemicalgroup=&f%E2%80%A6, downloaded 3 Aug, 2016. *
Watkins, Catherine L. et al, "Cellular uptake, distribution and cytotoxicity of the hydrophobic cell penetrating peptide sequence pfvyli linked to the proapoptoic domain pad." J. Cont. Rel. (2009) 140 p237-244 *
White, Stephen, "Experimentally determined hydrophobicity scales." http://blanco.biomol.uci.edu/hydrophobicity_scales.html, downloaded 4 Aug, 2016 *
Yampolsky, Lev Y. and Stoltzfus, ARlin, "THe exchangeability of amino acids in proteins." Genetics (2005) 170 p1459-1472 *

Cited By (19)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10961292B2 (en) 2014-08-27 2021-03-30 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
US10774123B2 (en) * 2014-08-27 2020-09-15 Cellivery Therapeutics, Inc. Cell-permeable bone morphogenetic protein (CP-BMP) recombinant protein and use thereof
US20180237485A1 (en) * 2014-08-27 2018-08-23 Cellivery Therapeutics, Inc. Cell-permeable bone morphogenetic protein (cp-bmp) recombinant protein and use thereof
US10781241B2 (en) 2014-08-27 2020-09-22 Cellivery Therapeutics, Inc. Cell-permeable (iCP)-SOCS3 recombinant protein and uses thereof
US10385103B2 (en) 2014-08-27 2019-08-20 Cellivery Therapeutics, Inc. Cell-permeable (ICP)-SOCS3 recombinant protein and uses thereof
US11279743B2 (en) 2014-08-27 2022-03-22 Cellivery Therapeutics, Inc. Cell-permeable bone morphogenetic protein (CPBMP) recombinant protein and use thereof
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
US10787492B2 (en) 2014-08-27 2020-09-29 Cellivery Therapeutics, Inc. Cell-permeable (iCP)-SOCS3 recombinant protein and uses thereof
WO2017180587A2 (fr) 2016-04-11 2017-10-19 Obsidian Therapeutics, Inc. Systèmes de biocircuits régulés
WO2018017747A3 (fr) * 2016-07-19 2018-04-12 University Of Pittsburgh - Of The Commonwealth System Of Higher Education Virus oncolytiques ciblant stat3
US11253559B2 (en) 2016-07-19 2022-02-22 University of Pittsburgh—of the Commonwealth System of Higher Education Oncolytic viruses targeting STAT3
CN108727469A (zh) * 2017-04-17 2018-11-02 北京翼方生物科技有限责任公司 一种介导药物递送的新型穿膜肽及其应用
US12036257B2 (en) 2017-10-31 2024-07-16 Kalivir Immunotherapeutics, Inc. Platform oncolytic vector for systemic delivery
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
WO2021046451A1 (fr) 2019-09-06 2021-03-11 Obsidian Therapeutics, Inc. Compositions et méthodes de régulation de protéine accordable dhfr
US11963990B2 (en) 2021-04-30 2024-04-23 Kalivir Immunotherapeutics, Inc. Oncolytic viruses for modified MHC expression
US12016893B2 (en) 2021-04-30 2024-06-25 Kalivir Immunotherapeutics, Inc. Oncolytic viruses for modified MHC expression

Also Published As

Publication number Publication date
EP3341395A1 (fr) 2018-07-04
US20170137482A1 (en) 2017-05-18
US20170198019A1 (en) 2017-07-13
US20160060319A1 (en) 2016-03-03
WO2017034349A1 (fr) 2017-03-02
WO2017034330A1 (fr) 2017-03-02
WO2017034335A1 (fr) 2017-03-02
EP3341396B1 (fr) 2021-04-07
US20170190754A1 (en) 2017-07-06
US10385103B2 (en) 2019-08-20
US10961292B2 (en) 2021-03-30
EP3341394A4 (fr) 2019-01-09
US20190359669A1 (en) 2019-11-28
US20160060312A1 (en) 2016-03-03
EP3341400A4 (fr) 2018-09-12
US20190338000A1 (en) 2019-11-07
EP3341394A1 (fr) 2018-07-04
US20160060310A1 (en) 2016-03-03
WO2017034344A1 (fr) 2017-03-02
US20170226168A1 (en) 2017-08-10
EP3341395B1 (fr) 2023-11-29
US20160060313A1 (en) 2016-03-03
EP3341395A4 (fr) 2018-08-08
US20200299348A1 (en) 2020-09-24
EP3341396A1 (fr) 2018-07-04
US10774123B2 (en) 2020-09-15
US10781241B2 (en) 2020-09-22
US11279743B2 (en) 2022-03-22
EP3341400B1 (fr) 2020-10-21
US20180237485A1 (en) 2018-08-23
WO2017034333A1 (fr) 2017-03-02
US20160060314A1 (en) 2016-03-03
EP3341400A1 (fr) 2018-07-04
US20180051060A1 (en) 2018-02-22
WO2017034347A1 (fr) 2017-03-02
US10975132B2 (en) 2021-04-13
EP3341394B1 (fr) 2021-07-28
EP3341396A4 (fr) 2019-03-06
US10787492B2 (en) 2020-09-29

Similar Documents

Publication Publication Date Title
US20160060311A1 (en) 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
Hotchkiss et al. TAT-BH4 and TAT-Bcl-xL peptides protect against sepsis-induced lymphocyte apoptosis in vivo
JP5734865B2 (ja) 選択性に優れた抗がんキメラペプチド
US8841414B1 (en) Targeted delivery of therapeutic peptides by thermally responsive biopolymers
EP3059242A1 (fr) Multimère de peptides de pénétration cellulaire à hélice alpha, procédé de préparation associé et utilisation associée
EA035463B1 (ru) Способы и композиции для лечения рака
US10259852B2 (en) Conjugate comprising P21 protein for the treatment of cancer
Li et al. Effective therapeutic drug delivery by GALA3, an endosomal escape peptide with reduced hydrophobicity
Watson et al. Shortened penetratin cell-penetrating peptide is insufficient for cytosolic delivery of a Grb7 targeting peptide
US9683025B2 (en) Methods and compositions for treating cancer and inflammatory diseases
JP5700409B2 (ja) Ｈｓｐ９０を標的にした新規抗がんキメラペプチド
US20160083441A1 (en) Development of Protein-Based Biotherapeutics That Penetrate Cell-Membrane and Induce Anti-Cancer Effect - Cell-Permeable Trefoil Factor 1 (CP-TFF1) in Gastrointestinal Track (GIT) Cancer, Polynucleotides Encoding The Same, and Anti-Cancer Compositions Comprising The Same
US20160068825A1 (en) Development of Protein-Based Biotherapeutics That Penetrate Cell-Membrane and Induce Anti-Cancer Effect- Cell-Permeable Glutathione Peroxidase7 (CP-GPX7) in Gastrointestinal Track (GIT), Polynucleotides Encoding the Same, and Anti-Cancer Compositions Comprising the Same
WO2018152446A2 (fr) Polypeptides atf5 de pénétration cellulaire et leurs utilisations
Hassanvand Jamadi et al. Anticancer activity of brevinin-2R peptide and its Two analogues against myelogenous leukemia cell line as natural treatments: An in vitro study
US20060198832A1 (en) Peptide drugs for chronic lymphocytic leukemia (CLL) and other cancers
US20220354957A1 (en) Peptides targeting macrophages, and conjugates, compositions, and uses thereof
CA3153060A1 (fr) Peptides ciblant les macrophages et conjugues, compositions et utilisations connexes
US20170267747A1 (en) Protein Therapeutant And Method For Treating Cancer
Eissa Enhancing the delivery of therapeutic cargo to cancer cells using cell penetrating peptides
Mao et al. C-Terminal p53 Palindromic Tetrapeptide Restores Full Apoptotic Function to Mutant p53 Cancer Cells in Vitro and in Vivo.

Legal Events

Date Code Title Description
AS Assignment

Owner name: CELLIVERY THERAPEUTICS, INC., KOREA, REPUBLIC OF

Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:JO, DAEWOONG;CHOI, YOUNG SIL;LEE, KYU SOOK;AND OTHERS;REEL/FRAME:036443/0688

Effective date: 20150827

Owner name: JO, DAEWOONG, TENNESSEE

Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:JO, DAEWOONG;CHOI, YOUNG SIL;LEE, KYU SOOK;AND OTHERS;REEL/FRAME:036443/0688

Effective date: 20150827

AS Assignment

Owner name: CELLIVERY THERAPEUTICS, INC., KOREA, REPUBLIC OF

Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNOR:JO, DAEWOONG;REEL/FRAME:041061/0842

Effective date: 20170123

STCB Information on status: application discontinuation

Free format text: ABANDONED -- FAILURE TO RESPOND TO AN OFFICE ACTION