Patent Document

CROSS-REFERENCE TO RELATED APPLICATIONS 
       [0001]    This application claims the benefit of the filing date of U.S. Provisional Application No. 62/042,493, filed on Aug. 27, 2014, in the United States Patent and Trademark Office, the disclosure of which is incorporated herein in its entirety by reference. 
     
    
     TECHNICAL FIELD 
       [0002]    The present invention pertains to have (i) improved cell-permeable SOCS3 (iCP-SOCS3) proteins as protein-based biotherapeutics, which are well-enhanced in their ability to transport biologically active SOCS3 proteins across the plasma membrane, to increase in its solubility and manufacturing yield, and to induce anti-hepatocellular carcinoma effect; (ii) polynucleotides that encode the same, and (iii) anti-hepatocellular carcinoma compositions that comprise the same. 
       BACKGROUND ART 
       [0003]    Hepatocellular carcinoma (HCC) is one of the most common cancers with high mobility/mortality rate and the tumor often develops after liver cirrhosis and advanced fibrosis. Cytokines including IL-6 and interferon-gamma (IFN-γ) activate the Janus kinase (JAK)/signal transducers and activators of transcription (STAT) signaling pathway, a vital role promoting the inflammation, fibrosis and carcinogenesis in the liver. STAT3, which functions as an oncogene downstream of IL-6/gp130, is hyper-activated in hepatocellular carcinoma contributes to increase cell proliferation and inhibits apoptosis by inducing c-MYC, Cyclin-D1, and Bcl-XL. 
         [0004]    Cytokine signaling is strictly regulated by the SOCS family proteins induced by different classes of agonists, including cytokines, hormones and infectious agents. Among them, 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. Furthermore, the SOCS box, another domain in SOCS proteins, interacts with E3 ubiquitin ligases and/or couples the SH2 domain-binding proteins to the ubiquitin-proteasome pathway. Therefore, SOCSs inhibit cytokine signaling by suppressing JAK kinase activity and degrading the activated cytokine receptor complex. 
         [0005]    In connection with SOCSs and HCC, the SOCS1 gene has been implicated as an anti-oncogene in the HCC development. Previous studies have reported that aberrant methylation in the CpG island of SOCS1 induces its transcriptional silencing in HCC cell lines, and SOCS1 heterozygous mice are hypersensitive to hepatocellular carcinogenesis. In addition, abnormalities of SOCS3 are also associated with the hepatocellular carcinoma. Hypermethylation of CpG islands in the SOCS3 promoter is correlated with its transcriptional silencing in tumors and in HCC cell lines. SOCS3 overexpression down-regulates active STAT3, induces apoptosis, and suppresses growth in cancer cells. The importance of STAT3 to inflammation-associated hepatocellular carcinogenesis is underlined by the previous study that hepatocyte-specific deletion of SOCS3 in a mouse hepatocellular carcinoma model results in larger and more numerous tumors. This means that SOCS3 plays a major role in the negative regulation of the JAK/STAT pathway in hepatocarcinogenesis and contributes to the suppression of HCC development by protecting the liver cells. 
         [0006]    To negatively control JAK/STAT signaling, recombinant SOCS3 proteins that contain a cell-penetrating peptide (CPP)—membrane-translocating motif (MTM) from fibroblast growth factor (FGF)-4 has been reported. These recombinant SOCS3 proteins inhibited STAT phosphorylation, inflammatory cytokines production and MHC-II expression in cultured and primary macrophages. In addition, SOCS3 fused to MTM protected mice challenged with a lethal dose of the SEB super-antigen, by suppressing apoptosis and hemorrhagic necrosis in multiple organs. However, the SOCS3 proteins fused to FGF4-derived MTM displayed extremely low solubility, poor yields and relatively low cell- and tissue-permeability. Therefore, the MTM-fused SOCS3 proteins were not suitable for further clinical development as therapeutic agents. To overcome these limitations, 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. 
         [0007]    In this new art of invention, aMTD/SD-fused iCP-SOCS3 recombinant proteins (iCP-SOCS3), much improved physicochemical characteristics (solubility &amp; yield) and functional activity (cell-/tissue-permeability) compared with the protein fused only to FGF-4-derived MTM. In addition, the newly developed iCP-SOCS3 proteins have now been demonstrated to have therapeutic application in treating hepatocellular carcinoma, 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 hepatocellular carcinoma. These findings suggest that restoration of SOCS3 by replenishing the intracellular SOCS3 with iCP-SOCS3 protein creates a new paradigm for anti-cancer therapy, and the intracellular protein replacement therapy with the SOCS3 recombinant protein fused to the combination of aMTD and SD pair may be useful to treat the HCC. 
       SUMMARY 
       [0008]    An aspect of the present invention relates to improved cell-permeable SOCS3 (iCP-SOCS3) recombinant proteins capable of mediating the transduction of biologically active macromolecules into live cells. 
         [0009]    According to an aspect of the present invention, iCP-SOCS3 recombinant proteins fused to novel hydrophobic CPPs—namely advanced macromolecule transduction domains (aMTDs)—greatly improve the efficiency of membrane penetrating ability in vitro and in vivo of the recombinant proteins. 
         [0010]    According to an aspect of the present invention, iCP-SOCS3 recombinant proteins fused to solubilization domains (SDs) greatly increase in their solubility and manufacturing yield when they are expressed and purified in the bacteria system. 
         [0011]    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. 
         [0012]    Other 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. 
         [0013]    The present invention provides improved cell-permeable SOCS3 as a biotherapeutics having improved solubility/yield and cell-/tissue-permeability and anti-hepatocellular carcinoma effects. Therefore, this would allow their practically effective applications in drug delivery and protein therapy including intracellular protein therapy and protein replacement therapy. 
     
    
     
       BRIEF DESCRIPTION OF DRAWINGS 
         [0014]    The above and other objects, features and other advantages of the present invention will be more clearly understood from the following detailed description taken in conjunction with the accompanying drawings. 
           [0015]      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 an aspect of the present invention. The his-tag for affinity purification (white), aMTD165 (black), SOCS3 (gray) and solubilization domain A and B (SDA &amp; SDB, hatched) are shown. 
           [0016]      FIG. 2  shows the construction of expression for SOCS3 recombinant proteins.  FIG. 2  shows the agarose gel electrophoresis analysis showing plasmid DNA fragments encoding SOCS3, aMTDs fused SOCS3 and SD cloned into the pET28 (+) vector according to an aspect of the present invention. 
           [0017]      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. 
           [0018]      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). 
           [0019]      FIG. 5  shows aMTD-mediated cell-permeability of SOCS3 recombinant proteins. RAW264.7 cells were exposed to FITC-labeled SOCS3 recombinant proteins (10 μM) 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. 
           [0020]      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 μM FITC-labeled SOCS3 protein. Cell-permeability of SOCS3 recombinant proteins was visualized by utilizing confocal microscopy LSM700 version. 
           [0021]      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 μg FITC-conjugated recombinant SOCS3 proteins, and were analyzed by fluorescence microscopy. 
           [0022]      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. 
           [0023]      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). 
           [0024]      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 μM antimycin for 2 hrs either without or with 1 mM supplemental ATP for 3 hrs. Cells were exposed for 1 hr to 10 μM 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 μM FITC-labeled HS3 (black), -HS3B (blue) or -HM165S3B (red), treated with proteinase K, and analyzed by flow cytometry. 
           [0025]      FIG. 11  shows aMTD-mediated cell-to-cell delivery. RAW264.7 cells exposed to 10 μM FITC-HS3B or FITC-HM 165 S3B for 2 hrs, were mixed with non-treated RAW264.7 cells pre-stained with Cy5.5 labeled anti-CD14 antibody, and analyzed by flow cytometry (left, top). 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. 
           [0026]      FIG. 12  shows the inhibition of STAT phosphorylation Induced by IFN-γ. Inhibition of STAT1 phosphorylation detected by immunoblotting analysis. The levels of phosphorylated STAT1 and STAT3 untreated and treated with IFN-γ were compared to the levels in IFN-γ-treated RAW 264.7 cells that were pulsed with 10 μM of indicated proteins. 
           [0027]      FIG. 13  shows the inhibition of cytokines secretion induced by LPS. Inhibition of TNF-α 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. 
           [0028]      FIG. 14  shows the cell-permeability of iCP-SOCS3 (HM 165 S3B) in hepatocellular carcinoma cell. RAW264.7 cells were exposed to FITC-labeled SOCS3 recombinant proteins (10 μM) 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. 
           [0029]      FIG. 15  shows the tissue distribution of iCP-SOCS3 (HM 165 S3B) into liver. Cryosections of saline-perfused organs were prepared from mice 1 hr after intraperitoneal injection of FITC only or 600 μg FITC-conjugated recombinant SOCS3 proteins, and were analyzed by fluorescence microscopy. 
           [0030]      FIG. 16  shows the inhibition of cancer cell proliferation with iCP-SOCS3 recombinant protein. Hep3B2.1-7 HCC cell was 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 h in the presence of serum (2%). Cell viability was evaluated with the CellTiter-Glo Cell Viability Assay. 
           [0031]      FIG. 17  shows the induction of apoptosis in hepatocellular carcinoma cell with iCP-SOCS3. HepG2 cells were treated for 24 hr with 10 μM HS3B or HM 165 S3B proteins and apoptotic cells were visualized by TUNEL staining. 
           [0032]      FIG. 18  shows the stimulation of apoptosis in hepatocellular carcinoma cell with iCP-SOCS3. HepG2 cells were treated for 24 hr with 10 μM HS3B or HM 165 S3B proteins and analyzed by flow cytometry of cells stained with annexin-V and 7-AAD. 
           [0033]      FIG. 19  shows the alternation of molecular mechanism in hepatocellular carcinoma Cell with iCP-SOCS3. HepG2 cells were treated for 24 hr with 10 μM 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. 
           [0034]      FIG. 20  shows the inhibition of hepatocellular carcinoma cell migration with iCP-SOCS3. HepG2 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. 
           [0035]      FIG. 21  shows the inhibition of migration/invasion in hepatocellular carcinoma cell with iCP-SOCS3. HepG2 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&lt;0.01. 
           [0036]      FIG. 22  shows the inhibition of tumor growth of human hepatocellular carcinoma in a mouse Xenograft model treated with iCP-SOCS3. Female Balb/c nu/nu mice were subcutaneously implanted with Hep3B2.1-7 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 1, 21 and 35 after starting protein therapy. 
           [0037]      FIG. 23  shows the inhibition of primary tumor on experimental in vivo model treated with iCP-SOCS3. Female Balb/c nu/nu mice were subcutaneously implanted with Hep3B2.1-7 tumor block (1 mm3) 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. 
           [0038]      FIG. 24  shows the changes in biomarker expression linked to SOCS3 signaling in tumor Xenograft. Female Balb/c nu/nu mice were subcutaneously implanted with Hep3B2.1-7 tumor block (1 mm3) 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. The expression of each protein was determined by immunoblotting with anti-p21 or Bax 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 Bax or VEGF. 
       
    
    
     DETAILED DESCRIPTION 
       [0039]    It has been hypothesized that exogenously administered SOCS3 proteins could compensate for the apparent inability of endogenously expressed members of this physiologic regulator to interrupt constitutively active cancer-initiating JAK/STAT signaling and excessive cell cycle, resulting in the inhibition of the tumorigenesis. To prove our hypothesis, the SOCS3 recombinant proteins fused to novel hydrophobic CPPs called aMTDs to improve their cell-/tissue-permeability and additionally adopted solubilization domains to increase their solubility/yield in physiological condition, and then tested whether exogenous administration of SOCS3 proteins can reconstitute their endogenous stores and restore their basic function as the negative feedback regulator that attenuates JAK/STAT signaling. This art of invention has demonstrated “intracellular protein therapy” by designing and introducing cell-permeable form of SOCS3 has a great potential of anti-cancer therapeutic applicability in hepatocellular carcinoma. 
       1. Novel Hydrophobic Cell-Penetrating Peptides—Advanced Macromolecule Transduction Domains 
       [0040]    To address the limitation of previously developed hydrophobic CPPs, novel sequences have been developed. To design new hydrophobic CPPs for intracellular delivery of cargo proteins such as SOCS3, identification of optimal common sequence and/or homologous structural determinants, namely critical factors (CFs), had been crucial. To do it, the physicochemical characteristics of previously published hydrophobic CPPs were analyzed. To keep the similar mechanism on cellular uptake, all CPPs analyzed were hydrophobic region of signal peptide (HRSP)-derived CPPs (e.g. MTS and MTD). 
       (1) Basic Characteristics of CPPs Sequence. 
       [0041]    These 17 hydrophobic CPPs published from 1995 to 2014 have been analyzed for their 11 different characteristics—sequence, amino acid length, molecular weight, pI value, bending potential, rigidity/flexibility, structural feature, hydropathy, residue structure, amino acid composition, and secondary structure of the sequences. Two peptide/protein analysis programs were used (ExPasy: http://web.expasy.org/protparam/, SoSui: http://harriernagahama-i-bio.ac.jp/sosui/sosui_submit.html) to determine various indexes, structural features of the peptide sequences and to design new sequence. Followings are important factors analyzed. 
         [0042]    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. 
       (2) Bending Potential (Proline Position: PP) 
       [0043]    Bending potential (Bending or No-Bending) 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. As indicated above, 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. 
       (3) Rigidity/Flexibility (Instability Index: II) 
       [0044]    Since one of the crucial structural features of any peptide is based on the fact whether the motif is rigid or flexible, which is an intact physicochemical characteristic of the peptide sequence, 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. 
       (4) Hydropathy (Grand Average of Hydropathy: GRAVY) and Structural Feature (Aliphatic Index: AI) 
       [0045]    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. 
         [0000]    (5) Secondary Structure (a-Helix) 
         [0046]    As explained above, the CPP sequences may be supposed to penetrate the plasma membrane directly after inserting into the membranes in a “bent” configuration with hydrophobic sequences adopting an a-helical conformation. In addition, 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. 
       (6) Determination of Critical Factors (CFs) 
       [0047]    In the 11 characteristics analyzed, the following 6 are selected namely “Critical Factors (CFs)” 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). 
       1-2. Analysis of Selected Hydrophobic CPPs to Optimize ‘Critical Factors’ 
       [0048]    Since the analyzed data of the 17 different hydrophobic CPPs (analysis A) previously developed during the past 2 decades showed high variation and were hard to make common- or consensus-features, additional analysis B and C was also conducted to optimize the critical factors for better design of improved CPPs-aMTDs. 
         [0049]    In analysis B, 8 CPPs used with each cargo in vivo were selected. Length was 11±3.2, but 3 out of 8 CPPs possessed little bending potential. Rigidity/Flexibility was 41±15, but removing one [MTD85: rigid, with minimal (II: 9.1)] of the peptides increased the overall instability index to 45.6±9.3. This suggested that higher flexibility (40 or higher II) is potentially be better. All other characteristics of the 8 CPPs were similar to the analysis A, including structural feature and hydropathy. 
         [0050]    To optimize the ‘Common Range and/or Consensus Feature of Critical Factor’ for the practical design of aMTDs and the random peptides, which were to prove that the ‘Critical Factors’ determined in the analysis A, B and C were correct to improve the current problems of hydrophobic CPPs—protein aggregation, low solubility/yield, and poor cell/tissue-permeability of the recombinant proteins fused to the MTS/MTM or MTD, and non-common sequence and non-homologous structure of the peptides, empirically selected peptides were analyzed for their structural features and physicochemical factor indexes. 
         [0051]    The peptides which did not have a bending potential, rigid or too flexible sequences (too low or too high Instability Index), or too low or too high hydrophobic CPP were unselected, but secondary structure was not considered because helix structure of sequence was not required. 8 selected CPP sequences that could provide a bending potential and higher flexibility were finally analyzed. Common amino acid length is 12 (11.6±3.0). Proline should be presence in the middle of and/or the end of sequence. Rigidity/Flexibility (II) is 45.5-57.3 (Avg: 50.1±3.6). AI and GRAVY representing structural feature and hydrophobicity of the peptide are 204.7±37.5 and 2.4±0.3, respectively. All peptides are consisted with hydrophobic and aliphatic amino acids (A, V, L, I, and P). Therefore, analysis C was chosen as a standard for the new design of new hydrophobic CPPs (TABLE 1).
       1. Amino Acid Length: 9-13   2. Bending Potential (Proline Position: PP): Proline presences in the middle (from 5′ to 8′ amino acid) and at the end of sequence   3. Rigidity/Flexibility (Instability Index: II): 40-60   4. Structural Feature (Aliphatic Index: AI): 180-220   5. Hydropathy (Grand Average of Hydropathy: GRAVY): 2.1-2.6   6. Amino Acid Composition: Hydrophobic and Aliphatic amino acids—A, V, L, I and P       
 
         [0000]                              TABLE 1                   [Universal structure of newly Develop Hydrophobic]       Summarized Critical Factors of aMTD                Newly Designed CPPs       Critical Factor   Range               Bending Potential   Proline presences in the middle (5′, 6′, 7′ or 8′)        (Proline Position: PP)   and at the end (12′) of peptides       Rigidity/Flexibility   40-60       (Instability Index: II)           Structural Feature   180-220       (Aliphatic Index: AI)           Hydropathy   2.1-2.6       (Grand Average of           Hydropathy GRAVY)           Length    9-13       (Number of Amino Acid)           Amino acid Composition   A, V, I, L, P                    
1-3. Determination of Critical Factors for Development of aMTDs
 
         [0058]    For confirming the validity of 6 critical factors providing the optimized cell-/tissue-permeability. 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. 
         [0059]    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 &amp; non-alipatic 12A/a length peptide) was significantly increased by up to 164 fold, with average increase of 19.6±1.6. Moreover, compared with reference CPPs (MTM and MTD), novel 240 aMTDs averaged of 13±1.1 (maximum 109.9) and 6.6±0.5 (maximum 55.5) fold higher cell-permeability, respectively. As a result, there were vivid association of cell-permeability of the peptides and critical factors. According to the result from the newly designed and tested novel 240 aMTDs, the empirically optimized critical factors are provided below.
       1. Amino Acid Length: 12   2. Bending Potential (Proline Position: PP): Proline presences in the middle (from 5′ to 8′ amino acid) and at the end of sequence   3. Rigidity/Flexibility (Instability Index: II): 41.3-57.3   4. Structural Feature (Aliphatic Index: AI): 187.5-220.0   5. Hydropathy (Grand Average of Hydropathy: GRAVY): 2.2-2.6   6. Amino Acid Composition: Hydrophobic and Aliphatic amino acids—A, V, L, I and P       
 
         [0000]    
       
         
               
             
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 2-1 
               
             
             
               
                   
               
               
                 [Summarized Critical Factor of aMTD After In-Depth 
               
               
                 Analysis of Experimental Result (aMTD 1-184)] 
               
             
          
           
               
                 Sequence 
                   
                   
                   
                 Rigidity/ 
                 Structural 
                   
                   
               
               
                 ID 
                   
                   
                   
                 Flexibility 
                 Feature 
                 Hydropathy 
                 Residue 
               
               
                 Number 
                 aMTD 
                 Sequences 
                 Length 
                 (II) 
                 (AI) 
                 (GRAVY) 
                 Structure 
               
               
                   
               
               
                  1 
                   1 
                 AAALAPVVLALP 
                 12 
                 57.3 
                 187.5 
                 2.1 
                 Aliphatic 
               
               
                   
               
               
                  2 
                   2 
                 AAAVPLLAVVVP 
                 12 
                 41.3 
                 195.0 
                 2.4 
                 Aliphatic 
               
               
                   
               
               
                  3 
                   3 
                 AALLVPAAVLAP 
                 12 
                 57.3 
                 187.5 
                 2.1 
                 Aliphatic 
               
               
                   
               
               
                  4 
                   4 
                 ALALLPVAALAP 
                 12 
                 57.3 
                 195.8 
                 2.1 
                 Aliphatic 
               
               
                   
               
               
                  5 
                   5 
                 AAALLPALVAP 
                 12 
                 57.3 
                 187.5 
                 2.1 
                 Aliphatic 
               
               
                   
               
               
                  6 
                  11 
                 VVALAPALAALP 
                 12 
                 57.3 
                 187.5 
                 2.1 
                 Aliphatic 
               
               
                   
               
               
                  7 
                  12 
                 LLAAVPAVLLAP 
                 12 
                 57.3 
                 211.7 
                 2.3 
                 Aliphatic 
               
               
                   
               
               
                  8 
                  13 
                 AAALVPVVALLP 
                 12 
                 57.3 
                 203.3 
                 2.3 
                 Aliphatic 
               
               
                   
               
               
                  9 
                  21 
                 AVALLPALLAVP 
                 12 
                 57.3 
                 211.7 
                 2.3 
                 Aliphatic 
               
               
                   
               
               
                 10 
                  22 
                 AVVLVPVLAAAP 
                 12 
                 57.3 
                 195.0 
                 2.4 
                 Aliphatic 
               
               
                   
               
               
                 11 
                  23 
                 VVLVLPAAAAVP 
                 12 
                 57.3 
                 195.0 
                 2.4 
                 Aliphatic 
               
               
                   
               
               
                 12 
                  24 
                 IALAAPALIVAP 
                 12 
                 50.2 
                 195.8 
                 2.2 
                 Aliphatic 
               
               
                   
               
               
                 13 
                  25 
                 IVAVAPALVALP 
                 12 
                 50.2 
                 203.3 
                 2.4 
                 Aliphatic 
               
               
                   
               
               
                 14 
                  42 
                 VAALPVVAVVAP 
                 12 
                 57.3 
                 186.7 
                 2.4 
                 Aliphatic 
               
               
                   
               
               
                 15 
                  43 
                 LLAAPLVVAAVP 
                 12 
                 41.3 
                 187.5 
                 2.1 
                 Aliphatic 
               
               
                   
               
               
                 16 
                  44 
                 ALAVPVALLVAP 
                 12 
                 57.3 
                 203.3 
                 2.3 
                 Aliphatic 
               
               
                   
               
               
                 17 
                  61 
                 VAALPVLLAALP 
                 12 
                 57.3 
                 211.7 
                 2.3 
                 Aliphatic 
               
               
                   
               
               
                 18 
                  62 
                 VALLAPVALAVP 
                 12 
                 57.3 
                 203.3 
                 2.3 
                 Aliphatic 
               
               
                   
               
               
                 19 
                  63 
                 AALLVPALVAVP 
                 12 
                 57.3 
                 203.3 
                 2.3 
                 Aliphatic 
               
               
                   
               
               
                 20 
                  64 
                 AIVALPVAVLAP 
                 12 
                 50.2 
                 203.3 
                 2.4 
                 Aliphatic 
               
               
                   
               
               
                 21 
                  65 
                 IAIVAPVVALAP 
                 12 
                 50.2 
                 203.3 
                 2.4 
                 Aliphatic 
               
               
                   
               
               
                 22 
                  81 
                 AALLPALAALLP 
                 12 
                 57.3 
                 204.2 
                 2.1 
                 Aliphatic 
               
               
                   
               
               
                 23 
                  82 
                 AVVLAPVAAVLP 
                 12 
                 57.3 
                 195.0 
                 2.4 
                 Aliphatic 
               
               
                   
               
               
                 24 
                  83 
                 LAVAAPLALALP 
                 12 
                 41.3 
                 195.8 
                 2.1 
                 Aliphatic 
               
               
                   
               
               
                 25 
                  84 
                 AAVAAPLLLALP 
                 12 
                 41.3 
                 195.8 
                 2.1 
                 Aliphatic 
               
               
                   
               
               
                 26 
                  85 
                 LLVLPAAALAAP 
                 12 
                 57.3 
                 195.8 
                 2.1 
                 Aliphatic 
               
               
                   
               
               
                 27 
                 101 
                 LVALAPVAAVLP 
                 12 
                 57.3 
                 203.3 
                 2.3 
                 Aliphatic 
               
               
                   
               
               
                 28 
                 102 
                 LALAPAALALLP 
                 12 
                 57.3 
                 204.2 
                 2.1 
                 Aliphatic 
               
               
                   
               
               
                 29 
                 103 
                 ALIAAPILALAP 
                 12 
                 57.3 
                 204.2 
                 2.2 
                 Aliphatic 
               
               
                   
               
               
                 30 
                 104 
                 AVVAAPLVLALP 
                 12 
                 41.3 
                 203.3 
                 2.3 
                 Aliphatic 
               
               
                   
               
               
                 31 
                 105 
                 LLALAPAALLAP 
                 12 
                 57.3 
                 204.1 
                 2.1 
                 Aliphatic 
               
               
                   
               
               
                 32 
                 121 
                 AIVALPALALAP 
                 12 
                 50.2 
                 195.8 
                 2.2 
                 Aliphatic 
               
               
                   
               
               
                 33 
                 123 
                 AAIIVPAALLAP 
                 12 
                 50.2 
                 195.8 
                 2.2 
                 Aliphatic 
               
               
                   
               
               
                 34 
                 124 
                 IAVALPALIAAP 
                 12 
                 50.3 
                 195.8 
                 2.2 
                 Aliphatic 
               
               
                   
               
               
                 35 
                 141 
                 AVIVLPALAVAP 
                 12 
                 50.2 
                 203.3 
                 2.4 
                 Aliphatic 
               
               
                   
               
               
                 36 
                 143 
                 AVLAVPAVLVAP 
                 12 
                 57.3 
                 195.0 
                 2.4 
                 Aliphatic 
               
               
                   
               
               
                 37 
                 144 
                 VLAIVPAVALAP 
                 12 
                 50.2 
                 203.3 
                 2.4 
                 Aliphatic 
               
               
                   
               
               
                 38 
                 145 
                 LLAVVPAVALAP 
                 12 
                 57.3 
                 203.3 
                 2.3 
                 Aliphatic 
               
               
                   
               
               
                 39 
                 161 
                 AVIALPALIAAP 
                 12 
                 57.3 
                 195.8 
                 2.2 
                 Aliphatic 
               
               
                   
               
               
                 40 
                 162 
                 AVVALPAALIVP 
                 12 
                 50.2 
                 203.3 
                 2.4 
                 Aliphatic 
               
               
                   
               
               
                 41 
                 163 
                 LALVLPAALAAP 
                 12 
                 57.3 
                 195.8 
                 2.1 
                 Aliphatic 
               
               
                   
               
               
                 42 
                 164 
                 LAAVLPALLAAP 
                 12 
                 57.3 
                 195.8 
                 2.1 
                 Aliphatic 
               
               
                   
               
               
                 43 
                 165 
                 ALAVPVALAIVP 
                 12 
                 50.2 
                 203.3 
                 2.4 
                 Aliphatic 
               
               
                   
               
               
                 44 
                 182 
                 ALIAPVVALVAP 
                 12 
                 57.3 
                 203.3 
                 2.4 
                 Aliphatic 
               
               
                   
               
               
                 45 
                 183 
                 LLAAPVVIALAP 
                 12 
                 57.3 
                 211.6 
                 2.4 
                 Aliphatic 
               
               
                   
               
               
                 46 
                 184 
                 LAAIVPAIIAVP 
                 12 
                 50.2 
                 211.6 
                 2.4 
                 Aliphatic 
               
               
                   
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 2-2 
               
             
             
               
                   
               
               
                 [Summarized Critical Factor of aMTD After In-Depth 
               
               
                 Analysis of Experimental Result (aMTD 185-401)] 
               
             
          
           
               
                 Sequence 
                   
                   
                   
                 Rigidity/ 
                 Structural 
                   
                   
               
               
                 ID 
                   
                   
                   
                 Flexibility 
                 Feature 
                 Hydropathy 
                 Residue 
               
               
                 Number 
                 aMTD 
                 Sequences 
                 Length 
                 (II) 
                 (AI) 
                 (GRAVY) 
                 Structure 
               
               
                   
               
               
                 47 
                 185 
                 AALVLPLIIAAP 
                 12 
                 41.3 
                 220.0 
                 2.4 
                 Aliphatic 
               
               
                   
               
               
                 48 
                 201 
                 LALAVPALAALP 
                 12 
                 57.3 
                 195.8 
                 2.1 
                 Aliphatic 
               
               
                   
               
               
                 49 
                 204 
                 LIAALPAVAALP 
                 12 
                 57.3 
                 195.8 
                 2.2 
                 Aliphatic 
               
               
                   
               
               
                 50 
                 205 
                 ALALVPAIAALP 
                 12 
                 57.3 
                 195.8 
                 2.2 
                 Aliphatic 
               
               
                   
               
               
                 51 
                 221 
                 AAILAPIVALAP 
                 12 
                 50.2 
                 195.8 
                 2.2 
                 Aliphatic 
               
               
                   
               
               
                 52 
                 222 
                 ALLIAPAAVIAP 
                 12 
                 57.3 
                 195.8 
                 2.2 
                 Aliphatic 
               
               
                   
               
               
                 53 
                 223 
                 AILAVPIAVVAP 
                 12 
                 57.3 
                 203.3 
                 2.4 
                 Aliphatic 
               
               
                   
               
               
                 54 
                 224 
                 ILAAVPIALAAP 
                 12 
                 57.3 
                 195.8 
                 2.2 
                 Aliphatic 
               
               
                   
               
               
                 55 
                 225 
                 VAALLPAAAVLP 
                 12 
                 57.3 
                 187.5 
                 2.1 
                 Aliphatic 
               
               
                   
               
               
                 56 
                 241 
                 AAAVVPVLLVAP 
                 12 
                 57.3 
                 195.0 
                 2.4 
                 Aliphatic 
               
               
                   
               
               
                 57 
                 242 
                 AALLVPALVAAP 
                 12 
                 57.3 
                 187.5 
                 2.1 
                 Aliphatic 
               
               
                   
               
               
                 58 
                 243 
                 AAVLLPVALAAP 
                 12 
                 57.3 
                 187.5 
                 2.1 
                 Aliphatic 
               
               
                   
               
               
                 59 
                 245 
                 AAALAPVLALVP 
                 12 
                 57.3 
                 187.5 
                 2.1 
                 Aliphatic 
               
               
                   
               
               
                 60 
                 261 
                 LVLVPLLAAAAP 
                 12 
                 41.3 
                 211.6 
                 2.3 
                 Aliphatic 
               
               
                   
               
               
                 61 
                 262 
                 ALIAVPAIIVAP 
                 12 
                 50.2 
                 211.6 
                 2.4 
                 Aliphatic 
               
               
                   
               
               
                 62 
                 263 
                 ALAVIPAAAILP 
                 12 
                 54.9 
                 195.8 
                 2.2 
                 Aliphatic 
               
               
                   
               
               
                 63 
                 264 
                 LAAAPVVIVIAP 
                 12 
                 50.2 
                 203.3 
                 2.4 
                 Aliphatic 
               
               
                   
               
               
                 64 
                 265 
                 VLAIAPLLAAVP 
                 12 
                 41.3 
                 211.6 
                 2.3 
                 Aliphatic 
               
               
                   
               
               
                 65 
                 281 
                 ALIVLPAAVAVP 
                 12 
                 50.2 
                 203.3 
                 2.4 
                 Aliphatic 
               
               
                   
               
               
                 66 
                 282 
                 VLAVAPALIVAP 
                 12 
                 50.2 
                 203.3 
                 2.4 
                 Aliphatic 
               
               
                   
               
               
                 67 
                 283 
                 AALLAPALIVAP 
                 12 
                 50.2 
                 195.8 
                 2.2 
                 Aliphatic 
               
               
                   
               
               
                 68 
                 284 
                 ALIAPAVALIVP 
                 12 
                 50.2 
                 211.7 
                 2.4 
                 Aliphatic 
               
               
                   
               
               
                 69 
                 285 
                 AIVLLPAAVVAP 
                 12 
                 50.2 
                 203.3 
                 2.4 
                 Aliphatic 
               
               
                   
               
               
                 70 
                 301 
                 VIAAPVLAVLAP 
                 12 
                 57.3 
                 203.3 
                 2.4 
                 Aliphatic 
               
               
                   
               
               
                 71 
                 302 
                 LALAPALALLAP 
                 12 
                 57.3 
                 204.2 
                 2.1 
                 Aliphatic 
               
               
                   
               
               
                 72 
                 304 
                 AIILAPIAAIAP 
                 12 
                 57.3 
                 204.2 
                 2.3 
                 Aliphatic 
               
               
                   
               
               
                 73 
                 305 
                 IALAAPILLAAP 
                 12 
                 57.3 
                 204.2 
                 2.2 
                 Aliphatic 
               
               
                   
               
               
                 74 
                 321 
                 IVAVALPALAVP 
                 12 
                 50.2 
                 203.3 
                 2.3 
                 Aliphatic 
               
               
                   
               
               
                 75 
                 322 
                 VVAIVLPALAAP 
                 12 
                 50.2 
                 203.3 
                 2.3 
                 Aliphatic 
               
               
                   
               
               
                 76 
                 323 
                 IVAVALPVALAP 
                 12 
                 50.2 
                 203.3 
                 2.3 
                 Aliphatic 
               
               
                   
               
               
                 77 
                 324 
                 IVAVALPAALVP 
                 12 
                 50.2 
                 203.3 
                 2.3 
                 Aliphatic 
               
               
                   
               
               
                 78 
                 325 
                 IVAVALPAVALP 
                 12 
                 50.2 
                 203.3 
                 2.3 
                 Aliphatic 
               
               
                   
               
               
                 79 
                 341 
                 IVAVALPAVLAP 
                 12 
                 50.2 
                 203.3 
                 2.3 
                 Aliphatic 
               
               
                   
               
               
                 80 
                 342 
                 VIVALAPAVLAP 
                 12 
                 50.2 
                 203.3 
                 2.3 
                 Aliphatic 
               
               
                   
               
               
                 81 
                 343 
                 IVAVALPALVAP 
                 12 
                 50.2 
                 203.3 
                 2.3 
                 Aliphatic 
               
               
                   
               
               
                 82 
                 345 
                 ALLIVAPVAVAP 
                 12 
                 50.2 
                 203.3 
                 2.3 
                 Aliphatic 
               
               
                   
               
               
                 83 
                 361 
                 AVVIVAPAVIAP 
                 12 
                 50.2 
                 195.0 
                 2.4 
                 Aliphatic 
               
               
                   
               
               
                 84 
                 363 
                 AVLAVAPALIVP 
                 12 
                 50.2 
                 203.3 
                 2.3 
                 Aliphatic 
               
               
                   
               
               
                 85 
                 364 
                 LVAAVAPALIVP 
                 12 
                 50.2 
                 203.3 
                 2.3 
                 Aliphatic 
               
               
                   
               
               
                 86 
                 365 
                 AVIVVAPALLAP 
                 12 
                 50.2 
                 203.3 
                 2.3 
                 Aliphatic 
               
               
                   
               
               
                 87 
                 381 
                 VVAIVLPAVAAP 
                 12 
                 50.2 
                 195.0 
                 2.4 
                 Aliphatic 
               
               
                   
               
               
                 88 
                 382 
                 AAALVIPAILAP 
                 12 
                 54.9 
                 195.8 
                 2.2 
                 Aliphatic 
               
               
                   
               
               
                 89 
                 383 
                 VIVALAPALLAP 
                 12 
                 50.2 
                 211.6 
                 2.3 
                 Aliphatic 
               
               
                   
               
               
                 90 
                 384 
                 VIVAIAPALLAP 
                 12 
                 50.2 
                 211.6 
                 2.4 
                 Aliphatic 
               
               
                   
               
               
                 91 
                 385 
                 IVAIAVPALVAP 
                 12 
                 50.2 
                 203.3 
                 2.4 
                 Aliphatic 
               
               
                   
               
               
                 92 
                 401 
                 AALAVIPAAILP 
                 12 
                 54.9 
                 195.8 
                 2.2 
                 Aliphatic 
               
               
                   
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 2-3 
               
             
             
               
                   
               
               
                 [Summarized Critical Factor of aMTD After In-Depth 
               
               
                 Analysis of Experimental Result (aMTD 402-602)] 
               
             
          
           
               
                 Sequence 
                   
                   
                   
                 Rigidity/ 
                 Structural 
                   
                   
               
               
                 ID 
                   
                   
                   
                 Flexibility 
                 Feature 
                 Hydropathy 
                 Residue 
               
               
                 Number 
                 aMTD 
                 Sequences 
                 Length 
                 (II) 
                 (AI) 
                 (GRAVY) 
                 Structure 
               
               
                   
               
               
                  93 
                 402 
                 ALAAVIPAAILP 
                 12 
                 54.9 
                 195.8 
                 2.2 
                 Aliphatic 
               
               
                   
               
               
                  94 
                 403 
                 AAALVIPAAILP 
                 12 
                 54.9 
                 195.8 
                 2.2 
                 Aliphatic 
               
               
                   
               
               
                  95 
                 404 
                 LAAAVIPAAILP 
                 12 
                 54.9 
                 195.8 
                 2.2 
                 Aliphatic 
               
               
                   
               
               
                  96 
                 405 
                 LAAAVIPVAILP 
                 12 
                 54.9 
                 211.7 
                 2.4 
                 Aliphatic 
               
               
                   
               
               
                  97 
                 421 
                 AAILAAPLIAVP 
                 12 
                 57.3 
                 195.8 
                 2.2 
                 Aliphatic 
               
               
                   
               
               
                  98 
                 422 
                 VVAILAPLLAAP 
                 12 
                 57.3 
                 211.7 
                 2.4 
                 Aliphatic 
               
               
                   
               
               
                  99 
                 424 
                 AVVVAAPVLALP 
                 12 
                 57.3 
                 195.0 
                 2.4 
                 Aliphatic 
               
               
                   
               
               
                 100 
                 425 
                 AVVAIAPVLALP 
                 12 
                 57.3 
                 203.3 
                 2.4 
                 Aliphatic 
               
               
                   
               
               
                 101 
                 442 
                 ALAALVPAVLVP 
                 12 
                 57.3 
                 203.3 
                 2.3 
                 Aliphatic 
               
               
                   
               
               
                 102 
                 443 
                 ALAALVPVALVP 
                 12 
                 57.3 
                 203.3 
                 2.3 
                 Aliphatic 
               
               
                   
               
               
                 103 
                 444 
                 LAAALVPVALVP 
                 12 
                 57.3 
                 203.3 
                 2.3 
                 Aliphatic 
               
               
                   
               
               
                 104 
                 445 
                 ALAALVPALVVP 
                 12 
                 57.3 
                 203.3 
                 2.3 
                 Aliphatic 
               
               
                   
               
               
                 105 
                 461 
                 IAAVIVPAVALP 
                 12 
                 50.2 
                 203.3 
                 2.4 
                 Aliphatic 
               
               
                   
               
               
                 106 
                 462 
                 IAAVLVPAVALP 
                 12 
                 57.3 
                 203.3 
                 2.4 
                 Aliphatic 
               
               
                   
               
               
                 107 
                 463 
                 AVAILVPLLAAP 
                 12 
                 57.3 
                 211.7 
                 2.4 
                 Aliphatic 
               
               
                   
               
               
                 108 
                 464 
                 AVVILVPLAAAP 
                 12 
                 57.3 
                 203.3 
                 2.4 
                 Aliphatic 
               
               
                   
               
               
                 109 
                 465 
                 IAAVIVPVAALP 
                 12 
                 50.2 
                 203.3 
                 2.4 
                 Aliphatic 
               
               
                   
               
               
                 110 
                 481 
                 AIAIAIVPVALP 
                 12 
                 50.2 
                 211.6 
                 2.4 
                 Aliphatic 
               
               
                   
               
               
                 111 
                 482 
                 ILAVAAIPVAVP 
                 12 
                 54.9 
                 203.3 
                 2.4 
                 Aliphatic 
               
               
                   
               
               
                 112 
                 483 
                 ILAAAIIPAALP 
                 12 
                 54.9 
                 204.1 
                 2.2 
                 Aliphatic 
               
               
                   
               
               
                 113 
                 484 
                 LAVVLAAPAIVP 
                 12 
                 50.2 
                 203.3 
                 2.4 
                 Aliphatic 
               
               
                   
               
               
                 114 
                 485 
                 AILAAIVPLAVP 
                 12 
                 50.2 
                 211.6 
                 2.4 
                 Aliphatic 
               
               
                   
               
               
                 115 
                 501 
                 VIVALAVPALAP 
                 12 
                 50.2 
                 203.3 
                 2.4 
                 Aliphatic 
               
               
                   
               
               
                 116 
                 502 
                 AIVALAVPVLAP 
                 12 
                 50.2 
                 203.3 
                 2.4 
                 Aliphatic 
               
               
                   
               
               
                 117 
                 503 
                 AAIIIVLPAALP 
                 12 
                 50.2 
                 220.0 
                 2.4 
                 Aliphatic 
               
               
                   
               
               
                 118 
                 504 
                 LIVALAVPALAP 
                 12 
                 50.2 
                 211.7 
                 2.4 
                 Aliphatic 
               
               
                   
               
               
                 119 
                 505 
                 AIIIVIAPAAAP 
                 12 
                 50.2 
                 195.8 
                 2.3 
                 Aliphatic 
               
               
                   
               
               
                 120 
                 521 
                 LAALIVVPAVAP 
                 12 
                 50.2 
                 203.3 
                 2.4 
                 Aliphatic 
               
               
                   
               
               
                 121 
                 522 
                 ALLVIAVPAVAP 
                 12 
                 57.3 
                 203.3 
                 2.4 
                 Aliphatic 
               
               
                   
               
               
                 122 
                 524 
                 AVALIVVPALAP 
                 12 
                 50.2 
                 203.3 
                 2.4 
                 Aliphatic 
               
               
                   
               
               
                 123 
                 525 
                 ALAIVVAPVAVP 
                 12 
                 50.2 
                 195.0 
                 2.4 
                 Aliphatic 
               
               
                   
               
               
                 124 
                 541 
                 LLALIIAPAAAP 
                 12 
                 57.3 
                 204.1 
                 2.1 
                 Aliphatic 
               
               
                   
               
               
                 125 
                 542 
                 ALAIIIVPAVAP 
                 12 
                 50.2 
                 211.6 
                 2.4 
                 Aliphatic 
               
               
                   
               
               
                 126 
                 543 
                 LLAALIAPAALP 
                 12 
                 57.3 
                 204.1 
                 2.1 
                 Aliphatic 
               
               
                   
               
               
                 127 
                 544 
                 IVALIVAPAAVP 
                 12 
                 43.1 
                 203.3 
                 2.4 
                 Aliphatic 
               
               
                   
               
               
                 128 
                 545 
                 VVLVLAAPAAVP 
                 12 
                 57.3 
                 195.0 
                 2.3 
                 Aliphatic 
               
               
                   
               
               
                 129 
                 561 
                 AAVAIVLPAVVP 
                 12 
                 50.2 
                 195.0 
                 2.4 
                 Aliphatic 
               
               
                   
               
               
                 130 
                 562 
                 ALIAAIVPALVP 
                 12 
                 50.2 
                 211.7 
                 2.4 
                 Aliphatic 
               
               
                   
               
               
                 131 
                 563 
                 ALAVIVVPALAP 
                 12 
                 50.2 
                 203.3 
                 2.4 
                 Aliphatic 
               
               
                   
               
               
                 132 
                 564 
                 VAIALIVPALAP 
                 12 
                 50.2 
                 211.7 
                 2.4 
                 Aliphatic 
               
               
                   
               
               
                 133 
                 565 
                 VAIVLVAPAVAP 
                 12 
                 50.2 
                 195.0 
                 2.4 
                 Aliphatic 
               
               
                   
               
               
                 134 
                 582 
                 VAVALIVPALAP 
                 12 
                 50.2 
                 203.3 
                 2.4 
                 Aliphatic 
               
               
                   
               
               
                 135 
                 583 
                 AVILALAPIVAP 
                 12 
                 50.2 
                 211.6 
                 2.4 
                 Aliphatic 
               
               
                   
               
               
                 136 
                 585 
                 ALIVAIAPALVP 
                 12 
                 50.2 
                 211.6 
                 2.4 
                 Aliphatic 
               
               
                   
               
               
                 137 
                 601 
                 AAILIAVPIAAP 
                 12 
                 57.3 
                 195.8 
                 2.3 
                 Aliphatic 
               
               
                   
               
               
                 138 
                 602 
                 VIVALAAPVLAP 
                 12 
                 50.2 
                 203.3 
                 2.4 
                 Aliphatic 
               
               
                   
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 2-4 
               
             
             
               
                   
               
               
                 [Summarized Critical Factor of aMTD After In-Depth 
               
               
                 Analysis of Experimental Result (aMTD 603-809)] 
               
             
          
           
               
                 Sequence 
                   
                   
                   
                 Rigidity/ 
                 Sturctural 
                   
                   
               
               
                 ID 
                   
                   
                   
                 Flexibility 
                 Feature 
                 Hydropathy 
                 Residue 
               
               
                 Number 
                 aMTD 
                 Sequences 
                 Length 
                 (II) 
                 (AI) 
                 (GRAVY) 
                 Structure 
               
               
                   
               
               
                 139 
                 603 
                 VLVALAAPVIAP 
                 12 
                 57.3 
                 203.3 
                 2.4 
                 Aliphatic 
               
               
                   
               
               
                 140 
                 604 
                 VALIAVAPAVVP 
                 12 
                 57.3 
                 195.0 
                 2.4 
                 Aliphatic 
               
               
                   
               
               
                 141 
                 605 
                 VIAAVLAPVAVP 
                 12 
                 57.3 
                 195.0 
                 2.4 
                 Aliphatic 
               
               
                   
               
               
                 142 
                 622 
                 ALIVLAAPVAVP 
                 12 
                 50.2 
                 203.3 
                 2.4 
                 Aliphatic 
               
               
                   
               
               
                 143 
                 623 
                 VAAAIALPAIVP 
                 12 
                 50.2 
                 187.5 
                 2.3 
                 Aliphatic 
               
               
                   
               
               
                 144 
                 625 
                 ILAAAAAPLIVP 
                 12 
                 50.2 
                 195.8 
                 2.2 
                 Aliphatic 
               
               
                   
               
               
                 145 
                 643 
                 LALVLAAPAIVP 
                 12 
                 50.2 
                 211.6 
                 2.4 
                 Aliphatic 
               
               
                   
               
               
                 146 
                 645 
                 ALAVVALPAIVP 
                 12 
                 50.2 
                 203.3 
                 2.4 
                 Aliphatic 
               
               
                   
               
               
                 147 
                 661 
                 AAILAPIVAALP 
                 12 
                 50.2 
                 195.8 
                 2.2 
                 Aliphatic 
               
               
                   
               
               
                 148 
                 664 
                 ILIAIAIPAAAP 
                 12 
                 54.9 
                 204.1 
                 2.3 
                 Aliphatic 
               
               
                   
               
               
                 149 
                 665 
                 LAIVIAAPVAVP 
                 12 
                 50.2 
                 203.3 
                 2.3 
                 Aliphatic 
               
               
                   
               
               
                 150 
                 666 
                 AAIAIIAPAIVP 
                 12 
                 50.2 
                 195.8 
                 2.3 
                 Aliphatic 
               
               
                   
               
               
                 151 
                 667 
                 LAVAIVAPALVP 
                 12 
                 50.2 
                 203.3 
                 2.3 
                 Aliphatic 
               
               
                   
               
               
                 152 
                 683 
                 LAIVLAAPAVLP 
                 12 
                 50.2 
                 211.7 
                 2.4 
                 Aliphatic 
               
               
                   
               
               
                 153 
                 684 
                 AAIVLALPAVLP 
                 12 
                 50.2 
                 211.7 
                 2.4 
                 Aliphatic 
               
               
                   
               
               
                 154 
                 685 
                 ALLVAVLPAALP 
                 12 
                 57.3 
                 211.7 
                 2.3 
                 Aliphatic 
               
               
                   
               
               
                 155 
                 686 
                 AALVAVLPVALP 
                 12 
                 57.3 
                 203.3 
                 2.3 
                 Aliphatic 
               
               
                   
               
               
                 156 
                 687 
                 AILAVALPLLAP 
                 12 
                 57.3 
                 220.0 
                 2.3 
                 Aliphatic 
               
               
                   
               
               
                 157 
                 703 
                 IVAVALVPALAP 
                 12 
                 50.2 
                 203.3 
                 2.4 
                 Aliphatic 
               
               
                   
               
               
                 158 
                 705 
                 IVAVALLPALAP 
                 12 
                 50.2 
                 211.7 
                 2.4 
                 Aliphatic 
               
               
                   
               
               
                 159 
                 706 
                 IVAVALLPAVAP 
                 12 
                 50.2 
                 203.3 
                 2.4 
                 Aliphatic 
               
               
                   
               
               
                 160 
                 707 
                 IVALAVLPAVAP 
                 12 
                 50.2 
                 203.3 
                 2.4 
                 Aliphatic 
               
               
                   
               
               
                 161 
                 724 
                 VAVLAVLPALAP 
                 12 
                 57.3 
                 203.3 
                 2.3 
                 Aliphatic 
               
               
                   
               
               
                 162 
                 725 
                 IAVLAVAPAVLP 
                 12 
                 57.3 
                 203.3 
                 2.3 
                 Aliphatic 
               
               
                   
               
               
                 163 
                 726 
                 LAVAIIAPAVAP 
                 12 
                 57.3 
                 187.5 
                 2.2 
                 Aliphatic 
               
               
                   
               
               
                 164 
                 727 
                 VALAIALPAVLP 
                 12 
                 57.3 
                 211.6 
                 2.3 
                 Aliphatic 
               
               
                   
               
               
                 165 
                 743 
                 AIAIALVPVALP 
                 12 
                 57.3 
                 211.6 
                 2.4 
                 Aliphatic 
               
               
                   
               
               
                 166 
                 744 
                 AAVVIVAPVALP 
                 12 
                 50.2 
                 195.0 
                 2.4 
                 Aliphatic 
               
               
                   
               
               
                 167 
                 746 
                 VAIIVVAPALAP 
                 12 
                 50.2 
                 203.3 
                 2.4 
                 Aliphatic 
               
               
                   
               
               
                 168 
                 747 
                 VALLAIAPALAP 
                 12 
                 57.3 
                 195.8 
                 2.2 
                 Aliphatic 
               
               
                   
               
               
                 169 
                 763 
                 VAVIIAVPALAP 
                 12 
                 57.3 
                 203.3 
                 2.3 
                 Aliphatic 
               
               
                   
               
               
                 170 
                 764 
                 AVALAVLPAVVP 
                 12 
                 57.3 
                 195.0 
                 2.3 
                 Aliphatic 
               
               
                   
               
               
                 171 
                 765 
                 AVALAVVPAVLP 
                 12 
                 57.3 
                 195.0 
                 2.3 
                 Aliphatic 
               
               
                   
               
               
                 172 
                 766 
                 IVVIAVAPAVAP 
                 12 
                 50.2 
                 195.0 
                 2.4 
                 Aliphatic 
               
               
                   
               
               
                 173 
                 767 
                 IVVAAVVPALAP 
                 12 
                 50.2 
                 195.0 
                 2.4 
                 Aliphatic 
               
               
                   
               
               
                 174 
                 783 
                 IVALVPAVAIAP 
                 12 
                 50.2 
                 203.3 
                 2.5 
                 Aliphatic 
               
               
                   
               
               
                 175 
                 784 
                 VAALPAVALVVP 
                 12 
                 57.3 
                 195.0 
                 2.4 
                 Aliphatic 
               
               
                   
               
               
                 176 
                 786 
                 LVAIAPLAVLAP 
                 12 
                 41.3 
                 211.7 
                 2.4 
                 Aliphatic 
               
               
                   
               
               
                 177 
                 787 
                 AVALVPVIVAAP 
                 12 
                 50.2 
                 195.0 
                 2.4 
                 Aliphatic 
               
               
                   
               
               
                 178 
                 788 
                 AIAVAIAPVALP 
                 12 
                 57.3 
                 187.5 
                 2.3 
                 Aliphatic 
               
               
                   
               
               
                 179 
                 803 
                 AIALAVPVLALP 
                 12 
                 57.3 
                 211.7 
                 2.4 
                 Aliphatic 
               
               
                   
               
               
                 180 
                 805 
                 LVLIAAAPIALP 
                 12 
                 41.3 
                 220.0 
                 2.4 
                 Aliphatic 
               
               
                   
               
               
                 181 
                 806 
                 LVALAVPAAVLP 
                 12 
                 57.3 
                 203.3 
                 2.3 
                 Aliphatic 
               
               
                   
               
               
                 182 
                 807 
                 AVALAVPALVLP 
                 12 
                 57.3 
                 203.3 
                 2.3 
                 Aliphatic 
               
               
                   
               
               
                 183 
                 808 
                 LVVLAAAPLAVP 
                 12 
                 41.3 
                 203.3 
                 2.3 
                 Aliphatic 
               
               
                   
               
               
                 184 
                 809 
                 LIVLAAPALAAP 
                 12 
                 50.2 
                 195.8 
                 2.2 
                 Aliphatic 
               
               
                   
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 2-5 
               
             
             
               
                   
               
               
                 [Summarized Critical Factor of aMTD After In-Depth 
               
               
                 Analysis of Experimental Result (aMTD 810-900)] 
               
             
          
           
               
                 Sequence 
                   
                   
                   
                 Rigidity/ 
                 Structural 
                   
                   
               
               
                 ID 
                   
                   
                   
                 Flexibility 
                 Feature 
                 Hydropathy 
                 Residue 
               
               
                 Number 
                 aMTD 
                 Sequences 
                 Length 
                 (II) 
                 (AI) 
                 (GRAVY) 
                 Structure 
               
               
                   
               
               
                 185 
                 810 
                 VIVLAAPALAAP 
                 12 
                 50.2 
                 187.5 
                 2.2 
                 Aliphatic 
               
               
                   
               
               
                 186 
                 811 
                 AVVLAVPALAVP 
                 12 
                 57.3 
                 195.0 
                 2.3 
                 Aliphatic 
               
               
                   
               
               
                 187 
                 824 
                 LIIVAAAPAVAP 
                 12 
                 50.2 
                 187.5 
                 2.3 
                 Aliphatic 
               
               
                   
               
               
                 188 
                 825 
                 IVAVIVAPAVAP 
                 12 
                 43.2 
                 195.0 
                 2.5 
                 Aliphatic 
               
               
                   
               
               
                 189 
                 826 
                 LVALAAPIIAVP 
                 12 
                 41.3 
                 211.7 
                 2.4 
                 Aliphatic 
               
               
                   
               
               
                 190 
                 827 
                 IAAVLAAPALVP 
                 12 
                 57.3 
                 187.5 
                 2.2 
                 Aliphatic 
               
               
                   
               
               
                 191 
                 828 
                 IALLAAPIIAVP 
                 12 
                 41.3 
                 220.0 
                 2.4 
                 Aliphatic 
               
               
                   
               
               
                 192 
                 829 
                 AALALVAPVIVP 
                 12 
                 50.2 
                 203.3 
                 2.4 
                 Aliphatic 
               
               
                   
               
               
                 193 
                 830 
                 IALVAAPVALVP 
                 12 
                 57.3 
                 203.3 
                 2.4 
                 Aliphatic 
               
               
                   
               
               
                 194 
                 831 
                 IIVAVAPAAIVP 
                 12 
                 43.2 
                 203.3 
                 2.5 
                 Aliphatic 
               
               
                   
               
               
                 195 
                 832 
                 AVAAIVPVIVAP 
                 12 
                 43.2 
                 195.0 
                 2.5 
                 Aliphatic 
               
               
                   
               
               
                 196 
                 843 
                 AVLVLVAPAAAP 
                 12 
                 41.3 
                 219.2 
                 2.5 
                 Aliphatic 
               
               
                   
               
               
                 197 
                 844 
                 VVALLAPLIAAP 
                 12 
                 41.3 
                 211.8 
                 2.4 
                 Aliphatic 
               
               
                   
               
               
                 198 
                 845 
                 AAVVIAPLLAVP 
                 12 
                 41.3 
                 203.3 
                 2.4 
                 Aliphatic 
               
               
                   
               
               
                 199 
                 846 
                 IAVAVAAPLLVP 
                 12 
                 41.3 
                 203.3 
                 2.4 
                 Aliphatic 
               
               
                   
               
               
                 200 
                 847 
                 LVAIVVLPAVAP 
                 12 
                 50.2 
                 219.2 
                 2.6 
                 Aliphatic 
               
               
                   
               
               
                 201 
                 848 
                 AVAIVVLPAVAP 
                 12 
                 50.2 
                 195.0 
                 2.4 
                 Aliphatic 
               
               
                   
               
               
                 202 
                 849 
                 AVILLAPLIAAP 
                 12 
                 57.3 
                 220.0 
                 2.4 
                 Aliphatic 
               
               
                   
               
               
                 203 
                 850 
                 LVIALAAPVALP 
                 12 
                 57.3 
                 211.7 
                 2.4 
                 Aliphatic 
               
               
                   
               
               
                 204 
                 851 
                 VLAVVLPAVALP 
                 12 
                 57.3 
                 219.2 
                 2.5 
                 Aliphatic 
               
               
                   
               
               
                 205 
                 852 
                 VLAVAAPAVLLP 
                 12 
                 57.3 
                 203.3 
                 2.3 
                 Aliphatic 
               
               
                   
               
               
                 206 
                 863 
                 AAVVLLPIIAAP 
                 12 
                 41.3 
                 211.7 
                 2.4 
                 Aliphatic 
               
               
                   
               
               
                 207 
                 864 
                 ALLVIAPAIAVP 
                 12 
                 57.3 
                 211.7 
                 2.4 
                 Aliphatic 
               
               
                   
               
               
                 208 
                 865 
                 AVLVIAVPAIAP 
                 12 
                 57.3 
                 203.3 
                 2.5 
                 Aliphatic 
               
               
                   
               
               
                 209 
                 867 
                 ALLVVIAPLAAP 
                 12 
                 41.3 
                 211.7 
                 2.4 
                 Aliphatic 
               
               
                   
               
               
                 210 
                 868 
                 VLVAAILPAAIP 
                 12 
                 54.9 
                 211.7 
                 2.4 
                 Aliphatic 
               
               
                   
               
               
                 211 
                 870 
                 VLVAAVLPIAAP 
                 12 
                 41.3 
                 203.3 
                 2.4 
                 Aliphatic 
               
               
                   
               
               
                 212 
                 872 
                 VLAAAVLPLVVP 
                 12 
                 41.3 
                 219.2 
                 2.5 
                 Aliphatic 
               
               
                   
               
               
                 213 
                 875 
                 AIAIVVPAVAVP 
                 12 
                 50.2 
                 195.0 
                 2.4 
                 Aliphatic 
               
               
                   
               
               
                 214 
                 877 
                 VAIIAVPAVVAP 
                 12 
                 57.3 
                 195.0 
                 2.4 
                 Aliphatic 
               
               
                   
               
               
                 215 
                 878 
                 IVALVAPAAVVP 
                 12 
                 50.2 
                 195.0 
                 2.4 
                 Aliphatic 
               
               
                   
               
               
                 216 
                 879 
                 AAIVLLPAVVVP 
                 12 
                 50.2 
                 219.1 
                 2.5 
                 Aliphatic 
               
               
                   
               
               
                 217 
                 881 
                 AALIVVPAVAVP 
                 12 
                 50.2 
                 195.0 
                 2.4 
                 Aliphatic 
               
               
                   
               
               
                 218 
                 882 
                 AIALVVPAVAVP 
                 12 
                 57.3 
                 195.0 
                 2.4 
                 Aliphatic 
               
               
                   
               
               
                 219 
                 883 
                 LAIVPAAIAALP 
                 12 
                 50.2 
                 195.8 
                 2.2 
                 Aliphatic 
               
               
                   
               
               
                 220 
                 885 
                 LVAIAPAVAVLP 
                 12 
                 57.3 
                 203.3 
                 2.4 
                 Aliphatic 
               
               
                   
               
               
                 221 
                 887 
                 VLAVAPAVAVLP 
                 12 
                 57.3 
                 195.0 
                 2.4 
                 Aliphatic 
               
               
                   
               
               
                 222 
                 888 
                 ILAVVAIPAAAP 
                 12 
                 54.9 
                 187.5 
                 2.3 
                 Aliphatic 
               
               
                   
               
               
                 223 
                 889 
                 ILVAAAPIAALP 
                 12 
                 57.3 
                 195.8 
                 2.2 
                 Aliphatic 
               
               
                   
               
               
                 224 
                 891 
                 ILAVAAIPAALP 
                 12 
                 54.9 
                 195.8 
                 2.2 
                 Aliphatic 
               
               
                   
               
               
                 225 
                 893 
                 VIAIPAILAAAP 
                 12 
                 54.9 
                 195.8 
                 2.3 
                 Aliphatic 
               
               
                   
               
               
                 226 
                 895 
                 AIIIVVPAIAAP 
                 12 
                 50.2 
                 211.7 
                 2.5 
                 Aliphatic 
               
               
                   
               
               
                 227 
                 896 
                 AILIVVAPIAAP 
                 12 
                 50.2 
                 211.7 
                 2.5 
                 Aliphatic 
               
               
                   
               
               
                 228 
                 897 
                 AVIVPVAIIAAP 
                 12 
                 50.2 
                 203.3 
                 2.5 
                 Aliphatic 
               
               
                   
               
               
                 229 
                 899 
                 AVVIALPAVVAP 
                 12 
                 57.3 
                 195.0 
                 2.4 
                 Aliphatic 
               
               
                   
               
               
                 230 
                 900 
                 ALVAVIAPVVAP 
                 12 
                 57.3 
                 195.0 
                 2.4 
                 Aliphatic 
               
               
                   
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 2-6 
               
             
             
               
                   
               
               
                 [Summarized Critical Factor of aMTD After In-Depth 
               
               
                 Analysis of Experimental Result (aMTD 901-912)] 
               
             
          
           
               
                 Sequence 
                   
                   
                   
                 Rigidity/ 
                 Structural 
                   
                   
               
               
                 ID 
                   
                   
                   
                 Flexibility 
                 Feature 
                 Hydropathy 
                 Residue 
               
               
                 Number 
                 aMTD 
                 Sequences 
                 Length 
                 (II) 
                 (AI) 
                 (GRAVY) 
                 Structure 
               
               
                   
               
               
                 231 
                 901 
                 ALVAVLPAVAVP 
                 12 
                 57.3 
                 195.0 
                 2.4 
                 Aliphatic 
               
               
                   
               
               
                 232 
                 902 
                 ALVAPLLAVAVP 
                 12 
                 41.3 
                 203.3 
                 2.3 
                 Aliphatic 
               
               
                   
               
               
                 233 
                 904 
                 AVLAVVAPVVAP 
                 12 
                 57.3 
                 186.7 
                 2.4 
                 Aliphatic 
               
               
                   
               
               
                 234 
                 905 
                 AVIAVAPLVVAP 
                 12 
                 41.3 
                 195.0 
                 2.4 
                 Aliphatic 
               
               
                   
               
               
                 235 
                 906 
                 AVIALAPVVVAP 
                 12 
                 57.3 
                 195 0 
                 2.4 
                 Aliphatic 
               
               
                   
               
               
                 236 
                 907 
                 VAIALAPVVVAP 
                 12 
                 57.3 
                 195.0 
                 2.4 
                 Aliphatic 
               
               
                   
               
               
                 237 
                 908 
                 VALALAPVVVAP 
                 12 
                 57.3 
                 195.0 
                 2.3 
                 Aliphatic 
               
               
                   
               
               
                 238 
                 910 
                 VAALLPAVVVAP 
                 12 
                 57.3 
                 195.0 
                 2.3 
                 Aliphatic 
               
               
                   
               
               
                 239 
                 911 
                 VALALPAVVVAP 
                 12 
                 57.3 
                 195.0 
                 2.3 
                 Aliphatic 
               
               
                   
               
               
                 240 
                 912 
                 VALLAPAVVVAP 
                 12 
                 57.3 
                 195.0 
                 2.3 
                 Aliphatic 
               
               
                   
                   
                   
                   
                 52.6 ± 5.1 
                 201.7 ± 7.8 
                 2.3 ± 0.1 
               
               
                   
               
             
          
         
       
     
         [0066]    These examined critical factors are within the range that we have set for our critical factors; therefore, we were able to confirm that the aMTDs that satisfy these critical factors have much higher cell-permeability (TABLE 3) and intracellular delivery potential compared to reference hydrophobic CPPs reported during the past two decades. 
         [0000]                              TABLE 3                   [Summarized Critical Factors of aMTD After In-Depth       Analysis of Experimental Results]       Summarized Critical Factors of aMTD                Analysis of Experimental Results       Critical Factor   Range               Bending Potential   Proline presences in the middle (5′, 6′, 7′ or 8′)       (Proline Position: PP)   and at the end (12′) of peptides       Rigidity/Flexibility   41.3-57.3       (Instability Index: II)           Structural Feature   187.5-220.0       (Aliphatic Index: AI)           Hydropathy   2.2-2.6       (Grand Average of           Hydropathy GRAVY)           Length   12       (Number of Amino Acid)           Amino acid Composition   A, V, I, L, P                    
2. Development of SOCS3 Recombinant Proteins Fused to aMTD and Solubilization Domain
 
2-1. Design of Novel Hydrophobic CPPs—aMTDs for Development of Recombinant SOCS3 Proteins
 
         [0067]    Based on these six critical factors proven by experimental data, newly designed advanced macromolecule transduction domains (aMTDs) have been developed, and optimized for their practical therapeutic usage to facilitate protein translocation across the membrane. For this present invention, cell-permeable SOCS3 recombinant proteins have been developed by adopting aMTD165 (TABLE 4) that satisfied all 6 critical factors (TABLE 5). 
         [0000]    
       
         
               
             
               
               
               
             
           
               
                 TABLE 4 
               
             
             
               
                   
               
               
                 [Amino Acid and Nucleotide Sequence of Newly 
               
               
                 Developed Advanced MTD 165 Which Follow All 
               
               
                 Critical Factors] 
               
             
          
           
               
                   
                 Amino Acid 
                   
               
               
                 ID 
                 Sequence 
                 Nucleotide Sequence 
               
               
                   
               
               
                 165 
                 ALAVPVALAIVP 
                 GCG CTG GCG GTG CCG GTG  
               
               
                   
                   
                 GCG CTG GCG ATT GTG CCG 
               
               
                   
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 5 
               
             
             
               
                   
               
               
                 [Critical Factors of aMTD165] 
               
             
          
           
               
                   
                   
                   
                   
                 Bending Potential  
                 Rigidity/ 
                 Sturctural 
                   
               
               
                   
                   
                 Theoretical 
                 M.W. 
                 Prolin Position 
                 Flexibility 
                 Feature 
                 Hydropathy 
               
             
          
           
               
                 ID 
                 Length 
                 pI 
                 (Da) 
                 5′ 
                 6′ 
                 12′ 
                 (II) 
                 ( Al) 
                 (GRAVY) 
               
               
                   
               
               
                 165 
                 12 
                 5.57 
                 1133.4 
                 — 
                 1 
                 1 
                 50.2 
                 195.8 
                 2.2 
               
               
                   
               
             
          
         
       
     
       2-2. Selection of Solubilization Domain (SD) for SOCS3 Recombinant Proteins 
       [0068]    In the previous study, 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.g. 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. 
         [0000]    
       
         
               
             
               
               
               
               
               
               
               
             
           
               
                 TABLE 6 
               
             
             
               
                   
               
               
                 [Information of Solublization Domains] 
               
             
          
           
               
                   
                   
                   
                 Protein 
                   
                 Instability 
                   
               
               
                 SD 
                 Genbank ID 
                 Origin 
                 (kDa) 
                 pI 
                 Index (II) 
                 GRAVY 
               
               
                   
               
               
                 A 
                 CP000113.1 
                 Bacteria 
                 23 
                 4.6 
                 48.1 
                 −0.1 
               
               
                 B 
                 BC086945.1 
                 Pansy 
                 11 
                 4.9 
                 43.2 
                 −0.9 
               
               
                 C 
                 CP012127.1 
                 Human 
                 12 
                 5.8 
                 30.7 
                 −0.1 
               
               
                 D 
                 CP012127.1 
                 Bacteria 
                 23 
                 5.9 
                 26.3 
                 −0.1 
               
               
                 E 
                 CP011550.1 
                 Human 
                 11 
                 5.3 
                 44.4 
                 −0.9 
               
               
                 F 
                 NG_034970 
                 Human 
                 34 
                 7.1 
                 56.1 
                 −0.2 
               
               
                   
               
             
          
         
       
     
         [0069]    According to the specific aim, solubilization domain A (SDA) and B (SDB) were first selected. We hypothesize that fusion of SOCS3 with SDs and novel hydrophobic CPP, aMTD, would greatly increase solubility/yield and cell-/tissue-permeability of recombinant cargo proteins -SOCS3—for the clinical application. SDA is a soluble tag, a tandem repeat of 2 N-terminal domain (NTD) sequences of CP 000113.1, which is a very stable soluble protein present in a spore surface coat of  Myxococcus xanthus . SDB, a heme-binding part of cytochrome, provides a visual aid for estimating expression level and solubility. Bacteria expressing SDB containing fusion proteins appears red when the fused proteins are soluble. 
       2-3. Preparation of SOCS3 Recombinant Proteins 
       [0070]    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 ). 
         [0071]    4PCR 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 450 (SDB), respectively. 
         [0000]    
       
         
               
             
           
               
                 SEQ ID NO: 481 [cDNA Sequence of Histidine 
               
               
                 Tag] 
               
               
                 atgggcagcagccatcatcatcatcatcacagcagcggcctggtgccgcg 
               
               
                   
               
               
                 cggcagc 
               
               
                   
               
               
                 SEQ ID NO: 482 [Amino Acid Sequence of 
               
               
                 Histidine Tag] 
               
               
                 Met Gly Ser Ser His His His His His His Ser 
               
               
                   
               
               
                 Ser Gly Leu Val Pro Arg Gly Ser 
               
               
                   
               
               
                 SEQ ID NO: 483 [cDNA Sequences of aMTDs] 
               
               
                 Please see TABLE 4 
               
               
                   
               
               
                 SEQ ID NO: 484 [Amino Acid Sequences of aMTDs] 
               
               
                 Please see TABLE 4 
               
               
                   
               
               
                 SEQ ID NO: 485 [cDNA Sequence of human SOCS3] 
               
               
                 ATGGTCACCC ACAGCAAGTT TCCCGCCGCC GGGATGAGCC 
               
               
                   
               
               
                 GCCCCCTGGA CACCAGCCTG CGCCTCAAGA CCTTCAGCTC 
               
               
                   
               
               
                 CAAGAGCGAG TACCAGCTGG TGGTGAACGC AGTGCGCAAG 
               
               
                   
               
               
                 CTGCAGGAGA GCGGCTTCTA CTGGAGCGCA GTGACCGGCG 
               
               
                   
               
               
                 GCGAGGCGAA CCTGCTGCTC AGTGCCGAGC CCGCCGGCAC 
               
               
                   
               
               
                 CTTTCTGATC CGCGACAGCT CGGACCAGCG CCACTTCTTC 
               
               
                   
               
               
                 ACGCTCAGCG TCAAGACCCA GTCTGGGACC AAGAACCTGC 
               
               
                   
               
               
                 GCATCCAGTG TGAGGGGGGC AGCTTCTCTC TGCAGAGCGA 
               
               
                   
               
               
                 TCCCCGGAGC ACGCAGCCCG TGCCCCGCTT CGACTGCGTG 
               
               
                   
               
               
                 CTCAAGCTGG TGCACCACTA CATGCCGCCC CCTGGAGCCC 
               
               
                   
               
               
                 CCTCCTTCCC CTCGCCACCT ACTGAACCCT CCTCCGAGGT 
               
               
                   
               
               
                 GCCCGAGCAG CCGTCTGCCC AGCCACTCCC TGGGAGTCCC 
               
               
                   
               
               
                 CCCAGAAGAG CCTATTACAT CTACTCCGGG GGCGAGAAGA 
               
               
                   
               
               
                 TCCCCCTGGT GTTGAGCCGG CCCCTCTCCT CCAACGTGGC 
               
               
                   
               
               
                 CACTCTTCAG CATCTCTGTC GGAAGACCGT CAACGGCCAC 
               
               
                   
               
               
                 CTGGACTCCT ATGAGAAAGT CACCCAGCTG CCGGGGCCCA 
               
               
                   
               
               
                 TTCGGGAGTT CCTGGACCAG TACGATGCCC CGCTT 
               
               
                   
               
               
                 SEQ ID NO: 486 [Amino Acid Sequence of 
               
               
                 human SOCS3] 
               
               
                 Met Val Thr His Ser Lys Phe Pro Ala Ala Gly 
               
               
                   
               
               
                 Met Ser Arg Pro Leu Asp Thr Ser Leu Arg Leu 
               
               
                   
               
               
                 Lys Thr Phe Ser Ser Lys Ser Glu Tyr Gln Leu 
               
               
                   
               
               
                 Val Val Asn Ala Val Arg Lys Leu Gln Glu Ser 
               
               
                   
               
               
                 Gly Phe Tyr Trp Ser Ala Val Thr Gly Gly Glu 
               
               
                   
               
               
                 Ala Asn Leu Leu Leu Ser Ala Glu Pro Ala Gly 
               
               
                   
               
               
                 Thr Phe Leu Ile Arg Asp Ser Ser Asp Gln Arg 
               
               
                   
               
               
                 His Phe Phe Thr Leu Ser Val Lys Thr Gln Ser 
               
               
                   
               
               
                 Gly Thr Lys Asn Leu Arg Ile Gln Cys Gly Gly 
               
               
                   
               
               
                 Gly Ser Phe Ser Leu Gln Ser Asp Pro Arg Ser 
               
               
                   
               
               
                 Thr Gln Pro Val Pro Arg Phe Asp Cys Val Leu 
               
               
                   
               
               
                 Lys Leu Val His His Tyr Met Pro Pro Pro Gly 
               
               
                   
               
               
                 Ala Pro Ser Phe Pro Ser Pro Pro Thr Glu Pro 
               
               
                   
               
               
                 Ser Ser Glu Val Pro Glu Gln Pro Ser Ala Gln 
               
               
                   
               
               
                 Pro Leu Pro Gly Ser Pro Pro Arg Arg Ala Tyr 
               
               
                   
               
               
                 Tyr Ile Tyr Ser Gly Gly Glu Lys Ile Pro Leu 
               
               
                   
               
               
                 Val Leu Ser Arg Pro Leu Ser Ser Asn Val Ala 
               
               
                   
               
               
                 Thr Leu Gln His Leu Cys Arg Lys Thr Val Asn 
               
               
                   
               
               
                 Gly His Leu Asp Ser Tyr Glu Lys Val Thr Gln 
               
               
                   
               
               
                 Leu Pro Gly Pro Ile Arg Glu Phe Leu Asp Gln 
               
               
                   
               
               
                 Tyr Asp Ala Pro Leu 
               
               
                   
               
               
                 SEQ ID NO: 487 [cDNA Sequences of SDA] 
               
               
                 ATGGCAAATATT ACCGTTTTCTAT AACGAAGACTTC CAGGGTAAGCAG 
               
               
                   
               
               
                 GTCGATCTGCCG CCTGGCAACTAT ACCCGCGCCCAG TTGGCGGCGCTG 
               
               
                   
               
               
                 GGCATCGAGAAT AATACCATCAGC TCGGTGAAGGTG CCGCCTGGCGTG 
               
               
                   
               
               
                 AAGGCTATCCTG TACCAGAACGAT GGTTTCGCCGGC GACCAGATCGAA 
               
               
                   
               
               
                 GTGGTGGCCAAT GCCGAGGAGTTG GGCCCGCTGAAT AATAACGTCTCC 
               
               
                   
               
               
                 AGCATCCGCGTC ATCTCCGTGCCC GTGCAGCCGCGC ATGGCAAATATT 
               
               
                   
               
               
                 ACCGTTTTCTAT AACGAAGACTTC CAGGGTAAGCAG GTCGATCTGCCG 
               
               
                   
               
               
                 CCTGGCAACTAT ACCCGCGCCCAG TTGGCGGCGCTG GGCATCGAGAAT 
               
               
                   
               
               
                 AATACCATCAGC TCGGTGAAGGTG CCGCCTGGCGTG AAGGCTATCCTC 
               
               
                   
               
               
                 TACCAGAACGAT GGTTTCGCCGGC GACCAGATCGAA GTGGTGGCCAAT 
               
               
                   
               
               
                 GCCGAGGAGCTG GGTCCGCTGAAT AATAACGTCTCC AGCATCCGCGTC 
               
               
                   
               
               
                 ATCTCCGTGCCG GTGCAGCCGAGG 
               
               
                   
               
               
                 SEQ ID NO: 488 [Amino Acid Sequences of SDA] 
               
               
                 Met Ala Asn Ile Thr Val Phe Tyr Asn Glu Asp 
               
               
                   
               
               
                 Phe Gln Gly Lys Gln Val Asp Leu Pro Pro Gly 
               
               
                   
               
               
                 Asn Tyr Thr Arg Ala Gln Leu Ala Ala Leu Gly 
               
               
                   
               
               
                 Ile Glu Asn Asn Thr Ile Ser Ser Val Lys Val 
               
               
                   
               
               
                 Pro Pro Gly Val Lys Ala Ile Leu Tyr Gln Asn 
               
               
                   
               
               
                 Asp Gly Phe Ala Gly Asp Gln Ile Glu Val Val 
               
               
                   
               
               
                 Ala Asn Ala Glu Glu Leu Gly Pro Leu Asn Asn 
               
               
                   
               
               
                 Asn Val Ser Ser Ile Arg Val Ile Ser Val Pro 
               
               
                   
               
               
                 Val Gln Pro Arg Met Ala Asn Ile Thr Val Phe 
               
               
                   
               
               
                 Tyr Asn Glu Asp Phe Gln Gly Lys Gln Val Asp 
               
               
                   
               
               
                 Leu Pro Pro Gly Asn Tyr Thr Arg Ala Gln Leu 
               
               
                   
               
               
                 Ala Ala Leu Gly Ile Glu Asn Asn Thr Ile Ser 
               
               
                   
               
               
                 Ser Val Lys Val Pro Pro Gly Val Lys Ala Ile 
               
               
                   
               
               
                 Leu Tyr Gln Asn Asp Gly Phe Ala Gly Asp Gln 
               
               
                   
               
               
                 Ile Glu Val Val Ala Asn Ala Glu Glu Leu Gly 
               
               
                   
               
               
                 Pro Leu Asn Asn Asn Val Ser Ser Ile Arg Val 
               
               
                   
               
               
                 Ile Ser Val Pro Val Gln Pro Arg 
               
               
                   
               
               
                 SEQ ID NO: 489 [cDNA Sequences of SDB] 
               
               
                 ATGGCA GAACAAAGCG ACAAGGATGT GAAGTACTAC 
               
               
                   
               
               
                 ACTCTGGAGG AGATTCAGAA GCACAAAGAC AGCAAGAGCA 
               
               
                   
               
               
                 CCTGGGTGAT CCTACATCAT AAGGTGTACG ATCTGACCAA 
               
               
                   
               
               
                 GTTTCTCGAA GAGCATCCTG GTGGGGAAGA AGTCCTGGGC 
               
               
                   
               
               
                 GAGCAAGCTG GGGGTGATGC TACTGAGAAC TTTGAGGACG 
               
               
                   
               
               
                 TCGGGCACTC TACGGATGCA CGAGAACTGT CCAAAACATA 
               
               
                   
               
               
                 CATCATCGGG GAGCTCCATC CAGATGACAG ATCAAAGATA 
               
               
                   
               
               
                 GCCAAGCCTT CGGAAACCCT T 
               
               
                   
               
               
                 SEQ ID NO: 490 [Amino Acid Sequences of SDB] 
               
               
                 Met Ala Glu Gln Ser Asp Lys Asp Val Lys Tyr 
               
               
                   
               
               
                 Tyr Thr Leu Glu Glu Ile Gln Lys His Lys Asp 
               
               
                   
               
               
                 Ser Lys Ser Thr Trp Val Ile Leu His His Lys 
               
               
                   
               
               
                 Val Tyr Asp Leu Thr Lys Phe Leu Glu Glu His 
               
               
                   
               
               
                 Pro Gly Gly Glu Glu Val Leu Gly Glu Gln Ala 
               
               
                   
               
               
                 Gly Gly Asp Ala Thr Glu Asn Phe Glu Asp Val 
               
               
                   
               
               
                 Gly His Ser Thr Asp Ala Arg Glu Leu Ser Lys 
               
               
                   
               
               
                 Thr Tyr Ile Ile Gly Glu Leu His Pro Asp Asp 
               
               
                   
               
               
                 Arg Ser Lys Ile Ala Lys Pro Ser Glu Thr Leu 
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
               
               
               
             
               
               
               
               
               
             
           
               
                 TABLE 7 
               
             
             
               
                   
               
               
                 [PCR Primers for His-tagged SOCS3 Proteins] 
               
             
          
           
               
                   
                 aMTD 
                 Recombinant  
                   
               
             
          
           
               
                 Cargo 
                 ID 
                 Protein 
                 5′ Primers 
                 3′ Primers 
               
               
                   
               
               
                 SOCS3 
                 — 
                 HS3 
                 5′-GGAATTCCATATGGTCACCCA 
                 5′-CCCGGATCCTTAAAGCGGGG 
               
               
                   
                   
                   
                 CAGCAAGTTTCCCGCCGCC-3′ 
                 CATCGTACTGGTCCAGGAA-3′ 
               
               
                   
               
               
                   
                 165 
                 HM 165 S3 
                 5′-GGAATTCCATATGGCGCTGG 
                   
               
               
                   
                   
                   
                 CGGTGCCGGTGGCGCTGGCGA 
                   
               
               
                   
                 165 
                 HM 165 S3A 
                 TTGTGCCGGTCACCCACAGCAA 
                   
               
               
                   
                   
                   
                 GTTTC-3′ 
                   
               
               
                   
               
               
                   
                 165 
                 HM 165 S3B 
                   
                 5′-CCGGATCCAAGCGGGGCATC 
               
               
                   
                   
                   
                   
                 GTACTGGTCCAGGAA-3′ 
               
               
                   
               
               
                   
                 — 
                 HS3B 
                 5′-GGAATTCCATATGGTCACCC 
                   
               
               
                   
                   
                   
                 ACAGCAAGITTCCCGCCGCC-3′ 
               
               
                   
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
               
               
               
               
             
           
               
                 TABLE 8 
               
             
             
               
                   
               
               
                 [PCR Primers for aMTD/SDA-Fused SOCS3 Proteins] 
               
             
          
           
               
                   
                   
                 Recombinant 
                   
                   
               
               
                 Cargo 
                 SD 
                 Protein 
                 5′ Primers 
                 3′ Primers 
               
               
                   
               
               
                 SOCS3 
                 SDA 
                 HM 165 S3A 
                 5′-CCCGGATCCATGGCAAATAT 
                 5′-CGCGTCGACTTACCTCGGC 
               
               
                   
                   
                   
                 TACCGTTTTCTATAACGAA-3′ 
                 TGCACCGGCACGGCGATGAC-3′ 
               
               
                   
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
               
               
               
               
             
           
               
                 TABLE 9 
               
             
             
               
                   
               
               
                 [PCR Primers for aMTD/SDB-Fused SOCS3 Proteins] 
               
             
          
           
               
                   
                   
                 Recombinant 
                   
                   
               
               
                 Cargo 
                 SD 
                 Protein 
                 5′ Primers 
                 3′ Primers 
               
               
                   
               
               
                 SOCS3 
                 SDB 
                 HM 165 S3B 
                 5′-CCCGGATCCGCAGAACAAA 
                 5′-CGCGTCGACTTAAAGGGTTT 
               
               
                   
                   
                 HS3B 
                 GCGACAAGGATGTGAAG-3′ 
                 CCGAAGGCTTGGC TATCTT-3′ 
               
               
                   
               
             
          
         
       
     
         [0072]    The SOCS3 recombinant proteins were expressed in  E. coli BL 21-CodonPlus (DE3) cells, grown to an OD 600  of 0.6 and induced for 3 hrs with 0.6 mM isopropyl-D-thiogalactopyranoside (IPTG). The proteins were purified by Ni2+ affinity chromatography and dissolved in a physiological buffer such as DMEM medium. 
       2-4 Determination of Solubility and Yield of Each SOCS3 Recombinant Protein 
       [0073]    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). 
         [0074]    SOCS3 recombinant proteins containing aMTD165 and solubilization domain (HM 165 S3A and HM 165 S3B) 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 ). 
         [0075]    Yields per L of  E. coli  for each recombinant protein (mg/L) 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). 
         [0000]    3. aMTD/SD-Fused SOCS3 Recombinant Proteins Significantly Increase Cell- and Tissue-Permeability
 
3-1. aMTD/SD-Fused SOCS3 Recombinant Proteins are Cell-Permeable
 
         [0076]    To examine protein uptake, SOCS3 recombinant proteins were conjugated to 5/6-fluorescein isothiocyanate (FITC). RAW 264.7 ( FIG. 5 ) or NIH3T3 cells ( FIG. 6 ) were treated with 10 μM FITC-labeled SOCS3 recombinant proteins. 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) efficiently entered the cells ( FIGS. 5 and 6 ) and were localized to various extents in cytoplasm ( FIG. 6 ). In contrast, SOCS3 protein (HS3) containing lacking aMTD did not appear to enter cells. While all SOCS3 proteins containing aMTD165 transduced into the cells, HM 165 S3B displayed more uniform cellular distribution, and protein uptake of HM 165 S3B was also very efficient. 
         [0000]    3-2. aMTD/SD-Fused SOCS3 Recombinant Proteins Enhance the Systemic Delivery to a Variety of Tissues 
         [0077]    To further investigate in vivo delivery of SOCS3 recombinant proteins, FITC-labeled SOCS3 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) were distributed to a variety of tissues (liver, kidney, spleen, lung, heart and, to a lesser extent, brain). Predictably, 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. These data suggest that 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. 
         [0000]    3-3. aMTD-Mediated Intracellular Delivery is Bidirectional Mode 
         [0078]    SOCS3 recombinant proteins lacking SD (HS3 and HM 165 S3) 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. 
         [0079]    We next investigated how of 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. Conversely, 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. 
         [0080]    Moreover, we also tested whether cells treated with aMTD165-fused SOCS3 protein could transfer the protein to neighboring cells. For this, cells transduced with FITC-HM 165 S3B (green) were mixed with CD14-labeled cells (red), and cell-to-cell protein transfer was assessed by flow cytometry, scoring for CD14/FITC double-positive cells. Efficient cell-to-cell transfer of HM 165 S3B, but not HS3 or HS3B ( FIG. 11 ), suggests that SOCS3 recombinant proteins containing aMTD165 are capable of bidirectional passage across the plasma membrane. 
         [0000]    4. aMTD/SD-Fused SOCS3 Protein Efficiently Inhibits Cellular Processes
 
4-1. aMTD/SD-Fused SOCS3 Protein Inhibits the Activation of STATs Induced by INF-γ
 
         [0081]    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-γ-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-γ-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. 
         [0000]    4-2. aMTD/SD-Fused SOCS3 Recombinant Protein Inhibits the Secretion of Inflammatory Cytokines TNF-α and IL-6 
         [0082]    We next investigated the effect of cell-permeable SOCS3 proteins on cytokines secretion. Treatment of C3H/HeJ primary peritoneal macrophages with SOCS3 proteins containing aMTD165 suppressed TNF-α and IL-6 secretion induced by the combination of IFN-γ and LPS by 50-90% during subsequent 9 hrs of incubation ( FIG. 13 ). In particular, aMTD165/SDB-fused SOCS3 recombinant protein showed the greatest inhibitory effect on cytokine secretion. In contrast, cytokine secretion in macrophages treated with non-permeable SOCS3 protein (HS3) was unchanged, indicating that recombinant SOCS3 lacking the aMTD doesn&#39;t affect intracellular signaling. Therefore, we conclude that differences in the biological activities of HM165S3B as compared to HS3B are due to the differences in protein uptake mediated by the aMTD sequence. In light of solubility/yield, cell-/tissue-permeability, and biological effect, SOCS3 recombinant protein containing aMTD and SDB (HM165S3B) is a prototype of a new generation of improved cell-permeable SOCS3 (iCP-SOCS3), and will be selected for further evaluation as a potential anti-tumor agent. 
         [0000]    5. iCP-SOCS3 Suppresses Pro-Tumorigenic Functions in Hepatocellular Carcinoma Cells
 
5-1. iCP-SOCS3 Enhances the Penetration into Hepatocellular Carcinoma Cells and Systemic Delivery to Liver
 
         [0083]    Although hepatocellular carcinoma (HCC) 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 types of tumors and SOCS3 is closely related to the development of hepatocellular carcinoma, we first chose the hepatocellular carcinoma as a primary indication of the iCP-SOCS3 as an anti-cancer agent. 
         [0084]    To determine the cell-permeability of iCP-SOCS3 in the hepatocellular carcinoma cells, cellular uptake of FITC-labeled SOCS3 recombinant proteins was quantitatively evaluated by flow cytometry. FITC-HM165S3B recombinant protein (iCP-SOCS3) promoted the transduction into cultured HCC HepG2 cells ( FIG. 14 ). In addition, 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 hepatocytes and liver tissue, contributing for beneficial biotherapeutic effects. 
         [0000]    5-2. iCP-SOCS3 Inhibits Hepatocellular Carcinoma Cell Viability 
         [0085]    Since the endogenous level of SOCS3 protein is reduced in hepatocellular carcinoma patient, and SOCS3 negatively regulates cell growth and motility in cultured HCC cells, we investigated whether iCP-SOCS3 inhibits cell viability through SOCS3 intracellular replacement in HCC cells. As shown in  FIG. 16 , SOCS3 recombinant proteins containing aMTD165 significantly suppressed cancer cell proliferation. HM165S3B (iCP-SOCS3) protein was the most cytotoxic to Hep3B2.1-7 hepatocellular carcinoma cells—over 80% in 10 μM treatment (p&lt;0.01)—especially compared to vehicle alone (i.e. exposure of cells to culture media without recombinant proteins;  FIG. 16 , left). However, neither cell-permeable SOCS3 protein adversely affected the cell viability of non-cancer cells (NIH3T3) even after exposing these cells to equal concentrations (10 μM) of protein over 4 days ( FIG. 16 , right). These results suggest that the iCP-SOCS3 protein is not overly toxic to normal cells and selectively kills tumor cells, and would have a great ability to inhibit cell survival-associated phenotypes in hepatocellular carcinoma without any severe aberrant effects as a protein-based biotherapeutics. 
         [0000]    5-3. iCP-SOCS3 Protein Induces Apoptosis in Hepatocellular Carcinoma Cells 
         [0086]    To further determine the effect of iCP-SOCS3 on the tumorigenicity of hepatocellular carcinoma cells, we subsequently investigated whether iCP-SOCS3 regulates apoptosis in HepG2 cells. HM165S3B protein (iCP-SOCS3) was a considerably efficient inducer of apoptosis in HepG2 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 HepG2 cells treated with HS3B compared to untreated cell (Vehicle). In addition, HepG2 cells treated with HM165S3B protein (iCP-SOCS3) dramatically reduced the expression of anti-apoptotic protein such as B-cell lymphoma 2 (Bcl-2) and increased the level of cleaved cysteine-aspartic acid protease (caspase-3;  FIG. 19 ). These results indicate that iCP-SOCS3 induces apoptosis of hepatocellular carcinoma cells and may suppress the cancer progression by this pathway. 
         [0000]    5-4. iCP-SOCS3 Inhibits Migration/Invasion of Hepatocellular Carcinoma Cells 
         [0087]    We next examined the ability of iCP-SOCS3 to influence cell migration. HepG2 cells were treated with recombinant proteins for 2 hrs, the monolayers were wounded, and cell migration in the wound was monitored after 72 hrs ( FIG. 20 ). HM 165 S3B protein (iCP-SOCS3) suppressed the repopulation of wounded monolayer although SOCS3 protein lacking aMTD165 (HS3B) had no effect on the cell migration. Consistent with this, HepG2 cells treated with HM 165 S3B recombinant protein (iCP-SOCS3) also showed significant inhibitory effect on their Transwell migration compared with untreated cells (Vehicle) and non-permeable SOCS3 protein-treated cells (HS3B;  FIG. 21 ). In addition, HepG2 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 hepatocellular carcinoma cells. 
         [0000]    6. iCP-SOCS3 Suppresses Pro-Tumorigenic Functions in Hepatocellular Carcinoma Cells
 
6-1. iCP-SOCS3 Suppresses the HCC Xenograft
 
         [0088]    We assessed the anti-tumor activity of iCP-SOCS3 against human cancer xenografts. Balb/c nu/nu mice were subcutaneously implanted with tumor block (1 mm3) of hepatocellular carcinoma cells into the left side of the back. Tumor-bearing mice were intravenously administered HM165S3B or control proteins (HS3B; 600 μg/head, respectively) for 21 days and observed for 2 weeks following the termination of the treatment ( FIG. 22 ). HM165S3B protein significantly suppressed the tumor growth (p&lt;0.05) during the treatment and the effect persisted for at least 2 weeks after the treatment was terminated (80% inhibition at day 21; 70% at day 35, respectively). Whereas, the growth of HS3B-treated tumors increased, matching the rates observed in control mice (Vehicle;  FIGS. 22 and 23 ). These results suggest that iCP-SOCS3 inhibits the growth of established tumors as well as the tumor growth of hepatocellular carcinoma cells. 
         [0000]    6-2. iCP-SOCS3 Regulates the Expression of Tumor-Associated Proteins in Human Tumor Xenograft 
         [0089]    The anti-tumor activity of HM165S3B at day 35 was accompanied by changes in the expression of biomarkers linked to SOCS3 signaling, including p21, Bax and VEGF ( FIG. 24 ). Expression of tumor suppressors (p21 and Bax) was dramatically enhanced in tumor tissues treated with HM165S3B recombinant protein ( FIG. 24 ), suggesting that iCP-SOCS3 inhibits tumor growth by regulating tumor-specific protein expression in vivo. In addition, the levels of vascular endothelial growth factor (VEGF), a pro-angiogenic factor, were inhibited in HM165S3B-treated tumors. In contrast, tumor biomarker expression was not affected in mice treated with the HS3B control protein, which lacks aMTD sequence. These in vivo results suggest that iCP-SOCS3 targets tumor cells directly and may be developed for use as novel therapy against hepatocellular carcinoma. 
       Example 
       [0090]    The following examples are presented to aid practitioners of the invention, to provide experimental support for the invention, and to provide model protocols. In no way are these examples to be understood to limit the invention. 
       Example 1 
     Development of Novel Advanced Macromolecule Transduction Domain (aMTD) 
       [0091]    H-regions of signal sequences (HRSP)-derived CPPs (MTM, MTS and MTD) do not have a common sequence, a sequence motif, and/or a common structural homologous feature. In this invention, the aim is to develop improved hydrophobic CPPs formatted in the common sequence and structural motif that satisfy newly determined ‘critical factors’ to have a ‘common function’, to facilitate protein translocation across the membrane with similar mechanism to the analyzed CPPs. 6 critical factors have been selected to artificially develop novel hydrophobic CPP, namely advanced macromolecule transduction domain (aMTD). These 6 critical factors include the followings: amino acid length of the peptides (ranging from 9 to 13 amino acids), bending potentials (dependent with the presence and location of proline in the middle of sequence (at 5′, 6′, 7′ or 8′ amino acid) and at the end of peptide (at 12′)), instability index (II) for rigidity/flexibility (II: 40-60), grand average of hydropathy (GRAVY) for hydropathy (GRAVY: 2.1-2.4), and aliphatic index (AI) for structural features (AI: 180-220). Based on these standardized critical factors, new hydrophobic peptide sequences, namely advanced macromolecule transduction domain peptides (aMTDs), in this invention have been developed and selected to be fused with the cargo protein, SOCS3, to develop improved cell-permeable SOCS3 recombinant protein (iCP-SOCS3). 
       Example 2 
     Construction of Expression Vectors for Recombinant SOCS3 Proteins 
       [0092]    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 μmol 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. The 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. 
       Example 3 
     Inducible Expression, Purification, and Preparation of Recombinant Proteins 
       [0093]    The recombinant proteins were purified from  E. coli  BL21-CodonPlus (DE3) cells grown to an A600 of 0.6 and induced for 3 hrs with 0.6 mM IPTG. Denatured recombinant proteins were purified by Ni2+ affinity chromatography as directed by the supplier (Qiagen, Hilden, Germany). After purification, they were dialyzed against a refolding buffer (0.55 M guanidine HCl, 0.44 M L-arginine, 50 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, 100 mM NDSB, 2 mM reduced glutathione, and 0.2 mM oxidized glutathione) and changed to a physiological buffer such as DMEM medium. 
       Example 4 
     Determination of Quantitative Cell-Permeability of Recombinant Proteins 
       [0094]    For quantitative cell-permeability, recombinant SOCS3 proteins were conjugated to 5/6-fluorescein isothiocyanate (FITC) according to the manufacturer&#39;s instructions (Sigma-Aldrich, St. Louis, Mo.). RAW 264.7 cells were treated with 10 μM FITC-labeled recombinant proteins for 1 hr at 37° C., washed three times with cold PBS, and treated with proteinase K (10 μg/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. 
       Example 5 
     Determination of Intracellular Localization of SOCS3 Recombinant Proteins 
       [0095]    For visual cell permeability, NIH3T3 cells were cultured on coverslips in 24-well plates and with 10 μM 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). 
       Example 6 
     Determination of Tissue Distribution of Recombinant SOCS3 Proteins 
       [0096]    ICR mice (6-week-old, female) were injected intraperitoneally (600 μg/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 μm) were analyzed by fluorescence microscopy (Carl Zeiss, Gottingen, Germany). 
       Example 7 
     Mechanism of aMTD-Mediated Intracellular Delivery 
       [0097]    RAW264.7 cells were pretreated with different agents to assess the effect of various conditions on protein uptake: (i) 5 μg/ml proteinase K for 10 min, (ii) 20 μM Taxol for 30 min, (iii) 10 μM 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 μM FITC-labeled proteins for 1 hr at 37° C., washed three times with ice-cold phosphate-buffered saline, treated with proteinase K (10 μg/ml for 5 min at 37° C.) to remove cell-surface bound proteins, and analyzed by flow cytometry. To assess cell-to-cell protein transfer, RAW264.7 cells containing FITC-conjugated protein were prepared in the same way and mixed with untreated cells labeled with PreCP-Cy5.5-CD14 antibody for 2 hrs. Cell-to-cell protein transfer, resulting in FITC-Cy5.5 double-positive cells, was monitored by flow cytometry. 
       Example 8 
     STAT Phosphorylation: Western Blot Analysis 
       [0098]    PANC-1 cells (Korean Cell Line Bank, Seoul, Korea) were cultured in modified Eagle&#39;s medium (DMEM; Welgene, Daege, Korea) supplemented with 10% (v/v) FBS, penicillin (100 units/ml), and streptomycin (10 μg/ml, Gibco BRL) and pretreated with 10 μM of SOCS3 recombinant proteins for 2 hrs followed by exposing the cells to agonists (100 ng/ml IFN-γ) for 15 min. Cells were lysed with 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) containing a protease inhibitor cocktail and then centrifuged at 13,000×g for 15 min at 4° C. Equal amounts of lysates were resolved by SDS-PAGE, transferred onto PVDF membranes, and probed with phospho (pY701)-specific STAT1 (Cell Signaling, Danvers, Mass.). 
       Example 9 
     Cytokine Measurement: Cytometric Bead Array (CBA) Assay 
       [0099]    Peritoneal macrophages were obtained from C3H/HeJ mice. Peritoneal macrophages were incubated with 10 μM 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-γ (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&#39;s instructions. 
       Example 10 
     Cell Proliferation: CellTiter-Glo Cell Viability Assay 
       [0100]    Cells originated from human hepatocellular carcinoma cell and mouse fibroblast (NIH3T3) were purchased (ATCC, Manassas, Va.) and maintained as recommended by the supplier. These cells (3×10 3 /well) were seeded in 96 well plates. The next day, cells were treated with DMEM (vehicle) or recombinant SOCS3 proteins for 96 hrs in the presence of serum (2%). Proteins were replaced daily. Cell growth and survival were evaluated with the CellTiter-Glo Cell Viability Assay (Promega, Madison, Wis.). Measurements using a Luminometer (Turner Designs, Sunnyvale, Calif.) were conducted following the manufacturer&#39;s protocol. 
       Example 11 
     Apoptosis: TUNEL Assay 
       [0101]    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 μM 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). 
       Example 12 
     Apoptosis: Annexin V/7-AAD Staining 
       [0102]    Annexin V/7-Aminoactinomycin D (7-AAD) staining was performed using flow cytometry according to the manufacturer&#39;s guidelines. Briefly, 1×10 6  cells were washed three times with ice-cold PBS. The cells were then resuspended in 100 μl of binding buffer and incubated with 1 μl of 7-AAD and 1 μl of annexin V-PE for 30 min in the dark at 37° C. Flow cytometric analysis was immediately performed using a guava easyCyte™ 8 Instrument (Merck Millipore). 
       Example 13 
     Molecular Mechanism: Western Blot Analysis 
       [0103]    Cells were treated with either DMEM (vehicle) or 10 μM 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. 
       Example 14 
     Cell Migration: Wound-Healing Assay 
       [0104]    Cells were seeded into 12-well plates, grown to 90% confluence, and incubated with 10 μM HS3, HM 165 S3, 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. 
       Example 15 
     Transwell Migration Assay 
       [0105]    The lower surface of Transwell inserts (Costar) was coated with gelatin (10 μg/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 μg/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. 
       Example 16 
     Invasion Assay 
       [0106]    The lower surface of Transwell inserts (Costar) was coated with gelatin (10 μg/ml), the upper surface of Transwell inserts was coated with matrigel (40 μg 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 μg/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. 
       Example 17 
     Xenograft Animal Models 
       [0107]    Female Balb/c nu/nu mice were subcutaneously implanted with Hep3B2.1-7 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 μg/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. 
         [0108]    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. 
       Example 18 
     Immunohistochemistry (IHC) 
       [0109]    Tissue samples were fixed in 4% Paraformaldehyde (Duksan) for 3 days, dehydrated, cleared with xylene and embedded in Paraplast. Sections (6 μm 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). 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 μg/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. After the slides are reacted with oxidized 3,3-diaminobenzidine as a chromogen, they were counterstained with Harris hematoxylin (Sigma-Aldrich). Permanently mounted slides were observed and photographed using a microscope equipped with a digital imaging system (ECLIPSE Ti, Nikon, Japan). 
       Example 19 
     Statistical Analysis 
       [0110]    All data are presented as mean±s.d. Differences between groups were tested for statistical significance using Student&#39;s t-test and were considered significant at p&lt;0.05 or p&lt;0.01. 
         [0111]    It will be apparent to those skilled in the art that various modifications can be made to the above-described exemplary embodiments of the present invention without departing from the spirit or scope of the invention. Thus, it is intended that the present invention covers all such modifications provided that they come within the scope of the appended claims and their equivalents.

Technology Category: 8