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

TECHNICAL FIELD

The present invention pertains to have (i) improved cell-permeable SOCS3 (iCP-SOCS3) proteins as protein-based biotherapeutics, which are well-enhanced in their ability to transport biologically active SOCS3 proteins across the plasma membrane, to increase in its solubility and manufacturing yield, and to induce anti-non-small cell lung carcinoma effect; (ii) polynucleotides that encode the same, and (iii) anti-lung cancer compositions that comprise the same.

BACKGROUND ART

Worldwide, lung cancer is the most common cause of cancer-related death in men and women, and was responsible for 1.56 million deaths annually. There are two main types of primary lung cancer: non-small cell lung cancer (NSCLC), small cell lung cancer (SCLC). About 85% of lung cancers are NSCLCs which is the most common type of lung cancer. Squamous cell carcinoma, adenocarcinoma, and large cell carcinoma are all subtypes of non-small cell lung cancer. Cytokines including IL-6 and interferon-gamma (IFN- custom-character ) activate the Janus kinase (JAK)/signal transducers and activators of transcription (STAT) signaling pathway, a vital role promoting the inflammation, carcinogenesis and metastasis in the lung. STAT3, which functions as an oncogene downstream of IL-6/gp130, is hyper-activated in lung cancer cells contributes to increase cell proliferation and inhibits apoptosis.

Cytokine signaling is strictly regulated by the SOCS family proteins induced by different classes of agonists, including cytokines, hormones and infectious agents. 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.

A previous study has confirmed that SOCS3 may significantly inhibit the proliferation of lung cancer cells in vitro and indicated that SOCS3 may act as an anti-oncogene involved in the development of tumors. Furthermore, SOCS3 may regulate the movement and migration of tumor cells. Methylation-mediated silencing of SOCS3 has been reported in non-small lung cancer (NSCLC) and other human cancers. In addition to the effect of SOCS3 in inflammation, abnormalities of the JAK/STAT pathway are also associated with cancer. It has been reported that methylationin of CpG islands in the functional SOCS3 promoter is correlated with its transcription silencing in the lung cancer cell lines. Restoration of SOCS3 in lung cancer cells where SOCS3 was methylation-silenced resulted in the down-regulation of active STAT3, induction of apoptosis, and growth suppression of cancer cells. It means that SOCS3 silencing is one of the important mechanisms of constitutive activation of the JAK/STAT pathway in cancer pathogenesis. Therefore, it can be suggested that intracellular SOCS3 protein replacement therapy may be useful in the treatment of lung cancer.

In the previous study, recombinant SOCS3 proteins that contain a cell-penetrating peptide (CPP)-membrane-translocating motif (MTM) from fibroblast growth factor (FGF)-4 has been reported to negatively control JAK/STAT signaling. 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 their solubility and manufacturing yield when expressed and purified from bacteria cells.

In this new art of invention, aMTD/SD-fused SOCS3 recombinant proteins (iCP-SOCS3) have much improved physicochemical characteristics (solubility & yield) and functional activity (cell-/tissue-permeability) compared to 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 the lung cancer, exploiting the ability of SOCS3 to suppress JAK/STAT signaling. The present invention represents that macromolecule intracellular transduction technology (MITT) enabled by the new hydrophobic CPPs that are aMTDs may provide novel protein therapy through SOCS3-intracellular protein replacement against the lung cancer. 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 lung cancer.

SUMMARY

An aspect of the present invention relates to improved cell-permeable SOCS3 (iCP-SOCS3) capable of mediating the transduction of biologically active macromolecules into live cells.

iCP-SOCS3 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.

iCP-SOCS3 fused to solubilization domains (SDs) greatly increase in their solubility and manufacturing yield when they are expressed and purified in the bacteria system.

An aspect of the present invention also relates to its therapeutic application for delivery of a biologically active molecule to a cell involving a cell-permeable SOCS3 recombinant protein, where the aMTD is attached to a biologically active cargo molecule.

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.

An aspect of the present invention provides improved cell-permeable SOCS3 as a biotherapeutics having improved solubility/yield, cell-/tissue-permeability and anti-lung cancer effects. Therefore, this would allow their practically effective applications in drug delivery and protein therapy including intracellular protein therapy and protein replacement therapy.

BRIEF DESCRIPTION OF DRAWINGS

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.

FIG. 1 shows the structure of SOCS3 recombinant proteins. A schematic diagram of the His-tagged SOCS3 recombinant protein is illustrated and constructed according to the present invention. The his-tag for affinity purification (white), aMTD165 (black), SOCS3 (gray) and solubilization domain A and B (SDA & SDB, hatched) are shown.

FIG. 2 shows the construction of expression for SOCS3 recombinant proteins This figure shows the agarose gel electrophoresis analysis showing plasmid DNA fragments encoding SOCS3, aMTDs fused SOCS3 and SD cloned into the pET28 (+) vector according to the present invention.

FIG. 3 shows inducible expression and purification of SOCS3 recombinant proteins. Expression of SOCS3 recombinant proteins in E. coli before (−) and after (+) induction with IPTG and purification by Ni2+ affinity chromatography (P) were monitored by SDS-PAGE, and stained with Coomassie blue.

FIG. 4 shows the improvement of solubility/yield with aMTD/SD-fusion. The solubility, yield and recovery (in percent) of soluble form from denatured form are indicated (left). Relative yield of recombinant proteins is normalized to the yield of HS3 protein (Right).

FIG. 5 shows aMTD-mediated cell-permeability of SOCS3 recombinant proteins. RAW264.7 cells were exposed to FITC-labeled SOCS3 recombinant proteins (10 custom-character M) for 1 hr, treated with proteinase K to remove cell-associated but non-internalized proteins and analyzed by flow cytometry. Untreated cells (gray) and equimolar concentration of unconjugated FITC (FITC only, green)-treated cells were served as control.

FIG. 6 shows aMTD-mediated intracellular delivery and localization of SOCS3 recombinant proteins. Each of NIH3T3 cells was incubated for 1 hour at 37° C. with 10 custom-character M FITC-labeled SOCS3 protein. Cell-permeability of SOCS3 recombinant proteins was visualized by utilizing confocal microscopy LSM700 version.

FIG. 7 shows the systemic delivery of aMTD/SD-fused SOCS3 recombinant proteins In vivo. Cryosections of saline-perfused organs were prepared from mice 1 hr after intraperitoneal injection of FITC only or 600 custom-character g FITC-conjugated recombinant SOCS3 proteins, and were analyzed by fluorescence microscopy.

FIG. 8 shows the structure of SDB-fused SOCS3 recombinant protein. A schematic diagram of the SOCS3 recombinant protein is illustrated and constructed according to the present invention. The his-tag for affinity purification (white), SOCS3 (gray) and solubilization domain B (SDB, hatched) are shown.

FIG. 9 shows the expression, purification and determination of solubility/yield of SD-fused SOCS3 recombinant protein. Expression of SOCS3 recombinant proteins in E. coli before (−) and after (+) induction with IPTG and purification by Ni2+ affinity chromatography (P) were monitored by SDS-PAGE, and stained with Coomassie blue (Left, top). The solubility, yield and recovery (in percent) of soluble form from denatured form are indicated (Left, bottom). Relative yield of recombinant proteins is normalized to the yield of HS3 protein (Right).

FIG. 10 shows the mechanism of aMTD-mediated SOCS3 protein uptake into cells. (A-D) RAW264.7 cells were treated with 100 mM EDTA for 3 hrs (A), 5 mg/ml Proteinase K for 10 mins (B), 20 mM taxol for 30 mins (C), or 10 μM antimycin for 2 hrs either without or with 1 mM supplemental ATP for 3 hrs. Cells were exposed for 1 hr to 10 custom-character M FITC-labeled HS3 (black), -HS3B (blue) or -HM165S3B (red), treated with proteinase K for 20 mins, and analyzed by flow cytometry. Untreated cells (gray) and equimolar concentration of unconjugated FITC (FITC only, green)-treated cells were served as control. (E) RAW264.7 cells were exposed for the indicated times to 10 μM FITC-labeled HS3 (black), -HS3B (blue) or -HM165S3B (red), treated with proteinase K, and analyzed by flow cytometry.

FIG. 11 shows aMTD-mediated cell-to-cell delivery. RAW264.7 cells exposed to 10 μM FITC-HS3B or FITC-HM165S3B 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-HM165S3B to Cy5.5 labeled cells and the remaining FITC and Cy5.5 single-positive cells. The bottom panels show FITC fluorescence profiles of cell populations before mixing (coded as before) and 1 hr after the same cells were mixed with Cy5.5-labeled cells.

FIG. 12 shows the inhibition of STAT phosphorylation induced by IFN- custom-character . Inhibition of STAT1 phosphorylation detected by immunoblotting analysis. The levels of phosphorylated STAT1 and STAT3 untreated and treated with IFN-were compared to the levels in IFN--treated RAW 264.7 cells that were pulsed with 10 M of indicated proteins.

FIG. 13 shows the inhibition of cytokines secretion induced by LPS. Inhibition of TNF- custom-character and IL-6 expression by recombinant SOCS3 proteins in primary macrophages isolated from peritoneal exudates of C3H/HeJ mice. Error bars indicate+s.d. of the mean value derived from each assay done in triplicate.

FIG. 14 shows the cell-permeability of iCP-SOCS3 (HM165S3B) in lung cancer cells. A549 lung cancer cells were exposed to FITC-labeled SOCS3 recombinant proteins (10 custom-character M) for 1 hr, treated with proteinase K to remove cell-associated proteins for 20 mins, and analyzed by flow cytometry. Untreated cells (gray) and equimolar concentration of unconjugated FITC (FITC only, green)-treated cells were served as control.

FIG. 15 shows the tissue distribution of iCP-SOCS3 (HM165S3B) into lung tissue. Cryosections of saline-perfused organs were prepared from mice 1 hr after intraperitoneal injection of FITC only or 600 custom-character g FITC-conjugated recombinant SOCS3 proteins, and were analyzed by fluorescence microscopy.

FIG. 16 shows the inhibition of proliferation in lung cancer cells with iCP-SOCS3. A549 lung cancer cells were seeded in 96 well plates. Next day, cells were treated with DMEM (V), HS3 (1), HM165S3 (2), HM165S3A (3) or HM165S3B (4) recombinant proteins for 96 hrs in the presence of serum (2%). Cell viability was evaluated with the CellTiter-Glo Cell Viability Assay.

FIG. 17 shows the induction of apoptosis in lung cancer cells with iCP-SOCS3. A549 lung cancer cells were treated for 24 hrs with 10 μM HS3B or HM165S3B proteins and apoptotic cells were visualized by TUNEL staining.

FIG. 18 shows the stimulation of apoptosis in lung cancer cells with iCP-SOCS3. A549 lung cancer cells were treated for 24 hr with 10 μM HS3B or HM165S3B proteins and analyzed by flow cytometry of cells stained with annexin-V and 7-AAD.

FIG. 19 shows the inhibition of migration in lung cancer cells with iCP-SOCS3. A549 lung cancer cells were grown to 100% confluence and these procedures were performed on wound-healing assays. The wound areas were examined and photographed at 0 and 48 hrs post-wounding.

FIG. 20 shows the inhibition of migration/invasion in lung cancer cells with iCP-SOCS3. A549 lung cancer cells were treated with SOCS3 recombinant proteins for 24 hrs, and migration/invasion were measured by Transwell assay. The data shown are representative of three independent experiments. **, p<0.01.

DETAILED DESCRIPTION

In this invention, 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 were fused to novel hydrophobic CPPs called aMTDs to improve their cell-/tissue-permeability, 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 that has a great potential of anti-cancer therapeutic applicability in lung cancer.

1. Novel Hydrophobic Cell-Penetrating Peptides—Advanced Macromolecule Transduction Domains

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.

These 17 hydrophobic CPPs published from 1995 to 2014 have been analyzed for their 11 different characteristics—sequence, amino acid length, molecular weight, pl 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://harrier.nagahama-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.

Average length, molecular weight and pl value of the peptides analyzed were 10.8±2.4, 1,011±189.6 and 5.6±0.1, respectively.

(2) Bending Potential (Proline Position: PP)

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)

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)

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.

(5) Secondary Structure (α-Helix)

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 α-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)

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’

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.

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.

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.

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

TABLE 1

[Universal Structure of Newly Develop Hydrophobic CPPs]

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: Al)

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

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.)

All 240 aMTDs (hydrophobic, flexible, bending, aliphatic and helical 12 a/a-length peptides) were practically confirmed by their quantitative and visual cell-permeability. To determine the cell-permeability of aMTDs and random peptides which do not satisfy one or more critical factors have also been designed and tested. Relative cell-permeability of 240 aMTDs to the negative control (random peptide, hydrophilic & non-aliphatic 12A/a length peptide) was significantly increased by up to 164 fold, with average increase of 19.6±1.6. 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

TABLE 2-1

[Newly Developed Hydrophobic CPPs-240 aMTDs That All Critical Factors Are

Considered and Satisfied (Sequence ID No. 1-46)]

Sequence

Rigidity/
Sturctural

ID

Flexibility
Feature
Hydropathy
Residue

Number
aMTD
Sequences
Length
(II)
(Al)
(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
AAALLPVALVAP
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
Aliphatio

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
Aliphatio

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

TABLE 2-2

[Newly Developed Hydrophobic CPPs-240 aMTDs That All Critical Factors

Are Considered and Satisfied (Sequence ID No. 47-92)]

Sequence

Rigidity/
Sturctural

ID

Flexibility
Feature
Hydropathy
Residue

Number
aMTD
Sequences
Length
(II)
(Al)
(GRAVY)
Structure

47
185
AALVLPLIIAAP
12
41.3
220.0
2.4
Aliphatic

48
201
LALAVPALAALP
12
57.3
195.5
2.1
Aliphatic

49
204
LIAALPAVAALP
12
57.3
195.5
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

TABLE 2-3

[Newly Developed Hydrophobic CPPs-240 aMTDs That All Critical Factors Are

Considered and Satisfied (Sequence ID No. 93-138)]

Sequence

Rigidity/
Sturctural

ID

Flexibility
Feature
Hydropathy
Residue

Number
aMTD
Sequences
Length
(II)
(Al)
(GRAVY)
Structure

93
402
ALAAVIPAAILP
12
54.9
195.8
2.2
Aliphatic

94
403
AAALVIPAAILP
2
54.9
195.6
2.2
Aliphatic

95
404
LAAAVIPAAILP
2
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
ALALIIVPAVAP
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

TABLE 2-4

[Newly Developed Hydrophobic CPPs-240 aMTDs That All Critical Factors Are

Considered and Satisfied (Sequence ID No. 139-184)]

Sequence

Rigidity/
Sturctural

ID

Flexibility
Feature
Hydropathy
Residue

Number
aMTD
Sequences
Length
(II)
(Al)
(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
LAIVLAAPVAVP
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
IAVLAVALAVLP
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
VAVLIAVPALAP
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

TABLE 2-5

[Newly Developed Hydrophobic CPPs-240 aMTDs That All Critical Factors Are

Considered and Satisfied (Sequence ID No. 185-230)]

Sequence

Rigidity/
Sturctural

ID

Flexibility
Feature
Hydropathy
Residue

Number
aMTD
Sequences
Length
(II)
(Al)
(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

TABLE 2-6

[Newly Developed Hydrophobic CPPs-240 aMTDs That All Critical Factors Are

Considered and Satisfied (Sequence ID No. 231-240)]

Sequence

Rigidity/
Sturctural

ID

Flexibility
Feature
Hydropathy
Residue

Number
aMTD
Sequences
Length
(II)
(Al)
(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

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.

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: Al)

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

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).

TABLE 4

[Amino Acid and Nucleotide Sequence of

Newly Developed Advanced MTD 165

Which Follow All Critical Factors]

ID
Amino Acid Sequence
Nucleotide Sequence

165
ALAVPVALAIVP
GCG CTG GCG GTG CCG GTG

GCG CTG GCG ATT GTG CCG

TABLE 5

[Critical Factors of aMTD165]

Bending

Potential

Prolin
Rigidity/
Sturctural

Theoretical
M.W.
Position
Flexibility
Feature
Hydropathy

ID
Length
pl
(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 50053 Recombinant Proteins

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.q. PBS, DMEM or RPMI1640 etc.) was highly insoluble and had extremely low. Therefore, an additional non-functional protein domain (solubilization domain: SD; TABLE 6) has been fused to the recombinant proteins at their C terminus to improve low solubility/yield and to enhance relative cell-/tissue-permeability.

TABLE 6

[Information of Solublization Domains]

Protein

Instability

SD
Genbank ID
Origin
(kDa)
pl
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

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

Histidine-tagged human SOCS3 proteins were designed (FIG. 1) and constructed by amplifying the SOCS3 cDNA (225 amino acids) from nt 4 to 678 using primers [TABLE 7] for SOCS3 cargo fused to aMTD. The PCR products were subcloned with NdeI (5′) and BamHI (3′) into pET-28a(+). Coding sequences for SDA or SDB were fused to the C terminus of his-tagged aMTD-fused SOCS3 and cloned at between the BamHI (5′) and SalI (3′) sites in pET-28a(+) (FIG. 2).

PCR primers for SOCS3 and SDA and/or SDB fused to SOCS3 are summarized in TABLES 7, 8 and 9, respectively. The cDNA and amino acid sequences of histidine tag are provided in SEQ ID NO: 481 and 482, and cDNA and amino acid sequences of aMTDs are indicated in SEQ ID NO: 483 and 484, respectively. The cDNA and amino acid sequences are displayed in SEQ ID NO: 485 and 486 (S0053); SEQ ID NO: 487 and 488 (SDA); and SEQ ID NO: 489 and 490 (SDB), respectively.

[cDNA Sequence of Histidine Tag]

SEQ ID NO: 481

ATGGGCAGCAGCCATCATCATCATCATCACAGCAGCGGCCTGGTGCCGCGCGGCAGC

[Amino Acid Sequence of Histidine Tag]

SEQ ID NO: 482

Met Gly Ser Ser His His His His His His Ser Ser Gly Leu Val Pro Arg Gly Ser

[cDNA Sequences of aMTDs]

SEQ ID NO: 483

Please see TABLE 4

[Amino Acid Sequences of aMTDs]

SEQ ID NO: 484

Please see TABLE 4

[cDNA Sequence of human SOC53]

SEQ ID NO: 485

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

[Amino Acid Sequence of human S0053]

SEQ ID NO: 486

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

[cDNA Sequences of SDA]

SEQ ID NO: 487

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

[Amino Acid Sequences of SDA]

SEQ ID NO: 488

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

[cDNA Sequences of SDB]

SEQ ID NO: 489

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

[Amino Acid Sequences of SDB]

SEQ ID NO: 490

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

The SOCS3 recombinant proteins were expressed in E. coli BL21-CodonPlus (DE3) cells, grown to an OD₆₀₀of 0.6 and induced for 3 hrs with 0.6 mM isopropyl-β-D-thiogalactopyranoside (IPTG). The proteins were purified by Ni2⁺ affinity chromatography and dissolved in a physiological buffer such as DMEM medium.

TABLE 7

[PCR Primers for His-Tagged SOCS3 Proteins]

aMTD
Recombinant

Cargo
ID
Protein
5′ Primers
3′ Primers

SOCS3
—
HS3
5′-GGAATTCCAT
5′-CCCSGATCCT

ATGGTCACCCACA
TAAAGCGGGGCAT

GCARGTTTCCCGC
CGTACTGGTCCAG

CGCC-3′
GAA-3′

165
HM₁₆₅S3
5′-GGAATTCCAT

165
HM₁₆₅S3A
ATGGCGCTGGCGG
5′-CCGGATCCAA

165
HM₁₆₅S3B
TGCCGGTGGCGCT
GCGGGGCATCGTA

GGCGATTGTGCCG
CTGGTCCAGGAA-3′

GTCACCCACAGCA

AGTTTC-3′

—
HS3B
5′-GGAATTCCAT

ATGGTCACCCACA

GCAAGTTTCCCGC

CGCC-3′

TABLE 8

[PCR Primers for aMTD/SDA-Fused SOCS3 Proteins]

Recombinant

Cargo
SD
Protein
5′ Primers
3′ Primers

SOCS3
SDA
HM₁₆₅S3A
5′-CCCGGATCCATG
5′-CGCGTCGACTTA

GCAAATATTACCGTT
CCTCGGCTGCACCGG

TTCTATAACGAA-3′
CACGGCGATGAC-3′

TABLE 9

[PCR Primers for aMTD/SDB-Fused SOCS3 Proteins]

Recombinant

Cargo
SD
Protein
5′ Primers
3′ Primers

SOCS3
SDB
HM₁₆₅S3B
5′-CCCGGATCCGC
5′-CGCGTCGACTTA

HS3B
AGAACAAAGCGACA
AAGGGTTTCCGAAGG

AGGATGTGAAG-3′
CTTGGCTATCTT-3′

2-4. Determination of Solubility and Yield of Each SOCS3 Recombinant Protein

The histidine-tagged SOCS3 proteins were expressed, purified, and prepared in soluble form (FIG. 3). The yield of each soluble SOCS3 recombinant proteins was determined by measuring absorbance (A450).

SOCS3 recombinant proteins containing aMTD165 and solubilization domain (HM₁₆₅S3A and HM₁₆₅S3B) had little tendency to precipitate whereas recombinant SOCS3 proteins lacking a solubilization domain (HS3 and HM₁₆₅S3) were largely insoluble. Solubility of aMTD/SD-fused SOCS3 proteins was scored on a 5 point scale compared with that of SOCS3 proteins lacking the solubilization domain (FIG. 4).

Yields per L of E. coli for each recombinant protein (mg/L) ranged from 1 to 47 mg/L (FIG. 4). Yields of SOCS3 proteins containing an aMTD and SDB (HM₁₆₅S3B) were 50% higher than his-tagged SOCS3 protein (HS3).

3. aMTD/SD-Fused SOCS3 Recombinant Proteins Significantly Increase Cell- and Tissue-Permeability

3-1. aMTD/SD-Fused 50053 Recombinant Proteins are Cell-Permeable

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 custom-character M 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 (HM165S3, HM165S3A and HM165S3B) 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, HM165S3B displayed more uniform cellular distribution, and protein uptake of HM165S3B was also very efficient.

3-2. aMTD/SD-Fused SOCS3 Recombinant Proteins Enhance the Systemic Delivery to a Variety of Tissues

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 (HM₁₆₅S3, HM₁₆₅S3A and HM₁₆₅S3B) 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 (HM₁₆₅S3B) showed the highest systemic delivery of SOCS3 protein to the tissues comparable to the SOCS3 containing only aMTD (HM₁₆₅S3) or aMTD/SDA (HM165S3A) 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.

3-3. aMTD-Mediated Intracellular Delivery is Bidirectional Mode

SOCS3 recombinant proteins lacking SD (HS3 and HM165S3) 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.

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.

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-HM165S3B (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 HM165S3B, but not HS3 or HS3B (FIG. 11), suggests that SOCS3 recombinant proteins containing aMTD165 are capable of bidirectional passage across the plasma membrane.

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

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 (HM₁₆₅S3, HM₁₆₅S3A and HM₁₆₅S3B), 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.

4-2. aMTD/SD-Fused SOCS3 Recombinant Protein Inhibits the Secretion of Inflammatory Cytokines TNF-α and IL-6

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'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.

5. iCP-SOCS3 Suppresses Pro-Tumorigenic Functions in Lung Cancer Cells

5-1. iCP-50053 Enhances the Cellular Uptake into Lung Cancer Cells and the Systemic Delivery to the Lung Tissue

Although lung cancer is one of the most common cancers with a high mortality rate, there are few drugs for treating this lethal disorder. Since constitutive activation of STAT3 is found in various cancers and SOCS3 is closely related to the development of lung cancer, we first chose lung cancer as a primary indication of the iCP-SOCS3 as an anti-cancer agent.

To determine the cell-permeability of iCP-SOCS3 in the lung cancer 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 A549 lung cancer cells (FIG. 14). In addition, iCP-SOCS3 proteins enhanced the systemic delivery to lung tissue after intraperitoneal injection (FIG. 15). Therefore, these data indicate that iCP-SOCS3 protein could be intracellularly delivered and distributed to the lung cells and tissue, contributing for beneficial biotherapeutic effects.

5-2. iCP-50053 Inhibits Viability of Lung Cancer Cells

Since the endogenous level of SOCS3 protein is reduced in lung cancer patients, and SOCS3 negatively regulates cell growth and motility in cultured lung cancer cells, we investigated whether iCP-SOCS3 inhibits cell viability through SOCS3 intracellular delivery in lung cancer 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 A549 lung cancer cells—over 80% in 10 μM treatment (p<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 cancer cells, and would have a great ability to inhibit cell survival-associated phenotypes in lung cancer without any severe aberrant effects as a protein-based biotherapeutics.

5-3. iCP-SOCS3 Protein Induces Apoptosis in Lung Cancer Cells

To further determine the effect of iCP-SOCS3 on the tumorigenicity of lung cancer cells, we subsequently investigated whether iCP-SOCS3 regulates apoptosis in A549 cells. HM₁₆₅S3B protein (iCP-SOCS3) was a considerably efficient inducer of apoptosis in A549 cells, as assessed either by a fluorescent terminal dUTP nick-end labeling (TUNEL) assay (FIG. 17) and Annexin V staining (FIG. 18). Consistently, no changes in TUNEL and Annexin V staining were observed in A549 cells treated with HS3B compared to untreated cells (Vehicle).

5-4. iCP-SOCS3 Inhibits Migration/Invasion of Lung Cancer Cells

We next examined the ability of iCP-SOCS3 to influence cell migration. A549 cells were treated with recombinant proteins for 2 hrs, the monolayers were wounded, and cell migration in the wound was monitored after 48 hrs (FIG. 19). HM₁₆₅S3B 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, A549 cells treated with HM₁₆₅S3B 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. 20). In addition, A549 cells treated with HM₁₆₅S3B recombinant protein (iCP-SOCS3) caused remarkable decrease in invasion compared with the control proteins (HS3B; FIG. 21). Taken together, these data indicate that iCP-SOCS3 contributes to inhibit tumorigenic activities of lung cancer cells.

Example

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)

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

Histidine-tagged human SOCS3 proteins were constructed by amplifying the SOCS3 cDNA (225 amino acids) for aMTD fused to SOCS3 cargo. The PCR reactions (100 ng genomic DNA, 10 pmol each primer, each 0.2 mM dNTP mixture, lx reaction buffer and 2.5 U Pfu(+) DNA polymerase (Doctor protein, Korea)) were digested on the restriction enzyme site between Nde I (5′) and Sal I (3′) involving 35 cycles of denaturing (95° C.), annealing (62° C.), and extending (72° C.) for 45 sec each. For the last extension cycle, the PCR reactions remained for 10 min at 72° C. 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

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

For quantitative cell-permeability, recombinant SOCS3 proteins were conjugated to 5/6-fluorescein isothiocyanate (FITC) according to the manufacturer'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

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

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

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

PANC-1 cells (Korean Cell Line Bank, Seoul, Korea) were cultured in modified Eagle's medium (DMEM; Welgene, Daege, Korea) supplemented with 10% (v/v) FBS, penicillin (100 units/ml), and streptomycin (10 custom-character g/ml, Gibco BRL) and pretreated with 10 M of SOCS3 recombinant proteins for 2 hrs followed by exposing the cells to agonists (100 ng/ml IFN-) for 15 min. 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

Peritoneal macrophages were obtained from C3H/HeJ mice. Peritoneal macrophages were incubated with 10 μM recombinant proteins (1:HS3, 2:HM165S3, 3:HM165S3A and 4:HM165S3B, respectively) for 1 hr at 37° C. and then stimulated them with LPS (500 ng/ml) and/or IFN- custom-character (100 U/ml) without removing iCP-SOCS3 proteins for 3, 6, or 9 hrs. The culture media were collected, and the extracellular levels of cytokine were measured by a cytometric bead array (BD Biosciences, Pharmingen) according to the manufacturer's instructions.

Example 10
Cell Proliferation: CellTiter-Glo Cell Viability Assay

Cells originated from lung cancer and mouse fibroblast (NIH3T3) were purchased (ATCC, Manassas, Va.) and maintained as recommended by the supplier. These cells (3×103/well) were seeded in 96 well plates. The next day, cells were treated with DMEM (vehicle) or recombinant SOCS3 proteins for 96 hrs in the presence of serum (2%). Proteins were replaced daily. Cell growth and survival were evaluated with the CellTiter-Glo Cell Viability Assay (Promega, Madison, Wis.). Measurements using a Luminometer (Turner Designs, Sunnyvale, Calif.) were conducted following the manufacturer's protocol.

Example 11
Apoptosis: TUNEL Assay

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

Annexin V/7-Aminoactinomycin D (7-AAD) staining was performed using flow cytometry according to the manufacturer's guidelines. Briefly, 1×106 cells were washed three times with ice-cold PBS. The cells were then resuspended in 100 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
Cell Migration: Wound-Healing Assay

Cells were seeded into 12-well plates, grown to 90% confluence, and incubated with 10 μM HS3, HM16553, HM165S3A or HM165S3B in serum-free medium for 2 hrs prior to changing the growth medium. The cells were washed twice with PBS, and the monolayer at the center of the well was “wounded” by scraping with a pipette tip. Cells were cultured for an additional 48 hrs and cell migration was observed by phase contrast microscopy. The migration is quantified by counting the number of cells that migrated from the wound edge into the clear area.

Example 14
Transwell Migration Assay

The lower surface of Transwell inserts (Costar) was coated with gelatin (10 custom-character g/ml), and the membranes were allowed to dry for 1 hr at room temperature. The Transwell inserts were assembled into a 24-well plate, and the lower chamber was filled with growth media containing 10% FBS and FGF2 (10 g/ml). Cells (5×105) were added to each upper chamber, and the plate was incubated at 37° C. in a 5% CO2 incubator for 24 hrs. Migrated cells were stained with 0.6% hematoxylin and 0.5% eosin and counted.

Example 15
Invasion Assay

The lower surface of Transwell inserts (Costar) was coated with gelatin (10 custom-character g/ml), the upper surface of Transwell inserts was coated with matrigel (40 g per well; BD Biosciences), and the membranes were allowed to dry for 1 hr at room temperature. The Transwell inserts were assembled into a 24-well plate, and the lower chamber was filled with growth media containing 10% FBS and FGF2 (10 custom-character g/ml). Cells (5×105) were added to each upper chamber, and the plate was incubated at 37° C. in a 5% CO2 incubator for 24 hrs. Migrated cells were stained with 0.6% hematoxylin and 0.5% eosin and counted.

Example 16
Statistical Analysis

All data are presented as mean±s.d. Differences between groups were tested for statistical significance using Student's t-test and were considered significant at p<0.05 or p<0.01.

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.

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

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