Development of Protein-Based Biotherapeutics That Penetrate Cell-Membrane and Induce Anti-Cancer Effect - Cell-Permeable Trefoil Factor 1 (CP-TFF1) in Gastrointestinal Track (GIT) Cancer, Polynucleotides Encoding The Same, and Anti-Cancer Compositions Comprising The Same

TECHNICAL FIELD

In the present invention, we adopted the use of macromolecule intracellular transduction technology (MITT) to deliver biologically active TFF1 protein into gastric cancer cells, grown both in culture and as tumor xenografts and to investigate the feasibility of using TFF1 as a protein-based therapy for gastric cancer.

BACKGROUND ART

Gastric cancer remains the fourth most common cancer worldwide and the second leading cause of cancer-related deaths. The most common form of gastric cancer is intestinal-type gastric adenocarcinoma, which progresses through a cascade of gastric carcinogenesis from normal mucosa to chronic superficial gastritis, atrophic gastritis, intestinal metaplasia with low- and high-grade dysplasia (LGD and HGD, respectively), and invasive gastric adenocarcinoma finally.

Trefoil factor 1 (TFF1) is a member of the trefoil factor family peptides that are cysteine-rich proteins and form a characteristic trefoil domain. TFF1 is expressed predominantly in the gastric epithelia and secreted by the mucus-secreting pit cells of the corpus and antropyloric regions of the stomach. TFF1 has been reported as a gastric-specific tumour-suppressor gene. The TFF1 protein, which is secreted as a component of the protective mucus layer in the stomach, is highly expressed in response to mucosal injury. Previous reports have shown loss of TFF1 protein expression in more than two-thirds of gastric adenocarcinomas (AC) because of mutation-independent mechanisms. The silencing of the TFF1 gene expression in gastric cancer is predominantly induced by the loss of heterozygosity and hypermethylation of the TFF1 promoter. The TFF1-knockout mouse model provided first evidence supporting a tumor suppressor role of TFF1 in gastric tumorigenesis, demonstrating that it is essential for normal differentiation of the antral and pyloric gastric mucosa. The NF-κB transcription factor, regulated via the IκB kinase (IKK) complex, play a critical role in coupling inflammation and cancer. The activation of the NF-κB signaling pathway promotes the induction of inflammation-associated tumors and suppresses apoptosis in advanced tumors. In the regulation of the complex cancer-inducing NF-κB signaling, TFF1 plays an important role in NF-κB-mediated inflammatory response in the multistep gastric tumorigenesis cascade.

In principle, protein-based therapeutics offer to a way to control biochemical processes in living cells under non steady-state conditions and with fewer off target effects than conventional small molecule therapeutics. In practice, systemic protein delivery in animals has proven difficult due to poor tissue penetration and rapid clearance. Protein transduction exploits the ability of some cell-penetrating peptide (CPP) sequences to enhance the uptake of proteins and other macromolecules by mammalian cells. Previously developed hydrophobic CPPs, named membrane translocating sequence (MTS), membrane translocating motif (MTM) and macromolecule transduction domain (MTD), are able to deliver biologically active proteins into a variety of cells and tissues. Various cargo proteins fused to these CPPs have been used to test the functional and/or therapeutic efficacy of protein transduction. However, the recombinant proteins fused to previously develop hydrophobic CPPs displayed extremely low solubility, poor yields and relatively low cell- and tissue-permeability. Therefore, these recombinant proteins were not suitable for further clinical development as therapeutic agents. To overcome these limitations, cell-permeable TFF1 recombinant proteins (CP-TFF1) 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.

We have hypothesized that exogenously administered TFF1 proteins can compensate for the apparent inability of endogenously expressed members of this physiologic tumor suppressor in GIT to cure the established gastric cancers. We have tried to demonstrate that this approach namely “intracellular protein therapy” by designing and introducing cell-permeable form of TFF1 to determine its potential of anti-cancer therapeutic applicability. The present invention suggests that intracellular restoration of TFF1 with CP-TFF1 creates a new paradigm for anti-cancer therapy, and the intracellular protein replacement therapy with the TFF1 recombinant protein fused to the combination of aMTD and SDs pair may be useful to treat the cancer.

SUMMARY

An aspect of the present invention relates to cell-permeable TFF1 (CP-TFF1) recombinant proteins capable of mediating the transduction of biologically active macromolecules into live cells.

CP-TFF1 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.

CP-TFF1 fused to solubilization domains (SDs) greatly increase in their solubility and manufacturing yield when they are expressed and purified in the bacteria system.

The present invention also, relates to its therapeutic application for delivery of a biologically active molecule to a cell, involving a cell-permeable TFF1 recombinant protein, where the aMTD is attached to a biologically active cargo molecule.

Other aspects of the present invention relate to the development of TFF1 recombinant protein fused with aMTD sequences for drug delivery, protein therapy, intracellular protein therapy, protein replacement therapy and peptide therapy.

An aspect of the present invention provides cell-permeable TFF1 as a biotherapeutics having improved solubility/yield and cell-/tissue-permeability and anti-gastric 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 that the determination of aMTD43- and aMTD165-Mediated cell-permeability. uptake of aMTD-EGFP fusion proteins by RAW264.7 cells. Cells were exposed to the FITC conjugated proteins (10 μM) for 1 hour, treated to remove cell-associated but non-internalized protein and analyzed by flow cytometry. The EGFP protein cargos contained aMTDs (red), a SDA only (blue), FITC only (green), or vehicle (shaded). Lower Panel: EGFP protein uptake by NIH3T3 cells. Cells were visualized by fluorescence confocal laser scanning microscopy.

FIG. 2 shows that the determination of aMTD43- and aMTD165-mediated intracellular delivery and localization. Uptake of aMTD-EGFP fusion proteins by NIH3T3 cells. Cells were exposed to the FITC conjugated proteins (10 μM) for 1 hour, treated to remove cell-associated but non-internalized protein. Cells were visualized by fluorescence confocal laser scanning microscopy.

FIG. 3 shows that the structure of aMTD/SD-fused TFF1 recombinant proteins: Set 1. A schematic Diagram of aMTD/SD-fused TFF1 recombinant proteins containing, aMTD43 and SDA or SDB is illustrated and constructed (Set 1).

FIG. 4 shows that the inducible expression and purification of TFF1 recombinant proteins: Set 1. Proteins expressed in E. coli were determined by SDS-PAGE analysis of cell lysates before (−) and after (+) induction with IPTG; aliquots of Ni2+ affinity purified proteins (P); and size marker (M). Solubility was scored a 5-point scale ranging from highly soluble proteins with little tendency to precipitate (+++++) to largely insoluble proteins (+).

FIG. 5 shows that the structural change of aMTD/SD-fused TFF1 recombinant protein: Set 2. A schematic Diagram of aMTD/SD-fused TFF1 recombinant proteins containing, aMTD165 and SDC or SDD is illustrated and constructed (Set 2).

FIG. 6 shows that the inducible expression and purification of TFF1 recombinant proteins: Set 2. Proteins expressed in E. coli were determined by SDS-PAGE analysis of cell lysates before (−) and after (+) induction with IPTG; aliquots of Ni2+ affinity purified proteins (P); and size marker (M). Solubility was scored a 5-point scale ranging from highly soluble proteins with little tendency to precipitate (+++++) to largely insoluble proteins (+).

FIG. 7 shows that the structure of aMTD/SD-fused TFF1 recombinant protein: Set 3. A schematic Diagram of aMTD/SD-fused TFF1 recombinant proteins containing, aMTD165 and SDA and SDB is illustrated and constructed (Set 3).

FIG. 8 shows that the inducible expression and purification of TFF1 recombinant proteins: Set 3. Proteins expressed in E. coli were determined by SDS-PAGE analysis of cell lysates before (−) and after (+) induction with IPTG; aliquots of Ni2+ affinity purified proteins (P); and size marker (M). Solubility was scored a 5-point scale ranging from highly soluble proteins with little tendency to precipitate (+++++) to largely insoluble proteins (+).

FIG. 9 shows that aMTD-mediated cell-permeability of TFF1 recombinant proteins. RAW264.7 cells were exposed to 10 μM of the FITC conjugated TFF1 proteins containing aMTD165 and solubilization domains, lacking aMTD (HST1SB, HSsT1SB) or 10 μM of FITC alone (green) for 1 hour and analyzed by flow cytometry.

FIG. 10 shows that aMTD-mediated intracellular delivery and localization of recombinant proteins. NIH3T3 cells were exposed to 10 μM of the FITC conjugated TFF1 proteins containing aMTD165 and solubilization domains (HSAM165T1SB), lacking aMTD (HSAT1SB, HST1SB), lacking cargo (HSAM165SB) or 10 μM of FITC alone (green) for 1 hour and visualized by fluorescence confocal laser scanning microscopy.

FIG. 11 shows that the systemic delivery of aMTD/SD-fused TFF1 recombinant proteins In vivo. CP-TFF1 recombinant proteins were systemically delivered to various tissues. Tissue distribution of the recombinant proteins (green staining) was assessed by fluorescence microscopy.

FIG. 12 shows that the mechanism of aMTD-mediated TFF1 proteins uptake into cells. (A) Cell surface protein-independence of aMTD165-mediated protein uptake. (B) Endocytosis-independence of aMTD165-mediated protein uptake. (C) ATP source-independence of aMTD165-mediated protein uptake. (D) EDTA suppresses aMTD165-mediated protein uptake. (E) Temperature-dependence of aMTD165-stimulated protein uptake. Cells (shaded) were exposed for one hour to HSAM165T1SB (red), HSAT1SB (blue) and FITC (green) were processed as before to remove non-internalized protein and were analyzed by flow cytometry.

FIG. 13 shows that the cell-permeability of CP-TFF1 (HSAM165T1SB) in gastric cancer cells. Gastric cell lines (AGS and MKN75 cells) were exposed to 10 μM of the FITC conjugated TFF1 proteins containing aMTD165 and solubilization domains, lacking aMTD (HST1SB, HST1SB) or 10 μM of FITC alone (green) for 1 hour and analyzed by flow cytometry.

FIG. 14 shows that the tissue distribution of CP-TFF1 into stomach. Systemic TFF1 recombinant protein delivery to murine stomach. Tissue distribution of the recombinant proteins (green staining) was assessed by fluorescence microscopy.

FIG. 15 shows that the CP-TFF1 inhibits proliferation of gastric cancer cells. Various gastric cancer cells (AGS, MKN45 and NCI-N87 Cells) were treated with CP-TFF1 (HSAM165T1SB, 10 μM) for 72 hours. Cell viability assay was evaluated with Cell-Titer Glo luminescent cell viability assay. All experimental data obtained using cultured cells were expressed as means S.D. For the ATP-Glo cell viability assay, statistical significance was evaluated using a one-tailed Student t-test. Statistical significance was established at p<0.05.

FIGS. 16A to 16C show that the CP-TFF1 inhibits migration of gastric cancer cells. Representative photomicrographs of wound healing assays. After gastric cancer cell lines (AGS, MKN45 and STKM2 cells) were seeded into 12-well plates and grown to 90% confluence, wounds were made by scraping the cell layer with a sterile tip. Cells were treated with CP-TFF1 (HSAM165T1SB, 10 μM) for 2 hours before changing the growth medium. Photograph was taken after 24 hours of incubation. *: p<0.05.

FIG. 17 shows that the CP-TFF1 Inhibits transwell migration of gastric cancer cells. Representative photomicrographs of Transwell migration assay. Gastric cancer cells, AGS were treated with CP-TFF1 (HSAM165T1SB, 10 μM) proteins for 24 hour in serum-free media, and migration were measured by Transwell assay. Photograph was taken after 24 hours of incubation. *: p<0.05.

FIG. 18 shows that the CP-TFF1 induces expression of apoptosis-related protein in human gastric cancer cells. AGS and NCI-N87 cells were treated for 24 hours with 10 μM CP-TFF1 recombinant proteins. Equal amounts of cell lysate protein were immunoblotted with cleaved Caspase-3 antibody. β-actin was used for loading control.

FIG. 19 shows that the external appearances of human gastric tumor bearing mice. Tumor was induced by implanted with MKN45 tumor block. After tumor reached a size of 50-80 mm3 (start), the mice were injected daily (I.V.) for 3 weeks with the diluent alone, HSAT1SB (TFF1) or HSAM165T1SB (CP-TFF1) and observed for 3 weeks following the termination of the treatment.

FIG. 20 shows that the CP-TFF1 suppresses tumor growth in mouse xenograft model. Tumor was induced by implanted with MKN45 tumor block. After tumor reached a size of 50-80 mm3 (start), the mice were injected daily (I.V.) for 3 weeks with the diluent alone, HSAT1SB (TFF1) or HSAM165T1SB (CP-TFF1) and observed for 3 weeks following the termination of the treatment. *P<0.05 as determined by the Student t-test.

FIG. 21 shows that the CP-TFF1 inhibits tumor growth in xenograft model. Tumor was induced by implanted with MKN45 tumor block. After tumors reached a size of 50 to 80 m3 (start), mouse were injected daily (I. V.) for 3 weeks with diluent alone (black), 800 μg TFF1 (blue) or CP-TFF1 (red). Tumor growth was suppressed to varying degrees after the protein therapy ended (stop). *P<0.05 as determined by the Student t-test.

FIG. 22 shows that the CP-TFF1 regulates expression of tumor-associated proteins in human Tumor Xenograft. Tumor was induced by implanted with MKN45 tumor block. After tumors reached a size of 50 to 80 m3 (start), mouse were injected daily (I. V.) for 3 weeks with diluent alone, 800 μg TFF1 or CP-TFF1. Tumor tissues from mice treated daily for 3 weeks with indicated proteins and observed for 3 weeks following the termination of the treatment were sectioned and immunostained with antibodies against VEGF.

DETAILED DESCRIPTION

In this invention, it has been hypothesized that exogenously administered TFF1 proteins can compensate for the apparent inability of endogenously expressed members of this physiologic tumor suppressor in GIT to cure the established gastric cancers. To prove our hypothesis, the TFF1 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 TFF1 proteins can reconstitute their endogenous stores and restore their basic function as the tumor suppressor. This art of invention has demonstrated “intracellular protein therapy” by designing and introducing cell-permeable form of TFF1 has a great potential of anti-cancer therapeutic applicability in gastric cancer.

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

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 TFFF1, 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. membrane translocating sequence: MTS and macromolecule transduction domain: MTD) as explained previously.

(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.

TABLE 4

[Characteristics of aMTD43 and aMTD165]

Rigidity/
Structural
Hydrop-

Length
Flexibility
Feature
athy

No.
Sequences
(a.a)
(II)
(AI)
(GRAVY)

43
LLAPLVVAAVP
12
41.2
203.3
2.3

165
ALAVPVALAIVP
12
50.2
203.3
2.3

(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 (Al) were 2.5±0.4 and 217.9±43.6, respectively.

(5) Secondary Structure (Cc-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, but favored for membrane penetration.

(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 were used with each cargo in vivo. 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 (rPs or rPeptides), 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). Al 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 (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 Developed Hydrophobic CPPs]

Summarized Critical Factors of aMTD

Newly Designed CPPs

Critical Factor
Range

Bending Potential
Proline presences in the

(Proline Position: PP)
middle (5′, 6′, 7′ or 8′) 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

For confirming the validity of 6 critical factors providing the optimized cell-/tissue-permeability, 240 aMTD sequences have been designed and developed based on six critical factors (TABLES 2-1 to 2-6 and 3). All 240 aMTDs (hydrophobic, flexible, bending, aliphatic and helical 12 a/a-length peptides) are 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) was significantly increased by up to 164 fold, with average increase of 19.6±1.6. Moreover, 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 (CFs) 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 (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)]

Rigidity/
Sturctural

Sequence

Flexibility
Feature
Hydropathy
Residue

ID 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
283.3
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

TABLE 2-2

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

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

Rigidity/
Sturctural

Sequence

Flexibility
Feature
Hydropathy
Residue

ID 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
195.8
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

TABLE 2-3

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

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

Rigidity/
Sturctural

Sequence

Flexibility
Feature
Hydropathy
Residue

ID Number
aMTD
Sequences
Length
(II)
(Al)
(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
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)]

Rigidity/
Sturctural

Sequence

Flexibility
Feature
Hydropathy
Residue

ID 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
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
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)]

Rigidity/
Sturctural

Sequence

Flexibility
Feature
Hydropathy
Residue

ID 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)]

Rigidity/
Sturctural

Sequence

Flexibility
Feature
Hydropathy
Residue

ID 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

TABLE 3

[Summarized Critical Factors of aMTD after

In-Depth Analysis of Experimental Results]

Summarized Critical Factors of aMTD

Newly Designed CPPs

Critical Factor
Range

Bending Potential
Proline presences in the

(Proline Position: PP)
middle (5′, 6′, 7′ or 8′) 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
2.1-2.6

(Grand Average of

Hydropathy)

Length
12

(Number of Amino Acid)

Amino acid Composition
A, V, I, L, P

Secondary Structure
α-Helix is favored

but not required

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 and intracellular delivery potential compared to reference hydrophobic CPPs reported during the past two decades.

2. Development of TFF1 Recombinant Proteins Fused to aMTD and Solubilization Domain

High solubility, yield, stability, and cell-/tissue-permeability are essentially required together with their functional activity (anti-cancer effect) to determine whether they are eligible to have therapeutic applicability in clinic. Therefore, additional modifications were strongly recommended on the conventional recombinant TFF1 proteins fused to previously developed MTD.

2-1. The Advanced Hydrophobic CPP—aMTD43 and aMTD165

Based on these analytical data of hydrophobic CPPs published, 240 advanced MTDs (aMTDs) sequences have been designed and developed based on 6 critical factors (TABLES 2-1 to 2-6 and 3). Based on these six critical factors proven by experimental data, newly designed aMTD have been developed for their practical therapeutic usage to facilitate protein translocation across the plasma membrane.

For this present invention, aMTD/SD-fused TFF1 recombinant proteins have been developed by adopting aMTD43 and aMTD165 that satisfied all 6 critical factors. We found that aMTD43 and aMTD165 had a potential to enhance the uptake of a His-tagged enhanced green fluorescent protein (EGFP) in RAW264.7 cells as assessed by flow cytometry. Both peptides promoted greater cellular uptake of an EGFP cargo protein by cultured NIH3T3 cells than SDA only (FIGS. 1 and 2).

2-2. Selection of Solubilization Domain for Recombinant Proteins

In addition with aMTD, we adopted non-functional protein domain (solubilization domain: SD; TABLE 5) to improve solubility, yield, and stability of the recombinant proteins. In previous study, to develop recombinant aMTD/SD-fused TFF1 recombinant proteins as protein-based biotherapeutics to treat gastric cancer, recombinant cargo (TFF1) proteins fused to conventional hydrophobic CPPs—MTDs could be expressed in bacteria system, purified with single-step affinity chromatography, but protein dissolved in physiological buffers (e.q. PBS, DMEM or RPMI1640 etc.) was highly insoluble and had low yield as a soluble form. This problem is extremely crucial in terms of the fact whether these proteins could go ahead for further pre-clinical and clinical development.

According to this specific aim, 5 solubilization domains were selected and information of these SDs are shown TABLE 5.

TABLE 5

[Characteristics of Solubilization 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

2-3. Preparation of TFF1 Recombinant Proteins

To overcome the limitations of high insolubility and low yield, we developed a newly recombinant TFF1 fused to aMTD and the SDs. To develop stable aMTD/SD-fused TFF1 recombinant proteins with better yield and solubility, total of three sets of TFF1 recombinant protein clones were designed and developed. In Set 1, TFF1 recombinant protein clones fused with aMTD43 and either SDA or SDB fused to C-terminus of the protein were developed. Recombinant proteins in Set 1 have shown relatively weak expression with very low solubility and yield (FIG. 4). To develop more stable aMTD/SD-fused TFF1 recombinant proteins, we performed the cloning for aMTD/SD-fused TFF1 recombinant proteins containing aMTD165 and SDC or SDD (FIG. 5). Despite the fact that Set 2 has shown higher yield and solubility compared to set 1, we were not satisfied with the protein solubility and yield in Set 2 of aMTD/SD-fused TFF1 recombinant proteins (FIG. 6). Therefore, Set 3 recombinant protein clones have been designed and developed for more stable, improved solubility and yielding TFF1 recombinant protein. In Set 3, TFF1 recombinant proteins were developed by replacing the aMTD and SDs to the combination of aMTD165 and both SDA and SDB on each ends of the protein (FIG. 7). TFF1 protein has a signal sequence at the N terminal. From analyzing this signal sequence based on our critical factors, we determined that this signal sequence has a cell-permeable potential (TABLE 6). Therefore, we additionally developed TFF1 (sTFF1) by removing these 15 amino acid signal sequences. These recombinant protein clones were expressed in E. coli BL21-Codon plus (DE3). The recombinant proteins were purified under the naturing conditions and the yields of soluble protein 45 mg/L (FIG. 8). Set 3 aMTD/SD-TFF1 recombinant protein has been significantly enhanced (250 fold) in solubility and yield. Therefore, aMTD/SD-fused TFF1 recombinant protein 6 in Set 3, with highest solubility/yield, has been selected to determine the cell-/tissue-permeability in vitro and xenograft model experiments.

TABLE 6

[Characteristics of TFF1 signal sequence]

Rigidity/
Structural
Hydrop-

Length
Flexibility
Feature
athy

Group
Sequences
(a.a)
(II)
(AI)
(GRAVY)

Signal
VICALVLVSMLAL
13
9.23
232.31
3.04

sequence

PCR primers for the His-tagged solubilization domain recombinant proteins fused to aMTD43 and aMTD165 are summarized in TABLE 7. cDNA and amino acid sequences of histidine tag are provided in SEQ ID NO: 1 and 2, and cDNA and amino acid sequences of aMTDs and the peptides are indicated in SEQ ID NO: 3 and 4, respectively. cDNA and amino acid sequences are displayed in SEQ ID NO: 5 and 6 (TFF1), SEQ ID NO: 7 and 8 (SDA), SEQ ID NO: 9 and 10 (SDB), SEQ ID NO: 11 and 12 (SDC), SEQ ID NO: 13 and 14 (SDD), SEQ ID NO: 15 and 16 (SDE) and SEQ ID NO: 17 and 18 (SDF), respectively.

TABLE 7

[PCR Primers for His-tagged TFF1 Proteins Fused to aMTD]

Recombinant

Cargo
aMTD
Protein
5′ Primers
3′ Primers

TFF1
Non
HT1
C1/F:
C1/R:

5′-CCACCATGGAGAACAAGGT
5′-TTTGAATTCTCAGTAGGGC

GATCTGC-3′
CGCCACACGGCCT-3′

aMTD43
HM₄₃T1
C2/F:
C2/R:

5′-GGAATTCCATATGCTGCTGG
5′-CCCGGATCCTAAAAATTCA

CGGCGCCGCTGGTGGTGGCGGCG
CACTCCTCTTCTGGAGGGAC-3′

GTGCCGGCCACCATGGAGAACAA

GGTGATCTGC-3′

HM₄₃T1SA
C3/F:
C3/R:

5′-GGAATTCCATATGCTGCTGG
5′-CCCAAGCTTAAATTCACACT

CGGCGCCGCTGGTGGTGGCGGCG
CCTCTTCTGGAGGGAC-3′

GTGCCGGCCACCATGGAGAACAA

GGTGATCTGC-3′

HM₄₃T1SB
C4/F:
C4/R:

5′-GGAATTCCATATGCTGCTGG
5′-CCCAAGCTTAAATTCACACT

CGGCGCCGCTGGTGGTGGCGGCG
CCTCTTCTGGAGGGAC-3′

GTGCCGGCCACCATGGAGAACAA

GGTGATCTGC-3′

aMTD165
HSAM₁₆₅T1SB
C6/F:
C6/R:

5′-CGCGGATCCGCGCTGGCGG
5′-GGGTTTGTCGACAAATTCAC

TGCCGGTGGCGCTGGCGATTGTG
ACTCCTCTTC-3′

CCGATGGCCACCATGGAGAACAA

G-3′

HSCT1M₁₆₅
C7/F:
C7/R:

5′-CGCGGATCCATGGCCACCA
5′-CCCAAGCTTTTACGGCACAA

TGGAGAAC-3′
TCGCCAGCGCCACCGGCACCGCC

AGCGCAAATTCACACTCCTCTTC-

3′

HSDT1M165
C8/F:
C8/R:

5′-CGCGGATCCATGGCCACCAT
5′-CCCAAGCTTTTACGGCACAA

GGAGAAC-3′
TCGCCAGCGCCACCGGCACCGCC

AGCGCAAATTCACACTCCTCTTC-

3′

HSAT1SB
C6-1/F:
C6-1/6-2/6-3/6-5R:

5′-CGCGGATCCGCGCTGGCGG
5′-GGGTTTGTCGACAAATTCAC

TGCCGGTGGCGCTGGCGATTGTG
ACTCCTCTTC-3′

CCGATGGCCACCATGGAGAACAA

G-3′

HSAM165sT1SB
C6-2/F:

5′-CGCGGATCCGCGCTGGCGGT

GCCGGTGGCGCTGGCGATTGTGC

CGGGCACCCTGGCCGAGGCCCAG

ACAGAG-3′

HSAMsT1SB
C6-3/F:

5′-CGCGGATCCGGCACCCTGGCC

GAGGCCCAGACAGAG-3′

HSAM₁₆₅T1SBM₁₆₅
C6-5/F:

5′-CGCGGATCCATGGCCACCA

TGGAGAACAAGGTG-3′

3. aMTD/SDs-Fused TFF1 Recombinant Proteins Significantly Increase Cell- and Tissue-Permeability

3-1. aMTD/SDs-Fused TFF1 Recombinant Proteins are Cell-Permeable

Cell permeability of TFF1 recombinant protein was evaluated in RAW 264.7 cells after 1 hour of protein treatment. aMTD/SD-fused TFF1 recombinant proteins were conjugated to FITC, according to the manufacturer's instructions. Protein uptake of TFF1 proteins containing aMTD165 (HSAM165T1SB, HSAM165sT1SB, HSAM165T1SB M165) in RAW264.7 cell (FIG. 9).

In contrast, FITC-labeled TFF1 lacking aMTD (HSAT1SB, HSAsT1SB) was not detectable in RAW 264.7 cell. Similar Results were observed in NIH3T3 cells, using fluorescence confocal laser scanning microscopy to determine intracellular localization (FIGURE ID NO. 10). TFF1 proteins containing aMTD165 (HSAM₁₆₅T1SB, HSAM₁₆₅sT1SB, HSAM₁₆₅T1SB M₁₆₅) and efficiently entered the cells and were localized to various extents in the cytoplasm. In contrast, TFF1 protein (HSAT1SB, HSAsT1SB), containing only 6×His and the SDs, did not appear to enter the cells.

3-2. aMTD/SDs-Fused TFF1 Recombinant Proteins Enhance the Systemic Delivery to a Variety of Tissues

Next, to further investigate in vivo delivery of TFF1 recombinant proteins, FITC-labeled TFF1 proteins were monitored following intraperitoneal (IP) injections in mice. Tissue distributions of fluorescence-labeled-TFF1 proteins in different organs were analyzed by fluorescence microscopy. As shown in FIG. 11, aMTD165 enhanced the systemic delivery of TFF1 protein to variety of tissues including brain, heart, lung, liver, spleen and kidney. However, the recombinant TFF1 lacking aMTD (HSAT1SB) showed limited tissue-permeability in various organs. Therefore, the aMTD165/SD-fused TFF1 recombinant proteins have significantly higher cell- and tissue-permeability as compared to the recombinant protein lacking aMTD. Thus, we determined aMTD165/SD-fused TFF1 recombinant protein which shows cell-/tissue-permeability as cell-permeable TFF1 (CP-TFF1).

4. Membrane Integrity and Fluidity were Essential for aMTD165-Delivery Mechanism

Since the aMTD165 outperformed over other transduction domains tested, we next investigated the mechanism of aMTD165-mediated protein uptake. The hydrophobic aMTD is thought to enter the cells directly by penetrating the plasma membrane. Several lines of evidence suggested that endocytosis was not the major route of entry by aMTD165-fused TFF1 proteins. In particular, uptake was unaffected by the treatment of cells with proteases, microtubule inhibitors or the ATP-depleting agent, antimycin. Conversely, HSAM165T1SB uptake was blocked by the conditions affecting membrane fluidity (temperature) and integrity (EDTA) (FIG. 12).

5. CP-TFF1 Enhances the Penetration into Gastric Cancer Cells and Systemic Delivery to Stomach

To determine the cell-permeability of CP-TFF1 in the gastric cancer cells, cellular uptake of FITC-labeled TFF1 recombinant proteins was quantitatively evaluated by flow cytometry. FITC-HSAM165T1SB recombinant protein (CP-TFF1) promoted the transduction into cultured AGS and MKN75 gastric cancer cells (FIG. 13). In addition, CP-TFF1 enhanced the systemic delivery to stomach after intraperitoneal injection (FIG. 14). Therefore, these data indicate that CP-TFF1 could be intracellularly delivered and distributed to the target cells and stomach tissue, contributing for beneficial biotherapeutic effects.

6. CP-TFF1 Suppressed Proliferation of Gastric Cancer Cells

To examine the effect of CP-TFF1 on cancer cell proliferation, we performed the CellTiter-Glo Luminescent Cell Viability Assay. As shown in FIG. 15, CP-TFF1 (HSAM165T1SB) was the most potent inducer of cytotoxicity—over 60%˜80% in 10 μM treatment (p<0.05) than other proteins including HSAT1SB or vehicle alone (i.e. exposure of cells to culture media without recombinant proteins) in AGS and NCI-N87 cells. Also, CP-TFF1 mildly suppressed the proliferation in other gastric cancer cells (MKN45). However, CP-TFF1 proteins neither appeared to induce cytotoxicity in NIH3T3 cells nor were cell viability affected, even after exposing these cells to equal concentrations (10 μM) of protein over 3 days. These results suggest that the protein is merely non-toxic to cells and indicates that CP-TFF1 has a great ability to inhibit cell survival-associated phenotypes in gastric cancer cells.

7. CP-TFF1 Protein Inhibited Migration of Gastric Cancer Cells

We next examined the wound healing assay and Transwell assay to assess the effects of CP-TFF1 proteins on gastric cancer cell migration (AGS, MKN45 and STKM2 cells). The wounds were produced by scraping of the cell monolayer with sterile white tip. CP-TFF1 protein (HSAM165T1SB) suppressed repopulation of the wounded monolayer (FIGS. 16A to 16C). Consistent with this, AGS cells treated with CP-TFF1 (HSAM165T1SB) also showed significant inhibitory effect on their Transwell migration compared with untreated cells (Vehicle) (FIG. 17). However, TFF1 protein lacking aMTD165 (HST1SB) was not affected on gastric cancer cell migration. Taken together, these data indicate that CP-TFF1 contributes to inhibit tumorigenic activities of gastric cancer cells.

8. CP-TFF1 Protein Induced Apoptosis in Gastric Cancer Cells

To further examine the underlying mechanisms of the anti-cancer effect of CP-TFF1, we performed western blot analysis. Human gastric cancer cells (AGS, NCI-N87) were treated with 10 μM of CP-TFF1 proteins (HSAM165T1SB) for 24 hr. Compared to control recombinant protein (HST1SB)-treated cells, cells treated with CP-TFF1 showed significantly increased cleaved Caspase-3 which plays an important role in apoptosis (FIG. 18). Therefore, these data indicate that CP-TFF1 recombinant protein induce the apoptosis in gastric cancer cells.

9. CP-TFF1 Suppresses Pro-Tumorigenic Functions in Gastric Cancer Cells
9-1. CP-TFF1 Suppressed Tumor Growth in Tumor Block Implanted Xenograft

Next, we assessed the anti-tumor activity of CP-TFF1 against human cancer xenografts. Balb/c nu/nu mice were subcutaneously implanted with MKN45 tumor block (2 mm3) into the left back side of the mouse. Then, the mice were intravenously injected with 800 Ng/head recombinant TFF1 proteins (HSAT1SB or HSAM165T1SB) or diluent (DMEM) every day for 3 weeks. Mice were observed for an additional 3 weeks after the treatments ended. It shows phenotypic appearance of mouse treated with diluent, TFF1, CP-TFF1 at Day 0, 21, 42 (each group tested 7 mice) (FIG. 19). Tumor weight was significantly reduced in mice treated with CP-TFF1 (FIG. 20 lower left panel) and the diagram shows the isolated tumor of mice from each group at Day 42 (FIG. 20 lower right panel).

CP-TFF1 (HSAM165T1SB) protein significantly suppressed the tumor growth (p<0.05) during the treatment phase and persisted for at least 3 weeks after the treatment terminated (90% inhibition at day 42) (FIG. 21). While tumor growth was also reduced in mice treated with the HSAT1SB control protein, which lacked aMTD sequences, the effect was not significant. These results suggest that CP-TFF1 inhibits the persistence of established tumors as well as the tumor growth of cancer cells.

9-2. CP-TFF1 Regulates the Expression of Tumor-Associated Proteins in Human Tumor Xenograft

The anti-tumor activity of CP-TFF1 (HSAM165T1SB) at day 42 was accompanied by changes in the expression of biomarkers (FIG. 22). The expressions of vascular endothelial growth factor (VEGF), a pro-angiogenic factor, were inhibited in HSAM165T1SB)-treated tumors. While the expression of VEGF was also reduced in mice treated with the HSAT1SB control protein, which lacked aMTD sequences, the effect was not significant. These in vivo results suggest that CP-TFF1 targets tumor cells directly and may be developed for use as novel therapy against gastric cancer.

Examples

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
Construction of Expression Vectors for TFF1 Recombinant Proteins Fused to aMTDs

The expression vectors were designed for TFF1 recombinant protein fused with either one or both SDs (SDA, SDB, SDC, and SDD) and aMTD43 or aMTD165. To acquire expression vectors for TFF1 recombinant proteins, polymerase chain reaction (PCR) had been devised to amplify each designed aMTD43 or aMTD165 fused with TFF1.

The PCR reactions (100 ng genomic DNA, 10 pmol each primer, each 0.2 mM dNTP mixture, 1× reaction buffer and 2.5 U Pfu(+) DNA polymerase (Doctor Protein, Korea)) was digested on the different restriction enzyme site involving 40 cycles of denaturation (95° C.), annealing (58° C.), and extension (72° C.) for 30 seconds each. For the last extension cycle, the PCR reactions remained for 10 minutes at 72° C. TFF1 recombinant protein clones were produced in three sets. Set 1 coding sequence for SDA or SDB fused to C terminus of his-tagged aMTD/SD-TFF1 was cloned at NdeI (5′) and SalI (3′) in pET-28a(+) (Novagen, Madison, Wis., USA) from PCR-amplified DNA segments. PCR primers for the TFF1-fuseded SDA or SDB are summarized in TABLE 8, respectively. Set 2 coding sequence for SDC or SDD fused to N terminus of TFF1 recombinant protein was cloned at BamHI (5′) and HindIII (3′) in pET-32a(+) (Novagen, Madison, Wis., USA) or pET-39b(+) (Novagen, Madison, Wis., USA) from PCR-amplified DNA segments. PCR primers for the TFF1-fused to either SDC or SDD are summarized in TABLES 8 and 9, respectively. Set 3 coding sequence for SDA fused to N terminus and SDB fused to C terminus of aMTD/SD-TFF1 or aMTD/SD-sTFF1 was cloned at NdeI (5′) and XhoI (3′) in pET-28a(+) (Novagen, Madison, Wis., USA) from PCR-amplified DNA segments. DNA ligation was performed using T4 DNA ligase at 4° C. overnight. These plasmids were mixed with competent cells of E. coli DH5-alpha strain on the ice for 30 minutes. These mixture was placed in a water bath at 42° C. for 90 seconds and placed on ice for 2 minutes. Then, the mixture added with LB broth media was recovered in 37° C. shaking incubator for 1 hour. Transformant was plated on LB broth agar plate with kanamycin (50 μg/mL) (Biopure, Johnson, Tenn.) or ampicillin (100 μg/mL) (Biopure, Johnson, Tenn.) before incubating overnight at 37° C. From a single colony, plasmid DNA was extracted; and after the double digestion of Inserts fit restriction enzymes, digested DNA was confirmed at TFF1 (252 bp), sTFF1(207 bp), SDA (297 bp), and SDB (552 bp) by using 1.4% agarose gels electrophoresis.

TABLE 8

[PCR Primers for SD Fused to TFF1 Protein]

Recombinant

SD
aMTD
Protein
5′Primers
3′Primers

SDA
aMTD43
HM₄₃T1SA
C3/F:
C3/R:

5′-CGCGGATCCATGGCAAATATT
5′-ACGCGTCGACTTACCTCGGCT

ACCG-3′
GCACCGG-3′

aMTD165
HSAM₁₆₅T1SB
C6/F:
C6/6-1/6-2/6-3/R:

HSAT1SB
5′-GGGTTTCATATGATGGCAAATA
5′-CGCGGATCCCCTCGGCTGCAC

HSAM₁₆₅sT1SB
TTACCGTTTTC-3′
CGGCACGGC-3′

HSAMsT1SB

HSAM₁₆₅SB

C6-4/R:

5′-ACGCGTCGACCGGCACAATCGC

CAGCGCCACCGGCACCGCCAGCGC

CCTCGGCTGCACCGGCACGGA-3′

HSAM₁₆₅T1SBM₁₆₅

C6-5/R:

5′-CGCGGATCCCCTCGGCTGCACC

GGCACGGC-3′

SDB
aMTD43
HM₄₃T1SB
C4/F:
C3/R:

5′-CGCGGATCCATGGCAGAAC
5′-ACGCGTCGACTTACCTCG

AAAGCG-3′
GCTGCACCGG-3′

aMTD165
HSAM₁₆₅T1SB
C6/F:
C6/6-1/6-2/6-3/6-4/R:

HSAT1SB
5′ ACGCGTCGACATGGCAGAAC
5′-CCGCTCGAGGTTAAAGGGTTT

HSAM₁₆₅sT1SB
AAAGCGAC-3′
CCGAAGGCTTG-3′

HSAMsT1SB

HSAM₁₆₅SB

HSAM₁₆₅T1SBM₁₆₅

C6-5/R:

5′-CCGCTCGAGGTTACACAATCGC

CAGCGCCACCGGCACCGCCAGCGC

CGGAAGGGTTTCCGAAGGCTTGGC

TATC-3′

Example 2
Purification and Preparation of aMTD/SD-Fused TFF1 Recombinant Protein

Denatured recombinant proteins were lysed using denature lysis buffer (8 M Urea, 10 mM Tris, 100 mM NaH2PO4) and purified by adding Ni-NTA resin. Resin bound to proteins were washed 3 times with 30 mL of denature washing buffer (8 M Urea, 10 mM Tris, 20 m imidazole, 100 mM NaH2PO4). Proteins were eluted 3 times with 30 mL of denature elution buffer (8 M Urea, 10 mM Tris, 250 mM imidazole). After purification, they was dialyzed twice against a refolding buffer (550 mM Guanidine-HCl, 440 mM L-Arginine, 50 mM Tris, 100 mM NDSB, 150 mM NaCl, 2 mM reduced glutathione and 0.2 mM oxidized glutathione). Finally, they were dialyzed against a physiological buffer such as DMEM at 4° C. until the dialysis was over 300×105 times. Concentration of purified proteins was quantified using Bradford assay according to the manufacturer's instructions. After purification, they were dialyzed against DMEM as indicated above. Finally, SDS-PAGE analysis was conducted to confirm the presence of target protein (FIG. 6).

The His-tagged aMTD/SD-fused TFF1 recombinant proteins (FIG. 8, Set 3) were purified from E. coli BL21-Gold (DE3) competent cells (Agilent Technologies, Santa Clara, USA) grown to an A600 of 0.6 and induced overnight at 25° C. with 0.7 mM/IPTG. The E. coli cultures were harvested by centrifugation at 5,000× rpm for 10 minutes, and the supernatant was discarded. The pellet was resuspended in the lysis buffer (50 mM NaH2PO4, 10 mM Imidazol, 300 mM NaCl, pH 8.0). The cell lysates were sonicated on ice using a sonicator (Sonics and Materials, Inc., Newtowen, Conn.) equipped with a probe. After centrifuging the cell lysates at 5,000× rpm for 10 minutes to pellet the cellular debris, the supernatant was incubated with lysis buffer-equilibrated Ni-NTA resin (Qiagen, Hilden, Germany) gently by open-column system (Bio-rad, Hercules, Calif.). After washing protein-bound resin with 30 ml native wash buffer (50 mM NaH2PO4, 20 mM Imidazol, 300 mM NaCl, pH 8.0), the bounded proteins were eluted with 20 ml elution buffer (50 mM NaH2PO4, 250 mM Imidazol, 300 mM NaCl, pH 8.0). Total concentration of protein was quantified using Bradford assay according to the manufacturer's instructions. After purification, they were dialyzed against DMEM at 4° C. until the dialysis was over 300×105 times the protein volume. aMTD/SD-fused TFF1 recombinant proteins purified under natural condition were analyzed on 15% SDS-PAGE gel and stained with Coomassie Brilliant Blue.

Solubility will be scored on a 5 point scale ranging from highly soluble proteins with little tendency to precipitate (+++++) to largely insoluble proteins (+) by measuring their turbidity (A450). Yield (mg/L) in physiological buffer condition of each recombinant protein will also be determined.

Example 3
Determination of Cell-Permeability of aMTD/SD-Fused TFF1 Recombinant Proteins

For quantitative cell-permeability, the aMTD-fused recombinant proteins were conjugated to 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 hour at 37° C., washed three times with cold PBS, and treated with proteinase K (10 μg/mL) for 5 minutes at 37° C. to remove cell surface-bound proteins. Cell-permeability of these recombinant proteins were analyzed by flow cytometry (Guava, Millipore, Darmstadt, Germany) using the FlowJo cytometric analysis software.

Example 4
Cell-Permeability and Intracellular Localization of aMTD/SD-Fused TFF1 Recombinant Proteins

For a visual reference of cell-permeability, NIH3T3 cells were cultured for 24 hours on coverslip in 24-wells chamber slides, treated with 10 μM FITC-conjugated recombinant proteins for 1 hour at 37° C., and washed three times with cold PBS. Treated cells were fixed in 4% paraformaldehyde (PFA, Junsei, Tokyo, Japan) for 10 minutes at room temperature, washed three times with PBS, and mounted with VECTASHIELD Mounting Medium (Vector laboratories, Burlingame, Calif.) with DAPI (4′,6-diamidino-2-phenylindole) for nuclear staining. The intracellular localization of the fluorescent signal was determined by confocal laser scanning microscopy (LM700, Zeiss, Germany).

Example 5
Systemic Delivery of CP-TFF1 In Vivo

ICR mice (6-week-old, female) were injected intraperitoneally (750 μg/head) with FITC only or FITC-conjugated CP-TFF1 recombinant proteins. After 2 hours, 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.

Example 6
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 minutes, (ii) 20 μM Taxol for 30 minutes, (iii) 10 μM antimycin in the presence or absence of 1 mM ATP for 2 hours (iv) incubation on ice (or maintained at 37° C.) for 15, 30, or 60 minutes, and (v) 100 mM EDTA for 3 hours. 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 hour at 37° C., were washed three times with ice-cold PBS, treated with proteinase K (10 μg/ml for 5 minutes at 37° C.) to remove cell-surface bound proteins and analyzed by flow cytometry.

Example 7
Cell Proliferation Assay: CellTiter-Glo Cell Viability Assay

Cell viability assay was evaluated with Cell-Titer Glo luminescent cell viability assay. Various gastric cancer cell lines were treated with 10 μM CP-TFF1 recombinant proteins or buffer alone for 72 hours with 2% fetal bovine serum, and the luminescence was analyzed.

Example 8
Cell Migration Assay: Wound-Healing Assay

Cancer cell migration was determined using the wound healing assay. Briefly, cells were seeded into 12-well plates and grown to 90% confluence. The wounds were produced by scraping of the cell layer with a sterile white tip. For the CP-TFF1 recombinant protein treatment group, cells were treated with CP-TFF1 recombinant protein (10 μM) for 1 hour prior to changing the growth medium. Cells were cultured for an additional 24˜48 hours before being photographed. The migration is quantified by counting the number of cells that migrated from the wound edge into the clear area.

Example 9
Cell Migration Assay: Transwell Assay

The lower surface of Transwell inserts (Costar) was coated with gelatin (10 μg/ml), and the membranes were allowed to dry for 1 hour 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 bFGF (40 ng/ml). CP-TFF1-treated AGS Cells (5×105) were added to each upper chamber, and the plate was incubated at 37° C. in a 5% CO2 incubator for 24 hours. Migrated cells were fixed with 4% paraformaldehyde for 10 minutes, stained with 0.1% (w/v) crystal violet for 1 hour and counted.

Example 10
Western Blot Analysis

For western blot analysis, CP-TFF1 (10 μM)-treated gastric cells were washed with PBS and were lysed in a lysis buffer (RIPA buffer) containing a protease cocktail (Roche) and phosphatase inhibitor cocktail. Equal amounts of cell lysate protein were subjected to SDS-PAGE and transferred to nitrocellulose membranes (BioRad). The protein transferred membranes were incubated to block non-specific binding sites in immersing the membrane in 5% non-fat dried milk (BD Bioscience). The membranes were incubated with cleaved Caspase-3 (1:1000) (Cell Signaling) overnight at 4° C. and β-actin at room temperature and then incubated with the appropriate horseradish peroxidase-conjugated secondary antibodies for 1 hour at room temperature. The blots were developed using a chemiluminescence detection system (ECL kit; Amersham Pharmacia Biotech) and exposed to an x-ray film (AGFA).

Example 11
Xenograft Animal Models

Female Balb/c nu/nu mice (DooYeol Biotech., Seoul, Korea) were subcutaneously implanted with MKN45 tumor block (2 mm3) into the left back side of the mouse. Tumor-bearing mice were intravenously administered with 800 μg/head the recombinant protein (HSAM165T1SB, HSAMT1SB) 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 width2×length×0.5.

Example 12
Immunohistochemistry (IHC)

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 minutes with 3% H2O2 in methanol. After washing three times with PBS, slides were incubated for 30 minutes with blocking solution (5% fetal bovine serum in PBS). 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 hours. After washing three times with PBS, sections were incubated with biotinylated mouse and rabbit IgG (Vector Laboratories) at a 1:1000 dilution for 1 hour at room temperature, then incubated with avidin-biotinylated peroxidase complex using a Vectorstain ABC Kit (Vector Laboratories) for 30 minutes 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 13
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.

SEQUENCE LISTING

[cDNA Sequence of Histidine Tag]

SEQ ID NO: 481

ATGGGCAGCAGCCATCATCATCATCATCACAGCAGCGGCCTGGTGCCGCGCGGCAGC

[Amino Acid Sequence of Histidine Tag]

SEQ ID NO: 482

MetGlySerSerHisHisHisHisHisHisSerSerGlyLeuValProArgGlySer

[cDNA Sequences of aMTDs and Peptides]

SEQ ID NO: 483

aMTD43: CTGCTGGCGGCGCCGCTGGTGGTGGCGGCGGTGCCG

[cDNA Sequences of aMTDs and Peptides]

SEQ ID NO: 484

aMTD165: GCGCTGGCGGTGCCGGTGGCGCTGGCGATTGTGCCG

[Amino Acid Sequences of aMTDs and Peptides]

SEQ ID NO: 485

aMTD43: LLAAPLVVAAVP

[Amino Acid Sequences of aMTDs and Peptides]

SEQ ID NO: 486

aMTD165: ALAVPVALAIVP

[cDNA Sequence of human TFF1]

SEQ ID NO: 487

ATGGCCACCATGGAGAACAAGGTGATCTGCGCCCTGGTCCTGGTGTCCATGCTGGCCCTCGG

CACCCTGGCCGAGGCCCAGACAGAGACGTGTACAGTGGCCCCCCGTGAAAGACAGAATTGTG

GTTTTCCTGGTGTCACGCCCTCCCAGTGTGCAAATAAGGGCTGCTGTTTCGACGACACCGTT

CGTGGGGTCCCCTGGTGCTTCTATCCTAATACCATCGACGTCCCTCCAGAAGAGGAGTGTGA

ATTT

[Amino Acid Sequence of human TFF1]

SEQ ID NO: 488

MetAlaThrMetGluAsnLysVallleCysAlaLeuValLeuValSerMetLeuAlaLeuGly

ThrLeuAlaGluAlaGlnThrGluThrCysThrValAlaProArgGluArgGlnAsnCysGly

PheProGlyValThrProSerGlnCysAlaAsnLysGlyCysCysPheAspAspThrVal

ArgGlyValProTrpCysPheTyrProAsnThrIleAspValProProGluGluGluCysGlu

Phe

[cDNA Sequences of SDA]

SEQ ID NO: 489

ATGGCAAATA TTACCGTTTT CTATAACGAA GACTTCCAGG GTAAGCAGGT

CGATCTGCCG

CCTGGCAACT ATACCCGCGC CCAGTTGGCG GCGCTGGGCA TCGAGAATAA

TACCATCAGC

TCGGTGAAGG TGCCGCCTGG CGTGAAGGCT ATCCTGTACC AGAACGATGG

TTTCGCCGGC

GACCAGATCG AAGTGGTGGC CAATGCCGAG GAGTTGGGCC CGCTGAATAA

TAACGTCTCC

AGCATCCGCG TCATCTCCGT GCCCGTGCAG CCGCGCATGG CAAATATTAC

CGTTTTCTAT

AACGAAGACT TCCAGGGTAA GCAGGTCGAT CTGCCGCCTG GCAACTATAC

CCGCGCCCAG

TTGGCGGCGC TGGGCATCGA GAATAATACC ATCAGCTCGG TGAAGGTGCC

GCCTGGCGTG

AAGGCTATCC TCTACCAGAA CGATGGTTTC GCCGGCGACC AGATCGAAGT

GGTGGCCAAT

GCCGAGGAGC TGGGTCCGCT GAATAATAAC GTCTCCAGCA TCCGCGTCAT

CTCCGTGCCG

GTGCAGCCGA GG

[Amino Acid Sequences of SDA]

SEQ ID NO: 490

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: 491

ATGGCAGAAC AAAGCGACAA GGATGTGAAG TACTACACTC TGGAGGAGAT

TCAGAAGCAC

AAAGACAGCA AGAGCACCTG GGTGATCCTA CATCATAAGG TGTACGATCT

GACCAAGTTT

CTCGAAGAGC ATCCTGGTGG GGAAGAAGTC CTGGGCGAGC AAGCTGGGGG

TGATGCTACT

GAGAACTTTG AGGACGTCGG GCACTCTACG GATGCACGAG AACTGTCCAA

AACATACATC

ATCGGGGAGC TCCATCCAGA TGACAGATCA AAGATAGCCA AGCCTTCGGA

AACCCTT

[Amino Acid Sequences of SDB]

SEQ ID NO: 492

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 Lue 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 Lue

[cDNA Sequences of SDC]

SEQ ID NO: 493

ATGAGCGATA AAATTATTCA CCTGACTGAC GACAGTTTTG ACACGGATGT

ACTCAAAGCG

GACGGGGCGA TCCTCGTCGA TTTCTGGGCA GAGTGGTGCG GTCCGTGCAA

AATGATCGCC

CCGATTCTGG ATGAAATCGC TGACGAATAT CAGGGCAAAC TGACCGTTGC

AAAACTGAAC

ATCGATCAAA ACCCTGGCAC TGCGCCGAAA TATGGCATCC GTGGTATCCC

GACTCTGCTG

CTGTTCAAAA ACGGTGAAGT GGCGGCAACC AAAGTGGGTG CACTGTCTAA

AGGTCAGTTG

AAAGAGTTCC TCGACGCTAA

CCTGGCC

[Amino Acid Sequences of SDC]

SEQ ID NO: 494

Met Ser Asp Lys Ile Ile His Leu Thr Asp Asp Ser Phe Asp Thr

Asp Val Leu Lys Ala Asp Gly Ala Ile Leu Val Asp Phe Trp Ala

Glu Trp Cys Gly Pro Cys Lys Met Ile Ala Pro Ile Leu Asp Glu

Ile Ala Asp Glu Tyr Gln Gly Lys Leu Thr Val Ala Lys Leu Asn

Ile Asp Gln Asn Pro Gly Thr Ala Pro Lys Tyr Gly Ile Arg Gly

Ile Pro Thr Leu Leu Leu Phe Lys Asn Gly Glu Val Ala Ala Thr

Lys Val Gly Ala Leu Ser Lys Gly Gln Leu Lys Glu Phe Leu Asp

Ala Asn Leu Ala

[cDNA Sequences of SDD]

SEQ ID NO: 495

ATGAAAAAGA TTTGGCTGGC GCTGGCTGGT TTAGTTTTAG CGTTTAGCGC

ATCGGCGGCG

CAGTATGAAG ATGGTAAACA GTACACTACC CTGGAAAAAC CGGTAGCTGG

CGCGCCGCAA

GTGCTGGAGT TTTTCTCTTT CTTCTGCCCG CACTGCTATC AGTTTGAAGA

AGTTCTGCAT

ATTTCTGATA ATGTGAAGAA AAAACTGCCG GAAGGCGTGA AGATGACTAA

ATACCACGTC

AACTTCATGG GTGGTGACCT GGGCAAAGAT CTGACTCAGG CATGGGCTGT

GGCGATGGCG

CTGGGCGTGG AAGACAAAGT GACTGTTCCG CTGTTTGAAG GCGTACAGAA

AACCCAGACC

ATTCGTTCTG CTTCTGATAT CCGCGATGTA TTTATCAACG CAGGTATTAA

AGGTGAAGAG

TACGACGCGG CGTGGAACAG CTTCGTGGTG AAATCTCTGG TCGCTCAGCA

GGAAAAAGCT

GCAGCTGACG TGCAATTGCG TGGCGTTCCG GCGATGTTTG TTAACGGTAA

ATATCAGCTG

AATCCGCAGG GTATGGATAC CAGCAATATG GATGTTTTTG TTCAGCAGTA

TGCTGATACA

GTGAAATATC TGTCCGAGAA AAAA

[Amino Acid Sequences of SDD]

SEQ ID NO: 496

Met Lys Lys Ile Trp Leu Ala Leu Ala Gly Leu Val Leu Ala Phe

Ser Ala Ser Ala Ala Gln Tyr Glu Asp Gly Lys Gln Tyr Thr Thr

Leu Glu Lys Pro Val Ala Gly Ala Pro Gln Val Leu Glu Phe Phe

Ser Phe Phe Cys Pro His Cys Tyr Gln Phe Glu Glu Val Leu His

Ile Ser Asp Asn Val Lys Lys Lys Leu Pro Glu Gly Val Lys Met

Thr Lys Tyr His Val Asn Phe Met Gly Gly Asp Leu Gly Lys Asp

Leu Thr Gln Ala Trp Ala Val Ala Met Ala Leu Gly Val Glu Asp

Lys Val Thr Val Pro Leu Phe Glu Gly Val Gln Lys Thr Gln Thr

Ile Arg Ser Ala Ser Asp Ile Arg Asp Val Phe Ile Asn Ala Gly

Ile Lys Gly Glu Glu Tyr Asp Ala Ala Trp Asn Ser Phe Val Val

Lys Ser Leu Val Ala Gln Gln Glu Lys Ala Ala Ala Asp Val Gln

Leu Arg Gly Val Pro Ala Met Phe Val Asn Gly Lys Tyr Gln Leu

Asn Pro Gln Gly Met Asp Thr Ser Asn Met Asp Val Phe Val Gln

Gln Tyr Ala Asp Thr Val Lys Tyr Leu Ser Glu Lys Lys

[cDNA Sequences of SDE]

SEQ ID NO: 497

GGGTCCCTGC AGGACTCAGA AGTCAATCAA GAAGCTAAGC CAGAGGTCAA

GCCAGAAGTC AAGCCTGAGA CTCACATCAA TTTAAAGGTG TCCGATGGAT

CTTCAGAGAT CTTCTTCAAG ATCAAAAAGA CCACTCCTTT AAGAAGGCTG

ATGGAAGCGT TCGCTAAAAG ACAGGGTAAG GAAATGGACT CCTTAACGTT

CTTGTACGAC GGTATTGAAA TTCAAGCTGA TCAGACCCCT GAAGATTTGG

ACATGGAGGA TAACGATATT ATTGAGGCTC ACCGCGAACA GATTGGAGGT

[Amino Acid Sequences of SDE]

SEQ ID NO: 498

Gly Ser Leu Gln Asp Ser Glu Val Asn Gln Glu Ala Lys Pro Glu Val

Lys Pro Glu Val Lys Pro Glu Thr His Ile Asn Leu Lys Val Ser Asp

Gly Ser Ser Glu Ile Phe Phe Lys Ile Lys Lys Thr Thr Pro Leu Arg

Arg Leu Met Glu Ala Phe Ala Lys Arg Gln Gly Lys Glu Met Asp Ser

Leu Thr Phe Leu Tyr Asp Gly Ile Glu Ile Gln Ala Asp Gln Thr Pro

Glu Asp Leu Asp Met Glu Asp Asn Asp Ile Ile Glu Ala His Arg Glu

Gln Ile Gly Gly

[cDNA Sequences of SDF]

SEQ ID NO: 499

GGATCCGAAA TCGGTACTGG CTTTCCATTC GACCCCCATT ATGTGGAAGT

CCTGGGCGAG

CGCATGCACT ACGTCGATGT TGGTCCGCGC GATGGCACCC CTGTGCTGTT

CCTGCACGGT

AACCCGACCT CCTCCTACGT GTGGCGCAAC ATCATCCCGC ATGTTGCACC

GACCCATCGC

TGCATTGCTC CAGACCTGAT CGGTATGGGC AAATCCGACA AACCAGACCT

GGGTTATTTC

TTCGACGACC ACGTCCGCTT CATGGATGCC TTCATCGAAG CCCTGGGTCT

GGAAGAGGTC

GTCCTGGTCA TTCACGACTG GGGCTCCGCT CTGGGTTTCC ACTGGGCCAA

GCGCAATCCA

GAGCGCGTCA AAGGTATTGC ATTTATGGAG TTCATCCGCC CTATCCCGAC

CTGGGACGAA

TGGCCAGAAT TTGCCCGCGA GACCTTCCAG GCCTTCCGCA CCACCGACGT

CGGCCGCAAG

CTGATCATCG ATCAGAACGT TTTTATCGAG GGTACGCTGC CGATGGGTGT

CGTCCGCCCG

CTGACTGAAG TCGAGATGGA CCATTACCGC GAGCCGTTCC TGAATCCTGT

TGACCGCGAG

CCACTGTGGC GCTTCCCAAA CGAGCTGCCA ATCGCCGGTG AGCCAGCGAA

CATCGTCGCG

CTGGTCGAAG AATACATGGA CTGGCTGCAC CAGTCCCCTG TCCCGAAGCT

GCTGTTCTGG

GGCACCCCAG GCGTTCTGAT CCCACCGGCC GAAGCCGCTC GCCTGGCCAA

AAGCCTGCCT

AACTGCAAGG CTGTGGACAT CGGCCCGGGT CTGAATCTGC TGCAAGAAGA

CAACCCGGAC

CTGATCGGCA GCGAGATCGC GCGCTGGCTG TCTACTCTGG AGATTTCCGG

T

[Amino Acid Sequences of SDF]

SEQ ID NO: 500

Gly Ser Glu Ile Gly Thr Gly Phe Pro Phe Asp Pro His Tyr Val Glu

Val Leu Gly Glu Arg Met His Tyr Val Asp Val Gly Pro Arg Asp Gly

Thr Pro Val Leu Phe Leu His Gly Asn Pro Thr Ser Ser Tyr Val Trp

Arg Asn Ile Ile Pro His Val Ala Pro Thr His Arg Cys Ile Ala Pro

Asp Leu Ile Gly Met Gly Lys Ser Asp Lys Pro Asp Leu Gly Tyr Phe

Phe Asp Asp His Val Arg Phe Met Asp Ala Phe Ile Glu Ala Leu Gly

Leu Glu Glu Val Val Leu Val Ile His Asp Trp Gly Ser Ala Leu Gly

Phe His Trp Ala Lys Arg Asn Pro Glu Arg Val Lys Gly Ile Ala Phe

Met Glu Phe Ile Arg Pro Ile Pro Thr Trp Asp Glu Trp Pro Glu Phe

Ala Arg Glu Thr Phe Gln Ala Phe Arg Thr Thr Asp Val Gly Arg Lys

Leu Ile Ile Asp Gln Asn Val Phe Ile Glu Gly Thr Leu Pro Met Gly

Val Val Arg Pro Leu Thr Glu Val Glu Met Asp His Tyr Arg Glu Pro

Phe Leu Asn Pro Val Asp Arg Glu Pro Leu Trp Arg Phe Pro Asn Glu

Leu Pro Ile Ala Gly Glu Pro Ala Asn Ile Val Ala Leu Val Glu Glu

Tyr Met Asp Trp Leu His Gln Ser Pro Val Pro Lys Leu Leu Phe Trp

Gly Thr Pro Gly Val Leu Ile Pro Pro Ala Glu Ala Ala Arg Leu Ala

Lys Ser Leu Pro Asn Cys Lys Ala Val Asp Ile Gly Pro Gly Leu Asn

Leu Leu Gln Glu Asp Asn Pro Asp Leu Ile Gly Ser Glu Ile Ala Arg

Trp Leu Ser Thr Leu Glu Ile Ser Gly

Development of Protein-Based Biotherapeutics That Penetrate Cell-Membrane and Induce Anti-Cancer Effect - Cell-Permeable Trefoil Factor 1 (CP-TFF1) in Gastrointestinal Track (GIT) Cancer, Polynucleotides Encoding The Same, and Anti-Cancer Compositions Comprising The Same

Information

Publication Number

Date Filed

Date Published

Inventors

Original Assignees

CPC

International Classifications

Abstract

Description

Claims

CROSS-REFERENCE TO RELATED APPLICATIONS

Provisional Applications (1)