Fusion polypeptide in which anti-inflammatory polypeptide and ferritin monomer fragment are bound and pharmaceutical composition for preventing and treating inflammatory diseases, containing same as active ingredient

Abstract

The present invention relates to: a fusion polypeptide in which an anti-inflammatory polypeptide and a ferritin monomer fragment are bound; and a pharmaceutical composition for treating inflammatory diseases, containing the same as an active ingredient and, more specifically, to: a fusion polypeptide in which an anti-inflammatory polypeptide is fused to an N-terminus and/or a C-terminus of a ferritin monomer fragment from which a portion of a fourth loop and a fifth helix, of a human derived ferritin monomer, are removed; and a use thereof for treating inflammatory diseases. As in the above, the fusion polypeptide, which has an amino acid sequence represented by SEQ ID NO: 1 and in which an anti-inflammatory polypeptide is fused to an N-terminus and/or a C-terminus of a fragment of a human-derived ferritin monomer, can fuse two types of anti-inflammatory polypeptides, acting through different mechanisms, into a nano cage and administer the same, and thus the fusion polypeptide can exhibit an excellent effect in the treatment of inflammatory diseases including sepsis.

Claims

1. A fusion polypeptide comprising the amino acid sequence of SEQ ID NO: 14 or SEQ ID NO: 15.

2. A polynucleotide encoding the fusion polypeptide of claim 1.

3. An expression vector comprising the polynucleotide of claim 2.

4. A transformant transformed with the expression vector of claim 3.

5. A protein cage comprising the fusion polypeptide of claim 1, wherein an anti-inflammatory polypeptide protrudes outside the protein cage.

6. A pharmaceutical composition, the composition comprising the fusion polypeptide of claim 1 as an active ingredient.

7. A method for treating an inflammatory disease in a subject in need thereof, the method comprising administering an effective amount of a composition comprising the fusion polypeptide of claim 1 as an active ingredient to the subject in need thereof.

8. The method of claim 7, wherein the inflammatory disease is selected from the group consisting of inflammatory bowel disease, diabetic eye disease, peritonitis, osteomyelitis, cellulitis, meningitis, encephalitis, pancreatitis, traumatic shock, bronchial asthma, rhinitis, sinusitis, otitis, pneumonia, gastritis, enteritis, cystic fibrosis, apoplexy, bronchitis, bronchiolitis, hepatitis, nephritis, arthritis, gout, spondylitis, Reiter's syndrome, polyarteritis nodosa, irritable vasculitis, Lou Gehrig's granulomatosis, Polymyalgia rheumatica, arthritic arteritis, calcium crystal arthropathies, pseudogout, non-articular rheumatism, bursitis, tenosynovitis, epicondylitis (tennis elbow), neuropathic joint disease (Charcot's joint), hemarthrosis, Henoch-Schonlein purpura, hypertrophic osteoarthropathy, multicentric reticulohistiocytoma, surcoilosis, hemochromatosis, sickle cell disease and other hemochromatosis, hyperlipoproteinemia, hypogammaglobulinemia, hyperparathyroidism, acromegaly, familial Mediterranean fever, Behcet's disease, systemic lupus erythematosus, relapsing fever, psoriasis, multiple sclerosis, sepsis, septic shock, acute respiratory distress syndrome, multiple organ dysfunction syndrome, chronic obstructive pulmonary disease, acute lung injury and broncho-pulmonary dysplasia.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

(2) FIG. 1A and FIG. 1B are schematic diagrams showing the fusion of TFG and TFMG fusion polypeptides (FIG. 1A: Fusion schematic diagram of TFG and TFMG, FIG. 1B: 3D schematic diagram of fusion polypeptide and ferritin nanocage).

(3) FIG. 2A and FIG. 2B show the results of measurement of the molecular weights of TFG and TFMG with a MALDI-ToF mass spectrometer (FIG. 2A: TFG, FIG. 2B: TFMG).

(4) FIG. 3A and FIG. 3B are the result of TEM imaging observing that TFG and TFMG form a ferritin cage by self-assembling (FIG. 3A: TFG, FIG. 3B: TFMG).

(5) FIG. 4 shows the results of measuring the diameter of the ferritin cage formed by TFG and TFMG.

(6) FIG. 5A and FIG. 5B show the results of secretion of MMP-2 by CLP or LPS stimulation in HUVEC cells (FIG. 5A: results of Western-blot, FIG. 5B: results of zymography).

(7) FIG. 6 shows the result of observing whether PC-Gla is released by cleavage of the linker in TFMG by AMPA-activated MMP-2.

(8) FIG. 7 shows the HPLC analysis results of confirming the released PC-Gla after the linker of TFMG was cleaved by MMP-2.

(9) FIG. 8 shows the MALDI-ToF results of confirming the released PC-Gla after the linker of TFMG was cleaved by MMP-2.

(10) FIG. 9 shows the solid-phase ELISA results of evaluating the binding affinity of Protein C (PC), PC-Gla domain, TFG and TFMG to soluble EPCR.

(11) FIG. 10 shows the results of evaluating the binding affinity of PC-Gla, TFG and TFMG to HUVEC cells.

(12) FIG. 11 shows the in vivo immunohistological staining results of confirming the binding affinity of PC-Gla, TFG and TFME, respectively.

(13) FIG. 12A and FIG. 12B show the results of evaluating the degree of binding affinity of PC-Gla, TFG and TFMG to endothelial cells in vitro (HUVEC) or in vivo (mouse animal model) (FIG. 12A: in vitro, FIG. 5B: in vivo), respectively.

(14) FIG. 13 shows the results of evaluating the degree of activation of PAR-1 by TRAP peptide, TFG and TFMG, respectively.

(15) FIG. 14 shows the results of observing the survival rate of animals after TFG administration, TFMG administration, and co-administration of TRAP and PC-Gla to an animal model of CLP-induced sepsis, respectively.

(16) FIG. 15 shows the results of observing the infiltration of inflammatory cells into tissues and the damage of the lung tissues of the animal after TFG administration, TFMG administration, and co-administration of TRAP and PC-Gla to an animal model of CLP-induced sepsis, respectively.

(17) FIG. 16 shows the results of scoring the degree of lung damage in animals after TFG administration, TFMG administration, and co-administration of TRAP and PC-Gla to an animal model of CLP-induced sepsis, respectively.

(18) FIG. 17A, FIG. 17B, FIG. 17C and FIG. 17D show the results of the evaluating ALT or AST, creatinine, BUN and LDH levels after TFG administration, TFMG administration and co-administration of TRAP and PC-Gla to an animal model of CLP-induced sepsis, respectively.

(19) FIG. 18A and FIG. 18B show the results of evaluating the levels of inflammatory cytokines in blood after TFG administration, TFMG administration, and co-administration of TRAP and PC-Gla to an animal model of CLP-induced sepsis (FIG. 18A: IL-6, IL-10; FIG. 18B: TNF-a).

(20) FIG. 19 shows the results of measuring the permeability after TFG administration, TFMG administration and co-administration of TRAP and PC-Gla to LPS-stimulated HUVEC cells, respectively.

(21) FIG. 20 shows the result of evaluating vascular permeability after TFG administration, TFMG administration and co-administration of TRAP and PC-Gla to an animal model of CLP-induced sepsis, respectively.

(22) FIG. 21A and FIG. 21B show the results of evaluating the expression level of vascular cell adhesion factor-1 (VCAM-1) and the adhesion amount of leukocyte after TFG administration, TFMG administration and co-administration of TRAP and PC-Gla to LPS-stimulated HUVEC cells, respectively.

(23) FIG. 22 shows the results of evaluating the amount of leukocyte movement after TFG administration, TFMG administration, and co-administration of TRAP and PC-Gla to an animal model with CLP-induced sepsis, respectively.

MODE FOR CARRYING OUT INVENTION

(24) Hereinafter, the present invention will be described in detail.

(25) However, the following Examples are merely illustrative of the present invention, while the scope of the present invention is not limited to the following Examples.

(26) <Experimental Method>

(27) 1. Preparation of Reagents

(28) PC-Gla and TRAP were synthesized by Peptron Inc. (Daejeon, Republic of Korea) and Anygen Inc. (Gwangju, Republic of Korea). Peptides were labeled with FNG-456 NHS ester or FNI-675 NHS ester fluorescent dyes (Bioacts Inc., Incheon, Republic of Korea). Bacterial lipopolysaccharide (LPS, serotype: 0111:B4, L5293), antibiotics (penicillin G and streptomycin), -cyano-4-hydroxycinnamic acid (CHCA), sinapic acid, and p-aminophenylmercuric acetate were purchased from Sigma (St. Louis, Mo.). Anti-MMP-2 antibody (MAB13434) was purchased from Millipore, anti-mouse CD31 (553369) from BD Falcon, and anti-EPCR antibody (FL-238, sc-28978) from Santa Cruz.

(29) 2. Expression and Purification of TFG (TRAP-Ferritin Monomer Fragment-PC-Gla Fusion Polypeptide) and TFMG (TRAP-Ferritin Monomer Fragment-Linker-PC-Gla Fusion Polypeptide)

(30) A plasmid was constructed for the expression of short ferritin (sFn) by deleting the short E-helix from ferritin light chain. The DNA plasmids of TFG and TFMG were constructed by introducing TRAP (TFLLRN)(SEQ ID NO: 3) peptide sequence into the N-terminus of sFn with restriction sites, SpeI and XhoI, at 5- and 3-ends. In addition, the PC-Gla domain (ANSFLEELRHSSLERECIEEICDFEEAKEIFQNVDDTLAFWSKHV)(SEQ ID NO: 4) sequence or MMP-2 cleavage site (GPLGLAG)(SEQ ID NO: 13) were constructed in front of the PC-Gla domain to the C-terminus of sFn at 5- and 3-ends with restriction sites, SpeI and XhoI at 5- and 3-ends. Primers were designed as follows: (+) 5 CAC TTT TCT TCT TCG GAA CG 3(SEQ ID NO: 16) and () 5 CTA GCG TTC CGA AGA AGA AAA GTG GTA C 3(SEQ ID NO: 17) for TRAP; (+) 5 GAA ACT AGT GCC AAC TCC TTC CTG GAG G 3(SEQ ID NO: 18) and () 5 GAA CTC GAG GAC GTG CTT GGA CCA G 3(SEQ ID NO: 19) for PC-Gla domain; (+) 5 GAA ACT AGT GGT CCT CTA GGT CTA GCC GGT GCC AAC TCC TTC CTG G 3(SEQ ID NO: 20) and () 5 GAA CTC GAG GAC GTG CTT GGA CCA G 3 (SEQ ID NO: 21) for the MMP-2 cleavage site in front of the PC-Gla domain. TFG and TMFG plasmids were transformed into Escherichia coli (E. coli) expression strain BL21 (DE3). Cells were grown at 37 C. in LB medium containing 50 g/ml kanamycin until OD.sub.600 reached 0.5, and the expression of protein was induced by 0.1M IPTG treatment at 37 C. for 5 hours. After induction, cells were harvested by centrifugation, and the pellets were suspended in lysis buffer (20 mM Tris-HCl pH 8.0, 100 mM NaCl, 1% Triton X-100, 1 mM PMSF, 1 mM DTT 1:000 dilution protease inhibitor cocktail) and homogenized with an ultrasonic processor. The inclusion bodies from cell lysates were solubilized by incubating in binding buffer (20 mM Tris-HCl pH 8.0, 300 mM NaCl, 10 mM imidazole) containing 8 M urea at room temperature for 1 hour. Subsequently, the denatured protein was loaded onto a nickel ion chelate affinity column rinsed with a washing buffer (20 mM Tris-HCl pH 8.0, 500 mM NaCl, 30 mM imidazole) containing 8 M urea. The protein was eluted with elution buffer (20 mM Tris-HCl pH 8.0, 100 mM NaCl, 300 mM imidazole) and refolded by dialysis with a gradient of urea.

(31) 3. Characterization of TFMG and TFG

(32) The mass spectrum of each construct monomer was confirmed by matrix assisted laser desorption ionization time of flight (MALDI-ToF). MALDI-ToF MS was carried out using a Bruker Daltonics Microflex MALDI-ToF mass spectrometer (Bremen, Germany) with a 337 nm nitrogen laser. Mass spectra were obtained in the linear and positive-ion mode with an acceleration voltage of 20 kV. A saturated solution of cyano-4-hydroxycinnamic acid (CHCA) or sinapic acid in 50% acetonitrile, containing a final concentration of 0.1% trifluoroacetic acid, was used as the matrix solution. A CHCA matrix was chosen for analysis of fragments after enzyme digestion or sinapic acid for intact proteins. The analyte-matrix solution was prepared at a ratio of 1:2 (analyte:matrix, v/v). Each mixture was thoroughly mixed, and 1 L of the analyte-matrix solution was deposited onto the sample plate and dried by vacuum evaporation. The spectrometer was calibrated using bradykinin; cytochrome C and bovine serum albumin were run as close external standards. Transmission electron microscopy (TEM) images were recorded using an FEI Tecnai (Korea Basic Science Institute, KBSI). The size of nanocaged TFG and TFMG was measured using a DelsaMax Pro light scattering analyzer (Beckman Coulter).

(33) 4. Cell Culture

(34) The primary HUVECs were obtained from Cambrex BioScience (Charles City, Iowa) and maintained as previously described. All experiments were performed using HUVECs at passage 3-5. Human neutrophils were freshly isolated from whole blood (15 ml) obtained by venous venipuncture from five healthy volunteers, and maintained as previously described.

(35) 5. Animal Care

(36) Male C57BL/6 mice (6-7 weeks of age, 18-20 g) were purchased from Orient Biotech (Seongnam, Gyeonggi Province, Republic of Korea) and used after 12 days of acclimation. Five mice per cage were housed under the conditions of a controlled temperature (20-25 C.), humidity (40-45%), and 12:12 h day/night cycle, while being fed with a normal rodent pellet and water ad libitum. All animals were treated according to the Guidelines for the Care and Use of Laboratory Animals issued by Kyungpook National University.

(37) 6. Preparation of Cecal Ligation and Puncture (CLP) Sepsis Animal Models

(38) To induce inflammation, male mice were anesthetized with 2% isoflurane (JW Pharmaceutical, Republic of Korea) in oxygen delivered via a small rodent gas anesthesia machine (RC2, Vetequip, Pleasanton, Calif.), first in a breathing chamber and then via a facemask. They were allowed to breathe spontaneously during this procedure. The CLP-induced inflammation model was prepared as previously described. In brief, a 2-cm midline incision was made to expose the cecum and adjoining intestine. The cecum was then tightly ligated with a 3.0-silk suture at 5.0 mm from the cecal tip and punctured once using a 22-gauge needle for the induction of high grade inflammation. It was then squeezed gently to extrude a small amount of feces from the perforation site and returned to the peritoneal cavity. The laparotomy site was then sutured with 4.0-silk. In sham control animals, the cecum was exposed but not ligated or punctured and then returned to the abdominal cavity. This protocol was approved by the Animal Care Committee at Kyungpook National University prior to the conduct of the study (IRP No, KNU 2012-13).

(39) 7. Gelatin Zymography

(40) The activity of MMP-2 and MMP-9 enzymes in medium and plasma was determined by SDS-PAGE gelatin zymography. Gelatinases present in the plasma degrade the gelatin matrix, leaving a clear band after staining the gel for protein. Briefly, LPS time-dependently treated HUVECs media and albumin-derived septic mice plasma (normalized to an equal amount of protein [20 g]) were denatured in the absence of a reducing agent and electrophoresed using 10% SDS-PAGE containing 0.1% (w/v) gelatin. Gels were incubated in the presence of 2.5% Triton X-100 at room temperature for 2 h and subsequently at 37 C. overnight in a buffer containing 10 mM CaCl.sub.2, 0.15M NaCl, and 50 mM Tris (pH 7.5). Thereafter, gels were stained with 0.25% Coomassie Blue, and proteolysis was detected as a white band against a blue background.

(41) 8. Cleavage of Nanocaged TFMG by MMP-2

(42) To evaluate whether TFMG could be selectively cleaved by MMP2, TFMG was incubated with APMA-mediated activated MMP-2 in PBS at 37 C. for 3 h. The cleaved fragments of TFMG were detected by MALDI-ToF.

(43) 9. Enzyme-Linked Immunosorbent Assays (ELISA) for Evaluating EPCR (Endothelial Protein C Receptor) Binding Affinity

(44) To evaluate the interaction of the wild-type PC, PC-Gla peptides, TFG, and TFMG with EPCR, 96-well flat microtiter plates were coated with soluble EPCR in 20 mM carbonate-bicarbonate buffer (pH 9.6) containing 0.02% sodium azide, overnight at 4 C. After the plates were washed three times in TBS buffer (0.1 M NaCl, 0.02 M Tris-HCl, pH 7.4) containing 0.05% Tween 20, the plates were incubated with wild-type PC, PC-Gla peptides, TFG, and TFMG (7-1000 nM) diluted in the buffer for 1 h. After the plates were rinsed again, they were incubated with a goat anti-protein C polyclonal antibody (1:1000) for 1 h. Then, the plates were washed and incubated with rabbit anti-goat IgG (KPL, MD, 1:1000) for 1 h. After washing, the plates were incubated with 2,2-azino-di(3-ethylbenzthiazoline-6-sulfonate) (ABTS; KPL, Gaithersburg, Md.). Colorimetric analysis was performed by measuring absorbance values at 405 nm.

(45) 10. Isolation of Endothelial Cells from Mouse

(46) The endothelial cells were isolated according to the manufacturer's (Dynal Biotec, Lake Success, N.Y.) instructions, using Dynabeads coupled to anti-CD31 antibody and the Dynal Magnetic holder. Briefly, for endothelial cell isolation, four to six mice (6-10 weeks old) were anesthetized, followed by exposure of the peritoneal cavity. Excised lungs and hearts were put into RPMI media, followed by removing other tissues from the heart and lungs, and then rinsing once in PBS. The lungs and heart were incubated with 1.0 mg/mL of collagenase A in a 50 mL tube for 1 h at around 37 C. Every 5 min during this incubation, the tube was gently agitated for a few seconds, and then the suspension was transferred into a new 50 mL tube by passing it through the 70 um tissue sieve (BD Falcon). The filtered cell suspension was centrifuged for 10 min at 1000 rpm. After removal of the supernatant, the cell pellet was washed once with cold PBS in a new 15-mL tube. To prepare the Dynabead-coupled anti-mouse CD31 antibody, Dynabeads (60 l) were washed with MACS buffer (PBS, 0.5% BSA, 2 mM EDTA) on a magnetic holder (Invitrogen). The Dynabeads were resuspended with MACS buffer (600 al), anti-mouse CD31 (5 g of per 10 l of beads) was added, and the mixture was incubated for 12 h at 4 C. Cells were incubated with Dynabead-coupled anti-mouse CD31 antibody for 10 min at room temperature and then placed in a magnetic holder. Cell suspension was slowly added to a 15-mL tube by placing the pipette on the wall of the tube. After incubation for 5 min, PBS was carefully removed by aspiration. The Dynabead-coupled anti-mouse CD31 antibodies were washed three times in cold PBS, the pellet was resuspended in EBM-2 growth medium, and then harvested and lysed in RIPA buffer containing protease inhibitor cocktail on ice.

(47) 11. Fluorescence of PC-Gla Domain, TFG, and TFMG

(48) PC-Gla, TFG, and TFMG were labeled with FNG-456 NHS ester for in vitro assays or FNI-675 NHS ester for in vivo assays at a molar ratio of 1:3. Briefly, each molecule (10 M) was dissolved in PBS (1.5 mL), and FNG-456 NHS ester (30 M) or FNI-675 NHS ester (30 M) was dissolved in DMSO (0.2 mL). Each molecule and fluorescent dye was reacted at room temperature for 3 h. The reaction product was passed through a 0.2-m filtering unit, and the unreacted dye was separated on a PD midiTrap G-25 (GE Healthcare, UK) that had been pre-equilibrated in PBS with 2 mM sodium azide. This process yielded more than 2.17 M of each nanoparticle with more than 1.5 ratio of dye per protein.

(49) 12. HUVEC Cell-Binding Assay

(50) A direct cell-binding assay was performed on HUVECs and in vivo using fluorescence labeled-PC-Gla, TFG, and TFMG. The assay was performed with PC-Gla, TFG, or TFMG treated on HUVECs, intravenously injected mice, and isolated mouse endothelial cells. The fluorescence value of the HUVECs or endothelial cells were measured with tightly bound PC-Gla, TFG, and TFMG, respectively. The concentrations of PC-Gla, TFG, and TFMG were measured by using the nanoparticle ratio of fluorescent dye per protein.

(51) 13. PAR-1 Cleavage Assay

(52) HUVECs at 90% confluence in 24-well plates were transiently transfected with pRc/RSV containing ALP-PAR-1-TF cDNA in antibiotic-free Opti-MEM medium using Lipofectamine (Invitrogen) according to the manufacturer's instruction. On the following day, cells were washed and incubated in serum-free medium for 5 h. Cells were then incubated for an additional hour with thrombin, TRAP, TFG, or TFMG. Conditioned medium was collected and centrifuged to remove cellular debris. Supernatant was collected, and ALP (alkaline phosphatase) activity was measured using EnzoLyte p-nitrophenyl phosphate alkaline phosphatase assay kit (AnaSpec, San Jose, Calif.) according to the manufacturer's instructions.

(53) 14. H&E Staining and Histopathological Examination

(54) Male C57BL/6 mice underwent CLP and were administered PC-Gla with TRAP, TFG, or TFMG (200 nM) intravenously at 6 h after CLP (n=5). Mice were euthanized 96 h after CLP. To analyze the phenotypic change of the lungs in mice, lung samples were removed from each mouse, washed tree times in PBS (pH 7.4) to remove remaining blood, fixed in 4% formaldehyde solution (Junsei, Tokyo, Japan) in PBS, pH 7.4 for 20 h at 4 C. After fixation, the samples were dehydrated through ethanol series, embedded in paraffin, sectioned into 4-m sections, and placed on a slide. The slides were de-paraffinized in a 60 C. oven, rehydrated, and stained with hematoxylin (Sigma). To remove over-staining, the slides were quick dipped three times in 0.3% acid alcohol, and counterstained with eosin (Sigma). They are then washed in ethanol series and xylene, and then coverslipped. Light microscopic analysis of lung specimens was performed by blinded observation to evaluate pulmonary architecture, tissue edema, and infiltration of the inflammatory cells. The results were classified into four grades where Grade 1 represented normal histopathology; Grade 2 represented minimal neutrophil leukocyte infiltration; Grade 3 represented moderate neutrophil leukocyte infiltration, perivascular edema formation, and partial destruction of pulmonary architecture; and Grade 4 included dense neutrophil leukocyte infiltration, abscess formation, and complete destruction of pulmonary architecture.

(55) 15. Immunofluorescence Staining

(56) HUVECs were grown to confluence on glass cover slips coated with 0.05% poly-L-lysine in complete media containing 10% FBS and maintained for 48 h. Cells were then stimulated with LPS (100 g/ml) for 6 h, followed by treatment with PC-Gla with TRAP, TFG, or TFMG for 6 h. For cytoskeletal staining, the cells were fixed in 4% formaldehyde in TBS (v/v) for 15 min at room temperature, permeabilized in 0.05% Triton X-100 in TBS for 15 min, and blocked in blocking buffer (5% bovine serum albumin (BSA) in TBS) overnight at 4 C. Then, the cells were incubated with a rabbit anti-EPCR polyclonal antibody (Santa Cruz, Calif.). EPCR was visualized using an Alexa Fluor 647-conjugated secondary antibody (Molecular Probes, donkey anti-rabbit IgG) and observed by confocal microscopy at a magnification of 630 (TCS-Sp5, Leica Microsystems, Germany).

(57) 16. Histological Analysis of EPCR Binding In Vivo

(58) Twenty-four hours prior to CLP surgery, fluorescence labeled-PC-Gla, TFG, and TFMG (200 nM/mouse) was intravenously injected into the mice, respectively. After 24 h, mouse vena cava was enucleated and fixed in visikol for 24 h. Subsequently, vena cava was embedded in optimum cutting temperature (OCT) compound (Tissue Tek) at 80 C. Consecutive sections were incubated with anti-EPCR antibody (Santa Cruz, Calif.), anti-rabbit Alexa 488 (green), anti-CD31 antibody, and anti-rabbit Alexa 350 (blue), and observed by confocal microscopy at 63 magnification (TCS-SP5, Leica microsystem, Germany).

(59) 17. Analysis of Serum Components in Septic Animal Model

(60) Fresh serum was used for assaying aspartate transaminase (AST), alanine transaminase (ALT), blood urea nitrogen (BUN), creatinine, and LDH using biochemical kits (Mybiosource). To determine the concentrations of IL-6, IL-10, and TNF-, commercially available ELISA kits were used according to the manufacturer's protocol (R&D Systems). Values were measured using an ELISA plate reader (Tecan, Austria GmbH, Austria).

(61) 18. In Vitro Permeability Assay

(62) For spectrophotometric quantification of endothelial cell permeabilities in response to increasing concentrations of each molecule, the flux of Evans blue-bound albumin across functional cell monolayers was measured using a modified 2-compartment chamber model, as previously described. HUVECs were plated (510.sup.4/well) in 12-mm diameter Transwells with a pore size of 3 m for 3 days. Confluent monolayers of HUVECs were exposed to LPS (100 ng/mL) for 4 h before being subjected to PC-Gla with TRAP, TFG, or TFMG (up to 100 nM). Transwell inserts were then washed with TBS (pH 7.4), followed by the addition of Evans blue (0.5 mL; 0.67 mg/mL) diluted in a growth medium containing 4% BSA. Fresh growth medium was then added to the lower chamber, and the medium in the upper chamber was replaced with Evans blue/BSA. Ten minutes later, the optical density of the sample in the lower chamber was measured at 650 nm.

(63) 19. In Vivo Permeability and Leukocyte Migration Assays

(64) CLP-operated mice were injected with PC-Gla with TRAP, TFG, or TFMG intravenously. After 6 h, 1% Evans blue dye solution in normal saline was injected intravenously into each mouse. Thirty minutes later, the mice were sacrificed, and the peritoneal exudates were collected after being washed with normal saline (5 mL) and centrifuged at 200g for 10 min. The absorbance of the supernatant was read at 650 nm. The vascular permeability was expressed in terms of dye (mg/mouse), which leaked into the peritoneal cavity according to a standard curve of Evans blue dye, as previously described.

(65) For assessment of total leukocyte migration, CLP operated mice were treated with each nanoparticle (100 nM) 6 h after CLP surgery. The mice were then sacrificed and the peritoneal cavities were washed with 5 mL of normal saline. Peritoneal fluid (20 L) was mixed with Turk's solution (0.38 mL; 0.01% crystal violet in 3% acetic acid) and the number of leukocytes was counted under an optical microscope. The results were expressed as neutrophils10.sup.6 per peritoneal cavity.

(66) 20. Expression Analysis of Cell Adhesion Factor (CAM)

(67) The expression of vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1), and E-selectin on HUVECs were determined by a whole-cell ELISA as described. Briefly, confluent monolayers of HUVECs were treated with PC-Gla with TRAP, TFG, or TFMG for 6 h followed by LPS (100 ng/mL) for 16 h (VCAM-1 and ICAM-1) or 24 h (E-Selectin). After washing, mouse anti-human monoclonal VCAM-1 (100 M; clone; 6C7.1), ICAM-1 (clone; P2A4) and E-selectin (clone; P3H3) antibodies (Millipore Corporation, 1:50 each) were added. After 1 h (37 C., 5% CO.sub.2), the cells were washed three times and then 1:2000 peroxidase-conjugated anti-mouse IgG antibody (100 l; Sigma) was added for 1 h. The cells were washed again three times and developed using the o-phenylenediamine substrate (Sigma). Colorimetric analysis was performed by measuring absorbance at 490 nm. All measurements were performed in triplicate wells.

(68) 21. Cell-Cell Adhesion Assay

(69) Adherence of monocytes to endothelial cells was evaluated by fluorescent labeling of monocytes, as previously described. Briefly, monocytes were labeled with 5 M Vybrant DiD for 20 min at 37 C. in phenol red-free RPMI containing 5% FBS. After washing, the cells (1.510.sup.6 cells/mL, 200 L/well) were resuspended in adhesion medium (RPMI containing 2% fetal bovine serum and 20 mM HEPES). The cells were then added to confluent monolayers of HUVECs in 96-well plates. Prior to the addition of cells, HUVECs were treated PC-Gla with TRAP, TFG, or TFMG for 6 h, followed by treatment with LPS (100 ng/mL, 4 h). Quantification of cell adhesion was determined as previously described.

(70) 22. Statistical Analysis

(71) All experiments were performed independently at least three times. Values are expressed as meansSEM. The statistical significance of differences between test groups was evaluated using SPSS for Windows, version 16.0 (SPSS, Chicago, Ill.). Statistical relevance was determined by one-way analysis of variance (ANOVA) and Tukey's post-test. P values less than 0.05 were considered to indicate a statistical significance. Survival analysis of CLP-induced sepsis was performed using Kaplan-Meier analysis.

Experimental Results

Example 1

(72) Preparation and Characterization of Fusion Polypeptides

(73) A short ferriitin (sFn) was constructed by deleting the short helix E and loop from the ferritin light chain to make genetic modification more amenable. To prepare TRAP-ferritin-PC-Gla (TFG) protein, the sFn was genetically engineered, inserting the EPCR ligand at the C-terminus and PAR-1 activator (TRAP peptide) at the N-terminus (FIG. 1A & FIG. 1B). To prevent free PC-Gla from interfering with the remaining TRAP-ferritin during simultaneous double binding to each receptor, the matrix metalloproteinase (MMP)-2 binding site was inserted between the sFn and the PC-Gla domain (TFMG) so that PC-Gla was able to be released from nanocages when they reached MMP-activating sites.

(74) The molecular weights of purified TFG and TFMG monomers were 26,866 Da and 27,403.6 Da, respectively, as determined by the matrix-associated laser desorption ionization time-of-flight (MALDI-ToF) mass spectrometry (FIG. 2A & FIG. 2B). The lack of large aggregates in TEM images of negatively stained TFG and TFMG verifies this cage structure (FIG. 3A & FIG. 3B). The mean outer diameters of the TFG and TFMG nanocages were 15.5 nm and 16.3 nm, respectively (FIG. 4). The size distribution of each construct increased with the addition of MMP-2 binding sites between the C-terminus of sFn and the PC-Gla region.

Example 2

(75) Linker Cleavage of TFMG by MMP-2 and Evaluation on Secretion of PC-Gla

(76) It was verified that LPS- or CLP-induced MMP-2 was released from activated HUVECs using Western blotting and zymography (FIG. 5A & FIG. 5B). To evaluate whether PC-Gla could be selectively cleaved from TFMG by MMP-2 and whether the release of PC-Gla from TFMG was regulated, TFMG was incubated with p-aminophenylmercuric acetate (AMPA)-activated MMP-2. It was found that TFMG continuously released PC-Gla (FIG. 6), TFMG was cleaved by MMP-2 to release PC-Gla in agreement with the calculated mass (5,518.6 Da by MALDI-ToF) (FIG. 7 & FIG. 8).

Example 3

(77) Evaluation on Binding Affinity Toward EPCR and PAR-1 Cleavage Activity

(78) In the context of the binding properties of TFG or TFMG to EPCR and PAR-1, the present inventors sought to determine whether the attachment of multiple ligands to the ferritin scaffold affects the binding dynamics in vitro or in vivo.

(79) First, a solid-phase ELISA was used to measure the binding affinity of TFG or TFMG toward the PC-Gla receptor, EPCR. In this assay, the effciency of their binding to soluble EPCR (sEPCR) was compared. As shown in FIG. 9, wild type PC bound to sEPCR, with a K.sub.d(app) of approximately 31.3 nM, which is similar to previously reported binding afinity for PC to sEPCR (29 nM) in an aqueous solution. K.sub.d(app) of 32.0, 31.5 and 307 nM were obtained for the interaction between sEPCR and PC-Gla peptide, TFG and TFMG, respectively. These data suggest that each of TFG and TFMG construct could bind to EPCR as strongly as wild-type PC.

(80) Subsequently, the present inventors evaluated the degree of binding of PC-Gla, TFG and TFMG to HUVECs, respectively, and found that TFG and TFMG exhibited strong binding affinities which are equivalent to that of PC-Gla binding to HUVECs (FIG. l0). This suggests that binding to HUVEC was enhanced by TRAP which binds to PAR-1.

(81) The in vivo binding activities of TFG and TTMG were verified by immunohistochemistry, showing that injected TFG and TFMG were colocalized with EPCR and CD31 in the mouse vena cava. in addition, their binding seemed to be dominant over that of PC-Gla (FIG. ll).

(82) Next, the present inventors compared in vitro and in vivo binding of TFG and TFMG to endothelial cells with that of PC-Gla. As a result, as shown in FIG. 12A & FIG. 12B, it was confirmed that TFG and TFMG bound more strongly to, and stayed longer on, endothelial cells than PC-Gla did in both in vivo and in vitro.

(83) The present inventors also assessed the ability of TRAP peptides, TFG and TFMG to activate PAR-1. As shown in FIG. 13, the data showed strong similarities in the PAR-1 cleavage activities of TRAP peptides, TFG, and TFMG.

Example 4

(84) Evaluation of Efficacy in Animal Models of CLP-Induced Sepsis

(85) Based on the above described experimental results, the present inventors expected that TFG and TFMG would exhibit an excellent efficacy in an animal model of CLP-induced sepsis, and that TFG and TFMG would exhibit a better efficacy than the co-administration of PC-Gla and TRAP.

(86) (1) Assessment of Survival Rate

(87) As a result of TFG administration, TFMG administration or co-administration of PC-Gla and TRAP to an animal model of CLP-induced sepsis, as shown in FIG. 14, the survival rate was significantly improved in the TFG or TFMG administration groups, in comparison with the PC-Gla and TRAP co-administration group.

(88) (2) Assessment of the Degree of Tissue Edema and Lung Tissue Damage

(89) Meanwhile, as shown in FIG. 15, in an animal model of CLP-induced sepsis, interstitial edema with massive infiltration of inflammatory cells into the interstitium and alveolar spaces were observed and the pulmonary architecture was severely damaged. However, these pathologic conditions were significantly alleviated by TFG administration, TFMG administration or co-administration of PC-Gla and TRAP. Especially, TFG administration or TFMG administration was found to be more effective than the co-administration of PC-Gla and TRAP. In addition, the TFG administration or TFMG administration group showed a significantly superior effect in terms of a histopathologic score of pulmonary injury, in comparison with the co-administration of PC-Gla and TRAP (FIG. 16).

(90) (3) Assessment of General Indicators on Organ Toxicity

(91) Systemic inflammation during sepsis frequently causes multiple organ failure, in which the liver and kidneys are major target organs.

(92) As seen in FIG. 17A, FIG. 17B, FIG. 17C and FIG. 17D, it was observed in the animal model of CLP-induced sepsis that the levels of ALT and AST (markers of hepatic injury) were greatly increased, while those of creatinine and BUN levels (markers of renal injury) were also significantly increased. In addition, the level of LDH, which is another marker of general organ injury, increased significantly in the sepsis animal model. It was found that the toxicity indicators for liver, kidneys, and general organs were considerably improved by TFG administration, TFMG administration or co-administration of PC-Gla and TRAP. In particular, it was confirmed that such an effect was particularly prominent in the TFG administration group and the TFMG administration group, respectively.

(93) (4) Evaluation on the Level of Inflammatory Cytokine Secretion

(94) The present inventors evaluated the levels of IL-6, IL-10 and TNF-, inflammatory cytokines secreted during the course of sepsis. The IL-10 and TNF- appear to be essential mediators of sepsis-induced vascular inflammation. IL-6 blockade with neutralizing antibodies has been shown to be an important protective mechanism against sepsis mortality.

(95) As shown in FIG. 18A & FIG. 18B, the levels of IL-6, IL-10 and TNF- were significantly increased in the CLP-induced sepsis animal model, which such a condition was mitigated by the TFG administration, TFMG administration or co-administration of PC-Gla and TRAP, respectively. Especially, it was confirmed that such an effect was remarkably excellent in the TFG- and TFMG-treated groups.

(96) Taken together, the TFG- and TFMG-administered groups were found to be significantly effective in sepsis treatment in comparison with the PC-Gla and TRAP co-administered group, while TFMG was more effective antiseptic than TFG.

(97) The above results suggest that the linker in TFMG was cleaved by MMP-2 at the inflammation pathological site to release PC-Gla and the released PC-Gla then independently exhibits its physiological activity, while TFMG following the release of PC-Gla was able to exhibit TRAP activity superior to that of TFG due to a reduction in the steric hindrance.

Example 5

(98) Assessment of Endothelial Cell Permeability

(99) During severe vascular inflammatory reactions, overexpression of inflammatory cytokines/chemokines may irreversibly damage vascular integrity, and cause excessive circulatory fluid loss. This may lead to prolonged tissue hypoperfusion, organ dysfunction, and ultimately death. Therefore, vascular permeability plays a pivotal role in severe vascular inflammatory diseases.

(100) Results of evaluating in vitro and in vivo endothelial cell permeability by the TFG administration, TFMG administration or co-administration of PC-Gla and TRAP are shown in FIG. 19 and FIG. 20. It was found that LPS or CLP induction significantly increased the permeability in HUVECs and mice, and that this phenomenon was alleviated by the TFG administration, TFMG administration or co-administration of PC-Gla and TRAP, respectively.

Example 6

(101) Assessment of the Expression of Cell Adhesion Factor (CAM) and the Migration of Leukocytes

(102) The vascular inflammatory responses are known to be mediated by the increased expression of CAMs such as ICAM-1, VCAM-1 and E-selection on the surfaces of endothelial cells, thereby promoting the adhesion and migration of leukocytes across the endothelium to the sites of inflammation. Transendothelial migration of circulating leukocytes to the vascular endothelium is a fundamental step during the pathogenesis of vascular inflammatory diseases.

(103) It was observed that TFMG and TFG inhibited the LPS-mediated upregulation of CAMs (FIG. 21A), adhesion of leukocytes (FIG. 21B), and CLP-mediated migration of leukocytes (FIG. 22). The above results demonstrate the potential of TFMG and TFG for treating vascular inflammatory diseases.

INDUSTRIAL APPLICABILITY

(104) As described above, there is provided a fusion polypeptide, in which an anti-inflammatory polypeptide is fused to a N-terminus and/or a C-terminus of a human-derived ferritin monomer fragment having an amino acid sequence of SEQ ID NO: 1, may fuse two types of anti-inflammatory polypeptides which act through different mechanisms, respectively, into a nanocage for administration, thus the fusion polypeptide exhibiting an excellent effect in the treatment of an inflammatory disease including sepsis to be highly industrially applicable.