# Patent

## Title
Fusion protein comprising albumin and retinol-binding protein

## Abstract

There is provided a fusion protein comprising albumin and retinol-binding protein, which can be used for preventing or treating fibrotic diseases. The fusion protein, in which albumin and a retinol-binding protein (RBP) are bound together, induces the formation of cytoplasmic lipid droplets in stellate cells and returns the shape of activated stellate cells to the previous shape thereof before activation. Therefore, the fusion protein can be effectively used in preventing or treating fibrotic diseases occurring in the liver, pancreas, lungs, or other organs.

## Background
TECHNICAL FIELD
The present invention relates to a fusion protein comprising albumin and retinol-binding protein, which is capable of being used for preventing or treating fibrotic diseases occurring in the liver, pancreas, lungs, or other organs.
BACKGROUND ART
Tissue fibrosis leads to fatal defunctionalization of tissues. For example, liver fibrosis leads to defunctionalization of the liver, and subsequently progresses to hepatocirrhosis or liver cancer, and pancreas fibrosis is commonly observed in cases of chronic pancreatitis or pancreatic cancer. Nevertheless, up to now, there have been no drugs for treating the fibrosis, and tissue grafting is the only effective cure. The reason that there are no drugs for treating the fibrosis is because molecular mechanism of tissue fibrosis has not been identified.
Recently, it has been found that tissue fibrosis is caused by activating stellate cells, which are one type of cell constituting tissues, and thus excessively expressing and accumulating an extracellular matrix such as collagen. It has been reported that the stellate cells become distributed in the pancreas, lungs, kidneys, and intestines, in addition to the liver.
The stellate cells play a key role in controlling retinoid homeostasis in the whole body. Vitamin A (retinol) acquired from diet is bound to retinol-binding protein (RBP) in blood flow; circulated, transferred to the stellate cells through STRA6 as a RBP receptor, and then stored as retinyl ester in cytoplasmic fat droplets. The present inventors disclosed that albumin that is expressed in the stellate cells and has intact fatty acid binding sites is involved in formation of vitamin A—containing fat droplets, inhibits activation of the stellate cells, and returns albumin expressed in the activated stellate cells to its previous state before activation (Non-Patent Document 1: Kim N, Yoo W, Lee J, Kim H, Lee H, Kim Y, Kim D, Oh J.* (2009) Formation of vitamin A fat droplets in pancreatic stellate cells requires albumin. Gut 58(10), 1382-90; Non-Patent Document 2: Kim N, Choi S, Lim C, Lee H, Oh J. (2010) Albumin mediates PPAR-g and C/EBP-a-induced phenotypic changes in pancreatic stellate cells. Biochem. Biophys. Res. Commun. 391(1), 640-44.)
DISCLOSURE
Technical Problem
An object of the present invention is to treat fibrotic diseases by inhibiting activation of stellate cells through increasing the levels of albumin in the stellate cells, returning the activated stellate cells to the state of the previous stellate cells before activation, or inducing aging of the stellate cells.
Technical Solution
In order to achieve the above object, an exemplary embodiment of the present invention provides a fusion protein comprising albumin and a retinol-binding protein.
Albumin is a multifunctional plasma protein that is primarily synthesized by liver cells. Albumin has three domains, each of which consists of two small sub-domains: A and B. It is known that albumin plays a role in molecular migration by wrapping around hydrophobic substances including fatty acids, and then carrying the hydrophobic substances including fatty acids in the blood. According to a crystallographic analysis, five principal fatty acid binding sites are asymmetrically distributed within the albumin (one in sub-domain IB, one between IA and IIA, two in IIIA, and one in IIIB).
The present inventors hypothesized that albumin expressed in stellate cells may promote formation of intracellular fat droplets by stabilizing storage of retinyl ester. On the other hand, the present inventors noticed that a retinol-RBP complex prepared by combining retinol with a RBP receptor in the stellate cells may be internalized into a cell by endocytosis. In reference to this point, an expression vector encoding a fusion protein including albumin and a retinol-binding protein (RBP) was prepared in order to increase a level of albumin in the stellate cells, and then an effect according to the expression of the fusion protein was tested. As a result, with the wild-type albumin, the expression of recombinant fusion protein induced the formation of fat droplets in the stellate cells, and led to the phenotype reversion of activated stellate cells into quiescent cells. In addition, levels of α-SMA, an activation marker of stellate cells, were decreased. Furthermore, when conditioned medium from of 293 cells transfected with the fusion protein expression vector was added to the activated stellate cells, unlike the wild-type albumin, the fusion protein was found to be successfully incorporated into the stellate cells, induce the formation of fat droplets, and decrease the level of α-SMA. In addition, as the expression of mutated albumin with amino acid substitutions induces the senescence of stellate cells, the expression of mutant form of fusion protein also led to stellate cell senescence.
According to the present invention, the albumin used for the formation of fusion protein may be derived from any species, but may be preferably derived from humans, in order to avoid a risk of immunogenicity. Albumin may be encoded by a nucleic acid sequence as set forth in SEQ ID NO: 1, but the present invention is not limited thereto.
Since a high-affinity fatty acid binding site of albumin is present in an albumin I domain and an albumin III domain, it may be preferable to use the domains as a fusion partner, but the present invention is not limited thereto. Therefore, according to a specific example, the albumin used for the formation of fusion protein may be at least one of an albumin I domain and albumin III domain.
The albumin I domain may be encoded by a nucleic acid sequence (1st to 666th nucleic acids among the nucleic acids encoding albumin) as set forth in SEQ ID NO: 2, but the present invention is not limited thereto.
In addition, the albumin III domain may have a nucleic acid sequence (1216th to 1827th nucleic acids among the nucleic acids encoding albumin) as set forth in SEQ ID NO: 3, but the present invention is not limited thereto.
According to a specific example, in the case of locating the albumin III at a N-terminal side of the fusion protein, an albumin N-terminal including a secretory sequence may be added before the albumin III domain. The albumin N-terminal may be encoded by a nucleic acid sequence as set forth in SEQ ID NO: 4, but the present invention is not limited thereto.
Meanwhile, for the RBP bound to the albumin, the full sequence of the RBP or a part of the full sequence of the RBP may be used, and may be properly selected according to an albumin sequence to be bound or an order of binding with the albumin. The RBP may be encoded by a nucleic acid sequence (1st to 585th nucleic acids among the nucleic acid encoding the RBP) as set forth in SEQ ID NO: 5, a nucleic acid sequence (55th to 585th nucleic acids among the nucleic acid encoding the RBP) as set forth in SEQ ID NO: 6, or a nucleic acid sequence (55th to 603th nucleic acids among the nucleic acid encoding the RBP) as set forth in SEQ ID NO: 7, but the present invention is not limited thereto. For example, since when the RBP is bound to the C-terminal of the albumin, a secretory sequence may not be needed, the RBP peptide encoded by a nucleic acid as set forth in SEQ ID NO: 6 or SEQ ID NO: 7 may be used. In addition, in a case where an albumin domain is again bound to the C-terminal of the RBP, it may be preferable to use a partial peptide of the RBP encoded by a nucleic acid sequence as set forth in SEQ ID NO: 7 rather than the full sequence of the RBP.
According to a preferable specific example, the fusion protein may be albumin I domain-RBP-albumin III, albumin III-RBP-albumin I, RBP-albumin III, albumin III-RBP, an albumin-RBP, or RBP-albumin. The albumin I domain-RBP-albumin III is a type in which the N-terminal of the RBP is bound to the C-terminal of albumin I domain and the albumin III domain is bound to the C-terminal of the RBP. The RBP-albumin III, albumin III-RBP, albumin-RBP, and RBP-albumin are also interpreted in the same way. From the above-mentioned sequence analysis, the present inventors found that the fusion protein of the above-mentioned type does not inhibit a natural steric conformation of the albumin, and thus has no influence on a fatty acid binding site or on RBP binding through a RBP receptor. Therefore, the fusion protein may have any one amino acid sequence among amino acid sequences set forth in SEQ ID NO: 8 to SEQ ID NO: 13, but the present invention is not limited thereto. Specifically, the albumin I domain-RBP-albumin III, albumin III-RBP-albumin I, RBP-albumin III, albumin III-RBP, albumin-RBP, and RBP-albumin may have amino acid sequences set forth in SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, and SEQ ID NO: 13, respectively. Those sequences may include an amino acid in the protein to be fused, an amino acid in the nucleic acid encoding a restriction site, a His tag for purifying, and the like.
According to another specific example, wild-type albumin, or the wild-type albumin having partially substituted amino acids, may be used to induce aging of stellate cells by the variation of albumin. According to a specific example, for the albumin or albumin III domain included in the fusion protein, Arg410, Tyr411, and Lys525 may be substituted with Ala, but the present invention is not limited thereto.
In addition, the present invention provides a polynucleotide encoding the above-mentioned fusion protein including the albumin and retinol-binding protein (RBP), a recombinant vector including the polynucleotide, and a transformant transformed by the recombinant vector.
According to the present invention, the polynucleotide may have nucleic acid sequences set forth in SEQ ID NO: 14 to SEQ ID NO: 19, but the present invention is not limited thereto. Specifically, albumin I domain-RBP-albumin III, albumin III-RBP-albumin I, RBP-albumin III, albumin III-RBP, albumin-RBP, and RBP-albumin may be respectively encoded by nucleic acid sequences set forth in SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, and SEQ ID NO: 19, but the present invention is not limited thereto. Those sequences may include nucleic acids encoding the proteins to be fused, nucleic acids encoding restriction sites used for fusing proteins, a His tag for purifying, a stop codon, and the like.
According to another specific example, wild-type albumin, or the wild-type albumin having a partially substituted amino acid sequence among the amino acid sequences of the domain thereof may be used to induce aging of stellate cells by variation of the albumin. According to a specific example, for the albumin or albumin III domain included in the fusion protein, Arg410, Tyr411, and Lys525 may be substituted by Ala, but the present invention is not limited thereto.
Meanwhile, the recombinant vector including the polynucleotide may be prepared by inserting the polynucleotide into the known expression vector capable of being used for preparing a fusion protein. In the present invention, the term “vector” means a DNA construct including a DNA sequence operably bound to a proper regulatory sequence capable of expressing DNA in a proper host. A vector may be plasmid, phage particles, or simply a potential genome insert. In the case of transforming into a proper host, the vector can be replicated and can function regardless of a host genome, or may be integrated into the genome itself in some cases. Recently, plasmid is a type that is most generally used as a vector, so that in the present specification, “plasmid” and “vector” are used interchangeably. For purposes of the present invention, a plasmid vector is preferably used. A typical plasmid vector capable of being used for these purposes has (a) a replication origin that allows it to be effectively replicated to include hundreds of plasmid vectors per host cell, (b) antibiotic resistance genes allowing the host cell transformed into the plasmid vector to be selected, and (c) a structure including restriction enzyme cleavage sites capable of receiving insertion of an external DNA fragment. Even if there are no proper restriction enzyme cleavage sites, when a synthetic oligonucleotide adaptor or linker according to the general method is used, the vector and external DNA may be easily ligated.
Meanwhile, such a recombinant vector may include an expression vector allowing a His tag to be expressed at the end of a fusion protein in order to effectively isolate and purify a protein.
A host cell may be transformed by using a polynucleotide encoding a fusion protein including albumin and a retinol-binding protein (RBP) and a recombinant vector including the polynucleotide. The host cell used for expressing a fusion protein according to the present invention may include a cancer cell, but the present invention is not limited thereto.
In addition, the present invention provides a method of producing a fusion protein in which comprises albumin and a retinol-binding protein (RBP), comprising expressing the fusion protein in which comprises the albumin and the retinol-binding protein (RBP) from the transformant. The expression of the fusion protein from the transformant may be generally induced through culturing the host cell. The fusion protein comprising the albumin and retinol-binding protein (RBP) according to the present invention may be isolated from a culture medium by a known method for purifying a protein because the albumin and RBP themselves have a secretory signal, and thus the fusion protein is secreted out of the cell.
In the present invention, content in connection with genetic engineering technologies will be more clear by the content as disclosed in the document by Sambrook, et al. (Sambrook, et al. Molecular Cloning, A Laboratory Manual, Cold Spring Harbor laboratory Press, Cold Spring Harbor, N.Y. (2001)) and the document by Frederick, et al. (Frederick M. Ausubel et al., Current protocols in molecular biology volume 1, 2, 3, John Wiley & Sons, Inc. (1994)).
In addition, the present invention provides a pharmaceutical composition for preventing or treating fibrotic diseases, in which the composition comprises a fusion protein in which comprises albumin and a retinol-binding protein as an effective ingredient; a use of the fusion protein comprising the albumin and retinol-binding protein (RBP) for preparing a medicine for preventing or treating fibrotic diseases; and a method for preventing or treating fibrotic diseases, comprising administering to a subject the fusion protein comprising the albumin and retinol-binding protein (RBP) in a therapeutically effective dose.
According to a specific example, the fibrotic disease occurs in the liver, pancreas, lungs, kidneys, or intestines, but the present invention is not limited thereto. Examples of the fibrotic disease capable of being prevented or treated by the fusion protein according to the present invention include liver fibrosis, chronic hepatitis, cirrhosis, hepatic cancer, chemotherapy-associated steatohepatitis (CASH), lung fibrosis, renal fibrosis, renal failure, pancreatic fibrosis, chronic pancreatitis, and pancreatic cancer.
The pharmaceutical composition of the present invention may be preferably formulated into a pharmaceutical composition by further including at least one pharmaceutically acceptable carrier for administration in addition to an effective ingredient. Preferably, a liquid solution for an injection is suitable.
For the composition to be formulated in a liquid solution, the pharmaceutically acceptable carrier may include, to be suitable for sterilization and for a living body, saline solution, sterilized water, Ringer's solution, buffered saline, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, ethanol. In combination with at least one of these components, and if necessary, other general additives, such as antioxidant, a buffer solution, and bacteristat may be added. In addition, a form of dose to be injected, such as an aqueous solution, suspension, and an emulsion, may be formulated by further adding diluents, dispersing agents, surfactants, binding agents, and a lubricant. Furthermore, it may be preferably formulated according to a disease or components by using the method disclosed in Remington's Pharmaceutical Science, Mack Publishing Company, Easton 5 PA, as a proper method in the art.
The pharmaceutical composition of the present invention may be administrated in a general way through a route such as intravenous injection, intra-arterial injection, intraperitoneal injection, intramuscular injection, and intrasternal injection.
An effective dose of an effective ingredient of the pharmaceutical composition according to the present invention means the amount required for effectively preventing or treating diseases. Accordingly, the effective dose may be controlled according to various factors such as a type of disease, disease severity, types and contents of the effective ingredient and other ingredients of the composition, a form of administration, an age, body weight, general health conditions, sex and a diet of a patient, an administration time, an administration route, composition secretion rate, a treatment period, and drugs taken concurrently. For example, in the case of the adult, the fusion protein of the present invention may be administrated in doses of 10 ng/kg to 10 g/kg when administrated once a day or several times a day, but the present invention is not limited thereto.
According to the present invention, subjects may be human, orangutan, chimpanzee, mouse, rat, dog, cow, chicken, pig, goat, and sheep, but they are preferably used.
Effects of the Invention
The fusion protein comprising albumin and retinol-binding protein (RBP) according to the present invention can be used for preventing or treating fibrotic diseases occurring in the liver, pancreas, lungs, or other organs by promoting formation of fat droplets in stellate cells and converting activated stellate cells into non-activated state or by inducing cellular senescence of the stellate cells.



DESCRIPTION OF DRAWINGS
FIG. 1A is a schematic diagram illustrating production of the fusion protein of RBP-albumin406-608a.a. (domain III) (R-III) and albumin1-222 (domain I)-RBP-albumin406-608 (I-R-III), and FIG. 1B shows the result of western blot analysis assessing the effect of fusion protein expression on activated stellate cells.
FIGS. 2A to 2D show the effect of the expression of albumin or fusion protein on cell morphology of activated stellate cells; phase contrast image (the left top panel), autofluorescence image (the right top panel), immunofluorescence (the right bottom panel), and oil red O staining (the left bottom panel).
FIG. 3 shows a cytomorphological effect of the expression of the mutant fusion protein including albumin/albumin III domains having induced point mutation (R410A/Y411A/K525A).
FIG. 4A shows the result of western blot analysis illustrating that the fusion protein according to the present invention is taken into stellate cells and induces biochemical changes, FIG. 4B shows the result of an oil red O staining, describing morphological changes of cell type due to introduction of the fusion protein, and FIG. 4C shows the result of western blott analysis indicating that the fusion protein uptake is mediated through caveolae-mediated endocytosis.
FIG. 5A shows the purification process for the albumin-RBP fusion protein according to the present invention using an ammonium sulfate precipitation (lane 1), a His Trap affinity column (lane 2), and a Resource Q column (lane 3), FIG. 5B shows the result of western blot analysis of hepatic tissue lysate after an intravenous injection of the fusion protein for 1 week, and FIG. 5C shows the result of in vivo experiment illustrating tissue distribution.
FIG. 6 shows the results of immunohistochemical analysis and Sirius red staining, and a photomicrograph illustrating that the fusion protein according to the present invention alleviates liver fibrosis.
FIG. 7 shows the results of Sirius red staining, immunohistochemical staining and western blotting, illustrating that the fusion protein according to the present invention prevents liver fibrosis.
FIG. 8 shows the result of MT (Masson's trichrome) staining illustrating that the fusion protein according to the present invention reduces kidney fibrosis.



BEST MODE
The above and other objects, features and advantages of the present invention will become clear by describing Examples below in detail. However, the present invention is not limited to the Examples described below, and can be implemented in various different forms. The following Examples are provided so that this disclosure will completely enable those of ordinary skill in the art to embody and practice the present invention.
EXAMPLE
Experiment Method
Isolation and Culture of Pancreatic Stellate Cells (PSCs)
Rat pancreatic stellate cells were isolated according to the method disclosed in Apte, M. V. et al., Periacinar stellate shaped cells in rat pancreas: identification, isolation, and culture. Gut 43 (1), 128-133 (1998). In summary, pancreas was finely minced, placed in a Hank's buffer solution containing 0.05% collagenase, 0.02% protease, and 0.1% DNase, and then shaken at 37° C. for 20 minutes. After filtering through a 150 mm mesh, the cells were centrifuged by 13.2% Nycodenz gradient at 1400 g for 20 minutes. The pancreatic stellate cells were collected from the band just above the interface between the Nycodenz solution and aqueous layer, suspended in a DMEM (Dulbecco's modified Eagle's medium, Carlsbad, Calif.) supplemented with 10% fetal bovine serum, and then plated on a non-coated plastic dish. After reaching confluence in the primary culture, serial passages were obtained always applying 1:3 split.
Constitution of Expression Vector to Albumin-RBP Fusion Protein
Total RNA was extracted from a rat river tissue using a RNeasy kit (Qiagen, Valencia, Calif.) and reverse-transcribed into cDNA using GeneAmp RNA PCR (Applied Biosystems, Foster city, CA). The entire open reading frame (ORF) of albumin or RBP was amplified by polymerase chain reaction (PCR) with the designed primers and inserted into a pBluescript vector.
The expression vector encoding albumin I-RBP-albumin III (hereinafter, also referred to as 1R3 or I-RBP-III) was prepared as follows. A DNA fragment encoding albumin (domain I: 1-666) (SEQ ID NO: 2) or a RBP (55-585) (SEQ ID NO: 6) was amplified from the pBluescript-albumin or pBluescript-RBP by PCR with the primers:









(SEQ ID NO: 2)



Albumin (domain I: 1-666)



Sense primer:



(SEQ ID NO: 20)



5′ GGGGTACCCC ACCATGAAGT GGGTAACCTT TC 3′



 



Antisense primer:



(SEQ ID NO: 21)



5′ CCCCAATTGC ATCCTCTGAC GGACAGC 3′



 



(SEQ ID NO: 6)



RBP (55-585)



Sense primer:



(SEQ ID NO: 22)



5′ GGGCAATTGG AGCGCGACTG CAGGGTG 3′



 



Antisense primer:



(SEQ ID NO: 23)



5′ CCCCTCGAGT CTGCTTTGAC AGTAACC 3′.






The PCR products were double digested with KpnI/MFeI or MfeI/XhoI, respectively, and the DNA fragments purified by an agarose gel electrophoresis were ligated together and then cloned into KpnI/XhoI-cut pBluescript vector to yield pBluescript-1R.
A DNA fragment encoding albumin (domain III: 1216-1827) (SEQ ID NO: 3) was amplified with the following primers:









Sense primer:



(SEQ ID NO: 24)



5′ GGGCTCGAGGAAGAACCTAAGAACTTG 3′



 



Antisense primer:



(SEQ ID NO: 25)



5′ GGCTCTAGAT TAATGATGAT GATGATGATGGGCTAAGGCT



TCTTTGCT 3′.






A His-tag sequence was included in the antisense primer. The PCR products were double digested with XhoI/XbaI and then ligated with the DNA fragment of IR prepared above. The resulting DNA fragment 1R3 was inserted into expression vector pcDNA3.1+ at KpnI and XbaI sites to yield pcDNA3.1-1R3.
An expression vector encoding RBP-albumin III (hereinafter, also referred to as R3 or RBP-III) was prepared as follows. A DNA fragment encoding RBP (1-585) (SEQ ID NO: 5) was amplified with the following primers.









Sense primer:



(SEQ ID NO: 26)



5′ GCGGAATTCC ACCATGGAGT GGGTGTGGGC 3′



 



Antisense primer:



(SEQ ID NO: 27)



5′ CCCCTCGAGT CTGCTTTGAC AGTAACC 3′






The PCR products were double digested with EcoRI/XhoI, ligated with a DNA fragment encoding albumin (domain III: 1216-1827) (SEQ ID NO: 3), and then inserted into pcDNA3.1+ vector at EcoRI and XbaI sites to yield pcDNA3.1-R3. In the pcDNA3.1-1R3 or pcDNA3.1-R3, an albumin/RBP encoding region was located immediately upstream of 6-histidine tag encoding sequence and stop codon in the same reading frame.
Meanwhile, it has been reported that expression of mutant albumin, in which Arg410, Tyr411, and Lys525, amino acid residues of the albumin were substituted with Ala, leads to aging of stellate cells (Kim N, Yoo W, Lee J, Kim H, Lee H, Kim Y, Kim D, Oh J.* (2009) Formation of vitamin A fat droplets in pancreatic stellate cells requires albumin. Gut 58(10), 1382-90.). It means that direct interaction with fatty acids is an important mechanism for albumin function in stellate cells. Accordingly, in the present invention, a mutant fusion protein expression vector including albumin/albumin III domain having a point mutation (R410A/Y411A/K525A) induced, using a PCR-based method which was constructed using Muta-directTM Site-Directed Mutagenesis Kit (iNtRON, Korea) in order to prepare a fusion protein for inducing aging of stellate cells; the expression vector was transfected into the activated stellate cells; and then a change of phenotype was investigated.
All the constructs were sequenced by using an auto-sequencer to confirm an albumin/RBP encoding region.
Purification of His6 Tag Recombinant Fusion Protein
An expression vector encoding mouse R3 was prepared in the same manner as the rat fusion protein. Primers used for a PCR were as follows.









(SEQ ID NO: 3)



Albumin (domain III: 1216-1827)



Sense primer:



(SEQ ID NO: 28)



5′ GGGCTCGAGG AAGAGCCTAA GAACTTG 3′



 



Antisense primer:



(SEQ ID NO: 29)



5′ GGCTCTAGAT TAATGATGAT GATGATGATGGGCTAAGGTG



TCTTTGCA 3′



 



(SEQ ID NO: 5)



RBP (1-585)



Sense primer:



(SEQ ID NO: 30)



5′ GCGGAATTCC ACCATGGAGT GGGTGTGGGC 3′



 



Antisense primer:



(SEQ ID NO: 31)



5′ CCCCTCGAGC CTGCTTTGAC AGTAACC 3′






Clonal cell lines having high expression rate were selected by assessing a level of the secreted recombinant fusion protein by western blotting using an anti-His tag antibody after 293 cells were stably transfected with an expression vector encoding R3. A culture medium of 293 cells was fractionated with ammonium sulfate (55%), then subject to His Trap affinity column. The sample was further purified by a Resource Q. The purified proteins were dialyzed with deionized water, freeze-dried, and then dissolved in saline solution. As a result of measuring with a SDS-PAGE and protein staining, the purity of R-III was above 95%.
Transfection
Activated pancreas stellate cells (after passage 2) was transiently transfected using lipofectamine 2000 (Invitrogen, Carlsbad, Calif.), and then after 24 hours, the cells were analyzed.
Western Blotting Analysis
The cells were rinsed in ice-cold phosphate buffer saline (PBS) twice, and harvested by scraping in a lysis buffer solution. The equivalent amounts of proteins were separated by a SDS-PAGE, followed by immunoblot detection using a primary antibody. The primary antibodies were as follows: albumin (Santa Cruz, Santa Cruz, Calif.), α-SMA (Sigma, St. Louis, Mo.), α-tubulin (Cell signaling, Beverly, Mass.) and Type I collagen (Calbiochem, San Diego, Calif.), His-tag (AB Frontier, Seoul, Korea).
Immunofluorescence Analysis
Pancreatic stellate cells were plated on a glass cover slip coated with gelatin. The samples were fixed with paraformaldehyde, incubated with an albumin antibody (Santa Cruz #sc-58698) overnight at 4° C. in a moist chamber, and reacted with a secondary antibody bound with Alexa Fluor 568. The cells were washed with PBS and mounted onto a slide. The stained cells were visualized by using Zeiss AXIO Imager M1 microscope.
Oil Red O Staining
Fat droplets were visualized by staining the pancreatic stellate cells with an oil red O using the method disclosed by Koopman (Koopman, R., Schaart, G., & Hesselink, M. K., Optimisation of oil red O staining permits combination with immunofluorescence and automated quantification of lipids. Histochem Cell Biol 116 (1), 63-68 (2001)). The oil red O was diluted in triethyl phosphate instead of isopropane.
Preparation of Liver Fibrosis Model
BALB/c mice were injected intraperitoneally with 1 mL/kg CCl4 dissolved in a mineral oil at 1:1 three times per week for 7 weeks to induce liver damage and prepare liver fibrosis mouse model having damaged liver. The group administered only mineral oil in the same amount thereof was used as a control group. At 72 hours after final CCl4 injection, the mouse was sacrificed. The hepatic tissue was sectioned and fixed with 10% buffer formalin in order for histological analysis. The remnant was added to a Rnase-free tube and quickly frozen in liquid nitrogen.
Preparation of Kidney Fibrosis Model
UUO (unilateral ureteral obstruction) model was performed by using a BALB/c mouse. In summary, the abdominal cavity of the mouse was opened through a midline incision, and then the left ureter was isolated and tied up. By a similar method, sham-operated animals were subjected to the same surgical operation, but the ureter ligation was not performed. In order to test an effect of albumin-RBP fusion protein R-III on improving kidney fibrosis, the R-III was administrated every day for 7 days through the tail vein injection from starting on the sixth day after blocking. After completing the test, the mouse was sacrificed, and then the kidney tissues were removed. The half of the kidney was fixed with 10% buffer formalin in order for a histological study, and the other half was snap-frozen in liquid nitrogen to store at −80° C. in order for extractions of protein and RNA.
Immunohistological Analysis
The section (5 μm thickness) of formalin-fixed, paraffin-embedded liver tissues were prepared, stained with a H&E for a histological analysis and with Sirius red or Masson's trichrome for collagen deposition. In addition, the tissue sections were immunohistochemically stained with Type I collagen (Abeam, Cambridge, UK) antibody. In order to quantify the Sirius red staining, Image J software (NIH) was used.
Statistical Analysis
The results were expressed as mean±standard deviation (SD). A statistical analysis was performed by using t-tests. Comparisons were considered significant at P<0.05, and the P values were two-tailed.
Experimental Result
Facilitation of Formation of Fat Droplets in Pancreatic Stellate Cells of Albumin-RBP Fusion Protein
In the aforementioned RBP-albumin406-608 a.a. (domain III) (R-III) and albumin1-222 (domain I)-RBP-albumin406-608 (I-R-III) (FIG. 1A), the respective protein parts were linked through restriction enzyme recognition site linkers and a polyhistidine tag was located at the C-terminal of fusion protein. After the pancreatic stellate cells were activated by serial passages (two passages), the cells were transfected with an expression vector to wild-type albumin, R-III or I-R-III and then a change of phenotype was investigated. As a result of western blotting, the fusion protein having the expected size was expressed (R-III  ̃45 kDa and I-R-III  ̃68 kDa (FIG. 1B)).
FIGS. 2A to 2D show the results of analyzing a cell morphological effect of the albumin or fusion protein expression on the activated stellate cells; phase contrast image (left top panel), autofluorescens image (right top panel), immunofluorescence (Right bottom panel), and oil red O staining (left bottom panel). Activated pancreatic stellate cells under normal culture conditions show a shape of fibroblastoid (FIG. 2A), but expression of wild-type albumin, R-III or I-R-III induced formation of autofluorescent fat droplets, resulting in changing a cell shape into a polygonal shape (FIGS. 2B to 2D). Such a change of cell shape is accompanied with a decrease in levels α-SMA, a marker for the activated stellate cells, and type I collagen (FIG. 1B). This supports that, like albumin, expression of the fusion protein can inactivate the activated stellate cells.
Meanwhile, as a result of experimenting with a mutant fusion protein including the albumin/albumin III domain having an induced point mutation (R410A/Y411A/K525A) performed by the above-mentioned method, the expression of the mutant fusion protein led to a slight increase in lipid droplet formation and cellular senescence (FIG. 3).
Cellular Uptake of Albumin-RBP Fusion Protein into Stellate Cells
In order to investigate whether the RBP moiety can facilitate cellular uptake of the fusion protein, conditioned medium was prepared by incubating the 293 cells stably transfected with albumin, R-III, or I-R-III for 24 hours, and applied to activated pancreatic stellate cells. Western blotting with use of anti-His tag antibody revealed that fusion proteins, but not wild-type albumin, were successfully incorporated into stellate cells (FIG. 4A). In addition, fusion proteins induced phenotypic conversion (FIG. 4B), and reduced the expression levels of α-SMA and Type I collagen (FIG. 4A). We further investigated a mechanism of R-III internalization using an inhibitor of clathrin-mediated endocytosis (chlorpromazine) and an inhibitor of caveolae-mediated endocytosis (filipin). Western blotting analysis revealed that cellular uptake of R-III was largely inhibited by filipin pre-treatment (FIG. 4C). This result agrees with the previous report that RBP enters the cell through caveolae-mediated endocytosis. Therefore, the data shows that the constituent parts of the fusion protein are functionally important. In other words, in the fusion protein, the RBP performs a role in stellate cells-specific targeting moiety and albumin domain performs a role in stellate cells inactivating domain.
Absorption of Injected Albumin-RBP Fusion Protein into Liver
Since then, tissue distribution of albumin-RBP fusion protein was investigated in vivo. Since R-III was expressed and secreted more (data not shown) from the transfected 293 cells as compared with I-R-III, R-III was selected and purified using FPLC to >95% purity (FIG. 5A). The R-III (3 or 10 μg) dissolved in 0.1 ml of saline solution was injected every day into the tail vein of a BALB/c mouse for 7 days, and then liver lysate was analyzed by western blotting using an anti-His tag antibody. The distinct R-III protein band was observed in the R-III-injected mouse, and the band intensity thereof increased in dose dependent manner (FIG. 5B). When equivalent amounts of whole cell lysates obtained from different tissues was analyzed by western blotting, strong R-III signal was observed in liver, and also a weak signal was detected in the brain, lungs, spleen, pancreas, kidneys, and intestines (FIG. 5C). Such tissue distribution of R-III appears to be similar to that of RBP.
Improvement of Liver Fibrosis by R-III
The present inventors assessed therapeutic and preventive potentials of R-III using the carbon tetrachloride (CCl4)-induced liver fibrosis model. CCl4 was injected three time per week into the abdominal cavity of a BALB/c mouse for 7 weeks, and then R-III (10 μg; n=25), albumin (10 μg; n=7), RBP (5 μg; n=5), or saline solution alone (n=23) was intravenously administrated every day during the last 2 weeks in the CCl4 treatment (FIG. 6A). The mineral oil/saline solution control mice exhibited a normal liver structure (FIG. 6C). Meanwhile, severe liver fibrosis was observed in CCl4-treated mice. In other words, multiple nodules were found on the surface of the liver under a microscope (FIG. 6B), and destruction of the liver tissue architecture, fibrous expansion, and large fibrous septa formation were observed (FIG. 6C). In addition, the Sirius red staining and immunohistochemical analysis exhibited extensive collagen deposition in the liver tissue of CCl4-treated mice (FIG. 6C). However, the administration of R-III significantly reduced nodule incidence, histopathological alterations and collagen deposition, which were shown in the CCl4-treated group (FIGS. 6B and 6C). Quantification of Sirius red staining by Image J Software (NIH) showed that collagen content was decreased by R-III by  ̃35% (FIG. 6D). On the other hand, the administration of albumin or RBP did not influence progress of fibrosis by treating CCl4 (data not shown). As a result, it was identified that the administration of R-III was a remedy for liver fibrosis for the CCl4-induced liver fibrosis mouse model.
Effect of R-III on Inhibiting Liver Fibrosis
To examine whether R-III might have preventive effect on CCl4-induced liver fibrosis, mice (n=22) were treated with CCl4 and R-III (10 μg) three times per week over 7 weeks. The CCl4 and R-III were administrated on different days (FIG. 7A). H&E and Sirius red staining of liver sections revealed that R-III markedly reduced histopathological alterations and collagen deposition (FIG. 7B). As a result of quantitative analysis of Sirius red staining, it was confirmed that a content of collagen was decreased by  ̃38% (FIG. 7C). This result was further supported by immunohistochemical staining and western blotting (FIGS. 7B and 7D). Therefore, the present inventors' data showed that the albumin-RBP fusion protein has both a therapeutic and preventive effects on liver fibrosis.
Kidney Fibrosis Decrease by R-III
The therapeutic potential of R-III was estimated using a UUO (unilateral ureteral obstruction)-induced kidney fibrosis model. The mice were subjected to UUO and then intravenously administered R-III (10 μg; n=10) or saline solution (n=10) every day for 7 days (FIG. 8A). MT (Masson's trichrome) staining of kidney sections confirmed extensive collagen deposition in UUO-mediated fibrotic kidney and R-III markedly reduced collagen deposition (FIG. 8B). Therefore, from this result, it was confirmed that the RAH attenuated UUO-induced kidney fibrosis in the mouse.

## Claims

1. A fusion protein comprising albumin and a retinol-binding protein (RBP), wherein the fusion protein has any one of the amino acid sequences set forth in SEQ ID NOs: 8 to 13.

2. The fusion protein of claim 1, wherein the albumin is at least one of an albumin I domain and an albumin III domain.

3. The fusion protein of claim 1, wherein the fusion protein is albumin I domain-RBP-albumin III, albumin III-RBP-albumin I, RBP-albumin III, albumin III-RBP, albumin-RBP, or RBP-albumin.

4. A polynucleotide encoding the fusion protein of claim 1.

5. The polynucleotide of claim 4, wherein the polynucleotide has any one of nucleic acid sequences set forth in SEQ ID NO: 14 to SEQ ID NO: 19.

6. A fusion protein comprising albumin and a retinol-binding protein (RBP), wherein the fusion protein comprises the amino acid sequence selected from the croup consisting of SEQ ID NO 8, SEQ ID NO 9, SEQ ID NO 10, SEQ ID NO 11, SEQ ID NO 12, and SEQ ID NO 13.

7. The fusion protein of claim 6, wherein the albumin is at least one of an albumin I domain and an albumin III domain.

8. The fusion protein of claim 6, wherein the fusion protein comprises the amino acid sequence set forth in SEQ ID NO: 8.

9. The fusion protein of claim 6, wherein the fusion protein comprises the amino acid sequence set forth in SEQ ID NO: 9.

10. The fusion protein of claim 6, wherein the fusion protein comprises the amino acid sequence set forth in SEQ ID NO: 10.

11. The fusion protein of claim 6, wherein the fusion protein comprises the amino acid sequence set forth in SEQ ID NO: 11.

12. The fusion protein of claim 6, wherein the fusion protein comprises the amino acid sequence set forth in SEQ ID NO: 12.

13. The fusion protein of claim 6, wherein the fusion protein comprises the amino acid sequence set forth in SEQ ID NO: 13.