Pharmaceutical composition for preventing or treating fibrotic diseases

Abstract

The present invention relates to a novel fusion protein of albumin and retinol-binding protein, which can be used for preventing or treating fibrotic diseases occurring in the liver, pancreas, lungs, and the like. The fusion protein of the present invention can prevent or treat fibrotic diseases by inhibiting activation of stellate cells that cause tissue fibrosis or by inducing reversion of the activated state.

Description

Pharmaceutical composition for preventing or treating fibrotic diseases

[0001] The present invention relates to a novel fusion protein comprising a retinol binding protein and an albumin-derived peptide, and its use in the prevention or treatment of fibrotic diseases.

[0002] Fibrosis occurs in various tissues, including the liver, kidneys, pancreas, and lungs, and can lead to fatal consequences, including the loss of tissue function. For example, liver fibrosis, caused by excessive accumulation of extracellular matrix within the tissue, hardens the liver and can lead to cirrhosis and, in severe cases, liver cancer. Pulmonary fibrosis, caused by the hardening of lung tissue, can cause severe respiratory distress. While pulmonary fibrosis can be caused by inhalation of toxic substances or persistent inflammation, there are also cases of idiopathic pulmonary fibrosis, where the specific cause remains unknown. Currently, there is no method to restore tissues that have become significantly fibrotic due to fibrosis, and organ transplantation is currently the only option, necessitating the development of new treatments.

[0003] Although the molecular mechanisms of tissue fibrosis remain unclear, recent research has revealed that the activation and transdifferentiation of stellate cells, a type of cell constituting liver, pancreas, kidney, and lung tissues, play a crucial role in the development of tissue fibrosis. Specifically, stellate cells undergo activation and differentiate into myofibroblasts, which then overexpress extracellular matrix components such as collagen, accumulating them within the tissue.

[0004] Normal hepatic stellate cells play a central role in regulating vitamin A homeostasis throughout the body. Vitamin A (retinol), which enters the body through dietary intake, circulates in the bloodstream bound to retinol binding protein (RBP), and is then transported into stellate cells via STRA6, an RBP receptor located on the stellate cell membrane, where it is stored in the form of retinyl esters within intracytoplasmic lipid droplets. Meanwhile, albumin expression rapidly decreases as stellate cells undergo activation, and it has been revealed that forced albumin expression in already activated stellate cells reverts the stellate cells to their pre-activation state.

[0005] In a previous study, the inventors of the present invention produced a fusion protein of RBP and albumin that targets astrocytes via STRA6 to control the activation of astrocytes and confirmed its effect in preventing and treating fibrosis (KR 10-1395394).

[0006] The technical task to be achieved by the present invention is to provide a novel fusion protein having significantly improved expression / productivity and efficacy due to increased structural stability of the previously developed RBP and albumin fusion protein, and to provide a pharmaceutical composition for preventing and treating fibrotic diseases containing the fusion protein as an active ingredient.

[0007] However, the technical problems to be solved by the present invention are not limited to the problems mentioned above, and other problems not mentioned can be clearly understood by those skilled in the art from the description below.

[0008] To solve the above problem, the present invention provides a fusion protein in which a retinol binding protein (RBP) and an albumin-derived peptide are combined.

[0009] In the above fusion protein, the albumin-derived peptide comprises albumin domain IIIA and albumin domain IB, and the two domains are connected by a linker composed of hydrophobic amino acids.

[0010] As one embodiment of the present invention, the linker may be composed of 3 to 10 aa, preferably 3 to 7 aa, and more preferably 4 aa.

[0011] As another embodiment of the present invention, the linker may comprise or consist of one of the amino acid sequences of SEQ ID NOs: 17 to 19.

[0012] As another embodiment of the present invention, the fusion protein may have a structure of (N-terminal)-RBP-albumin-derived peptide-(C-terminal) or (N-terminal)-albumin-derived peptide-RBP-(C-terminal), and more specifically, may have a structure of (N-terminal)-RBP-albumin domain IIIA-linker-albumin domain IB-(C-terminal) or (N-terminal)-albumin domain IIIA-linker-albumin domain IB-RBP-(C-terminal).

[0013] In another embodiment of the present invention, the albumin domain IIIA may comprise or consist of an amino acid sequence of one of SEQ ID NO: 4 to SEQ ID NO: 13, and the albumin domain IB may comprise or consist of an amino acid sequence of SEQ ID NO: 14 or SEQ ID NO: 15.

[0014] As another embodiment of the present invention, the albumin domain IIIA may be derived from an albumin protein in which the 441st amino acid is glutamic acid or glutamine.

[0015] As another embodiment of the present invention, the fusion protein may comprise or consist of one amino acid sequence selected from the group consisting of SEQ ID NOs: 20 to 31, SEQ ID NOs: 44 to 63, and SEQ ID NOs: 84 to 88.

[0016] As another embodiment of the present invention, disulfide bonds may be additionally introduced into albumin domains IIIA and IB, and the number of such bonds may be one or more.

[0017] As another embodiment of the present invention, the fusion protein to which the disulfide bond is additionally introduced may comprise or consist of one amino acid sequence selected from the group consisting of SEQ ID NOs: 44 to 63 and SEQ ID NOs: 86 to 88.

[0018] In addition, the present invention provides an RBP-albumin fusion protein having the structure [N-terminal-RBP-albumin-derived peptide-C-terminal], wherein the RBP domain may be derived from RBP in which the 194th amino acid is substituted with lysine (K), and more specifically, may include or consist of the amino acid sequence of SEQ ID NO: 16.

[0019] As one embodiment of the present invention, the albumin-derived peptide may include albumin domain IIIA and albumin domain IB.

[0020] In another embodiment of the present invention, the albumin domain IIIA and the albumin domain IB may be connected by a linker consisting of 3 to 7 hydrophobic amino acids, and the linker may comprise or consist of one amino acid sequence selected from the group consisting of SEQ ID NOs: 17 to 19.

[0021] As another embodiment of the present invention, the albumin domain IIIA may comprise or consist of one amino acid sequence selected from the group consisting of SEQ ID NOs: 4 to 13.

[0022] As another embodiment of the present invention, the albumin domain IB may comprise or consist of the amino acid sequence of SEQ ID NO: 14 or SEQ ID NO: 15.

[0023] As another embodiment of the present invention, the fusion protein may comprise or consist of one amino acid sequence selected from the group consisting of SEQ ID NOs: 84 to 88.

[0024] Each of the above-described fusion proteins may additionally include a signal peptide at the N terminus.

[0025] As one embodiment of the present invention, the signal peptide may comprise or consist of the amino acid sequence of SEQ ID NO: 94 or SEQ ID NO: 95.

[0026] In addition, the present invention provides the fusion protein gene, an expression cassette including the gene, and a recombinant vector including the expression cassette.

[0027] In addition, the present invention provides a pharmaceutical composition for preventing or treating fibrotic diseases, which comprises the fusion protein as an active ingredient.

[0028] In addition, the present invention provides a pharmaceutical composition for preventing or treating fibrotic diseases, comprising a vector containing the fusion protein gene. The fusion protein gene refers to a base sequence encoding the fusion protein.

[0029] As one embodiment of the present invention, the composition can inhibit the expression of α-SMA (smooth muscle alpha-actin) and collagen I in stellate cells.

[0030] In another embodiment of the present invention, the composition can inhibit the activation of astrocytes or inactivate activated astrocytes.

[0031] In addition, the present invention provides a method for preventing or treating a fibrotic disease, comprising a step of administering the fusion protein to a subject.

[0032] The present invention also provides the use of the fusion protein for the manufacture of a drug for the prevention or treatment of a fibrotic disease.

[0033] The fusion protein of the present invention is capable of preventing or treating fibrotic diseases by inhibiting the activation or inducing the inactivation of stellate cells that cause tissue fibrosis, and, compared to conventional fusion proteins for the same purpose, is expected to be a universal basic technology that can drastically reduce the human administration dose / frequency and manufacturing cost of protein therapeutics by greatly improving the excellent efficacy and expression / productivity based on high stability.

[0034] Figure 1 shows the results of a western blot using a His-tag antibody to confirm the expression level of the fusion protein in Expi293 cells transfected with an expression vector for albumin-RBP fusion proteins 1-6 of the present invention. Samples in each lane are as follows:

[0035] Lane 1. Positive control for protein mass

[0036] Lane 2. RBP-albumin (IIIA-DGPG-IB);441E

[0037] Lane 3. RBP-albumin (IIIA-GGPA-IB);441E

[0038] Lane 4. RBP-albumin (IIIA-EVDD-IB);441E

[0039] Lane 5. RBP-albumin (IIIA-AAAA-IB);441E

[0040] Lane 6. Albumin (IIIA-DGPG-IB)-RBP;441E

[0041] Lane 7. Albumin (IIIA-GGPA-IB)-RBP;441E

[0042] Lane 8. Albumin (IIIA-EVDD-IB)-RBP;441E

[0043] Lane 9. Albumin (IIIA-AAAA-IB)-RBP;441E.

[0044]

[0045] Figure 2 shows the results of performing qPCR to determine the expression levels of α-SMA and collagen type I, markers of myofibroblasts, after transducing an expression vector for the fusion protein 1-6 of the present invention into activated stellate cells. The indices on the x-axis of the graph are as follows:

[0046] 1. Control

[0047] 2. RBP-albumin (IIIA-IIIB)

[0048] 3. RBP-albumin (IIIA-DGPG-IB);441E

[0049] 4. RBP-albumin (IIIA-GGPA-IB);441E

[0050] 5. RBP-albumin (IIIA-EVDD-IB);441E

[0051] 6. RBP-albumin (IIIA-AAAA-IB);441E

[0052] 7. Albumin (IIIA-DGPG-IB)-RBP;441E

[0053] 8. Albumin (IIIA-GGPA-IB)-RBP;441E

[0054] 9. Albumin (IIIA-AAAA-IB)-RBP;441E.

[0055]

[0056] Figure 3 shows the results of performing qPCR to determine the expression levels of α-SMA and collagen type I, markers of myofibroblasts, after transducing an expression vector for the fusion protein 7-12 of the present invention into activated stellate cells. The indices on the x-axis of the graph are as follows:

[0057] 1. Control

[0058] 2. RBP-albumin (IIIA-DGPG-IB);441Q

[0059] 3. RBP-albumin (IIIA-GGPA-IB);441Q

[0060] 4. RBP-albumin (IIIA-AAAA-IB);441Q

[0061] 5. Albumin (IIIA-DGPG-IB)-RBP;441Q

[0062] 6. Albumin (IIIA-GGPA-IB)-RBP;441Q

[0063] 7. Albumin (IIIA-AAAA-IB)-RBP;441Q.

[0064]

[0065] Figure 4 shows the results of qPCR performed to confirm the levels of α-SMA (A) and collagen type I (B) RNA expression after treatment of activated stellate cells with RBP-albumin(IIIA-IIIB);441E (black) and RBP-albumin(IIIA-AAAA-IB);441E (gray) at concentrations of 0.75 or 0.375 μM. PBS control (white).

[0066]

[0067] Figure 5 shows the results of H&E and Sirius red staining of liver tissue sections to confirm the effect of improving liver fibrosis following administration of a fusion protein (RBP-albumin (IIIA-AAAA-IB)) in an animal model of liver fibrosis induced by intraperitoneal injection of carbon tetrachloride.

[0068]

[0069] Figure 6 shows the results of statistical analysis using the Whitney U-test of the Sirius red staining results of Figure 5. (A) is the result of administration of 15 μg of the fusion protein, and (B) is the result of administration of 7.5 μg. The p-values ​​are 0.0484 (A) and 0.0472 (B), respectively.

[0070]

[0071] Figure 7 shows the results of confirming the cell morphology of activated stellate cells transformed with an expression vector for the RBP-albumin fusion protein 13-16 of the present invention with an additional CC bond introduced.

[0072]

[0073] Figure 8 shows the results of qPCR that confirmed the levels of α-SMA (A) and collagen type I (B) RNA expression in the stellate cells of Figure 7. The indices on the graph's x-axis are as follows:

[0074] 1. Control

[0075] 2. RBP-albumin (IIIA-AAAA-IB);441E

[0076] 3. RBP-albumin (IIIA-AAAA-IB): C144-C199;441E

[0077] 4. RBP-albumin (IIIA-AAAA-IB): C446-C487;441E

[0078] 5. RBP-albumin (IIIA-AAAA-IB): C453-C480;441E

[0079] 6. RBP-albumin (IIIA-AAAA-IB):C457-C476;441E.

[0080]

[0081] Figure 9 shows the results of qPCR performed after transfection of activated astrocytes with an expression vector for fusion protein 21-28, derived by substituting glutamine for albumin sequence number 441 along with the introduction of additional CC bonds, to determine the levels of α-SMA (A) and collagen type I (B) RNA expression. The indices on the x-axis of the graph are as follows:

[0082] 1. Control

[0083] 2. RBP-albumin (IIIA-AAAA-IB): C144-C199;441Q

[0084] 3. RBP-albumin (IIIA-AAAA-IB): C446-C487;441Q

[0085] 4. RBP-albumin (IIIA-AAAA-IB): C453-C480;441Q

[0086] 5. RBP-albumin (IIIA-AAAA-IB):C457-C476;441Q.

[0087] 6. Albumin (IIIA-AAAA-IB)-RBP:C144-C199;441Q

[0088] 7. Albumin (IIIA-AAAA-IB)-RBP:C446-C487;441Q

[0089] 8. Albumin (IIIA-AAAA-IB)-RBP:C453-C480;441Q

[0090] 9. Albumin (IIIA-AAAA-IB)-RBP:C457-C476;441Q.

[0091]

[0092] Figure 10 shows the results of qPCR performed after transfection of activated astrocytes with an expression vector for the albumin-RBP fusion protein 29-32, which has two additional CC bonds, to determine the levels of α-SMA (A) and collagen type I (B) RNA expression. The indices on the x-axis of the graph are as follows:

[0093] 1. Control

[0094] 2. RBP-albumin (IIIA-AAAA-IB);441Q

[0095] 3. RBP-albumin (IIIA-AAAA-IB): C144-C199, C453-C480; 441Q

[0096] 4. RBP-albumin (IIIA-AAAA-IB): C453-C480, C457-C476; 441Q

[0097] 5. Albumin (IIIA-AAAA-IB)-RBP: C144-C199, C453-C480;441Q

[0098] 6. Albumin (IIIA-AAAA-IB)-RBP: C453-C480,C457-C476;441Q.

[0099]

[0100] Figure 11 shows the results of confirming the cell morphology of activated stellate cells transformed with an expression vector for fusion protein 33-37 using an RBP-derived domain in which the 194th amino acid of the RBP amino acid sequence is substituted with lysine in the RBP-albumin fusion protein.

[0101]

[0102] Figure 12 shows the results of qPCR that confirmed the expression levels of α-SMA (A) and collagen type I (B) RNA in the stellate cells of Figure 11. The indices on the x-axis of the graph are as follows:

[0103] 1. Control

[0104] 2. RBP-albumin (IIIA-EVDD-IB);441Q;194K

[0105] 3. RBP-albumin (IIIA-AAAA-IB);441Q;194K

[0106] 4. RBP-albumin (IIIA-AAAA-IB): C453-C480; 441Q; 176K

[0107] 5. RBP-albumin (IIIA-AAAA-IB): C144-199, C453-C480; 441Q; 176K

[0108] 6. RBP-albumin (IIIA-AAAA-IB): C453-C480, C457-C476; 441Q; 176K

[0109]

[0110] Figure 13 shows the results of a western blot using a His-tag antibody to confirm the expression level of fusion proteins in Expi293 cells transfected with expression vectors for albumin-RBP fusion proteins 33-35 and 37. Samples in each lane are as follows:

[0111] Lane 1. RBP-albumin (IIIA-AAAA-IB);C453-480;441E

[0112] Lane 2. RBP-albumin (IIIA-EVDD-IB);441Q;194K

[0113] Lane 3. RBP-albumin (IIIA-AAAA-IB);441Q;194K

[0114] Lane 4. RBP-albumin (IIIA-AAAA-IB);C453-480;441Q;194K

[0115] Lane 5. RBP-albumin (IIIA-AAAA-IB); C453-480, C457-476; 441Q; 194K

[0116] Albumin is a multifunctional plasma protein synthesized primarily by hepatocytes. Albumin has three domains, each composed of two subdomains, A and B. Albumin is known to play a role in molecular transport, binding to various hydrophobic substances, including fatty acids and retinoic acid, and transporting them within the bloodstream. Crystallographic analysis reveals five strong fatty acid binding sites asymmetrically distributed within albumin (one in subdomains IB, one between IA and IIA, two in III, and one in III).

[0117] In a previous study, the present inventors developed a fusion protein combining albumin and retinol binding protein (RBP) for astrocyte targeting. The fusion protein was found to be able to enter activated astrocytes, reverting the cell morphology back to its pre-activation state, thereby enabling the prevention and treatment of fibrotic diseases. Furthermore, the present inventors elucidated that retinoic acid (RA) plays a crucial role in the astrocyte activation process, and the fusion protein controlled astrocyte activation by reducing intracellular levels of RA.

[0118] Accordingly, the present inventors confirmed in a previous study that an albumin-RBP fusion protein combining albumin sub-domains (IIIA and IB) and RBP can induce inhibition or deactivation of astrocyte activation by binding to retinoic acid (RA) (KR 10-2021-0133732).

[0119] To increase the structural stability and efficacy of the fusion proteins, the present inventors developed various albumin-RBP fusion protein variants (Table 3; fusion proteins 1-6) in which the albumin IIIA and IB domains were connected by a linker composed of hydrophobic amino acids in the albumin region. In addition, albumin-RBP fusion proteins in which the albumin amino acid sequence number 441 in fusion proteins 1-6 was replaced with glutamine (Table 4; fusion proteins 7-12) were also produced.

[0120] Among them, in particular, the albumin-RBP fusion protein in which the albumin IIIA and IB domains are connected by the linker sequence AAAA showed significantly improved expression / productivity compared to the fusion protein of RBP and albumin (IIIA-IB) that was previously produced, and also induced inactivation of stellate cells at an excellent level, and it was confirmed to inhibit liver fibrosis in a liver fibrosis animal model (Example 3-6).

[0121] Furthermore, the inventors of the present invention considered the introduction of additional disulfide bonds in the albumin domain to create fusion proteins with improved structural stability and efficacy of albumin-RBP fusion proteins. Eight albumin-RBP fusion proteins (fusion proteins 13 to 20) with one additional disulfide bond were constructed, and eight fusion protein variants (fusion proteins 21-28) were constructed in which the amino acid at position 441 of the albumin protein was substituted with glutamine. In addition, four albumin-RBP fusion proteins (fusion proteins 29-32) with two additional disulfide bonds were also developed. Among them, the most excellent stellate cell inactivation induction effect was confirmed in fusion protein 15 (RBP-albumin(IIIA-AAAA-IB):C453-C480;441E), fusion protein 23 {RBP-albumin(IIIA-AAAA-IB):C453-C480;441Q}, and fusion protein 30 {RBP-albumin(IIIA-AAAA-IB):C453-C480,C457-C476;441Q} (Example 7-10).

[0122] In addition, the present inventors developed RBP-albumin fusion proteins 33-37 using RBP-derived domains in which the 194th amino acid of RBP was substituted with lysine in order to create fusion proteins with improved structural stability of (N-terminal) - RBP - albumin domain IIIA - linker - albumin domain IB - (C-terminal) fusion proteins. Among them, the most excellent stellate cell inactivation inducing effect was confirmed in fusion proteins 34 {RBP-albumin(IIIA-AAAA-IB):441Q;194K), 35 {RBP-albumin(IIIA-AAAA-IB):C453-C480;441Q;194K} (Example 109).

[0123] Meanwhile, the inventors of the present invention transformed cells with vectors expressing fusion protein 15 and fusion proteins 33-35 and 37 to determine whether substitution of the 194th amino acid of RBP with lysine and addition of a CC bond affect the enhancement of fusion protein expression, and compared the expression levels. As a result, high expression levels were confirmed for fusion proteins 35 and 37, and in particular, the highest level of expression was confirmed for fusion protein 37 (Example 10-3).

[0124] As described above, the fusion proteins 1 to 37 of the present invention exhibit the effect of inhibiting astrocytes from being activated despite an activation signal and the activity of inducing deactivation of activated astrocytes to convert them to a state prior to activation, i.e., an inactive state.

[0125] Accordingly, the present inventors can provide a pharmaceutical composition for preventing or treating fibrous diseases comprising at least one fusion protein selected from the group consisting of the developed fusion proteins 1 to 37, and can provide a method for preventing or treating fibrous diseases comprising a step of administering to a subject at least one fusion protein selected from the group consisting of the developed fusion proteins 1 to 37.

[0126] It has been confirmed that the fusion proteins 1 to 37 of the present invention can show superior activity and, more importantly, greatly improved expression / productivity compared to the conventional fusion proteins of RBP and albumin. Therefore, it is expected that the pharmaceutical composition of the present invention can become a universal basic technology that can drastically reduce the human administration dose and administration frequency of protein therapeutics.

[0127] In the present invention, the term “fibrotic disease” refers to a disease in which an organ fails to function properly due to fibrosis, in which normal tissue is destroyed and replaced with fibrous connective tissue, and includes liver fibrosis, chronic hepatitis, cirrhosis, hepatic cancer, chemotherapy-associated steatohepatitis (CASH), pulmonary fibrosis, renal fibrosis, renal failure, pancreatic fibrosis, chronic pancreatitis, and pancreatic cancer, and is not limited thereto as long as it is a disease in which a part of an organ is hardened by fibrosis, but preferably, it may be liver fibrosis and pulmonary fibrosis in particular.

[0128] In the present invention, the term “subject” is not limited to a mammal, but may preferably be a human or a livestock.

[0129] In the present invention, “prevention” means any act of delaying the onset of a fibrotic disease or delaying fibrosis of a tissue by administering a pharmaceutical composition according to the present invention, and “treatment” means any act of improving or beneficially changing the symptoms of a fibrotic disease by administering a pharmaceutical composition according to the present invention.

[0130] In the present invention, the albumin used to form the fusion protein may be derived from any species, but it is preferably derived from the same species as the subject to which it is administered in order to avoid the risk of immunogenicity.

[0131] In the present invention, the pharmaceutical composition may further include one or more known substances capable of preventing or treating tissue fibrosis in addition to the fusion proteins 1 to 37, and may further include appropriate carriers, excipients, and diluents commonly used in the manufacture of pharmaceutical compositions.

[0132] In the present invention, the term "carrier" is also called a vehicle and refers to a compound that facilitates the addition of a protein or peptide into a cell or tissue. For example, dimethyl sulfoxide (DMSO) is a commonly used carrier that facilitates the introduction of many organic substances into the cells or tissues of a living organism.

[0133] In the present invention, a "diluent" is defined as a compound that is diluted in water, which not only stabilizes the biologically active form of the target protein or peptide, but also dissolves the protein or peptide. Salts dissolved in buffer solutions are used as diluents in the art. A commonly used buffer solution is phosphate-buffered saline, as it mimics the salt state of human fluids. Since buffer salts can control the pH of a solution at low concentrations, it is rare for a buffer diluent to alter the biological activity of the compound. The compounds containing azelaic acid used herein can be administered to a human patient on their own, or as a pharmaceutical composition mixed with other ingredients, such as in combination therapy, or with suitable carriers or excipients.

[0134] In addition, the pharmaceutical composition for preventing or treating fibrotic diseases according to the present invention can be formulated and used in the form of external preparations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc. and sterile injection solutions according to conventional methods, and the antibacterial composition of the present invention can be administered orally or parenterally (for example, intravenously, subcutaneously, intraperitoneally, or topically) according to the intended method, and the dosage varies depending on the patient's condition and weight, the degree of the disease, the drug form, the route and period of administration, but can be appropriately selected by those skilled in the art, and for example, about 0.001 mg to 1000 mg can be administered in a form mixed with a pharmaceutically acceptable carrier. The antibacterial composition of the present invention can be administered once or several times a day as needed, and can be used alone or in combination with methods using surgery, hormone therapy, drug therapy, and biological response modifiers.

[0135] Meanwhile, the amino terminal of the recombinant fusion protein of the present invention may be bound with a protecting group such as an acetyl group, a fluorenyl methoxy carbonyl group, a formyl group, a palmitoyl group, a myristyl group, a stearyl group, and polyethylene glycol (PEG), and the carboxy terminal of the peptide may be modified with a hydroxyl group (-OH), an amino group (-NH2), an azide group (-NHNH2), and the like.

[0136] In addition, fatty acids, oligosaccharide chains, all nanoparticles (gold particles, liposomes, heparin, hydrogels, etc.), amino acids, carrier proteins, etc. can be bound to the terminal of the fusion protein of the present invention or the R-group of the amino acid. The above-described amino acid modification has the effect of improving the potency and stability of the protein of the present invention.

[0137] The term “stability” in this specification means not only in vivo stability but also storage stability (including storage stability at room temperature, refrigerated, and frozen storage).

[0138] Meanwhile, the fusion protein of the present invention can be provided as a gene encoding it so that it can be expressed within a cell.

[0139] In this specification, the term "gene" should be considered in the broadest sense and includes, but is not limited to, a DNA fragment encoding one or more fusion proteins selected from the group consisting of fusion proteins 1 to 37 of the present disclosure, which encode a structural protein or a regulatory protein, and may specifically include one of the base sequences of SEQ ID NOs: 1 to 37.

[0140] Additionally, as used herein, the term "vector" refers to a DNA construct containing a DNA sequence operably linked to a suitable regulatory sequence capable of expressing the DNA in a suitable host. The vector may be a plasmid, a phage particle, or simply a potential genomic insert. Once transformed into a suitable host, the vector can replicate and function independently of the host genome, or in some cases, can integrate into the genome itself. Since plasmids are currently the most commonly used form of vector, the terms "plasmid" and "vector" are sometimes used interchangeably herein.

[0141] A sequence is "operably linked" when it is placed into a functional relationship with another sequence. This can be a gene and regulatory sequence(s) that are linked in such a way that gene expression is enabled when an appropriate molecule (e.g., a transcriptional activating protein) binds to the regulatory sequence(s). For example, DNA for a presequence or secretory leader is operably linked to DNA for a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it influences the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it influences the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation. Generally, "operably linked" means that the linked DNA sequences are in contact, and in the case of a secretory leader, are in contact and are in reading frame. However, an enhancer need not be in contact. These sequences are joined by ligation at convenient restriction enzyme sites. If such sites do not exist, synthetic oligonucleotide adaptors or linkers are used according to conventional methods.

[0142] The vector according to the present invention may preferably be a plasmid vector, a cosmid vector, a viral vector, or the like.

[0143] The plasmid vector or cosmid vector of the present invention can be introduced into a host cell using a cationic lipid carrier or cationic polymer carrier, or using LNP or electroporation.

[0144] Cationic lipid delivery systems utilize the positive charge of nanometer-sized liposomes or lipid-based nanoparticles, which are mainly composed of cationic lipids, to form a complex with negatively charged ribozyme RNA or genes, expression vectors containing genes, or nucleic acids, and then deliver this complex into cells through phagocytosis. The complex delivered into the cell is first transported from the endosome to the lysosome and then released into the cytoplasm for expression. Cationic polymer delivery systems deliver ribozyme RNA or genes in a similar manner to cationic lipid delivery systems, except that they use polymers instead of lipids. Representative cationic polymers include polyethyleneimine, poly-L-lysine, and chitosan.

[0145] Therefore, the recombinant vector of the present invention can be used as a gene delivery vehicle in the form of a complex formed by combining with a cationic lipid carrier or a cationic polymer carrier.

[0146] Additionally, the fusion protein of the present invention can be provided as mRNA that can be expressed within a cell.

[0147] Additionally, as used herein, “transformation” or “transfection” means introducing DNA or RNA into a host so that the DNA becomes replicable as an extrachromosomal element or by chromosomal integration.

[0148] The fusion protein of the present invention is a combination of a retinol binding protein (RBP) and an albumin-derived peptide, wherein the albumin-derived peptide includes albumin domain IIIA and albumin domain IB. The albumin-derived peptide may be a peptide in which each albumin domain is connected by a linker consisting of 3 to 10 hydrophobic amino acids. In addition, the albumin-derived peptide may be a peptide in which one or more amino acids are substituted, modified, and / or deleted within a range in which the function thereof is maintained, and preferably, two or more amino acids may be substituted with cysteine ​​within a range in which the function thereof is maintained so that a disulfide bond can be added in the formation of the tertiary structure. Finally, in the case of the (N-terminal) - RBP - albumin domain IIIA - linker - albumin domain IB - (C-terminal) fusion protein, the RBP-derived peptide may be a peptide in which one or more amino acids are substituted, modified, and / or deleted within a range in which the function is maintained, and in particular, the RBP-derived peptide may be derived from RBP in which the 194th amino acid is substituted with lysine.

[0149] In this specification, the expression of a fusion protein in which a retinol binding protein and an albumin-derived peptide are combined is “albumin-RBP fusion protein”, “albumin fusion protein”, “albumin-RBP” or “albumin”, and in the case of “albumin fusion protein” and “albumin”, it means that RBP is combined at the N-terminus of the albumin-derived peptide, and in the case of “albumin-RBP fusion protein” and “albumin-RBP”, it is used to mean all fusion proteins in which RBP is combined at the N-terminus or C-terminus of the albumin-derived peptide, unless it is intended to indicate whether RBP is combined at the N- or C-terminus of the albumin-derived peptide.

[0150] In the fusion protein of the present invention, the positions of amino acids are described by numbering the positions of amino acids based on the wild-type protein or peptide from which each domain in the fusion protein is derived. For example, SEQ ID NO: 4 is an amino acid sequence of “albumin domain IIIA (404-515): 441Q”. At this time, albumin domain IIIA means a region including the 404 to 515 amino acid sequence of the albumin protein, and 441Q means that the albumin domain IIIA is derived from an albumin protein in which the 441st amino acid is glutamine (Q), and is a domain including the 404 to 515 amino acid sequence of the albumin protein. As another example, SEQ ID NO: 16 is an amino acid sequence of “: 194K”. At this time, 194K means that the RBP domain of SEQ ID NO: 16 is derived from an RBP in which the 194th amino acid is substituted with lysine (K).

[0151] In the fusion protein of the present invention, the RBP and albumin domains are derived from RBP and albumin, respectively, and refer to domains that exhibit the function that the fusion protein of the present invention seeks to achieve.

[0152] In the present invention, the amino acid sequence is described with the following abbreviations according to the IUPAC-IUB nomenclature.

[0153] Arginine (Arg, R), lysine (Lys, K), histidine (His, H), serine (Ser, S), threonine (Thr, T), glutamine (Gln, Q), asparagine (Asp, N), methionine (Met, M), leucine (Leu, L), isoleucine (Ile, I), valine (Val, V), phenylalanine (Phe, F), tryptophan (Trp, W), tyrosine (Tyr, Y), alanine (Ala, A), glycine (Gly, G), proline (Pro, P), cysteine ​​(Cys, C), aspartic acid (Asp, D), glutamic acid (Glu, E), norleucine (Nle)

[0154]

[0155] The present invention is susceptible to various modifications and embodiments. Specific embodiments are illustrated in the drawings and described in detail in the following detailed description. However, this is not intended to limit the present invention to specific embodiments, but rather to encompass all modifications, equivalents, and alternatives falling within the spirit and technical scope of the present invention. In describing the present invention, detailed descriptions of related known technologies will be omitted if they are deemed to obscure the gist of the present invention.

[0156]

[0157] [Example]

[0158] Example 1. Isolation and culture of stellate cells (SCs)

[0159] 1-1. Preparation of hepatic stellate cells (HSCs)

[0160] The livers of 14-week-old male BALB / c mice were first perfused with phosphate-buffered saline (PBS) and then with Gey's balanced salt solution (GBSS) containing collagenase, pronase, and DNase, and the livers were extracted. The gallbladder and connective tissue attached to the liver were removed, and the liver cell suspension was placed in the same solution as the GBSS and treated at 37°C for 12 minutes, followed by centrifugation at 1400 g for 20 minutes on a 13.4% Nycodenz gradient. The stellate cells at the interface of the Nycodenz solution and the aqueous layer were collected and cultured in Dulbecco's modified Eagle's medium (DMEM) (Carlsbad, CA) supplemented with 10% fetal bovine serum (FBS). The purity of stellate cells was assessed by microscopic observation and Western blotting using an anti-tyrosine aminotransferase antibody. Once the cells reached confluency in the dish, they were passaged and used as activated stellate cells. Activation of hepatic stellate cells was confirmed by morphological changes and increased expression of α-SMA and collagen I.

[0161]

[0162] Example 2. Design and production of fusion proteins

[0163] 2-1. Basics of fusion proteins

[0164] Albumin protein is composed of three structurally similar homologous domains (I, II & III), each of which is composed of two sub-domains (A & B).

[0165] Table 1 below shows the sequences of the domains used in the production of the albumin-RBP fusion protein by the inventors of the present invention. RBP is located at the N-terminus or C-terminus of albumin (IIIA+IB).

[0166] Sequence number name sequence (N-terminal → C-terminal) 1 RBP domain (19~194 aa or 19~195 aa of RBP including signal peptide) ERDCRVSSFRVKENFDKARFSGTWYAMAKKDPEGLFLQDNIVAEFSVDETGQMSATAKGRVRLLNNWDVCADMVGTFTDTEDPAKFKMKYWGVASFLQKGNDDHWIVDTDYDTYAVQYSCRLLNLDGTCADSYSFVFSRDPNGLPPEAQKIVRQRQEELCLARQYRLIVHNGYCDG(R) 2 Albumin domain IIIA (404~517 aa of albumin protein including signal peptide) aa)LVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEV3Albumin domain IB (131-218 aa of albumin protein including signal peptide or 134-218 aa)(DDN)PNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSA

[0167]

[0168] 2-2. Design of each domain and linker of the novel fusion protein

[0169] We used the protein structure modeling programs Swiss-Model and AlphaFold2 to investigate whether albumin structural stability could be improved through sequence modification.

[0170] Table 2 below shows the sequences of RBP, albumin domain, and linker used in the production of the newly improved albumin-RBP fusion protein of the present invention.

[0171] A peptide linker composed of hydrophobic amino acids was inserted between albumin domains IIIA and IB in the albumin-RBP fusion protein. The linker between domains IIIA and IB is mainly composed of hydrophobic amino acids (e.g., A, G, P), and in the experimental examples below, linkers 1 to 3 below were used as examples.

[0172]

[0173] Sequence number name sequence (N-terminal → C-terminal) 4 Albumin domain IIIA (404-515): 441QLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVST PTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQL CVLHEKTPVSDRVTKCCTES LVNRRPCFSAL 5 Albumin domain IIIA (404-515): C446-C487; 441QLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPCLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVCHEKTPVSDRVTKCCTESLVNRRPCFSAL6Albumin domain IIIA (404-515): C453-C480; 441QLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRCLGKVGSKCCKHPEAKRMPCAEDYLSVCLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSAL7Albumin domain IIIA (404-515): C457-C476; 441QLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKCGSKCCKHPEAKRMPCAEDCLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSAL8Albumin domain IIIA (404-515): C453-C480, C457-C476; 441QLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRCLGKCGSKCCKHPEAKRMPCAEDCLSVCLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSAL9Albumin domain IIIA (404-515): 441ELVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPEVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSAL 10Albumin domain IIIA (404-515): C446-C487;441ELVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPEVSTPCLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVCHEKTPVSDRVTKCCTESLVNRRPCFSAL11Albumin domain IIIA (404-515): C453-C480; 441ELVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPEVSTPTLVEVSRCLGKVGSKCCKHPEAKRMPCAEDYLSVCLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSAL12Albumin domain IIIA (404-515): C457-C476; 441ELVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPEVSTPTLVEVSRNLGKCGSKCCKHPEAKRMPCAEDCLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSAL13Albumin domain IIIA (404-515): C453-C480, C457-C476;441ELVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPEVSTPTLVEVSRCLGKCGSKCCKHPEAKRMPCAEDCLSVCLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSAL14Albumin domain IB (133-218)NPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSA15Albumin domain IB (133-218) : C144-C199NPNLPRLVRPECDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKCACLLPKLDELRDEGKASSA16RBP : 194KERDCRVSSFRVKENFDKARFSGTWYAMAKKDPEGLFLQDNIVAEFSVDETGQMSATAKGRVRLLNNWDVCADMVGTFTDTEDPAKFKMKYWGVASFLQKGNDDHWIVDTDYDTYAVQYSCRLLNLDGTCADSYSFVFSRDPNGLPPEAQKIVRQRQEELCLARQYRLIVHNGYCDK(R)17Linker 1AAAA18Linker 2GGPA19Linker 3DGPG;

[0174]

[0175] The structure of the novel albumin-RBP fusion protein was designed as follows.

[0176] - RBP-albumin (IIIA-linker-IB)

[0177] - Albumin (IIIA-linker-IB)-RBP

[0178] Six types of albumin-RBP fusion proteins were produced (Table 3).

[0179] In Table 3 below, RBP domains are underlined and linkers are bold.

[0180]

[0181] Sequence number structure sequence (N-terminal → C-terminal) 20 Fusion protein 1: RBP-albumin (IIIA-DGPG-IB); 441EERDCRVSSFRVKENFDKARFSGTWYAMAKKDPEGLFLQDNIVAEFSVDETGQMSATAKGRVRLLNNWDVCADMVGTFTDTEDPAKFKMKYWGVASFLQKGNDDHWIVDTDYDTYAVQYSCRLLNLDGTCADSYSFVFSRDPNGLPPEAQKIVRQRQEELCLARQYRLIVHNGYCD GRLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPEVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRR PCFSALDGPGNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSA21Fusion Protein2:RBP -Albumin (IIIA-GGPA-IB);441EERDCRVSSFRVKENFDKARFSGTWYAMAKKDPEGLFLQDNIVAEFSVDETGQMSATAKGRVRLLNNWDVCADMVGTFTDTEDPAK FKMKYWGVASFLQKGNDDHWIVDTDYDTYAVQYSCRLLNNLDGTCADSYSFVFSRDPNGLPPEAQKIVRQRQEELCLARQYRLIVHNGYCDGRLVEEPQNLIKQNCELF EQLGEYKFQNALLVRYTKKVPEVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALGGPANPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSA22 fusion protein 3:RBP-albumin (IIIA-AAAA-IB);441EERDCRVSSFRVKENFDKARFSGTWYAMAKKDPEGLFLQDNIVAEFSVDETGQMSATAKGRVRLLNNWDVCADMVGTFTDTEDPAKFKMKYWGVASFLQKGNDDHWIVDTDYDTYAVQYSCRLLNLDGTCADSYSFVFSRDPNGLPPEAQKIVRQRQEELCLARQYRLIVHNGYCDGRLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPEVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALAAAANPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSA23융합단백질4:알부민(IIIA-DGPG-IB)-RBP;441ELVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPEVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALDGPGNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAERDCRVSSFRVKENFDKARFSGTWYAMAKKDPEGLFLQDNIVAEFSVDETGQMSATAKGRVRLLNNWDVCADMVGTFTDTEDPAKFKMKYWGVASFLQKGNDDHWIVDTDYDTYAVQYSCRLLNLDGTCADSYSFVFSRDPNGLPPEAQKIVRQRQEELCLARQYRLIVHNGYCDG24융합단백질5:알부민(IIIA-GGPA-IB)-RBP;441ELVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPEVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALGGPANPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRD EGKASSAERDCRVSSFRVKENFDKARFSGTWYAMAKKDPEGLFLQDNIVAEFSVDETGQMSATAKGRVRLLNNWDVCADMVGTFTDTEDPAKFKMKYWGVASFLQKGNDDHWIVDTDYDTYAVQYSCRLLNLDGTCADSYSFVFSRDPNGLPPEAQKIVRQRQEELCLARQYRLIVHNGYCDG25 fusion protein 6: albumin (III A-AAAA-IB)-RBP:441ELVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPEVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQ LCVLHEKTPVSDRVTKCCTESLVNRRPCFSALAAAANPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAA DKAACLLPKLDELRDEGKASSAERDCRVSSFRVKENFDKARFSGTWYAMAKKDPEGLFLQDNIVAEFSVDETGQMSATAKGRVRLLNNWDVCADMVGTF TDTEDPAKFKMKYWGVASFLQKGNDDHWIVDTDYDTYAVQYSCLLLNLDGTCADSYSFVFSRDPNGLPPEAQKIVRQRQEELCLARQYRLIVHNGYCDG;

[0182]

[0183] Fusion proteins 7-12 were produced using an albumin-derived domain in which the 441st amino acid in the above fusion proteins 1-6 was substituted with glutamine (Table 4).

[0184]

[0185] Sequence number structure sequence (N-terminal → C-terminal) 26 fusion protein 7: RBP-albumin (IIIA-DGPG-IB); 441QERDCRVSSFRVKENFDKARFSGTWYAMAKKDPEGLFLQDNIVAEFSVDETGQMSATAKGRVRLLNNWDVCADMVGTFTDTEDPAKFKMKYWGVASFLQKGNDDHWIVDTDYDTYAVQYSCRLLNLDGTCADSYSFVFSRDPNGLPPEAQKIVRQRQEELCLARQYRLIVHNGYCD GRLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRR PCFSALDGPGNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSA27Fusion Protein 8:RBP -Albumin (IIIA-GGPA-IB);441QERDCRVSSFRVKENFDKARFSGTWYAMAKKDPEGLFLQDNIVAEFSVDETGQMSATAKGRVRLLNNWDVCADMVGTFTDTEDPAK FKMKYWGVASFLQKGNDDHWIVDTDYDTYAVQYSCRLLNNLDGTCADSYSFVFSRDPNGLPPEAQKIVRQRQEELCLARQYRLIVHNGYCDGRLVEEPQNLIKQNCELF EQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALGGPANPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSA28 fusion protein 9:RBP-albumin (IIIA-AAAA-IB);441QERDCRVSSFRVKENFDKARFSGTWYAMAKKDPEGLFLQDNIVAEFSVDETGQMSATAKGRVRLLNNWDVCADMVGTFTDTEDPAKFKMKYWGVASFLQKGNDDHWIVDTDYDTYAVQYSCRLLNLDGTCADSYSFVFSRDPNGLPPEAQKIVRQRQEELCLARQYRLIVHNGYCDGRLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALAAAANPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSA29융합단백질10:알부민(IIIA-DGPG-IB)-RBP;441QLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALDGPGNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAERDCRVSSFRVKENFDKARFSGTWYAMAKKDPEGLFLQDNIVAEFSVDETGQMSATAKGRVRLLNNWDVCADMVGTFTDTEDPAKFKMKYWGVASFLQKGNDDHWIVDTDYDTYAVQYSCRLLNLDGTCADSYSFVFSRDPNGLPPEAQKIVRQRQEELCLARQYRLIVHNGYCDG30융합단백질11:알부민(IIIA-GGPA-IB)-RBP;441QLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALGGPANPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPCLDELRD EGKASSAERDCRVSSFRVKENFDKARFSGTWYAMAKKDPEGLFLQDNIVAEFSVDETGQMSATAKGRVRLLNNWDVCADMVGTFTDTEDPAKFKMKYWGVASFLQKGNDDHWIVDTDYDTYAVQYSCRLLNLDGTCADSYSFVFSRDPNGLPPEAQKIVRQRQEELCLARQYRLIVHNGYCDG31챠합단백에12:아부민(II IA-AAAA-IB)-RBP:441QLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALAAAANPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQA ADKAACLLPCLDELRDEGKASSAERDCRVSSFRVKENFDKARFSGTWYAMAKKDPEGLFLQDNIVAEFSVDETGQMSATAKGRVRLLNNWDVCADMVGTFTDTEDPAKFKMKYWGVASFLQKGNDDHWIVDTDYDTYAVQYSCRLLNLDGTCADSYSFVFSRDPNGLPPEAQKIVRQRQEELCLARQYRLIVHNGYCDG;

[0186]

[0187] Polynucleotides encoding fusion proteins 1 to 12, including signal peptides designed for the production of fusion proteins, were constructed. For the RBP-albumin fusion protein, MKWVWALLLLAAWAAA (SEQ ID NO: 94) was used as the signal peptide, and for the albumin-RBP fusion protein, MKWVTFISLLFLFSSAYS (SEQ ID NO: 95) was used as the signal peptide. Each DNA fragment was cloned into the XbaI / KpnI cut pcDNA3.1(+) vector to produce a recombinant expression vector. Specific information on the DNA fragment encoding each fusion protein, including the signal peptide, is summarized in Tables 5 and 6 below. Each vector was introduced into Expi293 cells using Lipofectamine 2000 (Invitrogen, Carlsbad, CA) to produce transformants expressing the fusion proteins. The transformant was cultured and the culture supernatant was analyzed by Western blot to confirm the level of expression of the fusion protein.

[0188] As a result, high expression of RBP-albumin(IIIA-DGPG-IB);441E, RBP-albumin(IIIA-GGPA-IB;441E), RBP-albumin(IIIA-EVDD-IB);441E, and RBP-albumin(IIIA-AAAA-IB);441E was confirmed (Fig. 1), and in particular, the expression of RBP-albumin(IIIA-AAAA-IB);441E was high (lane 5).

[0189] Polynucleotides encoding the fusion proteins of Tables 3 and 4 above are as shown in Tables 5 and 6 below, respectively.

[0190]

[0191]

[0192]

[0193]

[0194]

[0195] 2-3. Design and fabrication of fusion proteins with additional CC bonds

[0196] Albumin domain IIIA contains four disulfide bonds: C416-C462, C461-C472, C485-C501, and C500-C511. To enhance protein structural stability, the inventors substituted V457 and Y476 with cysteine ​​(C) to induce additional disulfide bond formation. For the same reason, N453 and V480, or T446 and L487, were substituted with C. By substituting amino acids with C, one or two additional disulfide bonds were introduced into the fusion protein, thereby improving protein structural stability.

[0197] Albumin domain IB has two disulfide bonds, C148-C193 and C192-C201, but V144 and A199 were substituted with C to induce additional disulfide bond formation to improve protein structural stability.

[0198] Eight types of albumin-RBP fusion proteins were produced (Table 7).

[0199]

[0200] Sequence number structure sequence (N-terminal → C-terminal) 44 fusion protein 13: RBP-albumin (IIIA-AAAA-IB): C144-C199; 441 EERDCRVSSFRVKENFDKARFSGTWYAMAKKDPEGLFLQDNIVAEFSVDETGQMSATAKGRVRLLNNWDVCADMVGTFTDTEDPAKFKMKYWGVASFLQKGNDDHWIVDTDYDTYAVQYSCRLLNLDGTCADSYSFVFSRDPNGLPPEAQKIVRQRQEELCLARQYRLIVHNG YCDGRLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPEVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALAAAANPNLPRLVRPECDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKCACLLPKLDELRDEGKASSA45 fusion protein 14:RBP-albumin( IIIA-AAAA-IB):C446-C487;441EERDCRVSSFRVKENFDKARFSGTWYAMAKKDPEGLFLQDNIVAEFSVDETGQMSATAKGRVRLLNNWDVCADMVGTFTDTEDPA KFKMKYWGVASFLQKGNDDHWIVDTDYDTYAVQYSCRLLNNLDGTCADSYSFVFSRDPNGLPPEAQKIVRQRQEELCLARQYRLIVHNGYCDGRLVEEPQNLIKQNCELFEQL GEYKFQNALLVRYTKKVPEVSTPCLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVCHEKTPVSDRVTKCCTESLVNRRPCFSALAAAANPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSA46 fusion protein 15:RBP-albumin (IIIA-AAAA-IB): C453-C480;441EERDCRVSSFRVKENFDKARFSGTWYAMAKKDPEGLFLQDNIVAEFSVDETGQMSATAKGRVRLLNNWDVCADMVGTFTDTEDPAKFKMKYWGVASFLQKGND DHWIVDTDYDTYAVQYSCRLLLNLDGTCADSYSFVFSRDPNGLPPEAQKIVRQRQEELCLARQYRLIVHNGYCDGRLVEEPQNLIKQNCELFEQLGEYKFQNALLVR YTKKVPEVSTPTLVEVSRCLGKVGSKCCKHPEAKRMPCAEDYLSVCLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALAAAANPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSA47 fusion protein 16:RBP-albumin (IIIA-AAAA-IB): C457-C476; 441EERDCRVSSFRVKENFDKARFSGTWYAMAKKDPEGLFLQDNIVAEFSVDETGQMSATAKGRVRLLNNWDVCADMVGTFTDTEDPAKFKMKYWGVASFLQKGND DHWIVDTDYDTYAVQYSCRLLLNLDGTCADSYSFVFSRDPNGLPPEAQKIVRQRQEELCLARQYRLIVHNGYCDGRLVEEPQNLIKQNCELFEQLGEYKFQNALLVR YTKKVPEVSTPTLVEVSRNLGKCGSKCCKHPEAKRMPCAEDCLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALAAAANPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSA48 fusion protein 17: albumin (IIIA-AAAA-IB)-RBP: C144-C199;441ELVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPEVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESL VNRRPCFSALAAAANPNLPRLVRPECDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKCACLLPKLDELRDEGKASSAERDCRV SSFRVKENFDKARFSGTWYAMAKKDPEGLFLQDNIVAEFSVDETGQMSATAKGRVRLLNNWDVCADMVGTFTDTEDPAKFKMKYWGVASFLQKGNDDHWIVDTDYDTYAVQYSCRLLNLDGTCADSYSFVFSRDPNGLPPEAQKIVRQRQEELCLARQYRLIVHNGYCDG49 fusion protein 18: albumin (IIIA-AAAA-IB)-RBP: C446-C487; 441ELVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPEVSTPCLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVCHEKTPVSDRVTKCCTESL VNRRPCFSALAAAANPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAERDCRV SSFRVKENFDKARFSGTWYAMAKKDPEGLFLQDNIVAEFSVDETGQMSATAKGRVRLLNNWDVCADMVGTFTDTEDPAKFKMKYWGVASFLQKGNDDHWIVDTDYDTYAVQYSCRLLNLDGTCADSYSFVFSRDPNGLPPEAQKIVRQRQEELCLARQYRLIVHNGYCDG50Fusion protein 19:Albumin(IIIA-AAAA-IB)-RBP:C453-C480;441ELVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPEVSTPTLVEVSRCLGKVGSKCCKHPEAKRMPCAEDYLSVCLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALAAAANPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEG KASSAERDCRVSSFRVKENFDKARFSGTWYAMAKKDPEGLFLQDNIVAEFSVDETGQMSATAKGRVRLLNNWDVCADMVGTFTDTEDPAKFKMKYWGVASFLQKGNDDHWIVDTDYDTYAVQYSCRLLNLDGTCADSYSFVFSRDPNGLPPEAQKIVRQRQEELCLARQYRLIVHNGYCDG51 fusion protein 20: albumin (IIIA-AA) AA-IB)-RBP:C457-C476;441ELVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPEVSTPTLVEVSRNLGKCGSKCCKHPEAKRMPCAEDCLSV VLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALAAAANPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECC QAADKAACLLPKLDELRDEGKASSAERDCRVSSFRVKENFDKARFSGTWYAMAKKDPEGLFLQDNIVAEFSVDETGQMSATAKGRVRLLNNWDVCADMVGT FTDTEDPAKFKMKYWGVASFLQKGNDDHWIVDTDYDTYAVQYSCRLLLNLDGTCADSYSFVFSRDPNGLPPEAQKIVRQRQEELCLARQYRLIVHNGYCDG;

[0201]

[0202] Fusion protein 21-36 was produced using a domain derived from albumin in which amino acid position 441 of the above fusion protein 13-20 was substituted with glutamine (Table 8).

[0203]

[0204] Sequence number structure sequence (N-terminal → C-terminal) 52 fusion protein 21: RBP-albumin (IIIA-AAAA-IB): C144-C199; 441QERDCRVSSFRVKENFDKARFSGTWYAMAKKDPEGLFLQDNIVAEFSVDETGQMSATAKGRVRLLNNWDVCADMVGTFTDTEDPAKFKMKYWGVASFLQKGNDDHWIVDTDYDTYAVQYSCRLLNLDGTCADSYSFVFSRDPNGLPPEAQKIVRQRQEELCLARQYRLIVHNG YCDGRLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALAAAANPNLPRLVRPECDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKCACLLPKLDELRDEGKASSA53 fusion protein 22:RBP-albumin( IIIA-AAAA-IB):C446-C487;441QERDCRVSSFRVKENFDKARFSGTWYAMAKKDPEGLFLQDNIVAEFSVDETGQMSATAKGRVRLLNNWDVCADMVGTFTDTEDPA KFKMKYWGVASFLQKGNDDHWIVDTDYDTYAVQYSCRLLNNLDGTCADSYSFVFSRDPNGLPPEAQKIVRQRQEELCLARQYRLIVHNGYCDGRLVEEPQNLIKQNCELFEQL GEYKFQNALLVRYTKKVPQVSTPCLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVCHEKTPVSDRVTKCCTESLVNRRPCFSALAAAANPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSA54 fusion protein 23:RBP-albumin (IIIA-AAAA-IB): C453-C480;441QERDCRVSSFRVKENFDKARFSGTWYAMAKKDPEGLFLQDNIVAEFSVDETGQMSATAKGRVRLLNNWDVCADMVGTFTDTEDPAKFKMKYWGVASFLQKGND DHWIVDTDYDTYAVQYSCRLLLNLDGTCADSYSFVFSRDPNGLPPEAQKIVRQRQEELCLARQYRLIVHNGYCDGRLVEEPQNLIKQNCELFEQLGEYKFQNALLVR YTKKVPQVSTPTLVEVSRCLGKVGSKCCKHPEAKRMPCAEDYLSVCLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALAAAANPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSA55 fusion protein 24:RBP-albumin (IIIA-AAAA-IB): C457-C476; 441QERDCRVSSFRVKENFDKARFSGTWYAMAKKDPEGLFLQDNIVAEFSVDETGQMSATAKGRVRLLNNWDVCADMVGTFTDTEDPAKFKMKYWGVASFLQKGND DHWIVDTDYDTYAVQYSCRLLLNLDGTCADSYSFVFSRDPNGLPPEAQKIVRQRQEELCLARQYRLIVHNGYCDGRLVEEPQNLIKQNCELFEQLGEYKFQNALLVR YTKKVPQVSTPTLVEVSRNLGKCGSKCCKHPEAKRMPCAEDCLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALAAAANPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSA56 fusion protein 25: albumin (IIIA-AAAA-IB)-RBP: C144-C199;441QLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESL VNRRPCFSALAAAANPNLPRLVRPECDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKCACLLPKLDELRDEGKASSAERDCRV SSFRVKENFDKARFSGTWYAMAKKDPEGLFLQDNIVAEFSVDETGQMSATAKGRVRLLNNWDVCADMVGTFTDTEDPAKFKMKYWGVASFLQKGNDDHWIVDTDYDTYAVQYSCRLLNLDGTCADSYSFVFSRDPNGLPPEAQKIVRQRQEELCLARQYRLIVHNGYCDG57Fusion protein 26:Albumin(IIIA-AAAA-IB)-RBP:C446-C487; 441QLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPCLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVCHEKTPVSDRVTKCCTESL VNRRPCFSALAAAANPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAERDCRV SSFRVKENFDKARFSGTWYAMAKKDPEGLFLQDNIVAEFSVDETGQMSATAKGRVRLLNNWDVCADMVGTFTDTEDPAKFKMKYWGVASFLQKGNDDHWIVDTDYDTYAVQYSCRLLNLDGTCADSYSFVFSRDPNGLPPEAQKIVRQRQEELCLARQYRLIVHNGYCDG58Fusion protein 27:Albumin(IIIA-AAAA-IB)-RBP:C453-C480;441QLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRCLGKVGSKCCKHPEAKRMPCAEDYLSVCLNQLCVLHEKTPVSDRVTK CCTESLVNRRPCFSALAAAANPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEG KASSAERDCRVSSFRVKENFDKARFSGTWYAMAKKDPEGLFLQDNIVAEFSVDETGQMSATAKGRVRLLNNWDVCADMVGTFTDTEDPAKFKMKYWGVASFLQKGNDDHWIVDTDYDTYAVQYSCRLLNLDGTCADSYSFVFSRDPNGLPPEAQKIVRQRQEELCLARQYRLIVHNGYCDG59 fusion protein 28: albumin (IIIA-AA) AA-IB)-RBP:C457-C476;441QLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKCGSKCCKHPEAKRMPCAEDCLSV VLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALAAAANPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECC QAADKAACLLPKLDELRDEGKASSAERDCRVSSFRVKENFDKARFSGTWYAMAKKDPEGLFLQDNIVAEFSVDETGQMSATAKGRVRLLNNWDVCADMVGT FTDTEDPAKFKMKYWGVASFLQKGNDDHWIVDTDYDTYAVQYSCRLLLNLDGTCADSYSFVFSRDPNGLPPEAQKIVRQRQEELCLARQYRLIVHNGYCDG;

[0205]

[0206] Fusion protein 29-22 was constructed with two additional disulfide bonds introduced (Table 9).

[0207]

[0208] Sequence number structure sequence (N-terminal → C-terminal) 60 fusion protein 29: RBP-albumin (IIIA-AAAA-IB): C144-C199, C453-C480; 441 QERDCRVSSFRVKENFDKARFSGTWYAMAKKDPEGLFLQDNIVAEFSVDETGQMSATAKGRVRLLNNWDVCADMVGTFTDTEDPAKFKMKYWGVASFLQKGNDDHWIVDTDYDTYAVQYSCRLLNLDGTCADSYSFVFSRDPNGLPPEAQKIVRQRQEELCLARQYRLIVH NGYCDGRLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRCLGKVGSKCCKHPEAKRMPCAEDYLSVCLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALAAAANPNLPRLVRPECDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKCACLLPKLDELRDEGKASSA61 fusion protein 30:RBP-albumin (IIIA- AAAA-IB):C453-C480,C457-C476;441QERDCRVSSFRVKENFDKARFSGTWYAMAKKDPEGLFLQDNIVAEFSVDETGQMSATAKGRVRLLNNWDVCADMVGTFTDTEDP AKFKMKYWGVASFLQKGNDDHWIVDTDYDTYAVQYSCRLLNLDGTCADSYSFVFSRDPNGLPPEAQKIVRQRQEELCLARQYRLIVHNGYCDGRLVEEPQNLIKQNCELFEQLGEY KFQNALLVRYTKKVPQVSTPTLVEVSRCLGKCGSKCCKHPEAKRMPCAEDCLSVCLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALAAAANPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSA62 Fusion protein 31: Albumin (IIIA-AAAA-IB)-RBP: C144-C199, C453-C480;441QLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRCLGKVGSKCCKHPEAKRMPCAEDYLSVCLNQLCVLHEKTPVSDRVTKCC TESLVNRRPCFSALAAAANPNLPRLVRPECDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKCACLLPKLDELRDEGKAS SAERDCRVSSFRVKENFDKARFSGTWYAMAKKDPEGLFLQDNIVAEFSVDETGQMSATAKGRVRLLNNWDVCADMVGTFTDTEDPAKFKMKYWGVASFLQKGNDDHWIVDTDYDTYAVQYSCRLLNLDGTCADSYSFVFSRDPNGLPPEAQKIVRQRQEELCLARQYRLIVHNGYCDG63 fusion protein 32: albumin (IIIA-AAAA-IB )-RBP:C453-C480,C457-C476;441QLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRCLGKCGSKCCKHPEAKRMPCAED CLSVCLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALAAAANPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFT ECCQAADKAACLLPKLDELRDEGKASSAERDCRVSSFRVKENFDKARFSGTWYAMAKKDPEGLFLQDNIVAEFSVDETGQMSATAKGRVRLLNNWDVCADMV GTFTDTEDPAKFKMKYWGVASFLQKGNDDHWIVDTDYDTYAVQYSCRLLLNLDGTCADSYSFVFSRDPNGLPPEAQKIVRQRQEELCLARQYRLIVHNGYCDG;

[0209]

[0210] The vector expressing the above fusion protein 13-32 (Table 7-9) was prepared in the same manner as the preparation of the fusion protein 1-12 expression vector in Example 2-2.

[0211] Polynucleotides encoding the fusion proteins of Table 7-9 linked to the signal peptides are as shown in Table 10-12 below, respectively.

[0212]

[0213]

[0214]

[0215]

[0216]

[0217]

[0218] 2-4. Design and production of a fusion protein using an RBP domain derived from RBP with lysine at position 194.

[0219] To improve the structural stability of the (N-terminal) - RBP - albumin domain IIIA - linker - albumin domain IB - (C-terminal) fusion protein, the present inventors developed albumin-RBP fusion protein variants (fusion proteins 33 to 37) in which the 194th amino acid of RBP was substituted with lysine.

[0220] A total of five albumin-RBP fusion proteins were produced (Table 13).

[0221]

[0222] Sequence number structure sequence (N-terminal → C-terminal) 84 fusion protein 33: RBP-albumin (IIIA-EVDD-IB); 441Q; 194KERDCRVSSFRVKENFDKARFSGTWYAMAKKDPEGLFLQDNIVAEFSVDETGQMSATAKGRVRLLNNWDVCADMVGTFTDTEDPAKFKMKYWGVASFLQKGNDDHWIVDTDYDTYAVQYSCRLLNLDGTCADSYSFVFSRDPNGLPPEAQKIVRQRQEELCLARQYRLIVHNGYCDKR LVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSA85 fusion protein 34:RBP-albumin(III A-AAAA-IB);441Q;194KERDCRVSSFRVKENFDKARFSGTWYAMAKKDPEGLFLQDNIVAEFSVDETGQMSATAKGRVRLLNNWDVCADMVGTFTDTEDPAKFKMKYW GVASFLQKGNDDHWIVDTDYDTYAVQYSCRLLNLDGTCADSYSFVFSRDPNGLPPEAQKIVRQRQEELCLARQYRLIVHNGYCDKRLVEEPQNLIKQNCELFEQLGEYKFQN ALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALAAAANPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSA86Fusion protein 35:RBP-albumin(IIIA-AAAA-IB):C453-C480;441Q;194KERDCRVSSFRVKENFDKARFSGTWYAMAKKDPEGLFLQDNIVAEFSVDETGQMSATAKGRVRLLNNWDVCADMVGTFTDTEDPAKFKMKYWGVASFLQKGNDDHWI VDTDYDTYAVQYSCLLLNLDGTCADSYSFVFSRDPNGLPPEAQKIVRQRQEELCLARQYRLIVHNGYCDKRLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQV STPTLVEVSRCLGKVGSKCCKHPEAKRMPCAEDYLSVCLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALAAAANPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSA87Fusion protein 36:RBP-albumin (IIIA-AAAA-IB):C144-C199,C453-C480;441Q; 194KERDCRVSSFRVKENFDKARFSGTWYAMAKKDPEGLFLQDNIVAEFSVDETGQMSATAKGRVRLLNNWDVCADMVGTFTDTEDPAKFKMKYWGVASFLQKGNDDHWI VDTDYDTYAVQYSCLLLNLDGTCADSYSFVFSRDPNGLPPEAQKIVRQRQEELCLARQYRLIVHNGYCDKRLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQV STPTLVEVSRCLGKVGSKCCKHPEAKRMPCAEDYLSVCLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALAAAANPNLPRLVRPECDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKCACLLPKLDELRDEGKASSA88Fusion protein 37:RBP-albumin (IIIA-AAAA-IB):C453-C480,C457-C476;441Q;194KERDCRVSSFRVKENFDKARFSGTWYAMAKKDPEGLFLQDNIVAEFSVDETGQMSATAKGRVRLLNNWDVCADMVGTFTDTEDPAKFKMKYWG VASFLQKGNDDHWIVDTDYDTYAVQYSCRLLLNLDGTCADSYSFVFSRDPNGLPPEAQKIVRQRQEELCLARQYRLIVHNGYCDKRLVEEPQNLIKQ NCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRCLGKCGSKCCKHPEAKRMPCAEDCLSVCLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALAAAANPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSA;

[0223]

[0224] The polynucleotides encoding the fusion proteins of Table 13 linked to the signal peptides are as shown in Table 14 below.

[0225]

[0226]

[0227]

[0228] Example 3. Comparison of the effects of fusion proteins 1 to 6 and conventional albumin-RBP fusion proteins in astrocytes.

[0229] The effects of RBP-albumin (IIIA-IIIB), which showed excellent effects in inhibiting or deactivating the activation of stellate cells in previous studies, and fusion proteins 1 to 6 produced in Example 2-2 were compared.

[0230] Specifically, after the expression vector of Example 2-2 was transduced into activated astrocytes, the RNA levels of alpha-smooth muscle actin (α-SMA) and collagen type I, which are astrocyte activation markers, were analyzed using qPCR. As a result, a decrease in the expression of α-SMA and collagen was confirmed, although the degree varied (Fig. 2). In other words, the expression of the above fusion protein induced astrocyte deactivation.

[0231]

[0232] Example 4. Analysis of the effect of fusion proteins 7-12, which are E441Q derivatives of fusion proteins 1-6.

[0233] In the same manner as in Example 3, activated astrocytes were transduced with expression vectors of fusion proteins 7-12, and then the RNA levels of α-SMA and collagen type I, markers of astrocyte activation, were analyzed using qPCR. As a result, it was confirmed that fusion proteins 7 to 12 reduced the expression of α-SMA and collagen in astrocytes to a level similar to that of fusion proteins 1 to 6 (Fig. 3).

[0234]

[0235] Example 5. In vitro assay for RBP-albumin (IIIA-AAAA-IB) activity

[0236] Considering the expression level in Expi293 cell line and the degree of activity in astrocytes, fusion protein 3 {RBP-albumin (IIIA-AAAA-IB)} was selected among the above fusion proteins, and the fusion protein with a purity of >95% was secured through an affinity and size exclusion chromatography purification process. First, the previously developed fusion protein RBP-albumin (IIIA-IIIB) and the novel fusion protein 3 {RBP-albumin (IIIA-AAAA-IB)} were treated with activated astrocytes for 20 hours (at concentrations of 0.75 and 0.375 μM), and then the RNA levels of α-SMA and collagen type I were examined using qPCR.

[0237] As a result, it was confirmed that the fusion protein 3 {RBP-albumin (IIIA-AAAA-IB)} effectively induced astrocyte inactivation at a higher level compared to the previously developed fusion protein (Fig. 4).

[0238]

[0239] Example 6. In vivo assay for RBP-albumin (IIIA-AAAA-IB) activity

[0240] A liver fibrosis mouse model was prepared by inducing liver damage by intraperitoneally injecting BALB / c mice with CCl4 dissolved in a 1:1 ratio in mineral oil at a concentration of 1 mL / kg three times a week for 7 weeks. Five weeks after the CCl4 intraperitoneal injection, 15 or 7.5 μg of fusion protein 3 was injected via tail vein three times a week for 2 weeks. The control group received the same amount of PBS alone. 48 hours after the final CCl4 injection, the mice were sacrificed, and the livers were removed, fixed in formalin, embedded in paraffin, and prepared for tissue sections. Each tissue section was stained with H&E and Sirius red for histological analysis and observed under a light microscope (Fig. 5).

[0241] As a result, it was confirmed that fusion protein 3 {RBP-albumin (IIIA-AAAA-IB)} statistically significantly improved liver fibrosis (Fig. 6).

[0242]

[0243] Example 7. Confirmation of the action of fusion proteins 13 to 20 in astrocytes.

[0244] 7-1. Morphological changes in astrocytes

[0245] Among the fusion proteins 13-20 that additionally induced CC disulfide bonds in the albumin domain, experiments were performed on RBP-albumin fusion proteins. Among the RBP-albumin fusion proteins, expression vectors for fusion proteins 13-16 in which V144-A199, T446-L487, N453-V480, or V457-Y476 were substituted with cysteine ​​for fusion protein 3 {RBP-albumin (IIIA-AAAA-IB)} in which each domain of albumin is connected by linker 1 were constructed, and after transduction into activated stellate cells, morphological changes in the cells were observed.

[0246] As a result, compared to fusion protein 3 {RBP-(albuminIIA-AAAA-IB)}, the cell morphological changes caused by the expression of fusion protein 15 {RBP-albumin(IIIA-AAAA-IB):C453-C480} and fusion protein 16 {RBP-albumin(IIIA-AAAA-IB):C457-C476} were more prominent (Fig. 7).

[0247]

[0248] 7-2. Changes in stellate cell activation markers

[0249] Next, qPCR was performed to analyze the RNA levels of α-SMA, a marker of stellate cell activation, and collagen type I. As a result, a decrease in α-SMA and collagen expression was confirmed, and in particular, the decrease due to the expression of fusion protein 15 {RBP-albumin (IIIA-AAAA-IB): C453-C480} was prominent (Fig. 8).

[0250]

[0251] Example 8. Confirmation of the action of fusion proteins 21 to 28 in astrocytes.

[0252] Among the fusion proteins 21-28, which are E441Q derivatives of fusion proteins 13-20, expression vectors for fusion proteins 21 to 24 were constructed and transduced into activated astrocytes. Then, qPCR was performed to analyze the RNA levels of α-SMA and collagen type I, which are astrocyte activation markers. As a result, a decrease in the expression of α-SMA and collagen was confirmed, and similar to the results of Example 7-2, a decrease in the expression of α-SMA and collagen was confirmed (Fig. 9).

[0253]

[0254] Example 9. Confirmation of the action of fusion proteins 29 to 32 in astrocytes.

[0255] Next, the expression vector for the fusion protein 29-32, which had two additional disulfide bonds introduced, was transduced into activated astrocytes, and the RNA levels of α-SMA and collagen type I were analyzed. As a result, it was confirmed that the expression of α-SMA and collagen type I was reduced in all transduced cells. In particular, the decrease in the expression of astrocyte activation markers due to the introduction of the fusion protein 30 {RBP-albumin(IIIA-AAAA-IB):C453-C480,C457-C476;441Q} was remarkable (Fig. 10).

[0256]

[0257] Example 10. Confirmation of the action of fusion proteins 33 to 37 in astrocytes.

[0258] 10-1. Morphological changes in astrocytes

[0259] Expression vectors for RRBP-albumin (IIIA-EVDD-IB), fusion protein 9 {RBP-albumin (IIIA-AAAA-IB)}, and G194K derivatives of fusion proteins 23, 29, and 30, fusion proteins 33 to 37, were constructed and transduced into activated stellate cells. The morphological changes of the transformed cells were observed. As a result, changes in cell morphology, such as oil droplet re-formation, due to expression of fusion proteins 33 to 37 were observed (Fig. 11).

[0260]

[0261] 10-2. Changes in stellate cell activation markers

[0262] Next, qPCR was performed to analyze the RNA levels of α-SMA, a marker of stellate cell activation, and collagen type I. As a result, a decrease in α-SMA and collagen expression was confirmed, and in particular, the decrease due to the expression of fusion proteins 34 {RBP-albumin(IIIA-AAAA-IB):441Q;194K} and 35 {RBP-albumin(IIIA-AAAA-IB):C453-C480;441Q;194K} was prominent (Fig. 12).

[0263]

[0264] 10-3. Comparison of expression productivity of fusion proteins

[0265] Vectors corresponding to fusion proteins 33-35 and 37 were introduced into Expi293 cells using Lipofectamine 2000 (Invitrogen, Carlsbad, CA), and the transformants were cultured. The culture supernatant was analyzed by Western blot to confirm the expression level of the fusion proteins.

[0266] Compared with RBP-albumin(IIIA-AAAA-IB):C453-480;441E, high expression of RBP-albumin(IIIA-AAAA-IB):C453-480;441Q;194K and RBP-albumin(IIIA-AAAA-IB):C453-480,C457-476;441Q;194K was confirmed (Fig. 13).

[0267]

[0268] Although the embodiments described above have been described with limited drawings, those skilled in the art will appreciate that various technical modifications and variations can be applied based on the above. For example, appropriate results can still be achieved even if the described techniques are performed in a different order than described, and / or components of the described systems, structures, devices, circuits, etc. are combined or combined in a different manner than described, or are replaced or substituted with other components or equivalents.

[0269] Therefore, other implementations, other embodiments, and equivalents to the claims also fall within the scope of the claims described below.

Claims

1. A fusion protein composed of retinol binding protein (RBP) and albumin-derived peptide. The above albumin-derived peptide comprises albumin domain IIIA and albumin domain IB, The above retinol binding protein is a fusion protein comprising an amino acid sequence of sequence number 16.

2. In paragraph 1, A fusion protein wherein the above albumin domain IIIA and albumin domain IB are connected by a linker consisting of 3 to 7 hydrophobic amino acids.

3. In paragraph 1, The above fusion protein is a fusion protein having the structure of [N-terminal-RBP-albumin-derived peptide-C-terminal].

4. In paragraph 1, The above albumin domain IIIA comprises one amino acid sequence selected from the group consisting of SEQ ID NOs: 4 to 13, A fusion protein wherein the above albumin domain IB comprises an amino acid sequence of SEQ ID NO: 14 or SEQ ID NO:

15.

5. In paragraph 2, A fusion protein, wherein the linker comprises one amino acid sequence selected from the group consisting of SEQ ID NOs: 17 to 19.

6. In paragraph 1, A fusion protein comprising an amino acid sequence selected from the group consisting of sequence numbers 84 to 88.

7. In paragraph 1, The above fusion protein is a fusion protein that additionally contains a signal peptide at the N-terminus.

8. In paragraph 7, A fusion protein wherein the signal peptide comprises an amino acid sequence of SEQ ID NO: 94 or SEQ ID NO:

95.

9. A vector containing the fusion protein gene of paragraph 1.

10. A pharmaceutical composition for preventing or treating fibrotic disease, comprising the fusion protein of paragraph 1 or the vector of paragraph 9 as an active ingredient.

11. In paragraph 10, A pharmaceutical composition characterized in that the composition inhibits the expression of α-SMA (smooth muscle alpha-actin) and collagen I in stellate cells.

12. In paragraph 10, A pharmaceutical composition characterized in that the composition inhibits the activation of stellate cells or deactivates activated stellate cells.

13. In paragraph 10, A pharmaceutical composition, wherein the fibrotic disease is at least one selected from the group consisting of liver fibrosis, cirrhosis, hepatic cancer, chemotherapy-associated steatohepatitis (CASH), pulmonary fibrosis, pulmonary cancer, renal fibrosis, renal failure, renal cancer, pancreatic fibrosis, chronic pancreatitis, and pancreatic cancer.

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