WO2023059160A1 - Protéine de fusion pour prévenir et traiter la fibrose - Google Patents

Protéine de fusion pour prévenir et traiter la fibrose Download PDF

Info

Publication number
WO2023059160A1
WO2023059160A1 PCT/KR2022/015214 KR2022015214W WO2023059160A1 WO 2023059160 A1 WO2023059160 A1 WO 2023059160A1 KR 2022015214 W KR2022015214 W KR 2022015214W WO 2023059160 A1 WO2023059160 A1 WO 2023059160A1
Authority
WO
WIPO (PCT)
Prior art keywords
albumin
fusion protein
fragment
amino acids
present
Prior art date
Application number
PCT/KR2022/015214
Other languages
English (en)
Korean (ko)
Inventor
오준서
Original Assignee
주식회사 세네릭스
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from KR1020220127783A external-priority patent/KR20230051415A/ko
Application filed by 주식회사 세네릭스 filed Critical 주식회사 세네릭스
Publication of WO2023059160A1 publication Critical patent/WO2023059160A1/fr

Links

Images

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00

Definitions

  • the present invention relates to novel fusion proteins of albumin and retinol binding proteins that can be used to prevent or treat fibrosis.
  • Tissue fibrosis results in fatal tissue loss of function.
  • pulmonary fibrosis is a respiratory disease in which lung tissue hardens and causes severe respiratory disorders.
  • Pulmonary fibrosis is a systemic hypoxia caused by reduced lung capacity and impaired oxygen diffusion, as well as pulmonary artery and capillaries as fibrous tissue contracts.
  • Pulmonary hypertension caused by blood vessels, heart failure caused by blood accumulation in the right ventricle due to pulmonary circulation disorder, and other diseases such as thrombosis and pneumonia can be accompanied.
  • Pulmonary fibrosis may be caused by inhalation of toxic substances or continuous inflammation, but there are also idiopathic pulmonary fibrosis for which a specific cause cannot be identified.
  • tissue fibrosis The molecular mechanism of tissue fibrosis has not yet been clearly identified, and research on the development of therapeutic agents for fibrotic diseases is needed. Recently, it has been found that fibrosis of liver, pancreas, and kidney tissue occurs by activation and transdifferentiation of stellate cells, which are one of the cells constituting the tissue. Astrocytes differentiate into myofibroblasts through an activation process, and excessively express extracellular matrix such as collagen to accumulate in tissues. Therefore, activated astrocytes play a crucial role in the development of tissue fibrosis.
  • Vitamin A obtained from food circulates in the bloodstream in combination with Retinol Binding Protein (RBP), moves to astrocytes through the RBP receptor STRA6, and is stored as retinyl ester in fat droplets in the cytoplasm. do.
  • RBP Retinol Binding Protein
  • albumin it is known that the expression of albumin inhibits the activation of astrocytes, and the forced expression of albumin in already activated astrocytes reversely converts the cells to the state before activation.
  • the present inventors prepared a fusion protein targeting astrocytes by combining RBP and albumin capable of moving into astrocytes through STRA6 to suppress or inversely activate the activation of astrocytes, which initiate tissue fibrosis, and to induce fibrosis thereof. Preventive and therapeutic effects were confirmed (KR 10-1395394).
  • An object of the present invention is to enhance the activation inhibitory or inactivating effect of astrocytes of a conventional RBP and albumin fusion protein, and the present invention is RBP4 (retinol-binding protein 4) RBP4 fragment, albumin IIIA It is to provide a fusion protein comprising the fragment and the albumin IB fragment.
  • RBP4 retinol-binding protein 4
  • Another object of the present invention is to provide a fusion protein in the form of an RBP4 fragment-albumin IIIA fragment-albumin IB fragment or an albumin IIIA fragment-albumin IB fragment-RBP4 fragment.
  • Another object of the present invention is to provide a polynucleotide encoding the fusion protein.
  • Another object of the present invention is to provide a vector containing the polynucleotide.
  • Another object of the present invention is to provide a host cell containing the vector.
  • Another object of the present invention is to provide a method for producing the fusion protein.
  • Another object of the present invention is to provide a pharmaceutical composition for preventing or treating fibrotic diseases, including the fusion protein, a polynucleotide encoding the fusion protein, and a vector containing the polynucleotide.
  • Another object of the present invention is to provide a method for preventing or treating fibrosis, comprising administering the fusion protein, a polynucleotide encoding the fusion protein, and a vector containing the polynucleotide to a subject.
  • Another object of the present invention is to provide the use of the fusion protein, a polynucleotide encoding the fusion protein, and a vector containing the polynucleotide for the production of a drug for preventing or treating fibrotic diseases.
  • the present invention is an RBP4 fragment comprising amino acids 19 to 194 or 19 to 195 of RBP4 (retinol-binding protein 4); Albumin IIIA fragment containing amino acids 404 to 517 of albumin protein; And an albumin IB fragment comprising amino acids 134 to 218 or amino acids 131 to 218 of the albumin protein; provides a fusion protein comprising a.
  • the fusion protein is a combination of RBP4 fragment-albumin IIIA fragment-albumin IB fragment or albumin IIIA fragment-albumin IB fragment-RBP4 fragment form.
  • the fusion protein is an RBP4 fragment (amino acids 19-195)-albumin IIIA fragment (amino acids 404-517)-albumin IB fragment (amino acids 131-218), an RBP4 fragment ( 19-195 amino acids)-Albumin IIIA fragment (404-517 amino acids)-Albumin IB fragment (134-218 amino acids), Albumin IIIA fragment (404-517 amino acids)-Albumin IB fragment (131 amino acids) -RBP4 fragment (amino acids 19-194) -RBP4 fragment (amino acids 19-194) and Albumin IIIA fragment (amino acids 404-517) -Albumin IB fragment (amino acids 134-218) -RBP4 fragment (amino acids 19-194) ) is selected from the group consisting of
  • the RBP4 fragment comprises or consists of an amino acid sequence represented by the following formula:
  • X19 is E or Q
  • X23 is R, G or P
  • X24 is V, L or M
  • X25 is S or N
  • X26 is S or G
  • X30 is K, E or R
  • X31 is E, Q or V
  • X32 is N or D
  • X33 is F or L
  • X37 is R or H
  • X39 is S or P
  • X40 is G or E
  • X41 is T or I
  • X44 is A, S or V
  • X45 is M, I, L or T
  • X48 is K or R
  • X51 is E, K or V
  • X53 is L or V
  • X56 is Q or R
  • X59 is I or L
  • X60 is V, I or L
  • X65 is V or M
  • X68 is T or N
  • X69 is G, C, D or S
  • X70 is Q, E or H
  • X71 is M or I
  • X72 is S, I or R
  • X79 is V or G
  • X80 is R or H
  • X81 is L or V
  • X85 is W or R
  • X86 is D, H or V
  • X89 is A or T
  • X90 is D or A
  • X91 is M or I
  • X93 is G or S
  • X99 is E or K
  • X101 is P, H, L or S
  • X102 is A, S or V;
  • X106 is M or L
  • X107 is K or E
  • X108 is Y or C
  • X113 is S or F
  • X116 is Q or H
  • X118 is G or E
  • X191 is N or I
  • X123 is W, C or R
  • X125 is V or I
  • X126 is D, H or N
  • X128 is D or N
  • X129 is Y or C
  • X130 is D or N
  • X131 is T, K or M
  • X132 is Y, F or N
  • X134 is V, E, L or M
  • X136 is Y or S
  • X139 is R or H
  • X141 is L or Q
  • X143 is L or F
  • X144 is D, H or N
  • X152 is S or F
  • X154 is V, L or M
  • X156 is S, A or F
  • X157 is R, G or W;
  • X158 is D, E or Y;
  • X159 is P, L or S
  • X160 is N, H or S
  • X161 is G, C, D or S;
  • X163 is P, L, S or T;
  • X164 is P, L, R or S
  • X165 is E or A
  • X166 is A, G or V;
  • X167 is Q or R
  • X169 is I, F, L, M, T or V;
  • X170 is V, A, G, I or L;
  • X173 is R, Q or W;
  • X175 is E or G
  • X176 is E, D or G
  • X177 is L or V
  • X181 is R, G or W;
  • X182 is Q or K
  • X184 is R, G or K
  • X186 is I, L or M
  • X187 is V or I
  • X188 is H or R
  • X189 is N, D or S
  • X190 is G or S
  • X193 is D or N.
  • the RBP4 fragment includes or consists of the amino acid sequence of SEQ ID NO: 1.
  • the RBP4 fragment comprises or consists of an amino acid sequence represented by the following formula:
  • X19 is E or Q
  • X23 is R, G or P
  • X24 is V, L or M
  • X25 is S or N
  • X26 is S or G
  • X30 is K, E or R
  • X31 is E, Q or V
  • X32 is N or D
  • X33 is F or L
  • X37 is R or H
  • X39 is S or P
  • X40 is G or E
  • X41 is T or I
  • X44 is A, S or V
  • X45 is M, I, L or T
  • X48 is K or R
  • X51 is E, K or V
  • X53 is L or V
  • X56 is Q or R
  • X59 is I or L
  • X60 is V, I or L
  • X65 is V or M
  • X68 is T or N
  • X69 is G, C, D or S
  • X70 is Q, E or H
  • X71 is M or I
  • X72 is S, I or R
  • X79 is V or G
  • X80 is R or H
  • X81 is L or V
  • X85 is W or R
  • X86 is D, H or V
  • X89 is A or T
  • X90 is D or A
  • X91 is M or I
  • X93 is G or S
  • X99 is E or K
  • X101 is P, H, L or S
  • X102 is A, S or V;
  • X106 is M or L
  • X107 is K or E
  • X108 is Y or C
  • X113 is S or F
  • X116 is Q or H
  • X118 is G or E
  • X191 is N or I
  • X123 is W, C or R
  • X125 is V or I
  • X126 is D, H or N
  • X128 is D or N
  • X129 is Y or C
  • X130 is D or N
  • X131 is T, K or M
  • X132 is Y, F or N
  • X134 is V, E, L or M
  • X136 is Y or S
  • X139 is R or H
  • X141 is L or Q
  • X143 is L or F
  • X144 is D, H or N
  • X152 is S or F
  • X154 is V, L or M
  • X156 is S, A or F
  • X157 is R, G or W;
  • X158 is D, E or Y;
  • X159 is P, L or S
  • X160 is N, H or S
  • X161 is G, C, D or S;
  • X163 is P, L, S or T;
  • X164 is P, L, R or S
  • X165 is E or A
  • X166 is A, G or V;
  • X167 is Q or R
  • X169 is I, F, L, M, T or V;
  • X170 is V, A, G, I or L;
  • X173 is R, Q or W;
  • X175 is E or G
  • X176 is E, D or G
  • X177 is L or V
  • X181 is R, G or W;
  • X182 is Q or K
  • X184 is R, G or K
  • X186 is I, L or M
  • X187 is V or I
  • X188 is H or R
  • X189 is N, D or S
  • X190 is G or S
  • X193 is D or N
  • X195 is R or K.
  • the RBP4 fragment includes or consists of the amino acid sequence of SEQ ID NO: 2.
  • the albumin IIIA fragment comprises or consists of an amino acid sequence represented by the following formula:
  • X404 is L, F or P
  • X406 is E, D, K or V;
  • X408 is P or S
  • X409 is Q, K or L
  • X411 is L or S
  • X412 is I or L
  • X414 is Q or E
  • X419 is F or Y
  • X421 is Q, K or R;
  • X423 is G or R
  • X424 is E or G
  • X425 is Y or H
  • X426 is K, E or R;
  • X427 is F or L
  • X429 is N, D, K or S;
  • X430 is A, E, T or V;
  • X431 is L or V
  • X433 is V or I
  • X434 is R, C or H
  • X440 is P or H
  • X441 is Q or E
  • X442 is V or M
  • X444 is T or S
  • X445 is P or L
  • X446 is T or A
  • X450 is V, F, I or L
  • X455 is G, A or R;
  • X456 is K or R
  • X458 is G or R
  • X459 is S or R
  • X460 is K or N
  • X464 is H or L
  • X465 is P, A, L, R or S;
  • X466 is E or V
  • X469 is R or I
  • X473 is A or E
  • X475 is D, A or Y;
  • X476 is Y or C
  • X479 is V or M
  • X483 is Q or R
  • X484 is L or F
  • X487 is L or S
  • X488 is H, Q or R;
  • X490 is K or E
  • X491 is T, K or M
  • X492 is P or S
  • X494 is S or C
  • X495 is D, E or G
  • X497 is V or A
  • X502 is T or I
  • X503 is E, D or K
  • X506 is V, A or M
  • X508 is R or S
  • X509 is R or Q
  • X511 is C or Y
  • X517 is V or F.
  • the albumin IIIA fragment comprises or consists of the amino acid sequence of SEQ ID NO: 3 or 4.
  • the albumin IB fragment comprises or consists of an amino acid sequence represented by the following formula:
  • X135 is N or D
  • X136 is L or V
  • X137 is P or S
  • X138 is R, G, P or Q
  • X139 is L or F
  • X140 is V, L or M
  • X143 is E or K
  • X144 is V or L
  • X145 is D, E or V;
  • X146 is V or E
  • X147 is M, I or T
  • X149 is T, A or I;
  • X150 is A, D, S or T;
  • X152 is H or R
  • X153 is D or E
  • X154 is N or S
  • X156 is E, A or G
  • X157 is T or K
  • X164 is Y, C or H
  • X170 is H or R
  • X171 is P or L
  • X174 is Y or D
  • X175 is A or D
  • X176 is P or L
  • X177 is E or K
  • X179 is L or R
  • X180 is F, L, S or Y;
  • X181 is F or Y
  • X183 is K or I
  • X184 is R, M or T;
  • X185 is Y, C or N;
  • X187 is A, D or S;
  • X188 is A, D or V;
  • X190 is T, I or S
  • X191 is E or G
  • X194 is G or L
  • X195 is A or T
  • X197 is D, G or Y;
  • X199 is A or G
  • X200 is A or V
  • X201 is C or F
  • X205 is K or R
  • X206 is L or F
  • X207 is D, E, N or V;
  • X209 is L or H
  • X210 is R, Q or W;
  • X211 is D, V or Y;
  • X212 is E or K
  • X213 is G or E;
  • X215 is A, T or V;
  • X216 is S or L
  • X218 is A or G.
  • the albumin IB fragment comprises or consists of the amino acid sequence of SEQ ID NO: 5.
  • the albumin IB fragment comprises or consists of an amino acid sequence represented by the following formula:
  • X132 is D or E
  • X133 is N or K
  • X135 is N or D
  • X136 is L or V
  • X137 is P or S
  • X138 is R, G, P or Q
  • X139 is L or F
  • X140 is V, L or M
  • X143 is E or K
  • X144 is V or L
  • X145 is D, E or V;
  • X146 is V or E
  • X147 is M, I or T
  • X149 is T, A or I;
  • X150 is A, D, S or T;
  • X152 is H or R
  • X153 is D or E
  • X154 is N or S
  • X156 is E, A or G
  • X157 is T or K
  • X164 is Y, C or H
  • X170 is H or R
  • X171 is P or L
  • X174 is Y or D
  • X175 is A or D
  • X176 is P or L
  • X177 is E or K
  • X179 is L or R
  • X180 is F, L, S or Y;
  • X181 is F or Y
  • X183 is K or I
  • X184 is R, M or T;
  • X185 is Y, C or N;
  • X187 is A, D or S;
  • X188 is A, D or V;
  • X190 is T, I or S
  • X191 is E or G
  • X194 is G or L
  • X195 is A or T
  • X197 is D, G or Y;
  • X199 is A or G
  • X200 is A or V
  • X201 is C or F
  • X205 is K or R
  • X206 is L or F
  • X207 is D, E, N or V;
  • X209 is L or H
  • X210 is R, Q or W;
  • X211 is D, V or Y;
  • X212 is E or K
  • X213 is G or E;
  • X215 is A, T or V;
  • X216 is S or L
  • X218 is A or G.
  • the albumin IB fragment comprises or consists of the amino acid sequence of SEQ ID NO: 6.
  • the fusion protein comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 7 to 12.
  • the present invention provides a polynucleotide encoding the fusion protein.
  • the present invention provides a host cell containing the vector.
  • the present invention provides a method for producing a fusion protein comprising the following steps:
  • the present invention provides a pharmaceutical composition for preventing or treating fibrotic diseases, including the fusion protein, a polynucleotide encoding the fusion protein, and a vector containing the polynucleotide.
  • the present invention provides a method for preventing or treating a fibrotic disease comprising administering the fusion protein, a polynucleotide encoding the fusion protein, and a vector including the polynucleotide to a subject.
  • the present invention provides the use of the fusion protein, a polynucleotide encoding the fusion protein, and a vector containing the polynucleotide for the production of a drug for preventing or treating fibrotic diseases.
  • the fibrotic disease is liver fibrosis, chronic hepatitis, cirrhosis, hepatic cancer, chemotherapy-associated steatohepatitis, CASH ), at least one selected from the group consisting of pulmonary fibrosis, renal fibrosis, renal failure, pancreatic fibrosis, chronic pancreatitis and pancreatic cancer could be a disease.
  • the fusion protein of the present invention can prevent or treat fibrotic diseases by suppressing or inducing inverse activation of the activation of astrocytes, which is the cause of tissue fibrosis, and has a better effect at lower concentrations than conventional fusion proteins for the same purpose. It is expected to be a universal base technology that can dramatically reduce the dose and frequency of administration of protein therapeutics to the human body.
  • FIG. 1 is a schematic diagram showing the mechanism in which a fusion protein in which Albumin III and RBP are combined acts by being embedded in activated astrocytes.
  • FIG. 2 is a schematic diagram of albumin protein and RBP and fusion proteins 4 and 6 of the present invention.
  • Figure 3 is a view comparing and confirming the inverse activation induction effect of fusion proteins 1 and 2 of the present invention and conventional fusion proteins (RBP-Albumin III and Albumin III-RBP) in activated hepatic stellate cells.
  • FIG. 4 is a diagram comparing and confirming the inverse activation inducing effects of fusion proteins 1 and 2 and fusion proteins 3 to 6 of the present invention in activated hepatic stellate cells.
  • FIG. 5 is a view confirming the effect of fusion protein 6 of the present invention on inducing roleization in activated lung astrocytes.
  • 6a and 6b are views comparing and confirming the degree of liver fibrosis according to the administration of fusion proteins 1 and 4 and RBP-albumin2 to liver fibrosis animal models through Sirius red tissue staining.
  • Figure 7 shows the outline of the lung fibrosis animal model experiments.
  • Figure 8 shows the weight change of mice during the test period in the lung fibrosis animal model.
  • the horizontal axis represents the passage of time (day) from the bleomacin treatment day to the reference day (day 0), and the vertical axis represents the body weight (mg) of the mouse.
  • FIG. 9 is a diagram confirming through hydroxyproline assay that collagen levels are reduced in lung tissue according to fusion protein 6 treatment in a lung fibrosis animal model.
  • FIG. 10 is a diagram confirming the degree of lung fibrosis according to fusion protein 6 treatment in a lung fibrosis animal model through H&E staining and immunohistochemical staining.
  • Albumin is a multifunctional plasma protein synthesized primarily by hepatocytes. Albumin has three domains, each consisting of two subdomains A and B. Albumin is known to perform a role of molecular movement by binding to various hydrophobic substances including fatty acids and retinoic acid and transporting them in the blood. According to crystallographic analysis, five strong fatty acid binding sites are asymmetrically distributed within albumin (one in subdomain IB, one between IA and IIA, two in IIIA, and one in IIIB).
  • the present inventors prepared a fusion protein in which albumin and retinol binding protein (RBP) for targeting astrocytes were combined, and the fusion protein was introduced into activated astrocytes to activate cell morphology. It was reversed to the previous one, and it was confirmed that it is possible to prevent and treat fibrotic diseases. Furthermore, the present inventors found that retinoic acid (RA) plays an important role in the process of activating astrocytes, and that the fusion protein controls the activation of astrocytes by reducing the intracellular level of RA.
  • RBP retinol binding protein
  • the inventors replaced the 526th amino acid residue (phenylalanine) and the 600th amino acid residue (valine) in albumin domain III with valine and alanine, respectively, to design and manufacture fusion proteins 1 and 2 as follows. did
  • fusion proteins 3 to 6 were designed and prepared as follows.
  • the present inventors treated activated hepatic stellate cells with the prepared fusion protein 1 or 2 and measured the expression levels of ⁇ -smooth muscle actin ( ⁇ -SMA) and collagen type I, which are markers for astrocyte activation, to develop conventionally developed hepatic stellate cells.
  • ⁇ -SMA smooth muscle actin
  • collagen type I which are markers for astrocyte activation
  • the inverse activation effect of fusion proteins (KR 10-1395394, RBP-albumin III and albumin III-RBP) and astrocytes was compared and confirmed.
  • the fusion proteins 1 and 2 of the present invention can reduce the expression of ⁇ -SMA and collagen I from a low concentration to a high level compared to previously developed fusion proteins (see Example 1).
  • the present inventors confirmed that when 526 a.a and 600 a.a of Albumin III are substituted with amino acids of small molecular weight, the binding force with retinoic acid is increased, thereby more effectively inducing an inactivated state of activated astrocytes.
  • the present invention can provide a fusion protein in which albumin III and RBP, in which the 526th and 600th amino acids are independently substituted, are combined for use in the treatment of fibrotic diseases, and in the fusion protein, the 526th amino acid residue of albumin III is located.
  • the possible amino acids may be valine, alanine, and glycine, and the amino acids that may be positioned as the 600th amino acid residue may be alanine and glycine, and the fusion protein may consist of or include the amino acid sequence of SEQ ID NO: 13 or 14. there is.
  • the present invention relates to an RBP4 fragment containing amino acids 19 to 194 or 19 to 195 of RBP4 (retinol-binding protein 4), albumin containing amino acids 404 to 517 of albumin protein.
  • RBP4 retinol-binding protein 4
  • albumin containing amino acids 404 to 517 of albumin protein.
  • a fusion protein comprising an IIIA fragment, an albumin IB fragment comprising amino acids 134 to 218 or amino acids 131 to 218 of an albumin protein is provided.
  • the fusion protein is a combination of RBP4 fragment-albumin IIIA fragment-albumin IB fragment or albumin IIIA fragment-albumin IB fragment-RBP4 fragment.
  • the RBP4 fragment of the present invention is a site not associated with a disease and may contain naturally occurring mutations.
  • the naturally occurring mutation can be found in the UniProt Database ( www.uniprot.org ), and the RBP4 fragment containing the mutation site is also included in the present invention:
  • the albumin IIIA fragment of the present invention may contain a naturally occurring mutation as a site not associated with a disease.
  • the naturally occurring mutation can be found in the UniProt Database ( www.uniprot.org ), and the albumin IIIA fragment containing the mutation site is also included in the present invention:
  • the albumin IB fragment of the present invention may contain a naturally occurring mutation as a site not associated with a disease.
  • the naturally occurring mutation can be found in the UniProt Database ( www.uniprot.org ), and the albumin IB fragment containing the mutation site is also included in the present invention:
  • the fusion protein is an RBP4 fragment (amino acids 19-195)-albumin IIIA fragment (amino acids 404-517)-albumin IB fragment (amino acids 131-218), an RBP4 fragment (amino acids 19-195)-albumin IIIA fragment (amino acids 404-517)-albumin IB fragment (amino acids 134-218), albumin IIIA fragment (amino acids 404-517)-albumin IB fragment (131 No.-218 amino acids)-RBP4 fragment (amino acids 19-194) and Albumin IIIA fragment (amino acids 404-517)-Albumin IB fragment (amino acids 134-218)-RBP4 fragment (19-194 It is selected from the group consisting of amino acids).
  • the RBP4 fragment is composed of an amino acid sequence represented by the following formula:
  • X19 is E or Q
  • X23 is R, G or P
  • X24 is V, L or M
  • X25 is S or N
  • X26 is S or G
  • X30 is K, E or R
  • X31 is E, Q or V
  • X32 is N or D
  • X33 is F or L
  • X37 is R or H
  • X39 is S or P
  • X40 is G or E
  • X41 is T or I
  • X44 is A, S or V
  • X45 is M, I, L or T
  • X48 is K or R
  • X51 is E, K or V
  • X53 is L or V
  • X56 is Q or R
  • X59 is I or L
  • X60 is V, I or L
  • X65 is V or M
  • X68 is T or N
  • X69 is G, C, D or S
  • X70 is Q, E or H
  • X71 is M or I
  • X72 is S, I or R
  • X79 is V or G
  • X80 is R or H
  • X81 is L or V
  • X85 is W or R
  • X86 is D, H or V
  • X89 is A or T
  • X90 is D or A
  • X91 is M or I
  • X93 is G or S
  • X99 is E or K
  • X101 is P, H, L or S
  • X102 is A, S or V;
  • X106 is M or L
  • X107 is K or E
  • X108 is Y or C
  • X113 is S or F
  • X116 is Q or H
  • X118 is G or E
  • X191 is N or I
  • X123 is W, C or R
  • X125 is V or I
  • X126 is D, H or N
  • X128 is D or N
  • X129 is Y or C
  • X130 is D or N
  • X131 is T, K or M
  • X132 is Y, F or N
  • X134 is V, E, L or M
  • X136 is Y or S
  • X139 is R or H
  • X141 is L or Q
  • X143 is L or F
  • X144 is D, H or N
  • X152 is S or F
  • X154 is V, L or M
  • X156 is S, A or F
  • X157 is R, G or W;
  • X158 is D, E or Y;
  • X159 is P, L or S
  • X160 is N, H or S
  • X161 is G, C, D or S;
  • X163 is P, L, S or T;
  • X164 is P, L, R or S
  • X165 is E or A
  • X166 is A, G or V;
  • X167 is Q or R
  • X169 is I, F, L, M, T or V;
  • X170 is V, A, G, I or L;
  • X173 is R, Q or W;
  • X175 is E or G
  • X176 is E, D or G
  • X177 is L or V
  • X181 is R, G or W;
  • X182 is Q or K
  • X184 is R, G or K
  • X186 is I, L or M
  • X187 is V or I
  • X188 is H or R
  • X189 is N, D or S
  • X190 is G or S
  • X193 is D or N.
  • the RBP4 fragment is composed of the amino acid sequence of SEQ ID NO: 1.
  • the RBP4 fragment is composed of an amino acid sequence represented by the following formula:
  • X19 is E or Q
  • X23 is R, G or P
  • X24 is V, L or M
  • X25 is S or N
  • X26 is S or G
  • X30 is K, E or R
  • X31 is E, Q or V
  • X32 is N or D
  • X33 is F or L
  • X37 is R or H
  • X39 is S or P
  • X40 is G or E
  • X41 is T or I
  • X44 is A, S or V
  • X45 is M, I, L or T
  • X48 is K or R
  • X51 is E, K or V
  • X53 is L or V
  • X56 is Q or R
  • X59 is I or L
  • X60 is V, I or L
  • X65 is V or M
  • X68 is T or N
  • X69 is G, C, D or S
  • X70 is Q, E or H
  • X71 is M or I
  • X72 is S, I or R
  • X79 is V or G
  • X80 is R or H
  • X81 is L or V
  • X85 is W or R
  • X86 is D, H or V
  • X89 is A or T
  • X90 is D or A
  • X91 is M or I
  • X93 is G or S
  • X99 is E or K
  • X101 is P, H, L or S
  • X102 is A, S or V;
  • X106 is M or L
  • X107 is K or E
  • X108 is Y or C
  • X113 is S or F
  • X116 is Q or H
  • X118 is G or E
  • X191 is N or I
  • X123 is W, C or R
  • X125 is V or I
  • X126 is D, H or N
  • X128 is D or N
  • X129 is Y or C
  • X130 is D or N
  • X131 is T, K or M
  • X132 is Y, F or N
  • X134 is V, E, L or M
  • X136 is Y or S
  • X139 is R or H
  • X141 is L or Q
  • X143 is L or F
  • X144 is D, H or N
  • X152 is S or F
  • X154 is V, L or M
  • X156 is S, A or F
  • X157 is R, G or W;
  • X158 is D, E or Y;
  • X159 is P, L or S
  • X160 is N, H or S
  • X161 is G, C, D or S;
  • X163 is P, L, S or T;
  • X164 is P, L, R or S
  • X165 is E or A
  • X166 is A, G or V;
  • X167 is Q or R
  • X169 is I, F, L, M, T or V;
  • X170 is V, A, G, I or L;
  • X173 is R, Q or W;
  • X175 is E or G
  • X176 is E, D or G
  • X177 is L or V
  • X181 is R, G or W;
  • X182 is Q or K
  • X184 is R, G or K
  • X186 is I, L or M
  • X187 is V or I
  • X188 is H or R
  • X189 is N, D or S
  • X190 is G or S
  • X193 is D or N
  • X195 is R or K.
  • the RBP4 fragment is composed of the amino acid sequence of SEQ ID NO: 2.
  • the albumin IIIA fragment is composed of an amino acid sequence represented by the following formula:
  • X404 is L, F or P
  • X406 is E, D, K or V;
  • X408 is P or S
  • X409 is Q, K or L
  • X411 is L or S
  • X412 is I or L
  • X414 is Q or E
  • X419 is F or Y
  • X421 is Q, K or R;
  • X423 is G or R
  • X424 is E or G
  • X425 is Y or H
  • X426 is K, E or R;
  • X427 is F or L
  • X429 is N, D, K or S;
  • X430 is A, E, T or V;
  • X431 is L or V
  • X433 is V or I
  • X434 is R, C or H
  • X440 is P or H
  • X441 is Q or E
  • X442 is V or M
  • X444 is T or S
  • X445 is P or L
  • X446 is T or A
  • X450 is V, F, I or L
  • X455 is G, A or R;
  • X456 is K or R
  • X458 is G or R
  • X459 is S or R
  • X460 is K or N
  • X464 is H or L
  • X465 is P, A, L, R or S;
  • X466 is E or V
  • X469 is R or I
  • X473 is A or E
  • X475 is D, A or Y;
  • X476 is Y or C
  • X479 is V or M
  • X483 is Q or R
  • X484 is L or F
  • X487 is L or S
  • X488 is H, Q or R;
  • X490 is K or E
  • X491 is T, K or M
  • X492 is P or S
  • X494 is S or C
  • X495 is D, E or G
  • X497 is V or A
  • X502 is T or I
  • X503 is E, D or K
  • X506 is V, A or M
  • X508 is R or S
  • X509 is R or Q
  • X511 is C or Y
  • X517 is V or F.
  • the albumin IIIA fragment is composed of the amino acid sequence of SEQ ID NO: 3 or 4.
  • the albumin IB fragment is composed of an amino acid sequence represented by the following formula:
  • X135 is N or D
  • X136 is L or V
  • X137 is P or S
  • X138 is R, G, P or Q
  • X139 is L or F
  • X140 is V, L or M
  • X143 is E or K
  • X144 is V or L
  • X145 is D, E or V;
  • X146 is V or E
  • X147 is M, I or T
  • X149 is T, A or I;
  • X150 is A, D, S or T;
  • X152 is H or R
  • X153 is D or E
  • X154 is N or S
  • X156 is E, A or G
  • X157 is T or K
  • X164 is Y, C or H
  • X170 is H or R
  • X171 is P or L
  • X174 is Y or D
  • X175 is A or D
  • X176 is P or L
  • X177 is E or K
  • X179 is L or R
  • X180 is F, L, S or Y;
  • X181 is F or Y
  • X183 is K or I
  • X184 is R, M or T;
  • X185 is Y, C or N;
  • X187 is A, D or S;
  • X188 is A, D or V;
  • X190 is T, I or S
  • X191 is E or G
  • X194 is G or L
  • X195 is A or T
  • X197 is D, G or Y;
  • X199 is A or G
  • X200 is A or V
  • X201 is C or F
  • X205 is K or R
  • X206 is L or F
  • X207 is D, E, N or V;
  • X209 is L or H
  • X210 is R, Q or W;
  • X211 is D, V or Y;
  • X212 is E or K
  • X213 is G or E;
  • X215 is A, T or V;
  • X216 is S or L
  • X218 is A or G.
  • the albumin IB fragment is composed of the amino acid sequence of SEQ ID NO: 5.
  • the albumin IB fragment is composed of an amino acid sequence represented by the following formula:
  • X132 is D or E
  • X133 is N or K
  • X135 is N or D
  • X136 is L or V
  • X137 is P or S
  • X138 is R, G, P or Q
  • X139 is L or F
  • X140 is V, L or M
  • X143 is E or K
  • X144 is V or L
  • X145 is D, E or V;
  • X146 is V or E
  • X147 is M, I or T
  • X149 is T, A or I;
  • X150 is A, D, S or T;
  • X152 is H or R
  • X153 is D or E
  • X154 is N or S
  • X156 is E, A or G
  • X157 is T or K
  • X164 is Y, C or H
  • X170 is H or R
  • X171 is P or L
  • X174 is Y or D
  • X175 is A or D
  • X176 is P or L
  • X177 is E or K
  • X179 is L or R
  • X180 is F, L, S or Y;
  • X181 is F or Y
  • X183 is K or I
  • X184 is R, M or T;
  • X185 is Y, C or N;
  • X187 is A, D or S;
  • X188 is A, D or V;
  • X190 is T, I or S
  • X191 is E or G
  • X194 is G or L
  • X195 is A or T
  • X197 is D, G or Y;
  • X199 is A or G
  • X200 is A or V
  • X201 is C or F
  • X205 is K or R
  • X206 is L or F
  • X207 is D, E, N or V;
  • X209 is L or H
  • X210 is R, Q or W;
  • X211 is D, V or Y;
  • X212 is E or K
  • X213 is G or E;
  • X215 is A, T or V;
  • X216 is S or L
  • X218 is A or G.
  • the albumin IB fragment is composed of the amino acid sequence of SEQ ID NO: 6
  • the fusion protein comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 7 to 12.
  • the present invention provides a polynucleotide encoding the fusion protein.
  • gene or “polynucleotide” should be regarded in the broadest sense, and as encoding a protein, it is not limited as long as it is a DNA fragment encoding the fusion proteins 1 to 6 of the present application, and specifically SEQ ID NOs: 15 to 15 One of 20 nucleotide sequences may be included.
  • the present invention provides a vector containing the polynucleotide.
  • vector means a DNA preparation containing a DNA sequence operably linked to suitable regulatory sequences capable of expressing the DNA in a suitable host.
  • Vectors can be plasmids, phage particles, viral particles or simply latent genomic inserts. 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. Because the plasmid is currently the most commonly used form of vector, plasmid and vector are sometimes used interchangeably in the context of the present invention.
  • a sequence is "operably linked" when it is placed into a functional relationship with another sequence. It can be genes and regulatory sequence(s) linked in such a way as to enable gene expression when appropriate molecules (eg, transcriptional activating proteins) are bound to the regulatory sequence(s).
  • DNA for a pre-sequence or secretory leader is operably linked to DNA for a polypeptide when expressed as a pre-protein that participates in secretion of the polypeptide;
  • a promoter or enhancer is operably linked to a coding sequence if it affects transcription of the sequence; or the ribosome binding site is operably linked to a coding sequence if it affects transcription of the sequence; or the ribosome binding site is operably linked to a coding sequence when positioned to facilitate translation.
  • "operably linked” means that the DNA sequences being linked are contiguous and, in the case of a secretory leader, contiguous and in reading frame. However, enhancers do not have to be in contact. Linkage of these sequences is accomplished by ligation (linkage) at convenient restriction enzyme sites. If such a site does not exist, a synthetic oligonucleotide adapter or linker according to a conventional method is used.
  • the present invention provides a host cell comprising the vector or transformed with the vector.
  • the term "cell” includes eukaryotic cells and prokaryotic cells, and refers to any transformable cell capable of replicating the vector or expressing a gene encoded by the vector.
  • a cell may be transfected, transduced, or transformed by the vector, which means a process in which an exogenous polynucleotide (nucleic acid molecule) is delivered or introduced into a host cell.
  • transformation is used as a meaning including the transfection (Transfected) and transduction (Transduced).
  • the (host) cells of the present invention are not limited, but preferably insect cells or mammalian cells, more preferably Sf9 in the case of insect cells, HEK293 cells in the case of mammalian cells, HeLa cells, ARPE-19 cells, RPE- 1 cell, HepG2 cell, Hep3B cell, Huh-7 cell, C8D1a cell, Neuro2A cell, CHO cell, MES13 cell, BHK-21 cell, COS7 cell, COP5 cell, A549 cell, MCF-7 cell, HC70 cell, HCC1428 cell , BT-549 cells, PC3 cells, LNCaP cells, Capan-1 cells, Panc-1 cells, MIA PaCa-2 cells, SW480 cells, HCT166 cells, LoVo cells, A172 cells, MKN-45 cells, MKN-74 cells, Kato-III cells, NCI-N87 cells, HT-144 cells, SK-MEL-2 cells, SH-SY5Y cells, C6 cells, HT-22 cells,
  • the host cell of the present invention is preferably an isolated host cell.
  • the present invention provides a pharmaceutical composition for preventing or treating fibrotic diseases, including the fusion protein, a polynucleotide encoding the fusion protein, and a vector containing the polynucleotide.
  • the present invention provides a method for preventing or treating a fibrotic disease comprising administering the fusion protein, a polynucleotide encoding the fusion protein, and a vector including the polynucleotide to a subject.
  • the present invention provides the use of the fusion protein, a polynucleotide encoding the fusion protein, and a vector containing the polynucleotide for the production of a drug for preventing or treating fibrotic diseases.
  • the present inventors compared and confirmed the inhibitory effects of ⁇ -SMA and collagen I expression of fusion proteins 1 and 2 and fusion proteins 3 to 6, which showed superior inhibitory effects on the activation process of hepatic stellate cells than conventional fusion proteins. As a result, it was confirmed that the fusion proteins 3 to 6 could effectively inhibit the activation of astrocytes even at lower concentrations (Example 2).
  • fusion protein 6 as a representative and conducted the same experiment in order to confirm whether the fusion protein of the present invention, which has an activity to induce the state before activation in hepatic stellate cells, exhibits the same activity in pulmonary stellate cells. It was performed in cells, and it was confirmed that fusion protein 6 had an inverse activating effect in lung astrocytes (Example 3).
  • the present inventors confirmed the effect of improving liver fibrosis of fusion proteins 1 and 4 and a conventional fusion protein (KR 10-1395394, RBP-albumin III) using an animal model of liver fibrosis caused by carbon tetrachloride, and as a result, fusion proteins 1 and 4 It was confirmed that fibrosis can be improved more effectively at a lower concentration than RBP-Albumin III (Example 4).
  • the present inventors administered fusion protein 6 to a bleomycin-induced lung fibrosis animal model in order to confirm that the fusion protein of the present invention, which exhibits a fibrotic disease effect in a liver fibrosis animal model, has the same effect in a lung fibrosis animal model. It was confirmed that the degree of fibrosis was improved (Example 5).
  • the fusion proteins 1 to 6 of the present invention have an effect of inhibiting inactivated astrocytes from being activated despite an activation signal and inducing inverse activation of activated astrocytes to a state before activation, that is, an inactive state. shows the conversion activity.
  • the inventors of the present invention can provide a pharmaceutical composition for preventing or treating fibrotic disease comprising one or more fusion proteins selected from the group consisting of fusion proteins 1 to 6 developed above, and from the group consisting of fusion proteins 1 to 6. It is possible to provide a method for preventing or treating a fibrotic disease comprising administering one or more selected fusion proteins to a subject.
  • the fusion proteins 1 to 6 of the present invention can effectively induce an inactive state of activated astrocytes at a lower concentration than the conventional fusion protein of RBP and albumin (KR 10-1395394).
  • the composition is expected to be a universal base technology that can dramatically reduce the dosage and frequency of administration of protein therapeutics to the human body.
  • fibrotic disease is a disease that occurs when 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 (pancreatic fibrosis), chronic pancreatitis (chronic pancreatitis) and pancreatic cancer (pancreatic cancer), including, but not limited to, any disease caused by hardened fibrosis of a part of an organ, but preferably liver fibrosis and lung fibrosis.
  • the term "individual” is not limited as long as it is a mammal, but may preferably be a human or livestock.
  • prevention refers to any action that delays the onset of fibrotic disease or delays tissue fibrosis by administration of the pharmaceutical composition according to the present invention
  • treatment refers to the pharmaceutical composition according to the present invention. It refers to all activities that improve or beneficially change the symptoms of fibrotic disease by administration of
  • the albumin used to form the fusion protein may be derived from any species, but is preferably from the same species as the subject to be administered to avoid the risk of immunogenicity.
  • 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 6, and is suitable for preparing pharmaceutical compositions. It may further include a carrier, an excipient, and a diluent.
  • carrier is also called a vehicle, and means a compound that facilitates the addition of proteins or peptides into cells or tissues.
  • DMSO dimethyl sulfoxide
  • carrier is a commonly used carrier that facilitates the entry of many organisms into cells or tissues.
  • diot is defined as a compound diluted in water that not only stabilizes the biologically active form of a protein or peptide of interest, 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 because it mimics the salt state of human solutions. Because buffer salts can control the pH of a solution at low concentrations, buffer diluents rarely modify the biological activity of a compound.
  • Compounds containing azelaic acid as used herein may be administered to human patients by themselves or as pharmaceutical compositions mixed with other ingredients, as in combination therapy, or with suitable carriers or excipients.
  • the pharmaceutical composition for preventing or treating fibrotic diseases according to the present invention is formulated in the form of external preparations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols and sterile injection solutions according to conventional methods, respectively.
  • the antibacterial composition of the present invention may be administered orally or parenterally (eg, intravenous, subcutaneous, intraperitoneal or topical application) depending on the desired method, and the dosage is the patient's Although it varies depending on the condition and body weight, the severity of the disease, the type of drug, the route and duration of administration, it can be appropriately selected by those skilled in the art, for example, about 0.001 mg to 1000 mg in a mixed form with a pharmaceutically acceptable carrier. can be administered.
  • the composition of the present invention can be administered once or several times a day, if necessary, and can be used alone or in combination with methods using surgery, hormone therapy, drug therapy, and biological response modifiers.
  • the amino terminus 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).
  • 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).
  • PEG polyethylene glycol
  • the carboxy terminal of the peptide may be modified with a hydroxy group (-OH), an amino group (-NH 2 ), an azide (-NHNH 2 ), and the like.
  • fatty acids, oligosaccharides chains, all nanoparticles (gold particles, liposomes, heparin, hydrogel, etc.), amino acids, carriers are added to the R-group of the fusion protein or amino acid of the present invention. It can bind to proteins (carrier proteins) and the like. Modification of the amino acids described above serves to improve the potency and stability of the protein of the present invention.
  • the term “stability” means not only in vivo stability, but also storage stability (including storage stability when stored at room temperature, refrigerated, or frozen).
  • the present invention provides a method for producing a fusion protein comprising the following steps:
  • a step of preparing a recombinant vector containing a gene encoding the fusion protein may additionally be included.
  • SCs stellate cells
  • 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. After perfusion, the liver was 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.
  • PBS phosphate-buffered saline
  • GBSS Gey's balanced salt solution
  • ⁇ -SMA and collagen I were taken and cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS) (Carlsbad, CA). Purity of astrocytes was evaluated by microscopic observation and western blotting using an anti-tyrosine aminotransferase antibody. When the cells became confluent in the dish, they were subcultured and used as activated astrocytes. Activation of hepatic stellate cells was confirmed through morphological changes and increased expression of ⁇ -SMA and collagen I.
  • DMEM Dulbecco's modified Eagle's medium
  • FBS fetal bovine serum
  • LSCs Lung stellate cells
  • the lungs of 14-week-old male BALB/c mice were extracted, and the lung cell suspension was placed in the same solution as GBSS and treated at 37°C for 12 minutes, followed by 20 min at 1400 g on a 13.4% Nycodenz gradient. After centrifugation for 10 minutes, astrocytes on the interface between the Nycodenz solution and the aqueous layer were taken and cultured.
  • the sequence of the albumin IB fragment including amino acids 134 to 218 or amino acids 131 to 218 is shown in Table 1 below.
  • fusion proteins 1 to 6 designed to enhance binding force with retinoic acid is shown in Table 2 below. Regions derived from RBP are underlined, and amino acids substituted in albumin III are bold and underlined. A signal peptide was added to the N-terminus of each fusion protein to induce secretion outside the transformant cells, and a signal peptide consisting of the amino acid sequence mkwvwallllaawaaa or mkwvtfisllflfssays was selected and added.
  • Fusion protein 1 RBP (19 ⁇ 195) + Albumin III ⁇ 404 ⁇ 526 (F ⁇ V) ⁇ 600 (V ⁇ A) ⁇ 601 ⁇ ERDCRVSSFRVKENFDKARFSGTWYAMAKKDPEGLFLQDNIVAEFSVDETGQMSATAKGRVRLLNNWDVCADMVGTFTDTEDPAKFKMKYWGVASFLQKGNDDHWIVDTDYDTYAVQYSCRLLNLDGTCADSYSFVFSRDPNGLPPEAQKIVRQRQEELCLARQYRLIVHNGYCDGR LVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPEVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKE V NAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADD
  • fusion proteins Preparation of fusion proteins
  • polynucleotides encoding fusion proteins 1 to 6 including signal peptides are prepared, and each DNA fragment is cloned into XbaI/KpnI cut pcDNA3.1(+) vector to obtain a recombinant expression vector. manufactured. Specific information of the DNA fragment encoding each fusion protein is summarized in Table 2 below.
  • Each of the above vectors was introduced into CHO cells using Lipofectamine 2000 (Invitrogen, Carlsbad, CA) to prepare transformants expressing the fusion proteins.
  • the fusion protein expressed and secreted by culturing the transformant was purified by affinity chromatography and HPLC before use.
  • Fusion protein 1 CCGCTCTGGTGGAGCTGGTGAAGCATAAGCCTAAGGCTACAAAGGAGCAGCTGAAGGCCGTGATGGACGATTTCGCTGCCTTTGTGGAGAAGTGCTGTAAGGCTGACGATAAGGAGACCTGTTTCGCCGAGGAGGGCAAGAAGCTGGCGGCT 16 fusion protein 2 TGGATGGCACCTGTGCCGACAGCTATTCTTTCGTGTTTTCCCGCGATCCTAATGGCCTGCCCTGAGGCTCAGAAGATCGTGAGGCAGAGGCAGGAGGAGCTGTGCCTGGCCAGGCAGTACCGGCTGATCGTGCATAATGGCTATTGTGACGGC 17 Fusion protein 3 atgagatcgccagacgccatccctacttttatgctcctgagctgtctttgccaagcggtacaaggctgcttcacagagtgctgtcaggct
  • a liver fibrosis mouse model in which liver damage was induced was prepared by injecting CCl 4 dissolved in 1:1 in mineral oil at a concentration of 1 mL/kg into the abdominal cavity of BALB/c mice 3 times a week for 6 weeks.
  • a control group was administered with the same amount of mineral oil alone. Mice were sacrificed 48 hours after the final CCl 4 injection.
  • liver tissue of each experimental group was fixed with 10% buffered formalin, and then paraffin-embedded to prepare tissue sections. Each tissue section was observed under an optical microscope after performing Sirius red staining for histological analysis.
  • Bleomycin which is commonly used as an anticancer drug, is well known to cause side effects of lung fibrosis, and is widely used in the production of lung fibrosis animal models. Specifically, lung fibrosis was induced by bronchial inhalation of bleomycin in 6-week-old BAL/c male mice.
  • the body weight of the mouse was measured every day. After the mouse was sacrificed, the lung was removed to evaluate fibrosis, and the amount of collagen in the tissue was measured by performing a hydroxyproline assay. In addition, the lung tissues of each experimental group were fixed with 10% buffered formalin, embedded in paraffin to prepare tissue sections, and each tissue section was observed under an optical microscope after performing H&E staining for histological analysis. All experimental data were shown in comparison with normal mice (control) in which pulmonary fibrosis was not induced.
  • Results are expressed as mean ⁇ standard deviation (SD). Statistical analysis was performed using t-tests. The comparison was tested for significance at P ⁇ 0.05, and a two-tailed test was performed for the P value.
  • RBP-Albumin III and Albumin III-RBP which showed excellent effects on suppressing or inversely activating astrocytes in previous studies, and fusion proteins 1 and 2 designed in Example 2 were compared. Specifically, hepatic stellate cells of activated mice were treated with 0.25 ⁇ M of each fusion protein for 20 hours, and the expression levels of ⁇ -SMA and collagen I were measured using real-time PCR.
  • Conventional fusion proteins (RBP-Albumin III and Albumin III-RBP) are R-III and III-R fusion proteins of KR 10-1395394.
  • fusion proteins 1 and 2 and fusion proteins 3 to 6 which were confirmed to be superior to those of conventional fusion proteins, were compared and confirmed in terms of the inverse activation effect of activated hepatic stellate cells. It was performed in the same manner as in Example 1, but the amount of fusion protein treated with activated hepatic stellate cells was 0.125 ⁇ M.
  • the fusion proteins 3 to 6 can reduce the expression of ⁇ -SMA and collagen I to a remarkably high level even with a smaller amount of protein.
  • fusion proteins 3 to 6 can induce inverse activation of activated hepatic stellate cells with a small amount of protein compared to fusion proteins 1 and 2 and convert them to an inactive state.
  • fusion protein 6 was randomly selected from among the fusion proteins whose effect was confirmed in hepatic stellate cells, and it was confirmed whether the same effect was exhibited in lung stellate cells.
  • Example 2 The experiment was performed in the same manner as in Example 2, and as can be seen in FIG. 5, when the astrocytes extracted from lung tissue were activated and then treated with fusion protein 6, it was confirmed that the cell shape was reversed to the initial stage of activation. In addition, it was confirmed that ⁇ -SMA expression was decreased by fusion protein 6 treatment in activated lung astrocytes.
  • the therapeutic effect of the fusion protein was comparatively evaluated using a liver fibrosis model induced by carbon tetrachloride (CCl 4 ) injection.
  • CCl 4 carbon tetrachloride
  • fusion protein 6 was administered intravenously 2, 3, 5, 6, 8, and 10 days after the bleomycin treatment day as the reference day (day 0), respectively, and the mice were sacrificed 24 hours after the final administration (FIG. 7).
  • Example 6-1 Liver fibrosis model
  • fusion proteins 3'(441Q), 4'(441Q), 4(441E), and 6(441E) were compared using a liver fibrosis model induced by intraperitoneal injection of carbon tetrachloride (CCl 4 ).
  • CCl 4 carbon tetrachloride
  • carbon tetrachloride dissolved 1:1 in mineral oil at a concentration of 1 ml/kg was intraperitoneally injected into BALB/c mice for 4 weeks, 3 times a week, and fusion proteins #3'(441Q), 4'(441Q), 4(441E) and 6(441E) were intravenously administered (12.5 ug) three times a week.
  • fusion proteins #3'(441Q), 4'(441Q), 4(441E) and 6(441E) were intravenously administered (12.5 ug) three times a week.
  • the same amount of physiological saline was intravenously administered.
  • Example 6-2 Lung fibrosis model
  • fusion proteins #3'(441Q), 4(441E), and 6(441E) were compared using a pulmonary fibrosis model induced by bronchial administration of bleomycin.
  • fusion proteins #3'(441Q), 4(441E), and 6(441E) were intravenously administered (12.5 ug), and the mice were sacrificed 24 hours after the final administration. In the negative control group, the same amount of physiological saline was intravenously administered.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • Veterinary Medicine (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Public Health (AREA)
  • Medicinal Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • General Health & Medical Sciences (AREA)
  • Epidemiology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Immunology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Organic Chemistry (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Pulmonology (AREA)
  • Peptides Or Proteins (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

La présente invention concerne une protéine de fusion d'albumine et une protéine de liaison au rétinol, qui peut être utilisée pour prévenir ou traiter des maladies fibrotiques se produisant dans le foie, le pancréas, le poumon ou similaire. La protéine de fusion de la présente invention peut prévenir ou traiter des maladies fibrotiques en inhibant l'activation des cellules stellaires, qui provoque une fibrose tissulaire, ou en induisant une activation inverse de celle-ci. La protéine de fusion de la présente invention devrait être une technologie à usage général qui peut réduire considérablement la dose et la fréquence d'administration de protéines thérapeutiques au corps humain, car la protéine de fusion présente un meilleur effet à une concentration plus faible par rapport aux protéines de fusion classiques pour le même objectif.
PCT/KR2022/015214 2021-10-08 2022-10-07 Protéine de fusion pour prévenir et traiter la fibrose WO2023059160A1 (fr)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
KR20210133732 2021-10-08
KR10-2021-0133732 2021-10-08
KR1020220127783A KR20230051415A (ko) 2021-10-08 2022-10-06 섬유증의 예방 또는 치료용 융합 단백질
KR10-2022-0127783 2022-10-06

Publications (1)

Publication Number Publication Date
WO2023059160A1 true WO2023059160A1 (fr) 2023-04-13

Family

ID=85803612

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/KR2022/015214 WO2023059160A1 (fr) 2021-10-08 2022-10-07 Protéine de fusion pour prévenir et traiter la fibrose

Country Status (1)

Country Link
WO (1) WO2023059160A1 (fr)

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20120101617A (ko) * 2011-02-28 2012-09-14 고려대학교 산학협력단 알부민과 레티놀 결합 단백질의 융합 단백질

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20120101617A (ko) * 2011-02-28 2012-09-14 고려대학교 산학협력단 알부민과 레티놀 결합 단백질의 융합 단백질

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
DATABASE Protein NCBI; 15 July 2006 (2006-07-15), ANONYMOUS : "Retinol binding protein 4, plasma [Homo sapiens]", XP093057428, Database accession no. AAH20633.1 *
DATABASE Protein NCBI; 25 September 2002 (2002-09-25), ANONYMOUS : "serum albumin [Homo sapiens] ", XP093057429, Database accession no. AAN17825.1 *
LEE HONGSIK, JEONG HYEYEUN, PARK SANGEUN, YOO WONBAEK, CHOI SOYOUNG, CHOI KYUNGMIN, LEE MIN‐GOO, LEE MIHWA, CHA DAERYONG, KIM YOUN: "Fusion protein of retinol-binding protein and albumin domain Ⅲ reduces liver fibrosis", EMBO MOLECULAR MEDICINE, vol. 7, no. 6, 1 June 2015 (2015-06-01), US , pages 819 - 830, XP055857683, ISSN: 1757-4676, DOI: 10.15252/emmm.201404527 *
PARK JI HOON, KIM JANGHYUN, CHOI SO-YOUNG, LEE BORAM, LEE JUNG-EUN, PARK HEEKYUNG, MOON JI WOOK, PARK SUN-HWA, LEE JAE MIN, LEE HO: "Albumin inhibits the nuclear translocation of Smad3 via interleukin-1beta signaling in hepatic stellate cells", SCIENTIFIC REPORTS, vol. 11, no. 1, XP093057424, DOI: 10.1038/s41598-021-82758-4 *

Similar Documents

Publication Publication Date Title
WO2017116205A1 (fr) Conjugué persistant d'un triple activateur activant le récepteur du glucagon, du glp-1 et du gip
AU2016346870B2 (en) Dual function proteins and pharmaceutical composition comprising same
WO2020050667A1 (fr) Récepteur antigénique chimérique pour un cancer solide et lymphocytes t exprimant ce récepteur chimérique de l'antigène
WO2017155288A1 (fr) Dérivé de polyéthylèneglycol et son utilisation
WO2021145552A1 (fr) Utilisation thérapeutique pour les maladies hépathiques, d'un triple agoniste ayant une activité par rapport à tous les récepteurs du glucagon, du glp-1 et du gip contre les maladies pulmonaires
WO2017116207A1 (fr) Analogue de fgf21, conjugué de fgf21 et leur utilisation
WO2022139493A1 (fr) NOUVEAU PEPTIDE POUVANT INHIBER LA SIGNALISATION DU TGF-β ET SON UTILISATION
WO2020130749A1 (fr) Composition pharmaceutique comprenant de l'insuline et un triple agoniste ayant une activité par rapport à l'ensemble des récepteurs glp1, glucagon et gip
WO2013039295A1 (fr) Nouveau variant d'alpha 1-antitrypsine, et ses procédés de préparation et d'utilisation
WO2022015082A1 (fr) Utilisation thérapeutique d'un dérivé du glucagon ou d'un conjugué de celui-ci pour une maladie hépatique
WO2021066600A1 (fr) Glucagon, composition comprenant un agoniste double du récepteur de glp-1 et du récepteur de gip et utilisation thérapeutique associée
WO2023059160A1 (fr) Protéine de fusion pour prévenir et traiter la fibrose
WO2023106845A1 (fr) Nouvel analogue et conjugué d'adiponectine
WO2022015115A1 (fr) Utilisation thérapeutique d'une combinaison contenant un conjugué à action prolongée triplement agoniste ou un triple agoniste
WO2017010790A1 (fr) Composition pour l'inhibition de l'angiogenèse contenant un complexe de nanoparticule-protéine à base de corps vitré en tant que substance active, et utilisation de celle-ci
WO2020263063A1 (fr) Utilisation thérapeutique, pour les maladies hépathiques, d'un triple agoniste ayant une activité par rapport à tous les récepteurs du glucagon, de glp-1 et de gip, ou conjugué de ceux-ci
WO2022119380A1 (fr) Nouveau variant d'eca2 et utilisation associée
WO2021215624A1 (fr) Nouveau dérivé de 2-arylthiazole ou sel de celui-ci, procédé de préparation associé et composition pharmaceutique les comprenant
WO2011111914A1 (fr) Cellules progénitrices endothéliales vasculaires introduites avec le gène d'haptoglobine et composition contenant celui-ci pour favoriser l'angiogenèse
WO2020214013A1 (fr) Utilisation thérapeutique, pour l'hyperlipidémie, d'un triple agoniste ayant une activité par rapport à tous les récepteurs du glucagon, glp -1 et gip, ou conjugué de ceux-ci
WO2022035302A1 (fr) Composition pharmaceutique à effet hypotenseur comprenant un activateur triple présentant une activité pour tous les récepteurs du glucagon, de la glp-1, et de la gip
WO2019035672A1 (fr) Analogue peptidique d'oxyntomoduline acylée
WO2023121371A1 (fr) Substance ciblant le foie et son utilisation
WO2022080991A1 (fr) Utilisation d'un triple activateur ayant une activité sur tous les récepteurs du glucagon, du glp-1 et du gip pour le traitement des séquelles d'une maladie infectieuse respiratoire
WO2021187904A1 (fr) Protéine de fusion comprenant une protéine il-2 et une protéine cd80 et utilisations associées

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 22878981

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE