Classifications

• • 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
  • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • A61K38/19—Cytokines; Lymphokines; Interferons
  • A61K38/21—Interferons [IFN]
  • A61K38/212—IFN-alpha
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
  • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
  • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
  • A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
  • A61K47/60—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
  • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/12—Antivirals
  • A61P31/14—Antivirals for RNA viruses
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/52—Cytokines; Lymphokines; Interferons
  • C07K14/555—Interferons [IFN]
  • C07K14/56—IFN-alpha
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2319/00—Fusion polypeptide
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2319/00—Fusion polypeptide
  • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
  • C07K2319/10—Fusion polypeptide containing a localisation/targetting motif containing a tag for extracellular membrane crossing, e.g. TAT or VP22

Definitions

• the present invention relates to an interferon-alpha fusion protein in which a cytoplasmic transduction peptide (CTP) and polyethylene glycol are bound together to an interferon-alpha (IFNa) protein.
• CTP cytoplasmic transduction peptide
• IFNa interferon-alpha
• Interferon is used to treat hepatitis. It is sold as an interferon-alpha preparation (Intron-A: Schering, Roferon-A: Roche) and a PEGylated IFN preparation (PEG-Intron: Schering, Pegasys: Roche), which reduces the frequency of administration than the interferon-alpha preparation.
• Interferon-alpha preparation Intron-A: Schering, Roferon-A: Roche
• PEG-Intron Schering, Pegasys: Roche
• interferon As described above, as the side effects of the dosage of interferon are known, the use of interferon as a therapeutic agent is required to reduce the dose to minimize the side effects.
• the present inventors have tried to develop an interferon-alpha fusion protein which improves the pharmaceutical efficacy of the conventional interferon through protein modification such as binding of foreign peptide and polyethylene glycol.
• protein modification such as binding of foreign peptide and polyethylene glycol.
• CTP cytoplasmic residual cell membrane-penetrating peptide
• an object of the present invention is to provide an interferon-alpha fusion protein which can be expected to reduce the interferon dosage as a result of improving the liver migration and retention characteristics of the existing interferon fusion protein.
• Another object of the present invention is to provide a nucleic acid molecule ' encoding the interferon-alpha fusion protein.
• Another object of the present invention to provide a vector comprising the nucleic acid molecule *
• Another object of the present invention to provide a transformant comprising the vector.
• Another object of the present invention to provide a pharmaceutical composition for preventing or treating liver disease.
• the invention is an interferon-alpha fusion protein represented by CTP-X-IFNa-Y-PEG, wherein the CTP is a cytoplasmic transduction peptide; X is a peptide linker consisting of 1-10 glycines; Y is 1-100 cysteine, glycine, or a peptide linker consisting of glycine and cysteine; IFNa is interferon-alpha 2a or interferon-alpha 2b;
• the PEG provides an interferon-alpha fusion protein, characterized in that polyethylene glycol.
• the present inventors have tried to develop an interferon-alpha fusion protein which improves the pharmaceutical efficacy of the conventional interferon through protein modification such as binding of foreign peptide and polyethylene glycol.
• protein modification such as binding of foreign peptide and polyethylene glycol.
• CTP cytoplasmic residual membrane permeation peptide
• interferon-alpha (IFNa) fusion protein refers to a protein in which only CTP is bound to an interferon-alpha protein or a protein in which CTP and PEG are simultaneously bound.
• IFNa fusion protein refers to a protein in which only CTP is bound to an interferon-alpha protein or a protein in which CTP and PEG are simultaneously bound.
• CTP-X-IFNa-Y refers to a protein in which only CTP is bound to the interferon-alpha protein.
• CTP cytoplasmic residual cell membrane permeation peptide
• CTP included in the IFNa fusion protein of the present invention was filed by the present inventors as a delivery peptide developed by the present inventors to solve the problem of PTD, and has been patented (Korean Patent No. 0608558, US Patent No. 7101844 and Japanese Patent No. 4188909), and the contents of the CTP disclosed in the patent document are inserted as the contents of the present invention.
• the length of the peptide corresponds to the general length accepted in the art, preferably 9-20 amino acids, more preferably 9-15 amino acids, most preferably 11 amino acids.
• the CTP included in the IFNa fusion protein of the present invention comprises an amino acid sequence selected from the group consisting of the sequences set forth in SEQ ID NO: 1 to SEQ ID NO: 14.
• the CTP in the present invention comprises the sequence of SEQ ID NO: 1.
• CTP has a high cell membrane permeability and has a property of inhibiting migration to the nucleus even after being introduced into a cell, and thus, cytotoxicity is higher than that of other transporters (especially, PTE protein transduction domain or polyarginine) in vivo. It is low and has the unique property of transporting fused proteins to the liver, making it an effective drug delivery peptide for the treatment of diseases targeted to the liver.
• IFNa interferon-alpha
• Interferon-alpha (IFNa) included in the IFNa fusion protein of the present invention is a purified mixture of recombinant IFNa 2b, recombinant IFNa 2a, recombinant IFNa 2c, native alpha-interferon, IFNa-nl, consensus alpha-interferon (US Patent No. 4,897,471 and 4695623) or mixtures of natural alpha-interferons, IFNa-n3, more preferably IFNa 2a or IFNa 2b, most preferably IFNa 2b.
• the process for the preparation of IFNa 2b is described in detail in US Pat. No. 4,530901.
• IFNa included in the IFNa fusion protein of the present invention comprises the amino acid sequence set forth in SEQ ID NO: 21.
• X is a peptide linker consisting of 1-10 glycine residues. This peptide linker binds CTP to the N-terminus of IFNa.
• CTP peptides By linking between CTP and IFNa with a peptide linker X, CTP peptides can be flexibly and freely rotated to allow CTP to perform its original functions.
• Purification yield can be increased by minimizing the precipitation of fusion proteins caused by CTP.
• precipitation occurred when the CFN1 protein (CTP-IFNa) was purified, but precipitation did not occur with the CFN8 protein (CTP-GGGG-IFNa) to which the peptide linker was bound.
• the depth linker X may be 1-10 glycine residues, preferably 1-8 glycine residues, more preferably 1-7 glycine residues, more preferably 1-5. Preferably 3-5 glycine residues, and may be four.
• Y is a peptide linker consisting of 1-100 cysteine residues, glycine residues or glycine and cysteine residues. Through this peptide linker, polyethylene glycol (PEG) is bound to the C-terminus of IFNa.
• the peptide linker Y may be composed of 0-10 glycine residues and 1-80 cysteine residues, more preferably 0-10 glycine residues and 1-60 cysteine residues, more preferably 1-10 glycine Residues and 1-10 cysteine residues, even more preferably 1-5 glycine residues and 1-5 cysteine residues.
• the peptide linker Y may be composed of 0-10 glycine residues or 1-80 cysteine residues, more preferably 1-10 glycine residues or 1-60 cysteine residues, and more preferably May consist of 1-10 glycine residues or 1-10 cysteine residues, more preferably 1-5 glycine residues or 1-5 cysteine residues, and most preferably 3-5 glycine residues or It may consist of 3-5 cysteine residues.
• the peptide moiety represented by "CTP-X-IFNa- ⁇ in the interferon-alpha (IFNa) fusion protein of the present invention is a sequence disclosed in SEQ ID NO: 15 to SEQ ID NO: 20 It is a peptide consisting of the amino acid sequence selected from the group consisting of, more preferably a peptide consisting of the amino acid sequence of SEQ ID NO: 18.
• the amino acid sequence of the peptide moiety represented by "CTP-X-IFNa-Y” is interpreted to include an amino acid sequence showing substantial identity with respect to any of the amino acid sequences of SEQ ID NO: 15 to SEQ ID NO: 20.
• the above substantial identity is at least 80 when the amino acid sequence of the present invention and any other sequence are maximally aligned and analyzed using an algorithm commonly used in the art.
• amino acid sequence it represents% identity, more preferably at least 90% identity, and most preferably at least 95% identity.
• Polyethylene glycol (PEG) is bound to the C-terminus of the IFNa fusion protein of the invention.
• PEG has the effect of reducing the dose of the fusion protein of the present invention by reducing the mechanism of renal clearance to prolong half-life in the blood.
• the molecular weight of the polyethylene glycol (PEG) is 20-60 kDa, more preferably 20-50 kDa, even more preferably 30-45 KDa, more preferably 35-45 KDa, most preferably 40-45 kDa.
• the use of 40-45 KDa PEG has the greatest hepatic mobility enhancement effect.
• the polyethylene glycol (PEG) may be linear PEG (linear PEG) or branched PEG branched PEG (PEG).
• linear PEG refers to a PEG form in which PEG is composed of a single chain without branches
• branched PEG refers to a central core group in which two to ten PEG chains are formed. It is a form branching out from).
• the PEG of the present invention is a linear PEG, specifically bound to a specific amino acid residue of the interferon alpha protein, it is easy to separate and purify the final PEG-interferon alpha fusion protein, which is advantageous in quality control of the protein.
• Pegylation reaction to bind the PEG to the IFNa fusion protein of the present invention can be carried out through a method known in the art [MJ Roberts, MD Bent ley et al. , Chemistry for peptide and protein PEGylat ion, Advanced Drug Delivery Reviews 54: 459476 (2002); Francesco M., Peptide and protein PEGylation: a review of problems and solut ions, Veronese Biomaterials 22: 405—417 (2001).
• the present invention provides a nucleic acid molecule encoding a peptide moiety represented by "CTP-X-IFNa-Y" in the above-described interferon-alpha (IFNa) fusion protein. do.
• nucleic acid molecule is meant to encompass DNA (gDNA and cDNA) and RNA molecules inclusively, and the nucleotides, which are the basic structural units in nucleic acid molecules, are modified from sugar or base sites, as well as natural nucleotides. Analogs are also included (Scheit, Nucleotide Analogs, John Wiley, New York (1980); Uhlman and Peyman, Chemical Reviews, 90: 543-584 (1990)).
• the nucleic acid molecule encoding the peptide moiety represented by "CTP-X-IFNa-Y" in the IFN- ⁇ fusion protein of the present invention is any one of SEQ ID NO: 15 to SEQ ID NO: 20
• a nucleic acid molecule encoding a peptide consisting of an amino acid sequence more preferably a nucleic acid molecule encoding a peptide consisting of an amino acid sequence of SEQ ID NO: 18, most preferably a nucleic acid molecule having a DNA base sequence of SEQ ID NO: 22.
• the invention provides a vector comprising a nucleic acid molecule encoding a peptide moiety represented by "CTP-X-IFNa-Y" in an IFN-a fusion protein.
• the vector system of the present invention can be constructed through various methods known in the art, and specific methods thereof are described in Sambrook et al. , Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory
• Vectors of the invention can typically be constructed as vectors for cloning or vectors for expression.
• the vector of the present invention can be constructed using prokaryotic or eukaryotic cells as hosts.
• the nucleotide sequence of the present invention is a prokaryotic cell-derived, it is preferable to use a prokaryotic cell as a host in consideration of the convenience of culture.
• a strong promoter capable of promoting transcription e.g., tac promoter, lac promoter, lacUV5 promoter, lpp promoter, ⁇ promoter pR promoter, rac5 promoter, amp promoters, recA promoters, SP6 promoters, trp promoters and T7 promoters, etc.
• ribosomal binding sites and transcription / detox termination sequences for initiation of translation.
• E. coli is used as the host cell, the promoter and operator site of the E. coli tryptophan biosynthesis pathway (Yanofsky, C., J.
• vectors that can be used in the present invention are often used in the art, such as polasmids (eg pSClOl, ColEl, pBR322, pUC8 / 9, pHC79, pUC19, pET, etc.), phage (eg Xgt4 B, ⁇ -Charon , ⁇ ⁇ and M13, etc.) or viruses (eg SV40, etc.).
• polasmids eg pSClOl, ColEl, pBR322, pUC8 / 9, pHC79, pUC19, pET, etc.
• phage eg Xgt4 B, ⁇ -Charon , ⁇ ⁇ and M13, etc.
• viruses eg SV40, etc.
• a promoter derived from the genome of the mammalian cell for example, a metallothionine promoter
• a promoter derived from a mammalian virus Examples: adenovirus late promoter, vaccinia virus 7.5K promoter, SV40 promoter, cytomegalovirus promoter and tk promoter of HSV can be used and generally have a polyadenylation sequence as a transcription termination sequence.
• the sequence to be fused for example, glutathione S-transferase (Pharmacia, USA), maltose binding protein (NEB, USA), FLAG (IBI, USA), 6x His (hexahistidine; Quiagen, USA) and the like can be used. It is not limited to this.
• the peptide represented by "CTP-X-IFNa-Y" expressed by the vector of the present invention is purified by cation exchange chromatography and gel filtration chromatography.
• the expression vector of the present invention as an optional marker, and may include antibiotic resistance genes commonly used in the art, for example, ampicillin, gentamicin, carbenicillin, chloramphenicol, straptomycin, kanamycin, geneeti There are genes resistant to leucine, neomycin and tetracycline.
• the present invention provides a transformant comprising the vector of the present invention described above.
• Host cells capable of stably and continuously cloning and expressing the vector of the present invention may use any host cell known in the art, for example, E. coli JM109, E. coli BL2KDE3), E. coli RR1, E . coli LE392, E. coli B, E. coli X 1776, E. coli W3110, Bacillus subtilis, bunch, Bacillus spp, and Salmonella typhimurium as Bacillus Chuo ringen cis Titanium, Serratia, and various Pseudomonas species Marseille CL Enterobacteria and strains, such as.
• a host cell when transforming the vector of the present invention into a eukaryotic cell, as a host cell, it can be used as a host cell, including oSaccharomyce cerevisiae, gonocytes and human cells (eg, CH0 cell line (Chinese hamster ovary), W138, BHK, COS -7, 293, HepG2, 3T3, RIN and MDCK cell lines] and the like can be used.
• the method of carrying the vector of the present invention into a host cell is performed by the CaCl 2 method (Cohen, SN et al., Proc. Natl. Acac. Sci. USA, 9: 2110-2114 (1973), when the host cell is a prokaryotic cell). ), One method (Cohen, SN et al., Proc. Natl. Acac. Sci. USA, 9: 2110-2114 (1973); and Hanahan, D., J. Mo J. Biol., 166: 557- 580 (1983)) and electroporation methods (Dower, WJ et al., Nucleic. Acids Res., 16: 6127-6145 (1988)) and the like.
• the host cell is a eukaryotic cell
• a micro-injection method Capecchi® MR, Cell, 22: 479 (1980)
• calcium phosphate precipitation method Graham, FL et al., Virology, 52: 456 (1973)
• Electroporation Necmann, E. et al., EMBO J., 1: 841 (1982)
• liposome-mediated transfection Wong, TK et al., Gene, 10:87 (1980)
• DEAE- Dextran treatment Gopal, Mo J. Cell Biol., 5: 1188-1190 (1985)
• gene balm Yang et al., Proc. Natl. Acad.
• Vector can be injected into the host cell.
• Vectors injected into a host cell can be expressed in the host cell, in which case a large amount of "CTP-X-IFNa-Y" peptide is obtained.
• the expression vector contains a lac promoter
• the host cell may be treated with ' IPTG to induce gene expression.
• the method for preparing the IFN-a fusion protein of the present invention includes the above-described method for preparing the peptide represented by "CTP-X—IFNa-Y" of the present invention. In order to avoid excessive complexity of the specification according to the description, the description is omitted.
• the present invention provides a method for preparing an IFNa fusion protein, comprising the following steps: (a) represented by "CTP-X-IFNa-Y" operably linked to a promoter.
• promoter refers to a DNA base sequence that regulates the expression of a coding sequence or functional RNA.
• operably linked refers to a functional bond between a nucleic acid expression control sequence (eg, an array of promoters, signal sequences, or transcriptional regulator binding sites) and other nucleic acid sequences, whereby the regulatory sequence Will regulate transcription and / or translation of said other nucleic acid sequences.
• a nucleic acid expression control sequence eg, an array of promoters, signal sequences, or transcriptional regulator binding sites
• the medium for transformant culture may be any natural medium or synthetic medium as long as it contains a carbon source, nitrogen source, inorganic salt, etc. which can be efficiently used by prokaryotic or eukaryotic cells.
• Cultivation of the transformants is usually carried out under aerobic conditions such as by shaking culture or rotation by a rotator.
• Incubation temperature is preferably carried out at 15 to 50 ° C, incubation time is generally 5 hours To 7 days.
• the pH of the medium is preferably maintained in the range of 3.0 to 9.0 in culture.
• the pH of the medium can be adjusted with inorganic or organic acids, alkaline solutions, urea, calcium carbonate, ammonia, and the like.
• antibiotics such as ampicillin and tetracycline can be added if necessary.
• the method for isolating the foreign recombinant protein expressed from the cells of the transformant cultured in the present invention can be carried out using a method for isolating and purifying proteins commonly used in the art. For example, solubility fractionation using ammonium sulfate or PEG, ultrafiltration separation according to molecular weight, separation by various chromatography (manufactured for separation according to size, charge, hydrophobicity or affinity), etc. Various methods may be used, and are typically separated and purified using a combination of the above methods.
• the present invention provides a pharmaceutical composition
• a pharmaceutical composition comprising (a) a pharmaceutically effective amount of the above-described IFNQ fusion protein of the present invention; And (b) provides a pharmaceutical composition for preventing or treating liver disease comprising a pharmaceutically acceptable carrier.
• Liver diseases prevented or treated by the composition of the present invention include liver cancer, hepatitis, cirrhosis and other liver diseases, preferably liver cancer and hepatitis, for example, the hepatitis is acute viral hepatitis, chronic hepatitis, It may be a hepatitis or the like, more preferably hepatitis B or C, and most preferably hepatitis C by HCV (Hepatitis C virus) hepatitis.
• HCV Hepatitis C virus
• the pharmaceutical composition for treating liver disease of the present invention may be preferably administered parenterally, and even more preferably subcutaneously.
• the term “pharmaceutically effective amount” means an amount sufficient to achieve the efficacy or activity of the fusion proteins of the invention described above.
• compositions of this invention are administered orally, parenterally, by in-home spray, rectally or topically as dosage unit preparations containing conventional, non-toxic carriers, adjuvants and excipients which are pharmaceutically acceptable as necessary. It may be administered, preferably parenterally. Topical administration may also include the use of transdermal administration or iontophoretic devices such as transdermal patches.
• parenteral administration includes subcutaneous injection, intravenous, intramuscular, intrasternal injection or infusion techniques.
• Drug formulations are published, for example, in Hoover, John E. Remington's Pharmaceutical Sciences, Mack Publishing Co., East on, Pennsylvania, 1975.
• For drug formulations see also Lierman, H.A. And Lachman, L., Eds., Pharmaceutical Dosage Forms, Marcel Decker, New York, N.Y. , 198.
• the pharmaceutically acceptable contained in the pharmaceutical compositions of this invention lactose, and text Ross, sucrose, know sorbitan, the manni, starch, gum acacia, calcium phosphate, carbonate, Gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyridone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil Including, but not limited to.
• the pharmaceutical composition of the present invention may further include a lubricant, a humectant, a sweetener, a flavoring agent, an emulsifier, a suspending agent, a preservative, and the like.
• a lubricant e.g., a talc, a kaolin, a kaolin, a kaolin, a kaolin, a kaolin, a kaolin, kaolin, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, a talcrate, a glycerol, a sorbitol, a sorbitol, a talcrate, a sorbitol, sorbitol, sorbi
• Suitable dosages of the pharmaceutical compositions of the present invention may vary depending on factors such as the formulation method, mode of administration, age, weight, sex, morbidity, food, time of administration, route of administration, rate of excretion and reaction in response to the patient. It may be prescribed. Preferably, the dosage of the pharmaceutical composition of the present invention may be 0.001-100 / ig / kg (body weight) on an adult basis.
• compositions of the present invention may be prepared in unit dosage form by formulating with a pharmaceutically acceptable carrier and / or excipient according to methods which can be easily carried out by those skilled in the art. Or may be prepared by incorporation into a multi-dose container.
• the formulation may be in the form of solutions, suspensions, syrups or emulsions in oils or aqueous media or in the form of extracts, powders, powders, granules, tablets or capsules, and may further comprise dispersants or stabilizers.
• the present invention relates to IFNa fusion proteins in which cytoplasmic persistent cell membrane permeation peptide (CTP) and polyethylene glycol (PEG) are bound to IFNa protein.
• CTP cytoplasmic persistent cell membrane permeation peptide
• PEG polyethylene glycol
• the IFNa fusion protein of the present invention maintains high intrinsic activity of interferon.
• the IFNa fusion protein of the present invention has an extended half-life and improved liver mobility upon in vivo administration. ⁇
• the IFNa fusion protein of the present invention can be used for the development of protein pharmaceuticals effective for the prevention or treatment of liver diseases including various viral infections.
• FIG. 1 shows the structure of the INFa fusion protein of the invention wherein CTP is bound via a linker.
• CFN7 CTP-GGGGGG-IFNa, SEQ ID NO: 32
• CFN11 SEQ ID NO: 34
• CFN12C SEQ ID NO: 35 CFN13 (SEQ ID NO: 36) with Heps in Cleavage linker ) Is shown.
• Figure 2 is a result of analyzing the secondary structure of the IFNa fusion protein of the present invention prepared by binding the linker using a CD spectrophotometer (Jasco-815).
• FIG. 3 shows CTP-IF a -PEG fusion proteins in which PEG is bound to CTP—INFa fusion protein in various forms.
• Figure 4 shows the comparison of preclinical pharmacokinetic measurements using the monkey of the IFNa fusion protein 401C and PEGASYS (Hoffman LaRoche Ltd.) of the present invention. The samples used in the experiment were administered at a dose of 300 ⁇ g / kg body weight, respectively, for PEGASYS and IFNa fusion protein 401C. Activity was measured for 6 days after the sample was administered once. IFNa fusion protein 401C of the present invention has an IFNa activity It was confirmed that the activity was higher and maintained better or equivalent to PEGASYS until 6 days.
• FIG. 5a and 5b show the results of comparing the liver mobility of the fusion protein of the present invention and the control drug.
• a CTP peptide was inserted at the N-terminus of human-derived interferon-alpha (IFNa), and a glycine linker consisting of 4 or 6 glycines was inserted between CTP and IFNa.
• IFNa human-derived interferon-alpha
• the glycine linker provides flexibility to the CTP peptide to facilitate its function, and also minimizes precipitation due to hydrophobic CTP during purification.
• cysteine cysteine
• PEG was able to attach PEG to C- ' end in response to Maleimide PEG. By attaching linear PEG at the C-terminus, half-life in the body could be greatly improved.
• PCR was performed using the primers described in Table 1 below.
• IFNa was used as a template and primary PCR was performed using PR2 and PR3 primers.
• the PCR product was again templated and subjected to secondary PCR using PR1 and PR2 primers. This gave a nucleic acid molecule encoding peptide CTP-GGGGGG-IFNa linked to a linker consisting of six glycine as a final product.
• the final product CTP-GGGGGG-IFNa protein was named CFN7 (SEQ ID NO: 32).
• CTP-GGGG-IFN a was used as a template and primary PCR was performed using PR2 and PR4 primers. The resulting PCR product was again used as a template and subjected to secondary PCR using PR1 and PR2 primers. As a result, a nucleic acid molecule encoding CTP-GGGG-IFN a linked with a linker composed of four glycines was obtained as a final product. This final product CTP-GGGG-IFN a protein was named CFN8 (SEQ ID NO: 33).
• PCR was performed using the primers described in Table 1 below. IFNa was used as a template and primary PCR was performed using PR2 and PR5 primers. The resulting PCR product was again used as a template and subjected to secondary PCR using PR1 and PR2 primers. As a result, a nucleic acid molecule encoding peptide CTP-KQLRWNG-IFNa linked to a linker composed of hepsin cleavage sequences was obtained. The final product CTP-KQLRWNG-IFNa protein was named CFN11 (SEQ ID NO: 34).
• PCR was performed using the primers described in Table 1 below. IFNA * template was used and primary PCR was performed using PR2 and PR7 primers. The resulting PCR product was again used as a template and subjected to secondary PCR using PR1 and PR2 primers.
• CTP-GGGGKQLRWNGGGG-IFNa a nucleic acid molecule encoding the peptide CTP-GGGGKQLRWNGGGG-IFNa, which was linked to a hepsin cleavage sequence and a linker consisting of four glycines and three glycines, respectively, was obtained as a final product.
• the final product CTP-GGGGKQLRWNGGGG-IFNa protein was named CFN12 (SEQ ID NO: 35).
• PCR was performed using the primers shown in Table 1 below. IFNa was used as a template and primary PCR was performed using PR2 and PR8 primers. Regenerate PCR product As a template, secondary PCR was performed using the PR1 and PR2 primers. As a result, a nucleic acid molecule encoding the peptide CTP-AKTKQLRWNGGGG-IFNa linked to a hepacyte growth factor activator and a linker consisting of three glycines was obtained as a final product. The final product CTP-AKTKQLRWNGGGG-IFNa protein was named CFN13 (SEQ ID NO: 36).
• Example 2 Preparation of E. coli expression vector of interferon-alpha fusion protein CFN7CCTP-GGGGGG-IFN ⁇ ), CFN8 (CTP-GGGG-IFN ⁇ ), CFN8C (CTP-GGGG-IFNa-C), CFNlKCTP obtained by PCR Cloning the respective genes in pCFM536, an E. coli expression vector, to express the respective genes encoding -KQLRWNG-IFN a), CFN12 (CTP-GGGG QLRWNGGGG-IFNa), and CFN13 (CTP-MTKQLRWNGGGG-IFN a) in E. coli.
• PCFM536-CFN7, pCFM536-CFN8, pCFM536-CFN8C, PCFM536-CFN11, pCFM536-CFN12, and pCFM536-CFN13 expression vectors were prepared. First, CFN7 (CTP-GGGGGG-IFNa), CFN8CCTP-GGGG-IFN a), CFN8CCCTP-
• GGGG-IFNa-C CFNll (CTP-KQLRWNG-IFN a), CFN 12 (CTP-GGGG QLRWNGGGG- IFNa), and CFN13 (CTP-AKTKQLRWNGGGG-IFNa) PCR products were cloned into the pGEM-T vector and sequenced, respectively.
• inserts were obtained using Xba ⁇ and Bar restriction enzymes contained in the primers during PCR, and these were introduced into the pCFM536 vector to express the expression vector pCFM536-CFN7, P. CFM536-CFN8, pCFM536-CFN8C, pCFM536-CFNll, PCFM536-CFN12, and pCFM536-CFN13 were completed, respectively.
• Example 3 Expression and Identification of Interferon-alpha Fusion Proteins in Escherichia Coli P0FM236 vector cloned with the CFN7, CFN8, CFN8C, CFNll, CFN12, and CFN13 genes prepared in Example 2 was converted into P0P2136 competent cells After transfection, the cells were plated on solid medium containing ampicillin. Colonies grown on solid media were obtained and placed on LB broth. After inoculation, the cells were incubated at 30 ° C for 18 hours. When the absorbance at 600 nm was 0.4 to 0.6, the temperature was raised to 42 ° C. to induce expression. After further incubation for 4 hours, the expression of protein was confirmed by SDS-PAGE analysis from the cultured cells.
• Example 4 Mass Production of Interferon-alpha Fusion Proteins
• a mass culture was performed using a 5 L fermentation tank (Biostat B, B. Braun Biotech Internat ional).
• the expression was stored at -80 ° C the identified strains each produced by a MCB (Master cell bank) and WCB (Working cell bank) eu 2xYT medium with 100 ⁇ before the present day culture of IFNa fusion protein (tryptone 16 g / L, yeast 'extract 10 g / L, NaCl 5 g / L) was added so that the ampicillin concentration was 5 (ig / ml) and then inoculated with the stored WCB was incubated for 16 hours at 30 ° C.
• MCB Master cell bank
• WCB Working cell bank
• the culture medium was prepared by adding 3 L 2xYT medium (tryptone 16 g / L, yeast extract 10 g / L, NaCl 5 g / L) to an ampicillin concentration of 50 yg / ml.
• the silver temperature was not lowered below 30 ° C, pH 6.8, wm 1, DO 50%, and the 0D value was measured at 1 hour intervals.When 0D value was 10, additional medium was added and the incubation temperature was 42 ° C. Expression was measured at 0 hour intervals even after expression induction so that the 0D value no longer increased.
• the culture was terminated in the section.
• washing solution 4 50 mM Tris, 1% Triton X-100, 0.3 M
• NaCl (pH8.0) was used to sequence cell suspension, agitation, washing and centrifugation.
• Washing process was carried out in order to recover the insoluble aggregate fraction.
• the aggregates are dissolved in each solubilization complete solution optimized by type.
• Protein recovery was solubilized after adding 0.5 mi of tertiary distilled water and suspending the cells.
• Acetic acid was added to adjust the pH to 4.0. More than 2 hours
• the refolded CTP-X-IFNa-Y protein was added to the column binding buffer (50 ⁇
• the secondary structure of the IFNa fusion protein prepared by binding the linker as described in the above example was analyzed.
• the secondary structure was analyzed as IFNa (interferon-a), CFNKCTP-IFN a), CFN7 (CTP-GGGGGG-IFN a), CFN8 (CTP-GGGG-IFNa), CFNll with Heps in Cleavage linker, CFN12 and CFN13 were carried out.
• Secondary structure analysis of the protein was performed using a CD spectrophotometer (Jasco-815).
• the protein to be analyzed was subjected to a final concentration of 1 mg / ml in 50 mM Sodium Citrate-Citric acid, 1 mM EDTA (pH 4.0) at 25 ° C, 0.2 nm of resolut ion and 1.0 nm. bandwidth and has a scan rate of 50 nm / min, the path length to obtain a "spectrum in the range of 190 nm to 250 nm by using a quartz cell 0.1cm.
• N-terminal pegylation is a form of PEG attached to the front of CTP of IFNa fusion protein
• internal pegylation random pegylation
• C-terminal pegylation is a form in which a linear PEG or a branched PEG is attached to the C-terminus of the IFNa fusion protein (see FIG. 3).
• IFNa fusion protein CFN8 (CTP-GGGG-IFNa, SEQ ID NO: 33) was replaced with 50 mM sodium phosphate (pH 4.5) buffer.
• IFNa fusion protein CFN8 and A 1 dehycle-PEG were mixed at 1:10.
• 1M NaBH 3 CN was added to the mixture as a reducing agent so that the final concentration was 20 mM, and the mixture was reacted at room temperature for 2 hours.
• the degree of PEGylation was confirmed by SDS-PAGE, and the PEGylated protein was finally separated by cation exchange chromatography and gel filtration chromatography.
• the PEGylation yield was about 40%.
• C-terminal pegylation was performed on CFN8C (CTP-GGGG-IFN a ′ SEQ ID NO: 18) protein with cysteine added to the C-terminus of IFNa among IFNa fusion proteins.
• PEG was attached using Maleimide-PEG to bind to the cysteine produced at C-malt of CFN8C (CTP-GGGG-IFNa-C).
• the ratio of PEG was mixed to 1:10 and 1M sodium phosphate (pH 8.0) buffer was added to a final concentration of 100 mM and reacted for 2 hours.
• the degree of PEGylation was confirmed by SDS-PAGE, and the PEGylated protein was finally separated by cation exchange chromatography and gel filtration chromatography. Pegyration yield was about 25%.
• Table 4 below shows the yield of each preparation according to the PEGylation method.
• Example 7 Determination of anti-viral activity of interferon-alpha fusion proteins
• Antiviral activity of the prepared interferon-alpha fusion proteins was determined in vitro. Antiviral activity is MDBK (Madin-Darby)
• VB cells Bovine Kidney (VB) cells (Vesicular stomatitis virus) (Korea Cell Line Bank) was measured by identifying how much of the interferon-alpha fusion proteins can alleviate it. .
• MDBK cells are lysed to 1.0 X 10 5 cells in DMEM medium, and then 200 ⁇ aliquots are generated in each well of a 96-well plate and incubated for about 18 hours in a 37 ° C, 5% CO 2 incubator.
• interferon-alpha at a concentration of 5000 pg / ml
• blood diluted to 1/50, and liver sample diluted to ⁇ 0 were prepared, and then half-diluted and treated with 100 cells.
• the cells were fixed for 30 minutes. After washing twice with PBS, 0.05% crystal violet was added to each well for 100 years and stained for 30 minutes.
• the interferon-alpha fusion protein CFN 7, CFN 8 the interferon-alpha fusion protein CFN 7, CFN 8 ,
• the anti-viral activity of the interferon-alpha fusion protein according to the PEG size and the PEGylation method was measured. Determination of anti-viral activity is based on the standard IFNa as an interferon alpha protein without CTP and PEG attached; IFNa fusion protein (CTP-X-IFNa-Y protein) with CTP attached to N-terminus but not PEG attached; Di-PEG, 202, [2 + 4] K, 401C, and 202C, which are PEG-attached IFNa fusion proteins in various forms at the N-terminus or C-terminus of the CTP-X-IFNa-Y protein, were carried out.
• Di-PEG fusion protein two linear PEGs (30 kDa) are bound to the amine group (NH 2 ) of the CFN8 protein N-terminal amino acid and the amine group (N) of the lysine residue inside the protein, respectively.
• 202K fusion proteins are the two linear PEG (20 kDa) is a CFN8 protein i of IFNa fused form which is bonded at the same time to the amine group (N3 ⁇ 4) of the internal lysine protein
• the [2 + 4JK fusion proteins are the two linear PEG (5 kDa) and four linear PEG (7.5 kDa) are IFNa fusion proteins in the form of simultaneous binding to the amine group (NH 2 ) of the internal lysine residue of CFN8 protein
• the 401C fusion protein is a linear PEG ( 40 kDa) is an IFNa fusion protein in the form of a sulfhydryl (SH) group of the C-terminal cysteine residue of CFN8C protein
• the 202C fusion protein has two linear PEG (20 kDa) C-terminus of CFN8C protein.
• Cysteine sulfide (SH) groups are in the form of IFNa fused protein to which
• mice were used as one experimental or control group (hours (4 h,
• each interferon-alpha fusion protein sample was injected subcutaneously in the back of the mouse.
• the sample used in the experiment was a fusion protein of 202K, 202C and 401C as a test group, and PEGASYS (Hoffman LaRoche Ltd.), a PEG-attached IFNa drug, was used as a control group.
• PEGASYS is an IFNa fusion protein in which 20 kDa branched PEG is randomly bound to IFNa protein.
• Two monkeys in one group were injected subcutaneously in the back of the monkey with 300 g / kg of each sample of time (4h, 8h, 12h, Id, 2d, 3d, 4d, 5d, 6d).
• the sample used the PEG-attached interferon-alpha fusion protein (401C) and the commercially available IFN- ⁇ drug PEGASYS with PEG as a control group.
• About 2.5 ⁇ blood was collected from the monkey hind limb vein before and after injection, and then placed on ice for 1 hour and centrifuged (13,000 rpm, 4 ° C, 0.5h) to recover only serum. Serum was diluted 1/50 to measure antiviral activity. The measurement results are shown in FIG. 4.
• mice Three mice were taken as one group and each sample was injected subcutaneously in the back of the mouse at a time of (4h, 8h, 12h ( Id, 2d, 3d, 4d, 5d) at a time.
• a fusion protein of 202K, 202C and 401C was used, and PEGASYS (Hoffman LaRoche Ltd.), a PEG-attached IFNa drug, was used as a control group PBS stored in the blood vessels of the liver after dissection of the mouse's belly over time.
• Hepatic perfusion was performed by injection, and when the blood was removed from the liver, the whole liver was cut and placed in a 10% sucrose buffer and washed twice with Ganol PBS.
• the liver contained a protease inhibitor. Put in PBS buffer and crushed with a grinder.
• About 1.2 ml per sample was dispensed into three 1.5 ml tubes and stored at 4 ° C. for 12 hours. After centrifugation (13,000 rpm, 4 ° C., 0.5 h), only 700 ul of supernatant was collected for each tube, and about 2 ml of each sample was collected.
• the results of the measurement of liver mobility using the mouse showed that the liver mobility of the interferon-alpha fusion protein of the present invention was about 13 times higher than that of PEGASYS and about 6 times higher than that of 202K. .
• PEGASYS has high hepatic mobility property by self PEG
• hepatic mobility of the interferon-alpha fusion protein of the present invention is remarkably high.
• 202N it was found that the mobility of the liver almost does not appear. This is probably because PEG in the form of 202N inhibited the liver mobility of CTP by PEGylating to C-terminal CTP.
• Table 7 below shows the best pharmacokinetic test groups in blood and liver.
• 401C and 202K were compared with those of PEGASYS, a control. As a result, 401C showed the best pharmacokinetic activity in blood and liver as well as the control group.
• the present invention relates to an interferon alpha fusion protein with improved liver migration and residual properties, and induces a structural change based on the structure of the interferon alpha fusion protein according to the type and length of linkers linking the fusion proteins.
• the invention by screening for non-linkers and demonstrating that the in vitro (//?) And in vivo (/ y? Vivo) properties of these fusion proteins have improved the activity, liver migration and residual properties of the interferon alpha protein. Completed.
• the fusion protein according to an embodiment of the present invention is not only improved activity and half-life compared to the existing fusion protein, it is easy to maintain and manufacture quality, it can be useful in the composition for treating liver disease.

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