Long-acting recombinant alpha-1 antitrypsin and use thereof

Abstract

The present invention relates to a recombinant alpha-1 antitrypsin and use thereof for preventing and treating alpha-1 antitrypsin deficiency, the antitrypsin having improved in vivo persistence by having a collagen mimetic peptide bound thereto. The recombinant protein according to the present invention can be mass-produced at a low cost in Escherichia coli, eliminates the concern of pathogen transmission, which conventionally occurred when supplied from human blood, and has a collagen mimetic peptide fused thereto so as to increase persistence in the blood when administered into an organ and significantly increase delivery ability to the lungs, which is the target organ.

Description

Sustained recombinant alpha-1 antitrypsin and uses thereof

[0001] The present invention relates to a sustained-release recombinant alpha-1 antitrypsin and its use, and more particularly, to a recombinant alpha-1 antitrypsin with enhanced in vivo persistence by binding a collagen-mimetic peptide, and its use for preventing and treating alpha-1 antitrypsin deficiency.

[0002] Alpha-1 antitrypsin deficiency (AATD), an autosomal dominant inherited disorder caused by point mutations, insertions and deletions, or deletions in the gene, is estimated to affect more than 3 million people worldwide and is known to present with lower extremity emphysema at a relatively young age. Most cases are first diagnosed in infants with long-term obstructive jaundice, but severe liver dysfunction can also occur in growth and adulthood. In these patients, serum alpha-1 antitrypsin levels are approximately 35% lower than in normal individuals (Brantly M, Nukiwa T, Crystal RG. Molecular basis of alpha-1-antitrypsin deficiency. The American journal of medicine. 1988;84(6a):13-31.).

[0003] Alpha-1 antitrypsin (AAT or A1AT) is a 52-kDa glycoprotein encoded by the 12.2-kb SERPINA1 gene, consisting of seven exons and six introns, located on chromosome 14q32.1 and synthesized primarily in hepatocytes. It belongs to the serpin family, is normally present in serum levels in the range of 1.5–3.5 g / L, and belongs to the largest class of protease inhibitors, functioning as a regulatory enzyme that protects the lung from post-inflammatory proteolytic damage caused by the proteolytic enzyme neutrophil elastase.

[0004] Among the A1AT alleles, the Z allele (Glu342Lys) causes the most severe AATD in plasma. In contrast, the S allele (Glu264Val), another allele frequently observed in the Americas and Europe, causes milder AATD. Looking at the average prevalence of SZ by European region, Portugal has the highest prevalence at 1:205, while Russia has the lowest prevalence at 1:25,000. Compared to normal M homozygotes, MZ heterozygotes produce 60% of the A1AT level, and Z homozygotes exhibit liver dysfunction more than 90% of the time, showing symptoms similar to those of SERPINA1 gene deficiency. Since the clinical manifestations of AATD and emphysema were first described by Laurel and Eriksson in 1963, the association between AATD and liver disease has been defined.

[0005] To treat AATD, augmentation therapy is applied by increasing the body's concentration using intravenous injection of A1AT. This augmentation therapy can 1) delay the progression of emphysema, 2) improve the frequency of exacerbations, and 3) improve the quality of life.

[0006] Currently, the only drugs approved by the Food and Drug Administration (FDA) for the treatment of AATD include Glassia (Kamada Ltd.), Aralast NP (Baxter Healthcare Corp.), and Prolastin-C (Talecris biotherapeutics, Inc.). These are all therapies based on proteins isolated from donor blood. However, production of these protein therapies is limited, and there is a risk that the infectious agent can be transmitted to AATD patients if the donor is infected.

[0007] Meanwhile, various methods are being developed to extend the in vivo half-life of protein pharmaceuticals. First, glycosylation is essential for protein folding and tertiary structure formation, and it is also essential for protein stability for specific interactions. Glycans block protease degradation, which can positively affect protein half-life. Furthermore, modifying or adding glycosylation sites to glycoproteins can decrease glomerular filtration, extending in vivo half-life. Second, recombinant proteins can be produced by fusion with an Fc domain. Fc-fused recombinant proteins bind to the neonatal Fc receptor (FcRn) via the Fc domain of human immunoglobulin G1 (IgG1). pH-dependent binding to FcRn has been demonstrated to extend the half-life of Fc-fused recombinant proteins, demonstrating their safety and efficacy. Finally, covalent linkage between recombinant proteins and polyethylene glycol (PEG) can be applied to physiologically active molecules such as proteins and peptides, which are limited by poor pharmacokinetic (PK) properties in vivo. This can block the access of other molecules, increase drug half-life, and reduce immunogenicity. Furthermore, renal clearance is influenced by protein size, so PEG-linked proteins are eliminated from the body at a much slower rate. Currently, more than 10 PEGylated drugs are FDA-approved, but some patients exhibit adverse reactions due to PEG sensitivity.

[0008] The present inventors have developed a method for mass-producing a recombinant protein in which a collagen-mimetic peptide is combined with human-derived A1AT in E. coli in order to solve the problems of excessively high production cost of alpha-1 antitrypsin, which was previously separated from donor blood, and the risk of transmission of infectious agents, thereby completing the present invention.

[0009] The present invention aims to provide a novel recombinant protein having improved productivity and in vivo persistence, a nucleic acid molecule encoding the same, a recombinant vector comprising the nucleic acid molecule, a recombinant cell into which the recombinant vector has been introduced, and a method for producing the recombinant protein using the recombinant cell.

[0010] The present invention also aims to provide a use of the recombinant protein for preventing and / or treating alpha-1 antitrypsin deficiency.

[0011] To achieve the above purpose, the present invention provides a recombinant protein in which a collagen-mimetic peptide is fused to alpha-1 antitrypsin.

[0012] In the present invention, the collagen-mimetic peptide may be (Xaa-Yaa-Gly)n (wherein Xaa and Yaa are each independently any one amino acid selected from the group consisting of proline, 4S-hydroxyproline, fluoroproline, chloroproline, lysine, glutamic acid, cysteine, and methionine, and n is an integer from 5 to 15) or (Gly-Pro-Pro)n (wherein n is an integer from 5 to 15).

[0013] In the present invention, the collagen-mimetic peptide may be fused to the N-terminus and / or C-terminus of alpha-1 antitrypsin.

[0014] In the present invention, the recombinant protein may be used for preventing or treating alpha-1 antitrypsin deficiency.

[0015] In the present invention, the recombinant protein may have an extended half-life in the body by fusing a collagen-mimetic peptide to alpha-1 antitrypsin.

[0016] In the present invention, the recombinant protein may be administered intravenously to extend its half-life in the body.

[0017] The present invention also provides a nucleic acid molecule encoding the recombinant protein.

[0018] The present invention also provides a recombinant vector comprising the nucleic acid molecule.

[0019] The present invention also provides a recombinant cell into which the recombinant vector has been introduced.

[0020] The present invention also provides a method for producing the recombinant protein, comprising the following steps:

[0021] (a) a step of culturing the recombinant cells; and

[0022] (b) a step of recovering the recombinant protein from the recombinant cell.

[0023] In the present invention, the recombinant cell may be E. coli.

[0024] In the present invention, the step (b) may be to recover the recombinant protein by crushing and purifying the recombinant cell.

[0025] The present invention also provides a pharmaceutical composition for preventing or treating alpha-1 antitrypsin deficiency, comprising the recombinant protein as an active ingredient.

[0026] In the present invention, the pharmaceutical composition may be characterized by intratracheal administration.

[0027] The present invention also provides a method for preventing or treating alpha-1 antitrypsin deficiency, comprising administering the recombinant protein or the pharmaceutical composition to a subject in need thereof.

[0028] The present invention also provides a use of the recombinant protein or the pharmaceutical composition for preventing or treating alpha-1 antitrypsin deficiency.

[0029] The present invention also provides the use of the recombinant protein or the pharmaceutical composition for the manufacture of a drug for preventing or treating alpha-1 antitrypsin deficiency.

[0030] The recombinant protein according to the present invention has the advantages of being able to be mass-produced at low cost in E. coli, eliminating the concern of infectious agent transmission that occurred when supplied from human blood in the past, and increasing blood persistence and significantly increasing delivery to the target organ, the lungs, when administered intravenously due to the fusion of a collagen-mimetic peptide.

[0031] Figure 1 shows the structure in which the hA1AT gene and the CMP-hA1AT-CMP gene are inserted into the pET-28b vector.

[0032] Figure 2A shows the expected structures of hA1AT protein and CMP-hA1AT-CMP protein, Figure 2B shows the western blot results of purified hA1AT protein and CMP-hA1AT-CMP protein, Figure 2C shows the MALDI-TOF analysis results of hA1AT protein, and Figure 2D shows the MALDI-TOF analysis results of CMP-hA1AT-CMP protein.

[0033] Figure 3 shows the results of measuring the elastase activity of hA1AT protein and CMP-hA1AT-CMP protein.

[0034] Figure 4 shows the results comparing the collagen binding ability of CMP-hA1AT-CMP protein with hA1AT protein.

[0035] Figure 5A shows the results of analyzing the blood concentrations after intravenous administration of hA1AT protein and CMP-hA1AT-CMP protein to mice, respectively, and Figure 5B shows the results of analyzing the blood concentrations after intratracheal administration of hA1AT protein and CMP-hA1AT-CMP protein to mice, respectively.

[0036] Figure 6A shows the results of analyzing the concentrations in tissues (lung, liver, kidney) and blood 30 minutes after intravenous or intratracheal administration of hA1AT protein and CMP-hA1AT-CMP protein to mice, respectively, and Figure 6B shows the results of analyzing the concentrations in tissues (lung, liver, kidney) and blood 4 hours after intravenous or intratracheal administration of hA1AT protein and CMP-hA1AT-CMP protein to mice, respectively.

[0037] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. In general, the nomenclature used herein and the experimental methods described below are well known and commonly used in the art.

[0038]

[0039] Alpha-1 antitrypsin deficiency (AATD) occurs when alpha-1 antitrypsin (A1AT) production is reduced or altered, leading to impaired lung function and altered collagen structure. To treat AATD, alpha-1 antitrypsin isolated and purified from donor blood is administered. However, this drug is expensive to produce and carries the risk of transmitting infectious agents.

[0040] To compensate for these shortcomings, the present invention developed a technology for producing alpha-1 antitrypsin in E. coli, and when producing alpha-1 antitrypsin in E. coli, the protein can be produced in a short period of time through a simple process, resulting in excellent efficiency and economic feasibility in drug manufacturing.

[0041] Meanwhile, in the present invention, a method for extending the half-life in vivo was sought to improve the efficacy of alpha-1 antitrypsin, and specifically, when producing alpha-1 antitrypsin by fusing a collagen mimetic peptide (CMP), not only was the half-life in the blood improved, but it was also confirmed that it could bind to both normal and destroyed collagen structures in the lungs, and the molecular weight of the recombinant protein was increased, thereby reducing renal clearance.

[0042] Specifically, the collagen-mimetic peptide according to the present invention is one of the best peptide sequences that binds to natural collagen, and can strongly bind with high affinity to both normal and disrupted collagen structures, and it was confirmed that it can maintain high resistance to serum degradation, a high level of stability in vivo, and can improve the half-life. In addition, it was confirmed that the collagen-mimetic peptide according to the present invention can increase the protein size by increasing the molecular weight by about 8 kDa, thereby reducing the renal clearance rate, and when bound to collagen in the body, it can be eliminated more slowly.

[0043] Accordingly, the present invention relates, in one aspect, to a recombinant protein in which a collagen-mimetic peptide is fused to alpha-1 antitrypsin.

[0044] In the present invention, “alpha-1 antitrypsin is fused with a collagen-mimicking peptide” can be used with the same meaning as “alpha-1 antitrypsin is bonded with a collagen-mimicking peptide” or “alpha-1 antitrypsin is linked with a collagen-mimicking peptide” or “alpha-1 antitrypsin is conjugated with a collagen-mimicking peptide.”

[0045] In the present invention, the alpha-1 antitrypsin and collagen mimetic peptide may be directly linked and expressed as a single fusion peptide in one embodiment, and may be linked via a linker in another embodiment.

[0046] In the present invention, the linker may be a known linker used in producing a fusion peptide, for example, a known peptide linker, but is not limited thereto. For example, the peptide linker may be a sequence in which 1 to 5 amino acids selected from glycine, alanine, leucine, isoleucine, proline, serine, threonine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, lysine, and arginine are repeated 1 to 10 times.

[0047] In the present invention, the alpha-1 antitrypsin may be of human origin and may be characterized by the amino acid sequence represented by SEQ ID NO: 1, but is not limited thereto, and alpha-1 antitrypsin is interpreted to mean that it includes isoforms or SNPs thereof, and also includes variants in which amino acid residues are conservatively substituted at specific amino acid residue positions.

[0048] In the present invention, the alpha-1 antitrypsin may be a codon-optimized amino acid sequence having the signal peptide excluded from the amino acid sequence of SEQ ID NO: 1.

[0049] In the present invention, the signal peptide sequence may be MPSSVSWGILLLAGLCCLVPVSLA.

[0050]

[0051] Sequence number 1. Human alpha-1 antitrypsin

[0052]

[0053]

[0054] Sequence number 2. Gene sequence of human alpha-1 antitrypsin (K01396.1)

[0055]

[0056]

[0057] As used herein, “conservative substitution” means a modification of alpha-1 antitrypsin that involves replacing one or more amino acids with amino acids having similar biochemical properties that do not result in loss of biological or biochemical function of the alpha-1 antitrypsin.

[0058] A "conservative amino acid substitution" is a substitution that replaces an amino acid residue with an amino acid residue having a similar side chain. Classes of amino acid residues with similar side chains are well-defined and known in the art. These classes include amino acids with basic side chains (e.g., lysine, arginine, histidine), amino acids with acidic side chains (e.g., aspartic acid, glutamic acid), amino acids with uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), amino acids with non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), amino acids with beta-branched side chains (e.g., threonine, valine, isoleucine), and amino acids with aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).

[0059] It is expected that the alpha-1 antitrypsin of the present invention may still retain activity even if it has conservative amino acid substitutions.

[0060] In addition, the human alpha-1 antitrypsin according to the present invention is interpreted to mean an alpha-1 antitrypsin having substantially the same function and / or effect as the alpha-1 antitrypsin according to the present invention and having an amino acid sequence homology of 80% or 85% or more, preferably 90% or more, more preferably 95% or more, and most preferably 99% or more.

[0061] It should be understood that the alpha-1 antitrypsin according to the present invention also includes those having substantially the same effect as the alpha-1 antitrypsin according to the present invention, even if some amino acid residues in the N-terminal, C-terminal or internal amino acid sequence are truncated or substituted.

[0062] In the present invention, the collagen-mimetic peptide may be characterized by being selected from the group consisting of (GPP)n (wherein n is a natural number from 5 to 15), but is not limited thereto.

[0063] In another embodiment, the collagen mimetic peptide comprises, consists essentially of, or consists of an amino acid sequence which is a multimeric repeat of a specific tripeptide having the sequence (Xaa-Yaa-Gly)n, wherein Xaa is independently selected from the group consisting of proline, 4S-hydroxyproline, fluoroproline, chloroproline, lysine, glutamic acid, cysteine, and methionine; wherein Yaa is independently selected from the group consisting of proline, 4R-hydroxyproline, fluoroproline, chloroproline, lysine, glutamic acid, cysteine, and methionine; wherein Gly is a glycine residue; and wherein n is an integer in the range of 1 to 20, such as 3 to 15, 5 to 15, or 5 to 10, and preferably 5, 6, 7, 8, 9, or 10.

[0064] In the present invention, the collagen-mimetic peptide may be characterized in that it is fused to the N-terminus and / or C-terminus of alpha-1 antitrypsin.

[0065] In a preferred embodiment, the collagen mimetic peptide is (GPP) 10 and may be characterized by being fused to the N-terminus and C-terminus of alpha-1 antitrypsin, but is not limited thereto.

[0066] In the present invention, the recombinant protein may be characterized as being for the prevention or treatment of alpha-1 antitrypsin deficiency.

[0067] In particular, in the present invention, the recombinant protein has the characteristic of extending the half-life in the body by fusing a collagen-mimetic peptide to alpha-1 antitrypsin, and can have the characteristic of extending the half-life in the body when administered intravenously.

[0068] The above characteristics may be due to the property of collagen-mimicking peptides to effectively bind to collagen existing in the body (collagen with normal structure and collagen with destroyed structure).

[0069] In another aspect, the present invention relates to a nucleic acid molecule encoding the recombinant protein.

[0070] In another aspect, the present invention relates to a recombinant vector comprising the nucleic acid molecule.

[0071] In another aspect, the present invention relates to a recombinant cell into which the recombinant vector has been introduced.

[0072] As used herein, the term “nucleic acid molecule” has a comprehensive meaning including DNA (gDNA and cDNA) and RNA molecules, and nucleotides, which are the basic structural units of nucleic acid molecules, include not only natural nucleotides but also analogues in which sugar or base moieties are modified (Scheit, Nucleotide Analogs, John Wiley, New York (1980); Uhlman and Peyman, Chemical Reviews. 1990; 90:543-84). The sequence of a nucleic acid molecule encoding the human A1AT fusion protein of the present invention may be modified, and the modifications include additions, deletions, or non-conservative or conservative substitutions of nucleotides.

[0073] The term “vector” as used herein refers to a means for expressing a target gene in a host cell, including a plasmid vector; a cosmid vector; and a viral vector such as a bacteriophage vector, an adenovirus vector, a retrovirus vector, and an adeno-associated virus vector, and is preferably a plasmid vector, but is not limited thereto.

[0074] In the vector of the present invention, the nucleic acid molecule encoding the fusion protein comprising alpha-1 antitrypsin may be operatively linked to a promoter.

[0075] As used herein, the term “operably linked” means a functional linkage between a nucleic acid expression regulatory sequence (e.g., a promoter, a signal sequence, or an array of transcription factor binding sites) and another nucleic acid sequence, whereby the regulatory sequence regulates transcription and / or translation of the other nucleic acid sequence.

[0076] The recombinant vector system of the present invention can be constructed through various methods known in the art, and specific methods thereof are disclosed in Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press (2001), which is incorporated herein by reference.

[0077] The vector of the present invention can typically be constructed as a vector for cloning or as a vector for expression. In addition, the vector of the present invention can be constructed using a prokaryotic or eukaryotic cell as a host.

[0078] For example, when the vector of the present invention is an expression vector and uses a prokaryotic cell as a host, it generally includes a strong promoter capable of initiating transcription (e.g., tac promoter, lac promoter, lacUV5 promoter, lpp promoter, pLλ promoter, pRλ promoter, rac5 promoter, amp promoter, recA promoter, SP6 promoter, trp promoter, and T7 promoter, etc.), a ribosome binding site for initiating translation, and a transcription / translation termination sequence. When E. coli (e.g., HB101, BL21, DH5α, etc.) is used as a host cell, E. The promoter and operator regions of the tryptophan biosynthetic pathway of E. coli (Yanofsky, C., J. Bacteriol., (1984) 158:1018-1024) and the left-hand promoter of phage λ (pLλ promoter, Herskowitz, I. and Hagen, D., Ann. Rev. Genet., (1980) 14:399-445) can be used as regulatory regions. When Bacillus is used as the host cell, the promoter of the toxin protein gene of Bacillus thuringiensis (Appl. Environ. Microbiol. (1998) 64:3932-3938; Mol. Gen. Genet. (1996) 250:734-741) or any promoter that can be expressed in Bacillus can be used as regulatory regions.

[0079] Meanwhile, the recombinant vector of the present invention can be produced by manipulating plasmids (e.g., pCL, pSC101, pGV1106, pACYC177, ColE1, pKT230, pME290, pBR322, pUC8 / 9, pUC6, pBD9, pHC79, pIJ61, pLAFR1, pHV14, pGEX series, pET series, and pUC19, etc.), phages (e.g., λgt4.λB, λ-Charon, λΔz1, and M13, etc.), or viruses (e.g., SV40, etc.) that are frequently used in the art. For example, the recombinant vector of the present invention can be produced by manipulating a pCL expression vector, specifically, a pCLS05 (Korean Patent No. 10-1420274) expression vector, but is not limited thereto.

[0080] In addition, when the vector of the present invention is an expression vector and uses a eukaryotic cell as a host, a promoter derived from the genome of a mammalian cell (e.g., metallothionine promoter, β-actin promoter, human hemoglobin promoter, and human muscle creatine promoter) or a promoter derived from a mammalian virus (e.g., adenovirus late promoter, vaccinia virus 7.5K promoter, SV40 promoter, cytomegalovirus (CMV) promoter, HSV tk promoter, mouse mammary tumor virus (MMTV) promoter, HIV LTR promoter, Moloney virus promoter, Epstein-Barr virus (EBV) promoter, and Rous sarcoma virus (RSV) promoter) can be used, and generally has a polyadenylation sequence as a transcription termination sequence. Specifically, the recombinant vector of the present invention includes a CMV promoter.

[0081] The recombinant vector of the present invention can be fused with other sequences to facilitate the purification of recombinant proteins expressed therefrom. Examples of the fused sequences include glutathione S-transferase (Pharmacia, USA), maltose binding protein (NEB, USA), FLAG (IBI, USA), and 6x His (hexahistidine; Quiagen, USA).

[0082] Meanwhile, the recombinant vector of the present invention includes an antibiotic resistance gene commonly used in the art as a selection marker, and may include, for example, a resistance gene for ampicillin, gentamicin, carbenicillin, chloramphenicol, streptomycin, kanamycin, geneticin, neomycin, and tetracycline.

[0083] Any recombinant cell capable of stably and continuously cloning and expressing the vector of the present invention can be used as a host cell known in the art, and includes, but is not limited to, prokaryotic host cells such as Bacillus strains such as Escherichia coli, Bacillus subtilis and Bacillus thuringiensis, Streptomyces, Pseudomonas (e.g., Pseudomonas putida), Proteus mirabilis or Staphylococcus (e.g., Staphylococcus carnosus).

[0084] Suitable eukaryotic host cells for the above vector include fungi such as Aspergillus species, yeasts such as Pichia pastoris, Saccharomyces cerevisiae, Schizosaccharomyces and Neurospora crassa, other lower eukaryotic cells, higher eukaryotic cells such as insect-derived cells, and cells derived from plants or mammals.

[0085] Specifically, the host cell may be monkey kidney cells 7 (COS7), NSO cells, SP2 / 0, Chinese hamster ovary (CHO) cells, W138, baby hamster kidney (BHK) cells, MDCK, a myeloma cell line, HuT 78 cells, or 293 cells.

[0086] In the present invention, "transformation" and / or "transfection" into a host cell includes any method for introducing a nucleic acid into an organism, cell, tissue, or organ, and can be performed by selecting a standard technique suitable for the host cell as known in the art. Such methods include, but are not limited to, electroporation, protoplast fusion, calcium phosphate (CaPO4) precipitation, calcium chloride (CaCl2) precipitation, stirring using silicon carbide fibers, Agrobacterium-mediated transformation, PEG, dextran sulfate, lipofectamine, and desiccation / inhibition-mediated transformation methods.

[0087]

[0088] Meanwhile, in the present invention, it was confirmed that, particularly when using microorganisms such as E. coli, productivity is significantly higher than that of animal cells, and, compared to commercialized protein therapeutics, elastase inhibitory activity is maintained while increasing the stability of the drug in the body.

[0089] Specifically, the present invention establishes a method for effectively producing the recombinant protein using an E. coli protein expression system.

[0090] In order to improve the expression of a recombinant protein, the growth of related biomass, such as cell stress, and the formation of the recombinant protein must be balanced. Therefore, in the present invention, the growth temperature of E. coli was maintained at less than 37°C to prevent the formation of inclusion bodies of the recombinant protein and induce accurate folding.

[0091] In addition, in the present invention, the expression of the recombinant protein was induced by IPTG using the E. coli T7 promoter system under the control of the lac operon present in the pET vector, and the temperature and IPTG concentration for recombinant protein expression were set to about 25°C and about 0.1 mM, respectively.

[0092] To purify the expressed protein with a 6x His tag, affinity chromatography was performed using Ni-NTA agarose resin. Since non-target proteins were eluted in addition to the target protein during the first purification using Ni-NTA affinity chromatography, additional purification was required. Subsequently, a second purification was performed using size exclusion chromatography, and a third purification was performed using a dialysis membrane with a cut-off of 25 kDa.

[0093] Accordingly, the present invention relates to a method for producing a recombinant protein, comprising the following steps:

[0094] (a) a step of culturing the recombinant cells; and

[0095] (b) a step of recovering the recombinant protein from the recombinant cell.

[0096] In the present invention, the recombinant cell may be characterized as being E. coli, but is not limited thereto.

[0097] In the present invention, the step (b) may be characterized by recovering the recombinant protein by crushing and purifying the recombinant cell.

[0098] Preferably, the purification may be carried out in a three-step purification process, but such a three-step purification process is not essential.

[0099] In one embodiment, the purification may be performed in the following steps: first purification by affinity chromatography, second purification by size exclusion chromatography, and third purification using a dialysis membrane.

[0100] The affinity chromatography may be, but is not limited to, Ni-NTA affinity chromatography, and it is well known in the art that purification is possible using affinity chromatography suitable for a tag labeled at the end of the recombinant protein for purification.

[0101] In order to purify a target protein having a size of 52.5 kDa through the above size exclusion chromatography, a fraction having a size in the range of about 45 kDa to about 60 kDa, preferably in the range of about 50 kDa to about 55 kDa, may be obtained.

[0102] The above permeation may be performed using a dialysis membrane with a cut-off of about 25 kDa to remove impurities of 25 kDa.

[0103] In the process of manufacturing the above recombinant protein, the transformed recombinant cells can be cultured according to appropriate media and culture conditions known in the art. Those skilled in the art can easily adjust this culture process according to the selected strain. Various culture methods are disclosed in various references (e.g., James M. Lee, Biochemical Engineering, Prentice-Hall International Editions, 138-176). Cell culture is classified into suspension culture and adherent culture depending on the cell growth pattern, and batch, fed-batch, and continuous culture methods depending on the culture method. The culture medium used must appropriately satisfy the requirements of the specific strain.

[0104] In animal cell culture, the medium contains various carbon sources, nitrogen sources, and trace element components. Examples of carbon sources that can be used include carbohydrates such as glucose, sucrose, lactose, fructose, maltose, starch, and cellulose; fats such as soybean oil, sunflower oil, castor oil, and coconut oil; fatty acids such as palmitic acid, stearic acid, and linoleic acid; alcohols such as glycerol and ethanol; and organic acids such as acetic acid. These carbon sources can be used alone or in combination.

[0105] Nitrogen sources that can be used in the present invention include organic nitrogen sources such as peptone, yeast extract, meat juice, malt extract, corn steep liquor (CSL), and soybean meal, and inorganic nitrogen sources such as urea, ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate, and ammonium nitrate, and these nitrogen sources can be used alone or in combination. The medium can include potassium dihydrogen phosphate, dipotassium hydrogen phosphate, and the corresponding sodium-containing salts as phosphorus. It can also include metal salts such as magnesium sulfate or iron sulfate. In addition, amino acids, vitamins, and appropriate precursors can be included.

[0106] During cultivation, compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid, and sulfuric acid can be appropriately added to the culture to adjust the pH of the culture. Furthermore, foaming can be suppressed during cultivation using antifoaming agents such as fatty acid polyglycol esters. Furthermore, oxygen or an oxygen-containing gas (e.g., air) is injected into the culture to maintain an aerobic state. The culture temperature is usually between 20°C and 45°C, preferably between 25°C and 40°C.

[0107] The recombinant protein obtained by culturing transformed recombinant cells can be used in an unpurified state, and can be further purified to a high purity using various conventional methods such as dialysis, salt precipitation, and chromatography. Among these, the method using chromatography is the most commonly used, and the type and order of the column can be selected from ion exchange chromatography, size exclusion chromatography, and affinity chromatography, depending on the characteristics of the recombinant protein, the culture method, etc.

[0108]

[0109] Meanwhile, in the present invention, the activity of the recombinant protein produced by the above method was analyzed in vitro as an anti-elastase activity. Since both the recombinant alpha-1 antitrypsin (rhA1AT) produced in E. coli and the recombinant alpha-1 antitrypsin fusion protein (rhA1AT-CMP) in which a collagen-mimetic peptide is fused were designed and expressed based on human alpha-1 antitrypsin, it was expected that these recombinant proteins would be able to inhibit elastase, and as a result of conducting an experiment, it was confirmed that the recombinant protein according to the present invention could inhibit elastase at a level similar to that of alpha-1 antitrypsin isolated from human blood, thereby proving that the recombinant protein of the present invention exhibits an effect that can replace currently commercialized AATD augmentation therapy treatments.

[0110] In addition, in the present invention, it was confirmed that rhA1AT-CMP has excellent binding affinity to collagen. In particular, by changing the pH of the buffer solution to change the structure of collagen, the binding affinity of rhA1AT-CMP to collagen with a normal structure and a destroyed structure was confirmed. As a result, it was shown that rhA1AT-CMP effectively binds to both of these structures, proving that rhA1AT-CMP can work effectively even in patients whose collagen structure is destroyed due to AATD.

[0111] Collagen and collagen-mimetic peptides with triple helix structures are resistant to most proteases except collagen-specific degrading enzymes such as MMP and cathepsin K. Therefore, it is expected that recombinant proteins fused with CMP will be less susceptible to protease degradation in vivo and thus be retained for a longer period of time.

[0112] Meanwhile, pharmacokinetic analysis was conducted to determine the efficacy of rhA1AT and rhA1AT-CMP according to the drug administration route (intravenous and intratracheal administration). Two types of recombinant proteins, rhA1AT or rhA1AT-CMP, were administered once at a dose of 1 mg / kg via the tail vein or intratracheal, respectively.

[0113] As a result of intravenous administration, rhA1AT-CMP and rhA1AT showed C max While no significant difference was observed between the C and half-lives of rhA1AT-CMP and rhA1AT in intravenous administration, max There was no significant difference in the half-life of rhA1AT-CMP, but the half-life of rhA1AT was observed to be more than 2.6 times longer than that of rhA1AT. MRT, which indicates the mean residence time of the drug INFWhile no significant difference was observed between the two drugs when administered intravenously, a significant difference of over 3.7 times was observed when administered intratracheally. These results confirm that rhA1AT-CMP is maintained in vivo for a longer period when administered intratracheally.

[0114] Finally, after removing all blood from each organ by cardiac perfusion, the distribution concentration of the drug in each tissue according to the route of administration was measured. When rhA1AT-CMP was administered intratracheally, the highest concentration was maintained in the lung, the organ where the drug acts. This was expected to be due to CMP binding to collagen present in the lung and its prolonged retention. Residual drug concentrations in liver tissues were measured, indicating that each drug was metabolized to a similar degree regardless of the route of administration. Meanwhile, the drug bound to CMP was detected at a lower concentration in the kidney than the drug not bound to CMP after intratracheal administration. This is expected to be the result of a gradual drug loss process in vivo through CMP binding to proteins in the bloodstream and collagen in the target organ, the lung.

[0115] Therefore, the present invention, from another aspect, relates to a composition comprising the recombinant protein as an active ingredient.

[0116] In another aspect, the present invention relates to a method for preventing and / or treating alpha-1 antitrypsin deficiency, comprising administering the recombinant protein or the composition to a subject in need thereof.

[0117] In another aspect, the present invention relates to the use of the recombinant protein or the composition for preventing and / or treating alpha-1 antitrypsin deficiency.

[0118] In another aspect, the present invention relates to the use of the recombinant protein or the composition for the manufacture of a drug for preventing and / or treating alpha-1 antitrypsin deficiency.

[0119] The above composition may be used for preventing and / or treating alpha-1 antitrypsin deficiency.

[0120] The above composition may be in the form of a pharmaceutical composition, a quasi-drug composition, or a health food composition.

[0121] Preferably, the composition may be characterized by intratracheal administration, but is not limited thereto.

[0122] Through intravenous administration, the recombinant protein, which is an active ingredient in the composition, can have increased persistence in the body and an increased degree of targeting to the lungs.

[0123] The disease prevention or treatment composition of the present invention may additionally include a pharmaceutically acceptable carrier.

[0124] In the present invention, the term "pharmaceutically acceptable carrier" refers to a carrier or diluent that does not stimulate a living organism and does not inhibit the biological activity and properties of the administered compound. In a composition formulated as a liquid solution, the pharmaceutically acceptable carrier is sterile and biocompatible, and may include saline solution, sterile water, buffered saline, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, and a mixture of one or more of these components. If necessary, other conventional additives such as antioxidants, buffers, and bacteriostatic agents may be added. In addition, diluents, dispersants, surfactants, binders, and lubricants may be additionally added to formulate the composition into injectable formulations such as aqueous solutions, suspensions, and emulsions, pills, capsules, granules, or tablets.

[0125] The pharmaceutical composition of the present invention may be in various oral or parenteral dosage forms. When formulated, it is prepared using diluents or excipients such as commonly used fillers, bulking agents, binders, wetting agents, disintegrating agents, and surfactants. Solid preparations for oral administration include tablets, pills, powders, granules, and capsules, and these solid preparations are prepared by mixing one or more compounds with at least one excipient, such as starch, calcium carbonate, sucrose or lactose, gelatin, etc. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid preparations for oral administration include suspensions, oral solutions, emulsions, and syrups, and in addition to commonly used simple diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, fragrances, and preservatives may be included. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized preparations, and suppositories. Non-aqueous solutions and suspensions can include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. Suppository bases include witepsol, macrogol, tween, cacao butter, laurin, and glycerogelatin.

[0126] These pharmaceutically acceptable carriers and formulations are described in detail in Remington's Pharmaceutical Sciences (19th ed., 1995).

[0127] The composition of the present invention can be administered orally or parenterally, and in the case of parenteral administration, it can be administered intravenously, subcutaneously, intramuscularly, intraperitoneally, intradermally, topically, intranasally, intrapulmonary, and intrarectally. When administered orally, since proteins or peptides are digested, the oral composition can be formulated to coat the active agent or protect it from degradation in the stomach, and the composition of the present invention can be administered by any device that allows the active agent to travel to target cells.

[0128] The appropriate dosage of the disease prevention or treatment composition of the present invention varies depending on factors such as the formulation method, administration method, patient's age, weight, sex, pathological condition, food, administration time, administration route, excretion rate, and response sensitivity, and a generally skilled physician can easily determine and prescribe a dosage effective for the desired treatment or prevention.

[0129] According to one embodiment of the present invention, the daily dosage of the pharmaceutical composition of the present invention may be 0.001-100 mg / kg. As used herein, the term "pharmaceutically effective amount" means an amount sufficient to treat, prevent, and diagnose diseases such as emphysema and neuromuscular disease.

[0130] The disease prevention or treatment composition of the present invention can be manufactured in the form of a unit dose by formulating it using a pharmaceutically acceptable carrier and / or excipient according to a method that can be easily performed by a person having ordinary skill in the art to which the present invention pertains, or can be manufactured by placing it in a multi-dose container. In this case, the formulation may be in the form of a solution, suspension or emulsion in an oil or aqueous medium, or in the form of an extract, powder, suppository, powder, granules, tablet or capsule, and may additionally include a dispersing agent or stabilizer.

[0131] Preferably, it may be formulated in an injectable form, and the injectable form may be a form that is reconstituted in a lyophilized form and used, or a liquid formulation in a ready-to-injection (RTI) form, but is not limited thereto.

[0132] The composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, and may be administered sequentially or simultaneously with conventional therapeutic agents.

[0133] In the present invention, the term "subject" may be used interchangeably with terms such as "object" and "patient," and the subject may refer to a vertebrate, such as a mammal. The mammal includes, but is not limited to, humans, farm animals, and pets.

[0134]

[0135] In the present invention, a recombinant protein (rhA1AT-CMP) conjugated with a collagen mimetic peptide (CMP) was designed to enhance the pharmacological efficacy of recombinant human alpha-1 antitrypsin (rhA1AT). Fibrous collagen, a component of the extracellular matrix (ECM), plays a crucial role in maintaining normal lung structure. Type I collagen accounts for 50-60% of the major structural components of the ECM. AATD causes lung tissue destruction, leading to emphysema. Excessive protein degradation leads to the destruction of elastin and collagen fibers, resulting in restructuring of the respiratory tract.

[0136] The structural characteristics of collagen begin with the formation of a triple helix structure through the interaction of three polypeptide strands rich in proline, which are then formed into a structure in which the Gly-XY structure is repeated. The CMP-binding recombinant human alpha-1 antitrypsin (rhA1AT-CMP) according to the present invention was developed by binding to a Gly-Pro-Pro repeat peptide (GPP), which is one of the most well-conserved amino acid sequences of natural collagen, and it was confirmed that it exhibits a strong affinity for collagen.

[0137] Natural collagen has a loose network structure, making it less effective at retaining passively adsorbed substances. In the presence of regenerated type I collagen fibers, binding occurred when the molten CMP folded. When protecting the lungs from protease damage, CMP was shown to be conjugated to alpha-1 antitrypsin (rhA1AT), enhancing this effect.

[0138] In the present invention, conditions for the soluble protein production of rhA1AT and rhA1AT-CMP were established using an Escherichia coli protein expression system. Specifically, purification was performed using nickel-nitrilotriacetic acid (Ni-NTA) affinity chromatography and size exclusion chromatography (SEC). The molecular weights of the purified rhA1AT and rhA1AT-CMP were evaluated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and matrix-assisted laser desorption / ionization-time of flight (MALDI-TOF). The activity and collagen-binding affinity of rhA1AT and rhA1AT-CMP were evaluated using an elastase inhibition assay by ELISA and a collagen type I binding assay.

[0139] Augmentation therapy, which involves intravenous administration of purified A1AT from blood, is currently the only disease-specific treatment approved for AATD. However, more than 80% of A1AT is produced and released by liver cells and acts in the lungs.

[0140] In the present invention, we developed rhA1AT-CMP, which effectively binds to collagen, and clearly confirmed its therapeutic efficacy compared to intravenous administration by improving the administration route for direct intratracheal administration. Specifically, CMP, which exhibits strong affinity for natural collagen, was fused to rhA1AT, expressed, and purified. Efficacy was evaluated after injection into mice via two administration routes. The results confirmed that the recombinant protein fused with CMP targets collagen in the body and exerts long-term effects when administered intratracheally.

[0141]

[0142] Example

[0143]

[0144] Hereinafter, the present invention will be described in more detail through examples. These examples are intended solely to illustrate the present invention, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples.

[0145]

[0146] Example 1. Experimental materials

[0147] Human plasma Alpha-1 antitrypsin (molecular weight 52 kDa) was purchased from Athens Research & Technology (Athens, GA, USA). E. coli strains BL21(DE3) CodonPlus RIL and Rosetta gami-2 TM (DE3) pLysS was purchased from Agilent Technologies, Inc. (Santa Clara, CA, USA) and Milipore (Darmstadt, Germany), respectively. Ni-NTA agarose resin was purchased from QIAGEN (Hilden, Germany). HiLoad TM 16 / 600 Superdex TM200 pg was purchased from GE Healthcare (Chicago, IL, USA). Porcine pancreatic elastase (PPE) and N-succinyl-Ala-Ala-Ala-p-nitroanilide (SApNA) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Collagen solution (type I rat tail) was purchased from Gibco (New York, USA). Human Alpha-1 antitrypsin ELISA kit was purchased from Abcam (Cambridge, UK). Excelband TM The three-color universal-range protein marker was purchased from SMOBIO Technology (Hsinchu, Taiwan). The Model FMJ-250 high-pressure syringe and the Model IA-1C intratracheal aerosolizer were purchased from PENN-CENTURY, INC. (Wyndmoor, PA, USA). All chemical solvents used were of analytical grade.

[0148]

[0149] Example 2. Production of recombinant proteins

[0150]

[0151] 2-1. Expression

[0152] To express human alpha-1 antitrypsin protein in E. coli, the signal peptide sequence was excluded and the codon was optimized as shown in SEQ ID NO: 3 and cloned into the pET28b vector.

[0153]

[0154] Sequence number 3. Human alpha-1 antitrypsin codon-optimized sequence

[0155]

[0156]

[0157] pET-28b-rhA1AT and pET-28b-rhA1AT-CMP vectors were transformed into BL21 (DE3) CodonPlus RIL electrocompetent cells and Rosetta gami-2™ (DE3) pLysS using a Micropulser (Bio-rad, Hercules, CA, USA). The transformed rhA1AT and rhA1AT-CMP were cultured overnight at 37°C on LB+ agar plates containing kanamycin. One colony was selected and cultured in LB medium containing kanamycin at 37°C with shaking at 200 rpm for 8 h. After culture, the cells were inoculated into LB medium at a ratio of 1:100. When the O.D600 value reached 0.4 to 0.6, 0.1 mM isopropyl β-thiogalactopyranoside (IPTG) was added and cultured at 25°C and 200 rpm for 16 hours.

[0158]

[0159] 2-2. Obtaining the dissolved substance

[0160] Cultured E. coli was centrifuged at 4°C and 6,000 rpm for 20 min. The supernatant was removed, and the pellet was suspended in a resuspension buffer containing 20 mM Tris-HCl (pH 8.0) + 100 mM NaCl + protease inhibitor cocktail (Roche, Basel, Switzerland). The E. coli was then disrupted on ice using an ultrasonicator (Cole-Parmer, Vernon Hills, IL, USA) at 60% amplitude, 3 s / on and 3 s / off, 600,000 J, and 30 min.

[0161] The disrupted cells were centrifuged at 4°C and 10,000 rpm for 20 min. The supernatant was collected and filtered using a 0.45 μm syringe filter (Sartorius, Göttingen, Germany) to obtain a cell lysate.

[0162]

[0163] 2-3. Refining

[0164] A three-step purification process for rhA1AT and rhA1AT-CMP was established as follows.

[0165] A column filled with Ni-NTA agarose resin was equilibrated with PBS + 10 mM imidazole buffer (pH 7.4). Cell lysate and PBS + 10 mM imidazole buffer (pH 7.4) were mixed in a 1:1 ratio and loaded onto the equilibrated column for protein binding. Nontarget proteins were removed using PBS + 50 mM imidazole buffer (pH 7.4), and the target protein was eluted using PBS + 250 mM imidazole buffer (pH 7.4). Proteins of 45.3 kDa and 52.5 kDa, the sizes of the target proteins, were eluted.

[0166] Proteins eluted using Ni-NTA affinity chromatography prior to size exclusion chromatography (SEC) were purified using Amicon 　 Concentration was performed using ultra-15 centrifugal filter-15 K (Merck, Darmstadt, IN, USA).

[0167] HiLoad TM 16 / 600 Superdex TM 200 pg was equilibrated with PBS (pH 7.4). The concentrated sample was injected into the equilibrated column, and protein size fractions were obtained using PBS (pH 7.4). The protein solution eluted from the fractions was confirmed using SDS-PAGE, and the eluted fractions of the desired protein size were collected for the next process.

[0168] In addition to the target protein, a protein of about 25 kDa was eluted together and dialyzed. Specifically, after SEC, rhA1AT and rhA1AT-CMP were purified using Spectra / Por with PBS (pH 7.4) buffer. 　 7 Dialysis was performed using pretreated RC tubing MWCO: 25 kD (Repligen, Waltham, MA, USA).

[0169]

[0170] 2-4. Protein identification (SDS-PAGE & CBB staining)

[0171] Proteins were mixed with Tris-glycine SDS sample buffer (Bio-rad, Hercules, CA, USA) and denatured. Protein samples were separated using a 10% Tris-glycine SDS-PAGE gel. Electrophoresis was performed at 60 V for 30 minutes, followed by 1 hour and 10 minutes at 120 V. The gel was stained with Coomassie Brilliant Blue R-250 (Biosolution, SEOUL, Korea) to confirm the purified protein (Fig. 2b).

[0172]

[0173] 2-5. Protein molecular weight measurement (MALDI-TOF analysis)

[0174] rhA1AT and rhA1AT-CMP samples dissolved in PBS (pH 7.4) were dialyzed using deionized distilled water as an external buffer to remove salts. 0.1% trifluoroacetic acid / acetonitrile (1:1, v / v) was added to 10 mg / mL sinapinic acid to form a matrix. 2 μL of each solution was mixed to make a mixture, which was then placed on a MALDI target and vacuum-dried. Molecular weights were measured using an autoflex maX TOF / TOF (Bruker Daltonics, Billerica, MA, USA) with the Flex control 3.4 program (Bruker Daltonics, Billerica, MA, USA). MALDI-TOF analysis was performed at the Gyeonggi Economic and Science Accelerator (GBSA, Suwon, Korea).

[0175] rhA1AT and rhA1AT-CMP were found to have molecular masses of 45.3 kDa and 52.5 kDa, respectively (Figs. 2c and 2d). After confirming that the molecular weights were consistent with the amino acid sequence analysis results, subsequent experiments were conducted.

[0176]

[0177] 2-6. Concentration measurement

[0178] The concentration of purified protein was determined by Pierce according to the manufacturer's protocol. TM It was measured using a BCA protein assay kit (Thermo Scientific, Waltham, MA, USA).

[0179]

[0180] Example 3. In vitro elastase inhibitory activity of rhA1AT and rhA1AT-CMP

[0181] The elastase inhibitory activities of rhA1AT and rhA1AT-CMP purified from Escherichia coli and hA1AT purified from human blood were measured by PPE. Elastase regulation is known to be one of the major functions of A1AT (Sun Z, Yang P. Role of imbalance between neutrophil elastase and α1-antitrypsin in cancer development and progression. The Lancet Oncology. 2004;5(3):182-190.).

[0182] Proteins at various concentrations (10–100 nM) were mixed with 0.2 M Tris-HCl (pH 8.0) buffer. hA1AT, rhA1AT, and rhA1AT-CMP were mixed with 10 nM PPE and incubated at 25°C for 15 min. Afterwards, SApNA was added to assess activity. To measure activity, fluorescence was measured at 25°C and 410 nm using a BioTek Hybrid reader (BioTek Instruments, Inc., Winooski, VT, USA).

[0183] A1AT, rhA1AT, and rhA1AT-CMP isolated from human blood were all found to inhibit the activity of PPE in a concentration-dependent manner (Fig. 3).

[0184]

[0185] Example 4. Collagen binding analysis of rhA1AT and rhA1AT-CMP

[0186] Collagen self-assembles in a pH-dependent manner. At pH 3, collagen molecules do not exhibit a distinct fiber arrangement in the form of spherical protrusions, whereas at pH 6, collagen molecules exhibit a fiber arrangement.

[0187] Therefore, for binding analysis of collagen samples of various structures, 3 mg / mL collagen solution (type I rat tail) was diluted to 0.4 mg / mL in 0.1 M citrate buffer with different pH values ​​(pH 3 or pH 6).

[0188] After adding 50 μL to a 96-well plate (Costar, Washington DC, USA) and air-drying, a collagen film was formed. The collagen film was blocked with 1% BSA-containing PBS for 1 hour and washed three times with PBS. 2.5 μg each of rhA1AT-CMP and rhA1AT were incubated with HRP-conjugated anti-A1AT antibody (Abcam, Cambridge, UK) on the collagen film at 25°C with agitation for 1 hour and washed five times with PBS. After incubation with 3,3',5,5'-tetramethylbenzidine (TMB) liquid substrate (Sigma-Aldrich, St. Louis, MO, USA) at 25°C for 15 minutes, 2 M H2SO4 was treated in the same manner. Absorbance was detected using a BioTek Hybrid reader set to 450 nm.

[0189] As a result, rhA1AT-CMP was found to effectively bind to both collagen with a normal structure and collagen with a destroyed structure, but rhA1AT was found to not bind to collagen with either structure (Fig. 4).

[0190]

[0191] Example 5. Pharmacokinetic analysis

[0192] Four-week-old male ICR mice weighing approximately 28–32 g were purchased from Orient Bio Korea (Seongnam, Korea). All mice were individually housed in an air-purified clean room at the Laboratory Animal Research Center of Ajou University Medical Center (Suwon, Korea) under conditions of 45–55% humidity, 21–23°C, and a 12-h day–night cycle, with free access to food and water.

[0193] All experimental protocols and animal manipulations were approved by the Institutional Animal Care and Use Committee of the Laboratory Animal Research Center of Ajou University Medical Center and were performed in accordance with the standard operating procedures of the animal laboratory.

[0194] Mice were anesthetized using isoflurane. Mice were randomly assigned to groups of four per experimental group.

[0195] rhA1AT (1 mg / kg) and rhA1AT-CMP (1 mg / kg) were administered intratracheally using a Model FMJ-250 high-pressure syringe (PENN-CENTURY, INC., Wyndmoor, PA, USA). Blood samples were collected at 1, 5, 15, and 30 minutes and 1, 2, 4, 8, 24, and 48 hours after intratracheal administration.

[0196] Meanwhile, an equal amount of protein was administered intravenously via the tail vein of mice using a 1 mL syringe (Korea Vaccine, Seoul, Korea). Blood samples were collected at 1, 5, 15, and 30 minutes and 1, 2, 4, and 8 hours after intravenous drug administration.

[0197] Blood samples were centrifuged at 12,000 rpm for 5 minutes, and plasma samples were collected and stored at -20°C until analysis using a human anti-A1AT ELISA kit.

[0198] Pharmacokinetic analysis of recombinant protein concentrations in plasma was performed using Pharsight WinNonlin Version 3.0 (Pharsight Corporation, CA, USA).

[0199] In the pharmacokinetic analysis, the following pharmacokinetic parameters were calculated: AUC INF (Total area under the plasma concentration versus time curve from 0 to infinity), AUC last (Area under the plasma concentration-time curve from 0 to the last sampling time), t 1 / 2 (Drug elimination half-life), C max (peak plasma concentration), t max (C max (Time to reach) MRT INF (average residence time), Cl (clearance).

[0200] In the first blood draw after drug administration, if rhA1AT was administered into the tail vein, C max was 4445.77±601.39ng / mL, and C was observed in cases where rhA1AT-CMP was administered into the tail vein. max was found to be 5479.80±871.81ng / mL.

[0201] When administered intravenously via the tail vein, both rhA1AT and rhA1AT-CMP exhibited peak concentrations at the beginning of plasma collection, and the plasma concentration of rhA1AT-CMP showed a steeper initial decline slope than that of rhA1AT. However, rhA1AT-CMP allowed quantification of plasma concentrations for up to 8 hours, and its in vivo half-life was longer than that of rhA1AT.

[0202] AUC INF In case of intravenous administration, there was no significant difference between rhA1AT and rhA1AT-CMP.

[0203]

[0204]

[0205]

[0206] Meanwhile, when the drug was administered via the intravenous route and blood concentrations were measured, enhanced efficacy was expected. rhA1AT-CMP and rhA1AT showed the highest plasma concentrations at 120 and 60 minutes, respectively, after administration at a concentration of 1 mg / kg.

[0207] For rhA1AT-CMP, C 120 minutes after drug administration max was 2421.00 ± 358.77 ng / mL, and in the case of rhA1AT, C was 60 minutes after drug administration. max was found to be 2395.86 ± 293.96 ng / mL.

[0208] AUC when administered through an organ INF In the case of rhA1AT, there was a significant difference of 615539.5±117909.00 ngmin / mL and rhA1AT-CMP was 2894168±769743.10 ngmin / mL, and rhA1AT-CMP showed an AUC that was more than 4 times higher. The half-life was 349.19±72.03 minutes (5.82 hours) versus 938.41±322.98 minutes (15.63 hours), indicating that when rhA1AT-CMP was administered intravenously, the half-life was significantly increased compared to when administered intravenously, and rhA1AT-CMP remained in the body for a longer time than rhA1AT.

[0209]

[0210] This suggests that the half-life of rhA1AT-CMP increases approximately 1.5 times when administered intravenously, which is the commercially available route of administration for the drug, whereas the drug remains in the body for a longer time (approximately 2.7 times) and exhibits efficacy when administered intratracheally, which may enhance the efficacy of the drug in vivo.

[0211] The results of the pharmacokinetic analysis, drug concentration-time profiles and pharmacokinetic parameters are shown in Figure 5 and Table 1.

[0212]

[0213] Example 6. Distribution within the tissue

[0214] Pharmacokinetic analysis predicted that rhA1AT conjugated to CMP would bind to collagen present in the lungs and alveoli and be slowly released when administered directly to the lungs through the trachea. To confirm this, a tissue distribution analysis of the drug was conducted as a follow-up experiment.

[0215] Male 4-week-old ICR mice (3 per experimental group) were administered rhA1AT and rhA1AT-CMP intravenously and intratracheally, respectively, at a concentration of 1 mg / kg, respectively. Blood was collected 30 minutes and 4 hours later, and the mice were sacrificed. Blood in the organs was removed by cardiac perfusion with 0.1% PBS-T at a rate of 1.5 mL / min for 15 minutes, and the lungs, liver, and kidneys were removed. Plasma was separated and stored at -20°C, and the weight of the removed lungs, liver, and kidneys was measured. Homogenization was performed using a homogenizer in 1x PBS containing protease inhibitors and 0.3% Triton-X100. The homogenized tissue samples were centrifuged, and only the supernatant was collected and stored at -80°C until analysis.

[0216] The concentration-time profiles of the tissues are shown in Fig. 6 and Table 2.

[0217]

[0218]

[0219]

[0220] First, a comparison of tissue distribution 30 minutes after drug administration showed that the recombinant protein conjugated to CMP exhibited higher lung tissue retention regardless of the route of administration. The amount of rhA1AT-CMP administered through the trachea was the highest (12192.00 ± 2825.31 ng / g tissue), followed by rhA1AT administered through the trachea (5773.33 ± 768.42 ng / g tissue), rhA1AT-CMP administered through the tail vein (4645.33 ± 773.65 ng / g tissue), and rhA1AT (2912.00 ± 925.04 ng / g tissue).

[0221] Additionally, even 4 hours after drug administration, rhA1AT-CMP showed a higher distribution in the lungs (6298.67 ± 916.46 ng / g tissue) than rhA1AT when administered intratracheally.

[0222]

[0223] Example 7. Statistical Analysis

[0224] Data were analyzed using GraphPad Prism version 7.0 (Graphpad Software, La Jolla, CA, USA). Data are expressed as mean ± standard deviation (SD), and statistical analysis was performed using two-way analysis of variance (ANOVA). A p value less than 0.05 was considered statistically significant.

[0225]

[0226] While specific aspects of the present invention have been described in detail above, it will be apparent to those skilled in the art that these specific descriptions merely represent preferred embodiments and are not intended to limit the scope of the present invention. Therefore, the substantial scope of the present invention is defined by the appended claims and their equivalents.

[0227]

[0228] [National Research and Development Project Supporting This Invention]

[0229]

[0230] [Project ID] 1711188494

[0231] [Task Number] 2022R1A2C1004714

[0232] [Ministry Name] Ministry of Science and ICT

[0233] [Name of Project Management (Specialist) Institution] National Research Foundation of Korea

[0234] [Research Project Name] Individual Basic Research (Ministry of Science and ICT)

[0235] [Research Project Name] Development of Novel Ovarian Cancer Treatment Agents and Combination Therapy Research for Gene Editing-Immunotherapy Dual Therapy for Ovarian Cancer

[0236] [Name of Project Performing Organization] Ajou University

[0237] [Research Period] March 1, 2022 - February 28, 2027

[0238]

[0239] [Project ID]1711176277

[0240] [Task Number] 2022K2A9A1A01098000

[0241] [Ministry Name] Ministry of Science and ICT

[0242] [Name of Project Management (Specialist) Institution] National Research Foundation of Korea

[0243] [Research Project Name] National Research Foundation of Korea Research Operational Expense Support (Main Project Expenses)

[0244] [Research Project Name] Accelerating the Body's Elimination of MRI Contrast Agents through the Development of Gadolinium-Mining Biomaterials and Gadolinium Bio-Mining Research

[0245] [Name of Project Performing Organization] Ajou University

[0246] Research Period: December 1, 2022 - November 30, 2023

Claims

1. A recombinant protein in which a collagen-mimicking peptide is fused to alpha-1 antitrypsin.

2. In the first paragraph, the collagen mimetic peptide is a recombinant protein which is (Xaa-Yaa-Gly)n (wherein Xaa and Yaa are each independently any one amino acid selected from the group consisting of proline, 4S-hydroxyproline, fluoroproline, chloroproline, lysine, glutamic acid, cysteine, and methionine, and n is an integer from 5 to 15) or (Gly-Pro-Pro)n (wherein n is an integer from 5 to 15).

3. A recombinant protein in claim 1, wherein the collagen mimetic peptide is fused to the N-terminus and / or C-terminus of alpha-1 antitrypsin.

4. In the first paragraph, the recombinant protein is a recombinant protein for preventing or treating alpha-1 antitrypsin deficiency.

5. In the first paragraph, the recombinant protein is a recombinant protein in which a collagen-mimetic peptide is fused to alpha-1 antitrypsin, thereby extending the half-life in the body.

6. In paragraph 5, the recombinant protein is administered intravenously and has an extended half-life in the body.

7. A nucleic acid molecule encoding a recombinant protein of any one of claims 1 to 6.

8. A recombinant vector comprising the nucleic acid molecule of clause 7.

9. A recombinant cell into which the recombinant vector of clause 8 has been introduced.

10. A method for producing a recombinant protein according to claim 1, comprising the following steps: (a) a step of culturing the recombinant cell of clause 9; and (b) a step of recovering the recombinant protein from the recombinant cell.

11. A method for producing a recombinant protein in claim 10, wherein the recombinant cell is Escherichia coli.

12. In paragraph 10, The step (b) above is a method for producing a recombinant protein, wherein the recombinant cell is crushed and then purified to recover the recombinant protein.

13. A pharmaceutical composition for preventing or treating alpha-1 antitrypsin, comprising a recombinant protein of any one of claims 1 to 6 as an active ingredient.

14. A pharmaceutical composition according to claim 13, characterized in that the pharmaceutical composition is administered intratracheally.

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