KR101947341B1 - A method for extending half-life of FSH beta - Google Patents

Abstract

The present invention relates to a method for increasing the half-life of follicle stimulating hormone? Or a folate stimulating hormone? Having an increased half-life, including substituting one or more lysine residues present in the amino acid sequence of follicle stimulating hormone? The residue-substituted follicle stimulating hormone? Remains in the human body for a long time and has excellent therapeutic effect.

Description

Methods for increasing the half-life of FSH [beta]

The present invention relates to a method for increasing the half-life of a protein or (poly) peptide by substituting at least one amino acid residue of the protein or (poly) peptide. Further, the present invention relates to a protein or (poly) peptide having an increased half-life produced by such a method.

Intracellular proteolysis occurs through two pathways: lysosomes and proteasomes. The lysosomal pathway, which degrades 10-20% of the protein, lacks substrate specificity and elaborate temporal regulation. In other words, it is a process of decomposing mostly extracellular or membrane proteins as the cell surface proteins incorporated into cells by endocytosis are degraded in lysosomes. However, in order for proteins to be selectively degraded in eukaryotic cells, a process in which ubiquitin is bound to a target protein by a ubiquitin-binding enzyme to form a polyubiquitin chain, which is recognized and degraded by proteasome, that is, ubiquitin- The ubiquitin-proteasome pathway (UPP). More than 80% to 90% of eukaryotic proteins are degraded through this process, and the ubiquitin-proteasome pathway regulates protein degradation and protein homeostasis by regulating protein degradation in eukaryotic cells.

Ubiquitin is a highly conserved 76 amino acid protein that is present in almost all eukaryotic cells, of which 6, 11, 27, 29, 33, 48 and 63 amino acid residues are lysine (Lysine, Lys, K) 48 and 63 play a major role in the formation of the poly ubiquitin chain. The ubiquitination of ubiquitin involves a series of enzymes (E1, E2, E3), which are degraded by the ATP-dependent protease complex 26S proteasome. The ubiquitin-proteasome pathway involves two separate, sequential processes, the first of which is the process of labeling multiple ubiquitin molecules with a covalent bond to the substrate, and the second is that the protein labeled by ubiquitin is a 26S pro It is a process that is decomposed by a teasome complex. The binding of ubiquitin and substrate takes place via an isopeptide bond between the lysine residue of the substrate molecule and the glycine at the C-terminal of ubiquitin, and the ubiquitin-activating enzyme E1, the ubiquitin-binding enzyme E2 and the ubiquitin ligase E3 The formation of thiol esters between ubiquitin and enzymes. E1 (ubiquitin-activating enzyme) activates ubiquitin by ATP-dependent reaction. E2 (ubiquitin-conjugating enzyme) takes ubiquitin activated from E1 to cysteine residue in ubiquitin-conjugated domain and transfers it to E3 ligase or directly to substrate protein. The E3 enzyme also catalyzes stable isopeptide binding between the lysine residue of the substrate protein and the glycine residue of ubiquitin. Another ubiquitin may be linked to the C-terminal lysine residue of the ubiquitin bound to the substrate protein. When this process is repeated and several ubiquitin molecules are ligated to the substrate protein in a branched form to form a polyubiquitin chain, 26S proteasome and is selectively degraded.

On the other hand, various kinds of proteins and (poly) peptides having a therapeutic effect in vivo are known. Such a protein or (poly) peptide having a therapeutic effect in vivo can be used as a therapeutic agent for the treatment of diseases such as growth hormone releasing hormone (GHRH), growth hormone releasing peptide, interferons, interferon-α or interferon-β, interferon receptors, colony stimulating factors (CSFs), glucagon-like peptides, interleukins, interleukin receptors, Human interleukin binding proteins, cytokine binding proteins, G-protein-coupled receptors, human growth hormone (hGH), and the like. Macrophage activating factor, macrophage peptide, B cell factor, T cell factor, protein A (prot ein A), allergy inhibitor, cell necrosis glycoproteins, G-protein-coupled receptor, immunotoxin, lymphotoxin, tumor necrosis Tumor necrosis factor, tumor suppressors, metastasis growth factor, alpha-1 antitrypsin, albumin, alpha-lactalbumin, Apolipoprotein-E, erythropoietin, highly glycosylated erythropoietin, angiopoietins, hemoglobin, thrombin, and the like. Thrombin receptor activating peptide, thrombomodulin, factor VII, factor VIIa, factor VIII, factor IX, factor IX, Factor XIII, A plasminogen activating factor, a urokinase, a streptokinase, a hirudin, a protein C, a C-reactive protein, a renin inhibitor renin inhibitor, collagenase inhibitor, superoxide dismutase, leptin, platelet-derived growth factor, epithelial growth factor, , Epidermal growth factor, angiostatin, angiotensin, bone growth factor, bone stimulating protein, calcitonin, insulin, art Atriopeptin, a cartilage inducing factor, a fibrin-binding peptide, elcatonin, a connective tissue activating factor, A tissue factor pathway inhibitor, a follicle stimulating hormone, a luteinizing hormone, a luteinizing hormone releasing hormone, a nerve growth factor, a parathyroid hormone parathyroid hormone, relaxin, secretin, somatomedin, insulin-like growth factor, adrenocortical hormone, glucagon, cholecytokinin, cholesterol, cholecystokinin, pancreatic polypeptide, gastrin releasing peptide, corticotropin releasing factor, thyroid stimulating hormone, autotaxin, lactoferrin lactoferrin, myostatin, receptors, receptor antagonists, cell surface antigens, ns, virus derived vaccine antigens, monoclonal antibodies, polyclonal antibodies, and antibody fragments.

Follicle-stimulating hormone (FSH) is a glycoprotein polypeptide in the form of a 35.5 kDa polypeptide, which is a heterodimer of two polypeptides, alpha and beta, It is one of the gonadotrophins that promotes and maintains female follicular growth and male spermatogenesis. It is synthesized and secreted in the gonadal cells of the anterior pituitary gland and regulates the body's reproductive process, development, growth, pubertal maturation (Annu Rev Biochem., 50: 465-495, 1981; Proc Natl Acad Sci USA., 109 (31): 12491-12496, 2012). In general, follicle stimulating hormone (IVF) is used to induce ovarian hyperstimulation during in vitro fertilization (IVF). In the case of solid tumors, FSH receptors have been reported to be highly expressed in tumor vascular endothelium and are involved in the regeneration of neovascularization, and thus can be used as anticancer therapy when FSH and receptor antagonists are developed (N Engl J Med., 363 ): 1621-1630, 2010).

The present invention aims to provide a method for increasing the half-life of a protein.

The present invention also aims to provide a protein having one or more lysine residues substituted in the amino acid sequence, wherein the protein has an increased half-life.

It is also an object of the present invention to provide a pharmaceutical composition comprising a protein having an increased half-life.

In order to achieve the above object, the present invention provides a method for increasing the half-life of a protein, including replacing one or more lysine residues present in the amino acid sequence of the protein.

In the present invention, lysine residues of proteins may be substituted with conservative amino acids. In the present invention, "conservative amino acid substitution" means that an amino acid residue is substituted by another amino acid residue having a similar side chain, e.g., a charge or hydrophobic chemical character. In general, conservative amino acid substitutions do not substantially change the functional properties of the protein. Examples of amino acid groups having side chains with similar chemical properties include: 1) aliphatic side chains: glycine, alanine, valine, leucine and isoleucine; 2) aliphatic-hydroxyl side chain: serine and threonine; 3) Amide-containing side chains: asparagine and glutamine; 4) aromatic side chains: phenylalanine, tyrosine and tryptophan; 5) Basic side chains: lysine, arginine and histidine; 6) Acid side chains: aspartate and glutamate 7) Sulfur-containing side chains: include cysteine and methionine.

In the present invention, the lysine residue of the protein may be substituted with arginine or histidine containing a basic side chain, preferably arginine residue.

According to the present invention, a protein in which at least one lysine residue present in the amino acid sequence of a protein is substituted with arginine may have an increased half-life and may remain in the body for a long time.

Figure 1 shows the structure of FSH-beta expression vector.
Figure 2 shows the FSH-beta gene size and PCR results.
Fig. 3 shows the expression of FSH-β through the plasmid in HEK-293T cells.
Figure 4 shows the degradation pathway of FSH-beta through ubiquitination assay.
Figure 5 shows the degree of ubiquitination of the FSH-beta substituent in which the lysine residue was replaced with arginine as compared to the wild type.
Figure 6 shows the half-life change of FSH-beta after treatment with the protein synthesis inhibitor cycloheximide (CHX).
Figure 7 shows the results for effects such as JAK-STAT, PI3K-AKT and MAPK / ERK signal induction.

In one embodiment of the invention, the protein is FSH- ?. At least one of the 67, 104 and 128 lysine residues from the N-terminus in the FSH-beta amino acid sequence represented by SEQ ID NO: 1 is substituted with an arginine residue. Accordingly, there is provided a pharmaceutical composition for the treatment of infertility and / or solid cancer, which has increased half-life, FSH for inducing ovarian hyperstimulation containing the same, and /

In the present invention, site-directed mutagenesis was used to replace the lysine residue present in the amino acid sequence of the protein with the arginine (R) residue. In this method, a primer is prepared using a DNA sequence that induces a specific mutation, and then a PCR is carried out under specific conditions to prepare a plasmid DNA in which a specific amino acid residue is substituted.

In the present invention, the target protein was transfected into the cell line by immunoprecipitation analysis and precipitated to confirm the degree of ubiquitination. As a result of treatment with the MG132 (proteasome inhibitor) reagent, the degree of ubiquitination was increased, - proteasome degradation pathway.

In the present invention, the pharmaceutical composition may be delivered into the body by various routes including oral, transcutaneous, subcutaneous, intravenous or intramuscular administration and may be administered as a syringe formulation . In addition, the pharmaceutical composition of the present invention can be formulated according to methods well known to those skilled in the art, such as rapid release, delayed release or slow release after administration according to the method. The formulations may be in the form of tablets, pills, powders, sachets, elixirs, suspensions, emulsions, solutions, syrups, Aerosol, soft and hard gelatin capsules, sterile injectable solutions, sterile packaged powders, and the like. Suitable carriers, excipients and diluents include, but are not limited to, lactose, dextrose, sucrose, mannitol, xylitol, erythritol, maltitol, carbohydrates, Gum acacia, alginates, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoates, propylhydroxybenzoates, talc, magnesium stearate, and mineral oil. . In addition, the formulations can additionally include fillers, anti-agglutinating agents, lubricating agents, wetting agents, flavoring agents, emulsifiers, preservatives, and the like. have.

In the present invention, the singular forms include plural forms unless expressly stated otherwise. Furthermore, terms such as "comprising" in the present invention are interpreted to have a similar meaning to "including". In the present invention, " physiologically active (poly) peptide or protein " means a (poly) peptide or protein which exhibits biological activity useful when administered to a mammal including a human.

Hereinafter, the present invention will be described in more detail based on examples. The following examples are for illustrative purposes only and are not intended to limit the scope of the present invention.

Example 1: FSH -β protein Ubiquitination  Analysis and confirmation of increased half-life and confirmation of intracellular signaling

1. As an expression vector Cloning  And protein expression confirmation

(1) expression vector Cloning

The FSH-β DNA amplification product by polymerase chain reaction and pcDNA3-myc (5.6 kb) were ligated with restriction enzymes BamHI and XhoI and then cloned (FIG. 1, FSH-β amino acid sequence: SEQ No.1 ), And the result was confirmed by agarose gel electrophoresis after restriction enzyme digestion (Fig. 2). In addition, the underlined and bolded parts on the nucleotide sequence of FIG. 1 are part of the primer set used for confirmation of the cloned site through polymerase chain reaction, and the results are shown in the agarose gel electrophoresis (Fig. 2). The polymerase chain reaction conditions were as follows; The initial denaturation was carried out at 94 ° C for 3 minutes, followed by repeated cycles of 25 cycles of denaturation at 94 ° C for 30 seconds, annealing at 56 ° C for 30 seconds, and extension at 72 ° C for 1 minute, Thereafter, reaction was carried out at 72 ° C for 10 minutes. In order to confirm whether the DNA thus prepared properly expressed in the protein, myc present in the pcDNA3-myc vector shown in the map of Fig. 1 was Western blotted using an anti-myc (9E10, Santa Cruz Biotechnology, sc- Lt; / RTI > expression. (Fig. 3). It was confirmed that the FSH-beta protein bound to myc was well expressed and the quantification was loaded through the blot confirmed by actin (Fig. 3).

(2) Lysine  (Lysine, K) Residue  substitution

The lysine residue was substituted with arginine using site-directed mutagenesis and the primer FSH-beta K67R FP 5'-GGCCCAAAATCCAGAGAACATGTACCTT-3 '(SEQ ID NO: 2) ), RP 5'-AAGGTACATGTTCTCTGGATTTTGGGCC-3 '(SEQ ID No. 3), FSH-β K104R FP 5'-GTCACTGTGGCAGGTGTGACAGCGA-3' (SEQ ID No. 4), RP 5'- TCGCTGTCACACCTGCCACAGTGAC- (SEQ. ID. No. 6) and RP 5'-CCGCGTACGCGTTTCTCTCATTTCACC-3 '(SEQ. ID No. 7) were prepared and PCR was performed under specific conditions to replace a specific amino acid residue Was prepared. Three plasmid DNAs each with pcDNA3-myc-FSH-β as templates and Lysine residues substituted with arginine (K → R) were prepared (Table 1).

Lysine (K) residue position Lysine (K) was substituted with Arginine (R) and FSH-β construct 67 pcDNA3-myc-FSH-beta (K67R) 104 pcDNA3-myc-FSH-beta (K104R) 128 pcDNA3-myc-FSH-beta (K128R)

2. In vivo Ubiquitination  analysis

HEK 293T cells were infected with a plasmid encoding pcDNA3-myc-FSH-beta WT and pMT123-HA-ubiquitin DNA. To confirm the ubiquitination process, 3 μg of pcDNA3-myc-FSH-β WT and 1 μg of pMT123-HA-ubiquitin DNA were co-transfected into the cells. After 24 hours, MG132 (proteasome inhibitor, 5 μg / Ml, Sigma-Aldrich) for 6 hours, followed by immunoprecipitation analysis (FIG. 4). In order to compare the degree of ubiquitination between WT and the substituents, we used pcDNA3-myc-FSH-β substitution (K67R), pcDNA3-myc-FSH-β substituent (K104R), pcDNA3-myc- (Co-transfection) of HEK 293T cells (ATCC, CRL-3216) together with 1 쨉 g of pMT123-HA-ubiquitin DNA and subjected to immunoprecipitation analysis after 24 hours (Fig. 5).

Samples obtained for immunoprecipitation were dissolved in lysis buffer (1% Triton X, 150 mM NaCl, 50 mM Tris-HCl, pH 8 and 1 mM PMSF (phenylmethanesulfonyl fluoride) and mixed with anti-myc (9E10) And incubated overnight at 4 ° C. Immunoprecipitates were separated by reaction with protein A / G beads (Santa Cruz Biotechnology) at 4 ° C. for 2 hours and then washed twice with lysis buffer. The protein samples were mixed with 2X SDS buffer, heated at 100 ° C for 7 minutes, and separated by SDS-PAGE. The separated proteins were transferred to a PVDF membrane and then subjected to anti-myc (9E10, Santa Cruz Biotechnology, blocking solution containing anti-beta-actin (sc-40), anti-HA (Santa Cruz Biotechnology, sc-7392) and anti- beta -actin (Santa Cruz Biotechnology, labeled antibody to mouse IgG (H + L), KPL, 074-1806) using an ECL system Western blot detection kit, ABfrontier, Seoul, Korea).

As a result, when immunoprecipitation was carried out with anti-myc (9E10, Santa Cruz Biotechnology, sc-40), ubiquitin-binding to ubiquitin was detected in pcDNA3-myc-FSH-β WT, The band appeared thick (Fig. 4, lanes 3 and 4). In addition, treatment with MG132 (protease inhibitor, 5 / / ml) for 6 hours resulted in an increase in polyubiquitin formation and a darker band in which ubiquitin was detected (Fig. 4, lane 4). In the case of pcDNA3-myc-FSH-β substituent (K67R), pcDNA3-myc-FSH-β substituent (K104R) and pcDNA3-myc-FSH-β substituent (K128R) (Fig. 5, lanes 3 to 5) were detected because FSH-beta substituent (K67R), pcDNA3-myc-FSH-beta substituent (K104R), and pcDNA3-myc-FSH-beta substituent (K128R) failed to bind to ubiquitin ). These results show that FSHβ binds to ubiquitin and is degraded by polyubiquitination through the ubiquitin-proteasome system.

3. Protein production inhibitors cycloheximide  ( CHX )On by FSH -β Half-life of

3 μg of pcDNA3-myc-FSH-β WT, pcDNA3-myc-FSH-β substituent (K67R), pcDNA3-myc-FSH-β substituent (K104R) and pcDNA3-myc-FSH-β substituent (K128R) And transfected. After 48 h of transfection, the protein production inhibitor cycloheximide (CHX) (Sigma-Aldrich) (100 μg / ml) was treated and half-life was measured over 30 min, 60 min and 90 min. As a result, it was confirmed that decomposition of human FSH-β was inhibited (FIG. 6). The half-life of human pcDNA3-myc-FSH-β substituent (K104R) was more than 60 minutes, while the half-life of human FSH-β was within 30 minutes. The half-life of the substituent (K128R) was longer than 90 minutes and longer than the WT, and the result was plotted (Fig. 6).

4. Intracellular FSH -β Wow FSH - beta Substituents  Confirmation of signaling by

FSH signaling has been reported to increase intracellular cAMP, thereby regulating the phosphorylation of transcription factors such as CREBP when PKA is activated, and is also associated with Erk and PI3K / AKT signaling (Front Endocrinol (Lausanne), 6 : 142, 2015; Biochem J., 473 (11): 1483-1501, 2016).

In this example, signal transduction by FSH-beta and FSH-beta substituents was observed in the cells. 3 μg each of pcDNA3-myc-FSH-β WT, pcDNA3-myc-FSH-β substitution (K67R), pcDNA3-myc-FSH-β substituent (K104R) and pcDNA3-myc-FSH-β substitution To infect HeLa cells. After 2 days of infection, proteins were extracted from the cells, quantified, and Western blotting was carried out to confirm intracellular signal transduction. In this process, HeLa infected with pcDNA3-myc-FSH-beta WT, pcDNA3-myc-FSH-beta substituent (K67R), pcDNA3-myc-FSH-beta substituent (K104R), pcDNA3-myc-FSH-beta substituent (K128R) (9E10, Santa Cruz Biotechnology, sc-40), anti-STAT3 (Santa Cruz Biotechnology, sc-21876), and then transferred to polyvinylidene difluoride (PVDF) ), Anti-phospho-STAT3 (Y705, cell signaling 9131S), anti-AKT (H-136, Santa Cruz Biotechnology, sc-8312), anti-phospho-AKT (S473, cell signaling 9271S) (9B3, Abfrontier LF-MAO134), anti-phospho-Erk1 / 2 (Thr202 / Tyr204, Abfrontier LF- PA0090) and anti- beta -actin (Santa Cruz Biotechnology, sc- (Goat anti-rabbit IgG-HRP, Santa Cruz Biotechnology, sc-2004) and anti-mouse (Peroxidase-labeled antibody to mouse IgG (H + L), KPL, 074-1806 ) Secondary antibodies were used for Western blot detection (ECL) kit, ABfrontier, Seoul, Korea). As a result, the expression of pcDNA3-myc-FSH-β substituent (K67R), pcDNA3-myc-FSH-β substituent (K104R) and pcDNA3-myc- Phospho-STAT3, phospho-AKT and phospho-Erk1 / 2 signaling similar or increased to WT (Fig. 7).

According to the present invention, a folate stimulating hormone? Having an increased half-life is provided. Accordingly, the present invention relates to a follicle-stimulating hormone? Which can be used as a therapeutic agent, and thus may be usefully used in the pharmaceutical industry.

&Lt; 110 > UbiProtein. Corp <120> A method for extending half-life of FSH beta <130> UBPRN17P-0007 <160> 7 <170> KoPatentin 3.0 <210> 1 <211> 129 <212> PRT <213> Artificial Sequence <220> <223> Follicle-stimulating hormone beta <400> 1 Met Lys Thr Leu Gln Phe Phe Phe Leu Phe Cys Cys Trp Lys Ala Ile   1 5 10 15 Cys Cys Asn Ser Cys Glu Leu Thr Asn Ile Thr Ile Ala Ile Glu Lys              20 25 30 Glu Glu Cys Arg Phe Cys Ile Ser Ile Asn Thr Thr Trp Cys Ala Gly          35 40 45 Tyr Cys Tyr Thr Arg Asp Leu Val Tyr Lys Asp Pro Ala Arg Pro Lys      50 55 60 Ile Gln Lys Thr Cys Thr Phe Lys Glu Leu Val Tyr Glu Thr Val Arg  65 70 75 80 Val Pro Gly Cys Ala His His Ala Asp Ser Leu Tyr Thr Tyr Pro Val                  85 90 95 Ala Thr Gln Cys His Cys Gly Lys Cys Asp Ser Asp Ser Thr Asp Cys             100 105 110 Thr Val Gly Leu Gly Pro Ser Tyr Cys Ser Phe Gly Glu Met Lys         115 120 125 Glu     <210> 2 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 2 ggcccaaaat ccagagaaca tgtacctt 28 <210> 3 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 3 aaggtacatg ttctctggat tttgggcc 28 <210> 4 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 4 gtcactgtgg caggtgtgac agcga 25 <210> 5 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 5 tcgctgtcac acctgccaca gtgac 25 <210> 6 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 6 ggtgaaatga gagaaacgcg tacgcgg 27 <210> 7 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 7 ccgcgtacgc gtttctctca tttcacc 27

Claims (5)

Wherein the lysine residue at positions 67, 104 and 128 from the N-terminus of follicle-stimulating hormone beta (FSH beta) having the amino acid sequence of SEQ ID NO: 1 is substituted with arginine Follicle - stimulating hormone with half - life β.
delete (a) a promoter; And (b) an expression vector comprising a nucleotide sequence encoding the follicle stimulating hormone? Of claim 1, wherein the promoter and the nucleotide sequence are operatively linked. A host cell comprising the expression vector of claim 3.
delete

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