KR20180122898A - A method for extending half-life of GMCSF - Google Patents

Abstract

The present invention relates to a method for extending the half-life of GM-CSF, including the substitution of one or more lysine residues present in the amino acid sequence of GM-CSF, or to a GM-CSF with extended half-life. The lysine residue-substituted GM-CSF of the present invention remains in the human body for a long time and has excellent therapeutic effect.

Description

A method for increasing the half-life of GM-CSF (GMCSF)

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.

The granulocyte-macrophage colony-stimulating factor (GM-CSF) is a protein secreted by macrophages, T cells, mast cells, natural killer cells, endothelial cells and fibroblasts, Is a cyto-Cain. In addition, stem cells stimulate the production of granulocytes (neutrophils, basophils, eosinophils) and monocytes, and act on immune / infectious responses by rapidly increasing the number of macrophages and allowing them to counteract the infection response (Med Oncol. 1): 774, 2014; Blood, 77 (6): 1131-1145,1991). It has been reported that recombinant GM-CSF can be used as a vaccine adjuvant in HIV-infected patients (Hum Vaccin Immunother., 8 (11): 16541658, 2012; Vaccines (Basel), 2 (1): 160178, 2014). It is also reported that patients with non-Hodgkin's lymphoma, lymphoma, leukemia or Hawkins are effective in increasing leukocyte count when treated with bone marrow transplantation, but they are highly expressed in the joints of patients with rheumatoid arthritis, thus reducing inflammation or damage if GM-CSF is reduced (Expert Rev Neurother., 13 (3): 313-335, 2013).

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 a GM-CSF expression vector.
Figure 2 shows the size of the GM-CSF gene and the PCR result.
Figure 3 shows the expression of the protein through plasmid of GM-CSF in HEK-293T cells.
Figure 4 shows the degradation pathway of GM-CSF through ubiquitination assay.
Figure 5 shows the degree of ubiquitination of a GM-CSF substituent in which the lysine residue is replaced with arginine as compared to the wild type.
Figure 6 shows the half-life change of GM-CSF 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 GM-CSF. At least one of the 89, 91, and 102 lysine residues from the N-terminus in the amino acid sequence of GM-CSF represented by SEQ ID NO: 1 is substituted with an arginine residue. Accordingly, there is provided a pharmaceutical composition for preventing and / or treating cancer and / or rheumatoid arthritis comprising GM-CSF having an increased half-life and neutropenia prevention thereof and / or an immunological disease and / or a solid cancer and a blood cancer.

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: Preparation of GM- CSF  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 GM-CSF DNA amplification product by polymerase chain reaction and pcDNA3-myc (5.6 kb) were digested with restriction enzymes BamHI and XhoI, and then ligated and cloned (Fig. 1, GM-CSF amino acid sequence: ), And the result was confirmed by agarose gel electrophoresis after restriction enzyme digestion (Fig. 2). Also, the underlined and bolded parts on the nucleotide sequence of Figure 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). 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 at 94 ° C for 30 seconds for denaturation, 30 seconds at 60 ° C for annealing, and 30 seconds at 72 ° C for extension reaction, Thereafter, reaction was carried out at 72 ° C for 10 minutes. In order to confirm whether the DNA prepared as described above is properly expressed in the protein, myc present in the pcDNA3-myc vector shown in the map of Fig. 15 was expressed by Western blotting using an anti-myc (9E10, sc-40) , Confirming that the GM-CSF protein bound to myc was well expressed and quantitatively loaded through the blot confirmed by actin (FIG. 3).

(2) Lysine  (Lysine, K) Residue  substitution

The lysine residue was replaced with arginine using site-directed mutagenesis and the primer (GM-CSF K89R FP 5'-GGCAGCCTCACCAGGCTCAAGGGCC-3 '(SEQ. 2), RP 5'-GGCCCTTGAGCCTGGTGAGGCTGCC-3 '(SEQ No. 3); GM-CSF K91R FP 5'-CTC ACCAAGCTCAGGGGCCCCTTGACC-3' (SEQ ID No. 4); RP 5'-GGTCAAGGGGCCCCTGAGCTTGGTGAG-3 ' (SEQ ID No. 6) and RP 5'-AGGGCAGTGCTGTCTGTAGTGGCTAGC-3 '(SEQ No. 7), and then PCR was carried out under specific conditions to obtain a specific amino acid Three plasmid DNAs with lysine residues replaced with arginine (K → R) were prepared using pcDNA3-myc-GM-CSF as a template (Table 1).

Lysine (K) residue position GM-CSF constructs in which Lysine (K) was replaced with Arginine (R) 89 pcDNA3-myc-GM-CSF (K89R) 91 pcDNA3-myc-GM-CSF (K91R) 102 pcDNA3-myc-GM-CSF (K102R)

2. In vivo Ubiquitination  analysis

HEK 293T cells were infected with a plasmid encoding pcDNA3-myc-GM-CSF WT and pMT123-HA-ubiquitin DNA. To confirm the ubiquitination process, 3 μg of pcDNA3-myc-GM-CSF 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 substituent, pcDNA3-myc-GM-CSF WT, pcDNA3-myc-GM-CSF substitution (K89R), pcDNA3-myc- GM- CSF substituent (K91R) 3 μg of each GM-CSF substitute (K102R) was co-transfected with HEK 293T cells (ATCC, CRL-3216) together with 1 μg 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 then mixed with anti-myc (9E10) Immunoprecipitates were isolated by reacting with protein A / G beads (Santa Cruz Biotechnology) at 4 ° C for 2 hours, followed by washing twice with lysis buffer. Immunoblotting Protein samples were mixed with 2X SDS buffer, boiled at 100 ° C for 7 minutes and then separated by SDS-PAGE. The separated proteins were transferred to PVDF membrane and incubated with anti-myc (9E10, Santa Cruz Biotechnology, sc- (Peroxidase-labeled antibody), anti-mouse (Santa Cruz Biotechnology, sc-7392) and anti-beta-actin (Santa Cruz Biotechnology, to mouse IgG (H + L), KPL, 074-1806) secondary antibody to Western blot The results showed that when immunoprecipitation was performed with anti-myc (9E10, sc-40), pcDNA3-myc-GM-CSF WT was bound to ubiquitin and poly (Fig. 4, lanes 3 and 4). In addition, in the case of treatment with MG132 (protease inhibitor, 5 / / ml) for 6 hours, the ubiquitin- (K89R), pcDNA3-myc-GM-CSF substitution (K91R), pcDNA3-myc-GM (SEQ ID NO: In the case of the -CSF substituent (K102R), the band was longer than that of WT, and pcDNA3-myc-GM-CSF WT, pcDNA3-myc-GM- CSF substituent (K89R), pcDNA3- -myc-GM-CSF substituent (K102R) failed to bind to ubiquitin, resulting in a small amount of ubiquitin detected (Fig. 5). These results show that GM-CSF binds to ubiquitin and is degraded by polyubiquitination through the ubiquitin-proteasome system.

3. Protein production inhibitor cycloheximide  ( CHX ) In the GM- CSF's  Verify half-life

3 g of each of the pcDNA3-myc-GM-CSF WT, pcDNA3-myc-GM-CSF substituent (K89R), pcDNA3-myc-GM- CSF substituent (K91R) 293T cells. After 48 hours of transfection, half-life was measured for 30 minutes, 60 minutes, and 90 minutes after treatment with the protein production inhibitor cycloheximide (CHX) (Sigma-Aldrich) (100 μg / (Fig. 6). The half-life of human GM-CSF is within 30 minutes, while the half-lives of human pcDNA3-myc-GM-CSF substituent (K91R) and pcDNA3-myc-GM- CSF substituent (K102R) are longer than 60 minutes and 90 minutes And the results are shown graphically (Figure 6).

4. Intracellular GM- CSF  And GM- CSF  Identification of signaling by substituents

It has been reported that the intracellular role of GM-CSF is mediated by JAK / STAT, MAPK and PI3K / AKT signaling (Blood, 119 (15): 3383-3393, 2012).

In this example, signal transduction by GM-CSF and GM-CSF substituents was confirmed in the cells. Two days after infecting HeLa cells with 3 ㎍ each of pcDNA3-myc-GM-CSF substitution (K89R), pcDNA3-myc-GM-CSF substitution (K91R) and pcDNA3-myc-GM- CSF substitution (K102R) After the protein was extracted from the infected cells, Western blotting was performed to confirm intracellular signal transduction. Proteins isolated from HeLa cells infected with pcDNA3-myc-GM-CSF substituent (K89R), pcDNA3-myc-GM-CSF substituent (K91R) and pcDNA3-myc-GM-CSF substituent (K102R) (970, Santa Cruz Biotechnology, sc-40), anti-STAT3 (Santa Cruz Biotechnology, sc-21876), anti-phospho-STAT3 (Y705, cell signaling 9131S) 136, Santa Cruz Biotechnology, sc-8312), anti-phospho-AKT (S473, cell signaling 9271S), anti-Erk1 / 2 (9B3, Abfrontier LF- MA0134), anti- phospho-Erk1 / 2 (Thr202 / Tyr204, Anti-rabbit IgG-HRP, Santa Cruz Biotechnology, Santa Cruz Biotechnology, sc-47778) at a weight ratio of 1: 1000 to 1: (Western blot detection kit, AB Frontier, Seoul, Korea) using a secondary antibody (Peroxidase-labeled antibody to mouse IgG (H + L), KPL, 074-1806) Respectively. As a result, pcDNA3-myc-GM-CSF substituent (K89R), pcDNA3-myc-GM-CSF substituent (K91R) and pcDNA3-myc- GM- CSF substituent (K102R) Showed the same or increased phospho-STAT3 and phospho-AKT signaling as WT (Fig. 7).

According to the present invention, an GM-CSF with an increased half-life is provided. Therefore, the present invention relates to GM-CSF which can be used as a therapeutic agent, and can be usefully used in the pharmaceutical industry.

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Claims (4)

Wherein one of the lysine residues at positions 91 and 102 from the N-terminus of the granulocyte-macrophage colony-stimulating factor (GM-CSF) having the amino acid sequence of SEQ ID NO: 1 is substituted with arginine GM-CSF having an increased half-life. A pharmaceutical composition for preventing, treating, or preventing and treating cancer, rheumatoid arthritis, or cancer and rheumatoid arthritis, comprising the GM-CSF of claim 1 and a pharmaceutically acceptable excipient. (a) a promoter; (b) a base sequence encoding the GM-CSF of claim 1; And an optional linker, wherein the promoter and the base sequence are operatively linked. A host cell comprising the expression vector of claim 3.

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