Angiopoietin-1 fusion protein and use thereof

Abstract

The present invention relates to an angiopoietin-1 fusion protein and a use thereof. The novel angiopoietin-1 fusion protein according to the present invention is structurally stable, and can enhance the vascular regenerative capability of peripheral blood stem cells, thereby promoting angiogenesis and vascular maturation. Therefore, the protein is expected to be effectively utilized for the development of therapeutic agents for ischemic diseases.

Description

Angiopoietin-1 fusion protein and uses thereof

[0001] The present invention relates to angiopoietin-1 fusion protein and uses thereof.

[0002] This invention claims priority to Republic of Korea Patent Application No. 10-2023-0120512, filed September 11, 2023, the entire disclosure of which is incorporated herein by reference.

[0003]

[0004] With the recent rise in mortality from cardiovascular disease, many treatments for myocardial infarction, the leading cause, are being developed. As myocardial infarction worsens and can lead to heart failure, prevention is crucial. However, no clear treatment is currently available. While stem cell-based therapies are being developed, methods like bone marrow aspiration to obtain stem cells pose a burden on both patients and medical staff, limiting their use.

[0005] In addition to myocardial infarction and heart failure, the increase in cerebral infarction and lower limb ischemic disease is also serious, and it is predicted that all of these diseases can be treated by implementing angiogenesis using the angiopoietin protein.

[0006] Meanwhile, natural angiopoietin is a protein that is active only when it exists as a triplex or higher multimer, as it has a coiled coil domain that can form multimers. Previously, when natural angiopoietin was expressed in animal cells, there were problems with aggregation and solubility, so COMP-Ang1 was developed that can stably form multimers by replacing the coiled coil domain with cartilage oligomeric matrix protein (COMP).

[0007] However, the developed COMP-Ang1 is also difficult to apply to patients due to the difficulty in purifying clinical-grade proteins. Therefore, there is a need to develop a novel protein that has structural stability and activity equivalent to or higher than COMP-Ang1 while meeting the standards for clinical-grade protein purification.

[0008]

[0009] The present inventors conducted extensive research to commercialize angiopoietin-1, which had been difficult to use clinically due to its low solubility and unstable activity due to structural problems, and as a result, they discovered that a fusion protein that fuses angiopoietin-1 with vasodilator-stimulated phosphoprotein (VASP), which can form pentamers, is formed into a structurally stable multimer, and that when peripheral blood stem cells are primed using this, angiogenesis is promoted, thereby completing the present invention.

[0010] Accordingly, an object of the present invention is to provide a fusion protein comprising vasodilator-stimulated phosphoprotein (VASP) or a fragment thereof; and angiopoietin-1 protein or a fragment thereof.

[0011] Another object of the present invention is to provide a polynucleotide encoding the fusion protein.

[0012] Another object of the present invention is to provide an expression vector comprising the polynucleotide.

[0013] Another object of the present invention is to provide a cell into which the polynucleotide or an expression vector comprising the same has been introduced.

[0014] Another object of the present invention is to provide a method for producing the fusion protein, comprising a step of culturing a cell containing a polynucleotide encoding the fusion protein or an expression vector containing the same.

[0015] Another object of the present invention is to provide a composition for promoting the activity of peripheral blood stem cells, which comprises the above fusion protein as an active ingredient.

[0016] Another object of the present invention is to provide a method for promoting the activity of peripheral blood stem cells, which comprises a step of treating the fusion protein to isolated peripheral blood stem cells.

[0017] Another object of the present invention is to provide a pharmaceutical composition for preventing or treating ischemic disease, which comprises the fusion protein or peripheral blood stem cells treated therewith as an active ingredient.

[0018]

[0019] However, the technical problems to be solved by the present invention are not limited to the problems mentioned above, and other problems not mentioned can be clearly understood by a person having ordinary skill in the technical field to which the present invention belongs from the description below.

[0020]

[0021] To achieve the above purpose, the present invention provides a fusion protein comprising vasodilator-stimulated phosphoprotein (VASP) or a fragment thereof; and angiopoietin-1 protein or a fragment thereof.

[0022] In one embodiment of the present invention, the vasodilator-stimulated phosphoprotein or fragment thereof may further comprise, but is not limited to, a disulfide bonding domain.

[0023] In another embodiment of the present invention, the vasodilator-stimulated phosphoprotein or fragment thereof may comprise any one amino acid sequence selected from the group consisting of SEQ ID NOs: 3 to 5, but is not limited thereto.

[0024] In another embodiment of the present invention, the angiopoietin-1 protein or fragment thereof may include, but is not limited to, the amino acid sequence of SEQ ID NO: 1 or 2.

[0025] In another embodiment of the present invention, the vasodilator-stimulated phosphoprotein or fragment thereof; and the angiopoietin-1 protein or fragment thereof; may be sequentially linked from the N-terminus to the C-terminus of the vasodilator-stimulated phosphoprotein or fragment thereof, but is not limited thereto.

[0026] In another embodiment of the present invention, the vasodilator-stimulated phosphoprotein or fragment thereof; and the angiopoietin-1 protein or fragment thereof may be linked by a linker, but are not limited thereto.

[0027] In another embodiment of the present invention, the fusion protein may further comprise, but is not limited to, a signal peptide.

[0028] In another embodiment of the present invention, the fusion protein may comprise, but is not limited to, the amino acid sequence of SEQ ID NO: 9.

[0029] In another embodiment of the present invention, the fusion protein can enhance the angiogenic capacity of peripheral blood stem cells, but is not limited thereto.

[0030] Additionally, the present invention provides a polynucleotide encoding the fusion protein.

[0031] In addition, the present invention provides an expression vector comprising the polynucleotide.

[0032] In addition, the present invention provides a cell into which the polynucleotide or an expression vector comprising the same has been introduced.

[0033] In one embodiment of the present invention, the cell may be a CHO-K1 cell, but is not limited thereto.

[0034] In another embodiment of the present invention, the polynucleotide or the vector expressing the same may be introduced into a hot-spot 13 region of a cell, but is not limited thereto.

[0035] In addition, the present invention provides a method for producing the fusion protein, comprising a step of culturing a cell comprising a polynucleotide encoding the fusion protein; or an expression vector comprising the same.

[0036] In one embodiment of the present invention, the cell may be a CHO-K1 cell, but is not limited thereto.

[0037] In another embodiment of the present invention, the culture may be a suspension culture, but is not limited thereto.

[0038] In addition, the present invention provides a composition for promoting the activity of peripheral blood stem cells, which comprises the fusion protein as an active ingredient.

[0039] In addition, the present invention provides a use of the fusion protein or a composition containing the fusion protein as an active ingredient for promoting peripheral blood stem cell activity.

[0040] In addition, the present invention provides a use for producing a peripheral blood stem cell activity promoter using the fusion protein or a composition containing the fusion protein as an active ingredient.

[0041] In addition, the present invention provides a method for promoting the activity of peripheral blood stem cells, comprising a step of treating the fusion protein to isolated peripheral blood stem cells.

[0042] In one embodiment of the present invention, the step of treating the fusion protein may be performed for, but is not limited to, 0.25 to 5 hours.

[0043] In addition, the present invention provides a pharmaceutical composition for preventing or treating ischemic disease, which comprises the fusion protein or peripheral blood stem cells treated therewith as an active ingredient.

[0044] In addition, the present invention provides a method for preventing or treating ischemic disease, comprising administering to a subject in need thereof a pharmaceutically effective amount of the fusion protein; peripheral blood stem cells treated with the fusion protein; or a composition comprising the fusion protein or the peripheral blood stem cells as an active ingredient.

[0045] In addition, the present invention provides a use for preventing or treating ischemic disease using the fusion protein; peripheral blood stem cells treated with the fusion protein; or a composition comprising the fusion protein or the peripheral blood stem cells as an active ingredient.

[0046] In addition, the present invention provides a use for manufacturing an agent for preventing or treating ischemic disease, comprising the fusion protein; peripheral blood stem cells treated with the fusion protein; or a composition comprising the fusion protein or the peripheral blood stem cells as an active ingredient.

[0047] In one embodiment of the present invention, the fusion protein can enhance the angiogenic capacity of peripheral blood stem cells, but is not limited thereto.

[0048] In another embodiment of the present invention, the treatment of the fusion protein may be performed for, but is not limited to, 0.25 to 5 hours.

[0049] In another embodiment of the present invention, the ischemic disease may be selected from the group consisting of ischemic heart disease, ischemic myocardial infarction, ischemic heart failure, ischemic enteritis, ischemic vascular disease, ischemic eye disease, ischemic retinopathy, ischemic glaucoma, ischemic renal failure, ischemic cerebral disease, and ischemic lower extremity disease, but is not limited thereto.

[0050]

[0051] The novel angiopoietin-1 fusion protein according to the present invention is structurally stable and can promote the formation and maturation of blood vessels by enhancing the vascular regeneration capacity of peripheral blood stem cells, and thus is expected to be useful in the development of ischemic disease treatment agents.

[0052]

[0053] Figure 1 is a schematic diagram of the vectors in Table 1.

[0054] Figure 2 shows the results of temporarily expressing the vectors in Table 1.

[0055] Figure 3 illustrates a method for introducing the FVA3-Ang1 gene into CHO-KIDW cells for mass production of FVA3-Ang1.

[0056] Figure 4 briefly illustrates the cell line selection process for mass production of FVA3-Ang1.

[0057] Figure 5 shows the results of confirming the dose-dependent effect of FVA3-Ang1 through phosphorylation of Akt in HUVEC cells (*: p<0.05, **: p<0.01, ***: p<0.001, hereinafter the same).

[0058] Figure 6 shows the results of confirming the time-dependent effect of FVA3-Ang1 through CD31 and VECAD expression in HUVEC cells.

[0059] Figures 7a and 7b are naive Mob PBSC group, primed with FVA3-Ang1 for 1 hour MobPBSC group, primed with FVA3-Ang1 for 24 hours Mob This shows the results of gene expression analysis in the PBSC group. Figure 7a shows a gene expression analysis protocol using microarray, and Figure 7b shows a heat map showing the results of gene expression analysis.

[0060] Figure 8: PBMC group, naive Mob PBSC group, primed with FVA3-Ang1 for 1 hour Mob PBSC group, primed with FVA3-Ang1 for 24 hours Mob This shows the results of confirming RNA expression of CD31, CXCR4, VE CAD, and Tie2 in the PBSC group.

[0061] Figures 9a and 9b show the PBMC group, naive Mob PBSC group, primed with FVA3-Ang1 for 1 hour Mob PBSC group, primed with FVA3-Ang1 for 24 hours Mob This shows the results of flow cytometry analysis to confirm the protein expression of CD31, CXCR4, VE CAD, CD45, and Annexin V in the PBSC group.

[0062] Figure 10 is primed with FVA3-Ang1. Mob This shows the experimental protocol in a hindlimb vascular occlusion model to confirm the infarct treatment effect of PBSC.

[0063] Figure 11 shows the FVA3-Ang1-primed model of hindlimb vascular occlusion. Mob This shows the results of visually observing the infarct area treatment effect of PBSC.

[0064] Figure 12 shows the FVA3-Ang1-primed model of hindlimb vascular occlusion using laser Doppler flow imaging (LDPI). Mob This shows the results of confirming the infarct area treatment effect of PBSC.

[0065] Figure 13 is primed with FVA3-Ang1. MobThis shows the experimental protocol in a myocardial infarction model to confirm the therapeutic effect of PBSC on the infarct area.

[0066] Figure 14 is primed with FVA3-Ang1. Mob To confirm the therapeutic effect of PBSC on the infarct area, FVA3-Ang1 was primed in a myocardial infarction model. Mob This shows the results of checking the overall survival rate after PBSC treatment.

[0067] Figure 15 shows the priming with FVA3-Ang1 in a myocardial infarction model. Mob This shows the results of echocardiography after PBSC treatment (LVESD (left ventricular end systolic diameter): left ventricular end systolic diameter, LEVDD (left ventricular end diastolic diameter): left ventricular end diastolic diameter, LVFS (left ventricular fractional shortening): left ventricular fractional shortening, LVEF (left ventricular ejection fraction): left ventricular ejection fraction).

[0068] Figure 16 shows the priming with FVA3-Ang1 in a myocardial infarction model. Mob This shows the results of confirming the fibrotic area after PBSC treatment.

[0069] Figure 17 shows the sequence of a novel angiopoietin-1 fusion protein (FVA3-Ang1) of the present invention.

[0070]

[0071] The present inventors conducted extensive research to commercialize angiopoietin-1, which had been difficult to use clinically due to its low solubility and unstable activity due to structural problems, and as a result, they confirmed that a fusion protein in which angiopoietin-1 is fused with vasodilator-stimulated phosphoprotein (VASP), which can form pentamers, is formed into a structurally stable multimer, and that this promotes angiogenesis by priming peripheral blood stem cells, thereby completing the present invention.

[0072]

[0073] Hereinafter, the present invention will be described in detail.

[0074]

[0075] The present invention provides a fusion protein comprising vasodilator-stimulated phosphoprotein (VASP) or a fragment thereof; and angiopoietin-1 protein or a fragment thereof; wherein the fusion protein may be referred to as an "angiopoietin-1 fusion protein" in the present invention.

[0076] The term "vasodilator-stimulated phosphoprotein (VASP)" as used herein refers to a protein that inhibits platelet activation by aggregating and forming a network of adhesion molecule glycoprotein GP IIb-IIIa (glycoprotein).

[0077] The above "vasodilator-stimulated phosphoprotein or fragment thereof" means a native or recombinant full-length vasodilator-stimulated phosphoprotein, or a protein having substantially equivalent physiological activity thereto, and the protein having substantially equivalent physiological activity may include not only a native or recombinant vasodilator-stimulated phosphoprotein, but also fragments thereof, functional equivalents, and functional derivatives thereof. According to one embodiment of the present invention, the fragment of the vasodilator-stimulated phosphoprotein may be a VASP domain, and the VASP domain may be comprised of amino acids 340 to 377 from the N-terminus of a vasodilator-stimulated phosphoprotein comprising or consisting of the amino acid sequence of SEQ ID NO: 3 or consisting of the amino acid sequence of SEQ ID NO: 4, but is not limited thereto.

[0078] In the present invention, the vasodilator-stimulated phosphoprotein or a fragment thereof may further include a disulfide bonding domain capable of stabilizing a structure, and the disulfide bonding domain may include or consist of the amino acid sequence of SEQ ID NO: 6, but is not limited thereto.

[0079] According to one embodiment of the present invention, the disulfide bond domain may be in a form linked to the C-terminus of a vasodilator-stimulated phosphoprotein or a fragment thereof, and in the present invention, the form in which the disulfide bond domain is linked to the vasodilator-stimulated phosphoprotein or a fragment thereof may be referred to as "VASP2" or "VASP2 domain". Preferably, the VASP2 may include the amino acid sequence of SEQ ID NO: 5 or consist of the amino acid sequence of SEQ ID NO: 5 in a form in which the disulfide bond domain is linked to the C-terminus of the fragment of the vasodilator-stimulated phosphoprotein, but is not limited thereto.

[0080] In the present invention, the vasodilator-stimulated phosphoprotein or a fragment thereof may include any one amino acid sequence selected from the group consisting of SEQ ID NOs: 3 to 5 or may be composed of any one amino acid sequence selected from the group consisting of SEQ ID NOs: 3 to 5, but is not limited thereto, and variants of the amino acid sequence may be included within the scope of the present invention.

[0081] The term "angiopoietin-1 protein (Ang-1)" used herein refers to a vascular growth factor that acts directly on vascular endothelial cells of the vascular system to create blood vessels, as a protein that promotes blood vessel formation. The angiopoietin-1 protein or a fragment thereof may include the amino acid sequence of SEQ ID NO: 1 or 2 or may be composed of the amino acid sequence of SEQ ID NO: 1 or 2, but is not limited thereto.

[0082] The above "angiopoietin-1 protein or fragment thereof" refers to a native or recombinant full-length angiopoietin-1 protein, or a protein having a physiological activity substantially equivalent thereto, and the protein having a physiological activity substantially equivalent thereto may include a native or recombinant angiopoietin-1 protein as well as a fragment thereof, a functional equivalent thereof, and a functional derivative thereof. According to one embodiment of the present invention, the fragment of the angiopoietin-1 protein may be an Angiopoietin-1 domain, and the Angiopoietin-1 domain may be comprised of amino acids 260 to 497 from the N-terminus of an angiopoietin-1 protein comprising the amino acid sequence of SEQ ID NO: 1 or consisting of the amino acid sequence of SEQ ID NO: 1 or consisting of the amino acid sequence of SEQ ID NO: 2, but is not limited thereto.

[0083] In the present invention, the fusion protein may be a vasodilator-stimulated phosphoprotein or a fragment thereof; and angiopoietin-1 protein or a fragment thereof; sequentially linked from the N-terminus to the C-terminus, and the vasodilator-stimulated phosphoprotein or a fragment thereof; and angiopoietin-1 protein or a fragment thereof; may be linked by a linker (GS), that is, the vasodilator-stimulated phosphoprotein or a fragment thereof; a linker (GS); and angiopoietin-1 protein or a fragment thereof may be sequentially linked from the N-terminus to the C-terminus in this order, but is not limited thereto.

[0084] In the present invention, the fusion protein may further include a signal peptide, and the signal peptide may be a signal peptide or a FLAG domain of an influenza A virus. According to one embodiment of the present invention, the fusion protein may include a signal peptide and a FLAG domain of an influenza A virus as a signal peptide, and may be connected in the order of the signal peptide and the FLAG domain of the influenza A virus from the N-terminus to the C-terminus, but is not limited thereto. The signal peptide of the influenza A virus may include the amino acid sequence of SEQ ID NO: 7 or may consist of the amino acid sequence of SEQ ID NO: 7, and the FLAG domain may include the amino acid sequence of SEQ ID NO: 8 or may consist of the amino acid sequence of SEQ ID NO: 8.

[0085] According to one embodiment of the present invention, the signal peptide and the fusion protein may be in a form in which the signal peptide and the fusion protein are sequentially connected. That is, the signal peptide, FLAG domain, and fusion protein of the influenza A virus may be sequentially connected from the N-terminus to the C-terminus, respectively, and the signal peptide and the fusion protein may be in a form in which they are sequentially connected via a linker (GIL), i.e., the signal peptide, the linker (GIL), and the fusion protein may be sequentially connected from the N-terminus to the C-terminus, respectively, but are not limited thereto.

[0086] According to one embodiment of the present invention, the fusion protein may comprise or consist of the following structure and may be referred to as “FVA3-Ang1”:

[0087] N'-influenza A virus signal peptide-FLAG domain-linker (GIL)-VASP2 domain [fragment of vasodilator-stimulated phosphoprotein-disulfide bond domain]-linker (GS)-fragment of angiopoietin-1 protein-C'

[0088] (In the above, N' is the N-terminus and C' is the C-terminus.)

[0089] In the present invention, the fusion protein "FVA3-Ang1" may include the amino acid sequence of SEQ ID NO: 9 or may consist of the amino acid sequence of SEQ ID NO: 9.

[0090] The protein or polypeptide comprising or consisting of any one of the amino acid sequences represented by SEQ ID NOS: 1 to 9 of the present invention includes a functional equivalent of the polypeptide constituting it, for example, a variant that can perform the same function as the polypeptide even though a part of the amino acid sequence of the polypeptide has been modified by deletion, substitution or insertion. Specifically, any one of the amino acid sequences represented by SEQ ID NOS: 1 to 9 may include an amino acid sequence having a sequence identity of 70% or more, more preferably 80% or more, still more preferably 90% or more, and most preferably 95% or more with any one of the amino acid sequences represented by SEQ ID NOS: 1 to 9. For example, polypeptides having sequence identity of 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%. The "% sequence identity" for a polypeptide is determined by comparing a comparison region with two optimally aligned sequences, wherein the portion of the sequence of the polypeptide in the comparison region may include additions or deletions (i.e., gaps) compared to the reference sequence for the optimal alignment of the two sequences (which does not include additions or deletions).

[0091] According to one embodiment of the present invention, when priming peripheral blood stem cells with the fusion protein, preferably, when priming peripheral blood stem cells with the fusion protein for 1 hour, it was confirmed that the expression of Tie2, a receptor for angiopoietin-1, increases and the vascular regeneration capacity of peripheral blood stem cells is improved without changing the gene expression characteristics of peripheral blood stem cells, i.e., without changing the type of cell (Example 3-5). Therefore, the fusion protein of the present invention is characterized by improving the vascular regeneration capacity of peripheral blood stem cells.

[0092] As used herein, the term "priming" refers to enhancing cell efficacy by enhancing cell reactivity (activity). Priming can alter cell characteristics to enhance specific cell capabilities. In the present invention, priming promotes the activity of peripheral blood stem cells, thereby enhancing their vascular regeneration capacity.

[0093] As used herein, the term "blood vessel formation" encompasses both "vasculogenesis" and "angiogenesis." Angiogenesis is characterized by the in situ differentiation of endothelial precursor cells into mature endothelial cells and the assembly of these cells to form new blood vessels, as occurs during the formation of the primary vascular bed in the early embryo. Angiogenesis is caused by the growth and branching of pre-existing blood vessels. Alternatively, angiogenesis may be derived from pre-existing blood vessels or may be generated from stem cells, hemangioblasts, or other precursor cells.

[0094]

[0095] Additionally, the present invention provides a polynucleotide encoding the fusion protein.

[0096] The term "polynucleotide" as used herein refers to a polymer compound in which nucleotides, which are chemical monomers composed of three elements: base, sugar, and phosphate, are linked in a chain form via a plurality of phosphate ester bonds, and is a polymer of deoxyribonucleotides or ribonucleotides that exists in a single-stranded or double-stranded form.

[0097] The polynucleotide may be an RNA genome sequence, DNA (gDNA or cDNA), an RNA sequence transcribed therefrom, etc., and may include not only a nucleotide sequence encoding the amino acid sequence of the fusion protein, but also a sequence complementary to the sequence.

[0098]

[0099] In addition, the present invention provides an expression vector comprising the polynucleotide.

[0100] An expression vector is a vector capable of expressing the fusion protein in a host cell, and refers to a genetic construct comprising a gene sequence and an expression control sequence that are operably linked to enable expression of the gene insert. The term "operably linked" refers to a functional linkage between a nucleic acid expression control sequence and a nucleic acid sequence encoding a desired protein so that the nucleic acid expression control sequence can perform a general function. The term "expression control sequence" refers to a DNA sequence that regulates the expression of an operably linked polynucleotide sequence in a specific host cell. Such expression vectors can be produced using genetic recombination techniques well known in the art, and enzymes commonly used in the art for site-specific DNA cleavage and ligation can be used.

[0101] The above expression vector includes, in addition to expression control elements such as a promoter, operator, initiation codon, termination codon, polyadenylation signal, and enhancer, a signal sequence or leader sequence for membrane targeting or secretion, and can be manufactured in various ways depending on the purpose. In addition, the expression vector includes a selection marker for selecting a host cell containing the expression vector, and in the case of a replicable expression vector, it can self-replicate by including a replication origin or can be integrated into host DNA. The expression vector can use any vector commonly used in the art for external gene expression without limitation, and examples thereof may include a plasmid vector, a cosmid vector, a viral vector, etc.

[0102]

[0103] In addition, the present invention provides a cell into which the polynucleotide or an expression vector comprising the same has been introduced.

[0104] As used herein, the term "introduction" refers to the introduction (transfer) of a polynucleotide (e.g., a polynucleotide according to the present invention or an expression vector comprising the same) into a cell or organism. The nucleic acid of the polynucleotide may be in the form of naked DNA or RNA, linked to various proteins, or integrated into a vector. The term "introduction" as used herein conveys the broadest possible meaning and includes, for example, introduction by transfection methods (methods in which a polynucleotide is introduced into a eukaryotic cell by physical and / or chemical treatment), transformation methods (methods in which a polynucleotide is introduced into a prokaryotic cell by physical and / or chemical treatment), viral methods / viral transduction methods (methods in which a polynucleotide is introduced into a eukaryotic and / or prokaryotic cell by a virus or viral vector), conjugation methods (methods in which a polynucleotide is introduced from one cell to another by direct cell-to-cell contact or by a cytoplasmic bridge between cells), and fusion methods (methods in which two cells are fused, including homotypic cell fusion and heterotypic cell fusion). Preferably, the introduction can be achieved via a nickase enzyme system (e.g., Cas9D10A).

[0105] The cell can be selected to regulate the expression of the inserted sequence or to process the gene product in a specific, desirable manner. Different cells have distinct and specific mechanisms for protein translation and post-translational processing and modification. A suitable cell line or host system can be selected that provides the desired modification and processing of the expressed heterologous protein. Expression in yeast can produce a biologically active product. Expression in eukaryotic cells can increase the folding potential of the peptide chain constituting the polypeptide or protein.

[0106] The above cells can be used without limitation as cells known in the art that can stably and continuously clone and express a vector, and examples thereof include Escherichia coli such as E. coli JM109, E. coli BL21DE, E. coli DH5, E. coli RR1, E. coli LE392, E. coli B, E. coli X 1776, E. coli W3110, Agrobacterium strains such as Agrobacterium A4, bacilli such as Bacillus subtilis, enteric bacteria such as Salmonella typhimurium, Serratia marcescens, and various Pseudomonas strains. In addition, when transfecting an expression vector into a eukaryotic cell, yeast (Saccharomyces cerevisiae), insect cells, human cells (e.g., CHO (Chinese hamster ovary), W138, BHK, COS-7, 293, HepG2, 3T3, RIN, MDCK cell lines) etc. can be used as host cells, and preferably, the cell can be a CHO-K1 cell.

[0107] Additionally, according to one embodiment of the present invention, the expression vector can be introduced into a hot-spot 13 region of a cell, and the hot-spot 13 region can be located in the euchromatin of the cell, but is not limited thereto.

[0108] In this specification, the term "hot-spot" region is defined as a transcriptionally active zone capable of expressing genes at a high level for some epigenetic reason. The hot-spot region is a region for high expression of recombinant genes in eukaryotic cells, and is a region of the genome originally existing within a eukaryotic cell, and the hot-spot 13 region refers to a region located at the 13th position among the hot-spot regions of euchromatin.

[0109] According to one embodiment of the present invention, a cell line capable of mass-producing the fusion protein was constructed using CHO-K1 cells and CDM4CHO medium, and named "CHO-K1DW".

[0110] The above CHO-K1DW may be adapted to suspension culture, which is advantageous for mass production, and a polynucleotide encoding the fusion protein or an expression vector containing the same may be introduced into a hot-spot 13 region located in the euchromatin of the CHO-K1DW via a nickase enzyme system (e.g., Cas9D10A), but is not limited thereto.

[0111]

[0112] In addition, the present invention provides a method for producing the fusion protein, comprising a step of culturing a cell comprising a polynucleotide encoding the fusion protein; or an expression vector comprising the same; wherein the fusion protein can be obtained from the cultured cell or a culture medium thereof.

[0113] In the present invention, the cell may be a CHO-K1 cell, preferably a CHO-K1 cell adapted to suspension culture, but is not limited thereto.

[0114] According to one embodiment of the present invention, the culture of cells containing a polynucleotide encoding the fusion protein or an expression vector containing the same can be suspended in suspension for mass production.

[0115] In this specification, the term "suspension culture" refers to culturing cells to be cultured in a floating state in a culture medium without being fixed to a substrate or the like. To make the cells in a suspended state, SR (serum replacement) may be added to the medium, or the cells may be cultured using a low-attachment culture dish or a bioreactor, but is not limited thereto and various suspension culture methods used in the art may be used.

[0116]

[0117] In addition, the present invention provides a composition for promoting the activity of peripheral blood stem cells, which comprises the fusion protein as an active ingredient.

[0118] As used herein, the term "peripheral blood stem cell activity" may refer to the angiogenic capacity of peripheral blood stem cells. Therefore, in the present invention, promoting peripheral blood stem cell activity means enhancing the vascular regenerative capacity of peripheral blood stem cells by priming peripheral blood stem cells by treating them with the fusion protein.

[0119] The above-mentioned composition for promoting activity can not only enhance the vascular regeneration effect of peripheral blood stem cells by mixing it with peripheral blood stem cells and injecting it into a living body, but can also be used as a method for transplanting peripheral blood stem cells with enhanced vascular regeneration ability into a living body after treating the peripheral blood stem cells themselves with the composition.

[0120] In the present invention, the peripheral blood stem cell (PBSC) is a peripheral blood stem cell with improved angiogenic ability disclosed in Korean Patent No. 10-1544674. mobPBSC) but is not limited thereto.

[0121]

[0122] In addition, the present invention provides a method for promoting the activity of peripheral blood stem cells, including a step of treating (priming) the separated peripheral blood stem cells with the fusion protein.

[0123] In the present invention, the step of treating the fusion protein may be performed for 0.25 to 5 hours, for 0.25 to 4 hours, for 0.25 to 3 hours, for 0.25 to 2 hours, for 0.25 to 1 hour, for 0.5 to 5 hours, for 0.5 to 3 hours, for 0.5 to 2 hours, for 0.5 to 1 hour, for 0.75 to 3 hours, for 0.75 to 2 hours, for 0.75 to 1 hour, for 1 to 2 hours, for 0.25 hours, for 0.5 hours, for 0.75 hours, for 1 hour, or for 2 hours, but may be performed without limitation within a range in which the vascular regeneration ability is improved without changing the gene expression characteristics of peripheral blood stem cells. According to one embodiment of the present invention, the step of treating the fusion protein may be performed for 1 hour.

[0124]

[0125] In addition, the present invention provides a pharmaceutical composition for preventing or treating ischemic disease, which comprises the fusion protein or peripheral blood stem cells treated therewith as an active ingredient.

[0126] In the present invention, the fusion protein can enhance the angiogenic capacity of peripheral blood stem cells, but is not limited thereto.

[0127] In the present invention, the treatment of the fusion protein may be performed for 0.25 to 5 hours, for 0.25 to 4 hours, for 0.25 to 3 hours, for 0.25 to 2 hours, for 0.25 to 1 hour, for 0.5 to 5 hours, for 0.5 to 3 hours, for 0.5 to 2 hours, for 0.5 to 1 hour, for 0.75 to 3 hours, for 0.75 to 2 hours, for 0.75 to 1 hour, for 1 to 2 hours, for 0.25 hours, for 0.5 hours, for 0.75 hours, for 1 hour, or for 2 hours, and according to one embodiment of the present invention, the treatment of the fusion protein may be performed for 1 hour, but is not limited thereto.

[0128] According to one embodiment of the present invention, the fusion protein can stimulate (prime) peripheral blood stem cells to enhance the vascular regeneration ability of peripheral blood stem cells, thereby promoting the formation and maturation of blood vessels to restore vascular tissue or increase the number of blood vessels to restore blood flow, and thus can be effective in treating ischemic diseases in which blood flow is reduced due to loss of vascular tissue, deficiency of angiogenesis, or formation of abnormal blood vessels.

[0129] The term "ischemic disease" as used herein refers to a disease in which blood flow is reduced due to blood leakage, embolism, or infarction caused by blood vessel damage, leading to necrosis of tissue whose blood supply is blocked. The ischemic disease may be selected from the group consisting of ischemic heart disease, ischemic myocardial infarction, ischemic heart failure, ischemic enteritis, ischemic vascular disease, ischemic eye disease, ischemic retinopathy, ischemic glaucoma, ischemic renal failure, ischemic cerebral disease, and ischemic lower extremity disease, but is not limited thereto.

[0130] The term "ischemic heart disease" in this specification refers to a disease caused by a decrease in blood flow to the heart muscle due to damage, narrowing, or occlusion of the coronary artery supplying blood to the heart, and may include angina, myocardial infarction, and heart failure.

[0131] In this specification, the term "ischemic cerebral disease" refers to a general term for diseases in which pathological abnormalities occur in blood vessels supplying blood flow to the brain due to thrombosis and embolism, etc., and may include ischemic stroke, vascular dementia, Alzheimer's dementia, Huntington's disease, Parkinson's disease, etc.

[0132] In the present invention, "pharmaceutical composition" means a composition manufactured for the purpose of preventing or treating a disease, and may be formulated and used in various forms according to conventional methods. For example, it may be formulated in oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, and syrups, and may be formulated and used in the form of topical preparations, suppositories, and sterile injectable solutions.

[0133] The pharmaceutical composition according to the present invention may further comprise suitable carriers, excipients, and diluents commonly used in the manufacture of pharmaceutical compositions. The excipients may be, for example, one or more selected from the group consisting of diluents, binders, disintegrants, lubricants, adsorbents, moisturizers, film-coating materials, and controlled-release additives.

[0134] The pharmaceutical composition according to the present invention may be formulated and used in the form of external preparations such as powders, granules, sustained-release granules, enteric-coated granules, liquids, eye drops, ellipsoids, emulsions, suspensions, alcohols, troches, aromatic waters, limonades, tablets, sustained-release tablets, enteric-coated tablets, sublingual tablets, hard capsules, soft capsules, sustained-release capsules, enteric capsules, pills, tinctures, soft extracts, dry extracts, fluid extracts, injections, capsules, irrigation solutions, ointments, lotions, pastes, sprays, inhalants, patches, sterile injection solutions, or aerosols, according to a conventional method, and the external preparations may have formulations such as creams, gels, patches, sprays, ointments, ointments, lotions, liniments, pastes, or cataplasmas.

[0135] Carriers, excipients and diluents that may be included in the pharmaceutical composition according to the present invention include lactose, dextrose, sucrose, oligosaccharides, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.

[0136] When formulating, it is usually prepared using diluents or excipients such as fillers, bulking agents, binders, wetting agents, disintegrants, and surfactants.

[0137] The additives of the tablets, powders, granules, capsules, pills, and troches according to the present invention include excipients such as corn starch, potato starch, wheat starch, lactose, sucrose, glucose, fructose, D-mannitol, precipitated calcium carbonate, synthetic aluminum silicate, calcium hydrogen phosphate, calcium sulfate, sodium chloride, sodium bicarbonate, purified lanolin, microcrystalline cellulose, dextrin, sodium alginate, methylcellulose, sodium carboxymethylcellulose, kaolin, urea, colloidal silica gel, hydroxypropyl starch, hydroxypropyl methylcellulose (HPMC) 1928, HPMC 2208, HPMC 2906, HPMC 2910, propylene glycol, casein, calcium lactate, and Primogel; Gelatin, gum arabic, ethanol, agar powder, cellulose acetate phthalate, carboxymethylcellulose, calcium carboxymethylcellulose, glucose, purified water, sodium caseinate, glycerin, stearic acid, sodium carboxymethylcellulose, sodium methylcellulose, methylcellulose, microcrystalline cellulose, dextrin, hydroxycellulose, hydroxypropyl starch, hydroxymethylcellulose, refined shellac, starch starch, hydroxypropyl cellulose, hydroxypropyl methylcellulose, polyvinyl alcohol, polyvinyl pyrrolidone, and binders such as hydroxypropyl methylcellulose, corn starch, agar powder, methylcellulose, bentonite, hydroxypropyl starch, sodium carboxymethylcellulose, sodium alginate, Disintegrants such as carboxymethylcellulose calcium, calcium citrate, sodium lauryl sulfate, anhydrous silicic acid, 1-hydroxypropyl cellulose, dextran, ion exchange resin, polyvinyl acetate, formaldehyde-treated casein and gelatin, alginic acid, amylose, guar gum, baking soda, polyvinylpyrrolidone, calcium phosphate, gelled starch, gum arabic, amylopectin, pectin, sodium polyphosphate, ethylcellulose, sucrose, magnesium aluminum silicate, di-sorbitol solution, and light anhydrous silicic acid;Lubricants such as calcium stearate, magnesium stearate, stearic acid, hydrogenated vegetable oil, talc, lycopodium dentata, kaolin, petrolatum, sodium stearate, cacao butter, sodium salicylate, magnesium salicylate, polyethylene glycol (PEG) 4000, PEG 6000, liquid paraffin, hydrogenated soybean oil (Lubri wax), aluminum stearate, zinc stearate, sodium lauryl sulfate, magnesium oxide, macrogol, synthetic aluminum silicate, anhydrous silicic acid, higher fatty acids, higher alcohols, silicone oil, paraffin oil, polyethylene glycol fatty acid ether, starch, sodium chloride, sodium acetate, sodium oleate, dl-leucine, and light anhydrous silicic acid can be used.

[0138] As additives of the liquid formulation according to the present invention, water, dilute hydrochloric acid, dilute sulfuric acid, sodium citrate, monostearate sucrose, polyoxyethylene sorbitol fatty acid esters (twin esters), polyoxyethylene monoalkyl ethers, lanolin ethers, lanolin esters, acetic acid, hydrochloric acid, ammonia water, ammonium carbonate, potassium hydroxide, sodium hydroxide, prolamine, polyvinylpyrrolidone, ethylcellulose, sodium carboxymethylcellulose, etc. can be used.

[0139] The syrup according to the present invention may include a solution of white sugar, other sugars, or sweeteners, and may also include a fragrance, a coloring agent, a preservative, a stabilizer, a suspending agent, an emulsifier, a viscosity modifier, and the like, as needed.

[0140] Purified water may be used in the emulsion according to the present invention, and emulsifiers, preservatives, stabilizers, fragrances, etc. may be used as needed.

[0141] The suspension according to the present invention may include suspending agents such as acacia, tragacanth, methylcellulose, carboxymethylcellulose, sodium carboxymethylcellulose, microcrystalline cellulose, sodium alginate, hydroxypropylmethylcellulose (HPMC), HPMC 1828, HPMC 2906, and HPMC 2910, and surfactants, preservatives, stabilizers, colorants, and fragrances may be used as needed.

[0142] The injection according to the present invention includes a solvent such as distilled water for injection, 0.9% sodium chloride injection, Ringer's injection, dextrose injection, dextrose + sodium chloride injection, PEG, lactated Ringer's injection, ethanol, propylene glycol, non-volatile oils - sesame oil, cottonseed oil, peanut oil, soybean oil, corn oil, ethyl oleate, isopropyl myristate, and benzene benzoate; a solubilizing agent such as sodium benzoate, sodium salicylate, sodium acetate, urea, urethane, monoethylacetamide, butazolidine, propylene glycol, tween, nitrile acid amide, hexamine, and dimethylacetamide; a buffer such as weak acids and their salts (acetic acid and sodium acetate), weak bases and their salts (ammonia and ammonium acetate), organic compounds, proteins, albumin, peptone, and gums; It may include isotonic agents such as sodium chloride; stabilizers such as sodium bisulfite (NaHSO3), carbon dioxide gas, sodium metabisulfite (Na2S2O5), sodium sulfite (Na2SO3), nitrogen gas (N2), and ethylenediaminetetraacetic acid; oxidizing agents such as sodium bisulfite 0.1%, sodium formaldehyde sulfoxylate, thiourea, disodium ethylenediaminetetraacetic acid, and acetone sodium bisulfite; analgesics such as benzyl alcohol, chlorobutanol, procaine hydrochloride, glucose, and calcium gluconate; and suspending agents such as sodium cis-methylenediamine, sodium alginate, Tween 80, and aluminum monostearate.

[0143] The suppository according to the present invention comprises cocoa butter, lanolin, withepsol, polyethylene glycol, glycerogelatin, methylcellulose, carboxymethylcellulose, a mixture of stearic acid and oleic acid, Subanal, cottonseed oil, peanut oil, palm oil, cocoa butter + cholesterol, lecithin, ranet wax, glycerol monostearate, Tween or Span, Imhausen, monolene (propylene glycol monostearate), glycerin, Adeps solidus, Buytyrum Tego-G, Cebes Pharma 16, hexalide base 95, Cotomar, Hydroxocote SP, S-70-XXA, S-70-XX75 (S-70-XX95), Mechanisms such as Hydrokote 25, Hydrokote 711, Idropostal, Massa estrarium (A, AS, B, C, D, E, I, T), Massa-MF, Masupol, Masupol-15, Neosupostal-N, Paramound-B, Suposiro (OSI, OSIX, A, B, C, D, H, L), Suppository type IV (AB, B, A, BC, BBG, E, BGF, C, D, 299), Supostal (N, Es), Wecovi (W, R, S, M, Fs), Tezester triglyceride basis (TG-95, MA, 57) can be used.

[0144] Solid dosage forms for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid dosage forms are prepared by mixing the extract 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.

[0145] Liquid preparations for oral administration include suspensions, solutions, emulsions, and syrups. In addition to commonly used simple diluents such as water and liquid paraffin, they may contain various excipients such as wetting agents, sweeteners, flavoring agents, and preservatives. Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, and suppositories. Non-aqueous solvents and suspensions can include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate.

[0146] The pharmaceutical composition according to the present invention is administered in a pharmaceutically effective amount. In the present invention, "pharmaceutically effective amount" means an amount sufficient to treat a disease with a reasonable benefit / risk ratio applicable to medical treatment. The effective dosage level can be determined based on factors including the type and severity of the patient's disease, drug activity, drug sensitivity, administration time, administration route and excretion rate, treatment period, concurrently used drugs, and other factors well known in the medical field.

[0147] The pharmaceutical composition according to the present invention can be administered as an individual therapeutic agent or in combination with other therapeutic agents. It can be administered sequentially or simultaneously with conventional therapeutic agents, or in single or multiple doses. Taking all of the above factors into account, it is important to administer an amount that achieves maximum efficacy with minimal side effects. This amount can be readily determined by those skilled in the art to which the present invention pertains.

[0148] The pharmaceutical composition of the present invention can be administered to a subject via various routes. All modes of administration are conceivable, including oral ingestion, subcutaneous injection, intraperitoneal administration, intravenous injection, intramuscular injection, intrathecal injection, sublingual administration, buccal mucosal administration, rectal insertion, vaginal insertion, ocular administration, otic administration, nasal administration, inhalation, oral or nasal spraying, dermal administration, and transdermal administration.

[0149] The pharmaceutical composition of the present invention is determined according to the type of drug as an active ingredient along with various related factors such as the disease to be treated, route of administration, age, sex, weight of the patient, and severity of the disease.

[0150] In addition, the present invention provides a method for preventing or treating ischemic disease, comprising administering the fusion protein; peripheral blood stem cells treated with the fusion protein; or the pharmaceutical composition; to a subject in need thereof.

[0151] In addition, the present invention provides a use of the fusion protein; peripheral blood stem cells treated therewith; or the pharmaceutical composition; for preventing or treating ischemic disease.

[0152] In addition, the present invention provides a use of the fusion protein; peripheral blood stem cells treated therewith; or the pharmaceutical composition; for the manufacture of an agent for preventing or treating ischemic disease.

[0153] In the present invention, the term "subject" means a subject requiring treatment for a disease, and more specifically, a mammal such as a human or non-human primate, mouse, rat, dog, cat, horse, and cow.

[0154] In the present invention, “administration” means providing a predetermined composition of the present invention to an individual by any appropriate method.

[0155] In the present invention, “prevention” means any action that suppresses or delays the onset of a target disease, “treatment” means any action that improves or beneficially changes a target disease and its metabolic abnormality symptoms by administering a pharmaceutical composition according to the present invention, and “improvement” means any action that reduces a parameter related to a target disease, for example, the severity of a symptom, by administering a composition according to the present invention.

[0156]

[0157] Hereinafter, preferred examples are presented to aid in understanding the present invention. However, the following examples are provided solely to facilitate a better understanding of the present invention, and the scope of the present invention is not limited by the following examples.

[0158]

[0159] [Experimental Method]

[0160] 1. Collection of MobPBSC (Peripheral blood stem cells)

[0161] Peripheral blood stem cells (PBSCs) were mobilized by administering erythropoietin (5 ug / kg) subcutaneously for 1 day and cytokine (Granulocyte colony stimulating factor; G-CSF) for 3 days. Afterwards, on the 4th day, blood apheresis was performed. Mob PBSCs were collected. The subject's plasma and Dulbecco's Phosphate-Buffered Saline (DPBS) were mixed in a 1:1 ratio to prepare a washing solution, and the prepared washing solution was collected using an apheresis device. Mob Add the same amount of PBSCs and centrifuge at 3000 RPM for 25 minutes. Mob PBSCs were isolated.

[0162]

[0163] 2. Collection of PBMCs

[0164] From a healthy control group, 20 mL of peripheral blood was collected, mixed with 10 mL of DPBS and 12 mL of Ficoll Paqur Plus, and centrifuged at 2,500 rpm for 30 minutes at room temperature. The buffy coat layer beneath the separated plasma was collected with a 1 mL pipette and transferred to a new 50 mL sterile tube. After diluting with PBS, the mixture was centrifuged, and the supernatant was removed. This process was repeated to obtain peripheral blood mononuclear cells (PBMCs).

[0165]

[0166] 3. FVA3-Ang1 priming and washing protocol in MobPBSC

[0167] FVA3-Ang1 was used at a concentration of 400 ng / mL and primed for 1 or 24 hours in a 37°C incubator. After priming, the cells were washed twice using a washing solution made by mixing DPBS and patient plasma in a 1:1 ratio to remove FVA3-Ang1 residues. The cells were separated and washed in this manner. Mob Plasma pre-stored in PBSCs was used as a resuspension solution for animal experiments.

[0168]

[0169] 4. Total RNA sequencing

[0170] Libraries were prepared from total RNA using the NEBNext Ultra II Directional RNA-Seq Kit. rRNA was removed using the RIBO COP rRNA depletion kit, and cDNA synthesis and shearing were performed using the remaining RNA according to the manufacturer's instructions. The average fragment size was assessed using an Agilent 2100 bioanalyzer and quantified using a library quantification kit. Paired-end 100 sequencing was then performed using a NovaSeq 6000. Quality control of raw sequencing data was performed using FastQC, and adapter and low-quality reads ( <Q20)를 제거하였다. TopHat을 사용하여 정제된 리드를 참고 유전체에 매핑하였다. Cufflinks를 사용하여 FPKM 값으로 유전자, 이소폼 및 IncRNA의 유전자 발현 수준을 추정하였다. EdgeR 내에서 TMM+CPM 방법을 사용하여 FRKM 값을 정규화하였다.

[0171]

[0172] 5. Flow cytometry

[0173] The cells primed with FVA3-Ang1 were washed by centrifuging at 1000 g for 3 minutes at room temperature in a 50 mL sterile tube filled with PBS. The supernatant was removed, the cells were resuspended in 1 mL PBS, centrifuged under the same conditions, and the cells were resuspended in FACS buffer. Then, FITC-conjugated anti-CD31, PE-conjugated anti-VE-cadherin, APC-conjugated anti-CXCR4, APC-conjugated anti-CD45, FITC-conjugated anti-Annexin V antibodies, and Propidium Iodide (PI) were used. Mob Cell characteristics of PBSCs and PBMCs were confirmed.

[0174]

[0175] 6. Real-time mRNA expression quantification

[0176] The RNA solution was heated at 65°C for 5 minutes and then cooled sufficiently on ice. 5x RT Master Mix was added to adjust the final volume to 20 μL. cDNA synthesis was completed by incubating the reaction at 37°C for 15 minutes, 50°C for 5 minutes, and 98°C for 5 minutes. mRNA expression was quantified using the 7500 qRT-PCR system according to an established protocol. Real-time reactions were performed in triplicate in 96-well plates in a final volume of 20 μL and normalized to GAPDH RNA. Quantitative real-time PCR for CD31, CXCR4, VE-cadherin (VE CAD), and Tie2 was performed using the TOYOBO protocol.

[0177]

[0178] 7. Protein immunoblot

[0179] The FVA-Ang1-treated and control groups were lysed in RIPA buffer containing protease / phosphatase inhibitors and centrifuged at 15,000 rpm for 30 min at 4°C. The separated proteins were subjected to 4–15% SDS-PAGE and transferred to PVDF membranes. The membranes were blocked with 5% skim milk for 1 h at room temperature and incubated with primary antibodies at 4°C. Subsequently, the membranes were reacted with secondary antibodies, and immunoblot signals were detected with Amersham 680 and quantified using ImageJ.

[0180]

[0181] 8. Immunohistochemical staining

[0182] Paraffin-fixed sections from each group were stained with CD31 antibody to measure capillary density and laminin antibody to stain muscle structure. Transplanted cells were stained with anti-HLA-ABC as the primary antibody, and secondary antibodies were used with Alexa-488 and Alexa-555 for 1 hour at room temperature. The sections were then visualized using a Leica confocal microscope.

[0183]

[0184] 9. Animal disease models

[0185] 9-1. Hindlimb vascular occlusion model

[0186] Balb / c nude mice (male, 8 weeks old) were used. They were anesthetized by intraperitoneal injection of 100 μL of alpha-Zalone and 5 μL of Rompun, and maintained under respiratory anesthesia with 1.5-2% isoflurane. A hindlimb vascular occlusion model was created by ligating the femoral artery, and PBS, naive Mob PBSCs, FVA3-Ang1 primed Mob The experiment was conducted by dividing PBSCs into three groups: primed or unprimed. Mob PBSCs (cell count 1Х10 5 ) was injected into the muscles surrounding the ligated blood vessels in a volume of 30 μL. Laser Doppler flow imaging (LDPI) was performed four times in total on days 0, 3, 7, and 14 from the date of cell injection.

[0187]

[0188] 9-2. Myocardial infarction model

[0189] Balb / c nude mice (male, 8 weeks old) were used. They were anesthetized by intraperitoneal injection of 100 μL of alpha-Zalone and 5 μL of Rompun, and maintained under respiratory anesthesia with 1.5–2% isoflurane. A 1-cm incision was made in the left thorax, and the thoracic cavity was opened under a microscope. The left descending coronary artery was ligated with surgical thread to block retrograde blood flow. PBS, naive MobPBSCs, FVA3-Ang1 primed Mob The experiment was conducted by dividing the PBSCs into three groups. After injecting the experimental solution into the corresponding myocardial area of ​​the left descending coronary artery, the skin was sutured with 6-0 suture, and intraperitoneal gas was removed using a 5 mL syringe connected to the catheter to create negative pressure before completing the suture.

[0190]

[0191] 10. Apoptosis analysis

[0192] Cells treated with FVA3-Ang1 for 1 hour were washed twice and resuspended in saline. The resuspended cells were stained with 0.4% trypan blue for 3 minutes, and viable cells were counted using a cell counter. The percentage of viable cells was then calculated.

[0193]

[0194] [Example]

[0195] Example 1. Development of a novel form of Ang1 (angiopoietin-1) as a priming agent: FVA3-Ang1

[0196] We developed a novel form of Ang1 that can function as a stable ex vivo priming agent. Azurocidin or influenza A virus was used as a signal peptide together with FLAG to enhance the expression of proteins attached to the N-terminal region. Because purification of COMP-Ang1 is difficult at clinical-grade levels, domains such as Phe14 and human vasodilator-stimulated phosphoprotein (VASP) were evaluated as alternatives to COMP. In particular, VASP, which has a domain primarily expressed as a tetramer, was hypothesized to contribute more to structural stability than COMP.

[0197] As shown in Table 1 and Fig. 1, various Ang1 overexpression vectors were constructed using COMP, Phe14, and VASP2 (a VASP domain with a disulfide bonding domain that stabilizes the structure). As a result of transient expression analysis with various combinations, it was confirmed that Phe14 and COMP had low oligomerization efficiency, whereas VASP2 did not form monomers or dimers but formed tetramers of Ang1, as shown in Fig. 2. In addition, Hot-spot site 13 with high GFP expression was used for final verification. As a result, the combination of Hot-spot site 13, influenza A virus, and VASP2 bound to Ang1 was confirmed to be the optimal combination, and was named FVA3-Ang1.

[0198] Finally, to promote high protein expression, genome integration was performed at hot-spot sites using the Cas9D10A system (Sigma-Aldrich), as shown in Fig. 3.

[0199] Vector informationFCA(Vector)Influenza A virus + FLAG + COMP + Ang 1FCA (HT-int)Hot spot 13 + Influenza A virus + FLAG + COMP + Ang1FPA (HT-int)Hot spot 13 + Influenza A virus + FLAG + Phe14 + Ang1FPA2 (HT-int)Hot spot 13 + Influenza A virus + FLAG + Phe14 with disulfide bridge + Ang1FVA2 (HT-int)Hot spot 13 + Azurocidin + FLAG + VASP2 + Ang1FVA3 (HT-int)Hot spot 13 + Influenza A virus + FLAG + VASP2 + Ang1

[0200]

[0201] Example 2. Mass production and evaluation of FVA3-Ang1 overexpressing cell lines.

[0202] As shown in Fig. 4, a cell line capable of mass-producing FVA3-Ang1 was established using CHO-K1 cells, which are suitable for high-yield production of proteins and have proven stability because they do not contain pathogens such as viruses and prions, and CDM4CHO medium (Cytiva™). Specifically, CHO-K1 cells were adapted to suspension culture for mass production using a bioreactor before use. CHO-K1 cells adapted to suspension culture were cultured to form clonal colonies, and colonies were then selected using ClonePix FL. After confirming the growth rate and cell characteristics, the final cells were selected and named CHO-K1DW. The FVA3-Ang1 overexpressing cell line was developed by introducing the FVA3-Ang1 gene into a hot spot in CHO-K1DW cells. The FVA3-Ang1 gene was synthesized after codon optimization to ensure that there were no problems with expression in CHO-K1DW cells. As a result, a cell line that can be used for stable mass production of FVA3-Ang1 was successfully established.

[0203] The novel FVA3-Ang1 protein at 200 ng / mL and 400 ng / mL was treated to HUVEC cells to determine its effects on cell characteristics. As shown in Figure 5, after dose-dependent priming in HUVECs for 1 hour, phosphorylation of AKT (p-AKT) showed a concentration-dependent relationship, and was found to be superior in FVA3-Ang1 compared to COMP-Ang1. Furthermore, as shown in Figure 6, the time-dependent effect of priming was confirmed, and when treated for 24 hours, endothelial cell lineage characteristics in HUVEC cells were enhanced, which was confirmed by up-regulated CD31 and VECAD.

[0204]

[0205] Example 3. Gene expression in MobPBSCs regulated by FVA3-Ang1 priming.

[0206] We evaluated the FVA3-Ang1 priming strategy focusing on safety aspects for altering cell characteristics and efficacy aspects for increasing angiogenic potential. Mob PBSCs were collected from patients with acute myocardial infarction primed with FVA3-Ang1.

[0207] Gene expression pattern is naive Mob PBSC group, primed for 1 hour Mob PBSC group, 24-hour primed Mob The total RNA sequencing was performed in the PBSC group. As a result, as shown in Figures 7a and 7b, the gene expression pattern among the 26,137 reliable probes was 1 hour primed. Mob PBSC and naive Mob It was similar in the PBSC group. On the other hand, naive Mob PBSC group and 1 hour primed Mob 24-hour primed compared to PBSC group MobIn the PBSC group, gene expression patterns were found to have changed, with 59 genes being upregulated and 243 genes being downregulated.

[0208] From the above results, it was confirmed that the characteristics of cells primed with FVA3-Ang1 for a short period of 1 hour did not change compared to cells before priming, but the characteristics of cells primed for a long period of 24 hours changed.

[0209]

[0210] Example 4. RNA and protein expression regulated by FVA3-Ang1 priming in MobPBSC.

[0211] RNA-seq analysis was performed to evaluate the effect of FVA3-Ang1 priming on RNA expression. As a result, as shown in Figure 8, the negative control PBMC and naive Mob Compared to the PBSC group, 1-hour primed Mob In the PBSC group, RNA expression of CD31, CXCR4, and VE-CAD did not increase, but after 24 hours of priming Mob In the PBSC group, RNA expression of CXCR4 and VE-CAD was significantly increased. However, the level of Tie2, a receptor for Ang1, was increased by 1-hour FVA3-Ang1 priming.

[0212] As shown in Figures 9a and 9b, the flow cytometry results showed that after 1 hour of priming, Mob In the PBSC group, the protein expression of CD31, CXCR4, VE-CAD, and CD45 did not show a significant increase or decrease. In contrast, in the 24-hour primed group Mob In the PBSC group, CD31, CXCR4, and VE-CAD expression significantly increased along with a decrease in CD45, indicating a change in cell characteristics from hematopoietic stem cells to vascular endothelial cell lineage. Annexin V protein expression showed no difference regardless of the priming period.

[0213]

[0214] Example 5. Confirmation of the regenerative efficacy of MobPBSCs in hindlimb vascular occlusion models and myocardial infarction models.

[0215] Primed with FVA3-Ang1 using hindlimb vascular occlusion model and myocardial infarction model Mob The vascular regeneration effect of PBSC was confirmed. When priming for 24 hours, the cell characteristics were changed, so when priming for 1 hour, the cell characteristics were not changed. Mob Experiments were conducted using PBSC.

[0216] As shown in Figure 10, after ligation of the right femoral artery in the hindlimb vascular occlusion mouse model, 1Х10 5 The dog's naive Mob PBSC or 1Х10 5 1 hour primed dog Mob PBSCs were injected into the peripheral muscles. PBS was used as a negative control. LDPI was performed on days 0, 3, 7, and 14 after surgery. As a result, as shown in Figures 11 and 12, FVA3-Ang1-primed Mob The PBSC group showed the best recovery in long-term regeneration, which was related to blood flow recovery.

[0217] Next, the efficacy of cell therapy was evaluated in a myocardial infarction model, as shown in Fig. 13. After ligation of the left anterior descending artery of the mouse, 1Х10 5 The dog's naive Mob PBSC or 1Х10 5 1 hour primed dog Mob PBSCs were injected into the infarcted anterior myocardium, and echocardiographic and histological evaluations were performed on the 14th day. PBS was used as a negative control. As shown in Figure 14, PBS and naive Mob Compared with the PBSC group, primed with FVA3-Ang1 MobIn the PBSC group, we were able to confirm that the overall survival rate was significantly improved. As shown in Figure 15, the echocardiographic results showed that the left ventricular end-systolic diameter (LVESD), left ventricular end-diastolic diameter (LVEDD), left ventricular fractional shortening (LVFS), and left ventricular ejection fraction (LVEF) were significantly improved, indicating that the left ventricular function and size were significantly improved after priming with FVA3-Ang1. Mob The PBSC group showed excellent functional improvement. As shown in Fig. 16, the histological evaluation results showed that the fibrotic area was priming with FVA3-Ang1. Mob It was confirmed that there was a decrease in the PBSC group.

[0218] From the above results, it was confirmed that FVA3-Ang1 enhances angiogenic capacity without changing the gene expression characteristics of peripheral blood stem cells, i.e., the characteristics of the cells.

[0219]

[0220] 서열번호이름서열1Angiopoietin-1 domainLVNLCTKEGVLLKGGKREEEKPFRDCADVYQAGFNKSGIYTIYINNMPEPKKVFCNMDVNGGGWTVIQHREDGSLDFQRGWKEYKMGFGNPSGEYWLGNEFIFAITSQRQYMLRIELMDWEGNRAYSQYDRFHIGNEKQNYRLYLKGHTGTAGKQSSLILHGADFSTKDADNDNCMCKCALMLTGGWWFDACGPSNLNGMFYTAGQNHGKLNGIKWHYFKGPSYSLRSTTMMIRPLDF2Angiopoietin-1MTVFLSFAFLAAILTHIGCSNQRRSPENSGRRYNRIQHGQCAYTFILPEHDGNCRESTTDQYNTNALQRDAPHVEPDFSSQKLQHLEHVMENYTQWLQKLENYIVENMKSEMAQIQQNAVQNHTATMLEIGTSLLSQTAEQTRKLTDVETQVLNQTSRLEIQLLENSLSTYKLEKQLLQQTNEILKIHEKNSLLEHKILEMEGHKEELDTLKEEKENLQGLVTRQTYIIQELEKQLNRATTNNSVLQKQQLELMDTVHNLVNLCTKEGVLLKGGKREEEKPFRDCADVYQAGFNKSGIYTIYINNMPEPKKVFCNMDVNGGGWTVIQHREDGSLDFQRGWKEYKMGFGNPSGEYWLGNEFIFAITSQRQYMLRIELMDWEGNRAYSQYDRFHIGNEKQNYRLYLKGHTGTAGKQSSLILHGADFSTKDADNDNCMCKCALMLTGGWWFDACGPSNLNGMFYTAGQNHGKLNGIKWHYFKGPSYSLRSTTMMIRPLDF3VASPdomainDYSDLQRVKQELLEEVKKELQKVKEEIIEAFVQELRKR4VASPMSETVICSSRATVMLYDDGNKRWLPAGTGPQAFSRVQIYHNPTANSFRVVGRKMQPDQQVVINCAIVRGVKYNQATPNFHQWRDARQVWGLNFGSKEDAAQFAAGMASALEALEGGGPPPPPALPTWSVPNGPSPEEVEQQKRQQPGPSEHIERRVSNAGGPPAPPAGGPPPPPGPPPPPGPPPPPGLPPSGVPAAAHGAGGGPPPAPPLPAAQGPGGGGAGAPGLAAAIAGAKLRKVSKQEEASGGPTAPKAESGRSGGGGLMEEMNAMLARRRKATQVGEKTPKDESANQEEPEARVPAQSESVRRPWEKNSTTLPRMKSSSSVTTSETQPCTPSSSDYSDLQRVKQELLEEVKKELQKVKEEIIEAFVQELRKRGSP5VASP2 domainDYSDLQRVKQELLEEVKKELQKVKEEIIEAFVQELRKRECDACG6disulfide bonding domainECDACG7signal peptide of Influenza A virusMKTIIALSYIFCLVFA8FLAG domainDYKDDDDK9FVA3-Ang1MKTIIALSYIFCLVFADYKDDDDKGILDYSDLQRVKQELLEEVKKELQKVKEEIIEAFVQELRKRECDACGGSLVNLCTKEGVLLKGGKREEEKPFRDCADVYQAGFNKSGIYTIYINNMPEPKKVFCNMDVNGGGWTVIQHREDGSLDFQRGWKEYKMGFGNPSGEYWLGNEFIFAITSQRQYMLRIELMDWEGNRAYSQYDRFHIGNEKQNYRLYLKGHTGTAGKQSSLILHGADFSTKDADNDNCMCKCALMLTGGWWFDACGPSNLNGMFYTAGQNHGKLNGIKWHYFKGPSYSLRSTTMMIRPLDF

[0221]

[0222] The foregoing description of the present invention is provided for illustrative purposes only. Those skilled in the art will readily appreciate that the present invention can be readily modified into other specific forms without altering the technical spirit or essential characteristics of the present invention. Therefore, the embodiments described above should be understood as illustrative in all respects and not restrictive.

[0223]

[0224] The novel angiopoietin-1 fusion protein according to the present invention is structurally stable and can promote the formation and maturation of blood vessels by enhancing the vascular regeneration capacity of peripheral blood stem cells, and thus can be usefully utilized in the development of a treatment for ischemic diseases, and thus has industrial applicability.

Claims

1. A fusion protein comprising vasodilator-stimulated phosphoprotein (VASP) or a fragment thereof; and angiopoietin-1 protein or a fragment thereof.

2. In paragraph 1, A fusion protein, wherein the vasodilator-stimulated phosphoprotein or fragment thereof further comprises a disulfide bonding domain.

3. In paragraph 1, A fusion protein, characterized in that the vasodilator-stimulated phosphoprotein or a fragment thereof comprises any one amino acid sequence selected from the group consisting of SEQ ID NOs: 3 to 5.

4. In paragraph 1, A fusion protein, characterized in that the angiopoietin-1 protein or a fragment thereof comprises the amino acid sequence of SEQ ID NO: 1 or 2.

5. In paragraph 1, A fusion protein characterized in that the vasodilator-stimulated phosphoprotein or a fragment thereof; and the angiopoietin-1 protein or a fragment thereof; are sequentially linked from the N-terminus to the C-terminus.

6. In paragraph 1, A fusion protein characterized in that the vasodilator-stimulated phosphoprotein or a fragment thereof; and angiopoietin-1 protein or a fragment thereof; are connected by a linker.

7. In paragraph 1, A fusion protein, characterized in that the fusion protein further comprises a signal peptide.

8. In paragraph 1, A fusion protein characterized in that the fusion protein comprises the amino acid sequence of sequence number 9.

9. In paragraph 1, The above fusion protein is a fusion protein characterized in that it enhances the angiogenic capacity of peripheral blood stem cells.

10. A polynucleotide encoding a fusion protein of any one of claims 1 to 9.

11. An expression vector comprising the polynucleotide of clause 10.

12. A cell into which the polynucleotide of Article 10 or an expression vector containing the same has been introduced.

13. In paragraph 12, A cell characterized in that the above cell is a CHO-K1 cell.

14. In paragraph 12, A cell characterized in that the polynucleotide or the vector expressing the same is introduced into a hot-spot 13 region of the cell.

15. A method for producing a fusion protein according to any one of claims 1 to 9, comprising the step of culturing a cell comprising a polynucleotide encoding the fusion protein according to any one of claims 1 to 9; or an expression vector comprising the same.

16. In paragraph 15, A method for producing a fusion protein, characterized in that the above cells are CHO-K1 cells.

17. In paragraph 15, A method for producing a fusion protein, characterized in that the above culture is a suspension culture.

18. A composition for promoting the activity of peripheral blood stem cells, comprising the fusion protein of any one of claims 1 to 9 as an active ingredient.

19. A method for promoting the activity of peripheral blood stem cells, comprising the step of treating isolated peripheral blood stem cells with a fusion protein of any one of claims 1 to 9.

20. In paragraph 19, A method for promoting the activity of peripheral blood stem cells, characterized in that the step of processing the above fusion protein is performed for 0.25 to 5 hours.

21. A pharmaceutical composition for preventing or treating ischemic disease, comprising a fusion protein of any one of claims 1 to 9 or peripheral blood stem cells treated therewith as an active ingredient.

22. In paragraph 21, A pharmaceutical composition characterized in that the above fusion protein enhances the angiogenic capacity of peripheral blood stem cells.

23. In paragraph 21, A pharmaceutical composition, characterized in that the treatment of the fusion protein is performed for 0.25 to 5 hours.

24. In paragraph 21, A pharmaceutical composition characterized in that the above ischemic disease is selected from the group consisting of ischemic heart disease, ischemic myocardial infarction, ischemic heart failure, ischemic enteritis, ischemic vascular disease, ischemic eye disease, ischemic retinopathy, ischemic glaucoma, ischemic renal failure, ischemic cerebral disease, and ischemic lower extremity disease.

25. Use of a fusion protein according to any one of claims 1 to 9; or a composition comprising the same as an active ingredient for promoting peripheral blood stem cell activity.

26. Use of a fusion protein according to any one of claims 1 to 9; or a composition comprising the same as an active ingredient for producing a peripheral blood stem cell activity promoter.

27. A method for preventing or treating ischemic disease, comprising administering to a subject in need thereof a pharmaceutically effective dose of a fusion protein according to any one of claims 1 to 9; peripheral blood stem cells treated with the fusion protein; or a composition comprising the fusion protein or the peripheral blood stem cells as an active ingredient.

28. Use of a fusion protein according to any one of claims 1 to 9; peripheral blood stem cells treated with the fusion protein; or a composition comprising the fusion protein or the peripheral blood stem cells as an active ingredient; for preventing or treating ischemic disease.

29. Use of a fusion protein according to any one of claims 1 to 9; peripheral blood stem cells treated with the fusion protein; or a composition comprising the fusion protein or the peripheral blood stem cells as an active ingredient; for the manufacture of an agent for preventing or treating ischemic disease.

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