Recombinant protein for treating thrombocytopenia, and use thereof

Abstract

The present invention relates to a recombinant protein for treating thrombocytopenia, and a use thereof, and, specifically, to developing a biobetter (NT-202) of romiplostim by using an NT-CoreX system. The present invention relates to: producing of an NT-202 recombinant protein, which is a TPO-RA containing EBPn (NT-202An or NT-202Gn, wherein n is the number of repeats of an EBP basic unit, A is VPAXG, and G is VPGXG) having different sequences and repeat frequency, by using the NT-Corex (EBPn-Fc) system; and a use as a therapeutic agent for thrombocytopenia. The NT-202 recombinant protein of the present invention exhibits, in vitro, activity and signaling mechanism as a TPO-RA, and exhibits platelet differentiation-promoting activity in normal mice, an anticancer drug-induced thrombocytopenia mouse model and an immune thrombocytopenia mouse model, and thus can be clinically applied as a biobetter of romiplostim.

Description

Recombinant protein for the treatment of thrombocytopenia and its use

[0001] The present invention relates to a recombinant protein for treating thrombocytopenia and its use, and relates to a recombinant protein therapeutic agent (NT-202) for treating thrombocytopenia and its use, using the EBPn-Fc recombinant protein animal cell expression platform (NT-CoreX) system previously developed by the present inventors.

[0002] Elastin is a major component of extracellular matrix (ECM) proteins and is composed of an elastomeric domain and a crosslinking domain. The elastomeric domain is composed of hydrophobic amino acids and repetitive peptides such as VPGG, VPGVG, and APGVGV, and based on this, elastin-based polypeptides (EBPs) with stimuli-responsive, biocompatible, biodegradable, and non-immunogenic properties have been designed. Elastin-based polypeptides (EBPs) with pentapeptide repeating units are thermoresponsive biopolymers, and many studies have been conducted because Val-Pro-(Gly or Ala)-Xaa-Gly (wherein Xaa is any amino acid except Pro) can regulate responsiveness to environmental changes.

[0003] EBPs have the advantage of being able to be purified by centrifugation without chromatography based on their property of reversible aggregation by heat stimulation, and their low immunogenicity makes them useful as biomaterials. Since size control and nanocomplex formation are possible through genetic recombination, there is also a possibility of increasing the in vivo half-life of protein therapeutics and using them as drug delivery platforms. However, there have been limitations such as the formation of intracellular inclusion bodies when expressed in E. coli or insufficient purification efficiency when expressed in animal cells. To overcome these limitations, the present inventors developed the NT-CoreX system, an animal cell expression platform technology for EBPn-Fc recombinant proteins, by linking an antibody constant region (Fc) to EBPn (a polypeptide in which EBP, a repeating unit of Val-Pro-(Ala or Gly)-Xaa-Gly pentapeptide, is repeated n times) in a previous invention (Korean Patent Application No. 10-2023-0121573).

[0004] EBPs can be fused with other functional peptides or proteins to impart functional multivalency. Peptides, as signaling molecules, have the advantages of low toxicity, a low incidence of side effects, and highly selective intracellular effects. However, their short half-lives pose a challenge for bioapplication. To extend the half-lives of these short-lived peptides, PEGylation, which involves modifying proteins with poly(ethylene glycol) (PEG), is being utilized. However, PEGylation involves chemical modification after recombinant protein production, requiring multiple purification steps, which increases production costs. Furthermore, long-term use can lead to kidney morphological changes and immune responses to PEG.

[0005] The purpose of the present invention is to provide a recombinant protein for promoting platelet differentiation comprising a hydrophilic elastin-like polypeptide (EBPn), an antibody constant region (Fc) and a thrombopoietin receptor stimulating peptide (TPOR Stimulating Peptide; TSP), a recombinant expression vector comprising a gene encoding the recombinant protein for promoting platelet differentiation, a cell line transfected with the recombinant expression vector and a method for producing the recombinant protein for promoting platelet differentiation using the cell line.

[0006] In addition, another object of the present invention is to provide a pharmaceutical composition for preventing or treating thrombocytopenia, which comprises the recombinant protein for promoting platelet differentiation as an active ingredient.

[0007] In addition, another object of the present invention is to provide a pharmaceutical composition for preventing or treating thrombocytopenia, which comprises the recombinant protein for promoting platelet differentiation and an anticancer agent as active ingredients.

[0008] In addition, another object of the present invention is to provide a health functional food composition for preventing or improving thrombocytopenia, which contains the recombinant protein for promoting platelet differentiation as an active ingredient.

[0009] To achieve the above purpose, the present invention provides a recombinant protein for promoting platelet differentiation, comprising a hydrophilic elastin-like polypeptide (EBPn), an antibody constant region (Fc), and a thrombopoietin receptor stimulating peptide (TPOR Stimulating Peptide; TSP).

[0010] In addition, the present invention provides a pharmaceutical composition for preventing or treating thrombocytopenia, which comprises the recombinant protein for promoting platelet differentiation as an active ingredient.

[0011] In addition, the present invention provides a pharmaceutical composition for preventing or treating thrombocytopenia, comprising the recombinant protein for promoting platelet differentiation and an anticancer agent as active ingredients.

[0012] In addition, the present invention provides a health functional food composition for preventing or improving thrombocytopenia, which comprises the recombinant protein for promoting platelet differentiation as an active ingredient.

[0013] In addition, the present invention provides a recombinant expression vector comprising a gene encoding the recombinant protein for promoting platelet differentiation.

[0014] Additionally, the present invention provides a cell line transfected with the recombinant expression vector.

[0015] In addition, the present invention provides a method for producing a recombinant protein for promoting platelet differentiation, comprising the steps of culturing the cell line and recovering the culture medium; and isolating and purifying the recombinant protein for promoting platelet differentiation from the recovered culture medium.

[0016] The present invention relates to a recombinant protein for treating thrombocytopenia and its use, and more particularly, to the development of a biobetter (NT-202) of romiplostim using the NT-CoreX system. The present invention relates to a recombinant protein, NT-202, which is a TPO-RA comprising a basic sequence of an elastin-based polypeptide (NT-202An or NT-202Gn, where n is the number of repeats of the EBP basic unit, A is VPAXG, G is VPGXG), and its use. The NT-202 recombinant protein acts as a TPO-RA in bone marrow, promotes platelet differentiation, and has therapeutic efficacy for various thrombocytopenias.

[0017] Figure 1A is a diagram schematically showing the structure of NT-202 protein, in which TPOR Stimulating Peptide (TSP) is fused to the EBPn-Fc basic structure, according to the order of expression, and Figure 1B is a diagram showing the amino acid sequence of each structure of NT-202 protein.

[0018] Figure 2 is a drawing showing the results of confirming the expression and purity of NT-202 protein produced and purified through affinity chromatography using the Expi293 Expression System, analyzed by SDS-PAGE and Western blot.

[0019] Figure 3 is a diagram showing the results of confirming the activity as a TPO receptor (TPOR) agonist through cell proliferation assessment in M-07e cells, which are TPOR endogenous expression cells.

[0020] Figure 4 is a diagram showing the results of confirming the mechanism for promoting cell proliferation of M-07e cells through changes in phosphorylation of signaling proteins.

[0021] Figure 5 is a diagram showing the results of evaluating the platelet increasing efficacy in normal mice.

[0022] Figure 6 is a diagram showing the results of evaluating the preventive effect and in vivo efficacy of NT-202-G3, G6, and G12 in a 5-fluorouracil (5-FU)-induced chemotherapy-induced thrombocytopenia (CIT) mouse model.

[0023] Figure 7 is a diagram showing the results of evaluating the preventive and platelet recovery efficacy of NT-202-G12 in an immune thrombocytopenia (ITP) mouse model induced by anti-CD41 antibody.

[0024] Romiplate (romiplostim) is a peptide drug that can activate the thrombopoietin receptor activator (TPO-RA) that promotes platelet differentiation and is used for chronic immune thrombocytopenia. In the present invention, a biobetter of romiplostim was produced using the NT-CoreX system (EBPn-Fc), and its clinical applicability was confirmed in a preclinical model, thereby completing the present invention.

[0025]

[0026] The present invention provides a recombinant protein for promoting platelet differentiation comprising a hydrophilic elastin-like polypeptide (EBPn), an antibody constant region (Fc), and a thrombopoietin receptor stimulating peptide (TPOR Stimulating Peptide; TSP).

[0027] Preferably, the hydrophilic elastin-like polypeptide (EBPn) is composed of [VPAXG VPAXG VPAXG VPAXG VPAXG VPAXG VPAXG (SEQ ID NO: 1)]n or [VPGXG VPGXG VPGXG VPGXG VPGXG VPGXG (SEQ ID NO: 2)]n, wherein n is an integer greater than or equal to 1 and is the number of repetitions of SEQ ID NO: 1 or SEQ ID NO: 2, and wherein X may be selected from any amino acid except proline, but is not limited thereto.

[0028] More preferably, the hydrophilic elastin-like polypeptide (EBPn) may be represented by any one amino acid sequence selected from SEQ ID NO: 3 to SEQ ID NO: 8, but is not limited thereto.

[0029] In the present invention, EBPn is a polypeptide in which EBP, which is a repeating unit of Val-Pro-(Ala or Gly)-Xaa-Gly pentapeptide, is repeated n times, and depending on the sequence of the repeating unit, one is EBPA with the sequence Val-Pro-Ala-Xaa-Gly, and the other is EBPG with the sequence Val-Pro-Gly-Xaa-Gly.

[0030] Previously, the present inventors developed an EBPn-Fc recombinant protein animal cell expression platform technology by linking an antibody constant region (Fc) to the EBPn (Korean Patent Application No. 10-2023-0121573).

[0031] Preferably, the antibody constant region (Fc) has the cysteine ​​residue of the Fc hinge portion of human IgG removed (named CoreX), and more preferably, the antibody constant region (Fc) may be represented by the amino acid sequence of SEQ ID NO: 9, but is not limited thereto.

[0032] Preferably, the TSP is synthesized as a 36-mer nucleotide (named TSP36) and may be represented by the amino acid sequence of SEQ ID NO: 10, but is not limited thereto.

[0033] Preferably, the recombinant protein may be represented by any one amino acid sequence selected from the group consisting of SEQ ID NO: 11 to SEQ ID NO: 16, but is not limited thereto.

[0034] Preferably, the recombinant protein may be, but is not limited to, a thrombopoietin receptor activator (TPO-RA).

[0035]

[0036] In addition, the present invention provides a pharmaceutical composition for preventing or treating thrombocytopenia, which comprises a recombinant protein for promoting platelet differentiation as an active ingredient.

[0037] In addition, the present invention provides a pharmaceutical composition for preventing or treating thrombocytopenia, comprising a recombinant protein for promoting platelet differentiation and an anticancer agent as active ingredients.

[0038] Preferably, the anticancer agent may be, but is not limited to, 5-fluorouracil.

[0039] The pharmaceutical composition of the present invention can be prepared using pharmaceutically suitable and physiologically acceptable adjuvants in addition to the active ingredient, and the adjuvants may include solubilizers such as excipients, disintegrants, sweeteners, binders, coating agents, swelling agents, lubricants, glidants, or flavoring agents. The pharmaceutical composition of the present invention can be preferably formulated as a pharmaceutical composition by additionally including one or more pharmaceutically acceptable carriers in addition to the active ingredient for administration. In the composition formulated as a liquid solution, acceptable pharmaceutical carriers are sterile and biocompatible, and may include saline solution, sterile water, Ringer's solution, buffered saline, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, ethanol, and a mixture of one or more of these components. If necessary, other conventional additives such as antioxidants, buffers, and bacteriostatic agents may be added. In addition, diluents, dispersants, surfactants, binders and lubricants can be additionally added to formulate the composition into injectable formulations such as aqueous solutions, suspensions and emulsions, pills, capsules, granules or tablets.

[0040] The pharmaceutical formulation form of the pharmaceutical composition of the present invention may be granules, powders, coated tablets, tablets, capsules, suppositories, syrups, juices, suspensions, emulsions, drops or injectable solutions, and sustained-release formulations of the active compound, etc. The pharmaceutical composition of the present invention may be administered in a conventional manner via intravenous, intraarterial, intraperitoneal, intramuscular, intraarterial, intraperitoneal, intrasternal, transdermal, intranasal, inhalation, topical, rectal, oral, intraocular or intradermal routes. The effective amount of the active ingredient of the pharmaceutical composition of the present invention refers to the amount required for the prevention or treatment of a disease. Therefore, it can be adjusted according to various factors including the type of disease, the severity of the disease, the types and contents of the active ingredient and other ingredients contained in the composition, the type of formulation, and the patient's age, body weight, general health condition, sex and diet, administration time, administration route and secretion rate of the composition, treatment period, and concurrently used drugs.

[0041]

[0042] In addition, the present invention provides a health functional food composition for preventing or improving thrombocytopenia, which comprises the recombinant protein for promoting platelet differentiation as an active ingredient.

[0043] The health functional food composition of the present invention may be provided in the form of a powder, granules, tablets, capsules, syrup, or beverage. The health functional food composition may be used in combination with other foods or food additives in addition to the active ingredient, and may be appropriately used according to conventional methods. The amount of the active ingredient mixed may be appropriately determined depending on the intended use, for example, for preventive, health, or therapeutic treatment.

[0044] The effective dosage of the active ingredient contained in the above health functional food composition may be used in accordance with the effective dosage of the above pharmaceutical composition, but in the case of long-term intake for the purpose of health and hygiene or health control, it may be below the above range. It is certain that the active ingredient may be used in an amount exceeding the above range because there is no problem in terms of safety.

[0045] There are no special restrictions on the types of the above health foods, and examples include meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gum, dairy products including ice cream, various soups, beverages, tea, drinks, alcoholic beverages, and vitamin complexes.

[0046]

[0047] In addition, the present invention provides a recombinant expression vector comprising a gene encoding the recombinant protein for promoting platelet differentiation.

[0048] In the present invention, "vector" means a self-replicating DNA molecule used to carry a clone gene (or other piece of clone DNA).

[0049] In the present invention, an “expression vector” refers to a recombinant DNA molecule containing a desired coding sequence and an appropriate nucleic acid sequence essential for expressing the coding sequence operably linked to a specific host organism. The expression vector may preferably include one or more selectable markers. The markers are typically nucleic acid sequences having properties that can be selected by a chemical method, and include all genes that can distinguish transformed cells from non-transformed cells. Examples include, but are not limited to, antibiotic resistance genes such as ampicillin, kanamycin, geneticin (G418), bleomycin, hygromycin, and chloramphenicol, and can be appropriately selected by those skilled in the art.

[0050] Additionally, the present invention provides a cell line transfected with the recombinant expression vector.

[0051] In addition, the present invention provides a method for producing a recombinant protein for promoting platelet differentiation, comprising the steps of culturing the cell line and recovering the culture medium; and isolating and purifying the recombinant protein for promoting platelet differentiation from the recovered culture medium.

[0052] Hereinafter, to aid understanding of the present invention, examples will be given in detail. However, the following examples are intended only to illustrate the scope of the present invention and are not intended to limit its scope. These examples are provided to more fully explain the present invention to those of average skill in the art.

[0053]

[0054] <Example 1> Fabrication of NT-202 protein structure

[0055] TPOR Stimulating Peptide (TSP) was synthesized as a 36-mer nucleotide (TSP36) and fused to the C-terminus of the EBPn-Fc basic structure, producing a total of six structures.

[0056] At this time, the EBPn-Fc basic structure was constructed using EBPn, in which the EBPA and EBPG repeat sequences were repeated 3, 6, and 12 times, respectively, and CoreX, in which the cysteine ​​residues in the hinge region of human IgG Fc were removed, as Fc.

[0057] In Figure 1A, the structure of NT-202, a TSP fusion EBPn-Fc protein, is schematically illustrated in the order of expression, with names for each type.

[0058] Additionally, the amino acid sequence for each NT-202 structure is described in Figure 1B.

[0059]

[0060] <Example 2> Production and purification of NT-202 protein

[0061] Recombinant DNA for NT-202 expression was produced using the Expi293 Expression System (Thermo Fisher Scientific) via transient expression in animal cells. The protein in the produced cell culture was confirmed for expression through SDS-PAGE and Western blot analysis, and then concentrated to less than 50 mL using Amicon Ultra-15 30K (Millipore).

[0062] The concentrated culture medium was purified through affinity chromatography using protein A resin. Specifically, the concentrate was mixed with 0.1 M sodium phosphate (pH 7.0) to a final concentration of 20 mM sodium phosphate, and then passed twice through a HiTrap rProtein A FF (GE Healthcare) column connected to a Peristaltic Pump P-1 (GE Healthcare). The column was then washed with 20 mM sodium phosphate buffer, and the NT-202 protein bound to the column was separated and purified using 0.1 M sodium citrate buffer (pH 4.0).

[0063] The purified protein was buffer exchanged using a PD-10 Desalting Column (GE Healthcare), and then its concentration was quantified using a BCA Protein Assay Kit (Thermo Fisher Scientific).

[0064] To verify the size and purity of the protein, 0.5 μg of purified protein was used for SDS-PAGE analysis, followed by electrophoresis and staining with EZ Gel Staining Solution (Daeil Lab Service). As a result, the purified protein was confirmed as a strong single band in the expected molecular weight region. Although some non-specific bands were observed, it was confirmed that the protein was obtained overall with high purity. For Western blot analysis, 0.05 μg of purified protein was used, and the protein was detected by targeting the Fc region with an anti-human IgG-HRP antibody (Jackson ImmunoResearch), confirming that it was an Fc fusion protein with the expected molecular weight. Considering the SDS-PAGE and Western blot results comprehensively, it was confirmed that the protein was purified with relatively high purity (Fig. 2).

[0065]

[0066] <Example 3> Evaluation of cell proliferation through TPOR agonist activity

[0067] The activity of NT-202 protein as a TPO receptor (TPOR) agonist was confirmed by increased proliferation of M-07e cells endogenously expressing TPOR (Fig. 3). 5 × 10⁴ M-07e cells per well were seeded in 96-well plates and cultured in the presence or absence of various concentrations of TPOR agonists for 48 h. After 3 h of incubation using Cell Counting Kit-8 (CCK-8, Dojindo), the absorbance was measured at 450 nm using a Victor Nivo™ Plate Reader (PerkinElmer). Human TPO synthetic protein (rhTPO) was used as a positive control.

[0068] As a result, both rhTPO and NT-202 promoted the growth of M-07e cells in a concentration-dependent manner. When the relative cell proliferation ability was calculated based on 100 nM rhTPO, which showed the maximum proliferation ability, the EC of rhTPO was 50 was measured as 0.0820 nM.

[0069] As a result of evaluating the activity of six NT-202 proteins with different sequences and lengths of EBP, A3, A6, and A12 had EC 50 The values ​​were measured as 2.13 nM, 3.28 nM, and 237 nM, and A12 showed a significant decrease in activity compared to A3 and A6.

[0070] Additionally, G3, G6, and G12 are each EC 50 The values ​​were measured as 0.0383 nM, 0.110 nM, and 0.516 nM, and showed better activity than A3 and A6.

[0071]

[0072] <Example 4> Evaluation of intracellular signaling pathway activation according to TPOR action

[0073] TPO is known to bind to TPOR and activate intracellular signaling pathways, including the JAK / STAT pathway. In the present invention, to confirm the proliferation-promoting mechanism of NT-202-G3, G6, and G12, which showed excellent cell proliferation activity in M-07e cells, the phosphorylation of JAK2, STAT5, STAT3, AKT, and ERK proteins was investigated.

[0074] M-07e cells (1 × 10 6 Cells (10 μg / mL) were washed with PBS, resuspended in RPMI-1640 medium containing 0.5% FBS, and cultured overnight in a serum-starved condition. The cells were then stimulated by treating them with various concentrations of TPOR agonists for 30 minutes. The stimulated cells were lysed using RIPA buffer (Elpis Biotech) supplemented with protease inhibitors and protein phosphatase inhibitors, and the dissolved proteins were quantified using a BCA Protein Assay Kit (Thermo Fisher Scientific), mixed with SDS-PAGE sample buffer, and heated at 95°C for 5 minutes. The prepared samples (10 μg / well) were electrophoresed on an SDS-PAGE gel (Bio-Rad), and the proteins were transferred to a nitrocellulose membrane (Millipore).

[0075] The transferred membranes were sequentially reacted with primary antibodies against JAK2, p-JAK2, STAT5, p-STAT5 (Y925), STAT3, p-STAT3 (Y705), AKT, p-AKT (S473), ERK and p-ERK1 / 2 (Thr202 / Tyr204), and GAPDH, and anti-rabbit IgG-HRP secondary antibodies; all antibodies were purchased from Cell Signaling Technology. Chemiluminescence signals were detected using a Thermo ECL kit (Thermo Fisher Scientific) and visualized with Amersham™ ImageQuant 800 (Cytiva) (Fig. 4).

[0076] As positive controls, rhTPO and Romiplostim were used, and both treatment groups confirmed the activation of phosphorylation of major signaling pathways. Similarly, NT-202-G3, G6, and G12 induced the phosphorylation of JAK2, STAT5, STAT3, AKT, and ERK in a concentration-dependent manner. These results confirmed that NT-202-G3, G6, and G12 activate TPOR, stimulate downstream signaling pathways, and thereby induce the proliferation of M-07e cells.

[0077]

[0078] <Example 5> Evaluation of platelet-increasing efficacy in normal mice

[0079] To evaluate the platelet-increasing effects of NT-202-G3, G6, and G12 in normal mice, experiments were conducted on 8- to 10-week-old BALB / c wild-type (WT) female mice (Nara Biotech, Seoul, Korea). NT-202 G3, G6, and G12 (500 μg / kg) were each injected subcutaneously once. Romiplostim (500 μg / kg) was injected as a positive control, and PBS containing BSA (PBS-BSA) was injected as a negative control under the same conditions. Each group consisted of four mice. Blood was collected from the orbital venous plexus on days 7, 14, and 21 after drug administration (Fig. 5A). Blood sampling was performed after anesthesia with intraperitoneal injection of ketamine (90 mg / kg) and xylazine (10 mg / kg), and the collected blood (50 μL) was stored in EDTA tubes containing 2.5 mM EDTA buffer (450 μL). Platelet count analysis was performed at the EONE Research Institute (Incheon, Korea), and all animal experiments were approved by the University College of Medicine Institutional Animal Care and Use Committee (Approval No. 2022-003).

[0080] As a result, NT-202-G3, G6, and G12 all showed the effect of significantly increasing the platelet count on day 7, similar to Romiplostim. The platelet count reached the maximum on day 7 and then gradually decreased on days 14 and 21 (Fig. 5B). In particular, the NT-202-G6 and G12 administration groups showed a significant increase in platelet count even on day 14, demonstrating sustained activity compared to Romiplostim. This demonstrated that NT-202-G3, G6, and G12 could significantly increase the platelet count in WT mice.

[0081]

[0082] <Example 6> Evaluation of the preventive effect and in vivo efficacy of chemotherapy-induced thrombocytopenia (CIT).

[0083] To evaluate the preventive effect of NT-202-G3, G6, and G12 on chemotherapy-induced thrombocytopenia (CIT), animal experiments were conducted on 8- to 10-week-old BALB / c wild-type (WT) female mice (Nara Biotech, Seoul, Korea). The thrombocytopenia model was induced by a single intraperitoneal injection of 5-fluorouracil (5-FU, 150 mg / kg), followed by a single subcutaneous injection of NT-202 G3, G6, and G12 (each 500 μg / kg). Romiplostim (500 μg / kg) was administered as a positive control, and PBS containing BSA (PBS-BSA) was administered as a negative control under the same conditions. Each group consisted of four mice.

[0084] Drug administration and blood sampling were performed according to the schedule shown in Figure 6A, and blood was collected periodically from the orbital venous plexus. Blood sampling was performed after anesthesia with an intraperitoneal injection of ketamine (90 mg / kg) and xylazine (10 mg / kg). The collected blood (50 μL) was stored in EDTA tubes containing 2.5 mM EDTA buffer (450 μL). Platelet count analysis was performed at the EONE Research Institute (Incheon, Korea), and all animal experiments were approved by the INJE University College of Medicine Institutional Animal Care and Use Committee (Approval No. 2022-003).

[0085] As a result, the platelet count significantly decreased on the 4th day after 5-FU administration, and the NT-202 G6 administration group showed a significant increase in the platelet count on the 7th day, similar to Romiplostim. The platelet count reached the maximum on the 14th day and then showed a gradual decrease on the 21st day (Fig. 6B). In particular, the NT-202-G3 and G12 administration groups showed a significant increase in the platelet count even on the 14th day, showing sustained activity similar to Romiplostim. These results demonstrated that NT-202-G3, G6, and G12 could effectively increase the platelet count in the CIT mouse model.

[0086]

[0087] <Example 7> Evaluation of the efficacy of preventing immune thrombocytopenia (ITP) and recovering platelets.

[0088] To evaluate the immune thrombocytopenia (ITP) prevention effect of NT-202-G12, animal experiments were conducted on 8- to 10-week-old BALB / c wild-type (WT) female mice (Nara Biotech, Seoul, Korea). The ITP model was induced by daily intraperitoneal injection of anti-CD41 antibody, followed by a single subcutaneous injection of NT-202-G12 (500 μg / kg). Romiplostim (500 μg / kg) was injected as a positive control group, and PBS containing BSA (PBS-BSA) was injected as a negative control group under the same conditions. The isotype control group consisted of 4 mice, and the anti-CD41 administration group consisted of 8 mice each.

[0089] Drug administration and blood sampling were performed according to the schedule shown in Figure 7A, and blood was collected periodically from the orbital venous plexus. Blood sampling was performed after anesthesia with an intraperitoneal injection of ketamine (90 mg / kg) and xylazine (10 mg / kg). The collected blood (50 μL) was stored in EDTA tubes containing 2.5 mM EDTA buffer (450 μL). Platelet count analysis was performed at the EONE Research Institute (Incheon, Korea), and all animal experiments were approved by the INJE University College of Medicine Institutional Animal Care and Use Committee (Approval No. 2022-003).

[0090] As a result, in the ITP model induced by anti-CD41 antibody injection, the platelet count showed a rapid decrease, but in the Romiplostim and NT-202-G12 administration groups, the platelet count recovered to a level similar to the normal group from day 4 (Figures 7B and 7C). In particular, while the efficacy of Romiplostim was mostly lost on day 9, NT-202-G12 showed a significant increase in platelet count, demonstrating sustained efficacy (Figure 7D). These results demonstrated that NT-202-G12 can effectively increase platelet count in the ITP mouse model.

[0091]

[0092] While specific aspects of the present invention have been described in detail above, it will be apparent to those skilled in the art that these specific descriptions are merely preferred embodiments and are not intended to limit the scope of the present invention. Therefore, the substantial scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A recombinant protein for promoting platelet differentiation comprising a hydrophilic elastin-like polypeptide (EBPn), an antibody constant region (Fc), and a thrombopoietin receptor stimulating peptide (TPOR Stimulating Peptide; TSP).

2. A recombinant protein for promoting platelet differentiation, characterized in that in the first paragraph, the hydrophilic elastin-like polypeptide (EBPn) is composed of [VPAXG VPAXG VPAXG VPAXG VPAXG VPAXG VPAXG (SEQ ID NO: 1)]n or [VPGXG VPGXG VPGXG VPGXG VPGXG VPGXG (SEQ ID NO: 2)]n, wherein n is an integer greater than or equal to 1 and is the number of repetitions of SEQ ID NO: 1 or SEQ ID NO: 2, and X is selected from any amino acid except proline.

3. A recombinant protein for promoting platelet differentiation, characterized in that in the second paragraph, the hydrophilic elastin-like polypeptide (EBPn) is represented by any one amino acid sequence selected from SEQ ID NO: 3 to SEQ ID NO:

8.

4. A recombinant protein for promoting platelet differentiation, characterized in that the antibody constant region (Fc) in the first paragraph is represented by the amino acid sequence of sequence number 9.

5. A recombinant protein for promoting platelet differentiation, characterized in that the TSP in the first paragraph is represented by the amino acid sequence of sequence number 10.

6. A recombinant protein for promoting platelet differentiation, characterized in that the recombinant protein in claim 1 is represented by any one amino acid sequence selected from the group consisting of SEQ ID NO: 11 to SEQ ID NO:

16.

7. A recombinant protein for promoting platelet differentiation, characterized in that the recombinant protein in claim 1 is a thrombopoietin receptor activator (TPO-RA).

8. A pharmaceutical composition for preventing or treating thrombocytopenia, comprising a recombinant protein for promoting platelet differentiation according to any one of claims 1 to 6 as an active ingredient.

9. A pharmaceutical composition for preventing or treating thrombocytopenia, comprising a recombinant protein for promoting platelet differentiation according to any one of claims 1 to 6 and an anticancer agent as active ingredients.

10. A pharmaceutical composition for preventing or treating thrombocytopenia, characterized in that the anticancer agent in claim 9 is 5-fluorouracil.

11. A health functional food composition for preventing or improving thrombocytopenia, comprising a recombinant protein for promoting platelet differentiation according to any one of claims 1 to 6 as an active ingredient.

12. A recombinant expression vector comprising a gene encoding a recombinant protein for promoting platelet differentiation according to any one of claims 1 to 6.

13. A cell line transfected with a recombinant expression vector according to Article 12.

14. A step of culturing the cell line according to Article 13 and recovering the culture solution; and A method for producing a recombinant protein for promoting platelet differentiation, comprising a step of isolating and purifying a recombinant protein for promoting platelet differentiation from the recovered culture solution.

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