Preparation of recombinant protein based on elastin-like polypeptide and animal cell expression platform thereof

Abstract

The present invention relates to the preparation of a recombinant protein based on an elastin-like polypeptide and an animal cell expression platform thereof, and specifically relates to an animal cell expression platform using a fusion recombinant protein comprising: a repetitive elastin-based polypeptide (EBPn, where n represents the number of repetitive base units of EBP); and an antibody constant region (Fc) linked to the EBPn; the present invention relates to an EBPn-Fc recombinant protein animal cell expression platform and, more specifically, to a technology for an EBPn-Fc recombinant protein animal cell expression platform capable of fusing various proteins, including peptides, proteins or antibody fragments, on the front and rear sides of EBPn-Fc or on the front and rear sides of EBPn-Fc. In the EBPn-Fc animal cell expression platform, a protein (such as a peptide, a protein, an antibody fragment and the like) is expressed in a form fused with EBPn-Fc, so that high-purity purification can be realized, and a nanostructure can be formed while the function of the fusion protein is maintained. Therefore, the EBPn-Fc animal cell expression platform can be used for research and treatment application of recombinant protein.

Description

Technical Field

[0001] The present disclosure relates to the development and utilization of recombinant proteins based on a fusion recombinant protein, the fusion recombinant protein comprising a repetitive hydrophilic elastin-like polypeptide (EBPn), and an antibody constant region (Fc) linked to the EBPn; and an animal cell expression platform. Background Art

[0002] Elastin is a major component of the extracellular matrix (ECM) protein and consists of an elastomeric domain and a cross-linking domain. The elastomeric domain is composed of hydrophobic amino acids and repetitive peptides (such as VPGG, VPGVG, and APGVGV). Based on this, elastin-based polypeptides (EBPs) that are stimulus-responsive, biocompatible, biodegradable, and non-immunogenic have been designed. The elastin-based polypeptides (EBPs) with pentapeptide repeat units are thermoresponsive biopolymers, and many studies have been conducted because Val-Pro-(Gly or Ala)-Xaa-Gly (where Xaa is any amino acid other than Pro) can regulate the responsiveness to environmental changes.

[0003] The EBPs undergo a reversible phase transition at the lower critical solution temperature (LCST), which is called the transition temperature (Tt). They are in a soluble state below Tt, but become insoluble when the temperature rises above Tt. These thermoreversible phase transitions enable the use of simple purification methods, such as inverse transition cycling (ITC), and self-assembly into particles, gels, fibers, and other structures by heat triggering. However, for purification to a purity suitable for therapeutic use, ITC purification requires multiple purification processes and may involve processes such as endotoxin removal, depending on the protein expression system.

[0004] Generally, the physicochemical properties of EBPs are mainly regulated by the combination of the pentapeptide repeat unit Val-Pro-(Gly or Ala)-Xaa-Gly. Specifically, the third amino acid in the repeat unit shows mechanical properties, such as the elasticity of Gly and the plasticity of Ala; while the fourth amino acid Xaa and the polymerization of the pentapeptide repeat unit affect Tt. EBPs with different physicochemical properties and Tt can be prepared according to the combination of the pentapeptide repeat units.

[0005] EBPs can be fused with other functional peptides or proteins to provide functional multivalency. For peptides, they have low toxicity as signal transduction molecules, a low incidence of side effects, and high selectivity in inducing intracellular effects, but their short half-life limits their in vivo applicability. Cytokines also have high functional utility, but their utilization is limited due to their short in vivo half-life. To extend the half-life of peptides and recombinant proteins with short half-lives, polyethylene glycol (PEG) modification of proteins, i.e., PEGylation, is being applied. However, since PEGylation is a chemical modification after the production of recombinant proteins, multiple purification processes are required, which increases the production cost, changes the elongated shape upon long-term use, and elicits an immune response to PEG.

[0006] EBPs are used as inactive protein-based biomaterials such as PEG in advanced drug delivery systems, regenerative medicine, and tissue engineering, and are also used as drug delivery carriers together with drugs or other functional proteins.

[0007] Based on their property of reversible aggregation by heat stimulation, EBPs have the advantage of being purified by centrifugation without chromatography, and EBPs are also used as biomaterials due to their low immunogenicity, and allow size control and the formation of nanocomposites through genetic recombination, making them also suitable for increasing the half-life of protein therapeutics in vivo and as a drug delivery platform. However, there are limitations, such as the formation of intracellular inclusion bodies when they are expressed in Escherichia coli, or the difficulty in providing sufficient purification efficiency when they are expressed in animal cells. Summary of the Invention

[0008] Technical Problem

[0009] An object of the present disclosure is to provide an animal cell expression cassette comprising a gene encoding a hydrophilic elastin-like polypeptide (EBPn) and a gene encoding a protein for expression connected to the front side and / or the rear side of a gene encoding an antibody constant region (Fc); a recombinant expression vector comprising the animal cell expression cassette; and a cell line transfected with the recombinant expression vector.

[0010] Furthermore, another object of the present disclosure is to provide a method for producing a protein for expression, comprising culturing the cell line and recovering the culture solution; and separating and purifying the protein for expression fused with EBPn and Fc from the recovered culture solution.

[0011] Technical Solution

[0012] To achieve the above object, the present disclosure provides an animal cell expression cassette, which comprises a gene encoding a hydrophilic elastin-like polypeptide (EBPn), a gene encoding an antibody constant region (Fc), and a gene encoding a first protein for expression, and provides an animal cell expression cassette, which further comprises a gene encoding a second protein for expression.

[0013] In addition, the present disclosure provides a recombinant expression vector, which comprises the animal cell expression cassette; and a cell line transfected with the recombinant expression vector.

[0014] In addition, the present disclosure provides a method for producing a protein for expression, which includes culturing the cell line and recovering the culture solution, and separating and purifying the protein for expression fused with EBPn and Fc from the recovered culture solution.

[0015] Beneficial effects

[0016] The present disclosure relates to the production of elastin-like polypeptide-based recombinant proteins and their animal cell expression platforms. More specifically, as an animal cell expression platform for using a fusion recombinant protein comprising a repetitive elastin-like polypeptide (EBPn, where n is the number of repetitions of the EBP basic unit) and an antibody constant region (Fc) linked to EBPn, the present disclosure relates to the animal cell expression platform technology for EBPn-Fc recombinant proteins, which can fuse various proteins (such as peptides, proteins, and antibody fragments) to the front side, back side, or both sides of EBPn-Fc. The EBPn-Fc animal cell expression platform of the present disclosure expresses proteins (such as peptides, proteins, and antibody fragments) in the form of fusion with EBPn-Fc, allows for high-purity purification, and forms nanostructures while maintaining the function of the fusion protein, thereby enabling the recombinant protein to be applied in the research and therapeutic fields. Brief description of the drawings

[0017] Figure 1 Shows the structure and amino acid sequence of the EBPn-Fc fusion protein, where Figure 1 A shows a schematic structure of the EBPn-Fc fusion recombinant protein using the pSecTag2 vector, Figure 1 B and Figure 1 C show the amino acid sequence of EBPn-Fc, which is the basic structure generated for protein fusion, and Figure 1 D shows the amino acid sequences of peptides, proteins, and antibody fragments fused with EBPn-Fc.

[0018] Figure 2Shows the results of a purity comparison of the purification of each EBPn-Fc fusion protein produced using animal cells by ITC purification (which is a conventional EBP purification method) and affinity chromatography purification using a protein A column.

[0019] Figure 3 shows the results of identifying the size and purity by purifying each fusion protein produced using animal cells, and shows the results obtained by SDS-PAGE and Western blotting after the following purifications: the basic EBPn-Fc protein in Figure 3A, the peptide fusion proteins in Figures 3B and 3C, the cytokine in Figure 3D, the enzyme fusion protein in Figure 3E, and the antibody fragment fusion proteins in Figures 3F, 3G, and 3H.

[0020] Figure 4 shows the results of identifying the functions of each fusion protein. Figure 4A shows the results of identifying the receptor recognition and cell inhibitory ability of the anti-Flt1 peptide fusion protein, and Figure 4B shows the results of identifying the cell proliferation ability of the TPO-stimulating peptide 14 (TSP14) fusion protein. Figure 4C shows the results of identifying the ability of the interferon-β (IFN-β) cytokine fusion protein to increase intracellular receptor expression and regulate gene expression, and Figure 4D shows the results of identifying the enzyme activity of the adenosine deaminase 2 (ADA2) enzyme fusion protein and the antigen recognition ability after the addition of antibody fragments. Figures 4E, 4F, and 4G respectively show the results of identifying the antigen recognition abilities of the 7.16.4, α-PD-L1, and Erbitux antibody fragment fusion proteins.

[0021] Figure 5 Shows the results of identifying the formation of the nanostructure of the EBPn-Fc protein, where Figure 5 A shows the results of measuring the turbidity according to the change in salt concentration at room temperature, and Figure 5 B shows the results of measuring the protein nanoparticle size according to the change in salt concentration and temperature using a nanoparticle size analyzer (Zetasizer), which measures the particle size by dynamic light scattering (DLS). Detailed Description

[0022] In the present disclosure, an attempt is made to overcome the drawbacks of ITC purification by developing an animal cell expression platform for EBPn-Fc recombinant proteins, in which a hydrophilic EBP is fused with the constant region (Fc) of an antibody, enabling high-purity purification using protein A or protein G. In addition, since EBPn-Fc exhibits the property of forming nanostructures in response to salt concentration and temperature stimuli, and is expressed as a recombinant protein in the form of a fusion with functional proteins (such as peptides, proteins, and antibody fragments), thereby achieving functional stability or targeting antigens, the present disclosure is thus completed.

[0023] The present disclosure provides an animal cell expression cassette, which comprises a gene encoding a hydrophilic elastin-like polypeptide (EBPn), a gene encoding an antibody constant region (Fc), and a gene encoding a first protein for expression.

[0024] In addition, the animal cell expression cassette may further comprise a gene encoding a second protein for expression.

[0025] Preferably, the hydrophilic elastin-like polypeptide (EBPn) consists of [VPAXG VPAXG VPAXG VPAXG VPAXGVPAXG (SEQ ID NO: 1)]n or [VPGXG VPGXG VPGXG VPGXG VPGXG VPGXG (SEQ ID NO: 2)]n, where n can be an integer greater than or equal to 1 and is the number of repeats of SEQ ID NO: 1 or SEQ ID NO: 2, and X can be selected from any amino acid other than proline, but is not limited thereto.

[0026] More preferably, the hydrophilic elastin-like polypeptide (EBPn) can be represented by any one of the amino acid sequences selected from SEQ ID NOs: 3 to SEQ ID NO: 8, but is not limited thereto.

[0027] In the present disclosure, EBPn is a polypeptide, wherein EBP (a repeating unit of the pentapeptide Val-Pro-(Ala or Gly)-Xaa-Gly) repeats n times, and according to 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. The inventors developed an animal cell expression platform technology for EBPn-Fc recombinant proteins by linking the antibody constant region (Fc) to the EBPn.

[0028] Preferably, the antibody constant region (Fc) may comprise the hinge region and CH2-CH3 domains of IgG, and the cysteine residues in the hinge region of IgG may be additionally replaced by serine residues, but is not limited thereto.

[0029] More preferably, the antibody constant region (Fc) can be represented by any one of the amino acid sequences selected from SEQ ID NOs: 9 to SEQ ID NO: 12, but is not limited thereto.

[0030] Preferably, the protein for expression may be a peptide, a protein, or an antibody fragment, and more preferably, the protein for expression may be anti-Flt1 shown by the amino acid sequence of SEQ ID NO: 13, TSP14 shown by the amino acid sequence of SEQ ID NO: 14, IFN-β shown by the amino acid sequence of SEQ ID NO: 15, ADA2 shown by the amino acid sequence of SEQ ID NO: 16, 7.16.4 scFv shown by the amino acid sequence of SEQ ID NO: 17, α-PD-L1 scFv shown by the amino acid sequence of SEQ ID NO: 18, or Erbitux scFv shown by the amino acid sequence of SEQ ID NO: 19, but not limited thereto.

[0031] In addition, the present disclosure provides a recombinant expression vector containing the animal cell expression cassette.

[0032] As used herein, the term "vector" refers to a self-replicating DNA molecule for carrying a cloned gene (or other fragment of cloned DNA).

[0033] As used herein, the term "expression vector" refers to a recombinant DNA molecule containing a desired coding sequence and the appropriate nucleic acid sequences necessary for the coding sequence to be operably linked for expression in a particular host organism. The expression vector preferably may contain one or more selectable markers. The marker is a nucleic acid sequence having a characteristic that can generally be selected chemically and includes any gene capable of distinguishing transformed cells from untransformed cells. Examples include but are not limited to antibiotic resistance genes such as ampicillin, kanamycin, geneticin (G418), bleomycin, hygromycin, and chloramphenicol, which can be appropriately selected by those skilled in the art.

[0034] In addition, the present disclosure provides a cell line transfected with the recombinant expression vector.

[0035] In addition, the present disclosure provides a method for producing a protein for expression, including culturing the cell line and recovering the culture medium; and separating and purifying the protein for expression fused with EBPn and Fc from the recovered culture medium.

[0036] Preferably, the purification can be performed by affinity chromatography using a protein A column, but not limited thereto.

[0037] Examples

[0038] The present disclosure will be described in more detail below by way of examples. These examples are only intended to illustrate the present disclosure more specifically, and it is obvious to those skilled in the art that the scope of the present disclosure is not limited by these examples according to the gist of the present disclosure.

[0039] <Example 1> Generation of the EBP-Fc fusion recombinant protein structure

[0040] For EBP, the first cloning is performed after nucleotide synthesis; for Fc, the CH2-CH3 domain of the Fc portion of a murine or human antibody is used as a template, and after PCR, the first cloning is performed similarly to EBP. The gene sequences of each EBP and Fc are identified by sequencing (Bioneer). For two types of EBP with the pentapeptide repeat unit Val-Pro-(Ala or Gly)-Xaa-Gly [VP(A or G)XG], peptides are synthesized by constructing the amino acid sequence such that the ratio of Ala, Gly, and Ile at the fourth guest residue position (Xaa) is 1:4:1. After the first cloning, EBPn is generated by repeated fusion using restriction enzymes and T4 DNA ligase. For Fc, the CH2-CH3 domain of the Fc portion of a murine or human IgG antibody is used as a template, and after PCR, the first cloning is performed. At this time, after differently preparing the structures with two cysteine amino acid residues of the Fc hinge region remaining intact and the structure becoming serine amino acids, the gene base sequences of EBPn and Fc are identified by sequencing (Bioneer).

[0041] For EBPn and Fc, they are cloned into the pSecTag2-Hygro B vector for animal cell expression using restriction enzymes and T4 DNA ligase to form EBPn-Fc. The base sequence of the recombinant EBPn-Fc is identified by sequencing (Bioneer).

[0042] Subsequently, genes of various proteins (such as peptides, proteins, and antibody fragments) are used as the basic structure for fusion to the front or back side of EBPn-Fc ( Figure 1 )). In Figure 1 A, each fusion protein is labeled as A, B, C, and D, and the structure of the EBPn-Fc fusion recombinant protein is simplified according to the expression order. The protein amino acid sequence of EBPn used in the basic structure is as described in Figure 1 B.

[0043] For peptide fusion, anti-Flt1 is cloned to the front side of the EBPn-Fc basic structure after nucleotide synthesis, and the TPO-stimulating peptide (TSP) is cloned to the front side of the EBPn-Fc basic structure after synthesizing 14mer nucleotides (TSP14).

[0044] For protein fusion, after PCR using the cDNA of THP-1 cells as a template, IFN-β was cloned in two forms to the front and back sides of EBPn-Fc. The ADA2 enzyme was produced in two forms: one was obtained by cloning it to the front side of EBPn-Fc after PCR using the cDNA of MCF7 cells as a template; the other was obtained by adding the 7.16.4 antibody fragment to the front side of EBPn-Fc and then cloning the ADA2 enzyme to the back side to produce a bifunctional protein.

[0045] For antibody fragment fusion, PCR was performed using the 7.16.4 antibody fragment portion as a template and then cloned to the front side of EBPn-Fc. After PCR using the antibody fragment portion as a template, α-PD-L1 and Erbitux were also cloned to the front side of EBPn-Fc.

[0046] <Example 2> Labeling of EBPn-Fc Fusion Protein

[0047] EBP is the VP(A or G)XG pentapeptide repeat unit, and VPAXG is labeled with EBPA, while VPGXG is labeled with EBPG. The number n in EBP(A or G)n represents the number of repetitions of SEQ ID NO: 1 [VPAXG VPAXG VPAXG VPAXG VPAXG VPAXG] or SEQ ID NO: 2 [VPGXG VPGXG VPGXG VPGXG VPGXG VPGXG] ( Figure 1 B). When the cysteine residues in the Fc hinge region of human IgG are removed, Fc is named CoreX and CoreX2; when the cysteine residues in the Fc hinge region of mouse IgG are removed, Fc is named CoreXm. In addition, CoreXC represents Fc that keeps the cysteine residues in the Fc hinge region of human IgG intact. The name of the final completed recombinant protein structure is used by sequentially connecting the EBPn and Fc names, which are named differently according to the name and type of the fusion protein. The amino acid sequences of the peptides, proteins, and antibody fragments fused to the EBPn-Fc structure are as Figure 1 shown in D.

[0048] <Example 3> Comparison between ITC Purification and Affinity Chromatography Purification

[0049] The constructed recombinant DNA for expressing anti-Flt1-EBPA12-CoreXm was transfected into mammalian HEK293E cells to produce the EBPn-Fc fusion protein. For the resulting cell culture medium, purification was performed by ITC purification (the existing EBP purification method) and affinity chromatography using a protein A column, respectively. The purified protein was subjected to SDS-PAGE, and the gel was stained with EZGel staining solution (Daeil Lab Service) to determine the size and purity of the protein. In addition, Western blotting was performed using an anti-mouse IgG-HRP antibody (Jackson ImmunoResearch) targeting the Fc segment for identification ( Figure 2 ).

[0050] For ITC purification, the cell culture medium was mixed with a high-salt buffer (2.25 M glycine, 4.5 M NaCl, pH 8.9) at a ratio of 1:2 (culture medium: buffer) and reacted at 37 °C for 45 minutes to aggregate the EBPn-Fc fusion protein. Then, it was precipitated by centrifugation at room temperature for 10 minutes, resuspended in PBS, and then centrifuged at 4 °C for 10 minutes to isolate only the dissolved protein. To improve the purity, the above process was repeated three times in total ( Figure 2 A).

[0051] In affinity chromatography purification, the cell culture medium was mixed with 0.1 M sodium phosphate at pH 7.0 to a final concentration of 20 mM sodium phosphate, and then passed through a protein A (HiTrap rProtein A FF, GE) column connected to a peristaltic pump P-1 (GE) twice. After washing the column with 20 mM sodium phosphate buffer, the EBPn-Fc fusion protein bound to the column was separated and purified with 0.1 M sodium citrate buffer at pH 4.0 ( Figure 2 B).

[0052] In both purification attempts, it was found that when expressed in animal cells, affinity chromatography purification using the antibody constant region (Fc) of the EBPn-Fc recombinant protein had higher purification efficiency and purity than ITC purification.

[0053] <Example 4> Production of EBPn-Fc Fusion Protein

[0054] Each recombinant DNA for expression was used to produce the EBPn-Fc fusion protein using the transient expression system of animal cells, the Expi293 expression system (Thermo). The expression of the protein in the resulting cell culture supernatant was detected by performing SDS-PAGE and Western blotting. The SDS-PAGE gel was stained with EZ Gel Staining Solution (Daeil Lab Service) to detect protein expression, and Western blotting was performed by targeting the Fc portion with anti-mouse IgG-HRP antibody (Jackson ImmunoResearch) or anti-human IgG-HRP antibody (Jackson ImmunoResearch) for detection. Additionally, the culture supernatant in which protein expression was identified was concentrated to less than 50 mL using Amicon Ultra-15, 30k (Millipore), mixed with 0.1 M sodium phosphate at pH 7.0 to bring the final concentration of sodium phosphate to 20 mM, and purified using a Protein A (HiTrap rProtein A FF, GE) column connected to a peristaltic pump P-1 (GE). The purified protein was buffer-exchanged using a PD-10 desalting column (GE), and its concentration was quantified using a BCA Protein Assay Kit (Thermo). The size and purity of the purified protein were verified using 0.5 μg to 1 μg of the quantified protein (for SDS-PAGE) and 0.05 μg to 0.1 μg of the quantified protein (for Western blotting) (Figure 3). Figure 3A shows the identification results of the protein purified by expressing EBP-Fc in its basic structure without the fusion protein, and Figures 3B and 3C show the identification results of the proteins fused with anti-Flt1 peptide and TSP14 peptide, respectively. Figures 3D and 3E show the identification results of the proteins fused with IFN-β cytokine and ADA2 enzyme, and Figures 3F, 3G, and 3H show the identification results of the proteins fused with antibody fragments of 7.16.4, αPD-L1, and Erbitux, respectively.

[0055] <Example 5> Functional Identification of Recombinant Proteins

[0056] The experimental methods and results for identifying the functions of each recombinant fusion protein are described in detail for each protein below.

[0057] For the function of the anti-Flt1 peptide fusion protein, its anti-angiogenic effect was identified by inhibiting VEGF-induced HUVEC tube formation. HUVECs were seeded at 4×10 per well 4were seeded onto 8-well chamber slides coated with Matrigel, stimulated with 50 ng / mL human VEGF 165, and then treated with different concentrations of anti-Flt1-EBPA12-CoreXm. The anti-angiogenic effect was observed by randomly selecting three sites and dividing the length of the connected cells counted by the total number of cells in the same field of view. The VEGF receptor inhibitor Avastin (bevacizumab) was used as a positive control. The results in the upper left of Figure 4A showed that VEGF-induced HUVEC tube formation was significantly inhibited by more than 50% at concentrations of anti-Flt1-EBPA12-CoreXm from 0.01 μg / mL to 1 μg / mL. In addition, the bar graph in the upper right of Figure 4A showed that competitive ELISA revealed that anti-Flt1-EBPA12-CoreX / Xm was able to bind to the VEGF receptor VEGFR, thereby inhibiting its binding to VEGF in a concentration-dependent manner. Competitive ELISA was performed as follows: human Flt1-Fc chimeric protein (0.5 μg / mL) was reacted with different concentrations of anti-Flt1-EBPA12-CoreX / Xm at room temperature for 2 hours and then added to a VEGF-coated 96-well plate to determine the amount of human Flt1-Fc chimeric protein bound to VEGF. Compared with the control group, the anti-Flt1 peptide-treated groups (0.013 μg / mL, 0.13 μg / mL) did not inhibit the binding of VEGF to VEGFR1, but the anti-Flt1-EBPA12-CoreX-treated group significantly inhibited (by about 50%) the binding of VEGF to VEGFR1 at concentrations from 0.001 μg / mL to 1 μg / mL, and there was no inhibitory effect at a high concentration of 10 μg / mL. In the anti-Flt1-EBPA12-CoreXm-treated group, the binding of VEGF to VEGFR1 was significantly inhibited at concentrations from 0.1 μg / mL to 10 μg / mL. Anti-Flt1-EBPA12-CoreX and anti-Flt1-EBPA12-CoreXm showed similar efficacy, but anti-Flt1-EBPA12-CoreXm was more effective when used at high concentrations.

[0058] The anti-angiogenic effect of the anti-Flt1 peptide fusion protein was identified by a reduction in the area of choroidal neovascularization (CNV) in a laser-induced CNV mouse model. Under anesthesia, choroidal neovascular lesions were induced in the retinas of C57BL / 6 mice (n = 3 - 4) using a 532 nm wavelength diode laser, and on the day of laser injury, 2 μL each of the anti-Flt1 peptide and anti-Flt1-EBPA12-CoreX were injected into the vitreous of one eye as a single dose. Aflibercept (Eylea) was used as a positive control. As a result, as shown in the CNV lesion images and area graphs in Figure 4A, the group treated with the anti-Flt1 peptide did not show an inhibitory effect on the CNV area, but intravitreal administration of different concentrations of anti-Flt1-EBPA12-CoreX significantly inhibited the CNV area. In addition, it also showed a therapeutic effect similar to that of the existing therapy, aflibercept.

[0059] The activity of the TSP14 peptide fusion protein as a thrombopoietin receptor (TPOR) agonist was identified by an increase in the proliferation of BaF3 / MPL cells overexpressing TPOR (Figure 4B). BaF3 / MPL cells were seeded at 5 × 10 3 cells per well in a 96-well plate and then cultured for 48 hours in the presence or absence of different concentrations of the TPOR agonist to measure the luminescence signal on a Victor 3 1420 multilabel counter (Perkin Elmer) using the Cell Titer-Glo luminescent cell viability assay kit (Promega). Recombinant human thrombopoietin (rhTPO) was used as a positive control. Both TSP14 and rhTPO stimulated the growth of BaF3 / MPL cells in a concentration-dependent manner. As a result of calculating the relative cell proliferation ability based on 100 ng / mL rhTPO, which showed the maximum proliferation ability, the EC50 values of TSP14 and rhTPO were 211.5 ng / mL and 1.7 ng / mL, respectively.

[0060] The immune activation effect of IFN-β1-EBPA12-CoreX showed an increase in the expression of MHC I. A549 cells were seeded at 0.3 × 10 5Cells were seeded at 2×10 per well in 6-well plates and treated with the protein for 48 hours. The cells were collected and bound to human HLA antibodies, and the expression level of MHC I was detected using flow cytometry. Human IFN-β1 synthetic protein was used as a positive control. As shown in the left histogram of Fig. 4C, compared with the MHC I expression (green) in untreated A549 cells, the expression increased when treated with IFN-β1-EBPA12-CoreX and human IFN-β1 synthetic protein, and the two proteins showed similar expression levels. The gene expression regulatory effect of IFN-β1-EBPA12-CoreXm was identified by the increase in TRAIL gene through treatment with the recombinant protein. A549 cells were seeded at 2×10 5 per well in 6-well plates, and RNA was extracted after treatment with the protein for 24 and 48 hours. cDNA was synthesized from the extracted RNA, and primers specific for the TRAIL gene (F: 5'-ACCAACGAGCTGAAGCAGAT-3', R: 5'-ACGGAGTTGCCACTTGACTT-3') and the GAPDH gene (F: 5'-GGAGCGAGATCCCTCCAAAAT-3', R:5'-GGCTGTTGTCATACTTCTCATGG-3') were used. PCR premix (Bioneer) was used to denature at 94°C for 5 minutes, and then 20 PCR cycles were performed under the temperature conditions of 94°C for 30 seconds, 57°C for 30 seconds, and 72°C for 60 seconds. The PCR products were electrophoresed on a 1.5% agarose gel. The electrophoresis results on the right side of Fig. 4C showed that the expression of the TRAIL gene increased with the prolongation of the treatment time of IFN-β1-EBPA12-CoreXm and human IFN-β1 synthetic protein, and a greater increase was shown by IFN-β1-EBPA12-CoreXm.

[0061] The enzymatic activity of the ADA2 enzyme fusion protein was identified by the degree of activity in decomposing adenosine into inosine. After the reaction of adenosine with the fusion protein, the amount of inosine formed by decomposition was quantified using an adenosine deaminase activity assay kit (BioVisin, 328-100). The results are shown in the bar graph of Fig. 4D. When the ADA2 enzyme fusion protein was added, the amount of inosine increased significantly. On the other hand, there was no significant difference in the amount of inosine whether the 7.16.4 antibody fragment was present or not.

[0062] The antigen recognition ability of the ADA2 enzyme fusion protein fused with the 7.16.4 antibody fragment was detected by the concentration-dependent increase in binding to 4T1-neu cells overexpressing the neu antigen. At concentrations from 0.05 μg / mL to 12.5 μg / mL, in combination with 5×10 5After reacting 4T1-neu cells at 4 °C for 30 minutes, binding to the cells was detected by flow cytometry using anti-human IgG antibody (ebioscience). As a result, when comparing the antigen recognition ability of ADA2 fused with the 7.16.4 antibody fragment using an anti-neu antibody, it can be seen from the linear graph obtained from the fluorescence values in Figure 4D that the degree of binding to the neu antigen was similar to that of the control anti-neu antibody.

[0063] The antigen recognition ability of the 7.16.4 antibody fragment fusion protein was identified by a concentration-dependent increase in binding to 4T1-neu cells overexpressing the neu antigen (Figure 4E). 3×10 5 4T1-neu cells were reacted with 1 ng to 500 ng of the fusion protein at room temperature for 20 minutes, and binding to the cells was detected by flow cytometry using anti-human IgG antibody (ebioscience). Fluorescence values were calculated and plotted, and an anti-neu antibody was used as a control. As a result, when comparing the antigen recognition ability of the anti-neu antibody with that of the 7.16.4 antibody fragment fusion protein, it was found that at low concentrations, its degree of binding to the neu antigen was similar to that of the control anti-neu antibody, while the fluorescence value of the 7.16.4 antibody fragment fusion protein further increased at 50 ng and above. A similar pattern was observed between CoreX and CoreXC, which differ in the amino acid residues of the Fc.

[0064] The antigen recognition ability of the α-PD-L1 antibody fragment fusion protein was identified by a concentration-dependent increase in binding to breast cancer cell line MDA-MB-231 cells (Figure 4F). After reacting with 5×10 5 MDA-MB-231 cells at a concentration of 0.01 μg / mL to 2.5 μg / mL at 4 °C for 30 minutes, binding to the cells was detected by flow cytometry using anti-human IgG antibody (ebioscience). Fluorescence values were calculated and plotted, and compared with atezolizumab used clinically. The IC50 values were 0.34 and 0.2 respectively, indicating that the IC50 value of the α-PD-L1 antibody fragment fusion protein was approximately 0.1 lower.

[0065] The antigen recognition ability of the Erbitux antibody fragment fusion protein was identified by binding to lung cancer cell line A549 cells (Figure 4G). After reacting with 2×10 5 A549 cells at room temperature for 1 hour, binding to the cells was detected by flow cytometry using anti-mouse IgG antibody (ebioscience). As a result, when A549 cells were treated with the Erbitux antibody fragment fusion protein, the fluorescence value increased compared with the control group, indicating that it binds by recognizing EGFR on A549 cells.

[0066] <Example 6> Identification of the Formation of Nanostructures of Recombinant Proteins

[0067] The reactivity of the EBPn-Fc protein (control of the fusion protein) to salt was identified by the increase in its turbidity with the increase in salt concentration. After mixing the protein with salts at concentrations ranging from 0.25 M to 3.0 M, it was vortexed for 1 minute at room temperature, and the absorbance was measured at a wavelength of 300 nm using a micro-spectrophotometer (nano-drop) ( Figure 5 A). As a result, EBPA12 showed a concentration-dependent increase in absorbance values at high salt concentrations above 2 M, while EBPG12 showed a first increase and then decrease in absorbance values at 2.5 M. This indicates that both types of EBPn-Fc are reactive to salt, but the reactivity varies depending on the type. Then, using the fusion protein, 10 μM of the protein was mixed with salts at 1.5 M to 2.0 M, the temperature was raised from 25 °C to 50 °C at 1.0 °C increments each time, and the change in protein particle size was measured by dynamic light scattering (DLS) using a nanoparticle size analyzer (Zetasizer). As a result, even though they have the same EBPn-Fc, the change pattern of the temperature-dependent protein size varies depending on the type of fusion protein, and when a certain temperature is reached at high salt concentrations, the size of the protein increases and then tends to be uniform. This indicates that the EBPn-Fc fusion protein can form nanostructures while maintaining the inherent properties of EBPn that are reactive to salt and temperature ( Figure 5 B).

[0068] Although specific parts of the present disclosure have been described in detail above, it is clear to those skilled in the art that this specific description is only a preferred exemplary embodiment, but the scope of the present disclosure is not limited thereto. In other words, the substantial scope of the present disclosure is defined by the appended claims and their equivalents.

Claims

1. An animal cell expression cassette, the animal cell expression cassette comprising: a gene encoding a hydrophilic elastin-like polypeptide (EBPn), a gene encoding an antibody constant region (Fc), and a gene encoding a first protein for expression.

2. The animal cell expression cassette according to claim 1, wherein, The animal cell expression cassette further comprises a gene encoding a second protein for expression.

3. The animal cell expression cassette according to claim 1 or 2, wherein The hydrophilic elastin-like polypeptide (EBPn) consists of [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 repeats of SEQ ID NO: 1 or SEQ ID NO: 2, and X is selected from any amino acid other than proline.

4. The animal cell expression cassette according to claim 3, wherein, The hydrophilic elastin-like polypeptide (EBPn) is represented by any one of the amino acid sequences selected from SEQ ID NO: 3 to SEQ ID NO:

8.

5. The animal cell expression cassette according to claim 1 or 2, wherein The antibody constant region (Fc) comprises the hinge region and CH2-CH3 domains of IgG.

6. The animal cell expression cassette according to claim 5, wherein, The cysteine residue in the hinge region of the antibody constant region (Fc) is additionally replaced by a serine residue.

7. The animal cell expression cassette according to claim 5, wherein, The antibody constant region (Fc) is represented by any one of the amino acid sequences selected from SEQ ID NO: 9 to SEQ ID NO:

12.

8. The animal cell expression cassette according to claim 1 or 2, wherein The protein for expression is a peptide, a protein or an antibody fragment.

9. The animal cell expression cassette according to claim 8, wherein, The protein for expression is anti-Flt1 shown by the amino acid sequence of SEQ ID NO: 13, TSP14 shown by the amino acid sequence of SEQ ID NO: 14, IFN-β shown by the amino acid sequence of SEQ ID NO: 15, ADA2 shown by the amino acid sequence of SEQ ID NO: 16, 7.16.4 scFv shown by the amino acid sequence of SEQ ID NO: 17, α-PD-L1 scFv shown by the amino acid sequence of SEQ ID NO: 18, or Erbitux scFv shown by the amino acid sequence of SEQ ID NO:

19.

10. A recombinant expression vector, the recombinant expression vector comprising the animal cell expression cassette according to claim 1 or 2.

11. A cell line transfected with the recombinant expression vector according to claim 10.

12. A method for producing a protein for expression, the method comprising: culturing the cell line according to claim 11 and recovering the culture solution; and isolating and purifying the protein for expression fused with EBPn and Fc from the recovered culture solution.

13. The method according to claim 12, wherein, The purification is carried out by affinity chromatography using a protein A column.

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