Long-acting insulin gene construct and use thereof

Abstract

The present invention relates to a long-acting insulin gene construct and a use thereof and to: a gene construct designed for long-acting insulin production based on recombinant gene technology; and recombinant long-acting insulin production through the high expression of the construct in plants. The gene construct comprising immunoglobulin Fc region mutant genes of the present invention forms a heterodimer by the simultaneous expression of mutually different Fc region mutant genes, and thus forms the mature form of insulin, thereby enabling the sustained expression of insulin. Accordingly, the present invention has the advantage of enabling the production of recombinant long-acting insulin through the high expression of such long-acting insulin gene constructs in plants.

Description

Long-acting insulin gene construct and uses thereof

[0001] The present invention relates to a long-acting insulin gene construct and its use, and relates to a gene construct designed for long-acting insulin production based on recombinant gene technology and to the production of recombinant long-acting insulin through high expression of the construct in plants.

[0002] Diabetes is a metabolic disorder characterized by impaired ability to regulate blood sugar levels. The number of people with diabetes is rapidly increasing worldwide, particularly in low-income countries and the Asia-Pacific region (Chatterjee et al., 2017; Cheng et al., 2019). The number of people with diabetes is projected to increase to approximately 643 million by 2030 and 783 million by 2045 (Pouya et al., 2019). However, current manufacturing technologies are projected to fail to meet this demand due to limitations in production capacity, high costs, and production volume.

[0003] There are two main types of diabetes. Type I (DM) is a completely insulin-dependent diabetes caused by the destruction of pancreatic β-cells, the insulin-producing cells. The other is Type II (DM), a non-insulin-dependent diabetes caused by inadequate insulin secretion or insulin resistance. Type II diabetes is caused by several key factors, including high calorie intake due to physical inactivity, obesity, multi-organ dysfunction, and especially coronary heart disease and chronic kidney disease (American Diabetes Association, 2018; Chatterjee et al., 2017; Forbes and Cooper, 2013). Both Type I and Type II diabetes patients require regular insulin therapy.

[0004] In 1922, the first commercial insulin was produced and used, obtained from the pancreas of cows and pigs, but the pig-derived insulin exhibited immunogenic effects. Thirty years later, in the 1970s, scientists resolved this problem when they were able to obtain the amino acid sequence of recombinant human insulin. In the early 1980s, this knowledge was used to apply recombinant genetic techniques to insulin production for the first time, and in 1982, insulin was finally produced in Escherichia coli using recombinant DNA technology. However, as the demand for insulin continued to grow, technologies for producing insulin in other recombinant protein expression systems, such as the yeast Pichia pasteris or Sacharomyces cerevisiae, were developed in addition to E. coli (Baeshen et al., 2014; Polez et al., 2016).

[0005] Insulin is originally made as a single polypeptide, preproinsulin, but the ER targeting signal sequence is first cleaved in the ER to create proinsulin, which is known to have three intramolecular disulfide bonds. Then, in the Golgi apparatus, Zn2+ and Ca2+ bind to this hexameric proinsulin to form a complex, and this complexed proinsulin is cleaved into A, B, and C chains by a processing protease, convertase PC1 or convertase PC2. Among these chains, the A and B chains are connected through C-C disulfide bonds, and the remaining arginine and lysine residues at the C-terminus of the B chain are removed by carboxypeptidase H to create mature insulin. On the other hand, technology has been developed to produce recombinant insulin using E. coli or yeast, and it has been announced that active insulin can be produced in plants as well. Various design methods have been used. Genentech uses a method to separate and purify the A and B chains and then induce cross-linking between them in vitro. Eli Lilly uses a single polypeptide to produce proinsulin and then removes the C-peptide from it in vitro. At this time, trypsin is used to remove the C-peptide, and the missing threonine residue in the B chain is added through an in vitro reaction to produce mature insulin. On the other hand, when producing insulin using yeast, the small C-chain is removed from proinsulin using an enzyme called Kex, and a threonine residue is then added to produce mature insulin.The reaction that adds this threonine residue requires a compound called H-thre(tBu)-OtBu, which is added during the trypsin cleavage reaction to add to the B29 lysine residue. The resulting pre-insulin-thre(tBu)-OBu is then purified and the tertiary butyl group is removed from the threonine residue using trifluoroacetic acid to produce mature insulin. Therefore, a fairly complex process is required.

[0006] Recently, various types of insulin have become commercially available. These include insulin analogs with altered amino acid sequences, such as lispro (short-acting insulin with an onset of action of 1–2 h) (Chance et al., 1999; Glidden et al., 2018) and aspart (short-acting insulin with an onset of action of 2–5 h) (Brange et al., 1988, 1990). Furthermore, to avoid multiple daily injections, Aventis Pharmaceuticals developed a long-acting insulin called glargine. Glargine is produced by replacing asparagine, the C-terminal amino acid of the A chain of insulin, with glycine (N / G). It also adds an arginine residue, RR, to the C-terminus of the B chain. Attempts have also been made to produce long-acting insulin by fusing the Fc of an IgG antibody to insulin to dramatically increase its in vivo half-life. One of them was developed by fusing a minimized form of insulin C chain to an Fc chain, targeting canine insulin, and fused two insulins to the N-terminus of a mature Fc in a homodimeric form. Another study developed a two-chain heterodimeric Fc (tcI-Fc) fusion insulin (Faust et al., 2020). In this case, the A and B chains of insulin were fused to the N-terminus of each chain of the heterodimeric Fc via a linker, so that one mature insulin was linked to the heterodimeric Fc. In the case of this tcI-Fc, the insulin efficacy was sustained for 2 days, but it was found that blood glucose was no longer lowered on the third day.

[0007] Accordingly, in the present invention, to produce insulin for sustained expression, a long-acting insulin recombinant protein was designed using an IgG Fc fusion domain, known to extend the in vivo half-life of recombinant proteins, and a recombinant gene encoding this recombinant protein was designed. To induce high expression of the designed recombinant gene in plants and to mass-produce the recombinant protein at low cost, a high-expression vector for plant cells was constructed.

[0008] An object of the present invention is to provide a gene construct for sustained expression of an insulin protein, wherein the following (i) and (ii) are simultaneously expressed to form a heterodimer: (i) a first insulin analogue comprising a first immunoglobulin Fc region variant gene; and (ii) a second insulin analogue comprising a second immunoglobulin Fc region variant gene.

[0009] Another object of the present invention is to provide a recombinant expression vector for sustained expression of insulin protein, comprising the above genetic construct.

[0010] Another object of the present invention is to provide a transformant, a cell line or a whole plant transformed with the recombinant expression vector for sustained expression of an insulin protein.

[0011] Another object of the present invention is to provide a plant cell into which the transformant has been introduced.

[0012] Another object of the present invention is to provide a plant into which the transformant has been introduced.

[0013] Another object of the present invention is to provide a pharmaceutical composition for preventing or treating diabetes, comprising a genetic construct for sustained expression of the insulin protein.

[0014] The present invention provides a genetic construct for sustained expression of an insulin protein, wherein the following (i) and (ii) are simultaneously expressed to form a heterodimer: (i) a first insulin analogue comprising a first immunoglobulin Fc region variant gene; and (ii) a second insulin analogue comprising a second immunoglobulin Fc region variant gene.

[0015] In addition, the present invention provides a recombinant expression vector for sustained expression of insulin protein, comprising the above genetic construct.

[0016] In addition, the present invention provides a transformant, cell line or whole plant for sustained expression of insulin protein, transformed with the recombinant expression vector.

[0017] Additionally, the present invention provides a plant cell into which the transformant has been introduced.

[0018] In addition, the present invention provides a plant into which the transformant has been introduced.

[0019] In addition, the present invention provides a pharmaceutical composition for preventing or treating diabetes, comprising a genetic construct for sustained expression of the insulin protein.

[0020] The genetic construct comprising the immunoglobulin Fc region variant gene of the present invention enables sustained expression of insulin, as different Fc region variant genes are simultaneously expressed to form heterodimers, thereby forming the mature form of insulin. Therefore, there is an advantage in that high expression of these sustained-acting insulin gene constructs in plants enables the production of recombinant sustained-acting insulin.

[0021] Figure 1 is a schematic diagram of insulin A chain and B chain constructs fused to the knob and hole chains of Fc in a heterodimeric form.

[0022] Figure 2 is a schematic diagram of a construct according to the substitution of the Fc chains of Fc:hInA-2 and Fc:hInB-2 with electrostatic heterodimer type Fc (eFcA and eFcB).

[0023] Figure 3 shows the expression of recombinant proteins after introducing FchInA-1 and FchInB-1, constructs of the A chain and B chain of the long-acting insulin recombinant gene, separately (A) or simultaneously (B) into N. benthaimana leaf tissues via Agrobacterium-mediated infiltration, as confirmed by Western blot analysis and CBB staining (NT in B, unmodified sample; NR, unmodified sample; R, denatured sample).

[0024] Figure 4 shows the results of CBB staining (A) and Western blot analysis (B) of the purified Fc:hInA / Fc:hInB heterodimer complex of human insulin from the total soluble protein extract of leaf tissue of Nicotiana benthamiana using protein A beads (M, protein size standard marker; NT, unmodified sample; T, total soluble protein extract; FT, flow-through fraction; W1, W2, W3; washed-off fractions; E, eluted fraction; B, protein remaining without being released from beads).

[0025] Figure 5 shows the results of Western blot analysis (A) and CBB staining (B) confirming that mature insulin is released from Fc heterodimers after treatment with enterokinase after isolating Fc:hInA / Fc:hInB heterodimer complexes from total available protein extracts (M, standard marker of protein molecular weight; C, no EK; T, + EK; RE, unmodified sample).

[0026] Figure 6 shows the activity of insulin produced by treating a plant-produced Fc:hInA / Fc:hInB heterodimer complex with EK, and comparing it with the activity of Lantus, a positive control.

[0027] Figure 7 is a schematic diagram showing the construction of Fc:bInA and Fc:bInB by fusing bovine insulin A chain and B chain to the knob chain and hole chain of the heterodimer Fc, respectively.

[0028] Figure 8 shows the expression of Fc:bInA and Fc:bInB, fusion protein genes of bovine insulin, introduced into N. benthamiana individually or simultaneously, and confirmed through Western blot analysis (A, B) and CBB staining (B) (NT, unmodified sample; NR, unmodified sample; R, denatured sample).

[0029] Figure 9 shows the results of CBB staining (A) and Western blot analysis (B) of the Fc:bInA / Fc:bInB heterodimer complex purified using protein A beads (M, protein size standard marker; NT, unmodified sample; T, total soluble protein extract; FT, flow-through fraction; W1, W2, W3; wash-off fractions; E, elution fraction; B, protein remaining without being released from the beads).

[0030] Figure 10 shows the separation of Fc and mature bovine insulin when the purified Fc:bInA / Fc:bInB heterodimer complex was treated with EK, as confirmed by Western blot analysis (A) and CBB staining (B) (arrow in A, position of Fc monomer part; M, standard marker for protein molecular weight; C1, no EK at -20 o C; C2, no EK at 25 o C; T, EK-treated sample; RE, unmodified sample. Red arrows, mature bovine insulin) (red arrows in B, mature insulin).

[0031] Figure 11 is a schematic diagram of constructing Fc:cInA and Fc:cInB by fusing canine insulin A and B chains to the knob and hole chains of heterodimeric Fc, respectively, to produce long-acting canine insulin.

[0032] Figure 12 shows the results of confirming expression through Western blot analysis after simultaneously introducing Fc:cInA and Fc:cInB constructs into N. benthamiana.

[0033] Figure 13 shows the expression patterns in N. benthamiana of constructs in which the human insulin A chain is linked to the eFcA chain and the human insulin B chain is linked to the eFcB chain.

[0034] Figure 14 shows the results of SDS-PAGE analysis of Fc:cInA and Fc:cInB heterotypic fusion protein complexes purified using protein A beads, followed by CBB staining of the gel (M, protein size standard marker; WT, unmodified sample; T, total soluble protein extract; FT, flow-through fraction; W1, W2; wash-off fractions; E, elution fraction).

[0035] Figure 15 shows the results of purely isolating Fc:hInA / Fc:hInB and Fc:bInA / Fc:bInB produced in N. benthamiana and comparing their insulin activity with that of the positive control, Lantus.

[0036] Figure 16 shows the activity over time after treatment of cells with Fc-fused bovine insulin compared to that of the control, Lantus. p-IR and p-AKT were confirmed on the same membrane using a Phos-tag acrylamide gel by mixing the two Abs in a 1:1 ratio.

[0037] Figure 17 shows the results of examining the degree to which blood sugar levels are lowered over time after intraperitoneal injection using Fc:hInA / Fc:hInB produced in N. benthamiana.

[0038]

[0039] Hereinafter, the present invention will be described in detail.

[0040]

[0041] The present invention provides a genetic construct for sustained expression of an insulin protein, wherein the following (i) and (ii) are simultaneously expressed to form a heterodimer: (i) a first insulin, a wild type thereof, or an analog thereof, comprising a first immunoglobulin Fc region variant gene; and (ii) a second insulin, a wild type thereof, or an analog thereof, comprising a second immunoglobulin Fc region variant gene.

[0042] In the present invention, the first immunoglobulin Fc region and the second immunoglobulin Fc region may be selected from the group consisting of CH1, CH2, CH3, and CH4 domains, and preferably, the first immunoglobulin Fc region and the second immunoglobulin Fc region may be a CH3 domain.

[0043] In the present invention, the first immunoglobulin Fc region and the second immunoglobulin Fc region may be Fc regions derived from IgG, IgA, IgD, IgE, or IgM. Preferably, the first immunoglobulin Fc region and the second immunoglobulin Fc region may be Fc regions derived from IgM.

[0044] In the present invention, the first immunoglobulin Fc region variant gene and the second immunoglobulin Fc region variant gene may have a CSWHLCEQ sequence introduced into the C-terminus of the Fc region.

[0045] In the present invention, the first immunoglobulin Fc region variant may be a CH3 domain mutant, and the second immunoglobulin Fc region variant may be a CH3 domain mutant.

[0046] In the present invention, the formation of the heterodimer may be for forming a mature form of insulin.

[0047] In the present invention, the formation of the heterodimer may be due to electrostatic interaction.

[0048] In the present invention, the first insulin, its wild type, or its analog may sequentially comprise a linker, an enterokinase-encoding gene, and a first insulin-encoding gene at the C-terminus of the first immunoglobulin Fc region variant gene, and the second insulin, its wild type, or its analog may sequentially comprise a linker, an enterokinase-encoding gene, and a second insulin-encoding gene at the C-terminus of the second immunoglobulin Fc region variant gene.

[0049] In a specific embodiment of the present invention, the inventors introduced multiple mutations in the knob and hole portion of the human IgG-γ Fc region to form a heterodimer of Fc. First, to create an Fc having a knob, two mutations (383S / C, 395T / W) were introduced into the CH3 domain of Fc (Fcγ[CH3A]), and to create an Fc having a hole, four mutations (349Y / C, 366T / S, 368L / A, 407Y / A) were introduced into the CH3 domain (Fcγ[CH3B]). These Fcγ[CH3A] and Fcγ[CH3B] do not form homodimers of Fcγ[CH3A] or Fcγ[CH3B], but form heterodimers of Fcγ[CH3A] / Fcγ[CH3B].

[0050] In another specific embodiment of the present invention, a long-acting insulin analog was constructed by fusing insulin to the C-terminus of the Fc in the heterodimeric form. The insulin gene used in the present invention is human insulin, and the Glargine sequence, known as long-acting insulin, was used. One of the most important factors when creating such a fusion protein is whether the insulin A chain and the insulin B chain fused to the C-terminus of the Fc accurately form a mature dimer form and whether the formed insulin properly binds to the insulin receptor to induce insulin signaling. Therefore, when fusing the two functional domains, it is important to use a flexible linker so that the insulin portion can act independently from the Fc portion. For this purpose, a flexible linker, GGGGSGGGGS (LK2), was used. In addition, Fcγ[CH3A]:LK2:EK:hInA (Fc:hInA) and Fcγ[CH3B]:LK2:EK:hInB (Fc:hInB) were constructed by fusing the insulin A chain to the Fc knob and the insulin B chain to the Fc hole.

[0051] In another specific embodiment of the present invention, these Fc:hInA and Fc:hInB fusion proteins were fused to the leader sequence, which is an ER targeting signal of Arabidopsis BiP, to target them to the ER. The 5'-UTR sequence, which increases translation efficiency, was fused to the 5' of the fusion gene thus created, thereby completing the recombinant gene. These recombinant genes used the MacT promoter and the 3PR terminator. In addition, to further increase their expression level, the UBQ10 intron was included in the 5' UTR to complete the recombinant gene. In addition, to further increase the expression level, a DNA sequence linked to the 35S terminator, the terminator of PINII, a soybean protease inhibitor, and the matrix attachment sequence RB7 was used as a terminator (3PRt).

[0052] In the present invention, the insulin may be mammalian insulin. In one embodiment of the present invention, it has been confirmed that long-acting human, bovine, and canine insulin can be produced by producing a recombinant construct for insulin production.

[0053] According to a preferred embodiment of the present invention, the first immunoglobulin Fc region variant gene may include the base sequence of SEQ ID NO: 1, and the second immunoglobulin Fc region variant gene may include the base sequence of SEQ ID NO: 3.

[0054] According to a preferred embodiment of the present invention, the linker may include a base sequence of SEQ ID NO: 5, and the enterokinase coding gene may include a base sequence of SEQ ID NO: 7.

[0055] According to a preferred embodiment of the present invention, the first insulin coding gene may include any one base sequence selected from the group consisting of SEQ ID NO: 9, SEQ ID NO: 26, and SEQ ID NO: 36, and the second insulin coding gene may include any one base sequence selected from the group consisting of SEQ ID NO: 11, SEQ ID NO: 28, and SEQ ID NO: 38.

[0056] In the present invention, the base sequence and amino acid sequence can be interpreted to be extended to a sequence having 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more homology with the provided sequence. The "% of sequence homology" can be determined by comparing two optimally aligned sequences and the comparison region, and a part of the base sequence in the comparison region may include additions or deletions (i.e., gaps) compared to the reference sequence (which does not include additions or deletions) for the optimal alignment of the two sequences.

[0057] In addition, the present invention provides a recombinant expression vector for sustained expression of insulin protein, comprising the above genetic construct.

[0058] In the present invention, the recombinant expression vector refers to a plasmid, virus, or other medium known in the art into which the various types of genetic constructs described above can be inserted or introduced. The various types of genetic constructs according to the present invention can be operably linked. The operably linked genetic constructs can be contained within a single expression vector that also includes a selection marker and a replication origin.

[0059] In the present invention, the term "operably linked" may refer to a gene and an expression control sequence that are linked in a manner that enables gene expression when an appropriate molecule is bound to the expression control sequence. The term "expression control sequence" refers to a DNA sequence that controls the expression of an operably linked base sequence in a specific host cell. Such control sequences include a promoter for initiating transcription, an optional operator sequence for regulating transcription, a sequence encoding a suitable mRNA ribosome binding site, and a sequence that controls the termination of transcription and translation.

[0060] In the present invention, the recombinant expression vector may be at least one selected from the group consisting of all plasmids, phages, yeast plasmids, plant cell viruses, mammalian cell viruses, and other vectors known in the art into which a gene sequence or base sequence can be inserted or introduced, and in general, any plasmid and vector can be used without particular limitation as long as it can replicate and stabilize in a plant cell or plant host. Suitable vectors for introducing the various forms of gene constructs described above in the present invention include Ti plasmid and plant virus vectors. Examples of known vectors include pBI121, pHellsgate8, pROKII, pBI76, pET21, pSK(+), pLSAGPT, pUC, and pGEM. In addition, vectors that are expressed in plants, including the CMV35s promoter, may be used, for example, but are not limited to, the pCAMBIA series (pCAMBIA1200, 1201, 1281, 1291, 1300, 1301, 1302, 1303, 1304, 1380, 1381, 2200, 2201, 2300, 2301, 3200, 3201, 3300), pMDC32, and pC-TAPapYL436. Those skilled in the art can select a vector suitable for introducing the gene construct of the present invention, and in the present invention, any vector that can introduce the various types of gene constructs described above into plant cells can be used.

[0061] In addition, the present invention provides a transformant, cell line or whole plant for sustained expression of insulin protein, transformed with the recombinant expression vector.

[0062] According to a preferred embodiment of the present invention, the transformant may be Agrobacterium.

[0063] Additionally, the present invention provides a plant cell into which the transformant has been introduced.

[0064] In the present invention, the plant cell may be a callus, rice callus, cell line, carrot cell line or BY-2 cell.

[0065] In addition, the present invention provides a plant into which the transformant has been introduced.

[0066] According to a preferred embodiment of the present invention, the plant may be selected from food crops including rice, wheat, barley, corn, soybeans, potatoes, wheat, red beans, oats, and sorghum; vegetable crops including Arabidopsis, cabbage, radish, pepper, strawberry, tomato, watermelon, cucumber, cabbage, melon, pumpkin, green onion, onion, and carrot; specialty crops including ginseng, tobacco, cotton, sesame, sugarcane, sugar beet, perilla, peanut, and rapeseed; fruit trees including apple trees, pear trees, jujube trees, peaches, grapes, citrus fruits, persimmons, plums, apricots, lemons, and bananas; and floriculture including roses, carnations, chrysanthemums, lilies, sunflowers, cosmos, and tulips; and Nicotiana benthamiana and Nicotiana tabacum.

[0067] In addition, the present invention provides a pharmaceutical composition for preventing or treating diabetes, comprising a genetic construct for sustained expression of the insulin protein.

[0068] In the present invention, treatment means any action that improves or beneficially changes the symptoms of a cancer disease by administering the composition of the present invention.

[0069] In the present invention, prevention means any act of suppressing or delaying the possibility of developing a cancer disease or disease by administering the composition of the present invention.

[0070] The pharmaceutical composition may include a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable carrier" may refer to a carrier or diluent that does not irritate the organism and does not inhibit the biological activity or properties of the compound being injected. Here, "pharmaceutically acceptable" means that it does not inhibit the activity of the active ingredient and does not exhibit toxicity beyond what the intended subject can tolerate.

[0071] Any carrier that is commonly used in the art and is pharmaceutically acceptable may be used as the carrier for the present invention. Non-limiting examples of the carrier include saline solution, sterile water, Ringer's solution, buffered saline, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, ethanol, etc. These may be used alone or in combination of two or more. The pharmaceutical composition may be prepared as an oral formulation or a parenteral formulation according to the route of administration by a conventional method known in the art, including a pharmaceutically acceptable carrier in addition to the active ingredient.

[0072] The above pharmaceutical composition may be formulated and used in the form of oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc., external preparations, suppositories, or sterile injectable solutions, respectively, according to conventional methods. When formulating the above pharmaceutical composition, it may be prepared by adding diluents or excipients such as fillers, bulking agents, binders, wetting agents, disintegrants, or surfactants that are commonly used.

[0073] When the above pharmaceutical composition is manufactured into an oral dosage form, it can be manufactured into a dosage form such as powder, granules, tablets, pills, dragees, capsules, liquids, gels, syrups, suspensions, wafers, etc., using a suitable carrier according to a method known in the art. At this time, examples of suitable pharmaceutically acceptable carriers include sugars such as lactose, glucose, sucrose, dextrose, sorbitol, mannitol, and xylitol; starches such as corn starch, potato starch, and wheat starch; cellulosics such as cellulose, methylcellulose, ethylcellulose, sodium carboxymethylcellulose, and hydroxypropylmethylcellulose; polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, magnesium stearate, mineral oil, malt, gelatin, talc, polyols, and vegetable oils. In case of formulation, the formulation may include diluents and / or excipients such as fillers, bulking agents, binders, wetting agents, disintegrants, and surfactants, as needed.

[0074] When the above pharmaceutical composition is prepared as a parenteral dosage form, it can be formulated in the form of injections, transdermal administration, nasal inhalation, and suppositories using a suitable carrier according to a method known in the art. When formulated as an injection, suitable carriers include sterile water, ethanol, polyols such as glycerol or propylene glycol, or mixtures thereof, and preferably, Ringer's solution, phosphate buffered saline (PBS) containing triethanolamine, sterile water for injection, and isotonic solutions such as 5% dextrose can be used. When formulated as a transdermal dosage form, it can be formulated in the form of ointments, creams, lotions, gels, external solutions, pastes, liniments, aerosols, etc. In the case of nasal inhalation, it can be formulated in the form of an aerosol spray using a suitable propellant such as dichlorofluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, or carbon dioxide, and in the case of formulating it as a suppository, the base can be witepsol, tween 61, polyethylene glycol, cacao butter, laurin butter, polyoxyethylene sorbitan fatty acid ester, polyoxyethylene stearate, sorbitan fatty acid ester, etc.

[0075] The above pharmaceutical composition may be administered in a pharmaceutically effective amount, wherein the term "pharmaceutically effective amount" means an amount sufficient to treat or prevent a disease at a reasonable benefit / risk ratio applicable to medical treatment or prevention, and the effective dosage level may be determined according to the severity of the disease, the activity of the drug, the patient's age, weight, health, sex, the patient's sensitivity to the drug, the time of administration of the composition of the present invention used, the route of administration and the excretion rate, the treatment period, the drug used in combination with or concurrently with the composition of the present invention used, and other factors well known in the medical field. The pharmaceutical composition may be administered alone or in combination with a component known to exhibit a therapeutic effect on a known cancer disease. It is important to take all of the above factors into consideration and administer an amount that can achieve the maximum effect with the minimum amount without side effects.

[0076]

[0077] 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.

[0078]

[0079] Example 1. Recombinant construct for producing long-acting human insulin.

[0080]

[0081] 1.1. Construction of recombinant genes Fc:hInA and Fc:hInB for human insulin production

[0082]

[0083] In the present invention, a method for producing recombinant insulin using the Fc of immunoglobulin IgG in a plant system was completed. In the present invention, an Fc variant having the CSWHLCEQ sequence at the terminal of the Ig gamma-1 Fc was used (Borrok et al., 2015). It is known that Fc variants with this modified sequence have high binding affinity for the FcRN receptor under both acidic pH and pH 7.4 in vivo. Fc is a protein that originally forms a homodimer, and by introducing multiple mutations in the regions called the knob and hole of Fc, an Fc that forms a heterodimer can be created (Ha et al., 2016).

[0084] First, a recombinant fusion gene was designed to sequentially have a BamHI site at the N-terminus, a linker GGGGSGGGGS (LK2), an enterokinase site, a human insulin A chain, and an XhoI site at the C-terminus in addition to the sequence of an Fc mutant having a CSWHLCEQ sequence introduced at the Fc C-terminus of human Ig gamma-1 and a mutation of knob type (CH3A: 383S / C, 395T / W), and this was synthesized by a chemical method. For the insulin B chain, a Fc variant in which the CSWHLCEQ sequence was introduced at the C-terminus of the Fc of human Ig gamma-1 was introduced, and after introducing mutations of the Fc hole type (CH3B: 349Y / C, 366T / S, 368L / A, 407Y / A), a fusion gene sequentially having a BamHI site at the N-terminus and GGGGSGGGGS (LK2), an enterokinase site, human insulin B chain, and an XhoI site at the C-terminus was designed and chemically synthesized. The recombinant genes thus created were inserted into an expression vector containing the cDNA of the leader sequence of BiP that induces ER targeting, the 5' UTR translational enhancer sequence, and the Ubiquitin 10 intron in the 5' UTR, and Fcγ[CH3A]:LK2:EK:hInA (Fc:hInA-2) and Fcγ[CH3B]:LK2:EK:InB (Fc:hInB-2) using BamH1 and XhoI. Then, these recombinant genes were introduced into an expression vector having MacT and 3PR as a promoter and terminator, respectively, to complete the recombinant expression vector (Fig. 1).

[0085] Additionally, we constructed constructs linking hInA and InB chains using Fc fragments that form heterodimers (termed eFcA and eFcB) through electrostatic interactions in the Fc region. The electrostatic eFcA chain contains mutations K409D and K392D, and the eFcB chain contains mutations D399K and D356K, which form heterodimers through positive and negative charge interactions (K. Gunasekaran et al. Enhancing Antibody Fc Heterodimer Formation through Electrostatic Steering Effects. J. Biol Chem. 25. (2010)). The eFcA and eFcB chain genes were synthesized by geneuniversal, and the constructs were completed by replacing the Fc chains of Fcγ[CH3A]:LK2:EK:hInA (Fc:hInA-2) and Fcγ[CH3B]:LK2:EK:InB (Fc:hInB-2) using BamH1 and XmaI (Fig. 2).

[0086]

[0087]

[0088]

[0089] 1.2. Confirmation of single or simultaneous expression of human insulin A and B chain fusion genes Fc:hInA and Fc:hInB in plants

[0090]

[0091] The above-mentioned expression constructs, Fc:hInA and Fc:hInB, were introduced into N. benthamiana leaf tissue using Agrobacterium, and then transient expression was induced to confirm expression.

[0092] The expression vectors of Fc:hInA and Fc:hInB were first introduced into Agrobacterium (GV3101) to obtain transformed Agrobacterium colonies, which were then cultured to prepare an infiltration suspension. The Agrobacterium suspension containing these constructs was prepared by adjusting the concentration to OD600 of 0.75, and the Agrobacterium culture transformed with the gene silencing repressor p38 was cultured at OD 600 The ratio of Agrobacterium to 0.75 was adjusted to 0.75, and the two Agrobacterium suspensions were mixed in a 1:1 ratio to prepare an infiltration suspension. After infiltrating the leaves with these Agrobacterium suspensions, the leaves were harvested on the 3rd, 5th, and 7th days, and total soluble protein was extracted from the leaf tissue using 400 ml of extraction buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 1% protease inhibitor cocktail, 0.1% tween 20). 15 μg of total soluble protein was developed by SDS-PAGE and Western blot analysis was performed using HRP-conjugated anti-human IgG antibody as a secondary antibody. The same gel was stained with CBB to confirm the protein bands (Fig. 3A).

[0093] To simultaneously express Fc:hInA and Fc:hInB, Agrobacterium suspension transformed with Fc:hInA, Fc:hInB and p38 (OD 1.0) 600 ) were mixed in a ratio of 1:1:1 to prepare an infiltration suspension. These infiltration suspensions were infiltrated using a needle-less syringe. Leaf tissues were harvested on days 3, 5, and 7 after infiltration (dpi), and total soluble protein extracts were prepared. 15 μg of total protein was developed through SDS / PAGE, followed by Western blot analysis using a secondary antibody. The same gel was stained with CBB to identify the proteins (Fig. 3B).

[0094] Each construct was expressed at a high level, and the size of these fusion proteins appeared at approximately 35 kD position, with Fc:hInA being slightly larger than Fc:hInB. When the two genes were introduced simultaneously to induce coexpression, both genes were expressed at a high level. When these two genes were coexpressed, they appeared as a doublet at the 35 kD position. And it is important to know whether they actually form a complex when coexpressed. Even when the total soluble extract was boiled in the sample buffer of SDS-PAGE, some proteins appeared at a position of dimer size, and when the proteins were not boiled in the sample buffer, most of the monomers disappeared and these proteins appeared at a position larger than the dimer size. Based on this, it was interpreted that these two fusion proteins form a heterodimeric complex or a more complex complex.

[0095]

[0096] 1.3. Isolation and purification of Fc:hInA / Fc:hInB heterodimers from plant total soluble protein extracts

[0097]

[0098] Leaf tissues (40 g) co-expressing recombinant fusion protein genes Fc:hInA and Fc:hInB were harvested on the 6th day, and total soluble protein extracts were prepared using 400 ml of extraction buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 1% protease inhibitor cocktail, 0.1% tween 20), which were loaded onto a column made of protein A beads (1 ml). A total of 5 washings (washing buffer: 140 mM NaCl, 10 mM NaPO 4, Nonspecific proteins were removed by performing a lysis with 1.8 mM KH2PO4, 2.7 mM KCl, pH 7.3), and the Fc:hInA / Fc:hInB heterodimer bound to protein A beads was eluted using 200 mM glycine buffer (pH 2.3) and neutralized using 1 M Tris buffer (pH 8.5). All separation and purification processes were performed at 4 o C was performed. In order to analyze the separation and purification steps, total extract, flow-through fraction, washed-off fraction (1-3 times), and eluted fraction were developed by SDS-PAGE and confirmed through CBB staining. Western blot analysis was also performed on these samples (Fig. 4).

[0099]

[0100] 1.4. Confirmation of release of mature insulin from Fc:hInA / Fc:hInB heterodimers using enterokinase

[0101]

[0102] The purified Fc:hInA / Fc:hInB complex protein (4 μg) was prepared by adding (EK+) or not (EK-) EK (0.5 U) in EK cleavage buffer (50 mM NaCl, 20 mM Tris, pH 7.4, 2 mM CaCl2) and lysing them at 25 o After incubation for 12 h at C, the cells were denatured by heating in SDS-PAGE sample buffer, developed through SDS-PAGE, and subjected to Western blot analysis using secondary antibodies. As a negative control for EK treatment, -20 o A sample stored in C was loaded onto the same gel. (B) The result of the sample treated with EK being developed by SDS-PAGE in an undenatured state and confirmed through CBB staining (Fig. 5).

[0103] To confirm that the Fc:hInA / Fc:hInB fusion protein manufactured by the above method is active, first, Fc:hInA / Fc:hInB was isolated and purified, and then the protein was treated with enterokinase (EK). Western blot analysis was performed with a secondary antibody. The size of the monomeric FchInA and FchInB was reduced, confirming that the insulin moiety was released from Fc. Then, when Fc:hInA / Fc:hINB was treated with EK again, developed on SDS-PAGE without denaturation, and stained with Coomassie brilliant blue (CBB), a band of 6 kD in size clearly appeared below 10 kD, so it can be assumed that mature insulin was produced.

[0104]

[0105] 1.5. Confirmation of the activity of human insulin isolated from plant-produced Fc:hInA / Fc:hInB heterodimers

[0106]

[0107] It was confirmed that the insulin fused to the C-terminus of a long-acting insulin analog produced in plants, the Fc:In fusion protein, has a mature insulin form and that this part is active.

[0108]

[0109] After Fc:hInA / Fc:hInB was separated and purified, the C-terminal insulin portion was released from Fc by treating with enterokinase, and the activity was confirmed. As a positive control, Lantus was used to confirm the activity using hIR cells, which are cells expressing the human insulin receptor. In addition, using these insulins, the phosphorylation levels of IR and AKT, known as markers of insulin signaling, were confirmed through Western blot analysis.

[0110] hIR cells were cultured in growth medium containing DMEM supplemented with 10% fetal bovine serum (FBS) containing 100 U / mL penicillin and 100 mg / mL streptomycin at 37 o C (incubator containing 5% CO2 in air). After 2 days, the cells were confirmed to have grown well enough to cover 80% of the plate, the medium was removed, and the cells were washed with PBS. To detach the cells from the polystyrene-coated plate, a 0.5% trypsin-EDTA solution was used and 37 o C in a CO2 incubator for 1.30 min. After counting the cells using a hemocytometer, 2.5 x 10 were added to each well of a 6-well plate. 5 Put the cells in 37 o C was maintained in a 5% CO2 incubator for 24 hours. Afterwards, the media was removed and washed with PBS buffer. 37 o After culturing in FBS-free media for 4 h at C, the media was removed again and washed with PBS. 37 oCells were treated with commercially available Lantus and plant-produced insulin at concentrations of 1 nM, 10 nM, 25 nM, or 100 nM for 10 min at C, then the medium was removed, washed with PBS, and 100 μl (lysis buffer + sample buffer) was added and incubated overnight at 4 o C. Then, the cells were collected from the plate into a 1 ml tube, sonicated for 1.30 seconds, and centrifuged at 13,000 rpm for 15 minutes to obtain the supernatant, which was finally collected at 95 o Samples were obtained by heating at C for 10 minutes. Then, these samples were developed by SDS-PAGE and Western blot was performed using anti-pIR, anti-IR, anti-pAKT, anti-AKT, and anti-Actin antibodies.

[0111] As a result, when plant-produced Fc:hInA / Fc:hInB was treated with EK or Lantus, the amount of phosphorylated IR and AKT was the same in a concentration-dependent manner. Through this, it was confirmed that in the case of Fc:hInA, a recombinant human insulin A chain gene, and Fc:hInB, a recombinant B chain gene, heterodimers were efficiently formed, and thus, insulin A and B chains were formed into functional mature forms through dimerization (Fig. 6).

[0112] In addition, constructs linking human insulin A chain to eFcA chain and human insulin B chain to eFcB chain were expressed individually or together in N. benthamiana, and it was confirmed that target proteins were expressed and that they formed heterodimers (Fig. 13).

[0113]

[0114] Example 2. Recombinant construct for producing long-acting bovine insulin.

[0115]

[0116] 2.1. Construction of recombinant genes Fc:bInA and Fc:bInB for bovine insulin production

[0117]

[0118] Based on the above results, we attempted to produce long-acting bovine insulin as well as human insulin using the same method.

[0119] Fc:bInA and Fc:bInB were constructed by substituting the bovine insulin A chain and B chain in Fc:hInA and Fc:hInB, which are human insulin production constructs. PCR was used to substitute the human insulin A chain and B chain with the corresponding bovine chains in these constructs. For the A chain, PCR was performed using Fc:hInA as a template using the BamHI-FcRn:F primer and XhoI-bIn-A:R primer, and for the B chain, PCR was performed using Fc:hInB as a template using the BamHI-FcRn:F primer and XhoI-bIn-B:R primer. These PCR products were then digested with BamH1 and XhoI and ligated to Fc:hInA and Fc:hInB digested with the same restriction enzymes to complete the bovine insulin expression vector (Fig. 7).

[0120]

[0121] Name Base sequence / amino acid sequence Sequence number BamHI-FcRn:F Primer GCggatccctccgtccgtcttcctctt23 XhoI-bIn-A:R Primer cctcgagtcaGCCGcagtagttTtccaactggtacaaggagcagaCggaggCgcagcactg24 XhoI-bIn-B:R Primer aCCTCGAGtcaccggcgggccttgggcgtgtagaag25 Bovine insulin A chain GGCATCGTGGAGCAGTGCTGCGCCTCCGTCTGCTCCTTGTACCAGTTGGAAAACTACTGTGGTtga26 Bovine insulin A chain GIVEQCCASVCSLYQLENYCG27 Bovine insulin B Chain TTCGTCAACCAGCACCTGTGCGGCTCCCACCTGGTGGAGGCGCTGTACCTGGTGTGCGGAGAGCGCGGCTTCTTCTACACGCCCAAGGCCCGCCGGTGA28 Bovine insulin B chain FVNQHLCGSHLVEALYLVCGERGFFYTPKARR29

[0122]

[0123] 2.2. Confirmation of expression of bovine insulin A and B chain fusion genes Fc:bInA and Fc:bInB, individually or together, in plants

[0124]

[0125] And each of these expressions or simultaneous expressions was induced in N. benthamiana leaf tissue and their expression levels were confirmed.

[0126] Expression of each of the Fc:bInA and Fc:bInB expression vectors was confirmed in N. benthamiana leaf tissues using the method used in Example 1. Leaf tissues were harvested on days 3, 5, and 7 after infiltration (dpi) to prepare total soluble protein extracts. 15 μg of total protein was developed through SDS / PAGE, followed by Western blot analysis using HRP-conjugated anti-human IgG antibody as a secondary antibody. CBB-stained RbcL was used as a loading control. Expression of these proteins was confirmed after simultaneously introducing Fc:bInA and Fc:bInB into N. bethamiana leaf tissues. Total soluble protein was added to buffered sample buffer, heated (R), and not heated (NR), and then developed through SDS-PAGE. The resulting mixture was subjected to Western blot analysis using secondary antibodies and stained with CBB to identify the bands of these proteins.

[0127] As a result, it was confirmed that these small constructs were also well expressed in N. benthamiana. They were slightly larger in size than the human A chain and B chain fusion proteins, and both appeared slightly below the 45 kD position (Fig. 8).

[0128]

[0129] 2.3. Isolation and purification of Fc:bInA / Fc:bInB heterodimers from plant total water-soluble protein extracts

[0130]

[0131] Total soluble protein extracts were prepared from leaf tissues (6 dpi, 10 g) co-expressing Fc:bInA and Fc:bInB using 100 ml of extraction buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% protease inhibitor cocktail, 0.1% Tween 20), and purified using protein A beads (300 μl). The purified proteins were developed by SDS / PAGE and subjected to CBB staining (A) or Western blot analysis (B) using secondary antibodies (Fig. 9).

[0132]

[0133] 2.4. Confirmation of release of bovine mature insulin from Fc:bInA / Fc:bInB heterodimers using EK

[0134]

[0135] The purified Fc:bInA / Fc:bInB complex protein (4 μg) was treated with EK (0.3 U), then resolved on SDS / PAGE and subjected to Western blot analysis using a secondary antibody. The purified Fc:bInA / Fc:bInB complex sample treated with EK was resolved on SDS / PAGE under non-denaturing conditions and analyzed by CBB staining.

[0136] And when the Fc:bInA / Fc:bInB heterodimeric complex was purified using protein A beads and then treated with EK, the monomer size of these two fusion proteins was reduced, and when stained with CBB without denaturation, it was confirmed that it appeared similar to that of human insulin at a position of about 6 kD (Fig. 10). Based on these results, it was inferred that mature bovine insulin was formed.

[0137]

[0138] Example 3. Recombinant construct for producing long-acting canine insulin.

[0139]

[0140] 3.1. Construction of recombinant genes Fc:cInA and Fc:cInB for canine insulin production

[0141]

[0142] In order to additionally produce canine insulin in a long-acting form by utilizing the method of designing long-acting insulin analogs by fusing human and bovine insulin to Fc, canine insulin A chain and insulin B chain were fused to Fc[CH3A] and Fc[CH3B], respectively, using the same method, and Fc:cInA and Fc:cInB constructs were constructed and expressed simultaneously to confirm expression (Fig. 11).

[0143] Fc:cInA and Fc:cInB recombinant genes were constructed by replacing the canine insulin A and B chains in each of the human insulin production constructs Fc:hInA and Fc:hInB using overlap PCR. For the A chain replacement, PCR was performed using four types of primers: BamHI-FcRn:F primer, CIA-R primer, CIA-OF primer, and CIA-OR primer, and for the B chain replacement, four types of primers: BamHI-FcRn:F primer, CIB-OF primer, CIB-OR primer, and CIB-R primer. These PCR products were digested with BamH1 and XhoI and ligated into an expression vector digested with the same restriction enzymes.

[0144]

[0145] Name Base sequence / Amino acid sequence Sequence number CIA-R primer ggctcgagtcagccgcagtagttctccag 30 CIA-OF primer tccGacgacgatgacaagggcatcgtggagcagtgctgc 31 CIA-OR primer gcagcactgctccacgatgcccttgtcatcgtcgtCgga 32 CIB-OF primer tccGacgacgatgacaagTTCGTCAACCAGCACCTGTGC 33 CIB-OR primer GCACAGGTGCTGGTTGACGAActtgtcatcgtcgtCgga 34 CIB-R primer ggctcgagTCACCGGCGGGCCTTGGGCG 35 Insulin A chain ggcatcgtggagcagtgctgcacctccatctgctccctctaccagctggagaactactgcggctga 36 Insulin A chain GIVEQCCTSICSLYQLENYCG 37 Insulin B Chain TTCGTCAACCAGCACCTGTGCGGCTCCCACCTGGTGGAGGCGCTGTACCTGGTGTGCGGAGAGCGCGGCTTCTTCTACACGCCCAAGGCCCGCCGGTGA38 insulin B chainsFVNQHLCGSHLVEALYLVCGERGFFYTPKARR39

[0146]

[0147] 3.2. Confirmation of simultaneous expression of canine insulin A and B chain fusion genes Fc:cInA and Fc:cInB in plants

[0148]

[0149] After simultaneously introducing Fc:cInA and Fc:cInB into N. bethamiana leaf tissues, leaves were harvested on the 3rd and 5th days, and total soluble proteins were collected to confirm the expression of these proteins. Total soluble proteins were added to sample buffer, and either heated (reducing) or not (non-reducing) were developed using SDS-PAGE, and then Western blot analysis was performed using secondary antibodies to confirm the bands of these proteins.

[0150] We confirmed that these two recombinant fusion genes were highly expressed in N. benthamiana and that they also formed heterodimers. This confirmed that, as in the case of human insulin, they can be produced in N. benthamiana (Fig. 12).

[0151]

[0152] 3.3. Isolation and purification of Fc:cInA / Fc:cInB heterodimers from plant total soluble protein extracts

[0153]

[0154] Total soluble protein extracts were prepared from leaf tissues (6 dpi, 10 g) co-expressing Fc:cInA and Fc:cInB using 100 ml of extraction buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% protease inhibitor cocktail, 0.1% Tween 20), and purified using protein A beads (300 μl). The purified proteins were analyzed by SDS / PAGE, and the degree of protein purification was analyzed by CBB staining (Fig. 14).

[0155]

[0156] Example 4. Confirmation of insulin activity of recombinant constructs for long-acting insulin production.

[0157]

[0158] 4.1. Confirmation of insulin activity of plant-produced Fc:hInA / Fc:hInB and Fc:bInA / Fc:bInB heterodimers

[0159]

[0160] We aimed to confirm the activity of these long-acting insulin analogs designed by fusing them to human, bovine, and canine Fc in plants. To this end, the long-acting human and bovine forms, Fc:hInA / Fc:hInB and FcbInA / FcbInB, were isolated and purified, and their activity was confirmed in vitro using hR cells.

[0161] To confirm the activity of plant-produced insulin, long-acting insulin Lantus was used as a positive control at 1 nM, 50 nM, and 100 nM, and plant-produced Fc:hInA / Fc:hInB and Fc:bInA / Fc:bINB were treated to cells at 50 nM, 100 nM, 250 nM, and 500 nM. After developing cell extracts with SDS / PAGE, Western blotting was performed using anti-pIR, anti-pAKT, and anti-AKT antibodies.

[0162] As a result, nearly identical results were obtained for the two types of insulin. Compared to Lantus, used as a positive control, it was confirmed to exhibit approximately 10% the activity. Based on this, it can be assumed that canine insulin will exhibit similar trends (Figure 15).

[0163]

[0164] To analyze the effect of time on the long-acting effect produced in plants, bovine insulin (B-insulin) fused to Fc was treated in animal cells, and the activity was measured for up to 72 hours. HiRcb was seeded in 12-well plates. The media was DMEM + 10% FBS + 1% PS, and the cells were cultured for 24 hours to reach approximately 60% confluence. After 4 hours of culture in DMEM + 1% PS, the cells were replaced with media (DMEM + 5% FBS + 1% PS) containing 5 nM Lantus and 100 nM B-insulin, and the culture was continued, and the data were harvested hourly. The negative control was harvested immediately after 4 hours of starvation with PBS. All samples were quantified to 5 mg of total protein using Bradford, and 10 ml each was loaded. After unfolding this using SDS-PAGE, Western blot analysis was performed using anti-p-IR, anti-p-AKT, and anti-IR (Fig. 16).

[0165] As a result, it can be seen that the dimer-dimer bovine insulin of the present invention can maintain long-term activity for 24 hours, or up to 72 hours, compared to Lantus, which was used as a positive control.

[0166]

[0167] 4.2. Confirmation of the in vivo hypoglycemic effect of plant-produced Fc:hInA / Fc:hInB long-acting insulin

[0168]

[0169] To confirm the activity in vivo using Fc:hInA / Fc:hInB, which is designed as a long-acting form of human insulin, the effect of lowering blood sugar was measured by intraperitoneally injecting Fc:hInA / Fc:hInB into mice and checking the activity at different time points.

[0170] The experimental groups were composed of 1. Vehicle, PBS; 2. Lantus, 1 U / kg; 3. Fc:hInA / Fc:hInB, 1 U / kg), 4. Fc:hInA / Fc:hInB, 0.2 U / kg. To accurately measure the amount of plant insulin to be used in the experiment, the ALPCO mouse insulin ELISA kit, which has cross-activity to several types of insulin and insulin analogues, was used for measurement, and the same dose of insulin was administered. Specifically, 8-week-old C57BL / 6 mice (Hana Biotech, Korea) that maintained normal blood glucose levels were prepared per group, with 4 mice. The mice were allowed free access to food and water throughout the experimental period. The experimental substances were prepared at 0.2 ml per mouse and injected intraperitoneally. Blood samples were collected from the tail vein of mice at hourly intervals (0 min, 15 min, 30 min, 1 h, 3 h, 6 h, 24 h, 48 h, and 72 h after administration) and blood glucose levels were measured using an Accuchek (Roche, Switzerland) blood glucose meter.

[0171] As a result, Lantus (Lantus 1U = 0.0364 mg) administered at a dose of 1 U / kg maintained blood sugar levels at 50-60% of the initial blood sugar level for up to 1 hour after administration and then returned to levels higher than those of the vehicle-administered group, but in the Fc:hInA / Fc:hInB-administered group at the same insulin dose, blood sugar levels decreased somewhat more slowly, but lowered blood sugar levels were maintained for a much longer period of time (48 hours after administration) than the vehicle. A concentration-dependent hypoglycemic effect of plant insulin could also be observed. This confirmed that in the Fc-fused protein Fc:hInA / Fc:hInB of the present invention, activity was maintained for more than 48 hours and the activity almost disappeared at 72 hours, demonstrating that it exhibited significantly longer activity as a long-acting insulin analog compared to the positive control group (Fig. 17).

[0172]

[0173] 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 merely represent 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 gene construct for sustained expression of an insulin protein, wherein the following (i) and (ii) are simultaneously expressed to form a heterodimer: (i) a first insulin comprising a first immunoglobulin Fc region variant gene, a wild type thereof, or an analogue thereof; and (ii) a second insulin comprising a second immunoglobulin Fc region variant gene, a wild type thereof, or an analogue thereof.

2. In paragraph 1, A genetic construct wherein the first immunoglobulin Fc region and the second immunoglobulin Fc region are selected from the group consisting of hinge, CH1, CH2, CH3 and CH4 domains.

3. In paragraph 1, A genetic construct wherein the first immunoglobulin Fc region and the second immunoglobulin Fc region are Fc regions derived from IgG, IgA, IgD, IgE or IgM.

4. In paragraph 1, The above first immunoglobulin Fc region variant gene and the second immunoglobulin Fc region variant gene are gene constructs in which a CSWHLCEQ sequence is introduced into the C-terminus of the Fc region.

5. In paragraph 1, A genetic construct wherein the first immunoglobulin Fc region variant is a CH3 domain mutation and the second immunoglobulin Fc region variant is a CH3 domain mutation.

6. In paragraph 1, A genetic construct in which the above heterodimer formation is due to electrostatic interaction.

7. In paragraph 1, The above heterodimer formation is a genetic construct for forming the mature form of insulin.

8. In paragraph 1, A genetic construct wherein the first insulin, its wild type, or an analog thereof comprises a linker, an enterokinase-encoding gene, and a first insulin-encoding gene sequentially at the C-terminus of the first immunoglobulin Fc region variant gene, and the second insulin, its wild type, or an analog thereof comprises a linker, an enterokinase-encoding gene, and a second insulin-encoding gene sequentially at the C-terminus of the second immunoglobulin Fc region variant gene.

9. In paragraph 1, The above insulin is a genetic construct that is mammalian insulin.

10. In paragraph 1, The first immunoglobulin Fc region variant gene comprises the base sequence of sequence number 1, A gene construct comprising a second immunoglobulin Fc region variant gene having a base sequence of sequence number 3.

11. In paragraph 1, A genetic construct wherein the linker comprises a base sequence of SEQ ID NO: 5, and the enterokinase coding gene comprises a base sequence of SEQ ID NO:

7.

12. In paragraph 1, The first insulin coding gene comprises any one base sequence selected from the group consisting of SEQ ID NO: 9, SEQ ID NO: 26 and SEQ ID NO: 36, A genetic construct wherein the second insulin coding gene comprises any one base sequence selected from the group consisting of SEQ ID NO: 11, SEQ ID NO: 28, and SEQ ID NO:

38.

13. A genetic construct comprising any one of claims 1 to 12, Recombinant expression vector for sustained expression of insulin protein.

14. A transformant, cell line or whole plant transformed with the recombinant expression vector of Article 13 for sustained expression of insulin protein.

15. In paragraph 14, A transformant, wherein the above transformant is Agrobacterium.

16. A plant cell into which the transformant of clause 14 has been introduced.

17. In paragraph 16, The plant cell is a plant cell that is a callus, rice callus, a cell line, a carrot cell line or a BY-2 cell.

18. Plants into which the transformant of Article 14 has been introduced.

19. In paragraph 18, The plant is selected from food crops including rice, wheat, barley, corn, soybeans, potatoes, wheat, red beans, oats and sorghum; vegetable crops including Arabidopsis, cabbage, radish, pepper, strawberry, tomato, watermelon, cucumber, cabbage, melon, pumpkin, green onion, onion and carrot; specialty crops including ginseng, tobacco, cotton, sesame, sugarcane, sugar beet, perilla, peanut and rapeseed; fruit trees including apple trees, pear trees, jujube trees, peaches, grapes, citrus fruits, persimmons, plums, apricots, lemons and bananas; floriculture including roses, carnations, chrysanthemums, lilies, sunflowers, cosmos and tulips; and Nicotiana benthamiana and Nicotiana tabacum.

20. A pharmaceutical composition for preventing or treating diabetes, comprising a gene construct for sustained expression of an insulin protein according to any one of claims 1 to 12.

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