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WO2025135700A1 - Construction de gène d'insuline à action prolongée et son utilisation - Google Patents

Construction de gène d'insuline à action prolongée et son utilisation Download PDF

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Publication number
WO2025135700A1
WO2025135700A1 PCT/KR2024/020414 KR2024020414W WO2025135700A1 WO 2025135700 A1 WO2025135700 A1 WO 2025135700A1 KR 2024020414 W KR2024020414 W KR 2024020414W WO 2025135700 A1 WO2025135700 A1 WO 2025135700A1
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insulin
gene
immunoglobulin
paragraph
region
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Korean (ko)
Inventor
황인환
엔테사리메흐나즈
모하세파테매 차마니
김재윤
강주현
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Bioin Inc
POSTECH Research and Business Development Foundation
Bion Inc
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Bioin Inc
POSTECH Research and Business Development Foundation
Bion Inc
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C07K14/62—Insulins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
    • C12N15/8202—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation by biological means, e.g. cell mediated or natural vector
    • C12N15/8205—Agrobacterium mediated transformation
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
    • C12N15/8257—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits for the production of primary gene products, e.g. pharmaceutical products, interferon
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
    • C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
    • C12N9/6424—Serine endopeptidases (3.4.21)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
    • C12Y304/21—Serine endopeptidases (3.4.21)
    • C12Y304/21009—Enteropeptidase (3.4.21.9), i.e. enterokinase

Definitions

  • the present invention relates to a long-acting insulin gene construct and its use, and more particularly, 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.
  • Diabetes is a metabolic disorder characterized by impaired ability to regulate blood sugar levels in the bloodstream.
  • the number of people with diabetes is increasing rapidly worldwide, especially in low-income countries and the Asia- Pacific region (Chatterjee et al., 2017; Cheng et al., 2019).
  • the number of people with diabetes is expected to increase to about 643 million in 2030 and 783 million in 2045 (Pouya et al., 2019), but current manufacturing technologies are not expected to meet this demand due to limitations in production capacity, high costs, and production volume.
  • Type-I diabetes There are two main types of diabetes. One of them, Type-I (DM), is a completely insulin-dependent diabetes due to the destruction of the pancreatic ⁇ -cells, the insulin-producing cells. The other is Type-II (DM), which is a non-insulin-dependent diabetes due to inadequate insulin secretion or insulin resistance. Type II diabetes is caused by several major factors such as lack of physical activity, obesity, multi-organ dysfunction, and high calorie intake, 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 insulin regularly.
  • Insulin is originally made as a single polypeptide, preproinsulin, but the ER targeting signal sequence is first cut off 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 complex-type proinsulin is cut into A, B, and C chains by a processing protease, convertase PC1 or convertase PC2.
  • the A and B chains are connected through a C-C disulfide bond, and the arginine and lysine residues remaining at the C-terminus of the B chain are removed by carboxypeptidase H to create mature insulin.
  • 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; in the case of Genentech, a method was used to separate and purify the A and B chains and then induce cross-linking in vitro; and in the case of Lilly, a method was used to produce proinsulin using a single polypeptide and remove the C peptide from it in vitro.
  • trypsin is used to remove the C peptide, and the threonine residue that is missing from the B chain is additionally added through an in vitro reaction to produce mature insulin.
  • the small C chain is removed from proinsulin with trypsin using an enzyme called Kex, which is present in the yeast, and then a threonine residue is added to produce mature insulin.
  • Kex an enzyme responsible for producing insulin.
  • the reaction of adding this threonine residue requires a compound called H-thre(tBu)-OtBu, which is added during the trypsin cleavage reaction so that it is added to the B29 lysine residue.
  • insulin analogues with changed 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) that have been developed to increase the absorption time (Brange et al., 1988, 1990).
  • Aventis Pharmaceuticals developed a long-acting insulin called glargine to avoid multiple daily injections. Glargine was created by replacing asparagine, the C-terminal amino acid of the A chain of insulin, with glycine (N/G).
  • the A and B chains of insulin were fused to the N-terminus of each chain of the heterodimeric Fc through a linker, so that one mature insulin was linked to the heterodimeric Fc.
  • the insulin efficacy was sustained for 2 days, but it was found that blood sugar was no longer lowered on the 3rd day.
  • a long-acting insulin recombinant protein was designed using IgG Fc, which is known to extend the in vivo half-life of recombinant proteins, as a fusion domain, and a recombinant gene encoding the recombinant protein was designed.
  • IgG Fc IgG Fc
  • a high-expression vector for plant cells was constructed.
  • 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.
  • Another object of the present invention is to provide a recombinant expression vector for sustained expression of insulin protein, comprising the genetic construct.
  • 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 insulin protein.
  • Another object of the present invention is to provide a plant cell into which the transformant has been introduced.
  • Another object of the present invention is to provide a plant into which the transformant has been introduced.
  • 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.
  • 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.
  • the present invention provides a recombinant expression vector for sustained expression of insulin protein, comprising the genetic construct.
  • the present invention provides a transformant, cell line or whole plant for sustained expression of insulin protein, transformed with the recombinant expression vector.
  • the present invention provides a plant cell into which the transformant has been introduced.
  • the present invention provides a plant into which the transformant has been introduced.
  • the present invention provides a pharmaceutical composition for preventing or treating diabetes, comprising a genetic construct for sustained expression of the insulin protein.
  • the gene construct containing the immunoglobulin Fc region variant gene of the present invention enables sustained expression of insulin because different Fc region variant genes are simultaneously expressed to form a heterodimer, thereby forming a mature form of insulin. Accordingly, there is an advantage in that production of recombinant sustained insulin is possible through high expression of these sustained insulin gene constructs in plants.
  • Figure 2 is a schematic diagram of the constructs according to the substitution of the Fc chains of Fc:hInA-2 and Fc:hInB-2 with electrostatic heterodimer type Fc (eFcA and eFcB).
  • Figure 3 shows that the expression of recombinant proteins, FchInA-1 and FchInB-1, constructs of the A chain and B chain of the long-acting recombinant insulin gene, were introduced separately (A) or simultaneously (B) into N. benthaimana leaf tissues via Agrobacterium-mediated infiltration, and then the expression of the recombinant proteins was confirmed by Western blot analysis and CBB staining (NT, unmodified sample in B; NR, unmodified sample; R, denatured sample).
  • NT unmodified sample in B
  • NR unmodified sample
  • R denatured sample
  • 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 release to 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 release to beads
  • Figure 5 shows the results of Western blot analysis (A) and CBB staining (B) that mature insulin is released from Fc heterodimers after treatment with enterokinase following separation of 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).
  • A Western blot analysis
  • B CBB staining
  • Figure 6 shows a comparison analysis of the activity of insulin using plant-produced Fc:hInA/Fc:hInB heterodimer complexes treated with EK with the activity of Lantus, a positive control.
  • 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 heterodimer Fc, respectively.
  • 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).
  • 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; washed-off fractions; E, eluted fraction; B, protein remaining without release to the 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 release to the beads
  • Figure 10 shows that when the purified Fc:bInA/Fc:bInB heterodimer complex was treated with EK, Fc and mature bovine insulin were separated, as confirmed by Western blot analysis (A) and CBB staining (B).
  • A Western blot analysis
  • B CBB staining
  • Figure 11 is a schematic diagram of constructing Fc:cInA and Fc:cInB by fusing the canine insulin A chain and B chain to the knob and hole chains of heterodimeric Fc, respectively, to produce long-acting canine insulin.
  • Figure 12 shows the results of confirming expression through Western blot analysis after simultaneously introducing Fc:cInA and Fc:cInB constructs into N. benthamiana.
  • Figure 13 shows the expression patterns in N. benthamiana of constructs linking human insulin A chain to an eFcA chain and human insulin B chain to an eFcB chain.
  • 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).
  • 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
  • Figure 16 shows the activity over time after treatment of cells with Fc-fused bovine insulin compared to the control, Lantus.
  • p-IR and p-AKT were confirmed on the same membrane by mixing the two Abs in a 1:1 ratio using a Phos-tag acrylamide gel.
  • Figure 17 shows the results of examining the degree to which blood glucose levels are lowered over time after intraperitoneal injection using Fc:hInA/Fc:hInB produced in N. benthamiana.
  • the first immunoglobulin Fc region and the second immunoglobulin Fc region may be Fc regions derived from IgG, IgA, IgD, IgE or IgM.
  • the first immunoglobulin Fc region and the second immunoglobulin Fc region may be Fc regions derived from IgM.
  • the formation of the heterodimer may be for forming a mature form of insulin.
  • the inventors introduced multiple mutations in the knob and hole of the human IgG- ⁇ Fc portion for the formation of a heterodimer of Fc.
  • two mutations (383S/C, 395T/W) were introduced into the CH3 domain of Fc (Fc ⁇ [CH3A])
  • four mutations (349Y/C, 366T/S, 368L/A, 407Y/A) were introduced into the CH3 domain (Fc ⁇ [CH3B]).
  • 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, to complete the recombinant gene.
  • These recombinant genes used the MacT promoter and the 3PR terminator.
  • the recombinant gene was completed by including the UBQ10 intron in the 5' UTR.
  • a DNA sequence in which the 35S terminator, the terminator of PINII, a soybean protease inhibitor, and RB7, a matrix attachment sequence, are linked was used as a terminator (3PRt).
  • the insulin may be mammalian insulin.
  • long-acting human, bovine, and canine insulin can be produced by producing a recombinant construct for insulin production.
  • the first immunoglobulin Fc region variant gene may comprise the base sequence of SEQ ID NO: 1
  • the second immunoglobulin Fc region variant gene may comprise the base sequence of SEQ ID NO: 3.
  • the linker may comprise a base sequence of SEQ ID NO: 5
  • the enterokinase coding gene may comprise a base sequence of SEQ ID NO: 7.
  • 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 confirmed by comparing two optimally aligned sequences with 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.
  • the present invention provides a recombinant expression vector for sustained expression of insulin protein, comprising the genetic construct.
  • the recombinant expression vector refers to a plasmid, virus or other vector known in the art into which various types of gene constructs described above can be inserted or introduced.
  • Various types of gene constructs according to the present invention can be operably linked.
  • the operably linked gene constructs can be included in one expression vector that includes a selection marker and a replication origin together.
  • operably linked may refer to a gene and an expression control sequence that are linked in such a way that gene expression is enabled when an appropriate molecule is bound to the expression control sequence.
  • 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 inducing transcription, an optional operator sequence for controlling transcription, a sequence encoding a suitable mRNA ribosome binding site, and a sequence for controlling the termination of transcription and translation.
  • 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 a base sequence can be inserted or introduced, and in general, any plasmid and vector may be used without particular limitation as long as it can replicate and stabilize in a plant cell or a plant host.
  • Suitable vectors for introducing various forms of gene constructs described above in the present invention include Ti plasmid and plant virus vectors.
  • vectors examples include pBI121, pHellsgate8, pROKII, pBI76, pET21, pSK(+), pLSAGPT, pUC, and pGEM.
  • vectors that contain the CMV35s promoter and are expressed in plants 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.
  • pCAMBIA series pCAMBIA1200, 1201, 1281, 1291, 1300, 1301, 1302, 1303, 1304, 1380, 1381, 2200, 2201, 2300, 2301, 3200, 3201, 3300
  • pMDC32 and pC-TAPapYL436.
  • the present invention provides a transformant, cell line or whole plant for sustained expression of insulin protein, transformed with the recombinant expression vector.
  • the transformant may be Agrobacterium.
  • the present invention provides a plant cell into which the transformant has been introduced.
  • the plant cell may be callus, rice callus, a cell line, a carrot cell line, or BY-2 cells.
  • the present invention provides a plant into which the transformant has been introduced.
  • 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.
  • food crops including rice, wheat, barley, corn, soybeans, potatoes, wheat, red beans, oats and sorghum
  • vegetable crops including Arabid
  • the present invention provides a pharmaceutical composition for preventing or treating diabetes, comprising a genetic construct for sustained expression of the insulin protein.
  • treatment means any act of improving or beneficially changing the symptoms of a cancer disease by administering the composition of the present invention.
  • prevention means any act in which the possibility of onset of a cancer disease or disease is suppressed or delayed by administration of the composition of the present invention.
  • the pharmaceutical composition may include a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable carrier may refer to a carrier or diluent that does not stimulate a living organism and does not inhibit the biological activity and properties of the compound to be injected.
  • pharmaceutically acceptable means that it does not inhibit the activity of the active ingredient and does not have toxicity that is more than the target of application (prescription) can adapt to.
  • the type of the carrier that can be used in the present invention may be any carrier that is commonly used in the relevant technical field and is pharmaceutically acceptable.
  • 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 administration route by a conventional method known in the art, including a pharmaceutically acceptable carrier in addition to an active ingredient.
  • the above pharmaceutical composition can 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 injection solutions, respectively, according to conventional methods.
  • oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc., external preparations, suppositories, or sterile injection solutions, respectively, according to conventional methods.
  • diluents or excipients such as fillers, bulking agents, binders, wetting agents, disintegrating agents, or surfactants that are commonly used.
  • the above pharmaceutical composition when manufactured as an oral dosage form, it can be manufactured as a dosage form such as powder, granules, tablets, pills, dragees, capsules, liquids, gels, syrups, suspensions, wafers, etc., using a suitable carrier and a method known in the art.
  • suitable pharmaceutically acceptable carriers include sugars such as lactose, glucose, sucrose, dextrose, sorbitol, mannitol, xylitol, starches such as corn starch, potato starch, wheat starch, etc., celluloses 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.
  • the formulation may include diluents and/or excipients such as fillers, bulking agents, binders, wetting agents, disintegrants, and surfactants, as needed.
  • 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.
  • PBS phosphate buffered saline
  • transdermal dosage form 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.
  • nasal inhalers they 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 suppositories, the bases that can be used include witepsol, Tween 61, polyethylene glycols, cacao butter, laurin butter, polyoxyethylene sorbitan fatty acid esters, polyoxyethylene stearates, and sorbitan fatty acid esters.
  • the 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 factors including 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 or simultaneously 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 administer an amount that can obtain the maximum effect with the minimum amount without side effects by considering all of the above factors.
  • an Fc variant having a CSWHLCEQ sequence at the end of the Fc of Ig gamma-1 was used (Borrok et al., 2015). It is known that an Fc variant having such a modified sequence has high binding affinity to the FcRN receptor under both acidic pH and pH 7.4 conditions in vivo.
  • Fc is a protein that originally forms a homodimer, and by introducing multiple mutations into the parts called knob and hole of Fc, an Fc that forms a heterodimer can be created (Ha et al., 2016).
  • a recombinant fusion gene that sequentially had 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 into the Fc C-terminus of human Ig gamma-1 and a knob-type (CH3A: 383S/C, 395T/W) mutation, and synthesized it by a chemical method.
  • LK2 linker GGGGSGGGGS
  • a fusion gene that sequentially had a BamHI site at the N-terminus and GGGGSGGGGS (LK2), an enterokinase site, human insulin B chain, and XhoI site at the C-terminus of an Fc mutant of human Ig gamma-1, into which a CSWHLCEQ sequence was introduced at the C-terminus of the Fc, and then introduced mutations in the hole type (CH3B: 349Y/C, 366T/S, 368L/A, 407Y/A). This was then chemically synthesized.
  • the recombinant genes thus produced 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, using BamH1 and XhoI to construct Fc ⁇ [CH3A]:LK2:EK:hInA (Fc:hInA-2) and Fc ⁇ [CH3B]:LK2:EK:InB (Fc:hInB-2). 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).
  • eFcA and eFcB Fc fragments that form heterodimers
  • the electrostatic eFcA chain has mutations K409D and K392D
  • the eFcB chain has mutations D399K and D356K, which form heterodimers through interactions of positive and negative charges (K. Gunasekaran et al. Enhancing Antibody Fc Heterodimer Formation through Electrostatic Steering Effects. J. Biol Chem. 25. (2010)).
  • eFcA and eFcB chain genes were synthesized from geneuniversal, and the constructs were completed by replacing the Fc chains of Fc ⁇ [CH3A]:LK2:EK:hInA (Fc:hInA-2) and Fc ⁇ [CH3B]:LK2:EK:InB (Fc:hInB-2) using BamH1 and XmaI (Fig. 2).
  • Fc:hInA and Fc:hInB constructed above were introduced into N. benthamiana leaf tissue using Agrobacterium, and then transient expression was induced to confirm expression.
  • the expression vectors of Fc:hInA and Fc:hInB were first introduced into Agrobacterium (GV3101), transformed Agrobacterium colonies were secured, and these were cultured to prepare an infiltration suspension.
  • the Agrobacterium suspension containing these constructs was prepared by adjusting the concentration to an OD600 of 0.75, and an Agrobacterium culture transformed with gene silencing repressor p38 was adjusted to an OD600 of 0.75, and these two Agrobacterium suspensions were mixed in a 1:1 ratio to prepare an infiltration suspension.
  • the leaves were harvested on days 3, 5, and 7, and total soluble proteins were extracted from the leaf tissues using 400 ml of extraction buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 1% protease inhibitor cocktail, 0.1% Tween 20). 15 ⁇ g of total soluble proteins were resolved 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).
  • washing buffer 140 mM NaCl, 10 mM Na 2 PO 4 , 1.8 mM KH 2 PO 4 , 2.7 mM KCl, pH 7.3
  • washing buffer 140 mM NaCl, 10 mM Na 2 PO 4 , 1.8 mM KH 2 PO 4 , 2.7 mM KCl, pH 7.3
  • All separation and purification processes were performed at 4 o C.
  • total extract, flow-through fraction, washing-off fraction (1-3 times), and elution fraction were developed by SDS-PAGE, and confirmed through CBB staining.
  • Western blot analysis was performed on these samples (Fig. 4).
  • the purified Fc:hInA/Fc:hInB complex protein (4 ⁇ g) was incubated with (EK+) or without (EK-) EK (0.5 U) in EK cleavage buffer (50 mM NaCl, 20 mM Tris, pH 7.4, 2 mM CaCl2) as samples. These were incubated at 25 o C for 12 h, denatured by heating in SDS-PAGE sample buffer, developed by SDS-PAGE, and subjected to Western blot analysis using a secondary antibody. One sample stored at -20 o C as a negative control for EK treatment was loaded on the same gel. (B) The results confirmed by CBB staining after developing SDS-PAGE in an undenatured state of the EK-treated sample (Fig. 5).
  • the Fc:In fusion protein has a mature insulin form and that this part is active.
  • the C-terminal insulin portion was released from Fc by treating with enterokinase, and the activity was confirmed.
  • enterokinase enterokinase
  • Lantus was used to confirm the activity using hIR cells, which are cells expressing the human insulin receptor.
  • hIR cells which are cells expressing the human insulin receptor.
  • the phosphorylation levels of IR and AKT known as markers of insulin signaling, were confirmed through Western blot analysis.
  • hIR cells were cultured in growth medium containing DMEM medium supplemented with 10% fetal bovine serum (FBS) containing 100 U/mL penicillin and 100 mg/mL streptomycin at 37 o C (in an 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, 0.5% trypsin-EDTA solution was treated and incubated at 37 o C for 1.30 min in a CO2 incubator.
  • FBS fetal bovine serum
  • 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).
  • Fc:bInA and Fc:bInB were constructed by substituting the bovine insulin A chain and B chain in each of the human insulin production constructs, Fc:hInA and Fc:hInB. PCR was used to substitute the human insulin A chain and B chain in these constructs with the corresponding bovine chains. For the A chain, PCR was performed using Fc:hInA as a template with 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 with the BamHI-FcRn:F primer and XhoI-bIn-B:R primer.
  • canine insulin A chain and insulin B chain were fused to Fc[CH3A] and Fc[CH3B], respectively, using the same method to construct Fc:cInA and Fc:cInB constructs, which were then co-expressed to confirm expression (Fig. 11).
  • 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.
  • 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 replacement of the B chain, four types of primers: BamHI-FcRn:F primer, CIB-OF primer, CIB-OR primer, and CIB-R primer.
  • long-acting insulin Lantus was used as a positive control at 1 nM, 50 nM, and 100 nM, and in the case of plant-produced Fc:hInA/Fc:hInB and Fc:bInA/Fc:bINB, 50 nM, 100 nM, and 500 nM were treated to the cells.
  • Western blotting was performed using anti-pIR, anti-pAKT, and anti-AKT antibodies.
  • bovine Insulin fused to Fc was treated with animal cells, and the activity was measured for up to 72 hours. HiRcb was seeded in 12-well plates. At this time, DMEM + 10% FBS + 1% PS was used as the media and cultured for 24 hours until the cell confluent was about 60%. After culturing the cells in DMEM + 1% PS for 4 hours, the media (DMEM + 5% FBS + 1% PS) containing 5 nM Lantus and 100 nM B-insulin was changed and the culture was continued and the data were obtained over time. The negative control group was obtained immediately after starvation with PBS for 4 hours.
  • 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.
  • Fc:hInA/Fc:hInB which is designed as a long-acting form of human insulin
  • the effect of lowering blood glucose was measured by intraperitoneally injecting Fc:hInA/Fc:hInB into mice and checking the activity at different time points.
  • the experimental groups consisted 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.
  • 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.
  • 8-week-old C57BL/6 mice (Hana Biotech, Korea) that maintain normal blood sugar levels were prepared, 4 per group. The mice were allowed free access to and consumption of water and food throughout the experimental period.
  • the experimental substances were prepared at 0.2 ml per mouse and injected into the peritoneal cavity. A small amount of blood was collected from the tail vein of the mice at regular intervals (0 min, 15 min, 30 min, 1 h, 3 h, 6 h, 24 h, 48 h, and 72 h after administration), and the blood sugar level was measured using an Accuchek (Roche, Switzerland) blood glucose meter.

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Abstract

La présente invention concerne une construction de gène d'insuline à action prolongée et son utilisation et : une construction de gène conçue pour la production d'insuline à action prolongée basée sur la technologie du gène recombinant ; et la production d'insuline à action prolongée recombinante par l'expression élevée de la construction dans des plantes. La construction de gène comprenant des gènes mutants de région Fc d'immunoglobuline de la présente invention forme un hétérodimère par l'expression simultanée de gènes mutants de région Fc mutuellement différents, et forme ainsi la forme mature de l'insuline, ce qui permet l'expression prolongée d'insuline. En conséquence, la présente invention présente l'avantage de permettre la production d'insuline à action prolongée recombinante par l'expression élevée de telles constructions de gènes d'insuline à action prolongée dans des plantes.
PCT/KR2024/020414 2023-12-21 2024-12-16 Construction de gène d'insuline à action prolongée et son utilisation Pending WO2025135700A1 (fr)

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Citations (3)

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Publication number Priority date Publication date Assignee Title
KR910007612A (ko) * 1989-10-11 1991-05-30 구니오 아지미 호울 소오
KR20150138101A (ko) * 2014-05-29 2015-12-09 한미약품 주식회사 지속형 인슐린 아날로그 결합체 및 지속형 인슐린 분비 펩타이드 결합체를 포함하는 당뇨병 치료용 조성물
WO2023004368A1 (fr) * 2021-07-21 2023-01-26 Trutino Biosciences Inc. Polypeptides lieurs

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KR20240000403A (ko) 2022-06-22 2024-01-02 한국생명공학연구원 단백질 융합 기술을 이용하여 효모에서 인슐린을 대량 생산하는 방법

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KR910007612A (ko) * 1989-10-11 1991-05-30 구니오 아지미 호울 소오
KR20150138101A (ko) * 2014-05-29 2015-12-09 한미약품 주식회사 지속형 인슐린 아날로그 결합체 및 지속형 인슐린 분비 펩타이드 결합체를 포함하는 당뇨병 치료용 조성물
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HA, J.-H. ET AL.: "Immunoglobulin Fc heterodimer platform technology: from design to applications in therapeutic antibodies and proteins.", FRONTIERS IN IMMUNOLOGY., vol. 7, no. 394, 2016, pages 1 - 16, XP055377975, DOI: 10.3389/fimmu.2016.00394 *

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