WO2021230705A1 - Nouvelle protéine de fusion recombinante et utilisation associée - Google Patents

Nouvelle protéine de fusion recombinante et utilisation associée Download PDF

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WO2021230705A1
WO2021230705A1 PCT/KR2021/006069 KR2021006069W WO2021230705A1 WO 2021230705 A1 WO2021230705 A1 WO 2021230705A1 KR 2021006069 W KR2021006069 W KR 2021006069W WO 2021230705 A1 WO2021230705 A1 WO 2021230705A1
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fusion protein
protein
bispecific fusion
glp
amino acid
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PCT/KR2021/006069
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Korean (ko)
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최보경
진현탁
남은주
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주식회사 프로젠
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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
    • C07K14/605—Glucagons
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • 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
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/715—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons
    • 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
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/715—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons
    • C07K14/7155—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons for interleukins [IL]
    • 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
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/52—Constant or Fc region; Isotype
    • 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

Definitions

  • the present invention relates to a novel recombinant fusion protein and its use, and specifically, to a recombinant bispecific fusion protein comprising a glucagon-like peptide 2 analog, and use of the fusion protein for treatment and prevention of inflammatory bowel disease or short bowel syndrome is about
  • GLP-2 is a 33 aa peptide corresponding to amino acid positions 126-158 of proglucagon and is produced by enzymatic cleavage of proglucagon (Drucker, Endocrinol. 142(2): 521-527, 2001). GLP-2 binds to GLP-2R, a member of the gut-expressed G-protein-coupled receptor superfamily (Monroe et al ., Proc. Natl. Acad. Sci. USA 96: 1569-1573, 1999). GLP-2 has a half-life in serum of only 7 minutes due to cleavage by dipeptidyl peptidase-IV (DPP-IV) and clearance by the kidney (Brubaker, Endocrinol .
  • DPP-IV dipeptidyl peptidase-IV
  • Teduglutide has been approved for marketing by the US FDA in several rodent models and clinical trials in patients with short bowel syndrome (Kochar and Herfarth, Expert Opin. Drug Saf ., 17(7): 733-739, 2018). However, since the in vivo half-life of teduglutide is still short, it has a problem that it must be administered frequently.
  • GLP-2-Mimetibody, etc. fused with the Fc domain of IgG has been developed to further extend the half-life of Teduglutide (US7812121B2).
  • GLP-2 is known to form a dimer by non-covalent bonding again between the dimer upon fusion with the Fc domain, which has the disadvantage of reducing the yield of protein production.
  • IL-10 is also known as cytokine synthesis inhibitory factor (CSIF) as one of the cytokines.
  • IL-10 is an anti-inflammatory cytokine and synthesizes pro-inflammatory cytokines such as IFN- ⁇ , IL-2, IL-3, TNF ⁇ and GM-CSF produced by macrophages and Th1 T cells.
  • pro-inflammatory cytokines such as IFN- ⁇ , IL-2, IL-3, TNF ⁇ and GM-CSF produced by macrophages and Th1 T cells.
  • Th2 T cells and mast cells are known to activate certain immune cells. Because of this immunosuppressive activity, clinical trials for the use of recombinant IL-10 for the treatment of various autoimmune diseases are being attempted, but, contrary to expectations, recombinant human IL-10 is used in Crohn's disease (Schreiber et al ., Gastroenterol.
  • An object of the present invention is to solve various problems, including the above-mentioned problems, and to provide a novel recombinant fusion protein capable of more efficiently treating inflammatory bowel disease or short bowel syndrome, and uses thereof.
  • the protection scope of the present invention is not limited to the above purpose.
  • a recombinant bispecific fusion protein in which GLP-2 or an analog thereof is linked to an IL-10 protein.
  • a recombinant bispecific fusion protein in which GLP-2 or an analog thereof is linked to the N-terminus of the antibody Fc region and IL-10 protein is linked to the C-terminus of the antibody Fc.
  • a bispecific fusion protein dimer produced by dimerization of the recombinant bispecific fusion protein.
  • a recombinant vector comprising the polynucleotide.
  • composition for treating and preventing inflammatory bowel disease comprising the recombinant bispecific fusion protein or the bispecific fusion protein dimer as an active ingredient.
  • composition for treating and preventing short bowel syndrome comprising the recombinant bispecific fusion protein or the bispecific fusion protein dimer as an active ingredient.
  • a method of treatment comprising administering a therapeutically effective amount of the bispecific fusion protein or the bispecific fusion protein dimer to the subject suffering from inflammatory bowel disease.
  • a method of treating short bowel syndrome in an individual comprising administering a therapeutically effective amount of the bispecific fusion protein or the bispecific fusion protein dimer to an individual suffering from short bowel syndrome this is provided
  • the recombinant dual specificity fusion protein according to an embodiment of the present invention properly exerts the function of GLP-2 and has an increased in vivo half-life, and has immunomodulatory functions such as inhibition of inflammation, short bowel syndrome and various inflammatory bowel diseases. can be used as a treatment for
  • FIG. 1 is a schematic diagram schematically showing the structure of a dual specificity fusion protein according to an embodiment of the present invention.
  • FIG. 2 is an SDS-PAGE gel photograph (left) showing the production result of the fusion protein (PG-72) prepared according to an embodiment of the present invention and the image analysis program (Image J) for the left gel photograph of the protein. It is a figure (right) showing the result of measuring purity.
  • FIG. 3 is an SDS-PAGE gel photograph (left) showing the production result of the fusion protein (PG-73) prepared according to an embodiment of the present invention and the image analysis program (Image J) for the left gel photograph of the protein. It is a figure (right) showing the result of measuring purity.
  • FIGS. 2 and 3 are analysis diagrams showing the analysis results for the cleaved form of the fusion protein from the results of FIGS. 2 and 3 .
  • FIG. 5 is a SDS-PAGE gel photograph (left) and size exclusion high performance liquid chromatography (SEC-HPLC) results showing the production results of the dual specificity fusion protein (PG-210-5) prepared according to an embodiment of the present invention; (right) is shown.
  • FIG. 6 is a SDS-PAGE gel photograph (left) and size exclusion high performance liquid chromatography (SEC-HPLC) results showing the production results of the dual specificity fusion protein (PG-210-6) prepared according to an embodiment of the present invention; (right) is shown.
  • Figure 7a is a photograph showing the result of performing SDS-PAGE analysis after primary purification of the dual specificity fusion protein (PG-210-8) prepared according to an embodiment of the present invention by Protein A affinity chromatography;
  • Figure 7b is a chromatogram showing the result of analyzing the first purified fusion protein (PG-210-8) by HPLC analysis,
  • Figure 7c is the size exclusion of the first purified fusion protein (PG-210-8)
  • SEC-HPLC high performance liquid chromatography
  • FIG. 7d is a chromatogram showing the result of high performance liquid chromatography to confirm the purity of the secondary purified fusion protein.
  • FIG. 8 is a graph showing the results of analyzing the GLP-2 activity of the dual specificity fusion proteins (PG-210-5 and PG-210-6) according to an embodiment of the present invention by cAMP Hunter TM eXpress GPCR assay.
  • the graphs on the left and right show the results of two analyzes, respectively.
  • Figure 9a is a schematic diagram showing an experimental schedule for the STAT3 reporter analysis performed to investigate the IL-10 activity of the dual specificity fusion protein according to an embodiment of the present invention
  • Figure 9b is a dual according to an embodiment of the present invention It is a graph showing the results of STAT3 reporter analysis after treatment with specific fusion proteins (PG-210-6 and PG-210-8).
  • FIG. 10 is a graph showing the results of analyzing the immunosuppressive activity by IL-10 of the dual specificity fusion protein (PG-210-6) according to an embodiment of the present invention by measuring the degree of TNF- ⁇ secretion in macrophages; am.
  • FIG. 11a schematically shows an experimental schedule for examining the binding activity of the dual specificity fusion proteins (PG-210-6 and PG-210-7) to the GLP-2 receptor (GLP-2R) according to an embodiment of the present invention
  • FIG. 11b is the result of measuring the binding activity of the dual specificity fusion proteins (PG-210-6 and PG-210-7) to the GLP-2 receptor according to an embodiment of the present invention at various concentrations. It is a graph representing
  • FIG. 12 is a graph showing the results of pharmacokinetics analysis of dual specificity fusion proteins (PG-210-6 and PG-210-8) according to an embodiment of the present invention.
  • FIG. 13A is a schematic diagram schematically showing an experimental schedule for examining the distribution in vivo after administration of a dual specificity fusion protein (PG-210) according to an embodiment of the present invention
  • FIG. 13B is a dual specificity fusion protein according to an embodiment of the present invention. It is a graph showing the biodistribution analysis result after administration of the protein (PG-210).
  • Figure 15a is an animal for analyzing whether colitis symptoms are improved after administration of the dual specificity fusion protein (PG-210) according to an embodiment of the present invention to a disease model animal induced by colitis by administration of T reg CD4 + T cells. It is a schematic diagram schematically showing the experimental schedule, and FIG. 15b is a control group and a positive control group (anti-TNF ⁇ antibody administered group) and an embodiment of the present invention on the 20th (D20) and 27th (D27) of the experiment in the animal experiment. It is a graph showing the result of measuring the diarrhea index after administration of the dual specificity fusion protein (PG-210), and FIG. 15c is a graph recording the change in body weight over time of the experimental animal.
  • PG-210 dual specificity fusion protein
  • Figure 16a is a representative photograph for each group taken by excision of the colon after administration of the dual specificity fusion protein (PG-210) according to an embodiment of the present invention to an inflammatory bowel disease (IBD) model animal for 4 weeks
  • Figure 16b is It is a graph showing the result of measuring the length of the large intestine in each experimental group in the animal experiment
  • FIG. 16C is a representative photograph showing the result of performing histochemical analysis on a slice of colon tissue in each experimental group.
  • GLP-2 is a 33 amino acid long peptide produced by a special post-translational cleavage process of proglucagon like GLP-1. It is produced by enteroendocrine L cells of the small intestine and various neurons of the central nervous system. becomes this GLP-2 is secreted together with GLP-1 when food is ingested. In the case of GLP-2, when administered, it is known to improve small intestine growth and function, reduce bone destruction, and act as a neuroprotective agent.
  • GLP-2 analogue or "GLP-2 receptor agonist” refers to a protein that biologically performs the function of GLP-2 and the GLP-2 receptor. It refers to a protein capable of binding to and mediating downstream signaling.
  • Teduglutide is a GLP-2 analogue sold under the trade name of Gattex in the United States and Revestive in Europe as a treatment for short bowel syndrome.
  • Glepaglutide used in this document is a GLP-2 analogue with improved half-life, which was developed as a treatment for short bowel syndrome and is currently undergoing phase 3 clinical trials for short bowel syndrome.
  • GLP-2 analogue 10 is one of the GLP-2 analogues, and is an intramolecular lipid lipidated through the thiol groups of two substituted cysteines by replacing the 11th and 18th amino acids of GLP-2 with cysteines. It has a stabilized structure by linking with a crosslinking agent, and is characterized by adding 9 amino acids at the C-terminus of Exendin 4 to the C-terminus (Yang et al ., J. Med. Chem . 61: 3218-3223, 2018).
  • fusion protein refers to a recombinant protein in which two or more proteins or domains responsible for a specific function in the protein are linked so that each protein or domain takes on its original function.
  • antibody Fc region refers to a crystallized fragment among fragments generated when an antibody is cleaved with papain, and a cell surface receptor called Fc receptor and some of the complement system. interacts with proteins.
  • the antibody Fc region exhibits a homodimeric structure in which fragments comprising the second and third constant regions (CH2 and CH3) of the heavy chain are linked by intermolecular disulfide bonds at the hinge region.
  • the Fc region of IgG has a number of N-glycan attachment sites, which are known to play an important role in Fc receptor-mediated action, and IgG1, IgG2, IgG3, IgG4, and IgD may be used.
  • modified antibody Fc region refers to an Fc region peptide produced by a combination of parts of various subtypes of Ig Fc region, and Fc receptors and complement and It may exhibit a difference from the wild-type antibody Fc region in its binding ability, and may be representative of those disclosed in Korean Patent No. 897938.
  • linker peptide is an unstructured peptide used to prepare a fusion protein by linking two or more proteins or peptides having different biological activities.
  • the term “dual specificity fusion protein” refers to a protein in which polypeptides or domains having two or more different biological functions are linked directly or by a linker peptide to simultaneously exert two biological functions.
  • the term “dual-specificity fusion protein dimer” refers to a homologous or heterodimeric protein having a structure similar to that of an antibody generated by intermolecular interaction of the bispecific fusion protein. Such dimerization is known to be achieved by intermolecular disulfide bonds generated in the hinge region present in the antibody Fc region and intermolecular hydrophobic interactions by CH2 and CH3 domains.
  • NTIG refers to the modified Fc region described in WO2020231199A1.
  • the NTIG is a fusion of two or more isotypes of Ig Fc regions in a hybrid form, and while increasing affinity for FcRn, inhibits effector functions such as ADCC or CDC, thereby minimizing side effects and fused to a physiologically active protein ( It is a platform protein that can significantly increase the half-life of API).
  • a recombinant bispecific fusion protein in which GLP-2 or an analog thereof is linked to an IL-10 protein.
  • the GLP-2 or its analog may include an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 7.
  • the IL-10 protein may be a mutant and/or a monomeric mutant in which an immune activity enhancing action is suppressed.
  • the mutant in which the immune activity enhancing action is suppressed may be one in which isoleucine (I), the 87th amino acid of the wild-type human IL-10 protein, is substituted with alanine (A), and the monomeric mutant is a wild-type human IL-10 protein Length 6 to 12 aa, 7 to 11 aa, 8 to 10 aa between asparagine (N), the 134th amino acid, and lysine (K), the 135th amino acid of
  • the linker peptide of 9 a.a. may be inserted.
  • the IL-10 protein may include an amino acid sequence selected from the group consisting of SEQ ID NOs: 8 to 11.
  • a recombinant bispecific fusion protein in which GLP-2 or an analog thereof is linked to the N-terminus of the antibody Fc region and IL-10 protein is linked to the C-terminus of the antibody Fc.
  • the GLP-2 or an analog thereof may include an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 7.
  • the IL-10 protein may be a mutant and/or a monomeric mutant in which an immune activity enhancing action is suppressed.
  • the mutant in which the immune activity enhancing action is suppressed may be one in which isoleucine (I), which is the 87th amino acid of the wild-type human IL-10 protein, is substituted with alanine (A), and the monomeric mutant is a wild-type human IL-10 protein
  • a linker peptide having a length of 6 to 12 aa may be inserted between asparagine (N), the 134th amino acid, and lysine (K), the 135th amino acid.
  • the IL-10 protein may include an amino acid sequence selected from the group consisting of SEQ ID NOs: 8 to 11.
  • the monomeric variant IL-10 protein (IL-10Vm) is described in detail in Korean Patent Application Laid-Open No. 10-2021-0006302. This document is incorporated by reference in the above patent document.
  • the antibody Fc region may be an IgG1, IgG2, IgG3, IgG4, or IgD Fc region or a hybrid antibody Fc region in which Fc regions of two or more isotypes are mixed.
  • the antibody Fc region may include an amino acid sequence selected from the group consisting of SEQ ID NOs: 12 to 16, 63 and 64.
  • a linker peptide having a generally flexible structure may be inserted between two or more proteins or domains in the fusion protein according to an embodiment of the present invention.
  • the linker peptide is GGGGSGGGGSGGGGSEKEKEEQEERTHTCPPCP (SEQ ID NO: 17), RNTGRGGEEKKGSKEKEEQEERETKTPECP (SEQ ID NO: 18), GGGGSGGGGSGGGGSEPKSCDKTHTCPPCP (SEQ ID NO: 19), GSGGGSGTLVTVSSESKYGPPCPPCP (SEQ ID NO: 20), GGGGSGGGGSGGGGSEPKSSDKTHTCPPCP (SEQ ID NO: 21), EPKSSDKTHTCPPCP (SEQ ID NO: 22), EPKSCDKTHTCPPCP (SEQ ID NO: 23), GGGGSGGGGSGGGGSAKNTTAPATTRNTTRGGEEKKKEKEKEKEEQEERTHTCPPCP (SEQ ID NO: 24), A (EAAAK) 4 ALEA (EAAAK) 4 A
  • the recombinant bispecific fusion protein of the present invention may be in the form of a first linker peptide between the GLP-2 analogue and the antibody Fc region and a second linker peptide between the antibody Fc region and the IL-10 protein.
  • the first linker peptide may be any of the above linker peptides, but it may be preferable to use the linker peptide set forth in SEQ ID NO: 29, and the second linker peptide has a length of 1 to 20 aa. It was confirmed that there was no significant difference in the stability and activity of the protein no matter which one was used, and it was confirmed that there was no significant difference in protein production even when the 40 aa linker was applied.
  • the second linker has a length of 1 to 40 aa. Any peptide having a
  • a bispecific fusion protein dimer produced by dimerization of the recombinant bispecific fusion protein.
  • a recombinant vector comprising the polynucleotide.
  • the treatment of inflammatory bowel disease in an individual comprising administering a therapeutically effective amount of the bispecific fusion protein or the bispecific fusion protein dimer to the subject suffering from inflammatory bowel disease
  • a method is provided.
  • a bispecific fusion protein dimer produced by dimerization of the bispecific fusion protein.
  • a recombinant vector comprising the polynucleotide.
  • the polynucleotide may be included in the form of a gene construct operably linked to a regulatory sequence.
  • operably linked to means that a nucleic acid sequence of interest (eg, in an in vitro transcription/translation system or in a host cell) is regulated in such a way that its expression can be achieved. It means that it is connected to the sequence.
  • regulatory sequence is meant to include promoters, enhancers and other regulatory elements (eg, polyadenylation signals). Regulatory sequences include instructing that a target nucleic acid can be constitutively expressed in many host cells, instructing the expression of a target nucleic acid only in specific tissue cells (eg, tissue-specific regulatory sequences), and This includes directing expression to be induced by a specific signal (eg, an inducible regulatory sequence). It can be understood by those skilled in the art that the design of the expression vector may vary depending on factors such as the selection of the host cell to be transformed and the level of desired protein expression.
  • the expression vector of the present invention can be introduced into a host cell to express the fusion protein.
  • Regulatory sequences enabling expression in eukaryotic and prokaryotic cells are well known to those skilled in the art. As described above, they usually contain regulatory sequences responsible for initiation of transcription and, optionally, poly-A signals responsible for termination and stabilization of the transcript. Additional regulatory sequences may include, in addition to transcriptional regulators, translation enhancers and/or natively-combined or heterologous promoter regions.
  • Possible regulatory sequences enabling expression in, for example, mammalian host cells are the CMV-HSV thymidine kinase promoter, SV40, RSV-promoter (Rous sarcoma virus), human kidney element 1 ⁇ -promoter, glucocorticoid-inducible MMTV- promoters (Moloni mouse tumor virus), metallothionein-inducible or tetracycline-inducible promoters, or amplifying agents such as CMV amplifiers or SV40-amplifiers.
  • neurofilament-promoter For intraneuronal expression, it is contemplated that neurofilament-promoter, PGDF-promoter, NSE-promoter, PrP-promoter or thy-1-promoter may be used.
  • Such promoters are known in the art and are described in Charron, J. Biol. Chem. 270: 25739-25745, 1995.
  • a number of promoters have been disclosed, including the lac-promoter, the tac-promoter or the trp promoter.
  • the regulatory sequences include transcription termination signals such as SV40-poly-A site or TK-poly-A site downstream of the polynucleotide according to an embodiment of the present invention.
  • suitable expression vectors are known in the art, examples of which are Okayama-Berg cDNA expression vectors pcDV1 (Parmacia), pRc/CMV, pcDNA1, pcDNA3 (Invitrogen), pSPORT1 (GIBCO BRL), pGX-27 (Patent No.
  • the vector may further comprise a polynucleotide encoding a secretion signal.
  • the secretion signals are well known to those skilled in the art.
  • a leader sequence capable of guiding the recombinant protein to the cell compartment is combined with the coding sequence of the polynucleotide according to an embodiment of the present invention, preferably the translated protein or its It is a leader sequence capable of direct secretion of proteins into the periplasmic or extracellular medium.
  • the vector of the present invention can be prepared by, for example, standard recombinant DNA techniques, and standard recombinant DNA techniques include, for example, blunt-end and adherent ligation, treatment with restriction enzymes to provide appropriate ends, inappropriate In order to prevent binding, phosphate group removal by alkaline forstase treatment and enzymatic linkage by T4 DNA ligase are included.
  • the vector of the present invention is prepared by recombination of a DNA encoding a signal peptide obtained by chemical synthesis or genetic recombination technology, a recombinant fusion protein of the present invention, or a DNA encoding a fusion protein containing the same into a vector containing an appropriate regulatory sequence.
  • the vector containing the control sequence can be purchased or prepared commercially, and in an embodiment of the present invention, pBispecific backbone vector (Genexine, Inc., Korea) or pAD15 vector was used as a backbone vector.
  • the expression vector may further include a polynucleotide encoding a secretion signal sequence, wherein the secretion signal sequence induces secretion of a recombinant protein expressed in the cell out of the cell, and a tissue plasminogen activator (tPA) signal sequence; It may be an HSV gDs (herpes simplex virus glycoprotein Ds) signal sequence or a growth hormone signal sequence.
  • tPA tissue plasminogen activator
  • the expression vector according to an embodiment of the present invention may be an expression vector capable of expressing the protein in a host cell, and the expression vector is a plasmid vector, a viral vector, a cosmid vector, a phagemid vector, an artificial human chromosome. It is free to show any form, etc.
  • composition comprising the recombinant bispecific fusion protein or the bispecific fusion protein dimer as an active ingredient.
  • the composition may be a pharmaceutical composition for use in the treatment or prevention of short bowel syndrome or inflammatory bowel disease.
  • the composition may include a pharmaceutically acceptable carrier, and may further include a pharmaceutically acceptable adjuvant, excipient or diluent in addition to the carrier.
  • the term “pharmaceutically acceptable” refers to a composition that is physiologically acceptable and does not normally cause gastrointestinal disorders, allergic reactions such as dizziness or similar reactions when administered to humans.
  • examples of such carriers, excipients and diluents include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.
  • fillers, anti-agglomeration agents, lubricants, wetting agents, fragrances, emulsifiers and preservatives may be further included.
  • compositions according to an embodiment of the present invention may be formulated using a method known in the art to enable rapid, sustained or delayed release of the active ingredient when administered to a mammal.
  • Formulations include powders, granules, tablets, emulsions, syrups, aerosols, soft or hard gelatin capsules, sterile injectable solutions, and sterile powder forms.
  • composition according to an embodiment of the present invention may be administered by various routes, for example, oral, parenteral, for example, suppository, transdermal, intravenous, intraperitoneal, intramuscular, intralesional, nasal, intravertebral administration may be administered, and may also be administered using an implantable device for sustained release or continuous or repeated release.
  • the number of administration may be administered once a day or divided into several times within a desired range, and the administration period is not particularly limited.
  • composition according to an embodiment of the present invention may be formulated in a suitable form together with a commonly used pharmaceutically acceptable carrier.
  • Pharmaceutically acceptable carriers include, for example, carriers for parenteral administration such as water, suitable oils, saline, aqueous glucose and glycol, and may further include stabilizers and preservatives. Suitable stabilizers include antioxidants such as sodium hydrogen sulfite, sodium sulfite or ascorbic acid. Suitable preservatives are benzalkonium chloride, methyl- or propyl-paraben and chlorobutanol.
  • composition according to the present invention may contain a suspending agent, a solubilizing agent, a stabilizer, an isotonic agent, a preservative, an adsorption inhibitor, a surfactant, a diluent, an excipient, a pH adjuster, an analgesic agent, a buffer, Antioxidants and the like may be included as appropriate.
  • a suspending agent e.g., a solubilizing agent, a stabilizer, an isotonic agent, a preservative, an adsorption inhibitor, a surfactant, a diluent, an excipient, a pH adjuster, an analgesic agent, a buffer, Antioxidants and the like may be included as appropriate.
  • Pharmaceutically acceptable carriers and agents suitable for the present invention including those exemplified above, are described in detail in Remington's Pharmaceutical Sciences, latest edition.
  • the dosage of the composition to a patient will depend on many factors, including the patient's height, body surface area, age, the particular compound being administered, sex, time and route of administration, general health, and other drugs being administered concurrently.
  • the pharmaceutically active protein may be administered in an amount of 100 ng/body weight (kg) - 10 mg/body weight (kg), more preferably 1 to 500 ⁇ g/kg (body weight), and most Preferably, it may be administered at 5 to 50 ⁇ g/kg (body weight), and the dosage may be adjusted in consideration of the above factors.
  • a therapeutically effective amount of any one or more of the above bispecific fusion proteins or the bispecific fusion protein dimer comprising administering to an individual suffering from inflammatory bowel disease.
  • the inflammatory bowel disease may be Crohn's disease, ulcerative colitis, or Behcet's disease.
  • the present invention comprising administering to an individual suffering from short bowel syndrome a therapeutically effective amount of any one or more of the bispecific fusion proteins or the bispecific fusion protein dimer.
  • a method of treating the syndrome is provided.
  • short bowel syndrome is a malabsorption disease, which may be caused by surgical removal of a part of the small intestine or dysfunction of a segment of the intestine.
  • Crohn's disease inflammatory disorders of the digestive tract, volvulus, spontaneous twisting of the small intestine resulting in tissue death due to an interruption of blood supply, tumors of the small intestine, small intestine
  • Most disorders are caused by surgery related to wounds or traumas, necrotizing enterocolitis, bypass surgery to treat obesity, disease of the small intestine, and surgery to remove the damaged area. Also, some infants are born with short intestines.
  • malabsorption refers to a disease caused by partial or complete non-absorption of nutrients in the intestinal tract, particularly the small intestine.
  • causes are: First, as a primary congenital abnormality, a deficiency of disaccharide degrading enzymes such as lactose and sugar, a deficiency of digestive enzymes such as pancreas and small intestine, and transmission disorders of the small intestine mucosa such as glucose and vitamin B12. Second, as secondary causes In case of continuous malabsorption due to intestinal disease, etc.
  • inflammatory bowel disease used in this document is a disease in which abnormal chronic inflammation in the intestinal tract repeats improvement and recurrence. have. Diagnosis of inflammatory bowel disease is made by synthesizing clinical symptoms, endoscopy and histopathological findings, blood test findings, and radiology findings. The purpose of treatment is to control symptoms, prevent complications, and improve quality of life rather than cure.
  • the term "ulcerative colitis” is one of inflammatory bowel diseases along with Crohn's disease and Behcet's disease, and is a disease in which inflammation or ulceration of unknown cause occurs chronically in the large intestine. The exact cause is still unknown. In most cases, symptoms worsen and improve repeatedly, and it is an incurable disease that is difficult to cure with modern medicine.
  • Bechete's disease is an autoimmune disease in which the mucous membranes in the body, especially the mucous membranes of the gastrointestinal tract, are broken by autoimmunity. It is an intractable disease that can develop fatally when it appears in areas such as the nervous system, gastrointestinal system, eye system, and vascular system, and there is no fundamental treatment until now.
  • the term "therapeutically effective amount” means an amount sufficient to treat a disease with a reasonable benefit/risk ratio applicable to medical treatment, and the effective dose level depends on the type and severity of the subject; Age, sex, drug activity, sensitivity to drug, administration time, administration route and excretion rate, duration of treatment, factors including concomitant drugs and other factors well known in the medical field.
  • the therapeutically effective amount of the composition of the present invention may be 0.1 mg/kg to 1 g/kg, more preferably 1 mg/kg to 500 mg/kg, but the effective dosage may vary depending on the age, sex and condition of the patient. can be appropriately adjusted.
  • IL-10 as a candidate material for treating inflammatory bowel disease, but there were side effects such as anemia and thrombocytopenia due to systemic reactions. This was a case of oral administration of genetically modified Lactobacillus lactis lactic acid bacteria, which was genetically engineered to prevent adverse effects.
  • the present inventors have focused on the fact that the receptor of GLP-2, which is being studied as a candidate for the treatment of IBD, is most expressed in the intestine, and minimized the side effects that occur during systemic administration of IL-10, and
  • the present invention by designing a dual specificity fusion protein in which GLP-2 and IL-10 are linked to the N-terminus and C-terminus of NTIG, an immunoglobulin Fc domain mutant protein developed by the present inventors, respectively, in anticipation of a synergistic effect according to the present invention was completed.
  • the fusion protein of the present invention has a high specific binding degree to FcRn and a very low binding affinity to other Fc ⁇ receptors. Therefore, for short bowel syndrome or inflammatory bowel disease, it is expected to be more effective than conventional biopharmaceuticals for these diseases, and the possibility of side effects is expected to be low.
  • NTIG immunoglobulin Fc domain variant protein
  • Example code construct (SEQ ID NO:) GLP-2 (SEQ ID NO:) IL-10 (SEQ ID NO:) Total Protein (after Protein A) Purity (SE-HPLC)
  • GLP-2 SEQ ID NO:
  • IL-10 SEQ ID NO:
  • Total Protein After Protein A) Purity (SE-HPLC)
  • Comparative examples include GLP-2G-NTIG fusion protein (PG-22, Comparative Example 1), GLP-2V-NTIG fusion protein (PG-22V, Comparative Example 2) as a fusion protein to which IL-10 protein is not added, and addition NTIG-IL-10Vm (PG-010, Comparative Example 3), which is not a fusion protein, was used.
  • GLP-2V a 3-point mutation
  • Comparative Example 3 only IL-10Vm was linked to NTIG protein.
  • the fusion protein is a first linker peptide having the amino acid sequence shown in SEQ ID NO: 17 at the N-terminus of the NTIG protein shown in SEQ ID NO: 13 by using a GLP-2 (HA deleted) variant having the amino acid sequence shown in SEQ ID NO: 2 and a second linker peptide having an amino acid sequence set forth in SEQ ID NO: 29 to human wild-type IL-10 protein (IL-10wt) having an amino acid sequence set forth in SEQ ID NO: 8 at the C-terminus of the NTIG protein.
  • GLP-2 HA deleted
  • IL-10wt human wild-type IL-10 protein
  • GLP-2 sub-vector After synthesizing a polynucleotide encoding each component peptide of the fusion protein or amplifying it by PCR and preparing it, GLP-2 sub-vector, NTIG sub-vector, and IL-10 sub-vector and backbone vector After subcloning in (pBispecific vector, Genexine, Inc.), it was reacted in one tube to prepare a final vector construct.
  • Recombinant expression vector constructs capable of expressing the fusion proteins of Examples 2 to 4 were prepared in the same manner as above.
  • Example 2 the difference from the fusion protein of Example 1 was that Teduglutide (SEQ ID NO: 3) in which the second amino acid was substituted with glycine was used, not the form in which the two amino acids at the N-terminus of GLP-2 were removed.
  • Examples 3 and 4 are 3-point mutants (L17Q) in which the 16th amino acid, asparagine, is substituted with glycine (N16G) and the 17th amino acid, leucine, is substituted with glutamine to prevent self-dimer formation in the Teduglutide (L17Q) SEQ ID NO: 4) was used.
  • Example 5 Teduglutide of SEQ ID NO: 3 was used instead of the 3-point mutant of GLP-2 in Example 4, and in Example 6, KIH (Knobs-into-Holes) in which the GLP-2 3-point mutant was introduced into NTIG Designed to form a heterodimer by the structure, IL-10Vm was linked to a hybrid Fc region (NTIG Knob ) into which Knob was introduced by a linker peptide of SEQ ID NO: 29 with a length of 20 aa, and a hybrid Fc region into which a Hole was introduced.
  • NTIG Knob hybrid Fc region
  • NTIG Hole was designed to connect only GLP-2V and not IL-10Vm.
  • the linker lengths in the heterodimeric fusion protein of Example 6 are 1 aa (G), 5 aa (GGGGS, SEQ ID NO: 26) and 10 aa (GGGGSGGGGS, SEQ ID NO: 65), respectively. will be replaced with Among the vector constructs prepared as described above, the constructs of Examples 1 and 2 were transiently expressed using Thermo Fisher's ExpiCHO kit.
  • the supernatant obtained through the above culture was appropriately diluted with 4X LDS sample buffer and water for injection with fusion proteins purified through Protein A column and secondary column (referred to as 'PG-72' and 'PG-73', respectively). It was prepared so that it might become the final 3-10 microgram/20 microliters.
  • 4X LDS sample buffer, 10X reducing agent, and water for injection were appropriately diluted to make a final 3-10 ⁇ g/20 ⁇ L, and heated in a heating block at 70° C. for 10 minutes.
  • 20 ⁇ L of the prepared sample was loaded into each well of the gel fixed on the pre-installed electrophoresis equipment. For size markers, 3-5 ⁇ L/well were loaded. After setting the power supply to 120 V, 90 minutes, electrophoresis was performed. After the electrophoresis was completed, the gel was separated and stained using a staining solution and a de-staining solution, and the results were analyzed.
  • the present inventors replaced the linker used between GLP-2 and NTIG in PG-72 and PG-73 with the amino acid sequence shown in SEQ ID NOs: 20 and 21, respectively, and formed a dimer upon expression of the existing protein.
  • IL-10wt which is known to cause a decrease in protein production yield
  • IL-10Vm monomeric IL-10 variant designed by the present inventors
  • the protein production yield is significantly increased by changing the linker between the GLP-2 analog and the NTIG protein and changing the IL-10 protein type to the monomeric variant.
  • the obtained gene construct encoding the bispecific fusion protein was inserted into the pAD15 expression vector (WO2015/009052A), and then transfected into CHO DG44 (from Dr. Chasin, Columbia University, USA) cells, A cell line stably expressing the dual specificity fusion protein was constructed.
  • the present inventors prepared the gene construct encoding the fusion protein (PG-210-8) of Example 6 in the same manner as described above, and then transduced it into HEK-293F host cells, followed by transient gene expression was induced.
  • the culture supernatant was first purified by chromatography using a Protein A column as described above, followed by SDS PAGE analysis and HPLC analysis. did.
  • the present inventors further purified the first purified fusion protein using size exclusion HPLC (SEC-HPLC), and the purified protein was analyzed by SDS PAGE and HPLC analysis as described above ( FIGS. 7c and 7d ).
  • SEC-HPLC size exclusion HPLC
  • FIGS. 7c and 7d the recovery rate of the protein was 61.3% and the purity was 96.72%.
  • Example 1 In order to confirm whether the fusion protein purified in Example 1 normally exhibits GLP-2 activity, the present inventors analyzed the GLP-2 in vitro activity of the dual specificity fusion protein prepared in Example 1 with cAMP. was investigated by analysis. Specifically, in order to evaluate the degree of cAMP induction by GLP-2 specific reaction, cAMP Hunter TM eXpress GLP2R CHO-K1 GPCR Assay kit (DiscoverX, Cat# 95-0062E2CP2M) was used.
  • cAMP Hunter TM eXpress Assay cells were inoculated into 96-well plates and incubated for 16 hours, fusion proteins of Examples 3 and 4 (PG-210-5 and PG-210-6) and PG-22 used as Comparative Examples were 0.1 Each was treated at a concentration of 10 to 10 nM and incubated for 30 minutes. The biological activity (EC 50 ) of the drug was evaluated by measuring the luminescent signal 1 hour after adding the anti-cAMP antibody and the cAMP detection reagent.
  • PG210-6 among the fusion proteins prepared according to an embodiment of the present invention had almost similar activity to PG-22 and GLP-2, which is a comparative example, and PG-210- 5 was confirmed to exhibit half the activity compared to PG-210-6.
  • PG-210-5 is a protein to which the linker peptide used in the prior patent document (US7812121B2) is applied, and the productivity of PG-210-6 to which the (G 4 S) 3 + IgG1 hinge mixed linker peptide used in the present invention is applied It can be said that the excellent GLP-2 activity is a very encouraging phenomenon.
  • Example 3 (PG-210-5) 4 (PG-210-6) comparative example (PG-22) Primary EC 50 (nM) 7.44 2.46 2.84 Relative activity (%) 38.1% 115% 100% Secondary EC 50 (nM) 5.95 2.93 2.65 Relative activity (%) 44.5% 90% 100% Average activity (%) 41% 102.5% 100%
  • the STAT3 reporter gene which is a sub-signal of the IL-10 receptor and IL-10, was HEK-Blue IL-10 Cells (invivogen, Cat# hkb-IL10) using the transfected HEK-293 cell line were used.
  • HEK-Blue IL-10 Cells invivogen, Cat# hkb-IL10
  • fusion proteins PG210-6 and PG210-8
  • recombinant IL-10 protein as a positive control were treated at a concentration of 0.001 to 10 nM, respectively, and Incubated for 18 hours in a 37 °C incubator (Fig. 9a).
  • the reacted supernatant was transferred to a new 96-well plate, a detection reagent was added, and the luminescence signal was measured at 630 nm after 1 hour to evaluate the biological activity (EC 50 ) of the drug.
  • PG-210-6 showed about 1/3 activity compared to the control recombinant IL-10 protein, and in the case of PG-210-8, PG- It exhibited a 3.4-fold lower activity compared to 210-6.
  • the present inventors analyzed the in vitro immunosuppressive activity of the dual specificity fusion protein (PG-210-6) according to an embodiment of the present invention.
  • the PG-210-6 prepared in Example 4 was sequentially diluted to various concentrations (0 to 1,000 nM), and 50 ⁇ l of each of the Raw264.7 macrophages was treated in a 96-well plate. After 20 minutes, the final concentration of LPS (lipopolysaccharide) was 400 ng/ml, treated with 50 ⁇ l each, and incubated at 37° C. for 16 hours. The next day, after collecting the supernatant, TNF- ⁇ ELISA analysis was performed according to the protocol of the manufacturer (Biolegend, USA).
  • the dual specificity fusion protein according to an embodiment of the present invention inhibited the secretion of TNF- ⁇ in macrophages in a concentration-dependent manner. This means that even if the monomeric IL-10 variant protein (IL-10Vm) linked to the dual specificity fusion protein according to an embodiment of the present invention is linked to the C-terminus of the NTIG protein, it works properly.
  • IL-10Vm monomeric IL-10 variant protein
  • the present inventors evaluated the binding ability of the PG-210 fusion protein prepared in the above Example using FACS (flow cytometry) to determine whether it binds to the GLP-2 receptor (GLP-2R).
  • the present inventors confirmed the binding using the HEK-293 cell line expressing the GLP-2 receptor.
  • the HEK-293 cell line was cultured and then the fusion protein (PG210-6) according to an embodiment of the present invention.
  • the negative control PG-11 did not bind to the GLP-2 receptor at 1-1000 nM concentration, and PG-210 and PG-22 did not bind the GLP-2 receptor. It was confirmed that binding to a similar level to HEK-293 cells expressed on the cell membrane surface.
  • the present inventors performed pharmacokinetic analysis to confirm how stable the fusion protein of the present invention is when administered in vivo.
  • the fusion proteins (PG-210-6 and PG-210-8) of the present invention were injected intravenously at a dose of 1 mg/kg for 3 SD rats in each group for 0 min, 5 min. Serum was collected at 1 hour, 5 hours, 10 hours, 24 hours, 48 hours, 72 hours, 120 hours, and 168 hours, and the fusion protein was quantified using human IgG4 Fc ELISA.
  • the fusion protein of the present invention showed a modest decrease over time, indicating that the fusion protein is stably maintained in the body for a considerable period of time.
  • CF750 fluorophore was conjugated to PG-210-6 according to an embodiment of the present invention using the VivoBrite rapid antibody labeling kit (Biotium, Cat 92161).
  • the fluorescence/protein molar ratio (degree of labeling, DOL) was measured according to the expected ratio recommended according to the formula provided in the kit.
  • Normal C57BL/6 mice were intravenously injected with CF750 fluorescence-labeled PG-010 and PG-210-6 at a dose of 100 ⁇ g/100 ⁇ l ( FIG. 13a ).
  • the fluorescence signal emitted from the mouse was detected using a small animal bioimaging system. It was confirmed, and fluorescence signal analysis was performed using living imaging software.
  • PG-210-6 showed a lower fluorescence intensity in blood compared to PG-010, and it was confirmed that it was more distributed in the liver and intestines.
  • the present inventors collected a blood sample (200 ⁇ l) and measured the fluorescence intensity with an optical imaging device.
  • the present inventors performed the analysis on the 12th day after intravenous administration of PG-210 at 29.4 nmol/kg to 8-week-old male C57BL/6 mice to confirm the GLP-2 activity of PG-210-6 in vivo. did.
  • the fusion protein according to an embodiment of the present invention actually exhibits GLP-2 activity in vivo.
  • Figure 16a is a schematic diagram of this analysis. Using Rag1 ko mice, 5x10 5 naive CD4 + T reg cells were intravenously administered on day 0 to induce a colitis model, and at the same time various doses of PG-210-6 and a positive control, TNF ⁇ . The antibody was administered once a week for 3 weeks to confirm the efficacy of the administered drug (FIG. 15a).
  • the efficacy of the administered drug was evaluated by measuring a diarrhea score and a weight loss rate, which was evaluated according to the criteria described at the bottom of FIG. 15B .
  • FIG. 15b it was confirmed that the diarrhea index decreased in a concentration-dependent manner in the PG-210 administration group compared to the disease induction group and the anti-TNF ⁇ antibody administration group.
  • FIG. 15c the disease induction group and It was confirmed that there was less weight loss in a concentration-dependent manner in the PG-210-6 administration group compared to the anti-TNF ⁇ antibody administration group.
  • the present inventors evaluated the GLP-2 activity by examining the change in the length of the colon during in vivo administration of the fusion protein (PG-210-6) according to an embodiment of the present invention.
  • mice after administration of an anti-TNF antibody as a positive control and a fusion protein (PG-210) according to an embodiment of the present invention at various concentrations to mice (manufactured by Pohang University of Science and Technology) for IBD disease model once a week for 4 weeks , After sacrificing the mice, the length of the changed intestine for each experimental group was measured, and tissue staining was performed on the section of the intestinal tissue to observe the change in the thickness of the colon.
  • PG-210 fusion protein
  • the fusion protein according to an embodiment of the present invention was able to confirm a statistically significant increase in intestinal length in the 10 mg/kg body weight administration group, and as shown in FIG. 16c, PG -210-6 3 and 10 mg/kg body weight administration group showed a decrease in intestinal thickness.
  • the recombinant dual specificity fusion protein according to an embodiment of the present invention can be used in the pharmaceutical field, in particular, for the development of a therapeutic agent for short bowel syndrome or inflammatory bowel disease.

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Abstract

La présente invention concerne une nouvelle protéine de fusion recombinante qui est utilisée pour traiter et prévenir une entéropathie inflammatoire ou un syndrome de l'intestin grêle court et qui comprend le GLP-2 ou un analogue de celui-ci lié à la protéine IL-10 et, plus particulièrement, une protéine de fusion bispécifique recombinée, qui comprend le GLP-2 ou un analogue de celui-ci lié à l'extrémité N-terminale d'une région Fc d'anticorps et la protéine IL-10 liée à l'extrémité C-terminale de l'anticorps Fc, et une composition pharmaceutique comprenant celle-ci.
PCT/KR2021/006069 2020-05-14 2021-05-14 Nouvelle protéine de fusion recombinante et utilisation associée WO2021230705A1 (fr)

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Citations (3)

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Publication number Priority date Publication date Assignee Title
US7867491B2 (en) * 2007-05-30 2011-01-11 Genexine Co., Ltd. Immunoglobulin fusion proteins
KR20150038012A (ko) * 2012-08-08 2015-04-08 로슈 글리카트 아게 인터루킨-10 융합 단백질 및 그의 용도
KR101825048B1 (ko) * 2014-12-31 2018-02-05 주식회사 제넥신 GLP 및 면역글로불린 하이브리드 Fc 융합 폴리펩타이드 및 이의 용도

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US7867491B2 (en) * 2007-05-30 2011-01-11 Genexine Co., Ltd. Immunoglobulin fusion proteins
KR20150038012A (ko) * 2012-08-08 2015-04-08 로슈 글리카트 아게 인터루킨-10 융합 단백질 및 그의 용도
KR101825048B1 (ko) * 2014-12-31 2018-02-05 주식회사 제넥신 GLP 및 면역글로불린 하이브리드 Fc 융합 폴리펩타이드 및 이의 용도

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