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WO2012053828A2 - Protéine de fusion fc hybride - antagoniste du récepteur humain de l'interleukine-1 - Google Patents

Protéine de fusion fc hybride - antagoniste du récepteur humain de l'interleukine-1 Download PDF

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WO2012053828A2
WO2012053828A2 PCT/KR2011/007809 KR2011007809W WO2012053828A2 WO 2012053828 A2 WO2012053828 A2 WO 2012053828A2 KR 2011007809 W KR2011007809 W KR 2011007809W WO 2012053828 A2 WO2012053828 A2 WO 2012053828A2
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fusion protein
hybrid
receptor
protein
present
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PCT/KR2011/007809
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Korean (ko)
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WO2012053828A9 (fr
WO2012053828A3 (fr
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조영규
신혜정
이운영
장우익
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주식회사 한독약품
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Priority to US13/880,175 priority Critical patent/US8883134B2/en
Priority claimed from KR1020110107194A external-priority patent/KR101333958B1/ko
Publication of WO2012053828A2 publication Critical patent/WO2012053828A2/fr
Publication of WO2012053828A9 publication Critical patent/WO2012053828A9/fr
Publication of WO2012053828A3 publication Critical patent/WO2012053828A3/fr

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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
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/54—Interleukins [IL]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • 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 fusion protein in which a human interleukin-1 receptor antagonist and a hybrid Fc are bound.
  • autoimmune inflammatory diseases include inflammatory autoimmune disease, inflammatory bowel disease (Crohn's disease and ulcerative bowel disease), rheumatoid arthritis, psoriasis and the like.
  • TNF inhibitors are difficult to treat severe autoimmune diseases, and the number of patients treated with TNF inhibitors who do not respond to or maintain the existing drug is increasing and is expected to continue to increase.
  • TNF- ⁇ plays an important role in host defense against bacterial or viral infections, inhibition of TNF- ⁇ is likely to increase the risk of infections such as severe infections, opportunistic infections and tuberculosis reactivation. Therefore, there is a need for the development of a new mechanism of autoimmune inflammatory therapy that can replace TNF inhibitor.
  • IL-1 receptor antagonist is an IL-1 inhibitor that exists naturally in our body and competitively prevents IL-1 from binding to IL-1 receptor. Inhibit the effects of IL-1 (Protein Engineering, Design & Selection, Zoey L. Fredericks et al., 2004).
  • genetically engineered human IL-1 receptor antagonists e.g. anakinra
  • anakinra has been developed and are known to have good effects in patients with rheumatoid arthritis who do not respond to TNF- ⁇ inhibitors (Ann. Rheum. Dis., Bresnihan). , 2002; J. Rheumatol., St. Clair, EW, 2002).
  • anakinra has the major disadvantage of having to be administered once a day because of its high immunogenicity in microorganisms and its short half-life that degrades within 6 hours.
  • anti-IL-1 receptor-hybrid Fc fusion proteins have been expected to effectively treat autoimmune diseases by inhibiting IL-1, which plays an important role in sustaining inflammation.
  • IgG1 Fc is used to prepare a conventional Fc fusion protein, and in the case of IgG1 Fc, antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) Causing the target cell to die. That is, a target cell to which agonistic proteins and peptides bind is dead, resulting in a fatal problem that does not result in a therapeutic effect.
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • CDC complement-dependent cytotoxicity
  • the conventional antibody fusion technology has a problem that when the antibody fusion structurally two therapeutic proteins bind to one Fc to form a dimer, thereby reducing the activity of the bound protein and peptide therapeutics.
  • the present inventors have developed an anti-IL-1 receptor-hybrid Fc fusion protein to develop an excellent drug for treating autoimmune diseases.
  • the present invention provides a fusion protein, wherein the human interleukin-1 receptor antagonist is bound to a human immunoglobulin (Ig) hybrid Fc fragment.
  • Ig immunoglobulin
  • the hybrid Fc fragment includes IgD and IgG4.
  • the present invention also provides a pharmaceutical composition comprising the fusion protein.
  • the pharmaceutical composition is used for the treatment of autoimmune diseases.
  • autoimmune diseases include rheumatoid arthritis, inflammatory bowel disease (eg Crohn's disease, ulcerative bowel disease), psoriasis, diabetes and the like.
  • the present invention also provides a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 6, encoding a human interleukin-1 receptor antagonist-hybrid Fc fusion protein.
  • the present invention also provides a human interleukin-1 receptor antagonist-hybrid Fc fusion protein comprising the amino acid sequence of SEQ ID NO: 7.
  • the anti-IL-1 receptor-hybrid Fc fusion protein of the present invention is enhanced in vivo stabilization, bioavailability, target directivity, non-toxicity, and compared to the conventional recombinant human IL-1 receptor antagonist and anti IL-1 receptor type Fc fusion protein. It has technical advantages such as non-immunogenicity, and therefore, it is expected to provide a new autoimmune disease treatment agent with reduced side effects while maintaining excellent efficacy compared to conventional autoimmune disease treatment agents.
  • FIG. 1 schematically shows the fusion of a human IL-1 receptor antagonist (IL-1RA) to the Fc portion of a human immunoglobulin (hybrid Fc).
  • IL-1RA human IL-1 receptor antagonist
  • hybrid Fc human immunoglobulin
  • Figure 2 graphically shows the recombinant plasmid finally constructed by inserting DNA encoding the anti-IL-1 receptor-hybrid Fc fusion protein into a vector.
  • Figure 3 shows the expression of the anti-IL-1 receptor-hybrid Fc fusion protein of the present invention in CHO DG44 cells.
  • Figure 4 shows the productivity of selected cells expressing the anti IL-1 receptor-hybrid Fc fusion protein of the present invention.
  • Figure 5 shows the qualitative and quantitative confirmation of the anti-IL-1 receptor-hybrid Fc fusion protein of the present invention expressed in each cell line.
  • Figure 7 shows the results of qualitatively and quantitatively confirming the protein samples of the fraction identified as the peak after the antibody affinity column chromatography, Figure 10a in the reducing conditions of 12% SDS-PAGE, Figure 10b non reducing It was confirmed under conditions.
  • FIG. 8A shows a chromatogram of anion exchange resin chromatography
  • FIG. 8B shows a result of qualitatively and quantitatively confirming a protein sample of a fraction identified as a peak after anion exchange resin chromatography.
  • FIG. 9a shows a chromatogram of hydroxyapatite column chromatography
  • FIG. 9b shows a result of qualitatively and quantitatively confirming a protein sample of a fraction identified as a peak after hydroxyapatite column chromatography.
  • Figure 10a is denatured electrophoresis
  • 10b shows the analysis of the anti-IL-1 receptor-hybrid Fc fusion protein by Western blotting.
  • Figure 11 shows the results of analysis of anti-IL-1 receptor-hybrid Fc fusion protein via isoelectric focus electrophoresis.
  • FIG. 12 shows SE-HPLC chromatogram of purified anti-IL-1 receptor-hybrid Fc fusion protein.
  • Figure 13 shows the binding value of the protein fusion of IgG1 Fc to the purified anti-IL-1 receptor-hybrid Fc fusion protein of the present invention or a control anti-IL-1 receptor is bound to the IL-1 receptor.
  • Figure 14 shows the results confirming the effect of the anti-IL-1 receptor-hybrid Fc fusion protein of the present invention to suppress the immune response in human T cells or peripheral blood monocytes.
  • Figure 15 shows the pharmacokinetic values of the anti-IL-1 receptor-hybrid Fc fusion protein of the invention or the IL-1 receptor anakinra sold in Europe as a control.
  • 16 and 17 show the results confirming the effect of the anti-IL-1 receptor-hybrid Fc fusion protein of the present invention expressed in the medium to block hIL-1 ⁇ in cells.
  • Figure 18 shows the results confirming the effect of the anti-IL-1 receptor-hybrid Fc fusion protein of the present invention or the control IL-1 receptor and the hybrid Fc fusion protein block hIL-1 ⁇ in the cell.
  • Figure 19 shows the results confirming the effect of the anti-IL-1 receptor-hybrid Fc fusion protein of the present invention or the IL-1 receptor anakinra (anakinra) sold in Europe as a control block the hIL-1 ⁇ in the cell will be.
  • Figure 20 shows the anti-IL-1 receptor-hybrid Fc fusion protein of the present invention or interleukin, an inflammation-induced cytokine, by blocking hIL-1 ⁇ in cells by IL-1 receptor anakinra sold in Europe as a control group. The results show the effect of blocking -8.
  • Figure 21 shows that the anti-IL-1 receptor-hybrid Fc fusion protein of the present invention or the protein fusion of IgG1 Fc to the anti-IL-1 receptor as a control block hIL-1 ⁇ in the cell to block interleukin-8, an inflammation-inducing cytokine. It shows the result of confirming the blocking effect.
  • FIG. 22 shows the effects of the anti-IL-1 receptor-hybrid Fc fusion protein of the present invention or anakinra, an IL-1 receptor sold in Europe, as a control in the collagen-induced arthritis rat model. It shows the result confirmed.
  • the present invention provides an anti IL-1 receptor-hybrid Fc fusion protein, wherein the fusion protein comprises a human IL-1 receptor antagonist (IL-1RA) in the Fc portion of a human immunoglobulin. It is composed in the form fused to (see Fig. 1).
  • the human IL-1 receptor antagonist is a protein that contains the entire gene sequence, the human IL-1 receptor antagonist may be coupled to each other with the hybrid Fc through a linker.
  • the linker molecule is a synthetic linker.
  • the synthetic linker may be a peptide of amino acid residues consisting of Gly and Ser residues. In one specific embodiment, such Gly-Ser linker is GGS.
  • the hybrid Fc comprises a hinge region, a CH2 domain and a CH3 domain in an N-terminal to C-terminal direction, the hinge region comprising at least a human IgD hinge region, and the CH2 domain at least amino acids of human IgD and IgG4 CH2 domains.
  • IL-1 is an important inflammatory mediator that amplifies the inflammatory response in the pathogenesis of autoimmune inflammatory diseases.
  • neutrophils gather at the site of inflammation, activate macrophages, promote the growth and differentiation of T cells and B cells, and also play an important role in inducing various enzyme secretions that degrade the extracellular matrix.
  • IL-1 receptor antagonists IL-1 RA
  • IL-1 RA are naturally occurring IL-1 inhibitors in our bodies that inhibit the effects of IL-1 by competitively interfering with the binding of IL-1 to IL-1 receptors. .
  • IgG1 Fc portion was used in the preparation of the conventional IL-1 RA fusion protein. Therefore, as described above, the antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) caused by IgG1 Fc causes the target cells to die. There was this.
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • CDC complement-dependent cytotoxicity
  • a hybrid Fc was selected using a combination of IgD and IgG4 Fc without using an IgG1 Fc portion.
  • Table 1 compares the characteristics of IgG subclasses and IgD.
  • the hybrid Fc of the present invention has an increased potency due to the flexibility of the hinge region of IgD, and there is no site that binds to Fc ⁇ R, which is a binding receptor for Fc of neutrophil cells, thereby reducing side effects.
  • IgG4 does not have an effector function such as complement-depent cytotoxicity (CDC), IgG4 can reduce unintended immune responses and the ability to bind FcRn associated with intracellular recycling. Best shown increased stability and increased serum half-life. Also with respect to in vivo half-life, IgG1, IgG2, and IgG4 have a long half-life of 21 days, while other immunoglobulins have a short half-life of relatively less than a week.
  • the fusion protein of the present invention has the effect of increasing the therapeutic efficacy and reducing the side effects of autoimmune diseases by using the hybrid Fc in combination with the IgD and IgG4 Fc, while also increasing the half-life thereof.
  • the ADCC and CDC will not be induced, and thus, it will be suitable for interfering with cytokine.
  • the anti-IL-1 receptor-hybrid Fc fusion protein of the present invention has low immunogenicity by being expressed in CHO cells, which are mammalian cells.
  • HIL1 RA DNA for gene cloning was codon optimized (codon optimization) and then synthesized the gene into several pieces to prepare the DNA of hIL1 RA through sewing PCR.
  • Hybrid Fc DNA was obtained from Genexine and PCR was performed with DNA of hIL1RA to obtain the entire gene sequence DNA encoding the final anti IL-1 receptor-hybrid Fc fusion protein.
  • pAD15 vector was used for gene construction, and the clean DNA was extracted by cutting the insert DNA and the vector with EcoRI and XbaI restriction enzymes.
  • the final product inserted into the pAD15 vector was obtained by ligation of the obtained vector and the inserted DNA.
  • the final product was inserted into efficient DH5 ⁇ competent cells, smeared on an agar plate containing ampicillin antibiotics for antibiotic resistance genes in the vector, colonies were selected, and sequencing was confirmed.
  • the synthesized gene sequence and the nucleotide sequence of the final product obtained are as follows.
  • the synthetic DNA used to obtain the entire gene sequence of the final product is a gene encoding hIL-1 RA, the synthetic gene
  • the sequences F-1, R-2, F-3, R-4, F-5 are shown in SEQ ID NOs: 1-5 (SEQ. ID. NOs: 1-5), respectively.
  • SEQ ID NO: 6 The entire gene sequence of the final product is shown in SEQ ID NO: 6 (SEQ. ID. NO: 6), and the amino acid sequence of the anti-IL-1 receptor-hybrid Fc fusion protein of the present invention is shown in SEQ ID NO: 7 (SEQ. ID. NO. : 7).
  • Figure 2 shows the final constructed recombinant plasmid
  • a 6610bp sized vector was constructed by inserting the DNA of the anti-IL-1 receptor-hybrid Fc obtained through synthesis and PCR into the cloning site of the pAD 15 vector.
  • the final vector obtained through the cloning of Example 1 was introduced into CHO DG44 mammalian cells to express the fusion protein.
  • PEI Polyethylenimine
  • DNA was mixed at a Nitrogen / Phosphate ratio of 1:20, incubated at room temperature for 5-10 minutes, and then added to CHO DG44 mammalian cells. After 12 hours it was replaced with fresh medium.
  • HT Hypoxanthine
  • CHO DG44 cells transduced with the vector containing the dihydrofolate reductase (DHFR) gene and the fusion protein gene of the present invention obtained in Example 1 were replaced with a medium without HT after 24 hours. The medium was changed every 3-4 days until colonies were formed, and each colony was fractionated and incubated on a new plate.
  • DHFR dihydrofolate reductase
  • FIG. 3 is a result of Western blotting confirming that the anti-IL-1 receptor-hybrid Fc fusion protein of the present invention is expressed in cells.
  • the cell transfected with the vector encoding the fusion protein when compared with the medium (lane 1) and cell lysate (lane 3) of the cell without transducing the vector encoding the fusion protein.
  • the medium lane 2
  • cell lysate lane 4
  • the protein-expressing medium was collected 24 hours later, and the number of cells was counted.
  • ELISA quantitation kit Bethyl lab., Inc., E80-104. In this experiment, a total of 20 cell lines were used, and randomly numbered from 1 to 20 times.
  • the expressed protein was verified by westernblotting.
  • a medium sample of the protein expressed on the E-tube was added, 1 ⁇ loading dye, and a 12% SDS-PAGE gel was mounted in a running tank. I made a 1X running buffer, filled it inside the gel, filled about 1/3 of the outside, loaded the sample into the comb with a hamilton syringe, and connected the electrodes to flow the current. The dye was run between 80v and 130v until the dye stayed at the gel end. The protein of the running SDA-PAGE gel was transferred to the nitro cellulose membrane using a blotting system (Invitrogen, iBlot gel transfer machine).
  • the transferred nitro cellulose membrane was taken out, placed in a wide dish, and shaken with a rocker for 1 hour at room temperature using a blocking buffer (PBST + 5% skim milk). After diluting the anti-hIL-1RA antibody to 0.2 ⁇ g / ml in 1X PBST, it was added to nitro cellulose membrane for 3 hours at room temperature or overnight at 4 °C, and washed 5 times in rocker for 10 minutes in 1X PBST to remove antibody. .
  • HRP-fused anti-rabbit secondary antibody (diluted 1: 2000) in 1X PBST was added to nitro cellulose membrane, shaken for 1 hour at room temperature, and washed 5 times in rocker for 10 minutes at 1X PBST to remove antibody. It was. Place the nitro cellulose membrane on the exposer cassette and spread the substrate evenly so that the gel doc. Photos were taken after being placed in the camera, and the results are shown in FIG. 5.
  • the fusion protein expressed in each cell line had a molecular weight of about 55 kDa when the disulfide bond was removed, and compared with hIL1 RA, the molecular weight of 30 kDa or more was increased due to hybrid Fc binding. have.
  • the thickness and density of bands are exactly different in concentration.
  • the anti-IL-1 receptor-hybrid Fc fusion protein obtained by suspension cell production was filtered through a cellulose filtration membrane having a pore size of 0.2 ⁇ m to remove impurities, and the filtered cells were stored at 4 ° C. or in ice until loaded into a column. It was.
  • the column was filled with Mabselect Sure (GE), a type of antibody-affinity column resin labeled with Protein A protein, and a mobile phase was prepared.
  • GE Mabselect Sure
  • 50 mM sodium phosphate (pH 3.0) was prepared. Then, 0.5M arginine (pH3.0), 0.1M sodium chloride solution (buffer C) and 0.5N sodium hydroxide solution (buffer D) were prepared to strongly elute the less eluted protein from the resin. After washing the lines of the chromatography (AKTA Purifier, GE healthcare) system with four solutions from buffer A to buffer D, the antibody affinity column filled with 20 ml volume was connected and the resin was equilibrated to 10 CV (column volume). .
  • AKTA Purifier GE healthcare
  • antibody affinity chromatography was performed by loading the pre-prepared anti IL-1 receptor-hybrid Fc fusion protein onto the column by pump loading in FPLC. After lowering the pH with buffer B, the eluted protein was recovered by fractionation by 3 ml. Fractionated proteins were collected by including the band at the 50kda position on 12% SDS-PAGE (reducing) and the protein amount was measured using the Bradford protein quantification method (see FIGS. 6 and 7).
  • Figure 6 shows the chromatogram of antibody affinity column chromatography, showing that the peak of the fusion protein eluted by lowering the pH with buffer B was identified between fractions 30 to 40.
  • Figure 7a is a 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis to qualitatively and quantitatively confirm the protein sample of the fraction identified as the peak after the antibody affinity column chromatography , SDS-PAGE), the fusion protein at the position of 50kDa seen in the loading solution was mostly present in the fraction of the peak (peak) eluted by binding to the column and the purity was confirmed to be about 95% or more.
  • 7B shows a dimer molecular weight of 110 kda and a non-reducing sodium dodecyl sulfate polyacrylamide gel electrophoresis on Fc fusion protein, thus forming a dimer by disulfide bonds.
  • Protein solution obtained through antibody affinity chromatography was adjusted to the final 50mM Tris-HCl (pH8.0) with 1M Tirs-HCl (pH8.0) solution, and left for 30 minutes at room temperature to inactivate the virus.
  • a 50 mM Tris-HCl (pH 8.0) solution was added to twice the volume of the protein solution to load the anion exchange resin to lower the sodium chloride concentration.
  • anion exchange resins Q HP, GE
  • elution buffer (buffer B) 50 mM Tris-HCl (pH8.0), and 1 M sodium chloride in the AKTA Purifier line. After filling the protein solution was loaded.
  • Dimers and multimers of the anti-IL-1 receptor-hybrid Fc fusion protein were isolated while flowing in a sodium chloride concentration gradient.
  • the peak near 300 mM sodium chloride concentration (peak 1 in Fig. 8a) is mainly present in the dimer-type anti-IL-1 receptor-hybrid Fc fusion protein and the peak eluted at a concentration above 500 mM sodium chloride (peak 2 in Fig. 8a) Heterogeneous fractions in which the multimeric forms are present together.
  • peak 1 of FIG. 8A mainly in the dimer form confirmed the fraction of peak1 and peak2 under non-reducing conditions of SDS-PAGE as shown in FIG. 8B (see FIG. 8).
  • the mobile phase of the hydroxyapatite column (CHTTMCeramic Hydorxyapatite, BIO-RAD) is column equilibration buffer with 10 mM sodium phosphate (pH6.5) and protein elution buffer with 10 mM sodium phosphate (pH6.5), 2M sodium chloride and 500 mM sodium phosphate (pH6. 5) It flowed in two ways.
  • the dimer fraction separated from the anion exchange column was added to the equilibrated hydroxyapatite column by adding about 4 times the volume of the protein solution with equilibration buffer and diluting the sodium chloride solution by 5 times.
  • FIGS. 10 to 12 The results are shown in FIGS. 10 to 12.
  • a 10% Polyacrylamide gel was used and a band located at 150 kda of the protein molecular weight marker (GE Healthcare, AmershamTM HMW Clibration Kit For Native Electrophoresis) had an anti IL-1 receptor-hybrid Fc fusion It was confirmed that the protein showed a dimer form in an undenatured state.
  • FIG. 10B Western blotting was able to qualitatively identify the anti-IL-1 receptor-hybrid Fc fusion protein when detected by anti-hIL-1RA antibody.
  • the theoretical isoelectric point of the anti-IL-1 receptor-hybrid Fc fusion protein has a value of 6.01 and is widely distributed from pI5.3 to 6.0 at the initial stage of purification as shown in isoelectric focus electrophoresis of FIG. As a result, it was confirmed that the protein was purified into a uniform form.
  • Size exclusion HPLC in a SEC column (G3000SWXL, 5micron, 7.8 * 300, TSK) in a mobile phase 50mM sodium phosphate (pH7.5), while loading 20 ⁇ L of sample while flowing 50mM NaCl as shown in Figure 12
  • the retention time was found to be eluted with a purity of about 99.9% at a peak of 7.6 minutes, and the SEC column was confirmed that the purified protein was present in high purity.
  • the surface plasma was analyzed using a Biacore (GE healthcare) instrument. Surface plasmon resonance (SPR) values were measured.
  • the Biacore instrument was equipped with a CM5 chip and flowed through a solution of Phosphate buffered saline (PBS).
  • PBS Phosphate buffered saline
  • 1-ethyl-3-dimethylaminopropyl carbodiimide (EDC) / N-hydroxy succinimide (N-hydroxy succinimide) , NHS) was flowed to the chip to activate the amine group.
  • EDC 1-ethyl-3-dimethylaminopropyl carbodiimide
  • NHS N-hydroxy succinimide
  • the hIL-1RA and IgG1 Fc fusion proteins to be immobilized or the hIL-1RA-hybrid Fc fusion protein expressed in the present invention are injected into the chip to form a covalent bond with an activated amine group, and immobilized with ethanol amine.
  • Kd dissociation constant
  • the anti-IL-1 receptor-hybrid Fc fusion protein of the present invention had a dissociation constant of 186 pM and the control group had a dissociation constant of 1.02 nM.
  • Hybrid Fc fusion protein was confirmed to have a better binding than the control.
  • hIL-1RA-hybrid Fc fusion protein expressed in the present invention was assessed for the effect of hIL-1RA-hybrid Fc fusion protein expressed in the present invention to suppress the immune response.
  • human T cell proliferation inhibition assay and inflammatory cytokine secretion assay were performed. Inhibition of T cell proliferation was achieved by diluting PBMCs from blood and diluting the cells at a concentration of 1 ⁇ 10 5 cells / ml in RPMI-1640 medium, and 100 ng / ml lipopolysaccharide (LPS) or 1 ug / ml of anti- Cells were stimulated with CD3 antibody for 3 days. At this time, various concentrations were added to evaluate the activity of the hIL1RA-hyFc fusion protein of the present invention.
  • LPS lipopolysaccharide
  • Inflammatory cytokine secretion was isolated from PBMC from blood and diluted in cells of RPMI-1640 at a concentration of 1 ⁇ 10 6 cells / ml and stimulated with 100 ng / ml LPS.
  • the hIL1RA-hyFc fusion protein of the present invention was added at various concentrations. After 48 hours, only the culture medium was taken, and the concentrations of interleukin-17 cancer cell death factor-a, nuclear factor kappa-B receptor activator (RANKL), and vascular endothelial growth factor (VEGF) were quantified by ELISA.
  • RTKL nuclear factor kappa-B receptor activator
  • VEGF vascular endothelial growth factor
  • FIG. 14 shows the anti-IL-1 receptor-hybrid Fc fusion protein of the present invention as a result of inhibiting the immune response in human T cells or peripheral blood mononuclear cells.
  • the group which put the group was used.
  • FIG. 14 it was confirmed that the proliferation of human T cells was effectively inhibited when the fusion protein was treated, and cytokines involved in inflammation-induced cytokines and bone destruction in human peripheral blood mononuclear cells. Inhibitory effect was shown.
  • TBST Tris-Buffered Saline Tween-20
  • BSA Bovine serum albumin
  • FIG. 15 shows pharmacokinetic values of the anti-IL-1 receptor-hybrid Fc fusion protein of the present invention or the IL-1 receptor anakinra sold in Europe as a control, as can be seen in FIG. 15. Likewise, it was confirmed that the treatment of the fusion protein persisted in blood for a longer time than the treatment with Anakinra control.
  • Luciferase assay was performed to see the cell signal transduction blocking effect of hIL-1 ⁇ by the fusion protein of the present invention obtained above.
  • hIL-1 ⁇ delivers cellular signals by binding to hIL-1 receptor 1 to activate NFkB.
  • the vector transformed into the luciferase gene having the NFkB binding site in the cell line expressing the hIL-1 receptor 1 was treated with hIL-1 ⁇ and the NFkB was activated to express the luciferase protein.
  • IL-1 RA when IL-1 RA is treated, IL-1 RA binds to hIL-1 receptor 1, thereby blocking hIL-1 ⁇ from binding to this receptor, thereby preventing intracellular signal transduction and thus not luciferase protein expression. Therefore, experiments were performed to determine whether the fusion protein functions properly in the cell and exhibits its effects.
  • HeLa cells were dispensed into 2 ⁇ 10 4 water in Nunc 48 well plates.
  • the plasmid containing the NFkB binding site, the firefly luciferase gene, and the renilla luciferase gene with the CMV promoter required to normalize the results were transduced into HeLa cells. After 3 hours and replaced with fresh medium and stabilized for about 24 hours.
  • the transfected HeLa cells were incubated for 6 hours with hIL-1 ⁇ alone or hIL-1RA (R & D systems). After 6 hours the medium was removed from the cultured cells and washed with the addition of sufficient PBS. After the PBS was completely removed, the emission value was measured by performing a manual using a dual-luciferase reporter assau sustem (promega, E1960).
  • 16 and 17 are a result of confirming whether the fusion protein expressed in the medium in the cell can properly block hIL-1 ⁇ in the cell and the degree of the effect, the group containing only the medium without any treatment as a negative control and hIL1 Only the RA group was used, and the positive control group was compared with the hIL-1 ⁇ group.
  • Figure 16 when treated with hIL-1 ⁇ media obtained from several cell lines expressing the fusion protein showed a similar or better cell signal blocking effect than the commercially available hIL1 RA.
  • Figure 17 when treated with the concentrated media obtained from these cell lines showed a more certain cell signal blocking effect.
  • hIL-1RI anakinra protein
  • HeLa cells were dispensed into 2 ⁇ 10 4 water in Nunc 48 well plates.
  • the plasmid containing the NFkB binding site, the firefly luciferase gene, and the renilla luciferase gene with the CMV promoter required to normalize the results were transduced into HeLa cells. After 3 hours and replaced with fresh medium and stabilized for about 24 hours.
  • hIL-1 ⁇ alone or an existing commercially available anakinra protein or hIL-1RI a receptor that binds to hIL-1 ⁇ , the hybrid Fc fusion protein and the protein expressed in the present invention were added to the transfected HeLa cells. Incubated for 6 hours.
  • the medium was removed from the cultured cells and washed with the addition of sufficient PBS. After the PBS was completely removed, the luminescence value was measured using a Dual-luciferase reporter assay system (promega, E1960) as described in the manual.
  • FIG. 18 and 19 show whether the fusion protein can properly block hIL-1 ⁇ in cells than the control IL-1 receptor and hybrid Fc fusion protein or commercially available anti-IL-1 receptor Anakinra.
  • the fusion protein or the control IL-1 receptor and the hybrid Fc fusion protein were treated with hIL-1 ⁇ , the fusion protein had a better cell signal blocking effect than the control. Indicated.
  • the fusion protein exhibited a more obvious cell signal blocking effect than the commercially available hIL-1 RA.
  • Interleukin-8 detection ELISA was performed to compare the secretion blocking effect of interleukin-8 by the protein expressed in the present invention and the protein fused with the normal IgG1 Fc to the conventionally sold anakinra protein or hIL1RA.
  • HeLa cells were dispensed into 2 ⁇ 10 4 water in Nunc 48 well plates.
  • the next day after replacement with fresh medium, hIL-1 ⁇ alone or hIL-1 ⁇ and the conventionally marketed anakinra protein or hIL1RA fused with the normal IgG1 Fc and the protein expressed in the present invention were added by concentration 24 Time incubation. After 24 hours, the medium was collected and the amount of interleukin-8 secreted in the medium was measured using a Duoset ELISA development quantitation kit (R & D systems., Inc., DY208).
  • 100 ⁇ l of the coated antibody diluted in the coating solution was added to Nunc 96 well plates and incubated at room temperature.
  • the plate was washed three times with a TBST (Tris-Buffered Saline Tween-20) washing solution, and then 300 ⁇ l of blocking buffer containing 1% BSA (Bovine serum albumin) was added per well and blocked at room temperature for 1 h. After the plate was washed three times with TBST washing solution, 100 ⁇ l of the standard solution diluted in 1/2 to 2000 pg / ml ⁇ 0 pg / ml or a medium sample diluted in an appropriate ratio was added.
  • TBST Tris-Buffered Saline Tween-20
  • FIGS. 20 and 21 show the effect of the fusion protein blocking hIL-1 ⁇ in the cell to block the inflammation-inducing cytokine interleukin-8 by measuring the concentration of interleukin-8.
  • the fusion protein showed a better interleukin-8 secretion blocking effect than the commercially available hIL1 RA when treated with hIL-1 ⁇ .
  • the fusion protein was treated with hIL-1 ⁇ in FIG. 21, it was confirmed that the IL-1RA and immunoglobulin 1 (IgG1) Fc fusion protein showed better interleukin-8 secretion blocking effect.
  • FIG. 22 is a result of confirming the fusion protein by inhibiting arthritis when the concentration of the fusion protein administered in each model rat having collagen-induced arthritis.
  • mice administered the fusion protein inhibited arthritis in a concentration-dependent manner, and was even more at the same concentration than the commercially available hIL-1 RA, a positive control. It showed a good inhibitory effect on arthritis and a better effect than the rats administered with the higher concentration of the control group.
  • the present invention is expected to be used in the field of new autoimmune disease therapeutics with reduced side effects while maintaining excellent efficacy compared to conventional autoimmune disease therapeutics.

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Abstract

La présente invention concerne une protéine de fusion résultant du couplage d'un antagoniste du récepteur humain de l'interleukine-1 et d'un Fc hybride. Plus particulièrement, l'invention concerne une protéine de fusion dans laquelle un antagoniste du récepteur humain de l'interleukine-1 est couplé à un fragment Fc hybride d'immunoglobuline (Ig) humaine. Le fragment Fc hybride comprend IgD et IgG4. L'invention concerne également une composition pharmaceutique comprenant la protéine de fusion. La composition pharmaceutique de la présente invention peut être utilisée pour le traitement de maladies auto-immunes, comprenant la polyarthrite rhumatoïde, une maladie intestinale inflammatoire (par exemple la maladie de Crohn et la colite ulcéreuse), le psoriasis, le diabète, et similaire. La protéine de fusion de la présente invention peut être attendue d'être utilisable dans le développement de nouveaux médicaments contre des maladies auto-immunes qui conservent une efficacité largement meilleure et ont moins d'effets secondaires par rapport aux médicaments existants contre des maladies auto-immunes.
PCT/KR2011/007809 2010-10-20 2011-10-19 Protéine de fusion fc hybride - antagoniste du récepteur humain de l'interleukine-1 WO2012053828A2 (fr)

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US13/880,175 US8883134B2 (en) 2010-10-20 2011-10-19 Human interleukin-1 receptor antagonist—hybrid Fc fusion protein

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KR10-2010-0102492 2010-10-20
KR20100102492 2010-10-20
KR10-2011-0107194 2011-10-19
KR1020110107194A KR101333958B1 (ko) 2010-10-20 2011-10-19 인간 인터루킨-1 수용체 길항제-하이브리드 Ｆｃ 융합단백질

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WO2022010319A1 (fr) * 2020-07-10 2022-01-13 (주)지아이이노베이션 Protéine de fusion comprenant un peptide-1 de type glucagon et un antagoniste de récepteur de l'interleukine-1 et utilisation associée
EP3998279A4 (fr) * 2019-07-08 2023-01-18 GI Innovation, Inc. Dimère polypeptidique à haute teneur en acide sialique, comprenant un domaine extracellulaire de sous-unité alpha du récepteur fc d'ige, et composition pharmaceutique le comprenant

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP3998279A4 (fr) * 2019-07-08 2023-01-18 GI Innovation, Inc. Dimère polypeptidique à haute teneur en acide sialique, comprenant un domaine extracellulaire de sous-unité alpha du récepteur fc d'ige, et composition pharmaceutique le comprenant
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WO2022010319A1 (fr) * 2020-07-10 2022-01-13 (주)지아이이노베이션 Protéine de fusion comprenant un peptide-1 de type glucagon et un antagoniste de récepteur de l'interleukine-1 et utilisation associée

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