TYPE 1 INTERFERON NEUTRALIZING FC-FUSION PROTEIN AND USE THEREOF

Abstract

The present invention relates to a type 1 interferon neutralizing FC-fusion protein and a use thereof and, more specifically, to: a dimer-type polypeptide to which a monomer comprising an interferon receptor fragment or an antibody Fc fragment is bound; a preparation method there for; and a pharmaceutical composition comprising same. The type 1 interferon neutralizing FC-fusion protein of the present invention blocks binding between type 1 interferon and an interferon receptor, and has an excellent ability of inhibiting the signaling and biological activities of interferon, thereby enabling diseases mediated by a type 1 interferon to be effectively treated.

Claims

1. A dimeric polypeptide in which a monomer comprising an interferon receptor fragment or an antibody Fc fragment is bound, wherein the monomer is a polypeptide selected from the group consisting of (i), (ii) and (iii): (i) a monomer comprising an interferon receptor 1 (IFNAR1) fragment and an antibody Fc fragment; (ii) a monomer comprising an interferon receptor 2 (IFNAR2) fragment and an antibody Fc fragment; and (iii) an antibody Fc fragment.

2. The dimeric polypeptide according to claim 1, wherein the polypeptide neutralizes type 1 interferon.

3. The dimeric polypeptide according to claim 1, wherein the polypeptide comprises (i) and (ii).

4. The dimeric polypeptide according to claim 1, wherein the interferon receptor 1 fragment comprises the amino acid sequence of SEQ ID NO: 4.

5. The dimeric polypeptide according to claim 1, wherein the interferon receptor 2 fragment comprises the amino acid sequence of SEQ ID NO: 6.

6. The dimeric polypeptide according to claim 1, wherein the antibody Fc fragment comprises the amino acid sequence of SEQ ID NO: 12.

7. A polynucleotide encoding the polypeptide of claim 1.

8. A vector comprising the polynucleotide of claim 7.

9. A host cell transformed with the vector of claim 8.

10. A method for producing the polypeptide of claim 1 comprising: (a) providing a host cell transformed with a vector comprising a polynucleotide encoding the polypeptide of claim 1; (b) culturing the provided cells; and (c) preparing the polypeptide by recovering the polypeptide from the cell or culture medium.

11. A pharmaceutical composition for prevention or treatment of type 1 interferon-mediated diseases or disorders comprising the polypeptide of claim 1 as an active ingredient.

12. The pharmaceutical composition according to claim 11, wherein the type 1 interferon-mediated disease or disorder is selected from the group consisting of systemic lupus erythematosus, Sjogren's syndrome, systemic sclerosis, insulin-dependent diabetes mellitus (IDDM), inflammatory bowel disease (IBD) (including Crohn's disease, ulcerative colitis and celiac disease), multiple sclerosis (MS), psoriasis, autoimmune thyroiditis, rheumatoid arthritis, inflammatory Myositis, glomerulonephritis HIV infection, AIDS, transplant rejection and graft-versus-host reaction (GVHD).

13. Use of the polypeptide of claim 1 for the preparation of an agent for the prevention or treatment of a type 1 interferon-mediated disease or disorder.

14. A method of treating a type 1 interferon-mediated disease or disorder, comprising administering to a subject in need thereof an effective amount of a composition comprising the polypeptide of claim 1 as an active ingredient.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0133] FIG. 1 shows a schematic diagram of the preparation of an expression vector for the production of type 1 interferon Fc fusion receptor protein.

[0134] FIG. 2a is a result of comparing the size of each protein through size exclusion chromatography.

[0135] FIG. 2b is a result confirming the binding of IFNAR1/2 hetero Fc fusion protein and human IFN 13 under native conditions and reducing conditions.

[0136] FIG. 2c is a schematic diagram showing the type 1 interferon Fc fusion receptor protein.

[0137] FIG. 3 is a diagram showing the results of SPR analysis of the type 1 interferon Fc fusion receptor protein.

[0138] FIGS. 4a to 4c are results confirming the type 1 interferon neutralizing ability of the type 1 interferon Fc fusion receptor protein of the present invention.

[0139] FIG. 4a is a result showing the cell viability in Daudi cells treated with human IFN β-1a alone or type 1 interferon Fc fusion receptor protein.

[0140] FIG. 4b is a result showing the IC50 value treated with human IFN β-1a alone or type 1 interferon Fc fusion receptor protein.

[0141] FIG. 4c is a result showing the cell viability of Daudi cells treated with human IFN β-1a according to the concentration of the protein of the present invention.

[0142] FIG. 5 confirms the neutralizing ability of the IFNAR1/2-Fc heterodimer (4GS*3) protein of the present invention for the biological activity of type 1 interferon (IFN-α 1, IFN-α 2a, IFN-α 2b, IFN-α 5, IFN-α 8, IFN-α 10, IFN-β 1a, IFN-ω), and the cell viability of Daudi cells in the group to which type 1 interferon was added and the group in which each interferon was treated with IFNAR1/2-Fc heterodimer (4GS*3) protein was shown.

[0143] FIGS. 6a and 6b are the results of confirming the neutralizing ability of the IFNAR1/2-Fc heterodimer (4GS*3) protein of the present invention for the signaling mechanism of type 1 interferon, and the results show the phosphorylation change of STAT1 protein in Daudi cells of the group to which type 1 interferon (IFN-α 1, IFN-α 2a, IFN-α 2b, IFN-α 5, IFN-α 8, IFN-α 10, IFN-β 1a, IFN-ω, IFN-ε) was added and the group in which each interferon was treated with IFNAR1/2-Fc heterodimer (4GS*3) protein.

MODE FOR CARRYING OUT INVENTION

[0144] Hereinafter, the present invention will be described in detail.

[0145] However, the following examples are only illustrative of the present invention, and the content of the present invention is not limited to the following examples.

Example 1: Preparation of Expression Vector for Production of Type 1 Interferon Fc Fusion Receptor Protein

[0146] Human IFNAR1 (P17181, 28-436 aa.), IFNAR2 (P48552, 27-243 aa.) and IGHG (P01857, 100-330 aa) amino acid sequences were used to develop an Fc fusion protein capable of binding or neutralizing type 1 interferon protein. As shown in FIG. 1 (left), Fc were labeled with a polypeptide linker (L; IEGRMD) at the C-terminus of the expression vector in all Fc fusion protein expression vectors, it is linked by IFNAR1 or IFNAR2 between the C-terminus and the N-terminus including the Kozac sequence (1) and the extracellular secretion inducing signal sequence (S), or directly linked.

[0147] As shown in FIG. 1 (right), the sequence including each gene was linked between restriction enzymes NheI and XhoI. Expression vectors were named pIFNAR1-FcWT, pIFNAR2-FcWT, pIFNAR1-FcA, pIFNAR2-FcB, pIFNAR1-FcB and pLeader-FcA, respectively.

[0148] Codon optimization of the gene comprising the polynucleotide sequence (SEQ ID NO: 1), the extracellular domain of IFNAR1 (SEQ ID NO: 3), the extracellular domain of IFNAR2 (SEQ ID NO: 5), a polypeptide linker (SEQ ID NO: 7), and the Fc portion of human IGHG (SEQ ID NO: 11) and variants (SEQ ID NO: 13, SEQ ID NO: 15) comprising the Kozac sequence and the signal sequence was performed to synthesize the gene to prepare the nucleotide sequence fragments e to g of FIG. 1. An expression vector was prepared using T4 DNA ligase (RBC) between NheI and Xho I among the restriction enzyme sites of the expression vector (pOptiVec-TOPO) for each nucleotide sequence fragment.

[0149] In addition, the expression vector was prepared in the same manner except that the linker of SEQ ID NO: 33 was used instead of the polypeptide linker of SEQ ID NO: 7. In other words, the gene comprising the polynucleotide sequence (SEQ ID NO: 1), the extracellular domain of IFNAR1 (SEQ ID NO: 3), the extracellular domain of IFNAR2 (SEQ ID NO: 5), a polypeptide linker (SEQ ID NO: 33), and the Fc portion of human IGHG (SEQ ID NO: 11) and variants (SEQ ID NO: 13, SEQ ID NO: 13) number 15) comprising the Kozac sequence and the signal sequence was codon-optimized to synthesize the gene to prepare the nucleotide sequence fragments e to g of FIG. 1. An expression vector was prepared using T4 DNA ligase (RBC) between NheI and Xho I among the restriction enzyme sites of the expression vector (pOptiVec-TOPO) for each nucleotide sequence fragment.

Example 2: Protein Expression Purification, Structure Confirmation and Binding Confirmation

[0150] The following expression and purification processes were performed in order to produce a protein having the structure shown in FIG. 2c using expression vectors into which the nucleotide sequence fragments of each combination were inserted.

[0151] In order to maintain the native post-translational modification (PTM) of each type 1 interferon receptor, all recombinant protein expression use Expi293TM expression medium in 300-600 mL according to the protocol of the transient expression system, using the human-derived expression cell, Expi293 cell line (Thermo Fisher Scientific). Depending on the final protein, one or two expression vectors were transfected into cells, and then Enhancers 1 and 2 were added according to the manufacturer's protocol. Thereafter, the culture medium cultured for 6 days was centrifuged at 6580 rpm, 20 minutes, and 8° C., and then filtered using a 0.22 μm polytyrene filter (Corning). All proteins were purified in the AKTA avant purification system (GE healthcare Life Sciences), and the column filled with MabSelect SuRe (GE healthcare Life Science) was equilibrated as much as 2 CV using 20 mM sodium phosphate, pH 7.2 and 150 mM NaCl buffer at a rate of 5 ml/min, and then the culture solution was flowed to bind to the resin.

[0152] Thereafter, the recombinant protein was eluted with 100 mM citrate buffer, pH 3.5 buffer after a column wash process using 5 CV of 35 mM sodium phosphate, pH 7.2, 500 mM NaCl buffer and 1 CV of 20 mM sodium phosphate, pH 7.2 buffer. The eluted protein was concentrated to 3 ml using an Amicon Ultra Centrifugal filter (50K-cut off, Merck Millipore) after dialysis for 12 hours and 3 times using 4L of 1×PBS buffer (Biosesang). The concentrated recombinant protein was additionally separated from the target protein using the same buffer used for dialysis on a Hiload Superdex 200 pg (GE healthcare Life Sciences) column.

[0153] As a result, as shown in FIG. 2a, the size of each Fc fusion protein was compared through size exclusion chromatography, and it was expected to have the protein structure shown in FIG. 2c.

[0154] In addition, as shown in FIG. 2b, binding between the IFNAR1/2 hetero Fc fusion protein and human IFN β-1a was confirmed, respectively in native conditions (7.5% Mini-PROTEIN® TGXTM Precase protein gels), reducing conditions (Any kDTM Mini-PROTEIN® TGXTM Precast protein gel, Bio-rad)

[0155] The FC portion of the amino acid sequence of the present invention includes the HINGE portion of IGHG1 (100-330a.a.), and a disulfide bond is formed by amino acids 109-109 and 112-112 to form a dimer. In more detail, the formation of a heterodimer occurs dominantly from a homodimer to a heterodimer due to a change in the specific amino acid sequence of the CH3 portion of FC.

Example 3: SPR Analysis Using Fc Fusion Protein

[0156] Binding affinity and kinetics of Fc fusion proteins (IFNAR2-Fc heterodimer, IFNAR1/2-Fc heterodimer, IFNAR1-Fc heterodimer) expressed/purified through the expression vector combination of FIG. 1 and hIFN β-1a among Type I IFNs were analyzed.

[0157] In this experiment, (IFNAR1-Fc EW+-Fc RVT), (IFNAR2-Fc EW+-Fc RVT) and (IFNAR1-Fc EW+IFNAR2Fc RVT) were used to analyze binding strength and kinetics. When expressing (IFNAR1-Fc RVT+IFNAR2-Fc EW), as it was confirmed that the problem that the homodimer form appeared more than (IFNAR1-Fc EW+IFNAR2-Fc RVT) was confirmed, so (IFNAR1-Fc RVT+IFNAR2-Fc EW) was not used.

[0158] Based on Biacore T200 (GE Healthcare Life Sciences), 25° C., 30 μl/min conditions and 0.005% DPBST (DPBS/modified, Hyclone and Tween20, Signal) running buffer were used, and the gold sensor chip was amine-coupled with an anti-human Fcγ capture antibody (AffiniPure Goat Anti-Human IgG, Fcγ fragment specific, Jackson ImmunoResearch). Each analyte was tested under 180 seconds, association and 600 seconds, and dissociation conditions.

[0159] As shown in FIG. 3, the curve fitting was performed according to hIFNb-1a-IFNAR2 (1:1 fitting model), hIFNb-1a-IFNAR1/2 (heterogeneous ligand model), hIFNb-1a-IFNAR1 (two-state binding models) sensorgram shape and the docking model prediction. In particular, the IFNAR1/2 hetero Fc fusion protein showed a low KD value. (See Table 2 and Table 3)

TABLE-US-00002 TABLE 2 k.sub.1a × 10.sup.5 k.sub.1d × 10.sup.−3 K.sub.1D k.sub.2a × 10.sup.7 k.sub.2d × 10.sup.−3 K.sub.2D Ligand Analyte (M.sup.−1 s.sup.−1) (s.sup.−1) (nM) (M.sup.−1 s.sup.−1) (s.sup.−1) (pM) IFNAR2-Fc IFN-β-1a.sup.a 75.45 3.556 0.47 heterodimer IFN-β-1a.sup.b 4.26 IFNAR1/2-Fc IFN-β-1a.sup.c 76.85 0.0044 0.0006 1.57 3.109 198.6 heterodimer IFN-β-1a.sup.b 4.775 IFNAR1-Fc IFN-β-1a.sup.a 0.2368 0.9723 41.11 heterodimer IFN-β-1a.sup.b 81.51 .sup.aSensorgrams fit with one to one binding model (A + B     AB) .sup.bthe steady state equilibrium analysis were estimated using each parameter at 179.04 sec. .sup.cSensorgrams fit with heterogeneous ligand model (A + B     AB + C     ABC)

TABLE-US-00003 TABLE 3 k.sub.1a × 10.sup.5 k.sub.1d × 10.sup.−3 k.sub.2a × 10.sup.−3 k.sub.2d × 10.sup.−3 K.sub.2D Ligand Analyte (M.sup.−1 s.sup.−1) (s.sup.−1) (s.sup.−1) (s.sup.−1) (nM) IFNAR1-Fc heterodimer IFN-β-1a.sup.a 5.426 129.7 1.776 45.75 48.97 IFNAR2-Fc heterodimer IFN-β-1a.sup.a 2.23E+06 8.81E+05 22.04 3.444 0.5342 .sup.aSensorgrams fit with two-state binding model (A + B     AB     AB*)

Example 4: Confirmation of Biological Activity Neutralizing Ability by Ligand of Fc Fusion Protein

[0160] The following experiment was conducted to confirm the neutralizing ability of the Fc fusion protein to the biological activity caused by the ligand. As in Example 3, (IFNAR1-Fc EW+-Fc RVT), (IFNAR2-Fc EW+-Fc RVT) and (IFNAR1-Fc EW+IFNAR2Fc RVT) were used in this experiment.

[0161] All experiments were designed based on the anti-proliferative effect of hIFN-β using the Ez-cytox cell viability assay kit (Deaillab) in Daudi cells, where the expression of each receptor is high. This test method showed test results that well reflect the biophysical characteristics of the ligand, similar to the results of the kinetics analysis of SPR.

[0162] Specifically, in a 96-well plate (SPL), the concentration of hIFN-β-1a in cells of 3×10.sup.3 cells was determined by concentration (10 nM of IFNAR1-Fc heterodimer, IFNAR2-Fc heterodimer, IFNAR1/2-Fc heterodimer; 6 pM of IFNAR1-Fc heterodimer; IFNAR1/2-Fc heterodimer), and then the reaction was carried out in a cell incubator at 37° C. and 5% CO.sub.2 for 72 hours. Then, after adding the Ex-cytox reagent according to the manufacturer's protocol, after an additional 3 hours of reaction, it was measured at 450 nm with a Microplate reader (Genios Pro, Tecan), and IC.sub.50 values were compared and analyzed using nonlinear regression analysis (GraphPad Prism version 7.0 software, san diego, Ca, USA). The neutralizing ability of each protein of the present invention was confirmed for statistical significance through One-way ANOVA, Bonferroni's multiple comparisons post hoc test.

[0163] As shown in FIG. 4a, it was confirmed that anti-cell proliferation by interferon was significantly reduced as compared to the case of a fusion protein that blocks IFNAR1 and IFNAR2, respectively, when interferon is treated with an Fc fusion protein that blocks both IFNAR1 and IFNAR2 binding.

[0164] As shown in FIG. 4b, the IC50 value was the highest when the Fc fusion protein blocking both IFNAR1 and IFNAR2 according to the present invention was added. Therefore, it was confirmed that anti-cell proliferation by interferon was superior to cell viability when blocking IFNAR1 or IFNAR2, respectively.

[0165] As shown in FIG. 4c, it was confirmed that the cell viability increased in a concentration-dependent manner when the IFNAR1/2-Fc heterodimer with the highest neutralizing ability was treated.

Example 5: Confirmation of Signal Mechanism Activity Neutralizing Ability by Ligand of IFNAR1/2-Fc Heterodimer

[0166] In the same manner as in Example 4, except for the IFNAR1/2-Fc heterodimer mentioned below, the ability to neutralize the biological activity by the ligand of the IFNAR1/2-Fc heterodimer was confirmed.

[0167] Specifically, the group added IFNs (IFN-α 1 (pbl assay science_cat #11125-1), IFN-α 2a (pbl assay science_cat #11100-1), IFN-α 2b (pbl assay science_cat #11105-1), IFN-α 5 (pbl assay science_cat #11135-1), IFN-α 8 (pbl assay science_cat #11115-1), IFN-α 10 (pbl assay science_cat #11120-1), IFN-β 1a (pbl assay science_cat #11415-1), IFN-ω (pbl assay science_cat #11395) -One) at a concentration of 1 nM and the group that each interferon was treated with a type 1 interferon Fc fusion receptor protein at a concentration of 10 nm in cells of 3×10.sup.3 cells were reacted in a cell incubator at 37° C. and 5% CO.sub.2 for 72 hours. Then, after adding the Ex-cytox reagent according to the manufacturer's protocol, and after an additional 2 hours of reaction, it was measured at 450 nm with a Microplate reader (Genios Pro, Tecan), and IC.sub.50 values were compared and analyzed using nonlinear regression analysis (GraphPad Prism version 7.0 software, san diego, Ca, USA).

[0168] As a result, as shown in FIG. 5, it was confirmed that the cell viability was significantly superior as compared with the cell viability treated only with interferon, when IFN-α 1, IFN-α 2a, IFN-α 2b, IFN-α 5, IFN-α 8, IFN-α 10, IFN-β 1a, IFN-ω was treated with an IFNAR1/2-Fc heterodimer (using 4GS*3 as a linker) that blocks both the binding of IFNAR1 and IFNAR2.

[0169] Through these results, it was found that the regulation of excessive interferon signals for cells is possible by the dimer-type polypeptide to which the monomer (monomer) comprising the interferon receptor fragment or antibody Fc fragment of the present invention is bound, and through this, it was confirmed that it would show excellent pharmacological effects on patients with type 1 interferon-mediated diseases such as systemic lupus erythematosus, Sjogren's syndrome, systemic sclerosis, myositis and rheumatoid arthritis in which interferon-inducible genes expressed in response to excessive interferon signals are high.

Example 6: Confirmation of Chemical Signal Inhibition Ability by Ligand of IFNAR1/2-Fc Heterodimer

[0170] All experiments were performed to confirm changes in STAT1 and its phosphorylated pSTAT1 protein by Type I IFNs using Western blot in Daudi cells, in which the expression of each receptor is high.

[0171] Specifically, in a 6-well plate (SPL_cat #30006), the group added IFNs (IFN-α 1 (pbl assay science_cat #11125-1), IFN-α 2a (pbl assay science_cat #11100-1), IFN-α 2b (pbl assay science_cat #11105-1), IFN-α 5 (phi assay science_cat #11135-1), IFN-α 8 (01 assay science_cat #11115-1), IFN-α 10 (phi assay science_cat #11120-1), IFN-β 1a (phi assay science_cat #11415-1), IFN-ω (phi assay science_cat #11395-) 1), IFN-ε (R&D systems_cat #9667-ME) at a concentration of 1 nM and the group that each interferon was treated with IFNAR1/2-Fc heterodimer protein at a concentration of 10 nM in the cells of 2×10.sup.6 cells were reacted in a cell incubator at 37° C. and 5% CO.sub.2 for 72 hours. Thereafter, cells were collected from the plate, total protein was extracted and determined with BCA protein assay kit (Thermo scientific_cat #23227). Total protein was separated on a 10% gel by sodium-dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transcribed onto a PVDF membrane (BIO RAD_cat #1620177). The membrane was blocked with 5% skim milk, 3% bovine serum albumin, 10 mmol/L Tris-HCL (pH 8.0), 150 mmol/L NaCl, and 0.05% Tween-20 for 1 hour at room temperature. The blocked membrane was treated with STAT1 antibody (cell signaling_cat #06-501), pSTAT1 antibody (cell signaling_cat #58D6), and beta actin antibody (santa cruz_cat #sc 47778) as primary antibodies (1:3000 dilution) overnight at 4° C. Thereafter, a secondary HRP-conjugated antibody, goat anti rabbit antibody (Invitrogen_cat #31460) and goat anti mouse antibody (invitrogen_cat #G21040), was treated at room temperature for 1 hour. After treating the membrane with ECL solution (BIO RAD_cat #1705061), it was visualized on a film (Agfa healthcare_cat #EA8EC) using a developer and fixer from poohung.

[0172] As a result, as shown in FIGS. 6a and 6b, it was confirmed that phosphorylation of the pSTAT1 protein was significantly reduced as compared with the high phosphorylation of pSTAT1 protein when only interferon was treated, when the IFNAR1/2-Fc heterodimer (4GS*3) protein, which blocks both the binding of IFNAR1 and IFNAR2, was treated with interferon (IFN-α 1, IFN-α 2a, IFN-α 2b, IFN-α 5, IFN-α 8, IFN-α 10, IFN-β 1a, IFN-ω, IFN-ε). This indicates that the IFNAR1/2-Fc heterodimer protein binds to type 1 interferon and exhibits neutralizing ability, indicating that interferon-related signaling in the cell is inhibited.

INDUSTRIAL APPLICABILITY

[0173] The type 1 interferon neutralizing Fc-fusion protein of the present invention has excellent industrial applicability as while blocking the binding of type 1 interferon to the interferon receptor, has excellent initiation and biological activity inhibition of signaling mechanisms, so it can be very usefully used in the development of therapeutic agents for preventing or treating type 1 interferon-mediated diseases.