Neutralizing Fc fusion protein of type I interferon and use thereof

Abstract

The present invention relates to a type I interferon neutralizing Fc fusion protein and uses thereof, more particularly to a dimeric polypeptide containing a monomer comprising an interferon receptor fragment or an antibody Fc fragment, a method for producing the same, and a pharmaceutical composition containing the same. The type I interferon neutralizing Fc fusion protein of the present invention has excellent ability to inhibit the signaling and biological activity of interferon by blocking the binding of type I interferon to interferon receptors, and can effectively treat type I interferon-mediated diseases.

Description

[Technical field]

[0001] This application claims priority to Korean Patent Application No. 10-2018-0168801, filed on December 24, 2018, the entire specification of which is incorporated herein by reference.

[0002] The present invention relates to a neutralizing Fc fusion protein of type I interferon and uses thereof, and more particularly to a dimeric polypeptide in which a monomer containing an interferon receptor fragment or an antibody Fc fragment is bound, a method for producing the same, and a composition for preventing or treating diseases associated with the abnormal expression of type I interferon or interferon-inducible genes, comprising the same. [Background technology]

[0003] Type I interferons (IFNs) (IFN-α, IFN-β, IFN-ω, IFN-τ) are a group of structurally linked cytokines with antiviral, antitumor and immunomodulatory effects. The human IFNα locus has two subgroups. The first subgroup has 14 nonallelic genes and four pseudogenes with at least 80% identity. In the second subgroup, αII or omega (ω) has one functional gene and five pseudogenes that show 70% identity with the IFNα gene. The IFNα subtypes have different specific activities, but show the same biological spectrum and share the same cellular receptor. Interferon-β (IFNβ) is encoded by a single gene with approximately 50% identity with the IFNα gene. Gamma interferon, produced by activated lymphocytes, does not share identity with alpha / beta interferons and does not react with their receptors.

[0004] All human type I interferons bind to a cell surface receptor (IFN alpha receptor, IFNAR) composed of two membrane proteins, IFNAR-1 and IFNAR-2. IFNAR-1 is required for high affinity binding of the IFNAR complex with distinct specificity. Although the functional differences between the individual type I IFN subtypes remain unclear, it is thought that each exerts distinct functions on components of the IFNAR receptor, potentially leading to different signaling events.

[0005] Early studies into the function of type I IFNs focused on innate defense against viral infections, but more recent studies have implicated type I IFNs as immunoregulatory cytokines with effects on adaptive immune responses. In particular, type I IFNs have been shown to facilitate the maturation of naive T cells in the Th1 pathway, enhance antibody production, and support the functional activity and survival of memory T cells.

[0006] Recent studies from several groups have suggested that IFN-α can enhance the maturation or activation of dendritic cells (DCs), and increased expression of type I interferons has been described in the literature in association with a number of autoimmune diseases. The best-studied examples include insulin-dependent diabetes mellitus (IDDM), which is all associated with elevated levels of IFN-α (Foulis et al. (1987) Lancet 2:1423), systemic lupus erythematosus (SLE) (Hooks et al. (1982) Arthritis Rheum. 25:396), Sjögren's syndrome (Yihong Yao et al. (2013) Autoimmun Rev. 12(5):558-66), inflammatory myositis (Baechler et al. (2007) Mol Med. 13(1-2): 59-68), and rheumatoid arthritis (RA), in which IFN-β plays an even more important role (Hertzog et al. (1988) Clin. Immunol. Immunopathol. 48:192), Hopkins and Megol (1988) Clin. Exp. Immunol. 73:88; Alvin and Miller (1984) Arthritis Rheum. 27:582).

[0007] It has also been shown that administration of interferon α, a representative type of type I interferon, aggravates the underlying disease in patients with psoriasis and multiple sclerosis, and induces symptoms similar to SLE in patients with no prior history of autoimmune disease. Interferon α has also been shown to induce glomerulonephritis in normal mice and accelerate the development of concurrent autoimmune disease in NZB / W mice. Furthermore, it has been found that IFN-α therapy induces undesirable side effects, including fever and neuropathy, in some cases.

[0008] Although there are various types of type I interferons, and recent studies on SLE patients have shown that not only IFN-α but also IFN-β act as a cause of mucocutaneous diseases, there is a problem that type I interferon-mediated disease treatments are only focused on IFN-α, such as vaccines (IFN-kinoid) or anti-IFN-α monoclonal neutralizing antibodies (Sifalimumab, Rontalizumab, AGS-009) treatments. Another problem with such neutralizing antibodies is that they cannot neutralize all of the parts that bind to the two different receptors of interferon, and only have the ability to bind to IFNAR1 or IFNAR2. To address this issue, Anifrolumab (MAbs. 2015 March-April; 7(2):428-439.), which specifically binds to the type I interferon receptor IFNAR1 and shows neutralizing ability, is being developed. However, the characteristics of these neutralizing antibody therapeutics are limited by the complex signaling mechanisms of type I interferons, such as the non-canonical pathway mediated by IFNAR1 (Nat Immunol. 2013 September; 14(9):901-7.) or IFNAR2 (Sci Signal. 2014 May 27; 7 (327):ra50; PLoS One. 2017; 12(8):e0182866), other than the canonical pathway mediated by IFNAR1 and IFNAR2, and the inability to remove type I interferons from the body. Summary of the Invention [Problem to be solved by the invention]

[0009] Detailed Description of the Invention technical challenges The inventors of the present invention, while developing a therapeutic agent for type I interferon-mediated diseases, found that a dimeric polypeptide comprising a fragment of interferon receptor 1 and a fragment of interferon receptor 2 not only binds to type I interferon, but also significantly inhibits the initiation of signaling mechanisms and biological activity upon treatment, and thus completed the present invention.

[0010] Therefore, the object of the present invention is to As a dimeric polypeptide to which a monomer containing an interferon receptor fragment or an antibody Fc fragment is bound, The monomer is a polypeptide selected from the group consisting of (i), (ii) and (iii): (i) a monomer comprising a fragment of interferon receptor 1 (IFNAR1) and an Fc fragment of an antibody; (ii) a monomer comprising a fragment of interferon receptor 2 (IFNAR2) and an Fc fragment of an antibody; and (iii) It provides an Fc fragment of an antibody.

[0011] Yet another object of the present invention is to provide A polynucleotide encoding the polypeptide is provided.

[0012] A further object of the present invention is to provide a vector comprising the polynucleotide.

[0013] A further object of the present invention is to provide a host cell transfected with said vector.

[0014] Yet another object of the present invention is to provide (a) providing a host cell; (b) culturing the designated cells; and (c) recovering the polypeptide from the cells or the culture medium thereby producing the polypeptide.

[0015] Yet another object of the present invention is to provide The present invention provides a pharmaceutical composition for preventing or treating a type I interferon-mediated disease or disorder, which comprises a polypeptide as an active ingredient. The present invention also provides a pharmaceutical composition for preventing or treating a type I interferon-mediated disease or disorder, comprising the polypeptide as an active ingredient. The present invention also provides a pharmaceutical composition for preventing or treating type I interferon-mediated diseases or disorders, which essentially comprises a polypeptide as an active ingredient.

[0016] Yet another object of the present invention is to provide The present invention also provides a use of the polypeptide for the manufacture of a medicament for the prevention or treatment of a type I interferon mediated disease or disorder.

[0017] Yet another object of the present invention is to provide The present invention also provides a method for treating a type I interferon-mediated disease or disorder, comprising the step of administering to an individual in need thereof an effective amount of a composition containing the polypeptide as an active ingredient. [Means for solving the problem]

[0018] Therefore, in order to achieve the object of the present invention, As a dimeric polypeptide to which a monomer containing an interferon receptor fragment or an antibody Fc fragment is bound, The monomer is a polypeptide selected from the group consisting of (i), (ii) and (iii): (i) a monomer containing a fragment of interferon receptor 1 (IFNAR1) and an Fc fragment of an antibody; (ii) a monomer comprising a fragment of interferon receptor 2 (IFNAR2) and an Fc fragment of an antibody; and (iii) providing an Fc fragment of an antibody;

[0019] In order to achieve yet another object of the present invention, A polynucleotide encoding the polypeptide is provided.

[0020] In order to achieve yet another object of the present invention, there is provided a vector comprising the above polynucleotide.

[0021] In order to achieve yet another object of the present invention, there is provided a host cell transformed with the above vector.

[0022] In order to achieve yet another object of the present invention, (a) providing a host cell; (b) culturing the designated cells; and (c) recovering the polypeptide from said cells or from the culture medium thereby producing the polypeptide.

[0023] In order to achieve yet another object of the present invention, The present invention provides a pharmaceutical composition for preventing or treating a type I interferon-mediated disease or disorder, comprising the polypeptide as an active ingredient. Also provided is a pharmaceutical composition for preventing or treating a type I interferon-mediated disease or disorder, comprising the polypeptide as an active ingredient. The present invention also provides a pharmaceutical composition for preventing or treating a type I interferon-mediated disease or disorder, which essentially comprises the polypeptide as an active ingredient.

[0024] In order to achieve yet another object of the present invention, Use of the polypeptide for the manufacture of a medicament for the prevention or treatment of a type I interferon mediated disease or disorder is provided.

[0025] In order to achieve yet another object of the present invention, The present invention provides a method for treating a type I interferon-mediated disease or disorder, comprising the step of administering to an individual in need thereof an effective amount of a composition containing the polypeptide as an active ingredient.

[0026] The present invention will be described in detail below.

[0027] Therefore, the present invention relates to a dimeric polypeptide comprising a monomer containing an interferon receptor fragment or an antibody Fc fragment, The monomer provides a polypeptide selected from the group consisting of: (i), (ii) and (iii) below. (i) a monomer comprising a fragment of interferon receptor 1 (IFNAR1) and an Fc fragment of an antibody; (ii) a monomer comprising a fragment of interferon receptor 2 (IFNAR2) and an Fc fragment of an antibody; and (iii) An Fc fragment of an antibody.

[0028] In this specification, including the claims, the term "fragment of an interferon receptor" is meant to include all polypeptides that have all or part of the amino acid sequence derived from a naturally occurring human interferon receptor and have the activity of a human interferon receptor.

[0029] The terms "interferon receptor 1", "IFNAR 1" and "IFNAR-1 antigen" are used interchangeably and include variants, isomorphs, species homomeric forms and analogs of human IFNAR 1 that contain at least one common epitope of IFNAR 1. Thus, the polypeptides of the invention may in some cases cross-react with IFNAR 1 from non-human species or other proteins structurally linked to human IFNAR 1 (e.g., human IFNAR-1 homomeric forms). In other cases, the polypeptides may be completely specific for human IFNAR-1 and do not show species or other types of cross-reactivity. The complete cDNA sequence of human IFNAR 1 has genbank accession numbers XM_005260964.2, NM_000629.2, or XM_011529552.2. The base sequence used in the present invention may include the extracellular domain (28-436a.a; P17181-1) in the sequence of IFNAR 1 isoform 1 (genebank accession number: NM_000629.2), or may include a part of the base sequence. The IFNAR 1 fragment of the present invention may be an interferon receptor 1 comprising the amino acid sequence shown in SEQ ID NO:4, and more preferably an interferon receptor 1 consisting of the amino acid sequence shown in SEQ ID NO:4.

[0030] In one embodiment of the present invention, the polypeptide was produced using interferon receptor 1 containing the amino acid sequence represented by SEQ ID NO: 4 (polynucleotide sequence represented by SEQ ID NO: 3. The polynucleotide sequence may include a termination codon. The same applies to all DNA base sequences below).

[0031] The terms "interferon receptor 2", "IFNAR 2" and "IFNAR-2 antigen" are used interchangeably and include variants, isomorphs, species homomers and analogs of human IFNAR 2 that contain at least one common epitope of IFNAR 2. Thus, the polypeptides of the invention may in some cases cross-react with IFNAR 2 from non-human species or other proteins structurally linked to human IFNAR 2 (e.g., human IFNAR-2 homomers). In other cases, the polypeptides may be completely specific for human IFNAR-2 and do not show species or other types of cross-reactivity. The complete cDNA sequence of human IFNAR 2 has genbank accession numbers NM_000874.4, NM_001289125.1, or NM_001289126.1. The nucleotide sequence used in the present invention may include the extracellular domain (27-243a.a; P48551-1) of the sequence of human IFNAR2 isoform 1 (genebank accession number: NM_001289125.1), and may include a part of the above-mentioned nucleotide sequence. The IFNAR2 fragment of the present invention may be an interferon receptor 2 comprising the amino acid sequence shown in SEQ ID NO:6, and more preferably an interferon receptor 2 consisting of the amino acid sequence shown in SEQ ID NO:6.

[0032] In one embodiment of the present invention, the polypeptide was prepared using a fragment of interferon receptor 2 containing the amino acid sequence shown in SEQ ID NO:6 (the polynucleotide sequence shown in SEQ ID NO:5).

[0033] Furthermore, the above-mentioned "polypeptide having all or part of the amino acid sequence derived from a fragment of an interferon receptor" means a polypeptide that contains all or a substantial part of the amino acid sequence of SEQ ID NO: 4 or SEQ ID NO: 6, which is the amino acid sequence of a fragment of a natural interferon receptor, or a polypeptide that is substantially similar to these polypeptides.

[0034] Here, the "polypeptide comprising a substantial portion of the entire amino acid sequence of SEQ ID NO:4 or SEQ ID NO:6" is defined as a polypeptide comprising a portion of the amino acid sequence of SEQ ID NO:4 or SEQ ID NO:6, which has the same or greater activity as a naturally occurring interferon receptor having the amino acid sequence of SEQ ID NO:4 or SEQ ID NO:6, or which retains the activity of a human interferon receptor even if the activity is lower, and the "polypeptide substantially similar to the entire amino acid sequence disclosed in SEQ ID NO:4 or SEQ ID NO:6, or a substantial portion thereof, which comprises multiple substituted amino acids, while having the same or greater activity as the interferon receptor of SEQ ID NO:4 or SEQ ID NO:6, or which still retains the activity of an interferon receptor even if the activity is lower.

[0035] An example of a polypeptide comprising a substantial portion of the entire amino acid sequence disclosed in SEQ ID NO:4 or SEQ ID NO:6 is a polypeptide comprising the amino acid sequence disclosed in SEQ ID NO:4 or SEQ ID NO:6 in which the N-terminal portion and / or the C-terminal portion has been deleted. An example of a polypeptide substantially similar to the entire amino acid sequence disclosed in SEQ ID NO:4 or SEQ ID NO:6 or a substantial portion thereof is a polypeptide in which multiple amino acids have been substituted but the amino acids before the substitution are chemically equivalent to the substituted amino acids, for example, when the hydrophobic amino acid alanine has been substituted with another hydrophobic amino acid, particularly when the hydrophobic amino acid has been substituted with a more hydrophobic amino acid, such as valine, leucine or isoleucine.

[0036] In the present invention, "fusion" refers to the integration of two molecules that are different or identical in function or structure, and may be fusion by any physical, chemical, or biological method that allows the antibody Fc portion or each monomer to bind to the interferon receptor. The fusion is preferably via a linker peptide, which can be attached, for example, to the C-terminus of an Fc fragment of an antibody.

[0037] The "Fc fragment of an antibody" may be an Fc fragment of an IgA, IgM, IgE, IgD, or IgG antibody, or a variant thereof. In one embodiment, the fragment is an Fc fragment of an IgG antibody (e.g., an Fc fragment of an IgG1, IgG2a, IgG2b, IgG3, or IgG4 antibody). In addition, a polypeptide comprising an Fc fragment of the present invention may be partially or entirely non-glycosylated or may be glycosylated. In addition, a polypeptide may be added to the Fc fragment to include multiple regions derived from an antibody. Additionally, the polypeptide may include an antigen binding domain derived from an antibody, and multiple polypeptides may form an antibody or an antibody-type protein.

[0038] In the present specification, the amino acid residue numbers of the Fc fragment of an antibody are based on the Kabat numbering system commonly used in the art (EU index as in Kabat et al., in Proteins of Immunological Interest 5th Ed., US Department of Health and Human Services, NIH Publication No. 91-3242, 1991).

[0039] In addition to including the wild-type Fc fragments of the present invention, one embodiment of the present invention can also include substituted Fc variants. The wild-type Fc fragment sequence of the present invention may comprise the amino acids shown in SEQ ID NO:8, and may preferably consist of the amino acids shown in SEQ ID NO:8. The substituted Fc fragment of the present invention comprises multiple amino acid substitutions selected from the group consisting of Q347R, K360E, D399V, F405T and K409W according to the Kabat numbering system. The substituted Fc fragment of the present invention may comprise K360E and K409W amino acid substitutions, and may preferably comprise the amino acid sequence represented by SEQ ID NO:12, and more preferably consist of the amino acid sequence represented by SEQ ID NO:12. The substituted Fc fragment of the present invention may contain Q347R, D399V, and F405T amino acid substitutions, and may preferably consist of the amino acid sequence represented by SEQ ID NO:14, and more preferably consist of SEQ ID NO:14. The Fc fragment of the antibody as a monomer forming a dimer may comprise SEQ ID NO:8, SEQ ID NO:12 or SEQ ID NO:14, with a linker or other short fragment peptide attached to the N-terminus or C-terminus. The linker may comprise an amino acid sequence selected from the group consisting of SEQ ID NO:10, SEQ ID NO:34, SEQ ID NO:53 to SEQ ID NO:76. The Fc fragment of the antibody as a monomer may comprise an amino acid sequence selected from the group consisting of SEQ ID NO:8, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:36, SEQ ID NO:38 and SEQ ID NO:40.

[0040] The "monomer comprising a fragment of interferon receptor 1 (IFNAR1) and an Fc fragment of an antibody" of the present invention may preferably comprise i) an interferon receptor 1 (IFNAR1) fragment comprising the amino acid sequence represented by SEQ ID NO:4, and ii) an antibody Fc fragment comprising an amino acid sequence selected from the group consisting of SEQ ID NO:8, SEQ ID NO:12, and SEQ ID NO:14, more preferably i) an interferon receptor 1 (IFNAR1) fragment comprising the amino acid sequence represented by SEQ ID NO:4, ii) a linker comprising an amino acid sequence selected from the group consisting of SEQ ID NO:10, SEQ ID NO:34, SEQ ID NO:53 to SEQ ID NO:76, and iii) an antibody Fc fragment comprising an amino acid sequence selected from the group consisting of SEQ ID NO:8, SEQ ID NO:12, and SEQ ID NO:14, and most preferably a monomer comprising or consisting of amino acids selected from the group consisting of SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:42, SEQ ID NO:44, and SEQ ID NO:46.

[0041] The "monomer comprising a fragment of interferon receptor 2 (IFNAR2) and an Fc fragment of an antibody" of the present invention may preferably comprise i) a fragment of interferon receptor 2 (IFNAR2) comprising the amino acid sequence represented by SEQ ID NO:6, and ii) an Fc fragment of an antibody comprising an amino acid sequence selected from the group consisting of SEQ ID NO:8, SEQ ID NO:12, and SEQ ID NO:14, more preferably i) a fragment of interferon receptor 2 (IFNAR2) comprising the amino acid sequence represented by SEQ ID NO:6, ii) a linker comprising an amino acid sequence selected from the group consisting of SEQ ID NO:10, SEQ ID NO:34, SEQ ID NO:53 to SEQ ID NO:76, and iii) an Fc fragment of an antibody comprising an amino acid sequence selected from the group consisting of SEQ ID NO:8, SEQ ID NO:12, and SEQ ID NO:14, and most preferably comprises or is composed of amino acids selected from the group consisting of SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:48, SEQ ID NO:50, and SEQ ID NO:52.

[0042] The polypeptide of the present invention may neutralize type I interferon, and therefore can be used as a type I interferon neutralizing antibody. The term "type I interferon" as used herein is intended to refer to the large type I interferon population of molecules that are ligands for IFNAR 1 and IFNAR 2 (i.e., the large type I interferon population of molecules that can bind to IFNAR 1 or IFNAR 2). Examples of type I interferon ligands include interferon alpha 1, 2a, 2b, 4, 5, 6, 7, 8, 10, 14, 16, 17, 21, interferon beta, interferon omega, and interferon epsilon.

[0043] A person of ordinary skill in the art can use the polypeptides without restriction in carrying out the present invention.

[0044] The present invention also provides a dimeric polypeptide in which the monomers are linked to the antibody or fragment thereof by a peptide linker. The peptide linker means a molecule that is a short fragment of an amino acid or an amino acid analog in which amino acids or substances similar to amino acids are connected to each other by peptide bonds, and serves to connect multiple separate substances to each other. Glycine, serine, alanine, etc. are used as the main constituent amino acids, and a glycine-serine linker, a glycine-serine-alanine linker, etc. can be used. According to a preferred embodiment of the present invention, the linker is composed of or includes the amino acid sequences shown in SEQ ID NO: 8 and SEQ ID NO: 10.

[0045] The polypeptides of the present invention may preferably include flexible linker sequences inserted between the monomers. The linker means a peptide linker of natural origin or a peptide linker of synthetic origin. The peptide linker is composed of a linear amino acid chain, in which the 20 naturally occurring amino acids are monomeric components. The linker may have a repeated amino acid sequence, and may have the sequence of a naturally occurring polypeptide, for example, a polypeptide with a hinge function. All peptide linkers may be encoded by a nucleic acid molecule, and therefore may be expressed by recombinant methods. Since the linker is itself a peptide, each monomer may be connected to the linker through a peptide bond to form a dimer.

[0046] The linker is composed of amino acids connected together by peptide bonds, preferably 1-20 amino acids connected by peptide bonds, where the amino acids are preferably selected from among the 20 naturally occurring amino acids, where one or more of these amino acids are glycosylated as understood by one of ordinary skill in the art. Preferably, but not limited to, the 1-20 amino acids are selected from glycine, alanine, proline, asparagine, glutamine, and lysine.

[0047] Suitable linkers include, for example, cleavable linkers and non-cleavable linkers. Cleavable linkers are typically easily cleaved under intracellular conditions. Suitable cleavable linkers include, for example, peptide linkers that are cleavable by intracellular proteases, such as lysosomal or endosomal proteases.

[0048] The linker is, for example, connected at its N-terminus to the C-terminus of the interferon receptor. Preferably, the linker can be directly connected to the C-terminus of the interferon receptor by connecting a base sequence encoding the linker sequence to an expression vector that expresses the protein of the present invention so that the expression frame of the protein matches, and then the linker can be directly connected to the antibody expressed by the expression vector. Alternatively, the N-terminus of the Fc portion of the antibody of the present invention is connected to the C-terminus of the linker.

[0049] The peptide linker of the present invention may be any peptide linker known in the art, but is preferably a glycine-serine linker, a helix-forming peptide linker, or a peptide linker that may contain or consist of the amino acid sequence of SEQ ID NO:8 or SEQ ID NO:10.

[0050] Preferably, a gly-ser linker, e.g., (Gly x Ser y ) z (x is an integer from 1 to 5, y is an integer from 1 to 2, and z is an integer from 1 to 6), for example, (gly 4 ser 1 ) 3 Or (gly 3 ser 2 ) 3 In the case of a helix-forming peptide linker (or rigid helical linker), A(EAAAK) n A (n = an integer from 2 to 5) may also be used.

[0051] Preferably, the peptide linker of the present invention is GGGGS, (GGGGS) 3, (GGGGS) n (n = 1, 2, 4), (Gly) 6 , (Gly) 8 , AEAAAKEAAAKA, A(EAAAK) 4 ALEA(EAAAK) 4 A, (EAAAK) 3 , EAAAK, (EAAAK) 2 , (Ala-Pro) n (n is 5 to 17, and the length is 10 to 34 aa), PAPAP, or a peptide linker consisting of an amino acid sequence selected from SEQ ID NO:10, SEQ ID NO:34, and SEQ ID NO:53 to SEQ ID NO:76.

[0052] In the present invention, the peptide linker may be appropriately configured by overlapping the above-exemplified peptide linkers or combining them with other linkers as necessary.

[0053] In the present invention, Fc heterodimerization techniques known in the art can be used for Fc heterodimerization. The Fc heterodimerization techniques are shown in Table 1 below.

[0054] [Table 1] The present invention also provides a polypeptide characterized in that the polypeptide comprises the amino acid sequence of SEQ ID NO: 4 or SEQ ID NO: 6, or comprises both of them.

[0055] The present invention also provides a method for producing a method for manufacturing a semiconductor device comprising the steps of: (a) a monomer comprising a fragment of interferon receptor 1 represented by the amino acid sequence of SEQ ID NO:4 and an Fc fragment of an antibody; (b) providing a dimeric polypeptide comprising a monomer comprising a fragment of interferon receptor 2 represented by the amino acid sequence of SEQ ID NO:6 and an Fc fragment of an antibody;

[0056] The monomer (a) comprises a fragment of interferon receptor 1 represented by the amino acid sequence of SEQ ID NO: 4 and an Fc fragment of an antibody, and may preferably be composed of or comprise an amino acid sequence selected from the group consisting of SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 42, SEQ ID NO: 44 and SEQ ID NO: 46.

[0057] The monomer (b) comprises a fragment of interferon receptor 2 represented by the amino acid sequence of SEQ ID NO: 6 and an Fc fragment of an antibody, and may preferably be composed of or comprise an amino acid sequence selected from the group consisting of SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 48, SEQ ID NO: 50 and SEQ ID NO: 52.

[0058] The polypeptide of the present invention may preferably be an antibody that mediates type I interferon.

[0059] The present invention also provides a polynucleotide encoding the polypeptide.

[0060] As used herein, "polynucleotide" or "nucleic acid" refers to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) in either single-stranded or double-stranded form, and includes known analogs of natural nucleotides that hybridize to nucleic acids in a manner similar to naturally occurring nucleotides, unless otherwise limited.

[0061] A monomer comprising a fragment of interferon receptor 1 represented by the amino acid sequence of SEQ ID NO:4 and an Fc fragment of an antibody may preferably consist of or comprise a DNA sequence selected from the group consisting of SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:41, SEQ ID NO:43, and SEQ ID NO:45.

[0062] The monomer comprising a fragment of interferon receptor 2 represented by the amino acid sequence of SEQ ID NO:6 and an Fc fragment of an antibody may preferably consist of or comprise a DNA sequence selected from the group consisting of SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:47, SEQ ID NO:49, and SEQ ID NO:51.

[0063] The polynucleotide may be any that encodes the polypeptide of the present invention, and includes all of DNA, cDNA, and RNA sequences. It means a polynucleotide that encodes a peptide having an amino acid sequence represented by the sequence or an amino acid sequence having at least 70% homology with the amino acid sequence, which may be isolated from nature or produced by genetic engineering methods known in the art.

[0064] The present invention also provides a vector comprising the polynucleotide.

[0065] The vector refers to an expression vector prepared by a person skilled in the art by inserting the polynucleotide of the present invention into the vector by any method known in the art and using appropriate transcription / decoding regulatory sequences to express the polypeptide of the present invention.

[0066] The polynucleotide sequence cloned according to the present invention may be operably linked to an appropriate expression control sequence, and the operably linked gene sequence and the expression control sequence may be included in one expression vector that includes a selection marker and a replication origin together. "Operably linked" means that the polynucleotide sequence is connected to the expression control sequence in a manner that allows gene expression. The "expression control sequence" means a DNA sequence that controls the expression of the operably linked polynucleotide sequence in a specific host cell. Such a control sequence may include one or more selected from the group consisting of a promoter for performing transcription, an optional operator sequence for controlling transcription, a sequence encoding a suitable mRNA ribosome binding site, and a sequence controlling the termination of transcription and translation.

[0067] The vector used as the mother vector of the expression vector is not particularly limited, and all plasmids, viruses, or other mediators commonly used for expression in microorganisms used as host cells in the technical field to which the present invention pertains can be used. For example, the plasmids include E. coli-derived plasmids (pBR322, pBR325, pUC118 and pUC119, pET-22b(+)), Bacillus subtilis-derived plasmids (pUB110 and pTP5), and yeast-derived plasmids (YEp13, YEp24, and YCp50), and the viruses include retroviruses, adenoviruses, or animal viruses such as vaccinia viruses, and insect viruses such as baculoviruses, but are not limited thereto.

[0068] The present invention also provides a host cell transformed with the vector.

[0069] A host cell can be chosen to regulate the expression of the inserted sequence or to direct the gene product in a particular manner as desired. Different host cells have characteristic and specific mechanisms for protein translation and post-translational processing and modification. An appropriate cell line or host system can be chosen to provide for the desired modification and processing of the expressed heterologous protein. Expression in yeast can produce a biologically active product. Expression in eukaryotic cells can increase the likelihood of "native" folding.

[0070] Any host cell known in the art may be used as a host cell capable of stably and continuously cloning and expressing the vector of the present invention, such as E. coli JM109, E. coli BL21DE, E. coli DH5, E. coli RR1, E. coli LE392, E. coli B, E. coli X1776, E. coli W3110, etc., and also include strains of the genus Agrobacterium, such as Agrobacterium A4, bacilli, such as Bacillus subtilis, other enterobacteria, such as Salmonella typhimurium or Serratia marcescens, and various strains of the genus Pseudomonas.

[0071] Furthermore, when the vector of the present invention is transformed into a eukaryotic cell, yeast (Saccharomyces cerevisiae), insect cells, and human cells (e.g., CHO cell lines (Chinese hamster ovary), Expi293, W138, BHK, COS-7, 293, HepG2, 3T3, RIN, and MDCK cell lines) can be used as host cells.

[0072] In the present invention, the host cell may preferably be an Expi293 or CHO cell line.

[0073] The method of transferring a vector into a host cell to transform the host cell may be any known method, and is not particularly limited. For example, transformation can be performed by calcium phosphate precipitation, DEAE-dextran method, electroporation, direct microinjection, DNA-loaded liposome method, liofectamine-DNA complex method, cell sonication, gene bombardment using high velocity microprojectile, polycation method, and receptor-mediated transfection. Some of these techniques can be modified for in vivo or in vitro use.

[0074] The present invention also provides a method for producing a dimeric polypeptide, comprising the steps of: (a) providing a host cell; (b) culturing the provided cell; and (c) recovering the polypeptide from the cell or the culture medium, thereby producing the polypeptide of the present invention.

[0075] The transformed cells are cultured under suitable conditions that allow the expression of the fusion protein (or fusion polypeptide), and these conditions can be performed based on methods already known to those skilled in the art. The transformed cells can be cultured in large quantities by a conventional culture method. As the culture medium, a medium composed of a carbon source, a nitrogen source, vitamins and minerals can be used, for example, 2XYT medium can be used. The cells can be cultured under conventional cell culture conditions, for example, at a temperature range of 15°C to 45°C for 10 to 40 hours. In order to remove the cells in the culture solution and recover only the culture medium, a centrifugation or filtration process can be performed, and these steps can be performed by those skilled in the art as necessary. The culture medium (filtrate) from which the cells have been removed can be refrigerated in a conventional manner and stored for a short period of time so as not to lose its activity.

[0076] The fusion protein expressed in the transformed cell (or transformant) can be purified by a conventional method, for example, salting out (e.g., ammonium sulfate precipitation, sodium phosphate precipitation), solvent precipitation (protein fraction precipitation using acetone, ethanol, etc.), dialysis, gel filtration, ion exchange, column chromatography such as reverse phase column chromatography, affinity chromatography, and ultrafiltration, either alone or in combination, to purify the fusion protein of the present invention.

[0077] The interferon receptor of the present invention is a hyper-glycosylated protein that contains a large amount of glycosylation in the post-translational modification (PTM), and the sugars cause problems in structural stability, so it may not be suitable for cultivation in microorganisms. Therefore, the polypeptide of the present invention is preferably one in which the host cell is an animal cell.

[0078] In this regard, the present invention provides a pharmaceutical composition for preventing or treating a type I interferon-mediated disease or disorder, which comprises a polypeptide as an active ingredient.

[0079] By "type I interferon-mediated disease or disorder" according to the present invention is meant a disease or disorder mediated by type I interferon or a disease associated with the aberrant expression (e.g., overexpression or suppression) of an interferon-inducible gene. For example, systemic lupus erythematosus (SLE, J Immunol. 2014 Jun 15; 192(12):5459-5468.), Sjogren's syndrome (Autoimmun Rev. 2013 Mar; 12 (5):558-66), 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 (RA, Front Immunol. 2017; 8:2007), inflammatory myositis (Arthritis Rheum 2009; 60:181524; Mol Med 2007; 13:5968). and glomerulonephritis, but are not limited to these. Additionally, the compositions of the present invention may be used to inhibit or prevent transplant rejection, or to treat graft-versus-host disease (GVHD), or to treat HIV infection / AIDS.

[0080] Meanwhile, the pharmaceutical composition according to the present invention can be provided by formulating the polypeptide in a pure form or in a suitable form together with a pharma- ceutically acceptable carrier. The term "pharma-ceutically acceptable" means a non-toxic composition that is physiologically acceptable and does not normally cause allergic reactions such as gastrointestinal disorders or dizziness or similar reactions when administered to humans. The carrier includes any kind of solvent, dispersion medium, oil-in-water or oil-in-heavy water emulsion, aqueous composition, liposome, microbeads and microsomes.

[0081] Meanwhile, the pharmaceutical composition according to the present invention may be formulated together with a suitable carrier depending on the administration route. The administration route of the pharmaceutical composition according to the present invention may be, but is not limited to, oral or parenteral. The parenteral administration route includes several routes such as transdermal, nasal, abdominal, intramuscular, subcutaneous, or intravenous.

[0082] When the pharmaceutical composition of the present invention is orally administered, it can be formulated into the form of powder, granules, tablets, pills, sugar-coated tablets, capsules, liquids, gels, syrups, suspensions, wafers, etc., together with a suitable carrier for oral administration according to a method known in the art. Examples of suitable carriers include sugars including lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, etc., starches including corn starch, wheat starch, rice starch, potato starch, etc., celluloses including cellulose, methylcellulose, sodium carboxymethylcellulose, hydroxypropylmethylcellulose, etc., and fillers such as gelatin and polyvinylpyrrolidone. In addition, cross-linked polyvinylpyrrolidone, agar, alginic acid or sodium alginate, etc., can be added to the disintegrant, if necessary. In addition, the pharmaceutical composition can further include anti-agglomerating agents, lubricants, wetting agents, flavorings, emulsifiers, preservatives, etc.

[0083] In addition, when administered parenterally, the pharmaceutical composition of the present invention can be formulated in the form of an injection, a transdermal agent, or a nasal inhaler together with a suitable parenteral carrier according to a method known in the art. The injection must be sterilized and must be protected from contamination by microorganisms such as bacteria and fungi. In the case of an injection, examples of suitable carriers include, but are not limited to, solvents or dispersion media including water, ethanol, polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycol, etc.), mixtures thereof, and / or vegetable oils. More preferably, suitable carriers include Hank's solution, Ringer's solution, PBS (phosphate buffered saline) containing triethanolamine, or isotonic solutions such as sterile water for injection, 10% ethanol, 40% propylene glycol, and 5% dextrose. In order to protect the injection from microbial contamination, various antibacterial and antifungal agents such as paraben, chlorobutanol, phenol, sorbic acid, and thimerosal can be further included. In most cases, the injections may further contain sugar or sodium chloride.

[0084] The transdermal formulations include ointments, creams, lotions, gels, external liquids, pastes, liniments, aerosols, etc. The term "transdermal administration" refers to topical administration of a pharmaceutical composition to the skin, so that an effective amount of the active ingredient contained in the pharmaceutical composition is delivered into the skin. These dosage forms are described in prescriptions that are generally known in the pharmaceutical and chemical fields.

[0085] For inhalation administration, the compounds used according to the present invention can be conveniently delivered in the form of an aerosol spray from a pressurized pack or nebulizer using a suitable propellant, for example, dichlorofluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol, the dosage unit can be determined by providing a valve to deliver a metered amount. For example, gelatin capsules and cartridges used in an inhaler or blower can be formulated to contain a powder mix of the compound and a suitable powdered base, such as lactose or starch. Other pharma-ceutically acceptable carriers may be referred to as those known in the art.

[0086] In addition, the pharmaceutical composition of the present invention may additionally contain a plurality of buffering agents (e.g., physiological saline or PBS), carbohydrates (e.g., glucose, mannose, sucrose or dextran), stabilizers (sodium bisulfite, sodium sulfite or ascorbic acid), antioxidants, bacteriostatic agents, chelating agents (e.g., EDTA or glutathione), adjuvants (e.g., aluminum hydroxide), suspending agents, thickening agents and / or preservatives (benzalkonium chloride, methyl- or propyl-paraben, and chlorobutanol).

[0087] In addition, the pharmaceutical composition of the present invention can be formulated using methods known in the art so as to provide quick, sustained or delayed release of the active ingredient after administration to a mammal.

[0088] The pharmaceutical composition formulated by the above method can be administered in an effective amount via multiple routes including oral, transdermal, subcutaneous, intravenous or intramuscular. The "effective amount" refers to the amount of compound or extract that allows the diagnosis or treatment effect to be tracked when administered to a patient. The dosage of the pharmaceutical composition according to the present invention can be appropriately selected depending on the administration route, the subject, the target disease and its severity, age, sex, weight, individual differences and the state of the disease. Preferably, the pharmaceutical composition containing the polypeptide of the present invention can have a different content of the active ingredient depending on the degree of the disease, but can usually be repeatedly administered several times a day with an effective dose of 10 μg to 10 mg per administration based on an adult.

[0089] Furthermore, the present invention provides a vaccine containing the polypeptide as an active ingredient, and the vaccine according to the present invention is characterized in that it is a vaccine for preventing or treating a type I interferon-mediated disease or disorder.

[0090] In the present invention, the term "vaccine" refers to a biological preparation containing an antigen that confers immunity to a living body, and refers to an immunogen or antigenic substance that induces immunity in a living body by injecting or orally administering to humans or animals for the prevention of infectious diseases. Immunity in a living body can be broadly divided into autoimmunity, in which immunity is automatically acquired after infection with a pathogen, and passive immunity, which is acquired by an externally injected vaccine. Autoimmunity is characterized by a long period of production of antibodies related to immunity and continuous immunity, while passive immunity by a vaccine has the disadvantage of being less durable, although it immediately acts to treat infectious diseases.

[0091] The vaccine composition of the present invention may contain a pharma- ceutically acceptable carrier, which means any component suitable for delivering an antigenic substance to an in vivo site, including, but not limited to, water, saline, phosphate buffered saline, Ringer's solution, dextrose solution, serum-containing solution, Hans solution, other aqueous physiologically balanced solutions, oils, esters, and glycols.

[0092] The carrier of the present invention may contain suitable auxiliary ingredients and preservatives to enhance chemical stability and isotonicity, and may contain stabilizers such as trehalose, glycine, sorbitol, lactose, or monosodium glutamate (MSG) to protect the vaccine composition against temperature changes or lyophilization. The vaccine composition of the present invention may include suspensions such as sterile water or saline (preferably buffered saline).

[0093] The vaccine compositions of the present invention can contain any adjuvant in an amount sufficient to enhance the immune response to the immunogen. Suitable adjuvants are known in the art and include, but are not limited to, aluminum salts (aluminum phosphate or aluminum hydroxide), squalene mixture (SAF-1), muramyl peptides, saponin derivatives, mycobacterium cell wall products, monophosphoryl lipid A, mycolic acid derivatives, non-ionic block copolymer surfactants, Quil A, cholera toxin B subunit, polyphosphazene and derivatives, and immune-stimulating complexes (ISCOMs).

[0094] As with all vaccine compositions, the immunologically effective amount of immunogen must be empirically determined, where factors that may be considered include immunogenicity, route of administration, and number of immunization doses administered, and may be adjusted according to the progression and metastatic status of the patient's disease, the type of formulation, the patient's age, sex, weight, health condition, diet, and the time and method of administration.

[0095] The dimeric polypeptide in the vaccine composition of the present invention may be present in a variety of concentrations within the composition of the present invention, but typically the antigenic substance is included at a concentration necessary to induce the formation of an appropriate level of antibodies in vivo.

[0096] In the present invention, the term "administration" means introducing a given substance into a patient by any suitable method, and the route of administration of the vaccine of the present invention can be via any common route as long as it can reach the target tissue.

[0097] The vaccine compositions of the present invention can be used to treat type I interferon-mediated diseases or disorders by administration via systemic or mucosal routes.

[0098] Administration of the vaccine composition may include, but is not limited to, injection via intramuscular, intraperitoneal, intradermal or subcutaneous routes, oral / dietary, respiratory, or genitourinary tract mucosal administration.

[0099] In addition, to enhance the efficacy of the vaccine, it may be possible to administer cytokines that help activate T cells, such as IL-12, at the time of vaccine injection, or to use vaccines transformed with these cytokine genes.

[0100] Since the cells containing the dimeric polypeptide, which is the active ingredient of the vaccine produced by the present invention, are inoculated into the human body as a therapeutic vaccine, it is possible to eliminate the cell proliferation ability in order to enhance safety. For example, in order to selectively use the cells as a cell vaccine more safely, the cells can be treated with heat treatment, radiation treatment, mitomycin C (MMC) treatment, etc., to eliminate the proliferation ability while retaining the function as a vaccine. For example, when X-ray irradiation is used, irradiation can be performed with a total radiation dose of 1000 to 3300 Rad. In the mitomycin C treatment method, for example, 25 to 50 μg / ml of mitomycin C of the polypeptide can be added, and the cells can be incubated at 37° C. for 30 to 60 minutes. In the cell treatment method using heat, for example, heat treatment can be performed at 50° C. to 65° C. for 20 minutes.

[0101] As used herein, "treatment" refers to a clinical procedure that alters the natural process of the individual or cell being treated, and may also be performed for the prevention of clinical pathology. The favorable effects of treatment include suppression of onset or recurrence of a disease, alleviation of symptoms, reduction of any direct or indirect pathological consequences of a disease, reduction in the rate of progression of a disease, amelioration, reversal, palliative or improved prognosis of a disease state, etc. In addition, the term "prevention" refers to any action that inhibits the onset of a disease or delays its progression.

[0102] In this specification, the term "comprising" is used interchangeably with "including" or "characterized by" and does not exclude additional components or method steps, etc. that are not specifically recited in the composition or method of the present invention. In addition, the term "consisting of" means excluding additional elements, steps, ingredients, etc. that are not specifically recited. The term "essentially consisting of" means that the scope of a composition or method can include, in addition to the recited substances or steps, substances or steps that do not substantially affect its basic characteristics.

[0103] The present invention also provides the use of said polypeptide for the manufacture of a medicament for the prevention or treatment of a type I interferon mediated disease or disorder.

[0104] The present invention also provides a method for treating a type I interferon-mediated disease or disorder, comprising the step of administering an effective amount of a composition containing the polypeptide as an active ingredient to an individual in need thereof.

[0105] The "effective amount" of the present invention means an amount that, when administered to an individual, shows an effect of improving, treating, preventing, detecting, diagnosing, or suppressing or reducing a type I interferon-mediated disease or disorder, and the "individual" may be an animal, preferably a mammal, particularly an animal including a human, or a cell, tissue, organ, etc. derived from an animal. The individual may be a patient in need of the effect. The term "treatment" as used herein refers generally to improving a type I interferon-mediated disease or disorder, or a symptom of a type I interferon-mediated disease or disorder, which may include curing, substantially preventing, or improving the condition of the disease, including, but not limited to, alleviating one or most of the symptoms of the disease, or curing or preventing the disease. Effect of the Invention

[0106] The neutralizing Fc fusion protein of type I interferon of the present invention is excellent at initiating the signaling mechanism and inhibiting biological activity, and can effectively treat diseases mediated by type I interferon by blocking the binding of type I interferon to interferon receptors. [Brief description of the drawings]

[0107] [Figure 1] FIG. 1 shows a schematic diagram of the construction of an expression vector for producing an Fc fusion receptor protein of type I interferon. [Figure 2a] FIG. 2a shows the comparison of the sizes of each protein through size exclusion chromatography. [Figure 2b] FIG. 2b shows the results of binding between IFNAR1 / 2 heterologous Fc fusion protein and human IFN-β under natural and reducing conditions. [Figure 2c] FIG. 2c is a schematic diagram showing an Fc fusion receptor protein of type I interferon. [Diagram 3] FIG. 3 shows the results of SPR analysis of type I interferon Fc fusion receptor proteins. [Figure 4] 4a to 4c show the results of confirming the neutralizing ability of type I interferon by the Fc fusion receptor protein of type I interferon of the present invention. [Figure 4a] FIG. 4a shows the results showing cell viability in Daudi cells treated with human IFNβ-1a alone or together with an Fc fusion receptor protein of type I interferon. [Figure 4b] FIG. 4b shows the IC50 values ​​obtained by treating with human IFNβ-1a alone or together with an Fc fusion receptor protein of type I interferon. [Figure 4c]FIG. 4c shows the cell viability of Daudi cells treated with human IFNβ-1a as a function of the concentration of the protein of the present invention. [Diagram 5] FIG. 5 confirms the neutralizing ability of the IFNAR1 / 2-Fc heterodimer (4GS*3) protein of the present invention to the biological activity of type I interferons (IFN-α 1, IFN-α 2a, IFN-α 2b, IFN-α 5, IFN-α 8, IFN-α 10, IFN-β 1a, IFN-ω), and shows the cell viability of Daudi cells in the group treated with type I interferon and the group treated with each interferon and the IFNAR1 / 2-Fc heterodimer (4GS*3) protein. [Figure 6a] 6a and 6b show the results of confirming the neutralizing ability of the IFNAR1 / 2-Fc heterodimer (4GS*3) protein of the present invention against the signaling mechanism of type I interferon, and show the change in phosphorylation of STAT1 protein in Daudi cells in a group treated with type I interferons (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) and a group treated with each interferon and IFNAR1 / 2-Fc heterodimer (4GS*3) protein. [Figure 6b] 6a and 6b show the results of confirming the neutralizing ability of the IFNAR1 / 2-Fc heterodimer (4GS*3) protein of the present invention against the signaling mechanism of type I interferon, and show the change in phosphorylation of STAT1 protein in Daudi cells in a group treated with type I interferons (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) and a group treated with each interferon and IFNAR1 / 2-Fc heterodimer (4GS*3) protein. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0108] The present invention will be described in detail below. However, the following examples are merely illustrative of the present invention, and the contents of the present invention are not limited to the following examples.

[0109] Example 1: Construction of an expression vector for the production of Fc fusion receptor protein of type I interferon To develop Fc fusion proteins capable of binding to or neutralizing type I interferon proteins, human IFNAR1 (P17181, 28-436 aa.), IFNAR2 (P48552, 27-243 aa.) and IGHG (P01857, 100-330 aa.) amino acid sequences were used. As shown in Figure 1 (left), all Fc fusion protein expression vectors have Fc labeled at the C-terminus with a polypeptide linker (L; IEGRMD), and this C-terminus is connected to IFNAR1 or IFNAR2 between the N-terminus containing the Kozak sequence (▲) and the extracellular secretion-inducing signaling sequence (S), or directly connected. As shown in Figure 1 (right), the sequences containing each gene were connected between the restriction enzymes NheI and XhoI. The expression vectors were named pIFNAR1-FcWT, pIFNAR2-FcWT, pIFNAR1-FcA, pIFNAR2-FcB, pIFNAR1-FcB and pLeader-FcA, respectively.

[0110] The polynucleotide sequence (SEQ ID NO: 1) containing the Kozak sequence and the signaling sequence, the extracellular domain of IFNAR1 (SEQ ID NO: 3), the extracellular domain of IFNAR2 (SEQ ID NO: 5), the polypeptide linker (SEQ ID NO: 7), and the Fc portion of human IGHG (SEQ ID NO: 11) and mutants (SEQ ID NO: 13, SEQ ID NO: 15) were codon optimized and the gene was synthesized to prepare the base sequence fragments of eg in Figure 1. Each base sequence fragment was inserted between the NheI and XhoI restriction enzyme sites of the expression vector (pOptiVec-TOPO) using T4 DNA ligase (RBC) to prepare an expression vector.

[0111] In addition, an expression vector was prepared in the same manner, except that the polypeptide linker was SEQ ID NO: 33 instead of SEQ ID NO: 7. That is, a gene including a polynucleotide sequence (SEQ ID NO: 1) containing a Kozak sequence and a signaling sequence, an extracellular domain of IFNAR1 (SEQ ID NO: 3), an extracellular domain of IFNAR2 (SEQ ID NO: 5), a polypeptide linker (SEQ ID NO: 33), and an Fc portion (SEQ ID NO: 11) and mutants (SEQ ID NO: 13 and SEQ ID NO: 15) of human IGHG was codon-optimized and then synthesized to prepare the base sequence segment eg in FIG. 1. An expression vector was prepared by inserting each base sequence segment between NheI and XhoI of the restriction enzyme sites of the expression vector (pOptiVec-TOPO) using T4 DNA ligase (RBC).

[0112] Example 2: Protein expression and purification, structural confirmation, and binding confirmation Using expression vectors into which the base sequence fragments of each combination had been inserted, the following expression and purification steps were carried out to produce proteins having the structures shown in FIG. 2c. To maintain the unique PTM of each type I interferon receptor, all recombinant proteins were expressed using human-derived Expi293 cell line (Thermo Fisher Scientific), and 300-600 mL of Expi293® expression medium was used according to the protocol of the transient expression system. Depending on the final protein, one or two expression vectors were transfected into the cells, and enhancers 1 and 2 were inserted according to the manufacturer's protocol. After 6 days of incubation, the culture medium was centrifuged at 6580 rpm for 20 min at 8°C and then filtered through a 0.22 μm polystyrene filter (Corning). All proteins were purified using an AKTA avant purification system (GE Healthcare Life Sciences). A column packed with MabSelect SuRe (GE Healthcare Life Sciences) was equilibrated for about 2 CV with 20 mM sodium phosphate, pH 7.2, 150 mM NaCl buffer at a rate of 5 ml / min, and the culture medium was then passed through the column to bind to the resin. The column was then washed with 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, and the recombinant protein was then released with 100 mM citrate buffer, pH 3.5 buffer. The released protein was dialyzed three times for 12 hours using 4 L of 1X PBS buffer (Biosesang) and concentrated to 3 ml using an Amicon Ultra Centrifugal filter (50K-cut off, Merck Millipore). The concentrated recombinant protein was then separated on a Hiload Superdex 200 pg (GE Healthcare Life Sciences) column using the same buffer used for dialysis to isolate only the target protein.

[0113] As a result, the sizes of the Fc fusion proteins were compared through size exclusion chromatography as shown in FIG. 2a, and the proteins are predicted to have the structures shown in FIG. 2c.

[0114] As shown in FIG. 2b, the binding between IFNAR1 / 2 heterologous Fc fusion protein and human IFN β-1a was analyzed under native conditions (7.5% Mini-PROTEIN® TGX® precast protein gels), reducing conditions (Any kD® Mini-PROTEIN®TGX Each was confirmed by precast protein gels (Bio-Rad).

[0115] The FC portion of the amino acid sequence of the present invention contains the hinge portion of IGHG1 (100-330 a.a.), and disulfide bonds are formed between the 109-109 and 112-112 amino acids to form a dimer. More specifically, the formation of a heterodimer is dominant over a homodimer due to a change in a specific amino acid sequence in the CH3 portion of the FC.

[0116] Example 3: SPR analysis using Fc fusion proteins The binding affinity and kinetics of the Fc fusion proteins (IFNAR2-Fc heterodimer, IFNAR1 / 2-Fc heterodimer, IFNAR1-Fc heterodimer) expressed / purified using the expression vector combinations in Figure 1 and type I IFNs hIFN β-1a were analyzed.

[0117] In this experiment, binding strength and kinetic analysis were performed using (IFNAR1-Fc EW + -Fc RVT), (IFNAR2Fc EW + -Fc RVT) and (IFNAR1-Fc EW + IFNAR2Fc RVT). When (IFNAR1-Fc RVT + IFNAR2-Fc EW) was expressed, it was confirmed that there was a problem in that more homodimeric forms were shown than in (IFNAR1-Fc EW + IFNAR2-Fc RVT), so (IFNAR1-Fc RVT + IFNAR2-Fc EW) was not used.

[0118] The Biacore T200 (GE Healthcare Life Sciences) was used at 25°C, 30 μl / min, and 0.005% DPBST (DPBS / modified, Hyclone and Tween 20, Signal) running buffer. The gold sensor chip was amine-coupled with anti-human Fcγ capture antibody (AffiniPure Goat Anti-Human IgG, Fcγfragment specific, Jackson ImmunoResearch). Each analyte was subjected to an experiment under the conditions of 180 seconds for binding and 600 seconds for dissociation. As shown in Figure 3, curve fitting was performed based on the sensorgram shapes and docking model predictions for hIFNb-1a-IFNAR2 (1:1 fitting model), hIFNb-1a-IFNAR1 / 2 (heterogeneous ligand model), and hIFNb-1a-IFNAR1 (two-state binding models). In particular, the IFNAR1 / 2 heterologous Fc fusion protein showed a low K D The values ​​were shown (see Tables 2 and 3).

[0119] [Table 2]

[0120] [Table 3]

[0121] Example 4: Confirmation of ability to neutralize biological activity of Fc fusion protein by ligand The following experiment was carried out to confirm the neutralizing ability of the Fc fusion proteins against biological activity caused by ligands. As in Example 3, (IFNAR1-Fc EW + -Fc RVT), (IFNAR2Fc EW + -Fc RVT), and (IFNAR1-Fc EW + IFNAR2Fc RVT) were used in this experiment. All experiments were designed based on the anti-proliferative effect of hIFN-β in Daudi cells expressing all receptors abundantly using the Ez-cytox cell viability assay kit (Deaillab). The assay results were consistent with the biophysical characteristics of the ligands, as well as the SPR kinetic analysis results. Specifically, 3 × 10 of a 96 well plate (SPL) 3 Cells were then incubated with hIFN-β-1a at different concentrations (10 nM IFNAR1-Fc heterodimer, IFNAR2-Fc heterodimer, IFNAR1 / 2-Fc heterodimer; 6 pM IFNAR1 / 2-Fc heterodimer) for 72 h at 37°C and 5% CO 2 After that, Ex-cytox reagent was added according to the manufacturer's protocol, and the reaction was continued for an additional 3 hours. The IC was then measured at 450 nm using a microplate reader (Genios Pro, Tecan) and calculated using nonlinear regression analysis (GraphPad Prism version 7.0 software, San Diego, CA, USA). 50The neutralizing activity of each protein of the present invention was confirmed to have statistical significance through one-way ANOVA and Bonferroni's multiple comparisons post hoc test.

[0122] As shown in Figure 4a, when treated with Fc fusion protein that completely blocks the binding of interferon to IFNAR1 and IFNAR2, the anti-cell proliferation induced by interferon was significantly reduced compared to the cases of fusion proteins that block IFNAR1 and IFNAR2, respectively. As shown in Figure 4b, the IC50 value was highest when the Fc fusion protein according to the present invention, which blocks both IFNAR1 and IFNAR2, was added, demonstrating that anti-cell proliferation by interferon is superior to blocking IFNAR1 or IFNAR2 individually in terms of cell viability. As shown in FIG. 4c, it was confirmed that the cell viability increased in a concentration-dependent manner when the IFNAR1 / 2-Fc heterodimer, which has the highest neutralizing activity, was treated.

[0123] Example 5: Confirmation of the ability of IFNAR1 / 2-Fc heterodimer ligands to neutralize the activity of signaling mechanisms The ability of the IFNAR1 / 2-Fc heterodimer to neutralize the biological activity of the ligand was confirmed in the same manner as in Example 4, except for the IFNAR1 / 2-Fc heterodimer described below. Specifically, 3 x 10 cells in a 96-well plate (SPL_cat#30096) 3The cells were treated with 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), and IFN-β The group that received 1nM of IFN-ω (pbl assay science cat #11415-1) and 1nM of IFN-ω (pbl assay science cat #11395-1) and the group that received 10nM of Fc fusion receptor protein of type I interferon were incubated at 37°C for 72 hours in 5% CO. 2 After that, Ex-cytox reagent was added according to the manufacturer's protocol, and the reaction was continued for an additional 2 hours. The IC was then measured at 450 nm using a microreader (Genios Pro, Tecan) and calculated using nonlinear regression analysis (GraphPad Prism version 7.0 software, San Diego, CA, USA). 50 The values ​​were comparatively analyzed. As a result, as shown in FIG. 5, the IFNAR1 / 2-Fc heterodimer (containing 4GS as a linker) was synthesized, which completely blocks the binding of IFNAR1 and IFNAR2. * When cells were treated with IFN-α 1, IFN-α 2a, IFN-α 2b, IFN-α 5, IFN-α 8, IFN-α 10, IFN-β 1a, or IFN-ω, the cell viability was significantly higher than that of cells treated with interferon alone.

[0124] These results indicate that the dimeric polypeptide of the present invention, which is bound to a monomer containing an interferon receptor fragment or an antibody Fc fragment, can regulate excessive interferon signaling in cells, and thus can have excellent pharmacological effects on patients with type I interferon-mediated diseases such as systemic lupus erythematosus, Sjogren's syndrome, systemic sclerosis, myositis, and rheumatoid arthritis, in which interferon-inducible genes are expressed at high levels due to excessive interferon signaling.

[0125] Example 6: Confirmation of the ability of IFNAR1 / 2-Fc heterodimer ligands to inhibit chemical signaling All experiments were performed in Daudi cells expressing all receptors at high levels, using Western blot to confirm the changes in STAT1 and its phosphorylated pSTAT1 protein induced by type I IFNs. Specifically, 2 × 10 cells were placed in a 6-well plate (SPL_cat#30006). 6 The cells were then treated with 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 #11395-1), IFN-ε (R&D Systems Cat #11415-1), and IFN-β 1b (pbl Assay Science Cat #11415-1). The group was treated with either interferon or IFNAR1 / 2-Fc heterodimer protein at a concentration of 10 nM for 72 hours at 37°C, 5% CO. 2The cells were incubated in a cell culture vessel at 4°C for 12 h. The cells were then harvested from the plate, and total protein was extracted and quantified using a 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 transferred to 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 at room temperature for 1 h. The blocked membrane was incubated overnight at 4°C with primary antibodies (1:3000 dilution), namely STAT1 antibody (cell signaling_cat #06-501), pSTAT1 antibody (cell signaling_cat #58D6), and β-actin antibody (santa cruz_cat #sc 47778), and then incubated at room temperature for 1 hour with secondary HRP-conjugated antibodies, goat anti rabbit antibody (Invitrogen_cat #31460) and goat anti mouse antibody (Invitrogen_cat #G21040). After treating the membrane with ECL solution (Bio-Rad Cat #1705061), the membrane was visualized on film (Agfa Healthcare Cat #EA8EC) using Poohung developer & fixer. As a result, as shown in Figures 6a and 6b, the IFNAR1 / 2-Fc heterodimer (4GS *3) When the protein was treated with interferons (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), the phosphorylation of pSTAT1 protein was significantly decreased compared to the high phosphorylation of pSTAT1 protein when the protein was treated with interferons alone. This indicates that the IFNAR1 / 2-Fc heterodimer protein binds to and neutralizes type I interferons, thereby inhibiting interferon-related signaling in cells. [Industrial Applicability]

[0126] The type I interferon neutralizing Fc fusion protein of the present invention is excellent in inhibiting the initiation of signaling mechanisms and biological activity while blocking the binding of type I interferon to interferon receptors, and can be extremely useful in the development of therapeutic agents for preventing or treating diseases mediated by type I interferon, and therefore has excellent industrial applicability.

Claims

1. A dimeric polypeptide comprising a monomer comprising a fragment of interferon receptor 1 (IFNAR1) and an Fc fragment of an antibody comprising the amino acid sequence of SEQ ID NO: 12 linked to a monomer comprising a fragment of interferon receptor 2 (IFNAR2) and an Fc fragment of an antibody comprising the amino acid sequence of SEQ ID NO: 14, wherein the fragment of interferon receptor 1 comprises the amino acid sequence of SEQ ID NO:4, and the fragment of interferon receptor 2 comprises the amino acid sequence of SEQ ID NO:6; Dimeric polypeptide.

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

3. A polynucleotide encoding the dimeric polypeptide of claim 1.

4. A vector comprising the polynucleotide of claim 3.

5. A host cell transformed with the vector of claim 4.

6. (a) providing a host cell of claim 5; (b) culturing the provided cells; and The method for producing the dimeric polypeptide according to claim 1, further comprising the step of (c) recovering the dimeric polypeptide from the cells or the culture medium, thereby preparing the dimeric polypeptide.

7. A pharmaceutical composition for preventing or treating a type I interferon-mediated disease or disorder, comprising the dimeric polypeptide according to claim 1 as an active ingredient.

8. 8. The pharmaceutical composition of claim 7, wherein the type I 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).

9. 13. Use of the dimeric polypeptide according to claim 1 for preparing a preparation for the prevention or treatment of a type I interferon mediated disease or disorder.

10. A method for treating a type I interferon-mediated disease or disorder in a subject other than a human, comprising administering to an individual in need thereof an effective amount of a composition comprising the dimeric polypeptide of claim 1 as an active ingredient.

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