Fusion protein comprising IGE FC receptor alpha subunit extracellular domain and anti-IL-4 antibody, and use of the fusion protein

Abstract

The present invention relates to a fusion protein dimer comprising an IgE Fc receptor alpha subunit extracellular domain (FcεRIα-ECD) and a fragment of an anti-IL-4R antibody; and a composition for treating allergic diseases comprising the fusion protein dimer. The fusion protein dimer according to the present invention exhibits excellent IgE-binding ability and an excellent effect of reducing serum IgE levels. In addition, the present invention has an excellent effect of inducing an IgE hyperimmune response and thus inhibiting the activity of cytokines that induce allergic diseases, such as IL-4 and IL-13, and therefore can be applied as a pharmaceutical for treating or preventing IgE-mediated allergic diseases.

Description

[Technical Field]

[0001] The present invention relates to a fusion protein dimer comprising an IGE Fc receptor alpha subunit extracellular domain (FcεRIα-ECD) and a fragment of an anti-IL-4R antibody, and to a composition for treating allergic diseases comprising the fusion protein dimer. [Background technology]

[0002] Allergic diseases, including asthma, allergic rhinitis, atopic dermatitis, and food allergies, are rapidly increasing in modern industrialized and Westernized societies, and the incidence of anaphylaxis, a severe allergic disease, is also increasing. These chronic immune diseases severely impair the quality of life of individuals, and corresponding socioeconomic costs are skyrocketing. Therefore, there is a pressing need for measures to solve such diseases.

[0003] Most allergic diseases are caused by an excessive immune response of immunoglobulin E (IgE). IgE is an antibody normally present in serum at extremely low concentrations. IgE is also normally produced in response to harmless antigens. However, IgE levels can increase without any specific stimulus, leading to allergic diseases. Abnormally elevated levels of IgE can bind to high-affinity IgE Fc receptors (FcεRIs) expressed on the surface of mast cells, basophils, and other cells. Such binding induces the mast cells or basophils to release chemical mediators, such as histamine, leukotrienes, prostaglandins, bradykinin, and platelet-activating factor. The release of these chemical mediators leads to allergic symptoms. In particular, allergic diseases may exhibit exacerbated symptoms due to the binding between IgE and FcεRI.

[0004] Currently, various methods have been proposed to treat allergic diseases, such as avoiding allergens, administering anti-allergic drugs, regulating IgE synthesis in the body, and developing anti-IgE antibodies. However, the known treatment methods have many drawbacks, such as being unable to cure the underlying cause of allergy, poor drug efficacy, and the occurrence of severe side effects.

[0005] On the other hand, the biological activity of the proinflammatory cytokine IL-4 is mediated by specific IL-4 receptors (interleukin-4 receptors, IL-4R) on the cell surface. There are two types of IL-4 receptors: type 1, in which the IL-4 receptor α chain and γc chain form a complex, and type 2, in which the IL-4 receptor α chain and IL-13 receptor α1 chain form a complex. In this regard, human monoclonal antibodies against the IL-4R α chain have been demonstrated to be clinically effective in alleviating and treating symptoms such as asthma, eczema, and atopic dermatitis.

[0006] To date, dupilumab, an anti-hIL-4Rα antibody developed by Regeneron Pharmaceuticals Inc., was approved by the FDA in 2017 and has been used to treat allergic diseases (U.S. Patent No. 7,605,237). No other anti-hIL-4Rα antibodies have yet been approved. Summary of the Invention

[0007] [Technical Issues] Therefore, the present inventors conducted research to develop a novel combination of fusion proteins for the effective treatment and prevention of allergic diseases. As a result, it was confirmed that a fusion protein dimer comprising the IgE Fc receptor alpha subunit extracellular domain (FcεRIα-ECD) and a fragment of an anti-IL-4R antibody exhibits excellent IgE binding ability and an excellent effect of reducing serum IgE levels. In addition, it was confirmed that the activity of IL-4 and IL-13, cytokines that induce IgE hyperimmune responses and induce allergic diseases, was inhibited in a concentration-dependent manner. Based on the above, the present inventors confirmed that the fusion protein dimer can be useful as a therapeutic agent for allergic diseases such as asthma and atopic dermatitis, thereby completing the present invention.

[0008] [Problem Resolution] To achieve the above object, one aspect of the present invention provides a fusion protein dimer comprising an IgE Fc receptor alpha subunit extracellular domain (FcεRIα-ECD) and a fragment of an anti-IL-4R antibody.

[0009] In another aspect, the present invention provides a polynucleotide encoding the fusion protein, an expression vector containing the polynucleotide, and a transformed cell into which the expression vector has been introduced.

[0010] In another aspect of the present invention, there is provided a method for producing a fusion protein dimer, comprising the steps of: culturing a transformed cell; and recovering the fusion protein dimer.

[0011] In another aspect of the present invention, there is provided a pharmaceutical composition for preventing or treating an allergic disease, comprising the fusion protein dimer.

[0012] In another aspect of the present invention, there is provided a food composition for improving or alleviating allergic symptoms, comprising the fusion protein dimer.

[0013] In another aspect of the present invention, there is provided use of a fusion protein dimer comprising FcεRIα-ECD and a fragment of an anti-IL-4R antibody for the manufacture of a medicament for the treatment or prevention of an allergic disease.

[0014] In another aspect of the present invention, there is provided the use of a fusion protein dimer comprising FcεRIα-ECD and a fragment of an anti-IL-4R antibody for the treatment or prevention of an allergic disease.

[0015] In another aspect of the present invention, there is provided a method for treating or preventing an allergic disease, comprising the step of administering to a subject a fusion protein dimer comprising FcεRIα-ECD and a fragment of an anti-IL-4R antibody.

[0016] [Effects of the invention] The fusion protein dimer of the present invention, which comprises an IgE Fc receptor alpha subunit extracellular domain (FcεRIα-ECD) and a fragment of an anti-IL-4R antibody, exhibits excellent IgE-binding ability and an excellent effect of reducing serum IgE levels. In addition, the fusion protein dimer of the present invention has an excellent effect of inducing an IgE hyperimmune response and thus inhibiting the activity of cytokines, such as IL-4 and IL-13, that induce allergic diseases. Furthermore, functionally, the fusion protein dimer acts in a complex manner as an IgE trap and an anti-IL-4R antibody. As a result, the fusion protein dimer can be useful as a novel pharmaceutical composition for the treatment of allergic diseases, which can replace the conventional individual therapeutic agents of anti-IgE antibody and anti-IL-4R antibody. [Brief explanation of the drawings]

[0017] [Figure 1] FIG. 1 illustrates the structure of the fusion protein GI-305 (FcεRIα ECD-Fc-anti-IL-4RscFv) according to the present invention. [Figure 2] FIG. 1 illustrates the results obtained by checking the obtained fusion protein GI-305 by SDS-PAGE. [Figure 3]FIG. 1 illustrates the results obtained by checking the resulting fusion protein GI-305 by Western blot. [Figure 4] FIG. 1 is a graph showing the results obtained by measuring the binding capacity of the fusion protein GI-305 according to the invention to human IgE. [Figure 5] FIG. 1 is a graph showing the results obtained by confirming the effectiveness of the fusion protein GI-305 according to the present invention as an anti-IL-4R antibody. [Figure 6] FIG. 1 is a graph showing the results obtained by confirming the serum IgE level-reducing effect of the fusion protein GI-305 according to the present invention. [Figure 7] FIG. 1 is a graph showing the results obtained by confirming the serum IgE level-reducing effect of the fusion protein GI-305 according to the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0018] [Best Mode for Carrying Out the Invention] Fusion protein containing FcεRIα-ECD and a fragment of an anti-IL-4R antibody In one aspect of the present invention, a fusion protein is provided comprising an IgE Fc receptor alpha subunit extracellular domain (FcεRIα-ECD) and a fragment of an anti-IL-4R antibody.

[0019] As used herein, the term "IgE" refers to an antibody protein known as immunoglobulin E. IgE has affinity for mast cells, basophils in the blood, and the like. In addition, the reaction between IgE antibodies and the antigens (allergens) that correspond to IgE antibodies causes inflammatory reactions. In addition, IgE is known to be an antibody that causes anaphylaxis, which occurs due to the rapid secretion of IgE from mast cells or basophils.

[0020] As used herein, the term "IgE Fc receptor," also known as the Fcε receptor, binds to the Fc portion of IgE. There are two types of receptors: the receptor with high affinity for IgE Fc is called Fcε receptor I (FcεRI). The receptor with low affinity for IgE Fc is called Fcε receptor II (FcεRII). FcεRI is expressed on mast cells and basophils. When IgE antibodies bound to FcεRI are crosslinked by polyvalent antigens, degranulation occurs in mast cells and basophils, resulting in the release of various chemical mediators, including histamine. This release leads to an immediate allergic reaction.

[0021] FcεRI is a membrane protein composed of one α chain, one β chain, and two γ chains linked by disulfide bonds. The IgE-binding portion of these chains is the α chain (FcεRIα), which is approximately 60 kDa in size and consists of a hydrophobic domain located on the inner side of the cell membrane and a hydrophilic domain located on the outer side of the cell membrane. IgE specifically binds to the extracellular domain of the α chain. "FcεRIα" can be used interchangeably with "alpha subunit of the IgE Fc receptor."

[0022] In particular, the alpha subunit of the IgE Fc receptor can have the amino acid sequence set forth in NP_001992.1. Additionally, the extracellular domain of the alpha subunit of the IgE Fc receptor (FcεRIα-ECD) can have the amino acid sequence of SEQ ID NO: 2. As used herein, the extracellular domain of the alpha subunit of the IgE Fc receptor can be a fragment or variant of the extracellular domain of the alpha subunit of the IgE Fc receptor, so long as the fragment or variant is capable of binding to IgE.

[0023] Variants can be generated by substituting, deleting, or adding one or more proteins in the wild-type FcεRIα-ECD (extracellular domain), so long as the method described below does not alter the function of the α-chain of FcεRI. Such different proteins or peptides may be 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence of SEQ ID NO: 2. In addition, the FcεRIα ECD of SEQ ID NO: 2 can be encoded by a polynucleotide having the sequence of SEQ ID NO: 11.

[0024] As used herein, the term "IL-4R (interleukin-4 receptor)" or "interleukin-4 receptor" refers to a cytokine receptor to which interleukin-4 (IL-4) specifically binds, and exists as type 1 IL-4 receptor and type 2 IL-4 receptor. Type 1 IL-4 receptor is a dimeric receptor comprising an IL-4Rα chain and a γc chain, while type 2 IL-4 receptor is a dimeric receptor comprising an IL-4Rα chain and an IL-13Rα1 chain. Type 1 IL-4 receptor interacts with and is stimulated by IL-4, while type 2 IL-4 receptor interacts with and is stimulated by both IL-4 and IL-13.

[0025] The Th2 cytokines IL-4 and IL-13 are known to play important roles in allergic diseases such as asthma, atopic dermatitis, and allergic rhinitis, and are involved in T lymphocyte proliferation, differentiation into Th2 cells, and conversion of B lymphocyte products to IgE. In addition, IL-4 and IL-13 share the IL-4 receptor α (IL-4Rα) and have similar biological properties. In particular, IL-4 is essential for the conversion of B lymphocyte products to IgE and promotes eosinophil chemotaxis and adhesion. In addition, IL-13 functions as an important mediator of IgE production and maintenance. Therefore, IL-4 and IL-13 induce IgE hyperimmune responses, leading to allergic diseases.

[0026] Here, the anti-IL-4R antibody may be an antibody that specifically binds to the IL-4 receptor. In particular, the anti-IL-4R antibody may be an antibody that specifically binds to the type 2 IL-4 receptor. In addition, any form of antibody fragment can be used as long as it contains an antigen-binding domain capable of specifically binding to the IL-4 receptor. In addition, the anti-IL-4R antibody fragment may be in the form of an scFv. As used herein, the term "scFv" is an abbreviation for single-chain variable fragment, and refers to a form in which the heavy chain variable region and the light chain variable region are combined via a peptide linker.

[0027] The heavy chain variable region of the scFv of an anti-IL-4R antibody used in one embodiment of the present invention may have the amino acid sequence of SEQ ID NO: 7 or 26. In particular, HCDR1, HCDR2, and HCDR3 of the heavy chain variable region may be SEQ ID NOs: 20, 21, and 22, respectively. In addition, the light chain variable region of the scFv of an anti-IL-4R antibody may have the amino acid sequence of SEQ ID NO: 8 or 27. In particular, LCDR1, LCDR2, and LCDR3 of the light chain variable region may be SEQ ID NOs: 23, 24, and 25, respectively. In addition, in one embodiment, the peptide linker connecting the heavy chain variable region and the light chain variable region may have the amino acid sequence of SEQ ID NO: 9. In addition, the scFv of an anti-IL-4R antibody used in one embodiment of the present invention may have the amino acid sequence of SEQ ID NO: 6.

[0028] On the other hand, the heavy chain variable region of the scFv of the anti-IL-4R antibody of SEQ ID NO: 7 used in one embodiment of the present invention may be obtained by mutating the 44th amino acid in the amino acid sequence of dupilumab scFv (SEQ ID NO: 26) from G to C in order to stabilize the scFv structure through disulfide bond formation. In addition, the light chain variable region of the anti-IL-4R antibody of SEQ ID NO: 8 used in one embodiment of the present invention may be obtained by mutating the 105th amino acid in the amino acid sequence of dupilumab scFv (SEQ ID NO: 27) from Q to C in order to stabilize the scFv structure through disulfide bond formation.

[0029] Here, the fusion protein can contain an immunoglobulin Fc region. Here, the immunoglobulin Fc domain refers to a protein containing the immunoglobulin heavy chain constant region 2 (CH2) and heavy chain constant region 3 (CH3), but not the immunoglobulin heavy chain variable region and light chain variable region and light chain constant region (CL). The immunoglobulin can be IgG, IgA, IgE, IgD, or IgM, and is preferably IgG4.

[0030] In addition, the Fc domain of an immunoglobulin may be a wild-type Fc domain as well as an Fc domain variant. Additionally, as used herein, "Fc domain variant" refers to a form that differs from the wild-type Fc domain in terms of glycosylation pattern, or has higher glycosylation than the wild-type Fc domain, or has lower glycosylation than the wild-type Fc domain, or a deglycosylated form. Additionally, an aglycosylated Fc domain is included in the Fc domain variant. The Fc domain or its variant can be configured to have a controlled number of sialic acids, fucosylation, or glycosylation through culture conditions or genetic manipulation of the host.

[0031] In addition, glycosylation of the Fc domain of an immunoglobulin can be modified by standard methods such as chemical methods, enzymatic methods, and genetic engineering methods using microorganisms. Furthermore, the Fc domain variant may be a hybrid of the Fc regions of immunoglobulins IgG, IgA, IgE, IgD, or IgM. Furthermore, the Fc domain variant may be a form in which some amino acids in the Fc domain are substituted with other amino acids.

[0032] In addition, the modified Fc region may have native glycosylation or may have increased glycosylation compared to the native type. The glycosylation of the Fc domain of an immunoglobulin can be modified by standard methods such as chemical methods, enzymatic methods, and genetic engineering methods using microorganisms.

[0033] In one embodiment, the Fc domain variant may have the amino acid sequence of SEQ ID NO:4. In particular, the fusion protein has the following structural formula (I) or (II): N'-X-linker (1)-Fc region fragment or variant thereof-linker (2)-Y-C'(I) N'-Y-linker (1)-Fc region fragment or variant thereof-linker (2)-X-C'(II) and In structural formulas (I) and (II), N' is the N-terminus of the fusion protein; C' is the C-terminus of the fusion protein; X is FcεRIα-ECD or a fragment thereof; Y is a fragment of an anti-IL-4R antibody, and Linker (1) and linker (2) are peptide linkers.

[0034] Here, the FcεRIα-ECD or a fragment thereof and the anti-IL-4R antibody fragment are as described above.

[0035] Here, the peptide linker (1) may consist of 5 to 80 consecutive amino acids, 10 to 70 consecutive amino acids, 15 to 60 consecutive amino acids, 20 to 50 consecutive amino acids, 25 to 40 consecutive amino acids, or 25 to 35 amino acids. In one embodiment, the peptide linker (1) may consist of 30 amino acids. In addition, the peptide linker (1) may contain at least one cysteine. In particular, the peptide linker (1) may contain one, two, or three cysteines. In addition, the peptide linker (1) may be derived from an immunoglobulin hinge. In one embodiment, the peptide linker (1) may be a peptide linker consisting of the amino acid sequence of SEQ ID NO: 3.

[0036] The peptide linker (2) can consist of 1 to 50 consecutive amino acids, or 3 to 30 consecutive amino acids, or 5 to 15 amino acids. The peptide linker (2) may contain (G4S)n (wherein n is an integer from 1 to 10) or may further contain (G)n. Here, in (G4S)n and (G)n, n may be 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, respectively. The peptide linker (2) may be a peptide linker consisting of the amino acid sequence of SEQ ID NO:5.

[0037] The amino acid sequences of each of the fusion proteins are shown below in Table 1. Additionally, in one embodiment, the amino acid sequence of GI-305 (FcεRIα ECD-Fc-anti-IL-4R scFv) can have the amino acid sequence of SEQ ID NO: 10.

[0038] [Table 1] TIFF0007784412000002.tif194149

[0039] Fusion protein dimer In another aspect of the present invention, there is provided a fusion protein dimer in which the two fusion proteins described above are linked together. The fusion proteins can be linked together via a cysteine ​​contained in a linker connecting the FcεRIα-ECD or a fragment thereof to the Fc region of an immunoglobulin. The linker can include the hinge region of an immunoglobulin.

[0040] Polynucleotide encoding the fusion protein Another aspect of the present invention provides a polynucleotide encoding a fusion protein comprising FcεRIα-ECD and an anti-IL-4R antibody fragment, wherein the FcεRIα-ECD and the anti-IL-4R antibody fragment are as described above.

[0041] The polynucleotide may have the sequence of SEQ ID NO:19.

[0042] When a polynucleotide encodes the same polypeptide, one or more nucleotides can be mutated by substitution, deletion, insertion, or a combination thereof. When a polynucleotide sequence is produced by chemical synthesis, a synthesis method well known in the art, such as the method described in Engels and Uhlmann, Angew Chem Int Ed Engl., 37:73-127, 1988, can be used. Well-known synthesis methods include the triester method, the phosphite method, the phosphoramidite method, the H-phosphate method, PCR and other autoprimer methods, and oligonucleotide synthesis on a solid support.

[0043] According to one embodiment, the polynucleotide can comprise a nucleic acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% identity to the nucleotide sequence of SEQ ID NO:19.

[0044] The polynucleotide may further comprise a signal sequence or leader sequence. As used herein, the term "signal sequence" refers to a nucleic acid encoding a signal peptide that directs the secretion of a target protein. The signal peptide is translated in the host cell and then cleaved. In particular, a signal sequence of the present invention is a nucleotide that encodes an amino acid sequence that initiates the translocation of a protein across the endoplasmic reticulum (ER) membrane.

[0045] The characteristics of signal sequences are well known in the art. Such signal sequences typically contain 16-30 amino acid residues, but may contain more or fewer. A typical signal peptide consists of three regions: a basic N-terminal region, a central hydrophobic region, and a more polar C-terminal region. The central hydrophobic region contains 4-12 hydrophobic residues that anchor the signal sequence through the membrane lipid bilayer during translocation of the immature polypeptide.

[0046] After initiation, the signal sequence is cleaved in the lumen of the ER by a cellular enzyme commonly known as a signal peptidase. Here, the signal sequence may be a secretory signal sequence of tissue plasminogen activator (tPa), herpes simplex virus glycoprotein D (HSV gD), IgG signal sequence, or growth hormone. Preferably, a secretory signal sequence used in cells of higher eukaryotes, including mammals, can be used.

[0047] Useful signal sequences in the present invention include antibody light chain signal sequences such as antibody 14.18 (Gillies et al., J. Immunol. Meth. 1989, 125:191-202), antibody heavy chain signal sequences such as the MOPC141 antibody heavy chain signal sequence (Sakano et al., Nature, 1980, 286:676-683), and other signal sequences known in the art (see, for example, Watson et al., Nucleic Acid Research, 1984, 12:5145-5164). In one embodiment, a signal sequence consisting of the amino acids of SEQ ID NO: 1 can be used as the signal sequence.

[0048] Polynucleotide-loaded vector Another aspect of the present invention provides an expression vector comprising a polynucleotide encoding a fusion protein comprising FcεRIα-ECD and a fragment of an anti-IL-4R antibody, wherein the polynucleotide can have the sequence of SEQ ID NO:19.

[0049] As used herein, the term "vector" is intended to mean a vector that can be introduced into a host cell and can be integrated into the genome of the host cell. Alternatively, a vector can be understood as a nucleic acid vector containing a nucleotide sequence that is capable of autonomous replication as an episome. Vectors include linear nucleic acids, plasmids, phagemids, cosmids, RNA vectors, viral vectors, minichromosomes, and analogs thereof. Examples of viral vectors include, but are not limited to, retroviruses, adenoviruses, and adeno-associated viruses.

[0050] In particular, the vector may be a plasmid DNA, a phage DNA, or the like, a commercially developed plasmid (pUC18, pBAD, pIDTSAMRT-AMP, etc.), an Escherichia coli (E. coli)-derived plasmid (pYG601BR322, pBR325, pUC118, pUC119, etc.), a Bacillus subtilis-derived plasmid (pUB110, pTP5, etc.), a yeast-derived plasmid (YEp13, YEp24, YCp50, etc.), a phage DNA (Charon4A, Charon21A, EMBL3, EMBL4, λgt10, λgt11, λZAP, etc.), an animal virus vector (retrovirus, adenovirus, vaccinia virus, etc.), an insect virus vector (baculovirus, etc.), etc. Since vectors exhibit various protein expression levels and modifications depending on the host cell, it is preferable to select and use the most suitable host cell for the purpose.

[0051] In addition, the plasmid may contain a selectable marker, such as an antibiotic resistance gene, so that host cells maintaining the plasmid can be cultured under selective conditions.

[0052] As used herein, the term "gene expression" or "expression" of a target protein is understood to mean the transcription of a DNA sequence, the translation of an mRNA transcript, and the secretion of a fusion protein product or fragment thereof. A useful expression vector may be RcCMV (Invitrogen, Carlsbad) or a variant thereof. The expression vector may contain a human cytomegalovirus (CMV) promoter to promote continuous transcription of the target gene in mammalian cells and a bovine growth hormone polyadenylation signal sequence to improve the level of RNA stability after transcription.

[0053] Transformed cells expressing the fusion protein Another aspect of the present invention provides a transformed cell into which an expression vector containing a polynucleotide encoding a fusion protein comprising FcεRIα-ECD and a fragment of an anti-IL-4R antibody has been introduced.

[0054] As used herein, the term "transformed cell" refers to prokaryotic and eukaryotic cells into which a recombinant expression vector can be introduced. A transformed cell can be prepared by introducing a vector into a host cell and transforming the host cell. In addition, a fusion protein of the present invention can be produced by expressing a polynucleotide contained in a vector.

[0055] Transformation can be carried out by various methods. As long as the transformation can produce the fusion protein of the present invention, the transformation method is not particularly limited. In particular, transformation methods that can be used include the CaCl2 precipitation method, the Hanahan method, the efficiency of which is improved by using a reducing agent such as dimethyl sulfoxide (DMSO) in the CaCl2 precipitation method, electroporation, calcium phosphate precipitation, protoplast fusion, agitation using silicon carbide fibers, Agrobacterium-mediated transformation, transformation methods using PEG, dextran sulfate, or lipofectamine, and desiccation / inhibition-mediated transformation. In addition, by using infection as a means, the target substance can be delivered into cells using virus particles. In addition, vectors can be introduced into host cells using a gene gun or the like.

[0056] In addition, the host cell used to create the transformed cell is not particularly limited as long as it is capable of producing the fusion protein of the present invention. In particular, host cells may include, but are not limited to, prokaryotic cells, eukaryotic cells, and cells of mammalian, plant, insect, fungal, or bacterial origin. An example of a prokaryotic cell is Escherichia coli. An example of a eukaryotic cell is yeast. Mammalian cells include, but are not limited to, CHO cells, F2N cells, COS cells, BHK cells, Bowes melanoma cells, HeLa cells, 911 cells, AT1080 cells, A549 cells, SP2 / 0 cells, human lymphoblastoid cells, NSO cells, HT-1080 cells, PERC.6 cells, HEK293 cells, and HEK293T cells. Any cells known to those skilled in the art as suitable for use as mammalian host cells may be used.

[0057] As described above, to optimize the properties of the fusion protein as a therapeutic agent, or for any other purpose, the glycosylation pattern (e.g., sialic acid, fucosylation, glycosylation) of the fusion protein can be adjusted by manipulating glycosylation-related genes carried by the host cell through methods known to those skilled in the art.

[0058] Methods for producing fusion proteins In another aspect of the present invention, a method for producing a fusion protein dimer comprising FcεRIα-ECD and a fragment of an anti-IL-4R antibody is provided.

[0059] A method for producing the fusion protein can include: i) culturing the transformed cells; and ii) recovering the fusion protein dimer of the present invention comprising FcεRIα-ECD and a fragment of an anti-IL-4R antibody.

[0060] As used herein, the term "cultivation" refers to a method of growing microorganisms under appropriately artificially controlled environmental conditions.

[0061] The method for culturing the transformed cells can be carried out using methods well known in the art. In particular, the culture is not particularly limited as long as the culture can express and produce the fusion protein of the present invention. In particular, the culture can be carried out by a batch method or continuously by a fed-batch method or a repeated fed-batch method.

[0062] In addition, the step of recovering the fusion protein dimer from the culture can be carried out by a method known in the art. In particular, the recovery method is not particularly limited as long as it can recover the produced fusion protein of the present invention. Preferably, the recovery method may be centrifugation, filtration, extraction, spraying, drying, evaporation, precipitation, crystallization, electrophoresis, differential dissolution (e.g., ammonium sulfate precipitation), chromatography (e.g., ion exchange, affinity, hydrophobic, and size exclusion), etc.

[0063] Use of fusion proteins In another aspect of the present invention, there is provided a pharmaceutical composition for preventing or treating an allergic disease, comprising a fusion protein dimer comprising FcεRIα-ECD and a fragment of an anti-IL-4R antibody.

[0064] As used herein, the term "allergic disease" refers to a pathological condition caused by an allergic reaction mediated by mast cell activation, such as mast cell degranulation. Such allergic diseases include food allergies, atopic dermatitis, asthma, allergic rhinitis, allergic conjunctivitis, allergic dermatitis, allergic contact dermatitis, anaphylaxis, urticaria, pruritus, insect allergies, chronic idiopathic urticaria, chronic spontaneous urticaria, drug allergies, etc. In particular, allergic diseases may be IgE-mediated.

[0065] The term "prevention" refers to any action that inhibits the occurrence or delays the onset of an allergic disease by administering a pharmaceutical composition. The term "treatment" refers to any action that improves or beneficially alters the symptoms of an allergic disease by administering a pharmaceutical composition.

[0066] In the pharmaceutical composition of the present invention for treating or preventing allergic diseases, the fusion protein dimer can be contained in any amount (effective amount) depending on the use, formulation, purpose of incorporation, etc., as long as the fusion protein dimer can exhibit anti-allergic activity. A conventional effective amount can be determined within the range of 0.001% to 20.0% by weight based on the total weight of the composition. Here, the "effective amount" refers to the amount of active ingredient capable of inducing an anti-allergic effect. Such an effective amount can be determined experimentally within the scope of the general knowledge of those skilled in the art.

[0067] Here, the pharmaceutical composition can further comprise a pharmaceutically acceptable carrier. As long as the pharmaceutically acceptable carrier is a non-toxic substance suitable for delivery to a patient, the pharmaceutically acceptable carrier can be any carrier. Distilled water, alcohol, fat, wax, and inert solids can be included as carriers. Pharmaceutically acceptable auxiliary substances (buffers, dispersants) can also be included in the pharmaceutical composition.

[0068] In particular, the pharmaceutical composition of the present invention contains a pharmaceutically acceptable carrier in addition to the fusion protein dimer, and can be prepared into a parenteral formulation by a standard method known in the art depending on the administration route. Here, the term "pharmaceutically acceptable" means that the subject to which it is applied (prescribed) does not have any greater toxicity than can be tolerated without inhibiting the activity of the fusion protein dimer.

[0069] When the pharmaceutical composition of the present invention is prepared as an oral formulation, the pharmaceutical composition can be prepared with a suitable carrier in the form of powder, granules, tablets, pills, sugar-coated tablets, capsules, liquids, gels, syrups, suspensions, wafers, etc., by a method known in the art. Examples of suitable pharmaceutically acceptable carriers include sugars such as lactose, glucose, sucrose, dextrose, sorbitol, mannitol, and xylitol; starches such as corn starch, potato starch, and wheat starch; celluloses such as methylcellulose, ethylcellulose, sodium carboxymethylcellulose, and hydroxypropylmethylcellulose; polyvinylpyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, magnesium stearate, mineral oil, malt, gelatin, talc, polyols, vegetable oils, etc. In the case of formulations, diluents and / or excipients such as fillers, extenders, binders, wetting agents, disintegrants, and surfactants can be included, as necessary, to formulate the formulation.

[0070] When the pharmaceutical composition of the present invention is prepared into a parenteral preparation, it can be formulated into the form of an injection, a transdermal drug, a nasal inhaler, and a suppository together with a suitable carrier by a method known in the art.When formulated into an injection, sterile water, ethanol, a polyhydric alcohol such as glycerol or propylene glycol, or a mixture thereof can be used as a suitable carrier.As for the carrier, an isotonic solution such as Ringer's solution, phosphate buffered saline (PBS) containing triethanolamine, sterile water for injection, 5% dextrose, etc. can be preferably used.

[0071] The formulation of pharmaceutical compositions is known in the art, and reference may be made in particular to Remington's Pharmaceutical Sciences (19th Edition, 1995), which is incorporated herein by reference.

[0072] Meanwhile, the pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount.As used herein, the term "administration" means introducing a predetermined substance into a subject by an appropriate method, and as long as the composition can reach the target tissue, the composition can be administered via any common route.The route of administration may include, but is not limited to, oral administration, intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, topical administration, intranasal administration, and rectal administration.

[0073] The term "pharmaceutically effective amount" refers to an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to any medical treatment and which does not cause side effects. The effective dose level can be readily determined by one skilled in the art depending on factors such as the patient's sex, age, weight, and health condition, the type and severity of the disease, the activity of the drug, sensitivity to the drug, the method, time of administration, route of administration and excretion rate, duration of treatment, concomitant or simultaneous drugs used, and other factors well known in the medical field.

[0074] The preferred daily dosage of the pharmaceutical composition of the present invention may range from 0.01 μg / kg to 10 g / kg per day, preferably from 0.01 mg / kg to 1 g / kg per day, depending on the patient's condition, body weight, sex, age, severity of the disease, or route of administration. Administration may be once a day or several times a day. Such dosages should not be construed as limiting the scope of the present invention in any way.

[0075] The term "subject" refers to a subject to which the composition of the present invention can be applied (prescribed), and may be a mammal such as a human, rat, mouse, or livestock. Preferably, the subject may be a human, but is not limited to this. In addition to the fusion protein dimer, the anti-allergic composition of the present invention may further contain any compound or natural extract known to have anti-allergic activity and whose safety has already been verified for enhancing and strengthening the anti-allergic activity. Here, the fusion protein dimer and the compound or natural extract having anti-allergic activity may be administered simultaneously or sequentially.

[0076] In another aspect of the present invention, there is provided a food composition for improving or alleviating allergic symptoms, comprising a fusion protein dimer comprising FcεRIα-ECD and a fragment of an anti-IL-4R antibody.

[0077] Here, the fusion protein dimer can be combined with an appropriate delivery means for efficient delivery to the intestine. Additionally, the food composition of the present invention can be prepared in any form, such as beverages, e.g., tea, juice, carbonated drinks, and electrolyte drinks; processed dairy products, e.g., milk and yogurt; and health functional food preparations, e.g., tablets, capsules, pills, granules, liquids, powders, flakes, pastes, syrups, gels, jellies, and bars. Additionally, as long as the food composition of the present invention complies with the applicable regulations when manufactured and distributed, the food composition can fall into any product category in legal or functional classifications. For example, the food composition may be a health functional food in accordance with the Health Functional Food Act, or the food composition may be a confectionery, bean, tea, beverage, food for special dietary uses, or the like, according to the food type in the Food Standards Regulations (Food and Drug Administration Safety Notification, Food Standards and Specifications) of the Food Sanitation Act. For other food additives that can be included in the food composition of the present invention, reference may be made to the Food Safety Regulations or Food Additive Regulations in accordance with the Food Sanitation Act.

[0078] Another aspect of the present invention provides use of a fusion protein dimer comprising FcεRIα-ECD and a fragment of an anti-IL-4R antibody for the manufacture of a medicament for treating or preventing an allergic disease, wherein FcεRIα-ECD, anti-IL-4R antibody, allergic disease, treatment and prevention are as defined above.

[0079] Another aspect of the present invention provides use of a fusion protein dimer comprising FcεRIα-ECD and a fragment of an anti-IL-4R antibody for the treatment or prevention of an allergic disease, wherein FcεRIα-ECD, anti-IL-4R antibody, allergic disease, treatment and prevention are as defined above.

[0080] In another aspect, the present invention provides a method for treating or preventing an allergic disease, comprising administering to a subject a fusion protein dimer comprising FcεRIα-ECD and a fragment of an anti-IL-4R antibody, wherein FcεRIα-ECD, anti-IL-4R antibody, administration, allergic disease, treatment, and prevention are as described above.

[0081] The subject may be a mammal, preferably a human. In addition, the subject may be a patient suffering from an allergic disease or a subject at high risk of suffering from an allergic disease.

[0082] The route of administration, dosage, and frequency of administration of a fusion protein dimer comprising an FcεRIα-ECD and an anti-IL-4R antibody fragment can vary depending on the patient's condition and the presence or absence of side effects; therefore, the fusion protein dimer may be administered to a subject in various ways and amounts. Those skilled in the art can select the optimal administration method, dosage, and frequency of administration within an appropriate range. In addition, the fusion protein dimer comprising an FcεRIα-ECD and an anti-IL-4R antibody fragment may be administered in combination with other drugs or physiologically active substances known to have therapeutic effects on the disease being treated, or may be formulated in the form of a combination drug with other drugs. [Example]

[0083] [Mode for Carrying Out the Invention] The present invention will now be described in more detail with reference to the following examples, which will be apparent to those skilled in the art, as they are intended to illustrate the invention by reference and should not be construed as limiting the scope of the present invention.

[0084] Preparation Example 1. Preparation of FcεRIα ECD-Fc-anti-IL-4R scFv fusion protein: GI-305 To generate a fusion protein containing the FcεRI α-chain extracellular domain, an Fc domain, and an antibody that specifically binds to the IL-4 receptor (IL-4R), a polynucleotide containing a nucleotide sequence (SEQ ID NO: 19) encoding a fusion protein containing, from the N-terminus, the FcεRI α-chain extracellular domain (SEQ ID NO: 2), a linker (SEQ ID NO: 3), an IgG4 Fc domain (SEQ ID NO: 4), a linker (SEQ ID NO: 5), and an scFv of an antibody that specifically binds to the IL-4 receptor (SEQ ID NO: 6) in this order was synthesized and loaded into a pcDNA3.4 vector (Genscript).

[0085] The vector was introduced into CHO cells (ExpiCHO-S cells). The cells were then cultured at 37°C, 8% CO2, in serum-free ExpiCHO™ Expression Medium (Thermo Fisher Scientific) for 14 days. The culture medium was then harvested, and the fusion protein was purified using affinity chromatography (affinity purification column).

[0086] The molecular weight and purity of the purified fusion protein were confirmed by SDS-PAGE and Western blot analysis. SDS-PAGE and Western blot analysis confirmed that the protein was detected under both non-reducing and reducing conditions. This confirmed that the purified fusion protein formed a dimer (Figures 2 and 3). The fusion protein dimer was named "GI-305 (also known as FcεRIα ECD-Fc-anti-IL-4R scFv)."

[0087] Preparation Example 2. Preparation of FcεRIα ECD-Fc fusion protein as a control Preparation Example 2.1. Preparation of GI-301 To generate a control fusion protein containing the FcεRI α-chain extracellular domain and Fc domain, a polynucleotide containing a nucleotide sequence (SEQ ID NO: 30) encoding a fusion protein containing, from the N-terminus, the FcεRI α-chain extracellular domain (SEQ ID NO: 2), a linker (SEQ ID NO: 28), and a modified IgG4 Fc domain (SEQ ID NO: 29), in this order, was synthesized and loaded into a pcDNA3.4 vector (Genscript).

[0088] The vector was introduced into CHO cells (ExpiCHO-S cells). The cells were then cultured at 37°C, 8% CO2, in serum-free ExpiCHO™ Expression Medium (Thermo Fisher Scientific) for 14 days. The culture medium was then harvested, and the fusion protein dimer was purified using affinity chromatography (affinity purification column).

[0089] Preparation Example 2.2. Preparation of GI-305CN To generate a fusion protein containing the FcεRI α-chain extracellular domain and the Fc domain, a polynucleotide containing a nucleotide sequence (SEQ ID NO: 31) encoding a fusion protein containing, in this order from the N-terminus, the FcεRI α-chain extracellular domain (SEQ ID NO: 2), a linker (SEQ ID NO: 3), and the IgG4 Fc domain (SEQ ID NO: 4) was synthesized and loaded into the pcDNA3.4 vector (Genscript).

[0090] The vector was introduced into CHO cells (ExpiCHO-S cells). The cells were then cultured at 37°C, 8% CO2, in serum-free ExpiCHO™ Expression Medium (Thermo Fisher Scientific) for 14 days. The culture medium was then harvested, and the fusion protein dimer was purified using affinity chromatography (affinity purification column).

[0091] Preparation Example 3. Preparation of Fc-anti-IL4Rα scFv: GI-305C1 as a control To produce a fusion protein containing an Fc domain and an antibody that specifically binds to IL-4 receptor (IL-4R), a polynucleotide containing a nucleotide sequence (SEQ ID NO: 32) encoding a fusion protein containing, from the N-terminus, an IgG4 Fc domain (SEQ ID NO: 4), a linker (SEQ ID NO: 5), and an scFv (SEQ ID NO: 6) of an antibody that specifically binds to IL-4 receptor (IL-4R), in this order, was synthesized and loaded into a pcDNA3.4 vector (Genscript).

[0092] The vector was introduced into CHO cells (ExpiCHO-S cells). The cells were then cultured at 37°C, 8% CO2, in serum-free ExpiCHO™ Expression Medium (Thermo Fisher Scientific) for 14 days. The culture medium was then harvested, and the fusion protein dimer was purified using affinity chromatography (affinity purification column).

[0093] Experimental Example 1: Confirmation of the binding ability of GI-305 fusion protein dimer to human IgE The IgE-binding ability of GI-305, a fusion protein containing FcεRIα ECD and an anti-IL-4R antibody fragment obtained by the method described in Example 1 above, was measured. At this point, the IgE-binding ability was confirmed using Octet RED384 (ForteBio). An AHC (anti-human IgG capture) biosensor was used, immersed in 1× kinetic buffer for 10 minutes. GI-305 was prepared at a concentration of 10 μg / mL and coated onto the biosensor. IgE was serially diluted from 1.6 to 100 nM, and the binding ability was confirmed at each concentration. The binding ability between the GI-305 fusion protein dimer and IgE was measured as shown in Figure 4 and Table 2.

[0094] [Table 2]

[0095] Experimental Example 2: Confirmation of the inhibitory effect of GI-305 fusion protein dimer on the activity of IL-4 and IL-13 To confirm the activity of the GI-305 fusion protein dimer as an anti-IL-4R antibody, experiments were performed using TF-1 cells, a cytokine-dependent cell line for cell proliferation. TF-1 cells respond to several cytokines, including IL-4 and IL-13. We examined whether the GI-305 fusion protein dimer inhibited IL-4- or IL-13-mediated cell proliferation of TF-1 cells.

[0096] TF-1 cells (ATCC, #CRL-2003) were cultured in medium (RPMI-1640 + 10% FBS + 1% penicillin / streptomycin) containing human GM-CSF (4 ng / mL, R&D Systems). Prior to analysis, TF-1 cells were pelleted by centrifugation at 1,500 rpm for 5 minutes, the medium was removed, and the cells were resuspended in assay medium without GM-CSF. 5 × 10 resuspended cells were added to each well of a 96-well plate. 4 Aliquots were taken at a cell / well density. GI-305 fusion protein dimers at various concentrations were mixed with IL-4 (100 ng / mL) and IL-13 (100 ng / mL) and added to the wells. The assay plate was incubated at 37°C in 5% CO for 24 hours. Then, 10 μL of WST-1 (Roche) was added to each well and incubated for 4 hours.

[0097] The absorbance at 450 nm was then recorded using a plate reader, and the data were analyzed using GraphPad Prism software.The results showed that GI-305 inhibited IL-4- or IL-13-induced proliferation of TF-1 cells (Figure 5).

[0098] Experimental Example 3: Confirmation of the serum IgE level-reducing effect of GI-305 fusion protein dimer IL-4 and IL-13 promote B cell proliferation, costimulate CD40 / CD40L, and induce IgG4 and IgE class switching. The effect of GI-305 fusion protein dimers on IL-4-induced IgE release from B cells was evaluated. Peripheral blood mononuclear cells (PBMCs) were purchased from Cellular Technology Limited and used for the experiment. PBMCs were cultured in assay medium (RPMI-1640 + 10% FBS + 1% penicillin / streptomycin).

[0099] Prior to analysis, PBMCs stored in LN2 tanks were pelleted by centrifugation at 1,500 rpm for 5 minutes, the medium was removed by aspiration, and the cells were then resuspended in assay medium. Cells were plated at 2 × 10 cells per well of a 96-well plate in assay medium. 5 Aliquots were taken at a cell / well density. The commercially available anti-IgE antibody omalizumab (trade name: Xolair), the control anti-IL-4Rα antibody dupilumab (trade name: Dupixent), GI-301 (FcεRIα ECD-modified Fc), GI-305CN (FcεRIα ECD)-IgG4 Fc), and GI-305C1 (IgG4 Fc-IL-4Rα scFv) prepared in the above preparation examples, and the test substance GI-305 were mixed with IL-4 (30 ng / mL) and anti-human CD40 antibody (1 μg / mL), respectively, and added to the plate wells. The control and test substances were prepared and used at concentrations of 0.008 nM or 0.04 nM. The assay plate was incubated at 37°C in 5% CO2 for 12 days. The cells were then pelleted by centrifugation at 1,500 rpm, and the IgE levels present in the supernatant were measured using an R-PLEX assay kit (MSD).

[0100] Specifically, 25 μL of biotinylated capture antibody was aliquoted into each well of an MSD GOLD 96-well Small Spot Streptavidin Plate. After 1 hour of incubation at room temperature, each well was washed five times with 150 μL of PBST (PBS containing 0.05% Tween 20). Then, 25 μL of sample was added to each well and then incubated for 1 hour at room temperature. Each well was then washed five times with 150 μL of PBST per well. After washing, 150 μL of sulfotag detection antibody was aliquoted into each well. After 1 hour of incubation at room temperature, each well was washed five times with 150 μL of PBST per well. Then, 150 μL of MSD GOLD lead buffer was aliquoted into each well and measured on a MESO QuickPlex SQ120. Data were analyzed using GraphPad Prism software.

[0101] As a result, the GI-305 fusion protein dimer was found to exhibit significantly superior effects in inhibiting IgE production through the inhibition of B cell activity compared to the anti-IgE antibody omalizumab, the anti-IL-4Rα antibody dupilumab, GI-301 (FcεRIα ECD-modified Fc), GI-305CN (FcεRIα ECD-IgG4Fc), and GI-305C1 (IgG4Fc-IL-4Rα scFv) (Figures 6 and 7).

Claims

1. a fusion protein dimer in which two fusion proteins are linked together, each fusion protein comprising an IgE Fc receptor alpha subunit extracellular domain (FcεRIα-ECD), an immunoglobulin Fc region, and a fragment of an anti-IL-4R antibody; the FcεRIα-ECD consists of the amino acid sequence of SEQ ID NO: 2; A fusion protein dimer, wherein the fragment of the anti-IL-4R antibody comprises a heavy chain variable region comprising HCDR1 of SEQ ID NO: 20, HCDR2 of SEQ ID NO: 21, and HCDR3 of SEQ ID NO: 22, and a light chain variable region comprising LCDR1 of SEQ ID NO: 23, LCDR2 of SEQ ID NO: 24, and LCDR3 of SEQ ID NO:

25.

2. The fusion protein dimer of claim 1, wherein the anti-IL-4R antibody fragment comprises a heavy chain variable region of SEQ ID NO: 7 and a light chain variable region of SEQ ID NO:

8.

3. The fusion protein dimer of claim 2, wherein the heavy chain variable region of SEQ ID NO: 7 and the light chain variable region of SEQ ID NO: 8 are linked by a peptide linker.

4. The fusion protein has the following structural formula (I) or (II): N'-X-linker (1)-Fc region fragment or variant thereof-linker (2)-Y-C'(I) N'-Y-linker (1)-Fc region fragment or variant thereof-linker (2)-X-C' (II) It consists of In the structural formulas (I) and (II), N' is the N-terminus of the fusion protein; C' is the C-terminus of the fusion protein; X is FcεRIα-ECD; Y is a fragment of the anti-IL-4R antibody, and The linker (1) and the linker (2) are peptide linkers. The fusion protein dimer of claim 1.

5. The fusion protein dimer according to claim 4, wherein the Fc region of the fusion protein is derived from human IgG4.

6. A polynucleotide encoding a fusion protein, the fusion protein comprises an IgE Fc receptor alpha subunit extracellular domain (FcεRIα-ECD), an immunoglobulin Fc region, and a fragment of an anti-IL-4R antibody; the FcεRIα-ECD consists of the amino acid sequence of SEQ ID NO: 2; A polynucleotide wherein the fragment of the anti-IL-4R antibody comprises a heavy chain variable region comprising HCDR1 of SEQ ID NO: 20, HCDR2 of SEQ ID NO: 21, and HCDR3 of SEQ ID NO: 22, and a light chain variable region comprising LCDR1 of SEQ ID NO: 23, LCDR2 of SEQ ID NO: 24, and LCDR3 of SEQ ID NO:

25.

7. An expression vector comprising the polynucleotide of claim 6.

8. A transformed cell into which the vector according to claim 7 has been introduced.

9. i) culturing the transformed cells of claim 8; and ii) recovering the fusion protein dimer; Including, Methods for generating fusion protein dimers.

10. A pharmaceutical composition for preventing or treating an allergic disease, comprising the fusion protein dimer of claim 1.

11. 11. The pharmaceutical composition according to claim 10, wherein the allergic disease is selected from the group consisting of food allergy, atopic dermatitis, asthma, allergic rhinitis, allergic conjunctivitis, allergic dermatitis, chronic idiopathic urticaria, and allergic contact dermatitis.

12. A food composition for improving or alleviating allergic symptoms, comprising the fusion protein dimer of claim 1.

13. 10. Use of the fusion protein dimer of claim 1 for the manufacture of a medicament for the treatment or prevention of an allergic disease.

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