RU2796162C2 - EXTRACELLULAR DOMAIN OF ALPHA SUBUNIT OF IgE Fc RECEPTOR, A PHARMACEUTICAL COMPOSITION CONTAINING IT AND A METHOD FOR ITS PRODUCTION - Google Patents

Abstract

FIELD: pharmaceuticals.

SUBSTANCE: invention relates to a polypeptide dimer capable of binding to IgE. The polypeptide dimer contains two monomers, each containing the extracellular domain of the alpha subunit of the IgE Fc receptor (FcεRIa-ECD). Also a polynucleotide that encodes a monomer contained in the said polypeptide dimer is proposed, wherein the monomer contains the extracellular domain of the alpha subunit of the IgE Fc receptor (FcεRIa-ECD). Also an expression vector, a host cell, a pharmaceutical composition, a food composition, a method for producing a polypeptide dimer, and a method for treating or preventing an allergic disease are proposed. The polypeptide dimer binds to IgE and exhibits fewer side effects due to the lack of ADCC and CDC functions.

EFFECT: invention can be used to treat or prevent an IgE-mediated allergic disease.

10 cl, 9 dwg, 3 tbl, 7 ex

Description

FIELD OF TECHNOLOGY

The present invention relates to modified IgE Fc receptors and their uses.

BACKGROUND OF THE INVENTION

Allergic diseases such as allergic rhinitis, atopic dermatitis, and food allergies, including asthma, are spreading at a high rate in industrialized and Europeanized modern societies, and the incidence of anaphylaxis, a severe allergic disease, is also on the rise. These chronic immune diseases greatly impair the quality of life of individuals and the socioeconomic costs increase accordingly. Thus, there is an urgent need for measures to overcome such diseases.

Most allergic diseases are caused by an excessive immune response due to immunoglobulin E (IgE). IgE is an antibody that is present in serum at a very low concentration under normal conditions. IgE is also usually produced as a result of exposure to harmless antigens. There is a case where the level of IgE increases without any specific stimulus. Such a case can lead to allergic diseases. Abnormally increased amounts of IgE can bind to high affinity IgE Fc receptors (FcεRI) that are expressed on the surface of mast cells, basophils, and the like.

This binding causes the mast cells or basophils to release chemical mediators such as histamine, leukotriene, prostaglandin, bradykinin, and platelet activating factors. The release of these chemical mediators results in allergic symptoms. In particular, allergic diseases may show more severe symptoms due to the binding between IgE and FcεRI. It is known that the level of FcεRI-expressing cells increases in allergic patients.

At present, various methods have been proposed for the treatment of allergic diseases, such as allergen avoidance, administration of antiallergic agents, modulation of IgE synthesis in the body, and development of anti-IgE antibodies. However, the therapeutic methods known so far have many disadvantages, such as failure to treat the root cause of allergy, lack of efficacy of drugs, and the occurrence of serious side effects.

In addition, immunoglobulin compositions capable of binding to IgE and FcγRIIb with high affinity and inhibiting cells expressing IgE (KR10-1783272B1) have been investigated. Such compositions are described as useful in the treatment of IgE-mediated disorders including allergy and asthma. In addition, omalizumab (trade name: Xolair), which targets the Fc portion of an IgE antibody, has been developed and used as a therapeutic agent for refractory severe asthma and refractory urticaria.

However, administration of high dose omalizumab to maintain therapeutic effects results in high cost and side effects such as angioedema and anaphylactic reaction (The Journal of Clinical Investigation Volume 99, Number 5, March 1997, 915-925). In addition, based on the results of a post-marketing study, allergic granulomatous vasculitis and idiopathic severe thrombocytopenia have been described. Thus, there is a growing need to develop therapeutic agents capable of effectively treating allergic diseases without side effects.

Although the main mechanism by which omalizumab causes side effects has not yet been identified, it can be expected that it is due to the fact that omalizumab is an IgG1 antibody. A study in a mouse model showed that a large number of antigen-specific IgG1 antibodies can induce passive systemic anaphylaxis (PSA) via FcγRIII, a low affinity IgG receptor, and platelet-activating factors in an antigen-rich environment. In addition, such IgG-mediated anaphylaxis occurs to an excessive extent due to the lack of FcεRI signaling. Thus, passive systemic anaphylaxis may occur when a significant amount of omalizumab is injected into a patient with an allergic disease who exhibits a high level of IgE. It has recently been described that FcγRIIA, a low affinity IgG receptor expressed in humans, is also associated with IgG-mediated anaphylaxis. In addition, post-marketing studies have shown abnormal reactions such as allergic granulomatous vasculitis and idiopathic severe thrombocytopenia.

Technical problem

An object of the present invention is to provide a polypeptide dimeric protein for the treatment of an IgE-mediated allergic disease. Another object of the present invention is to provide a nucleic acid molecule encoding a protein, an expression vector containing the nucleic acid molecule, and a host cell containing the expression vector. Another object of the present invention is to provide a method for producing a polypeptide dimer.

Solution

To achieve the objectives described above, a polypeptide dimer is provided, containing two monomers, each of which contains the extracellular domain of the alpha subunit of the IgE Fc receptor. The monomer contains a modified Fc region and the modified Fc region and the extracellular domain of the alpha subunit of the Fc receptor of the IgE are linked through the hinge region of the IgD antibody. In another aspect, a pharmaceutical composition for treating or preventing an allergic disease is provided, comprising a polypeptide dimer as an active ingredient.

Advantageous Effects of the Invention

The polypeptide dimeric protein of the present invention not only has excellent safety and body retention compared to conventionally used anti-IgE antibodies, but also binds very strongly to IgE due to having an ability to bind to IgE that is 70 times higher than that of conventionally used antibodies against IgE omalizumab, which allows a longer course of administration. In addition, the polypeptide dimeric protein of the present invention is a substance obtained using a modified Fc that has only IgE as the only target and does not bind to the Fc-gamma receptor, and thus lacks the functions of antibody-dependent cellular cytotoxicity (ADCC). ) and complement dependent cytotoxicity (CDC). Thus, unlike conventional anti-IgE antibodies containing the Fc region of IgG1, the polypeptide dimeric protein does not bind to the Fc-gamma receptor and thus can inhibit the release of mediators caused by binding to the Fc-gamma receptor on the surface of mast cells, so that severe side effects, such as the occurrence of anaphylaxis, which can be caused by binding between IgG1 and the Fc-gamma III receptor on mast cells, can be minimized. Thus, the polypeptide dimeric protein of the present invention can be used as a novel pharmaceutical composition that can replace conventional anti-IgE antibody therapeutics.

Brief description of the drawings

Figure 1 presents the results of SDS-PAGE and isoelectric gel focusing (IEF) for proteins produced in each cell line. In this case, it can be seen that the truncated form was not formed under either reducing or non-reducing conditions, and that the content of acidic proteins increases due to the increase in the content of sialic acids caused by the introduction of the sialic acid transferase gene.

2 illustrates SDS-PAGE results for unreduced and reduced forms of a polypeptide dimeric protein in accordance with an embodiment of the present invention. In particular, it can be seen that the polypeptide dimer is of high purity even in the culture supernatant, which corresponds to the input material.

Figure 3 illustrates a graph showing the binding ability of omalizumab to IgE. The graph shows the results obtained by immobilizing omalizumab and analyzing its binding capacity as a function of the IgE concentrations used.

Figure 4 illustrates a graph showing the IgE binding ability of a polypeptide dimeric protein in accordance with one embodiment of the present invention. The graph shows the results obtained by immobilizing the dimeric protein and analyzing its binding capacity as a function of the IgE concentrations used.

Figure 5 illustrates the results obtained by identifying the interactions of the polypeptide dimeric protein (IgE _TRAP ), which is an embodiment of the present invention, and omalizumab with IgG receptors FcγRI (Fig.5A), FcγRIIA (Fig.5B), FcγRIIB (Fig.5C ), FcγRIIIA (FIG. 5D) and FcγRIIIB (FIG. 5E) using biolayer interferometry (BLI) analysis.

Figure 6 illustrates a graph obtained by quantifying the binding capacity between IgE _TRAP and IgG receptors, and between omalizumab and IgG receptors.

FIG. 7 is a graph showing the inhibitory activity of mouse-derived mast cell polypeptide dimeric protein (IgE _TRAP ) according to an embodiment of the present invention as a function of its concentrations.

FIG. 8 is a graph showing a comparison between the inhibitory activity of human FcεRI-expressing mouse-derived mast cell polypeptide dimeric protein (IgE _TRAP ) according to an embodiment of the present invention and Xolair (omalizumab) as a function of their concentration.

9 is a graph showing the effects of administration of a polypeptide dimeric protein according to one embodiment of the present invention in a food allergy model.

Detailed description of the invention

The present invention relates to a polypeptide dimer containing two monomers, each of which contains the extracellular domain (FcεRIa-ECD) of the alpha subunit of the Fc receptor of IgE, where the monomer contains a modified Fc region and a modified Fc region and FcεRIa-ECD are connected through the hinge region IgD antibodies.

As used herein, the term "IgE" refers to an antibody protein known as immunoglobulin E. IgE has an affinity for mast cells, blood basophils, and the like. In addition, the reaction between an IgE antibody and an antigen (allergen) corresponding to it causes an inflammatory reaction. In addition, IgE is known to be an antibody that causes anaphylaxis.

As used herein, the term "IgE Fc receptor" is also referred to as the Fcε receptor and it binds to the Fc portion of IgE. There are two types of this receptor. The receptor with high affinity for Fc IgE is called Fcε receptor I (FcεRI). The receptor with low affinity for Fc IgE is called the Fcε receptor II (FcεRII). FcεRI is expressed in mast cells and basophils. When IgE antibodies bound to FcεRI are bound by polyvalent antigens, degranulation of mast cells or basophils occurs, thereby releasing various carrier chemicals, including histamine. This release leads to an immediate allergic reaction.

FcεRI is a membrane protein consisting of one α-chain, one β-chain and two γ-chains linked by a disulfide bond. Among these chains, the part to which IgE binds is the α-chain (FcεRIα). FcεRIα has a size of approximately 60 kDa and consists of a hydrophobic domain that exists inside the cell membrane and a hydrophilic domain that exists outside the cell membrane. In particular, IgE binds to the extracellular domain of the α chain.

In particular, the alpha subunit of the IgE Fc receptor may have the amino acid sequence specified in NP_001992.1. In addition, the extracellular domain (FcεRIa-ECD) of the alpha subunit of the IgE Fc receptor may have the amino acid sequence of SEQ ID NO: 1. In this application, the extracellular domain of the alpha subunit of the Fc receptor of IgE may be a fragment or variant of the extracellular domain of the alpha subunit An IgE Fc receptor provided that the fragment or variant is capable of binding to IgE.

A variant can be generated by a method of substitution, deletion or insertion of one or more proteins into the wild-type FcεRIa-ECD (extracellular domain), provided that the method does not alter the function of the FcεRI α-chain. 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: 1. In addition , FcεRia-ECD SEQ ID NO: 1 may be encoded by a polynucleotide having the sequence of SEQ ID NO: 5.

In addition, as used herein, the term "modified Fc region" means a region in which a portion of the Fc region of an antibody has been modified. As used herein, the term “Fc region” refers to a protein that contains the heavy chain constant region 2 (CH2) and the heavy chain constant region 3 (CH3) of an immunoglobulin, and does not contain the heavy and light chain variable regions and the light chain constant region 1 (CH1) immunoglobulin. In particular, a modified Fc region means a region obtained by substituting certain amino acids in an Fc region or by combining different types of Fc regions. In particular, the modified Fc region may have the amino acid sequence of SEQ ID NO: 2. In addition, the modified Fc region of SEQ ID NO: 2 may be encoded by a polynucleotide having the sequence of SEQ ID NO: 6.

In addition, the modified Fc region of the present invention may be in a form having sugar chains in native form, an increased number of sugar chains relative to native form, or a reduced number of sugar chains relative to native form, or it may be in a form from which the sugar chain has been removed. The Fc sugar chains of immunoglobulins can be modified by conventional methods such as chemical methods, enzymatic methods, and genetic engineering methods using microorganisms.

As used herein, a "modified Fc region" of the present invention may be a region that lacks antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) functions due to the absence of FcγR or C1q binding sites. In addition, the modified Fc region and the FcεRIα-ECD can be linked through the hinge region of an IgD antibody. The hinge region of an IgD antibody consists of 64 amino acids and can selectively comprise 20-60 consecutive amino acids, 25-50 consecutive amino acids, or 30-40 amino acids. In one embodiment, the hinge region of an IgD antibody may contain 30 or 49 amino acids, as shown below. In addition, the hinge region of the IgD antibody may be a variant of the hinge region obtained by modifying the hinge region, where the hinge region may contain at least one cysteine residue. Within the scope of the present invention, a hinge region variant can be obtained by modifying certain residues in the sequence of the hinge region of an IgD antibody to minimize the formation of truncated forms during the protein production process.

In one embodiment, the hinge region may comprise the following sequence:

Arg Asn Thr Gly Arg Gly Gly Glu Glu Lys Lys Xaa1 Xaa2 Lys Glu Lys Glu Glu Gln Glu Glu Arg Glu Thr Lys Thr Pro Glu Cys Pro (SEQ ID NO: 17), where Xaa1 may be Lys or Gly and Xaa2 may be Glu, Gly or Ser. In particular, the hinge region may have the amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 19, thereby minimizing the formation of truncated forms during protein production.

In another embodiment, the hinge region may comprise the following sequence:

( SEQ ID NO: 18) wherein Xaa3 may be Lys or Gly and Xaa4 may be Glu, Gly or Ser. In particular, the hinge region may have the amino acid sequence of SEQ ID NO: 4, thereby minimizing the formation of truncated forms during protein production.

In particular, in the hinge region having the sequence of SEQ ID NO: 4, at least one of the Thr may be glycosylated. In particular, among the amino acids of SEQ ID NO: 18th, 13th, 14th, 18th or 19th Thr may be glycosylated. Preferably, all four amino acids may be glycosylated. Within the scope of the present application, the glycosylation may be an O-glycosylation.

In addition, as described above, the polypeptide dimer according to the present invention may have a form in which two monomers are linked to each other and each monomer is obtained by linking between the extracellular domain of the IgE Fc receptor alpha subunit and the modified Fc region. The polypeptide dimer may be in the form in which two identical monomers are linked to each other via a cysteine located in the linker region. In addition, the polypeptide dimer may be in the form in which two different monomers are linked to each other. For example, in the case where two monomers are different from each other, the polypeptide dimer may be in the form in which one monomer contains the extracellular domain of the IgE Fc receptor alpha subunit and the other monomer contains a fragment of the extracellular domain of the IgE Fc receptor alpha subunit. Within the scope of this application, an embodiment of a monomer may have the amino acid sequence of SEQ ID NO: 20, SEQ ID NO: 21, or SEQ ID NO: 22.

In addition, the polypeptide dimer according to the present invention exhibits an IgE binding capacity that is 10-100 times, 20-90 times, 20-70 times, 30-70 times, or 40-70 times higher than that of omalizumab, an anti-IgE antibody, and preferably it can show an IgE binding ability that is 70 times higher than that of omalizumab.

In another aspect of the invention, a polynucleotide is provided that encodes a monomer that contains the extracellular domain of the alpha subunit of an IgE receptor Fc to which a modified Fc region is associated.

Meanwhile, the polynucleotide may also contain a signal sequence or a leader sequence. As used herein, the term "signal sequence" means a nucleic acid encoding a signal peptide that directs the secretion of a target protein. The signal peptide is translated and then cleaved off in the host cell. In particular, the signal sequence of the present invention is a nucleotide encoding an amino acid sequence that initiates the movement of a protein across the membrane of the endoplasmic reticulum (ER). Suitable signal sequences for the present invention include antibody light chain sequences, e.g. antibody 14.18 (Gillies et al., J. Immunol. Meth 1989. 125:191-202), antibody heavy chain signal sequences, e.g. antibody heavy chain signal sequence MOPC141 (Sakano et al., Nature, 1980. 286:676-683), and other signal sequences known in the art (see, e.g., Watson et al., Nucleic Acid Research, 1984. 12:5145-5164) .

The characteristics of the signal sequences are well known in the art. Signal sequences typically contain 16-30 amino acid residues, and may contain more or fewer amino acid residues. A typical signal peptide consists of three regions, which are a major N-terminal region, a central hydrophobic region, and a more polar C-terminal region. The central hydrophobic region contains 4-12 hydrophobic residues that immobilize the signal sequence on the lipid bilayer of the membrane during the movement of the immature polypeptide.

After initiation, the signal sequence is cleaved in the ER lumen by cellular enzymes commonly known as signal peptidases. Within the scope of the present invention, the signal sequence may be a secretory signal sequence for tissue plasminogen activator (tPA), herpes simplex virus (HSV gD) glycoprotein D, or growth hormone. Preferably, secretory signal sequences used in higher eukaryotic cells, including mammals and the like, may be used. In addition, a signal sequence can be used in which codons are substituted for codons that are highly expressed in the host cell.

Meanwhile, the extracellular domain of the IgE Fc receptor alpha subunit monomer to which the signal sequence and the modified Fc region are associated may have the amino acid sequence of SEQ ID NO: 11 or SEQ ID NO: 13. Proteins of SEQ ID NO: 11 and SEQ ID NO: 13 can be encoded by polynucleotides having the sequences SEQ ID NO: 12 and SEQ ID NO: 14, respectively.

In another aspect of the present invention, an expression vector is provided into which a polynucleotide encoding a monomer is inserted. Within the scope of the present invention, the polynucleotide may have the sequence of SEQ ID NO: 12 or SEQ ID NO: 14.

As used herein, the term "vector" is intended to be a structure that is intended to be introduced into a host cell and is capable of recombining with and integrating into the host cell's genome. Alternatively, the vector is an episome and is understood as a nucleic acid element containing a nucleotide sequence that can autonomously replicate. Vectors include linear nucleic acids, plasmids, phagemids, cosmids, RNA vectors, viral vectors, and their analogs. Examples of viral vectors include, but are not limited to, retroviruses, adenoviruses, and adeno-associated viruses. In addition, plasmids may contain a selectable marker such as an antibiotic resistance gene, and host cells containing the plasmids may be cultured under selective conditions.

As used herein, the term "genetic expression" or "expression" of a target protein refers to transcription of a DNA sequence, translation of an mRNA transcript, and secretion of a fusion protein product or fragment thereof. A suitable expression vector may be RcCMV (Invitrogen, Carlsbad) or a variant thereof. The expression vector may contain a human cytomegalovirus (CMV) promoter to ensure continuous transcription of the target gene in mammalian cells, and a bovine growth hormone ladenylation signal sequence to increase the level of RNA stability after transcription.

In another aspect of the present invention, there is provided a host cell into which an expression vector has been introduced. As used herein, the term "host cell" refers to a prokaryotic or eukaryotic cell into which a recombinant expression vector can be introduced. As used herein, the terms "transduced", "transformed", and "transfected" mean the introduction of a nucleic acid (eg, vector) into a cell using methods known in the art.

Preferred host cells that can be used in the present invention include immortalized hybridoma cells, NS/0 myeloma cells, 293 cells, Chinese hamster ovary cells (CHO cells), HeLa cells, human cells derived from amniotic fluid (CapT cells) , or COS cells. Preferably, the host cells may be CHO cells. On the other hand, the host cell may be a cell into which said vector and a vector loaded with a sialic acid transferase gene have been introduced. Within the scope of the present invention, the sialic acid transferase may be a 2,3-sialic acid transferase or a 2,6-sialic acid transferase. Within the scope of the present invention, the 2,6-sialic acid transferase may have the amino acid sequence of SEQ ID NO: 15.

In another aspect of the present invention, a pharmaceutical composition for the treatment or prevention of an allergic disease is provided, comprising a polypeptide dimer as an active ingredient.

As used herein, the term "allergic disease" means a pathological symptom caused by an allergic reaction mediated by mast cell activation, such as degranulation of mast cells. Such allergic diseases include food allergy, atopic dermatitis, asthma, allergic rhinitis, allergic conjunctivitis, allergic dermatitis, allergic contact dermatitis, anaphylaxis, urticaria, pruritus, insect sting allergy, chronic idiopathic urticaria, drug allergy, and the like. In particular, allergic diseases can be IgE-mediated.

In the composition for treating or preventing an allergic disease of the present invention, the active ingredient may be contained in any amount (effective amount) depending on the use, formulation, purpose of mixing, and the like, as long as the active ingredient can exhibit antiallergic activity. A typical effective amount of the active ingredient is in the range of 0.001% by weight to 20.0% by weight, based on the total weight of the composition. As used herein, an "effective amount" refers to an amount of an active ingredient that is capable of inducing an antiallergic effect. Such an effective amount can be determined experimentally by one skilled in the art.

Within the scope of the present invention, the pharmaceutical composition may also contain a pharmaceutically acceptable carrier. Any carrier may be used as a pharmaceutically acceptable carrier, provided that the carrier is a non-toxic substance suitable for delivery to a patient. Carriers may include distilled water, alcohol, fat, wax, and an inert solid. The pharmaceutical composition may also contain pharmaceutically acceptable adjuvants (buffers and dispersing agents).

In particular, the pharmaceutical composition of the present invention contains, in addition to the active ingredient, a pharmaceutically acceptable carrier, and it can be formulated into an oral or parenteral formulation, depending on the route of administration, in a conventional manner known in the art. As used herein, the term "pharmaceutically acceptable" means having no greater toxicity than can be tolerated by the individual who will use (to whom) the agent, without inhibiting the activity of the active ingredient.

In the case where the pharmaceutical composition of the present invention is in the form of an oral formulation, the pharmaceutical composition may be in the form of formulations such as powders, granules, tablets, pills, sugar-coated tablets, capsules, liquids, gels, syrups, suspensions, and wafers. , together with suitable carriers, in accordance with methods known in this field. Within the scope of the present invention, examples of suitable pharmaceutically acceptable carriers may 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 cellulose, methyl cellulose , ethylcellulose, sodium carboxymethylcellulose, and hydroxypropylmethylcellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, magnesium stearate, mineral oil, malt, gelatin, talc, polyol, vegetable oil, and the like. In the case of preparation in the form of preparations, the preparation can be carried out, if necessary, by including diluents and/or excipients, such as a filler, a dry diluent, a binder, a wetting agent, a disintegrant and a surfactant.

In the case where the pharmaceutical composition of the present invention is formulated as a parenteral formulation, the pharmaceutical composition may be formulated as injections, transdermal drugs, nasal inhalers and suppositories, together with suitable carriers, in accordance with methods known in the art. . In the case of an injection, sterilized water, ethanol, a polyol such as glycerol and propylene glycol, or a mixture thereof can be used as a suitable carrier. As a carrier, isotonic solutions such as Ringer's solution, phosphate buffered saline (PBS) containing triethanolamine, sterile water for injection and 5% dextrose, and the like can preferably be used.

The preparation of a pharmaceutical composition is known in the art, and in particular Remington's Pharmaceutical Sciences (19th ed., 1995) and the like may be mentioned. This document is considered part of this specification.

The preferred daily dosage of the pharmaceutical composition of the present invention is in the range of 0.01 µg/kg to 10 g/kg, and preferably 0.01 mg/kg to 1 g/kg, depending on the condition of the patient, body weight, sex, age, disease severity, or route of administration. The introduction can be carried out once or several times a day. Such a dosage should in no way be interpreted as limiting the scope of the present invention.

The subject to whom the composition of the present invention can be administered (may be administered) is a mammal and a human, with a human being particularly preferred. The anti-allergy composition of the present invention may further comprise, in addition to the active ingredient, any compound or natural extract that has already been proven safe and known to have anti-allergic activity, to increase and enhance the anti-allergic activity.

In another aspect of the present invention, a food composition for alleviating or alleviating an allergy symptom is provided, comprising a polypeptide dimer as an active ingredient.

Within the scope of the present invention, the polypeptide dimer can be linked to an appropriate delivery structure for efficient delivery to the intestine. The food composition of the present invention can be made in any form and can be obtained, for example, in the form of beverages such as tea, juice, carbonated drinks and ionic drinks, processed dairy products such as milk and yogurt, dietary functional food preparations such as as tablets, capsules, pills, granules, liquids, powders, flakes, pastes, syrups, gels, jellies and bars, etc. In addition, the food composition of the present invention may be classified in any category of products according to the official or functional classification, provided that the food composition satisfies the requirements of regulatory authorities at the time of production and distribution. For example, the food composition may be a dietary functional food under the Functional Dietary Products Act, or may relate to confectionery, legume based products, teas, beverages, specialty foods, and the like. according to each type of food in the Food Sanitation Act Code of Foods (standards and specifications for food writings registered with the Food and Drug Administration). With respect to other food additives that may be contained in the food composition of the present invention, the Food Code or Supplementary Food Code under the Food Sanitation Act may be mentioned.

In another aspect of the present invention, a method for producing a polypeptide dimer is provided, comprising the step of culturing a host cell into which a polynucleotide encoding a monomer and a sialic acid transferase gene are introduced; and the step of isolating the polypeptide dimer.

Within the scope of the present invention, a polynucleotide encoding a monomer can be introduced into a host cell while loaded onto an expression vector. In addition, the sialic acid transferase gene can be introduced into the host cell while loaded on a vector.

First, the step of introducing into the host cell a vector into which a polynucleotide encoding a monomer is inserted and a vector into which a sialic acid transferase gene is inserted is carried out. Within the scope of this invention, the sialic acid transferase may be a 2,3-sialic acid transferase or a 2,6-sialic acid transferase.

Next, carry out the stage of cultivation of the transformed cells.

Finally, the step of isolating the polypeptide dimer is carried out. Within the framework of the present application, the polypeptide dimer can be purified from the culture medium or cell extract. For example, after obtaining the supernatant of the culture medium in which the polypeptide dimer has been secreted, the supernatant can be concentrated using a commercially available protein concentration filter, such as an Amicon or Millipore Pellicon ultrafiltration element. The concentrate can then be purified by methods known in the art. For example, purification can be carried out using a matrix associated with protein A.

In another aspect of the present invention, there is provided a polypeptide dimer produced as described above for producing a dimer.

Within the scope of the present invention, the polypeptide dimer has a high sialic acid content and thus has a very high acidity relative to the theoretical pI value.

In another aspect of the present invention, there is provided a pharmaceutical composition for the treatment or prevention of an allergic disease, comprising, as an active ingredient, a polypeptide dimer obtained by the method for producing a dimer as described above.

In another aspect of the present invention, there is provided a food composition for alleviating or alleviating an allergy symptom, comprising, as an active ingredient, a polypeptide dimer obtained by the dimer production method described above.

In a further aspect of the present invention, a method for treating or preventing an allergic disease is provided, comprising the step of administering to an individual a polypeptide dimer that contains two monomers, each containing an extracellular domain (FcεRIa-ECD) of an IgE Fc receptor alpha subunit.

The subject may be a mammal, preferably a human. Within the scope of the present invention, administration can be carried out orally or parenterally. Within the scope of the present invention, parenteral administration can be carried out by methods such as subcutaneous administration, intravenous administration, mucosal administration, and muscular administration.

Hereinafter, the present invention is described in more detail using the following examples. However, the following examples are only intended to illustrate the present invention and the scope of the present invention is not limited to them.

Example 1 Preparation of a Polypeptide Containing FcεRIα-ECD and a Modified Fc Region

The C-terminal modified extracellular domain (FcεRIα-ECD) polypeptide of the Fc receptor alpha subunit of IgE was prepared by the method described in IgE US Pat. No. 7,867,491.

First, to express a protein (FcεRIαECD-Fc1), a protein (FcεRIαECD-Fc2) and a protein (FcεR1αECD-Fc3) in which the extracellular domain of the FcεRI α-chain having the amino acid sequence of SEQ ID NO: 1 and the Fc-modified immunoglobulin SEQ ID NO: 2 linked through the hinge region of SEQ ID NO: 19, the hinge region of SEQ ID NO: 3 and the hinge region of SEQ ID NO: 4, respectively, the cassettes obtained by linking the gene encoding each protein were cloned into pAD15 vectors (Genexin, Inc.), with a construct expressing the FcεRIαECD-Fc protein. Each of the expression vectors was then transduced into CHO DG44 cells (from Dr. Chasin, Columbia University, USA).

In this case, when transduced into a cell line, an expression vector obtained by cloning the α-2,6-sialic acid transferase gene into the pCI Hygro vector (Invitrogen) was simultaneously transduced to obtain separately cell lines that are capable of expressing the FcεRIαECD-Fc2ST and FcεRIα proteins. ECD-Fc3ST to which sialic acid has been added.

As a primary screening procedure, HT selection was performed using 5-hydroxytryptamine (HT) free medium with 10% dFBS (Gibco, USA, 30067-334), MEMα medium (Gibco, 12561, USA, catalog number 12561-049 ) and HT+ media (Gibco, USA, 11067-030). Amplification was then performed with methotrexate (MTX) using HT-selected clones to increase productivity using the "dihydrofolate reductase (DHFR)-" system.

After completion of the MTX amplification, subculture was performed approximately 1-5 times to stabilize the cells in order to evaluate productivity. After that, the productivity was evaluated in units for cells amplified with MTX. The results are presented in table 1 below.

[Table 1]

Version Wednesday MTX concentration Productivity Cultivation for 3 days Fed Culture (mg/mL) mcg/ml µg/10 ⁶ cells FcεRIαECD-Fc2 Ex-cell DHFR 500 nM 37.23 20.9 225 FcεRIαECD-Fc2+a2,6-ST 100 nM 45.4 25.1 338.2 FcεRIαECD-Fc3 2 µM 27.0 16.9 180.4 FcεRIαECD-Fc3+a2,6-ST 1 µM 17.5 10.2 101.7

As shown in Table 1, the FcεRIαECD-Fc3 cell line showed a productivity of 16.9 μg/10 ⁶ cells after amplification with methotrexate at a concentration of 2 μM. On the other hand, the cell line FcεRIαECD-Fc3 (FcεRIαECD-Fc3ST) co-transduced with 2,6-sialic acid transferase showed a productivity of 17.5 μg/10 ⁶ cells after amplification with methotrexate at a concentration of 1 μM. In addition, the FcεRIαECD-Fc2 cell line showed a productivity of 20.9 μg/10 ⁶ cells under amplification conditions with methotrexate at a concentration of 0.5 μM. In addition, the cell line FcεRIαECD-Fc2 (FcεRIαECD-Fc2ST) co-transduced with 2,6-sialic acid transferase showed a productivity of 25.1 µg/10 ⁶ cells after amplification with methotrexate at a concentration of 0.1 µM. Therefore, it was identified that the FcεRIαECD-Fc2 cell line co-transfected with 2,6-sialic acid transferase, which was selected under amplification conditions with methotrexate at a concentration of 0.1 μM, showed the best productivity.

Example 2 Purification of the FcεRIα ECD Fusion Protein and Determination of Its Purity

Among the cell lines selected according to Example 1 above, i) FcεRIαECD-Fc3, ii) FcεRIαECD-Fc3ST and iii) FcεRIαECD-Fc2ST were cultured at a 60 ml scale by batch culture method. The resulting cultures were purified using a protein A affinity column, and then the purified proteins were subjected to SDS-PAGE and size exclusion HPLC (SE-HPLC) to determine the purity of the proteins.

As shown in Figure 1, all relevant proteins purified by SE-HPLC were identified to be 93% pure or higher. In addition, as a result of SDS-PAGE analysis, it was identified that proteins of approximately 150 kDa and approximately 75 kDa were detected, respectively, in non-reducing and reducing conditions (figure 1, lanes 1-6). Based on this, it was found that associated with Fc FcεRIαECD forms a dimer. In addition, no impurities such as the truncated form were observed in the SDS-PAGE results. In particular, even after the thaw/freeze process (FIG. 1, lanes 7-8), all proteins were identified to be 93% pure or higher and free of impurities. From this, the production of truncated protein forms was found to be reduced relative to FcεRIαECD-Fc1, which used a wild-type IgD hinge region.

In this case, IEF gel was performed under the following test conditions to identify the degree of sialic acid content in proteins after the introduction of sialic acid transferase. From this, it was identified that the content of acidic proteins increased due to the increase in the content of sialic acids.

[Table 2]

Test conditions Gel Gel IEF pH3-10 1.0 mm sample buffer IEF sample buffer (2X) Download conditions 100V 1h, 200V 1h, 500V 2h

To determine the reproducibility of the purification yield, the FcεRIαECD-Fc2ST cell line was batch cultured in a 1 L 250 ml scale flask and purified using a protein A affinity column. The culture supernatant and purified product were then separated on a 4%-15% TGX ^™ gel ( Bio-Rad Laboratories, Inc.) for 30 minutes under Tris-Glycine SDS (TGS) and 200 V buffer conditions and then subjected to SDS PAGE analysis. As a result, it was identified that not only very high purity proteins (98% or higher) were purified even by only the first purification step, but also proteins were expressed with very high purity even in the culture supernatant. This indicates that the process development steps can be simplified when developing the FcεRIαECD-Fc protein, which is expressed in the cell line in question, as a medical product, and as a result, it is likely that the cost of developing a medical product will be significantly reduced.

Example 3 Determination of IgE FcεRIα ECD Fusion Protein Binding Ability

IgE binding capacity was compared for four proteins: i) FcεRIαECD-Fc2, ii) FcεRIαECD-Fc2ST, iii) FcεRIαECD-Fc3 and iv) FcεRIαECD-Fc3ST, which were purified by the method of Example 2 above, and a commercially available anti-IgE antibody omalizumab (trade name: Xolair). In particular, the ability to bind to IgE was determined by applying IgE to the channel of the Protein GLC sensor chip (Bio-Rad Laboratories, Inc., cat. no. 176-5011) and allowing omalizumab or each FceR1αECD-Fc protein at various concentrations to flow at a rate of 30 µl in a minute.

The experiments were carried out by identifying the zero background using 25 mm NaOH as a regenerating buffer, and then repeating the above steps. Thereafter, the binding curve was identified using a protein binding analyzer (ProteOn XPR36, Bio-Rad Laboratories, Inc., USA). The results are presented in table 3, and in figures 3 and 4.

[Table 3]

Samples
Regulations FcεRIa ECD-Fc Omalizumab Notes Type of drug Fc fusion protein Ab vs. IgE Binding affinity ka (association rate) Fc2 2.14×10 ⁵ 4.05×10 ⁵ 1.9 times weaker than omalizumab Fc2ST 2.64×10 ⁵ 1.5 times weaker than omalizumab Fc3 1.98×10 ⁵ 2.0 times weaker than omalizumab Fc3ST 2.40×10 ⁵ 1.7 times weaker than omalizumab kd (rate of dissociation) Fc2 8.2×10 ^-5 6.02×10 ^-3 73 times better than omalizumab Fc2ST 5.69×10 ^-5 106 times better than omalizumab Fc3 1.33×10 ^-4 45 times better than omalizumab Fc3ST 1.49×10 ^-4 40 times better than omalizumab KD (kd/ka) Fc2 3.88×10 ^-10 1.49×10 ^-8 38 times better than omalizumab Fc2ST 2.16×10 ^-10 69 times better than omalizumab Fc3 6.72×10 ^-10 22 times better than omalizumab Fc3ST 6.21×10 ^-10 24 times better than omalizumab

As shown in Table 3, the association rate (ka) value of the polypeptide dimer according to one embodiment of the present invention was determined to be 1.5-2.0 times lower than that of omalizumab. Thus, its binding affinity for substances other than IgE was found to be 1.5-2.0 lower than that of omalizumab. In addition, the dissociation rate (kd) value of the polypeptide dimer according to one embodiment of the present invention was determined to be 40-106 times higher than that of omalizumab. In addition, as shown in Figures 3 and 4, it could be identified that omalizumab lost its binding to IgE when a certain period of time elapsed after binding, while after binding of the polypeptide dimer of the FcεRIαECD fusion protein of the present invention to IgE the polypeptide dimer was not separated from IgE.

Thus, it can be seen that the polypeptide dimer of the present invention is not readily separated from IgE and has a significantly better ability to retain its bound state than omalizumab. As a result, the polypeptide dimer according to one embodiment of the present invention was found to have an equilibrium dissociation constant value (KD<kd/ka>) that is 22-69 times higher than that of omalizumab. From this, it can be seen that the FcεRIαECD fusion protein of the present invention has a significantly increased ability to bind to IgE compared to omalizumab. In particular, it was identified that FcεRIαECD-Fc2 (FcεRIαECD-Fc2ST) to which sialic acid was added exhibited the highest IgE binding capacity, which was 69 times higher than that of omalizumab.

Example 4 Identification of the Ability of the FcεRIα ECD Fusion Protein to Bind to the IgG Receptor

The degree of binding of IgE _TRAP and omalizumab to IgG receptors was determined using the Octet RED384 system (ForteBio, CA, USA). Recombinant proteins FcγRI, FcγRIIA, FcγRIIB, FcγRIIIA, and FcγRIIIB (R&D Systems Inc., 5 μg/mL) were immobilized in 300 mM acetate buffer (pH 5) on an activated AR2G biosensor. PBS containing 0.1% Tween-20 and 1% bovine serum was used as running buffer. All experiments were performed at 30° C. using a plate shaker at 1000 rpm. The results are shown in FIGS. 5A-5E, and the ability of omalizumab and IgE _TRAP to bind to IgG receptors was quantified and shown in FIG. 6.

Example 5 Determination of FcεRIα ECD fusion protein activity using beta-hexosaminidase assay in mouse bone marrow-derived mast cells

A beta-hexosaminidase assay was performed to investigate the in vitro activity of the FcεRIαECD fusion bean of the present invention. In particular, the FcεRIαECD-Fc2 protein according to one embodiment of the present invention was mixed at each concentration with mouse IgE (1 μg/ml) and incubated at room temperature (20°C) for 30 minutes to obtain samples. The bone marrow-derived mast cells in the mast cell activation culture were washed with Hank's balanced salt solution (HBSS) buffer to remove the medium, and the cell number was determined. Then the adjustment was carried out so that 5×10 ⁵ cells were injected into 40 μl of HBSS buffer.

Then, 50 μl of the sample solution obtained by pre-incubation was added to the activated mast cells. The resulting material was then incubated in a 5% CO ₂ incubator at 37°C for 30 minutes. Then, after adding in each case 10 μl of DNP (2,4-dinitrophenol, 100 ng/ml), which is a foreign antigen, incubation was again carried out at 37°C for 30 minutes in 5% CO ₂ , and then 30 μl was separated supernatant. 30 μl of the separated supernatant and 30 μl of substrate (4-nitrophenyl N-acetyl-β-D-glucosaminide, 5.84 mm) were mixed well and then incubated at 37° C. for 20 minutes in 5% CO ₂ . Then, 140 μl of 0.1 M sodium carbonate buffer (pH 10) was added as a stop solution to complete the reaction. Thereafter, absorbance at 405 nm was determined to identify the secreted amount of β-hexosaminidase secreted by the foreign antigen in the activated mast cells. The results are shown in Fig.7.

As shown in FIG. 7, the polypeptide dimer according to one embodiment of the present invention exhibited a mast cell inhibition fraction of approximately 49.4% at half (0.5 μg/mL) mouse IgE concentration, and exhibited a mast cell inhibition fraction of approximately 99.4 % in the case of the same concentration (1 µg/ml) of mouse IgE. Thus, it can be seen that the IgE-induced activity of bone marrow-derived mast cells is significantly suppressed by the FceRIa-ECD polypeptide dimer of the present invention.

Example 6 Comparison of activity of FcεRIα ECD fusion protein and anti-human IgE antibody using β-hexosaminidase assay in human FcεRI-expressing bone marrow-derived mast cells

A β-hexosaminidase assay was performed to identify superiority of the FcεRIα ECD fusion protein over Xolair by in vitro activity assay. The respective drugs, FcεRIαECD-Fc2ST (IgE _TRAP ) and Xolair, were prepared at each concentration and then mixed with human IgE (1 μg/ml). Then, incubation was carried out at room temperature for 30 minutes. During pre-incubation with the drug, the human FcεRI gene was introduced and mast cells derived and differentiated from mouse bone marrow in which the mouse FcεRI gene was deleted were obtained. The obtained mast cells were washed with HBSS buffer, and then 5×10 ⁵ cells were injected into 60 μl of HBSS buffer. 20 µl of the pre-incubated sample was added to the obtained mast cells, and then incubated in a 5% CO ₂ incubator at 37° C. for 30 minutes.

Then 20 μl of anti-human IgE antibody (BioLegend, catalog number 325502, 0.5 μg/ml) was added, and then the resulting material was again incubated in a 5% CO ₂ incubator at 37°C for 30 minutes. Then, after centrifugation at 1500 rpm at 4°C, 30 μl of the supernatant was separated. 30 μl of the separated supernatant and 30 μl of substrate (4-nitrophenyl N-acetyl-β-glucosaminide, 5.84 mm) were mixed well and then incubated in a 5% CO ₂ incubator at 37° C. for 25 minutes. Then 140 μl of 0.1 M sodium carbonate buffer (pH 10) was added to complete the reaction.

Then, the absorbance at 405 nm was determined to compare the relative amounts of β-hexosaminidase secreted, and the effect of mast cell inhibition was identified depending on each drug concentration. The results are presented in Fig.8. As shown in FIG. 8, the IC ₅₀ of the FcεRIα ECD fusion protein was determined to be approximately 11.16 ng/mL and the IC ₅₀ of the Xolair protein was determined to be approximately 649.8 ng/mL. Thus, the FcεRIα ECD fusion protein was identified to have a 58-fold higher inhibitory activity on mast cell activity than Xolair.

Example 7 Analysis in vivo fusion protein FcεRIα ECD (food allergy model)

50 μg of ovalbumin (OVA) and 1 mg of alum were intraperitoneally administered to Balb/c mice (Orientbio Inc.) twice with an interval of 14 days to induce sensitization. Thereafter, 50 mg OVA was orally administered five times in total on days 28, 30, 32, 34 and 36 to induce food allergy in the intestine.

After oral administration of OVA twice, ie. on day 31, the mice were divided into three groups, each containing 7 mice. The three divided groups were as follows: the first group received high concentration FcεRIαECD-Fc2ST fusion protein (200 µg), the second group received low concentration FcεRIαECD-Fc2ST fusion protein (20 µg), and the third group received nothing. When OVA was administered orally, it was determined whether diarrhea was due to food allergy induction. The mice were sacrificed on day 37, and the number of mast cells in the small intestine, the concentration of IgE in the blood, and the concentration of the enzyme (mast cell protease-1 (MCPT-1)) with degranulation of mast cells in the blood of mice belonging to each group were analyzed.

As shown in Fig. 9, it was identified that mice belonging to the group administered with FcεRIαECD-Fc2ST, which is a polypeptide dimer, at a high concentration showed the effect of alleviating food allergy in a concentration-dependent manner, compared with mice belonging to the group in which nothing was entered.

[Sequence List]

SEQ ID NO: 1

VPQKPKVSLN PPWNRIFKGE NVTLTCNGNN FFEVSSTKWF HNGSLSEETN

SSLNIVNAKF EDSGEYKCQH QQVNESEPVY LEVFSDWLLL QASAEVVMEG

QPLFLRCHGW RNWDVYKVIY YKDGEALKYW YENHNISITN ATVEDSGTYY

CTGKVWQLDY ESEPLNITVI KAPREKYWLQ

SEQ ID NO: 2

SHTQPLGVFL FPPKPKDTLM ISRTPEVTCV VVDVSQEDPE VQFNWYVDGV

EVHNAKTKPR EEQFNSTYRV VSVLTVLHQD WLNGKEYKCK VSNKGLPSSI

EKTISKAKGQ PREPQVYTLP PSQEEMTKNQ VSLTCLVKGF YPSDIAVEWE

SNGQPENNYK TTPPVLDSDG SFFLYSRLTV DKSRWQEGNV FSCSVMHEAL

HNHYTQKSLS LSLGK

SEQ ID NO: 3

RNTGRGGEEK KGSKEKEEEQE ERETKTPECP

SEQ ID NO: 4

AQPQAEGSLA KATTAPATTR NTGRGGEEKK GSKEKEEQEE RETKTPECP

SEQ ID NO: 5

gtgccccaga agcccaaggt gagcctgaac cctccctgga acagaatctt

caagggcgag aacgtgaccc tgacctgcaa cggcaacaac ttcttcgagg

tgagcagcac caagtggttc cacaatggca gcctgagcga ggagaccaac

agctccctga acatcgtgaa cgccaagttc gaggacagcg gcgagtacaa

gtgccagcac cagcaggtga acgagagcga gcccgtgtac ctggaggtgt

tcagcgactg gctgctgctg caggccagcg ccgaggtggt gatggagggc

cagcccctgt tcctgagatg ccacggctgg agaaactggg acgtgtacaa

ggtgatctac tacaaggatg gcgaggccct gaagtactgg tacgagaacc

acaacatctc catcaccaac gccaccgtgg aggacagcgg cacctactac

tgcacaggca aggtgtggca gctggactac gagagcgagc ccctgaacat

caccgtgatc aaggctccca gagagaagta ctggctgcag

SEQ ID NO: 6

tgcgtggtcg tggatgtgag cggaagat cccgaagtgc agttcaactg

gtacgtggat ggcgtggaag tgcacaacgc caagaccaag cccagagaag

agcagttcaa ctccacctac agagtggtga gcgtgctgac cgtgctgcac

caggactggc tgaacggcaa ggagtacaag tgcaaggtgt ccaacaaagg

cctgcccagc tccatcgaga agaccatcag caaagccaaa ggccagccca

gagaacccca ggtgtacacc ctgcctccca gccaggaaga gatgaccaag

aaccaggtgt ccctgacctg cctggtgaaa ggcttctacc ccagcgacat

cgccgtggag tgggaaagca acggccagcc cgagaacaat tacaagacaa

cccctcccgt gctggatagc gatggcagct tctttctgta cagcagactg

accgtggaca agagcagatg gcaggaaggc aacgtgttca gctgcagcgt

gatgcacgaa gccctgcaca accactacac ccagaagagc ctgtccctga

gcctgggcaa g

SEQ ID NO: 7

aggaacaccg gcagaggagg cgaggaaaag aaaggaagca aggagaagga

ggagcaggag gaaagagaaa ccaagacccc cgagtgcccc agccacaccc

agcccctggg cgtgttcctg ttccccccca agcccaagga caccctgatg

atcagcagaa cccccgaggt gacc

SEQ ID NO: 8

gcccagcccc aggccgaggg cagcctggct aaggccacca cagctcccgc

caccaccagg aacaccggca gaggaggcga ggaaaagaaa ggaagcaagg

agaaggagga gcaggaggaa agagaaacca agacccccga gtgccccagc

cacacccagc ccctgggcgt gttcctgttc ccccccaagc ccaaggacac

cctgatgatc agcagaaccc ccgaggtgac c

SEQ ID NO: 9

MDAMLRGLCC VLLLCGAVFV SPSHA

SEQ ID NO: 10

atggacgcca tgctgagagg cctgtgctgt gtgctgctgc tgtgcggcgc

cgtgttcgtg tcccctagcc acgcc

SEQ ID NO: 11

MDAMLRGLCC VLLLCGAVFV SPSHAVPQKP KVSLNPPWNR IFKGENVTLT

CNGNNFFEVS STKWFHNGSL SEETNSSLNI VNAKFEDSGE YKCQHQQVNE

SEPVYLEVFS DWLLLQASAE VVMEGQPLFL RCHGWRNWDV YKVIYYKDGE

ALKYWYENHN ISITNATVED SGTYYCTGKV WQLDYESEPL NITVIKAPRE

KYWLQRNTGR GGEEKKGSKE KEEQEERETK TPECPSHTQP LGVFLFPPKP

KDTLMISRTP EVTCVVVDVS QEDPEVQFNW YVDGVEVHNA KTKPREEQFN

STYRVVSVLT VLHQDWLNGK EYKCKVSNKG LPSSIEKTIS KAKGQPREPQ

VYTLPPSQEE MTKNQVSLTC LVKGFYPSDI AVEWESNGQP ENNYKTTPPV

LDSDGSFFLY SRLTVDKSRW QEGNVFSCSV MHEALHNHYT QKSLSLSLGK

SEQ ID NO: 12

atggacgcca tgctgagagg cctgtgctgt gtgctgctgc tgtgcggcgc

cgtgttcgtg tcccctagcc acgccgtgcc ccagaagccc aaggtgagcc

tgaaccctcc ctggaacaga atcttcaagg gcgagaacgt gaccctgacc

tgcaacggca acaacttctt cgaggtgagc agcaccaagt ggttccacaa

tggcagcctg agcgaggaga ccaacagctc cctgaacatc gtgaacgcca

agttcgagga cagcggcgag tacaagtgcc agcaccagca ggtgaacgag

agcgagcccg tgtacctgga ggtgttcagc gactggctgc tgctgcaggc

cagcgccgag gtggtgatgg agggccagcc cctgttcctg agatgccacg

gctggagaaa ctgggacgtg tacaaggtga tctactacaa ggatggcgag

gccctgaagt actggtacga gaaccacaac atctccatca ccaacgccac

cgtggaggac agcggcacct actactgcac aggcaaggtg tggcagctgg

actacgagag cgagcccctg aacatcaccg tgatcaaggc tcccagagag

aagtactggc tgcagaggaa caccggcaga ggaggcgagg aaaagaaagg

aagcaaggag aaggaggagc aggaggaaag agaaaccaag acccccgagt

gccccagcca cacccagccc ctgggcgtgt tcctgttccc ccccaagccc

aaggacaccc tgatgatcag cagaaccccc gaggtgacct gcgtggtcgt

ggatgtgagc caggaagatc ccgaagtgca gttcaactgg tacgtggatg

gcgtggaagt gcacaacgcc aagaccaagc ccagagaaga gcagttcaac

tccacctaca gagtggtgag cgtgctgacc gtgctgcacc aggactggct

gaacggcaag gagtacaagt gcaaggtgtc caacaaaggc ctgcccagct

ccatcgagaa gaccatcagc aaagccaaag gccagcccag agaaccccag

gtgtacaccc tgcctcccag cggaagag atgaccaaga accaggtgtc

cctgacctgc ctggtgaaag gcttctaccc cagcgacatc gccgtggagt

gggaaagcaa cggccagccc gagaacaatt acaagacaac ccctcccgtg

ctggatagcg atggcagctt ctttctgtac agcagactga ccgtggacaa

gagcagatgg caggaaggca acgtgttcag ctgcagcgtg atgcacgaag

ccctgcacaa ccactacacc cagaagagcc tgtccctgag cctgggcaag

SEQ ID NO: 13

MDAMLRGLCC VLLLCGAVFV SPSHAVPQKP KVSLNPPWNR IFKGENVTLT

CNGNNFFEVS STKWFHNGSL SEETNSSLNI VNAKFEDSGE YKCQHQQVNE

SEPVYLEVFS DWLLLQASAE VVMEGQPLFL RCHGWRNWDV YKVIYYKDGE

ALKYWYENHN ISITNATVED SGTYYCTGKV WQLDYESEPL NITVIKAPRE

KYWLQAQPQA EGSLAKATTA PATTRNTGRG GEEKKGSKEK EEQEERETKT

PECPSHTQPL GVFLFPPKPK DTLMISRTPE VTCVVVDVSQ EDPEVQFNWY

VDGVEVHNAK TKPREEQFNS TYRVVSVLTV LHQDWLNGKE YKCKVSNKGL

PSSIEKTISK AKGQPREPQV YTLPPSQEEM TKNQVSLTCL VKGFYPSDIA

VEWESNGQPE NNYKTTPPVL DSDGSFFLYS RLTVDKSRWQ EGNVFSCSVM

HEALHNHYTQ KSLSLSLGK

SEQ ID NO: 14

atggacgcca tgctgagagg cctgtgctgt gtgctgctgc tgtgcggcgc

cgtgttcgtg tcccctagcc acgccgtgcc ccagaagccc aaggtgagcc

tgaaccctcc ctggaacaga atcttcaagg gcgagaacgt gaccctgacc

tgcaacggca acaacttctt cgaggtgagc agcaccaagt ggttccacaa

tggcagcctg agcgaggaga ccaacagctc cctgaacatc gtgaacgcca

agttcgagga cagcggcgag tacaagtgcc agcaccagca ggtgaacgag

agcgagcccg tgtacctgga ggtgttcagc gactggctgc tgctgcaggc

cagcgccgag gtggtgatgg agggccagcc cctgttcctg agatgccacg

gctggagaaa ctgggacgtg tacaaggtga tctactacaa ggatggcgag

gccctgaagt actggtacga gaaccacaac atctccatca ccaacgccac

cgtggaggac agcggcacct actactgcac aggcaaggtg tggcagctgg

actacgagag cgagcccctg aacatcaccg tgatcaaggc tcccagagag

aagtactggc tgcaggccca gccccaggcc gagggcagcc tggctaaggc

caccacagct cccgccacca cggaacac cggcagagga ggcgaggaaa

agaaaggaag caaggagaag gaggagcagg aggaaagaga aaccaagacc

ccggagtgcc ccagccacac ccagcccctg ggcgtgttcc tgttcccccc

caagcccaag gacaccctga tgatcagcag aacccccgag gtgacctgcg

tggtcgtgga tgtgagccag gaagatcccg aagtgcagtt caactggtac

gtggatggcg tggaagtgca caacgccaag accaagccca gagaagagca

gttcaactcc acctacagag tggtgagcgt gctgaccgtg ctgcaccagg

actggctgaa cggcaaggag tacaagtgca aggtgtccaa caaaggcctg

cccagctcca tcgagaagac catcagcaaa gccaaaggcc agcccagaga

accccaggtg tacaccctgc ctcccagcca ggaagagatg accaagaacc

aggtgtccct gacctgcctg gtgaaaggct tctaccccag cgacatcgcc

gtggagtggg aaagcaacgg ccagcccgag aacaattaca agacaacccc

tcccgtgctg gatagcgatg gcagcttctt tctgtacagc agactgaccg

tggacaagag cagatggcag gaaggcaacg tgttcagctg cagcgtgatg

cacgaagccc tgcacaacca ctacacccag aagagcctgt ccctgagcct

gggcaag

SEQ ID NO: 15

MIHTNLKKKF SCCVLVFLLF AVICVWKEKK KGSYYDSFKL QTKEFQVLKS

LGKLAMGSDS QSVSSSSTQD PHRGRQTLGS LRGLAKAKPE ASFQVWNKDS

SSKNLIPRLQ KIWKNYLSMN KYKVSYKGPG PGIKFSAEAL RCHLRDHVNV

SMVEVTDFPF NTSEWEGYLP KESIRTKAGP WGRCAVVSSA GSLKSSQLGR

EIDDHDAVLR FNGAPTANFQ QDVGTKTTIR LMNSQLVTTE KRFLKDSLYN

EGILIVWDPS VYHSDIPKWY QNPDYNFFNN YKTYRKLHPN QPFYILKPQM

PWELWDILQE ISPEEIQPNP PSSGMLGIII MMTLCDQVDI YEFLPSKRKT

DVCYYYQKFF DSACTMGAYH PLLYEKNLVK HLNQGTDEDI YLLGKATLPG

FRTIHC

SEQ ID NO: 16

atgatccaca ccaacctgaa gaagaagttc agctgctgcg tgctggtgtt

cctgctgttc gccgtgatct gcgtgtggaa ggagaagaag aaaggcagct

actacgacag cttcaagctg cagaccaagg agttccaggt gctgaagagc

ctgggcaagc tggccatggg cagcgacagc cagagcgtgt ccagctcctc

cacccaggat ccccacagag gcagacagac cctgggcagc ctgagaggcc

tggccaaggc caagcccgag gccagcttcc aggtgtggaa caaggacagc

agcagcaaga acctgatccc cagactgcag aagatctgga agaactacct

gagcatgaac aagtacaagg tgagctacaa aggacccggga cccggcatca

agttcagcgc cgaggccctg aggtgccacc tgagagacca cgtgaacgtg

agcatggtgg aagtgaccga cttccccttc aacaccagcg agtgggaagg

ctacctgcc aaggagagca tcaggaccaa ggctggcccc tggggcagat

gcgccgtggt gagcagcgct ggcagcctga agagctccca gctgggcaga

gagatcgacg accacgatgc cgtgctgagg ttcaatggcg ctcccaccgc

caacttccag caggacgtgg gcaccaagac cacaatccgg ctgatgaaca

gccagctggt gacaaccgag aagcggttcc tgaaggacag cctgtacaac

gagggcatcc tgatcgtgtg ggatcccagc gtgtaccaca gcgacatccc

caagtggtac cagaatcccg actacaactt cttcaacaac tacaagacct

atagaaagct gcaccccaac cagcccttct acatcctgaa gccccagatg

ccctgggagc tgtgggacat cctgcaggag atcagccctg aagagatcca

gcccaaccct ccctccagcg gcatgctggg cattatcatc atgatgaccc

tgtgcgacca ggtggacatc tacgagttcc tgcccagcaa gagaaagacc

gacgtgtgct actactatca gaagttcttc gacagcgcct gcaccatggg

cgcctaccac cccctgctgt acgagaagaa cctggtgaag cacctgaacc

agggcaccga cgaggacatc tacctgctgg gcaaagccac cctgcccggc

ttcagaacca tccactgc

SEQ ID NO: 17

RNTGRGGEEK KXXKEKEEQE ERETKTPECP

SEQ ID NO: 18

AQPQAEGSLA KATTAPATTR NTGRGGEEKK XXKEKEEQEE RETKTPECP

SEQ ID NO: 19

RNTGRGGEEK KKEKEKEEQE ERETKTPECP

SEQ ID NO: 20

VPQKPKVSLN PPWNRIFKGE NVTLTCNGNN FFEVSSTKWF HNGSLSEETN

SSLNIVNAKF EDSGEYKCQH QQVNESEPVY LEVFSDWLLL QASAEVVMEG

QPLFLRCHGW RNWDVYKVIY YKDGEALKYW YENHNISITN ATVEDSGTYY

CTGKVWQLDY ESEPLNITVI KAPREKYWLQ RNTGRGGEEK KKEKEKEEQE

ERETKTPECP SHTQPLGVFL FPPKPKDTLM ISRTPEVTCV VVDVSQEDPE

VQFNWYVDGV EVHNAKTKPR EEQFNSTYRV VSVLTVLHQD WLNGKEYKCK

VSNKGLPSSI EKTISKAKGQ PREPQVYTLP PSQEEMTKNQ VSLTCLVKGF

YPSDIAVEWE SNGQPENNYK TTPPVLDSDG SFFLYSRLTV DKSRWQEGNV

FSCSVMHEAL HNHYTQKSLS LSLGK

SEQ ID NO: 21

VPQKPKVSLN PPWNRIFKGE NVTLTCNGNN FFEVSSTKWF HNGSLSEETN

SSLNIVNAKF EDSGEYKCQH QQVNESEPVY LEVFSDWLLL QASAEVVMEG

QPLFLRCHGW RNWDVYKVIY YKDGEALKYW YENHNISITN ATVEDSGTYY

CTGKVWQLDY ESEPLNITVI KAPREKYWLQ RNTGRGGEEK KGSKEKEEEQE

ERETKTPECP SHTQPLGVFL FPPKPKDTLM ISRTPEVTCV VVDVSQEDPE

VQFNWYVDGV EVHNAKTKPR EEQFNSTYRV VSVLTVLHQD WLNGKEYKCK

VSNKGLPSSI EKTISKAKGQ PREPQVYTLP PSQEEMTKNQ VSLTCLVKGF

YPSDIAVEWE SNGQPENNYK TTPPVLDSDG SFFLYSRLTV DKSRWQEGNV

FSCSVMHEAL HNHYTQKSLS LSLGK

SEQ ID NO: 22

VPQKPKVSLN PPWNRIFKGE NVTLTCNGNN FFEVSSTKWF HNGSLSEETN

SSLNIVNAKF EDSGEYKCQH QQVNESEPVY LEVFSDWLLL QASAEVVMEG

QPLFLRCHGW RNWDVYKVIY YKDGEALKYW YENHNISITN ATVEDSGTYY

CTGKVWQLDY ESEPLNITVI KAPREKYWLQ AQPQAEGSLA KATTAPATTR

NTGRGGEEKK GSKEKEEQEE RETKTPECPS HTQPLGVFLF PPKPKDTLMI

SRTPEVTCVV VDVSQEDPEV QFNWYVDGVE VHNAKTKPRE EQFNSTYRVV

SVLTVLHQDW LNGKEYKCKV SNKGLPSSIE KTISKAKGQP REPQVYTLPP

SQEEMTKNQV SLTCLVKGFY PSDIAVEWES NGQPENNYKT TPPVLDSDGS

FFLYSRLTVD KSRWQEGNVF SCSVMHEALH NHYTQKSLSL SLGK

--->

SEQUENCE LIST

<110> PROGEN CO., LTD.

<120> EXTRACELLULAR DOMAIN OF IGE FC RECEPTOR ALPHA SUBUNIT,

PHARMACEUTICAL COMPOSITION CONTAINING IT AND METHOD FOR ITS PRODUCTION

<130> PCB812140PRG/PCT

<150> KR 10-2018-0002248

<151> 2018-01-08

<160> 22

<170> KopatentIn 2.0

<210> 1

<211> 180

<212> PROTEIN

<213> Artificial sequence

<220>

<223> FCeRI1 ECD

<400> 1

Val Pro Gln Lys Pro Lys Val Ser Leu Asn Pro Pro Trp Asn Arg Ile

1.5 10 15

Phe Lys Gly Glu Asn Val Thr Leu Thr Cys Asn Gly Asn Asn Phe Phe

20 25 30

Glu Val Ser Ser Thr Lys Trp Phe His Asn Gly Ser Leu Ser Glu Glu

35 40 45

Thr Asn Ser Ser Leu Asn Ile Val Asn Ala Lys Phe Glu Asp Ser Gly

50 55 60

Glu Tyr Lys Cys Gln His Gln Gln Val Asn Glu Ser Glu Pro Val Tyr

65 70 75 80

Leu Glu Val Phe Ser Asp Trp Leu Leu Leu Gln Ala Ser Ala Glu Val

85 90 95

Val Met Glu Gly Gln Pro Leu Phe Leu Arg Cys His Gly Trp Arg Asn

100 105 110

Trp Asp Val Tyr Lys Val Ile Tyr Tyr Lys Asp Gly Glu Ala Leu Lys

115 120 125

Tyr Trp Tyr Glu Asn His Asn Ile Ser Ile Thr Asn Ala Thr Val Glu

130 135 140

Asp Ser Gly Thr Tyr Tyr Cys Thr Gly Lys Val Trp Gln Leu Asp Tyr

145 150 155 160

Glu Ser Glu Pro Leu Asn Ile Thr Val Ile Lys Ala Pro Arg Glu Lys

165 170 175

Tyr Trp Leu Gln

180

<210> 2

<211> 215

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Modified Fc

<400> 2

Ser His Thr Gln Pro Leu Gly Val Phe Leu Phe Pro Pro Lys Pro Lys

1.5 10 15

Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val

20 25 30

Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp

35 40 45

Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe

50 55 60

Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp

65 70 75 80

Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu

85 90 95

Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg

100 105 110

Glu Pro Gln Val Tyr Thr Leu Pro Ser Gln Glu Glu Met Thr Lys

115 120 125

Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp

130 135 140

Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys

145 150 155 160

Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser

165 170 175

Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser

180 185 190

Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser

195 200 205

Leu Ser Leu Ser Leu Gly Lys

210 215

<210> 3

<211> 30

<212> PROTEIN

<213> Artificial sequence

<220>

<223> IgD hinge variant

<400> 3

Arg Asn Thr Gly Arg Gly Gly Glu Glu Lys Lys Gly Ser Lys Glu Lys

1.5 10 15

Glu Glu Gln Glu Glu Arg Glu Thr Lys Thr Pro Glu Cys Pro

20 25 30

<210> 4

<211> 49

<212> PROTEIN

<213> Artificial sequence

<220>

<223> IgD hinge variant

<400> 4

Ala Gln Pro Gln Ala Glu Gly Ser Leu Ala Lys Ala Thr Thr Ala Pro

1.5 10 15

Ala Thr Thr Arg Asn Thr Gly Arg Gly Gly Glu Glu Glu Lys Lys Gly Ser

20 25 30

Lys Glu Lys Glu Glu Gln Glu Glu Arg Glu Thr Lys Thr Pro Glu Cys

35 40 45

Pro

<210> 5

<211> 540

<212> DNA

<213> Artificial sequence

<220>

<223> nucleotide sequence of FCeRI1 ECD

<400> 5

gtgccccaga agcccaaggt gagcctgaac cctccctgga acagaatctt caagggcgag 60

aacgtgaccc tgacctgcaa cggcaacaac ttcttcgagg tgagcagcac caagtggttc 120

cacaatggca gcctgagcga ggagaccaac agctccctga acatcgtgaa cgccaagttc 180

gaggacagcg gcgagtacaa gtgccagcac cagcaggtga acgagagcga gcccgtgtac 240

ctggaggtgt tcagcgactg gctgctgctg caggccagcg ccgaggtggt gatggagggc 300

cagcccctgt tcctgagatg cccacggctgg agaaactggg acgtgtacaa ggtgatctac 360

tacaaggatg gcgaggccct gaagtactgg tacgagaacc acaacatctc catcaccaac 420

gccaccgtgg aggacagcgg cacctactac tgcacaggca aggtgtggca gctggactac 480

gagagcgagc ccctgaacat caccgtgatc aaggctccca gagagaagta ctggctgcag 540

540

<210> 6

<211> 561

<212> DNA

<213> Artificial sequence

<220>

<223> nucleotide sequence Modified Fc

<400> 6

tgcgtggtcg tggatgtgag cggaagat cccgaagtgc agttcaactg gtacgtggat 60

ggcgtggaag tgcacaacgc caagaccaag cccagagaag agcagttcaa ctccacctac 120

agagtggtga gcgtgctgac cgtgctgcac caggactggc tgaacggcaa ggagtacaag 180

tgcaaggtgt ccaacaaagg cctgcccagc tccatcgaga agaccatcag caaagccaaa 240

ggccagccca gagaacccca ggtgtacacc ctgcctccca gccaggaaga gatgaccaag 300

aaccaggtgt ccctgacctg cctggtgaaa ggcttctacc ccagcgacat cgccgtggag 360

tgggaaagca acggccagcc cgagaacaat tacaagacaa cccctcccgt gctggatagc 420

gatggcagct tctttctgta cagcagactg accgtggaca agagcagatg gcaggaaggc 480

aacgtgttca gctgcagcgt gatgcacgaa gccctgcaca accactacac ccagaagagc 540

ctgtccctga gcctgggcaa g 561

<210> 7

<211> 174

<212> DNA

<213> Artificial sequence

<220>

<223> nucleotide sequence of an IgD hinge variant

<400> 7

aggaacaccg gcagaggagg cgaggaaaag aaaggaagca aggagaagga ggagcaggag 60

gaaagagaaa ccaagacccc cgagtgcccc agccacaccc agcccctggg cgtgttcctg 120

ttccccccca agcccaagga caccctgatg atcagcagaa cccccgaggt gacc 170

<210> 8

<211> 231

<212> DNA

<213> Artificial sequence

<220>

<223> nucleotide sequence of an IgD hinge variant

<400> 8

gcccagcccc aggccgaggg cagcctggct aaggccacca cagctcccgc caccaccagg 60

aacaccggca gaggaggcga ggaaaagaaa ggaagcaagg agaaggagga gcaggaggaa 120

agagaaacca agacccccga gtgccccagc cacacccagc ccctgggcgt gttcctgttc 180

ccccccaagc ccaaggacac cctgatgatc agcagaaccc ccgaggtgac c 231

<210> 9

<211> 25

<212> PROTEIN

<213> Artificial sequence

<220>

<223> signal peptide

<400> 9

Met Asp Ala Met Leu Arg Gly Leu Cys Cys Val Leu Leu Leu Cys Gly

1.5 10 15

Ala Val Phe Val Ser Pro Ser His Ala

20 25

<210> 10

<211> 75

<212> DNA

<213> Artificial sequence

<220>

<223> signal peptide nucleotide sequence

<400> 10

atggacgcca tgctgagagg cctgtgctgt gtgctgctgc tgtgcggcgc cgtgttcgtg 60

tcccctagcc acgcc 75

<210> 11

<211> 450

<212> PROTEIN

<213> Artificial sequence

<220>

<223> FceRIa ECD-hinge region-Fc2

<400> 11

Met Asp Ala Met Leu Arg Gly Leu Cys Cys Val Leu Leu Leu Cys Gly

1.5 10 15

Ala Val Phe Val Ser Pro Ser His Ala Val Pro Gln Lys Pro Lys Val

20 25 30

Ser Leu Asn Pro Pro Trp Asn Arg Ile Phe Lys Gly Glu Asn Val Thr

35 40 45

Leu Thr Cys Asn Gly Asn Asn Phe Phe Glu Val Ser Ser Thr Lys Trp

50 55 60

Phe His Asn Gly Ser Leu Ser Glu Glu Thr Asn Ser Ser Leu Asn Ile

65 70 75 80

Val Asn Ala Lys Phe Glu Asp Ser Gly Glu Tyr Lys Cys Gln His Gln

85 90 95

Gln Val Asn Glu Ser Glu Pro Val Tyr Leu Glu Val Phe Ser Asp Trp

100 105 110

Leu Leu Leu Gln Ala Ser Ala Glu Val Val Met Glu Gly Gln Pro Leu

115 120 125

Phe Leu Arg Cys His Gly Trp Arg Asn Trp Asp Val Tyr Lys Val Ile

130 135 140

Tyr Tyr Lys Asp Gly Glu Ala Leu Lys Tyr Trp Tyr Glu Asn His Asn

145 150 155 160

Ile Ser Ile Thr Asn Ala Thr Val Glu Asp Ser Gly Thr Tyr Tyr Cys

165 170 175

Thr Gly Lys Val Trp Gln Leu Asp Tyr Glu Ser Glu Pro Leu Asn Ile

180 185 190

Thr Val Ile Lys Ala Pro Arg Glu Lys Tyr Trp Leu Gln Arg Asn Thr

195 200 205

Gly Arg Gly Gly Glu Glu Lys Lys Gly Ser Lys Glu Lys Glu Glu Gln

210 215 220

Glu Glu Arg Glu Thr Lys Thr Pro Glu Cys Pro Ser His Thr Gln Pro

225 230 235 240

Leu Gly Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

245 250 255

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu

260 265 270

Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

275 280 285

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg

290 295 300

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

305 310 315 320

Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu

325 330 335

Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr

340 345 350

Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu

355 360 365

Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

370 375 380

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

385 390 395 400

Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp

405 410 415

Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His

420 425 430

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu

435 440 445

Gly Lys

450

<210> 12

<211> 1350

<212> DNA

<213> Artificial sequence

<220>

<223> nucleotide sequence FceRIa ECD-hinge region-Fc2

<400> 12

atggacgcca tgctgagagg cctgtgctgt gtgctgctgc tgtgcggcgc cgtgttcgtg 60

tcccctagcc acgccgtgcc ccagaagccc aaggtgagcc tgaaccctcc ctggaacaga 120

atcttcaagg gcgagaacgt gaccctgacc tgcaacggca acaacttctt cgaggtgagc 180

agcaccaagt ggttccacaa tggcagcctg agcgaggaga ccaacagctc cctgaacatc 240

gtgaacgcca agttcgagga cagcggcgag tacaagtgcc agcaccagca ggtgaacgag 300

agcgagcccg tgtacctgga ggtgttcagc gactggctgc tgctgcaggc cagcgccgag 360

gtggtgatgg agggccagcc cctgttcctg agatgccacg gctggagaaa ctgggacgtg 420

tacaaggtga tctactacaa ggatggcgag gccctgaagt actggtacga gaaccacaac 480

atctccatca ccaacgccac cgtggaggac agcggcacct actactgcac aggcaaggtg 540

tggcagctgg actacgagag cgagcccctg aacatcaccg tgatcaaggc tcccagagag 600

aagtactggc tgcagaggaa caccggcaga ggaggcgagg aaaagaaagg aagcaaggag 660

aaggaggagc aggaggaaag agaaaccaag acccccgagt gccccagcca cacccagccc 720

ctgggcgtgt tcctgttccc ccccaagccc aaggacaccc tgatgatcag cagaaccccc 780

gaggtgacct gcgtggtcgt ggatgtgagc caggaagatc ccgaagtgca gttcaactgg 840

tacgtggatg gcgtggaagt gcacaacgcc aagaccaagc ccagagaaga gcagttcaac 900

tccacctaca gagtggtgag cgtgctgacc gtgctgcacc aggactggct gaacggcaag 960

gagtacaagt gcaaggtgtc caacaaaggc ctgcccagct ccatcgagaa gaccatcagc 1020

1080

atgaccaaga accaggtgtc cctgacctgc ctggtgaaag gcttctaccc cagcgacatc 1140

gccgtggagt gggaaagcaa cggccagccc gagaacaatt acaagacaac ccctcccgtg 1200

ctggatagcg atggcagctt ctttctgtac agcagactga ccgtggacaa gagcagatgg 1260

caggaaggca acgtgttcag ctgcagcgtg atgcacgaag ccctgcacaa ccactacacc 1320

cagaagagcc tgtccctgag cctgggcaag 1350

<210> 13

<211> 469

<212> PROTEIN

<213> Artificial sequence

<220>

<223> FceRIa ECD-hinge-Fc3

<400> 13

Met Asp Ala Met Leu Arg Gly Leu Cys Cys Val Leu Leu Leu Cys Gly

1.5 10 15

Ala Val Phe Val Ser Pro Ser His Ala Val Pro Gln Lys Pro Lys Val

20 25 30

Ser Leu Asn Pro Pro Trp Asn Arg Ile Phe Lys Gly Glu Asn Val Thr

35 40 45

Leu Thr Cys Asn Gly Asn Asn Phe Phe Glu Val Ser Ser Thr Lys Trp

50 55 60

Phe His Asn Gly Ser Leu Ser Glu Glu Thr Asn Ser Ser Leu Asn Ile

65 70 75 80

Val Asn Ala Lys Phe Glu Asp Ser Gly Glu Tyr Lys Cys Gln His Gln

85 90 95

Gln Val Asn Glu Ser Glu Pro Val Tyr Leu Glu Val Phe Ser Asp Trp

100 105 110

Leu Leu Leu Gln Ala Ser Ala Glu Val Val Met Glu Gly Gln Pro Leu

115 120 125

Phe Leu Arg Cys His Gly Trp Arg Asn Trp Asp Val Tyr Lys Val Ile

130 135 140

Tyr Tyr Lys Asp Gly Glu Ala Leu Lys Tyr Trp Tyr Glu Asn His Asn

145 150 155 160

Ile Ser Ile Thr Asn Ala Thr Val Glu Asp Ser Gly Thr Tyr Tyr Cys

165 170 175

Thr Gly Lys Val Trp Gln Leu Asp Tyr Glu Ser Glu Pro Leu Asn Ile

180 185 190

Thr Val Ile Lys Ala Pro Arg Glu Lys Tyr Trp Leu Gln Ala Gln Pro

195 200 205

Gln Ala Glu Gly Ser Leu Ala Lys Ala Thr Thr Ala Pro Ala Thr Thr

210 215 220

Arg Asn Thr Gly Arg Gly Gly Glu Glu Lys Lys Gly Ser Lys Glu Lys

225 230 235 240

Glu Glu Gln Glu Glu Arg Glu Thr Lys Thr Pro Glu Cys Pro Ser His

245 250 255

Thr Gln Pro Leu Gly Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr

260 265 270

Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val

275 280 285

Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val

290 295 300

Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser

305 310 315 320

Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu

325 330 335

Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser

340 345 350

Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro

355 360 365

Gln Val Tyr Thr Leu Pro Ser Gln Glu Glu Met Thr Lys Asn Gln

370 375 380

Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala

385 390 395 400

Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr

405 410 415

Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu

420 425 430

Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser

435 440 445

Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser

450 455 460

Leu Ser Leu Gly Lys

465

<210> 14

<211> 1407

<212> DNA

<213> Artificial sequence

<220>

<223> nucleotide sequence of FceRIa ECD-hinge-Fc3

<400> 14

atggacgcca tgctgagagg cctgtgctgt gtgctgctgc tgtgcggcgc cgtgttcgtg 60

tcccctagcc acgccgtgcc ccagaagccc aaggtgagcc tgaaccctcc ctggaacaga 120

atcttcaagg gcgagaacgt gaccctgacc tgcaacggca acaacttctt cgaggtgagc 180

agcaccaagt ggttccacaa tggcagcctg agcgaggaga ccaacagctc cctgaacatc 240

gtgaacgcca agttcgagga cagcggcgag tacaagtgcc agcaccagca ggtgaacgag 300

agcgagcccg tgtacctgga ggtgttcagc gactggctgc tgctgcaggc cagcgccgag 360

gtggtgatgg agggccagcc cctgttcctg agatgccacg gctggagaaa ctgggacgtg 420

tacaaggtga tctactacaa ggatggcgag gccctgaagt actggtacga gaaccacaac 480

atctccatca ccaacgccac cgtggaggac agcggcacct actactgcac aggcaaggtg 540

tggcagctgg actacgagag cgagcccctg aacatcaccg tgatcaaggc tcccagagag 600

aagtactggc tgcaggccca gccccaggcc gagggcagcc tggctaaggc caccacagct 660

cccgccacca cggaacac cggcagagga ggcgaggaaa agaaaggaag caaggagaag 720

gaggagcagg aggaaagaga aaccaagacc cccgagtgcc ccagccacac ccagcccctg 780

ggcgtgttcc tgttcccccc caagcccaag gacaccctga tgatcagcag aacccccgag 840

gtgacctgcg tggtcgtgga tgtgagccag gaagatcccg aagtgcagtt caactggtac 900

gtggatggcg tggaagtgca caacgccaag accaagccca gagaagagca gttcaactcc 960

acctacagag tggtgagcgt gctgaccgtg ctgcaccagg actggctgaa cggcaaggag 1020

tacaagtgca aggtgtccaa caaaggcctg cccagctcca tcgagaagac catcagcaaa 1080

gccaaaggcc agcccagaga accccaggtg tacaccctgc ctcccagcca ggaagagatg 1140

accaagaacc aggtgtccct gacctgcctg gtgaaaggct tctaccccag cgacatcgcc 1200

1260

gatagcgatg gcagcttctt tctgtacagc agactgaccg tggacaagag cagatggcag 1320

gaaggcaacg tgttcagctg cagcgtgatg cacgaagccc tgcacaacca ctacacccag 1380

aagagcctgt ccctgagcct gggcaag 1407

<210> 15

<211> 406

<212> PROTEIN

<213> Artificial sequence

<220>

<223> human sialic acid transferase a-2,6

<400> 15

Met Ile His Thr Asn Leu Lys Lys Lys Phe Ser Cys Cys Val Leu Val

1.5 10 15

Phe Leu Leu Phe Ala Val Ile Cys Val Trp Lys Glu Lys Lys Lys Gly

20 25 30

Ser Tyr Tyr Asp Ser Phe Lys Leu Gln Thr Lys Glu Phe Gln Val Leu

35 40 45

Lys Ser Leu Gly Lys Leu Ala Met Gly Ser Asp Ser Gln Ser Val Ser

50 55 60

Ser Ser Ser Thr Gln Asp Pro His Arg Gly Arg Gln Thr Leu Gly Ser

65 70 75 80

Leu Arg Gly Leu Ala Lys Ala Lys Pro Glu Ala Ser Phe Gln Val Trp

85 90 95

Asn Lys Asp Ser Ser Ser Lys Asn Leu Ile Pro Arg Leu Gln Lys Ile

100 105 110

Trp Lys Asn Tyr Leu Ser Met Asn Lys Tyr Lys Val Ser Tyr Lys Gly

115 120 125

Pro Gly Pro Gly Ile Lys Phe Ser Ala Glu Ala Leu Arg Cys His Leu

130 135 140

Arg Asp His Val Asn Val Ser Met Val Glu Val Thr Asp Phe Pro Phe

145 150 155 160

Asn Thr Ser Glu Trp Glu Gly Tyr Leu Pro Lys Glu Ser Ile Arg Thr

165 170 175

Lys Ala Gly Pro Trp Gly Arg Cys Ala Val Val Ser Ser Ala Gly Ser

180 185 190

Leu Lys Ser Ser Gln Leu Gly Arg Glu Ile Asp Asp His Asp Ala Val

195 200 205

Leu Arg Phe Asn Gly Ala Pro Thr Ala Asn Phe Gln Gln Asp Val Gly

210 215 220

Thr Lys Thr Thr Ile Arg Leu Met Asn Ser Gln Leu Val Thr Thr Glu

225 230 235 240

Lys Arg Phe Leu Lys Asp Ser Leu Tyr Asn Glu Gly Ile Leu Ile Val

245 250 255

Trp Asp Pro Ser Val Tyr His Ser Asp Ile Pro Lys Trp Tyr Gln Asn

260 265 270

Pro Asp Tyr Asn Phe Phe Asn Asn Tyr Lys Thr Tyr Arg Lys Leu His

275 280 285

Pro Asn Gln Pro Phe Tyr Ile Leu Lys Pro Gln Met Pro Trp Glu Leu

290 295 300

Trp Asp Ile Leu Gln Glu Ile Ser Pro Glu Glu Ile Gln Pro Asn Pro

305 310 315 320

Pro Ser Ser Gly Met Leu Gly Ile Ile Ile Met Met Thr Leu Cys Asp

325 330 335

Gln Val Asp Ile Tyr Glu Phe Leu Pro Ser Lys Arg Lys Thr Asp Val

340 345 350

Cys Tyr Tyr Tyr Gln Lys Phe Phe Asp Ser Ala Cys Thr Met Gly Ala

355 360 365

Tyr His Pro Leu Leu Tyr Glu Lys Asn Leu Val Lys His Leu Asn Gln

370 375 380

Gly Thr Asp Glu Asp Ile Tyr Leu Leu Gly Lys Ala Thr Leu Pro Gly

385 390 395 400

Phe Arg Thr Ile His Cys

405

<210> 16

<211> 1218

<212> DNA

<213> Artificial sequence

<220>

<223> nucleotide sequence of a-2,6-sialic transferase

human acid

<400> 16

atgatccaca ccaacctgaa gaagaagttc agctgctgcg tgctggtgtt cctgctgttc 60

gccgtgatct gcgtgtggaa ggagaagaag aaaggcagct actacgacag cttcaagctg 120

cagaccaagg agttccaggt gctgaagagc ctgggcaagc tggccatggg cagcgacagc 180

cagagcgtgt ccagctcctc cacccaggat ccccacagag gcagacagac cctgggcagc 240

ctgagaggcc tggccaaggc caagcccgag gccagcttcc aggtgtggaa caaggacagc 300

agcagcaaga acctgatccc cagactgcag aagatctgga agaactacct gagcatgaac 360

aagtacaagg tgagctacaa aggacccgga cccggcatca agttcagcgc cgaggccctg 420

aggtgccacc tgagagacca cgtgaacgtg agcatggtgg aagtgaccga cttccccttc 480

aacaccagcg agtgggaagg ctacctgccc aaggagagca tcaggaccaa ggctggcccc 540

tggggcagat gcgccgtggt gagcagcgct ggcagcctga agagctccca gctgggcaga 600

gagatcgacg accacgatgc cgtgctgagg ttcaatggcg ctcccaccgc caacttccag 660

caggacgtgg gcaccaagac cacaatccgg ctgatgaaca gccagctggt gacaaccgag 720

aagcggttcc tgaaggacag cctgtacaac gagggcatcc tgatcgtgtg ggatcccagc 780

gtgtaccaca gcgacatccc caagtggtac cagaatcccg actacaactt cttcaacaac 840

tacaagacct atagaaagct gcaccccaac cagcccttct acatcctgaa gccccagatg 900

ccctgggagc tgtgggacat cctgcaggag atcagccctg aagagatcca gcccaaccct 960

ccctccagcg gcatgctggg cattatcatc atgatgaccc tgtgcgacca ggtggacatc 1020

tacgagttcc tgcccagcaa gagaaagacc gacgtgtgct actactatca gaagttcttc 1080

gacagcgcct gcaccatggg cgcctaccac cccctgctgt acgagaagaa cctggtgaag 1140

cacctgaacc agggcaccga cgaggacatc tacctgctgg gcaaagccac cctgcccggc 1200

ttcagaacca tccactgc 1218

<210> 17

<211> 30

<212> PROTEIN

<213> Artificial sequence

<220>

<223> IgD hinge variant

<400> 17

Arg Asn Thr Gly Arg Gly Gly Glu Glu Lys Lys Xaa Xaa Lys Glu Lys

1.5 10 15

Glu Glu Gln Glu Glu Arg Glu Thr Lys Thr Pro Glu Cys Pro

20 25 30

<210> 18

<211> 49

<212> PROTEIN

<213> Artificial sequence

<220>

<223> IgD hinge variant

<400> 18

Ala Gln Pro Gln Ala Glu Gly Ser Leu Ala Lys Ala Thr Thr Ala Pro

1.5 10 15

Ala Thr Thr Arg Asn Thr Gly Arg Gly Gly Glu Glu Lys Lys Xaa Xaa

20 25 30

Lys Glu Lys Glu Glu Gln Glu Glu Arg Glu Thr Lys Thr Pro Glu Cys

35 40 45

Pro

<210> 19

<211> 30

<212> PROTEIN

<213> Artificial sequence

<220>

<223> IgD hinge region

<400> 19

Arg Asn Thr Gly Arg Gly Gly Glu Glu Lys Lys Lys Glu Lys Glu Lys

1.5 10 15

Glu Glu Gln Glu Glu Arg Glu Thr Lys Thr Pro Glu Cys Pro

20 25 30

<210> 20

<211> 425

<212> PROTEIN

<213> Artificial sequence

<220>

<223> FceRIa ECD-hinge-Fc1

<400> 20

Val Pro Gln Lys Pro Lys Val Ser Leu Asn Pro Pro Trp Asn Arg Ile

1.5 10 15

Phe Lys Gly Glu Asn Val Thr Leu Thr Cys Asn Gly Asn Asn Phe Phe

20 25 30

Glu Val Ser Ser Thr Lys Trp Phe His Asn Gly Ser Leu Ser Glu Glu

35 40 45

Thr Asn Ser Ser Leu Asn Ile Val Asn Ala Lys Phe Glu Asp Ser Gly

50 55 60

Glu Tyr Lys Cys Gln His Gln Gln Val Asn Glu Ser Glu Pro Val Tyr

65 70 75 80

Leu Glu Val Phe Ser Asp Trp Leu Leu Leu Gln Ala Ser Ala Glu Val

85 90 95

Val Met Glu Gly Gln Pro Leu Phe Leu Arg Cys His Gly Trp Arg Asn

100 105 110

Trp Asp Val Tyr Lys Val Ile Tyr Tyr Lys Asp Gly Glu Ala Leu Lys

115 120 125

Tyr Trp Tyr Glu Asn His Asn Ile Ser Ile Thr Asn Ala Thr Val Glu

130 135 140

Asp Ser Gly Thr Tyr Tyr Cys Thr Gly Lys Val Trp Gln Leu Asp Tyr

145 150 155 160

Glu Ser Glu Pro Leu Asn Ile Thr Val Ile Lys Ala Pro Arg Glu Lys

165 170 175

Tyr Trp Leu Gln Arg Asn Thr Gly Arg Gly Gly Glu Glu Glu Lys Lys Lys

180 185 190

Glu Lys Glu Lys Glu Glu Gln Glu Glu Arg Glu Thr Lys Thr Pro Glu

195 200 205

Cys Pro Ser His Thr Gln Pro Leu Gly Val Phe Leu Phe Pro Pro Lys

210 215 220

Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val

225 230 235 240

Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr

245 250 255

Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu

260 265 270

Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His

275 280 285

Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys

290 295 300

Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln

305 310 315 320

Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Ser Gln Glu Glu Met

325 330 335

Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro

340 345 350

Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn

355 360 365

Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu

370 375 380

Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val

385 390 395 400

Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln

405 410 415

Lys Ser Leu Ser Leu Ser Leu Gly Lys

420 425

<210> 21

<211> 425

<212> PROTEIN

<213> Artificial sequence

<220>

<223> FceRIa ECD-hinge-Fc2

<400> 21

Val Pro Gln Lys Pro Lys Val Ser Leu Asn Pro Pro Trp Asn Arg Ile

1.5 10 15

Phe Lys Gly Glu Asn Val Thr Leu Thr Cys Asn Gly Asn Asn Phe Phe

20 25 30

Glu Val Ser Ser Thr Lys Trp Phe His Asn Gly Ser Leu Ser Glu Glu

35 40 45

Thr Asn Ser Ser Leu Asn Ile Val Asn Ala Lys Phe Glu Asp Ser Gly

50 55 60

Glu Tyr Lys Cys Gln His Gln Gln Val Asn Glu Ser Glu Pro Val Tyr

65 70 75 80

Leu Glu Val Phe Ser Asp Trp Leu Leu Leu Gln Ala Ser Ala Glu Val

85 90 95

Val Met Glu Gly Gln Pro Leu Phe Leu Arg Cys His Gly Trp Arg Asn

100 105 110

Trp Asp Val Tyr Lys Val Ile Tyr Tyr Lys Asp Gly Glu Ala Leu Lys

115 120 125

Tyr Trp Tyr Glu Asn His Asn Ile Ser Ile Thr Asn Ala Thr Val Glu

130 135 140

Asp Ser Gly Thr Tyr Tyr Cys Thr Gly Lys Val Trp Gln Leu Asp Tyr

145 150 155 160

Glu Ser Glu Pro Leu Asn Ile Thr Val Ile Lys Ala Pro Arg Glu Lys

165 170 175

Tyr Trp Leu Gln Arg Asn Thr Gly Arg Gly Gly Glu Glu Glu Lys Lys Gly

180 185 190

Ser Lys Glu Lys Glu Glu Gln Glu Glu Arg Glu Thr Lys Thr Pro Glu

195 200 205

Cys Pro Ser His Thr Gln Pro Leu Gly Val Phe Leu Phe Pro Pro Lys

210 215 220

Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val

225 230 235 240

Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr

245 250 255

Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu

260 265 270

Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His

275 280 285

Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys

290 295 300

Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln

305 310 315 320

Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Ser Gln Glu Glu Met

325 330 335

Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro

340 345 350

Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn

355 360 365

Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu

370 375 380

Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val

385 390 395 400

Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln

405 410 415

Lys Ser Leu Ser Leu Ser Leu Gly Lys

420 425

<210> 22

<211> 444

<212> PROTEIN

<213> Artificial sequence

<220>

<223> FceRIa ECD-hinge-Fc3

<400> 22

Val Pro Gln Lys Pro Lys Val Ser Leu Asn Pro Pro Trp Asn Arg Ile

1.5 10 15

Phe Lys Gly Glu Asn Val Thr Leu Thr Cys Asn Gly Asn Asn Phe Phe

20 25 30

Glu Val Ser Ser Thr Lys Trp Phe His Asn Gly Ser Leu Ser Glu Glu

35 40 45

Thr Asn Ser Ser Leu Asn Ile Val Asn Ala Lys Phe Glu Asp Ser Gly

50 55 60

Glu Tyr Lys Cys Gln His Gln Gln Val Asn Glu Ser Glu Pro Val Tyr

65 70 75 80

Leu Glu Val Phe Ser Asp Trp Leu Leu Leu Gln Ala Ser Ala Glu Val

85 90 95

Val Met Glu Gly Gln Pro Leu Phe Leu Arg Cys His Gly Trp Arg Asn

100 105 110

Trp Asp Val Tyr Lys Val Ile Tyr Tyr Lys Asp Gly Glu Ala Leu Lys

115 120 125

Tyr Trp Tyr Glu Asn His Asn Ile Ser Ile Thr Asn Ala Thr Val Glu

130 135 140

Asp Ser Gly Thr Tyr Tyr Cys Thr Gly Lys Val Trp Gln Leu Asp Tyr

145 150 155 160

Glu Ser Glu Pro Leu Asn Ile Thr Val Ile Lys Ala Pro Arg Glu Lys

165 170 175

Tyr Trp Leu Gln Ala Gln Pro Gln Ala Glu Gly Ser Leu Ala Lys Ala

180 185 190

Thr Thr Ala Pro Ala Thr Thr Arg Asn Thr Gly Arg Gly Gly Glu Glu

195 200 205

Lys Lys Gly Ser Lys Glu Lys Glu Glu Gln Glu Glu Arg Glu Thr Lys

210 215 220

Thr Pro Glu Cys Pro Ser His Thr Gln Pro Leu Gly Val Phe Leu Phe

225 230 235 240

Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val

245 250 255

Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe

260 265 270

Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro

275 280 285

Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr

290 295 300

Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val

305 310 315 320

Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala

325 330 335

Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln

340 345 350

Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly

355 360 365

Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro

370 375 380

Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser

385 390 395 400

Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu

405 410 415

Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His

420 425 430

Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys

435 440

<---

Claims (27)

1. Polypeptide dimer capable of binding to IgE, containing: two monomers, each containing the extracellular domain of the IgE Fc receptor alpha subunit (FcεRIa-ECD), where FcεRIa-ECD consists of the amino acid sequence of SEQ ID NO: 1, where the monomer contains a modified Fc region consisting of the amino acid sequence of SEQ ID NO: 2, and the modified Fc region and the FcεRIa-ECD are linked through the hinge region, where the IgD-derived hinge region is Arg Asn Thr Gly Arg Gly Gly Glu Glu Lys Lys Xaa1 Xaa2 Lys Glu Lys Glu Glu Gln Glu Glu Arg Glu Thr Lys Thr Pro Glu Cys Pro (SEQ ID NO: 17) or Ala Gln Pro Gln Ala Glu Gly Ser Leu Ala Lys Ala Thr Thr Ala Pro Ala Thr Thr Arg Asn Thr Gly Arg Gly Gly Glu Glu Glu Lys Lys Xaa3 Xaa4 Lys Glu Lys Glu Glu Gln Glu Glu Arg Glu Thr Lys Thr Pro Glu Cys Pro (SEQ ID NO: 18), where Xaa1 and Xaa3 represent Gly and Xaa2 and Xaa4 are Gly or Ser. 2. Polypeptide dimer according to claim 1, where the hinge region has any of the amino acid sequences selected from the group consisting of SEQ ID NO: 3 and SEQ ID NO: 4. 3. A polynucleotide that encodes a monomer contained in a polypeptide dimer according to claim 1, where the monomer contains the extracellular domain of the alpha subunit of the IgE Fc receptor (FcεRIa-ECD), where the monomer consists of the amino acid sequence of SEQ ID NO: 21 or SEQ ID NO: 22. 4. An expression vector into which the polynucleotide of claim 3 is inserted. 5. A host cell that contains a polynucleotide encoding a polypeptide dimer according to claim 1 and produces a polypeptide dimer according to claim 1. 6. Pharmaceutical composition for the treatment or prevention of an allergic disease, containing any of the polypeptide dimers according to claims 1, 2 as an active ingredient and a pharmaceutically acceptable carrier, where the polypeptide dimer is present in an effective amount of from 0.001% by weight to 20.0% by weight. 7. Pharmaceutical composition according to claim 6, wherein the allergic disease is any disease selected from the group consisting of food allergy, atopic dermatitis, asthma, allergic rhinitis, allergic conjunctivitis, allergic dermatitis, chronic idiopathic urticaria, and allergic contact dermatitis. 8. A food composition for alleviating an allergy symptom, comprising any of the polypeptide dimers according to claims 1 to 2 as an active ingredient and an acceptable carrier, where the polypeptide dimer is present in an effective amount of from 0.001% by weight to 20.0% by weight, where the allergic symptom is a symptom of any of the diseases selected from the group consisting of food allergy, atopic dermatitis, asthma, allergic rhinitis, allergic conjunctivitis, allergic dermatitis, chronic idiopathic urticaria and allergic contact dermatitis. 9. A method for producing a polypeptide dimer, including: a cell culture step into which a polynucleotide encoding a polypeptide dimer according to claim 1 and a sialic acid transferase gene are introduced; And the step of isolating the polypeptide dimer. 10. A method for treating or preventing an allergic disease, including: the step of administering to the individual the polypeptide dimer of claim 1.

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