TW201930343A - Pharmaceutical composition comprising recombinant extracellular domain of IgE receptor - Google Patents

Abstract

The present invention provides uses of a polypeptide dimeric protein containing two monomers, each of which contains an extracellular domain (FceRIa-ECD) of an alpha subunit of an IgE Fc receptor. The fusion protein according to the present invention has advantages that an excellent binding ability to IgE is exhibited as compared with a conventional therapeutic agent containing an anti-IgE antibody, and less other side effects are exhibited due to lack of ADCC and CDC functions. Thus, the fusion protein can be applied to a medical product for treating or preventing an IgE-mediated allergic disease.

Description

Pharmaceutical composition comprising a recombinant extracellular region of an IgE receptor

FIELD OF THE INVENTION The invention relates to the use of a modified IgE Fc receptor.

BACKGROUND OF THE INVENTION Allergic diseases such as allergic rhinitis, atopic dermatitis and food allergies, including asthma, spread rapidly in modern society of industrialization and westernization, and systemic allergic reactions (anaphylaxis), a serious allergic disease, It has also increased. These chronic immune diseases seriously impair the quality of life of individuals and the corresponding increase in social and economic costs. Therefore, measures are urgently needed to overcome these diseases.

Most allergic diseases are caused by excessive immune responses to immunoglobulin E (IgE). IgE is an antibody that is normally present in serum at very low concentrations. IgE is also usually produced from harmless antigens. There is a case where the number of IgE increases without any specific stimuli. This condition may cause an allergic disease. An abnormally increased number of IgE binds to high affinity IgE Fc receptors (FceRIs) that appear on the surface of mast cells, basophils, and the like. This combination results in the release of chemical mediators such as histamine, leukotrienes, prostaglandins, bradykinin and platelet active factors by mast cells or basophils. The release of these chemical mediators causes allergic symptoms. In particular, allergic diseases may show worse symptoms due to the combination of IgE and FceRI. FceRI-expressing cells are known to increase in allergic diseases.

At present, various methods for treating allergic diseases have been proposed, such as avoiding allergens, administering anti-allergic drugs, adjusting the synthesis of IgE in vivo, and developing anti-IgE antibodies. However, the treatment methods known so far have a number of disadvantages, such as the inability to treat the underlying cause of allergies, insufficient efficacy, and serious side effects.

Further, an immunoglobulin composition (KR10-1783272B1) capable of binding IgE and FceRIIb with high affinity and inhibiting cells expressing IgE has been studied. This composition has been reported to be useful in the treatment of IgE mediated disorders, including allergies and asthma. In addition, omalizumab (trade name: Xolair), which targets the Fc portion of the IgE antibody, has been developed as a therapeutic agent for refractory severe asthma and refractory urticaria.

However, administration of high doses of omalizumab to maintain therapeutic efficacy results in high cost burdens and side effects such as angioedema and systemic allergic reactions (The Journal of Clinical Investigation Volume 99, Number 5, March 1997, 915- 925). In addition, post-marketing results have been reported for allergic granulomatous vasculitis and idiopathic thrombocytopenia. Accordingly, there is an increasing need to develop therapeutic agents that are effective in treating allergic diseases without side effects.

Technical Problem An object of the present invention is to provide a pharmaceutical composition and a food composition comprising a polypeptide dimeric protein as an active ingredient for the treatment of an IgE-mediated allergic disease.
Ways to solve the problem

In one aspect of the invention, a pharmaceutical composition for treating or preventing an allergic disease comprising a polypeptide dimer comprising two monomers each containing IgE Fc is provided The extracellular region of the subunit of the body, wherein the monomer comprises a modified Fc region, and the modified Fc region and the extracellular region of the subunit of the IgE Fc receptor are permeable to the IgD antibody. connection. In another aspect, a food composition for improving or alleviating allergy symptoms comprising the polypeptide dimer as an active ingredient is provided.
Advantageous effects of the present invention

Compared with the conventional anti-IgE antibody, the polypeptide dimeric protein according to the present invention not only has excellent safety and persistence in vivo, but also has 70 times larger than the conventional anti-IgE antibody, omalimumab, because of its IgE binding ability. Therefore, it will be strongly combined with IgE, which allows for an extended dosing cycle. Furthermore, the polypeptide dimeric protein according to the present invention is a substance obtained by using a modified Fc which has only IgE as a single target and does not bind to an Fcg receptor, and thus has no antibody-dependent cytotoxicity (ADCC) And the function of complement dependent cytotoxicity (CDC). Therefore, unlike the conventional anti-IgE antibody containing the IgG1 Fc region, the polypeptide dimer does not bind to the Fcg receptor, thereby inhibiting the release of the mediator due to binding to the Fcg receptor on the surface of the mast cell. Thus, the occurrence of serious side effects such as systemic allergic reactions due to binding between IgG1 and Fcg receptor III on mast cells can be minimized. Therefore, the polypeptide dimeric protein according to the present invention can be used as a novel pharmaceutical composition for substituting a therapeutic agent containing a conventional anti-IgE antibody.

DETAILED DESCRIPTION OF THE INVENTION The present invention provides a pharmaceutical composition for treating or preventing an allergic disease, comprising a polypeptide dimer comprising two monomers each containing an IgE Fc receptor The extracellular region of the subunit (FceRIa-ECD), wherein the monomer comprises a modified Fc region, and the modified Fc region is joined to the hinge of the FceRIa-ECD through the IgD antibody.

The term "IgE" as used herein refers to an antibody protein called immunoglobulin E. IgE has affinity for mast cells, blood basophils, and the like. In addition, the reaction between the IgE antibody and its corresponding antigen (allergen) causes an inflammatory response. In addition, IgE is known as an antibody that causes a systemic allergic reaction due to sudden secretion of mast cells or basophils.

The term "IgE Fc receptor" as used herein, also referred to as the Fce receptor, binds to the Fc portion of IgE. There are two types of receptors. A receptor with high IgE Fc affinity is called Fce receptor I (FceRI). A receptor with low IgE Fc affinity is called Fce receptor II (FceRII). FceRI is expressed in mast cells and basophils. In the case where the binding of the IgE antibody to FceRI is cross-linked via a multivalent antigen, the mast cells or basophilic balls undergo a degranulation reaction, thereby releasing various chemical delivery substances, including histamine. This release can cause an immediate allergic reaction.

FceRI is a membrane protein consisting of an a-chain, a b-chain and two g-chains linked by disulfide bonds. In these chains, the part to which IgE binds is the a chain (FceRIa). FceRIa is about 60 kDa large and consists of a hydrophobic region existing inside the cell membrane and a hydrophilic region existing outside the cell membrane. In particular, IgE binds to the extracellular region of the a chain.

Specifically, the a-unit of the IgE Fc receptor may have the amino acid sequence described in NP_001992.1. Furthermore, the extracellular region of the subunit of the IgE Fc receptor (FceRIa-ECD) may have the amino acid sequence of SEQ ID NO: 1. In the present specification, the extracellular region of the subunit of the IgE Fc receptor may be a fragment or variant of the extracellular region of the subunit of the IgE Fc receptor, as long as the fragment or variant is capable of binding IgE.

This variant can be made by substituting, deleting or adding one or more proteins in the wild type FceIa-ECD (extracellular region) as long as the method does not alter the function of the a chain of FceRI. The various proteins or peptides may be 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher with the amino acid sequence of Sequence Identification Number 1. Consistency. Further, FceRIa-ECD of Sequence Identification Number 1 can be encoded by a polynucleotide having Sequence Identification Number 5.

Furthermore, the term "modified Fc region" as used herein means a region in which a portion of the Fc portion of an antibody has been modified. Here, the term "Fc region" means a heavy chain constant region 2 (CH2) and a heavy chain constant region 3 (CH3) containing an immunoglobulin, but does not contain a variable region of a heavy chain and a light chain of an immunoglobulin. Part of the light chain constant region 1 (CH1). In particular, the modified Fc region means a region obtained by substituting some of the amino acids in the Fc region or by binding different types of Fc regions. Specifically, the modified Fc region may have the amino acid sequence of SEQ ID NO: 2. Furthermore, a modified Fc region having sequence identification number 2 can be encoded by a polynucleotide having the sequence of SEQ ID NO: 6.

Furthermore, the "modified Fc region" of the present invention may be in the form of a sugar chain having a native form, increased relative to a natural form of a sugar chain or reduced relative to a native form, or may be in the form of a sugar chain removed. The immunoglobulin Fc sugar chain can be modified by conventional methods such as chemical methods, enzyme methods, and genetic engineering methods using microorganisms.

Here, the "modified Fc region" of the present invention may be a region free of antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) function since it has no binding site of FcgR or C1q. Furthermore, the modified Fc region and the FceRIa-ECD are ligated via a hinge of an IgD antibody. The hinge of the IgD antibody consists of 64 amino acids and optionally contains 20 to 60 contiguous amino acids, 25 to 50 contiguous amino acids or 30 to 40 amino acids. In one embodiment, the hinge of the IgD antibody can be composed of 30 or 49 amino acids, as described below. Here, the hinge may be a variant obtained by modifying the hinge region, wherein the hinge may contain at least one cysteine. Here, in order to minimize the production of truncation during the protein production process, the hinge variant can be obtained by modifying some of the hinge sequences in the IgD antibody.

In an embodiment, the hinge may contain 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 can be Lys or Gly, and Xaa2 can be Glu , Gly or Ser. In particular, the hinge may have the amino acid sequence of Sequence Identification Number 3 or Sequence Identification Number 19 to minimize the production of truncated forms during the protein production process.

In another embodiment, the hinge can contain the following sequences
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 Lys Lys Xaa3 Xaa4 Lys Glu Lys Glu Glu Gln Glu Glu Arg Glu Thr Lys Thr Pro Glu Cys Pro ( Sequence identification number 18), wherein Xaa3 can be Lys or Gly, and Xaa4 can be Glu, Gly or Ser. In particular, the hinge may have the amino acid sequence of SEQ ID NO: 4 to minimize the production of truncation during the protein production process.

In particular, in the hinge with sequence number 4, at least one Thr can be glycosylated. Specifically, in the amino acid of SEQ ID NO: 18, the 13th, 14th, 18th or 19th Thr can be glycosylated. Preferably, all four amino acids are glycosylated. Here, glycosylation can be O-glycosylated.

Furthermore, as described above, the polypeptide dimer provided by the present invention may be one in which two monomers are bound to each other and each monomer can pass through the extracellular region of the subunit of the IgE Fc receptor and the modified Fc region. The form obtained by the combination. The polypeptide dimer can be in the form in which the same two monomers are bound to each other by a cysteine at the hinge. Further, the polypeptide dimer may be in a form in which two different monomers are combined with each other. For example, in the case where the two monomers are different from each other, the polypeptide dimer may be an extracellular region in which one monomer contains the a-unit of the IgE Fc receptor, and the other contains the IgE Fc receptor a times. The form of a fragment of the extracellular region of the unit. Here, an embodiment of the monomer may have an amino acid sequence of sequence identification number 20, sequence identification number 21 or sequence identification number 22.

Furthermore, the polypeptide dimer provided by the present invention exhibits 10 to 100 times, 20 to 90 times, 20 to 70 times, 30 to 70 times or 40 to 70 times higher than omalizumab, an anti-IgE antibody. The IgE binding ability preferably exhibits 70-fold higher IgE binding ability than omalizumab.

Further, in the present specification, the term "allergic disease" means a pathological condition caused by mast cell activation, such as an allergic reaction mediated by a mast cell degranulation reaction. This allergic disease includes food allergies, atopic dermatitis, asthma, allergic rhinitis, allergic conjunctivitis, allergic dermatitis, allergic contact dermatitis, systemic allergic reactions, urticaria, pruritus, insect allergies, Chronic spontaneous urticaria, drug allergies, etc. In particular, the allergic disease can be IgE mediated.

In the composition for treating or preventing allergic diseases of the present invention, any amount of the active ingredient (effective amount) may be included depending on the use, formulation, blending purpose, etc., as long as the active ingredient can exhibit anti-allergic activity. can. The effective amount of the active ingredient is generally determined to be in the range of from 0.001% by weight to 20.0% by weight based on the weight of the composition. As used herein, "effective amount" means the amount of active ingredient that is capable of inducing an anti-allergic effect. This effective amount can be determined experimentally by those skilled in the art.

Here, the pharmaceutical composition may further comprise a pharmaceutically acceptable carrier. For the pharmaceutically acceptable carrier, any carrier can be used as long as the carrier is a non-toxic substance suitable for delivery to a patient. Distilled water, alcohol, fat, wax and inert solids may be included as a carrier. Pharmaceutically acceptable adjuvants (buffers and dispersing agents) may also be included in the pharmaceutical composition.

Specifically, in addition to the active ingredient, the pharmaceutical compositions of the present invention also contain a pharmaceutically acceptable carrier, and can be formulated into oral or parenteral formulations by conventional methods known in the art, depending on the route of administration. As used herein, the term "pharmaceutically acceptable" means having a toxicity that is acceptable to a subject that does not exceed the administration (prescription) and does not inhibit the activity of the active ingredient.

In the case where the pharmaceutical composition of the present invention is formulated into an oral formulation, the pharmaceutical composition can be formulated into a powder, granule, lozenge, pill, according to methods known in the art. Formulations for dragees, capsules, liquids, gels, syrups, suspensions and flakes. Here, examples of suitable pharmaceutically acceptable carriers may include sugars such as lactose, glucose, sucrose, dextrose, sorbitol, mannose and xylitol; starches such as corn starch, potato starch and Wheat starch; cellulose, such as cellulose, methyl cellulose, ethyl cellulose, sodium carboxymethyl cellulose and hydroxypropyl methyl cellulose; polyvinylpyrrolidone, water, methyl hydroxybenzoate, hydroxybenzene Propyl formate, magnesium stearate, mineral oil, malt, gelatin, talc, polyol, vegetable oil, and the like. In the case of a preparation, the preparation may be carried out by including a diluent and/or an excipient such as a filler, a bulking agent, a binder, a wetting agent, a disintegrating agent, and a surfactant, as necessary.

In the case where the pharmaceutical composition of the present invention is formulated into a parenteral formulation, the pharmaceutical composition can be formulated into an injection, a transdermal drug, a nasal inhalation, and a suppository in accordance with a method known in the art. Preparation. In the case of injection preparation, sterile water, ethanol, a polyol such as glycerin and propylene glycol, or a mixture thereof may be used as a suitable carrier. As the carrier, an isotonic solution such as Ringer's solution, phosphate buffered saline (PBS) containing triethanolamine, sterile water for injection, 5% dextrose, or the like can be preferably used.

The preparation of such pharmaceutical compositions is known in the art, and specifically reference is made to Remington's Pharmaceutical Sciences (19th edition, 1995) and the like. This document is considered to be part of the invention.

Preferred daily doses of the pharmaceutical compositions of the present invention range from 0.01 ug/kg to 10 g/kg, preferably from 0.01 mg/kg to 1 g/kg, depending on the condition, weight, sex, age, and severity of the disease. Sexual or medication route. You can take it once or several times a day. This dose should in no way be construed as limiting the scope of the invention.

The subject to which the composition of the present invention can be administered (prescription) is a mammal and a human, and a human is most suitable. In addition to the active ingredient, the anti-allergic composition of the present invention may further comprise any compound or natural extract which has been certified to have anti-allergic activity and is intended to enhance and enhance anti-allergic activity. .

In another aspect of the invention, a food composition for improving and alleviating allergy symptoms comprising the polypeptide dimer as an active ingredient is provided.

Here, the polypeptide dimer can be bound to a suitable delivery unit for efficient delivery to the intestine. The food composition of the present invention can be made into any form, for example, in the form of a beverage, such as tea, fruit juice, carbonated beverage and ionic beverage, processed dairy product, such as milk and yogurt, health functional food preparation, such as lozenge, Capsules, pills, granules, liquids, powders, flakes, pastes, syrups, gels, jellies and sticks, and the like. Furthermore, the food composition of the present invention can be classified into any legal product category or functional classification as long as the food composition meets the law enforcement requirements at the time of manufacture and distribution. For example, the food composition may be a health functional food according to the health functional food law, or a confectionery dessert of each food type according to the Food Code of Food Sanitation Law (Food Standards and Regulations notified by the Food and Drug Administration), Beans, tea, beverages, special-purpose foods, etc. As for other food additives which may be included in the food composition of the present invention, reference may be made to the Codex Alimentarius or Food Additive Code in accordance with the Food Sanitation Law.

In still another aspect of the invention, a polynucleotide encoding a monomer comprising an extracellular region of a subunit of an IgE Fc receptor that binds to a modified Fc region is provided.

At the same time, the polynucleotide may additionally contain a message sequence or a leader sequence. The term "message sequence" as used herein refers to a nucleotide that encodes a message peptide that directs secretion of a protein of interest. The message peptide is translated in the host cell and then cleaved. Specifically, the message sequence of the present invention is a nucleotide encoding an amino acid sequence capable of initiating translocation of a protein across the endoplasmic reticulum (ER) membrane. Useful message sequences of the invention include antibody light chain message sequences, for example, antibody 14.18 (Gillies et al., J. Immunol. Meth 1989. 125:191-202), antibody heavy chain message sequences, such as MOPC141 antibody heavy chain messages. Sequences (Sakano et al., Nature, 1980. 286: 676-683) and other sequences of information known in the art (see, for example, Watson et al., Nucleic Acid Research, 1984. 12: 5145-5164). ).

The characteristics of the message sequence are well known in the art. The message sequence typically contains from 16 to 30 amino acid residues and may contain more or fewer amino acid residues. A typical message peptide consists of three regions, which are the basic N-terminal region, the central hydrophobic region, and the more polar C-terminal region. The central hydrophobic region contains 4 to 12 hydrophobic residues that fix the message sequence through the membrane lipid bilayer during translocation of the immature polypeptide.

After initiation, the message sequence is cleaved by a cell enzyme called the message peptidase in the lumen of the endoplasmic reticulum. Here, the message sequence may be a secretion message sequence of tissue plasminogen activator (tPA), herpes simplex virus glycoprotein D (HSV gD) or growth hormone. Preferably, higher eukaryotic cells, including secretory message sequences for use by mammals and the like, can be used. Furthermore, the secretion message sequence can be replaced with a codon having a high expression frequency in the host cell and used.

Meanwhile, the message sequence and the extracellular region monomer of the subunit of the IgE Fc receptor to which the modified Fc region binds may have the amino acid sequence of SEQ ID NO: 11 or SEQ ID NO: 13. The proteins of sequence identification number 11 and sequence identification number 13 can be encoded by a polynucleotide having a sequence of sequence identification number 12 and sequence identification number 14, respectively.

In still another aspect of the invention, an expression vector for loading a polynucleotide encoding the monomer is provided. Here, the polynucleotide may have a sequence of sequence identification number 12 or sequence identification number 14.

The term "vector," as used herein, is intended to be introduced into a host cell and capable of being recombined with and inserted into the host cell genome. Alternatively, the vector is an episomal genome and is understood to be a nucleic acid unit containing an automatically replicable nucleotide sequence. Vectors include linear nucleic acids, plastids, phagemids, plastids, RNA vectors, viral vectors, and the like. Examples of viral vectors include, but are not limited to, retroviruses, adenoviruses, and adeno-associated viruses. In addition, the plastid may contain a selectable marker, such as an antibiotic resistance gene, so that host cells carrying the plastid can be cultured under selective conditions.

As used herein, the term "gene expression" or "expression" of a protein of interest 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 can be RcCMV (Invitrogen, Carlsbad) or variants thereof. The expression vector may contain a human large cell virus (CMV) promoter for promoting continuous transcription of a target gene in mammalian cells, and a bovine growth hormone polyadenylation message sequence for increasing the stability level of post-transcriptional RNA. .

In still another aspect of the invention, a host cell into which an expression vector is introduced is provided. The term "host cell" as used herein refers to a prokaryotic or eukaryotic cell into which a recombinant expression vector can be introduced. The terms "transduction", "transformation" and "transfection" as used herein mean the introduction of a nucleic acid (eg, a vector) into a cell using techniques known in the art.

Preferred host cells for use in the present invention include immortalized fusion tumor cells, NS/0 myeloma cells, 293 cells, Chinese hamster ovary cells (CHO cells), HeLa cells, human amniotic fluid-derived cells (CapT cells), or COS cells. Preferably, the host cell can be a CHO cell. In another aspect, the host cell can be a host cell into which the vector and the vector carrying the sialyltransferase gene are introduced. Here, the sialyltransferase may be a 2,3-sialyltransferase or a 2,6-sialyltransferase. Here, the 2,6-sialyltransferase may have the amino acid sequence of SEQ ID NO: 15.

In still another aspect of the present invention, a pharmaceutical composition for treating or preventing an allergic disease, comprising a polypeptide dimer as an active ingredient, the polypeptide dimer comprising two monomers, Each of the extracellular regions (FceRIa-ECD) containing a subunit of the IgE Fc receptor, the polypeptide dimer is obtained by: 1) cultivating a polynucleotide encoding a polypeptide monomer and a chymase transferase. a step of cells of a gene; and 2) a step of recovering a polypeptide dimer.

In still another aspect of the present invention, there is provided a food composition for improving or alleviating allergy symptoms comprising a polypeptide dimer produced by the method for producing a dimer described above as an active ingredient.

In still another aspect of the present invention, a method for treating or preventing an allergic disease, comprising the step of administering to a subject a polypeptide dimer comprising two monomers, each monomer comprising The extracellular domain of the subunit of the IgE Fc receptor (FceRIa-ECD).

The subject can be a mammal, preferably a human. Here, administration can be achieved orally or parenterally. Here, non-oral administration can be carried out by methods such as subcutaneous administration, intravenous administration, mucosal administration, and muscle administration.

Hereinafter, the present invention will be described in detail with reference to the following examples. However, the following examples are only intended to illustrate the invention, and the scope of the invention is not limited only.
Example 1. Preparation of polypeptides comprising Fc eRI a-ECD and modified Fc regions

The C-terminally modified polypeptide of the extracellular region of the subunit of the IgE Fc receptor (FceRIa-ECD) is prepared according to the method disclosed in U.S. Patent No. 7,867,491.

First, in order to express the protein (FceRIaECD-Fc1), protein (FceRIaECD-Fc2) and protein (FceR1aECD-Fc3), the extracellular region of the a chain of FceRI having the amino acid sequence of sequence identification number 1 and the sequence identification number 2 The modified immunoglobulin Fc line is ligated through the hinge of SEQ ID NO: 19, the hinge of SEQ ID NO: 3, and the hinge of SEQ ID NO: 4, and the cassette obtained by ligating the gene encoding each protein is inserted into the pAD15 vector. (Genexin, Inc.), a FceRIaECD-Fc protein expression vector was constructed. Thereafter, each expression vector was transfected into CHO DG44 cells (from Dr. Chasin, Columbia University, USA).

Here, at the time of transfection into the cell line, the expression vector obtained by entering the a-2,6- serotonyltransferase gene into the pCI Hygro vector (Invitrogen) can be expressed separately and transfected into separate preparations. A cell line of FceRIaECD-Fc2ST and FceRIaECD-Fc3ST protein in which sialic acid was added.

Use serotonin (HT) - no 10% dFBS medium (Gibco, USA, 30067-334), MEMa medium (Gibco, 12561, USA, Cat No. 12561-049) and HT+ medium (Gibco, USA, 11067-030) ) Perform HT selection as the primary screening program. Thereafter, the HT-selected selection strain was used for aminoguanidine (MTX) amplification to amplify the yield using the dihydrofolate reductase (DHFR) system.

After completion of MTX amplification, subculture was performed about 1 to 5 times to stabilize the cells for evaluation of the yield. Thereafter, an evaluation of the unit yield of the MTX-amplified cells was performed. The results are shown in Table 1 below.
[Table 1]

As shown in Table 1, the FceRIaECD-Fc3 cell line showed a yield of 16.9 ug/10 ⁶ cells after amplification of 2 uM of the amine guanidine. On the other hand, the FceRIaECD-Fc3 cell line (FceRIaECD-Fc3ST) co-transfected with 2,6-valerate transferase showed 17.5 ug/10 ⁶ cells after amplification of 1 uM of the amine formazan. rate. Furthermore, the FceRIaECD-Fc2 cell line exhibited a yield of 20.9 ug/10 ⁶ cells under 0.5 uM amine formazan amplification. Furthermore, the FceRIaECD-Fc2 cell line (FceRIaECD-Fc2ST) co-transfected with 2,6- chymotransferase showed a yield of 25.1 ug/10 ⁶ cells after amplification of 1 uM of the amine guanidine. Namely, the FceRIaECD-Fc2 cell line co-transfected with 2,6- succinate transferase was determined, which had been selected under the conditions of 1 uM of the amine formazan amplification, showing the best yield.
Example 2 : Purification of Fc eRI aECD fusion protein and identification of its purity

For the cell strain selected in the above Example 1, i) FceRIaECD-Fc3, ii) FceRIaECD-Fc3ST and iii) FceRIaECD-Fc2ST were cultured on a 60 ml scale by a batch culture method. The resulting culture was purified using Protein A affinity column, and the purified protein was subjected to SDS-PAGE and particle size exclusion HPLC (SE-HPLC) to identify the purity of the protein.

As shown in Figure 1, all of the proteins purified by the SE-HPLC method were each identified to have a purity of 93% or higher. Further, based on the results of the SDS-PAGE analysis, it was confirmed that a protein having a size of about 150 kDa and about 75 kDa was detected (Fig. 1, lanes 1 to 6). It was found here that Fc binds to FceRIaECD to form a dimer. In addition, no impurities such as truncated type were observed in the SDS-PAGE results. In particular, even after the thawing/freezing process (Fig. 1, lanes 7 to 8), all proteins were identified to have a purity of 93% or higher and no impurities. It was found here that the yield of the truncated protein type was reduced relative to FceRIaECD-Fc1 using the wild type IgD hinge.

Here, Gel-IEF was carried out under the following test conditions to identify the degree of sialytic acid content in the protein after introduction of the sputum acid transferase. It was confirmed from this that the content of the acidic protein increased due to an increase in the content of the limulus acid.
[Table 2]

To determine the reproducibility of the purification yield, the FceRIaECD-Fc2ST cell line was batch-cultured in a 1 L flask on a 250 ml scale and then purified using a Protein A affinity column. Thereafter, the culture supernatant and the product was purified on a 4% to 15% TGX ^TM gels (Bio-Rad Laboratories, Inc.) , at 30 electrophoresis Tris- Glycine SDS (TGS) and buffer conditions 200V Minutes, then processed by SDS PAGE analysis. As a result, it was confirmed that not only a protein having a very high purity (98% or higher) was purified (even if it was only purified by the first step), but also a protein of a very high purity was exhibited in the culture supernatant. This indicates that the development of the FceRIaECD-Fc protein that has been expressed in the cell line in question as a medical product simplifies the process development steps and, therefore, is highly likely to significantly reduce the development cost of medical products.
Example 3 , Identification of IgE Binding Ability of Fc eRI aECD Fusion Protein

Comparison of the following four proteins and commercially available anti-IgE antibodies, IgE binding capacity of omalizumab (trade name: Xolair): i) FceRIaECD-Fc2, ii) FceRIaECD-Fc2ST, iii) FceRIaECD-Fc3 and iv) FceRIaECD- Fc3ST, which has been purified by the method of Example 2 above. Specifically, IgE was coated on a tube of a protein GLC sensing wafer (Bio-Rad Laboratories, Inc., Cat # 176-5011), and then omalizumab or various concentrations of each FceR1aECD-Fc protein was 30 μl/ The rate of fractionation flows through to measure the binding capacity of IgE.

The experiment was performed using 25 mM NaOH as the regeneration buffer, determining the zero baseline, and then repeating the above steps. Thereafter, the binding curve was determined using a protein binding analyzer (ProteOn XPR36, Bio-Rad Laboratories, Inc., USA). The results are shown in Table 3 and Figures 3 and 4.
[table 3]

As shown in Table 3, the binding rate (ka) measured by the polypeptide dimer according to one embodiment of the present invention was 1.5 to 2.0 times lower than that of omalizumab. That is, it was found to have a binding ability to substances other than IgE which is 1.5 to 2.0 times lower than that of omalizumab. Furthermore, the dissociation rate (kd) value measured by the polypeptide dimer according to an embodiment of the present invention is 40 to 106 times higher than that of omalizumab. Furthermore, as shown in Figures 3 and 4, it can be determined that omalizumab loses its binding to IgE after a period of time after binding, whereas once the polypeptide dimer of the FceRIaECD fusion protein of the present invention binds to IgE, The polypeptide dimer is not separated from IgE.

That is, it can be seen that the polypeptide dimer of the present invention is not easily separated from IgE, and the ability to maintain its binding state is much better than that of omalizumab. As a result, it can be seen that the polypeptide dimer according to an embodiment of the present invention has an equilibrium dissociation constant (KD <kd/ka>) value which is 22 to 69 times higher than that of omalizumab. From this, it was confirmed that the IgE binding ability of the FceRIaECD fusion protein of the present invention was significantly increased as compared with omalizumab. In particular, it was confirmed that FceRIaECD-Fc2 (FceRIaECD-Fc2ST) to which sialic acid was added exhibited the highest IgE binding ability, which was 69 times higher than that of omalizumab.
Example 4 : Identification of IgG receptor binding ability of Fc eRI aECD fusion protein

The degree of IgG receptor binding of IgE _TRAP and omalizumab was identified using the Octet RED384 system (ForteBio, CA, USA). FcgRI, FcgRIIA, FcgRIIB, FcgRIIIA and FcgRIIIB recombinant proteins (R & D Systems Inc., 5 μg/ml) were immobilized on activated AR2G biosensor under 300 mM acetate buffer (pH 5). PBS containing 0.1% Tween-20 and 1% bovine serum was used as a running buffer. All experiments were performed at 30 ° C with a sample disk oscillator at a rate of 1,000 rpm. The results are shown in Figures 5A to 5E, and the IgG receptor binding ability of omalizumab and IgE _{TRAP was} quantified and shown in Fig. 6.
Example 5 : Identification of the activity of Fc eRI aECD fusion protein in mouse bone marrow-derived mast cells by b -hexosaminidase assay

The b-hexosaminidase assay was performed to analyze the in vitro activity of the FceRIaECD fusion protein of the present invention. Specifically, each concentration of the FceRIaECD-Fc2 fusion protein according to an embodiment of the present invention was mixed with mouse IgE (1 ug/mL), and incubated at room temperature (20 ° C) for 30 minutes to prepare a sample. The mouse bone marrow-derived mast cells activated by mast cells were washed with Hank's balanced salt solution (HBSS) buffer, the medium was removed, and the number of cells was measured. Thereafter, the number of cells was adjusted so as to be injected into 40 μL of HBSS buffer in a number of 5 × 10 ⁵ cells.

Thereafter, 50 uL of the sample solution prepared by pre-incubation was added to the activated mast cells. The product was then incubated in a 5% CO ₂ incubator at 37 ° C for 30 minutes. After adding 10 μL of the foreign antigen DNP (2,4-dinitrobenzene, 100 ng/mL), the cells were again incubated at 5% CO ₂ at 37 ° C for 30 minutes, and then 30 μL of the supernatant was separated. The 30 uL of the separated supernatant was thoroughly mixed with a 30 uL substrate (4-nitrophenyl N-ethinyl-β-D-glucoside, 5.84 mM), and then incubated at 37 ° C for 20 minutes at 5% CO ₂ . Thereafter, 140 μL of a 0.1 M sodium carbonate buffer (pH 10) as a stop solution was added to terminate the reaction. Thereafter, the absorbance at 405 nm was measured to identify the amount of β-hexosaminidase secreted by the foreign antigen in the activated mast cells. The results are shown in Fig. 7.

As shown in Figure 7, the polypeptide dimer of one embodiment of the present invention exhibited a mast cell inhibition rate of about 49.4% at half the concentration of mouse IgE (0.5 ug/mL), and at the same concentration. In the case of mouse IgE (1 ug/mL), a mast cell inhibition rate of about 99.4% was exhibited. Namely, it can be seen that the activity of IgE-induced bone marrow-derived mast cells is largely inhibited by the FceRIa-ECD polypeptide dimer of the present invention.
Example 6. Comparison of the activity of Fc eRI aECD fusion protein and anti-human IgE antibody in Fc eRI bone marrow derived mast cells using β -hexosaminidase assay

The β-hexosaminidase assay was performed to determine the advantage of the FceRIaECD fusion protein over Xolair by in vitro activity assay. Individual drugs, various concentrations of FceRIaECD-Fc2ST (IgETRAP) and Xolair were prepared and then mixed with human IgE (1 ug/mL). Thereafter, it was incubated at room temperature for 30 minutes. During the pre-incubation of the drug, the human FceRI gene is introduced and mast cells derived and differentiated from mouse bone marrow (where the mouse FceRI gene has been removed) are prepared. The prepared 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 prepared mast cells, and then incubated for 30 minutes in a 5% CO ₂ , 37 ° C incubator.

Subsequently, after adding 20 uL of anti-human IgE antibody (BioLegend, Cat No. 325502, 0.5 ug/mL), the product was again cultured in a 5% CO ₂ , 37 ° C incubator for 30 minutes. Thereafter, after centrifugation at 1,500 rpm and 4 ° C, 30 uL of the supernatant was separated. The 30 uL of the separated supernatant was thoroughly mixed with 30 uL of the substrate (4-nitrophenyl N-ethinyl-β-glucoside, 5.84 mM), and then incubated in a 5% CO ₂ incubator at 37 ° C for 25 minutes. Thereafter, the reaction was terminated by adding 140 uL of 0.1 M sodium carbonate buffer (pH 10).

Thereafter, the absorbance at 405 nm was measured, the relative amount of secreted β-hexosaminidase was compared, and the inhibitory effect of mast cells depending on the respective drug concentrations was determined. The results are shown in Fig. 8. 8, FceRIaECD IC fusion proteins was measured about ₅₀ 11.16ng / mL, and Xolair proteins was measured IC ₅₀ of about 649.8ng / mL. Therefore, it was confirmed that the FceRIaECD fusion protein was 58-fold more potent than the Xolair in inhibiting mast cell activity.
Example 7 , In Vivo Analysis of Fc eRI aECD Fusion Protein ( Food Allergy Model)

Balb/c mice (Orientbio Inc.) were administered 50 ug of ovalbumin (OVA) and 1 mg of aluminum hydroxide twice daily at 14-day intervals to induce sensitization. Thereafter, 50 mg of OVA was orally administered five times on days 28, 30, 32, 34, and 36 to induce intestinal food allergy.

After the oral administration of OVA twice, that is, on the 31st day, the mice were divided into three groups, each having 7 mice. The three groups that were separated were as follows: the first group received a high concentration (200 ug) of the FceRIaECD-Fc2ST fusion protein, and the second group received a low concentration (20 ug) of the FceRIaECD-Fc2ST fusion protein, while the third group did not receive anything. At the same time as oral administration of OVA, it was identified whether diarrhea occurred due to induction of food allergy. On the 37th day, the mice were sacrificed, 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)) produced by degranulation of mast cells in the blood were analyzed. .

As shown in Fig. 9, a group of mice receiving a high concentration of FceRIaECD-Fc2ST (which is a polypeptide dimer) was determined to alleviate food allergy in a concentration-dependent manner compared to a group of mice that did not receive anything. .

Sequence identification number 1
VPQKPKVSLN PPWNRIFKGE NVTLTCNGNN FEVSSTKWF HNGSLSEETN SSLNIVNAKFEDSGEYKCQH QQVNESEPVY LEVFSDWLLL QASAEVVMEG QPLFLRCHGW RNWDVYKVIY
YKDGEALKYW YENHNISITN ATVEDSGTYY CTGKVWQLDY ESEPLNITVI KAPREKYWLQ

Sequence identification number 2
SHTQPLGVFL FPPKPKDTLM ISRTPEVTCV VVDVSQEDPE VQFNWYVDGV EVHNAKTKPR
EEQFNSTYRV VSVLTVLHQD WLNGKEYKCK VSNKGLPSSI EKTISKAKGQ PREPQVYTLP
PSQEEMTKNQ VSLTCLVKGF YPSDIAVEWE SNGQPENNYK TTPPVLDSDG SFFLYSRLTV
DKSRWQEGNV FSCSVMHEAL HNHYTQKSLS LSLGK

Sequence identification number 3
RNTGRGGEEK KGSKEKEEQE ERETKTPECP

Sequence identification number 4
AQPQAEGSLA KATTAPATTR NTGRGGEEKK GSKEKEEQEE RETKTPECP

Sequence identification number 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

Sequence Identification Number 6
Tgcgtggtcg tggatgtgag ccaggaagat cccgaagtgc agttcaactg gtacgtggat
Ggcgtggaag tgcacaacgc caagaccaag cccagagaag agcagttcaa ctccacctac
Agaggtgga 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

Sequence Identification Number 7
Aggaacaccg gcagaggagg cgaggaaaag aaaggaagca aggagaagga ggagcaggag
Gaaagagaaa ccaagacccc cgagtgcccc agccacaccc agcccctggg cgtgttcctg
Ttccccccca agcccaagga caccctgatg atcagcagaa cccccgaggt gacc

Sequence Identification Number 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

Sequence Identification Number 9
MDAMLRGLCC VLLLCGAVFV SPSHA

Sequence identification number 10
Atggacgcca tgctgagagg cctgtgctgt gtgctgctgc tgtgcggcgc cgtgttcgtg
Tcccctagcc acgcc

Sequence identification number 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

Sequence Identification Number 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 ccaggaagag
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

Sequence Identification Number 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

Sequence Identification Number 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 ccaggaacac cggcagagga ggcgaggaaa agaaaggaag caaggagaag
Gaggagcagg aggaaagaga aaccaagacc cccgagtgcc 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

Sequence identification number 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

Sequence Identification Number 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 aggacccgga cccggcatca agttcagcgc cgaggccctg
Aggtgccacc tgagagacca cgtgaacgtg agcatggtgg aagtgaccga cttccccttc
Aacaccagcg agtgggaagg ctacctgccc 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

Sequence Identification Number 17
RNTGRGGEEK KXXKEKEEQE ERETKTPECP

Sequence Identification Number 18
AQPQAEGSLA KATTAPATTR NTGRGGEEKK XXKEKEEQEE RETKTPECP

Sequence identification number 19
RNTGRGGEEK KKEKEKEEQE ERETKTPECP

Sequence identification number 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

Sequence identification number 21
VPQKPKVSLN PPWNRIFKGE NVTLTCNGNN FFEVSSTKWF HNGSLSEETN SSLNIVNAKF
EDSGEYKCQH QQVNESEPVY LEVFSDWLLL QASAEVVMEG QPLFLRCHGW RNWDVYKVIY
YKDGEALKYW YENHNISITN ATVEDSGTYY CTGKVWQLDY ESEPLNITVI KAPREKYWLQ
RNTGRGGEEK KGSKEKEEQE ERETKTPECP SHTQPLGVFL FPPKPKDTLM ISRTPEVTCV
VVDVSQEDPE VQFNWYVDGV EVHNAKTKPR EEQFNSTYRV VSVLTVLHQD WLNGKEYKCK
VSNKGLPSSI EKTISKAKGQ PREPQVYTLP PSQEEMTKNQ VSLTCLVKGF YPSDIAVEWE
SNGQPENNYK TTPPVLDSDG SFFLYSRLTV DKSRWQEGNV FSCSVMHEAL HNHYTQKSLS
LSLGK

Sequence Identification Number 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

Figure 1 shows SDS-PAGE and gel isoelectric focusing (IEF) results of proteins produced in each cell line. Here, it can be seen that no truncation type is produced under both the reducing and non-reducing conditions, and the content of the acidic protein is increased due to an increase in the lysolic acid content caused by the introduction of the sputum acid transferase gene.

Figure 2 shows the results of SDS-PAGE of the polypeptide dimeric protein in a non-reduced and reduced form according to an embodiment of the present invention. In particular, it can be seen that the polypeptide dimer has high purity even in the culture supernatant corresponding to the input.

Figure 3 shows a graph of the binding ability of omalizumab to IgE. The figure shows the results obtained by immobilizing omalizumab and analyzing its binding capacity to the treated IgE concentration.

Figure 4 depicts a graph showing the IgE binding ability of a polypeptide dimeric protein according to an embodiment of the present invention. The figure shows the results obtained by immobilizing the dimeric protein and analyzing its binding ability to the treated IgE concentration.

Figure 5 depicts the identification of polypeptide dimeric protein (IgE _TRAP ) and IgG receptor FcgRI (Figure 5A), FcgRIIA (Figure 5B), FcgRIIB of an embodiment of the invention by biofilm interference technique (BLI) analysis. (Fig. 5C), the results obtained by the interaction of FcgRIIIA (Fig. 5D) and FcgRIIIB (Fig. 5E).

Figure 6 depicts a graph obtained by quantifying the binding ability of IgE _TRAP to IgG receptor and omalizumab to IgG receptor.

Figure 7 depicts a graph showing the ability of a polypeptide dimeric protein (IgE _TRAP ) to inhibit the activity of mouse-derived mast cells according to its concentration in accordance with an embodiment of the present invention.

Figure 8 depicts a comparison of the ability of polypeptide dimeric protein (IgE _TRAP ) and Xolair (omalizumab) according to an embodiment of the present invention to inhibit the activity of mouse mast cells derived from human FceRI. Figure.

Figure 9 depicts a graph showing the effect of administration of a polypeptide dimeric protein in a food allergy model in accordance with an embodiment of the present invention.

Claims (11)

A pharmaceutical composition for treating or preventing an allergic disease, comprising a polypeptide dimer as an active ingredient, the polypeptide dimer comprising two monomers each containing a cell of a subunit of the IgE Fc receptor Outer area (FceRIa-ECD), Wherein the monomer comprises a modified Fc region, and The modified Fc region is joined to the FceRIa-ECD by a hinge. The pharmaceutical composition according to claim 1, wherein the extracellular region of the subunit of the IgE Fc receptor is the amino acid sequence of Sequence Identification No. 1 or a fragment thereof. The pharmaceutical composition of claim 1, wherein the modified Fc region is sequence identification number 2. The pharmaceutical composition of claim 1, wherein the hinge is a hinge region derived from immunoglobulin IgD or a variant thereof. The pharmaceutical composition of claim 4, wherein the hinge region derived from immunoglobulin IgD or a variant thereof contains at least one cysteine. The pharmaceutical composition according to claim 4, wherein the hinge region derived from immunoglobulin IgD or a variant thereof 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 (Serial Identification Number 17), Xaa1 is Lys or Gly, and Xaa2 is Glu, Gly or Ser. The pharmaceutical composition according to claim 4, wherein the hinge region derived from immunoglobulin IgD or a variant thereof is 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 Lys Lys Xaa3 Xaa4 Lys Glu Lys Glu Glu Gln Glu Glu Arg Glu Thr Lys Thr Pro Glu Cys Pro (Serial Identification No. 18), Xaa1 is Lys or Gly, and Xaa2 is Glu, Gly or Ser. The pharmaceutical composition of claim 1, wherein the hinge has an amino acid sequence selected from any one of sequence identification number 3, sequence identification number 4, and sequence identification number 19. The pharmaceutical composition according to claim 1, wherein the allergic disease is selected from any one of the group consisting of: food allergy, atopic dermatitis, asthma, allergic rhinitis, allergic conjunctivitis, allergic Dermatitis, chronic spontaneous urticaria and allergic contact dermatitis. A food composition for improving or alleviating allergy symptoms, comprising a polypeptide dimer as an active ingredient, the polypeptide dimer comprising two monomers each containing an extracellular unit of the IgE Fc receptor Area (FceRIa-ECD), Wherein the monomer comprises a modified Fc region, and The modified Fc region is joined to the FceRIa-ECD by a hinge. A pharmaceutical composition for treating or preventing an allergic disease, comprising a polypeptide dimer as an active ingredient, the polypeptide dimer comprising two monomers each containing a cell of a subunit of the IgE Fc receptor The outer region (FceRIa-ECD), the polypeptide dimer is obtained by: 1) cultivating a step in which a polynucleotide encoding a polypeptide monomer and a cell of a chyluate transferase gene are introduced; 2) the step of recovering the polypeptide dimer, Wherein the monomer comprises a modified Fc region, and The modified Fc region and the FceRIa-ECD are linked by a hinge.