WO2021006375A1 - Polypeptide dimer containing high amount of sialic acid and including extracellular domain of alpha subunit of ige fc receptor, and pharmaceutical composition containing same - Google Patents

Abstract

The present invention pertains to a modified IgE Fc receptor containing a high amount of sialic acid, and a pharmaceutical composition containing same. A polypeptide dimer containing a high amount of sialic acid according to the present invention not only has excellent persistence in the body and safety compared to conventional anti-IgE antibodies, but also strongly binds to IgE, and thus has the advantage of allowing the extension of dosing intervals. In addition, the polypeptide dimer containing a high amount of sialic acid according to the present invention is an IgE single target substance, and does not bind to Fc gamma receptors unlike conventional anti-IgE antibodies to which Fc of IgG1 is applied. As a result, the release of mediators due to the binding with Fc gamma receptors of the surfaces of mast cells can be inhibited, and side effects such as anaphylaxis, which can be caused by the binding of IgG1 to Fc gamma receptor III of mast cells, can thus be minimized. Therefore, the polypeptide dimer containing a high amount of sialic acid according to the present invention can be advantageously used for preventing or treating allergic diseases.

Description

Polypeptide dimer with high sialic acid content comprising the extracellular domain of the alpha subunit of the IGE FC receptor and pharmaceutical composition comprising the same

The present invention relates to a polypeptide dimer comprising the IgE Fc receptor and a high sialic acid content, and a pharmaceutical composition comprising the same.

As the modern society becomes industrialized and dietary habits become westernized, the incidence of allergic diseases such as asthma, allergic rhinitis, atopic dermatitis, and food allergy is increasing. The incidence of anaphylaxis, a severe allergic disease, is also increasing. These allergic diseases degrade the quality of life of individuals and increase economic costs, so measures to overcome them are urgently needed.

Most allergic diseases are caused by an excessive immune response of immunoglobulin E (IgE). IgE is an antibody that is present in very low concentrations in the blood under normal conditions. IgE is generally produced by innocuous antigens, and there are cases where IgE is increased without any special stimulation. In this case, allergic diseases can be caused. Abnormally increased IgE can bind to high-affinity IgE receptor (hereinafter, FcεRI) expressed on the surface of mast cells and basophils. By this binding, mast cells or basophilic granulocytes release chemical mediators such as histamine, leukotriene, prostaglandin, bradykinin, and platelet-activating factors. Allergic symptoms are caused by the released chemical mediators. In other words, allergic diseases may worsen symptoms due to the combination of IgE and FcεRI. In addition, it is known that cells expressing FcεRI are increased in patients with allergies.

Currently, methods such as allergen avoidance, anti-allergic drug administration, regulation of IgE synthesis in the body, and anti-IgE antibodies have been proposed to treat allergic diseases. However, problems such as insufficient efficacy of the drug or serious side effects remain.

Recently, a composition capable of inhibiting cells expressing IgE that binds to IgE and FcγRIIb with high affinity and which is immobilized on a membrane has been studied. Such compositions have been reported to be useful in treating disorders mediated by IgE, including allergies and asthma (KR10-1783272B1). In particular, Omalizumab (trade name: Xolair), which targets the Fc portion of IgE, has been developed and is used as a treatment for refractory severe asthma and refractory urticaria. However, in order to maintain the effect, it has to be administered at a high dose, so the cost burden is high, and it has been reported that there are side effects such as angioedema and anaphylactic reaction (The Journal of Clinical Investigation Volume 99, Number 5, March 1997, 915-925; The Journal of Clinical Investigation, Volume 99, Number 5, March 1997, 901-914). In addition, serious adverse reactions such as allergic granulomatous vasculitis and idiopathic severe thrombocytopenia have been reported after the marketing.

Accordingly, the present inventors studied to develop a safe and effective treatment for allergic diseases. As a result, when the sialic acid content of the polypeptide dimer containing two monomers (FcεRIα-ECD) containing the extracellular domain of the alpha subunit of the IgE Fc receptor is high, it was found that the polypeptide dimer has excellent binding ability with IgE. I did. In particular, the present invention was completed by confirming that effective delivery into the body is possible even when the polypeptide dimer having a high sialic acid content is administered subcutaneously.

One aspect of the present invention is a polypeptide dimer comprising two monomers comprising the extracellular domain (FcεRIα-ECD) of the alpha subunit of the IgE Fc receptor, the monomer comprising a modified Fc region, the Fc The region and FcεRIα-ECD are bound through a hinge, and the polypeptide dimer provides a polypeptide dimer, characterized in that the molar ratio of sialic acid/polypeptide dimer is 8 or more.

Another aspect of the present invention provides a pharmaceutical composition for preventing or treating allergic diseases comprising a polypeptide dimer having a high sialic acid content as an active ingredient.

Another aspect of the present invention provides a transdermal patch comprising the pharmaceutical composition.

Another aspect of the present invention provides a topical patch comprising the pharmaceutical composition.

Another aspect of the present invention provides a food composition for improving or alleviating symptoms of allergies comprising the polypeptide dimer having a high sialic acid content as an active ingredient.

Another aspect of the present invention provides a method for preventing or treating an allergic disease comprising administering to an individual a polypeptide dimer having a high sialic acid content.

Another aspect of the present invention provides a use of the polypeptide dimer having a high sialic acid content for preventing or treating allergic diseases for preventing or treating allergic diseases.

The polypeptide dimer having a high sialic acid content according to the present invention not only has superior persistence and safety in the body compared to the conventional anti-IgE antibody, but also strongly binds to IgE, so it has the advantage of extending the administration cycle. In addition, the polypeptide dimer having a high sialic acid content according to the present invention is an IgE single target material, and does not bind to the Fc gamma receptor, unlike the conventional anti-IgE antibody to which Fc of IgG1 is applied. Due to this, it is possible to inhibit the release of mediators due to the binding of the Fc gamma receptor on the mast cell surface, thereby minimizing side effects such as anaphylaxis that may be caused by the binding of IgG1 to the Fc gamma receptor III of mast cells. Therefore, the polypeptide dimer having a high sialic acid content according to the present invention can be usefully used in the prevention or treatment of allergic diseases.

1 is a diagram showing the results of SDS-PAGE of polypeptides produced in each cell line.

2 is a diagram showing the results of SDS-PAGE of the non-reduced and reduced forms of a polypeptide dimer according to an embodiment. In particular, it can be seen that the purity of the polypeptide dimer is high even in the culture supernatant corresponding to the input, and the sample obtained by passing this culture supernatant through an affinity column (FT; Flow Through) contains no polypeptide at all. It is not detected, but is detected in the eluted sample. In addition, when the eluted sample has a low pH, a large difference is not observed even when a sample (Elute_N) that is quickly neutralized with 1M Tris buffer (pH 9.0) is compared with a sample that is not (Elute).

Figure 3 is a diagram showing the binding ability of omalizumab to IgE.

4 is a diagram showing the binding ability of a polypeptide dimer (IgE _TRAP ) to IgE according to an embodiment.

Figure 5a is a diagram showing the binding ability of a specific example of a polypeptide dimer (IgE _TRAP ) or omalizumab and IgG Fc gamma receptor I (FcγRI).

Figure 5b is a diagram showing the binding ability of a specific example of the polypeptide dimer (IgE _TRAP ) or omalizumab and IgG Fc gamma receptor IIA (FcγRIIA).

Figure 5c is a diagram showing the binding ability of a specific example of the polypeptide dimer (IgE _TRAP ) or omalizumab and IgG Fc gamma receptor IIB (FcγRIIB).

Figure 5d is a diagram showing the binding ability of a specific example of the polypeptide dimer (IgE _TRAP ) or omalizumab and IgG Fc gamma receptor IIIA (FcγRIIIA).

Figure 5e is a diagram showing the binding ability of the Fc gamma receptor IIIB (FcγRIIIB) of a specific example polypeptide dimer (IgE _TRAP ) or omalizumab and IgG.

FIG. 6 is a graph showing the quantification of the binding power of an exemplary polypeptide dimer (IgE _TRAP ) or omalizumab to the Fc gamma receptors of IgG.

7 is a diagram showing the degree of binding to IgE according to the concentration of the polypeptide dimer according to an embodiment.

FIG. 8 is a view comparing the activity inhibitory ability of mast cells derived from human FcεRI-expressing mice according to the concentration of the polypeptide dimer protein (IgE _TRAP ) and Xolere (omalizumab) according to an embodiment.

9 is a view confirming the anti-allergic effect of the polypeptide dimer according to an embodiment through the measurement of the incidence of diarrhea in a food allergy model.

10 is a view showing the results of SDS-PAGE of the non-reduced and reduced forms of the polypeptide dimers (SA ^low , SA ^medi and SA ^high ) according to an embodiment.

11 is a diagram illustrating the isoelectric point of a polypeptide dimer (SA ^low , SA ^medi and SA ^high ) according to an embodiment.

12 is a view confirming the anti-allergic effect according to subcutaneous injection of the polypeptide dimer (SA ^low and SA ^high ) of one embodiment through the measurement of the incidence of diarrhea in a food allergy model.

13 is a view confirming the anti-allergic effect of the polypeptide dimers (SA ^low and SA ^high ) according to an embodiment through the measurement of blood IgE concentration in a food allergy model.

14 is a view confirming the anti-allergic effect of the polypeptide dimers (SA ^low and SA ^high ) according to an embodiment through the measurement of blood MCPT-1 concentration in a food allergy model.

One aspect of the present invention is a polypeptide dimer comprising two monomers containing the extracellular domain (FcεRIa-ECD) of the alpha subunit of the IgE Fc receptor, the monomer comprising an Fc region, the Fc region and FcεRIa-ECD is bound through a hinge, and the polypeptide dimer provides a polypeptide dimer having a high sialic acid content, characterized in that the molar ratio of sialic acid/polypeptide dimer is 8 or more.

The term "IgE" used in the present invention refers to an antibody known as immunoglobulin E. IgE has affinity for mast cells and basophils. In addition, when IgE and its corresponding antigen (allergen) react, it causes an inflammatory reaction. In addition, IgE is known to be the main cause of anaphylaxis.

The term "IgE Fc receptor" used in the present invention is also called an Fcε receptor, and refers to a receptor that binds to the Fc portion of IgE. There are two types of receptors. Receptors having high affinity for Fc of IgE are called Fcε receptor I (FcεRI). Receptors with low affinity for IgE Fc are referred to as Fcε receptor II (FcεRII). FcεRI is expressed in mast cells and basophils. When IgE antibodies bound to FcεRI are crosslinked by multivalent antigens, degranulation occurs in mast cells or basophils, releasing various chemicals such as histamine. This release causes an immediate allergic reaction.

The FcεRI is a membrane protein composed of one α-chain, one β-chain, and two γ-chains bonded by disulfide bonds. Among them, the part to which IgE binds is an α chain (FcεRIα), and FcεRIα has a size of about 60 kDa, and is composed of a hydrophobic domain present in the cell membrane and a hydrophilic domain present outside the cell membrane. In particular, IgE binds to the extracellular domain of the α chain. The FcεRIα may be used interchangeably as an alpha subunit of the IgE Fc receptor.

Specifically, the alpha subunit of the IgE Fc receptor may consist of the amino acid sequence described in NP_001992.1. In addition, the extracellular domain (FcεRIa-ECD) of the alpha subunit of the IgE Fc receptor may be composed of the amino acid sequence of SEQ ID NO: 1. The FcεRIa-ECD may be a fragment of FcεRIa-ECD or a variant thereof, as long as it can bind to IgE. In addition, FcεRIa-ECD of SEQ ID NO: 1 may be encoded by a polynucleotide represented by SEQ ID NO: 5.

The variant may be performed through a method of substituting, deleting or adding one or more amino acids in wild-type FcεRIa-ECD, as long as the function of FcεRIa-ECD is not modified. The variant may have 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more homology with the amino acid sequence of SEQ ID NO: 1.

In this case, the Fc region may be a wild-type Fc region or a modified Fc region. In addition, the term "modified Fc region" used in the present invention refers to a region in which part of the Fc portion of an antibody has been modified. In this case, the Fc region includes a heavy chain constant region 2 (CH2) and a heavy chain constant region 3 (CH3) of an immunoglobulin, and does not include the variable regions of the heavy and light chain and the light chain constant region 1 (CH1) of the immunoglobulin. Say. In particular, the modified Fc region may be prepared by substituting some amino acids in the Fc region or combining different types of Fc regions. Specifically, the modified Fc region may be composed of 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 represented by SEQ ID NO: 6.

In addition, the modified Fc region may be a natural sugar chain or an increased sugar chain compared to the natural type. The immunoglobulin Fc sugar chain can be modified by conventional methods such as chemical methods, enzymatic methods, and genetic engineering methods using microorganisms. In addition, the modified Fc region does not have a binding site for the Fc gamma receptor (FcγR) or C1q (complement component 1q), and thus may be lacking the functions of ADCC (antibody dependent cellular cytotoxicity) and CDC (Complement dependent Cytotoxicity). .

In one embodiment, the modified Fc region may be coupled through the hinge of FcεRIα-ECD and IgD. The hinge may be a hinge region derived from immunoglobulin IgD or a variant thereof. The hinge region of native IgD consists of 64 amino acids. The hinge region derived from the immunoglobulin IgD or a variant thereof may be composed of 20 to 60 consecutive amino acids, 25 to 50 consecutive amino acids, or 30 to 40 amino acids. In this case, the hinge variant may be a partial modification of the amino acid sequence of the hinge region of IgD in order to minimize the occurrence of a truncated form during protein production.

In one embodiment, the hinge region derived from the immunoglobulin IgD or a variant thereof may be composed of 30 or 49 amino acids. The hinge region derived from the immunoglobulin IgD or a variant thereof may include at least one cysteine.

In one embodiment, the hinge region or a variant thereof derived from the immunoglobulin IgD may include 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 is Glu , Gly or Ser. Specifically, the hinge region derived from the immunoglobulin IgD or a variant thereof may have an amino acid sequence of SEQ ID NO: 3 and SEQ ID NO: 19, thereby minimizing the occurrence of a truncated form during protein production.

In addition, in another embodiment, the hinge region or a variant thereof derived from the immunoglobulin IgD may include the following sequence:

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 ( SEQ ID NO: 18), in which case Xaa3 is Lys or Gly, and Xaa4 may be Glu, Gly or Ser. Specifically, the hinge region derived from the immunoglobulin IgD or a variant thereof may have the amino acid sequence of SEQ ID NO: 4, thereby minimizing the occurrence of a truncated form during protein production.

In particular, in the hinge region derived from the immunoglobulin IgD consisting of the amino acid represented by SEQ ID NO: 4 or a variant thereof, at least one of threonine (Thr) may be glycosylation. Specifically, threonine of the 13th, 14th, 18th and 19th amino acid sequence represented by SEQ ID NO: 18 may be glycosylated. Preferably, all four threonines can be glycosylated. At this time, the saccharification may be O-glycosylation.

The term "sialic acid" used in the present invention may include N-acetylneuraminic acid (Neu5Ac) of Formula 1 and N-glycorylneuraminic acid (Neu5Gc) of Formula 2:

[Formula 1]

[Formula 2]

In this case, in one embodiment, the polypeptide dimer having a high sialic acid content may have a high content of N-acetylneuraminic acid.

In addition, the sialic acid content of the polypeptide dimer can be increased through a purification method. In addition, by producing a polypeptide dimer in a cell into which a sialic acid transferase gene has been introduced, the sialic acid content of the polypeptide dimer can be increased.

The polypeptide dimer having a high sialic acid content may be characterized in that the molar ratio of sialic acid/polypeptide dimer is 8 or more. For example, the sialic acid-rich polypeptide dimer has a sialic acid/polypeptide dimer molar ratio of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, It may be 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30. In one embodiment, the polypeptide dimer having a high sialic acid content may be characterized in that the molar ratio of the sialic acid/polypeptide dimer is 8 to 30 or 12 to 25. Specifically, the polypeptide dimer having a high sialic acid content may be characterized in that the molar ratio of sialic acid/polypeptide dimer is 19 to 22. Further, the polypeptide dimer having a high sialic acid content may be characterized in that the molar ratio of sialic acid/polypeptide dimer is 19 to 20. In this case, the sialic acid may be N-acetylneuraminic acid.

In addition, the sialic acid may bind at least 8 positions to a monomer including an extracellular domain (FcεRIa-ECD) and an Fc region of the alpha subunit of the IgE Fc receptor. At this time, sialic acid may be bonded to at least four or more of the positions. Specifically, the sialic acid may be bonded to the monomer at 4, 5, 6, 7 or 8 positions.

As described above, the polypeptide dimer having a high sialic acid content provided by the present invention may be in a form in which two monomers to which an extracellular domain of the alpha subunit of the IgE Fc receptor and a modified Fc region are bound are combined. The polypeptide dimer having a high sialic acid content may be a form in which two identical monomers are bonded by a cysteine located at a hinge site. In addition, the polypeptide dimer having a high sialic acid content may be in a form in which two different monomers are combined.

In this case, each of the polypeptide monomers may include sialic acid at the same position, but may include sialic acid at different positions. In addition, when the molar ratio of the sialic acid/polypeptide dimer is 8 or more, each of the monomers constituting the dimer may have a different molar ratio of sialic acid/polypeptide.

In addition, the polypeptide dimer may contain two different monomers. Specifically, one monomer may include the extracellular domain of the alpha subunit of the IgE Fc receptor, and the other monomer may be a form including a fragment of the extracellular domain of the alpha subunit of the IgE Fc receptor. In this case, one specific example of the monomer may be composed of an amino acid sequence represented by SEQ ID NO: 20, SEQ ID NO: 21, or SEQ ID NO: 22.

In addition, the polypeptide dimer having a high sialic acid content provided by the present invention is 10 to 100 times, 20 to 90 times, 20 to 70 times, 30 to 70 times or 40 to 70 times higher IgE than omalizumab, an anti-IgE antibody. Represents binding power, and preferably can exhibit IgE binding power 70 times higher than that of omalizumab.

Another aspect of the present invention provides a pharmaceutical composition for preventing or treating allergic diseases comprising a polypeptide dimer having a high sialic acid content as an active ingredient.

The polypeptide dimer is the same as described above. In particular, the pharmaceutical composition may be characterized in that it is for subcutaneous injection. In the case of a polypeptide dimer having a high sialic acid content, it was confirmed that it is effective in the treatment and/or prevention of allergic diseases compared to a polypeptide dimer having a low sialic acid content upon subcutaneous injection.

The term "allergic disease" used in the present invention refers to a pathological condition caused by an allergic reaction mediated by the activation of mast cells, such as degranulation of mast cells. For example, the allergic disease is food allergy, atopic dermatitis, asthma, allergic rhinitis, allergic conjunctivitis, allergic dermatitis, chronic idiopathic urticaria. (Chronic idiopathic urticarial), and allergic contact dermatitis (allergic contact dermatitis) may be one selected from the group consisting of, but is not limited thereto. In particular, the allergic disease may be a disease mediated by IgE.

The active ingredient polypeptide dimer in the pharmaceutical composition may be included in an arbitrary amount (effective amount) depending on the use, formulation, purpose of combination, etc., as long as it can exhibit anti-allergic activity, and a typical effective amount is based on the total weight of the composition. When it can be determined in the range of 0.001% by weight to 20.0% by weight. The effective amount means an amount capable of inducing an anti-allergic effect. Such an effective amount can be determined by one of skill in the art.

The pharmaceutical composition may additionally include a pharmaceutically acceptable carrier. Specifically, the pharmaceutical composition may be prepared in an oral dosage form or a parenteral dosage form according to an administration route by a conventional method known in the art, including a pharmaceutically acceptable carrier in addition to the active ingredient.

When the pharmaceutical composition is prepared in an oral dosage form, powders, granules, tablets, pills, dragees, capsules, solutions, gels, syrups, suspensions, wafers, etc., according to a method known in the art together with a suitable carrier. It can be prepared in a formulation. At this time, examples of suitable pharmaceutically acceptable carriers include sugars such as lactose, glucose, sucrose, dextrose, sorbitol, mannitol, and xylitol, corn starch, potato starch, wheat starch, and other starches, cellulose, methylcellulose, and ethylcellulose. , Celluloses such as sodium carboxymethylcellulose, hydroxypropylmethylcellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, magnesium stearate, mineral oil, malt, gelatin, talc, polyol, Vegetable oils and the like. In the case of formulation, if necessary, it may include diluents and/or excipients such as fillers, extenders, binders, wetting agents, disintegrants, and surfactants.

When the pharmaceutical composition is prepared in a parenteral formulation, it may be formulated in the form of an injection, transdermal administration, topical administration, nasal inhalation and suppository according to a method known in the art together with a suitable carrier. When formulated as an injection, sterile water, ethanol, polyols such as glycerol or propylene glycol, or mixtures thereof may be used as suitable carriers, preferably Ringer's solution, PBS (phosphate buffered saline) containing triethanol amine, or sterilization for injection Water, isotonic solutions such as 5% dextrose can be used.

The formulation of the pharmaceutical composition is known in the art, and specifically, Remington's Pharmaceutical Sciences (19th ed., 1995) may be referred to.

The preferred dosage of the pharmaceutical composition is in the range of 0.01 µg/kg to 10 g/kg per day, preferably 0.01 mg/kg to 1 g, depending on the patient's condition, weight, sex, age, patient severity, and route of administration. May be in the range of /kg. The pharmaceutical composition may be administered once a day or several times a day if the dosage is large.

Subjects to which the pharmaceutical composition can be applied (prescribed) are mammals and humans, particularly preferably humans. In addition to the active ingredient, the pharmaceutical composition may further include any known compounds or natural extracts that have already been verified for safety and have anti-allergic activity in order to increase or reinforce anti-allergic activity.

As a result of researching to develop a safe and effective therapeutic agent for allergic diseases, the present inventors confirmed that a polypeptide dimer having a high sialic acid content has better binding ability with IgE than a polypeptide dimer having a low sialic acid content (Table 3). In addition, when a polypeptide dimer having a high sialic acid content is administered subcutaneously to an individual suffering from an allergic disease, it effectively reduces the concentration of IgE and MCPT-1 in the blood than a polypeptide dimer having a low sialic acid content, and a predetermined time has passed. It was confirmed that the effect persisted even after (FIGS. 13 and 14 ).

Another aspect of the present invention is a monomer comprising an extracellular domain (FcεRIa-ECD) of the alpha subunit of the IgE Fc receptor, the monomer comprising an Fc region, and the Fc region and FcεRIa-ECD are bound through a hinge In addition, the monomer provides a polypeptide monomer having a high sialic acid content, characterized in that the molar ratio of sialic acid/polypeptide monomer is 4 or more. At this time, the FcεRIa-ECD, the Fc region and the hinge are as described above. In addition, the molar ratio of the sialic acid/polypeptide monomer of the monomer may be 4, 5, 6, 7, 8, 9, 10 or 11.

Accordingly, another aspect of the present invention provides a transdermal patch comprising the pharmaceutical composition. In addition, another aspect of the present invention provides a topical patch comprising a pharmaceutical composition.

Another aspect of the present invention provides a food composition for improving or alleviating symptoms of allergies comprising the polypeptide dimer having a high sialic acid content as an active ingredient.

The polypeptide dimer having a high sialic acid content is the same as described above. In addition, the sialic acid-rich polypeptide dimer can be combined with an appropriate delivery means for efficient delivery into the intestine.

In addition, the food composition may be prepared in any form, such as beverages such as tea, juice, carbonated beverages, ion beverages, processed oils such as milk, yogurt, tablets, capsules, pills, granules, liquids, powders, It can be prepared as a health functional food preparation such as flannel, paste, syrup, gel, jelly, bar, etc.

The food composition may be classified as an arbitrary product as long as it conforms to the enforcement regulations at the time of manufacture and distribution in terms of legal and functional classification. For example, it may be a health functional food according to the Health Functional Food Act, or confectionery, beans, tea, beverages, special purpose foods, etc., according to each food type according to the food code of the Food Sanitation Act (notified by the Ministry of Food and Drug Safety, food standards and standards). .

Regarding other food additives that may be included in the food composition, reference may be made to the food code or food additive code according to the Food Sanitation Act.

Another aspect of the present invention provides a method for preventing or treating an allergic disease comprising administering to an individual a polypeptide dimer having a high sialic acid content. The polypeptide dimer having a high sialic acid content is the same as described above.

The individual may be a mammal, preferably a human. In this case, administration may be administered orally or parenterally. In this case, parenteral administration may be performed by subcutaneous administration, intravenous administration, mucosal administration, and intramuscular administration.

The allergic diseases are food allergy, atopic dermatitis, asthma, allergic rhinitis, allergic conjunctivitis, allergic dermatitis, chronic idiopathic urticaria (Chronic idiopathic urticaria). ), and allergic contact dermatitis may be one selected from the group consisting of.

Another aspect of the present invention provides the use of the sialic acid-rich polypeptide dimer for preventing or treating allergic diseases.

Hereinafter, the present invention will be described in more detail by the following examples. However, the following examples are for illustrative purposes only, and the scope of the present invention is not limited thereto.

Example 1. Preparation of a polypeptide containing FcεRIα-ECD and an Fc region

A polypeptide having a modified C-terminus of the extracellular domain of the alpha subunit of the IgE Fc receptor (FcεRIα ECD) was prepared according to the method disclosed in US Pat. No. 7,867,491.

First, the extracellular domain of the α chain of FcεRI having the amino acid sequence of SEQ ID NO: 1 and the modified immunoglobulin Fc of SEQ ID NO: 2 are respectively linked by a linker of SEQ ID NO: 19 (FcεRIαECD-Fc1), a linker of SEQ ID NO: 3 In order to express the protein (FcεRIαECD-Fc2) and the linker-linked protein of SEQ ID NO: 4 (FcεR1αECD-Fc3), a cassette connecting the gene encoding each protein was cloned into the pAD15 vector (Genexine), and FcεRIαECD -Fc protein expression vector was constructed. Then, the expression vector was transfected into CHO DG44 (from Dr. Chasin, Columbia University, USA) cells, respectively.

At this time, when transfecting the cell line, the expression vector in which the α-2,6-sialic acid transferase gene was cloned was simultaneously transfected into the pCI Hygro vector (Invitrogen) to express the sialic acid-added FcεRIαECD-Fc2ST and FcεRIαECD-Fc3ST proteins. A capable cell line was prepared separately.

As a first screening process, 10% dFBS (Gibco, USA, 30067-334) without HT (5-hydroxytrypamine), MEMα (Gibco, 12561, USA, Cat No. 12561-049), HT+ (Gibco, USA, 11067- 030)) HT selection was performed using the medium. Thereafter, in order to amplify productivity using the DHFR (dihydrofolate reductase)-system, methotrexat (MTX) amplification was performed using HT-selected clones.

After MTX amplification was completed, passage culture was performed about 1 to 5 times to stabilize cells for productivity evaluation. Thereafter, the unit productivity evaluation of the MTX amplified cells was performed, and the results are shown in Table 1 below.

Version	Media	MTX conc.	Productivity
			3-day culture		Batch culture (mg/ml)
			Μg/ml	㎍/10 6 cells
FcεRIαECD-Fc2	Ex-cellDHFR	500nM	37.23	20.9	225
FcεRIαECD-Fc2+a2,6-ST		100nM	45.4	25.1	338.2
FcεRIαECD-Fc3		2uM	27.0	16.9	180.4
FcεRIαECD-Fc3+a2,6-ST		1uM	17.5	10.2	101.7

As described in Table 1, the FcεRIαECD-Fc3 cell line exhibited 16.9 μg/10 ⁶ cell productivity after 2 μM methotrexat amplification. On the other hand, the FcεRIαECD-Fc3 cell line (FcεRIαECD-Fc3ST) co-transfected with 2,6-sialic acid transferase showed 17.5 μg/10 ⁶ cells productivity after 1 μM methotrexat amplification. In addition, the FcεRIαECD-Fc2 cell line showed a productivity of 20.9 μg/10 ⁶ cells under 0.5 μM methotrexat amplification conditions. In addition, the FcεRIαECD-Fc2 cell line (FcεRIαECD-Fc2ST) co-transfected with 2,6-sialic acid transferase showed a productivity of 25.1 μg/10 ⁶ cells after 0.1 μM methotrexat amplification. That is, it was confirmed that the FcεRIαECD-Fc2ST cell line co-transfected with 2,6-sialic acid transferase selected under 0.1 μM methotrexat amplification conditions had the best productivity.

The polypeptide produced from the FcεRIαECD-Fc2 cell line (FcεRIαECD-Fc) was named "FcεRIαECD-Fc2", and the polypeptide produced from the FcεRIαECD-Fc2+a2,6-ST cell line was named "FcεRIαECD-Fc2ST". In addition, the polypeptide produced from the FcεRIαECD-Fc3 cell line was named "FcεRIαECD-Fc3", and the polypeptide produced from the FcεRIαECD-Fc3+a2,6-ST cell line was named "FcεRIαECD-Fc3ST".

Example 2. Purification and purity confirmation of polypeptide (FcεRIα ECD-Fc)

Among the cell lines selected in Example 1, i) the FcεRIαECD-Fc3 cell line, ii) the FcεRIαECD-Fc3+a2,6-ST cell line, iii) the FcεRIαECD-Fc2+a2,6-ST cell line was batch-cultured in a 60 ml medium volume (batch culture). After purifying FcεRIα ECD-Fc using a Protein-A affinity column of the culture medium, the purified FcεRIα ECD-Fc was subjected to size-exclusion high performance liquid chromatography (SE-HPLC) and SDS-PAGE to obtain the purity of the polypeptide. Was confirmed.

Specifically, SDS-PAGE was performed under non-reducing conditions. The non-reducing conditions are, after mixing each purified polypeptide with a non-reducing sample buffer, in Mini-Protean TGX ^TM gels (Bio-Rad) TGS (Tris Glycine SDS) buffer, under 200V conditions Electrophoresis was performed for 30 minutes. After electrophoresis, the protein was stained with Coomassie Brilliant Blue solution. The results are shown in Table 2 and FIG. 1.

Lane #	Sample	Purification	Purity(SE-HPLC)	Sample condition
M	Protein standard	One-step(Protein-A affinity column) Purification	-	-	-
One	FcεRIαECD-Fc3		94.5%	-	Non-reducing
2	FcεRIαECD-Fc3ST		93.7%
3	FcεRIαECD-Fc2ST		93.2%
4	FcεRIαECD-Fc3		94.5%	Freezing/Thawingtest
5	FcεRIαECD-Fc3ST		93.7%
6	FcεRIαECD-Fc2ST		93.2%

As shown in Table 2, it was confirmed that the purity of each polypeptide purified by the SE-HPLC method was 93% or more. In the non-reducing condition, impurities such as cut form did not appear, and in particular, it was confirmed that the purity was more than 93% even after the process of thawing/freezing, and no impurities were present. .

Example 3. Confirmation of Dimer Formation of Polypeptide (FcεRIα ECD-Fc)

Among the cell lines selected in Example 1, i) the FcεRIαECD-Fc3 cell line, ii) the FcεRIαECD-Fc3+a2,6-ST cell line, iii) the FcεRIαECD-Fc2+a2,6-ST cell line was batch-cultured in a 60 ml medium volume I did. Thereafter, the purified product obtained by purifying the polypeptide using the culture supernatant and the Protein-A affinity column was subjected to SE-HPLC and SDS-PAGE. At this time, the culture supernatant and the purified product were performed under non-reducing conditions and reducing conditions, respectively.

The non-reduction conditions were carried out in the same manner as in Example 2. On the other hand, in reducing conditions, each purified polypeptide was mixed with a reducing sample buffer containing 2-mercaptoethanol, and then denatured for 5 minutes at 100°C. . Thereafter, electrophoresis was performed on Mini-Protean TGX ^TM gels (Bio-Rad) for 30 minutes at 200V using a TGS buffer. After electrophoresis, the protein was stained with Coomassie Brilliant Blue solution.

As a result, a polypeptide having a size of about 150 kDa was detected under non-reducing conditions, and a polypeptide having a size of about 75 kDa was detected under reducing conditions. Through this, it was confirmed that the polypeptide forms a dimer (Fig. 2). In addition, it was confirmed that not only the polypeptide of very high purity (98% or more) was purified from the purified product, but also the polypeptide was expressed with very high purity in the culture supernatant. This means that in developing the polypeptides expressed in the cell lines as pharmaceuticals, the process development step can be simplified, and as a result, there is a very high possibility that the development cost as a pharmaceutical can be significantly reduced.

Example 4. Confirmation of IgE binding ability of polypeptide dimer

The polypeptides of i) FcεRIαECD-Fc2, ii) FcεRIαECD-Fc2ST, iii) FcεRIαECD-Fc3 and iv) FcεRIαECD-Fc3ST produced in Example 1 were purified by the same method as in Example 2 and are commercially available. IgE binding strength was compared and measured with respect to the anti-IgE antibody, Omalizumab (trade name: Xolair).

Specifically, IgE binding ability is determined by coating IgE on the channel of the Protein GLC sensor chip (Bio-Rad, Cat #. 176-5011), and omalizumab or each FceR1αECD-Fc protein at various concentrations at a rate of 30 μl/min. I spilled it. At this time, after confirming the zero-base using a sodium hydroxide solution having a concentration of 25 mM as a regeneration buffer, the above steps were repeated to measure. Thereafter, a binding curve was confirmed using a protein binding analyzer (Proteon XPR36, BIO-RAD, USA), and the results are shown in Table 3.

SamplesItems		FcεRIa ECD-Fc		Omalizumab	Remark
Drug type		Fc fusion protein		Anti-IgE Ab
Bindingaffinity	ka (Association rate)	Fc2	2.14 x 10 5	4.05 x 10 5	1.9 times weaker than omalizumab
		Fc2ST	2.64 x 10 5		1.5 times weaker than omalizumab
		Fc3	1.98 x 10 5		2.0 times weaker than omalizumab
		Fc3ST	2.40 x 10 5		1.7 times weaker than omalizumab
	kd (Dissociation rate)	Fc2	8.29 x 10 -5	6.02 x 10 -3	73 times better than omalizumab
		Fc2ST	5.69 x 10 -5		106 times better than omalizumab
		Fc3	1.33 x 10 -4		45 times better than omalizumab
		Fc3ST	1.49 x 10 -4		40 times better than omalizumab
	KD (kd/ka)	Fc2	3.88 x 10 -10	1.49 x 10 -8	38 times better than omalizumab
		Fc2ST	2.16 x 10 -10		69 times better than omalizumab
		Fc3	6.72 x 10 -10		22 times better than omalizumab
		Fc3ST	6.21 x 10 -10		24 times better than omalizumab

As shown in Table 3, the association rate (ka) value of the polypeptide dimer and IgE according to an embodiment of the present invention was measured to be 1.5 to 2.0 times smaller than that of omalizumab. That is, it was found that the binding force with substances other than IgE was 1.5 to 2.0 times lower than that of omalizumab. In addition, the dissociation rate (kd) value of the polypeptide dimer according to an embodiment of the present invention was measured to be 40 to 106 times greater than that of omalizumab.

As a result, it can be seen that the value of the equilibrium dissociation constant (KD <kd/ka>) of the polypeptide dimer according to an embodiment of the present invention is 22 to 69 times higher than that of omalizumab. Through this, it can be seen that the polypeptide dimer according to an embodiment of the present invention has a remarkably increased binding capacity to IgE compared to omalizumab. In particular, it was confirmed that the polypeptide dimer to which sialic acid was added (FcεRIαECD-Fc2ST) had the best IgE binding ability, 69 times as compared to omalizumab.

In addition, as shown in Figures 3 and 4, omalizumab is degraded after a certain time after binding to IgE, whereas the polypeptide dimer (FcεRIαECD-Fc2ST, IgE _TRAP ) according to an embodiment of the present invention is once IgE After combining with, it was confirmed that it did not separate from IgE. That is, it was confirmed that the polypeptide dimer according to an embodiment of the present invention was not easily separated from IgE, and the ability to remain in a bound state was superior to omalizumab.

Example 5. Confirmation of binding ability of polypeptide dimer and IgG receptor

The ability of the polypeptide dimer (FcεRIαECD-Fc2ST, IgE _TRAP ) or omalizumab (Xolair) to bind to the Fc gamma receptor of the polypeptide according to an embodiment of the present invention was measured using the Octet RED384 system (Pall ForteBio, CA, USA) And confirmed.

Specifically, Fc gamma receptors FcγRI, FcγRIIA, FcγRIIB, FcγRIIIA or FcγRIIIB recombinant protein (R & D system, 5 μg/ml) were immobilized in 300 mM acetate buffer (pH 5) on the activated AR2G biosensor. At this time, PBS containing 0.1% Tween-20 and 1% bovine serum (BS) was used as a running buffer. All measurements were performed at a temperature of 30° C. at a speed of 1,000 rpm on a sample plate shaker. The measurement results are shown in FIGS. 5A to 5E, and the binding ability of omalizumab and the polypeptide dimer to the IgG Fc gamma receptor is quantified and shown in FIG. 6.

As a result, it was confirmed that omalizumab showed a high binding ability to the Fc gamma receptor of IgG, whereas the polypeptide dimer had a significantly low binding ability to the Fc gamma receptor of IgG. Through this, it was confirmed that the polypeptide dimer did not bind to the Fc gamma receptor of IgG.

Example 6. Confirmation of the activity of a polypeptide dimer through beta-hexosaminidase assay in mouse bone marrow-derived mast cells

In order to analyze the in vitro activity of the polypeptide dimers according to an embodiment of the present invention, beta-hexosaminidase analysis was performed.

Specifically, in one embodiment of the present invention, a polypeptide dimer (FcεRIαECD-Fc2) was mixed with IgE (1 μg/ml) by concentration, and incubated at a temperature of 20° C. for 30 minutes to prepare a sample. In addition, the cultured mouse bone marrow-derived mast cells for mast cell activation were washed with HBSS (Hank's balanced salt solution) buffer to remove the medium. After measuring the number of cells, 5x10 ⁵ cells were removed in 40 μl Resuspended in HBSS buffer.

50 µl of the prepared sample was added to the activated mast cells. Then, incubation was performed for 30 minutes in a 5% CO ₂ incubator for 30 minutes at a temperature of 37°C. Thereafter, 10 µl of a foreign antigen (DNP (2,4-dinitrophenol), 100 ng/ml) was added each, followed by incubation at 37° C. for 30 minutes 5% CO ₂ , and then 30 µl of the supernatant was separated.

30 µl of the separated supernatant and 30 µl of the substrate (4-Nitrophenyl N-acetyl-β-D-glucosaminide, 5.84 mM) were well mixed, and incubated at 37° C. for 20 minutes and 5% CO ₂ . Then, 140 µl of a 0.1 M sodium carbonate buffer (pH 10), which is a stop solution, was added to terminate the reaction. Thereafter, absorbance was measured at a wavelength of 405 nm to confirm the secretion amount of beta-hexosaminidase secreted by foreign antigens in activated mast cells, and the results are shown in FIG. 7.

As shown in FIG. 7, it was confirmed that the activity of IgE was greatly inhibited from the case where the concentration of IgE and the polypeptide dimer of the present invention was 1:1. That is, it was confirmed that the polypeptide dimer of one embodiment of the present invention reacts even when there is the same concentration as IgE.

Example 7. Comparison of the activity of a polypeptide dimer and a human anti-IgE antibody using beta-hexosaminidase assay in mast cells derived from human FcεRI mouse bone marrow

In order to compare the activity of the polypeptide dimer (FcεRIαECD-Fc2ST, IgE _TRAP ) and the human anti-IgE antibody Xolair (omalizumab) according to an embodiment of the present invention, beta-nuclear sosaminidase analysis was performed. .

Each of the polypeptide dimers and Xolere were prepared by concentration, mixed with human IgE (1 µg/ml), and incubated at room temperature for 30 minutes to prepare a sample. In addition, mast cells derived and differentiated from the bone marrow of a mouse into which the human FcεRI gene was introduced and the mouse FcεRI gene was removed were prepared. After the prepared mast cells were washed with HBSS buffer, ⁵ ×10 ⁵ cells were resuspended in 60 μl of HBSS buffer.

Thereafter, 20 µl of the prepared sample was added to the mast cells, and then incubated in a 5% CO ₂ incubator at 37°C for 30 minutes. Thereafter, 20 µl of a human anti-IgE antibody (Biolegend, Cat No. 325502, 0.5 µg/ml) was added, and then incubated in a 5% CO ₂ incubator at 37°C for 30 minutes. After centrifugation at 1,500 rpm and 4° C. temperature conditions, 30 μl of the supernatant was separated. 30 µl of the separated supernatant and 30 µl of the substrate (4-Nitrophenyl N-acetyl-β-glucosaminide, 5.84 mM) were well mixed and incubated in a 5% CO ₂ incubator at 37°C for 25 minutes. Thereafter, 140 µl of 0.1 M sodium carbonate buffer (pH10) was added to terminate the reaction. Thereafter, absorbance was measured at a wavelength of 405 nm, and the relative amount of secreted beta-hexosaminidase was compared to confirm the mast cell inhibitory effect according to the concentration of each sample. The results are shown in FIG. 8.

As shown in Figure 8, the IC ₅₀ of the polypeptide dimer was measured to be approximately 11.16 ng/ml, and the IC ₅₀ of Xolair was measured to be approximately 649.8 ng/ml. Therefore, it was confirmed that the polypeptide dimer has a mast cell activity inhibitory ability that is 58 times higher than that of Xolair.

Example 8. In a food allergy model in vivo Confirmation of the activity of the polypeptide dimer through assay

To Balb/c mice (Orientbio), sensitization was induced by intraperitoneal administration of 50 µg of OVA (ovalbumin) and 1 mg of Alum at 14-day intervals. Thereafter, on days 28, 30, 32, 34, and 36, OVA 50 mg was orally administered over a total of 5 times to induce food allergy in the intestine.

After oral administration of the OVA twice, the mice were classified into three groups of 7 mice each on the 31st day. The first group, a group administered with a high concentration (200 μg) of the polypeptide dimer (FcεRIαECD-Fc2ST), a second group, a group administered with a low concentration (20 μg), and a third group, a group not administered were classified.

While the OVA was administered orally, it was confirmed whether diarrhea occurred according to the induction of food allergy. In addition, the mice were sacrificed on day 37 to analyze the number of mast cells in the small intestine, the concentration of IgE in the blood, and the concentration of degranulation enzymes (MCPT-1, Mast cell protease-1) of the mast cells in the blood for mice belonging to each group.

As a result, as shown in FIG. 9, diarrhea occurred in the mice of the third group after oral administration of the second OVA. On the other hand, the mice of the first and second groups developed diarrhea after oral administration of the third OVA. In particular, the mice of the first group had a lower incidence of diarrhea than the mice of the second group, and through this, it was confirmed that the food allergic effect increased in proportion to the concentration of the polypeptide dimer.

Example 9. Analysis of Sialic Acid Content of Polypeptide Dimer

Prepared in Example 1 above The production rate of the polypeptide produced from the FcεRIαECD-Fc2+a2,6-ST cell line is the highest, and in Examples 6 to 8, the anti-allergic effect of the polypeptide dimer (FcεRIαECD-Fc2ST) is excellent. The sialic acid content of the polypeptide dimer (FcεRIαECD-Fc2ST) was analyzed to investigate the anti-allergic efficacy of the polypeptide dimer.

Specifically, prepared in Example 1 In order to measure the sialic acid content contained in the glycan structure of the polypeptide dimer (FcεRIαECD-Fc2ST) produced from the FcεRIαECD-Fc2+a2,6-ST cell line, first, sialidase, a sialic acid-degrading enzyme. ) To separate sialic acid. Then, the separated sialic acid was separated, detected, and quantified using HPLC (waters, alliance e2659).

The polypeptide dimer was separated and purified by dividing into three samples according to the pH gradient. Three samples were placed in an AmiconUltra 10K (Millipore, UFC501096) filter, centrifuged for 10 minutes under conditions of 13,000 rpm and 4°C, and repeated 5 times to exchange the concentrate with deionized water and concentrated. The concentration of the sample was 10 mg/ml or more when measured at a wavelength of 280 nm.

Thereafter, 0.5 mg of each sample was taken and placed in an EP tube, 1 μl of Cialidate (Roche, 10 269 611 001) and 40 μl of a 10 mM sodium phosphate buffer (pH 7.0) were added. Deionized water was added so that the final volume became 100 μl. 2 µl of each sample was taken and the concentration was measured at a wavelength of 280 nm, and the confirmed concentration value was used as the final analysis concentration.

The sample was reacted in an incubator at 37° C. for 18 hours, then placed in an AmiconUltra 10K filter, centrifuged at 13,000 rpm and 4° C. for 15 minutes, and the filtrate exiting the filter was used for analysis. HPLC analysis conditions are shown in Table 4 below.

Analysis column	RHM-monosaccharide H+ (8%) 300 X 7.8 mm (Rezex); Analysis column RHM-monosaccharide H+ (8%) 50 Х 7.8 mm (Rezex); Guard column
Standard material	NGNA: 2 to 40 μMNANA: 100 to 2000 μM
flux	0.55 ml/min
Column temperature	50℃
Detection	206 nm
Injection volume	5 μl
Mobile phase	5 mN sulfuric acid in water
Gradient / time	Isotropic solvent / 45 minutes

The standard material for calculating the sialic acid content is a mixture of N-acetylneuraminic acid (N-acetylneuraminic acid, hereinafter referred to as NANA) and N-glycolylneuraminic acid (N-glycolylneuraminic acid, hereinafter referred to as NGNA). A linear regression equation was obtained to calculate the molar concentrations of NGNA and NANA in the analyzed sample. The mixtures used are shown in Table 5 below.

mixture	One	2	3	4	5
NANA (μM)	2,000	1,000	500	300	200
NGNA (μM)	40	20	10	6	4

The sialic acid content of the sample was calculated using the NANA content. The sialic acid content of the sample was expressed as a sialic acid/sample mole ratio (mol/mol).

In addition, the NGNA content of the sample was calculated using the NGNA content. The content of NGNA in the sample was expressed as the NGNA/analytical sample molar ratio (mol/mol).

The results are shown in Table 6 below.

sample		NGNA/Protein (mol/mol)	NANA/Protein (mol/mol)
FcεRIαECD-Fc2ST	Sample 1 (SA low )	0.13	7.7
	Sample 2 (SA medi )	0.17	12.0
	Sample 3 (SA high )	0.27	19.1

As shown in Table 6, the sialic acid content of the samples separated and purified by dividing into three samples according to the pH gradient was measured differently. In order to compare the anti-allergic efficacy according to the sialic acid content, it was named "SA ^low ", "SA ^medi " and "SA ^high " in the order of sialic acid content.

Example 10. Confirmation of changes in the formation of dimers of polypeptides according to sialic acid content

In order to confirm whether there is a change in the formation of a dimer of the polypeptide depending on the sialic acid content, SA ^low , SA ^medi and SA ^high isolated in Example 9 were analyzed by SDS-PAGE under non-reducing and reducing conditions in the same manner as in Example 3. Was performed.

As a result, a polypeptide having a size of about 150 kDa was detected under non-reducing conditions in all of SA ^low , SA ^medi and SA ^high, and a polypeptide having a size of about 75 kDa was detected under reducing conditions. Through this, it was confirmed that the polypeptide forms a dimer (FIG. 10).

Example 11. Identification of the dimer isoelectric point of the polypeptide according to the sialic acid content

To check whether the dimer isoelectric point of the polypeptide changes depending on the sialic acid content, SA ^low , SA ^medi and SA ^high isolated in Example 9 were mixed with the IEF sample solution, and then in a pH 3-7 IEF gel (Invitrogen). After loading, electrophoresis was carried out sequentially for 1 hour at 100V condition, 1 hour at 200V condition, and 30 minutes at 500V condition. After the electrophoresis was completed, the gel was reacted with a fixed solution containing 12% trichloroacetic acid and 3.5% sulfosalicylic acid for 30 minutes, and then washed with distilled water. Proteins were stained with Coomassie Brilliant Blue solution.

As a result, the isoelectric points of SA ^low , SA ^medi, and SA ^high were slightly different depending on the sialic acid content, and were respectively about 5.3, 4.9 and 4.7 (FIG. 11).

Example 12. Confirmation of anti-allergic activity of polypeptide dimers according to sialic acid content in an animal model of food allergy: measurement of diarrhea frequency

In order to confirm whether the activity of the polypeptide dimer varies depending on the sialic acid content, sensitization was induced by intraperitoneal administration of 50 µg of OVA and 1 mg of Alum to Balb/c mice (Orientbio) at 14 days intervals. Thereafter, on days 28, 30, 32, 34, 36, 38, and 40, OVA 50 mg was orally administered over a total of 7 times to induce food allergy. At this time, oral administration of OVA was performed after fasting for 4 hours.

After oral administration of the OVA twice, the mice were classified into 3 groups of 7 mice each on the 40th day. The first group, a group in which a polypeptide dimer having a high sialic acid content (SA ^high ) is administered subcutaneously at a high concentration (200 μg), and a second group, a polypeptide dimer having a low sialic acid content (SA ^low ), is administered at a high concentration (200 μg) It was classified into a subcutaneous administration group and a third group, a subcutaneous administration group. While the OVA was administered orally, it was confirmed whether diarrhea occurred according to the induction of food allergy.

As a result, the mice of the third group had diarrhea after oral administration of the fourth OVA. The second group of mice developed diarrhea after oral administration of the fifth OVA. In particular, the incidence of diarrhea rapidly increased in mice in the second group after oral administration of the 6th OVA. On the other hand, in the first group of mice, diarrhea occurred after oral administration of the 6th OVA, but the incidence of diarrhea did not increase even after oral administration of the 7th OVA (FIG. 12 ). Through this, it was confirmed that a polypeptide dimer having a high sialic acid content (SA ^high ) has an excellent anti-allergic effect than a polypeptide dimer having a low sialic acid content (SA ^low ).

Example 13. Confirmation of anti-allergic activity of a polypeptide dimer according to sialic acid content in an animal model of food allergy: measurement of IgE concentration in blood

Blood was collected from mice of each group to which OVA of Example 12 was administered orally by the orbital blood sampling method. After reacting at room temperature for 30 minutes, the serum was separated by centrifugation for 15 minutes at 13,000 rpm at 4°C. To measure the free IgE (free IgE) concentration in blood, a mouse total IgE ELISA kit (BioLegend) was used, and at this time, the material coated on the 96-well-plate was an embodiment of the present invention instead of an anti-IgE antibody. ELISA was performed according to the manufacturer's protocol, except that polypeptide dimers (SA ^low and SA ^high ) were used.

Specifically, a 96-well plate was coated with a polypeptide dimer diluted in PBS (SA ^low and SA ^high ), and reacted at 4°C overnight. The next day, after washing with PBS containing 0.05% tween 20 (hereinafter, washing buffer), a blocking buffer (assay diluent) was added and reacted for 1 hour. Thereafter, after washing with a washing buffer, mouse IgE to be used as a standard solution and a serum sample of a mouse were diluted in 1X assay diluent, placed in a plate, and reacted for 2 hours. After washing again with a washing buffer, a mouse anti-IgE antibody labeled with biotin was added and reacted for 1 hour. After washing with a washing buffer, HRP (horse radish peroxidase)-labeled avidin (Avidin-HRP) was added and reacted for 30 minutes. After washing with a washing buffer, a substrate solution was added and reacted for 20 minutes while blocking light, and then a stop solution (1 MH ₂ SO ₄ ) was added to stop the reaction. Then, the concentration was calculated by measuring the absorbance value at 450 nm wavelength with a microplate reader (Epoch Microplate Spectrophotometer).

As a result, in the case of the mice of the third group to which PBS was administered subcutaneously, the blood IgE concentration was calculated to be about 8,000 ng/ml. In addition, in the case of the second group of mice administered subcutaneously with a polypeptide dimer having a low sialic acid content (SA ^low ), the concentration of IgE in the blood was calculated to be about 7,000 ng/ml. On the other hand, in the case of the mice of the first group administered subcutaneously with a polypeptide dimer (SA ^high ) having a high sialic acid content, about 4,900 ng/ml was calculated, and the values of IgE concentration in the blood of the mice of the second and third groups were significant. The difference was shown (Fig. 13).

From these results, it was found that when a polypeptide dimer having a high sialic acid content was administered subcutaneously, the IgE content in the blood decreased. In addition, due to the decrease in IgE in blood, it could be predicted that the polypeptide dimer having a high sialic acid content is effective in treating allergy.

Example 14. Confirmation of anti-allergic activity of polypeptide dimers according to sialic acid content in an animal model of food allergy: measurement of blood MCPT-1 concentration

Two days after the food allergy experiment was over, blood was collected from mice in each group to prepare serum. In order to measure the concentration of MCPT-1 (Mast Cell Protease-1) in the blood, the MCPT-1 ELISA kit (Invitrogen) was used and according to the manufacturer's protocol.

Specifically, a 96-well-immune plate was coated with a mouse anti-MCPT-1 antibody and reacted at 4°C overnight.

The next day, after washing with PBS containing 0.05% tween 20 (hereinafter, washing buffer), PBS containing 1% BSA (Bovine serum albumin) (hereinafter referred to as blocking buffer, assay diluent) was added and reacted for 1 hour. Thereafter, after washing with a washing buffer, MCPT-1 to be used as a standard solution and a serum sample of a mouse were diluted in 1X assay diluent, placed in a plate, and reacted for 2 hours. After 2 hours, biotin-labeled mouse anti-MCPT-1 antibody was added and reacted for 1 hour. After washing with a washing buffer, HRP (horse radish peroxidase)-labeled avidin (Avidin-HRP) was added and reacted for 30 minutes. After washing with a washing buffer, a substrate solution was added and reacted for 20 minutes while blocking the light, and then a stop solution (1 MH ₂ SO ₄ ) was added to stop the reaction. Then, the concentration was calculated by measuring the absorbance value excluding the 570 nm wavelength at 450 nm wavelength using a microplate reader (Epoch Microplate Spectrophotometer).

As a result, in the case of the mice of the third group to which PBS was administered subcutaneously, the concentration of MCPT-1 in the blood was calculated to be about 4,000 ng/ml. In addition, in the case of the second group of mice administered subcutaneously with a polypeptide dimer having a low sialic acid content (SA ^low ), the concentration of MCPT-1 in blood was calculated to be about 4,200 ng/ml. On the other hand, in the case of mice of the first group administered subcutaneously with a polypeptide dimer (SA ^high ) having a high sialic acid content, about 2,800 ng/ml was calculated, and the values of IgE concentration in the blood of the mice of the second and third groups were significant. It showed a difference (Fig. 14).

Claims (21)

1. As a polypeptide dimer comprising two monomers containing the extracellular domain (FcεRIa-ECD) of the alpha subunit of the IgE Fc receptor,

   The monomer comprises an Fc region,

   The Fc region and FcεRIa-ECD are coupled through a hinge,

   The polypeptide dimer is a polypeptide dimer having a high sialic acid content, characterized in that the molar ratio of sialic acid/polypeptide dimer is 8 or more.
2. The method of claim 1,

   The extracellular domain of the alpha subunit of the IgE Fc receptor consists of the amino acid sequence of SEQ ID NO: 1, a polypeptide dimer having a high sialic acid content.
3. The method of claim 1,

   The Fc region is a polypeptide dimer having a high sialic acid content, which consists of an amino acid sequence represented by SEQ ID NO: 2.
4. The method of claim 1,

   The hinge is a hinge region derived from immunoglobulin IgD or a variant thereof, a polypeptide dimer having a high sialic acid content.
5. The method of claim 1,

   The sialic acid is N-acetylneuraminic acid, the polypeptide dimer having a high sialic acid content.
6. The method of claim 1,

   The polypeptide dimer is a polypeptide dimer having a high sialic acid content, characterized in that the molar ratio of sialic acid/polypeptide dimer is 8 or more.
7. The method of claim 6,

   The polypeptide dimer is characterized in that the molar ratio of sialic acid/polypeptide dimer is 8 to 30, a polypeptide dimer having a high sialic acid content.
8. The method of claim 6,

   The polypeptide dimer is characterized in that the molar ratio of sialic acid/polypeptide dimer is 12 to 25. Polypeptide dimer having high sialic acid content.
9. The method of claim 6,

   The polypeptide dimer is a polypeptide dimer having a high sialic acid content, characterized in that the molar ratio of sialic acid/polypeptide dimer is 19 to 22.
10. The method of claim 4,

    The hinge region derived from the immunoglobulin IgD or a variant thereof comprises at least one cysteine, a polypeptide dimer having a high sialic acid content.
11. The method of claim 4,

    The hinge region derived from the immunoglobulin IgD or a variant thereof

    Arg Asn Thr Gly Arg Gly Gly Glu Glu Lys Lys Xaa1 Xaa2 Lys Glu Lys Glu Glu Glu Gln Glu Glu Arg Glu Thr Lys Thr Pro Glu Cys Pro (SEQ ID NO: 17),

    At this time, Xaa1 is Lys or Gly, and Xaa2 is Glu, Gly or Ser; 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 Lys Lys Xaa3 Xaa4 Lys Glu Lys Glu Glu Glu Gln Glu Glu Arg Glu Thr Lys Thr Pro Glu Cys Pro ( SEQ ID NO: 18),

    At this time, Xaa3 is Lys or Gly, and Xaa4 is Glu, Gly or Ser,

    Polypeptide dimer with high sialic acid content.
12. The method of claim 4,

    The hinge region derived from the immunoglobulin IgD or a variant thereof is composed of any one amino acid sequence selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 19, a polypeptide dimer having a high sialic acid content.
13. A pharmaceutical composition for preventing or treating allergic diseases comprising a polypeptide dimer having a high sialic acid content according to any one of claims 1 to 12 as an active ingredient.
14. The pharmaceutical composition for preventing or treating allergic diseases according to claim 13, wherein the pharmaceutical composition is for subcutaneous injection.
15. The method of claim 13,

    The allergic diseases are food allergy, atopic dermatitis, asthma, allergic rhinitis, allergic conjunctivitis, allergic dermatitis, chronic idiopathic urticaria (Chronic idiopathic urticaria). ), and allergic contact dermatitis (allergic contact dermatitis), characterized in that one selected from the group consisting of, allergic disease prevention or treatment pharmaceutical composition.
16. A transdermal patch comprising the pharmaceutical composition of claim 13.
17. A topical patch comprising the pharmaceutical composition of claim 13.
18. A food composition for improving or alleviating allergic symptoms comprising the polypeptide dimer having a high sialic acid content according to any one of claims 1 to 12 as an active ingredient.
19. A method for preventing or treating an allergic disease comprising administering to an individual the polypeptide dimer having a high sialic acid content according to any one of claims 1 to 12.
20. The method of claim 19,

    The administration is characterized in that the subcutaneous administration, allergic disease prevention or treatment method.
21. Use of a polypeptide dimer having a high sialic acid content according to any one of claims 1 to 12 for preventing or treating allergic diseases for preventing or treating allergic diseases.

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