阅读说明：本技术 IgE Fc受体的α亚基的胞外域、包含其的药物组合物及其制备方法 (Extracellular domain of alpha subunit of IgE Fc receptor, pharmaceutical composition comprising the same, and preparation method thereof ) 是由 成永哲 梁祯允 于 2019-01-08 设计创作，主要内容包括：本发明提供了一种包含两个单体的多肽二聚体蛋白,每个单体包含IgE Fc受体的α亚基的胞外域(FcεRIa-ECD)。与包含抗IgE抗体的常规治疗剂相比,本发明的二聚体蛋白显示出具有对IgE优异结合能力的优点,并且由于其缺乏ADCC和CDC功能而表现出较少的其他副作用。因此,该二聚体蛋白质可以应用于治疗或预防IgE介导的过敏性疾病的医疗产品。(The present invention provides a polypeptide dimeric protein comprising two monomers, each monomer comprising the extracellular domain of the alpha subunit of the IgE Fc receptor (FcRIa-ECD). The dimeric protein of the present invention shows the advantage of having excellent binding ability to IgE and shows less other side effects due to its lack of ADCC and CDC functions, compared to conventional therapeutic agents comprising anti-IgE antibodies. Therefore, the dimeric protein may be applied to medical products for treating or preventing IgE-mediated allergic diseases.)

1. A polypeptide dimer, comprising:

two monomers, each monomer comprising the extracellular domain of the alpha subunit of the IgE Fc receptor (FcRIa-ECD),

wherein said monomer comprises a modified Fc region, and

said modified Fc region and said FcRIA-ECD are hingedly connected.

2. The polypeptide dimer of claim 1,

wherein the extracellular domain of the alpha subunit of the IgE Fc receptor is an amino acid sequence shown as SEQ ID NO. 1 or a fragment thereof.

3. The polypeptide dimer of claim 1,

wherein the modified Fc region is shown as SEQ ID NO. 2.

4. The polypeptide dimer of claim 1,

wherein said hinge is a hinge region derived from an immunoglobulin IgD or a variant thereof.

5. The polypeptide dimer of claim 4,

wherein said hinge region derived from an immunoglobulin IgD or a variant thereof comprises at least one cysteine.

6. The polypeptide dimer of claim 4,

wherein said hinge region derived from an immunoglobulin IgD or a variant thereof is Arg Asn Thr Gly Arg GlyGly Glu Glu Lys Lys Xaa1 Xaa2 Lys Glu Lys Glu Glu GlnGlu Glu Arg Glu Thr LysThr Pro Glu Cys Pro (SEQ ID NO:17),

xaa1 is Lys or Gly, and

xaa2 is Glu, Gly, or Ser.

7. The polypeptide dimer of claim 4,

wherein said hinge region derived from an immunoglobulin IgD or a variant thereof is Ala Gln Pro Gln Ala GluGly Ser Leu Ala Lys Ala Thr Thr Ala Pro Ala Thr Thr Arg Asn Thr Gly Arg GlyGly Glu Glu Lys Lys Xaa3 Xaa4 Lys Glu Lys Glu Glu Gln Glu Glu Arg Glu Thr LysThr Pro Glu Cys Pro (SEQ ID NO:18),

xaa1 is Lys or Gly, and

xaa2 is Glu, Gly, or Ser.

8. The polypeptide dimer of claim 1,

wherein the hinge has any one amino acid sequence selected from the group consisting of SEQ ID NO 3, SEQ ID NO 4 and SEQ ID NO 19.

9. A polynucleotide encoding a monomer as claimed in claim 1, comprising the extracellular domain of the alpha subunit of the IgE Fc receptor (FcRIa-ECD).

10. An expression vector carrying the polynucleotide of claim 9.

11. A host cell comprising the expression vector of claim 10.

12. A pharmaceutical composition for treating or preventing allergic diseases, which comprises the polypeptide dimer according to any one of claims 1 to 8 as an active ingredient.

13. The pharmaceutical composition according to claim 12, wherein the compound is selected from the group consisting of,

wherein the allergic disease is any one selected from the group consisting of food allergy, atopic dermatitis, asthma, allergic rhinitis, allergic conjunctivitis, allergic dermatitis, chronic idiopathic urticaria and allergic contact dermatitis.

14. A food composition for ameliorating or alleviating allergic symptoms, comprising the polypeptide dimer according to any one of claims 1 to 8 as an active ingredient.

15. A method of producing a polypeptide dimer, comprising:

culturing a cell into which the polynucleotide of claim 9 and a sialyltransferase gene have been introduced; and

and (3) recovering the polypeptide dimer.

16. A polypeptide dimer obtained according to claim 15.

17. A pharmaceutical composition for treating or preventing allergic diseases, which comprises the polypeptide dimer according to claim 16 as an active ingredient.

18. A food composition for ameliorating or alleviating allergic symptoms, comprising the polypeptide dimer according to claim 16 as an active ingredient.

19. A method of treating or preventing an allergic disease, comprising:

a step of administering the polypeptide dimer of claim 1 and/or claim 16 to a subject.

Technical Field

The present invention relates to a modified IgE Fc receptor and uses thereof.

Background

Allergic diseases such as allergic rhinitis, atopic dermatitis and food allergy (including asthma) are rapidly spreading in industrialized and westernized modern society, and anaphylactic shock is a serious allergic disease, and the number of the onset thereof is also increasing. These chronic immune diseases seriously impair the quality of life of people, and the economic cost of society is soaring. Therefore, methods of treating these diseases are urgently needed.

Most allergic diseases are caused by an excessive immune response by immunoglobulin e (ige). IgE is an antibody that is present in serum at very low concentrations under normal conditions. IgE is also usually produced by harmless antigens. In some cases, the amount of IgE will increase without any specific stimulus. This condition may lead to allergic diseases. Abnormally increased amounts of IgE may bind to the high affinity IgE Fc receptor (FcRI) expressed on the surface of mast cells, basophils, etc. This binding results in the mast cells or basophils releasing chemical mediators such as histamine, leukotrienes, prostaglandins, bradykinin, and platelet activating factor. The release of these chemical mediators leads to allergic symptoms. Symptoms of allergic diseases may be exacerbated, particularly due to the binding between IgE and FcRI. FcRI-expressing cells are known to be increased in allergic patients.

Various methods for treating allergic diseases have been proposed, such as avoidance of allergens, administration of anti-allergic agents, regulation of IgE synthesis in vivo, and development of anti-IgE antibodies. However, the treatment methods known to date have numerous disadvantages, such as an inability to cure the underlying cause of the allergy, insufficient drug efficacy and the occurrence of serious side effects.

Furthermore, immunoglobulin compositions have been developed which are capable of binding IgE and FcRIIb with high affinity and inhibiting IgE expressing cells (KR10-1783272B 1). Such compositions are reported to be useful in the treatment of IgE-mediated diseases, including allergy and asthma. In addition, omalizumab (trade name: Xolair) targeted to the Fc portion of IgE antibody has been developed and used as a therapeutic agent for refractory severe asthma and refractory urticaria.

However, administration of large doses of omalizumab to maintain therapeutic effect results in a high cost burden, as well as side effects such as angioedema and anaphylaxis (The Journal of Clinical Investigation Volume 99, Number 5, March 1997, 915-925). In addition, from the results after marketing, the development of allergic granulomatous vasculitis and idiopathic severe thrombocytopenia has been reported. Therefore, there is an urgent need to develop a therapeutic agent capable of effectively treating allergic diseases without side effects.

Technical problem

The object of the present invention is to provide a polypeptide dimeric protein for use in the treatment of IgE-mediated allergic diseases. It is another object of the present invention to provide a nucleic acid molecule encoding the protein, an expression vector comprising the nucleic acid molecule, and a host cell comprising the expression vector. It is still another object of the present invention to provide a method for preparing the polypeptide dimer.

Means for solving the problems

To achieve the above object, the present invention provides a polypeptide dimer comprising two monomers, each monomer comprising the extracellular domain of the alpha subunit of the IgE Fc receptor. The monomer comprises a modified Fc region, and the modified Fc region and the extracellular domain of the alpha subunit of the IgE Fc receptor are connected by a hinge of an IgD antibody. In another aspect, the present invention provides a pharmaceutical composition for treating or preventing allergic diseases, which comprises a polypeptide dimer as an active ingredient.

Advantageous effects of the invention

Compared with the conventionally used anti-IgE antibody, the polypeptide dimeric protein of the invention not only has excellent safety and durability in vivo, but also has very strong binding with IgE, and the binding capacity with the IgE is 70 times higher than that with the conventionally used anti-IgE antibody omalizumab (omalizumab), so that the administration period can be prolonged. In addition, the polypeptide dimer protein of the present invention is a product obtained by employing a modified Fc that has only IgE as a single target and does not bind to Fc single receptors, so that the polypeptide dimer protein of the present invention lacks antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) functions. Therefore, unlike conventional anti-IgE antibodies comprising the Fc region of IgG1, the polypeptide dimeric protein does not bind to Fc-non-receptors, and thus can inhibit mediator release due to binding to Fc-inhibitory receptors on mast cell surfaces, minimizing severe side effects such as anaphylactic shock due to binding between IgG1 and Fc large receptor III on mast cells. Accordingly, the polypeptide dimer protein of the present invention can be used as a novel pharmaceutical composition which can replace a therapeutic agent containing a conventional anti-IgE antibody.

Drawings

FIG. 1 shows the results of SDS-PAGE and isoelectric focusing (IEF) of the proteins produced in each cell line. It can be seen that no truncated forms were produced under both reducing and non-reducing conditions and that the introduction of the sialyltransferase gene resulted in an increase in sialic acid content and thus in an increase in the content of acidic proteins.

FIG. 2 illustrates SDS-PAGE results of polypeptide dimer proteins in non-reduced and reduced forms in one embodiment of the 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 omalizumab's binding ability to IgE. The figure shows the results obtained by immobilizing omalizumab and analyzing its binding capacity according to the treated IgE concentration.

FIG. 4 is a graph showing the binding ability of a polypeptide dimer protein to IgE according to one embodiment of the present invention. The figure shows the results obtained by immobilizing the dimeric protein and analyzing its binding capacity according to the treated IgE concentration.

FIG. 5 shows the determination of polypeptide dimer protein (IgE) by biolayer interferometry (BLI) in one embodiment of the invention_TRAP) And the interaction of omalizumab with the IgG receptors Fc γ RI (fig. 5A), Fc γ RIIA (fig. 5B), Fc γ RIIB (fig. 5C), Fc γ RIIIA (fig. 5D), and Fc γ RIIIB (fig. 5E).

FIG. 6 shows the quantification of IgE_TRAPAnd the binding capacity between IgG receptors and between omalizumab and IgG receptors.

FIG. 7 shows a polypeptide dimer protein (IgE) according to an embodiment of the present invention_TRAP) Graph of inhibitory ability on mouse-derived mast cell activity at different concentrations.

FIG. 8 shows a polypeptide dimer protein (IgE) according to an embodiment of the present invention_TRAP) And Xolair (omalizumab) for the inhibition of the activity of mouse-derived mast cells expressing human FcRI.

FIG. 9 is a graph showing the effect of administering a polypeptide dimer protein in a food allergy model in one embodiment of the present invention.

Detailed Description

The present invention relates to a polypeptide dimer comprising two monomers, each monomer comprising the extracellular domain of the alpha subunit of the IgE Fc receptor (FcRIa-ECD), wherein said monomer comprises a modified Fc region, and the modified Fc region and the FcRIa-ECD are connected by a hinge of an IgD antibody.

As used herein, the term "IgE" refers to an antibody protein known as immunoglobulin E. IgE has affinity for mast cells, basophils, and the like. In addition, the reaction between IgE antibodies and their corresponding antigens (allergens) causes an inflammatory reaction. In addition, IgE is known to be an antibody that causes anaphylactic shock (anaphylaxis), which usually occurs due to the sudden secretion of mast cells or basophils.

As used herein, the term "IgE Fc receptor" is also referred to as an Fc receptor and binds to the Fc portion of IgE. There are two types of receptors. Receptors with high affinity for IgE Fc are called Fc receptor i (Fc ri). Receptors with low affinity for IgE Fc are called Fc receptor ii (fcrii). FcRI is expressed in mast cells and basophils. In the case where IgE antibodies bound to FcRI are cross-linked by multivalent antigens, degranulation occurs in mast cells or basophils, thereby releasing various chemical mediators including histamine. This release results in an immediate allergic reaction.

FcRI is a membrane protein consisting of one alpha, one beta and two gamma chains linked by disulfide bonds. Among these chains, the moiety that binds IgE is the alpha chain (FcRI α). FcRI α is approximately 60kDa in size and consists of a hydrophobic domain present within the cell membrane and a hydrophilic domain present outside the cell membrane. In particular, IgE binds to the extracellular domain of the alpha chain.

Specifically, the alpha subunit of the IgE Fc receptor may have an amino acid sequence as shown in NP _ 001992.1. In addition, the extracellular domain of the alpha subunit of the IgE Fc receptor (FcRIa-ECD) may have the amino acid sequence shown in SEQ ID NO: 1. In the present specification, the extracellular domain of the α subunit of the IgE Fc receptor may be a fragment or variant of the extracellular domain of the α subunit of the IgE Fc receptor, as long as the fragment or variant is capable of binding IgE.

Variants may be prepared by methods in which one or more proteins are substituted, deleted or inserted in the wild-type FcRIa-ECD (extracellular domain), provided that the method does not alter the function of the alpha chain of the FcRI. Such various proteins or peptides may have 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more homology with the amino acid sequence of SEQ ID NO. 1. In addition, the FcRIA-ECD shown in SEQ ID NO. 1 may be encoded by a polynucleotide having a sequence shown in SEQ ID NO. 5.

In addition, as used herein, the term "modified Fc region" refers to a region in which a portion of the Fc portion of an antibody has been modified. The term "Fc region" refers to a protein that comprises heavy chain constant region 2(CH2) and heavy chain constant region 3(CH3) of an immunoglobulin, and does not comprise the heavy and light chain variable regions and light chain constant region 1(CH1) of an immunoglobulin. In particular, a modified Fc region refers to a region obtained by making some amino acid substitutions in the Fc region or by combining different types of Fc regions. Specifically, the modified Fc region can have an amino acid sequence as shown in SEQ ID NO. 2. In addition, the modified Fc region shown as SEQ ID NO. 2 can be encoded by a polynucleotide having a sequence shown as SEQ ID NO. 6.

In addition, the "modified Fc region" in the present invention may be in a form having a natural form of sugar chains, having an increased number of sugar chains relative to the natural form or having fewer sugar chains relative to the natural form, or may be in a form in which sugar chains are removed. The sugar chain of the immunoglobulin Fc can be modified by a conventional method such as a chemical method, an enzymatic method, and a genetic engineering method using a microorganism.

Among them, the "modified Fc region" in the present invention may be a region lacking antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) functions due to lack of binding site to fcyr or C1 q. In addition, the modified Fc region and FcRI α -ECD may be linked by a hinge of an IgD antibody. The hinge of an IgD antibody consists of 64 amino acids, and may selectively comprise 20-60 contiguous amino acids, 25-50 contiguous amino acids, or 30-40 amino acids. In one embodiment, the hinge of an IgD antibody may consist of 30 or 49 amino acids, as shown below. In addition, the hinge of the IgD antibody may be a hinge variant obtained by modifying the hinge region, wherein the hinge may comprise at least one cysteine. Here, variants of the hinge can be obtained by modifying some amino acids in the hinge sequence of the IgD antibody such that the production of truncated forms is minimized during protein production.

In one embodiment, the hinge may comprise the sequence:

arg Asn Thr Gly Arg Gly Gly Glu Glu Lys Lys Xaa1 Xaa2 Lys Glu Lys GluGlu Gln Glu Glu Arg Glu Thr Lys Thr Pro Glu Cys Pro (SEQ ID NO:17), wherein Xaa1 can be Lys or Gly and Xaa2 can be Glu, Gly or Ser. In particular, the hinge may have an amino acid sequence as shown in SEQ ID NO 3 or SEQ ID NO 19, thereby minimizing the production of truncated forms of the protein during production.

In another embodiment, the hinge may comprise the sequence:

ala Gln Pro Gln Ala Glu Gly Ser Leu Ala Lys Ala Thr Thr Ala Pro AlaThr Thr Arg Asn Thr Gly Arg Gly Gly Glu Glu Lys Lys Xaa3 Xaa4 Lys Glu Lys GluGlu Gln Glu Glu Arg Glu Thr Lys Thr Pro Glu Cys Pro (SEQ ID NO: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 shown in SEQ ID NO 4, thereby minimizing the production of truncated forms of the protein during production.

In particular, at least one Thr can be glycosylated in the hinge having the sequence shown in SEQ ID NO. 4. Specifically, the 13 th, 14 th, 18 th or 19 th Thr among the amino acids shown in SEQ ID NO:18 may be glycosylated. Preferably, all four amino acids may be glycosylated. Wherein the glycosylation may be O-glycosylation.

In addition, as described above, the polypeptide dimers provided by the present invention may be in the form of: wherein two monomers bind to each other and each monomer is derived by binding the extracellular domain of the alpha subunit of the IgE Fc receptor to the modified Fc region. Polypeptide dimers may be in the form of two identical monomers bound to each other via a cysteine at the hinge site. In addition, the polypeptide dimer may be in the form of two different monomers bound to each other. For example, where the two monomers are different from each other, the polypeptide dimer may be in the form of a monomer wherein one monomer comprises the extracellular domain of the alpha subunit of the IgE Fc receptor and the other monomer comprises a fragment of the extracellular domain of the alpha subunit of the IgE Fc receptor. Wherein, in one embodiment, the monomer may have an amino acid sequence as shown in SEQ ID NO 20, SEQ ID NO 21 or SEQ ID NO 22.

In addition, the polypeptide dimer provided by the present invention exhibits a binding ability to IgE, which is 10-100 times, 20-90 times, 20-70 times, 30-70 times or 40-70 times higher than that of omalizumab, which is an anti-IgE antibody, and may preferably exhibit a binding ability to IgE, which is 70 times higher than that of omalizumab.

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

Meanwhile, the polynucleotide may additionally comprise a signal sequence or a leader sequence. As used herein, the term "signal sequence" refers to a nucleic acid encoding a signal peptide that directs secretion of a target protein. The signal peptide is translated and then cleaved in the host cell. In particular, the signal sequences of the present invention are nucleotides that encode amino acid sequences that initiate protein translocation across the Endoplasmic Reticulum (ER) membrane. Signal sequences useful in the present invention include antibody light chain signal sequences, such as antibody 14.18(Gillies et al, J.Immunol. meth 1989.125:191-202), antibody heavy chain signal sequences, such as MOPC141 antibody heavy chain signal sequence (Sakano et al, Nature,1980.286:676-683), and other signal sequences known in the art (see, e.g., Watson et al, Nucleic Acid Research,1984.12: 5145-5164).

The nature of signal sequences is well known in the art. The signal sequence typically comprises 16-30 amino acid residues, and may comprise more or fewer amino acid residues. A typical signal peptide consists of three regions: a basic N-terminal region, a middle hydrophobic region, and a more polar C-terminal region. The middle hydrophobic region contains 4-12 hydrophobic residues that fix the signal sequence through the membrane lipid bilayer during translocation of the immature polypeptide.

After initiation, cellular enzymes, commonly referred to as signal peptidases, cleave the signal sequence in the lumen of the ER. Wherein the signal sequence can be tissue plasminogen activator (tPA), herpes simplex virus glycoprotein D (HSV gD) or secretion signal sequence of growth hormone. Preferably, a secretory signal sequence used in higher eukaryotic cells including mammals and the like can be used. In addition, the secretory signal sequence may be substituted and used by a codon having a high expression frequency in the host cell.

Meanwhile, the ectodomain monomer of the α subunit of the IgE Fc receptor, which binds to the signal sequence and the modified Fc region, may have an amino acid sequence as shown in SEQ ID No. 11 or SEQ ID No. 13. The proteins shown as SEQ ID NO. 11 and SEQ ID NO. 13 can be encoded by polynucleotides having the sequences shown as SEQ ID NO. 12 and SEQ ID NO. 14, respectively.

In yet another aspect of the invention, an expression vector is provided loaded with a polynucleotide encoding the monomer. Wherein, the polynucleotide can have a sequence shown as SEQ ID NO. 12 or SEQ ID NO. 14.

As used herein, the term "vector" is intended to be introduced into a host cell and capable of recombining with and inserting into the genome of the host cell. Alternatively, the vector is episomal and is understood as a nucleic acid unit comprising a nucleotide sequence that can replicate autonomously. Such vectors include linear nucleic acids, plasmids, phagemids, cosmids, RNA vectors, viral vectors, and the like. Examples of such viral vectors include, but are not limited to, retroviruses, adenoviruses, and adeno-associated viruses. In addition, the plasmid may contain a selectable marker such as an antibiotic resistance gene, and the host cell carrying the plasmid may be cultured under selective conditions.

As used herein, the term "genetic expression" or "expression" of a target protein is understood to refer to the transcription of a DNA sequence, the translation of an mRNA transcript, and the secretion of the product of a fusion protein or fragment thereof. Useful expression vectors may be RcCMV (Invitrogen, Carlsbad) or variants thereof. The expression vector may comprise a human Cytomegalovirus (CMV) promoter for promoting continuous transcription of a target gene in mammalian cells, and a bovine growth hormone polyadenylation signal sequence for increasing the stability of the transcribed RNA.

In yet another aspect of the present invention, there is provided a host cell into which the expression vector is introduced. The term "host cell" as used herein refers to a prokaryotic or eukaryotic cell into which a recombinant expression vector may be introduced. As used herein, the terms "transduction," "transformation," and "transfection" refer to the introduction of a nucleic acid (e.g., a vector) into a cell using techniques known in the art.

Preferred host cells useful in the present invention include immortal hybridoma 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 may be a CHO cell. On the other hand, the host cell may be one into which the vector and the vector carrying the sialyltransferase gene are introduced. Wherein the sialyltransferase may be a2, 3-sialyltransferase or a2, 6-sialyltransferase. Wherein, the 2, 6-sialyltransferase can have an amino acid sequence shown as SEQ ID NO. 15.

In still another aspect of the present invention, there is provided a pharmaceutical composition for treating or preventing allergic diseases, which comprises the polypeptide dimer as an active ingredient.

In the present specification, the term "allergic disease" refers to a pathological condition caused by an allergic reaction mediated by mast cell activation such as mast cell degranulation. Such allergic diseases include food allergy, atopic dermatitis, asthma, allergic rhinitis, allergic conjunctivitis, allergic dermatitis, allergic contact dermatitis, anaphylactic shock, urticaria, pruritus, insect allergy, chronic idiopathic urticaria, drug allergy, etc. In particular, allergic diseases may be IgE-mediated.

In the composition for treating or preventing allergic diseases of the present invention, any amount (effective amount) of the active ingredient may be contained according to its use, formulation, blending purpose, etc., as long as the active ingredient can exhibit anti-allergic activity. The conventional effective amount of the active ingredient may be determined to be in the range of 0.001% to 20.0% by weight of the total composition. Wherein "effective amount" refers to the amount of active ingredient that is capable of eliciting an anti-allergic effect. Such effective amounts can be determined experimentally within the ordinary skill of those in the art.

Wherein, the pharmaceutical composition may further comprise a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier may be any carrier used so 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 carriers. Pharmaceutically acceptable adjuvants (buffers and dispersants) may also be included in the pharmaceutical composition.

Specifically, the pharmaceutical composition of the present invention comprises a pharmaceutically acceptable carrier in addition to the active ingredient, and can be prepared into oral or parenteral formulations based on the route of administration of the drug according to conventional methods known in the art. The term "pharmaceutically acceptable" refers to a toxicity not exceeding that which can be tolerated by the subject to whom it is administered (prescribed), without inhibiting the activity of the active ingredient.

In the case of preparing the pharmaceutical composition of the present invention into an oral preparation, the pharmaceutical composition may be prepared into powders, granules, tablets, pills, dragees, capsules, liquids, gels, syrups, suspensions, wafers, and suitable carriers according to the methods known in the art. Among them, examples of suitable pharmaceutically acceptable carriers may include sugars such as lactose, glucose, sucrose, dextrose, sorbitol, mannitol, and xylitol, starches such as corn starch, potato starch, and wheat starch, celluloses such as cellulose, methyl cellulose, ethyl cellulose, sodium carboxymethyl cellulose, and hydroxypropyl methyl cellulose, polyvinylpyrrolidone, water, methyl hydroxybenzoate (methyl hydroxybenzoate), propyl hydroxybenzoate (propyl hydroxybenzoate), magnesium stearate, mineral oil, malt, gelatin, talc, polyols, vegetable oils, and the like. In the case of formulation, it can be prepared by adding diluents and/or excipients such as filler, extender, binder, wetting agent, disintegrant and surfactant as necessary.

In the case of preparing the pharmaceutical composition of the present invention into parenteral formulations, the pharmaceutical composition may be prepared in the form of injections, transdermal drugs, nasal inhalants and suppositories, together with suitable carriers according to methods known in the art. In the case of injection preparations, sterile water, ethanol, polyhydric alcohols such as glycerol and propylene glycol, or mixtures 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, etc. can be preferably used.

The preparation of Pharmaceutical compositions is known in the art, and in particular reference may be made to Remington's Pharmaceutical Sciences (19 th edition, 1995) et al. This document is to be considered as part of this specification.

The preferred daily dose range of the pharmaceutical composition of the present invention is 0.01. mu.g/kg to 10g/kg, and preferably 0.01mg/kg to 1g/kg, depending on the condition, body weight, sex, age, severity of disease or administration route of the patient. The administration may be carried out one or more times per day. Such dosages should in no way be construed as limiting the scope of the invention.

The subject to which the composition of the invention can be administered (prescribed) can be mammals and humans, with particular preference being given to humans. The composition for anti-allergy of the present invention may further comprise any compound or natural extract, the safety of which has been verified and which is known to have anti-allergic activity, in addition to the active ingredient, thereby enhancing and potentiating the anti-allergic activity thereof.

Another aspect of the present invention provides a food composition for improving and alleviating allergic symptoms, which comprises a polypeptide dimer as an active ingredient.

Wherein the polypeptide dimer can be combined with a suitable delivery unit for efficient delivery into the intestine. The food composition of the present invention can be prepared in any form, and can be prepared, for example, in the form of beverages such as tea, fruit juice, carbonated beverages and ionic beverages, processed dairy products such as milk and yogurt, food preparations having health functions such as tablets, capsules, pills, granules, liquid preparations, powders, flakes, pastes, syrups, jellies, gels, and sticks, and the like. In addition, the food compositions of the present invention may fall into any product category or functional classification that is legitimate, so long as the food composition complies with regulatory compliance at the time of manufacture and distribution. For example, the Food composition may be a Health functional Food described in Health functional foods Act, or may be various types of foods belonging to candies, beans, teas, beverages, special-purpose foods, and the like described in Food Code of Food Sanitation Act (Food standards and specifications, promulgated by the Food and drug administration). With respect to other Food additives that may be included in the Food composition of the present invention, reference may be made to Food Code or Food Additive Code of the Food Sanitation Act (Food Sanitation Act).

Another aspect of the present invention provides a method for producing a polypeptide dimer, the method comprising the step of culturing a host cell into which a polynucleotide encoding a monomer and a sialyltransferase gene is introduced; and a step of recovering the polypeptide dimer.

Wherein the polynucleotide encoding the monomer may be introduced into the host cell in a form loaded on an expression vector. Alternatively, the sialyltransferase gene may be introduced into a host cell in a form loaded on a vector.

First, a vector loaded with a polynucleotide encoding a monomer and a vector loaded with a sialyltransferase gene are introduced into a host cell. Wherein the sialyltransferase may be a2, 3-sialyltransferase or a2, 6-sialyltransferase.

Subsequently, a step of culturing the transformed cells is performed.

Finally, a step of recovering the polypeptide dimer is performed. Wherein the polypeptide dimer may be purified from the culture medium or cell extract. For example, after obtaining the supernatant of the medium in which the polypeptide dimer is secreted, the supernatant can be concentrated using a commercially available protein concentration filter, such as an Amicon or Millipore Pellicon ultrafiltration unit. The concentrate can then be purified by methods known in the art. For example, purification can be achieved using a matrix coupled to protein a.

In still another aspect of the present invention, there is provided a polypeptide dimer produced by the above-mentioned method for producing a dimer.

Wherein the polypeptide dimer has a high sialic acid content and thus a very high acidic protein content relative to the theoretical pI value.

In still another aspect of the present invention, there is provided a pharmaceutical composition for treating or preventing allergic diseases, which comprises the polypeptide dimer produced by the above-described method for producing a dimer as an active ingredient.

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

In yet another aspect of the present invention, there is provided a method for treating or preventing an allergic disease, comprising the step of administering to a subject a polypeptide dimer comprising two monomers, each comprising the extracellular domain of the alpha subunit of the IgE Fc receptor (FcRIa-ECD).

The subject may be a mammal, preferably a human. Wherein, the administration can be oral or parenteral. The parenteral administration may be performed by subcutaneous administration, intravenous administration, mucosal administration, intramuscular administration, or the like.

Detailed Description

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, the following examples are only intended to explain the present invention, and the scope of the present invention is not limited thereto.