HK1244016B - Fc fusion high affinity ige receptor α-chain - Google Patents

Description

Fc fusion high affinity IgE receptor α chain

Technical Field

The present invention relates to an Fc-fused high affinity IgE receptor α chain for use as a drug.

More specifically, the present invention relates to an Fc-fused high-affinity IgE receptor α chain having excellent stability at low pH values, and its pharmaceutical use.

Background Art

Immunoglobulin E (IgE) is one of the immunoglobulins responsible for allergic reactions. IgE secreted by B cells or expressed on the surface of B cells binds to the known high-affinity IgE receptor (FcεRI) on the surface of mast cells and basophils. When antigen proteins bind to IgE on the mast cell surface receptor, a cross-linked structure is formed between IgE and the antigen. Subsequently, chemical mediators such as histamine and serotonin stored in intracellular granules are released. As a result, an inflammatory response is induced, causing type I allergic symptoms such as capillary dilation and increased vascular permeability (Non-Patent Document 1).

Therefore, compounds or proteins that inhibit the binding of IgE to FcεRI are expected to be therapeutic agents for type I allergic diseases such as bronchial asthma, allergic rhinitis, and allergic conjunctivitis because they inhibit the binding of IgE to the known FcεRI on the surface of mast cells and basophils (Non-Patent Document 2).

In recent years, in addition to the development of drugs based on existing low-molecular-weight compounds as active ingredients, protein drugs have also been developed that strongly bind to specific receptors in the body and exhibit excellent therapeutic effects. For example, etanercept is known as a therapeutic agent for rheumatoid arthritis. Etanercept is a fully human soluble TNFα/LTα receptor preparation developed with the goal of inhibiting the effects of TNF through soluble tumor necrosis factor (TNF) receptors in the body.

Protein drugs are expected to have high therapeutic effects, but on the other hand, problems unique to protein drugs may arise during their production process.

Typically, purification methods using Protein A are used to develop antibodies and Fc fusion proteins into pharmaceuticals. In this method, a low pH buffer is used to elute the target protein bound to Protein A. Furthermore, to inactivate viruses, it is desirable to treat the target protein at a low pH for a specific period of time.

Proteins with poor stability at low pH levels are prone to aggregate formation. High aggregate concentrations can reduce purification efficiency and yield in protein drug manufacturing. Furthermore, the presence of aggregates in drugs can trigger immune responses, leading to serious side effects such as allergic reactions.

Therefore, instability of target proteins at low pH values becomes a problem in the manufacture of protein drugs.

Patent Document 1 describes a polypeptide (immunoadhesin) comprising an immunoglobulin and an extracellular region. Patent Document 1 describes a high-affinity IgE receptor as an example of an immunoadhesin. However, the document does not specifically describe a fusion protein of a high-affinity IgE receptor and an immunoglobulin.

Non-Patent Document 3 describes a fusion protein (hereinafter referred to as "Fusion Protein A") of the high-affinity IgE receptor α chain (FcεRIα-chain, hereinafter referred to as "FCER1A") and immunoglobulin G1 (IgG1). However, the binding pattern of FCER1A to IgG1 (Fc) differs significantly between the fusion protein A described in the aforementioned document and the protein of the present invention. Specifically, the protein of the present invention has a characteristic amino acid sequence in the linker region between FcεRI and IgG1.

Patent Document 2 describes a fusion protein (NPB301) consisting of a water-soluble fragment of the high-affinity IgE receptor (FcεRI) and a human Fc region linked via a linker peptide. However, the protein of the present invention does not contain the linker peptide described in Patent Document 2. Furthermore, Patent Document 2 does not describe or suggest the characteristic amino acid sequence of the linker fragment region of the present invention.

Patent Document 3 describes a fusion protein of FCER1A and immunoglobulin G2 (IgG2). However, this fusion protein differs from the aforementioned IgG2 fusion protein and the protein of the present invention in the amino acid sequence in the linker region and the Fc region.

Patent Document 4 describes a fusion protein of non-human primate FCER1A and IgG1. Furthermore, Patent Documents 5 to 7 describe fusion proteins of FCER1A and IgG1. However, these documents do not describe or suggest the characteristic amino acid sequence of the linker fragment region of the present invention.

The aforementioned Non-Patent Document 3 and Patent Documents 1 to 7 do not describe or suggest the protein of the present invention.

Prior art literature

Patent Literature

Patent Document 1: U.S. Patent No. 5,565,335

Patent Document 2: International Publication No. 2012/169735

Patent Document 3: Chinese Patent Application Publication No. 101633698

Patent Document 4: International Publication No. 2008/028068

Patent Document 5: International Publication No. 2011/056606

Patent Document 6: International Publication No. 2008/099178

Patent Document 7: International Publication No. 2008/099188

Non-patent literature

Non-patent literature 1: Luo Zhijing, “Molecular and cellular mechanisms of Alergi,” Bio Industry, 2008, Vol. 25, No. 9, P. 23-39

Non-patent document 2: Chisei Ra et al., "International Immunology", 1993, Vol. 5, No. 1, P. 47-54

Non-patent document 3: M. Haak-Frendscho et al., “Journal of Immunology”, 1993, Vol. 151, No. 1, pp. 351-358

Summary of the Invention

Problems to be solved by the invention

The present invention aims to provide an Fc-fused high-affinity IgE receptor α chain having excellent stability at low pH.

Means of solving problems

The present inventors conducted intensive research to obtain an Fc-fused, high-affinity IgE receptor α chain with high stability at low pH and heat. As a result, they discovered that using a linker fragment containing three Cys residues in a fusion protein comprising the high-affinity IgE receptor α chain and the Fc region of IgG1 enables the production of a highly stable Fc-fused, high-affinity IgE receptor α chain, leading to the completion of the present invention. Specifically, the present invention is as follows.

The present invention relates to the following [1] to [5] and the like.

[1] An Fc fusion protein, characterized by comprising:

(i) high-affinity IgE receptor α chain, and

(ii) the Fc region of IgG1,

And the linker fragment region between (i) and (ii) is the amino acid sequence of SEQ ID NO: 2.

[2] An Fc fusion protein, which is the Fc fusion protein according to claim 1, characterized in that the protein is a protein comprising the amino acid sequence of SEQ ID NO: 3; or it is an Fc fusion protein comprising the amino acid sequence of SEQ ID NO: 3 with lysine (K) deleted from the C-terminus.

[3] The Fc fusion protein according to [1] or [2], which is a dimer.

[4] The Fc fusion protein according to [3], wherein the cysteine residues in the linker fragment region form three disulfide bonds with each other.

[5] A pharmaceutical composition comprising the Fc fusion protein of any one of [1] to [4] as an active ingredient.

This specification incorporates the disclosures of Japanese Patent Application Nos. 2015-032231 and 2015-252231 on which the present application claims priority.

Effects of the Invention

The protein of the present invention has excellent stability at low pH values. In addition, the protein of the present invention has excellent neutralizing activity against IgE. Therefore, the protein of the present invention can be used as a protein drug for preventing and treating type I allergic diseases mediated by IgE.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows the inhibitory activity against human IgE binding. The horizontal axis represents the concentration of each drug (mol/L), and the vertical axis represents the percentage of IgE bound to Protein 1 immobilized on the plate (free IgE (% relative to control)), based on the binding amount when a certain amount of IgE is added. Circles represent Protein 1, and squares represent the values for omalizumab.

Figure 2 shows the evolution of aggregate content (%) at low pH. The horizontal axis represents days (days), and the vertical axis represents aggregate content (%). Circles represent protein 1, and squares represent fusion protein A.

Figure 3 shows the transition of aggregate content changes (%) under heat treatment. In the figure, the horizontal axis represents days (days), and the vertical axis represents the aggregate content change (%). In the figure, circles represent protein 1, and squares represent the values of fusion protein A.

DETAILED DESCRIPTION

The embodiments of the present invention are described in detail below.

In the present invention, unless otherwise specified, each term has the following meaning.

In the present invention, "high-affinity IgE receptor α chain (FCER1A)" refers to a protein comprising the α chain portion, which is the extracellular region of the high-affinity IgE receptor. For example, the high-affinity IgE receptor α chain refers to the protein represented by the following SEQ ID NO: 1.

SEQ ID NO: 1:

VPQKPKVSLNPPWNRIFKGENVTLTCNGNNFFEVSSTKWFHNGSLSEETNSSLNIVNAKFEDSGEYKCQHQQVNESEPVYLEVFSDWLLLQASAEVVMEGQPLFLRCHGWRNWDVYKVIYYKDGEALKYWYENHNISITNATVEDSGTYYCTGKVWQLDYESEPLNITVIKAPREKYWL

The high-affinity IgE receptor α chain includes proteins having an amino acid sequence with 90% or greater, 95% or greater, 97% or greater, or 99% or greater identity to the amino acid sequence set forth in SEQ ID NO: 1, when calculated using, for example, BLAST (Basic Local Alignment Search Tool at the National Center for Biological Information) (e.g., using default, i.e., initially set, parameters), and having the ability to bind to IgE. Furthermore, proteins having an amino acid sequence with a substitution, deletion, and/or addition of one or more or several (1 to 10, preferably 1 to 5, more preferably 1 or 2) amino acids to the amino acid sequence set forth in SEQ ID NO: 1 and having the ability to bind to IgE are also included.

In the present invention, "IgG1 Fc region" refers to the Fc fragment of immunoglobulin G1, that is, the CH2 and CH3 constant regions of natural immunoglobulin G1. The IgG1 Fc region includes any of natural mutants, artificial variants, and truncated forms.

In the present invention, the "linker fragment region between the high affinity IgE receptor α chain and the IgG1 Fc region" refers to a region of 14 amino acid residues extending from the junction of the high affinity IgE receptor α chain and the IgG1 Fc region toward the Fc region.

In the present invention, "Fc fusion protein" refers to a recombinant protein comprising a high-affinity IgE receptor α chain and an immunoglobulin Fc fragment.

The protein of the present invention is characterized in that the linker fragment region between the high-affinity IgE receptor α chain and the Fc region of IgG1 is the amino acid sequence shown in the following SEQ ID NO: 2.

SEQ ID NO: 2:

EPKSCDKTHTCPPC

The protein of the present invention is preferably an Fc fusion protein comprising the amino acid sequence shown in the following SEQ ID NO: 3 (hereinafter referred to as "Protein 1").

SEQ ID NO: 3:

VPQKPKVSLNPPWNRIFKGENVTLTCNGNNFFEVSSTKWFHNGSLSEETNSSLNIVNAKFEDSGEYKCQHQQVNESEPVYLEVFSDWLLLQASAEVVMEGQP LFLRCHGWRNWDVYKVIYYKDGEALKYWYENHNISITNATVEDSGTYYCTGKVWQLDYESEPLNITVIKAPREKYWLEPKSCDKTHTCPPCPAPELLGGPSVF LFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPR EPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

The amino acid sequence shown in SEQ ID NO: 3 comprises a sequence formed by fusion of the amino acid sequence shown in SEQ ID NO: 1 (Val at position 1 to Leu at position 179 in the amino acid sequence shown in SEQ ID NO: 3), the amino acid sequence shown in SEQ ID NO: 2 (Glu at position 180 to Cys at position 193 in the amino acid sequence shown in SEQ ID NO: 3), and the amino acid sequence of the Fc fragment of an immunoglobulin (Pro at position 194 to Lys at position 411 in the amino acid sequence shown in SEQ ID NO: 3), in the following order. The Fc fusion protein includes an amino acid sequence having 90% or more, 95% or more, 97% or more, or 99% or more identity to the amino acid sequence of SEQ ID NO: 3, excluding the amino acid sequence from Glu 180 to Cys 193 corresponding to the amino acid sequence of SEQ ID NO: 2, when calculated using, for example, BLAST (Basic Local Alignment Search Tool at the National Center for Biological Information) (e.g., using default, i.e., initially set parameters), and a protein having IgE binding ability. Furthermore, the protein includes an amino acid sequence having substitutions, deletions, and/or additions of one or more or several (1 to 10, preferably 1 to 5, more preferably 1 or 2) amino acids to the amino acid sequence of SEQ ID NO: 3, excluding the amino acid sequence from Glu 180 to Cys 193 corresponding to the amino acid sequence of SEQ ID NO: 2, and a protein having IgE binding ability.

When producing recombinant antibodies, the C-terminal lysine may be deleted due to post-translational modification. Therefore, the protein of the present invention can also be an Fc fusion protein comprising an amino acid sequence in which the C-terminus of the above-mentioned protein 1 is deleted from lysine (K). For example, an Fc fusion protein comprising an amino acid sequence in which the C-terminus of the protein 1 of SEQ ID NO: 3 is deleted from lysine (K) is composed of the amino acid sequence of positions 1 to 410 of the amino acid sequence of SEQ ID NO: 3.

The protein of the present invention, its high affinity IgE receptor α chain and the Fc region of IgG1 include both monomers of the Fc fusion protein formed by the linker fragment composed of the amino acid sequence of SEQ ID NO: 2, and also dimers. There are 3 Cys residues in the above-mentioned linker fragment region (Cys at positions 184, 190 and 193 of the amino acid sequence shown in SEQ ID NO: 3), which can form dimers through disulfide bonds. Typically, two Fc fusion protein monomers form dimers by forming 3 disulfide bonds between the Cys at the same position of the above-mentioned 3 Cys. The above-mentioned 3 disulfide bonds make the dimer stable and have higher stability for low pH and heat. High stability to low pH and heat, for example, refers to the formation of aggregates rarely under low pH conditions and heating. For example, after low pH treatment or heat treatment, it can be confirmed by measuring the content of aggregates using gel filtration chromatography. For example, even when the Fc fusion protein of the present invention is stored at a low temperature of 2-8°C, preferably 4°C; at a pH of 1-5, preferably pH 2-4, for 1 day to 1 month, preferably 1 day to 14 days, more preferably 5-12 days, or at 25-45°C, preferably 30-40°C, for 1 day to 1 month, preferably 1 day to 14 days, more preferably 1 day to 7 days, the aggregate content rate changes little. For example, when the aggregate content rate of the protein of the present invention is calculated based on the peak area of gel filtration chromatography, it is less than 10%, preferably less than 8%. In addition, the aggregate content rate of the protein of the present invention changes less than that of Fc fusion proteins having linker fragments containing two or fewer Cys as linkers.

The protein of the present invention can be produced, for example, by the following method or a method based thereon, or by a method described in the literature or a method based thereon.

The proteins of the present invention can be produced using genetic recombination techniques known to those skilled in the art. For example, DNA encoding the protein of the present invention can be prepared, and an expression vector containing the DNA can be constructed. The vector can then be used to transform or transfect prokaryotic or eukaryotic cells, and the target protein can be isolated or purified from the resulting cell culture supernatant.

The protein of the present invention can be produced using protein-expressing cells known to those skilled in the art. For example, a cDNA encoding the amino acid sequence of SEQ ID NO: 3 can be transferred into a mammalian expression plasmid vector to prepare a protein expression plasmid, which can then be introduced into animal cells such as Chinese hamster ovary (CHO) cells to establish a stable expression cell line. The cells can be cultured to obtain the protein of the present invention from the culture supernatant.

If desired, the protein of the present invention can be isolated and purified using separation and purification methods known to those skilled in the art. For example, examples of separation and purification methods include affinity chromatography, ion exchange chromatography, gel filtration chromatography, hydrophobic chromatography, mixed-mode chromatography, dialysis, fractional sedimentation, and electrophoresis. These methods can also be appropriately combined to isolate or purify the protein of the present invention.

The protein of the present invention may also be subjected to chemical modifications known to those skilled in the art, for example, glycosylation, polyethylene glycol (PEGylation), acetylation, amidation, and the like.

Since the proteins of the present invention have excellent neutralizing activity against IgE, they can be used as preventive or therapeutic agents for various diseases mediated by IgE. For example, the proteins of the present invention can be used as preventive or therapeutic agents for diseases associated with type I allergies, such as bronchial asthma, eosinophilic otitis media, eosinophilic sinusitis, allergic conjunctivitis, allergic rhinitis, hay fever, food allergies, tick allergies, urticaria, and anaphylactic shock.

The proteins of the present invention have excellent affinity for IgE. Therefore, like antibody-drug conjugates (ADCs), the proteins of the present invention can also be used as "protein-drug conjugates" that utilize this affinity. For example, "protein 1-drug" and "protein 1-linker-drug" are examples of usage. Antiallergic agents can be used as drugs, and conjugates can be produced using methods known to those skilled in the art.

The pharmaceutical compositions of the present invention can be used in various dosage forms depending on the intended use. For example, oral dosage forms include tablets, powders, granules, fine granules, and capsules. Parenteral dosage forms include injections, inhalation powders, inhalation liquids, eye drops, liquids, lotions, sprays, nasal drops, drops, ointments, suppositories, and patches.

The pharmaceutical composition of the present invention can be administered by various methods depending on the intended use, for example, oral administration, intravenous administration, intraperitoneal administration, subcutaneous administration, topical administration, intramuscular administration, etc.

The pharmaceutical composition of the present invention is prepared using the protein of the present invention and at least one pharmaceutical additive. Depending on its dosage form, the pharmaceutical composition of the present invention can be prepared by known methods of pharmaceutical pharmaceutics. For example, pharmaceutical additives include: excipients, disintegrants, binders, lubricants, diluents, buffers, isotonic agents, preservatives, stabilizers, cosolvents and the like. The pharmaceutical additives may also contain physiological saline, water for injection, etc. The pharmaceutical composition of the present invention can be prepared by mixing, diluting or dissolving the pharmaceutical additives.

When the pharmaceutical composition of the present invention is used for prevention or treatment, the dosage of its active ingredient, i.e., the protein of the present invention, should be appropriately determined based on the patient's age, sex, weight, disease severity, dosage form, route of administration, etc. For adults, the dosage can be determined, for example, within the range of 0.1 μg/kg to 1000 mg/kg/day in the case of oral administration. Depending on the dosage form, the daily dosage for oral administration is preferably in the range of 0.1 mg/kg to 10 mg/kg/day. The daily dosage can also be divided into one, two, or three doses. In addition, the dosage for adults, in the case of parenteral administration, can also be determined within the range of 0.01 μg/kg to 1000 mg/kg/day. Depending on the dosage form, the daily dosage for parenteral administration is preferably in the range of 0.1 μg/kg to 10 μg/kg/day, 1 μg/kg to 100 μg/kg/day, or 10 μg/kg to 1000 μg/kg/day.

Example

The present invention will be described in more detail below by the following examples and test examples. However, the present invention is not limited to these.

Example 1

Expression and preparation of protein 1

(1) Preparation of protein 1 expression vector

The cDNA encoding the amino acid sequence of SEQ ID NO: 3 was transferred into a mammalian expression plasmid vector to prepare a protein 1 expression plasmid.

(2) Preparation of protein 1-expressing cells

The protein 1 expression plasmid was introduced into Chinese hamster ovary (CHO) cells to establish a cell line stably expressing protein 1. The secretion of protein 1 in the culture supernatant was confirmed by SDS-PAGE.

Test Example 1

IgE binding inhibitory activity (IgE neutralizing activity)

(1) Preparation of ELISA plate

Protein 1 was dissolved in coating buffer and added to a microplate. After incubation at 4°C for at least 18 hours, the plate was washed with wash buffer (PBS-Tween 20) and blocked with Assay Diluent (BD Biosciences). After incubation at room temperature for 1 hour, the blocking buffer was removed and the plate was washed with wash buffer for binding inhibition assay.

(2) Method for determining binding inhibitory activity

Using omalizumab (anti-human IgE antibody) as a positive control, the IgE binding inhibitory activity of protein 1 was determined by the following method.

A certain amount of human IgE (ANTIBODYSHOP) was mixed with any concentration of protein 1 or omalizumab (Novartis), added to the well plate prepared in (1) above, and left at room temperature for about 2 hours. After discarding the mixed solution, the plate was washed with washing buffer, and HRP-labeled anti-human IgE antibody (BD Biosciences) was added and left at room temperature for about 1 hour. After discarding the antibody solution, the plate was washed with washing buffer. TMB (3,3',5,5'-tetramethylbenzidine) solution was added, and after a certain period of time, phosphoric acid was added to stop the color reaction. Thereafter, the absorbance (OD450) was measured using a plate reader. Based on the amount of IgE bound to protein 1 immobilized on the well plate, the IgE binding inhibitory activity (IgE neutralization activity) of protein 1 and omalizumab was evaluated (Figure 1).

(3) Results

The protein 1 of the present invention inhibits the binding of IgE to FCER1A in a concentration-dependent manner.

Test Example 2

Stability test at low pH

(1) Sample preparation

The purification operation was carried out using AKTA Explorer 10S (GE Healthcare). Protein 1 was expressed by the same method as described in Example 1, and the culture supernatant was diluted 2-fold with D-PBS (-) (Dulbecco's phosphate buffered saline) and loaded onto HiTrap rProtein A FF (GE Healthcare, 17-5079-01). After washing the chromatography column with D-PBS (-), elution was performed with 100mM glycine-HCl buffer (pH 2.2), and the protein A adsorption fraction was separated in an amount of 1.0mL/tube. The peak fractions were mixed and used as a low pH treatment sample (pH 2.9). The low pH treatment sample was stored at 4°C, and this sample was used as an evaluation sample. Within a specified time (5 to 12 days after storage at 4°C), 0.25mL was sampled from the evaluation sample and neutralized by adding 0.05mL of 1M Tris-HCl buffer (pH 9.0) to obtain a neutralized sample.

As a comparative control, fusion protein A described in Non-Patent Document 3 was used. Fusion protein A was expressed using the same method as in Example 1, and a neutralization-treated sample was obtained using the same method as described above. It should be noted that the fusion protein A used in this experiment is the protein represented by the amino acid sequence of SEQ ID NO:4. The amino acid sequence of SEQ ID NO:4 contains a linker fragment consisting of two Cys residues from Asp at position 180 to Cys at position 188.

SEQ ID NO: 4:

VPQKPKVSLNPPWNRIFKGENVTLTCNGNNFFEVSSTKWFHNGSLSEETNSSLNIVNAKFEDSGEYKCQHQQVNESEPVYLEVFSDWLLLQASAEVVMEGQ PLFLRCHGWRNWDVYKVIYYKDGEALKYWYENHNISITNATVEDSGTYYCTGKVWQLDYESEPLNITVIKAPREKYWLDKTHTCPPCPAPELLGGPSVFLFP PKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPRE PQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

(2) Analysis method of aggregate content

Aggregate analysis was performed using an AKTA Explorer 10S (GE Healthcare) and Superdex 200 10/300GL (GE Healthcare, 17-5175-01) with a mobile phase of D-PBS(-). The peak areas representing monomers eluting in the monomeric region and aggregates eluting in the high-molecular-weight region were calculated, and the changes in aggregate content (%) following low-pH treatment were evaluated for protein 1 and fusion protein A (Figure 2).

(3) Results

Compared to fusion protein A, the protein of the present invention exhibits significantly less aggregate formation with prolonged low pH treatment, demonstrating high stability when exposed to low pH. Therefore, the protein of the present invention exhibits excellent stability at low pH, potentially leading to improved purification efficiency and yield in the production process.

Test Example 3

Thermal stability test

(1) Sample preparation

Purification operation is implemented using AKTA Explorer 10S (GE Healthcare company). Protein 1 is expressed by the same method as described in Example 1, and its culture supernatant is loaded on HiTrap MabSelect SuRe (GE Healthcare company, 17-0034-94). After washing the chromatography column with D-PBS (-) and 100mM citrate buffer (pH value 4.0), the protein A adsorbate is eluted with 100mM glycine-hydrochloric acid buffer (pH value 3.3). 1/10 capacity of 1M Tris-HCl buffer (pH value 9.0) is added to the recovered fraction for neutralization to obtain protein A purified protein. The protein A purified protein is adjusted to pH value 4.0 with 1N HCl, loaded into a chromatography column filled with a mixed mode resin of hydrophobic interaction cation exchange. After washing the non-adsorbed protein with 50mM acetate buffer (pH 4.0), perform 100% linear gradient elution with 50mM Tris-HCl buffer (pH 9.0), recover the peak fraction, and obtain the purified protein. The obtained protein was separated by gel filtration using HiLoad 16/60 Superdex200prep grade (GE Healthcare, 17-1069-01) with D-PBS(-) as the mobile phase. Recover the peak fraction equivalent to the monomer to obtain a gel filtration purified sample. Prepare the gel filtration purified sample again using D-PBS(-) and dispense it into a microtube, and use the sample incubated at 37°C as an evaluation sample. Sampling is performed from the evaluation sample within the specified time (1 to 7 days after storage at 37°C) to obtain a heat-treated sample.

As a comparative control, fusion protein A described in Non-Patent Document 3 was used. Fusion protein A was expressed using the same method as in Example 1, and heat-treated samples were obtained using the same method as described above. It should be noted that the fusion protein A used in the experiment had the same amino acid sequence as the protein used in Experimental Example 2.

(2) Analytical method for aggregate content

Aggregate analysis was performed using an AKTA Explorer 10S (GE Healthcare) and Superdex 200 10/300GL (GE Healthcare, 17-5175-01) with a mobile phase of D-PBS(-). The peak area representing the monomer eluting position and the peak area representing the aggregate eluting in the high molecular weight region were calculated, and the change in aggregate content (%) of protein 1 and fusion protein A under heat treatment was evaluated (Figure 3).

(3) Results

Compared to fusion protein A, the protein of the present invention exhibits less increase in aggregate content when stored at 37°C, demonstrating greater stability when exposed to 37°C. Therefore, the protein of the present invention exhibits excellent stability not only at low pH but also to heat, and is expected to improve purification efficiency and productivity in the production process.

Industrial Applicability

Since the protein of the present invention has excellent neutralizing activity against IgE, it can be used as a protein drug for preventing or treating various diseases mediated by IgE.

Sequence Listing Free Text

SEQ ID NO:2 synthesis

All publications, patents, and patent applications cited in this specification are incorporated herein by direct reference.

Sequence Listing

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20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

Tyr Trp Leu Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu

180 185 190

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

195 200 205

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

210 215 220

Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly

225 230 235 240

Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

Ser Leu Ser Pro Gly Lys

405

Claims (6)

1. An Fc fusion protein, characterized in that the protein contains: (i) the high-affinity IgE receptor α chain, and (ii) The Fc region of IgG1, Furthermore, the linker fragment region between (i) and (ii) is the amino acid sequence of SEQ ID NO:2. 2. An Fc fusion protein, which is the Fc fusion protein of claim 1, wherein the Fc fusion protein has neutralizing activity against IgE. 3. An Fc fusion protein, which is the Fc fusion protein of claim 2, characterized in that the protein is a protein containing the amino acid sequence of SEQ ID NO:3; or it is an Fc fusion protein containing an amino acid sequence of SEQ ID NO:3 with a lysine (K) residue deleted from the C-terminus of the amino acid sequence. 4. The Fc fusion protein according to any one of claims 1 to 3, wherein it is a dimer. 5. The Fc fusion protein according to claim 4, wherein the cysteine residues in the linker fragment region form three disulfide bonds with each other. 6. A pharmaceutical composition comprising the Fc fusion protein as any one of claims 1 to 5 as an active ingredient.