CD300c, which is receptor of dimeric TCTP, and use thereof

Abstract

The present invention identifies for the first time that the receptor of dimeric TCTP is CD300C, and relates to an anti-CD300C antibody, a pharmaceutical use thereof, and a drug screening method and a disease diagnosis method using CD300C. In the present invention, it was confirmed that an extracellular domain of CD300C interacts with dimeric TCTP. Accordingly, CD300C can be effectively used as a target for the development of preventive or therapeutic agents for allergic diseases, inflammatory diseases, or malaria. The anti-CD300C antibody of the present invention can be effectively used for preventing or treating allergic diseases, inflammatory diseases, or malaria, or can be effectively used for drug screening or diagnosis related to allergic diseases, inflammatory diseases, or malaria.

Description

CD300C, a receptor for dimeric TCTP, and uses thereof

[0001] The present invention is the first to identify that the receptor for dimeric TCTP is CD300c, and relates to an anti-CD300c antibody, its medical use, and a drug screening method and a disease diagnosis method using CD300c.

[0002] TCTP (Translationally Controlled Tumor Protein) is an IgE-dependent histamine-releasing factor that was known to induce histamine release from basophils in the presence of specific IgE. However, it was later observed that it regulates the secretion of histamine, IL-4, and IL-13 from inflammatory cells regardless of the presence of FcεR, the IgE receptor. In other words, the possibility that TCTP may act by binding to a specific cell membrane receptor other than IgE has been suggested, but such a receptor has not yet been identified (Blood 2000 Sep 15;96(6):2191-8).

[0003] Through prior research, the present inventors have confirmed that extracellularly secreted TCTP forms dimers, activating bronchial epithelial cells and other inflammatory cells to secrete various cytokines and induce an inflammatory response. As a result, we were the first to demonstrate that dimeric TCTP is an allergen (Korean Patent No. 10-0780255, U.S. Patent No. 7,772,368, Japanese Patent No. 4,564,926, European Patent No. 1,683,866). We have conducted various studies targeting dimeric TCTP to treat related allergic and inflammatory diseases. As structural parts of dimeric TCTP, the flexible loop domain (Korean Patent No. 10-1804291) or helix 2 domain (Korean Patent No. 10-1843051) and C-terminus (Korean Patent No. 10-1804285) that bind to the receptor of dimeric TCTP were identified, and peptides, natural products, and monoclonal antibodies that bind to dimeric TCTP and have anti-allergic effects were identified (Korean Patent No. 10-1830838, Korean Patent No. 10-1875317, Korean Patent Application No. 10-2023-0087414).

[0004] Accordingly, the present inventors identified CD300c, a receptor for dimeric TCTP associated with various diseases, and confirmed that the cytokine-like activity of dimeric TCTP can be suppressed by inhibiting CD300c. Therefore, they confirmed that inhibiting CD300c can prevent or treat allergic diseases, inflammatory diseases, or malaria, and completed the present invention.

[0005] One object of the present invention is to provide an anti-CD300c antibody or an antigen-binding fragment thereof, characterized in that it specifically binds to the extracellular domain of CD300c and inhibits the binding of TCTP (Translationally Controlled Tumor Protein) to CD300c.

[0006] Another object of the present invention is to provide a use of a CD300c inhibitor for the prevention or treatment of allergic diseases, inflammatory diseases, or malaria.

[0007] Another object of the present invention is to provide a nucleic acid molecule encoding the antibody or an antigen-binding fragment thereof, an expression vector comprising the same, and a host cell into which the expression vector has been introduced.

[0008] Another object of the present invention is to provide a method for screening drugs using CD300c, a method for diagnosing diseases, and a diagnostic composition.

[0009] This is explained in detail as follows. Meanwhile, each description and embodiment disclosed in the present invention can also be applied to each other description and embodiment. In other words, all combinations of the various elements disclosed in the present invention fall within the scope of the present invention. Furthermore, the scope of the present invention should not be considered limited by the specific descriptions described below.

[0010] To achieve the above purpose, the present invention provides a medicinal use of a CD300c inhibitor for preventing or treating allergic diseases, inflammatory diseases, or malaria.

[0011] According to one specific example of the present invention, the present invention provides a pharmaceutical composition for preventing or treating allergic diseases, inflammatory diseases, or malaria, comprising a CD300c inhibitor as an active ingredient.

[0012] Also, according to one embodiment of the present invention, there is provided a use of a CD300c inhibitor for use in the manufacture of a medicament for use in the treatment or prevention of an allergic disease, an inflammatory disease, or malaria.

[0013] In addition, according to one embodiment of the present invention, the present invention provides a method for preventing or treating an allergic disease, an inflammatory disease, or malaria, comprising administering a therapeutically effective amount of a CD300c inhibitor to a subject in need thereof.

[0014] In the present invention, the term "CD300c (CD300 antigen-like family member C)" refers to a cell membrane protein also called CLM-6 (CMRF35-like molecule 6). The present inventors have confirmed that CD300c is the receptor for the TCTP (Translationally Controlled Tumor Protein) dimer, which directly affects diseases such as allergic diseases, inflammatory diseases, and malaria, thereby proving that CD300c can be usefully used as a therapeutic target for the above diseases. TCTP is a unique protein secreted outside of cells, stored in small secretory vesicles and then secreted through an unconventional pathway. It has been reported that a p53-inducible membrane protein called TSAP6 is involved in this process (Amzallag et al., J Biol Chem, 279, 46104-46112, 2004) and that H,K-ATPase is involved (Choi et al., PLoS One, 4(6), 5732-5739, 2009). It has been reported that the secreted TCTP dimer stimulates IgE-sensitized basophils to release histamine, interleukin-4, and interleukin-13, thereby causing late-onset allergic diseases such as allergic rhinitis, asthma, and atopic dermatitis (MacDonald et al., Science, 269, 688-690, 1995).

[0015] In the present invention, through specific examples, it was confirmed that CD300c can activate the NF-κB signaling pathway through binding to TCTP dimers and secrete cytokines such as IL-6 and IL-8. In addition, it was confirmed that the signaling pathway and cytokine secretion were suppressed by inhibiting the expression of CD300c through an antibody that specifically binds to the extracellular domain of CD300c or siRNA. Furthermore, when an antibody that specifically binds to the extracellular domain of CD300c was administered to a mouse model of asthma, the effect of suppressing airway inflammatory response through reduction of immune cell infiltration, mucus secretion, and cytokine secretion was confirmed. Therefore, CD300c has sufficient potential to become a novel therapeutic target for various diseases previously known to be related to TCTP.

[0016] In the present invention, the term "CD300c inhibitor" is used to collectively refer to all agents that reduce the expression or activity of CD300c, and specifically, may include all agents that reduce the expression level or activity of CD300c by directly acting on CD300c or indirectly acting on its ligand, thereby reducing the expression of CD300c at the transcriptional level or interfering with its activity. However, in the present invention, the CD300c inhibitor does not mean an antibody against TCTP.

[0017] The CD300c inhibitor described above can be used without limitation in the form of a compound, natural product, protein, nucleic acid, peptide, virus, or vector containing the nucleic acid that can target CD300c and inhibit the expression or activity of CD300c. The CD300c inhibitor is not limited thereto, but may be an oligonucleotide that specifically inhibits the expression of CD300c, or an antibody or antigen-binding fragment thereof that inhibits the activity of the CD300c protein. More specifically, the oligonucleotide that inhibits the expression of CD300c may be, but is not limited to, an antisense oligonucleotide, shRNA, or siRNA specific for CD300c mRNA.

[0018] As used herein, the term "antisense oligonucleotide" refers to DNA, RNA, or derivatives thereof containing a nucleic acid sequence complementary to the sequence of a specific mRNA, which binds to the complementary sequence within the mRNA and inhibits the translation of the mRNA into protein. The antisense oligonucleotide sequence refers to a DNA or RNA sequence that is complementary to and capable of binding to the CD300c mRNA. This can inhibit essential activities for translation, cytoplasmic translocation, maturation, or any other overall biological function of the CD300c mRNA. The antisense oligonucleotide may be 6 to 100 bases in length, preferably 8 to 60 bases, and more preferably 10 to 40 bases. The antisense oligonucleotide may be synthesized in vitro by conventional methods and administered in vivo, or the antisense oligonucleotide may be synthesized in vivo. One example of synthesizing an antisense oligonucleotide in vitro is using RNA polymerase I. One example of in vivo synthesis of antisense RNA involves using a vector with the origin of the multiple cloning site (MCS) in the opposite direction to allow antisense RNA to be transcribed. Preferably, the antisense RNA sequence includes a translation stop codon, preventing translation into a peptide sequence. The design of antisense oligonucleotides useful in the present invention can be readily produced using methods known in the art, with reference to the base sequence of CD300c.

[0019] In the present invention, the terms "siRNA" and "shRNA" refer to nucleic acid molecules capable of mediating RNA interference or gene silencing, and are used as efficient gene knockdown methods or gene therapy methods because they can suppress the expression of target genes. shRNA forms a hairpin structure by binding between complementary sequences within a single-stranded oligonucleotide, and in vivo, the shRNA is cleaved by Dicer into small RNA fragments of 21 to 25 nucleotides in size, which become siRNA, a double-stranded oligonucleotide, and can specifically bind to mRNA with a complementary sequence to suppress its expression. Therefore, which means between shRNA and siRNA to use can be determined by the choice of those skilled in the art, and if the mRNA sequences they target are the same, a similar expression reduction effect can be expected. For the purpose of the present invention, CD300c can be inhibited by specifically acting on CD300c to cleave CD300c mRNA molecules and inducing RNA interference (RNAi). siRNA can be synthesized chemically or enzymatically. The method for producing siRNA is not particularly limited, and methods known in the art can be used. For example, there are, but are not limited to, a method of directly chemically synthesizing siRNA, a method of synthesizing siRNA using in vitro transcription, a method of cleaving long double-stranded RNA synthesized by in vitro transcription using an enzyme, an expression method through intracellular delivery of an shRNA expression plasmid or viral vector, and an expression method through intracellular delivery of a PCR (polymerase chain reaction)-induced siRNA expression cassette.

[0020] In addition, in the present invention, the antibody or antigen-binding fragment thereof that inhibits the activity of the CD300c protein may be characterized by specifically binding to the extracellular domain of CD300c (e.g., amino acids 1 to 183 of SEQ ID NO: 3 or amino acids 22 to 188 of SEQ ID NO: 56) and inhibiting the binding of TCTP (Translationally Controlled Tumor Protein) to CD300c.

[0021] As used herein, the term "antibody" refers to a protein molecule that acts as a receptor that specifically recognizes an antigen, including an immunoglobulin molecule that immunologically has reactivity with a specific antigen, and examples thereof may include monoclonal antibodies, polyclonal antibodies, full-length antibodies, and antibody fragments. The term may also include bivalent or dual-specific molecules (e.g., bispecific antibodies), diabodies, triabodies, or tetrabodies.

[0022] As used herein, the term "monoclonal antibody" refers to an antibody molecule of a single molecular composition obtained from a substantially identical antibody population, and such monoclonal antibodies exhibit a single binding specificity and affinity for a specific epitope. As used herein, the term "full-length antibody" has a structure having two full-length light chains and two full-length heavy chains, each light chain being linked to a heavy chain by a disulfide bond. The heavy chain constant region has gamma (γ), mu (μ), alpha (α), delta (δ), and epsilon (ε) types and has gamma1 (γ1), gamma2 (γ2), gamma3 (γ3), gamma4 (γ4), alpha1 (α1), and alpha2 (α2) as subclasses. The constant region of the light chain has kappa (κ) and lambda (λ) types. IgG includes IgG1, IgG2, IgG3, and IgG4 as subtypes.

[0023] In the present invention, the terms "fragment," "antibody fragment," and "antigen-binding fragment" are used interchangeably to refer to any fragment of an antibody of the present invention that retains the antigen-binding function of the antibody. Exemplary antigen-binding fragments include, but are not limited to, Fab, Fab', F(ab')2, Fv, and the like.

[0024] The Fab has a structure with variable regions of the light and heavy chains, a constant region of the light chain, and the first constant region (CH1 domain) of the heavy chain, and has one antigen-binding site. An antigen-binding fragment of an antibody molecule or antibody fragment refers to a fragment that has an antigen-binding function, and Fab' differs from Fab in that it has a hinge region containing one or more cysteine ​​residues at the C-terminus of the CH1 domain of the heavy chain. F(ab')2 antibody is produced when the cysteine ​​residues in the hinge region of Fab' form a disulfide bond. Fv is the minimum antibody fragment that has only a heavy chain variable region and a light chain variable region, and recombinant techniques for producing Fv fragments are disclosed in PCT International Patent Publication Nos. WO 88 / 10649, WO 88 / 106630, WO 88 / 07085, WO 88 / 07086, and WO 88 / 09344, etc. A two-chain Fv has a heavy chain variable region and a light chain variable region linked non-covalently, and a single-chain Fv generally has a heavy chain variable region and a single chain variable region covalently linked via a peptide linker or directly linked at the C-terminus, so that they can form a dimer-like structure like a two-chain Fv. Although not limited thereto, these antibody fragments can be obtained using proteolytic enzymes (for example, restriction digestion of a whole antibody with papain can yield a Fab fragment, and digestion with pepsin can yield an F(ab')2 fragment), or can be produced through genetic recombination technology.

[0025] In the present invention, the allergic disease may be, but is not limited to, asthma, bronchitis, chronic obstructive pulmonary disease, bronchiectasis, rhinitis, atopic dermatitis, urticaria, hay fever, conjunctivitis, or anaphylaxis.

[0026] In the present invention, the inflammatory disease may be, but is not limited to, rheumatoid arthritis, bronchitis, pneumonia, arthritis, nephritis, psoriasis, dermatitis, Crohn's disease, enteritis, gingivitis, arteriosclerosis, coronary arteritis, hepatitis, Behcet's disease, bladder cancer, prostatitis, pyelonephritis, glomerulonephritis, osteomyelitis, thyroiditis, uveitis, peritonitis, meningitis, or pulmonary fibrosis.

[0027] IL-8 increased by TCTP is associated with asthma or bronchitis (Chanez et al., Int Arch Allergy Immunol, 111, 83-88, 1996), chronic obstructive pulmonary disease (Nocker et al., Int Arch Allergy Immunol, 109, 183-191, 1996), bronchiectasis (Simpson et al., Thorax, 62, 211-218, 2007), rhinitis (Benson et al., Pediatr Allergy Immunol, 10, 178-185, 1999; Kuna et al., J Allergy Clin Immun, 97, 104-112, 1996), atopic dermatitis (Kimata & Lindley, Arch Dis Child 70,119-122, 1994), It is known to be involved in various allergic diseases such as urticaria (Choi et al., J Clin Immunol, 28, 244-249, 2008), hay fever (Ciprandi et al., Otolaryngol Head Neck Surg, 133, 429-435, 2005), conjunctivitis (Miyoshi et al., Cornea, 20, 743-747, 2001), and anaphylaxis (Cho et al., Front Pharmacol., 12, 764321-764329, 2021).

[0028] In addition, IL-8 has been shown to be involved in chronic inflammatory bronchial diseases such as chronic bronchitis (Richman-Eisenstat et al., Am J Physiol, 264, L413-418, 1993), inflammatory lung diseases such as pneumonia (Erger and Casale, Eur Respir J, 11, 299-305, 1998; Pease & Sabroe, Am J Respir Med, 1, 19-25, 2002), arthritis or nephritis (Harada et al., J Leukoc Biol, 56, 559-564, 1994), psoriasis (Schulz et al., J Immunol, 151, 4399-4406, 1993; Bruch-Gerharz et al., J Exp Med, 184, 2007-2012, 1996), and dermatitis. (Sticherling et al., Arch Dermatol Res, 284, 82-85, 1992), Crohn's disease (Izutani et al., Inflamm Bowel Dis, 1, 37-47, 1995), inflammatory bowel disease (Mitsuyama et al., Clin Exp Immunol, 96, 432-436, 1994), gingivitis (Haake & Huang, Clinical Periodontology, 9th Edition. Philadelphia: WBSaunders Co. 2002. page 162), cardiovascular diseases such as arteriosclerosis and coronary artery disease (Apostolakis et al., Cardiovasc Res, 84, 353-360, 2009; Boekholdt et al., Arterioscler Thromb Vasc Biol, 24, 1503-1508, 2004), chronic liver disease (Zimmermann et al., PLoS ONE, 6, e21381, 2011), Behcet's disease (Katsantonis et al., Dermatology, 201, 37-39, 2000), bladder cancer, prostatitis, pyelonephritis or osteomyelitis (Shahzad et al., Int arch med, 3, 11, 2010), thyroid disease (Kobawala et al., J Thyroid Res, 8, 270149, 2011), uveitis (Klok et al., Br J Ophthalmol, 82, 871-874, 1998), glomerulonephritis, peritonitis, meningitis, and pulmonary fibrosis (Harada et al., Mol Med Today, 2, 482-489, 1996). Therefore, inhibition of IL-8 has been suggested as a therapeutic strategy in inflammatory diseases such as lung disease, rheumatoid arthritis, inflammatory bowel disease, psoriasis, chronic inflammatory skin diseases such as palmoplantar pustulosis, and ocular inflammation (Mukaida, Am J Physiol Lung Cell Mol Physiol, 284, L566-L577, 2003; Skov et al., J Immunol, 181, 669-679, 2008; Harada et al., J Leukoc Biol, 56, 559-564, 1994). Treatments that block IL-8 antibodies or inhibit the gene encoding the IL-8 receptor have anti-inflammatory effects (Harada et al., Mol Med Today, 2, 482-489, 1996), and for example, administration of antibodies against IL-8 reduced inflammation in patients with chronic inflammatory skin diseases (Skov et al., J Immunol, 181, 669-679, 2008). GM-CSF, whose secretion is increased by TCTP, is also associated with various inflammatory diseases (Hamilton, Trends Immunol, 23, 403-408, 2002), and GM-CSF has also been suggested as a target for inflammatory diseases such as rheumatoid arthritis (Cornish et al., Nat Rev Rheumatol, 5, 554-559, 2009).

[0029] In addition, it was discovered in 1998 that the antimalarial drug artemisinin acts by binding to the malarial protein TCTP (Bhisutthibhan et al., J Biol Chem, 273, 16192-16198, 1998). IL-8 is also secreted by malaria patients (Friedland et al., Trans R Soc Trop Med Hyg, 87, 54-55, 1993), and it has been reported that HRF of malaria promotes IL-8 secretion (MacDonald et al., Proc Natl Acad Sci USA, 98, 10829-32, 2001).

[0030] The above-mentioned previous research results show that targeting CD300c, the receptor for TCTP, can prevent and treat various allergic diseases, inflammatory diseases, and malaria.

[0031] As used herein, the term "treatment" refers to clinical intervention to alter the natural processes of an individual or cell to be treated, and may be performed during the progression of a clinical pathological condition or to prevent it. The desired therapeutic effect includes preventing the occurrence or recurrence of a disease, alleviating symptoms, reducing all direct or indirect pathological consequences of the disease, preventing metastasis, slowing the progression of the disease, alleviating or temporarily alleviating the disease state, and causing remission or improving the prognosis. Preferably, the present invention includes all actions that improve the course of a disease by administering a composition comprising a substance that inhibits CD300c. In addition, "prevention" refers to all actions that inhibit or delay the onset of the disease by administering a composition comprising a substance that inhibits CD300c according to the present invention.

[0032] The pharmaceutical composition of the present invention may additionally include a suitable carrier, excipient, or diluent commonly used in the manufacture of pharmaceutical compositions. A composition including a pharmaceutically acceptable carrier may be in various oral or parenteral dosage forms. When formulated, the composition may be prepared using diluents or excipients such as fillers, bulking agents, binders, wetting agents, disintegrants, and surfactants commonly used. Solid preparations for oral administration may include tablets, pills, powders, granules, capsules, etc., and such solid preparations may be prepared by mixing one or more compounds with at least one excipient, such as starch, calcium carbonate, sucrose or lactose, gelatin, etc. In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used. Liquid preparations for oral administration include suspensions, solutions, emulsions, and syrups. In addition to commonly used simple diluents such as water and liquid paraffin, they may contain various excipients such as wetting agents, sweeteners, fragrances, and preservatives. Preparations for parenteral administration may include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, and suppositories. Non-aqueous solvents and suspensions may include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. Suppository bases may include witepsol, macrogol, Tween 61, cacao butter, laurin butter, and glycerogelatin.

[0033] In addition, the pharmaceutical composition of the present invention may have any one dosage form selected from the group consisting of, but not limited to, tablets, pills, powders, granules, capsules, suspensions, liquid solutions, emulsions, syrups, sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized preparations, and suppositories.

[0034]

[0035] Another aspect of the present invention for achieving the above purpose is an anti-CD300c antibody or an antigen-binding fragment thereof, characterized in that it specifically binds to the extracellular domain of CD300c (amino acids 1 to 183 of SEQ ID NO: 3 or amino acids 22 to 188 of SEQ ID NO: 56) and inhibits the binding of TCTP (Translationally Controlled Tumor Protein) to CD300c.

[0036] According to a specific example of the present invention, the antibody or antigen-binding fragment thereof,

[0037] (a) a light chain variable region comprising a light chain CDR1 as set forth in SEQ ID NO: 9 or SEQ ID NO: 34; a light chain CDR2 as set forth in SEQ ID NO: 10 or SEQ ID NO: 35; and a light chain CDR3 as set forth in SEQ ID NO: 11 or SEQ ID NO: 36, and a heavy chain variable region comprising a heavy chain CDR1 as set forth in SEQ ID NO: 12 or SEQ ID NO: 27; a heavy chain CDR2 as set forth in SEQ ID NO: 13 or SEQ ID NO: 28; and a heavy chain CDR3 as set forth in SEQ ID NO: 14, SEQ ID NO: 22, SEQ ID NO: 44, or SEQ ID NO: 51, or

[0038] (b) at least one amino acid sequence in (a) is substituted, wherein the F at the 2nd position of the heavy chain CDR1 set forth in SEQ ID NO: 27 is replaced with Y; the N at the 3rd position is replaced with T; the R at the 5th position is replaced with G, T, or S; the N at the 6th position is replaced with G or S; or the A at the 8th position is replaced with G; the N at the 2nd position of the heavy chain CDR2 set forth in SEQ ID NO: 28 is replaced with S; the G at the 3rd position is replaced with W or V; the R at the 4th position is replaced with N, Y, or S; the G at the 5th position is replaced with N; the D at the 6th position is replaced with G; the T at the 7th position is replaced with D, N, or S; or the T at the 8th position is replaced with I; or the R at the 2nd position of the heavy chain CDR3 set forth in SEQ ID NO: 51 is replaced with S; the G at the 3rd position is replaced with E; the P at the 4th position is replaced with A or D; the Y at the 5th position is replaced with S; the Y at the 6th position is replaced with SS; and the F at the 7th position is replaced with Y. An anti-CD300c antibody or antigen-binding fragment thereof, wherein D at position 8 is replaced with N, wherein the heavy chain CDR1 comprises 0 to 5 substitutions, the heavy chain CDR2 comprises 0 to 6 substitutions, and the heavy chain CDR3 comprises 0 to 4 substitutions (provided that the heavy chain CDR1, the heavy chain CDR2, or the heavy chain CDR3 comprises at least one substitution).

[0039] In the present invention, the term "heavy chain" may include both a full-length heavy chain and fragments thereof, which include a variable domain VH comprising an amino acid sequence having sufficient variable region sequence to confer specificity to an antigen, and three constant region domains CH1, CH2, and CH3. In addition, the term "light chain" in the present invention may include both a full-length light chain and fragments thereof, which include a variable domain VL comprising an amino acid sequence having sufficient variable region sequence to confer specificity to an antigen, and a constant region domain CL.

[0040] In the present invention, the antibody may include both mouse antibodies produced from mice and variants in which a portion of the amino acid sequence of the parent antibody is substituted, added, and / or deleted to improve the affinity, immunogenicity, etc. of the antibody. The variants are not limited thereto, but examples thereof may include chimeric antibodies, humanized antibodies, affinity-optimized antibodies, etc.

[0041] In the present invention, the above variants comprehensively refer to antibodies that contain the same CDRs as the parent antibody, or in which a portion of the parent antibody CDR amino acid sequence is mutated (substituted, added, or deleted) under the condition that they target the same epitope. Such variants can be appropriately adjusted by those skilled in the art to improve the affinity and immunogenicity of the antibody, etc., within the range where binding ability to the same epitope is maintained.

[0042] In other words, the antibody or antigen-binding fragment thereof of the present invention may include not only the sequence of the anti-CD300c antibody described herein, but also biological equivalents thereof, as long as it can specifically recognize CD300c. For example, additional changes may be made to the amino acid sequence of the antibody to further improve the binding affinity and / or other biological properties of the antibody. Such modifications include, for example, deletions, insertions, and / or substitutions of amino acid sequence residues of the antibody. Such amino acid mutations are made based on the relative similarity of the amino acid side chain substituents, such as hydrophobicity, hydrophilicity, charge, size, etc. Analysis of the size, shape, and type of amino acid side chain substituents reveals that arginine, lysine, and histidine are all positively charged residues; alanine, glycine, and serine have similar sizes; and phenylalanine, tryptophan, and tyrosine have similar shapes. Therefore, based on these considerations, arginine, lysine, and histidine; Alanine, glycine and serine; and phenylalanine, tryptophan and tyrosine are biologically functional equivalents.

[0043] In the present invention, the term "chimeric antibody" is an antibody obtained by recombining the variable region of a mouse antibody and the constant region of a human antibody, and is an antibody with a greatly improved immune response compared to a mouse antibody.

[0044] As used herein, the term "humanized antibody" refers to an antibody in which the protein sequence of an antibody derived from a non-human species is modified to resemble an antibody variant naturally produced in humans. For example, the humanized antibody can be produced by recombining a mouse-derived CDR with a human antibody-derived FR to produce a humanized variable region, which is then recombined with a desired human antibody constant region. However, since simple CDR grafting reduces the affinity of the humanized antibody, the affinity of the humanized antibody can be raised to the same level as the original mouse antibody by grafting several key FR amino acid residues, which are thought to affect the three-dimensional structure of the CDR, to those of a mouse antibody.

[0045] In one embodiment of the present invention, the antibody may include a light chain variable region comprising a light chain CDR1 as set forth in SEQ ID NO: 9 or SEQ ID NO: 34; a light chain CDR2 as set forth in SEQ ID NO: 10 or SEQ ID NO: 35; and a light chain CDR3 as set forth in SEQ ID NO: 11 or SEQ ID NO: 36; and a heavy chain variable region comprising a heavy chain CDR1 as set forth in SEQ ID NO: 12, SEQ ID NO: 20, SEQ ID NO: 27, SEQ ID NO: 37, or SEQ ID NO: 49; a heavy chain CDR2 as set forth in SEQ ID NO: 13, SEQ ID NO: 21, SEQ ID NO: 28, SEQ ID NO: 38, or SEQ ID NO: 50; and a heavy chain CDR3 as set forth in SEQ ID NO: 14, SEQ ID NO: 22, SEQ ID NO: 29, SEQ ID NO: 39, SEQ ID NO: 44, or SEQ ID NO: 51, but is not limited thereto.

[0046] In another embodiment of the present invention, the antibody may include, but is not limited to, a light chain variable region as set forth in SEQ ID NO: 7, SEQ ID NO: 18, SEQ ID NO: 25, SEQ ID NO: 32, SEQ ID NO: 42, SEQ ID NO: 47, or SEQ ID NO: 54, and a heavy chain variable region as set forth in SEQ ID NO: 8, SEQ ID NO: 19, SEQ ID NO: 26, SEQ ID NO: 33, SEQ ID NO: 43, SEQ ID NO: 48, or SEQ ID NO: 55.

[0047] In another embodiment of the present invention, the antibody may further comprise a linker as set forth in SEQ ID NO: 15, and may be specifically composed of an amino acid sequence as set forth in SEQ ID NO: 6, SEQ ID NO: 17, SEQ ID NO: 24, SEQ ID NO: 31, SEQ ID NO: 41, SEQ ID NO: 46, or SEQ ID NO: 53, but is not limited thereto.

[0048] According to a specific example of the present invention, the antibody or antigen-binding fragment thereof,

[0049] Light chain CDR1 described by SEQ ID NO: 61;

[0050] light chain CDR2 as described by SEQ ID NO: 62; and

[0051] A light chain variable region comprising a light chain CDR3 as set forth in SEQ ID NO: 63; and

[0052] Heavy chain CDR1 as described by SEQ ID NO: 64;

[0053] Heavy chain CDR2 as described by SEQ ID NO: 65; and

[0054] It may include a heavy chain variable region comprising a heavy chain CDR3 described by sequence number 66.

[0055] As one embodiment of the present invention, the antibody may include, but is not limited to, a light chain variable region as described in SEQ ID NO: 59 and a heavy chain variable region as described in SEQ ID NO: 60.

[0056] In another embodiment of the present invention, the antibody may further comprise a linker as set forth in SEQ ID NO: 15, and may be specifically composed of an amino acid sequence as set forth in SEQ ID NO: 58, but is not limited thereto.

[0057] Another aspect of the present invention is a nucleic acid molecule encoding the antibody or an antigen-binding fragment thereof, an expression vector comprising the nucleic acid molecule, and a host cell into which the expression vector has been introduced.

[0058] The above antibodies and antigen-binding fragments thereof are as described above.

[0059] The term "nucleic acid molecule" used in the present invention has a meaning that comprehensively includes DNA and RNA molecules, and the nucleotides, which are the basic structural units of the nucleic acid molecule, include not only natural nucleotides but also analogs in which the sugar or base portion is modified (Scheit, Nucleotide Analogs, John Wiley, New York (1980); Uhlman and Peyman, Chemical Reviews, (1990) 90:543-584). The sequence of the nucleic acid molecule encoding the heavy and light chain variable regions of the present invention may be modified, and the modifications include additions, deletions, or non-conservative or conservative substitutions of nucleotides.

[0060] The nucleic acid molecule of the present invention is also interpreted to include a nucleotide sequence that exhibits substantial identity with the nucleotide sequence described above. In the present invention, substantial identity means a nucleotide sequence that exhibits at least 80% homology, specifically at least 90% homology, and more specifically at least 95% homology, when the nucleotide sequence of the present invention is aligned with any other sequence to the greatest extent possible and the aligned sequence is analyzed using an algorithm commonly used in the art.

[0061] The term "vector" as used herein refers to a means for expressing a target gene in a host cell, including, but not limited to, a plasmid vector; a cosmid vector; and a viral vector such as a bacteriophage vector, an adenovirus vector, a retrovirus vector, and an adeno-associated virus vector, and may specifically be a plasmid vector.

[0062] In the vector of the present invention, the nucleic acid molecule encoding the light chain variable region and the nucleic acid molecule encoding the heavy chain variable region may be operably linked to a promoter.

[0063] As used herein, the term "operably linked" means a functional linkage between a nucleic acid expression regulatory sequence (e.g., a promoter, a signal sequence, or an array of transcription regulatory factor binding sites) and another nucleic acid sequence, whereby the regulatory sequence regulates transcription and / or translation of the other nucleic acid sequence.

[0064] The recombinant vector system of the present invention can be constructed using various methods known in the art. For example, specific methods thereof are disclosed in Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press (2001), which is incorporated herein by reference.

[0065] The vector of the present invention can typically be constructed as a vector for cloning or as a vector for expression. In addition, the vector of the present invention can be constructed using a prokaryotic cell or a eukaryotic cell as a host.

[0066] For example, when the vector of the present invention is an expression vector and uses a prokaryotic cell as a host, it is common to include a strong promoter capable of initiating transcription (e.g., tac promoter, lac promoter, lacUV5 promoter, lpp promoter, pLλ promoter, pRλ promoter, rac5 promoter, amp promoter, recA promoter, SP6 promoter, trp promoter, and T7 promoter, etc.), a ribosome binding site for initiating translation, and a transcription / translation termination sequence. When E. coli (e.g., HB101, BL21, DH5α, etc.) is used as a host cell, the promoter and operator region of the E. coli tryptophan biosynthetic pathway (Yanofsky, C., J. Bacteriol., (1984) 158:1018-1024) and the left-hand promoter of phage λ (pLλ promoter, Herskowitz, I. and Hagen, D., Ann. Rev. Genet., (1980) 14:399-445) can be used as regulatory regions. When Bacillus is used as a host cell, the promoter of the toxin protein gene of Bacillus thuringiensis (Appl. Environ. Microbiol. (1998) 64:3932-3938; Mol. Gen. Genet. (1996) 250:734-741) or any promoter that can be expressed in Bacillus can be used as a regulatory region.

[0067] Meanwhile, the recombinant vector of the present invention can be produced by manipulating plasmids (e.g., pCL, pSC101, pGV1106, pACYC177, ColE1, pKT230, pME290, pBR322, pUC8 / 9, pUC6, pBD9, pHC79, pIJ61, pLAFR1, pHV14, pGEX series, pET series, and pUC19, etc.), phages (e.g., λgt4·λB, λ-Charon, λΔz1, and M13, etc.), or viruses (e.g., SV40, etc.) that are frequently used in the art. For example, the recombinant vector of the present invention can be produced by manipulating a pCL expression vector, specifically, a pCLS05 (Korean Patent No. 10-1420274) expression vector, but is not limited thereto.

[0068] Meanwhile, when the vector of the present invention is an expression vector and uses a eukaryotic cell as a host, a promoter derived from the genome of a mammalian cell (e.g., metallothionine promoter, β-actin promoter, human hemoglobin promoter, and human muscle creatine promoter) or a promoter derived from a mammalian virus (e.g., adenovirus late promoter, vaccinia virus 7.5K promoter, SV40 promoter, cytomegalovirus (CMV) promoter, HSV tk promoter, mouse mammary tumor virus (MMTV) promoter, HIV LTR promoter, Moloney virus promoter, Epstein-Barr virus (EBV) promoter, and Rous sarcoma virus (RSV) promoter) can be used, and generally has a polyadenylation sequence as a transcription termination sequence. Specifically, the recombinant vector of the present invention includes a CMV promoter.

[0069] The recombinant vector of the present invention can be fused with other sequences to facilitate the purification of antibodies expressed therefrom. Examples of such fusion sequences include glutathione S-transferase (Pharmacia, USA), maltose binding protein (NEB, USA), FLAG (IBI, USA), and 6x His (hexahistidine; Quiagen, USA). Furthermore, since the protein expressed by the vector of the present invention is an antibody, the expressed antibody can be easily purified using a protein A column or the like, without the need for additional purification sequences.

[0070] Meanwhile, the recombinant vector of the present invention includes an antibiotic resistance gene commonly used in the art as a selection marker, and may include, for example, a resistance gene for ampicillin, gentamicin, carbenicillin, chloramphenicol, streptomycin, kanamycin, geneticin, neomycin, and tetracycline.

[0071] The vector expressing the antibody of the present invention can be either a vector system in which the light chain and the heavy chain are simultaneously expressed from a single vector, or a system in which the light chain and the heavy chain are each expressed from separate vectors. In the latter case, the two vectors can be introduced into a host cell, for example, through co-transformation or targeted transformation. Co-transformation is a method in which vector DNA encoding the light chain and the heavy chain are simultaneously introduced into a host cell, and then cells expressing both the light chain and the heavy chain are selected. Targeted transformation is a method in which cells transformed with a vector containing a light chain (or heavy chain) are selected, and the selected cells are transformed again with a vector containing a heavy chain (or light chain), thereby finally selecting cells expressing both the light chain and the heavy chain.

[0072] Any host cell known in the art that can stably and continuously clone and express the vector of the present invention may be used, and examples thereof include, but are not limited to, prokaryotic host cells such as Bacillus strains such as Escherichia coli, Bacillus subtilis, and Bacillus thuringiensis, Streptomyces, Pseudomonas (e.g., Pseudomonas putida), Proteus mirabilis, or Staphylococcus (e.g., Staphylococcus carnosus).

[0073] Suitable eukaryotic host cells for the above vector may include fungi such as Aspergillus species, yeasts such as Pichia pastoris, Saccharomyces cerevisiae, Schizosaccharomyces and Neurospora crassa, other lower eukaryotic cells, higher eukaryotic cells such as insect-derived cells, and cells derived from plants or mammals.

[0074] Specifically, the host cell may be COS7 cells (monkey kidney cells), NSO cells, SP2 / 0, Chinese hamster ovary (CHO) cells, W138, baby hamster kidney (BHK) cells, MDCK, a myeloma cell line, HuT 78 cells or 293 cells, and more specifically, may be Chinese hamster ovary cells, but is not limited thereto.

[0075] In the present invention, "transformation" and / or "transfection" into a host cell includes any method for introducing a nucleic acid into an organism, cell, tissue, or organ, and can be performed by selecting a standard technique suitable for the host cell as known in the art. Such methods include, but are not limited to, electroporation, protoplast fusion, calcium phosphate (CaPO4) precipitation, calcium chloride (CaCl2) precipitation, stirring using silicon carbide fibers, Agrobacterium-mediated transformation, PEG, dextran sulfate, lipofectamine, and desiccation / inhibition-mediated transformation methods.

[0076]

[0077] Another aspect of the present invention is a method for screening a drug for preventing or treating an allergic disease, an inflammatory disease, or malaria, comprising the steps of: (a) treating isolated cells expressing CD300c with dimeric Translationally Controlled Tumor Protein (dTCTP) and a candidate substance; (b) measuring the activity of the NF-κB signaling pathway or the level of IL-8 secretion in the isolated cells treated with the candidate substance; and (c) comparing the activity of the NF-κB signaling pathway or the level of IL-8 secretion measured in step (b) with the level in isolated cells not treated with the candidate substance, thereby selecting a candidate substance that reduces the activity of the NF-κB signaling pathway or the level of IL-8 secretion. Here, the expression of CD300c includes both naturally expressing CD300c and artificially expressing CD300c.

[0078] In the absence of a candidate substance capable of preventing or treating an allergic disease, an inflammatory disease, or malaria, the activity of the NF-κB signaling pathway or the level of IL-8 secretion in a cell is measured, and further, in the presence of the candidate substance, the activity of the signaling pathway or the level of IL-8 secretion of the present invention is measured and compared, and a substance that reduces the activity of the signaling pathway or the level of IL-8 secretion of the present invention in the presence of the candidate substance compared to the level in the absence of the candidate substance can be predicted as an agent for preventing or treating an allergic disease, an inflammatory disease, or malaria.

[0079] Candidate substances may be known substances or novel substances, and large-scale screening can be performed, for example, using plant extracts or chemical libraries.

[0080]

[0081] Another aspect of the present invention is a method for providing information for diagnosing an allergic disease, an inflammatory disease, or malaria, comprising the steps of: (a) measuring the expression or secretion level of CD300c from an isolated biological sample; and (b) comparing the expression or secretion level with the CD300c expression or secretion level of a normal control sample.

[0082] Another aspect of the present invention is a composition for diagnosing allergic diseases, inflammatory diseases, or malaria, comprising a preparation for measuring the expression or secretion level of CD300c.

[0083] As confirmed through specific examples of the present invention, by treating with dimeric TCTP, the expression of CD300c increases, and conversely, when the expression or activity of CD300c is inhibited, the signal transduction pathway and cytokine secretion induced by the TCTP dimer decrease, so that by confirming the expression or secretion level of CD300c from a separated biological sample, it is possible to diagnose TCTP-related diseases, i.e., allergic diseases, inflammatory diseases, or malaria.

[0084] In the present invention, the term "diagnosis" means confirming the presence or characteristics of an allergic disease, an inflammatory disease, or malaria by measuring the presence or absence of CD300c of the present invention in a biological sample or tissue sample. In addition, a "marker or diagnostic marker" is a substance that can diagnose an individual with an allergic disease, an inflammatory disease, or malaria by distinguishing it from a normal cell or a normal individual, and includes organic biomolecules such as polypeptides, proteins, or nucleic acids (e.g., mRNA, etc.), lipids, glycolipids, glycoproteins, or sugars (monosaccharides, disaccharides, oligosaccharides, etc.) that show an increase or decrease in cells or individuals with an allergic disease, an inflammatory disease, or malaria compared to normal cells. For the purposes of the present invention, the diagnostic marker for an allergic disease, an inflammatory disease, or malaria of the present invention is CD300c that shows a specifically high level of expression in cells with an allergic disease, an inflammatory disease, or malaria compared to cells of normal cells or tissues.

[0085] In the present invention, measuring the CD300c expression level may be measuring the mRNA expression level of CD300c or measuring the CD300c protein expression level.

[0086] The above "measurement of mRNA expression level" refers to the process of confirming the presence and expression level of mRNA of an allergic disease, inflammatory disease, or malaria marker gene in a biological sample for the purpose of diagnosing allergic disease, inflammatory disease, or malaria, and can be determined by measuring the amount of mRNA. Analysis methods for this include, but are not limited to, RT-PCR, competitive RT-PCR, real-time RT-PCR, RNase protection assay (RPA), Northern blotting, DNA chips, etc.

[0087] The above "measurement of protein expression level" refers to a process of confirming the presence and expression level of a protein expressed in an allergic disease, inflammatory disease, or malaria marker gene in a biological sample in order to diagnose an allergic disease, inflammatory disease, or malaria, and confirming the amount of the protein using an antibody that specifically binds to the protein of the gene. Analysis methods for this purpose include, but are not limited to, Western blot, ELISA (enzyme linked immunosorbent assay), radioimmunoassay (RIA), radioimmunodiffusion, Ouchterlony immunodiffusion, rocket immunoelectrophoresis, tissue immunostaining, immunoprecipitation assay, complement fixation assay, FACS, protein chip, etc.

[0088] For example, the agent for measuring the mRNA level may be a primer pair, probe, or antisense nucleotide for the mRNA of CD300c of the present invention, and a person skilled in the art can easily design the primer, probe, or antisense nucleotide sequence based on the polynucleotide sequence of CD300c of the present invention. As another example, the agent for measuring the protein level may be an antibody.

[0089]

[0090] The embodiments of the present invention may be modified in various ways, and the scope of the present invention is not limited to the embodiments described below. Furthermore, the embodiments of the present invention are provided to more completely explain the present invention to those skilled in the art. Furthermore, throughout the specification, the term "including" a certain component does not exclude other components, but rather implies the inclusion of other components, unless specifically stated otherwise.

[0091]

[0092] In the present invention, it was confirmed that the CD300c extracellular domain interacts with dimeric TCTP, and thus CD300c can be usefully used in the development of agents for the prevention or treatment of allergic diseases, inflammatory diseases, or malaria. Furthermore, the anti-CD300c antibody of the present invention can be usefully used in the prevention or treatment of allergic diseases, inflammatory diseases, or malaria, or can be usefully used in the screening or diagnosis of drugs related to allergic diseases, inflammatory diseases, or malaria.

[0093]

[0094] Figure 1 shows the results of a Western blot analysis of the interaction of the CD300c extracellular domain or control protein with dimeric or monomeric TCTP through a pull-down analysis.

[0095] Figure 2 is a graph showing the IL-8 secretion ability of bronchial epithelial cells treated after preincubation with dimeric TCTP and various concentrations of CD300c extracellular domain.

[0096] Figure 3 shows the results of Western blot analysis of changes in CD300c expression over time in bronchial epithelial cells treated with dimeric TCTP.

[0097] Figure 4a shows the results of Western blot analysis to confirm the knockdown efficiency of CD300c in bronchial epithelial cells.

[0098] Figure 4b is a graph showing the results of comparing the cytokine secretion capacity by dimeric TCTP treatment according to the knockdown of CD300c in bronchial epithelial cells.

[0099] Figure 5 shows the results of measuring NF-κB activity by dimeric TCTP treatment following overexpression of CD300c in a cell line expressing an NF-κB activity-dependent reporter gene.

[0100] Figure 6a is a graph showing the results of a primary screening to confirm the inhibitory effect of anti-CD300c scFv-Fc antibody on dimeric TCTP in bronchial epithelial cells.

[0101] Figure 6b is a graph showing the results of comparing the binding affinity of four anti-CD300c scFv-Fc antibodies to CD300c protein through ELISA.

[0102] Figure 7a is a graph showing the results of a secondary screening to confirm the inhibitory effect of anti-CD300c scFv-Fc antibody on dimeric TCTP in bronchial epithelial cells.

[0103] Figure 7b is a graph showing the results comparing the binding affinity of three anti-CD300c scFv-Fc antibodies to CD300c protein with the JEWR-195 antibody.

[0104] Figure 8 is a diagram showing the amino acid sequences of seven types of anti-CD300c scFv-Fc antibodies.

[0105] Figure 9 is a diagram showing the results of SDS-PAGE and Coomassie brilliant blue staining of the JEWR-195 antibody under reducing and non-reducing conditions.

[0106] Figure 10 shows the results of flow cytometry analysis confirming that the JEWR-195 antibody binds to CD300c on the cell surface in HEK293T cells overexpressing CD300c.

[0107] Figure 11 shows the results of a Western blot analysis of the effect of JEWR-195 antibody on the NF-κB signaling pathway of dimeric TCTP in bronchial epithelial cells.

[0108] Figure 12 is a graph showing the results of comparing the binding affinity of four anti-CD300c scFv-Fc antibodies to mouse CD300c protein through ELISA.

[0109] Figure 13 is a diagram showing the results of SDS-PAGE and Coomassie Brilliant Blue staining of the JEWR-M10 antibody under reducing and non-reducing conditions.

[0110] Figure 14 is a diagram showing the method for producing an asthma mouse model and the timing of JEWR-M10 antibody administration.

[0111] Figure 15 is a graph showing the results of staining cells in the bronchoalveolar lavage fluid of a mouse with Diff-Quick and measuring the total number of cells.

[0112] Figure 16a is a diagram showing the results of analyzing mouse lung tissue using PAS staining.

[0113] Figure 16b is a graph showing the results of measuring the amount of MUC5AC produced in the bronchoalveolar lavage fluid of a mouse using ELISA.

[0114] Figure 17a is a graph showing the results of measuring the production of IL-4, IL-5, and IL-13 in the lung tissue of a mouse using ELISA.

[0115] Figure 17b is a graph showing the results of measuring the production of IL-4, IL-5, and IL-13 in the bronchoalveolar lavage fluid of mice using ELISA.

[0116] The present inventors have identified CD300c, a receptor for dimeric TCTP (Translationally Controlled Tumor Protein), as a cell membrane receptor for the first time. Inhibiting CD300c effectively suppresses the cytokine-like activity of dimeric TCTP by inhibiting its interaction with the protein. Therefore, it can be used to target various cells expressing CD300c and effectively treat diseases associated with dimeric TCTP, such as allergic diseases, inflammatory diseases, and malaria.

[0117]

[0118] Hereinafter, the composition and effects of the present invention will be described in more detail through examples. These examples are intended solely to illustrate the present invention and are not intended to limit the scope of the present invention.

[0119]

[0120] <Example 1> Expression and purification of recombinant dimeric TCTP protein

[0121] To produce a dimeric TCTP protein, a pRSET A (Invitrogen) expression vector containing a gene (SEQ ID NO: 2) with 10 N-terminal amino acids deleted from TCTP (SEQ ID NO: 1) was constructed, and this was transformed into E. coliBL21(DE3)pLysS (Merck) and cultured to induce synthesis of the recombinant protein.

[0122] After centrifugation of the culture medium, the resulting pellet was resuspended in a solution of 20 mM Tris-HCl, 300 mM NaCl, 5 mM Imidazole, and 1 mM PMSF (pH 8.0), and the cells were disrupted using an ultrasonicator. The supernatant obtained through centrifugation was purified using Ni-NTA Agarose (Qiagen). To obtain high-purity protein, the protein was further purified using a HiTrap Q HP anion exchange chromatography column (Cytiva). The column was washed with distilled water and then stabilized by flowing 20 mM Tris-HCl, 50 mM NaCl, and 1 mM EDTA (pH 7.4) for approximately 20 minutes. The previously purified protein was injected into the column, and the protein was eluted at a rate of 0.8 ml / min according to a concentration gradient of 0 to 1 M NaCl. The eluted protein was purified by non-reducing SDS-PAGE and Coomassie Brilliant Blue staining to identify fractions containing dimeric TCTP. The buffer was exchanged for phosphate-buffered saline (PBS) using a PD-10 column (Cytiva), and the protein was concentrated using Amicon Ultra Centrifugal Filters (Millipore).

[0123]

[0124] <Example 2> Confirmation of the interaction between dimeric TCTP and CD300c protein

[0125] To confirm the interaction between dimeric TCTP and CD300c protein, a pull-down assay was performed using Dynabeads Protein A (Invitrogen). A recombinant human CD300c-Fc chimeric protein (BioLegend) was used for the analysis. This protein contains the sequence Met29-Arg183 corresponding to the extracellular domain of CD300c (SEQ ID NO: 3) and a hIgG Fc at the C-terminus, and is expressed in 293E cells. Recombinant human IgG1 Fc protein (BioLegend) was used as a control. Because human immunoglobulin (Ig) binds to protein A beads, the beads and the aforementioned proteins were incubated at room temperature for 30 minutes. After washing three times with PBS containing 0.02% Tween-20, the protein-bound beads were incubated with recombinant dimeric or monomeric TCTP for 1 hour at room temperature. Unbound proteins were removed by three washes, and proteins bound to the beads were eluted and analyzed by Western blot. The assay was performed under non-reducing conditions and detected with an anti-TCTP antibody (Santa Cruz Biotechnology).

[0126] As shown in Fig. 1, the interaction between the CD300c extracellular domain and dimeric TCTP was confirmed by the detection of higher levels of dimeric TCTP than monomeric TCTP on beads bound to the CD300c-Fc protein. In the control beads bound to the IgG1-Fc protein, both monomeric and dimeric TCTP were barely detectable.

[0127]

[0128] <Example 3> Confirmation of the IL-8 secretion inhibitory effect of the CD300c extracellular domain on dimeric TCTP in bronchial epithelial cells.

[0129] The above results confirmed the binding of dimeric TCTP to CD300c protein. Since BEAS-2B cells, a bronchial epithelial cell line, are activated by dimeric TCTP and secrete IL-8 cytokine, the inhibitory efficacy of the CD300c extracellular domain on dimeric TCTP was evaluated using this cell line. BEAS-2B cells were cultured in DMEM medium containing 10% FBS and 1% penicillin-streptomycin at 37°C in the presence of 5% carbon dioxide. Recombinant CD300c protein (Sino Biological) was expressed in HEK293 cells as the extracellular domain Met1-Arg183 of CD300c (SEQ ID NO: 3). CD300c extracellular domain (0–32 μg / ml) was preincubated with dimeric TCTP (8 μg / ml) for 30 min at room temperature and treated onto BEAS-2B cells, which were then cultured for 20 h. The levels of secreted IL-8 in the cell supernatant were quantified using an IL-8 ELISA kit (Biolegend).

[0130] As shown in Fig. 2, the increased IL-8 secretion amount when BEAS-2B cells were treated with dimeric TCTP alone was confirmed to be dose-dependently reduced by pre-incubation with the CD300c extracellular domain. This suggests that the CD300c extracellular domain inhibits intracellular activity by inhibiting the interaction between dimeric TCTP and CD3000c on the cell membrane.

[0131]

[0132] <Example 4> Confirmation of changes in CD300c expression following treatment with dimeric TCTP in bronchial epithelial cells.

[0133] To confirm the change in CD300c protein expression by treatment with dimeric TCTP, BEAS-2B cells were cultured in 6-well plates and treated with dimeric TCTP (8 μg / ml) for 0.5, 1, 2, 4, 8, 20, and 24 h, respectively. Afterwards, the cells were washed with PBS, and the cells were lysed in a buffer containing 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM EDTA (pH 8.0), 0.25% deoxycholate, 1% Triton X-100, phosphatase inhibitor cocktail (Sigma-Aldrich), and protease inhibitor (Roche). The supernatant obtained by centrifugation was concentrated using the Bradford assay, and Western blot analysis was performed using anti-CD300c antibody (Abcam) and anti-GAPDH antibody (Cell Signaling Technology).

[0134] As shown in Fig. 3, it was confirmed that the expression of CD300c increased over time by dimeric TCTP treatment.

[0135]

[0136] <Example 5> Confirmation of changes in cytokine secretion by dimeric TCTP following knockdown of CD300c

[0137] To determine whether the inflammatory response induced by dimeric TCTP is mediated by CD300c, CD300c was knocked down in BEAS-2B cells. CD300c siRNA (Thermo Fisher Scientific) or control siRNA (Santa Cruz Biotechnology) was transfected into cells using Lipofectamine RNAiMAX (Thermo Fisher Scientific) according to the manufacturer's recommended procedure. After 24 h, cells were treated with dimeric TCTP (0, 5, or 10 μg / ml) and cultured for 20 h. The levels of IL-8 and IL-6 secreted into the cell supernatant were quantified using an ELISA kit.

[0138] As shown in Fig. 4a, knockdown of CD300c in BEAS-2B cells resulted in a 50% decrease in CD300c expression.

[0139] As shown in Fig. 4b, the secretion of IL-8 and IL-6, which increased due to dimeric TCTP treatment in the control group (siCtrl), was significantly reduced due to CD300c knockdown. These results confirmed that dimeric TCTP induces an inflammatory response mediated by the CD300c receptor.

[0140]

[0141] <Example 6> Confirmation of NF-κB activation by dimeric TCTP following overexpression of CD300c

[0142] To confirm the regulation of the cell signaling pathway by dimeric TCTP by overexpression of CD300c, HEK293 cells expressing an NF-κB activity-dependent reporter gene were used. Cells were cultured in MEM medium (Cytiva) containing 10% FBS, 1% nonessential amino acids, 1 mM Na pyruvate, 1% penicillin / streptomycin, and 50 μg / ml hygromycin B at 37°C in the presence of 5% carbon dioxide. The pCMV6-CD300c vector (ORIGENE) containing CD300c (SEQ ID NO: 4) was transfected into cells using Lipofectamine 2000 (Thermo Fisher Scientific) according to the manufacturer's recommended procedure, and after 24 h, cells were cultured in medium supplemented with 400 μg / ml G418. The selected cells were cultured in 96-well white plates and treated with dimeric TCTP (0, 8, or 16 μg / ml) for 6 h, and luciferase activity was measured using the ONE-Glo™ Luciferase Assay System (Promega) according to the manufacturer's recommended procedure.

[0143] As shown in Fig. 5, it was confirmed that NF-κB activity significantly increased according to the concentration of dimeric TCTP treatment in cells overexpressing CD300c. From the above results, it was confirmed that dimeric TCTP activates the NF-κB signaling pathway through CD300c.

[0144]

[0145] <Example 7> Preparation of anti-CD300c monoclonal antibody

[0146] 7-1. Anti-CD300c monoclonal antibody screening

[0147] The scFv (single-chain variable fragment) clone was isolated from a human scFv phage library through a panning process using a recombinant CD300c protein (Sino Biological), which is the human CD300c extracellular domain Met1-Arg183. 5 μg of the CD300c extracellular domain antigen was adsorbed onto the surface of the immunoassay tube. The following day, the cells were washed three times with PBST (phosphate-buffered saline-Tween 20), blocked with 3% skimmed milk for 1 h at room temperature, and then incubated at 10 13The pfu antibody phage library was added and reacted with the antigen. After washing with PBST, 100 mM TEA was added and allowed to stand to recover phages that specifically bound to the antigen. The solution was neutralized with 1 M Tris-HCl (pH 7.4). To determine the number of recovered phages, E. coli was infected and stored in SB culture medium. The phage solution from the previous round was reinoculated into E. coli and amplified by adding helper phage. The panning process was repeated three times to secure antibody candidates that specifically bind to the CD300c antigen. To identify clones that bind to the antigen, 96-well plates were coated with 1 μg / ml of antigen in sodium bicarbonate buffer (pH 9.6). The plates were washed three times with PBST and blocked with 3% skimmed milk for 1 hour. The obtained periplasmic region extract was then added and allowed to react at room temperature for 1 hour to allow for antigen binding. The secondary antibody for detection was diluted 1:2,000 and incubated for 1 hour, followed by the addition of TMB substrate. The reaction was stopped after 5 minutes with 1 N HCl, and the absorbance at 450 nm was measured to select positive clones. Based on the results, 148 clones that showed a positive reaction to CD300c were selected from 480 clones. The selected positive clones were subjected to DNA sequencing to analyze the base sequence of the clones. As a result, 20 types of anti-CD300c monoclonal antibodies with different amino acid sequences were obtained.

[0148] Seven anti-CD300c scFv antibodies with excellent efficacy were identified through cell experiments, and their sequences are shown in Figure 8.

[0149] Additionally, the DNA sequences and amino acid sequences of seven anti-CD300c scFv antibodies are shown in Table 1.

[0150]

[0151]

[0152]

[0153]

[0154]

[0155]

[0156]

[0157]

[0158] 7-2. Production and purification of anti-CD300c monoclonal antibodies

[0159] To produce an anti-CD300c monoclonal antibody, human scFv and IgG1 were inserted into a fused vector, and expression was induced by transfection of 293F cells. The supernatant obtained by centrifugation of 50 ml of culture medium was purified using protein G agarose (GE) resin, and 20 types of antibodies with different amino acid sequences were selected and purified through base sequence analysis and production processes. To confirm the purity of the antibody, 2 μg of antibody was subjected to SDS-PAGE and Coomassie Brilliant Blue staining.

[0160] As exemplarily shown in Fig. 9, it was confirmed that seven types of anti-CD300c scFv-Fc antibodies were produced and purified with a purity of 95% or higher in the reduced and non-reduced states.

[0161] 7-3. Confirmation of the IL-8 secretion inhibitory effect of anti-CD300c monoclonal antibodies against dimeric TCTP in bronchial epithelial cells.

[0162] To confirm the efficacy of the monoclonal antibody, BEAS-2B cells were used. BEAS-2B cells were pretreated with anti-CD300c monoclonal antibody (50 μg / ml) at 37°C for 30 minutes, then dimeric TCTP (4 μg / ml) was added and cultured for 20 hours. Cell supernatants were collected, and the level of secreted IL-8 was determined by ELISA.

[0163] As shown in Figs. 6a and 7a, it was confirmed that four anti-CD300c monoclonal antibodies, JEWR-22, JEWR-139, JEWR-195, and JEWR-235 antibodies (Fig. 6a) through the primary screening, and three anti-CD300c monoclonal antibodies, JEWR-314, JEWR-332, and JEWR-375 antibodies (Fig. 7a) through the secondary screening, had the efficacy of inhibiting IL-8 secretion by dimeric TCTP.

[0164] 7-4. Confirmation of antigen binding affinity of anti-CD300c monoclonal antibody

[0165] To select monoclonal antibodies with high antigen binding affinity among the purified antibodies in the above examples, a binding assay was performed using ELISA. CD300c antigen at a concentration of 1 μg / ml was coated on a 96-well plate with PBS. The next day, after washing three times with PBST, the plate was blocked with 1% skim milk for 1 hour at room temperature, and seven types of anti-CD300c antibodies were reacted at concentrations of 0 to 1 μg / ml for 1 hour at room temperature. Afterwards, HRP-conjugated anti-human IgG antibody was diluted 1:20,000 and reacted for 1 hour. TMB substrate was added to induce color development, and 1 N HCl solution was added to terminate the color development reaction. The binding affinity of the antibodies was confirmed by measuring the absorbance at 450 nm, and the results for the seven types of anti-CD300c antibodies are shown in Table 2.

[0166]

[0167]

[0168] As shown in Figures 6b and 7b, the binding affinity for CD300c was confirmed to be high in the order of JEWR-195 > JEWR-22 > JEWR-139 > JEWR-235 through the first screening (Figure 6b), and the binding affinity was confirmed to be high in the order of JEWR-195 > JEWR-332 > JEWR-375 > JEWR-314 through the second screening including the JEWR-195 antibody with the highest binding affinity (Figure 7b).

[0169] To confirm the binding affinity of CD300c present on the cell membrane and monoclonal antibody, a HEK293T cell line overexpressing CD300c was used. The cell line overexpressing human CD300c was transfected with the pCMV6-CD300c vector using Lipofectamine 2000 according to the manufacturer's recommended procedure. After 24 h of culture, the cells were detached from the plate with accutase (Innovative Cell Technologies), washed with 1% BSA, and reacted with anti-CD300c monoclonal antibody (10 μg / ml) at 4 °C for 30 min. After washing away unbound antibody, the cells were stained with FITC-labeled anti-human IgG Fc secondary antibody (Invitrogen) at 4 °C for 30 min, washed, and analyzed by flow cytometry.

[0170] As shown in Fig. 10, overexpression of CD300c was confirmed using PE anti-CD300c TX45 antibody (BD Biosciences), and increased fluorescence intensity confirmed that JEWR-195 antibody bound to CD300c expressed on the cell membrane.

[0171]

[0172] <Example 8> Confirmation of the action of anti-CD300c monoclonal antibody on the NF-κB signaling pathway of dimeric TCTP in bronchial epithelial cells.

[0173] In order to confirm the mechanism of action of the JEWR-195 antibody, which has excellent antigen binding affinity and activity inhibitory effect of dimeric TCTP in the above example, Western blotting was performed. BEAS-2B cells were cultured in 6-well plates, and the cells were pretreated with JEWR-195 antibody (0, 30, or 50 μg / ml) for 30 minutes, followed by treatment with dimeric TCTP (4 μg / ml) and incubation for 1 hour. The expression of phosphorylated IκBα (p-IκBα), IκBα, phosphorylated p65, p65, and GAPDH (Cell signaling) was confirmed through Western blotting.

[0174] As shown in Fig. 11, it was confirmed that p-IκBα and p-p65 increased by dimeric TCTP were concentration-dependently decreased by pretreatment with JEWR-195 antibody.

[0175] Through the above results, it was confirmed that the anti-CD300c monoclonal antibody of the present invention exhibits an effect of suppressing allergic reactions by inhibiting NF-κB signal transduction by dimeric TCTP in bronchial epithelial cells and reducing the secretion of IL-8 cytokines.

[0176]

[0177] <Example 9> Confirmation of the anti-inflammatory effect of anti-CD300c monoclonal antibody in an asthma mouse model.

[0178] 9-1. Screening of anti-mouse CD300c monoclonal antibodies

[0179] To determine whether CD300c inhibition exerts anti-inflammatory effects in a mouse model of asthma, we generated a novel antibody that specifically binds to mouse CD300c.

[0180] A panning process was performed using a recombinant CD300c protein (MyBioSource) with the sequence His22-His188 corresponding to the extracellular domain of mouse CD300c (SEQ ID NO: 56), and scFv clones were isolated from a human scFv phage library. This process was performed in the same manner as Example 7-1, and 14 clones that showed a positive reaction to mouse CD300c were selected from a total of 288 clones. Subsequently, the base sequences of the selected clones were analyzed through DNA sequencing, and as a result, four anti-mouse CD300c monoclonal antibodies with different amino acid sequences were obtained.

[0181] The four monoclonal antibodies obtained were produced and purified using the same method as in Example 7-2, and ELISA was performed using the same method as in Example 7-4 to select monoclonal antibodies with high antigen binding affinity among the purified antibodies. As shown in Fig. 12, the binding affinity for mouse CD300c was confirmed to be high in the order of JEWR-M10 > JEWR-M161 > JEWR-M79 > JEWR-M116.

[0182] In addition, as shown in Fig. 13, it was confirmed that the monoclonal antibody JEWR-M10 with excellent antigen binding affinity was purified to a purity of 95% or more in both reduced and non-reduced states. Table 3 shows the DNA sequence and amino acid sequence of JEWR-M10.

[0183]

[0184]

[0185] 9-2. Creation of an asthma mouse model

[0186] To confirm the anti-inflammatory effect of anti-CD300c scFv-Fc antibody in vivo, an ovalbumin-induced asthma model was created using 6-week-old BALB / c mice, as shown in Figure 14. To induce allergic asthma, 50 μg ovalbumin (Thermo Scientific) and 1 mg alum (Invivogen) were injected intraperitoneally for primary sensitization, and an immune response was induced through secondary sensitization using the same method on day 14. Thereafter, 20 μg ovalbumin was administered intranasally on days 21, 22, 23, and 26 to induce an allergic response (challenge). The JEWR-M10 antibody was administered four times in total, and the antibody dose was 200 μg / mouse, administered intraperitoneally 30 minutes before intranasal ovalbumin administration. Forty-eight hours after the last antibody administration, mice were sacrificed under anesthesia with Zoletil® (Virbac) and Rompun® (Bayer).

[0187] 9-3. Confirmation of the anti-inflammatory effect by administration of anti-CD300c monoclonal antibody

[0188] In a mouse model of asthma induced by repeated sensitization to ovalbumin, immune cells and inflammatory cytokines are increased in bronchoalveolar lavage fluid and lung tissue. Therefore, bronchoalveolar lavage fluid and lung tissue were obtained to confirm the anti-inflammatory effects of JEWR-M10.

[0189] After anesthetizing the mice and opening the thorax, a 20-gauge intravascular catheter was inserted into the trachea, and 1 ml of PBS was injected and aspirated three times to collect the lavage fluid. The collected bronchoalveolar lavage fluid was centrifuged at 2,000 rpm for 10 minutes, and the supernatant was subjected to ELISA for the measurement of MUC5AC and Th2 cytokines. The cell sediment was resuspended in 100 μl of PBS for total cell count, and the cells were spread on glass slides using a Cytospin, and stained with a Diff-Quick staining kit (Sysmex).

[0190] As shown in Fig. 15, the total cell number in the bronchoalveolar lavage fluid increased in the airway inflammation-induced group (OVA) compared to the control group, and it was confirmed that the cell number decreased with the administration of JEWR-M10.

[0191] For histological analysis of lung tissue, some tissues were fixed in 4% formalin solution, dehydrated in ethanol, paraffin blocks were made, and sections were cut into 5 μm thick sections and stained with Periodic acid-Schiff (PAS).

[0192] As shown in Fig. 16a, images obtained by an optical microscope confirmed an increase in peribronchial immune cell infiltration and PAS-positive cells in the lung tissue of mice in which airway inflammation was induced by ovalbumin, and it was confirmed that these changes were alleviated by administration of JEWR-M10, resulting in a decrease in immune cell infiltration and goblet cell area.

[0193] Consistent with the above results, as shown in Fig. 16b, it was confirmed that the amount of MUC5AC produced in bronchoalveolar lavage fluid was significantly reduced in the antibody treatment group compared to the airway inflammation-induced group.

[0194] As shown in Figures 17a and 17b, the production of Th2 cytokines IL-4, IL-5, and IL-13 increased in the airway inflammation-induced group, and it was confirmed that the administration of JEWR-M10 significantly decreased the production in lung homogenate (Figure 17a) and bronchoalveolar lavage fluid (Figure 17b).

[0195] Through the above results, it was confirmed that the anti-CD300c monoclonal antibody of the present invention has the effect of suppressing airway inflammatory response by reducing immune cell infiltration, mucus secretion, and cytokine secretion in an asthma disease mouse model.

[0196]

[0197] From the above description, those skilled in the art will understand that the present invention can be implemented in other specific forms without altering its technical spirit or essential characteristics. In this regard, it should be understood that the embodiments described above are illustrative in all respects and not restrictive. The scope of the present invention should be interpreted as encompassing all changes or modifications derived from the meaning and scope of the following claims and their equivalent concepts, rather than the detailed description above.

Claims

1. An anti-CD300c antibody or an antigen-binding fragment thereof, characterized by specifically binding to the extracellular domain of CD300c and inhibiting the binding of TCTP (Translationally Controlled Tumor Protein) to CD300c.

2. In paragraph 1, (a) a light chain CDR1 as set forth in SEQ ID NO: 9 or SEQ ID NO: 34; A light chain CDR2 as set forth in SEQ ID NO: 10 or SEQ ID NO: 35; and A light chain variable region comprising a light chain CDR3 as set forth in SEQ ID NO: 11 or SEQ ID NO: 36, and Heavy chain CDR1 as set forth in SEQ ID NO: 12 or SEQ ID NO: 27; A heavy chain CDR2 as set forth in SEQ ID NO: 13 or SEQ ID NO: 28; and Comprising a heavy chain variable region comprising a heavy chain CDR3 set forth in SEQ ID NO: 14, SEQ ID NO: 22, SEQ ID NO: 44 or SEQ ID NO: 51, or (b) at least one amino acid sequence in (a) is substituted, In the heavy chain CDR1 of SEQ ID NO: 27, F at position 2 is replaced with Y; N at position 3 is replaced with T; R at position 5 is replaced with G, T or S; N at position 6 is replaced with G or S; or A at position 8 is replaced with G; In the heavy chain CDR2 of SEQ ID NO: 28, N at position 2 is replaced with S; G at position 3 is replaced with W or V; R at position 4 is replaced with N, Y or S; G at position 5 is replaced with N; D at position 6 is replaced with G; T at position 7 is replaced with D, N or S; or T at position 8 is replaced with I; or In the heavy chain CDR3 described in SEQ ID NO: 51, R at the 2nd position is replaced with S; G at the 3rd position is replaced with E; P at the 4th position is replaced with A or D; Y at the 5th position is replaced with S; Y at the 6th position is replaced with SS; F at the 7th position is replaced with Y; and D at the 8th position is replaced with N. An anti-CD300c antibody or an antigen-binding fragment thereof, wherein the heavy chain CDR1 comprises 0 to 5 substitutions, the heavy chain CDR2 comprises 0 to 6 substitutions, and the heavy chain CDR3 comprises 0 to 4 substitutions, wherein the heavy chain CDR1, the heavy chain CDR2 or the heavy chain CDR3 comprises at least one substitution.

3. In paragraph 1, A light chain CDR1 as set forth in SEQ ID NO: 9 or SEQ ID NO: 34; A light chain CDR2 as set forth in SEQ ID NO: 10 or SEQ ID NO: 35; and A light chain variable region comprising a light chain CDR3 as set forth in SEQ ID NO: 11 or SEQ ID NO: 36; and A heavy chain CDR1 set forth in SEQ ID NO: 12, SEQ ID NO: 20, SEQ ID NO: 27, SEQ ID NO: 37 or SEQ ID NO: 49; A heavy chain CDR2 set forth in SEQ ID NO: 13, SEQ ID NO: 21, SEQ ID NO: 28, SEQ ID NO: 38 or SEQ ID NO: 50; and An anti-CD300c antibody or an antigen-binding fragment thereof, comprising a heavy chain variable region comprising a heavy chain CDR3 set forth in SEQ ID NO: 14, SEQ ID NO: 22, SEQ ID NO: 29, SEQ ID NO: 39, SEQ ID NO: 44 or SEQ ID NO:

51.

4. In paragraph 1, A light chain variable region set forth in SEQ ID NO: 7, SEQ ID NO: 18, SEQ ID NO: 25, SEQ ID NO: 32, SEQ ID NO: 42, SEQ ID NO: 47 or SEQ ID NO: 54, and An anti-CD300c antibody or an antigen-binding fragment thereof, comprising a heavy chain variable region set forth in SEQ ID NO: 8, SEQ ID NO: 19, SEQ ID NO: 26, SEQ ID NO: 33, SEQ ID NO: 43, SEQ ID NO: 48 or SEQ ID NO:

55.

5. In paragraph 4, An anti-CD300c antibody or an antigen-binding fragment thereof, further comprising a linker as described in SEQ ID NO:

15.

6. In paragraph 1, An anti-CD300c antibody or an antigen-binding fragment thereof, comprising an amino acid sequence set forth in SEQ ID NO: 6, SEQ ID NO: 17, SEQ ID NO: 24, SEQ ID NO: 31, SEQ ID NO: 41, SEQ ID NO: 46 or SEQ ID NO:

53.

7. In paragraph 1, Light chain CDR1 described by SEQ ID NO: 61; Light chain CDR2 as described by SEQ ID NO: 62; and A light chain variable region comprising a light chain CDR3 as set forth in SEQ ID NO: 63; and Heavy chain CDR1 described by SEQ ID NO: 64; Heavy chain CDR2 as described by SEQ ID NO: 65; and An anti-CD300c antibody or an antigen-binding fragment thereof, comprising a heavy chain variable region comprising a heavy chain CDR3 set forth in SEQ ID NO:

66.

8. The light chain variable region described by sequence number 59, and An anti-CD300c antibody or an antigen-binding fragment thereof, comprising a heavy chain variable region represented by SEQ ID NO:

60.

9. In paragraph 7, An anti-CD300c antibody or an antigen-binding fragment thereof, comprising an amino acid sequence set forth in SEQ ID NO:

58.

10. In paragraph 7, An anti-CD300c antibody or an antigen-binding fragment thereof, further comprising a linker as described in SEQ ID NO:

15.

11. A nucleic acid molecule encoding an antibody or an antigen-binding fragment thereof according to any one of claims 1 to 10.

12. An expression vector comprising the nucleic acid molecule of claim 11.

13. A host cell into which the expression vector of clause 12 has been introduced.

14. A pharmaceutical composition for preventing or treating allergic diseases, inflammatory diseases, or malaria, comprising a CD300c inhibitor as an active ingredient.

15. A pharmaceutical composition according to claim 14, wherein the CD300c inhibitor is an antibody or an antigen-binding fragment thereof according to any one of claims 1 to 10.

16. A pharmaceutical composition according to claim 15, wherein the allergic disease is selected from the group consisting of asthma, bronchitis, chronic obstructive pulmonary disease, bronchiectasis, rhinitis, atopic dermatitis, urticaria, hay fever, conjunctivitis, and anaphylaxis.

17. A pharmaceutical composition according to claim 15, wherein the inflammatory disease is selected from the group consisting of rheumatoid arthritis, bronchitis, pneumonia, arthritis, nephritis, psoriasis, dermatitis, Crohn's disease, enteritis, gingivitis, arteriosclerosis, coronary arteritis, hepatitis, Behcet's disease, bladder cancer, prostatitis, pyelonephritis, glomerulonephritis, osteomyelitis, thyroiditis, uveitis, peritonitis, meningitis, and pulmonary fibrosis. 18.(a) A step of treating dTCTP (dimeric Translationally Controlled Tumor Protein) and a candidate substance to separated cells expressing CD300c; (b) a step of measuring the activity of the NF-κB signaling pathway or the level of IL-8 secretion in the separated cells treated with the candidate substance; and (c) a step of selecting a candidate substance that reduces the activity of the NF-κB signaling pathway or the level of IL-8 secretion by comparing the activity of the NF-κB signaling pathway or the level of IL-8 secretion measured in step (b) with the level in isolated cells that have not been treated with the candidate substance. A method for screening a drug for preventing or treating an allergic disease, an inflammatory disease, or malaria. 19.(a) a step of measuring the expression or secretion level of CD300c from a separated biological sample; and (b) a method for providing information for diagnosing an allergic disease, an inflammatory disease, or malaria, comprising the step of comparing the expression or secretion level with the CD300c expression or secretion level of a normal control sample.

20. A composition for diagnosing allergic diseases, inflammatory diseases, or malaria, comprising a preparation for measuring the expression or secretion level of CD300c.

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