PEGYLATED IGE-DEPENDENT HISTAMINE-RELEASING FACTOR (HRF)-BINDING PEPTIDE AND USE THEREOF

Abstract

The present invention relates to a modified HRF binding peptide. The modified HRF-binding peptide according to the present invention can exert drug efficacy with high stability in vivo, can be effectively used as a drug because the number of administrations can be reduced due to its longer half-life, and can prevent or treat allergies, malaria, autoimmune diseases, acute or chronic inflammatory diseases, hypertension, and cancer in humans as well as animals by effectively inhibiting histamine secretion in cells.

Claims

1.-23. (canceled)

24. A PEGylated HRF-binding peptide in which polyethylene glycol is bound to the HRF peptide comprising a sequence of amino acids wherein the first amino acid is selected from the group consisting of A, L and W; the second amino acid is selected from the group consisting of V, Y, E and A; the third amino acid is selected from the group consisting of T, V, F and A; the fourth amino acid is selected from the group consisting of Y, P and A; the fifth amino acid is selected from the group consisting of P, G and K; the sixth amino acid is selected from the group consisting of A, L, S and W; and the seventh amino acid consists of a sequence of amino acids selected from the group consisting of A, P and M.

25. The PEGylated HRF-binding peptide according to claim 24, wherein the polyethylene glycol or the derivative thereof has a molecular weight of 1 kDa to 50 kDa.

26. The PEGylated HRF-binding peptide according to claim 24, wherein the polyethylene glycol is covalently bound to a carboxyl group or an amino group of the HRF-binding peptide.

27. The PEGylated HRF-binding peptide according to claim 24, wherein the polyethylene glycol has a functional group including an aldehyde group, a carboxyl group, an amino group or a hydrazide group bound to a terminal thereof.

28. The PEGylated HRF-binding peptide according to claim 24, wherein the polyethylene glycol or the derivative thereof is bound to the N-terminal or C-terminal of the peptide.

29. The PEGylated HRF-binding peptide according to claim 24, wherein the HRF-binding peptide consists of the amino acid sequence W-Y-V-Y-P-S-M.

30. The PEGylated HRF-binding peptide according to claim 24, wherein the PEGylated HRF-binding peptide has an inhibitory effect on the secretion of IL-8.

31. The PEGylated HRF-binding peptide according to claim 24, wherein the PEGylated HRF-binding peptide has an inhibitory effect on the increase of eosinophils.

32. The PEGylated HRF-binding peptide according to claim 24, wherein the PEGylated HRF-binding peptide has an inhibitory effect on the secretion of IL-5.

33. The PEGylated HRF-binding peptide according to claim 24, wherein the PEGylated HRF-binding peptide has an inhibitory effect on the secretion of IL-4.

34. The PEGylated HRF-binding peptide according to claim 24, wherein the PEGylated HRF-binding peptide has an inhibitory effect on the secretion of IL-13.

35. The PEGylated HRF-binding peptide according to claim 24, wherein the PEGylated HRF-binding peptide has an inhibitory effect on the ovalbumin-specific secretion of IgE.

36. A method for preventing or treating allergy comprising a step of administering the PEGylated HRF-binding peptide of claim 24 to an individual or subject in need thereof.

37. The method according to claim 36, wherein the allergy is asthma, rhinitis, atopy, urticaria, anaphylaxis, allergic bronchiectasis, allergy caused by food, drug, pollen, mold or insects, allergic conjunctivitis, hay fever, cold urticaria, or atopic dermatitis.

38. A method for preventing or treating malaria comprising a step of administering the PEGylated HRF-binding peptide of claim 24 to an individual or subject in need thereof.

39. The method according to claim 38, wherein the malaria is Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale, or Plasmodium malariae.

40. A method for preventing or treating autoimmune disease comprising a step of administering the PEGylated HRF-binding peptide of claim 24 to an individual or subject in need thereof.

41. The method according to claim 40, wherein the autoimmune disease is rheumatoid arthritis, Sjogrean's disease, systemic sclerosis, polymyositis, systemic angitis, mixed connective tissue disease, Crohn's disease, Hashimoto's disease, Grave's disease, Goodpasture's syndrome, Guillain-Barre syndrome, idiopathic thrombocytopenic purpura, irritable bowel syndrome, myasthenia gravis, narcolepsy, pemphigus vulgaris, pernicious anemia, primary biliary cirrhosis, ulcerative colitis, vasculitis, Wegener's granulomatosis, psoriasis, alopecia areata, rheumatic fever, systemic lupus erythematosus, or multiple scleorosis.

42. A method for preventing or treating acute or chronic inflammatory disease comprising a step of administering the PEGylated HRF-binding peptide of claim 24 to an individual or subject in need thereof.

43. The method according to claim 42, wherein the acute or chronic inflammatory disease is conjunctivitis, periodontitis, rhinitis, otitis media, pharyngitis, tonsillitis, dermatitis, gastritis, colitis, ankylosing spondylitis, psoriatic arthritis, osteoarthritis, rheumatoid arthritis, periarthritis, tendinitis, xerosis, periostitis, myositis, hepatitis, cystitis, nephritis, pneumonia, gastric ulcer, Crohn's disease, Sjogrean's disease, gout, fibromyalgia, lupus, bursitis, or systemic lupus erythematodes.

44. A method for preventing or treating hypertension comprising a step of administering the PEGylated HRF-binding peptide of claim 24 to an individual or subject in need thereof.

45. A method for preventing or treating cancer comprising a step of administering the PEGylated HRF-binding peptide of claim 24 to an individual or subject in need thereof.

46. The method according to claim 45, wherein the cancer is oral cancer, liver cancer, stomach cancer, colon cancer, breast cancer, lung cancer, bone cancer, pancreatic cancer, skin cancer, head and neck cancer, skin cancer, cervical cancer, ovarian cancer, colorectal cancer, small intestine cancer, rectal cancer, fallopian tube carcinoma, perianal cancer, endometrial carcinoma, vaginal carcinoma, vulvar carcinoma, Hodgkin's disease, esophageal cancer, lymph adenocarcinoma, bladder cancer, gallbladder cancer, endocrine adenocarcinoma, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, prostate cancer, chronic leukemia, acute leukemia, lymphocytic lymphoma, renal cancer, ureter cancer, renal cell carcinoma, renal pelvic carcinoma, central nervous system tumor, primary central nervous system lymphoma, spinal cord tumor, brainstem glioma or pituitary adenoma.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0046] FIG. 1 is a graph showing the HPLC results of the PEGylated HRF-binding peptide of Example 1 according to the present invention.

[0047] FIG. 2 is a diagram showing the MALDI-TOF mass spectra of the compounds of Preparative Example 1 (top) and Example 1 (bottom) according to the present invention.

[0048] FIG. 3 is a graph showing the HPLC results of the PEGylated HRF-binding peptide of Example 2 according to the present invention.

[0049] FIG. 4 is a diagram showing the MALDI-TOF mass spectra of the compounds of Preparative Example 2 (top) and Example 2 (bottom) according to the present invention.

[0050] FIG. 5 is a graph showing the HPLC results of the PEGylated HRF-binding peptide of Example 3 according to the present invention.

[0051] FIG. 6 is a diagram showing the MALDI-TOF mass spectrum of the compound of Example 3 according to the present invention.

[0052] FIG. 7 is a diagram showing the experimental results for the inhibitory effect of the compounds of Preparative Example 4, Example 1 and Example 2 according to the present invention by HRF on IL-8 secretion in BEAS-2B cells.

[0053] FIG. 8 is a diagram showing the simple schematic diagram of an experiment of Experimental Example 4 according to the present invention.

[0054] FIG. 9 is a diagram showing the experimental results for the inhibitory effect of the normal control (NC), positive control (PC), dexamethasone, and the compounds of Preparative Example 4 and Example 3 according to the present invention on eosinophil increase.

[0055] FIG. 10 is a diagram showing the experimental results for the inhibitory effect of the normal control (NC), positive control (PC), and the compounds of Preparative Example 4 and Example 3 on IL-5 secretion (administration of the compounds of Preparative Example 4 and Example 3 once a day for a total of 8 times).

[0056] FIG. 11 is a diagram showing the experimental results for the inhibitory effect of the normal control (NC), positive control (PC), and the compounds of Preparative Example 4 and Example 3 on IL-5 secretion (administration of the compounds of Preparative Example 4 and Example 3 once in 4 days for a total of 2 times).

[0057] FIG. 12 is a diagram showing the experimental results for the inhibitory effect of the normal control (NC), positive control (PC), and the compounds of Preparative Example 4 and Example 3 on IL-5 secretion (administration of the compounds of Preparative Example 4 and Example 3 once on the 1.sup.st day of 8 days inducing bronchial asthma and rhinitis).

[0058] FIG. 13 is a diagram showing the experimental results for the inhibitory effect of the normal control (NC), positive control (PC), and the compounds of Preparative Example 4 and Example 3 on IL-5 secretion (administration of the compounds of Preparative Example 4 and Example 3 once on the 5.sup.th day of 8 days inducing bronchial asthma and rhinitis).

[0059] FIG. 14 is a diagram showing the experimental results for the inhibitory effect of the normal control (NC), positive control (PC), and the compounds of Preparative Example 4 and Example 3 on IL-4 secretion (administration of the compounds of Preparative Example 4 and Example 3 once a day for a total of 8 times).

[0060] FIG. 15 is a diagram showing the experimental results for the inhibitory effect of the normal control (NC), positive control (PC), and the compounds of Preparative Example 4 and Example 3 on IL-4 secretion (administration of the compounds of Preparative Example 4 and Example 3 once in 4 days for a total of 2 times).

[0061] FIG. 16 is a diagram showing the experimental results for the inhibitory effect of the normal control (NC), positive control (PC), and the compounds of Preparative Example 4 and Example 3 on IL-4 secretion (administration of the compounds of Preparative Example 4 and Example 3 once on the 1.sup.st day of 8 days inducing bronchial asthma and rhinitis).

[0062] FIG. 17 is a diagram showing the experimental results for the inhibitory effect of the normal control (NC), positive control (PC), and the compounds of Preparative Example 4 and Example 3 on IL-4 secretion (administration of the compounds of Preparative Example 4 and Example 3 once on the 5.sup.th day of 8 days inducing bronchial asthma and rhinitis).

[0063] FIG. 18 is a diagram showing the experimental results for the inhibitory effect of the normal control (NC), positive control (PC), and the compounds of Preparative Example 4 and Example 3 on IL-13 secretion (administration of the compounds of Preparative Example 4 and Example 3 once a day for a total of 8 times).

[0064] FIG. 19 is a diagram showing the experimental results for the inhibitory effect of the normal control (NC), positive control (PC), and the compounds of Preparative Example 4 and Example 3 on IL-13 secretion (administration of the compounds of Preparative Example 4 and Example 3 once in 4 days for a total of 2 times).

[0065] FIG. 20 is a diagram showing the experimental results for the inhibitory effect of the normal control (NC), positive control (PC), and the compounds of Preparative Example 4 and Example 3 on IL-13 secretion (administration of the compounds of Preparative Example 4 and Example 3 once on the 1.sup.st day of 8 days inducing bronchial asthma and rhinitis).

[0066] FIG. 21 is a diagram showing the experimental results for the inhibitory effect of the normal control (NC), positive control (PC), and the compounds of Preparative Example 4 and Example 3 on IL-13 secretion (administration of the compounds of Preparative Example 4 and Example 3 once on the 5.sup.th day of 8 days inducing bronchial asthma and rhinitis).

[0067] FIG. 22 is a diagram showing the experimental results for the inhibitory effect of the normal control (NC), positive control (PC), and the compounds of Preparative Example 4 and Example 3 on ovalbumin-specific IgE secretion (administration of the compounds of Preparative Example 4 and Example 3 once a day for a total of 8 times).

[0068] FIG. 23 is a diagram showing the experimental results for the inhibitory effect of the normal control (NC), positive control (PC), and the compounds of Preparative Example 4 and Example 3 on ovalbumin-specific IgE secretion (administration of the compounds of Preparative Example 4 and Example 3 once in 4 days for a total of 2 times).

[0069] FIG. 24 is a diagram showing the experimental results for the inhibitory effect of the normal control (NC), positive control (PC), and the compounds of Preparative Example 4 and Example 3 on ovalbumin-specific IgE secretion (administration of the compounds of Preparative Example 4 and Example 3 once on the 1.sup.st day of 8 days inducing bronchial asthma and rhinitis).

[0070] FIG. 25 is a diagram showing the experimental results for the inhibitory effect of the normal control (NC), positive control (PC), and the compounds of Preparative Example 4 and Example 3 on ovalbumin-specific IgE secretion (administration of the compounds of Preparative Example 4 and Example 3 once on the 5.sup.th day of 8 days inducing bronchial asthma and rhinitis).

[0071] FIG. 26 is a diagram showing the results of confirming the concentration changes of the compounds of Preparative Example 4 and Example 3 in male mice over time.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0072] Hereinafter, the present invention is described in detail.

[0073] The present invention provides a PEGylated HRF-binding peptide in which polyethylene glycol is bound to the HRF peptide comprising a sequence of amino acids wherein the first amino acid is selected from the group consisting of A, L and W; the second amino acid is selected from the group consisting of V, Y, E and A; the third amino acid is selected from the group consisting of T, V, F and A; the fourth amino acid is selected from the group consisting of Y, P and A; the fifth amino acid is selected from the group consisting of P, G and K; the sixth amino acid is selected from the group consisting of A, L, S and W; and the seventh amino acid consists of a sequence of amino acids selected from the group consisting of A, P and M.

[0074] The molecular weight of the polyethylene glycol is not limited as long as it is a molecular weight capable of exhibiting the activity of the HRF-binding peptide according to the present invention, but can be 1 kDa to 50 kDa, 2 kDa to 45 kDa, 3 kDa to 40 kDa, 4 kDa to 35 kDa, 5 kDa to 30 kDa, 3 kDa to 25 kDa, 3 kDa to 20 kDa, 3 kDa to 15 kDa, 3 kDa to 13 kDa, 4 kDa to 6 kDa, 5 kDa, 5 kDa to 10 kDa, 9 kDa to 11 kDa, or 10 kDa.

[0075] The binding of the HRF-binding peptide to polyethylene is due to the covalent bond of polyethylene glycol to the carboxyl group or amino group of the HRF-binding peptide.

[0076] The polyethylene glycol can be connected with a functional group including an aldehyde group, a carboxyl group, an amino group, or a hydrazide group at the terminal.

[0077] The functional group can be connected to the terminal oxygen of polyethylene glycol through C.sub.1-6 alkylene.

[0078] The other terminal of the polyethylene glycol can be capped. At this time, the terminal can be capped with an alkoxy group, for example, can be capped with a methoxy group.

[0079] The polyethylene glycol may be methoxy polyethylene glycol propion aldehyde (5000), methoxy polyethylene glycol hydrazide (5000), or methoxy polyethylene glycol propion aldehyde (1000).

[0080] The polyethylene glycol or a derivative thereof can be bound to the N-terminal or C-terminal of the peptide.

[0081] The HRF-binding peptide can be composed of a sequence of amino acids in which the first amino acid is selected from A or W; the second amino acid is selected from Y or A; the third amino acid is selected from V or A; the fourth amino acid is selected from Y or A; the fifth amino acid is selected from P or K; the sixth amino acid is selected from S or A; and the seventh amino acid is selected from M or A.

[0082] Or, the HRF-binding peptide can be composed of a sequence of amino acids in which the first amino acid is W; and the seventh amino acid is M.

[0083] Preferably, the HRF-binding peptide can be composed of the amino acid sequence W-Y-V-Y-P-S-M; A-Y-V-Y-P-S-M; or W-Y-V-A-P-S-M.

[0084] Most preferably, the HRF-binding peptide can be composed of the amino acid sequence W-Y-V-Y-P-S-M.

[0085] The HRF-binding peptide can be a peptide consisting of L-, D-, or L- and D-amino acids.

[0086] The HRF-binding peptide can be a peptide comprising one or more modified amino acids.

[0087] The modified amino acid can be an amino acid derivative or an alkylated amino acid.

[0088] The HRF-binding peptide has an effect of inhibiting IL-8 secretion.

[0089] The HRF-binding peptide has an effect of inhibiting eosinophil increase.

[0090] The HRF-binding peptide has an effect of inhibiting IL-5 secretion.

[0091] The HRF-binding peptide has an effect of inhibiting IL-4 secretion.

[0092] The HRF-binding peptide has an effect of inhibiting IL-13 secretion.

[0093] The HRF-binding peptide has an effect of inhibiting ovalbumin-specific IgE secretion.

[0094] The peptide comprising the amino acid sequence W-Y-V-Y-P-S-M is a heptamer named dTBP2 [dTCTP (dimerized translationally controlled tumor protein) binding peptide 2] prepared by the present inventors (Korean Laid-Open Patent Publication No. 10-2017-0004906), and was confirmed by inhibiting the action of dTCTP. The chemical structure of dTBP2 is as follows.

##STR00001##

[0095] As described in Korean Laid-Open Patent Publication No. 10-2017-0004906, it was confirmed that the activity of dTBP2 was equally maintained even if the first amino acid tryptophan and the fourth amino acid tyrosine were substituted with alanine.

[0096] The present invention provides a composition for preventing or treating allergy containing the PEGylated HRF-binding peptide. The allergy can be asthma, rhinitis, atopy, urticaria, anaphylaxis, allergic bronchiectasis, allergy caused by food, drug, pollen, or insects, allergic conjunctivitis, hay fever, cold urticaria, or atopic dermatitis.

[0097] The present invention provides a composition for preventing or treating malaria containing the PEGylated HRF-binding peptide. The malaria can be caused by Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale, or Plasmodium malariae.

[0098] The present invention provides a composition for preventing or treating autoimmune disease containing the PEGylated HRF-binding peptide. The autoimmune disease can be rheumatoid arthritis, Sjogrean's disease, systemic sclerosis, polymyositis, systemic angitis, mixed connective tissue disease, Crohn's disease, Hashimoto's disease, Grave's disease, Goodpasture's syndrome, Guillain-Barre syndrome, idiopathic thrombocytopenic purpura, irritable bowel syndrome, myasthenia gravis, narcolepsy, pemphigus vulgaris, pernicious anemia, primary biliary cirrhosis, ulcerative colitis, vasculitis, Wegener's granulomatosis, psoriasis, alopecia areata, rheumatic fever, systemic lupus erythematosus, or multiple scleorosis.

[0099] The present invention provides a composition for preventing or treating acute or chronic inflammatory disease containing the PEGylated HRF-binding peptide. The acute or chronic inflammatory disease can be conjunctivitis, periodontitis, rhinitis, otitis media, pharyngitis, tonsillitis, dermatitis, gastritis, colitis, ankylosing spondylitis, psoriatic arthritis, osteoarthritis, rheumatoid arthritis, periarthritis, tendinitis, xerosis, periostitis, myositis, hepatitis, cystitis, nephritis, pneumonia, gastric ulcer, Crohn's disease, Sjogrean's disease, gout, fibromyalgia, lupus, bursitis, or systemic lupus erythematodes.

[0100] The present invention provides a composition for preventing or treating hypertension containing the PEGylated HRF-binding peptide.

[0101] The present invention provides a composition for preventing or treating cancer containing the PEGylated HRF-binding peptide. The cancer can be oral cancer, liver cancer, stomach cancer, colon cancer, breast cancer, lung cancer, bone cancer, pancreatic cancer, skin cancer, head and neck cancer, skin cancer, cervical cancer, ovarian cancer, colorectal cancer, small intestine cancer, rectal cancer, fallopian tube carcinoma, perianal cancer, endometrial carcinoma, vaginal carcinoma, vulvar carcinoma, Hodgkin's disease, esophageal cancer, lymph adenocarcinoma, bladder cancer, gallbladder cancer, endocrine adenocarcinoma, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, prostate cancer, chronic leukemia, acute leukemia, lymphocytic lymphoma, renal cancer, ureter cancer, renal cell carcinoma, renal pelvic carcinoma, central nervous system tumor, primary central nervous system lymphoma, spinal cord tumor, brainstem glioma or pituitary adenoma.

[0102] In addition to the method of modification by pegylating the HRF-binding peptide with polyethylene glycol or its derivative as described above, a technique using cyclotide or cyclic peptide as a scaffold can be applied to the HRF-binding peptide according to the present invention.

[0103] That is, in one aspect of the present invention, there is provided a conjugate peptide in which the HRF-binding peptide and cyclotide are combined, the cyclotide contains 25 to 35 amino acids, and a head and a tail are cyclized.

[0104] The cyclotide contains three disulfide bonds.

[0105] The three disulfide bonds may be interlocking.

[0106] The cyclotide may be derived from a plant.

[0107] The plant can be at least one selected from the group consisting of C. parcifolia, P. longipes, V. odorata, O. affinis, P. condensata, V. tricolor, V. arvensis and M. cochinchinensis.

[0108] The conjugate may be an oral formulation.

[0109] In addition to the method of modification by pegylating the HRF-binding peptide according to the present invention with polyethylene glycol or its derivative as described above, the following compounds can be used instead of polyethylene glycol.

[0110] For example, biocompatible polymers such as poly(glycerol), poly(oxazoline), poly(vinylpyrrolidone), poly(acrylamide), poly(peptide), poly(2-alkyl-2-oxazoline), polysarcosine, poly(vinyl alcohol), polyzwitterion, and the like can be bound to the HRF-binding peptide according to the present invention directly or via a linker. However, as a method for modifying the HRF-binding peptide according to the present invention, the compound capable of replacing polyethylene glycol is not limited to the above, and as a compound having biocompatibility, it is not limited as long as it is a compound that can compensate for the disadvantages of a peptide drug.

[0111] That is, in one aspect of the present invention, there is provided an HRF-binding peptide modified with one compound selected from the group consisting of poly(glycerol), poly(oxazoline), poly(vinylpyrrolidone), poly(acrylamide), poly(peptide), poly(2-alkyl-2-oxazoline), polysarcosine, poly(vinyl alcohol) and polyzwitterion.

[0112] In addition, the HRF-binding peptide according to the present invention can be modified using a polypeptide, which is one of the polymers that can replace the polyethylene glycol. For example, the HRF-binding peptide according to the present invention can be linked (bound) to the peptide in which proline-alanine-serine residues are repeated (PASylation). In this case, the PASylated HRF-binding peptide has similar properties to the PEGylated HRF-binding peptide according to the present invention.

[0113] That is, in one aspect of the present invention, there is provided an HRF-binding peptide to which the peptide having a repeating amino acid sequence P-A-S is bound or linked (PASylation).

[0114] In addition, as another example in which the HRF-binding peptide according to the present invention can be modified using a polypeptide, ELPs (elastin like polypeptides) can be bound to the HRF-binding peptide according to the present invention. ELPs are peptides in which valine-proline-glycine-Xaa-glycine residues are repeated (In this case, Xaa is an amino acid except for proline (P)).

[0115] That is, in one aspect of the present invention, there is provided a composition for diagnosing, preventing or treating allergy, malaria, autoimmune disease, acute or chronic inflammatory disease, hypertension, or cancer, containing the HRF-binding peptide and at least 60 elastin-like peptide (ELP) structural units selected from the group consisting of the sequences represented by SEQ. ID. NO: 1 to NO: 13 disclosed in Korean Laid-Open Patent Publication No. 10-2019-0005171.

[0116] In another aspect, there is provided a sustained release pharmaceutical formulation comprising the composition.

[0117] The formulation provides slow absorption from the site of injection upon administration.

[0118] The formulation provides a flat PK profile upon administration when compared to the pharmacokinetic (PK) profile of the HRF-binding peptide in the absence of the elastin-like peptide.

[0119] (Korean Laid-Open Patent Publication No. 10-2019-0005171)

[0120] In addition, as another example in which the HRF-binding peptide according to the present invention can be modified using a polypeptide, the HRF-binding peptide can be modified through XTEN technology. XTEN technology, which is a technology of Amunix, has the effect of extending the half-life of drugs in the body and has the advantage of low immunogenicity.

[0121] That is, in one aspect of the present invention, there is provided a recombinant fusion protein comprising the HRF-binding peptide and an extended recombinant polypeptide (XTEN), wherein the fusion protein exhibits an apparent molecular weight coefficient of at least about 4 and exhibits an effect of diagnosing, preventing or treating allergy, malaria, autoimmune disease, acute or chronic inflammatory disease, hypertension, cancer when administered to a subject by using a therapeutically effective amount. In this case, the XTEN has the following characteristics. [0122] (a) XTEN contains at least 36 amino acid residues; [0123] (b) the sum of glycine (G), alanine (A), serine (S), threonine (T), glutamate (E) and proline (P) residues account for greater than about 80% of the total amino acid residues of XTEN; [0124] (c) (i) XTEN does not contain three contiguous amino acids that are identical unless the amino acid is serine, (ii) at least about 80% of the XTEN sequence consists of non-overlapping sequence motifs comprising each of about 9 to about 14 amino acid residues composed of 4 to 6 amino acids selected from glycine (G), alanine (A), serine (S), threonine (T), glutamate (E) and proline (P), wherein no two contiguous amino acid residues occur more than twice in each non-overlapping sequence motif, or (iii) XTEN is substantially non-repeatable such that the XTEN sequence has a subsequence score of less than 10; [0125] (d) XTEN has greater than 90% random coil formation when measured by GOR algorithm; [0126] (e) XTEN has less than 2% alpha helices and 2% beta-sheets when measured by Chou-Fasman algorithm; and [0127] (f) XTEN lacks the predicted T cell epitope when analyzed by the TEPITOPE algorithm, with the TEPITOPE threshold score of −9 for that prediction by the algorithm.

[0128] (Korean Laid-Open Patent Publication No. 10-2014-0069131, Korean Laid-Open Patent Publication No. 10-2011-01276969)

[0129] In addition, the HRF-binding peptide according to the present invention can be modified using albumin fusion technology. That is, the recombinant albumin can be linked to the HRF-binding peptide.

[0130] That is, in one aspect of the present invention, there is provided an albumin-fused HRF-binding peptide in which the HRF-binding peptide and the recombinant albumin are linked by a cleavable peptide linker.

[0131] In addition, antibody-based biopersistence drug platform technology can be applied to the HRF-binding peptide according to the present invention. That is, lapscovery (Long Acting Protein/Peptide Discovery Platform Technology, Hanmi Pharm. Co., Ltd.) or HyFc (Hybid Fc) technology (Genexine, Inc.) can be applied to the HRF-binding peptide according to the present invention.

[0132] That is, in one aspect of the present invention, there is provided an HRF-binding peptide conjugate in which the HRF-binding peptide and an immunoglobulin Fc region are linked via a non-peptidyl polymer selected from the group consisting of polyethylene glycol, polypropylene glycol, ethylene glycol-propylene glycol copolymer, polyoxyethylated polyol, polyvinyl alcohol, polysaccharide, dextran, polyvinyl ethyl ether, biodegradable polymer, lipid polymer, chitin, hyaluronic acid and a combination thereof.

[0133] (Korean Laid-Open Patent Publication No. 10-2008-0064750)

[0134] That is, in one aspect of the present invention, there is provided a conjugate in which the HRF-binding peptide is bound to a hybrid Fc, wherein the hybrid Fc is derived from a combination of human IgG subclass or a combination of human IgD and IgG.

[0135] Specifically, it is a hybrid human immunoglobulin Fc fragment comprising a hinge region, a CH2 domain and a CH3 domain in the N-terminal to C-terminal direction. The hinge region is at least a partial amino acid sequence of a human IgD hinge region or a human IgG1 hinge region. The CH2 domain is a human IgG4 CH2 domain in which the N-terminal region is substituted with 4-37 amino acid residues of the N-terminal region of a human IgG2 CH2 or human IgD CH2 domain.

[0136] The conjugate is a conjugate in which the HRF-binding peptide is linked to the polypeptide represented by the following formula: (Korean Laid-Open Patent Publication No. 10-2008-0094781)

N′-(Z1)p-Y-Z2-Z3-Z4-C′

[0137] Wherein, N′ is the N-terminus of the polypeptide and C′ is the C-terminus of the polypeptide;

[0138] p is an integer of 0 or 1; and

[0139] (i) Z1 is an amino acid sequence having the amino acid residues at positions 90 to 98 of SEQ. ID. NO: 11,

[0140] Y is an amino acid sequence having the amino acid residues at positions 99 to 113 of SEQ. ID. NO: 11.

[0141] Z2 is an amino acid sequence having the amino acid residues at positions 111 to 116 of SEQ. ID. NO: 12,

[0142] Z3-Z4 is an amino acid sequence consisting of continuous amino acid sequences having the amino acid residues at positions 118 to 220 of SEQ. ID. NO: 13 and the amino acid residues at positions 221 to 327 of SEQ. ID. NO: 13, or

[0143] (ii) Z1 is an amino acid sequence having the amino acid residues at positions 90 to 98 of SEQ. ID. NO: 14,

[0144] Y is an amino acid sequence having the amino acid residues at positions 158 to 162 of SEQ. ID. NO: 14, the amino acid residues at positions 153 to 162 of SEQ. ID. NO: 14, the amino acid residues at positions 148 to 162 of SEQ. ID. NO: 14, the amino acid residues at positions 143 to 162 of SEQ. ID. NO: 14, the amino acid residues at positions 133 to 162 of SEQ. ID. NO: 14, or the amino acid residues at positions 99 to 162 of SEQ. ID. NO: 14,

[0145] Z2 is an amino acid sequence having the amino acid residues at positions 163 to 170 of SEQ. ID. NO: 14,

[0146] Z3-Z4 is an amino acid sequence consisting of continuous amino acid sequences having the amino acid residues at positions 121 to 220 of SEQ. ID. NO: 13 and the amino acid residues at positions 221 to 327 of SEQ. ID. NO: 13.

[0147] The HRF-binding peptide can be fused to the N-terminus or C-terminus of the hybrid Fc, and the HRF-binding peptide may exhibit an increased circulating half-life compared to the circulating half-life of the native form of the HRF-binding peptide.

[0148] The HRF-binding peptide and the hybrid Fc can be linked by a linker.

[0149] The linker can be an albumin linker or a synthetic linker,

[0150] The albumin linker may include the residues at positions 321 to 323, 318 to 325, 316 to 328, 313 to 330, 311 to 333, or 306 to 338 of the sequence represented by SEQ. ID. NO: 25 disclosed in Korean Laid-Open Patent Publication No. 10-2008-0094781.

[0151] The synthetic linker can be a peptide of 10 to 20 amino acid residues composed of Gly and Ser residues.

[0152] The synthetic linker can be GGGGSGGGGSGGGSG.

[0153] (Korean Laid-Open Patent Publication No. 10-2008-0094781)

[0154] In addition, the HRF-binding peptide according to the present invention can be administered by using nanoparticles (liposomes, exosomes, etc.), or by coating or loading on the skin through a patch or microneedle.

[0155] In addition, the HRF-binding peptide according to the present invention can be used for drug delivery using a cell-penetrating peptide. For example, the peptide disclosed in Korean Laid-Open Patent Publication No. 10-2017-0114997 can be used.

[0156] That is, in one aspect of the present invention, there is provided a composition for diagnosing, preventing or treating allergy, malaria, autoimmune disease, acute or chronic inflammatory disease, hypertension or cancer, containing the HRF-binding peptide and the peptide consisting of the following amino acid sequence:

R1-R2-R3-R4-R5-R6-R7-R8-R9-R10 [0157] R1 is any one amino acid selected from M, L or P, [0158] R2 is any one amino acid selected from I, A, L, P or H, [0159] R3 is any one amino acid selected from I, L, A, P, or H, [0160] R4 is any one amino acid selected from F, E, A, L, P or H, [0161] R5 is any one amino acid selected from R or K, [0162] R6 is any one amino acid selected from A, M, I, P, H or L, [0163] R7 is any one amino acid selected from L, A, P or H, [0164] R8 is any one amino acid selected from I, L, A, P or H, [0165] R9 is any one amino acid selected from S, E or Y, [0166] R10 is any one amino acid selected from H, K, R, P or L, and any one amino acid selected from KK, KKK, and KKKK can be added thereto. However, except when the sequence is MIIFRIAASHKK, MIIFRALISHKK, MIIFRAAASHKK, LIIFRIAASHKK, MIIFRIAAYHKK, MIIFKIAASHKK, LIIFRILISHKK, or MIIFRILISHKK.

[0167] Meanwhile, the amino acid sequence of the peptide according to the present invention can be modified according to the conventional techniques known in the art. For example, the peptide of the present invention can be modified by increasing or decreasing the number of amino acids. In addition, within a range that does not reduce the activity of the peptide according to the present invention, the peptide can be modified by changing the order of specific residue components except for the residues that are directly involved in binding or must be conserved. Modifiable amino acids can be modified not only with natural L-α-amino acids, but also with β, γ, δ amino acids as well as D-α-amino acid derivatives. Typically, as a result of examining the effect of electrostatic force or hydrophilicity on binding using a peptide in which one amino acid is substituted, it can be seen that the sensitivity changes when positively charged amino acids (for example, Lys, Arg, His) or negatively charged amino acids (for example, Glu, Asp, Asn, Gln) are substituted. As such, the number or type of the residues to be substituted or added is determined by the required space between the essential binding points and the required functions such as hydrophilicity or hydrophobicity. By such substitution, the affinity of the peptide according to the present invention to the target protein can be further increased. Significant changes in function can also result from the substitution. The selection of the residue to be modified has a great influence on the electrical conductivity, hydrophobicity and maintaining the basic backbone of the peptide such as side chain change or helix structure change of the molecule present at the target position. In general, substitution of a hydrophilic residue such as serine with a hydrophobic residue such as leucine, isoleucine, phenylalanine, valine or alanine, substitution of an electrically positive residue such as lysine, arginine or histidine with an electrically negative residue such as glutamic acid or aspartic acid, or substitution of an amino acid having no side chain, such as glycine, with a residue having a bulky side chain causes a significant change in the properties of a peptide. In consideration of the above-mentioned facts, those of ordinary skill in the art can modify the specific peptide using conventional techniques within the range of maintaining, or not increasing/impairing its binding ability to HRF and the histamine secretion inhibitory activity, and this is within the scope of the present invention.

[0168] Hereinafter, the present invention will be described in detail by the following examples and experimental examples. However, the following examples and experimental examples are only for illustrating the present invention, and the contents of the present invention are not limited thereto.

<Preparative Example 1> Preparation of Methoxy Polyethylene Glycol Propionaldehyde (5000) (aldehyde-PEG (5000))

[0169] Methoxy polyethylene glycol propionaldehyde (5 kDa) was purchased from NOF CORPORATION (Japan) and prepared.

<Preparative Example 2> Preparation of Methoxy Polyethylene Glycol Hydrazide (5000) (hydrazide-PEG (5000))

[0170] Methoxy polyethylene glycol hydrazide (5 kDa) was purchased from SunBio (Korea) and prepared.

<Preparative Example 3> Preparation of Methoxy Polyethylene Glycol Propionaldehyde (10000) (aldehyde-PEG (10000))

[0171] Methoxy polyethylene glycol propionaldehyde (10 kDa) was purchased from NOF CORPORATION (Japan) and prepared.

Preparative Example 4

[0172] A peptide having an amino acid sequence of WYVYPSM (dTBP2) was prepared according to Example 1 of Korean Laid-Open Patent Publication No. 10-2017-0004906.

<Example 1> Preparation of N-Terminal 5K PEG-Conjugated dTBP2 (aldehyde-PEG (5000)-dTBP2)

[0173] The N-terminal 5K PEG-conjugated dTBP2 (aldehyde-PEG (5000)-dTBP2) prepared by the method described below was supplied and used. Specifically, 5 mg of dTBP2 prepared in Preparative Example 4 was dissolved in purified water to prepare a peptide solution at a concentration of 10 mg/mL, and 53 mg of methoxy polyethylene glycol propionaldehyde prepared in Preparative Example 1 was dissolved in 0.1 M acetate buffer (40 mM NaCNBH.sub.3 (pH 5.5)) to prepare a PEG solution. After mixing the solution of Preparative Example 4 and the solution of Preparative Example 1 (reaction molar ratio of 1:2), the mixture was reacted at 4° C. for 18 hours to prepare the N-terminal 5K PEG-conjugated dTBP2 of Example 1.

<Example 2> Preparation of C-Terminal 5K PEG-Conjugated dTBP2 (hydrazide-PEG (5000)-dTBP2)

[0174] The C-terminal 5K PEG-conjugated dTBP2 (hydrazide-PEG (5000)-dTBP2) prepared by the method described below was supplied and used. Specifically, 2 mg of dTBP2 prepared in Preparative Example 4 was dissolved in purified water to prepare a peptide solution at a concentration of 5 mg/mL, and 52.2 mg of methoxy polyethylene hydrazide prepared in Preparative Example 2 was dissolved in 50 mM MES buffer (pH 4.4) to prepare a PEG solution. Then, the solution of Preparative Example 4 and the solution of Preparative Example 2 were added (reaction molar ratio of 1:5) to the dTBP2 peptide solution, followed by stirring at room temperature for 10 minutes. The C-terminal 5K PEG-conjugated dTBP2 according to Example 2 was prepared by reacting 4 mg of EDAC (N-(3-methylaminopropyl)-N-ethylcarbodiimide hydrochloride) at room temperature for 1 hour and 30 minutes.

<Example 3> Preparation of N-Terminal 10K PEG-Conjugated dTBP2 (aldehyde-PEG (10000)-dTBP2)

[0175] The N-terminal 10K PEG-conjugated dTBP2 (aldehyde-PEG (10000)-dTBP2) prepared by the method described below was supplied and used.

[0176] Specifically, 50 mg of dTBP2 prepared in Preparative Example 4 was dissolved in purified water to prepare a peptide solution at a concentration of 10 mg/mL, and 1060 mg of methoxy polyethylene hydrazide prepared in Preparative Example 3 was dissolved in 0.1 M acetate buffer (40 mM NaCNBH.sub.3 (pH 5.5)) to prepare a PEG solution. After mixing the solution of Preparative Example 4 and the solution of Preparative Example 3 (reaction molar ratio of 1:2), the mixture was reacted at 4° C. for 18 hours.

<Experimental Example 1> Separation and Purification of PEG (5000)-dTBP2 According to Examples 1 and 2, HPLC Analysis, and Mass Spectrometry

[0177] The PEG (5000)-dTBP2 prepared in Examples 1 and 2 above was dialyzed in 10 mM Tris buffer using Centricon-30, and each of the PEG (5000)-dTBP2 prepared in Examples 1 and 2 was separated using an anion exchange resin (mono-Q; Pharmacia, Sweden). The sodium salt used in the separation process was used in a concentration gradient from 0 to 300 mM. The amount of the PEG (5000)-dTBP2 according to Examples 1 and 2 separated was confirmed by size-exclusion HPLC (High Performance Liquid Chromatography) and MALDI-TOF mass spectrometry. The results are shown in FIGS. 1 to 4.

<Experimental Example 2> Separation and Purification of PEG (10000)-dTBP2 According to Example 3, HPLC Analysis, and Mass Spectrometry

[0178] The PEG (10000)-dTBP2 prepared in Example 3 above was dialyzed in 10 mM Tris buffer using Centricon-30, and the aldehyde-PEG (10000)-dTBP2 prepared in Example 3 was separated using an anion exchange resin (mono-Q; Pharmacia, Sweden). The sodium salt used in the separation process was used in a concentration gradient from 0 to 300 mM. The amount of the aldehyde-PEG (10000)-dTBP2 according to Example 3 separated was confirmed by size-exclusion HPLC (High Performance Liquid Chromatography) and MALDI-TOF mass spectrometry. The results are shown in FIGS. 5 and 6.

<Experimental Example 3> Confirmation of Inhibitory Effect of dTBP2 and PEG (5000)-dTBP2 According to Examples 1 and 2 on IL-8 Secretion by HRF in BEAS-2B Cells

[0179] The PEG (5000)-dTBP2 prepared in Preparative Example 4, Example 1, and Example 2 was diluted with 1% penicillin streptomycin/DMEM for each concentration (0, 0.75 nM, 7.5 nM), incubated with 75 nM recombinant HRF at room temperature for 15 minutes, and then treated to BEAS-2B cells grown to about 70% on a 48 well plate. The cells were cultured in a 37° C., 5% CO.sub.2 incubator for 24 hours, and the supernatant was obtained. Then, the released IL-8 was quantified by enzyme immunosorbent detection (Biolegend). The IL-8 enzyme immunosorbent detection was performed according to the protocol of Biolegend Human IL-8 ELISA kit with pre coated plates (431508). 300 μl of wash buffer was added to each well of the plate pre-coated with the IL-8 antibody, and the plate was washed a total of 4 times. Then, 50 μl of the supernatant was added to each well of the plate and left at room temperature for 2 hours. After 2 hours, the supernatant in the plate was removed, and 300 μl of wash buffer was added to each well of the plate. The plate was washed a total of 4 times, and then 100 μl of a detection antibody solution was added to each well of the plate and left at room temperature for 1 hour. Thereafter, 300 μl of wash buffer was added to each well of the plate, and the plate was washed a total of 4 times. Then, 100 μl of Avidin-HRP A solution was added to each well of the plate and left at room temperature for 30 minutes. Finally, 300 μl of wash buffer was added to each well of the plate, and after washing the plate 5 times, 100 μl of substrate solution F was added to each well of the plate and left at room temperature in a dark room. 100 μl of a stop solution was additionally added to each well of the plate, and absorbance was measured at 450 nm and 570 nm. The wash buffer, detection antibody solution, avidin HRP A solution, substrate solution F, and stop solution were all used as reagents included in a kit (431508, Biolegend). The results are shown in FIG. 7.

[0180] As a result, as shown in FIG. 7, it was confirmed that the dTBP2 of Preparative Example 1, Example 1 and Example 2 were all effective in the inhibition of IL-8 secretion by HRF in BEAS-2B cells. In particular, it was found that the inhibitory effect of the N-terminal 5K PEG-conjugated dTBP2 according to Example 1 on the IL-8 secretion by HRF was the best.

<Experimental Example 4> Confirmation of Inhibitory Effect of dTBP2 and N-Terminal 10K PEG-Conjugated dTBP2 on Eosinophil Increase in Bronchoalveolar Lavage Fluid

[0181] 4-1. Preparation of Ovalbumin-Induced Bronchial Asthma and Rhinitis Mouse Model and Administration of dTBP2 and N-Terminal 10K PEG-Conjugated dTBP2

[0182] A bronchial asthma and rhinitis animal model was prepared by the following method. An ovalbumin solution was prepared by dissolving ovalbumin in an aluminum hydroxide solution (1 mg/ml) 28 and 14 days before the start of inducing bronchial asthma and rhinitis, and 200 μl of the solution was intraperitoneally injected into each mouse for sensitization. After 14 days of final sensitization, 20 μl of saline/PBS (normal control; NC) or 20 μl of 10 mg/mL ovalbumin solution (positive control; PC) was instilled into the nasal cavity of each lightly anesthetized mouse 4 times every other day for 8 days to induce bronchial asthma and rhinitis. 200 μl of the dTBP2 according to Preparative Example 4 or the N-terminal 10K PEG-conjugated dTBP2 according to Example 3 (1 mg/kg) was injected intraperitoneally to each mouse while inducing bronchial asthma and rhinitis under the same conditions as the PC group. Dexamethasone (D2915, Sigma Aldrich) was dissolved in sterile tertiary distilled water, and it was administered to each mouse at the concentration of 1 mg/kg the same number of times as the dTBP2 according to Preparative Example 4 and the N-terminal 10K PEG-conjugated dTBP2 according to Example 3 (FIG. 8).

TABLE-US-00001 TABLE 1 Sensitization Instillation Treatment Group (IP) (IN) (IP) 1 NC PBS PBS PBS, IP 2 PC OVA/alum OVA PBS, IP 3 dexamethasone OVA/alum OVA 1 mg/kg, IP 4 Preparative OVA/alum OVA 1 mg/kg, IP Example 4 5 Example 3 OVA/alum OVA 1 mg/kg, IP

[0183] 4-2. Confirmation of Inhibitory Effect of dTBP2 and N-Terminal 10K PEG-Conjugated dTBP2 on Eosinophil Increase in Bronchoalveolar Lavage Fluid

[0184] In order to confirm the inhibitory effect of the dTBP2 according to Preparative Example 4 and the N-terminal 10K PEG-conjugated dTBP2 according to Example 3 on eosinophil increase in the bronchial asthma and rhinitis model, the dTBP2 of Preparative Example 4 and Example 3 was administered to the mouse model of Example 4-1 through intraperitoneal injection a total of 8 times daily for 8 days inducing bronchial asthma and rhinitis. After anesthetizing the mouse by injecting a mixture of zoletil (250 mg/kg) and rompun (50 mg/kg) intraperitoneally, a 20-gauge catheter was inserted into the trachea by puncturing the trachea with a knife, and 0.6 mL of PBS at a time was injected-collected three times, and the collection rate was adjusted to 80%. The collected bronchoalveolar lavage fluid was centrifuged at 4° C., 3000 rpm for 10 minutes, and the cell precipitate was resuspended in 0.1 mL of PBS. 20 μL of the suspension was analyzed for the composition of blood cells using a fully automatic hematology analyzer (HEMAVET 950 FS, Drew Scientific, Inc.). The results are shown in FIG. 9.

[0185] As a result, as shown in FIG. 9, it was confirmed that eosinophils, which are inflammatory cells, were decreased in both groups treated with the dTBP2 according to Preparative Example 4 and the N-terminal 10K PEG-conjugated dTBP2 according to Example 3 when compared with the positive control group (PC). Particularly, it was confirmed that the dTBP2 according to Preparative Example 4 reduced the number of eosinophils increased to about 0.75×10.sup.5 cells/mL by the ovalbumin solution by about 40%. It was also confirmed that the N-terminal 10K PEG-conjugated dTBP2 according to Example 3 reduced the increased eosinophils by about 55% with a superior effect. From the above results, it was confirmed that the eosinophil reduction effect could be further improved by pegylation of dTBP2 using the aldehyde-PEG (10000).

<Experimental Example 5> Confirmation of Inhibitory Effect of dTBP2 and N-Terminal 10K PEG-Conjugated dTBP2 on IL-5 Secretion in Bronchoalveolar Lavage Fluid

[0186] In order to confirm the inhibitory effect of the dTBP2 according to Preparative Example 4 and the N-terminal 10K PEG-conjugated dTBP2 according to Example 3 on IL-5 secretion in the bronchial asthma and rhinitis model, the dTBP2 of Preparative Example 4 and Example 3 was administered to the mouse model of Example 4-1 through intraperitoneal injection a total of 8 times daily for 8 days inducing bronchial asthma and rhinitis. After the end of the experiment, the amount of IL-5 in the supernatant obtained by centrifuging bronchoalveolar lavage fluid was measured and quantified by enzyme immunosorbent detection using mouse IL-5 ELISA kit (Biolegend, USA). The results are shown in FIG. 10.

[0187] As a result, as shown in FIG. 10, it was confirmed that the dTBP2 according to Preparative Example 4 and the N-terminal 10K PEG-conjugated dTBP2 according to Example 3 effectively inhibited IL-5 secretion in bronchoalveolar lavage fluid. Specifically, the dTBP2 according to Preparative Example 4 reduced IL-5 secretion by about 25%, and the N-terminal 10K PEG-conjugated dTBP2 according to Example 3 reduced IL-5 secretion by about 50%.

[0188] Next, in order to confirm whether there is an effect of inhibiting IL-5 secretion even when administered in a small number of times, the dTBP2 of Preparative Example 4 and Example 3 was administered to the mouse model of Example 4-1 through intraperitoneal injection once in 4 days for a total of 2 times for 8 days inducing bronchial asthma and rhinitis. The amount of IL-5 was measured and quantified by the enzyme immunosorbent detection method in the same manner. The results are shown in FIG. 11.

[0189] In addition, the dTBP2 of Preparative Example 4 and Example 3 was administered to the mouse model of Example 4-1 through intraperitoneal injection once on the 1st day of 8 days inducing bronchial asthma and rhinitis. The amount of IL-5 was measured and quantified by the enzyme immunosorbent detection method in the same manner. The results are shown in FIG. 12.

[0190] As a result, as shown in FIGS. 11 and 12, it was confirmed that the N-terminal 10K PEG-conjugated dTBP2 according to Example 3 was effective in inhibiting IL-5 secretion even when administered only once within the induction period of bronchial asthma and rhinitis.

[0191] Next, in order to confirm whether there is an inhibitory effect on IL-5 secretion even when administered after inducing bronchial asthma and rhinitis, the dTBP2 of Preparative Example 4 and Example 3 was administered to the mouse model of Example 4-1 through intraperitoneal injection once on the 5th day of 8 days inducing bronchial asthma and rhinitis. The amount of IL-5 was measured and quantified by the enzyme immunosorbent detection method in the same manner. The results are shown in FIG. 13.

[0192] As a result, as shown in FIG. 13, it was confirmed that the N-terminal 10K PEG-conjugated dTBP2 according to Example 3 effectively inhibited IL-5 secretion in bronchoalveolar lavage fluid. The dTBP2 according to Preparative Example 4 showed a weak IL-5 inhibitory effect, whereas the N-terminal 10K PEG-conjugated dTBP2 according to Example 3 had an IL-5 secretion inhibitory effect even after only one administration after inducing bronchial asthma and rhinitis. From the above results, it was confirmed that the IL-5 secretion inhibitory effect could be further improved by pegylation of dTBP2 using the aldehyde-PEG (10000).

<Experimental Example 6> Confirmation of Inhibitory Effect of dTBP2 and N-Terminal 10K PEG-Conjugated dTBP2 on IL-4 Secretion in Bronchoalveolar Lavage Fluid

[0193] In order to confirm the inhibitory effect of the dTBP2 according to Preparative Example 4 and the N-terminal 10K PEG-conjugated dTBP2 according to Example 3 on IL-4 secretion in the bronchial asthma and rhinitis model, the dTBP2 of Preparative Example 4 and Example 3 was administered to the mouse model of Example 4-1 through intraperitoneal injection a total of 8 times daily for 8 days inducing bronchial asthma and rhinitis. After the end of the experiment, the amount of IL-4 in the supernatant obtained by centrifuging bronchoalveolar lavage fluid was measured and quantified by enzyme immunosorbent detection using mouse IL-4 ELISA kit (Biolegend, USA). The results are shown in FIG. 14.

[0194] As a result, as shown in FIG. 14, it was confirmed that the dTBP2 according to Preparative Example 4 and the N-terminal 10K PEG-conjugated dTBP2 according to Example 3 effectively inhibited IL-4 secretion in bronchoalveolar lavage fluid. Specifically, the dTBP2 according to Preparative Example 4 reduced IL-4 secretion by about 50%, and the N-terminal 10K PEG-conjugated dTBP2 according to Example 3 reduced IL-4 secretion by about 60%.

[0195] Next, in order to confirm whether there is an effect of inhibiting IL-4 secretion even when administered in a small number of times, the dTBP2 of Preparative Example 4 and Example 3 was administered to the mouse model of Example 4-1 through intraperitoneal injection once in 4 days for a total of 2 times for 8 days inducing bronchial asthma and rhinitis. The amount of IL-4 was measured and quantified by the enzyme immunosorbent detection method in the same manner. The results are shown in FIG. 15.

[0196] In addition, the dTBP2 of Preparative Example 4 and Example 3 was administered to the mouse model of Example 4-1 through intraperitoneal injection once on the 1st day of 8 days inducing bronchial asthma and rhinitis. The amount of IL-4 was measured and quantified by the enzyme immunosorbent detection method in the same manner. The results are shown in FIG. 16.

[0197] As a result, as shown in FIGS. 15 and 16, it was confirmed that the N-terminal 10K PEG-conjugated dTBP2 according to Example 3 was effective in inhibiting IL-4 secretion even when administered only once within the induction period of bronchial asthma and rhinitis.

[0198] Next, in order to confirm whether there is an inhibitory effect on IL-4 secretion even when administered after inducing bronchial asthma and rhinitis, the dTBP2 of Preparative Example 4 and Example 3 was administered to the mouse model of Example 4-1 through intraperitoneal injection once on the 5th day of 8 days inducing bronchial asthma and rhinitis. The amount of IL-4 was measured and quantified by the enzyme immunosorbent detection method in the same manner. The results are shown in FIG. 17.

[0199] As a result, as shown in FIG. 17, it was confirmed that the N-terminal 10K PEG-conjugated dTBP2 according to Example 3 effectively inhibited IL-4 secretion in bronchoalveolar lavage fluid. The dTBP2 according to Preparative Example 4 showed a weak IL-4 inhibitory effect, whereas the N-terminal 10K PEG-conjugated dTBP2 according to Example 3 had an IL-4 secretion inhibitory effect even after only one administration after inducing bronchial asthma and rhinitis. From the above results, it was confirmed that the IL-4 secretion inhibitory effect could be further improved by pegylation of dTBP2 using the aldehyde-PEG (10000).

<Experimental Example 7> Confirmation of Inhibitory Effect of dTBP2 and N-Terminal 10K PEG-Conjugated dTBP2 on IL-13 Secretion in Bronchoalveolar Lavage Fluid

[0200] In order to confirm the inhibitory effect of the dTBP2 according to Preparative Example 4 and the N-terminal 10K PEG-conjugated dTBP2 according to Example 3 on IL-13 secretion in the bronchial asthma and rhinitis model, the dTBP2 of Preparative Example 4 and Example 3 was administered to the mouse model of Example 4-1 through intraperitoneal injection a total of 8 times daily for 8 days inducing bronchial asthma and rhinitis. After the end of the experiment, the amount of IL-13 in the supernatant obtained by centrifuging bronchoalveolar lavage fluid was measured and quantified by enzyme immunosorbent detection using mouse IL-13 ELISA kit (R&D system, USA). The results are shown in FIG. 18.

[0201] As a result, as shown in FIG. 18, it was confirmed that the dTBP2 according to Preparative Example 4 and the N-terminal 10K PEG-conjugated dTBP2 according to Example 3 effectively inhibited IL-13 secretion in bronchoalveolar lavage fluid. Specifically, the dTBP2 according to Preparative Example 4 reduced IL-13 secretion by about 30%, and the N-terminal 10K PEG-conjugated dTBP2 according to Example 3 reduced IL-13 secretion by about 20%.

[0202] Next, in order to confirm whether there is an effect of inhibiting IL-13 secretion even when administered in a small number of times, the dTBP2 of Preparative Example 4 and Example 3 was administered to the mouse model of Example 4-1 through intraperitoneal injection once in 4 days for a total of 2 times for 8 days inducing bronchial asthma and rhinitis. The amount of IL-13 was measured and quantified by the enzyme immunosorbent detection method in the same manner. The results are shown in FIG. 19.

[0203] In addition, the dTBP2 of Preparative Example 4 and Example 3 was administered to the mouse model of Example 4-1 through intraperitoneal injection once on the 1st day of 8 days inducing bronchial asthma and rhinitis. The amount of IL-13 was measured and quantified by the enzyme immunosorbent detection method in the same manner. The results are shown in FIG. 20.

[0204] As a result, as shown in FIGS. 19 and 20, it was confirmed that the N-terminal 10K PEG-conjugated dTBP2 according to Example 3 was effective in inhibiting IL-13 secretion even when administered only once within the induction period of bronchial asthma and rhinitis.

[0205] Next, in order to confirm whether there is an inhibitory effect on IL-13 secretion even when administered after inducing bronchial asthma and rhinitis, the dTBP2 of Preparative Example 4 and Example 3 was administered to the mouse model of Example 4-1 through intraperitoneal injection once on the 5th day of 8 days inducing bronchial asthma and rhinitis. The amount of IL-13 was measured and quantified by the enzyme immunosorbent detection method in the same manner. The results are shown in FIG. 21.

[0206] As a result, as shown in FIG. 21, it was confirmed that the N-terminal 10K PEG-conjugated dTBP2 according to Example 3 had an IL-13 secretion inhibitory effect even after only one administration after inducing bronchial asthma and rhinitis. From the above results, it was confirmed that the IL-13 secretion inhibitory effect could be further improved by pegylation of dTBP2 using the aldehyde-PEG (10000).

<Experimental Example 8> Confirmation of Inhibitory Effect of dTBP2 and N-Terminal 10K PEG-Conjugated dTBP2 on Ovalbumin-Specific IgE Secretion in Mouse Plasma

[0207] Ovalbumin is an allergy-causing substance mainly contained in egg whites. In order to confirm the inhibitory effect of the dTBP2 according to Preparative Example 4 and the N-terminal 10K PEG-conjugated dTBP2 according to Example 3 on ovalbumin-specific IgE secretion in plasma in the bronchial asthma and rhinitis model, the dTBP2 of Preparative Example 4 and Example 3 was administered to the mouse model of Example 4-1 through intraperitoneal injection a total of 8 times daily for 8 days inducing bronchial asthma and rhinitis. After completion of the experiment, the mouse was anesthetized by injecting a mixture of zoletil (250 mg/kg) and rompun (50 mg/kg) intraperitoneally, and the chest was opened. 0.8 mL blood was collected by puncture of the heart with a 26 gauge 1 mL syringe coated with heparin. The collected blood was centrifuged at 4° C., 3000 rpm for 10 minutes to obtain plasma components. The amount of ovalbumin-specific IgE in the blood serum obtained through the above process was measured and quantified by enzyme immunosorbent detection method using mouse OVA specific IgE kit (Biolegend, USA). The results are shown in FIG. 22.

[0208] As a result, as shown in FIG. 22, it was confirmed that the dTBP2 according to Preparative Example 4 and the N-terminal 10K PEG-conjugated dTBP2 according to Example 3 effectively inhibited ovalbumin-specific IgE secretion in plasma. Specifically, the dTBP2 according to Preparative Example 4 reduced ovalbumin-specific IgE secretion by about 50%, and the N-terminal 10K PEG-conjugated dTBP2 according to Example 3 reduced ovalbumin-specific IgE secretion by about 50%.

[0209] From the above results, it was confirmed that the dTBP2 and N-terminal 10K PEG-conjugated dTBP2 were very effective in inhibiting ovalbumin-specific IgE secretion in plasma.

[0210] Next, in order to confirm whether there is an effect of inhibiting ovalbumin-specific IgE secretion in plasma even when administered in a small number of times, the dTBP2 of Preparative Example 4 and Example 3 was administered to the mouse model of Example 4-1 through intraperitoneal injection once in 4 days for a total of 2 times for 8 days inducing bronchial asthma and rhinitis. The amount of ovalbumin-specific IgE secretion was measured and quantified by the enzyme immunosorbent detection method in the same manner. The results are shown in FIG. 23.

[0211] In addition, the dTBP2 of Preparative Example 4 and Example 3 was administered to the mouse model of Example 4-1 through intraperitoneal injection once on the 1st day of 8 days inducing bronchial asthma and rhinitis. The amount of ovalbumin-specific IgE secretion was measured and quantified by the enzyme immunosorbent detection method in the same manner. The results are shown in FIG. 24.

[0212] As a result, as shown in FIGS. 23 and 24, it was confirmed that the N-terminal 10K PEG-conjugated dTBP2 according to Example 3 was effective in inhibiting ovalbumin-specific IgE secretion even when administered only once within the induction period of bronchial asthma and rhinitis. It was also confirmed that the N-terminal 10K PEG-conjugated dTBP2 of Example 3 had better inhibitory ability than the dTBP2 of Preparative Example 4.

[0213] Next, in order to confirm whether there is an inhibitory effect on ovalbumin-specific IgE secretion in plasma even when administered after inducing bronchial asthma and rhinitis, the dTBP2 of Preparative Example 4 and Example 3 was administered to the mouse model of Example 4-1 through intraperitoneal injection once on the 5th day of 8 days inducing bronchial asthma and rhinitis. The amount of ovalbumin-specific IgE secretion was measured and quantified by the enzyme immunosorbent detection method in the same manner. The results are shown in FIG. 25.

[0214] As a result, as shown in FIG. 25, it was confirmed that the N-terminal 10K PEG-conjugated dTBP2 according to Example 3 had an ovalbumin-specific IgE secretion inhibitory effect even after only one administration after inducing bronchial asthma and rhinitis. From the above results, it was confirmed that the ovalbumin-specific IgE secretion inhibitory effect could be further improved by pegylation of dTBP2 using the aldehyde-PEG (10000).

<Experimental Example 9> Pharmacokinetic Analysis of dTBP2 and N-Terminal 10K PEG-Conjugated dTBP2

[0215] The dTBP2 according to Preparative Example 4 and the N-terminal 10K PEG-conjugated dTBP2 according to Example 3 were administered to 7-8 weeks old ICR mice that had undergone the acclimatization period at the concentration of 10 mg/kg IV, respectively, and then blood was collected according to a predetermined time. The dTBP2 of Preparative Examples 4 and Example 3 was added to a solution prepared in DMSO:PEG400:DW=5:55:40, and this was administered at the concentration of 5 mL/kg. Blood samples were taken at 0.083, 0.25, 0.5, 1, 2, 4, and 8 hours post injection. The blood was centrifuged at 4° C., 3000 rpm for 10 minutes, to which cold acetonitrile containing internal standard was added in an amount 4 times the plasma component, and then deproteinization was carried out. After centrifugation again at 4° C., 13000 rpm for 10 minutes, the supernatant was collected and analyzed by Agilent 6460 LC-MS/MS. Pharmacokinetic analysis was performed with Phoenix WinNonlin (Pharsight ver 6.4, USA) using a non-compartmental analysis model. The results are shown in FIG. 26, and the pharmacokinetic analysis results were analyzed and shown in Table 2 below.

TABLE-US-00002 TABLE 2 Preparative Example 4, Example 3, Parameter IV, 10 mg/kg IV, 10 mg/kg T.sub.max (h) NA NA C.sub.max (μg/mL) NA NA T.sub.1/2 (h) 1.91 3.18 AUC.sub.last (μg .Math. h/mL) 0.02 2.83 AUC.sub.∞ (μg .Math. h/mL) 0.03 3.15 CL (L/h/kg) 292.34 3.29 V.sub.ss (L/kg) 782.86 7.37 F.sub.t (%) NA NA NA: not applicable T.sub.max: time to C.sub.max C.sub.max: maximum plasma concentration T.sub.1/2: half life AUC.sub.last: area of concentration-time curve from beginning to the maximum time at which the concentration can be measured AUC.sub.∞: area of concentration-time curve from beginning to infinity CL: clearance from plasma V.sub.ss: steady-state volume of distribution F.sub.t: bioavailability (AUC.sub.P.O./AUC.sub.I.V.) × 100

[0216] As a result, as shown in FIG. 26, it was confirmed that the stability of the N-terminal 10K PEG-conjugated dTBP2 according to Example 3 in plasma was improved by the dTBP2 according to Preparative Example 4. In addition, as shown in Table 1, it was confirmed that the half-life of the PEGylated dTBP2 of Example 3 was increased by about 1.6 times compared to the dTBP2 of Preparative Example 4. In addition, as shown in FIG. 18, it was confirmed that the initial plasma concentration of the N-terminal 10K PEG-conjugated dTBP2 according to Example 3 was about 1000 times higher than that of the dTBP2 according to Preparative Example 4. From the above results, it was found that the stability was increased by about 1000 times by pegylation of the peptide of Preparative Example 4.

<Experimental Example 10> Confirmation of Metabolic Stability of 10K PEG-Conjugated dTBP2

[0217] In order to evaluate the first-step metabolic stability of the N-terminal 10K PEG-conjugated dTBP2 according to Example 3 in the liver, the following evaluation was performed using mouse and human liver microsomes. After incubating the mouse and human liver microsomes diluted with 0.5 M potassium phosphate buffer (pH 7.4) at 37° C. for 5 minutes, NADPH for activating the metabolic enzyme system was reacted with the peptide of Example 3 at 37° C. for 30 minutes. Then, the amount of the peptide of Example 3 remaining was analyzed. Upon completion of the reaction, cold acetonitrile containing internal standard was added, and then deproteinization was carried out. The reactant was centrifuged at 4° C., 4000 rpm for 15 minutes, and the supernatant was analyzed by LC-MS/MS. The system was verified with buspirone, a reference material. The results are shown in Table 3.

TABLE-US-00003 TABLE 3 Compound Mouse (%) Human (%) Example 3 75.26 75.77 Buspirone 0.10 6.15

[0218] In the evaluation of the experiment, the microsome stability evaluation criteria according to the % value, which is the result of the reaction experiment for 30 minutes, are as follows (R. SCOTT OBACH, Prediction of human clearance of twenty-nine drugs form hepatic microsomal intrinsic clearance data: An examination of in vitro half-life approach and nonspecific binding to microsomes, 1999, 27, (11): 1350-59).

[0219] >90%: very stable compound with a half-life longer than 3 hours

[0220] 70˜90%: stable compound with a half-life of 1 to 3 hours

[0221] 50˜70%: relatively stable compound with a half-life of 30 to 60 minutes

[0222] 30˜50%: relatively unstable compound with a half-life of 15 to 30 minutes

[0223] <30%: unstable compound with a half-life of less than 15 minutes

[0224] Referring to the above evaluation criteria, the N-terminal 10K PEG-conjugated dTBP2 according to Example 3 of the present invention was confirmed to be a stable compound with a half-life of about 1 to 3 hours since the remaining drug was more than 75% after 30 minutes in both mice and humans. Therefore, since the peptide can exert a drug effect with high stability in vivo, the number of administration can be reduced, so that it can be effectively used as a drug.

[0225] As mentioned above, the present invention has been described in detail through the preferred preparative examples, examples and experimental examples, but the scope of the present invention is not limited to the specific examples, and should be interpreted by the appended claims. In addition, those of ordinary skill in the art should understand that many modifications and variations are possible without departing from the scope of the present invention.