Klk5 inhibitor and method for producing the same

Abstract

To provide a novel peptide.SOLUTION: A peptide contains the amino acid sequence represented by SEQ ID NO:61 and inhibits protease.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to peptides, polynucleotides, vectors, cells, methods for producing peptides, and A peptide, a conjugate containing a peptide, a peptide or a conjugate obtained by the method Compositions containing gates, pharmaceutical compositions, pharmaceutical compositions for treating or preventing various diseases, and compositions for treating or preventing various diseases Use of peptides or conjugates for the treatment or prevention of diseases, and methods for treating various diseases including the step of administering a peptide or a conjugate. The present invention relates to compositions for diagnosing or testing diseases. [Background technology]

[0002] Kallikrein 5 (KLK5) is a trypsin-like serine protease (Clan PA, family S1) and stratum corneu It is also called SCTE (serotype 7 tryptic enzyme). likrein7 (KLK7) is a chymotrypsin-like protease (Clan PA, f amily S1) and stratum corneum chymotrypti It is also called SCCE (SEQ ID NO: 14). ein14;KLK14) is a trypsin-like protease (Clan PA, family KLK5, KLK7, and KLK14 are highly conserved transcription factors in 15 species. The tissue kallikrein family consists of trypsin- or chymotrypsin-like serine proteases After being expressed in cells, KLK5 is converted to active KLK5 by autoactivation. KLK7 and KLK14 are expressed as inactive preproenzymes, whereas KLK7 and KLK14 are expressed as inactive preproenzymes. The prepro sequence is cleaved by proteases such as KLK5, converting it into an active form. KLK5 is expressed in the skin and is involved in the regulation of Desmog It has been reported that it degrades factors involved in cell adhesion, such as lein and desmocollin. KLK5, KLK7, and KLK14 are thought to be important in skin desquamation (Non-Patent Document 2). Furthermore, protease-activated receptor 2 (Proteinase-activated receptor 2) It is involved in the activation of PAR-2 ​​receptor 2 (PAR-2) (Non-patent Document 3). Activation induces cytokines and chemokines, enhancing immune and inflammatory responses.

[0003] Netherton syndrome is an autosomal recessive disorder It can cause severe skin inflammation, scaling, hair abnormalities, and allergies such as asthma and allergic dermatitis. It is a rare symptomatic ichthyosis syndrome (OMIM256500) (non-patent literature) Reference 4). Exfoliative dermatitis occurs from birth, and is caused by severe damage to the skin barrier function. It can cause dehydration and infections, and may also be accompanied by growth retardation. Netherton syndrome is a condition characterized by the deficiency of serine 1, a nucleotide polynucleotide expressed in skin epithelial cells. associated with mutations in the gene (SPINK5) encoding the protease inhibitor (LEKTI) It is caused by a loss of function of LEKTI. TI consists of 15 Kazal-like inhibitor domains, but in patients with Netherton syndrome Multiple mutations were found in SPINK5, which encodes each domain. Symptoms and severity vary depending on the location (Non-patent documents 4, 5).

[0004] SPINK5-deficient mice (Spink5 - / - ) shows skin symptoms similar to Netherton syndrome. In addition, high protease activity of KLK5 and KLK7 is observed in skin epithelium (non-patented Reference 1). SPINK5-deficient mice die within a few hours after birth, but Mice were crossed with KLK5-deficient mice (Spink5 - / - Klk5 - / - ) Improvement in neonatal mortality rate has been reported (Non-patent Document 6). - / - Kl k5 - / - In mice, Spink5 - / - The severe skin barrier defects and epithelial Structural defects and skin inflammation are resolved. SPINK5 mutant Spink5 A135X / A135X Mice with Netherton syndrome The mice exhibited skin symptoms similar to those of the neutrophil group and died within 12 hours after birth, whereas the mice lacking KLK5 showed similar skin symptoms. Klk5, a cross between a mouse and a mouse - / - Spink5 A135X / A135X So the skin barrier Furthermore, the treatment of KLK7-deficient mice improved the severity of severe skin symptoms (Non-Patent Document 7). By combining (Klk5 - / - Klk7 - / - Spink5 A135X / A135X ), and abnormal skin symptoms are no longer observed. It has been reported that Netherton syndrome-like skin symptoms are present (Non-patent document 8). In the stratum corneum of patients with Netherton syndrome and Netherton syndrome model mice, trypsin-like and kymotomycin-like proteins were detected. It has been shown to have high liposin-like protease activity, and in addition to KLK5, KLK7 and KL Kallikrein family members located downstream, such as K14, are involved in the protease activity of the stratum corneum These findings suggest that Netherton syndrome is caused by a mutation in the SPINK5 gene. The cause is abnormally increased expression of KLK5, KLK7, and KLK14 proteases in the keratinocyte. Currently, symptomatic treatments include applying moisturizers. There is no cure for the disease.

[0005] KLK5 has also been implicated in rosacea, a chronic inflammatory disease of the face. Increased expression of KLK5 and the antimicrobial peptide Cathelicidin has been reported. Although the details of the pathogenesis of this disease are unknown, it is thought that increased expression of KLK5 degrades cathelicidin. It is believed that this produces peptides that cause rosacea (Non-patent Document 9 ).

[0006] There are several reports that SPINK5 polymorphisms are associated with the severity of atopic dermatitis. (Non-Patent Documents 10 to 14) In the skin of atopic dermatitis patients who have both alleles of the SPINK5 gene, which regulates glutamate production, Desmoglein1 protein levels were significantly higher than when the amino acid was encoded biallelically. The expression of KLK5 and KLK7 was decreased and the activity of proteases such as KLK6 and KLK7 was increased. It has been reported that the activation of these proteases leads to the formation of a barrier around the skin. This weakening of the immune system makes it easier for allergens to invade, and also creates conditions that make it more susceptible to inflammatory reactions. It is thought that they will release it.

[0007] SPINK2(Serine Protease Inhibitor Kazal- type 2) is a Kazal-like domain with three disulfide bonds, and It functions as a ypsin / acrosin inhibitor (Non-patent Document 16). SPINK2 and its mutations in diseases such as Netherton syndrome, rosacea, and atopic dermatitis The relationship has not been revealed. [Prior art documents] [Non-patent literature]

[0008] [Non-Patent Document 1] Ovaere P, et al. (2009) Trends Biochem Sci. 34(9): 453-463 [Non-patent document 2] Descargues P, et al. (2005) Nature Genetics, Vol. 37(1): pp. 56-65 [Non-patent document 3] Rattenholl A, et al. (2008) Drug News and Perspectives, Vol. 21 (No. 7): pp. 369-381 [Non-patent document 4] Hovnanian A. (2013) Cell Tissue Res. 351(2):289-300 [Non-patent document 5] Sarri CA, et al. (2017) Mol Diagn Ther. Vol. 21(2): pp. 137-152 [Non-patent document 6] Furio L, et al. (2015) PLoS Genet. 11(9): e1005389 [Non-Patent Document 7] Kasparek P, et al. (2017) PLoS Genet. 13(1): e1006566 [Non-patent document 8] Furio L, et al. (2014) Journal of Experimental Medicine (J Exp Med.) Vol. 211(3): pp. 499-513 [Non-Patent Document 9] Yamasaki K, et al. (2007) Nature Medicine (Nat Med.) Vol. 13 (No. 8): pp. 975-980 [Non-Patent Document 10] Nishio Y, et al. (2003) Genes and Immunity, Vol. 4 (No. 7): pp. 515-517 [Non-Patent Document 11] Kusunoki T, et al. (2005) J Allergy Clin Immunol. Vol. 4 (No. 7): pp. 515-517 [Non-Patent Document 12] Lan CC, et al. (2011) Experimental Dermatology, Vol. 20(12): 975-979 [Non-Patent Document 13] Kato A, et al. (2003) British Journal of Dermatology, Vol. 148 (No. 4): pp. 665-669 [Non-Patent Document 14] Zhao LP, et al. (2012) J Eur Acad Dermatol Venereol. 26(5):572-577 [Non-Patent Document 15] Fortugno P, et al. (2012) Hum Mol Genet. 21(19): 4187-4200 [Non-Patent Document 16] Chen T, et al. (2009) Proteins. 77(1):209-219 Summary of the Invention [Problem to be solved by the invention]

[0009] Novel KLK5 inhibitory peptide, conjugate containing said peptide, said peptide or and providing a pharmaceutical composition containing the conjugate. [Means for solving the problem]

[0010] The present invention (1) A polypeptide comprising the amino acid sequence shown in SEQ ID NO: 61 (Figure 69) and an active form of human KLK5. SPINK2 mutant peptides that inhibit the protease activity of (2) The method according to (1), which inhibits the protease activity of human KLK7 or human KLK14. peptide, (3) (1) The inhibition is selective for human KLK5 and optionally human KLK7 or KLK14. The peptides listed above (4) Xaa 16 (X1) is Ala, Asp, Gly, Gln, Leu, Ser, or Thr The peptide according to any one of (1) to (3), (5) Xaa 17 (X2) is Arg, Glu, Asn, Gln, or Ser; (1) The peptide according to any one of (1) to (4), (6) Xaa at position 18 (X3) is Asp, Gln, Ile, Thr, Trp, or Tyr; A peptide according to any one of (1) to (5). (7) Xaa 19 (X4) is Arg, Gly, Met, Gln, or Thr; (1) The peptide according to any one of (1) to (6). (8) Xaa at position 20 (X5) is Asp, Glu, Leu, Lys, Thr, or Tyr; A peptide according to any one of (1) to (7). (9) Xaa at position 21 (X6) is Glu, Gly, His, Leu, Ser, Gln, or Tyr The peptide according to any one of (1) to (8), (10) Xaa at position 22 (X7) is Asp, Gly, Gln, Sey, or Tyr; (1) The peptide according to any one of (9) to (9). (11) Xaa 24 (X8) is Ala, Asp, Glu, Gly, Asn, Ser, or Thr The peptide according to any one of (1) to (10), (12) Xaa at position 25 (X9) is Arg or Lys; any one of (1) to (11) a peptide according to (13) No. 26 Xaa(X 10 ) is Asp, Glu, Gln, Ser, or Val, (1) (12) A peptide according to any one of (11) to (12). (14) No. 27 Xaa(X 11 ) is Phe or Tyr, any one of (1) to (13). a peptide according to any one of the preceding paragraphs; (15) No. 28 Xaa(X 12 ) is Asp or Glu, any one of (1) to (14). a peptide according to any one of the preceding paragraphs; (16) SEQ ID NOs: 6, 8, 10, 12, 14, 16, 18 and 20 (Fig. 14, 16, 18, 20 , 22, 24, 26 and 28) The peptide according to any one of (1) to (15), including any one of (1) to (63). (17) Any of (1) to (3) in which Xaa at position 16 (X1) is Gly, Met, or Tyr. a peptide according to any one of (18) Xaa 17 (X2) is Glu, Gln or Thr, (1) to (3) and (1 7) A peptide according to any one of (19) Xaa at position 18 (X3) is His, Met or Tyr, (1) to (3), (17 (18) A peptide according to any one of (17) and (18). (20) Xaa at position 19 (X4) is Ala, Arg, Lys, or Gln; (1) to (3) and a peptide according to any one of (17) to (19). (twenty one) Xaa at position 20 (X5) is Gly, Arg, or Ser; (1) to (3) and (1 The peptide according to any one of (7) to (20). (twenty two) Xaa at position 21 (X6) is Arg, Lys, Gln, or Ser; (1) to (3) and a peptide according to any one of (17) to (21). (twenty three) Xaa at position 22 (X7) is Gly. (1) to (3) and (17) to (22) The peptide according to any one of (twenty four) Xaa at position 24 (X8) is His, Thr, or Tyr; (1) to (3) and (1 The peptide according to any one of (7) to (23). (twenty five) Xaa at position 25 (X9) is His or Tyr; (1) to (3) and (17) to (19) (24) The peptide according to any one of (24). (26) No. 26 Xaa(X 10 ) is Asp, Glu or His, (1) to (3) and ( The peptide according to any one of (17) to (25). (27) No. 27 Xaa(X 11 ) is Tyr, (1) to (3) and (17) to (26) The peptide according to any one of (28) No. 28 Xaa(X 12 ) is Asp or Glu, (1) to (3) and (17) The peptide according to any one of (27) to (27). (29) Any one of SEQ ID NOs: 22, 24, 26 and 28 (Figures 30, 32, 34 and 36) (1) to (3) and (17) containing amino acid numbers 1 to 63 of the amino acid sequence shown. (28) A peptide according to any one of (10) to (28). (30) Any of (1) to (3) in which Xaa at position 16 (X1) is Gly, Ser, or Tyr. a peptide according to any one of (31) Xaa at position 17 (X2) is Asp or Gln; any of (1) to (3) and (30) The peptide according to any one of the preceding claims. (32) Xaa at position 18 (X3) is Thr or Val, (1) to (3), (30) and ( 31) A peptide according to any one of (33) Xaa at position 19 (X4) is Thr or Val, (1) to (3) and (30) to (4) (32) A peptide according to any one of (32). (34) Xaa at position 20 (X5) is Glu or Thr; (1) to (3) and (30) to (4) (33) A peptide according to any one of (33). (35) Xaa at position 21 (X6) is His or Thr; (1) to (3) and (30) to (33) (34) A peptide according to any one of (34). (36) Xaa at position 22 (X7) is Tyr. The peptide according to any one of (37) Xaa at position 24 (X8) is Asn or Ser; (1) to (3) and (30) to (31) (36) A peptide according to any one of (36) above. (38) Xaa at position 25 (X9) is Arg. (1) to (3) and (30) to (37) The peptide according to any one of (39) No. 26 Xaa(X 10 ) is Asp or Glu, (1) to (3) and (30) The peptide according to any one of (38) to (39). (40) No. 27 Xaa(X 11 ) is Tyr, (1) to (3) and (30) to (39) The peptide according to any one of (41) No. 28 Xaa(X 12 ) is Asp, (1) to (3) and (30) to (40) The peptide according to any one of (42) The amino acid sequence shown in either one of SEQ ID NOs: 30 and 32 (Figures 38 and 40) Any of (1) to (3) and (30) to (41) containing amino acid numbers 1 to 63 a peptide according to any one of (43) It has three disulfide bonds and contains a loop structure, an α-helix, and a β-sheet. A peptide according to any one of (1) to (42), having a characteristic three-dimensional structure. , (44) A compound encoding an amino acid sequence contained in a peptide according to any one of (1) to (43). a polynucleotide comprising a nucleotide sequence (45) A vector comprising the polynucleotide according to (44). (46) (44) or (45) comprising the polynucleotide according to (44) or the vector according to (1) A cell producing the peptide according to any one of (43) to (43). (47) A method for producing a SPINK2 mutant peptide, comprising the following steps (i) and (ii): (i) culturing the cell according to (46); (ii) recovering the SPINK2 mutant peptide from the culture; (48) The peptide according to any one of (1) to (43) is chemically synthesized or in vitro translated. a method for producing the peptide, comprising the step of preparing the peptide by (49) A SPINK2 mutant peptide obtained by the method according to (47) or (48). (50) One or more peptides according to any one of (1) to (43) and (49) a conjugate comprising any moiety of (51) (50) wherein one optional portion comprises a second peptide that is not a SPINK2 mutant. Conjugates of (52) The construct according to (51), wherein the second peptide is located on the amino terminal side of the SPINK2 mutant. Jugate, (53) The second peptide is located on the carboxyl-terminal side of the SPINK2 mutant, as described in (51). Conjugates of (54) The second peptide is an antibody or a fragment thereof and contains one or more Fc regions; The conjugate according to any one of (51) to (53), (55) The conjugate according to (54), wherein the Fc region is an Fc region of a human immunoglobulin or a fragment thereof. dugete, (56) The Fc region is IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, (54) or (55), which is an Fc region of IgD and / or IgE or a fragment thereof. Conjugates of (57) Any of (54) to (56), wherein the Fc region is a human IgG1 Fc region or a fragment thereof. a conjugate according to any one of (58) the Fc region of human IgG1 comprises the amino acid sequence set forth in SEQ ID NO: 87 (Figure 95); (57) The conjugate according to (57), (59) (54) The Fc region according to any one of (54) to (57), wherein the Fc region is a wild type or a mutant type. Indugete, (60) One to several aspartic acid and / or glutamic acid residues are added to the amino terminus. The conjugate according to any one of (51) to (59). (61) Any of (50) to (60) containing the amino acid sequence described in (i) or (ii) below: or a conjugate according to any one of the following: (i) SEQ ID NOs: 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60 and 96 (Figures 42, 44, 46, 48, 50, 52, 54, 56, 58 , 60, 62, 64, 66, 68 and 106); (ii) an amino acid sequence that is 90% or more identical to the amino acid sequence described in (i) above and has the amino acid sequence of KLK5; an amino acid sequence contained in a conjugate that inhibits protease activity; (62) The SPINK2 mutant and the second peptide are linked via a linker. (51) The conjugate according to any one of (61) to (61). (63) The linker is a third peptide that is not a SPINK2 mutant or a second peptide. 2) The conjugate according to (64) The method according to any one of (50) to (63), comprising the following steps (i) and (ii): How to make Conjugate: (i) comprising a nucleotide sequence encoding the amino acid sequence contained in the conjugate; and culturing cells containing a polynucleotide comprising the polynucleotide or a vector into which the polynucleotide has been inserted. the process of (ii) extracting a SPINK2 mutant peptide conjugate or the conjugate from the culture. recovering the peptide moiety contained in (65) (63) A SPINK2 mutant peptide conjugate according to any one of (50) to (63). The peptide moiety contained in the conjugate or the conjugate is synthesized by chemical synthesis or in vitro translation. a method for producing the conjugate, the method comprising the step of preparing the conjugate by (66) SPINK2 mutant peptide conjugate obtained by the method according to (64) or (65). gate, (67) An antibody that binds to the peptide according to any one of (1) to (43) and (49), or its binding fragments, (68) The peptide according to any one of (1) to (43) and (49), the peptide according to (44), a oligonucleotide, the vector according to (45), the cell according to (46), or any of (50) to (63). and (66) a conjugate according to any one of (66) and (66), and / or (67) an antibody according to (67). or a composition comprising a binding fragment thereof; (69) The peptide according to any one of (1) to (43) and (49), the peptide according to (44), (45) A vector according to (46), a cell according to (50), and / or a (63) to (66). A pharmaceutical composition comprising the conjugate according to any one of (63) to (66). , (70) The pharmaceutical composition according to (69) for treating or preventing a KLK5-related disease. (71) KLK5-related diseases include Netherton syndrome, atopic dermatitis, rosacea, and UV-induced skin damage (70) The pharmaceutical composition according to (70), wherein the disease is a spinal cord injury, psoriasis, asthma, spinal cord injury, cancer, or Barrett's esophagus. Composition, (72) Any of the drugs listed in (69) to (71) used in combination with other drugs The pharmaceutical composition described above, (73) The peptide according to any one of (1) to (43) and (49), the peptide according to (44), a oligonucleotide, the vector according to (45), the cell according to (46), or any of (50) to (63). and (66) a conjugate according to any one of (66) and (66), and / or (67) an antibody according to (67). or a binding fragment thereof; (74) (47), which comprises an affinity purification step using the antibody or binding fragment thereof according to (67). ), (48), (64) and (65), (75) A method for producing a KLK5-inhibiting SPINK2 mutant peptide, comprising the steps of: Determination method: (i) KLK5 protease and SPINK2 mutant peptides in the presence and absence of the test peptides. and incubating the substrate; (ii) KLK5 proteases in the presence and absence of test SPINK2 mutant peptides measuring the enzyme activity; and (iii) the KLK5 protease activity in the presence of the peptide is higher than that in the absence of the peptide; determining the peptide as positive when the activity is smaller than the KLK5 protease activity; (76) The method comprises the following steps (i) to (iii), and 8, 20, 22, 24, 26, 28, 30 and 32 (Figs. 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38 and 40) A peptide containing the amino acid sequence or a conjugate containing the peptide is used as a reference compound. The method for identifying KLK5 inhibitor compounds is used: (i) incubating KLK5 protease and a substrate in the presence and absence of a test compound ; (ii) measuring KLK5 protease activity in the presence and absence of the compound and (iii) the KLK5 protease activity in the presence of the compound is higher than that in the absence of the compound; determining the compound as positive if the activity is smaller than the K5 protease activity; (77) A method for identifying a KLK5 inhibitory compound, comprising the following steps (i) to (iii): (i) measuring the KLK5 protease inhibitory activity of a test compound; (ii) SEQ ID NOs: 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 2 8, 30 and 32 (Figs. 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38 and 40), or The KLK5 protease inhibitory activity of a reference compound, which is a conjugate containing the peptide, was measured. determining the amount of the compound; and (iii) The KLK5 protease inhibitory activity of the test compound is higher than that of the reference compound. determining the compound as positive if the enzyme inhibitory activity is equal to or stronger than that of the compound; (78) The method comprises the following steps (i) and (ii), and , 20, 22, 24, 26, 28, 30 and 32 (Figs. 14, 16, 18, 20, 22, 2 4, 26, 28, 30, 32, 34, 36, 38 and 40) A peptide containing the amino acid sequence or a conjugate containing the peptide is used as a reference compound. Methods for measuring KLK5 protease activity include: (i) incubating the KLK5 protease, substrate, and optionally other components; (ii) after step (i), measuring the amount of substrate and / or product; (79) KLK5-inhibiting SPINK2 mutant peptide or SPINK2 mutant peptide conjugate and immobilizing the peptide or conjugate in surface plasmon resonance analysis. The dissociation constant (K D ) is 1 x10 -9 the peptide or conjugate, which has a molecular weight of M or less; (80) a conjugate comprising the amino acid sequence shown in SEQ ID NO: 34 (Figure 42); (81) a conjugate comprising the amino acid sequence shown in SEQ ID NO: 36 (Figure 44); (82) a conjugate comprising the amino acid sequence shown in SEQ ID NO: 38 (Figure 46); (83) A conjugate comprising the amino acid sequence shown in SEQ ID NO: 40 (Figure 48), (84) a conjugate comprising the amino acid sequence shown in SEQ ID NO: 42 (Figure 50); (85) a conjugate comprising the amino acid sequence set forth in SEQ ID NO: 44 (Figure 52); (86) a conjugate comprising the amino acid sequence set forth in SEQ ID NO: 46 (Figure 54); (87) a conjugate comprising the amino acid sequence set forth in SEQ ID NO: 48 (Figure 56); (88) a conjugate comprising the amino acid sequence set forth in SEQ ID NO: 50 (Figure 58); (89) a conjugate comprising the amino acid sequence set forth in SEQ ID NO: 52 (Figure 60); (90) a conjugate comprising the amino acid sequence set forth in SEQ ID NO: 54 (Figure 62); (91) a conjugate comprising the amino acid sequence set forth in SEQ ID NO: 56 (Figure 64); (92) a conjugate comprising the amino acid sequence set forth in SEQ ID NO: 58 (Figure 66); (93) a conjugate comprising the amino acid sequence set forth in SEQ ID NO: 60 (Figure 68); and, (94) a conjugate comprising the amino acid sequence set forth in SEQ ID NO: 96 (Figure 106), etc. [Effects of the Invention]

[0011] The peptides provided by the present invention, conjugates containing the peptides, and Alternatively, a pharmaceutical composition containing the conjugate has KLK5 inhibitory activity and is a KLK5-related It is useful for treating or preventing diseases (described below). [Brief explanation of the drawings]

[0012] [Figure 1] A diagram comparing the amino acid sequence similarity of human KLK5, KLK7, and KLK14. [Figure 2] This figure shows the KLK5 inhibitory activity (50% inhibitory concentration: IC50) of KLK5 inhibitor peptides, using the degradation rate of the peptide substrate as an indicator. KLK5 at a final concentration of 10 nM and Boc-Val-Pro-Arg-AMC (R&D Systems; ES011) at a final concentration of 100 μM were used to evaluate KLK5 inhibitory activity. [Figure 3(A)] The cross-reactivity of each inhibitor peptide with each protease was evaluated using the degradation of the peptide substrate. For bovine trypsin inhibition, trypsin (Pierce; 20233) was used at a final concentration of 5 nM, and the substrate peptide Boc-Val-Pro-Arg-AMC (R&D Systems; ES011) was used at a final concentration of 100 μM. For human trypsin inhibition, trypsin (Sigma-Aldrich; T6424) was used at a final concentration of 1 nM, and the substrate peptide Boc-Val-Pro-Arg-AMC (R&D Systems; ES011) was used at a final concentration of 100 μM. For bovine α-chymotrypsin inhibition, chymotrypsin (Worthington Biochemical Corporation; LS001434) was used at a final concentration of 10 nM, and the substrate peptide Suc-Leu-Leu-Val-Tyr-MCA (Peptide Institute, Inc.; 3120-v) was used at a final concentration of 100 μM. [Figure 3(B)]The cross-reactivity of each inhibitor peptide with each protease was evaluated using the degradation of the peptide substrate as an indicator. For the evaluation of human chymotrypsin inhibitory activity, chymotrypsin (Sigma-Aldrich; C8946) was used at a final concentration of 10 nM, and the substrate peptide Suc-Leu-Leu-Val-Tyr-MCA (Peptide Institute, Inc.; 3120-v) was used at a final concentration of 10 μM. For the evaluation of human tryptase inhibitory activity, tryptase (Sigma-Aldrich; T7063) was used at a final concentration of 1 nM, and the substrate peptide Boc-Phe-Ser-Arg-MCA (Peptide Institute, Inc.; 3107-v) was used at a final concentration of 100 μM. To evaluate the inhibitory activity against human chymase, chymase (Sigma-Aldrich; C8118) at a final concentration of 100 nM and the substrate peptide Suc-Leu-Leu-Val-Tyr-MCA (Peptide Institute, Inc.; 3120-v) at a final concentration of 100 μM were used. [Figure 3(C)] The cross-reactivity of each inhibitory peptide with each protease was evaluated using the degradation of the peptide substrate as an indicator. Plasmin (Sigma-Aldrich; P1867) at a final concentration of 50 nM and the substrate peptide Boc-Val-Leu-Lys-MCA (Peptide Institute, Inc.; 3104-v) at a final concentration of 100 μM were used to evaluate human plasmin inhibitory activity. Thrombin (Sigma-Aldrich; T6884) at a final concentration of 1 nM and the substrate peptide Boc-Val-Pro-Arg-AMC (R&D Systems; ES011) at a final concentration of 100 μM were used to evaluate human thrombin inhibitory activity. To evaluate the inhibitory activity against human neutrophil elastase, we used neutrophil elastase (Enzo Life Sciences; BML-SE284) at a final concentration of 0.00001 U / μL and the substrate peptide Suc(OMe)-Ala-Ala-Pro-Val-MCA (Peptide Institute, Inc.; 3153-v) at a final concentration of 100 μM. [Figure 3(D)]The cross-reactivity of each inhibitor peptide with each protease was evaluated using the degradation of the peptide substrate. Human matriptase inhibitory activity was evaluated using 1 nM matriptase (R&D Systems; 3946-SE) at a final concentration and 100 μM substrate peptide Boc-Gln-Ala-Arg-AMC (R&D Systems; ES014) at a final concentration. Human protein C inhibitory activity was evaluated using 100 nM protein C (Sigma-Aldrich; P2200) at a final concentration and 100 μM substrate peptide Boc-Leu-Ser-Thr-Arg-MCA (Peptide Institute, Inc.; 3112-v) at a final concentration. Human tPA inhibitory activity was evaluated using 10 nM tPA (Sigma-Aldrich; T0831) at a final concentration and 100 μM substrate peptide Pyr-Gly-Arg-MCA (Peptide Institute, Inc.; 3145-v) at a final concentration. [Figure 3(E)] The cross-reactivity of each inhibitor peptide with each protease was evaluated using the degradation of the peptide substrate as an indicator. For the evaluation of human uPA inhibitory activity, uPA (Sigma-Aldrich; U0633) was used at a final concentration of 2 nM, and the substrate peptide Pyr-Gly-Arg-MCA (Peptide Institute, Inc.; 3145-v) was used at a final concentration of 100 μM. For the evaluation of human plasma kallikrein inhibitory activity, plasma kallikrein (R&D Systems; 2497-SE) was used at a final concentration of 0.125 μg / mL, and the substrate peptide Z-Phe-Arg-MCA (Peptide Institute, Inc.; 3095-v) was used at a final concentration of 100 μM. [Figure 3(F)]The cross-reactivity of each inhibitor peptide with each protease was evaluated using the degradation of the peptide substrate as an indicator. For human KLK1 inhibitory activity, hKLK1 (R&D Systems; 2337-SE) was used at a final concentration of 0.1 μg / mL, and the substrate peptide Pro-Phe-Arg-MCA (Peptide Institute, Inc.; 3096-v) was used at a final concentration of 100 μM. For human KLK2 inhibitory activity, hKLK2 (R&D Systems; 2337-SE) was used at a final concentration of 2 μg / mL, and the substrate peptide Pro-Phe-Arg-MCA (Peptide Institute, Inc.; 3096-v) was used at a final concentration of 100 μM. For human KLK4 inhibitory activity, hKLK4 (R&D Systems; 1719-SE) was used at a final concentration of 1 μg / mL, and the substrate peptide Boc-Val-Pro-Arg-AMC (R&D Systems; ES011) was used at a final concentration of 100 μM. [Figure 3(G)] This figure shows the cross-reactivity of each inhibitor peptide with each protease, using the degradation of the peptide substrate as an indicator. For the evaluation of human KLK7 inhibitory activity, a final concentration of 1 μg / mL hKLK7 and a final concentration of 20 μM substrate peptide Mca-Arg-Pro-Lys-Pro-Val-Glu-Nval-Trp-Arg-Lys(Dnp)-NH2 (R&D Systems; ES002) were used. For the evaluation of human KLK8 inhibitory activity, a final concentration of 5 nM hKLK8 (UniProt: O60259, prepared by the inventors) and a final concentration of 100 μM substrate peptide Boc-Val-Pro-Arg-AMC (R&D Systems; ES011) were used. To evaluate the inhibitory activity against human KLK12, hKLK12 (R&D Systems; 3095-SE) at a final concentration of 0.1 μg / mL and the substrate peptide Boc-Val-Pro-Arg-AMC (R&D Systems; ES011) at a final concentration of 100 μM were used. [Figure 3(H)]The cross-reactivity of each inhibitor peptide with each protease was evaluated using the degradation of the peptide substrate. For human KLK13 inhibitory activity, a final concentration of 0.5 μg / mL hKLK13 (R&D Systems; 2625-SE) and a final concentration of 100 μM substrate peptide Boc-Val-Pro-Arg-AMC (R&D Systems; ES011) were used. Fluorescence signals were excitation 380 nm and emission 460 nm. For human KLK14 inhibitory activity, a final concentration of 0.2 μg / mL hKLK14 and a final concentration of 100 μM substrate peptide Boc-Val-Pro-Arg-AMC (R&D Systems; ES011) were used. [Figure 4] 1 shows the KLK5 / KLK5 inhibitory peptide complex obtained by X-ray crystal structure analysis, in which the inhibitory peptide K51034 bound to a region containing the KLK5 active center. [Figure 5] This figure shows the KLK5 inhibitory activity (IC50) of KLK5 inhibitory peptide-Fc fusions, using the degradation rate of the peptide substrate as an indicator. KLK5 at a final concentration of 10 nM and Boc-Val-Pro-Arg-AMC (R&D Systems; ES011) at a final concentration of 100 μM were used to evaluate the KLK5 inhibitory activity. [Figure 6(A)] The cross-reactivity of each inhibitory peptide-Fc fusion to each protease was evaluated using the degradation of the peptide substrate as an indicator. The inhibitory activity of bovine trypsin, human trypsin, and bovine α-chymotrypsin was evaluated under the same conditions as in Figure 3(A). [Figure 6(B)] The cross-reactivity of each inhibitory peptide-Fc fusion to each protease was evaluated using the degradation of the peptide substrate. The inhibitory activity against human chymotrypsin, human tryptase, and human chymase was evaluated under the same conditions as in Figure 3(B). [Figure 6(C)]This figure shows the cross-reactivity of each inhibitory peptide-Fc fusion to each protease, assessed using the degradation of the peptide substrate as an indicator. The inhibitory activity against human plasmin, human thrombin, and human neutrophil elastase was evaluated under the same conditions as in Figure 3(C). [Figure 6(D)] This figure shows the cross-reactivity of each inhibitory peptide-Fc fusion to each protease, using the degradation of the peptide substrate as an indicator. The inhibitory activity against human matriptase, human protein C, and human tPA was evaluated under the same conditions as in Figure 3(D). [Figure 6(E)] The cross-reactivity of each inhibitory peptide-Fc fusion to each protease was evaluated using the degradation of the peptide substrate as an indicator. The inhibitory activity of human uPA and human plasma kallikrein was evaluated under the same conditions as in Figure 3(E). [Figure 6(F)] This figure shows the cross-reactivity of each inhibitory peptide-Fc fusion to each protease, assessed using the degradation of the peptide substrate as an indicator. The inhibitory activity of human KLK1, human KLK2, and human KLK4 was evaluated under the same conditions as in Figure 3(F). [Figure 6(G)] This figure shows the cross-reactivity of each inhibitory peptide-Fc fusion to each protease, assessed using the degradation of the peptide substrate as an indicator. The inhibitory activity of human KLK7, human KLK8, and human KLK12 was evaluated under the same conditions as in Figure 3(G). [Figure 6(H)] This figure shows the cross-reactivity of each inhibitory peptide-Fc fusion to each protease, assessed using the degradation of the peptide substrate as an indicator. The inhibitory activity of human KLK13 and human KLK14 was assessed under the same conditions as in Figure 3(H). [Figure 7]This figure shows that administration of a KLK5 inhibitor peptide-Fc fusion suppressed transepidermal water loss (TEWL) in Crusty2 model mice. When the SPINK5 mutation was heterozygous (+ / -), no skin inflammation developed, but when the SPINK5 mutation was homozygous (+ / +), severe skin inflammation developed and TEWL increased. Administration of the peptide-Fc fusion to Crusty2 mice homozygous for the SPINK5 mutation (+ / +) improved dermatitis and reduced TEWL. Regardless of whether the PBS-treated group or the D1-K50055-Fc-treated group was used, there were 9 Crusty2(+ / -) mice and 12 Crusty2(+ / +) mice. Error bars in the figure indicate standard error. [Figure 8] A diagram comparing the sequence identities of human SPINK2, D8 and D9 of human SPINK5, and human SPINK9. Human SPINK5 D8 and D9 and human SPINK9 are known to have human KLK5 inhibitory activity, but the amino acid sequence identity with human SPINK2, which has not been reported to have human KLK5 inhibitory activity, is low. [Figure 9] Amino acid sequence of human SPINK2 (SEQ ID NO: 1) [Figure 10] Amino acid sequence of human KLK5 (SEQ ID NO: 2) [Figure 11] Amino acid sequence of human KLK7 (SEQ ID NO: 3) [Figure 12] Amino acid sequence of human KLK14 (SEQ ID NO: 4) [Figure 13] Nucleotide sequence of KLK5 inhibitor peptide K50032 (SEQ ID NO: 5) [Figure 14] Amino acid sequence of KLK5 inhibitor peptide K50032 (SEQ ID NO: 6) [Figure 15] Nucleotide sequence of KLK5 inhibitor peptide K50055 (SEQ ID NO: 7) [Figure 16] Amino acid sequence of KLK5 inhibitor peptide K50055 (SEQ ID NO: 8) [Figure 17] Nucleotide sequence of KLK5 inhibitor peptide K51072 (SEQ ID NO: 9) [Figure 18]Amino acid sequence of KLK5 inhibitor peptide K51072 (SEQ ID NO: 10) [Figure 19] Nucleotide sequence of KLK5 inhibitor peptide K50016 (SEQ ID NO: 11) [Figure 20] Amino acid sequence of KLK5 inhibitor peptide K50016 (SEQ ID NO: 12) [Figure 21] Nucleotide sequence of KLK5 inhibitor peptide K51034 (SEQ ID NO: 13) [Figure 22] Amino acid sequence of KLK5 inhibitor peptide K51034 (SEQ ID NO: 14) [Figure 23] Nucleotide sequence of KLK5 inhibitor peptide K50062 (SEQ ID NO: 15) [Figure 24] Amino acid sequence of KLK5 inhibitor peptide K50062 (SEQ ID NO: 16) [Figure 25] Nucleotide sequence of KLK5 inhibitor peptide K51090 (SEQ ID NO: 17) [Figure 26] Amino acid sequence of KLK5 inhibitor peptide K51090 (SEQ ID NO: 18) [Figure 27] Nucleotide sequence of KLK5 inhibitor peptide K50098 (SEQ ID NO: 19) [Figure 28] Amino acid sequence of KLK5 inhibitor peptide K50098 (SEQ ID NO: 20) [Figure 29] Nucleotide sequence of KLK5 / KLK7 inhibitory peptide K51028 (SEQ ID NO: 21) [Figure 30] Amino acid sequence of KLK5 / KLK7 inhibitor peptide K51028 (SEQ ID NO: 22) [Figure 31] Nucleotide sequence of KLK5 / KLK7 inhibitory peptide K51005 (SEQ ID NO: 23) [Figure 32] Amino acid sequence of KLK5 / KLK7 inhibitor peptide K51005 (SEQ ID NO: 24) [Figure 33] Nucleotide sequence of KLK5 / KLK7 inhibitor peptide K50031 (SEQ ID NO: 25) [Figure 34] Amino acid sequence of KLK5 / KLK7 inhibitor peptide K50031 (SEQ ID NO: 26) [Figure 35] Nucleotide sequence of KLK5 / KLK7 inhibitory peptide K51057 (SEQ ID NO: 27) [Figure 36] Amino acid sequence of KLK5 / KLK7 inhibitor peptide K51057 (SEQ ID NO: 28) [Figure 37] Nucleotide sequence of KLK5 / KLK14 inhibitor peptide K51069 (SEQ ID NO: 29) [Figure 38] Amino acid sequence of KLK5 / KLK14 inhibitor peptide K51069 (SEQ ID NO: 30) [Figure 39] Nucleotide sequence of KLK5 / KLK14 inhibitor peptide K50015 (SEQ ID NO: 31) [Figure 40] Amino acid sequence of KLK5 / KLK14 inhibitor peptide K50015 (SEQ ID NO: 32) [Figure 41] Nucleotide sequence of KLK5 inhibitory peptide Fc fusion D3-K50032dN-Fc (SEQ ID NO: 33) [Figure 42] Amino acid sequence of KLK5 inhibitory peptide Fc fusion D3-K50032dN-Fc (SEQ ID NO: 34) [Figure 43] Nucleotide sequence of KLK5 inhibitory peptide Fc fusion D3-K50055-Fc (SEQ ID NO: 35) [Figure 44] Amino acid sequence of KLK5 inhibitory peptide Fc fusion D3-K50055-Fc (SEQ ID NO: 36) [Figure 45] Nucleotide sequence of KLK5 inhibitory peptide Fc fusion D3-K51072-Fc (SEQ ID NO: 37) [Figure 46] Amino acid sequence of KLK5 inhibitory peptide Fc fusion protein D3-K51072-Fc (SEQ ID NO: 38) [Figure 47] Nucleotide sequence of KLK5 inhibitory peptide Fc fusion D3-K50016dN-Fc (SEQ ID NO: 39) [Figure 48] Amino acid sequence of KLK5 inhibitory peptide Fc fusion D3-K50016dN-Fc (SEQ ID NO: 40) [Figure 49] Nucleotide sequence of KLK5 inhibitory peptide Fc fusion D3-K51034-Fc (SEQ ID NO: 41) [Figure 50] Amino acid sequence of KLK5 inhibitory peptide Fc fusion D3-K51034-Fc (SEQ ID NO: 42) [Figure 51] Nucleotide sequence of KLK5 inhibitory peptide Fc fusion D3-K50062-Fc (SEQ ID NO: 43) [Figure 52] Amino acid sequence of KLK5 inhibitory peptide Fc fusion D3-K50062-Fc (SEQ ID NO: 44) [Figure 53] Nucleotide sequence of KLK5 inhibitory peptide Fc fusion D3-K51090-Fc (SEQ ID NO: 45) [Figure 54] Amino acid sequence of KLK5 inhibitory peptide Fc fusion D3-K51090-Fc (SEQ ID NO: 46) [Figure 55] Nucleotide sequence of KLK5 inhibitory peptide Fc fusion D3-K50098dN-Fc (SEQ ID NO: 47) [Figure 56] Amino acid sequence of KLK5 inhibitory peptide Fc fusion D3-K50098dN-Fc (SEQ ID NO: 48) [Figure 57] Nucleotide sequence of KLK5 / KLK7 inhibitory peptide Fc fusion D3-K51028-Fc (SEQ ID NO: 49) [Figure 58] Amino acid sequence of KLK5 / KLK7 inhibitory peptide Fc fusion D3-K51028-Fc (SEQ ID NO: 50) [Figure 59] Nucleotide sequence of KLK5 / KLK7 inhibitory peptide Fc fusion D3-K51005-Fc (SEQ ID NO: 51) [Figure 60] Amino acid sequence of KLK5 / KLK7 inhibitory peptide Fc fusion D3-K51005-Fc (SEQ ID NO: 52) [Figure 61] Nucleotide sequence of KLK5 / KLK7 inhibitory peptide Fc fusion D3-K50031-Fc (SEQ ID NO: 53) [Figure 62] Amino acid sequence of KLK5 / KLK7 inhibitory peptide Fc fusion D3-K50031-Fc (SEQ ID NO: 54) [Figure 63]Nucleotide sequence of KLK5 / KLK7 inhibitory peptide Fc fusion D3-K51057-Fc (SEQ ID NO: 55) [Figure 64] Amino acid sequence of KLK5 / KLK7 inhibitory peptide Fc fusion D3-K51057-Fc (SEQ ID NO: 56) [Figure 65] Nucleotide sequence of KLK5 / KLK14 inhibitory peptide Fc fusion D3-K51069dN-Fc (SEQ ID NO: 57) [Figure 66] Amino acid sequence of KLK5 / KLK14 inhibitory peptide Fc fusion D3-K51069dN-Fc (SEQ ID NO: 58) [Figure 67] Nucleotide sequence of KLK5 / KLK14 inhibitory peptide Fc fusion D3-K50015-Fc (SEQ ID NO: 59) [Figure 68] Amino acid sequence of KLK5 / KLK14 inhibitory peptide Fc fusion D3-K50015-Fc (SEQ ID NO: 60) [Figure 69] General formula of SPINK2 mutant peptide (SEQ ID NO: 61) [Figure 70] Nucleotide sequence of primer 1 (SEQ ID NO: 62) [Figure 71] Nucleotide sequence of primer 2 (SEQ ID NO: 63) [Figure 72] Nucleotide sequence of primer 3 (SEQ ID NO: 64) [Figure 73] Nucleotide sequence of primer 4 (SEQ ID NO: 65) [Figure 74] Nucleotide sequence of primer 5 (SEQ ID NO: 66) [Figure 75] Nucleotide sequence of primer 6 (SEQ ID NO: 67) [Figure 76] Nucleotide sequence of primer 7 (SEQ ID NO: 68) [Figure 77] Nucleotide sequence of primer 8 (SEQ ID NO: 69) [Figure 78] Nucleotide sequence of primer 9 (SEQ ID NO: 70) [Figure 79] Nucleotide sequence of primer 10 (SEQ ID NO:71) [Figure 80]Nucleotide sequence of primer 11 (SEQ ID NO: 72) [Figure 81] Nucleotide sequence of primer 12 (SEQ ID NO: 73) [Figure 82] Nucleotide sequence of primer 13 (SEQ ID NO: 74) [Figure 83] Nucleotide sequence of primer 14 (SEQ ID NO: 75) [Figure 84] Nucleotide sequence of primer 15 (SEQ ID NO: 76) [Figure 85] KLK7 substrate peptide (amino acid sequence SEQ ID NO: 77) [Figure 86] Bovine α-chymotrypsin substrate peptide (amino acid sequence SEQ ID NO: 78) [Figure 87] Neutrophil elastase substrate peptide (amino acid sequence SEQ ID NO: 79) [Figure 88] Human protein C substrate peptide (amino acid sequence SEQ ID NO: 80) [Figure 89] Nucleotide sequence of primer 16 (SEQ ID NO:81) [Figure 90] Nucleotide sequence of primer 17 (SEQ ID NO:82) [Figure 91] Nucleotide sequence of primer 18 (SEQ ID NO: 83) [Figure 92] Nucleotide sequence of primer 19 (SEQ ID NO: 84) [Figure 93] Nucleotide sequence of primer 20 (SEQ ID NO: 85) [Figure 94] Nucleotide sequence of primer 21 (SEQ ID NO: 86) [Figure 95] Amino acid sequence of human IgG1 Fc (SEQ ID NO: 87) [Figure 96] Amino acid sequence of D8 of human SPINK5 (SEQ ID NO: 88) [Figure 97] Amino acid sequence of D9 of human SPINK5 (SEQ ID NO: 89) [Figure 98] Amino acid sequence of human SPINK9 (SEQ ID NO: 90) [Figure 99] Amino acid sequence of mouse KLK5 (SEQ ID NO: 91) [Figure 100] Amino acid sequence of mouse KLK7 (SEQ ID NO: 92) [Figure 101] Amino acid sequence of mouse KLK14 (SEQ ID NO: 93) [Figure 102] The KLK5 inhibitory activity (n = 3, Mean ± SD) of KLK5 inhibitory peptide-Fc fusions was evaluated using the rate of decomposition of the peptide substrate as an index to calculate the inhibition constant Ki. KLK5 inhibitory activity was evaluated using KLK5 at a final concentration of 10 nM and Boc-Val-Pro-Arg-AMC (R&D Systems; ES011) at a final concentration of 100 μM. [Figure 103(A)] The KLK5 inhibitory activity of KLK5 inhibitor peptide-Fc fusions was evaluated using human desmoglein-1 degradation as an indicator. KLK5 (final concentration 1 μM) and Recombinant Human Desmoglein-1 Fc Chimera Protein (R&D Systems; 944-DM-100) (final concentration 1 μM) were used to evaluate KLK5 inhibitory activity. Western blot analysis was performed using Desmoglein 1 Antibody (aa471-499) (LSBio; LS-C167542) and Anti-Rabbit IgG, HRP-Linked F(ab')2 Fragment Donkey (GE Healthcare; NA9340V). [Figure 103(B)] This figure shows the KLK5 inhibitory activity of KLK5 inhibitor peptide-Fc fusions evaluated using the degradation of human desmocollin-1 as an indicator. KLK5 at a final concentration of 0.2 μM and recombinant human desmocollin-1 protein with a C-terminal His tag (R&D Systems; 4955-DC-050) at a final concentration of 2 μM were used to evaluate KLK5 inhibitory activity. Analysis was performed by Western blotting using Penta His HRP Conjugate (QIAGEN; 34460). [Figure 104] Nucleotide sequence of primer 22 (SEQ ID NO: 94) [Figure 105]Nucleotide sequence of KLK5 inhibitory peptide Fc fusion D1-K50055-Fc (SEQ ID NO: 95) [Figure 106] Amino acid sequence of KLK5 inhibitory peptide Fc fusion D1-K50055-Fc (SEQ ID NO: 96) [Figure 107(A)] This figure shows the KLK5 inhibitory activity (IC50) of the KLK5 inhibitory peptide-Fc fusion product D1-K50055-Fc, using the degradation rate of the peptide substrate as an indicator. KLK5 at a final concentration of 10 nM and Boc-Val-Pro-Arg-AMC (R&D Systems; ES011) at a final concentration of 100 μM were used to evaluate the KLK5 inhibitory activity. [Fig. 107(B)] This figure shows the cross-reactivity of the KLK5 inhibitory peptide Fc fusion product D1-K50055-Fc with KLK7, evaluated using the degradation rate of the peptide substrate as an indicator. The same conditions as in Figure 3(G) were used. [Fig. 107(C)] This figure shows the cross-reactivity of the KLK5 inhibitory peptide Fc fusion product D1-K50055-Fc with KLK14, evaluated using the degradation rate of the peptide substrate as an indicator, under the same conditions as in Figure 3(H). [Figure 108] Sequence listing for Patent Application No. 2018-209729, filed November 7, 2018. DETAILED DESCRIPTION OF THE INVENTION

[0013] 1.Definition In the present invention, a "gene" refers to a nucleotide sequence that encodes an amino acid sequence contained in a protein. It means a nucleic acid molecule containing a nucleotide sequence or its complementary strand, and may be single-stranded, double-stranded, triple-stranded or more. It becomes an assembly of DNA and RNA chains, and ribonucleotides and deoxyribonucleotides on a single chain. Nucleic acid molecules containing a mixture of nucleic acids and double-stranded or triple- or more-stranded nucleic acid molecules containing such strands are also considered "genes." It is included in the meaning of "denshi".

[0014] In the present invention, the terms "gene," "polynucleotide," and "nucleic acid molecule" are synonymous. Their building blocks are ribonucleotides, deoxyribonucleotides, and nucleotides. There is no limitation on the number of nucleosides, etc., and examples thereof include DNA, RNA, mRNA, This includes cDNA, cRNA, probes, oligonucleotides, primers, etc. "Nucleic acid molecule" may be abbreviated to "nucleic acid."

[0015] In the present invention, the terms "polypeptide," "peptide," and "protein" have the same meaning.

[0016] In the present invention, a molecule that recognizes or binds to a target molecule X (hereinafter referred to as the recognition molecule) (The binding activity is collectively referred to as "X-binding activity") Peptides are called "X-binding peptides." Furthermore, it is possible to recognize or bind to target molecule X and to Inhibiting or suppressing one or more activities or functions of a compound (hereinafter, the term "inhibition or suppression" is used to refer to the inhibition or suppression of such activities or functions). The peptides used for production are collectively referred to as "X inhibitory activity") and are referred to as "X inhibitor peptides." can.

[0017] In the present invention, "SPINK2" refers to a serine protease inhibitor Itor Kazal-type 2, which has three disulfide bonds It is a 7 kDa protein consisting of an al-like domain. The preferred SPINK2 is of human origin. In the present invention, unless otherwise stated, human SPINK2 (SEQ ID NO: 1: Figure 9) ) is simply called "SPINK2".

[0018] In the present invention, "KLK5" consists of an N-terminal propeptide and a protease active domain. It has trypsin-like and chymotrypsin-like protease activity with three N-glycosylation sites. A preferred KLK5 is of human origin. Unless otherwise specified, human KLK5 (SEQ ID NO: 2: Figure 10) may be referred to simply as "KLK5". be.

[0019] In the present invention, "KLK7" is a peptide consisting of an N-terminal propeptide and a tripeptide having protease activity. It is a protein consisting of a scin-like domain and N-glycosylated. The preferred KLK7 is derived from human In the present invention, unless otherwise stated, human KLK7 (SEQ ID NO: 3) is used. 11) is sometimes simply referred to as "KLK7".

[0020] In the present invention, "KLK14" is also called neuropsin, and is composed of an N-terminal propeptide and a pro- It is a protein with a trypsin-like domain that has protease activity and is N-glycosylated. The preferred KLK14 is of human origin. In the present invention, unless otherwise stated, Human KLK14 (SEQ ID NO: 4: Figure 12) may be simply referred to as "KLK14."

[0021] In the present invention, "precursor KLK5" means pro-KLK5, and includes the propeptide and It is composed of a domain with protease activity. It refers to KLK5, which is composed of a domain with protease activity. LK5 is of human origin.

[0022] In the present invention, "precursor KLK7" means pro-KLK7, and includes the propeptide and It is composed of a domain with protease activity. KLK7 is composed of a domain with protease activity. LK7 is of human origin.

[0023] In the present invention, "precursor KLK14" means pro-KLK14, and and a domain with protease activity. It means ive KLK14 and is composed of a domain having protease activity. The active form of KLK14 is derived from humans.

[0024] In the present invention, "KLK5 inhibitory peptide", "KLK5 / KLK7 inhibitory peptide" or The "KLK5 / KLK14 inhibitor peptide" refers to a peptide that inhibits KLK5, KLK5 and KLK7, or A peptide that inhibits or suppresses one or more activities or functions of KLK5 and KLK14. Each of them means petite.

[0025] "KLK5 inhibitor peptide," "KLK5 / 7 inhibitor peptide," and "KLK5 / KLK1 The scope of "inhibitory peptide" includes fragments of the peptide, other moieties of the peptide, Among the conjugates formed by adding or binding to a peptide or a fragment thereof, those that inhibit KLK5 (binding ) activity, KLK5 / KLK7 inhibitory (binding) activity, or KLK5 / KLK14 inhibitory (binding) activity ) activity. KLK5 inhibitory (binding) activity and KLK7 inhibitory (binding) activity, or KLK5 inhibitory (binding) activity Fragments, adducts and modifications of the peptide that maintain the activity and KLK14 inhibitory (binding) activity (Conjugate) also includes "KLK5 inhibitor peptide" and "KLK5 / KLK7 inhibitor peptide" or "KLK5 / KLK14 inhibitory peptides," respectively.

[0026] In the present invention, the "site" to which the peptide binds, i.e., the "site" recognized by the peptide, The term "peptide" refers to a continuous or discontinuous partial amino acid sequence on a target molecule that a peptide binds to or recognizes. In the present invention, such a site is referred to as an end site on the target molecule. It can be called a pitope or a binding site.

[0027] In the present invention, the term "cells" includes various cells derived from individual animals, subcultured cells, primary cultured cells, and the like. Also included are cultured cells, cell lines, recombinant cells, yeast, microorganisms, and the like.

[0028] In the present invention, a "SPINK2 mutant" refers to a mutant having the same amino acid sequence as wild-type SPINK2. In the sequence, one or more amino acids are substituted with amino acids different from the wild type, One or more wild-type amino acids are deleted, and one or more amino acids that are not present in the wild-type are added. The term "peptide" refers to a peptide containing an amino acid sequence in which a amino acid has been inserted (hereinafter collectively referred to as "mutation"). Among the "SPINK2 mutants," KLK5 inhibitory activity, KLK5 inhibitory activity and KLK7 Inhibitory activity (KLK5 / KLK7 inhibitory activity), or KLK5 inhibitory activity and KLK14 inhibitory activity Those having KLK5 / KLK14 inhibitory activity are KLK5 inhibitor peptides, KLK These are included in the KLK5 / KLK7 inhibitory peptides and KLK5 / KLK14 inhibitory peptides. In the present invention, "insertion" can also be included in the scope of "addition."

[0029] In the present invention, the term "several" in "one to several" refers to three to ten.

[0030] In the present invention, "hybridizing under stringent conditions" means hybridizing under stringent conditions, such as 5xSS Hybridization was carried out at 65°C in a solution containing 2×SSC-0.1 0.5×SSC-0.1%SDS at 65°C for 20 minutes. in aqueous solution at 65°C for 20 minutes, and in aqueous solution containing 0.2xSSC-0.1%SDS Hybridization was performed under conditions equivalent to those described above, with washing in a 500 ml buffer at 65°C for 20 minutes. SSC means 150mM NaCl-15mM sodium citrate. It is an aqueous solution, and n×SSC means n-fold concentrated SSC.

[0031] In the present invention, the terms "specific" and "specificity" are interchangeable with "selective" and "selectivity," respectively. For example, a KLK5-specific inhibitory peptide is a KLK5-selective inhibitor. The inhibitory peptide specific to KLK5 and KLK7 was They are synonymous with K7-selective inhibitory peptides.

[0032] 2. Peptides 2-1. Amino acids "Amino acid" refers to an organic compound containing an amino group and a carboxyl group, preferably a protein. The term "amino acids" refers to α-amino acids contained as building blocks in proteins, more preferably in natural proteins. In the present invention, more preferred amino acids are Ala, Arg, Asn, Asp, Cys, G ln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, S er, Thr, Trp, Tyr, and Val, and unless otherwise specified, "amino acids" are These 20 amino acids are called "natural amino acids." The KLK5 inhibitory peptide, KLK5 / KLK7 inhibitory peptide, or KLK5 / KLK14 inhibitory peptides preferably contain natural amino acids.

[0033] In the present invention, "amino acid residue" may be abbreviated as "amino acid".

[0034] In the present invention, the amino acids may be L-amino acids, D-amino acids, or mixtures thereof ( DL-amino acids), but unless otherwise specified, L-amino acids are meant.

[0035] Natural amino acids can be divided into groups based on their common side chain properties, for example: It is possible. (1) Hydrophobic amino acid group: Met, Ala, Val, Leu, Ile (2) Neutral hydrophilic amino acid group: Cys, Ser, Thr, Asn, Gln (3) Acidic amino acid group: Asp, Glu (4) Basic amino acid group: His, Lys, Arg (5) Group of amino acids that influence the direction of the main chain: Gly, Pro (6) Aromatic amino acid group: Trp, Tyr, Phe However, the classification of natural amino acids is not limited to these.

[0036] In the present invention, natural amino acids may be subjected to conservative amino acid substitutions.

[0037] "Conservative amino acid substitutions" "Titation" means substitution with a functionally equivalent or similar amino acid. Conservative amino acid substitutions in a peptide result in a silent change in the amino acid sequence of the peptide. For example, one or more amino acids of a similar polarity act functionally equivalently; In general, substitutions within a group result in a static change in the amino acid sequence of the resulting peptide. However, it is obvious to those skilled in the art that As such, the role of a particular amino acid residue depends on the three-dimensional structure of the molecule that contains that amino acid. For example, cysteine ​​residues can be determined in their reduced form (thiol ) form, which is less polar than the oxidized (disulfide) form. The long aliphatic portion of the arginine side chain may constitute an important structural and functional feature. In addition, side chains containing aromatic rings (tryptophan, tyrosine, phenylalanine) are ionic. In such cases, this may contribute to cation-aromatic interactions or cation-pi interactions. The amino acids having these side chains can be replaced by amino acids belonging to the acidic or non-polar groups. They may be structurally and functionally conservative. Proline, glycine, cysteine ​​(disulfide Residues such as hydroxyl groups (e.g., hydroxyl groups) can have a direct effect on the main-chain conformation and are often cannot be replaced without structural distortion.

[0038] Conservative amino acid substitutions are specific substitutions based on side chain similarity (Leni), as shown below. L. Lehning, Biochemistry, 2nd revised edition, 1975, pp. 73-75: er, Biochemistry, 2 nd edition, pp73-75, Worth Publisher, New York (1975)) and typical substitutions Includes. (1) Nonpolar amino acid group: alanine (hereinafter referred to as "Ala" or simply "A"); Valine (hereinafter referred to as "Val" or simply "V"), leucine (hereinafter referred to as "Leu" or simply isoleucine (hereinafter referred to as "Ile" or simply "I"), proline (hereinafter referred to as "Pro" or simply "P"), phenylalanine (hereinafter referred to as "Phe" or simply " F), tryptophan (hereinafter referred to as "Trp" or simply "W"), methionine (Hereafter referred to as "Met" or simply "M") (2) Uncharged polar amino acid group: glycine (hereinafter referred to as "Gly" or simply "G") ), serine (hereinafter referred to as "Ser" or simply "S"), threonine (hereinafter referred to as "Thr" or simply "T"), cysteine ​​(hereinafter referred to as "Cys" or simply "C"), tyrosine (hereinafter referred to as "Cys" or simply "C"), Tyr (hereinafter referred to as "Tyr" or simply "Y"), asparagine (hereinafter referred to as "Asn" or (hereinafter referred to as "N"), glutamine (hereinafter referred to as "Gln" or simply "Q") (3) Acidic amino acid group: aspartic acid (hereinafter referred to as "Asp" or simply "D") ), glutamic acid (hereinafter referred to as "Glu" or simply "E") (4) Basic amino acid group: lysine (hereinafter referred to as “Lys” or simply “K”), arginine (hereinafter referred to as "Arg" or simply "R"), histidine (hereinafter referred to as "His" or is simply written as "H") In the present invention, the amino acid may be an amino acid other than a natural amino acid. For example, Selenocysteine ​​and N-formylmethionine found in natural peptides and proteins , pyrrolidine, pyroglutamic acid, cystine, hydroxyproline, hydroxylysine, Thyroxine, O-phosphoserine, desmosine, β-alanine, sarcosine, ornithine, Creatine, gamma-aminobutyric acid, opine, theanine, tricholominic acid, kainic acid, domo Examples of the amino acids include norleucine, Ac-amino acids, Boc- N-terminal protected amino acids such as amino acids, Fmoc-amino acids, Trt-amino acids, and Z-amino acids Acids, amino acid t-butyl esters, benzyl esters, cyclohexyl esters, fluoro C-terminal protected amino acids such as lenyl esters, diamines, ω amino acids, β amino acids, γ amino acids Other naturally occurring compounds include amino acids, Tc derivatives of amino acids, and aminophosphonic acids. Examples of amino acids that are not naturally occurring include, but are not limited to, the above 20 "natural amino acids." For convenience, other amino acids are collectively referred to as "unnatural amino acids" in the present invention.

[0039] 2-2. KLK5 inhibitory peptide, KLK5 / KLK7 inhibitory peptide, and KLK5 / K LK14 inhibitor peptide The peptide of the present invention has KLK5 inhibitory activity, KLK5 / KLK7 inhibitory activity, or KLK 5 / KLK14 inhibitory activity.

[0040] The KLK5 inhibitory peptide, KLK5 / KLK7 inhibitory peptide, and KLK5 / KLK7 inhibitor of the present invention The targets of the KLK14 inhibitor peptide, KLK5, KLK7 and KLK14, are preferably It is derived from a vertebrate, more preferably a mammal, even more preferably a primate, and most preferably a human. LK5, KLK7, and KLK14 are purified from tissues and cells or recombinantly expressed. Proteins can be prepared by methods known to those skilled in the art, such as in vitro translation and peptide synthesis. KLK5, KLK7, and KLK14 have a signal sequence The Fc region of immunoglobulin, a tag, a label, etc. may be linked. The KLK5 / KLK7 inhibitory activity and the KLK5 / KLK14 inhibitory activity were The protease activity of KLK5 and KLK7, as well as KLK5 and KLK14, was used as an index. For example, KLK5, KLK5 and KLK7, or KLK5 and KLK7 can be evaluated. and KLK14, or functional fragments thereof, substrates and the KLK5 inhibitor peptide of the present invention, KLK 5 / KLK7 inhibitory peptide or KLK5 / KLK14 inhibitory peptide, or a candidate thereof When coexisting, compared to the presence of a control or the absence of the inhibitor or candidate thereof, The protease activity of KLK5, KLK5 and KLK7, or KLK5 and KLK14 70% or less, 50% or less, 30% or less, 20% or less, 10% or less, 5% or less, 1% or less If the ratio is 0%, the results are consistent with KLK5 inhibition, KLK5 / KLK7 inhibition, or KLK5 / KLK14 inhibition. The inhibitory activity was 30% or more, 50% or more, 70% or more, and 80% or more, respectively. or more, 90% or more, 95% or more, 99% or more, or 100%. The LK5 / KLK7 inhibitory activity and the KLK5 / KLK14 inhibitory activity depend on the reaction conditions and the substrate. The reaction conditions may vary depending on the type, concentration, etc. The reaction conditions are exemplified by those described in the Examples. A certain concentration of KLK5, KLK5 and KLK7, or The substrate peptide or substrate protein was added to KLK5 and KLK14, and the mixture was allowed to react for a certain period of time. Then, the fluorescence of the substrate peptide is detected, or the substrate protein is analyzed by SDS-PAGE or Western Enzyme activity can be evaluated by detecting it using the n blot method, liquid chromatography, etc. Examples of buffer solutions include phosphate buffer saline (phosphate buffer). te buffer saline (hereinafter referred to as "PBS"), Tris buffer (50 1 mM Tris, pH 7 to 8.5, for example, pH 7.5), etc. can be used, (0 to 200 mM, e.g., 200 mM), CaCl2 (0 to 10 mM, e.g., 2 mM ), ZnCl 2、 Salts such as, but not limited to, Brij-35 may also be added. stomach.

[0041] KLK5 inhibitory activity, KLK5 / KLK7 inhibitory activity, and KLK5 / KLK14 inhibitory activity The inhibitory constant K i The substrate peptide is added to a fixed concentration of the enzyme, and the After the reaction time, the fluorescence of the substrate peptide is detected to measure the protease activity. The protease activity at a given concentration was calculated using the Michaelis-Menten equation (Mich aelis L, et al. (2011) Biochemistry. 50:39 No. 8264-8269, the maximum reaction rate V max and Michaelis constant K m Calculate Furthermore, the protease activity when an inhibitor is added to a fixed concentration of the enzyme was determined. Morrison's formula (Morrison JF. (1969) Biochim Biop 185, No. 2, pp. 269-286) and the inhibition constant K i Calculate The software used for the calculation was GraphPad Prism (GraphPad Software Inc.) can be mentioned as an example.

[0042] Proteases of KLK5, KLK7 and KLK5, or KLK5 and KLK14 The enzyme substrate is not particularly limited and may be an endogenous substrate, an exogenous substrate, a synthetic substrate, or the like. Human endogenous substrates of KLK5 include low molecular weight kininogen, kallistatin, and collagen. Examples include desmoglein, desmocollin, and cathelicidin. The endogenous substrates of KLK7 in humans include Pro-KLK3 and Fibroblast Growth Factor-1 (FGF1). Examples include pronectin and collagen. Examples of such substances include tPA, fibronectin, and collagen. Gelatin obtained by thermally denaturing gelatin can also be used as a substrate. Although not particularly limited, examples include PFR-AMC and Boc-VPR-AMC. The KLK5 inhibitory activity (IC 50 or K i ), KLK5 KLK5 inhibitory activity of KLK7 inhibitor peptides and KLK5 / KLK14 inhibitor peptides The KLK5 inhibitory activity and KLK14 inhibitory activity of the amide are each 1 μM or less, preferably 300 μM or less. nM or less, more preferably 100 nM or less, even more preferably 30 nM or less, and even more preferably The KLK7 inhibitory activity of the KLK5 / KLK7 inhibitory peptide is preferably Preferably, 1000 nM or less, more preferably 300 nM or less, even more preferably 100 nM or less, Even more preferably, it is 30 nM or less. The KLK5 / KLK14 inhibitor peptide has KLK5 inhibitory activity and KLK7 inhibitory activity. K14 inhibitory activity (both IC 50 or K i ) can be classified according to their relative size. Preferably, (i) the KLK5 inhibitory activity is 0.5 times that of the KLK7 inhibitory activity or the KLK14 inhibitory activity. (ii) KLK5 inhibitory activity is less than 0.5 times that of KLK7 inhibitory activity or KLK14 inhibitory activity. (iii) KLK5 inhibitory activity is greater than or equal to 5-fold but less than 2-fold, and (iv) KLK5 inhibitory activity is greater than or equal to KLK7 inhibitory activity or KLK5 / KL and K7 inhibitory activity more than twice that of other compounds. The peptide may be selected from

[0043] In addition, the KLK5 inhibitory peptide, KLK5 / KLK7 inhibitory peptide, or KL The K5 / KLK14 inhibitor peptides inhibit KLK5, KLK5 and KLK7, or K It does not inhibit or suppress the activity of proteases other than LK5 and KLK14, or In other words, it is preferable that the degree of inhibition or suppression of KLK5 inhibitory peptide, KLK5 / KLK7 inhibitory peptide, or KLK5 / KLK1 4. The protease inhibitory activity of the inhibitor peptide is preferably KLK5 specific, KLK5 / KL The preferred peptides of the present invention are K7 specific or KLK5 / KLK14 specific. LK1, KLK2, KLK3, KLK4, KLK6, KLK8, KLK9 to KLK13 , KLK15, chymotrypsin, tryptase, chymase, plasmin, thrombin, plasminogen activator, matriptase, protein C, tissue plasminogen activator (tPA), urokinase plasma Inhibits or inhibits the activity of proteases such as uPA and plasma kallikrein. They do not inhibit or the degree of inhibition or suppression is relatively weak. Preferred peptides of the present invention are those that inhibit or suppress the activity of other proteases. It does not show any side effects and is suitable for use as a therapeutic or preventive drug for KLK5-related diseases (described below). Furthermore, the preferred KLK5-specific inhibitory peptide of the present invention is KLK7 and KLK14. does not inhibit or suppress the protease activity of The preferred KLK5 / KLK7-specific inhibitory peptides of the present invention have a relatively weak inhibitory effect on KL It does not inhibit or suppress the protease activity of K14, or The preferred KLK5 / KLK14 inhibitor peptides of the present invention have a relatively weak inhibitory effect on K It does not inhibit or suppress the protease activity of LK7, or inhibits or suppresses it. The degree of inhibition is relatively weak.

[0044] Low specificity for KLK5, KLK5 and KLK7, or KLK5 and KLK14 In addition to KLK5, KLK5 and KLK7, or KLK5 and KLK14, other K Inhibitors that also inhibit the protease activity of LK, i.e., non-selective inhibitors, are It can cause side effects when administered to animals (Coussens, LM et al., Science, 2014). Volume 95 (No. 5564), pp. 2387-2392 (2002): Bissett, D et al. J. Clin. Oncol., Vol. 23 (No. 4), pp. 842-849 (2005)). one On the other hand, it has high specificity for KLK5, KLK5 / KLK7, or KLK5 / KLK14. Inhibitors, i.e., KLK5-specific inhibitory peptides, KLK5 / KLK7-specific inhibitory peptides Alternatively, KLK5 / KLK14-specific inhibitory peptides may avoid the aforementioned side effects. Therefore, they can be suitably used for the treatment or prevention of KLK5-related diseases (described below). can.

[0045] The KLK5 inhibitory peptide, KLK5 / KLK5 inhibitory peptide, or KLK5 / The KLK14 inhibitor peptide is a peptide that inhibits KLK5, KLK5 and / or KLK7, or KLK The binding of the protease substrate to KLK14 and / or KLK5 may be competitive. stomach.

[0046] As described above, KLK5, KLK7 and KLK14, which are targets of the peptides of the present invention, are expressed in the spinal cord. derived from a vertebrate, preferably a mammal, more preferably a primate, even more preferably a human, Non-human animals, for example, rodents such as rats and mice, cynomolgus monkeys, and common marmosets KLK5, KLK5 and KLK6 derived from non-human animals may also be derived from primates such as rhesus monkeys. Peptides having inhibitory activity against KLK7, or KLK5 and KLK14, are useful for the treatment of such non- It can be used for the diagnosis, testing, treatment, or prevention of diseases related to KLK5 in humans and animals. In addition, such peptides may be used in combination with human KLK5, KLK5 and KLK7, or K When LK5 and KLK14 are also inhibited, it can be used as a therapeutic or preventive agent for KLK5-related diseases (described below). In the non-clinical research and development of the peptide as an animal pathological model, such non-human animals are used. Pharmacological and pharmacokinetic studies, safety and toxicity studies using healthy animals, etc. This can be done.

[0047] Furthermore, the KLK5 inhibitory peptide, KLK5 / KLK7 inhibitory peptide, and KLK The 5 / KLK14 inhibitor peptide is used in addition to antibodies and other compounds used in the relevant fields as pharmaceuticals and diagnostic agents. It has a smaller molecular weight than other biopolymers, is relatively easy to manufacture (described later), and is stable in storage. It has excellent physical properties such as viscosity and thermal stability, and is suitable for use as a pharmaceutical composition (described later). It has the advantage of being able to choose from a wide range of administration routes, administration methods, and formulations. The molecular weight of the peptide of the present invention can be increased by applying known methods such as the addition of a ribonucleotide. This also makes it possible to adjust the blood half-life to be longer when used as a pharmaceutical composition. The KLK5 inhibitory peptides, KLK5 / KLK7 inhibitory peptides, and KLK The molecular weight of the 5 / KLK14 inhibitor peptide is less than 10,000, preferably less than 8,000, more preferably More preferably, it is about 7,000 to 7,200. A variable loop region consisting of Cys to Cys at position 31 or a region consisting of Cys at position 15 to Cys at position 63 Among the six Cys-containing moieties, the KLK5 inhibitory activity and KLK5 / K Those having KLK7 inhibitory activity or KLK5 / KLK14 inhibitory activity are also included in the KLK of the present invention. KLK5 inhibitor peptide, KLK5 / KLK7 inhibitor peptide, or KLK5 / KLK14 inhibitor peptide The molecular weight of the variable loop portion is less than 2,500, preferably about 1, The molecular weight of the portion containing six Cys is less than 6,000, preferably is approximately 5,300 to 5,500.

[0048] The KLK5 inhibitory peptide, KLK5 / KLK7 inhibitory peptide, or KLK5 / KLK7 inhibitor of the present invention The KLK14 inhibitor peptide at least partially retained the SPINK2 scaffold. SPINK2 mutant (hereinafter abbreviated as "SPINK2 mutant"), preferably K Partial peptides of LK5, KLK5 and KLK7, or KLK5 and KLK14, partial peptides (hereinafter, such recognition or binding action is collectively referred to as "target binding activity").

[0049] The binding of the SPINK2 mutant to KLK5, KLK7, or KLK14 in the present invention is LISA method, surface plasmon resonance e: SPR analysis, BioLayer Interference (BioLayer Interference) erferometry (hereinafter referred to as "BLI"), isothermal titration calorimetry (Isoth Thermal Titration Calorimetry (hereinafter referred to as "ITC") , flow cytometry, immunoprecipitation, etc., using methods known to those skilled in the art. It can be determined.

[0050] For the ELISA method, KLK5, KLK5 / KLK7, or KLK5 inhibitor peptide, KLK5 / KLK14, which recognizes and binds to KLK5 / KLK14 7. Methods for detecting inhibitory peptides or KLK5 / KLK14 inhibitory peptides For immobilization of KLK5, KLK5 / KLK7, or KLK5 / KLK14, biotin was used. - streptavidin, KLK5, KLK5 / KLK7, or KLK5 / KL K14, or fused to KLK5, KLK5 / KLK7, or KLK5 / KLK14 A solid-phase antibody that recognizes the tag can be used. To detect the K5 / KLK7 inhibitory peptide or the KLK5 / KLK14 inhibitory peptide, a target In addition to the recognized streptavidin, KLK5 inhibitor peptide, KLK5 / KLK7 inhibitor peptide KLK5 / KLK14 inhibitory peptide, or KLK5 inhibitory peptide, fused to an LK5 / KLK7 inhibitory peptide or a KLK5 / KLK14 inhibitory peptide A labeled detection antibody that recognizes the tag can be used. In addition, methods that can be used for biochemical analysis, such as HRP, alkaline phosphatase, and FITC, are also available. For detection using enzyme labeling, TMB (3,3',5,5'-tet ramethylbenzidine), BCIP(5-bromo-4-chloro -3-indolyl phosphate), p-NPP(p-nitropheny l phosphate), OPD (o-Phenylenediamine), ABT S(3-Ethylbenzothiazoline-6-sulfonic acid ), SuperSignal ELISA Pico Chemiluminescen Development of t Substrate (Thermo Fisher Scientific) etc. Color substrates and QuantaBlu® Fluorogenic Peroxidas Fluorescent Substrate (Thermo Fisher Scientific) Photoluminescent and chemiluminescent substrates can be used. The detection signal is measured using an absorbance plate. plate reader, fluorescence plate reader, luminescence plate reader, RI liquid scintillation Counters can be used.

[0051] The SPR analysis method involves immobilizing SPINK2 mutant peptides on a sensor chip. and measuring the binding between them by adding a target molecule such as KLK5. Target molecules such as LK5 are immobilized on a sensor chip, and SPINK2 mutant peptides are added. The former is preferred. Immobilization of the SPINK2 mutant contained in the peptide or conjugate of the present invention is directly Either the direct method or the capture method can be used, preferably the latter. Direct immobilization using the hydrophobicity of the variant and the amino and carboxyl groups of SPINK2 In the capture method, biotin-streptavidin and other conjugates thereof can be used. an antibody, protein A, protein G, or the like that recognizes the tag fused to the conjugate The SPINK2 mutant can be immobilized on a sensor chip. Target molecules such as KLK5 diluted with measurement buffer were added, and the SPR signal was measured over time. Then, a sensorgram of binding is obtained by observing the binding of the target molecule such as KLK5. The test buffer was added, and the SPR signal was observed over time to obtain a sensorgram of dissociation. The binding affinity was analyzed using the acquired sensorgram, and the dissociation constant KD Calculate S The equipment used for PR analysis was BIAcore (registered trademark) (GE Healthcare re), ProteOn® (BioRad), SPR-Navi® (BioNavisOy), Spreeta® (Texas Instruments ents), SPRi-PlexII® (Horiba), Autolab SPR (registered trademark) (Metrohm) and the like. An example of such a material is Octet (registered trademark) (Pall).

[0052] For immunoprecipitation, we used a KLK5 inhibitor peptide, KLK5 / KL, immobilized on beads. Recognized and bound by K7 inhibitor peptide or KLK5 / KLK14 inhibitor peptide Methods for detecting KLK5, KLK5 and KLK7, or KLK5 and KLK14 are also provided. Magnetic beads, agarose beads, etc. can be used as beads. KLK5 inhibitor peptide, KLK5 / KLK7 inhibitor peptide, or KLK5 / KLK14 inhibitor peptide For immobilizing the peptide, in addition to biotin-streptavidin, the peptide or the peptide itself may be immobilized using a method other than the above. An antibody that recognizes the fused tag, such as protein A or protein G, can be used. The beads were separated using a magnet or centrifugation, and the KLK5 and KLK5 precipitated with the beads were collected. and KLK7, or KLK5 and KLK14, were analyzed by SDS-PAGE or Western b Detected by lot method. KLK5, KLK5 and KLK7, or KLK5 and KLK14 In addition to labeled streptavidin, KLK5, KLK7, or KLK1 4, or a labeled antibody that recognizes a tag fused to KLK5, KLK7, or KLK14. Detection antibodies and the like can be used. Labels include biotin, HRP, alkaline phosphatase, and the like. Methods that can be used for biochemical analysis, such as enzymes, FITC, etc., can be used. For detection using a label, the same substrate as in ELISA can be used. ChemiDoc (registered trademark) (BioRad) and LuminoGraph are used for the measurement of the leukocyte count. (ATTO) etc. can be used.

[0053] In the present invention, "specific recognition", i.e., "specific binding" means non-specific adsorption. The criteria for determining whether binding is specific or not include, for example, ELI Binding activity EC in SA method 50 Other criteria include: , dissociation constant (hereinafter referred to as "K D ") The KLK5 inhibitory peptide of the present invention has the K D value, KLK5 / KLK7 inhibitor peptides D Value or KLK K5 / KLK14 inhibitor peptides for KLK5 and KLK14 D The value is 1 x 10 -5 M Below, 5 x 10 -6 M or less, 2×10 -6 M or less or 1 x 10 -6 M or less, more preferably 5×10 -7 M or less, 2×10 -7 M or less or 1 x 10 -7 M or less, more preferably 5 x10 -8 M or less, 2×10 -8 M or less or 1 x 10 -8 M or less, even more suitable is 5 x 10 -9 M or less, 2×10-9 M or less or 1 x 10 -9 M or less. Other criteria An example of the criteria is the results of analysis by immunoprecipitation. Suitable KLK5 inhibitory peptides, KLK5 / KLK7 inhibitory peptides, or KLK5 / KLK 14 inhibitory peptides were immobilized on beads, and KLK5, KLK5 and KLK7, or After adding KLK5 and KLK14, the beads were separated, and the KLKs that precipitated with the beads were analyzed. 5, KLK5 and KLK7, or KLK5 and KLK14, Signals of KLK5 and KLK7, or KLK5 and KLK14, are detected.

[0054] The KLK5 inhibitory peptide, KLK5 / KLK7 inhibitory peptide, or KLK5 / K of the present invention The SPINK2 mutants as LK14 inhibitor peptides have the protease inhibitory activity described above. While it may have the same properties, functions, characteristics, etc. as the full-length amino acid sequence, The sequence has high sequence identity to the amino acid sequence of human wild-type SPINK2. The SPINK2 mutant has a 60% identical amino acid sequence to that of human SPINK2 (SEQ ID NO: 1: Figure 7). Above, 70% or above, 75% or above, 80% or above, 85% or above, 90% or above, 95% or above, 9 The sequence has 8% or more or 99% or more sequence identity.

[0055] "Identity" refers to the property of the degree of similarity or relationship between two sequences. The percent identity of amino acid sequences is the number of identical amino acids or amino acid residues. It is calculated by dividing the number by the total number of amino acid residues and multiplying the result by 100.

[0056] A "gap" refers to a deletion and / or addition in at least one of two or more sequences. This refers to gaps in the alignment between the sequences that are the result of

[0057] The identity between two amino acid sequences that have completely identical amino acid sequences is 100%. However, one amino acid sequence is compared with the other to find one or more amino acids or amino acid residues If there are substitutions, deletions, or additions, the identity between the two will be less than 100%. Gaps are also taken into consideration. There are standard algorithms and programs for determining identity between two sequences using BLAST (Altschul, et al. Nucleic Acids Res. 25, 3389-3402, 1997), BLAST2 (Altschul, et al. J.Mol.Biol. 215, 403-4 10 pages, 1990), Smith-Waterman (Smith, et al. J. Mol. Biol. vol. 147, pp. 195-197, 1981) and other methods well known to those skilled in the art. Examples of such materials include:

[0058] In the present invention, "mutated" refers to a nucleic acid molecule or peptide that is mutated in a manner that is comparable to that of a naturally occurring nucleic acid molecule or peptide. In a nucleotide or amino acid sequence, one or more nucleotides or Nucleotide residues or amino acids or amino acid residues have been substituted, deleted or inserted The amino acid sequence of the SPINK2 mutant of the present invention is the same as that of human SPINK2. Compared to the amino acid sequence, one or more amino acids or amino acid residues have been mutated. .

[0059] In one embodiment of the invention, the amino acid sequence of the SPINK2 variant is The amino acid sequence of (SEQ ID NO: 1: Figure 9): One, two, three, four, five, six or seven amino acids from Ser 16 to Gly 22 the amino acid is replaced by another amino acid or amino acid residue; One, two, three, four or five amino acids from 24th Pro to 28th Asn are different from other amino acids. substituted with amino acid or amino acid residues; Cys at position 15, Cys at position 23, Cys at position 31, Cys at position 42, Cys at position 45, and C at position 63 Cys must be Cys as in the wild type to maintain the native disulfide bond. is preferred, and the natural disulfide bonds are eliminated or non-natural disulfide bonds are formed. To produce or produce one, two, three, four, five or six of them, Some of the preferred SPINK2 mutants of the present invention may be substituted with KLK5-inhibiting amino acids. Peptide, KLK5 / KLK7 inhibitory peptide, or KLK5 / KLK14 inhibitory peptide In this case, Cys is maintained at the six positions, just like in the natural form, and the disulfide bonds are maintained. In some more preferred embodiments of such peptides, the amino acid sequence is Cys-15-45. Cys, Cys23-Cys42, and Cys31-Cys63, respectively. It forms a disulfide bond.

[0060] The amino acid sequence of such a SPINK2 mutant is a KLK5 inhibitor peptide, KLK When contained in a KLK5 / KLK7 inhibitory peptide or a KLK5 / KLK14 inhibitory peptide, The wild-type SPINK2 amino acid sequence contains a loop consisting of Ser 16 to Val 30. The loop structure, β-strand (1) consisting of Cys31 and Gly32, and Il57 β-strand (2) consisting of β-sheet consisting of β-strands (e) to (59) Arg, and β-strand (41) Glu an α-helix consisting of Gly 51 to 53, or a helix similar thereto or It consists of loop structures, β-sheets, α-helices, etc. that at least partially correspond to (positions of) The resulting three-dimensional structure has KLK5 inhibitory activity, KLK5 / KLK7 inhibitory activity, or KLK5 It is preferable that the amount of KLK14 is maintained to an extent that it can exert its inhibitory activity against KLK14.

[0061] Among the SPINK2 mutants of the present invention, some KLK5 inhibitor peptides, KLK5 / KLK 7. Amino acid sequence of inhibitory peptide or KLK5 / KLK14 inhibitory peptide As mentioned above, in the present invention, "amino acid residue" is simply referred to as "amino acid." It may be written down.

[0062] In the amino acid sequence (general formula) shown in SEQ ID NO: 61 (Figure 69), X1 to X 12 Insofar as it inhibits KLK5, KLK5 and KLK7, or KLK5 and KLK14, There is no particular limitation as long as each of these amino acids is an arbitrary amino acid. 12 Suitable for The following describes amino acids that are naturally occurring, i.e., wild-type human amino acids. It may contain amino acids identical to those in the amino acid sequence of SPINK2.

[0063] The amino acid sequence shown in SEQ ID NO: 61 (Figure 69) contained in the KLK5 inhibitor peptide and preferably: Xaa 16 (X1) is Ala, Asp, Gly, Gln, Leu, Ser, or Thr ; Xaa 17 (X2) is Arg, Glu, Asn, Gln, or Ser; Xaa 18 (X3) is Asp, Gln, Ile, Thr, Trp, or Tyr; Xaa at position 19 (X4) is Arg, Gly, Met, Gln, or Thr; Xaa at position 20 (X5) is Asp, Glu, Leu, Lys, Thr, or Tyr; Xaa at position 21 (X6) is Glu, Gly, His, Leu, Ser, Gln, or Tyr ; Xaa at position 22 (X7) is Asp, Gly, Gln, Sey, or Tyr; Xaa 24 (X8) is Ala, Asp, Glu, Gly, Asn, Ser, or Thr ; Xaa 25 (X9) is Arg or Lys; No. 26 Xaa(X 10 ) is Asp, Glu, Gln, Ser, or Val; No. 27 Xaa(X 11 ) is Phe or Tyr; and No. 28 Xaa(X 12 ) is Asp or Glu is.

[0064] The amino acid sequence shown in SEQ ID NO: 61 (FIG. 69) contained in the KLK5 / KLK7 inhibitory peptide In the acid sequence, preferably: Xaa 16 (X1) is Gly, Met, or Tyr; Xaa 17 (X2) is Glu, Gln, or Thr; Xaa 18 (X3) is His, Met, or Tyr; Xaa at position 19 (X4) is Ala, Arg, Lys, or Gln; Xaa 20 (X5) is Gly, Arg, or Ser; Xaa at position 21 (X6) is Arg, Lys, Gln, or Ser; Xaa 22 (X7) is Gly; Xaa at position 24 (X8) is His, Thr, or Tyr; Xaa 25 (X9) is His or Tyr; No. 26 Xaa(X 10 ) is Asp, Glu, or His; No. 27 Xaa(X 11 ) is Tyr; and No. 28 Xaa(X12 ) is Asp or Glu is.

[0065] The KLK5 / KLK14 inhibitor peptide contains the amino acid sequence shown in SEQ ID NO: 61 (Figure 69). In the amino acid sequence, preferably: Xaa 16 (X1) is Gly, Ser, or Tyr; Xaa 17 (X2) is Asp or Gln; Xaa 18 (X3) is Thr or Val; Xaa at position 19 (X4) is Thr or Val; Xaa 20 (X5) is Glu or Thr; Xaa at position 21 (X6) is His or Thr; 22nd Xaa (X7) is Tyr; Xaa 24 (X8) is Asn or Ser; 25th Xaa (X9) is Arg; No. 26 Xaa(X 10 ) is Asp or Glu; No. 27 Xaa(X 11 ) is Tyr; No. 28 Xaa(X 12 ) is Asp is.

[0066] In addition, the wild-type Xaa 16 to 22 and 24 to 28 (X1 to X 12 ) is that Ser, Gln, Tyr, Arg, Leu, Pro, Gly, Pro, Arg, Hi respectively s, Phe and Asn.

[0067] In the present invention, one to several or more amino acids may be further added to the N-terminal side of the first amino acid. may have 1 to 5 amino acids added. Such amino acid additions include: Preferably, 1 to 5 Asp and / or Glu are added (including both Asp and Glu). more preferably, one to five Asps or one to five Glus are added. Examples include those with additional units.

[0068] In the present invention, the N-terminal and / or C-terminal adduct of the SPINK2 mutant peptide (hereinafter referred to as In the additional portion of the parent peptide, one or more amino acids are substituted or added. and / or deleted, and having part or all of the activity of the SPINK2 mutant peptide. Peptides that maintain the same structure may be referred to as "derivatives of the parent peptide" or "parent peptide derivatives." Such "derivatives" are also included within the scope of the "peptide" of the present invention.

[0069] The KLK5 inhibitory peptide, KLK5 / KLK7 inhibitory peptide, or KLK5 / KLK7 inhibitor of the present invention The amino acid sequence of the SPINK2 mutant included in the range of the KLK14 inhibitor peptide In this case, X1 to X 12 The portion other than the amino acid sequence of wild-type human SPINK2 In SEQ ID NO: 1: Figure 9, Pro at position 2 to Cys at position 15, Cys at position 23 and Pro at position 29 At positions ~63Cys, a natural amino acid or a mutated amino acid or an amino acid For example, SPINK2 variants may have KLK5 inhibitory activity, KLK 5 / KLK7 inhibitory activity, or KLK5 / KLK14 inhibitory activity or folding in one or more positions, provided that they do not at least partially obstruct or interfere with Such mutations can be made using standard methods known to those skilled in the art. Typical mutations in the amino acid sequence include the substitution of one or more amino acids. Examples of substitutions include substitutions, deletions, and insertions. Examples of substitutions include conservative substitutions. By conservative substitution, certain amino acid residues can be made to have a larger polarity as well as a larger bulkiness. Conservative substitutions are made with amino acid residues having similar chemical characteristics. Examples of conservative substitutions are described herein. On the other hand, X1 to X 12 The remaining part has KLK5 inhibitory activity , KLK5 / KLK7 inhibitory activity, or KLK5 / KLK14 inhibitory activity or fold one or more, as long as they do not at least partially impede or interfere with the operation of the Non-conservative substitutions of amino acids may also be tolerated.

[0070] The KLK5 inhibitory peptide, KLK5 / KLK7 inhibitory peptide, or KLK5 / KLK7 inhibitor of the present invention The amino acid sequence of the SPINK2 mutant as a KLK14 inhibitor peptide is X 12 However, preferably, SEQ ID NOs: 6, 8, 10, 12, 14, 16, 18 and 20 (Figure 14 , 16, 18, 20, 22, 24, 26 and 28), SEQ ID NOs: 22, 24, 26 and 28 (Figures 30, 32, 34 and 36), or SEQ ID NOs: 30 and 32 (Figures 38 and 40) X1 to X in any one 12 and X1 to X 12 Other departments The compound has KLK5 inhibitory activity, KLK5 / KLK7 inhibitory activity, or KLK5 / KLK14 inhibitory activity. Amino acids or amino acids that do not at least partially prevent or interfere with activity or folding can have an amino acid sequence.

[0071] In addition, the KLK5 inhibitory peptide, KLK5 / KLK7 inhibitory peptide, or KL Examples of amino acid sequences of SPINK2 mutants as K5 / KLK14 inhibitory peptides include: Any of the following (a1) to (a4), (b1) to (b4), or (c1) to (c4) The amino acid sequences described herein are: (a1) SEQ ID NOs: 6, 8, 10, 12, 14, 16, 18 and 20 (Figures 14, 16, 18 , 20, 22, 24, 26 and 28) an amino acid sequence consisting of numbers 1 to 63; (a2) A nucleotide sequence complementary to the nucleotide sequence encoding the amino acid sequence described in (a1). hybridizes to the nucleotide sequence under stringent conditions and has KLK5 inhibitory activity. The peptide is encoded by a nucleotide sequence that encodes the amino acid sequence contained in the peptide. amino acid sequence; (a3) In the amino acid sequence of (a1), 1 to 20, 1 to 15, 1 to 1 0, 1 to 8, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 or 2 or one amino acid is substituted, deleted, added and / or inserted, and has KLK5 inhibitory activity an amino acid sequence contained in a peptide having the property; and (a4) The amino acid sequence of (a1) and 60%, 70%, 80%, 85%, 90%, 9 2%, 94%, 96%, 97%, 98% or 99% or more identical and have KLK5 inhibitory activity an amino acid sequence contained in a peptide having the property (b1) any of SEQ ID NOs: 22, 24, 26, and 28 (Figures 30, 32, 34, and 36) an amino acid sequence consisting of amino acid numbers 1 to 63 of an amino acid sequence represented by one of the following: (b2) A nucleotide sequence complementary to the nucleotide sequence encoding the amino acid sequence described in (b1). hybridizes under stringent conditions with the KLK5 / KLK7 inhibitor The coding sequence is a nucleotide sequence that encodes the amino acid sequence contained in the peptide having anti-inflammatory activity. the amino acid sequence to be encoded; (b3) In the amino acid sequence of (b1), 1 to 20, 1 to 15, or 1 to 1 0, 1 to 8, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 or 2 or one amino acid substitution, deletion, addition and / or insertion, and KLK5 / KL an amino acid sequence contained in a peptide having K7 inhibitory activity; and (b4) The amino acid sequence of (b1) and 60%, 70%, 80%, 85%, 90%, 9 2%, 94%, 96%, 97%, 98% or 99% or more identical and KLK5 / KL an amino acid sequence contained in a peptide having K7 inhibitory activity, or (c1) an amino acid sequence shown in any one of SEQ ID NOs: 30 and 32 (Figures 38 and 40) an amino acid sequence consisting of amino acid numbers 1 to 63 of the sequence; (c2) A nucleotide sequence complementary to the nucleotide sequence encoding the amino acid sequence described in (c1). hybridizes under stringent conditions with the nucleotide sequence KLK5 / KLK14 The nucleotide sequence encoding the amino acid sequence contained in the peptide having inhibitory activity the encoded amino acid sequence; (c3) In the amino acid sequence of (c1), 1 to 20, 1 to 15, or 1 to 1 0, 1 to 8, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 or 2 or one amino acid substitution, deletion, addition and / or insertion, and KLK5 / KL an amino acid sequence contained in a peptide having K14 inhibitory activity; and (c4) The amino acid sequence of (c1) and 60%, 70%, 80%, 85%, 90%, 9 2%, 94%, 96%, 97%, 98% or 99% or more identical and KLK5 / KL Amino acid sequence contained in a peptide with K14 inhibitory activity.

[0072] In addition, SEQ ID NOs: 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 2 8, 30 and 32 (Figs. 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, Amino acids 64 and 65 of wild-type human SPINK2 ( 1, FIG. 9: consisting of 63 amino acids), but is not an amino acid corresponding to the In one embodiment, it is added to express the peptide of the present invention.

[0073] The peptide of the present invention is characterized by its folding stability, thermal stability, storage stability, and blood half-life. Mutations are introduced to improve the molecular structure, water solubility, biological activity, pharmacological activity, side effects, etc. For example, polyethylene glycol (PEG), hydroxyethyl starch (H Cytochrome P450 (Cytochrome P450), ... New reactive groups such as s can be introduced by mutation.

[0074] In the present invention, a KLK5 inhibitory peptide, a KLK5 / KLK7 inhibitory peptide, or a K The LK5 / KLK14 inhibitor peptide may be linked or attached to other moieties, such as The conjugates are called "KLK5 inhibitor peptide conjugates" and "KLK5 / KLK7 Inhibitory peptide conjugate" or "KLK5 / KLK14 inhibitory peptide conjugate" In the present invention, the term "conjugate" refers to a peptide of the present invention. or a fragment thereof to which another moiety is attached. "Jugation" involves the transfer of a part to an amino acid via a chemical substance such as a cross-linking agent. The N-terminus and / or C-terminus of the peptide of the present invention may be linked to a suitable agent or the like via a side chain. The peptide of the present invention is linked or bonded to the Such "moieties" include polyethylene glycols and other polyisoprene derivatives that improve blood half-life. Polyalkylene glycol molecules such as ethylene glycol (PEG), hydroxyethyl starch fatty acid molecules such as palmitic acid, Fc regions of immunoglobulins (e.g., human Fc region of immunoglobulin G1: its amino acid sequence is shown in SEQ ID NO: 87, Figure 95), CH3 domain of immunoglobulin, CH4 domain of immunoglobulin, albumin or its fragments, albumin-binding peptides, albumin-binding proteins such as streptococcal protein G, Other examples of "moieties" include phenanthrene, ... The peptides of the present invention can be linked via a linker, such as a peptide linker.

[0075] In one embodiment of the invention, the conjugate comprises a SPINK2 mutant peptide of the invention. The antibody is a fusion of the Fc region of an antibody or a fragment thereof. The antibody origin is human or non-human. Animals, for example, rodents such as mice, rats, and rabbits, cows, pigs, dogs, and cynomolgus monkeys , marmosets, rhesus monkeys and other mammals, and chickens and other birds. Preferably, the antibody is human. Examples of antibodies include IgG1, IgG2, IgG3, IgG4, and IgM. Examples include IgA1, IgA2, IgD, and IgE, with IgG1 being preferred. More preferably, the conjugate comprises a peptide of the present invention and an Fc region of human IgG1 or or a fusion with a fragment thereof. is sometimes referred to as an "Fc fusion protein" or a "conjugate," but these are synonymous. .

[0076] The Fc region of human IgG1 includes, for example, the amino acid sequence shown in SEQ ID NO: 87 (FIG. 95). Examples of antibodies include, but are not limited to, antibodies that contain or consist of the nucleic acid sequence. The Fc region may be either wild-type or mutant.

[0077] In addition, the KLK5 inhibitory peptide, KLK5 / KLK7 inhibitory peptide, or KL K5 / KLK14 inhibitor peptides may be used in combination with other drugs to exert or enhance their pharmacological activity. In the field of antibodies, antibody-drug conjugates (antibodies) are used. Techniques and embodiments known to those skilled in the art as antibody-drug conjugates (ADCs) include: Replacing the antibody with the peptide of the present invention can be an embodiment of the present invention.

[0078] The KLK5 inhibitory peptide, KLK5 / KLK7 inhibitory peptide, or KLK5 / KLK7 inhibitor of the present invention The KLK14 inhibitor peptides inhibit KLK5, KLK5 and KLK7, or KLK5 and binding affinity, inhibitory activity, antagonistic activity, agonistic activity, etc. for target molecules other than KLK14. and further comprising or conjugated to one or more moieties that exhibit Such "portions" may include antibodies or fragments thereof, SPINK2 mutants, etc. Examples of such proteins include proteins or fragments thereof that have a framework other than that of an antibody. Multispecific and bispecific antibodies The techniques and embodiments known to those skilled in the art as antibodies (bispecific antibodies, etc.) are At least one of the two or more "antibodies" contained therein is replaced with the peptide of the present invention. This constitutes some aspects of the conjugates of the present invention.

[0079] The KLK5 inhibitory peptide, KLK5 / KLK7 inhibitory peptide, or KLK The 5 / KLK14 inhibitory peptide or its precursor may contain a signal sequence. A signal sequence present at or added to the N-terminus of a polypeptide or its precursor may be used to express the polypeptide. peptides into specific compartments of the cell, e.g., the periplasm in E. coli or the endoplasmic reticulum in eukaryotic cells. Many signal sequences are known to those skilled in the art and are useful for delivering the gene to the host cell. A signal sequence for secreting the desired peptide into the periplasm of E. coli can be selected. An example of such a conjugate is OmpA. A form containing a signal sequence can also be used in the conjugates of the present invention. It may be included as part of the gateway.

[0080] In addition, the KLK5 inhibitory peptide, KLK5 / KLK7 inhibitory peptide, or By adding a tag to the KLK5 / KLK14 inhibitor peptide in advance, affinity The peptide can be purified by chromatography.

[0081] The peptide of the present invention may have, for example, biotin, Strep tag (registered trademark), Strep tag II (registered trademark), oligohistidine such as His6, polyhistidine, immunoglobulin immunoglobulin domain, maltose-binding protein, glutathione-S-transferase (GST), calmodulin-binding peptide (CBP), digoxigenin and dinitrophen Haptens such as hydroxyl, epitope tags such as FLAG (registered trademark), myc tags, HA tags, etc. (hereinafter collectively referred to as "affinity tags"). Tagged products can also be The conjugate of the present invention may include the compound as a part of its embodiment. may be a peptide (polypeptide) as a whole.

[0082] The KLK5 inhibitory peptide, KLK5 / KLK7 inhibitory peptide, or KLK5 / K of the present invention The LK14 inhibitor peptides can include a moiety for labeling, specifically an enzyme label, Radioactive labels, colored labels, fluorescent labels, chromogenic labels, luminescent labels, haptens, digoxigenin, Labeling moieties such as thiocin, metal complexes, metals, colloidal gold, etc. may be conjugated. The conjugate of the present invention may also include a portion thereof. .

[0083] Conjugates of the KLK5 inhibitory peptide of the present invention, KLK5 / KLK7 inhibitory peptide Conjugate or amino acid of KLK5 / KLK14 inhibitor peptide conjugate Examples of the arrangement are the following (a1) to (a4), (b1) to (b4), or (c1) to (c2). c4) can each be an amino acid sequence according to any one of: (a1) SEQ ID NOs: 34, 36, 38, 40, 42, 44, 46, 48 and 96 (Figure 42, 44, 46, 48, 50, 52, 54, 56 and 106) No acid sequence; (a2) A nucleotide sequence complementary to the nucleotide sequence encoding the amino acid sequence described in (a1). hybridizes to the nucleotide sequence under stringent conditions and has KLK5 inhibitory activity. The peptide is encoded by a nucleotide sequence that encodes the amino acid sequence contained in the peptide. amino acid sequence; (a3) In the amino acid sequence of (a1), 1 to 20, 1 to 15, 1 to 1 0, 1 to 8, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 or 2 or one amino acid is substituted, deleted, added and / or inserted, and has KLK5 inhibitory activity an amino acid sequence contained in a peptide having the property; and (a4) The amino acid sequence of (a1) and 60%, 70%, 80%, 85%, 90%, 9 2%, 94%, 96%, 97%, 98% or 99% or more identical and have KLK5 inhibitory activity an amino acid sequence contained in a peptide having the property (b1) any of SEQ ID NOs: 50, 52, 54, and 56 (Figures 58, 60, 62, and 64) The amino acid sequence shown in one; (b2) A nucleotide sequence complementary to the nucleotide sequence encoding the amino acid sequence described in (b1). hybridizes under stringent conditions with the KLK5 / KLK7 inhibitor The coding sequence is a nucleotide sequence that encodes the amino acid sequence contained in the peptide having anti-inflammatory activity. the amino acid sequence to be encoded; (b3) In the amino acid sequence of (b1), 1 to 20, 1 to 15, or 1 to 1 0, 1 to 8, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 or 2 or one amino acid substitution, deletion, addition and / or insertion, and KLK5 / KL an amino acid sequence contained in a peptide having K7 inhibitory activity; and (b4) The amino acid sequence of (b1) and 60%, 70%, 80%, 85%, 90%, 9 2%, 94%, 96%, 97%, 98% or 99% or more identical and KLK5 / KL an amino acid sequence contained in a peptide having K7 inhibitory activity, or (c1) an amino acid sequence shown in any one of SEQ ID NOs: 58 and 60 (Figures 66 and 68) column; (c2) A nucleotide sequence complementary to the nucleotide sequence encoding the amino acid sequence described in (c1). hybridizes under stringent conditions with the nucleotide sequence KLK5 / KLK14 The nucleotide sequence encoding the amino acid sequence contained in the peptide having inhibitory activity the encoded amino acid sequence; (c3) In the amino acid sequence of (c1), 1 to 20, 1 to 15, or 1 to 1 0, 1 to 8, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 or 2 or one amino acid substitution, deletion, addition and / or insertion, and KLK5 / KL an amino acid sequence contained in a peptide having K14 inhibitory activity; and (c4) The amino acid sequence of (c1) and 60%, 70%, 80%, 85%, 90%, 9 2%, 94%, 96%, 97%, 98% or 99% or more identical and KLK5 / KL Amino acid sequence contained in a peptide with K14 inhibitory activity.

[0084] The KLK5 inhibitory peptide, KLK5 / KLK7 inhibitory peptide, or KLK5 / K of the present invention The LK14 inhibitor peptide (amino acid sequence) contains both natural and unnatural amino acids. Natural amino acids may include both L-amino acids and D-amino acids. It can also include:

[0085] The KLK5 inhibitory peptide, KLK5 / KLK7 inhibitory peptide, or KLK5 / K of the present invention The LK14 inhibitor peptide exists as a monomer, dimer, trimer or higher oligomer or multimer. Dimers, trimers and higher oligomers and polymers are composed of a single monomer. It can be either homo or hetero, which is composed of two or more different monomers. For example, dimers and oligomers may diffuse rapidly and have excellent tissue penetration. The multimers may, for example, have high affinity or avidity for a target molecule locally. or have a slow dissociation rate, or have high KLK5 inhibitory activity, KLK5 / KLK7 inhibitory activity, It may have advantageous aspects such as exhibiting anti-inflammatory activity or KLK5 / KLK14 inhibitory activity. In addition to natural dimerization, oligomerization, and multimerization, Dimerization can also be achieved by introducing a jun-fos domain, a leucine zipper, or the like into the peptide of the present invention. This can be achieved by doing so.

[0086] The KLK5 inhibitory peptide, KLK5 / KLK7 inhibitory peptide, or KLK5 / KLK7 inhibitor of the present invention The KLK14 inhibitor peptide is a monomer, dimer, trimer or higher oligomer or multimer. The antibody can bind to or inhibit the activity of one or more target molecules.

[0087] The KLK5 inhibitory peptide, KLK5 / KLK7 inhibitory peptide, or KLK5 / KLK7 inhibitor of the present invention The KLK14 inhibitor peptide can be in an isolated form (lyophilized sample, solution, etc.). etc.), the above-mentioned conjugates, forms bound to other molecules (immobilized forms, forms combined with foreign molecules) Examples of such structures include, but are not limited to, aggregates of the target molecule and the like. It is not a simple substance, and any form suitable for expression, purification, use, storage, etc. can be selected. .

[0088] 3. KLK5 inhibitory peptide, KLK5 / KLK7 inhibitory peptide, and KLK5 / KLK 14. Identification of Inhibitory Peptides KLK5 inhibitory peptide, KLK5 / KLK7 inhibitory peptide, and KLK5 / KLK1 The inhibitory peptide has the amino acid sequence of SPINK2 or the KLK5 inhibitor peptide of the present invention. Amino acids possessed by LK5 / KLK7 inhibitory peptides and KLK5 / KLK14 inhibitory peptides an amino acid sequence (for example, an amino acid sequence described in (a1), (b1), or (c1) above), Starting from a nucleotide sequence encoding an amino acid sequence, a nucleic acid molecule containing the nucleotide sequence, etc. The material can be identified by a method well known to those skilled in the art. From the SPINK2 mutant library, we identified KLK5 inhibitory activity and KLK5 / KLK7 inhibitory activity. The inhibitory activity against KLK5 / KLK14 can be used as an index to identify the inhibitory activity against KLK5 / KLK14. The binding activity to KLK5, KLK5 / KLK7, or KLK5 / KLK14 was also They may be combined as indicators.

[0089] For example, the starting nucleic acid molecule may be subjected to mutagenesis and transformed using recombinant DNA techniques. The SPINK2 mutant library can be introduced into a suitable bacterial or eukaryotic host. It is a well-known technique for identifying binders and inhibitors of target molecules, for example, WO2 The disclosure in Publication No. 014 / 024914 is also incorporated herein by reference in its entirety. After expressing the nucleotide sequence subjected to mutagenesis in a suitable host, SPINK2 mutants with desired properties, activities, functions, etc. are linked to their genetic traits Clones can be enriched and / or selected from the library and identified. For enrichment and / or selection of clones, bacterial display methods (Francisco, JA , et al. (1993) Proc. Natl. Acad. Sci. U. SA Vol. 90, pp. 10444-10448), yeast display method (Boder, E .T., et al. (1997) Nat. Biotechnol. vol. 15, 5 pp. 53-557), mammalian cell display method (Ho M, et al. (2009 (Methods Mol Biol. 525, 337-352), phage Display method (Smith, GP (1985) Science. Vol. 228, 13 15-1317), ribosome display method (Mattheakis LC, et al. (1994) Proc. Natl. Acad. Sci. USA 91, No. 19, pp. 9022-9029), nucleic acid displays such as mRNA display Nemoto N, et al. (1997) FEBS Lett. 414 Vol. 2, No. 405-408), colony screening method (Pini, A. et al (2002) Comb. Chem. High Throughput Scre Selection can be performed by using methods known to those skilled in the art, such as the method described in the Japanese Society of Microbiology, Vol. 5, pp. 503-510. The nucleotide sequences of the SPINK2 mutants contained in the clones identified were then determined. The amino acid sequence encoded by the nucleotide sequence is then extracted from the clone. SPINK2 mutants, i.e., KLK5 inhibitor peptides, KLK5 / KLK7 inhibitor peptides The amino acid sequence of the KLK5 / KLK14 inhibitor peptide is determined as follows: can be done.

[0090] The SPINK2 mutant of the present invention can be prepared by, for example, inducing a mutation in a native SPINK2. "Mutation induction" refers to the process of introducing one or more amino acids into each position of a given amino acid sequence. One or more amino acids may be substituted with other amino acids or deleted, or This means that it is possible to insert amino acids that do not exist in the amino acid sequence. Such deletions or insertions may alter the length of the sequence. In this case, the mutagenesis is preferably carried out at the position: X1 to X 12 It may occur at one or more positions.

[0091] However, after such suitable mutagenesis, X1 to X 12 One or more positions of In the formula, a naturally occurring amino acid, i.e., an amino acid present at a specific position in a naturally occurring amino acid sequence, is Even if the same amino acids are maintained, at least one amino acid is mutated overall. Similarly, in one embodiment of the present invention, X1 to X1 After inducing mutations at one or more positions in the portion other than 2, naturally occurring amino acids at those positions, That is, the same amino acid that is present at a particular position in the naturally occurring amino acid sequence is maintained. However, if at least one amino acid is mutated, the mutation is included in the range of the mutant. do.

[0092] "Random mutagenesis" refers to the induction of one or more different mutations at specific positions in a sequence. This means that the amino acid to be introduced into the corresponding position is introduced with a certain probability by mutagenesis. However, the probabilities of introducing at least two different amino acids may not all be the same. In the present invention, at least two different amino acids are selected from a group consisting of natural amino acids (one type). This does not preclude the inclusion of such a mutation, and such a case is also within the scope of "induction of random mutations." Included.

[0093] Random mutations at specific positions can be induced using standard methods known to those skilled in the art. For example, synthetic sequences containing degenerate nucleotide compositions at specific positions in the sequence can be used. PCR (polymerase chain reaction) using a mixture of oligonucleotides For example, mutations can be induced by the codons NNK or NNS ( N = adenine, guanine, cytosine, or thymine; K = guanine or thymine; S = adenine or cytosine) introduces all 20 natural amino acids plus a stop codon. Mutations are induced by using the codon VVS (V = adenine, guanine, or cytosine). Cys, Ile, Leu, Met, Phe, Trp, Tyr and Val can be introduced. The remaining 12 natural amino acids are mutagenized to introduce the desired amino acids. If the codon NMS (M = adenine or cytosine) is used, Arg, Cys, Gly, I There is no possibility of introducing Le, Leu, Met, Phe, Trp, or Val, and the remaining 11 To introduce a natural amino acid, Special codons, artificial codons, etc. can be used.

[0094] Site-directed mutagenesis involves targeting a target containing a higher-order structure and / or a peptide or polypeptide directed against that target. Alternatively, the structural information of the wild-type peptide from which the peptide is derived can be used. Specifically, the target KLK5, KLK7, or KLK14, and / or K SPINK for LK5, KLK5 and KLK7, or KLK5 and KLK14 Structural information, including high-level information, of mutant or wild-type SPINK2, or of the complex of both. For example, KLK5 inhibitory activity, K SPINK2 having LK5 / KLK7 inhibitory activity or KLK5 / KLK14 inhibitory activity The mutants are identified and then KLK5, KLK7 or KLK14 and the SPINK2 mutant are isolated. The crystals of the complex are obtained and subjected to X-ray crystal structure analysis. Epitopes on the KLK5, KLK7 or KLK14 molecule to which K2 mutants bind and The paratope on the SPINK2 mutant corresponding to the paratope is identified. The structural information and KLK5 inhibitory activity, KLK5 / KLK7 inhibitory activity, or KLK5 / KLK7 inhibitory activity are In some cases, a correlation can be found between the structure-activity relationship and the K14 inhibitory activity. Based on this, substitution of specific amino acids at specific positions, insertion of amino acids at specific positions, We designed insertions or deletions, and actually demonstrated KLK5 inhibitory activity, KLK5 / KLK7 inhibitory activity, or can confirm KLK5 / KLK14 inhibitory activity.

[0095] In addition, for example, nucleotide building blocks with modified base pair specificity, such as inosine, may be used. This can induce mutations.

[0096] Furthermore, some enzymes, such as Taq DNA polymerase, lack proofreading function and have a high error rate. Randomization is performed using error-prone PCR with DNA polymerase, chemical mutagenesis, etc. Mutagenesis at specific positions is possible.

[0097] KLK5 inhibitory peptide, KLK5 / KLK7 inhibitory peptide, or KLK5 / KLK1 4. Inhibitory peptides were used in bacterial display, yeast display, mammalian cell display, and phagocytic display. page display, ribosome display, nucleic acid display, colony screening Using techniques such as screening of phage libraries and colony libraries, The libraries can be enriched and / or selected from libraries known to those skilled in the art that are suitable for screening methods. Among these libraries, phage libraries include phagemids, colony screens, and For screening, vectors and vectors known to those skilled in the art, such as cosmids, suitable for each library are used. Such vectors can be constructed by any suitable method and can be transformed into either prokaryotic or eukaryotic cells. The recombinant vector may be a virus or viral vector that infects the gene. They can be prepared by methods known to those skilled in the art, such as genetic manipulation.

[0098] Bacterial display is, for example, a portion of the outer membrane lipoprotein (Lpp) of Escherichia coli and outer membrane proteins. This technology fuses the protein OmpA with a desired protein and displays the desired protein on the surface of E. coli. Random mutations are introduced into the nucleotide sequence that encodes the amino acid sequence of a protein. The DNA fragments obtained by the induction are introduced into a vector suitable for bacterial display, and the vector If bacterial cells are transformed with the A library of quality groups can be obtained (Francisco, JA, e t al. (1993) Proc. Natl. Acad. Sci. USA . Volume 90, pages 10444-10448).

[0099] Yeast display targets proteins such as α-agglutinin, which are present on the outer surface of yeast cells. This technology fuses the proteins and displays them on the yeast surface. A putative C-terminal cosylphosphatidylinositol (GPI) anchor attachment signal and It contains and manipulates aqueous regions, signal sequences, activation domains, cell wall domains, etc. By this method, it is possible to display a desired protein on the cell surface of yeast. The nucleotide sequence encoding the amino acid sequence of the protein is randomly mutated. The DNA group to be displayed is introduced into a vector suitable for yeast display, and yeast cells are grown using the vector. Once transformed, the transformed yeast cells display a group of randomly mutated proteins on their cell surface. A library containing the above information can be obtained (Ueda, M. & Tanaka, A., Biot echnol.Adv., Vol. 18, pp. 121-, 2000: Ueda, M. & Ta naka, A., J.Biosci.Bioeng., vol. 90, p. 125~, 2000 (published, etc.).

[0100] Animal cell display has been used to display, for example, platelet-derived growth factor receptor (PDGFR). The transmembrane domain of a membrane protein is fused to the desired protein, and the protein is then transformed into HEK293 or Chinese hamster ovary. This is a technology for displaying desired proteins on the surface of mammalian cells such as CHO cells. Random mutations are introduced into the nucleotide sequence that encodes the amino acid sequence of a protein. The DNA group obtained by the expression is introduced into a vector suitable for animal cell display, and the vector If animal cells are transformed with the gene, randomly mutated proteins will be generated on the surface of the transformed animal cells. A library representing white matter groups can be obtained (Ho M, et al. (200 9) Methods Mol Biol. Vol. 525, pp. 337-352).

[0101] The desired library displayed on cells such as yeast, bacteria, and animal cells is displayed based on the presence of the target molecule. The target molecule can be modified with, for example, biotin or the like. The cells containing the library were incubated with the selected KLK5, KLK7, or KLK14 for a certain period of time. Then, carriers such as magnetic beads are added to separate the cells from the carriers, and the carriers are then washed. The non-specific adsorbed and bound substances are removed, and the carrier (bound KLK5, KLK7 or KLK) is then purified. LK14)-bound peptides, peptide ensembles, or enriched peptide ensembles are presented. Similarly, after adding magnetic beads, magnetic cell separation (MA) can be performed. CS), or cell culture using anti-KLK5 antibody, anti-KLK7 antibody, or anti-KLK14 antibody. After cell staining, FACS was performed to identify KLK5, KLK7, or KLK2 bound to a carrier. KLK14) or a peptide or peptides bound to KLK5, KLK7 or KLK14 A population of cells presenting a collection of peptides or an enriched collection of peptides can be collected. The specific adsorbent and / or binding sites can be, for example, blocked. A blocking step may also be incorporated if appropriate. A vector expressing an enriched peptide, collection of peptides, or collection of enriched peptides is The nucleotide sequence of the polynucleotide inserted into the vector is determined, and The amino acid sequence encoded by the nucleotide sequence can be determined. The vector is then introduced into the host cell again, and the above-mentioned procedure is repeated once or several times as a cycle. The collection of peptides that bind to the target molecule can be more highly enriched.

[0102] In the case of phage display, for example, the phagemid contains, in addition to a plasmid replication origin, It is a bacterial plasmid that contains a second origin of replication derived from a single-stranded bacteriophage. The phagemid-carrying cells are then transfected with M13 or a similar helper bacteriophage. In superinfection with the phagemid, the phagemid can replicate via a single-stranded replication mode. That is, a single-stranded phagocytic protein is contained in an infectious particle coated with a bacteriophage coat protein. In this way, the phagemid DNA is packaged into the infected bacteria. Clone the phagemid as a double-stranded DNA plasmid into a culture of cells that are co-infected with the phagemid. They can be formed from the supernatant as bacteriophage-like particles, respectively. The phage-like particles are then introduced into bacteria having F pili to infect the bacteria with the DNA. By injecting the particles into the host, the particles themselves can be reformed as plasmids.

[0103] A polynucleotide having a nucleotide sequence encoding the amino acid sequence of the test peptide and a bacteriophage coat protein gene. The fusion gene is inserted into the phagemid, bacteria are infected, and the cells are cultured. The peptide is expressed or displayed (synonymous with display) on the bacterium or phage-like particle. ) or as a fusion protein with the coat protein in a phage particle or in the bacterium. It can be produced in the culture supernatant.

[0104] For example, a vector containing the polynucleotide and the bacteriophage coat protein gene gpIII The fusion gene consisting of the gene for the phagemid is inserted into M13 or a similar helper vector. When E. coli is co-infected with the phage, a compound comprising the peptide and the coat protein is produced. The fusion protein can be produced in the culture supernatant of the E. coli.

[0105] Instead of phagemids, various vectors, circular or non-circular, such as viral vectors, can be used. When using a vector, the polynucleotide inserted into the vector can be prepared according to methods known to those skilled in the art. A peptide having an amino acid sequence encoded by a nucleotide sequence of the vector is The vector is expressed or displayed on the cell or virus-like particle into which the vector is introduced, or It can be produced in the culture supernatant of the strain.

[0106] The resulting peptide-expressing library is then subjected to a step of inducing a nucleotide sequence in the presence of a target molecule. The cells can be incubated with or contacted with a target molecule. For example, KLK5, KLK5 and / or KLK7, or KLK5 and / or KLK14 immobilized on a carrier After incubation with the mobile phase containing the library for a certain period of time, the mobile phase is separated from the carrier, and then The carrier is washed to remove non-specific adsorbed and bound substances, and the KL bound to the carrier is removed. K5, KLK5 and / or KLK7, or KLK5 and / or KLK14) The bound peptides, peptide populations, or concentrated peptide populations are recovered by elution. Elution can be achieved by relatively high ionic strength, low pH, moderately denaturing conditions, It can be carried out non-selectively, for example, in the presence of a dihydrotropic salt, or by using KLK5, KLK7, KLK1 Add soluble target molecules such as 4, antibodies that bind to the target molecules, natural ligands, substrates, etc. This can be achieved selectively by competing with the immobilized target molecule. The adhesion site and / or the binding site may be subjected to, for example, a blocking treatment. A suitable method for incorporating a coating step may also be used.

[0107] The peptide, collection of peptides, or concentrated collection of peptides thus obtained is The expression vector is recovered, and the nucleotide sequence of the polynucleotide inserted into the vector is analyzed. The nucleotide sequence can be determined to determine the amino acid sequence encoded by the nucleotide sequence. Alternatively, the vector can be reintroduced into the host cell, and the above-mentioned procedure can be repeated once or several times as a cycle. By repeating the procedure several times, the peptide population that binds to the target molecule can be further enriched. can.

[0108] Ribosome display, for example, encodes a desired protein without a stop codon. By using mRNA and a cell-free protein synthesis system, desired proteins and their corresponding proteins can be synthesized in vitro. It is a technology to synthesize molecules that contain mRNA and ribosomes. mRNA obtained by inducing random mutations in the nucleotide sequence encoding the amino acid sequence By using a group of proteins and a cell-free protein synthesis system, randomly mutated proteins were synthesized using ribosomal Libraries displayed on a display system can be obtained (Mattheakis LC, e t al. (1994) Proc. Natl. Acad. Sci. USA . Volume 91, Issue 19, Pages 9022-9029).

[0109] Nucleic acid display is also called mRNA display, and for example, tyrosyl tRNA By using a linker such as puromycin with a similar structure at the 3' end, the desired protein can be It is a technology to synthesize molecules that are linked together with the mRNA that encodes it and the ribosomes. This technology uses a cell-free protein synthesis system rather than living cells, so synthesis can be performed in a test tube. It is possible to randomly select a nucleotide sequence that encodes the amino acid sequence of a protein. The mRNA group obtained by inducing mutations and linkers such as puromycin, and the cell-free protein By utilizing the protein synthesis system, a group of randomly mutated proteins is displayed on the ribosome. A library containing the nucleotide sequences can be obtained (Nemoto N, et al. (1997) FEBS Lett. Vol. 414, No. 2, pp. 405-408).

[0110] Peptides obtained through cell-free synthesis systems such as ribosome display and nucleic acid display The library expressing the peptides is incubated in the presence of the target molecule or in contact with the target molecule. For example, KLK5, KLK5 and / or KLK7, or The carrier on which KLK5 and / or KLK14 is immobilized is mixed with a mobile phase containing the library. After incubation for a certain period of time, the mobile phase is separated from the carrier, and then the carrier is washed to remove nonspecific and removing the adsorbed and bound substances, and Peptides bound to KLK7, or KLK5 and / or KLK14, and collections of peptides The mixture or concentrated peptide collection can be recovered by elution. High ionic strength, low pH, moderate denaturing conditions, and the presence of chaotropic salts are common. Alternatively, soluble target molecules such as KLK5, KLK7, and KLK14 may be used. Antibodies, natural ligands, substrates, etc. that bind to the target molecule are added to compete with the immobilized target molecule. Non-specific adsorbent and / or binding sites can be selectively removed by For example, blocking treatment is also possible, and the blocking step can be carried out by an appropriate method. It may be incorporated.

[0111] The peptide, collection of peptides, or concentrated collection of peptides thus obtained is Expressed nucleic acids are collected, and in the case of mRNA, the nucleotide sequence is determined after reverse transcription to cDNA. The sequence can then be determined to determine the amino acid sequence encoded by the nucleotide sequence. In addition, mRNA is transcribed from the collected nucleic acid, and the above-mentioned procedure is repeated once or several times as a cycle. By returning the target molecule to the target molecule, the peptide population can be more highly enriched. .

[0112] A peptide, a collection of peptides, or an enriched collection of peptides is pre-treated with affinity By conjugating the peptide to a target molecule, it is possible to efficiently purify the peptide or a collection of peptides. For example, a substrate for a protease can be conjugated to a peptide assembly in advance as a tag. If gated, peptides can be eluted by cleavage with the protease activity. It is possible.

[0113] Based on the obtained sequence information and peptide functions, the obtained clones or libraries Further mutations are induced in the resulting library, and the function (e.g., K KLK5 inhibitory activity, KLK5 / KLK7 inhibitory activity, or KLK5 / KLK14 inhibitory activity) Peptides with improved physical properties (thermal stability, storage stability, etc.), pharmacokinetics (distribution, blood half-life, etc.) It is also possible to obtain chido.

[0114] The resulting peptide has KLK5 inhibitory activity, KLK5 / KLK7 inhibitory activity, or KLK By determining whether or not a KLK5 inhibitor peptide has KLK5 / KLK14 inhibitory activity, KLK5 / KLK7 inhibitory peptide or KLK5 / KLK14 inhibitory peptide Each can be identified.

[0115] Also, KLK5 inhibitory peptides, KLK5 / KLK7 inhibitory peptides, or KLK5 / K The LK14 inhibitor peptide is preferably a peptide having 16 amino acids contained in the amino acid sequence of wild-type SPINK2. A loop structure consisting of Ser at position 30 to Val at position 31, Cys at position 32 and Gly at position 33 β strand (1) and β strand (2) consisting of Ile 57 to Arg 59 The β-sheet is composed of 41 Glu to 51 Gly amino acids, and the α-helix is ​​composed of 41 Glu to 51 Gly amino acids. or similar to or at least partially corresponding to (the location of) them The three-dimensional structure consisting of loop structures, β-sheets, α-helices, etc. that interact with the KLK5 inhibitory activity exhibits KLK5 / KLK7 inhibitory activity, or KLK5 / KLK14 inhibitory activity, respectively Such a three-dimensional structure (whole structure or partial structure) can be used as a part of the index. As the inhibitory peptide of the present invention, a more preferred inhibitory peptide of the present invention is a KLK5 inhibitory peptide, a KLK5 / KLK7 inhibitory peptide, or a K It is also possible to identify LK5 / KLK14 inhibitory peptides.

[0116] Furthermore, the present invention provides a method for the production of KLK5 inhibitory peptides using a SPINK2 mutant library. Identification of KLK5 / KLK7 inhibitory peptides or KLK5 / KLK14 inhibitory peptides Regarding the method, the method described in (75) above can be exemplified. Using the seed compound library, KLK5 inhibitor compounds and KLK5 / KLK7 inhibitor compounds were identified. or a method for identifying a KLK5 / KLK14 inhibitor compound, Use of the peptides or conjugates of the invention as reference compounds, controls, etc. As such a method, the above (76) can be exemplified. In this case, the enzyme inhibitory activity of the test compound is equivalent to or greater than that of the reference compound or control. If the signal is stronger, the compound is judged as positive, and if it is weaker, the compound is judged as negative. An example of a method involving such a comparison is (77) above. On the other hand, the protease activity of KLK5 and optionally KLK7 or KLK14 is measured. In any test involving the steps, the peptide or conjugate of the present invention may be used as a reference compound, Such test methods are also included in the present invention. Such a test is not particularly limited, and the above (78) can be exemplified. methods, testing methods, detection methods, and methods for identifying individuals to whom pharmaceutical compositions are administered (all of which are described below) ) can also be mentioned as a suitable example.

[0117] 4. Nucleic acid molecules encoding the peptides or conjugates of the present invention, vectors containing the same, and Cells containing the same, and methods for producing recombinant peptides or conjugates The present invention relates to a KLK5 inhibitory peptide, a KLK5 / KLK7 inhibitory peptide, or a KLK5 / Contains a nucleotide sequence encoding the amino acid sequence contained in the KLK14 inhibitor peptide Polynucleotides (hereinafter referred to as "nucleic acid molecules encoding KLK5 inhibitory peptides" and " "Nucleic acid molecule encoding a KLK5 / KLK7 inhibitory peptide" or "KLK5 / KLK14 inhibitory peptide" a nucleic acid molecule encoding the anti-inflammatory peptide), a recombinant vector into which the gene has been inserted, Cells into which a gene or vector has been introduced (hereinafter referred to as "cells encoding KLK5 inhibitory peptides") Cells containing a nucleic acid molecule, cells containing a nucleic acid molecule encoding a KLK5 / KLK7 inhibitory peptide " or "cells containing a nucleic acid molecule encoding a KLK5 / KLK14 inhibitory peptide"); KLK5 inhibitory peptide, KLK5 / KLK7 inhibitory peptide, or KLK5 / KLK14 The cells that produce the inhibitory peptide (hereinafter referred to as "KLK5 inhibitory peptide-producing cells" and "K "LK5 / KLK7 inhibitory peptide producing cells" or "KLK5 / KLK14 inhibitory peptide producing cells" The present invention also provides a method for producing a human ovarian cancer cell.

[0118] Nucleic acid molecules encoding the KLK5 inhibitor peptides of the present invention, KLK5 / KLK7 inhibitor peptides a nucleic acid molecule encoding a KLK5 / KLK14 inhibitor peptide; Suitable examples of the child include the following (a1) to (a4), (b1) to (b4), or ( nucleotide sequence set forth in any one of (c1) to (c4) (hereinafter referred to as "KLK") Nucleotide sequence of KLK5 inhibitor peptide, Nucleotide sequence of KLK5 / KLK7 inhibitor peptide nucleotide sequence of the KLK5 / KLK14 inhibitor peptide) or a nucleotide sequence of a KLK5 inhibitory peptide, a KLK5 / KLK7 inhibitory peptide, Nucleotide sequence of the KLK5 / KLK14 inhibitor peptide or the nucleotide sequence of a KLK5 inhibitor peptide, Nucleotide sequence of KLK5 / KLK7 inhibitory peptide or KLK5 / KLK14 inhibitor Examples include peptides consisting of nucleotide sequences: (a1) SEQ ID NOs: 6, 8, 10, 12, 14, 16, 18 and 20 (Figures 14, 16, 18 , 20, 22, 24, 26 and 28) Nucleotide numbers of the nucleotide sequence encoding the amino acid sequence consisting of numbers 1 to 63 nucleotide sequence consisting of nucleotides 1 to 189, or SEQ ID NOs: 5, 7, 9, 11, 13, 15 , 17 and 19 (Figures 13, 15, 17, 19, 21, 23, 25 and 27) a nucleotide sequence set forth in any one of (a2) A nucleotide sequence complementary to the nucleotide sequence described in (a1) and The present invention provides a peptide that hybridizes under favorable conditions and has KLK5 inhibitory activity. a nucleotide sequence encoding an amino acid sequence; (a3) In the nucleotide sequence according to (a1), 1 to 20, 1 to 15, 1 or more up to 10, 1 to 8, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 or Substitution, deletion, addition and / or insertion of two or one nucleotide or nucleotide residue and encoding an amino acid sequence contained in a peptide having KLK5 inhibitory activity. a nucleotide sequence; and (a4) The nucleotide sequence described in (a1) and 60%, 70%, 80%, 85%, 90% , 92%, 94%, 96%, 97%, 98% or 99% or more identical and a KLK5 inhibitor Nucleotide sequence encoding the amino acid sequence contained in the peptide having anti-cancer activity: (b1) any of SEQ ID NOs: 22, 24, 26, and 28 (Figures 30, 32, 34, and 36) The amino acid sequence shown in the figure encodes an amino acid sequence consisting of amino acid numbers 1 to 63. or the nucleotide sequences of SEQ ID NOs: 21, 23, 25 and 27 (Figs. 29, 31, 33 and nucleotide numbers 1 to 189 of the nucleotide sequence set forth in any one of a nucleotide sequence comprising: (b2) A nucleotide sequence complementary to the nucleotide sequence described in (b1) and The peptides hybridized under the appropriate conditions and had KLK5 / KLK7 inhibitory activity. a nucleotide sequence encoding the amino acid sequence involved; (b3) In the nucleotide sequence according to (b1), 1 to 20, 1 to 15, 1 or more up to 10, 1 to 8, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 or Substitution, deletion, addition and / or insertion of two or one nucleotide or nucleotide residue and an amino acid sequence contained in a peptide having KLK5 / KLK7 inhibitory activity. a nucleotide sequence encoding (b4) The nucleotide sequence described in (b1) and 60%, 70%, 80%, 85%, 90% , 92%, 94%, 96%, 97%, 98% or 99% or more identical, and KLK5 / Nucleotide sequence encoding the amino acid sequence contained in the peptide with KLK7 inhibitory activity Column: (c1) an amino acid sequence shown in any one of SEQ ID NOs: 30 and 32 (Figures 38 and 40) a nucleotide sequence encoding the amino acid sequence consisting of amino acid numbers 1 to 63 of the sequence; or , the nucleotide number of the nucleotide sequence set forth in SEQ ID NO: 29 or 31 (Figure 37 or 39) the nucleotide sequence consisting of 1 to 189; (c2) A nucleotide sequence complementary to the nucleotide sequence described in (c1) and Peptides that hybridize under favorable conditions and have KLK5 / KLK14 inhibitory activity a nucleotide sequence encoding the amino acid sequence contained in (c3) In the nucleotide sequence according to (c1), 1 to 20, 1 to 15, 1 or more up to 10, 1 to 8, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 or Substitution, deletion, addition and / or insertion of two or one nucleotide or nucleotide residue and an amino acid sequence contained in a peptide having KLK5 / KLK14 inhibitory activity. a nucleotide sequence encoding (c4) The nucleotide sequence described in (c1) and 60%, 70%, 80%, 85%, 90% , 92%, 94%, 96%, 97%, 98% or 99% or more identical, and KLK5 / Nucleotides encoding amino acid sequences contained in peptides with KLK14 inhibitory activity array.

[0119] Any of the above (a1) to (a4), (b1) to (b4), or (c1) to (c4) or consisting of the amino acid sequence encoded by the nucleotide sequence set forth in any one of the preceding claims. SPINK2 variant peptides containing the amino acid sequence include KLK5, KLK5 and KLK7, or , KLK5 and KLK14, and preferably inhibits the protease activity of the proteases Specifically inhibits activity.

[0120] SEQ ID NOs: 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, and 27 , 29 and 31 (Figs. 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 3 Nucleotides 190 to 195 of the sequences (3, 35, 37, and 39) are wild-type human SPIN corresponds to the nucleotide sequence encoding K2 (SEQ ID NO: 1, FIG. 9: consisting of 63 amino acids) In some embodiments of the present invention, the peptides of the present invention are not nucleotides that This was added for this purpose.

[0121] However, KLK5 inhibitory peptides, KLK5 / KLK7 inhibitory peptides, or KL The nucleic acid molecule encoding the K5 / KLK14 inhibitory peptide is (a1) to (a4), (b1) (c1) to (c4), but are not limited to (b4) to (c4), KLK5 inhibitory activity, SPINK having KLK5 / KLK7 inhibitory activity or KLK5 / KLK14 inhibitory activity The amino acid sequence contained in the two variants, preferably the amino acid sequence shown in SEQ ID NO: 61 (Figure 69) Nucleic acid molecules containing nucleotide sequences encoding KLK5 inhibitory peptides, KLK Nucleic acids encoding KLK5 / KLK7 inhibitory peptides or KLK5 / KLK14 inhibitory peptides It is included in the scope of the molecule.

[0122] The present invention also provides a conjugate of a KLK5 inhibitory peptide, a KLK5 / KLK7 inhibitory peptide, and a or a conjugate of a KLK5 / KLK14 inhibitor peptide. A polynucleotide containing a nucleotide sequence encoding the amino acid sequence contained therein (hereinafter referred to as the They are "nucleic acid molecule encoding a KLK5 inhibitor conjugate" and "KLK5 / KLK7 inhibitor," respectively. "Nucleic acid molecule encoding an inhibitory conjugate" or "KLK5 / KLK14 inhibitory conjugate" a nucleic acid molecule encoding the gene), a recombinant vector into which the gene has been inserted, or cells into which a vector has been introduced (hereinafter referred to as "nucleic acid encoding a KLK5 inhibitor conjugate"). "Cells containing nucleic acid molecules," "cells containing nucleic acid molecules encoding KLK5 / KLK7 inhibitory conjugates," "Cells" or "Cells containing a nucleic acid molecule encoding a KLK5 / KLK14 inhibitory conjugate" KLK5 inhibitory peptide conjugates, KLK5 / KLK7 inhibitory peptide conjugates, Producing a conjugate or a conjugate of a KLK5 / KLK14 inhibitor peptide cells (hereinafter referred to as "KLK5 inhibitor conjugate-producing cells" and "KLK5 / KLK7 cells" respectively) Inhibitory conjugate-producing cells" or "KLK5 / KLK14 inhibitory conjugate-producing cells" ") is also provided.

[0123] Nucleic acid molecules encoding the KLK5 inhibitor conjugates of the present invention, KLK5 / KLK7 inhibitors Nucleic acid molecules encoding conjugates or KLK5 / KLK14 inhibitory conjugates Suitable examples of nucleic acid molecules encoding the above include the following (a1) to (a4) and (b1), respectively: A nucleotide sequence according to any one of (b1) to (b4) or (c1) to (c4) (hereinafter , "nucleotide sequence of KLK5 inhibitor conjugate" and "KLK5 / KLK7 "Nucleotide sequence of inhibitory conjugate" or "KLK5 / KLK14 inhibitory conjugate" the nucleotide sequence of the KLK5 inhibitor conjugate Nucleotide sequence of the KLK5 / KLK7 inhibitor conjugate or K From the nucleotide sequence containing the nucleotide sequence of the LK5 / KLK14 inhibitor conjugate or the nucleotide sequence of the KLK5 inhibitor conjugate, the KLK5 / KLK7 inhibitor Nucleotide sequence of the inhibitor conjugate or the KLK5 / KLK14 inhibitor peptide Examples include those consisting of the nucleotide sequence: (a1) SEQ ID NOs: 34, 36, 38, 40, 42, 44, 46, 48 and 96 (Figure 42, 44, 46, 48, 50, 52, 54, 56 and 106) Nucleotide sequence encoding the amino acid sequence, or SEQ ID NO: 33, 35, 37, 39, 41 , 43, 45, 47 and 95 (Figures 41, 43, 45, 47, 49, 51, 53, 55 and 105); (a2) A nucleotide sequence complementary to the nucleotide sequence described in (a1) and A peptide or conjugate that hybridizes under favorable conditions and has KLK5 inhibitory activity. a nucleotide sequence encoding the amino acid sequence contained in the gate; (a3) In the nucleotide sequence according to (a1), 1 to 20, 1 to 15, 1 or more up to 10, 1 to 8, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 or Substitution, deletion, addition and / or insertion of two or one nucleotide or nucleotide residue and an amino acid sequence contained in the peptide or conjugate having KLK5 inhibitory activity. a nucleotide sequence encoding the nucleic acid sequence; and (a4) The nucleotide sequence described in (a1) and 60%, 70%, 80%, 85%, 90% , 92%, 94%, 96%, 97%, 98% or 99% or more identical and a KLK5 inhibitor Nucleotides encoding the amino acid sequences contained in the antimicrobial peptides or conjugates Otide sequence: (b1) any of SEQ ID NOs: 50, 52, 54, and 56 (Figures 58, 60, 62, and 64) A nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 49, 51 nucleotide sequence shown in any one of Figures 57, 59, 61 and 63 column; (b2) A nucleotide sequence complementary to the nucleotide sequence described in (b1) and and a peptide or a peptide which hybridizes under suitable conditions and has KLK5 / KLK7 inhibitory activity. is a nucleotide sequence encoding the amino acid sequence contained in the conjugate; (b3) In the nucleotide sequence according to (b1), 1 to 20, 1 to 15, 1 or more up to 10, 1 to 8, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 or Substitution, deletion, addition and / or insertion of two or one nucleotide or nucleotide residue and the peptide or conjugate having KLK5 / KLK7 inhibitory activity a nucleotide sequence encoding the amino acid sequence to be (b4) The nucleotide sequence described in (b1) and 60%, 70%, 80%, 85%, 90% , 92%, 94%, 96%, 97%, 98% or 99% or more identical, and KLK5 / Encoding an amino acid sequence contained in a peptide or conjugate having KLK7 inhibitory activity The nucleotide sequence: (c1) an amino acid sequence shown in any one of SEQ ID NOs: 58 and 60 (Figures 66 and 68) The nucleotide sequence encoding the sequence is set forth in SEQ ID NO: 57 or 59 (Figure 65 or 67). nucleotide sequence of; (c2) A nucleotide sequence complementary to the nucleotide sequence described in (c1) and Peptides that hybridize under favorable conditions and have KLK5 / KLK14 inhibitory activity or a nucleotide sequence encoding the amino acid sequence contained in the conjugate; (c3) In the nucleotide sequence according to (c1), 1 to 20, 1 to 15, 1 or more up to 10, 1 to 8, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 or Substitution, deletion, addition and / or insertion of two or one nucleotide or nucleotide residue and a peptide or conjugate having KLK5 / KLK14 inhibitory activity. a nucleotide sequence encoding the amino acid sequence contained therein; and (c4) The nucleotide sequence described in (c1) and 60%, 70%, 80%, 85%, 90% , 92%, 94%, 96%, 97%, 98% or 99% or more identical, and KLK5 / The amino acid sequence contained in the peptide or conjugate having KLK14 inhibitory activity is encoded. The nucleotide sequence to be encoded.

[0124] Any of the above (a1) to (a4), (b1) to (b4), or (c1) to (c4) or consisting of the amino acid sequence encoded by the nucleotide sequence set forth in any one of the preceding claims. SPINK2 variant peptides containing the amino acid sequence include KLK5, KLK5 and KLK7, or , KLK5 and KLK14, and preferably inhibits the protease activity of the proteases Specifically inhibits activity.

[0125] However, KLK5 inhibitory peptides, KLK5 / KLK7 inhibitory peptides, or KL The nucleic acid molecule encoding the K5 / KLK14 inhibitory peptide is (a1) to (a4), (b1) (c1) to (c4), but are not limited to (b4) to (c4), KLK5 inhibitory activity, SPINK having KLK5 / KLK7 inhibitory activity or KLK5 / KLK14 inhibitory activity The amino acid sequence contained in the two variants, preferably the amino acid sequence shown in SEQ ID NO: 61 (Figure 69) a nucleotide sequence encoding the amino acid sequence contained in the conjugate comprising the sequence The nucleic acid molecule encodes a universal KLK5 inhibitor conjugate, KLK5 / KLK7. Nucleic acid molecules encoding inhibitory conjugates or KLK5 / KLK14 inhibitory conjugates The term "nucleic acid molecule" as used herein encompasses nucleic acid molecules encoding the polypeptides.

[0126] To design a nucleotide sequence that encodes an amino acid sequence, the code for each amino acid is One or more of the above-mentioned compounds can be used. Therefore, the single compound possessed by a certain peptide can be used. The base sequence encoding the amino acid sequence may have multiple variations. In selecting the above, a polynucleotide containing the nucleotide sequence or a vector containing the same is is introduced, depending on the codon usage of the host cell for expression. Selecting codons and adjusting the frequency or proportion of multiple codon usage accordingly For example, when E. coli is used as a host cell, The nucleotide sequence may be designed using new codons.

[0127] Nucleic acid molecules encoding the peptides or conjugates of the present invention may be prepared in one or more preparations. The term "operably linked" refers to a nucleic acid that is linked to a nucleic acid sequence. A molecule can be expressed or a nucleotide sequence contained in the molecule can be expressed. The regulatory sequence contains information for transcriptional and / or translational regulation. Regulatory sequences vary by species but generally include a promoter. -35 / -10 box and Shine-Dalgarno sequence in prokaryotes, TAT in eukaryotes A box, CAAT sequence, and 5' capping sequence, etc. including 5' non-coding sequences involved in initiation. Such sequences may include enhancer elements and / or or repressor elements, and the native or mature forms of the proteins to specific compartments inside or outside the host cell. It may contain a translatable signal sequence, leader sequence, etc. for delivering the peptide. In addition, regulatory sequences may include 3' non-coding sequences, such as sequences that regulate transcription termination or transcription. However, the sequence for transcription termination may be particularly If it does not function satisfactorily in a particular host cell, it can be substituted with a sequence appropriate for that cell.

[0128] Promoter sequences include the tet promoter and lacUV5 promoter in prokaryotes. In eukaryotic cells, the SV40 promoter and CMV promoter are used. The following can be exemplified.

[0129] Nucleic acid molecules encoding the peptides or conjugates of the present invention may be used in isolated form, vectors, or other media. vectors or other cloning vehicles (hereinafter simply referred to as "vectors": plasmids, Phagemid, phage, baculovirus, cosmid, etc.) or in a chromosome The vector may be in any form, but is not limited to these forms. Replication sequences and control sequences suitable for the host cell currently used, and nucleic acid sequences obtained by transformation or the like The vector may contain a selectable marker that confers a selectable phenotype on the introduced cell.

[0130] Nucleic acid molecules encoding the peptides or conjugates of the invention, and peptides of the invention or a vector containing the nucleotide sequence of the conjugate. or by transforming the nucleotide sequence into a host cell capable of expressing the nucleotide sequence, by methods known to those skilled in the art. The host cell into which the nucleic acid molecule or vector has been introduced can express the peptide. The host cell may be prokaryotic or eukaryotic, or may be cultured under conditions suitable for expression of the nucleotide sequence. Any of the following may be used: prokaryotic bacteria such as Escherichia coli and Bacillus subtilis; eukaryotic bacteria such as Saccharomyces cerevisiae. yeast such as Pichia pastoris, insect cells such as SF9 and High5, HeLa cells, Examples of such cells include animal cells such as O cells, COS cells, and NS0 cells. By using the peptide as a cell, it is possible to subject the expressed peptide of the present invention to desired post-translational modification. Post-translational modifications include the addition of functional groups such as sugar chains, the addition of peptides or proteins, and amino acid sequences. Examples of the method include the conversion of the chemical properties of the peptide or conjugate of the present invention. It is also possible to artificially introduce desired modifications to the gate. Modified forms of conjugates are also included within the scope of the "peptide" or "conjugate" of the present invention. .

[0131] The present invention also provides a method for producing a peptide or conjugate, the method comprising: Cells containing nucleic acid molecules encoding KLK5 inhibitory peptides (or KLK5 inhibitory conjugates) or KLK5 inhibitory peptide (or KLK5 inhibitory conjugate) producing cells, KL K5 / KLK7 inhibitory peptide (or KLK5 / KLK7 inhibitory conjugate) Cells containing nucleic acid molecules or KLK5 / KLK7 inhibitory peptides (or KLK5 / KLK7 inhibitory peptides) KLK7 inhibitor conjugate) producing cells or KLK5 / KLK14 inhibitor peptide (young or a cell containing a nucleic acid molecule encoding a KLK5 / KLK14 inhibitory conjugate. KLK5 / KLK14 inhibitory peptide (or KLK5 / KLK14 inhibitory conjugate) g) Step 1 of culturing the producing cells, and / or SPINK2 from the culture obtained in step 1 Step 2 includes recovering the mutants. Step 2 includes fractionation, chromatographic techniques known to those skilled in the art. For example, the antibody or its binding site of the present invention described below can be used. Purification by affinity chromatography using fragments is possible.

[0132] In some embodiments of the present invention, the peptide or peptides included in the conjugate are Peptides with intramolecular disulfide bonds have signal In some cases, it is preferable to deliver the compound to a cell compartment having an oxidizing redox environment using a sequence or the like. The oxidative environment is found in the periplasm of gram-negative bacteria such as E. coli and in the extracellular space of gram-positive bacteria. It can be provided by the environment, the lumen of the endoplasmic reticulum of a eukaryotic cell, etc., and under such an environment, In addition, the formation of structural disulfide bonds can be promoted in the cytoplasm of host cells such as E. coli. It is also possible to prepare peptides with intramolecular disulfide bonds. The peptides are obtained directly in a soluble, folded state or recovered in the form of inclusion bodies. Furthermore, host cells with an oxidative intracellular environment can be reconstituted in vitro. The cells are selected and a peptide having an intramolecular disulfide bond is produced in the cytoplasm. On the other hand, if the peptide does not have an intramolecular disulfide bond, it can be oxidized by reducing It can be produced in a cellular compartment that has a reducing environment, for example, the cytoplasm of gram-negative bacteria.

[0133] The peptide or conjugate (or the peptide moiety contained therein) of the present invention may be Other solid-phase peptide synthesis methods, as well as t-butoxycarb 9-Fluorenylmethoxycarbonyl (9-Fluorenylm Organic synthetic peptide synthesis using ethoxycarbonyl (Fmoc) Other methods known to those skilled in the art, such as chemical synthesis and in vitro translation, are also possible. It can be manufactured.

[0134] In some aspects, the present invention provides a peptide contained in the peptide or conjugate of the present invention. The present invention provides antibodies and binding fragments thereof that bind to the peptide. The antibodies include polyclonal antibodies and monoclonal antibodies. The monoclonal antibody may be either an immunoglobulin or a monoclonal antibody. There is no particular limitation as long as it is derived from the antibody. The antibody-binding fragment has antigen-binding activity, i.e., There is no limitation as to the extent to which the compound has binding activity to the peptide, and both heavy and light chains may be used. or one of them or a fragment thereof, lacking the constant region or Fc region, or with other proteins or labeling substances Such antibodies and binding fragments thereof can be prepared by methods known to those skilled in the art. The peptide can be prepared by affinity chromatography. and the use of said peptide in a pharmaceutical composition containing said peptide or in clinical tests, diagnoses, etc. related to its use. The antibody or binding fragment thereof of the present invention is useful for detecting nucleotides, immunoassays, etc. or fragments thereof by affinity chromatography using the peptides of the present invention to which they bind. It can be further refined.

[0135] 5. Pharmaceutical Compositions The present invention also provides pharmaceutical compositions comprising the or conjugates thereof.

[0136] A pharmaceutical composition comprising the peptide of the present invention or a conjugate thereof is By inhibiting or suppressing the expression or function of KLK5, Various drugs that can suppress the onset or worsening of diseases, cure diseases, maintain or improve symptoms, and prevent secondary diseases. Treatment and / or administration of diseases (hereinafter referred to as "KLK5-related diseases" or "KLK5-associated diseases") Examples of diseases associated with KLK5 include Netherton syndrome (NLS). etherton syndrome) (Furio, L., et al. (2015) )PLoS. Genet. Vol. 11, e1005389), atopic dermatitis (Fo rtugno, P., et al. (2012) Hum. Mol. Genet. 21, pp. 4187-4200), rosacea (Yamasaki, K., et al. (20 2007) Nat. Med. Vol. 13, pp. 975-980), UV-induced skin damage (N in, M., et al. (2009) J. Dermatol. Sci. Volume 54 , pp. 17-24), psoriasis (Komatsu, N., et al. (2007) Br. J. Dermatol. Vol. 156, pp. 875-883), asthma (Grunberg, M., et al. (2018) Eur. J. Immunol. 48, 159 2-1594), spinal cord injury (Radulovic, M., et al. (2013) J. Neuropathol. Exp. Neurol. 72, 1072-10 89), cancer (e.g., uterine cancer, bladder urothelial cancer, colon cancer, oral squamous cell carcinoma, breast cancer, head Cervical cancer, melanoma, prostate cancer, glioma, etc.) (Emami, N., et al. (2 007) Mol. Oncol. vol. 1, pp. 269-287), Barrett's esophagus ( Gene Expression Omnibus, accession #GSE1 3083) and the like.

[0137] KLK5 is thought to be the main factor in the development of Netherton syndrome-like skin symptoms. It is an active protease and is also involved in the activation of KLK7 and KLK14. In the stratum corneum of patients with Netherton syndrome and Netherton syndrome model mice, trypsin-like and chymotrypsin-like high protease activity has been observed. The downstream kallikrein family, including K7 and KLK14, is involved in the proteolytic activity of the stratum corneum. In addition to inhibiting KLK5, it also inhibits KLK7 or KLK14. By doing so, it may be possible to more effectively suppress Netherton syndrome-like skin symptoms. It is expected that the SPINK5 mutation associated with Netherton syndrome is There are over 70 mutations listed in the Gene Mutation Database (HGMD) It has been reported that this is associated with the severity of Netherton syndrome. Mutations in genes 1 to 9 are associated with a more severe form of Netherton syndrome. By examining the mutation of SPINK5, the present invention can be used to treat or prevent Netherton syndrome. It is possible to determine whether or not a pharmaceutical composition containing the peptide or conjugate should be used. become.

[0138] The pharmaceutical composition of the present invention contains a therapeutically or prophylactically effective amount of a peptide or conjugate and a drug. and / or a pharmaceutical acceptable diluent, carrier, solubilizer, emulsifier, preservative, and / or adjuvant. It is possible.

[0139] "Therapeutically or prophylactically effective amount" means an amount effective for treating or preventing a particular disease, dosage form, and administration route. It means an amount that produces a preventive effect, and is synonymous with "pharmacologically effective amount."

[0140] The pharmaceutical composition of the present invention may be prepared by adjusting the pH, osmolality, viscosity, transparency, color, isotonicity, sterility, and the like of the composition. The stability, solubility, sustained release, and absorbability of the substance or the peptides and conjugates contained therein, To change, maintain, or preserve permeability, dosage form, strength, properties, shape, etc. The pharmaceutical composition may contain a substance (hereinafter referred to as "substance for formulation"). There are no particular limitations on the substance, as long as it is a pharmacologically acceptable substance. Alternatively, low toxicity is a property that a substance for use in a pharmaceutical preparation preferably possesses.

[0141] Examples of formulation materials include, but are not limited to: Glycine, alanine, glutamine, asparagine, histidine, arginine Amino acids such as nin or lysine, antibacterial agents, ascorbic acid, sodium sulfate or sulfite water Antioxidants such as sodium chloride, phosphoric acid, citric acid, borate buffer, sodium bicarbonate Buffers such as Tris-HCl solution, filling agents such as mannitol and glycine chelating agents such as ethylenediaminetetraacetic acid (EDTA), caffeine, polyvinylpyrrolidone Complexes of lysine, β-cyclodextrin, hydroxypropyl-β-cyclodextrin, etc. bulking agents such as glucose, mannose or dextrin; monosaccharides, disaccharides and glucose; Other carbohydrates such as mannose and dextrin, colorants, flavorings, diluents, emulsifiers and polysaccharides Hydrophilic polymers such as divinylpyrrolidine, low molecular weight polypeptides, salt-forming counterions, chloride ions Alkonium, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methyl Preservatives such as propylparaben, propylparaben, chlorexidine, sorbic acid, or hydrogen peroxide a solvent such as glycerin, propylene glycol, or polyethylene glycol (PEG); Sugar alcohols such as mannitol or sorbitol, suspending agents, sorbitan esters, polyisoprene Polysorbates such as Polysorbate 20 and Polysorbate 80, Triton, surfactants such as tromethamine, lecithin, or cholesterol , stabilizing agents such as sucrose and sorbitol, sodium chloride, potassium chloride, mannitol elasticity enhancers, such as ethanol and sorbitol, transport agents, diluents, excipients, and / or pharmaceutical additives Auxiliary agent.

[0142] The amounts of these substances added for formulation may vary depending on the peptide or conjugate of the present invention. The weight of the peptide is 0.001 to 1000 times, preferably 0.01 to 100 times, more preferably More preferably, it is 0.1 to 10 times.

[0143] A liposome containing the peptide or conjugate of the present invention, The pharmaceutical composition of the present invention also includes a pharmaceutical composition containing a modified form in which the liposome is bound to the It can be enjoyed.

[0144] The excipient or carrier is usually a liquid or solid, and may be water for injection, saline, artificial cerebrospinal fluid, Other substances that can be used in oral or parenteral administration preparations are not particularly limited. Examples of physiological saline include neutral saline and saline containing serum albumin. .

[0145] The buffering agent is T, which is prepared so that the final pH of the pharmaceutical composition is 7.0 to 8.5. ris buffer, acetate buffer adjusted to 4.0 to 5.5, Citrate buffer prepared to a pH of 5.0 to 8.0, An example is a histidine buffer prepared to have a pH of 0.0.

[0146] The pharmaceutical composition of the present invention may be a solid, liquid, suspension, etc. Other examples of pharmaceutical compositions of the present invention include: To form a freeze-dried preparation, sucrose or the like is used. The following excipients can be used:

[0147] The pharmaceutical composition of the present invention can be administered by eye drop, enteral administration, topical administration, and parenteral administration. Any of these may be used, for example, by instillation onto the conjunctiva, intravitreal administration, intravenous administration, intraarterial administration, or intramuscular administration. Examples include intramuscular administration, intradermal administration, subcutaneous administration, intraperitoneal administration, transdermal administration, intraosseous administration, and intra-articular administration. It is possible.

[0148] The composition of such pharmaceutical compositions includes the method of administration, the peptide or conjugate of the present invention. The peptides against KLK5, KLK5 and KLK7, or KLK5 and KLK14 The target of the inhibitory peptide of the present invention can be determined based on the inhibitory activity, binding affinity, etc. It has strong inhibitory activity (IC 50 value or K i value) or high affinity (K D Small value The smaller the dose, the smaller the efficacy can be achieved.

[0149] The amount of the peptide or conjugate of the present invention to be administered is not limited as long as it is a pharmacologically effective amount. The individual's species, type of disease, symptoms, sex, age, chronic illness, and inhibition of the peptide's target It can be determined appropriately depending on the activity, binding affinity, and other factors, but is usually 0.01 to 1000 mg / kg, preferably 0.1 to 100 mg / kg, for 1 to 180 days. It can be administered once, or two or more times daily.

[0150] The pharmaceutical composition may be in the form of an injection (including freeze-dried preparations and drip infusions), a suppository, or a nasal inhaler. Extractable preparations, transdermal absorption preparations, sublingual preparations, capsules, tablets, ointments, granules, aerosols, pills Examples of the formulation include pharmaceutical preparations, powders, suspensions, emulsions, eye drops, and bioimplantable preparations.

[0151] A pharmaceutical composition containing the peptide or conjugate of the present invention as an active ingredient can be administered in combination with other drugs. They can be administered simultaneously or separately. For example, the peptides of the present invention can be administered after other drugs. or administering a pharmaceutical composition containing the compound or a conjugate thereof as an active ingredient. After administering the compound, another drug is administered, or the pharmaceutical composition and the other drug are administered simultaneously. When administered simultaneously, the peptide or conjugate of the present invention and the other drug may be administered simultaneously. The above may be contained in either a single preparation or separate preparations (multiple preparations).

[0152] These other medications may be administered in one, two, three or more doses. These are collectively referred to as the pharmaceutical composition of the present invention and "combined use with other drugs" or is called "combination with other drugs" and refers to the combination of a drug with other drugs in addition to the peptide or conjugate thereof of the present invention. The pharmaceutical compositions of the present invention that contain other drugs or are used in combination with other therapies are also referred to as "other drugs." These are included in the present invention as embodiments of "combination with other drugs" or "combination with other drugs."

[0153] For example, for Netherton syndrome, moisturizers, steroids, antibiotics, etc. For atopic dermatitis, steroid drugs, calcineurin inhibitors, and PDE4 inhibitors are used. , immunosuppressants, IL-4 / IL-13 inhibitors, phototherapy, etc. These include doxycycline, minocycline, azelaic acid, and brimonidine. For psoriasis, TNFα inhibitors, IL-12 / 23 inhibitors, IL-17 inhibitors, PDE4, antimetabolites, calcineurin inhibitors, fumaric acid esters, retinoid preparations, Examples of treatments include steroids, vitamin D3 analogs, and phototherapy. Examples include steroid drugs, β2 agonists, etc. Uterine cancer, bladder urothelial carcinoma, colon cancer For cancers such as oral squamous cell carcinoma, breast cancer, head and neck cancer, melanoma, prostate cancer, and glioma, Examples include various anticancer drugs.

[0154] A method for treating a disease associated with KLK5, comprising administering the peptide or conjugate of the present invention. The present invention provides a method for treating or preventing the disease, a pharmaceutical composition for treating or preventing the disease, and Use of the peptide or conjugate, said peptide or conjugate for the treatment or prevention of said disease The present invention also provides a therapeutic agent comprising the peptide or conjugate. Alternatively, a preventive kit is also included in the present invention.

[0155] Furthermore, a gene encoding the amino acid sequence of the peptide of the present invention or a conjugate thereof is a polynucleotide comprising the nucleotide sequence, a vector comprising the polynucleotide, or A cell containing the polynucleotide or the vector, or a peptide of the present invention or Also provided are pharmaceutical compositions comprising cells expressing the conjugates. The peptide and vector are used for gene therapy of diseases related to KLK5, and the cells express KLK5. These can be applied to cell therapy for diseases related to the immune system using known techniques. For example, such polynucleotides or vectors can be introduced into autologous cells or allogeneic cells (allogeneic cells). By introducing such polynucleoside, cells for cell therapy can be prepared. The peptides and vectors are also encompassed by the present invention as compositions for preparing cell therapy drugs. However, the embodiment of the pharmaceutical composition containing the polynucleotide, vector, cell, etc. of the present invention is the above-mentioned Not limited to:

[0156] The K of the peptide or its conjugate contained as an active ingredient in the pharmaceutical composition of the present invention Animal models can be used as a means of evaluating the therapeutic efficacy of LK5-related diseases. For example, in Netherton syndrome, a mutation in the SPINK5 gene, which is the causative gene, As a model of this condition, SPINK5 gene-deficient mice (Descargues, P., et al. t al. (2004) Nat. Genet. 37, 56-65), SPIN K5 gene conditional knockout mice (Petrova, E., et al. (2019) 8th International Symposium on K allikreins and Kallikrein-Related Peptid Oral presentation title at the symposium: Abstract Book, page 28), Crusty 2 Mouse (Mutagenetix database) and others can be mentioned. Not limited to these.

[0157] 6. Diagnostic Compositions A test or diagnostic composition containing the peptide of the present invention or a conjugate thereof (hereinafter, The present invention provides a diagnostic composition (hereinafter referred to as a "diagnostic composition").

[0158] The diagnostic composition of the present invention is useful for detecting diseases associated with KLK5, KLK5 expression, KLK7 expression, and KLK The present invention is useful for testing or diagnosing K14 expression, etc. Determining or measuring the risk of disease, determining whether or not a disease has occurred, measuring the degree of progression or worsening, Measuring or determining the effect of drug treatment with a pharmaceutical composition containing a peptide or conjugate, This includes measuring or assessing the effectiveness of treatment other than medical treatment, measuring the risk of recurrence, and determining whether or not recurrence has occurred. However, it is not limited to these as long as it is an examination or diagnosis.

[0159] The diagnostic composition of the present invention includes the peptide of the present invention or a conjugate thereof, a composition containing the peptide or a conjugate thereof, and a composition containing the peptide or a conjugate thereof. These are useful for identifying individuals to whom the compositions and pharmaceutical compositions containing them should be administered.

[0160] Such diagnostic compositions may contain pH buffers, osmolality adjusters, salts, stabilizers, preservatives, color developers, etc. The ink may contain additives such as a binder, a sensitizer, and an anti-aggregating agent.

[0161] The present invention relates to a method for testing or diagnosing a disease associated with KLK5, and a method for preparing a diagnostic composition for the disease. use of the peptide of the present invention for the examination or diagnosis of said disease; The present invention also provides a test or diagnostic kit comprising the peptide of the present invention. .

[0162] A preferred testing or diagnostic method involving the peptides of the present invention is sandwich ELISA. However, conventional ELISA, RIA, and ELISPOT (Enzyme-Linked Immunosorbent Assay) ImmunoSpot) method, dot blot method, Ouchterlony method, CIE (Count erimmunoelectrophoresis) method, CLIA (Chemilumi (nescent immunoassay), FCM (Flow Cytometry) For detection, an antibody or a binding fragment thereof, or the peptide or peptides of the present invention can be used. Labeling methods include biotin and the like. or HRP, alkaline phosphatase, fluorophores such as FITC, radioisotopes, etc. Labeling methods that can be used for biochemical analysis such as bell are available. For detection using enzyme labels, TM B(3,3',5,5'-tetramethylbenzidine), BCIP(5 -bromo-4-chloro-3-indolyl phosphate), pN PP (p-nitrophenyl phosphate), OPD (o-phenyl phosphate) enediamine), ABTS(3-Ethylbenzothiazoline- 6-sulfonic acid), SuperSignal ELISA Pico Chemiluminescent Substrate(Thermo Fisher Chromogenic substrates such as QuantaBlu® Fluor genic Peroxidase Substrate(Thermo Fishe) In addition to fluorescent substrates, chemiluminescent substrates can also be used. The determination includes samples derived from humans or non-human animals, as well as artificially processed proteins such as recombinant proteins. A sample can also be provided. Examples of test samples derived from an individual organism include blood and synovial fluid. , ascites, lymph, cerebrospinal fluid, alveolar lavage fluid, saliva, sputum, tissue homogenate supernatant, tissue sections These can include, but are not limited to, the following.

[0163] The sandwich ELISA kit for testing or diagnosis containing the peptide of the present invention includes the Protein standard solutions of clear peptides or conjugates, color reagents, dilution buffers, solid-phase proteins The amount of protein bound to the antigen may be measured. Suitable methods include absorption, fluorescence, luminescence, and RI (Radioisotope) methods. The measurement is performed using an absorbance plate reader, a fluorescence plate reader, or a luminescence plate reader. A detector, an RI liquid scintillation counter, or the like is preferably used.

[0164] Testing or diagnosis is also possible using immunoprecipitation.

[0165] The present invention also relates to a method for detecting KLK5, KLK5 and KLK7, or KLK5 and KLK7 in a test sample. The present invention provides methods for detecting or measuring KLK14 and KLK14. The peptides of the present invention or their conjugates can be used for diagnostic purposes. The test sample is contacted (step 1), and then the KLK bound to the peptide or conjugate is 5. Measure the amount or measurement of KLK5 and KLK7, or KLK5 and KLK14 (process By step 2), KLK5, KLK5 and KLK7, or KLK5 and K In step 1, for example, a method for detecting LK14 is performed by immunizing a subject with the peptide of the present invention. The Fc region of riboglobulin was conjugated to magnetic beads via protein G. In step 2, for example, the magnetic beads are separated and the test sample is added thereto. The soluble proteins precipitated with the beads were analyzed by SDS-PAGE and Western blotting. and detect KLK5, KLK5 and KLK7, or KLK5 and KLK14. In addition to samples derived from humans or non-human animals, this measurement can also be performed using recombinant proteins. Artificially processed samples such as white matter can also be provided. Examples include blood, synovial fluid, ascites, lymphatic fluid, cerebrospinal fluid, alveolar lavage fluid, saliva, sputum, tissue Examples of the tissue homogenate supernatant, tissue slices, etc., but are not limited to these. do not have.

[0166] The detection of KLK5, KLK5 and KLK7, or KLK5 and KLK14 is It can be performed in vitro as well as in vivo. The peptides of the present invention or their conjugates are labeled with environmentally acceptable radionuclides or luminescent materials. In step 1, for example, a subject is given a labeled peptide. or a conjugate thereof. Step 2 may involve administering, for example, imaging such as PET / CT. Using imaging techniques, images are taken of KLK5, KLK5 and / or KLK7, or Examples of such methods include determining or testing the presence of KLK5 and / or KLK14.

[0167] The peptide or its conjugate contained in the diagnostic composition of the present invention is KLK5, KLK 5 and KLK7, or KLK5 and KLK14, preferably KLK5, KLK5 and and KLK7, or KLK5 and KLK14 specific binding activity.

[0168] A method for identifying an individual to whom the pharmaceutical composition of the present invention is to be administered is also encompassed by the present invention. In the method, KLK5, KLK5 and / or KLK7 are detected in a sample derived from the individual. or KLK5 and / or KLK14 are measured, and KLK5, KLK14, or KLK5 and / or KLK14 are detected in the sample. Were K5 and / or KLK7, or KLK5 and / or KLK14 detected? or KLK5, KLK5 and / or KLK detected in a sample from a healthy individual 7, or more KLK5, KLK14 compared to the amount of KLK5 and / or KLK14 If K5 and / or KLK7, or KLK5 and / or KLK14 are detected, In this case, the individual can be determined to be positive. It can be used.

[0169] In a preferred embodiment of the identification method, the individual is affected with a KLK5-related disease. There is or is a risk of

[0170] Furthermore, in one embodiment, the pharmaceutical composition of the present invention is It can be administered to the determined individual.

[0171] 7. Method for isolating KLK5, KLK5 and KLK7, or KLK5 and KLK14 The peptide or conjugate of the present invention is preferably KLK5, KLK5 and / or has specific binding activity to KLK7, or KLK5 and / or KLK14. Therefore, the peptides of the present invention or conjugates thereof can be used to detect KLK5, KLK5 and / or KLK7, or KLK5 and / or KLK14 mixed with other KLKs KLK5, KLK5 and / or KLK7, or KLK5 and / or can specifically isolate KLK14 from peptides or conjugates. K5, KLK5 and / or KLK7, or KLK5 and / or KLK14 The separation is due to relatively high ionic strength, low pH, moderate denaturing conditions, and the presence of chaotropic salts. KLK5, KLK5 and / or KLK7, Alternatively, the protease activity of KLK5 and / or KLK14 is not attenuated. It is preferable. [Example]

[0172] The following examples further illustrate some aspects of the invention. Not limited to:

[0173] In the following examples, unless otherwise specified, each procedure related to genetic manipulation was performed using the "Moreki" method. Molecular Cloning" (Sambrook, J ., Fritsch, E.F. and Maniatis, T., Cold Spring Published by Harbor Laboratory Press in 1982 or 1989 ) and other methods used in experimental manuals by those skilled in the art, or When commercially available reagents or kits were used, the procedures were carried out according to the instructions provided with the products.

[0174] Example 1. Preparation of KLK5 inhibitory peptides (1-1) Construction of KLK5 inhibitor peptide expression vector The nucleotide sequences of each inhibitory peptide (SEQ ID NOs: 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31) and the nucleotide sequence of SPINK2 as a template. PCR was performed using the following primers and KOD-plus-(TOYOBO) ( 94℃ 15 seconds, 60℃ 30 seconds, 68℃ 20 seconds) × 30 cycles) to detect inhibitory peptides. The resulting fragment was amplified. Primer 1: 5'-AAAAGGATCCCTGGACAAACGTGGCCCGCA GTTTGGTCTGTTTAG-3' (SEQ ID NO: 62: Figure 70) Primer 2: 5'-AAAACTCGAGTTAGCCGCCGCACGGACCAT TGCGAATAA-3' (SEQ ID NO: 63: Figure 71) The amplified fragment is subjected to agarose gel electrophoresis, and the desired DNA fragment is excised. DNA extraction was performed using the QIAquick Gel Extraction Kit (QIAGEN). A was prepared. The prepared DNA fragment and pET 32a (Novagen) were digested with restriction enzyme B The mixture was treated with amHI (NEB) and XhoI (NEB) at 37°C for at least 1 hour, and then agarose was added. After gel electrophoresis, the desired DNA fragment was excised and purified using QIAquick PCR Pro. Purification was performed using the LigaFast purification kit (QIAGEN). Rapid DNA Ligation System (Promega) was used. The ligation reaction was carried out by incubating each purified fragment at room temperature for 10 minutes. The ligation solution was added to E. coli JM109 (TOYOBO) and incubated on ice for 30 minutes. After standing, the solution was heat-treated at 42°C for 45 seconds, and then left on ice for 5 minutes. After seeding on a 2YT plate containing ampicillin, E. coli was grown by static cultivation overnight at 37°C. The next day, the transformed E. coli was cultured in Tetrahymena thaliana containing 0.1 mg / mL ampicillin. The bacteria were inoculated into a ferric broth medium (Invitrogen) and cultured overnight at 37°C. QIAprep 96 Turbo Miniprep Kit (Qiagen) was used. Plasmid DNA was recovered (hereinafter referred to as "miniprep treatment") and sequenced. By carrying out this study, the pET 32a_Kex2_KLK5 inhibitor peptide was constructed.

[0175] (1-2) Preparation of KLK5 inhibitor peptides The vector constructed in (1-1) was transformed into E. coli Origami B(DE3) (Novage n) was transformed and cultured at 37°C in 2YT medium containing 0.1 mg / mL ampicillin. After the incubation, IPTG (final concentration 1 mM) was added and the mixture was cultured overnight at 16°C. After harvesting the bacteria by centrifugation at 3,000g for 20 minutes at 4°C, the bacteria were then immersed in BugBuster Master Lysate was prepared using TALON Metal Aff His-tagged target proteins were isolated using Inity Resin (Clontech). Next, Kex2 (Saccharomyces cerevisiae:Ac cession CAA96143) to bind the thioredoxin tag and the desired The protein was cleaved and purified using TALON. (Superdex75 10 / 300 GL) or reversed phase chromatography (YMC- Pack ODS-AM) to prepare 14 KLK5 inhibitory peptides. The amino acid sequences of the derivatives are SEQ ID NOs: 6, 8, 10, 12, 14, 16, 18, and 20. , 22, 24, 26, 28, 30, 32, 34 (Figs. 14, 16, 18, 20, 22, 24 , 26, 28, 30, 32, 34, 36, 38, 40).

[0176] Example 2. Preparation of KLK5, KLK7, and KLK14 (2-1) Construction of human KLK5, human KLK7, and human KLK14 expression vectors Cloning of human pro-KLK5, human pro-KLK7, and human pro-KLK14 The primers and PCR conditions used for the PCR were as follows: PCR using us-(TOYOBO) (94℃ 15 seconds, 60℃ 30 seconds, 68℃ Fragment A was amplified by PCR using PCR amplification with PCR amplification for 10 seconds x 30 cycles. Primer 3: 5'-GGCGATTATAAAGATGACGATGATAAACAC CATCACCACCATC-3' (SEQ ID NO: 64: Figure 72) Primer 4: 5'-GTTTAAACTCAATGATGGTGGTGATGGTGT TTATCATCGTCAT-3' (SEQ ID NO: 65: Figure 73) Next, human pro-KLK5 (Uniprot: Q9Y337), human pro-KLK 7 (Uniprot: P49862), human pro-KLK14 (Uniprot: Q9 The nucleotide sequences encoding P0G3) were used as templates, and the following primers and K PCR using OD-plus (TOYOBO) (94°C for 15 seconds, 60°C for 30 seconds) The fragment was amplified by 30 cycles of PCR amplification (10 cycles at 68°C for 60 seconds, 68°C for 60 seconds).

[0177] Human pro-KLK5 amplification primers Primer 5: 5'-AAAATCTAGAGCCGCCACCATGGCCACAGC TAGACCCCCT-3' (SEQ ID NO: 66: Figure 74) Primer 6: 5'-CGTCATCTTTATAATCGCCGCTGTTGGCCT GGATGGTTTCCTG-3' (SEQ ID NO: 67: Figure 75)

[0178] Human pro-KLK7 amplification primers Primer 7: 5'-AAAATCTAGAGCCGCCACCATGGCCAGATC TCTGCTGCTGCCC-3' (SEQ ID NO: 68: Figure 76) Primer 8: 5'-CGTCATCTTTATAATCGCCCCGGTGTTTCT TCATGGTGTCGTT-3' (SEQ ID NO: 69: Figure 77)

[0179] Human pro-KLK14 amplification primers Primer 9: 5'-AAAATCTAGAGCCGCCACCATGTTCCTCCT CCTCACCGCCCTC-3' (SEQ ID NO: 70: Figure 78) Primer 10: 5'-CGTCATCTTTATAATCGCCCTTGTCGCGC ATGGTCTCCTCGAT-3' (SEQ ID NO: 71: Figure 79)

[0180] The amplified fragments and fragment A, the following primers, and KOD-plus-(TOYOBO The desired DNA fragment was amplified by overlap PCR using the following primers: Primer 5 (SEQ ID NO: 66: Figure 74) or Primer 7 (SEQ ID NO: 68: Figure 76) or Primer 9 (SEQ ID NO: 70: Figure 78) Primer 11: 5'-AAAAGTTTAAACTCAATGATGGTGGTGAT GGTGT-3' (SEQ ID NO: 72: Figure 80)

[0181] Next, mouse pro-KLK7 (Uniprot:Q91VE3), mouse pro-K The nucleotide sequence encoding LK14 (Uniprot: Q8CGR5) was templated. The PCR method using the following primers and KOD-plus- (TOYOBO) was used as the template. The fragments were amplified by 30 cycles of 94°C for 15 seconds, 60°C for 30 seconds, and 68°C for 60 seconds. It was wide.

[0182] Mouse pro-KLK7 amplification primers Primer 12: 5'-AAAATCTAGAGCCGCCACCATGGGAGTGT GGCTGCTGAGCCTG-3' (SEQ ID NO: 73: Figure 81) Primer 13: 5'-AAAAGTTTAAACTCAATGATGGTGGTGAT GGTGCCGGTGGGTCTTCATGGTTTCCATG-3' (SEQ ID NO: 74: Figure 82)

[0183] Mouse pro-KLK14 amplification primers Primer 14: 5'-AAAATCTAGAGCCGCCACCATGTTTCTGC TGCTGATCATCCTG-3' (SEQ ID NO: 75: Figure 83) Primer 15: 5'-AAAAGTTTAAACTCAATGATGGTGGTGAT GGTGGTTGCTCTGCATGGTCCGCTGAA-3' (SEQ ID NO: 76: Figure 8 4)

[0184] The amplified desired DNA fragment was amplified using the restriction enzymes XbaI (NEB) and PmeI (NEB). By cloning using the same method, mammalian cell expression vectors with His tags added to the C-terminus of each gene were obtained. pCMA_pro-hKLK5, pCMA_pro-hKLK7, pCMA_pr o-hKLK14, pCMA_pro-mKLK7, pCMA_pro-mKLK14 The construction was carried out according to the method described in (1-1).

[0185] (2-2) Human KLK5, human pro-KLK7, human pro-KLK14, mouse pro-KLK Expression and purification of o-KLK7 and mouse pro-KLK14 The expression vector constructed in (2-1) was transformed into PEI MAX 40000 (Polysci ences) were used to culture Expi293F cells (Thermo Fisher Scientific) The HisTr tific was transfected into the cells, and the culture supernatant was collected after 3 days of culture. The desired His-protein complex was extracted from the culture supernatant using a sap excel (GE Healthcare). The tagged protein was collected and purified using an Amicon Ultra NMWL 10,000 ( The buffer was exchanged into PBS using a Merck Millipore 5, human pro-KLK7, human pro-KLK14, mouse pro-KLK7, mouse pro-KLK14 was purified separately.

[0186] (2-3) Human KLK5, human KLK7, human KLK14, mouse KLK5, mouse KL Preparation of K7 and mouse KLK14 KLK activation buffer (50 mM Tris-HCl, 150 mM NaCl, 10 20 mM CaCl2, 0.05% (w / w) Brij-35, pH 7.5 0 μg / mL pro-KLK7 or 14 with an equivalent volume of 20 μg / mL thermolys After adding in and incubating at 37°C for a certain period of time, an equal volume of 100mM EDTA was added to activate the Activated human KLK7, activated human KLK14, activated mouse KLK7, activated mouse KLK 14 was prepared.

[0187] In addition, activation buffer (50 mM Tris-HCl, 0.005% (w / w) Br 200 μg / mL of mouse KLK5 (R&D Sy) prepared in ij-35, pH 8.0 Equal volumes of 1000kJ / mL human KLK5 were mixed with 2 μg / mL human KLK5 and incubated at 37°C for 24 hours. Activated mouse KLK5 was prepared by time reaction.

[0188] Example 3. Evaluation of KLK5 inhibitory peptides (3-1) KLK5 inhibitor peptides: human / mouse KLK5, human / mouse KLK7, and human Evaluation of human / mouse KLK14 inhibitory activity The substrate peptide was dissolved in DMSO to a concentration of 10 mM and added to Assay buffer (5 0 mM Tris-HCl, 150 mM NaCl, pH 8.0) before use. Human / mouse KLK5, human / mouse KLK7, or 25 μL of human / mouse KLK14 and inhibitor peptide were mixed and incubated at 37°C for 20 minutes. After the reaction, 50 μL of the substrate diluted with Assay buffer was added. The fluorescent signal was measured using a PerkinElmer chromatograph. The inhibitory peptides were used as follows. The final concentrations of each inhibitor were 0.098 to 1,000 nM. ProteoSave (registered trademark) SS96F black plates (Sumitomo Bakelite Co., Ltd.) were used for reaction and measurement. The company was used.

[0189] Human KLK5 inhibitory activity evaluation: final concentration 10 nM hKLK5, final concentration 100 μM Substrate peptide Boc-Val-Pro-Arg-AMC (R&D Systems), firefly Optical signal excitation 380nm / emission 460nm

[0190] Human KLK7 inhibitory activity evaluation: final concentration 1 μg / mL hKLK7, final concentration 20 μM Substrate peptide Mca-Arg-Pro-Lys-Pro-Val-Glu-Nval- Trp-Arg-Lys(Dnp)-NH2 (R&D Systems: Figure 85, amino The sequence is SEQ ID NO: 77), and the fluorescence signal excitation is 320 nm / emiss ion 405nm

[0191] Evaluation of human KLK14 inhibitory activity: final concentration 0.2 μg / mL hKLK14, final concentration 100μM substrate peptide Boc-Val-Pro-Arg-AMC (R&D Syst ems), fluorescence signal excitation 380nm / emission 460 nm

[0192] Mouse KLK5 inhibitory activity evaluation: final concentration 0.25 μg / mL mouse KLK5 Final concentration: 100 μM Substrate peptide Boc-Val-Pro-Arg-AMC (R&D S systems), fluorescent signal excitation 380nm / emission 460nm

[0193] Mouse KLK7 inhibitory activity evaluation; final concentration 0.5μg / mL mouseKLK7, final Concentration 7 μM Substrate peptide Mca-Arg-Pro-Lys-Pro-Val-Glu- Nval-Trp-Arg-Lys(Dnp)-NH2(R&D Systems: Figure 8 5, amino acid sequence is SEQ ID NO: 77), fluorescent signal excitation 320 nm / emission 405nm

[0194] Mouse KLK14 inhibitory activity assessment: final concentration 0.1 μg / mL mouse KLK14 , final concentration 100 μM substrate peptide Boc-Val-Pro-Arg-AMC (R&D Systems), fluorescent signal excitation 380nm / emission 460nm

[0195] The substrate peptide degradation rate of each inhibitory peptide at each concentration was calculated, and the inhibitory peptide concentration of 0 The decomposition rate was calculated using GraphPad Prism (version 5) with nM as the decomposition rate. 50% inhibitory concentration (IC ) was calculated using GraphPad Software Inc. 50 ) were calculated, and it was found that all inhibitory peptides inhibited human KLK5 enzyme activity at low concentrations. It was revealed that some inhibitory peptides inhibited human KLK7 or human KLK LK14 enzyme activity was inhibited at low concentrations, and some inhibitory peptides inhibited these proteases. The inhibitory peptide showed weak inhibitory activity against mouse KLK5 and KLK (Table 1). 7 or KLK14 showed similar activity (Table 2). 50 To calculate the value, The mean values ​​of three independent experiments were used.

[0196] [Table 1]

[0197] [Table 2]

[0198] (3-2) Cross-reactivity evaluation of KLK5 inhibitor peptides The specificity of the enzyme relative to other proteases was evaluated using the degradation of the substrate peptide as an indicator. (3-1 ) and the sample ( Mix 25 μL of each solution (final concentration 1 μM) and incubate at 37°C for 20 minutes. 50 μL of the substrate diluted with buffer was added and incubated with Enspire (PerkinElm) The fluorescent signal was measured using Assay Buffer. Reaction and measurement were performed using er (50 mM Tris, 150 mM NaCl, pH 8.0). ProteoSave (registered trademark) SS96F black plate (Sumitomo Bakelite Co., Ltd.) The combinations of proteases and substrates used for specificity evaluation were as follows:

[0199] Bovine trypsin inhibitory activity evaluation: final concentration 5 nM trypsin (Pier ce;20233), final concentration 100 μM substrate peptide Boc-Val-Pro-Arg -AMC (R&D Systems; ES011), fluorescent signal excitation 380nm / emission 460nm

[0200] Human trypsin inhibitory activity evaluation; final concentration 1nM trypsin (Sigma -Aldrich;T6424), final concentration 100μM substrate peptide Boc-Val-P ro-Arg-AMC (R&D Systems; ES011), fluorescent signal exci Station 380nm / emission 460nm

[0201] Bovine α-chymotrypsin inhibitory activity assessment; final concentration 10 nM chym otrypsin(Worthington Biochemical Corpora tion;LS001434), final concentration 100 μM substrate peptide Suc-Leu-Le u-Val-Tyr-MCA (Peptide Institute, Inc.; 3120-v: Figure 86, Amino The sequence is SEQ ID NO: 78), and the fluorescence signal excitation is 380 nm / emiss ion 460nm

[0202] Human chymotrypsin inhibitory activity evaluation; final concentration 10nM chymotr ypsin (Sigma-Aldrich; C8946), final concentration 10 μM substrate peptide Suc-Leu-Leu-Val-Tyr-MCA (Peptide Institute, Inc.; 312 0-v: Figure 87, amino acid sequence is SEQ ID NO: 79), fluorescent signal excitation 380nm / emission 460nm

[0203] Human tryptase inhibitory activity assessment: final concentration 1 nM tryptase (Sig ma-Aldrich;T7063), final concentration 100μM substrate peptide Boc-Phe -Ser-Arg-MCA (Peptide Institute, Inc.; 3107-v), fluorescent signal e xcitation 380nm / emission 460nm

[0204] Human chymase inhibitory activity evaluation: final concentration 100 nM chymase (Sig ma-Aldrich;C8118), final concentration 100μM substrate peptide Suc-Leu -Leu-Val-Tyr-MCA (Peptide Institute, Inc.; 3120-v: Figure 87, The amino acid sequence is SEQ ID NO: 79), and the fluorescence signal excitation is 380 nm / em Issue 460nm

[0205] Human plasmin inhibitory activity evaluation; final concentration 50 nM Plasmin (Sigm a-Aldrich;P1867), final concentration 100μM substrate peptide Boc-Val- Leu-Lys-MCA (Peptide Institute, Inc.; 3104-v), fluorescent signal ex Citation 380nm / emission 460nm

[0206] Human thrombin inhibitory activity evaluation; final concentration 1 nM thrombin (Sig ma-Aldrich;T6884), final concentration 100μM substrate peptide Boc-Val -Pro-Arg-AMC (R&D Systems; ES011), fluorescent signal ex Citation 380nm / emission 460nm

[0207] Human neutrophil elastase inhibitory activity; final concentration 0.00001 U / μL Neutrophil elastase(Enzo Life Science ces), final concentration 100 μM substrate peptide Suc(OMe)-Ala-Ala-Pro -Val-MCA (Peptide Institute, Inc.; 3153-v: Figure 88; amino acid sequence is Column number 80), fluorescence signal excitation 380nm / emission 4 60nm

[0208] Human matriptase inhibitory activity assessment: final concentration 1nM matriptase (R&D Systems;3946-SE), final concentration 100μM substrate peptide Boc -Gln-Ala-Arg-AMC (R&D Systems; ES014), fluorescent signal Excitation 380nm / emission 460nm

[0209] Human protein C inhibitory activity assessment: final concentration 100nM protein C (Sigma-Aldrich;P2200), final concentration 100μM substrate peptide Boc -Leu-Ser-Thr-Arg-MCA (Peptide Institute, Inc.; 3112-v: Figure 89, amino acid sequence is SEQ ID NO: 81) Fluorescence signal excitation 380nm / emission 460nm

[0210] Human tPA inhibitory activity evaluation: final concentration 10 nM tPA (Sigma-Aldric h; T0831), final concentration 100 μM substrate peptide Pyr-Gly-Arg-MCA ( Peptide Institute, Inc.; 3145-v), Fluorescent Signal Excitation 380 nm / emission 460nm

[0211] Human uPA inhibitory activity evaluation: final concentration 2 nM uPA (Sigma-Aldrich ;U0633), final concentration 100 μM substrate peptide Pyr-Gly-Arg-MCA (Lab) Peptide Research Institute, Inc.; 3145-v), Fluorescent Signal Excitation 380n m / emission 460nm

[0212] Evaluation of human plasma kallikrein inhibitory activity; final concentration: 0.125 μg / mL plasma kallikrein(R&D Systems;2497-S E), final concentration 100 μM substrate peptide Z-Phe-Arg-MCA (Peptide Corporation) Laboratory; 3095-v), fluorescent signal excitation 380nm / emiss ion 460nm

[0213] Evaluation of human KLK1 inhibitory activity: final concentration 0.1 μg / mL KLK1 (R&D Sy stems;2337-SE), final concentration 100μM substrate peptide Pro-Phe-Ar g-MCA (Peptide Institute, Inc.; 3096-v), fluorescent signal excitation on 380nm / emission 460nm

[0214] Evaluation of human KLK2 inhibitory activity: final concentration 2 μg / mL KLK2 (R&D Syst ems;2337-SE), final concentration 100 μM substrate peptide Pro-Phe-Arg- MCA (Peptide Institute, Inc.; 3096-v), fluorescent signal excitation 380nm / emission 460nm

[0215] Evaluation of human KLK4 inhibitory activity: final concentration 1 μg / mL KLK4 (R&D Syst ems;1719-SE), final concentration 100 μM substrate peptide Boc-Val-Pro- Arg-AMC (R&D Systems; ES011), fluorescent signal excitation Ion 380nm / emission 460nm

[0216] Evaluation of human KLK7 inhibitory activity: final concentration 1 μg / mL KLK7, final concentration 20 μM Substrate peptide Mca-Arg-Pro-Lys-Pro-Val-Glu-Nval-T rp-Arg-Lys(Dnp)-NH2 (R&D Systems: Figure 85, Amino Acid The sequence is SEQ ID NO: 77), and the fluorescence signal excitation is 320 nm / emissivity. on 405nm

[0217] Evaluation of human KLK8 inhibitory activity; final concentration 5nM KLK8 (UniProt: O60 259, prepared by the inventors), final concentration 100 μM substrate peptide Boc-Val-Pro- Arg-AMC (R&D Systems; ES011), fluorescent signal excitation Ion 380nm / emission 460nm

[0218] Evaluation of human KLK12 inhibitory activity: final concentration 0.1 μg / mL KLK12 (R&D Systems;3095-SE), final concentration 100μM substrate peptide Boc-Val- Pro-Arg-AMC (R&D Systems; ES011), fluorescent signal exc Illumination 380nm / emission 460nm

[0219] Evaluation of human KLK13 inhibitory activity: final concentration 0.5 μg / mL KLK13 (R&D Systems;2625-SE), final concentration 100μM substrate peptide Boc-Val- Pro-Arg-AMC (R&D Systems; ES011), fluorescent signal exc Illumination 380nm / emission 460nm

[0220] Evaluation of human KLK14 inhibitory activity: final concentration 0.2 μg / mL hKLK14, final concentration 100μM substrate peptide Boc-Val-Pro-Arg-AMC (R&D Syst ems), fluorescence signal excitation 380nm / emission 460 nm

[0221] As in (3-1), the degradation of the peptide substrate was used as an indicator to determine the activity of KLK5 inhibitor peptides. Cross-reactivity with other proteases was evaluated. Some inhibitory peptides were tested using Chymotryp The inhibitor peptide showed weak cross-reactivity with sin at a final concentration of 1 μM (IC 50 The value is less than 1 μM. Most of the inhibitory peptides were KLKn (n=1, 2, 4, 5, 7, 8, 12, or 14). It did not show any inhibitory activity against any proteases other than those mentioned above (Fig. 3). The inhibitory peptide exhibited inhibitory activity against KLK4 or KLK12 at a final concentration of 1 μM. (IC 50 Although the inhibitory activity of KLK5, KLK7 and KLK8 was less than 1 μM, most inhibitory peptides It does not exhibit protease inhibitory activity against KLKn except for LK14, and the inhibitory peptides are highly It was revealed that it has specificity.

[0222] (3-3) Evaluation of KLK5 binding activity of KLK5 inhibitor peptides To measure the binding affinity of the KLK5 inhibitor peptides, a BIAcore T 200 (G Surface plasmon resonance analysis was performed using a fluorochrome plated polymerase (FGP) manufactured by E Healthcare. Single-stranded DNA The sensor chip cap (GE healthcare) on which the Capture of complementary strand DNA by hybridization with streptavidin conjugate Next, EZ-Link NHS-PEG4-Biotin (Thermo F Biotinylated KLK5 was incubated at a flow rate of 10 μL / min using a centrifuge tube (Division of Biological Sciences). Approximately 10RU was immobilized by capturing at 1 min. Two-fold serial dilutions of KLK5 inhibitor peptide (0.625-10 nM) were run as analytes. The flow rate was 30 μL / min. BIAcore T 200 Evaluation In software (version 2.0), simple one-to- Single cycle with one Langmuir binding model The dissociation constant K D teeth k off / k on Furthermore, the Biotin CAPture Ki t (GE healthcare) with the regeneration buffer provided. The sensor chip CAP is regenerated and biotinylated KLK5 is repeatedly captured. By doing so, multiple KLK5 inhibitory peptides were measured.

[0223] All 14 KLK5 inhibitor peptides tested had K values ​​below 1 nM. D The value is shown It was revealed that the binding strength was very strong (Table 3(A)).

[0224] [Table 3(A)]

[0225] (3-4) Evaluation of KLK5-binding activity of KLK5 inhibitory peptide-Fc fusion Measure the binding affinity of the KLK5 inhibitor peptide-Fc fusion prepared in (5-2) below. To investigate the effect of surface plasma on the chromatogram, a BIAcore T 200 (GE Healthcare) was used. Montmron resonance analysis was performed.

[0226] Sensor Chip CM5 (GE he) with immobilized anti-human IgG (Fc) antibody The KLK5 inhibitor peptide-Fc fusion was carried out at a flow rate of 20 μL / min. Approximately 30-50RU were immobilized by immersion in HBS-EP. Serially diluted KLK5 (0.625-10 nM) was used as the analyte at a flow rate of 30 μL / min. The BIAcore T 200 Evaluation software ( In version 2.0), simple one-to-one Langm Single cycle kinetics with uir binding model The dissociation constant K D is k off / k on of The ratio was calculated. (GE healthcare) Regeneration buffer was used to The Sensor Chip CM5 with immobilized human IgG (Fc) antibody was regenerated and used for KLK. By repeatedly capturing KLK5 inhibitor peptide-Fc fusions, multiple KLK5 inhibitor peptides were identified. The binding activity of KLK5 to the peptide Fc fusion was measured.

[0227] All 14 KLK5 inhibitor peptide-Fc fusions measured had a K value of less than 1 nM. D Value It was revealed that the binding strength was very strong (Table 3(B)).

[0228] [Table 3(B)]

[0229] Example 4. Analysis of KLK5 inhibitor peptide using X-ray crystal structure (4-1) Preparation of KLK5 / KLK5 inhibitor peptide complex According to the methods described in (1-2) and (2-2), the amino acid sequence shown in SEQ ID NO: The KLK5 inhibitor peptides K51034 and KLK5, each having the formula: After mixing the two under the conditions of 10 mM Tris-HCl, 150 mM NaCl, pH 8.0, Complexes were isolated by gel filtration chromatography (Superdex 200 10 / 300 GL). The product was isolated and purified.

[0230] (4-2) X-ray crystal structure analysis The complex solution prepared in (4-1) was concentrated to 12 mg / mL and then added to the reservoir solution (0. 2M Magnesium Chloride hexahydrate,20%PEG 3350) in a 1:1 ratio and crystallized by vapor diffusion. The resulting cubic single crystals The tissue was immersed in a reservoir solution containing 20% ​​glycerol and then frozen in liquid nitrogen. The frozen crystal was irradiated with X-rays under a cryo-air flow, and diffraction images were obtained (Hypixel 600 0HE / MicroMax007). Analysis using CrysAlisPro revealed that We obtained scaling data with a high resolution of 1.7 Å. KLK5 alone (PDB ID: 2P SX) and SPINK2 alone (PDB ID: 2JXD) as templates by molecular replacement. After the phase was determined and the structure was refined, the KLK5 / peptide K51034 was determined at a resolution of 1.8 Å. The complex crystal was determined. The unit cell contained one molecule each of KLK5 and SPINK2. For the SPINK2 molecule, we identified KLK5 based on the sequence information and the observed electron density. A partial molecular model was constructed that includes the interaction site with the KLK5 inhibitor peptide K5. It was found that 1034 binds to the region containing the KLK5 enzyme active center (Fig. 4).

[0231] Example 5. Preparation of KLK5 inhibitory peptide Fc fusion (5-1) Construction of KLK5 inhibitory peptide-Fc fusion expression vector The nucleotide sequences of each inhibitory peptide (SEQ ID NOs: 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31) as templates, and the following primers and KOD PCR method using -plus- (TOYOBO) (94℃ 15 seconds, 60℃ 30 seconds, The inhibitory peptide fragment was amplified by PCR using 30 cycles of PCR amplification at 68°C for 20 seconds. Primer 16: 5'-AGATGGGTGTTGTCTGATGACGACGGCCC TCAGTTCGGCCTGTTC-3' (SEQ ID NO: 81: Figure 89) Primer 17: 5'-GCAGGGGCCATTCCGGAT-3' (SEQ ID NO: 82: Figure 90)

[0232] PCR (94℃) using the following primers and KOD-plus-(TOYOBO) Fragment B was amplified by 30 cycles of PCR amplification at 60°C for 15 seconds, 60°C for 30 seconds, and 68°C for 10 seconds. . Primer 18: 5'-AAAATCTAGAGCCGCCACCATGAAGCACC TGTGGTTCTTTCTGCTGCT-3' (SEQ ID NO: 83: Figure 91) Primer 19: 5'-AGACAACACCCATCTAGGAGCGGCCACCA GCAGCAGAAAGAACC-3' (SEQ ID NO: 84: Figure 92)

[0233] The Fc region of human IgG1 (SEQ ID NO: 87) was used as a template, and the following primers and KOD-p PCR using lus-(TOYOBO) (94℃ 15 seconds, 60℃ 30 seconds, 68 Fragment C containing the Fc region of human IgG1 was amplified by PCR using PCR amplification at 100°C for 30 seconds (30 cycles). Primer 20: 5'-ATCCGGAATGGCCCCTGCGAACCCAAGAG CTGCGAC-3' (SEQ ID NO: 85: Figure 93) Primer 21: 5'-AAAAGTTTAAACTCATTTGCCGGGGCTCA G-3' (SEQ ID NO: 86: Figure 94)

[0234] The inhibitory peptide fragment amplified above, fragment B, fragment C, primer 18, and primer 21 and the desired DNA was obtained by overlap PCR using KOD-plus- (TOYOBO). The DNA fragment was amplified.

[0235] Furthermore, cloning using the restriction enzymes XbaI (NEB) and PmeI (NEB) The mammalian cell expression vector pCMA_KLK5 inhibitory peptide-Fc fusion was constructed using the above. The procedure was carried out in accordance with the method described in (1-1).

[0236] (5-2) Preparation of KLK5 inhibitory peptide-Fc fusion The expression vector constructed in (5-1) was transformed into PEI MAX 40000 (Polysci ences) were used to culture Expi293F cells (Thermo Fisher Scientific) The cells were transfected into MabSe (tificial), and the culture supernatant was collected after 6 days of culture. The desired Fc was extracted from the culture supernatant using the lect SuRe (GE Healthcare). The combined material was collected and analyzed using Amicon Ultra NMWL 10,000 (Merck Mi The KLK5 inhibitor peptide F was then buffer exchanged into PBS using a PBS buffer (Lithium Ion Cells). The clones containing the glycosylation sequence in the KLK5 inhibitor peptide were The glycosylation sequence was removed by group substitution, and the ID "dN" was added to indicate the deglycosylated form. Furthermore, modification of the glycosylation sequence does not affect any activity, such as KLK5 inhibitory activity or cross-reactivity.

[0237] (5-3) Construction of KLK5 inhibitory peptide-Fc fusion D1-K50055-Fc expression vector Construction Using the nucleotide sequence of the KLK5 inhibitor peptide K50055 (SEQ ID NO: 7) as a template, PCR (94℃) using the following primers and KOD-plus-(TOYOBO) 15 seconds, 60°C 30 seconds, 68°C 20 seconds) × 30 cycles) to isolate inhibitory peptide fragments. Amplified.

[0238] Primer 22: 5'-AGATGGGTGTTGTCTGACGGCCCTCAGT TCGGCCTGTTC-3' (SEQ ID NO: 94: Figure 104) Primer 17: 5'-GCAGGGGCCATTCCGGAT-3' (SEQ ID NO: 82 :Figure 90)

[0239] The inhibitory peptide fragment amplified above, fragment B and fragment C amplified in (5-1), and primers 18, overlap using primer 21 and KOD-plus-(TOYOBO) The desired DNA fragment was amplified by PCR.

[0240] Furthermore, cloning using the restriction enzymes XbaI (NEB) and PmeI (NEB) The mammalian cell expression vector pCMA_KLK5 inhibitory peptide-Fc fusion was constructed using the above. The procedure was carried out in accordance with the method described in (1-1).

[0241] (5-4) Preparation of KLK5 inhibitory peptide-Fc fusion complex D1-K50055-Fc The expression vector constructed in (5-3) was transformed into PEI MAX 40000 (Polysci ences) were used to culture Expi293F cells (Thermo Fisher Scientific) The cells were transfected into MabSe (tificial), and the culture supernatant was collected after 6 days of culture. The desired Fc was extracted from the culture supernatant using the lect SuRe (GE Healthcare). The combined material was collected and analyzed using Amicon Ultra NMWL 10,000 (Merck Mi The KLK5 inhibitor peptide F was then buffer exchanged into PBS using a PBS buffer (Lithium Ion Cells). c The fusion D1-K50055-Fc was prepared.

[0242] Example 6. Evaluation of KLK5 inhibitory peptide Fc fusions (6-1) Human / mouse KLK5 and human / mouse KLK5 inhibitory peptide-Fc fusion Evaluation of inhibitory activity of K7 and human / mouse KLK14 According to the method described in Example 3-1, human / mouse KLK5 inhibitor peptide-Fc fusion Evaluated inhibitory activity against human KLK5, human / mouse KLK7, and human / mouse KLK14 The rate of substrate peptide degradation for each inhibitory peptide-Fc fusion at each concentration was calculated. The decomposition rate at a peptide-Fc fusion concentration of 0 nM was set as 100%. m (version 5.0; GraphPad Software Inc.) 50% inhibitory concentration (IC 50 ) was calculated, and it was found that all inhibitory peptide-Fc fusions exhibited low concentrations. It was revealed that the enzyme activity of human KLK5 was inhibited at low concentrations (Table 4, Figure 5, Figure 107). Some inhibitory peptide-Fc fusions inhibit the enzyme activity of human KLK7 or human KLK14 at low concentrations. Furthermore, some inhibitory peptide-Fc fusions have weak inhibitory activity against these proteases. The inhibitory peptide-Fc fusion showed activity against mouse KLK5, KLK7, or KLK14. The same activity was observed even when the IC 50 The values ​​were calculated based on three independent experiments. The average value was used.

[0243] [Table 4]

[0244] [Table 5]

[0245] (6-2) Cross-reactivity of KLK5 inhibitory peptide-Fc fusion Similar to the results in (3-2), some inhibitory peptide-Fc fusions inhibited bovine trypsin Weak cross-reactivity against chymotrypsin and plasmin at a final concentration of 1 μM inhibitor peptide showed the effect (IC 50 The value was less than 1 μM, but most of the inhibitory peptides were not related to any of the KLKs. The inhibitory peptides were also shown to have no inhibitory activity against the proteases (Fig. 6). The complex exhibited inhibitory activity against KLK4 or KLK12 at a final concentration of 1 μM (IC 50 The value is Although most inhibitory peptide-Fc fusions inhibited KLKs other than KLK7 or KLK14, the inhibitory peptide-Fc fusions inhibited KLKs less than 1 μM. Therefore, like the inhibitory peptide, it did not show any protease inhibitory activity against s. It was revealed that the peptide-Fc fusion had high specificity.

[0246] (6-3) Evaluation of KLK5 inhibitory activity of KLK5 inhibitor peptide-Fc fusions using peptide substrates Titer (inhibitory constant K i (Calculation of The inhibitory activity of the KLK5 inhibitory peptide-Fc fusion against human KLK5 was evaluated. number K i The substrate peptide Boc-Val-Pro-Arg-AMC (R&D S systems; ES011) was dissolved in DMSO to a concentration of 10 mM, and the assay was Dilute with 50 mM Tris-HCl, 150 mM NaCl, pH 8.0 The final concentration of human KLK diluted in assay buffer was 25-200 μM. 25 μL of 5 and KLK5 inhibitor peptide Fc fusion were mixed and incubated at 37°C for 20 minutes. After incubation, 50 μL of the substrate diluted with Assay buffer was added and Enspire Fluorescence signal (excitation 380nm / emission 460nm) The final concentration of human KLK5 was 10 nM, and the final concentration of KLK5 inhibitor peptide-Fc fusion was 0.5 to 25 nM, ProteoSave (registered trademark) SS96F black plate for reaction and measurement (Sumitomo Bakelite Co., Ltd.) was used.

[0247] The rate of substrate peptide degradation for each inhibitory peptide-Fc fusion at each concentration was calculated. The decomposition rate of each inhibitory peptide Fc fusion was calculated by setting the decomposition rate at 0 nM of the inhibitory peptide Fc fusion as 100%. The KLK5 inhibitory activity of each compound was evaluated (Figure 102). ion 5.0; GraphPad Software Inc.) According to the Aelis-Menten equation, the maximum reaction rate at an enzyme concentration of 10 nM is V max and Michaelis constant K m Furthermore, M was calculated using GraphPad Prism. According to the Orrison equation, the inhibition constant K at a substrate concentration of 100 μM i As a result of calculating Both KLK5 inhibitory peptide-Fc fusions inhibited human KLK5 enzyme activity at low concentrations. It was revealed that (Table 6). i The values ​​were calculated using the average of three independent experiments. Used.

[0248] [Table 6]

[0249] (6-4) Evaluation of KLK5 inhibitory activity of KLK5 inhibitor peptide-Fc fusions using protein substrates Price Human Desmoglein1 and human Desmocollin1 were used as protein substrates. The KLK5 inhibitory activity of the KLK5 inhibitory peptide-Fc fusion was evaluated using Assay b. Human KLK5 and each KLK5 inhibitor peptide-Fc fusion (D3-K50) diluted in water 032dN-Fc, D3-K50055-Fc, D3-K51072-Fc, or D3- K50016dN-Fc) and incubated at 37°C for 1 hour. The protein substrate diluted with PBS was added and reacted at 37°C for 4 hours. The enzyme reaction was stopped by adding SDS sample buffer and treating at 99°C for 5 minutes. Subsequently, SDS-PAGE (reducing conditions) and Western blot analysis were performed to confirm the The degradation of the protein substrate was evaluated using each substrate and enzyme, each inhibitor peptide Fc fusion, Western The antibody combinations used for n blot analysis are as follows:

[0250] Evaluation using human desmoglein 1; final concentration 1 μM hKLK5, final concentration 0.001-10 μM inhibitory peptide-Fc fusion, final concentration 1 μM Recombinant t Human Desmoglein-1 Fc Chimera Protein( R&D Systems), Desmoglein 1 Antibody (aa471 -499) (LSBio) and Anti-Rabbit IgG, HRP-Linked d F(ab')2 Fragment Donkey(GE healthcare)

[0251] Evaluation using human desmocollin1; final concentration 0.2 μM hKLK5 Final concentration 0.0002~2μM Inhibitory peptide Fc fusion, final concentration 2μM Recombi nant Human Desmocollin-1 Protein with C- terminal His tag(R&D Systems), Penta His HRP Conjugate (QIAGEN)

[0252] Human Desmoglein1 and human Desmocollin1 are expressed in the absence of human KLK5. In the presence of human KLK5, it was not degraded, but was completely degraded. The results of preincubation with KLK5 inhibitor peptide-Fc fusion and evaluation showed that all inhibitors Peptide-Fc fusions also bind to human Desmoglein 1 and human Desmoglein 2 by the human KLK5 enzyme. It was revealed that the inhibitory effect of human KLK5 on the degradation of smocollin1 was inhibited. At equal concentrations of the peptide-Fc fusion, human Desmoglein 1 and human Degradation of Desmocollin1 was completely inhibited (Figure 103).

[0253] Example 7. Transduction of KLK5 inhibitor peptide-Fc fusion in Netherton syndrome model mice Inhibits the increase in transepidermal water loss (TEWL) (7-1) Netherton syndrome model mouse Crusty2 mice carrying a mutation in SPINK5, the gene responsible for Netherton syndrome Crusty2 is a mouse model of Netherton syndrome, and its homozygous counterpart, Crusty2 ( + / +) presents with skin symptoms (Mutagenetix database).

[0254] (7-2) T of KLK5 inhibitory peptide-Fc fusion in Netherton syndrome model mice EWL increase suppression effect Crusty2(+ / -) mice and Crusty2(+ / +) mice were artificially inseminated. The resulting offspring (Crusty2(+ / -) mice or Crusty2(+ / +) mice) were then mixed and analyzed. The KLK5 inhibitory peptide Fc fusion protein D1-K50 prepared in (5-4) was used. 055-Fc was evaluated. From day 0 or 1 after birth, PBS or 100 mg / kg D1- K50055-Fc was administered subcutaneously every other day for 4 weeks. Three times the dose of D1-K50055-Fc was administered as a stimulant. T100RS, Asahi Techno Lab Co., Ltd.) at 2 and 4 weeks after administration. The TEWL in the skin of the back or buttocks was measured (Fig. 7). Regardless of the K50055-Fc administration group, the number of Crusty2(+ / -) mice was 9, and the number of Crusty2(+ / -) mice was 1. There were 12 usty2(+ / +) mice.

[0255] Statistically, Crusty2(+ / +) mice showed significantly higher levels of β-glucan than Crusty2(+ / -) mice. A significant increase in TEWL was confirmed, and it was more pronounced in the 4-week-old rats than in the 2-week-old rats. Four weeks after administration, the D1-K50055-Fc group showed significantly more severe back and ankle pain than the PBS-administered group. A statistically significant decrease in TEWL was observed in both the more severe buttocks. Therefore, although SPINK5 mutations cause increased TEWL in mice, In contrast, it was revealed that D1-K50055-Fc exhibited an inhibitory effect, and Peptides and conjugates of the present invention containing 55-Fc are useful for the treatment of skin symptoms of Netherton syndrome. It has been shown to be useful in reducing [Industrial Applicability]

[0256] The peptides and conjugates provided by the present invention, as well as pharmaceutical compositions containing them, are It is useful in treating various diseases. [Sequence List Free Text]

[0257] SEQ ID NO: 1: Amino acid sequence of human SPINK2 (Figure 9) SEQ ID NO: 2: Amino acid sequence of human KLK5 (Figure 10) SEQ ID NO: 3: Amino acid sequence of human KLK7 (Figure 11) SEQ ID NO: 4: Amino acid sequence of human KLK14 (Figure 12) SEQ ID NO: 5: Nucleotide sequence of KLK5 inhibitor peptide K50032 (Figure 13) SEQ ID NO: 6: Amino acid sequence of KLK5 inhibitor peptide K50032 (Figure 14) SEQ ID NO: 7: Nucleotide sequence of KLK5 inhibitor peptide K50055 (Figure 15) SEQ ID NO: 8: Amino acid sequence of KLK5 inhibitor peptide K50055 (Figure 16) SEQ ID NO: 9: Nucleotide sequence of KLK5 inhibitor peptide K51072 (Figure 17) SEQ ID NO: 10: Amino acid sequence of KLK5 inhibitor peptide K51072 (Figure 18) SEQ ID NO: 11: Nucleotide sequence of KLK5 inhibitor peptide K50016 (Figure 19) SEQ ID NO: 12: Amino acid sequence of KLK5 inhibitor peptide K50016 (Figure 20) SEQ ID NO: 13: Nucleotide sequence of KLK5 inhibitor peptide K51034 (Figure 21) SEQ ID NO: 14: Amino acid sequence of KLK5 inhibitor peptide K51034 (Figure 22) SEQ ID NO: 15: Nucleotide sequence of KLK5 inhibitor peptide K50062 (Figure 23) SEQ ID NO: 16: Amino acid sequence of KLK5 inhibitor peptide K50062 (Figure 24) SEQ ID NO: 17: Nucleotide sequence of KLK5 inhibitor peptide K51090 (Figure 25) SEQ ID NO: 18: Amino acid sequence of KLK5 inhibitor peptide K51090 (Figure 26) SEQ ID NO: 19: Nucleotide sequence of KLK5 inhibitor peptide K50098 (Figure 27) SEQ ID NO: 20: Amino acid sequence of KLK5 inhibitor peptide K50098 (Figure 28) SEQ ID NO: 21: Nucleotide sequence of KLK5 / KLK7 inhibitory peptide K51028 (Figure 2 9) SEQ ID NO: 22: Amino acid sequence of KLK5 / KLK7 inhibitory peptide K51028 (Figure 30) SEQ ID NO: 23: Nucleotide sequence of KLK5 / KLK7 inhibitory peptide K51005 (Figure 3 1) SEQ ID NO: 24: Amino acid sequence of KLK5 / KLK7 inhibitory peptide K51005 (Figure 32) SEQ ID NO: 25: Nucleotide sequence of KLK5 / KLK7 inhibitory peptide K50031 (Figure 3 3) SEQ ID NO: 26: Amino acid sequence of KLK5 / KLK7 inhibitory peptide K50031 (Figure 34) SEQ ID NO: 27: Nucleotide sequence of KLK5 / KLK7 inhibitory peptide K51057 (Figure 3 5) SEQ ID NO: 28: Amino acid sequence of KLK5 / KLK7 inhibitory peptide K51057 (Figure 36) SEQ ID NO: 29: Nucleotide sequence of KLK5 / KLK14 inhibitor peptide K51069 (Figure 37) SEQ ID NO: 30: Amino acid sequence of KLK5 / KLK14 inhibitor peptide K51069 (Figure 38 ) SEQ ID NO: 31: Nucleotide sequence of KLK5 / KLK14 inhibitor peptide K50015 (Figure 39) SEQ ID NO: 32: Amino acid sequence of KLK5 / KLK14 inhibitor peptide K50015 (Figure 40 ) SEQ ID NO: 33: Nucleotide sequence of KLK5 inhibitory peptide-Fc fusion D3-K50032dN-Fc Otide sequence (Figure 41) SEQ ID NO: 34: Amino acid sequence of KLK5 inhibitory peptide Fc fusion D3-K50032dN-Fc Acid sequence (Figure 42) SEQ ID NO: 35: Nucleotide sequence of KLK5 inhibitory peptide Fc fusion D3-K50055-Fc Code arrangement (Figure 43) SEQ ID NO: 36: Amino acid sequence of KLK5 inhibitory peptide Fc fusion D3-K50055-Fc Column (Figure 44) SEQ ID NO: 37: Nucleotide sequence of KLK5 inhibitory peptide Fc fusion D3-K51072-Fc Code arrangement (Figure 45) SEQ ID NO: 38: Amino acid sequence of KLK5 inhibitory peptide Fc fusion D3-K51072-Fc Column (Figure 46) SEQ ID NO: 39: Nucleotide sequence of KLK5 inhibitory peptide-Fc fusion D3-K50016dN-Fc Otide sequence (Figure 47) SEQ ID NO: 40: Amino acid sequence of KLK5 inhibitory peptide Fc fusion D3-K50016dN-Fc Acid sequence (Figure 48) SEQ ID NO: 41: Nucleotide sequence of KLK5 inhibitory peptide-Fc fusion D3-K51034-Fc Code arrangement (Figure 49) SEQ ID NO: 42: Amino acid sequence of KLK5 inhibitory peptide Fc fusion D3-K51034-Fc Column (Figure 50) SEQ ID NO: 43: Nucleotide sequence of KLK5 inhibitory peptide Fc fusion D3-K50062-Fc Code arrangement (Figure 51) SEQ ID NO: 44: Amino acid sequence of KLK5 inhibitory peptide Fc fusion D3-K50062-Fc Column (Figure 52) SEQ ID NO: 45: Nucleotide sequence of KLK5 inhibitory peptide Fc fusion D3-K51090-Fc Code arrangement (Figure 53) SEQ ID NO: 46: Amino acid sequence of KLK5 inhibitory peptide Fc fusion D3-K51090-Fc Column (Figure 54) SEQ ID NO: 47: Nucleotide sequence of KLK5 inhibitory peptide-Fc fusion D3-K50098dN-Fc Otide sequence (Figure 55) SEQ ID NO: 48: Amino acid sequence of KLK5 inhibitory peptide Fc fusion D3-K50098dN-Fc Acid sequence (Figure 56) SEQ ID NO: 49: KLK5 / KLK7 inhibitory peptide Fc fusion D3-K51028-Fc Nucleotide sequence (Figure 57) SEQ ID NO: 50: KLK5 / KLK7 inhibitory peptide Fc fusion D3-K51028-Fc Amino acid sequence (Figure 58) SEQ ID NO: 51: KLK5 / KLK7 inhibitory peptide Fc fusion D3-K51005-Fc Nucleotide sequence (Figure 59) SEQ ID NO: 52: KLK5 / KLK7 inhibitory peptide Fc fusion D3-K51005-Fc Amino acid sequence (Figure 60) SEQ ID NO: 53: KLK5 / KLK7 inhibitory peptide Fc fusion D3-K50031-Fc Nucleotide sequence (Figure 61) SEQ ID NO: 54: KLK5 / KLK7 inhibitory peptide Fc fusion D3-K50031-Fc Amino acid sequence (Figure 62) SEQ ID NO: 55: KLK5 / KLK7 inhibitory peptide Fc fusion D3-K51057-Fc Nucleotide sequence (Figure 63) SEQ ID NO: 56: KLK5 / KLK7 inhibitory peptide Fc fusion D3-K51057-Fc Amino acid sequence (Figure 64) SEQ ID NO: 57: KLK5 / KLK14 inhibitory peptide Fc fusion D3-K51069dN- Nucleotide sequence of Fc (Figure 65) SEQ ID NO: 58: KLK5 / KLK14 inhibitory peptide Fc fusion D3-K51069dN- Amino acid sequence of Fc (Figure 66) SEQ ID NO: 59: KLK5 / KLK14 inhibitory peptide Fc fusion D3-K50015-Fc The nucleotide sequence of (Figure 67) SEQ ID NO: 60: KLK5 / KLK14 inhibitory peptide Fc fusion D3-K50015-Fc The amino acid sequence of (Figure 68) SEQ ID NO: 61: General formula of SPINK2 variant peptides (Figure 69) SEQ ID NO: 62: Nucleotide sequence of primer 1 (Figure 70) SEQ ID NO: 63: Nucleotide sequence of primer 2 (Figure 71) SEQ ID NO: 64: Nucleotide sequence of primer 3 (Figure 72) SEQ ID NO: 65: Nucleotide sequence of primer 4 (Figure 73) SEQ ID NO: 66: Nucleotide sequence of primer 5 (Figure 74) SEQ ID NO: 67: Nucleotide sequence of primer 6 (Figure 75) SEQ ID NO: 68: Nucleotide sequence of primer 7 (Figure 76) SEQ ID NO: 69: Nucleotide sequence of primer 8 (Figure 77) SEQ ID NO: 70: Nucleotide sequence of primer 9 (Figure 78) SEQ ID NO: 71: Nucleotide sequence of primer 10 (Figure 79) SEQ ID NO: 72: Nucleotide sequence of primer 11 (Figure 80) SEQ ID NO: 73: Nucleotide sequence of primer 12 (Figure 81) SEQ ID NO: 74: Nucleotide sequence of primer 13 (Figure 82) SEQ ID NO: 75: Nucleotide sequence of primer 14 (Figure 83) SEQ ID NO: 76: Nucleotide sequence of primer 15 (Figure 84) SEQ ID NO: 77: Amino acid sequence in KLK7 substrate peptide (Figure 85) SEQ ID NO: 78: Amino acid sequence of bovine α-chymotrypsin substrate peptide (Figure 86) SEQ ID NO: 79: Amino acid sequence in neutrophil elastase substrate peptide (Figure 87) column SEQ ID NO: 80: Amino acid sequence in human protein C substrate peptide (Figure 88) SEQ ID NO: 81: Nucleotide sequence of primer 16 (Figure 89) SEQ ID NO: 82: Nucleotide sequence of primer 17 (Figure 90) SEQ ID NO: 83: Nucleotide sequence of primer 18 (Figure 91) SEQ ID NO: 84: Nucleotide sequence of primer 19 (Figure 92) SEQ ID NO: 85: Nucleotide sequence of primer 20 (Figure 93) SEQ ID NO: 86: Nucleotide sequence of primer 21 (Figure 94) SEQ ID NO: 87: Amino acid sequence of Fc of human IgG1 (Figure 95) SEQ ID NO: 88: Amino acid sequence of D8 of human SPINK5 (Figure 96) SEQ ID NO: 89: Amino acid sequence of D9 of human SPINK5 (Figure 97) SEQ ID NO: 90: Amino acid sequence of human SPINK9 (Figure 98) SEQ ID NO: 91: Amino acid sequence of mouse KLK5 (Figure 99) SEQ ID NO: 92: Amino acid sequence of mouse KLK7 (Figure 100) SEQ ID NO: 93: Amino acid sequence of mouse KLK14 (Figure 101) SEQ ID NO: 94: Nucleotide sequence of primer 22 (Figure 104) SEQ ID NO: 95: Nucleotide sequence of KLK5 inhibitory peptide Fc fusion D1-K50055-Fc Code arrangement (Figure 105) SEQ ID NO: 96: Amino acid sequence of KLK5 inhibitory peptide Fc fusion D1-K50055-Fc Column (Figure 106)

Claims

1. A SPINK2 mutant peptide comprising the amino acid sequence (i), (ii), or (iii) below, which selectively inhibits the protease activity of activated human KLK5: (i) an amino acid sequence in which three or fewer amino acids have been substituted in an amino acid sequence (reference sequence) consisting of amino acid numbers 1 to 63 of an amino acid sequence represented by any one of SEQ ID NOs: 6, 8, 10, 12, 14, 16, 18, and 20 (FIGS. 14, 16, 18, 20, 22, 24, 26, and 28); (ii) an amino acid sequence in which no more than two amino acids are substituted in a reference sequence; (iii) an amino acid sequence in which one amino acid is substituted in the reference sequence; A SPINK2 variant peptide wherein the amino acid sequences corresponding to amino acid positions 16 to 22 and 24 to 28 in the reference sequence are substituted in the amino acid sequence of (i), (ii), or (iii).

2. 2. The peptide of claim 1, having three disulfide bonds and a three-dimensional structure characterized by including a loop structure, an alpha helix, and a beta sheet.

3. A polynucleotide comprising a nucleotide sequence encoding the amino acid sequence contained in the peptide of claim 1 or 2.

4. A vector comprising the polynucleotide of claim 3.

5. A cell comprising the polynucleotide of claim 3 or the vector of claim 4, or producing the peptide of claim 1 or 2.

6. A method for producing a SPINK2 mutant peptide that inhibits the protease activity of activated KLK5, comprising the following steps (i) and (ii): (i) culturing the cell according to claim 5; (ii) recovering the SPINK2 variant peptide from the culture.

7. A method for producing a SPINK2 mutant peptide that inhibits the protease activity of activated KLK5, comprising the step of preparing the peptide according to claim 1 or 2 by chemical synthesis or in vitro translation.

8. 8. A SPINK2 variant peptide obtainable by the method of claim 6 or 7.

9. A conjugate comprising one or more arbitrary moieties attached to a first peptide according to any one of claims 1, 2 and 8.

10. 10. The conjugate of claim 9, wherein one optional moiety comprises a second peptide that is not a SPINK2 variant.

11. The conjugate of claim 10 , wherein the second peptide is located amino-terminally to the first peptide.

12. The conjugate of claim 10 , wherein the second peptide is located carboxyl-terminal to the first peptide.

13. 13. The conjugate of claim 12, wherein the second peptide is an antibody or a fragment thereof and comprises one or more Fc regions.

14. 14. The conjugate of claim 13, wherein the Fc region is a human immunoglobulin Fc region or a fragment thereof.

15. 15. The conjugate of claim 13 or 14, wherein the Fc region is an Fc region of IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD, and / or IgE or a fragment thereof.

16. 16. The conjugate according to any one of claims 13 to 15, wherein the Fc region is a human IgG1 Fc region or a fragment thereof.

17. The conjugate of claim 16, wherein the Fc region of human IgG1 comprises the amino acid sequence shown in SEQ ID NO: 87 (Figure 95).

18. 16. The conjugate of any one of claims 13 to 15, wherein the Fc region is wild-type or mutated.

19. 19. The conjugate according to any one of claims 9 to 18, wherein one to several aspartic acid and / or glutamic acid residues are added to the amino terminus.

20. A conjugate that selectively inhibits the protease activity of human KLK5, described in any one of claims 9, 10, and 12 to 19, which comprises an amino acid sequence that is 90% or more identical to the amino acid sequence shown in any one of SEQ ID NOs: 34, 36, 38, 40, 42, 44, 46, 48 and 96 (Figures 42, 44, 46, 48, 50, 52, 54, 56 and 106).

21. 21. The conjugate of any one of claims 9, 10 and 12 to 20, wherein the first peptide and the second peptide are linked via a linker.

22. 22. The conjugate of claim 21, wherein the linker is a third peptide that is not the first peptide or the second peptide.

23. A method for producing a conjugate according to any one of claims 9, 10 and 12 to 22, comprising the steps (i) and (ii) of: (i) culturing a cell containing a polynucleotide comprising a nucleotide sequence encoding the amino acid sequence contained in the conjugate or a vector into which the polynucleotide has been inserted; (ii) recovering the SPINK2 variant peptide conjugate from the culture.

24. 23. A method for producing a SPINK2 variant peptide conjugate according to any one of claims 9, 10 and 12 to 22, or a peptide moiety contained in said conjugate, by chemical synthesis or in vitro translation.

25. 25. A SPINK2 variant peptide conjugate obtainable by the method of claim 23 or 24.

26. A composition comprising a peptide according to claim 1, 2 or 8, a polynucleotide according to claim 3, a vector according to claim 4, a cell according to claim 5, and / or a conjugate according to any one of claims 9, 10, 12 to 22 and 25.

27. A pharmaceutical composition comprising a peptide according to claim 1, 2 or 8, a polynucleotide according to claim 3, a vector according to claim 4, a cell according to claim 5, and / or a conjugate according to any one of claims 9, 10, 12 to 22 and 25.

28. 28. The pharmaceutical composition according to claim 27, for the treatment or prevention of Netherton syndrome, atopic dermatitis, rosacea, skin damage caused by ultraviolet light, psoriasis, asthma, spinal cord injury, cancer or Barrett's esophagus.

29. 29. The pharmaceutical composition of claim 27 or 28, used in combination with other pharmaceutical agents.

30. 25. The method of any one of claims 6, 7, 23 and 24, comprising an affinity purification step using an antibody or binding fragment thereof that specifically binds to the peptide of claim 1.

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