KLK5 Inhibitor and Method for Producing the Same

Abstract

To provide a novel KLK5 inhibitory peptide, a conjugate containing the peptide, a pharmaceutical composition containing the peptide or the conjugate, and the like.SOLUTION: The present invention provides a peptide that comprises a specific amino acid sequence and inhibits protease.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to peptides, polynucleotides, vectors, cells, methods for producing peptides, peptides obtained by such methods, conjugates containing peptides, compositions containing peptides or conjugates, pharmaceutical compositions, pharmaceutical compositions for the treatment or prevention of various diseases, the use of peptides or conjugates for the treatment or prevention of various diseases, methods for treating various diseases including the step of administering a peptide or conjugate, and compositions for the diagnosis or examination of various diseases containing a peptide or conjugate, etc.

Background Art

[0002] Kallikrein 5 (KLK5) is a trypsin-like serine protease (Clan PA, family S1) and is also called stratum corneum tryptic enzyme (SCTE). Kallikrein 7 (KLK7) is a chymotrypsin-like protease (Clan PA, family S1) and is also called stratum corneum chymotryptic enzyme (SCCE). Also, Kallikrein 14 (KLK14) is a trypsin-like protease (Clan PA, family S1). KLK5, KLK7, and KLK14 belong to the tissue kallikrein family consisting of 15 highly conserved trypsin or chymotrypsin-like serine proteases. KLK5 is converted into active KLK5 by autoactivation after being expressed in cells, whereas KLK7 and KLK14 are expressed as inactive preproenzymes. ​​​​​​​​​​​​​​The prepro sequence is cleaved by proteases typified by KLK5 and converted into the active form (Non-Patent Document 1). KLK5 is expressed in the skin and has been reported to degrade factors involved in cell adhesion such as desmoglein and desmocollin (Non-Patent Document 2). KLK5, KLK7, and KLK14 are considered important for skin desquamation and are further involved in the activation of protease-activated receptor 2 (PAR-2) (Non-Patent Document 3). Activation of PAR-2 induces cytokines and chemokines, enhancing immune and inflammatory responses. Netherton syndrome is an autosomal recessive genetic disorder and is one of the ichthyosis syndromes accompanied by severe skin inflammation, desquamation, hair abnormalities, and allergic symptoms such as asthma and atopic dermatitis. It is a rare disease (OMIM256500) (Non-Patent Document 4). Since exfoliative dermatitis develops at birth, it shows dehydration and infections due to significant damage to the skin barrier function and may be accompanied by growth retardation. Although detailed epidemiological data are not available, it is said to have a high postnatal mortality rate. Netherton syndrome is caused by a loss of function of LEKTI due to mutations in the gene (SPINK5) encoding serine protease inhibitor (LEKTI) expressed in skin epithelial cells. LEKTI consists of 15 Kazal-like inhibitor domains, and multiple mutation sites have been found in SPINK5 encoding each domain in patients with Netherton syndrome, and the symptoms and severity vary depending on the mutation sites (Non-Patent Documents 4 and 5).

[0003]

[0004] ​​​​​​​​​​​​​​​​SPINK5-deficient mice (Spink5 - / - ) exhibit Netherton syndrome-like skin symptoms and high protease activities of KLK5 and KLK7 are observed in the skin epithelium (Non-Patent Literature 1). SPINK5-deficient mice die within a few hours after birth, but mice obtained by mating SPINK5-deficient mice with KLK5-deficient mice (Spink5 - / - Klk5 - / - ) have been reported to show improved neonatal lethality (Non-Patent Literature 6). In addition, in Spink5 Klk5 - / - Kl k5 - / - mice, severe skin barrier defects, epithelial - / - structural defects, and skin inflammation observed in Spink5 have recovered. Similarly, mice carrying the SPINK5 mutation Spink5 observed in patients with Netherton syndrome show Netherton syndrome A135X / A135X -like skin symptoms and die within 12 hours after birth, whereas in Klk5 Spink5 obtained by crossing with KLK5-deficient mice, the skin barrier - / - Spink5 A135X / A135X and severe skin symptoms are improved (Non-Patent Literature 7). Furthermore, by crossing with KLK7-deficient mice (Klk5 Klk7 Spink5 - / - Klk7 - / - Spink5 A135X / A135X ), abnormal skin symptoms are no longer observed. KLK5 transgenic mice have also been reported to show Netherton syndrome -like skin symptoms (Non-Patent Literature 8). In the stratum corneum of patients with Netherton syndrome and Netherton syndrome model mice, high protease activities of trypsin-like and chymotrypsin-like are observed, and in addition to KLK5, kallikrein family members located downstream such as KLK7 and KL K14 are involved in the protease activity of the stratum corneum . This is suggested. From the above, Netherton syndrome is considered to be caused by genetic mutations in SPINK5, and is thought to develop due to abnormally increased protease activities of KLK5, KLK7, and KLK14 in the stratum corneum. Currently, symptomatic treatments such as application of moisturizers are available, but there is no radical treatment. KLK5 has also been suggested to be associated with rosacea, a chronic inflammatory disease of the face. In patients with rosacea, elevated expression of KLK5 and the antimicrobial peptide Cathelicidin has been reported. Although the details of its etiology are unknown, it is thought that increased expression of KLK5 degrades Cathelicidin, thereby producing peptides that cause rosacea (Non-Patent Document 9).

[0005] resulting in the production of peptides that cause rosacea (Non-Patent Document 9). There are multiple reports that polymorphisms in SPINK5 are associated with the severity of atopic dermatitis (Non-Patent Documents 10 to 14). In the skin of atopic dermatitis patients who have the SPINK5 gene encoding LEKTI with lysine as the 420th amino acid residue on both alleles, compared to the case where both alleles encode glutamic acid, a decrease in the protein level of Desmoglein1 and increased protease activities including KLK5 and KLK7 have been reported (Non-Patent Document 15). Activation of these proteases is thought to facilitate the entry of allergens due to a decrease in the skin barrier function and create a state in which an inflammatory reaction is likely to occur.

[0006] There are multiple reports suggesting that polymorphisms in SPINK5 are associated with the severity of atopic dermatitis (Non-Patent Documents 10 to 14). In the skin of atopic dermatitis patients who have the SPINK5 gene encoding LEKTI with lysine as the 420th amino acid residue on both alleles, compared to the case where both alleles encode glutamic acid, a decrease in the protein level of Desmoglein1 and increased protease activities including KLK5 and KLK7 have been reported (Non-Patent Document 15). Activation of these proteases is thought to facilitate the entry of allergens due to a decrease in the skin barrier function and create a state in which an inflammatory reaction is likely to occur. This activation of proteases is thought to facilitate the entry of allergens due to a decrease in the skin barrier function and create a state in which an inflammatory reaction is likely to occur.

[0007] SPINK2 (Serine Protease Inhibitor Kazal- type 2) is a Kazal-like domain having three disulfide bonds, and tr ​​​It functions as a trypsin / acrosin inhibitor (Non-Patent Document 16), but the relationship between SPINK2 and its variants and diseases such as Netherton syndrome, atopic dermatitis, and ichthyosis has not been clarified.

Prior Art Documents

Non-Patent Documents

[0008]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

Non-Patent Document 4

Non-Patent Document 5

Non-Patent Document 6

Non-Patent Document 7

Non-Patent Document 8

Non-Patent Document 9

Non-Patent Document 10

Non-Patent Document 11

Non-Patent Document 12

Non-Patent Document 13

Non-Patent Document 14

Non-Patent Document 15

Non - Patent Document 16

Summary of the Invention

Problems to be Solved by the Invention

[0009] To provide a novel KLK5 - inhibiting peptide, a conjugate containing the peptide, a pharmaceutical composition containing the peptide or the conjugate, etc.

Means for Solving the Problems

[0010] The present invention provides (1) An SPINK2 variant peptide that contains the amino acid sequence shown in SEQ ID NO: 61 (Figure 69) and inhibits the protease activity possessed by active - form human KLK5 (2) The peptide according to (1), which inhibits the protease activity possessed by human KLK7 or human KLK14 (3) The peptide according to (1), wherein the inhibition is selective for human KLK5 and optionally human KLK7 or KLK14 (4) Xaa (X1) at position 16 is Ala, Asp, Gly, Gln, Leu, Ser or Thr The peptide according to any one of (1) to (3) (5) Xaa (X2) at position 17 is Arg, Glu, Asn, Gln or Ser The peptide according to any one of (1) to (4) (6) ​Xaa(X3) at position 18 is Asp, Gln, Ile, Thr, Trp or Tyr, the peptide according to any one of (1) to (5), (7) Xaa(X4) at position 19 is Arg, Gly, Met, Gln or Thr, the peptide according to any one of (1) to (6), (8) Xaa(X5) at position 20 is Asp, Glu, Leu, Lys, Thr or Tyr, the peptide according to any one of (1) to (7), (9) Xaa(X6) at position 21 is Glu, Gly, His, Leu, Ser, Gln or Tyr the peptide according to any one of (1) to (8), (10) Xaa(X7) at position 22 is Asp, Gly, Gln, Sey or Tyr, the peptide according to any one of (1) to (9), (11) Xaa(X8) at position 24 is Ala, Asp, Glu, Gly, Asn, Ser or Thr the peptide according to any one of (1) to (10), (12) Xaa(X9) at position 25 is Arg or Lys, the peptide according to any one of (1) to (11) described, (13) Xaa(X 10 ) at position 26 is Asp, Glu, Gln, Ser or Val, (1) to any one of (12) described peptide, (14) Xaa(X 11 ) at position 27 is Phe or Tyr, any one of (1) to (13) described peptide, (15) Xaa(X 12 ) at position 28 is Asp or Glu, any one of (1) to (14) The peptide described in (16) A peptide according to any one of (1) to (15), comprising amino acid numbers 1 to 63 of the 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), (17) A peptide according to any one of (1) to (3), wherein Xaa (X1) at position 16 is Gly, Met or Tyr, (18) A peptide according to any one of (1) to (3) and (17), wherein Xaa (X2) at position 17 is Glu, Gln or Thr, (19) A peptide according to any one of (1) to (3), (17) and (18), wherein Xaa (X3) at position 18 is His, Met or Tyr, (20) A peptide according to any one of (1) to (3) and (17) to (19), wherein Xaa (X4) at position 19 is Ala, Arg, Lys or Gln, (21) A peptide according to any one of (1) to (3) and (17) to (20), wherein Xaa (X5) at position 20 is Gly, Arg or Ser, (22) A peptide according to any one of (1) to (3) and (17) to (21), wherein Xaa (X6) at position 21 is Arg, Lys, Gln or Ser, (23) A peptide according to any one of (1) to (3) and (17) to (22), wherein Xaa (X7) at position 22 is Gly, (24) A peptide according to any one of (1) to (3) and (17) to (24), wherein Xaa (X8) at position 24 is His, Thr or Tyr, ​​​​​​​​The peptide according to any one of (7) to (23), (25) wherein Xaa (X9) at position 25 is His or Tyr, the peptide according to any one of (1) to (3) and (17) to (24); (26) wherein Xaa (X 10 ) at position 26 is Asp, Glu or His, the peptide according to any one of (1) to (3) and ( 17) to (25); (27) wherein Xaa (X 11 ) at position 27 is Tyr, the peptide according to any one of (1) to (3) and (17) to (26) ; (28) wherein Xaa (X 12 ) at position 28 is Asp or Glu, the peptide according to any one of (1) to (3) and (17) to (27); (29) comprising amino acid numbers 1 to 63 of the amino acid sequence shown by any one of SEQ ID NOs: 22, 24, 26 and 28 (Figures 30, 32, 34 and 36), the peptide according to any one of (1) to (3) and (17) to (28); (30) wherein Xaa (X1) at position 16 is Gly, Ser or Tyr, the peptide according to any one of (1) to (3) ; (31) wherein Xaa (X2) at position 17 is Asp or Gln, the peptide according to any one of (1) to (3) and (30) ; (32) wherein Xaa (X3) at position 18 is Thr or Val, the peptide according to any one of (1) to (3), (30) and ( (31); (33) wherein Xaa (X4) at position 19 is Thr or Val, the peptide according to any one of (1) to (3) and (30) to (32); The peptide according to any one of (32), (34) At position 20, Xaa (X5) is Glu or Thr, (1) to (3) and (30) to The peptide according to any one of (33), (35) At position 21, Xaa (X6) is His or Thr, (1) to (3) and (30) to The peptide according to any one of (34), (36) At position 22, Xaa (X7) is Tyr, (1) to (3) and (30) to (35) of The peptide according to any one of them, (37) At position 24, Xaa (X8) is Asn or Ser, (1) to (3) and (30) to The peptide according to any one of (36), (38) At position 25, Xaa (X9) is Arg, (1) to (3) and (30) to (37) of The peptide according to any one of them, (39) At position 26, Xaa (X 10 ) is Asp or Glu, (1) to (3) and (30) to The peptide according to any one of (38), (40) At position 27, Xaa (X 11 ) is Tyr, (1) to (3) and (30) to (39) The peptide according to any one of them, (41) At position 28, Xaa (X 12 ) is Asp, (1) to (3) and (30) to (40) The peptide according to any one of them, (42) The amino acid numbers 1 to 63 of the amino acid sequence represented by any one of SEQ ID NOs: 30 and 32 (FIGS. 38 and 40), (1) to (3) and (30) to (41) of the peptide according to any one of the descriptions, The peptide according to any one of them, (43) Having three disulfide bonds and characterized by a three-dimensional structure including a loop structure, an α-helix, and a β-sheet, the peptide according to any one of (1) to (42) 、 、 (44) A polynucleotide encoding an amino acid sequence contained in the peptide according to any one of (1) to (43) 、 (45) A vector containing the polynucleotide according to (44) (46) A cell containing the polynucleotide according to (44) or the vector according to (45), or producing the peptide according to any one of (1) 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) A method for producing the peptide according to any one of (1) to (43), comprising preparing the peptide by chemical synthesis or in vitro translation 、 (49) A SPINK2 mutant peptide obtained by the method according to (47) or (48) (50) (1) to (43) and (49), a conjugate formed by binding one or more arbitrary moieties to the peptide according to any one of (51) A conjugate according to (50), wherein one arbitrary moiety comprises a second peptide that is not a SPINK2 mutant 、 (52) The conjugate according to (51), wherein the second peptide is located on the amino-terminal side of the SPINK2 mutant 、 (53) The conjugate according to (51), wherein the second peptide is located on the carboxyl-terminal side of the SPINK2 variant (54) The conjugate according to any one of (51) to (53), wherein the second peptide is an antibody or a fragment thereof and comprises one or more Fc regions (55) The conjugate according to (54), wherein the Fc region is an Fc region of human immunoglobulin or a fragment thereof (56) The conjugate according to (54) or (55), wherein the Fc region is an Fc region of IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2 , IgD, and / or IgE or a fragment thereof (57) The conjugate according to any one of (54) to (56), wherein the Fc region is an Fc region of human IgG1 or a fragment thereof (58) The conjugate according to (57), wherein the Fc region of human IgG1 comprises the amino acid sequence shown in SEQ ID NO: 87 (FIG. 95) (59) The conjugate according to any one of (54) to (57), wherein the Fc region is wild-type or mutant (60) The conjugate according to any one of (51) to (59), wherein one to several aspartic acids and / or glutamic acids are added to the amino terminus (61) The conjugate according to any one of (50) to (60), comprising the amino acid sequence described in the following (i) or (ii): (i) SEQ ID NO: 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54 ​​​​​​​​​Any one of 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 at least 90% identical to the amino acid sequence described in (i) above and that is contained in a conjugate that inhibits the protease activity of KLK5, (62) (62) A conjugate according to any one of (51) to (61), wherein the SPINK2 variant and the second peptide are linked via a linker, (63) (63) A conjugate according to (62), wherein the linker is a third peptide that is not the SPINK2 variant and the second peptide, (64) (64) A method for producing a conjugate according to any one of (50) to (63), comprising the following steps (i) and (ii): (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 or the peptide moiety contained in the conjugate from the culture, (65) (65) (iii) A method for producing the conjugate, comprising preparing the SPINK2 variant peptide conjugate or the peptide moiety contained in the conjugate by chemical synthesis or in vitro translation, (66) (66) A SPINK2 variant peptide conjugate according to any one of (50) to (63) or a peptide moiety contained in the conjugate, (67) (67) (68) A SPINK2 variant peptide conjugate obtained by the method described in (64) or (65), (69) (69) An antibody that binds to the peptide according to any one of (1) to (43), (49), or its binding fragment, (68) a peptide according to any one of (1) to (43), (49), a olynucleotide according to (44), a vector according to (45), a cell according to (46), a conjugate according to any one of (50) to (63) and (66), and / or an antibody according to (67) or a composition comprising its binding fragment, (69) a peptide according to any one of (1) to (43), (49), a olynucleotide according to (44), a vector according to (45), a cell according to (46), and / or a conjugate according to any one of (50 ) to (63) and (66), , (70) a pharmaceutical composition according to (69) for the treatment or prevention of KLK5-related diseases, (71) wherein the KLK5-related disease is Netherton syndrome, atopic dermatitis, rosacea, skin injury by ultraviolet rays , psoriasis, asthma, spinal cord injury, cancer or Barrett's esophagus, a pharmaceutical composition according to (70), (72) a pharmaceutical composition according to any one of (69) to (71) for use in combination with other pharmaceuticals, (73) a peptide according to any one of (1) to (43), (49), a olynucleotide according to (44), a vector according to (45), a cell according to (46), a conjugate according to any one of (50) to (63) and (66), and / or an antibody according to (67) or its binding fragment, (74) (67) An affinity purification step using the antibody or its binding fragment described, (47 ) The method according to any one of (48), (64) and (65), (75) A method for identifying a KLK5-inhibiting SPINK2 mutant peptide, comprising the following steps (i) to (iii): : (i) Incubating KLK5 protease and a substrate in the presence and absence of a test SPINK2 mutant peptide; (ii) Measuring the KLK5 protease activity in the presence and absence of the test SPINK2 mutant peptide; and (iii) When the KLK5 protease activity in the presence of the peptide is lower than the KLK5 protease activity in the absence of the peptide, determining the peptide as positive. (76) A method for identifying a KLK5-inhibiting compound, comprising the following steps (i) to (iii), wherein a peptide having an amino acid sequence represented by any one of SEQ ID NOs: 6, 8, 10, 12, 14, 16, 1 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) or a conjugate containing the peptide is used as a reference compound: (i) Incubating KLK5 protease and a substrate in the presence and absence of a test compound ; (ii) Measuring the KLK5 protease activity in the presence and absence of the compound; and (iii) When the KLK5 protease activity in the presence of the compound is lower than the KLK5 protease activity in the absence of the compound, determining the compound as positive. (77) A method for identifying a KLK5-inhibiting compound, comprising the following steps (i) to (iii):​​​​​​​ (i) Measuring the KLK5 protease inhibitory activity of the test compound; (ii) Measuring the KLK5 protease inhibitory activity of a reference compound that is a peptide comprising an amino acid sequence represented by any one of SEQ ID NOs: 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 2 8, 30, and 32 (Figures 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, and 40) or a conjugate comprising the peptide; and (iii) When the KLK5 protease inhibitory activity of the test compound is equal to or stronger than the KLK5 protease inhibitory activity of the reference compound, determining that the compound is positive, (78) (iv) A method for measuring KLK5 protease activity, comprising the following steps (i) and (ii), wherein a peptide comprising an amino acid sequence represented by any one of SEQ ID NOs: 6, 8, 10, 12, 14, 16, 18 , 20, 22, 24, 26, 28, 30, and 32 (Figures 14, 16, 18, 20, 22, 2 (78) 4, 26, 28, 30, 32, 34, 36, 38, and 40) or a conjugate comprising the peptide is used as the reference compound: (i) Incubating KLK5 protease, a substrate, and optionally other components; (ii) After step (i), measuring the amount of the substrate and / or the amount of the product, (79) A KLK5 inhibitory SPINK2 mutant peptide or an SPINK2 mutant peptide conjugate, wherein in surface plasmon resonance analysis, the dissociation constant (K for KLK5 measured by immobilizing the peptide or conjugate and adding KLK5 is 1 × 10 (79) M or less, (80) the peptide or conjugate, D (81) × 10 -9 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 shown in SEQ ID NO: 44 (Figure 52), (86) A conjugate comprising the amino acid sequence shown in SEQ ID NO: 46 (Figure 54), (87) A conjugate comprising the amino acid sequence shown in SEQ ID NO: 48 (Figure 56), (88) A conjugate comprising the amino acid sequence shown in SEQ ID NO: 50 (Figure 58), (89) A conjugate comprising the amino acid sequence shown in SEQ ID NO: 52 (Figure 60), (90) A conjugate comprising the amino acid sequence shown in SEQ ID NO: 54 (Figure 62), (91) A conjugate comprising the amino acid sequence shown in SEQ ID NO: 56 (Figure 64), (92) A conjugate comprising the amino acid sequence shown in SEQ ID NO: 58 (Figure 66), (93) A conjugate comprising the amino acid sequence shown in SEQ ID NO: 60 (Figure 68), and, (94) A conjugate comprising the amino acid sequence shown in SEQ ID NO: 96 (Figure 106), etc.

Advantages of the Invention

[0011] The peptide provided by the present invention, the conjugate containing the peptide, and the peptide or the pharmaceutical composition containing the conjugate has KLK5 inhibitory activity and is useful for the treatment or prevention of KLK5-related diseases (described below).

Brief Description of the Drawings

[0012]

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BEST MODE FOR CARRYING OUT THE INVENTION

[0013] 1. Definitions In the present invention, the "gene" means a nucleic acid molecule containing a nucleotide sequence encoding an amino acid sequence contained in a protein or its complementary strand, which may be single-stranded, double-stranded or triple-stranded or more, and includes DNA-RNA hybrids, molecules in which ribonucleotides and deoxyribonucleotides are mixed on a single strand, and double-stranded or triple-stranded or more nucleic acid molecules containing such strands are also included in the meaning of "gene". In the present invention, "gene", "polynucleotide" and "nucleic acid molecule" are synonymous,

[0014] ​​​Ribonucleotides, deoxyribonucleotides, nucleotides, etc., which are their constituent units, are not limited by the number of them, for example, DNA, RNA, mRNA, cDNA, cRNA, probes, oligonucleotides, primers, etc. are also included in the scope. "Nucleic acid molecule" may be abbreviated as "nucleic acid".

[0015] In the present invention, "polypeptide", "peptide" and "protein" are synonymous.

[0016] In the present invention, a peptide that recognizes a target molecule X or binds to a target molecule X (hereinafter, the recognition or binding action is collectively referred to as "X-binding activity") can be called an "X-binding peptide". Furthermore, a peptide that recognizes a target molecule X or binds to a target molecule X and inhibits or suppresses one or more activities or functions of the target molecule X (hereinafter, the inhibition or suppression action is collectively referred to as "X-inhibitory activity") can be called an "X-inhibitory peptide". can be called an "X-inhibitory peptide". In the present invention,

[0017] In the present invention, "SPINK2" means Serine Protease Inhib itor Kazal-type 2, and is a 7 kDa protein consisting of a Kaz al-like domain having three disulfide bonds. A preferred SPINK2 is of human origin. In the present invention, unless otherwise specified, human SPINK2 (SEQ ID NO: 1: FIG. 9 ) is simply referred to as "SPINK2".

[0018] In the present invention, "KLK5" consists of an N-terminal propeptide and a protease active domain, and has trypsin-like and chymotrypsin-like protease activities with three N-glycans added. It is a protein shown. Suitable KLK5 is derived from human. In the present invention, unless otherwise specified, human KLK5 (SEQ ID NO: 2: Figure 10) is simply referred to as "KLK5". There is.

[0019] In the present invention, "KLK7" consists of an N-terminal propeptide and a trypsin-like domain having protease activity, and is a protein to which an N-linked sugar chain is added. Suitable KLK7 is derived from human. In the present invention, unless otherwise specified, human KLK7 (SEQ ID NO: 3: Figure 11) is simply referred to as "KLK7". There is.

[0020] In the present invention, "KLK14" is also called neuropsin, consists of an N-terminal propeptide and a trypsin-like domain having protease activity, and is a protein to which an N-linked sugar chain is added. Suitable KLK14 is derived from human. In the present invention, unless otherwise specified, human KLK14 (SEQ ID NO: 4: Figure 12) is simply referred to as "KLK14". There is.

[0021] In the present invention, "pro-KLK5" means pro-KLK5 and is composed of a propeptide and a domain having protease activity. "Active KLK5" means active KLK5 and is composed of a domain having protease activity. Suitable active KLK5 is derived from human.

[0022] In the present invention, "pro-KLK7" means pro-KLK7 and is composed of a propeptide and a domain having protease activity. "Active KLK7" means active KLK7 and is composed of a domain having protease activity. Suitable active KLK7 is derived from human. ​​​​​​​​​​​

[0023] In the present invention, "pro-KLK14" means pro-KLK14 and is composed of a propeptide and a domain having protease activity. "Active KLK14" means act ive KLK14 and is composed of a domain having protease activity. A preferred active KLK14 is derived from a human.

[0024] In the present invention, "KLK5 inhibitory peptide", "KLK5 / KLK7 inhibitory peptide" or "KLK5 / KLK14 inhibitory peptide" means a peptide that inhibits or suppresses one or more activities or functions of KLK5, KLK5 and KLK7, or KLK5 and KLK14. Each of the peptides means a peptide that inhibits or suppresses one or more activities or functions of KLK5, KLK5 and KLK7, or

[0025] "KLK5 inhibitory peptide", "KLK5 / 7 inhibitory peptide" and "KLK5 / KLK1 4 inhibitory peptide" include fragments of the peptide and conjugates formed by adding or binding other moieties to the peptide or its fragments, among which the KLK5 inhibitory (binding ) activity, KLK5 / KLK7 inhibitory (binding) activity, or KLK5 / KLK14 inhibitory (binding ) activity is maintained. That is, fragments, adducts and modified forms (conjugates) of the peptide that maintain KLK5 inhibitory (binding) activity, KLK5 inhibitory (binding) activity and KLK7 inhibitory (binding) activity, or KLK5 inhibitory (binding ) activity and KLK14 inhibitory (binding) activity are also included in "KLK5 inhibitory peptide", "KLK5 / KLK7 inhibitory peptide" or "KLK5 / KLK14 inhibitory peptide", respectively. or "KLK5 / KLK14 inhibitory peptide", respectively.

[0026] In the present invention, the "site" to which the peptide binds, that is, the "site" recognized by the peptide It refers to a continuous or intermittent partial amino acid sequence or partial higher-order structure on a target molecule to which a peptide binds or recognizes. In the present invention, such a site can be referred to as an epitope or a binding site on the target molecule. In the present invention, "cell" includes various cells derived from an animal individual, subcultured cells, primary cultured cells, cell lines, recombinant cells, yeast, microorganisms, and the like.

[0027] In the present invention, "SPINK2 variant" means a peptide containing an amino acid sequence in which one or more amino acids are substituted with amino acids different from the wild type in the amino acid sequence of wild-type SPINK2, one or more wild-type amino acids are deleted, and one or more amino acids not present in the wild type are inserted (hereinafter collectively referred to as "mutation"). Among the "SPINK2 variants", those having KLK5 inhibitory activity, KLK5 inhibitory activity and KLK7

[0028] inhibitory activity (KLK5 / KLK7 inhibitory activity), or KLK5 inhibitory activity and KLK14 inhibitory activity (KLK5 / KLK14 inhibitory activity) are included in KLK5 inhibitory peptides, KLK 5 / KLK7 inhibitory peptides, or KLK5 / KLK14 inhibitory peptides. In addition, in the present invention, "insertion" may also be included in the scope of "addition". In the present invention, "several" in "one to several" refers to 3 to 10. In the present invention, "hybridize under stringent conditions" means performing hybridization at 65 °C in a solution containing 5×SS C, and then at 65 °C for 20 minutes in an aqueous solution containing 2×SSC - 0.1 % SDS, and in an aqueous solution containing 0.5×SSC - 0.1% SDS

[0029]

[0030] In the present invention, "several" in "one to several" refers to 3 to 10.

[0030] In the present invention, "hybridize under stringent conditions" means performing hybridization at 65 °C in a solution containing 5×SS C, and then at 65 °C for 20 minutes in an aqueous solution containing 2×SSC - 0.1 % SDS, and in an aqueous solution containing 0.5×SSC - 0.1% SDS In an aqueous solution at 65 °C for 20 minutes, and an aqueous solution containing 0.2×SSC - 0.1% SDS Hybridize under the conditions of washing in each at 65 °C for 20 minutes or equivalent conditions thereto This means. SSC is an aqueous solution of 150 mM NaCl - 15 mM sodium citrate, and n×SSC means SSC at n-fold concentration

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

[0032] 2. Peptide 2-1. Amino acid "Amino acid" is an organic compound containing an amino group and a carboxyl group, preferably an α-amino acid contained as a constituent unit in a protein, more preferably a natural protein In the present invention, more preferred amino acids are Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr and Val, and unless otherwise specified, "amino acid" means these 20 amino acids in total. These 20 amino acids in total can be called "natural amino acids". The KLK5 inhibitory peptide, KLK5 / KLK7 inhibitory peptide, or KLK5 / KLK14 inhibitory peptide of the present invention preferably contains natural amino acids

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

[0034] ​​​​​​​​​​ In the present invention, the amino acid is an L-amino acid, D-amino acid, or a mixture thereof ( DL-amino acid), and means an L-amino acid unless otherwise specified.

[0035] Natural amino acids can be classified into the following groups, for example, based on the properties of their common side chains as follows. (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 affect the backbone conformation: 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 can undergo conservative amino acid substitutions.

[0037] "Conservative amino acid substitution" means substitution with an amino acid that is functionally equivalent or similar. A conservative amino acid substitution in a peptide results in a static change in the amino acid sequence of the peptide. For example, one or more amino acids having similar polarity act functionally equivalently and result in a static change in the amino acid sequence of such a peptide. Generally, substitutions within a group can be considered to be conservative with respect to structure and function. However, as will be apparent to those skilled in the art, the role played by a particular amino acid residue depends on the three-dimensional structure of the molecule containing the amino acid and function. However, as will be apparent to those skilled in the art, the role played by a particular amino acid residue depends on the three-dimensional structure of the molecule containing the amino acid and function. However, as will be apparent to those skilled in the art, the role played by a particular amino acid residue depends on the three-dimensional structure of the molecule containing the amino acid can be determined in terms of meaning. For example, a cysteine residue can take an oxidized (disulfide) form that is less polar compared to the reduced (thiol) form. The long aliphatic portion of the arginine side chain can constitute structurally and functionally important features. Also, side chains containing aromatic rings (tryptophan, tyrosine, phenylalanine) can contribute to ion-aromatic interactions or cation-π interactions. In such cases, substituting an amino acid having these side chains with an amino acid belonging to an acidic or nonpolar group may be structurally and functionally conservative. Residues such as proline, glycine, cysteine (disulfide form), etc. may have a direct effect on the conformation of the main chain and often cannot be substituted without structural distortion.

[0038] Conservative amino acid substitutions include specific substitutions based on side chain similarity (L. Lehninger, Biochemistry, 2nd edition, pp73-75, Worth Publisher, New York (1975)) and typical substitutions as shown below. nd (1) Nonpolar amino acid groups: 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 "L"), isoleucine (hereinafter referred to as "Ile" or simply "I"), proline (hereinafter referred to as "Pro" or simply "P"), phenylalanine ("Phe" or simply "F"), tryptophan (hereinafter referred to as "Trp" or simply "W"), methionine ​​​​​​​​​​​​​​​​​(Hereinafter, referred to as "Met" or simply "M") (2) Non-charged polar amino acid group: Glycine (hereinafter, referred to as "Gly" or simply "G") , Serine (hereinafter, referred to as "Ser" or simply "S"), Threonine (hereinafter, "Thr" or simply "T"), Cysteine (hereinafter, referred to as "Cys" or simply "C"), Tyr rosine (hereinafter, referred to as "Tyr" or simply "Y"), Asparagine (hereinafter, "Asn" or simply "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"), A rgining (hereinafter, referred to as "Arg" or simply "R"), Histidine (hereinafter, "His" or simply "H") In the present invention, the amino acid may be an amino acid other than a natural amino acid. For example, selenocysteine, N-formylmethionine found in natural peptides and proteins , pyrrolidine, pyroglutamic acid, cystine, hydroxyproline, hydroxylysine, thyroxine, O-phosphoserine, desmosine, β-alanine, sarcosine, ornithine, creatine, γ-aminobutyric acid, opine, theanine, tricholomic acid, kainic acid, domo ic acid, acromelic acid, etc. can be mentioned, norleucine, Ac-amino acid, Boc- amino acid, Fmoc-amino acid, Trt-amino acid, N-terminal protected amino of Z-amino acid acid, amino acid t-butyl ester, benzyl ester, cyclohexyl ester, fluoro renyl ester and other C-terminal protected amino acids, diamine, ω-amino acid, β-amino acid, γ-amino Acids, Tic derivatives of amino acids, other amino acids such as aminophosphonic acids, etc., which are not found in nature can be mentioned, but are not limited to these, and amino acids other than the above 20 "natural amino acids" are collectively referred to as "unnatural amino acids" for convenience in the present invention.

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

[0040] The target of the KLK5 inhibitory peptide, KLK5 / KLK7 inhibitory peptide, and KLK5 / KLK14 inhibitory peptide of the present invention, KLK5, KLK7, and KLK14 are preferably derived from vertebrates, more preferably mammals, even more preferably primates, and most preferably humans. K LK5, KLK7, and KLK14 can be purified from tissues or cells, or by methods known to those skilled in the art as protein preparation methods such as gene recombination , in vitro translation, peptide synthesis, etc., and can be prepared. To KLK5, KLK7, and KLK14, a signal sequence , the Fc region of immunoglobulin, a tag, a label, etc. may be linked. KLK5 inhibitory activity, K LK5 / KLK7 inhibitory activity, and KLK5 / KLK14 inhibitory activity can be evaluated using the protease activities of KLK5, KLK 5 and KLK7, and KLK5 and KLK14 as indicators . For example, KLK5, KLK5 and KLK7 or KLK5 and KLK14, or functional fragments, substrates thereof, and the KLK5 inhibitory peptide, KLK 5 / KLK7 inhibitory peptide or KLK5 / KLK14 inhibitory peptide of the present invention, or candidates thereof When co - existing, compared with the case in the presence of a control or in the absence of the inhibitor or its candidate, the protease activities of KLK5, KLK5 and KLK7, or KLK5 and KLK14 are 70% or less, 50% or less, 30% or less, 20% or less, 10% or less, 5% or less, 1% or less, or 0%, KLK5 inhibition, KLK5 / KLK7 inhibition or KLK5 / KLK14 inhibition has occurred, and the inhibition activities are 30% or more, 50% or more, 70% or more, 80% or more, 90% or more, 95% or more, 99% or more, or 100% respectively. The KLK5 inhibition activity, K LK5 / KLK7 inhibition activity, and KLK5 / KLK14 inhibition activity may vary depending on reaction conditions, the type and concentration of the substrate, etc. Examples of the reaction conditions include those described in the Examples, but are not limited thereto. Substrate peptides or substrate proteins are added to a certain concentration of KLK5, KLK5 and KLK7, or , KLK5 and KLK14, and after reacting for a certain period of time, the fluorescence of the substrate peptide is detected, or the substrate protein is detected by SDS - PAGE, Western n blot method, liquid chromatography, etc., and the enzyme activity can be evaluated. As the buffer, for example, phosphate buffer saline (hereinafter referred to as "PBS"), Tris buffer (50 mM Tris, pH 7 to 8.5, for example, pH 7.5), etc. can be used, and NaCl (0 to 200 mM, for example, 200 mM), CaCl2 (0 to 10 mM, for example, 2 mM ), ZnCl Brij - 35 and other salts can also be added, but are not limited to 2、 these.

[0041] The KLK5 inhibition activity, KLK5 / KLK7 inhibition activity, and KLK5 / KLK14 inhibition activity The property can be represented by the inhibition constant K i It can be shown as follows. A substrate peptide is added to an enzyme at a certain concentration, and after reacting for a certain time, the fluorescence of the substrate peptide is detected to measure the protease activity. From the protease activity at each substrate concentration, according to the Michaelis-Menten equation (Mich aelis L, et al. (2011) Biochemistry. 50(39 ), 8264-8269), the maximum reaction rate V max and the Michaelis constant K m are calculated. Furthermore, from the protease activity when an inhibitor is added to an enzyme at a certain concentration, according to Morrison's equation (Morrison JF. (1969) Biochim Biop hys Acta. 185(2), 269-286), the inhibition constant K is calculated. i Examples of the software used for the calculation include GraphPad Prism (GraphPad Software Inc.). The substrates of the proteases possessed by KLK5, KLK7, and KLK5, or KLK5 and KLK14 are not particularly limited, such as endogenous substrates, exogenous substrates, synthetic substrates, etc.

[0042] Examples of the human endogenous substrates of KLK5 include low molecular weight kininogen, calistatin, collagen, Desmoglein, Desmocollin, Cathelicidin, etc. Examples of the human endogenous substrates of KLK7 include Pro-KLK3, fibronectin, collagen, etc. Examples of the human endogenous substrates of KLK14 include tPA, fibronectin, collagen, etc. Examples can be shown as follows. Examples of the human endogenous substrates of KLK7 include Pro-KLK3, fibrinogen, collagen, etc. can be exemplified. Examples of the human endogenous substrates of KLK14 include tPA, fibrinogen, collagen, etc. can be exemplified. Gelatin obtained by thermally denaturing gelatin can also be used as a substrate. Examples include, but are not limited to, 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 the KLK14 inhibitory activity of the compound 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 inhibitor peptide is preferably Preferably, the concentration is 1000 nM or less, more preferably, 300 nM or less, and 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 or KL 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-fold lower than 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 KLK7 inhibitory activity or less than KLK5 / KL and K7 inhibitory activity 2-fold or more. The peptide may be selected from

[0043] In addition, the KLK5 inhibitor peptide, KLK5 / KLK7 inhibitor peptide, or KL The K5 / KLK14 inhibitor peptides inhibit KLK5, KLK5 and KLK7, or K It is preferable that it does not inhibit or suppress protease activities other than LK5 and KLK14, or that the degree of inhibition or suppression thereof is relatively weak. In other words, the protease inhibitory activity of the KLK5 inhibitory peptide, KLK5 / KLK7 inhibitory peptide, or KLK5 / KLK1 4 inhibitory peptide of the present invention preferably has high KLK5 specificity, KLK5 / KL K7 specificity, or KLK5 / KLK14 specificity. Preferred peptides of the present invention are K LK1, KLK2, KLK3, KLK4, KLK6, KLK8, KLK9 to KLK13 、KLK15, chymotrypsin, tryptase, chymase, plasmin, thrombin, e lasterase, matriptase, protein C, tissue (tPA), urokinase plasmi nogen activator (uPA), plasma kallikrein and other proteases do not inhibit or suppress their activities, or the degree of inhibition or suppression thereof is relatively weak. Such preferred peptides of the present invention do not show side effects caused by inhibiting or suppressing other protease activities, and are preferably used as therapeutic or prophylactic agents for KLK5-related diseases (described later). Furthermore, the preferred KLK5-specific inhibitory peptide of the present invention does not inhibit or suppress the protease activities of KLK7 and KLK14, or the degree of inhibition or suppression thereof is relatively weak; the preferred KLK5 / KLK7-specific inhibitory peptide of the present invention does not inhibit or suppress the protease activity of KL K14, or the degree of inhibition or suppression thereof is relatively weak; the preferred KLK5 / KLK14 inhibitory peptide of the present invention does not inhibit or suppress the protease activity of K LK7, or the degree of inhibition or suppression thereof 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 LK inhibitors that also inhibit the protease activity of other KLKs, i.e., non-selective inhibitors, may cause side effects when administered to humans (Coussens, LM et al., Science, 2 95(5564), 2387-2392 (2002): Bissett, D et al., J.Clin.Oncol., 23(4), 842-849 (2005)). On the other hand, inhibitors with high specificity for KLK5, KLK5 / KLK7, or KLK5 / KLK14, i.e., KLK5-specific inhibitory peptides, KLK5 / KLK7-specific inhibitory peptides or KLK5 / KLK14-specific inhibitory peptides can avoid the above-mentioned side effects and can thus be preferably used for the treatment or prevention of KLK5-related diseases (described below). The KLK5 inhibitory peptide, KLK5 / KLK5 inhibitory peptide, or KLK5 / KLK14 inhibitory peptide of the present invention may be competitive in binding of protease substrates to KLK5, KLK5 and / or KLK7, or KLK

[0045] 5 and / or KLK14. As described above, KLK5, KLK7 and KLK14, which are the targets of the peptides of the present invention, are derived from vertebrates, preferably mammals, more preferably primates, and even more preferably humans, but may also be derived from non-human animals, such as rodents such as rats and mice, and primates such as cynomolgus monkeys and common marmosets . KLK5, KLK5 and K derived from non-human animals

[0046] As described above, KLK5, KLK7 and KLK14, which are the targets of the peptides of the present invention, are derived from vertebrates, preferably mammals, more preferably primates, and even more preferably humans, but may also be derived from non-human animals, such as rodents such as rats and mice, and primates such as cynomolgus monkeys and common marmosets , rhesus monkeys, etc. KLK5, KLK5 and K derived from non-human animals , and may also be derived from primates such as cynomolgus monkeys and common marmosets, rhesus monkeys, etc. KLK5, KLK5 and K derived from non-human animals Peptides having inhibitory activity against LK7, or KLK5 and KLK14 can be used for the diagnosis, examination, treatment, prevention, etc. of diseases related to KLK5 in non- human animals. Also, when such a peptide inhibits human KLK5, KLK5 and KLK7, or KLK 5 and KLK14 as well, in the non-clinical research and development of the peptide as a therapeutic or preventive agent for KLK5-related diseases (described later), such non-human animals can be used as animal disease models in efficacy and pharmacological tests, pharmacokinetic tests, safety tests and toxicity tests using healthy animals, etc. Moreover, the KLK5 inhibitory peptide, KLK5 / KLK7 inhibitory peptide, and KLK 5 / KLK14 inhibitory peptide of the present invention have a smaller molecular weight compared to other biopolymers such as antibodies used in the art as pharmaceuticals and diagnostic agents, and their production (described later) is relatively easy, and they are excellent in terms of physical properties such as storage stability and thermal stability. When used as a pharmaceutical composition (described later), they have advantages such as a wide range of choices for administration routes, administration methods, formulations, etc. Also, by increasing the molecular weight of the peptide of the present invention by applying known methods such as the addition of biopolymers or polymers, the blood half-life when used as a pharmaceutical composition can also be adjusted to be longer. The molecular weights of the KLK5 inhibitory peptide, KLK5 / KLK7 inhibitory peptide, and KLK 5 / KLK14 inhibitory peptide of the present invention are less than 10,000, preferably less than 8,000, and more preferably about 7,000 to 7,200. Also, among the variable loop portion consisting of Cys at position 15 to Cys at position 31 or the portion consisting of Cys at position 15 to Cys at position 63 (hereinafter referred to as the "portion containing 6 Cys") of SEQ ID NO: 61 (Figure 69), the KLK5 inhibitory activity, KLK5 / K

[0047] Furthermore, the KLK5 inhibitory peptide, KLK5 / KLK7 inhibitory peptide, and KLK 5 / KLK14 inhibitory peptide of the present invention are compared with other biopolymers such as antibodies used in the art as pharmaceuticals and diagnostic agents. The molecular weight is small, its production (described later) is relatively easy, and it is excellent in terms of physical properties such as storage stability and thermal stability. When used as a pharmaceutical composition (described later), it has advantages such as a wide range of choices for administration routes, administration methods, formulations, etc. Also, by increasing the molecular weight of the peptide of the present invention by applying known methods such as the addition of biopolymers or polymers, the blood half-life when used as a pharmaceutical composition can also be adjusted to be longer. The molecular weights of the KLK5 inhibitory peptide, KLK5 / KLK7 inhibitory peptide, and KLK 5 / KLK14 inhibitory peptide of the present invention are less than 10,000, preferably less than 8,000, and more preferably about 7,000 to 7,200. Also, among the variable loop portion consisting of Cys at position 15 to Cys at position 31 or the portion consisting of Cys at position 15 to Cys at position 63 (hereinafter referred to as the "portion containing 6 Cys") of SEQ ID NO: 61 (Figure 69), the KLK5 inhibitory activity, KLK5 / K The molecular weights of the KLK5 inhibitory peptide, KLK5 / KLK7 inhibitory peptide, and KLK5 / KLK14 inhibitory peptide of the present invention are less than 10,000, preferably less than 8,000, and more preferably about 7,000 to 7,200. Also, among the variable loop portion consisting of Cys at position 15 to Cys at position 31 or the portion consisting of Cys at position 15 to Cys at position 63 (hereinafter referred to as the "portion containing 6 Cys") of SEQ ID NO: 61 (Figure 69), the KLK5 inhibitory activity, KLK5 / K The KLK5 inhibitory peptide, KLK5 / KLK7 inhibitory peptide, and KLK5 / KLK14 inhibitory peptide of the present invention have the following advantages: The molecular weight is less than 10,000, preferably less than 8,000, and more preferably about 7,000 to 7,200. Also, among the variable loop portion consisting of Cys at position 15 to Cys at position 31 or the portion consisting of Cys at position 15 to Cys at position 63 (hereinafter referred to as the "portion containing 6 Cys") of SEQ ID NO: 61 (Figure 69), the KLK5 inhibitory activity, KLK5 / K The molecular weights of the KLK5 inhibitory peptide, KLK5 / KLK7 inhibitory peptide, and KLK5 / KLK14 inhibitory peptide of the present invention are less than 10,000, preferably less than 8,000, and more preferably about 7,000 to 7,200. Also, among the variable loop portion consisting of Cys at position 15 to Cys at position 31 or the portion consisting of Cys at position 15 to Cys at position 63 (hereinafter referred to as the "portion containing 6 Cys") of SEQ ID NO: 61 (Figure 69), the KLK5 inhibitory activity, KLK5 / K ys to Cys at position 31 or the portion consisting of Cys at position 15 to Cys at position 63 (hereinafter referred to as the "portion containing 6 Cys") of SEQ ID NO: 61 (Figure 69), the KLK5 inhibitory activity, KLK5 / K Among the variable loop portion consisting of Cys at position 15 to Cys at position 31 or the portion consisting of Cys at position 15 to Cys at position 63 (hereinafter referred to as the "portion containing 6 Cys") of SEQ ID NO: 61 (Figure 69), the KLK5 inhibitory activity, KLK5 / K ​Those having LK7 inhibitory activity or KLK5 / KLK14 inhibitory activity are also the KLK of the present invention 5 inhibitory peptides, KLK5 / KLK7 inhibitory peptides, or KLK5 / KLK14 inhibitory pep tides are respectively included, and the molecular weight of the variable loop portion is less than 2,500, preferably about 1, 800 to 2,000, and the molecular weight of the portion containing 6 Cys is less than 6,000, preferably is about 5,300 to 5,500.

[0048] The KLK5 inhibitory peptide, KLK5 / KLK7 inhibitory peptide, or KLK5 / KLK14 inhibitory peptide of the present invention is a SPINK2 variant (hereinafter abbreviated as "SPINK2 variant") in which at least part of the backbone of SPINK2 is maintained, and preferably K LK5, KLK5 and KLK7, or partial peptides of KLK5 and KLK14, partial sec ondary structures, etc. are recognized or bound to them (hereinafter, such recognition or binding action is collectively referred to as "target binding activity").

[0049] The binding of the SPINK2 variant and KLK5, KLK7 or KLK14 in the present invention is E LISA method, surface plasmon resonance (Surface Plasmon Resonanc e: hereinafter referred to as "SPR") analysis method, biolayer interferometry (BioLayer Int erferometry: hereinafter referred to as "BLI") method, isothermal titration calorimetry (Isoth ermal Titration Calorimetry: hereinafter referred to as "ITC") , flow cytometry, immunoprecipitation method, etc., using methods known to those skilled in the art, can be measured or determined.

[0050] As the ELISA method, KLK5, KLK5 / KLK7, or ​includes a method for detecting a KLK5 inhibitory peptide that recognizes and binds to KLK5 / KLK14, a KLK5 / KLK 7 inhibitory peptide, or a KLK5 / KLK14 inhibitory peptide. For immobilization of KLK5, KLK5 / KLK7, or KLK5 / KLK14, in addition to biotin -streptavidin, solid-phase antibodies that recognize tags fused to KLK5, KLK5 / KLK7, or KLK5 / KL K14, or KLK5, KLK5 / KLK7, or KLK5 / KLK14 can be used. For detection of a KLK5 inhibitory peptide, a KLK5 / KLK7 inhibitory peptide, or a KLK5 / KLK14 inhibitory peptide, in addition to labeled streptavidin, labeled detection antibodies that recognize tags fused to a KLK5 inhibitory peptide, a KLK5 / KLK7 inhibitory peptide, or a KLK5 / KLK14 inhibitory peptide, or a KLK5 inhibitory peptide, a KLK5 / KLK7 inhibitory peptide, or a KLK5 / KLK14 inhibitory peptide can be used. For labeling, in addition to biotin methods applicable to biochemical analysis such as HRP, alkaline phosphatase, FITC, etc. can be used. For detection using an enzyme label, TMB (3,3',5,5'-tet ramethylbenzidine), BCIP (5-bromo-4-chloro -3-indolyl phosphate), p-NPP (p-nitropheny l phosphate), OPD (o-Phenylenediamine), ABTS (3-Ethylbenzothiazoline-6-sulfonic acid ), SuperSignal ELISA Pico Chemiluminescen t Substrate (Thermo Fisher Scientific), etc. can be used. In addition to biotin, methods applicable to biochemical analysis such as HRP, alkaline phosphatase, FITC, etc. can be used. For detection using an enzyme label, TMB (3,3',5,5'-tetramethylbenzidine), BCIP (5-bromo-4-chloro-3-indolyl phosphate), p-NPP (p-nitrophenyl phosphate), OPD (o-Phenylenediamine), ABTS (3-Ethylbenzothiazoline-6-sulfonic acid), SuperSignal ELISA Pico Chemiluminescent Substrate (Thermo Fisher Scientific), etc. can be used. For detection using an enzyme label, TMB (3,3',5,5'-tetramethylbenzidine), BCIP (5-bromo-4-chloro-3-indolyl phosphate), p-NPP (p-nitrophenyl phosphate), OPD (o-Phenylenediamine), ABTS (3-Ethylbenzothiazoline-6-sulfonic acid), SuperSignal ELISA Pico Chemiluminescent Substrate (Thermo Fisher Scientific), etc. can be used. ramethylbenzidine), BCIP (5-bromo-4-chloro -3-indolyl phosphate), p-NPP (p-nitropheny l phosphate), OPD (o-Phenylenediamine), ABTS (3-Ethylbenzothiazoline-6-sulfonic acid )、SuperSignal ELISA Pico Chemiluminescen t Substrate (Thermo Fisher Scientific), etc. can be used. Chromogenic substrates and QuantaBlu (registered trademark) Fluorogenic Peroxidase e Substrate (Thermo Fisher Scientific), and other fluorescent substrates, and chemiluminescent substrates can be used. For measuring the detection signal, an absorption plate reader, a fluorescence plate reader, a luminescence plate reader, an RI liquid scintillation counter, etc. can be utilized.

[0051] The measurement method by SPR analysis is a method of immobilizing the SPINK2 mutant peptide on a sensor chip and measuring the binding between the two by adding a target molecule such as KLK5, and a method of immobilizing a target molecule such as K LK5 on a sensor chip and measuring the binding between the two by adding the SPINK2 mutant peptide can be either method, and preferably the former. For immobilizing the SPINK2 mutant contained in the peptide or conjugate of the present invention, a direct method or a capture method can be utilized, and preferably the latter. In the direct method, the hydrophobicity of the SPINK2 mutant, the amino group or carboxyl group of SPINK2, etc. can be utilized for direct immobilization . In the capture method, in addition to biotin-streptavidin, an antibody, protein A or protein G, etc. that recognizes a tag fused to the conjugate or the conjugate can be utilized for immobilization. To the sensor chip on which the SPINK2 mutant is immobilized, a target molecule such as KLK5 diluted with a measurement buffer is added, and the SPR signal is observed over time to obtain a sensorgram of the binding. Subsequently, a measurement buffer not containing a target molecule such as KLK5 is added, and the SPR signal is observed over time to obtain a sensorgram of the dissociation . The binding affinity is analyzed using the obtained sensorgram, and the dissociation constant K ​​​​​​D Calculate S Examples of the equipment used for PR analysis include BIAcore (registered trademark) (GE healthcare re), ProteOn (registered trademark) (BioRad), SPR-Navi (registered trademark) (BioNavis Oy), Spreeta (registered trademark) (Texas Instrum ents), SPRi-PlexII (registered trademark) (Horiba), Autolab SPR (registered trademark) (Metrohm), etc. Examples of the equipment used for the BLI method include Octet (registered trademark) (Pall). As the immunoprecipitation method, KLK5, KLK5 and KLK7, or KLK5 and KLK14 recognized and bound by the KLK5 inhibitory peptide, KLK5 / KLK

[0052] 7 inhibitory peptide, or KLK5 / KLK14 inhibitory peptide immobilized on beads can be detected. Magnetic beads, agarose beads, etc. can be used for the beads. For the immobilization of the KLK 5 inhibitory peptide, KLK5 / KLK7 inhibitory peptide, or KLK5 / KLK14 inhibitory peptide, in addition to biotin-streptavidin, an antibody that recognizes the peptide or a tag fused to the peptide, protein A, protein G, etc. can be used . The beads are separated by a magnet, centrifugation, etc., and KLK5, KLK5 and KLK7, or KLK5 and KLK14 precipitated with the beads are detected by SDS-PAGE or Western b lot method. For the detection of KLK5, KLK5 and KLK7, or KLK5 and KLK14 , in addition to labeled streptavidin, a labeled antibody that recognizes a tag fused to KLK5, KLK7 or KLK1 4, or KLK5, KLK7 or KLK14 can be used . and KLK7, or KLK5 and KLK14 are detected by SDS-PAGE or Western blot method. For the detection of KLK5, KLK5 and KLK7, or KLK5 and KLK14 , in addition to labeled streptavidin, a labeled antibody that recognizes a tag fused to KLK5, KLK7 or KLK1 4, or KLK5, KLK7 or KLK14 can be used Detection antibodies and the like can be used. As labels, in addition to biotin, HRP, alkaline phosphatase, FITC, and other methods that can be used in biochemical analysis can be utilized. For detection using enzyme labels, substrates similar to those used in the ELISA method can be used. For measuring detection signals, ChemiDoc (registered trademark) (BioRad), LuminoGraph (ATTO), etc. can be used. (ATTO) and the like can be used.

[0053] In the present invention, "specific recognition", that is, "specific binding" means binding without non-specific adsorption. As criteria for determining whether the binding is specific, for example, the binding activity EC in the ELISA method can be cited. As other criteria, for example the dissociation constant (hereinafter referred to as "K 50 ") can be cited. The K value of the KLK5 inhibitory peptide against KLK5 in the present invention, D the K value of the KLK5 / KLK7 inhibitory peptide against KLK5 and KLK7, or the K D value of the KLK 5 / KLK14 inhibitory peptide against KLK5 and KLK14 is 1×10 D M or less, 5×10 D M or less, 2×10 -5 M or less, or 1×10 M or less, more preferably -6 5×10 -6 M or less, 2×10 -6 M or less, or 1×10 M or less, even more preferably 5 -7 ×10 -7 M or less, 2×10 -7 M or less, or 1×10 ×10 -8 M or less, 2×10 -8 M or less, or 1×10 -8 M or less, even more preferably 5×10 -9 M or less, 2×10-9 M or less or 1×10 -9 is less than or equal to M. As another determination criterion, for example, the analysis result by immunoprecipitation can be cited. In the present invention, preferred suitable KLK5 inhibitory peptides, KLK5 / KLK7 inhibitory peptides, or KLK5 / KLK 14 inhibitory peptides are immobilized on beads, and after adding KLK5, KLK5 and KLK7, or KLK5 and KLK14 respectively, the beads are separated, and when KLK 5, KLK5 and KLK7, or KLK5 and KLK14 precipitated with the beads are detected, the signals of KLK5, KLK5 and KLK7, or KLK5 and KLK14 are detected.

[0054] The SPINK2 variant as the KLK5 inhibitory peptide, KLK5 / KLK7 inhibitory peptide, or KLK5 / K LK14 inhibitory peptide of the present invention may have protease inhibitory activity, target binding activity, and other properties, functions, characteristics, etc. as described above, while its full-length amino acid sequence has a high sequence identity with the amino acid sequence of human wild-type SPINK2. The SPINK2 variant of the present invention has 60% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 9 8% or more or 99% or more sequence identity with the amino acid sequence of human SPINK2 (SEQ ID NO: 1: FIG. 7).

[0055] "Identity" means a property indicating the degree of similarity or relationship between two sequences. The identity (%) of an amino acid sequence is calculated by multiplying the number of identical amino acids or amino acid residues by 100 and dividing by the total number of amino acids or amino acid residues.

[0056] "Gap" means deletions and / or additions in at least one of two or more sequences It means the gaps in the alignment between the arrays, which is the result of

[0057] The identity between two amino acid sequences having exactly the same amino acid sequence is 100% However, if there is one or more amino acid or amino acid residue substitution, deletion or addition in one amino acid sequence compared to the other, the identity between the two will be less than 100%. Algorithms and programs for determining the identity between two sequences considering gaps include those well-known to those skilled in the art, such as BLAST (Altschul, et al. Nucleic Acids Res. Vol. 25, pp. 3389 - 3402, 1997) using standard parameters, BLAST2 (Altschul, et al. J.Mol.Biol. Vol. 215, pp. 403 - 4 10, 1990), Smith - Waterman (Smith, et al. J.Mol.Biol. Vol. 147, pp. 195 - 197, 1981), etc. can be exemplified.

[0058] In the present invention, "mutated" means that in the nucleotide sequence or amino acid sequence compared to a naturally occurring nucleic acid molecule or peptide, 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 variant of the present invention has one or more amino acids or amino acid residues mutated compared to the amino acid sequence of human SPINK2 .

[0059] In one aspect of the present invention, the amino acid sequence of the SPINK2 variant is that of human SPINK2 (SEQ ID NO: 1: Figure 9): One, two, three, four, five, six, or seven of the amino acids from Ser at position 16 to Gly at position 22 are substituted with other amino acids or amino acid residues; One, two, three, four, or five of the amino acids from Pro at position 24 to Asn at position 28 are substituted with other amino acids or amino acid residues; One, two, three, four, or five of the amino acids from Pro at position 24 to Asn at position 28 are substituted with other amino acids or amino acid residues; One, two, three, four, or five of the amino acids from Pro at position 24 to Asn at position 28 are substituted with other amino acids 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 Cys at position 63 are preferably Cys as in the wild type in order to maintain the native disulfide bond, and in order to eliminate the native disulfide bond or generate a non-native disulfide bond, one, two, three, four, five, or six of them may be substituted with other amino acids. In some preferred KLK5 inhibitory peptides, KLK5 / KLK7 inhibitory peptides, or KLK5 / KLK14 inhibitory peptides among the SPINK2 mutants of the present invention, Cys is maintained at the same six positions as in the native type, and the disulfide bond is retained. In some more preferred embodiments of such peptides, Cys at position 15 - Cys at position 45, Cys at position 23 - Cys at position 42, and Cys at position 31 - Cys at position 63 form disulfide bonds respectively. In order to maintain the native disulfide bond, it is preferable that they are Cys as in the wild type, and in order to eliminate the native disulfide bond or generate a non-native disulfide bond, one, two, three, four, five, or six of them may be substituted with other amino acids. In some preferred KLK5 inhibitory peptides, KLK5 / KLK7 inhibitory peptides, or KLK5 / KLK14 inhibitory peptides among the SPINK2 mutants of the present invention, Cys is maintained at the same six positions as in the native type, and the disulfide bond is retained. In some more preferred embodiments of such peptides, Cys at position 15 - Cys at position 45, Cys at position 23 - Cys at position 42, and Cys at position 31 - Cys at position 63 form disulfide bonds respectively. In order to maintain the native disulfide bond, it is preferable that they are Cys as in the wild type, and in order to eliminate the native disulfide bond or generate a non-native disulfide bond, one, two, three, four, five, or six of them may be substituted with other amino acids. In some preferred KLK5 inhibitory peptides, KLK5 / KLK7 inhibitory peptides, or KLK5 / KLK14 inhibitory peptides among the SPINK2 mutants of the present invention, Cys is maintained at the same six positions as in the native type, and the disulfide bond is retained. In some more preferred embodiments of such peptides, Cys at position 15 - Cys at position 45, Cys at position 23 - Cys at position 42, and Cys at position 31 - Cys at position 63 form disulfide bonds respectively. In order to maintain the native disulfide bond, it is preferable that they are Cys as in the wild type, and in order to eliminate the native disulfide bond or generate a non-native disulfide bond, one, two, three, four, five, or six of them may be substituted with other amino acids. In some preferred KLK5 inhibitory peptides, KLK5 / KLK7 inhibitory peptides, or KLK5 / KLK14 inhibitory peptides among the SPINK2 mutants of the present invention, Cys is maintained at the same six positions as in the native type, and the disulfide bond is retained. In some more preferred embodiments of such peptides, Cys at position 15 - Cys at position 45, Cys at position 23 - Cys at position 42, and Cys at position 31 - Cys at position 63 form disulfide bonds respectively. In some preferred KLK5 inhibitory peptides, KLK5 / KLK7 inhibitory peptides, or KLK5 / KLK14 inhibitory peptides among the SPINK2 mutants of the present invention, Cys is maintained at the same six positions as in the native type, and the disulfide bond is retained. In some more preferred embodiments of such peptides, Cys at position 15 - Cys at position 45, Cys at position 23 - Cys at position 42, and Cys at position 31 - Cys at position 63 form disulfide bonds respectively. In some preferred KLK5 inhibitory peptides, KLK5 / KLK7 inhibitory peptides, or KLK5 / KLK14 inhibitory peptides among the SPINK2 mutants of the present invention, Cys is maintained at the same six positions as in the native type, and the disulfide bond is retained. In some more preferred embodiments of such peptides, Cys at position 15 - Cys at position 45, Cys at position 23 - Cys at position 42, and Cys at position 31 - Cys at position 63 form disulfide bonds respectively. In some preferred KLK5 inhibitory peptides, KLK5 / KLK7 inhibitory peptides, or KLK5 / KLK14 inhibitory peptides among the SPINK2 mutants of the present invention, Cys is maintained at the same six positions as in the native type, and the disulfide bond is retained. In some more preferred embodiments of such peptides, Cys at position 15 - Cys at position 45, Cys at position 23 - Cys at position 42, and Cys at position 31 - Cys at position 63 form disulfide bonds respectively. In some more preferred embodiments of such peptides, Cys at position 15 - Cys at position 45, Cys at position 23 - Cys at position 42, and Cys at position 31 - Cys at position 63 form disulfide bonds respectively. In some more preferred embodiments of such peptides, Cys at position 15 - Cys at position 45, Cys at position 23 - Cys at position 42, and Cys at position 31 - Cys at position 63 form disulfide bonds respectively. In some more preferred embodiments of such peptides, Cys at position 15 - Cys at position 45, Cys at position 23 - Cys at position 42, and Cys at position 31 - Cys at position 63 form disulfide bonds respectively.

[0060] When the amino acid sequence of such an SPINK2 mutant is included in a KLK5 inhibitory peptide, a KLK5 / KLK7 inhibitory peptide, or a KLK5 / KLK14 inhibitory peptide, the loop structure consisting of Ser at position 16 to Val at position 30, the β-strand (1) consisting of Cys at position 31 and Gly at position 32, and the β-sheet composed of the β-strand (2) consisting of Ile at position 57 to Arg at position 59, which are included in the amino acid sequence of wild-type SPINK2, When the amino acid sequence of such an SPINK2 mutant is included in a KLK5 inhibitory peptide, a KLK5 / KLK7 inhibitory peptide, or a KLK5 / KLK14 inhibitory peptide, the loop structure consisting of Ser at position 16 to Val at position 30, the β-strand (1) consisting of Cys at position 31 and Gly at position 32, and the β-sheet composed of the β-strand (2) consisting of Ile at position 57 to Arg at position 59, which are included in the amino acid sequence of wild-type SPINK2, the loop structure consisting of Ser at position 16 to Val at position 30, the β-strand (1) consisting of Cys at position 31 and Gly at position 32, and the β-sheet composed of the β-strand (2) consisting of Ile at position 57 to Arg at position 59, which are included in the amino acid sequence of wild-type SPINK2, the loop structure consisting of Ser at position 16 to Val at position 30, the β-strand (1) consisting of Cys at position 31 and Gly at position 32, and the β-sheet composed of the β-strand (2) consisting of Ile at position 57 to Arg at position 59, which are included in the amino acid sequence of wild-type SPINK2, an α-helix consisting of Gly up to 51, or a loop structure, β-sheet, α-helix, etc. that is similar to or at least partially corresponds to them at their positions (in position) is preferably maintained to such an extent that it is composed of a three-dimensional structure formed from a loop structure, β-sheet, α-helix, etc. that exhibits KLK5 inhibitory activity, KLK5 / KLK7 inhibitory activity, or KLK5 / KLK14 inhibitory activity. Regarding the amino acid sequences of some of the KLK5 inhibitory peptides, KLK5 / KLK7 inhibitory peptides, or KLK5 / KLK14 inhibitory peptides among the SPINK2 variants of the present invention, they are described below. As described above, in the present invention, "amino acid residue" may simply be referred to as "amino acid". In the amino acid sequence (general formula) shown by SEQ ID NO: 61 (FIG. 69), X1 to X

[0061] are not particularly limited as long as they inhibit KLK5, KLK5 and KLK7, or KLK5 and KLK14, and are each arbitrary amino acids. Hereinafter, preferred amino acids will be described, but among these amino acids, there may be amino acids that are the same as those in the amino acid sequence of natural-type, i.e., wild-type human SPINK2. In the amino acid sequence shown by SEQ ID NO: 61 (FIG. 69) included in the KLK5 inhibitory peptide, preferably:

[0062] Xaa (X1) at position 16 is Ala, Asp, Gly, Gln, Leu, Ser or Thr 12 ; Xaa (X2) at position 17 is Arg, Glu, Asn, Gln or Ser; 12 preferred amino acids will be described, but among these amino acids, there may be amino acids that are the same as those in the amino acid sequence of natural-type, i.e., wild-type human SPINK2.

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

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

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

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

[0067] In the present invention, one or several or more, preferably 1 to 5 amino acids may be added to the N-terminal side of the 1st amino acid. Such amino acid addition includes preferably those with 1 to 5 Asp and / or Glu added (both Asp and Glu may be included ), more preferably those with 1 to 5 Asp added or those with 1 to 5 Glu Examples of additional ones can be cited.

[0068] In the present invention, in the added portion of the N-terminus and / or C-terminus adduct of the SPINK2 variant peptide (hereinafter, referred to as the "parent peptide"), one or more amino acids are substituted, added and / or deleted, and a peptide that maintains some or all of the activity of the SPINK2 variant peptide is referred to as a "derivative of the parent peptide" or "parent peptide derivative". Such a "derivative" is also included in the scope of the "peptide" of the present invention.

[0069] In the amino acid sequence of the SPINK2 variant included in the scope of the KLK5 inhibitory peptide, KLK5 / KLK7 inhibitory peptide, or KLK5 / KLK14 inhibitory peptide of the present invention, in the portion other than X1 to X , that is, in the amino acid sequence of wild-type human SPINK2 12 (SEQ ID NO: 1: FIG. 9), at positions 2nd Pro to 15th Cys, 23rd Cys, and 29th Pro ~63rd Cys, it can contain natural amino acids, mutated amino acids, or amino acid sequences. For example, the SPINK2 variant can have a KLK5 inhibitory activity, KLK 5 / KLK7 inhibitory activity, or KLK5 / KLK14 inhibitory activity or folding, as long as it does not at least partially interfere with or interfere with it, and can be mutated at one or more positions. Such mutations can be made using standard methods known to those skilled in the art. Typical mutations in the amino acid sequence can include substitution, deletion, or insertion of one or more amino acids, and examples of substitution can include conservative substitution. By conservative substitution, an amino acid residue not only has a bulky size but also has a polar surface For example, conservative substitution can be exemplified. By conservative substitution, an amino acid residue has not only a large size but also a polar surface It is substituted by amino acid residues with similar chemical characteristics. Examples of conservative substitutions are described in other parts of this specification while the parts other than X1 to X 12 can tolerate non-conservative substitutions of one or more amino acids, provided that they do not substantially interfere with or prevent the KLK5 inhibitory activity , KLK5 / KLK7 inhibitory activity, or KLK5 / KLK14 inhibitory activity or folding, at least partially .

[0070] The amino acid sequence of the SPINK2 variant as the KLK5 inhibitory peptide, KLK5 / KLK7 inhibitory peptide, or KLK5 / KLK14 inhibitory peptide of the present invention is such that X1 to X 12 is preferably each amino acid of X1 to X in 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), SEQ ID NOs: 22, 24, 26 and 28 (FIGS. 30, 32, 34 and 36), or SEQ ID NOs: 30 and 32 (FIGS. 38 and 40), and the part other than X1 to X has an amino acid sequence that does not substantially interfere with or prevent the KLK5 inhibitory activity, KLK5 / KLK7 inhibitory activity, or KLK5 / KLK14 inhibitory 12 activity or folding, at least partially 12 . In addition, as examples of the amino acid sequence of the SPINK2 variant as the KLK5 inhibitory peptide, KLK5 / KLK7 inhibitory peptide, or KLK5 / KLK14 inhibitory peptide of the present invention, the amino acid sequences described in any one of the following (a1) to (a4), (b1) to (b4) or (c1) to (c4) can be respectively cited:

[0071] Also, as examples of the amino acid sequence of the SPINK2 variant as the KLK5 inhibitory peptide, KLK5 / KLK7 inhibitory peptide, or KLK5 / KLK14 inhibitory peptide of the present invention, the amino acid sequences described in any one of the following (a1) to (a4), (b1) to (b4) or (c1) to (c4) can be respectively cited: ​​​​(a1) An amino acid sequence consisting of amino acid numbers 1 to 63, 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); ; (a2) An amino acid sequence encoded by a nucleotide sequence complementary to the nucleotide sequence encoding the amino acid sequence described in (a1), which hybridizes under stringent conditions with the nucleotide sequence and has KLK5 inhibitory activity and is encoded by a nucleotide sequence encoding an amino acid sequence contained in a peptide having KLK5 inhibitory activity; ; ; (a3) An amino acid sequence in which 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 amino acids are substituted, deleted, added and / or inserted, and which has KLK5 inhibitory activity; and, ; (a4) An amino acid sequence that is 60%, 70%, 80%, 85%, 90%, 9 2%, 94%, 96%, 97%, 98% or 99% or more identical to the amino acid sequence described in (a1), and which has KLK5 inhibitory activity, ; (b1) An amino acid sequence consisting of amino acid numbers 1 to 63, represented by any one of SEQ ID NOs: 22, 24, 26, and 28 (FIGS. 30, 32, 34, and 36); ; (b2) An amino acid sequence encoded by a nucleotide sequence complementary to the nucleotide sequence encoding the amino acid sequence described in (b1), which hybridizes under stringent conditions with the nucleotide sequence and has KLK5 / KLK7 inhibitory activity and is encoded by a nucleotide sequence encoding an amino acid sequence contained in a peptide having KLK5 / KLK7 inhibitory activity; ; ; (b3) An amino acid sequence in which 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, or 1 or 2 wherein one amino acid is substituted, deleted, added and / or inserted, and the amino acid sequence contained in the peptide having KLK5 / KL K7 inhibitory activity; and, (b4)(b1) the amino acid sequence that is 60%, 70%, 80%, 85%, 90%, 9 2%, 94%, 96%, 97%, 98% or 99% or more identical, and the amino acid sequence contained in the peptide having KLK5 / KL K7 inhibitory activity, or, (c1) the amino acid sequence consisting of amino acid numbers 1 to 63 of the amino acid sequence shown by any one of SEQ ID NOs: 30 and 32 (FIGS. 38 and 40); the amino acid sequence consisting of amino acid numbers 1 to 63 of the amino acid sequence shown by any one of SEQ ID NOs: 30 and 32 (FIGS. 38 and 40); (c2) a nucleotide sequence complementary to the nucleotide sequence encoding the amino acid sequence described in (c1) hybridizes under stringent conditions, and the nucleotide sequence encoding the amino acid sequence contained in the peptide having KLK5 / KLK14 inhibitory activity; the amino acid sequence encoded by the nucleotide sequence encoding the amino acid sequence contained in the peptide having KLK5 / KLK14 inhibitory activity; (c3) in the amino acid sequence described in (c1), 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, or 1 or 2 wherein one amino acid is substituted, deleted, added and / or inserted, and the amino acid sequence contained in the peptide having KLK5 / KL K14 inhibitory activity; and, (c4)(c1) the amino acid sequence that is 60%, 70%, 80%, 85%, 90%, 9 2%, 94%, 96%, 97%, 98% or 99% or more identical, and the amino acid sequence contained in the peptide having KLK5 / KL K14 inhibitory activity.

[0072] Note that SEQ ID NOs: 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 2 8, 30, and 32 (Figures 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, and 40) at amino acid numbers 64 and 65 are not the amino acids corresponding to wild-type human SPINK2 ( consisting of 63 amino acids in SEQ ID NO: 1, Figure 9) but are added for expressing the peptide of the present invention in certain embodiments of the present invention.

[0073] Mutations can be introduced into the peptide of the present invention for the purpose of improving its folding stability, thermal stability, storage stability, plasma half-life, water solubility, biological activity, pharmacological activity, side effects, etc. For example, new reactive groups such as Cy s can be introduced by mutation for conjugation to other substances such as polyethylene glycol (PEG), hydroxyethyl starch (H ES), biotin, peptides, or proteins.

[0074] In the present invention, the KLK5 inhibitory peptide, the KLK5 / KLK7 inhibitory peptide, or the K LK5 / KLK14 inhibitory peptide may be linked or attached to other moieties, and such conjugates are collectively referred to as "conjugates of KLK5 inhibitory peptides", "conjugates of KLK5 / KLK7 inhibitory peptides", or "conjugates of KLK5 / KLK14 inhibitory peptides", respectively. In the present invention, "conjugate" means a molecule in which another moiety is bound to the peptide of the present invention or a fragment thereof. "Conjugate" or "conjugation" includes a moiety being linked or bound to the peptide of the present invention at the N-terminus and / or C-terminus of the peptide of the present invention by a chemical substance such as a cross-linking agent or an agent suitable for linking a moiety to the side chain of an amino acid via a synthetic chemical method or a genetic engineering method, etc. forms are included. Such "moieties" include, as those that improve the plasma half-life, polyalkylene glycol molecules such as polyethylene glycol (PEG), hydroxyethyl starch (HES), fatty acid molecules such as palmitic acid, the Fc region of an immunoglobulin (e.g., the Fc region of human immunoglobulin G1; its amino acid sequence is shown in SEQ ID NO: 87 and FIG. 95), the CH3 domain of an immunoglobulin, the CH4 domain of an immunoglobulin, albumin or a fragment thereof, an albumin-binding peptide, an albumin-binding protein such as streptococcal protein G, transferrin, etc. can be exemplified. As other "moieties", such "moieties" can be linked to the peptide of the present invention via a linker such as a peptide linker. polyalkylene glycol molecules such as polyethylene glycol (PEG), hydroxyethyl starch (HES), fatty acid molecules such as palmitic acid, the Fc region of an immunoglobulin (e.g., the Fc region of human immunoglobulin G1; its amino acid sequence is shown in SEQ ID NO: 87 and FIG. 95), the CH3 domain of an immunoglobulin, the CH4 domain of an immunoglobulin, albumin or a fragment thereof, an albumin-binding peptide, an albumin-binding protein such as streptococcal protein G, transferrin, etc. can be exemplified. polyalkylene glycol molecules such as polyethylene glycol (PEG), hydroxyethyl starch (HES), fatty acid molecules such as palmitic acid, the Fc region of an immunoglobulin (e.g., the Fc region of human immunoglobulin G1; its amino acid sequence is shown in SEQ ID NO: 87 and FIG. 95), the CH3 domain of an immunoglobulin, the CH4 domain of an immunoglobulin, albumin or a fragment thereof, an albumin-binding peptide, an albumin-binding protein such as streptococcal protein G, transferrin, etc. can be exemplified. polyalkylene glycol molecules such as polyethylene glycol (PEG), hydroxyethyl starch (HES), fatty acid molecules such as palmitic acid, the Fc region of an immunoglobulin (e.g., the Fc region of human immunoglobulin G1; its amino acid sequence is shown in SEQ ID NO: 87 and FIG. 95), the CH3 domain of an immunoglobulin, the CH4 domain of an immunoglobulin, albumin or a fragment thereof, an albumin-binding peptide, an albumin-binding protein such as streptococcal protein G, transferrin, etc. can be exemplified. polyalkylene glycol molecules such as polyethylene glycol (PEG), hydroxyethyl starch (HES), fatty acid molecules such as palmitic acid, the Fc region of an immunoglobulin (e.g., the Fc region of human immunoglobulin G1; its amino acid sequence is shown in SEQ ID NO: 87 and FIG. 95), the CH3 domain of an immunoglobulin, the CH4 domain of an immunoglobulin, albumin or a fragment thereof, an albumin-binding peptide, an albumin-binding protein such as streptococcal protein G, transferrin, etc. can be exemplified. polyalkylene glycol molecules such as polyethylene glycol (PEG), hydroxyethyl starch (HES), fatty acid molecules such as palmitic acid, the Fc region of an immunoglobulin (e.g., the Fc region of human immunoglobulin G1; its amino acid sequence is shown in SEQ ID NO: 87 and FIG. 95), the CH3 domain of an immunoglobulin, the CH4 domain of an immunoglobulin, albumin or a fragment thereof, an albumin-binding peptide, an albumin-binding protein such as streptococcal protein G, transferrin, etc. can be exemplified. polyalkylene glycol molecules such as polyethylene glycol (PEG), hydroxyethyl starch (HES), fatty acid molecules such as palmitic acid, the Fc region of an immunoglobulin (e.g., the Fc region of human immunoglobulin G1; its amino acid sequence is shown in SEQ ID NO: 87 and FIG. 95), the CH3 domain of an immunoglobulin, the CH4 domain of an immunoglobulin, albumin or a fragment thereof, an albumin-binding peptide, an albumin-binding protein such as streptococcal protein G, transferrin, etc. can be exemplified. polyalkylene glycol molecules such as polyethylene glycol (PEG), hydroxyethyl starch (HES), fatty acid molecules such as palmitic acid, the Fc region of an immunoglobulin (e.g., the Fc region of human immunoglobulin G1; its amino acid sequence is shown in SEQ ID NO: 87 and FIG. 95), the CH3 domain of an immunoglobulin, the CH4 domain of an immunoglobulin, albumin or a fragment thereof, an albumin-binding peptide, an albumin-binding protein such as streptococcal protein G, transferrin, etc. can be exemplified.

[0075] In certain embodiments of the present invention, the conjugate is a fusion of the SPINK2 variant peptide of the present invention with the Fc region or a fragment thereof of an antibody. The origin of the antibody can be human and non-human animals, such as rodents such as mice, rats, rabbits, etc., cows, pigs, dogs, cynomolgus monkeys, marmosets, rhesus monkeys and other mammals, birds such as chickens, etc., preferably human. Examples of antibodies include IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD and IgE, preferably IgG1. More preferably, the conjugate is a fusion of the peptide of the present invention with the Fc region or a fragment thereof of human IgG1. The fusion of the peptide of the present invention with the Fc region or a fragment thereof of an antibody may be described as an "Fc fusion" or a "conjugate", both of which are synonymous. In certain embodiments of the present invention, the conjugate is a fusion of the SPINK2 variant peptide of the present invention with the Fc region or a fragment thereof of an antibody. The origin of the antibody can be human and non-human animals, such as rodents such as mice, rats, rabbits, etc., cows, pigs, dogs, cynomolgus monkeys, marmosets, rhesus monkeys and other mammals, birds such as chickens, etc., preferably human. In certain embodiments of the present invention, the conjugate is a fusion of the SPINK2 variant peptide of the present invention with the Fc region or a fragment thereof of an antibody. The origin of the antibody can be human and non-human animals, such as rodents such as mice, rats, rabbits, etc., cows, pigs, dogs, cynomolgus monkeys, marmosets, rhesus monkeys and other mammals, birds such as chickens, etc., preferably human. In certain embodiments of the present invention, the conjugate is a fusion of the SPINK2 variant peptide of the present invention with the Fc region or a fragment thereof of an antibody. The origin of the antibody can be human and non-human animals, such as rodents such as mice, rats, rabbits, etc., cows, pigs, dogs, cynomolgus monkeys, marmosets, rhesus monkeys and other mammals, birds such as chickens, etc., preferably human. In certain embodiments of the present invention, the conjugate is a fusion of the SPINK2 variant peptide of the present invention with the Fc region or a fragment thereof of an antibody. The origin of the antibody can be human and non-human animals, such as rodents such as mice, rats, rabbits, etc., cows, pigs, dogs, cynomolgus monkeys, marmosets, rhesus monkeys and other mammals, birds such as chickens, etc., preferably human. In certain embodiments of the present invention, the conjugate is a fusion of the SPINK2 variant peptide of the present invention with the Fc region or a fragment thereof of an antibody. The origin of the antibody can be human and non-human animals, such as rodents such as mice, rats, rabbits, etc., cows, pigs, dogs, cynomolgus monkeys, marmosets, rhesus monkeys and other mammals, birds such as chickens, etc., preferably human. In certain embodiments of the present invention, the conjugate is a fusion of the SPINK2 variant peptide of the present invention with the Fc region or a fragment thereof of an antibody. The origin of the antibody can be human and non-human animals, such as rodents such as mice, rats, rabbits, etc., cows, pigs, dogs, cynomolgus monkeys, marmosets, rhesus monkeys and other mammals, birds such as chickens, etc., preferably human. In certain embodiments of the present invention, the conjugate is a fusion of the SPINK2 variant peptide of the present invention with the Fc region or a fragment thereof of an antibody. The origin of the antibody can be human and non-human animals, such as rodents such as mice, rats, rabbits, etc., cows, pigs, dogs, cynomolgus monkeys, marmosets, rhesus monkeys and other mammals, birds such as chickens, etc., preferably human. In certain embodiments of the present invention, the conjugate is a fusion of the SPINK2 variant peptide of the present invention with the Fc region or a fragment thereof of an antibody. The origin of the antibody can be human and non-human animals, such as rodents such as mice, rats, rabbits, etc., cows, pigs, dogs, cynomolgus monkeys, marmosets, rhesus monkeys and other mammals, birds such as chickens, etc., preferably human. In certain embodiments of the present invention, the conjugate is a fusion of the SPINK2 variant peptide of the present invention with the Fc region or a fragment thereof of an antibody. The origin of the antibody can be human and non-human animals, such as rodents such as mice, rats, rabbits, etc., cows, pigs, dogs, cynomolgus monkeys, marmosets, rhesus monkeys and other mammals, birds such as chickens, etc., preferably human.

[0076] As the Fc region of human IgG1, for example, the amino acid shown in SEQ ID NO: 87 (FIG. 95) Examples include, but are not limited to, those containing or consisting of an acid sequence. Antibodies The Fc region of the may be either wild-type or mutant.

[0077] In addition, the KLK5 inhibitory peptide, KLK5 / KLK7 inhibitory peptide, or KL K5 / KLK14 inhibitory peptide of the present invention may be conjugated with other drugs in order to exert or enhance pharmacological activity. In the field of antibodies, techniques and embodiments known to those skilled in the art as antibody-drug conjugates (ADCs) may become part of the present invention by replacing the antibody with the peptide of the present invention.

[0078] The KLK5 inhibitory peptide, KLK5 / KLK7 inhibitory peptide, or KLK5 / KLK14 inhibitory peptide of the present invention may further include one or more moieties that exhibit binding affinity, inhibitory activity, antagonistic activity, agonistic activity, etc. for target molecules other than KLK5, KLK5 and KLK7, or KLK5 and KLK14, or may be conjugated to such moieties. Examples of such "moieties" include antibodies or fragments thereof, proteins having a backbone other than antibodies such as SPINK2 variants or fragments thereof. In the field of antibodies Techniques and embodiments known to those skilled in the art as multispecific antibodies and bispecific antibodies in the art may become part of the conjugates of the present invention by replacing at least one of the two or more "antibodies" included therein with the peptide 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 is useful for delivering the polypeptide to a specific compartment of a cell, e.g., the periplasm in Escherichia coli or the endoplasmic reticulum in eukaryotic cells. Many signal sequences are known to those skilled in the art and can be selected according to the host cell. As a signal sequence for secreting a desired peptide into the periplasm of Escherichia coli, OmpA can be exemplified. A form containing a signal sequence may also be included as a part of the conjugate of the present invention.

[0080] Also, by pre-attaching a tag to the KLK5 inhibitory peptide, KLK5 / KLK7 inhibitory peptide, or KLK5 / KLK14 inhibitory peptide of the present invention, the peptide can be purified by affinity chromatography.

[0081] The peptide of the present invention can contain, for example, at its C-terminus, biotin, Strep tag (registered trademark), Strep tag II (registered trademark), oligo-histidine such as His6, poly-histidine, immune globulin domain, maltose binding protein, glutathione-S-transferase (GST), calmodulin binding peptide (CBP), haptens such as digoxigenin and dinitrophenol, epitope tags such as FLAG (registered trademark), myc tag, HA tag, etc. (hereinafter collectively referred to as "affinity tag"). The tagged adduct can also be included as a part of the conjugate of the present invention. The conjugate of the present invention may be a peptide (polypeptide) as a whole.

[0082] ​​​​​​​​​​​​​​​ The KLK5 inhibitory peptide, KLK5 / KLK7 inhibitory peptide, or KLK5 / K LK14 inhibitory peptide of the present invention may contain a moiety for labeling, specifically, an enzyme label, a radioactive label, a coloring label, a fluorescent label, a chromogenic label, a luminescent label, a hapten, digoxigenin, bi otin, a metal complex, a metal, a colloidal gold, or other labeling moieties may be conjugated. An embodiment containing a moiety for labeling may also be included as a part of the conjugate of the present invention. .

[0083] Examples of the amino acid sequences of the conjugate of the KLK5 inhibitory peptide, the conjugate of the KLK5 / KLK7 inhibitory peptide, or the conjugate of the KLK5 / KLK14 inhibitory peptide of the present invention include, for example, any one of the following (a1) to (a4), (b1) to (b4), or (c1) to ( c4): (a1) An amino acid sequence represented by any one of SEQ ID NOs: 34, 36, 38, 40, 42, 44, 46, 48, and 96 (FIGS. 42, 44, 46, 48, 50, 52, 54, 56, and 106); (a2) An amino acid sequence that hybridizes under stringent conditions to a nucleotide sequence complementary to the nucleotide sequence encoding the amino acid sequence described in (a1), and is encoded by a nucleotide sequence encoding an amino acid sequence contained in a peptide having KLK5 inhibitory activity; (a3) In the amino acid sequence described in (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 amino acids are substituted, deleted, added, and / or inserted, and the resulting amino acid sequence has KLK5 inhibitory activity; The amino acid sequence contained in the peptide having the property; and, (a4)(a1) The amino acid sequence described above is 60%, 70%, 80%, 85%, 90%, 9 2%, 94%, 96%, 97%, 98% or 99% or more identical, and the KLK5 inhibitory activity The amino acid sequence contained in the peptide having the property, (b1) Any one of SEQ ID NOs: 50, 52, 54 and 56 (Figs. 58, 60, 62 and 64) The amino acid sequence represented by one of them; (b2) A nucleotide sequence complementary to the nucleotide sequence encoding the amino acid sequence described in (b1) Hybridizes under stringent conditions with the nucleotide sequence, and the nucleotide sequence encoding the amino acid sequence contained in the peptide having KLK5 / KLK7 inhibitory activity Encoded amino acid sequence; (b3) In the amino acid sequence described in (b1), 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 1 amino acid is substituted, deleted, added and / or inserted, and the amino acid sequence contained in the peptide having KLK5 / KL K7 inhibitory activity; and, (b4)(b1) The amino acid sequence described above is 60%, 70%, 80%, 85%, 90%, 9 2%, 94%, 96%, 97%, 98% or 99% or more identical, and the KLK5 / KL K7 inhibitory activity-containing peptide amino acid sequence, or, (c1) The amino acid sequence represented by any one of SEQ ID NOs: 58 and 60 (Figs. 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, and KLK5 / KLK14 And the nucleotide sequence encoding the amino acid sequence contained in the peptide having inhibitory activity By a nucleotide sequence encoding an amino acid sequence contained in a peptide having inhibitory activity The encoded amino acid sequence; (c3)(c1) In the amino acid sequence described above, 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 KLK5 / KL The amino acid sequence contained in the peptide having K14 inhibitory activity; and, (c4)(c1) 60%, 70%, 80%, 85%, 90%, 9 2%, 94%, 96%, 97%, 98% or 99% or more identical, and KLK5 / KL The amino acid sequence contained in the peptide having K14 inhibitory activity.

[0084] The KLK5 inhibitory peptide, KLK5 / KLK7 inhibitory peptide, or KLK5 / K LK14 inhibitory peptide (amino acid sequence) of the present invention may contain any of natural amino acids and non-natural amino acids, and natural amino acids may include any of L-amino acids and D-amino acids.

[0085] The KLK5 inhibitory peptide, KLK5 / KLK7 inhibitory peptide, or KLK5 / K LK14 inhibitory peptide of the present invention may exist as a monomer, dimer, trimer or higher oligomer or multimer. Dimers, trimers or higher oligomers and multimers may be either homo composed of a single monomer or hetero composed of two or more different monomers. Monomers For example, may rapidly diffuse and be excellent in penetrating into tissues. Dimers, oligomers And multimers, for example, have high affinity or binding activity for target molecules locally ​​​​either have a slow dissociation rate or have excellent aspects such as high KLK5 inhibitory activity, KLK5 / KLK7 inhibitory activity or KLK5 / KLK14 inhibitory activity. In addition to spontaneous dimerization, oligomerization and multimerization, intentional dimerization, oligomerization and multimerization can also be achieved by introducing a jun-fos domain, leucine zipper, etc. into the peptides of the present invention. The KLK5 inhibitory peptide, KLK5 / KLK7 inhibitory peptide or KLK5 / KLK14 inhibitory peptide of the present invention can be a monomer, dimer, trimer or higher oligomer or multimer, and can bind to one or more target molecules or inhibit the activity of target molecules. In addition to spontaneous dimerization, oligomerization and multimerization, intentional dimerization, oligomerization and multimerization can also be achieved by introducing a jun-fos domain, leucine zipper, etc. into the peptides of the present invention. The KLK5 inhibitory peptide, KLK5 / KLK7 inhibitory peptide or KLK5 / KLK14 inhibitory peptide of the present invention can be a monomer, dimer, trimer or higher oligomer or multimer, and can bind to one or more target molecules or inhibit the activity of target molecules. In addition to spontaneous dimerization, oligomerization and multimerization, intentional dimerization, oligomerization and multimerization can also be achieved by introducing a jun-fos domain, leucine zipper, etc. into the peptides of the present invention.

[0086] The KLK5 inhibitory peptide, KLK5 / KLK7 inhibitory peptide or KLK5 / KLK14 inhibitory peptide of the present invention can be a monomer, dimer, trimer or higher oligomer or multimer, and can bind to one or more target molecules or inhibit the activity of target molecules. The forms that the KLK5 inhibitory peptide, KLK5 / KLK7 inhibitory peptide or KLK5 / KLK14 inhibitory peptide of the present invention can take include isolated forms (lyophilized preparations, solutions, etc.), the above-mentioned conjugate forms, forms bound to other molecules (immobilized forms, aggregates with different molecules, forms bound to target molecules, etc.), etc., but are not limited thereto, and forms suitable for expression, purification, use, storage, etc. can be arbitrarily selected. The KLK5 inhibitory peptide, KLK5 / KLK7 inhibitory peptide or KLK5 / KLK14 inhibitory peptide of the present invention can be a monomer, dimer, trimer or higher oligomer or multimer, and can bind to one or more target molecules or inhibit the activity of target molecules.

[0087] The forms that the KLK5 inhibitory peptide, KLK5 / KLK7 inhibitory peptide or KLK5 / KLK14 inhibitory peptide of the present invention can take include isolated forms (lyophilized preparations, solutions, etc.), the above-mentioned conjugate forms, forms bound to other molecules (immobilized forms, aggregates with different molecules, forms bound to target molecules, etc.), etc., but are not limited thereto, and forms suitable for expression, purification, use, storage, etc. can be arbitrarily selected. The forms that the KLK5 inhibitory peptide, KLK5 / KLK7 inhibitory peptide or KLK5 / KLK14 inhibitory peptide of the present invention can take include isolated forms (lyophilized preparations, solutions, etc.), the above-mentioned conjugate forms, forms bound to other molecules (immobilized forms, aggregates with different molecules, forms bound to target molecules, etc.), etc., but are not limited thereto, and forms suitable for expression, purification, use, storage, etc. can be arbitrarily selected. The forms that the KLK5 inhibitory peptide, KLK5 / KLK7 inhibitory peptide or KLK5 / KLK14 inhibitory peptide of the present invention can take include isolated forms (lyophilized preparations, solutions, etc.), the above-mentioned conjugate forms, forms bound to other molecules (immobilized forms, aggregates with different molecules, forms bound to target molecules, etc.), etc., but are not limited thereto, and forms suitable for expression, purification, use, storage, etc. can be arbitrarily selected. The forms that the KLK5 inhibitory peptide, KLK5 / KLK7 inhibitory peptide or KLK5 / KLK14 inhibitory peptide of the present invention can take include isolated forms (lyophilized preparations, solutions, etc.), the above-mentioned conjugate forms, forms bound to other molecules (immobilized forms, aggregates with different molecules, forms bound to target molecules, etc.), etc., but are not limited thereto, and forms suitable for expression, purification, use, storage, etc. can be arbitrarily selected. The forms that the KLK5 inhibitory peptide, KLK5 / KLK7 inhibitory peptide or KLK5 / KLK14 inhibitory peptide of the present invention can take include isolated forms (lyophilized preparations, solutions, etc.), the above-mentioned conjugate forms, forms bound to other molecules (immobilized forms, aggregates with different molecules, forms bound to target molecules, etc.), etc., but are not limited thereto, and forms suitable for expression, purification, use, storage, etc. can be arbitrarily selected.

[0088] 3. Identification of KLK5 inhibitory peptide, KLK5 / KLK7 inhibitory peptide, and KLK5 / KLK14 inhibitory peptide The KLK5 inhibitory peptide, KLK5 / KLK7 inhibitory peptide, and KLK5 / KLK14 inhibitory peptide are based on the amino acid sequence of SPINK2 or the amino acids of the KLK5 inhibitory peptide, KLK5 / KLK7 inhibitory peptide, and KLK5 / KLK14 inhibitory peptide of the present invention. The KLK5 inhibitory peptide, KLK5 / KLK7 inhibitory peptide, and KLK5 / KLK14 inhibitory peptide are based on the amino acid sequence of SPINK2 or the amino acids of the KLK5 inhibitory peptide, KLK5 / KLK7 inhibitory peptide, and KLK5 / KLK14 inhibitory peptide of the present invention. The KLK5 inhibitory peptide, KLK5 / KLK7 inhibitory peptide, and KLK5 / KLK14 inhibitory peptide are based on the amino acid sequence of SPINK2 or the amino acids of the KLK5 inhibitory peptide, KLK5 / KLK7 inhibitory peptide, and KLK5 / KLK14 inhibitory peptide of the present invention. The KLK5 inhibitory peptide, KLK5 / KLK7 inhibitory peptide, and KLK5 / KLK14 inhibitory peptide are based on the amino acid sequence of SPINK2 or the amino acids of the KLK5 inhibitory peptide, KLK5 / KLK7 inhibitory peptide, and KLK5 / KLK14 inhibitory peptide of the present invention. ​An amino acid sequence (for example, the amino acid sequence described in the above (a1), (b1), or (c1)), the nucleotide sequence encoding the amino acid sequence, a nucleic acid molecule containing the nucleotide sequence, etc. can be identified by methods well known to those skilled in the art starting from the material. As a preferred example, from a human SPINK2 mutant library, KLK5 inhibitory activity, KLK5 / KLK7 inhibitory activity or KLK5 / KLK14 inhibitory activity can be respectively identified using them as indicators, and K LK5, KLK5 / KLK7, or the binding activity to KLK5 / KLK14 can also be used as indicators in combination respectively.

[0089] For example, the nucleic acid molecule as the starting material is subjected to mutagenesis and can be introduced into an appropriate bacterial host or eukaryotic host using recombinant DNA technology. The SPINK2 mutant library is known as a technique for identifying binders and inhibitors of target molecules. For example, the disclosure in WO2 014 / 024914 is also included in the disclosure of the present invention by reference in its entirety. After expressing the nucleotide sequence subjected to mutagenesis in an appropriate host, clones in which an SPINK2 mutant having the desired properties, activities, functions, etc. is linked to its genetic traits can be concentrated and / or selected from the above library and identified. For the concentration and / or selection of clones, the bacterial display method (Francisco, J.A. , et al. (1993) Proc. Natl. Acad. Sci. U. S.A. 90, 10444 - 10448), the yeast display method (Boder, E .T., et al. (1997) Nat. Biotechnol. 15, 5 53 - 557), the mammalian cell display method (Ho M, et al. (2009 , et al. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 10444 - 10448), the yeast display method (Boder, E.T., et al. (1997) Nat. Biotechnol. 15, 553 - 557), the mammalian cell display method (Ho M, et al. (2009 S.A. 90, 10444 - 10448), the yeast display method (Boder, E.T., et al. (1997) Nat. Biotechnol. 15, 553 - 557), the mammalian cell display method (Ho M, et al. (2009 .T., et al. (1997) Nat. Biotechnol. 15, 5 53 - 557), the mammalian cell display method (Ho M, et al. (2009 (Year) Methods Mol Biol. Vol. 525, pp. 337-352), phage de isplay method (Smith, G.P. (1985) Science. Vol. 228, 13 15-1317), ribosome display method (Mattheakis LC, et al. (1994) Proc. Natl. Acad. Sci. U.S.A . Vol. 91, No. 19, pp. 9022-9029), nucleic acid display such as mRNA display method (Nemoto N, et al. (1997) FEBS Lett. 414 Vol. 2, pp. 405-408), colony screening method (Pini, A. et al . (2002) Comb. Chem. High Throughput Scre en. Vol. 5, pp. 503-510), etc. By using methods known to those skilled in the art, the selection The nucleotide sequence of the SPINK2 variant contained in the identified clone is determined By this, the amino acid sequence encoded by the nucleotide sequence is included in the clone The SPINK2 variant, that is, the KLK5 inhibitory peptide, the KLK5 / KLK7 inhibitory peptide Or, it can be determined as the amino acid sequence of the KLK5 / KLK14 inhibitory peptide .

[0090] The SPINK2 variant of the present invention can be obtained, for example, by inducing mutations in the natural-type SPINK2 . "Inducing mutations" means substituting or deleting one Or two or more amino acids present at each position of a certain amino acid sequence with other amino acids, or deleting them, or inserting amino acids that do not exist in the Amino acid sequence so that it can be inserted. Due to such deletions or insertions, the sequence length may change. In the SPINK2 variant of the present invention ​ wherein the induction of mutations preferably occurs at one or more positions among X1 to X in the amino acid sequence represented by SEQ ID NO: 61 (FIG. 69). 12 can occur at one or two or more positions of

[0091] However, after such a preferred induction of mutations, X1 to X 12 at one or two or more positions of wherein the same amino acid as that present at a specific position in the natural amino acid, i.e., the natural amino acid sequence, is maintained, and at least one amino acid is mutated as a whole is included in the range of mutants. Similarly, in certain embodiments of the present invention, after inducing mutations at one or more positions other than X1 to X1 2, if the same amino acid as that present at a specific position in the natural amino acid, i.e., the natural amino acid sequence, is maintained at that position, and at least one amino acid is mutated as a whole is included in the range of mutants. That is, those in which the same amino acid as that present at a specific position in the natural amino acid sequence is maintained are also included in the range of mutants as long as at least one amino acid is mutated as a whole.

[0092] "Induction of random mutations" means introducing one or two or more different amino acids at a specific position on the sequence with a certain probability by inducing mutations, but the probabilities of introducing at least two different amino acids do not all have to be the same. Also in the present invention, the inclusion of a natural amino acid (one kind) in at least two different amino acids is not precluded, and such cases are also included in the scope of "induction of random mutations".

[0093] As a method for inducing random mutations at a specific position, standard methods known to those skilled in the art can be used. For example, a synthetic nucleotide composition containing degenerate nucleotides is introduced at a specific position in the sequence. ​​​Mutations can be induced by PCR (polymerase chain reaction) using a mixture of oligonucleotides. For example, if the codon NNK or NNS ( N = adenine, guanine, cytosine or thymine; K = guanine or thymine; S = adenine or cytosine) is used, in addition to all 20 natural amino acids, a stop codon is introduced and mutations are induced, whereas if the codon VVS (V = adenine, guanine or cytosine) is used Cys, Ile, Leu, Met, Phe, Trp, Tyr and Val cannot be introduced, and mutations are induced for the introduction of the remaining 12 natural amino acids. Also, for example, if the codon NMS (M = adenine or cytosine) is used, Arg, Cys, Gly, I le, Leu, Met, Phe, Trp and Val cannot be introduced, and mutations are induced for the introduction of the remaining 11 natural amino acids. To induce mutations for the introduction of unnatural amino acids, special codons, artificial codons, etc. can be used. Site-directed mutagenesis can also be performed using a target including a higher-order structure and / or structural information of a peptide or a wild-type peptide from which the peptide is derived with respect to the target. In the present invention, structural information including higher-order information of the target KLK5, KLK7, or KLK14, and / or SPINK

[0094] 2 variants or wild-type SPINK2 with respect to KLK5, KLK5 and KLK7, or KLK5 and KLK14, or a complex of both can be used to introduce site-directed mutations. For example, SPINK2 having KLK5 inhibitory activity, K LK5 / KLK7 inhibitory activity, or KLK5 / KLK14 inhibitory activity ​​​​​Identify a variant, and then obtain crystals of a complex of KLK5, KLK7, or KLK14 and the SPINK2 variant and perform X-ray crystallographic analysis. Based on the analysis results, identify the epitope on the KLK5, KLK7, or KLK14 molecule to which the SPIN K2 variant binds and the paratope on the SPINK2 variant corresponding to the epi tope. Structural information obtained through such means as the above, and the correlation between the KLK5 inhibitory activity, KLK5 / KLK7 inhibitory activity, or KLK5 / KL K14 inhibitory activity may be found. Based on such structure-activity correlations , substitutions to specific amino acids at specific positions, insertions or deletions of amino acids at specific positions, etc. are designed, and the KLK5 inhibitory activity, KLK5 / KLK7 inhibitory activity, or KLK5 / KLK14 inhibitory activity can actually be confirmed. Moreover, for example, nucleotide constituent units with modified base pair specificities such as inosine can be used to induce mutations.

[0095] Furthermore, for example, error-prone PCR methods using DNA polymerases lacking proofreading functions and having a high error rate, such as Taq DNA polymerase, chemical mutagenesis, etc., can induce mutations at random positions.

[0096] The KLK5 inhibitory peptide, KLK5 / KLK7 inhibitory peptide, or KLK5 / KLK1 4 inhibitory peptide can be screened using phage display, yeast display, mammalian cell display, pha ge display, ribosome display, nucleic acid display, colony screening, etc., for each of the phage library, colony library, etc.

[0097] ​​​​​It can be concentrated and / or selected from libraries known to those skilled in the art suitable for the phage display method . Among those libraries, phagemid is included in the phage library, and cosmid etc. is included in the cosmid library. Vectors known to those skilled in the art suitable for each library and methods can be used to construct them. Such vectors may be viruses or viral vectors that infect prokaryotic cells or eukaryotic cells. Those recombinant vectors can be prepared by methods known to those skilled in the art such as genetic engineering etc.

[0098] Bacterial display is a technique in which, for example, a part of the outer membrane lipoprotein (Lpp) of Escherichia coli and the outer membrane protein OmpA are fused with a desired protein to present the desired protein on the surface of Escherichia coli. Random mutations are induced in the nucleotide sequence encoding the amino acid sequence of a certain protein, and the obtained group of DNAs is introduced into a vector suitable for bacterial display, and the bacterial cells are transformed with the vector , then a library presenting a group of randomly mutagenized proteins can be obtained on the surface of the transformed bacterial cells (Francisco, J. A., et al. (1993) Proc. Natl. Acad. Sci. U.S.A . Vol. 90, pp. 10444 - 10448).

[0099] Yeast display is a technique in which a desired protein is fused with a protein such as α - agglutinin on the outer shell of the cell surface of yeast and presented on the surface of yeast. α - agglutinin includes a C - terminal hydrophobic region presumed to be a glycosylphosphatidylinositol (GPI) anchor attachment signal, a signal sequence, an active domain, a cell wall domain, etc., and by operating them ​​​​​​​​As a result, a desired protein can be displayed on the cell surface of yeast. By inducing random mutations in the nucleotide sequence encoding the amino acid sequence of a certain protein, a group of DNAs obtained can be introduced into a vector suitable for yeast display, and if yeast cells are transformed with the vector, a library presenting a group of proteins induced with random mutations can be obtained on the surface of the transformed yeast cells (Ueda, M. & Tanaka, A., Biotechnol. Adv., Vol. 18, pp. 121-, published in 2000; Ueda, M. & Tanaka, A., J. Biosci. Bioeng., Vol. 90, pp. 125-, published in 2000, etc.). Animal cell display is a technique in which, for example, the transmembrane region of a membrane protein typified by platelet-derived growth factor receptor (PDGFR) is fused with a desired protein, and the desired protein is presented on the surface of mammalian cells such as HEK293 and Chinese hamster ovary (CHO) cells. By inducing random mutations in the nucleotide sequence encoding the amino acid sequence of a certain protein, a group of DNAs obtained can be introduced into a vector suitable for animal cell display, and if animal cells are transformed with the vector, a library presenting a group of proteins induced with random mutations can be obtained on the surface of the transformed animal cells (Ho M, et al. (2009) Methods Mol Biol. 525, 337 - 352). A desired library presented on cells such as yeast, bacteria, and animal cells can be incubated in the presence of a target molecule or contacted with the target molecule. For example, it can be modified with biotin or the like.

[0100]

[0101] ​​​​​​​​​​​​​​​Cells containing the produced KLK5, KLK7 or KLK14 and a library were incubated for a certain period of time. After that, a carrier such as magnetic beads is added, the cells are separated from the carrier, and then the carrier is washed. To remove more non-specific adsorbates and conjugates, the peptide, peptide aggregate or concentrated peptide aggregate bound to the carrier (KLK5, KLK7 or K LK14) can be presented. The cell population with the presented can be recovered. Similarly, after adding magnetic beads, magnetic cell separation (MA CS) is performed, or after cell staining with an anti-KLK5 antibody, anti-KLK7 antibody or anti-KLK14 antibody, FACS is carried out, and the carrier (KLK5, KLK7 or K LK14) bound to the carrier, or the cell population with the peptide, peptide aggregate or concentrated peptide aggregate bound to KLK5, KLK7 or KLK14 can be recovered. Non specific adsorption sites and / or binding sites can, for example, also be blocked, and the blocking step can be incorporated if it is an appropriate method. The peptide, peptide aggregate or concentrated peptide aggregate obtained in this way expressing vector is recovered, 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. Also, the vector is introduced into the host cell again, and the above operations are repeated one to several times as a cycle, so that the peptide aggregate binding to the target molecule can be concentrated to a higher degree. In the case of phage display, for example, a phagemid is a bacterial plasmid containing, in addition to a plasmid replication origin, a second replication origin derived from a single-stranded bacteriophage. By introducing the vector into the host cell again and repeating the above operations one to several times as a cycle, the peptide aggregate binding to the target molecule can be concentrated to a higher degree.

[0102] In the case of phage display, for example, a phagemid is a bacterial plasmid containing, in addition to a plasmid replication origin, a second replication origin derived from a single-stranded bacteriophage. Cells having phagemids can replicate the phagemids via a single-stranded replication mode in superinfection by M13 or a helper bacteriophage similar thereto. That is, single-stranded phagemid DNA is packaged into infectious particles coated with bacteriophage coat proteins. In this way, the phagemid DNA can be formed as a cloned double-stranded DNA plasmid in infected bacteria, and the phagemid can be formed as bacteriophage-like particles from the culture supernatant of superinfected cells, respectively. By injecting the bacteriophage-like particles into bacteria to infect the DNA of bacteria having F pili, the particles themselves can be re-formed as plasmids. A polynucleotide having a nucleotide sequence encoding the amino acid sequence of a test peptide and a fusion gene composed of a bacteriophage coat protein gene are inserted into the phagemid and the bacteria are infected, and if the cells are cultured, such a peptide can be expressed or presented (synonymous with display) on the bacteria or phage-like particles, or produced as a fusion protein with the coat protein in phage particles or in the culture supernatant of the bacteria. For example, a fusion gene composed of the polynucleotide and the bacteriophage coat protein gene gpIII is inserted into the phagemid and co-infected with M13 or a helper phage similar thereto into Escherichia coli, and the fusion protein composed of the peptide and the coat protein can be produced in the culture supernatant of the Escherichia coli.

[0103]

[0104]

[0105] ​​​​​​​​​​​​​​​Instead of phagemids, various circular or linear vectors, such as viral vectors can be used. According to methods known to those skilled in the art, a peptide having an amino acid sequence encoded by the nucleotide sequence of the polynucleotide inserted into the vector is expressed or presented on the cells or virus-like particles into which the vector has been introduced, or can be produced in the culture supernatant of the cells. The library expressing the peptide thus obtained can be incubated in the presence of a target molecule or contacted with the target molecule. For example, a carrier immobilized with KLK5, KLK5

[0106] and / or KLK7, or KLK5 and / or KLK14 is incubated with a mobile phase containing the library for a certain period of time, then the mobile phase is separated from the carrier, and then the carrier is washed to remove non-specific adsorbates and conjugates, and the peptide, peptide aggregate or concentrated peptide aggregate bound to the carrier (KLK5, KLK5 and / or KLK7, or KLK5 and / or KLK14 immobilized thereon) can be recovered by elution. Elution can be performed non-selectively under relatively high ionic strength, low pH, medium denaturing conditions, in the presence of chaotropic salts, etc., or selectively by adding soluble target molecules such as KLK5, KLK7, KLK1 4, antibodies that bind to the target molecule, natural ligands, substrates, etc. to compete with the immobilized target molecule. Non-specific adsorption sites and / or binding sites can also be blocked, for example, and the blocking step can be incorporated if an appropriate method is used.

[0107] ​​​​​​The peptide, aggregate of peptides, or concentrated aggregate of peptides thus obtained is used to recover the vector expressing it, determine the nucleotide sequence of the polynucleotide inserted into the vector, and determine the amino acid sequence encoded by the nucleotide sequence. Also, the vector is introduced back into the host cell, and the above operations are repeated once to several times as a cycle, so that the aggregate of peptides binding to the target molecule can be concentrated to a higher degree.

[0108] Ribosome display is a technique for synthesizing a molecule in which a desired protein, the corresponding mRNA, and a ribosome are linked in vitro by using mRNA encoding a desired protein having no stop codon and a cell-free protein synthesis system. By using a group of mRNAs obtained by inducing random mutations in the nucleotide sequence encoding the amino acid sequence of a certain protein and a cell-free protein synthesis system, a library in which a group of randomly mutated proteins are presented on ribosomes can be obtained (Mattheakis LC, et al. (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 9022-9029). .

[0109] Nucleic acid display, also called mRNA display, is a technique for synthesizing a molecule in which a desired protein, the mRNA encoding it, and a ribosome are linked by using a linker such as puromycin having a structure similar to the 3'-end of tyrosyl tRNA. Since this technique uses a cell-free protein synthesis system instead of living cells, it can be synthesized in vitro. By introducing random mutations into the nucleotide sequence encoding the amino acid sequence of a certain protein, ​​​​​​​​​​​​​​An mRNA group obtained by inducing mutations, a linker such as puromycin, and a cell-free protein synthesis system can be used to obtain a library in which a group of randomly mutated proteins are presented on ribosomes (Nemoto N, et al. (1997) FEBS Lett. Vol. 414, No. 2, pp. 405-408).

[0110] A library expressing peptides obtained through a cell-free synthesis system such as ribosome display or nucleic acid display can be incubated in the presence of a target molecule or contacted with the target molecule. For example, a carrier immobilized with KLK5, KLK5 and / or KLK7, or KLK5 and / or KLK14 is incubated with a mobile phase containing the library for a certain period of time, then the mobile phase is separated from the carrier, and then the carrier is washed to remove non-specific adsorbates and conjugates, and the peptides, peptide aggregates or concentrated peptide aggregates bound to the carrier (KLK5, KLK5 and / or K LK7, or KLK5 and / or KLK14 bound thereto) can be recovered by elution. Elution can be carried out non-selectively under relatively high ionic strength, low pH, medium denaturing conditions, in the presence of chaotropic salts, etc., or selectively by adding a soluble target molecule such as KLK5, KLK7, KLK14, an antibody that binds to the target molecule, a natural ligand, a substrate, etc. to compete with the immobilized target molecule . Non-specific adsorption sites and / or binding sites can also be blocked, for example, and the blocking step can be incorporated in an appropriate manner if appropriate.

[0111] ​​​​​The peptide, aggregate of peptides, or concentrated aggregate of peptides thus obtained is used to recover the nucleic acid that is expressed. In the case of mRNA, after reverse transcription into cDNA, the nucleotide sequence can be determined, and the amino acid sequence encoded by the nucleotide sequence can be determined. Further, mRNA can be transcribed from the recovered nucleic acid, and the above operations can be repeated once or several times as a cycle to more highly concentrate the aggregate of peptides that binds to the target molecule.

[0112] If an affinity tag is conjugated in advance to the peptide, aggregate of peptides, or concentrated aggregate of peptides, the peptide or the aggregate thereof can be efficiently purified. For example, if a substrate of a protease is conjugated in advance to the aggregate of peptides as a tag, the peptide can be eluted by cleavage with the protease activity.

[0113] Based on the obtained sequence information and functions of the peptides, etc., further mutations are induced in the obtained clone or library, and from the library into which mutations have been introduced, peptides having improved functions (for example, KLK5 inhibitory activity, KLK5 / KLK7 inhibitory activity, or KLK5 / KLK14 inhibitory activity), physical properties (thermal stability, storage stability, etc.), pharmacokinetics (distribution, blood half-life), etc. can also be obtained.

[0114] By determining whether the obtained peptide has KLK5 inhibitory activity, KLK5 / KLK7 inhibitory activity, or KLK5 / KLK14 inhibitory activity, a KLK5 inhibitory peptide, a KLK5 / KLK7 inhibitory peptide, or a KLK5 / KLK14 inhibitory peptide can be identified, respectively. ​

[0115] In addition, a KLK5 inhibitor peptide, a KLK5 / KLK7 inhibitor peptide, or a KLK5 / K The LK14 inhibitor peptide is preferably a peptide having a length of 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 and Gly at position 32 β strand (1) and β strand (2) consisting of Ile 57 to Arg 59 The β-sheet is composed of Glu41 and the α-helix is ​​composed of Glu41 to Gly51. or similar thereto or at least partially corresponding thereto (the location of) The three-dimensional structure consisting of loop structures, β-sheets, α-helices, etc. that act as agonists for KLK5 inhibition. 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 above, more preferred KLK5 inhibitor peptides, KLK5 / KLK7 inhibitor peptides, or K It is also possible to identify LK5 / KLK14 inhibitory peptides.

[0116] Furthermore, the present invention provides a method for the production of KLK5 inhibitor peptides using a SPINK2 mutant library. Identification of KLK5 / KLK7 inhibitory peptide or KLK5 / KLK14 inhibitory peptide Regarding the method, the method described in (75) above can be exemplified. Using the seed compound library, KLK5 inhibitors and KLK5 / KLK7 inhibitors 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. then, if the enzyme inhibitory activity of the test compound is equal to or stronger than the enzyme inhibitory activity of the reference compound or control, the compound is determined to be positive, and if it is weaker, the compound is determined to be negative. This may be done, and as a method involving such comparison, the above (77) can be exemplified. On the other hand, in any test involving the step of measuring the protease activity of KLK5 and optionally KLK7 or KLK14, the peptide or conjugate of the present invention can be used as a reference compound, control, etc., and such test methods are also included in the present invention. Such tests are not particularly limited, and the above (78) can be exemplified, but the diagnostic methods, inspection methods, detection methods, and methods for identifying individuals to whom the pharmaceutical composition is administered (all of which will be described later ) can also be mentioned as preferred examples.

[0117] 4. Nucleic acid molecules encoding the peptide or conjugate of the present invention, vectors containing the same, cells containing them, and methods for producing recombinant peptides or conjugates The present invention relates to polynucleotides containing nucleotide sequences encoding the amino acid sequences contained in KLK5 inhibitory peptides, KLK5 / KLK7 inhibitory peptides, or KLK5 / KLK14 inhibitory peptides (hereinafter, each referred to as "nucleic acid molecule encoding KLK5 inhibitory peptide", " nucleic acid molecule encoding KLK5 / KLK7 inhibitory peptide" or "nucleic acid molecule encoding KLK5 / KLK14 inhibitory peptide"), recombinant vectors into which the gene has been inserted, and cells into which the gene or vector has been introduced (hereinafter, "nucleic acid molecule-containing cell encoding KLK5 inhibitory peptide", "nucleic acid molecule-containing cell encoding KLK5 / KLK7 inhibitory peptide "), "nucleic acid molecule-containing cell encoding KLK5 / KLK14 inhibitory peptide "), 」or「a cell containing a nucleic acid molecule encoding a KLK5 / KLK14 inhibitory peptide」) A KLK5 inhibitory peptide, a KLK5 / KLK7 inhibitory peptide, or a KLK5 / KLK14 inhibitory peptide-producing cell (hereinafter, each referred to as a「KLK5 inhibitory peptide-producing cell」,「K LK5 / KLK7 inhibitory peptide-producing cell」or「KLK5 / KLK14 inhibitory peptide-producing cell」) is also provided.

[0118] As suitable examples of the nucleic acid molecule encoding the KLK5 inhibitory peptide, the KLK5 / KLK7 inhibitory peptide or the nucleic acid molecule encoding the KLK5 / KLK14 inhibitory peptide of the present invention, respectively, the following (a1) to (a4), (b1) to (b4) or (c1) to (c4) any one of the nucleotide sequences described (hereinafter, each referred to as a「nucleotide sequence of the KLK 5 inhibitory peptide」,「nucleotide sequence of the KLK5 / KLK7 inhibitory peptide」or「nucleotide sequence of the KLK5 / KLK14 inhibitory peptide」) is included, consists of a nucleotide sequence including the nucleotide sequence of the KLK5 inhibitory peptide, the nucleotide sequence of the KLK5 / KLK7 inhibitory peptide or the nucleotide sequence of the KLK5 / KLK14 inhibitory peptide, or consists of a nucleotide sequence consisting of the nucleotide sequence of the KLK5 inhibitory peptide, the nucleotide sequence of the KLK5 / KLK7 inhibitory peptide or the nucleotide sequence of the KLK5 / KLK14 inhibitory peptide can be mentioned: (a1) The nucleotide number of the nucleotide sequence encoding the amino acid sequence consisting of amino acid numbers 1 to 63 of the 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) The nucleotide sequence of the KLK5 inhibitory peptide, the nucleotide sequence of the KLK5 / KLK7 inhibitory peptide or the KLK5 / KLK14 inhibitory peptide nucleotide sequence consisting of (a1) The nucleotide sequence encoding the amino acid sequence consisting of amino acid numbers 1 to 63 of the 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) The nucleotide sequence encoding the amino acid sequence consisting of amino acid numbers 1 to 63 of the amino acid sequence represented by any one of SEQ ID NOs: 6, 8, 10, 12, 14, 16, 18 and 20 (FIGS. 14, 16, 18 A nucleotide sequence consisting of 1 to 189, or SEQ ID NO: 5, 7, 9, 11, 13, 15 , any one of 17 and 19 (FIGS. 13, 15, 17, 19, 21, 23, 25 and 27) the nucleotide sequence described in any one of them; (a2)(a1) A nucleotide sequence complementary to the nucleotide sequence described in (a1) hybridizes under stringent conditions and encodes an amino acid sequence contained in a peptide having KLK5 inhibitory activity; a nucleotide sequence; (a3)(a1) In the nucleotide sequence described in (a1), 1 to 20, 1 to 15, 1 to 10, 1 to 8, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 or 2, or 1 nucleotide or nucleotide residue is substituted, deleted, added and / or inserted and encodes an amino acid sequence contained in a peptide having KLK5 inhibitory activity a nucleotide sequence; and, (a4)(a1) 60%, 70%, 80%, 85%, 90% with the nucleotide sequence described in (a1), 92%, 94%, 96%, 97%, 98% or 99% or more identical, and KLK5 inhibition a nucleotide sequence encoding an amino acid sequence contained in a peptide having activity: (b1) A nucleotide sequence encoding an amino acid sequence consisting of amino acid numbers 1 to 63 of the amino acid sequence represented by any one of SEQ ID NOs: 22, 24, 26 and 28 (FIGS. 30, 32, 34 and 36), or SEQ ID NOs: 21, 23, 25 and 27 (FIGS. 29, 31, 33 and 35) any one of the nucleotide sequences described in any one of them, consisting of nucleotide numbers 1 to 189 a nucleotide sequence; from; a nucleotide sequence; (b2)(b1) A nucleotide sequence complementary to the nucleotide sequence described in (b1) hybridizes under stringent conditions and has a peptide having KLK5 / KLK7 inhibitory activity A nucleotide sequence encoding the amino acid sequence contained therein; (b3)(b1) In the nucleotide sequence described, 1 to 20, 1 to 15, 1 to 10, 1 to 8, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 or 2, or 1 nucleotide or nucleotide residue is substituted, deleted, added and / or inserted and a nucleotide sequence encoding the amino acid sequence contained in a peptide having KLK5 / KLK7 inhibitory activity; and, A nucleotide sequence encoding the amino acid sequence contained in a peptide having KLK5 / KLK7 inhibitory activity; and, (b4)(b1) 60%, 70%, 80%, 85%, 90% with, 92%, 94%, 96%, 97%, 98% or 99% or more identity, and KLK5 / A nucleotide sequence encoding the amino acid sequence contained in a peptide having KLK7 inhibitory activity: Column: (c1) A nucleotide sequence encoding the amino acid sequence consisting of amino acid numbers 1 to 63 of the amino acid sequence shown by any one of SEQ ID NOs: 30 and 32 (FIGS. 38 and 40), or A nucleotide sequence consisting of nucleotide numbers 1 to 189 of the nucleotide sequence described in SEQ ID NO: 29 or 31 (FIGS. 37 or 39); A nucleotide sequence complementary to the nucleotide sequence described in (c1) hybridizes under stringent conditions and encodes the amino acid sequence contained in a peptide having KLK5 / KLK14 inhibitory activity; A nucleotide sequence consisting of nucleotide numbers 1 to 189 of the nucleotide sequence described in SEQ ID NO: 29 or 31 (FIGS. 37 or 39); (c2)(c1) A nucleotide sequence complementary to the nucleotide sequence described in (c1) hybridizes under stringent conditions and encodes the amino acid sequence contained in a peptide having KLK5 / KLK14 inhibitory activity; A nucleotide sequence complementary to the nucleotide sequence described in (c1) hybridizes under stringent conditions and encodes the amino acid sequence contained in a peptide having KLK5 / KLK14 inhibitory activity; A nucleotide sequence encoding the amino acid sequence contained in a peptide having KLK5 / KLK14 inhibitory activity; (c3)(c1) In the nucleotide sequence described, 1 to 20, 1 to 15, 1 to 10, 1 to 8, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 or 2, or 1 nucleotide or nucleotide residue is substituted, deleted, added and / or inserted and a nucleotide sequence encoding the amino acid sequence contained in a peptide having KLK5 / KLK14 inhibitory activity A nucleotide sequence encoding the same; and, (c4)(c1) The nucleotide sequence described above is 60%, 70%, 80%, 85%, 90% , 92%, 94%, 96%, 97%, 98% or 99% or more identical, and KLK5 / A nucleotide encoding an amino acid sequence contained in a peptide having KLK14 inhibitory activity sequence.

[0119] Any one of the above (a1) to (a4), (b1) to (b4) or (c1) to (c4) The amino acid sequence encoded by the nucleotide sequence described in any one of the above or a SPINK2 variant peptide containing the amino acid sequence inhibits the protease activities of KLK5, KLK5 and KLK7, or , KLK5 and KLK14, and preferably specifically inhibits the protease activity.

[0120] Note that nucleotide numbers 190 to 195 of SEQ ID NOs: 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27 , 29 and 31 (Figs. 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 3 3, 35, 37 and 39) do not correspond to the nucleotide sequence encoding wild-type human SPIN K2 (consisting of 63 amino acids of SEQ ID NO: 1, Fig. 9), but are added in order to express the peptide of the present invention in certain embodiments of the present invention. For purposes.

[0121] However, a nucleic acid molecule encoding a KLK5 inhibitory peptide, a KLK5 / KLK7 inhibitory peptide, or a KLK K5 / KLK14 inhibitory peptide is not limited to (a1) to (a4), (b1) to (b4) or (c1) to (c4), and has KLK5 inhibitory activity, SPINK having KLK5 / KLK7 inhibitory activity or KLK5 / KLK14 inhibitory activity The amino acid sequence contained in the 2 mutant, preferably the amino acid sequence shown in SEQ ID NO: 61 (Figure 69). Nucleic acid molecules containing a nucleotide sequence encoding the sequence are everywhere KLK5 inhibitory peptide, KLK 5 / KLK7 inhibitory peptide, or nucleic acid encoding KLK5 / KLK14 inhibitory peptide molecules are included within the scope.

[0122] In addition, the present invention provides conjugates of KLK5 inhibitory peptides, conjugates of KLK5 / KLK7 inhibitory peptides, or conjugates of KLK5 / KLK14 inhibitory peptides polynucleotides containing a nucleotide sequence encoding the amino acid sequence contained therein (hereinafter, each referred to as "nucleic acid molecule encoding KLK5 inhibitory conjugate", "nucleic acid molecule encoding KLK5 / KLK7 inhibitory conjugate" or "nucleic acid molecule encoding KLK5 / KLK14 inhibitory conjugate"), recombinant vectors into which the gene has been inserted, cells into which the gene or vector has been introduced (hereinafter, "nucleic acid molecule-containing cell encoding KLK5 inhibitory conjugate", "nucleic acid molecule-containing cell encoding KLK5 / KLK7 inhibitory conjugate " or "nucleic acid molecule-containing cell encoding KLK5 / KLK14 inhibitory conjugate"), and cells producing conjugates of KLK5 inhibitory peptides, conjugates of KLK5 / KLK7 inhibitory peptides, or conjugates of KLK5 / KLK14 inhibitory peptides (hereinafter, each referred to as "KLK5 inhibitory conjugate-producing cell", "KLK5 / KLK7 inhibitory conjugate-producing cell" or "KLK5 / KLK14 inhibitory conjugate-producing cell ") are also provided.

[0123] ​​​​​​A nucleic acid molecule encoding a KLK5 inhibitory conjugate of the present invention, KLK5 / KLK7 inhibition A nucleic acid molecule encoding a conjugate, or a KLK5 / KLK14 inhibitory conjugate As preferred examples of the nucleic acid molecule encoding the conjugate, each of the following (a1) to (a4), (b1) to (b4) or (c1) to (c4) (hereinafter , each referred to as the "nucleotide sequence of the KLK5 inhibitory conjugate", "KLK5 / KLK7 inhibitory conjugate nucleotide sequence" or "KLK5 / KLK14 inhibitory conjugate nucleotide sequence"), or consists of a nucleotide sequence containing the nucleotide sequence of the KLK5 inhibitory conjugate, the nucleotide sequence of the KLK5 / KLK7 inhibitory conjugate or the nucleotide sequence of the KLK5 / KLK14 inhibitory conjugate or can be exemplified by a nucleotide sequence consisting of the nucleotide sequence of the KLK5 inhibitory conjugate, the nucleotide sequence of the KLK5 / KLK7 inhibitory conjugate or the nucleotide sequence of the KLK5 / KLK14 inhibitory peptide: (a1) The nucleotide sequence encoding the amino acid sequence shown by any one of SEQ ID NOs: 34, 36, 38, 40, 42, 44, 46, 48 and 96 (FIGS. 42, 44, 46, 48, 50, 52, 54, 56 and 106), or the nucleotide sequence described in any one of SEQ ID NOs: 33, 35, 37, 39, 41 , 43, 45, 47 and 95 (FIGS. 41, 43, 45, 47, 49, 51, 53, 55 and 105); (a2) A nucleotide sequence that hybridizes under stringent conditions with a nucleotide sequence complementary to the nucleotide sequence described in (a1), and has a peptide or conjugate having KLK5 inhibitory activity under stringent conditions, and has KLK5 inhibitory activity ; (a2) A nucleotide sequence that hybridizes with a nucleotide sequence complementary to the nucleotide sequence described in (a1) under stringent conditions and has KLK5 inhibitory activity peptide or conjugate The nucleotide sequence encoding the amino acid sequence contained in the gate; (a3)(a1) In the nucleotide sequence described, 1 to 20, 1 to 15, 1 to 10, 1 to 8, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 or 2, or 1 nucleotide or nucleotide residue is substituted, deleted, added and / or inserted and the nucleotide sequence encoding the amino acid sequence contained in the peptide or conjugate having KLK5 inhibitory activity; and, (a4)(a1) The nucleotide sequence described is 60%, 70%, 80%, 85%, 90% 92%, 94%, 96%, 97%, 98% or 99% or more identical, and the nucleotide encoding the amino acid sequence contained in the peptide or conjugate having KLK5 inhibitory activity sequence: (b1) The nucleotide sequence encoding any one of the amino acid sequences shown in SEQ ID NOs: 50, 52, 54 and 56 (Figures 58, 60, 62 and 64), or the nucleotide sequence described in any one of SEQ ID NOs: 49, 51 53 and 55 (Figures 57, 59, 61 and 63); (b2) A nucleotide sequence complementary to the nucleotide sequence described in (b1) hybridizes under stringent conditions, and the nucleotide sequence encoding the amino acid sequence contained in the peptide or conjugate having KLK5 / KLK7 inhibitory activity or conjugate; (b3)(b1) In the nucleotide sequence described, 1 to 20, 1 to 15, 1 to 10, 1 to 8, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 or 2, or 1 nucleotide or nucleotide residue is substituted, deleted, added and / or inserted and the nucleotide sequence encoding the amino acid sequence contained in the peptide or conjugate having KLK5 / KLK7 inhibitory activity or conjugate; (b3)(b1) In the nucleotide sequence described, 1 to 20, 1 to 15, 1 to 10, 1 to 8, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 or 2, or 1 nucleotide or nucleotide residue is substituted, deleted, added and / or inserted and contained in the peptide or conjugate having KLK5 / KLK7 inhibitory activity A nucleotide sequence encoding the amino acid sequence; and, (b4)(b1) The nucleotide sequence described above is 60%, 70%, 80%, 85%, 90% , 92%, 94%, 96%, 97%, 98% or 99% or more identical, and KLK5 / A nucleotide sequence encoding an amino acid sequence contained in a peptide or conjugate having KLK7 inhibitory activity: (c1) A nucleotide sequence encoding the amino acid sequence represented by any one of SEQ ID NOs: 58 and 60 (FIGS. 66 and 68), or the nucleotide sequence described in SEQ ID NO: 57 or 59 (FIGS. 65 or 67); (c2) A nucleotide sequence that hybridizes under stringent conditions with a nucleotide sequence complementary to the nucleotide sequence described in (c1), and encodes an amino acid sequence contained in a peptide or conjugate having KLK5 / KLK14 inhibitory activity; (c3) In the nucleotide sequence described in (c1), 1 to 20, 1 to 15, 1 to 10, 1 to 8, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 or 2, or 1 nucleotide or nucleotide residue is substituted, deleted, added and / or inserted, and encodes an amino acid sequence contained in a peptide or conjugate having KLK5 / KLK14 inhibitory activity; and, (c4) The nucleotide sequence described in (c1) is 60%, 70%, 80%, 85%, 90% , 92%, 94%, 96%, 97%, 98% or 99% or more identical, and KLK5 / A nucleotide sequence encoding an amino acid sequence contained in a peptide or conjugate having KLK14 inhibitory activity.

[0124] Any one of the above (a1) to (a4), (b1) to (b4), or (c1) to (c4) The amino acid sequence encoded by the nucleotide sequence described in any one of them, or the SPINK2 mutant peptide containing the amino acid sequence inhibits the protease activities of KLK5, KLK5 and KLK7, or KLK5 and KLK14, and preferably specifically inhibits the protease activity.

[0125] However, the nucleic acid molecule encoding the KLK5 inhibitory peptide, the KLK5 / KLK7 inhibitory peptide, or the KL K5 / KLK14 inhibitory peptide is not limited to (a1) to (a4), (b1) to (b4), or (c1) to (c4). The SPINK 2 mutants having KLK5 inhibitory activity, KLK5 / KLK7 inhibitory activity, or KLK5 / KLK14 inhibitory activity, preferably the amino acid sequence shown in SEQ ID NO: 61 (FIG. 69) The nucleic acid molecule encoding the amino acid sequence contained in the conjugate containing the amino acid sequence is ubiquitously a nucleic acid molecule encoding a KLK5 inhibitory conjugate, a KLK5 / KLK7 inhibitory conjugate, or a nucleic acid molecule encoding a KLK5 / KLK14 inhibitory conjugate is included in the range.

[0126] To design a nucleotide sequence encoding an amino acid sequence, one or more codons corresponding to each amino acid can be used. Therefore, the base sequence encoding a single amino acid sequence of a peptide can have multiple variations. When selecting such codons, the codon usage of the host cell for expression, into which the polynucleotide containing the nucleotide sequence or the vector containing it is introduced, is appropriately considered accordingly. ​ selecting a codon or appropriately adjusting the frequency or ratio of use of multiple codons is possible. For example, when using Escherichia coli as a host cell, a nucleotide sequence may be designed using codons with high usage frequency in Escherichia coli.

[0127] The nucleic acid molecule encoding the peptide or conjugate of the present invention may be functionally linked to one or more regulatory sequences. "Functionally linked" means that the linked nucleic acid molecule can be expressed or enables the expression of the nucleotide sequence contained in the molecule. Regulatory sequences include sequence elements containing information related to transcriptional regulation and / or translational regulation. Regulatory sequences vary depending on the species, but generally include a promoter, the -35 / -10 box and Shine-Dalgarno sequence of prokaryotes, and the TATA box, CAAT sequence, and 5' capping sequence of eukaryotes, etc., and include 5' non-coding sequences involved in the initiation of transcription and translation. Such sequences may include enhancer elements and / or repressor elements, as well as signal sequences, leader sequences, etc. that can be translated for delivering the native or mature peptide to specific compartments inside and outside the host cell. Furthermore, the regulatory sequence may include a 3' non-coding sequence, and such a sequence may include elements involved in transcription termination or polyadenylation, etc. However, if the sequence related to transcription termination does not function sufficiently in a specific host cell, it may be replaced with a sequence suitable for that cell.

[0128] Examples of promoter sequences include the tet promoter, lacUV5 promoter, T7 promoter, etc. in prokaryotes, and the SV40 promoter, CMV promoter, etc. in eukaryotic cells. The following can be given as examples:

[0129] Nucleic acid molecules encoding the peptides or conjugates of the invention may be administered in an isolated form, as a vector, or in a recombinant form. Vectors or other cloning vehicles (hereinafter simply referred to as "vectors": plasmids, In a form contained in a 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 being used, and nucleic acid sequences that have been synthesized by transformation or the like The progeny may contain a selectable marker which 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 nucleotide sequence by methods known to those skilled in the art, such as transformation into a host cell capable of expressing the sequence. The host cell into which the nucleic acid molecule or vector has been introduced can express the peptide. The host cell 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 the host cell 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 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 apply a desired modification to the gate. Such a peptide or conjugate of the gate is also included in 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 includes culturing a nucleic acid molecule-containing cell encoding a KLK5 inhibitory peptide (or a KLK5 inhibitory conjugate) or a KLK5 inhibitory peptide (or a KLK5 inhibitory conjugate)-producing cell, a nucleic acid molecule-containing cell encoding a KLK5 / KLK7 inhibitory peptide (or a KLK5 / KLK7 inhibitory conjugate) or a KLK5 / KLK7 inhibitory peptide (or a KLK5 / KLK7 inhibitory conjugate)-producing cell, or a nucleic acid molecule-containing cell encoding a KLK5 / KLK14 inhibitory peptide (or a KLK5 / KLK14 inhibitory conjugate) or a KLK5 / KLK14 inhibitory peptide (or a KLK5 / KLK14 inhibitory conjugate)-producing cell in step 1, and / or step 2 of recovering a SPINK2 variant from the culture obtained in step 1. Step 2 can apply operations such as fractionation, chromatography known to those skilled in the art, purification, etc. For example, purification by affinity chromatography using the antibody of the present invention or a binding fragment thereof described later can be applied. In some embodiments of the present invention, the peptide contained in the peptide or conjugate has an intramolecular disulfide bond. A peptide having an intramolecular disulfide bond is preferably delivered to a cellular compartment having an oxidative redox environment using a signal sequence or the like . In some embodiments of the present invention, the peptide contained in the peptide or conjugate has an intramolecular disulfide bond. A peptide having an intramolecular disulfide bond is preferably delivered to a cellular compartment having an oxidative redox environment using a signal sequence or the like

[0132] In some aspects of the present invention, the peptide contained in the peptide or conjugate has an intramolecular disulfide bond. A peptide having an intramolecular disulfide bond is preferably delivered to a cellular compartment having an oxidative redox environment using a signal sequence or the like and the like, and is preferably delivered to a cell section having an oxidative redox environment There is a combination. The oxidative environment can be provided by the periplasm of Gram-negative bacteria such as Escherichia coli, the extracellular environment of Gram-positive bacteria, the lumen of the endoplasmic reticulum of eukaryotic cells, etc. Under such an environment, the formation of structural disulfide bonds can be promoted. Also, it is possible to produce a peptide having an intramolecular disulfide bond in the cytoplasm of a host cell such as Escherichia coli. In that case, the peptide is directly obtained in a soluble folded state or recovered in the form of inclusion bodies and then can be refolded in vitro. Furthermore, a host cell having an oxidative intracellular environment can be selected, and a peptide having an intramolecular disulfide bond can be produced in its cytoplasm. On the other hand, when the peptide does not have an intramolecular disulfide bond, it can be produced in a cell compartment having a reducing oxidative reducing environment, for example, in the cytoplasm of Gram-negative bacteria. The peptide or conjugate (peptide moiety contained therein) of the present invention can be produced by chemical synthesis exemplified by Merrifield and other peptide solid-phase synthesis methods, as well as organic synthetic chemical peptide synthesis methods using t-butoxycarbonyl (t-Butoxycarb onyl: Boc), 9-fluorenylmethoxycarbonyl (9-Fluorenylm

[0133] ethoxycarbonyl: Fmoc), etc., and other methods known to those skilled in the art such as in vitro translation. The present invention provides, as some of its aspects, an antibody that binds to the peptide contained in the peptide or conjugate of the present invention and a binding fragment thereof. The antibody can be either a polyclonal antibody or a monoclonal antibody. As the monoclonal antibody, it can be an immunoglobulin or a monoclonal antibody. The present invention can be produced by other methods known to those skilled in the art, such as chemical synthesis exemplified by Merrifield and other peptide solid-phase synthesis methods, as well as organic synthetic chemical peptide synthesis methods using t-butoxycarbonyl (t-Butoxycarb onyl: Boc), 9-fluorenylmethoxycarbonyl (9-Fluorenylm

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

[0135] 5. Pharmaceutical Compositions The present invention also provides a pharmaceutical composition comprising the compound or a conjugate thereof.

[0136] The 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 aggravation of diseases, cure diseases, maintain or improve symptoms, and avoid secondary diseases. Treatment and / or administration of diseases (hereinafter referred to as "KLK5-related diseases" or "KLK5-associated diseases") The disease associated with KLK5 is, for example, 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), Sake (Yamasaki, K., et al. (20 07) Nat. Med. 13, pp. 975-980), Skin damage by ultraviolet rays (N in, M., et al. (2009) J. Dermatol. Sci. 54 , pp. 17-24), Psoriasis (Komatsu, N., et al. (2007) Br. J. Dermatol. 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, colorectal cancer, oral squamous cell carcinoma, breast cancer, head neck cancer, melanoma, prostate cancer, glioma, etc.) (Emami, N., et al. (2 007) Mol. Oncol. 1, pp. 269-287), Barrett esophagus( Gene Expression Omnibus, accession #GSE1 3083, etc.), but are not limited thereto.

[0137] KLK5 is considered to be a major factor in the development of Netherton syndrome-like skin symptoms. KLK5 is a self active protease and is also involved in the activation of KLK7 and KLK14. On the other hand, in the stratum corneum of patients with Netherton syndrome and Netherton syndrome model mice, trypsin-like and chymotrypsin-like high protease activities are observed. In addition to KLK5, KL K7 and KLK14 and other kallikrein family members located downstream are involved in the protease activity of the stratum corneum. is suggested to be involved in properties. In addition to inhibiting KLK5, inhibiting KLK7 or KLK14 may be expected to more strongly suppress Netherton syndrome-like skin symptoms. Mutations in SPINK5 related to Netherton syndrome are listed in the Human Gene Mutation Database (HGMD) at 70 or more, and it has been reported to be related to the severity of Netherton syndrome. When having mutations in exons 1 to 9 of SPINK5, it is related to the pathological condition of more severe Netherton syndrome. By examining mutations in SPINK5, it is possible to determine whether to use the pharmaceutical composition containing the peptide or conjugate of the present invention for the treatment or prevention of Netherton syndrome. The pharmaceutical composition of the present invention can contain a therapeutically or prophylactically effective amount of a peptide or conjugate and a pharmaceutically acceptable diluent, carrier, solubilizer, emulsifier, preservative and / or adjuvant. The "therapeutically or prophylactically effective amount" means an amount that exhibits a therapeutic or prophylactic effect for a specific disease, dosage form and administration route, and is synonymous with the "pharmacologically effective amount". The pharmaceutical composition of the present invention can contain substances (hereinafter referred to as "substances for formulation") for changing, maintaining or retaining the pH, osmotic pressure, viscosity, transparency, color, isotonicity, sterility, stability, solubility, sustained release property, absorbability, permeability, dosage form, strength, properties, shape, etc. of the composition or the peptide, conjugate, etc. contained therein. The substances for formulation are not particularly limited as long as they are pharmacologically acceptable substances. For example, non-toxic

[0138]

[0139]

[0140] ​​​​​​​​​​​​ Alternatively, low toxicity is a property that a substance for use in a formulation preferably possesses.

[0141] Examples of formulation materials include, but are not limited to, the following: 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, and fillers such as mannitol and glycine agents, 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 trivinylpyrrolidine, low molecular weight polypeptides, salt-forming counterions, and chloride ions. Alkonium, Benzoic Acid, Salicylic Acid, Thimerosal, Phenethyl Alcohol, Methyl Preservatives such as ruparaben, 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, polyiso Polysorbate 20, Polysorbate 80, Triton, Surfactants such as tromethamine, lecithin or cholesterol , stabilizing agents such as sucrose and sorbitol, sodium chloride, potassium chloride, mannitol, etc. Elasticity enhancers such as tallose and sorbitol, transport agents, diluents, excipients, and / or pharmaceutical supplements Auxiliary agents.

[0142] The addition amount of these substances for the preparation is 0.001 to 1000 times, preferably 0.01 to 100 times, more preferably 0.1 to 10 times the weight of the peptide contained in the peptide or conjugate of the present invention.

[0143] A pharmaceutical composition containing the peptide or conjugate of the present invention in liposomes, and the peptide A modified product formed by binding with liposomes is also included in the pharmaceutical composition of the present invention.

[0144] Excipients and carriers are usually liquid or solid, and are not particularly limited as long as they are substances used in preparations for oral administration or parenteral administration, such as water for injection, physiological saline, artificial cerebrospinal fluid, and others. Examples of physiological saline include neutral ones and those containing serum albumin.

[0145] Examples of the buffer include Tris buffer prepared so that the final pH of the pharmaceutical composition is 7.0 to 8.5, acetic acid buffer prepared so that it is also 4.0 to 5.5, citric acid buffer prepared so that it is also 5.0 to 8.0, and histidine buffer prepared so that it is also 5.0 to 8 .0.

[0146] The pharmaceutical composition of the present invention is a solid, liquid, suspension, etc. Another example of the pharmaceutical composition of the present invention is a lyophilized preparation. To form a lyophilized preparation, excipients such as sucrose can be used.

[0147] ​As the administration route of the pharmaceutical composition of the present invention, any of instillation, enteral administration, topical administration, and parenteral administration may be used. For example, instillation onto the conjunctiva, intravitreal administration, intravenous administration, intraarterial administration, intramuscular administration, intradermal administration, subcutaneous administration, intraperitoneal administration, transdermal administration, intraosseous administration, intraarticular administration, etc. can be mentioned. Any of these may be used, for example, instillation onto the conjunctiva, intravitreal administration, intravenous administration, intraarterial administration, intramuscular administration, intradermal administration, subcutaneous administration, intraperitoneal administration, transdermal administration, intraosseous administration, intraarticular administration, etc. can be mentioned.

[0148] The composition of such a pharmaceutical composition can be determined according to the administration method, the peptide contained in the peptide or conjugate of the present invention, or the inhibitory activity, binding affinity, etc. against KLK5, KLK5 and KLK7, or KLK5 and KLK14 of the peptide contained therein. The stronger the inhibitory activity (the smaller the IC value or K value) or the higher the affinity (the smaller the K value) of the inhibitory peptide of the present invention for the target, the more effective the drug effect can be exerted at a lower dose. The composition of such a pharmaceutical composition can be determined according to the administration method, the peptide contained in the peptide or conjugate of the present invention, or the inhibitory activity, binding affinity, etc. against KLK5, KLK5 and KLK7, or KLK5 and KLK14 of the peptide contained therein. The stronger the inhibitory activity (the smaller the IC value or K value) or the higher the affinity (the smaller the K value) of the inhibitory peptide of the present invention for the target, the more effective the drug effect can be exerted at a lower dose. The composition of such a pharmaceutical composition can be determined according to the administration method, the peptide contained in the peptide or conjugate of the present invention, or the inhibitory activity, binding affinity, etc. against KLK5, KLK5 and KLK7, or KLK5 and KLK14 of the peptide contained therein. The stronger the inhibitory activity (the smaller the IC value or K value) or the higher the affinity (the smaller the K value) of the inhibitory peptide of the present invention for the target, the more effective the drug effect can be exerted at a lower dose. The composition of such a pharmaceutical composition can be determined according to the administration method, the peptide contained in the peptide or conjugate of the present invention, or the inhibitory activity, binding affinity, etc. against KLK5, KLK5 and KLK7, or KLK5 and KLK14 of the peptide contained therein. The stronger the inhibitory activity (the smaller the IC value or K value) or the higher the affinity (the smaller the K value) of the inhibitory peptide of the present invention for the target, the more effective the drug effect can be exerted at a lower dose. 50 value or K i value is small) or the higher the affinity (the smaller the K D value is small the more effective the drug effect can be exerted at a lower dose.

[0149] The dose of the peptide or conjugate of the present invention is not limited as long as it is a pharmacologically effective amount, and can be appropriately determined according to the species of the individual, the type of disease, symptoms, sex, age, pre-existing disease, inhibitory activity against the target of the peptide, binding affinity, and other factors. Usually, it can be administered once, or twice or three times or more a day for 1 to 180 days at 0.01 to 1000 mg / kg, preferably 0.1 to 100 mg / kg. The dose of the peptide or conjugate of the present invention is not limited as long as it is a pharmacologically effective amount, and can be appropriately determined according to the species of the individual, the type of disease, symptoms, sex, age, pre-existing disease, inhibitory activity against the target of the peptide, binding affinity, and other factors. Usually, it can be administered once, or twice or three times or more a day for 1 to 180 days at 0.01 to 1000 mg / kg, preferably 0.1 to 100 mg / kg. The dose of the peptide or conjugate of the present invention is not limited as long as it is a pharmacologically effective amount, and can be appropriately determined according to the species of the individual, the type of disease, symptoms, sex, age, pre-existing disease, inhibitory activity against the target of the peptide, binding affinity, and other factors. Usually, it can be administered once, or twice or three times or more a day for 1 to 180 days at 0.01 to 1000 mg / kg, preferably 0.1 to 100 mg / kg. to 1000 mg / kg, preferably 0.1 to 100 mg / kg, for 1 to 180 days once, or twice or three times or more a day.

[0150] Examples of the form of the pharmaceutical composition include injections (including freeze-dried preparations and drip preparations), suppositories, nasal absorption preparations, transdermal absorption preparations, sublingual preparations, capsules, tablets, ointments, granules, aerosol preparations, pills, powders, suspensions, emulsions, eye drops, bio-implantable preparations, etc. Examples of the form of the pharmaceutical composition include injections (including freeze-dried preparations and drip preparations), suppositories, nasal absorption preparations, transdermal absorption preparations, sublingual preparations, capsules, tablets, ointments, granules, aerosol preparations, pills, powders, suspensions, emulsions, eye drops, bio-implantable preparations, etc. can be exemplified.

[0151] The pharmaceutical composition containing the peptide or conjugate of the present invention as an active ingredient can be administered simultaneously or separately with other pharmaceuticals. For example, after administering other pharmaceuticals, the pharmaceutical composition containing the peptide or conjugate of the present invention as an active ingredient is administered, or after administering such a pharmaceutical composition, other pharmaceuticals are administered, or the pharmaceutical composition and other pharmaceuticals may be administered simultaneously. When administered simultaneously, the peptide or conjugate of the present invention and other pharmaceuticals may be contained in either a single formulation or separate formulations (multiple formulations).

[0152] Those other pharmaceuticals may be administered or received, in some cases one, and in other cases two, three or more. Collectively, they are referred to as "combined use with other pharmaceuticals" or "combination with other pharmaceuticals" with the pharmaceutical composition of the present invention, and the pharmaceutical composition of the present invention containing other pharmaceuticals or used in combination with other therapies in addition to the peptide or its conjugate of the present invention is also included in the present invention as an aspect of "combined use with other pharmaceuticals" or "combination with other pharmaceuticals".

[0153] For example, in Netherton syndrome, emollients, steroid drugs, antibacterial agents, etc. can be mentioned. In atopic dermatitis, steroid drugs, calcineurin inhibitors, PDE4 inhibitors, immunosuppressive drugs, IL-4 / IL-13 inhibitors, phototherapy, etc. can be mentioned. In rosacea, doxycycline, minocycline, azelaic acid, brimonidine, etc. can be mentioned. In psoriasis, TNFα inhibitors, IL-12 / 23 inhibitors, IL-17 inhibitors, PDE4, antimetabolites, calcineurin inhibitors, fumaric acid esters, retinoid formulations, steroid drugs, vitamin D3 analogs, phototherapy, etc. can be mentioned. In asthma, ​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 relates to a method for treating or preventing a 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 method for treating a disease comprising administering to a patient a therapeutic agent comprising said peptide or said conjugate. A preventive kit is also included in the present invention.

[0155] Furthermore, a nucleic acid sequence encoding the amino acid sequence of the peptide of the present invention or its conjugate 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 peptides and vectors are useful for gene therapy of diseases related to KLK5, and the cells express KLK5. The present invention can be applied to cell therapy for diseases related to the present invention using known techniques. For example, such polynucleotides or vectors can be introduced into autologous or heterologous (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 the preparation of 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 As a means for evaluating the therapeutic effect of diseases related to LK5, an animal model can be used For example, in Netherton syndrome, as a model having a mutation in the causative gene, SPINK5 gene, SPINK5 gene-deficient mice (Descargues, P., et al. (2004) Nat. Genet. Vol. 37, pp. 56-65), SPIN K5 gene conditional knockout mice (Petrova, E., et al. (2019) Oral presentation at the 8th International Symposium on K allikreins and Kallikrein-Related Peptid ases: Abstract Book p. 28), Crusty 2 mice (Mutagenetix database), etc. can be mentioned, but are not limited thereto.

[0157] 6. Diagnostic composition Provided is a test or diagnostic composition (hereinafter collectively referred to as "diagnostic composition") containing the peptide of the present invention or its conjugate.

[0158] The diagnostic composition of the present invention is useful for the examination or diagnosis of diseases related to KLK5, KLK5 expression, KLK7 expression, KL K14 expression, etc. In the present invention, the examination or diagnosis includes, for example, Determination or measurement of the risk of suffering, determination of the presence or absence of suffering, measurement of the degree of progression or exacerbation, measurement or determination of the effect of drug treatment with the pharmaceutical composition containing the peptide or conjugate of the present invention, drug Measurement or determination of the effect of treatment other than drug treatment, measurement of the risk of recurrence, determination of the presence or absence of recurrence, etc. are included. However, it is not limited to these if it is inspection or diagnosis.

[0159] The diagnostic composition of the present invention is useful for identifying an individual to whom the peptide of the present invention or its conjugate, a composition containing them, or a pharmaceutical composition containing them is administered. It is useful for identifying an individual to whom a composition containing the peptide of the present invention or its conjugate, or a pharmaceutical composition containing them is administered.

[0160] Such a diagnostic composition can contain a pH buffer, an osmotic pressure regulator, salts, a stabilizer, a preservative, a chromogenic agent, a sensitizer, an anti-aggregation agent, etc. Such a diagnostic composition can contain a pH buffer, an osmotic pressure regulator, salts, a stabilizer, a preservative, a chromogenic agent, a sensitizer, an anti-aggregation agent, etc.

[0161] The present invention also provides a method for inspecting or diagnosing a disease related to KLK5, the use of the peptide of the present invention for preparing a diagnostic composition for the disease, and the use of the peptide of the present invention for inspecting or diagnosing the disease. An inspection or diagnostic kit containing the peptide of the present invention is also included in the present invention. The present invention also provides a method for inspecting or diagnosing a disease related to KLK5, the use of the peptide of the present invention for preparing a diagnostic composition for the disease, and the use of the peptide of the present invention for inspecting or diagnosing the disease. An inspection or diagnostic kit containing the peptide of the present invention is also included in the present invention. The present invention also provides a method for inspecting or diagnosing a disease related to KLK5, the use of the peptide of the present invention for preparing a diagnostic composition for the disease, and the use of the peptide of the present invention for inspecting or diagnosing the disease. An inspection or diagnostic kit containing the peptide of the present invention is also included in the present invention.

[0162] As a method for inspecting or diagnosing using the peptide of the present invention, sandwich ELISA is desirable, but ordinary ELISA methods, RIA methods, ELISPOT (Enzyme-Linked ImmunoSpot) methods, dot blot methods, Ouchterlony methods, CIE (Counterimmunoelectrophoresis) methods, CLIA (Chemiluminescent immunoassay), FCM (Flow Cytometry), etc. can be used. For detection, an antibody or its binding fragment, or a labeled peptide of the present invention or its conjugate, etc. is used. As a labeling method, biotin, a fluorescent group such as HRP, alkaline phosphatase, FITC, a label such as a radioisotope, etc., which can be used in biochemical analysis, can be used. For detection using an enzyme label, TM However, sandwich ELISA is desirable as a method for inspecting or diagnosing using the peptide of the present invention, but ordinary ELISA methods, RIA methods, ELISPOT (Enzyme-Linked ImmunoSpot) methods, dot blot methods, Ouchterlony methods, CIE (Count erimmunoelectrophoresis) methods, CLIA (Chemilumi nescent immuno assay), FCM (Flow Cytometry ) and other detection methods can be used. For detection, an antibody or its binding fragment, or a labeled peptide of the present invention or its conjugate, etc. is used. As a labeling method, biotin, a fluorescent group such as HRP, alkaline phosphatase, FITC, a label such as a radioisotope, etc., which can be used in biochemical analysis, can be used. For detection using an enzyme label, TM However, sandwich ELISA is desirable as a method for inspecting or diagnosing using the peptide of the present invention, but ordinary ELISA methods, RIA methods, ELISPOT (Enzyme-Linked ImmunoSpot) methods, dot blot methods, Ouchterlony methods, CIE (Count erimmunoelectrophoresis) methods, CLIA (Chemiluminescent immunoassay), FCM (Flow Cytometry), etc. can be used. For detection, an antibody or its binding fragment, or a labeled peptide of the present invention or its conjugate, etc. is used. As a labeling method, biotin, a fluorescent group such as HRP, alkaline phosphatase, FITC, a label such as a radioisotope, etc., which can be used in biochemical analysis, can be used. For detection using an enzyme label, TM B(3,3’,5,5’-tetramethylbenzidine), BCIP(5 -bromo-4-chloro-3-indolyl phosphate), p-N PP(p-nitrophenyl phosphate), OPD(o-Phenyl enediamine), ABTS(3-Ethylbenzothiazoline- 6-sulfonic acid), SuperSignal ELISA Pico Chemiluminescent Substrate(Thermo Fisher Scientific), etc., chromogenic substrates, and QuantaBlu® Fluor ogenic Peroxidase Substrate(Thermo Fishe r Scientific) fluorescent substrates, and chemiluminescent substrates can also be used. In addition to samples derived from humans or non-human animals, samples subjected to artificial treatments such as recombinant proteins can also be used for this measurement. Examples of test samples derived from biological individuals include, but are not limited to, blood, synovial fluid , ascites, lymph fluid, cerebrospinal fluid, bronchoalveolar lavage fluid, saliva, sputum, tissue homogenate supernatant, tissue sections , etc.

[0163] The sandwich ELISA kit for inspection or diagnosis containing the peptide of the present invention may include a protein standard solution of the peptide or conjugate of the present invention, a chromogenic reagent, a dilution buffer, a solid-phase protein , a detection protein, and a washing solution, etc. As a method for measuring the amount of protein bound to the antigen, absorbance measurement, fluorescence measurement, luminescence measurement, RI (Radioisotope) method, etc. are preferably applicable, and for the measurement, an absorbance plate reader, a fluorescence plate reader, a luminescence plate reader , an RI liquid scintillation counter, etc. are preferably used. ​

[0164] In addition, inspection or diagnosis can also be performed by a method using immunoprecipitation.

[0165] In addition, the present invention provides a method for detecting or measuring KLK5, KLK5 and KLK7, or KLK5 and KLK14 in a test sample. These detection or measurement methods can use the diagnostic composition of the present invention. The peptide of the present invention or its conjugate is brought into contact with a test sample (step 1), and then the amount or measurement of KLK 5, KLK5 and KLK7, or KLK5 and KLK14 bound to the peptide or conjugate is measured (step 2), whereby KLK5, KLK5 and KLK7, or KLK5 and KLK14 in the sample can be detected. As step 1, for example, a conjugate of the peptide of the present invention and the Fc region of immunoglobulin is immobilized on magnetic beads via Protein G, and a test sample is added thereto. As step 2, for example, the magnetic beads are separated, and the soluble proteins precipitated together with the beads are analyzed by SDS-PAGE or Western blot to detect KLK5, KLK5 and KLK7, or KLK5 and KLK14. This measurement can be applied to samples derived from human or non-human animals, as well as samples subjected to artificial treatments such as recombinant proteins. Examples of test samples derived from biological individuals include, but are not limited to, blood, synovial fluid, ascites, lymph fluid, cerebrospinal fluid, alveolar lavage fluid, saliva, sputum, tissue homogenate supernatant, tissue sections, etc. The detection of the above-mentioned KLK5, KLK5 and KLK7, or KLK5 and KLK14 is performed by not limited thereto.

[0166] It can be carried out not only in vitro but also in vivo. In the case of imaging diagnosis, the peptide of the present invention or its conjugate labeled with a pharmaceutically acceptable radionuclide or phosphor can be used. As step 1, for example, the labeled peptide or its conjugate is administered to a subject. As step 2, for example, an image is taken using an imaging diagnosis technique such as PET / CT, and the presence of KLK5, KLK5 and / or KLK7, or KLK5 and / or KLK14 is determined or examined, etc. can be mentioned. The peptide of the present invention or its conjugate contained in the diagnostic composition of the present invention binds to KLK5, KLK5 and KLK7, or KLK5 and KLK14, and preferably has specific binding activity to KLK5, KLK5 and KLK7, or KLK5 and KLK14. As step 1, for example, the labeled peptide or its conjugate is administered to a subject. As step 2, for example, an image is taken using an imaging diagnosis technique such as PET / CT. And the presence of KLK5, KLK5 and / or KLK7, or KLK5 and / or KLK14 is determined or examined. Examples thereof can include the like.

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

[0168] A method for identifying an individual to whom the pharmaceutical composition of the present invention is administered is also included in the present invention. In such an identification method, KLK5, KLK5 and / or KLK7, or KLK5 and / or KLK14 in a sample derived from the individual is measured, and whether KLK5, KLK 5 and / or KLK7, or KLK5 and / or KLK14 is detected in the sample, or Compared with the amount of KLK5, KLK5 and / or KLK7, or KLK5 and / or KLK14 detected in a sample derived from a healthy individual, when more KLK5, KL K5 and / or KLK7, or KLK5 and / or KLK14 is detected, The individual can be determined to be positive. The diagnostic composition of the present invention can be used in the method. In this case, the individual can be determined to be positive. The diagnostic composition of the present invention can be used in the method. When more KLK5, KL K5 and / or KLK7, or KLK5 and / or KLK14 is detected, The diagnostic composition of the present invention can be used in the method.

[0169] Also, in a preferred embodiment of such an identification method, the individual is suffering from or at risk of suffering from a KLK5-related disease. or is at risk thereof.

[0170] Furthermore, in one embodiment, the pharmaceutical composition of the present invention can be administered to an individual determined to be positive in such an identification method. or is at risk thereof.

[0171] 7. Method for Separating KLK5, KLK5 and KLK7, or KLK5 and KLK14 The peptide or conjugate of the present invention preferably has specific binding activity to KLK5, KLK5 and / or KLK7, or KLK5 and / or KLK14. Thus, using the peptide or conjugate of the present invention, KLK5, KLK5 and / or KLK7, or KLK5 and / or KLK14 can be specifically separated from a sample in which KLK5, KLK5 and / or KLK7, or KLK5 and / or KLK14 are mixed with other KLKs. Release of KLK5, KLK5 and / or KLK7, or KLK5 and / or KLK14 from the peptide or conjugate can be carried out non-selectively under relatively high ionic strength, low pH, medium denaturing conditions, in the presence of chaotropic salts, etc., but it is preferably carried out within a range that does not attenuate the protease activity of KLK5, KLK5 and / or KLK7, or KLK5 and / or KLK14. or is at risk thereof. Therefore, using the peptide or conjugate of the present invention, KLK5, KLK5 and / or KLK7, or KLK5 and / or KLK14 can be specifically separated from a sample in which KLK5, KLK5 and / or KLK7, or KLK5 and / or KLK14 are mixed with other KLKs. Release of KLK5, KLK5 and / or KLK7, or KLK5 and / or KLK14 from the peptide or conjugate can be carried out non-selectively under relatively high ionic strength, low pH, medium denaturing conditions, in the presence of chaotropic salts, etc., but it is preferably carried out within a range that does not attenuate the protease activity of KLK5, KLK5 and / or KLK7, or KLK5 and / or KLK14. or is at risk thereof. from a sample in which KLK5, KLK5 and / or KLK7, or KLK5 and / or KLK14 are mixed with other KLKs. from a sample in which KLK5, KLK5 and / or KLK7, or KLK5 and / or KLK14 are mixed with other KLKs. KLK5, KLK5 and / or KLK7, or KLK5 and / or KLK14 can be specifically separated. Release of KLK5, KLK5 and / or KLK7, or KLK5 and / or KLK14 from the peptide or conjugate can be carried out non-selectively under relatively high ionic strength, low pH, medium denaturing conditions, in the presence of chaotropic salts, etc., but it is preferably carried out within a range that does not attenuate the protease activity of KLK5, KLK5 and / or KLK7, or KLK5 and / or KLK14. can be carried out non-selectively under relatively high ionic strength, low pH, medium denaturing conditions, in the presence of chaotropic salts, etc., but it is preferably carried out within a range that does not attenuate the protease activity of KLK5, KLK5 and / or KLK7, or KLK5 and / or KLK14. or KLK5 and / or KLK14 can be specifically separated. Release of KLK5, KLK5 and / or KLK7, or KLK5 and / or KLK14 from the peptide or conjugate is preferably carried out within a range that does not attenuate the protease activity of KLK5, KLK5 and / or KLK7, or KLK5 and / or KLK14.

Examples

[0172] In the following examples, some embodiments of the present invention will be described in more detail, but the present invention is not limited thereto. Thereafter, it is not limited thereto.

[0173] In the following examples, unless otherwise specified, each operation related to genetic manipulation was carried out according to the methods described in "Molecular Cloning" (Sambrook, J ., Fritsch, E.F. and Maniatis, T., published by Cold Spring Harbor Laboratory Press in 1982 or 1989 ), or according to the methods described in other experimental manuals used by those skilled in the art, or when using commercially available reagents and kits, it was carried out according to the instructions of the commercial products.

[0174] Example 1. Preparation of KLK5 inhibitory peptide (1-1) Construction of KLK5 inhibitory peptide expression vector Using 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 templates, the following primers and KOD-plus- (TOYOBO) were used for PCR (( 94°C for 15 seconds, 60°C for 30 seconds, 68°C for 20 seconds) × 30 cycles) to amplify the inhibitory peptide fragment. Primer 1: 5’-AAAAGGATCCCTGGACAAACGTGGCCCGCA GTTTGGTCTGTTTAG-3’ (SEQ ID NO: 62: Figure 70) Primer 2: 5’-AAAACTCGAGTTAGCCGCCGCACGGACCAT TGCGAATAA-3’ (SEQ ID NO: 63: Figure 71) After subjecting the amplified fragment to agarose gel electrophoresis, the desired DNA fragment was excised, and DNA was prepared using the QIAquick Gel Extraction Kit (QIAGEN). The prepared DNA fragment and pET 32a (Novagen) were digested with restriction enzyme B ​​Treat with amHI (NEB) and XhoI (NEB) at 37°C for 1 hour or more, and then after agarose gel electrophoresis, cut out the desired DNA fragment and purify it using the QIAquick PCR P urification Kit (QIAGEN). Perform the ligation reaction by reacting each purified fragment at room temperature for 10 minutes using the LigaFast Rapid DNA Ligation System (Promega). The ligation solution was added to Escherichia coli JM109 (TOYOBO), allowed to stand on ice for 30 minutes, then heat-treated at 42°C for 45 seconds, further allowed to stand on ice for 5 minutes, seeded on a 2YT plate containing 0.1 mg / mL ampicillin, and then statically cultured at 37°C overnight to transform Escherichia coli. The next day, the transformed Escherichia coli was inoculated into Terrific Broth medium (Invitrogen) containing 0.1 mg / mL ampicillin, cultured overnight at 37°C, and then the plasmid DNA was recovered using the QIAprep 96 Turbo Miniprep Kit (Qiagen) (hereinafter referred to as "miniprep treatment"), and pET 32a_Kex2_KLK5 inhibitory peptide was constructed by performing sequence analysis. (1 - 2) Preparation of KLK5 inhibitory peptide (1 - 1) Escherichia coli Origami B (DE3) (Novagen n) was transformed with the vector constructed in (1 - 1), cultured at 37°C using 2YT medium containing 0.1 mg / mL ampicillin, then IPTG (final concentration 1 mM) was added and cultured overnight at 16°C. The next day, after collecting the bacteria by centrifugation (

[0175] (1 - 2) Preparation of KLK5 inhibitory peptide (1 - 1) Escherichia coli Origami B (DE3) (Novagen n) was transformed with the vector constructed in (1 - 1), cultured at 37°C using 2YT medium containing 0.1 mg / mL ampicillin, then IPTG (final concentration 1 mM) was added and cultured overnight at 16°C. The next day, after collecting the bacteria by centrifugation ( 3,000 g, 20 minutes, 4°C), lysate was prepared using BugBuster Master Mi x (Novagen), and TALON Metal Aff​ The His-tagged target protein was purified using ininity Resin (Clontech). Next, Kex2 (Saccharomyces cerevisiae: Ac cession CAA96143) was used to cleave the thioredoxin tag and the desired protein, and purification was performed using TALON. Furthermore, 14 KLK5 inhibitory peptides were prepared by subjecting them to gel filtration chromatography (Superdex75 10 / 300 GL) or reverse phase chromatography (YMC- Pack ODS-AM). The amino acid sequences of the derivatives are set forth in SEQ ID NOs: 6, 8, 10, 12, 14, 16, 18, 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 The primers and PCR conditions used for cloning human pro-KLK5, human pro-KLK7 and human pro-KLK14 were as follows. Fragment A was amplified by PCR ((94°C for 15 seconds, 60°C for 30 seconds, 68°C for 10 seconds) × 30 cycles) using the following primers and KOD-plus- (TOYOBO). Primer 3: 5'-GGCGATTATAAAGATGACGATGATAAACAC CATCACCACCATC-3' (SEQ ID NO: 64: FIG. 72) Primer 4: 5'-GTTTAAACTCAATGATGGTGGTGATGGTGT TTATCATCGTCAT-3' (SEQ ID NO: 65: FIG. 73) ​Next, using the nucleotide sequences encoding human pro-KLK5 (Uniprot: Q9Y337), human pro-KLK 7 (Uniprot: P49862), and human pro-KLK14 (Uniprot: Q9 P0G3) as templates respectively, the following primers and K OD-plus-(TOYOBO) were used for PCR ((94°C for 15 seconds, 60°C for 30 seconds, 68°C for 60 seconds) × 30 cycles) to amplify the fragments.

[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 fragment above, fragment A, the following primers, and KOD-plus- (TOYOBO ) were used to amplify the desired DNA fragment by the overlap PCR method. 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, the nucleotide sequences encoding mouse pro-KLK7 (Uniprot: Q91VE3) and mouse pro-K LK14 (Uniprot: Q8CGR5) were each used as templates, and the following primers and KOD-plus- (TOYOBO) were used for PCR ( (94°C for 15 seconds, 60°C for 30 seconds, 68°C for 60 seconds) × 30 cycles) to amplify the fragments. (94°C for 15 seconds, 60°C for 30 seconds, 68°C for 60 seconds) × 30 cycles) to amplify the fragments. amplified.

[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 desired amplified DNA fragment, restriction enzymes XbaI (NEB) and PmeI (NEB) were used for cloning, and mammalian cell expression vectors pCMA_pro-hKLK5, pCMA_pro-hKLK7, pCMA_pr o-hKLK14, pCMA_pro-mKLK7, pCMA_pro-mKLK14 with His tag added to the C-terminus of each gene were constructed. The operations were carried out according to the method described in (1-1).

[0185] (2-2) Expression and purification of human KLK5, human pro-KLK7, human pro-KLK14, mouse pr o-KLK7, mouse pro-KLK14 (2-1) The expression vectors constructed were transfected into Expi293F cells (Thermo Fisher Scien tific) using PEI MAX 40000 (Polysciences), and the culture supernatant was collected 3 days after culturing. The desired His tag fusion proteins were recovered from the culture supernatant using HisTrap excel (GE healthcare), and buffer-exchanged into PBS using Amicon Ultra NMWL 10,000( Merck Millipore), thereby purifying KLK 5, human pro-KLK7, human pro-KLK14, mouse pro-KLK7, mouse pro-KLK14 respectively.

[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 mM CaCl2, 0.05% (w / w) Brij-35, pH 7.5) was used to prepare 20 μg / mL of pro-KLK7 or 14 was added with an equal amount of 20 μg / mL of thermolys in, and after reacting at 37°C for a certain period of time, 100 mM EDTA was added in an equal amount to activate human KLK7, activated human KLK14, activated mouse KLK7, and activated mouse KLK 14 were prepared.

[0187] Also, 200 μg / mL of mouse KLK5 (R&D Sy stems; 7236-SE) prepared with activation buffer (50 mM Tris-HCl, 0.005% (w / w) Br ij-35, pH 8.0) and 2 μg / mL of human KLK5 were mixed in equal amounts and reacted at 37°C for 24 hours to prepare activated mouse KLK5.

[0188] Example 3. Evaluation of KLK5 Inhibitory Peptide (3-1) Evaluation of the Inhibitory Activity of KLK5 Inhibitory Peptide against Human / Mouse KLK5, Human / Mouse KLK7, and Hu man / Mouse KLK14 The substrate peptide was dissolved in DMSO to a concentration of 10 mM and diluted with Assay buffer (5 0 mM Tris-HCl, 150 mM NaCl, pH 8.0) for use. Human / mouse KLK5, human / mouse KLK7, or human / mouse KLK14 diluted with Assay buffer and the inhibitory peptide were each mixed in 25 μL and reacted at 37°C for 20 minutes. Then, 50 μL of the substrate diluted with Assay buffer was added, and the fluorescence signal was measured with Enspi re (PerkinElmer). For each combination of enzyme and substrate It was used as follows. Each inhibitory peptide had a final concentration of 0.098 - 1,000 nM, and for the reaction and measurement, ProteoSave (registered trademark) SS96F black plates (Sumitomo Bakelite Co., Ltd.) were used. For the evaluation of human KLK5 inhibitory activity; final concentration of 10 nM hKLK5, final concentration of 100 μM substrate peptide Boc-Val-Pro-Arg-AMC (R&D Systems), fluorescence signal excitation 380 nm / emission 460 nm

[0189] For the evaluation of human KLK7 inhibitory activity; final concentration of 1 μg / mL hKLK7, final concentration of 20 μM substrate peptide Mca-Arg-Pro-Lys-Pro-Val-Glu-Nval- Trp-Arg-Lys(Dnp)-NH2 (R&D Systems: Figure 85, amino acid sequence is SEQ ID NO: 77), fluorescence signal excitation 320 nm / emission

[0190] 405 nm For the evaluation of human KLK14 inhibitory activity; final concentration of 0.2 μg / mL hKLK14, final concentration of 100 μM substrate peptide Boc-Val-Pro-Arg-AMC (R&D Systems), fluorescence signal excitation 380 nm / emission 460 nm

[0191] For the evaluation of mouse KLK5 inhibitory activity; final concentration of 0.25 μg / mL mouseKLK5, final concentration of 100 μM substrate peptide Boc-Val-Pro-Arg-AMC (R&D S ystems), fluorescence signal excitation 380 nm / emission 460 nm

[0192]

[0193] ​​​​​Mouse KLK7 inhibitory activity evaluation; final concentration 0.5 μg / mL mouse KLK7, 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), fluorescence signal excitation 320 nm / emission 405 nm

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

[0195] The degradation rate of the substrate peptide by each inhibitory peptide at each concentration was calculated, and the degradation rate at an inhibitory peptide concentration of 0 nM was taken as 100%, and the 50% inhibitory concentration (IC 50 ) was calculated using GraphPad Prism (version 5 .0; GraphPad Software Inc.). As a result, it was revealed that all the inhibitory peptides inhibited the human KLK5 enzyme activity at low concentrations (Table 1, Figure 2). Some of the inhibitory peptides inhibited the human KLK7 or human K LK14 enzyme activity at low concentrations, and some of the inhibitory peptides showed weak inhibitory activity against these proteases (Table 1). The inhibitory peptides showed similar activity against mouse KLK5, KLK 7 or KLK14 (Table 2). In addition, for the calculation of the IC 50 value, the average value of three independent experiments was used.

[0196]

Table 1

[0197]

Table 2

[0198] (3-2) Cross-reactivity evaluation of KLK5 inhibitory peptide Using the degradation of the substrate peptide as an index, the specificity for other proteases was evaluated. (3-1 ) In the same manner as the method described in (3-1), 25 μL each of the protease and the sample ([[]] final concentration 1 μM) diluted with Assay buffer were mixed, reacted at 37 °C for 20 minutes, and then 50 μL of the substrate diluted with Assay buffer was added, and the fluorescence signal was measured with Enspire (PerkinElm er). For the protease activity evaluation, Assay buff er (50 mM Tris, 150 mM NaCl, pH 8.0) was used, and for the reaction and measurement ProteoSave (registered trademark) SS96F black plate (Sumitomo Bakelite Co., Ltd.) was used. The combinations of proteases and substrates used for the specificity evaluation are 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), fluorescence signal excitation 380 nm / emission 460 nm

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

[0201] Evaluation of inhibitory activity against bovine α-chymotrypsin; final concentration of chymotrypsin 10 nM otrypsin (Worthington Biochemical Corporation ; LS001434), final concentration of substrate peptide Suc-Leu-Le u-Val-Tyr-MCA (Peptide Institute, Inc.; 3120-v: Figure 86, amino acid sequence is SEQ ID NO: 78), fluorescence signal excitation 380nm / emiss ion 460nm Excitation 380nm / Emission 460nm

[0202] Evaluation of inhibitory activity against human chymotrypsin; final concentration of chymotrypsin 10 nM ypsin (Sigma-Aldrich; C8946), final concentration of substrate peptide Suc-Leu-Leu-Val-Tyr-MCA (Peptide Institute, Inc.; 312 0-v: Figure 87, amino acid sequence is SEQ ID NO: 79), fluorescence signal excitation 380nm / Emission 460nm Excitation 380nm / Emission 460nm

[0203] Evaluation of inhibitory activity against human tryptase; final concentration of tryptase 1 nM (Sig ma-Aldrich; T7063), final concentration of substrate peptide Boc-Phe -Ser-Arg-MCA (Peptide Institute, Inc.; 3107-v), fluorescence signal e xcitation 380nm / Emission 460nm

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

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

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

[0207] Human neutrophil elastase inhibitory activity; final concentration 0.00001 U / μL Neutrophil elastase (Enzo Life Scien ces), final concentration 100 μM substrate peptide Suc(OMe)-Ala-Ala-Pro -Val-MCA (Peptide Institute, Inc.; 3153-v: Figure 88, the amino acid sequence is as arranged SEQ ID NO: 80), fluorescence signal excitation 380 nm / emission 4 60 nm

[0208] Human matriptase inhibitory activity evaluation; final concentration 1 nM matriptase (R&D Systems; 3946-SE), substrate peptide Boc at a final concentration of 100 μM -Gln-Ala-Arg-AMC (R&D Systems; ES014), fluorescence signal nal excitation 380 nm / emission 460 nm

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

[0210] Evaluation of human tPA inhibitory activity; tPA at a final concentration of 10 nM (Sigma-Aldric h; T0831), substrate peptide Pyr-Gly-Arg-MCA at a final concentration of 100 μM ( Peptide Institute, Inc.; 3145-v), fluorescence signal excitation 380 nm / emission 460 nm

[0211] Evaluation of human uPA inhibitory activity; uPA at a final concentration of 2 nM (Sigma-Aldrich ; U0633), substrate peptide Pyr-Gly-Arg-MCA at a final concentration of 100 μM (stock Peptide Institute, Inc.; 3145-v), fluorescence signal excitation 380 n m / emission 460 nm

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

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

[0214] Human KLK2 inhibitory activity evaluation; 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), fluorescence signal excitation 380 nm / emission 460 nm

[0215] Human KLK4 inhibitory activity evaluation; 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), fluorescence signal excitat ion 380 nm / emission 460 nm

[0216] Human KLK7 inhibitory activity evaluation; 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 Array with array number 77), fluorescence signal excitation 320nm / emissi 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), fluorescence signal excitat 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 100 μM substrate peptide Boc-Val- Pro-Arg-AMC (R&D Systems; ES011), fluorescence signal exc itation 380nm / emission 460nm

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

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

[0221] Similar to (3-1), using the degradation of the peptide substrate as an index, the cross-reactivity of the KLK5 inhibitory peptide against proteases other than KLK5 was evaluated. Some of the inhibitory peptides showed weak cross-reactivity against Chymotrypsin at a final inhibitory peptide concentration of 1 μM (IC value was less than 1 μM), but most of the inhibitory peptides did not show inhibitory activity against any protease other than KLKn (n = 1, 2, 4, 5, 7, 8, 12 or 14) (Figure 3). On the other hand, some inhibitory peptides showed inhibitory activity against KLK4 or KLK12 at a final inhibitory peptide concentration of 1 μM (IC 50 value was less than 1 μM), but many inhibitory peptides did not show protease inhibitory activity against KLKn except for KLK5, KLK7 and KLK14, indicating that the inhibitory peptide has high specificity. (3-3) Evaluation of KLK5 binding activity of KLK5 inhibitory peptide To measure the binding affinity of the KLK5 inhibitory peptide, surface plasmon resonance analysis was performed using a BIAcore T 200 (G 50 E healthcare). Complementary strand DNA of streptavidin conjugate was captured by hybridization to a Sensor Chip CAP (GE healthcare) immobilized with single-stranded DNA. Next, biotinylated KLK5 was captured at a flow rate of 10 μL / min using EZ-Link NHS-PEG4-Biotin (Thermo F isher Scientific) to immobilize approximately 10 RU. Then, the KLK5 inhibitory peptide (0.625 - 10 nM) serially diluted 2-fold with HBS-EP was used as an analyte and flowed

[0222] (3-3) KLK5 binding activity evaluation of KLK5 inhibitory peptide To measure the binding affinity of the KLK5 inhibitory peptide, surface plasmon resonance analysis was performed using a BIAcore T 200 (G E healthcare). Complementary strand DNA of streptavidin conjugate was captured by hybridization to a Sensor Chip CAP (GE healthcare) immobilized with single-stranded DNA. Next, biotinylated KLK5 was captured at a flow rate of 10 μL / min using EZ-Link NHS-PEG4-Biotin (Thermo F isher Scientific) to immobilize approximately 10 RU. Then, the KLK5 inhibitory peptide (0.625 - 10 nM) serially diluted 2-fold with HBS-EP was used as an analyte and flowed -captured. Next, KLK5 was captured at a flow rate of 10 μL / min using EZ-Link NHS-PEG4-Biotin (Thermo Fisher Scientific) to immobilize approximately 10 RU. Then, the KLK5 inhibitory peptide (0.625 - 10 nM) serially diluted 2-fold with HBS-EP was used as an analyte and flowed at a flow rate of 30 μL / min. captured. Then, the KLK5 inhibitory peptide (0.625 - 10 nM) serially diluted 2-fold with HBS-EP was used as an analyte and flowed at a flow rate of 30 μL / min. (0.625 - 10 nM) serially diluted 2-fold with HBS-EP was used as an analyte and flowed at a flow rate of 30 μL / min. It was added at a speed of 30 μL / min. BIAcore T 200 Evaluation software (version 2.0), single one-to- one Langmuir binding model was used to analyze single cycle kinetics, and kon and koff were calculated. The dissociation constant K D was k off / k on was calculated as the ratio of. Furthermore, the Biotin CAPture Ki t (GE healthcare)-attached Regeneration buffer was used to regenerate the S ensor Chip CAP, and by repeatedly capturing biotinylated KLK5 several KLK5 inhibitory peptides were measured.

[0223] All 14 KLK5 inhibitory peptides measured showed K D values below 1 nM and it was revealed that they had a very strong binding force (Table 3(A)).

[0224]

Table 3(A)

[0225] (3-4) Evaluation of the KLK5 binding activity of the KLK5 inhibitory peptide Fc fusion To measure the binding affinity of the KLK5 inhibitory peptide Fc fusion prepared in (5-2) described below surface plasmon resonance analysis was performed using BIAcore T 200 (GE healthcare).

[0226] On the Sensor Chip CM5 (GE he althcare) immobilized with anti-human IgG (Fc) antibody, the KLK5 inhibitory peptide Fc fusion was added at a flow rate of 20 μL / min By subjecting it to patching, approximately 30 - 50 RU was immobilized. Subsequently, KLK5 (0.625 - 10 nM) serially diluted 2-fold with HBS-EP was added as an analyte at a flow rate of 30 μL / min. In BIAcore T 200 Evaluation software ( version 2.0), single cycle kinetics was analyzed using the simple one-to-one Langmuir binding model to calculate kon and koff. The dissociation constant K was calculated as the ratio of k D to off k on . Furthermore, the Sensor Chip CM5 immobilized with anti-human IgG (Fc) antibody was regenerated with the Regeneration buffer attached to the Human Antibody Capture Kit (GE healthcare), and the binding activity of KLK5 to multiple KLK5 inhibitory peptide Fc fusions was measured by repeatedly capturing the KLK5 inhibitory peptide Fc fusions. (GE healthcare) and the KLK 5 inhibitory peptide Fc fusions were repeatedly captured. All 14 KLK5 inhibitory peptide Fc fusions measured showed K values below 1 nM, indicating a very strong binding affinity (Table 3(B)).

[0227] D It was revealed that they have a very strong binding force (Table 3(B)).

[0228]

Table 3(B)

[0229] Example 4. Analysis of KLK5 inhibitory peptides using X-ray crystal structure (4-1) Preparation of KLK5 / KLK5 inhibitory peptide complex According to the methods described in (1-2) and (2-2), the amino acid sequences represented by the sequence numbers ​​The KLK5 inhibitory peptide K51034 having [specific content] and KLK5 were each prepared. 50 m After mixing the two under the conditions of 50 mM Tris-HCl, 150 mM NaCl, pH 8.0, the complex was isolated and purified by gel filtration chromatography (Superdex 200 10 / 300 GL).

[0230] (4-2) X-ray crystal structure analysis After concentrating the complex solution prepared in (4-1) to 12 mg / mL, the reservoir solution (0. 2 M Magnesium Chloride hexahydrate, 20% PEG 3350) was mixed with it in a 1:1 ratio and crystallized by the vapor diffusion method. The obtained cubic single crystal was immersed in the reservoir solution containing 20% glycerol and then frozen in liquid nitrogen. The frozen crystal was irradiated with X-rays under a cryogenic airflow to obtain diffraction images (Hypixel 600 0HE / MicroMax007). By analysis using CrysAlisPro, scaling data with a maximum resolution of 1.7 Å was obtained. Using the molecular replacement method with the KLK5 monomer (PDB ID: 2P SX) and the SPINK2 monomer (PDB ID: 2JXD) as templates, the phase was determined. After structure refinement, the complex crystal of KLK5 / the peptide K51034 was determined at a resolution of 1.8 Å. Each unit cell contained one molecule of KLK5 and one molecule of SPINK2. For the SPINK2 molecule, a partial molecular model including the interaction site with KLK5 was constructed based on the sequence information and the observed electron density. It was confirmed that the KLK5 inhibitory peptide K5 1034 binds to the region containing the KLK5 enzyme active center (Figure 4).

[0231] Example 5. Preparation of KLK5 inhibitory peptide Fc fusion ​​(5-1) Construction of KLK5 Inhibitory Peptide Fc Fusion Protein Expression Vector Using 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, the following primers and KOD -plus- (TOYOBO) were used for PCR ((94°C for 15 seconds, 60°C for 30 seconds, 68°C for 20 seconds) × 30 cycles) to amplify the inhibitory peptide fragments. Primer 16: 5’-AGATGGGTGTTGTCTGATGACGACGGCCC TCAGTTCGGCCTGTTC-3’ (SEQ ID NO: 81: Figure 89) Primer 17: 5’-GCAGGGGCCATTCCGGAT-3’ (SEQ ID NO: 82: Figure 90)

[0232] Using the following primers and KOD-plus- (TOYOBO) for PCR ((94°C for 15 seconds, 60°C for 30 seconds, 68°C for 10 seconds) × 30 cycles) to amplify fragment B . 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] Using the Fc region of human IgG1 (SEQ ID NO: 87) as a template, the following primers and KOD-p lus- (TOYOBO) were used for PCR ((94°C for 15 seconds, 60°C for 30 seconds, 68 °C for 30 seconds) × 30 cycles) to amplify fragment C containing the Fc region of human IgG1. 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 KOD-plus-(TOYOBO) were used to amplify the desired DNA fragment by overlap PCR.

[0235] Furthermore, a mammalian cell expression vector pCMA_KLK5 inhibitory peptide Fc fusion was constructed by cloning using restriction enzymes XbaI (NEB) and PmeI (NEB). Note that the operation was performed according to the method described in (1-1).

[0236] (5-2) Preparation of KLK5 inhibitory peptide Fc fusion (5-1) The expression vector constructed above was transfected into Expi293F cells (Thermo Fisher Scientific) using PEI MAX 40000 (Polysciences). After 6 days of culture, the culture supernatant was collected. The desired Fc fusion was recovered from the culture supernatant using MabSelect SuRe (GE healthcare), and buffer-exchanged into PBS using Amicon Ultra NMWL 10,000 (Merck Millipore) to prepare the KLK5 inhibitory peptide F c fusion. For clones having a sugar chain addition sequence in the KLK5 inhibitory peptide, the sugar chain addition sequence was removed by a single residue substitution, and "dN" was added as an ID indicating the sugar chain-removed form. Note that the modification of the sugar chain addition sequence does not affect any activity such as KLK5 inhibitory activity or cross-reactivity.

[0237] ​​​​​​​ (5-3) Construction of KLK5 inhibitory peptide Fc fusion protein D1-K50055-Fc expression vector Construction Using the nucleotide sequence of KLK5 inhibitory peptide K50055 (SEQ ID NO: 7) as a template, the inhibitory peptide fragment was amplified by PCR ((94°C for 15 seconds, 60°C for 30 seconds, 68°C for 20 seconds) × 30 cycles) using the following primers and KOD-plus- (TOYOBO). 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, fragment C amplified in (5-1), primers 18, primer 21 and KOD-plus- (TOYOBO) were used for overlap PCR to amplify the desired DNA fragment.

[0240] Furthermore, cloning using restriction enzymes XbaI (NEB) and PmeI (NEB) was used to construct the mammalian cell expression vector pCMA_KLK5 inhibitory peptide Fc fusion protein. Note that the operation was performed according to the method described in (1-1).

[0241] (5-4) Preparation of KLK5 inhibitory peptide Fc fusion protein D1-K50055-Fc (5-3) The expression vector constructed was transfected into Expi293F cells (Thermo Fisher Scien tific) using PEI MAX 40000 (Polysci ences), and the culture supernatant was collected 6 days after culturing. MabSe Using lect SuRe (GE healthcare), the desired Fc fusion complex was recovered from the culture supernatant and buffer-exchanged into PBS using Amicon Ultra NMWL 10,000 (Merck Mi llipore), thereby preparing the KLK5 inhibitory peptide F c fusion D1-K50055-Fc.

[0242] Example 6. Evaluation of KLK5 inhibitory peptide Fc fusion (6-1) Evaluation of inhibitory activities of KLK5 inhibitory peptide Fc fusion against human / mouse KLK5, human / mouse KL K7 and human / mouse KLK14 According to the method described in Example 3-1, the inhibitory activities of the KLK5 inhibitory peptide Fc fusion against human / mouse KL K5, human / mouse KLK7 and human / mouse KLK14 were evaluated. The substrate peptide degradation rate of each inhibitory peptide Fc fusion at each concentration was calculated, and the inhibitory peptide Fc fusion concentration of 0 nM was taken as 100%, and GraphPad Pris m (version 5.0; GraphPad Software Inc.) was used to calculate the 50% inhibitory concentration (IC 50 50). As a result, it was revealed that all inhibitory peptide Fc fusions inhibited human KLK5 enzyme activity at low concentrations (Table 4, Figures 5 and 107). Some inhibitory peptide Fc fusions inhibited human KLK7 or human KLK14 enzyme activity at low concentrations, and some inhibitory peptide Fc fusions showed weak inhibitory activity against these proteases. The inhibitory peptide Fc fusions showed similar activities against mouse KLK5, KLK7 or KLK14 (Table 5). Incidentally, the average value of three independent experiments was used for the calculation of the IC 50 value. Some inhibitory peptide Fc fusions inhibited human KLK7 or human KLK14 enzyme activity at low concentrations, and some inhibitory peptide Fc fusions showed weak inhibitory activity against these proteases. The inhibitory peptide Fc fusions showed similar activities against mouse KLK5, KLK7 or KLK14 (Table 5). Incidentally, the average value of three independent experiments was used for the calculation of the IC 50 value. The inhibitory peptide Fc fusions showed similar activities against mouse KLK5, KLK7 or KLK14 (Table 5). Incidentally, the average value of three independent experiments was used for the calculation of the IC 50 50 value. The average value of three independent experiments was used.

[0243]

Table 4

[0244]

Table 5

[0245] (6 - 2) Cross - reactivity evaluation of KLK5 - inhibiting peptide Fc - fusion (3 - 2) Similar to the results of (3 - 2), some of the inhibitory peptide Fc - fusions showed weak cross - reactivity against bovine trypsin, chymotrypsin, and plasmin at an inhibitory peptide final concentration of 1 μM (IC 50 value < 1 μM), but most of the inhibitory peptides showed no inhibitory activity against any proteases other than KLKs (Figure 6). Some of the inhibitory peptide Fc - fusions showed inhibitory activity against KLK4 or KLK12 at a final concentration of 1 μM (IC 50 value < 1 μM), but many of the inhibitory peptide Fc - fusions showed no protease inhibitory activity against KLKs except for KLK7 or KLK14. Therefore, similar to the inhibitory peptides, it was revealed that the inhibitory peptide Fc - fusions have high specificity.

[0246] (6 - 3) Evaluation of KLK5 inhibitory activity of KLK5 - inhibiting peptide Fc - fusion using peptide substrate (Calculation of inhibition constant K i ) The inhibitory activity of the KLK5 - inhibiting peptide Fc - fusion against human KLK5 was evaluated, and the inhibition constant K was calculated. The substrate peptide Boc - Val - Pro - Arg - AMC (R&D S i ystems; ES011) was dissolved in DMSO to a concentration of 10 mM, and Assay bu was used to make a 10 mM solution. Dilute with buffer (50 mM Tris-HCl, 150 mM NaCl, pH 8.0) and used at a final concentration of 25 - 200 μM. Human KLK5 and the KLK5 inhibitory peptide Fc fusion were each mixed with 25 μL, reacted at 37 °C for 20 minutes, and then 50 μL of the substrate diluted with assay buffer was added, and the fluorescence signal (excitation 380 nm / emission 460 nm) was measured with Enspire. Human KLK5 was used at a final concentration of 10 nM, and the KLK5 inhibitory peptide Fc fusion was used at a final concentration of 0.5 - 25 nM.

[0247] The substrate peptide degradation rate in each inhibitory peptide Fc fusion at each concentration was calculated, and the inhibitory activity of each inhibitory peptide Fc fusion against human KLK5 was evaluated with the degradation rate at an inhibitory peptide Fc fusion concentration of 0 nM set as 100% (Figure 102). Using max GraphPad Prism (version 5.0; GraphPad Software Inc.), the maximum m reaction rate V and Michaelis constant K at an enzyme concentration of 10 nM were calculated according to the Michaelis-Menten equation. i Furthermore, using GraphPad Prism, the inhibition constant K at a substrate concentration of 100 μM was calculated according to Morrison's equation. As a result, it was revealed that all KLK5 i inhibitory peptide Fc fusions inhibited human KLK5 enzyme activity at low concentrations (Table 6). For the calculation of the K value,

[0248]

Table 6

[0249] (6-4) Evaluation of KLK5 inhibitory activity of KLK5 inhibitory peptide Fc fusions using a protein substrate Valence Using human Desmoglein1 and human Desmocollin1 as protein substrates the KLK5 inhibitory activity of the KLK5 inhibitory peptide Fc fusions was evaluated. Assay b Human KLK5 diluted with buffer and each KLK5 inhibitory peptide Fc fusion (D3-K50 032dN-Fc, D3-K50055-Fc, D3-K51072-Fc, or D3- K50016dN-Fc) were mixed and reacted at 37 °C for 1 hour. Next, Assay b The protein substrate diluted with buffer was added and reacted at 37 °C for 4 hours, and then a SDS sample buffer containing a reducing agent was added, and the enzyme reaction was stopped by treating at 99 °C for 5 minutes. Thereafter, the degradation of the protein substrate was evaluated by SDS-PAGE (reducing conditions) and Western blot analysis. The combinations of each substrate and enzyme, each inhibitory peptide Fc fusion, and the antibody for Western blot analysis were as follows.

[0250] Evaluation using Human Desmoglein1; final concentration 1 μM hKLK5, final concentration 0.001 - 10 μM inhibitory peptide Fc fusion, final concentration 1 μM Recombinan t Human Desmoglein-1 Fc Chimera Protein( R&D Systems), Desmoglein 1 Antibody (aa471 -499) (LSBio) and Anti-Rabbit IgG, HRP-Linke d F(ab’)2 Fragment Donkey(GE healthcare)

[0251] Evaluation using Human Desmocollin1; final concentration 0.2 μM hKLK5, final concentration 0.0002 - 2 μM inhibitory peptide Fc-fusion, final concentration 2 μM 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 were not degraded in the absence of human KLK5, but were completely degraded in the presence of human KLK5. As a result of pre-incubating human KLK5 with the KLK5 inhibitory peptide Fc-fusion for evaluation, it was revealed that all inhibitory peptide Fc-fusions inhibited the degradation activity of human KLK5 enzyme on human Desmoglein1 and human De smocollin1. Under the condition where the human KLK5 concentration and the concentration of the inhibitory peptide Fc-fusion were equal, the degradation of human Desmoglein1 and human Desmocollin1 was completely inhibited. (Figure 103).

[0253] Example 7. Inhibitory effect of KLK5 inhibitory peptide Fc-fusion on the increase in transepidermal water loss (TEWL) in Netherton syndrome model mice (7-1) Netherton syndrome model mice Crusty2 mice with a mutation in SPINK5, the causative gene of Netherton syndrome, are known as a model mouse for Netherton syndrome, and its homozygous mouse Crusty2( (+ / +) shows skin symptoms (Mutagenetix database). + / +) shows skin symptoms (Mutagenetix database).​

[0254] (7-2) T of KLK5 inhibitory peptide Fc fusion in Netherton syndrome model mice Effect of suppressing the increase in EWL Crusty2(+ / -) mice and Crusty2(+ / +) mice were crossbred by artificial insemination and the resulting offspring (Crusty2(+ / -) mice or Crusty2(+ / +) mice) were used to evaluate the KLK5 inhibitory peptide Fc fusion D1-K50 055-Fc prepared in (5-4). From postnatal day 0 or 1, PBS or 100 mg / kg of D1- K50055-Fc was subcutaneously administered every other day for 4 weeks. Only at the first administration, a triple dose of D1-K50055-Fc was administered as a loading dose VAPO SCAN (AS-V T100RS, Asahi TechnoLab Co., Ltd.) was used to measure the TEWL of the skin on the back or hip of the mice at 2 and 4 weeks after administration (Figure 7). In addition, regardless of the PBS administration group or the D1-K50055-Fc administration group, the number of Crusty2(+ / -) mice was 9 and the number of Crusty2(+ / +) mice was 12 In Crusty2(+ / +) mice, a statistically significant increase in TEWL was confirmed compared to Crusty2(+ / -) mice, and it was more prominent at 4 weeks of age than at 2 weeks of age. Four weeks after administration, in the D1-K50055-Fc administration group, a statistically significant decrease in TEWL was observed in both the back and the more severe hip compared to the PBS administration group. From the above it was found that an increase in TEWL caused by a mutation in SPINK5 was observed in mice while D1-K50055-Fc showed an inhibitory effect, and it became clear that D1-K500

[0255] Statistically significant increase in TEWL was confirmed in Crusty2(+ / +) mice compared to Crusty2(+ / -) mice, and it was more prominent at 4 weeks of age than at 2 weeks of age. Four weeks after administration, in the D1-K50055-Fc administration group, a statistically significant decrease in TEWL was observed in both the back and the more severe hip compared to the PBS administration group. From the above it was found that an increase in TEWL caused by a mutation in SPINK5 was observed in mice while D1-K50055-Fc showed an inhibitory effect, and it became clear that D1-K500 In contrast, in the D1-K50055-Fc administration group, a statistically significant decrease in TEWL was observed in both the back and the more severe hip compared to the PBS administration group. From the above it was found that an increase in TEWL caused by a mutation in SPINK5 was observed in mice while D1-K50055-Fc showed an inhibitory effect, and it became clear that D1-K500 The peptides and conjugates of the present invention containing 55-Fc have been shown to be useful for alleviating the skin symptoms of Netherton syndrome. It has been shown to be useful for alleviation.

Industrial Applicability

[0256] The peptides and conjugates provided by the present invention, and pharmaceutical compositions containing the same, are useful for the treatment of various diseases. It is useful for the treatment of various diseases.

Sequence Listing 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 inhibitory peptide K50032 (Figure 13) SEQ ID NO: 6: Amino acid sequence of KLK5 inhibitory peptide K50032 (Figure 14) SEQ ID NO: 7: Nucleotide sequence of KLK5 inhibitory peptide K50055 (Figure 15) SEQ ID NO: 8: Amino acid sequence of KLK5 inhibitory peptide K50055 (Figure 16) SEQ ID NO: 9: Nucleotide sequence of KLK5 inhibitory peptide K51072 (Figure 17) SEQ ID NO: 10: Amino acid sequence of KLK5 inhibitory peptide K51072 (Figure 18) SEQ ID NO: 11: Nucleotide sequence of KLK5 inhibitory peptide K50016 (Figure 19) SEQ ID NO: 12: Amino acid sequence of KLK5 inhibitory peptide K50016 (Figure 20) SEQ ID NO: 13: Nucleotide sequence of KLK5 inhibitory peptide K51034 (Figure 21) SEQ ID NO: 14: Amino acid sequence of KLK5 inhibitory peptide K51034 (Figure 22) SEQ ID NO: 15: Nucleotide sequence of KLK5 inhibitory 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 inhibitor peptide K51028 (Figure 2 9) SEQ ID NO: 22: Amino acid sequence of KLK5 / KLK7 inhibitor peptide K51028 (Figure 30) SEQ ID NO: 23: Nucleotide sequence of KLK5 / KLK7 inhibitor peptide K51005 (Figure 3 1) SEQ ID NO: 24: Amino acid sequence of KLK5 / KLK7 inhibitor peptide K51005 (Figure 32) SEQ ID NO: 25: Nucleotide sequence of KLK5 / KLK7 inhibitor peptide K50031 (Figure 3 3) SEQ ID NO: 26: Amino acid sequence of KLK5 / KLK7 inhibitor peptide K50031 (Figure 34) SEQ ID NO: 27: Nucleotide sequence of KLK5 / KLK7 inhibitor peptide K51057 (Figure 3 5) SEQ ID NO: 28: Amino acid sequence of KLK5 / KLK7 inhibitor 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 ) Accession No. 33: Nucleotide sequence of KLK5 inhibitory peptide Fc fusion protein D3-K50032dN-Fc Oligonucleotide sequence (Figure 41) Accession No. 34: Amino acid sequence of KLK5 inhibitory peptide Fc fusion protein D3-K50032dN-Fc (Figure 42) Accession No. 35: Nucleotide sequence of KLK5 inhibitory peptide Fc fusion protein D3-K50055-Fc (Figure 43) Accession No. 36: Amino acid sequence of KLK5 inhibitory peptide Fc fusion protein D3-K50055-Fc (Figure 44) Accession No. 37: Nucleotide sequence of KLK5 inhibitory peptide Fc fusion protein D3-K51072-Fc (Figure 45) Accession No. 38: Amino acid sequence of KLK5 inhibitory peptide Fc fusion protein D3-K51072-Fc (Figure 46) Accession No. 39: Nucleotide sequence of KLK5 inhibitory peptide Fc fusion protein D3-K50016dN-Fc Oligonucleotide sequence (Figure 47) Accession No. 40: Amino acid sequence of KLK5 inhibitory peptide Fc fusion protein D3-K50016dN-Fc (Figure 48) Accession No. 41: Nucleotide sequence of KLK5 inhibitory peptide Fc fusion protein D3-K51034-Fc (Figure 49) Accession No. 42: Amino acid sequence of KLK5 inhibitory peptide Fc fusion protein D3-K51034-Fc (Figure 50) Accession No. 43: Nucleotide sequence of KLK5 inhibitory peptide Fc fusion protein D3-K50062-Fc (Figure 51) Accession No. 44: Amino acid sequence of KLK5 inhibitory peptide Fc fusion protein D3-K50062-Fc (Figure 52) Accession No. 45: Nucleotide sequence of KLK5 inhibitory peptide Fc fusion protein D3-K51090-Fc (Figure 53) Accession No. 46: Amino acid sequence of KLK5 inhibitory peptide Fc fusion protein D3-K51090-Fc Column (Figure 54) Array number 47: Nucleotide sequence of KLK5 inhibitory peptide Fc fusion protein D3-K50098dN-Fc Oligonucleotide sequence (Figure 55) Array number 48: Amino acid sequence of KLK5 inhibitory peptide Fc fusion protein D3-K50098dN-Fc (Figure 56) Array number 49: Nucleotide sequence of KLK5 / KLK7 inhibitory peptide Fc fusion protein D3-K51028-Fc (Figure 57) Array number 50: Amino acid sequence of KLK5 / KLK7 inhibitory peptide Fc fusion protein D3-K51028-Fc (Figure 58) Array number 51: Nucleotide sequence of KLK5 / KLK7 inhibitory peptide Fc fusion protein D3-K51005-Fc (Figure 59) Array number 52: Amino acid sequence of KLK5 / KLK7 inhibitory peptide Fc fusion protein D3-K51005-Fc (Figure 60) Array number 53: Nucleotide sequence of KLK5 / KLK7 inhibitory peptide Fc fusion protein D3-K50031-Fc (Figure 61) Array number 54: Amino acid sequence of KLK5 / KLK7 inhibitory peptide Fc fusion protein D3-K50031-Fc (Figure 62) Array number 55: Nucleotide sequence of KLK5 / KLK7 inhibitory peptide Fc fusion protein D3-K51057-Fc (Figure 63) Array number 56: Amino acid sequence of KLK5 / KLK7 inhibitory peptide Fc fusion protein D3-K51057-Fc (Figure 64) Array number 57: Nucleotide sequence of KLK5 / KLK14 inhibitory peptide Fc fusion protein D3-K51069dN-Fc (Figure 65) Array number 58: Amino acid sequence of KLK5 / KLK14 inhibitory peptide Fc fusion protein D3-K51069dN-Fc (Figure 66) Accession No. 59: KLK5 / KLK14 Inhibitory Peptide Fc Fusion Protein D3-K50015-Fc Nucleotide sequence (Figure 67) Accession No. 60: KLK5 / KLK14 Inhibitory Peptide Fc Fusion Protein D3-K50015-Fc Amino acid sequence (Figure 68) Accession No. 61: General formula of SPINK2 variant peptide (Figure 69) Accession No. 62: Nucleotide sequence of Primer 1 (Figure 70) Accession No. 63: Nucleotide sequence of Primer 2 (Figure 71) Accession No. 64: Nucleotide sequence of Primer 3 (Figure 72) Accession No. 65: Nucleotide sequence of Primer 4 (Figure 73) Accession No. 66: Nucleotide sequence of Primer 5 (Figure 74) Accession No. 67: Nucleotide sequence of Primer 6 (Figure 75) Accession No. 68: Nucleotide sequence of Primer 7 (Figure 76) Accession No. 69: Nucleotide sequence of Primer 8 (Figure 77) Accession No. 70: Nucleotide sequence of Primer 9 (Figure 78) Accession No. 71: Nucleotide sequence of Primer 10 (Figure 79) Accession No. 72: Nucleotide sequence of Primer 11 (Figure 80) Accession No. 73: Nucleotide sequence of Primer 12 (Figure 81) Accession No. 74: Nucleotide sequence of Primer 13 (Figure 82) Accession No. 75: Nucleotide sequence of Primer 14 (Figure 83) Accession No. 76: Nucleotide sequence of Primer 15 (Figure 84) Accession No. 77: Amino acid sequence in KLK7 substrate peptide (Figure 85) Accession No. 78: Amino acid sequence in bovine α-chymotrypsin substrate peptide (Figure 86) Accession No. 79: Amino acid sequence in neutrophil elastase substrate peptide (Figure 87) Sequence Accession 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 the Fc region 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 the KLK5 inhibitory peptide Fc fusion D1-K50055-Fc sequence (Figure 105) SEQ ID NO: 96: Amino acid sequence of the KLK5 inhibitory peptide Fc fusion D1-K50055-Fc sequence (Figure 106)

Claims

1. A SPINK2 variant peptide that contains the amino acid sequence described in the following (i) or (ii) and selectively inhibits the protease activity of active human KLK5: (i) An amino acid sequence consisting of amino acid numbers 1 to 63 of the 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) (reference sequence) and having an amino acid sequence that is 96% or more identical; (ii) An amino acid sequence that is 98% or more identical to the reference sequence, where the amino acid sequences of amino acid numbers 1 Gly to 15 Cys, 23 Cys, and 29 Pro to 63 Cys in the reference sequence of (i) or (ii) are 100% identical to the reference sequence, and the disulfide bonds formed by 15 Cys - 45 Cys, 23 Cys - 42 Cys, and 31 Cys - 63 Cys are retained, a SPINK2 variant peptide.

2. The peptide according to claim 1, having a three-dimensional structure characterized by having three disulfide bonds and including a loop structure, an α-helix, and a β-sheet.

3. A polynucleotide that contains a nucleotide sequence encoding the amino acid sequence included in the peptide according to claim 1 or 2.

4. A vector that contains the polynucleotide according to claim 3.

5. A cell that contains the polynucleotide according to claim 3 or the vector according to claim 4 or produces the peptide according to claim 1 or 2.

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

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

8. A SPINK2 variant peptide obtained by the method according to claim 6 or 7.

9. A conjugate formed by binding one or more arbitrary moieties to the first peptide according to any one of claims 1, 2, and 8.

10. The conjugate according to claim 9, wherein one arbitrary moiety comprises a second peptide that is not a SPINK2 variant.

11. The conjugate according to claim 10, wherein the second peptide is located on the amino-terminal side of the first peptide.

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

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

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

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

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

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

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

20. The conjugate according to any one of claims 9, 10, and 12 to 19, which selectively inhibits the protease activity of human KLK5 and comprises an amino acid sequence that is at least 90% identical to the amino acid sequence represented by 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. The conjugate according to any one of claims 9, 10, and 12 to 20, wherein the first peptide and the second peptide are linked via a linker.

22. The conjugate according to claim 21, wherein the linker is a third peptide that is not the first peptide and the second peptide.

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

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

25. The SPINK2 variant peptide conjugate obtained by the method according to claim 23 or 24. Claim 26 A composition comprising the peptide according to claim 1, 2 or 8, the polynucleotide according to claim 3, the vector according to claim 4, the cell according to claim 5, and / or the conjugate according to any one of claims 9, 10, 12 to 22 and 25. Claim 27 A pharmaceutical composition comprising the peptide according to claim 1, 2 or 8, the polynucleotide according to claim 3, the vector according to claim 4, the cell according to claim 5, and / or the conjugate according to any one of claims 9, 10, 12 to 22 and 25. Claim 28 The pharmaceutical composition according to claim 27, for the treatment or prevention of Netherton syndrome, atopic dermatitis, urticaria, skin damage by ultraviolet rays, psoriasis, asthma, spinal cord injury, cancer or Barrett's esophagus. Claim 29 The pharmaceutical composition according to claim 27 or 28, for use in combination with other pharmaceuticals. Claim 30 A method according to any one of claims 6, 7, 23 and 24, comprising an affinity purification step using an antibody or a binding fragment thereof that specifically binds to the peptide according to claim 1.

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