STATEMENT REGARDING SEQUENCE LISTING

The sequence listing associated with this application is provided in text format in lieu of a paper copy and is hereby incorporated by reference into the specification. The name of the text file containing the sequence listing is DAISAN174089_Seq_List_FINAL_20210519_ST25.txt. The text file is 155 KB; was created on May 19, 2021; and is being submitted via EFS-Web with the filing of the specification.

TECHNICAL FIELD

The present invention relates to a peptide, a polynucleotide, a vector, a cell, a method for producing the peptide, a peptide obtained by such a method, a conjugate containing the peptide, a composition containing the peptide or the conjugate, a pharmaceutical composition, the pharmaceutical composition for treating or preventing various diseases, use of the peptide or the conjugate for treating or preventing various diseases, a method for treating various diseases that includes the step of administering the peptide or the conjugate, a composition for diagnosing or testing various diseases that contains the peptide or the conjugate, and the like.

BACKGROUND ART

Kallikrein 5 (KLK5) is a trypsin-like serine protease (Clan PA, family S1) and is also called a stratum corneum tryptic enzyme (SCTE). Kallikrein 7 (KLK7) is a chymotrypsin-like protease (Clan PA, family S1) and is also called a stratum corneum chymotryptic enzyme (SCCE). Further, kallikrein 14 (KLK14) is a trypsin-like protease (Clan PA, family S1). KLK5, KLK7, and KLK14 belong to a tissue kallikrein family composed of 15 types of highly conserved trypsin- or chymotrypsin-like serine proteases. KLK5 is expressed in cells and thereafter converted into active KLK5 by autoactivation, whereas KLK7 and KLK14 are expressed as inactive preproenzymes and converted into active forms by cleavage of their preprosequences by proteases represented by KLK5 (Non Patent Literature 1). Expression of KLK5 has been observed in the skin, and it is reported to degrade factors related to cell adhesion such as Desmoglein and Desmocollin (Non Patent Literature 2). KLK5, KLK7, and KLK14 are important for skin desquamation and are further involved in activation of proteinase-activated receptor 2 (PAR-2) (Non Patent Literature 3). Activation of PAR-2 induces cytokines and chemokines and enhances immunity, inflammation reactions, and the like.

Netherton syndrome is one of the ichthyosis syndromes with severe skin inflammation, desquamation, abnormal hair, and allergic symptoms such as asthma and allergic dermatitis due to autosomal recessive inheritance, and is a rare disease (0=256500) (Non Patent Literature 4). Since exfoliative dermatitis develops from the time of birth, dehydration, infection, or the like may be caused by significant damage to the skin barrier function and may be accompanied by developmental delay. Although detailed epidemiological data is not available, it is said that high postnatal mortality is exhibited. Netherton syndrome develops due to the loss of function of a serine protease inhibitor (LEKTI) following a mutation of a gene (SPINK5) encoding LEKTI expressed in skin epithelial cells. LEKTI is composed of 15 Kazal-like inhibitor domains. In patients with Netherton syndrome, a plurality of mutation sites has been found in SPINK5 across the sequence encoding each domain, and symptoms and severity change in association with the mutation sites (Non Patent Literatures 4 and 5).

SPINK5-deficient mice (Spink5−/−) exhibit Netherton syndrome-like skin symptoms, and high protease activity of KLK5 and KLK7 is observed in the skin epithelium (Non Patent Literature 1). Although SPINK5-deficient mice die within several hours after birth, improvement of neonatal mortality is reported in mice (Spink5−/−Klk5−/−) obtained by crossing KLK5-deficient mice with SPINK5-deficient mice (Non Patent Literature 6). Further, in Spink5−/−Klk5−/−mice, severe skin barrier defects, epithelium structural defects, skin inflammation, and the like observed in Spink5−/−have been recovered. Likewise, mice with the SPINK5 mutation Spink5A135X/A135Xobserved in patients with Netherton syndrome show Netherton syndrome-like skin symptoms, and death is observed within 12 hours after birth, whereas skin barrier and severe skin symptoms are improved in Klk5−/−Spink5A135X/A135Xobtained by crossing with KLK5-deficient mice (Non Patent Literature 7). Further, abnormal skin symptoms are eliminated by crossing with KLK7-deficient mice (Klk5−/−Klk7−/−Spink5A135X/A135X). It has been reported that KLK5 transgenic mice also show Netherton syndrome-like skin symptoms (Non Patent Literature 8). High protease activity like trypsin and chymotrypsin is observed in the stratum corneum of patients with Netherton syndrome and Netherton syndrome model mice, and it is suggested that the kallikrein family located downstream, such as KLK7 and KLK14, are related to the protease activity in the stratum corneum, in addition to KLK5. From the above, it is considered that Netherton syndrome is caused by a gene mutation of SPINKS and develops with abnormally elevated KLK5, KLK7, or KLK14 protease activity in the corneum. Currently, there is no fundamental therapeutic agent but only symptomatic treatment such as application of humectants.

KLK5 has also been suggested to be associated with rosacea, which is a chronic inflammatory disease of the face. In rosacea patients, increased expressions of KLK5 and the antimicrobial peptide Cathelicidin have been reported. Although the details of its etiology are unknown, it is considered that KLK5 with elevated expression degrades Cathelicidin, thereby producing a peptide that causes rosacea (Non Patent Literature 9).

There are multiple reports that SPINK5 polymorphisms are associated with the severity of atopic dermatitis (Non Patent Literatures 10 to 14). It has been reported that, in the skin of atopic dermatitis patients having SPINK5 genes encoding LEKTI in which amino acid residue 420 is lysine in both alleles, the expression of Desmogleinl is decreased at the protein level, and the activity of proteases, including KLK5 and KLK7, is increased, as compared with the case in which glutamic acid is encoded in both alleles (Non Patent Literature 15). It is considered that the activation of these proteases facilitates the invasion of allergens due to a decrease in skin barrier function and creates a condition where an inflammation reaction easily occurs.

SPINK2 (Serine Protease Inhibitor Kazal-type 2) is composed of a Kazal-like domain having three disulfide bonds and functions as a trypsin/acrosin inhibitor (Non Patent Literature 16), but the relationship of SPINK2 and its mutants to diseases such as Netherton syndrome, rosacea, and atopic dermatitis has not been clarified.

CITATION LIST

Non Patent Literatures

Non Patent Literature 1: Ovaere P, et al., published in 2009, Trends Biochem Sci., Vol. 34, No. 9: pp. 453-463Non Patent Literature 2: Descargues P, et al., published in 2005, Nat Genet., Vol. 37, No. 1: pp. 56-65Non Patent Literature 3: Rattenholl A, et al., published in 2008, Drug News Perspect., Vol. 21, No. 7: pp. 369-381Non Patent Literature 4: Hovnanian A., published in 2013, Cell Tissue Res., Vol. 351, No. 2: pp. 289-300Non Patent Literature 5: Sarri C A, et al., published in 2017, Mol Diagn Ther., Vol. 21, No. 2: pp. 137-152Non Patent Literature 6: Furio L, et al., published in 2015, PLoS Genet., Vol. 11, No. 9: e1005389Non Patent Literature 7: Kasparek P, et al., published in 2017, PLoS Genet., Vol. 13, No. 1: e1006566Non Patent Literature 8: Furio L, et al., published in 2014, J Exp Med., Vol. 211, No. 3: pp. 499-513Non Patent Literature 9: Yamasaki K, et al., published in 2007, Nat Med., Vol. 13, No. 8: pp. 975-980Non Patent Literature 10: Nishio Y, et al., published in 2003, Genes Immun., Vol. 4, No. 7: pp. 515-517Non Patent Literature 11: Kusunoki T, et al., published in 2005, J Allergy Clin Immunol., Vol. 4, No. 7: pp. 515-517Non Patent Literature 12: Lan C C, et al., published in 2011, Exp Dermatol., Vol. 20, No. 12: pp. 975-979Non Patent Literature 13: Kato A, et al., published in 2003, Br J Dermatol., Vol. 148, No. 4: pp. 665-669Non Patent Literature 14: Zhao L P, et al., published in 2012, J Eur Acad Dermatol Venereol., Vol. 26, No. 5: pp. 572-577Non Patent Literature 15: Fortugno P, et al., published in 2012, Hum Mol Genet., Vol. 21, No. 19: pp. 4187-4200Non Patent Literature 16: Chen T, et al., published in 2009, Proteins., Vol. 77, No. 1: pp. 209-219

SUMMARY OF INVENTION

Technical Problem

An object of the present invention is 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 to Problem

The present invention relates to:

(1) A SPINK2 mutant peptide that comprises the amino acid sequence set forth in SEQ ID NO: 61 (FIG. 69) and inhibits the protease activity of active human KLK5;

(2) The peptide according to (1), wherein the peptide inhibits the protease activity of human KLK7 or human KLK14;

(3) The peptide according to (1), wherein the peptide selectively inhibits human KLK5 and optionally human KLK7 or KLK14;

(4) The peptide according to any one of (1) to (3), wherein Xaa16 (X1) is Ala, Asp, Gly, Gln, Leu, Ser, or Thr;

(5) The peptide according to any one of (1) to (4), wherein Xaa17 (X2) is Arg, Glu, Asn, Gln, or Ser;

(6) The peptide according to any one of (1) to (5), wherein Xaa18 (X3) is Asp, Gln, Ile, Thr, Trp, or Tyr;

(7) The peptide according to any one of (1) to (6), wherein Xaa19 (X4) is Arg, Gly, Met, Gln, or Thr;

(8) The peptide according to any one of (1) to (7), wherein Xaa20 (X5) is Asp, Glu, Leu, Lys, Thr, or Tyr;

(9) The peptide according to any one of (1) to (8), wherein Xaa21 (X6) is Glu, Gly, His, Leu, Ser, Gln, or Tyr;

(10) The peptide according to any one of (1) to (9), wherein Xaa22 (X7) is Asp, Gly, Gln, Ser, or Tyr;

(11) The peptide according to any one of (1) to (10), wherein Xaa24 (X8) is Ala, Asp, Glu, Gly, Asn, Ser, or Thr;

(12) The peptide according to any one of (1) to (11), wherein Xaa25 (X9) is Arg or Lys;

(13) The peptide according to any one of (1) to (12), wherein Xaa26 (X10) is Asp, Glu, Gln, Ser, or Val;

(14) The peptide according to any one of (1) to (13), wherein Xaa27 (X11) is Phe or Tyr;

(15) The peptide according to any one of (1) to (14), wherein Xaa28 (X12) is Asp or Glu;

(16) The peptide according to any one of (1) to (15), wherein the peptide comprises the amino acids at positions 1 to 63 in the amino acid sequence set forth 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);
(17) The peptide according to any one of (1) to (3), wherein Xaa16 (X1) is Gly, Met, or Tyr;
(18) The peptide according to any one of (1) to (3) and
(17), wherein Xaa17 (X2) is Glu, Gln, or Thr;
(19) The peptide according to any one of (1) to (3), (17), and (18), wherein Xaa18 (X3) is His, Met, or Tyr;
(20) The peptide according to any one of (1) to (3) and (17) to (19), wherein Xaa19 (X4) is Ala, Arg, Lys, or Gln;
(21) The peptide according to any one of (1) to (3) and (17) to (20), wherein Xaa20 (X5) is Gly, Arg, or Ser;
(22) The peptide according to any one of (1) to (3) and (17) to (21), wherein Xaa21 (X6) is Arg, Lys, Gln, or Ser;
(23) The peptide according to any one of (1) to (3) and (17) to (22), wherein Xaa22 (X7) is Gly;
(24) The peptide according to any one of (1) to (3) and (17) to (23), wherein Xaa24 (X8) is His, Thr, or Tyr;
(25) The peptide according to any one of (1) to (3) and (17) to (24), wherein Xaa25 (X9) is His or Tyr;
(26) The peptide according to any one of (1) to (3) and (17) to (25), wherein Xaa26 (X10) is Asp, Glu, or His;
(27) The peptide according to any one of (1) to (3) and (17) to (26), wherein Xaa27 (X11) is Tyr;
(28) The peptide according to any one of (1) to (3) and (17) to (27), wherein Xaa28 (X12) is Asp or Glu;
(29) The peptide according to any one of (1) to (3) and (17) to (28), wherein the peptide comprises the amino acids at positions 1 to 63 in the amino acid sequence set forth in any one of SEQ ID NOs: 22, 24, 26, and 28 (FIGS. 30, 32, 34, and 36);
(30) The peptide according to any one of (1) to (3), wherein Xaa16 (X1) is Gly, Ser, or Tyr;
(31) The peptide according to any one of (1) to (3) and (30), wherein Xaa17 (X2) is Asp or Gln;
(32) The peptide according to any one of (1) to (3), (30), and (31), wherein Xaa18 (X3) is Thr or Val;
(33) The peptide according to any one of (1) to (3) and (30) to (32), wherein Xaa19 (X4) is Thr or Val;
(34) The peptide according to any one of (1) to (3) and (30) to (33), wherein Xaa20 (X5) is Glu or Thr;
(35) The peptide according to any one of (1) to (3) and (30) to (34), wherein Xaa21 (X6) is His or Thr;
(36) The peptide according to any one of (1) to (3) and (30) to (35), wherein Xaa22 (X7) is Tyr;
(37) The peptide according to any one of (1) to (3) and (30) to (36), wherein Xaa24 (X8) is Asn or Ser;
(38) The peptide according to any one of (1) to (3) and (30) to (37), wherein Xaa25 (X9) is Arg;
(39) The peptide according to any one of (1) to (3) and (30) to (38), wherein Xaa26 (X10) is Asp or Glu;
(40) The peptide according to any one of (1) to (3) and (30) to (39), wherein Xaa27 (X11) is Tyr;
(41) The peptide according to any one of (1) to (3) and (30) to (40), wherein Xaa28 (X18) is Asp;
(42) The peptide according to any one of (1) to (3) and (30) to (41), wherein the peptide comprises the amino acids at positions 1 to 63 in the amino acid sequence set forth in any one of SEQ ID NOs: 30 and 32 (FIGS. 38 and 40);
(43) The peptide according to any one of (1) to (42), wherein the peptide has three disulfide bonds and has a three-dimensional structure characterized by including a loop structure, an α-helix, and a β-sheet;
(44) A polynucleotide comprising a nucleotide sequence encoding the amino acid sequence of the peptide according to any one of (1) to (43);
(45) A vector comprising the polynucleotide according to (44);
(46) A cell that comprises the polynucleotide according to (44) or the vector according to (45) or produces the peptide according to any one of (1) to (43);
(47) A method for producing a SPINK2 mutant peptide, comprising the steps (i) and (ii) below: (i) culturing the cell according to (46); and (ii) collecting the SPINK2 mutant peptide from the culture;
(48) A method for producing the peptide according to any one of (1) to (43), comprising a step of 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) A conjugate comprising the peptide according to any one of (1) to (43) and (49) wherein the peptide is bound by one and optionally more than one moiety;
(51) The conjugate according to (50), wherein the one or more optional moiety comprises a second peptide that is not the 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 mutant;
(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 or each Fc region is an Fc region of human immunoglobulin, or a fragment thereof;
(56) The conjugate according to (54) or (55), wherein the or each 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 or each Fc region is an Fc region of human IgG1, or a fragment thereof;
(58) The conjugate according to (57), wherein the or each Fc region of human IgG1 comprises the amino acid sequence set forth in SEQ ID NO: 87 (FIG. 95);
(59) The conjugate according to any one of (54) to (57), wherein the or each Fc region is a wild type or a mutant Fc region;
(60) The conjugate according to any one of (51) to (59), wherein the conjugate comprises one to several aspartic acids and/or glutamic acids added to the amino terminus thereof;
(61) The conjugate according to any one of (50) to (60), wherein the conjugate comprises the amino acid sequence described in (i) or (ii) below: (i) the amino acid sequence set forth in any one of SEQ ID NOs: 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, and 96 (FIGS. 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, and 106); or (ii) the amino acid sequence of a conjugate that is 90% or more identical to the amino acid sequence described in (i) above and inhibits the protease activity of KLK5;
(62) The conjugate according to any one of (51) to (61), wherein the SPINK2 mutant and the second peptide are linked to each other via a linker;
(63) The conjugate according to (62), wherein the linker is a third peptide that is not the SPINK2 mutant or the second peptide;
(64) A method for producing the conjugate according to any one of (50) to (63), comprising the steps (i) and (ii) below: (i) culturing a cell comprising a polynucleotide comprising a nucleotide sequence encoding an amino acid sequence of the conjugate or a vector in which the polynucleotide is inserted; and (ii) collecting a SPINK2 mutant peptide conjugate or a peptide moiety comprised in the conjugate from the culture;
(65) A method for producing the SPINK2 mutant peptide conjugate according to any one of (50) to (63), comprising a step of preparing the conjugate or a peptide moiety comprised in the conjugate by chemical synthesis or in-vitro translation;
(66) A SPINK2 mutant peptide conjugate obtained by the method according to (64) or (65);
(67) An antibody or a binding fragment thereof that binds to the peptide according to any one of (1) to (43) and (49);
(68) A composition comprising the peptide according to any one of (1) to (43) and (49), the polynucleotide according to (44), the vector according to (45), the cell according to (46), the conjugate according to any one of (50) to (63) and (66), and/or the antibody or the binding fragment thereof according to (67);
(69) A pharmaceutical composition comprising the peptide according to any one of (1) to (43) and (49), the polynucleotide according to (44), the vector according to (45), the cell according to (46), and/or the conjugate according to any one of (50) to (63) and (66);
(70) The pharmaceutical composition according to (69), for treating or preventing a KLK5-related disease;
(71) The pharmaceutical composition according to (70), wherein the KLK5-related disease is Netherton syndrome, atopic dermatitis, rosacea, UV-induced skin injury, psoriasis, asthma, spinal cord injury, cancer, or Barrett's esophagus;
(72) The pharmaceutical composition according to any one of (69) to (71), for use in combination with an additional pharmaceutical product;
(73) A composition for testing or diagnosis comprising the peptide according to any one of (1) to (43) and (49), the polynucleotide according to (44), the vector according to (45), the cell according to (46), the conjugate according to any one of (50) to (63) and (66), and/or the antibody or the binding fragment thereof according to (67);
(74) The method according to any one of (47), (48), (64), and (65), comprising a step of performing affinity purification using the antibody or the binding fragment thereof according to (67);
(75) A method for identifying a KLK5 inhibitory SPINK2 mutant peptide, comprising the steps (i) to (iii) below: (i) incubating a KLK5 protease and a substrate in the presence and absence of a test SPINK2 mutant peptide; (ii) measuring the protease activity of KLK5 in the presence and absence of the test SPINK2 mutant peptide; and (iii) determining the peptide to be positive when the protease activity of KLK5 in the presence of the peptide is lower than the protease activity of KLK5 in the absence of the peptide;
(76) A method for identifying a KLK5 inhibitory compound, comprising the steps (i) to (iii) below, using a peptide comprising the amino acid sequence set forth in any one of SEQ ID NOs: 6, 8, 10, 12, 14, 16, 18, 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 comprising the peptide as a reference compound: (i) incubating a KLK5 protease and a substrate in the presence and absence of a test compound; (ii) measuring the protease activity of KLK5 in the presence and absence of the test compound; and (iii) determining the test compound to be positive when the protease activity of KLK5 in the presence of the test compound is lower than the protease activity of KLK5 in the absence of the test compound;
(77) A method for identifying a KLK5 inhibitory compound, comprising the steps (i) to (iii) below: (i) measuring a protease inhibitory activity against KLK5 of a test compound; (ii) measuring a protease inhibitory activity against KLK5 of a reference compound that is a peptide comprising the amino acid sequence set forth in any one of SEQ ID NOs: 6, 8, 10, 12, 14, 16, 18, 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 comprising the peptide; and (iii) determining the test compound to be positive when the protease inhibitory activity against KLK5 of the test compound is equivalent to or higher than the protease inhibitory activity against KLK5 of the reference compound;
(78) A method of measuring a KLK5 protease activity, comprising the steps (i) and (ii) below, using a peptide comprising the amino acid sequence set forth in any one of SEQ ID NOs: 6, 8, 10, 12, 14, 16, 18, 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 comprising the peptide as a reference compound: (i) incubating a KLK5 protease with a substrate, and optionally another component; and (ii) measuring an amount of the substrate and/or an amount of a product after step (i);
(79) A KLK5 inhibitory SPINK2 mutant peptide or a SPINK2 mutant peptide conjugate, having a dissociation constant (KD) for KLK5, as measured by surface plasmon resonance analysis by immobilizing the peptide or the conjugate and adding KLK5 thereto, of 1×10−9M or less;
(80) A conjugate comprising the amino acid sequence set forth in SEQ ID NO: 34 (FIG. 42);
(81) A conjugate comprising the amino acid sequence set forth in SEQ ID NO: 36 (FIG. 44);
(82) A conjugate comprising the amino acid sequence set forth in SEQ ID NO: 38 (FIG. 46);
(83) A conjugate comprising the amino acid sequence set forth in SEQ ID NO: 40 (FIG. 48);
(84) A conjugate comprising the amino acid sequence set forth in SEQ ID NO: 42 (FIG. 50);
(85) A conjugate comprising the amino acid sequence set forth in SEQ ID NO: 44 (FIG. 52);
(86) A conjugate comprising the amino acid sequence set forth in SEQ ID NO: 46 (FIG. 54);
(87) A conjugate comprising the amino acid sequence set forth in SEQ ID NO: 48 (FIG. 56);
(88) A conjugate comprising the amino acid sequence set forth in SEQ ID NO: 50 (FIG. 58);
(89) A conjugate comprising the amino acid sequence set forth in SEQ ID NO: 52 (FIG. 60);
(90) A conjugate comprising the amino acid sequence set forth in SEQ ID NO: 54 (FIG. 62);
(91) A conjugate comprising the amino acid sequence set forth in SEQ ID NO: 56 (FIG. 64);
(92) A conjugate comprising the amino acid sequence set forth in SEQ ID NO: 58 (FIG. 66);
(93) A conjugate comprising the amino acid sequence set forth in SEQ ID NO: 60 (FIG. 68); and
(94) A conjugate comprising the amino acid sequence set forth in SEQ ID NO: 96 (FIG. 106).

Advantageous Effects of Invention

A peptide provided by the present invention, a conjugate containing the peptide, and a pharmaceutical composition containing the peptide or the conjugate have KLK5 inhibitory activity and are useful for treating or preventing KLK5-related diseases (which will be described below).

