Peptides for the treatment of retinitis pigmentosa

Abstract

A novel pharmaceutical use of a peptide is provided. A pharmaceutical composition for treating or preventing retinitis pigmentosa, comprising a peptide having an amino acid sequence represented by SEQ ID NO: 30 and inhibiting protease activity.

Description

Technical Field

[0001] The present invention relates to a pharmaceutical composition for treating or preventing various diseases comprising a peptide, a conjugate thereof, etc., a method for identifying a peptide for treating or preventing various diseases, etc. Background Art

[0002] High temperature requirement A serine peptidase 1 (HTRA1) is a trypsin-like serine protease (PRSS11; ClanPA, family S1), and is composed of an N-terminal domain, a protease domain, and a C-terminal PDZ domain, and the N-terminal domain is composed of an IGFBP-like module and a Kazal-like module. HTRA1 belongs to the HTRA family including HTRA2, HTRA3, and HTRA4, and reversibly exhibits active and inactive structures in the same manner as other HTRA molecules (non-patent literature 1 and 2). Its expression is unevenly distributed in the human body, and is found at relatively high levels in cartilage, synovium, placenta, etc. It is known that HTRA1 cuts many extracellular matrix components as substrates, such as amyloid precursor protein, fibromodulin, cluster protein, ADAM9, and vitronectin, and is associated with diseases represented by arthritis and bone calcification (non-patent literature 3, 4, 5, and 6). It is further known that if the HTRA1 promoter region has a gene polymorphism (rs11200638), the HTRA1 transcription level increases. Genome-wide association analysis has also revealed that this polymorphism is strongly associated with age-related macular degeneration (hereinafter referred to as AMD) (Non-patent Documents 7 and 8). However, the association with retinitis pigmentosa has not been fully reported.

[0003] Retinitis pigmentosa is a progressive retinal degenerative disease, which begins with the degeneration and loss of rod photoreceptor cells. Due to the degeneration of photoreceptor cells, progressive night blindness, narrow visual field and photophobia are observed, resulting in decreased visual acuity and possible blindness. Retinitis pigmentosa is known as a hereditary disease, and 3,000 or more gene mutations causing retinitis pigmentosa have been identified so far (non-patent literature 9). Among them, for the gene mutation of single rhodopsin (which represents a high ratio), 120 or more have been confirmed in people, and 11 mechanisms causing rod loss have been proposed according to classification (non-patent literature 10). In this case, it is extremely difficult to narrow the target molecule for drug discovery, and it is considered to be a factor that makes the development of therapeutic drugs for retinitis pigmentosa difficult. Therefore, it is expected to develop a method of treating without directly targeting genes (non-patent literature 11).

[0004] There is currently no established therapeutic drug for pigmentary retinitis. However, from many animal experiments and human clinical trials, the following concept has been established as a potential treatment for pigmentary retinitis (Non-patent Document 9). That is:

[0005] a) Even small improvements in rod survival lead to cone protection

[0006] b) Even with dysfunctional rods, cones can survive

[0007] c) If only a few cones can be maintained even in the macula, minimal visual acuity may be maintained, for example to be able to walk well on one's own.

[0008] Based on these concepts, clinical trials have been conducted for the purpose of protecting rods or cones on nutritional factors such as CNTF, valproic acid, vitamin A, docosahexaenoic acid (DHA), etc. However, so far, no clear drug effects have been reported and no compound has been approved by the FDA.

[0009] On the other hand, regenerative medicine by transplanting stem cells or cells differentiated into rods is currently being actively studied in non-clinical and clinical stages. However, there are many problems to be solved, such as immune rejection, low survival rate of transplanted cells, retention rate and biosafety (Non-Patent Document 12).

[0010] Citation List

[0011] Non-patent literature

[0012] Non-patent document 1: Truebestein L, et al., 2011, Nat Struct Mol Biol., Vol. 18(No. 3): p. 386-8

[0013] Non-patent document 2: Eigenbrot C, et al., 2012, Structure, Vol. 20 (No. 6): p.1040-50

[0014] Non-patent document 3: Grau S, et al., 2005, Proc Natl Acad Sci US A., Vol. 102(No. 17): p. 6021-26

[0015] Non-patent document 4: Grau S, et al., 2006, J Biol Chem., Vol. 281 (No. 10): p.6124-29

[0016] Non-patent document 5: Hadfield KD, et al., 2008, J Biol Chem., Vol. 283, (No. 9): p. 5928-38

[0017] Non-patent document 6: An E, et al., 2010, Invest Ophthalmol Vis Sci., Vol. 51(No. 7): p. 3379-86

[0018] Non-patent document 7: Yang Z, et al., 2006, Science, Vol. 314 (No. 5801): p. 992-93

[0019] Non-patent document 8: Tang NP, et al., 2009, Ann Epidemiol., Vol. 19 (No. 10): p.740-45

[0020] Non-patent document 9: Guadagni V, et al., 2015, Prog Retin Eye Res., Vol. 48: p.62-81

[0021] Non-patent document 10: Memdes HF et al., 2005, TRENDS Mol. Medicine, Vol. 11: p.177-185

[0022] Non-patent document 11: Farrar GJ, et al., 2002, EMBO J., Vol. 21 (No. 5): p. 857-864

[0023] Non-patent literature 12: He Y, et al., 2014, Int. J. Mol. Sci., Vol. 15 (No. 8): p.14456-14474. SUMMARY OF THE INVENTION

[0025] Technical issues

[0026] Provided are novel pharmaceutical compositions for treating or preventing retinitis pigmentosa.

[0027] Solution to the problem

[0028] The present invention relates to: (1)

[0030] A pharmaceutical composition for treating or preventing pigmentary retinitis, a genetic disease involving photoreceptor cell degeneration and / or a disease associated with PDE6 protein dysfunction, comprising a SPINK2 mutant peptide, wherein the SPINK2 mutant peptide comprises SEQ ID NO: 30 ( Fig.42 ) and inhibits the protease activity of human HTRA1; (2)

[0032] The pharmaceutical composition according to (1), wherein the peptide comprises SEQ ID NO: 30 ( Fig.42 ), the first Xaa (X1) is Asp, Glu, Ser, Gly or Ile, the second Xaa (X2) is Ala, Gly, Leu, Ser or Thr, the third Xaa (X3) is Asp, His, Lys, Met or Gln, the fourth Xaa (X4) is Asp, Phe, His, Ser or Tyr, the fifth Xaa (X5) is Ala, Asp, Glu, Met or Asn, the sixth Xaa (X6) is Met or Trp, the seventh Xaa (X7) is Gln, Trp, Tyr or Val, the eighth Xaa (X8) is Phe, Leu or Tyr, the ninth Xaa (X9) is Phe or Tyr, the tenth Xaa (X10) is Asp, His, Lys, Met or Gln, the fourth Xaa (X11) is Asp, His, Lys, Met or Gln, the fifth Xaa (X12) is Asp, His, Lys, Met or Asn, the sixth Xaa (X13) is Met or Trp, the seventh Xaa (X14) is Gln, Trp, Tyr or Val, the eighth Xaa (X15) is Phe, Leu or Tyr, the ninth Xaa (X16) is Phe or Tyr, the tenth Xaa (X17) is Asp, His, Lys, Met or Gln, the fourth Xaa (X18) is Asp, His, Lys, Met or Gln, the fifth Xaa (X19) is Ala, Asp, Glu, Met or Asn, the sixth Xaa (X10) is Met or Trp, the seventh Xaa (X11) is Gln, Trp, Tyr or Val, the eighth Xaa (X12) is Phe, Leu or Tyr, the ninth Xaa (X13) is Phe or Tyr 10 ) is Ala, Glu, Met or Val, and the eleventh Xaa (X 11 ) is Ala, Thr or Val; (3)

[0034] The pharmaceutical composition according to (1) or (2), wherein the peptide comprises SEQ ID NO: 3, 5, 7, 9, 11, 13, 15, 17, 19, 21 and 23 to 29 ( Fig.15 , 17 , 19, 21, 23, 25, 27, 29, 31, 33, and 35 to 41); (4)

[0036] The pharmaceutical composition according to any one of (1) to (3), wherein the peptide comprises a peptide bond connected via a peptide bond to SEQ ID NO: 30 ( Fig.42 ) is an amino acid sequence prepared by adding 1 to 3 amino acids to the amino terminal side of the amino acid sequence shown in ); (5)

[0038] The pharmaceutical composition according to any one of (1) to (4), wherein the peptide comprises a peptide bond connected via a peptide bond to SEQ ID NO: 30 ( Fig.42 ) is an amino acid sequence prepared by adding 1 or 2 amino acids to the carboxyl terminal side of the amino acid sequence shown in ); (6)

[0040] The pharmaceutical composition according to any one of (1) to (5), wherein the peptide has a three-dimensional structure characterized by having three disulfide bonds and comprising a loop structure, an α-helix, and a β-sheet; (7)

[0042] A pharmaceutical composition for treating or preventing retinitis pigmentosa, a genetic disease involving photoreceptor cell degeneration, and / or a disease associated with PDE6 protein dysfunction, comprising a polynucleotide comprising a nucleotide sequence encoding an amino acid sequence contained in the peptide according to any one of (1) to (6); (8)

[0044] A pharmaceutical composition for treating or preventing retinitis pigmentosa, a genetic disease involving photoreceptor cell degeneration, and / or a disease associated with PDE6 protein dysfunction, comprising a vector comprising a nucleotide sequence encoding an amino acid sequence contained in the peptide according to any one of (1) to (6); (9)

[0046] A pharmaceutical composition for treating or preventing retinitis pigmentosa, a genetic disease involving photoreceptor cell degeneration, and / or a disease associated with PDE6 protein dysfunction, comprising: a cell containing a polynucleotide comprising a nucleotide sequence encoding an amino acid sequence contained in the peptide according to any one of (1) to (6), or a vector containing the nucleotide sequence, or a cell producing the peptide according to any one of (1) to (6); (10)

[0048] A pharmaceutical composition for treating or preventing retinitis pigmentosa, a genetic disease involving photoreceptor cell degeneration, and / or a disease associated with PDE6 protein dysfunction, comprising a conjugate comprising the peptide according to any one of (1) to (6) linked to an additional moiety; (11)

[0050] The pharmaceutical composition according to (10), wherein the conjugate is a polypeptide; (12)

[0052] The pharmaceutical composition according to any one of (1) to (11), which is used for treating or preventing retinitis pigmentosa; (13)

[0054] The pharmaceutical composition according to any one of (1) to (11), which is used for treating or preventing a genetic disease involving photoreceptor cell degeneration; (14)

[0056] The pharmaceutical composition according to (13), wherein the hereditary disease involving photoreceptor cell degeneration is macular dystrophy; (15)

[0058] The pharmaceutical composition according to any one of (1) to (11), which is used for treating or preventing a disease associated with PDE6 protein dysfunction; (16)

[0060] The pharmaceutical composition according to (15), wherein the disease associated with PDE6 protein dysfunction is achromatopsia or autosomal dominant congenital stationary night blindness; (17)

[0062] The pharmaceutical composition according to any one of (1) to (16), comprising one or two or more additional drugs; (18)

[0064] The pharmaceutical composition according to any one of (1) to (17), which is used in combination with one or two or more additional drugs; and (19)

[0066] A method for identifying a therapeutic or preventive drug for retinitis pigmentosa, a genetic disease involving photoreceptor cell degeneration, and / or a disease associated with PDE6 protein dysfunction, comprising the following steps 1 to 3:

[0067] [Step 1] Incubate HTRA1 protease and substrate in the presence and absence of a test substance, wherein the test substance is a protein comprising SEQ ID NO: 30 ( Fig.42 ) SPINK2 mutant peptide having the amino acid sequence shown in;

[0068] [Step 2] detecting HTRA1 protease activity in the presence and absence of the test substance; and

[0069] [Step 3] When the HTRA1 protease activity in the presence of the test substance is lower than the HTRA1 protease activity in the absence of the test substance, the test substance is determined to be positive.

[0070] Advantageous Effects of the Invention

[0071] The peptide and the pharmaceutical composition comprising the peptide provided by the present invention have HTRA1 inhibitory activity and can be used for the treatment or prevention of retinitis pigmentosa, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0073] [Fig. 1(A)] Fig. 1(A) is a diagram showing the results of comparing the sequence similarities among human, mouse, rat and monkey HTRA1. The dotted line depicts the enzymatically active domain (204Gly to 364Leu).

[0074] [Fig. 1(B)] Fig. 1(B) is a diagram showing the results of comparing the sequence similarities among human, mouse, rat and monkey HTRA1 (continued).

[0075] [ Figure 2 ] Figure 2 It is a figure which shows the result of evaluating the HTRA1(cat) inhibitory activity of HTRA1-inhibitory peptides by using the decomposition rate of peptide substrate as an index. Panels A to C each show the evaluation result of each inhibitory peptide, and panel D shows the evaluation result of control wild-type SPINK2.

[0076] [ Figure 3 ] Figure 3 are graphs showing the results of evaluating the HTRA1 (full length) inhibitory activity of the HTRA1-inhibitory peptide by using the decomposition rate of the peptide substrate as an index (panels A to C).

[0077] [Fig. 4(A)] Fig. 4(A) is a diagram showing the results of evaluating the HTRA1 (cat) inhibitory activity of HTRA1-inhibitory peptides by using the degradation of human vitronectin as an index. The analysis was performed by Western blotting using a human vitronectin antibody (R&D Systems, Inc.; MAB2349).

[0078] [ Fig. 4(B)] Fig. 4(B) is a graph showing the results of evaluating the HTRA1 (cat) inhibitory activity of the HTRA1-inhibitory peptide by using the degradation of human vitronectin as an index (continued).

[0079] [Fig. 5(A)] Fig. 5(A) is a diagram showing the results of evaluating the cross-reactivity of the HTRA1-inhibitory peptide with each protease by using the decomposition of the peptide substrate as an index (Part 1). For the name of each protease used and its concentration, the name of the substrate and its concentration, etc., see Example 3.

[0080] [ Fig. 5(B)] Fig. 5(B) is a diagram showing the results of evaluating the cross-reactivity of the HTRA1-inhibitory peptide with each protease by using the decomposition of the peptide substrate as an index (part 2).

[0081] [ Fig. 5(C)] Fig. 5(C) is a diagram showing the results of evaluating the cross-reactivity of the HTRA1-inhibitory peptide with each protease by using the decomposition of the peptide substrate as an index (part 3).

[0082] [ Fig. 5(D)] Fig. 5(D) is a diagram showing the results of evaluating the cross-reactivity of the HTRA1-inhibitory peptide with each protease by using the decomposition of the peptide substrate as an index (part 4).

[0083] [ Figure 6 ] Figure 6 is a diagram showing an HTRA1 (cat) / HTRA1-inhibitory peptide complex obtained by X-ray crystallography. The inhibitory peptide binds to each molecule of the HTRA1 trimer formed by HTRA1 (cat).

[0084] [ Figure 7 ] Figure 7 is a diagram showing the HTRA1 (cat) / HTRA1-inhibitory peptide complex obtained as a monomer by X-ray crystallography. The inhibitory peptide binds to the region containing the active center of HTRA1 (cat).

[0085] [ Figure 8 ] Figure 8The amino acid sequence of H2-Opt is shown (SEQ ID NO: 54). The N-terminal "Mca-I" means N-(4-methylcoumaryl-7-amide)-isoleucine, and the C-terminal "(Dnp)K" means Nε-(2,4-dinitrophenyl)-lysine.

[0086] [ Fig. 9 ] Fig. 9 This is a graph showing that the expression of HTRA1 in the vitreous humor of a rat model of retinal damage induced by light exposure is increased. The analysis was performed by Western blotting using a human HTRA1 / PRSS11 antibody (R&D Systems, Inc.; AF2916).

[0087] [ Fig.10 ] Fig.10 It is a graph showing that the HTRA1-inhibitory peptide administration group of the rat model of retinal damage induced by light exposure inhibited the decrease in the nuclear count in the outer nuclear layer on the cross section of the retina. For the saline administration group, n = 4, and for the other groups, n = 5.

[0088] [ Fig.11 ] Fig.11 It is a graph showing the results of evaluating the HTRA1 (cat) inhibitory activities of five HTRA1-inhibitory peptide derivatives by using the decomposition rate of the peptide substrate as an index.

[0089] [ Fig.12 ] Fig.12 is a diagram showing the results of evaluating the binding of three HTRA1-inhibitory peptides to HTRA1 (cat) by an immunoprecipitation method.

[0090] [ Fig.13 ] Fig.13 The amino acid sequence of human SPINK2 is shown (SEQ ID NO: 1).

[0091] [ Fig.14 ] Fig.14 The nucleotide sequence encoding the amino acid sequence of human SPINK2 is shown (SEQ ID NO: 2).

[0092] [ Fig.15 ] Fig.15 The amino acid sequence of peptide H218 is shown (SEQ ID NO: 3).

[0093] [ Fig.16 ] Fig.16 The nucleotide sequence encoding the amino acid sequence of peptide H218 is shown (SEQ ID NO: 4).

[0094] [ Fig.17 ] Fig.17 The amino acid sequence of peptide H223 is shown (SEQ ID NO: 5).

[0095] [ Fig.18 ] Fig.18 The nucleotide sequence encoding the amino acid sequence of peptide H223 is shown (SEQ ID NO: 6).

[0096] [ Fig.19 ] Fig.19 The amino acid sequence of peptide H228 is shown (SEQ ID NO: 7).

[0097] [ Fig. 20 ] Fig. 20 The nucleotide sequence encoding the amino acid sequence of peptide H228 is shown (SEQ ID NO: 8).

[0098] [ Fig.21 ] Fig.21 The amino acid sequence of peptide H308 is shown (SEQ ID NO: 9).

[0099] [ Fig. 22 ] Fig. 22 The nucleotide sequence encoding the amino acid sequence of peptide H308 is shown (SEQ ID NO: 10).

[0100] [ Fig.23 ] Fig.23 The amino acid sequence of peptide H321 is shown (SEQ ID NO: 11).

[0101] [ Fig.24 ] Fig.24 The nucleotide sequence encoding the amino acid sequence of peptide H321 is shown (SEQ ID NO: 12).

[0102] [ Fig.25 ] Fig.25 The amino acid sequence of peptide H322 is shown (SEQ ID NO: 13).

[0103] [ Fig.26 ] Fig.26 The nucleotide sequence encoding the amino acid sequence of peptide H322 is shown (SEQ ID NO: 14).

[0104] [ Fig. 27 ] Fig. 27 The amino acid sequence of the peptide derivative H308AT is shown (SEQ ID NO: 15).

[0105] [ Fig.28 ] Fig.28The nucleotide sequence encoding the amino acid sequence of the peptide derivative H308AT is shown (SEQ ID NO: 16).

[0106] [ Fig.29 ] Fig.29 The amino acid sequence of the peptide derivative H321AT is shown (SEQ ID NO: 17).

[0107] [ Fig.30 ] Fig.30 The nucleotide sequence encoding the amino acid sequence of the peptide derivative H321AT is shown (SEQ ID NO: 18).

[0108] [ Fig.31 ] Fig.31 The amino acid sequence of the peptide derivative H322AT is shown (SEQ ID NO: 19).

[0109] [ Fig.32 ] Fig.32 The nucleotide sequence encoding the amino acid sequence of the peptide derivative H322AT is shown (SEQ ID NO: 20).

[0110] [ Fig.33 ] Fig.33 The amino acid sequence of peptide M7 is shown (SEQ ID NO: 21).

[0111] [ Fig.34 ] Fig.34 The nucleotide sequence encoding the amino acid sequence of peptide M7 is shown (SEQ ID NO: 22).

[0112] [ Fig.35 ] Fig.35 The amino acid sequence of the peptide derivative H308_S16A is shown (SEQ ID NO: 23).

[0113] [ Fig.36 ] Fig.36 The amino acid sequence of the peptide derivative H308_D1G_S16A is shown (SEQ ID NO: 24).

[0114] [ Fig.37 ] Fig.37 The amino acid sequence of the peptide derivative H308_D1S_S16A is shown (SEQ ID NO: 25).

[0115] [ Fig.38 ] Fig.38 The amino acid sequence of the peptide derivative H308_D1E_S16A is shown (SEQ ID NO: 26).

[0116] [ Fig.39 ] Fig.39 The amino acid sequence of the peptide derivative H308_D1SLI_S16A is shown (SEQ ID NO: 27).

[0117] [ Fig.40 ] Fig.40 The amino acid sequence of the peptide derivative H321AT_D1G_S16A is shown (SEQ ID NO: 28).

[0118] [ Fig.41 ] Fig.41 The amino acid sequence of the peptide derivative H322AT_D1G_S16A is shown (SEQ ID NO: 29).

[0119] [ Fig.42 ] Fig.42 The general formula of the HTRA1-inhibitory peptide is shown (SEQ ID NO: 30). 11 Each represents any amino acid.

[0120] [ Fig.43 ] Fig.43 The amino acid sequence consisting of the S tag and the linker is shown (SEQ ID NO: 31).

[0121] [ Fig.44 ] Fig.44 The amino acid sequence of the C-terminal hexamer is shown (SEQ ID NO: 32).

[0122] [ Fig.45 ] Fig.45 The nucleotide sequence of primer 1 is shown (SEQ ID NO: 33).

[0123] [ Fig.46 ] Fig.46 The nucleotide sequence of primer 2 is shown (SEQ ID NO: 34).

[0124] [ Fig.47 ] Fig.47 The nucleotide sequence of primer 3 is shown (SEQ ID NO: 35).

[0125] [ Fig.48 ] Fig.48 The nucleotide sequence of primer 4 is shown (SEQ ID NO: 36).

[0126] [ Fig.49 ] Fig.49 The nucleotide sequence of primer 5 is shown (SEQ ID NO: 37).

[0127] [ Fig.50 ] Fig.50The nucleotide sequence of primer 6 is shown (SEQ ID NO: 38).

[0128] [ Fig.51 ] Fig.51 The nucleotide sequence of primer 7 is shown (SEQ ID NO: 39).

[0129] [ Fig.52 ] Fig.52 The nucleotide sequence of primer 8 is shown (SEQ ID NO: 40).

[0130] [ Fig.53 ] Fig.53 The nucleotide sequence of primer 9 is shown (SEQ ID NO: 41).

[0131] [ Fig.54 ] Fig.54 The nucleotide sequence of primer 10 is shown (SEQ ID NO: 42).

[0132] [ Fig.55 ] Fig.55 The nucleotide sequence of primer 11 is shown (SEQ ID NO: 43).

[0133] [ Fig.56 ] Fig.56 The nucleotide sequence of primer 12 is shown (SEQ ID NO: 44).

[0134] [ Fig.57 ] Fig.57 The nucleotide sequence of primer 13 is shown (SEQ ID NO: 45).

[0135] [ Fig.58 ] Fig.58 The nucleotide sequence of primer 14 is shown (SEQ ID NO: 46).

[0136] [ Fig.59 ] Fig.59 The nucleotide sequence of primer 15 is shown (SEQ ID NO: 47).

[0137] [ Fig.60 ] Fig.60 The nucleotide sequence of primer 16 is shown (SEQ ID NO: 48).

[0138] [ Fig.61 ] Fig.61 The nucleotide sequence of primer 17 is shown (SEQ ID NO: 49).

[0139] [ Fig.62 ] Fig.62The nucleotide sequence of primer 18 is shown (SEQ ID NO: 50).

[0140] [ Fig.63 ] Fig.63 The nucleotide sequence of primer 19 is shown (SEQ ID NO: 51).

[0141] [ Fig.64 ] Fig.64 The nucleotide sequence of primer 20 is shown (SEQ ID NO: 52).

[0142] [ Fig.65 ] Fig.65 The amino acid sequence of human HTRA1 (full length) is shown (SEQ ID NO: 53).

[0143] [ Fig.66 (A)] Fig.66 (A) is a graph showing the results of evaluating the HTRA1 (cat) inhibitory activity of HTRA1-inhibitory peptides by using the decomposition rate of the peptide substrate as an index.

[0144] [ Fig.66 (B)] Fig.66 (B) is a graph showing the results of evaluating the HTRA1 (full length) inhibitory activity of the HTRA1-inhibitory peptide by using the decomposition rate of the peptide substrate as an index.

[0145] [ Fig.67 ] Fig.67 This is a diagram showing the results of evaluating the HTRA1 (cat) inhibitory activity of HTRA1-inhibitory peptides by using the degradation of human vitronectin as an index. The analysis was performed by Western blotting using a human vitronectin antibody (R&D Systems, Inc.; MAB2349).

[0146] [ Fig. 68(A)] Fig. 68(A) is a diagram showing the results of evaluating the cross-reactivity of the HTRA1-inhibitory peptide with each protease by using the decomposition of the peptide substrate as an index (part 1).

[0147] [ Fig. 68(B)] Fig. 68(B) is a diagram showing the results of evaluating the cross-reactivity of the HTRA1-inhibitory peptide with each protease by using the decomposition of the peptide substrate as an index (part 2).

[0148] [ Fig. 68(C)] Fig. 68(C) is a diagram showing the results of evaluating the cross-reactivity of the HTRA1-inhibitory peptide with each protease by using the decomposition of the peptide substrate as an index (part 3).

[0149] [ Fig. 68(D)] Fig. 68(D) is a diagram showing the results of evaluating the cross-reactivity of the HTRA1-inhibitory peptide with each protease by using the decomposition of the peptide substrate as an index (part 4).

[0150] [ Fig. 68(E)] Fig. 68(E) is a diagram showing the results of evaluating the cross-reactivity of the HTRA1-inhibitory peptide with each protease by using the decomposition of the peptide substrate as an index (part 5).

[0151] [ Fig.69 ] Fig.69 is a diagram showing the results of evaluating the binding of three HTRA1-inhibitory peptides to HTRA1 (cat) by an immunoprecipitation method.

[0152] [ Fig.70 (A)] Fig.70 (A) is a graph showing that the HTRA1-inhibitory peptide H308_D1G_S16A administration group of the rat model of retinal damage induced by light exposure inhibited the decrease in the nuclear count in the outer nuclear layer on the cross section of the retina. For all groups, n = 6. The doses of HTRA1-inhibitory peptide H308_D1G_S16A were 0.2 and 1 μg / eye.

[0153] [ Fig.70 (B)] Fig.70 (B) is a graph showing that the HTRA1-inhibitory peptide H321AT_D1G_S16A administration group of the rat model of retinal damage induced by light exposure inhibited the decrease in the nuclear count in the outer nuclear layer on the cross section of the retina. For all groups, n = 6. The doses of HTRA1-inhibitory peptide H321AT_D1G_S16A were 0.2 and 1 μg / eye.

[0154] [ Fig.70 (C)] Fig.70 (C) is a graph showing that the HTRA1-inhibitory peptide H322AT_D1G_S16A administration group of the rat model of retinal damage induced by light exposure inhibited the decrease in the nuclear count in the outer nuclear layer on the cross section of the retina. For all groups, n = 6. The doses of the HTRA1-inhibitory peptide H322AT_D1G_S16A were 0.2 and 1 μg / eye.

[0155] [ Fig.71 (A)] Fig.71 (A) is a graph showing the results of immunostaining of RPE cells in 12-week-old rabbits, 3-year-old rabbits, and 3-year-old rabbits loaded with HFD-HQ using ZO-1 antibody (Thermo Fisher Scientific Inc.).

[0156] [ Fig.71 (B)] Fig.71 (B) shows the average area of ​​RPE cells in 12-week-old rabbits, 3-year-old rabbits, and 3-year-old rabbits loaded with HFD-HQ.

[0157] [ Fig.71 (C)] Fig.71 (C) is a graph showing increased expression of mRNA of complement component 3 "C3", an AMD-related factor, in retinal tissues of 3-year-old rabbits loaded with HFD-HQ.

[0158] [ Fig.71 (D)] Fig.71 (D) is a graph showing increased expression of mRNA of complement component 3 "C3", an AMD-related factor, in RPE / choroid tissues of 3-year-old rabbits loaded with HFD-HQ.

[0159] [ Fig.71 (E)] Fig.71 (E) is a graph showing the results of measuring the HTRA1 concentration of the vitreous humor in 12-week-old rabbits, 3-year-old rabbits, and 3-year-old rabbits loaded with HFD-HQ by LC-MS / MS.

[0160] [ Fig.72 (A)] Fig.72 (A) is a graph showing that the HTRA1-inhibitory peptide H308-administered group of 3-year-old rabbits loaded with HFD-HQ exhibited an inhibitory effect on the hypertrophy of RPE cells. For all groups, n = 5. The average area was used as an indicator.

[0161] [ Fig.72 (B)] Fig.72 (B) is a graph showing that the HTRA1-inhibitory peptide H308-administered group of 3-year-old rabbits loaded with HFD-HQ exhibited an inhibitory effect on RPE cell hypertrophy. For all groups, n = 5. 2 The number of RPE cells with a cell area of ​​100 μg / cm2 or larger was used as an indicator.

[0162] [ Fig.73 ] Fig.73 It is a diagram showing the results of comparing the sequence similarities among the enzymatically active domains of human, monkey, rabbit, mouse and rat HTRA1.

[0163] [ Fig.74 ] Fig.74 is a graph showing that HTRA1-inhibitory peptides exhibited an inhibitory effect on VEGF mRNA induced in a human retinal pigment epithelial cell line ARPE-19 by the addition of H2O2, normal human serum complement, and HTRA1.

[0164] [ Fig.75 ] Fig.75This is a graph showing that the HTRA1-inhibitory peptide exhibits an inhibitory effect on the migration of human umbilical vein endothelial cells (HUVEC) induced by serum.

[0165] [ Fig.76 ] Fig.76 The nucleotide sequence of primer 21 is shown.

[0166] [ Fig.77 ] Fig.77 The nucleotide sequence of primer 22 is shown.

[0167] [ Fig.78 ] Fig.78 is a graph showing that the ONL / INL ratio in the central part of the 160 μg / eye administration group was significantly increased compared with that in the control PBS group, and when the 160 μg / eye administration group was administered to Rd10 mice (B6.CXB1-Pde6b rd10 / J) When the HTRA1-inhibiting peptide was administered, the ONL / INL ratio in the peripheral part also tended to increase compared with that in the control group. *Indicates p<0.05 in Student's paired-t test. See Example 15.

[0168] In the present invention, the phrase "SEQ ID NO: X (Figure Y)" or "Figure Y (SEQ ID NO: X)" means that the sequence is shown in SEQ ID NO: X or is shown in Figure Y.

[0169] Description of the implementation plan

[0170] 1. Definition

[0171] In the present invention, the term "gene" is used to refer to a nucleic acid molecule comprising a nucleotide sequence or its complementary chain that encodes an amino acid sequence contained in a protein. The gene is composed of a single strand, a double strand, or a triple strand or more strands. The associate of a DNA strand and an RNA strand, ribonucleotides and deoxyribonucleotides coexisting on one strand, and a double strand or triple strand or more strand of nucleic acid molecules comprising such a strand are also included in the meaning of the term "gene".

[0172] In the present invention, the terms "gene", "polynucleotide" and "nucleic acid molecule" are synonymous with each other and are not limited in any way by the number of their constituent units, such as ribonucleotides, deoxyribonucleotides, nucleotides and nucleosides. For example, DNA, RNA, mRNA, cDNA, cRNA, probes, oligonucleotides, primers, etc. are also included in its scope. The term "nucleic acid molecule" is also abbreviated as "nucleic acid".

[0173] In the present invention, the terms "polypeptide", "peptide" and "protein" are synonymous with each other. A peptide that inhibits or suppresses one or two or more activities or functions of a target molecule X (hereinafter, these inhibitory or suppressive effects are collectively referred to as "X inhibitory activity") can be referred to as an "X-inhibitory peptide".

[0174] The term "SPINK2" is used to refer to the serine protease inhibitor Kazal-2 type. This 7 kDa protein consists of a Kazal-like domain with three disulfide bonds. SPINK2 is preferably of human origin. In the present invention, human SPINK2 is referred to as "SPINK2" for short, unless otherwise specified.

[0175] The term "HTRA1" is used to refer to high temperature requirement A serine peptidase 1. The protein is composed of an N-terminal domain consisting of an IGFBP-like module and a Kazal-like module, a protease domain, and a C-terminal PDZ domain, and the protein belongs to the HTRA family. HTRA1 is preferably of human origin. In the present invention, human HTRA1 is also referred to as "HTRA1" unless otherwise specified.

[0176] The term "HTRA1-inhibitory peptide" is used to refer to a peptide that inhibits or suppresses one or two or more activities or functions of HTRA1. The term "HTRA1-inhibitory peptide" includes within its scope a fragment of the peptide that maintains HTRA1 inhibitory activity, an adduct of the peptide with an additional moiety, or a conjugate of the peptide. Specifically, the term "HTRA1-inhibitory peptide" also includes fragments, adducts, and modified forms of the peptide that maintain HTRA1 inhibitory activity.

[0177] In the present invention, the term "cell" is used to include various cells derived from an animal individual, subcultured cells, primary cultured cells, cell lines, recombinant cells, yeast, microorganisms and the like.

[0178] In the present invention, the term "site" for peptide binding, i.e., the "site" recognized by the peptide, is used to refer to a continuous or discontinuous partial amino acid sequence or partial conformation on a target molecule to be bound or recognized by the peptide. In the present invention, such a site may be referred to as an epitope or binding site on a target molecule.

[0179] The term "SPINK2 mutant" is used to refer to a peptide comprising an amino acid sequence derived from the amino acid sequence of wild-type SPINK2 by substitution of one or two or more amino acids with an amino acid different from the wild-type amino acid, deletion of one or two or more wild-type amino acids, insertion of one or two or more amino acids not present in the wild-type, and / or addition of one or more amino acids not present in the wild-type to the amino terminus (N-terminus) and / or carboxyl terminus (C-terminus) of the wild-type (hereinafter collectively referred to as "mutation"). "SPINK2 mutants" having HTRA1 inhibitory activity are included in HTRA1-inhibitory peptides. In the present invention, "insertion" may also be included in "addition".

[0180] In the present invention, the term “several” in the phrase “one or several” means 3 to 10.

[0181] In the present invention, the phrase "hybridize under stringent conditions" is used to mean that hybridization is performed in a solution containing 5 × SSC at 65° C. and then the resultant is washed in an aqueous solution containing 2 × SSC and 0.1% SDS at 65° C. for 20 minutes, in an aqueous solution containing 0.5 × SSC and 0.1% SDS at 65° C. for 20 minutes, and in an aqueous solution containing 0.2 × SSC and 0.1% SDS at 65° C. for 20 minutes, or under conditions equivalent thereto. SSC means an aqueous solution of 150 mM NaCl and 15 mM sodium citrate, and "n × SSC" means n-fold concentration of SSC.

[0182] In the present invention, the terms "specific" and "specificity" are synonymous with and interchangeable with the terms "selective" and "selectivity," respectively. For example, an HTRA1-specific inhibitory peptide is synonymous with an HTRA1-selective inhibitory peptide.

[0183] 2. Peptides

[0184] 2-1. Amino Acids

[0185] The term "amino acid" is used to refer to an organic compound containing an amino group and a carboxyl group, and refers to an α-amino acid contained as a constituent unit in a preferred protein and more preferably a natural protein. In the present invention, the amino acid is more preferably Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr and Val. The term "amino acid" is used to refer to these 20 amino acids, unless otherwise specified. These 20 amino acids can be referred to as "natural amino acids". The HTRA1-inhibitory peptide of the present invention preferably contains natural amino acids.

[0186] In the present invention, the term "amino acid residue" is also referred to as "amino acid".

[0187] In the present invention, the amino acid may be an L-amino acid, a D-amino acid or a mixture thereof (DL-amino acid), and means an L-amino acid unless otherwise specified.

[0188] Based on common properties of their side chains, natural amino acids can be divided into groups such as:

[0189] (1) Hydrophobic amino acid group: Met, Ala, Val, Leu and Ile;

[0190] (2) Neutral hydrophilic amino acid group: Cys, Ser, Thr, Asn and Gln;

[0191] (3) Acidic amino acid group: Asp and Glu;

[0192] (4) Basic amino acid group: His, Lys and Arg;

[0193] (5) amino acid groups that affect the orientation of the main chain: Gly and Pro; and

[0194] (6) Aromatic amino acid group: Trp, Tyr and Phe.

[0195] However, the classification of natural amino acids is not limited thereto.

[0196] In the present invention, each natural amino acid can be subjected to conservative amino acid substitution.

[0197] The term "conservative amino acid substitution" means substitution by an amino acid that is functionally equivalent or similar. Conservative amino acid substitutions in a peptide bring about static changes in the amino acid sequence of the peptide. For example, one or two or more amino acid substitutions with similar polarity function equivalently and bring about static changes in the amino acid sequence of the peptide. Generally, substitutions within a particular group can be considered conservative in terms of structure and function. However, it will be apparent to those skilled in the art that the role of a particular amino acid residue can be determined by its position in the three-dimensional structure of the molecule containing the amino acid. For example, a cysteine ​​residue can take an oxidized (disulfide) form that is less polar than the reduced (thiol) form. The long aliphatic portion of the arginine side chain can constitute important features in structure and function. Alternatively, side chains containing aromatic rings (tryptophan, tyrosine and phenylalanine) can contribute to ion-aromatic interactions or cation-π interactions. In this case, even amino acids with these side chains are replaced by amino acids belonging to acidic or non-polar groups and can be conservative in structure and function. Residues such as proline, glycine and cysteine ​​(in disulfide form) can have a direct impact on the three-dimensional structure of the backbone and often cannot be substituted without distorting the structure.

[0198] Conservative amino acid substitutions include specific substitutions based on side chain similarity as shown below (L. Lehninger, Biochemistry, 2nd edition, pp. 73-75, Worth Publisher, New York (1975)) and typical substitutions.

[0199] (1) Non-polar amino acid group: alanine (hereinafter referred to as "Ala" or "A"), valine (hereinafter referred to as "Val" or "V"), leucine (hereinafter referred to as "Leu" or "L"), isoleucine (hereinafter referred to as "Ile" or "I"), proline (hereinafter referred to as "Pro" or "P"), phenylalanine (hereinafter referred to as "Phe" or "F"), tryptophan (hereinafter referred to as "Trp" or "W"), and methionine (hereinafter referred to as "Met" or "M")

[0200] (2) Uncharged polar amino acids group: glycine (hereinafter referred to as "Gly" or "G" for short), serine (hereinafter referred to as "Ser" or "S" for short), threonine (hereinafter referred to as "Thr" or "T" for short), cysteine ​​(hereinafter referred to as "Cys" or "C" for short), tyrosine (hereinafter referred to as "Tr" or "Y" for short), asparagine (hereinafter referred to as "Asn" or "N" for short) and glutamine (hereinafter referred to as "Gln" or "Q" for short)

[0201] (3) Acidic amino acid group: Aspartic acid (hereinafter referred to as "Asp" or "D") and Glutamic acid (hereinafter referred to as "Glu" or "E")

[0202] (4) Basic amino acid group: lysine (hereinafter referred to as "Lys" or "K" for short), arginine (hereinafter referred to as "Arg" or "R" for short), and histidine (hereinafter referred to as "His" or "H" for short)

[0203] In the present invention, amino acid can be amino acid other than natural amino acid. Its example can include selenocysteine, N-formylmethionine, pyrrolysine, pyroglutamic acid, cystine, hydroxyproline, hydroxylysine, thyroxine, O-phosphoserine, desmosine, β-alanine, sarcosine, ornithine, creatine, γ-aminobutyric acid, opine, theanine, mushroom amino acid, kainic acid, domoic acid and acromelic acid (which is found in natural peptides or proteins), and can include: N-terminal protected amino acids, such as norleucine, Ac-amino acids, Boc-amino acids, Fmoc-amino acids, Trt-amino acids and Z-amino acids; C-terminal protected amino acids, such as tert-butyl esters, benzyl esters, cyclohexyl esters and fluorene esters of amino acids; and other amino acids not found in nature, including diamines, ω amino acids, β amino acids, γ amino acids, Tic derivatives of amino acids and aminophosphonic acids. For convenience, amino acids other than 20 kinds of "natural amino acids" are not limited thereto, and are collectively referred to as "non-natural amino acids" in the present invention.

[0204] 2-2. HTRA1-inhibitory peptide

[0205] The HTRA1-inhibitory peptide of the present invention is a SPINK2 mutant that at least partially maintains the framework of SPINK2 (hereinafter referred to as a "SPINK2 mutant"), and inhibits or suppresses the protease activity of HTRA1 or a fragment that retains its enzymatic activity (hereinafter referred to as a "functional fragment") (hereinafter, such inhibition and suppression are collectively referred to as "HTRA1 inhibitory activity").

[0206] HTRA1 as a target of the inhibitory peptide of the present invention is preferably mammalian HTRA1, more preferably primate HTRA1, and still more preferably human HTRA1. The amino acid sequence of full-length mature human HTRA1 (hereinafter referred to as "HTRA1 (full-length)") has SEQ ID NO: 53 ( Fig.65) is an amino acid sequence shown in SEQ ID NO: 53 ( Fig.65 ) and a functional fragment consisting of 136Gly to 351Lys of HTRA1 or its functional fragment as the target of the inhibitory peptide of the present invention is also referred to as HTRA1 protease. HTRA1 protease is preferably of vertebrate origin, more preferably of mammalian origin, still more preferably of primate origin, and most preferably of human origin, and can be prepared by purification from tissues or cells of any of these animals or by methods known to those skilled in the art as protein preparation methods (such as gene recombination, in vitro translation or peptide synthesis). HTRA1 or its functional fragment can be connected to a signal sequence, an immunoglobulin Fc region, a tag, a marker, etc.

[0207] HTRA1 inhibitory activity can be evaluated by using the protease activity of HTRA1 as an index. For example, HTRA1 or its functional fragment, substrate and inhibitory peptide of the present invention or its candidate are coexisted with each other. In this case, when the protease activity of HTRA1 is 70% or less, 50% or less, 30% or less, 20% or less, 10% or less, 5% or less, 1% or less or 0% compared with the protease activity in the presence of a control or in the absence of an inhibitor or its candidate, inhibition of HTRA1 occurs, wherein the inhibitory activity is 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 HTRA1 inhibitory activity can be different according to reaction conditions, substrate type, concentration, etc. Examples of reaction conditions can include, but are not limited to, those described in the embodiments. The enzyme activity can be evaluated by adding a substrate peptide or substrate protein to a given concentration of HTRA1, reacting the mixture for a given time, and then detecting the fluorescence of the substrate peptide or detecting the substrate protein by SDS-PAGE, Western blotting, liquid chromatography, etc. For example, phosphate buffered saline (hereinafter referred to as "PBS"), borate buffer (50 mM borate, pH 7 to 9, for example, pH 8.5) or Tris buffer (50 mM Tris, pH 6 to 9, for example, pH 8.0) can be used as a buffer solution. NaCl (50 to 300 mM, for example, 150 mM) or a surfactant such as CHAPS or octyl β-D-pyranoglucoside can be added to the reaction system, although the additive is not limited thereto.

[0208] Examples of substrates of HTRA1 protease include, but are not particularly limited to, endogenous substrates, exogenous substrates, and synthetic substrates. Examples of human endogenous substrates may include vitronectin. Examples of synthetic substrates may include, but are not particularly limited to, H2-Opt (Mca-IRRVSYSFK(Dnp)K), β-casein, and other HTRA1 substrates. The HTRA1 inhibitory activity (IC 50 or K i ) is 1 μM or lower, more preferably 100 nM or lower.

[0209] Preferably, the inhibitory peptide of the present invention should not inhibit or suppress the activity of proteases other than HTRA1, or should have a relatively weak degree of inhibition or suppression of such activity. In other words, the inhibitory peptide of the present invention preferably has high HTRA1 specificity. Preferably, the inhibitory peptide of the present invention does not inhibit or suppress the activity of proteases such as trypsin, α-chymotrypsin, tryptase, plasmin, thrombin, matriptase, protein C, tissue plasminogen activator (tPA), urokinase (uPA), plasmin or plasma kallikrein, or has a relatively weak degree of inhibition or suppression of such activity. Such preferred peptides of the present invention do not show adverse reactions caused by the inhibition or suppression of the activity of other proteases, and can be preferably used as therapeutic drugs or preventive drugs for HTRA1-related diseases (mentioned above).

[0210] As mentioned above, HTRA1, which is the target of the peptide of the present invention, is preferably of vertebrate origin, more preferably of mammalian origin, still more preferably of primate origin, and most preferably of human origin. Alternatively, HTRA1, which is the target of the peptide of the present invention, may be derived from non-human animals, for example, rodents such as rats or mice, or primates such as cynomolgus monkeys, common marmosets or macaques. Peptides having inhibitory activity against HTRA1 of non-human animal origin can be used for diagnosis, examination, treatment or prevention of HTRA1-related diseases in non-human animals, etc. When such a peptide also inhibits human HTRA1, the non-human animal can be used for: non-clinical research and development of peptides as therapeutic drugs or preventive drugs for human HTRA1-related diseases; or using non-human animals as animal models of diseases for pharmacological tests or pharmacokinetic tests; or using non-human animals as healthy animals for safety tests, toxicity tests, etc.

[0211] The HTRA1-inhibitory peptide of the present invention has advantages such as: a molecular weight lower than that of other biomolecules (such as antibodies used as pharmaceutical agents and diagnostic drugs in the art); its production is relatively easy (mentioned later); excellent physical properties in terms of tissue permeability, storage stability, thermal stability, etc.; and a wide range of choices for administration routes, administration methods, preparation, etc. for use as pharmaceutical compositions (mentioned later). The half-life of the peptide of the present invention used as a pharmaceutical composition in the blood can be adjusted to be longer by applying known methods, such as adding biomolecules or polymers, and thereby increasing the molecular weight of the peptide. The molecular weight of such an HTRA1-inhibitory peptide of the present invention is less than 10,000, preferably less than 8,000, and more preferably about 7,000 to 7,200. The HTRA1-inhibitory peptide of the present invention also includes a peptide consisting of SEQ ID NO: 23 ( Fig.35 ) or a portion consisting of 15Cys to 63Cys thereof (hereinafter referred to as "a portion containing six Cys residues") and having HTRA1 inhibitory activity. The molecular weight of the variable loop portion is less than 2,500, and preferably about 1,800 to 2,000. The molecular weight of the portion containing six Cys residues is less than 6,000, and preferably about 5,300 to 5,500.

[0212] The SPINK2 mutants (hereinafter referred to as "SPINK2 mutants") included in the scope of the HTRA1-inhibitory peptides of the present invention that at least partially maintain the framework of SPINK2 can bind to HTRA1, and are capable of binding to preferably mammalian HTRA1, more preferably primate HTRA1, and still more preferably human HTRA1. Such peptides that bind to HTRA1 recognize or bind to a partial peptide, a partial conformation, etc. of HTRA1 (hereinafter, such recognition and binding effects are collectively referred to as "target binding activity").

[0213] In one aspect, the inhibitory peptides of the present invention are capable of binding to immunogenic fragments of HTRA1. Immunogenic fragments of HTRA1 have one or two or more epitopes, mimotopes or other antigenic determinants and are thus capable of inducing an immune response or of eliciting antibodies against the fragment to be generated.

[0214] The binding of the SPINK2 mutant according to the present invention to HTRA1 or to an immunogenic fragment thereof can be evaluated, measured or determined by using methods known to those skilled in the art, such as measuring detectable binding affinity (ELISA, surface plasmon resonance (hereinafter referred to as "SPR") analysis (also referred to as "BIAcore" method), isothermal titration calorimetry (hereinafter referred to as "ITC"), flow cytometry, immunoprecipitation methods, etc.).

[0215] Examples of ELISA include methods involving detection of HTRA1-inhibitory peptides that have been recognized and bound to HTRA1 fixed on a plate. HTRA1 can be fixed using biotin-streptavidin and, for example, antibodies for fixing that recognize a tag fused to HTRA1. Detection of HTRA1-inhibitory peptides can use labeled streptavidin and, for example, labeled antibodies for detecting that recognize a tag fused to HTRA1-inhibitory peptides. Labeling can use biotin and methods that are feasible in biochemical analysis, such as HRP, alkaline phosphatase or FITC. Detection using enzymatic labels can employ chromogenic substrates, such as 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) or SuperSignalELISA Pico chemiluminescent substrate (Thermo Fisher Scientific Inc.), fluorescent substrates, such as QuantaBlu (TM) fluorescent peroxidase substrate (Thermo Fisher Scientific Inc.), and chemiluminescent substrates. Measurement of the detection signal can employ an absorbance plate reader, a fluorescence plate reader, a luminescence plate reader, an RI liquid scintillation counter, etc.

[0216] Examples of instruments used in SPR analysis include BIAcore (TM) (GE Healthcare), ProteOn (TM) (Bio-Rad Laboratories, Inc.), SPR-Navi (TM) (Oy BioNavis Ltd.), Spreeta (TM) (Texas Instruments Inc.), SPRi-PlexII (TM) (HORIBA, Ltd.), and Autolab SPR (TM) (Metrohm AG). Examples of instruments used in BLI may include Octet (TM) (Pall Corp.).

[0217] Examples of immunoprecipitation methods include methods involving detection of HTRA1 that has been identified and bound to an HTRA1-inhibitory peptide fixed on beads. Magnetic beads, agarose beads, etc. can be used as beads. The fixation of HTRA1-inhibitory peptides can use biotin-streptavidin and antibodies that recognize the peptide or the label fused with the peptide, protein A or protein G, etc. Beads are separated using magnets, centrifugation, etc., and HTRA1 precipitated with beads is detected by SDS-PAGE or Western blot. The detection of HTRA1 can use labeled streptavidin and antibodies for detection of labels fused with HTRA1, such as labeled recognition. Labeling can use biotin and methods feasible in biochemical analysis, such as HRP, alkaline phosphatase or FITC. The detection using enzymatic labeling can use the same substrate as in ELISA. The measurement of the detection signal can use ChemiDoc (TM) (Bio-Rad Laboratories, Inc.), LuminoGraph (ATTO Corp.), etc.

[0218] In the present invention, the term "specific recognition", that is, "specific binding", is used to mean binding that is not non-specific adsorption. Examples of criteria for determining whether binding is specific include binding activity EC in ELISA 50 Other examples of determination criteria may include the dissociation constant (hereinafter referred to as "K D ”). The HTRA1-inhibitory peptide according to the present invention has an inhibitory effect on the K of HTRA1 D The value is 1 × 10 -4 M or less, 1 × 10 -5 M or less, 5 × 10 -6 M or less, 2 × 10 -6 M or less or 1 × 10 -6 M or less, more preferably 5 × 10 -7 M or less, 2 × 10 -7 M or less or 1 × 10 -7 M or less, still more preferably 5 × 10 -8 M or less, 2 × 10 -8 M or less or 1 × 10 -8 M or less, more preferably 5× 10 -9 M or less, 2 × 10 -9 M or less or 1 × 10 -9M or less. Other examples of determination criteria may include analysis results by an immunoprecipitation method. A preferred HTRA1-inhibitory peptide according to the present invention is immobilized on beads, and HTRA1 is added thereto. Then, the beads are separated, and HTRA1 precipitated with the beads is detected. In this case, the signal of HTRA1 is detected.

[0219] Despite the above description, for a SPINK2 mutant to serve as an inhibitory peptide of the present invention, the ability to bind to HTRA1 or an immunogenic fragment thereof is not necessary, as long as the SPINK2 mutant has HTRA1 inhibitory activity.

[0220] The inhibitory peptides of the present invention may be competitive in the binding of protease substrates to HTRA1.

[0221] In some preferred aspects, the inhibitory peptides of the present invention have a retinal protective effect. For example, the preferred inhibitors of the present invention can suppress the light exposure-induced reduction of nuclear counts in the outer nuclear layer in a model of retinal damage induced by light exposure, which is described in detail in the Examples. Compared with the non-exposed group, a greater amount of HTRA1 protein has been detected in the vitreous fluid of the light-exposed group of the model. Therefore, the present invention discloses that: HTRA1 is involved in retinal damage; and HTRA1 inhibitory activity brings about a retinal protective effect.

[0222] The SPINK2 mutants serving as inhibitory peptides of the present invention may have the activities, properties, functions, characteristics, etc. as described above, and their full-length amino acid sequences have high sequence identity with the amino acid sequence of human wild-type SPINK2. The amino acid sequence of the SPINK2 mutants of the present invention and human SPINK2 (SEQ ID NO: 1: Fig.13 ) have 60% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 98% or more or 99% or more sequence identity.

[0223] The term "identity" is used to refer to a property indicating the degree of similarity or relationship between two sequences. The identity (%) of amino acid sequences is calculated by dividing the number of identical amino acids or amino acid residues by the total number of amino acids or amino acid residues and multiplying the resulting value by 100.

[0224] The term "gap" is used to refer to a space in an alignment between two or more sequences that is a result of a deletion and / or addition in at least one of the two or more sequences.

[0225] The identity between two amino acid sequences with identical amino acid sequences is 100%. If one of the amino acid sequences has a replacement, deletion or addition of one, two or more amino acids or amino acid residues compared to another amino acid sequence, the identity between the two amino acid sequences is less than 100%. The example of the algorithm or program for determining the identity between the two sequences in the case of considering a room can include those known to those skilled in the art, such as BLAST (Altschul, et al., Nucleic Acids Res., Vol. 25, p. 3389-3402, 1997), BLAST2 (Altschul, et al., J. Mol. Biol., Vol. 215, p. 403-410, 1990) and Smith-Waterman (Smith, et al., J. Mol. Biol., Vol. 147, p. 195-197, 1981).

[0226] In the present invention, the term "mutated" is used to mean that one or two or more nucleotides or nucleotide residues or amino acids or amino acid residues are substituted, deleted or inserted in a nucleotide sequence or an amino acid sequence compared to a naturally occurring nucleic acid molecule or peptide. Compared to the amino acid sequence of human SPINK2, the amino acid sequence of the SPINK2 mutant of the present invention has one or two or more mutated amino acids or amino acid residues.

[0227] In one aspect of the present invention, the amino acid sequence of the SPINK2 mutant may have any of the following:

[0228] The amino acid sequence of human SPINK2 (SEQ ID NO: 1: Fig.13 ) wherein 1, 2, 3, 4, 5, 6 or 7 amino acids from 16Ser to 22Gly are substituted by other amino acids or amino acid residues;

[0229] The amino acid sequence of human SPINK2 (SEQ ID NO: 1: Fig.13 ) wherein 1, 2, 3, 4 or 5 amino acids from 24Pro to 28Asn are substituted by other amino acids or amino acid residues;

[0230] The amino acid sequence of human SPINK2 (SEQ ID NO: 1: Fig.13 ) wherein 1 or 2 amino acids at 39Ala and 43Thr are replaced by other amino acids or amino acid residues,

[0231] Or these amino acids may be present as wild-type amino acids (including 2 amino acids or amino acid residues in the α helix), as long as the tertiary structure of the main chain of the ring composed of amino acid residues at positions 16 to 30, etc. can at least partially exert HTRA1 inhibitory activity; in order to maintain the natural disulfide bonds, 15Cys, 23Cys, 31Cys, 42Cys, 45Cys and 63Cys are each preferably Cys, as in the wild type. Among them, 1, 2, 3, 4, 5 or 6 may be substituted by other amino acids to delete the natural disulfide bonds or generate non-natural disulfide bonds. Some preferred HTRA1-inhibitory peptides in the SPINK2 mutants of the present invention maintain the Cys at these 6 positions, as in the natural SPINK2, and retain the disulfide bonds. In some more preferred forms of such inhibitory peptides, 15Cys-45Cys, 23Cys-42Cys and 31Cys-63Cys form disulfide bonds, respectively.

[0232] When the amino acid sequence of such a SPINK2 mutant is included in an HTRA1-inhibitory peptide, it is preferred that the three-dimensional structure contained in the amino acid sequence of wild-type SPINK2, which is composed of, for example, a loop structure consisting of 16Ser to 30Val, a β-sheet consisting of a β-strand (1) consisting of 31Cys and 32Gly and a β-strand (2) consisting of 57Ile to 59Arg, an α-helix consisting of 41Glu to 51Gly, or a loop structure, β-sheet or α-helix similar thereto or at least partially corresponding thereto (or to these positions), should be maintained to the extent that the HTRA1 inhibitory activity can be exerted.

[0233] The amino acid sequences of some HTRA1-inhibitory peptides in the SPINK2 mutants of the present invention will be mentioned below. As mentioned above, in the present invention, the term "amino acid residue" is also referred to as "amino acid" for short.

[0234] SEQ ID NO:30( Fig.42 ) in the amino acid sequence shown in , each of which is the same as X1 to X11, is any arbitrary amino acid without particular limitation, as long as the resulting mutant binds to HTRA1 and inhibits HTRA1 activity. Hereinafter, preferred amino acids for X1 to X11 will be described. However, these amino acids may include natural amino acids, i.e., amino acids identical to those in the amino acid sequence of wild-type human SPINK2.

[0235] X1 at position 1 is preferably Asp, Glu, Gly, Ser or Ile, more preferably Asp or Gly, and still more preferably Gly;

[0236] X2 at position 16 is preferably Ala, Asp, Glu, Phe, Gly, His, Lys, Leu, Met, Gln, Arg, Ser, Thr or Tyr, more preferably Ala, Asp, Gly, His, Lys, Leu, Met, Gln, Arg, Ser or Thr, still more preferably Ala, Gly, Lys, Leu, Ser or Thr, further preferably Ala, Gly, Leu, Ser or Thr, and still further preferably Ala or Ser;

[0237] X3 at position 17 is preferably Ala, Asp, Glu, Gly, His, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr or Tyr, more preferably Asp, Gly, His, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr or Tyr, still more preferably Asp, His, Lys, Met or Gln, and further preferably Asp or Gln;

[0238] X4 at position 18 is preferably Ala, Asp, Glu, Phe, Gly, His, Ile, Leu, Lys, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp or Tyr, more preferably Asp, Phe, His, Met, Asn, Gln, Ser or Tyr, still more preferably Asp, Phe, His, Ser or Tyr, and further preferably Phe or His;

[0239] X5 at position 19 is preferably Ala, Asp, Glu, Gly, His, Ile, Lys, Met, Asn, Gln, Arg, Ser, Thr, Val or Tyr, more preferably Ala, Asp, Glu, Gly, His, Lys, Met, Asn, Gln, Arg, Ser or Val, still more preferably Ala, Asp, Glu, Met or Asn, and further preferably Ala, Asp or Glu;

[0240] X6 at position 21 is preferably Ala, Glu, Phe, Gly, Ile, Leu, Met, Gln, Arg, Ser, Trp or Tyr, more preferably Glu, Phe, Ile, Leu, Met, Gln, Arg or Trp, still more preferably Met or Trp, further preferably Met;

[0241] X7 at position 24 is preferably Ala, Asp, Glu, Phe, Gly, His, Lys, Leu, Met, Pro, Gln, Arg, Ser, Thr, Val, Trp or Tyr, more preferably Asp, Glu, His, Pro, Gln, Ser, Thr, Val, Trp or Tyr, still more preferably Gln, Trp, Tyr or Val, and further preferably Tyr or Val;

[0242] X8 at position 26 is preferably Ala, Asp, Glu, Phe, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Val or Tyr, more preferably Ala, Phe, His, Ile, Leu, Met, Gln, Arg, Ser, Val or Tyr, still more preferably Phe, Leu or Tyr, and further preferably Phe or Leu;

[0243] X9 at position 27 is preferably Glu, Phe, Leu, Ser, Thr or Tyr, more preferably Phe, Leu, Ser, Thr or Tyr, still more preferably Phe or Tyr, further preferably Tyr;

[0244] X10 at position 39 is preferably Ala, Glu, Met or Val, and more preferably Ala or Glu; and

[0245] X11 at position 43 is preferably Ala, Thr or Val, more preferably Thr or Val.

[0246] The wild-type X1 to X11 are Asp, Ser, Gln, Tyr, Arg, Pro, Pro, His, Phe, Ala and Thr, respectively. Position 20 is Leu, position 22 is Gly, position 25 is Arg, and position 28 is Asn.

[0247] In the present invention, one or more or more amino acids may be further added to the N-terminal side of the first amino acid. Examples of such amino acids to be added may include Ser-Leu, and an amino acid sequence consisting of an S tag and a linker (SEQ ID NO: 31: Fig.43 ).

[0248] One or more amino acids may be further added to 63Cys at the C-terminus. Examples of such amino acid sequences may include an amino acid sequence having 64Gly at the C-terminus by adding Gly-Gly and an amino acid sequence having 65Gly at the C-terminus. Examples of such amino acids to be added may include Gly-Gly, and a C-terminal hexamer (SEQ ID NO: 32: Fig.44).

[0249] By adding other amino acids to SEQ ID NO:30 ( Fig.42 ) or a more preferred form of the amino acid sequence derived from SEQ ID NO: 30 includes SEQ ID NOs: 3, 5, 7, 9, 11, 13, 15, 17, 19, 21 and 23 to 29 ( Fig.15 , 17 , 19, 21, 23, 25, 27, 29, 31, 33 and 35 to 41), and the amino acid sequence shown in any one of SEQ ID NOs: 4, 6, 8, 10, 12, 14, 16, 18, 20 and 22 ( Fig.16 , 18 , 20, 22, 24, 26, 28, 30, 32 and 34). Still more preferred forms thereof include SEQ ID NOs: 24, 28 and 29 ( Fig.36 , 40 and 41) as shown in any one of the amino acid sequences.

[0250] In the present invention, a peptide prepared by replacing, adding and / or deleting one or two or more amino acids in, to or from the N-terminal and / or C-terminal adduct of a SPINK2 mutant peptide or a SPINK2 mutant peptide (hereinafter referred to as a "parent peptide") is referred to as a "derivative of the parent peptide" or a "parent peptide derivative" (e.g., Example 6). Such "derivatives" are also included within the scope of the "peptide" of the present invention.

[0251] The amino acid sequence of the SPINK2 mutant included in the scope of the inhibitory peptide of the present invention may be within the amino acid sequence of wild-type human SPINK2 (SEQ ID NO: 1: Fig.13) in the portion other than X1 to X11, i.e., 2Pro to 15Cys, 20Leu, 22Gly, 23Cys, 25Arg, 28Asn to 38Tyr, 41Glu, 42Cys and 44Thr to 63Cys, contains a natural amino acid or a mutated amino acid or amino acid sequence. For example, the SPINK2 mutant may have a mutation at one or two or more positions, as long as at least part of it neither hinders nor interferes with the HTRA1 inhibitory activity or folding. Such mutations are obtained by using standard methods known to those skilled in the art. Typical examples of mutations in the amino acid sequence may include substitution, deletion or addition of one, two or more amino acids. Examples of substitutions may include conservative substitutions. Conservative substitutions replace certain amino acid residues with amino acid residues that are chemically similar not only in volume but also in polarity. Examples of conservative substitutions are described in other parts of this specification. On the other hand, the portion other than X1 to X11 may accept non-conservative substitutions of one or two or more amino acids, as long as at least part of it neither hinders nor interferes with the HTRA1 inhibitory activity or folding.

[0252] In the amino acid sequences of the SPINK2 mutants serving as the inhibitory peptides of the present invention, in SEQ ID NOs: 3, 5, 7, 9, 11, 13, 15, 17, 19, 21 and 23 to 29 ( Fig.15 , 17 , 19, 21, 23, 25, 27, 29, 31, 33 and 35 to 41) and more preferably SEQ ID NO: 24, 28 and 29 ( Fig.36 , 40 In any of 41), X1 to X11 are preferably amino acids X1 to X11, respectively, and the portion other than X1-X11 may have at least a portion of amino acids or amino acid sequences that neither hinder nor interfere with the HTRA1 inhibitory activity or folding.

[0253] Examples of the amino acid sequence of the SPINK2 mutant serving as the HTRA1-inhibiting peptide of the present invention may include any one of the following amino acid sequences (a) to (e):

[0254] (a) SEQ ID NO: 3, 5, 7, 9, 11, 13, 15, 17, 19, 21 and 23 to 29 ( Fig.15 , 17 , 19, 21, 23, 25, 27, 29, 31, 33, and 35 to 41);

[0255] (b) an amino acid sequence encoded by a nucleotide sequence which hybridizes under stringent conditions with a nucleotide sequence complementary to the nucleotide sequence encoding the amino acid sequence (a) and encodes an amino acid sequence contained in a peptide having HTRA1 inhibitory activity;

[0256] (c) an amino acid sequence derived from the amino acid sequence (a) by substitution, deletion, addition and / or insertion of 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 amino acid and comprised in a peptide having HTRA1 inhibitory activity;

[0257] (d) an amino acid sequence that has 60%, 70%, 80%, 85%, 90%, 92%, 94%, 96%, 97%, 98% or 99% or more identity with the amino acid sequence (a) and is contained in a peptide having HTRA1 inhibitory activity; and

[0258] (e) consisting of SEQ ID NO: 4, 6, 8, 10, 12, 14, 16, 18, 20 and 22 ( Fig.16 , 18 , 20, 22, 24, 26, 28, 30, 32 to 34) encoded by the amino acid sequence.

[0259] However, the amino acid sequence of the HTRA1-inhibitory peptide is not limited to (a) to (e). Each amino acid sequence contained in the SPINK2 mutant having HTRA1 inhibitory activity and preferably SEQ ID NO: 30 ( Fig.42 ) is included in the range of the amino acid sequence of the HTRA1-inhibiting peptide.

[0260] Mutations can be introduced into the inhibitory peptides of the present invention for the purpose of improving their folding stability, thermal stability, storage stability, half-life in blood, water solubility, biological activity, pharmacological activity, secondary effects, etc. For example, new reactive groups such as Cys can be introduced therein by mutation so as to conjugate the inhibitory peptides with additional substances such as polyethylene glycol (PEG), hydroxyethyl starch (HES), biotin, peptides or proteins. In the present invention, HTRA1-inhibitory peptides can be added, connected or bound to additional parts. Such conjugates are collectively referred to as "conjugates of HTRA1-inhibitory peptides". In the present invention, the term "conjugate" is used to refer to molecules of the peptides of the present invention or fragments thereof that are added, connected or bound to additional parts. The term "conjugate" or "conjugation" includes forms in which a specific part is connected or bound to the N-terminus and / or C-terminus of the peptide of the present invention via reagents suitable for connecting a specific part to the side chain of an amino acid, etc. (including chemical substances such as cross-linking agents) through synthetic chemistry methods, genetic engineering methods, etc. Examples of such "parts" for increasing the half-life in the blood may include polyalkylene glycol molecules, such as polyethylene glycol (PEG), hydroxyethyl starch, fatty acid molecules, such as palmitic acid, immunoglobulin Fc regions, immunoglobulin CH3 domains, immunoglobulin CH4 domains, albumin or fragments thereof, albumin binding peptides, albumin binding proteins, such as streptococcal protein G and transferrin. Alternatively, the "part" may be linked to the peptide of the present invention via a linker, such as a peptide linker.

[0261] To exert or enhance pharmacological activity, the HTRA1-inhibiting peptides of the present invention can be conjugated with additional drugs. In the field of antibodies, by replacing antibodies with the peptides of the present invention, techniques or formats known to those skilled in the art as antibody-drug conjugates (ADCs) constitute some aspects of the present invention.

[0262] The HTRA1-inhibiting peptides of the present invention may further comprise one or two or more moieties that exert binding affinity, inhibitory activity, antagonistic activity, agonistic activity, etc., against target molecules other than HTRA1, or may be conjugated to such moieties. Examples of "moieties" may include antibodies or fragments thereof, and proteins having a non-antibody framework, such as SPINK2 mutants, or fragments thereof. In the field of antibodies, by replacing at least one or two or more "antibodies" contained therein with the peptides of the present invention, the techniques or forms known to those skilled in the art as multispecific antibodies and bispecific antibodies constitute some aspects of the conjugates of the present invention.

[0263] The peptide of the present invention or its precursor may include a signal sequence. The signal sequence present at the N-terminus of a specific polypeptide or its precursor or added thereto can be used for delivering the polypeptide to a specific compartment of a cell, such as the periplasm of Escherichia coli or the endoplasmic reticulum of a eukaryotic cell. Many signal sequences are known to those skilled in the art, and signal sequences can be selected according to the host cell. Examples of signal sequences for secreting the desired peptide into the periplasm of Escherichia coli may include OmpA. Forms comprising signal sequences may also be included in some aspects of the conjugate of the present invention.

[0264] The peptides of the invention may be pre-labeled and thus purified by affinity chromatography. The peptides of the invention may contain, for example, biotin, Strep Tag (TM), Strep Tag II (TM), oligohistidine such as His6, polyhistidine, immunoglobulin domains, maltose-binding protein, glutathione-S-transferase (GST), calmodulin-binding peptide (CBP), haptens such as digoxigenin or dinitrophenol, epitope tags such as FLAG (TM), myc tag or HA tag (hereinafter collectively referred to as "affinity tags") at their C-terminus. Labeled forms may also be included in some aspects of the conjugates of the invention. The conjugates of the invention as a whole may be peptides (polypeptides).

[0265] The peptides of the present invention may include a moiety for labeling. Specifically, the peptides of the present invention may be conjugated with a labeling moiety such as an enzymatic label, a radioactive label, a colored label, a fluorescent label, a chromogenic label, a luminescent label, a hapten, digoxigenin, biotin, a metal complex, a metal or colloidal gold. The form including a moiety for labeling may also be included in some aspects of the conjugates of the present invention.

[0266] The inhibitory peptide of the present invention may contain any of natural amino acids and unnatural amino acids in its peptide portion, and may contain L-amino acids and D-amino acids as natural amino acids.

[0267] The amino acid sequence of the inhibitory peptide of the present invention may contain any of natural amino acids and unnatural amino acids, and may contain L-amino acids and D-amino acids as natural amino acids.

[0268] The inhibitory peptide of the present invention can exist as a monomer, dimer or trimer or higher oligomer or polymer. The dimer or trimer or higher oligomer or polymer can be any of a homotypic form consisting of a single monomer and a heterotypic form consisting of two or more different monomers. For example, the monomer can diffuse rapidly and is excellent in penetrating into tissues. The dimer, oligomer and polymer can have excellent aspects, for example, high local affinity or binding activity for target molecules, slow dissociation rate, or high HTRA1 inhibitory activity. In addition to spontaneous dimerization, oligomerization and multimerization, the intended dimerization, oligomerization and multimerization are also achieved by introducing jun-fos domains, leucine zippers, etc. into the inhibitory peptide of the present invention.

[0269] The inhibitory peptide of the present invention may bind to one or two or more target molecules in the form of a monomer, dimer, trimer, or higher oligomer or multimer, or inhibit the activity of the target molecule.

[0270] Examples of forms that the inhibitory peptides of the present invention may take include, but are not limited to, isolated forms (lyophilized preparations, solutions, etc.), the above-mentioned conjugates, and forms bound to other molecules (immobilized forms, forms associated with different molecules, forms bound to target molecules, etc.). Any form compatible with expression, purification, use, storage, etc. may be selected.

[0271] 3. Identification of HTRA1-inhibitory peptides

[0272] The amino acid sequence of the HTRA1-inhibitory peptide of the present invention (e.g., selected from SEQ ID NO: 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, and 23 to 29, or ... Fig.15 , 17 , 19, 21, 23, 25, 27, 29, 31, 33 and 35 to 41 amino acid sequences), nucleotide sequences encoding the amino acid sequences or nucleic acid molecules comprising the nucleotide sequences to identify HTRA1-inhibitory peptides. As a preferred example, HTRA1-inhibitory peptides can be identified from a human SPINK2 mutant library by using HTRA1 inhibitory activity as an indicator (which can be combined with HTRA1 binding activity as another indicator).

[0273] For example, nucleic acid molecules serving as starting materials can be mutated using recombinant DNA techniques and transferred to appropriate bacterial hosts or eukaryotic hosts. SPINK2 mutant libraries are known as techniques for identifying binding agents or inhibitors of target molecules. For example, the disclosure of WO2012 / 105616 is also incorporated herein by reference in its entirety. After expressing the mutagenized nucleotide sequence in an appropriate host, clones of SPINK2 mutants with desired properties, activities, functions, etc. connected to their genotypes can be enriched and / or selected from the library and identified. Enrichment and / or selection of clones are performed by methods known to those skilled in the art, such as bacterial display methods (Francisco, JA, et al., (1993), Proc. Natl. Acad. Sci. USA Vol. 90, p. 10444-10448), yeast display methods (Boder, ET, et al., (1997), Nat. Biotechnol., Vol. 15, p. 553-557), mammalian cell display methods (Ho M, et al., (2009), Methods Mol Biol., Vol. 525: p. 337-52), phage display methods (Smith, GP (1985), Science., Vol. 228, p. 1315-1317), ribosome display methods (Mattheakis LC, et al., (1994), Proc. Natl. Acad. Sci. USA Vol. 91, No. 19, p. 9022-9029), nucleic acid display methods such as mRNA display (Nemoto N, et al., (1997), FEBS Lett., Vol. 414, No. 2, p. 405-408), or colony screening methods (Pini, A. et al., (2002), Comb. Chem. High Throughput Screen., Vol. 5, p. 503-510). The nucleotide sequence of the SPINK2 mutant contained in the clone thus selected and identified can be determined so that the amino acid sequence encoded by the nucleotide sequence can be determined as the amino acid sequence of the SPINK2 mutant contained in the clone, i.e., the HTRA1-inhibitory peptide.

[0274] The SPINK2 mutant of the present invention can be obtained, for example, by mutagenesis of natural SPINK2. The term "mutagenesis" is used to mean that one or two or more amino acids at their respective positions in a specific amino acid sequence can be substituted by other amino acids, or deleted, or an amino acid not present in the amino acid sequence can be added or inserted therein. Such deletion or such addition or insertion can change the sequence length. In the SPINK2 mutant of the present invention, mutagenesis can preferably occur in SEQ ID NO: 30 ( Fig.42 ) at one or two or more positions X1 to X11 in the amino acid sequence shown in ).

[0275] However, the mutant also includes within its scope a form in which the natural amino acid at one or two or more positions of X1 to X11 is maintained after such preferred mutagenesis, i.e., the same amino acid as the amino acid present at a specific position in the natural amino acid sequence, as long as at least one amino acid is mutated as a whole. Similarly, in one aspect of the present invention, the mutant also includes within its scope a form in which the natural amino acid at one or more positions of a portion other than X1 to X11 is maintained after mutagenesis, i.e., the same amino acid as the amino acid present at a specific position in the natural amino acid sequence, as long as at least one amino acid is mutated as a whole.

[0276] The term "random mutagenesis" is used to mean that for a specific position on a sequence, one or two or more different amino acids are introduced into the position with a given probability by mutagenesis. The probability of introducing at least two different amino acids may not always be the same. The present invention does not exclude that the at least two different amino acids include naturally occurring amino acids (as one of the amino acids). This situation is also included within the scope of the term "random mutagenesis".

[0277] Standard methods well known to those skilled in the art can be used as the method for random mutagenesis at a specific position.Mutagenesis can be achieved by, for example, using a PCR (polymerase chain reaction) of a mixture of synthetic oligonucleotides composed of degenerate nucleotides at a specific position in the sequence.For example, codons NNK or NNS (N=adenine, guanine, cytosine or thymine; K=guanine or thymine; and S=guanine or cytosine) are used to cause mutagenesis to introduce any of all 20 natural amino acids and terminator codons, and codons VVS (V=adenine, guanine or cytosine) are used to eliminate the possibility of introducing Cys, Ile, Leu, Met, Phe, Trp, Tyr and Val, and mutagenesis is caused to introduce any remaining 12 natural amino acids. For example, the codon NMS (M = adenine or cytosine) is used to eliminate the possibility of introducing Arg, Cys, Gly, Ile, Leu, Met, Phe, Trp and Val, and cause mutagenesis to introduce any of the remaining 11 natural amino acids. Specific codons, artificial codons, etc. can be used for mutagenesis to introduce non-natural amino acids.

[0278] Site-directed mutagenesis can also be performed by using structural information about a target containing a conformation and / or a peptide directed to the target or a wild-type peptide derived from the peptide. In the present invention, site-directed mutations can be introduced by using structural information about the target HTRA1 and / or a SPINK2 mutant directed to the target or a wild-type SPINK2 or a complex therebetween, the structural information including high-order information. In one example, a SPINK2 mutant having HTRA1 inhibitory activity is identified. Subsequently, a crystal complex of HTRA1 and SPINK2 mutants is obtained and subjected to X-ray crystallographic analysis. Based on the analysis results, the site on the HTRA1 molecule to which the SPINK2 mutant binds, and the amino acid residues in the SPINK2 mutant that participate in the interaction with the site are identified. The correlation between the structural information obtained by such methods and the HTRA1 inhibitory activity can be found. Based on this structure-activity correlation, it is possible to design that the amino acid at a specific position is replaced by a specific amino acid, the insertion or deletion of an amino acid at a specific position, etc., to actually confirm the HTRA1 activity.

[0279] Mutagenesis can also be achieved using, for example, nucleotide building blocks with modified base pair specificity, such as inosine.

[0280] Furthermore, mutagenesis at random positions is achieved by, for example, error-prone PCR or chemical mutagenesis using a DNA polymerase that lacks an error correction function and produces a high error rate, such as Taq DNA polymerase.

[0281] The HTRA1-inhibitory peptide can be enriched and / or selected from libraries suitable for respective screening methods and known to those skilled in the art, such as phage libraries or colony libraries, by using bacterial display, yeast display, mammalian cell display, phage display, ribosome display, nucleic acid display, colony screening, etc. These libraries can be constructed using vectors and methods, such as phagemids for phage libraries or cosmids for colony screening (which are suitable for each library and known to those skilled in the art). Such vectors can be viruses or viral vectors that infect prokaryotic cells or eukaryotic cells. These recombinant vectors can be prepared by methods known to those skilled in the art, such as genetic manipulation.

[0282] Bacterial display is a technique for fusing a desired protein with, for example, a portion of an E. coli outer membrane lipoprotein (Lpp) and an outer membrane protein OmpA and displaying the desired protein on the surface of E. coli. The DNA group obtained by random mutagenesis of a nucleotide sequence encoding an amino acid sequence of a specific protein is inserted into a vector suitable for bacterial display, and the bacterial cells are transformed with the vector to obtain a library of randomly mutagenized protein groups displayed on the surface of the transformed bacterial cells (Francisco, JA, et al., (1993), Proc. Natl. Acad. Sci. USA Vol. 90, p. 10444-10448).

[0283] Yeast display is a technique of fusing a desired protein with a coat protein (such as, α-lectin on the cell surface of yeast) and displaying the desired protein on the surface of yeast. α-lectin comprises a C-terminal hydrophobic region with a putative glycosylphosphatidylinositol (GPI) anchor attachment signal, a signal sequence, an active domain, a cell wall domain, etc. By operating it, the desired protein can be displayed on the cell surface of yeast. The DNA group obtained by random mutagenesis of the nucleotide sequence encoding the amino acid sequence of a specific protein is inserted into a vector suitable for yeast display, and the yeast cells are transformed with the vector to obtain a library of randomly mutagenized protein groups displayed on the cell surface of the transformed yeast (Ueda, M. & Tanaka, A., Biotechnol. Adv., Vol. 18, p. 121-, 2000; Ueda, M. & Tanaka, A., J. Biosci. Bioeng., Vol. 90, p. 125-, 2000; etc.).

[0284] Animal cell display is a technique of fusing a desired protein with a transmembrane region of a membrane protein represented by platelet-derived growth factor receptor (PDGFR) and displaying the desired protein on the surface of mammalian cells (e.g., HEK293 and Chinese hamster ovary (CHO) cells). A DNA group obtained by random mutagenesis of a nucleotide sequence encoding an amino acid sequence of a specific protein is inserted into a vector suitable for animal cell display, and yeast cells can be transformed with the vector to obtain a library of randomly mutagenized protein groups displayed on the surface of transformed animal cells (Ho M, et al., (2009), Methods Mol Biol. Vol. 525: p. 337-52).

[0285] The desired library displayed on cells, such as yeast cells, bacterial cells or animal cells, can be incubated or contacted with the target molecule in the presence of the target molecule. For example, the cells related to the library are incubated with HTRA1 modified with biotin etc. for a given time. Then, a support, such as magnetic beads, is added thereto, and the cells are separated from the support. Subsequently, the support can be washed to remove non-specific adsorption substances and binding substances to recover the cell population of the peptide, peptide set or enriched peptide set combined with the support (it has the HTRA1 combined therewith). Similarly, the cell population of the peptide, peptide set or enriched peptide set combined with the support (it has the HTRA1 combined therewith) can be recovered by magnetic activated cell sorting (MACS) or by FACS after cell staining with anti-HTRA1 antibodies, or the cell population of the peptide, peptide set or enriched peptide set combined with HTRA1 can be recovered. For example, non-specific adsorption sites and / or binding sites can be closed (saturated). This article can be incorporated into a blocking step, as long as blocking is performed by an appropriate method. The carrier of the expressed peptide, peptide set or enriched peptide set thus obtained is recovered, and the nucleotide sequence of the polynucleotide inserted into the carrier can be determined to determine the amino acid sequence encoded by the nucleotide sequence. In addition, the carrier can be transferred to a host cell again, and the above-mentioned operation cycle can be repeated once to several times to highly enrich the peptide set binding target molecule.

[0286] In the case of phage display, for example, a phagemid is a bacterial plasmid containing a plasmid replication origin and a second replication origin derived from a single-stranded bacteriophage. Cells with phagemids can replicate phagemids via a single-stranded replication mode by co-infection with M13 or a helper phage similar thereto. Specifically, single-stranded phagemid DNA is packaged into infectious particles with phage coat protein as an outer shell. In this way, phagemid DNA can be formed into cloned double-stranded DNA plasmids in infected bacteria, and phagemids can be formed into bacteriophage-like particles from the culture supernatant of co-infected cells. Bacteriophage-like particles are injected into bacteria with F-pilus for infection of bacteria with DNA so that the particles themselves can be re-formed into plasmids.

[0287] A fusion gene comprising a polynucleotide having a nucleotide sequence encoding the amino acid sequence of a test peptide and a phage coat protein gene is inserted into a phagemid, and the resulting phagemid is used to infect bacteria. The cells are cultured so that the peptide can be expressed or displayed on the bacteria or on phage-like particles, or can be produced as a fusion protein with a coat protein into phage particles or in the culture supernatant of bacteria.

[0288] For example, a fusion gene comprising the polynucleotide and the bacterial phage coat protein gene gpIII is inserted into a phagemid, and Escherichia coli is co-infected with the resulting phagemid and M13 or a helper phage similar thereto, so that a fusion protein comprising the peptide and the coat protein can be produced and released into the culture supernatant of Escherichia coli.

[0289] In the case of using various circular or non-circular vectors (e.g., viral vectors) instead of phagemids, a peptide having an amino acid sequence encoded by the nucleotide sequence of a polynucleotide inserted into the vector can be expressed or displayed on cells or virus-like particles carrying the vector, or can be produced and released into the culture supernatant of cells according to methods known to those skilled in the art.

[0290] The library of expressed peptides thus obtained can be incubated in the presence of a target molecule, or contacted with the target molecule. For example, a HTRA1-fixed support is incubated for a given time with a mobile phase containing the library. Then, the mobile phase is separated from the support. Subsequently, the support is washed to remove non-specific adsorption substances and binding substances. Peptides, peptide sets or enriched peptide sets bound to a support (which has HTRA1 bound thereto) can be recovered by elution. The elution can be performed non-selectively, for example, in the presence of relatively high ionic strength, low pH, medium denaturing conditions or chaotropic salts, or can be selectively performed by adding soluble target molecules (such as HTRA1), antibodies, natural ligands, substrates, etc. that bind to target molecules and competing with fixed target molecules. For example, non-specific adsorption sites and / or binding sites can be blocked. The blocking step can be incorporated herein, as long as blocking is performed by an appropriate method.

[0291] The vector of the expressed peptide, peptide set or enriched peptide set thus obtained is recovered, and the nucleotide sequence of the polynucleotide inserted into the vector can be determined to determine the amino acid sequence encoded by the nucleotide sequence. In addition, the vector can be transferred into a host cell again, and the above-mentioned operation cycle can be repeated once to several times to highly enrich the peptide set binding to the target molecule.

[0292] Ribosome display is such a technology, it uses mRNA and cell-free protein synthesis system that for example encodes the protein of expectation and lacks stop codon, and synthesizes the molecule of the protein of expectation in vitro, its corresponding mRNA and ribosome connected to each other.By using the mRNA group and cell-free protein synthesis system that obtain by the nucleotide sequence of the amino acid sequence of the specific protein of random mutagenesis encoding, it is possible to obtain the library (Mattheakis LC, et al., (1994) Proc. Natl. Acad. Sci. USA Vol. 91, No. 19, p. 9022-9029) of the protein group that displays random mutagenesis on ribosome.

[0293] Nucleic acid display, also referred to as mRNA display, is a technique that uses, for example, a linker, such as puromycin similar to the 3' end of tyrosyl-tRNA in structure, and synthesizes molecules of desired protein, its encoding mRNA, and ribosomes connected to each other. This technique uses a cell-free protein synthesis system, rather than living cells, and therefore allows in vitro synthesis. By using mRNA groups, linkers (such as puromycin) and cell-free protein synthesis systems obtained by random mutagenesis of the nucleotide sequence encoding the amino acid sequence of a specific protein, it is possible to obtain a library of protein groups displaying random mutagenesis on ribosomes (Nemoto N, et al., (1997), FEBS Lett., Vol. 414, No. 2, p. 405-408).

[0294] The library of display peptides obtained via cell-free synthesis system, such as ribosome display or nucleic acid display, can be incubated in the presence of target molecules, or contacted with target molecules. For example, the support fixed by HTRA1 is incubated for a given time with the mobile phase containing the library. Then, the mobile phase is separated from the support. Subsequently, the support is washed to remove non-specific adsorption substances and binding substances. The peptides, peptide sets or enriched peptide sets bound to the support (which has HTRA1 bound thereto) can be recovered by elution. The elution can be performed non-selectively, for example, in the presence of relatively high ionic strength, low pH, medium denaturing conditions or chaotropic salts, or can be selectively performed by adding soluble target molecules (such as HTRA1), antibodies, natural ligands, substrates, etc. that bind to target molecules and competing with fixed target molecules. For example, non-specific adsorption sites and / or binding sites can be blocked. This article can be incorporated into a blocking step, as long as it is blocked by an appropriate method.

[0295] Reclaim the nucleic acid of the peptide, peptide set or enriched peptide set thus obtained. In the case of mRNA, the nucleotide sequence can be determined after the reverse transcription reaction into cDNA to determine the amino acid sequence encoded by the nucleotide sequence. In addition, the recovered nucleic acid is transcribed into mRNA, and the above-mentioned operation cycle can be repeated once to several times to highly enrich the peptide set binding target molecule.

[0296] If the peptide, peptide pool or enriched peptide pool is pre-conjugated with an affinity tag, the peptide or pool can be effectively purified. For example, the peptide pool is pre-conjugated with a protease substrate as a tag so that the peptide can be eluted by cleavage via protease activity.

[0297] Based on the obtained sequence information and the function of the peptide, etc., the obtained clones or libraries can be further mutated, and peptides with improved functions (e.g., HTRA1 inhibitory activity), physical properties (thermal stability, storage stability, etc.), in vivo dynamics (distribution in blood and half-life), etc. can be obtained from the mutated library.

[0298] HTRA1-inhibitory peptides can be identified by determining whether the obtained peptides have HTRA1 inhibitory activity.

[0299] To the extent that the HTRA1 inhibitory activity can be exerted, the HTRA1-inhibitory peptide is preferably able to maintain the three-dimensional structure contained in the amino acid sequence of wild-type SPINK2, which is composed of, for example, a loop structure consisting of 16Ser to 30Val, a β-sheet consisting of a β-strand (1) consisting of 31Cys and 32Gly and a β-sheet consisting of a β-strand (2) consisting of 57Ile to 59Arg, and an α-helix consisting of 41Glu to 51Gly, or a loop structure, β-sheet or α-helix similar to or at least partially corresponding to them (or to these positions). A more preferred HTRA1-inhibitory peptide can be identified by using such a three-dimensional structure (whole structure or partial structure) as part of the index.

[0300] 4. Nucleic acid molecules encoding HTRA1-inhibitory peptides, vectors comprising said nucleic acid molecules, cells comprising said nucleic acid molecules or said vectors, and methods for producing recombinant HTRA1-inhibitory peptides

[0301] The present invention also provides a polynucleotide comprising a nucleotide sequence encoding an amino acid sequence contained in an HTRA1-inhibitory peptide (hereinafter referred to as a "nucleic acid molecule encoding an HTRA1-inhibitory peptide"), a recombinant vector having an insert of the gene, a cell carrying the gene or the vector (hereinafter referred to as a "cell containing a nucleic acid molecule encoding an HTRA1-inhibitory peptide") or a cell producing an HTRA1-inhibitory peptide (hereinafter referred to as a "cell producing an HTRA1-inhibitory peptide").

[0302] Some preferred examples of the nucleic acid molecules encoding the HTRA1-inhibitory peptide of the present invention may include a nucleic acid molecule comprising any one of the following nucleotide sequences (a) to (e) (hereinafter referred to as “the nucleotide sequence of the HTRA1-inhibitory peptide”), a nucleic acid molecule consisting of a nucleotide sequence comprising the nucleotide sequence of the HTRA1-inhibitory peptide, and a nucleic acid molecule consisting of the nucleotide sequence of the HTRA1-inhibitory peptide:

[0303] (a) a nucleotide sequence encoding SEQ ID NOs: 3, 5, 7, 9, 11, 13, 15, 17, 19, 21 and 23 to 29 ( Fig.15 , 17, 19, 21, 23, 25, 27, 29, 31, 33, and 35 to 41);

[0304] (b) SEQ ID NO: 4, 6, 8, 10, 12, 14, 16, 18, 20 and 22 ( Fig.16 , 18 , 20, 22, 24, 26, 28, 30, 32 to 34);

[0305] (c) a nucleotide sequence which hybridizes under stringent conditions with a nucleotide sequence complementary to the nucleotide sequence (a) or (b) and encodes an amino acid sequence contained in a peptide having HTRA1 inhibitory activity;

[0306] (d) a nucleotide sequence which is derived from the nucleotide sequence (a) or (b) by substitution, deletion, addition and / or insertion of 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 nucleotides or nucleotide residues, and encodes an amino acid sequence comprised in a peptide having HTRA1 inhibitory activity;

[0307] (e) a nucleotide sequence having 60%, 70%, 80%, 85%, 90%, 92%, 94%, 96%, 97%, 98% or 99% or more identity with the nucleotide sequence of (a) or (b) and encoding an amino acid sequence contained in a peptide having HTRA1 inhibitory activity.

[0308] However, the nucleic acid molecule encoding the HTRA1-inhibitory peptide is not limited to the nucleotide sequences (a) to (e). The nucleic acid molecule encoding the amino acid sequence contained in the SPINK2 mutant having HTRA1 inhibitory activity and preferably SEQ ID NO: 30 ( Fig.42 Each nucleic acid molecule having a nucleotide sequence of the amino acid sequence shown in ) is included in the scope of the nucleic acid molecule encoding the HTRA1-inhibitory peptide.

[0309] The nucleotide sequence encoding the amino acid sequence can be designed using one or two or more codons corresponding to each amino acid. Therefore, the nucleotide sequence encoding the single amino acid sequence of a specific peptide can have multiple variations. In order to select such codons, codons can be appropriately selected according to the codon usage of the host cell for expression, the host cell being used to carry a polynucleotide comprising a nucleotide sequence or a vector comprising the polynucleotide, or the frequency or rate of the multiple codons used can be appropriately adjusted. For example, in the case of using Escherichia coli cells as host cells, codons with high usage frequencies in Escherichia coli can be used to design the nucleotide sequence.

[0310] The nucleic acid molecule encoding the HTRA1-inhibitory peptide can be functionally connected to one or two or more regulatory sequences. The phrase "functional connection" is used to mean that the nucleic acid molecule connected can be expressed, or the nucleotide sequence contained in the molecule is expressible. The regulatory sequence includes sequence elements that provide information about transcriptional regulation and / or translational regulation. Although the regulatory sequence varies according to the species, the regulatory sequence generally includes a promoter and includes a 5' non-coding sequence involved in the initiation of transcription and translation, such as a prokaryotic-35 / -10 box and a Shine-Dalgarno sequence or a eukaryotic TATA box, a CAAT sequence, and a 5' capping sequence. The sequence may include an enhancer element and / or a repressor element, and a translatable signal sequence, a leader sequence, etc., for delivering a natural or mature peptide to a specific compartment inside or outside a host cell. The regulatory sequence may further include a 3' non-coding sequence, and the sequence may include elements involved in transcriptional termination or polyadenylation, etc. However, when the function is insufficient in a specific host cell, the sequence associated with transcriptional termination may be replaced by a sequence suitable for the cell.

[0311] Examples of the promoter sequence may include a prokaryotic tet promoter, a lacUV5 promoter, and a T7 promoter, and an SV40 promoter and a CMV promoter for eukaryotic cells.

[0312] The nucleic acid molecule encoding the HTRA1-inhibiting peptide may be in an isolated form or in a form contained in a vector or another cloning vehicle (hereinafter, referred to as "vector"; plasmid, phagemid, phage, baculovirus, cosmid, etc.) or in a chromosome, but the form is not limited thereto. In addition to the nucleotide sequence and regulatory sequence of the HTRA1-inhibiting peptide, the vector may also contain a replication sequence and a control sequence suitable for the host cell used for expression, and a selection marker that confers a phenotype that allows selection of cells carrying the nucleic acid molecule by transformation, etc.

[0313] A nucleic acid molecule encoding an HTRA1-inhibiting peptide or a vector containing a nucleotide sequence of an HTRA1-inhibiting peptide can be transferred to a host cell capable of expressing the peptide or nucleotide sequence by methods known to those skilled in the art, such as transformation. The host cell carrying the nucleic acid molecule or vector can be cultured under conditions suitable for expressing the peptide or nucleotide sequence. The host cell can be any of a prokaryotic and eukaryotic cell. Examples of prokaryotes can include Escherichia coli and Bacillus subtilis. Examples of eukaryotic cells can include yeast cells, such as Saccharomyces cerevisiae and Pichia pastoris, insect cells, such as SF9 and High5, and animal cells, such as HeLa cells, CHO cells, COS cells, and NS0. The peptides of the present invention expressed by using host cells, such as eukaryotic cells, can undergo desired post-translational modifications. Examples of post-translational modifications can include adding functional groups, such as sugar chains, adding peptides or proteins, and converting the chemical properties of amino acids. Alternatively, the peptides of the present invention can be artificially modified as desired. Such modified forms of the peptides are also included within the scope of the "peptide" of the present invention.

[0314] The present invention also includes a method for producing an HTRA1-inhibitory peptide. The method includes: step 1: culturing a host cell carrying a nucleic acid molecule encoding the HTRA1-inhibitory peptide or a vector containing a nucleotide sequence of the HTRA1-inhibitory peptide, or a cell expressing the HTRA1-inhibitory peptide; and / or step 2: recovering the HTRA1-inhibitory peptide from the culture obtained in step 1. Operations known to those skilled in the art (such as fractionation, chromatography or purification) can be applied to step 2. For example, affinity purification using the antibody of the present invention mentioned later can be applied here.

[0315] In some aspects of the present invention, the HTRA1-inhibitory peptide has an intramolecular disulfide bond. It may be preferred that the peptide with an intramolecular disulfide bond should be delivered to a cell compartment with an oxidative redox environment by using a signal sequence or the like. The oxidative environment can be provided by Gram-negative bacteria, such as the periplasm of Escherichia coli, the extracellular environment of Gram-positive bacteria, the endoplasmic reticulum cavity of eukaryotic cells, and the like. This environment can promote the formation of structural disulfide bonds. Alternatively, a peptide with an intramolecular disulfide bond can be prepared in the cytoplasm of a host cell, such as an Escherichia coli cell. In this case, the peptide can be directly obtained in a soluble folded state or recovered in the form of an inclusion body and subsequently reconstituted in vitro. In addition, a host cell with an oxidative intracellular environment is selected, and a peptide with an intramolecular disulfide bond can also be prepared in its cytoplasm. On the other hand, when the HTRA1-inhibitory peptide does not have an intramolecular disulfide bond, the peptide can be prepared in a cell compartment with a reducing redox environment, such as the cytoplasm of Gram-negative bacteria.

[0316] The HTRA1-inhibitory peptides of the present invention can also be produced by other methods known to those skilled in the art, such as chemical synthesis, for example, the solid phase peptide synthesis method of Merrifield et al., and organic synthetic chemistry peptide synthesis methods using tert-butyloxycarbonyl (Boc) or 9-fluorenylmethoxycarbonyl (Fmoc), and in vitro translation.

[0317] 5. Pharmaceutical Composition

[0318] The present invention also provides a pharmaceutical composition comprising the HTRA1-inhibitory peptide or its conjugate.

[0319] The pharmaceutical composition comprising the HTRA1-inhibitory peptide of the present invention or its conjugate can be used to treat and / or prevent various diseases, which are induced or aggravated by HTRA1 and allow the induction or aggravation to be suppressed by inhibiting or suppressing the expression or function of HTRA1, cure, maintain or improve symptoms, avoid secondary diseases, etc. (hereinafter, these diseases are referred to as "HTRA1-related diseases"). The HTRA1-related diseases also include various diseases, which allow the induction or aggravation to be suppressed by suppressing the degeneration of photoreceptor cells, cure, maintain or improve symptoms, avoid secondary diseases, etc. The HTRA1-inhibitory peptide of the present invention has an inhibitory effect on the degeneration of photoreceptor cells and can be used to treat and / or prevent these HTRA1-related diseases.

[0320] Examples of HTRA1-related diseases include, but are not limited to, various hereditary and non-hereditary eye diseases, such as retinitis pigmentosa, hereditary diseases involving photoreceptor degeneration, and diseases involving photoreceptor degeneration caused by PDE6 gene mutations (e.g., mutations of the human PDE6B gene corresponding to the Pde6b gene encoding the mouse PDE6β subunit) or PDE6 protein dysfunction (hereinafter collectively referred to as "diseases associated with PDE6 protein dysfunction"). Examples of retinitis pigmentosa include, but are not limited to, atypical retinitis pigmentosa, typical retinitis pigmentosa, and retinitis pigmentosa associated with systemic diseases. Examples of hereditary diseases involving photoreceptor degeneration include, but are not limited to, diseases other than retinitis pigmentosa, such as macular dystrophy. Examples of diseases associated with PDE6 protein dysfunction include, but are not limited to, color blindness, autosomal dominant congenital fixed night blindness, and retinitis pigmentosa. In addition, the pharmaceutical composition of the present invention can be used as a photoreceptor degeneration inhibitor.

[0321] By observing, for example, an increase in the thickness of the outer nuclear layer of the retina compared to a control group when the HTRA1-inhibiting peptide of the present invention is administered to Rd10 retinitis pigmentosa model mice (in which the Pde6b gene encoding the PDE6β subunit has a mutation), it can be confirmed that the pharmaceutical composition of the present invention can be used to treat or prevent HTRA1-related diseases (Example 15). In addition, an increase in HTRA1 gene expression in model animals with rhodopsin gene mutations is associated with photoreceptor cell degeneration (Example 16). The pharmaceutical composition of the present invention can be used to treat or prevent retinitis pigmentosa, hereditary (not limited to rhodopsin gene) diseases involving photoreceptor cell degeneration other than retinitis pigmentosa, and / or diseases associated with PDE6 protein dysfunction.

[0322] The HTRA1-inhibitory peptide of the present invention is excellent in tissue permeability (Example 11), and is also excellent in its physical properties, stability, safety, kinetics after administration, productivity, etc., and therefore can be preferably contained in a pharmaceutical composition as an active ingredient.

[0323] The pharmaceutical composition of the present invention may contain a therapeutically or prophylactically effective amount of an HTRA1-inhibitory peptide or a conjugate thereof and a pharmaceutically acceptable diluent, carrier, solubilizer, emulsifier, preservative and / or adjuvant.

[0324] The term "therapeutically or preventively effective amount" is used to mean an amount that exerts a therapeutic or preventive effect on a specific disease through a dosage form and an administration route, and is synonymous with a "pharmacologically effective amount."

[0325] The pharmaceutical composition of the present invention may contain a substance (hereinafter referred to as "drug substance") for changing, maintaining or preserving the pH, osmotic pressure, viscosity, transparency, color, isotonicity, sterility or stability, solubility, sustained release rate, absorbability, permeability, dosage form, strength, characteristics, shape, etc. of the composition or the peptide of the present invention or its conjugate contained therein. The drug substance is not particularly limited as long as the substance is pharmacologically acceptable. For example, non-toxicity or low toxicity is a property that the drug substance preferably has.

[0326] Examples of the drug substance may include, but are not limited to, the following: amino acids such as glycine, alanine, glutamine, asparagine, histidine, arginine and lysine; antimicrobial agents; antioxidants such as ascorbic acid, sodium sulfate and sodium bisulfite; buffers such as phosphate, citrate or borate buffers, sodium bicarbonate and Tris-HCl solution; fillers such as mannitol and glycine; chelating agents such as ethylenediaminetetraacetic acid (EDTA); complexing agents such as caffeine, polyvinylpyrrolidone, β-cyclodextrin and hydroxypropyl-β-cyclodextrin; bulking agents such as glucose, mannose and dextrin; other hydrocarbons such as monosaccharides, disaccharides, glucose, mannose and dextrin; colorants; flavoring agents; diluents; emulsifiers agents; hydrophilic polymers such as polyvinyl pyrrolidone; low molecular weight polypeptides; salt-forming counterions; preservatives such as benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenylethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid and hydrogen peroxide; solvents such as glycerol, propylene glycol and polyethylene glycol; sugar alcohols such as mannitol and sorbitol; suspending agents; surfactants such as PEG, sorbitan esters, polysorbates such as polysorbate 20 and polysorbate 80, triton, tromethamine, lecithin and cholesterol; stability enhancers such as sucrose and sorbitol; elasticity enhancers such as sodium chloride, potassium chloride, mannitol and sorbitol; transport agents; diluents; excipients; and / or pharmaceutical adjuvants.

[0327] This drug substance is added to the HTRA1-inhibiting peptide in an amount of 0.001 to 1000 times, preferably 0.01 to 100 times, and more preferably 0.1 to 10 times the weight of the HTRA1-inhibiting peptide.

[0328] Also included in the pharmaceutical composition of the present invention is a liposome containing the HTRA1-inhibitory peptide or its conjugate, or a pharmaceutical composition containing a modified form of the HTRA1-inhibitory peptide or its conjugate conjugated to the liposome.

[0329] The excipient or carrier is not particularly limited as long as the excipient or carrier is generally liquid or solid and is water for injection, physiological saline, artificial cerebrospinal fluid or other substances used in preparations for oral administration or parenteral administration. Examples of physiological saline may include neutral physiological saline or physiological saline containing serum albumin.

[0330] Examples of the buffer may include a Tris buffer prepared so that the final pH of the pharmaceutical composition reaches 7.0 to 8.5, an acetate buffer prepared so that the final pH reaches 4.0 to 5.5, a citrate buffer prepared so that the final pH reaches 5.0 to 8.0, and a histidine buffer prepared so that the final pH reaches 5.0 to 8.0.

[0331] The pharmaceutical composition of the present invention is a solid, a liquid, a suspension, etc. Another example of the pharmaceutical composition of the present invention may include a freeze-dried preparation. A freeze-dried preparation may be formed using an excipient such as sucrose.

[0332] The administration route of the pharmaceutical composition of the present invention can be any one of eye instillation, enteral administration, topical administration and parenteral administration. Examples thereof can include conjunctival instillation, intravitreal administration, intravenous administration, intraarterial administration, intramuscular administration, intradermal administration, subcutaneous administration, intraperitoneal administration, transdermal administration, intraosseous administration and intraarticular administration.

[0333] The formulation of the pharmaceutical composition can be determined according to the administration method, the HTRA1 binding affinity of the HTRA1-inhibitory peptide, etc. 50 value) or have a higher affinity for HTRA1 protein (lower K D The HTRA1-inhibitory peptide of the present invention having a value of (a) can exert its pharmaceutical effect at a lower dose.

[0334] The dosage of the HTRA1-inhibitory peptide or its conjugate of the present invention is not limited as long as the dosage is a pharmacologically effective amount. The dosage can be appropriately determined according to the species of the individual, the type of disease, symptoms, sex, age, pre-existing conditions, the binding affinity of the peptide to the HTRA1 peptide or its biological activity and other factors. The dosage is usually 0.01 to 1000 mg / kg and preferably 0.1 to 100 mg / kg, which can be administered once a day to every 180 days or twice or three times a day or more.

[0335] Examples of the form of the pharmaceutical composition may include injections (including freeze-dried preparations and drip infusions), suppositories, nasal absorption preparations, percutaneous absorption preparations, sublingual preparations, capsules, tablets, ointments, granules, aerosols, pills, powders, suspensions, emulsions, eye drops, and biological implant preparations.

[0336] The pharmaceutical composition comprising the HTRA1-inhibitory peptide or its conjugate as an active ingredient can be administered simultaneously with an additional drug or separately from the additional drug. For example, the pharmaceutical composition comprising the HTRA1-inhibitory peptide or its conjugate as an active ingredient can be administered after the additional drug is administered, or the additional drug can be administered after the pharmaceutical composition is administered. Alternatively, the pharmaceutical composition and the additional drug can be administered simultaneously. For simultaneous administration, the HTRA1-inhibitory peptide or its conjugate and the additional drug can be contained in a single preparation, or can be contained in separate preparations (multiple preparations).

[0337] Examples of additional drugs used in combination with the pharmaceutical composition of the present invention may include anti-VEGF agents, anti-inflammatory agents, inflammatory cytokine-neutralizers and complement activation pathway inhibitors. The anti-VEGF agent is classified into anti-VEGF antibodies, VEGF inhibitors, VEGF receptor antagonists and soluble VEGF receptors, etc., and includes bevacizumab, ranibizumab, aflibercept, pegaptanib, brolucizumab, etc. The anti-inflammatory agent is not particularly limited as long as the anti-inflammatory agent can be topically applied to curb intraocular or intra-articular inflammation. Examples of the inflammatory cytokine-neutralizer include anti-TNFα antibodies, anti-interleukin-6 (hereinafter referred to as "IL-6") antibodies, anti-IL-6 receptor antibodies and soluble TNF receptors, and may specifically include infliximab, adalimumab, golimumab, certolizumab pegaptanib, tocilizumab and etanercept. Examples of the complement activation pathway inhibitor may include lampalizumab. These drugs are suitable for treating or preventing HTRA1-related diseases, and can also be combined with the pharmaceutical composition of the present invention to treat or prevent diseases other than HTRA1-related diseases.

[0338] One of these additional drugs may be used, or two or three or more of them may be administered or received. These methods are collectively referred to as "use in combination with an additional drug and the pharmaceutical composition of the present invention" or "combination with an additional drug and the pharmaceutical composition of the present invention". Also included within the present invention are pharmaceutical compositions of the present invention comprising additional drugs other than the antibodies of the present invention, binding fragments thereof, or modified forms of antibodies or fragments, or pharmaceutical compositions of the present invention comprising additional drugs other than the antibodies of the present invention, binding fragments thereof, or modified forms of antibodies or fragments used in combination with additional therapies as an aspect of "use in combination with an additional drug" or "combination with an additional drug".

[0339] The present invention also provides a method for treating or preventing HTRA1-related diseases, such as retinitis pigmentosa, comprising the following steps: administering an HTRA1-inhibitory peptide or a conjugate thereof, using the HTRA1-inhibitory peptide or a conjugate thereof of the present invention for preparing a pharmaceutical composition for treating or preventing the disease, and using the HTRA1-inhibitory peptide or a conjugate thereof for treating or preventing the disease. The present invention also includes a kit for treatment or prevention, which comprises the HTRA1-inhibitory peptide or a conjugate thereof of the present invention.

[0340] The present invention further provides a polynucleotide comprising a nucleotide sequence encoding an amino acid sequence of an HTRA1-inhibiting peptide or its conjugate, a vector comprising the polynucleotide, and a pharmaceutical composition comprising the polynucleotide or the vector or a cell expressing an HTRA1-inhibiting peptide or its conjugate of the present invention. For example, the polynucleotide and the vector can be applied to gene therapy for HTRA1-related diseases using known methods. The cells can be applied to cell therapy for HTRA1-related diseases using known methods. Moreover, the polynucleotide or the vector can be transferred to, for example, autologous cells or allogeneic cells (homologous cells) to prepare cells for cell therapy. Such polynucleotides and vectors are also included in the present invention as compositions for the preparation of cell therapy drugs. However, the form of the pharmaceutical composition of the present invention comprising the polynucleotide, vector, cell, etc. is not limited to those described above.

[0341] 6. Compositions for diagnosis and detection of HTRA1

[0342] The HTRA1-inhibitory peptide of the present invention or its conjugate may have HTRA1 binding activity in addition to HTRA1 protease inhibitory activity, and may be used in various studies, such as positive control in the search for HTRA1 inhibitors, detection of HTRA1, examination and diagnosis using the detection, separation of HTRA1, reagents and other purposes. In order to detect or separate HTRA1, at least one of the peptide of the present invention and HTRA1 may be immobilized.

[0343] The present invention provides a composition for detection or diagnosis (hereinafter collectively referred to as "composition for diagnosis"), which comprises the peptide of the present invention that binds to HTRA1 or a conjugate thereof.

[0344] The composition for diagnosis of the present invention can be used to examine or diagnose HTRA1-related diseases, HTRA1 expression, etc. In the present invention, examples of examination or diagnosis include, but are not limited to, determining or measuring the risk of acquiring a disease, determining the presence or absence of a disease, measuring the degree of progression or deterioration, measuring or determining the effect of medication with a pharmaceutical composition comprising an HTRA1-inhibitory peptide or a conjugate thereof, measuring or determining the effect of treatment other than a drug, measuring the risk of relapse, and determining the presence or absence of relapse.

[0345] The composition for diagnosis of the present invention can be used to identify a recipient individual for the peptide or its conjugate, a composition comprising the peptide or its conjugate, or a pharmaceutical composition comprising the peptide or its conjugate.

[0346] The composition for diagnosis may contain a pH buffer, an osmotic pressure regulator, a salt, a stabilizer, a preservative, a developer, a sensitizer, an aggregation inhibitor, and the like.

[0347] The present invention also provides a method for detecting or diagnosing a disease associated with HTRA1, the use of the peptide of the present invention for preparing a composition for diagnosing a disease, and the use of the peptide of the present invention binding to HTRA1 or its conjugate for detecting or diagnosing a disease. The present invention also includes a kit for detection or diagnosis, which comprises the peptide of the present invention or its conjugate.

[0348] The test or diagnosis method comprising the peptide binding to HTRA1 of the present invention is desirably a sandwich ELISA. Alternatively, conventional detection methods such as ELISA, RIA, ELISPOT, dot blot, Ouchterlony method, CIE, CLIA or flow cytometry can be used. The test or diagnosis is also achieved by a method based on an immunoprecipitation method.

[0349] The present invention also provides a method for detecting or measuring HTRA1 in a test sample. This detection or measurement method can use the composition for diagnosis of the present invention. HTRA1 in the test sample can be detected as follows: contacting an HTRA1-inhibitory peptide or its conjugate with the test sample (step 1); and then measuring the amount of HTRA1 bound to the peptide (step 2). Step 1 can involve, for example, fixing the conjugate of the HTRA1-inhibitory peptide and the immunoglobulin Fc region to magnetic beads via protein G, and adding the test sample thereto. Step 2 can involve, for example, separating the magnetic beads, and analyzing the soluble proteins precipitated with the beads by SDS-PAGE or Western blot to detect HTRA1. In addition to samples of human or non-human animal origin, this measurement can even be performed on artificially processed samples, such as recombinant proteins. Examples of test samples derived from individual organisms can include, but are not limited to, blood, synovial fluid, ascites, lymph fluid, cerebrospinal fluid, bronchoalveolar lavage fluid, saliva, sputum, tissue homogenate supernatant, and tissue sections.

[0350] HTRA1 detection can be performed not only in vitro but also in vivo. In the case of diagnostic imaging, an HTRA1-inhibiting peptide or its conjugate labeled with a pharmaceutically acceptable radionuclide or light emitter can be used. Step 1 can involve, for example, administering the labeled peptide or its conjugate to a test subject. Step 2 can involve, for example, obtaining an image by using diagnostic imaging technology such as PET / CT, and determining or examining the presence of HTRA1.

[0351] The peptide or its conjugate contained in the composition for diagnosis of the present invention binds to HTRA1, and preferably has HTRA1-specific binding activity.

[0352] The present invention also includes a method for identifying a recipient individual for the pharmaceutical composition of the present invention. In this identification method, HTRA1 in a sample derived from an individual is measured using the HTRA1-binding peptide of the present invention, and when HTRA1 is detected in the sample or when a greater amount of HTRA1 is detected therein than in a sample derived from a healthy individual, the individual can be determined to be positive. The method can employ the composition for diagnosis of the present invention.

[0353] In a preferred aspect of the identification method, the individual has an HTRA1-related disease or is at risk of acquiring the disease.

[0354] In one aspect, the pharmaceutical compositions of the invention can be administered to individuals determined to be positive in the identification method.

[0355] Using the peptide or its conjugate having HTRA1-specific binding activity of the present invention, HTRA1 can be specifically separated from a sample in which HTRA1 coexists with other components. The release of HTRA1 from the peptide can be carried out non-selectively, for example, under relatively high ionic strength, low pH, moderate denaturing conditions or in the presence of chaotropic salts, and preferably without reducing the protease activity of HTRA1.

[0356] 7. Method for identifying therapeutic or preventive drugs for HTRA1-related diseases

[0357] In one aspect, the present invention provides a method for identifying HTRA1-related diseases and preferably retinitis pigmentosa, hereditary diseases involving photoreceptor degeneration other than retinitis pigmentosa, and / or therapeutic drugs or preventive drugs or candidates thereof for diseases associated with PDE6 protein dysfunction by using HTRA1 inhibitory activity as an index. The method may include: step 1: incubating HTRA1 protease and substrate in the presence or absence of a test substance (or in the presence of a vehicle); step 2: determining HTRA1 protease activity in the presence and absence of the test substance; and / or step 3: when the HTRA1 protease activity in the presence of the test substance is lower than the HTRA1 protease activity in the absence of the test substance, the test substance is determined as a therapeutic drug or preventive drug or candidate thereof for retinitis pigmentosa, hereditary diseases involving photoreceptor degeneration other than retinitis pigmentosa, and / or diseases associated with PDE6 protein dysfunction. The test substance may be peptide or non-peptide. Peptide test substances are not limited to SPINK2 mutants. Examples thereof may include, but are not limited to, antibodies, peptides other than SPINK2 mutants having a non-immunoglobulin protein framework, and HTRA1 substrate analogs. Examples of non-peptide test substances may include, but are not limited to, synthetic low molecular weight compounds and nucleic acids. One or two or more of the above steps may also be preferably included in the method for identifying a substance having an inhibitory effect on photoreceptor cell degeneration or a candidate thereof. The present invention also relates to a method for identifying a substance having an inhibitory effect on photoreceptor cell degeneration or a candidate thereof. Example

[0358] Hereinafter, some aspects of the present invention will be described in more detail with reference to Examples. However, the present invention is not limited to these Examples.

[0359] In the following examples, unless otherwise indicated, individual operations regarding gene manipulation were performed according to the methods described in "Molecular Cloning" (Sambrook, J., Fritsch, EF and Maniatis, T., published by Cold Spring Harbor Laboratory Press in 1982 or 1989) or other methods described in experimental manuals used by those skilled in the art, or when commercially available reagents or kits have been used, the examples have been performed according to the instructions contained in the commercially available products.

[0360] Example 1. Preparation of HTRA1-inhibitory peptide

[0361] (1-1) Construction of HTRA1-inhibitory peptide expression vector

[0362] (1-1-1) Construction of pET 32a (modified)_HTRA1-inhibitory peptide

[0363] First, an HTRA1-inhibitory peptide expression vector with SPINK2 scaffold as the backbone was constructed. The inhibitor fragment was amplified by PCR ((94°C for 15 seconds, 60°C for 30 seconds, and 68°C for 30 seconds) × 30 cycles) using the nucleotide sequence of each inhibitory peptide (SEQ ID NO: 4, 6, 8, 10, 12, 14, 16, 18, 20 and 22) and the nucleotide sequence of SPINK2 (SEQ ID NO: 2) as templates using the following primers and KOD-plus- (Toyobo Co., Ltd.).

[0364] Primer 1: 5'-AAAAGAATTCTGATCCGCAGTTTGGTCTGTTTAG-3' (SEQ ID NO: 33)

[0365] Primer 2: 5'-AAAACTCGAGTTATGCGGCCCGCAGACGCGCCGCACGGACC-3' (SEQ ID NO:34)

[0366] Each amplified fragment was subjected to agarose gel electrophoresis. Then, the desired DNA fragment was cut from the gel, and DNA was prepared using a QIAquick gel extraction kit (Qiagen NV). The prepared DNA fragment and pET 32a (modified) were each treated with restriction enzymes EcoRI (New England BioLabs Inc.) and XhoI (New England BioLabs Inc.) at 37°C for 1 hour or longer. After agarose gel electrophoresis, the desired DNA fragment was cut from the gel and purified using a QIAquick PCR purification kit (Qiagen NV). Using T4 DNA ligase (New England BioLabs Inc.), the purified fragment was reacted overnight at 16°C for ligation. The ligation solution was added to Escherichia coli JM109 (Toyobo Co., Ltd.), and the mixture was 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, and inoculated on a 2YT plate containing 0.1 mg / ml ampicillin, followed by static culture overnight at 37°C to transform Escherichia coli. The next day, the transformed Escherichia coli was inoculated into Terrific Broth medium (Invitrogen Corp.) containing 0.1 mg / ml ampicillin and cultured overnight at 37°C. Then, the plasmid DNA was recovered using a QIAprep 96Turbo Miniprep kit (Qiagen NV) (hereinafter, this treatment is referred to as "mini-prep treatment"), and sequence analysis was performed to construct "pET 32a (modified)_HTRA1-inhibitory peptide".

[0367] (1-1-2) Construction of pET 32a_HTRA1-inhibitory peptide_Kex2

[0368] Likewise, the inhibitor fragment was amplified by PCR ((94°C for 15 sec, 60°C for 30 sec, and 68°C for 30 sec) × 30 cycles) using the sequence of each inhibitory peptide (Sequence Listing) and the nucleotide sequence of SPINK2 as a template using the following primers and KOD-plus- (Toyobo Co., Ltd.).

[0369] Primer 3: 5'-AAAAGGATCCCTGGACAAACGTGATCCGCAGTTTGGTCTGTTTAG-3' (SEQ ID NO:35)

[0370] Primer 4: 5'-AAAACTCGAGTTAGCCGCCGCACGGACCATTGCGAATAATTTTA-3' (SEQ ID NO:36)

[0371] Each amplified fragment is subjected to agarose gel electrophoresis. Then, the desired DNA fragment is cut from the gel, and DNA is prepared using a QIAquick gel extraction kit (Qiagen NV). The prepared DNA fragment and pET 32a (Novagen) are each treated with restriction enzymes BamHI (New England BioLabs Inc.) and XhoI (New England BioLabs Inc.) at 37°C for 1 hour or longer. After agarose gel electrophoresis, the desired DNA fragment is cut from the gel and purified using a QIAquick PCR purification kit (Qiagen NV). Using T4 DNA ligase (New England BioLabs Inc.), the purified fragment is reacted overnight at 16°C for ligation. The ligation solution was added to Escherichia coli JM109 (Toyobo Co., Ltd.), and the mixture was 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, and inoculated onto a 2YT plate containing 0.1 mg / ml ampicillin, followed by static culture overnight at 37°C to transform Escherichia coli. The transformed Escherichia coli was cultured, and then small-scale preparation and sequence analysis were performed to construct "pET 32a_HTRA1-inhibitory peptide_Kex2". The operation was performed according to the method described in (1-1-1).

[0372] (1-2) Expression and purification of HTRA1-inhibitory peptide

[0373] E. coli Origami B (DE3) (Novagen) was transformed with the vector pET 32a (modified)_HTRA1-inhibitory peptide constructed in (1-1-1) and cultured at 37°C using 2YT medium containing 0.1 mg / ml ampicillin. Then, IPTG (final concentration: 1 mM) was added thereto, and E. coli was cultured overnight at 16°C. The next day, after harvesting by centrifugation (3,000 g, 20 min, 4°C), lysates were prepared using BugBuster Master Mix (Novagen), and the target His tag fusion protein was purified using TALON metal affinity resin (Clontech Laboratories, Inc.). Next, the thioredoxin tag and the desired protein were cut using a thrombin cleavage capture kit (Novagen) and purified using TALON. The resulting product was subjected to gel filtration chromatography (Superdex 75 10 / 300 GL) or reverse phase chromatography (YMC-PackODS-AM) to prepare HTRA1-inhibitory peptides. The obtained peptide was fused at its N-terminus with a moiety consisting of an S tag and a linker (SEQ ID NO: 31: Fig.43 ) and conjugated at its C-terminus to a C-terminal hexamer (SEQ ID NO: 32: Fig.44 ), rather than Gly-Gly conjugation.

[0374] Similarly, E. coli Origami B (DE3) (Novagen) was transformed with the vector pET 32a_HTRA1-inhibitory peptide_Kex2 constructed in (1-1-2) and cultured at 37°C using 2YT medium containing 0.1 mg / ml ampicillin. Then, IPTG (final concentration: 1 mM) was added thereto, and E. coli was cultured overnight at 16°C. The next day, after harvesting by centrifugation (3,000 g, 20 min, 4°C), lysates were prepared using BugBuster Master Mix (Novagen), and the target His-tag fusion protein was purified using TALON metal affinity resin (Clontech Laboratories, Inc.). Next, the thioredoxin tag and the desired protein were cut using Kex2 (Saccharomyces cerevisiae: Accession CAA96143) and purified using TALON. The resultant was subjected to gel filtration chromatography (Superdex 75 10 / 300 GL) or reverse phase chromatography (YMC-PackODS-AM) to prepare an HTRA1-inhibitory peptide (in which both the N-terminus and the C-terminus are not conjugated to a tag, a linker, etc.).

[0375] Example 2. Evaluation of HTRA1-inhibitory peptides for HTRA1 inhibitory activity

[0376] The sequence similarity between human, mouse, rat and monkey HTRA1 is shown in Figure 1. The primary sequence constituting the HTRA1 protease domain (204Gly to 364Leu) (which is the enzymatically active domain) is identical between humans and monkeys. The sequence of the human and mouse or rat HTRA1 protease domain differs by 1 residue. However, this residue is structurally located on the side opposite to the active center of the enzyme, and it is therefore inferred that it has no effect on the active center of the enzyme (Figure 1). Therefore, the HTRA1 protease domain effectively has the same sequence, regardless of species (human / mouse / rat / monkey). Therefore, no special mention is made about species.

[0377] (2-1) Preparation of HTRA1 protease domain HTRA1 (cat)

[0378] (2-1-1) Construction of pET 21b_HTRA1 (cat)

[0379] The protease domain (158Gly to 373Lys) of human HTRA1 (Q92743) excluding the N-terminal domain and the PDZ domain was used as HTRA1 (cat) to construct an HTRA1 (cat) expression vector. The desired DNA fragment was amplified by PCR ((94°C for 15 seconds, 60°C for 30 seconds, and 68°C for 45 seconds) × 30 cycles) using a human HTRA1-inserted plasmid (GeneCopoeia, Inc.; GC-M0558) as a template using the following primers and KOD-plus- (Toyobo Co., Ltd.).

[0380] Primer 5: 5'-AAACATATGGGGCAGGAAGATCCCAACAGTTTGC-3' (SEQ ID NO: 37)

[0381] Primer 6: 5'-AAACTCGAGTTTGGCCTGTCGGTCATGGGACTC-3' (SEQ ID NO: 38)

[0382] The amplified fragments were subjected to agarose gel electrophoresis. Then, the desired DNA fragments were cut from the gel, and DNA was prepared using a QIAquick gel extraction kit (Qiagen NV). The prepared DNA fragments and pET 32a (Novagen) were each treated with restriction enzymes NdeI (New England BioLabs Inc.) and XhoI (New England BioLabs Inc.) at 37°C for 1 hour or longer. After agarose gel electrophoresis, the desired DNA fragments were cut from the gel, and purified using a QIAquick PCR purification kit (Qiagen NV). Using T4 DNA ligase (New England BioLabs Inc.), the purified fragments were reacted overnight at 16°C for ligation. The ligation solution was added to Escherichia coli JM109 (Toyobo Co., Ltd.), and the mixture was 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, and inoculated onto a 2YT plate containing 0.1 mg / ml ampicillin, followed by static culture overnight at 37°C to transform Escherichia coli. The transformed Escherichia coli was cultured, and then small-scale preparation and sequence analysis were performed to construct "pET 21b_HTRA1 (cat)". The operation was performed according to the method described in (1-1-1).

[0383] (2-1-2) Preparation of HTRA1 (cat)

[0384] E. coli BL21 (DE3) (Novagen) was transformed with constructed pET 21b_HTRA1 (cat) and cultured at 37°C using 2YT medium containing 0.1 mg / ml ampicillin. Then, IPTG (final concentration: 1 mM) was added thereto and the E. coli was cultured overnight at 28°C. After harvesting, lysates were prepared by suspending in phosphate buffer (50 mM sodium phosphate and 300 mM NaCl) containing 1 mg / ml lysozyme and sonicating, and the desired His tag fusion protein was recovered using TALON (Clontech Laboratories, Inc.). The resulting product was subjected to gel filtration chromatography (Superdex 200 10 / 300 GL) to purify HTRA1 (cat).

[0385] (2-2) Preparation of full-length HTRA1 (HTRA1 (full-length))

[0386] (2-2-1) Construction of pcDNA3.1_HTRA1 (full length)_His

[0387] The desired DNA fragment was amplified by PCR ((94°C for 15 seconds, 60°C for 30 seconds, and 68°C for 90 seconds)×30 cycles) with synthetic human HTRA1 (Q92743) DNA (GeneArt) as a template using the following primers and KOD-plus- (Toyobo Co., Ltd.).

[0388] Primer 7: 5'-AAAAGAATTCGCCACCATGCAGATTCCTAGAGCCG-3' (SEQ ID NO: 39)

[0389] Primer 8: 5'-AAAACTCGAGTCAGTGGTGATGGTGGTGGTGGCCGG-3' (SEQ ID NO:40)

[0390] The amplified fragments were subjected to agarose gel electrophoresis. Then, the desired DNA fragments were cut from the gel, and DNA was prepared using a QIAquick gel extraction kit (Qiagen NV). The prepared DNA fragments and pcDNA3.1 (ThermoFisher Scientific Inc.) were each treated with restriction enzymes EcoRI (New England BioLabs Inc.) and XhoI (New England BioLabs Inc.) at 37°C for 1 hour or longer. After agarose gel electrophoresis, the desired DNA fragments were cut from the gel, and purified using a QIAquick PCR purification kit (Qiagen NV). Using T4 DNA ligase (New England BioLabs Inc.), the purified fragments were reacted overnight at 16°C for ligation. The ligation solution was added to E. coli JM109 (Toyobo Co., Ltd.), and the mixture was 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, and inoculated onto a 2YT plate containing 0.1 mg / ml ampicillin, followed by static culture overnight at 37°C to transform E. coli. The transformed E. coli was cultured, and then small-scale preparation and sequence analysis were performed to construct "pcDNA3.1_HTRA1 (full length)_His". The operation was performed according to the method described in (1-1-1).

[0391] (2-2-2) Construction of pcDNA3.3_HTRA1 (full length)_FLAG_His

[0392] Fragment A was amplified by PCR ((94°C for 15 seconds, 60°C for 30 seconds, and 68°C for 90 seconds)×30 cycles) using the pcDNA3.1_HTRA1(full length)_His constructed in (2-2-1) as a template using the following primers and KOD-plus- (Toyobo Co., Ltd.).

[0393] Primer 7

[0394] Primer 9: 5'-CTTGTCGTCATCGTCCTTGTAGTCGCCGGGGTCGATTTCCTC-3' (SEQ ID NO:41)

[0395] Next, fragment B was amplified by PCR ((94° C. for 15 seconds, 60° C. for 30 seconds, and 68° C. for 10 seconds)×30 cycles) using the following primers and KOD-plus- (Toyobo Co., Ltd.).

[0396] Primer 10: 5'-GCGACTACAAGGACGATGACGACAAGCACCACCACCATCATCAC-3' (SEQ ID NO:42)

[0397] Primer 11: 5'-AAAAACTCGAGCTAGTGATGATGGTGGTGGTGCTTGTCGTC-3' (SEQ ID NO:43)

[0398] The desired DNA fragment was amplified by PCR ((94°C for 15 seconds, 60°C for 30 seconds, and 68°C for 90 seconds) × 30 cycles) using fragment A and fragment B as templates using primers 7 and 11 and KOD-plus- (Toyobo Co., Ltd.). The amplified fragment was subjected to agarose gel electrophoresis. Then, the desired DNA fragment was cut from the gel, and DNA was prepared using a QIAquick gel extraction kit (Qiagen NV). The prepared DNA fragment and pcDNA3.3 (Thermo Fisher Scientific Inc.) were each treated with restriction enzymes EcoRI (New England BioLabs Inc.) and XhoI (New England BioLabs Inc.) at 37°C for 1 hour or longer. After agarose gel electrophoresis, the desired DNA fragment was cut from the gel and purified using a QIAquick PCR purification kit (Qiagen NV). Using T4 DNA ligase (New England BioLabs Inc.), the purified fragment was reacted overnight at 16°C for ligation. The ligation solution was added to Escherichia coli JM109 (Toyobo Co., Ltd.), and the mixture was left on ice for 30 minutes, then heat treated at 42°C for 45 seconds, further left on ice for 5 minutes, and inoculated on a 2YT plate containing 0.1 mg / ml ampicillin, followed by static culture overnight at 37°C to transform Escherichia coli. The transformed Escherichia coli was cultured, and then a small amount of preparation and sequence analysis were performed to construct "pcDNA3.3_HTRA1 (full length)_FLAG_His". The method described in (1-1-1) was used.

[0399] (2-2-3) Preparation of HTRA1 (full length)

[0400] FreeStyle 293F (Thermo Fisher Scientific Inc.) was transfected with pcDNA3.3_HTRA1 (full length)_FLAG_His constructed in (2-2-2) using Polyethylenimine Max (Polysciences, Inc.). After 6 days, the culture supernatant was recovered. His tag fusion protein was recovered using HisTrap excel (GE Healthcare), and HTRA1 (full length) was further purified using anti-FLAG M2 affinity agarose gel (Sigma-Aldrich Co. LLC).

[0401] (2-3) Preparation of the inactive HTRA1 mutant HTRA1 (S328A)

[0402] (2-3-1) Construction of pcDNA3.3_HTRA1(S328A)_FLAG_His

[0403] In order to construct an inactive HTRA1 mutant HTRA1 (S328A) expression vector, PCR ((95°C for 30 seconds, 55°C for 1 min, and 68°C for 7 min) × 18 cycles) was performed using the vector "pcDNA3.3_HTRA1 (full length)_FLAG_His" constructed in Example (2-2-2) as a template using the following primers and the QuikChange II Site-Directed Mutagenesis Kit (Agilent Technologies Japan, Ltd.).

[0404] Primer 21: 5'-CCATCATCAACTACGGCAACGCGGGCGGACCCCTCGTGAACC-3' (SEQ ID NO:55: Fig.76 )

[0405] Primer 22: 5'-GGTTCACGAGGGGTCCGCCCGCGTTGCCGTAGTTGATGATGG-3' (SEQ ID NO: 56: Fig.77 )

[0406] After the PCR reaction, the PCR reaction solution treated with DpnI was used to transform E. coli JM109 (Toyobo Co., Ltd.) according to the protocol attached to the kit. The transformed E. coli was cultured, and then a small amount of preparation and sequence analysis were performed to construct "pcDNA3.3_HTRA1(S328A)_FLAG_His". The operation was performed according to the method described in (1-1-1).

[0407] (2-3-2) Preparation of HTRA1 (S328A)

[0408] According to the method described in (2-2-3), HTRA1 (S328A) was expressed using FreeStyle 293F and prepared by affinity purification.

[0409] Example 3. Evaluation of HTRA1-inhibitory peptides for HTRA1 inhibitory activity

[0410] (3-1) Evaluation of HTRA1-inhibitory peptides for HTRA1 inhibitory activity using peptide substrates

[0411] The substrate peptide H2-Opt (Mca-IRRVSYSFK(Dnp)K) (Peptide Institute, Inc.: SEQ ID NO: 54, Figure 8 ) was dissolved in DMSO at 10 mM, diluted with assay buffer (50 mM borate and 150 mM NaCl, pH 8.5), and used at a final concentration of 10 μM. HTRA1 (HTRA1 (cat) or HTRA1 (full length)) diluted with assay buffer and each HTRA1-inhibitory peptide were mixed with 25 μL each and reacted at 37°C for 20 minutes. Then, 50 μL of substrate diluted with assay buffer was added thereto. The fluorescence signal (excitation at 328 nm / emission at 393 nm) was measured using Enspire (PerkinElmer, Inc.). The final concentration of HTRA1 was 100 nM, and the final concentration of HTRA1-inhibitory peptide was 1.875 to 1,000 nM. PROTEOSAVE(R) SS96F blackboard (Sumitomo BakeliteCo., Ltd.) was used in the reaction and measurement.

[0412] The substrate peptide decomposition rate of each HTRA1-inhibitory peptide concentration was calculated. When the decomposition rate at an inhibitor concentration of 0 nM was defined as 100%, the HTRA1 (cat) and HTRA1 (full length) inhibitory activities of each HTRA1-inhibitory peptide were evaluated ( Figure 2 and 3 As a result of calculating the 50% inhibitory concentration (IC50) using GraphPad Prism (version 5.0; GraphPad Software Inc.), it was found that all HTRA1-inhibitory peptides inhibited the enzyme activities of HTRA1 (cat) and HTRA1 (full length) at low concentrations ( Figure 2 A to 2C and Figure 3 By contrast, wild-type SPINK2 (wt) showed no HTRA1 inhibitory activity ( Figure 2 D).

[0413]

[0414] (3-2) Evaluation of HTRA1-inhibitory peptides for HTRA1 inhibitory activity using protein substrates

[0415] The HTRA1 inhibitory activity of HTRA1-inhibitory peptides was evaluated using human vitronectin as a protein substrate. HTRA1 (cat) diluted with assay buffer (50 mM Tris and 150 mM NaCl, pH 8.0) and each HTRA1-inhibitory peptide were mixed and reacted at 37°C for 1 hour. Next, human vitronectin (BD Biosciences; 354238) diluted with assay buffer was added thereto and reacted at 37°C for 2 hours. SDS sample buffer was added thereto, and the enzyme reaction was terminated by treating at 99°C for 5 minutes. Then, the decomposition of human vitronectin was evaluated by SDS-PAGE and Western blot analysis. The final concentration of HTRA1-inhibitory peptides was 0 to 25 μM, the final concentration of HTRA1 (cat) was 1 μM, and the final concentration of human vitronectin was 1 μM. For Western blot analysis, human vitronectin antibody (R&D Systems, Inc.; MAB2349) was used as the primary antibody, and anti-mouse IgG, HRP-linked total Ab sheep (GE Healthcare; NA931) was used as the secondary antibody.

[0416] As in (3-1), when human vitronectin was used as a substrate, the HTRA1-inhibitory peptide also strongly exhibited inhibition of HTRA1 (cat) ( FIG. 4 ).

[0417] (3-3) Specificity evaluation of HTRA1-inhibitory peptides

[0418] The specificity for other proteases was evaluated by using the cleavage of the substrate peptide as an indicator. In the same manner as described in (3-1), each protease diluted with assay buffer and each sample (final concentration: 1 μM) were mixed with 25 μL each and reacted at 37°C for 20 minutes. Then, 50 μL of each substrate diluted with assay buffer was added thereto. The fluorescence signal (excitation at 380 nm / emission at 460 nm) was measured using Enspire (PerkinElmer, Inc.). The same assay buffer (50 mM borate and 150 mM NaCl, pH 8.5) as in Example 2 was used to evaluate HTRA2 activity. The assay buffer (50 mM Tris and 150 mM NaCl, pH 8.0) was used to evaluate protease activity other than HTRA2 activity. PROTEOSAVE(R) SS96F blackboard (Sumitomo BakeliteCo., Ltd.) was used in the reaction and measurement. The combination of protease and substrate used in the specificity evaluation is as follows.

[0419] Evaluation of bovine trypsin inhibitory activity; 5 nM (final concentration) trypsin (Pierce; 20233) and 100 μM (final concentration) substrate peptide Boc-VPR-AMC fluorescent peptide substrate (R&D Systems, Inc.; ES011)

[0420] Evaluation of bovine α-chymotrypsin inhibitory activity; 10 nM (final concentration) chymotrypsin (Worthington Biochemical Corporation; LS001434) and 100 μM (final concentration) substrate peptide Suc-Leu-Leu-Val-Tyr-MCA (Peptide Institute, Inc.; 3120-v)

[0421] Evaluation of human trypsin inhibitory activity; 1 nM (final concentration) tryptase (Sigma-Aldrich Co. LLC; T7063) and 100 μM (final concentration) substrate peptide Boc-Phe-Ser-Arg-MCA (Peptide Institute, Inc.; 3107-v)

[0422] Evaluation of human chymotrypsin inhibitory activity; 100 nM (final concentration) chymotrypsin (Sigma-Aldrich Co. LLC; C8118) and 100 μM (final concentration) substrate peptide Suc-Leu-Leu-Val-Tyr-MCA (Peptide Institute, Inc.; 3120-v)

[0423] Evaluation of human plasmin inhibitory activity; 50 nM (final concentration) plasmin (Sigma-Aldrich Co. LLC; P1867) and 100 μM (final concentration) substrate peptide Boc-Val-Leu-Lys-MCA (Peptide Institute, Inc.; 3104-v)

[0424] Evaluation of human thrombin inhibitory activity; 1 nM (final concentration) thrombin (Sigma-Aldrich Co. LLC; T6884) and 100 μM (final concentration) substrate peptide Boc-VPR-AMC fluorescent peptide substrate (R&D Systems, Inc.; ES011)

[0425] Evaluation of human matriptase inhibitory activity; 1 nM (final concentration) matriptase (R&D Systems, Inc.; E3946-SE) and 100 μM (final concentration) substrate peptide Boc-QAR-AMC fluorescent peptide substrate (R&D Systems, Inc.; ES014)

[0426] Evaluation of human protein C inhibitory activity; 100 nM (final concentration) protein C (Sigma-Aldrich Co. LLC; P2200) and 100 μM (final concentration) substrate peptide Boc-Leu-Ser-Thr-Arg-MCA (Peptide Institute, Inc.; 3112-v)

[0427] Evaluation of human tPA inhibitory activity; 10 nM (final concentration) tPA (Sigma-Aldrich Co. LLC; T0831) and 100 μM (final concentration) substrate peptide Pyr-Gly-Arg-MCA (Peptide Institute, Inc.; 3145-v)

[0428] Evaluation of human uPA inhibitory activity; 10 nM (final concentration) uPA (Sigma-Aldrich Co. LLC; T0831) and 100 μM (final concentration) substrate peptide Pyr-Gly-Arg-MCA (Peptide Institute, Inc.; 3145-v)

[0429] Evaluation of human plasma kallikrein inhibitory activity; 0.125 μg / ml (final concentration) plasma kallikrein (Sigma-Aldrich Co. LLC; T0831) and 100 μM (final concentration) substrate peptide Z-Phe-Arg-MCA (PeptideInstitute, Inc.; 3095-v)

[0430] Evaluation of human HTRA2 inhibitory activity; 200 nM (final concentration) HTRA2 (R&D Systems, Inc.; 1458-HT) and 50 μM (final concentration) substrate peptide H2-Opt (Peptide Institute, Inc.)

[0431] The cross-reactivity with proteases other than HTRA1 was evaluated in the same manner as in (3-2) by using the decomposition of the peptide substrate as an index. At a final inhibitor concentration of 1 μM, each HTRA1-inhibitory peptide did not inhibit the protease activity of any protease, indicating that each HTRA1-inhibitory peptide had an HTRA1-specific inhibitory effect ( FIG. 5 ).

[0432] Example 4. Analysis of HTRA1-inhibitory peptides using X-ray crystallography

[0433] (4-1) Preparation of HTRA1 (cat) / HTRA1-inhibitory peptide complex

[0434] According to the methods described in (1-2) and (2-1), HTRA1 (cat) and an HTRA1-inhibitory peptide having the amino acid sequence shown in SEQ ID NO: 3 were prepared respectively. These were mixed under the conditions of 20 mM Tris-HCl and 150 mM NaCl, pH 7.6. Then, the complex was separated and purified by gel filtration chromatography (Superdex 200 10 / 300 GL).

[0435] (4-2) X-ray crystallography

[0436] The complex solution prepared in (4-1) was concentrated to 18 mg / ml and then mixed with a storage solution (1.0 M LiCl, 7.5% PEG6000 and 0.1 M Tris / HCl (pH 8.5)) at a ratio of 1:1, and the mixture was crystallized by a vapor diffusion method. The obtained cubic single crystal was immersed in a storage solution containing 20% ​​ethylene glycol and then frozen in liquid nitrogen. The frozen crystal was exposed to X-rays under a low-temperature air flow to obtain a diffraction image (Photon Factory BL5A: High Energy Accelerator Research Organization). The scaled data with a maximum resolution of 2.6 angstroms were obtained by analysis using HKL2000. The phase was determined by a molecular replacement method using serine protease HTRA1 (PDB ID: 3NZI) as a template. After structural refinement, the crystal complex of HTRA1 (cat) and the peptide was determined at a resolution of 2.6 angstroms. One HTRA1 and one SPINK2 molecule were contained in the unit cell. Regarding the SPINK2 molecule, a partial molecular model containing the interaction site with HTRA1 (cat) was constructed based on the sequence information and observed electron density. It was confirmed that the HTRA1-inhibitory peptide binds to the region containing the active center of the HTRA1 enzyme ( Figure 6 and 7 ).

[0437] Example 5. Retinal protection by inhibition of HTRA1 in a rat model of retinal damage induced by light exposure

[0438] (5-1) Preparation of a rat model of retinal damage induced by light exposure

[0439] The rat model of retinal damage induced by light exposure is a model of cell death of retinal photoreceptor cells induced by light exposure, and is generally used as a model animal of retinal degeneration (Daniel T. Organisciak et al., (1996) Invest Ophthalmol Vis Sci. Vol. 37 (No. 11): p. 2243-2257). Under dark adaptation conditions, 0.5% (W / V) tropicamide-0.5% phenylephrine hydrochloride ophthalmic solution is instilled into the eyes of rats adapted to the dark for 72 hours. Then, the rats are exposed to white light of 5500 Lux for 3 hours. The rats thus exposed are adapted to the dark again for about 24 hours, and then raised under light-dark conditions of ordinary feeding for 2 days. After euthanasia, the eyeballs are cut off and fixed by immersion in 3.7% (W / V) formaldehyde-0.5 to 1% (W / V) methanol-0.2% (W / V) picric acid fixative for 24 hours or more. After paraffin embedding, thin sections were prepared. The sections were stained with hematoxylin-eosin, and the nuclear counts in the outer nuclear layer of the cross-section of the retina were determined to assess retinal damage. It was found that the rat model of retinal damage induced by light exposure had a significant decrease in the nuclear count in the outer nuclear layer due to light exposure.

[0440] (5-2) Confirmation of extracellular HTRA1 expression during retinal injury

[0441] To examine the involvement of HTRA1 in a rat model of retinal damage induced by light exposure, vitreous humor was collected from the model rats prepared in (5-1), and the HTRA1 expression level was evaluated by Western blot analysis. The vitreous humor was subjected to SDS-PAGE under reducing conditions. Rat HTRA1 was detected using a human HTRA1 / PRSS11 antibody (R&D Systems, Inc.; AF2916) as a primary antibody and a sheep IgG horseradish peroxidase-conjugated antibody (R&D Systems, Inc.; HAF016) as a secondary antibody. An increase in the amount of HTRA1 in the vitreous humor was confirmed in the light-exposed group compared to the non-exposed group, indicating that in this model, HTRA1 is involved in the process of retinal damage induced by light exposure ( Fig. 9 ).

[0442] (5-3) Retinal protective effect of HTRA1-inhibitory peptide in a rat model of retinal damage induced by light exposure

[0443] Immediately before light exposure, 5 µL of HTRA1-inhibitory peptide H308 at a concentration of 0.04 mg / mL or 0.2 mg / mL was intravitreally injected under anesthesia in the rats. For the saline administration group, n = 4, and for the other groups, n = 5. Light exposure reduced the nuclear count in the outer nuclear layer on the cross section of the retina in the saline administration group, while the effect of suppressing the reduction of the nuclear count in the outer nuclear layer was confirmed in the HTRA1-inhibitory peptide administration group ( Fig.10 ). These results indicate that the HTRA1-inhibitory peptide exhibits a pharmacological effect on tissue damage caused by HTRA1.

[0444] Example 6. Evaluation of HTRA1-inhibitory peptide derivatives

[0445] (6-1) Construction of pET 32a_HTRA1-inhibitory peptide H308_S16A_Kex2

[0446] Using HTRA1-inhibitory peptide H308 as a template, a derivative S16A having an amino acid sequence in which SEQ ID NO: 9 ( Fig.21 ) was substituted with Ala. Fragment C was amplified by PCR ((94°C for 15 seconds, 60°C for 30 seconds, and 68°C for 15 seconds)×30 cycles) using the following primers and KOD-plus-(Toyobo Co., Ltd.).

[0447] Primer 12: 5'-CCGCAGTTTGGTCTGTTTAGCAAATATCGTAACCCCGAATTGT-3' (SEQ ID NO:44)

[0448] Primer 13: 5'-GCCATACCAGCATGGTCCGCACAATTCGGGGTACGATATTTGC-3' (SEQ ID NO:45)

[0449] Next, fragment D was amplified by PCR ((94°C for 15 seconds, 60°C for 30 seconds, and 68°C for 20 seconds)×30 cycles) with the HTRA1-inhibitory peptide H308 as a template using the following primers and KOD-plus- (Toyobo Co., Ltd.).

[0450] Primer 14: 5'-GCGGACCATGCTGGTATGGCATGTGTTGCTCTGTATGAAC-3' (SEQ ID NO:46)

[0451] Primer 15: 5'-AAAACTCGAGTTAGCCGCCGCACGGACCATTGCGAATAA-3' (SEQ ID NO:47)

[0452] The desired DNA fragment was amplified by PCR ((94°C for 15 seconds, 60°C for 30 seconds, and 68°C for 20 seconds)×30 cycles) using fragments C and D, the following primers, and KOD-plus- (Toyobo Co., Ltd.).

[0453] Primer 16: 5'-AAAAGGATCCCTGGACAAACGTGATCCGCAGTTTGGTCTGTTTAG-3' (SEQ IDNO: 48)

[0454] Primer 15

[0455] The amplified fragments were subjected to agarose gel electrophoresis. Then, the desired DNA fragments were cut from the gel, and DNA was prepared using a QIAquick gel extraction kit (Qiagen NV). The prepared DNA fragments and pET 32a (Novagen) were each treated with restriction enzymes BamHI (New England BioLabs Inc.) and XhoI (New England BioLabs Inc.) at 37°C for 1 hour or longer. After agarose gel electrophoresis, the desired DNA fragments were cut from the gel, and purified using a QIAquick PCR purification kit (Qiagen NV). Using T4 DNA ligase (New England BioLabs Inc.), the purified fragments were reacted overnight at 16°C for ligation. The ligation solution was added to E. coli JM109 (Toyobo Co., Ltd.), and the mixture was 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, and inoculated onto a 2YT plate containing 0.1 mg / ml ampicillin, followed by static culture overnight at 37°C to transform E. coli. The transformed E. coli was cultured, and then small-scale preparation and sequence analysis were performed to construct "pET 32a_HTRA1-inhibitory peptide H308_S16A_Kex2". The operation was performed according to the method described in (1-1-1).

[0456] (6-2) Preparation of HTRA1-inhibitory peptide_N-terminal derivative expression vector

[0457] In order to prepare a preparation having an amino acid sequence (wherein SEQ ID NO:9 ( Fig.21Four N-terminal sequence derivatives of the HTRA1-inhibitory peptide (designated as D1G, D1S, D1E, and D1SLI, respectively) in which 1Asp in the amino acid sequence shown in (6-1) was replaced by Gly, Ser, Glu, or Ser-Leu-Ile, and expression vectors were constructed by the same method as in (6-1). The four target fragments were amplified by PCR ((94°C for 15 seconds, 60°C for 30 seconds, and 68°C for 20 seconds) × 30 cycles) using fragments C and D, the following primers, and KOD-plus- (Toyobo Co., Ltd.).

[0458] D1G primer preparation

[0459] Primer 17: 5'-AAAAGGATCCCTGGACAAACGTGGCCCGCAGTTTGGTCTGTTTAG-3' (SEQ IDNO: 49)

[0460] Primer 15

[0461] D1S primer preparation

[0462] Primer 18: 5'-AAAAGGATCCCTGGACAAACGTAGCCCGCAGTTTGGTCTGTTTAG-3' (SEQ IDNO: 50)

[0463] Primer 15

[0464] D1E primer preparation

[0465] Primer 19: 5'-AAAAGGATCCCTGGACAAACGTGAACCGCAGTTTGGTCTGTTTAG-3' (SEQ IDNO: 51)

[0466] Primer 15

[0467] D1SLI primer preparation

[0468] Primer 20: 5'-AAAAGGATCCCTGGACAAACGTAGCCTGATTCCGCAGTTTGGTCTGTTTAG-3' (SEQID NO:52)

[0469] Primer 15

[0470] Four kinds of amplified fragments are subjected to agarose gel electrophoresis respectively.Then, the desired DNA fragment is cut from the gel, and DNA is prepared using QIAquick gel extraction kit (Qiagen NV). The prepared DNA fragment and pET 32a (Novagen) are each treated with restriction enzymes BamHI (New England BioLabs Inc.) and XhoI (New England BioLabs Inc.) at 37°C for 1 hour or longer. After agarose gel electrophoresis, the desired DNA fragment is cut from the gel, and purified using QIAquick PCR purification kit (Qiagen NV). Using T4 DNA ligase (New England BioLabs Inc.), the purified fragment is reacted overnight at 16°C for ligation. The ligation solution was added to Escherichia coli JM109 (Toyobo Co., Ltd.), and the mixture was 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, and inoculated onto a 2YT plate containing 0.1 mg / ml ampicillin, followed by static culture overnight at 37° C. to transform Escherichia coli. The transformed Escherichia coli was cultured, and then small-scale preparation and sequence analysis were performed to construct "pET 32a_HTRA1-inhibitory peptide H308_D1G_S16A_Kex2", "pET 32a_HTRA1-inhibitory peptide H308_D1S_S16A_Kex2", "pET 32a_HTRA1-inhibitory peptide H308_D1E_S16A_Kex2", and "pET 32a_HTRA1-inhibitory peptide H308_D1SLI_S16A_Kex2". The operation was carried out according to the method described in (1-1-1).

[0471] (6-3) Preparation of HTRA1-inhibitory peptide derivatives

[0472] E. coli Origami B (DE3) (Novagen) was transformed with each of the five vectors constructed in (6-1) and (6-2) and cultured at 37°C using 2YT medium containing 0.1 mg / ml ampicillin. Then, IPTG (final concentration: 1 mM) was added thereto and the E. coli was cultured overnight at 16°C. The next day, after harvesting by centrifugation (3,000 g, 20 min, 4°C), lysates were prepared using BugBuster Master Mix (Novagen) and the target His tag fusion protein was purified using TALON metal affinity resin (Clontech Laboratories, Inc.). Next, the thioredoxin tag and the desired protein were cut using Kex2 (mentioned above) and purified using TALON. The resulting product was subjected to gel filtration chromatography (Superdex 75 10 / 300 GL) or reverse phase chromatography (YMC-Pack ODS-AM) to prepare five HTRA1-inhibitory peptide derivatives. The amino acid sequences of the derivatives are shown in SEQ ID NOs: 23 to 27 ( Figures 35 to 39 )middle.

[0473] (6-4) Evaluation of HTRA1-inhibitory peptide derivatives

[0474] As a result of measuring the HTRA1 (cat) inhibitory activity according to the method described in (3-1), all the derivatives had inhibitory activities equivalent to that of H308 ( Fig.11 ).

[0475] Example 7. Evaluation of HTRA1-inhibitory peptides for binding activity against HTRA1 (cat)

[0476] The binding activity was evaluated by immunoprecipitation using the three HTRA1-inhibitory peptides (H308, H321AT, and H322AT) prepared in Example (1-2) and HTRA1 (cat) prepared in (2-1). 2.5 μg of each HTRA1-inhibitory peptide and 10 μg of HTRA1 (cat) were reacted for 30 minutes at room temperature. Then, 10 μL of TALON metal affinity resin (Clontech Laboratories, Inc.) was added thereto. After further reaction for 30 minutes, the resin was recovered as an immunoprecipitation (IP) fraction and subjected to SDS-PAGE to evaluate the binding activity. PBS was used as a buffer in the reaction.

[0477] When the three HTRA1-inhibitory peptides or HTRA1 (cat) were each reacted with TALON, only the band of His tag-fused HTRA1 (cat) was detected in the input lane. On the other hand, the bands of each inhibitory peptide and enzyme were detected only in the IP lane where the inhibitory peptide was reacted with HTRA1 (cat). Therefore, it was confirmed that the three HTRA1-inhibitory peptides each bound to HTRA1 (cat) ( Fig.12 ).

[0478] Example 8. Evaluation of HTRA1-inhibitory peptides for HTRA1 inhibitory activity

[0479] (8-1) Evaluation of HTRA1-inhibitory peptides for HTRA1 inhibitory activity using peptide substrates

[0480] The HTRA1 (cat) or HTRA1 (full length) inhibitory activity of the three HTRA1-inhibitory peptides (H308_D1G_S16A, H321AT_D1G_S16A, and H322AT_D1G_S16A) constructed in Example 6 was evaluated using the substrate peptide H2-Opt (n = 3). The substrate peptide H2-Opt (Mca-IRRVSYSFK(Dnp)K) (Peptide Institute, Inc.: SEQ ID NO: 54, Figure 8 ) was dissolved in DMSO at 10 mM, diluted with assay buffer (50 mM Tris, 150 mM NaCl and 0.25% CHAPS, pH8.0), and used at a final concentration of 10 μM. HTRA1 (HTRA1 (cat) or HTRA1 (full length); Example 2) diluted with assay buffer and each HTRA1-inhibitory peptide were mixed with 25 μL each and reacted at 37°C for 20 minutes. Then, 50 μL of substrate diluted with assay buffer was added thereto. Fluorescence signals (excitation at 328 nm / emission at 393 nm) were measured using Enspire (PerkinElmer, Inc.). The final concentration of HTRA1 was 100 nM, and the final concentration of HTRA1-inhibitory peptide was 1.875 to 1,000 nM. PROTEOSAVE (R) SS96F blackboard (Sumitomo Bakelite Co., Ltd.) was used in the reaction and measurement.

[0481] The substrate peptide decomposition rate of each HTRA1-inhibitory peptide concentration was calculated. When the decomposition rate at an inhibitor concentration of 0 nM was defined as 100%, the HTRA1 (cat) and HTRA1 (full length) inhibitory activities of each HTRA1-inhibitory peptide were evaluated.

[0482] As a result of calculating the 50% inhibitory concentration (IC50) using GraphPad Prism (version 5.0; GraphPad Software Inc.), it was found that all HTRA1-inhibitory peptides inhibited the enzyme activities of HTRA1 (cat) and HTRA1 (full length) at low concentrations ( Fig.66 ).

[0483]

[0484] (8-2) Evaluation of HTRA1-inhibitory peptides for HTRA1 inhibitory activity using protein substrates

[0485] The HTRA1 inhibitory activity of the HTRA1-inhibitory peptides was evaluated using human vitronectin as a protein substrate. The procedure was in accordance with Example (3-2).

[0486] As in (8-1), when human vitronectin was used as a substrate, the HTRA1-inhibitory peptide also strongly exhibited inhibition of HTRA1 (cat) ( Fig.67 ).

[0487] (8-3) Specific evaluation of HTRA1-inhibitory peptides

[0488] The specificity to other proteases was assessed by using the cleavage of the substrate peptide as an index. The operation of bovine trypsin, bovine α-chymotrypsin, protein C, tryptase, chymotrypsin, thrombin, plasmin, tPA, plasma kallikrein, matriptase, uPA and HTRA2 followed the method described in Example (3-3) (n = 3). The procedure for measuring the inhibitory activity against other proteases and the combination of proteases and substrates are as follows.

[0489] PROTEOSAVE(R) SS96F black board (Sumitomo Bakelite Co., Ltd.) was used for the reaction and measurement. Each protease diluted with the assay buffer and each sample (final concentration: 1 μM) were mixed at 25 μL each and reacted at 37°C for 20 minutes. Then, 50 μL of each substrate diluted with the assay buffer was added thereto. The fluorescence signal was measured using Enspire (PerkinElmer, Inc.).

[0490] Evaluation of human trypsin inhibitory activity; 1 nM (final concentration) trypsin (Sigma-Aldrich Co. LLC; T6424) and 100 μM (final concentration) substrate peptide Boc-VPR-AMC fluorescent peptide substrate (R&D Systems, Inc.; ES011), fluorescence signal: excitation at 380 nm / emission at 460 nm.

[0491] Evaluation of human chymotrypsin inhibitory activity; 10 nM (final concentration) chymotrypsin (Sigma-AldrichCo. LLC; C8946) and 10 μM (final concentration) substrate peptide Suc-Leu-Leu-Val-Tyr-MCA (PeptideInstitute, Inc.; 3120-v), fluorescence signal: excitation at 380 nm / emission at 460 nm.

[0492] Evaluation of human factor XIIa inhibitory activity; 100 nM (final concentration) factor α-XIIa (Enzyme Research Laboratories Inc.) and 100 μM (final concentration) substrate peptide Pyr-Gly-Arg-MCA (Peptide Institute, Inc.; 3145-v), fluorescence signal: excitation at 380 nm / emission at 460 nm.

[0493] Evaluation of human MMP-2 inhibitory activity; 1 nM (final concentration) MMP-2 (Calbiochem; PF023) and 100 μM (final concentration) substrate peptide MOCAx-KPLGL-A2pr(Dnp)-AR (Peptide Institute, Inc.; 3226-v), fluorescence signal: excitation at 328 nm / emission at 393 nm.

[0494] Evaluation of human TPP1 inhibitory activity; 0.5 μg / ml (final concentration) TPP1 (Calbiochem; 2237-SE) and 200 μM (final concentration) substrate peptide AAF-MCA (Peptide Institute, Inc.; 3201-v), fluorescence signal: excitation at 380 nm / emission at 460 nm.

[0495] Cross-reactivity with proteases other than HTRA1 was evaluated by using decomposition of peptide substrates as an index. At a final concentration of 1 μM, each HTRA1-inhibitory peptide did not inhibit the protease activity of any protease, indicating that the HTRA1-inhibitory peptide had an HTRA1-specific inhibitory effect ( FIG. 68 ).

[0496] Example 9. Evaluation of HTRA1-inhibitory peptides for binding activity against HTRA1 (cat)

[0497] Using the three HTRA1-inhibitory peptides prepared in Example 6 and HTRA1 (cat) prepared in (2-1), the binding activity was evaluated by the immunoprecipitation method according to the procedure of Example 7.

[0498] When the three HTRA1-inhibitory peptides or HTRA1 (cat) were each reacted with TALON, only the band of His tag-fused HTRA1 (cat) was detected in the input lane. On the other hand, the bands of each inhibitory peptide and enzyme were detected only in the IP lane where the inhibitory peptide was reacted with HTRA1 (cat). Therefore, it was confirmed that the three HTRA1-inhibitory peptides each bound to HTRA1 (cat) ( Fig.69 ).

[0499] Example 10. Retinal protection by inhibition of HTRA1 in a rat model of retinal damage induced by light exposure (Part 2)

[0500] The retinal protective effects of the three HTRA1-inhibitory peptides prepared in Example 6 were evaluated using the rat model of retinal damage induced by light exposure constructed in Example (5-1). The operation followed Example 5. For all groups, n=6.

[0501] The results of pathological evaluation of the retina are shown in Fig.70 The three HTRA1-inhibitory peptides showed a significant inhibitory effect on the decrease in nuclear count in the outer nuclear layer induced by light exposure.

[0502] Example 11. Protection of retinal pigment epithelial cells by inhibiting HTRA1 in a rat model of retinal damage induced by high-fat diet loading with hydroquinone

[0503] (11-1) Preparation of a rabbit retinal injury model by feeding a high-fat diet containing hydroquinone

[0504] The retinal injury model prepared using a high-fat diet (HFD) and hydroquinone (HQ) is a model in which oxidative stress is induced by prooxidants to cause retinal injury. These models have only been reported for mice (Diego G. Espinosa-Heidmann et al., (2006) Invest Ophthalmol Vis Sci., Vol. 47 (No. 2): p. 729-737). Therefore, RC4 (Oriental Yeast Co., Ltd.) diet containing 1.5% (W / V) coconut oil-0.25% (W / V) cholesterol-1.5% (W / V) peanut oil-2.4% (W / V) hydroquinone (HFD-HQ) was fed to 3-year-old JW rabbits for 4 months to construct a rabbit retinal injury model. After euthanasia, the eyeball was cut off and the anterior segment of the eye was removed through an external incision of about 5 mm from the limbus. The vitreous body was further separated. Then, retina-choroid-sclera is fixed by immersion in 4% (W / V) paraformaldehyde fixative for 24 hours or longer.After fixing, choroid is separated and uses as primary antibody ZO-1 monoclonal antibody (ZO1-1A12) (Thermo Fisher Scientific Inc.; 33-9100) and as secondary antibody chicken anti-mouse IgG (H+L) cross-adsorbed secondary antibody Alexa Fluor 594 (Thermo Fisher Scientific Inc.; A-21201) to carry out immunostaining.Under fluorescence microscope (BZ-9000; Keyence Corp.), observe the choroid of dyeing.Determine the area of ​​retinal pigment epithelium (RPE) cell of dyeing, to assess RPE cell injury.

[0505] Fig.71 The staining images of RPE cells of 12-week-old rabbits, 3-year-old rabbits, and 3-year-old rabbits loaded with HFD-HQ are shown ( Fig.71 (A)) and the average area of ​​RPE cells ( Fig.71 (B)). RPE cells were confirmed to be more hypertrophic in 3-year-old rabbits than in 12-week-old rabbits, and were further hypertrophic by HFD-HQ loading. It was confirmed that the damage occurred in RPE cells. Similar changes have been observed in the eyes of patients with age-related macular degeneration (Ding JD et al., (2011) Proc Natl Acad Sci U SA., Vol. 108 (No. 28): p. 279-87).

[0506] (11-2) Increased expression of AMD-related factor C3 in retinal damage

[0507] In order to assess the expression of AMD-related factors, tissues were collected from the retina and RPE / choroid of the rabbit retinal injury model respectively. mRNA was extracted using RNeasy mini test kit (Qiagen NV), and then reverse transcription was performed using TaqMan GeneExpression Master Mix (Thermo Fisher Scientific Inc.). 7900HT fast real-time PCR system (Applied Biosystems, Inc.) was used to measure (Oc03397832_g1 and Oc03824857_g1 by TaqMan gene expression; Thermo Fisher Scientific Inc.) quantitative analysis of complement component 3 (C3) and internal standard beta-actin mRNA levels. For 3-year-old rabbits, analysis was performed with n=4, and for 3-year-old rabbits loaded with HFD-HQ, analysis was performed with n=10.

[0508] The C3 expression levels in the retina as well as in RPE cells and choroid are shown in Fig.71 (C) and (D). For both tissues, the expression level of C3 was confirmed to be increased in the rabbit group given HFD-HQ.

[0509] (11-3) Increased HTRA1 protein levels in retinal damage

[0510] To examine the involvement of HTRA1 in the rabbit retinal injury model, vitreous fluid was collected from the rabbit model prepared in (11-1) and enzymatically digested with a trypsin / Lys-C mixture (Promega Corp.). Then, peptide fragments of HTRA1 were quantified using LC (EASY-nLC 1000; Thermo Fisher Scientific Inc.)-MS (TripleTOF 6600; AB Sciex Pte. Ltd). In the vitreous fluid of rabbits given HFD-HQ, an increase in HTRA1 protein levels was found ( Fig.71 (E)). From these results, hypertrophy of RPE cells and increased expression of AMD-related factors C3 and HTRA1 were confirmed, indicating that the rabbit retinal injury model can be used for the study of age-related retinal diseases.

[0511] (11-4) Retinal protective effect of HTRA1 inhibitor in a rabbit retinal injury model

[0512] The retinal protective effect of the HTRA1-inhibitory peptide H308 prepared in Example 1 was evaluated using a rabbit model. Two months after the start of HFD-HQ feeding, 50 μL of a 40 mg / mL H308 solution was administered into the vitreous of one eye under anesthesia. Physiological saline was injected into the vitreous of the other eye. For all groups, n = 5.

[0513] Four months after the start of feeding, RPE cell hypertrophy in the model animals was evaluated. The results are shown in Fig.72 In the case of two indicators (i.e., the average area of ​​RPE cells ( Fig.72 (A)) and with 1500 μm 2 or larger cell area. Fig.72 (B))) shows that HTRA1 inhibitors showed an inhibitory effect on RPE cell hypertrophy. Fig.71 As shown in (E), an increase in HTRA1 was confirmed in the vitreous humor of the model, indicating that HTRA1 is involved in the damage process of RPE cells due to HFD-HQ. Therefore, the HTRA1-inhibitory peptide can be used as an anti-age-related macular degeneration agent. This test shows that the HTRA1-inhibitory peptide is particularly useful for preventing dry age-related macular degeneration.

[0514] The presence of the HTRA1-inhibitory peptide was confirmed in the retina of normal rabbits to which the HTRA1-inhibitory peptide was administered, indicating high tissue permeability of the HTRA1-inhibitory peptide.

[0515] Example 12. Inhibitory effect of HTRA1-inhibitory peptide in VEGF mRNA induction assay using human retinal pigment epithelial cells ARPE-19

[0516] ARPE-19 cells were cultured in 12 mm Transwells with 0.4 mm pore sterile polyester membrane inserts (Corning Inc.) using DMEM / F-12 medium (Wako Pure Chemical Industries, Ltd.) containing 10% fetal bovine serum (FBS) and penicillin-streptomycin (Thermo Fisher Scientific Inc.) at 37°C and 5% CO2 until confluence. Then, the cells were cultured in DMEM / F-12 without FBS for 5 days. H2O2 (final concentration: 500 μM) was added to the upper and lower layers of the chamber, and normal human serum complement (Quidel Corp.) (final concentration: 25%) was added to the upper layer of the chamber. HTRA1 (Example 2-2), inactive HTRA1 protease mutant HTRA1 (S328A) (Example 2-3) or HTRA1-inhibitory peptide H308_D1G_S16A (Example 6) were added to the upper and lower layers of the chamber at a final concentration of 1 μM, respectively. After 4 hours, the culture supernatant was removed and the cells were washed with PBS. Then, mRNA was extracted using the SuperPrep (TM) cell lysis and RT kit for qPCR (Toyobo Co., Ltd.) and reverse transcription reaction was performed. The mRNA level of VEGF was quantitatively analyzed by TaqMan gene expression assay (Hs000900055_m1 and Hs02786624_g1; Thermo Fisher Scientific Inc.) using the 7900HT fast real-time PCR system (Applied Biosystems, Inc.). GAPDH was used to correct mRNA levels.

[0517] The results are shown in Fig.74Compared with the conditions involving the addition of H2O2, normal human serum complement and the inactive HTRA1 mutant HTRA1 (S328A), the addition of H2O2, normal human serum complement and HTRA1 significantly increased the mRNA level of VEGF. It was confirmed that the co-addition of HTRA1-inhibitory peptide H308_D1G_S16A inhibited the expression of VEGF. VEGF induction from retinal pigment epithelial cells is reported to be important for the pathogenesis of wet age-related macular degeneration (Klettner A. et al., (2009) Graefes Arch Clin Exp Ophthalmol., Vol. 247: p. 1487-1492). In addition, it is believed that this pathological VEGF induction is not only involved in the pathogenesis, but also in maintaining the pathological condition. Therefore, the administration of the peptides of the present invention, such as HTRA1-inhibitory peptide H308_D1G_S16A, is effective for preventing and treating wet age-related macular degeneration.

[0518] Example 13. Inhibitory effect of HTRA1-inhibitory peptide H308_D1G_S16A on migration of human umbilical vein endothelial cells (HUVEC)

[0519] (13-1) HUVEC migration test

[0520] HUVEC (Kurabo Industries Ltd.) were cultured for 18 hours at 37°C and 5% CO2 in EBM(TM)-2 basal medium (LonzaWalkersville, Inc.) containing 0.1% BSA supplemented with EGM(TM)-2 SingleQuots(TM) supplement factor set (serum-free EGM containing 0.1% BSA) in addition to serum and VEGF. The cells were then adjusted to 4 × 10 5 cells / mL. 4 × 10 5 The HUVEC suspension of cells / mL was added to the upper layer of the chamber of a Corning FluoroBlok HTS 96-well porous permeability support system with a 3.0 μm high-density PET film (Corning Inc.) with a gelatin-coated membrane at 50 μL / well. Then, each sample described below (culture medium 1, 2, or 3) was added to the lower layer of the chamber at 210 μL / well (n = 3). Without adding HUVEC, serum-free EGM containing 0.1% BSA was added to the upper layer of the chamber at 50 μL / well, and serum-free EGM containing 0.1% BSA was added to the lower layer of the chamber at 210 μL / well (n = 3).

[0521] Medium 1: serum-free EGM containing 0.1% BSA

[0522] Medium 2; EBM(TM)-2 medium supplemented with all EGM(TM)-2 SingleQuots(TM) supplemental factors (EGM growth medium)

[0523] Medium 3; EGM growth medium containing 300 nM H308_D1G_S16A

[0524] FluoroBlok HTS 96-well multi-well support system supplemented with cells and samples was incubated for 2 hours at 37°C and 5% CO2. HUVEC migrated to the lower layer were washed with PBS and then stained with serum-free EGM (containing 4 μg / mL calcein-AM (Thermo Fisher Scientific Inc.) containing 0.1% BSA) for 15 minutes. Then, the culture medium was replaced with PBS. The fluorescence intensity (excitation wavelength / fluorescence wavelength: 485 nm / 535 nm) of each well was measured using a plate reader (ARVO-MX, PerkinElmer, Inc.), and the migrated cells were counted for each well according to the following expression.

[0525] Migrated cells = mean fluorescence intensity of wells containing HUVECs (n = 3) - mean fluorescence intensity of blank wells (n = 3).

[0526] Results are shown in Fig.75 It was confirmed that the HTRA1-inhibitory peptide H308_D1G_S16A has an inhibitory effect on the migration of HUVEC induced in a serum-containing medium. Therefore, it was found that the peptide of the present invention exhibits an inhibitory effect on angiogenesis, a characteristic of wet age-related macular degeneration.

[0527] Example 14. Retinal protective effect of HTRA1-inhibitory peptide in a rabbit retinal injury model (Part 2)

[0528] The rabbit retinal injury model prepared and evaluated in Examples (11-1) to (11-3) was used to evaluate the therapeutic effect of the HTRA1-inhibitory peptide H308 prepared in Example 1 or one of the three HTRA1-inhibitory peptides prepared in Example 6 on retinal injury. 50 μL of a 40 mg / mL inhibitory peptide solution was administered intravitreally to one eye of each model animal under anesthesia, and the animals were housed for 2 months. Physiological saline was administered intravitreally to the other eye. For all groups, n = 5.

[0529] In the physiological saline application group, the increase of RPE cell area or the increase of RPE cell count should be found, while in the HTRA1-inhibitory peptide application group, the increase of RPE cell area or the increase of RPE cell count should be suppressed. Therefore, it can be confirmed that the HTRA1-inhibitory peptide can be used as an anti-aging related macular degeneration agent. In the present embodiment, it can be confirmed that the HTRA1-inhibitory peptide is particularly useful for treating dry age-related macular degeneration.

[0530] Example 15. Study on the protective effect of HTRA1-inhibitory peptide on photoreceptor cells in Rd10 retinitis pigmentosa model mice

[0531] (15-1) Feeding of Rd10 mice

[0532] B6. CXB1-Pde6brd10 / J (hereinafter referred to as Rd10) mice have a mutation in the Pde6b gene and are a model of spontaneously induced photoreceptor cell death. It is widely used as a model animal for retinitis pigmentosa (Chang B1, Hawes NL, Hurd RE, Davisson MT, Nusinowitz S, and Heckenlivery JR., Vision Res., February 2002, 42 (No. 4): p.517-25). Mice were raised under normal feeding conditions.

[0533] (15-2) Administration of test substances to the vitreous of mice on postnatal days 14 and 19

[0534] On P14 and 19, 0.5 µL of liquid drug was administered intravitreally using a 33G needle under ketamine anesthesia. PBS was administered to one eye and HTRA1-inhibitory peptide (H308_D1G_S16A: SEQ ID NO: 24, Fig.36 ).

[0535] (15-3) Assessment and analysis of photoreceptor cell death

[0536] After euthanasia, the eyeball was excised and fixed by immersing it in 3.7% (W / V) formaldehyde-0.5-1% (W / V) methanol-0.2% (W / V) picric acid fixative for 24 hours or longer. After paraffin embedding, thin slices were prepared. The photoreceptor cell protective effect was assessed by staining the slices with hematoxylin-eosin and correcting the thickness of the outer nuclear layer (ONL layer) of the retinal slices with the thickness of the inner nuclear layer (INL layer). The two points at 0.6 mm from the mastoid and on either side of the mastoid were measured as the central part, and the two points at 1.8 mm from the mastoid and on either side of the mastoid were measured as the peripheral part, and then the mean value of the two central parts and the mean value of the two peripheral parts were calculated. Student's paired t-test method was used for statistical analysis.

[0537] (15-4) Results

[0538] The thickness of the outer nuclear layer (where the nuclei of photoreceptor cells are concentrated) of the eyes to which 160 μg / eye of the HTRA1-inhibitory peptide was administered in the central part was statistically significantly increased compared to the eyes to which PBS was administered. Similar drug effects were not observed in the eyes to which 32 μg / eye was administered, confirming dose dependence ( Fig.78 ).

[0539] Example 16 Analysis of HTRA1 Expression in Mutant Rhodopsin Gene [P347L]-Introduced Rabbits

[0540] (16-1) Analysis of HTRA1 mRNA expression in rabbit retina

[0541] RNA was extracted from the retina of male 15-week-old wild-type (WT) and mutant rhodopsin gene [P347L]-introduced (Tg) rabbits using RNeasy Mini kit (QIAGEN) and reverse transcribed into cDNA using High Capacity cDNA Reverse Transcription Kit (ThermoFisher). Then, the mRNA expression of HTRA1 was analyzed using TaqMan probe (Thermo Fisher) and TaqMan gene expression assay (Thermo Fisher).

[0542] (16-2) Site-specific analysis of HTRA1 mRNA expression using laser microdissection

[0543] The retina of 24-week-old WT and Tg rabbits of males was fixed with 10% neutral buffered formalin. Paraffin-embedded samples were prepared and then sliced ​​and stained with Nissl. LMD6500 (Leica) was used to cut the ganglion cell layer, inner nuclear layer and outer nuclear layer from the stained sections, and RNA was extracted using QIAGEN RNeasy FFPE Mini Kit (QIAGEN). Then, SMARTer Stranded Total RNA-Seq Kit-Pico Input Mammalian (Clontech) was used to create libraries for each. Nextseq (Illumina) was used to comprehensively analyze the sequence of the library. By analyzing the marker genes specifically expressed in each layer, it was confirmed that there was no problem with excision. The copy number of HTRA1 mRNA was calculated as TPM (transcripts per million) value.

[0544] (16-3) Results

[0545] (16-3-1)

[0546] At 15 weeks of age, the retina of Tg rabbits expressed about 1.5 times more HTRA1 mRNA than that of wild-type rabbits. In addition, photoreceptor cell degeneration occurred in the outer nuclear layer (the number was 5 in Tg rabbits and 4 in wild-type rabbits).

[0547] (16-3-2)

[0548] When site-specific analysis was performed by laser microdissection in wild-type rabbits and Tg rabbits at 24 weeks of age, the TPM values ​​in (a) the ganglion cell layer were 11.76±1.47 and 9.99±3.94, (b) the inner nuclear layer were 75.79±22.02 and 54.55±13.12, and (c) the outer nuclear layer were 6.21±3.33 and 25.37±6.45, respectively (there was a statistically significant difference between the two values) (n=3 to 6).

[0549] (16-3-3)

[0550] P347L transgenic rabbits carry the same rhodopsin gene mutation as in human retinitis pigmentosa patients. Photoreceptor cell death was induced in the same manner as in human retinitis pigmentosa patients, and photoreceptor cell degeneration was observed at 15 to 24 weeks of age, which is the condition used here (Kondo M1, Sakai T, Komeima K, Kurimoto Y, Ueno S, Nishizawa Y, Usukura J, Fujikado T, Tano Y, and Terasaki H., Invest OphthalmolVis Sci., May 2009, Vol. 50 (No. 3): p. 1371-7).

[0551] The outer nuclear layer is the site where the nuclei of photoreceptor cells accumulate, and a correlation was observed between photoreceptor cell degeneration and increased HTRA1 gene expression, which, if considered in conjunction with the other disclosures of the present invention, suggests that it would be a useful therapeutic agent for retinitis pigmentosa with rhodopsin gene mutations, such as P347L.

[0552] Industrial Applicability

[0553] The peptide or its conjugate provided by the present invention and the pharmaceutical composition comprising the peptide or its conjugate can be used for the treatment or prevention of retinitis pigmentosa and the like.

[0554] Sequence Listing Free Text

[0555] SEQ ID NO:1 - Amino acid sequence of human SPINK2 ( Fig.13 )

[0556] SEQ ID NO:2 - Nucleotide sequence encoding the amino acid sequence of human SPINK2 ( Fig.14 )

[0557] SEQ ID NO:3 - Amino acid sequence of peptide H218 ( Fig.15 )

[0558] SEQ ID NO:4 - Nucleotide sequence encoding the amino acid sequence of peptide H218 ( Fig.16 )

[0559] SEQ ID NO:5 - Amino acid sequence of peptide H223 ( Fig.17 )

[0560] SEQ ID NO:6 - Nucleotide sequence encoding the amino acid sequence of peptide H223 ( Fig.18 )

[0561] SEQ ID NO:7 - Amino acid sequence of peptide H228 ( Fig.19 )

[0562] SEQ ID NO:8 - Nucleotide sequence encoding the amino acid sequence of peptide H228 ( Fig. 20 )

[0563] SEQ ID NO:9 - Amino acid sequence of peptide H308 ( Fig.21 )

[0564] SEQ ID NO:10 - Nucleotide sequence encoding the amino acid sequence of peptide H308 ( Fig. 22 )

[0565] SEQ ID NO:11 - Amino acid sequence of peptide H321 ( Fig.23 )

[0566] SEQ ID NO:12 - Nucleotide sequence encoding the amino acid sequence of peptide H321 ( Fig.24 )

[0567] SEQ ID NO:13 - Amino acid sequence of peptide H322 ( Fig.25 )

[0568] SEQ ID NO:14 - Nucleotide sequence encoding the amino acid sequence of peptide H322 ( Fig.26 )

[0569] SEQ ID NO:15 - Amino acid sequence of peptide derivative H308AT ( Fig. 27 )

[0570] SEQ ID NO:16 - Nucleotide sequence encoding the amino acid sequence of the peptide derivative H308AT ( Fig.28 )

[0571] SEQ ID NO:17 - Amino acid sequence of peptide derivative H321AT ( Fig.29 )

[0572] SEQ ID NO:18 - Nucleotide sequence encoding the amino acid sequence of the peptide derivative H321AT ( Fig.30 )

[0573] SEQ ID NO:19 - Amino acid sequence of peptide derivative H322AT ( Fig.31 )

[0574] SEQ ID NO:20 - Nucleotide sequence encoding the amino acid sequence of the peptide derivative H322AT ( Fig.32 )

[0575] SEQ ID NO:21 - Amino acid sequence of peptide M7 ( Fig.33 )

[0576] SEQ ID NO:22 - Nucleotide sequence encoding the amino acid sequence of peptide M7 ( Fig.34 )

[0577] SEQ ID NO:23 - Amino acid sequence of peptide derivative H308_S16A ( Fig.35 )

[0578] SEQ ID NO:24 - Amino acid sequence of peptide derivative H308_D1G_S16A ( Fig.36 )

[0579] SEQ ID NO:25 - Amino acid sequence of peptide derivative H308_D1S_S16A ( Fig.37 )

[0580] SEQ ID NO:26 - Amino acid sequence of peptide derivative H308_D1E_S16A ( Fig.38 )

[0581] SEQ ID NO:27 - Amino acid sequence of peptide derivative H308_D1SLI_S16A ( Fig.39 )

[0582] SEQ ID NO:28 - Amino acid sequence of peptide derivative H321AT_D1G_S16A ( Fig.40 )

[0583] SEQ ID NO:29 - Amino acid sequence of peptide derivative H322AT_D1G_S16A ( Fig.41 )

[0584] SEQ ID NO:30 - General formula of HTRA1-inhibitory peptide ( Fig.42 )

[0585] SEQ ID NO:31 - Amino acid sequence consisting of an S tag and a linker ( Fig.43 )

[0586] SEQ ID NO:32 - Amino acid sequence of the C-terminal hexamer ( Fig.44 )

[0587] SEQ ID NO:33 - Nucleotide sequence of primer 1 ( Fig.45 )

[0588] SEQ ID NO:34 - Nucleotide sequence of primer 2 ( Fig.46 )

[0589] SEQ ID NO:35 - Nucleotide sequence of primer 3 ( Fig.47 )

[0590] SEQ ID NO:36 - Nucleotide sequence of primer 4 ( Fig.48 )

[0591] SEQ ID NO:37 - Nucleotide sequence of primer 5 ( Fig.49 )

[0592] SEQ ID NO:38 - Nucleotide sequence of primer 6 ( Fig.50 )

[0593] SEQ ID NO:39 - Nucleotide sequence of primer 7 ( Fig.51 )

[0594] SEQ ID NO:40 - Nucleotide sequence of primer 8 ( Fig.52 )

[0595] SEQ ID NO:41 - Nucleotide sequence of primer 9 ( Fig.53 )

[0596] SEQ ID NO:42 - Nucleotide sequence of primer 10 ( Fig.54 )

[0597] SEQ ID NO:43 - Nucleotide sequence of primer 11 ( Fig.55 )

[0598] SEQ ID NO:44 - Nucleotide sequence of primer 12 ( Fig.56 )

[0599] SEQ ID NO:45 - Nucleotide sequence of primer 13 ( Fig.57 )

[0600] SEQ ID NO:46 - Nucleotide sequence of primer 14 ( Fig.58 )

[0601] SEQ ID NO:47 - Nucleotide sequence of primer 15 ( Fig.59 )

[0602] SEQ ID NO:48 - Nucleotide sequence of primer 16 ( Fig.60 )

[0603] SEQ ID NO:49 - Nucleotide sequence of primer 17 ( Fig.61 )

[0604] SEQ ID NO:50 - Nucleotide sequence of primer 18 ( Fig.62 )

[0605] SEQ ID NO:51 - Nucleotide sequence of primer 19 ( Fig.63 )

[0606] SEQ ID NO:52 - Nucleotide sequence of primer 20 ( Fig.64 )

[0607] SEQ ID NO:53 - Amino acid sequence of human HTRA1 (full length) ( Fig.65 )

[0608] SEQ ID NO:54 - Amino acid sequence of H2-Opt ( Figure 8 )

[0609] SEQ ID NO:55 - Nucleotide sequence of primer 21 ( Fig.76 )

[0610] SEQ ID NO:56 - Nucleotide sequence of primer 22 ( Fig.77 ).

Claims

1. Use of a pharmaceutical composition for preparing a medicament for treating or preventing retinitis pigmentosa, a hereditary disease involving photoreceptor cell degeneration and / or a disease involving photoreceptor cell degeneration caused by PDE6 protein dysfunction, the pharmaceutical composition comprising a SPINK2 mutant peptide consisting of an amino acid sequence as shown in any one of SEQ ID NOs: 24, 29, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23 and 25 to 28, And wherein the peptide inhibits the protease activity of human HTRA1.

2. Use of the pharmaceutical composition according to claim 1, wherein the peptide has a three-dimensional structure characterized by having three disulfide bonds and comprising a loop structure, an α-helix and a β-sheet.

3. Use of the pharmaceutical composition according to claim 1, wherein the drug comprises one or two or more additional drugs.

4. Use of a pharmaceutical composition for preparing a medicament for treating or preventing retinitis pigmentosa, a hereditary disease involving photoreceptor cell degeneration and / or a disease involving photoreceptor cell degeneration caused by dysfunction of the PDE6 protein, the pharmaceutical composition comprising a polynucleotide comprising a nucleotide sequence encoding the amino acid sequence contained in the peptide according to claim 1 or 2.

5. Use of a pharmaceutical composition for the preparation of a medicament for treating or preventing retinitis pigmentosa, a genetic disease involving photoreceptor cell degeneration and / or a disease involving photoreceptor cell degeneration caused by dysfunction of the PDE6 protein, the pharmaceutical composition comprising a vector comprising a nucleotide sequence encoding the amino acid sequence contained in the peptide according to claim 1 or 2.

6. Use of a pharmaceutical composition for preparing a medicament for treating or preventing retinitis pigmentosa, a hereditary disease involving photoreceptor cell degeneration and / or a disease involving photoreceptor cell degeneration caused by dysfunction of the PDE6 protein, the pharmaceutical composition comprising: a cell containing a polynucleotide comprising a nucleotide sequence encoding the amino acid sequence contained in the peptide according to claim 1 or 2, or a vector comprising the nucleotide sequence, or a cell producing the peptide according to claim 1 or 2.

7. Use of a pharmaceutical composition for the preparation of a medicament for the treatment or prevention of retinitis pigmentosa, a genetic disease involving photoreceptor cell degeneration and / or a disease involving photoreceptor cell degeneration caused by dysfunction of the PDE6 protein, the pharmaceutical composition comprising a conjugate comprising a peptide according to claim 1 or 2 linked to an additional part.

8. Use of the pharmaceutical composition according to claim 7, wherein the conjugate is a polypeptide.

9. Use of the pharmaceutical composition according to claim 1 in the preparation of a medicament for treating or preventing retinitis pigmentosa.

10. Use of the pharmaceutical composition according to claim 1 in the preparation of a medicament for treating or preventing a genetic disease involving photoreceptor cell degeneration.

11. The use of the pharmaceutical composition according to claim 10, wherein the hereditary disease involving photoreceptor cell degeneration is macular dystrophy.

12. Use of the pharmaceutical composition according to claim 1 in the preparation of a medicament for treating or preventing a disease involving photoreceptor cell degeneration caused by PDE6 protein dysfunction.

13. The use of the pharmaceutical composition according to claim 12, wherein the disease involving photoreceptor cell degeneration caused by PDE6 protein dysfunction is color blindness or autosomal dominant congenital stationary night blindness.

14. Use of the pharmaceutical composition according to claim 4 in the preparation of a medicament for treating or preventing retinitis pigmentosa.

15. Use of the pharmaceutical composition according to claim 4 in the preparation of a medicament for treating or preventing a genetic disease involving photoreceptor cell degeneration.

16. Use of the pharmaceutical composition according to claim 15, wherein the hereditary disease involving photoreceptor cell degeneration is macular dystrophy.

17. Use of the pharmaceutical composition according to claim 4 in the preparation of a medicament for treating or preventing a disease involving photoreceptor cell degeneration caused by PDE6 protein dysfunction.

18. Use of the pharmaceutical composition according to claim 17, wherein the disease involving photoreceptor cell degeneration caused by PDE6 protein dysfunction is color blindness or autosomal dominant congenital stationary night blindness.

19. Use of the pharmaceutical composition according to claim 5 in the preparation of a medicament for treating or preventing retinitis pigmentosa.

20. Use of the pharmaceutical composition according to claim 5 in the preparation of a medicament for treating or preventing a genetic disease involving photoreceptor cell degeneration.

21. The use of the pharmaceutical composition according to claim 20, wherein the hereditary disease involving photoreceptor cell degeneration is macular dystrophy.

22. Use of the pharmaceutical composition according to claim 5 in the preparation of a medicament for treating or preventing a disease involving photoreceptor cell degeneration caused by PDE6 protein dysfunction.

23. The use of the pharmaceutical composition according to claim 22, wherein the disease involving photoreceptor cell degeneration caused by PDE6 protein dysfunction is color blindness or autosomal dominant congenital stationary night blindness.

24. Use of the pharmaceutical composition according to claim 6 in the preparation of a medicament for treating or preventing retinitis pigmentosa.

25. Use of the pharmaceutical composition according to claim 6 in the preparation of a medicament for treating or preventing a genetic disease involving photoreceptor cell degeneration.

26. Use of the pharmaceutical composition according to claim 25, wherein the inherited disease involving photoreceptor cell degeneration is macular dystrophy.

27. Use of the pharmaceutical composition according to claim 6 in the preparation of a medicament for treating or preventing a disease involving photoreceptor cell degeneration caused by PDE6 protein dysfunction.

28. Use of the pharmaceutical composition according to claim 27, wherein the disease involving photoreceptor cell degeneration caused by PDE6 protein dysfunction is color blindness or autosomal dominant congenital stationary night blindness.

29. Use of the pharmaceutical composition according to claim 7 in the preparation of a medicament for treating or preventing retinitis pigmentosa.

30. Use of the pharmaceutical composition according to claim 7 in the preparation of a medicament for treating or preventing a genetic disease involving photoreceptor cell degeneration.

31. Use of the pharmaceutical composition according to claim 30, wherein the inherited disease involving photoreceptor cell degeneration is macular dystrophy.

32. Use of the pharmaceutical composition according to claim 7 in the preparation of a medicament for treating or preventing a disease involving photoreceptor cell degeneration caused by PDE6 protein dysfunction.

33. The use of the pharmaceutical composition according to claim 32, wherein the disease involving photoreceptor cell degeneration caused by PDE6 protein dysfunction is color blindness or autosomal dominant congenital stationary night blindness.

34. The use according to any one of claims 1, 2 and 4 to 33, wherein the drug is used in combination with one or two or more additional drugs.

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