WO2018117244A1 - 加齢黄斑変性症治療用ペプチド - Google Patents
加齢黄斑変性症治療用ペプチド Download PDFInfo
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- WO2018117244A1 WO2018117244A1 PCT/JP2017/046044 JP2017046044W WO2018117244A1 WO 2018117244 A1 WO2018117244 A1 WO 2018117244A1 JP 2017046044 W JP2017046044 W JP 2017046044W WO 2018117244 A1 WO2018117244 A1 WO 2018117244A1
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Definitions
- the present invention relates to peptides, polynucleotides, vectors, cells, methods for producing peptides, peptides obtained by such methods, compositions containing peptides, pharmaceutical compositions containing peptides, and treatment or prevention of various diseases containing peptides.
- the present invention relates to a pharmaceutical composition, use of a peptide for treating or preventing various diseases, a method for treating various diseases including a step of administering a peptide, and the like.
- HTRA1 is a trypsin-like serine protease (PRSS11; Clan PA, family S1), an N-terminal domain consisting of an IGFBP-like module and a Kazal-like module, a protease domain and a C-terminal PD Is done.
- HTRA1 belongs to the HTRA family including HTRA2, HTRA3, and HTRA4, and reversibly shows active and inactive structures like other HTRA molecules (Non-patent Documents 1 and 2). Its expression is ubiquitous in the human body, and relatively high expression is observed in cartilage, synovium, placenta and the like.
- HTRA1 is known to cleave many extracellular matrix components such as Amyloid precursor protein, Fibromodulin, Clusterin, ADAM9, and Vitronectin as substrates, and to be associated with diseases typified by arthritis and bone mineralization (non-patent literature) 3, 4, 5, 6). Furthermore, it is known that when the HTRA1 promoter region has a gene polymorphism (rs11200638), the amount of HTRA1 transcription increases, and the polymorphism and age-related macular degeneration (hereinafter referred to as AMD). ”) Is strongly correlated with genome-wide association analysis (Non-patent Documents 7 and 8). AMD is a chronic degenerative disease associated with aging and is characterized by decreased central vision. The number of patients with acquired blindness is No.
- Non-patent Document 12 HTRA11 is elevated in mRNA and protein levels in lymphocytes or retinal pigment epithelial cells of AMD patients (Non-patent Document 13). In addition, it has been reported that the expression of HTRA1 protein is elevated in drusen, which is a precursor lesion of AMD, degenerated retinal pigment epithelial cells, or neovascular membranes (Non-patent Documents 11 and 14 to 16).
- HTRA1 protein has been detected in vitreous humor such as retinal detachment, retinal vein occlusion, vitreous hemorrhage and macular hole, and there is a report that the value is linked to VEGF as an angiogenesis marker. (Non-patent document 17). Furthermore, in HTRA1 transgenic mice, degradation of proteins constituting the basement membrane such as Fibulin 5 and Tropoelastin and fragmentation of Bruch's membrane Elastic layer have been observed (Non-patent Document 9). However, there is no direct indication as to whether inhibition of protease activity of HTRA1 is effective for treating the above diseases as well as for protecting the retina.
- SPINK2 Serine Protease Inhibitor Kazal-type 2
- SPINK2 Serine Protease Inhibitor Kazal-type 2
- RPE cells retinal pigment epithelial cells
- HTRA1 serine peptidase 1
- the present invention is (1) A SPINK2 mutant peptide comprising the amino acid sequence shown by SEQ ID NO: 30 (FIG. 42) and inhibiting the protease activity of human HTRA1; (2)
- the first Xaa (X 1) is Asp, Glu, Ser, Gly, or Ile
- 2 th Xaa (X 2) is Ala, Gly, Leu, Ser or Thr
- 3-th Xaa (X 3) is Asp, His, Lys, Met or Gln
- 4th Xaa (X 4 ) is Asp, Phe, His, Ser or Tyr
- 5th Xaa (X 5 ) is Ala, Asp, Glu, Met or Asn
- 6th Xaa (X 6 ) is Met or Trp
- 7th Xaa (X 7 ) is Gln, Trp, Tyr or Val
- 8th Xaa (X 8 ) is Phe, Leu or Tyr
- the peptide according to (1) or (2) comprising the amino acid sequence shown in any one of FIG. 31, FIG. 33 and FIGS. (4)
- the peptide according to any one of (1) to (3) comprising an amino acid sequence in which 1 to 3 amino acids are peptide-bonded to the amino terminal side of the amino acid sequence represented by SEQ ID NO: 30 (FIG. 42).
- the peptide according to any one of (1) to (4) comprising an amino acid sequence in which one or two amino acids are peptide-bonded to the carboxyl terminal side of the amino acid sequence represented by SEQ ID NO: 30 (FIG. 42).
- peptide according to any one of (1) to (5) having a three-dimensional structure characterized by having three disulfide bonds and including a loop structure, an ⁇ helix, and a ⁇ sheet; (7) (1) A polynucleotide comprising a nucleotide sequence encoding an amino acid sequence contained in the peptide according to any one of (6), (8) (7) a vector comprising the polynucleotide according to claim, (9) A cell comprising the polynucleotide according to (7) or the vector according to (8) or producing the peptide according to any one of (1) to (6), (10) A method for producing a SPINK2 mutant peptide that inhibits the protease activity of HTRA1, comprising the following steps (i) and (ii): (I) a step of culturing the cell according to (9); (Ii) recovering the SPINK2 mutant peptide from the culture, (11) (10) SPINK2 mutant peptide obtained by the method described
- the pharmaceutical composition according to (16) for the treatment or prevention of an HTRA1-related disease (18) A group in which HTRA1-related diseases are wet age-related macular degeneration, atrophic age-related macular degeneration, map-like atrophy, diabetic retinopathy, retinopathy of prematurity, polypoidal choroidal vasculopathy, rheumatoid arthritis, and osteoarthritis
- a method for identifying a retinal protective agent comprising the following steps 1 to 3: [Step 1] Incubate the HTRA1 protease and the substrate in the presence or absence of the test substance; [Step 2] Detect HTRA1 protease activity in the presence and absence of the test substance; [Step 3] If the HTRA1 protease activity in the presence of the test substance is small compared to the HTRA1 protease activity in the absence of the test substance, the test substance is determined to be positive.
- a method for producing a retinal dysfunction model rabbit comprising a step of feeding a high fat diet containing hydroquinone for 3 to 6 months, wherein the retinal pigment epithelial cells of the model rabbit are enlarged compared to retinal pigment epithelial cells of a normal rabbit
- a method for identifying a therapeutic or prophylactic agent for age-related macular degeneration, or a retinal protective agent comprising the following steps (i) and (ii): (I) a step of measuring retinal pigment epithelial cell hypertrophy in the rabbit described in (25) with and without administration of a test substance; and (ii) retinal pigment epithelial cell hypertrophy under administration of the test substance.
- a step of determining that the test substance is positive when it is suppressed as compared with non-administration (27) The method according to (26), wherein the measurement in step (i) is a measurement of the average area of retinal pigment epithelial cells or the number of enlarged retinal pigment epithelial cells, (28) The pharmaceutical composition according to any one of (16) to (18), for the treatment or prevention of atrophic age-related macular degeneration, and (29) The pharmaceutical composition according to any one of (16) to (18), for treating or preventing wet age-related macular degeneration, Etc.
- the peptide provided by the present invention and a pharmaceutical composition containing the peptide have HTRA1 inhibitory activity and are useful for the treatment or prevention of age-related macular degeneration.
- the dashed line indicates the enzyme active domain (204Gly-364Leu). Comparison of human / mouse / rat / monkey HTRA1 sequence similarity (continued).
- index panel A thru
- the figure which evaluated the HTRA1 (cat) inhibitory activity of the HTRA1 inhibitory peptide using degradation of human Vitronectin as an index The analysis was performed by Western blot using Human Vitronectin Antibody (R & D Systems; MAB2349).
- the figure which evaluated the HTRA1 (cat) inhibitory activity of the HTRA1 inhibitory peptide using degradation of human Vitronectin as an index (continuation).
- index the 1). See Example 3 for the names and concentrations of each protease used, and the names and concentrations of the substrates.
- index (the 2).
- index (the 3).
- index the 4
- HTRA1 trimer formed by HTRA1 (cat).
- the inhibitory peptide was bound to a region containing the HTRA1 (cat) active center.
- Amino acid sequence of H2-Opt SEQ ID NO: 54).
- N-terminal “Mca-I” is N- (4-methylcoumeric-7-amide) -isoleucine
- C-terminal “(Dnp) K” is N epsilon- (2,4-dinitrophenyl) -lysine, Each means.
- Amino acid sequence of human SPINK2 (SEQ ID NO: 1) Nucleotide sequence encoding the amino acid sequence of human SPINK2 (SEQ ID NO: 2) Amino acid sequence of peptide H218 (SEQ ID NO: 3) Nucleotide sequence encoding the amino acid sequence of peptide H218 (SEQ ID NO: 4) Amino acid sequence of peptide H223 (SEQ ID NO: 5) Nucleotide sequence encoding the amino acid sequence of peptide H223 (SEQ ID NO: 6) Amino acid sequence of peptide H228 (SEQ ID NO: 7) Nucleotide sequence encoding the amino acid sequence of peptide H228 (SEQ ID NO: 8) Amino acid sequence of peptide H308 (SEQ ID NO: 9) Nucleotide sequence encoding the amino acid sequence of peptide H308 (SEQ ID NO: 10) Amino acid sequence of peptide H321 (SEQ ID NO:
- X 1 to X 11 represent any amino acid.
- Amino acid sequence consisting of S tag and linker (SEQ ID NO: 31) C-terminal 6-mer amino acid sequence (SEQ ID NO: 32) Nucleotide sequence of primer 1 (SEQ ID NO: 33) Nucleotide sequence of primer 2 (SEQ ID NO: 34) Nucleotide sequence of primer 3 (SEQ ID NO: 35) Nucleotide sequence of primer 4 (SEQ ID NO: 36) Nucleotide sequence of primer 5 (SEQ ID NO: 37) Nucleotide sequence of primer 6 (SEQ ID NO: 38) Nucleotide sequence of primer 7 (SEQ ID NO: 39) Nucleotide sequence of primer 8 (SEQ ID NO: 40) Nucleotide sequence of primer 9 (SEQ ID NO: 41) Nucleotide sequence of primer 10 (SEQ ID NO: 42) Nucleotide sequence of primer 11 (SEQ ID NO: 43) Nucleotide sequence of primer 12 (SEQ ID NO:
- index The figure which evaluated the HTRA1 (cat) inhibitory activity of the HTRA1 inhibitory peptide using degradation of human Vitronectin as an index. The analysis was performed by Western blot using Human Vitronectin Antibody (R & D Systems; MAB2349). The figure which evaluated the cross property of the HTRA1 inhibitory peptide with respect to each protease using decomposition
- index the 1).
- index (the 2).
- index (the 3).
- index the 4).
- index (the 5).
- the number of cases in each group was 6, and the HTRA1 inhibitory peptide H308_D1G_S16A dosage was 0.2 and 1 ⁇ g / eye.
- the number of cases in each group was 6, and the HTRA1 inhibitory peptide H321AT_D1G_S16A dosage was 0.2 and 1 ⁇ g / eye.
- the number of cases in any group was 6, and the HTRA1 inhibitory peptide H322AT_D1G_S16A dosage was 0.2 and 1 ⁇ g / eye.
- the number of cases was 5, and the average area was used as an index.
- the number of cases was 5, and the number of RPE cells having a cell area of 1500 ⁇ m 2 or more was used as an index.
- the dashed line indicates the enzyme active domain (204Gly-364Leu).
- HTRA1 inhibitory peptide showed an inhibitory effect FIG.
- FIG. The figure which showed the inhibitory effect of the HTRA1 inhibitory peptide with respect to the migration of the human umbilical vein endothelial cells (HUVEC) induced by serum.
- Nucleotide sequence of primer 21 Figure 66
- Nucleotide sequence of primer 22 FIG. 67
- “gene” means a nucleic acid molecule comprising a nucleotide sequence that encodes an amino acid sequence contained in a protein or a complementary strand thereof, consisting of a single strand, a double strand, a triple strand or more, and a DNA
- the term “gene” also includes an assembly of strands and RNA strands, a mixture of ribonucleotides and deoxyribonucleotides on one strand, and a double-stranded or triple-stranded nucleic acid molecule containing such a strand.
- nucleic acid molecule is synonymous, and are not limited at all depending on the number of ribonucleotides, deoxyribonucleotides, nucleotides, nucleosides, etc., which are their constituent units. RNA, mRNA, cDNA, cRNA, probes, oligonucleotides, primers and the like are also included in the scope. “Nucleic acid molecule” is sometimes referred to as “nucleic acid” for short.
- polypeptide In the present invention, “polypeptide”, “peptide” and “protein” are synonymous.
- a peptide that inhibits or suppresses one or more activities or functions of the target molecule X (hereinafter, the inhibition or suppression action is collectively referred to as “X inhibitory activity”) is referred to as “X inhibitory peptide”. be able to.
- SPINK2 means Serine Protease Inhibitor Kazal-type 2 and is a 7 kDa protein composed of a Kazal-like domain having three disulfide bonds.
- Preferred SPINK2 is human.
- human SPINK2 is simply referred to as “SPINK2” unless otherwise specified.
- HTRA1 means high temperature requirement A serine peptidase 1 and is a protein belonging to the HTRA family composed of an N-terminal domain, a protease domain, and a C-terminal PDZ domain consisting of an IGFBP-like module and a module for Kazal. Preferred HTRA1 is human. In the present invention, human HTRA1 may be simply referred to as “HTRA1” unless otherwise specified.
- HTRA1 inhibitory peptide means a peptide that inhibits or suppresses one or more activities or functions of HTRA1.
- the range of “HTRA1 inhibitory peptide” includes fragments of the peptide, adducts of other moieties (moiety), or conjugates that maintain HTRA1 inhibitory activity. That is, fragments, adducts, and modifications of the peptide that maintain HTRA1 inhibitory activity are also included in the “HTRA1 inhibitory peptide”.
- cells include various cells derived from individual animals, subculture cells, primary culture cells, cell lines, recombinant cells, yeast, microorganisms, and the like.
- the “site” to which the peptide binds that is, the “site” recognized by the peptide means a continuous or intermittent partial amino acid sequence or partial higher order structure on the target molecule to which the peptide binds or recognizes. .
- such a site can be referred to as an epitope or binding site on the target molecule.
- a “SPINK2 mutant” is an amino acid sequence possessed by wild-type SPINK2 in which one or more amino acids are replaced with amino acids different from the wild-type, and one or more wild-type amino acids are deleted. 1 or 2 or more amino acids not present in the wild type are inserted and / or amino acids not present in the wild type are added to the amino terminus (N terminus) and / or the carboxyl terminus (C terminus) of the wild type. (Hereinafter collectively referred to as “mutation”).
- mutation those having HTRA1 inhibitory activity are encompassed by HTRA1 inhibitory peptides.
- “insertion” can also be included in the range of “addition”.
- “several” in “1 to several” refers to 3 to 10.
- hybridize under stringent conditions means hybridization at 65 ° C. in a solution containing 5 ⁇ SSC, and then in an aqueous solution containing 2 ⁇ SSC-0.1% SDS. 20 minutes at 65 ° C. in an aqueous solution containing 0.5 ⁇ SSC-0.1% SDS for 20 minutes at 65 ° C. and 65 ° C. in an aqueous solution containing 0.2 ⁇ SSC-0.1% SDS Means to hybridize under the conditions of washing for 20 minutes or under equivalent conditions.
- SSC is an aqueous solution of 150 mM NaCl-15 mM sodium citrate, and nx SSC means n-fold concentration of SSC.
- an HTRA1-specific inhibitory peptide is synonymous with an HTRA1-selective inhibitory peptide.
- amino acid is an organic compound containing an amino group and a carboxyl group, and preferably means an ⁇ -amino acid contained as a structural unit in a protein, more preferably in a natural protein.
- more preferred amino acids are Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr and Val. Yes, unless otherwise specified, “amino acid” means a total of 20 amino acids. These 20 amino acids in total can be referred to as “natural amino acids”.
- the HTRA1 inhibitory peptides of the present invention preferably contain natural amino acids.
- amino acid residue may be abbreviated as “amino acid”.
- the amino acid is an L-amino acid, a D-amino acid, or a mixture thereof (DL-amino acid), and unless otherwise specified, means an L-amino acid.
- Natural amino acids can be divided, for example, into the following groups based on their common side chain properties.
- Hydrophobic amino acid group Met, Ala, Val, Leu, Ile
- Neutral hydrophilic amino acid group Cys, Ser, Thr, Asn, Gln
- Acidic amino acid group Asp, Glu
- Basic amino acid group His, Lys, Arg
- Amino acid groups that affect the direction of the main chain Gly, Pro
- Aromatic amino acid group Trp, Tyr, Phe
- the classification of natural amino acids is not limited to these.
- natural amino acids can undergo conservative amino acid substitutions.
- Constant amino acid substitution means a functionally equivalent or similar amino acid substitution.
- a conservative amino acid substitution in a peptide results in a static change in the amino acid sequence of the peptide.
- one or more amino acids with similar polarity act functionally equivalent, resulting in a static change in the amino acid sequence of such peptides.
- substitutions within a group can be considered conservative in structure and function.
- the role played by a particular amino acid residue can be determined in the context of the three-dimensional structure of the molecule containing that amino acid.
- cysteine residues can take the oxidized (disulfide) form, which is less polar compared to the reduced (thiol) form.
- the long aliphatic part of the arginine side chain can constitute structurally and functionally important features.
- side chains containing aromatic rings can contribute to ion-aromatic interactions or cation-pi interactions.
- substitution of amino acids having these side chains with amino acids belonging to acidic or non-polar groups can be structurally and functionally conservative.
- Residues such as proline, glycine, and cysteine (disulfide form) can directly affect the conformation of the main chain and often cannot be substituted without structural distortion.
- Nonpolar amino acid groups alanine (hereinafter “Ala” or simply “A”), valine (hereinafter “Val” or simply “V”), leucine (hereinafter “Leu” or simply “V”) L ”), isoleucine (hereinafter“ Ile ”or simply“ I ”), proline (hereinafter“ Pro ”or simply“ P ”), phenylalanine (“ Phe ”or simply“ F ”) ), Tryptophan (hereinafter referred to as “Trp” or simply “W”), methionine (hereinafter referred to as “Met” or simply “M”)
- Uncharged polar amino acid group glycine (hereinafter “Gly” or simply “G”), serine (hereinafter “Ser” or simply “S”), threonine (hereinafter “Thr” or simply “G”) "T”), cysteine (hereinafter “Cys” or simply “C”), tyrosine (hereinafter “Ty
- selenocysteine, N-formylmethionine, pyrrolidine, pyroglutamic acid, cystine, hydroxyproline, hydroxylysine, thyroxine, O-phosphoserine, desmosine, ⁇ -alanine, sarcosine, ornithine, creatine, ⁇ found in natural peptides and proteins Examples include aminobutyric acid, opain, theanine, tricolominic acid, kainic acid, domoic acid, acromelic acid, and the like. Protected amino acid, amino acid t-butyl ester, benzyl ester, cyclohexyl ester, fluorenyl ester, etc.
- amino acids other than the above-mentioned “natural amino acids” include, but are not limited to, the amino acids other than the above-mentioned “natural amino acids” in the present invention for convenience. Collectively referred to as “natural amino acids”.
- HTRA1 inhibitory peptide The HRTA1 inhibitory peptide of the present invention is a SPINK2 mutant in which the backbone of SPINK2 is at least partially maintained (hereinafter abbreviated as “SPINK2 mutant”), and retains HTRA1 or its enzyme activity. Protease activity of a fragment (hereinafter referred to as “functional fragment”) is inhibited or suppressed (hereinafter, such inhibition or suppression is collectively referred to as “HTRA1 inhibitory activity”).
- HTRA1 which is the target of the inhibitory peptides of the present invention, is preferably mammalian, more preferably primate, and even more preferably human HTRA1, full-length mature human HTRA1 (hereinafter “HTRA1 (full)”).
- HTRA1 (full) has the amino acid sequence shown by SEQ ID NO: 53 (FIG. 65).
- the amino acid sequence consists of numbers 23 to 480 and does not include a signal sequence consisting of numbers 1 to 22.
- the amino acid sequence of a functional fragment of human HTRA1 (hereinafter referred to as “HTRA1 (cat)”) is not particularly limited as long as the protease activity is retained, but the 158th Gly to 373th of SEQ ID NO: 53 (FIG. 65).
- HTRA1 and its functional fragments which are targets of the inhibitory peptides of the present invention, are also referred to as HTRA1 protease, preferably derived from vertebrates, more preferably mammals, even more preferably primates, optimally humans, It can be purified from those tissues and cells, or can be prepared by methods known to those skilled in the art as protein preparation methods such as gene recombination, in vitro translation, and peptide synthesis.
- a signal sequence, an immunoglobulin Fc region, a tag, a label, and the like may be linked to HTRA1 and functional fragments thereof.
- the HTRA1 inhibitory activity can be evaluated using the protease activity of HTRA1 as an index. For example, when HTRA1 or a functional fragment thereof, a substrate, and the inhibitory peptide of the present invention or a candidate thereof are present together, the protease activity of HTRA1 is higher than that in the presence of a control or in the absence of the inhibitor or the candidate. When it is 70% or less, 50% or less, 30% or less, 20% or less, 10% or less, 5% or less, 1% or less, or 0%, HTRA1 inhibition has occurred, and its inhibitory activity is 30% or more, It is 50% or more, 70% or more, 80% or more, 90% or more, 95% or more, 99% or more, or 100%.
- HTRA1 inhibitory activity may vary depending on the reaction conditions, the type and concentration of the substrate, and the like. Examples of the reaction conditions include those described in the examples, but are not limited thereto.
- the fluorescence of the substrate peptide is detected, or the substrate protein is detected by SDS-PAGE, Western blotting, liquid chromatography, etc.
- the enzyme activity can be evaluated.
- buffer solution examples include phosphate buffer saline (hereinafter referred to as “PBS”), borate buffer (50 mM boric acid, pH 7 to 9, for example, pH 8.5), Tris buffer (50 mM Tris, pH 6 to 9 (for example, pH 8.0) can be used, and surfactants such as NaCl (50 to 300 mM, for example 150 mM), CHAPS, and Octyl ⁇ -D-glucopyranoside can be added, but are not limited thereto.
- PBS phosphate buffer saline
- borate buffer 50 mM boric acid, pH 7 to 9, for example, pH 8.5
- Tris buffer 50 mM Tris, pH 6 to 9 (for example, pH 8.0)
- surfactants such as NaCl (50 to 300 mM, for example 150 mM), CHAPS, and Octyl ⁇ -D-glucopyranoside can be added, but are not limited thereto.
- the protease substrate of HTRA1 is not particularly limited, such as an endogenous substrate, an exogenous substrate, or a synthetic substrate.
- An example of a human endogenous substrate is vitronectin.
- the synthetic substrate is not particularly limited, and examples thereof include H2-Opt (Mca-IRRVSYSFK (Dnp) K), ⁇ -Casein, and other HTRA1 substrates.
- the HTRA1 inhibitory activity (IC 50 or K i ) of the peptide of the present invention is 1 ⁇ M or less, preferably 100 nM or less.
- the inhibitory peptide of this invention does not inhibit or suppress protease activities other than HTRA1, or the degree of inhibition or suppression with respect to them is relatively weak.
- the inhibitory peptide of the present invention preferably has high HTRA1 specificity.
- Suitable inhibitor peptides of the present invention include proteases such as trypsin, ⁇ -chymotrypsin, tryptase, plasmin, thrombin, matriptase, protein C, tissue plasminogen activator (tPA), urokinase (uPA), plasmin, plasma kallikrein and the like The activity is not inhibited or suppressed, or the degree of inhibition or suppression against them is relatively weak.
- HTRA1 which is the target of the peptides of the present invention, is derived from a vertebrate, preferably a mammal, more preferably a primate, and even more preferably a human, but a non-human animal such as a rat, It may be derived from rodents such as mice, primates such as cynomolgus monkeys, common marmosets, and rhesus monkeys.
- Peptides having inhibitory activity against non-human animal-derived HTRA1 can be used for diagnosis, examination, treatment or prevention of diseases related to HTRA1 in such non-human animals.
- a peptide also inhibits human HTRA1
- a medicinal pharmacology using such a non-human animal as an animal pathological model Tests pharmacokinetic tests, safety tests used as healthy animals, toxicity tests, etc. can be performed.
- the HTRA1 inhibitory peptide of the present invention has a small molecular weight compared to other biopolymers such as antibodies used in the art as pharmaceuticals and diagnostic agents, and its production (described later) is relatively easy. It has excellent physical properties such as penetration into the skin, storage stability, and heat stability, and has a wide range of choices for administration route, administration method, formulation, etc. when used as a pharmaceutical composition (described later). Has advantages.
- by increasing the molecular weight of the peptide of the present invention by applying a known method such as addition of a biopolymer or polymer the blood half-life when used as a pharmaceutical composition can be adjusted to be longer. it can.
- the molecular weight of such HTRA1 inhibitory peptides of the present invention is less than 10,000, preferably less than 8,000, more preferably about 7,000-7,200.
- the variable loop part consisting of 15th Cys to 31st Cys or the part consisting of 15th Cys to 63Cys (hereinafter referred to as “part containing 6 Cys”) of SEQ ID NO: 23 (FIG. 29) inhibition of HTRA1 Those having activity are also included in the HTRA1 inhibitory peptide of the present invention, the molecular weight of the variable loop portion is less than 2,500, preferably about 1,800 to 2,000, and the molecular weight of the portion containing 6 Cys is Less than 6,000, preferably about 5,300-5,500.
- SPINK2 mutants in which the backbone of SPINK2 included in the HTRA1 inhibitory peptide of the present invention is maintained at least partially may bind to HTRA1.
- HTRA1 Preferably mammalian, more preferably primate, and even more preferably human HTRA1.
- target binding activity a partial higher-order structure or the like
- the inhibitory peptides of the invention can bind to an immunogenic fragment of HTRA1.
- An immunogenic fragment of HTRA1 has one or more epitopes, mimotopes or other antigenic determinants so that it can elicit an immune response or an antibody against the fragment can be produced.
- the binding of SPINK2 mutant to HTRA1 or an immunogenic fragment thereof is performed by measuring a detectable binding affinity (ELISA method, surface plasmon resonance (hereinafter referred to as “SPR”) analysis method (“BIAcore”). ), Isothermal titration calorimetry (hereinafter referred to as “ITC” method, flow cytometry, immunoprecipitation method, etc.), etc., and evaluation, measurement or determination using methods known to those skilled in the art can do.
- ELISA method detectable binding affinity
- SPR surface plasmon resonance
- ITC Isothermal titration calorimetry
- flow cytometry flow cytometry
- immunoprecipitation method etc.
- Examples of the ELISA method include a method of detecting an HTRA1-inhibiting peptide that recognizes and binds to HTRA1 immobilized on a plate.
- an antibody for solid phase that recognizes a tag fused to HTRA1 can be used.
- a labeled detection antibody that recognizes a tag fused to the HTRA1 inhibitory peptide can be used for detection of the HTRA1 inhibitory peptide.
- methods that can be used for biochemical analysis such as HRP, alkaline phosphatase, and FITC can be used.
- 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), a chromogenic substrate and QuantaBlu (TM) such as SuperSignal ELISA Pico Chemiluminescent substrate (Thermo Fisher Scientific) Fluorogenic Peroxidase substrate (Thermo Fisher Cientific) fluorescent substrate, and the like, and can be used chemiluminescent substrate.
- an absorption plate reader, a fluorescence plate reader, a light emission plate reader, an RI liquid scintillation counter, or the like can be used.
- Devices used for SPR analysis include BIAcore (trademark) (GE Healthcare), ProteOn (trademark) (BioRad), SPR-Navi (trademark) (BioNavisOy), Spreeta (trademark) (Texas Instruments), and SPri-PlexII (trademark). (Horiba), Autolab SPR (trademark) (Metrohm), etc. can be illustrated.
- BIAcore trademark
- BioRad ProteOn
- SPR-Navi trademark
- BioNavisOy SPR-Navi
- Spreeta trademark
- Texas Instruments Texas Instruments
- SPri-PlexII trademark
- Examples of the immunoprecipitation method include a method of detecting HTRA1 recognized and bound by an HTRA1 inhibitory peptide immobilized on a bead. Magnetic beads, agarose beads, etc. can be used as the beads. In addition to biotin-streptavidin, an antibody that recognizes the peptide or a tag fused to the peptide, protein A, protein G, or the like can be used to immobilize the HTRA1-inhibiting peptide. The beads are separated by a magnet, centrifugation, etc., and HTRA1 precipitated together with the beads is detected by SDS-PAGE or Western blot method.
- a labeled detection antibody that recognizes a tag fused to HTRA1 can be used for detection of HTRA1.
- methods that can be used for biochemical analysis such as HRP, alkaline phosphatase, and FITC can be used.
- HRP alkaline phosphatase
- FITC FITC
- the same substrate as in the ELISA method can be used for detection using an enzyme label.
- ChemiDoc (trademark) (BioRad), LuminoGraph (ATTO), etc. can be utilized for the measurement of a detection signal.
- “specific recognition”, that is, “specific binding” means binding that is not non-specific adsorption.
- the binding activity EC 50 in the ELISA method can be mentioned.
- a dissociation constant hereinafter referred to as “K D ”.
- K D values for HTRA1 of HTRA1 inhibitory peptide of the present invention 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, even more preferably 5 ⁇ 10 ⁇ 8 M or less, 2 ⁇ 10 ⁇ 8 M Or 1 ⁇ 10 ⁇ 8 M or less, and even more preferably 5 ⁇ 10 ⁇ 9 M or less, 2 ⁇ 10 ⁇ 9 M or less, or 1 ⁇ 10 ⁇ 9 M or less.
- an analysis result by an immunoprecipitation method can be given.
- a suitable HTRA1 inhibitory peptide in the present invention is immobilized on beads, each HTRA1 is added and then the beads are separated, and when HTRA1 precipitated together with the beads is detected, an HTRA1 signal is detected.
- the ability to bind to HTRA1 or an immunogenic fragment thereof is not essential for the SPINK2 mutant as the inhibitory peptide of the present invention as long as it has HTRA1 inhibitory activity.
- the inhibitory peptide of the present invention may be competitive in the binding of a protease substrate to HTRA1.
- the inhibitory peptide of the present invention has a retinal protective effect.
- the preferred inhibitor of the present invention can suppress a decrease in the number of nuclei contained in the outer granule layer due to light irradiation.
- a larger amount of HTRA1 protein was detected in the vitreous humor of the group irradiated with light compared to the non-irradiated group, and HTRA1 was involved in retinal damage, and HTRA1 inhibitory activity had a retinal protective effect.
- the present invention discloses.
- the SPINK2 mutant as the inhibitory peptide of the present invention may have the activity, properties, functions, features and the like as described above, while its full-length amino acid sequence has high sequence identity to the amino acid sequence of human wild-type SPINK2.
- the SPINK2 variant of the present invention has a human SPINK2 amino acid sequence (SEQ ID NO: 1 FIG. 13) and 60% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, Has sequence identity of 98% or more or 99% or more.
- Identity means a property indicating the degree of similarity or relationship between two sequences. Amino acid sequence identity (%) is calculated by multiplying the numerical value obtained by dividing the number of identical amino acids or amino acid residues by the total number of amino acids or amino acid residues by 100.
- Gap means a gap in alignment between the sequences as a result of deletion and / or addition in at least one of the two or more sequences.
- the identity between two amino acid sequences having the completely identical amino acid sequence is 100%, but the substitution or deletion of one or more amino acids or amino acid residues compared to one amino acid sequence compared to the other. If there is a loss or addition, the identity of both is less than 100%.
- BLAST Altschul, et al. Nucleic Acids Res. 25, 3389-3402, 1997) using standard parameters is used as an algorithm or program for determining identity between two sequences in consideration of gaps.
- BLAST2 Altschul, et al. J. Mol. Biol. 215, 403-410, 1990
- Smith-Waterman Smith, et al. J. Mol. Biol. 147, 195-197) , 1981
- the like can be exemplified.
- mutated refers to a substitution of one or more nucleotides or nucleotide residues or amino acid or amino acid residues in a nucleotide sequence or amino acid sequence compared to a naturally occurring nucleic acid molecule or peptide. Means that a deletion or insertion has been made.
- amino acid sequence of the SPINK2 variant of the present invention one or more amino acids or amino acid residues are mutated as compared to the amino acid sequence of human SPINK2.
- the amino acid sequence of the SPINK2 variant is that of human SPINK2 (SEQ ID NO: 1 FIG. 13): One, two, three, four, five, six, or seven amino acids of No. 16 Ser to No. 22 Gly are substituted with other amino acids or amino acid residues; One, two, three, four or five amino acids from No. 24 Pro to No. 28 Asn are substituted with other amino acids or amino acid residues; Even if one or two amino acids of No. 39 Ala and No. 43 Thr are substituted with other amino acids or amino acid residues, or in the wild type, it is composed of amino acid residues such as Nos. 16-30.
- the two structures or amino acid residues are included in the ⁇ helix; 15th Cys, 23rd Cys, 31st Cys, 42nd Cys, 45th Cys and 63rd Cys are preferably Cys as well as the wild type in order to maintain the natural disulfide bond. In order to eliminate disulfide bonds or to generate unnatural disulfide bonds, one, two, three, four, five or six of them can be replaced with other amino acids. Also good.
- Cys is maintained at the same 6 positions as the natural type, and a disulfide bond is maintained.
- the 15th Cys-45Cys, 23rd Cys-42Cys, and 31Cys-63Cys each form a disulfide bond.
- a loop structure consisting of 16th Ser to 30th Val included in the amino acid sequence of wild-type SPINK2 a ⁇ structure consisting of 31Cys and 32Gly ⁇ sheet composed of strand (1) and ⁇ strand (2) consisting of 57 Ile to 59 Arg, ⁇ helix consisting of 41 Glu amino acid to 51 Gly, or similar to them
- the three-dimensional structure composed of a loop structure, a ⁇ sheet, an ⁇ helix, etc. corresponding at least partially to the position of (1) is maintained to such an extent that it can exhibit HTRA1 inhibitory activity.
- amino acid residue may be simply expressed as “amino acid”.
- the first to eleventh Xaa are arbitrary amino acids as long as they bind to HTRA1 and inhibit HTRA1 activity. If there is no particular limitation.
- preferred amino acids of X1 to X11 will be described, and these amino acids may contain the same amino acid as that in the amino acid sequence of the natural type, that is, wild type human SPINK2.
- # 1 X1 is preferably Asp, Glu, Gly, Ser or Ile, more preferably Asp or Gly, even more preferably Gly; 16 X2 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, even more preferred Ala, Gly, Lys, Leu, Ser or Thr, even more preferred Ala, Gly, Leu, Ser or Thr, and even more Preferably Ala or Ser; No.
- 17 X3 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, even more preferably Asp, His, Lys, Met or Gln, even more preferably Asp or Gln; 18th X4 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, even more preferably Asp, Phe, His, Ser or Tyr, even more preferably Asp, Phe, His, Ser or Tyr, even more preferably Phe or His; No.
- 19 X5 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, even more preferably Ala, Asp, Glu, Met or Asn, even more preferably Ala, Asp or Glu; 21st X6 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, more preferably Met or Trp, even more preferably Met; No.
- 24 X7 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, even more preferably Gln, Trp, Tyr or Val, even more preferably Tyr or Val;
- 26th X8 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, even more preferably Phe, Leu or Tyr, even more preferably Phe or Leu;
- 27th X9 is preferably Glu, Phe, Leu, Ser, Thr or Tyr, more preferably Phe, Leu, Ser, Thr or Tyr, even more
- wild-type X1 to X11 are Asp, Ser, Gln, Tyr, Arg, Pro, Pro, His, Phe, Ala, and Thr, respectively. Also, No. 20 is Leu, No. 22 is Gly, No. 25 is Arg, and No. 28 is Asn.
- amino acids may be further added to the N-terminal side of the first amino acid.
- added amino acids include Ser-Leu, S An amino acid sequence comprising a tag and a linker (SEQ ID NO: 31: FIG. 43) and the like can be mentioned.
- 1 to several amino acids may be added to the 63rd Cys located at the C-terminus.
- an amino acid sequence having a Gly-Gly of C-terminal 64th Gly and the C-terminal 65th Gly is added.
- Some amino acid sequences can be mentioned. Examples of such added amino acids include Gly-Gly, C-terminal 6-mer (SEQ ID NO: 32: FIG. 44), and the like.
- amino acid sequences obtained by adding other amino acids to the N-terminal and / or C-terminal of the amino acid sequence shown in SEQ ID NO: 30 (FIG. 42) or the amino acid sequence derived from SEQ ID NO: 30, SEQ ID NOs: 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, and 23 to 29 (FIGS. 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, and 35 to 41) and SEQ ID NOs: 4, 6,
- SEQ ID NOs: 4, 6 The nucleotide sequence represented by any one of 8, 10, 12, 14, 16, 18, 20, and 22 (FIGS. 16, 18, 20, 22, 24, 26, 28, 30, 32, and 34) encodes Amino acid sequences are included in more preferred embodiments, and SEQ ID NOs: 24, 28, and 29 (FIGS. 36, 40, and 41) are included in even more preferred embodiments, respectively.
- one or two or more amino acids are substituted, added, and / or added to the SPINK2 mutant peptide or the N-terminal and / or C-terminal adduct of the SPINK2 mutant peptide (hereinafter referred to as “parent peptide”).
- the deleted peptide may be referred to as “parent peptide derivative” or “parent peptide derivative” (eg, Example 6).
- parent peptide derivative eg, Example 6
- Such “derivatives” are also included in the scope of the “peptide” of the present invention.
- a portion other than X1 to X11 that is, No. 2 Pro in the amino acid sequence of wild-type human SPINK2 (SEQ ID NO: 1 FIG. 13).
- Natural amino acids at positions 15 to Cys, 20 Pro, 22 Gly, 23 Cys, 25 Arg, 28 Asn to 38 Tyr, 41 Glu, 42 Cys and 44 Thr to 63 Cys Alternatively, it can contain mutated amino acids or amino acid sequences.
- a SPINK2 variant may be mutated at one or more positions as long as it does not at least partially prevent or interfere with HTRA1 inhibitory activity or folding.
- Such mutations can be made by using standard methods known to those skilled in the art.
- Typical mutations in the amino acid sequence can include substitutions, deletions or additions of one or more amino acids, and examples of substitutions include conservative substitutions.
- conservative substitution certain amino acid residues are replaced by amino acid residues that have similar chemical characteristics not only in bulk but also in terms of polarity. Examples of conservative substitutions are described elsewhere in this specification.
- moieties other than X1-X11 may tolerate non-conservative substitutions of one or more amino acids as long as they do not at least partially prevent or interfere with HTRA1 inhibitory activity or folding.
- the amino acid sequence possessed by the SPINK2 variant as the inhibitory peptide of the present invention is such that X1 to X11 are preferably SEQ ID NOs: 3, 5, 7, 9, 11, 13, 15, 17, 19, 21 and 23 to 29 (FIGS. 15, 17, 19, 21, 23, 25, 27, 29, 31, 33 and 35 to 41), more preferably of SEQ ID NOS: 24, 28 and 29 (FIGS. 36, 40 and 41).
- Each of the amino acids X1 to X11 in any one of them, and a portion other than X1 to X11 can have an amino acid or an amino acid sequence that does not at least partially prevent or interfere with HTRA1 inhibitory activity or folding.
- amino acid sequence of the SPINK2 mutant as the HTRA1 inhibitory peptide of the present invention can include the amino acid sequences described in any one of the following (a) to (e): (A) SEQ ID NOs: 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23 to 29 (FIGS.
- the nucleic acid molecule encoding the HTRA1 inhibitory peptide is not limited to (a) to (e), and the amino acid sequence contained in the SPINK2 mutant having HTRA1 inhibitory activity, preferably SEQ ID NO: 30 (FIG. 42)
- a nucleic acid molecule comprising a nucleotide sequence that encodes the amino acid sequence represented by is ubiquitously encompassed within the scope of a nucleic acid molecule that encodes an HTRA1 inhibitory peptide.
- introducing a mutation into the inhibitory peptide of the present invention for the purpose of improving its folding stability, heat stability, storage stability, blood half-life, water solubility, biological activity, pharmacological activity, side effects, etc. Can do.
- new reactive groups such as Cys can be introduced by mutation to conjugate to other substances such as polyethylene glycol (PEG), hydroxyethyl starch (HES), biotin, peptides or proteins.
- PEG polyethylene glycol
- HES hydroxyethyl starch
- biotin peptides or proteins.
- the HTRA1 inhibitory peptide may be added, linked or bound to other moieties, and such conjugates are collectively referred to as “conjugates of HTRA1 inhibitory peptides”.
- the “conjugate” means a molecule obtained by adding, linking or binding other parts to the peptide of the present invention or a fragment thereof.
- the peptide of the present invention is formed through a chemical substance such as a cross-linking agent, an agent suitable for linking a certain moiety to the side chain of an amino acid, or the like.
- the N-terminal and / or C-terminal of the present invention includes a form linked or bound to the peptide of the present invention by a synthetic chemical method, a genetic engineering method, or the like.
- portions include polyalkylene glycol molecules such as polyethylene glycol (PEG), fatty acid molecules such as hydroxyethyl starch and palmitic acid, Fc region of immunoglobulin, immunoglobulin And CH3 domain of immunoglobulin, CH4 domain of immunoglobulin, albumin or a fragment thereof, albumin binding peptide, albumin binding protein such as streptococcal protein G, and transferrin.
- PEG polyethylene glycol
- fatty acid molecules such as hydroxyethyl starch and palmitic acid
- Fc region of immunoglobulin immunoglobulin And CH3 domain of immunoglobulin, CH4 domain of immunoglobulin, albumin or a fragment thereof
- albumin binding peptide such as streptococcal protein G
- transferrin transferrin.
- the peptide of the present invention can be linked to the “portion” via a linker such as a peptide linker.
- the HTRA1 inhibitory peptide of the present invention may be conjugated with other drugs in order to exert or enhance pharmacological activity.
- Techniques and embodiments known to those skilled in the art as antibody-drug conjugates (ADCs) in the antibody field become a part of the present invention by replacing antibodies with the peptides of the present invention.
- the HTRA1 inhibitory peptide of the present invention further comprises one or two or more moieties that exhibit binding affinity, inhibitory activity, antagonistic activity, agonistic activity, etc. to target molecules other than HTRA1, or are conjugated to such moieties May be.
- the “portion” include an antibody or a fragment thereof, a protein having a skeleton other than an antibody such as a SPINK2 mutant, or a fragment thereof.
- multispecific antibodies and bispecific antibodies are at least one of two or more “antibodies” included in the present invention. By substituting these peptides, some aspects of the conjugates of the present invention are obtained.
- the peptide of the present invention or a precursor thereof may contain a signal sequence.
- a signal sequence present at or added to the N-terminus of a polypeptide or its precursor is used to place the polypeptide in a specific compartment of the cell, for example, periplasm in the case of E. coli, or endoplasmic reticulum in the case of a eukaryotic cell.
- Many signal sequences are known to those skilled in the art and can be selected depending on the host cell.
- OmpA can be exemplified, and a form containing a signal sequence can also be included as a part of the embodiment of the conjugate of the present invention. .
- the peptide can be purified by affinity chromatography.
- the peptide of the present invention has, for example, a biotin, a Strep tag (trademark), a Strep tag II (trademark), an oligohistidine such as His6, a polyhistidine, an immunoglobulin domain, a maltose binding protein, a glutathione-S-transferase. (GST), calmodulin-binding peptide (CBP), haptens such as digoxigenin and dinitrophenol, epitope tags such as FLAG TM, myc tag, HA tag, etc.
- GST calmodulin-binding peptide
- CBP calmodulin-binding peptide
- haptens such as digoxigenin and dinitrophenol
- epitope tags such as FLAG TM, myc tag, HA tag, etc.
- the conjugate of the present invention may be a peptide (polypeptide) as a whole.
- the peptide of the present invention may contain a moiety for labeling, specifically, enzyme label, radioactive label, colored label, fluorescent label, chromogenic label, luminescent label, hapten, digoxigenin, biotin, metal complex, Labeling moieties such as metals, colloidal gold, etc. can be conjugated.
- a moiety for labeling can also be included as part of the embodiment of the conjugate of the invention.
- the inhibitory peptide of the present invention can contain both natural amino acids and non-natural amino acids in the peptide portion, and natural amino acids can contain both L-amino acids and D-amino acids.
- the amino acid sequence of the inhibitory peptide of the present invention can include both natural amino acids and unnatural amino acids, and natural amino acids can include both L-amino acids and D-amino acids.
- the inhibitory peptide of the present invention may exist as a monomer, dimer, trimer or higher oligomer or multimer.
- the dimer, trimer or higher oligomer and multimer may be either a homopolymer composed of a single monomer or a heteropolymer composed of two or more different monomers.
- the monomer may diffuse quickly and excel in tissue penetration.
- Dimers, oligomers and multimers have excellent aspects such as having high affinity or binding activity to the target molecule locally, having a slow dissociation rate, or exhibiting high HTRA1 inhibitory activity. Can do.
- intended dimerization, oligomerization, and multimerization may be performed by introducing a jun-fos domain, a leucine zipper, or the like into the inhibitory peptide of the present invention. Can be done.
- the inhibitory peptides of the present invention are monomers, dimers, trimers or oligomers or multimers that can bind to or inhibit the activity of one or more target molecules. .
- Possible forms of the inhibitory peptide of the present invention include isolated forms (lyophilized preparations, solutions, etc.), the above-mentioned conjugates, forms bound to other molecules (solid-phase forms, foreign molecules and And the like, and the like are not limited to these, and a form suitable for expression, purification, use, storage and the like can be arbitrarily selected.
- HTRA1 Inhibitory Peptide is an amino acid sequence of SPINK2 or an amino acid sequence of the HTRA1 inhibitory peptide of the present invention (for example, SEQ ID NOs: 3, 5, 7, 9, 11, 13, 15, 17, 19, 21 and 23 to 29, or an amino acid sequence selected from the group consisting of FIGS. 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, and 35 to 41), and encodes the amino acid sequence
- a nucleotide sequence, a nucleic acid molecule containing the nucleotide sequence, or the like can be used as a starting material for identification by methods well known to those skilled in the art.
- HTRA1 inhibitory activity can be identified from a human SPINK2 mutant library as an index, and HTRA1 binding activity may also be combined as an index.
- the starting nucleic acid molecule can be subjected to mutagenesis and introduced into a suitable bacterial or eukaryotic host using recombinant DNA technology.
- the SPINK2 mutant library is known as a technique for identifying a target molecule binder or inhibitor.
- the disclosure in WO2012 / 105616 is also included in the disclosure of the present invention by referring to the entirety thereof.
- a clone in which a SPINK2 mutant having a desired property, activity, function, etc. is linked to the genetic trait is enriched from the library. / Or can be selected and identified.
- the bacterial display method (Francisco, JA, et al. (1993) Proc. Natl. Acad. Sci. USA 90, 10444-10448) Yeast display method (Boder, ET, et al. (1997) Nat. Biotechnol. 15, 553-557), mammalian cell display method (Ho M, et al. (2009) Methods Mol. Biol. 525: 337-52), phage display method (Smith, GP (1985) Science. 228, 1315-1317), ribosome display method ((Mattheakis LC, et al. (1994)). ) Proc Natl. Acad. Sci.
- nucleic acid display methods such as mRNA display (Nemoto N, et al. (1997) FEBS Lett. 414, 2) 405-408), colony screening method (Pini, A. et al. (2002) Comb. Chem. High Throughput Screen. 5, 503-510), etc.
- the amino acid sequence encoded by the nucleotide sequence is converted into the amino acid of the SPINK2 variant, ie, the HTRA1 inhibitory peptide contained in the clone. It can be determined as an array.
- the SPINK2 mutant of the present invention can be obtained, for example, by inducing mutation in natural type SPINK2.
- “Induction of mutagenesis” refers to substitution or deletion of one or more amino acids present at each position of a certain amino acid sequence with other amino acids, or amino acids that are not present in the amino acid sequence. It means to be able to add or insert. Such deletion or addition or insertion can change the sequence length.
- mutagenesis can preferably occur at one or more positions from X1 to X11 in the amino acid sequence shown in SEQ ID NO: 30 (FIG. 42).
- a natural amino acid that is, the same amino acid present at a specific position in the natural amino acid sequence
- a natural amino acid is present at that position, that is, at a specific position in the natural amino acid sequence.
- those in which the same amino acid is maintained are also included in the range of the mutant as long as at least one amino acid is mutated as a whole.
- Random mutagenesis means that for a particular position on a sequence, one or more different amino acids are introduced at that position with a certain probability by mutagenesis, but at least 2 The probability that two different amino acids are introduced may not all be the same. Further, in the present invention, it does not prevent at least two different amino acids from containing a natural amino acid (one kind), and such a case is also included in the range of “induction of random mutation”.
- Standard methods known to those skilled in the art can be used as a method for inducing random mutation at a specific position.
- a mutation can be induced at a specific position in the sequence by PCR (polymerase chain reaction) using a mixture of synthetic oligonucleotides containing a degenerate nucleotide composition.
- PCR polymerase chain reaction
- a mutation of a stop codon is introduced.
- Site-directed mutagenesis can also be performed using the structural information of a target including a higher-order structure and / or a peptide for the target or a wild-type peptide from which the peptide is derived.
- site-specific mutation is introduced using structural information including higher-order information of target HTRA1 and / or target SPINK2 mutant or wild type SPINK2 or a complex of both. be able to.
- a SPINK2 mutant having HTRA1 inhibitory activity is identified, then a crystal of a complex of HTRA1 and the SPINK2 mutant is obtained and X-ray crystal structure analysis is performed, and the SPINK2 mutant binds based on the analysis result
- it is possible to find a correlation between the structural information obtained through specifying the amino acid residue in the SPINK2 mutant involved in the interaction with the site on the HTRA1 molecule and the HTRA1 molecule, and the HTRA1 inhibitory activity Based on such structure-activity relationship, substitution with a specific amino acid at a specific position, insertion or deletion of an amino acid at a specific position, etc. can be designed to actually confirm HTRA1 activity.
- mutation can be induced using a nucleotide constituent unit in which the specificity of base pair such as inosine is modified.
- mutagenesis to random positions is possible by, for example, error-prone PCR method using a DNA polymerase having a high error rate, such as Taq DNA polymerase, which has a high error rate, and chemical mutagenesis.
- HTRA1 inhibitory peptides can be used by those skilled in the art suitable for each screening method such as phage library and colony library using bacterial display, yeast display, mammalian cell display, phage display, ribosome display, nucleic acid display, colony screening, etc. It can be enriched and / or selected from known libraries.
- phage libraries can be constructed by vectors and methods known to those skilled in the art suitable for each library, such as phagemids for phage libraries and cosmids for colony screening.
- Such vectors may be viruses or viral vectors that infect prokaryotic or eukaryotic cells.
- These recombinant vectors can be prepared by methods known to those skilled in the art, such as gene manipulation.
- Bacteria display is, for example, a technique in which a part of the outer membrane lipoprotein (Lpp) of E. coli and the outer membrane protein OmpA are fused with the desired protein to display the desired protein on the E. coli surface.
- Lpp outer membrane lipoprotein
- OmpA outer membrane protein
- a DNA group obtained by inducing random mutations in the nucleotide sequence encoding the amino acid sequence of a protein is introduced into a vector suitable for bacterial display, and bacterial cells are transformed with the vector, the surface of the transformed bacterial cell
- a library that presents a group of randomly mutagenized proteins can be obtained (Francisco, JA, et al. (1993) Proc. Natl. Acad. Sci. USA, 90 volumes. , 10444-10448).
- Yeast display is a technique in which a desired protein is fused to a protein such as ⁇ -agglutinin in the outer shell of the yeast cell surface and displayed on the yeast surface.
- ⁇ -Agglutinin includes a C-terminal hydrophobic region presumed to be a glycosylphosphatidylinositol (GPI) anchor attachment signal, a signal sequence, an active domain, a cell wall domain, and the like. Desired proteins can be displayed.
- GPI glycosylphosphatidylinositol
- a DNA group obtained by inducing random mutations in the nucleotide sequence encoding the amino acid sequence of a protein is introduced into a vector suitable for yeast display, and the yeast cell is transformed with the vector, the surface of the transformed yeast cell
- a library presenting a randomly mutagenized protein group can be obtained (Ueda, M. & Tanaka, A., Biotechnol. Adv., 18, 121-, 2000 edition: Ueda, M. & Tanaka, A., J. Biosci. Bioeng., 90, 125-, 2000, etc.).
- Animal cell display is performed on the surface of a mammalian cell such as HEK293 or Chinese hamster ovary (CHO) cell by fusing a transmembrane region of a membrane protein typified by platelet-derived growth factor receptor (PDGFR) with a desired protein.
- PDGFR platelet-derived growth factor receptor
- the surface of a transformed animal cell can be obtained by introducing a DNA group obtained by inducing random mutation into a nucleotide sequence encoding an amino acid sequence of a protein into a vector suitable for animal cell display and transforming the animal cell with the vector.
- a library presenting randomly mutagenized proteins can be obtained (Ho M, et al. (2009) Methods Mol Biol. 525: 337-52).
- Desired libraries displayed on cells such as yeast, bacteria, animal cells can be kept warm in the presence of the target molecule or brought into contact with the target molecule.
- a carrier such as magnetic beads is added, the cells are separated from the carrier, and then the carrier is washed, thereby nonspecific adsorption. And the cell group in which the peptide bound to the carrier (bound to HTRA1), the peptide aggregate, or the concentrated peptide aggregate is displayed can be recovered.
- a population of cells displaying aggregates or enriched peptide aggregates can be recovered.
- Non-specific adsorbate sites and / or binding sites can be, for example, blocked and a blocking step can be incorporated if appropriate.
- a vector expressing the peptide thus obtained, a collection of peptides or a concentrated peptide collection is recovered, the nucleotide sequence of the polynucleotide inserted into the vector is determined, and the nucleotide sequence encodes The amino acid sequence to be determined can be determined.
- the peptide aggregate that binds to the target molecule can be highly concentrated.
- a phagemid is a bacterial plasmid that contains a second origin of replication derived from a single-stranded bacteriophage in addition to a plasmid origin of replication.
- Cells carrying the phagemid can replicate the phagemid through a single-stranded replication mode in superinfection with M13 or similar helper bacteriophage. That is, single-stranded phagemid DNA is packaged in infectious particles coated with bacteriophage coating protein.
- phagemid DNA can be formed as a clone double-stranded DNA plasmid in the infected bacterium, and phagemid can be formed as bacteriophage-like particles from the culture supernatant of superinfected cells.
- the particles themselves can be reformed as plasmids by injecting the bacteriophage-like particles into the bacteria in order to infect the bacteria with F-fibrotic bacteria.
- a fusion gene comprising a polynucleotide having a nucleotide sequence encoding the amino acid sequence of the test peptide and a bacteriophage coat protein gene is inserted into the phagemid to infect bacteria, and the cells are cultured.
- such peptides are expressed or displayed (synonymous with display) on the bacteria or phage-like particles, or produced in the phage particles or in the culture supernatant of the bacteria as a fusion protein with the coating protein. be able to.
- a fusion gene comprising the polynucleotide and the bacteriophage coat protein gene gpIII is inserted into a phagemid and co-infected with E. coli with M13 or similar helper phage, the peptide and the coat protein are contained. Can be produced in the culture supernatant of the E. coli.
- phagemids When various circular or non-circular vectors such as viral vectors are used instead of phagemids, they have an amino acid sequence encoded by the nucleotide sequence of the polynucleotide inserted into the vector according to a method known to those skilled in the art. Peptides can be expressed or presented on cells or virus-like particles into which the vector has been introduced, or can be produced in the culture supernatant of the cells.
- the library expressing the peptide thus obtained can be kept warm in the presence of the target molecule or brought into contact with the target molecule.
- a carrier on which HTRA1 is immobilized is incubated for a certain period of time with a mobile phase containing a library, then the mobile phase is separated from the carrier, and then the carrier is washed to remove nonspecific adsorbate and bound substance.
- Peptides, peptide aggregates or concentrated peptide aggregates that have been removed and bound to the carrier (HTRA1 bound to) can be recovered by elution.
- Elution is carried out non-selectively in the presence of relatively high ionic strength, low pH, moderate denaturation conditions, the presence of chaotropic salts, etc., or soluble target molecules such as HTRA1, antibodies that bind to target molecules Alternatively, it can be carried out selectively by adding a natural ligand, a substrate or the like to compete with the immobilized target molecule.
- Non-specific adsorbate sites and / or binding sites can be subjected to blocking treatment, for example, and the blocking step can be incorporated if appropriate.
- the peptide aggregate that binds to the target molecule can be highly concentrated.
- Ribosome display uses, for example, mRNA encoding a desired protein having no stop codon and a cell-free protein synthesis system to synthesize a desired protein, its corresponding mRNA, and a ribosome-linked molecule in vitro.
- Technology A library in which random mutagenized proteins are presented on a ribosome by using a group of mRNAs obtained by inducing random mutations in the nucleotide sequence encoding the amino acid sequence of a protein and a cell-free protein synthesis system. (Mattheakis LC, et al. (1994) Proc. Natl. Acad. Sci. USA 91, 19: 9022-9029).
- Nucleic acid display is also called mRNA display.
- a linker such as puromycin having a similar structure at the 3 ′ end of tyrosyl-tRNA
- a desired protein mRNA encoding the molecule
- a molecule linked with ribosome are synthesized.
- Technology Since this technique uses a cell-free protein synthesis system rather than living cells, it can be synthesized in vitro.
- a linker such as puromycin, and a cell-free protein synthesis system, the randomly mutagenized protein group can be obtained.
- the library presented on the ribosome can be obtained (Nemoto N, et al. (1997) FEBS Lett. 414, 2, 405-408).
- a library expressing peptides obtained via a cell-free synthesis system such as ribosome display or nucleic acid display can be kept warm in the presence of the target molecule or brought into contact with the target molecule.
- a carrier on which HTRA1 is immobilized is incubated for a certain period of time with a mobile phase containing a library, then the mobile phase is separated from the carrier, and then the carrier is washed to remove nonspecific adsorbate and bound substance.
- Peptides, peptide aggregates or concentrated peptide aggregates that have been removed and bound to the carrier (HTRA1 bound to) can be recovered by elution.
- Elution is carried out non-selectively in the presence of relatively high ionic strength, low pH, moderate denaturation conditions, the presence of chaotropic salts, etc., or soluble target molecules such as HTRA1, antibodies that bind to target molecules Alternatively, it can be carried out selectively by adding a natural ligand, a substrate or the like to compete with the immobilized target molecule.
- Non-specific adsorbate sites and / or binding sites can be subjected to blocking treatment, for example, and the blocking step can be incorporated if appropriate.
- the nucleic acid expressing the peptide, the peptide aggregate or the concentrated peptide aggregate thus obtained is recovered, and in the case of mRNA, the nucleotide sequence is determined after reverse transcription reaction to cDNA.
- the encoding amino acid sequence can be determined.
- the peptide aggregate that binds to the target molecule can be concentrated to a higher degree by transcribing mRNA from the collected nucleic acid and repeating the above operation once or several times as a cycle.
- an affinity tag is conjugated in advance to a peptide, a collection of peptides, or a concentrated peptide collection, the peptide or a collection thereof can be efficiently purified.
- the peptide can be eluted by cleaving with the protease activity.
- the obtained clone or library is induced to further mutation, and the function (for example, HTRA1 inhibitory activity), physical properties (thermal stability) from the library into which the mutation has been introduced.
- the function for example, HTRA1 inhibitory activity
- physical properties for example, thermal stability
- the HTRA1 inhibitory peptide can be identified by determining whether or not the obtained peptide has HTRA1 inhibitory activity.
- the HTRA1 inhibitory peptide is preferably a loop structure consisting of 16th Ser to 30th Val included in the amino acid sequence of wild type SPINK2, a ⁇ strand (1) consisting of 31Cys and 32Gly, and 57th Ile.
- a ⁇ -sheet composed of ⁇ -strand (2) consisting of No. 59 to Arg, and an ⁇ -helix consisting of 41Glu amino acid to No. 51 Gly, or similar to them or at least partially Can be maintained to the extent that HTRA1 inhibitory activity can be exerted. It is also possible to identify a more suitable HTRA1 inhibitory peptide using such a three-dimensional structure (total structure or partial structure) as a part of the index.
- Nucleic acid molecule encoding HTRA1 inhibitory peptide, vector containing the same, cell containing them, and method for producing recombinant HTRA1 inhibitory peptide The present invention relates to a polynucleotide comprising a nucleotide sequence encoding an amino acid sequence contained in an HTRA1 inhibitory peptide (Hereinafter referred to as “nucleic acid molecule encoding HTRA1 inhibitory peptide”), a recombinant vector into which the gene has been inserted, a cell into which the gene or vector has been introduced (hereinafter referred to as “cell containing nucleic acid molecule encoding HTRA1 inhibitory peptide”) Or a cell that produces an HTRA1-inhibiting peptide (hereinafter referred to as an “HTRA1-inhibiting peptide-producing cell”).
- a polynucleotide comprising a nucleotide sequence encoding an amino acid sequence contained in an HTRA1 inhibitory
- the nucleotide sequence described in any one of the following (a) to (e) (hereinafter referred to as “the nucleotide sequence of the HTRA1 inhibitory peptide” Or consisting of an antibody gene sequence, or consisting of an antibody gene sequence: (A) SEQ ID NOs: 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23 to 29 (FIGS. 15, 17, 19, 21, 23, 25, 25, 29, 31, 33, A nucleotide sequence encoding the amino acid sequence represented by any one of 35 to 41); (B) any one of SEQ ID NOs: 4, 6, 8, 10, 12, 14, 16, 18, 20, and 22 (FIGS.
- C a nucleotide sequence that hybridizes with a nucleotide sequence complementary to the nucleotide sequence described in (a) or (b) under a stringent condition and encodes an amino acid sequence contained in a peptide having HTRA1 inhibitory activity;
- D 1 to 20, 1 to 15, 1 to 10, 1 to 8, 1 to 6, 1 to 5, 1 to 4 in the nucleotide sequence described in (a) or (b) 1 to 3, 1 or 2, or 1 nucleotide or nucleotide residue is substituted, deleted, added and / or inserted, and encodes an amino acid sequence contained in a peptide having HTRA1 inhibitory activity
- E 60%, 70%, 80%, 85%, 90%, 92%, 94%, 96%, 97%, 98% or 99% or more identical to the nucleotide sequence described in (a) or (b) And a nucleotide sequence represented by one;
- E 60%, 70%, 80%, 85%,
- the nucleic acid molecule encoding the HTRA1 inhibitory peptide is not limited to (a) to (e), and the amino acid sequence contained in the SPINK2 mutant having HTRA1 inhibitory activity, preferably SEQ ID NO: 30 (FIG. 42)
- a nucleic acid molecule comprising a nucleotide sequence that encodes the amino acid sequence represented by is ubiquitously encompassed within the scope of a nucleic acid molecule that encodes an HTRA1 inhibitory peptide.
- nucleotide sequence encoding an amino acid sequence one or more codons corresponding to each amino acid can be used. Therefore, the base sequence encoding a single amino acid sequence possessed by a certain peptide can have a plurality of variations.
- a codon is appropriately selected according to the codon usage of the host cell for expression into which a polynucleotide containing the nucleotide sequence or a vector containing the polynucleotide is introduced, or a plurality of codons are selected. The frequency or rate of use of can be adjusted as appropriate. For example, when E. coli is used as a host cell, a nucleotide sequence may be designed using codons that are frequently used in E. coli.
- the nucleic acid molecule encoding the HTRA1 inhibitory peptide may be operably linked to one or more regulatory sequences.
- operably linked is meant that the linked nucleic acid molecule can be expressed or allows the expression of the nucleotide sequence contained in the molecule.
- Regulatory sequences include sequence elements that contain information regarding transcriptional regulation and / or translational regulation. Regulatory sequences vary from species to species, but generally include promoters and are exemplified by prokaryotic -35 / -10 boxes and Shine Dalgarno sequences, eukaryotic TATA boxes, CAAT sequences, and 5 'capping sequences, etc. 5 'non-coding sequences involved in transcription and translation initiation.
- sequences may include enhancer elements and / or repressor elements and signal sequences, leader sequences, etc. that can be translated to deliver native or mature peptides to specific compartments inside or outside the host cell.
- regulatory sequences may include 3 'non-coding sequences, such sequences may include elements involved in transcription termination or polyadenylation. However, if the sequence for termination of transcription does not function sufficiently in a particular host cell, it can be replaced with a sequence suitable for that cell.
- promoter sequences include tet promoter, lacUV5 promoter, T7 promoter and the like in prokaryotes, and SV40 promoter and CMV promoter in eukaryotic cells.
- Nucleic acid molecules encoding HTRA1 inhibitory peptides are contained in isolated form, vectors or other cloning vehicles (hereinafter simply referred to as “vectors”: plasmids, phagemids, phages, baculoviruses, cosmids, etc.) or in chromosomes. Although it may be a form, it is not limited to those forms.
- the vector also includes replication and control sequences suitable for the host cell used for expression, and expression capable of selecting a cell into which a nucleic acid molecule has been introduced by transformation or the like. It may contain a selection marker that gives the mold.
- a nucleic acid molecule encoding an HTRA1 inhibitory peptide and a vector containing the nucleotide sequence of the HTRA1 inhibitory peptide can be introduced into a host cell capable of expressing the peptide or nucleotide sequence by methods known to those skilled in the art such as transformation.
- Host cells into which the nucleic acid molecule or vector has been introduced can be cultured under conditions suitable for expression of the peptide or nucleotide sequence.
- the host cell may be either prokaryotic or eukaryotic.
- yeasts such as Saccharomyces cerevisiae and Pichia pastoris
- insect cells such as SF9 and High5, HeLa cells, CHO cells, and COS. Examples thereof include animal cells such as cells and NS0.
- post-translational modifications include addition of functional groups such as sugar chains, addition of peptides or proteins, conversion of chemical properties of amino acids, and the like. It is also possible to artificially apply desired modifications to the peptides of the present invention. Such modified peptides are also encompassed within the scope of the “peptide” of the present invention.
- the present invention also includes a method for producing an HTRA1 inhibitory peptide.
- a method for producing an HTRA1 inhibitory peptide in the method, in Step 1 and / or Step 1 of culturing a host cell into which a nucleic acid molecule encoding an HTRA1 inhibitory peptide or a vector containing a nucleotide sequence of an HTRA1 inhibitory peptide is introduced, or a cell that expresses an HTRA1 inhibitory peptide Step 2 of recovering the HTRA1 inhibitory peptide from the resulting culture is included.
- steps known to those skilled in the art such as fractionation, chromatography, and purification can be applied.
- affinity purification using the antibody of the present invention described later can be applied.
- the HTRA1 inhibitory peptide has an intramolecular disulfide bond. It may be preferable to deliver a peptide having an intramolecular disulfide bond to a cell section having an oxidizing redox environment using a signal sequence or the like.
- the oxidizing environment can be provided by the periplasm of Gram-negative bacteria such as E. coli, the extracellular environment of Gram-positive bacteria, the endoplasmic reticulum lumen of eukaryotic cells, etc. Under such circumstances, structural disulfide Bond formation can be promoted. It is also possible to produce a peptide having an intramolecular disulfide bond in the cytoplasm of a host cell such as E.
- the peptide in which case the peptide can be obtained directly in a soluble folded state or of inclusion bodies. It can be recovered in form and then reconstituted in vitro. Furthermore, a host cell having an oxidative intracellular environment can be selected to produce a peptide having an intramolecular disulfide bond in the cytoplasm. On the other hand, when the HTRA1 inhibitory peptide does not have an intramolecular disulfide bond, it can be produced in a cell section having a reducing redox environment, for example, in the cytoplasm of a gram-negative bacterium.
- the HTRA1 inhibitory peptide of the present invention utilizes Merrifield et al.'S solid phase peptide synthesis method, t-butoxycarbonyl (Boc), 9-fluorenylmethoxycarbonyl (9-Fluorenylmethoxycarbonyl: Fmoc), etc. It can also be produced by other methods known to those skilled in the art, such as chemical synthesis exemplified by organic synthetic chemical peptide synthesis methods, in vitro translation, and the like.
- the present invention provides, as some aspects thereof, an antibody that binds to a SPINK2 mutant peptide having HTRA1 inhibitory activity and a functional fragment thereof.
- the antibody may be either a polyclonal antibody or a monoclonal antibody, and the monoclonal antibody is not particularly limited as long as it is an immunoglobulin or derived from it.
- the functional fragment of the antibody is not limited as long as it has antigen-binding activity, that is, binding activity to the SPINK2 mutant peptide, and lacks heavy chain and / or light chain, or a fragment thereof, constant region and Fc region. And conjugates with other proteins and labeling substances.
- Such antibodies and functional fragments thereof can be prepared by methods known to those skilled in the art, and purification of the SPINK2 mutant peptide by affinity chromatography, a pharmaceutical composition containing the peptide or a clinical test related to its use, It is useful for detection of the peptide in diagnosis and the like, immunoassay and the like.
- the antibody of the present invention can be purified by affinity chromatography using the peptide of the present invention to which the antibody binds.
- composition comprising an HTRA1 inhibitory peptide or a conjugate thereof.
- HTRA1 inhibitory peptide of the present invention or the conjugate pharmaceutical composition thereof is induced or exacerbated by HTRA1, and inhibition or suppression of the expression or function of HTRA1 suppresses the induction or exacerbation, cure, symptoms It is useful for the treatment and / or prevention of various diseases (hereinafter referred to as “diseases associated with HTRA1” or “HTRA1-related diseases”) that can maintain or improve the disease, avoid secondary diseases, and the like.
- Diseases related to HTRA1 include various diseases that can be caused by retinal protective effects, such as suppression of induction or exacerbation, healing, maintenance or improvement of symptoms, avoidance of secondary diseases, and the like.
- HTRA1 inhibitory peptides have a retinal protective effect and are useful for the treatment and / or prevention of diseases associated with HTRA1.
- Examples of diseases associated with HTRA1 include age-related macular degeneration, retinopathy of prematurity, polypoidal choroidal vasculopathy, rheumatoid arthritis, or osteoarthritis.
- age-related macular degeneration exudation Examples include age-related macular degeneration, atrophic age-related macular degeneration, and map-like atrophy, but are not limited thereto.
- the pharmaceutical composition of the present invention can also be used as a photoreceptor protecting agent, retinal protecting agent, etc. (collectively referred to as “retinal protecting agent”).
- Age-related macular degeneration is divided into wet type and atrophic type.
- the exudative type is further classified into typical age-related macular degeneration (typical AMD), polypoidal choroidal vasculopathy (PCV), retinal hemangiomatous proliferation (RAP). Treatments such as anti-VEGF drugs and photodynamic therapy (PDT) exist for the exudative type, but they are not always sufficient.
- PDT photodynamic therapy
- AREDS Age-Released Eye Study Study
- the pharmaceutical composition of the present invention can be used for the treatment or prevention of age-related macular degeneration, for example, by administering the HTRA1 inhibitory peptide of the present invention before or after light irradiation in a rat light irradiation retinopathy model, respectively.
- the number of nuclei contained in the outer granule layer decreased in the control group, whereas the decrease in the number of nuclei contained in the outer granule layer was suppressed in the administration group (Examples 5, 10 and 14).
- the area of the retinal pigment epithelial cells (RPE cells) or a certain amount is administered in the control group by administering the HTRA1 inhibitory peptide of the present invention.
- the number of RPE cells having the above cell area increases, whereas this increase is suppressed in the administration group (Example 11).
- the rabbit retinopathy model is particularly excellent as a model for atrophic age-related macular degeneration because it incorporates risk factors for human atrophic age-related macular degeneration.
- VEGF mRNA induction test using retinal pigment epithelial cells (RPE) cells suppression of VEGF mRNA expression was observed when an HTRA1 inhibitory peptide was administered (Example 12).
- RPE retinal pigment epithelial cells
- VEGF induction from retinal pigment epithelial cells Klettner A. et al., (2009) Grafes Arch Clin Exp Ophthalmol.
- the pathological VEGF induction is also involved in the maintenance of the disease state, and the HTRA1 inhibitory peptide of the present invention is capable of atrophic age-related macular degeneration and exudative age-related macular degeneration. It is shown to be useful for the treatment and prevention.
- HTRA1-inhibitory peptide was administered (Example 13). Therefore, treatment for wet age-related macular degeneration characterized by angiogenesis And is useful for prevention. Wet age-related macular degeneration is progressive.
- HTRA1-inhibiting peptide of the present invention alone or in combination with an anti-VEGF drug has a therapeutic effect on these wet age-related macular degeneration (including polypoidal choroidal vasculopathy and intraretinal hemangiomatous proliferation). Can play.
- prophylactic administration of the peptide can prevent or delay the recurrence of the disease state, delay the start or resumption of treatment with an anti-VEGF drug, and the like.
- the peptide works to suppress atrophy formation in patients with exudative age-related macular degeneration, it can bring about long-term visual benefits.
- the HTRA1 inhibitory peptide of the present invention has excellent tissue permeability (Example 11), physical properties / stability, safety, post-administration kinetics, productivity, and the like. It can be made to contain suitably.
- the pharmaceutical composition of the present invention contains a therapeutically or prophylactically effective amount of an HTRA1 inhibitory peptide or conjugate thereof and a pharmaceutically acceptable diluent, carrier, solubilizer, emulsifier, preservative and / or adjuvant. It can be shown.
- “Therapeutically or prophylactically effective amount” means an amount that exhibits a therapeutic or prophylactic effect for a specific disease, administration form, and administration route, and is synonymous with “pharmacologically effective amount”.
- the pharmaceutical composition of the present invention includes pH, osmotic pressure, viscosity, transparency, color, isotonicity, sterility, stability of the composition or peptide of the present invention, conjugate thereof, solubility, It may contain substances that change, maintain, or maintain release, absorbability, permeability, dosage form, strength, properties, shape, etc. (hereinafter referred to as “substances for formulation”) it can.
- the substance for the preparation is not particularly limited as long as it is a pharmacologically acceptable substance. For example, non-toxicity or low toxicity is a property that a drug substance preferably has.
- Examples of the substance for formulation include, but are not limited to, the following: amino acids such as glycine, alanine, glutamine, asparagine, histidine, arginine or lysine, antibacterial agent, ascorbic acid Antioxidants such as sodium sulfate or sodium bisulfite, phosphoric acid, citric acid, boric acid buffer, sodium hydrogen carbonate, buffer such as tris-hydrochloric acid (Tris-Hcl) solution, filler such as mannitol and glycine, ethylenediamine Chelating agents such as tetraacetic acid (EDTA), caffeine, polyvinylpyrrolidine, complexing agents such as ⁇ -cyclodextrin and hydroxypropyl- ⁇ -cyclodextrin, bulking agents such as glucose, mannose or dextrin, monosaccharides, disaccharides, Glucose, mannose and dextrin Other carbohydrates, coloring agents, flavoring agents, diluents, hydrophil
- the amount of these substances for formulation is 0.001 to 1000 times, preferably 0.01 to 100 times, more preferably 0.1 to 10 times the weight of the HTRA1 inhibitory peptide.
- the pharmaceutical composition of the present invention also includes a pharmaceutical composition containing a modified product obtained by binding an HTRA1 inhibitory peptide or its conjugate in a liposome, or an HTRA1 inhibitory peptide or its conjugate and a liposome. .
- the excipient and carrier are usually liquid or solid, and are not particularly limited as long as they are water used for injection, physiological saline, artificial cerebrospinal fluid, and other substances used for oral or parenteral administration.
- physiological saline examples include neutral ones and those containing serum albumin.
- the buffer examples include Tris buffer prepared so that the final pH of the pharmaceutical composition is 7.0 to 8.5, acetate buffer prepared so as to be 4.0 to 5.5, and 5. Examples thereof include a citrate buffer prepared to be 0 to 8.0, a histidine buffer prepared to be 5.0 to 8.0, and the like.
- the pharmaceutical composition of the present invention is a solid, liquid, suspension or the like. Freeze-dried preparations can be mentioned. An excipient such as sucrose can be used to mold the lyophilized preparation.
- the administration route of the pharmaceutical composition of the present invention may be any of instillation, enteral administration, topical administration and parenteral administration.
- instillation on the conjunctiva intravitreal administration, intravenous administration, intraarterial administration, muscle
- examples thereof include internal administration, intradermal administration, subcutaneous administration, intraperitoneal administration, transdermal administration, intraosseous administration, and intraarticular administration.
- composition of such a pharmaceutical composition can be determined according to the administration method, the HTRA1 binding affinity of the HTRA1 inhibitory peptide, and the like.
- the dosage of the HTRA1 inhibitory peptide of the present invention or a conjugate thereof is not limited as long as it is a pharmacologically effective amount.
- the species of the individual, the type of disease, symptoms, sex, age, disease, HTRA1 protein of the peptide Although it can be appropriately determined depending on the binding affinity or its biological activity and other factors, it is usually 0.01 to 1000 mg / kg, preferably 0.1 to 100 mg / kg, 1 to 180 days. Can be administered once, or twice or more times a day.
- the form of the pharmaceutical composition includes injections (including lyophilized preparations and infusions), suppositories, nasal absorption preparations, transdermal absorption preparations, sublingual preparations, capsules, tablets, ointments, granules, aerosols. Examples thereof include pills, pills, powders, suspensions, emulsions, eye drops, and implantable preparations.
- a pharmaceutical composition containing an HTRA1 inhibitory peptide or a conjugate thereof as an active ingredient can be administered simultaneously with or separately from other drugs.
- a pharmaceutical composition containing an HTRA1 inhibitory peptide or a conjugate thereof as an active ingredient is administered after administration of another pharmaceutical agent, or another pharmaceutical agent is administered after such pharmaceutical composition is administered, or the pharmaceutical agent You may administer a composition and another pharmaceutical simultaneously.
- the HTRA1 inhibitory peptide or its conjugate and the other drug may be contained in either a single preparation or separate preparations (plural preparations).
- Examples of other medicaments used in combination with the pharmaceutical composition of the present invention include anti-VEGF agents, anti-inflammatory agents, inflammatory cytokine neutralizing agents, complement activation pathway inhibitors and the like.
- Anti-VEGF agents are classified into anti-VEGF antibodies, VEGF inhibitors, VEGF receptor antagonists, soluble VEGF receptors and the like, and include bevacizumab, ranibizumab, aflibercept, pegaptanib, brolucitumab and the like.
- the anti-inflammatory agent is not particularly limited as long as it can be locally administered to suppress intraocular or intra-articular inflammation.
- inflammatory cytokine neutralizing agents include anti-TNF ⁇ antibody, anti-interleukin-6 (hereinafter referred to as “IL-6”) antibody, anti-IL-6 receptor antibody, soluble TNF receptor, and the like.
- IL-6 anti-interleukin-6
- examples include infliximab, adalimumab, golimumab, certolizumab, tocilizumab, etanercept, and the like.
- the complement activation pathway inhibitor include lamparizumab. They are suitable for the treatment or prevention of diseases relating to HTRA1, but can also be combined with the pharmaceutical composition of the present invention in the treatment or prevention of diseases other than those relating to HTRA1.
- These other medications may be one, and two, three or more can be administered or received. These are collectively referred to as the pharmaceutical composition of the present invention and “combination with other pharmaceuticals” or “combination with other pharmaceuticals”, and in addition to the antibody of the present invention, a binding fragment thereof or a modified form thereof, other pharmaceuticals are used.
- Pharmaceutical compositions of the present invention that are included or used in combination with other therapies are also included in the present invention as embodiments of “combination with other medicaments” or “combination with other medicaments”.
- the present invention provides a method for treating or preventing a disease associated with HTRA1, such as age-related macular degeneration, and a pharmaceutical composition for treating or preventing the disease, comprising the step of administering an HTRA1-inhibiting peptide or a conjugate thereof.
- a disease associated with HTRA1 such as age-related macular degeneration
- a pharmaceutical composition for treating or preventing the disease comprising the step of administering an HTRA1-inhibiting peptide or a conjugate thereof.
- the use of the HTRA1 inhibitory peptide of the present invention or a conjugate thereof, and the use of the HTRA1 inhibitory peptide or a conjugate thereof for the treatment or prevention of the disease are also provided.
- a therapeutic or prophylactic kit containing the HTRA1 inhibitory peptide of the present invention or a conjugate thereof is also included in the present invention.
- the present invention includes a polynucleotide comprising a nucleotide sequence encoding an amino acid sequence of an HTRA1 peptide or conjugate thereof, a vector comprising the polynucleotide, or comprising the polynucleotide or the vector or inhibiting HTRA1 of the present invention
- pharmaceutical compositions comprising cells that express the peptide or conjugate thereof.
- such polynucleotides and vectors can be applied to gene therapy for diseases related to HTRA1, and such cells can be applied to cell therapy for diseases related to HTRA1, using known techniques.
- cells for cell therapy can be prepared by introducing such polynucleotides or vectors into autologous cells or allogeneic cells (allogeneic cells).
- polynucleotides and vectors are also encompassed by the present invention as compositions for preparing cell therapeutics.
- the embodiment of the pharmaceutical composition containing the polynucleotide, vector, cell, etc. of the present invention is not limited to the above.
- HTRA1 inhibitory peptide of the present invention or a conjugate thereof may have HTRA1 binding activity in addition to HTRA1 protease inhibitory activity.
- HTRA1 inhibitor search studies It can be used in various studies such as the use of HTRA1, detection of HTRA1, testing and diagnosis utilizing the detection, separation of HTRA1, reagents and other applications.
- detecting or separating HTRA1 at least one of the peptide of the present invention and HTRA1 can be immobilized.
- the present invention provides a test or diagnostic composition (hereinafter collectively referred to as “diagnostic composition”) comprising the peptide of the present invention or a conjugate thereof, which binds to HTRA1.
- the diagnostic composition of the present invention is useful for examination or diagnosis of diseases relating to HTRA1, expression of HTRA1 and the like.
- the examination or diagnosis in the present invention includes, for example, determination or measurement of morbidity risk, determination of the presence or absence of morbidity, measurement of the degree of progression or deterioration, and the effect of drug treatment with a pharmaceutical composition containing an HTRA1 inhibitory peptide or a conjugate thereof.
- the diagnostic composition of the present invention is useful for identifying an individual to which the peptide of the present invention or a conjugate thereof, a composition containing them, or a pharmaceutical composition containing them is administered.
- a diagnostic composition may contain a pH buffer, an osmotic pressure regulator, salts, a stabilizer, an antiseptic, a developer, a sensitizer, an aggregation inhibitor, and the like.
- the present invention relates to a method for testing or diagnosing a disease associated with HTRA1, use of the peptide of the present invention for preparing a diagnostic composition for the disease, the present invention binding to HTRA1 for testing or diagnosing the disease Of peptides or conjugates thereof are also provided.
- a test or diagnostic kit containing the peptide of the present invention or a conjugate thereof is also included in the present invention.
- a sandwich ELISA is preferable as a test or diagnostic method containing the peptide of the present invention that binds to HTRA1, but a normal ELISA method, RIA method, ELISPOT method, dot blot method, octalony method, CIE method, CLIA, flow cytometry, etc. Detection methods are available.
- the present invention also provides a method for detecting or measuring HTRA1 in a test sample.
- the diagnostic composition of the present invention can be used for these detection or measurement methods.
- HTRA1 in the sample is detected by contacting the HTRA1 inhibiting peptide or its conjugate with a test sample (step 1) and then measuring the amount or measurement of HTRA1 bound to the peptide (step 2). be able to.
- Step 1 for example, HTRA1 inhibitor peptide conjugated with Fc region of immunoglobulin is immobilized on magnetic beads via protein G, and a test sample is added thereto.
- HTRA1 is detected.
- a sample to which an artificial treatment such as a recombinant protein has been added can be provided.
- test samples derived from living organisms include blood, joint fluid, ascites, lymph fluid, cerebrospinal fluid, alveolar lavage fluid, saliva, sputum, tissue homogenate supernatant, tissue sections, and the like. It is not limited.
- the detection of HTRA1 can be performed not only in vitro but also in vivo.
- a pharmaceutically acceptable radionuclide or HTRA1 inhibitory peptide labeled with a luminescent material, or a conjugate thereof can be used.
- the subject is administered the labeled peptide or its conjugate.
- an image is taken using an imaging technique such as PET / CT, and the presence of HTRA1 is confirmed. Judgment or inspection can be given.
- the peptide or its conjugate contained in the diagnostic composition of the present invention binds to HTRA1, and preferably has HTRA1-specific binding activity.
- a method of identifying an individual to which a pharmaceutical composition of the present invention is administered is also encompassed by the present invention.
- HTRA1 in the sample derived from the individual is measured using the HTRA1 binding peptide of the present invention, and HTRA1 is detected in the sample, or HTRA1 detected in the sample derived from a healthy individual. If more HTRA1 is detected compared to the amount, the individual can be determined to be positive.
- the diagnostic composition of the present invention can be used.
- the individual suffers from or is at risk of an HTRA1-related disease.
- the pharmaceutical composition of the present invention can be administered to an individual who has been determined to be positive in such an identification method.
- HTRA1 can be specifically separated from a sample in which HTRA1 and other components are mixed, using the peptide of the present invention having a binding activity specific to HTRA1 or a conjugate thereof. Release of HTRA1 from peptides can be performed non-selectively under relatively high ionic strength, low pH, moderate denaturation conditions, the presence of chaotropic salts, etc., but does not reduce the protease activity of HTRA1 It is preferable to carry out with.
- the therapeutic or preventive agent for diseases related to HTRA1 preferably age-related macular degeneration, or the like, using HTRA1 inhibitory activity as an index
- a method for identifying candidates is provided.
- HTRA1 protease and substrate are incubated in the presence or absence of a test substance (or in the presence of a vehicle), step 1, HTRA1 protease activity in the presence and absence of the test substance is determined And / or when the HTRA1 protease activity in the presence of the test substance is small compared to the HTRA1 protease activity in the absence of the test substance, the test substance is treated with age-related macular degeneration.
- the step 3 which determines with a therapeutic agent or a preventive agent, or its candidate may be included.
- the test substance may be either peptidic or non-peptidic.
- Peptidic properties are not limited to SPINK2 mutants, but antibodies, peptides other than SPINK2 mutants having a protein backbone other than immunoglobulin, HTRA1 Substrate analogs and the like can be exemplified, and examples of non-peptide properties include, but are not limited to, synthetic low molecular weight compounds and nucleic acids.
- the above-described one or more steps can be suitably included in a method for identifying a substance having a retinal protective effect or a candidate thereof.
- the present invention also relates to a method for identifying a substance having a retinal protective effect or a candidate thereof.
- Rabbit retinopathy model The present invention uses a rabbit retinal disorder model due to a high fat diet (HGIh Fat Diet: hereinafter referred to as “HFD”) containing hydroquinone (hereinafter referred to as “HQ”), a method for producing the same, and the use of this model.
- HGIh Fat Diet hereinafter referred to as “HFD”
- HQ hydroquinone
- This model can be any white rabbit, such as NZW, JW, etc.
- HFD includes 0.1-2% (w / v) cholesterol, 1-10% (w / v) coconut oil, 1-10% (w / v) peanut oil, 1-10% (wv) soybean oil Add 10 to 20% (w / v) beef tallow, 10 to 50% (w / v) lard, 1 to 10% (w / v) corn oil in any amount However, it is not limited to these as long as it is suitable for the production of the model.
- the amount of HQ contained in HQ-HFD is 0 to 4% (w / v), preferably 0.5 to 3.5% (w / v), more preferably 1.0 to 3. It is 0% (w / v), more preferably 2.2 to 2.8% (w / v), and most preferably 2.4% (w / v).
- the feeding period of HQ-HFD is 3-6 months, preferably 3.5-5 months, more preferably 4 months. Continued feeding for more than 8 months should be avoided.
- the model of the present invention is preferable in terms of the size and function of the eyeball, as compared to a mouse model (Non-patent Documents 18 and 19).
- the rabbit model (Non-patent Document 20) required 8 months for its production, but the model of the present invention can be produced in a shorter period of time, and the RPE cells that were unclear in the conventional model Hypertrophy can be induced and is more preferable as a model of age-related macular degeneration.
- retinal pigment epithelial cells RPE cells
- RPE cells retinal pigment epithelial cells
- Such an identification method comprises (i) a step of measuring hypertrophy of retinal pigment epithelial cells in a rabbit of this model with and without administration of a test substance; and (ii) retinal pigment epithelial cells under administration of the test substance. A step of determining that the test substance is positive when hypertrophy is suppressed as compared to non-administration. For the measurement in step (i), the average area of retinal pigment epithelial cells and / or the number of enlarged retinal pigment epithelial cells is suitable.
- each operation relating to genetic manipulation is from “Molecular Cloning” (Sambrook, J., Fritsch, EF, and Maniatis, T., Cold Spring Harbor ab Press). Published in 1982 or published in 1989) and other methods described in experiments used by those skilled in the art, or in the case of using commercially available reagents and kits, according to the instructions for commercial products .
- Example 1 Preparation of HTRA1 Inhibitory Peptide (1-1) Construction of HTRA1 Inhibitory Peptide Expression Vector (1-1-1) Construction of pET 32a (modified) _HTRA1 Inhibitory Peptide
- an expression vector for HTRA1 inhibitory peptide with SPIK2 scaffold was used as a backbone .
- SEQ ID NOs: 4, 6, 8, 10, 12, 14, 16, 18, 20, and 22 an expression vector for HTRA1 inhibitory peptide with SPIK2 scaffold was used as a backbone .
- SEQ ID NOs: 4 the nucleotide sequence of each inhibitory peptide
- SPINK2 SEQ ID NO: 2
- the following primers and KOD-plus- Inhibitor fragments were amplified by PCR using (TOYOBO) ((94 ° C. 15 seconds, 60 ° C.
- Primer 1 5′-AAAAGAAATTCTGATCCGCAGTTTGGTCTGTTTAG-3 ′
- Primer 2 5'-AAAACTCGAGTTATGCGGCCGCAACGCGCCGCCACGGACC-3 '
- QIAGEN QIAquick Gel Extraction Kit
- Primer 3 5′-AAAAGGATCCCTGGACAAACGGTGATCCGCAGTTTGGTCTGTTTAG-3 ′
- Primer 4 5′-AAAACTCGAGTTTAGCCGCCGCACGGACATTTGCGAATAATTTTTA-3 ′
- pET 32a_HTRA1 inhibitory peptide_Kex2 was constructed by performing miniprep and sequence analysis. The operation was performed according to the method described in (1-1-1).
- (1-2) Expression Purification of HTRA1 Inhibitory Peptide The vector pET 32a (modified) _HTRA1 inhibitory peptide constructed in (1-1-1) was transformed into E. coli Origami B (DE3) (Novagen) and 0.1 mg / ml After culturing at 37 ° C. using 2YT medium containing ampicillin, IPTG (final concentration 1 mM) was added, and the cells were cultured at 16 ° C. overnight.
- lysate is prepared using BugBuster Master Mix (Novagen), and His tag fusion target protein is prepared using TALON Metal Affinity Resin (Clontech). Purified.
- thioredoxin tag and the desired protein were cleaved using a Thrombin Cleavage Capture Kit (Novagen) and purified using TALON.
- the HTRA1 inhibitory peptide was prepared by subjecting it to gel filtration chromatography (Superdex 75 10/300 GL) or reverse phase chromatography (YMC-Pack ODS-AM). A portion consisting of S tag and a linker (SEQ ID NO: 31: FIG. 43) at the N-terminus of the obtained peptide, a C-terminal 6-mer (SEQ ID NO: 32: FIG. 44) instead of Gly-Gly at the C-terminus, Each is conjugated.
- the vector pET 32a_HTRA1 inhibitory peptide_Kex2 constructed in (1-1-2) was transformed into E. coli Origami B (DE3) (Novagen) and used at 37 ° C. using 2YT medium containing 0.1 mg / ml ampicillin. After culturing, IPTG (final concentration 1 mM) was added and cultured overnight at 16 ° C. The next day, after collection by centrifugation (3,000 g, 20 minutes, 4 ° C.), a lysate is prepared using BugBuster Master Mix (Novagen), and a His tag fusion target protein is prepared using TALON Metal Affinity Resin (Clontech). Purified.
- thioredoxin tag and the desired protein were cleaved using Kex2 (Saccharomyces cerevisiae: Access CAA96143) and purified using TALON. Furthermore, the HTRA1 inhibitory peptide (tag, linker, etc. is conjugated to the N-terminus and C-terminus) by subjecting it to gel filtration chromatography (Superdex 75 10/300 GL) or reverse phase chromatography (YMC-Pack ODS-AM). Not) was prepared.
- Example 2 Evaluation of HTRA1 inhibitory activity of HTRA1 inhibitory peptide The sequence similarity of human / mouse / rat / monkey HTRA1 is shown in FIG.
- the primary sequences constituting the HTRA1 protease domain (204Gly-364Leu), which is an enzyme active domain, are completely identical in humans and monkeys.
- the human and mouse or rat HTRA1 protease domain sequences differ by one residue, it is speculated that the residue is located on the opposite side of the enzyme active center because of its structure. (FIG. 1). Therefore, since the HTRA1 protease domain has the same sequence regardless of human / mouse / rat / monkey species, the species is not clearly shown.
- HTRA1 protease domain HTRA1 (2-1-1) Construction of pET 21b_HTRA1 (cat) Protease donmain (158Gly-373Lys) excluding the N-terminal domain and PDZ domain of human HTRA1 (Q92743) HTRA1 (cat) expression vector was constructed using HTRA1 (cat). PCR method using human HTRA1 insertion plasmid (GeneCopoia; GC-M0558) as a template and the following primers and KOD-plus- (TOYOBO) (94 ° C. for 15 seconds, 60 ° C. for 30 seconds, 68 ° C. for 45 seconds) ⁇ 30 cycles) To amplify the desired DNA fragment.
- Primer 5 5'-AAACATATGGGGCAGGAAGATCCCAACAGTTTGC-3 ' Primer 6: 5′-AAACTCGAGTTTGGCCTGTCGGTCATGGGAACTC-3 ′
- QIAGEN QIAquick Gel Extraction Kit
- the prepared DNA fragment and pET 32a were treated with restriction enzymes NdeI (NEB) and XhoI (NEB) at 37 ° C. for 1 hour or longer, and after agarose gel electrophoresis, the desired DNA fragment was excised and QIAquick PCR Purified by Purification Kit (QIAGEN).
- ligation reaction was performed by reacting each purified fragment overnight at 16 ° C.
- Ligation solution was added to Escherichia coli JM109 (TOYOBO), allowed to stand on ice for 30 minutes, then heat-treated at 42 ° C. for 45 seconds, then allowed to stand on ice for 5 minutes, and placed on a 2YT plate containing 0.1 mg / ml ampicillin.
- E. coli was transformed by stationary culture at 37 ° C. overnight.
- pET 21b_HTRA1 (cat) was constructed by performing miniprep and sequence analysis. The operation was performed according to the method described in (1-1-1).
- HTRA1 (cat) The constructed pET 21b_HTRA1 (cat) was transformed into E. coli BL21 (DE3) (Novagen) at 37 ° C. using 2YT medium containing 0.1 mg / ml ampicillin. After culturing, IPTG (final concentration 1 mM) was added and cultured overnight at 28 ° C. After collection, the cells were suspended in a phosphate buffer (50 mM sodium phosphate, 300 mM NaCl) containing 1 mg / ml lysozyme, and lysate was prepared by ultrasonic disruption.
- phosphate buffer 50 mM sodium phosphate, 300 mM NaCl
- the desired His tag fusion protein was recovered using TALON (Clontech) and subjected to gel filtration chromatography (Superdex 200 10/300 GL) to purify HTRA1 (cat).
- TALON Click-ontech
- Superdex 200 10/300 GL gel filtration chromatography
- HTRA1 cat.
- 2 Preparation of full-length HTRA1 HTRA1 (full)
- 2-2-1 Construction of pcDNA3.1_HTRA1 (full) _His Using synthesized human HTRA1 (Q92743) DNA (GENEART) as a template, the following primers and KOD-plus
- the desired DNA fragment was amplified by PCR using (TOYOBO) ((94 ° C. 15 seconds, 60 ° C. 30 seconds, 68 ° C. 90 seconds) ⁇ 30 cycles).
- Primer 7 5'-AAAAGAAATTCGCCACCATGCAGATTCCTAGAGCCG-3 ' Primer 8: 5′-AAAAACTCGAGTCAGTGGTGATGGGTGGGTGGGCCGG-3 ′
- QIAGEN QIAquick Gel Extraction Kit
- the prepared DNA fragment and pcDNA3.1 are treated with restriction enzymes EcoRI (NEB) and XhoI (NEB) at 37 ° C. for 1 hour or longer.
- EcoRI EcoRI
- XhoI XhoI
- the prepared DNA fragment and pcDNA3.3 (vector using ThermoFisher Scientific) as a template were treated with restriction enzymes EcoRI (NEB) and XhoI (NEB) at 37 ° C. for 1 hour or longer, and after agarose gel electrophoresis, desired The DNA fragment was excised and purified by QIAquick PCR Purification Kit (QIAGEN). Using T4 DNA Ligase (NEB), ligation reaction was performed by reacting each purified fragment overnight at 16 ° C.
- Primer 21 5′-CCATCATCAACTACGGCAACGCCGGGCGGACCCCTCGTGAACC-3 ′ (SEQ ID NO: 55: FIG. 76)
- Primer 22 5'-GGTTCACGAGGGGTCCGCCCCGGTTGCCGTTAGTTGATGTGGG-3 '(SEQ ID NO: 56: FIG. 77)
- Escherichia coli JM109 TOYOBO
- pcDNA3.3_HTRA1 (S328A) _FLAG_His was constructed by performing miniprep and sequence analysis.
- HTRA1 inhibitory activity of HTRA1 inhibitory peptide (3-1) Evaluation of HTRA1 inhibitory activity of HTRA1 inhibitory peptide using peptide substrate Substrate peptide H2-Opt (Mca-IRRVSYSFK (Dnp) K) (Peptide Institute: SEQ ID NO: 54) 8) was dissolved in DMSO to a concentration of 10 mM, diluted with Assay buffer (50 mM forate, 150 mM NaCl, pH 8.5) and used at a final concentration of 10 ⁇ M.
- Assay buffer 50 mM forate, 150 mM NaCl, pH 8.5
- HTRA1 HTRA1 (cat) or HTRA1 (full)
- Assay buffer 50 ⁇ L
- HTRA1 inhibitory peptide 25 ⁇ L
- Assay buffer 50 ⁇ L
- HTRA1 inhibitory peptide 25 ⁇ L
- Assay buffer 50 ⁇ L
- HTRA1 inhibitory peptide 25 ⁇ L
- Assay buffer 50 ⁇ L
- Proteo Save registered trademark
- the substrate peptide degradation rate of HTRA1 inhibitory peptide at each concentration was calculated, and the HTRA1 (cat) and HTRA1 (full) inhibitory activity of each HTRA1 inhibitory peptide was evaluated with the degradation rate of the inhibitor concentration of 0 nM as 100% (FIG. 2 and 3).
- IC50 50% inhibitory concentration
- GraphPad Prism version 5.0; GraphPad Software Inc.
- Figs. 2A to C and Fig. 3 did not show HTRA1 inhibitory activity (FIG. 2D).
- HTRA1 inhibitory activity of HTRA1 inhibitory peptide was evaluated.
- HTRA1 cat
- Assay buffer 50 mM Tris, 150 mM NaCl, pH 8.0
- human Vitronectin BD Biosciences; 354238
- Assay buffer 50 mM Tris, 150 mM NaCl, pH 8.0
- human Vitronectin BD Biosciences; 354238
- Assay buffer 50 mM Tris, 150 mM NaCl, pH 8.0
- HTRA1 inhibitory peptide was 0 to 25 ⁇ M
- the final concentration of HTRA1 (cat) was 1 ⁇ M
- the final concentration of human Vitronectin was 1 ⁇ M.
- Western blot analysis Human Vibrin Antibody (R & D Systems; MAB2349) was used as the primary antibody, and Anti-Mouse IgG and HRP-Linked Whole Ab Sheep (GE Health 93) were used as the secondary antibody.
- HTRA1 inhibitory peptide strongly inhibited HTRA1 (cat) even when human vitronetin was used as a substrate (FIG. 4).
- (3-3) Specificity evaluation of HTRA1-inhibiting peptides was evaluated using the cleavage of the substrate peptide as an index. Similarly to the method described in (3-1), 25 ⁇ L each of protease diluted with Assay buffer and sample (final concentration: 1 ⁇ M) were mixed, reacted at 37 ° C. for 20 minutes, and then added with 50 ⁇ L of substrate diluted with Assay buffer. The fluorescence signal (excitation 380 nm / emission 460 nm) was measured with Enspire (PerkinElmer).
- Proteo Save (registered trademark) SS96F black plate (Sumitomo Bakelite Co., Ltd.) was used for the reaction and measurement.
- the combinations of protease and substrate used for the specificity evaluation are as follows.
- Bovine trypsin inhibitory activity evaluation of Bovine trypsin inhibitory activity; final concentration 5 nM trypsin (Pierce; 20233), final concentration 100 ⁇ M Substrate peptide Boc-VPR-AMC Fluorogenic Peptide Substrate (R & D Systems; ES011) Evaluation of Bovine ⁇ -chymotrypsin inhibitory activity; final concentration of 10 nM chymotrypsin (Worthington Biochemical Corporation; LS001434), final concentration of 100 ⁇ M substrate peptide Suc-Leu-Leu-Val-Tyr-MCA (Peptide Institute, Inc.) 3120 Human tryptase inhibitory activity evaluation; final concentration 1 nM tryptase (Sigma-Aldrich; T7063), final concentration 100 ⁇ M substrate peptide Boc-Phe-Ser-Arg-MCA (Peptide Institute, Inc .; 3107-v) Evaluation of human chymase inhibitory activity; final concentration 100
- each HTRA1 inhibitory peptide did not suppress protease activity against any protease, indicating that the HTRA1 inhibitory peptide has an inhibitory action specific to HTRA1 (FIG. 5).
- Example 4 Analysis of HTRA1 inhibitory peptide using X-ray crystal structure (4-1) Preparation of HTRA1 (cat) / HTRA1 inhibitory peptide complex According to the method 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 was prepared. Both were mixed under the conditions of 20 mM Tris-HCl, 150 mM NaCl, pH 7.6, and the complex was isolated and purified by gel filtration chromatography (Superdex 200 10/300 GL).
- the phase was determined by a molecular replacement method using Serine protein HTRA1 (PDB ID: 3NZI) as a template, and after structural refinement, a complex crystal of HTRA1 (cat) / the peptide was determined with a resolution of 2.6 A.
- the unit cell contained one molecule each of HTRA1 and SPINK2.
- SPINK2 molecule a partial molecular model including an interaction site with HTRA1 (cat) was constructed based on the sequence information and the observed electron density. It was confirmed that the HTRA1 inhibitory peptide was bound to a region containing the HTRA1 enzyme activity center (FIGS. 6 and 7).
- FIG. Retinal protection effect by HTRA1 inhibition in rat light irradiation retinal damage model (5-1) Preparation of rat light irradiation retinal damage model
- Rat light irradiation retinal damage model is a model that induces cell death of retinal photoreceptor cells by light irradiation, and retina It is widely used as a model animal for degeneration (Daniel T. Organisciak et al., (1996) Invest Ophthalmol Vis Sci. 37 (11): 2243-2257).
- Rats dark-adapted for 72 hours were instilled with 0.5% (W / V) tropicamide-0.5% phenylephrine hydrochloride ophthalmic solution under dark adaptation and then irradiated with 5500 Lux white light for 3 hours. After irradiation, dark adaptation was carried out again for about 24 hours, and after that, the condition was returned to normal breeding conditions and reared for 2 days. After euthanasia, the eyeball was removed and immersed in 3.7% (W / V) formaldehyde-0.5-1% (W / V) methanol-0.2% (W / V) picric acid fixative for 24 hours or more. Fixed. After embedding in paraffin, sliced sections were prepared.
- Sections were stained with hematoxylin-eosin, and retinal damage was evaluated by counting the number of nuclei contained in the outer granular layer of the retinal tomography. It was revealed that the number of nuclei contained in the outer granule layer was significantly reduced by light irradiation in the rat model of light irradiation retinopathy.
- (5-2) Confirmation of extracellular HTRA1 expression during retinal injury
- vitreous humor was collected from the model rat prepared in (5-1) and subjected to Western blot analysis. HTRA1 expression level was evaluated.
- the vitreous humor is subjected to SDS-PAGE under reducing conditions, using the primary antibody Human HTRA1 / PRSS11 Antibody (R & D Systems; AF2916) and the secondary antibody Sheep IgG Horsadish Peroxidase-ConjudationATR did.
- R & D Systems R & D Systems; AF2916
- Sheep IgG Horsadish Peroxidase-ConjudationATR did.
- an increase in the amount of HTRA1 in the vitreous humor was observed in the light-irradiated group. Therefore, in this model, it is considered that HTRA1 is involved in the process of retinal damage caused by light irradiation (Fig. 9).
- HTRA1 inhibitory peptide H308 Retinal protective effect of HTRA1 inhibitory peptide in rat light irradiation retinal injury model 5 ⁇ L of HTRA1 inhibitory peptide H308 at a concentration of 0.04 mg / mL or 0.2 mg / mL under anesthesia immediately before light irradiation to rats It was administered intravitreally.
- the number of cases in the physiological saline administration group was 4, and the number of cases in the other groups was 5.
- Example 6 Evaluation of HTRA1 Inhibitory Peptide Derivatives (6-1) Construction of pET 32a_HTRA1 Inhibitory Peptide H308_S16A_Kex2 Using HTRA1 Inhibitory Peptide H308 as a template, amino acid in which 16th Ser in the amino acid sequence shown in SEQ ID NO: 9 (FIG. 21) was substituted with Ala A derivative S16A having the sequence was prepared. Fragment C was amplified by the PCR method ((94 ° C. 15 seconds, 60 ° C. 30 seconds, 68 ° C. 15 seconds) ⁇ 30 cycles) using the following primers and KOD-plus- (TOYOBO).
- Fragment D was obtained by PCR ((94 ° C. 15 seconds, 60 ° C. 30 seconds, 68 ° C. 20 seconds) ⁇ 30 cycles) using the HTRA1 inhibitory peptide H308 as a template and the following primers and KOD-plus- (TOYOBO). was amplified.
- Primer 14 5′-GCGGACCATGCTGGTATGGCATGGTTGCTCTGTATGAAC-3 ′
- Primer 15 5′-AAAACTCGAGTAGTAGCCGCCGCACGGCACCTGGCGAATAA-3 ′
- the desired DNA fragment was amplified by PCR using fragments C and D, the following primers, and KOD-plus- (TOYOBO) (94 ° C. for 15 seconds, 60 ° C. for 30 seconds, 68 ° C. for 20 seconds) ⁇ 30 cycles).
- Primer 16 5′-AAAAGGATCCCTGGACAAACGTGATCCGCAGTTTGGTCTGTTTAG-3 ′ Primer 15
- the prepared DNA fragment and pET 32a (Novagen) were treated with restriction enzymes BamHI (NEB) and XhoI (NEB) at 37 ° C. for 1 hour or longer.
- BamHI NEB
- XhoI NEB
- the desired DNA fragment was excised, and QIAquick PCR Purified by Purification Kit (QIAGEN).
- ligation reaction was performed by reacting each purified fragment overnight at 16 ° C.
- Ligation solution was added to Escherichia coli JM109 (TOYOBO), allowed to stand on ice for 30 minutes, then heat-treated at 42 ° C. for 45 seconds, then allowed to stand on ice for 5 minutes, and placed on a 2YT plate containing 0.1 mg / ml ampicillin. After seeding, E. coli was transformed by stationary culture at 37 ° C. overnight. After culturing the transformed E. coli, “pET 32a_HTRA1 inhibitory peptide H308_S16A_Kex2” was constructed by performing miniprep and sequence analysis.
- Fragments C and D 4 types of target fragments were amplified by PCR using the following primers and KOD-plus- (TOYOBO) (94 ° C. 15 seconds, 60 ° C. 30 seconds, 68 ° C. 20 seconds) ⁇ 30 cycles) .
- TOYOBO KOD-plus-
- D1G production primer primer 17 5′-AAAAGGATCCCTGGACAAACGTGGCCCGCAGTTTGTCTGTTTAG-3 ′
- D1S production primer 18 5′-AAAAGGATCCCTGGACAAACGTAGCCCCCAGTTTGGTCTGTTTAG-3 ′
- Primer 15 D1E preparation primer 19 5′-AAAAGGATCCCTGGACAAACGGTGAACCGCAGTTTGGTCTGTTTTAG-3 ′
- Primer 15 D1SLI production primer primer 20 5′-AAAAGGATCCCTGGACAAACGTAGCCCTATTCCGCAGTTTGGTCTGTTTTAG-3 ′
- Primer 15 The four amplified fragments were subjected to agarose gel electrophoresis, and then the desired DNA fragment was excised and DNA was prepared by QIAquick Gel Extraction Kit (QIAGEN).
- the prepared DNA fragment and pET 32a were treated with restriction enzymes BamHI (NEB) and XhoI (NEB) at 37 ° C. for 1 hour or longer. After agarose gel electrophoresis, the desired DNA fragment was excised, and QIAquick PCR Purified by Purification Kit (QIAGEN). Using T4 DNA Ligase (NEB), ligation reaction was performed by reacting each purified fragment overnight at 16 ° C. Ligation solution was added to Escherichia coli JM109 (TOYOBO), allowed to stand on ice for 30 minutes, then heat-treated at 42 ° C.
- BamHI BamHI
- XhoI XhoI
- E. coli was transformed by stationary culture at 37 ° C. overnight. After culturing the transformed E. coli, miniprep and sequence analysis were performed, so that “pET 32a_HTRA1 inhibitory peptide H308_D1G_S16A_Kex2” “pET 32a_HTRA1 inhibitory peptide H308_D1S_S16A_Kex2” “pET 32a_HSL1E2A32 did. The operation was performed according to the method described in (1-1-1).
- HTRA1 inhibitory peptide derivatives were prepared by subjecting to gel filtration chromatography (Superdex 75 10/300 GL) or reverse phase chromatography (YMC-Pack ODS-AM). The amino acid sequences of the derivatives are described in SEQ ID NOs: 23 to 27 (FIGS. 35 to 39).
- 6-4 Evaluation of HTRA1 Inhibitory Peptide Derivatives According to the method described in (3-1), HTRA1 (cat) inhibitory activity was measured. As a result, all the derivatives were equivalent to H308 (FIG. 11).
- Example 7 Evaluation of binding property between HTRA1 inhibitory peptide and HTRA1 (cat) Using the three HTRA1 inhibitory peptides (H308, H321AT, H322AT) prepared in Example (1-2) and HTRA1 (cat) prepared in (2-1) The binding was evaluated by immunoprecipitation. After 2.5 ⁇ g of each HTRA1 inhibitory peptide and 10 ⁇ g of HTRA1 (cat) were reacted at room temperature for 30 minutes, 10 ⁇ L of TALON Metal Affinity Resin (Clontech) was added. Furthermore, Resin after 30 minutes of reaction was recovered as an immunoprecipitation (IP) fraction and subjected to SDS-PAGE to evaluate binding. PBS was used as a reaction buffer.
- IP immunoprecipitation
- Substrate peptide H2-Opt (Mca-IRRVSYSFK (Dnp) K) (Peptide Institute, Inc .: SEQ ID NO: 54, FIG.
- HTRA1 diluted with Assay buffer (HTRA1 (cat) or HTRA1 (full); Example 2) and HTRA1 inhibitory peptide were mixed at 37 ° C. for 20 minutes, and then 50 ⁇ L of substrate diluted with Assay buffer was added.
- the fluorescence signal (excitation 328 nm / emission 393 nm) was measured with Enspire (PerkinElmer).
- the final concentration of HTRA1 was 10 nM, the final concentration of HTRA1 inhibitory peptide was 1.875 to 1,000 nM, and Proteo Save (registered trademark) SS96F black plate (Sumitomo Bakelite Co., Ltd.) was used for the reaction and measurement.
- the substrate peptide degradation rate of HTRA1 inhibitory peptide at each concentration was calculated, and the HTRA1 (cat) and HTRA1 (full) inhibitory activity of each HTRA1 inhibitory peptide was evaluated with the degradation rate at an inhibitor concentration of 0 nM as 100%.
- IC50 50% inhibitory concentration
- GraphPad Prism version 5.0; GraphPad Software Inc.
- Proteo Save (registered trademark) SS96F black plate (Sumitomo Bakelite Co., Ltd.) was used for the reaction and measurement, and 25 ⁇ L each of the protease diluted with Assay buffer and the sample (final concentration 1 ⁇ M) were mixed and reacted at 37 ° C. for 20 minutes. After that, 50 ⁇ L of the substrate diluted with Assay buffer was added, and the fluorescence signal was measured with Enspire (PerkinElmer).
- Human chymotrypsin inhibitory activity evaluation final concentration 10 nM chymotrypsin (Sigma-Aldrich; C8946), final concentration 10 ⁇ M substrate peptide Suc-Leu-Leu-Val-Tyr-MCA (Peptide Institute, Inc .; 3120-v), fluorescence signal excitation 3 / Emission 460 nm.
- Human MMP-2 inhibitory activity evaluation final concentration 1 nM tryptase (Calbiochem; PF023), final concentration 100 ⁇ M substrate peptide MOCAx-KPLGL-A2pr (Dnp) -AR (Peptide Institute, Inc .; 3226-v), fluorescence signal excitation 328 nm / emission 393nm.
- TPP1 inhibitory activity evaluation final concentration 0.5 ⁇ g / mL TPP1 (Calbiochem; 2237-SE), final concentration 200 ⁇ M substrate peptide AAF-MCA (Peptide Institute, Inc .; 3201-v), fluorescence signal excitation 380 nm / emission 460 nm.
- the cross-reactivity to proteases other than HTRA1 was evaluated using the degradation of the peptide substrate as an index. For any protease, protease activity was not suppressed at a final concentration of 1 uM of each HTRA1 inhibitory peptide, indicating that the HTRA1 inhibitory peptide has an inhibitory action specific to HTRA1 (FIG. 68).
- Example 9 Evaluation of binding between HTRA1 inhibitory peptide and HTRA1 (cat) Immunoprecipitation using the three HTRA1 inhibitory peptides prepared in Example 6 and HTRA1 (cat) prepared in (2-1) according to the procedure of Example 7 The binding was evaluated by the method.
- TALON was reacted with each of the three types of HTRA1 inhibitory peptides or HTRA1 (cat)
- a band of only HTRA1 (cat) fused with His tag was detected in the input lane.
- bands of inhibitory peptides and enzymes were detected only in the IP lane in which each inhibitory peptide was reacted with HTRA1 (cat). Therefore, it was confirmed that each of the three HTRA1 inhibitory peptides binds to HTRA1 (cat) (FIG. 69).
- Example 10 Retinal protective effect by inhibition of HTRA1 in rat model of light irradiation retinal injury (Part 2) Using the rat light irradiation retinal injury model constructed in Example (5-1), the retinal protective effect of the three HTRA1 inhibitor peptides prepared in Example 6 was evaluated. The operation followed Example 5. In each group, the number of cases was 6. The results of retinal pathology evaluation are shown in FIG. The three HTRA1 inhibitory peptides showed a marked inhibitory effect on the decrease in the number of nuclei contained in the outer granule layer caused by light irradiation.
- Example 11 Protective effect of retinal pigment epithelial cells by HTRA1 inhibition in rabbit retinal injury model with hydroquinone-containing high fat diet load (11-1) Preparation of rabbit retinal injury model with hydroquinone-containing high fat diet load High fat diet (HFD) And hydroquinone (HQ) are retinal disorder models that induce oxidative stress by pro-oxidants and cause retinal disorders, and models have been reported only in mice (Diego G. Espinosa-Heidmann et al. (2006) Invest Ophthalmol Vis Sci. 47 (2): 729-737).
- HFD High fat diet
- HQ hydroquinone
- FIG. 71 shows stained images of RPE cells (FIG.
- FIG. 72 shows the result of evaluating RPE cell hypertrophy of the model animal 4 months after the start of feeding.
- the average area of RPE cells FIG. 72 (A)
- the number of enlarged RPE cells with a cell area of 1500 ⁇ m 2 or more FIG. 72
- HTRA1 inhibitor against RPE cell hypertrophy in any index The inhibitory effect was shown.
- FIG. 71 (E) an increase in HTRA1 was observed in the vitreous humor of the model, suggesting the involvement of HTRA1 in the RPE cell damage process by HFD-HQ.
- the HTRA1 inhibitory peptide is useful as an anti-aging macular degeneration agent, and this test has been shown to be particularly useful for the prevention of atrophic age-related macular degeneration.
- the presence of HTRA1 inhibitory peptide was observed in the normal rabbit retina administered with HTRA1 inhibitory peptide, indicating a high tissue permeability of HTRA1 inhibitory peptide.
- Example 12 Suppressive effect of HTRA1 inhibitory peptide in VEGF mRNA induction test using human retinal pigment epithelial cells
- ARPE-19 ARPE-19 cells were treated with 10% Fetal bovine serum (FBS), Pencillin-Streptomycin (DRM / French Scientific) -containing DMEM / FEM Using 12 medium (Wako Pure Chemical Industries, Ltd.), the cells were cultured at 37 ° C. and 5% CO 2 in 12 mm Transwell with 0.4 ⁇ m Pole Polymer Membrane Insert, Sterile (Corning) until confluent.
- FBS Fetal bovine serum
- DRM / French Scientific Pencillin-Streptomycin
- HTRA1 (Example 2-2), HTRA1 protease inactive mutant HTRA1 (S328A) (Example 2-3), or HTRA1 inhibitory peptide H308_D1G_S16A (Example 6) is each at a final concentration of 1 ⁇ M in the upper and lower layers of the chamber. Added as follows.
- HTRA1 inactive mutant HTRA1 S328A
- H 2 O 2 Normal Human Serum Complement and HTRA1 inactive mutant HTRA1
- H308_D1G_S16A HTRA1 inhibitory peptide H308_D1G_S16A
- VEGF induction from retinal pigment epithelial cells is considered to be important for the formation of wet age-related macular degeneration (Klettner A. et al., (2009) Grafes Arch Clin Exp Ophthalmol. 247: 1487-1492).
- pathological VEGF inductions are involved in the maintenance of the pathology, and administration of the peptide of the present invention such as HTRA1 inhibitor peptide H308_D1G_S16A is exudative age-related macular degeneration It is effective for prevention and treatment.
- HUVEC Human Umbilical Vein Endothelial Cell (HUVEC) Migration Inhibitory Effect by HTRA1 Inhibitory Peptide H308_DIG_S16A (13-1) HUVEC Migration Test HUVEC (Kurabo Industries) was added to 0.1% BSA-containing EBM TM- 2 basal medium (Lonza Walkersville) with serum and VEGF After culturing at 37 ° C. under 5% CO 2 for 18 hours in medium supplemented with EGM TM -2 SingleQuots TM addition factor set (serum-free EGM containing 0.1% BSA), 0.1% BSA contained Serum-free EGM was prepared to 4 ⁇ 10 5 cells / mL.
- Example 14 Retinal protective effect of HTRA1 inhibitory peptide in rabbit retinopathy model (2) Using the rabbit retinal injury model prepared and evaluated in Examples (11-1) to (11-3), the HTRA1 inhibitory peptide H308 prepared in Example 1 or the three HTRA1 inhibitory peptides prepared in Example 6 The therapeutic effect on one type of retinal disorder is evaluated. 50 ⁇ L of a 40 mg / mL inhibitory peptide solution is administered intravitreally to one eye of a model animal under anesthesia and reared for 2 months. A saline solution is administered intravitreally to the fellow eyes. In each group, the number of cases is 5.
- the HTRA1-inhibiting peptide is useful as an anti-aging macular degeneration agent, and particularly useful in the treatment of atrophic age-related macular degeneration in this Example.
- the peptide provided by the present invention and a pharmaceutical composition containing the peptide are useful for the treatment or prevention of age-related macular degeneration and the like.
- SEQ ID NO: 1 amino acid sequence of human SPINK2 (FIG. 13)
- SEQ ID NO: 2 nucleotide sequence encoding the amino acid sequence of human SPINK2 (FIG. 14)
- SEQ ID NO: 3 amino acid sequence of peptide H218 (FIG. 15)
- SEQ ID NO: 4 Nucleotide sequence encoding the amino acid sequence of peptide H218 (FIG. 16)
- SEQ ID NO: 6 nucleotide sequence encoding the amino acid sequence of peptide H223 (FIG. 18)
- SEQ ID NO: 7 amino acid sequence of peptide H228 (FIG.
- SEQ ID NO: 8 nucleotide sequence encoding the amino acid sequence of peptide H228 (FIG. 20)
- SEQ ID NO: 9 Amino acid sequence of peptide H308 (FIG. 21)
- SEQ ID NO: 10 nucleotide sequence encoding the amino acid sequence of peptide H308 (FIG. 22)
- SEQ ID NO: 11 amino acid sequence of peptide H321 (FIG. 23)
- SEQ ID NO: 12 nucleotide sequence encoding the amino acid sequence of peptide H321 (FIG. 24)
- SEQ ID NO: 14 nucleotide sequence encoding the amino acid sequence of peptide H322 (FIG. 26)
- SEQ ID NO: 15 amino acid sequence of peptide derivative H308AT (FIG. 27)
- SEQ ID NO: 16 nucleotide sequence encoding the amino acid sequence of peptide derivative H308AT (FIG. 28)
- SEQ ID NO: 17 Amino acid sequence of peptide derivative H321AT (FIG. 29)
- SEQ ID NO: 18 nucleotide sequence encoding the amino acid sequence of peptide derivative H321AT (FIG. 30)
- SEQ ID NO: 20 nucleotide sequence encoding the amino acid sequence of peptide derivative H322AT (FIG. 32)
- SEQ ID NO: 21 amino acid sequence of peptide M7 (FIG. 33)
- SEQ ID NO: 22 nucleotide sequence encoding the amino acid sequence of peptide M7 (FIG. 34)
- SEQ ID NO: 23 Amino acid sequence of peptide derivative H308_S16A (FIG. 35)
- SEQ ID NO: 24 Amino acid sequence of peptide derivative H308_D1G_S16A (FIG. 36)
- SEQ ID NO: 25 Amino acid sequence of peptide derivative H308_D1S_S16A (FIG.
- SEQ ID NO: 26 Amino acid sequence of peptide derivative H308_D1E_S16A (FIG. 38)
- SEQ ID NO: 27 Amino acid sequence of peptide derivative H308_D1SLI_S16A (FIG. 39)
- SEQ ID NO: 28 Amino acid sequence of peptide derivative H321AT_D1G_S16A (FIG. 40)
- SEQ ID NO: 29 Amino acid sequence of peptide derivative H322AT_D1G_S16A (FIG. 41)
- SEQ ID NO: 30 General formula of HTRA1 inhibitory peptide (FIG. 42)
- SEQ ID NO: 31 amino acid sequence consisting of S tag and linker (FIG.
- SEQ ID NO: 32 amino acid sequence of C-terminal 6-mer (FIG. 44)
- SEQ ID NO: 33 nucleotide sequence of primer 1 (FIG. 45)
- SEQ ID NO: 34 nucleotide sequence of primer 2 (FIG. 46)
- SEQ ID NO: 35 nucleotide sequence of primer 3 (FIG. 47)
- SEQ ID NO: 36 nucleotide sequence of primer 4 (FIG. 48)
- SEQ ID NO: 38 nucleotide sequence of primer 6 (FIG. 50)
- SEQ ID NO: 39 nucleotide sequence of primer 7 (FIG.
- SEQ ID NO: 40 nucleotide sequence of primer 8 (FIG. 52)
- SEQ ID NO: 41 nucleotide sequence of primer 9 (FIG. 53)
- SEQ ID NO: 42 nucleotide sequence of primer 10 (FIG. 54)
- SEQ ID NO: 43 nucleotide sequence of primer 11 (FIG. 55)
- SEQ ID NO: 44 nucleotide sequence of primer 12 (FIG. 56)
- SEQ ID NO: 47 nucleotide sequence of primer 15 (FIG.
- SEQ ID NO: 48 nucleotide sequence of primer 16 (FIG. 60)
- SEQ ID NO: 49 nucleotide sequence of primer 17 (FIG. 61)
- SEQ ID NO: 50 nucleotide sequence of primer 18 (FIG. 62)
- SEQ ID NO: 51 nucleotide sequence of primer 19 (FIG. 63)
- SEQ ID NO: 52 nucleotide sequence of primer 20 (FIG. 64)
- SEQ ID NO: 54 Amino acid sequence of H2-Opt (FIG. 8)
- SEQ ID NO: 56 nucleotide sequence of primer 22 (FIG. 77)
Abstract
Description
AMDは加齢に伴う慢性変性疾患であり、中心視力の低下を特徴とする。後天的失明原因疾患として欧米では第1位、日本では緑内障、糖尿病網膜症、網膜色素変性に次いで第4位の患者数である(非特許文献12)。AMD患者のリンパ球、あるいは網膜色素上皮細胞でmRNAおよび蛋白質のレベルでHTRA11が上昇している(非特許文献13)。また、AMDの前駆病変であるドルーゼン、変性した網膜色素上皮細胞、あるいは新生血管膜において、HTRA1の蛋白質の発現が上昇しているとの報告がある(非特許文献11及び14~16)。また、網膜剥離、網膜静脈閉塞症、硝子体出血、黄斑円孔等の硝子体液中にHTRA1蛋白質が検出されており、その値は血管新生マーカーであるVEGFと連動しているとする報告もある(非特許文献17)。さらに、HTRA1トランスジェニックマウスでは、Fibulin5やTropoelastinといった基底膜を構成する蛋白質の分解およびブルッフ膜のElastic layerの断片化が観察されている(非特許文献9)。しかし、上記疾患を治療するため、並びに網膜を保護するために、HTRA1のプロテアーゼ活性の阻害が有効であるか否かについては直接的に示されていない。
AMDを評価する動物モデルとしては、マウスとウサギのものが知られているが(非特許文献18、19、20)。ヒト眼疾患に外挿し得るモデルとしてより好ましいウサギについて(非特許文献21)、従来のモデルでは、網膜・脈絡膜内の異常所見を観察するのみで、網膜色素上皮細胞(RPE細胞)の機能異常などを評価することは難しい上、モデル形成に8ヶ月という長期間を要する等問題点があった(非特許文献20)。
(1)
配列番号30(図42)で示されるアミノ酸配列を含み、且つ、ヒトHTRA1の有するプロテアーゼ活性を阻害する、SPINK2変異体ペプチド、
(2)
1番目のXaa(X1)はAsp、Glu、Ser、Gly,又はIle、2番目のXaa(X2)はAla、Gly、Leu、Ser又はThr、3番目のXaa(X3)はAsp、His、Lys、Met又はGln、4番目のXaa(X4)はAsp、Phe、His、Ser又はTyr、5番目のXaa(X5)はAla、Asp、Glu、Met又はAsn、6番目のXaa(X6)はMet又はTrp、7番目のXaa(X7)はGln、Trp、Tyr又はVal、8番目のXaa(X8)はPhe、Leu又はTyr、9番目のXaa(X9)はPhe又はTyr、10番目のXaa(X10)はAla、Glu、Met又はVal、並びに、11番目のXaa(X11)はAla、Thr又はValである、(1)記載のペプチド、
(3)
配列番号3、5、7、9、11、13、15、17、19、21及び23乃至29(図15、図17、図19、図21、図23、図25、図27、図29、図31、図33及び、図35乃至41)のいずれか一つで示されるアミノ酸配列を含む、(1)又は(2)記載のペプチド、
(4)
配列番号30(図42)で示されるアミノ酸配列のアミノ末端側に1乃至3個のアミノ酸がペプチド結合してなるアミノ酸配列を含む、(1)乃至(3)のいずれか一つに記載のペプチド、
(5)
配列番号30(図42)で示されるアミノ酸配列のカルボキシル末端側に1又は2個のアミノ酸がペプチド結合してなるアミノ酸配列を含む、(1)乃至(4)のいずれか一つに記載のペプチド、
(6)
3つのジスルフィド結合を有し、ループ構造、αへリックス及びβシートを含むことで特徴付けられる立体構造を有する、(1)乃至(5)のいずれか一つに記載のペプチド、
(7)
(1)乃至(6)のいずれか一つに記載のペプチドに含まれるアミノ酸配列をコードするヌクレオチド配列を含むポリヌクレオチド、
(8)
(7)記載のポリヌクレオチドを含むベクター、
(9)
(7)記載のポリヌクレオチド若しくは(8)記載のベクターを含むか又は(1)乃至(6)のいずれか一つに記載のペプチドを産生する細胞、
(10)
下記工程(i)及び(ii)を含む、HTRA1の有するプロテアーゼ活性を阻害するSPINK2変異体ペプチドの製造方法:
(i)(9)記載の細胞を培養する工程;
(ii)該培養物からSPINK2変異体ペプチドを回収する工程、
(11)
(10)記載の方法により得られるSPINK2変異体ペプチド、
(12)
(1)乃至(6)及び(11)のいずれか一つに記載のペプチドに他の部分が連結してなるコンジュゲート、
(13)
ポリペプチドである、(12)記載のコンジュゲート、
(14)
(1)乃至(6)及び(11)のいずれか一つに記載のペプチドに結合する、抗体又はその機能断片、
(15)
(1)乃至(6)及び(11)のいずれか一つに記載のペプチド、(7)記載のポリヌクレオチド、(8)記載のベクター、(9)記載の細胞、(12)若しくは(13)記載のコンジュゲート、及び/又は、(14)記載の抗体もしくはその機能断片を含む組成物、
(16)
(1)乃至(6)及び(11)のいずれか一つに記載のペプチド及び/又は(12)又は(13)に記載のコンジュゲートを含む医薬組成物。
(17)
HTRA1関連疾患の治療又は予防のための、(16)記載の医薬組成物、
(18)
HTRA1関連疾患が滲出型加齢黄斑変性症、萎縮型加齢黄斑変性症、地図状萎縮、糖尿病網膜症、未熟児網膜症、ポリープ状脈絡膜血管症、関節リウマチ、及び変形性関節症よりなる群から選択される1つ又は2つ以上である、(17)記載の医薬組成物、
(19)
1つ又は2つ以上の他の医薬を含む、(16)乃至(18)のいずれか一つに記載の医薬組成物、
(20)
1つ又は2つ以上の他の医薬と組み合わせて使用される、(16)乃至(18)のいずれか一つに記載の医薬組成物、
(21)
網膜保護剤である、(16)乃至(20)のいずれか一つに記載の医薬組成物、
(22)
下記の工程1乃至工程3を含む、加齢黄斑変性症の治療薬又は予防薬を同定する方法:
[工程1]HTRA1プロテアーゼ及び基質を、被検物質の存在下又は非存在下で保温する;
[工程2]被検物質の存在下及び非存在下でのHTRA1プロテアーゼ活性を検出する;
[工程3]被検物質の存在下でのHTRA1プロテアーゼ活性が、被検物質の非存在下でのHTRA1プロテアーゼ活性と比較して小さい場合、該被検物質を陽性と判定する、
(23)
下記の工程1乃至工程3を含む、網膜保護剤を同定する方法:
[工程1]HTRA1プロテアーゼ及び基質を、被検物質の存在下又は非存在下で保温する;
[工程2]被検物質の存在下及び非存在下でのHTRA1プロテアーゼ活性を検出する;
[工程3]被検物質の存在下でのHTRA1プロテアーゼ活性が、被検物質の非存在下でのHTRA1プロテアーゼ活性と比較して小さい場合、該被検物質を陽性と判定する、
(24)
ハイドロキノンを含む高脂肪食を3乃至6ヶ月摂食させる工程を含む、網膜障害モデルウサギの作製方法であって、該モデルウサギの網膜色素上皮細胞が正常ウサギの網膜色素上皮細胞に比して肥大していることを特徴とする方法、
(25)
(24)記載の方法により作製され、正常ウサギに比して肥大した網膜色素上皮細胞を有していることを特徴とする、網膜障害モデルウサギ、
(26)
下記工程(i)及び(ii)を含む、加齢黄斑変性症の治療薬若しくは予防薬、又は、網膜保護剤を同定する方法:
(i)(25)記載のウサギにおける網膜色素上皮細胞の肥大を、被検物質投与下及び非投与下で測定する工程;及び
(ii)該被検物質投与下における網膜色素上皮細胞の肥大が非投与下と比較して抑制された場合、該検物質を陽性と判定する工程、
(27)
工程(i)における測定が、網膜色素上皮細胞の平均面積又は肥大した網膜色素上皮細胞数の測定である、(26)記載の方法、
(28)
萎縮型加齢黄斑変性症の治療又は予防のための、(16)乃至(18)のいずれか一つに記載の医薬組成物、及び、
(29)
滲出型加齢黄斑変性症の治療又は予防のための、(16)乃至(18)のいずれか一つに記載の医薬組成物、
等に関する。
本発明において、「遺伝子」とは、蛋白質に含まれるアミノ酸配列をコードするヌクレオチド配列を含む核酸分子又はその相補鎖を意味し、一本鎖、二本鎖又は三本鎖以上からなり、DNA鎖とRNA鎖の会合体、一本の鎖上にリボヌクレオチドとデオキシリボヌクレオチドが混在するもの及びそのよう鎖を含む二本鎖又は三本鎖以上の核酸分子も「遺伝子」の意味に含まれる。
「アミノ酸」は、アミノ基及びカルボキシル基を含む有機化合物であり、好適にはタンパク質に、より好適には天然のタンパク質に、構成単位として含まれるα-アミノ酸を意味する。本発明において、より好適なアミノ酸は、Ala、Arg、Asn、Asp、Cys、Gln、Glu、Gly、His、Ile、Leu、Lys、Met、Phe、Pro、Ser、Thr、Trp、Tyr及びValであり、特に明記しない限り「アミノ酸」はこれらの計20アミノ酸を意味する。それらの計20アミノ酸を「天然アミノ酸」と呼ぶことができる。本発明のHTRA1阻害ペプチドは、好適には天然アミノ酸を含有する。
(1)疎水性アミノ酸グループ:Met、Ala、Val、Leu、Ile
(2)中性親水性アミノ酸グループ:Cys、Ser、Thr、Asn、Gln
(3)酸性アミノ酸グループ:Asp、Glu
(4)塩基性アミノ酸グループ:His、Lys、Arg
(5)主鎖の方角に影響を与えるアミノ酸のグループ:Gly、Pro
(6)芳香族アミノ酸グループ:Trp、Tyr、Phe
ただし、天然アミノ酸の分類はこれらに限定されるものではない。
(1)非極性アミノ酸グループ:アラニン(以下、「Ala」または単に「A」と記す)、バリン(以下、「Val」または単に「V」と記す)、ロイシン(以下、「Leu」または単に「L」と記す)、イソロイシン(以下、「Ile」または単に「I」と記す)、プロリン(以下、「Pro」または単に「P」と記す)、フェニルアラニン(「Phe」または単に「F」と記す)、トリプトファン(以下、「Trp」または単に「W」と記す)、メチオニン(以下、「Met」または単に「M」と記す)
(2)非荷電極性アミノ酸グループ:グリシン(以下、「Gly」または単に「G」と記す)、セリン(以下、「Ser」または単に「S」と記す)、スレオニン(以下、「Thr」または単に「T」と記す)、システイン(以下、「Cys」または単に「C」と記す)、チロシン(以下、「Tyr」または単に「Y」と記す)、アスパラギン(以下、「Asn」または単に「N」と記す)、グルタミン(以下、「Gln」または単に「Q」と記す)
(3)酸性アミノ酸グループ:アスパラギン酸(以下、「Asp」または単に「D」と記す)、グルタミン酸(以下、「Glu」または単に「E」と記す)
(4)塩基性アミノ酸グループ:リジン(以下、「Lys」または単に「K」と記す)、アルギニン(以下、「Arg」または単に「R」と記す)、ヒスチジン(以下、「His」または単に「H」と記す)
本発明において、アミノ酸は、天然アミノ酸以外のアミノ酸であってもよい。例えば、天然のペプチドや蛋白質において見出されるセレノシステイン、N-ホルミルメチオニン、ピロリジン、ピログルタミン酸、シスチン、ヒドロキシプロリン、ヒドロキシリジン、チロキシン、O-ホスホセリン、デスモシン、β-アラニン、サルコシン、オルニチン、クレアチン、γアミノ酪酸、オパイン、テアニン、トリコロミン酸、カイニン酸、ドウモイ酸、アクロメリン酸等を挙げることができ、ノルロイシン、Ac-アミノ酸、Boc-アミノ酸、Fmoc-アミノ酸、Trt-アミノ酸、Z-アミノ酸等のN末端保護アミノ酸、アミノ酸t-ブチルエステル、ベンジルエステル、シクロヘキシルエステル、フルオレニルエステル等のC末端保護アミノ酸、ジアミン、ωアミノ酸、βアミノ酸、γアミノ酸、アミノ酸のTic誘導体、アミノフォスフォン酸を含むその他の天然界には見出されないアミノ酸等を挙げることができるが、それらに限らず上記20の「天然アミノ酸」以外のアミノ酸を、本発明では便宜的に「非天然アミノ酸」と総称する。
本発明のHRTA1阻害ペプチドは、SPINK2の有する骨格が少なくとも部分的に維持されたSPINK2変異体(以下、「SPINK2変異体」と略記する)であり、HTRA1又はその酵素活性が保持された断片(以下、「機能断片」という)の有するプロテアーゼ活性を阻害又は抑制する(以下、かかる阻害又は抑制をまとめて「HTRA1阻害活性」という)。
前述の通り、本発明のペプチドの標的であるHTRA1は、脊椎動物、好適には哺乳動物、より好適には霊長類、より一層好適にはヒトに由来するが、非ヒト動物、例えば、ラット、マウス等のげっ歯類、カニクイザル、コモンマーモセット、アカゲザル等の霊長類に由来してもよい。非ヒト動物由来のHTRA1に対して阻害活性を有するペプチドは、かかる非ヒト動物におけるHTRA1に関わる疾患の診断、検査、治療又は予防等に使用することができる。また、そのようなペプチドが、ヒトHTRA1も阻害する場合、ヒトHTRA1に関わる疾患の治療薬又は予防薬としての該ペプチドの非臨床研究開発において、かかる非ヒト動物を動物病態モデルとして使用した薬効薬理試験や薬物動態試験、健常動物として使用した安全性試験や毒性試験等を行うことができる。
また、ある態様における本発明の阻害ペプチドは、HTRA1の免疫原性断片に結合し得る。HTRA1の免疫原性断片は、1つ又は2つ以上のエピトープ、ミモトープ又はその他の抗原決定基を有するので、免疫応答を誘発し得るか又は当該断片に対する抗体が産生され得る。
本発明におけるSPINK2変異体とHTRA1又はその免疫原性断片の結合は、検出可能な結合親和性の測定(ELISA法、表面プラズモン共鳴(Surface Plasmon Resonance :以下、「SPR」という)解析法(「BIAcore」法ともいう)、等温滴定熱量測定(Isothermal Titration Calorimetry:以下「ITC」という)法、フローサイトメトリー、免疫沈降法等)等により、当業者に公知の方法を用いて、評価、測定又は判定することができる。
前述の記載にもかかわらず、HTRA1阻害活性を有する限りにおいて、HTRA1又はその免疫原性断片への結合能は本発明の阻害ペプチドとしてのSPINK2変異体にとって必須ではない。
16番Ser~22番Glyのうち1つ、2つ、3つ、4つ、5つ、6つ又は7つのアミノ酸が他のアミノ酸又はアミノ酸残基に置換されており;
24番Pro~28番Asnのうち1つ、2つ、3つ、4つ又は5つのアミノ酸が他のアミノ酸又はアミノ酸残基に置換されており;
39番Ala及び43番Thrのうち1つ又は2つのアミノ酸が他のアミノ酸又はアミノ酸残基に置換されていても、野生型であっても、16番~30番等のアミノ酸残基から構成されるループ主鎖3次構造が、HTRA1阻害活性を少なくとも部分的に発揮し得る限りにおいて、いずれでもよく(当該2アミノ酸又はアミノ酸残基はαへリックスに含まれる);
15番Cys、23番Cys、31番Cys、42番Cys、45番Cys及び63番Cysは、天然型のジスルフィド結合を維持するためには野生型と同じくCysであることが好ましく、天然型のジスルフィド結合を消失させたり、非天然型のジスルフィド結合を生じさせたりするためには、それらのうち1つ、2つ、3つ、4つ、5つ又は6つを他のアミノ酸に置換してもよい。本発明のSPINK2変異体うち一部の好適なHTRA1阻害ペプチドにおいては、天然型と同じ当該6箇所にCysが維持され、ジスルフィド結合が保持されている。かかる阻害ペプチドのうちより好適な一部の態様においては、15番Cys-45番Cys、23番Cys-42番Cys、及び、31番Cys-63番Cysが、それぞれジスルフィド結合を形成している。
16番X2は、好適にはAla、Asp、Glu、Phe、Gly、His、Lys、Leu、Met、Gln、Arg、Ser、Thr又はTyr、より好適にはAla、Asp、Gly、His、Lys、Leu、Met、Gln、Arg、Ser又はThr、より一層好適にはAla、Gly、Lys、Leu、Ser又はThr、さらにより一層好適にはAla、Gly、Leu、Ser又はThr、その上さらにより一層好適にはAla又はSerであり;
17番X3は、好適には、Ala、Asp、Glu、Gly、His、Lys、Leu、Met、Asn、Gln、Arg、Ser、Thr又はTyr、より好適にはAsp、Gly、His、Lys、Leu、Met、Asn、Gln、Arg、Ser、Thr又はTyr、より一層好適にはAsp、His、Lys、Met又はGln、さらにより一層好適にはAsp又はGlnであり;
18番X4は、好適にはAla、Asp、Glu、Phe、Gly,His、Ile、Leu、Lys、Met、Asn、Gln、Arg、Ser、Thr、Val、Trp又はTyr、より好適にはAsp、Phe、His、Met、Asn、Gln、Ser又はTyr、より一層好適にはAsp、Phe、His、Ser又はTyr、さらにより一層好適にはPhe又はHisであり;
19番X5は、好適にはAla、Asp、Glu、Gly、His、Ile、Lys、Met、Asn、Gln、Arg、Ser、Thr、Val又はTyr、より好適にはAla、Asp、Glu、Gly、His、Lys、Met、Asn、Gln、Arg、Ser又はVal、より一層好適にはAla、Asp、Glu、Met又はAsn、さらにより一層好適にはAla、Asp又はGluであり;
21番X6は、好適にはAla、Glu,Phe、Gly、Ile、Leu、Met、Gln、Arg、Ser,Trp又はTyr、より好適にはGlu,Phe、Ile、Leu、Met、Gln、Arg又はTrp、より一層好適にはMet又はTrp、さらにより一層好適にはMetであり;
24番X7は、好適にはAla、Asp、Glu、Phe、Gly、His、Lys、Leu、Met、Pro、Gln、Arg、Ser、Thr,Val、Trp又はTyr、より好適にはAsp、Glu、His、Pro、Gln、Ser、Thr、Val、Trp又はTyr、より一層好適にはGln、Trp、Tyr又はVal、さらにより一層好適にはTyr又はValであり;
26番X8は、好適にはAla、Asp、Glu、Phe、His、Ile、Lys、Leu、Met、Asn、Gln、Arg、Ser、Val又はTyrであり、より好適にはAla、Phe、His、Ile、Leu、Met、Gln、Arg、Ser、Val又はTyrであり、より一層好適にはPhe、Leu又はTyrであり、さらにより一層好適にはPhe又はLeuであり;
27番X9は、好適にはGlu、Phe、Leu、Ser、Thr又はTyrであり、より好適にはPhe、Leu、Ser、Thr又はTyrであり、より一層好適にはPhe又はTyr、さらにより一層好適にはTyrであり;
39番X10は、好適にはAla、Glu、Met又はVal、より好適にはAla又はGluであり;
43番X11は、好適にはAla、Thr又はVal、より好適にはThrまたはValである。
また、本発明のHTRA1阻害ペプチドとしてのSPINK2変異体のアミノ酸配列の例として、次の(a)乃至(e)のいずれか一つに記載のアミノ酸配列をそれぞれあげることができる:
(a)配列番号3、5、7、9、11、13、15、17、19、21、23乃至29(図15、17、19、21、23、25、25、29、31、33、35乃至41)のいずれか一つで示されるアミノ酸配列;
(b)(a)に記載のアミノ酸配列をコードするヌクレオチド配列に相補的なヌクレオチド配列とストリンジェントな条件下でハイブリダイズし、且つHTRA1阻害活性を有するペプチドに含まれるアミノ酸配列をコードするヌクレオチド配列によりコードされるアミノ酸配列;
(c)(a)に記載のアミノ酸配列において1乃至20個、1乃至15個、1乃至10個、1乃至8個、1乃至6個、1乃至5個、1乃至4個、1乃至3個、1若しくは2個、又は1個のアミノ酸が置換、欠失、付加及び/又は挿入してなり、且つHTRA1阻害活性を有するペプチドに含まれるアミノ酸配列;
(d)(a)に記載のアミノ酸配列と60%、70%、80%、85%、90%、92%、94%、96%、97%、98%又は99%以上同一であり、且つHTRA1阻害活性を有するペプチドに含まれるアミノ酸配列;及び、
(e)配列番号4、6、8、10、12、14、16、18、20及び22(図16、18、20、22、24、26、28、30、32及び34)のいずれか一つで示されるヌクレオチド配列によりコードされるアミノ酸配列。
HTRA1阻害ペプチドは、SPINK2のアミノ酸配列又は本発明のHTRA1阻害ペプチドの有するアミノ酸配列(例えば、配列番号3、5、7、9、11、13、15、17、19、21及び23乃至29からなる群、又は、図15、17、19、21、23、25、27、29、31、33及び35乃至41からなる群から選択されるアミノ酸配列)、該アミノ酸配列をコードするヌクレオチド配列、該ヌクレオチド配列を含む核酸分子等を出発材料として、当業者に周知の方法により、同定することができる。好適な一例として、ヒトSPINK2変異体ライブラリーより、HTRA1阻害活性を指標として同定することができ、HTRA1結合活性も指標として組み合わせてもよい。
本発明は、HTRA1阻害ペプチドに含まれるアミノ酸配列をコードするヌクレオチド配列を含むポリヌクレオチド(以下、「HTRA1阻害ペプチドをコードする核酸分子」という)、該遺伝子が挿入された組換えベクター、該遺伝子もしくはベクターが導入された細胞(以下、「HTRA1阻害ペプチドをコードする核酸分子含有細胞」という)、又はHTRA1阻害ペプチドを産生する細胞(以下、「HTRA1阻害ペプチド産生細胞」という)をも提供する。
(a)配列番号3、5、7、9、11、13、15、17、19、21、23乃至29(図15、17、19、21、23、25、25、29、31、33、35乃至41)のいずれか一つで示されるアミノ酸配列をコードするヌクレオチド配列;
(b)配列番号4、6、8、10、12、14、16、18、20及び22(図16、18、20、22、24、26、28、30、32及び34)のいずれか一つで示されるヌクレオチド配列;
(c)(a)又は(b)に記載のヌクレオチド配列に相補的なヌクレオチド配列とストリンジェントな条件下でハイブリダイズし、且つHTRA1阻害活性を有するペプチドに含まれるアミノ酸配列をコードするヌクレオチド配列;
(d)(a)又は(b)に記載のヌクレオチド配列において1乃至20個、1乃至15個、1乃至10個、1乃至8個、1乃至6個、1乃至5個、1乃至4個、1乃至3個、1若しくは2個、または1個のヌクレオチド又はヌクレオチド残基が置換、欠失、付加及び/又は挿入してなり、且つHTRA1阻害活性を有するペプチドに含まれるアミノ酸配列をコードするヌクレオチド配列;および、
(e)(a)又は(b)に記載のヌクレオチド配列と60%、70%、80%、85%、90%、92%、94%、96%、97%、98%又は99%以上同一であり、且つHTRA1阻害活性を有するペプチドに含まれるアミノ酸配列をコードするヌクレオチド配列。
本発明はHTRA1阻害ペプチド、又は、そのコンジュゲートを含む医薬組成物をも提供する。
加齢性黄斑変性症は、滲出型および萎縮型に分かれる。滲出型は、さらに典型加齢性黄斑変性症(典型AMD)、ポリープ状脈絡膜血管症(PCV)、網膜血管腫状増殖(RAP)に分類される。滲出型に対しては、抗VEGF薬や光線力学療法(PDT)等の治療法が存在するが必ずしも十分ではなく、萎縮型に対しては、食生活の改善やAge-Related Eye Disease Study (AREDS)に基づくサプリメント摂取が行われているに過ぎない。
本発明の医薬組成物が、加齢黄斑変性症の治療又は予防に使用し得ることは、例えば、ラット光照射網膜障害モデルにおいて、光照射前又は後に、それぞれ本発明のHTRA1阻害ペプチドを投与することにより、対照群では外顆粒層に含まれる核の数が減少するのに対し、投与群では外顆粒層に含まれる核の数の減少を抑制したことから(実施例5、10及び14)、あるいは高脂肪食及びハイドロキノンの摂食により作製されるウサギ網膜障害モデル(後述)において、本発明のHTRA1阻害ペプチドを投与することにより、対照群では網膜色素上皮細胞(RPE細胞)の面積又は一定以上の細胞面積を持つRPE細胞数が増加するのに対し、投与群ではかかる増加が抑制されること(実施例11)から示される。ウサギ網膜障害モデルは、ヒト萎縮型加齢黄斑変性症のリスク因子を加味していることから、とりわけ萎縮型加齢黄斑変性症のモデルとして優れている。
網膜色素上皮細胞(RPE)細胞を用いたVEGF mRNA誘導試験において、HTRA1阻害ペプチドを投与すると、VEGF mRNAの発現抑制が認められた(実施例12)。網膜色素上皮細胞からのVEGF誘導は滲出型加齢性黄斑変性病態形成に関与していることに加え(Klettner A. et al.,(2009) Graefes Arch Clin Exp Ophthalmol.247巻:1487-1492頁)、病態の維持にもそれらの病的なVEGF誘導が関与していると考えられており、本発明のHTRA1阻害ペプチドは、萎縮型加齢性黄斑変性症ならびに滲出型加齢性黄斑変性症の治療及び予防に対して有用であることが示される。
ヒト臍帯静脈内皮細胞(HUVEC)の遊走試験において、HTRA1阻害ペプチドを投与すると、遊走の抑制が認められたことから(実施例13)、血管新生を特徴とする滲出型加齢黄斑変性症の治療及び予防に有用であることが示される。
滲出型加齢性黄斑変性症は進行性である。また抗VEGF薬の投与により病状を一時的に抑えることができるものの、高頻度に再発を繰り返すことが問題となっている(Yand S他、Drug Des Devel Ther.、2巻10号:1857-67頁、2016年刊)。また、近年では滲出型加齢性黄斑変性症患者に新たに萎縮を発症し視力低下を引き起こすことが問題となっている(Berg K他、Acta Ophthalmologica、95巻8号:796-802頁、2017年刊)。本発明のHTRA1阻害ペプチドの投与は、単独又は抗VEGF薬と併用することにより、これら滲出型加齢性黄斑変性症(ポリープ状脈絡膜血管症、網膜内血管腫状増殖を含む)に対する治療効果を奏し得る。加えて、該ペプチドを予防的に投与することにより、病状の再発を阻止するか又は遅らせ、抗VEGF薬による治療の開始又は再開を遅らせること等が可能である。さらに、滲出加齢性黄斑変性症患者における萎縮の形成に対しても該ペプチドは抑制的に働くことから、長期的な視力上のベネフィットをもたらし得る。
本発明のHTRA1阻害ペプチドは、組織への浸透性に優れ(実施例11)、物性・安定性、安全性、投与後の動態、生産性等にも優れており、医薬組成物に有効成分として好適に含有せしめることができる。
本発明のHTRA1阻害ペプチド又はそのコンジュゲートは、HTRA1プロテアーゼ阻害活性に加え、HTRA1結合活性を有していてもよく、HTRA1阻害剤探索研究のポジティブコントロールとしての使用等の様々な研究、HTRA1の検出、該検出を利用した検査及び診断、HTRA1の分離、試薬及びその他の用途で使用され得る。HTRA1の検出や分離の際には、本発明のペプチド及びHTRA1のうち少なくとも一方が固相化され得る。
本発明は、HTRA1に結合する本発明のペプチド又はそのコンジュゲートを含む検査用又は診断用組成物(以下、まとめて「診断用組成物」という)を提供する。
本発明の診断用組成物は、HTRA1に関わる疾患、HTRA1発現等の検査又は診断に有用である。本発明において検査又は診断には、例えば、罹患リスクの判定又は測定、罹患の有無の判定、進行や増悪化の程度の測定、HTRA1阻害ペプチド又はそのコンジュゲートを含む医薬組成物による薬物治療の効果の測定又は判定、薬物治療以外の治療の効果の測定又は判定、再発リスクの測定、再発の有無の判定等が含まれるが、検査又は診断であればそれらに限定されるものではない。
本発明の診断用組成物は、本発明のペプチド又はそのコンジュゲート、それらを含む組成物、それらを含む医薬組成物を投与する個体の同定に有用である。
かかる診断用組成物には、pH緩衝剤、浸透圧調節剤、塩類、安定化剤、防腐剤、顕色剤、増感剤、凝集防止剤等を含有せしめることができる。
本発明はHTRA1に関わる疾患、の検査方法又は診断方法、該疾患の診断用組成物を調製するための本発明のペプチドの使用、該疾患の検査又は診断のための、HTRA1に結合する本発明のペプチド又はそのコンジュゲートの使用、をも提供する。かかる本発明のペプチド又はそのコンジュゲートを含む検査又は診断用キットも本発明に含まれる。
HTRA1に結合する本発明のペプチドを含む検査又は診断の方法としてはサンドウィッチELISAが望ましいが、通常のELISA法やRIA法、ELISPOT法、ドットブロット法、オクタロニー法、CIE法、CLIA、フローサイトメトリー等の検出方法が利用可能である。免疫沈降法を利用した方法でも検査又は診断が可能である。
また、本発明は被検サンプル中のHTRA1を検出又は測定する方法を提供する。これらの検出又は測定方法には、本発明の診断用組成物を使用することができる。HTRA1阻害ペプチド、又は、そのコンジュゲートを被検試料と接触させ(工程1)、次いで該ペプチドに結合したHTRA1の量又は測定を測定する(工程2)ことにより、該試料中のHTRA1を検出することができる。工程1としては、例えば、HTRA1阻害ペプチドに免疫グロブリンのFc領域をコンジュゲートしたものを、プロテインGを介して磁気ビーズに固相化し、そこに被検試料を添加する等、工程2としては、例えば、磁気ビーズを分離し、ビーズと共に沈殿した可溶性蛋白質をSDS-PAGEやWestern blot法で解析し、HTRA1を検出する等をあげることができる。本測定には、ヒト又は非ヒト動物由来の試料に加え、組換え蛋白質等の人工的な処理を加えた試料をも供することができる。生物個体由来の被検試料としては、例えば、血液、関節液、腹水、リンパ液、脳脊髄液、肺胞洗浄液、唾液、痰、組織ホモジネート上清、組織切片等をあげることができるが、それらに限定されるものではない。
前記のHTRA1の検出は、イン・ビトロのみならず、イン・ビボでも実施することができる。画像診断の場合は、薬学的に許容可能な放射性核種や発光体で標識されたHTRA1阻害ペプチド、又は、そのコンジュゲートを利用することができる。工程1としては、例えば、被験者に、標識された該ペプチドもしくはそのコンジュゲートを投与する、工程2としては、例えば、PET/CT等の画像診断技術を使用して画像を取り、HTRA1の存在を判定又は検査する等をあげることができる。
本発明の診断用組成物に含まれるペプチド又はそのコンジュゲートはHTRA1に結合し、好適にはHTRA1特異的結合活性を有する。
本発明の医薬組成物が投与される個体を同定する方法も本発明に包含される。かかる同定方法においては、該個体由来サンプル中のHTRA1を本発明のHTRA1結合ペプチドを用いて測定し、該サンプル中に、HTRA1が検出されたか、又は、健常個体由来サンプル中に検出されたHTRA1の量と比較してより多くのHTRA1が検出された場合に、該個体を陽性と判定することができる。当該方法には、本発明の診断用組成物を使用することができる。
また、かかる同定方法の好適な一態様において、該個体はHTRA1関連疾患に罹患しているか又はそのリスクがある。
さらに、本発明の医薬組成物は、その一態様において、かかる同定方法において陽性と判定された個体に投与され得る。
本発明はそのある態様において、HTRA1阻害活性を指標として、HTRA1に関わる疾患、好適には加齢黄斑変性症の治療薬もしくは予防薬、又はその候補を同定する方法を提供する。該方法には、HTRA1プロテアーゼ及び基質を、被検物質の存在下又は非存在下(若しくはヴィークル存在下)で保温する工程1、被検物質の存在下及び非存在下でのHTRA1プロテアーゼ活性を決定する工程2、及び/又は、被検物質の存在下でのHTRA1プロテアーゼ活性が、検物質の非存在下でのHTRA1プロテアーゼ活性と比較して小さい場合、該被検物質を加齢黄斑変性症の治療薬もしくは予防薬、又はその候補と判定する工程3、を含み得る。被検物質は、ペプチド性、非ペプチド性のいずれであってもよく、ペプチド性としてはSPINK2変異体に限定されず、抗体、免疫グロブリンではない蛋白質の骨格を有するSPINK2変異体以外のペプチド、HTRA1基質アナローグ等を例示することができ、非ペプチド性としては合成低分子化合物、核酸等を例示することができるが、それらに限定されない。また、上記の1つ又は2つ以上の工程は、網膜保護効果を有する物質又はその候補の同定方法にも、好適に含まれ得る。本発明は網膜保護効果を有する物質又はその候補の同定方法にも関する。
本発明は、ハイドロキノン(Hydriguinone:以下「HQ」という)を含有する高脂肪食(Hgih Fat Diet:以下「HFD」という)負荷によるウサギ網膜障害モデル、その作製方法、当該モデルを用いた試験方法等も提供する。
本発明のモデルは、網膜色素上皮細胞(RPE細胞)が、正常ウサギに比して肥大しており、加齢黄斑変性症の初期病態を発症している。一方、従来のウサギモデル(非特許文献20)に近い10週齢のウサギにHQ-HFDを4ヶ月間摂取させたが、RPE細胞肥大は視覚的に確認できなかった。
病態を発症した本モデルを使用することにより、加齢黄斑変性症の治療薬及び/若しくは予防薬、又は網膜保護剤を同定することができる。かかる同定方法は(i)本モデルのウサギにおける網膜色素上皮細胞の肥大を、被検物質投与下及び非投与下で測定する工程;及び(ii)該被検物質投与下における網膜色素上皮細胞の肥大が非投与下と比較して抑制された場合、該検物質を陽性と判定する工程、を含むことができる。工程(i)における測定は、網膜色素上皮細胞の平均面積、及び/又は、肥大した網膜色素上皮細胞数が好適である。
(1-1)HTRA1阻害ペプチド発現ベクターの構築
(1-1-1)pET 32a(改変)_HTRA1阻害ペプチドの構築
まず、SPINK2scaffoldを骨格としたHTRA1阻害ペプチドの発現ベクターを構築した。各阻害ペプチドのヌクレオチド配列(配列番号4、6、8、10、12、14、16、18、20及び22)とSPINK2のヌクレオチド配列(配列番号2)を鋳型として、下記プライマーおよびKOD-plus-(TOYOBO)を用いたPCR法((94℃ 15秒、60℃ 30秒、68℃ 30秒)×30サイクル)により阻害剤断片を増幅した。
プライマー1:5’-AAAAGAATTCTGATCCGCAGTTTGGTCTGTTTAG-3’
プライマー2:5’-AAAACTCGAGTTATGCGGCCGCAGACGCGCCGCACGGACC-3’
増幅した断片をアガロースゲル電気泳動に供した後に、所望のDNA断片を切り出し、QIAquick Gel Extraction Kit(QIAGEN)によりDNAを調製した。調製したDNA断片およびpET 32a(改変)を制限酵素EcoRI(NEB)およびXhoI(NEB)を用いて、37℃で1時間以上処理し、アガロースゲル電気泳動後に、所望のDNA断片を切り出し、QIAquick PCR Purification Kit(QIAGEN)により精製した。T4 DNA Ligase(NEB)を用いて、精製した断片を16℃で一晩反応させることでligation反応を実施した。Ligation溶液は、大腸菌JM109(TOYOBO)に添加し、氷上で30分間静置した後、42℃で45秒の熱処理、さらに氷上で5分間静置し、0.1mg/mlアンピシリンを含む2YTプレートに播種後、37℃で一晩静置培養することで、大腸菌へ形質転換した。翌日、形質転換した大腸菌を、0.1mg/mlアンピシリンを含むTerrific Broth培地(Invitrogen)に植菌し、37℃で一晩培養後、QIAprep 96 Turbo Miniprep Kit(Qiagen)を用いてプラスミドDNAを回収し(以下、「miniprep処理」という)、配列解析を実施することで「pET 32a(改変)_HTRA1阻害ペプチド」を構築した。
(1-1-2)pET 32a_HTRA1阻害ペプチド_Kex2の構築
同様に、各阻害剤の配列(配列表)とSPINK2を鋳型として、下記プライマーおよびKOD-plus-(TOYOBO)を用いたPCR法((94℃ 15秒、60℃ 30秒、68℃ 30秒)×30サイクル)により阻害剤断片を増幅した。
プライマー3:5’-AAAAGGATCCCTGGACAAACGTGATCCGCAGTTTGGTCTGTTTAG-3’
プライマー4:5’-AAAACTCGAGTTAGCCGCCGCACGGACCATTGCGAATAATTTTA-3’
増幅した断片をアガロースゲル電気泳動に供した後に、所望のDNA断片を切り出し、QIAquick Gel Extraction Kit(QIAGEN)によりDNAを調製した。調製したDNA断片およびpET 32a(Novagen)を制限酵素BamHI(NEB)およびXhoI(NEB)を用いて、37℃で1時間以上処理し、アガロースゲル電気泳動後に、所望のDNA断片を切り出し、QIAquick PCR Purification Kit(QIAGEN)により精製した。T4 DNA Ligase(NEB)を用いて、それぞれの精製断片を16℃で一晩反応させることでligation反応を実施した。Ligation溶液は、大腸菌JM109(TOYOBO)に添加し、氷上で30分間静置した後、42℃で45秒の熱処理、さらに氷上で5分間静置し、0.1mg/mlアンピシリンを含む2YTプレートに播種後、37℃で一晩静置培養することで、大腸菌を形質転換した。形質転換された大腸菌を培養した後に、miniprepおよび配列解析を実施することで、「pET 32a_HTRA1阻害ペプチド_Kex2」を構築した。尚、操作は(1-1-1)に記載の方法に準じて行った。
(1-2)HTRA1阻害ペプチドの発現精製
(1-1-1)で構築したベクターpET 32a(改変)_HTRA1阻害ペプチドを大腸菌Origami B (DE3)(Novagen)へ形質転換し、0.1mg/mlアンピシリンを含む2YT培地を用いて37℃で培養後、IPTG(最終濃度1mM)を添加し、16℃で一晩培養した。翌日、遠心分離(3,000g、20分、4℃)により集菌後、BugBuster Master Mix(Novagen)を用いてlysateを調製し、TALON Metal Affinity Resin(Clontech)を用いてHis tag融合目的蛋白質を精製した。次に、Thrombin Cleavage Capture Kit(Novagen)を用いてthioredoxin tagと所望の蛋白質とを切断し、TALONを用いて精製した。さらに、ゲルろ過クロマトグラフィー(Superdex75 10/300 GL)または逆相クロマトグラフィー(YMC-Pack ODS-AM)に供することで、HTRA1阻害ペプチドを調製した。得られたペプチドのN末端にはS tag及びリンカーからなる部分(配列番号31:図43)が、C末端にはGly-Glyの代わりにC末6マー(配列番号32:図44)が、それぞれコンジュゲートされている。
ヒト/マウス/ラット/サルHTRA1の配列類似性を図1に示す。酵素活性ドメインであるHTRA1プロテアーゼドメイン(204Gly-364Leu)を構成する一次配列はヒトとサルにおいて完全に一致している。ヒトとマウスまたはラットのHTRA1プロテアーゼドメイン配列は1残基異なっているが、構造上、当該残基は酵素活性中心の反対側に位置していることから酵素活性中心に影響を与えないと推測された(図1)。よって、HTRA1プロテアーゼドメインはヒト/マウス/ラット/サルの種によらず同配列であることから、種に関しては明示しない。
(2-1)HTRA1プロテアーゼドメインHTRA1(cat)の調製
(2-1-1)pET 21b_HTRA1(cat)の構築
ヒトHTRA1(Q92743)のN末ドメインおよびPDZドメインを除いたプロテアーゼドンメイン(158Gly-373Lys)をHTRA1(cat)として、HTRA1(cat)発現ベクターを構築した。ヒトHTRA1挿入プラスミド(GeneCopoeia;GC-M0558)を鋳型として下記プライマーおよびKOD-plus-(TOYOBO)を用いたPCR法((94℃ 15秒、60℃ 30秒、68℃ 45秒)×30サイクル)により所望のDNA断片を増幅した。
プライマー5:5’-AAACATATGGGGCAGGAAGATCCCAACAGTTTGC-3’
プライマー6:5’-AAACTCGAGTTTGGCCTGTCGGTCATGGGACTC-3’
増幅した断片をアガロースゲル電気泳動に供した後に、所望のDNA断片を切り出し、QIAquick Gel Extraction Kit(QIAGEN)によりDNAを調製した。調製したDNA断片およびpET 32a(Novagen)を制限酵素NdeI(NEB)およびXhoI(NEB)を用いて、37℃で1時間以上処理し、アガロースゲル電気泳動後に、所望のDNA断片を切り出し、QIAquick PCR Purification Kit(QIAGEN)により精製した。T4 DNA Ligase(NEB)を用いて、それぞれの精製断片を16℃で一晩反応させることでligation反応を実施した。Ligation溶液は、大腸菌JM109(TOYOBO)に添加し、氷上で30分間静置した後、42℃で45秒の熱処理、さらに氷上で5分間静置し、0.1mg/mlアンピシリンを含む2YTプレートに播種後、37℃で一晩静置培養することで、大腸菌を形質転換した。形質転換された大腸菌を培養した後に、miniprepおよび配列解析を実施することで、「pET 21b_HTRA1(cat)」を構築した。尚、操作は(1-1-1)に記載の方法に準じて行った。
(2-1-2)HTRA1(cat)の調製
構築したpET 21b_HTRA1(cat)は大腸菌BL21 (DE3)(Novagen)へ形質転換し、0.1mg/mlアンピシリンを含む2YT培地を用いて37℃で培養後、IPTG(最終濃度1mM)を添加し、28℃で一晩培養した。集菌後に1mg/mlリゾチームを含むリン酸バッファー(50mM Sodium phosphate,300mM NaCl)で懸濁し、超音波破砕によりlysateを調製した。TALON(Clontech)を用いて所望のHis tag融合タンパクを回収し、ゲルろ過クロマトグラフィー(Superdex 200 10/300 GL)に供することでHTRA1(cat)を精製した。
(2-2)HTRA1全長HTRA1(full)の調製
(2-2-1)pcDNA3.1_HTRA1(full)_Hisの構築
合成したヒトHTRA1(Q92743)DNA(GENEART)を鋳型とし、下記プライマーおよびKOD-plus-(TOYOBO)を用いたPCR法((94℃ 15秒、60℃ 30秒、68℃ 90秒)×30サイクル)により所望のDNA断片を増幅した。
プライマー7:5’-AAAAGAATTCGCCACCATGCAGATTCCTAGAGCCG-3’
プライマー8:5’-AAAACTCGAGTCAGTGGTGATGGTGGTGGTGGCCGG-3’
増幅した断片をアガロースゲル電気泳動に供した後に、所望のDNA断片を切り出し、QIAquick Gel Extraction Kit(QIAGEN)によりDNAを調製した。調製したDNA断片とpcDNA3.1(Thermo Fisher Scientific)を制限酵素EcoRI(NEB)およびXhoI(NEB)を用いて、37℃で1時間以上処理し、アガロースゲル電気泳動後に、所望のDNA断片を切り出し、QIAquick PCR Purification Kit(QIAGEN)により精製した。T4 DNA Ligase(NEB)を用いて、それぞれの精製断片を16℃で一晩反応させることでligation反応を実施した。Ligation溶液は、大腸菌JM109(TOYOBO)に添加し、氷上で30分間静置した後、42℃で45秒の熱処理、さらに氷上で5分間静置し、0.1mg/mlアンピシリンを含む2YTプレートに播種後、37℃で一晩静置培養することで、大腸菌を形質転換した。形質転換された大腸菌を培養した後に、miniprepおよび配列解析を実施することで、「pcDNA3.1_HTRA1(full)_His」を構築した。尚、操作は(1-1-1)に記載の方法に準じて行った。
(2-2-2)pcDNA3.3_HTRA1(full)_FLAG_Hisの構築
(2-2-1)で構築したpcDNA3.1_HTRA1(full)_Hisを鋳型として、下記プライマーおよびKOD-plus-(TOYOBO)を用いたPCR法((94℃ 15秒、60℃ 30秒、68℃ 90秒)×30サイクル)により断片Aを増幅した。
プライマー7
プライマー9:5’-CTTGTCGTCATCGTCCTTGTAGTCGCCGGGGTCGATTTCCTC-3’
次に、下記プライマーおよびKOD-plus-(TOYOBO)を用いたPCR法((94℃ 15秒、60℃ 30秒、68℃ 10秒)×30サイクル)により断片Bを増幅した。
プライマー10:5’-GCGACTACAAGGACGATGACGACAAGCACCACCACCATCATCAC-3’
プライマー11:5’-AAAAACTCGAGCTAGTGATGATGGTGGTGGTGCTTGTCGTC-3’
断片Aおよび断片Bを鋳型として、プライマー7および11、KOD-plus-(TOYOBO)を用いたPCR法((94℃ 15秒、60℃ 30秒、68℃ 90秒)×30サイクル)により所望のDNA断片を増幅した。増幅した断片をアガロースゲル電気泳動に供した後に、所望のDNA断片を切り出し、QIAquick Gel Extraction Kit(QIAGEN)によりDNAを調製した。調製したDNA断片とpcDNA3.3(ThermoFisher Scientific)を鋳型としたベクター)を制限酵素EcoRI(NEB)およびXhoI(NEB)を用いて、37℃で1時間以上処理し、アガロースゲル電気泳動後に、所望のDNA断片を切り出し、QIAquick PCR Purification Kit(QIAGEN)により精製した。T4 DNA Ligase(NEB)を用いて、それぞれの精製断片を16℃で一晩反応させることでligation反応を実施した。Ligation溶液は、大腸菌JM109(TOYOBO)に添加し、氷上で30分間静置した後、42℃で45秒の熱処理、さらに氷上で5分間静置し、0.1mg/mlアンピシリンを含む2YTプレートに播種後、37℃で一晩静置培養することで、大腸菌を形質転換した。形質転換された大腸菌を培養した後に、miniprepおよび配列解析を実施することで、「pcDNA3.3_HTRA1(full)_FLAG_His」を構築した。尚、操作は(1-1-1)に記載の方法に準じて行った。
(2-2-3)HTRA1(full)の調製
(2-2-2)で構築したpcDNA3.3_HTRA1(full)_FLAG_HisはPolyethylenimine Max(Polysciences,Inc.)を用いてFreeStyle 293F(Thermo Fisher Scientific)にtransfectionし、6日後に培養上精を回収した。HisTrap excel(GE Healthcare)によりHis tag融合タンパク質を回収し、さらにANTI-FLAG M2 Affinity Agarose Gel(Sigma-Aldrich)を用いることでHTRA1(full)を精製した。
(2-3-1)pcDNA3.3_HTRA1(S328A)_FLAG_Hisの構築
HTRA1不活性変異体HTRA1(S328A)発現ベクターを構築するため、実施例(2-2-2)で構築したベクター「pcDNA3.3_HTRA1(full)_FLAG_His」を鋳型として、下記プライマーおよびQuikChange II Site-Directed Mutagenesis Kits(アジレント・テクノロジー株式会社)を用いたPCR法((95℃ 30秒、55℃ 1分、68℃ 7分)×18サイクル)を実施した。
プライマー21:5’-CCATCATCAACTACGGCAACGCGGGCGGACCCCTCGTGAACC-3’ (配列番号55:図76)
プライマー22:5’-GGTTCACGAGGGGTCCGCCCGCGTTGCCGTAGTTGATGATGG-3’(配列番号56:図77)
PCR反応後、キット付属のプロトコールに従い、DpnI処理したPCR反応液を用いて大腸菌JM109(TOYOBO)を形質転換した。形質転換された大腸菌を培養した後に、miniprepおよび配列解析を実施することで、「pcDNA3.3_HTRA1(S328A)_FLAG_His」を構築した。尚、操作は(1-1-1)に記載の方法に準じて行った。
(2-3-2)HTRA1(S328A)の調製
(2-2-3)に記載の方法に従い、FreeStyle 293Fを用いてHTRA1(S328A)を発現し、Affinity精製によりHTRA1(S328A)を調製した。
実施例3.HTRA1阻害ペプチドのHTRA1阻害活性評価
(3-1)ペプチド基質を用いたHTRA1阻害ペプチドのHTRA1阻害活性評価
基質ペプチドH2-Opt(Mca-IRRVSYSFK(Dnp)K)(株式会社ペプチド研究所:配列番号54、図8)を10mMになるようDMSOで溶解し、Assay buffer(50mM borate,150mM NaCl,pH8.5)で希釈して終濃度10μMで使用した。Assay bufferで希釈したHTRA1(HTRA1(cat)またはHTRA1(full))とHTRA1阻害ペプチドをそれぞれ25μLずつ混ぜ、37℃で20分反応させた後にAssay bufferで希釈した基質を50μL加えて、Enspire(PerkinElmer)で蛍光シグナル(excitation 328nm/emission 393nm)を測定した。HTRA1は終濃度100nM、HTRA1阻害ペプチドは終濃度1.875~1,000nM、反応および測定にはプロテオセーブ(登録商標)SS96F黒プレート(住友ベークライト株式会社)を使用した。
ヒトVitronectinをタンパク基質として、HTRA1阻害ペプチドのHTRA1阻害活性を評価した。Assay buffer(50mM Tris,150mM NaCl,pH8.0)で希釈したHTRA1(cat)と各HTRA1阻害ペプチドを混合し、37℃で1時間反応した。次に、Assay bufferで希釈したヒトVitronectin(BD Biosciences;354238)を加えて37℃で2時間反応させ、SDSサンプルバッファーを添加し、99℃で5分処理することで酵素反応を停止した。その後、SDS-PAGEおよびWestern blot解析により、ヒトVitronectinの分解を評価した。HTRA1阻害ペプチドの終濃度は0~25μM、HTRA1(cat)の終濃度は1μM、ヒトVitronectinの終濃度は1μMであった。また、Western blot解析では、一次抗体にHuman Vitronectin Antibody(R&D Systems;MAB2349)、二次抗体にAnti-Mouse IgG,HRP-Linked Whole Ab Sheep(GE Healthcare;NA931)を使用した。
(3-3)HTRA1阻害ペプチドの特異性評価
基質ペプチドの切断を指標に、他のプロテアーゼに対する特異性を評価した。(3-1)に記載の方法と同様、Assay bufferで希釈したプロテアーゼとサンプル(終濃度1μM)をそれぞれ25μLずつ混ぜ、37℃で20分反応させた後にAssay bufferで希釈した基質を50μL加えて、Enspire(PerkinElmer)で蛍光シグナル(excitation 380nm/emission 460nm)を測定した。尚、HTRA2活性評価では実施例2と同様のAssay buffer(50mM borate,150mM NaCl,pH8.5)、HTRA2以外のプロテアーゼ活性評価にはAssay buffer(50mM Tris,150mM NaCl,pH8.0)を用い、反応および測定にはプロテオセーブ(登録商標)SS96F黒プレート(住友ベークライト株式会社)を使用した。特異性評価に用いたプロテアーゼおよび基質の組み合わせは以下の通り。
Bovine trypsin阻害活性評価;終濃度5nM trypsin(Pierce;20233)、終濃度100μM 基質ペプチドBoc-VPR-AMC Fluorogenic Peptide Substrate(R&D Systems;ES011)
Bovine α-chymotrypsin阻害活性評価;終濃度10nM chymotrypsin(Worthington Biochemical Corporation;LS001434)、終濃度100μM 基質ペプチドSuc-Leu-Leu-Val-Tyr-MCA(株式会社ペプチド研究所;3120-v)
Human tryptase阻害活性評価;終濃度1nM tryptase(Sigma-Aldrich;T7063)、終濃度100μM 基質ペプチドBoc-Phe-Ser-Arg-MCA(株式会社ペプチド研究所;3107-v)
Human chymase阻害活性評価;終濃度100nM chymase(Sigma-Aldrich;C8118)、終濃度100μM 基質ペプチドSuc-Leu-Leu-Val-Tyr-MCA(株式会社ペプチド研究所;3120-v)
Human plasmin阻害活性評価;終濃度50nM Plasmin(Sigma-Aldrich;P1867)、終濃度100μM基質 ペプチドBoc-Val-Leu-Lys-MCA(株式会社ペプチド研究所;3104-v)
Human thrombin阻害活性評価;終濃度1nM thrombin(Sigma-Aldrich;T6884)、終濃度100μM 基質ペプチドBoc-VPR-AMC Fluorogenic Peptide Substrate(R&D Systems;ES011)
Human matriptase阻害活性評価;終濃度1nM matriptase(R&D Systems;E3946-SE)、終濃度100μM 基質ペプチドBoc-QAR-AMC Fluorogenic Peptide Substrate(R&D Systems;ES014)
Human protein C阻害活性評価;終濃度100nM protein C(Sigma-Aldrich;P2200)、終濃度100μM 基質ペプチドBoc-Leu-Ser-Thr-Arg-MCA(株式会社ペプチド研究所;3112-v)
Human tPA阻害活性評価;終濃度10nM tPA(Sigma-Aldrich;T0831)、終濃度100μM 基質ペプチドPyr-Gly-Arg-MCA(株式会社ペプチド研究所;3145-v)
Human uPA阻害活性評価;終濃度10nM uPA(Sigma-Aldrich;T0831)、終濃度100μM 基質ペプチドPyr-Gly-Arg-MCA(株式会社ペプチド研究所;3145-v)
Human plasma kallikrein阻害活性評価;終濃度0.125μg/ml plasma kallikrein(Sigma-Aldrich;T0831)、終濃度100μM 基質ペプチドZ-Phe-Arg-MCA(株式会社ペプチド研究所;3095-v)
Human HTRA2阻害活性評価;終濃度200nM HTRA2(R&D Systems;1458-HT)、終濃度50μM 基質ペプチドH2-Opt(株式会社ペプチド研究所)
(3-2)と同様、ペプチド基質の分解を指標にHTRA1以外のプロテアーゼへの交差性を評価した。阻害剤の終濃度1uMにおいては、いずれのプロテアーゼに対しても各HTRA1阻害ペプチドはプロテアーゼ活性を抑制せず、HTRA1阻害ペプチドはHTRA1特異的な阻害作用を有することが示された(図5)。
(4-1)HTRA1(cat)/HTRA1阻害ペプチド複合体の調製
(1-2)および(2-1)に記載した方法に従って、HTRA1(cat)および配列番号3で示されるアミノ酸配列を有するHTRA1阻害ペプチドをそれぞれ調製した。20mM Tris-HCl,150mM NaCl,pH7.6の条件下で両者を混合後、ゲルろ過クロマトグラフィー(Superdex 200 10/300 GL)により複合体を単離精製した。
(4-2)X線結晶構造解析
(4-1)で調製した複合体溶液を18mg/mlまで濃縮後、リザーバー溶液(LiCl 1.0M,7.5%PEG6000,0.1M Tris/HCl(pH8.5))と1対1で混合し、蒸気拡散法により結晶化した。得られた立方体状の単結晶を、20%のエチレングリコールを含むリザーバー溶液に浸漬した後液体窒素にて凍結した。凍結結晶をクライオ気流下でX線照射し、回折イメージを得た(photon factory BL5A:高エネルギー加速器研究機構)。HKL2000を使用した解析により、最大分解能2.6Aのスケーリングデータを取得した。Serine protease HTRA1(PDB ID:3NZI)を鋳型とした分子置換法により位相を決定し、構造精密化後、分解能2.6AでHTRA1(cat)/該ペプチドの複合体結晶を決定した。単位格子中にはHTRA1とSPINK2が各1分子ずつ含まれていた。SPINK2分子については、配列情報と観測された電子密度に基づき、HTRA1(cat)との相互作用部位を含む部分的な分子モデルを構築した。当該HTRA1阻害ペプチドはHTRA1酵素活性中心を含む領域へ結合していることが認められた(図6及び7)。
(5-1)ラット光照射網膜障害モデルの作製
ラット光照射網膜障害モデルは光照射により網膜視細胞の細胞死を誘発させるモデルであり、網膜変性のモデル動物として汎用されている(Daniel T. Organisciak et al., (1996) Invest Ophthalmol Vis Sci. 37巻(11号):2243-2257頁)。72時間暗順応させたラットに対し、0.5%(W/V)トロピカミド-0.5%塩酸フェニレフリン点眼液を暗順応下で点眼し、その後5500Luxの白色光を3時間照射した。照射後、再び約24時間暗順応させ、その後は通常飼育の明暗条件に戻して2日間飼育した。安楽殺後に眼球を摘出し、3.7%(W/V)ホルムアルデヒド-0.5~1%(W/V)メタノール-0.2%(W/V)ピクリン酸固定液で24時間以上浸漬させ固定した。パラフィン包埋後、薄切切片を作製した。切片はヘマトキシリン-エオジン染色を行い、網膜断層の外顆粒層に含まれる核の数を数えることで、網膜障害を評価した。ラット光照射網膜障害モデルは光照射により、外顆粒層に含まれる核の数が顕著に減少することが明らかになった。
(5-2)網膜障害時の細胞外HTRA1の発現確認
ラット光照射網膜障害モデルにおけるHTRA1の関与を調べるため、(5-1)で作製したモデルラットから硝子体液を採取し、Western blot解析によりHTRA1発現量を評価した。硝子体液は還元条件下でSDS-PAGEに供し、一次抗体Human HTRA1/PRSS11 Antibody(R&D Systems;AF2916)および二次抗体Sheep IgG Horseradish Peroxidase-conjugated Antibody(R&D Systems;HAF016)を用いてラットHTRA1を検出した。非照射群と比較し、光照射した群においては硝子体液中のHTRA1量の増加が認められたことから、当該モデルでは、光照射による網膜障害の過程にHTRA1が関与するものと考えられる(図9)。
(5-3)ラット光照射網膜障害モデルにおけるHTRA1阻害ペプチドの網膜保護効果
ラットへの光照射直前に麻酔下で、0.04mg/mLまたは0.2mg/mLの濃度のHTRA1阻害ペプチドH308を5uL硝子体内に投与した。尚、生理食塩水投与群の例数は4であり、その他の群の例数は5であった。生理食塩水投与群は光照射により網膜断層の外顆粒層に含まれる核が減少したのに対し、HTRA1阻害ペプチド投与群では外顆粒層に含まれる核の減少を抑制する効果が認められた(図10)。以上より、HTRA1により引き起こされた組織障害に対し、HTRA1阻害ペプチドは薬効を示すことが明らかになった。
(6-1)pET 32a_HTRA1阻害ペプチドH308_S16A_Kex2の構築
HTRA1阻害ペプチドH308を鋳型として、配列番号9(図21)で示されるアミノ酸配列中の16番SerがAlaに置換されたアミノ酸配列を有する誘導体S16Aを調製した。下記プライマーおよびKOD-plus-(TOYOBO)を用いたPCR法((94℃ 15秒、60℃ 30秒、68℃ 15秒)×30サイクル)により断片Cを増幅した。
プライマー12:5’-CCGCAGTTTGGTCTGTTTAGCAAATATCGTACCCCGAATTGT-3’
プライマー13:5’-GCCATACCAGCATGGTCCGCACAATTCGGGGTACGATATTTGC-3’
次に、HTRA1阻害ペプチドH308を鋳型として、下記プライマーおよびKOD-plus-(TOYOBO)を用いたPCR法((94℃ 15秒、60℃ 30秒、68℃ 20秒)×30サイクル)により断片Dを増幅した。
プライマー14:5’-GCGGACCATGCTGGTATGGCATGTGTTGCTCTGTATGAAC-3’
プライマー15:5’-AAAACTCGAGTTAGCCGCCGCACGGACCATTGCGAATAA-3’
断片CとD、下記プライマー、KOD-plus-(TOYOBO)を用いたPCR法((94℃ 15秒、60℃ 30秒、68℃ 20秒)×30サイクル)により所望のDNA断片を増幅した。
プライマー16:5’-AAAAGGATCCCTGGACAAACGTGATCCGCAGTTTGGTCTGTTTAG-3’
プライマー15
増幅した断片をアガロースゲル電気泳動に供した後に、所望のDNA断片を切り出し、QIAquick Gel Extraction Kit(QIAGEN)によりDNAを調製した。調製したDNA断片およびpET 32a(Novagen)を制限酵素BamHI(NEB)およびXhoI(NEB)を用いて、37℃で1時間以上処理し、アガロースゲル電気泳動後に、所望のDNA断片を切り出し、QIAquick PCR Purification Kit(QIAGEN)により精製した。T4 DNA Ligase(NEB)を用いて、それぞれの精製断片を16℃で一晩反応させることでligation反応を実施した。Ligation溶液は、大腸菌JM109(TOYOBO)に添加し、氷上で30分間静置した後、42℃で45秒の熱処理、さらに氷上で5分間静置し、0.1mg/mlアンピシリンを含む2YTプレートに播種後、37℃で一晩静置培養することで、大腸菌を形質転換した。形質転換された大腸菌を培養した後に、miniprepおよび配列解析を実施することで、「pET 32a_HTRA1阻害ペプチドH308_S16A_Kex2」を構築した。尚、操作は(1-1-1)に記載の方法に準じて行った。
(6-2)HTRA1阻害ペプチド_N末誘導体発現ベクターの調製
配列番号9(図21)で示されるアミノ酸配列中、1番AspがGly、Ser、Glu又はSer-Leu-Ileで置換されたアミノ酸配列を有する、HTRA1阻害ペプチドのN末端配列誘導体4種(それぞれD1G,D1S,D1E又はD1SLIと表記する)を調製するため、(6-1)と同様の手法で発現ベクターを構築した。断片CおよびD、下記プライマー、KOD-plus-(TOYOBO)を用いたPCR法((94℃ 15秒、60℃ 30秒、68℃ 20秒)×30サイクル)により目的断片4種をそれぞれ増幅した。
D1G作製プライマー
プライマー17:5’-AAAAGGATCCCTGGACAAACGTGGCCCGCAGTTTGGTCTGTTTAG-3’
プライマー15
D1S作製プライマー
プライマー18:5’-AAAAGGATCCCTGGACAAACGTAGCCCGCAGTTTGGTCTGTTTAG-3’
プライマー15
D1E作製プライマー
プライマー19:5’-AAAAGGATCCCTGGACAAACGTGAACCGCAGTTTGGTCTGTTTAG-3’
プライマー15
D1SLI作製プライマー
プライマー20:5’-AAAAGGATCCCTGGACAAACGTAGCCTGATTCCGCAGTTTGGTCTGTTTAG-3’
プライマー15
増幅した4種の断片をアガロースゲル電気泳動に供した後に、所望のDNA断片を切り出し、QIAquick Gel Extraction Kit(QIAGEN)によりDNAを調製した。調製したDNA断片およびpET 32a(Novagen)を制限酵素BamHI(NEB)およびXhoI(NEB)を用いて、37℃で1時間以上処理し、アガロースゲル電気泳動後に、所望のDNA断片を切り出し、QIAquick PCR Purification Kit(QIAGEN)により精製した。T4 DNA Ligase(NEB)を用いて、それぞれの精製断片を16℃で一晩反応させることでligation反応を実施した。Ligation溶液は、大腸菌JM109(TOYOBO)に添加し、氷上で30分間静置した後、42℃で45秒の熱処理、さらに氷上で5分間静置し、0.1mg/mlアンピシリンを含む2YTプレートに播種後、37℃で一晩静置培養することで、大腸菌を形質転換した。形質転換された大腸菌を培養した後に、miniprepおよび配列解析を実施することで、「pET 32a_HTRA1阻害ペプチドH308_D1G_S16A_Kex2」「pET 32a_HTRA1阻害ペプチドH308_D1S_S16A_Kex2」「pET 32a_HTRA1阻害ペプチドH308_D1E_S16A_Kex2」「pET 32a_HTRA1阻害ペプチドH308_D1SLI_S16A_Kex2」を構築した。尚、操作は(1-1-1)に記載の方法に準じて行った。
(6-3)HTRA1阻害ペプチド誘導体の調製
(6-1)および(6-2)で構築したベクター5種をそれぞれ大腸菌Origami B (DE3)(Novagen)へ形質転換し、0.1mg/mlアンピシリンを含む2YT培地を用いて37℃で培養後、IPTG(最終濃度1mM)を添加し、16℃で一晩培養した。翌日、遠心分離(3,000g、20分、4℃)により集菌後、BugBuster Master Mix(Novagen)を用いてlysateを調製し、TALON Metal Affinity Resin(Clontech)を用いてHis tag融合目的蛋白質を精製した。次に、Kex2(前述)を用いてthioredoxin tagと所望の蛋白質とを切断し、TALONを用いて精製した。さらに、ゲルろ過クロマトグラフィー(Superdex75 10/300 GL)または逆相クロマトグラフィー(YMC-Pack ODS-AM)に供することで、HTRA1阻害ペプチド誘導体5種を調製した。該誘導体の有するアミノ酸配列は、配列番号23乃至27(図35乃至39)に記載されている。
(6-4)HTRA1阻害ペプチド誘導体の評価
(3-1)記載の方法に従い、HTRA1(cat)阻害活性を測定した結果、いずれの誘導体も阻害活性はH308と同等であった(図11)。
実施例(1-2)で調製した3種のHTRA1阻害ペプチド(H308、H321AT、H322AT)および(2-1)で調製したHTRA1(cat)を用いて、免疫沈降法により結合性を評価した。2.5μgの各HTRA1阻害ペプチドと10μgのHTRA1(cat)を室温で30分反応した後、10μLのTALON Metal Affinity Resin(Clontech)を添加した。さらに30分の反応後のResinをImmunoprecipitation(IP)画分として回収し、SDS-PAGEに供することで結合性を評価した。尚、反応のバッファーにはPBSを用いた。
(8-1)ペプチド基質を用いたHTRA1阻害ペプチドのHTRA1阻害活性評価
実施例6で構築した3種のHTRA1阻害ペプチド(H308_D1G_S16A、H321AT_D1G_S16A、H322AT_D1G_S16A)について、基質ペプチドH2-Optを用いて、HTRA1(cat)またはHTRA1(full)阻害活性を評価した(n=3)。基質ペプチドH2-Opt(Mca-IRRVSYSFK(Dnp)K)(株式会社ペプチド研究所:配列番号54、図8)を10mMになるようDMSOで溶解し、Assay buffer(50mM Tris,150mM NaCl,0.25% CHAPS,pH8.0)で希釈して終濃度10μMで使用した。Assay bufferで希釈したHTRA1(HTRA1(cat)またはHTRA1(full);実施例2)とHTRA1阻害ペプチドをそれぞれ25μLずつ混ぜ、37℃で20分反応させた後にAssay bufferで希釈した基質を50μL加えて、Enspire(PerkinElmer)で蛍光シグナル(excitation 328nm/emission 393nm)を測定した。HTRA1は終濃度10nM、HTRA1阻害ペプチドは終濃度1.875~1,000nM、反応および測定にはプロテオセーブ(登録商標)SS96F黒プレート(住友ベークライト株式会社)を使用した。
GraphPad Prism (version 5.0; GraphPad Software Inc.)を用いて50%阻害濃度(IC50)を算出した結果、いずれのHTRA1阻害ペプチドも低濃度でHTRA1(cat)およびHTRA1(full)酵素活性を阻害することが明らかになった(図66)。
ヒトVitronectinをタンパク基質として、HTRA1阻害ペプチドのHTRA1阻害活性を評価した。操作は実施例(3-2)に従った。
(8-1)と同様、ヒトitronectinを基質とした場合でもHTRA1阻害ペプチドは強力にHTRA1(cat)阻害を示した(図67)。
基質ペプチドの切断を指標に、他のプロテアーゼに対する特異性を評価した。Bovine trypsin、Bovine α-chymotrypsin、Protein C、Tryptase、Chymase、Thrombin、Plasmin、tPA、Plasma kallikrein、Matriptase、uPA、HTRA2については実施例(3-3)に記載の方法に従った(n=3)。また、他のプロテアーゼに対する阻害活性の手順、プロテアーゼおよび基質の組み合わせは以下の通り。
実施例6で調製した3種のHTRA1阻害ペプチドおよび(2-1)で調製したHTRA1(cat)を用いて、実施例7の操作に従い、免疫沈降法により結合性を評価した。
3種のHTRA1阻害ペプチドまたはHTRA1(cat)それぞれとTALONを反応させた場合、His tagが融合したHTRA1(cat)のみのバンドがinputレーンに検出された。一方、各阻害ペプチドとHTRA1(cat)を反応させたIPレーンのみで、阻害ペプチドと酵素のバンドが検出された。よって、3種のHTRA1阻害ペプチドはそれぞれHTRA1(cat)に結合することが確認された(図69)。
実施例(5-1)で構築したラット光照射網膜障害モデルを用いて、実施例6で作製した3種のHTRA1阻害ペプチドの網膜保護効果を評価した。操作は実施例5に従った。いずれの群も例数は6であった。
網膜病理評価の結果を図70に示す。3種のHTRA1阻害ペプチドは、光照射によって引き起こされる外顆粒層に含まれる核の数の減少に対して顕著な抑制作用を示した。
(11-1)ハイドロキノン含有高脂肪食負荷によるウサギ網膜障害モデルの作製
高脂肪食(High Fat Diet;HFD)とハイドロキノン(Hydroquinone;HQ)を用いた網膜障害モデルは、酸化促進物質により酸化ストレスを惹起し網膜障害を引き起こすモデルであり、マウスにおいてのみモデルが報告されている(Diego G. Espinosa-Heidmann et al.,(2006) Invest Ophthalmol Vis Sci. 47巻(2号):729-737頁)。そこで、1.5%(W/V)ココナッツオイル-0.25%(W/V)コレステロール-1.5%(W/V)ピーナッツオイル-2.4%(W/V)ハイドロキノン含有RC4(オリエンタル酵母)食(HFD-HQ)を3歳齢JWウサギに4ヶ月間摂食させることでウサギ網膜障害モデルを構築した。安楽殺後に眼球を摘出し、角膜輪部より5mm程度外側で切開して前眼部を取り除き、さらに硝子体を分離した後、網膜-脈絡膜-強膜を4%(W/V)パラホルムアルデヒド固定液で24時間以上浸漬させ固定した。固定後、脈絡膜を分離し、一次抗体ZO-1 Monoclonal Antibody (ZO1-1A12)(Themo Fisher SCIENTIFIC;33-9100)および二次抗体Chicken anti-Mouse IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 594(Themo Fisher SCIENTIFIC;A-21201)を用いて免疫染色を行った。蛍光顕微鏡(BZ-9000;KEYENCE)を用いて染色した脈絡膜を観察し、染色された網膜色素上皮(RPE)細胞の面積を求め、RPE細胞障害を評価した。
図71に、12週齢ウサギ、3歳齢ウサギおよび、HFD-HQ負荷3歳齢ウサギのRPE細胞の染色像(図71(A))とRPE細胞の平均面積(図71(B))のグラフを示す。3歳齢のウサギでは12週齢のウサギよりもRPE細胞が肥大しており、HFD-HQ負荷でさらに肥大することが確認され、RPE細胞に障害が現れていることを認めた。加齢黄斑変性症患者の眼球においても同様の変化が観察されている(Ding JD et al.,(2011) Proc Natl Acad Sci U S A. 108巻(28号):279-87頁)。
AMD関連因子の発現を評価するため、当該ウサギ網膜障害モデルから網膜およびRPE/脈絡膜からそれぞれ組織を採取し、RNeasy mini kit (QIAGEN)を用いてmRNAを抽出後、TaqMan Gene Expression Master Mix(Thermo Fisher Scientific)を用いて逆転写反応を行った。TaqMan Gene Expression Assay(Oc03397832_g1およびOc03824857_g1;Thermo Fisher Scientific)を用いて、7900HT Fast Real-Time PCR System(Applied Biosystems)により補体第三因子C3および内部標準β-actinのmRNA量を定量解析した。尚、3歳齢ウサギの例数は4、HFD-HQ負荷3歳齢ウサギの例数は10として解析を実施した。
ウサギ網膜障害モデルにおけるHTRA1の関与を調べるため、(6-1)で作製したモデルウサギから硝子体液を採取し、Trypsin/Lys-C Mix(Promega)を用いて酵素消化後、LC(EASY-nLC 1000; Thermo Fisher Scientific)-MS(TripleTOF 6600;SCIEX)を用いてHTRA1のペプチド断片を定量した。HFD-HQを与えたウサギの硝子体液中ではHTRA1タンパク量が増加していることが明らかになった(図71(E))。以上のことから、RPE細胞肥大化およびAMD関連因子C3、HTRA1の発現亢進が認められたことから、当該ウサギ網膜障害モデルが加齢性網膜疾患研究に有用であることが示された。
当該ウサギモデルを用いて、実施例1で作製したHTRA1阻害ペプチドH308の網膜保護効果を評価した。HFD-HQの給餌開始から2ヶ月後に、40mg/mLのH308溶液50μLを麻酔下で片眼に硝子体内に投与した。僚眼には生理食塩水を硝子体内投与した。いずれの群も例数は5であった。
給餌開始4ヶ月後に、モデル動物のRPE細胞肥大を評価した結果を図72に示す。RPE細胞の平均面積(図72(A))または細胞面積が1500μm2以上の肥大したRPE細胞数(図72(B))、いずれの指標においても、HTRA1阻害剤はRPE細胞の肥大に対して抑制効果を示した。図71(E)に示す通り、当該モデルの硝子体液中ではHTRA1の増加が認められ、HFD-HQによるRPE細胞の障害過程に対してHTRA1の関与が示唆された。このように、HTRA1阻害ペプチドは、抗加齢黄斑変性症剤として有用であり、本試験ではとりわけ萎縮型の加齢黄斑変性症の予防に有用であることが示された。
また、HTRA1阻害ペプチドを投与した正常ウサギ網膜においてHTRA1阻害ペプチドの存在が認められ、HTRA1阻害ペプチドの高い組織浸透性が示された。
ARPE-19細胞を10%Fetal bovine serum(FBS)、Penicillin-Streptomycin(Thermo Fisher Scientific)含有のDMEM/F-12培地(和光純薬工業株式会社)を用いて37℃、5%CO2条件下で、12mm Transwell with 0.4μm Pore Polyester Membrane Insert,Sterile(Corning)中にてコンフルエントになるまで培養した。その後、FBS非含有のDMEM/F-12で5日間培養し、チャンバー上下層に終濃度が500μMのH2O2を、チャンバー上層に終濃度25%のNormal Human Serum Complement(Quidel)をそれぞれ添加した。さらにチャンバー上下層にHTRA1(実施例2-2)、HTRA1プロテアーゼ不活性変異体HTRA1(S328A)(実施例2-3)、または、HTRA1阻害ペプチドH308_D1G_S16A(実施例6)をそれぞれ終濃度1μMとなるように加えた。4時間後に培養上清を除去し、PBSで洗浄後、SuperPrepTM Cell Lysis & RT Kit for qPCR(東洋紡株式会社)によりmRNAを抽出し、逆転写反応を行った。TaqMan Gene Expression Assays(Hs000900055_m1およびHs02786624_g1;Thermo Fisher Scientific)を用いて、7900HT Fast Real-Time PCR System(Applied Biosystems)によりVEGFのmRNA量を定量解析した。尚、mRNA量の補正にはGAPDHを用いた。
結果を図74に示す。H2O2、Normal Human Serum ComplementおよびHTRA1不活性変異体HTRA1(S328A)添加条件と比べて、H2O2、Normal Human Serum ComplementおよびHTRA1を添加することで、VEGFのmRNA量が顕著に増加した。さらに、HTRA1阻害ペプチドH308_D1G_S16Aを共添加することにより、VEGFの発現抑制が認められた。網膜色素上皮細胞からのVEGF誘導は滲出型加齢性黄斑変性病態形成に重要とされている(Klettner A. et al.,(2009) Graefes Arch Clin Exp Ophthalmol. 247巻:1487-1492頁)。また、病態形成だけでなく、病態の維持にもこれらの病的なVEGF誘導が関与していると考えられており、HTRA1阻害ペプチドH308_D1G_S16A等本発明のペプチドの投与は滲出型加齢性黄斑変性の予防および治療に対して有効である。
(13-1)HUVEC遊走試験
HUVEC(Kurabo Industries)は0.1%BSA含有EBMTM-2基本培地(Lonza Walkersville)に血清とVEGFを除いたEGMTM-2 SingleQuotsTM添加因子セットを添加した培地(0.1%BSA含有無血清EGM)で37℃、5%CO2条件下で18時間培養した後、0.1%BSA含有無血清EGMで4×105個/mLとなるよう調製した。メンブレンをゼラチンコートしたCorning FluoroBlok HTS 96 Well Multiwell Permeable Support System with 3.0 μm High Density PET Membrane(Corning)のチャンバー上層に、4×105個/mLのHUVEC懸濁液を50μL/ウェルで添加した後、チャンバー下層に210μL/ウェルで下記サンプル(培地1または2、3)を添加した(n=3)。また、HUVECを添加していないチャンバー上層には、0.1%BSA含有無血清EGMを50μL添加し、そのチャンバー下層には、0.1%BSA含有無血清EGMを210μL添加した(n=3)。
培地1;0.1%BSA含有無血清EGM
培地2;全てのEGMTM-2 SingleQuotsTM 添加因子を添加したEBMTM-2培地(EGM増殖培地)
培地3;300nMのH308_DIG_S16Aを含むEGM増殖培地
細胞とサンプルを添加したFluoroBlok HTS 96 Well Multiwell Support Systemを37℃、5%CO2条件下で2時間インキュベーションし、下層へ遊走したHUVECをPBSで洗浄後、4μg/mLのCalcein-AM(Thermo Fisher Scientific)含有0.1%BSA含有無血清EGMで15分間染色した。その後、培地をPBSに置換し、各ウェルの蛍光強度(励起波長/蛍光波長:485nm/535nm)をプレートリーダー(ARVO-MX、PerkinElmer)で測定し、次式で各ウェルの遊走細胞を算出した。
遊走細胞=HUVEC存在ウェルの蛍光強度の平均(n=3)-ブランクウェルの蛍光強度の平均(n=3)。
結果を図75に示す。HTRA1阻害ペプチドH308_DIG_S16Aは血清含有培地で誘導されたHUVECの遊走に対して抑制効果を認めた。よって、本発明のペプチドは、滲出型加齢黄斑変性症の特徴である血管新生に対して抑制効果を示すことが明らかになった。
実施例(11-1)~(11-3)で作製、評価したウサギ網膜障害モデルを用いて、実施例1で作製したHTRA1阻害ペプチドH308又は実施例6で作製した3種のHTRA1阻害ペプチドのうち1種の網膜障害に対する治療効果を評価する。40mg/mLの阻害ペプチド溶液50μLを麻酔下でモデル動物の片眼に硝子体内に投与し2ヶ月間飼育する。僚眼には生理食塩水を硝子体内投与する。いずれの群も例数は5とする。
生理食塩水投与群ではRPE細胞面積の増大又は細胞数の増加が見られるのに対し、HTRA1阻害ペプチド投与群ではRPE細胞面積の増大又は細胞数の増加が抑制されるであろう。このように、HTRA1阻害ペプチドは、抗加齢黄斑変性症剤として有用であること、本実施例ではとりわけ萎縮型の加齢黄斑変性症の治療に有用であることを確認することができる。
配列番号2:ヒトSPINK2のアミノ酸配列をコードするヌクレオチド配列(図14)
配列番号3:ペプチドH218のアミノ酸配列(図15)
配列番号4:ペプチドH218のアミノ酸配列をコードするヌクレオチド配列(図16)
配列番号5:ペプチドH223のアミノ酸配列(図17)
配列番号6:ペプチドH223のアミノ酸配列をコードするヌクレオチド配列(図18)
配列番号7:ペプチドH228のアミノ酸配列(図19)
配列番号8:ペプチドH228のアミノ酸配列をコードするヌクレオチド配列(図20)
配列番号9:ペプチドH308のアミノ酸配列(図21)
配列番号10:ペプチドH308のアミノ酸配列をコードするヌクレオチド配列(図22)
配列番号11:ペプチドH321のアミノ酸配列(図23)
配列番号12:ペプチドH321のアミノ酸配列をコードするヌクレオチド配列(図24)
配列番号13:ペプチドH322のアミノ酸配列(図25)
配列番号14:ペプチドH322のアミノ酸配列をコードするヌクレオチド配列(図26)
配列番号15:ペプチド誘導体H308ATのアミノ酸配列(図27)
配列番号16:ペプチド誘導体H308ATのアミノ酸配列をコードするヌクレオチド配列(図28)
配列番号17:ペプチド誘導体H321ATのアミノ酸配列(図29)
配列番号18:ペプチド誘導体H321ATのアミノ酸配列をコードするヌクレオチド配列(図30)
配列番号19:ペプチド誘導体H322ATのアミノ酸配列(図31)
配列番号20:ペプチド誘導体H322ATのアミノ酸配列をコードするヌクレオチド配列(図32)
配列番号21:ペプチドM7のアミノ酸配列(図33)
配列番号22:ペプチドM7のアミノ酸配列をコードするヌクレオチド配列(図34)
配列番号23:ペプチド誘導体H308_S16Aのアミノ酸配列(図35)
配列番号24:ペプチド誘導体H308_D1G_S16Aのアミノ酸配列(図36)
配列番号25:ペプチド誘導体H308_D1S_S16Aのアミノ酸配列(図37)
配列番号26:ペプチド誘導体H308_D1E_S16Aのアミノ酸配列(図38)
配列番号27:ペプチド誘導体H308_D1SLI_S16Aのアミノ酸配列(図39)
配列番号28:ペプチド誘導体H321AT_D1G_S16Aのアミノ酸配列(図40)
配列番号29:ペプチド誘導体H322AT_D1G_S16Aのアミノ酸配列(図41)
配列番号30:HTRA1阻害ペプチドの一般式(図42)
配列番号31:S tag及びリンカーからなるアミノ酸配列(図43)
配列番号32:C末6マーのアミノ酸配列(図44)
配列番号33:プライマー1のヌクレオチド配列(図45)
配列番号34:プライマー2のヌクレオチド配列(図46)
配列番号35:プライマー3のヌクレオチド配列(図47)
配列番号36:プライマー4のヌクレオチド配列(図48)
配列番号37:プライマー5のヌクレオチド配列(図49)
配列番号38:プライマー6のヌクレオチド配列(図50)
配列番号39:プライマー7のヌクレオチド配列(図51)
配列番号40:プライマー8のヌクレオチド配列(図52)
配列番号41:プライマー9のヌクレオチド配列(図53)
配列番号42:プライマー10のヌクレオチド配列(図54)
配列番号43:プライマー11のヌクレオチド配列(図55)
配列番号44:プライマー12のヌクレオチド配列(図56)
配列番号45:プライマー13のヌクレオチド配列(図57)
配列番号46:プライマー14のヌクレオチド配列(図58)
配列番号47:プライマー15のヌクレオチド配列(図59)
配列番号48:プライマー16のヌクレオチド配列(図60)
配列番号49:プライマー17のヌクレオチド配列(図61)
配列番号50:プライマー18のヌクレオチド配列(図62)
配列番号51:プライマー19のヌクレオチド配列(図63)
配列番号52:プライマー20のヌクレオチド配列(図64)
配列番号53:ヒトHTRAI(full)のアミノ酸配列(図65)
配列番号54:H2-Optのアミノ酸配列(図8)
配列番号55:プライマー21のヌクレオチド配列(図76)
配列番号56:プライマー22のヌクレオチド配列(図77)
Claims (29)
- 配列番号30(図42)で示されるアミノ酸配列を含み、且つ、ヒトHTRA1の有するプロテアーゼ活性を阻害する、SPINK2変異体ペプチド。
- 1番目のXaa(X1)はAsp、Glu、Ser、Gly,又はIle、2番目のXaa(X2)はAla、Gly、Leu、Ser又はThr、3番目のXaa(X3)はAsp、His、Lys、Met又はGln、4番目のXaa(X4)はAsp、Phe、His、Ser又はTyr、5番目のXaa(X5)はAla、Asp、Glu、Met又はAsn、6番目のXaa(X6)はMet又はTrp、7番目のXaa(X7)はGln、Trp、Tyr又はVal、8番目のXaa(X8)はPhe、Leu又はTyr、9番目のXaa(X9)はPhe又はTyr、10番目のXaaX10)はAla、Glu、Met又はVal、並びに、11番目のXaa(X11)はAla、Thr又はValである、請求項1記載のペプチド。
- 配列番号3、5、7、9、11、13、15、17、19、21及び23乃至29(図15、図17、図19、図21、図23、図25、図27、図29、図31、図33及び、図35乃至41)のいずれか一つで示されるアミノ酸配列を含む、請求項1又は2記載のペプチド。
- 配列番号30(図42)で示されるアミノ酸配列のアミノ末端側に1乃至3個のアミノ酸がペプチド結合してなるアミノ酸配列を含む、請求項1乃至3のいずれか一つに記載のペプチド。
- 配列番号30(図42)で示されるアミノ酸配列のカルボキシル末端側に1又は2個のアミノ酸がペプチド結合してなるアミノ酸配列を含む、請求項1乃至4のいずれか一つに記載のペプチド。
- 3つのジスルフィド結合を有し、ループ構造、αへリックス及びβシートを含むことで特徴付けられる立体構造を有する、請求項1乃至5のいずれか一つに記載のペプチド。
- 請求項1乃至6のいずれか一つに記載のペプチドに含まれるアミノ酸配列をコードするヌクレオチド配列を含むポリヌクレオチド。
- 請求項7記載のポリヌクレオチドを含むベクター。
- 請求項7記載のポリヌクレオチド若しくは請求項8記載のベクターを含むか又は請求項1乃至6のいずれか一つに記載のペプチドを産生する細胞。
- 下記工程(i)及び(ii)を含む、HTRA1の有するプロテアーゼ活性を阻害するSPINK2変異体ペプチドの製造方法:
(i)請求項9記載の細胞を培養する工程;
(ii)該培養物からSPINK2変異体ペプチドを回収する工程。 - 請求項10記載の方法により得られるSPINK2変異体ペプチド。
- 請求項1乃至6及び11のいずれか一つに記載のペプチドに他の部分が連結してなるコンジュゲート。
- ポリペプチドである、請求項12記載のコンジュゲート。
- 請求項1乃至6及び11のいずれか一つに記載のペプチドに結合する抗体又はその機能断片。
- 請求項1乃至6及び11のいずれか一つに記載のペプチド、請求項7記載のポリヌクレオチド、請求項8記載のベクター、請求項9記載の細胞、請求項12若しくは13記載のコンジュゲート、及び/又は、請求項14記載の抗体もしくはその機能断片を含む組成物。
- 請求項1乃至6及び11のいずれか一つに記載のペプチド及び/又は請求項12又は13に記載のコンジュゲートを含む医薬組成物。
- HTRA1関連疾患の治療又は予防のための、請求項16記載の医薬組成物。
- HTRA1関連疾患が滲出型加齢黄斑変性症、萎縮型加齢黄斑変性症、地図状萎縮、糖尿病網膜症、未熟児網膜症、ポリープ状脈絡膜血管症、関節リウマチ、及び変形性関節症よりなる群から選択される1つ又は2つ以上である、請求項17記載の医薬組成物。
- 1つ又は2つ以上の他の医薬を含む、請求項16乃至18のいずれか一つに記載の医薬組成物。
- 1つ又は2つ以上の他の医薬と組み合わせて使用される、請求項16乃至18のいずれか一つに記載の医薬組成物。
- 網膜保護剤である、請求項16乃至20のいずれか一つに記載の医薬組成物。
- 下記の工程1乃至工程3を含む、加齢黄斑変性症の治療薬又は予防薬を同定する方法:
[工程1]HTRA1プロテアーゼ及び基質を、被検物質の存在下又は非存在下で保温する;
[工程2]被検物質の存在下及び非存在下でのHTRA1プロテアーゼ活性を検出する;
[工程3]被検物質の存在下でのHTRA1プロテアーゼ活性が、被検物質の非存在下でのHTRA1プロテアーゼ活性と比較して小さい場合、該被検物質を陽性と判定する。 - 下記の工程1乃至工程3を含む、網膜保護剤を同定する方法:
[工程1]HTRA1プロテアーゼ及び基質を、被検物質の存在下又は非存在下で保温する;
[工程2]被検物質の存在下及び非存在下でのHTRA1プロテアーゼ活性を検出する;
[工程3]被検物質の存在下でのHTRA1プロテアーゼ活性が、被検物質の非存在下でのHTRA1プロテアーゼ活性と比較して小さい場合、該被検物質を陽性と判定する。 - ハイドロキノンを含む高脂肪食を3乃至6ヶ月摂食させる工程を含む、網膜障害モデルウサギの作製方法であって、該モデルウサギの網膜色素上皮細胞が正常ウサギの網膜色素上皮細胞に比して肥大していることを特徴とする方法。
- 請求項24記載の方法により作製され、正常ウサギに比して肥大した網膜色素上皮細胞を有していることを特徴とする、網膜障害モデルウサギ。
- 下記工程(i)及び(ii)を含む、加齢黄斑変性症の治療薬若しくは予防薬、又は、網膜保護剤を同定する方法:
(i)請求項25記載のウサギにおける網膜色素上皮細胞の肥大を、被検物質投与下及び非投与下で測定する工程;及び
(ii)該被検物質投与下における網膜色素上皮細胞の肥大が非投与下と比較して抑制された場合、該検物質を陽性と判定する工程。 - 工程(i)における測定が、網膜色素上皮細胞の平均面積又は肥大した網膜色素上皮細胞数の測定である、請求項26記載の方法。
- 萎縮型加齢黄斑変性症の治療又は予防のための、請求項16乃至18のいずれか一つに記載の医薬組成物。
- 滲出型加齢黄斑変性症の治療又は予防のための、請求項16乃至18のいずれか一つに記載の医薬組成物。
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