WO2019049933A1 - Klk1、klk4、又は、klk4及びklk8を阻害するペプチド - Google Patents
Klk1、klk4、又は、klk4及びklk8を阻害するペプチド Download PDFInfo
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- WO2019049933A1 WO2019049933A1 PCT/JP2018/033037 JP2018033037W WO2019049933A1 WO 2019049933 A1 WO2019049933 A1 WO 2019049933A1 JP 2018033037 W JP2018033037 W JP 2018033037W WO 2019049933 A1 WO2019049933 A1 WO 2019049933A1
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- C07K14/8107—Endopeptidase (E.C. 3.4.21-99) inhibitors
- C07K14/811—Serine protease (E.C. 3.4.21) inhibitors
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- C12N9/14—Hydrolases (3)
- C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
- C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
- C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
- C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
- C12N9/6424—Serine endopeptidases (3.4.21)
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Definitions
- the present invention relates to a peptide, a polynucleotide, a vector, a cell, a method for producing a peptide, a peptide obtained by such a method, a conjugate obtained by binding another moiety to the peptide, a composition comprising the peptide or conjugate, a peptide or a conjugate Pharmaceutical composition comprising a gate, said pharmaceutical composition for treatment or prevention of various diseases comprising a peptide or conjugate, use of a peptide or conjugate for treatment or prevention of various diseases, administration of a peptide or conjugate
- the present invention relates to methods for treating various diseases including steps, compositions for diagnosing or examining various diseases including peptides or conjugates, and the like.
- KLK1 inhibitors by human monoclonal antibodies have been reported, and it has been clarified that they show efficacy in sheep asthma models (Non-patent Document 3).
- low-molecular-weight proteins other than antibodies or fragments thereof for example, proteins that do not contain immunoglobulin variable regions
- KLK8 is also called neuropsin and is composed of an N-terminal propeptide and a trypsin-like domain having a protease activity, and is known to be added with an N-type sugar chain (Non-patent Document 1).
- KLK8 is expressed in the hippocampus, amygdala, cerebral limbic system, etc. as a probody having a propeptide, and is cleaved by a protease to become activated KLK8.
- SPINK2 Serine Protease Inhibitor Kazal-type 2
- Kazal-type domain with three disulfide bonds. It is expressed in testis and seminal vesicles in humans and functions as a trypsin / acrosin inhibitor (Non-patent Document 8).
- SPINK2 itself can be KLK1, KLK4, or KLK4 and KLK8 dual inhibitors.
- a SPINK2 variant peptide comprising the amino acid sequence shown in SEQ ID NO: 23 (FIG. 29) and specifically inhibiting the protease activity possessed by KLK4.
- the peptide provided by the present invention or the pharmaceutical composition containing the same has KLK1 inhibitory activity, KLK4 inhibitory activity, or KLK4 / KLK8 inhibitory activity, and it is a KLK1-related disease, a KLK4-related disease, or a KLK4 / KLK8-related disease ( All are useful for the treatment or prevention etc. of below-mentioned each.
- a final concentration of 1 nM matriptase (R & D Systems; E3946-SE) and a final concentration of 100 ⁇ M substrate peptide Boc-QAR-AMC Fluogenic Peptide Substrate (ES014) were used for human matriptase inhibitory activity.
- human protein C inhibitory activity use a final concentration of 100 nM protein C (Sigma-Aldrich; P2200) and a final concentration of 100 ⁇ M substrate peptide Boc-Leu-Ser-Thr-Arg-MCA (Peptide Research Institute, Inc .; 3112-v) It was.
- a final concentration of 10 nM uPA (Sigma-Aldrich; T0831) and a final concentration of 100 ⁇ M substrate peptide Pyr-Gly-Arg-MCA (Peptide Research Laboratories; 3145-v) were used for Human uPA inhibitory activity.
- a final concentration of 0.125 ⁇ g / ml plasma kallikrein (Sigma-Aldrich; T0831) and a final concentration of 100 ⁇ M substrate peptide Z-Phe-Arg-MCA (Peptide Research Institute, Inc .; 3095-v) are used for human plasma kallikrein inhibitory activity. It was.
- the inhibitory peptide was linked to the region containing the KLK4 active center.
- Amino acid sequence of human SPINK2 (SEQ ID NO: 1) Amino acid sequence of human KLK1 (SEQ ID NO: 2) Amino acid sequence of human KLK4 (SEQ ID NO: 3) Amino acid sequence of human KLK8 (SEQ ID NO: 4) Amino acid sequence of KLK1 inhibitory peptide K10061 (SEQ ID NO: 5) Amino acid sequence of KLK1 inhibitory peptide K10062 (SEQ ID NO: 6) Amino acid sequence of KLK1 inhibitory peptide K10066 (SEQ ID NO: 7) Amino acid sequence of KLK1 inhibitory peptide K10071 (SEQ ID NO: 8) Amino acid sequence of KLK4 inhibitory peptide K40001 (SEQ ID NO: 9) Amino acid sequence of KLK4 inhibitory peptide K40003 (SEQ ID NO: 10) Amino acid sequence of KLK4 inhibitory peptide K40004 (SEQ ID NO: 11) Amino acid sequence of KLK
- polypeptide In the present invention, “polypeptide”, “peptide” and “protein” are synonymous.
- a peptide that recognizes or binds to target molecule X (hereinafter, the recognition or binding action is collectively referred to as “X binding activity”) can be referred to as “X binding peptide”.
- X binding activity a peptide that recognizes or binds to target molecule X
- the recognition or binding action is collectively referred to as “X binding activity”.
- the target molecule X is recognized or bound to the target molecule X, and one or more activities or functions possessed by the target molecule X are inhibited or suppressed (hereinafter, their inhibitory or suppressive actions are summarized The "X inhibitory activity”.
- the peptide can be called “X inhibitory peptide”.
- SPINK2 means Serine Protease Inhibitor Kazal-type 2 and is a 7 kDa protein consisting of a Kazal-like domain having three disulfide bonds.
- Preferred SPINK2 is from human.
- human SPINK2 is simply referred to as "SPINK2" unless otherwise stated.
- KLK1 is a protein consisting of an N-terminal propeptide and a protease activity domain and exhibiting trypsin-like and chymotrypsin-like protease activities to which three N-type sugar chains are added.
- Preferred KLK1 is of human origin.
- human KLK1 may be simply referred to as "KLK1" unless otherwise stated.
- precursor KLK1 means pro-KLK1 and is composed of a domain having propeptide and protease activity.
- Active KLK1 means active KLK1 and is composed of domains having protease activity. Preferred active forms of KLK1 are of human origin.
- precursor KLK4 means pro-KLK4 and is composed of a domain having propeptide and protease activity.
- Active KLK4 means active KLK4 and is composed of domains having protease activity. Preferred active forms of KLK4 are of human origin.
- precursor KLK8 means pro-KLK8, which is composed of a domain having propeptide and protease activity.
- Active KLK8 means active KLK8, which is composed of domains having protease activity. Preferred active forms of KLK8 are of human origin.
- KLK1 inhibitory peptide Within the scope of “KLK1 inhibitory peptide”, “KLK4 inhibitory peptide” and “KLK4 / KLK8 inhibitory peptide”, a conjugate comprising a fragment of the peptide, other moiety (moiety) added to or bound to the peptide or a fragment thereof Among them, those which maintain the KLK1 inhibitory (binding) activity, the KLK4 inhibitory (binding) activity, or the KLK4 / KLK8 inhibitory (binding) activity are included, respectively.
- fragments, adducts and modifications (conjugates) of the peptide that maintain KLK1 inhibitory (binding) activity, KLK4 inhibitory (binding) activity, or KLK4 inhibitory (binding) activity and KLK8 inhibitory (binding) activity are also “ KLK1 inhibitory peptide ",” KLK4 inhibitory peptide "or” KLK4 / KLK8 inhibitory peptide "is respectively contained.
- cells include various cells derived from an individual animal, subcultured cells, primary cultured cells, cell lines, recombinant cells, yeast, microorganisms and the like.
- SPINK2 variant refers to one or more wild type amino acid sequences in which one or more amino acids are replaced with an amino acid different from the wild type in the amino acid sequence of wild type SPINK2 An amino acid is deleted, an amino acid which is not in one or more wild types is inserted, and / or an amino acid which is not in wild type is wild type amino terminal (N terminal) and / or carboxyl terminal (C (Hereinafter referred to as "mutation”) is meant to be a peptide containing an amino acid sequence.
- KLK4 / KLK8 inhibitory activity those having KLK1 inhibitory activity, KLK4 inhibitory activity, or KLK4 inhibitory activity and KLK8 inhibitory activity are KLK1 inhibitory peptides, KLK4 inhibitory peptides, or KLK4 / KLK8 Included in inhibitory peptides.
- “insertion” may also be included in the scope of “addition”.
- hybridize under stringent conditions refers to hybridization in a solution containing 5 ⁇ SSC at 65 ° C., and then in an aqueous solution containing 2 ⁇ SSC-0.1% SDS 20 minutes at 65 ° C., 20 minutes at 65 ° C. in an aqueous solution containing 0.5 ⁇ SSC-0.1% SDS, and 65 ° C. in an aqueous solution containing 0.2 ⁇ SSC-0.1% SDS The hybridization is performed under the conditions for washing for 20 minutes, respectively, or conditions equivalent thereto.
- SSC is an aqueous solution of 150 mM NaCl-15 mM sodium citrate, and n ⁇ SSC means n-fold concentration of SSC.
- KLK1-specific inhibitory peptides are synonymous with KLK1-selective inhibitory peptides.
- amino acid residue may be abbreviated as “amino acid”.
- Natural amino acids can be divided, for example, into the following groups based on the nature of their common side chains.
- 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 (4) Basic amino acid group: His, Lys, Arg
- Group of amino acids affecting the direction of main chain Gly, Pro
- Aromatic amino acid group Trp, Tyr, Phe
- classification of natural amino acids is not limited to these.
- naturally occurring amino acids can be subjected to conservative amino acid substitutions.
- Constant amino acid substitution means substitution with a functionally equivalent or similar amino acid.
- Conservative amino acid substitutions in a peptide result in static changes in the amino acid sequence of the peptide.
- one or more amino acids of similar polarity act functionally equivalently, resulting in static changes in the amino acid sequence of such peptides.
- substitutions within a group can be considered as conservative as to 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 comprising the amino acid.
- cysteine residues can take the less polar, oxidized (disulfide) form as compared to the reduced (thiol) form.
- the long aliphatic portion of the arginine side chain can constitute structurally and functionally important features.
- side chains containing an aromatic ring 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 nonpolar groups may be structurally and functionally conservative.
- Residues such as proline, glycine, cysteine (disulfide form) can have a direct effect on the conformation of the main chain and often can not be substituted for structural distortion.
- conservative amino acid substitution is specific substitution based on the similarity of side chains (Reinja, Biochemistry, Second Revised Edition, 1975, pp. 73 to 75: L. Lehninger, Biochemistry, 2 nd edition, pp 73-75, Worth Publisher, New York (1975)) and typical substitutions.
- Nonpolar amino acid group alanine (hereinafter referred to as “Ala” or simply “A”), valine (hereinafter referred to as “Val” or simply “V”), leucine (hereinafter referred to as “Leu” or simply “ Described as L “), isoleucine (hereinafter referred to as” Ile “or simply” I “), proline (hereinafter referred to as” Pro “or simply as” P “), phenylalanine (hereinafter referred to as” 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 referred to as “Gly” or simply “G”), serine (hereinafter referred to as “Ser” or simply “S”), threonine (hereinafter referred to as "Thr” or simply “M
- KLK1 Inhibitory Peptide KLK4 Inhibitory Peptide
- KLK4 / KLK8 Inhibitory Peptide The peptide of the present invention has KLK1 inhibitory activity, KLK4 inhibitory activity, or KLK4 / KLK8 inhibitory activity.
- the substrates of the proteases possessed by KLK1, KLK4, or KLK4 and KLK8 are not particularly limited, such as endogenous substrates, exogenous substrates, synthetic substrates and the like.
- KLK1 endogenous substrates for humans include low molecular weight kininogen, calistatin, collagen and the like.
- endogenous human substrates of KLK4 include Pro-KLK3, fibronectin, collagen and the like.
- As an endogenous human substrate for KLK8, tPA, fibronectin, collagen and the like can be exemplified. Gelatin obtained by heat denaturation of collagen can also be used as a substrate.
- the KLK1 inhibitory peptide, the KLK4 inhibitory peptide, or the KLK4 / KLK8 inhibitory peptide of the present invention does not inhibit or inhibit KLK1, KLK4 or protease activities other than KLK4 and KLK8, respectively, or inhibit or inhibit them. It is preferred that the degree is relatively weak.
- the protease inhibitory activity of the KLK1 inhibitory peptide, the KLK4 inhibitory peptide, or the KLK4 / KLK8 inhibitory peptide of the present invention is preferably high in KLK1 specificity, KLK4 specificity or KLK4 / KLK8 specificity.
- Such preferred peptides of the present invention do not show any side effects due to inhibition or suppression of other protease activities, and diseases associated with KLK1, diseases associated with KLK4, or diseases associated with KLK4 / KLK8 (any one It can be suitably used as a therapeutic agent or prophylactic agent described below).
- an inhibitor with high specificity for KLK1, KLK4 or KLK4 / KLK8 that is, a KLK1-specific inhibition peptide, a KLK4-specific inhibition peptide or a KLK4 / KLK8-specific inhibition peptide avoids the side effects as described above Therefore, they can be suitably used for the treatment or prevention of diseases associated with KLK1, diseases associated with KLK4, or diseases associated with KLK4 / KLK8, respectively.
- the KLK1 inhibitory peptide, the KLK4 inhibitory peptide, or the KLK4 / KLK8 inhibitory peptide of the present invention may be competitive in binding of a protease substrate to KLK1, KLK4, or KLK4 and / or KLK8.
- KLK1, KLK4 and KLK8 which are targets for the peptides of the invention, are derived from vertebrates, preferably mammals, more preferably primates, even more preferably humans, but non-human animals, For example, they may be derived from rodents such as rats and mice, and primates such as cynomolgus monkeys, common marmosets, and rhesus monkeys.
- a peptide having inhibitory activity against KLK1, KLK4 or KLK4 and KLK8 derived from non-human animals can be used to diagnose, test, treat or prevent a disease associated with KLK1, KLK4 or KLK4 and KLK8 in such non-human animals, etc.
- the KLK1 inhibitory peptide, the KLK4 inhibitory peptide, and the KLK4 / KLK8 inhibitory peptide of the present invention are smaller in molecular weight compared to other biopolymers such as antibodies used in the field as drugs and diagnostic agents, and their production It is relatively easy (described later), is excellent in physical properties such as storage stability and thermal stability, and can be selected as an administration route, administration method, preparation, etc. when used as a pharmaceutical composition (described later) It has advantages such as wide width. In addition, it is also possible to adjust the blood half life longer when used as a pharmaceutical composition by increasing the molecular weight of the peptide of the present invention by applying known methods such as addition of biopolymers and polymers. it can.
- the molecular weight of such KLK1 inhibitory peptide, KLK4 inhibitory peptide, and KLK4 / KLK8 inhibitory peptide of the present invention is less than 10,000, preferably less than 8,000, more preferably about 7,000 to 7,200. . Further, among the variable loop portion consisting of No. 15 Cys to No. 31 Cys of SEQ ID NO: 23 or the part consisting of No. 15 Cys to No.
- the part containing 6 Cys KLK1 inhibition
- Those having the activity, KLK4 inhibitory activity or KLK4 / KLK8 inhibitory activity are also included in the KLK1 inhibitory peptide, KLK4 inhibitory peptide or KLK4 / KLK8 inhibitory peptide of the present invention, respectively, and the molecular weight of the variable loop portion is 2,500
- the molecular weight of the portion containing six Cys is less than, preferably about 1,800 to 2,000, and the molecular weight of the portion containing six Cys is less than 6,000, preferably about 5,300 to 5,500.
- the KLK1 inhibitory peptide, the KLK4 inhibitory peptide, or the KLK4 / KLK8 inhibitory peptide of the present invention is a SPINK2 variant (hereinafter abbreviated as “SPINK2 variant”) in which the backbone of SPINK2 is at least partially maintained,
- SPINK2 variant a SPINK2 variant in which the backbone of SPINK2 is at least partially maintained
- target binding activity a recognition or binding action
- the ELISA method includes a method of detecting KLK1, KLK4 or KLK4 inhibitory peptide or KLK4 inhibitory peptide or KLK4 / KLK8 inhibitory peptide which is recognized and bound to KLK1, KLK4 or KLK4 / KLK8 immobilized on a plate. .
- KLK1, KLK4 or KLK4 / KLK8 in addition to biotin-streptavidin, KLK1, KLK4 or KLK4 / KLK8, or a tag fused to KLK1, KLK4 or KLK4 / KLK8 is recognized
- a solid phase antibody etc. can be used.
- KLK1 inhibitory peptide For detection of KLK1 inhibitory peptide, KLK4 inhibitory peptide, or KLK4 / KLK8 inhibitory peptide, besides labeled streptavidin, KLK1 inhibitory peptide, KLK4 inhibitory peptide, or KLK4 / KLK8 inhibitory peptide, or KLK1 inhibitory peptide, A KLK4 inhibitory peptide, or a labeled detection antibody that recognizes a tag fused to the KLK4 / KLK8 inhibitory peptide can be used.
- labeling other than biotin, HRP, alkaline phosphatase, FITC and other methods that can be used for biochemical analysis can be used.
- the instruments used for SPR analysis include BIAcore (registered trademark) (GE Healthcare), ProteOn (registered trademark) (BioRad), SPR-Navi (registered trademark) (BioNavisOy), Spreeta (registered trademark) (Texas Instruments), SPRi- Plex II (registered trademark) (Horiba), Autolab SPR (registered trademark) (Metrohm) and the like can be exemplified.
- Octet registered trademark
- Pall can be illustrated as an apparatus used for the BLI method.
- Examples of the immunoprecipitation method include a method for detecting KLK1, KLK4 or KLK4 or KLK8 which is recognized and bound by KLK1 inhibitory peptide, KLK4 inhibitory peptide or KLK4 / KLK8 inhibitory peptide immobilized on beads.
- Be Magnetic beads, agarose beads and the like can be used as the beads.
- KLK1, KLK4 or KLK4 and KLK8 precipitated with the beads are detected by SDS-PAGE or Western blot.
- KLK1, KLK4 or KLK4 and KLK8 besides labeled streptavidin, KLK1, KLK4 or KLK8, or a labeled detection antibody that recognizes a tag fused to KLK1, KLK4 or KLK8, etc. It can be used.
- labeling other than biotin, HRP, alkaline phosphatase, FITC and other methods that can be used for biochemical analysis can be used.
- a substrate similar to the ELISA method can be used. ChemiDoc (registered trademark) (BioRad) or LuminoGraph (ATTO) can be used to measure the detection signal.
- binding activity EC 50 in ELISA
- K D dissociation constant
- the KLK1 inhibitory peptide, the KLK4 inhibitory peptide, or the SPINK2 variant as the KLK4 / KLK8 inhibitory peptide of the present invention may have the above-mentioned protease inhibitory activity, target binding activity, other properties, functions, features, etc. 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 is 60% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more of the amino acid sequence of human SPINK2 (SEQ ID NO: 1: FIG. 7). It has 98% or more or 99% or more sequence identity.
- the identity between two amino acid sequences having a completely identical amino acid sequence is 100%, but substitution of one or more amino acids or amino acid residues, one missing, compared to one amino acid sequence with the other, missing 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.
- BLAST 2 Altschul, et al. J. Mol. Biol. 215: 403-410, 1990
- Smith-Waterman Smith, et al. J. Mol. Biol. 147, 195-197. , 1981
- 1981 and the like can be exemplified.
- the amino acid sequence of the SPINK2 variant is of the amino acid sequence of human SPINK2 (SEQ ID NO: 1: FIG. 7) One, two, three, four, five, six or seven amino acids of the 16th to 22nd Glys are substituted with another amino acid or amino acid residue; One, two, three, four or five amino acids of No. 24 Pro to No.
- Wild-type No. 1, 16 to 22 and 24 to 28 Xaa are Asp, Ser, Gln, Tyr, Arg, Leu, Pro, Gly, Pro, Arg, His, respectively. Phe and Asn.
- one to several or more amino acids may be further added to the N-terminal side of the first amino acid, and such an added amino acid is, for example, an amino acid consisting of a Stag + linker Sequence (SEQ ID NO: 26: FIG. 32) and the like can be mentioned.
- Stag + linker Sequence SEQ ID NO: 26: FIG. 32
- amino acids may be added to the 63rd Cys located at the C-terminal, and for example, an amino acid sequence having Gly-Gly and a C-terminal of 65th GIy can be mentioned.
- added amino acids include C-terminal 6-mer (SEQ ID NO: 27: FIG. 33), Gly-Gly-Gly, Gly-Gly and the like.
- portions other than X 1 to X 13 ie, amino acids of wild type human SPINK2 Containing a naturally occurring amino acid or a mutated amino acid or amino acid sequence at the positions of No. 2 Pro to No. 15 Cys, No. 23 Cys and No. 29 Pro to No. 63 Cys in the sequence (SEQ ID NO: 1: FIG. 7) it can.
- a SPINK2 mutant is mutated at one or more positions, as long as it does not at least partially prevent or interfere with KLK1 inhibitory activity, KLK4 inhibitory activity, or KLK4 / KLK8 inhibitory activity or folding.
- Exemplary mutations in the amino acid sequence may include substitution, deletion or addition of one or more amino acids, and examples of the substitution may include conservative substitution.
- conservative substitution an amino acid residue is replaced by an amino acid residue having similar chemical characteristics not only in bulk but also in terms of polarity. Examples of conservative substitutions are described elsewhere in this specification.
- portions other than X 1 to X 13 are one or two unless they at least partially prevent or interfere with KLK1 inhibitory activity, KLK4 inhibitory activity, or KLK4 / KLK8 inhibitory activity or folding.
- Non-conservative substitutions of the above amino acids are also permissible.
- the amino acid sequences possessed by the KLK1 inhibitory peptide of the present invention, the KLK4 inhibitory peptide, or the SPINK2 variant as the KLK4 / KLK8 inhibitory peptide, preferably X 1 to X 13 are SEQ ID NO: 5 to 8 (FIGS. 11 to 14) ), SEQ ID NO: 9 to 12 (15 to 18), or a respective amino acid of X 1 to X 13 in any one of SEQ ID NO: 13 to 22 (19 to 28), and, X 1 to X
- the portion other than 13 can have an amino acid or amino acid sequence that does not at least partially prevent or interfere with KLK1 inhibitory activity, KLK4 inhibitory activity, or KLK4 / KLK8 inhibitory activity or folding.
- A2 A nucleotide sequence encoding an amino acid sequence which is hybridized under stringent conditions with a nucleotide sequence complementary to the nucleotide sequence encoding the amino acid sequence described in (a1) and which is contained in a peptide having KLK1 inhibitory activity
- the amino acid sequence encoded by (A3) 1 to 20, 1 to 15, 1 to 10, 1 to 8, 1 to 6, 1 to 5, 1 to 4, 1 to 3 of the amino acid sequences described in (a3)
- (a1) An amino acid sequence which is obtained by substitution, deletion, addition and / or insertion of one, one or two or one amino acid and which is contained in a peptide having KLK1 inhibitory activity;
- (B1) an amino acid sequence represented by any one of SEQ ID NOs
- (B2) A nucleotide sequence encoding an amino acid sequence which is hybridized under stringent conditions with a nucleotide sequence complementary to the nucleotide sequence encoding the amino acid sequence described in (b1) and which is contained in a peptide having KLK4 inhibitory activity
- the amino acid sequence encoded by (B3) 1 to 20, 1 to 15, 1 to 10, 1 to 8, 1 to 6, 1 to 5, 1 to 4, 1 to 3 of the amino acid sequences described in (b1) and (b1)
- (C1) an amino acid sequence represented by any one of SEQ ID NOs: 13
- (C2) encodes an amino acid sequence contained in a peptide which hybridizes under stringent conditions with a nucleotide sequence complementary to the nucleotide sequence encoding the amino acid sequence described in (c1) and which is contained in a peptide having KLK4 / KLK8 inhibitory activity
- An amino acid sequence encoded by the nucleotide sequence (C3) 1 to 20, 1 to 15, 1 to 10, 1 to 8, 1 to 6, 1 to 5, 1 to 4, 1 to 3 of the amino acid sequences described in (c3) and (c1)
- the KLK1 inhibitory peptide, the KLK4 inhibitory peptide, or the KLK4 / KLK8 inhibitory peptide may be linked or added to another moiety, and such a conjugate is referred to as “a conjugate of the KLK1 inhibitory peptide”, Collectively referred to as “conjugate of KLK4 inhibitory peptide” or “conjugate of KLK4 / KLK8 inhibitory peptide”, respectively.
- conjugate means a molecule formed by binding another part to the peptide of the present invention or a fragment thereof.
- conjugate refers to a peptide of the present invention via an agent or the like suitable for linking a moiety to a side chain of an amino acid via a chemical moiety such as a crosslinking agent.
- agent or the like suitable for linking a moiety to a side chain of an amino acid via a chemical moiety such as a crosslinking agent.
- forms to be linked or bound to the peptide of the present invention by synthetic chemical techniques, genetic engineering techniques and the like are included.
- portions include polyalkylene glycol molecules such as polyethylene glycol (PEG), fatty acid molecules such as hydroxyethyl starch and palmitic acid, and Fc regions of immunoglobulins (for example, Human immunoglobulin G1: its amino acid sequence is shown in SEQ ID NO: 28, shown in FIG. 34), CH3 domain of immunoglobulin, CH4 domain of immunoglobulin, albumin or its fragment, albumin binding peptide, albumin binding protein such as streptococcal protein G, Transferrin can be exemplified.
- the other "moiety” such "moiety” can be linked to the peptide of the present invention via a linker such as a peptide linker.
- ADCs antibody-drug conjugates
- the KLK1 inhibitory peptide, the KLK4 inhibitory peptide, or the KLK4 / KLK8 inhibitory peptide of the present invention exerts binding affinity, inhibitory activity, antagonistic activity, agonistic activity, etc. to KLK1, KLK4 or target molecules other than KLK4 and KLK8, respectively. It may further comprise one or more moieties or be conjugated to such moieties. Such "portions" can be exemplified by antibodies or fragments thereof, proteins having a backbone other than antibodies such as SPINK2 variants or fragments thereof.
- the techniques and embodiments known to those skilled in the art as multispecific antibodies and bispecific antibodies in the antibody field involve the invention of at least one of two or more of the "antibodies" contained therein.
- the substitution of the peptide of ⁇ 1> results in some aspects of the conjugate of the present invention.
- the KLK1 inhibitory peptide, the KLK4 inhibitory peptide, or the KLK4 / KLK8 inhibitory peptide of the present invention or a precursor thereof may contain a signal sequence.
- a signal sequence which is present or added at the N-terminal of a certain polypeptide or its precursor can be added to a specific compartment of the cell, for example periplasmic if E. coli, endoplasmic reticulum if eukaryotic cell Useful for delivery, many signal sequences are known to those skilled in the art and can be selected depending on the host cell.
- a form containing a signal sequence which can exemplify OmpA can also be included as a part of the aspect of the conjugate of the present invention .
- GST calmodulin binding peptide
- CBP calmodulin binding peptide
- hapten such as digoxigenin and dinitrophenol
- epitope tag such as FLAG (registered trademark)
- myc tag myc tag
- HA tag HA tag etc.
- Tag adducts may also be included in the conjugates of the present invention as some aspects thereof.
- the conjugate of the present invention may be a peptide (polypeptide) as a whole.
- the KLK1 inhibitory peptide, the KLK4 inhibitory peptide, or the KLK4 / KLK8 inhibitory peptide of the present invention may exist as a monomer, a dimer, an oligomer or multimer of trimers or more.
- the dimer, trimer or higher oligomer and multimer may be either a homo monomer composed of a single monomer or a hetero monomer composed of two or more different monomers.
- the monomer may, for example, diffuse rapidly and be better for tissue penetration.
- the dimers, oligomers and multimers have, for example, high affinity or binding activity to the target molecule at a local site, slow dissociation rate, or high KLK1 inhibitory activity, KLK4 inhibitory activity or KLK4 / KLK8 It can have superior aspects such as exhibiting inhibitory activity.
- intended dimerization, oligomerization and multimerization are also achieved by introducing jun-fos domain, leucine zipper, etc. into the peptide of the present invention. You can do it.
- the KLK1 inhibitory peptide, the KLK4 inhibitory peptide, or the KLK4 / KLK8 inhibitory peptide of the present invention is a monomer, dimer, trimer or higher oligomer or multimer and binds to one or more target molecules Or the activity of the target molecule can be inhibited.
- nucleic acid molecules as starting materials can be subjected to mutagenesis and introduced into suitable bacterial or eukaryotic hosts using recombinant DNA technology.
- the SPINK2 variant library is known as a technique for identifying binders and inhibitors of target molecules, and the disclosure in, for example, WO 2012/105616 is also included in the disclosure of the present invention by reference in its entirety.
- clones consisting of SPINK2 mutants having desired properties, activities, functions, etc. linked to their genetic traits are enriched from the above library and It can be selected and / or identified.
- the SPINK2 mutant of the present invention can be obtained, for example, by mutating natural SPINK2.
- the “mutation induction” refers to substitution or deletion of one or more amino acids present at each position of a certain amino acid sequence with another amino acid, or an amino acid not present in the amino acid sequence. It means making it possible to add or insert. Such deletions or additions or insertions can alter the length of the sequence.
- mutagenesis can suitably occur at one or more positions X 1 to X 13 in the amino acid sequence shown in SEQ ID NO: 23 (FIG. 29).
- N adenine, guanine, cytosine or thymine
- K guanine or thymine
- S adenine or cytosine
- VVS adenine, guanine or cytosine
- codon NMS adenine or cytosine
- Arg, Cys, Gly, Ile, Leu, Met, Phe, Trp and Val are introduced, and the remaining 11 natural amino acids are Introduction is mutagenized.
- Specialized codons, artificial codons, etc. can be used to mutagenize the introduction of unnatural amino acids.
- Site-directed mutagenesis can also be performed using structural information of the target including higher order structure and / or the peptide for the target or the wild-type peptide from which the peptide is derived.
- structural information of the target including higher order structure and / or the peptide for the target or the wild-type peptide from which the peptide is derived.
- higher-order information of the target KLK1, KLK4 or KLK8 and / or KLK1, KLK4 or SPINK2 mutant or wild type SPINK2 against KLK4 and KLK8, or a complex of both is disclosed.
- Site-specific mutations can be introduced using structural information involved.
- a SPINK2 mutant having KLK1 inhibitory activity, KLK4 inhibitory activity or KLK4 / KLK8 inhibitory activity is identified, and then a crystal of a complex of KLK1, KLK4 or KLK8 and the SPINK2 mutant is obtained to obtain an X-ray crystal structure Analysis is performed, and an epitope on the KLK1, KLK4 or KLK8 molecule to which the SPINK2 variant binds based on the analysis result, and structural information obtained through specifying the paratope on the SPINK2 variant corresponding to the epitope, etc.
- correlations with KLK1 inhibitory activity, KLK4 inhibitory activity, or KLK4 / KLK8 inhibitory activity can be found.
- mutations can be induced, for example, using nucleotide building blocks with altered specificity of base pairs, such as inosine.
- the desired library presented on cells such as yeast, bacteria, animal cells, etc. can be incubated in the presence of the target molecule or contacted with the target molecule.
- a carrier such as magnetic beads
- cells are separated from the carrier, and then the carrier is washed. Removing a specific adsorbate and a conjugate, and recovering a cell group in which a peptide, a collection of peptides or a collection of concentrated peptides is bound to a carrier (bound to KLK1, KLK4 or KLK8) it can.
- the phagemid is a bacterial plasmid which contains, in addition to a plasmid origin of replication, a second origin of replication derived from a single-stranded bacteriophage.
- Cells with phagemid can replicate phagemid through single-stranded replication mode in superinfection with M13 or similar helper bacteriophage. That is, single-stranded phagemid DNA is packaged into infectious particles coated with bacteriophage coat proteins.
- the vector expressing the thus-obtained peptide, a collection of peptides, or a concentrated collection of peptides is recovered, the nucleotide sequence of the polynucleotide inserted into the vector is determined, and the nucleotide sequence is a code. Amino acid sequence can be determined. Also, by introducing the vector into the host cell again and repeating the above-described operation once to several times as a cycle, the peptide assembly that binds to the target molecule can be highly enriched.
- Nucleic acid display is also referred to as mRNA display, for example, using a linker such as puromycin having a similar structure at the 3 'end of tyrosyl-tRNA, to synthesize a desired protein, an mRNA encoding it and a molecule linked to a ribosome Technology. Since this technique is not a living cell but utilizes a cell-free protein synthesis system, it can be synthesized in vitro. A group of randomly mutagenized proteins can be obtained by using a group of mRNAs obtained by causing random mutations in a nucleotide sequence encoding an amino acid sequence possessed by a certain protein, a linker such as puromycin, and a cell-free protein synthesis system. Libraries presented on ribosomes can be obtained (Nemoto N, et al. (1997) FEBS Lett. 414: 2 p. 405-408).
- a linker such as puromycin having a similar structure at the 3 'end of
- Elution is carried out nonselectively in relatively high ionic strength, low pH, moderate denaturing conditions, in the presence of chaotropic salts, etc. or soluble target molecules such as KLK1, KLK4, KLK8, target molecules It can be selectively carried out by adding an antibody that binds to H, a natural ligand, a substrate and the like and competing with the immobilized target molecule. Nonspecific adsorbate sites and / or binding sites can, for example, also be blocked and blocking steps can be incorporated if appropriate.
- an affinity tag is previously conjugated to a peptide, a collection of peptides or a concentrated collection of peptides, the peptides or the collection thereof can be efficiently purified.
- a protease substrate is previously conjugated to a peptide assembly as a tag, the peptide can be eluted by cleavage with the protease activity.
- a KLK1 inhibitory peptide, a KLK4 inhibitory peptide, or a KLK4 / KLK8 inhibitory peptide can be obtained. Each can be identified.
- the KLK1 inhibitory peptide, the KLK4 inhibitory peptide, or the KLK4 / KLK8 inhibitory peptide is preferably a loop structure consisting of # 16 Ser to # 30 Val contained in the amino acid sequence of wild type SPINK2, # 31 Cys and # 32 ⁇ -sheet composed of ⁇ -strand (1) consisting of Gly and ⁇ -strand (2) consisting of 57-Ile to 59-Arg, and ⁇ -helix consisting of 41-Glu amino acid to 51-Gly, or similar Or a three-dimensional structure composed of a ⁇ -sheet, an ⁇ -helix or the like corresponding at least partially to (the position of) KLK1 inhibitory activity, KLK4 inhibitory activity, or KLK4 / KLK8 inhibitory activity, respectively It can be maintained to the extent that it can be demonstrated.
- KLK1 inhibitory peptide, KLK4 inhibitory peptide, or nucleic acid molecule encoding KLK4 / KLK8 inhibitory peptide, vector containing the same, cells containing them, and recombinant KLK1 inhibitory peptide, KLK4 inhibitory peptide, or KLK4 / KLK8 inhibition Method for producing peptide
- the present invention relates to a polynucleotide comprising a nucleotide sequence encoding an amino acid sequence contained in a KLK1 inhibitory peptide, a KLK4 inhibitory peptide, or a KLK4 / KLK8 inhibitory peptide (hereinafter referred to as “a nucleic acid molecule encoding a KLK1 inhibitory peptide”, “KLK4 A nucleic acid molecule encoding the inhibitory peptide or a nucleic acid molecule encoding the KLK4 / KLK8 inhibitory peptide), a recombinant vector into which the gene is inserted, a cell into which the gene or vector is introduced (hereinafter referred to as "KLK1 inhibitory peptide Nucleic Acid Molecule-Containing Cell "," A Nucleic Acid Molecule-Containing Cell Encoding a KLK4 Inhibitory Peptide ", or” A Nucleic Acid Molecule-Con
- nucleic acid molecule encoding the KLK1 inhibitory peptide of the present invention the nucleic acid molecule encoding the KLK4 inhibitory peptide, or the nucleic acid molecule encoding the KLK4 / KLK8 inhibitory peptide, the following (a1) to (a4), The nucleotide sequence according to any one of (b1) to (b4) or (c1) to (c4) (hereinafter, “nucleotide sequence of KLK1 inhibitory peptide”, “nucleotide sequence of KLK4 inhibitory peptide” or “KLK4 / The nucleotide sequence of KLK8 inhibitory peptide (referred to as “the nucleotide sequence of KLK8 inhibitory peptide”), the nucleotide sequence of KLK1 inhibitory peptide, the nucleotide sequence of KLK4 inhibitory peptide or the nucleotide sequence comprising the nucleotide sequence of KLK4 / K
- (A2) a nucleotide sequence that hybridizes with a nucleotide sequence complementary to the nucleotide sequence described in (a1) under stringent conditions and encodes an amino acid sequence contained in a peptide having KLK1 inhibitory activity; (A3) 1 to 20, 1 to 15, 1 to 10, 1 to 8, 1 to 6, 1 to 5, 1 to 4, 1 to 3 of the nucleotide sequences described in (a3) (a1) A nucleotide sequence encoding an amino acid sequence contained in a peptide having substitution, deletion, addition and / or insertion of one, one or two or one nucleotide or nucleotide residue and having KLK1 inhibitory activity; , (A4) 60%, 70%, 80%, 85%, 90%, 92%, 94%, 96%, 97%, 98% or 99% or more identical to the nucleotide sequence described in (a1), and Nucleotide sequence encoding the amino acid sequence contained in a peptide having KLK1 inhibitory activity: (A3)
- (B2) a nucleotide sequence encoding an amino acid sequence which is hybridized under stringent conditions with a nucleotide sequence complementary to the nucleotide sequence described in (b1) and which is contained in a peptide having KLK4 inhibitory activity; (B3) 1 to 20, 1 to 15, 1 to 10, 1 to 8, 1 to 6, 1 to 5, 1 to 4, 1 to 3 in the nucleotide sequence described in (b1)
- (B4) 60%, 70%, 80%, 85%, 90%, 92%, 94%, 96%, 97%, 98% or 99% or more identical to the nucleotide sequence described in (b1), and Nucleotide sequence encoding the amino acid sequence contained in a peptide having KLK4 inhibitory activity: (C
- C2 a nucleotide sequence encoding an amino acid sequence which is hybridized under stringent conditions with a nucleotide sequence complementary to the nucleotide sequence described in (c1) and which is contained in a peptide having KLK4 / KLK8 inhibitory activity; (C3) 1 to 20, 1 to 15, 1 to 10, 1 to 8, 1 to 6, 1 to 5, 1 to 4, 1 to 3 in the nucleotide sequence described in (c1) Nucleotide sequence encoding an amino acid sequence contained in a peptide consisting of substitution, deletion, addition and / or insertion of one, one or two or one nucleotide or nucleotide residue and having KLK4 / KLK8 inhibitory activity ;as well as, (C4) 60%, 70%, 80%, 85%, 90%, 92%, 94%, 96%, 97%, 98% or 99% or more identical to the nucleotide sequence described in (c1), and A nucleotide sequence encoding an amino acid sequence contained
- a SPINK2 consisting of the amino acid sequence encoded by the nucleotide sequence described in any one of (a1) to (a4), (b1) to (b4) or (c1) to (c4) above, or comprising the amino acid sequence
- the variant peptide inhibits the protease activity possessed by KLK1, KLK4, or KLK4 and KLK8, and preferably specifically inhibits the protease activity.
- nucleic acid molecules encoding KLK1 inhibitory peptides, KLK4 inhibitory peptides or KLK4 / KLK8 inhibitory peptides are (a1) to (a4), (b1) to (b4) or (c1) to (c4) (d)
- the amino acid sequence contained in the SPINK2 variant having KLK1 inhibitory activity, KLK4 inhibitory activity, or KLK4 / KLK8 inhibitory activity preferably the amino acid sequence shown by SEQ ID NO: 23 (FIG. 29).
- Nucleic acid molecules comprising a nucleotide sequence encoding are ubiquitously included within the scope of KLK1 inhibitory peptides, KLK4 inhibitory peptides, or nucleic acid molecules encoding KLK4 / KLK8 inhibitory peptides.
- a base sequence encoding a single amino acid sequence possessed by a certain peptide may 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 same is introduced, or a plurality of codons are selected. The frequency or rate of use can be adjusted as appropriate. For example, when E. coli is used as a host cell, nucleotide sequences may be designed using codons frequently used in E. coli.
- a KLK1 inhibitory peptide, a KLK4 inhibitory peptide, or a nucleic acid molecule encoding a KLK4 / KLK8 inhibitory peptide may be operably linked to one or more regulatory sequences.
- "Operably linked” means capable of expressing linked nucleic acid molecules or allowing expression of nucleotide sequences contained in the molecules.
- Regulatory sequences include sequence elements that contain information regarding transcriptional and / or translational regulation.
- the regulatory sequences vary depending on species, but generally include promoters and are exemplified by prokaryotic -35 / -10 box and Shine-Dalgarno sequences, eukaryotic TATA box, CAAT sequences, and 5 'capping sequences etc.
- sequences involved in the initiation of transcription and translation.
- sequences may include enhancer elements and / or repressor elements, as well as signal sequences, leader sequences, etc. that can be translated to deliver the native or mature peptides to specific compartments inside or outside the host cell.
- regulatory sequences may include 3 'non-coding sequences, which may include elements involved in transcription termination or polyadenylation and the like. However, if the sequence for transcription termination does not function well in a particular host cell, it may 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, CMV promoter and the like in eukaryotic cells.
- a nucleic acid molecule encoding a KLK1 inhibitory peptide, a nucleic acid molecule encoding a KLK4 inhibitory peptide, or a nucleic acid molecule encoding a KLK4 / KLK8 inhibitory peptide may be in isolated form, a vector or other cloning vehicle (hereinafter simply referred to as “vector” May be in the form contained in a plasmid, phagemid, phage, baculovirus, cosmid etc.) or in a chromosome, but is not limited to these forms.
- the vector contains, in addition to the above-mentioned regulatory sequences, replication sequences and regulatory sequences suitable for the host cell to be used for expression, and a selective marker which confers a selectable phenotype on cells into which the nucleic acid molecule has been introduced by transformation or the like. It may be
- the vector comprising the vector or the nucleotide sequence of the KLK4 / KLK8 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 may be cultured under conditions suitable for expression of the peptide or nucleotide sequence.
- Host cells may be either prokaryotic or eukaryotic, and for prokaryotic, E. coli, Bacillus subtilis, etc., for eukaryotic cells, yeasts such as Saccharomyces cerevisiae, Pichia pastoris, etc., insect cells such as SF9, High5, HeLa cells, CHO cells, COS Cells, animal cells such as NS0 can be exemplified.
- a eukaryotic cell or the like as a host cell, the desired post-translational modification can be performed on the expressed peptide of the present invention.
- Such antibodies and their functional fragments can be prepared by methods known to those skilled in the art, and purification of the SPINK2 variant peptide by affinity chromatography, pharmaceutical composition containing the peptide or clinical examination related to its use, It is useful for the detection of the said peptide in a diagnosis etc., an immunoassay, etc.
- the antibodies of the invention can be purified by affinity chromatography using the peptides of the invention to which the antibodies bind.
- the diseases associated with KLK1, the diseases associated with KLK4, and the diseases associated with KLK4 / KLK8 are not limited to the diseases exemplified herein.
- the pharmaceutical composition of the present invention may have pH, osmotic pressure, viscosity, transparency, color, isotonicity, sterility, stability of the composition or the peptide contained therein, solubility, sustained release, etc.
- a substance for changing, maintaining, or holding the absorbability, permeability, dosage form, strength, properties, shape, etc. (hereinafter, referred to as “prescription substance”) can be contained.
- the substance for preparation is not particularly limited as long as it is a pharmacologically acceptable substance.
- non-toxic or low toxicity is a property which the substance for formulation suitably comprises.
- Examples of the substance for preparation include, but are not limited to, the following: amino acids such as glycine, alanine, glutamine, asparagine, histidine, arginine or lysine, antibacterial agents, ascorbic acid Antioxidants such as sodium sulfate or sodium bisulfite, buffers such as phosphoric acid, citric acid, borate buffer, sodium hydrogencarbonate, tris-hydrochloric acid (Tris-HCl) solution, fillers such as mannitol or glycine, ethylene diamine Chelating agents such as tetraacetic acid (EDTA), caffeine, polyvinyl pyrrolidine, complexing agents such as ⁇ -cyclodextrin and hydroxypropyl- ⁇ -cyclodextrin, extenders such as glucose, mannose or dextrin, monosaccharides, disaccharides Glucose, mannose and dextrin Other carbohydrates such as coloring agents, flavoring agents, diluents, emuls
- the administration route of the pharmaceutical composition of the present invention may be instillation, enteral administration, topical administration or parenteral administration, for example, instillation on the conjunctiva, intravitreal administration, intravenous administration, intraarterial administration, muscle Intravenous administration, intradermal administration, subcutaneous administration, intraperitoneal administration, transdermal administration, intraosseous administration, intraarticular administration and the like can be mentioned.
- the KLK1 inhibitory peptide, the KLK4 inhibitory peptide, or the pharmaceutical composition containing the KLK4 / KLK8 inhibitory peptide as an active ingredient can be administered simultaneously with or separately from other drugs.
- administering a KLK1 inhibitory peptide, a KLK4 inhibitory peptide, or a pharmaceutical composition containing the KLK4 / KLK8 inhibitory peptide as an active ingredient, or after administering such a pharmaceutical composition the other pharmaceutical is administered.
- the pharmaceutical composition may be administered simultaneously with the other pharmaceutical agent.
- pET 32a (modified) _KLK1 inhibitory peptide “pET 32a (modified) _KLK4 inhibitory peptide”, and “pET 32a (modified) _KLK4 / KLK8 inhibitory peptide” were constructed by performing sequence analysis.
- KLK4 inhibitory peptides and KLK4 / KLK8 inhibitory peptides were shown to be potent inhibitors with K i values below 1 nM.
- the average value of three independent experiments was used to calculate IC 50 and K i values.
- Binding Affinity of KLK4 Inhibitory Peptide In order to determine the binding affinity of KLK4 inhibitory peptide by Single cycle kinetics, surface plasmon resonance analysis was performed using BIAcore T 200 (GE healthcare). A complementary strand DNA of a streptavidin conjugate was captured by hybridization on Sensor Chip CAP (GE healthcare) on which single-stranded DNA was immobilized. Next, about 5 RU was immobilized by capturing biotinylated KLK4 at a flow rate of 10 ⁇ L / min using EZ-Link NHS-PEG4-Biotin (Thermo Fisher Scientific).
- the peptide provided by the present invention, and the pharmaceutical composition and the diagnostic composition containing the same are used for the treatment, prevention, examination or diagnosis of diseases associated with KLK1, diseases associated with KLK4, or diseases associated with KLK4 / KLK8. It is useful.
- SEQ ID NO: 8 amino acid sequence of KLK1 inhibitory peptide K10071 (FIG. 14)
- SEQ ID NO: 9 amino acid sequence of KLK4 inhibitory peptide K40001 (FIG. 15)
- SEQ ID NO: 10 amino acid sequence of KLK4 inhibitory peptide K40003 (FIG. 16)
- SEQ ID NO: 11 amino acid sequence of KLK4 inhibitory peptide K40004 (FIG. 17)
- SEQ ID NO: 12 amino acid sequence of KLK4 inhibitory peptide K40005 (FIG. 18)
- SEQ ID NO: 13 Amino acid sequence of KLK4 / KLK8 inhibitory peptide K41021 (FIG.
Abstract
Description
(1)
配列番号23(図29)で示されるアミノ酸配列を含み、且つ、KLK1の有するプロテアーゼ活性を特異的に阻害するSPINK2変異体ペプチド、
(2)
1乃至13番目のXaa(X1乃至X13)がCys及びPro以外のアミノ酸である、(1)記載のペプチド、
(3)
1番目のXaa(X1)がAsp又はGlyである、(1)又は(2)記載のペプチド、
(4)
2番目のXaa(X2)はAla、Asp又はSer、3番目のXaa(X3)はIle、Gln、Arg又はVal、4番目のXaa(X4)はAla、Asn又はTyr、5番目のXaa(X5)はLeu、Lys、Asn又はGln、6番目のXaa(X6)はIle、Arg、Tyr又はVal、7番目のXaa(X7)はAsp、Arg又はVal、8番目のXaa(X8)はAsp、Ile又はArg、9番目のXaa(X9)はPhe、His又はTrp、10番目のXaa(X10)はTyr又はTrp、11番目のXaa(X11)はAla、Thr又はTyr、12番目のXaa(X12)はSer又はTyr、並びに、13番目のXaa(X13)はGlu、Lys又はGlnである、(1)乃至(3)のいずれか一つに記載のペプチド、
(5)
配列番号5乃至8(図11乃至14)のいずれか一つで示され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)
配列番号23(図29)で示されるアミノ酸配列を含み、且つ、KLK4の有するプロテアーゼ活性を特異的に阻害するSPINK2変異体ペプチド、
(7)
1乃至13番目のXaa(X1乃至X13)がCys及びPro以外のアミノ酸である、(6)記載のペプチド、
(8)
1番目のXaa(X1)がAsp又はGlyである、(6)又(7)記載のペプチド、
(9)
2番目のXaa(X2)はGlu、Arg又はSer、3番目のXaa(X3)はHis、Lys、Leu又はGln、4番目のXaa(X4)はAla、Gln又はTyr、5番目のXaa(X5)はAla、Glu、Gln又はVal、6番目のXaa(X6)はGlu、Leu、Met又はTyr、7番目のXaa(X7)はAsp又はGly、8番目のXaa(X8)はAla又はVal、9番目のXaa(X9)はGln、10番目のXaa(X10)はLys又はArg、11番目のXaa(X11)はIle、Leu又はThr、12番目のXaa(X12)はPhe又はTyr、並びに、X13)はLys、Leu又はGlnである、(6)乃至(8)のいずれか一つに記載のペプチド、
(10)
配列番号9乃至12(図15乃至18)のいずれか一つで示されるアミノ酸配列を含む、(6)乃至(9)のいずれか一つに記載のペプチド、
(11)
配列番号23(図29)で示されるアミノ酸配列を含み、且つ、KLK4の有するプロテアーゼ活性及びKLK8の有するプロテアーゼ活性を特異的に阻害するSPINK2変異体ペプチド、
(12)
1乃至13番目のXaa(X1乃至X13)がCys及びPro以外のアミノ酸である、(11)記載のペプチド、
(13)
1番目のXaa(X1)がAsp又はGlyである、(11)又(12)記載のペプチド、
(14)
2番目のXaa(X2)はGly、Met、Gln、Arg、Ser又はThr、3番目のXaa(X3)はLys又はArg、4番目のXaa(X4)はPhe、His、Gln又はTyr、5番目のXaa(X5)はHis、Lys、Arg、Ser、Thr、Val又はTyr、6番目のXaa(X6)はIle、Lys、Leu、Met、Gln、Arg、Ser、Val又はTrp、7番目のXaa(X7)はAsp、Glu、Gly、His、Asn、Arg、Val又はTrp、8番目のXaa(X8)はGly又はTrp、9番目のXaa(X9)はAla、Phe、Asn、Ser又はThr、10番目のXaa(X10)はLys又はArg、11番目のXaa(X11)はIle、Met、Gln、Ser又はVal、12番目のXaa(X12)はPhe、Leu又はTyr、並びに、13番目のXaa(X13)はAla、Asp、Glu又はAsnである、(11)乃至(13)のいずれか一つに記載のペプチド、
(15)
配列番号13乃至22(図19乃至28)のいずれか一つで示されるアミノ酸配列を含む、(11)乃至(14)のいずれか一つに記載のペプチド、
(16)
配列番号23(図29)で示されるアミノ酸配列のアミノ末端側に、1乃至3個のアミノ酸残基が付加してなるアミノ酸配列、又は、配列番号26(図32)で示されるアミノ酸配列が付加してなるアミノ酸配列を含む、(1)乃至(15)のいずれか一つに記載のペプチド、
(17)
配列番号23(図29)で示されるアミノ酸配列のカルボキシル末端側に、1乃至6個のアミノ酸からなるアミノ酸配列が付加してなるアミノ酸配列を含む、(1)乃至(16)のいずれか一つに記載のペプチド、
(18)
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(17)のいずれか一つに記載のペプチド、
(19)
(1)乃至(18)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、
(20)
(19)記載のポリヌクレオチドを含むベクター、
(21)
(19)記載のポリヌクレオチド若しくは(20)記載のベクターを含むか又は(1)乃至(18)のいずれか一つに記載のペプチドを産生する細胞、
(22)
下記工程(i)及び(ii)を含む、SPINK2変異体ペプチドの製造方法:
(i)(21)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、
(23)
(1)乃至(18)のいずれか一つに記載のペプチドを化学合成又はイン・ビトロ翻訳により調製する工程を含む、該ペプチドの製造方法、
(24)
(22)又は(23)記載の方法により得られるSPINK2変異体ペプチド、
(25)
(1)乃至(18)及び(24)のいずれか一つに記載のペプチドに他の部分が結合してなるコンジュゲート、
(26)
ペプチドである、(25)記載のコンジュゲート、
(27)
免疫グロブリンのFc領域又はその機能断片を含む、(25)又は(26)記載のコンジュゲート、
(28)
下記工程(i)及び(ii)を含む、(25)乃至(27)のいずれか一つに記載のSPINK2変異体ペプチドコンジュゲートの製造方法:
(i)該コンジュゲート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んでなるポリヌクレオチド又は該ポリヌクレオチドが挿入されたベクターを含む細胞を培養する工程:
(ii)該培養物からSPINK2変異体ペプチドコンジュゲート又は該コンジュゲートに含まれるペプチド部分を回収する工程、
(29)
(25)乃至(27)のいずれか一つに記載のSPINK2変異体ペプチドコンジュゲート又は該コンジュゲートに含まれるペプチド部分を化学合成又はイン・ビトロ翻訳により調製する工程を含む、該コンジュゲートの製造方法、
(30)
(28)又は(29)記載の方法により得られるSPINK2変異体ペプチドコンジュゲート、
(31)
(1)乃至(18)及び(24)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結合断片、
(32)
(1)乃至(18)及び(24)のいずれか一つに記載のペプチド、(19)記載のポリヌクレオチド、(20)記載のベクター、(21)記載の細胞、(25)乃至(27)及び(30)のいずれか一つに記載のコンジュゲート、及び/又は、(31)記載の抗体もしくはその結合断片を含む組成物、
(33)
(1)乃至(18)及び(24)のいずれか一つに記載のペプチド、(19)記載のポリヌクレオチド、(20)記載のベクター、(21)記載の細胞、及び/又は、(25)乃至(27)及び(30)のいずれか一つに記載のコンジュゲートを含む医薬組成物、
(34)
KLK1関連疾患、KLK4関連疾患及び/又はKLK8関連疾患の治療用又は予防用である、(33)記載の医薬組成物、
(35)
(1)乃至(18)及び(24)のいずれか一つに記載のペプチド、(19)記載のポリヌクレオチド、(20)記載のベクター、(21)記載の細胞、(25)乃至(27)及び(30)のいずれか一つに記載のコンジュゲート、及び/又は、(31)記載の抗体もしくはその結合断片を含む、検査用又は診断用組成物、及び、
(36)
(31)記載の抗体又はその結合断片を用いたアフィニティー精製工程を含む、(22)、(23)、(28)又は(29)に記載の製造方法、
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。
「KLK1阻害ペプチド」、「KLK4阻害ペプチド」及び「KLK4/KLK8阻害ペプチド」の範囲には、当該ペプチドの断片、他の部分(moiety)が当該ペプチド又はその断片に付加又は結合してなるコンジュゲートのうち、KLK1阻害(結合)活性、KLK4阻害(結合)活性、又は、KLK4/KLK8阻害(結合)活性を維持しているものがそれぞれ含まれる。すなわち、KLK1阻害(結合)活性、KLK4阻害(結合)活性、又は、KLK4阻害(結合)活性及びKLK8阻害(結合)活性を維持する当該ペプチドの断片、付加体及び修飾体(コンジュゲート)も「KLK1阻害ペプチド」、「KLK4阻害ペプチド」又は「KLK4/KLK8阻害ペプチド」にそれぞれ含まれる。
2-1.アミノ酸
「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、好適には蛋白質に、より好適には天然の蛋白質に、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KLK1阻害ペプチド、KLK4阻害ペプチド、又は、KLK4/KLK8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」又は単に「A」と記す)、バリン(以下、「Val」又は単に「V」と記す)、ロイシン(以下、「Leu」又は単に「L」と記す)、イソロイシン(以下、「Ile」又は単に「I」と記す)、プロリン(以下、「Pro」又は単に「P」と記す)、フェニルアラニン(「Phe」又は単に「F」と記す)、トリプトファン(以下、「Trp」又は単に「W」と記す)、メチオニン(以下、「Met」又は単に「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」又は単に「G」と記す)、セリン(以下、「Ser」又は単に「S」と記す)、スレオニン(以下、「Thr」又は単に「T」と記す)、システイン(以下、「Cys」又は単に「C」と記す)、チロシン(以下、「Tyr」又は単に「Y」と記す)、アスパラギン(以下、「Asn」又は単に「N」と記す)、グルタミン(以下、「Gln」又は単に「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」又は単に「D」と記す)、グルタミン酸(以下、「Glu」又は単に「E」と記す)
(4)塩基性アミノ酸グループ:リジン(以下、「Lys」又は単に「K」と記す)、アルギニン(以下、「Arg」又は単に「R」と記す)、ヒスチジン(以下、「His」又は単に「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あげることができ、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あ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。
本発明のペプチドは、KLK1阻害活性、KLK4阻害活性、又は、KLK4/KLK8阻害活性を有する。
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;
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KLK1阻害ペプチド、KLK4阻害ペプチド、又は、KLK4/KLK8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。
1番Xaa(X1)はAsp又はGly;
16番Xaa(X2)はAla、Asp又はSer;
17番Xaa(X3)はIle、Gln、Arg又はVal;
18番Xaa(X4)はAla、Asn又はTyr;
19番Xaa(X5)はLeu、Lys、Asn又はGln;
20番Xaa(X6)はIle、Arg、Tyr又はVal;
21番Xaa(X7)はAsp、Arg又はVal;
22番Xaa(X8)はAsp、Ile又はArg;
24番Xaa(X9)はPhe、His又はTrp;
25番Xaa(X10)はTyr又はTrp;
26番Xaa(X11)はAla、Thr又はTyr;
27番Xaa(X12)はSer又はTyr;及び
28番Xaa(X13)はGlu、Lys又はGln
である。
1番Xaa(X1)はAsp又はGly;
16番Xaa(X2)はGlu、Arg又はSer;
17番Xaa(X3)はHis、Lys、Leu又はGln;
18番Xaa(X4)はAla、Gln又はTyr;
19番Xaa(X5)はAla、Glu、Gln又はVal;
20番Xaa(X6)はGlu、Leu、Met又はTyr;
21番Xaa(X7)はAsp又はGly;
22番Xaa(X8)はAla又はVal;
24番Xaa(X9)はGln;
25番Xaa(X10)はLys又はArg;
26番Xaa(X11)はIle;
27番Xaa(X12)はPhe又はTyr;及び
28番Xaa(X13はLys、Leu又はGln
である。
1番Xaa(X1)はAsp又はGly;
16番Xaa(X2)はGly、Met、Gln、Arg、Ser又はThr;
17番Xaa(X3)はLys又はArg、X4はPhe、His、Gln又はTyr;
18番Xaa(X4)はPhe、His、Gln又はTyr;
19番Xaa(X5)はHis、Lys、Arg、Ser、Thr、Val又はTyr;
20番Xaa(X6)はIle、Lys、Leu、Met、Gln、Arg、Ser、Val又はTrp;
21番Xaa(X7)はAsp、Glu、Gly、His、Asn、Arg、Val又はTrp;
22番Xaa(X8)はGly又はTrp;
24番Xaa(X9)はAla、Phe、Asn、Ser又はThr;
25番Xaa(X10)はLys又はArg;
26番Xaa(X11)はIle、Met、Gln、Ser又はVal;
27番Xaa(X12)はPhe、Ler又はTyr;及び
28番Xaa(X13)はAla、Asp、Glu又はAsn
である。
(a1)配列番号5乃至8(図11乃至14)のいずれか一つで示されるアミノ酸配列;
(a2)(a1)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KLK1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;
(a3)(a1)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KLK1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、
(a4)(a1)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KLK1阻害活性を有するペプチドに含まれるアミノ酸配列、
(b1)配列番号9乃至12(図15乃至18)のいずれか一つで示されるアミノ酸配列;
(b2)(b1)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KLK4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;
(b3)(b1)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KLK4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、
(b4)(b1)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KLK4阻害活性を有するペプチドに含まれるアミノ酸配列、又は、
(c1)配列番号13乃至22(図19乃至28)のいずれか一つで示されるアミノ酸配列;
(c2)(c1)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KLK4/KLK8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;
(c3)(c1)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KLK4/KLK8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、
(c4)(c1)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KLK4/KLK8阻害活性を有するペプチドに含まれるアミノ酸配列、
本発明のKLK1阻害ペプチド、KLK4阻害ペプチド、又は、KLK4/KLK8阻害ペプチドに、そのフォールディング安定性、熱安定性、保存安定性、血中半減期、水溶性、生物活性、薬理活性、副次的作用等を改善する目的で、変異を導入することができる。例えば、ポリエチレングルコール(PEG)、ヒドロキシエチルデンプン(HES)、ビオチン、ペプチド又は蛋白質等の他の物質にコンジュゲートさせるため、Cysのような新たな反応性基を変異により導入することができる。
本発明のペプチドは、例えば、そのC末に、ビオチン、Strepタグ(登録商標)、StrepタグII(登録商標)、His6等のオリゴヒスチジン、ポリヒスチジン、免疫グロブリンドメイン、マルトース結合蛋白質、グルタチオン-S-トランスフェラーゼ(GST)、カルモジュリン結合ペプチド(CBP)、ジゴキシゲニンやジニトロフェノール等のハプテン、FLAG(登録商標)等のエピトープタグ、mycタグ、HAタグ等(以下まとめて「アフィ二ティー・タグ」と呼ぶ)を含むことができる。タグ付加体も、本発明のコンジュゲートに、その一部の態様として含まれ得る。本発明のコンジュゲートは、全体としてペプチド(ポリペプチド)であってもよい。
KLK1阻害ペプチド、KLK4阻害ペプチド、及び、KLK4/KLK8阻害ペプチドは、SPINK2のアミノ酸配列又は本発明のKLK1阻害ペプチド、KLK4阻害ペプチド、及び、KLK4/KLK8阻害ペプチドの有するアミノ酸配列(例えば、配列番号5乃至8:図11乃至14からなる群、配列番号9乃至12:図15乃至18からなる群、又は、配列番号13乃至22:図19乃至28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、KLK1阻害活性、KLK4阻害活性、又は、KLK4/KLK8阻害活性を指標としてそれぞれ同定することができ、KLK1、KLK4、又は、KLK4/KLK8に対する結合活性もそれぞれ指標として組み合わせてもよい。
(a1)配列番号5乃至8(図11乃至14)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;
(a2)(a1)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KLK1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;
(a3)(a1)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、又は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KLK1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;及び、
(a4)(a1)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KLK1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:
(b1)配列番号5乃至8(図11乃至14)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;
(b2)(b1)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KLK4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;
(b3)(b1)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、又は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KLK4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;及び、
(b4)(b1)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KLK4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:
(c1)配列番号13乃至22(図19乃至28)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;
(c2)(c1)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KLK4/KLK8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;
(c3)(c1)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、又は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KLK4/KLK8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;及び、
(c4)(c1)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KLK4/KLK8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KLK1阻害ペプチド、KLK4阻害ペプチド、若しくは、KLK4/KLK8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。
本発明のKLK1阻害ペプチド、KLK4阻害ペプチド、若しくは、KLK4/KLK8阻害ペプチド、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。
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。
本発明のKLK1阻害ペプチド、KLK4阻害ペプチド、若しくは、KLK4/KLK8阻害ペプチドペプチドは、KLK1、KLK4、又は、KLK4及びKLK8に特異的な結合活性を有する。したがって、本発明の好適なKLK1阻害ペプチド、KLK4阻害ペプチド、若しくは、KLK4/KLK8阻害ペプチド又はそのコンジュゲートを用いて、KLK1、KLK4、又は、KLK4及びKLK8と他のKLKが混在した試料中から、KLK1、KLK4、又は、KLK4及びKLK8を特異的に分離することができる。ペプチドからのKLK1、KLK4、又は、KLK4及びKLK8の遊離は、相対的に高いイオン強度、低いpH、中程度の変性条件、カオトロピック塩の存在下等で、非選択的に行うことができるが、KLK1、KLK4、又は、KLK4及びKLK8のプロテアーゼ活性を減弱させない範囲で行うことが好ましい。
pro-KLK1(配列番号2:図8,UniProt;P06870)をコードするヌクレオチド配列、pro-KLK4(配列番号3:図9,UniProt;Q9Y5K2)をコードするヌクレオチド配列、pro-KLK8(配列番号4:図10、UniProt;O60259)をコードするヌクレオチド配列を鋳型として、KOD-plus-(TOYOBO)を用いたオーバーラップPCR法((94℃ 15秒、60℃ 30秒、68℃ 30秒)×30サイクル)により各断片をそれぞれ増幅し、各遺伝子のC末端にHis tagを付加した哺乳細胞発現ベクターpCMA_pro-KLK1、pCMA_pro-KLK4、pCMA_pro-KLK8を構築した。
(1-1)で構築した発現ベクターは、PEI MAX 40000(Polysciences)を用いてExpi293F細胞(Thermo Fisher Scientific)にトランスフェクションし、培養3日後に培養上清を回収した。HisTrap excel(GE healthcare)を用いて培養上精から所望のHis tag融合タンパク質を回収し、Amicon Ultra NMWL 10,000(Merck Millipore)を用いてPBSにバッファー交換することで、pro-KLK1、pro-KLK4、pro-KLK8をそれぞれ精製した。
KLK活性化バッファー(50mM Tris-HCl,150mM NaCl,10mM CaCl2,pH7.5)で調製した200μg/mLのpro-KLK1に等量の2μg/mLのthermolysinを添加し、37°Cで一定時間反応後、20mM 1,10-phenanthrolineを等量混合することで活性化KLK1を調製した。同様に、KLK活性化バッファーで調製した200μg/mLのpro-KLK4またはpro-KLK8に等量の8μg/mL Thermolysinを添加し、37°Cで一定時間反応させた後、20mM 1,10-phenanthrolineを等量混合することで活性化KLK4またはKLK8を調製した。
SPINK2 scaffoldを骨格としたKLK1阻害ペプチド、KLK4阻害ペプチド及びKLK4/KLK8阻害ペプチドの発現ベクターを構築した。各阻害ペプチドのアミノ酸配列(配列番号5乃至22:図11乃至28))をコードするヌクレオチド配列とヒトSPINK2(配列番号1:図7)をコードするヌクレオチド配列を鋳型として、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)により阻害ペプチド断片を増幅した。
プライマー1:5’-AAAAGAATTCTGATCCGCAGTTTGGTCTGTTTAG-3’(配列番号24:図30)
プライマー2:5’-AAAACTCGAGTTATGCGGCCGCAGACGCGCCGCACGGACC-3’(配列番号25:図31)
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、大腸菌を形質転換した。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、配列解析を実施することで「pET 32a(改変)_KLK1阻害ペプチド」「pET 32a(改変)_KLK4阻害ペプチド」「pET 32a(改変)_KLK4/KLK8阻害ペプチド」を構築した。
(2-1)で構築したベクターでそれぞれ大腸菌Origami B (DE3)(Novagen)を形質転換し、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)に供することで、N末にS tagおよびリンカー(配列番号26:図32)、C末に6残基(配列番号27:図33)を含むKLK1阻害ペプチド、KLK4阻害ペプチド、KLK4/KLK8阻害ペプチドを精製した。
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、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。
Single cycle kineticsによるKLK4阻害ペプチドの結合親和性を測定するため、BIAcore T 200(GE healthcare)を用いて表面プラズモン共鳴分析を実施した。一本鎖DNAが固定化されたSensor Chip CAP(GE healthcare)に、ストレプトアビジンコンジュゲートの相補鎖DNAをハイブリダイゼーションでキャプチャーさせた。次に、EZ-Link NHS-PEG4-Biotin(Thermo Fisher Scientific)を用いてビオチン化したKLK4を流速10μL/minでキャプチャーさせることで、約5RUを固定化した。その後、HBS-EPで3倍段階希釈したKLK4阻害ペプチド(0.08~20nM)をアナライトとして流速10μL/minで添加した。BIAcore T 200 Evaluation software(version 2.0)を用いて解析し、simple one-to-one Langmuir binding modelを用いてkonおよびkoffを算出した。尚、平衡定数KDはkoff/konの比率として算出した。さらに、Biotin CAPture Kit(GE healthcare)付属のRegeneration bufferでSensor Chip CAPを再生し、ビオチン化KLK4を繰り返しキャプチャーさせることで、複数のKLK4阻害ペプチドを測定した。測定した3つのKLK4阻害ペプチドはいずれも1nMを下回るKD値を示しており、非常に強い結合力であることが明らかとなった(図5)。
(1-3)および(2-2)に記載した方法に従って、KLK4およびK41043(配列番号19:図25)で示されるアミノ酸配列を有するKLK4阻害ペプチドをそれぞれ調製した。50mM Tris-HCl,150mM NaCl,pH8.0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。
(5-1)で調製した複合体溶液を30mg/mLまで濃縮し、終濃度16.7U/uLとなるようにEKMax(Invitrogen社)を添加後、リザーバー溶液(LiCl 0.2M,20%PEG3350)と1対1で混合し、蒸気拡散法により結晶化した。得られた単結晶をクライオ溶液(20%グリセロール、PBS、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 NE3A:高エネルギー加速器研究機構)。imosflmを使用した解析により、最大分解能1.9Å(オングストローム)のスケーリングデータを取得した。KLK4単体(PDB ID:4K1E)、およびSPINK2単体(PDB ID:2JXD)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2.0AでKLK4/該ペプチドの複合体結晶を決定した。当該KLK4阻害ペプチドはKLK4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)。
配列番号2:ヒトKLK1のアミノ酸配列(図8)
配列番号3:ヒトKLK4のアミノ酸配列(図9)
配列番号4:ヒトKLK8のアミノ酸配列(図10)
配列番号5:KLK1阻害ペプチドK10061のアミノ酸配列(図11)
配列番号6:KLK1阻害ペプチドK10062のアミノ酸配列(図12)
配列番号7:KLK1阻害ペプチドK10066のアミノ酸配列(図13)
配列番号8:KLK1阻害ペプチドK10071のアミノ酸配列(図14)
配列番号9:KLK4阻害ペプチドK40001のアミノ酸配列(図15)
配列番号10:KLK4阻害ペプチドK40003のアミノ酸配列(図16)
配列番号11:KLK4阻害ペプチドK40004のアミノ酸配列(図17)
配列番号12:KLK4阻害ペプチドK40005のアミノ酸配列(図18)
配列番号13:KLK4/KLK8阻害ペプチドK41021のアミノ酸配列(図19)
配列番号14:KLK4/KLK8阻害ペプチドK41024のアミノ酸配列(図20)
配列番号15:KLK4/KLK8阻害ペプチドK41025のアミノ酸配列(図21)
配列番号16:KLK4/KLK8阻害ペプチドK41026のアミノ酸配列(図22)
配列番号17:KLK4/KLK8阻害ペプチドK41041のアミノ酸配列(図23)
配列番号18:KLK4/KLK8阻害ペプチドK41042のアミノ酸配列(図24)
配列番号19:KLK4/KLK8阻害ペプチドK41043のアミノ酸配列(図25)
配列番号20:KLK4/KLK8阻害ペプチドK41045のアミノ酸配列(図26)
配列番号21:KLK4/KLK8阻害ペプチドK41046のアミノ酸配列(図27)
配列番号22:KLK4/KLK8阻害ペプチドK41047のアミノ酸配列(図28)
配列番号23:KLK1阻害結合ペプチド、KLK4阻害ペプチド又はKLK4/KLK8阻害ペプチドの一般式(図29)
配列番号24:プライマー1のヌクレオチド配列(図30)
配列番号25:プライマー2のヌクレオチド配列(図31)
配列番号26:Stag+リンカー1のアミノ酸配列(図32)
配列番号27:C末6マーのアミノ酸配列(図33)
配列番号28:ヒトIgG1 Fcのアミノ酸配列(図34)
Claims (36)
- 配列番号23(図29)で示されるアミノ酸配列を含み、且つ、KLK1の有するプロテアーゼ活性を特異的に阻害するSPINK2変異体ペプチド。
- 1乃至13番目のXaa(X1乃至X13)がCys及びPro以外のアミノ酸である、請求項1記載のペプチド。
- 1番目のXaa(X1)がAsp又はGlyである、請求項1又は2記載のペプチド。
- 2番目のXaa(X2)はAla、Asp又はSer、3番目のXaa(X3)はIle、Gln、Arg又はVal、4番目のXaa(X4)はAla、Asn又はTyr、5番目のXaa(X5)はLeu、Lys、Asn又はGln、6番目のXaa(X6)はIle、Arg、Tyr又はVal、7番目のXaa(X7)はAsp、Arg又はVal、8番目のXaa(X8)はAsp、Ile又はArg、9番目のXaa(X9)はPhe、His又はTrp、10番目のXaa(X10)はTyr又はTrp、11番目のXaa(X11)はAla、Thr又はTyr、12番目のXaa(X12)はSer又はTyr、並びに、13番目のXaa(X13)はGlu、Lys又はGlnである、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5乃至8(図11乃至14)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乃至4のいずれか一つに記載のペプチド。
- 配列番号23(図29)で示されるアミノ酸配列を含み、且つ、KLK4の有するプロテアーゼ活性を特異的に阻害するSPINK2変異体ペプチド。
- 1乃至13番目のXaa(X1乃至X13)がCys及びPro以外のアミノ酸である、請求項6記載のペプチド。
- 1番目のXaa(X1)がAsp又はGlyである、請求項6又7記載のペプチド。
- 2番目のXaa(X2)はGlu、Arg又はSer、3番目のXaa(X3)はHis、Lys、Leu又はGln、4番目のXaa(X4)はAla、Gln又はTyr、5番目のXaa(X5)はAla、Glu、Gln又はVal、6番目のXaa(X6)はGlu、Leu、Met又はTyr、7番目のXaa(X7)はAsp又はGly、8番目のXaa(X8)はAla又はVal、9番目のXaa(X9)はGln、10番目のXaa(X10)はLys又はArg、11番目のXaa(X11)はIle、Leu又はThr、12番目のXaa(X12)はPhe又はTyr、並びに、X13)はLys、Leu又はGlnである、請求項6乃至8のいずれか一つに記載のペプチド。
- 配列番号9乃至12(図15乃至18)のいずれか一つで示されるアミノ酸配列を含む、請求項6乃至9のいずれか一つに記載のペプチド。
- 配列番号23(図29)で示されるアミノ酸配列を含み、且つ、KLK4の有するプロテアーゼ活性及びKLK8の有するプロテアーゼ活性を特異的に阻害するSPINK2変異体ペプチド。
- 1乃至13番目のXaa(X1乃至X13)がCys及びPro以外のアミノ酸である、請求項11記載のペプチド。
- 1番目のXaa(X1)がAsp又はGlyである、請求項11又12記載のペプチド。
- 2番目のXaa(X2)はGly、Met、Gln、Arg、Ser又はThr、3番目のXaa(X3)はLys又はArg、4番目のXaa(X4)はPhe、His、Gln又はTyr、5番目のXaa(X5)はHis、Lys、Arg、Ser、Thr、Val又はTyr、6番目のXaa(X6)はIle、Lys、Leu、Met、Gln、Arg、Ser、Val又はTrp、7番目のXaa(X7)はAsp、Glu、Gly、His、Asn、Arg、Val又はTrp、8番目のXaa(X8)はGly又はTrp、9番目のXaa(X9)はAla、Phe、Asn、Ser又はThr、10番目のXaa(X10)はLys又はArg、11番目のXaa(X11)はIle、Met、Gln、Ser又はVal、12番目のXaa(X12)はPhe、Leu又はTyr、並びに、13番目のXaa(X13)はAla、Asp、Glu又はAsnである、請求項11乃至13のいずれか一つに記載のペプチド。
- 配列番号13乃至22(図19乃至28)のいずれか一つで示されるアミノ酸配列を含む、請求項11乃至14のいずれか一つに記載のペプチド。
- 配列番号23(図29)で示されるアミノ酸配列のアミノ末端側に、1乃至3個のアミノ酸残基が付加してなるアミノ酸配列、又は、配列番号26(図32)で示されるアミノ酸配列が付加してなるアミノ酸配列を含む、請求項1乃至15のいずれか一つに記載のペプチド。
- 配列番号23(図29)で示されるアミノ酸配列のカルボキシル末端側に、1乃至6個のアミノ酸からなるアミノ酸配列が付加してなるアミノ酸配列を含む、請求項1乃至16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17のいずれか一つに記載のペプチド。
- 請求項1乃至18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19記載のポリヌクレオチドを含むベクター。
- 請求項19記載のポリヌクレオチド若しくは請求項20記載のベクターを含むか又は請求項1乃至18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、SPINK2変異体ペプチドの製造方法:
(i)請求項21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1乃至18のいずれか一つに記載のペプチドを化学合成又はイン・ビトロ翻訳により調製する工程を含む、該ペプチドの製造方法。
- 請求項22又は23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18及び24のいずれか一つに記載のペプチドに他の部分が結合してなるコンジュゲート。
- ペプチドである、請求項25記載のコンジュゲート。
- 免疫グロブリンのFc領域又はその機能断片を含む、請求項25又は26記載のコンジュゲート。
- 下記工程(i)及び(ii)を含む、請求項25乃至27のいずれか一つに記載のSPINK2変異体ペプチドコンジュゲートの製造方法:
(i)該コンジュゲート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んでなるポリヌクレオチド又は該ポリヌクレオチドが挿入されたベクターを含む細胞を培養する工程:
(ii)該培養物からSPINK2変異体ペプチドコンジュゲート又は該コンジュゲートに含まれるペプチド部分を回収する工程。 - 請求項25乃至27のいずれか一つに記載のSPINK2変異体ペプチドコンジュゲート又は該コンジュゲートに含まれるペプチド部分を化学合成又はイン・ビトロ翻訳により調製する工程を含む、該コンジュゲートの製造方法。
- 請求項28又は29記載の方法により得られるSPINK2変異体ペプチドコンジュゲート。
- 請求項1乃至18及び24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結合断片。
- 請求項1乃至18及び24のいずれか一つに記載のペプチド、請求項19記載のポリヌクレオチド、請求項20記載のベクター、請求項21記載の細胞、請求項25乃至27及び30のいずれか一つに記載のコンジュゲート、及び/又は、請求項31記載の抗体もしくはその結合断片を含む組成物。
- 請求項1乃至18及び24のいずれか一つに記載のペプチド、請求項19記載のポリヌクレオチド、請求項20記載のベクター、請求項21記載の細胞、及び/又は、請求項25乃至27及び30のいずれか一つに記載のコンジュゲートを含む医薬組成物。
- KLK1関連疾患、KLK4関連疾患及び/又はKLK8関連疾患の治療用又は予防用である、請求項33記載の医薬組成物。
- 請求項1乃至18及び24のいずれか一つに記載のペプチド、請求項19記載のポリヌクレオチド、請求項20記載のベクター、請求項21記載の細胞、請求項25乃至27及び30のいずれか一つに記載のコンジュゲート、及び/又は、請求項31記載の抗体もしくはその結合断片を含む、検査用又は診断用組成物。
- 請求項31記載の抗体又はその結合断片を用いたアフィニティー精製工程を含む、請求項22、23、28又は29に記載の製造方法。
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AU2018330584A AU2018330584A1 (en) | 2017-09-07 | 2018-09-06 | Peptides inhibiting KLK1, KLK4, or KLK4 and KLK8 |
JP2019540996A JP7191834B2 (ja) | 2017-09-07 | 2018-09-06 | Klk1、klk4、又は、klk4及びklk8を阻害するペプチド |
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US11208467B2 (en) | 2017-09-07 | 2021-12-28 | Daiichi Sankyo Company, Limited | Peptides inhibiting KLK1, KLK4, or KLK4 and KLK8 |
US11845786B2 (en) | 2017-09-07 | 2023-12-19 | Daiichi Sankyo Company, Limited | Peptides inhibiting KLK1, KLK4, or KLK4 and KLK8 |
WO2020095922A1 (ja) | 2018-11-07 | 2020-05-14 | 第一三共株式会社 | ペプチドの血中動態改善方法 |
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JP2023025223A (ja) | 2023-02-21 |
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