Breast cancer therapeutic agent containing PHB2-derived peptide that inhibits BIG3-PHB2 interaction

Abstract

To provide peptides that have the ability to bind to BIG3, which is a protein highly expressed specifically in estrogen receptor-positive cancer, and have excellent growth suppressive effects on BIG3-positive cancer cells, so that the peptides are useful as therapeutic agents for breast cancer which can avoid expression of side effects, and to provide pharmaceutical compositions comprising the peptides.SOLUTION: The present invention provides peptides with a specific sequence comprising the BIG3 polypeptide-binding site in a PHB2 polypeptide, which inhibit the binding between the PHB2 polypeptide and the BIG3 polypeptide, and pharmaceutical compositions comprising the peptides.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a PHB2-derived peptide that inhibits BIG3-PHB2 interaction and a breast cancer therapeutic agent containing the same. [Background technology]

[0002] Estrogen receptor α (ERα) plays a central role in the development and progression of breast cancer. Recent endocrine therapies for breast cancer primarily target ERα signaling using selective ERα modulators (e.g., tamoxifen and raloxifene), ERα downregulators (e.g., fulvestrant), and aromatase inhibitors (AIs) (Non-Patent Documents 1-3). Among these therapies, tamoxifen, which inhibits breast cancer cell proliferation through competitive binding to ERα, is the standard treatment for patients with ERα-positive breast cancer. However, tamoxifen therapy is often ineffective, and patients may die from endocrine therapy-resistant tumor recurrence (Non-Patent Documents 4 and 5). AIs, which block estrogen synthesis, offer substantial clinical benefits compared to tamoxifen, including better efficacy, significantly longer recurrence-free survival, and prolonged recurrence time in postmenopausal women. However, some patients who receive AI therapy experience recurrence (Non-Patent Documents 6 and 7). The exact molecular events that influence the efficacy of these endocrine therapies remain to be elucidated.

[0003] The complex between the cancer-specific protein brefeldin A-inhibited guanine nucleotide-exchange protein 3 (BIG3) and the tumor suppressor prohibitin 2 (PHB2) plays a central role in regulating estrogen signaling in ERα-positive breast cancer (Non-Patent Documents 8, 9). BIG3 binds to PHB2 and inhibits its ability to suppress estrogen-dependent transcriptional activation, resulting in constitutive ERα activity. Based on these findings, a strategy of inhibiting the BIG3-PHB2 interaction to release PHB2 from its complex with BIG3 and thereby exerting the tumor-suppressing activity of PHB2 could be a novel therapy for breast cancer. Based on this strategy, the present inventors previously developed a BIG3 dominant-negative peptide that specifically inhibits the BIG3-PHB2 interaction (Patent Document 1). This peptide has been confirmed to reactivate the tumor-suppressing activity of PHB2, thereby inhibiting the ERα signaling pathway that leads to breast cancer proliferation and suppressing breast cancer proliferation (Patent Document 1). [Prior art documents] [Patent documents]

[0004] [Patent Document 1] WO2013 / 018690 [Non-patent literature]

[0005] [Non-Patent Document 1] Johnston, SR, Clin. Cancer Res. 16(7), 1979-87 (2010). [Non-patent document 2] Fisher, B. et al., J. Natl. Cancer Inst. 97(22), 1652-62 (2005). [Non-patent document 3] Jordan, V. C., Nature Rev. Drug Discov. 2(3), 205-13 (2003).

Non-Patent Document 4

Non-Patent Document 5

Non-Patent Document 6

Non-Patent Document 7

Non-Patent Document 8

Non-Patent Document 9

Non-Patent Document 10

Summary of the Invention

Problems to be Solved by the Invention

[0006] As mentioned above, it has been revealed that the estrogen receptor (ER) activation control molecule BIG3 interacts with the inhibitory factor PHB2 to lead to constitutive activation of ER, and that the BIG3-PHB2 interaction target inhibitory peptide (ERAP: amino acid sequence from positions 165 to 177 of the BIG3 protein (QMLSDLTLQLRQR: SEQ ID NO: 33) has an inhibitory effect on the proliferation of estrogen (E2)-dependent breast cancer cells (Patent Document 1, WO2017 / 126461). However, although ERAP derived from the BIG3 sequence inhibits the interaction by binding to PHB2, since PHB2 is expressed in organs throughout the human body, the occurrence of non-selective effects in organs other than cancer tissue cannot be denied.

[0007] Therefore, an objective of the present invention is to provide a therapeutic strategy that targets the BIG3-PHB2 interaction and is expected to have high selectivity for breast cancer. [Means for solving the problem]

[0008] The present inventors designed several PHB2-derived peptides (PHB2 peptides) based on data on protein interaction candidate regions on the PHB2 amino acid sequence predicted through in silico analysis, and used them to proceed with screening to identify the interaction region using cell proliferation inhibitory effects as an indicator. As a result, PHB2 peptide No. 1 (11-RLPAGPRGMGTA-22 (SEQ ID NO: 1)) and PHB2 peptide No. 5 (76-QYPIIYDIRARPRKI-90 (SEQ ID NO: 5)) each showed a proliferation inhibitory effect of approximately 50%, and in particular, the combined use of PHB2 peptides No. 1 and No. 5 showed a similar effect to ERAP. Over 90% We successfully demonstrated growth inhibitory effects and inhibitory effects on BIG3-PHB2 interaction. Furthermore, these effects were also observed with peptides consisting of sequences surrounding PHB2 peptides No. 1 and No. 5, as well as peptides with amino acid substitutions at various positions in PHB2 peptides No. 1 and No. 5.

[0009] On the other hand, PHB2 peptides No. 1 and No. 5 also showed cell growth inhibitory effects in triple-negative breast cancer cells, which do not express estrogen receptors but do express BIG3. Furthermore, the combined use of these peptides enhanced the effect. Triple-negative breast cancer grows independently of hormones and other growth signals, and proliferation is thought to be activated by the binding of PHB2 and BIG3 without the receipt of these signals within the cells. These results suggest that PHB2-derived peptides may inhibit cell growth by inhibiting the binding of BIG3 and PHB2, at least in breast cancers that express BIG3.

[0010] Thus, the present inventors have discovered PHB2-derived peptides that inhibit BIG3-PHB2 interaction and exert antitumor effects against E2-dependent breast cancer and triple-negative breast cancer, and have completed the present invention. Specifically, the present invention provides the following peptides and uses thereof.

[0011] [1] A peptide comprising a binding site in a PHB2 polypeptide for a BIG3 polypeptide, and inhibiting the binding of the PHB2 polypeptide to a BIG3 polypeptide. [2] The peptide according to [1], comprising any one or a combination of all or a part of the amino acid sequence consisting of amino acids 11 to 21, all or a part of the amino acid sequence consisting of amino acids 76 to 88, and all or a part of the amino acid sequence consisting of amino acids 44 to 57 in the amino acid sequence of SEQ ID NO: 28 (full length of PHB2 polypeptide). [3] A peptide that inhibits binding between a PHB2 polypeptide and a BIG3 polypeptide, comprising an amino acid sequence selected from the group consisting of the following (a) to (f): (a) an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 and 36 to 41 (PHB2 sequence-derived peptide Nos. 1 and 36 to 41); (b) an amino acid sequence in which one, two, or several amino acids have been substituted, deleted, inserted, and / or added to the amino acid sequence selected from the group consisting of SEQ ID NOs: 1 and 36 to 41 (PHB2 sequence-derived peptide Nos. 1, 36 to 41); (c) an amino acid sequence selected from the group consisting of SEQ ID NOs: 5 and 47 to 53 (PHB2 sequence-derived peptide Nos. 5 and 47 to 53); (d) an amino acid sequence in which one, two, or several amino acids have been substituted, deleted, inserted, and / or added to the amino acid sequence selected from the group consisting of SEQ ID NOs: 5 and 47 to 53 (PHB2 sequence-derived peptide Nos. 5, 47 to 53); (e) an amino acid sequence selected from the group consisting of SEQ ID NOs: 82 to 83 (PHB2 sequence-derived peptide Nos. 82 to 83); and (f) An amino acid sequence in which one, two, or several amino acids have been substituted, deleted, inserted, and / or added to the amino acid sequence selected from the group consisting of SEQ ID NOs: 82 to 83 (PHB2 sequence-derived peptide Nos. 82 to 83). [4] The peptide according to [3], which contains an amino acid sequence selected from the group consisting of the following (a') to (b'): (a') an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 and 36 to 41 (PHB2 sequence-derived peptide Nos. 1, 36 to 41), in which one, two, or several amino acid residues other than those corresponding to the 15th glycine (Gly / G) and the 18th glycine in the amino acid sequence of SEQ ID NO: 28 (full-length PHB2 polypeptide) are substituted with other amino acid residues; and (b') An amino acid sequence selected from the group consisting of SEQ ID NOs: 5 and 47 to 53 (PHB2 sequence-derived peptide Nos. 5, 47 to 53), in which one, two, or several amino acid residues other than those corresponding to the 82nd aspartic acid (Asp / D) in the amino acid sequence of SEQ ID NO: 28 (full length PHB2 polypeptide) are substituted with other amino acid residues. [5] The peptide according to any one of [1] to [4], which consists of 80 or less amino acid residues. [6] The peptide according to any one of [1] to [5], which consists of 25 or less amino acid residues. [7] The peptide according to any one of [1] to [6], which consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 5, 36 to 41, 47 to 53, and 82 to 83 (PHB2 sequence-derived peptide Nos. 1, 5, 36 to 41, 47 to 53, 82 to 83). [8] The peptide according to any one of [1] to [7], which is modified with a cell membrane-permeable substance. [9] The peptide according to any one of [1] to [8], which is a cyclic peptide.

[10] The peptide according to any one of [1] to [9], which is a cross-linked type.

[11] The peptide according to any one of [1] to

[10] , which has one or both of the following properties (i) and (ii): (i) inhibiting cell proliferation of BIG3-positive cells; and (ii) It promotes phosphorylation of serine residues in PHB2 polypeptide in BIG3-positive cells.

[12] A polynucleotide encoding the peptide according to any one of [1] to

[11] .

[13] A pharmaceutical composition comprising at least one component selected from the group consisting of one or more peptides according to any one of [1] to

[11] , polynucleotides encoding the peptides, and pharmaceutically acceptable salts of the peptides, and a pharmaceutically acceptable carrier.

[14] The pharmaceutical composition according to

[13] , comprising any one or a combination of a peptide comprising all or part of the amino acid sequence consisting of amino acids at positions 11 to 21 in the amino acid sequence of SEQ ID NO: 28 (full-length PHB2 polypeptide), a peptide comprising all or part of the amino acid sequence consisting of amino acids at positions 44 to 57 in the amino acid sequence of SEQ ID NO: 28 (full-length PHB2 polypeptide), and a peptide comprising all or part of the amino acid sequence consisting of amino acids at positions 76 to 88 in the amino acid sequence of SEQ ID NO: 28 (full-length PHB2 polypeptide).

[15] The pharmaceutical composition according to

[13] or

[14] , for inhibiting the proliferation of cancer cells or for treating and / or preventing cancer.

[16] The pharmaceutical composition according to

[15] , wherein the cancer is a BIG3-positive cancer.

[17] The pharmaceutical composition according to

[15] or

[16] , wherein the cancer is breast cancer.

[18] The pharmaceutical composition according to any one of

[15] to

[17] , wherein the cancer is an estrogen receptor-positive cancer.

[19] A method for either or both of treating and preventing cancer, comprising the step of administering to a subject at least one selected from the group consisting of one or more peptides according to any one of [1] to

[11] , polynucleotides encoding the peptides, and pharmaceutically acceptable salts of the peptides.

[20] The method according to

[19] , comprising the step of administering any one or a combination of a peptide comprising all or part of the amino acid sequence consisting of amino acids at positions 11 to 21 in the amino acid sequence of SEQ ID NO: 28 (full-length PHB2 polypeptide), a peptide comprising all or part of the amino acid sequence consisting of amino acids at positions 44 to 57 in the amino acid sequence of SEQ ID NO: 28 (full-length PHB2 polypeptide), and a peptide comprising all or part of the amino acid sequence consisting of amino acids at positions 76 to 88 in the amino acid sequence of SEQ ID NO: 28 (full-length PHB2 polypeptide).

[21] A method for either or both of treating and preventing chemotherapeutic-resistant breast cancer (e.g., triple-negative breast cancer), comprising the steps of selecting a chemotherapeutic-resistant breast cancer patient (e.g., a triple-negative breast cancer patient) and administering to the subject at least one selected from the group consisting of one or more peptides according to any one of [1] to

[11] , polynucleotides encoding the peptides, and pharmaceutically acceptable salts of the peptides.

[0012] Alternatively, the present invention provides use of at least one selected from the group consisting of one or more peptides described in any one of [1] to

[11] above, polynucleotides encoding the peptides, and pharmaceutically acceptable salts of the peptides, in the manufacture of a pharmaceutical composition for either or both of cancer treatment and prevention. The present invention also relates to at least one selected from the group consisting of one or more peptides described in any one of [1] to

[11] above, polynucleotides encoding the peptides, and pharmaceutically acceptable salts of the peptides, for use in either or both of cancer treatment and prevention. Furthermore, the present invention relates to a method for producing a pharmaceutical composition for either or both of cancer treatment and prevention, comprising the step of mixing or blending at least one selected from the group consisting of one or more peptides described in any one of [1] to

[11] above, polynucleotides encoding the peptides, and pharmaceutically acceptable salts of the peptides, with a carrier. [Effects of the Invention]

[0013] The peptide of the present invention has the ability to bind to BIG3, a protein that is specifically highly expressed in estrogen receptor-positive cancers, rather than PHB2, which is expressed in all organs of the human body, and can inhibit the BIG3-PHB2 interaction. Therefore, the peptide of the present invention is expected to have high selectivity for estrogen receptor-positive cancers. Furthermore, the peptides of the present invention exhibit growth-inhibitory effects not only on estrogen-dependent breast cancer cells but also on triple-negative breast cancer cells. Until now, there have been no effective molecular-targeted therapeutic agents for triple-negative breast cancer, and the only treatment options have been existing anticancer drugs with strong side effects. On the other hand, the cell growth-inhibitory effect of the peptides of the present invention was not observed in normal mammary epithelial cells in which BIG3 expression is not observed. These findings suggest that the peptides of the present invention are useful as therapeutic agents for BIG3-positive cancers, regardless of whether they are hormone-dependent or not. [Brief explanation of the drawings]

[0014] [Figure 1-1] Screening of peptides derived from the PHB2 sequence that inhibit estrogen-dependent cell proliferation is shown. (A) Human breast cancer cell line MCF-7 was treated with 10 μM of each peptide derived from the PHB2 sequence, followed immediately by stimulation with 10 nM estrogen. Cell numbers were assessed by MTT assay at 24 hours. Data represent the mean ± standard deviation of three independent experiments. The full-length sequence of human PHB2 protein is shown below the graph. A synthetic peptide with eight arginine residues added to the C-terminus of the underlined sequence was used in the assay. The bolded amino acids indicate amino acids implicated in interaction with BIG3 by in silico analysis. The boxed "S" indicates the phosphorylation site of PHB2. The dashed line indicates the region of proven BIG3 binding. [Figure 1-2] (B, C) MCF-7 cells were treated with 10 μM of peptides derived from the PHB2 sequence, centered on aa 11-22 (B) and aa 76-90 (C) (SEQ ID NO: 1, 34-43, 5, 44-55), followed immediately by 10 nM estrogen stimulation. Cell numbers were assessed by MTT assay 24 hours later. Data represent the mean ± standard deviation of three independent experiments. The left side of the graph indicates the position of the PHB2-derived sequence contained in the synthetic peptides used in the assay. [Figure 1-3] (D) 10 μM of peptides derived from the 11-22 aa PHB2 sequence and peptides derived from each of the PHB2 sequences, mainly consisting of aa 76-90, were combined, followed immediately by 10 nM estrogen stimulation. Cell numbers were assessed by MTT assay for 24 hours. (E) 10 μM of peptides derived from the 76-90 aa PHB2 sequence and peptides derived from each of the PHB2 sequences, mainly consisting of aa 11-22, were combined, followed immediately by 10 nM estrogen stimulation. Cell numbers were assessed by MTT assay for 24 hours. Data represent the mean ± standard deviation of three independent experiments. [Figure 2]The combined effect of PHB2 peptides 11-22aa and 76-90aa on estrogen-dependent growth inhibition is shown. (A) Human breast cancer cell line MCF-7 was treated with 10 μM each of ERAP (positive control), PHB2 peptides 11-22aa, 76-90aa, and 86-100aa (negative control), and the combination of 11-22aa and 76-90aa, or the combination of 76-90aa and 86-100aa. Immediately thereafter, 10 nM estrogen was added, and cell numbers were assessed 24 hours later by MTT assay. Data represent the mean ± standard deviation of three independent experiments. (B) The inhibitory effect of PHB2 peptides 11-22aa, 76-90aa, and their combination on the interaction between BIG3 and PHB2 in MCF-7 cells was assessed by Western blot. MCF-7 cells were treated with 20 μM or 50 μM of each PHB2 peptide for 24 hours, then lysed and immunoprecipitated with an anti-BIG3 antibody. Immunoblot analysis was performed using the indicated antibodies. The binding inhibition rate was expressed as a ratio to the PHB2 band area in untreated cells, which was set to 100. (C) Human breast cancer cell line MCF-7 was treated with 10 μM each of ERAP, PHB2 peptides 11-22aa, 76-90aa, 86-100aa, a combination of 11-22aa and 76-90aa, and a combination of 76-90aa and 86-100aa. Immediately thereafter, stimulation with 10 nM estrogen was performed, and 24 hours later, phosphorylation of PHB2 (Ser39) was evaluated by Western blotting. The phosphorylation intensity was expressed as a ratio to the area of the phosphorylated band after ERAP treatment in the presence of estrogen, which was set to 1.0. [Figure 3]The effects of PHB2 peptide 11-90aa on estrogen-dependent proliferation and the interaction between BIG3 and PHB2 are shown. (A) Human breast cancer cell line MCF-7 was treated with PHB2 peptide 11-90aa (10, 20, 50μM), 11-22aa (50μM), 76-90aa (50μM), or a combination of 11-22aa and 76-90aa. Immediately thereafter, 10nM estrogen was added and cell numbers were assessed 24 hours later by MTT assay. Data represent the mean ± standard deviation of three independent experiments. (B) The inhibitory effects of PHB2 peptide 11-90aa and 10μM ERAP (positive control) on the interaction between BIG3 and PHB2 in MCF-7 cells were assessed by Western blot. (C) Human breast cancer cell line MCF-7 was treated with PHB2 peptide 11-90aa (20, 50, 100μM) and 10μM ERAP, and then immediately stimulated with 10nM estrogen. 24 hours later, phosphorylation of PHB2 (Ser39) was assessed by Western blot. The phosphorylation intensity was expressed as a ratio, with the untreated phosphorylated band area set at 1.0. [Figure 4] Schematic diagrams of branched and cyclic PHB2 peptides. (A) Linear PHB2 peptide. (B) Branched PHB2 peptide. (C) Cyclic PHB2 peptide. [Figure 5]The inhibitory effect of branched and cyclic PHB2 peptides on estrogen-dependent proliferation is shown. (A) Human breast cancer cell line MCF-7 was treated with 10 μM each of the linear peptides 11-22aa, 76-90aa, a combination of 11-22aa and 76-90aa, linear, branched, and cyclic peptides 11-21aa, cyclic 76-88aa, and a combination of cyclic peptides. Immediately after treatment with 10 nM estrogen, cell numbers were assessed 24 hours later by MTT assay. Data represent the mean ± standard deviation of three independent experiments. (B) MTT assay results showing that the linear 11-22aa, linear 76-90aa, linear, branched, and cyclic peptides 11-21aa, and cyclic 76-88aa peptides had no effect on the proliferation of normal breast epithelial cells MCF-10A. (C) The inhibitory effect on the interaction between BIG3 and PHB2 was evaluated by Western blotting when the human breast cancer cell line MCF-7 was treated with 10 μM each of the linear peptides 11-22aa, 76-90aa, a combination of 11-22aa and 76-90aa, linear, branched, and cyclic peptides 11-21aa, cyclic 76-88aa, and a combination of cyclic peptides. [Figure 6] The combined effect of cyclic PHB2 peptides on the inhibition of estrogen-dependent proliferation is shown. (A) Human breast cancer cell line MCF-7 was treated with linear 11-22aa and cyclic 11-21aa peptides (left panel) or linear 76-90aa and cyclic 76-88aa peptides (right panel). Immediately thereafter, 10 nM estrogen was added and the inhibitory effects were assessed by MTT assay every 24 hours for up to 96 hours. (B) MTT assay results show that the cyclic 11-21aa and cyclic 76-88aa peptides have no effect on the proliferation of normal breast epithelial cells MCF-10A. Data represent the mean ± standard deviation of three independent experiments. [Figure 7]The concentration-dependent inhibitory effect of the cyclic PHB2 peptide on estrogen-dependent proliferation is shown. (A) Human breast cancer cell line MCF-7 was treated with cyclic 11-21aa and cyclic 76-88aa at 0.1, 0.5, 1, 2.5, 5, 10, and 20 μM, respectively, and then immediately stimulated with 10 nM estrogen. 24 hours later, cell numbers were assessed by MTT assay. Data represent the mean ± standard deviation of three independent experiments. (B) Human breast cancer cell line MCF-7 was treated with cyclic 11-21aa (4 μM), cyclic 76-88aa (2 μM), or their combination. Immediately stimulated with 10 nM estrogen, cell numbers were assessed by MTT assay every 24 hours for up to 96 hours. Data represent the mean ± standard deviation of three independent experiments. (C) The results of an MTT assay showing that the cyclic type 11-21aa (4 μM), the cyclic type 76-88aa (2 μM), and their combination did not affect the proliferation of normal mammary epithelial cells MCF-10A. [Figure 8-1] The amino acids of PHB2 important for the suppression of estrogen-dependent proliferation are shown. (A) Alanine-mutated PHB2 peptides No. 1 (aa 11-22) and No. 5 (aa 76-90) are shown. (B, C) Human breast cancer cell line MCF-7 was treated with 10 μM of each alanine-mutated peptide derived from the PHB2 sequence, followed immediately by stimulation with 10 nM estrogen. Cell numbers were assessed by MTT assay 24 hours later. Data represent the mean ± standard deviation of three independent experiments. [Figure 8-2] (D) PHB2 peptides consisting of peptides No. 2 (42-50aa) and No. 3 (38-50aa) with aa 51-57 added. (E) 10 μM of each of the peptides derived from the PHB2 sequence in (D) was added, followed immediately by stimulation with 10 nM estrogen. Cell counts were assessed by MTT assay 24 hours later. Data represent the mean ± standard deviation of three independent experiments. [Figure 9](A) and (B) show the cell growth inhibitory effects of PHB2 peptides 11-22aa (A) and 76-90aa (B) on triple-negative breast cancer cells. Each peptide was added to the breast cancer cell line MDA-MB-231 at 11 concentrations, with 3-fold serial dilutions starting from 20 μM. 96 hours after peptide addition, viable cell counts were measured, and the relative values were calculated relative to the negative control cells without peptide addition. Data represent the mean ± standard deviation of three independent experiments. (C) The combined effect of PHB2 peptides 11-22aa and 76-90aa on MDA-MB-231 cell growth was investigated. Peptides were added individually or in combination at IC50 values. 96 hours later, viable cell counts were measured, and the relative values were calculated relative to the negative control cells treated with phosphate-buffered saline (PBS). Data represent the mean ± standard deviation of three independent experiments. [Figure 10-1] The inhibitory effect of cross-linked PHB2 peptides on estrogen-dependent proliferation is shown. (A) Schematic diagram of the cross-linked form. (B) PHB2 peptides cross-linked by adding cysteines to both ends of the 11-21aa and 76-88aa PHB2 peptides (SEQ ID NOs: 106-108, 110-112, 115-117, 119-121). The PHB2 peptides of SEQ ID NOs: 109, 113, 114, 118, and 122 were made non-cross-linked by adding alanines to both ends. The PHB2 peptides of SEQ ID NOs: 106-114 had polyarginine added to the C-terminus. Furthermore, to avoid oxidation during synthesis of the 11-21aa PHB2 peptide, the methionine at position 19 in the amino acid sequence of SEQ ID NO: 28 (full-length PHB2 polypeptide) was replaced with norleucine (Nle). [Figure 10-2] (C) Human breast cancer cell line MCF-7 was treated with the 11-21aa crosslinked PHB2 peptide (left panel) or the 76-88aa crosslinked PHB2 peptide (right panel), followed immediately by stimulation with 10 nM estrogen. 24 hours later, cell numbers were assessed by MTT assay. Data represent the mean ± standard deviation of three independent experiments. [Figure 11]The inhibitory effect of cyclic PHB2 peptides on estrogen-dependent proliferation is shown. (A) Crosslinked forms of the cyclic 11-21aa (SEQ ID NO: 25) and 76-88aa (SEQ ID NO: 26) PHB2 peptides are shown. The cyclic PHB2 peptides of SEQ ID NOs: 125 and 128 were converted to non-crosslinked cyclic peptides by adding alanine residues to both ends of the 11-21aa and 76-88aa PHB2 peptides. All cyclic PHB2 peptides contained an unnatural amino acid and multiple consecutive arginine residues at the C-terminus. Furthermore, to avoid oxidation during synthesis, the 11-21aa cyclic PHB2 peptide contained norleucine (Nle) instead of methionine at position 19 in the amino acid sequence of SEQ ID NO: 28 (full-length PHB2 polypeptide). (B) Human breast cancer cell line MCF-7 was treated with cyclic PHB2 peptide and immediately stimulated with 10 nM estrogen. 24 hours later, cell numbers were assessed by MTT assay. Data represent the mean ± standard deviation of three independent experiments. [Figure 12] The effect of altering the 11-22aa PHB2 peptide on estrogen-dependent proliferation is shown. (A) This shows the PHB2 peptide of SEQ ID NO: 1 (11-22aa), in which the 15th and 18th glycines in the amino acid sequence of SEQ ID NO: 28 (full-length PHB2 polypeptide) were substituted with D-alanine and D-leucine, respectively. (B) The human breast cancer cell line MCF-7 was treated with the 11-22aa altered PHB2 peptide, immediately followed by stimulation with 10 nM estrogen. 24 hours later, cell numbers were assessed by MTT assay. Data represent the mean ± standard deviation of three independent experiments. [Figure 13] Figure 1 shows the effect of cross-linked PHB2 peptide on the proliferation of normal mammary epithelial cells MCF-10A. Figure 2 shows the results of an MTT assay, which demonstrated that cross-linked and cyclic PHB2 peptides had no effect on the proliferation of normal mammary epithelial cells MCF-10A. Data represent the mean ± standard deviation of three independent experiments. DETAILED DESCRIPTION OF THE INVENTION

[0015] Although any methods and materials similar or equivalent to those described herein can be used in practicing or testing embodiments of the present invention, the preferred methods, devices, and materials are described herein. However, before describing the materials and methods of the present invention, it should be understood that the present invention is not limited to the specific sizes, shapes, dimensions, materials, methodologies, protocols, etc. described herein, as these may vary depending on routine experimentation and optimization. It should also be understood that the terminology used in this description is for the purpose of describing particular versions or embodiments only, and is not intended to limit the scope of the present invention, which is limited solely by the appended claims.

[0016] definition As used herein, the words "a," "an," and "the" mean "at least one" unless otherwise specified.

[0017] In this specification, unless otherwise specified, amino acids written in capital letters represent L-amino acids. On the other hand, amino acids written in lowercase represent D-amino acids. In addition, the L-amino acids and D-amino acids written in this specification may also include those in which any of the amino group, carboxyl group, and side chain has been modified. Preferred examples of modifications include acetylation of the amino group, amidation of the carboxyl group, and addition of tag peptides such as FLAG tag and HA tag.

[0018] Furthermore, in this specification, unless otherwise specified, numbers indicating the positions of amino acid residues in an amino acid sequence are assigned in order toward the C-terminus, with the N-terminal amino acid residue being numbered 1.

[0019] As used herein, the term "BIG3" refers to brefeldin A-inhibited guanine nucleotide-exchange protein 3. BIG3 forms a complex with PHB2, thereby inhibiting the function of PHB2 in suppressing estrogen-dependent transcriptional activation. BIG3 is also referred to as "ARFGEF3 (ARFGEF family member 3)" or "A7322." A representative example of the nucleotide sequence of the human BIG3 gene is shown in SEQ ID NO: 31 (GenBank Accession No. NM_020340.4), and the amino acid sequence encoded thereby is shown in SEQ ID NO: 32. In the present invention, BIG3 is not limited to that encoded by the nucleotide sequence, but also includes isoforms and mutants thereof.

[0020] As used herein, the term "PHB2" refers to prohibitin 2. PHB2 binds to the estrogen receptor, inhibits the estrogen receptor signaling pathway, and suppresses estrogen-dependent cell proliferation. PHB2 is also referred to as "REA (Repressor of Estrogen Activity)." Representative examples of the nucleotide sequences of the human PHB2 gene are shown in SEQ ID NO: 27 (GenBank Accession No. NM_001144831.1) and SEQ ID NO: 29 (GenBank Accession No. NM_001267700.1), and the amino acid sequences encoded thereby are shown in SEQ ID NO: 28 and SEQ ID NO: 30, respectively. In the present invention, PHB2 is not limited to those encoded by the above nucleotide sequences, but also includes their isoforms and mutants.

[0021] The term "estrogen receptor" as used herein encompasses both estrogen receptor α (ERα) and estrogen receptor β (ERβ). ERα and ERβ are encoded by the ESR1 gene and ESR2 gene, respectively. The nucleotide sequence of a representative human ESR1 gene and the amino acid sequence of human ERα are shown in SEQ ID NO: 86 (GenBank Accession No. NM_000125.3) and SEQ ID NO: 87 (GenBank Accession No. NP_000116.2), respectively. The nucleotide sequence of a representative human ESR2 gene and the amino acid sequence of human ERβ are shown in SEQ ID NO: 88 (GenBank Accession No. NM_001437.2) and SEQ ID NO: 89 (GenBank Accession No. NP_001428.1), respectively. However, in the present invention, the nucleotide sequence and amino acid sequence of the estrogen receptor are not limited to these and include their isoforms and variants. In a preferred embodiment, the estrogen receptor is ERα. It has been reported that the transcriptional activation of both ERα and ERβ is controlled by the PHB2 polypeptide (Montano MM, et al., Proc Natl Acad Sci USA. 96(12): 6947-52(1999)). As used herein, the term "estrogen receptor-positive" in reference to cells or cancer means that the cells or cancer cells that constitute the cancer express the estrogen receptor. Whether or not a cell is estrogen receptor-positive can be confirmed by known methods such as ELISA or immunohistochemical staining. Furthermore, as used herein, the term "estrogen receptor-negative" in reference to cells or cancer means that the cells or cancer cells that constitute the cancer do not express the estrogen receptor.

[0022] The term "ERAP" as used herein refers to a peptide consisting of the amino acid sequence set forth in SEQ ID NO: 33. The amino acid sequence set forth in SEQ ID NO: 33 is a sequence consisting of amino acid residues at positions 165 to 177 of the amino acid sequence of BIG3 (SEQ ID NO: 32), and contains amino acid residues important for binding to PHB2 (glutamine (Q) at position 165, aspartic acid (D) at position 169, and glutamine (Q) at position 173 in the amino acid sequence set forth in SEQ ID NO: 32). ERAP has the ability to bind to PHB2, and inhibits the formation of a complex between BIG3 and PHB2 by competitively binding to PHB2.

[0023] The term "treatment" as used herein includes alleviating or improving at least one symptom caused by a target disease, inhibiting the progression of the disease, inhibiting the spread of the disease site, etc. For example, "treatment of cancer" includes inhibiting the proliferation of cancer cells, inhibiting the progression of cancer, inducing regression or remission of cancer, alleviating or improving symptoms associated with cancer, inhibiting cancer metastasis, inhibiting postoperative recurrence, and inducing an extension of survival time.

[0024] 1. PHB2 peptide The present invention provides peptides that contain the binding site of a PHB2 polypeptide to a BIG3 polypeptide and inhibit the binding between the PHB2 polypeptide and a BIG3 polypeptide. The peptides of the present invention are also referred to herein as "PHB2 peptides," "PHB2-derived peptides," or "PHB2 sequence-derived peptides." The peptides of the present invention have the ability to bind to BIG3 polypeptides by containing the binding site of the PHB2 polypeptide with BIG3 polypeptides. As a result, they competitively inhibit the binding of PHB2 polypeptides to BIG3 polypeptides. The PHB2 peptides of the present invention can also be in the form of salts, so long as they have the ability to inhibit the binding between PHB2 polypeptides and BIG3 polypeptides. For example, they can be salts with acids (inorganic acids, organic acids, etc.) or bases (alkali metals, alkaline earth metals, amines, etc.). Examples of salts with acids include salts with inorganic acids (e.g., hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid, acetic acid, etc.) and salts with organic acids (e.g., acetic acid, formic acid, propionic acid, fumaric acid, maleic acid, succinic acid, tartaric acid, citric acid, malic acid, oxalic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid, meglumine acid, etc.). Examples of salts with bases include salts with sodium, potassium, calcium, and ammonium. Preferred examples of the salt of the peptide of the present invention include acetate, hydrochloride, meglumine, and ammonium salt.

[0025] The term "binding site in a PHB2 polypeptide with a BIG3 polypeptide" refers to an amino acid residue in the amino acid sequence constituting a PHB2 polypeptide that is involved in binding to a BIG3 polypeptide. Examples of such amino acid residues include glycines at positions 15 and 18 and aspartic acid at position 82 in the amino acid sequence set forth in SEQ ID NO: 28 (full-length PHB2 polypeptide). Therefore, in a preferred embodiment, the peptide of the present invention is a peptide that contains glycines at positions 15 and 18 and aspartic acid at position 82 in the amino acid sequence set forth in SEQ ID NO: 28 (full-length PHB2 polypeptide), and inhibits binding between a PHB2 polypeptide and a BIG3 polypeptide. In this specification, the number of a specific amino acid residue in an amino acid sequence indicates the number of the amino acid residue counted from the N-terminus.

[0026] Examples of amino acid sequences in the PHB2 polypeptide that contain the binding site with the BIG3 polypeptide include (a) all or part of the amino acid sequence consisting of amino acids 11 to 21 (SEQ ID NO: 84), (b) all or part of the amino acid sequence consisting of amino acids 76 to 88 (SEQ ID NO: 85), and (c) all or part of the amino acid sequence consisting of amino acids 44 to 57 (SEQ ID NO: 82) in the amino acid sequence of SEQ ID NO: 28 (full-length PHB2 polypeptide). Therefore, preferred examples of the peptides of the present invention include peptides containing an amino acid sequence selected from the group consisting of the following (a) to (c): (a) an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 and 36 to 41 (PHB2 sequence-derived peptide Nos. 1 and 36 to 41); (b) an amino acid sequence selected from the group consisting of SEQ ID NOs: 5 and 47 to 53 (PHB2 sequence-derived peptide Nos. 5, 47 to 53); and (c) An amino acid sequence selected from the group consisting of SEQ ID NOs: 82 to 83 (PHB2 sequence-derived peptide Nos. 82 to 83). However, the peptides of the present invention are not limited to these, and the amino acid sequence constituting the peptide is not particularly limited as long as it contains the binding site in the PHB2 polypeptide with the BIG3 polypeptide and has the activity of inhibiting the binding between the PHB2 polypeptide and the BIG3 polypeptide.

[0027] It is generally known that modification of one or more amino acids in a peptide does not affect the function of the peptide. In fact, it is known that peptides having an amino acid sequence modified by substitution, deletion, insertion, and / or addition of one or more amino acid residues retain the biological activity of the original peptide (Mark et al., Proc Natl Acad Sci USA 81(18): 5662-6(1984); Zoller and Smith, Nucleic Acids Res 10(20): 6487-500(1982); Dalbadie-McFarland et al., Proc Natl Acad Sci USA 79(21): 6409-13(1982)). The peptides of the present invention may contain, for example, substitutions or deletions of amino acid residues at positions other than the BIG3 polypeptide-binding site in the PHB2 polypeptide, or insertions or additions of amino acid residues at positions that do not affect binding to the BIG3 polypeptide, as long as they contain the binding site in the PHB2 polypeptide and have the activity of inhibiting binding between the PHB2 polypeptide and the BIG3 polypeptide. In fact, in the Examples herein, it has been shown that peptides having amino acid sequences in which amino acid residues other than the BIG3 polypeptide-binding site in the PHB2 polypeptide are substituted with other amino acid residues also retain biological activity equivalent to that of peptides without such substitutions. Thus, the peptides of the present invention include peptides comprising an amino acid sequence selected from the group consisting of the following (a') to (b') and having the activity of inhibiting binding between the PHB2 polypeptide and the BIG3 polypeptide: (a') an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 and 36 to 41 (PHB2 sequence-derived peptide Nos. 1 and 36 to 41), in which one, two or several amino acid residues other than those corresponding to the 15th glycine and the 18th glycine in the amino acid sequence of SEQ ID NO: 28 (full-length PHB2 polypeptide) are substituted with other amino acid residues; and (b') An amino acid sequence selected from the group consisting of SEQ ID NOs: 5 and 47 to 53 (PHB2 sequence-derived peptide Nos. 5, 47 to 53), in which one, two or several amino acid residues other than those corresponding to the 82nd aspartic acid in the amino acid sequence of SEQ ID NO: 28 (full length PHB2 polypeptide) are substituted with other amino acid residues.

[0028] In the above (a') to (b'), the amino acid residues to be substituted may be any amino acid residues, as long as the ability to inhibit the binding between the PHB2 polypeptide and the BIG3 polypeptide is maintained. The amino acid residues to be substituted can also be determined by predicting amino acid residues not involved in the binding with the BIG3 peptide, for example, using a calculation method such as PSIVER. The number of amino acid residues to be substituted is also not particularly limited, as long as the ability to inhibit the binding between the PHB2 polypeptide and the BIG3 polypeptide is maintained; one, two, or several amino acid residues may be substituted. "Several" preferably refers to six, five, four, or three amino acid residues.

[0029] It is generally recognized that substitution of an original amino acid residue with another amino acid residue that conserves the properties of the amino acid side chain tends not to affect the function of the original peptide. Such substitutions are often referred to as "conservative substitutions" or "conservative modifications." Therefore, the substitutions in (a') to (b') above are preferably conservative substitutions. Conservative substitution tables showing functionally similar amino acids are well known in the art. Examples of amino acid side chain properties that are desirable to conserve include, for example, hydrophobic amino acids (A, I, L, M, F, P, W, Y, V), hydrophilic amino acids (R, D, N, C, E, Q, G, H, K, S, T), and side chains that share the following functional groups or features: aliphatic side chains (G, A, V, L, I, P); hydroxyl-containing side chains (S, T, Y); sulfur-containing side chains (C, M); carboxylic acid- and amide-containing side chains (D, N, E, Q); base-containing side chains (R, K, H); and aromatic-containing side chains (H, F, Y, W). In addition, the following eight groups each contain amino acids that are recognized in the art as conservative substitutions for one another: 1) Alanine (A), Glycine (G); 2) aspartic acid (D), glutamic acid (E); 3) asparagine (N), glutamine (Q); 4) arginine (R), lysine (K); 5) isoleucine (I), leucine (L), methionine (M), valine (V); 6) phenylalanine (F), tyrosine (Y), tryptophan (W); 7) serine (S), threonine (T); and 8) Cysteine (C), Methionine (M) (see, e.g., Creighton, Proteins 1984).

[0030] However, the substitutions in (a') to (b') above are not limited to these, and non-conservative substitutions may also be used as long as the activity of inhibiting the binding between the PHB2 polypeptide and the BIG3 polypeptide is maintained. The peptides of the present invention may contain amino acid residues other than those at the binding site with the BIG3 polypeptide in the PHB2 polypeptide, so long as they maintain the activity of inhibiting the binding between the PHB2 polypeptide and the BIG3 polypeptide. For example, a fragment of the PHB2 polypeptide containing the binding site with the BIG3 polypeptide in the PHB2 polypeptide is suitable as a peptide of the present invention. Therefore, preferred examples of the peptides of the present invention include PHB2 polypeptides containing glycine at position 15 and glycine at position 18 and their surrounding sequences in the amino acid sequence of SEQ ID NO: 28 (full-length PHB2 polypeptide) (SEQ ID NOs: 1 and 36-41 (PHB2 sequence-derived peptide Nos. 1 and 36-41)) and PHB2 polypeptides containing aspartic acid at position 82 and its surrounding sequences in the amino acid sequence of SEQ ID NO: 28 (full-length PHB2 polypeptide) (SEQ ID NOs: 5 and 47-53 (PHB2 sequence-derived peptide Nos. 5 and 47-53)). Another preferred example of the peptide of the present invention is a PHB2 polypeptide (SEQ ID NOS: 82 to 83 (PHB2 sequence-derived peptide Nos. 82 to 83) comprising amino acids 44 to 57 in the amino acid sequence of SEQ ID NOS: 28 (full-length PHB2 polypeptide) and their surrounding sequences.

[0031] For example, the PHB2 peptides of the present invention include peptides comprising the amino acid sequences of SEQ ID NOS: 1, 5, 36-41, 47-53, and 82-83 (PHB2 sequence-derived peptide Nos. 1, 5, 36-41, 47-53, and 82-83) and consisting of, for example, 30 or 20 residues, typically 19 residues, preferably 18 residues, and more preferably 17 or fewer amino acid residues. Examples of such peptides include peptides comprising the amino acid sequences (9 residues) of SEQ ID NOS: 1, 5, 36-41, 47-53, and 82-83 (PHB2 sequence-derived peptide Nos. 1, 5, 36-41, 47-53, and 82-83) and an amino acid sequence selected from the full-length amino acid sequence constituting the PHB2 polypeptide, and consisting of 30 or 20 residues, typically 19 residues, preferably 18 residues, and more preferably 17 or fewer amino acid residues.

[0032] In a preferred embodiment of the present invention, the amino acids added to the amino acid sequences of SEQ ID NOs: 1, 5, 36-41, 47-53, and 82-83 (PHB2 sequence-derived peptide Nos. 1, 5, 36-41, 47-53, and 82-83) can be one or more consecutive amino acid sequences selected from 0 (i.e., the amino acid sequence of SEQ ID NOs: 1, 5, 36-41, 47-53, and 82-83 (PHB2 sequence-derived peptide Nos. 1, 5, 36-41, 47-53, and 82-83)) and the full-length amino acid sequence constituting the PHB2 polypeptide (SEQ ID NO: 28 (full-length PHB2 polypeptide)). The amino acid sequences of SEQ ID NOs: 1, 5, 36-41, 47-53, and 82-83 (PHB2 sequence-derived peptide Nos. 1, 5, 36-41, 47-53, and 82-83) are amino acid sequences containing an amino acid sequence consisting of glycine at position 15, glycine at position 18, aspartic acid at position 82, or amino acids at positions 44 to 57 in the full-length amino acid sequence constituting the PHB2 polypeptide (SEQ ID NO: 28 (full-length PHB2 polypeptide)). Therefore, in a preferred embodiment of the present invention, the amino acid residue or amino acid sequence to be added to SEQ ID NOs: 1, 5, 36-41, 47-53, and 82-83 (PHB2 sequence-derived peptide Nos. 1, 5, 36-41, 47-53, and 82-83) can be selected from amino acid sequences adjacent to the amino acid sequence consisting of glycine at position 15, glycine at position 18, aspartic acid at position 82, or amino acids at positions 44 to 57 in the amino acid sequence of SEQ ID NO: 28 (full-length PHB2 polypeptide).

[0033] The peptide of the present invention preferably has, in addition to the activity of inhibiting the binding between a PHB2 polypeptide and a BIG3 polypeptide, either or both of the following properties (i) and (ii): (i) promoting nuclear translocation of PHB2 polypeptide in estrogen receptor-positive cells expressing BIG3 polypeptide; and (ii) BIG3 In estrogen receptor-positive cells expressing the polypeptide, the binding of the PHB2 polypeptide to estrogen receptors present in the nucleus and / or cell membrane is promoted. By possessing either or both of the above properties (i) and (ii), the peptide of the present invention suppresses estrogen receptor activation in BIG3-expressing cells, resulting in the inhibition of cell proliferation of estrogen receptor-positive cells. Both of the properties (i) and (ii) of the PHB2 peptide can be evaluated according to methods known to those skilled in the art.

[0034] PHB2 polypeptide is known to be an estrogen receptor-selective coregulator, and by interacting with the estrogen receptor, it suppresses the transcriptional activation of the estrogen receptor (Kasashima K, J Biol Chem 281(47): 36401-10(2006)). On the other hand, BIG3 polypeptide binds to PHB2 polypeptide, preventing its nuclear translocation and inhibiting its interaction with the estrogen receptor in the nucleus. It also prevents the binding of PHB2 polypeptide to the estrogen receptor present on the cell membrane. As a result of these actions, in cells overexpressing BIG3 polypeptide, the PHB2 polypeptide's suppression of estrogen receptor activation is insufficient, leading to increased cell proliferation.

[0035] The peptides of the present invention are characterized by competitively inhibiting the binding between a BIG3 polypeptide and a PHB2 polypeptide, thereby restoring the estrogen receptor activation inhibitory function of a PHB2 polypeptide inhibited by its binding to a BIG3 polypeptide. Meanwhile, a PHB2 polypeptide inhibits estrogen receptor activation through its binding to an estrogen receptor. Therefore, it is desirable that the peptides of the present invention inhibit the binding between a BIG3 polypeptide and a PHB2 polypeptide but do not interfere with the binding between an estrogen receptor and a PHB2 polypeptide, thereby preventing the inhibition of estrogen receptor activation by a PHB2 polypeptide. As described above, a fragment of a PHB2 polypeptide containing the binding site for a BIG3 polypeptide is suitable as a peptide of the present invention. However, a peptide close to the full-length PHB2 polypeptide may interfere with the binding of an endogenous PHB2 polypeptide to an estrogen receptor, thereby potentially preventing the inhibition of estrogen receptor activation by an endogenous PHB2 polypeptide. Therefore, the partial amino acid sequence of a PHB2 polypeptide contained in a peptide of the present invention preferably has 100 or fewer residues, more preferably 80 or fewer residues, and even more preferably 70 or fewer residues. In a more preferred embodiment, the partial amino acid sequence of the PHB2 polypeptide contained in the peptide of the present invention is 50 or fewer residues, 40 or fewer residues, 30 or fewer residues, 25 or fewer residues, or 20 or fewer residues. The estrogen receptor-binding site in PHB2 is the site consisting of amino acids 175 to 198 in the amino acid sequence of SEQ ID NO: 28 (full-length PHB2 polypeptide). Therefore, it is preferable that the peptide of the present invention does not contain this site. In such a case, the partial amino acid sequence of the PHB2 polypeptide contained in the peptide of the present invention is desirably 100 or fewer residues, excluding the amino acid sequence of amino acids 175 to 198, more preferably 80 or fewer residues, and even more preferably 70 or fewer residues. In a more preferred embodiment, the partial amino acid sequence of the PHB2 polypeptide contained in the peptide of the present invention is 50 or fewer residues, 40 or fewer residues, 30 or fewer residues, 25 or fewer residues, or 20 or fewer residues, excluding the amino acid sequence of amino acids 175 to 198.

[0036] Furthermore, the peptides of the present invention may contain an additional amino acid sequence other than the amino acid sequence derived from the PHB2 polypeptide, as long as the peptide maintains its activity of inhibiting the binding between the BIG3 polypeptide and the PHB2 polypeptide and does not interfere with the suppression of estrogen receptor activation by the PHB2 polypeptide. In this case, it is desirable that the additional amino acid sequence does not interfere with the binding of the endogenous PHB2 polypeptide to the estrogen receptor. Therefore, the peptides of the present invention are preferably peptides of 100 residues or less, 80 residues or less, or 70 residues or less. In more preferred embodiments, the peptides of the present invention are peptides of 50 residues or less, 40 residues or less, or 30 residues or less. Preferred examples of amino acid sequences contained in the peptides of the present invention include, but are not limited to, the amino acid sequences constituting the cell-penetrating peptides described below and linker sequences for binding other substances.

[0037] The peptides of the present invention may also be modified with other substances. As used herein, the term "modified" in reference to a peptide refers to the direct or indirect binding of other substances to the peptide. Examples of other substances that modify the peptides of the present invention include, but are not limited to, peptides, lipids, sugars, and natural or synthetic polymers. The peptides of the present invention may have any modification as long as they maintain the activity of inhibiting the binding between a BIG3 polypeptide and a PHB2 polypeptide. The peptides of the present invention may also be conferred additional functions by modification. Examples of additional functions include, but are not limited to, targeting, stability, and cell membrane permeability.

[0038] In the present invention, a preferred example of modification is the introduction of a cell membrane-permeable substance. Normally, intracellular structures are isolated from the outside world by a cell membrane. Therefore, it is difficult to efficiently introduce extracellular substances into cells. However, certain substances have cell membrane permeability and can be introduced into cells without being blocked by the cell membrane. By modifying with such a substance having cell membrane permeability (a cell membrane-permeable substance), it is possible to confer cell membrane permeability to a substance that does not have cell membrane permeability. Therefore, by modifying the peptide of the present invention with a cell membrane-permeable substance, the peptide of the present invention can be efficiently introduced into cells. Note that, as used herein, "cell membrane permeability" refers to the property of being able to permeate the mammalian cell membrane and enter the cytoplasm. Furthermore, a "cell membrane-permeable substance" refers to a substance that has "cell membrane permeability."

[0039] Examples of cell membrane-permeable substances include, but are not limited to, membrane-fusogenic liposomes and cell membrane-permeable peptides. For example, membrane-fusogenic liposomes fuse with the cell membrane and release their contents into the cell. For example, membrane-fusogenic liposomes are formed by modifying the liposome surface with a membrane-fusogenic substance. preparation Examples of fusogenic liposomes include pH-sensitive liposomes (Yuba E, et al., J. Control. Release, 149, 72-80 (2011)), Sendai virus fusogenic liposomes (WO97 / 016171), and liposomes modified with cell membrane-penetrating peptides. The peptides of the present invention may be encapsulated in fusogenic liposomes for efficient introduction into cells. In the present invention, encapsulation of a peptide in a fusogenic liposome is also included in the "modification" of the peptide.

[0040] Various natural and artificially synthesized cell membrane-permeable peptides have been reported (Joliot A. & Prochiantz A., Nat Cell Biol. 2004; 6: 189-96). Examples of cell membrane-permeable peptides include, but are not limited to, the following peptides:

[0041] Polyarginine (Matsushita et al., J. Neurosci.; 21(16), 6000-7(2003)); Tat / RKKRRQRRR (SEQ ID NO: 90) (Frankel et al., Cell 55(6), 1189-93 (1988)., Green & Loewenstein Cell 55, 1179-88 (1988)); Penetratin / RQIKIWFQNRRMKWKK (SEQ ID NO: 103) (Derossi et al., J. Biol. Chem. 269(14), 10444-50 (1994)); Buforin II / TRSSRAGLQFPVGRVHRLLRK (SEQ ID NO: 91) (Park et al., Proc. Natl Acad. Sci. USA 97(15), 8245-50(2000)); Transportan / GWTLNSAGYLLGKINLKALAALAKKIL (SEQ ID NO: 92) (Pooga et al., FASEB J. 12(1), 67-77(1998)); MAP (Model Amphipathic Peptide) / KLALKLALKALKAALKLA (SEQ ID NO: 93) (Oehlke et al., Biochim. Biophys. Acta. 1414(1-2), 127-39(1998)); K-FGF / AAVALLPAVLLALLAP (SEQ ID NO: 94) (Lin et al., J. Biol. Chem. 270(24), 14255-8 (1995)); Ku70 / VPMLK (SEQ ID NO: 95) (Sawada et al., Nature Cell Biol. 5(4), 352-7(2003)); Ku70 / PMLKE (SEQ ID NO: 96) (Sawada et al., Nature Cell Biol. 5(4), 352-7(2003)); Prion / MANLGYWLLALFVTMWTDVGLCKKRPKP (SEQ ID NO: 97) (Lundberg et al., Biochem. Biophys. Res. Commun. 299(1), 85-90(2002)); pVEC / LLIILRRRIRKQAHAHSK (SEQ ID NO: 98) (Elmquist et al., Exp. Cell Res. 269(2), 237-44(2001)); Pep-1 / KETWWETWWTEWSQPKKKRKV (SEQ ID NO: 99) (Morris et al., Nature Biotechnol. 19(2), 1173-6(2001)); SynB1 / RGGRLSYSRRRFSTSTGR (SEQ ID NO: 100) (Rousselle et al., Mol. Pharmacol. 57(4), 679-86(2000)); Pep-7 / SDLWEMMMVSLACQY (SEQ ID NO: 101) (Gao et al., Bioorg. Med. Chem. 10(12), 4057-65(2002)); and HN-1 / TSPLNIHNGQKL (SEQ ID NO: 102); (Hong & Clayman Cancer Res. 60(23), 6551-6(2000)).

[0042] The polyarginines listed above may be composed of any number of arginine residues. For example, they may be composed of 5 to 20 arginine residues. The number of arginine residues constituting the polyarginine is not particularly limited, as long as it does not interfere with the activity of the present peptide in inhibiting the binding between the BIG3 polypeptide and the PHB2 polypeptide.

[0043] Furthermore, it is known in the art to introduce various particularly useful amino acid mimetics or unnatural amino acids (e.g., by substitution, addition, or insertion) to enhance the in vivo stability of peptides. Examples of introduced amino acid mimetics or unnatural amino acids include, but are not limited to, β-amino acids, D-amino acids, and N-methylamino acids. Thus, the peptides of the present invention can incorporate such amino acid mimetics or unnatural amino acids to enhance their in vivo stability. Techniques for substituting azapeptides, in which the α-carbon of an amino acid is replaced with an amino group, and for substituting amide bonds in peptides with their equivalents (e.g., esters, sulfonamides, alkene isosteres, etc.) are also known in the art. Peptide stability can be confirmed, for example, using peptidases and various biological media, such as human plasma and serum (see, for example, Coos Verhoef et al. Eur. J. Drug Metab. Pharmacokin. 11(4): 291-302 (1986)).

[0044] Thus, the present invention provides peptides that contain the binding site in a PHB2 polypeptide with a BIG3 polypeptide and inhibit the binding between the PHB2 polypeptide and the BIG3 polypeptide, and that contain at least one amino acid mimetic or unnatural amino acid (e.g., β-amino acid, D-amino acid, or N-methyl amino acid). In a specific embodiment, the peptides of the present invention include an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 5, 36-41, 47-53, and 82-83 (PHB2 sequence-derived peptide Nos. 1, 5, 36-41, 47-53, and 82-83) in which one, two, or several amino acids have been substituted with the corresponding amino acid mimetic or unnatural amino acid (e.g., β-amino acid, D-amino acid, or N-methyl amino acid). The present invention also provides peptides that include the binding site in a PHB2 polypeptide with a BIG3 polypeptide and inhibit the binding between the PHB2 polypeptide and a BIG3 polypeptide, in which at least one amide bond has been replaced with an equivalent thereof (e.g., an ester, a sulfonamide, or an alkene isostere).

[0045] Cyclic and bridged peptides In certain embodiments, the peptides of the present invention may be cyclized, and cyclization can improve the stability of the peptides of the present invention. Methods for introducing a ring structure into the peptides of the present invention are well known. For example, a peptide can be cyclized by adding cysteines to the N- and C-termini of a linear peptide and forming a disulfide bond between these cysteines. Herein, a structure in which the side chains of two (a pair of) amino acid residues in the amino acid sequence constituting the peptide are crosslinked (stapled) can be referred to as a "stapled structure." A crosslinked peptide into which one or more stapled structures have been introduced is also referred to as a "stapled peptide." The positions of amino acid residues forming such intramolecular bridges are not limited to the N- and C-termini of the original linear peptide. The intramolecular bridges may be formed by amino acid residues present in the original linear peptide, or by amino acid residues introduced (e.g., by substitution, addition, or insertion) into the original linear peptide. The amino acid residues forming the intramolecular bridges are not limited to natural amino acids, but may also be amino acid mimetics or unnatural amino acids as described above. Furthermore, methods for cross-linking peptides are not limited to disulfide bond formation, but also include cross-linking between cysteine residues via fluorobenzene (e.g., using hexafluorobenzene or decafluorobiphenyl), thioether bond formation, ester bond formation, and hydrocarbon stapling techniques such as ring-closing olefin metathesis (e.g., as described in WO2017 / 126461).

[0046] Furthermore, methods for cyclizing peptides are not limited to the above-described methods of forming intramolecular crosslinks, and include the formation of an amide bond between the C-terminal amino acid residue and the N-terminal amino acid residue of the peptide. Peptides cyclized by these various methods are referred to herein as cyclic peptides, and include both cyclic peptides containing intramolecular crosslinks (i.e., crosslinked) (e.g., SEQ ID NOS: 25, 26, 123-124, and 126-127 in Figures 4 and 11) and cyclic peptides without intramolecular crosslinks (e.g., SEQ ID NOS: 125 and 128 in Figure 11). Thus, the present invention provides a cyclic peptide comprising a binding site in a PHB2 polypeptide to a BIG3 polypeptide and inhibiting the binding between the PHB2 polypeptide and the BIG3 polypeptide, the cyclic peptide being cyclized by at least one intramolecular bond. Examples of the intramolecular bond include, but are not limited to, a disulfide bond, a crosslink between cysteine residues via a fluorobenzene (e.g., using hexafluorobenzene or decafluorobiphenyl), a thioether bond, an ester bond, a thioester bond, a bond via a hydrocarbon chain (e.g., olefin, aryl, etc.), a bond via a heterocycle (e.g., triazole, oxazole, thiazole, etc.), an amide bond, and combinations thereof.

[0047] Such an intramolecular bond may be formed by amino acid residues at both ends of the linear peptide from which the cyclic peptide is derived, or by amino acid residues within the linear peptide. Furthermore, the intramolecular bond may be formed by amino acid residues in an amino acid sequence derived from a PHB2 polypeptide, or by amino acid residues introduced into the amino acid sequence (e.g., by substitution, addition, or insertion). Preferred examples of such cyclic peptides of the present invention include 11-21 aa and 76-88 aa cyclic PHB2 peptides (cyclic peptides consisting of SEQ ID NOs: 25, 106-108, 115-117, or 123-124; and SEQ ID NOs: 26, 110-112, 119-121, or 126-127, respectively). These exemplary cyclic peptides have been modified by adding two cysteine residues that form an intramolecular bond (intramolecular bridge) to linear peptides of 11-21 aa and 76-88 aa derived from the PHB2 sequence (SEQ ID NOS: 25-26, 106-108, 110-112, 115-117, 119-121, 123-124, 126-127); adding or substituting unnatural amino acids (SEQ ID NOS: 25-26, 106-108, 115-117, 123-124, 126-127); and The peptides were cyclized by the addition of multiple consecutive arginine residues (SEQ ID NOS: 25-26, 106-108, 110-112, 123-124, and 126-127), which form intramolecular bridges between the two cysteine residues (SEQ ID NOS: 25-26, 106-108, 110-112, 115-117, 119-121, 123-124, and 126-127), and by the formation of an amide bond between the C-terminal and N-terminal amino acid residues (SEQ ID NOS: 25-26, 123-124, and 126-127) (Figures 4C, 10A, and 11A). These exemplary cyclic peptides (especially the cyclic and cross-linked peptides) exhibited enhanced growth inhibitory effects compared to the original linear peptides (Figures 5A, 10C, and 11B), and the inhibitory effects were also shown to be long-lasting (Figure 6A).

[0048] The present invention also relates to a method for producing a cyclic peptide, comprising the steps of: (a) providing a linear peptide comprising a binding site in a PHB2 polypeptide for a BIG3 polypeptide, and inhibiting the binding of the PHB2 polypeptide to the BIG3 polypeptide; and (b) forming at least one intramolecular bond in said linear peptide, thereby cyclizing said linear peptide. The intramolecular bond is optionally selected from the group consisting of a disulfide bond, a crosslink between cysteine residues via a fluorobenzene (e.g., by using hexafluorobenzene or decafluorobiphenyl), a thioether bond, an ester bond, a thioester bond, a bond via a hydrocarbon chain (e.g., olefin, aryl, etc.), a bond via a heterocycle (e.g., triazole, oxazole, thiazole, etc.), an amide bond, and combinations thereof. The method optionally includes introducing (e.g., by substitution, addition, or insertion) at least one selected from the group consisting of a cysteine residue, an amino acid mimetic or an unnatural amino acid, and a series of arginine residues into the linear peptide.

[0049] The peptides of the present invention are characterized by their activity of inhibiting the binding between PHB2 polypeptide and BIG3 polypeptide. Whether or not a prepared peptide has the activity of inhibiting the binding between PHB2 polypeptide and BIG3 polypeptide can be confirmed by comparing the binding level between PHB2 polypeptide and BIG3 polypeptide in the presence and absence of the peptide. That is, if the binding level in the presence of the peptide is lower than the binding level in the absence of the peptide, the peptide can be determined to have "activity of inhibiting the binding between PHB2 polypeptide and BIG3 polypeptide."

[0050] The binding level between the PHB2 polypeptide and the BIG3 polypeptide can be measured using various known methods, such as immunoprecipitation using an anti-PHB2 antibody or an anti-BIG3 antibody, affinity chromatography, or a biosensor using surface plasmon resonance.

[0051] Specifically, for example, PHB2 polypeptide and BIG3 polypeptide are incubated in the presence and absence of a test peptide. The reaction solution is then immunoprecipitated with an anti-PHB2 antibody or an anti-BIG3 antibody, and the immunoprecipitate is subjected to Western blot analysis. The binding level between PHB2 polypeptide and BIG3 polypeptide can be confirmed by detecting at least one of the levels of BIG3 polypeptide immunoprecipitated with the anti-PHB2 antibody and the level of PHB2 polypeptide immunoprecipitated with the anti-BIG3 antibody. The PHB2 polypeptide and BIG3 polypeptide used here can be prepared by known genetic engineering techniques. preparation Alternatively, cell lysates of cells producing these polypeptides can be used. As cells producing these polypeptides, cell lines such as those described in the Examples of the present specification can be used.

[0052] Alternatively, the method described in the Examples section of the present specification can be used. Specifically, estrogen receptor-positive cells are cultured in the presence and absence of a test peptide. The cells are then lysed in an appropriate lysis buffer, and the cell lysate may be used to carry out immunoprecipitation and Western blot analysis in the same manner as described above. A peptide confirmed to have "activity to inhibit the binding between PHB2 polypeptide and BIG3 polypeptide" by any of the above-mentioned methods is determined to have "activity to inhibit the binding between PHB2 polypeptide and BIG3 polypeptide."

[0053] Furthermore, the peptide of the present invention may have either or both of the following properties (i) and (ii) as preferred properties: (i) promoting nuclear translocation of PHB2 polypeptide in estrogen receptor-positive cells expressing BIG3 polypeptide; and (ii) In estrogen receptor-positive cells expressing the BIG3 polypeptide, it promotes the binding of the PHB2 polypeptide to estrogen receptors present in the nucleus and / or cell membrane.

[0054] Whether the peptide of the present invention has the above-mentioned properties can be confirmed by comparing (i) the nuclear translocation level of the PHB2 polypeptide and / or (ii) the binding level of the estrogen receptor to the PHB2 polypeptide in the presence and absence of the peptide of the present invention. That is, if the level in the presence of the peptide of the present invention is higher than the level in the absence of the peptide of the present invention, the peptide of the present invention can be determined to have the above-mentioned properties (i) and / or (ii).

[0055] Specific methods for determining the presence or absence of properties (i) and / or (ii) above can be methods well known to those skilled in the art. Specifically, when examining property (i) above, estrogen receptor-positive cells are stimulated with estradiol for 24 hours with or without the addition of the peptide of the present invention. The cells are then fractionated by density centrifugation, and the PHB2 polypeptide present in the nuclear fraction is detected by Western blot analysis or the like. If the level of PHB2 polypeptide detected in the nuclear fraction is increased when the peptide of the present invention is added compared to when it is not added, the peptide of the present invention is determined to have property (i) above. The level of PHB2 polypeptide present in the nucleus can also be detected by immunocytochemical staining.

[0056] To examine the above property (ii), estrogen receptor-positive cells are stimulated with estradiol for 24 hours with or without the addition of the peptide of the present invention. The cells are then fractionated by density centrifugation, and the cytoplasmic and nuclear fractions are immunoprecipitated with an anti-estrogen receptor antibody or an anti-PHB2 antibody. The immunoprecipitates are then subjected to Western blot analysis. If the results show an increased level of binding between the estrogen receptor and the PHB2 polypeptide in the cytoplasmic and / or nuclear fractions when the peptide of the present invention is added compared to when it is not added, the peptide of the present invention is determined to have the above property (ii).

[0057] The peptides of the present invention can be produced using methods well known to those skilled in the art. For example, the peptides of the present invention can be obtained by chemical synthesis based on the amino acid sequence. Methods for chemically synthesizing peptides are known, and those skilled in the art can chemically synthesize the peptides of the present invention based on the amino acid sequence selected for the peptide of the present invention. Methods for chemically synthesizing peptides are described in, for example, the following documents: (i)Peptide Synthesis, Interscience, New York, 1966; (ii)The Proteins, Vol. 2, Academic Press, New York, 1976; (iii) Peptide Synthesis, Maruzen, 1975; (iv) Fundamentals and Experiments of Peptide Synthesis, Maruzen, 1985; (v) Pharmaceutical Development, Vol. 14 (Peptide Synthesis), Hirokawa Shoten, 1991; (vi) WO99 / 67288; and (vii) Barany G. & Merrifield RB, Peptides Vol. 2, "Solid Phase Peptide Synthesis", Academic Press, New York, 1980, 100-118.

[0058] Alternatively, the peptides of the present invention can be obtained by genetic engineering techniques (e.g., Morrison J, J Bacteriology, 132(1): 349-51 (1977); Clark-Curtiss & Curtiss, Methods in Enzymology (eds. Wu et al.), 101: 347-62 (1983)). For example, a polynucleotide encoding the peptide of the present invention is inserted into an appropriate expression vector, which is then introduced into an appropriate host cell to prepare a transformed cell. The host cell is then cultured to produce the peptide of the present invention, and the cell extract is then used for the preparation of a transformant. preparation The peptides of the present invention can be purified from the cell extract using standard protein purification techniques. For example, the peptides can be purified by appropriately selecting and combining column chromatography, filter filtration, ultrafiltration, salting out, solvent precipitation, solvent extraction, distillation, immunoprecipitation, SDS-polyacrylamide gel electrophoresis, isoelectric focusing, dialysis, and recrystallization. The peptides of the present invention can also be synthesized using an in vitro translation system in which the elements necessary for protein synthesis are reconstituted in vitro.

[0059] When genetic engineering techniques are used, the peptide of the present invention can also be expressed as a fusion protein with another peptide. A polynucleotide encoding the peptide of the present invention and a polynucleotide encoding the other peptide are ligated in-frame, inserted into an appropriate expression vector, and then introduced into an appropriate host cell to produce a transformed cell. preparation The host cells are then cultured to produce a fusion protein of the peptide of the present invention and another peptide, and the cell extract is then subjected to preparationThe fusion protein can be purified from the cell extract by capturing the fusion protein by affinity chromatography using a column bound to a substance having affinity for the fusion protein. Furthermore, if the peptide of the present invention is linked to another peptide via a linker sequence that can be cleaved by enzymes such as peptidases, proteases, and proteasomes, the fusion protein captured on the column can be treated with these enzymes to separate the peptide from the column. Other peptides that can be used to form fusion proteins include, but are not limited to, the following: FLAG(Hopp et al., Bio / Technology 6, 1204-10(1988)); 6xHis or 10xHis consisting of histidine (His) residues; influenza hemagglutinin (HA); human c-myc fragment, VSV-GP fragment; p18 HIV fragment; T7 tag; HSV tag; E tag; SV40 T antigen fragment; lcktag; α-tubulin fragment; B-tag; Protein C fragment; GST (glutathione-S-transferase); HA (influenza hemagglutinin); immunoglobulin constant region; β-galactosidase; and; MBP (maltose-binding protein).

[0060] 2. Polynucleotides encoding the peptides of the present invention, vectors, and host cells The present invention also provides polynucleotides encoding the peptides of the present invention. The present invention also provides vectors containing the polynucleotides and host cells containing the vectors. The polynucleotides, vectors, and host cells can be used to produce the peptides of the present invention.

[0061] The polynucleotides of the present invention can be prepared by methods known to those skilled in the art. For example, the polynucleotides of the present invention can be synthesized using solid-phase techniques such as those described in Beaucage SL & Iyer RP, Tetrahedron, 48: 2223-311 (1992); Matthes et al., EMBO J, 3(4): 801-5 (1984). Alternatively, the polynucleotides of the present invention can be synthesized using genetic engineering techniques. preparation For example, primers are prepared based on the partial nucleotide sequence of the PHB2 gene (SEQ ID NO: 27) encoding the amino acid sequence selected as the peptide of the present invention, and reverse transcription PCR is performed using mRNA extracted from cells expressing the PHB2 polypeptide as a template. This allows the polynucleotide of the present invention to be amplified.

[0062] The polynucleotide of the present invention can be inserted into an appropriate expression vector and introduced into an appropriate host cell to produce the peptide of the present invention in the host cell.

[0063] For example, when E. coli is selected as the host cell and a vector is to be amplified in large quantities within E. coli (e.g., JM109, DH5α, HB101, or XL1Blue), the vector must have an "ori" for amplification within E. coli and a marker gene for selecting transformed E. coli (e.g., a drug resistance gene selectable with drugs such as ampicillin, tetracycline, kanamycin, or chloramphenicol). For example, M13 series vectors, pUC series vectors, pBR322, pBluescript, and pCR-Script can be used. When using vectors to produce the peptides of the present invention, expression vectors are particularly useful. For example, expression vectors intended for expression in E. coli must have the above-mentioned characteristics for amplification within E. coli. When E. coli such as JM109, DH5α, HB101, or XL1Blue is used as a host cell, the vector must have a promoter capable of efficiently expressing the desired gene in E. coli, such as the lacZ promoter (Ward et al., Nature 341(6242): 544-6 (1989); FASEB J 6(7): 2422-7 (1992)), the araB promoter (Better et al., Science 240(4855): 1041-3 (1988)), or the T7 promoter. Furthermore, the vector may also contain a signal sequence for polypeptide secretion. An example of a signal sequence that directs polypeptide secretion into the periplasm of E. coli is the pelB signal sequence (Lei et al., J Bacteriol 169(9): 4379-83 (1987)). Means for introducing vectors into target host cells include, for example, the calcium chloride method and electroporation.

[0064] In addition to E. coli, for example, expression vectors derived from mammalian cells (e.g., pcDNA3 (Invitrogen) and pEGF-BOS (Mizushima S., Nucleic Acids Res 18(17): 5322 (1990)), pEF, pCDM8), expression vectors derived from insect cells (e.g., "Bac-to-BAC Baculovirus Expression System" (GIBCO BRL), pBacPAK8), expression vectors derived from plants (e.g., pMH1, pMH2), expression vectors derived from animal viruses (e.g., pHSV, pMV, pAdexLcw), expression vectors derived from retroviruses (e.g., pZIpneo), expression vectors derived from yeast (e.g., "Pichia Expression Kit" (Invitrogen), pNV11, SP-Q01), and expression vectors derived from Bacillus subtilis (e.g., pPL608, pKTH50) can also be used.

[0065] To express a vector in animal cells such as CHO cells, COS cells, or NIH3T3 cells, the vector must contain a promoter necessary for expression in these cells, such as the SV40 promoter (Mulligan et al., Nature 277(5692): 108-14 (1979)), the MMLV-LTR promoter, the EF1α promoter (Mizushima et al., Nucleic Acids Res 18(17): 5322 (1990)), or the CMV promoter. It is also preferable for the vector to contain a marker gene for selecting transformants (e.g., a drug resistance gene selectable with a drug, such as neomycin or G418). Examples of known vectors having these characteristics include pMAM, pDR2, pBK-RSV, pBK-CMV, pOPRSV, and pOP13.

[0066] Alternatively, the polynucleotide of the present invention may be inserted into an appropriate vector and introduced into the target cell to produce the peptide of the present invention in the target cell. The peptide of the present invention produced in the target cell inhibits the binding of the PHB2 polypeptide to the BIG3 polypeptide, thereby inducing suppression of cell proliferation of the target cell. In this case, the vector into which the polynucleotide of the present invention is inserted may be a vector for stably inserting the polynucleotide of the present invention into the genome of the target cell (see, for example, Thomas KR & Capecchi MR, Cell, 51(3): 503-12 (1987) for a description of homologous recombination cassette vectors). See, for example, Wolff et al., Science, 247: 1465-8 (1990); U.S. Patent No. 5,580,895; U.S. Patent No. 5,589,466; U.S. Patent No. 5,804,566; U.S. Patent No. 5,739,118; U.S. Patent No. 5,736,524; U.S. Patent No. 5,679,647; and International Publication No. WO 98 / 04720.

[0067] The polynucleotides of the present invention can also be inserted into expression vectors, such as viral or bacterial vectors. Examples of expression vectors include attenuated viral hosts such as cowpox or fowlpox (see, for example, U.S. Pat. No. 4,722,858). Another example of a vector that can be used is Bacille Calmette-Guerin (BCG) (Stover et al., Nature, 351(6326): 456-60(1991)). Other examples include adenovirus and adeno-associated virus vectors, retrovirus vectors, Salmonella typhi vectors, and detoxified anthrax toxin vectors (Shata et al., Mol Med Today, 6(2): 66-71(2000); Shedlock et al., J Leukoc Biol, 68(6): 793-806(2000); and Hipp et al., In Vivo, 14(5): 571-85(2000)).

[0068] The peptides of the present invention also include those in which either or both of the N-terminal and C-terminal amino acid residues have been modified. The type of modification is not particularly limited, but it is preferable that it does not affect the affinity for BIG3. Preferred examples of modifications include acetylation of the N-terminal amino acid residue, amidation of the C-terminal amino acid residue, and addition of tag peptides such as HA tag and FLAG tag.

[0069] The peptides of the present invention are not limited to those composed of L-amino acids, but may also contain one or more D-amino acids. The ratio of L-amino acids to D-amino acids in the peptide is not particularly limited, and may be any of the following: all amino acid residues are L-type (hereinafter referred to as "L-peptide"), all amino acid residues are D-type (hereinafter referred to as "D-peptide"), or only amino acid residues at specific positions are D-type. One preferred embodiment of the peptides of the present invention includes any of the above-mentioned peptides of the present invention in which all amino acid residues are replaced with D-type amino acid residues. Another preferred embodiment of the peptides of the present invention includes peptides in which amino acid residues at specific positions important for binding to BIG3 are replaced with the corresponding D-type amino acid residues. Examples of such positions include the positions corresponding to glycine 15, glycine 18, and aspartic acid 82 in the amino acid sequence of SEQ ID NO: 28.

[0070] Furthermore, the peptides of the present invention may be retroinverse peptides of any of the above-mentioned peptides of the present invention. In retroinverse peptides, the amino acid sequence is reversed from that of the original peptide, and all amino acid residues are replaced with D-amino acid residues. In other words, retroinverse peptides are D-peptides having an amino acid sequence reversed from that of the original peptide. Therefore, peptides that are retroinverse peptides of any of the above-mentioned peptides of the present invention are also preferred embodiments of the peptides of the present invention.

[0071] The peptides of the present invention may also be in the form of a salt. The salt form is not particularly limited, but a pharmaceutically acceptable salt is preferred. As used herein, the term "pharmaceutically acceptable salt" refers to a salt that retains the pharmacological or pharmaceutical effectiveness and properties of the peptide. Preferred examples of salts include salts with alkali metals (lithium, potassium, sodium, etc.), salts with alkaline earth metals (calcium, magnesium, etc.), salts with other metals (copper, iron, zinc, manganese, etc.), salts with organic bases, salts with amines, salts with organic acids (acetic acid, formic acid, propionic acid, fumaric acid, maleic acid, succinic acid, tartaric acid, citric acid, malic acid, oxalic acid, benzoic acid, methanesulfonic acid, etc.), and salts with inorganic acids (hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid, nitric acid, etc.). These salts can be prepared according to known methods.

[0072] 3. Pharmaceutical compositions containing the peptides or polynucleotides of the present invention and uses thereof The present invention also provides a pharmaceutical composition comprising the peptide of the present invention or a salt thereof, or a polynucleotide encoding the peptide of the present invention, together with a pharmaceutically acceptable carrier. The peptide of the present invention inhibits the binding between the PHB2 polypeptide and the BIG3 polypeptide, thereby inducing the suppression of estrogen receptor activation by the PHB2 polypeptide, thereby leading to the suppression of cell proliferation in estrogen receptor-positive cells. Therefore, the pharmaceutical composition of the present invention is useful for either or both of the treatment and prevention of cell proliferative disorders caused by estrogen receptor activation. Such cell proliferative disorders include, for example, cancer.

[0073] Among cancers, breast cancer, in particular, is known to be closely related to estrogen receptor activation. BIG3 polypeptide is a novel estrogen receptor activation regulator that has been confirmed to be frequently expressed in many breast cancer specimens and breast cancer cells, while its expression is rarely observed in normal tissues (Kim JW, Akiyama M, Park JH, et al. Cancer Sci.; 100(8):1468-78(2009)). Therefore, in breast cancer, expression of BIG3 polypeptide inhibits the function of PHB2 polypeptide in suppressing estrogen receptor activation, which is thought to result in promotion of breast cancer cell proliferation. Therefore, the pharmaceutical composition of the present invention is particularly suitable for either or both of the treatment and prevention of breast cancer. Furthermore, it is particularly useful for breast cancers that are estrogen receptor-positive and express BIG3 polypeptide. However, the pharmaceutical composition of the present invention is not limited to use in breast cancer; it can be used for any cancer that is estrogen receptor-positive and expresses BIG3 polypeptide. Examples of estrogen receptor-positive cancers other than breast cancer include, but are not limited to, uterine cancer, ovarian cancer, and prostate cancer (Nelles JL, et al., Expert Rev Endocrinol Metab.; 6(3):437-51(2011)), and lung cancer (particularly non-small cell lung cancer) (Stabile LP, et al., Cancer Res.; 65(4):1459-70(2005); Marquez-Garban DC, et al., Steroids.; 72(2):135-43(2007)). It is preferable that the cancer to which the pharmaceutical composition of the present invention is applied expresses BIG3 and PHB2, but estrogen receptor-positive cancers generally express BIG3 and PHB2. Whether a cancer is estrogen receptor-positive can be confirmed by known methods such as ELISA or immunohistochemical staining.

[0074] Furthermore, the peptides of the present invention also exhibited excellent cell growth inhibitory effects in triple-negative breast cancer cells, which are estrogen receptor-negative breast cancer cells (Figure 9). Triple-negative generally refers to breast cancer cells lacking expression of HER2, estrogen receptors, and progesterone receptors, which are major targets of drug therapy. Therefore, triple-negative breast cancers are usually resistant to drug therapy. Therefore, the pharmaceutical compositions of the present invention can be used for either or both of the treatment and prevention of estrogen receptor-negative breast cancer and are also useful as pharmaceutical compositions for administration to patients with such treatment-resistant breast cancer. That is, the present invention provides a pharmaceutical composition comprising the peptide of the present invention, the pharmaceutical composition being for administration to a patient with drug therapy-resistant breast cancer. The present invention also relates to the peptide of the present invention for use in either or both of treating and preventing a patient with drug therapy-resistant breast cancer. Furthermore, the present invention relates to the use of the peptide of the present invention in the manufacture of a pharmaceutical composition for either or both of treating and preventing a patient with drug therapy-resistant breast cancer. Alternatively, the present invention also provides a method for either or both of treating and preventing breast cancer, comprising the steps of selecting a patient with drug therapy-resistant breast cancer and administering a peptide of the present invention to the selected patient. Chemotherapy-resistant breast cancer patients can be identified by monitoring the treatment outcomes after standard drug therapy. Specifically, therapy resistance can be determined when no clear regression of lesions is observed after treatment. The state in which lesion growth is prevented is also considered to be tumor regression. Patients with triple-negative breast cancer, as mentioned above, are also considered to be resistant to chemotherapy. Triple-negative breast cancer is characterized by the lack of expression of estrogen receptors and progesterone receptors in addition to HER2. These chemotherapy-resistant markers can be quantitatively evaluated by immunohistochemistry or gene expression analysis. For example, a marker is considered negative if its expression level is similar to that of a negative control. A therapy-resistant cancer cell line lacking expression of these markers can be used as a negative control.

[0075] The pharmaceutical composition of the present invention can be produced by blending the peptide of the present invention or its salt with a pharmaceutically acceptable carrier using known formulation techniques. As used herein, the term "pharmaceutically acceptable carrier" refers to an inactive substance used as a diluent or solvent for a drug. The pharmaceutically acceptable carrier used in the pharmaceutical composition of the present invention may be appropriately selected from carriers used in general pharmaceuticals depending on the dosage form of the pharmaceutical composition to be prepared.

[0076] The dosage form of the pharmaceutical composition of the present invention is not particularly limited, and can be appropriately selected from dosage forms commonly used for pharmaceuticals such as liquids, tablets, elixirs, capsules, granules, powders, etc. Furthermore, additives such as excipients, stabilizers, suspensions, preservatives, surfactants, solubilizers, pH adjusters, and aggregation inhibitors can be added as appropriate depending on the selected dosage form.

[0077] The pharmaceutical composition of the present invention contains, as an active ingredient, a pharmaceutically effective amount of the peptide of the present invention or a salt thereof, or a polynucleotide encoding the peptide. A "pharmaceutically effective amount" refers to an amount sufficient for the pharmaceutical composition of the present invention to achieve its intended purpose. For example, when the pharmaceutical composition of the present invention is a pharmaceutical composition for either or both of the treatment and prevention of cancer, an example of a pharmaceutically effective amount would be an amount that, when administered to a patient, induces a reduction in the rate of cancer growth, a reduction in metastatic potential, an extension of survival time, a reduction or delay in the onset of cancer, or an alleviation of various clinical symptoms associated with cancer. The reduction in the rate of cancer growth can be, for example, about 5% or more compared to the rate when the pharmaceutical composition of the present invention is not administered. Preferably, the reduction in the rate of cancer growth can be about 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 75% or more, 80% or more, 90% or more, or 100% or more.

[0078] The pharmaceutically effective amount can be appropriately selected depending on the dosage form of the pharmaceutical composition, the administration interval, the age, sex, weight, body surface area, type of disease of the subject, etc. Examples of the content of the peptide of the present invention or a salt thereof in the pharmaceutical composition of the present invention include, but are not limited to, 0.001 mg to 1000 mg, 0.01 mg to 100 mg, 0.1 mg to 30 mg, and 0.1 mg to 10 mg.

[0079] The pharmaceutical composition of the present invention may also optionally contain other drugs. Examples of other drugs include anti-inflammatory agents, analgesics, antipyretics, other cancer therapeutic agents, etc. The other cancer therapeutic agents that can be used in the pharmaceutical composition of the present invention are not particularly limited. However, when used for estrogen-positive cancers, examples include hormone therapy agents such as selective ERα modulators (e.g., tamoxifen and raloxifene), ERα downregulators (e.g., fulvestrant), aromatase inhibitors, LH-RH agonist preparations, and progesterone preparations. These drugs may also be formulated in the form of prodrugs or pharmaceutically acceptable salts.

[0080] The pharmaceutical composition of the present invention can be administered to a subject by selecting an appropriate administration route depending on the dosage form. The administration route is not particularly limited, but examples include oral administration, as well as intradermal, subcutaneous, intramuscular, intraosseous, peritoneal, and intravenous injection. Systemic administration and local administration near the diseased site are both acceptable, with local administration being preferred. More specifically, the pharmaceutical composition of the present invention can be administered to cancer tissue or its vicinity by injection or other means. Alternatively, the pharmaceutical composition of the present invention can be administered surgically to cancer tissue or its vicinity. The pharmaceutical composition of the present invention can also be formulated into a sustained-release formulation by blending with an appropriate carrier.

[0081] The administration interval of the pharmaceutical composition of the present invention can also be appropriately selected depending on the age, sex, weight, body surface area, type of disease, etc. of the subject to be administered, as well as the dosage form and administration route of the pharmaceutical composition, etc. Examples of administration intervals include, but are not limited to, every day, every 4 days, every 7 days, etc.

[0082] The dose of the pharmaceutical composition of the present invention can also be appropriately selected depending on the age, sex, weight, body surface area, type of disease, etc. of the subject to be administered, as well as the dosage form and administration route of the pharmaceutical composition, etc. Examples of the dose of the peptide of the present invention or a salt thereof include, but are not limited to, 0.001 to 1000 mg / kg / day, 0.005 to 500 mg / kg / day, and 0.01 to 250 mg / kg / day.

[0083] The pharmaceutical composition of the present invention may be used in combination with other pharmaceuticals depending on the condition of the subject. The pharmaceuticals to be used in combination are not particularly limited, but when used for estrogen receptor-positive cancer, examples of the pharmaceuticals include hormone therapy agents such as selective ERα modulators (e.g., tamoxifen and raloxifene), ERα downregulators (e.g., fulvestrant), aromatase inhibitors, LH-RH agonist preparations, and progesterone preparations. Among these hormone therapy agents, tamoxifen and fulvestrant are particularly preferred.

[0084] When the pharmaceutical composition of the present invention is used for cancer treatment, it may be possible to examine whether the cancer to be treated expresses BIG3 and PHB2 before administration. Whether the cancer to be treated expresses BIG3 and PHB2 can be confirmed by detecting the transcription products or translation products of these genes in a sample collected from the subject. Known detection methods can be used, such as methods for detecting transcription products using probes or PCR (e.g., cDNA microarray, Northern blotting, RT-PCR, etc.), and methods for detecting translation products using antibodies, etc. (e.g., Western blotting, immunostaining, etc.).

[0085] The present invention also provides a product or kit containing the pharmaceutical composition of the present invention. The product or kit of the present invention may include a container containing the pharmaceutical composition of the present invention. Examples of suitable containers include, but are not limited to, bottles, vials, and test tubes. The container may be made of various materials, such as glass or plastic. A label may be attached to the container, and the label may describe the disease or disease state for which the pharmaceutical composition of the present invention should be used. The label may also indicate instructions for administration, etc.

[0086] In addition to the container containing the pharmaceutical composition of the invention, the article of manufacture or kit of the invention may optionally further comprise a second container containing a pharmaceutically acceptable diluent. The article of manufacture or kit of the invention may further include other materials desirable from a commercial and user standpoint, such as other buffers, diluents, filters, needles, syringes, and package inserts with instructions for use.

[0087] The pharmaceutical compositions of the present invention may also, if desired, be presented in a pack or dispenser device which may contain one or more unit dosage forms containing the active ingredient. The pack may, for example, comprise metal or plastic foil, such as a blister pack. The pack or dispenser device may be accompanied by instructions for administration.

[0088] In another aspect, the present invention also provides the following uses and methods: (a) use of the peptide of the present invention or a salt thereof, or a polynucleotide encoding the peptide, in the manufacture of a pharmaceutical composition for either or both of the treatment and prevention of cancer; (b) the peptide of the present invention or a salt thereof, or a polynucleotide encoding the peptide, for use in either or both of the treatment and prevention of cancer; (c) a method or process for producing a pharmaceutical composition for either or both of the treatment and prevention of cancer, the method or process comprising a step of formulating the peptide of the present invention or a salt thereof, or a polynucleotide encoding the peptide, and a pharmaceutically acceptable carrier; (d) a method or process for producing a pharmaceutical composition for either or both of treating and preventing cancer, the method or process comprising the step of mixing the peptide of the present invention or a salt thereof, or a polynucleotide encoding the peptide, with a pharmaceutically acceptable carrier; and (e) A method for either or both of treating and preventing cancer, which comprises administering to a subject the peptide of the present invention or a salt thereof, or a polynucleotide encoding the peptide. In the above uses and methods, the cancer is preferably a BIG3-positive cancer, and may be an estrogen receptor-positive cancer or an estrogen receptor-negative cancer (e.g., triple-negative breast cancer). A preferred example of such a cancer is breast cancer.

[0089] The present invention will now be described in more detail with reference to examples. However, the following materials, methods and examples, while useful for assisting those skilled in the art in making and using certain aspects of the present invention, are merely intended to illustrate aspects of the present invention and are therefore not intended to limit the scope of the present invention in any way. Those skilled in the art can use methods and materials similar or equivalent to those described herein in the practice or testing of the present invention.

[0090] All prior art documents cited in this specification are hereby incorporated by reference. [Example]

[0091] [Example 1] Effect on estrogen-dependent breast cancer 1. Materials and Methods Cell lines and culture conditions The human breast cancer cell line MCF-7 was purchased from the JCRB Cell Bank (Osaka, Japan) and maintained in MEM (Thermo Fisher Scientific) supplemented with 10% FBS (Nichirei Biosciences, Tokyo, Japan), 1% Antibiotic / Antimycotic solution (Thermo Fisher Scientific, Waltham, MA, USA), 0.1 mM NEAA (Thermo Fisher Scientific), 1 mM sodium pyruvate (Thermo Fisher Scientific), and 10 μg / mL insulin (Sigma, St. Louis, MO, USA) at 37°C with 5% CO . The normal mammary epithelial cell line MCF-10A was purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA) and maintained in MEBM (Lonza) supplemented with the SingleQuots kit (BPE, hydrocortisone, hEGF, insulin, gentamicin / amphotericin B) (Lonza, Walkersville, MD, USA) and 100 ng / mL cholera toxin at 37°C in 5% CO .

[0092] Cell proliferation assay MCF-7 proliferation assays were performed in 48-well plates (2 × 10 4 MCF-10A cells were seeded in 200 μL of 48-well plates (2 × 10 cells / 200 μL). The next day, the medium was changed to phenol red-free DMEM / F12 (Thermo Fisher Scientific) supplemented with 10% FBS, 1% Antibiotic / Antimycotic solution, 0.1 mM NEAA, 1 mM sodium pyruvate, and 10 μg / mL insulin. After 24 hours, the cells were treated with 10 nM 17β-estradiol (estrogen, Sigma) alone or with 10 nM estrogen and a peptide derived from the PHB2 sequence. MCF-10A proliferation assays were performed in 48-well plates (2 × 10 cells / 200 μL). 4The cells were seeded in 200 μL of PBS and 24 hours later, a peptide derived from the PHB2 sequence was added. Proliferation assays were performed using Cell Counting Kit-8 (CCK-8, Dojindo, Kumamoto, Japan). Data are shown as the mean ± standard deviation of three independent experiments.

[0093] Antibodies and immunoblot analysis For immunoblotting analysis, the membranes containing the blotted proteins after SDS-PAGE were blocked with 4% Block Ace solution (Dainippon Pharmaceutical, Osaka, Japan) for 3 hours, then incubated with antibodies against BIG3 (1:1,000), PHB2 (1:1,000, Abcam, Cambridge, UK), and phosphorylated PHB2 (Ser39, Scrum, Tokyo, Japan) for 12 hours. Subsequently, the blots were incubated with HRP-conjugated secondary antibodies (anti-rat IgG-HRP, 1:5,000 for BIG3; anti-rabbit IgG-HRP, 1:1,000 for PHB2 and phosphorylated PHB2, Santa Cruz Biotechnology, Dallas, TX, USA) for 1 hour. The blots were developed using an enhanced chemiluminescence (ECL) system (GE Healthcare, Buckinghamshire, UK) and scanned using an Image Reader LAS-3000 mini (Fujifilm, Tokyo, Japan).

[0094] Immunoprecipitation Immunoprecipitation was performed using MCF-7 cells in a 10 cm dish (2 × 10 6MCF-7 cells were seeded at 1000kJ / 1000kcal (1000kJ / 1000kcal) and treated with 10 nM estrogen alone or 10 nM estrogen and a peptide derived from the PHB2 sequence, as in the cell proliferation assay. For immunoprecipitation analysis, cell lysates were lysed in cell lysis buffer (50 mM Tris-HCl; pH 8.0, 150 mM NaCl, 0.1% NP-40, and 0.5% CHAPS, 0.1% protease inhibitor cocktail III) and precleared with rat IgG antibody and rec-Protein G Sepharose 4B (Thermo Fisher Scientific) for 3 hours at 4°C. The supernatant was then reacted with 5 μg of antibody against BIG3 for 12 hours at 4°C. The antigen-antibody complexes were then precipitated with rec-Protein G Sepharose 4B for 1 hour at 4°C. The immunoprecipitated protein complexes were washed four times with cell lysis buffer and subjected to SDS-PAGE and immunoblot analysis.

[0095] Peptide synthesis All peptides were synthesized by Fmoc solid-phase synthesis. NovaSyn TGR resin (0.25 mmol amine / g) or Rink Amide AM resin (0.62 mmol amine / g) were used, and manual Fmoc solid-phase synthesis was used. The Fmoc group was removed by treatment with 20% (v / v) piperidine / DMF solution at room temperature for 10 minutes. The resin was washed 5 to 10 times with DMF, and then 3 equivalents of the Fmoc amino acid were added to the reaction mixture in DMF with N,N-diisopropyl ether. Carbodiimide (DIPCDI, 3.0 equivalents) and 1-hydroxybenzotriazole hydrate (HOBt HO, 3.3 equivalents) or N,N-diisopropylethylamine (DIPEA, 3.0 equivalents) and N,N,N,N-tetramethyl-O-( BenzotriazoleCoupling was performed at room temperature for 90 minutes using (-1-yl)uronium hexafluorophosphate (HBTU, 2.9 equivalents). After washing with DMF, methanol, and ethanol and drying, 100 mg of the protected peptide resin was treated with 5 mL of a cocktail of TFA: thioanisole: m-cresol: 1,2-ethanedithiol: water (80:5:5:5:5) at room temperature for 90 minutes. After concentrating the TFA under a nitrogen stream, the residue was precipitated with ether. The precipitate was washed with ether, dissolved in an appropriate aqueous solvent, and purified by HPLC.

[0096] 2.Results Screening of peptides derived from PHB2 sequence We investigated the inhibitory effects of 20 peptides derived from the PHB2 protein sequence (Figure 1A) on estrogen (E2)-dependent proliferation of MCF-7 cells (treated at 10 μM for 24 hours). While MCF-7 cells showed significant proliferation enhancement upon E2 stimulation, treatment with PHB2-derived peptides No. 1 (11-22 aa: SEQ ID NO: 1) and No. 5 (76-90 aa: SEQ ID NO: 5) significantly inhibited E2-dependent proliferation by approximately 50% (No. 1: 58% inhibition, No. 5: 49% inhibition). These peptides closely matched the predicted BIG3 binding regions (bold amino acids) predicted by in silico analysis. Furthermore, peptides No. 2 (42-50 aa: SEQ ID NO: 2) and No. 3 (38-50 aa: SEQ ID NO: 3) also inhibited E2-dependent proliferation by 22% and 23%, respectively. However, each PHB2 sequence-derived peptide had a lower E2-dependent proliferation inhibitory effect compared to ERAP, suggesting the possibility that there are multiple BIG3-binding regions on the PHB2 side. Next, we synthesized two peptides derived from the PHB2 sequence, primarily No. 1 (11-22aa) and No. 5 (76-90aa), (Fig. 1B, C). We examined the effects of each peptide on E2-dependent proliferation after 24 hours of treatment at 10 μM. The PHB2 (11-22aa) peptide exhibited the highest inhibitory effect (63%) near No. 1 (11-22aa), with the inhibitory effect gradually decreasing (Fig. 1B). The PHB2 (76-90aa) peptide also exhibited a high inhibitory effect (51%) near No. 5 (76-90aa), and No. 50 (75-89aa) had a similar inhibitory effect, but the inhibitory effect decreased around these peptides (Fig. 1C). What these data have in common is that they contain amino acids 11-21aa and 76-88aaa, which showed high scores in in silico analysis as predicted binding sites for BIG3, suggesting that PHB2 has two binding sites for BIG3. We therefore investigated the inhibitory effects of peptides No. 1 (11-22aa) and No. 5 (76-90aa), and the combination of peptides No. 5 (76-90aa) and No. 1 (11-22aa), on E2-dependent proliferation in MCF-7 cells. The results showed that the No. 1 (11-22aa) peptide alone inhibited E2-dependent proliferation by 65%, almost completely, whereas the combination of peptide No. 5 (76-90aa) and peptide No. 50 (75-89aa) with peptide No. 5 (76-90aa) and peptide No. 50 (75-89aa) showed almost complete inhibition, with 100% and 97%, respectively (Fig. 1D). Similarly, treatment with No. 5 (76-90aa) alone showed a 55% inhibition rate, while combination with PHB2 peptides centered on No. 1 (11-22aa) almost completely inhibited the binding. When combined with peptides consisting of the 5-26aa region (Nos. 36, 37, 38, 39, and 40), the inhibition rate reached over 90% (Fig. 1E). These results suggest that the PHB2 regions of No. 1 (11-22aa) and No. 5 (76-90aa) are important for binding to BIG3, and that the development of dominant-negative peptides taking these regions into consideration is necessary.

[0097] Inhibitory effect of a peptide derived from PHB2 sequence on E2-dependent proliferation Next, we investigated the inhibitory effect of two peptides (No. 1 and No. 5) derived from the PHB2 sequence on E2-dependent proliferation of MCF-7 cells. The combined use of No. 1 and No. 5 significantly enhanced the inhibitory effect, achieving 88% inhibition compared with either peptide alone (Fig. 2A). Furthermore, the combined use of No. 5 and No. 6 (86-100 aa, approximately 10% inhibition), which showed no inhibitory effect when administered alone, did not enhance the inhibitory effect. This suggests that the BIG3-binding domains may be present in the 11-22 aa and 76-90 aa regions of PHB2, respectively. Next, we examined the inhibition of BIG3-PHB2 binding by No. 1 and No. 5 at concentrations of 20 μM and 50 μM using immunoprecipitation with a BIG3 antibody. Both No. 1 and No. 5 inhibited BIG3-PHB2 binding in a concentration-dependent manner, with No. 1 inhibiting 64% at 50 μM and No. 5 inhibiting 80% at 50 μM (Figure 2B). Furthermore, the combined use of both peptides at 50 μM resulted in an 87% inhibition rate (Figure 2B). We then examined the effect of each peptide (No. 1, No. 5, No. 6) on the phosphorylation of Ser39 of PHB2. Compared to the phosphorylation of PHB2 Ser39 by ERAP treatment (the positive control), treatment with No. 1 and No. 5 alone resulted in only 40% and 20% phosphorylation band intensity, respectively (Fig. 2C). The combined use of both peptides also resulted in 70% phosphorylation intensity (Fig. 2C). On the other hand, No. 6 barely induced PHB2 phosphorylation, with a band intensity of 10% (Fig. 2C). The combined use of No. 5 and No. 6 also resulted in a band intensity of 20% (Fig. 2C), suggesting that the binding of PHB2 and BIG3 spans multiple regions of No. 1 and No. 5.

[0098] Inhibitory effect of a novel peptide (11-90aa) encompassing the BIG3-binding region of PHB2 peptides No. 1 and No. 5 on E2-dependent proliferation Because the PHB2 peptides (No. 1 and No. 5) could only inhibit E2-dependent proliferation and induce PHB2 Ser39 phosphorylation by 50%, we synthesized a new 11-90aa PHB2 peptide containing these two regions and examined its effect on E2-dependent proliferation of MCF-7 cells. The 11-90aa PHB2 peptide concentration-dependently inhibited MCF-7 cells, which had doubled in proliferation after 24 hours of E2 stimulation. However, even at 50 μM, the inhibition rate was only 57%, which was almost the same as that of peptides No. 1 and No. 5 (Fig. 3A). Therefore, we evaluated whether the 11-90aa peptide could inhibit the binding of BIG3 to PHB2 and induce PHB2 phosphorylation. As a result, the 11-90aa PHB2 peptide inhibited the binding of BIG3 to PHB2 more than the untreated control, but did not achieve sufficient binding inhibition (Figure 3B). Furthermore, phosphorylation of Ser39 of PHB2 was induced in a concentration-dependent manner by the 11-90aa PHB2 peptide, but was only 30% of the phosphorylation obtained by ERAP treatment, failing to induce sufficient phosphorylation (Figure 3C). This may be due to the large number of amino acids (80) that the peptide has, resulting in insufficient binding to the α-helical structure of BIG3.

[0099] Inhibitory effects of linear, branched, and cyclic PHB2 on E2-dependent proliferation Since the combined use of the PHB2 peptides 11-22aa and 76-90aa enhanced estrogen-dependent proliferation by 88% (Fig. 2A), we further investigated the inhibitory effects of the linear and branched PHB2 peptides (Fig. 4A, 4B, 4C ... Furthermore, the 11-21aa and 76-88aa cyclic PHB2 peptides exhibited enhanced inhibitory effects compared with the non-cyclic peptide (Fig. 5A). Furthermore, the combined use of the cyclic peptides achieved almost complete inhibitory effects (96% inhibition, Fig. 5A), but no apoptosis-like phenomenon, such as cell floating, was observed. Next, we examined the effects of the cyclic peptides on the proliferation of normal mammary epithelial cells MCF-10A, which do not express ERα or BIG3 (treated at 10 μM for 24 hours). Although slight inhibitory effects were observed with the linear and cyclic PHB2 peptides (Fig. 5B, linear: 10% inhibition, cyclic 11-21aa: 14% inhibition, cyclic 76-88aa: 15% inhibition), most PHB2 peptides had little effect on MCF-10A proliferation, suggesting that they specifically inhibit E2-dependent proliferation. Next, we examined whether these PHB2 peptides could inhibit the interaction between BIG3 and PHB2. While BIG3 and PHB2 bound strongly in untreated and E2-stimulated cells (Fig. 5C), treatment with each PHB2 peptide alone barely inhibited the BIG3-PHB2 interaction (Fig. 5C). However, combined administration of the linear and cyclic PHB2 peptides significantly inhibited the interaction (Fig. 5C; linear and cyclic PHB2 peptides combined: 67% inhibition, 81% inhibition), suggesting the existence of two PHB2-binding domains for BIG3. On the other hand, the linear and branched PHB2 peptides were unable to cover the two BIG3-binding domains on PHB2.

[0100] Long-term stability of the inhibitory effect of cyclic PHB2 peptide on E2-dependent proliferation Because the cyclic PHB2 peptide may be stable at low concentrations due to its enhanced membrane permeability and structural immobilization, we investigated the long-term stability of 10 μM cyclic PHB2 peptide alone for up to 96 hours. The linear PHB2 peptides 11-22aa and 76-90aa exhibited 40% and 61% inhibition after 24 hours of treatment, respectively, but significantly reduced to 31% and 24% after 96 hours (Figure 6A). The cyclic peptides, 11-21aa and 76-88aa, exhibited 67% inhibition (53% after 24 hours) and 72% inhibition (58% after 24 hours) after 96 hours, respectively (Figure 6A). The inhibitory effect was stably maintained for up to 96 hours. These data suggest that the cyclic PHB2 peptide, like the cross-linked PHB2 peptide, maintains a stable conformation, contributing to its long-term inhibitory effect. Next, because the inhibitory effect of the cyclic PHB2 peptides was sustained for up to 96 h, we examined the effects of 1 and 10 μM of each cyclic PHB2 peptide on the proliferation of MCF-10A cells, which do not express ERα or BIG3. The results showed that the cyclic PHB2 peptides, which have the PHB2 sequence 11-21aa and 76-88aa, had little effect at 1 μM (5-7% inhibition for both), but showed 10-15% inhibition at 10 μM (Fig. 6B). This suggests that they have a slight nonspecific inhibitory effect, but the inhibition of E2-dependent proliferation of MCF-7 cells by the cyclic PHB2 peptides is thought to be mainly due to the inhibition of BIG3-PHB2 binding.

[0101] Concentration-dependent inhibitory effect of cyclic PHB2 peptide on E2-dependent proliferation The 50% inhibitory concentration (IC) of the cyclic PHB2 peptide on E2-dependent MCF-7 proliferation was 50 ) and calculate the IC 50 The synergistic inhibitory effect of each cyclic PHB2 peptide on E2-dependent proliferation was examined. As a result, each cyclic PHB2 peptide inhibited E2-dependent proliferation in a concentration-dependent manner, with an IC of 4.06 μM for the cyclic 11-21aa peptide and 2.11 μM for the cyclic 76-88aa peptide. 50 We then investigated the long-term effects of the combination of 4 μM cyclic 11-21aa and 2 μM cyclic 76-88aa. A synergistic inhibitory effect of 82% was observed after 24 hours of combined administration, and this inhibitory effect persisted for up to 96 hours (Figure 7B, combined administration: 88% inhibition, cyclic 11-21aa: 41% inhibition, cyclic 76-88aa: 59% inhibition). Furthermore, these concentrations had little effect on the proliferation of MCF-10A cells (Figure 7C).

[0102] Identification of amino acids in the PHB2 peptide sequence that are important for binding to BIG3 Since the PHB2-derived peptides No. 1 (11-22 aa: SEQ ID NO: 1) and No. 5 (76-90 aa: SEQ ID NO: 5) inhibited E2-dependent cell proliferation by approximately 50%, we created peptides in which each amino acid in the peptide sequences of No. 1 and No. 5 was mutated to alanine (Figure 8A) to identify the amino acids important for growth inhibition. Forty-eight hours after seeding, MCF-7 cells were seeded with 10 μM of each PHB2 peptide and 10 nM estrogen, and cell numbers were monitored after 24 hours. First, we evaluated the amino acids in the PHB2 sequence 11-22 aa. No. 1 (11-22 aa) inhibited estrogen-dependent cell proliferation by 65%, while the alanine-mutated peptides No. 59 and No. 62 (SEQ ID NO: 59, 62) only reduced the inhibition to 19% and 8% (Figure 8B). On the other hand, the other alanine-mutated peptides had almost the same inhibitory rate as No. 1 (Figure 8B), suggesting that the 15th and 18th glycines are important for binding to BIG3, and that converting these sites to the isomeric form of D-amino acids may improve inhibitory activity. Next, we evaluated the amino acids in the 76-90aa region of the PHB2 sequence. The inhibitory rate of No. 5 (76-90aa) against estrogen-dependent proliferation was almost reproduced at 54% (Figure 8C). However, the inhibitory rates of the alanine mutant peptides No. 71-73 (sequence numbers 71-73) were weaker than No. 5 (76-90aa), at 38%, 37%, and 13%, respectively (Figure 8C). This suggests that the aspartic acid at position 82 in particular is necessary for the binding of BIG3 to PHB2. Furthermore, in addition to peptides No. 1 (11-22aa) and No. 5 (76-90aa), peptides No. 2 (42-50aa: SEQ ID NO: 2) and No. 3 (38-50aa: SEQ ID NO: 3) showed inhibitory effects (Fig. 1A). In addition, peptides No. 82 and No. 83 (SEQ ID NO: 82, 83) were used to examine their effects on estrogen-dependent cell proliferation. These peptides contained 51-57aa, including 53-57aa (Fig. 1A), which were predicted by in silico analysis to be involved in the interaction between BIG3 and PHB2 (Fig. 8D). As a result, the inhibition rates of No. 2 and No. 3 were 20% and 17%, respectively, whereas the inhibition rates of No. 82 and No. 83, which had amino acids up to the 57th position, were improved to 59% and 61% (Fig. 8E). This suggests that by having amino acids from glutamic acid at the 44th position to glycine at the 57th position, PHB2 peptides with inhibition rates comparable to those of No. 1 and No. 5 can be produced.

[0103] [Example 2] Effect on triple-negative breast cancer 1. Materials and Methods Cell lines and culture conditions The human breast cancer cell line MDA-MB-231 was purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA) and cultured in Leibovitz's L-15 medium (Thermo Fisher Scientific) supplemented with 10% FBS (Thermo Fisher Scientific, Waltham, MA, USA) and 1% antibiotic-antimycotic solution (Wako Pure Chemical Industries, Osaka, Japan) at 37°C without CO2 regulation.

[0104] Cell proliferation assay MDA-MB-231 cells were plated in a 48-well plate at 1 × 10 4 cells / 200 μL After 48 hours, the medium in each well was replaced with medium containing PHB2 peptide 11-22aa or 76-90aa (20 μMAfter further culturing for 96 hours, the cell proliferation level was measured using Cell Counting Kit-8 (Dojindo, Kumamoto, Japan). Data were obtained from three independent experiments, and graphs (mean ± standard deviation) and the 50% inhibitory concentration (IC) of the peptide on cell proliferation were calculated using the graphing and data analysis software SigmaPlot (Systat Software, San Jose, CA, USA). 50 ) was calculated.

[0105] Combination Assays MDA-MB-231 cells were plated in a 48-well plate at 1 × 10 4 cells / 200 μL After 48 hours, the medium in each well was diluted with PHB2 peptide 11-22aa (addition concentration IC 50 value), PHB2 peptide 76-90aa (addition concentration IC 50 value), a mixture of both peptides (addition concentration is each IC 50 The media were replaced with media containing either phosphate-buffered saline (PBS) or phosphate-buffered saline (PBS) as a negative control, and after 96 hours of culture, cell proliferation levels were measured using Cell Counting Kit-8 (Dojindo, Kumamoto, Japan). The data obtained were used to calculate relative values based on the proliferation level when PBS was added, and a graph was created.

[0106] 2.Results Growth inhibition of breast cancer cell lines by PHB2 peptide To investigate the cell growth inhibitory effect of PHB2 peptides 11-22aa and 76-90aa on the breast cancer cell line MDA-MB-231, a serial dilution series of the peptides was prepared, and the proliferation level was measured 96 hours after addition of the peptides to the cells. As a result, as shown in Figure 9A and B, a concentration-dependent cell growth inhibitory effect was observed for both peptides. The 50% inhibitory concentration (IC 50 ) was 0.462 μM for peptide 11-22aa and 0.273 μM for peptide 76-90aa, with peptide 76-90aa showing a higher growth inhibitory effect.

[0107] Combined effect of PHB2 peptides 11-22aa and 76-90aa To investigate the combined effect of PHB2 peptides 11-22aa and 76-90aa on cell proliferation inhibition, both peptides were administered at IC 50 When the peptides were mixed at the IC value and added individually, 50 The cell proliferation levels were compared when each peptide was added alone, as shown in Figure 9C. While each peptide alone inhibited proliferation by approximately 50% compared to the negative control, phosphate-buffered saline (PBS), the inhibitory effect was enhanced to approximately 62% when used in combination.

[0108] [Example 3] Effect of cross-linked PHB2 peptide on estrogen-dependent breast cancer 1. Materials and Methods Cell lines and culture conditions The human breast cancer cell line MCF-7 was purchased from the JCRB Cell Bank (Osaka, Japan) and maintained in MEM (Thermo Fisher Scientific) supplemented with 10% FBS (Nichirei Biosciences, Tokyo, Japan), 1% Antibiotic / Antimycotic solution (Thermo Fisher Scientific, Waltham, MA, USA), 0.1 mM NEAA (Thermo Fisher Scientific), 1 mM sodium pyruvate (Thermo Fisher Scientific), and 10 μg / mL insulin (Sigma, St. Louis, MO, USA) at 37°C in 5% CO2. The normal mammary epithelial cell line MCF-10A was purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA) and maintained in MEBM (Lonza) supplemented with the SingleQuots kit (BPE, hydrocortisone, hEGF, insulin, gentamicin / amphotericin B) (Lonza, Walkersville, MD, USA) and 100 ng / mL cholera toxin at 37°C in 5% CO .

[0109] Cell proliferation assay MCF-7 proliferation assays were performed in 48-well plates (2 × 10 4 MCF-10A cells were seeded in 200 μL of 48-well plates (2 × 10 cells / 200 μL). The next day, the medium was changed to phenol red-free DMEM / F12 (Thermo Fisher Scientific) supplemented with 10% FBS, 1% Antibiotic / Antimycotic solution, 0.1 mM NEAA, 1 mM sodium pyruvate, and 10 μg / mL insulin. After 24 hours, the cells were treated with 10 nM 17β-estradiol (estrogen, Sigma) alone or with 10 nM estrogen and a peptide derived from the PHB2 sequence. MCF-10A proliferation assays were performed in 48-well plates (2 × 10 cells / 200 μL). 4 The cells were seeded in 200 μL of PBS and 24 hours later, a peptide derived from the PHB2 sequence was added. Proliferation assays were performed using Cell Counting Kit-8 (CCK-8, Dojindo, Kumamoto, Japan). Data are shown as the mean ± standard deviation of three independent experiments.

[0110] 2.Results Inhibitory effect of cross-linked PHB2 peptide on estrogen-dependent proliferation We crosslinked the PHB2 peptides 11-21aa and 76-88aa using three different crosslinking methods (Fig. 10A: hexafluorobenzene crosslinking, decafluorobiphenyl crosslinking, and disulfide crosslinking) to prepare stapled PHB2 peptides (Fig. 10B). We then examined the inhibitory effects of these peptides on estrogen-dependent proliferation. Forty-eight hours after seeding, breast cancer cells MCF-7 were seeded with 10 μM of each PHB2 peptide and 10 nM estrogen. After 24 hours, cell numbers were assessed using an MTT assay. The 11-22aa and 76-88aa cross-linked PHB2 peptides showed approximately 1.5-fold improved inhibitory effects against estrogen-dependent proliferation compared with the uncross-linked PHB2 peptides (SEQ ID NOs: 109, 113, 114, 118, and 122) (Figure 10C, left; without polyarginine, right). The cross-linking method (stapling) did not affect the inhibitory effects. Furthermore, the cross-linked PHB2 peptides without polyarginine at the C-terminus (SEQ ID NOs: 115, 116, 117, 119, 120, and 121) showed slightly higher inhibitory effects than the polyarginine-linked peptides (with polyarginine: approximately 60% inhibitory rate, without polyarginine: approximately 70% inhibitory rate), suggesting that polyarginine may interfere with the function of the cross-linked structure.

[0111] Inhibitory effect of cyclic PHB2 peptide on estrogen-dependent proliferation To improve the cell membrane permeability and structural stability of the cyclic PHB2 peptide (Fig. 4C, SEQ ID NOS: 25 and 26), we examined its inhibitory effect on estrogen-dependent proliferation by varying the crosslinking structure. In addition to the disulfide crosslink (SEQ ID NOS: 25 and 26), we also evaluated hexafluorobenzene crosslinks (Fig. 11A: SEQ ID NOS: 123 and 126) and decafluorobiphenyl crosslinks (Fig. 11A: SEQ ID NOS: 124 and 127). The cyclic PHB2 peptides crosslinked with fluorobenzene (Fig. 11A: SEQ ID NOS: 123, 124, 126, 127) showed slightly improved growth inhibitory activity compared with the disulfide crosslink (Fig. 11B), with the cyclic PHB2 peptide 11-21aa exhibiting approximately 70% and the cyclic PHB2 peptide 76-88aa exhibiting approximately 80%. There was no significant difference between the single (hexafluorobenzene) and double (decafluorobiphenyl) fluorobenzene crosslinks.

[0112] Effect of alteration of PHB2 peptide 11-22aa on estrogen-dependent proliferation For the PHB2 peptide 11-22aa, the glycines at positions 15 and 18 in the amino acid sequence of SEQ ID NO: 28 (full-length PHB2 polypeptide) were thought to be important for binding to BIG3 (Figure 8). Therefore, we investigated whether the inhibitory activity of this peptide would be enhanced by substituting D-alanine and D-leucine at these positions (Figure 12A). The PHB2 peptide in which the glycines at positions 15 and 18 were substituted with D-leucine (SEQ ID NO: 134) exhibited approximately 65% inhibitory activity (Figure 12B), whereas substitution with D-alanine (SEQ ID NO: 133) had no inhibitory effect (Figure 12B). However, since the inhibitory activity of the PHB2 peptide 11-22aa (SEQ ID NO: 1, Figure 8B) was approximately 65%, substitution with D-leucine did not significantly improve the inhibitory activity.

[0113] Effect of cross-linked PHB2 peptide on mammary epithelial cell proliferation We investigated the effects of cross-linked and cyclic PHB2 peptides on the proliferation of normal mammary epithelial cells MCF-10A, which do not express ERα or BIG3 (treated with 10 μM of each PHB2 peptide for 24 hours). As a result, all PHB2 peptides evaluated had no effect on the proliferation of MCF-10A cells (Figure 13). [Industrial Applicability]

[0114] According to the present invention, a peptide derived from the amino acid sequence of PHB2 is provided, which exhibits an inhibitory effect on the BIG3-PHB2 interaction and is useful as a therapeutic agent for breast cancer. The peptide provided by the present invention is useful for treating cancers such as breast cancer. More specifically, the peptide of the present invention is useful for treating BIG3-positive and / or estrogen receptor-positive cancers. The peptide of the present invention targets BIG3, a protein that is specifically and highly expressed in estrogen receptor-positive cancers, rather than PHB2, which is expressed in organs throughout the human body, and is therefore expected to have high selectivity against estrogen receptor-positive cancers. Furthermore, the peptide of the present invention also exhibits antitumor effects against triple-negative breast cancer.

[0115] Sequence information SEQUENCE LISTING <110> TOKUSHIMA UNIVERSITY ONCOTHERAPY SCIENCE, INC. <120> THERAPEUTIC AGENTS FOR BREAST CANCER COMPRISING PHB2-DERIVED PEPTIDE INHIBITING BIG3-PHB2 INTERACTION <150> JP 2018-225660 <151> 2018-11-30 <160> 136 <170> PatentIn version 3.5 <210> 1 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> PHB2 peptide No. 1 <400> 1 Arg Leu Pro Ala Gly Pro Arg Gly Met Gly Thr Ala 1 5 10 <210> 2 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> PHB2 peptide No. 2 <400> 2 Thr Val Glu Gly Gly His Arg Ala Ile 1 5 <210> 3 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> PHB2 peptide No. 3 <400> 3 Glu Ser Val Phe Thr Val Glu Gly Gly His Arg Ala Ile 1 5 10 <210> 4 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> PHB2 peptide No. 4 <400> 4 Tyr Gly Val Arg Glu Ser Val Phe Thr Val Glu Gly Gly His Arg Ala 1 5 10 15 Ile <210> 5 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> PHB2 peptide No. 5 <400> 5 Gln Tyr Pro Ile Ile Tyr Asp Ile Arg Ala Arg Pro Arg Lys Ile 1 5 10 15 <210> 6 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> PHB2 peptide No. 6 <400> 6 Arg Pro Arg Lys Ile Ser Ser Pro Thr Gly Ser Lys Asp Leu Gln 1 5 10 15 <210> 7 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> PHB2 peptide No. 7 <400> 7 Ser Lys Asp Leu Gln Met Val Asn Ile Ser Leu Arg Val Leu Ser 1 5 10 15 <210> 8 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> PHB2 peptide No. 8 <400> 8 Leu Arg Val Leu Ser Arg Pro Asn Ala Gln Glu Leu Pro Ser Met 1 5 10 15 <210> 9 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> PHB2 peptide No. 9 <400> 9 Glu Leu Pro Ser Met Tyr Gln Arg Leu Gly Leu Asp Tyr Glu Glu 1 5 10 15 <210> 10 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> PHB2 peptide No. 10 <400> 10 Leu Asp Tyr Glu Glu Arg Val Leu Pro Ser Ile Val Asn Glu Val 1 5 10 15 <210> 11 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> PHB2 peptide No. 11 <400> 11 Ile Val Asn Glu Val Leu Lys Ser Val Val Ala Lys Phe Asn Ala 1 5 10 15 <210> 12 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> PHB2 peptide No. 12 <400> 12 Ala Lys Phe Asn Ala Ser Gln Leu Ile Thr Gln Arg Ala Gln Val 1 5 10 15 <210> 13 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> PHB2 peptide No. 13 <400> 13 Gln Arg Ala Gln Val Ser Leu Leu Ile Arg Arg Glu Leu Thr Glu 1 5 10 15 <210> 14 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> PHB2 peptide No. 14 <400> 14 Arg Glu Leu Thr Glu Arg Ala Lys Asp Phe Ser Leu Ile Leu Asp 1 5 10 15 <210> 15 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> PHB2 peptide No. 15 <400> 15 Ser Leu Ile Leu Asp Asp Val Ala Ile Thr Glu Leu Ser Phe Ser 1 5 10 15 <210> 16 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> PHB2 peptide No. 16 <400> 16 Glu Leu Ser Phe Ser Arg Glu Tyr Thr Ala Ala Val Glu Ala Lys 1 5 10 15 <210> 17 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> PHB2 peptide No. 17 <400> 17 Ala Val Glu Ala Lys Gln Val Ala Gln Gln Glu Ala Gln Arg Ala 1 5 10 15 <210> 18 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> PHB2 peptide No. 18 <400> 18 Glu Ala Gln Arg Ala Gln Phe Leu Val Glu Lys Ala Lys Gln Glu 1 5 10 15 <210> 19 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> PHB2 peptide No. 19 <400> 19 Gln Phe Leu Val Glu Lys Ala Lys Gln Glu Gln Arg Gln Lys Ile 1 5 10 15 <210> 20 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> PHB2 peptide No. 20 <400> 20 Phe Leu Val Glu Lys Ala Lys Gln Glu Gln Arg Gln Lys Ile 1 5 10 <210> 21 <211> 80 <212> PRT <213> Artificial Sequence <220> <223> PHB2 derived peptide 11-90aa <400> 21 Arg Leu Pro Ala Gly Pro Arg Gly Met Gly Thr Ala Leu Lys Leu Leu 1 5 10 15 Leu Gly Ala Gly Ala Val Ala Tyr Gly Val Arg Glu Ser Val Phe Thr 20 25 30 Val Glu Gly Gly His Arg Ala Ile Phe Phe Asn Arg Ile Gly Gly Val 35 40 45 Gln Gln Asp Thr Ile Leu Ala Glu Gly Leu His Phe Arg Ile Pro Trp 50 55 60 Phe Gln Tyr Pro Ile Ile Tyr Asp Ile Arg Ala Arg Pro Arg Lys Ile 65 70 75 80 <210> 22 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> PHB2 derived peptide 11-22aa <220> <221> MOD_RES <222> (1)..(1) <223> ACETYLATION <400> 22 Arg Leu Pro Ala Gly Pro Arg Gly Met Gly Thr Ala 1 5 10 <210> 23 <211> 23 <212> PRT <213> Artificial Sequence <220> <223> PHB2 derived peptide 76-90aa + 8 Arg residues <220> <221> MOD_RES <222> (23)..(23) <223> AMIDATION <400> 23 Gln Tyr Pro Ile Ile Tyr Asp Ile Arg Ala Arg Pro Arg Lys Ile Arg 1 5 10 15 Arg Arg Arg Arg Arg Arg Arg 20 <210> 24 <211> 22 <212> PRT <213> Artificial Sequence <220> <223> PHB2 derived peptide 76-89aa + 8 Arg residues <220> <221> MOD_RES <222> (1)..(1) <223> ACETYLATION <220> <221> MOD_RES <222> (22)..(22) <223> AMIDATION <400> 24 Gln Tyr Pro Ile Ile Tyr Asp Ile Arg Ala Arg Pro Arg Lys Arg Arg 1 5 10 15 Arg Arg Arg Arg Arg Arg 20 <210> 25 <211> 19 <212> PRT <213> Artificial Sequence <220> <223> cyclic peptide containing PHB2 derived peptide 11-21aa <220> <221> DISULFID <222> (1)..(13) <220> <221> MISC_FEATURE <222> (10)..(10) <223> Xaa = Nle <220> <221> MISC_FEATURE <222> (15)..(15) <223> Xaa = Nal <400> 25 Cys Arg Leu Pro Ala Gly Pro Arg Gly Xaa Gly Thr Cys Phe Xaa Arg 1 5 10 15 Arg Arg Arg <210> 26 <211> 21 <212> PRT <213> Artificial Sequence <220> <223> cyclic peptide containing PHB2 derived peptide 76-88aa <220> <221> DISULFID <222> (1)..(15) <220> <221> MISC_FEATURE <222> (17)..(17) <223> Xaa = Nal <400> 26 Cys Gln Tyr Pro Ile Ile Tyr Asp Ile Arg Ala Arg Pro Arg Cys Phe 1 5 10 15 Xaa Arg Arg Arg Arg 20 <210> 27 <211> 1457 <212> DNA <213> Homo sapiens <220> <221> CDS <222> (211)..(1110) <400> 27 tccgtatgcg cgattcctgt gcgcgaagtt cgggtccgta gtgggctaag ggggagggtt 60 tcaaagggag cgcacttccg ctgccctttc tttcgccagc cttacgggcc cgaaccctcg 120 tgtgaagggt gcagtaccta agccggagcg gggtagaggc gggccggcac ccccttctga 180 cctccagtgc cgccggcctc aagatcagac atg gcc cag aac ttg aag gac ttg 234 Met Ala Gln Asn Leu Lys Asp Leu 1 5 gcg gga cgg ctg ccc gcc ggg ccc cgg ggc atg ggc acg gcc ctg aag 282 Ala Gly Arg Leu Pro Ala Gly Pro Arg Gly Met Gly Thr Ala Leu Lys 10 15 20 ctg ttg ctg ggg gcc ggc gcc gtg gcc tac ggt gtg cgc gaa tct gtg 330 Leu Leu Leu Gly Ala Gly Ala Val Ala Tyr Gly Val Arg Glu Ser Val 25 30 35 40 ttc acc gtg gaa ggc ggg cac aga gcc atc ttc ttc aat cgg atc ggt 378 Phe Thr Val Glu Gly Gly His Arg Ala Ile Phe Phe Asn Arg Ile Gly 45 50 55 gga gtg cag cag gac act atc ctg gcc gag ggc ctt cac ttc agg atc 426 Gly Val Gln Gln Asp Thr Ile Leu Ala Glu Gly Leu His Phe Arg Ile 60 65 70 cct tgg ttc cag tac ccc att atc tat gac att cgg gcc aga cct cga 474 Pro Trp Phe Gln Tyr Pro Ile Ile Tyr Asp Ile Arg Ala Arg Pro Arg 75 80 85 aaa atc tcc tcc cct aca ggc tcc aaa gac cta cag atg gtg aat atc 522 Lys Ile Ser Ser Pro Thr Gly Ser Lys Asp Leu Gln Met Val Asn Ile 90 95 100 tcc ctg cga gtg ttg tct cga ccc aat gct cag gag ctt cct agc atg 570 Ser Leu Arg Val Leu Ser Arg Pro Asn Ala Gln Glu Leu Pro Ser Met 105 110 115 120 tac cag cgc cta ggg ctg gac tac gag gaa cga gtg ttg ccg tcc att 618 Tyr Gln Arg Leu Gly Leu Asp Tyr Glu Glu Arg Val Leu Pro Ser Ile 125 130 135 gtc aac gag gtg ctc aag agt gtg gtg gcc aag ttc aat gcc tca cag 666 Val Asn Glu Val Leu Lys Ser Val Val Ala Lys Phe Asn Ala Ser Gln 140 145 150 ctg atc acc cag cgg gcc cag gta tcc ctg ttg atc cgc cgg gag ctg 714 Leu Ile Thr Gln Arg Ala Gln Val Ser Leu Leu Ile Arg Arg Glu Leu 155 160 165 aca gag agg gcc aag gac ttc agc ctc atc ctg gat gat gtg gcc atc 762 Thr Glu Arg Ala Lys Asp Phe Ser Leu Ile Leu Asp Asp Val Ala Ile 170 175 180 aca gag ctg agc ttt agc cga gag tac aca gct gct gta gaa gcc aaa 810 Thr Glu Leu Ser Phe Ser Arg Glu Tyr Thr Ala Ala Val Glu Ala Lys 185 190 195 200 caa gtg gcc cag cag gag gcc cag cgg gcc caa ttc ttg gta gaaaaa 858 Gln Val Ala Gln Gln Glu Ala Gln Arg Ala Gln Phe Val Val Glu Lys 205 210 215 gca aag cag gaa cag cgg cag aaa att gtg cag gcc gag ggt gag gcc 906 Ala Lys Gln Glu Gln Arg Gln Lys Ile Val Gln Ala Glu Gly Glu Ala 220 225 230 gag gct gcc aag atg ctt gga gaa gca ctg agc aag aac cct ggc tac 954 Glu Ala Ala Lys Met Leu Gly Glu Ala Leu Ser Lys Asn Pro Gly Tyr 235 240 245 atc aaa ctt cgc aag att cga gca gcc cag aat atc tcc aag acg atc 1002 Lys Ile Leu Arg Lys Ile Arg Only Only Gln Asn Only Served Lys Thr Only 250 255 260 gcc aca tca cag aat cgt atc tat ctc aca gct gac aac ctt gtg ctg 1050 Only Ser Gln Asn Arg With Leu Tyr Thr Only Asp Asn Leu Val Val Leu 265 270 275 280 aac cta cag gat gaa agt ttc acc agg gga agt gac agc ctc atc aag 1098 Asn Leu Gln Asp Glu Ser Phe Thr Arg Gly Ser Asp Ser Leu Ile Lys 285 290 295 ggt aag aaa tga gcctagtcac caagaactcc acccccagag gaagtggatc 1150 Gly Lys Lys tgcttctcca gttttgagg agccagccag gggtccagca cagccctacc ccgccccagt 1210 atcatgcgat ggtcccccac accggttccc tgaacccctc ttggattaag gaagactgaa 1270 gactagcccc tttctgggg aattactttc ctcctccctg tgttaactgg ggctgttggg 1330 gacagtgcgt gatttctcag tgattccta cagtgttgtt ccctccctca aggctgggag 1390 gagataaaca ccaacccagg aattctcaat aaattttat tacttaacct gaaaaaaaaa 1450 aaaaaaa 1457 <210> 28 <211> 299 <212> PRT <213> Homo sapiens <400> 28 Met Ala Gln Asn Leu Lys Asp Leu Ala Gly Arg Leu Pro Ala Gly Pro 1 5 10 15 Arg Gly Met Gly Thr Ala Leu Lys Leu Leu Leu Gly Ala Gly Ala Val 20 25 30 Ala Tyr Gly Val Arg Glu Ser Val Phe Thr Val Glu Gly Gly His Arg 35 40 45 Ala Ile Phe Phe Asn Arg Ile Gly Gly Val Gln Gln Asp Thr Ile Leu 50 55 60 Ala Glu Gly Leu His Phe Arg Ile Pro Trp Phe Gln Tyr Pro Ile Ile 65 70 75 80 Tyr Asp Ile Arg Ala Arg Pro Arg Lys Ile Ser Ser Pro Thr Gly Ser 85 90 95 Lys Asp Leu Gln Met Val Asn Ile Ser Leu Arg Val Leu Ser Arg Pro 100 105 110 Asn Ala Gln Glu Leu Pro Ser Met Tyr Gln Arg Leu Gly Leu Asp Tyr 115 120 125 Glu Glu Arg Val Leu Pro Ser Ile Val Asn Glu Val Leu Lys Ser Val 130 135 140 Val Ala Lys Phe Asn Ala Ser Gln Leu Ile Thr Gln Arg Ala Gln Val 145 150 155 160 Ser Leu Leu Ile Arg Arg Glu Leu Thr Glu Arg Ala Lys Asp Phe Ser 165 170 175 Leu Ile Leu Asp Asp Val Ala Ile Thr Glu Leu Ser Phe Ser Arg Glu 180 185 190 Tyr Thr Ala Ala Val Glu Ala Lys Gln Val Ala Gln Gln Glu Ala Gln 195 200 205 Arg Ala Gln Phe Leu Val Glu Lys Ala Lys Gln Glu Gln Arg Gln Lys 210 215 220 Ile Val Gln Ala Glu Gly Glu Ala Glu Ala Ala Lys Met Leu Gly Glu 225 230 235 240 Ala Leu Ser Lys Asn Pro Gly Tyr Ile Lys Leu Arg Lys Ile Arg Ala 245 250 255 Ala Gln Asn Ile Ser Lys Thr Ile Ala Thr Ser Gln Asn Arg Ile Tyr 260 265 270 Leu Thr Ala Asp Asn Leu Val Leu Asn Leu Gln Asp Glu Ser Phe Thr 275 280 285 Arg Gly Ser Asp Ser Leu Ile Lys Gly Lys Lys 290 295 <210> 29 <211> 1343 <212> DNA <213> Homo sapiens <220> <221> CDS <222> (211)..(996) <400> 29 tccgtatgcg cgattcctgt gcgcgaagtt cgggtccgta gtgggctaag ggggagggtt 60 tcaaagggag cgcacttccg ctgccctttc tttcgccagc cttacgggcc cgaaccctcg 120 tgtgaagggt gcagtaccta agccggagcg gggtagaggc gggccggcac ccccttctga 180 cctccagtgc cgccggcctc aagatcagac atg gcc cag aac ttg aag gac ttg 234 Met Ala Gln Asn Leu Lys Asp Leu 1 5 gcg gga cgg ctg ccc gcc ggg ccc cgg ggc atg ggc acg gcc ctg aag 282 Ala Gly Arg Leu Pro Ala Gly Pro Arg Gly Met Gly Thr Ala Leu Lys 10 15 20 ctg ttg ctg ggg gcc ggc gcc gtg gcc tac ggt gtg cgc gaa tct gtg 330 Leu Leu Leu Gly Ala Gly Ala Val Ala Tyr Gly Val Arg Glu Ser Val 25 30 35 40 ttc acc gtg gaa ggc ggg cac aga gcc atc ttc ttc aat cgg atc ggt 378 Phe Thr Val Glu Gly Gly His Arg Ala Ile Phe Phe Asn Arg Ile Gly 45 50 55 gga gtg cag cag gac act atc ctg gcc gag ggc ctt cac ttc agg atc 426 Gly Val Gln Gln Asp Thr Ile Leu Ala Glu Gly Leu His Phe Arg Ile 60 65 70 cct tgg ttc cag tac ccc att atc tat gac att cgg gcc aga cct cga 474 Pro Trp Phe Gln Tyr Pro Ile Ile Tyr Asp Ile Arg Ala Arg Pro Arg 75 80 85 aaa atc tcc tcc cct aca ggc tcc aaa gac cta cag atg gtg aat atc 522 Lys Ile Ser Ser Pro Thr Gly Ser Lys Asp Leu Gln Met Val Asn Ile 90 95 100 tcc ctg cga gtg ttg tct cga ccc aat gct cag gag ctt cct agc atg 570 Ser Leu Arg Val Leu Ser Arg Pro Asn Ala Gln Glu Leu Pro Ser Met 105 110 115 120 tac cag cgc cta ggg ctg gac tac gag gaa cga gtg ttg ccg tcc att 618 Tyr Gln Arg Leu Gly Leu Asp Tyr Glu Glu Arg Val Leu Pro Ser Ile 125 130 135 gtc aac gag gtg ctc aag agt gtg gtg gcc aag ttc aat gcc tca cag 666 Val Asn Glu Val Leu Lys Ser Val Val Ala Lys Phe Asn Ala Ser Gln 140 145 150 ctg atc acc cag cgg gcc cag gta tcc ctg ttg atc cgc cgg gag ctg 714 Leu Ile Thr Gln Arg Ala Gln Val Ser Leu Leu Ile Arg Arg Glu Leu 155 160 165 aca gag agg gcc aag gac ttc agc ctc atc ctg gat gat gtg gcc atc 762 Thr Glu Arg Ala Lys Asp Phe Ser Leu Ile Leu Asp Asp Val Ala Ile 170 175 180 aca gag ctg agc ttt agc cga gag tac aca gct gct gta gaa gcc aaa 810 Thr Glu Leu Ser Phe Ser Arg Glu Tyr Thr Ala Ala Val Glu Ala Lys 185 190 195 200 caa gtg gca ctg agc aag aac cct ggc tac atc aaa ctt cgc aag att 858 Gln Val Ala Leu Ser Lys Asn Pro Gly Tyr Ile Lys Leu Arg Lys Ile 205 210 215 cga gca gcc cag aat atc tcc aag acg atc gcc aca tca cag aat cgt 906 Arg Ala Ala Gln Asn Ile Ser Lys Thr Ile Ala Thr Ser Gln Asn Arg 220 225 230 atc tat ctc aca gct gac aac ctt gtg ctg aac cta cag gat gaa agt 954 Ile Tyr Leu Thr Ala Asp Asn Leu Val Leu Asn Leu Gln Asp Glu Ser 235 240 245 ttc acc agg gga agt gac agc ctc atc aag ggt aag aaa tga 996 Phe Thr Arg Gly Ser Asp Ser Leu Ile Lys Gly Lys Lys 250 255 260 gcctagtcac caagaactcc acccccagag gaagtggatc tgcttctcca gttttgagg 1056 agccagccag gggtccagca cagccctacc ccgccccagt atcatgcgat ggtcccccac 1116 accggttccc tgaacccctc ttggattaag gaagactgaa gactagcccc ttttctgggg 1176 aattactttc ctcctccctg tgttaactgg ggctgttggg gacagtgcgt gatttctcag 1236 tgattccta cagtgttgtt ccctccctca aggctgggag gagataaaca ccaacccagg 1296 aattctcaat aaattttat tacttaacct gaaaaaaaa aaaaaaa 1343 <210> 30 <211> 261 <212> PRT <213> Homo sapiens <400> 30 Met Ala Gln Asn Leu Lys Asp Leu Ala Gly Arg Leu Pro Ala Gly Pro 1 5 10 15 Arg Gly Met Gly Thr Ala Leu Lys Leu Leu Leu Gly Ala Gly Ala Val 20 25 30 Ala Tyr Gly Val Arg Glu Ser Val Phe Thr Val Glu Gly Gly His Arg 35 40 45 Ala Ile Phe Phe Asn Arg Ile Gly Gly Val Gln Gln Asp Thr Ile Leu 50 55 60 Ala Glu Gly Leu His Phe Arg Ile Pro Trp Phe Gln Tyr Pro Ile Ile 65 70 75 80 Tyr Asp Ile Arg Ala Arg Pro Arg Lys Ile Ser Ser Pro Thr Gly Ser 85 90 95 Lys Asp Leu Gln Met Val Asn Ile Ser Leu Arg Val Leu Ser Arg Pro 100 105 110 Asn Ala Gln Glu Leu Pro Ser Met Tyr Gln Arg Leu Gly Leu Asp Tyr 115 120 125 Glu Glu Arg Val Leu Pro Ser Ile Val Asn Glu Val Leu Lys Ser Val 130 135 140 Val Ala Lys Phe Asn Ala Ser Gln Leu Ile Thr Gln Arg Ala Gln Val 145 150 155 160 Ser Leu Leu Ile Arg Arg Glu Leu Thr Glu Arg Ala Lys Asp Phe Ser 165 170 175 Leu Ile Leu Asp Asp Val Ala Ile Thr Glu Leu Ser Phe Ser Arg Glu 180 185 190 Tyr Thr Val Val Glu Val Val Lys Gln Val Val Leu Ser Lys Asn Pro 195 200 205 Gly Tyr Containing Arg Lys With Arg Only Only Gln Asn With Lys Ser 210 215 220 Only Gln Asn Arg and Tyr Leu Only Asp Asn Leu 225 230 235 240 Val Leu Asn Leu Gln Asp Glu Ser Phe Thr Arg Gly Ser Asp Ser Leu 245 250 255 Ile Lys Lys Lys 260 <210> 31 <211> 14895 <212> DNA <213> Homo sapiens <220> <221> CDS <222> (172)..(6705) <400> 31 gtggcccgcg gcatggagcg ggcgtgattc atcagcatcc gcgccggggc ggcatggggg 60 cgcgcgcggc gccgcctag gcgcccaggg ccaggcagcg gcggctccc cggcccggct 120 cgccccgcgct tctctccctg tgggcggcgg cccggcgcct ggaggtcaa g atg gaa 177 Met Glu 1 gaa atc ctg agg aag ctg cag aag gag gcg tcc ggg agc aag tac aaa 225 Glu Ile Leu Arg Lys Leu Gln Lys Glu Ala Ser Gly Ser Lys Tyr Lys 5 10 15 gcc atc aag gag agc tgc acc tgg gcc ctg gaa act cta ggt ggt ctg 273 Ala Ile Lys Glu Ser Cys Thr Trp Ala Leu Glu Thr Leu Gly Gly Leu 20 25 30 gat acc att gtc aag atc cct cca cat gta ctg agg gag aaa tgc ctg 321 Asp Thr Ile Val Lys Ile Pro Pro His Val Leu Arg Glu Lys Cys Leu 35 40 45 50 ctg cct ctc cag ttg gct ttg gaa tcc aag aat gtg aag ctg gcc caa 369 Leu Pro Leu Gln Leu Ala Leu Glu Ser Lys Asn Val Lys Leu Ala Gln 55 60 65 cat gct ttg gca ggg atg cag aag ctt ctg tcg gaa gag agg ttt gta 417 His Ala Leu Ala Gly Met Gln Lys Leu Leu Ser Glu Glu Arg Phe Val 70 75 80 tcc atg gaa aca gat tct gat gag aag cag ctg ctc aat cag ata ctg 465 Ser Met Glu Thr Asp Ser Asp Glu Lys Gln Leu Leu Asn Gln Ile Leu 85 90 95 aat gcc gtg aaa gtg acg cct tcg ctc aac gag gac ctg cag gtg gaa 513 Asn Ala Val Lys Val Thr Pro Ser Leu Asn Glu Asp Leu Gln Val Glu 100 105 110 gtg atg aag gtt tta cta tgc atc acc tac acg cca aca ttt gat ctg 561 Val Met Lys Val Leu Leu Cys Ile Thr Tyr Thr Pro Thr Phe Asp Leu 115 120 125 130 aat ggg agt gcc gtg ctg aag atc gcg gag gtg tgc att gag acg tac 609 Asn Gly Ser Ala Val Leu Lys Ile Ala Glu Val Cys Ile Glu Thr Tyr 135 140 145 ata agc agc tgt cac cag cgt agc ata aac act gct gtg cgg gca act 657 Ile Ser Ser Cys His Gln Arg Ser Ile Asn Thr Ala Val Arg Ala Thr 150 155 160 ctc agt caa atg ctg agt gac ttg act tta cag tta cga cag agg cag 705 Leu Ser Gln Met Leu Ser Asp Leu Thr Leu Gln Leu Arg Gln Arg Gln 165 170 175 gag aat acg ata att gaa aac cca gat gtc cca cag gat ttc ggg aat 753 Glu Asn Thr Ile Ile Glu Asn Pro Asp Val Pro Gln Asp Phe Gly Asn 180 185 190 caa ggg tca aca gta gag tcc ctc tgt gat gat gtt gtc tct gta ctc 801 Gln Gly Ser Thr Val Glu Ser Leu Cys Asp Asp Val Val Ser Val Leu 195 200 205 210 acc gtc ctg tgt gag aag ctg caa gcc gcc ata aat gac agc cag cag 849 Thr Val Leu Cys Glu Lys Leu Gln Ala Ala Ile Asn Asp Ser Gln Gln 215 220 225 ctg cag ctt ctc tac ctg gag tgc atc ctg tct gtg ctc agc agc tcc 897 Leu Gln Leu Leu Tyr Leu Glu Cys Ile Leu Ser Val Leu Ser Ser Ser 230 235 240 tcc tcc tcc atg cac ctg cac agg cgc ttc acg gac ctg atc tgg aaa 945 Ser Ser Ser Met His Leu His Arg Arg Phe Thr Asp Leu Ile Trp Lys 245 250 255 aac ctc tgc cct gct ctc atc gtg atc ttg ggg aat cca att cat gac 993 Asn Leu Cys Pro Ala Leu Ile Val Ile Leu Gly Asn Pro Ile His Asp 260 265 270 aaa acc atc acc tct gct cac acc agc agc acc agt acc agc ctg gag 1041 Lys Thr Ile Thr Ser Ala His Thr Ser Ser Thr Ser Thr Ser Leu Glu 275 280 285 290 tcg gac tct gcg tct ccg gga gtg tct gac cac ggc cga gga tca ggc 1089 Ser Asp Ser Ala Ser Pro Gly Val Ser Asp His Gly Arg Gly Ser Gly 295 300 305 tgc tcc tgc act gcg ccg gcc ctg agc gga cct gtg gct cgg act atc 1137 Cys Ser Cys Thr Ala Pro Ala Leu Ser Gly Pro Val Ala Arg Thr Ile 310 315 320 tat tac atc gca gcc gag ctg gtc cgg ctg gtg ggg tct gtg gac tcc 1185 Tyr Tyr Ile Ala Ala Glu Leu Val Arg Leu Val Gly Ser Val Asp Ser 325 330 335 atg aag ccc gtg ctc cag tcc ctc tac cac cga gtg ctg ctc tac ccc 1233 Met Lys Pro Val Leu Gln Ser Leu Tyr His Arg Val Leu Leu Tyr Pro 340 345 350 cca ccc cag cac cgg gtg gaa gcc atc aaa ata atg aaa gag ata ctt 1281 Pro Gln His Arg Val Glue Ala Ile Lys Ile Met Lys Glue Ile Leu 355 360 365 370 ggg agc cca cag cgt ctc tgt gac ttg gca gga ccc agc tcc act gaa 1329 Gly Ser Pro Gln Arg Leu Cys Asp Leu Ala Gly Pro Ser Ser Thr Glu 375 380 385 tca gag tcc aga aaa aga tca att tca aaa aga aag tct cat ctg gat Ser Glu Ser Arg Lys Arg Ser Ile Ser Ser Lys Arg Lys Ser His Leu Asp 390,395,400 ctc ctc aaa ctc atc atg gat ggc atg acc ga gca tgc atc aag ggt 1425 Leu Leu Lys Leu Ile Met Asp Gly Met Thr Glu Ala Cys Ile Lys Gly 405 410 415 ggc atc gaa gct tgc tat gca gcc gtg tcc tgt gtc tgc acc ttg ctg 1473 Gly Ile Glu Ala Cys Tyr Ala Ala Val Ser Cys Val Cys Thr Leu Leu 420 425 430 ggt gcc ctg gat gag ctc agc cag ggg aag ggc tg agc gaa ggt cag 1521 Gly Ala Leu Asp Glu Leu Ser Gln Gly Lys Gly Leu Ser Glu Gly Gln 435 440 445 450 gtg caa ctg ctg ctt ctg cgc ctt gag gag ctg aag gat ggg gct gag 1569 Val Gln Leu Leu Leu Leu Arg Leu Glu Glu Leu Lys Asp Gly Ala Glu 455 460 465 tgg agc cga gat tcc atg gag atc aat gag gct gac ttc cgc tgg cag 1617 Trp Ser Arg Asp Ser Met Glu Ile Asn Glu Ala Asp Phe Arg Trp Gln 470 475 480 cgg cga gtg ctg tcc tca gaa cac acg ccg tgg gag tca ggg aac gag 1665 Arg Arg Val Leu Ser Ser Glu His Thr Pro Trp Glu Ser Gly Asn Glu 485 490 495 agg agc ctt gac atc agc atc agt gtc acc aca gac aca ggc cag acc 1713 Arg Ser Leu Asp Ile Ser Ile Ser Val Thr Thr Asp Thr Gly Gln Thr 500 505 510 act ctc gag gga gag ttg ggt cag act aca ccc gag gac cat tcg gga 1761 Thr Leu Glu Gly Glu Leu Gly Gln Thr Thr Pro Glu Asp His Ser Gly 515 520 525 530 aac cac aag aac agt ctc aag tcg cca gcc atc cca gag ggt aag gag 1809 Asn His Lys Asn Ser Leu Lys Ser Pro Ala Ile Pro Glu Gly Lys Glu 535 540 545 acg ctg agc aaa gta ttg gaa aca gag gcg gta gac cag cca gat gtc 1857 Thr Leu Ser Lys Val Leu Glu Thr Glu Ala Val Asp Gln Pro Asp Val 550 555 560 gtg cag aga agc cac acg gtc cct tac cct gac ata act aac ttc ctg 1905 Val Gln Arg Ser His Thr Val Pro Tyr Pro Asp Ile Thr Asn Phe Leu 565 570 575 tca gta gac tgc agg aca agg tcc tat gga tct agg tat agt gag agc 1953 Ser Val Asp Cys Arg Thr Arg Ser Tyr Gly Ser Arg Tyr Ser Glu Ser 580 585 590 aat ttt agc gtt gat gac caa gac ctt tct agg aca gag ttt gat tcc 2001 Asn Phe Ser Val Asp Asp Gln Asp Leu Ser Arg Thr Glu Phe Asp Ser 595 600 605 610 tgt gat cag tac tct atg gca gca gaa aag gac tcg ggc agg tcc gac 2049 Cys Asp Gln Tyr Ser Met Ala Ala Glu Lys Asp Ser Gly Arg Ser Asp 615 620 625 gtg tca gac att ggg tcg gac aac tgt tca cta gcc gat gaa gag cag 2097 Val Ser Asp Ile Gly Ser Asp Asn Cys Ser Leu Ala Asp Glu Glu Gln 630 635 640 aca ccc cgg gac tgc cta ggc cac cgg tcc ctg cga act gcc gcc ctg 2145 Thr Pro Arg Asp Cys Leu Gly His Arg Ser Leu Arg Thr Ala Ala Leu 645 650 655 tct cta aaa ctg ctg aag aac cag gag gcg gat cag cac agc gcc agg 2193 Ser Leu Lys Leu Leu Lys Asn Gln Glu Ala Asp Gln His Ser Ala Arg 660 665 670 ctg ttc ata cag tcc ctg gaa ggc ctc ctc cct cgg ctc ctg tct ctc 2241 Leu Phe Ile Gln Ser Leu Glu Gly Leu Leu Pro Arg Leu Leu Ser Leu 675 680 685 690 tcc aat gta gag gag gtg gac acc gct ctg cag aac ttt gcc tct act 2289 Ser Asn Val Glu Glu Val Asp Thr Ala Leu Gln Asn Phe Ala Ser Thr 695 700 705 ttc tgc tca ggc atg atg cac tct cct ggc ttt gac ggg aat agc agc 2337 Phe Cys Ser Gly Met Met His Ser Pro Gly Phe Asp Gly Asn Ser Ser 710 715 720 ctc agc ttc cag atg ctg atg aac gca gac agc ctc tac aca gct gca 2385 Leu Ser Phe Gln Met Leu Met Asn Ala Asp Ser Leu Tyr Thr Ala Ala 725 730 735 cac tgc gcc ctg ctc ctc aac ctg aag ctc tcc cac ggt gac tac tac 2433 His Cys Ala Leu Leu Leu Asn Leu Lys Leu Ser His Gly Asp Tyr Tyr 740 745 750 agg aag cgg ccg acc ctg gcg cca ggc gtg atg aag gac ttc atg aag 2481 Arg Lys Arg Pro Thr Leu Ala Pro Gly Val Met Lys Asp Phe Met Lys 755 760 765 770 cag gtg cag acc agc ggc gtg ctg atg gtc ttc tct cag gcc tgg att 2529 Gln Val Gln Thr Ser Gly Val Leu Met Val Phe Ser Gln Ala Trp Ile 775 780 785 gag gag ctc tac cat cag gtg ctc gac agg aac atg ctt gga gag gct 2577 Glu Glu Leu Tyr His Gln Val Leu Asp Arg Asn Met Leu Gly Glu Ala 790 795 800 ggc tat tgg ggc agc cca gaa gat aac agc ctt ccc ctc atc aca atg 2625 Gly Tyr Trp Gly Ser Pro Glu Asp Asn Ser Leu Pro Leu Ile Thr Met 805 810 815 ctg acc gat att gac ggc tta gag agc agt gcc att ggt ggc cag ctg 2673 Leu Thr Asp Ile Asp Gly Leu Glu Ser Ser Ala Ile Gly Gly Gln Leu 820 825 830 atg gcc tcg gct gct aca gag tct cct ttc gcc cag agc agg aga to 2721 Met Ala Ser Ala Ala Thr Glu Ser Pro Phe Ala Gln Ser Arg Arg Ile 835 840 845 850 gat gac tcc aca gtg gca ggc gtg gca ttt gct cgc tat att ctg gtg 2769 Asp Asp Ser Thr Val Ala Gly Val Ala Phe Ala Arg Tyr Ile Leu Val 855 860 865 ggc tgc tgg aag aac ttg atc gat act tta tca acc cca ctg act ggt 2817 Gly Cys Trp Lys Asn Leu Ile Asp Thr Leu Ser Thr Pro Leu Thr Gly 870 875 880 cga atg gcg ggg agc tcc aaa ggg ctg gcc ttc att ctg gga gct gaa 2865 Arg Met Ala Gly Ser Ser Lys Gly Leu Ala Phe Ile Leu Gly Ala Glu 885 890 895 ggc atc aaa gag cag aac cag aag gag cgg gac gcc atc tgc atg agc 2913 Gly Ile Lys Glu Gln Asn Gln Lys Glu Arg Asp Ala Ile Cys Met Ser 900 905 910 ctc gac ggg ctg cgg aaa gcc gca cgg ctg agc tgc gct cta ggc gtt 2961 Leu Asp Gly Leu Arg Lys Ala Ala Arg Leu Ser Cys Ala Leu Gly Val 915 920 925 930 gct gct aac tgc gcc tca gcc ctt gcc cag atg gca gct gcc tcc tgt 3009 Ala Ala Asn Cys Ala Ser Ala Leu Ala Gln Met Ala Ala Ala Ser Cys 935 940 945 gtc caa gaa gaa aaa gaa gag agg gag gcc caa gaa ccc agt gat gcc 3057 Val Gln Glu Glu Lys Glu Glu Arg Glu Ala Gln Glu Pro Ser Asp Ala 950 955 960 atc aca caa gtg aaa cta aaa gtg gag cag aaa ctg gag cag att ggg 3105 Ile Thr Gln Val Lys Leu Lys Val Glu Gln Lys Leu Glu Gln Ile Gly 965 970 975 aag gtg cag ggg gtg tgg ctg cac act gcc cac gtc ttg tgc atg gag 3153 Lys Val Gln Gly Val Trp Leu His Thr Ala His Val Leu Cys Met Glu 980 985 990 gcc atc ctc agc gta ggc ctg gag atg gga agc cac aac ccg gac 3198 Ala Ile Leu Ser Val Gly Leu Glu Met Gly Ser His Asn Pro Asp 995 1000 1005 tgc tgg cca cac gtg ttc agg gtg tgt gaa tac gtg ggc acc ctg 3243 Cys Trp Pro His Val Phe Arg Val Cys Glu Tyr Val Gly Thr Leu 1010 1015 1020 gag cac aac cac ttc agc gat ggt gcc tcg cag ccc cct ctg acc 3288 Glu His Asn His Phe Ser Asp Gly Ala Ser Gln Pro Pro Leu Thr 1025 1030 1035 atc agc cag ccc cag aag gcc act gga agc gct ggc ctc ctt ggg 3333 Ile Ser Gln Pro Gln Lys Ala Thr Gly Ser Ala Gly Leu Leu Gly 1040 1045 1050 gac ccc gag tgt gag ggc tcg ccc ccc gag cac agc ccg gag cag 3378 Asp Pro Glu Cys Glu Gly Ser Pro Pro Glu His Ser Pro Glu Gln 1055 1060 1065 ggg cgc tcc ctg agc acg gcc cct gtc gtc cag ccc ctg tcc atc 3423 Gly Arg Ser Leu Ser Thr Ala Pro Val Val Gln Pro Leu Ser Ile 1070 1075 1080 cag gac ctc gtc cgg gaa ggc agc cgg ggt cgg gcc tcc gac ttc 3468 Gln Asp Leu Val Arg Glu Gly Ser Arg Gly Arg Ala Ser Asp Phe 1085 1090 1095 cgc ggc ggg agc ctc atg agc ggg agc agc gcg gcc aag gtg gtg 3513 Arg Gly Gly Ser Leu Met Ser Gly Ser Ser Ala Ala Lys Val Val 1100 1105 1110 ctc acc ctc tcc acg caa gcc gac agg ctc ttt gaa gat gct acg 3558 Leu Thr Leu Ser Thr Gln Ala Asp Arg Leu Phe Glu Asp Ala Thr 1115 1120 1125 gat aag ttg aac ctc atg gcc ttg gga ggt ttt ctt tac cag ctg 3603 Asp Lys Leu Asn Leu Met Ala Leu Gly Gly Phe Leu Tyr Gln Leu 1130 1135 1140 aag aaa gca tcg cag tct cag ctt ttc cat tct gtt aca gat aca Lys Lys Ala Ser Gln Ser Gln Leu Phe His Ser Val Thr Asp Thr 1145 1150 1155 gtt gat tac tct ctg gca atg cca gga gaa gtt aaa tcc act caa 3693 Val Asp Tyr Ser Leu Ala Met Pro Gly Val Lys Ser Thr Gln 1160 1165 1170 gac cga aaa agc gcc ctc cac ctg ttc cgc ctg ggg aat gcc atg 3738 Asp Arg Lys Ser Ala Leu His Leu Phe Arg Leu Gly Asn Ala Met 1175 1180 1185 ctg agg att gtg cgg agc aaa gca cgg ccc ctc cac gtg atg 3783 Leu Arg Ile Val Arg Ser Lys Ala Arg Pro Leu Leu His Val Met 1190 1195 1200 cgc tgc tgg agc ctt gtg gcc cca cac ctg gtg gag gct gct tgc 3828 Arg Cys Trp Ser Leu Val Wing Pro His Leu Val Glu Wing Wing Cys 1205 1210 1215 cat aag gaa aga cat gtg tct cag aag gct gtt tcc ttc atc cat 3873 His Lys Glu Arg His Val Ser Gln Lys Ala Val Ser Phe Ile His 1220 1225 1230 gac ata ctg aca gaa gtc ctc act gac tgg aat gag cca cct cat 3918 Asp Ile Leu Thr Glu Val Leu Thr Asp Trp Asn Glu Pro Pro His 1235 1240 1245 ttt cac ttc aat gaa gca ctc ttc cga cct ttc gag cgc att atg 3963 Phe His Phe Asn Glu Ala Leu Phe Arg Pro Phe Glu Arg Ile Met 1250 1255 1260 cag ctg gaa ttg tgt gat gag gac gtc caa gac cag gtt gtc aca 4008 Gln Leu Glu Leu Cys Asp Glu Asp Val Gln Asp Gln Val Val Thr 1265 1270 1275 tcc att ggt gag ctg gtt gaa gtg tgt tcc acg cag atc cag tcg 4053 Ser Ile Gly Glu Leu Val Glu Val Cys Ser Thr Gln Ile Gln Ser 1280 1285 1290 gga tgg aga ccc ttg ttc agt gcc ctg gaa aca gtg cat ggc ggg 4098 Gly Trp Arg Pro Leu Phe Ser Ala Leu Glu Thr Val His Gly Gly 1295 1300 1305 aac aag tca gag atg aag gag tac ctg gtt ggt gac tac tcc atg 4143 Asn Lys Ser Glu Met Lys Glu Tyr Leu Val Gly Asp Tyr Ser Met 1310 1315 1320 gga aaa ggc caa gct cca gtg ttt gat gta ttt gaa gct ttt ctc 4188 Gly Lys Gly Gln Ala Pro Val Phe Asp Val Phe Glu Ala Phe Leu 1325 1330 1335 aat act gac aac atc cag gtc ttt gct aat gca gcc act agc tac 4233 Asn Thr Asp Asn Ile Gln Val Phe Ala Asn Ala Ala Thr Ser Tyr 1340 1345 1350 atc atg tgc ctt atg aag ttt gtc aaa gga ctg ggg gag gtg gac 4278 Ile Met Cys Leu Met Lys Phe Val Lys Gly Leu Gly Glu Val Asp 1355 1360 1365 tgt aaa gag att gga gac tgt gcc cca gca ccc gga gcc ccg tcc 4323 Cys Lys Glu Ile Gly Asp Cys Ala Pro Ala Pro Gly Ala Pro Ser 1370 1375 1380 aca gac ctg tgc ctc ccg gcc ctg gat tac ctc agg cgc tgc tct 4368 Thr Asp Leu Cys Leu Pro Ala Leu Asp Tyr Leu Arg Arg Cys Ser 1385 1390 1395 cag tta ttg gcc aaa atc tac aaa atg ccc ttg aag cca ata ttc 4413 Gln Leu Leu Ala Lys Ile Tyr Lys Met Pro Leu Lys Pro Ile Phe 1400 1405 1410 ctt agt ggg aga ctt gcc ggc ttg cct cga aga ctt cag gaa cag 4458 Leu Ser Gly Arg Leu Ala Gly Leu Pro Arg Arg Leu Gln Glu Gln 1415 1420 1425 tca gcc agc agt gag gat gga att gaa tca gtc ctg tct gat ttt 4503 Ser Ala Ser Ser Glu Asp Gly Ile Glu Ser Val Leu Ser Asp Phe 1430 1435 1440 gat gat gac acc ggt ctg ata gaa gtc tgg ata atc ctg ctg gag 4548 Asp Asp Asp Thr Gly Leu Ile Glu Val Trp Ile Ile Leu Leu Glu 1445 1450 1455 cag ctg aca gcg gct gtg tcc aat tgt cca cgg cag cac caa cca 4593 Gln Leu Thr Ala Ala Val Ser Asn Cys Pro Arg Gln His Gln Pro 1460 1465 1470 cca act ctg gat tta ctc ttt gag ctg ttg aga gat gtg acg aaa 4638 Pro Thr Leu Asp Leu Leu Phe Glu Leu Leu Arg Asp Val Thr Lys 1475 1480 1485 aca cca gga cca ggg ttt ggt atc tat gca gtg gtt cac ctc ctc 4683 Thr Pro Gly Pro Gly Phe Gly Ile Tyr Ala Val Val His Leu Leu 1490 1495 1500 ctt cct gtg atg tcc gtt tgg ctc cgc cgg agc cat aaa gac cat 4728 Leu Pro Val Met Ser Val Trp Leu Arg Arg Ser His Lys Asp His 1505 1510 1515 tcc tac tgg gat atg gcc tct gcc aat ttc aag cac gct att ggt 4773 Ser Tyr Trp Asp Met Ala Ser Ala Asn Phe Lys His Ala Ile Gly 1520 1525 1530 ctg tcc tgt gag ctg gtg gtg gag cac att caa agc ttt cta cat 4818 Leu Ser Cys Glu Leu Val Val Glu His Ile Gln Ser Phe Leu His 1535 1540 1545 tca gat atc agg tac gag agc atg atc aat acc atg ctg aag gac 4863 Ser Asp Ile Arg Tyr Glu Ser Met Ile Asn Thr Met Leu Lys Asp 1550 1555 1560 ctc ttt gag ttg ctg gtc gcc tgt gtg gcc aag ccc act gaa acc 4908 Leu Phe Glu Leu Leu Val Ala Cys Val Ala Lys Pro Thr Glu Thr 1565 1570 1575 atc tcc aga gtg ggc tgc tcc tgt att aga tac gtc ctt gtg aca 4953 Ile Ser Arg Val Gly Cys Ser Cys Ile Arg Tyr Val Leu Val Thr 1580 1585 1590 gcg ggc cct gtg ttc act gag gag atg tgg agg ctt gcc tgc tgt 4998 Ala Gly Pro Val Phe Thr Glu Glu Met Trp Arg Leu Ala Cys Cys 1595 1600 1605 gcc ctg caa gat gcg ttc tct gcc aca ctc aag cca gtg aag gac 5043 Ala Leu Gln Asp Ala Phe Ser Ala Thr Leu Lys Pro Val Lys Asp 1610 1615 1620 ctg ctg ggc tgc ttc cac agc ggc acg gag agc ttc agc ggg gaa 5088 Leu Leu Gly Cys Phe His Ser Gly Thr Glu Ser Phe Ser Gly Glu 1625 1630 1635 ggc tgc cag gtg cga gtg gcg gcc ccg tcc tcc tcc cca agt gcc 5133 Gly Cys Gln Val Arg Val Ala Wing Pro Be Being Pro Being Wing 1640 1645 1650 gag gcc gag tac tgg cgc atc cga gcc atg gcc cag cag gtg ttt 5178 Glu Ala Glu Tyr Trp Arg And Arg Ala Met Ala Gln Gln Val Phe 1655 1660 1665 atg ctg gac acc cag tgc tca cca aag aca cca aac aac ttt gac 5223 Met Leu Asp Thr Gln Cys Ser Pro Lys Thr Pro Asn Asn Phe Asp 1670 1675 1680 cac gct cag tcc tgc cag ctc att att gag ctg cct cct gat gaa 5268 His Ala Gln Ser Cys Gln Leu Ile Ile Glu Leu Pro Pro Asp Glu 1685 1690 1695 aaa cca aat gga cac acc aag aaa agc gtg tct ttc agg gaa att Lys Pro Asn Gly His Thr Lys Ser Val Ser Phe Arg Glu Ile 1700 1705 1710 gtg gtg agc ctg ctg tct cat cag gtg tta ctc cag aac tta tat 5358 Val Val Ser Leu Leu Ser His Gln Val Leu Leu Gln Asn Leu Tyr 1715 1720 1725 gac atc ttg tta gaa gag ttt gtc aaa ggc ccc tct cct gga gag 5403 Asp Ile Leu Leu Glu Glu Phe Val Lys Gly Pro Ser Pro Gly Glu 1730 1735 1740 gaa aag acg ata caa gtg cca gaa gcc aag ctg gct ggc ttc ctc 5448 Glu Lys Thr Ile Gln Val Pro Glu Ala Lys Leu Ala Gly Phe Leu 1745 1750 1755 aga tac atc tct atg cag aac ttg gca gtc ata ttc gac ctg ctg 5493 Arg Tyr Ile Ser Met Gln Asn Leu Ala Val Ile Phe Asp Leu Leu 1760 1765 1770 ctg gac tct tat agg act gcc agg gag ttt gac acc agc ccc ggg 5538 Leu Asp Ser Tyr Arg Thr Ala Arg Glu Phe Asp Thr Ser Pro Gly 1775 1780 1785 ctg aag tgc ctg ctg aag aaa gtg tct ggc atc ggg ggc gcc gcc 5583 Leu Lys Cys Leu Leu Lys Lys Val Ser Gly Ile Gly Gly Ala Ala 1790 1795 1800 aac ctc tac cgc cag tct gcg atg agc ttt aac att tat ttc cac 5628 Asn Leu Tyr Arg Gln Ser Ala Met Ser Phe Asn Ile Tyr Phe His 1805 1810 1815 gcc ctg gtg tgt gct gtt ctc acc aat caa gaa acc atc acg gcc 5673 Ala Leu Val Cys Ala Val Leu Thr Asn Gln Glu Thr Ile Thr Ala 1820 1825 1830 gag caa gtg aag aag gtc ctt ttt gag gac gac gag aga agc acg 5718 Glu Gln Val Lys Lys Val Leu Phe Glu Asp Asp Glu Arg Ser Thr 1835 1840 1845 gat tct tcc cag cag tgt tca tct gag gat gaa gac atc ttt gag 5763 Asp Ser Ser Gln Gln Cys Ser Ser Glu Asp Glu Asp Ile Phe Glu 1850 1855 1860 gaa acc gcc cag gtc agc ccc ccg aga ggc aag gag aag aga cag 5808 Glu Thr Ala Gln Val Ser Pro Pro Arg Gly Lys Glu Lys Arg Gln 1865 1870 1875 tgg cgg gca cgg atg ccc ttg ctc agc gtc cag cct gtc agc aac 5853 Trp Arg Ala Arg Met Pro Leu Leu Ser Val Gln Pro Val Ser Asn 1880 1885 1890 gca gat tgg gtg tgg ctg gtc aag agg ctg cac aag ctg tgc atg 5898 Ala Asp Trp Val Trp Leu Val Lys Arg Leu His Lys Leu Cys Met 1895 1900 1905 gaa ctg tgc aac aac tac atc cag atg cac ttg gac ctg gag aac 5943 Glu Leu Cys Asn Asn Tyr Ile Gln Met His Leu Asp Leu Glu Asn 1910 1915 1920 tgt atg gag gag cct ccc atc ttc aag ggc gac ccg ttc ttc atc 5988 Cys Met Glu Glu Pro Pro Ile Phe Lys Gly Asp Pro Phe Phe Ile 1925 1930 1935 ctg ccc tcc ttc cag tcc gag tca tcc acc cca tcc acc ggg ggc 6033 Leu Pro Ser Phe Gln Ser Glu Ser Ser Thr Pro Ser Thr Gly Gly 1940 1945 1950 ttc tct ggg aaa gaa acc cct tcc gag gat gac aga agc cag tcc 6078 Phe Ser Gly Lys Glu Thr Pro Ser Glu Asp Asp Arg Ser Gln Ser 1955 1960 1965 cgg gag cac atg ggc gag tcc ctg agc ctg aag gcc ggt ggt ggg 6123 Arg Glu His Met Gly Glu Ser Leu Ser Leu Lys Ala Gly Gly Gly 1970 1975 1980 gac ctg ctg ctg ccc ccc agc ccc aaa gtg gag aag aag gat ccc 6168 Asp Leu Leu Leu Pro Ser Pro Ser Pro Lys Val Glu Lys Lys Asp Pro 1985 1990 1995 agc cgg aag aag gag tgg tgg gag at gcg ggg aac aaa atc tac 6213 Ser Arg Lys Glu Trp Trp Glu Asn Ala Gly Asn Lys Ile Tyr 2000 2005 2010 acc atg gca gcc gac aag acc att tca aag ttg atg acc gaa tac 6258 Thr Met Ala Asp Lys Thr Ile Ser Lys Leu Met Thr Glu Tyr 2015 2020 2025 aaa aag agg aaa cag cag cac aac ctg tcc gcg ttc ccc aaa gag Lys Lys Arg Lys Gln Gln His Asn Leu Ser Ala Phe Pro Lys Glu 2030 2035 2040 gtc aaa gtg gag aag aaa gga gag cca ctg ggt ccc agg ggc cag 6348 Val Lys Val Glu Lys Lys Gly Glu Pro Leu Gly Pro Arg Gly Gln 2045 2050 2055 gac tcc ccg ctg ctt cag cgt ccc cag cac ttg atg gac caa ggg 6393 Asp Ser Pro Leu Leu Gln Arg Pro Gln His Leu Met Asp Gln Gly 2060 2065 2070 caa atg cgg cat tcc ttc agc gca ggc ccc gag ctg ctg cga cag 6438 Gln Met Arg His Ser Phe Ser Ala Gly Pro Glu Leu Leu Arg Gln 2075 2080 2085 gac aag agg ccc cgc tca ggc tcc acc ggg agc tcc ctc agt gtc 6483 Asp Lys Arg Pro Arg Ser Gly Ser Thr Gly Ser Ser Leu Ser Val 2090 2095 2100 tcg gtg aga gac gca gaa gca cag atc cag gca tgg acc aac atg 6528 Ser Val Arg Asp Ala Glu Ala Gln Ile Gln Ala Trp Thr Asn Met 2105 2110 2115 gtg cta aca gtt ctc aat cag att cag att ctc cca gac cag acc 6573 Valley Leu Thr Val Leo Asn Gln Ile Leo Pro Asp Gln Thr 2120 2125 2130 ttc acg gcc ctc cag ccc gca gtg ttc ccg tgc atc agt cag ctg 6618 Phe Thr Ala Leu Gln Pro Ala Val Phe Pro Cys Ile Ser Gln Leu 2135 2140 2145 acc tgt cac gtg acc gac atc aga gtt cgc cag gct gtg agg gag 6663 Thr Cys His Is Thr Asp With Arg Org Gln Ala And Arg Glu 2150 2155 2160 tgg ctg ggc agg gtg ggc cgt gtc tat gac atc att gtg tag 6705 Trp Leu Gly Arg Val Gly Arg Val Tyr Asp Ile Ile Val 2165 2170 2175 ccgactcctg ttctactctc ccaccaata acagtagtga gggttagagt cctgccaata 6765 cagctgttgc atttcccca ccactagccc cacttaact actactactg tctcagagaa 6825 cagtgtttcc taatgtaaaa agcctttcca accactgatc agcattgggg ccatactaag 6885 gtttgtatct agatgacaca aacgatattc tgattttgca cattattata gaagaatcta 6945 taatccttga tatgtttcta actcttgaag tatatttccc atgctttg cttacagtgt 7005 tgtccccaaa tggtcattt tcaggatta ctcatttgaa aacactatat tgatccattt gatccatcat ttaaaaaata atacaatc ctaggcaat atctgctggt aagtcaagct 7125 gataaacact cagacatcta gtaccaggga ttattaattg gaggagatt tattggttatg 7185 ggtctggctg ggagagac aacttaaat acatattctt gggtgtcata atcaagaag 7245 aggtgacttc tgttgtaaaa taatccagaa cactcaaa ttattcctaa atcattaaga 7305 tttcaggta ttcaccaatt tcccatgta aggtactgtg ttgtaccttt atttctgtat 7365 ttctaaaaga agaaagttct ttcctagcag ggtttgaagt ctgtggctta tcagcctgtg 7425 acacagagta cccagtgaaa gtggctgta cgtagattgt caagacat aagaccgacc 7485 agccaccctg gctgttcttg tggtgttgt ttccaccc aaggcaaca aggaaaggaa 7545 aggaaagaag aaaagtgcc ttagtccttt gttgcactc catttccattg ccccacatt 7605 gtctgaacat aaggtatagc atttggtttt tagaaaaaaacattaag acgcaactca 7665 ttttatatca acacgcttgg aggaaaggga ctcagggaag ggagcaggga gtgtggggtg 7725 gggatggatt atgatgaaat cattttcaat cttaaaatat aatacaacaa tcttgcaaaa 7785 ttatggtgtc agttacacaa gctctagtct caaaatgaaa gtaatggaga aagacactga 7845 aatttagaaa attttgtcga tttaaaatat ttctcctatc taccaagtaa agttacccta 7905 tgtttgatgt ctttgcattc agaccaatat ttcaggtgga tatttctaag tattactaga 7965 aaatacgttt gaaagcttta tcttattatt tacagtattt ttatatttct tacattatcc 8025 taatgattga aaactcctca atcaagctta cttacacaca ttctacagag ttatttaagg 8085 catacattat aatctcccag ccccattcat aatgaataag tcacccttta aatataagac 8145 acaaattcta cagtattgaa ataaggattt aaaggggtat ttgtaaactt tgccctcctt 8205 gagaaatatg gaactacctt agaggttaag aggaaggcag tgttctgact tctttaggtg 8265 atctgaaaaa aacaccctta tcatccagtg taccatctag agatcaccac agaatccatt 8325 tttttcccag ttccacaaaa cactctgttt gccttcagtt tttactcact agacaataat 8385 tcaagtttag aaacaggtaa tcagctattt gatcttaaaa ggcaatgaat tgttgggata 8445 tcagtgaact atgttgtata cttttgaatt tttacattt ataaatggaa ttgaaagttg 8505 gataactgct ttttttaaat tttccaacag aagtaacacc acagttgctt tgtttcttt 8565 tatagcttac ctgaggttca gttcttcttt gtgaacctgt gagtactcca cagtttactg 8625 ggggaaaagg cttcagtaaa gcagaggcta gaattacagt atttatacat agcaactttt 8685 cataaagtag aaaaattcaa aggaagctgt ctcaatttga gaataccagc tgggcacggt 8745 ggctcacgcc tgtaatccca gcacttactt tgggaggcca aggtgggcag ataacctgcg 8805 gtcaggagtt tgagaccagg ctggacaaca tggtgaaacc tcgtctctac taaaaataca 8865 aaaattagcc aggtgtggta ggatgcacct gtaatcccag ctacttagga ggccgagaca 8925 ggagaatcgc tcgaacccag gaggcggacg ttgcagtgag ccaagattgc accattgcac 8985 tccagactgg gtgacaagag tgaaactcca tctaaaaaaa aaaaaaaaaa aaagtgaata 9045 ctgtatccca aagtatgtta gttgtttgtt tggaaatcag cattctcccc gatgctctat 9105 tatgggatcc aaaattctg aacataagtt taccctgtac tgtgtccaaa cactgttcta 9165 gttctagcct gattatgggt cccaagaata aaaggatgag taggtgtaca gagctcttga 9225 cctacaattt tttaagagtg ttttggtacc ttcccattgt cttctctata actcagtcct 9285 aacatactct gcactcgagt taccagccat ccacactgac atcagatttc aaccagaacc 9345 atcactgagt gacagcagta cttctcagag gtatttgcag cttgatgcaa agtagtctct 9405 aatgagtagg cattcaggtg gttcttccca gcaggtggag aagaaaggga ggagatgaag 9465 aacactgaga ggggagtggc accttcccag gctgcccagc tcagtctctt gccctgttcc 9525 tgtgactcag ctgcccactc ccccaacttt gttccctcc ctcccagtct ctgaaagtgt 9585 caggtgtttc tctcctcaca gtctcttttg cagcaacagt aagacaaaat tcaaggcagc 9645 cttttaaagt tacgaacagt tattagcatg tatttacaga cctaagcaga atgagagttt 9705 atacattgtt tttagttgcc tgtatttata gccaaaagta tattacctta aagttgagat 9765 ctttctcttc ttttcctaaa ttttggtaaa gtgtgcttca tgaaacaaac atctggaaaa 9825 ctccaagtat aagagaccct ggactgatga tggcccagcc aagtatatgg agggacagag 9885 ttctctctgt cattaatgag gacatcggtt ttcacaattg aacctcatgc actgtccaca 9945 gcatctcacc tagctcctgt atctcctgat ctgcttttaa aaatagttag ttaggctgcc 10005 ttttacacc accttctctc tctccccttg tggtaattttt ccagccttcc ccatagatat 10065 aaaactagaa cacctttatg atttggggtc tatgtaatga ctgaccgata agaacccagg 10125 cagatgctaa catacttaac agctcgcatt aaaatacttt aaatcaggcg tgatggctca 10185 ttcctgtaat ctcaagcact ttgggaggct aaggtgggtg gatctcttga ggtcaggagt 10245 tcgagaccaa cctggccaac gtggtgaaac cccgtctcta ctaaaaatac aaaattagcc 10305 gggcatggtg gcagctgcct gtaatcccag ctactcggga agctgaggca ggagaattgc 10365 ttgaacctgg gaggtgggga ttgcagtcag ccaagattgt tctgcagcat gggtgacaaa 10425 gtgagacttc gtctcaagta aataaaacta aaatttttaa atcaaacatg acaaaaatgt 10485 tatataatt cagaagtacc ttgaaattga aacatatttg tgcaatgatc attaggcttt 10545 ttgtccttgt tgttttaaaa tgaggcttat acagagtgag tgagagtca agtagccttc 10605 gctgtgagac ggtaatgcag ttatatatata gataccttg actttgccag attcatcaca 10665 atactgctta tacaggaag ttttctcaga aaggaaaatc cattagtac agtcccatca 10725 agccaaacag atgaagacc tttagagta atagcaagg gttachaata gcagggagga 10785 ggcgagtagt gatgtcact gtgattgcaa acccttacct gtattatcac acgtagtcct 10845 cacacaacc ttgtgagaca agtgttgtgt tcctcatttt ttcagagggg aacacagacc 10905 cagagaggtt aagaatttg cccagaata caagtaaag gcaagttgg ttgcaaaga 10965 ggtgttctg aattcaaggg ccatactctc tctctgacaa catgctcta gtccatagag 11025 taagcactct agtatgaaa aaagtttca ggaacgaggc catgaaatg agactatttg 11085 acatctcaga tctgtctggg atgttatgga gttttttaa aaaagttg aaaaaagaaa 11145 atgaatcatg ttatacata aaaaaatcac atgtaacaca ttcaagtgt ttgaaaataa 11205 aaccaaaatc taaacttag tctcaagca gatacagt gttactttag aaaactcact 11265 gattaggtg gaatgatgg ataatacta ttcatggcca gctattaca cagagaaca 11325 tggcagtgtg tgtctggaac ggcatgcaca atttgtaaac ctttttcaa tatcattaa 11385 tcaactcaga ataagtgcc ctgtagccaa cagtgcctct ttacttgctt ctctgggaaa 11445 tacatggtac taaattagta gcacaaagtt tgggaatg caaataatg gataaccatt 11505 tttcaaatg tacattctt gagaggag cagctggttg gaggattt cttgagagc 11565 caggtgctaa gggcatcagg tcgacatcca tagtaccat gtgccataac atctacacat 11625 ttccactgt tttacagaca aggtaacagg cagaaggaaa atccagagtc ttgcagtaag 11685 cagatgacaa aacttcaata tgcttgggca ccacttaggt gaccccaggg agatttagtg 11745 tggccttagg aaagcaaag agcactttt attggaaata tgagcttgtc actgggaaag 11805 atttgtaaaa ttgatcaaga acttgattta taattatgcc tcaaaaaaa aagttctcat 11865 ttagtagtgg agcaatctag aaaacatacc ttttgttt gtttggaaga tcctctttcc 11925 ctggctgtat tgtagtgttt gctatttgat gtggaaataa ctaataactt aagattttgg 11985 aacagaacac cctttagatt tccaaaacac aattcttatt tcagggaaga cagaccaaaa 12045 atatctcctg agatcattgg tttctttata aattgtggta ccactccatc attgaagaga 12105 aaccactacc acaccactag caccatacag aaccttttct ctgtatcttt gtacaatact 12165 acaaaggggt accagggagg agagagtggc tgaccacttt agtgacaaaa cagcactcca 12225 ctgctggtga atcccatcta attatggtcc ttccaccctt ttcaaccacc aacaactgtt 12285 cgtactgtta attcctatcc tgaaggttta accagtggtt gtctagtatc ttctgtcttt 12345 agaacagtgg ttctcaaact ttagtacaca tcagcatcac ctggagggcc tttttttaaa 12405 ataagacaca gattgctggg ctcatggtca gagttcccag ttaagtaaat caggaaattt 12465 gtatttctaa caagtttata ggtgaggcca atactgctgt tttgggaact atgctttgag 12525 aaccactgcc ttgaaaaaat ttccaacttc tacctttaag atcagcctga cttatcaaac 12585 gctagagaaa aactgaatct acccttgggc agatgacttg ggattggatt ctatacagca 12645 gtcttgctca atcttcccag tttccagttt tattatacca acaattggtt tttacaagct 12705 agaagacaat gaatgtataa gttctatgga acagtgagat aaatctaagc ttcttgtctt 12765 tgtatttaga aacattgatt ctatggatga tcatttgtat catgttgacc ctttgacttg 12825 tactgaaggt gattttaaat ttaagtatgt agtgtttgaa tttcttccat ccatgtcgtt 12885 ttaatgagat gtttccatgt cagctccttt acagccttgg ctcctggctt acagattttt 12945 gaatagttgt ttgcttgcca gttgttttac atctttcatt ggccaccaaa atattagcca 13005 tttgagatga gatgagacta cttgttgtac cttcatcttt catttaattt tctggcgtaa 13065 attaacattt taatttcata tatatctgta aagagtctac ccaaaggctt cacggaaatt 13125 tgcaaaatga actaattccc ttttaagcag caggtgtgcc tgtttttgac ttttcagtaa 13185 atatgttgtt tgtgcacata tctacatggt ggagaccata ttcattattt catcttccaa 13245 ataatgggaa aaatataaaa gtgaatcagt gtgctttggg aattcagtga aatcatgtta 13305 actcatatag agggggcctt agtttatctc ttctttactg aattaattag ttttggaaat 13365 tcttttacca ttaaaaaaaa ttaaggacca tacagagaat gatttaagaa aaaacaagtc 13425 acttaaaaat catcacctat ttataaactg tattaattac acataatgct tattgattca 13485 atgaggttc tctaaagact tctgcttaat aaatatgctg acttcattta aattagttta 13545 gactattgta ggaatggaag gaaatgatta tatttactag aattagtgag atcagaaagc 13605 atatcagaat gttgatgata tcaaggagac aatctacaga gtttttgcct ctgtggatgg 13665 aaataagggt gtttttttt ggtttttt ttactttagt ttcccataat ttttggaaat 13725 tatgtgtgca tttagttctt ttagtaacac tgattttaaa attaaatttc aaaagtcaat 13785 ctctaagagt aatttatttt tgttttacca accagtgcca aaaaggagag gagggaatcc 13845 aaaagccaat cttttgaacc aatgtgtaaa agattatgtt ttttcttaaa gttagggagg 13905 ctcgggccct gacactgcca gccccagtga gcatccctgg ctacctcggg attatgtgca 13965 agctgctttg tcctacattt ctttcatctg gttcttattg ggagtgcttc tctctaataa 14025 aaattgattt cccacaaaat aggcaaagct gaacaaagat gaatgctttt gataagttgg 14085 gttcacttc agttgaaaca atgtgataga atatccaggt gtggcatgat ggggcaggag 14145 gaggtgccta gagggaaaag ttatttgtgt ttcttagtgt tgtgttgtgg ggatgggaca 14205 gataagaata agatgttat tgccctaatc atgctaagag actattattc aatatgcttt 14265 tcccgctttt ctaagaggaa taaacttaga caaattacat tataaacgt tcccctacta 14325 ctatctccca ctctagataa agccagtggg tggtatgggt ccttttattc cttatagtat 14385 tatgccaaag aatcaactta ttttcattga agattataaa taaatgaagc ttgttatagc 14445 cataatgatt tgagtcagta taccatttta cctataaaat gcaaaattca tccttgcaac 14505 cccattcacc aggagccttg aagcattttg tttactccaa aggccttgtc aaggaagcat 14565 aatttttgt tttgccttct tatttagtca gtttggtcat atttacttaa aaaaacaaac 14625 tgaaaatcac actcctttat atgttgatat aactgattttt atagaatctg tctgttcttt 14685 gttaacagg tctctgtaag caagcttgca agtgtatttt gtgtacattt tatctgaggt 14745 ggaaatgaaa attctaaaga gaaaatattt taaaagatat tgtatttatg ttgcttgtgt 14805 tgtagaataa agattcaaat gcattaaaaa tctggtacat gaaacaattg tgtttactga 14865 ataatatat ataaataaaa aaaaaaaaaa 14895 <210> 32 <211> 2177 <212> PRT <213> Homo sapiens <400> 32 Put Glu Glu Ile Leu Arg Lys Leu Gln Lys Glu Ala Ser Gly Ser Lys 1 5 10 15 Tyr Lys Ala Ile Lys Glu Ser Cys Thr Trp Ala Leu Glu Thr Leu Gly 20 25 30 Gly Leu Asp Thr Ile Val Lys Ile Pro Pro His Val Leu Arg Glu Lys 35 40 45 Cys Leu Leu Pro Leu Gln Leu Ala Leu Glu Ser Lys Asn Val Lys Leu 50 55 60 Ala Gln His Ala Leu Ala Gly Put Gln Lys Leu Leu Ser Glu Glu Arg 65 70 75 80 Phe Val Ser Met Glu Thr Asp Ser Asp Glu Lys Gln Leu Leu Asn Gln 85 90 95 Ile Leu Asn Ala Val Lys Val Thr Pro Ser Leu Asn Glu Asp Leu Gln 100 105 110 Val Glu Val Met Lys Val Leu Leu Cys Ile Thr Tyr Thr Pro Thr Phe 115 120 125 Asp Leu Asn Gly Ser Ala Val Leu Lys Ile Ala Glu Val Cys Ile Glu 130 135 140 Thr Tyr Ile Ser Ser Cys His Gln Arg Ser Ile Asn Thr Ala Val Arg 145 150 155 160 Ala Thr Leu Ser Gln Met Leu Ser Asp Leu Thr Leu Gln Leu Arg Gln 165 170 175 Arg Gln Glu Asn Thr Ile Ile Glu Asn Pro Asp Val Pro Gln Asp Phe 180 185 190 Gly Asn Gln Gly Ser Thr Val Glu Ser Leu Cys Asp Asp Val Val Ser 195 200 205 Val Leu Thr Val Leu Cys Glu Lys Leu Gln Ala Ala Ile Asn Asp Ser 210 215 220 Gln Gln Leu Gln Leu Leu Tyr Leu Glu Cys Ile Leu Ser Val Leu Ser 225 230 235 240 Ser Ser Ser Ser Met His Leu His Arg Arg Phe Thr Asp Leu Ile 245 250 255 Trp Lys Asn Leu Cys Pro Ala Leu Ile Val Ile Leu Gly Asn Pro Ile 260 265 270 His Asp Lys Thr Ile Thr Ser Ala His Thr Ser Ser Thr Ser Thr Ser 275 280 285 Leu Glu Ser Asp Ser Ala Ser Pro Gly Val Ser Asp His Gly Arg Gly 290 295 300 Ser Gly Cys Ser Cys Thr Ala Pro Ala Leu Ser Gly Pro Val Ala Arg 305 310 315 320 Thr Ile Tyr Tyr Ile Ala Ala Glu Leu Val Arg Leu Val Gly Ser Val 325 330 335 Asp Ser Met Lys Pro Val Leu Gln Ser Leu Tyr His Arg Val Leu Leu 340 345 350 Tyr Pro Pro Pro Gln His Arg Val Glu Ala Ile Lys Ile Met Lys Glu 355 360 365 Ile Leu Gly Ser Pro Gln Arg Leu Cys Asp Leu Ala Gly Pro Ser Ser 370 375 380 Thr Glu Ser Glu Ser Arg Lys Arg Ser Ile Ser Lys Arg Lys Ser His 385 390 395 400 Leu Asp Leu Leu Lys Leu Ile Met Asp Gly Met Thr Glu Ala Cys Ile 405 410 415 Lys Gly Gly Ile Glu Ala Cys Tyr Ala Ala Val Ser Cys Val Cys Thr 420 425 430 Leu Leu Gly Ala Leu Asp Glu Leu Ser Gln Gly Lys Gly Leu Ser Glu 435 440 445 Gly Gln Val Gln Leu Leu Leu Leu Arg Leu Glu Glu Leu Lys Asp Gly 450 455 460 Ala Glu Trp Ser Arg Asp Ser Met Glu Ile Asn Glu Ala Asp Phe Arg 465 470 475 480 Trp Gln Arg Arg Val Leu Ser Ser Glu His Thr Pro Trp Glu Ser Gly 485 490 495 Asn Glu Arg Ser Leu Asp Ile Ser Ile Ser Val Thr Thr Asp Thr Gly 500 505 510 Gln Thr Thr Leu Glu Gly Glu Leu Gly Gln Thr Thr Pro Glu Asp His 515 520 525 Ser Gly Asn His Lys Asn Ser Leu Lys Ser Pro Ala Ile Pro Glu Gly 530 535 540 Lys Glu Thr Leu Ser Lys Val Leu Glu Thr Glu Ala Val Asp Gln Pro 545 550 555 560 Asp Val Val Gln Arg Ser His Thr Val Pro Tyr Pro Asp Ile Thr Asn 565 570 575 Phe Leu Ser Val Asp Cys Arg Thr Arg Ser Tyr Gly Ser Arg Tyr Ser 580 585 590 Glu Ser Asn Phe Ser Val Asp Asp Gln Asp Leu Ser Arg Thr Glu Phe 595 600 605 Asp Ser Cys Asp Gln Tyr Ser Met Ala Ala Glu Lys Asp Ser Gly Arg 610 615 620 Ser Asp Val Ser Asp Ile Gly Ser Asp Asn Cys Ser Leu Ala Asp Glu 625 630 635 640 Glu Gln Thr Pro Arg Asp Cys Leu Gly His Arg Ser Leu Arg Thr Ala 645 650 655 Ala Leu Ser Leu Lys Leu Leu Lys Asn Gln Glu Ala Asp Gln His Ser 660 665 670 Ala Arg Leu Phe Ile Gln Ser Leu Glu Gly Leu Leu Pro Arg Leu Leu 675 680 685 Ser Leu Ser Asn Val Glu Glu Val Asp Thr Ala Leu Gln Asn Phe Ala 690 695 700 Ser Thr Phe Cys Ser Gly Met Met His Ser Pro Gly Phe Asp Gly Asn 705 710 715 720 Ser Ser Leu Ser Phe Gln Met Leu Met Asn Ala Asp Ser Leu Tyr Thr 725 730 735 Ala Ala His Cys Ala Leu Leu Leu Asn Leu Lys Leu Ser His Gly Asp 740 745 750 Tyr Tyr Arg Lys Arg Pro Thr Leu Ala Pro Gly Val Met Lys Asp Phe 755 760 765 Met Lys Gln Val Gln Thr Ser Gly Val Leu Met Val Phe Ser Gln Ala 770 775 780 Trp Ile Glu Glu Leu Tyr His Gln Val Leu Asp Arg Asn Met Leu Gly 785 790 795 800 Glu Ala Gly Tyr Trp Gly Ser Pro Glu Asp Asn Ser Leu Pro Leu Ile 805 810 815 Thr Met Leu Thr Asp Ile Asp Gly Leu Glu Ser Ser Ala Ile Gly Gly 820 825 830 Gln Leu Met Ala Ser Ala Ala Thr Glu Ser Pro Phe Ala Gln Ser Arg 835 840 845 Arg Ile Asp Asp Ser Thr Val Ala Gly Val Ala Phe Ala Arg Tyr Ile 850 855 860 Leu Val Gly Cys Trp Lys Asn Leu Ile Asp Thr Leu Ser Thr Pro Leu 865 870 875 880 Thr Gly Arg Met Ala Gly Ser Ser Lys Gly Leu Ala Phe Ile Leu Gly 885 890 895 Ala Glu Gly Ile Lys Glu Gln Asn Gln Lys Glu Arg Asp Ala Ile Cys 900 905 910 Met Ser Leu Asp Gly Leu Arg Lys Ala Ala Arg Leu Ser Cys Ala Leu 915 920 925 Gly Val Ala Ala Asn Cys Ala Ser Ala Leu Ala Gln Met Ala Ala Ala 930 935 940 Ser Cys Val Gln Glu Glu Lys Glu Glu Arg Glu Ala Gln Glu Pro Ser 945 950 955 960 Asp Ala Ile Thr Gln Val Lys Leu Lys Val Glu Gln Lys Leu Glu Gln 965 970 975 Ile Gly Lys Val Gln Gly Val Trp Leu His Thr Ala His Val Leu Cys 980 985 990 Met Glu Ala Ile Leu Ser Val Gly Leu Glu Met Gly Ser His Asn Pro 995 1000 1005 Asp Cys Trp Pro His Val Phe Arg Val Cys Glu Tyr Val Gly Thr 1010 1015 1020 Leu Glu His Asn His Phe Ser Asp Gly Ala Ser Gln Pro Pro Leu 1025 1030 1035 Thr Ile Ser Gln Pro Gln Lys Ala Thr Gly Ser Ala Gly Leu Leu 1040 1045 1050 Gly Asp Pro Glu Cys Glu Gly Ser Pro Pro Glu His Ser Pro Glu 1055 1060 1065 Gln Gly Arg Ser Leu Ser Thr Ala Pro Val Val Gln Pro Leu Ser 1070 1075 1080 Ile Gln Asp Leu Val Arg Glu Gly Ser Arg Gly Arg Ala Ser Asp 1085 1090 1095 Phe Arg Gly Gly Ser Leu Met Ser Gly Ser Ser Ala Ala Lys Val 1100 1105 1110 Val Leu Thr Leu Ser Thr Gln Ala Asp Arg Leu Phe Glu Asp Ala 1115 1120 1125 Thr Asp Lys Leu Asn Leu Met Ala Leu Gly Gly Phe Leu Tyr Gln 1130 1135 1140 Leu Lys Lys Ala Ser Gln Ser Gln Leu Phe His Ser Val Thr Asp 1145 1150 1155 Thr Val Asp Tyr Ser Leu Ala Met Pro Gly Glu Val Lys Ser Thr 1160 1165 1170 Gln Asp Arg Lys Ser Ala Leu His Leu Phe Arg Leu Gly Asn Ala 1175 1180 1185 Met Leu Arg Ile Val Arg Ser Lys Ala Arg Pro Leu Leu His Val 1190 1195 1200 Met Arg Cys Trp Ser Leu Val Ala Pro His Leu Val Glu Ala Ala 1205 1210 1215 Cys His Lys Glu Arg His Val Ser Gln Lys Ala Val Ser Phe Ile 1220 1225 1230 His Asp Ile Leu Thr Glu Val Leu Thr Asp Trp Asn Glu Pro Pro 1235 1240 1245 His Phe His Phe Asn Glu Ala Leu Phe Arg Pro Phe Glu Arg Ile 1250 1255 1260 Met Gln Leu Glu Leu Cys Asp Glu Asp Val Gln Asp Gln Val Val 1265 1270 1275 Thr Ser Ile Gly Glu Leu Val Glu Val Cys Ser Thr Gln Ile Gln 1280 1285 1290 Ser Gly Trp Arg Pro Leu Phe Ser Ala Leu Glu Thr Val His Gly 1295 1300 1305 Gly Asn Lys Ser Glu Met Lys Glu Tyr Leu Val Gly Asp Tyr Ser 1310 1315 1320 Met Gly Lys Gly Gln Ala Pro Val Phe Asp Val Phe Glu Ala Phe 1325 1330 1335 Leu Asn Thr Asp Asn Ile Gln Val Phe Ala Asn Ala Ala Thr Ser 1340 1345 1350 Tyr Ile Met Cys Leu Met Lys Phe Val Lys Gly Leu Gly Glu Val 1355 1360 1365 Asp Cys Lys Glu Ile Gly Asp Cys Ala Pro Ala Pro Gly Ala Pro 1370 1375 1380 Ser Thr Asp Leu Cys Leu Pro Ala Leu Asp Tyr Leu Arg Arg Cys 1385 1390 1395 Ser Gln Leu Leu Ala Lys Ile Tyr Lys Met Pro Leu Lys Pro Ile 1400 1405 1410 Phe Leu Ser Gly Arg Leu Ala Gly Leu Pro Arg Arg Leu Gln Glu 1415 1420 1425 Gln Ser Ala Ser Ser Glu Asp Gly Ile Glu Ser Val Leu Ser Asp 1430 1435 1440 Phe Asp Asp Asp Thr Gly Leu Ile Glu Val Trp Ile Ile Leu Leu 1445 1450 1455 Glu Gln Leu Thr Ala Ala Val Ser Asn Cys Pro Arg Gln His Gln 1460 1465 1470 Pro Pro Thr Leu Asp Leu Leu Phe Glu Leu Leu Arg Asp Val Thr 1475 1480 1485 Lys Thr Pro Gly Pro Gly Phe Gly Ile Tyr Ala Val Val His Leu 1490 1495 1500 Leu Leu Pro Val Met Ser Val Trp Leu Arg Arg Ser His Lys Asp 1505 1510 1515 His Ser Tyr Trp Asp Met Ala Ser Ala Asn Phe Lys His Ala Ile 1520 1525 1530 Gly Leu Ser Cys Glu Leu Val Val Glu His Ile Gln Ser Phe Leu 1535 1540 1545 His Ser Asp Ile Arg Tyr Glu Ser Met Ile Asn Thr Met Leu Lys 1550 1555 1560 Asp Leu Phe Glu Leu Leu Val Ala Cys Val Ala Lys Pro Thr Glu 1565 1570 1575 Thr Ile Ser Arg Val Gly Cys Ser Cys Ile Arg Tyr Val Leu Val 1580 1585 1590 Thr Ala Gly Pro Val Phe Thr Glu Glu Met Trp Arg Leu Ala Cys 1595 1600 1605 Cys Ala Leu Gln Asp Ala Phe Ser Ala Thr Leu Lys Pro Val Lys 1610 1615 1620 Asp Leu Leu Gly Cys Phe His Ser Gly Thr Glu Ser Phe Ser Gly 1625 1630 1635 Glu Gly Cys Gln Val Arg Val Ala Ala Pro Ser Ser Ser Pro Ser 1640 1645 1650 Ala Glu Ala Glu Tyr Trp Arg Ile Arg Ala Met Ala Gln Gln Val 1655 1660 1665 Phe Met Leu Asp Thr Gln Cys Ser Pro Lys Thr Pro Asn Asn Phe 1670 1675 1680 Asp His Ala Gln Ser Cys Gln Leu Ile Ile Glu Leu Pro Pro Asp 1685 1690 1695 Glu Lys Pro Asn Gly His Thr Lys Lys Ser Val Ser Phe Arg Glu 1700 1705 1710 Ile Val Val Ser Leu Leu Ser His Gln Val Leu Leu Gln Asn Leu 1715 1720 1725 Tyr Asp Ile Leu Leu Glu Glu Phe Val Lys Gly Pro Ser Pro Gly 1730 1735 1740 Glu Glu Lys Thr Ile Gln Val Pro Glu Ala Lys Leu Ala Gly Phe 1745 1750 1755 Leu Arg Tyr Ile Ser Met Gln Asn Leu Ala Val Ile Phe Asp Leu 1760 1765 1770 Leu Leu Asp Ser Tyr Arg Thr Ala Arg Glu Phe Asp Thr Ser Pro 1775 1780 1785 Gly Leu Lys Cys Leu Leu Lys Lys Val Ser Gly Ile Gly Gly Ala 1790 1795 1800 Ala Asn Leu Tyr Arg Gln Ser Ala Met Ser Phe Asn Ile Tyr Phe 1805 1810 1815 His Ala Leu Val Cys Ala Val Leu Thr Asn Gln Glu Thr Ile Thr 1820 1825 1830 Ala Glu Gln Val Lys Lys Val Leu Phe Glu Asp Asp Glu Arg Ser 1835 1840 1845 Thr Asp Ser Ser Gln Gln Cys Ser Ser Glu Asp Glu Asp Ile Phe 1850 1855 1860 Glu Glu Thr Ala Gln Val Ser Pro Pro Arg Gly Lys Glu Lys Arg 1865 1870 1875 Gln Trp Arg Ala Arg Met Pro Leu Leu Ser Val Gln Pro Val Ser 1880 1885 1890 Asn Ala Asp Trp Val Trp Leu Val Lys Arg Leu His Lys Leu Cys 1895 1900 1905 Met Glu Leu Cys Asn Asn Tyr Ile Gln Met His Leu Asp Leu Glu 1910 1915 1920 Asn Cys Met Glu Glu Pro Pro Ile Phe Lys Gly Asp Pro Phe Phe 1925 1930 1935 Ile Leu Pro Ser Phe Gln Ser Glu Ser Ser Thr Pro Ser Thr Gly 1940 1945 1950 Gly Phe Ser Gly Lys Glu Thr Pro Ser Glu Asp Asp Arg Ser Gln 1955 1960 1965 Ser Arg Glu His Met Gly Glu Ser Leu Ser Leu Lys Ala Gly Gly 1970 1975 1980 Gly Asp Leu Leu Leu Pro Pro Ser Pro Lys Val Glu Lys Lys Asp 1985 1990 1995 Pro Ser Arg Lys Lys Glu Trp Trp Glu Asn Ala Gly Asn Lys Ile 2000 2005 2010 Tyr Thr Met Ala Ala Asp Lys Thr Ile Ser Lys Leu Met Thr Glu 2015 2020 2025 Tyr Lys Lys Arg Lys Gln Gln His Asn Leu Ser Ala Phe Pro Lys 2030 2035 2040 Glu Val Lys Val Glu Lys Lys Gly Glu Pro Leu Gly Pro Arg Gly 2045 2050 2055 Gln Asp Ser Pro Leu Leu Gln Arg Pro Gln His Leu Met Asp Gln 2060 2065 2070 Gly Gln Met Arg His Ser Phe Ser Ala Gly Pro Glu Leu Leu Arg 2075 2080 2085 Gln Asp Lys Arg Pro Arg Ser Gly Ser Thr Gly Ser Ser Leu Ser 2090 2095 2100 Val Ser Val Arg Asp Ala Glu Ala Gln Ile Gln Ala Trp Thr Asn 2105 2110 2115 Met Val Leu Thr Val Leu Asn Gln Ile Gln Ile Leu Pro Asp Gln 2120 2125 2130 Thr Phe Thr Ala Leu Gln Pro Ala Val Phe Pro Cys Ile Ser Gln 2135 2140 2145 Leu Thr Cys His Val Thr Asp Ile Arg Val Arg Gln Ala Val Arg 2150 2155 2160 Glu Trp Leu Gly Arg Val Gly Arg Val Tyr Asp Ile Ile Val 2165 2170 2175 <210> 33 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> PHB2-binding peptide derived from BIG3 <400> 33 Gln Met Leu Ser Asp Leu Thr Leu Gln Leu Arg Gln Arg 1 5 10 <210> 34 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> PHB2 peptide No. 34 <400> 34 Met Ala Gln Asn Leu Lys Asp Leu Ala Gly Arg Leu 1 5 10 <210> 35 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> PHB2 peptide No. 35 <400> 35 Gln Asn Leu Lys Asp Leu Ala Gly Arg Leu Pro Ala 1 5 10 <210> 36 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> PHB2 peptide No. 36 <400> 36 Leu Lys Asp Leu Ala Gly Arg Leu Pro Ala Gly Pro 1 5 10 <210> 37 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> PHB2 peptide No. 37 <400> 37 Asp Leu Ala Gly Arg Leu Pro Ala Gly Pro Arg Gly 1 5 10 <210> 38 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> PHB2 peptide No. 38 <400> 38 Ala Gly Arg Leu Pro Ala Gly Pro Arg Gly Met Gly 1 5 10 <210> 39 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> PHB2 peptide No. 39 <400> 39 Pro Ala Gly Pro Arg Gly Met Gly Thr Ala Leu Lys 1 5 10 <210> 40 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> PHB2 peptide No. 40 <400> 40 Gly Pro Arg Gly Met Gly Thr Ala Leu Lys Leu Leu 1 5 10 <210> 41 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> PHB2 peptide No. 41 <400> 41 Arg Gly Met Gly Thr Ala Leu Lys Leu Leu Leu Gly 1 5 10 <210> 42 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> PHB2 peptide No. 42 <400> 42 Met Gly Thr Ala Leu Lys Leu Leu Leu Gly Ala Gly 1 5 10 <210> 43 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> PHB2 peptide No. 43 <400> 43 Thr Ala Leu Lys Leu Leu Leu Gly Ala Gly Ala Val 1 5 10 <210> 44 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> PHB2 peptide No. 44 <400> 44 Ile Leu Ala Glu Gly Leu His Phe Arg Ile Pro Trp Phe Gln Tyr 1 5 10 15 <210> 45 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> PHB2 peptide No. 45 <400> 45 Ala Glu Gly Leu His Phe Arg Ile Pro Trp Phe Gln Tyr Pro Ile 1 5 10 15 <210> 46 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> PHB2 peptide No. 46 <400> 46 Gly Leu His Phe Arg Ile Pro Trp Phe Gln Tyr Pro Ile Ile Tyr 1 5 10 15 <210> 47 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> PHB2 peptide No. 47 <400> 47 His Phe Arg Ile Pro Trp Phe Gln Tyr Pro Ile Ile Tyr Asp 1 5 10 <210> 48 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> PHB2 peptide No. 48 <400> 48 Arg Ile Pro Trp Phe Gln Tyr Pro Ile Ile Tyr Asp Ile Arg Ala 1 5 10 15 <210> 49 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> PHB2 peptide No. 49 <400> 49 Pro Trp Phe Gln Tyr Pro Ile Ile Tyr Asp Ile Arg Ala Arg Pro 1 5 10 15 <210> 50 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> PHB2 peptide No. 50 <400> 50 Phe Gln Tyr Pro Ile Ile Tyr Asp Ile Arg Ala Arg Pro Arg Lys 1 5 10 15 <210> 51 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> PHB2 peptide No. 51 <400> 51 Tyr Pro Ile Ile Tyr Asp Ile Arg Ala Arg Pro Arg Lys Ile Ser 1 5 10 15 <210> 52 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> PHB2 peptide No. 52 <400> 52 Ile Ile Tyr Asp Ile Arg Ala Arg Pro Arg Lys Ile Ser Ser Pro 1 5 10 15 <210> 53 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> PHB2 peptide No. 53 <400> 53 Tyr Asp Ile Arg Ala Arg Pro Arg Lys Ile Ser Ser Pro Thr Gly 1 5 10 15 <210> 54 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> PHB2 peptide No. 54 <400> 54 Ile Arg Ala Arg Pro Arg Lys Ile Ser Ser Pro Thr Gly Ser Lys 1 5 10 15 <210> 55 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> PHB2 peptide No. 55 <400> 55 Ala Arg Pro Arg Lys Ile Ser Ser Pro Thr Gly Ser Lys Asp Leu 1 5 10 15 <210> 56 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> PHB2 peptide No. 56 <400> 56 Ala Leu Pro Ala Gly Pro Arg Gly Met Gly Thr Ala 1 5 10 <210> 57 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> PHB2 peptide No. 57 <400> 57 Arg Ala Pro Ala Gly Pro Arg Gly Met Gly Thr Ala 1 5 10 <210> 58 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> PHB2 peptide No. 58 <400> 58 Arg Leu Ala Ala Gly Pro Arg Gly Met Gly Thr Ala 1 5 10 <210> 59 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> PHB2 peptide No. 59 <400> 59 Arg Leu Pro Ala Ala Pro Arg Gly Met Gly Thr Ala 1 5 10 <210> 60 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> PHB2 peptide No. 60 <400> 60 Arg Leu Pro Ala Gly Ala Arg Gly Met Gly Thr Ala 1 5 10 <210> 61 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> PHB2 peptide No. 61 <400> 61 Arg Leu Pro Ala Gly Pro Ala Gly Met Gly Thr Ala 1 5 10 <210> 62 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> PHB2 peptide No. 62 <400> 62 Arg Leu Pro Ala Gly Pro Arg Ala Met Gly Thr Ala 1 5 10 <210> 63 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> PHB2 peptide No. 63 <400> 63 Arg Leu Pro Ala Gly Pro Arg Gly Ala Gly Thr Ala 1 5 10 <210> 64 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> PHB2 peptide No. 64 <400> 64 Arg Leu Pro Ala Gly Pro Arg Gly Met Ala Thr Ala 1 5 10 <210> 65 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> PHB2 peptide No. 65 <400> 65 Arg Leu Pro Ala Gly Pro Arg Gly Met Gly Ala Ala 1 5 10 <210> 66 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> PHB2 peptide No. 66 <400> 66 Ala Leu Pro Ala Gly Pro Ala Gly Met Gly Thr Ala 1 5 10 <210> 67 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> PHB2 peptide No. 67 <400> 67 Ala Tyr Pro Ile Ile Tyr Asp Ile Arg Ala Arg Pro Arg Lys Ile 1 5 10 15 <210> 68 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> PHB2 peptide No. 68 <400> 68 Gln Ala Pro Ile Ile Tyr Asp Ile Arg Ala Arg Pro Arg Lys Ile 1 5 10 15 <210> 69 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> PHB2 peptide No. 69 <400> 69 Gln Tyr Ala Ile Ile Tyr Asp Ile Arg Ala Arg Pro Arg Lys Ile 1 5 10 15 <210> 70 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> PHB2 peptide No. 70 <400> 70 Gln Tyr Pro Ala Ile Tyr Asp Ile Arg Ala Arg Pro Arg Lys Ile 1 5 10 15 <210> 71 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> PHB2 peptide No. 71 <400> 71 Gln Tyr Pro Ile Ala Tyr Asp Ile Arg Ala Arg Pro Arg Lys Ile 1 5 10 15 <210> 72 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> PHB2 peptide No. 72 <400> 72 Gln Tyr Pro Ile Ile Ala Asp Ile Arg Ala Arg Pro Arg Lys Ile 1 5 10 15 <210> 73 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> PHB2 peptide No. 73 <400> 73 Gln Tyr Pro Ile Ile Tyr Ala Ile Arg Ala Arg Pro Arg Lys Ile 1 5 10 15 <210> 74 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> PHB2 peptide No. 74 <400> 74 Gln Tyr Pro Ile Ile Tyr Asp Ala Arg Ala Arg Pro Arg Lys Ile 1 5 10 15 <210> 75 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> PHB2 peptide No. 75 <400> 75 Gln Tyr Pro Ile Ile Tyr Asp Ile Ala Ala Arg Pro Arg Lys Ile 1 5 10 15 <210> 76 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> PHB2 peptide No. 76 <400> 76 Gln Tyr Pro Ile Ile Tyr Asp Ile Arg Ala Ala Pro Arg Lys Ile 1 5 10 15 <210> 77 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> PHB2 peptide No. 77 <400> 77 Gln Tyr Pro Ile Ile Tyr Asp Ile Arg Ala Arg Ala Arg Lys Ile 1 5 10 15 <210> 78 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> PHB2 peptide No. 78 <400> 78 Gln Tyr Pro Ile Ile Tyr Asp Ile Arg Ala Arg Pro Ala Lys Ile 1 5 10 15 <210> 79 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> PHB2 peptide No. 79 <400> 79 Gln Tyr Pro Ile Ile Tyr Asp Ile Arg Ala Arg Pro Arg Ala Ile 1 5 10 15 <210> 80 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> PHB2 peptide No. 80 <400> 80 Gln Tyr Pro Ile Ile Tyr Asp Ile Arg Ala Arg Pro Arg Lys Ala 1 5 10 15 <210> 81 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> PHB2 peptide No. 81 <400> 81 Gln Ala Pro Ile Ile Tyr Asp Ile Ala Ala Arg Pro Arg Lys Ile 1 5 10 15 <210> 82 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> PHB2 peptide No. 82 <400> 82 Glu Gly Gly His Arg Ala Ile Phe Phe Asn Arg Ile Gly Gly 1 5 10 <210> 83 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> PHB2 peptide No. 83 <400> 83 Glu Ser Val Phe Thr Val Glu Gly Gly His Arg Ala Ile Phe Phe Asn 1 5 10 15 Arg Ile Gly Gly 20 <210> 84 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> PHB2 derived peptide 11-21aa <400> 84 Arg Leu Pro Ala Gly Pro Arg Gly Met Gly Thr 1 5 10 <210> 85 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> PHB2 derived peptide 76-88aa <400> 85 Gln Tyr Pro Ile Ile Tyr Asp Ile Arg Ala Arg Pro Arg 1 5 10 <210> 86 <211> 6330 <212> DNA <213> Homo sapiens <220> <221> CDS <222> (235)..(2022) <400> 86 aggagctggc ggagggcgtt cgtcctggga ctgcacttgc tcccgtcggg tcgcccggct 60 tcaccggacc cgcaggctcc cggggcaggg ccggggccag agctcgcgtg tcggcgggac 120 atgcgctgcg tcgcctctaa cctcgggctg tgctcttttt ccaggtggcc cgccggtttc 180 tgagccttct gccctgcggg gacacggtct gcaccctgcc cgcggccacg gacc atg 237 Met 1 acc atg acc ctc cac acc aaa gca tct ggg atg gcc cta ctg cat cag 285 Thr Met Thr Leu His Thr Lys Ala Ser Gly Met Ala Leu Leu His Gln 5 10 15 atc caa ggg aac gag ctg gag ccc ctg aac cgt ccg cag ctc aag atc 333 Ile Gln Gly Asn Glu Leu Glu Pro Leu Asn Arg Pro Gln Leu Lys Ile 20 25 30 ccc ctg gag cgg ccc ctg ggc gag gtg tac ctg gac agc agc aag ccc 381 Pro Leu Glu Arg Pro Leu Gly Glu Val Tyr Leu Asp Ser Ser Lys Pro 35 40 45 gcc gtg tac aac tac ccc gag ggc gcc gcc tac gag ttc aac gcc gcg 429 Ala Val Tyr Asn Tyr Pro Glu Gly Ala Ala Tyr Glu Phe Asn Ala Ala 50 55 60 65 gcc gcc gcc aac gcg cag gtc tac ggt cag acc ggc ctc ccc tac ggc 477 Ala Ala Ala Asn Ala Gln Val Tyr Gly Gln Thr Gly Leu Pro Tyr Gly 70 75 80 ccc ggg tct gag gct gcg gcg ttc ggc tcc aac ggc ctg ggg ggt ttc 525 Pro Gly Ser Glu Ala Ala Ala Phe Gly Ser Asn Gly Leu Gly Gly Phe 85 90 95 ccc cca ctc aac agc gtg tct ccg agc ccg ctg atg cta ctg cac ccg 573 Pro Pro Leu Asn Ser Val Ser Pro Ser Pro Leu Met Leu Leu His Pro 100 105 110 ccg ccg cag ctg tcg cct ttc ctg cag ccc cac ggc cag cag gtg ccc 621 Pro Pro Gln Leu Ser Pro Phe Leu Gln Pro His Gly Gln Gln Val Pro 115 120 125 tac tac ctg gag aac gag ccc agc ggc tac acg gtg cgc gag gcc ggc 669 Tyr Tyr Leu Glu Asn Glu Pro Ser Gly Tyr Thr Val Arg Glu Ala Gly 130 135 140 145 ccg ccg gca ttc tac agg cca aat tca gat aat cga cgc cag ggt ggc 717 Pro Pro Ala Phe Tyr Arg Pro Asn Ser Asp Asn Arg Arg Gln Gly Gly 150 155 160 aga gaa aga ttg gcc agt acc aat gac aag gga agt atg gct atg gaa 765 Arg Glu Arg Leu Ala Ser Thr Asn Asp Lys Gly Ser Met Ala Met Glu 165 170 175 tct gcc aag gag act cgc tac tgt gca gtg tgc aat gac tat gct tca 813 Ser Ala Lys Glu Thr Arg Tyr Cys Ala Val Cys Asn Asp Tyr Ala Ser 180 185 190 ggc tac cat tat gga gtc tgg tcc tgt gag ggc tgc aag gcc ttc ttc 861 Gly Tyr His Tyr Gly Val Trp Ser Cys Glu Gly Cys Lys Ala Phe Phe 195 200 205 aag aga agt att caa gga cat aac gac tat atg tgt cca gcc acc aac 909 Lys Arg Ser Ile Gln Gly His Asn Asp Tyr Met Cys Pro Ala Thr Asn 210 215 220 225 cag tgc acc att gat aaa aac agg agg aag agc tgc cag gcc tgc cgg 957 Gln Cys Thr Ile Asp Lys Asn Arg Arg Lys Ser Cys Gln Ala Cys Arg 230 235 240 ctc cgc aaa tgc tac gaa gtg gga atg atg aaa ggt ggg ata cga aaa 1005 Leu Arg Lys Cys Tyr Glu Val Gly Met Met Lys Gly Gly Ile Arg Lys 245 250 255 gac cga aga gga ggg aga atg ttg aaa cac aag cgc cag aga gat gat 1053 Asp Arg Arg Gly Gly Arg Met Leu Lys His Lys Arg Gln Arg Asp Asp 260 265 270 ggg gag ggc agg ggt gaa gtg ggg tct gct gga gac atg aga gct gcc 1101 Gly Glu Gly Arg Gly Glu Val Gly Ser Ala Gly Asp Met Arg Ala Ala 275 280 285 aac ctt tgg cca agc ccg ctc atg atc aaa cgc tct aag aag aac agc 1149 Asn Leu Trp Pro Ser Pro Leu Met Ile Lys Arg Ser Lys Lys Asn Ser 290 295 300 305 ctg gcc ttg tcc ctg acg gcc gac cag atg gtc agt gcc ttg ttg gat 1197 Leu Ala Leu Ser Leu Thr Ala Asp Gln Met Val Ser Ala Leu Leu Asp 310 315 320 gct gag ccc ccc ata ctc tat tcc gag tat gat cct acc aga ccc ttc 1245 Ala Glu Pro Pro Ile Leu Tyr Ser Glu Tyr Asp Pro Thr Arg Pro Phe 325 330 335 agt gaa gct tcg atg atg ggc tta ctg acc aac ctg gca gac agg gag 1293 Ser Glu Ala Ser Met Met Gly Leu Leu Thr Asn Leu Ala Asp Arg Glu 340 345 350 ctg gtt cac atg atc aac tgg gcg aag agg gtg cca ggc ttt gtg gat 1341 Leu Val His Met Ile Asn Trp Ala Lys Arg Val Pro Gly Phe Val Asp 355 360 365 ttg acc ctc cat gat cag gtc cac ctt cta gaa tgt gcc tgg cta gag 1389 Leu Thr Leu His Asp Gln Val His Leu Leu Glu Cys Ala Trp Leu Glu 370 375 380 385 atc ctg atg att ggt ctc gtc tgg cgc tcc atg gag cac cca ggg aag 1437 Ile Leu Met Ile Gly Leu Val Trp Arg Ser Met Glu His Pro Gly Lys 390 395 400 cta ctg ttt gct cct aac ttg ctc ttg gac agg aac cag gga aaa tgt 1485 Leu Leu Phe Ala Pro Asn Leu Leu Leu Asp Arg Asn Gln Gly Lys Cys 405 410 415 gta gag ggc atg gtg gag atc ttc gac atg ctg ctg gct aca tca tct 1533 Val Glu Gly Met Val Glu Ile Phe Asp Met Leu Leu Ala Thr Ser Ser 420 425 430 cgg ttc cgc atg atg aat ctg cag gga gag gag ttt gtg tgc ctc aaa 1581 Arg Phe Arg Met Met Asn Leu Gln Gly Glu Glu Phe Val Cys Leu Lys 435 440 445 tct att att ttg ctt aat tct gga gtg tac aca ttt ctg tcc agc acc 1629 Ser Ile Ile Leu Leu Asn Ser Gly Val Tyr Thr Phe Leu Ser Ser Thr 450 455 460 465 ctg aag tct ctg gaa gag aag gac cat atc cac cga gtc ctg gac aag 1677 Leu Lys Ser Leu Glu Glu Lys Asp His Ile His Arg Val Leu Asp Lys 470 475 480 atc aca gac act ttg atc cac ctg atg gcc aag gca ggc ctg acc ctg 1725 Ile Thr Asp Thr Leu Ile His Leu Met Ala Lys Ala Gly Leu Thr Leu 485 490 495 cag cag cag cac cag cgg ctg gcc cag ctc ctc ctc atc ctc tcc cac 1773 Gln Gln Gln His Gln Arg Leu Ala Gln Leu Leu Leu Ile Leu Ser His 500 505 510 atc agg cac atg agt aac aaa ggc atg gag cat ctg tac agc atg aag 1821 Ile Arg His Met Ser Asn Lys Gly Met Glu His Leu Tyr Ser Met Lys 515 520 525 tgc aag aac gtg gtg ccc ctc tat gac ctg ctg ctg gag atg ctg gac 1869 Cys Lys Asn Val Val Pro Leu Tyr Asp Leu Leu Leu Glu Met Leu Asp 530 535 540 545 gcc cac cgc cta cat gcg ccc act agc cgt gga ggg gca tcc gtg gag 1917 Ala His Arg Leu His Ala Pro Thr Ser Arg Gly Gly Ala Ser Val Glu 550 555 560 gag acg gac caa agc cac ttg gcc act gcg ggc tct act tca tcg cat 1965 Glu Thr Asp Gln Ser His Leu Ala Thr Ala Gly Ser Thr Ser Ser His 565 570 575 tcc ttg caa aag tat tac atc acg ggg gag gca gag ggt ttc cct gcc 2013 Ser Leu Gln Lys Tyr Tyr Ile Thr Gly Glu Ala Glu Gly Phe Pro Ala 580 585 590 acg gtc tga gagctccctg gctcccacac ggttcagata atccctgctg 2062 Thr Val 595 cattttaccc tcatcatgca ccactttagc caaattctgt ctcctgcata cactccggca 2122 tgcatccaac accaatggct ttctagatga gtggccattc atttgcttgc tcagttctta 2182 gtggcacatc ttctgtcttc tgttgggaac agccaaaggg attccaaggc taaatctttg 2242 taacagctct ctttccccct tgctatgtta ctaagcgtga ggattcccgt agctcttcac 2302 agctgaactc agtctatggg ttggggctca gataactctg tgcatttaag ctacttgtag 2362 agacccaggc ctggagagta gacattttgc ctctgataag cactttttaa atggctctaa 2422 gaataagcca cagcaaagaa tttaaagtgg ctcctttaat tggtgacttg gagaaagcta 2482 ggtcaagggt ttattatagc accctcttgt attcctatgg caatgcatcc ttttatgaaa 2542 gtggtacacc ttaaagcttt tatatgactg tagcagagta tctggtgatt gtcaattcat 2602 tccccctata ggaatacaag gggcacacag ggaaggcaga tcccctagtt ggcaagacta 2662 ttttaacttg atacactgca gattcagatg tgctgaaagc tctgcctctg gctttccggt 2722 catgggttcc agttaattca tgcctcccat ggacctatgg agagcagcaa gttgatctta 2782 gttaagtctc cctatatgag ggataagttc ctgatttttg tttttatttt tgtgttacaa 2842 aagaaagccc tccctccctg aacttgcagt aaggtcagct tcaggacctg ttccagtggg 2902 cactgtactt ggatcttccc ggcgtgtgtg tgccttacac aggggtgaac tgttcactgt 2962 ggtgatgcat gatgagggta aatggtagtt gaaaggagca ggggccctgg tgttgcattt 3022 agccctgggg catggagctg aacagtactt gtgcaggatt gttgtggcta ctagagaaca 3082 agagggaaag tagggcagaa actggataca gttctgaggc acagccagac ttgctcaggg 3142 tggccctgcc acaggctgca gctacctagg aacattcctt gcagaccccg cattgccctt 3202 tgggggtgcc ctgggatccc tggggtagtc cagctcttct tcatttccca gcgtggccct 3262 ggttggaaga agcagctgtc acagctgctg tagacagctg tgttcctaca attggcccag 3322 caccctgggg cacgggagaa gggtggggac cgttgctgtc actactcagg ctgactgggg 3382 cctggtcaga ttacgtatgc ccttggtggt ttagagataa tccaaaatca gggtttggtt 3442 tggggaagaa aatcctcccc cttcctcccc cgccccgtc cctaccgcct ccactcctgc 3502 cagctcattt ccttcaattt cctttgacct ataggctaaa aaagaaaggc tcattccagc 3562 cacagggcag ccttccctgg gcctttgctt ctctagcaca attatgggtt acttcctttt 3622 tcttaacaaa aaagaatgtt tgatttcctc tgggtgacct tattgtctgt aattgaaacc 3682 ctattgagag gtgatgtctg tgttagccaa tgacccaggt gagctgctcg ggcttctct 3742 ggtatgtctt gtttggaaaaa gtggatttca ttcatttctg attgtccagt taagtgatca 3802 ccaaaggact gagaatctgg gagggcaaaa aaaaaaaaa agtttttatg tgcacttaaa 3862 tttggggaca attttatgta tctgtgttaa ggatatgttt aagaacataa ttctttttgtt 3922 gctgtttgtt taagaagcac cttagtttgt ttaagaagca ccttatatag tataatatat 3982 attttttga aattacattg cttgtttatc agacaattga atgtagtaat tctgttctgg 4042 atttaatttg actgggttaa catgcaaaaa ccaaggaaaa atatttagtt tttttttttt 4102 ttttgtata cttttcaagc taccttgtca tgtatacagt catttatgcc taaagcctgg 4162 tgattattca tttaaatgaa gatcacattt catatcaact tttgtatcca cagtagacaa 4222 aatagcacta atccagatgc ctattgttgg atactgaatg acagacaatc ttatgtagca 4282 aagattatgc ctgaaaagga aaattattca gggcagctaa ttttgctttt accaaaatat 4342 cagtagtaat atttttggac agtagctaat gggtcagtgg gttcttttta atgtttatac 4402 ttagatttc ttttaaaaaa attaaaataa aaaaaaaa aatttctagg actagacgat 4462 gtaataccag ctaaagccaa acaattatac agtggaaggt tttacattat tcatccaatg 4522 tgtttctatt catgttaaga tactactaca tttgaagtgg gcagagaaca tcagatgatt 4582 gaaatgttcg cccaggggtc tccagcaact ttggaaatct ctttgtattt ttacttgaag 4642 tgccactaat ggacagcaga tattttctgg ctgatgttgg tattgggtgt aggaacatga 4702 tttaaaaaaa aactcttgcc tctgctttcc cccactctga ggcaagttaa aatgtaaaag 4762 atgtgattta tctggggggc tcaggtatgg tggggaagtg gattcaggaa tctggggaat 4822 ggcaaatata ttaagaagag tattgaaagt atttggagga aaatggttaa ttctgggtgt 4882 gcaccagggt tcagtagagt ccacttctgc cctggagacc acaaatcaac tagctccatt 4942 tacagccatt tctaaaatgg cagcttcagt tctagagaag aaagaacaac atcagcagta 5002 aagtccatgg aatagctagt ggtctgtgtt tctttcgcc attgcctagc ttgccgtaat 5062 gattctataa tgccatcatg cagcaattat gagaggctag gtcatccaaa gagaagaccc 5122 tatcaatgta ggttgcaaaa tctaacccct aaggaagtgc agtctttgat ttgatttccc 5182 tagtaacctt gcagatatgt ttaaccaagc catagcccat gccttttgag ggctgaacaa 5242 ataagggact tactgataat ttactttga tcacattaag gtgttctcac cttgaaatct 5302 tatacactga aatggccatt gatttaggcc actggcttag agtactcctt cccctgcatg 5362 acactgatta caaatacttt cctattcata ctttccaatt atgagatgga ctgtgggtac 5422 tgggagtgat cactaacacc atagtaatgt ctaatattca caggcagatc tgcttgggga 5482 agctagttat gtgaaaggca atagagtca tacagtagct caaaaggcaa ccataattct 5542 ctttggtgca ggtcttggga gcgtgatcta gattacactg caccattccc aagttaatcc 5602 cctgaaaact tactctcaac tggagcaaat gaactttggt cccaaatatc catcttttca 5662 gtagcgttaa ttatgctctg tttccaactg catttccttt ccaattgaat taaagtgtgg 5722 cctcgttttt agtcatttaa aattgttttc tagtaattg ctgcctctat tatggcactt 5782 caattttgca ctgtcttttg agattcaaga aaaatttcta ttcttttttt tgcatccaat 5842 tgtgcctgaa cttttaaaat atgtaaatgc tgccatgttc caaacccatc gtcagtgtgt 5902 gtgtttagag ctgtgcaccc tagaaaac atattgtccc atgagcaggt gcctgagaca 5962 cagacccctt tgcattcaca gagaggtcat tggttataga gacttgaatt aataagtgac 6022 attatgccag tttctgttct ctcacaggtg ataaacaatg ctttttgtgc actacatact 6082 cttcagtgta gagctcttgt tttatgggaa aaggctcaaa tgccaaattg tgtttgatgg 6142 attaatatgc ccttttgccg atgcatacta ttactgatgt gactcggttt tgtcgcagct 6202 ttgctttgtt taatgaaaca cacttgtaaa cctcttttgc actttgaaaa agaatccagc 6262 gggatgctcg agcacctgta aacaattttc tcaacctatt tgatgttcaa ataaagaatt 6322 aaactaaa 6330 <210> 87 <211> 595 <212> PRT <213> Homo sapiens <400> 87 Met Thr Met Thr Leu His Thr Lys Ala Ser Gly Met Ala Leu Leu His 1 5 10 15 Gln Ile Gln Gly Asn Glu Leu Glu Pro Leu Asn Arg Pro Gln Leu Lys 20 25 30 Ile Pro Leu Glu Arg Pro Leu Gly Glu Val Tyr Leu Asp Ser Ser Lys 35 40 45 Pro Ala Val Tyr Asn Tyr Pro Glu Gly Ala Ala Tyr Glu Phe Asn Ala 50 55 60 Ala Ala Ala Ala Asn Ala Gln Val Tyr Gly Gln Thr Gly Leu Pro Tyr 65 70 75 80 Gly Pro Gly Ser Glu Ala Ala Ala Phe Gly Ser Asn Gly Leu Gly Gly 85 90 95 Phe Pro Pro Leu Asn Ser Val Ser Pro Ser Pro Leu Met Leu Leu His 100 105 110 Pro Pro Pro Gln Leu Ser Pro Phe Leu Gln Pro His Gly Gln Gln Val 115 120 125 Pro Tyr Tyr Leu Glu Asn Glu Pro Ser Gly Tyr Thr Val Arg Glu Ala 130 135 140 Gly Pro Pro Ala Phe Tyr Arg Pro Asn Ser Asp Asn Arg Arg Gln Gly 145 150 155 160 Gly Arg Glu Arg Leu Ala Ser Thr Asn Asp Lys Gly Ser Met Ala Met 165 170 175 Glu Ser Ala Lys Glu Thr Arg Tyr Cys Ala Val Cys Asn Asp Tyr Ala 180 185 190 Ser Gly Tyr His Tyr Gly Val Trp Ser Cys Glu Gly Cys Lys Ala Phe 195 200 205 Phe Lys Arg Ser Ile Gln Gly His Asn Asp Tyr Met Cys Pro Ala Thr 210 215 220 Asn Gln Cys Thr Ile Asp Lys Asn Arg Arg Lys Ser Cys Gln Ala Cys 225 230 235 240 Arg Leu Arg Lys Cys Tyr Glu Val Gly Met Met Lys Gly Gly Ile Arg 245 250 255 Lys Asp Arg Arg Gly Gly Arg Met Leu Lys His Lys Arg Gln Arg Asp 260 265 270 Asp Gly Glu Gly Arg Gly Glu Val Gly Ser Ala Gly Asp Met Arg Ala 275 280 285 Ala Asn Leu Trp Pro Ser Pro Leu Met Ile Lys Arg Ser Lys Lys Asn 290 295 300 Ser Leu Ala Leu Ser Leu Thr Ala Asp Gln Met Val Ser Ala Leu Leu 305 310 315 320 Asp Ala Glu Pro Pro Ile Leu Tyr Ser Glu Tyr Asp Pro Thr Arg Pro 325 330 335 Phe Ser Glu Ala Ser Met Met Gly Leu Leu Thr Asn Leu Ala Asp Arg 340 345 350 Glu Leu Val His Met Ile Asn Trp Ala Lys Arg Val Pro Gly Phe Val 355 360 365 Asp Leu Thr Leu His Asp Gln Val His Leu Leu Glu Cys Ala Trp Leu 370 375 380 Glu Ile Leu Met Ile Gly Leu Val Trp Arg Ser Met Glu His Pro Gly 385 390 395 400 Lys Leu Leu Phe Ala Pro Asn Leu Leu Leu Asp Arg Asn Gln Gly Lys 405 410 415 Cys Val Glu Gly Met Val Glu Ile Phe Asp Met Leu Leu Ala Thr Ser 420 425 430 Ser Arg Phe Arg Met Met Asn Leu Gln Gly Glu Glu Phe Val Cys Leu 435 440 445 Lys Ser Ile Ile Leu Leu Asn Ser Gly Val Tyr Thr Phe Leu Ser Ser 450 455 460 Thr Leu Lys Ser Leu Glu Glu Lys Asp His Ile His Arg Val Leu Asp 465 470 475 480 Lys Ile Thr Asp Thr Leu Ile His Leu Met Ala Lys Ala Gly Leu Thr 485 490 495 Leu Gln Gln Gln His Gln Arg Leu Ala Gln Leu Leu Leu Ile Leu Ser 500 505 510 His Ile Arg His Met Ser Asn Lys Gly Met Glu His Leu Tyr Ser Met 515 520 525 Lys Cys Lys Asn Val Val Pro Leu Tyr Asp Leu Leu Leu Glu Met Leu 530 535 540 Asp Ala His Arg Leu His Ala Pro Thr Ser Arg Gly Gly Ala Ser Val 545 550 555 560 Glu Glu Thr Asp Gln Ser His Leu Ala Thr Ala Gly Ser Thr Ser Ser 565 570 575 His Ser Leu Gln Lys Tyr Tyr Ile Thr Gly Glu Ala Glu Gly Phe Pro 580 585 590 Ala Thr Val 595 <210> 88 <211> 2169 <212> DNA <213> Homo sapiens <220> <221> CDS <222> (469)..(2061) <400> 88 ctcggtcttt aaaaggaaga aggggcttat cgttaagtcg cttgtgatct tttcagtttc 60 tccagctgct ggctttttgg acacccactc ccccgccagg aggcagttgc aagcgcggag 120 gctgcgagaa ataactgcct cttgaaactt gcagggcgaa gagcaggcgg cgagcgctgg 180 gccggggagg gaccacccga gctgcgacgg gctctggggc tgcggggcag ggctggcgcc 240 cggagcctga gctgcaggag gtgcgctcgc tttcctcaac aggtggcggc ggggcgcgcg 300 ccgggagacc ccccctaatg cgggaaaagc acgtgtccgc atttagaga aggcaaggcc 360 ggtgtgttta tctgcaagcc attatacttg cccacgaatc tttgagaaca ttataatgac 420 ctttgtgcct cttcttgcaa ggtgttttct cagctgttat ctcaagac atg gat ata 477 But Asp Ile 1 aaa aac tca cca tct agc ctt aat tct cct tcc tc tac aac tgc agt 525 Lys Asn Ser Pro Ser Ser Leu Asn Ser Pro Ser Ser Tyr Asn Cys Ser 5 10 15 caa tcc atc tta ccc ctg gag cac ggc tcc ata tac ata cct tcc tcc 573 Gln Ser Ile Leu Pro Leu Glu His Gly Ser Ile Tyr Ile Pro Ser Ser 20 25 30 35 tat gta gac agc cac cat gaa tat cca gcc atg aca ttc tat agc cct 621 Tyr Val Asp Ser His His Glu Tyr Pro Ala Met Thr Phe Tyr Ser Pro 40 45 50 gct gtg atg aat tac agc att ccc agc aat gtc act aac ttg gaa ggt 669 Ala Val Met Asn Tyr Ser Ile Pro Ser Asn Val Thr Asn Leu Glu Gly 55 60 65 ggg cct ggt cgg cag acc aca agc cca aat gtg ttg tgg cca aca cct 717 Gly Pro Gly Arg Gln Thr Thr Ser Pro Asn Val Leu Trp Pro Thr Pro 70 75 80 ggg cac ctt tct cct tta gtg gtc cat cgc cag tta tca cat ctg tat 765 Gly His Leu Ser Pro Leu Val Val His Arg Gln Leu Ser His Leu Tyr 85 90 95 gcg gaa cct caa aag agt ccc tgg tgt gaa gca aga tcg cta gaa cac 813 Ala Glu Pro Gln Lys Ser Pro Trp Cys Glu Ala Arg Ser Leu Glu His 100 105 110 115 acc tta cct gta aac aga gag aca ctg aaa agg aag gtt agt ggg aac 861 Thr Pro Leu Will Asn Arg Glu Thr Leu Lys Arg Lys Will Ser Gly Asn 120 125 130 cgt tgc gcc agc cct gtt act ggt cca ggt tca aag agg gat gct cac 909 Arg Cys Ala Ser Pro Val Thr Gly Pro Gly Ser Lys Arg Asp Ala His 135 140 145 ttc tgc gct gtc tgc agc gat tac gca tcg gga tat cac tat gga gtc 957 Phe Cys Ala Val Cys Ser Asp Tyr Ala Ser Gly Tyr His Tyr Gly Val 150 155 160 tgg tcg tgt gaa gga tgt aag gcc ttt ttt aaa aga agc att caa gga Trp Ser Cys Glu Gly Cys Lys Ala Phe Phe Lys Arg Ser Ile Gln Gly 165 170 175 cat aat gat tat att tgt cca gct aca aat cag tgt aca atc gat aaa 1053 His Asn Asp Tyr Ile Cys Pro Ala Thr Asn Gln Cys Thr Ile Asp Lys 180 185 190 195 aac cgg cgc aag agc tgc cag gcc tgc cga ctt cgg aag tgt tac gaa 1101 Asn Arg Arg Lys Ser Cys Gln Ala Cys Arg Leu Arg Lys Cys Tyr Glu 200 205 210 gtg gga atg gtg aag tgt ggc tcc cgg aga gag aga tgt ggg tac cgc 1149 Val Gly Met Val Lys Cys Gly Ser Arg Arg Glu Arg Cys Gly Tyr Arg 215 220 225 ctt gtg cgg aga cag aga agt gcc gac gag cag ctg cac tgt gcc ggc 1197 Leu Val Arg Arg Gln Arg Ser Ala Asp Glu Gln Leu His Cys Ala Gly 230 235 240 aag gcc aag aga agt ggc ggc cac gcg ccc cga gtg cgg gag ctg ctg 1245 Lys Ala Lys Arg Ser Gly Gly His Ala Pro Arg Val Arg Glu Leu Leu 245 250 255 ctg gac gcc ctg agc ccc gag cag cta gtg ctc acc ctc ctg gag gct 1293 Leu Asp Ala Leu Ser Pro Glu Gln Leu Val Leu Thr Leu Leu Glu Ala 260 265 270 275 gag ccg ccc cat gtg ctg atc agc cgc ccc agt gcg ccc ttc acc gag 1341 Glu Pro Pro His Val Leu Ile Ser Arg Pro Ser Ala Pro Phe Thr Glu 280 285 290 gcc tcc atg atg atg tcc ctg acc aag ttg gcc gac aag gag ttg gta 1389 Ala Ser Met Met Met Ser Leu Thr Lys Leu Ala Asp Lys Glu Leu Val 295 300 305 cac atg atc agc tgg gcc aag aag att ccc ggc ttt gtg gag ctc agc 1437 His Met Ile Ser Trp Ala Lys Lys Ile Pro Gly Phe Val Glu Leu Ser 310 315 320 ctg ttc gac caa gtg cgg ctc ttg gag agc tgt tgg atg gag gtg tta 1485 Leu Phe Asp Gln Val Arg Leu Leu Glu Ser Cys Trp Met Glu Val Leu 325 330 335 atg atg ggg ctg atg tgg cgc tca att gac cac ccc ggc aag ctc atc 1533 Met Met Gly Leu Met Trp Arg Ser Ile Asp His Pro Gly Lys Leu Ile 340 345 350 355 ttt gct cca gat ctt gtt ctg gac agg gat gag ggg aaa tgc gta gaa 1581 Phe Ala Pro Asp Leu Val Leu Asp Arg Asp Glu Gly Lys Cys Val Glu 360 365 370 gga att ctg gaa atc ttt gac atg ctc ctg gca act act tca agg ttt 1629 Gly Ile Leu Glu Ile Phe Asp Met Leu Leu Ala Thr Thr Ser Arg Phe 375 380 385 cga gag tta aaa ctc caa cac aaa gaa tat ctc tgt gtc aag gcc atg 1677 Arg Glu Leu Lys Leu Gln His Lys Glu Tyr Leu Cys Val Lys Ala Met 390 395 400 atc ctg ctc aat tcc agt atg tac cct ctg gtc aca gcg acc cag gat 1725 Ile Leu Leu Asn Ser Ser Met Tyr Pro Leu Val Thr Ala Thr Gln Asp 405 410 415 gct gac agc agc cgg aag ctg gct cac ttg ctg aac gcc gtg acc gat 1773 Ala Asp Ser Ser Arg Lys Leu Ala His Leu Leu Asn Ala Val Thr Asp 420 425 430 435 gct ttg gtt tgg gtg att gcc aag agc ggc atc tcc tcc cag cag caa 1821 Ala Leu Val Trp Val Ile Ala Lys Ser Gly Ile Ser Ser Gln Gln Gln 440 445 450 tcc atg cgc ctg gct aac ctc ctg atg ctc ctg tcc cac gtc agg cat 1869 Ser Met Arg You Ala Asn You Met You Met You Serve His Val Arg His 455 460 465 gcg agt aac aag ggc atg gaa cat ctg ctc aac atg aag tgc aaa aat 1917 Only Ser Asn Lys Gly Met Glu His Leu Leu Asn Met Lys Lys Lys Asn 470 475 480 gtg gtc cca gtg tat gac ctg ctg ctg gag atg ctg aat gcc cac gtg 1965 Val Val Pro Val Tyr Asp Leo Leo Leo Glue Met Leo Asn Ala His Val 485,490,495 ctt cgc ggg tgc aag tcc tcc atc acg ggg tcc gag tgc agc ccg gca 2013 Leu Arg Gly Cys Lys Ser Ser Ile Thr Gly Ser Glu Cys Ser Pro Ala 500 505 510 515 gag gac agt aaa agc aaa gag ggc tcc cag aac cca cag tct cag tga 2061 Glu Asp Ser Lys Ser Lys Glu Gly Ser Gln Asn Pro Gln Ser Gln 520 525 530 cgcctggccc tgaggtgaac tggcccacag aggtcacagg ctgaagcgtg aactccagtg 2121 tgtcaggagc ctgggcttca tctttctgct gtgtggtccc tcatttgg 2169 <210> 89 <211> 530 <212> PRT <213> Homo sapiens <400> 89 Met Asp Ile Lys Asn Ser Pro Ser Ser Leu Asn Ser Pro Ser Ser Tyr 1 5 10 15 Asn Cys Ser Gln Ser Ile Leu Pro Leu Glu His Gly Ser Ile Tyr Ile 20 25 30 Pro Ser Ser Tyr Val Asp Ser His His Glu Tyr Pro Ala Met Thr Phe 35 40 45 Tyr Ser Pro Ala Val Met Asn Tyr Ser Ile Pro Ser Asn Val Thr Asn 50 55 60 Leu Glu Gly Gly Pro Gly Arg Gln Thr Thr Ser Pro Asn Val Leu Trp 65 70 75 80 Pro Thr Pro Gly His Leu Ser Pro Leu Val Val His Arg Gln Leu Ser 85 90 95 His Leu Tyr Ala Glu Pro Gln Lys Ser Pro Trp Cys Glu Ala Arg Ser 100 105 110 Leu Glu His Thr Leu Pro Val Asn Arg Glu Thr Leu Lys Arg Lys Val 115 120 125 Ser Gly Asn Arg Cys Ala Ser Pro Val Thr Gly Pro Gly Ser Lys Arg 130 135 140 Asp Ala His Phe Cys Ala Val Cys Ser Asp Tyr Ala Ser Gly Tyr His 145 150 155 160 Tyr Gly Val Trp Ser Cys Glu Gly Cys Lys Ala Phe Phe Lys Arg Ser 165 170 175 Ile Gln Gly His Asn Asp Tyr Ile Cys Pro Ala Thr Asn Gln Cys Thr 180 185 190 Ile Asp Lys Asn Arg Arg Lys Ser Cys Gln Ala Cys Arg Leu Arg Lys 195 200 205 Cys Tyr Glu Val Gly Met Val Lys Cys Gly Ser Arg Arg Glu Arg Cys 210 215 220 Gly Tyr Arg Leu Val Arg Arg Gln Arg Ser Ala Asp Glu Gln Leu His 225 230 235 240 Cys Ala Gly Lys Ala Lys Arg Ser Gly Gly His Ala Pro Arg Val Arg 245 250 255 Glu Leu Leu Leu Asp Ala Leu Ser Pro Glu Gln Leu Val Leu Thr Leu 260 265 270 Leu Glu Ala Glu Pro Pro His Val Leu Ile Ser Arg Pro Ser Ala Pro 275 280 285 Phe Thr Glu Ala Ser Met Met Met Ser Leu Thr Lys Leu Ala Asp Lys 290 295 300 Glu Leu Val His Met Ile Ser Trp Ala Lys Lys Ile Pro Gly Phe Val 305 310 315 320 Glu Leu Ser Leu Phe Asp Gln Val Arg Leu Leu Glu Ser Cys Trp Met 325 330 335 Glu Val Leu Met Met Gly Leu Met Trp Arg Ser Ile Asp His Pro Gly 340 345 350 Lys Leu Ile Phe Ala Pro Asp Leu Val Leu Asp Arg Asp Glu Gly Lys 355 360 365 Cys Val Glu Gly Ile Leu Glu Ile Phe Asp Met Leu Leu Ala Thr Thr 370 375 380 Ser Arg Phe Arg Glu Leu Lys Leu Gln His Lys Glu Tyr Leu Cys Val 385 390 395 400 Lys Ala Met Ile Leu Leu Asn Ser Ser Met Tyr Pro Leu Val Thr Ala 405 410 415 Thr Gln Asp Ala Asp Ser Ser Arg Lys Leu Ala His Leu Leu Asn Ala 420 425 430 Val Thr Asp Ala Leu Val Trp Val Ile Ala Lys Ser Gly Ile Ser Ser 435 440 445 Gln Gln Gln Ser Met Arg Leu Ala Asn Leu Leu Met Leu Leu Ser His 450 455 460 Val Arg His Ala Ser Asn Lys Gly Met Glu His Leu Leu Asn Met Lys 465 470 475 480 Cys Lys Asn Val Val Pro Val Tyr Asp Leu Leu Leu Glu Met Leu Asn 485 490 495 Ala His Val Leu Arg Gly Cys Lys Ser Ser Ile Thr Gly Ser Glu Cys 500 505 510 Ser Pro Ala Glu Asp Ser Lys Ser Lys Glu Gly Ser Gln Asn Pro Gln 515 520 525 Ser Gln 530 <210> 90 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> A cell membrane permeable peptide <400> 90 Arg Lys Lys Arg Arg Gln Arg Arg Arg 1 5 <210> 91 <211> 21 <212> PRT <213> Artificial Sequence <220> <223> A cell membrane permeable peptide <400> 91 Thr Arg Ser Ser Arg Ala Gly Leu Gln Phe Pro Val Gly Arg Val His 1 5 10 15 Arg Leu Leu Arg Lys 20 <210> 92 <211> 27 <212> PRT <213> Artificial Sequence <220> <223> A cell membrane permeable peptide <400> 92 Gly Trp Thr Leu Asn Ser Ala Gly Tyr Leu Leu Gly Lys Ile Asn Leu 1 5 10 15 Lys Ala Leu Ala Ala Leu Ala Lys Lys Ile Leu 20 25 <210> 93 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> A cell membrane permeable peptide <400> 93 Lys Leu Ala Leu Lys Leu Ala Leu Lys Ala Leu Lys Ala Ala Leu Lys 1 5 10 15 Leu Ala <210> 94 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> A cell membrane permeable peptide <400> 94 Ala Ala Val Ala Leu Leu Pro Ala Val Leu Leu Ala Leu Leu Ala Pro 1 5 10 15 <210> 95 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> A cell membrane permeable peptide <400> 95 Val Pro Met Leu Lys 1 5 <210> 96 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> A cell membrane permeable peptide <400> 96 Pro Met Leu Lys Glu 1 5 <210> 97 <211> 28 <212> PRT <213> Artificial Sequence <220> <223> A cell membrane permeable peptide <400> 97 Met Ala Asn Leu Gly Tyr Trp Leu Leu Ala Leu Phe Val Thr Met Trp 1 5 10 15 Thr Asp Val Gly Leu Cys Lys Lys Arg Pro Lys Pro 20 25 <210> 98 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> A cell membrane permeable peptide <400> 98 Leu Leu Ile Ile Leu Arg Arg Arg Ile Arg Lys Gln Ala His Ala His 1 5 10 15 Ser Lys <210> 99 <211> 21 <212> PRT <213> Artificial Sequence <220> <223> A cell membrane permeable peptide <400> 99 Lys Glu Thr Trp Trp Glu Thr Trp Trp Thr Glu Trp Ser Gln Pro Lys 1 5 10 15 Lys Lys Arg Lys Val 20 <210> 100 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> A cell membrane permeable peptide <400> 100 Arg Gly Gly Arg Leu Ser Tyr Ser Arg Arg Arg Phe Ser Thr Ser Thr 1 5 10 15 Gly Arg <210> 101 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> A cell membrane permeable peptide <400> 101 Ser Asp Leu Trp Glu Met Met Met Val Ser Leu Ala Cys Gln Tyr 1 5 10 15 <210> 102 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> A cell membrane permeable peptide <400> 102 Thr Ser Pro Leu Asn Ile His Asn Gly Gln Lys Leu 1 5 10 <210> 103 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> A cell membrane permeable peptide <400> 103 Arg Gln Ile Lys Ile Trp Phe Gln Asn Arg Arg Met Lys Trp Lys Lys 1 5 10 15 <210> 104 <211> 32 <212> PRT <213> Artificial Sequence <220> <223> PHB2 derived peptide 1-32aa <400> 104 Met Ala Gln Asn Leu Lys Asp Leu Ala Gly Arg Leu Pro Ala Gly Pro 1 5 10 15 Arg Gly Met Gly Thr Ala Leu Lys Leu Leu Leu Gly Ala Gly Ala Val 20 25 30 <210> 105 <211> 37 <212> PRT <213> Artificial Sequence <220> <223> PHB2 derived peptide 63-99aa <400> 105 Ile Leu Ala Glu Gly Leu His Phe Arg Ile Pro Trp Phe Gln Tyr Pro 1 5 10 15 Ile Ile Tyr Asp Ile Arg Ala Arg Pro Arg Lys Ile Ser Ser Pro Thr 20 25 30 Gly Ser Lys Asp Leu 35 <210> 106 <211> 21 <212> PRT <213> Artificial Sequence <220> <223> cross-linked peptide containing PHB2 derived peptide 11-21aa <220> <221> MISC_FEATURE <222> (1)..(13) <223> linked by using hexafluorobenzene <220> <221> MISC_FEATURE <222> (10)..(10) <223> Xaa = Nle <400> 106 Cys Arg Leu Pro Ala Gly Pro Arg Gly Xaa Gly Thr Cys Arg Arg Arg 1 5 10 15 Arg Arg Arg Arg Arg 20 <210> 107 <211> 21 <212> PRT <213> Artificial Sequence <220> <223> cross-linked peptide containing PHB2 derived peptide 11-21aa <220> <221> MISC_FEATURE <222> (1)..(13) <223> linked by using decafluorobiphenyl <220> <221> MISC_FEATURE <222> (10)..(10) <223> Xaa = Nle <400> 107 Cys Arg Leu Pro Ala Gly Pro Arg Gly Xaa Gly Thr Cys Arg Arg Arg 1 5 10 15 Arg Arg Arg Arg Arg 20 <210> 108 <211> 21 <212> PRT <213> Artificial Sequence <220> <223> cross-linked peptide containing PHB2 derived peptide 11-21aa <220> <221> DISULFID <222> (1)..(13) <220> <221> MISC_FEATURE <222> (10)..(10) <223> Xaa = Nle <400> 108 Cys Arg Leu Pro Ala Gly Pro Arg Gly Xaa Gly Thr Cys Arg Arg Arg 1 5 10 15 Arg Arg Arg Arg Arg 20 <210> 109 <211> 21 <212> PRT <213> Artificial Sequence <220> <223> uncross-linked peptide containing PHB2 derived peptide 11-21aa <220> <221> MISC_FEATURE <222> (10)..(10) <223> Xaa = Nle <400> 109 Ala Arg Leu Pro Ala Gly Pro Arg Gly Xaa Gly Thr Ala Arg Arg Arg 1 5 10 15 Arg Arg Arg Arg Arg 20 <210> 110 <211> 23 <212> PRT <213> Artificial Sequence <220> <223> cross-linked peptide containing PHB2 derived peptide 76-88aa <220> <221> MISC_FEATURE <222> (1)..(15) <223> linked by using hexafluorobenzene <400> 110 Cys Gln Tyr Pro Ile Ile Tyr Asp Ile Arg Ala Arg Pro Arg Cys Arg 1 5 10 15 Arg Arg Arg Arg Arg Arg Arg 20 <210> 111 <211> 23 <212> PRT <213> Artificial Sequence <220> <223> cross-linked peptide containing PHB2 derived peptide 76-88aa <220> <221> MISC_FEATURE <222> (1)..(15) <223> linked by using decafluorobiphenyl <400> 111 Cys Gln Tyr Pro Ile Ile Tyr Asp Ile Arg Ala Arg Pro Arg Cys Arg 1 5 10 15 Arg Arg Arg Arg Arg Arg Arg 20 <210> 112 <211> 23 <212> PRT <213> Artificial Sequence <220> <223> cross-linked peptide containing PHB2 derived peptide 76-88aa <220> <221> DISULFID <222> (1)..(15) <400> 112 Cys Gln Tyr Pro Ile Ile Tyr Asp Ile Arg Ala Arg Pro Arg Cys Arg 1 5 10 15 Arg Arg Arg Arg Arg Arg Arg 20 <210> 113 <211> 23 <212> PRT <213> Artificial Sequence <220> <223> uncross-linked peptide containing PHB2 derived peptide 76-88aa <400> 113 Ala Gln Tyr Pro Ile Ile Tyr Asp Ile Arg Ala Arg Pro Arg Ala Arg 1 5 10 15 Arg Arg Arg Arg Arg Arg Arg 20 <210> 114 <211> 21 <212> PRT <213> Artificial Sequence <220> <223> uncross-linked peptide containing PHB2 derived peptide 11-21aa <400> 114 Ala Arg Leu Pro Ala Gly Pro Arg Gly Met Gly Thr Ala Arg Arg Arg 1 5 10 15 Arg Arg Arg Arg Arg 20 <210> 115 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> cross-linked peptide containing PHB2 derived peptide 11-21aa <220> <221> MISC_FEATURE <222> (1)..(13) <223> linked by using hexafluorobenzene <220> <221> MISC_FEATURE <222> (10)..(10) <223> Xaa = Nle <400> 115 Cys Arg Leu Pro Ala Gly Pro Arg Gly Xaa Gly Thr Cys 1 5 10 <210> 116 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> cross-linked peptide containing PHB2 derived peptide 11-21aa <220> <221> MISC_FEATURE <222> (1)..(13) <223> linked by using decafluorobiphenyl <220> <221> MISC_FEATURE <222> (10)..(10) <223> Xaa = Nle <400> 116 Cys Arg Leu Pro Ala Gly Pro Arg Gly Xaa Gly Thr Cys 1 5 10 <210> 117 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> cross-linked peptide containing PHB2 derived peptide 11-21aa <220> <221> DISULFID <222> (1)..(13) <220> <221> MISC_FEATURE <222> (10)..(10) <223> Xaa = Nle <400> 117 Cys Arg Leu Pro Ala Gly Pro Arg Gly Xaa Gly Thr Cys 1 5 10 <210> 118 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> uncross-linked peptide containing PHB2 derived peptide 11-21aa <220> <221> MISC_FEATURE <222> (10)..(10) <223> Xaa = Nle <400> 118 Ala Arg Leu Pro Ala Gly Pro Arg Gly Xaa Gly Thr Ala 1 5 10 <210> 119 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> cross-linked peptide containing PHB2 derived peptide 76-88aa <220> <221> MISC_FEATURE <222> (1)..(15) <223> linked by using hexafluorobenzene <400> 119 Cys Gln Tyr Pro Ile Ile Tyr Asp Ile Arg Ala Arg Pro Arg Cys 1 5 10 15 <210> 120 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> cross-linked peptide containing PHB2 derived peptide 76-88aa <220> <221> MISC_FEATURE <222> (1)..(15) <223> linked by using decafluorobiphenyl <400> 120 Cys Gln Tyr Pro Ile Ile Tyr Asp Ile Arg Ala Arg Pro Arg Cys 1 5 10 15 <210> 121 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> cross-linked peptide containing PHB2 derived peptide 76-88aa <220> <221> DISULFID <222> (1)..(15) <400> 121 Cys Gln Tyr Pro Ile Ile Tyr Asp Ile Arg Ala Arg Pro Arg Cys 1 5 10 15 <210> 122 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> uncross-linked peptide containing PHB2 derived peptide 76-88aa <400> 122 Ala Gln Tyr Pro Ile Ile Tyr Asp Ile Arg Ala Arg Pro Arg Ala 1 5 10 15 <210> 123 <211> 19 <212> PRT <213> Artificial Sequence <220> <223> cross-linked cyclic peptide containing PHB2 derived peptide 11-21aa <220> <221> MISC_FEATURE <222> (1)..(13) <223> linked by using hexafluorobenzene <220> <221> MISC_FEATURE <222> (10)..(10) <223> Xaa = Nle <220> <221> MISC_FEATURE <222> (15)..(15) <223> Xaa = Nal <400> 123 Cys Arg Leu Pro Ala Gly Pro Arg Gly Xaa Gly Thr Cys Phe Xaa Arg 1 5 10 15 Arg Arg Arg <210> 124 <211> 19 <212> PRT <213> Artificial Sequence <220> <223> cross-linked cyclic peptide containing PHB2 derived peptide 11-21aa <220> <221> MISC_FEATURE <222> (1)..(13) <223> linked by using decafluorobiphenyl <220> <221> MISC_FEATURE <222> (10)..(10) <223> Xaa = Nle <220> <221> MISC_FEATURE <222> (15)..(15) <223> Xaa = Nal <400> 124 Cys Arg Leu Pro Ala Gly Pro Arg Gly Xaa Gly Thr Cys Phe Xaa Arg 1 5 10 15 Arg Arg Arg <210> 125 <211> 19 <212> PRT <213> Artificial Sequence <220> <223> uncross-linked cyclic peptide containing PHB2 derived peptide 11-21aa <220> <221> MISC_FEATURE <222> (10)..(10) <223> Xaa = Nle <220> <221> MISC_FEATURE <222> (15)..(15) <223> Xaa = Nal <400> 125 Ala Arg Leu Pro Ala Gly Pro Arg Gly Xaa Gly Thr Ala Phe Xaa Arg 1 5 10 15 Arg Arg Arg <210> 126 <211> 21 <212> PRT <213> Artificial Sequence <220> <223> cross-linked cyclic peptide containing PHB2 derived peptide 76-88aa <220> <221> MISC_FEATURE <222> (1)..(15) <223> linked by using hexafluorobenzene <220> <221> MISC_FEATURE <222> (17)..(17) <223> Xaa = Nal <400> 126 Cys Gln Tyr Pro Ile Ile Tyr Asp Ile Arg Ala Arg Pro Arg Cys Phe 1 5 10 15 Xaa Arg Arg Arg Arg 20 <210> 127 <211> 21 <212> PRT <213> Artificial Sequence <220> <223> cross-linked cyclic peptide containing PHB2 derived peptide 76-88aa <220> <221> MISC_FEATURE <222> (1)..(15) <223> linked by using decafluorobiphenyl <220> <221> MISC_FEATURE <222> (17)..(17) <223> Xaa = Nal <400> 127 Cys Gln Tyr Pro Ile Ile Tyr Asp Ile Arg Ala Arg Pro Arg Cys Phe 1 5 10 15 Xaa Arg Arg Arg Arg 20 <210> 128 <211> 21 <212> PRT <213> Artificial Sequence <220> <223> uncross-linked cyclic peptide containing PHB2 derived peptide 76-88aa <220> <221> MISC_FEATURE <222> (17)..(17) <223> Xaa = Nal <400> 128 Ala Gln Tyr Pro Ile Ile Tyr Asp Ile Arg Ala Arg Pro Arg Ala Phe 1 5 10 15 Xaa Arg Arg Arg Arg 20 <210> 129 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> modified PHB2 peptide <220> <221> MISC_FEATURE <222> (8)..(8) <223> D-Ala <400> 129 Arg Leu Pro Ala Gly Pro Arg Ala Met Gly Thr Ala Arg Arg Arg Arg 1 5 10 15 Arg Arg Arg Arg 20 <210> 130 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> modified PHB2 peptide <220> <221> MISC_FEATURE <222> (5)..(5) <223> D-Ala <400> 130 Arg Leu Pro Ala Ala Pro Arg Gly Met Gly Thr Ala Arg Arg Arg Arg 1 5 10 15 Arg Arg Arg Arg 20 <210> 131 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> modified PHB2 peptide <220> <221> MISC_FEATURE <222> (8)..(8) <223> D-Leu <400> 131 Arg Leu Pro Ala Gly Pro Arg Leu Met Gly Thr Ala Arg Arg Arg Arg 1 5 10 15 Arg Arg Arg Arg 20 <210> 132 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> modified PHB2 peptide <220> <221> MISC_FEATURE <222> (5)..(5) <223> D-Leu <400> 132 Arg Leu Pro Ala Leu Pro Arg Gly Met Gly Thr Ala Arg Arg Arg Arg 1 5 10 15 Arg Arg Arg Arg 20 <210> 133 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> modified PHB2 peptide <220> <221> MISC_FEATURE <222> (5)..(5) <223> D-Ala <220> <221> MISC_FEATURE <222> (8)..(8) <223> D-Ala <400> 133 Arg Leu Pro Ala Ala Pro Arg Ala Met Gly Thr Ala Arg Arg Arg Arg 1 5 10 15 Arg Arg Arg Arg 20 <210> 134 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> modified PHB2 peptide <220> <221> MISC_FEATURE <222> (5)..(5) <223> D-Leu <220> <221> MISC_FEATURE <222> (8)..(8) <223> D-Leu <400> 134 Arg Leu Pro Ala Leu Pro Arg Leu Met Gly Thr Ala Arg Arg Arg Arg 1 5 10 15 Arg Arg Arg Arg 20 <210> 135 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> modified PHB2 peptide <220> <221> MISC_FEATURE <222> (5)..(5) <223> D-Ala <220> <221> MISC_FEATURE <222> (8)..(8) <223> D-Leu <400> 135 Arg Leu Pro Ala Ala Pro Arg Leu Met Gly Thr Ala Arg Arg Arg Arg 1 5 10 15 Arg Arg Arg Arg 20 <210> 136 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> modified PHB2 peptide <220> <221> MISC_FEATURE <222> (5)..(5) <223> D-Leu <220> <221> MISC_FEATURE <222> (8)..(8) <223> D-Ala <400> 136 Arg Leu Pro Ala Leu Pro Arg Ala Met Gly Thr Ala Arg Arg Arg Arg 1 5 10 15 Arg Arg Arg Arg 20

Claims

1. A peptide comprising a binding site in a PHB2 polypeptide with a BIG3 polypeptide and inhibiting the binding of the PHB2 polypeptide to a BIG3 polypeptide, the peptide having an amino acid sequence selected from the following (a): (a) an amino acid sequence selected from the group consisting of SEQ ID NOs: 26, 5, 23, 24, 47-53, 110-113, 119-122 and 126-128.

2. A peptide comprising a binding site in a PHB2 polypeptide with a BIG3 polypeptide and inhibiting the binding between the PHB2 polypeptide and a BIG3 polypeptide, the peptide having an amino acid sequence selected from the following (a'): (a') An amino acid sequence selected from the group consisting of SEQ ID NOs: 26, 5, 23, 24, 47-53, 110-113, 119-122, and 126-128, in which one amino acid residue other than the amino acid corresponding to the 82nd aspartic acid in the amino acid sequence of SEQ ID NO: 28 is substituted with another amino acid residue.

3. The peptide of claim 1, consisting of the amino acid sequence of SEQ ID NO:

26.

4. The peptide according to any one of claims 1 to 3, which is modified with a cell membrane-permeable substance.

5. 5. The peptide of claim 1, which is in cyclic form.

6. 6. The peptide according to any one of claims 1 to 5, which is in cross-linked form.

7. The peptide according to any one of claims 1 to 6, having one or both of the following properties (i) and (ii): (i) inhibiting cell proliferation of BIG3-positive cells; and (ii) It promotes phosphorylation of serine residues in PHB2 polypeptide in BIG3-positive cells.

8. A polynucleotide encoding the peptide of any one of claims 1 to 7.

9. A pharmaceutical composition comprising at least one component selected from the group consisting of one or more peptides according to any one of claims 1 to 7, polynucleotides encoding said peptides, and pharmaceutically acceptable salts of said peptides, and a pharmaceutically acceptable carrier.

10. The pharmaceutical composition described in claim 9, comprising a peptide having an amino acid sequence consisting of amino acids 76 to 88 in the amino acid sequence of SEQ ID NO:

28.

11. The pharmaceutical composition according to claim 9 or 10, for inhibiting the proliferation of cancer cells or for treating and / or preventing cancer.

12. The pharmaceutical composition according to claim 11, wherein the cancer is a BIG3-positive cancer.

13. 13. The pharmaceutical composition according to claim 11 or 12, wherein the cancer is breast cancer.

14. The pharmaceutical composition according to any one of claims 11 to 13, wherein the cancer is an estrogen receptor-positive cancer.

15. Use of an ingredient selected from the group consisting of one or more peptides according to any one of claims 1 to 7, polynucleotides encoding said peptides, and pharmaceutically acceptable salts of said peptides in the manufacture of a pharmaceutical composition for either or both of the treatment and prevention of cancer.

16. The use according to claim 15, wherein the peptide comprises an amino acid sequence consisting of amino acids 76 to 88 of the amino acid sequence of SEQ ID NO: 28.

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