WO2014178680A1

WO2014178680A1 - Peptide for inhibiting hsp expression and composition containing same

Info

Publication number: WO2014178680A1
Application number: PCT/KR2014/003948
Authority: WO
Inventors: 김상재; 김범준
Original assignee: 주식회사 카엘젬백스
Priority date: 2013-05-03
Filing date: 2014-05-02
Publication date: 2014-11-06
Also published as: KR20160009548A; TW201534725A; TWI634210B; KR102275912B1

Abstract

The present invention provides a composition for inhibiting an HSP expression, containing a peptide including an amino acid sequence of SEQ ID NO: 1, a peptide having a sequence similarity which is higher than or equal to 80% of the peptide sequence, or a HSP binding peptide, which is a fragment thereof, and a method for inhibiting an HSP expression using the same. Particularly, since an HSP specifically increases in a cancer cell, a method for inhibiting an HSP expression by using a peptide specifically bonding with the HSP can be provided.

Description

【Specification】

[Name of invention]

HSP expression inhibitory peptide and composition comprising same

Technical Field

The present invention relates to a peptide having HSP expression inhibitory activity and a composition comprising the same, and more particularly to a peptide having HSP expression inhibitory activity as a peptide derived from telomerase and a composition comprising the same, and It relates to a method of inhibiting HSP expression.

<2>

Background Art

Cancer cells express high levels of several Heat Shock Proteins (HSPs) to enhance tumor aggression and allow cells to survive mortality conditions, such as by treatment. In addition to imparting resistance to treatment, elevated HSP expression inhibits programmed cell death and promotes autologous growth resulting in cancer cell growth.

First reported in 1962, the thermal stratified protein has been known to be a cell constituent protein that acts as a molecular chaperone and protects cells in stress environments such as heat, hypoxia and radioactivity (Ritossa F. Experimentia 1962; 18: 571-3; Lindquist S et al., Craig Annu Rev Genet 988; 22: 631-77). Recently cell growth and

It has been speculated that heat shock proteins may play a role in tumorigenesis, as they are involved in apoptosis and overexpressed in various tumor tissues. HSP is important for cell survival in stress situations such as hypoxia. Thermal stratified proteins are classified into various groups (small HSP family, HSP 60 family, HSP 70 family, HSP 90 family) according to the molecular weight.

In relation to tumors, HSP 70 has increased expression in endometrial cancer, malignant osteosarcoma, and kidney cancer, which is thought to be due to the inhibition of apoptosis. HSP 90 accounts for 1- of normal cellular proteins and increased expression, particularly in stress environments. HSP 90 reacts with ATP to fold denatured proteins and has increased expression in lung cancer, leukemia, Hodgkin's disease, etc., and HSP 90 has also been reported to be associated with apoptosis, but the exact role has not been identified. not. <6> As the heat stratified protein was found to be involved in tumorigenesis, efforts were made to use an expression inhibitor for anticancer treatment, and the HSP 90 inhibitor 17AAGC 17-a 11 y 1 am i no 17-deme. t hyxy analogue of geldanamycin has been recognized for its potential as an anticancer drug and is in clinical trials. Quercetin

It is a phytochemical with a polyphenol group belonging to flavonoids and is known to inhibit cell proliferation in experiments involving several cancer cell lines. It is known to inhibit the production, and has been studied as a medicine that is treated in combination with hyperthermia therapy.

HSP70 is a highly conserved protein chaperone associated with many intracellular mechanisms.

HSP70 is induced by intracellular stress and suppresses stress-induced apoptosis

Suppress In addition, HSP70 has immunomodulatory capacity and is known to stimulate the production of anti-inflammatory cytokines (Van Eden, W. et al., Nat. Rev. Immunol.

5: 318-330 (2005)). In addition, it prevents inflammatory shock caused by tumor necrosis factor (TNF) and induces activation of Antigen presenting cells (APCs).

HSP70 (constitutively expressed Hsc70 and heat induced HSP70-1 and HSP70-6) is involved in the preliminary stage, while HSP90 is involved in the last stage. Their functions include a number of co-chaperones such as HSP70-regulators, HSP40, HSP110, BAG and HIP; Requires HSP-organizing protein (HOP), which is involved in the formation of intermediate molecular chaperone complexes, where the client passes from HSP70 to HSP90, and other such as p23, cdc37 and immunophilin in the final or mature HSP90 complex Works. HSP90 is recognized as a new anticancer target. HSP90 is a highly ubiquitous (1-2% of whole cell protein) essential protein that functions as a molecular chaperone to ensure morphological stability, shape and function of the client protein. Inhibition of its native ATPase activity of HSP90 disrupts HSP90-client protein interactions, leading to its degradation via the ubiquitin proteasome pathway. Subtypes of the HSP90 client protein, such as Raf, AKT, CDK4, and ErbB2, are EGFR family of oncogenic signaling molecules that are critically involved in cell growth, differentiation and apoptosis. It is important in Pau

Therefore, the development of a composition that can suppress the expression of HSP70 and HSP90 is expected to have a large ripple effect. [Preceding technical literature]

[Patent Documents]

KR2012-0117954A

KR2006-0023576A

[Non-Patent Documents]

Van Eden,. et al., Nat. Rev. Immunol. 5: 318-330 (2005)

Ritossa F. Experiment i a 1962; 18: 571-3.

Lindquist S et al. , Craig Annu Rev Genet 988; 22: 631-77

[Detailed Description of the Invention]

[Technical problem]

Accordingly, the present inventors have completed the present invention as a result of intensive efforts to develop a drug capable of specifically binding to HSP and inhibiting the expression of HSP.

The inventors have developed a peptide that specifically binds to HSP as a peptide derived from telomerase and can specifically inhibit the expression of HSP.

An object of the present invention is to provide a peptide that binds to HSP and inhibits the expression of HSP and a method for inhibiting HSP expression using the same.

Technical Solution

According to one aspect of the invention there is provided a composition for inhibiting HSP expression comprising a peptide comprising the amino acid sequence of SEQ ID NO: 1, a peptide having a sequence homology of 80% or more with the amino acid sequence or a fragment thereof.

In the HSP binding peptide according to an aspect of the present invention, the HSP may be HSP 70 or HSP 90.

In the HSP binding peptide according to an aspect of the present invention, the fragment may be a fragment consisting of three or more amino acids.

In the HSP binding peptide according to one aspect of the invention, the peptide may be composed of up to 30 amino acids.

In the composition for inhibiting angiogenesis according to an aspect of the present invention, the composition is tumor growth and metastasis, diabetic retinopathy, prematurity retinopathy, corneal graft rejection, neovascular glaucoma, melanoma proliferative retinopathy, psoriasis, macular degeneration ( macular degeneration, hemophilia Capillary proliferation in sexual joints, atherosclerotic plaques, keloids, wound granulation, vascular adhesion, rheumatoid arthritis, chronic inflammation, osteoarthritis, autoimmune disease, Crohn's disease, restenosis, atherosclerosis Hardening, intestinal adhesion, cat scratch disease, ulcers, cirrhosis, glomerulonephritis, diabetic nephropathy, malignant neurosis, thrombotic microangiopathy, organ transplant rejection, renal glomerulopathy, diabetes, inflammatory or neurodegenerative diseases Sex angiogenesis-may be used for the prevention or treatment of related diseases or disorders.

In the composition for inhibiting angiogenesis according to an aspect of the present invention, the composition may be used for the prevention or treatment of tumor growth and metastasis.

In the composition for inhibiting angiogenesis according to an aspect of the present invention, the composition may be a pharmaceutical composition.

In the composition for inhibiting angiogenesis according to an aspect of the present invention, the composition may be a food composition.

According to another aspect of the present invention, it may be a method for preventing and treating HSP expression related diseases, including a system for administering the above-mentioned composition to a subject.

<33>

Advantageous Effects

According to the present invention, it is possible to effectively inhibit HSP expression through a peptide that specifically binds to HSP. In particular, since HSP has an increasing characteristic in cancer cells, a method capable of effectively inhibiting tumor growth may be provided by using a template that specifically binds to HSP.

<35>

[Brief Description of Drawings]

1 is to identify a protein that interacts with PEP 1 as follows;

(A) Purified His-tag containing GFP (SEQ ID NOS: 3, 4), 11mer-GFP (GFP fused to short PEP 1), and PEP 1-GFP Protein is

Cultured with HEK293T cell lysate, each protein complex was purified using Ni-NTA chromatography and then subjected to SDS-PAGE and silver staining. Proteins specifically associated with PEP 1-GFP were confirmed by Maldi-TOF mass spectrometry after excision on gels. * Means GFP, llmer-GFP, PEP 1-GFP bands.

(B) Identified proteins associated with PEP 1 may be immunized with antibodies.

This is confirmed by blotting (i 誦 unoblotting, IB). (C) To confirm the interaction of PEP 1 and HSP70 in other cells,

Lysates of HepG2 cells were incubated with GST or PEP 1-GST proteins. The results of the pull down using glutathione resin and the results of HSP70 and PEP 1 were confirmed using immunoblotting and anti-HSP70 antibody.

(D) After membrane fragments of MCF7 were separated, GF? Or incubated with PEP 1-GFP. The combination of HSP70 was confirmed by immunoblotting after Ni-NTA chromatography.

2 to 3 show the down regulation of HSP70 and HSP90 according to PEP 1 treatment in cancer cells. Jurkat (FIG. 2) and MCF7 (FIG. 3) cells were treated with increasing concentrations of PEP 1 or scrambled peptide in serum-free medium for 2 hours. The amount of protein of HSP70 and HSP90 was analyzed by immunoblotting using antibodies to HSP70, HSP90 and GAPDH.

4 to 5 are experimental results showing hypoxia induced HSPs production by PEP 1. MCF7 and HeLa cells were treated with PEP 1 (20 μΜ) or a solvent and incubated at low oxygen for a specified time. Cell lysates were subjected to immunoblotting to analyze the amounts of HSP70 and HSP90.

FIG. 6 shows the results of treating solvent, PEP 1, 17-AAG, and KNK437 on Jurkat and MCF7 cells. Jurkat and MCF7 cells were treated with vehicle, PEP 1 (5 μΜ for Jurkat and 20 μΜ for MCF7), 17-AAG (1 μΜ), NK437 (1 μΜ) in serum-free medium for 2 hours. Cell lysates were analyzed by immunoblotting similar to the method used in FIG. 2.

FIG. 7 shows the results of treatment of MCF7 cells with PEP 1 or PBS in both MG132 (5 μΜ) and non-containing states. Intracellular HSPs and cell surface HSPs were stained by surface intracellular staining and surface staining staining, and analyzed using flow cytometry as described in the test materials and methods. Red means DMS0, Blue: DMS0CPEP 1 plus DMS0 with PEP 1; Orange: MG132 with PEP 1 (PEP 1 plus MG132); Green: Means MG132 do.

8 to 9 are graphs showing inhibition of cancer cell proliferation by PEP 1 in a low oxygen state. MCF7 and HeLa cells were cultured with or without PEP 1 in either normal oxygen (left panel) or low oxygen (right panel). At 2, 4 and 6 days of culture, the number of cells was measured. Data are mean 土 standard deviation (Data represent means 土 SD). *: p <0.05 was considered statistically significant. A one-way t-test was performed.

10 to 11 show the reduction of HSP70 and HSP90 protein levels of tumors by PEP 1 treatment. HSP70 and HSP90 protein levels in tumor sections were visualized by immunohistochemical staining with antibodies against HSP70 and HSP90 (FIG. 10) and quantified using Leica Qwin software (FIG. 10).

11). Randomly selected fields were quantified from six slides of each treatment group. Data represent mean 土 standard deviation. *** p <0.001, statistically significant, 2-way t-test was performed. Protein extracts from tumors were subjected to immunoblotting using antibodies against HSP70, HSP90, GRP78 and GAPDH (Fig.

12).

13 to 14 show the effect of PEP 1 on the HSP70 level secreted in the blood. In FIG. 13, HSP70 levels (left panel) and HSP90 (right panel) are from mouse models treated with PEP 1 (50 μg / kg) or PBS (10 mice per group; n = 20). The obtained serum was confirmed by performing ELISA. In FIG. 14, the correlation between HSP70 level in blood and tumor weight (left panel) or tumor size (right panel) was analyzed. A binary t-test was performed.

<48>

[Best form for implementation of the invention]

The present invention may be variously modified and have various embodiments. Hereinafter, the present invention will be described in more detail with reference to examples. However, these examples are not intended to limit the present invention to a particular embodiment, the present invention is capable of various examples and uses on the basis of the claims are included in the spirit and scope of the present invention It should be understood to include all transformations, equivalents, or substitutes that are made. In the following description of the present invention, if it is determined that the detailed description of the related known technology may obscure the gist of the present invention, the detailed description thereof will be omitted.

<50>

<5i> Telomere is a genetic material repeatedly present at the end of a chromosome and is known to prevent damage to the chromosome or binding to another chromosome. Each time a cell divides, the telomeres get a little shorter. In the presence of fever, telomeres become very short, and the cells stop dividing and die. On the other hand, elongation of telomeres is known to prolong cell life. For example, cancer cells secrete an enzyme called telomerase, which prevents telomeres from shortening _. Be known

<52>

As the compound that binds to the HSP90 family protein, benzoquinone anamycin-based antibiotics such as geldanamycin and habimycin, and radicicol are known. All these compounds are reported to exhibit pharmacological activity such as antitumor activity by binding to HSP90 family protein and inhibiting the function of HSP90 family protein. Therefore, compounds that bind to HSP90 family proteins are known to be useful as therapeutic agents for diseases involving HSP90 family proteins or proteins to which HSP90 family proteins bind (HSP90 client proteins).

<54>

One aspect of the present invention provides a peptide comprising a amino acid sequence of SEQ ID NO: 1, a polynucleotide encoding a peptide that is a peptide or fragment thereof having a sequence homology of 80% or more with the amino acid sequence. . The polynucleotides can be used to mass produce peptides. For example, a large amount of peptides can be produced by culturing a vector containing a polynucleotide encoding a peptide into a host cell.

<56>

The peptides disclosed herein may include peptides having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% homology. have. In addition, the peptide disclosed herein, the peptide comprising SEQ ID NO: 1 or fragments thereof and one or more amino acids, two or more amino acids, three or more amino acids, four or more amino acids, five or more amino acids, six or more amino acids Or peptides with seven or more amino acids changed.

In one aspect of the invention, amino acid changes belong to the property that allows the physicochemical properties of the peptide to be altered. For example, amino acid changes can be made, such as improving the thermal stability of the peptide, altering substrate specificity, changing the optimal pH, and the like.

In one aspect of the invention, a peptide comprising the sequence of SEQ ID NO: 1 or a peptide having at least 80% sequence homology with the peptide sequence is selected from telomerase, specifically ^ Homo sapiens telomerase. Peptides derived.

As used herein, the term "amino acid" refers to 22 amino acids that are naturally incorporated into a peptide. Standard amino acids as well as D-isomers and modified amino acids. Accordingly, in one aspect of the invention the peptide may be a peptide comprising D-amino acids. On the other hand, in another aspect of the invention, the peptide may include post-trans 1 at ional modification, non-standard amino acid and the like _. have. Of posttranslational _{modifications.} Examples include phosphorylation, glycosylation, acyllat ion (eg, acetylation, myristoylated yr istoylat ion) and

Palmitoylation), alkylat ions,

Carboxylation, hydroxy 1 at ion, glycation ion, biotinylation, ubiquit inylat ion, changes in chemical properties (e.g., beta- Removal deimidization, deamidation) and structural changes (eg, formation of disulfide bridges). In addition, to form a peptide conjugate

Changes in amino acids such as changes in amino acids, such as amino groups, carboxyl groups or side chains, caused by chemical reactions occurring in the process of bonding with crosslinkers.

Peptides disclosed herein can be wild-type peptides identified and isolated from a natural source. On the other hand, the peptides disclosed herein may be artificial variants comprising amino acid sequences in which one or more amino acids have been substituted, deleted and / or inserted compared to peptides that are fragments of SEQ ID NO: 1. Amino acid changes in wild type polypeptides as well as in artificial variants include conservative amino acid substitutions that have no significant effect on the folding and / or activity of the protein. Examples of conservative substitutions include basic amino acids (arginine, lysine and histidine), acidic amino acids (glutamic acid and aspartic acid), polar amino acids (glutamine and asparagine), hydrophobic amino acids (leucine, isoleucine, valine and methionine), aromatic amino acids ( Phenylalanine, tryptophan and tyrosine), and small amino acids (glycine, alanine, serine and threonine). Amino acid substitutions that generally do not alter specific activity are known in the art. The most common exchanges occur are Ala / Ser, Val / Ile, Asp / Glu, Thr / Ser, Ala / Gly, Ala / Thr, Ser / Asn, Ala / Val, Ser / Gly, Tyr / Phe, Ala / Pro, Lys / Arg, Asp / Asn, Leu / Ile, Leu / Val, Ala / Glu, and Asp / Gly, and vice versa. Other examples of conservative substitutions are shown in the following table.

<62> [Table 1]

Substantial modifications in the biological properties of the peptide include (a)

. Their effect in maintaining the structure of the peptide backbone, such as a sheet or helix conformation, (b) their effect in maintaining the charge or hydrophobicity of the molecule at the target site, or (C) side chains Their effect on maintaining the bulk of is accomplished by choosing significantly different substitutions. Natural residues are divided into the following groups based on conventional side chain properties:

(1) hydrophobicity: norleucine, met, ala, val, leu, ile;

(2) neutral hydrophilic: cys, ser, thr;

(3) acidic: asp, glu;

(4) basic: asn, gin, his, lys, arg;

(5) residues affecting chain orientation: gly, pro; And

(6) aromatic: trp, tyr, phe. ^'

Non-conservative substitutions may be made by exchanging a member of one of these classes for another class. Will be done. Any cysteine residue that is not involved in maintaining the proper conformation of the peptide can generally be substituted with serine to improve the oxidative stability of the molecule and to prevent abnormal crosslinking. Conversely, cysteine bond (s) can be added to the peptide to improve its stability.

Another type of amino acid variant of the peptide is that the glycosylation pattern of the antibody is altered.

^■ will. By change is meant deletion of one or more carbohydrate residues found in the peptide and / or addition of one or more glycosylation sites that are not present in the peptide.

Indicates.

Glycosylation of peptides is typically either N-linked or 0-linked. N-linked refers to a carbohydrate moiety attached to the side chain of an asparagine moiety. The tripeptide sequences asparagine -X-serine and asparagine -X-threonine, where X is any amino acid except proline, are recognition sequences for enzymatic attachment of carbohydrate moieties to the asparagine side chains. Thus, the presence of one of these tripeptide sequences in a polypeptide creates a potential glycosylation site. 0-linked glycosylation means attaching one of the sugars N-acetylgalactosamine, galactose or xylose to hydroxyamino acids, most commonly serine or threonine, but the 5-hydroxyproline is lean or 5-hydroxy Roxylysine can also be used.

The addition of glycosylation sites to the peptide is conveniently carried out by changing the amino acid sequence containing one or more of the above-mentioned tripeptide sequences (for N-linked glycosylation sites). Such changes may be made by adding one or more serine or threonine residues to the sequence of the original antibody or by substituting these residues (for a 0-linked glycosylation site).

In one aspect of the invention, the polynucleotide may be a naturally occurring or artificial DNA or RNA molecule as a nucleic acid molecule, and may be single stranded or double stranded. The nucleic acid molecule may be one or more, which may be nucleic acid molecules of the same type (eg, having the same nucleotide sequence), or may be nucleic acid molecules of other types. One or more of DNA, cDNA, decoy DNA, RNA, siRNA, miRNA, shRNA, stRNA, snoRNA, snRNA, PNA, antisense oligomer, plasmid and other modified nucleic acids, including It is not limited.

In one aspect of the present invention, a peptide comprising the amino acid sequence of SEQ ID NO: 1, a peptide having 80% or more sequence homology with the amino acid sequence, or a fragment thereof is administered to a subject in need of inhibition of HSP expression. HSP expression inhibition method comprising the. Another aspect of the present invention provides a peptide comprising an amino acid sequence of SEQ ID NO: 1 in preparing a composition for inhibiting HSP expression, a peptide having a sequence homology of 80% or more with the amino acid sequence, or a fragment thereof Phosphorus peptides.

SEQ ID NO: 1 (hereinafter 'PEP 1') in the present invention is a telomerase-derived peptide, a peptide consisting of 16 amino acids as described below.

SEQ ID NO: 1 EARPALLTSRLRFIPK

<79>

The peptides set forth in SEQ ID NO: 1 are shown in Table 2 below. "Name" in Table 2 below is named to distinguish the peptide. In one aspect of the invention, the peptide set forth in SEQ ID NO: 1 represents the entire peptide of human telomerase. In another aspect of the present invention, a peptide having a sequence of SEQ ID NO: 1, a peptide that is a fragment of the sequence of SEQ ID NO: 1, or a peptide having a sequence homology of 80% or more with the peptide sequence is a peptide contained in telomerase. It includes the "synthetic peptide" synthesized by selecting the peptide of the corresponding position. SEQ ID NO: 2 shows the amino acid sequence of the entire telomerase.

<81> [Table 2]

Marks ^ Sί ¾2 ^ ¾ ΞiΕ ^S 2><H_> -

An aspect of the present invention provides a method for inhibiting HSP expression for delivering at least one active ingredient of a peptide comprising SEQ ID NO: 1 or fragments thereof or a peptide having 80% or more sequence homology with the peptide sequence. It provides a composition for.

_<84>, in one aspect of the invention comprises a peptide or fragment thereof which are peptides de, or conjugates of HSP binding peptides as the active ingredient having the peptide sequence with at least 80% sequence homology comprising SEQ ID NO: 1, Provided is a pharmaceutical or food composition having an excellent effect of delivering an active ingredient into cells.

The composition according to one aspect of the present invention comprises a peptide comprising an amino acid sequence of SEQ ID NO: 1 in one aspect, a peptide having a sequence homology of 80% or more with the amino acid sequence, or a fragment thereof. jg / mg to 1 mg / mg, specifically 1 Ug / nig to 0.5 mg / mg, more specifically 10 // g / mg to 0.1 mg / mg. When included in the above range is not only suitable for showing the intended effect of the present invention, it can satisfy both the stability and safety of the composition, it may be appropriate to include in the above range in terms of cost-effectiveness.

The composition according to one aspect of the present invention may be applied to all animals including humans, dogs, chickens, pigs, cattle, sheep, guinea pigs or monkeys.

The pharmaceutical composition according to the uniaxial aspect of the present invention may be administered orally, rectally, transdermally, intravenously, intramuscularly, intraperitoneally, intramedullary, intradural or subcutaneously.

<88>^'formulation for oral administration is not the form of tablets, pills, soft or hard shell selje, granules, powders, solutions or emulsions, but can best, limited. Formulations for parenteral administration may be, but are not limited to, injections, drops, lotions, ointments, gels, creams, suspensions, emulsions, suppositories, patches or sprays.

The pharmaceutical composition according to one aspect of the present invention may include additives of diluents, excipients, lubricants, binders, disintegrants, laxatives, dispersants, surfactants, colorants, fragrances or sweeteners as needed. . Pharmaceutical compositions according to one aspect of the invention may be prepared by conventional methods in the art.

<90> The active ingredient of the pharmaceutical composition according to one aspect of the present invention

Age, sex, weight, pathology and severity, route of administration or prescriber's judgment will vary. Dosage determination based on these factors is within the level of one skilled in the art, the daily dosage of which is for example 0.1 ^ g / kg / day to 1 g / kg / day specifically 1; «g / kg / day to 10 mg / kg / day, more specifically 10 / g / kg / day to 1 mg / kg / day, even more specifically 50 zg / kg / day to 100 zg / kg / day, but is not limited thereto It is not. The pharmaceutical composition according to one aspect of the present invention may be administered once to three times a day, but is not limited thereto.

The formulation of the food composition according to one aspect of the present invention is not particularly limited, but may be, for example, formulated into a tablet, a granule, a powder, a liquid, a solid formulation, and the like. Each formulation can be appropriately selected and blended by those skilled in the art according to the formulation or purpose of use, in addition to the active ingredient, and synergistic effects may occur when applied simultaneously with other raw materials.

Determination of the dosage of the active ingredient is within the level of one skilled in the art, the daily dosage of which is for example specifically 1 / zg / kg / day to 10 mg / kg / day, more specifically 10 / kg / day to 1 mg / kg / day, and more specifically 50 / kg / day to 100 // g / kg / day, but is not limited thereto, and includes the age, health condition, It can depend on a variety of factors, including complications.

The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The term without a number before the noun is not intended to limit the quantity but rather to the presence of one or more of the noun articles mentioned. The terms "comprising", "having", and "comprising" 'are interpreted as open terms (ie, the meaning of "including, but not limited to").

Reference to a range of numbers is merely a substitute for the individual reference to each individual number within that range, unless otherwise indicated, each number being the same as stated individually in the specification. Applies to this specification. All range limits are included in the range and can be combined independently.

All methods mentioned herein may be performed in the proper order unless otherwise specified or clearly contradicted by context. The use of any one embodiment and all embodiments or exemplary language (such as "as") is merely intended to facilitate the description of the present invention, unless it is included in the claims. It is not intended to limit. No language in the specification should be construed as essential to the practice of the invention as to any unclaimed elements. Unless otherwise defined, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

<%> Preferred embodiments of the invention include the most optimal mode known to the inventors for carrying out the invention. Variations of the preferred embodiments may become apparent to those skilled in the art upon reading the foregoing description. We believe that those skilled in the art will appreciate such variations. In anticipation of use, the inventors expect the invention to be practiced in a manner different from that described herein. Accordingly, the invention includes all modifications and equivalents of the subject matter referred to in the appended claims, as permitted by patent law. Moreover, any and all combinations of the above-mentioned components within all possible variations are included in the present invention unless expressly stated to the contrary or apparently contradictory in context. While the invention has been particularly shown and described with reference to exemplary embodiments, those skilled in the art will appreciate that various changes in form and detail may be made without departing from the spirit and scope of the invention as defined by the following claims. .

<97>

Hereinafter, the configuration and effects of the present invention will be described in more detail with reference to Examples and Experimental Examples. However, the following Examples and Experimental Examples are provided only for the purpose of illustration to help the understanding of the present invention, but the scope and scope of the present invention is not limited thereto.

<99>

[Form for implementation of invention]

Example 1: Synthesis of Peptides

<ιοι> The peptide of SEQ ID NO: 1 (hereinafter referred to as "PEP 1 '') was prepared according to the conventionally known solid phase peptide synthesis method. Specifically, the peptides were prepared using Fmoc using ASP48S (Peptron, Inc., Daejeon, Korea). It was synthesized by coupling one amino acid from the C-terminus to one through solid phase peptide synthesis (SPPS), using the first amino acid at the C-terminus of the peptides attached to the resin, for example:

<i02> NH ₂ -Lys (Boc) -2-chloro-Trityl Resin

<i03> NH ₂ -A 1 a-2-chl oro-Tr i ty 1 Resin

<i04> NH ₂ -Arg (Pbf) -2-chl oro-Tr i ty 1 Resin

<105>

<i06> All amino acid raw materials used for peptide synthesis are N-term to Fmoc.

Trt, Boc, t-Bu protected and all residues removed from acid

(t-butylester) and Pbf (2,2,4,6, 7-pent amethy 1 dihydro-benzof uran-5-sul fonyl) were used. For example:

<i07> Fmoc-Ala-OH, Fmoc-Arg (Pbf) -0H, Fmoc-Glu (0tBu) -0H, Fmoc-Pro-OH,

Fmoc-Leu-OH, Fmoc-I le-OH, Fmoc-Phe-OH, Fmoc-Ser (tBu) -0H, Fmoc-Thr (tBu) ᅳ OH, Fffloc -L s (Boc) -OH, Fmoc-GlniT ^' r)-() H, Fmoc-Tr (Boc) -0H, Fmoc-Met-OH,

Fmoc ^to A sii (Tr t) ^to 0H, Fsnoc -Tyr (tBu) -OH, I¾ (} c— Ahx— OH, Tr t-Mercaptoacetic acid.

With Kerr Reagent (CoupHng reage!)

^{^{ΗΒ 'ί' ϋ [2- (}} lH-Benzot riazo! E- 1- y!) ~ 1, 1.3, 3- let ame thylainini um hexaf 1 uor ophosphat ej I HQBt [N-Hydroxxybenzot ri azo 1 e J /誦[4-Met ylmorphol i ne] was used, Fmoc removal was performed using piperidine (pi. Pe rfi ne in DMF) in 20% of DMF. Cleavage Cocktai! I TFA (tr I fiuo ro acetic acid) / ^' TIS (tri \ sopropy Isil ane) I EDT (eihanedi thiol) / ¾0 쒜 5 / 2.5 / 2.5 / 2.5] was used. When the starting amino acid with the amino acid protecting group is bound to the goring ^{and the} support, are the amino acids reacted with each other? Each peptide was synthesized by repeating the step of deprotection after washing with a solvent. The synthesized peptide was separated from the resin, purified by HPLC, and confirmed by MS and lyophilized.

The purity of all peptides was 95% or higher for the high titers used for the examples used in this example.

The specific process of manufacturing PEP 1 is as follows.

1) Copal ¾

NH Lys CBoc) -2-ch I oro-Tr t] dissolved in Resin and added to the DMF by dissolving amino acids (8 equivalents) and the coupling reagent ΗΒΠΚ8 equivalents) / H0Bt½ equivalents / NMMU6 equivalents) The phase was reacted for 2 hours at and washed with DMF, MeOR, and DMF in that order.

2) Fmoc Lal Protection

Piperidhie in DMF with 20% DMF was added and phase 2 was reacted for 5 minutes at the phase of Ji DMF MeDH. Washed in DMF order.

3) Repeated reaction of 1 and 2 to make the basic skeleton

(N¾— E (OtBu) -— / \ — R (Pbf) ᅳ P 一 A ᅳ L ᅳ L ᅳ T Bu) — -S (tBu) — ^

ro-Triiyi Resin)

4) Cleavage: Synthesis The cleavage cocktail (Cleavage C ckiaH.) Was added to the finished peptide resin to separate the peptide from the resin.

5) Cooling diethyl ether in the obtained mixture After addition, the peptide obtained by centrifugation is precipitated.

<i2i> 6) Purification by Prep-HPLC, molecular weight confirmed by LC / MS and frozen

Prepared as a powder.

<122>

PEP 1 prepared in the same manner as in Example 1 was used to experiment with binding to HSP and HSP inhibition.

<124>

Example 2: Cell Line Culture and Analysis Method

<126>

<127> cell line culture

Human breast cancer cell lines MCF7 (Human breast adenocarcinoma cell line), human T lymphocyte cell line (Jurkat) and MC38 (murine colon adenocarcinoma) cell lines were obtained from 10% fetal bovine Serum and 100 U / ml penicillin. And streptomycin added to RPM 11460 medium. HeLa (human cervical adenocarcinoma) cell line was supplemented with 10% fetal bovine serum and 100 U / ml penicillin and streptomycin.

It was maintained in Dulbecco's modified Eagle's medium (DMEM) medium.

<129>

<130> Confirmation of protein expression and cell growth in hypoxic state

<i3i> To test the effect of PEP 1 on the level of HSP at low oxygen,

MCF7 and HeLa cells were incubated with low oxygen and normal oxygen after treatment with 20μΜ PEP 1. Anoxia was induced using BBL GasPak (Becton Dickinson), which produced a catalyst reaction within 90 minutes to reduce oxygen to undetectable levels. Incubation time ranged from 2 to 24 hours. As described above, cells were harvested and subjected to immunoblotting using ci-HSP70, Q-HSP90, α-HIF-la, or a -GAPDH antibodies. a -GAPDH was used to normalize the amount of HSP70 / 90 to the amount of GAPDH for protein quant i f icat ion.

In order to investigate the effect of PEP 1 on the growth of cancer cells in the low oxygen state,

MCF7 and HeLa cells were inoculated in a 96 well plate to be ΙχΙΟ cells per well, and then cultured in 37 ^° C. and 5% C0 ₂ conditions in complete media with 10% FBS. After serum starvation treatment for 2 hours, the cells were cultured in all of the complete medium including PEP 1 (20 μΜ). As described above, the cells were low acid for 1 to 6 days. Cultures were carried out in small or normal oxygen. The number of viable cells was measured daily using trypan blue staining method. All calculation experiments were performed in duplicate.

<133>

<134> HSP70 and HSP90 protein level analysis method by immunoblotting

Jurkat and MCF7 cells (5 × 10 ⁵ ) were inoculated and cultured for 12 hours.

After 2 hours of starvation in OPT I-MEM medium, cells were treated with different concentrations of PEP 1, scrambled peptide and 17-AAG (1 μΜ) or N4437 (1 μΜ). After incubation for 2 hours, the cells were harvested and lysed using cell lysis buffer (Thermo Scientific, IL, USA). Bretford Protein. After quantifying the protein concentration using an assay (Bradford Protein Assay, Bio-Rad, USA), the samples were a-HSP70 (sc-32239 and sc-66048, Santa Cruz, CA, USA), a -HSP90 ( abl429, abeam, USA), α-GRP78 (sc-13968, Santa Cruz, CA, USA), α-HIF-la (sc-10790, Santa Cruz, CA, USA) or a -GAPDH (sc-25778, Santa Cruz, CA, USA) antibodies were used to perform SDS-PAGE and immunoblotting. Immune semi-male bands were visualized using an enhanced chemiluminescence kit (cha luminescence kit, iNtRoN Biotechnology, INC, Korea) and quantified using ImageQuantTM LAS-4000 (GE Healthcare Life Science, NJ, US).

<136>

<137> HSP70 and HSP90 protein level analysis method by flow cytometry

<i38> MCF7 cells were treated with PEP 1 or control. Proteasome Inhibition Test

To perform the proteasome inhibition test, cells were treated with 5 μM of proteasome inhibitor MG132 (Calboicam) during the culture. Cells were separated using trypsin and washed with cold PBS (phosphate buffered saline) and FACS buffer (PBS containing 1% BSA and 0.1% NaN ₃ ). Cells were treated with permeabilization buffer (eBioscience, CA, USA) according to the manufacturer's instructions for intracellular staining. Cells were reacted with a-HSP70-FTC (ab61907, Abeam) or a-HSP90-PE (ab65171, Abeam) at 4 ^° C for 30 minutes. Flow cytometry was performed using flow cytometry FACScan (FACScan flow cytometer, Bee ton Dickinson Co., CA, USA). Data was analyzed using Flovjo ™ software (version 10.0.5, Tree Star, Inc., OR, USA).

<139> <140>

<i4i> How to assess the effect of PEP 1 on tumor growth in vivo

7 week old BALB / c athymic (athymic) (Nu / Nu) mice (10 mice per group;

n = 20, dark, Orient Bio Co. Gyunggido, Korea) (murine colon

carcinoma) Subcutaneously inoculated with MC38 (5xl0 ⁵ cells / ml in 200 μl PBS per site) cells, and then randomly divided into two groups. Mice were injected with PEP 1 (50 ug / kg in 100 μl 0.9% NaCl solution (Solution)) or PBS intraperitoneally once every two days. When tumor size reached 10 mm 3, PEP 1 or PBS was administered by intratumoral injection. The size of the tumor was measured once, and the volume of the tumor was calculated using the following equation, volume (mm ³ ) = ((width ² x length) I 2). On day 14 of the experiment, mice were sacrificed and tumor weights were measured. All animal experiments were conducted at Seoul National University College of Medicine, Korea.

The Institute for Experimental Animals, Col lege of Medicine, Seoul

National University at Seoul, Korea).

<143>

Assessment of Proliferating Cells and Cell Death in Tumor Sections

DNA fragmentat ion through a Tunnel assay using formalin-fixed and paraffin-embedded tumor sections to evaluate apoptosis of tumors ) Was analyzed. Tumor sections were stained using ApopTag Peroxidase In Situ Apoptosis Detection Kit (Millipore) according to the manufacturer's instructions. Proliferating cells in tumors were detected using PCNA (Proliferating cell nuclear antigen). For antigen retrieval, tissue sections were deparaffinized, hydrated, and heated in 10 μΜ citrate buffer pH 6.0 for 40 minutes. Tissues were stained with an anti-mouse PCNA monoclonal antibody (ab29, Abeam). The tissues and sections were counterstained using H & E staining method after secondary antibody treatment and development. The fields were then randomly selected from six slides in each treatment group, and the fields were analyzed with Leica Qwin software for quantification.

<146>

<147> Immunoblotting Analysis of HSP Expression in Tumors

<i48> Expression of HSP70 and HSP90 proteins in tumors was evaluated using immunohistochemical staining (i 讓 unohistochemkal staining) in a similar manner to PCNA staining. Antibodies against heat stratified protein (HSP70; sc-7298, HSP90; abl429) were used as primary antibodies All. Expression of HSP70 and HSP90 proteins by tumors was assessed by immunoblotting using tumor lysate. After freezing with liquid oxygen, the tumors were ground using a mortar and pestle, followed by extract ion buffer, 20 mM HEPES, pH7.5, 100 mM NaCl, 0.05% Triton X-100, ImM DTT, 0.5 mM sodium orthovanadate, 1 mM EDTA, 0.5 mM PMSF, 10 μg / ml aprot inin, 5 μg / ml leupept in, 2 yg / ml pepstatin). After repeated centrifugation, the supernatant was subjected to SDS-PAGE and immunoblotting (i 隱 unoblotting) as described above.

<149>

<150> ELISA (Enzyme-1 inked immunosorbent assay) analysis

<i5i> VEGF secretion of cancer cells was confirmed by ELISA (Enzyme-1 inked i 睡 unosorbent assay). MCP7 and HeLa cells were cultured in low or normal oxygen state after adding PEP 1 or vehicle for 24 hours. The amount of VEGF in the cell supernatant was determined using the human VEGF immunoassay kit (R & D Systems, USA) according to the manufacturer's instructions. To analyze the concentrations of HSP70 and HSP90 in the blood, blood was collected from a mouse model with tumors. After serum preparation, the concentrations of HSP70 and HSP90 in the blood were confirmed using immunoassay kits of HSP70 (R & D systems, USA) and HSP90 (Cusabio Biotech co., Ltd, DE, USA.).

<152>

Confocal Microscopy Analysis

The sliced tumor sections were fixed for 15 minutes at 4 ° C. with paraformaldehyde at room temperature. After washing twice with PBS, it was placed in PBS containing 0.25% Triton X-100 for 10 minutes, and then washed three times again with PBS. After 30 min blocking of tissue with 1% BSA-PBST, 4 ^° C wet with a combination of mouse anti-Tie2 (557039, BD Pharmigen) and rat anti-CDllb antibodies (ab8878, abeam) Incubated in chamber. After washing, the tissues were incubated with a mixture of AlexaFlour 488 goat anti-mouse IgG and AelxaF lour 633 goat anti-rat IgG. In order to visualize the cell nuclei, the cells were treated with DAPI (Sigma Aldrich) for 1 minute and analyzed by confocal microscopy.

<155>

Statistical Analysis Method

Statistical comparisons between the controls and the experimental groups were analyzed using the student t-test, and the statistical significance level was determined when the value of the P-value was P <0.05 (*) or 0.01 (**). Deemed to be

<158>

Example 3 Screening of Binding Proteins of PEP 1 via Pull Down Assay The pool pull experiment to identify proteins interacting with PEP 1 comprises PEl—GFP. Was carried out using a His-tag. PEP 1-GFP was incubated with total cell lysates of HEK293T cells (Korea Cell Line Bank), followed by Ni-NTA affinity chromatography.

Treated. GFP protein and PEP 1 peptide fused with GFP were used as a control. Eluted protein was isolated by 12% SDS-PAGE and observed by silver staining (catalog # PR0T-SIL1, Sigma Aldrich, MO, USA). Protein bands showing specific interactions were excised from the gels and analyzed by MALDI-TOF (Proteomix, Seo, Korea) for their identification.

<i6i> Specifically, the GFP protein is bound to the N-terminus of PEP 1 is replicated,

Purified from £ \ // PEP 1-GFP, GFP-PEP 1 or GFP protein is incubated with whole cell lysate of HEK293T cells. Each protein complex is purified and subjected to SDS-PAGE and silver staining experiments. Several proteins were specifically found in the PEP 1-GFP protein complex. However, it was not found in the GFP protein or GFP-PEP 1 complex.

< ^| Next, experiments with mass spectrometry revealed that these materials were HSP90, HSP70, Enolase 1 and Creatinie Kinase B (FIG. 1A). PEP 1 interaction of these materials was also confirmed through immunoblotting experiments with antibodies (FIG. 1B).

PEP 1-GST was able to successfully pull down HSP70 from cell lysates of MCF7 cells and HepG2 cells (FIG. 1C). Subsequently, fragments of MCF7 cell membranes were purified and incubated with GFP or GFP-PEP 1. As can be seen in Figure 1D, HSP70 was associated with GFP-PEP 1 and precipitated with GFP-PEP 1. This results in the HSP70

Localization and confirm that PEP 1 and HSP70 interact.

<164>

Statistical Analysis Method

Statistical comparisons between the controls and the experimental groups were analyzed using the student t-test, and the statistical significance level was significant when the value of P—value was P <0.05 (*) or 0.01 (**). Considered.

<167> Example 4 Confirmation of Inhibition of HSP70 and HSP90 by PEP 1 Treatment

<i69> It was confirmed whether PEP 1 can affect the protein levels of HSP70 and HSP90. As can be seen in Figures 2 and 3, treatment with PEP 1 for 2 hours significantly reduced both HSP70 and HSP90 in Jurkat T-cell lymphoma cells and MCF7 breast cancer cells. In experiments with Jurkat cells, 5 μΜ of PEP 1 reduced HSP70 and HSP90 by more than 50%.

In MCF7 cells, HSP90 decreased by up to 20% in the PEP 1-treated group at 5 μΜ compared to the control. In the group treated with 20 μM of PEP 1, HSP70 decreased by about 50% compared to the control group. However, treatment with scrambled peptides similar to PEP 1 but with different sequences did not significantly affect the levels of HSP70 and HSP90 (see FIGS. 2 and 3).

<i7i> In addition, experiments to determine the effect of PEP 1 over time in hypoxic conditions were carried out on HSP70 and

It was performed on HSP90. The results are shown in FIGS. 4 and 5. As can be seen in FIGS. 4 and 5, MCF7 showed that in the mock treated control, the expression of HSP70 and HSP90 was not affected by time, but significantly decreased in the cells treated with PEP 1. It was confirmed in both and HeLa cells. This more clearly confirms that treatment of PEP 1 causes degradation of HSPs, which in turn can modulate their client proteins (see FIGS. 4 and 5). These results suggest that PEP 1 may affect the reaction of various cells associated with hypoxic conditions by reducing the protein levels of HSPs.

Next, we compared the activity of PEP 1 to inhibit HSPs and that of 17-AAG and KNK437, which are well known as inhibitors of HSP90 and HSP70, to HSPs. 17-AAG directly inhibits the action of HSP90 by inhibiting the activity of HSP90's ATPase [Uehara Y, Current cancer drug targets, 3: 325-30, 2003]; KNK437 is derived from stress Suppress the synthesis of HSPs. Experimental results showed that only PEP 1 reduced the levels of both HSP90 and HSP70 in Jurkat and MCF7 cells (see FIG. 6).

<i73> In Jurkat cells, only PEP 1 reduced the protein levels of HSP70 and HSP90.

17-AAG and NK437 reduced the amount of HSP90 but not HSP70. In MCF7 cells, PEP 1 and KNK437 reduced the amount of both HSP90 and HSP70, while 17-MG had little effect on the levels of HSP90 and HSP70.

<i74> Reduction of HSP90 and HSP70 by PEP 1 was confirmed by flow cytometry

Analysis can confirm more clearly. Surface staining and cells of HSP90 and HSP70 Intra-staining showed that the treatment of PEP 1 on HSPs on the cell surface was less than on HSPs on the cellular surface, but could reduce intracellular and cytoplasmic HSP90 and HSP70 (see FIG. 7). . Treatment with PEP 1 and MG132, a proteasome inhibitor, eliminated the effects of PEP 1, suggesting that PEP 1 may

Suggests that proteasome-dependent degradation can be induced (see FIG. 7).

<175>

Example 5: Confirmation of tumor cell growth under hypoxia (Hypoxia) and normal oxygen (Normoxia)

<177> Growth of Tumor Cells Under Hypoxia (Hypoxia) and Normal Oxygen (Normoxia)

The effect of PEP 1 was investigated. PEP 1 showed a weak inhibitory effect on MCF7 and HeLa cell growth under normal conditions (Normoxia, normal oxygen condition), but the inhibitory effect of PEP 1 was greatly enhanced in hypoxic conditions (see FIGS. 8 and 9).

<178>

Example 6: Reduction of HSP70 and HSP90 in Tumor by PEP 1 Treatment

<i80> In order to confirm whether PEP 1 inhibits the expression of HSP70 and HSP90 under in vivo experimental conditions, immunohistochemical staining using a-HSP70 or C1-HSP90 antibody was performed. Consistent with the data obtained from the cancer cell lines, the tumor part taken from the PEP 1 treated group showed a weak staining pattern compared to the PBS treated control group (see FIG. 10). The positively stained portion of the PEP 1 treated sample is very small compared to the control (see FIG. 11).

<i8i> Reduced HSP70 and HSP90 Protein Levels in PEP 1 Treated Tumor Samples

It is also confirmed by immunoblotting experiments with tumor lysates. Reductions in HSP70 and HSP90 were observed in all three PEP 1 treated tumor samples (see FIG. 12). In particular, HSP90 was rarely found in samples treated with PEP 1. GRP78, another family member of HSP, was also reduced in PEP 1 treated samples. Collectively, these results indicate that PEP 1 is capable of reducing HSPs in vivo and inhibiting tumor growth.

<182>

Example 7 Effect of PEP 1 on the Level of HSP70 Secreted in Blood

<i84> Both HSP70 and HSP90 can be secreted from tumor cells, and recent studies have shown some role in tumorigenicity and antitumor reaction. To further explain the role of PEP 1 in the secretion of HSP90 and HSP70, HSP70 and The concentration of HSP90 was measured. As a result of the measurement, it was observed that the PEP 1 treatment group had a great effect on the concentration decrease Λ of HSP70. (See Figure 13),

In addition, analysis of HSP70 concentration, tumor weight and tumor size showed that the tumor weight and concentration of HSP70 were statistically significant at P 0.037, and the tumor size and concentration of HSP70 were statistically significant at P = 0,039. (= 1). This indicates that the low number of HSP7O ': value correlates with tumor amount and tumor weight (see FIG. 14).

【Sequence List Free Text】

<ΐ¾6> <210> 0

<211> 239

PRT

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206> 50 55 6'j

<207>

<208> Leu Thr Tyr Giy Vai Gin Ser Arg Tyr Pro Asp His Met Lys

<20> 65 70 75 80

<210>

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ser 601

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Gu ⁱ p¾y Gha 2 G Gsse ^¾ SGV ⁱ n H ⁱ AH ⁱ Ler A ^pii Tr ^l n <243><211> 720

<244> <212> DNA

<245> <213> Artificial Sequence

<246>

<247> <220>

<248> <223> Green Fluorescent Protein

<249>

<250>

<25i> <400> 4

<252> atggtgagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac

60

<253>

<254> ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg gcgagggcga tgccacctac

120

<255>

<256> ggcaagctga ccctgaagtt catctgcacc accggcaagc tgcccgtgcc ctggcccacc

180

<257>

<258> ctcgtgacca ccctgaccta cggcgtgcag tgcttcagcc gctaccccga ccacatgaag

240

<259>

<260> cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatcttc

300

<261>

<262> ttcaaggacg acggcaacta caagacccgc gccgaggtga agttcgaggg cgacaccctg

360

<263>

<264> gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac

420

<265>

<266> aagctggagt acaactacaa cagccacaac gtctatatca tggccgacaa gcagaagaac

480 uz>< _■

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Claims

[Range of request]

[1] claims ^"

A peptide comprising the amino acid sequence of SEQ ID NO: 1, a peptide having a sequence homology of 8OT or more with the amino acid sequence, or a fragment thereof. A composition for inhibiting HSP expression.

[Claim 2]

The composition for inhibiting HSP expression according to claim U, wherein the HSP is HSP 70 or HSP 90.

[Claim 3]

The composition for inhibiting HSP expression according to claim U, wherein the HSP is HSP 70.

[Claim 4]

The composition of claim 1, wherein the fragment is a fragment consisting of three or more amino acids.

[Claim 5]

The composition for inhibiting HSP expression according to claim U, wherein the peptide consists of up to 30 amino acids.

[Claim 6]

According to claim 1, wherein the composition is tumor growth and metastasis, endometrial cancer, malignant bone sarcoma, kidney cancer., Leukemia, Hodgkin's disease, melanoma, psoriasis, macular degeneration

degeneration), hemophiliac joints, wound granulation, vascular adhesion, rheumatoid arthritis, chronic inflammation, osteoarthritis, autoimmune disease, Crohn's disease, restenosis, atherosclerosis, enterococcal cat scratch disease, ulcers, cirrhosis A composition for inhibiting HSP expression, which is used for the prevention or treatment of diseases or conditions, such as disease, glomerulonephritis, diabetic nephropathy, malignant neurosis, organ transplant rejection, renal glomerulopathy, diabetes mellitus, and inflammation.

[Claim 71

The method according to claim 6, wherein the tumor is a tumor, liver, kidney, pancreas, intestine, uterus, ovary, bladder, breast, prostate, thyroid, lymph, spine, brain, bone, esophagus, the composition for inhibiting HSP expression .

[Claim 8]

The composition for inhibiting HSP expression according to claim 1, wherein the composition is used for inhibiting tumor growth.

[Claim 91]

The composition of claim 1, wherein the composition is a pharmaceutical composition. [Claim 10]

The composition of claim 1, wherein the composition is a food composition. [Claim 11]

A method for preventing and treating HSP expression related diseases, comprising administering to the subject a composition for inhibiting HSP expression according to any one of claims 1 to 10.