WO2018008942A1 - Peptide for targeting breast cancer and use thereof - Google Patents

Peptide for targeting breast cancer and use thereof Download PDF

Info

Publication number
WO2018008942A1
WO2018008942A1 PCT/KR2017/007081 KR2017007081W WO2018008942A1 WO 2018008942 A1 WO2018008942 A1 WO 2018008942A1 KR 2017007081 W KR2017007081 W KR 2017007081W WO 2018008942 A1 WO2018008942 A1 WO 2018008942A1
Authority
WO
WIPO (PCT)
Prior art keywords
breast cancer
peptide
present
cells
drug
Prior art date
Application number
PCT/KR2017/007081
Other languages
French (fr)
Korean (ko)
Inventor
이병헌
이윤기
Original Assignee
경북대학교 산학협력단
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 경북대학교 산학협력단 filed Critical 경북대학교 산학협력단
Publication of WO2018008942A1 publication Critical patent/WO2018008942A1/en

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/42—Proteins; Polypeptides; Degradation products thereof; Derivatives thereof, e.g. albumin, gelatin or zein
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/06—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations
    • A61K49/08—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by the carrier
    • A61K49/10—Organic compounds
    • A61K49/14—Peptides, e.g. proteins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57415—Specifically defined cancers of breast
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere

Definitions

  • the present invention relates to a breast cancer target peptide and its use, and more particularly, to a breast cancer target peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 4 and its use.
  • Breast cancer is the most common cancer in women and the second most common cancer.
  • the prevalence of breast cancer in 2001 was 90-100 per 100 000 in the United States and 50-70 per 100,000 in Europe. The incidence of this disease is increasing worldwide.
  • Risk factors for breast cancer include race, age and mutations in the cancer suppressor genes BRCA-1 and BRCA-2 and p53. Alcohol consumption, high fat diet, lack of exercise, exogenous postmenopausal hormones and ionizing radiation also increase the risk of breast cancer.
  • Estrogen receptor and progesterone receptor negative breast cancers (" ER- " and R- ", respectively), large tumor size, high grade cytology results, and poor prognosis if under 35 years old (Goldhi rsch et al. (2001) J. Cl in.
  • the prognosis of all cancers cannot be judged, and their utility is also showing limitations.
  • hormone receptor positive breast cancer tamoxifen, a hormonal therapy
  • Herceptin an antibody therapy
  • triple negative breast cancer there is no target treatment yet, and conventional chemotherapy is mainly performed.
  • recurrence is common and the prognosis is poor because of poor response to drugs.
  • drug delivery systems or targeted therapies that selectively deliver drugs to specific tissues have received a lot of attention. Because the same amount of therapies can increase the effectiveness of the drug and at the same time significantly reduce the adverse effects on normal tissues.
  • an object of the present invention is to provide a breast cancer target peptide comprising amino acid sequences selected from the group consisting of SEQ ID NOs: 1 to 4.
  • Another object of the present invention is to provide a polynucleotide comprising a nucleotide sequence encoding the template.
  • Another object of the present invention is to provide a vector comprising the polynucleotide. Another object of the present invention is to provide a transformant transformed with the vector. It is another object of the present invention to provide a pharmaceutical composition for preventing or treating breast cancer, which comprises the tempide and a drug bound thereto as an active ingredient. Another object of the present invention is to provide a composition for drug delivery specific to breast cancer comprising the peptide. Another object of the present invention is to provide a breast cancer diagnostic composition comprising the peptide as an active ingredient. Another object of the present invention is to provide a kit for diagnosing breast cancer comprising the peptide. Another object of the invention to provide a marker for diagnosing breast cancer comprising the peptide.
  • Another object of the present invention is to provide a composition for imaging breast cancer comprising the peptide as an active ingredient. Another object of the present invention is to provide a use of the peptide for the preparation of an agent for the diagnosis, treatment or imaging of breast cancer. Another object of the present invention is to provide a method for diagnosing breast cancer, wherein the peptide and an effective amount are administered to a subject in need thereof. Another object of the present invention is to provide a method for treating breast cancer, characterized in that the effective amount of the peptide and the drug bound thereto is administered to a subject in need thereof.
  • the present invention provides a breast cancer target peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 4.
  • the present invention provides a polynucleotide comprising a nucleotide sequence encoding the template.
  • the present invention provides a vector comprising the polynucleotide.
  • the present invention provides a transformant transformed with the vector.
  • the present invention provides a pharmaceutical composition for the prevention or treatment of breast cancer comprising the peptide and a drug binding thereto as an active ingredient.
  • the present invention provides a composition for drug delivery specific to breast cancer comprising the temptide.
  • the present invention provides a composition for diagnosing breast cancer comprising the peptide as an active ingredient.
  • the present invention provides a kit for diagnosing breast cancer comprising the peptide.
  • the present invention provides a marker for diagnosing breast cancer comprising the tempide.
  • the present invention provides a composition for imaging breast cancer comprising the peptide as an active ingredient.
  • a use of the peptide for the preparation of an agent for the diagnosis, treatment or imaging of breast cancer comprising administering to a subject in need thereof an effective amount of the peptide.
  • a method for treating breast cancer comprising administering to the subject in need thereof an effective amount of the peptide and the drug bound thereto.
  • the present invention will be described in detail below.
  • the present invention provides a breast cancer target peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-4.
  • breast cancer has been considered a disease, but recently it has been found that there are various types of subtypes in breast cancer.
  • the breast cancer may be largely classified into three types according to proteins closely related to the onset of cancer. That is, HER-2 (human epidemal growth factor receptor 2) overexpressed breast cancer, estrogen receptor-positive luminal-type breast cancer, and trinegative breast mammin that does not express all of the estrogen receptor, progesterone receptor and HER-2. .
  • the peptide having the amino acid sequence of SEQ ID NO: 1 to 4 is treated to MCF7, which is a luminal breast cancer cell, Skbr3, which is a HER-2 breast cancer cell, or MDA-MB-231 cell, which is a triple negative breast cancer cell, respectively.
  • MCF7 luminal breast cancer cell
  • Skbr3 luminal breast cancer cell
  • MDA-MB-231 cell MDA-MB-231 cell
  • Triple-negative breast cancer is about 153 ⁇ 4 of the total breast cancer, but the frequency is low, but unlike other breasts, the target receptor is not expressed, and there is no suitable therapeutic agent, and after the chemotherapy, the recurrence is poor and the prognosis is poor.
  • the peptides of the present invention which exhibit higher specificity for triple negative breast cancer cells than HER-2 breast and luminal breast cancers, can be usefully developed for the development of effective therapeutics for triple negative breast cancers that currently lack effective therapeutic agents and are well relapsed. have. Therefore, in the present invention, the breast cancer may be luminal type, HER-2 type or triple negative breast cancer, and most preferably triple negative breast cancer.
  • Peptides of the invention can be readily prepared by chemical synthesis known in the art (Creighton, Proteins; Structures and Molecular Pr inciples, WH Freeman and Co., NY, 1983). Representative methods are limited to these.
  • the peptide of the present invention can be prepared by genetic engineering methods. First, a DNA sequence encoding the peptide is constructed according to a conventional method. DNA sequences can be constructed by PCR amplification using appropriate primers.
  • DNA sequences may be synthesized by standard methods known in the art, for example using an automated DNA synthesizer (available from Biosearch or Applied Biosys terns).
  • the constructed DNA sequence is 2.
  • Inserting into a vector comprising one or more expression control sequences (e.g., promoters, enhancers, etc.) that are operably linked to the DNA sequence and regulate expression of the DNA sequence The host cell is transformed with the recombinant expression vector formed therefrom. The resulting transformants are incubated under appropriate media and conditions to allow the DNA sequence to be expressed to recover substantially pure peptides encoded by the DNA sequences from the culture. The recovery can be carried out using methods known in the art (eg, chromatography).
  • substantially pure peptide means any other protein from which the peptide according to the invention is derived from a host. It does not mean to include.
  • Genetic engineering methods for peptide synthesis of the present invention reference may be made to Maniatis et al. Molecular Cloning; A laboratory Manual, Cold Spring Harbor laboratory, 1982; Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, NY, Second (1998) and Third (2000) Edition; Gene Expression Technology, Method in Enzymology, Genetics and Molecular Biology, Method in Enzymology, Guthrie & Fink (eds.), Academic Press, San Diego, Calif, 1991; and Hitzeman et al. , J. Biol. Chem.
  • the peptide having the amino acid sequence of SEQ ID NO: 1 to 4 is a concept including a functional variant thereof.
  • functional variant is meant any similar sequence in which substitution of some amino acids at the amino acid position has occurred that does not affect the properties of the peptides of the invention that bind to breast cancer cells.
  • the present invention provides a transfected with the expression vector, and the expression vector including a polynucleotide, the polynucleotide encoding an amino acid selected from the group standing column eojin Aru in SEQ ID NO: 1 to 4 switch transformants.
  • the polynucleotide may be obtained as described above, and preferably have a nucleotide sequence of SEQ ID NO: 5 to SEQ ID NO: 8, respectively.
  • Expression vector refers to a plasmid, viral vector or other medium known in the art capable of expressing a nucleic acid inserted in a host cell, and encodes the peptide of the present invention in a conventional expression vector known in the art.
  • the polynucleotide may be operably linked.
  • the expression vector is generally operatively associated with a replication origin that can proliferate in a host cell, one or more expression control sequences that regulate expression (eg, promoters, enhancers, etc.), selective markers, and expression control sequences.
  • the transformant may be transformed by the expression vector.
  • the transformant is a method known in the art, including, but not limited to, expression vectors comprising polynucleotides encoding peptides of the invention, such as transient transfection, microinjection, transformation Transduction ion, cell fusion, calcium phosphate precipitation, liposome-mediated transfection, DEAE dextran- Nucleic acid is introduced into mediated transfection (DEAE Dextran-mediated transfect ion), polybrene-mediated transfect ion, electroporation ion, gene gun and cells It can be obtained by introducing into host cells by other known methods for influx (Wu et al., J.
  • the present invention also provides a pharmaceutical composition for the prevention or treatment of breast cancer, comprising the template of the present invention and a drug binding thereto as an active ingredient.
  • the pharmaceutical composition according to the present invention may include a pharmaceutically effective amount of the peptide of the present invention alone or temide and a breast cancer therapeutic agent in combination therewith.
  • pharmaceutically effective amount refers to an amount that exhibits more reaction than the negative control, and preferably refers to a sufficient amount of Z-breast-cancer-or-Z.
  • Agents that can be linked to the peptides of the invention can be used without limitation as long as they are used in the treatment of conventional breast cancer.
  • examples include, but are not limited to, Raloxi fene hydrochloride, Tamoxi fen ci trate, which are licensed as drugs to prevent breast cancer, or drugs to treat breast cancer.
  • the binding of the peptide of the present invention to a therapeutic agent for breast cancer may be carried out through a method known in the art, for example, covalent bonding, crosslinking, etc. It may be combined indirectly.
  • the binding may be performed in vitro, but the peptide and the breast cancer therapeutic agent of the present invention may be administered separately to be bound in vivo.
  • the peptide of the present invention or the breast cancer therapeutic substance may be chemically modified (modi fi cat ion) in a range where its activity is not lost if necessary.
  • the peptide of the present invention is a chromophore, It may be provided in a state labeled with a radioisotope.
  • the amount of the peptide of the present invention and the breast cancer therapeutic material bound thereto may be included in the composition of the present invention, depending on the type and amount of the drug to be bound, and preferably, the amount is sufficient to be delivered to the breast cancer cells, thereby showing an effective effect. have.
  • the effective dosage for the patient is determined. In view of this, those skilled in the art will be able to determine an appropriate effective amount of a peptide of the present invention according to the use for treating breast cancer by combining the peptide with a medicament.
  • the pharmaceutical composition comprising the tempide according to the present invention has the effect according to the present invention.B. 1 is not limited to the type of administration route and administration method.
  • the lung peptide may be administered orally or parenterally.
  • the peptide of the present invention composed of L-type amino acids.
  • the parenteral administration includes subcutaneous injection, intramuscular injection, intravenous injection, and the like, preferably intravenous injection.
  • the pharmaceutical composition of the present invention may be prepared in powder, granule, tablet, pill, dragee, capsule, liquid, gel according to a method known in the art together with a suitable oral carrier.
  • suitable carriers include lactose, dextrose, sucrose, sorbobiol, manny, xylly, erythroli, malty, etc.
  • Layered agents such as starch, cellulose, methyl cellulose, sodium carboxymethyl salose and hydroxypropylmethyl-cellulose, including cellulose, gelatin, polyvinylpyrrolidone, and the like. have.
  • crosslinked polyvinylpyridone, agar, alginic acid or sodium alginate may be added as a disintegrating agent.
  • the pharmaceutical composition may further include an anticoagulant, a lubricant, a humectant, a fragrance, an emulsifier, and an antiseptic.
  • the pharmaceutical composition of the present invention when administered parenterally, the pharmaceutical composition of the present invention may be suitable. Together with parenteral carriers, for example in the form of injections It may be formulated according to a method known in the art. Such injections must be sterilized and protected from contamination of microorganisms such as bacteria and fungi.
  • suitable carriers for injectables include, but are not limited to, solvents including water, ethane, poly (e.g., glycerol, propylene glycol and liquid polyethylene glycol, etc.), combinations thereof and / or vegetable oils. Or dispersion medium. More preferably, suitable carriers include Hanks' solution, Ringer's solution, and triethane containing amines.
  • PBS Phosphate buf fered salin
  • isotonic solution such as 40% propylene glycol and 5% dextrose.
  • various antibacterial and antifungal agents such as paraben, chlorobutanol, phenol, sorbic acid ' , thimerosal, and the like may be further included.
  • the injection may in most cases further comprise an isotonic agent such as sugar or sodium chloride.
  • Other pharmaceutically acceptable carriers may be referred to those described in the following literature (Remington's Pharmaceutical Sciences, 19th ed., Mack Publ i shing Company, East on, PA, 1995).
  • composition of the present invention may further include a pharmaceutically acceptable carrier that is commonly added to the general pharmaceutical composition.
  • pharmaceutically acceptable means physiologically acceptable and does not cause allergic reactions or similar reactions such as gastrointestinal disorders, dizziness, etc. when administered to humans or animals.
  • complete agents e.g. saline or PBS
  • carbohydrates e.g. glucose, mannose, sucrose or textane
  • antioxidants e.g. bacteriostatic agents
  • chelating agents e.g.
  • compositions and formulations comprising the peptides of the present invention can be prepared in a variety of ways as described above, eg in the case of injections, unit dosage amps.
  • the drug delivery composition comprising the peptide of the present invention may be administered by conventional drug administration methods known in the art. Is formulated using methods known in the art to provide rapid, sustained or delayed release of the active ingredient after administration to a mammal. Can be converted.
  • the present invention also provides a composition for drug delivery specific to breast cancer comprising the peptide of the present invention.
  • the peptide of the present invention can be used as an intelligent drug carrier for selectively delivering a drug such as a breast cancer therapeutic agent to breast cancer tissue.
  • a drug such as a breast cancer therapeutic agent
  • the peptide of the present invention is used in the treatment of breast cancer in connection with a conventional breast cancer treatment agent, since the breast cancer treatment agent is selectively delivered only to breast cancer cells by the peptide of the present invention, the effect of the drug may be increased and at the same time, the side effects of the drug on normal tissues Can be significantly reduced.
  • the drug may include, but is not limited to, paclitaxal, doxorubicin, vincristine, daunorubicin, vinblastine, and actinomycin-
  • Actinomycin-D docetaxel, etoposide, etoniposide, teniposide, bisantrene, Jl ⁇ -3 ⁇ 4113 ⁇ 4 £ 'l d (homoharringtonine), gleevec; STI-571 ), Cisplain, 5-fluouraci 1, adriamycin, methotrexate, busul fan, chlorambucil, cyclophosphamide cyclophosphamide, melphalan, nitrogen mustard, nitrosourea, streptokinase, urokinase,reteplase, angiotensin II inhibitors , Aldosterone receptor inhibitor, erythropoietin, N-methyl-d-aspartate (NMDA) receptor inhibitor, lovastatin, rapamycin, Celebrex, Ticlopin Marimas Bit (Marimastat) and Trojan Cade (Trocade) and the like.
  • Cisplain 5-fluouraci 1, ad
  • the peptide of the present invention and the drug may be covalently linked, and in particular, may be linked by a linker (Linker), but is not limited thereto.
  • the present invention provides a composition for diagnosing breast cancer comprising the peptide of the present invention as an active ingredient.
  • diagnosis refers to confirming the presence or characteristics of a pathological state Means.
  • the diagnosis is to identify the presence or characteristics of breast cancer.
  • Peptides included in the diagnostic composition of the present invention may have an amino acid sequence selected from the group consisting of SEQ ID NO: 1 to 4, may be obtained by chemical or genetic engineering methods as described above.
  • the diagnostic composition may further include a complete layer solution or a semi-aqueous solution that maintains the peptide structure or physiological activity in a stable manner. State or 4 ° C. may be provided.
  • the peptide of the present invention may be provided in a labeled state.
  • the detectable label may be provided in a link (eg, covalently bonded or crosslinked).
  • the detectable label may be a colorase (e.g. peroxidase, alkaline phosphatase)
  • the detectable label can be an antibody epitope, substrate, cofactor, inhibitor or affinity ligand.
  • Such labeling may be performed in the course of synthesizing the peptide of the present invention, or may be performed in addition to the already synthesized peptide.
  • a fluorescent substance is used as a detectable label
  • breast cancer can be diagnosed by fluorescence mediated tomography (FMT).
  • FMT fluorescence mediated tomography
  • the peptide of the present invention labeled with a fluorescent material can be circulated into the blood and fluorescence by the peptide can be observed by fluorescence tomography. If fluorescence is observed, it is diagnosed as breast cancer.
  • a radioactive substance as a detectable label
  • breast cancer can be diagnosed by observing the radioactivity caused by the peptide by Positron emission tomography (PET).
  • PET Positron emission tomography
  • the present invention also provides a kit for diagnosing breast cancer comprising the tempide of the present invention.
  • the kit of the present invention may include a peptide labeled with a chromophore or a radioisotope as described above.
  • the kit of the present invention may further include a suitable monolayer solution or medium for binding reaction between the peptide and breast cancer cells and control cells (normal brain cells).
  • a detectable label for labeling the peptide may be further included in the kit.
  • Amount - it may include i secondary antibody, a chromogenic substrate contained in addition to the kit-of the present invention ⁇ the home iljeok-peptide for the eu taah- ⁇ antibody of eu eu labeled with a fluorescent substance.
  • Antibodies to the peptides of the present invention can be prepared according to conventional methods for preparing antibodies known in the art as described above.
  • the peptide of the present invention may be provided in a form coated on the surface of the plate. In this case, breast cancer cells can be diagnosed by observing the binding of the breast cancer cells and the peptide on the surface of the plate after inoculating breast cancer cells directly on the plate and reacting under appropriate conditions.
  • the present invention also provides a composition for imaging breast cancer comprising the peptide of the present invention as an active ingredient.
  • the composition for imaging breast cancer of the present invention may be used to image breast cancer by containing the peptide of the present invention.
  • the composition for imaging breast cancer comprising the tempide of the present invention may be usefully used for imaging, detecting, and quantifying the semi-shaded portion, and using the imaging technique of the conventional shaded portion, the relative size and extent of the semi-shaded portion and the shaded portion. It can also be useful for predicting prognosis through medication.
  • the peptide is not limited thereto, but is not limited thereto, a chromophore, a radioisotope, a chromophore, a luminescent material, a fluorescent substance (f luorescer), a paramagnetic particle (superparamagnetic part i cles) or a superparamagnetic It may be labeled with particles (ul trasuper paramagnet ic part ic les).
  • the present invention after transplanting MDA-MB-231 cells under the skin of a mouse, administering a peptide having the amino acid sequence of SEQ ID NOS: 1 to 4 by intravenous injection, extracting each tumor and fluorescence intensity As a result, the tumor tissue treated with the peptides of SEQ ID NO: 1 and SEQ ID NO: 4 showed higher fluorescence intensity than the control group, and the tumor tissue treated with the peptide of SEQ ID NO: 1 was applied to the tumor tissue treated with the other peptide. It was confirmed that the fluorescence intensity was higher than that.
  • the present invention provides the use of the peptides for the preparation of an agent for the diagnosis, treatment or imaging of breast cancer.
  • the present invention provides a method for diagnosing breast cancer, wherein the effective amount of the peptide is administered to a subject in need thereof.
  • the present invention provides a method for treating breast cancer, comprising administering to a subject in need thereof an effective amount of the peptide and the drug bound thereto.
  • the term 'effective amount' of the present invention when administered to an individual, refers to an amount showing improvement in breast cancer, treatment, prevention, detection or diagnosis, and the term 'object' is preferably an animal, preferably a mammal, especially a human. May be an animal containing, originating from an animal It may be a cell, tissue, organ. The subject may be a patient in need of the effect.
  • treatment' of the present invention refers generically to ameliorating symptoms of a breast cancer or breast cancer related disease or a breast cancer related disease, which may include treating, substantially preventing, or ameliorating the disease. And alleviating, healing or preventing one or most of the symptoms arising from breast or breast cancer related disorders.
  • the peptide of the present invention can specifically bind to breast cancer, it can be usefully used for the preparation of a composition for diagnosis, treatment or imaging of breast cancer.
  • FIG. 1 shows two strategies used in the present invention for phage library screening: negat ive subtract ion (A) and positive select ion (B) screening procedures.
  • Figure 2 shows the phage titer recovered in each round (Rl ⁇ R5) as a result of negative subtract ion screening for MCF7, SKBR3, MDA-MB-231 cells.
  • Figure 3 shows the phage titer recovered in each round (Rl ⁇ R5) as a result of positive select ion screening on MDA-MB-231 cells.
  • Figure 4 shows the peptide coding gene sequences inserted into phage clones selected through negat ive subtract ion screening (A) and posi tive select ion screening (B), and then converting to amino acid sequences and sequencing similar amino acid sequences Indicates some of the results.
  • 5A shows the results of investigating the binding capacity of six phage clones to MDA-MB-231 cells selected from a negat ive subtract ion screening strategy through plaque assay.
  • FIG. 5B shows the ability of 5-14 CVLKKPR) of SKBR3 breast cancer cells or MDA-MB-231 breast cancer cell lines of the six phage clones of FIG. 5A to be evaluated by plaque assay. Results are shown.
  • FIG. 6 shows the results of investigating the specific binding capacity of 7 phage clones selected from the positive selection screening strategy to MDA-MB231, SKBR3, and MCF7 cells by plaque assay.
  • FIG. 7 shows the results of investigating the binding capacity of four peptides (CVRAKGKPC, CVLKKPR, CSLKKPR, CRPMRPR) to MDA-MB231, MCF7, and SKBR3 cells, which were selected through a negative subtraction screening and a positive selection screening strategy. Indicates.
  • Figure 8 shows the results of investigating the binding capacity of four peptides (CVRAKGKPC, CVLKKPR, CSLKKPR, CRPMRPR) to MDA-MB231, MCF7, and SKBR3 cells using a flow cytometer (FACS).
  • Figure 9 shows cancer cells of different types of non-mammary cancer cells and normal lung and soybean cells (MDA-MB-231, MCF7, A549, BEAS-2B and HEK-293 cells) of four peptides (CVRAKGKPC, CVLKKPR, CSLKKPR, CRPMRPR) The binding ability to is shown by the flow cytometer (FACS).
  • a 11 is treated with fluorescent-labeled candidate peptide (CVRAKGKPC, CVLKKPR, CRPMRPR) in breast cancer mouse models created by administering the 'MDA-MB-231 cells was then thin sectioned the excised tumor and liver came up, fluorescence microscopy The result of verifying the binding capacity is shown.
  • Example 1 is merely to illustrate the present invention, the content of the present invention is not limited to the following embodiments.
  • Example 1
  • Phage screening was performed using a T7 phage hydrophobic amino acid library (1 ⁇ 10 7 pfu / ul).
  • MCF7 estrogen receptor / progesterone receptor positive, luminal type
  • SKBR3 Her2 receptor positive, Her2 type
  • Ngg t ⁇ e subTract ion screening is shown briefly in Figure 1. That is, a negative subtract ion that removes phages that bind to MCF7 and SKBR3 cells rather than triple negative breast cancer cells, and triple negative breast cancer The phage was recovered with a posi- tive select ion that recovers phage that binds to the MDA-MB231 cells.
  • T7 phages were injected into the MDA-MB231 cells prepared on a 35 ⁇ culture plate and combined with the cells for 1 hour at 4 ° C. After removing the supernatant, the cells were cultured to 0.D 1.0 in LB medium. After adding 300 ul of BL21 host Escherichia coli and reacting for 5 minutes, phages bound to MDA-MB-231 cells were eluted. The titer was calculated by performing a plaque assay using lOul of the recovered phage eluate 300ul. The remaining eluate was subjected to phage amplification with host E. coli to secure phage for the next round. This process was repeated up to five rounds.
  • the number of collected phages for each round was calculated and shown in FIG. 3. As each round proceeded, the number of eluted phages increased, and the number of eluted phages increased about 360 times in the fifth round compared to the titer of the first round.
  • For amino acid sequencing 20, 30, and 50 clones, respectively, were collected from the plaque assays used for 3, 4 and 5 round titers, and stored in lOul Tris-buffered saline complete fluid. PCR was performed to determine the nucleotide sequence of the gene encoding the peptides inserted into these phages and amino acid sequencing accordingly.
  • the nucleotide sequence of the peptide coding gene inserted into 70, 100 phage clones collected in Examples 1-1 and 1-2 was requested by Macrogen Corporation.
  • the analyzed base sequence was converted to the corresponding amino acid sequence, and the sequences were aligned using the Clustal X program to analyze similar sequences or motifs.
  • arginine (R) or lysine (K) which is an amino acid having basic properties, is commonly distributed.
  • Peptides having six or more residues on the amino acid sequence of the peptide were selected as experimental candidates for the next step.
  • the binding capacity of the selected phage to triple negative breast cancer cells was evaluated by measuring the number of phages bound to the cells.
  • each of six candidate phages was put in 10 9 pfu in 96-wel l di sh in which 50,000 cells were cultured, each was bound at 4 ° C for one hour, and the number of bound phages was eluted.
  • 3-6 and 5-14 phage clones among 6 candidate groups selected from negative subtract ion screening showed high binding to MDA-MB231 cells.
  • the 5-14 phage clone with the highest binding capacity to MDA-MB-231 cel l showed significantly lower binding to Skbr3 cells compared to MDA-MB-231 cells.
  • MDA-MB—231, Skbr3, and MCF7 cells of seven candidate groups selected from positive select ion screening were examined. As a result, all seven candidates had MDA compared to Skbr3 and MCF7 cells. It showed higher binding to -MB-231 cells.
  • 4R-28CCRPMRPR showed the highest binding of MDA-MB-231 cells to 7 candidates
  • 4R-12 CSLKKPR showed the highest binding capacity to MDA-MB-231 cells compared to MCF7 and Skbr3 cells. 11 times compared to MCF7 and 38 times compared to Skbr3. Based on this, CRPMRPR and CSLKKPR peptides were selected as further experiment candidates.
  • MDA-MB-231 cells which are triple negative breast cancer cells, and luminal A type, MCF7 cells, and HER2 type Skbr3 cells, which are non-triple negative breast cancer cells, are placed in a 4-wel l culture vessel.
  • CSLKKPR peptides bind relatively well to MDA-MB-231 cells as well as MCF7 and Skbr3 cells, resulting in less specificity for triple negative breast cancer cells.
  • the control peptide (NSSSVDK) was relatively weak in binding to the cells.
  • the FITC fluorescently labeled peptide was reacted at 4 ° C. for 1 hour, and then analyzed for binding ability of the peptide using a flow cytometer.
  • CVRAKGKPC, CVLKKPR, CSLKKPR, CRPMRPR peptides are compared to MCF7 cells, non-tri-negative breast cancer cells, lung cancer cells A549 cells and normal cells BEAS-2B cells and HKE-293 cells, Peptide binding and mean fluorescence intensity (MFI) were relatively higher in MDA—MB-231 cells, triple negative breast cancer cells. Through this, it was confirmed that these candidate peptides have a specific binding capacity to triple negative breast cancer cell line.
  • mice All animal experiments have been approved in accordance with the Institutional Guidelines and approved by the Guidelines of the Institutional Animal Care and Use Committee (IACUC) of Kyungpook National University (Permission No. KNU). 2014-0001-121). Eight-week-old Balb / c mice were purchased from Orient laboratories (CSeongnam, Korea) and were fed in a feed- and water-free diet and in SPF (specific-pathogen-free conditions).
  • IACUC Institutional Animal Care and Use Committee
  • MDA-MB-231 cells were transplanted under the skin of mice to grow the tumors. Thereafter, FITC fluorescently labeled control peptide NSSSVDK and the three peptides were injected intravenously into tumor mice, respectively. Two hours later, each tumor was extracted, and the fluorescence intensity of R0I (Region of Interest) was measured using a bio-optical imaging system (IVIS). The higher the R0I value, the stronger the fluorescence in the tumor tissue. Many photolabeled peptides are targeted and ' accumulated. As a result, as shown in Fig.
  • the tumor cells treated with CVRKGKPC and CRPMRPR peptides ⁇ showed higher fluorescence values than the tumor cells treated with NSSSVDK peptides, which were negative controls.
  • tumor cells treated with CVRAKGKPC peptide showed the highest fluorescence values.
  • tumor cells treated with the CVLKKPR peptide showed lower fluorescence values than the control group.
  • the CVRAKGKPC peptide has a relatively higher in vivo targeting ability than other peptides in the breast cancer mouse model, and CVLKKPR peptide was confirmed that the in vivo targeting ability is not superior to the control.
  • cryosections of the extracted tumor tissues were prepared and observed by fluorescence microscopy.
  • the fluorescence of liver tissue was observed to verify the tissue specificity of the peptide.
  • the tumor tissue of the mouse injected intravenously with the NSSSVDK peptide, a negative control showed a very low fluorescence, compared with other peptides in the tumor tissue of the rat injected with CVRAKGKPC peptide intravenously. The highest was observed. This was confirmed to be similar to the results of the experiment using the biofluorescence imaging system (IVIS).
  • the CVRAKGKPC peptide showed low fluorescence in liver tissues, unlike the fluorescence signal observed in tumors.
  • the peptide of the present invention can specifically bind to breast cancer, it can be usefully used for the production of a composition for diagnosing, treating or imaging breast cancer, and thus has excellent industrial applicability.

Abstract

The present invention relates to a peptide for targeting breast cancer and a use thereof and, more specifically, to: a breast cancer-targeting peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 4; and a use thereof. The peptide of the present invention can specifically bind to breast cancer, thereby being useful in the preparation of a composition for diagnosing, treating, or imaging breast cancer.

Description

【명세서】  【Specification】
【발명의 명칭】  [Name of invention]
유방암 표적용 펩타이드 및 이의 용도  Peptides for Breast Cancer Targeting and Uses thereof
【기술분야】 Technical Field
본 출원은 2016년 7월 4일에 출원된 대한민국 특허출원 계 10-2016-0084357호 를 우선권으로 주장하고， 상기 명세서 전체는 본 출원의 참고문헌이다. 본 발명은 유방암 표적용 펩타이드 및 이의 용도에 관한 것으로 보다 상세 하게는 서열번호 1 내지 4로 이루어진 군에서 선택된 아미노산 서열을 포함하는 유 방암 표적 펩타이드 및 이의 용도에 관한 것이다.  This application claims the priority of Korean Patent Application No. 10-2016-0084357, filed on July 4, 2016, the entirety of which is a reference of the present application. The present invention relates to a breast cancer target peptide and its use, and more particularly, to a breast cancer target peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 4 and its use.
【배경기술】 Background Art
유방암은 여성에 있어 가장 흔한 암이며, 두 번째로 사망률이 높은 암이다. 2001년 유방암의 유병를은 미국에서 100 , 000 명당 90-100이었으며, 유럽에서는 100,000 명당 50-70이었다. 이 질환의 발병은 세계적으로 점점 증가하는 추세에 있 다. 유방암의 위험 인자는 인종， 나이 및 암 억제 유전자 BRCA-1 및 BRCA-2 및 p53 에서의 돌연변이 등을 포함한다. 알코올 섭취， 고지방 식이， 운동 부족， 외인성 폐 경 후 호르몬 및 이온화 방사선 또한 유방암의 발명 위험을 증가시킨다. 에스트로 겐 수용체 및 프로게스테론 수용체 음성 유방암 (각각 "ER-" 및 R-" ) , 큰 종양 크 기, 높은 등급의 세포진단결과， 및 35세 이하인 경우 그 예후가 나쁘다 (Goldhi rsch et al . (2001) . J . Cl in. Oncol . 19： 3817-27) . 2005년 약 212, 000건의 새로운 침 습성 유방암 증례 및 58,000건의 새로운 비침습성 유방암증례가 진단될 것으로 추 산되며， 40 ,000명의 여성이 유방암으로사망할 것으로 예상된다. 현재의 유방암에 대한 치료 방법은 수술 이후， 항암 치료, 항호르몬 치료, 표적 치료 혹은 방사선 치료 등 향후 재발을 줄이기 위한 추가 보조적인 치료가 필 요한 경우가 많다， 유방암은 유방암 주요 수용체의 상태에 따라 암의 특성이 다르 며， 따라서 조직검사를 통해 호르몬 수용체 (ER 및 PR) 발현 유무와 'HER-2' 과발 현 여부 및 전이 상태 확인 및 림프절 전이 여부 (양성 또는 음성)를 고려하여 추후 치료 방법에 대한 근거를 확립하고 있다. 비록 이러한 임상적 정보들이 치료를 결 정짓는 지표로 널리 사용되고 있지만 임상적 지표의 표현형보다 암의 이질성이 더 커 모든 암의 예후를 판단할 수 없을뿐더러， 그 효용성에서도 한계를 드러내고 있 다. 호르몬 수용체 양성인 유방암의 경우 호르몬 치료제인 타목시펜 (Tamoxi fen) 이 효과적인 표적 치료제로 사용되고 있고, Her-2 과발현 유방암의 경우 항체치료 제인 허셉틴 (Hercept in)이 효과적인 표적 치료제로 사용되고 있다. 반면 삼중음성 유방암의 경우 표적 치료제가 아직 없으며 기존의 항암화학요법을 주로 시행하고 있으나 재발이 흔하고 재발시는 약물에 반응을 잘 하지 않아 예후가 나쁜 편이다. 한편， 약물을 특정 조직으로 선택적으로 전달하는 약물전달체계 또는 표적 치료는 많은 관심을 받아온 기술이다. 왜냐하면 동일 양의 치료제를 사용하더라도 약의 효력을 증가시킬 수 있고 동시에 정상조직에 미치는 부작용을 크게 줄일 수 —있—커-때문어—다. 또—한—유전자 -치료에 _적용할 경운 바이—러스를 -특정 -조최쒜ᅳ선택적_으 로 전달시킴으로써 치료 효율을 을리고 심각한 부작용을 줄일 수 있다. 이를 위해 지금까지는 주로 특정 조직에 특이한 항원 및 이를 표적하는 항체를 개발하여 왔 다. 그러나 항체의 경우 면역반응의 우려 및 조직 내 침투의 낮은 효율성 등의 문 제가 존재한다. 반면 펩타이드의 경우는 분자량이 작아 면역반웅의 우려가 적고 조 직 내 침투가 용이한 것이 장점이다. 따라서 표적 펩타이드를 기존의 치료제와 연 결하게 되면 특정 조직에 선택적으로 약물을 전달하는 지능형 약물전달체로 활용될 수 있으므로， 유방암의 표적 펩타이드 개발이 요구되고 있다. Breast cancer is the most common cancer in women and the second most common cancer. The prevalence of breast cancer in 2001 was 90-100 per 100 000 in the United States and 50-70 per 100,000 in Europe. The incidence of this disease is increasing worldwide. Risk factors for breast cancer include race, age and mutations in the cancer suppressor genes BRCA-1 and BRCA-2 and p53. Alcohol consumption, high fat diet, lack of exercise, exogenous postmenopausal hormones and ionizing radiation also increase the risk of breast cancer. Estrogen receptor and progesterone receptor negative breast cancers (" ER- " and R- ", respectively), large tumor size, high grade cytology results, and poor prognosis if under 35 years old (Goldhi rsch et al. (2001) J. Cl in. Oncol 19: 3817-27) In 2005, an estimated 212,000 new invasive breast cancer cases and 58,000 new non-invasive breast cancer cases were diagnosed, with 40,000 women Current treatments for breast cancer often require additional adjuvant treatments to reduce future recurrences, such as chemotherapy, anti-hormone therapy, targeted therapy or radiation therapy after surgery. The characteristics of the cancer differ according to the state of the major receptors of the breast cancer. Therefore, the biopsy confirms the presence of hormone receptors (ER and PR), 'HER-2' overexpression, metastasis status and lymph node metastasis. We consider the negative (positive or negative) to establish the rationale for future treatment methods, although these clinical information are widely used as an indicator for determining treatment, the heterogeneity of cancer is greater than the phenotype of clinical indicators. In addition, the prognosis of all cancers cannot be judged, and their utility is also showing limitations. In the case of hormone receptor positive breast cancer, tamoxifen, a hormonal therapy, is used as an effective target therapy, and in the case of Her-2 overexpressing breast cancer, Herceptin, an antibody therapy, is used as an effective target therapy. On the other hand, in the case of triple negative breast cancer, there is no target treatment yet, and conventional chemotherapy is mainly performed. However, recurrence is common and the prognosis is poor because of poor response to drugs. Meanwhile, drug delivery systems or targeted therapies that selectively deliver drugs to specific tissues have received a lot of attention. Because the same amount of therapies can increase the effectiveness of the drug and at the same time significantly reduce the adverse effects on normal tissues. In addition, by delivering a cultivation virus to a specific gene-treatment that can be used for gene therapy, treatment efficiency and significant side effects can be reduced. To this end, it has been mainly developed antigens specific to specific tissues and antibodies targeting them. However, for antibodies, there are concerns about the immune response and low efficiency of penetration into tissues. Peptides, on the other hand, have a low molecular weight and are less susceptible to immune reactions and are easier to penetrate into tissues. Therefore, when the target peptide is connected to an existing therapeutic agent, it can be used as an intelligent drug delivery agent that selectively delivers drugs to specific tissues, and thus, development of a target peptide for breast cancer is required.
【발명의 상세한 설명】 [Detailed Description of the Invention]
【기술적 과제】  [Technical problem]
이에, 본 발명자들은 유방암의 치료 및 진단에 사용될 수 있는 유방암 표적 펩타이드를 개발하기 위하여 연구들 거듭하던 중， 파지 펩타이드 디스플레이 기술 을 이용하여 유방암 조직에 특이적인 펩타이드를 탐색하고， 상기 펩타이드가 유방 암의 진단 마커 및 지능형 약물 전달체로서 유용하게 사용될 수 있음을 발견하고 본 발명을 완성하게 되었다. 따라서 본 발명의 목적은 서열번호 1 내지 4로 이루어진 군에서 선택된 아미 노산서열을 포함하는 유방암 표적 펩타이드를 제공하는 것이다. 본 발명의 다른 목적은 상기 템타이드를 암호화하는 염기서열을포함하는 폴 리뉴클레오티드를 제공하는 것이다. 본 발명의 다른 목적은 상기 폴리뉴클레오티드를 포함하는 백터를 제공하는 것이다. 본 발명의 다른 목적은 상기 백터로 형질전환된 형질전환체를 제공하는 것이 다. 본 발명의 다른 목적은 상기 템타이드 및 이와 결합하는 약물을 유효성분으 로포함하는유방암 예방또는 치료용 약학적 조성물을 제공하는 것이다. 본 발명의 다른 목적은 상기 펩타이드를 포함하는 유방암에 특이적인 약물 전달용조성물을 제공하는 것이다. 본 발명의 다른 목적은 상기 펩타이드를 유효성분으로 포함하는 유방암 진단 용조성물을 제공하는 것이다. 본 발명의 다른 목적은 상기 펩타이드를 포함하는 유방암 진단용 키트를 제 공하는 것이다. 본 발명의 다른 목적은 상기 펩타이드를 포함하는 유방암 진단용 마커를 제 공하는 것이다. 본 발명의 다른 목적은 상기 펩타이드를 유효성분으로 포함하는 유방암 영상 화용조성물을 제공하는 것이다. 본 발명의 다른 목적은 유방암의 진단， 치료 또는 영상화용 제제를 제조하기 위한상기 펩타이드의 용도를 제공하는 것이다. 본 발명의 다른 목적은 상기 펩타이드와 유효량을 이를 필요로 하는 개체에 투여하는 것을 특징으로 하는유방암의 진단 방법을 제공하는 것이다. 본 발명의 다른 목적은 상기 펩타이드 및 이와 결합하는 약물의 유효량을 이 를 필요로 하는 개체에 투여하는 것을 특징으로 하는 유방암의 치료 방법을 제공하 는 것이다. Accordingly, the inventors of the present invention, while developing studies to develop a breast cancer target peptide that can be used for the treatment and diagnosis of breast cancer, using a phage peptide display technology to search for a peptide specific to breast cancer tissue, the peptide is a The present invention has been accomplished by discovering that it can be usefully used as a diagnostic marker and intelligent drug carrier. Accordingly, an object of the present invention is to provide a breast cancer target peptide comprising amino acid sequences selected from the group consisting of SEQ ID NOs: 1 to 4. Another object of the present invention is to provide a polynucleotide comprising a nucleotide sequence encoding the template. Another object of the present invention is to provide a vector comprising the polynucleotide. Another object of the present invention is to provide a transformant transformed with the vector. It is another object of the present invention to provide a pharmaceutical composition for preventing or treating breast cancer, which comprises the tempide and a drug bound thereto as an active ingredient. Another object of the present invention is to provide a composition for drug delivery specific to breast cancer comprising the peptide. Another object of the present invention is to provide a breast cancer diagnostic composition comprising the peptide as an active ingredient. Another object of the present invention is to provide a kit for diagnosing breast cancer comprising the peptide. Another object of the invention to provide a marker for diagnosing breast cancer comprising the peptide. Another object of the present invention is to provide a composition for imaging breast cancer comprising the peptide as an active ingredient. Another object of the present invention is to provide a use of the peptide for the preparation of an agent for the diagnosis, treatment or imaging of breast cancer. Another object of the present invention is to provide a method for diagnosing breast cancer, wherein the peptide and an effective amount are administered to a subject in need thereof. Another object of the present invention is to provide a method for treating breast cancer, characterized in that the effective amount of the peptide and the drug bound thereto is administered to a subject in need thereof.
【가술적 해결방법】 Technical solution
상기한 본 발명의 목적을 달성하기 위하여 본 발명은 서열번호 1 내지 4로 이루어진 군에서 선택된 아미노산 서열을 포함하는 유방암 표적 펩타이드를 제공한 다. 본 발명의 다른 목적을 달성하기 위하여 본 발명은 상기 템타이드를 암호화 하는 염기서열을 포함하는 폴리뉴클레오티드를 제공한다. 본 발명의 다른 목적을 달성하기 위하여 본 발명은 상기 폴리뉴클레오티드를 포함하는 백터를 제공한다. 본 발명의 다른 목적을 달성하기 위하여 본 발명은 상기 백터로 형질전환된 형질전환체를 제공한다. 본 발명의 다른 목적을 달성하기 위하여 본 발명은 상기 펩타이드 및 이와 결합하는 약물을 유효성분으로 포함하는 유방암 예방 또는 치료용 약학적 조성물을 제공한다. 본 발명의 다른 목적을 달성하기 위하여 본 발명은 상기 템타이드를 포함하 는 유방암에 특이적인 약물 전달용 조성물을 제공한다. 본 발명의 다른 목적을 달성하기 위하여 본 발명은 상기 펩타이드를 유효성 분으로 포함하는 유방암 진단용 조성물을 제공한다. 본 발명의 다른 목적을 달성하기 위하여 본 발명은 상기 펩타이드를 포함하 는 유방암 진단용 키트를 제공한다. 본 발명의 다른 목적을 달성하기 위하여 본 발명은 상기 템타이드를 포함하 는 유방암 진단용 마커를 제공한다. 본 발명의 다른 목적을 달성하기 위하여 본 발명은 상기 펩타이드를 유효성 분으로포함하는 유방암 영상화용 조성물을 제공한다. 본 발명의 다른 목적을 달성하기 위하여 유방암의 진단， 치료 또는 영상화용 제제를 제조하기 위한 상기 펩타이드의 용도를 제공한다. 본 발명의 다른 목적을 달성하기 위하여 상기 펩타이드의 유효량을 이를 필 요로 하는 개체에 투여하는 것을 특징으로 하는 유방암의 진단 방법을 제공한다. 본 발명의 다른 목적을 달성하기 위하여 상기 펩타이드 및 이와 결합하는 약 물의 유효량을 이를 필요로 하는 개체에 투여하는 것을 특징으로 하는 유방암의 치 료 방법을 제공한다. 이하 본'발명에 대해 상세히 설명한다 . 본 발명은 서열번호 1 내지 4로 이루어진 군에서 선택된 아미노산 서열을 포 함하는 유방암 표적 펩타이드를 제공한다. 유방암은 하나의 질환으로 여겨져 왔지만， 최근 유방암에는 다양한 종류의 아형 (subtype)이 존재한다는 것이 밝혀졌다. 본 발명에서 상기 유방암은 암의 발병 과 밀접하게 관련이 되어 있는 단백질에 따라 크게 세 종류의 그룹으로 구분되어질 수 있다. 즉， HER-2(human epidemal growth factor receptor 2) 과발현 유방암， 에 스트로겐 수용체 양성인 루미날형 유방암 및 에스트로겐 수용체， 프로게스테론 수 용체 및 HER-2가 모두 발현되어 있지 않은 삼중음성 유방맘으로 구분될 수 있다. 본 발명의 일실시예에서는 상기 서열번호 1 내지 4의 아미노산 서열올 갖는 펩타이드를 루미날형 유방암 세포인 MCF7, HER-2형 유방암 세포인 Skbr3 또는 삼중 음성 유방암 세포인 MDA-MB-231세포에 각각 처리한 결과， 모든 아형의 유방암 세포 에 결합하는 것을 확인할 수 있었다. 그 중에서도， 상기 펩타이드는 삼중 음성 유 방암 세포에 더 높은 특이성을 나타내는 것으로 확인되었다. 삼중음성 유방암은 전체 유방암의 15¾정도로서 그 빈도는 낮은 편이나 다른 유방과는 달리 표적 수용체가 발현되어 있지 않아 마땅한 타겟 치료제가 없으며， 항암화학요법 이후 잘 재발하여 예후가 나쁜 편이다. 따라서, HER-2형 유방암 및 루미날형 유방암보다 삼중 음성 유방암 세포에 더 높은 특이성을 나타내는 본 발명 의 펩타이드들은 현재 효과적인 치료제가 부족하고 잘 재발하는 삼중음성 유방암에 대한 효율적인 치료제 개발에 유용하게 활용될 수 있다. 따라서， 본 발명에서 상기 유방암은 루미날형 , HER-2형 또는 삼중음성 유방 암일 수 있고, 가장 바람직하게는 삼중음성 유방암일 수 있다. 본 발명의 펩타이드는 당업계에 공지된 화학적 합성 (Creighton, Proteins ; Structures and Molecular Pr inciples , W. H. Freeman and Co . , NY, 1983)에 의해 쉽게 제조될 수 있다. 대표적인 방법으로서 이들로 한정되는 것은. 아니지만 액체 또는 고체상 합성， 단편 웅축， F-M0C 또는 T-B0C 화학법이 포함된다 (Chemical Approaches to the Synthesi s of Pept ides and Proteins , Wi l l iams et al . , Eds . , CRC Press , Boca Raton Florida, 1997； A Pract ical Approach, Athert on & Sheppard, Eds . , IRL Press , Oxford, England, 1989) . 또한 본 발명의 펩타이드는 유전공학적 방법에 의해 제조될 수 있다. 우선， 통상적인 방법에 따라 상기 펩타이 드를 코딩하는 DNA 서열을 작제한다. DNA 서열은 적절한 프라이머를 사용하여 PCR 증폭함으로써 작제할 수 있다. 다른 방법으로 당업계에 공지된 표준 방법에 의해， 예컨대， 자동 DNA 합성기 (Biosearch 또는 Appl ied Biosys terns 사에서 판매하는 것) 를 사용하여 DNA 서열을 합성할 수도 있다. 작제된 DNA 서열은 이 . DNA 서열에 작동 가능하게 연결되어 (operat ively l inked) 그 DNA 서열의 발현을 조절하는 하나 또는 그 이상의 발현 조절 서열 (expression control sequence) (예: 프로모터, 인핸서 등 )을 포함하는 백터에 삽입시키고， 이로부터 형성된 재조합 발현 백터로 숙주세포를 형질전환시킨다. 생성된 형질전환체를 상기 DNA 서열이 발현되도록 하기에 적절한 배지 및 조건 하에서 배양하여， 배양물로부터 상기 DNA 서열에 의해 코딩된 실질적 으로 순수한 펩타이드를 회수한다. 상기 회수는 당업계에 공지된 방법 (예컨대， 크 로마토그래피)올 이용하여 수행할 수 있다. 상기에서 '실질적으로 순수한 펩티드' 라 함은 본 발명에 따른 펩티드가 숙주로부터 유래된 어떠한 다른 단백질도 실질적 으로 포함하지 않는 것을 의미한다. 본 발명의 펩티드 합성을 위한 유전공학적 방 법은 다음의 문헌을 참고할 수 있다: Maniatis et al . , Molecular Cloning; A laboratory Manual , Cold Spring Harbor laboratory, 1982； Sambrook et al., Molecular Cloning: A Laboratory Manual , Cold Spring Harbor Press, N.Y. , Second (1998) and Third(2000) Edition; Gene Expression Technology , Method in Enzymology, Genetics and Molecular Biology, Method in Enzymology, Guthrie & Fink (eds. ) , Academic Press, San Diego, Calif, 1991； 및 Hitzeman et al . , J. Biol. Chem. , 255:12073—12080， 1990. 본 발명에서 상기 서열번흔 1 내지 4의 아미노산 서열을 갖는 펩타이드는 이 의 기능적 변이체를 포함하는 개념이다. "기능적 변이체" 란 유방암 세포에 결합 하는 본 발명의 펩타이드의 성질에는 영향을 미치지 아미노산 위치에서 일부 아미 노산의 치환이 발생된 모든 유사한 서열을 의미한다. 한편, 본 발명은 서열번호 1 내지 4로 아루어진 군에서 선택된 아미노산 서 열을 암호화하는 폴리뉴클레오티드, 상기 폴리뉴클레오티드를 포함하는 발현백터' 및 상기 발현백터로 형질전환된 형질전환체를 제공한다. 상기 폴리뉴클레오티드는 상기에서 기재한 바와 같이 수득할 수 있으며， 바 람직하게는 각각 서열번호 5 내지 서열번호 8의 염기서열을 가질 수 있다. 발현백 터는 숙주 세포 내에서 삽입된 핵산을 발현할 수 있는 당 분야에 공지된 플라스미 드， 바이러스 백터 또는 기타 매개체를 의미하는 것으로서, 당업계에 공지된 통상 의 발현백터에 본 발명의 펩타이드를 암호화하는 폴리뉴클레오티드가 작동가능하게 연결된 것일 수 있다. 상기 발현백터는 일반적으로 숙주세포에서 증식할 수 있는 복제원점, 발현을 조절하는 하나 이상의 발현 조절 서열 (예. 프로모터， 인핸서 등 )， 선별 마커 (selective marker) 및 발현 조절 서열과 작동가능하게 연결된 본 발 명의 펩타이드를 암호화하는 폴리뉴클레오티드를 포함할 수 있다. 형질전환체는 상 기 발현백터에 의해 형질전환된 것일 수 있다. 바람직하게는 형질전환체는 본 발명 의 펩타이드를 암호화하는 폴리뉴클레오티드를 포함하는 발현 백터를 당업계에 공 지된 방법， 예를 들어 이에 한정되지는 않으나， 일시적 형질감염 (transient transfection), 미세주사， 형질도입 (transduct ion)， 세포융합， 칼슘 포스페이트 침 전법， 리포좀 매개된 형질감염 (liposome-mediated transfection), DEAE 덱스트란- 매개된 형질감염 (DEAE Dextran- mediated transfect ion) , 폴리브렌-매개된 형질감 염 (polybrene一 medi ated transfect ion) , 전기침공법 (electropora t ion) , 유전자 종 (gene gun) 및 세포 내로 핵산을 유입시키기 위한 다른 공지의 방법에 의해 숙주세 포에 도입하여 수득할 수 있다 (Wu et al . , J . Bio . Chem. , 267 : 963-967， 1992 ; Wu and Wu, J . Bio . Chem. , 263 : 14621-14624 , 1988) . 본 발명은 또한 본 발명의 템타이드 및 이와 결합하는 약물을 유효성분으로 포함하는 유방암 예방 또는 치료용 약학적 조성물을 제공한다. 본 발명에 따른 약학적 조성물은 약학적으로 유효한 양의 본 발명의 펩타이 드 단독 또는 템타이드 및 이와 결합하는 유방암 치료물질을 포함할 수 있다. 상기 에서 "약학적으로 유효한 양" 이란 음성 대조군에 비해 그 이상의 반웅을 나타내 는 양을 말하며 바람직하게 _는_ -유방—암을 방 _또_는— 료하 Z 충분한 양을 말한.다. 본 발명의 펩타이드에 연결될 수 있는 약제는 종래 유방암의 치료에 사용되 는 것이라면 제한 없이 사용될 수 있다. 예를 들면， 이에 한정되지는 않으나， 유방 암을 예방하는 약물로 허가 받은 라록시펜 하이드로클로라이드 (Raloxi fene hydrochlor ide) , 타목시펜 시트레이트 (Tamoxi fen ci trate) 등 이거나， 또는 유방암 을 치료하는 약물로 허가 받은 메쏘트렉세이트 (Methotrexate) , 파클리탁셀 (Pacl i taxel ) , 트라스투주맙 (Trastuzumab)， 에버로리무스 (Everol imus) 등일 수 있 다. 본 발명의 펩타이드와 유방암 치료물질의 결합은 당업계에 공지된 방법， 예 컨대， 공유 결합, 가교 등을 통해 수행될 수 있으며， 본 발명의 펩타이드와 유방암 치료물질이 직접적으로 결합되거나 또는 매개체를 통하여 간접적으로 결합될 수도 있다. 아울러， 상기 결합은 생체 외에서 수행될 수도 있으나， 본 발명의 펩타이드 및 유방암 치료물질은 개별적으로 투여되어 생체 내에서 결합될 수도 있다. 이를 위해 본 발명의 펩타이드 또는 유방암 치료물질은 필요하다면 그 활성이 소실되지 않는 범위에서 화학적으로 수식 (modi f i cat ion)될 수 있다. 본 발명의 펩타이드가 유방암 세포에 결합되어 유방암 치료물질이 적절하게 투여되었는지 여부를 용이하게 확인하기 위하여， 본 발명의 펩타이드는 발색효소， 방사성 동위원소 등으로 표지된 상태로 제공될 수 있다. 본 발명의 조성물에 포함되는 본 발명의 펩타이드 및 이와 결합되는 유방암 치료물질의 양은 결합되는 약제의 종류 및 양에 따라 달라질 수 있으며， 바람직하 게는 유방암 세포에 충분히 전달되어, 유효한 효과를 보이는 양일 수 있다. 그러 나， 약물은 그 투여 경로 및 치료 횟수뿐 만 아니라 환자의 연령， 체중, 건강 상 태， 성별， 질환의 중증도， 식이 및 배설율 등 다양한 요인들을 고려하여 환자에 대 한 유효 투여량이 결정되는 것이므로， 이러한 점을 고려할 때 당 분야의 통상적인 지식을 가진 자라면 상기 펩타이드를 약제와 결합시켜 유방암을 치료하기 위한 용 도에 따른 본 발명의 펩타이드의 적절한 유효량을 결정할 수 있을 것이다. 본 발명에 따른 템타이드를 포함하는 약학적 조성물은 본 발명에 의한 효과 를.ᅵ보으 1는ᅳᅳ한ᅳ^ᅵ형 투여 경로ᅳ및„—투싀ᅳ방ᅳ법이ᅵ 별히 제한되지_아ᅳ니ᅳ한다—.—폐컨 대， 본 발명의 펩타이드는 경구 투여 또는 비경구 투여될 수 있다. 경구 투여 시에 는 위장관 내 소화효소에 의한 분해를 방지하기 위하여 본 발명의 펩타이드를 L형 아미노산으로 구성하여 사용하는 것이 바람직하다. 상기 비경구 투여로는 피하 주 사, 근육내 주사, 정맥 주사 등이 있는데， 바람직하게는 정맥 주사일 수 있다. 본 발명의 약학적 조성물을 경구 투여하는 경우 본 발명의 약학적 조성물은 적합한 경구 투여용 담체와 함께 당업계에 공지된 방법에 따라 분말， 과립, 정계， 환제, 당의정제， 캡슐제， 액제， 겔제, 시럽계, 현탁액， 웨이퍼 등의 형태로 제형화 될 수 있다. 적합한 담체의 예로는 락토즈， 덱스트로즈, 수크로즈, 솔비롤， 만니 를， 자일리를， 에리스리를 및 말티를 등을 포함하^ 당류와 옥수수 전분， 밀 전분， 쌀 전분 및 감자 전분 등을 포함하는 전분류， 셀를로즈， 메틸 셀를로즈， 나트륨 카 르복시메틸샐를로오즈 및 하이드록시프로필메틸-셀를로즈 등을 포함하는 셀를로즈 류， 젤라틴， 폴리비닐피롤리돈 등과 같은 층전제가 포함될 수 있다. 또한， 경우에 따라 가교결합 폴리비닐피를리돈， 한천， 알긴산 또는 나트륨 알기네이트 등을 붕해 제로 첨가할 수 있다. 나아가， 상기 약학적 조성물은 항웅집제， 윤활제, 습윤제， 향료, 유화제 및 방부제 등을 추가로 포함할 수 있다 또한, 본 발명의 약학적 조성물을 비경구적으로 투여하는 경우 본 발명의 약 학적 조성물은 적합한 비경구용 담체와 함께 예를 들면， 주사제의 형태로 당 업계 에 공지된 방법에 따라 제형화될 수 있다. 상기 주사제의 경우에는 반드시 멸균되 어야 하며 박테리아 및 진균과 같은 미생물의 오염으로부터 보호되어야 한다. 주사 제의 경우 적합한 담체의 예로는 이에 한정되지는 않으나， 물, 에탄을， 폴리을 (예 를 들어, 글리세를， 프로필렌 글리콜 및 액체 폴리에틸렌 글리콜 등)， 이들의 흔합 물 및 /또는 식물유를 포함하는 용매 또는 분산매질일 수 있다. 보다 바람직하게는， 적합한 담체로는 행크스 용액， 링거 용액， 트리에탄을 아민이 함유된In order to achieve the above object of the present invention, the present invention provides a breast cancer target peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 4. In order to achieve another object of the present invention, the present invention provides a polynucleotide comprising a nucleotide sequence encoding the template. In order to achieve another object of the present invention, the present invention provides a vector comprising the polynucleotide. In order to achieve another object of the present invention, the present invention provides a transformant transformed with the vector. In order to achieve the other object of the present invention, the present invention provides a pharmaceutical composition for the prevention or treatment of breast cancer comprising the peptide and a drug binding thereto as an active ingredient. In order to achieve the other object of the present invention, the present invention provides a composition for drug delivery specific to breast cancer comprising the temptide. In order to achieve the other object of the present invention, the present invention provides a composition for diagnosing breast cancer comprising the peptide as an active ingredient. In order to achieve the other object of the present invention, the present invention provides a kit for diagnosing breast cancer comprising the peptide. In order to achieve the another object of the present invention, the present invention provides a marker for diagnosing breast cancer comprising the tempide. In order to achieve the other object of the present invention, the present invention provides a composition for imaging breast cancer comprising the peptide as an active ingredient. To achieve another object of the present invention, there is provided a use of the peptide for the preparation of an agent for the diagnosis, treatment or imaging of breast cancer. In order to achieve another object of the present invention, there is provided a method for diagnosing breast cancer, comprising administering to a subject in need thereof an effective amount of the peptide. In order to achieve another object of the present invention, there is provided a method for treating breast cancer, comprising administering to the subject in need thereof an effective amount of the peptide and the drug bound thereto. The present invention will be described in detail below. The present invention provides a breast cancer target peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-4. Breast cancer has been considered a disease, but recently it has been found that there are various types of subtypes in breast cancer. In the present invention, the breast cancer may be largely classified into three types according to proteins closely related to the onset of cancer. That is, HER-2 (human epidemal growth factor receptor 2) overexpressed breast cancer, estrogen receptor-positive luminal-type breast cancer, and trinegative breast mammin that does not express all of the estrogen receptor, progesterone receptor and HER-2. . In one embodiment of the present invention, the peptide having the amino acid sequence of SEQ ID NO: 1 to 4 is treated to MCF7, which is a luminal breast cancer cell, Skbr3, which is a HER-2 breast cancer cell, or MDA-MB-231 cell, which is a triple negative breast cancer cell, respectively. As a result, it was confirmed that all subtypes bind to breast cancer cells. Among them, the peptide is a triple negative oil It has been shown to exhibit higher specificity to cancer cells. Triple-negative breast cancer is about 15¾ of the total breast cancer, but the frequency is low, but unlike other breasts, the target receptor is not expressed, and there is no suitable therapeutic agent, and after the chemotherapy, the recurrence is poor and the prognosis is poor. Therefore, the peptides of the present invention, which exhibit higher specificity for triple negative breast cancer cells than HER-2 breast and luminal breast cancers, can be usefully developed for the development of effective therapeutics for triple negative breast cancers that currently lack effective therapeutic agents and are well relapsed. have. Therefore, in the present invention, the breast cancer may be luminal type, HER-2 type or triple negative breast cancer, and most preferably triple negative breast cancer. Peptides of the invention can be readily prepared by chemical synthesis known in the art (Creighton, Proteins; Structures and Molecular Pr inciples, WH Freeman and Co., NY, 1983). Representative methods are limited to these. But liquid or solid phase synthesis, fragment expansion, F-M0C or T-B0C chemistry (Chemical Approaches to the Synthesis of Peptides and Proteins, Will iams et al., Eds., CRC Press, Boca Raton Florida , 1997; A Practical Approach, Athert on & Sheppard, Eds., IRL Press, Oxford, England, 1989). In addition, the peptide of the present invention can be prepared by genetic engineering methods. First, a DNA sequence encoding the peptide is constructed according to a conventional method. DNA sequences can be constructed by PCR amplification using appropriate primers. Alternatively, DNA sequences may be synthesized by standard methods known in the art, for example using an automated DNA synthesizer (available from Biosearch or Applied Biosys terns). The constructed DNA sequence is 2. Inserting into a vector comprising one or more expression control sequences (e.g., promoters, enhancers, etc.) that are operably linked to the DNA sequence and regulate expression of the DNA sequence, The host cell is transformed with the recombinant expression vector formed therefrom. The resulting transformants are incubated under appropriate media and conditions to allow the DNA sequence to be expressed to recover substantially pure peptides encoded by the DNA sequences from the culture. The recovery can be carried out using methods known in the art (eg, chromatography). As used herein, "substantially pure peptide" means any other protein from which the peptide according to the invention is derived from a host. It does not mean to include. For genetic engineering methods for peptide synthesis of the present invention, reference may be made to Maniatis et al. Molecular Cloning; A laboratory Manual, Cold Spring Harbor laboratory, 1982; Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, NY, Second (1998) and Third (2000) Edition; Gene Expression Technology, Method in Enzymology, Genetics and Molecular Biology, Method in Enzymology, Guthrie & Fink (eds.), Academic Press, San Diego, Calif, 1991; and Hitzeman et al. , J. Biol. Chem. , 255: 12073—12080, 1990. In the present invention, the peptide having the amino acid sequence of SEQ ID NO: 1 to 4 is a concept including a functional variant thereof. By "functional variant" is meant any similar sequence in which substitution of some amino acids at the amino acid position has occurred that does not affect the properties of the peptides of the invention that bind to breast cancer cells. On the other hand, the present invention provides a transfected with the expression vector, and the expression vector including a polynucleotide, the polynucleotide encoding an amino acid selected from the group standing column eojin Aru in SEQ ID NO: 1 to 4 switch transformants. The polynucleotide may be obtained as described above, and preferably have a nucleotide sequence of SEQ ID NO: 5 to SEQ ID NO: 8, respectively. Expression vector refers to a plasmid, viral vector or other medium known in the art capable of expressing a nucleic acid inserted in a host cell, and encodes the peptide of the present invention in a conventional expression vector known in the art. The polynucleotide may be operably linked. The expression vector is generally operatively associated with a replication origin that can proliferate in a host cell, one or more expression control sequences that regulate expression (eg, promoters, enhancers, etc.), selective markers, and expression control sequences. Polynucleotides encoding the peptides of the invention. The transformant may be transformed by the expression vector. Preferably the transformant is a method known in the art, including, but not limited to, expression vectors comprising polynucleotides encoding peptides of the invention, such as transient transfection, microinjection, transformation Transduction ion, cell fusion, calcium phosphate precipitation, liposome-mediated transfection, DEAE dextran- Nucleic acid is introduced into mediated transfection (DEAE Dextran-mediated transfect ion), polybrene-mediated transfect ion, electroporation ion, gene gun and cells It can be obtained by introducing into host cells by other known methods for influx (Wu et al., J. Bio. Chem., 267: 963-967, 1992; Wu and Wu, J. Bio.Chem. , 263: 14621-14624, 1988). The present invention also provides a pharmaceutical composition for the prevention or treatment of breast cancer, comprising the template of the present invention and a drug binding thereto as an active ingredient. The pharmaceutical composition according to the present invention may include a pharmaceutically effective amount of the peptide of the present invention alone or temide and a breast cancer therapeutic agent in combination therewith. As used herein, the term "pharmaceutically effective amount" refers to an amount that exhibits more reaction than the negative control, and preferably refers to a sufficient amount of Z-breast-cancer-or-Z. Agents that can be linked to the peptides of the invention can be used without limitation as long as they are used in the treatment of conventional breast cancer. Examples include, but are not limited to, Raloxi fene hydrochloride, Tamoxi fen ci trate, which are licensed as drugs to prevent breast cancer, or drugs to treat breast cancer. Authorized Methotrexate, Paclitaxel, Trastuzumab, Everol imus, and the like. The binding of the peptide of the present invention to a therapeutic agent for breast cancer may be carried out through a method known in the art, for example, covalent bonding, crosslinking, etc. It may be combined indirectly. In addition, the binding may be performed in vitro, but the peptide and the breast cancer therapeutic agent of the present invention may be administered separately to be bound in vivo. To this end, the peptide of the present invention or the breast cancer therapeutic substance may be chemically modified (modi fi cat ion) in a range where its activity is not lost if necessary. In order to easily confirm whether the peptide of the present invention is bound to breast cancer cells and the breast cancer therapeutic substance is properly administered, the peptide of the present invention is a chromophore, It may be provided in a state labeled with a radioisotope. The amount of the peptide of the present invention and the breast cancer therapeutic material bound thereto may be included in the composition of the present invention, depending on the type and amount of the drug to be bound, and preferably, the amount is sufficient to be delivered to the breast cancer cells, thereby showing an effective effect. have. However, since the drug is determined not only by the route of administration and the number of treatments, but also by various factors such as the age, weight, health status, sex, severity of the disease, diet and excretion rate, the effective dosage for the patient is determined. In view of this, those skilled in the art will be able to determine an appropriate effective amount of a peptide of the present invention according to the use for treating breast cancer by combining the peptide with a medicament. The pharmaceutical composition comprising the tempide according to the present invention has the effect according to the present invention.B. 1 is not limited to the type of administration route and administration method. The lung peptide may be administered orally or parenterally. In the case of oral administration, in order to prevent degradation by digestive enzymes in the gastrointestinal tract, it is preferable to use the peptide of the present invention composed of L-type amino acids. The parenteral administration includes subcutaneous injection, intramuscular injection, intravenous injection, and the like, preferably intravenous injection. In the case of oral administration of the pharmaceutical composition of the present invention, the pharmaceutical composition of the present invention may be prepared in powder, granule, tablet, pill, dragee, capsule, liquid, gel according to a method known in the art together with a suitable oral carrier. It can be formulated in the form of syrups, suspensions, wafers and the like. Examples of suitable carriers include lactose, dextrose, sucrose, sorbobiol, manny, xylly, erythroli, malty, etc. Sugars, corn starch, wheat starch, rice starch, potato starch, etc. Layered agents such as starch, cellulose, methyl cellulose, sodium carboxymethyl salose and hydroxypropylmethyl-cellulose, including cellulose, gelatin, polyvinylpyrrolidone, and the like. have. In some cases, crosslinked polyvinylpyridone, agar, alginic acid or sodium alginate may be added as a disintegrating agent. Furthermore, the pharmaceutical composition may further include an anticoagulant, a lubricant, a humectant, a fragrance, an emulsifier, and an antiseptic. In addition, when the pharmaceutical composition of the present invention is administered parenterally, the pharmaceutical composition of the present invention may be suitable. Together with parenteral carriers, for example in the form of injections It may be formulated according to a method known in the art. Such injections must be sterilized and protected from contamination of microorganisms such as bacteria and fungi. Examples of suitable carriers for injectables include, but are not limited to, solvents including water, ethane, poly (e.g., glycerol, propylene glycol and liquid polyethylene glycol, etc.), combinations thereof and / or vegetable oils. Or dispersion medium. More preferably, suitable carriers include Hanks' solution, Ringer's solution, and triethane containing amines.
PBS( phosphate buf fered sal ine) 또는 주사용 멸균수， 10% 에탄을， 40% 프로필렌 글리콜 및 5% 덱스트로즈와 같은 등장 용액 등을 사용할 수 있다. 상기 주사제를 미생물 오염으로부터 보호하기 위해서는 파라벤， 클로로부탄올， 페놀， 소르빈산'， 티메로살 등과 같은 다양한 항균제 및 항진균제를 추가로 포함할 수 있다. 또한， 상기 주사제는 대부분의 경우 당 또는 나트륨 클로라이드와 같은 등장화제를 추가 로 포함할 수 있다. 그 밖의 약학적으로 허용되는 담체로는 다음의 문헌에 기재되어 있는 것을 참고로 할 수 있다 (Remington ' sPharmaceut ical Sci ences , 19th ed. , Mack Publ i shing Company, East on, PA, 1995) . 이외에도 본 발명의 조성물에는 일반적인 약학적 조성물에 통상적으로 첨가되는 약학적으로 허용되는 담체가 추가로 포함될 수 있다. 상기 "약학적으로 허용되는' '이란 생리학적으로 허용되고 사람이나 동물에 투여될 때, 통상적으로 위장 장애， 현기증 등과 같은 알레르기 반응 또는 이와 유 사한 반응을 일으키지 않는 것을 말한다. 약학적으로 허용되는 담체는 주사제의 경 우에는, 예컨대 완층제 (예를 들어, 식염수 또는 PBS) , 카보하이트레이트 (예를 들 어, 글루코스， 만노즈， 슈크로즈 또는 텍스트란)， 항산화제， 정균제， 킬레이트화제 (예를 들어, EDTA 또는 글루타치온)， 아쥬반트 (예를 들어， 알루미늄 하이드톡사이 드)， 현탁제， 농후제， 보존제, 무통화제， 가용화제， 등장제 및 /또는 안정화제를 사 용할 수 있다. 이와 같이 본 발명의 펩타이드를 포함하는 조성물와 제형은 상술한 바와 같아 다양하게 제조될 수 있다. 예를 들어， 주사제의 경우에는 단위 투약 앰 플 또는 다수회 투약 포함제 형태로 제조할 수 있다. 본 발명의 펩타이드를 포함하 는 약물 전달용 조성물은 당업계에 공지된 통상적인 약물 투여 방법으로 투여될 수 있다. 또한， 본 발명의 약학적 조성물은 포유동물에 투여된 후 활성 성분의 신속， 지속 또는 지연된 방출을 제공할 수 있도록 당업계에 공지된 방법을 사용하여 제형 화될 수 있다. 본 발명은 또한 본 발명의 펩타이드를 포함하는 유방암에 특이적인 약물 전 달용 조성물을 제공한다. 본 발명의 펩타이드는 유방암 치료제와 같은 약물을 유방암 조직에 선택적으 로 전달하는 지능형 약물 전달체로서 사용될 수 있다. 본 발명의 펩타이드를 종래 유방암 치료제와 연결하여 유방암 치료에 이용한다면 본 발명의 펩타이드에 의해 유방암 치료제가 유방암 세포에만 선택적으로 전달되기 때문에 약의 효력을 증가시 킬 수 있고 동시에 정상조직에 미치는 약물의 부작용을 현저히 줄일 수 있다. 상기 약물은 이에 한정되지는 않으나， 파클리탁샐， 독소루비신, 빈크리스틴， 다우노루비신 (daunorubicin), 빈블라스틴 (vinblastine)， 액티노마이신-Phosphate buf fered salin (PBS) or sterile water for injection, 10% ethane, isotonic solution such as 40% propylene glycol and 5% dextrose. In order to protect the injection from microbial contamination, various antibacterial and antifungal agents such as paraben, chlorobutanol, phenol, sorbic acid ' , thimerosal, and the like may be further included. In addition, the injection may in most cases further comprise an isotonic agent such as sugar or sodium chloride. Other pharmaceutically acceptable carriers may be referred to those described in the following literature (Remington's Pharmaceutical Sciences, 19th ed., Mack Publ i shing Company, East on, PA, 1995). In addition, the composition of the present invention may further include a pharmaceutically acceptable carrier that is commonly added to the general pharmaceutical composition. The term “pharmaceutically acceptable” means physiologically acceptable and does not cause allergic reactions or similar reactions such as gastrointestinal disorders, dizziness, etc. when administered to humans or animals. In the case of injectables, for example, complete agents (e.g. saline or PBS), carbohydrates (e.g. glucose, mannose, sucrose or textane), antioxidants, bacteriostatic agents, chelating agents (e.g. For example, EDTA or glutathione), adjuvants (eg, aluminum hydride), suspending agents, thickening agents, preservatives, analgesics, solubilizers, isotonic and / or stabilizers can be used. Likewise, compositions and formulations comprising the peptides of the present invention can be prepared in a variety of ways as described above, eg in the case of injections, unit dosage amps. Alternatively, the drug delivery composition comprising the peptide of the present invention may be administered by conventional drug administration methods known in the art. Is formulated using methods known in the art to provide rapid, sustained or delayed release of the active ingredient after administration to a mammal. Can be converted. The present invention also provides a composition for drug delivery specific to breast cancer comprising the peptide of the present invention. The peptide of the present invention can be used as an intelligent drug carrier for selectively delivering a drug such as a breast cancer therapeutic agent to breast cancer tissue. When the peptide of the present invention is used in the treatment of breast cancer in connection with a conventional breast cancer treatment agent, since the breast cancer treatment agent is selectively delivered only to breast cancer cells by the peptide of the present invention, the effect of the drug may be increased and at the same time, the side effects of the drug on normal tissues Can be significantly reduced. The drug may include, but is not limited to, paclitaxal, doxorubicin, vincristine, daunorubicin, vinblastine, and actinomycin-
D(actinomycin-D), 도세탁셀 (docetaxel )， 에토포사이드 (etoposide)， 테니포사이드 (teniposide) , 비산트렌 (bisantrene)， Jl≤-¾11¾£'ld(homoharringtonine) , 글리백 (Gleevec; STI-571), 시스플라틴 (cisplain)， 5-플로오우라실 (5-fluouraci 1)， 아드 리아마이신 (adriamycin), 메토트렉세이트 (methotrexate)， 부설판 (busul fan)， 클로 람부실 (chlorambucil), 시클로포스파미드 (cyclophosphamide)， 멜팔란 (melphalan)， 니트로겐 무스타드 (nitrogen mustard), 니트로소우레아 (nitrosourea)， 스트렙토키 나제 (streptokinase), 유로키나제 (urokinase)， 알테플라제 (alteplase)， 안지오텐신 (angiotensin) II 억제제, 알도스테론 (aldosterone) 수용체 억제제， 에리트로포이 에틴 (erythropoietin), NMDA (N-methyl-d-aspartate) 수용체 억제제， 로바스타틴 (Lovastatin), 라파마이신 (Rapamycin)， 셀레브렉스 (Celebrex)， 티클로핀 (Ticlopin) 마리마스타트 (Marimastat)및 트로케이드 (Trocade) 등일 수 있다. 본 발명의 펩타이드와 상기 약물은 공유결합으로 연결될 수 있고， 특히， 링 커 (Linker)에 의해 연결될 수 있으나， 이에 제한을 두지 않는다. 또한， 본 발명은 본 발명의 펩타이드를 유효성분으로 포함하는 유방암 진단 용 조성물을 제공한다. 이 때， 본 발명에서 "진단 "이란 병리 상태의 존재 또는 특징을 확인하는 것 을 의미한다. 본 발명의 목적상， 진단은 유방암의 존재 또는 특징을 확인하는 것이 다. 본 발명의 진단용 조성물에 포함되는 펩타이드는 서열번호 1 내지 4로 이루어 진 군에서 선택된 아미노산 서열을 가질 수 있으며， 상기 기재한 바와 같이 화학적 또는 유전공학적 방법에 의해 수득된 것일 수 있다. 본 발명의 펩타이드는 유방암 세포에 대해서 유용하게 표적되므로 유방암 진단을 위하여 유용하게 사용될 수 있 다. 상기 진단용 조성물에는 본 발명의 펩타이드 이외에도 펩타이드 구조 또는 생리활성을 안정하게 유지시켜 주는 완층액 또는 반웅액이 추가로 포함될 수 있으 며, 또한， 안정성을 유지하기 위해， 분말 상태 또는 적절한 완층액에 용해된 상태 또는 4°C가 유지된 상태로 제공될 수 있다. 본 발명의 펩타이드가 유방암 조직에 결합하였는지 여부를 용이하게 확인， 검출 및 정량하기 위하여, 본 발명의 펩타이드는 표지된 상태로 제공될 수 있다. 즉， 검출가능한 표지에 링크 (예: 공유 결합 또는 가교)되어 제공될 수 있다. 상기 검출 가능한 표지는 발색효소 (예: 퍼옥시다제 (peroxidase) , 알칼라인 포스파타제 Actinomycin-D, docetaxel, etoposide, etoniposide, teniposide, bisantrene, Jl≤-¾11¾ £ 'l d (homoharringtonine), gleevec; STI-571 ), Cisplain, 5-fluouraci 1, adriamycin, methotrexate, busul fan, chlorambucil, cyclophosphamide cyclophosphamide, melphalan, nitrogen mustard, nitrosourea, streptokinase, urokinase, alteplase, angiotensin II inhibitors , Aldosterone receptor inhibitor, erythropoietin, N-methyl-d-aspartate (NMDA) receptor inhibitor, lovastatin, rapamycin, Celebrex, Ticlopin Marimas Bit (Marimastat) and Trojan Cade (Trocade) and the like. The peptide of the present invention and the drug may be covalently linked, and in particular, may be linked by a linker (Linker), but is not limited thereto. In addition, the present invention provides a composition for diagnosing breast cancer comprising the peptide of the present invention as an active ingredient. At this time, in the present invention, "diagnosis" refers to confirming the presence or characteristics of a pathological state Means. For the purposes of the present invention, the diagnosis is to identify the presence or characteristics of breast cancer. Peptides included in the diagnostic composition of the present invention may have an amino acid sequence selected from the group consisting of SEQ ID NO: 1 to 4, may be obtained by chemical or genetic engineering methods as described above. Since the peptide of the present invention is usefully targeted to breast cancer cells, it can be usefully used for breast cancer diagnosis. In addition to the peptide of the present invention, the diagnostic composition may further include a complete layer solution or a semi-aqueous solution that maintains the peptide structure or physiological activity in a stable manner. State or 4 ° C. may be provided. In order to easily identify, detect, and quantify whether the peptide of the present invention is bound to breast cancer tissue, the peptide of the present invention may be provided in a labeled state. In other words, the detectable label may be provided in a link (eg, covalently bonded or crosslinked). The detectable label may be a colorase (e.g. peroxidase, alkaline phosphatase)
12 125 111 99m 32 35 12 125 111 99m 32 35
(alkal ine phosphatase) ) , 방사성 동위원소 (예: I I In, Tc , P, S) , 크로모포어 (chromophore) , 바이오틴 (biot in)， 발광물질 또는 형광물질 (예 : FITC, RITC, 로다민 (rhodamine) , 텍사스레드 (Texas Red) , 플로레신 (f luorescein)， 피코에 리트린 (phycoerythrin) , 퀀텀닷 (quantum dots)) , 자기공명영상조영계 (예: 수퍼파라 마그네틱 산화철 (superparamagnet ic iron oxides , SPIO) , 을트라수퍼파라마그네틱 산화철 (ultrasuperparamagnet ic iron oxides , USPI0)) 등 일 수 있다. 유사하게， 상기 검출 가능한 표지는 항체 에피토프 (epi tope) , 기질 (substrate)， 보조인자 (cofactor) , 저해제 또는 친화 리간드일 수 있다. 이러한 표지는 본 발명의 펩타이 드를 합성하는 과정 중에 수행할 수도 있고， 이미 합성된 펩타이드에 추가로 수행 될 수도 있다. 만약 검출가능한 표지로 형광물질을 이용하는 경우에는 형광단층촬 영 (Fluorescence mediated tomography:FMT)으로 유방암을 진단할 수 있다. 예컨대， 형광물질로 표지된 본 발명의 펩타이드를 혈액 내로 순환시키고 형광단층촬영으로 펩타이드에 의한 형광을 관찰할 수 있다. 형광이 관찰된다면， 유방암으로 진단된 다. 또한 검출가능한 표지로 방사선 물질올 이용하는 경우에는 양전자단층촬영 (Posi tron emission tomography: PET)으로 펩타이드에 의한 방사능을 관찰하여 유방 암을 진단할 수 있다. 이 경우에는 보다 민감하게 유방암의 진단 및 분자영상이 가 능할 것이다. 본 발명은 또한 본 발명의 템타이드를 포함하는 유방암 진단용 키트를 제공 한다. 본 발명의 키트에는 상기한 바와 같은 발색효소 또는 방사선 동위원소 등으 로 표지된 펩타이드가 포함될 수 있다. 본 발명의 키트에는 본 발명의 펩타이드 이외에 상기 펩타이드와 유방암 세 포의 결합 반웅을 위한 적당한 완층용액 또는 배지 및 대조군 세포 (정상 뇌세포)를 추가로 포함할 수 있다. 또한 본 발명의 펩타이드가 표지되지 않은 채로 제공되는 경우에는， 펩타이드의 표지를 위한 검출가능한 표지가 추가로 키트에 포함될 수 있 다. 양—자택일적으로 ΤΓ본 발명의—펩타아 에ᅳ대한 -항체 Γᅳ형광물질로ᅳ표지된— 2차ᅵ항 체， 발색 기질 등이 상기 키트에 추가로 포함될 수도 있다. 본 발명의 펩티드에 대 한 항체는 상기 기재한 바와 같이 당업계에 공지된 통상적인 항체의 제조방법에 따 라 제조될 수 있다. 또한 본 발명의 펩타이드는 플레이트의 표면에 코팅된 형태로 제공될 수도 있다. 이 경우에는 상기 플레이트에 직접 유방암 세포를 접종하여 적당한 조건쎄서 반응시킨 후, 플레이트의 표면상에서의 유방암 세포와 펩타이드의 결합을 관찰하여 유방암을 진단할 수 있다. 본 발명은 또한 본 발명의 펩타이드를 유효성분으로 포함하는 유방암 영상화 용 조성물을 제공한다. 본 발명의 유방암 영상화용 조성물은 본 발명의 펩타이드를 함유하여 유방암 을 영상화하는데 사용될 수 있다. 본 발명의 템타이드를 포함하는 것을 특징으로 하는 유방암 영상화용 조성물 은 반음영부를 영상화하거나 검출 및 정량하는데 유용하게 사용될 수 있으며, 또한 종래 음영부의 영상화 기술을 이용하여 반음영부와 음영부의 상대적 크기 및 정도 를 측정함으로써 약물 치료를 통한 예후를 예측하는데도 유용하게 사용될 수 있다. 본 발명에서 상기 펩타이드는， 이에 한정되지는 않으나, 발색효소， 방사성동 위원소， 크로모포어 (chromophore) , 발광물질， 형광물질 ( f luorescer)， 상자성입자 (superparamagnet i c part i cles) 또는 초상자성입자 (ul trasuper paramagnet i c part ic les)로 표지된 것일 수 있다. 본 발명의 다른 일실시예에서는 상기 서열번호 1 내지 4의 아미노산 서열을 갖는 펩타이드를 루미날형 유방암 세포인 MCF7 , 삼중음성 유방암 세포인 MDA-MB- 231세포， 폐암 세포인 A549 세포, 정상 폐 세포인 BEAS-2B 세포， 정상 콩팔 세포임 HEK-293 세포에 각각 처리한 결과， 상기 펩타이드는 삼증음성유방암세포에 결합능 이 상대적으로 더 높은 것으로 보아， 삼중 음성 유방암 세포에 더 높은 특이성을 나타내는 것을 확인할 수 있었다. 본 발명의 또 다른 일실시예에서는 MDA-MB-231 세포를 마우스의 피부 아래 이식한 뒤 상기 서열번호 1 내지 4의 아미노산 서열을 갖는 펩타이드를 정맥 주사 로 투여한 뒤， 각 종양을 적출하여 형광 세기를 측정한 결과， 서열번호 1 및 서열 번호 4의 펩타이드를 처리한 종양 조직이 대조군에 비해 높은 형광 세기를 나타냈 으며, 서열번호 1의 펩타이드를 처리한 종양 조직이 다른 펩타이드를 처리한 종양 조직에 비하여 더 높은 형광 세기를 나타내는 것을 확인할 수 있었다. 본 발명은 유방암의 진단， 치료 또는 영상화용 제제를 제조하기 위한 상기 펩타이드의 용도를 제공한다. 본 발명은 상기 펩타이드의 유효량을 이를 필요로 하는 개체에 투여하는 것 을 특징으로 하는 유방암의 진단 방법을 제공한다. 본 발명은 상기 펩타이드 및 이와 결합하는 약물의 유효량을 이를 필요로 하 는 개체에 투여하는 것을 특징으로 하는 유방암의 치료 방법을 제공한다. 본 발명의 상기 '유효량' 이란 개체에게 투여하였을 때， 유방암의 개선， 치 료， 예방， 검출 또는 진단 효과를 나타내는 양을 말하며， 상기 '개체' 란 동물， 바 람직하게는 포유동물， 특히 인간올 포함하는 동물일 수 있으쪄， 동물에서 유래한 세포， 조직， 기관 등일 수도 있다. 상기 개체는 상기 효과가 필요한 환자 (pat ient ) 일 수 있다. (alkal ine phosphatase)), radioisotopes (eg II In, Tc, P, S), chromophore, biotin, luminescent or fluorescent substances (eg FITC, RITC, Rhodamine (rhodamine), Texas Red, fleuorescein, phycoerythrin, quantum dots, magnetic resonance imaging systems (e.g. superparamagnet ic iron oxides, SPIO), ultrasuperparamagnetic iron oxides (USPI0)), and the like. Similarly, the detectable label can be an antibody epitope, substrate, cofactor, inhibitor or affinity ligand. Such labeling may be performed in the course of synthesizing the peptide of the present invention, or may be performed in addition to the already synthesized peptide. If a fluorescent substance is used as a detectable label, breast cancer can be diagnosed by fluorescence mediated tomography (FMT). For example, the peptide of the present invention labeled with a fluorescent material can be circulated into the blood and fluorescence by the peptide can be observed by fluorescence tomography. If fluorescence is observed, it is diagnosed as breast cancer. In addition, in the case of using a radioactive substance as a detectable label, breast cancer can be diagnosed by observing the radioactivity caused by the peptide by Positron emission tomography (PET). In this case, breast cancer diagnosis and molecular imaging are more sensitive. Will be possible. The present invention also provides a kit for diagnosing breast cancer comprising the tempide of the present invention. The kit of the present invention may include a peptide labeled with a chromophore or a radioisotope as described above. In addition to the peptide of the present invention, the kit of the present invention may further include a suitable monolayer solution or medium for binding reaction between the peptide and breast cancer cells and control cells (normal brain cells). In addition, when the peptide of the present invention is provided unlabeled, a detectable label for labeling the peptide may be further included in the kit. Amount - it may include i secondary antibody, a chromogenic substrate contained in addition to the kit-of the present invention ΤΓ the home iljeok-peptide for the eu taah-Γ antibody of eu eu labeled with a fluorescent substance. Antibodies to the peptides of the present invention can be prepared according to conventional methods for preparing antibodies known in the art as described above. In addition, the peptide of the present invention may be provided in a form coated on the surface of the plate. In this case, breast cancer cells can be diagnosed by observing the binding of the breast cancer cells and the peptide on the surface of the plate after inoculating breast cancer cells directly on the plate and reacting under appropriate conditions. The present invention also provides a composition for imaging breast cancer comprising the peptide of the present invention as an active ingredient. The composition for imaging breast cancer of the present invention may be used to image breast cancer by containing the peptide of the present invention. The composition for imaging breast cancer comprising the tempide of the present invention may be usefully used for imaging, detecting, and quantifying the semi-shaded portion, and using the imaging technique of the conventional shaded portion, the relative size and extent of the semi-shaded portion and the shaded portion. It can also be useful for predicting prognosis through medication. In the present invention, the peptide is not limited thereto, but is not limited thereto, a chromophore, a radioisotope, a chromophore, a luminescent material, a fluorescent substance (f luorescer), a paramagnetic particle (superparamagnetic part i cles) or a superparamagnetic It may be labeled with particles (ul trasuper paramagnet ic part ic les). In another embodiment of the invention the peptide having the amino acid sequence of SEQ ID NOS: 1 to 4 luminal breast cancer cells MCF7, triple negative breast cancer cells MDA-MB- 231 cells, lung cancer cells A549 cells, normal lung cells Treatment with BEAS-2B cells and HEK-293 cells, which are normal soybean cells, showed that the peptides showed relatively higher binding capacity to trinegative breast cancer cells, indicating higher specificity to triple negative breast cancer cells. . In another embodiment of the present invention, after transplanting MDA-MB-231 cells under the skin of a mouse, administering a peptide having the amino acid sequence of SEQ ID NOS: 1 to 4 by intravenous injection, extracting each tumor and fluorescence intensity As a result, the tumor tissue treated with the peptides of SEQ ID NO: 1 and SEQ ID NO: 4 showed higher fluorescence intensity than the control group, and the tumor tissue treated with the peptide of SEQ ID NO: 1 was applied to the tumor tissue treated with the other peptide. It was confirmed that the fluorescence intensity was higher than that. The present invention provides the use of the peptides for the preparation of an agent for the diagnosis, treatment or imaging of breast cancer. The present invention provides a method for diagnosing breast cancer, wherein the effective amount of the peptide is administered to a subject in need thereof. The present invention provides a method for treating breast cancer, comprising administering to a subject in need thereof an effective amount of the peptide and the drug bound thereto. The term 'effective amount' of the present invention, when administered to an individual, refers to an amount showing improvement in breast cancer, treatment, prevention, detection or diagnosis, and the term 'object' is preferably an animal, preferably a mammal, especially a human. May be an animal containing, originating from an animal It may be a cell, tissue, organ. The subject may be a patient in need of the effect.
본 발명의 상기 '치료' 는 유방암 또는 유방암 관련 질환 또는 유방암 관련 질환의 증상을 개선시키는 것을 포괄적으로 지칭하고， 이는 이러한 질환을 치유하 거나， 실질적으로 예방하거나， 또는 상태를 개선시키는 것을 포함할 수 있으며, 유 방암 또는 유방암 관련 질환으로부터 비롯된 한 가지 증상 또는 대부분의 증상을 완화시키거나， 치유하거나 예방하는 것을 포함하나， 이에 제한되는 것은 아니다.  The term 'treatment' of the present invention refers generically to ameliorating symptoms of a breast cancer or breast cancer related disease or a breast cancer related disease, which may include treating, substantially preventing, or ameliorating the disease. And alleviating, healing or preventing one or most of the symptoms arising from breast or breast cancer related disorders.
【유리한 효과】 Advantageous Effects
본 발명의 펩타이드는 유방암에 특이적으로 결합할 수 있기 때문에, 유방암 의 진단， 치료 또는 영상화용 조성물의 제조에 유용하게 활용될 수 있다/  Since the peptide of the present invention can specifically bind to breast cancer, it can be usefully used for the preparation of a composition for diagnosis, treatment or imaging of breast cancer.
【도면의 간단한 설명】 [Brief Description of Drawings]
도 1은 파지 라이브러리 스크리닝을 위해 본 발명에서 사용된 두 가지 전략 인 negat ive subtract ion(A) 및 posit ive select ion(B) 스크리닝 과정을 나타낸다. 도 2는 MCF7, SKBR3, MDA-MB-231 세포를 대상으로 negat ive subtract ion 스 크리닝한 결과， 각 라운드 (Rl ~ R5)에서 회수된 파지 역가를 나타낸다. 도 3은 MDA-MB-231 세포를 대상으로 posi t ive select ion 스크리닝한 결과， 각 라운드 (Rl~ R5)에서 회수된 파지 역가를 나타낸다. 도 4는 negat ive subtract ion 스크리닝 (A) 및 posi t ive select ion 스크리닝 (B)을 통해 선별한 파지 클론에 삽입된 펩타이드 코딩 유전자 서열을 읽은 다음， 이^ 아미노산 서열로 전환 및 유사 아미노산 서열끼리 배열한 결과의 일부를 나타 낸다. 도 5A는 negat ive subtract ion 스크리닝 전략으로부터 선별된 6개의 파지 클 론의 MDA-MB-231 세포에 대한 결합능을 플라크 에세이를 통해 조사한 결과를 나타 낸다.  1 shows two strategies used in the present invention for phage library screening: negat ive subtract ion (A) and positive select ion (B) screening procedures. Figure 2 shows the phage titer recovered in each round (Rl ~ R5) as a result of negative subtract ion screening for MCF7, SKBR3, MDA-MB-231 cells. Figure 3 shows the phage titer recovered in each round (Rl ~ R5) as a result of positive select ion screening on MDA-MB-231 cells. Figure 4 shows the peptide coding gene sequences inserted into phage clones selected through negat ive subtract ion screening (A) and posi tive select ion screening (B), and then converting to amino acid sequences and sequencing similar amino acid sequences Indicates some of the results. 5A shows the results of investigating the binding capacity of six phage clones to MDA-MB-231 cells selected from a negat ive subtract ion screening strategy through plaque assay.
도 5B는 상기 도 5A의 6개의 파지 클론 중 5-14 CVLKKPR)의 SKBR3 유방암 세 포주 또는 MDA-MB-231 유방암 세포주에서의 결합능을 플라크 에세이를 통해 평가한 결과를 나타낸다. 도 6은 positive selection 스크리닝 전략으로부터 선별된 7개의 파지 클론 의 MDA-MB231, SKBR3, 및 MCF7 세포에 대한 각각의 특이적 결합능을 플라크 에세이 를 통해 조사한 결과를 나타낸다. 도 7은 negative subtraction 스크리닝 및 positive selection 스크리닝 전 략을 통해 선별해 낸 네 가지 펩타이드 (CVRAKGKPC, CVLKKPR, CSLKKPR, CRPMRPR)의 MDA-MB231, MCF7, 및 SKBR3 세포에 대한 결합능을 형광현미경을 통해 조사한 결과 를 나타낸다. 도 8은 네 가지 펩타이드 (CVRAKGKPC, CVLKKPR, CSLKKPR, CRPMRPR)의 MDA- MB231, MCF7, 및 SKBR3 세포에 대한 결합능을 유세포분석기 (FACS)를 이용해 조사한 결과를 나타낸다. 도 9는 네 가지 펩타이드 (CVRAKGKPC, CVLKKPR, CSLKKPR, CRPMRPR)의 유방암 세포가 아닌 다른 종류의 암세포와 정상 폐， 콩팔 세포 (MDA-MB-231, MCF7, A549, BEAS-2B 및 HEK-293 세포)에 대한 결합능을 .유세포분석기 (FACS)를 이용해 조사한 결과를 나타낸다. 도 10은 MDA-MB-231 세포를 투여하여 만든 유방암 마우스 모델에 형광표지된 후보 펩타이드 (CVRAKGKPC, CVLKKPR, CRPMRPR)를 처리하고 종양을 적출한 후， 생체 광학영상시스템 (IVIS)을 이용해 형광 세기를 측정한 결과를 나타낸다. 도 11은' MDA-MB-231 세포를 투여하여 만든 유방암 마우스 모델에 형광표지된 후보 펩타이드 (CVRAKGKPC, CVLKKPR, CRPMRPR)를 처리한 다음， 적출해낸 종양과 간 을 얇게 단면화한 후， 형광현미경을 이용해 그 결합능을 검증한 결과를 나타낸다. FIG. 5B shows the ability of 5-14 CVLKKPR) of SKBR3 breast cancer cells or MDA-MB-231 breast cancer cell lines of the six phage clones of FIG. 5A to be evaluated by plaque assay. Results are shown. FIG. 6 shows the results of investigating the specific binding capacity of 7 phage clones selected from the positive selection screening strategy to MDA-MB231, SKBR3, and MCF7 cells by plaque assay. FIG. 7 shows the results of investigating the binding capacity of four peptides (CVRAKGKPC, CVLKKPR, CSLKKPR, CRPMRPR) to MDA-MB231, MCF7, and SKBR3 cells, which were selected through a negative subtraction screening and a positive selection screening strategy. Indicates. Figure 8 shows the results of investigating the binding capacity of four peptides (CVRAKGKPC, CVLKKPR, CSLKKPR, CRPMRPR) to MDA-MB231, MCF7, and SKBR3 cells using a flow cytometer (FACS). Figure 9 shows cancer cells of different types of non-mammary cancer cells and normal lung and soybean cells (MDA-MB-231, MCF7, A549, BEAS-2B and HEK-293 cells) of four peptides (CVRAKGKPC, CVLKKPR, CSLKKPR, CRPMRPR) The binding ability to is shown by the flow cytometer (FACS). 10 is treated with fluorescently labeled candidate peptides (CVRAKGKPC, CVLKKPR, CRPMRPR) in a breast cancer mouse model prepared by administering MDA-MB-231 cells, tumors were extracted, and fluorescence intensity was measured using a bio-optic imaging system (IVIS). The result of the measurement is shown. A 11 is treated with fluorescent-labeled candidate peptide (CVRAKGKPC, CVLKKPR, CRPMRPR) in breast cancer mouse models created by administering the 'MDA-MB-231 cells was then thin sectioned the excised tumor and liver came up, fluorescence microscopy The result of verifying the binding capacity is shown.
【발명의 실시를 위한 형태】 [Form for implementation of invention]
이하 본 발명을 상세히 설명한다.  Hereinafter, the present invention will be described in detail.
단， 하기 실시예는 본 발명을 예시하는 것일 뿐， 본 발명의 내용이 하기 실 시예에 한정되는 것은 아니다. <실시예 1〉 However, the following examples are merely to illustrate the present invention, the content of the present invention is not limited to the following embodiments. Example 1
삼중음성유방암세포를 특이적으로 표적하는 펩타이드의 발굴  Identification of Peptides Targeting Negative Triple Breast Cancer Cells
<1-1> Negat ive subtract ion통한 파지 라이브러리의 스크리닝 <1-1> Screening of phage library through Negat ive subtract ion
파지 스크리닝은 T7 파지 소수성 아미노산 (hydrophobic amino acid) 라이브 러리 (1 X 107 pfu/ul )를 이용하여 수행하였다. 본 라이브러리는 양말단에 시스테인 과 중간에 7 개의 무작위 아미노산으로 구성된 CXXXXXXXC (X = random sequence)의 아미노산 서열을 가지면서， 7 개의 무작위 아미노산 중 최소 한 개 이상에서 소수 성 아미노산을 갖는 것을 특징으로 하고 있다. 유방암세포주로는 MCF7(에스트로젠 수용체 /프로제스테론 수용체 양성， luminal type) , SKBR3(Her2 수용체 양성, Her2 type) , 및 MDA-MB23K에스트론젠 수용체 /프로제스테론 수용체 /Her2 수용체 삼중음 성, basal type)—를 이용하였다. Ngg t† e subTract ion 스크라닝의 "과정—은 도 1에서 간단하게 도시하였다. 즉， 삼중음성유방암세포가 아닌 MCF7 및 SKBR3 세포에 결합 하는 파지를 제거하는 negat ive subtract ion을 하고， 삼중음성유방암세포인 MDA- MB231 세포에 결합하는 파지를 회수하는 posi t ive select ion으로 진행하였다. 먼저Phage screening was performed using a T7 phage hydrophobic amino acid library (1 × 10 7 pfu / ul). This library has an amino acid sequence of CXXXXXXXC (X = random sequence) consisting of cysteine and 7 random amino acids in the sock end, and at least one of 7 random amino acids has a hydrophobic amino acid. . Breast cancer cell lines using MCF7 (estrogen receptor / progesterone receptor positive, luminal type), SKBR3 (Her2 receptor positive, Her2 type), and MDA-MB23K estronegen receptor / progesterone receptor / Her2 receptor triple negative, basal type) It was. The " process " of Ngg t † e subTract ion screening is shown briefly in Figure 1. That is, a negative subtract ion that removes phages that bind to MCF7 and SKBR3 cells rather than triple negative breast cancer cells, and triple negative breast cancer The phage was recovered with a posi- tive select ion that recovers phage that binds to the MDA-MB231 cells.
109 pfu의 T7 파지를 80만개의 MCF7 세포가 부착 되어 있는 35 mm culture di sh에 넣고 4°C에서 30분간 결합시켰다. 이후 MCF7 세포에 결합하지 않은 파지가 있는 상 층액을 회수하여 SKBR3 세포가 부착되어 있는 35 mm culture dish에 옮겨 4°C에서— 30분간 결합시켰다. SKBR3 세포에 결합하지 않는 파지가 있는 상층액을 회수하여 MDA-MB231 세포가 부착,되어 있는 di sh에 상기 회수한 파지를 넣고 4°C에서 한 시간 동안 결합시켰다. 이후 상층액올 제거하여 MDA-MB231 세포에 결합하지 않은 파지를 제거한 후， 세포층에 0.D 1.0 까지 배양한 BL21 호스트 대장균을 300ul 넣고 5분간 반응시켜 MDA-MB231 세포에 결합된 파지를 용출 (elut ion)시키고 회수하였다. 회수 된 파지 용출액 300ul 중 lOul을 이용해 플라크 어세이 (plaque assay)를 수행하여 그 수 (t i ter)를 계산하였다. 나머지 용출액은 호스트 대장균을 이용해 파지 증폭을 수행함으로써 그 다음 라운드에 사용할 파지를 확보하였다. 이와 같은 과정을 5 라 운드까지 반복하여 수행하였다. 그 결과， 각 라운드 별 회수된 파지의 수를 계산하여 도 2에 나타내었다. 라 운드가 진행함에 따라， 용출해 낸 파지의 수가가 점진적으로 증가함을 볼 수 있었 다. 1 라운드를 수행했을 때， 용출된 파지의 수는 약 2.2 X 10개였고, 최종적으로 10 9 pfu of T7 phage was put into 35 mm culture dish to which 800,000 MCF7 cells were attached, and bound for 30 minutes at 4 ° C. Subsequently, the supernatant with phage that did not bind to MCF7 cells was recovered and transferred to a 35 mm culture dish to which SKBR3 cells were attached and bound at 4 ° C for 30 minutes. The supernatant with phage that did not bind to SKBR3 cells was recovered, and the recovered phage was added to di sh to which MDA-MB231 cells were attached and bound for 4 hours at 4 ° C. The supernatant was then removed to remove phages that did not bind to MDA-MB231 cells. Then, 300 μl of BL21 host Escherichia coli cultured to 0.D 1.0 was added to the cell layer, followed by reaction for 5 minutes to elute phages bound to MDA-MB231 cells. ) And recovered. The plater assay was performed using lOul in 300ul of the recovered phage eluate to calculate the number of titers. The remaining eluate was subjected to phage amplification with host E. coli to secure phage for the next round. This process was repeated up to 5 rounds. As a result, the number of collected phages for each round was calculated and shown in FIG. 2. As the round progressed, the number of eluted phages gradually increased. All. In the first round, the number of phages eluted was about 2.2 X 10, and finally
5라운드를 진행한 이후 2.3 X 108개의 파지가 용출되었음을 알 수 있었다. 즉， 1라 운드에서 5라운드 까지 진행한 결과， 약 10.4배의 파지 수의 증가를 볼 수 있었다. 아미노산 서열분석을 위해, 3， 4, 5 라운드의 역가 측정에 쓰인 플라크 어세이의 결과물로부터， 각각 10, 20， 40개의 클론들을 각각 취합하여 lOul Tris-buffered saline 완층액에 보관하였으며， PCR을 통해 이들 파지에 삽입된 펩타이드를 코딩하 는 유전자의 염기서열 파악과 이에 따른 아미노산 서열분석을 수행하였다. After 5 rounds, 2.3 x 10 8 phages were eluted. In other words, from 1 round to 5 rounds, the number of phages increased by about 10.4 times. For amino acid sequencing, 10, 20 and 40 clones, respectively, were collected from the results of plaque assays used for 3, 4 and 5 rounds of titer measurements and stored in lOul Tris-buffered saline supernatant, and PCR was used. The nucleotide sequence of the gene encoding the peptide inserted into these phage and amino acid sequencing accordingly were performed.
<1-2> Positive selection을 통한 파지 라이브러리의 스크리닝 <1-2> Screening of phage library through positive selection
35醒 culture plate에 준비된 MDA-MB231 세포에 109 개의 T7 파지를 주입하 여 4°C 조건 하에서 한 시간 동안 세포와 결합시킨 후， 상충액을 제거한 다음， LB 배지에서 0.D 1.0 까지 배양한 BL21 호스트 대장균을 300ul 넣고 5분간 반응시킨 후 MDA-MB-231 세포와 결합한 파지를 용출해 내었다. 회수된 파지 용출액 300ul 중 lOul을 이용해 플라크 어세이 (plaque assay)를 수행하여 그 수 (titer)를 계산하였 다. 나머지 용출액은 호스트 대장균을 이용해 파지 증폭을 수행함으로써 그 다음 라운드에 사용할 파지를 확보하였다. 이와 같은 과정을 5 라운드까지 반복하여 수 행하였다. 그 결과， 각 라운드 별 회수된 파지의 수를 계산하여 도 3에 나타내었다. 각 라운드를 진행함에 따라 용출된 파지의 숫자가 증가하였으며 1라운드의 역가에 비 해 5라운드에서 약 360배의 용출 파지 수의 증가를 관찰할 수 있었다. 아미노산 서 열분석을 위해， 3， 4, 5 라운드의 역가 측정에 쓰인 플라크 어세이의 결과물로부 터， 각각 20， 30， 50개의 클론들을 각각 취합하여 lOul Tris-buffered saline 완층 액에 보관하였으며， PCR을 통해 이들 파지에 삽입된 펩타이드를 코딩하는 유전자의 염기서열 파악과 이에 따른 아미노산 서열분석을 수행하였다. 10 9 T7 phages were injected into the MDA-MB231 cells prepared on a 35 醒 culture plate and combined with the cells for 1 hour at 4 ° C. After removing the supernatant, the cells were cultured to 0.D 1.0 in LB medium. After adding 300 ul of BL21 host Escherichia coli and reacting for 5 minutes, phages bound to MDA-MB-231 cells were eluted. The titer was calculated by performing a plaque assay using lOul of the recovered phage eluate 300ul. The remaining eluate was subjected to phage amplification with host E. coli to secure phage for the next round. This process was repeated up to five rounds. As a result, the number of collected phages for each round was calculated and shown in FIG. 3. As each round proceeded, the number of eluted phages increased, and the number of eluted phages increased about 360 times in the fifth round compared to the titer of the first round. For amino acid sequencing, 20, 30, and 50 clones, respectively, were collected from the plaque assays used for 3, 4 and 5 round titers, and stored in lOul Tris-buffered saline complete fluid. PCR was performed to determine the nucleotide sequence of the gene encoding the peptides inserted into these phages and amino acid sequencing accordingly.
<1-3>파지 클론의 유전자 및 번역된 아미노산의 서열 분석 <1-3> Sequence analysis of phage clone gene and translated amino acid
실시예 1-1， 1-2에서 각각 수집한 70， 100개의 파지 클론에 삽입된 펩타이드 코딩 유전자의 염기서열 파악을 마크로젠 (주) 사에 의뢰하여 수행였다. 분석된 염 기서열을 그에 상응하는 아미노산 서열로 변환하였으며， 유사한 서열 또는 모티프 를 분석하기 위하여 Clustal X프로그램을 이용하여 서열을 정렬하였다. 그 결과， 도 4에서 보듯이， 염기성 성질흘 가진 아미노산인 아르기닌 (R) 또 는 라이신 (K)이 공통적으로 많이 분포되어 있는 것을 확인할 수 있었다. 펩타이드 의 아미노산 서열 상 6개 이상 잔기를 지니는 펩타이드를 다음 단계의 실험 후보로 선별하였다. The nucleotide sequence of the peptide coding gene inserted into 70, 100 phage clones collected in Examples 1-1 and 1-2 was requested by Macrogen Corporation. The analyzed base sequence was converted to the corresponding amino acid sequence, and the sequences were aligned using the Clustal X program to analyze similar sequences or motifs. As a result, as shown in Fig. 4, it was confirmed that arginine (R) or lysine (K), which is an amino acid having basic properties, is commonly distributed. Peptides having six or more residues on the amino acid sequence of the peptide were selected as experimental candidates for the next step.
<1-4> 파지 클론의 삼중음성유방암세포에 대한 선택적 결합능 평가 <1-4> Evaluation of selective binding capacity of phage clones against triple negative breast cancer cells
삼중음성유방암세포에 대한 선별된 파지의 결합력을 세포에 결합된 파지의 수를 측정함으로써 평가하였다. 이를 위해 세포가 5만개 배양된 96-wel l di sh에 6 개의 후보 파지 각각을 109 pfu만큼 넣고 각각 한 시간 동안 4°C에서 결합시킨 다 음, 결합한 파지를 용출하여 그 수를 계산하였다. 그 결과, 도 5에서 보듯이, negat ive subtract ion 스크리닝으로부터 선별해 낸 6개 후보군 중 3-6 및 5-14 파지 클론이 MDA-MB231세포에 대한 높은 결합을 보 였다. 또한 MDA-MB-231 cel l에 가장 높은 결합력을 보인 5-14 파지 클론은 MDA-MB- 231 세포와 비교하여 Skbr3 세포에 현저히 낮은 결합을 보였다. 또한 도 6에서 보듯이， posit ive select ion 스크리닝으로부터 선별해 낸 7개 의 후보군의 MDA-MB— 231， Skbr3 , MCF7 세포에 대한 결합을 조사한 결과， 7개의 후 보 모두 Skbr3 및 MCF7 세포와 비교해 MDA-MB-231 세포에 더 높은 결합력을 보였 다. 특히 4R-28CCRPMRPR)의 경우 MDA-MB-231 세포에 7개 후보 중 가장 높은 결합을 보였 며， 4R-12 CSLKKPR)는 MCF7 및 Skbr3 세포 대비 MDA-MB-231 세포에 상대적으 로 가장 높은 결합력을 보였다 (MCF7 에 비해 약 11배 , Skbr3 에 비해 약 38배) . 이 를 바탕으로 하여 CRPMRPR 및 CSLKKPR 펩타이드를 추가 실험 후보로서 택하였다. The binding capacity of the selected phage to triple negative breast cancer cells was evaluated by measuring the number of phages bound to the cells. To this end, each of six candidate phages was put in 10 9 pfu in 96-wel l di sh in which 50,000 cells were cultured, each was bound at 4 ° C for one hour, and the number of bound phages was eluted. As a result, as shown in FIG. 5, 3-6 and 5-14 phage clones among 6 candidate groups selected from negative subtract ion screening showed high binding to MDA-MB231 cells. In addition, the 5-14 phage clone with the highest binding capacity to MDA-MB-231 cel l showed significantly lower binding to Skbr3 cells compared to MDA-MB-231 cells. In addition, as shown in FIG. 6, MDA-MB—231, Skbr3, and MCF7 cells of seven candidate groups selected from positive select ion screening were examined. As a result, all seven candidates had MDA compared to Skbr3 and MCF7 cells. It showed higher binding to -MB-231 cells. In particular, 4R-28CCRPMRPR) showed the highest binding of MDA-MB-231 cells to 7 candidates, and 4R-12 CSLKKPR) showed the highest binding capacity to MDA-MB-231 cells compared to MCF7 and Skbr3 cells. 11 times compared to MCF7 and 38 times compared to Skbr3. Based on this, CRPMRPR and CSLKKPR peptides were selected as further experiment candidates.
<실시예 2> <Example 2>
펩타이드의 삼중음성유방암세포에 대하 선택적 결합능 평가 <2-1> 펩타이드의 합성  Evaluation of Selective Binding Capacity of Peptides to Triple Negative Breast Cancer Cells <2-1> Synthesis of Peptides
(주) 펩트론 (Peptron Co, Daegeon, Korea. )에 의뢰하여 카복시말단에 형광물 질인 f luorescein isothiocyanate(FITC)가 접합된 펩타이드를 합성하였다. 각 펩타 이드들은 표준 Fmoc 방법에 의하여 합성되었고 질량분석기에 의하여 정제되었다. <2-2>면역형광현미경법을 이용한 펩타이드의 삼증음성유방암세포에 대한결 합능 평가 Peptron Co., Ltd. (Peptron Co, Daegeon, Korea.) By the fluoro terminal synthesized a peptide conjugated with the fluorescent substance f luorescein isothiocyanate (FITC). Each peptide was synthesized by standard Fmoc methods and purified by mass spectrometry. <2-2> Evaluation of Binding Activity of Peptides on Tri-negative Negative Breast Cancer Cells Using Immunofluorescence
Negat ive subtract ion 스크리닝에서 선별한 CVRAKGKPC 및 CVLKKPR 펩타이드 와 posi t ive select ion 스크리닝에서 선별한 CSLKKPR 및 CRPMRPR 펩타이드의 삼중 음성유방암세포 특이적인 결합올 확인하기 위해 면역형광현미경법올 수행하였다. 이를 위해 삼중음성유방암세포인 MDA-MB-231 세포 및 비삼중음성유방암세포인 luminal A type와 MCF7 세포와 HER2 type의 Skbr3 세포를 4-wel l 배양용기에 5 X Immunofluorescence microscopy was performed to identify the triple negative breast cancer cell specific binding of CVRAKGKPC and CVLKKPR peptides selected by Negat ive subtract ion screening and CSLKKPR and CRPMRPR peptides selected by posi- tive select ion screening. To this end, MDA-MB-231 cells, which are triple negative breast cancer cells, and luminal A type, MCF7 cells, and HER2 type Skbr3 cells, which are non-triple negative breast cancer cells, are placed in a 4-wel l culture vessel.
104 cel ls/wel l 배양하고 1 ) 소혈청알부민이 포함된 세포배양액을 전처리한 후， 10 μ Μ의 펩타이드를 4'C에서 한 시간 동안 결합시켰다. 이후 핵을 카운터염색한 후 형광현미경을 통한 촬영 및 Nuance program (Perkin elmer사)을 이용한 분석을 하 였다. 그 결과， 도 7에서 보듯이， CVRAKGKPC, CVLKKPR, 및 CRPMRPR 펩타이드는 MCF7 및 Skbr3 세포와 비교해 MDA-MB-231 세포에 더 강한 결합 및 FITC 형광 신호 를 ί였다. 특히， 이들 중 CRPMRPR 펩타이드는 MDA-MB-231 세포에 대해 가장 높은 특이성을 보였다. 반면， CSLKKPR 펩타이드는 MDA-MB-231 세포 뿐만아니라 MCF7 및 Skbr3 세포에도 비교적 잘 결합하여 삼중음성유방암세포에 대한 특이성은 적었다. 한편， 대조군 펩타이드 (NSSSVDK)는 상기 세포들에 대한 결합이 상대적으로 약하였 다. 10 4 cel ls / wel l culture and 1) pre-treat the cell culture medium containing bovine serum albumin, 10 μΜ peptide was bound for 1 hour at 4'C. After nucleating the nuclei, the fluorescence microscope photographed and analyzed using a Nuance program (Perkin elmer). As a result, as shown in FIG. 7, CVRAKGKPC, CVLKKPR, and CRPMRPR peptides showed stronger binding and FITC fluorescence signals to MDA-MB-231 cells compared to MCF7 and Skbr3 cells. In particular, the CRPMRPR peptide showed the highest specificity for MDA-MB-231 cells. CSLKKPR peptides, on the other hand, bind relatively well to MDA-MB-231 cells as well as MCF7 and Skbr3 cells, resulting in less specificity for triple negative breast cancer cells. On the other hand, the control peptide (NSSSVDK) was relatively weak in binding to the cells.
<2-3>유세포분석기법을 이용한펩타이드의 삼중음성유방암세포에 대한 결합 능 평가 <2-3> Evaluation of Binding Peptides to Triple Negative Breast Cancer Cells Using Flow Cytometry
펩타이드 (CVRAKGKPC, CVLKKPR, CSLKKPR, CRPMRPR)의 삼증음성유방암세포에 대한 선택적 결합력을 유세포분석기법을 이용하여 평가하였다. 이를 위해， 세포를 각각 1.5mL 튜브에 3 X 105 eel ls/tube로 준비한 후， 1% 소혈청알부민이 포함된 세 포배양액으로 37°C에서 30분간 전처리를 거친 다음， FITC 형광이 표지된 펩타이드 와 4°C에서 한 시간 동안 반웅하였다. 반응이 끝난후 유세포분석기를 이용하여 세 포에 대한 펩타이드의 결합 분석하였다. 그 결과， 도 8에서 보듯이, 상기 4개의 펩타이드 모두 MCF7 및 Skbr3 세포와 비교해 MDA-MB231 세포에 더욱 잘 결합하고 이에 따라 MFI (Mean Fluorescence Intensity: 평균형광강도) 값이 더 높은 것을 알 수 있었다. 한편， 각 펩타이드의 선택성을 보다 더 다양한 세포에서 검증하기 위하여， 유방암 세포인 MDA-MB-231 및 MCF7 세포， 폐암세포주인 A549 세포， 정상 폐. 세포주 인 BEAS-2B 세포 및 정상 콩팔 세포주인 HEK-293 세포를 이용하여 유세포분석기법 을 실시하였다. 세포를 각각 1.5mL 튜브에 3 X 105 eel ls/tube로 준비한 후， 1% 소 혈청알부민이 포함된 세포배양액으로 37°C에서 30분간 전처리를 실시하였다. 그 다 음， FITC 형광표지된 펩타이드를 4°C에서 한 시간 동안 반응시킨 후, 유세포분석기 를 이용하여 펩타이드의 결합력을 분석하였다. 그 결과, 도 9에 나타난 바와 같이， CVRAKGKPC, CVLKKPR, CSLKKPR, CRPMRPR 펩타이드는.비삼중음성유방암세포인 MCF7 세포， 폐암세포인 A549 세포 및 정상세포 인 BEAS-2B 세포와 HKE-293 세포에 비하여 , 삼중음성유방암세포인 MDA— MB-231 세포 에서 펩타이드의 결합력 및 평균형광강도 (MFI)가 상대적으로 더 높은 것으로 나타 났다. 이를 통해， 이들 후보 펩타이드가 삼중음성유방암세포주에 특이적인 결합능 을 가지는 것을 확인할 수 있었다. Selective binding of peptides (CVRAKGKPC, CVLKKPR, CSLKKPR, CRPMRPR) to trinegative breast cancer cells was evaluated using flow cytometry. To this end, cells were prepared with 3 X 10 5 eel ls / tube in 1.5 mL tubes each, followed by pretreatment at 37 ° C for 30 min with cell culture medium containing 1% bovine serum albumin, and then labeled with FITC fluorescence. The reaction was allowed to react with the peptide at 4 ° C. for one hour. After the reaction, the binding of peptides to cells was analyzed using a flow cytometer. As a result, as shown in Figure 8, all four peptides bind to MDA-MB231 cells better than MCF7 and Skbr3 cells and accordingly MFI (Mean Fluorescence) Intensity was found to be higher. On the other hand, to verify the selectivity of each peptide in more diverse cells, breast cancer cells MDA-MB-231 and MCF7 cells, lung cancer cell lines A549 cells, normal lung. Flow cytometry was performed using BEAS-2B cells, HEK-293 cells, and normal soybean cells. Cells were prepared in 3 x 10 5 eel ls / tube in 1.5 mL tubes, respectively, and pretreated at 37 ° C. for 30 minutes with a cell culture solution containing 1% bovine serum albumin. Then, the FITC fluorescently labeled peptide was reacted at 4 ° C. for 1 hour, and then analyzed for binding ability of the peptide using a flow cytometer. As a result, as shown in Figure 9, CVRAKGKPC, CVLKKPR, CSLKKPR, CRPMRPR peptides are compared to MCF7 cells, non-tri-negative breast cancer cells, lung cancer cells A549 cells and normal cells BEAS-2B cells and HKE-293 cells, Peptide binding and mean fluorescence intensity (MFI) were relatively higher in MDA—MB-231 cells, triple negative breast cancer cells. Through this, it was confirmed that these candidate peptides have a specific binding capacity to triple negative breast cancer cell line.
<2-4>마우스 종양모델을 이용한 펩타이드의 삼증음성유방암에 대한 생체내 표적능 평가 <2-4> In vivo Targetability Evaluation of Peptides for Trinegative Negative Breast Cancer Using Mouse Tumor Model
세 가지 펩타이드 (CVRAKGKPC, CVLKKPR, CRPMRPR)의 삼중음성유방암세포에 대 한 생체내 표적능을 종양 마우스 모델을 이용하여 검증하였다.  In vivo targeting of three peptides (CVRAKGKPC, CVLKKPR, CRPMRPR) against triple negative breast cancer cells was verified using a tumor mouse model.
모든 동물 실험은 위원회 지침서 (institutional guidelines)에 따라， 그리고 경북대학교 IACUC의 가이드라인 (guideline of the Institutional Animal Care and Use Committee (IACUC) of Kyungpook National University)에 의해 승인받은 동물 실험 프로토콜 (permission No. KNU 2014-0001-121)에 따라 수행되었다. 8 주령 Balb/c 마우스를 Orient laboratoriesCSeongnam, Korea)로부터 구입하였고， 사료 및 물을 자유식이하며 SPF (specific—pathogen- free condition)에서 사육되었다.  All animal experiments have been approved in accordance with the Institutional Guidelines and approved by the Guidelines of the Institutional Animal Care and Use Committee (IACUC) of Kyungpook National University (Permission No. KNU). 2014-0001-121). Eight-week-old Balb / c mice were purchased from Orient laboratories (CSeongnam, Korea) and were fed in a feed- and water-free diet and in SPF (specific-pathogen-free conditions).
MDA-MB-231 세포를 마우스의 피부 아래 이식하여 그 종양을 성장시켰다. 이 후 FITC 형광표지된 대조군 펩타이드 NSSSVDK와 상기 세 가지의 펩타이드를 종양 마우스에 각각 정맥 주사하였다. 이 후 두 시간이 지난 다음 각 종양을 적출해 내 어 이를 생체광학영상시스템 (IVIS)을 이용해 R0I (Region of Interest)의 형광 세기 를 측정하였다. R0I 값이 높을수록 종양조직에 형광이 강하며， 이는 종양조직에 형 광표지 펩타이드가 많이 표적 및 '축적되었음을 나타낸다. 그 결과 도 10에 나타난 바와 같이 , CVRKGKPC 및 CRPMRPR 펩타이드晕 처리 한 종양세포는 음성대조군인 NSSSVDK 펩타이드를 처리한 종양세포에 비해 형광 값 의 수치가 더 높은 것으로 나타났다. 특히， CVRAKGKPC 펩타이드를 처리한 종양세포 에서 형광 값의 수치가 가장 높은 것으로 나타났다. 반면, CVLKKPR 펩타이드를 처 리한 종양세포는 대조군에 비해 형광 값이 낮은 것으로 나타났다. MDA-MB-231 cells were transplanted under the skin of mice to grow the tumors. Thereafter, FITC fluorescently labeled control peptide NSSSVDK and the three peptides were injected intravenously into tumor mice, respectively. Two hours later, each tumor was extracted, and the fluorescence intensity of R0I (Region of Interest) was measured using a bio-optical imaging system (IVIS). The higher the R0I value, the stronger the fluorescence in the tumor tissue. Many photolabeled peptides are targeted and ' accumulated. As a result, as shown in Fig. 10, the tumor cells treated with CVRKGKPC and CRPMRPR peptides 晕 showed higher fluorescence values than the tumor cells treated with NSSSVDK peptides, which were negative controls. In particular, tumor cells treated with CVRAKGKPC peptide showed the highest fluorescence values. On the other hand, tumor cells treated with the CVLKKPR peptide showed lower fluorescence values than the control group.
이를 통해， CVRAKGKPC 펩타이드가 유방암 마우스 모델에서 다른 펩타이드에 비해 상대적으로 더 높은 생체내 표적능을 가지는 것을 확인할 수 있었고， CVLKKPR 펩타이드는 생체내 표적능이 대조군 대비 우수하지 않은 것을 확인할 수 있었다. 한편， 적출한 종양조직의 동결절편을 제작하고, 형광 현미경을 통해 관찰하 였다. 또한 펩타이드의 조직 특이성을 검증하기 위하여 간조직의 형광도 관측하였 다. 그 결과 도 11에 나타난 바와 같이， 음성 대조군인 NSSSVDK 펩타이드를 정 맥 주사한 마우스의 종양조직에서는 형광이 상당히 낮은 것으로 나타났으며， CVRAKGKPC 펩타이드를 정맥 주사한 쥐의 종양 조직에서 다른 펩타이드에 비해 형광 이 가장 높은 것으로 관측되었다. 이는 생체형광영상시스템 ( IVIS)을 이용한 실험의 결과와 유사한 것을 확인할 수 있었다. 또한 CVRAKGKPC 펩타이드는 종양에서 관찰 된 형광 신호와는 달리 간 조직에서는 형광신호가 낮은 것으로 나타났다.  Through this, it was confirmed that the CVRAKGKPC peptide has a relatively higher in vivo targeting ability than other peptides in the breast cancer mouse model, and CVLKKPR peptide was confirmed that the in vivo targeting ability is not superior to the control. On the other hand, cryosections of the extracted tumor tissues were prepared and observed by fluorescence microscopy. In addition, the fluorescence of liver tissue was observed to verify the tissue specificity of the peptide. As a result, as shown in Figure 11, the tumor tissue of the mouse injected intravenously with the NSSSVDK peptide, a negative control, showed a very low fluorescence, compared with other peptides in the tumor tissue of the rat injected with CVRAKGKPC peptide intravenously. The highest was observed. This was confirmed to be similar to the results of the experiment using the biofluorescence imaging system (IVIS). In addition, the CVRAKGKPC peptide showed low fluorescence in liver tissues, unlike the fluorescence signal observed in tumors.
【산업상 이용가능성】 Industrial Applicability
본 발명의 펩타이드는 유방암에 특이적으로 결합할 수 있기 때문에， 유방암 의 진단, 치료 또는 영상화용 조성물의 제조에 유용하게 활용될 수 있어 산업상 이 용가능성이 매우 우수하다.  Since the peptide of the present invention can specifically bind to breast cancer, it can be usefully used for the production of a composition for diagnosing, treating or imaging breast cancer, and thus has excellent industrial applicability.

Claims

【청구의 범위】 [Range of request]
【청구항 1】  [Claim 1]
서열번호 1 내지 4로 이루어진 군에서 선택된 아미노산 서열을 포함하는 유 방암 표적 펩타이드.  Breast cancer target peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1 to 4.
【청구항 2】 [Claim 2]
제 1항에 있어서， 상기 유방암은 삼증 음성 (tr iple-negat ive) 유방암인 것을 특징으로 하는 펩타이드.  The peptide of claim 1, wherein the breast cancer is tr iple-negat ive breast cancer.
【청구항 3] [Claim 3]
제 1항의 펩타이드를 암호화하는 염기서열을 포함하는 폴리뉴클레오티드.  A polynucleotide comprising a nucleotide sequence encoding the peptide of claim 1.
【청구항 4】 [Claim 4]
제 3항의 폴리뉴클레오티드를 포함하는 백터.  A vector comprising the polynucleotide of claim 3.
【청구항 5】. 【Claim 5】 .
제 4항의 백터로 형질전환된 형질전환체.  A transformant transformed with the vector of claim 4.
【청구항 6】 [Claim 6]
제 1항의 템타이 및 이와 결합하는 약물을 유효성분으로 포함하는 유방암 예방 또는 치료용 약학적 조성물.  The pharmaceutical composition for preventing or treating breast cancer comprising the tem tai of claim 1 and a drug coupled thereto as an active ingredient.
【청구항 7] [Claim 7]
제 1항의 펩타이드를 포함하는 유방암에 특이적인 약물 전달용 조성물.  Composition for drug delivery specific to breast cancer comprising the peptide of claim 1.
【청구항 8】 [Claim 8]
겨 17항에 있어서, 상기 약물은 파클리탁셀， 독소루비신， 빈크리스틴， 다우노 루비신 (daunorubicin) , 빈블라스틴 (vinblast ine)， 액티노마이신 -D(act inomycin-D)， 도세탁셀 (docetaxel ) , 에토포사이드 (etoposide) , 테니포사이드 (teniposide)， 비산 트렌 (bisantrene) , 호모해링토닌 (homoharr ingtonine) , 글리백 (Gleevec ; STI-571) , 시스플라틴 (cisplain) , 5-플로오우라실 (5-f luouraci 1 )， 아드리아마이신 According to claim 17, wherein the drug is paclitaxel, doxorubicin, vincristine, daunorubicin, vinblast ine, actinomycin-D (act inomycin-D), docetaxel (docetaxel), eto Epoposide, teniposide, bisantrene, homoharringtonin, gleevec (STI-571), cisplain, 5-flouracil (5-f luouraci) 1) ， Adriamycin
(adriamycin) , 메토트렉세이트 (methotrexate)， 부설판 (busul fan)， 클로람부실 (chlorambucil), 시클로포스파미드 (cyclophosphamide), 멜팔란 (melphalan) , 니트로 겐 무스타드 (nitrogen mustard), 니트로소우레아 (nitrosourea)， 스트렙토키나제 (streptokinase), 유로키나제 (urokinase), 알테플라제 (alteplase)， 안지오텐신 (angiotensin) II 억제제, 알도스테론 (aldosterone) 수용체 억제제， 에리트로포이 에틴 (erythropoietin), NMDA(N-methyl-d-aspartate) 수용체 억제제, 로바스타틴 (Lovastatin), 라파마이신 (Rapamycin) , 셀레브렉스 (Celebrex)， 티클로핀 (Ticlopin) 마리마스타트 (Marimastat)및 트로케이드 (Trocade) 등으로 이루어진 군에서 선택된 어느 하나인 것을 특징으로 하는 조성물. (adriamycin), methotrexate, busul fan, chlorambucil (chlorambucil), cyclophosphamide, melphalan, nitrogen mustard, nitrosourea, streptokinase, urokinase, alteplase ), Angiotensin II inhibitors, aldosterone receptor inhibitors, erythropoietin, N-methyl-d-aspartate (NMDA) receptor inhibitors, lovastatin, rapamycin, Celebrex ), Ticlopin Marimastat (Marimastat), Trocade (Trocade) and the like, characterized in that any one selected from the group consisting of.
【청구항 9] [Claim 9]
제 1항의 펩타이드를 유효성분으로 포함하는 유방암 진단용 조성물.  A composition for diagnosing breast cancer comprising the peptide of claim 1 as an active ingredient.
【청구항 10] [Claim 10]
제 1항의 펩타이드를 포함하는 유방암 진단용 키트.  Claim 1 breast cancer diagnostic kit comprising the peptide.
【청구항 11】 [Claim 11]
제 1항의 펩타이드를 포함하는 유방암 진단용 마커 .  A marker for diagnosing breast cancer comprising the peptide of claim 1.
【청구항 12】 [Claim 12]
제 1항의 펩타이드를 유효성분으로 포함하는 유방암 영상화용 조성물.  A composition for imaging breast cancer comprising the peptide of claim 1 as an active ingredient.
【청구항 13] [Claim 13]
제 12항에 있어서， 상기 펩타이드는 발색효소， 방사성동위원소， 크로모포어 (chromophore) , 발광물질 , 형광물질 (f luorescer)， 상자성입자 (superparamagnetic particles) 및 초상자성입자 (ultrasuper paramagnetic particles)로 이루어진 군에 서 선택되는 하나로 표지된 것을 특징으로 하는 조성물.  The method of claim 12, wherein the peptide is a chromophore, a radioisotope, a chromophore (chromophore), a luminescent material, a fluorescent substance (f luorescer), superparamagnetic particles (superparamagnetic particles) and ultrasuper paramagnetic particles (ultrasuper paramagnetic particles) Compositions characterized in that labeled with one selected from the group.
【청구항 14】 [Claim 14]
유방암의 진단, 치료 또는 영상화용 제제를 제조하기 위한 제 1항의 펩타이드 의 용도.  Use of the peptide of claim 1 for the preparation of an agent for the diagnosis, treatment or imaging of breast cancer.
【청구항 15] 제 1항의 펩타이드의 유효량을 이를 필요로 하는 개체에 투여하는 것을 특징 으로 하는 유방암의 진단 방법 . [Claim 15] A method for diagnosing breast cancer, comprising administering an effective amount of the peptide of claim 1 to a subject in need thereof.
[청구항 16】 [Claim 16]
거 11항의 템타이드 및 이와 결합하는 약물의 유효량을 이를 필요로 하는 개 체에 투여하는 것을 특징으로 하는 유방암의 치료 방법 .  A method of treating breast cancer, comprising administering to the subject in need thereof an effective amount of the Temptide of claim 11 and the drug bound thereto.
【청구항 17】 [Claim 17]
제 16항에 있어서， 상기 약물은 파클리탁셀， 독소루비신， 빈크리스틴， 다우노 루비신 (daunorubicin), 빈블라 ^틴： ！ ^ ine)， 액티노마이신 -D(actinomycin-D)， 도세탁셀 (docetaxel), 에토포사이드 (etoposide)， 테니포사이드 (teniposide)， 비산 트렌 (bisantrene), 호모해링토닌^^삿^^！ 어 ^ )， 글리백 (Gleevec; STI-571), 시스플라틴 (cisplain), 5-플로오우라실 (5-f luouraci 1 )， 아드리아마이신 The method of claim 16, wherein the drug is paclitaxel, doxorubicin, vincristine, daunorubicin, vinbla ^ tin:！ ^ ine, actinomycin-D, docetaxel, Etoposide, teniposide, bisantrene, homoharingtonin ^^ サ ッ ^^！ U ^, Gleevec (STI-571), cisplain, 5-fluoro Urasyl (5-f luouraci 1), Adriamycin
(adriamycin), 메토트렉세이트 (methotrexate)， 부설판 (busulfan) , 클로람부실 (chlorambucil), 시클로포스파미드 ( cyclophosphamide )， 멜팔란 (melphalan) , 니트로 겐 무스타드 (nitrogen mustard), 니트로소우레아 (nitrosourea)， 스트랩토키나제 (streptokinase), 유로키나제 (urokinase)， 알테플라제 (alteplase)， 안지오텐신 (angiotensin) II 억제제， 알도스테론 (aldosterone) 수용체 억제제， 에리트로포이 에틴 (erythropoietin), NMDA (N-methyl—d— aspartate) 수용체 억제제， 로바스타틴 (Lovastatin), 라파마이신 (Rapamycin)， 셀레브렉스 (Celebrex)， 티클로핀 (Ticlopin) 마리마스타트 (Marimastat)및 트로케이드 (Trocade) 등으로 이루어진 군에서 선택된 어느 하나인 것을 특징으로 하는 방법. (adriamycin), methotrexate, busulfan, chlorambucil, cyclophosphamide, melphalan, nitrogen mustard, nitrosourea ), Straptokinase, urokinase, alteplase, angiotensin II inhibitor, aldosterone receptor inhibitor, erythropoietin, NMDA (N-methyl—d— aspartate receptor inhibitor, lovastatin, rapamycin, rapamycin, celebrex, ticlopin marimastat, trocade, etc. How to.
PCT/KR2017/007081 2016-07-04 2017-07-04 Peptide for targeting breast cancer and use thereof WO2018008942A1 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
KR10-2016-0084357 2016-07-04
KR20160084357 2016-07-04

Publications (1)

Publication Number Publication Date
WO2018008942A1 true WO2018008942A1 (en) 2018-01-11

Family

ID=60912919

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/KR2017/007081 WO2018008942A1 (en) 2016-07-04 2017-07-04 Peptide for targeting breast cancer and use thereof

Country Status (2)

Country Link
KR (1) KR101921212B1 (en)
WO (1) WO2018008942A1 (en)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110746489A (en) * 2019-10-21 2020-02-04 清华-伯克利深圳学院筹备办公室 Polypeptide for specifically targeting triple-negative breast cancer and application thereof

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20040102382A1 (en) * 2002-11-27 2004-05-27 Transgene, S.A. Targeting peptides
US20160130365A1 (en) * 2013-05-13 2016-05-12 Tufts University Methods and compositions for prognosis, diagnosis, and treatment of ADAM8-expressing cancer

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20040102382A1 (en) * 2002-11-27 2004-05-27 Transgene, S.A. Targeting peptides
US20160130365A1 (en) * 2013-05-13 2016-05-12 Tufts University Methods and compositions for prognosis, diagnosis, and treatment of ADAM8-expressing cancer

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
ABBINENI: "Evolutionary selection of new breast cancer cell-targeting peptides and phages with the cell-targeting peptides fully displayed on the major coat and their effects on actin dynamics during cell internalization", MOLECULAR PHARMACEUTICS, vol. 7, no. 5, 2010, pages 1629 - 1642, XP055601598 *
WU: "Advancement and applications of peptide phage display technology in biomedical science", JOURNAL OF BIOMEDICAL SCIENCE, vol. 23, no. 8, 19 January 2016 (2016-01-19), pages 1 - 14, XP055448548 *
XIAO: "Peptide-based treatment: a promising cancer therapy", JOURNAL OF IMMUNOLOGY RESEARCH, vol. 2015, 2015, pages 1 - 13, XP055601607 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110746489A (en) * 2019-10-21 2020-02-04 清华-伯克利深圳学院筹备办公室 Polypeptide for specifically targeting triple-negative breast cancer and application thereof
CN110746489B (en) * 2019-10-21 2021-09-10 清华-伯克利深圳学院筹备办公室 Polypeptide for specifically targeting triple-negative breast cancer and application thereof

Also Published As

Publication number Publication date
KR20180004681A (en) 2018-01-12
KR101921212B1 (en) 2018-11-22

Similar Documents

Publication Publication Date Title
US20190209715A1 (en) Plectin-1 Targeted Agents for Detection and Treatment of Pancreatic Ductal Adenocarcinoma
JP5966000B2 (en) Tumor targeting tumor necrosis factor-related apoptosis-inducing ligand mutant and its application
KR20140085569A (en) Therapeutic agents and uses thereof
US7968081B2 (en) Peptide for diagnosing, preventing and treating atherosclerosis and uses thereof
US8188051B2 (en) Metadherin polypeptides, encoding nucleic acids and methods of use
EP1756166A1 (en) Prostate cancer diagnosis and treatment
US20220041696A1 (en) Polypeptide targeting aptamers for characterization, capture, and clinical management of circulating tumor cells
TW201124725A (en) Bladder cancer specific ligand peptides
WO2010019884A1 (en) Methods and compositions for the detection of cancer
MX2012000086A (en) Soricidin derived peptides and methods for the detection of trpv-6 cancers and drug delivery.
KR20140123311A (en) Targeting Peptide for Cancer and Medical Use Thereof
EP4103583A1 (en) Tandem repeat cancer-targeting peptides for molecular conjugation or engineering and uses thereof in cancer theranostics
KR101921212B1 (en) Peptides for targeting breast cancer and uses thereof
JP2020501599A (en) FSH hormone receptor ligands in tumor diagnosis and therapy
US7745410B2 (en) Bladder tumor-targeting peptide and use thereof
US9581598B2 (en) Diagnosis and treatment of brain tumor
EP2459584A1 (en) Homing peptide for tumor vasculature
CA2367256A1 (en) Surface localized colligin/hsp47 in carcinoma cells
CN111153967A (en) Polypeptide specifically binding to HPV16E5 protein and application thereof
KR100837341B1 (en) Bladder tumor-targeting peptides and uses thereof
KR102194026B1 (en) Peptides that specifically bind to TRAIL receptor and use thereof
Zeng et al. Identification of a novel peptide targeting TIGIT to evaluate immunomodulation of 125I seed brachytherapy in HCC by near-infrared fluorescence
CA2394914A1 (en) A novel method of diagnosing, monitoring, staging, imaging and treating cancer
WO2007037555A9 (en) Therapeutic or diagnostic application of dusp15 gene
WO2007037550A9 (en) Therapeutic or diagnostic application of tsta3 gene

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 17824498

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 17824498

Country of ref document: EP

Kind code of ref document: A1