KR20120094173A - Peptides targeting the cd44 protein as a biomarker for breast cancer stem cell and uses thereof - Google Patents

Abstract

PURPOSE: A peptide for cd 44 protein target which is a specific marker for breast cancer stem cell and a cancer diagnostic kit using the same are provided to diagnosis for expression of breast cancer cells. CONSTITUTION: A peptide for cd 44 protein target which is a specific marker for breast cancer stem cell is composed of amino acid sequence consisting of the sequence number 1 [SEQ ID NO:1] or 2. The polynucleotide which codes the amino acid sequence of the CD44 target is composed of the sequence number 3 or 4. The cancer diagnostic kit comprises the peptide for CD44 target. The peptide for CD44 target is marked by a color forming enzyme, a radioactive isotope, a chromopore, a luminescent material or a fluorescent material.

Description

PEPTIDES TARGETING THE CD44 PROTEIN AS A BIOMARKER FOR BREAST CANCER STEM CELL AND USES THEREOF}

The present invention relates to a CD44 protein targeting peptide, which is a breast cancer stem cell specific marker, and a cancer diagnostic kit using the same. More specifically, the present invention relates to a small molecule peptide for a diagnostic probe that specifically binds to CD44 protein, a breast cancer stem cell biomarker, and a cancer diagnostic kit using the same.

Because the human body is exposed to numerous external chemicals and internal factors, many genetic variations accumulate in the body. Some of these mutations work in concert with each other, which alone affects the body's internal signal transduction factors, leading to dysfunction, and when these mutations maintain continuous replication, abnormal colonies form within the cell. This builds up and causes cancer.

Among them, breast cancer is the number one cancer cancer incidence in Korea and breast cancer mortality rate among women aged 25-49 is the number one in the world. It is also announced that by 2020 breast cancer deaths will exceed 3,000. In addition, the number of patients is increasing rapidly and the age of patients is getting younger.

The treatment of breast cancer aims at finding breast cancer at an early stage and curing it by active treatment. Despite these efforts, however, there is a lack of treatment for patients who relapse after breast cancer surgery or after chemotherapy and hormonal therapy.

The organs that make up the body have their own adult stem cells. These cells are essential for the regeneration and maintenance of organs when they are damaged. However, cancer stem cells, which serve as preserving cancer tissues, exist in cancer tissues as well as general organs, and this is known to have a profound effect on cancer recurrence or metastasis by being involved in regenerating cancer cells reduced after cancer treatment. Cancer stem cells are known to exist in tissues of solid tumors such as breast cancer and brain cancer as well as blood cancers such as leukemia.

 Recently, breast cancer stem cells have been reported to be closely related to the development of breast cancer because they have the same characteristics in many ways. In addition, the concept of tissue engineering using breast cancer stem cells has been introduced in tissue reconstruction after breast cancer surgery, and it is important to study the existence and characteristics of breast cancer stem cells in the treatment of breast cancer.

Therefore, R & D should focus on cancer stem cells that occupy only a small portion of cancer tissues and play a key role in the development, maintenance, and recurrence of cancer, rather than conventional cancer treatments that use only general cancer cells, which occupy most cancer tissues. .

Since the presence of cancer stem cells in leukemia in 1997 by Dr. John E. Dick, cancer stem cells need markers to distinguish them from other cancer cells. Breast cancer stem cells are known to specifically express CD44. Muhammad Al-Hajj et al. Confirmed the breast cancer stem cells by transplanting human breast cancer cells using NOD / SCID mice (Proctive identification of tumorigenic breast cancer cells. Proc Natl Acad Sci USA 2003; 100: 3983-3988.). Furthermore, they demonstrated that transplantation of about 100-200 cancer cells with CD44 + / CD24- into immunodeficient mice causes cancer, but about 104-105 cells without these marker factors do not cause cancer in immunodeficient mice. .

Cancer can also recur after surgery and treatment with anticancer drugs, and experts believe the cause may be in cancer stem cells in the body.

For this reason, a study on CD44 protein, a breast cancer stem cell specific marker, is necessary. CD44 is known to be associated with breast cancer and may be associated with head and neck cancer as well as other forms of cancer.

CD44 is known to be associated with breast cancer. And with CD44, more research is needed to support the recently popular hypothesis: small groups of cancer cells play an important role in cancer growth and progression, and cancer stem cells must be killed to treat cancer.

In the early stages of cancer, there are often no symptoms, and the symptoms are nonspecific, making it difficult to distinguish them from other diseases. Therefore, using the CD44 as a breast cancer marker and discovering a peptide probe that recognizes the CD44 effectively and specifically, the disadvantage of the antibody can be compensated for.

New probe peptides for CD44 targets can be applied to a variety of applications. Specifically, the new probe peptide for CD44 targeting can act as a prognostic factor for breast cancer and can be used as a drug delivery agent for delivering drugs that actually block cancer cells.

The combination of target-aware probes with new imaging techniques and anticancer agents offers the opportunity to improve the treatment of early detection of cancer and develop potential diagnostic methods. In order to develop a probe for the early detection of cancer, it is necessary to find a probe with high affinity and selectively attach it to early cancer cells and use it for early diagnosis of cancer.

There is also a lot of research going on in the field of genes. Based on the results of this genetic analysis, genetic modification studies are underway to prevent the function of cancer genes to be cut off or expressed.

According to the prior art, there is a method using polymerase chain reaction (PCR) as the most common method for diagnosing a diagnosis subject. This method is a method of analyzing a small amount of a diagnostic subject by analyzing a unique gene possessed by the diagnostic subject and then amplifying it in vitro. In addition, the method uses a unique nucleotide sequence of the gene to analyze the unique nucleotide sequence of the diagnosis subject only. Such PCR method can be extended by securing extensive genetic information database through recent human genome projects. In addition, the PCR method uses SSCP (Single-strand conformation polymorphism), RFLP (Restriction fragment length polymorphism) and AFLP (Amplified fragment length polymorphism) methods to check the genetic variation of the amplified genes with various probes It provides useful information in conjunction.

However, the diagnostic method using PCR has the advantage of having a high sensitivity in obtaining a small amount of the unique nucleotide sequence that only the diagnostic subject has, but has a problem such as difficulty in pretreatment and in situ adaptation of the sample to the diagnostic system. There is a limit to use.

Diagnosis and early detection of breast cancer include screening and palpation, also used for self-examination. Examination, palpation, x-ray imaging (imaging), and ultrasonography are the most basic diagnostics. However, more than 90% of breast marrows are benign tumors, such as fibroadenoma, common in unmarried women and are not cancerous. Therefore, more accurate screening and early detection of cancer should be made.

Cancer marker markers that can detect cancer and detect it early are being progressed through studies of antibodies that can identify early development of tumors. Blood is the most representative body fluid and is a kind of 'in vivo barometer' whose components change sensitively during disease development and progression. Plasma proteins have an estimated 50,000 or more components, and the concentration of each protein component is also very dynamic (1-10 ^-12 mg / mL), with a detection limit of about 10 ^-6 -10 ^-9 mg / mL. Antigen-antibody reactions make detection and quantitation of biomarker proteins present in low and medium concentrations very difficult. However, diagnostic kits with high sensitivity, rapidity and field adaptability, and diagnostic probes with high physicochemical stability, sensitivity, specificity, and applicability have not yet been proposed.

Since antibodies have a high specificity for cancer, many studies are being conducted for cancer detection and drug delivery, but there may be a problem of generating immune reactivity in vivo. In addition, antibodies may be denatured by heat or chemical treatment.

Recently, Enzyme-Linked Immunosorbent Assay (ELISA) is a method for determining the increase and change in the expression level of a specific protein caused by disease. In order to recognize the specific protein, an antibody that specifically binds to the specific protein is used as a diagnostic probe to isolate the diagnostic subject from a biological sample such as blood or urine, and an enzyme-antibody reaction (ECL) is used for this purpose. However, nanomolar (10 ^-9 M) level of sensitivity has a disadvantage in that it has a low sensitivity compared to PCR diagnostics, despite the advantage that it can target the protein in vivo.

As a part of related research that can overcome the above disadvantages, surface enhanced Raman spectroscopy (SERS) and bio-barcode technology combined with nanotechnology (NT) based on current diagnostic methods such as PCR and ELISA methods As developed, analysis of diagnostic subjects at femtomol ( ^10-15 M) levels has proven experimentally feasible. Both of these methods are based on the signal characteristics of Raman spectroscopy (narrow Raman bands; low sensitivity to moisture, oxygen and soakers; and low photobleaching) and high sensitivity corresponding to fluorescence analysis. The amplification was performed to recognize femtomol level of a diagnosis subject, and an approach for prostate specific antigen (PSA) using easy sample such as blood was proposed. However, these methods are based on systems using antibodies that recognize biomarkers (diagnostic subjects), all of which are limited in number of bindings due to size limitations due to the physical and chemical structures of the antibodies, as well as various chemical reactions. There is a significant disadvantage of showing instability due to structural denaturation.

Therefore, there is a need for the development of new low molecular weight diagnostic probes differentiated from the above-mentioned antibody diagnostic system technology. The low molecular peptides can be used in such a low molecular diagnostic system.

Accordingly, the present inventors have developed a novel peptide as a new diagnostic substance that can replace the antibody currently used for cancer diagnosis. Specifically, we have developed a peptide that recognizes CD44 protein, a breast cancer stem cell specific biomarker that can be used to increase the chance of early detection of breast cancer.

It is an object of the present invention to provide a novel peptide that recognizes and specifically binds CD44 protein, a biomarker of breast cancer stem cells, for early diagnosis, prevention and treatment of breast cancer.

Another object of the present invention is to provide a CD44 protein targeting peptide consisting of the amino acid sequence consisting of SEQ ID NO: 1 or 2 and a polynucleotide encoding the amino acid sequence.

Another object of the present invention is to provide a cancer diagnostic kit comprising the peptide.

The present invention provides a peptide for targeting a CD44 protein which is a breast cancer stem cell specific marker consisting of an amino acid sequence consisting of SEQ ID NO: 1 or 2.

The CD44 protein is a protein well known in the art. Specifically, it may be made of a gene having a nucleotide sequence of SEQ ID NO: 5, but is not necessarily limited thereto.

In the present invention, in order to overcome the disadvantages of the above-mentioned antibody, a small molecule peptide is used as a target substance. These small molecule peptides have the advantage of being small in size and stabilized in three dimensions, easily passing through mucous membranes, and recognizing molecular targets deep in tissues. In addition, the small molecule peptide according to the present invention has the advantage of early diagnosis of cancer because stability can be secured through local injection and minimize the immune response. In addition, the low molecular weight peptides according to the present invention are relatively simple in mass production and weak in toxicity.

In addition, the low-molecular peptides according to the present invention have an advantage of having a higher binding strength to the target material than the antibodies, and do not cause denaturation even during thermal / chemical treatment. In addition, the small size of the molecule can be used as a fusion protein by attaching to other proteins. Specifically, since it can be used by attaching to a polymer protein chain, it is used as a diagnostic kit and drug delivery material.

The CD44 target peptide is characterized by binding to a specific site of the CD44 protein, consisting of 12 amino acids. This amino acid is a peptide having a high affinity for the CD44 protein and includes the amino acid sequence set forth in SEQ ID NO: 1 or 2.

Peptides of the invention can be prepared by chemical synthesis known in the art. Representative methods include, but are not limited to, liquid or solid phase synthesis, fragment condensation, F-MOC or T-BOC chemistry.

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, such as using automated DNA synthesizers (such as those sold by Biosearch or AppliedBiosystems). The constructed DNA sequence is a vector comprising one or more expression control sequences (e.g., promoters, enhancers, etc.) that are operatively linked to this DNA sequence to 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 so that the DNA sequences are 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 that the peptide according to the invention is substantially free of any other protein derived from the host. 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, 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; And Hitzeman et al., J. Biol. Chem., 255: 12073-12080, 1990.

In order to easily identify, detect, and quantify whether the peptide of the present invention has bound to cancer cell 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 is a chromophore (e.g. peroxidase, alkaline phosphatase), radioisotope (e.g. 124I, 125I, 111In, 99mTc, 32P, 35S), chromophore , Luminescent or fluorescent materials (e.g., FITC, RITC, rhodamine, Texas Red, fluorescein, phycoerythrin, quantum dots) .

Similarly, the detectable label can be an antibody epitope, substrate, cofactor, inhibitor or affinity ligand. Such labeling may be carried out in the course of synthesizing the peptide of the present invention, or may be performed in addition to the peptide already synthesized.

If fluorescent material is used as a detectable label, 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 cancer.

On the other hand, the present invention provides a polynucleotide encoding an amino acid sequence consisting of SEQ ID NO: 1 or 2.

The polynucleotide may be obtained as described above, preferably, bases of SEQ ID NO: 3 (base sequence corresponding to the amino acid of SEQ ID NO: 1) or 4 (base sequence corresponding to the amino acid of SEQ ID NO: 2), respectively May have a sequence.

Expression vectors refer to plasmids, viral vectors or other mediators known in the art capable of expressing inserted nucleic acids in a host cell, encoding the peptides of the invention in conventional expression vectors known in the art. The polynucleotides may be operably linked. The expression vector is generally operatively linked with a replication origin capable of proliferating in a host cell, one or more expression control sequences (eg, promoters, enhancers, etc.), selective markers, and expression control sequences that control expression. 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, Transduction, cell fusion, calcium phosphate precipitation, liposome-mediated transfection, DEAE dextran-mediated transfection, polybrene-mediated transfection ( It can be obtained by introducing into host cells by polybrene-mediated transfection, electroporation, gene gun and other known methods for introducing nucleic acids into cells (Wu et al., J Bio.Chem., 267: 963-967, 1992; Wu and Wu, J. Bio. Chem., 263: 14621-14624, 1988).

Host cells that can be used in the present invention may be selected from the group consisting of E. coli, yeast, heart cells, lymphocytes, liver cells, vascular endothelial cells, spleen cells, cancer cells, animal cell lines such as CHO, Cos7, NIH3T3, insect cells, Not limited to this, all possible cells can be used as host cells.

The present invention also provides an antibody specific for the CD44 protein targeting peptide. In the present invention, "antibody" refers to a protein molecule specific to the antigenic site. For the purposes of the present invention, an antibody refers to an antibody that specifically recognizes the CD44 protein targeting peptide, and includes both polyclonal and monoclonal antibodies. As described above, since the peptides specifically binding to the CD44 protein have been identified by the present invention, the production of antibodies using the same can be easily prepared using techniques well known in the art (Sambrook et al. , Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, NY, Second (1998) and Third (2000) Edition).

Monoclonal antibodies are known in the art by fusion methods (see Kohler and Milstein (1976) European Jounral of Immunology, 6: 511-519), recombinant DNA methods or phage antibody libraries (Clackson et al., Nature, 352: 624-628, 1991; Marks et al., J. Mol. Biol., 222: 58, 1-597, 1991).

Polyclonal antibodies can be produced by methods well known in the art for injecting a CD44 protein targeting peptide having the amino acid sequence of SEQ ID NO: 1 or 2 into an animal and collecting blood from the animal to obtain serum comprising the antibody. For example, the peptide may be prepared by injecting a goat or rabbit with CFA (Complete Freund's Adjuvant) by subcutaneous injection, and by booster injection with CFA subcutaneously or intraperitoneally. Such polyclonal antibodies can be prepared from any animal species host, such as sheep, monkeys, horses, pigs, bovine dogs, in addition to goat rabbits.

The antibody is preferably labeled with a marker, such as a radioactive or fluorescent marker. Antibodies can be indirectly labeled by binding to anti-goat or anti-rabbit antibodies covalently bound to the marker compound.

The present invention also provides a kit for diagnosing cancer comprising the peptide of the present invention. The peptide included in the kit may have a peptide of SEQ ID NO: 1 to 2 or a polynucleotide encoding SEQ ID NO: 3 to 4, and may be obtained by chemical or genetic engineering methods as described above. The diagnostic kit of the present invention may further include a buffer or a reaction solution that stably maintains the structure or physiological activity of the peptide. In addition, to maintain stability, it may be provided in a powder state or dissolved in a suitable buffer or maintained at 4 ° C.

At this time, "diagnosis" in the present invention means confirming the presence or characteristics of the pathological state. For the purposes of the present invention, the diagnosis is to identify the presence or characteristics of cancer.

In addition, in order to facilitate identification, detection and quantification of the peptide of the present invention bound to cancer cell tissue, the peptide of the present invention may be provided in a labeled state as described above.

 On the other hand, when the peptide of the present invention is provided unlabeled, the kit for diagnosing cancer of the present invention may be used to search for a cancer-producing site, in particular, whether to bind to a cancer cell or its location in vitro or in vivo of the peptide of the present invention. It may further comprise a component. Said component is a known compound for the labeling of the peptide of the invention, or an antibody against a peptide of the invention or a specific receptor which binds to the peptide of the invention or a secondary antibody against them for detection through an antigen-antibody reaction and It may be a reagent for its detection.

In addition, the peptide of the present invention may be provided in a form coated on the surface of the plate. In this case, after inoculating the target cells directly on the plate and reacted under appropriate conditions, cancer can be diagnosed by observing the binding of the peptide to the cancer cells on the surface of the plate.

Experimental procedures, reagents and reaction conditions that can be used in the above methods can utilize those conventionally known in the art, which is obvious to those skilled in the art.

Since the peptide of the present invention specifically binds to CD44 protein, which is a breast cancer stem cell specific marker, breast cancer can be diagnosed by checking the excessive activity of CD44 protein using the peptide of the present invention as a diagnostic probe. When used in the treatment of cancer by connecting drugs such as peptides and anticancer drugs of the present invention, the anticancer drugs are selectively delivered to cancer tissues to increase the effect of the drug and there are almost no side effects such as immune response or denaturation reaction.

1 is a PCR using a CD44 protein sequence according to an embodiment of the present invention to secure a gene (a), and inserts the obtained CD44 (ED1-CD44) gene into the recombinant expression vector of pGEX-4T-1 (B) shows the insertion of ED1-CD44 after treatment with restriction enzymes (BamHI, XhoI): P of (a) represents CD44 gene and P of (b) represents CD44 to expression vector pGEX-4T-1. In order to confirm this by inserting the gene, it was confirmed that the bands were identified at 5.0 and 1.8 Kb corresponding to the vector (V) and CD44 (I) sizes after restriction enzyme treatment, and M is a molecular weight marker.
Figure 2 shows the results of production, induction, isolation and purification of the CD44 protein cloned in the recombinant expression vector according to an embodiment of the present invention: M is the molecular weight marker; BI is before induction; After AI induction; S1 is a soluble protein; S2 is cultured and ground cells by sonication; FT is Flowthrough; F1-7 is a fraction containing the CD44 protein.
Figure 3 is an electrophoresis result indicating that GST-free CD44 protein was obtained before phage display: M is molecular weight marker; C is purified after Mono-Q after thrombin treatment; And P is GST-free protein CD44.
4 is a schematic of M13 phage screening for screening peptides that bind CD44.
FIG. 5 is a chart summarizing four-step biopanning conditions for detecting phage expressing peptides having high specificity and binding ability for CD44.
Figure 6 is a graph of amplifying peptide expression phage of the present invention and then the concentration was measured to analyze the binding force with CD44.
Figure 7 summarizes the binding peptide sequence and binding force of the peptide specifically binding to CD44 according to an embodiment of the present invention.
FIG. 8 shows the results of fluorescence microscopy confirming the binding ability of the synthesized FITC-labeled peptide (P7) with CD44 in the actual CD44 MCF7 cell system: (A) is a photograph without peptide in the normal cell line (MCF7). ; (B) is a fluorescence picture looked after the peptide according to the present invention in MCF7; (C) is a result of binding to the peptide after radiation treatment (3 times in 2gray) to express CD44. The binding of the fluorescent peptide was analyzed by fluorescence microscopy and Fluorescence Image Station 2000R (Olympus Ix71).
9 is a result obtained by performing FACS analysis to confirm that the peptide of the present invention specifically binds to normal MCF-7 cells and radiation treated CD44 MCF-7 cells. The red line shows that the graph is sheeted, indicating that the peptide selectively binds more with CD44 due to the increase in strength. Black line is normal MCF7 cell line.
10 is a result of Western blot using immunoprecipitation (IP) to confirm the specificity of the biotin-labeled peptide (P7) according to the present invention.
11 is a result of FACS analysis for comparing the binding capacity of the FITC-labeled peptide (P7) and the existing antibody (PE) to CD44 according to the present invention: (a) is a result taken by binding the CD44-PE antibody (b ) Is the result of the peptide (P7) of the present invention.
12 is an immunostaining photograph for showing the specific binding capacity of the FITC-labeled peptide (P7) and the existing antibody (PE) to CD44 according to the present invention.

Hereinafter, preferred examples are provided to aid the understanding of the present invention, but the following examples are merely illustrative of the present invention, and various changes and modifications can be made within the scope and spirit of the present invention. It is obvious to the skilled person, and it is natural that such variations and modifications fall within the scope of the appended claims.

Example  One: CD44  Mass production and separation of proteins

First, CD44 protein was expressed on E. coli and for mass production and isolation, CD44 gene (SEQ ID NO: 5) was amplified by PCR as follows: CD44 sense primer was 5'-AA GAA TTC (EcoRI) ATG GAC AAG TTT TGG TGG CAC GCA-3 ′ and CD44 antisense primer 5′-AA CTC GAG (XhoI) TTA TTC TGG AAT TTG GGG TGT CCT-3 ′. In a reaction solution containing 5% DMSO containing the synthetic primer and template DNA, pre-denaturation at 95 ° C. for 1 minute, denaturation at 94 ° C. for 30 seconds, annealing at 70 ° C. 45 The template DNA obtained after 20 cycles of PCR reaction was confirmed through the gene sequence by using 1 second at 72 ° C for 2 minutes and 45 seconds at extension (see FIG. 1). The amplified gene was cut with each restriction enzyme (EcoRI, XhoI), inserted into the bacterial expression vector pGEX-4T-1 (Promega, USA), cloned according to the manufacturer's manual, and the expression vector was expressed as the expression host protein. After transformation to E. coli BL21 (DE3), production of CD44 protein was induced for 12 hours under conditions of 0.5 mM IPTG, 18 ° C. The transformation method is briefly described as follows: First, 5 μl of DNA was added to 100 μl of competitive cells, mixed well, and left on ice for 30 minutes. Then heat shock was applied at 42 ° C. for 90 seconds. In order to prevent contamination, the alcohol lamp was first turned on, and then 800 µl of LB medium was added and incubated at 37 ° C for 45 minutes. Finally it was applied to the LBA plate and incubated overnight in a 37 ° C. incubator.

The bacterial cells were harvested by centrifugation to examine the solubility of the proteins. As a result, most of the proteins were dissolved. Therefore, water-soluble protein was obtained for the separation and purification of CD44, a GST fusion protein, which was then passed through a GSH-Sepharose column to bind the CD44 protein, and then eluted under 20 mM reduced form of glutathione (GSH) linear gradient. I was.

The obtained protein was removed by using a desalting column (Amersham Biosciences, USA), and the purified degree of the obtained protein was confirmed through SDS / PAGE (FIG. 2). Next, in order to proceed with the screening process to find a peptide that binds only to the CD44 protein, the GST-tag having a large molecular weight (26KDa) must be removed to find a peptide that specifically binds to the CD44 protein. To this end, GST-tags were cut using thrombin (5 units of thrombin / mg of protein). When thrombin treated, the GST fusion CD44 protein is divided into two fragments, GST-tag (26KDa) and CD44 (KDa), so that the same result is obtained by treating the purified CD44 protein separated by the above method with thrombin. It was. Thrombin treatment resulted in two protein fragments, GST-tag and CD44. 1 mM benzamidine was added to terminate the reaction. GST-free protein was obtained with a 0.5 M NaCl linear gradient via anion exchange chromatography (Mono Q HR 5/5 column) to remove the GST-tag and obtain only pure GST-free protein (FIG. 3).

Example  2: M13  scrap paper Peptides  Library Screening-Phage Display

In order to fix the protein obtained in Example 1 to a 96-well plate, a polystyrene plate (SPR) having a polystyrene surface was used. By fixing the protein to the bottom of the plate surface using a hydrophilic interaction between the protein and the plate surface, a random peptide library (designed to have about 2.7 billion different amino acid sequences through a random sequence of 12 amino acids) In the screening process using peptide library-Ph.DTM (purchased using phage display peptide library kit, New England Biolabs (NEB)), it was designed to enable higher specificity and binding analysis.

Specifically, in Figure 4, in order to screen peptides that bind to the breast cancer stem cell specific marker CD44, after binding CD44 to the surface of the plate (SPL), M13 phage peptide library (12 with about 2.7 billion different amino acid sequences Peptide-expressing phage that binds with high affinity through various binding times and washing conditions after binding of amino acid peptides into a library fused to M13 phage gp3 minor coat protein) It was. Screening schemes and reaction conditions in this regard are shown in FIGS. 4 and 5, respectively.

Specifically, FIG. 4 is a schematic diagram of M13 phage screening for peptide screening with CD44, comprising: (1) immobilizing the CD44 protein on the surface of a 96-well plate, and (2) consisting of 12 amino acids on the M13 phage surface. After binding the phage library expressing the peptide library with the CD44 protein, (3) it was washed under various conditions, and (4) finally bound phage was eluted to obtain phage. The phage obtained as described above is infected with E. coli, amplified, and then bound to CD44 protein, and then repeated under conditions such as higher washing conditions and shorter reaction times, thereby repeating the process of finding phages with stronger binding force. (Biopanning).

In addition, Figure 5 is a diagram of the four-step biopanning conditions for detecting phage expressing peptides with high specificity and binding capacity for the CD44 protein. In each step, the reaction conditions were variously changed by strong washing conditions and short reaction times. Thus, four biopannings were performed each to obtain phages having peptides that bind to the CD44 protein.

The phage obtained above was infected with Escherichia coli ER2738 cells, which are host cells, and amplified in LB medium. Then, about 45 phage plaques were selected to isolate and purify the M13 phage genomic DNA (single stranded circular DNA). As a result, the amino acid sequence of the peptide portion expressed on the phage surface protein (gp3 minor coat protein) and manufactured to bind CD44 was identified. The results are shown in Table 1 below.

Peptide number Peptide sequence P 1 HPLCTSHAPVPP P 2 FPTRFKKQWSLP P 3 NAGDNKFVRVSP P 4 SVSVGMKPSPRP P 5 WHKSEQGPRLYR P 6 FSRPPARHQPHQ P 7 FNLPLPSRPLLR

As a result of amino acid homology analysis of the peptides of P 1 to P 7, high similarity can be observed in the sequences of peptides belonging to the same group. The results suggest that these amino acids are residues that play an important role in the binding of the CD44 protein to the peptide.

Example  3: CD44 Combined with Peptide  Binding force analysis

For binding analysis of CD44 target specific peptides obtained by screening, amplify each phage expressing phage, determine the concentration of phage solution through titering and put it into the CD44 bound plate by concentration The binding capacity of the peptide was inferred by measuring the amount of bound phage.

Phage was put on the CD44 bound plate and washed to carry out ELISA (Biotrak multiwell plate reader, Amersham Bioscience, USA) using an antibody that recognizes phage surface protein. The ELISA signal intensity according to the concentration was indicated and the apparent Kd value was determined according to the saturation curve. Kd values were obtained using known methods (Kim JM et. Al, (2006) Journal of Microbiolgy and Biotechnology 16, 1784-1790.). Specifically, the CD44 protein (0.5 g / well) was fixed on a polystyrene plate (SPL), and then amplified phage was added and fixed for 1 hour. After washing 5 times with 1 X TBST buffer, anti-M13 antibody (mouse monoclonal antibody, diluted 1: 5000 in TBST, Amersham Bioscience) was attached for 1 hour. The antibody-labeled protein was then washed 10 times and the secondary antibody bound (anti-mouse IgG-HRP, diluted 1: 4000 in TBST, Santascruz) and then TMB substrate solution (3,3 '5,5). 15 minutes after the addition of '-tetramethylbenzidine (TMB) / H2O2, Chemicon), 1M sulfuric acid was added to terminate the reaction, and the saturation curve was determined using the value obtained at 450 nm, and the Kd value was determined. As shown in Fig. 7, all seven peptide groups showed binding capacity in the range of picomolar concentration (pM). In addition, P 6 (corresponding to P 6, SEQ ID NO: 1) and P 7 (corresponding to P 7, SEQ ID NO: 2) of Table 1 and FIG. 7 show excellent binding ability of 10 pM or less, thereby reducing the existing antibody-antigen reaction. The binding force corresponding to the binding force of was confirmed.

Example  4: synthetic Peptides and CD44 To verify that the in vivo  Test

The binding force was measured by attaching a fluorescent probe (FITC) and biotin directly to the peptide of SEQ ID NO: 2 and binding to CD44. Peptide binding capacity measurement using FITC and biotin used a method commonly used in the art.

SEQ ID NO: 2 (P 7) -FITC: Ac-FNLPLPSRPLLRGCGK-FITC

SEQ ID NO: 2 (P 7) -Biotin: Ac-FNLPLPSRPLLR-Biotin

Peptide SEQ ID NO 2-FITC was expected to diversify and increase the detection signal by binding the FITC fluorescent probe to the carboxy terminus, and put a Cys group to bind to the activated pLys (SPDP coupled pLYS).

In addition, SEQ ID NO: 2-Biotin by binding a biotin probe to the carboxy terminal to synthesize a peptide of commonly used biotin-avidin bonds as well as fluorescent probes. All peptides were prepared by acetylating the N-terminus (Ac) to remove the amino terminus reactivity.

First, in order to analyze the binding affinity of the FITC-bound peptide, each SEQ ID No. 2 peptide was put in CD44-bound plate by concentration, and the fluorescence intensity emitted from the fluorescent probe (FITC) of the bound peptide was measured. As a result, the binding force of the peptide was determined. The peptide was placed on a CD44 bound plate and washed to determine the maximum wavelength of the FITC fluorescent probe connected to the peptide of SEQ ID NO: 2 (Extinction 495 nm, Emission 510 nm), and fluorescence intensity measurement was performed using the fluorescence photometer. The signal intensity of the FITC fluorescence probe according to the concentration was indicated and the Kd value of the peptide was determined according to the saturation curve.

Then, for analysis of the binding capacity of the peptide of SEQ ID NO: 2 to which the biotin is bound, the peptide is put in a CD44 bound plate by concentration, and diluted with avidin (streptavidn-HRP, TBST 1: 4000 in TBST, BD) through the biotin of the bound peptide. After binding using antibody, TMB substrate solution (3,3 '5,5'-tetramethylbenzidine (TMB) / H2O2, Chemicon) was added 15 minutes later, 1M sulfuric acid was terminated, and measured at 450 nm. The saturation curve was drawn with the obtained value, and Kd value was determined.

As a result, as shown in Table 2, the synthesized FITC- or biotin labeling-peptide shows a binding capacity in the nanomolar concentration (nM) range.

Peptides Dissociation Constant (Kd) Ac-FNLPLPSRPLLRGCGK-FITC 115.8 ± 11.0 nM Ac-FNLPLPSRPLLR-Biotin 256 ± 17.5 nM

The human breast cancer stem cell line MCF-7 was used to see in vivo the binding ability of the peptide of the present invention (SEQ ID NO: 2) with CD44 in real animal cell lines. Cell culture was performed using human breast cancer stem cell line MCF-7 (Korea Cell Line Bank, No. 30022) with DMEM (Dulbecco's modified Eagle's medium) / F12 supplemented with 10% fat bovine serum (FBS) and 1% penicillin / streptomycin. (Gibco, invitrogen Corp, San Diego, Calif., USA) was used to culture using a wet incubator maintained at 37 ℃, 5% CO2.

To confirm peptide binding between normal cell line MCF-7 and breast cancer cell line CD44 MCF-7, FACS (Fluorescence activated cell sorter, FACSCalibur instrument, Becton Kickinson, Erembodegem, Belgium) analysis was performed using a fluorescence microscope and MitofluorTM. Radiation of the normal cell line MCF-7 to induce the expression of CD44, the peptide of the present invention is specific for CD44 through the difference in the expression of CD44 in normal cells and breast cancer cells by binding the peptide of SEQ ID NO: 2 with FITC Binding was confirmed in vivo (FIGS. 8 and 9). To this end, cells sufficiently cultured to trypsin treatment were isolated, washed with 1XPBS (phosphate buffer saline), and then the cells were divided, and each peptide was put in a peptide of SEQ ID No. 2 (FITC-labeled peptide) to react. After binding, the cells were washed three times, and the average fluorescence intensity was calculated using a flow cytometer (FIG. 9). The results show that the higher the amount of CD44, the more peptides bound and the stronger the fluorescence intensity, the more the graph shifts in change in intensity intensity. This is a result showing that the peptide specifically binds to CD44.

In addition, FITC-labeled peptide P7 (400 nM) was reacted with CD44 cell line MCF-7 for fluorescence microscopy, and then treated with 0.25% trypsin to grow on a 24-well chamber slide for 12 hours. Then, after washing three times with 1XPBS was attached to the cover slip on the slide glass fluorescence microscope (Olympus, Melvile, NY) observation was performed (Fig. 8).

Lysis buffer (40 mM Tris-HCl, pH8.0, 120 mM NaCl, 0.1% NP-4, proteinase inhibitor) was cultured in the cultured MCF-7 cell line for immunoprecipitation (IP) and western blot. After extracting the cell lysate using 400 nM of the peptide (P7) of the biotin-bound SEQ ID NO: 2 was shaken at 4 ℃ for 24 hours to immunoprecipitate. Streptavidin-conjugated agarose beads were added to immunoprecipitated CD44-peptide complexes, bound at 4 ° C. for 30 minutes, precipitated by centrifugation, and washed with cell lysis buffer. After washing, SDS sample buffer was added to boil and SDS-PAGE gel electrophoresis was performed. After transfer of the proteins separated by electrophoresis to the nitrocellulose membrane, HCAM (H-300) (Santa Cruz Biotechnology, INC.) As the primary antibody, anti-rabbit IgG HRP conjugate (Santa Cruz Biotechnology, INC) Was blotted using as a secondary antibody, and then protein bands were observed by ECL detection kit (Amersham Pharmacia Biotech). As shown in FIG. 10, when the CD44 is expressed by irradiation with radiation, a large amount of CD44 allows the peptide to bind relatively, resulting in a thicker band than the normal cell line (FIG. 10).

This result shows that the peptide of the present invention selectively binds to the target breast cancer stem cell specific marker CD44. The peptides of the present invention thus demonstrate the potential as novel breast cancer detection probes that can replace antibodies.

Example  5: present invention Peptide CD44 Good bonding strength for FACS  analysis

11 is a result demonstrating that the FITC-SEQ ID NO: 2 peptide (P7) according to the present invention has an excellent effect compared to an antibody used in the prior art. FACS was used to bind the actual CD44-PE (Phycoerythrin) (BD Biosciences Pharmingen) antibody (FIG. 11 (a)) and the peptide (P7) of the present invention to obtain FACS (FIG. 11 (b)). The result is taken. 10 shows the normal MCF-7 cell line, i.e., before CD44 expression prior to radiation, and the right graph shows CD44 expressed after irradiation. Referring to FIG. 10, looking at the values indicated by the red line in% Gated below the graph (3.73 and 8.68 in (a) and 2.38 and 8.54 in (b)), the antibodies and peptides are 8.68 and 8.54, respectively. Based on these results, the current antibody and the peptide discovered by the researchers found similar recognition of CD44.

Example  6: present invention Peptide CD44 Good binding capacity for immuno-staining images

In order to confirm whether the peptide according to the present invention actually binds to CD44, that is, specifically binds, an experiment was performed to show that an immunostaining image using a CD44 antibody overlaps with a fluorescent peptide P7 image. As shown in FIG. 12, the fluorescent peptide P7 according to the present invention is a FITC, and thus, when the CD44 immunostaining is overlaid with the FITC green image using the CD44-PE antibody showing a red image, a yellow image (marked by the arrow in FIG. 12) is obtained. I confirmed it. This results in co-localization of peptides and antibodies on CD44. The MCF7-cont of FIG. 12 is a result when there is a small amount of CD44 before irradiation as a control, and the following MCF7-2GyX3 is much more when the CD44 is expressed after irradiation by dividing the radiation three times by 2Gy. As more peptides bind to CD44, the fluorescence image can be seen better. This is a result showing that the peptide according to the present invention specifically binds the peptide to CD44.

<110> Industry-University Cooperation Foundation Hanyang University <120> PEPTIDES TARGETING THE CD44 PROTEIN AS A BIOMARKER FOR BREAST          CANCER STEM CELL AND USES THEREOF <130> P10-0444 / HYU <160> 5 <170> Kopatentin 1.71 <210> 1 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> P6 peptide targeting the CD44 <400> 1 Phe Ser Arg Pro Pro Ala Arg His Gln Pro His Gln   1 5 10 <210> 2 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> P7 peptide targeting the CD44 <400> 2 Phe Asn Leu Pro Leu Pro Ser Arg Pro Leu Leu Arg   1 5 10 <210> 3 <211> 36 <212> DNA <213> Artificial Sequence <220> P223 DNA <400> 3 tttagccgcc cgccggcgcg ccatcagccg catcag 36 <210> 4 <211> 36 <212> DNA <213> Artificial Sequence <220> P223 DNA <400> 4 tttaacctgc cgctgccgag ccgcccgctg ctgcgc 36 <210> 5 <211> 1821 <212> DNA <213> Homo sapiens <400> 5 atggacaagt tttggtggca cgcagcctgg ggactctgcc tcgtgccgct gagcctggcg 60 cagatcgatt tgaatataac ctgccgcttt gcaggtgtat tccacgtgga gaaaaatggt 120 cgctacagca tctctcggac ggaggccgct gacctctgca aggctttcaa tagcaccttg 180 cccacaatgg cccagatgga gaaagctctg agcatcggat ttgagacctg caggtatggg 240 ttcatagaag ggcatgtggt gattccccgg atccacccca actccatctg tgcagcaaac 300 aacacagggg tgtacatcct cacatccaac acctcccagt atgacacata ttgcttcaat 360 gcttcagctc cacctgaaga agattgtaca tcagtcacag acctgcccaa tgcctttgat 420 ggaccaatta ccataactat tgttaaccgt gatggcaccc gctatgtcca gaaaggagaa 480 tacagaacga atcctgaaga catctacccc agcaacccta ctgatgatga cgtgagcagc 540 ggctcctcca gtgaaaggag cagcacttca ggaggttaca tcttttacac cttttctact 600 gtacacccca tcccagacga agacagtccc tggatcaccg acagcacaga cagaatccct 660 gctaccagta cgtcttcaaa taccatctca gcaggctggg agccaaatga agaaaatgaa 720 gatgaaagag acagacacct cagtttttct ggatcaggca ttgatgatga tgaagatttt 780 atctccagca ccatttcaac cacaccacgg gcttttgacc acacaaaaca gaaccaggac 840 tggacccagt ggaacccaag ccattcaaat ccggaagtgc tacttcagac aaccacaagg 900 atgactgatg tagacagaaa tggcaccact gcttatgaag gaaactggaa cccagaagca 960 caccctcccc tcattcacca tgagcatcat gaggaagaag agaccccaca ttctacaagc 1020 acaatccagg caactcctag tagtacaacg gaagaaacag ctacccagaa ggaacagtgg 1080 tttggcaaca gatggcatga gggatatcgc caaacaccca gagaagactc ccattcgaca 1140 acagggacag ctgcagcctc agctcatacc agccatccaa tgcaaggaag gacaacacca 1200 agcccagagg acagttcctg gactgatttc ttcaacccaa tctcacaccc catgggacga 1260 ggtcatcaag caggaagaag gatggatatg gactccagtc atagtacaac gcttcagcct 1320 actgcaaatc caaacacagg tttggtggaa gatttggaca ggacaggacc tctttcaatg 1380 acaacgcagc agagtaattc tcagagcttc tctacatcac atgaaggctt ggaagaagat 1440 aaagaccatc caacaacttc tactctgaca tcaagcaata ggaatgatgt cacaggtgga 1500 agaagagacc caaatcattc tgaaggctca actactttac tggaaggtta tacctctcat 1560 tacccacaca cgaaggaaag caggaccttc atcccagtga cctcagctaa gactgggtcc 1620 tttggagtta ctgcagttac tgttggagat tccaactcta atgtcaatcg ttccttatca 1680 ggagaccaag acacattcca ccccagtggg gggtcccata ccactcatgg atctgaatca 1740 gatggacact cacatgggag tcaagaaggt ggagcaaaca caacctctgg tcctataagg 1800 acaccccaaa ttccagaata a 1821

Claims (5)

CD44 targeting peptide which is a breast cancer stem cell specific marker which consists of an amino acid sequence consisting of SEQ ID NO: 1 or 2.
A polynucleotide encoding the amino acid sequence of claim 1.
The polynucleotide of claim 2, wherein the polynucleotide consists of a base sequence consisting of SEQ ID NO: 3 or 4. 4.
Cancer diagnostic kit comprising the peptide of claim 1.
5. The kit for diagnosing cancer of claim 4, wherein the peptide is labeled with one selected from the group consisting of chromosome, radioisotope, chromopore, luminescent material and fluorescent material.

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