KR101860882B1 - Polypeptide Probe for Diagnosis of Breast Cancer - Google Patents

Abstract

The present invention relates to a polypeptide probe for the diagnosis of HER2-positive breast cancer that specifically binds to HER2. When the polypeptide probe for HER2 detection, the composition for diagnosing breast cancer or the diagnostic kit of the present invention is used, HER2-positive breast cancer with poor prognosis can be diagnosed early. Also, in the case of the polypeptide probe for HER2 detection of the present invention, it is possible to provide a small molecule probe which is easier to manufacture than conventional antibodies, has high physical and chemical stability, and is easily injected into the body.

Description

Polypeptide Probe for Diagnosis of Breast Cancer

The present invention relates to a polypeptide probe for the diagnosis of HER2-positive breast cancer that specifically binds to HER2.

Breast cancer, along with thyroid cancer, is one of the most common cancers in women. Early detection of breast cancer is known to be a disease with a recurrence rate of 30-40%, although the cure rate is 90%. In particular, HER2 / neu overexpressing breast cancer, which accounts for 20-30% of breast cancers, has a poorer prognosis. Therefore, it is necessary to develop a new conceptual diagnosis system that can effectively overcome the limitation of the existing system for the diagnosis of HER2 overexpressing breast cancer, and the discovery of HER2 specific binding peptides can contribute to the development of such a system.

Human Epidermal Growth Factor Receptor 2 (HER2) is a receptor protein on the cell surface that is involved in cell growth and division. When a specific ligand (Epidermal Growth Factor) binds to the HER2 receptor outside the cell, it transmits a signal to HER2 and cooperatively binds to form a heterodimer. After forming the heterodimer, it acts inside the cell using three different signaling pathways: PI3K, MAPK, and phospholipase C-γ (PLCγ) pathways. As a result, it induces cell proliferation and prevents cell apoptosis. The HER2 protein plays an important role in cell survival, but when the HER2 gene is amplified and the HER2 protein is overexpressed, malignant transformation occurs. In breast cancer patients, HER2 gene is amplified in cancer cells of 20 ~ 25% breast cancer patients, and HER2 protein is overexpressed in general. These patients are referred to as HER2-positive patients. In HER2-positive patients, the HER2 protein is overexpressed and sends more signals into the cell. As a result, more cells proliferate and grow faster than other cancer cells, so HER2-positive patients have shorter survival times and higher recurrence rates than patients who do not have HER2. Therefore, in order to improve the prognosis of patients with HER2 overexpressing breast cancer, diagnosis of HER2 overexpressing cancer cells is more important than anything else.

As a technique for detecting HER2 in the past, there is a Korean Patent Application No. 2014-7032719, which is a technology relating to an antibody binding to HER2, which has a disadvantage in that the manufacturing process is complicated and the manufacturing cost is high.

Numerous papers and patent documents are referenced and cited throughout this specification. The disclosures of the cited papers and patent documents are incorporated herein by reference in their entirety to better understand the state of the art to which the present invention pertains and the content of the present invention.

The present inventors have made extensive efforts to develop a peptide probe for detecting HER2, which is a breast cancer mark marker. As a result, the present inventors have completed the present invention by identifying a polypeptide sequence that specifically binds to HER2.

Therefore, an object of the present invention is to provide a polypeptide probe for HER2 detection.

It is another object of the present invention to provide a nucleic acid molecule encoding the above-mentioned polypeptide.

It is still another object of the present invention to provide an expression vector comprising the above-described nucleic acid molecule.

It is still another object of the present invention to provide a composition for the diagnosis of HER2-positive breast cancer comprising the polypeptide.

It is still another object of the present invention to provide a kit for the diagnosis of HER2-positive breast cancer comprising the polypeptide described above.

Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims and drawings.

According to one aspect of the present invention, there is provided a polypeptide probe for detecting human epidermal growth factor receptor (HER2) comprising a first sequence or a second sequence of the sequence listing.

The present inventors have made extensive efforts to develop a peptide probe for detecting HER2, which is a breast cancer mark marker. As a result, a polypeptide sequence specifically binding to HER2 was identified.

The amino acid sequence of the peptide probe consisting of the first sequence or the second sequence of the present invention is a random phage peptide library (randomized sequence consisting of 12 linear amino acids fused to M13 phage gp3 miner coat protein and 2.7 billion different amino acid sequences the produced phage peptide library to have -Ph.D ^TM to find the sequence that binds to the breast cancer marker labeled HER2 using (phage Display peptide library Kit, New England Biolabs). the present invention "peptide probe" is a low molecular peptide Dimensional molecular stability and can easily pass through the mucous membrane and recognize the molecular target even in deep tissues. The low molecular peptide according to the present invention is relatively simple in mass production compared to the antibody , And there is almost no toxicity. In addition, Tide has a high binding affinity for the target material advantages, does not cause degeneration even when thermal / chemical treatment. It is also possible the use of a fusion protein by attaching a different protein because of the small molecular size.

The peptides of the present invention can be produced by chemical synthesis known in the art. Representative methods include, but are not necessarily limited to, liquid or solid phase synthesis, fractional condensation, F-MOC or T-BOC chemistry. The peptides of the present invention can also be produced 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 automated DNA synthesizers (such as those sold by Biosearch or Applied Biosystems). The constructed DNA sequence is operatively linked to the DNA sequence and contains one or more expression control sequences (e.g., promoters, enhancers, etc.) that regulate the expression of the DNA sequence , And the host cells are transformed with the recombinant expression vector formed therefrom. The resulting transformant is cultured under appropriate medium and conditions so that the DNA sequence is expressed, and the substantially pure peptide encoded by the DNA sequence is recovered from the culture. The recovery can be performed using methods known in the art (e.g., chromatography). By "substantially pure peptide" herein is meant that the peptide according to the invention is substantially free of any other proteins derived from the host. Genetic engineering methods for peptide synthesis of the present invention can be found in the following references: 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. The peptides of the present invention are capable of known denaturation for eliminating the reactivity of the amino terminal of the N-terminus, for example, acetylation, no.

It will be apparent to those skilled in the art that the biological functional equivalents that may be included in the HER2 labeled peptide probe range of the present invention will be limited to include variations of amino acid sequences that exhibit equivalent biological activity to the peptide probes of the present invention.

Such amino acid variations are made based on the relative similarity of the amino acid side chain substituents, such as hydrophobicity, hydrophilicity, charge, size, and the like. By analysis of the size, shape and type of amino acid side chain substituents, arginine, lysine and histidine are both positively charged residues; Alanine, glycine and serine have similar sizes; Phenylalanine, tryptophan and tyrosine have similar shapes. Thus, based on these considerations, arginine, lysine and histidine; Alanine, glycine and serine; And phenylalanine, tryptophan and tyrosine are biologically functional equivalents.

In introducing the mutation, the hydrophobic index of the amino acid can be considered. Each amino acid is assigned a hydrophobic index according to its hydrophobicity and charge: isoruicin (+4.5); Valine (+4.2); Leucine (+3.8); Phenylalanine (+2.8); Cysteine / cysteine (+2.5); Methionine (+1.9); Alanine (+1.8); Glycine (-0.4); Threonine (-0.7); Serine (-0.8); Tryptophan (-0.9); Tyrosine (-1.3); Proline (-1.6); Histidine (-3.2); Glutamate (-3.5); Glutamine (-3.5); Aspartate (-3.5); Asparagine (-3.5); Lysine (-3.9); And arginine (-4.5).

The hydrophobic amino acid index is very important in imparting the interactive biological function of peptides. It is known that substitution with an amino acid having a similar hydrophobicity index can retain similar biological activities. When the mutation is introduced with reference to the hydrophobic index, substitution is made between amino acids showing preferably a hydrophobic index difference of within 2, more preferably within 1, even more preferably within 0.5.

On the other hand, it is also well known that the substitution between amino acids with similar hydrophilicity values leads to peptides with homogeneous biological activity. As disclosed in U.S. Patent No. 4,554,101, the following hydrophilicity values are assigned to each amino acid residue: arginine (+3.0); Lysine (+3.0); Aspartate (+3.0 ㅁ 1); Glutamate (+3.0 ㅁ 1); Serine (+0.3); Asparagine (+0.2); Glutamine (+0.2); Glycine (0); Threonine (-0.4); Proline (-0.5 ㅁ 1); Alanine (-0.5); Histidine (-0.5); Cysteine (-1.0); Methionine (-1.3); Valine (-1.5); Leucine (-1.8); Isoru Isin (-1.8); Tyrosine (-2.3); Phenylalanine (-2.5); Tryptophan (-3.4).

When a mutation is introduced with reference to the hydrophilicity value, the amino acid is substituted preferably within ± 2, more preferably within ± 1, even more preferably within ± 0.5.

Amino acid exchange in peptides that do not globally alter the activity of the molecule is known in the art (H. Neurath, R. L. Hill, The Proteins, Academic Press, New York, 1979). The most commonly occurring exchanges involve amino acid residues Ala / Ser, Val / Ile, Asp / Glu, Thr / Ser, Ala / Gly, Ala / Thr, Ser / Asn, Ala / Val, Ser / Gly, Thy / Pro, Lys / Arg, Asp / Asn, Leu / Ile, Leu / Val, Ala / Glu and Asp / Gly.

Considering the mutation having the above-mentioned biological equivalent activity, the peptide included in the HER2-labeled peptide probe of the present invention is interpreted to include a sequence showing substantial identity with the sequence described in the sequence listing. The above-mentioned substantial identity is determined by aligning the peptide probe sequence of the present invention with any other sequence as much as possible, and analyzing the aligned sequence using an algorithm commonly used in the art. Homology, more preferably 90% or more homology. Alignment methods for sequence comparison are well known in the art. Various methods and algorithms for alignment are described by Smith and Waterman, Adv. Appl. Math. 2: 482 (1981) ; Needleman and Wunsch, J. Mol. Bio. 48: 443 (1970); Pearson and Lipman, Methods in Mol. Biol. 24: 307-31 (1988); Higgins and Sharp, Gene 73: 237-44 (1988); Higgins and Sharp, CABIOS 5: 151-3 (1989); Corpet et al., Nuc. Acids Res. 16: 10881-90 (1988); Huang et al., Comp. Appl. BioSci. 8: 155-65 (1992) and Pearson et al., Meth. Mol. Biol. 24: 307-31 (1994). The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al., J. Mol. Biol. 215: 403-10 (1990)) is accessible from NCBI (National Center for Biological Information) It can be used in conjunction with sequence analysis programs such as blastx, tblastn and tblastx. BLAST is available at http://www.ncbi.nlm.nih.gov/BLAST/. A method for comparing sequence homology using this program can be found at http://www.ncbi.nlm.nih.gov/BLAST/blast_help.html.

According to one aspect of the present invention, the present invention provides a nucleic acid molecule encoding the above-mentioned polypeptide.

As used herein, the term "nucleic acid molecule" is intended to encompass DNA (gDNA and cDNA) and RNA molecules in a comprehensive sense. Nucleotides which are basic constituent units in nucleic acid molecules include not only natural nucleotides but also analogs (Scheit, Nucleotide Analogs, John Wiley, New York (1980); Uhlman and Peyman, Chemical Reviews , 90: 543-584 (1990)).

Variations in nucleotides do not cause changes in the protein. Such nucleic acids include functionally equivalent codons or codons that encode the same amino acid (e.g., by codon degeneration, six codons for arginine or serine), or codons that encode biologically equivalent amino acids ≪ / RTI >

Further, the mutation in the nucleotide may lead to a change in the amino acid sequence of the HER2 detecting peptide probe of the present invention. Even when the peptide probe of the present invention is a mutation which causes a change in the amino acid of the peptide probe, it can be obtained that exhibits almost the same activity as the HER2 detecting peptide of the present invention.

It is obvious to those skilled in the art that the biological functional equivalent that can be included in the probe range for HER2 detection of the present invention will be limited to the variation of the amino acid sequence exhibiting equivalent biological activity with the peptide of the present invention. Described above.

According to one aspect of the present invention, there is provided a recombinant expression vector comprising the above-described nucleic acid molecule.

The vector system of the present invention can be constructed through various methods known in the art, and specific methods for this are disclosed in Sambrook et al., Molecular Cloning, A Laboratory Manual , Cold Spring Harbor Laboratory Press (2001) This document is incorporated herein by reference.

The vector of the present invention can typically be constructed as a vector for cloning or as a vector for expression. In addition, the vector of the present invention can be constructed by using prokaryotic cells or eukaryotic cells as hosts. It is preferable that the nucleic acid molecule of the present invention is derived from a prokaryotic cell and that the prokaryotic cell is used as a host in consideration of the convenience of cultivation.

For example, the vector is an expression vector of the present invention, in the case of a prokaryotic cell as a host, the strong promoter that can proceed with the transfer (e.g., tac promoter, lac promoter, lac UV5 promoter, lpp promoter, p _L ^λ promoter , p _R ^? promoter, rac 5 promoter, amp promoter, recA promoter, SP6 promoter, trp promoter and T7 promoter, etc.), a ribosome binding site for initiation of decoding and a transcription / translation termination sequence. When E. coli is used as a host cell, the promoter and operator site of the E. coli tryptophan biosynthetic pathway (Yanofsky, C., J. Bacteriol. , 158: 1018-1024 (1984)) and the left promoter of phage l _L ^? Promoter, Herskowitz, I. and Hagen, D., Ann. Rev. Genet. , 14: 399-445 (1980)).

The vectors that can be used in the present invention include plasmids such as pSC101, ColE1, pBR322, pUC8 / 9, pHC79, pUC19 and pET which are frequently used in the art, phages such as λgt4 · λB, ,?? z1, and M13) or a virus (e.g., SV40 or the like).

On the other hand, when the vector of the present invention is an expression vector and a eukaryotic cell is used as a host, a promoter derived from a genome of a mammalian cell (for example, a metallothionein promoter) or a mammalian virus Virus late promoter, vaccinia virus 7.5K promoter, SV40 promoter, cytomegalovirus promoter, and tk promoter of HSV) can be used, and generally have a polyadenylation sequence as a transcription termination sequence.

The vector of the present invention may be fused with other sequences to facilitate purification of HER2 labeled peptide probes expressed therefrom. Fusion sequences include, for example, glutathione S-transferase (Pharmacia, USA), maltose binding protein (NEB, USA), FLAG (IBI, USA), and 6x His (hexahistidine; Quiagen, USA) Is 6x His. Because of the additional sequence for such purification, proteins expressed in the host are rapidly and easily purified through affinity chromatography.

On the other hand, the expression vector of the present invention includes, as a selection marker, an antibiotic resistance gene commonly used in the art and includes, for example, ampicillin, gentamycin, carbenicillin, chloramphenicol, streptomycin, kanamycin, There is a resistance gene for mitine and tetracycline.

According to one aspect of the present invention, the present invention provides a transformant comprising the recombinant expression vector described above.

The host cell capable of stably and continuously cloning and expressing the above-described expression vector can be any host cell known in the art, including E. coli Origami2, E. coli JM109, E. coli BL21 (DE3 ), E. coli RR1, E. coli LE392, E. coli B, E. coli X 1776, E.coli strain, Bacillus subtilis, Bacillus strains such as Bacillus Chuo ringen systems such as E. coli W3110, and Salmonella typhimurium, Serratia marcesensis, and various enterococci such as Pseudomonas species and strains.

In addition, Saccharomyce cerevisiae , insect cells and human cells (e.g., CHO cell line (Chinese hamster ovary), W138, BHK, COS-7, 293 , HepG2, 3T3, RIN and MDCK cell lines) and the like can be used.

The method of delivering the vector of the present invention into a host cell may be carried out by the CaCl ₂ method (Cohen, SN et al., Proc. Natl. Acac. Sci. USA , 9: 2110-2114 (1973) , Hanahan, D., J. MoI. Biol. , 166: 557-580 (1997)), one method (Cohen, SN et al., Proc. Natl. Acac. Sci. USA , 9: 2110-2114 (1983)) and electroporation (Dower, WJ et al., Nucleic Acids Res. , 16: 6127-6145 (1988)). In addition, when the host cell is a eukaryotic cell, microinjection (Capecchi, MR, Cell , 22: 479 (1980)), calcium phosphate precipitation (Graham, FL et al., Virology , 52: 456 electroporation (Neumann, E. et al, EMBO J., 1:. 841 (1982)), liposome-mediated transfection method (Wong, TK et al, Gene , 10:87 (1980).), DEAE- (Yang et al., Proc. Natl. Acad. Sci. , 87: 9568-9572 (1990)) and dextran treatment (Gopal, Mol. Cell Biol. , 5: 1188-1190 ) Or the like into the host cell.

The vector injected into the host cell can be expressed in the host cell, and in this case, a large amount of peptide probe for HER2 detection is obtained. For example, when the expression vector comprises a lac promoter, the host cell may be treated with IPTG to induce gene expression.

According to one aspect of the present invention, there is provided a composition for the diagnosis of HER2-positive breast cancer comprising the above-mentioned polypeptides.

The composition of the present invention is a composition for specifically detecting HER2 protein for HER2-positive breast cancer diagnosis, and includes a polypeptide probe for HER2 detection described above. Therefore, the above-mentioned contents are referred to for the HER2 detection polypeptide, and redundant contents are omitted in order to avoid the excessive complexity described in the present specification.

The term "diagnosing" in the context of the present invention includes determining the susceptibility of an object to a particular disease or disorder, determining whether an object currently has a particular disease or disorder, (Eg, determining the stage of breast cancer progression or determining the degree of efficacy of treatment for breast cancer), or determining the prognosis (eg, determining the level of efficacy of treatment for breast cancer) Lt; RTI ID = 0.0 > a < / RTI >

As described in the background section of this specification, HER2 is a representative biomarker of breast cancer. HER2 is known to be overexpressed in approximately 20% of breast cancer patients, and it is resistant to conventional systemic chemotherapy because it plays a role in promoting cell survival, proliferation, angiogenesis, invasion and metastasis, suggesting a poor prognosis.

Therefore, when it is detected that HER2 is overexpressed in a breast cancer cell, the individual from which the cell is derived can be diagnosed as having a poor prognosis of breast cancer. The "overexpression" of HER2 for the poor prognosis of breast cancer indicates a significant increase in expression compared to the mean HER2 expression in breast cancer patients who are not HER2-positive. As described above, breast cancer with HER2 overexpression is defined as "HER2-positive breast cancer ". By observing the overexpression of HER2 in breast cancer cells, it is possible to diagnose HER2-positive breast cancer with a high prognosis and a short survival time with poor prognosis. This is an important procedure for the prognosis of HER2-positive breast cancer patients.

When the composition is administered to a subject or brought into contact with a subject sample separated from the subject, the peptide in the composition specifically and selectively binds to HER2 expressing breast cancer cells, and the polypeptide tagged with the polypeptide The position or expression level of the peptide bound to the target as described above can be measured through the reaction occurring.

In one embodiment of the present invention, the peptide of the present invention may be labeled with at least one selected from the group consisting of chromogenic enzymes, radioactive isotopes, chromophores, luminescent materials and fluorescent materials.

The coloring enzyme may be, for example, peroxidase or alkaline phosphatase, and the radioisotope may be, for example, 124I, 125I, 111In, 99mTc, 32P, 35S, The luminescent material or fluorescent material can be, for example, FITC, RITC, rhodamine, Texas Red, fluorescein, phycoerythrin, quantum dots, and the like .

Similarly, the detectable label may be an antibody epitope, a substrate, a cofactor, an inhibitor or an affinity ligand. Such labeling may be performed during the synthesis of the peptide of the present invention, or may be performed in addition to the peptide already synthesized.

If a fluorescent substance is used as a detectable label, the fluorescence pattern of the peptide probe bound to HER2 can be observed by fluorescence-based tomography (FMT), and the presence or absence of HER2-positive Can be diagnosed.

 That is, when a large amount of the detection reaction occurs in the tissues or cells of the subject, HER2 is overexpressed in the breast cancer cells of the subject, the subject has breast cancer, and HER2 is positive and the prognosis is poor It can be diagnosed early. In addition, the composition can be non-invasively confirmed by HER2-positive by administering the composition in vivo.

According to another aspect of the present invention, there is provided a kit for the diagnosis of HER2-positive breast cancer comprising the above-mentioned peptide probe.

The breast cancer diagnostic kit of the present invention may include one or more other component compositions or apparatuses suitable for the method of assaying a peptide for HER2 detection and may further comprise a buffer or reaction solution for stably maintaining the structure or physiological activity of the peptide . Further, in order to maintain the stability, it may be provided at a temperature of 4 占 폚. The present invention relates to a kit comprising a polypeptide probe for detecting human epidermal growth factor receptor (HER2) comprising a first sequence or a second sequence of the sequence listing according to another aspect of the present invention, Will be omitted for avoiding the excessive complexity described herein.

In order to facilitate identification, detection and quantification of the peptides of the present invention bound to HER2, the peptides contained in the kit of the present invention may be provided in the labeled state as described above. When the peptide of the present invention is provided without labeling, the kit for breast cancer diagnosis of the present invention further includes a component for searching the binding degree or position of the peptide of the present invention in vitro or in vivo with HER2 can do. The components may be known compounds for the labeling of the peptides of the invention or may be conjugated to a peptide of the invention or an antibody against a particular receptor that binds to the peptide of the invention or a secondary antibody thereto, And may be a reagent for detection thereof. Specifically, the diagnosis of the present invention can be carried out by modifying a conventional immunoassay method or protocol. For example, the diagnosis of the present invention can be carried out by using the peptide of the present invention instead of the antibody in the conventional immunoassay, and the process is the same.

In addition, the peptide of the diagnostic kit of the present invention may be provided in a form coated on the surface of the plate. In this case, the sample containing the cancer cell tissue of the subject directly is reacted with the sample on the plate under a suitable condition, and then the binding with HER2 contained in the cancer cell tissue sample on the surface of the plate is quantitatively analyzed, Can be diagnosed.

The features and advantages of the present invention are summarized as follows:

(a) The present invention provides a polypeptide probe for HER2 detection.

(b) The present invention provides a nucleic acid molecule encoding the above-mentioned polypeptide.

(c) The present invention provides an expression vector comprising the aforementioned nucleic acid molecule.

(d) The present invention provides a composition for the diagnosis of HER2-positive breast cancer comprising the above-mentioned polypeptides.

(e) The present invention provides a kit for the diagnosis of HER2-positive breast cancer comprising the above-mentioned polypeptides.

(f) When a polypeptide probe for detecting HER2, a composition for diagnosing breast cancer or a diagnostic kit of the present invention is used, HER2-positive breast cancer with poor prognosis can be diagnosed early.

(g) In the case of the polypeptide probe for HER2 detection of the present invention, it is possible to provide a small molecule probe which is easier to manufacture than conventional antibodies, has high physical and chemical stability, and is easily injected into the body.

Figure 1 shows the gene cloning results of the breast cancer marker HER2 ECD. The "PCR product" in FIG. 1 (a) represents the HER2 ECD gene of 1.6 kbp. (b) shows (P1) and (P2, P3) before and after the restriction enzyme treatment to insert the HER2 gene into the expression vector pET-28a. The gene 1.6 kbp coding for HER2 ECD expresses the expression vector As shown in Fig. "M" represents a molecular weight marker.
FIG. 2 shows experimental results for confirming the protein expression ability and protein purification of the brain glioma stem cell marker marker HER2 ECD. FIG. 2 (a) is a graph showing protein (AI, after induction) overexpressed before incubation (BI, before induction) and overnight (O / N, overnight incubation) at 18 ° C, solubility soluble form protein and insoluble form of protein (InS). It was confirmed that the HER2 protein was expressed in an insoluble form. FIG. 2 (b) shows the result of screening of HER2 protein by affinity chromatography by performing denaturation purification to purify HER2 expressed in an insoluble protein form.
Figure 3 shows a plot of M13 page screening for screening page peptides binding to HER2 protein. The HER2 was fixed in each well of the plate ((1) in FIG. 3), and a page library expressing peptides composed of 12 amino acids was added to the surface of M13 (FIG. 3 (2) And binding time are applied, the page peptide binding to the HER2 protein under extreme conditions remains in the well ((3) in FIG. 3). Finally, when this is extracted ((4) in FIG. 3), only the page peptide that specifically binds to the HER2 protein is finally obtained ((4) in FIG. Repetition of the above rounds with one round as in (1) - (4) of FIG. 3 is referred to as biopanning. The present inventors carried out three rounds of bio-panning.
Figure 4 shows the biopanning conditions of each round for detecting page peptides with high specificity and binding affinity for the HER2 catalytic domain protein. Increasing the ratio of NaCl to Tween-20 per round, while shortening the binding time of the page peptide, detected page peptide binding to the HER2 protein even under extreme conditions.
Figure 5 shows the results of securing a total of 60 page plaques binding to HER2 protein. Sixty pages of plaques were classified into five different sequences of peptides.
Figure 6 shows the binding assay results of bacteriophage binding to HER2 protein. All five page-peptides were found to bind effectively to the HER2 protein at the picomolarity level.
Fig. 7 shows the result of binding assay by binding a fluorescent probe to a page binding to HER2 protein. FIG. 7A is a graph showing the binding force with HER2 against HBP1 (SEQ ID NO: 1), FIG. 7B is a graph showing the binding force with HER2 against HBP2 (SEQ ID NO: 2) , And HBP2, respectively.
FIG. 8 shows the result of measuring the binding ability at the cellular level to the HER2 positive cell line SKBR3 through the HER2 binding peptide. The specificity of HER2 binding antibody and sc peptide (sc peptide) composed of any amino acid sequence were simultaneously treated. As a result, HER2-binding peptides showed antibody-like binding at the cellular level, and the fluorescence signals of the sc-peptides were microscopically revealed, resulting in the binding of HER2 peptides to specific binding.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are only for describing the present invention in more detail and that the scope of the present invention is not limited by these embodiments in accordance with the gist of the present invention .

Example

Example 1: HER2 cell extracellular domain (ECD) gene cloning

First, the HER2 protein was expressed on E. coli, and the HER2 gene was amplified by PCR as follows: HER2 sense primer was 5'-ATAT GAATTC (EcoR1) ATG GAG CTG GCG GCC TTG-3 ' , HER2 antisense primer was 5'-ATAT CTCGAG (Xho1) TGA GTT GAC ACA CTG GGT-3 '. Denaturation at 95 DEG C for 1 minute, denaturation at 94 DEG C for 30 seconds, and annealing at 70 DEG C for 45 minutes in a reaction solution containing 5% DMSO containing the synthetic primer and template DNA. Sec, and extension at 72 ° C for 2 minutes and 45 sec. The template DNA obtained after 20 cycles of the PCR reaction was identified through the gene base sequence (see FIG. 1). The amplified gene was inserted into pET-28a (+), which is a bacterial expression vector, and cloned according to the manufacturer's manual. Then, the amplified gene was inserted into the expression host E. coli BL21 (SEQ ID NO: DE3), followed by induction of the production of HER2 protein at 0.5 mM IPTG at 18 DEG C for 12 hours. The transfection method is briefly described as follows: First, 5 μl of DNA is added to 100 μl of competent cells, mixed well and placed on ice for 30 minutes. Thereafter, heat shock was applied at 42 DEG C for 90 seconds. In order to prevent contamination, first, after turning on the alcohol lamp, 800 μl of LB medium was added and the mixture was incubated at 37 ° C for 45 minutes. Finally, it was applied to an LBA plate and incubated overnight in a 37 ° C incubator.

Example 2 Mass Production and Separation of HER2 Extracellular Domain (ECD)

The HER2 gene was amplified by PCR and inserted into the bacterial expression vector pET-28a. The cloned HER2-expressing plasmid was transformed into the expression host BL21 (DE3) to induce HER2 production at 0.5 mM IPTG and 18 ° C Respectively. The bacterial cells were harvested by centrifugation to examine the solubility of the proteins. As a result, it was confirmed that most of the proteins were insoluble (see FIG. 2). Therefore, proteins were isolated and purified under denaturing conditions to obtain such insoluble proteins.

The bacterial cells were harvested by centrifugation, and the solubility of the proteins was examined. As a result, it was confirmed that most of the proteins were insoluble. Therefore, only the intracellular insoluble protein was obtained for the HER2 separation purification, and the protein was dissolved in the denaturation condition (6 M guanidium-HCl) and then passed through a Sepharose column to bind the HER2 protein and denature HER2 was refolded by slowly removing the denature through a decreasing concentration of Urea in a state bound to the column to refold the HER2, and a 500 mM imidazole linear Elute under a gradient. The resulting protein was dialyzed against a dialysis buffer (50 mM Tris, 2 mM β-mercaptoethanol, pH 7.4) to remove excess imidazole, and the resulting protein was purified by SDS / PAGE (see FIG. 2) The amount was confirmed using Bradford reagent and 0.3 mg / L culture was obtained.

Example 3: M13 phage peptide library screening-phage display

The phage display begins with fixing the protein obtained in Example 1 to a 96-well plate. The 96-well plate used here was a polystyrene plate (SPR) made of polystyrene on its surface, which stably fixed the protein using a hydrophobic interaction between the protein and the plate surface, Thereby increasing the display efficiency. Thus the protein in a fixed random peptide library (manufactured so as to have 27 million different amino acid sequence by the random array of a 12 amino acid peptide library ^{-Ph.D TM (phage display peptide library kit} , New England Biolabs (NEB) The peptide display method was designed to select only specific peptides having a good binding force with proteins by inducing interaction between proteins and peptides by introducing the peptide into the sample.

Specifically, in FIG. 3, the HER2 protein is fixed on the surface of a plate (SPL) to screen for a peptide binding to the HER2 protein, and then the M13 phage peptide library (composed of 12 amino acids having about 2.7 billion different amino acid sequences And the peptide was fused to M13 phage gp3 minor coat protein) to induce binding. Thereafter, peptide expression phages binding with high affinity were selected through various binding times and washing conditions. The screening plan and the reaction conditions related thereto are shown in Fig. 3 and Fig. 4 (screening condition), respectively. Specifically, FIG. 3 is a plan view of M13 phage screening for peptide screening binding to HER2 protein, which comprises (1) immobilizing HER2 protein on the (SPL) surface of a 96-well plate, (2) After binding the phage library expressing the peptide library consisting of the amino acids to the HER2 protein, (3) washing it in various conditions, and (4) finally binding the phage was obtained by chemical elution or competitive elution. The thus obtained phage was infected with E. coli (ER2738) and amplified. After the amplified phage was bound to the HER2 protein again, it was repeated under the conditions such as the intensity of the washing condition and the short reaction time, Repeat the search process. This sequence of processes is called biopanning. FIG. 4 is a graph showing a total of three steps of bio-panning conditions for detecting a phage in which a peptide having high specificity and binding ability against HER2 protein is expressed. The reaction conditions were varied with strong washing conditions and short reaction times for each step, and four times of biopanning was performed to obtain a phage having a peptide binding to the HER2 protein. The thus obtained phage was infected with E. coli ER2738 cells, which were host cells, and amplified in LB medium. Then, about 60 phage plaques were selected and M13 phage genomic DNA (single stranded circular DNA) was isolated and purified, The amino acid sequence of the peptide fragment, which is expressed on the phage surface protein (gp3 minor coat protein) by the triplet code to bind to the HER2 protein, was identified. The results are shown in Fig.

Example 4: Analysis of Binding Ability of Peptides Binding to HER2

In order to analyze the binding force of the screened peptides, the phage expressing each of the peptides was amplified, and then the concentration of the phage solution was determined by titering. Then, the binding force of the peptide was deduced by measuring the amount of the phage bound by adding the phage to the plate to which the HER2 protein was bound.

Specifically, the phage was added to the plate to which the HER2 protein was bound, and washed after a certain period of time. Subsequently, an antibody recognizing the phage surface protein (mouse anti-M13 monoclonal antibody, diluted 1: 5000 in TBST, Amersham Bioscience) was added and reacted for 1 hour. After washing 5 times with washing solution, secondary antibody (mouse IgG-HRP, 1: 4000 diluted with TBST, Santacruz), which binds to mouse anti-M13 monoclonal antibody, was reacted for 1 hour. After 5 washing steps, TMB substrate solution (3,3 ', 5,5'-tetramethylbenzidine (TMB) / H ₂ O ₂ , Chemicon) was added and after 15 minutes, 1 M sulfuric acid was added to terminate the reaction . This whole process is called an enzyme-linked immunosorbant assay (ELISA) using antibodies. The K _d value obtained as a result of this experiment is an ELISA signal intensity according to the corresponding concentration of a page peptide reacted with a fixed protein in each well It is determined by the saturation curve measured at 450 nm.

As a result, as shown in FIG. 6, the binding force corresponding to the binding force of the existing antibody-antigen reaction was confirmed by showing the excellent binding force of the five page-peptides at the picomole level.

Example 5 Analysis of Binding Capability of Synthetic Peptide Sequence with Fluorescence Probe Binding to HER2

Sequences that specifically recognize the HER2 protein on the page peptide with high binding force were synthesized by attaching a fluorescent material (FITC; fluorescein isothiocyanate) to each signal, and the sequence is as follows.

H1 peptide: Ac - SQDIRTWNGTRS GCGK - FITC

H2 peptide: Ac - SLMVKAPASFWA GCGK - FITC

The indicated amino acid sequence represents the amino terminal. Acetylation was performed to remove the reactivity of the amino terminal at the N-terminal, and amino acid K (Lysine, lysine) was added to the C-terminal to attach FITC to the carboxy terminal. GCG was added near the carboxy terminus of each amino acid sequence for the process to maximize the sensitivity of the fluorescent peptide probe Ac- SQDIRTWNGTRS GCGK-FITC or Ac-SLMVKAPASFWA GCGK-FITC. To analyze the binding force of the peptides to the HER2 protein, each of the peptides was diluted by concentration on a plate fixed with HER2 protein. The method of inferring the binding force of each peptide by measuring the amount of the peptide bound to the HER2 protein in each well is similar to that described in Example 4. [ However, in the case of FITC-synthesized peptides, there is no need for a marker to indicate a signal as in the case of a page peptide test such as addition of a primary antibody, a secondary antibody, or a TMB substrate solution. HER2 protein and peptide are bound to each other, The fluorescence intensity of each peptide concentration was measured at the maximum wavelength of FITC (Excitation @ 495 nm, Emission @ 520 nm) connected to the peptide by a fluorometer.

Peptide The FITC signal intensity at the corresponding concentration was plotted to determine the Kd value of the peptide (see Figure 7).

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the same is by way of illustration and example only and is not to be construed as limiting the scope of the present invention. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

<110> IUCF-HYU (Industry-University Cooperation Foundation Hanyang University) <120> Polypeptide Probe for Diagnosis of Breast Cancer <130> PN150454 <160> 2 <170> Kopatentin 2.0 <210> 1 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> HER2 binding polypeptide probe <400> 1 Ser Gln Asp Ile Arg Thr Trp Asn Gly Thr Arg Ser   1 5 10 <210> 2 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> HER2 binding polypeptide probe <400> 2 Ser Leu Met Val Lys Ala Pro Ala Ser Phe Trp Ala   1 5 10

Claims (7)

A polypeptide probe for detection of Human Epidermal Growth Factor Receptor 2 (HER2) comprising a sequence of SEQ ID No. 2.
A nucleic acid molecule encoding the polypeptide of claim 1.
A recombinant expression vector comprising the nucleic acid molecule of claim 2.
A transformant comprising the recombinant expression vector of claim 3.
A composition for diagnosing HER2-positive breast cancer comprising the polypeptide probe of claim 1.
The composition according to claim 5, wherein the polypeptide probe is labeled with at least one selected from the group consisting of a chromogenic enzyme, a radioactive isotope, a chromophor, a luminescent material and a fluorescent material.
A kit for the diagnosis of HER2-positive breast cancer comprising the polypeptide probe of claim 1.

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