KR101636882B1

KR101636882B1 - A novel hEphA2-specific binding protein having human fibronectin III domain scaffold and use thereof

Info

Publication number: KR101636882B1
Application number: KR1020150090681A
Authority: KR
Inventors: 홍영진; 민정준; 박승환
Original assignee: 전남대학교 산학협력단
Priority date: 2015-06-25
Filing date: 2015-06-25
Publication date: 2016-07-06

Abstract

The present invention relates to a peptide derived from human fibronectin domain III (Fn3), which specifically binds to human ephrin A-type receptor 2 (hEphA2), and is formed of amino acid sequences represented by sequence number 1 or 2. The present invention further relates to a use thereof for diagnosing cancer.

Description

A novel human EphA2-specific binding protein of human fibronectin domain III basic skeleton and its use {A novel hEphA2-specific binding protein having human fibronectin III domain scaffold and use thereof}

FIELD OF THE INVENTION The present invention relates to a target binding protein having a basic framework of domain III (Fn3) of fibronectin and a use thereof, and more particularly to a peptide binding to an Fn3 derived peptide specifically binding to human ephrin type A receptor 2 (hEphA2) Lt; / RTI >

A variety of scaffold proteins known as antibody mimics have been studied for in vitro studies, diagnosis and treatment of human disease (Gebauer et al ., Curr. Opin. Chem. Biol ., 13 (3) : 245-255, 2009). A monobody, a binding protein with a backbone derived from the ternary domain III (Fn3 / FnIIIlO) of human fibronectin, is one of the proteins used as a specific binding agent for a target protein because of its high affinity and specificity for the target protein (Bloom, L. and V. Calabro, Drug Discov Today, 14 (19-20):..... 949-955, 2009; Lipovsek, D., Protein Eng Des Sel, 24 (1-2): 3 -9, 2011). The monobody serves as a binding agent for human clinical applications, such as small size (10 kDa) for tissue penetration, molecular stability with high melting point (82 ° C), efficient bacterial utilization, and low immunogenicity expected as a human origin protein (Bloom, L. and V. Calabro., Drug Discov. Today , 14 (19-20): 949-955, 2009). The structure of the Fn3 protein is well defined and binds directly to the target by the amino acid combination of the three exposed portions (BC, DE and FG loops) in a beta-sheet structure that constitutes the protein backbone (Main, AL et al ., Cell , 71 (4): 671-678, 1992; Dickinson, CD et al ., J. Mol. Biol ., 236 (4): 1079-1092,1994). (Lipovsek, D., Protein Eng. Des. Sel .) Have been developed for clinical purposes, such as diagnosis and treatment of diseases such as cancer, infection and so on. , 24 (1-2): 3-9, 2011). WO02010 / 051274A discloses the use of Fn3 monomers and Fn3-Fn3 repeats, including isolated nucleic acids, vectors, host cells, and methods for making and using them, which encode the protein backbone in human Fn3- And therapeutic compositions, methods and devices for diagnostic and / or therapeutic artificial proteins, such as, for example, < RTI ID = 0.0 > In particular, Fn3 proteins that bind IgG have been suggested to be useful in diagnostic and / or therapeutic applications. International patent application WO2012 / 016245A discloses fibronectin crayer molecules and libraries thereof. The invention described above is based on the use of CD and FG loops of multiple domain IIIs (e.g., FnIII7, Fn3 / FnIIIlO, and FnIuIl4) of the fibronectin protein with the surface exposed residues of the beta-sheet We present a combination of lower loop binder libraries. The patent also discloses a method of forming a library of Fn3 proteins useful for separating proteins that bind to a selected target protein.

On the other hand, human EphA2 (human ephrin type-A receptor 2, hEphA2) is a receptor protein belonging to the ephrin receptor subfamily of protein-tyrosine kinases and family. It is known as a diagnostic marker protein. (Mitra, S. et al ., Biochemistry 49 (31): 6687-6695, 2010), anti-hEphA2 monoclonal antibody (US7402298, US8449882), and antibody-derived artificial proteins (scFv, EP2595657A). However, the peptide has problems such as low binding force and high production cost for the antibody.

Disclosure of Invention Technical Problem [8] Accordingly, the present invention has been made to solve the above problems, and it is an object of the present invention to provide a peptide having the basic framework of human fibronectin domain III (Fn3) that specifically binds to human ephrin type A receptor 2 (hEphA2) .

The present invention also aims to provide an anticancer pharmaceutical composition comprising the peptide as an active ingredient.

The present invention aims at finally providing a molecular contrast agent for hEphA2 comprising the peptide as an active ingredient.

However, these problems are exemplary and do not limit the scope of the present invention.

According to one aspect of the present invention, there is provided a peptide derived from human fibronectin domain III (Fn3) comprising the amino acid sequence of SEQ ID NO: 1 or 2, which specifically binds to human ephrine type-A receptor 2 (hEphA2).

According to another aspect of the present invention, there is provided a pharmaceutical composition for treating cancer comprising the peptide as an active ingredient.

According to another aspect of the present invention, there is provided a pharmaceutical composition for treating cancer comprising, as an active ingredient, a peptide conjugate in which an anti-cancer drug or an anti-cancer peptide is covalently or non-covalently bound to the peptide.

According to another aspect of the present invention, there is provided a composition for molecular imaging for hEphA2 comprising the peptide as an active ingredient.

According to another aspect of the present invention, there is provided a composition for tumor screening, which comprises, as an active ingredient, a peptide conjugate in which a contrasting compound is bound to the peptide by covalent bond or noncovalent bond.

According to another aspect of the present invention, there is provided a method of treating cancer, And obtaining an image by the tumor imaging composition.

According to another aspect of the present invention there is provided a method of treating cancer, comprising administering the composition for tumor metastasis to a subject having cancer; Identifying a cancer tissue site through an image generated by the tumor imaging composition; And an intraoperative imaging method comprising the step of extracting the identified cancer tissue.

The novel Fn3-derived peptide according to one embodiment of the present invention as described above specifically binds to purified hEphA2 and hEphA2 on the surface of human cells, and thus is useful as a candidate probe for in vivo diagnosis and treatment of tumors Lt; / RTI > Of course, the scope of the present invention is not limited by these effects.

FIG. 1 shows the result of screening for the G4 yeast surface marker hEphA2 library according to an embodiment of the present invention. FIG. 1 (A) is a graph showing the results of screening of the yeast G4 library (left) and the 4.5- FACS profile, and B in Fig. 1 shows the amino acid sequence of the clone (EM1 represented by SEQ ID NO: 1 and EM2 represented by SEQ ID NO: 2) obtained in 4.5-fraction yeasts, with the original Fn3 amino acid sequence (SEQ ID NO: 3) Figure 1C is the FACS profile for hEphA2 of the isolated clones EM1 (left) and EM2 (right).
Fig. 2 shows the binding force between the Fn3 protein of the yeast transformed with the yeast surface expression vector pCT-Fn3-EphA2 and pCT-Fn3-EphA2 containing the gene encoding the two Fn3-derived proteins EM1 and EM2 and the target protein hEphA2 Graph.
Figure 3 is a FACS profile showing the binding of the EM1 and EM2 Fn3 proteins purified in E. coli to magnetic beads conjugated with the target protein hEphA2. H and amh in each Fn3 protein means a protein having His6 (HHHHHH, SEQ ID NO: 4) and Avidin-cMyc-His6 (GLNDIFEAQKIEWHEEQKLISEEDLRSHHHHHH, SEQ ID NO: 5) tag at the C terminus. As a control, magnetic beads with bead only and lysozyme or human antibody (IgG) were used.
FIG. 4 is an ELISA graph showing specific binding amounts of Fn3 proteins (EM1-amh and EM2-amh) having various concentrations of amh tag with proteins of the A-type Eph receptor group. The h, m, and r denoted at the start of each protein are respectively human, mouse, and rat derived proteins.
FIG. 5 shows the binding of hEphA2-specific Fn3 protein to cells. FIG. 5A shows the results of SDS-polyacrylamide gel electrophoresis (PAGE) of the whole protein of human prostate cancer PC3 and breast cancer SKBR3 cells, followed by western blotting with anti-hEphA2 antibody, western blotting of beta-actin protein as a quantitative control . 5B is a FACS profile observed after treatment with an anti-hEphA2 antibody and a fluorescent dye-conjugated secondary antibody to show the expression level of hEphA2 on the cell surface of the cells. FACS analysis was also performed on unstained cells as controls and cells treated with a fluorescent dye-conjugated secondary antibody (2nd Ab only). 5c is a graph showing the number of molecules of the surface hEphA2 of the cells. FIG. 5D is a FACS profile measuring the binding of His6-tagged EM1 Fn3 protein (Cy5.5-EM1-h, red solid line) bound to Cy5.5 fluorescent dye to the cells. FACS analysis of non-stained cells (black solid line) was performed as a control. FIG. 5E is a fluorescence microscope photograph of the cells obtained after staining with anti-hEphA2 antibody and Cy5.5-EM1-h, respectively. Each cell was stained with 4 ', 6-diamidino-2-phenylindole (DAPI), a fluorescent dye that stains the nucleus during staining.
FIG. 6 shows images obtained by intravenously injecting Cy5.5-EM1-h protein into a tumor model mouse in which hEphA2-overexpressing cells (PC3) have been transplanted subcutaneously into a tumor and in vivo. FIG. 6A is a photograph of a bio-optical image obtained on day 5 after re-administration of Cy5.5-EM1-h 1 day after (-) the EM1-h Fn3 protein administration (+ A PBS-administered mouse was used as a control. FIG. 6B is a confocal fluorescence microscope photograph showing the remaining Cy5.5 after staining the tumor tissue section obtained from the mouse described above with DAPI. FIG. 6C is optical image photographs of the tissues extracted from the mouse. FIG.

Definition of Terms:

"Fibronectin" is a glycoprotein that exists in the plasma and extracellular matrix (ECM), and contains two domains of type I, II, and III of the anti-parallel beta-sheet structure And has a plurality of connected structures. The I and II domains are characterized by the presence of disulfide bridges in the structure but not of the type III domain (FnIII). As used herein, the term "peptide derived from human fibronectin domain III (Fn3)" is defined as an artificial protein having a protein scaffold derived from the amino acid sequence of the 10th FnIII domain, , "Monobody ". In particular, the peptide derived from human fibronectin domain III (Fn3) specifically binding to human EphA2 according to an embodiment of the present invention is abbreviated as "anti-hEphA2 monobody ".

As used herein, the term "human ephrin type A receptor 2 " (hereinafter abbreviated as " hEphA2") refers to a protein belonging to the A type family (EphA) among the ephrin receptor family protein (Eph) in the protein- tyrosine kinase receptor group It is expressed on the cell surface as a membrane protein. Ephs are associated with embryonic development, stem cell differentiation, auto-apoptosis, and tumor development. Eph is structurally composed of an extracellular domain consisting of an ephrin-binding domain, a cysteine-rich region and two Fnlll-type repeats, an intracellular domain including a transmembrane domain and a kinase domain. Eph is divided into two groups, EphA and EphB, depending on affinity and amino acid similarity for ephrin ligand binding. EphA2 binds to the ephrin-A ligand. EphA2 is particularly useful as a marker protein for early onset of stomach, lung, breast and prostate cancer.

DETAILED DESCRIPTION OF THE INVENTION

According to one aspect of the present invention, there is provided a peptide derived from human fibronectin domain III (Fn3) consisting of the amino acid sequence of SEQ ID NO: 1 or 2, which specifically binds to human ephrin type-A receptor 2 (hEphA2).

In addition, the pharmaceutical composition according to an embodiment of the present invention may be a form in which another anti-cancer drug or anti-cancer protein is bound to the peptide by covalent or non-covalent bonds.

Therefore, according to another aspect of the present invention, there is provided a pharmaceutical composition for treating cancer comprising, as an active ingredient, a peptide conjugate wherein an anticancer drug or an anti-cancer protein is bound to the peptide by covalent bond or noncovalent bond.

Wherein the anticancer drug is selected from the group consisting of paclitaxel, 5-FU, docetaxel, tamoxifen, anastezole, carboplatin, topotecan, velotecan, imatinib, irinotecan, fluoxurdine, vinorelbine, gemcitabine But are not limited to, at least one selected from the group consisting of leuprolide, flutamide, zoledronate, cisplatin, doxorubicin, vincristine, hydroxyurea, streptozotocin and valvicin, The anti-cancer protein may be a protein toxin, an antibody specific for a cancer antigen, a fragment of the antibody, a tumor suppressor protein, or an antiangiogenic factor. At this time, the protein toxin is a botulinum toxin (Botulinum toxin), Te tanuseu toxin (Tetanus toxin), Shiga toxin (Shiga toxin), diphtheria toxin (Diphtheria toxin, DT), ricin (ricin), Pseudomonas exotoxin (Pseudomonas exotoxin, PE ), Cytolysin A (ClyA), and r-Gelonin. The tumor suppressor protein is a protein that inhibits tumor development. Typically, the tumor suppressor protein is VHL (von Hippel Lindau), APC (Adenomatous polyposis coli), CD95 (cluster of differentiation 95), ST5 (Suppression of tumorigenicity 5), YPEL3 ), p53, ST7 (Suppression of tumorigenicity 7) and ST14 (Suppression of tumorigenicity 14). The anti-angiogenic factor may be anti-VEGF antibody, angiostatin, endostatin, Kringle V domain of apolipoprotein, and the like.

In the pharmaceutical composition according to an embodiment of the present invention, the cancer may be any cancer as long as EhpA2 is a cancer over-expressed on the cell surface. Such cancer includes breast cancer, brain cancer, ovarian cancer, bladder cancer, pancreatic cancer, Cancer, prostate cancer, lung cancer, stomach cancer, and colon cancer. Cancer in which hEphA2 is overexpressed as described above is well described in the literature (Tandon, M. et al ., E xpert Opin. Ther. Targets , 15 (1): 31-51 , 2011)

The pharmaceutical composition according to an embodiment of the present invention may be administered to the individual by parenteral administration, and the parenteral administration may be intravenous injection, intraperitoneal injection, intramuscular administration injection may be subcutaneous injection, intraventricular injection, intracranial injection, intracerebrospinal injection, or intratumoral injection.

The pharmaceutical composition according to one embodiment of the present invention further comprises an inert ingredient, including a pharmaceutically acceptable carrier. As used herein, "pharmaceutically acceptable carrier" is a term referring to a composition, specifically a component other than the active ingredient of the pharmaceutical composition. Examples of pharmaceutically acceptable carriers include binders, disintegrants, diluents, fillers, lubricants, solubilizers or emulsifiers and salts.

In addition, the pharmaceutical composition according to one embodiment of the present invention may be administered at a dose of 0.1 mg / kg to 1 g / kg, more preferably at a dose of 0.1 mg / kg to 500 mg / kg . On the other hand, the dose can be appropriately adjusted according to the age, sex and condition of the patient.

According to another aspect of the present invention, there is provided a method of treating cancer comprising administering a therapeutically effective amount of the peptide to a cancerous individual. Said object comprising a rabbit neck, a horse, a donkey, a rhinoceros including a rabbit and a crying rabbit including a fowl, a rats, a hamster, a hamster, a mouse, and a guinea pig, including an animal including an animal, a dog, a lion, a tiger, , And headgear including cattle, cow, deer, goat, sheep, and nutrition, and equipment including elephants, and may be mammals other than humans.

In the composition for tumor screening, the contrasting compound may be a fluorescent protein, a fluorescent molecule, a radioisotope contrast agent for positron emission tomography (PET), a single photon emission computer tomography (SPECT) Radioactive isotope contrast agent, or magnetic resonance imaging (MRI) contrast agent.

In this case, the fluorescent protein may be selected from the group consisting of a green fluorescent protein (GFP), a yellow fluorescent protein (YFP), a red fluorescent protein (RFP), an orange fluorescent protein (OFP) , Cyan fluorescent protein (CFP), blue fluorescent protein (BFP), far-red fluorescent protein, or tetracystein motif. Herein, the green fluorescent protein is expressed by EGFP (Enhanced Green Fluorescent Protein), Emerald (Tsien, Annu. Rev. Biochem ., 67: 509-544, 1998), Superfolder (Pedelacq et al ., Nat. Biotech . -88, 2006), GFP (Prendergast et al ., Biochem ., 17 (17): 3448-3453, 1978), Azami Green (Karasawa, et al ., J. Biol. Chem ., 278: 34167-34171, 2003), TagGFP (Evrogen, Russia), TurboGFP (Shagin et al ., Mol Biol Evol ., 21 (5): 841-850, 2004), ZsGreen (Matz et al ., Nat Biotechnol . 3: 5, 2003), and the yellow fluorescent protein may be EYFP (enhanced yellow fluorescent protein, Tsien, Annu. Rev. 2003) or T-Sapphire (Zapata-Hommer et al ., BMC Biotechnol . Biochem ., 67: 509-544, 1998), Topaz (Hat et al ., Ann. NY Acad. Sci ., 1: 627-633, 2002), Venus (Nagai et al ., Nat Biotechnol . 1): 87-90, 2002), mCitrine (Griesbeck et al, J. Biol Chem, 276:... 29188-29194, 2001), Ypet (Nguyet and Daugherty, Nat Biotechnol, 23 (3):.. 355 -360, 2005), TagYFP (Evrogen , Russia), PhiYFP (Shagin e ... t al, Mol Biol Evol, 21 (5):.. 841-850, 2004), ZsYellow1 (Matz et al, Nat. Biotechnol. , 17: 969-973, 1999) or mBanana (Shaner et al., Nat Biotechnol ., 22: 1567-1572, 2004), and the red fluorescent protein is mRuby (Kredel et al ., PLoS ONE , 4 (2): e4391, 2009), mApple (Shaner et al ., Nat. Methods , 5 (6): 545-551, 2008), mStrawberry (Shaner et al ., Nat Biotechnol ., 22: 1567-1572, 2004), mRFP (Campbell et al ., Proc. Natl. Acad. Sci. USA , 99 (12): 7877-7882, 2004), AsRed2 (Shanner et al ., Nat Biotechnol ., 22: 1567-1572, 2002), and the orange fluorescent protein can be obtained from Kusabira Orange (Karawawa et al ., Biochem. J. , 381 (Pt 1): 307-312, 2004), Kusabira Orange 2 (MBL International Corp., Japan), mOrange Shaner et al, Nat Biotechnol, 22 :... 1567-1572, 2004), mOrange2 (Shaner et al, Nat Biotechnol, 22:..... 1567-1572, 2004), dTomato (Shaner et al, Nat Biotechnol Tetem (Shaner et al ., Nat. Biotechnol ., 22: 1567-1572, 2004), TagRFP (Merzlyak et al ., Nat. 555-557, 2007), TagRFP-T (Shaner et al ., Nat Methods , 5 (6): 545-551, 2008), DsRed (Baird et al ., Proc. Natl. Acad. Sci. USA , 97: 11984-11989, 1999) DsRed2 (Clontech, USA), DsRed-Express (Clontech, USA), DsRed-Monomer (Clontech, USA) or mTangerine (Shaner et al ., Nat. Biotechnol . (Cibitt et al ., Trends Biochem. Sci ., 20: 448-455, 1995), mECFP (Ai et al ., Biochem. J. , 91 (12): L99-L101, 2006), CyPet (Nguyet and Daugherty, Nat Biotechnol ., 2002), mCerulean (Koushik et al ., Biophys. 381 (1999)), Midori-Ishi Cyan (Karawawa et al ., Biochem ., 381 (1999)), AmCyan1 (Matz et al ., Nat Biotechnol ., 17: 969-973, 1999) Pt 1): 307-312, 2004) , TagCFP (Evrogen, Russia) or mTFP1 (Ai et al, Biochem J. , 400 (3):.. may be 531-540, 2006), the blue fluorescent protein is EBFP2 (Ai et al ., Biochemistry , 46 (20): 5904-5910, 2007), Azurite (Mena et al ., Nat. Biotechnol ., 24: 1569-1571 , 2006) or mTagBFP (Subach et al ., Chem. Biol ., 15 (10: 1116-1124, 2008), and the primary red fluorescent protein is mPlum (Wang et al ., Proc. Natl. Acad. Sci. USA , 101: 16745-16749, 2004), mCherry (Shanner et al ., Nat. Biotechnol ., 22: 1567-1572, 2004), dKeima-Tandem (Kogure et al . 226, 2008), JRed (Shagin et al ., Mol. Biol. Evol ., 21 (5): 841-850, 2004), mRaspberry (Shanner et al ., Nat Biotechnol ., 22: 1567-1572, 2004 ), HcRed1 (Fradkov et al ., Biochem. , 368 (Pt 1): 17-21, 2002), HcRed-Tandem (Fradkov et al ., Nat Biotechnol ., 22 (3): 289-296, sequences 649-654, 2005) may be, wherein the tetra-cysteine motif is Cys-Cys-Xaa-Xaa- Cys-Cys ( SEQ ID NO: 6): 2004) or AQ143 (Shkrob et al, Biochem J , 392... Wherein Xaa may be an amino acid other than cysteine.

Wherein the fluorescent molecule is selected from the group consisting of fluorescein, fluorescein isothiocyanate (FITC), Oregon green, and Texas red Xanthene) derivatives; (Iii) a compound selected from the group consisting of Cy2, Cy3, Cy3B, Cy3.5, Cy5, Cy5.5, Cy7, indocarbocyanine, rhodamine, oxacarbocyanine, thiacarbocyanine, Cyanene derivatives selected from the group consisting of merocyanine; An oxadiazole derivative selected from the group consisting of pyrodyloxazole, nitrobenzoxadiazole, and benzoxadiazole; An acridine derivative selected from the group consisting of Nile red, Nile orange, and acridine yellow; An arylmethine derivative selected from the group consisting of aumarine, crystal violet, and malachite green; A tetrapyrrole derivative selected from the group consisting of porphin, phthalocyanine and bilirubin; And X-SIGHT, Pz 247, DyLight 750, DyLight 800, Alexa Fluor 680, Alexa Fluor 750, IRDye 680, IRDye 800CW and indocyanine green and zwitterionic near-infrared fluorophores. And a near infrared light fluorescent material selected from the group consisting of NIR fluorophore. Particularly, in the case of the above near-infrared light fluorescent material, it is possible to perform imaging of biomolecules deeper in depth than fluorescence molecules emitting ordinary visible light rays, and thus it is a molecule suitable for in vivo fluorescence imaging of large animals such as humans.

In the tumor imaging composition, the PET contrasting radioisotope-containing contrast agent may be any compound containing any radionuclide used for PET imaging. Examples of the radioisotope include C-11, N A radioactive isotope having a short half life such as -13, O-15, F-18, Ru-82, Ga-68, Cu-60, Cu-61, Cu- -11, N-13, O-15, and F-18 can be included in the peptide as a member of any organic compound that is easy to bind to the peptide and the most frequently used F- There is a single-step F-18 labeling method using 4-nitro-3-trifluoromethyl arene as a precursor for F-18 labeling (Jacobson et al ., Bioconjug. Chem . 22 (3): 422-428, 2011). Metal radioactive isotopes such as Ru-82, Ga-68 and Cu-64 are known as DTPA (diethylenetriamimepentaacetic acid), EDTA (ethylenediaminetetraacetic acid), DOTA (1,4,7,10-tetraazacyclodocecane- N, N "-tetraacetic acid), DPTA (2- [Bis [2- [bis ( carboxymethyl) amino] ethyl] amino] acetic acid, BTS (thiosemicarbazone), PnAO (propyleneamine oxime), DADT (diaminedthiol), MAG3 (mercapto- acetylglycylglycylglycine) and AAZTA , 4-diazepine-tetraacetic acid) to form an anti-hEphA2 monobody according to one embodiment of the present invention.

Hereinafter, the present invention will be described in more detail with reference to Examples and Experimental Examples. It should be understood, however, that the invention is not limited to the disclosed embodiments, but may be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, Is provided to fully inform the user.

Example

Example 1: Isolation of hEphA2 specific Fn3 protein from yeast surface marker library

The yeast surface marker G4 library (2.5 × 10 ⁸ variety) was constructed by transforming EBY100 yeast with pCT, a surface marker expression vector containing the Fn3 gene with variants on three different binding loop sequences (BC, DE and FG) Hackel, BJ, et al ., J. Mol. Biol ., 401 (1): 84-96, 2010), and Sam Gambhir, Stanford University. The yeast library was screened as previously reported (Hackel, BJ, et al ., J. MoI. , Supra) in combination with magnetic beads and recombinant-activated cell sorting (FACS) using recombinant hEphA2 . Biol ., 401 (1): 84-96, 2010). Yeast separation of the primary is 6.7 pmole biotin-hEphA2 protein (R & D systems, USA) for one turn FACS for cMyc- positive yeast two times separated by the magnetic beads (Dynabeads ^ㄾ Biotin Binder, Life Technologies, USA) combined . A new library for the next order was constructed by performing error-prone PCR (Chao, G. et al ., Nat. Protoc . 1 (2): 755-768, 2006; Lipovsek, D. et al ., J. Mol. Biol ., 368 (4): 1024-1041, 2007). A total of four yeast isolations were performed to complete the screening. Yeast was further separated by double staining with hEphA2 and anti-cMyc antibodies using FACSAria Ⅲ (BD Biosciences, USA) after four separate rounds using magnetic beads as described above. Specifically, yeast was treated with 100 nM hEphA2 at room temperature for 2 hours. After washing with PBS (PBSA) containing 0.1% BSA, the cells were washed with mouse anti-hEphA2 antibody (R & D Systems, USA, 1:80 dilution) and chicken anti-cMyc IgY antibody (Invitrogen, USA, 1:80 dilution) Lt; RTI ID = 0.0 > 4 C < / RTI > Then, yeast Alexa 488- are coupled anti-mouse IgG Fab (Invitrogen, USA, 1 : 4000 dilution) and Alexa 555- coupled goat anti-chicken Ig antibodies in a ^{4 o C (Invitrogen, USA,} 1:80 dilution) And stained for 30 minutes. The yeast of the strongest hEphA2 signal region in the double positive region was isolated by FACSAria III. The isolated yeast was cultured in 5 ml of SD-CAA medium at 30 ° C, 250 rpm for 1 day, and the yeast centrifuged at 5,000 rpm for 1 minute was suspended in SG-CAA medium. The FACS isolates were stained twice with 10 and 1 nM EphA2, respectively, and the finally isolated yeast isolate was named 4.5 fractions (Fig. 1A). The 4.5-fold yeast expressed Fn3 protein on the yeast surface with a much greater affinity for hEphA2 than in the G4 library yeast (Fig. 1C).

The plasmid was isolated from the yeast of 4.5 fractions by a Zymoprep Yeast Plasmid Miniprep II kit and transformed into E. coli DH5a and amplified. Ten independent plasmids were isolated from independent E. coli colonies from an LB agar medium containing the antibiotic ampicillin and nucleotide sequencing was performed. Two groups (SEQ ID NO: 1 EM1 of SEQ ID NO: 2). Nine plasmids belong to the EM1 group and only one belong to the EM2 group. Among the plasmids contained in the EM1 group, there were one or two mutations in the framework amino acid sequence. Since the two plasmids pCT-EphA2 (EM1) and pCT-EphA2 (EM2) did not show any mutation in the Fn3 protein basic framework, they were selected as the representative plasmids of each group and the anti- EphA2 monobody Were named EM1 and EM2, respectively (Fig. 1B). Both EM1 and EM2 proteins have the same amino acid sequence except for the DE loop and their amino acid sequence and length in the BC and FG loops were completely different from wild-type Fn3 (SEQ ID NO: 3). Two plasmids were used to transform each wild-type EBY100 yeast. In the FACS analysis, the transformed yeasts were similar to the 4.5-fold yeasts isolated in staining with 1 nM hEphA2 (Fig. 1C).

Example 2 Measurement of Binding Capacity (Kd Dissociation Constant) of Yeast Expressing Fn3 Protein to hEphA2

The binding force of the Fn3 protein expressed in yeast to the target protein (hEphA2) was evaluated by measuring the dissociation constant (Kd). Yeast (2x10 ⁶ ) transformed with the plasmid cultured in the SGCAA medium were bound overnight with hEphA2 at a concentration of 0.03-100 nM at room temperature and analyzed by FACS after staining with the antibody combination described in Example 1. The average intensities of Alexa 488 and Alexa 555 in the double positive population of each yeast sample were obtained and plotted proportional to the concentration of hEphA2 used in each sample. The Kd values are given in Prism ver. Alexa 488] / mean [Alexa 555] to hEphA2 levels using a software (Graphpad, USA) (Fig. 2). The dissociation constants (Kd) were 1.84 nM and 2.092 nM for EM1 and EM2, respectively. These values were similar to the general affinity of commercial antibodies.

Example 3: E. coli From Fn3 protein purification

To construct an Escherichia coli expression vector for Fn3 protein, each Fn3 gene was fused with 94oldF primer (5'-TACATATG GCTAGC GTTTCTGATGTTCCGAG-3 ', SEQ ID NO: 7, SEQ ID NO: 7) for pCT-Fn3-EphA2 (EM1) and pCT- underlined was amplified with Nhe I recognition site) and 94oldR primer (5'-TACTGAGT GGATCC TGTTCGGTAATTAATGGAAATTGG-3 ', SEQ ID NO: 8, underlined is the polymerase chain reaction (PCR) as a Bam HI recognition site) primer combination. The amplified PCR fragments were cleaved at both ends with Nhe I and Bam HI restriction enzymes and bound to the same restriction sites of pETh or pETamh, an E. coli expression vector. pETh and pETamh were derived from the pET Escherichia coli expression vector (Novagen, USA), followed by the Nhe I restriction site immediately after the ATG start codon at the 5'- site and the Bam Hl restriction enzyme site at the 3'- Is an expression vector constructed to be located in front of the nucleotide sequence stop codon corresponding to h or amh tag, respectively. Thus, the present inventors have named the monobodies EM1, which are expressed by connecting the h and amh tags at the C-terminus, respectively, as EM1-h and EM1-amh, respectively. Likewise, EM2-h and EM2-amh were designated as monobody EM2 in which h and amh tags are linked to each other at the C-terminal of EM2. Fn3 protein purification was performed in E. coli BL21 (DE3) transformed with the pET expression vector described above. The grown single colonies were inoculated into 10 ml LB medium containing kanamycin (50 μg / ml). After overnight culture, the strain was inoculated with 1 L of LB medium and cultured at 37 DEG C and 250 rpm for 3 hours. 1 ml of 0.5 M IPTG was added to the cultured Escherichia coli culture, followed by further incubation at 37 ° C for 1-3 hours. The cultured Escherichia coli was centrifuged at 3,200 xg for 10 minutes to obtain an Escherichia coli precipitate. Cold Digestion Buffer _{3 ml [50 mM NaPO 4 (} pH 8.0), 0.5M NaCl, 5% glycerol, 5 mM CHAPS, 25 mM Imidazole , and EDTA-free protease inhibitor (Roche Applied Science, USA)] E. coli precipitate was suspended in Was disrupted four times with 60 W ultrasound and then centrifuged at 12,000 x g for 5 minutes. The obtained supernatant Fn3 protein was separated into an imidazole concentration gradient on an AKTA FPLC equipped with a HisTrap FF colume (GE Healthcare Biosciences, PA). The separated proteins were acidified with triflouroacetic acid and further purified by C4 semi-prep reverse phase chromotography and then dried. The dried Fn3 protein was dissolved in DMSO or PBS, and the concentration was measured using UV spectrometry. Purified Fn3 protein was obtained 2-5 mg in 1 L culture.

Example 4: Specific binding assay of Fn3 protein to hEphA2 target protein

The magnetic beads bound to the target protein were prepared as described in the yeast isolation method using the magnetic beads of Example 1. In 100 μl PBSA, hEphA2 conjugated magnetic beads were bound to 10 nM Fn3 protein for 30 min at room temperature. The magnetic beads were stained with 40 μl of FITC-conjugated anti-HisTag antibody (1: 100, Abcam, USA) and analyzed by FACS (FIG. After binding the human IgG or lysozyme protein conjugated magnetic beads used as a control with the EM1 and EM2 proteins, fluorescence levels similar to those of the original beads were shown. In contrast, the magnetic beads bound with hEphA2 showed high binding affinity to the EM1 and EM2 proteins regardless of the type of tag. Here, EM1 and EM2 proteins with two different tag types were used, but there was no significant difference in their binding to the hEphA2 target protein.

Since EphA2 is a member of the EphA receptor family and has high homology with other members in the amino acid sequence, the binding ability of Fn3 proteins to these member proteins was analyzed by ELISA analysis (Fig. 4). 100 μl of 1 μg / ml recombinant Eph receptor protein was dispensed into each well of a 96-well ELISA plate (Costar, Sigma, USA) and coated overnight at 4 ° C. The next day, each well was washed three times with PBS (PBST) containing 0.05% Tween 20 and treated with 300 μl of inhibition buffer (PBST containing 5% BSA) for 2 hours at room temperature. Fn3 proteins (EM1-amh and EM2-amh) with a 100 μl diluted concentration of amh tag were added to each well and allowed to bind at room temperature for 2 hours. Each well was washed with PBST and treated with 100 μl horse radish peroxidase (HRP) -conjugated mouse anti-cMyc antibody (1: 1,000, Invitrogen, USA) for 30 min at room temperature. After washing and drying, the wells were reacted with 100 μl of substrate [1: 1 mixture of substrate (TMB) and hydrogen peroxide, Thermo scientific, USA] at room temperature for 5 minutes. The reaction was stopped with 50 μl of 1 MH ₂ SO ₄ , and the degree of color development was read at 450 nm with an ELISA reader. EM1-h and EM2-h increased the binding strength to hEphA2 and mEphA2 in a concentration-dependent manner. Although EM1-h protein concentration of 100 nM did not show significant binding to other homologous proteins, EM2-h proteins also significantly bound to mEphA8 and mEphA6. These results suggest that EM1 binds more specifically to hEphA2 than EM2.

Example 5: Measurement of expression level of hEphA2 target protein on cell surface

The relative amount of total hEphA2 protein expressed in the cells was determined by Western blot analysis (Fig. 5A). The cultured cells (1 × 10 ⁶ ) were suspended in SDS sample buffer, boiled for several minutes, and the supernatant was separated on 10% SDS-PAGE. Proteins were transferred to the nitrocellulose membrane. hEphA2 was detected as a luminescence sample after staining with alkaline phosphatase (AP) -conjugated goat anti-mouse IgG after primary staining with mouse anti-hEphA2 antibody. Western blot for beta-actin protein was performed on the same membrane from which the antibody was removed for quantitative control of each sample. As a result, hEphA2 band was strongly detected in human-derived prostate cancer PC3 cells, but not in breast cancer SKBR3 cells, as shown in Fig. 5a.

The amount of hEphA on the cell surface was quantified as FACS compared to the Bangs bead standard (Bangs laboratories, IN) coated with an anti-rat antibody. Specifically, PC3 and SKBR3 cells (2.5 × 10 ⁵ ) were reacted with 100 μl mouse anti-hEphA2 antibody (1: 100, R & D systems, USA) on ice for 30 min, washed, suspended in 50 μl PBSA mu] l of FITC-conjugated rat anti-mouse IgG (1:50, BioLegend, USA) and left on ice for 30 minutes (Fig. At the same time, one drop (50 μl) of each anti-mouse-IgG standard Bangs beads was also mixed with 50 μl of FITC-conjugated rat anti-mouse IgG and left on ice for 30 minutes. After washing, the average amount of light was obtained from each cell and standard Bangs bead through FACS analysis (FIG. 5C). The standard curves of the average amount of light intensity versus concentration were prepared using the average amount of light of each Bangs bead, and the number of molecules of hEphA2 was calculated on the cell surface. As a result, as shown in Fig. 5C, the molecular number of hEphA2 on the surface of PC3 cells was about 6 times higher than that of SKBR3 cell surface.

Example 6: Specific binding of Fn3 protein to cell surface hEphA2 target protein

Binding of the Fn3 protein to the cell surface hEphA2 target protein was stained with Cy5.5-conjugated EM1 protein (Cy5.5-EM1-h) and analyzed by FACS and cell staining (Fig. 5d and 5e). Specifically, the EM1-h protein suspended in 0.1 M sodium carbonate buffer (pH 9.5) was chemically coupled with Cy5.5-NHS (BioActs, Korea) dissolved in DMSO at a ratio of 1: 2 for 16 hours at 4 ° C , Cy5.5-NH1 was removed using a PD-10 desalting column (GE Healthcare, USA), and purified by C18 analytical HPLC. The fluorescence labeling-EM1 protein thus generated was labeled with Cy5.5-EM1-h Respectively.

Then, human prostate cancer PC3 cells and breast cancer SKBR3 cells (0.5 x 10 ⁵ ), which had different surface expression levels of hEphA2 suspended in 100 μl of PBSA, were stained with 100 nM Cy5.5-EM1-h for 2 hours at room temperature FACS (Figure 5d). As a result, as shown in FIG. 5D, Cy5.5-EM1-h showed a high amount of fluorescence in the PC3 cells overexpressing hEphA2 on the cell surface, but in the low-expressed SKBR3 cells, There was no difference.

In addition, the present inventors stained cells incubated in the culture medium with an antibody combination such as FACS analysis (mouse anti-hEphA2 antibody and Alexa555-conjugated rat anti-mouse IgG) and Cy5.5-EM1-h and compared fluorescence microscope images (Fig. 5E). As a result, anti-hEphA2 antibody and Cy5.5-EM1-h stained PC3 cells stained similarly, but no fluorescence was observed in SKBR3 cells, as shown in Fig. 5e.

Example 7: Analysis of specific binding force of Fn3 protein on the cell surface hEphA2 target protein in vivo

Next, in order to confirm whether the Fn3 protein detects a target protein hEphA2 present in vivo, xenograft tumor model mice in which PC3 cells were transplanted to generate tumors were prepared, and Cy5.5-EM1 -h were intravenously injected to investigate whether fluorescence was observed in the tumor several days later (Fig. 6). Animal experiments were performed according to the protocol presented by Chonnam National University Animal Experiment Committee (Gwangju, Korea).

More specifically, 5-6-week-old male BALB / c athymic ^{(athymic) nu - / nu -} mouse (Orient Company, Korea) and then anesthetized with 2% isoflurane to avoid the PC3 cells (1x10 ^8/100 μl PBS) Transplantation A xenograft tumor model mouse was prepared. After 21-28 days transplantation, 150 mm ³ tumor size (0 day) was injected intravenously with unlabelled EM1-h (30 μg) or PBS as a control. On day 1, Cy5.5-EM1-h (6 μg) was intravenously injected to the same mouse. On the 6th day, optical images of whole body and extracted tumor tissues were obtained by IVIS 100 system (Caliper, USA) (FIG. In mice pretreated with unlabeled EM1-h protein, the optical image intensity (1.24) of tumor was lower than that of untreated mouse tumor (1.53). The tumor optical image intensity was a value obtained when the PBS treatment group was regarded as 1. These results demonstrate that unlabeled EM1-h and Cy5.5-EM1-h competitively bind to hEphA2, demonstrating that EM1-h specifically binds to hEphA2 in PC3 tumor tissue .

Next, the present inventors extracted tumor tissue sections from the model mice and observed the amount of residual Cy5.5 fluorescence on the flakes with a confocal fluorescence microscope (FIG. 6B). Cy5.5-fluorescently stained cells were observed in mouse tumor tissues not pretreated with unlabeled EM1-h protein, but fluorescently stained cells were rarely observed in pretreated mouse tumor tissues. This was the same as observed in the in vivo optical image.

On the other hand, organs including the tumor were excised from the sacrificed rats, and the remaining optical images of each organ were observed (Fig. 6C). As a result, fluorescence was detected in some of the irradiated organs other than tumor tissue, but the intensity was lower than that of tumor tissue.

As described above, according to one embodiment of the present invention, since the Fn3-derived polypeptide specifically binding to hEphA specifically binds to EphA2 or another kind of EphA protein, it is possible to inhibit hEphA2 in vivo or in vivo Of EphA2 protein and can be used effectively for the diagnosis of diseases such as tumors overexpressing hEphA2 through binding with various contrast agents and is very small in size compared to conventional antibodies. Therefore, hEphA2 overexpressing tumor And the like.

Although the present invention has been described with reference to the above embodiments, it is to be understood that various changes and modifications may be suggested to those skilled in the art without departing from the scope of the present invention. Accordingly, the true scope of the present invention should be determined by the technical idea of the appended claims.

<110> INDUSTRY FOUNDATION OF CHONNAM NATIONAL UNIVERSITY <120> A novel hEphA2-specific binding protein having human fibronectin III domain scaffold and use thereof <130> PD15-5204 <160> 8 <170> KoPatentin 3.0 <210> 1 <211> 90 <212> PRT <213> Artificial Sequence <220> <223> Anti-hEphA2 monobody EM1 <400> 1 Val Ser Asp Val Pro Arg Asp Leu Glu Val Val Ala Ala Thr Pro Thr 1 5 10 15 Ser Leu Leu Ile Ser Trp Tyr Tyr Pro Phe Cys Ala Phe Tyr Tyr Arg 20 25 30 Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro Val Gln Glu Phe Thr 35 40 45 Val Pro Arg Ser Pro Asp Thr Ala Thr Ile Ser Gly Leu Lys Pro Gly 50 55 60 Val Asp Tyr Thr Ile Thr Val Tyr Ala Val Thr Cys Leu Gly Ser Tyr 65 70 75 80 Ser Arg Pro Ile Ser Ile Asn Tyr Arg Thr 85 90 <210> 2 <211> 92 <212> PRT <213> Artificial Sequence <220> <223> anti-hEphA2 monobody EM2 <400> 2 Val Ser Asp Val Pro Arg Asp Leu Glu Val Val Ala Ala Thr Pro Thr 1 5 10 15 Ser Leu Leu Ile Ser Trp Tyr Tyr Pro Phe Cys Ala Phe Tyr Tyr Arg 20 25 30 Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro Val Gln Glu Phe Thr 35 40 45 Val Pro Arg Ser Tyr Lys Ala Pro Ser Ala Thr Ile Ser Gly Leu Lys 50 55 60 Pro Gly Val Asp Tyr Thr Ile Thr Val Tyr Ala Ala Thr Cys Leu Gly 65 70 75 80 Ser Tyr Ser Arg Pro Ile Ser Ile Asn Tyr Arg Thr 85 90 <210> 3 <211> 94 <212> PRT <213> Artificial Sequence <220> <223> 10th fibronectin domain III <400> 3 Val Ser Asp Val Pro Arg Asp Leu Glu Val Val Ala Ala Thr Pro Thr 1 5 10 15 Ser Leu Leu Ile Ser Trp Asp Ala Pro Ala Val Thr Val Arg Tyr Tyr 20 25 30 Arg Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro Val Gln Glu Phe 35 40 45 Thr Val Pro Gly Ser Lys Ser Thr Ala Thr Ile Ser Gly Leu Lys Pro 50 55 60 Gly Val Asp Tyr Thr Ile Thr Val Tyr Ala Val Thr Gly Arg Gly Asp 65 70 75 80 Ser Pro Ala Ser Ser Lys Pro Ile Ser Ile Asn Tyr Arg Thr 85 90 <210> 4 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> His6 tag <400> 4 His His His His His 1 5 <210> 5 <211> 33 <212> PRT <213> Artificial Sequence <220> <223> Avidin-cMyc-His6 (am) tag <400> 5 Gly Leu Asn Asp Ile Phe Glu Ala Gln Lys Ile Glu Trp His Glu Glu 1 5 10 15 Gln Lys Leu Ile Ser Glu Glu Asp Leu Arg Ser His His His His 20 25 30 His <210> 6 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> tetra-cysteine motif <400> 6 Cys Cys Xaa Xaa Cys Cys 1 5 <210> 7 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> 94oldF primer <400> 7 tacatatggc tagcgtttct gatgttccga g 31 <210> 8 <211> 38 <212> DNA <213> Artificial Sequence <220> <223> 94oldR primer <400> 8 tactgagtgg atcctgttcg gtaattaatg gaaattgg 38

Claims

1. A peptide derived from human fibronectin domain III (Fn3) comprising the amino acid sequence of SEQ ID NO: 1 or 2, which specifically binds to human ephrin type-A receptor 2 (hEphA2).

A peptide conjugate wherein the anti-cancer drug or the anti-cancer protein is bound to the peptide of claim 1 by covalent or non-covalent bond.

A pharmaceutical composition for treating cancer comprising the peptide of claim 1 or the peptide conjugate of claim 2 as an active ingredient.

The method of claim 3,
Wherein said cancer is cancer in which hEphA2 is overexpressed.

3. The method of claim 2,
The anticancer drugs include paclitaxel, 5-FU, docetaxel, tamoxifen, anastrozole, carboplatin, topotecan, velotecan, imatinib, irinotecan, fluoxidyne, vinorelbine, gemcitabine, leuprolide, Wherein the peptide conjugate is at least one selected from the group consisting of flutamide, zoledronate, cisplatin, doxorubicin, vincristine, hydroxyurea, streptozotocin, and valvicin.

3. The method of claim 2,
The anticancer protein may be a protein toxin, a cancer antigen-specific antibody or a fragment of the antibody, a tumor suppressor protein or an antiangiogenic factor,

A composition for molecular imaging of hEphA2 comprising the peptide of claim 1 as an active ingredient.

A peptide conjugate wherein the imaging compound is bound to the peptide of claim 1 by covalent or non-covalent association.

9. The method of claim 8,
The contrast agent may be a fluorescent protein, a fluorescent molecule, a radioisotope contrast agent for positron emission tomography (PET) contrast, a radioisotope contrast agent for single photon emission computer tomography (SPECT) Magnetic resonance imaging (MRI) contrast agent, a peptide conjugate.

A composition for detecting hEphA2 comprising the peptide conjugate of claim 8 as an active ingredient.

A composition for diagnosing or screening cancer, comprising the peptide conjugate of claim 8 as an active ingredient.

12. The method of claim 11,
Wherein said cancer is cancer in which hEphA2 is overexpressed.

Administering to the subject a cancer diagnostic or contrast composition of claim 11; And
A method of diagnosing a tumor in an animal other than a human, comprising the step of obtaining an image by said composition.

Administering the cancer diagnostic or contrast composition of claim 11 to a subject having cancer;
Obtaining an image by said composition; And
The method comprising the step of ablating the region in which the signal from the composition is displayed.