WO2014168230A1

WO2014168230A1 - CONJUGATE COMPOSED OF EpCAM-BINDING PEPTIDE APTAMER AND PHOSPHORYLCHOLINE POLYMER COPOLYMER

Info

Publication number: WO2014168230A1
Application number: PCT/JP2014/060458
Authority: WO
Inventors: 芝　清隆; 和浩日比野; 光孝吉田
Original assignee: 公益財団法人がん研究会
Priority date: 2013-04-12
Filing date: 2014-04-11
Publication date: 2014-10-16
Also published as: JPWO2014168230A1; JP5824596B2

Abstract

The present invention addresses the problem of providing a peptide conjugate, which is capable of functionalizing carriers formed of various materials by an EpCAM-binding peptide, a diagnostic device using the same and a separation device using the same. A solid phase can be easily coated by preparing a conjugate that is composed of a phosphorylcholine group-containing polymer [in particular, a copolymerized polymer comprising a 2-methacryloyloxyethylphosphorylcholine (MPC) and a hydrophobic unit] and a peptide capable of binding to EpCAM. The aforesaid conjugate also interacts with EpCAM molecules and exosomes and, therefore, is widely applicable as a diagnostic or therapeutic tool.

Description

Complex comprising peptide aptamer binding to EpCAM and phosphorylcholine polymer copolymer

The present invention relates to a method for preparing a complex of a peptide aptamer capable of binding to EpCAM and a phosphorylcholine polymer copolymer, and immobilizing the peptide aptamer using the complex to prepare a material surface having affinity for EpCAM. The present invention relates to a method and a tool for diagnosis and treatment using the complex.

Epidermal cell adhesion factor (EpCAM; Epithelial cell hesadhesion molecule, CD326, GA733-2, HEA125, KS1 / 4, MK-1, MH99, MOC31, 323 / A3, 17-1A, CO-17A, ESA, EGP-2, EGP34, EGP40, KSA, KS1 / 4, TROP-1, and TACST-1) are type I membrane proteins reported as cancer-specific cell surface antigens expressed in colorectal cancer (Non-patent Document 1). EpCAM is mainly expressed in the basement membrane of epithelial cells in normal tissues, but expression is observed in most cancer cells derived from epithelium, so that it is known as a so-called cancer antigen and is shown to be useful as a diagnostic marker. (Non-Patent Document 2).

As described above, since EpCAM is detected in most epithelial cancer cells, it has been attempted to collect cells having EpCAM antigen on the surface using an antibody, analyze this, and predict the prognosis. Since circulating tumor cells expressing EpCAM circulating in blood as a surface antigen are closely related to cancer metastasis, detection of circulating tumor cells expressing EpCAM is very important for prognosis prediction. Yes (Patent Document 1). In addition, attention has been focused on diagnostic methods using extracellular vesicles such as exosomes and microvesicles in body fluids, and here also the relationship between cancer and EpCAM positive extracellular vesicles has been reported (non-patented). Reference 3).

Furthermore, in recent years, the usefulness of EpCAM has been shown not only in diagnosis but also in treatment. Cancer vaccines using EpCAM have already been developed, and vaccine therapy using an anti-idiotype antibody that binds to the antigen recognition site of an EpCAM protein or anti-EpCAM antibody prepared using an insect cell expression system such as baculovirus is also available. It has been reported. In addition, anti-EpCAM antibodies for cancer treatment have been developed, and clinical trials using anti-EpCAM antibodies such as Adecatumumab (MT201) and ING-1 are being conducted. The treatment using these antibodies aims to reduce the cancer by binding the antibody to EpCAM on the surface of cancer cells and inducing cellular immunity (cytotoxic activity) by the in vivo immune system. Furthermore, for the purpose of enhancing the cytotoxic activity by the anti-EpCAM antibody, Proxinium Vivendiums (VB4-845) fused with anti-EpCAM antibody and Pseudomonas aeruginosa exotoxin, or EMD 273-066 (huKS-IL2 fused with IL-2) ), And an EU approved drug that has anti-CD3 activity, such as Katumamaximab (Non-patent Documents 4 and 5).

As described above, antibody molecules have been used for treatment and diagnosis targeting EpCAM. However, antibody molecules have the advantage of binding strongly to the target molecule, but this “strong binding” is often a drawback. For example, when an antibody binds to a target receptor on the cell surface, a signal transmission system downstream of the receptor is activated, or acid treatment for removing the bound antibody damages the cell. These drawbacks can be fatal when considering treatment using cells and regenerative medicine.

Therefore, specific but weak binding, not strong binding using an antibody, realizes cell culture, cell sorting, etc. in a flow channel system or macro flow channel system utilizing the dynamic properties of fluids. Needed above. In order to realize such specific and weak binding, a peptide aptamer is an effective technical means instead of an antibody.

The present inventors have already disclosed a peptide capable of binding to EpCAM, which can be easily prepared using chemical synthesis or genetic engineering techniques (Patent Document 2). In addition, the development of peptides having the ability to bind to EpCAM is being promoted.

Special table 2008-533587 gazette JP 2011-193728 A

When using a peptide capable of binding to EpCAM in a solid phase, there may be a method of directly binding or a method of increasing ligand binding using a linker. As a method for immobilizing an EpCAM-binding peptide, it is essential to establish a high-yield and stable binding method for binding to many solids of different materials. In fact, there are various materials and materials such as polycarbonate (PC), polydimethylsiloxane (PDMS), polystyrene (PS), polyethylene terephthalate (PET), epoxy resin, glass, and silicon in the flow path system of the target device. It is used. In addition, there are various shapes of materials such as beads, plates, films and filaments. Although it is possible to immobilize peptides on the surface of these materials using covalent bonds, it is necessary to consider a reaction system tailored to the material, and the solid phase on the surface of the material without reactive groups It becomes extremely difficult.

On the other hand, the “coating agent” approach using physical bonding has the advantage of wide material selectivity and high versatility. Therefore, the present invention has an object to develop a method for functionalizing a material as a material surface having affinity for a specific biomolecule by combining a peptide / aptamer with a coating agent, that is, a biointerface or a functional biosurface. .

Here, functional biosurfaces are created using peptides and aptamers that have affinity for EpCAM, but other aptamers and the like can be used to create functional biosurfaces that have affinity for molecules other than EpCAM. Can also be applied.

An object of the present invention is to create a material surface having affinity for EpCAM for use in cell therapy, regenerative medicine, diagnosis, and the like. For cell separation for cell therapy and regenerative medicine, a flow path system on the plate and a column filled with beads are used, and a solid phase such as beads and chips is used as a carrier for diagnosis etc. Therefore, there is a strong demand for a method for efficiently immobilizing a developed peptide aptamer capable of binding to EpCAM on a solid phase. An object of the present invention is to provide a complex of a peptide / aptamer capable of binding to EpCAM and a high molecular weight polymer, wherein the solid phase is simply a functional biosurface having the ability to bind EpCAM.

The realization of a material surface having affinity for specific biomolecules such as biointerfaces and functional biosurfaces according to the present invention is important for use in a wide range of fields such as diagnosis and treatment using biosensors and cells. Tool.

The present invention is characterized in that it is a complex of a polymer containing a phosphorylcholine group and a peptide capable of binding to EpCAM.

Since the phosphorylcholine group is a component of a biological membrane, it is very useful when creating a biofunctional surface that detects the interaction of biological components such as EpCAM molecules and EpCAM-expressing exosomes.

Further, the complex of the present invention is a complex of a copolymer having a phosphorylcholine group containing 2-methacryloyloxyethyl phosphorylcholine (MPC) and a hydrophobic unit, and a peptide capable of binding to EpCAM. It is characterized by that.

Among the polymers having a phosphorylcholine group, a high molecular polymer for immobilizing an EpCAM-binding peptide is, in particular, a copolymer containing 2-methacryloyloxyethyl phosphorylcholine (MPC) and a hydrophobic unit (for example, 2-methacryloyloxyethyl phosphorylcholine / n -Butyl methacrylate copolymer, 2-methacryloyloxyethyl phosphorylcholine / allylamine hydrochloride copolymer, etc.) are preferably used. This is because the high molecular weight polymer containing MPC has very good biocompatibility.

According to the complex of the present invention, a peptide capable of binding to EpCAM can be efficiently immobilized on a solid phase. Therefore, wide application to diagnosis and treatment using EpCAM as a target is possible. Furthermore, by using the MPC copolymer, various materials including glass, polycarbonate, and silica can be coated, and a biofunctional surface can be easily created.

In the complex of the present invention, the peptide having binding ability to EpCAM is an amino acid sequence represented by SEQ ID NO: 1 (KHLQCVRNICWS; Ep114), SEQ ID NO: 2 (EHLHCLGLSLCWP; Ep133), or SEQ ID NO: 3 (KSLQCINLCWP; Ep301). It is characterized by being a peptide having binding ability to EpCAM.

The peptide Ep114 of SEQ ID NO: 1 and the peptide Ep133 of SEQ ID NO: 2 disclosed by the present inventors show strong binding ability to EpCAM. These two peptides bind about 10 times stronger than the peptide Ep301 of SEQ ID NO: 3 disclosed by the present inventors in Patent Document 2. Therefore, EpCAM can be detected with high sensitivity.

In addition, if Ep301 having a relatively weak binding to the EpCAM molecule is used, it can be an effective tool when it is desired to avoid applying a strong stimulus to the cell.

Therefore, the composite material using the peptide aptamer is expected to have various applications in terms of diagnosis and treatment. In particular, if a synthetic peptide is used, it becomes easy to ensure safety in terms of treatment.

In addition, it is possible to easily produce a large amount of EpCAM-binding peptide by using a DNA or a recombinant vector encoding these peptides and using a known genetic engineering technique or a chemical synthesis technique. Therefore, it is possible to manufacture diagnostic materials and the like at low cost.

The composite material of the present invention is obtained by immobilizing the complex according to the present invention on a carrier and has an EpCAM affinity.

As described above, the composite of the present invention is spin-coated with various materials such as polycarbonate (PC), polydimethylsiloxane (PDMS), polystyrene (PS), polyethylene terephthalate (PET), epoxy resin, glass, and silicon glass. It can be easily coated by standing or standing. Therefore, it can be functionalized and applied to diagnosis, treatment, etc. by coating various shapes of carriers such as beads, plates, films, filaments, membranes, column carriers, flow paths, medical materials, etc. Be expected.

The separation apparatus of the present invention comprises a composite material having affinity for the EpCAM of the present invention, and is characterized by fractionating EpCAM-expressing cells, EpCAM-expressing exosomes, EpCAM-expressing microvesicles, or EpCAM molecules based on the expression of EpCAM And

For example, by coating the complex of the present invention on a channel system carrier, an apparatus for efficiently separating EpCAM-expressing cells, exosomes, and microvesicles can be produced. Therefore, circulating tumor cells, exosomes, and microvesicles expressing EpCAM can be detected with high sensitivity, and cancer can be diagnosed with high accuracy.

Further, separation that has been conventionally performed using antibodies, such as cell selection, exosomes, microvesicles, and purification of EpCAM, can be performed by the separation apparatus of the present invention. Furthermore, since it is possible to design the affinity for EpCAM as a peptide aptamer, it is possible to create a separation apparatus having a desired affinity. In other words, using a material surface with a moderate affinity, rather than strongly binding cells, exosomes, and microvesicles, the movement is delayed (retardation effect) while suppressing motility by weak interaction. A device that sorts in the flow path can be realized. In addition, in the case of such weak binding, it is possible to avoid damage caused to cells due to excessive stimulation, which is a problem with strong binding of antibodies, etc., and unexpected activation of signal transmission system It becomes possible.

Furthermore, since the retardation effect can be changed by adjusting the flow rate and flow pressure, the target substance can be selectively separated in various modes. This advantage is qualitatively different from conventional methods such as a method using a strong antigen antibody, a method of sieving by molecular weight, and a method of fractionating adsorption by charge or hydrophobicity.

The diagnostic apparatus of the present invention is characterized by comprising a measurement unit that measures the degree of EpCAM expression using the composite material of the present invention.

Detecting EpCAM expression by detecting circulating tumor cells, exosomes, microvesicles, etc. is very useful for cancer diagnosis and prognosis. By coating a carrier with the complex of the present invention, a highly accurate diagnostic apparatus can be easily prepared.

The figure which shows typically the preparation method of the composite_body | complex of the MPC copolymer and the peptide aptamer which has the binding ability to EpCAM. The PEG linker used for the coupling | bonding to the MPC copolymer of a peptide aptamer is shown. The figure which shows the amount of EpCAM binding peptide shown on the glass surface by the length of a linker. A linker (hereinafter referred to as PEG2 linker) structure containing two dPEG ₂ on the C-terminal side of the Ep114 peptide. The figure which shows the quantity of the EpCAM binding peptide shown on the glass surface at the time of using a PEG2 linker. The figure which shows the result of having coat | covered the polystyrene bead with the EpCAM binding peptide-MPC complex using a PEG2 linker. The figure which shows the result of the surface elemental analysis by X-ray photoelectron spectroscopy (XPS) of the glass surface which coated the EpCAM binding peptide-MPC complex. The figure which shows that various solid-phases can be coated by EpCAM binding peptide-MPC complex. The figure which shows that the support | carrier coated with the EpCAM binding peptide-MPC complex is recognized by an anti-EpCAM antibody, EpCAM, and an exosome. The figure which shows the example which coated the silica bead with the EpCAM binding peptide-MPC complex, and was packed in the flow-path system.

Hereinafter, the present invention will be described in detail with reference to examples.
[Example 1]
[Method for producing EpCAM-binding peptide-MPC complex]
FIG. 1 schematically shows a method for producing a complex of the MPC copolymer of the present invention and a peptide capable of binding to EpCAM. Conjugation of MPC with a peptide capable of binding to EpCAM via a polyethylene glycol (PEG) linker to an MPC copolymer polymer comprising a hydrophobic unit and a unit having a carboxyl group, that is, EpCAM-binding peptide-MPC complex Create a body.

The detailed peptide introduction method is as follows. MPC copolymer (NOF Corporation, MPC polymer, Lipidure-5903S) After adding 10 equivalents of PEG linker (Quantadesignbiodesign) to ethanol solution, triazine condensing agent 4- (4,6-Dimethoxy-1,3, 50 equivalents of 5-triazin-2-yl) -4-methylmorpholinium Chloride n-Hydrate (DMT-MM) is added and allowed to react overnight at room temperature. Thereafter, the solution is put into a dialysis tube (Spectra / Por (trademark) Dialysis Membrane MWCO: 8000), and the external solution is dialyzed for 2 days. After recovery from the dialysis tube, the solution is concentrated to the original reaction volume using an evaporator.

Subsequently, 2 ml of methanol and 2.5 ml of H ₂ O are added to 0.5 ml of the methanol solution of the MPC copolymer introduced with the linker. Subsequently, after adding 10 equivalent of CGK (FITC) GG (propargyl) introduced to the C-terminal side of the EpCAM-binding peptide, the peptide is dissolved by sonication.

Next, 0.5M sodium ascorbate aqueous solution (Wako Pure Chemical Industries, Ltd.) is added, and when the yellow color in the test tube becomes dark, 0.5M copper sulfate aqueous solution is added. When the color changes from yellow to black-brown, the test tube is sealed and heated at 50 ° C. for 30 minutes.

After completion of the reaction, add 0.5M ethylenediaminetetraacetic acid tetrasodium (Dojindo) aqueous solution, pH 8, and transfer to the above dialysis tube. Add 30 ml of 0.5 M ethylenediaminetetraacetate aqueous solution, pH 8 to 3 L of methanol / milli-Q water mixed solution as an external solution, and perform dialysis for 1 week. After dialysis for 2 days, changing the external solution to a solution not containing ethylenediaminetetraacetic acid aqueous solution, changing the external solution to methanol only, dialysis was performed for 24 hours, and then the solution in the dialysis tube was concentrated with an evaporator. EpCAM binding peptide-MPC complex was synthesized.

[Example 2]
[Examination of linker length for peptide introduction rate]
The length of the PEG linker used and the rate of peptide introduction were examined. Using the PEG linkers (Quanta biodesign) having different chain lengths shown in FIG. 2, the introduction rate of Ep114 into the MPC copolymer was determined.

The peptide introduction rate was measured as follows. The peptide introduced into the MPC copolymer was synthesized by synthesizing GGK (FITC) GG (propargyl), a peptide in which FITC was added to the C-terminal of the Ep114 peptide, and measuring the FITC absorbance in the Ep114 (FITC) peptide-MPC complex. The rate was determined.

Specifically, the spectrum was measured with an ultraviolet-visible spectrophotometer (UV-2550, Shimadzu Corporation), the absorption maximum value of FITC at 495 nm and the absorbance at 800 nm were measured, and a calibration curve prepared in advance was used. The peptide concentration was measured to determine the peptide introduction rate. The results are shown in Table 1.

As shown in Table 1, the amount of peptide introduced into the MPC copolymer varies depending on the length of the linker used. When a PEG linker with a short chain length, dPEG3, is used, the peptide introduction rate is extremely low, but when other linkers are used, the values are all 10% or more. A PEG linker with a short chain length has a low introduction rate, and as shown below, the binding property to EpCAM is also low. However, if the chain length is dPEG7 or more, a high introduction rate can form a functional surface material. it can.

[Example 3]
[Examination of linker length and amount of EpCAM-binding peptide presented as functional molecule]
The obtained EpCAM-binding peptide-MPC complex was immobilized on a solid phase, and the amount of functional EpCAM-binding peptide presented was examined. Immobilization to the solid phase was performed by the following two methods: spin coater method and stationary method.

(1) Spin coater method A glass plate is subjected to ultraviolet ozone treatment for 10 minutes, then immersed in methanol, sonicated for 5 minutes, and washed. 20 μl of EpCAM-binding peptide-MPC complex solution was spin-coated with a spin coater. Then, after air-drying for 30 minutes, it is vacuum-dried for 2 hours. Then, it leaves still at 4 degreeC in PBS (Phosphate buffered saline) for 15 hours.

(2) Standing method After washing the glass plate by the same method as described above, 50 μl of EpCAM-binding peptide-MPC complex solution is placed on the surface of the glass plate and left at 4 ° C. overnight. After standing, the excess EpCAM-binding peptide-MPC complex solution is removed with a spin coater, dried and treated with PBS as described above.

The amount of EpCAM-binding peptide after coating was compared by antibody staining. Here, Ep114 is used as the EpCAM-binding peptide. The glass on which the EpCAM-binding peptide-MPC complex was immobilized was blocked with a blocking agent (Nacalai, Blocking One) for 1 hour and washed three times with TBS (Tris buffered saline) containing 0.05% by weight of Tween20.

After blocking, anti-Ep114 rabbit IgG was used as the primary antibody, and HRP-labeled goat anti-rabbit IgG was used as the secondary antibody. After the reaction with each antibody, detection is performed by washing with TBS containing 0.05% by weight of Tween 20 and chemiluminescence with an ECL solution. The results are shown in FIG.

(A) shows the result of the spin coater method, and (B) shows the result of the stationary method. In the figure, B-3, B-7, B-11, B-23, and B-35 are dPEG3, dPEG7, dPEG11, dPEG23, and dPEG35, respectively. It shows that. MPC refers to an immobilized MPC copolymer not bound with a peptide.

In any of the spin coater method (A) and the stationary method (B), the reaction with the anti-Ep114 antibody is observed, so that the EpCAM-binding peptide may be immobilized on the glass carrier. Admitted. The short linker, dPEG3 (B-3), had a low introduction rate into the MPC copolymer, and no EpCAM bond was detected.

As shown in Table 1, the peptide introduction rate of the Ep114 peptide into the MPC copolymer showed the highest value when dPEG23 was used as a linker, but the EpCAM-binding peptide detected by the antibody is shown in FIG. As shown in FIG. 4, no significant difference is observed between dPEG7, dPEG11, dPEG23, and dPEG35.

The detection of the EpCAM-binding peptide shown in FIG. 3 by the antibody differs from the peptide introduction rate shown in Table 1 in that the peptide exists as a state recognized by the antibody, that is, as a state that can be recognized by the antibody as a functional molecule. Show. The difference between the amount of peptide introduced and the amount of peptide recognized by the antibody is because the amount of peptide introduced and the amount of peptide present as a functional molecule differ depending on the chain length of the linker. Seem. When the linker length is 3 (dPEG3), the peptide accessibility is poor, so the peptide introduction rate is low, and as a result, the amount of functional peptide recognized by the antibody is considered to be small.

From the above results, it was shown that the solid phase is efficiently coated with functional Ep114 by the EpCAM-binding peptide-MPC complex.

[Example 4]
The linker structure on the C-terminal side of the Ep114 peptide to be introduced was examined. Using the Fmoc-N-amido-dPEG ₂ -acid (manufactured by Quanta biodesign) at the C-terminus of the Ep114 peptide, the Ep114 peptide added with PEG was synthesized using a peptide synthesizer.

Peptide containing FITC, Ep114-dPEG ₂ -K (FITC) -dPEG ₂ -G (propargyl) -NH ₂ , and peptide not containing FITC, Ep114-dPEG ₂ -S-dPEG ₂ -G (propargyl) The structure of —NH ₂ is shown in FIG.

[Example 5]
The Ep114-MPC complex produced using the peptide produced in Example 4, Ep114-dPEG ₂ -K (FITC) -dPEG ₂ -G (propargyl) -NH ₂ , was spin-coated on a glass plate in the same manner as in Example 3. The samples were coated by the method and compared by antibody staining. Note that the Ep114 peptide is bound to MPC via dPEG7. The results are shown in FIG.

GGK (FITC) is Ep114-GGK (FITC) GG (propargyl) used in Example 2, and PEG2 (FITC) is Ep114-dPEG ₂ -K (FITC) -dPEG ₂ -produced in Example 4. G (propargyl) -NH ₂ , PEG ₂ represents Ep114-dPEG ₂ -S-dPEG ₂ -G (propargyl) -NH ₂ each formed into a complex with an MPC copolymer, and MPC represents a peptide A raw glass in which only an unbound MPC copolymer is fixed, and Raw glass indicate that nothing is fixed.

As shown in FIG. 5, Ep114-dPEG ₂ -K (FITC) -dPEG ₂ -G (propargyl) -NH ₂ represented by PEG2 (FITC) is particularly good for the antibody, and Ep114-dPEG represented by PEG 2 ₂ -S-dPEG ₂ -G (propargyl) -NH ₂ was shown to be almost as reactive as GGK (FITC) (Ep114-GGK (FITC) GG (propargyl).

Furthermore, the sample was coated on polystyrene beads and observed using a confocal microscope (OLYMPUS FLUOVIEW 1000). A complex of PEG2 (FITC) or GGK (FITC) and MPC is fixed to polystyrene beads, and an anti-Ep114 antibody prepared by immunizing rabbits is used, Zenon Rabbit IgG labeling reagent (Molecular-Probes) is used, and Alexa Labeled with Fluor (trademark) 594 and observed with a confocal microscope. Note that both use dPEG7 as a linker in preparing a complex of MPC and peptide. In addition, rabbit IgG (SANTA CRUZ BIOTECHNOLOGY) is used as a control. The results are shown in FIG.

When PEG2 (FITC) is compared with GGK (FITC), the staining property with the anti-Ep114 antibody is very good, indicating that the peptide appears efficiently on the surface of the MPC complex.

[Example 6]
[Surface elemental analysis using X-ray photoelectron spectroscopy (XPS)]
Surface analysis of the glass coated with EpCAM-binding peptide-MPC complex was performed by XPS.

A glass plate coated with the Ep114 peptide-MPC complex was measured using XPS (ULVAC-PHI® PHI5000® VersaProbe). Each sample is centered at three locations on both ends. Pass Energy: 117.4eV, Lower Energy: 0eV, Range: 1400eV, Energy step: 1eV, Time / step: 20ms, Total cycle: 10, X-ray width 100μm Scanned.

Figure 7 shows the results. Since the same result was obtained in three places where the measurement was performed, an example is shown. As shown in the enlarged portion, MPC-derived phosphorus (P) was detected on the glass surface, and it was confirmed that MPC was coated on the surface. However, the copper element used for the binding reaction between the linker and the peptide was not detected. The fact that copper is not detected indicates that the EpCAM-binding peptide-MPC complex of the present invention can be used safely in vivo, such as for treatment.

Also, the two large peaks shown in the enlarged portion indicate glass-derived silicon. In observation with a scanning electron microscope, the coated glass surface is smooth, but it is recognized that a portion where the glass surface is exposed remains. However, as a result of the film thickness measurement, the coated EpCAM-binding peptide-MPC complex had a film thickness of about 30 nm, so that the film thickness was not entirely thin and the glass surface was partially exposed. It is thought that there is a part.

[Example 7]
[Coating of various solid phases with EpCAM-binding peptide-MPC complex]
When a peptide capable of binding to EpCAM is used for diagnosis and treatment, it is expected that various materials such as beads and channels are used as a carrier. In addition to glass, various materials such as polypropylene and polycarbonate are known as materials used as these carriers. Therefore, it was investigated whether these materials could be coated with the prepared EpCAM-binding peptide-MPC complex.

As in the case of the glass carrier shown in Example 3, a carrier material such as polypropylene was coated with an EpCAM-binding peptide-MPC complex by a stationary method, and detection was performed using an anti-Ep114 antibody.

Figure 8 shows the results. The following materials are used as the carrier: (A) polypropylene, (B) vinyl chloride, (C) polycarbonate, (D) polystyrene, (E) polyethylene terephthalate (PET), (F) silicon, (G ) Hydrophilic polydimethylsiloxane (PDMS), (H) Hydrophobic polydimethylsiloxane.

In the figure, + indicates an Ep114-binding peptide-MPC complex, MPC only coats an MPC copolymer, and-indicates a region where nothing is coated. The upper row shows that the anti-Ep114 antibody was used as the primary antibody, and the lower row used the rabbit IgG antibody as a control as the primary antibody.

As shown in FIG. 8, any of these materials widely used as carriers could be efficiently coated with the EpCAM-binding peptide-MPC complex of the present invention. Although not shown here, it has been confirmed that gold is also efficiently coated. Furthermore, none of the carriers has any background and only specific binding has been observed.

In addition, it has been confirmed by fluorescence observation that when a silicon rubber-based channel or a polydimethylsiloxane-based channel is actually coated using the composite of the present invention, the channel can be efficiently coated.

Therefore, it was shown that the EpCAM-binding peptide-MPC complex of the present invention is very useful as a coating agent for a carrier material generally used for measurement, inspection, and treatment, such as a flow channel system and beads.
[Example 8]
[Application using gel carrier, binding to exosomes]
The gel carrier was coated with an EpCAM-binding peptide-MPC complex, and it was analyzed whether it interacted with an anti-EpCAM-binding peptide antibody, EpCAM, and exosome.

Specifically, Silica gel 60 (nacalai tesque) was coated with Ep114 peptide-MPC complex (dPEG7 was used as the linker). It was analyzed whether the gel carrier carrying the Ep114 peptide interacts with the anti-Ep114 antibody, purified EpCAM, and exosome.

(1) Interaction with anti-Ep114 antibody The gel carrier was blocked with 2% BSA for 30 minutes, washed with PBS three times, and immunized to a rabbit. The anti-Ep114 antibody prepared by Zenon Rabbit IgG labeling reagent (Molecular Probes) ) Was labeled with Alexa Fluor (trademark) 594, the gel carrier was labeled with this anti-Ep114-labeled antibody, and observed with a confocal microscope. Rabbit IgG (SANTA CRUZ BIOTECHNOLOGY) was used as a control. A result is shown to FIG. 9 (B) (1).

(2) Interaction with EpCAM After blocking and washing as described above, purified EpCAM was added, and the mixture was shaken and reacted for 30 minutes. After washing with PBS three times, the reaction was performed with a mouse anti-EpCAM antibody (Gene Tex, VU-1D9) antibody labeled with Alexa Fluor (trademark) 594 Zenon Mouse IgG Labeling Kits. After washing 3 times with PBS, it was observed with a confocal microscope. A result is shown to FIG. 9 (B) (2).

(3) Interaction with exosomes Cells release proteins and amino acids from the cytoplasm as small membrane vesicles called exosomes. It has been revealed that exosomes are involved in signal transmission between cells, and in recent years, it has been clarified that cancer cells release a large amount of exosomes and are deeply involved in cancer metastasis and formation. ing. Based on this phenomenon, it has been disclosed that exosomes are used for treatment and diagnosis (for example, see Non-Patent Documents 6 and 7). Therefore, it was analyzed whether the EpCAM-binding peptide-MPC complex of the present invention interacts with exosomes.

Isolation and labeling of exosomes were performed by the following method. HT-29 cells are cultured, the culture supernatant is centrifuged (5,500 g, 10 minutes), a microfiltration membrane Stericup filter unit (Membrane Type Millipore Express (R) PLUS (PES), Pore Size Rating 0.22μm) (Milliopore Corporation ) And ultracentrifuged at 160,000 g for 70 minutes to recover exosomes. Furthermore, sucrose density gradient centrifugation (100,000 g, 17 hours) of 0.25-2M sucrose, 20 mM Hepes, pH 7.2 was performed, and the exosome fraction was isolated. The isolated exosome fraction was washed with PBS and collected by ultracentrifugation (160,000 g, 2 hours). The collected exosomes were suspended in PBS and labeled by the following method.

Exosome labeling was performed using DiI (invitrogen). Dilute 20 μl of isolated exosome with 980 μl of PBS, add 5 μl of DiI, mix, and let stand at 37 ° C. for 1 hour. After ultracentrifugation (120,000 g, 90 minutes), the supernatant is removed, and 1% BSA / PBS is added and mixed. Ultracentrifugation (120,000 g, 90 minutes) was repeated twice to obtain labeled exosomes.

The labeled exosome was blocked and washed in the same manner as in (1), then allowed to interact with the carrier and observed with a confocal microscope. A result is shown to FIG. 9 (B) (3).

As shown in FIG. 9 (B), the interaction between the anti-EpCAM binding peptide antibody, EpCAM, and exosome and the EpCAM binding peptide on the carrier was confirmed in any case. The above results indicate that EpCAM-expressing cells and exosomes interact with the biofunctional surface coated with the complex of the present invention. In particular, the fact that interaction with exosomes has been demonstrated is very important in considering diagnostic and therapeutic applications targeting exosomes.

[Example 9]
[Application to flow path system]
An application example in which silica beads are coated with the EpCAM-binding peptide-MPC complex of the present invention and packed in a flow path system is shown.

Heat the silica gel at 115 ° C. for 15 minutes. Then, after cooling at room temperature for 15 minutes, the gel is replaced with methanol. Put Ep114 peptide-MPC complex and coat at 37 ° C. for 2 hours. After removing the solution, it is air-dried for 30 minutes and equilibrated with PBS for 15 hours.

The silica beads coated with the Ep114 peptide-MPC complex were packed in a microchannel (manufactured by microfluidic® ChipShop). The results are shown in FIG.

The upper part of FIG. 10 shows a transmitted light photograph in which silica beads are packed in a flow path. The middle and lower rows show FITC detected by a fluorescence microscope, while the middle row is filled with silica beads coated with PEG2 (FITC), and (C) is filled with uncoated silica beads. .

When the silica beads coated with PEG2 (FITC) were packed, fluorescence was observed, indicating that PEG2 (FITC) was bound to the silica beads. By coating beads and filling the flow path system, it can be used for a wide range of applications such as purification of EpCAM and diagnosis using patient specimens.

Although not shown here, the presence of the peptide on the carrier surface coated with the EpCAM-binding peptide-MPC complex as a functional molecule indicates that the QCM (crystal oscillator microbalance) sensor surface is attached to the EpCAM binding of the present invention. It has also been confirmed by coating with a peptide-MPC complex and interacting with exosomes.

In addition to the above-mentioned QCM, the measurement device to which the present invention is applied includes a fluorescence microscope, a fluorescence detection device such as a microplate reader, an optical detection device such as a dark field illumination microscope, a magnetic analysis device, and a zeta potential measurement. A known detection device such as a meter or an atomic force microscope can be used as the measurement unit.

As described above, the peptide 114 and the MPC complex have been studied. However, the peptide having a binding strength different from that of EpCAM, for example, the peptide Ep133 having a different affinity for the EpCAM already disclosed by the present inventors, By using Ep301, it is possible to create EpCAM-binding peptide-MPC complexes having different binding strengths to EpCAM. By using peptides having different binding capacities, it is possible to prepare EpCAM-binding peptide-MPC complexes that are suitable for applications such as cell separation and diagnosis.

According to the method of the present invention, it is possible to easily form various carriers as functionalized composite materials having EpCAM affinity with EpCAM-binding peptides. Therefore, various applications such as medical research, clinical measurement, testing, and development of therapeutic instruments are possible by using these for medical materials such as metallic titanium for flow channels, column carriers, magnetic beads, membranes, filters, etc. It becomes. The binding targets include all those in which EpCAM molecules are present, and include not only EpCAM-expressing cells but also EpCAM-expressing exosomes, microvesicles, and EpCAM molecules themselves. By arranging the functionalized composite material having the EpCAM affinity in a flow path system or the like and using it as a separation unit, those expressing the EpCAM molecule can be separated. EpCAM molecules have been found to release their extracellular domains into the blood under certain circumstances (see Non-Patent Document 8), and are also used to develop therapeutic devices that remove these extracellular domains from the blood. it can.

Furthermore, here, a composite material using an MPC copolymer has been examined using a peptide capable of binding to EpCAM, but various aptamers can be bound to the MPC copolymer by the method of the present invention. it can. As a result, it is possible to create a functional biosurface having affinity for various biomolecules, and a wide range of applications can be expected.

Claims

A complex of a polymer containing a phosphorylcholine group and a peptide capable of binding to EpCAM.
The complex according to claim 1,
A complex characterized in that the polymer containing a phosphorylcholine group is a copolymer polymer containing 2-methacryloyloxyethyl phosphorylcholine (MPC) and a hydrophobic unit.
The complex according to claim 1 or 2,
A complex characterized in that the peptide capable of binding to EpCAM is a peptide capable of binding to EpCAM consisting of the amino acid sequence shown in SEQ ID NO: 1 (KHLQCVRNICWS; Ep114).
The complex according to claim 1 or 2,
A complex characterized in that the peptide capable of binding to EpCAM is a peptide capable of binding to EpCAM consisting of the amino acid sequence shown in SEQ ID NO: 2 (EHLHCLGSLCWP; Ep133).
The complex according to claim 1 or 2,
A complex characterized in that the peptide capable of binding to EpCAM is a peptide capable of binding to EpCAM consisting of the amino acid sequence shown in SEQ ID NO: 3 (KSLQCINNLCWP; Ep301).
A composite material using the composite according to any one of claims 1 to 5,
A composite material having an affinity for EpCAM, wherein the complex is immobilized on a carrier.
A separation apparatus for separating an EpCAM-expressing cell, EpCAM-expressing exosome, EpCAM-expressing microvesicle, or EpCAM molecule using the composite material according to claim 6,
A separation apparatus comprising a separation unit including a composite material in which a complex having affinity for EpCAM is solid-phased.
A measuring device using the composite material according to claim 6,
A measuring apparatus comprising a measuring unit that measures the degree of EpCAM expression.