WO2014168230A1 - CONJUGÉ COMPOSÉ D'APTAMÈRE PEPTIDIQUE DE LIAISON À L'EpCAM ET DE COPOLYMÈRE DE POLYMÈRE DE PHOSPHORYLCHOLINE - Google Patents

CONJUGÉ COMPOSÉ D'APTAMÈRE PEPTIDIQUE DE LIAISON À L'EpCAM ET DE COPOLYMÈRE DE POLYMÈRE DE PHOSPHORYLCHOLINE Download PDF

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WO2014168230A1
WO2014168230A1 PCT/JP2014/060458 JP2014060458W WO2014168230A1 WO 2014168230 A1 WO2014168230 A1 WO 2014168230A1 JP 2014060458 W JP2014060458 W JP 2014060458W WO 2014168230 A1 WO2014168230 A1 WO 2014168230A1
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epcam
peptide
binding
complex
mpc
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Japanese (ja)
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芝　清隆
和浩 日比野
光孝 吉田
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Japanese Foundation for Cancer Research
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Japanese Foundation for Cancer Research
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M47/00—Means for after-treatment of the produced biomass or of the fermentation or metabolic products, e.g. storage of biomass
    • C12M47/04—Cell isolation or sorting
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54366—Apparatus specially adapted for solid-phase testing
    • G01N33/54386—Analytical elements
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/544—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being organic
    • G01N33/545—Synthetic resin
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/566—Immunoassay; Biospecific binding assay; Materials therefor using specific carrier or receptor proteins as ligand binding reagents where possible specific carrier or receptor proteins are classified with their target compounds
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/575—Immunoassay; Biospecific binding assay; Materials therefor for cancer

Definitions

  • 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 Non-Patent Document 2.
  • 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).
  • EpCAM EpCAM protein
  • 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.
  • antibody molecules have been used for treatment and diagnosis targeting EpCAM.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • shapes of materials such as beads, plates, films and filaments.
  • 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. .
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • MPC 2-methacryloyloxyethyl phosphorylcholine
  • hydrophobic unit for example, 2-methacryloyloxyethyl phosphorylcholine / n -Butyl methacrylate copolymer, 2-methacryloyloxyethyl phosphorylcholine / allylamine hydroch
  • 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.
  • 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.
  • Ep301 having a relatively weak binding to the EpCAM molecule it can be an effective tool when it is desired to avoid applying a strong stimulus to the cell.
  • the composite material using the peptide aptamer is expected to have various applications in terms of diagnosis and treatment.
  • a synthetic peptide is used, it becomes easy to ensure safety in terms of treatment.
  • EpCAM-binding peptide 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.
  • 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.
  • PC polycarbonate
  • PDMS polydimethylsiloxane
  • PS polystyrene
  • PET polyethylene terephthalate
  • epoxy resin glass
  • glass silicon glass
  • silicon glass silicon glass
  • 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
  • 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.
  • 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.
  • 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.
  • a highly accurate diagnostic apparatus can be easily prepared.
  • bonding to the MPC copolymer of a peptide aptamer is shown.
  • XPS X-ray photoelectron spectroscopy
  • 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.
  • PEG polyethylene glycol
  • 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.
  • PEG linker Quantadesignbiodesign
  • 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.
  • 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.
  • 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.
  • the amount of peptide introduced into the MPC copolymer varies depending on the length of the linker used.
  • a PEG linker with a short chain length dPEG3
  • 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.
  • 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.
  • 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.
  • a blocking agent Nacalai, Blocking One
  • (A) shows the result of the spin coater method
  • (B) shows the result of the stationary method.
  • 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.
  • 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.
  • 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.
  • 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 is Ep114-GGK (FITC) GG (propargyl) used in Example 2
  • PEG2 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
  • MPC represents a peptide A raw glass in which only an unbound MPC copolymer is fixed, and Raw glass indicate that nothing is fixed.
  • Ep114-dPEG 2 -K (FITC) -dPEG 2 -G (propargyl) -NH 2 represented by PEG2 (FITC) is particularly good for the antibody
  • 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).
  • 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 100 ⁇ m 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.
  • P MPC-derived phosphorus
  • the two large peaks shown in the enlarged portion indicate glass-derived silicon.
  • the coated glass surface is smooth, but it is recognized that a portion where the glass surface is exposed remains.
  • 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
  • various materials such as beads and channels are used as a carrier.
  • 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.
  • 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.
  • + 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.
  • 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.
  • 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.
  • a carrier material generally used for measurement, inspection, and treatment, such as a flow channel system and beads.
  • 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.
  • 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.
  • 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 0.22 ⁇ m) (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 ⁇ l of isolated exosome with 980 ⁇ l of PBS, add 5 ⁇ l 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.
  • DiI invitrogen
  • 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.
  • 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.
  • 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. .
  • 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.
  • 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 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 a magnetic analysis device
  • zeta potential measurement zeta potential measurement.
  • a known detection device such as a meter or an atomic force microscope can be used as the measurement unit.
  • the peptide 114 and the MPC complex have been studied.
  • 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.
  • Ep301 it is possible to create EpCAM-binding peptide-MPC complexes having different binding strengths to EpCAM.
  • 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.
  • 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.
  • 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.

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  • General Health & Medical Sciences (AREA)
  • Cell Biology (AREA)
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  • Medicinal Chemistry (AREA)
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  • Wood Science & Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biophysics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
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  • Gastroenterology & Hepatology (AREA)
  • Peptides Or Proteins (AREA)

Abstract

La présente invention aborde le problème de fourniture d'un conjugué peptidique, qui est apte à fonctionnaliser des supports formés de divers matériaux grâce à un peptide de liaison à l'EpCAM, concerne un dispositif de diagnostic l'utilisant et un dispositif de séparation l'utilisant. Une phase solide peut être facilement revêtue en préparant un conjugué qui est composé d'un polymère contenant un groupe phosphorylcholine [en particulier, un polymère copolymérisé comprenant une 2-méthacryloyloxyéthylphosphorylcholine (MPC) et un motif hydrophobe] et un peptide apte à lier à l'EpCAM. Le conjugué précédemment mentionné interagit également avec les molécules EpCAM et les exosomes et, par conséquent, est largement applicable pour servir d'outil de diagnostic ou d'outil thérapeutique.
PCT/JP2014/060458 2013-04-12 2014-04-11 CONJUGÉ COMPOSÉ D'APTAMÈRE PEPTIDIQUE DE LIAISON À L'EpCAM ET DE COPOLYMÈRE DE POLYMÈRE DE PHOSPHORYLCHOLINE Ceased WO2014168230A1 (fr)

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JP2018516358A (ja) * 2015-04-02 2018-06-21 バイオデシー， インコーポレイテッド 表面選択的非線形光学技法を使用してタンパク質構造を決定するための方法
JP7272590B2 (ja) 2017-07-11 2023-05-12 国立大学法人富山大学 細胞の選択的分離用又は細胞培養用ポリマーにより被覆された基体
WO2019013148A1 (fr) * 2017-07-11 2019-01-17 国立大学法人富山大学 Substrat recouvert de polymères pour la séparation sélective de cellules ou pour la culture cellulaire
CN110914309B (zh) * 2017-07-11 2023-10-24 国立大学法人富山大学 用于细胞的选择性分离或用于细胞培养的经聚合物被覆的基体
CN110914309A (zh) * 2017-07-11 2020-03-24 国立大学法人富山大学 用于细胞的选择性分离或用于细胞培养的经聚合物被覆的基体
JPWO2019013148A1 (ja) * 2017-07-11 2020-05-07 国立大学法人富山大学 細胞の選択的分離用又は細胞培養用ポリマーにより被覆された基体
TWI816680B (zh) * 2017-07-11 2023-10-01 國立大學法人富山大學 經細胞之選擇性分離用或細胞培養用聚合物被覆的基體
US11655273B2 (en) 2017-07-11 2023-05-23 National University Corporation University Of Toyama Substrates coated with selective cell separation or cell culture polymers
US20210130788A1 (en) * 2017-07-11 2021-05-06 National University Corporation University Of Toyama Substrates coated with selective cell separation or cell culture polymers
JPWO2019039179A1 (ja) * 2017-08-22 2020-06-18 国立大学法人広島大学 エクソソームの単離方法およびエクソソームの単離キット
WO2019039179A1 (fr) * 2017-08-22 2019-02-28 国立大学法人広島大学 Procédé d'isolement d'exosome et kit d'isolement d'exosome
US12117440B2 (en) 2017-08-22 2024-10-15 Hiroshima University Method for isolating exosome and exosome isolation kit
CN111393502B (zh) * 2019-01-03 2022-04-29 北京京东方技术开发有限公司 一种类肽及其制备方法和用途
CN111393502A (zh) * 2019-01-03 2020-07-10 北京京东方技术开发有限公司 一种类肽及其制备方法和用途
WO2020140530A1 (fr) * 2019-01-03 2020-07-09 京东方科技集团股份有限公司 Pseudopeptide, son procédé de préparation et ses applications
CN115053134A (zh) * 2019-11-11 2022-09-13 牛津大学创新有限公司 分离选择的外泌体群体的方法
CN115151822A (zh) * 2019-11-11 2022-10-04 牛津大学创新有限公司 用于预测和鉴定帕金森病的生物标志物
JP2023501479A (ja) * 2019-11-11 2023-01-18 オックスフォード ユニヴァーシティ イノヴェーション リミテッド 選択されたエキソソームの集団を単離する方法
WO2021094750A1 (fr) * 2019-11-11 2021-05-20 Oxford University Innovation Limited Procédé d'isolement d'une population sélectionnée d'exosomes
JP7680048B2 (ja) 2019-11-11 2025-05-20 オックスフォード ユニヴァーシティ イノヴェーション リミテッド 選択されたエキソソームの集団を単離する方法
WO2023157950A1 (fr) * 2022-02-18 2023-08-24 株式会社Ｌｓｉメディエンス Inhibiteur de réaction non spécifique

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