WO2016052971A1 - Conjugué herceptine-photosensibilisant destiné au diagnostic du cancer et son procédé de production - Google Patents

Conjugué herceptine-photosensibilisant destiné au diagnostic du cancer et son procédé de production Download PDF

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WO2016052971A1
WO2016052971A1 PCT/KR2015/010271 KR2015010271W WO2016052971A1 WO 2016052971 A1 WO2016052971 A1 WO 2016052971A1 KR 2015010271 W KR2015010271 W KR 2015010271W WO 2016052971 A1 WO2016052971 A1 WO 2016052971A1
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cancer
conjugate
maleimide
polyethylene glycol
herceptin
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PCT/KR2015/010271
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Korean (ko)
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나건
김경섭
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가톨릭대학교 산학협력단
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • 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
    • G01N33/531—Production of immunochemical test materials
    • G01N33/532—Production of labelled immunochemicals
    • G01N33/533—Production of labelled immunochemicals with fluorescent label
    • 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
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer

Definitions

  • the present invention relates to antibody-photosensitive agent conjugates for the diagnosis of cancer (particularly breast cancer) and a method for preparing the same. Specifically, the present invention relates to anti-HER2 antibodies (herceptin) and photosensitive agents specific to HER2 for diagnosing tumors expressing HER2. The present invention relates to a Herceptin-photosensitive agent conjugate that has shortened diagnosis time and improved tumor diagnosis ability by conjugation through chemical bonding.
  • Breast cancer is an advanced disease that is the most common cancer reported in the United States, and one in eight women can develop breast cancer during their lifetime. In Korea, after cancer, lung cancer, liver cancer and colorectal cancer, Korea accounted for 6.5 (5,444 patients) of all cancers in 2000. According to the Central Cancer Registration Report of the Ministry of Health and Welfare, women in 2002 were breast cancer. 16.8% of malignant tumors were ranked first. In breast cancer, early detection is more important than other cancers because it is difficult to cure when cancer cells invade surrounding tissues or begin to metastasize to lymph nodes.
  • Immunohistochemical staining a test method that complements these problems, confirms the origin of undifferentiated cells, the presence of enzymes, hormones, cancer markers and prognostic factors, the distinction between carcinomas and sarcomas, and the discrimination between benign and malignant tumors. It is efficiently used to estimate the primary origin of metastatic cancer.
  • a high sensitivity and specificity but high molecular weight (Antibody) is used in the conventional immunochemical staining method.
  • Antibody In order to label the tissue and cell permeability of the polymer antibody has a low problem.
  • conventional immunochemical staining is used to increase the tissue and cell membrane permeability of the polymer antibody by using a surfactant, but this may also lead to protein denaturation of the lesion tissue, there is a limit in certain disease detection.
  • it takes a long time to detect requires a high level of technology, and shows a disadvantage such as low accuracy, and thus cannot be used as the main method of cancer diagnosis.
  • the present inventors use a photosensitive agent (peophorbide and chlorine) that has been water solubilized using maleimide as a chemical linker, thereby recombining a herceptin (HER2 extracellular domain).
  • a photosensitive agent peophorbide and chlorine
  • maleimide as a chemical linker
  • Herceptin-photosensitizer conjugates are used in the field of immunochemistry because they can avoid the hassles of several steps of conventional immunochemical staining, block the problems of using surfactants, and increase tissue and cell permeability at the same time. You can expect a new large-scale market.
  • Herceptin-photosensitive agent conjugates as novel immunochemical staining materials that can enhance tissue and cell permeability of antibodies without the addition of surfactants.
  • Another object of the present invention a) connecting the biocompatible polymer and the optical sensor to prepare a primary conjugate; b) connecting the linker to the primary conjugate to produce a secondary conjugate; And c) linking the anti-HER2 antibody to the secondary conjugate, to provide a method for preparing a conjugate for diagnosing or treating cancer, wherein the antibody-linker-biocompatible polymer-photosensitive agent is sequentially connected.
  • the present invention provides a conjugate for diagnosing or treating cancer in which a biocompatible polymer and a photosensitive agent are sequentially connected to an anti-HER2 antibody.
  • the anti-HER2 antibody may be Trastuzumab.
  • the biocompatible polymer may be a polyethylene glycol having a molecular weight of 2,000 to 10,000.
  • the anti-HER2 antibody and the biocompatible polymer may be connected by a linker.
  • the linker may be maleimide.
  • the photosensitive agent is a porphyrin-based (phorphyrins) compounds, chlorins (chlorins) compounds, bacteriochlorins (bacteriochlorins) compounds, phthalocyanine (phtalocyanine) compounds, naphthalocyanines (naphthalocyanines) compounds
  • 5-aminolevulin ester-based (5-aminoevuline esters) compound may be selected from the group consisting of.
  • the cancer may be a cancer that overexpresses human epidermal growth factor receptor 2 (HER2).
  • HER2 human epidermal growth factor receptor 2
  • the cancer is breast cancer, squamous cell carcinoma, uterine cancer, cervical cancer, prostate cancer, head and neck cancer, pancreatic cancer, brain tumor, liver cancer, skin cancer, esophageal cancer, testicular cancer, kidney cancer, colon cancer, rectal cancer, It may be selected from the group consisting of gastric cancer, bladder cancer, ovarian cancer, bile duct cancer and gallbladder cancer.
  • the present invention comprises the steps of: a) connecting the biocompatible polymer and the photosensitizer to prepare a primary conjugate; b) connecting the linker to the primary conjugate to produce a secondary conjugate; And c) linking an anti-HER2 antibody to the secondary conjugate, wherein the antibody-linker-biocompatible polymer-photosensitive agent is sequentially connected to provide a diagnostic or therapeutic conjugate for cancer.
  • the biocompatible polymer may be a polyethylene glycol having a molecular weight of 2,000 to 10,000.
  • the linker may be maleimide.
  • the step a) is to increase the carboxyl reactivity of the photosensitizer by reacting the photosensitive agent in a solvent in the presence of a catalyst and then reacting by adding a solution in which the biocompatible polymer is dissolved It may be to include a process.
  • step a) and before the step b) further comprises the step of selectively separating and purifying only the conjugate having a one-to-one equivalent of the biocompatible polymer and the photosensitive agent as the primary conjugate It may be.
  • the step b) is to increase the carboxyl reactivity of the maleimide by reacting the linker maleimide dissolved in a solvent in the presence of a catalyst, and then reacted by adding a solution in which the primary conjugate is dissolved It may be to include a process.
  • step c) may include a process of reacting the secondary conjugates and the anti-HER2 antibody by dissolving each of them in a buffer.
  • the photosensitive agent is a porphyrin-based (phorphyrins) compounds, chlorins (chlorins) compounds, bacteriochlorins (bacteriochlorins) compounds, phthalocyanine (phtalocyanine) compounds, naphthalocyanines (naphthalocyanines) compounds It may be selected from the group consisting of 5-aminoevuline esters (5-aminoevuline esters) compound, pheophorbide a and chlorin e6.
  • chlorins chlorins
  • chlorins chlorins
  • bacteriochlorins bacteriochlorins
  • phthalocyanine phtalocyanine
  • naphthalocyanines naphthalocyanines
  • the anti-HER2 antibody may be trastuzumab (Trastuzumab).
  • the catalyst may be dicyclohexylcarbodiimide (N, N ⁇ -Dicyclohexylcarbodiimide, N-hydroxysuccinimide) alone or a mixture thereof.
  • a cancer diagnostic or therapeutic conjugate in which a biocompatible polymer and a photosensitive agent are sequentially connected to an anti-HER2 antibody expresses human epidermal growth factor receptor 2 (HER2) by including an anti-HER2 antibody in some components.
  • HER2 human epidermal growth factor receptor 2
  • Cancer can be targeted to form antibody-antigen bonds;
  • photosensitizers in some configurations activates the production of fluorescence and reactive oxygen (monotiary oxygen) in cancer cells to enable selective fluorescence imaging and photodynamic therapy of cancer;
  • polyethylene glycol in some components provides the hydrophilicity of photosensitive agents with poor solubility problems, and increases the tissue and solid permeability of antibodies with large molecular weights, thereby eliminating the need for the use of surfactants used in conventional immunochemical staining. Does not have the advantage.
  • the present invention when water-soluble polyethylene glycol is introduced into the photosensitive agent having a carboxyl group, the present invention shows higher solubility and higher generation efficiency of reactive oxygen species when irradiated with light, compared to the existing hydrophobic photosensitive agent. It is an immunochemical coloring material for breast cancer diagnosis that shortens the process and does not require a surfactant to compensate for the necessity of using a surfactant. It is expected to be applicable.
  • the conjugate (anti-HER2 antibody-maleimide-polyethylene glycol-photosensitive agent) of the present invention can be easily and easily conjugated to the anti-HER2 antibody through a stepwise and sequential method, In particular, by conjugating maleimide to a polyethylene glycol-photosensitive agent conjugated primary conjugate, stable anti-HER2 antibody (herceptin) conjugation is possible.
  • Figure 1 shows the 1H-NMR spectrum of HER-Mal-PEG-PheoA according to an embodiment of the present invention.
  • Figure 2 shows the 1H-NMR spectrum of the HER-Mal-PEG-Ce6 according to an embodiment of the present invention.
  • Figure 3 shows the molecular weight of HER-Mal-PEG-PheoA according to an embodiment of the present invention.
  • Figure 4 is an optical photograph showing the solubility before and after purification using hydrophobic chromatography of PEG-PheoA according to an embodiment of the present invention.
  • FIG 5 is an optical photograph showing the solubility of PEG2K-PheoA, PEG6K-PheoA, PEG2K-Ce6, PEG6K-Ce6 according to the polyethylene glycol molecular weight difference according to an embodiment of the present invention.
  • Figure 6 shows the fluorescence intensity of the HER-Mal-PEG-PheoA and HER-Mal-PEG-Ce6 in accordance with an embodiment of the present invention as a fluorescence imaging device picture.
  • Figure 7 is a mono-oxygen in the aqueous solution of HER-Mal-PEG2K-PheoA and HER-Mal-PEG6K-PheoA, HER-Mal-PEG2K-Ce6, HER-Mal-PEG6K-Ce6 according to an embodiment of the present invention ( singlet oxigen).
  • FIG. 8 shows breast cancer labeling ability of SK-BR-3 cells of Herceptin (8a), HER-Mal-PEG-PheoA (8b), and HER-Mal-PEG-Ce6 (8c) according to an embodiment of the present invention. To indicate.
  • FIG. 9 shows breast cancer tissue labeling ability of HER-Mal-PEG-PheoA according to an embodiment of the present invention.
  • Figure 10 shows the increase in tissue permeability of HER-Mal-PEG-PheoA in SK-BR-3 cancer model mice according to an embodiment of the present invention.
  • the present invention is characterized by providing a conjugate for diagnosing or treating cancer in which a biocompatible polymer and a photosensitive agent are sequentially connected to an anti-HER2 antibody.
  • the inventors of the present invention have the disadvantage of the conventional immunochemical coloring material, which has a characteristic that can generate monooxygen to overcome the problem of the fact that it takes several steps in the process of diagnosis and the feature of requiring a surfactant.
  • the sensory agent was intended to form a conjugate by chemical binding with an antibody against a target protein with increased expression in certain cancer cells (in the present invention, using Herceptin, an anti-HER2 antibody).
  • HER2 is an abbreviation of human epidermal growth factor receptor 2 and is an epidermal growth factor receptor (EGFR) family which is one of important signaling systems related to the proliferation and survival of breast cancer cells.
  • EGFR epidermal growth factor receptor
  • the tyrosine kinase receptors of the EGFR family are four, erb1, erb2 / HER2, erb3, and erb4.
  • erb1 erb1
  • erb2 / HER2 erb3
  • erb4 In addition to cell proliferation and survival, cell adhesion, migration and migration It is known to be involved in regulating differentiation.
  • no ligand binds erb2 / HER2, but it is known to be the most potent oncoprotein in breast cancer.
  • HER2 normal levels of HER2 are involved in the growth and development of normal mammary tissues, but abnormal HER2 overexpression or amplification leads to disruption of normal cell regulation, leading to the formation of aggressive cancer cells in mammary tissues. This process oligomerizes HER2 with other EGFR family members to phosphorylate many downstream molecules, which in turn activates several signaling cascades. Among them, the SOS-Ras-Raf-MEK-MAPK pathway involved in cell proliferation and the PI-3K / Akt pathway that inhibits apoptosis are representative mechanisms involved in cancer proliferation. Preclinical and clinical trials show that HER2 overexpression is an important phenomenon from the early stage of cancer development, which plays an important role in cancer growth and progression. HER2 overexpression is present in about 20-30% of invasive breast cancers. Overexpression is an aggressive cancer with higher malignancy and is associated with poor prognosis of breast cancer.
  • Herceptin is a trade name of Trastuzumab corresponding to a recombinant humanized monoclonal antibody targeting the extracellular domain of HER2.
  • the conjugate for diagnosing cancer of the present invention is composed of an anti-HER2 antibody-maleimide-polyethylene glycol-photosensitive agent, and the anti-HER2 antibody is preferably trastuzumab.
  • the polyethylene glycol may have a molecular weight of 2,000 to 10,000;
  • the photosensitizer is a compound having a carboxyl group, such as a porphyrin compound, a chlorins compound, a bacteriochlorins compound, a phthalocyanine compound, a naphthalocyanine compound, and 5-amino.
  • the cancer is a cancer that overexpresses human epidermal growth factor receptor 2 (HER2), breast cancer, squamous cell carcinoma, uterine cancer, cervical cancer, prostate cancer, head and neck cancer, pancreatic cancer, brain tumor, liver cancer, skin cancer, esophageal cancer, testicular cancer, kidney cancer, It may be selected from the group consisting of colorectal cancer, rectal cancer, gastric cancer, bladder cancer, ovarian cancer, cholangiocarcinoma and gallbladder cancer.
  • HER2 human epidermal growth factor receptor 2
  • the photosensitive agent is preferably pheophorbide A or chlorine e6 having a carboxyl group
  • the cancer is preferably breast cancer overexpressing HER2 (human epidermal growth factor receptor 2).
  • the conjugate of the present invention has a constitution of an anti-HER2 antibody-maleimide-polyethyleneglycol-photosensitive agent, and specifically, the composition of Herceptin-maleimide-polyethyleneglycol-pheformamide A / chlorine e6 is sequentially connected It relates to a conjugate for the diagnosis or treatment of cancer (particularly breast cancer, colon cancer, gastric cancer).
  • Such a conjugate of the present invention comprises the steps of: a) connecting the biocompatible polymer and the photosensitive agent to prepare a primary conjugate; b) connecting the linker to the primary conjugate to produce a secondary conjugate; And c) linking the anti-HER2 antibody to the secondary conjugate.
  • the present inventors used Herceptin as the anti-HER2 antibody to prepare a conjugate in which the biocompatible polymer and the photosensitive agent are sequentially connected to the anti-HER2 antibody, and the pheoformamide A having a carboxyl group as the photosensitive agent or Chlorine e6 was used (see Formula 1 below), and polyethylene glycol was used as the biocompatible polymer.
  • the molecular weight of polyethylene glycol used in the present invention can be flexibly adjusted, preferably those having a molecular weight of 2,000 to 6,000 can be used.
  • polyethylene glycol having two primary amine groups can be not only a one-to-one equivalent conjugation between the polyethylene glycol and the photosensitive agent but also a one-to-two equivalent conjugation. Only the product having the conjugation ratio was separated and purified by using hydrophobic chromatography with 1 to 1 equivalent of polyethylene glycol and 1 to 1 equivalent of the photosensitive agent by hydrophobic difference.
  • the primary amine group of the polyethylene glycol-photosensitive agent conjugate is first used.
  • a maleimide conjugate having one carboxyl group was prepared (see Formula 3 below).
  • maleimide-polyethylene glycol-photosensitive agent conjugate prepared through the above processes was conjugated through disulfide bond without using a catalyst in Herceptin and a buffer (see Formula 4).
  • the present invention also provides a composition for diagnosing or treating cancer comprising the conjugate (a conjugate in which anti-HER2 antibody-maleimide-polyethyleneglycol-photosensitive agent is sequentially connected) as an active ingredient.
  • the conjugate can form antibody-antigen binding by targeting cancer expressing human epidermal growth factor receptor 2 (HER2) by including anti-HER2 antibodies in some constructs;
  • HER2 human epidermal growth factor receptor 2
  • photosensitizers in some configurations activates the production of fluorescence and reactive oxygen (monotiary oxygen) in cancer cells, thereby enabling selective fluorescence imaging and photodynamic therapy of cancer;
  • polyethylene glycol in some components, it imparts hydrophilicity to photosensitive agents having poor solubility problems and increases the tissue and solid permeability of antibodies having high molecular weight, thus requiring the use of surfactants used in conventional immunochemical staining. I never do that.
  • the cancer is a cancer that expresses human epidermal growth factor receptor 2 (HER2), breast cancer, squamous cell carcinoma, uterine cancer, cervical cancer, prostate cancer, head and neck cancer, pancreatic cancer, brain tumor, liver cancer, skin cancer, esophageal cancer, testicular cancer , Kidney cancer, colorectal cancer, rectal cancer, gastric cancer, bladder cancer, ovarian cancer, cholangiocarcinoma and gallbladder cancer, but may be selected from the group.
  • HER2 human epidermal growth factor receptor 2
  • breast cancer squamous cell carcinoma
  • uterine cancer cervical cancer
  • prostate cancer head and neck cancer
  • pancreatic cancer brain tumor
  • liver cancer skin cancer
  • esophageal cancer testicular cancer
  • Kidney cancer colorectal cancer
  • rectal cancer gastric cancer
  • bladder cancer ovarian cancer
  • cholangiocarcinoma and gallbladder cancer but may be selected from the group.
  • the molecular weight of polyethylene glycol used in the present invention can be flexibly adjusted, preferably can be used with a molecular weight of 2,000 to 6,000.
  • the photosensitive agent of the present invention is excited when light of a specific wavelength is irradiated, and then generates a fluorescent signal or reacts with a surrounding substrate or oxygen to generate reactive oxygen species, Reactive oxygen species have the effect of suicide or necrosis of surrounding tumor cells.
  • the photosensitive agent used in the present invention is a compound containing a carboxyl group, and preferably pheoformamide A or chlorine e6 can be used.
  • composition comprising the conjugate of the present invention (a conjugate in which the anti-HER2 antibody-maleimide-polyethylene glycol-photosensitive agent is sequentially connected) can be used as a cancer diagnosis or treatment.
  • the conjugate of the present invention can be bound to the target cancer cells with high sensitivity, wherein the generation of fluorescence and reactive oxygen is activated to enable cancer selective fluorescence imaging and at the same time photodynamic therapy.
  • the cancer diagnostic or therapeutic composition may include a pharmaceutically effective amount of a conjugate (a conjugate in which an anti-HER2 antibody-maleimide-polyethylene glycol-photosensitive agent is sequentially connected), a pharmaceutically acceptable carrier,
  • the carrier may be a diluent.
  • the composition may further comprise adjuvants such as preservatives, wetting agents, emulsifiers and dispersants.
  • the term pharmaceutically effective amount means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment, and the effective dose level refers to an individual type and severity, age, sex, type of disease, drug Can be determined according to the activity of the drug, the sensitivity to the drug, the time of administration, the route of administration and the rate of release, the duration of treatment, factors including the drug used concurrently and other factors well known in the medical field. Taking all of the above factors into consideration, it is important to administer an amount that can obtain the maximum effect in a minimum amount without side effects, and can be easily determined by those skilled in the art.
  • Such cancer diagnostic or therapeutic compositions may be formulated to conform to the intended route of administration.
  • routes of administration include, but are not limited to, parenteral, such as intravenous, intradermal, subcutaneous, intranasal, transdermal (topical), mucosal penetration and rectal administration.
  • the composition for diagnosing or treating cancer of the present invention is intravenous injection, intraperitoneal injection, intramuscular injection, intracranial injection, intratumoral injection, intraepithelial injection, skin penetration delivery, esophageal administration, abdominal administration, arterial injection, intraarticular injection And it may be administered by a route selected from the group consisting of oral administration.
  • Suitable carriers that can be used for parenteral administration are well known to those skilled in the art.
  • Aqueous vehicles including but not limited to, for example, sodium chloride injection, Ringer's injection, dextrose injection, dextrose and sodium chloride injection, and lactate containing Ringer's injection;
  • Water-miscible carriers including but not limited to ethyl alcohol, polyethylene glycol, and polypropylene glycol;
  • non-aqueous carriers including but not limited to corn oil, cottonseed oil, peanut oil, sesame oil, ethyloleate, isopropyl myristate, and benzyl benzoate.
  • Such cancer diagnostic or therapeutic compositions can be used for tumor treatment or tumor diagnosis.
  • the effective amount can be determined by the physician on a case-by-case basis.
  • the age, sex, weight, or severity of the condition to be treated or diagnosed may be considered.
  • the conjugates of the present invention conjugates in which anti-HER2 antibody-maleimide-polyethyleneglycol-photosensitive agent is sequentially connected
  • the conjugate may be administered at 0.1 mg to 1 mg / kg based on the photosensitizer.
  • kits of the invention can be used to detect or diagnose breast cancer by identifying intracellular intracellular human epidermal growth factor receptor 2 (HER2) levels in patients suspected of cancer (particularly breast cancer).
  • the kit may include one or more other component compositions, solutions or devices suitable for the method of analysis as well as the conjugate of the present invention (anti-HER2 antibody-maleimide-polyethyleneglycol-photosensitive agent).
  • the present invention uses the conjugate (anti-HER2 antibody-maleimide-polyethyleneglycol-photosensitive agent) to antigen-antibody reaction of the HER2 protein of an isolated biological sample of an individual suspected of cancer (especially breast cancer) It provides a method of providing information for the diagnosis of breast cancer, including detecting through.
  • the conjugate (anti-HER2 antibody-maleimide-polyethyleneglycol-photosensitive agent) is processed to an isolated biological sample of a suspected breast cancer to detect the HER2 protein through an antigen-antibody reaction; Providing information for diagnosing breast cancer, comparing the level of HER2 protein detected in the step with a normal control group and determining that the patient is more likely to develop breast cancer if the HER2 protein level is higher than that of the normal control group. It may be a method.
  • biological sample includes tissue, cells, whole blood, serum, plasma, tissue autopsy samples (brain, skin, lymph nodes, spinal cord, etc.), cell culture supernatants, ruptured eukaryotic cells, and bacterial expression systems. It is not limited to this. These biological samples can be manipulated with or without manipulation of the biological sample to confirm the level of HER2 protein.
  • antigen-antibody complex means a combination of an HER2 protein antigen in a sample and an antibody according to the present invention which recognizes the same.
  • the formation of such antigen-antibody complex can be detected through the presence or absence of fluorescence of a photosensitizer. have.
  • Chloin e6 is purchased from Frontier Scientific, N, N'-Dicyclohexylcarbodiimide (DCC), N-hydroxysuccinimide (nhs), Dimethyl sulfoxide (DMSO), 9, 10-Dimethylanthracene, maleimide (4-Maleimidobutyric acid) is purchased from Sigma aldrich, human breast cancer cells (SK-BR-3) are purchased from Korea Cell Line Bank, Herceptin (Trastuzumab) is purchased from Roche, Korea Secondary antibody (Rabbit anti-Human IgG-h + I) was purchased from Bethyl Laboratories Inc.
  • Herceptin- of the present invention Maleimide - Polyethylene glycol - Peophorbide Preparation of A Conjugate (HER-Mal-PEG-PheoA)
  • pheophorbide a Prior to conjugation of polyethylene glycol with pheophorbide, pheophorbide a (pheophorbide a, PheoA, 0.146g) and N, N'-Dicyclohexylcarbodiimide (DCC, 0.0695g) were used to enhance the reactivity of the carboxyl group of pheophorbide A with a catalyst.
  • N-hydroxysuccinimide nhs, 0.0385g was dissolved in dimethyl sulfoxide (Dimethyl sulfoxide, DMSO, 25ml) and reacted at room temperature for 6 hours.
  • each of polyethylene glycol (polyethylene glycol-bisamine, PEG, 1g) having a molecular weight of 2,000 or 6,000 was dissolved in dimethyl sulfoxide (DMSO, 25ml), and then added to the above-mentioned pheophorbide solvent and reacted at room temperature for 24 hours. I was. After filtering the reactant, the reaction product was dialyzed with primary distilled water for 3 days using a dialysis membrane (Spectra / Por; mol. Wt. Cutoff size, 3,500) to remove dimethyl sulfoxide and the catalyst used in the reaction. The final reaction was dried via lyophilization. The results were confirmed by nuclear magnetic resonance spectrum (1H-NMR).
  • Hydrophobic chromatography (sephadex LH-20) was carried out to selectively separate and purify only the polyethylene glycol-phenophorbide A 1 to 1 equivalent of the conjugate product prepared in Example ⁇ 1-1>.
  • polyethylene glycol-pheophorbide A is dissolved in methanol at a concentration of 5 mg / ml, and then methanol containing polyethylene glycol-pheolovide A is injected into hydrophobic chromatography (sephadex LH-20), and The yield was recovered at 1 minute intervals, varying the ratio of methanol from 5 to 5 to 1 to 9 ratios.
  • the polyethylene glycol-phenophorbide conjugate (PEG-PheoA, 0.2 g) was dissolved in dimethyl sulfoxide (DMSO, 10 ml), added to the previous maleimide solvent, and reacted at room temperature for 24 hours. After filtering the reactant, the reaction product was dialyzed with primary distilled water for 3 days using a dialysis membrane (Spectra / Por; mol. Wt. Cutoff size, 3,500) to remove dimethyl sulfoxide and the catalyst used in the reaction. The final reaction was dried via lyophilization. The results were confirmed by nuclear magnetic resonance spectrum (1H-NMR).
  • Cutoff size 12,000 ⁇ 14,000
  • Herceptin- of the present invention Maleimide - Polyethylene glycol - Chlorine e6 conjugate (HER- Mal -PEG-Ce6) Preparation
  • Chlorine e6 Chlorin e6, Ce6, 0.146g), N, N'-Dicyclohexylcarbodiimide (DCC, 0.0695g), N-hydroxysuccinimide nhs, 0.0385g was dissolved in dimethyl sulfoxide (Dimethyl sulfoxide, DMSO, 25ml) and reacted for 6 hours at room temperature.
  • dimethyl sulfoxide Dimethyl sulfoxide, DMSO, 25ml
  • each of polyethylene glycol (polyethylene glycol-bisamine, PEG, 1g) having a molecular weight of 2,000 or 6,000 was dissolved in dimethyl sulfoxide (DMSO, 25ml), and then added to the above-mentioned pheophorbide solvent and reacted at room temperature for 24 hours. I was. After filtering the reactant, the reaction product was dialyzed with primary distilled water for 3 days using a dialysis membrane (Spectra / Por; mol. Wt. Cutoff size, 3,500) to remove dimethyl sulfoxide and the catalyst used in the reaction. The final reaction was dried via lyophilization. The results were confirmed by nuclear magnetic resonance spectrum (1H-NMR).
  • Hydrophobic chromatography (sephadex LH-20) was performed to selectively separate and purify only the polyethylene glycol-chlorine e6 one-to-one equivalent conjugate product prepared in Example ⁇ 2-1>.
  • polyethylene glycol-chlorine e6 is dissolved in methanol at a concentration of 5 mg / ml.
  • methanol containing polyethylene glycol-chlorine e6 is injected into hydrophobic chromatography (sephadex LH-20), and the ratio of water and methanol is adjusted. The yield was recovered at 1 minute intervals, varying from 5 to 5 at a ratio of 1 to 9.
  • the polyethyleneglycol-chlorine e6 conjugate (PEG-Ce6, 0.2 g) was dissolved in dimethyl sulfoxide (DMSO, 10 ml), added to the previous maleimide solvent, and reacted at room temperature for 24 hours. After filtering the reactant, the reaction product was dialyzed with primary distilled water for 3 days using a dialysis membrane (Spectra / Por; mol. Wt. Cutoff size, 3,500) to remove dimethyl sulfoxide and the catalyst used in the reaction. The final reaction was dried via lyophilization. The results were confirmed by nuclear magnetic resonance spectrum (1H-NMR).
  • maleimide-polyethylene glycol-chlorine e6 conjugate (0.010 g) and Herceptin (Trastuzumab, HER, 0.010 g), respectively, were prepared for Herceptin conjugation with the maleimide-polyethylene glycol-chlorine e6 conjugate prepared in Example ⁇ 2-3>. After dissolving in 0.5M MES buffer (2- (N-morpholino) ethanesulfonic acid, 1ml), the two solvents were mixed and reacted for 30 minutes. Primary distilled water was used for 24 hours using a dialysis membrane (Spectra / Por; mol. Wt.
  • Polyethylene glycol (molecular weight 2,000 / 6,000) -chlorine e6 with improved poor solubility was dissolved in distilled water at a concentration of 1 mg / ml and photographed with a digital camera.
  • Herceptin, Maleimide - Polyethylene glycol - A photosensor Junction Mal -PEG- PheoA Of Ce6
  • Herceptin-maleimide-polyethylene glycol-photosensitive agent conjugate HER- Mal -PEG- PheoA Of Ce6 Fluorescence intensity
  • Herceptin and maleimide-polyethylene glycol-photosensitive agent conjugate (Mal-PEG-PheoA / Ce6, 10 mg / ml) was dissolved in distilled water, and then Herceptin-maleimide-polyethylene glycol-photosensitive agent conjugate (HER-Mal-PEG- PheoA / Ce6) measured the fluorescence intensity using a fluorescence imaging device (12-bit CCD camera, Kodak) while the purification process through dialysis was completed.
  • the Herceptin-maleimide-polyethyleneglycol-photosensitive agent conjugated with the maleimide-polyethylene glycol-photosensitive agent having no fluorescence intensity but having no fluorescence intensity has a fluorescence intensity. I could confirm that.
  • Herceptin- of the present invention Maleimide - Polyethylene glycol - A photosensor Conjugate Monooxygen (Singlet oxigen) Check the generation ability
  • HER-Mal-PEG-PheoA and HER-Mal-PEG-Ce6 which are Herceptin-maleimide-polyethyleneglycol-photosensitive agent prepared according to Examples 1 and 2 of the present invention.
  • Single oxygen was measured by irradiating a 670 nm wavelength laser having a laser intensity of 10 mW using reagents (9,10-Dimethylanthracene, Singlet oxygen quencher, Sigma aldrich, USA).
  • Herceptin-maleimide-polyethylene glycol-photosensitive agent conjugate (HER-Mal-PEG-PheoA / Ce6) was used as Herceptin-maleimide-polyethylene glycol-feophore using polyethylene glycol having a molecular weight of 6,000.
  • Herceptin-maleimide-polyethylene glycol-feophore using polyethylene glycol having a molecular weight of 6,000.
  • Weed (HER-Mal-PEG6K-PheoA) was confirmed that the single-antioxidant production ability is the most excellent.
  • Herceptin- of the present invention Maleimide - Polyethylene glycol - A photosensor Identification of breast cancer cell discrimination ability of the conjugate
  • HER-Mal-PEG-PheoA and HER-Mal-PEG-Ce6 which are Herceptin-maleimide-polyethyleneglycol-photosensitive agent prepared according to Examples 1 and 2 of the present invention.
  • SK-BR-3 cells Human Breast Cancer Cell, Korea Cell Line Bank
  • SK-BR-3 cells were dispensed at a concentration of 1 ⁇ 10 6 cells / well in 2 mL of culture solution in a 6-well cell culture dish coated with glass glass for 12 hours. Incubated at 37% incubator at 5% CO 2 conditions.
  • the glass glass to which the SK-BR-3 cells were attached was stopped for 10 minutes in 4% Paraformaldehyde, washed three times with DPBS, and then 0.1% of Tween 20. After reacting for 30 minutes in 5% BSA (Bovine serum albumin) containing and washed three times with DPBS, Herceptin-maleimide-polyethyleneglycol-photosensitive agent conjugate (HER-Mal-PEG-PheoA / Ce6), respectively Cells were treated at concentrations of 10 ⁇ g, 1 ⁇ g, 0.1 ⁇ g and 0.01 ⁇ g and reacted for 1 hour. Thereafter, each cell treated with Herceptin-maleimide-polyethylene glycol-photosensitive agent conjugate (HER-Mal-PEG-PheoA / Ce6) was subjected to cell nuclei staining.
  • BSA Bovine serum albumin
  • Herceptin- of the present invention Maleimide - Polyethylene glycol - A photosensor Identification of Breast Cancer Tissue Discrimination in Conjugates
  • HER-Mal-PEG-PheoA and HER-Mal-PEG-Ce6 which are Herceptin-maleimide-polyethyleneglycol-photosensitive agent prepared according to Examples 1 and 2 of the present invention.
  • Human breast cancer tissues (Origene purchase, HER negative, HER positive, HER strong positive) were stopped for 10 minutes in 4% Paraformaldehyde and washed three times with DPBS, followed by 0.1% Tween 20.
  • Herceptin- of the present invention Maleimide - Polyethylene glycol - A photosensor Confirmation of increased tissue permeability of the conjugate
  • HER-Mal-PEG-PheoA and HER-Mal-PEG-Ce6 which are Herceptin-maleimide-polyethyleneglycol-photosensitive agent prepared according to Examples 1 and 2 of the present invention.
  • SK-BR-3 cells were transplanted into Athmic nude mice (Orient) mice to prepare a breast cancer model.
  • Herceptin-maleimide-polyethyleneglycol-photosensitive agent conjugate (HER-Mal-PEG-PheoA / Ce6) was injected at a concentration of 1 mg / kg to the mouse model cancer site prepared through the above procedures, and after 3 hours, 670 nm After giving a laser of the wavelength of 100J / cm 2 intensity, cancer tissue was removed, a section was prepared to a thickness of 10 ⁇ m and cell nuclei staining for each tissue.
  • PEG2K-PheoA Polyethyleneglycol-phephorbide A conjugate of molecular weight 2,000
  • PEG6K-PheoA Polyethyleneglycol-phephorbide A conjugate of molecular weight 6,000
  • PEG2K-Ce6 polyethyleneglycol-chlorine e6 conjugate of molecular weight 2,000
  • PEG6K-Ce6 polyethyleneglycol-chlorine e6 conjugate with molecular weight of 6,000
  • HER-Mal-PEG-PheoA Herceptin-maleimide-polyethyleneglycol-phephorbide A conjugate
  • HER-Mal-PEG-PheoA Herceptin-maleimide-polyethyleneglycol-chlorine e6 conjugate
  • HER-Mal-PEG2k-PheoA Herceptin-maleimide-molecular weight 2,000 polyethylene glycol-phephorbide A conjugate
  • HER-Mal-PEG6k-PheoA Herceptin-maleimide-molecular weight 6,000 polyethyleneglycol-phephorbide A conjugate
  • HER-Mal-PEG2k-Ce6 polyethylene glycol-chlorine e6 conjugate of Herceptin-maleimide-molecular weight 2,000
  • HER-Mal-PEG6k-Ce6 polyethylene glycol-chlorine e6 conjugate with Herceptin-maleimide-molecular weight 6,000

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Abstract

La présente invention concerne un conjugué anticorps-photosensibilisant destiné au diagnostic du cancer (en particulier le cancer du sein) et son procédé de production et, plus particulièrement, un conjugué herceptine-photosensibilisant qui raccourcit le délai de diagnostic et présente une meilleure capacité de diagnostic des tumeurs, le conjugué étant obtenu en fusionnant, par liaison chimique, un photosensibilisant et un anticorps anti-HER2 (herceptine) qui est spécifique à HER2, afin de diagnostiquer une tumeur qui exprime HER2. Il est attendu qu'un conjugué destiné au diagnostic ou au traitement du cancer, dans lequel du maléimide, du polyéthylène glycol et un photosensibilisant sont couplés de manière séquentielle à un anticorps anti-HER2, présente les avantages de : présenter, par comparaison avec un photosensibilisant hydrophobe existant, une meilleure solubilité et une plus grande efficacité de production de dérivés réactifs de l'oxygène au moment d'une irradiation lumineuse en raison de l'introduction de polyéthylène glycol soluble dans l'eau dans un photosensibilisant comportant un groupe carboxyle ; ne nécessiter aucun tensioactif en raison de la compensation des problèmes existants causés lorsqu'un anticorps anti-HER2 (herceptine) est utilisé seul, c'est-à-dire, les problèmes liés au fait que plusieurs étapes de transformation doivent être effectuées, et qu'il est essentiel d'utiliser un tensioactif ; et de pouvoir être appliqué comme matériau de coloration immunochimique pour le diagnostic du cancer du sein avec un temps réduit. De plus, un procédé de production d'un conjugué (anticorps anti-HER2-maléimide-polyéthylène glycol-photosensibilisant) selon la présente invention peut fusionner de façon simple et facile un photosensibilisant avec un anticorps anti-HER2 au moyen d'un procédé séquentiel et graduel et, en particulier, fusionne du maléimide avec un premier conjugué contenant du polyéthylène glycol-photosensibilisant combinés à l'intérieur de celui-ci, ce qui permet de fusionner de façon stable l'anticorps anti-HER2 (herceptine).
PCT/KR2015/010271 2014-09-30 2015-09-30 Conjugué herceptine-photosensibilisant destiné au diagnostic du cancer et son procédé de production WO2016052971A1 (fr)

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KR1020150136953A KR101756537B1 (ko) 2014-09-30 2015-09-25 유방암 진단용 허셉틴-광감각제 접합체 및 이의 제조 방법
KR10-2015-0136953 2015-09-25

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CN111423446A (zh) * 2020-04-14 2020-07-17 大连理工大学 具有光、声敏活性的二氢卟吩硝酸酯类化合物、制备方法与应用
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CN111454302A (zh) * 2020-04-30 2020-07-28 大连理工大学 具有光、声敏活性的二氢卟吩e6二茂铁结合物、制备方法与应用
CN111454302B (zh) * 2020-04-30 2022-07-05 大连理工大学 具有光、声敏活性的二氢卟吩e6二茂铁结合物、制备方法与应用
CN115804849A (zh) * 2023-01-08 2023-03-17 河北工业大学 一种光敏剂和一氧化氮前药的给药系统及其制备方法

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