WO2023204330A1 - Itgb2-mediated drug delivery system - Google Patents

Itgb2-mediated drug delivery system Download PDF

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WO2023204330A1
WO2023204330A1 PCT/KR2022/005783 KR2022005783W WO2023204330A1 WO 2023204330 A1 WO2023204330 A1 WO 2023204330A1 KR 2022005783 W KR2022005783 W KR 2022005783W WO 2023204330 A1 WO2023204330 A1 WO 2023204330A1
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itgb2
macrophages
agent
targeting
cancer
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PCT/KR2022/005783
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French (fr)
Korean (ko)
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배현수
한익환
강문규
이희경
최정윤
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트윈피그바이오랩(주)
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Priority to PCT/KR2022/005783 priority Critical patent/WO2023204330A1/en
Publication of WO2023204330A1 publication Critical patent/WO2023204330A1/en

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    • 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
    • A61K47/51—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 the non-active ingredient being a modifying agent
    • A61K47/62—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 the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • 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
    • 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
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids

Definitions

  • the present invention relates to a drug delivery system that specifically binds to ITGB2.
  • Anticancer immunotherapy which can minimize side effects compared to existing chemotherapy or radiation treatment methods, is a method of treating cancer using the body's immune system.
  • T cells which are therapeutic immune cells, are used to treat cancer. -T included), dendritic cells, natural killer cells, etc.
  • the activity of immune-activating substances and therapeutic immune cells is drastically reduced. Therefore, it is becoming important to develop treatments that have anti-cancer effects by controlling the microenvironment surrounding tumor cells without directly affecting tumor cells and immune cells, thereby blocking the supply of nutrients to tumor cells and angiogenesis around tumor cells.
  • the tumor microenvironment contributes to the proliferation and survival of malignant cells, angiogenesis, metastasis, abnormal adaptive immunity, and reduced response to hormonal and chemotherapy agents, and is therefore greatly considered as a therapeutic target.
  • tumor-associated macrophages play an important role in tumor growth.
  • Tumor-related macrophages play an important role in the overall tumor microenvironment, including cancer growth and metastasis, and are classified into two phenotypes: tumor suppressor M1 type macrophages or tumor supporting M2 type macrophages.
  • M1 type macrophages have a strong ability to present antigens, are generally activated by interferon- ⁇ , liposaccharide (LPS), and tumor necrosis factor (TNF)- ⁇ , and have pro-inflammatory and bactericidal effects.
  • LPS liposaccharide
  • TNF tumor necrosis factor
  • M2-type macrophages are known to promote immunosuppression, tumorigenesis, and angiogenesis by releasing various extracellular matrix components, angiogenic, and chemotactic factors. Generally, it is induced by IL-4 and IL-13 and expresses markers such as arginase-1, mannose (MMR, CD206), scavenger receptor (SR-A, CD204), CD163, and IL-10. This differentiates them from M1 type tumor-related macrophages. Tumor-related macrophages present around the tumor are closely related to the growth and metastasis of tumor cells. In cancer patients, if a large number of M2-type tumor-related macrophages are present around the tumor, the patient's prognosis and survival rate are poor.
  • M2-type macrophages produce cytokines such as IL-10, TGF ⁇ , and CCL18 that promote cancer growth, and receptors such as PDL1 and B7-1/2 present on the surface of M2-type tumor-related macrophages stimulate T cells. , it has been reported to inhibit the anti-tumor activity of NK cells. Since tumor growth, differentiation, and metastasis occur actively in a microenvironment where large amounts of M2-type tumor-related macrophages exist, the development of targeted therapeutic agents targeting M2-type tumor-related macrophages is necessary.
  • the object of the present invention is to provide a drug delivery system.
  • an object of the present invention is to provide a complex of a drug delivery system and a drug.
  • an object of the present invention is to provide an immuno-anticancer agent.
  • an object of the present invention is to provide a pharmaceutical composition for preventing or treating cancer.
  • an object of the present invention is to provide a composition for eliminating M2 tumor-related macrophages.
  • an object of the present invention is to provide a marker for diagnosing solid cancer and a composition containing the same.
  • an object of the present invention is to provide a method for screening immuno-anticancer agents.
  • the purpose of the present invention is to provide a method for predicting responsiveness to anti-cancer immunotherapy or diagnosing prognosis.
  • an object of the present invention is to provide a method for screening anticancer drugs targeting M2 macrophages.
  • the purpose of the present invention is to provide a method of providing information necessary for diagnosing cancer.
  • the present invention provides an ITGB2-mediated drug delivery system.
  • the present invention provides a complex in which a drug is bound to the ITGB2-mediated drug delivery system.
  • the present invention provides an anti-cancer immunological agent containing the above complex as an active ingredient.
  • the present invention provides a pharmaceutical composition for preventing or treating M2 tumor-related macrophage-mediated cancer, comprising the complex as an active ingredient.
  • the present invention provides a composition for eliminating M2 tumor-related macrophages containing the complex as an active ingredient.
  • the present invention provides a marker for diagnosing solid tumors containing the ITGB2 gene or a protein expressed from the gene and a composition containing the same.
  • the present invention provides a method for screening immuno-anticancer agents.
  • the present invention provides a method for predicting responsiveness to anti-cancer immunotherapy or diagnosing prognosis.
  • the present invention provides a method for screening anticancer agents targeting M2 macrophages.
  • the present invention provides a method of providing information necessary for diagnosing cancer.
  • ITGB2 was confirmed to be a specific marker for M2 macrophage-mediated solid cancer, and an ITGB2-mediated drug delivery system containing a targeting substance that specifically binds to ITGB2 (Integrin beta 2) of the present invention as an active ingredient and the drug thereto were developed.
  • the combined complex has the effect of reducing drug side effects and increasing drug efficacy by acting selectively (or specifically) only on M2 tumor-related macrophages, so it is useful as a drug carrier for apoptosis inducers, anticancer agents, contrast agents, etc. It can be used effectively.
  • Figure 1 is a diagram confirming the affinity of TAMpep for M2 macrophages:
  • Figure 2 is a diagram identifying proteins that bind to TAMpep in M2 macrophages:
  • Figure 3 is a diagram showing LC-MS chromatogram data obtained by reacting TAMpep826-biotin with cell membrane proteins extracted from M0, M1, and M2 macrophages, respectively.
  • Figure 4 is a diagram showing the identification of the target protein of TAMPep826 using LC-MS/MS proteomics.
  • Figure 5 is a diagram showing quantitative RT-PCR conditions.
  • Figure 6 is a diagram confirming ITGB2 mRNA expression in M2 macrophages.
  • Figure 7 is a diagram confirming ITGB2 protein expression in M2 macrophages.
  • Figure 8 is a diagram confirming the expression of ITGB2 protein in M2 macrophages by immunofluorescence analysis.
  • Figure 9 is a diagram confirming the expression of ITGB2 in the cell membrane of M2 macrophages.
  • Figure 10 is a diagram confirming the interaction of TAMpep826 and ITGB2 in M2 macrophages by pull-down assay.
  • Figure 11 is a diagram confirming the interaction between TAMpep826 and ITGB2 in M2 macrophages using immunofluorescence analysis.
  • Figure 12 is a diagram showing the immobilization and confirmation of ITGB2 on the surface of the sensor chip.
  • Figure 13 is a diagram confirming the binding force between anti-ITGB2 antibody and ITGB2 protein by SPR.
  • Figure 14 is a diagram confirming the binding force between Melittin-dKLA and ITGB2 protein by SPR.
  • Figure 15 is a diagram confirming the mRNA expression of ITGB2 in a cell model that suppresses ITGB2 expression established through Crispr/cas9 in M2 macrophages.
  • Figure 16 is a diagram confirming the effect of Melittin-dKLA on reducing apoptosis by suppressing ITGB2 expression in M2 macrophages.
  • Figure 17 is a diagram confirming the effect of TB511 (TAMpep826-dKLA) on reducing apoptosis induction by suppressing ITGB2 expression in M2 macrophages.
  • Figure 18 is a diagram confirming the effect of reducing apoptosis induction of TB511 by ITGB2 antibody in M2 macrophages.
  • amino acids referred to by abbreviations in the present invention are described according to the IUPAC-IUB nomenclature as follows:
  • the present invention relates to an ITGB2-mediated drug delivery system comprising a targeting substance that specifically binds to ITGB2 (Integrin beta 2) as an active ingredient.
  • the targeting agent can specifically bind to the ITGB2 protein present in the cell membrane of M2 tumor-related macrophages.
  • the targeting agent is a small molecule, virus, antibody, antibody fragment, aptamer, hormone, cytokine, chemokine, ligand, peptide that is a partial region of a cytokine, or a peptide that is a partial region of a ligand that specifically binds to ITGB2. It can be.
  • the targeting agent may further include a targeting sequence, tag, labeled residue, or amino acid sequence designed for the specific purpose of increasing half-life or stability.
  • RNA, DNA, antibodies, effectors, drugs, prodrugs, toxins, peptides, or delivery molecules may be additionally conjugated to the targeting agent (Shoari et al., Pharmaceutics 13:1391, pp 1-32 (2021)).
  • the targeting agent provides a targeting function by enabling selective binding to M2 tumor-related macrophages, which are target cells, through binding to ITGB2.
  • This cell-targeting substance specifically binds to ITGB2 on the surface of target cells and induces endocytosis, enabling the drug that binds to it to move into the cell.
  • antibodies, aptamers, hormones e.g., erythropoietin hormone
  • cytokines or chemokines e.g., IL13
  • Ligands which are biomolecules such as VEGF (Vascular endothelial growth factor) and BDNF (Brain-derived neurotrophic factor), that bind to target cell surface receptors, and peptides, which are some regions of these factors that have the ability to specifically bind to receptors, are used to target these cells. It can be used as a targeting agent.
  • antibodies or aptamers can be used as targeting substances, and antibodies as targeting substances include not only monoclonal antibodies and polyclonal antibodies, but also multispecific antibodies (i.e., antibodies that recognize two or more antigens or two or more epitopes). target antibodies, etc.), or antibody fragments, chemically modified antibodies, chimeric antibodies (human and mouse chimeric antibodies, human and monkey chimeric antibodies) as long as they have the ability to specifically bind to ITGB2. It may be any antibody, such as a humanized antibody or a human antibody with reduced immunogenicity (antibodies, etc.).
  • antibody fragments and chemically modified antibodies are known in the art, such as Fab, Fd, Fab', dAb, F(ab'), F(ab') 2 , scFv (single chain fragment variable), Examples include Fv, single-chain antibodies, Fv dimers, complementarity-determining region fragments, humanized antibodies, chimeric antibodies, and diabodies, and antibody fragments obtained by treating antibodies with protein cleavage enzymes such as papain and pepsin.
  • protein cleavage enzymes such as papain and pepsin.
  • the aptamer as the cell targeting region can be a single-stranded DNA aptamer or a single-stranded RNA aptamer.
  • an aptamer refers to a nucleic acid ligand that can specifically bind to a target molecule such as a target antigen. If it can specifically bind to a target molecule, the aptamer may be a double-stranded DNA or RNA aptamer. Methods for producing and selecting aptamers capable of specifically binding to such target molecules are all known in the art.
  • aptamers may be modified in sugars, phosphates and/or bases to improve half-life in vivo. Nucleotides modified from such sugars, phosphates and/or bases are specifically known in the art, including methods for their preparation.
  • a nucleotide modified from a sugar is one in which the hydroxyl group (OH group) of the sugar is modified with a halogen group, aliphatic group, ether group, amine group, etc., and the sugar ribose or deoxyribose itself is a sugar analogue that can replace it.
  • ⁇ -anomeric sugars, epimeric sugars such as arabinose, xyloses or lyxoses, pyranose sugars, furanos Examples include those substituted with furanose sugars, etc.
  • modifications in phosphate can cause phosphate to form P(O)S(thioate), P(S)S(dithioate), P(O)NR2(amidate), P(O)R, P(O)OR', CO Or transformation into CH2 (formacetal), etc.
  • R or R' is H or substituted or unsubstituted alkyl, etc.
  • the linking group becomes -O-, -N-, -S- or -C-, and the linking group becomes -O-, -N-, -S- or -C-, and the linking group is used to connect adjacent nucleotides. are combined with each other.
  • the drug delivery system of the present invention specifically and directly binds to ITGB2 expressed on the cell membrane of M2 tumor-related macrophages, so that the drug effect is non-selective, without the side effects of the target cell, M2 tumor-related macrophages, expressing ITGB2. It allows the drug effect to be displayed only selectively.
  • the present invention relates to a composition targeting M2 tumor-associated macrophages comprising an agent targeting ITGB2.
  • the present invention relates to a complex in which a drug is bound to a drug delivery system.
  • the complex can deliver a drug to M2 tumor-related macrophages by specifically binding to the ITGB2 protein present in the cell membrane of M2 tumor-related macrophages.
  • the drug may be a pro-apoptotic peptide, an immunogenic apoptosis inducer, or an anticancer agent.
  • the pro-apoptotic peptide is KLA, alpha-defensin-1, BMAP-28, Brevenin-2R, Buforin IIb, cecropin A-magainin 2(cecropin A-Magainin 2, CA-MA-2), Cecropin A, Cecropin B, chrysophsin-1, D-K6L9, gomesin (Gomesin), Lactoferricin B, LLL27, LTX-315, Magainin 2, Magainin II-bombesin conjugate (MG2B), Pardaxin and It may be selected from the group consisting of combinations of these.
  • the immunogenic apoptosis inducer is an anthracycline-based anticancer agent, taxane-based anticancer agent, anti-EGFR antibody, BK channel agonist, bortezomib, cardiac glycoside, cyclophosmide-based anticancer agent, GADD34 /PP1 inhibitor, LV-tSMAC, Measles virus, bleomycin, mitoxantrone, oxaliplatin, and combinations thereof.
  • the anticancer agent may be a cytotoxic anticancer agent, such as doxorubisin, paclitaxel, azithromycin, erythromycin, vinblastin, bleomycin, Dactinomycin, daunorubicin, idarubicin, mitoxantron, plicamycin, mitomycin, methotrexate, entinosthate ( Entinostat, Cladribine, Pralatrexate, Lorlatinib, Maytansine DM1, Maytansine DM3, Maytansine DM4 and combinations thereof.
  • doxorubisin paclitaxel
  • azithromycin erythromycin
  • vinblastin bleomycin
  • Dactinomycin daunorubicin
  • idarubicin mitoxantron
  • plicamycin mitomycin
  • methotrexate entinosthate ( Entinostat, Cladribine, Pralatrexate, Lorlatinib, Maytansine DM1, Maytansine
  • the targeting agent and the drug that specifically bind to Integrin beta 2 (ITGB2) of the drug delivery system may be covalently bound via a linker or non-covalently bound without the mediation of a linker.
  • the targeting agent and the drug that specifically bind to ITGB2 (Integrin beta 2) of the drug delivery system may be connected/combined via a linker, and the linker may be a protein such as a peptide, ligand, antibody, or antibody fragment. Any functional group that can bind through the amine group, carboxyl group, sulfhydryl group, or phosphate group or hydroxyl group of nucleic acids such as aptamers.
  • a linker can be used.
  • the linker may be selected and used appropriately depending on the drug.
  • a linker having an aldehyde reactive group can be connected to the drug and bound to the N-terminal amino group of the antibody (cell targeting region) of the targeting material of the drug delivery system.
  • linkers include isothiocyanate, isocyanates, acyl azide, NHS ester, sulfonyl chloride, aldehyde, and glyoxal ( glyoxal, epoxide, oxirane, carbonate, arylhalide, imidoester, carbodiimide, anhydride, fluorophenyl ester It may be (fluorophenyl ester), hydroxymethyl phosphine, maleimide, haloacetyl, pyridyldisulfide, thiosulfonate, or vinylsulfone. .
  • the linker may be cleavable by protease, cleavable under acid or base conditions, cleavable by high temperature or light irradiation, cleavable under reducing or oxidizing conditions, or a linker that is not cleavable under these conditions. It may be.
  • cleavable linkers include hydrazone linkers that are cleaved under acidic conditions, peptide linkers that are cleaved by proteases, and linkers with a disulfide functional group that are cleaved under reducing conditions.
  • MCC Maleimidomethyl cyclohexane-1-carboxylate
  • sMCC maleimidocaproyl
  • sMCC succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate
  • sulfo-sMCC imidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate
  • the linker may be a self-immolative linker or a traceless linker that leaves no trace after cleavage.
  • Self-immolative linkers include, for example, the linker disclosed in U.S. Pat. No. 9,089,614, entitled “Hydrophilic self-immolative linkers and conjugates thereof,” and the linker, entitled “SELF-IMMOLATIVE LINKERS CONTAINING MANDELIC ACID DERIVATIVES, DRUG-LIGAND CONJUGATES FOR TARGETED THERAPIES AND USES. THEREOF", the linker disclosed in International Publication No.
  • linkers that leave no trace after cutting include phenylhydrazide linker, aryl-triazene linker, and the literature [Blaney, et al., "Traceless solid-phase organic synthesis,” Chem Rev. 102: 2607-2024 (2002)], etc.
  • Biocompatible polymers refer to polymers that have tissue compatibility and anticoagulant properties that do not cause tissue necrosis or blood coagulation in contact with biological tissue or blood.
  • Synthetic polymers as biocompatible polymers include polyester, polyhydroxyalkanoate (PHAs), poly( ⁇ -hydroxyacid), poly( ⁇ -hydroxyacid), and poly(3-hydrosybutyrate-co).
  • PHAs polyhydroxyalkanoate
  • PHBV poly(3-hydroxypropionate
  • PHP poly(3-hydroxyhexanoate
  • PHH poly(4-hydroxy acid), poly(4-hydroxy butyrate), poly(4-hydroxyvalerate), poly(4-hydroxyhexanoate), poly(esteramide), polycaprolactone, polylactide, polyglycolide, poly(lactide-co-glycoside) ride;
  • PLGA polydioxanone, polyorthoester, polyanhydride, poly(glycolic acid-co-trimethylene carbonate), polyphosphoester, polyphosphoester urethane, poly(amino acid), polycyano Acrylate, poly(trimethylene carbonate), poly(iminocarbonate),
  • Vinyl ketone polyvinyl aromatics, polystyrene, polyvinyl ester, polyvinyl acetate, ethylene-methyl methacrylate copolymer, acrylonitrile-styrene copolymer, ABS resin and ethylene-vinyl acetate copolymer, polyamide, alkyd resin.
  • polyoxymethylene, polyimide, polyether, polyacrylate, polymethacrylate, polyacrylic acid-co-maleic acid or polyaminoamine, and natural polymers include chitosan, dextran, cellulose, heparin, hyaluronic acid, and alginate. , inulin, starch or glycogen.
  • the linker used to combine the drug delivery system with the drug is stable outside the target cell and is not cleaved, nor is it cleaved even in endosomes or rhizosomes under acidic conditions within the target cell.
  • This linker is stable outside the target cell and is not cleaved, allowing the drug to move into the cell, and is not cleaved in endosomes or rhizosomes under acidic conditions, allowing it to be moved from endosomes or rhizosomes to the cytoplasm.
  • the drug may be non-covalently bound to the drug delivery system.
  • intercalator agents such as doxorubicin, a type of anticancer drug that exerts its effect by intercalating with nucleic acids
  • doxorubicin a type of anticancer drug that exerts its effect by intercalating with nucleic acids
  • doxorubicin a type of anticancer drug that exerts its effect by intercalating with nucleic acids
  • the drug is not particularly limited as long as it is a drug that can move into cells and exert its effect.
  • drugs may be drugs made of any small molecule compounds such as cytotoxic anticancer drugs, recombinant proteins, or any biopharmaceuticals such as siRNA.
  • drugs include anti-inflammatory drugs, analgesics, anti-arthritis drugs, antispasmodics, antidepressants, antipsychotics, tranquilizers, anti-anxiety drugs, narcotic antagonists, anti-Parkinson's disease drugs, cholinergic agonists, anticancer drugs, angiogenesis inhibitors, immunosuppressants, etc.
  • Immunostimulants antivirals, antibiotics, appetite suppressants, painkillers, anticholinergics, antihistamines, anti-migraine drugs, hormones, coronary vasodilators, vasodilators, contraceptives, antithrombotic agents, diuretics, antihypertensive agents, cardiovascular disease treatments, contrast media, etc. It may be a diagnostic agent, etc.
  • the drug is preferably a cytotoxic anticancer agent.
  • Cytotoxic anticancer agents include antimetabolites, microtubulin targeting agents (Tubulin polymerase inhibitor and Tubulin depolymerisation), alkylating agents, antimitotic agents, DNA cleavage agents, and DNA. They can be roughly divided into DNA cross-linker agents, DNA intercalator agents, and DNA topoisomerase inhibitors.
  • folic acid such as methotrexate acid derivatives, purine derivatives such as Cladribine, pyrimidine derivatives such as Azacitidine, Doxifluridine, Fluorouracil, etc.
  • microtubule targeting agents include monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), auristatin series drugs such as dolastatin, and maytansines.
  • alkylating agents include alkyl sulfonate agents such as Busulfan and Treosulfan, nitrogen mustard derivatives such as Bendamustine, and cisplatin. ), Platinum preparations such as Heptaplatin, etc. are known in the art.
  • taxane preparations such as Docetaxel and Paclitaxel, Vinca alkalids such as Vinflunine, and Etoposide.
  • Podophyllotoxin derivatives such as podophyllotoxin are known in the art
  • Calichamicins, etc. are known as DNA cleavage agents
  • PBD is known as a DNA cross-linker agent.
  • Duplexes and the like are known.
  • doxorubicin and the like are known in the art as DNA intercalator agents
  • SN-28 and the like are known in the art as DNA topoisomerase inhibitors.
  • the drug may be a gene, plasmid DNA, antisense oligonucleotide, siRNA, peptide, ribozyme, viral particle, immunomodulator, protein, contrast agent, etc. More specifically, the drug may be a gene encoding Rb94, a variant of the retinoblastoma tumor suppressor gene, a gene encoding apoptin, which induces apoptosis only in tumor cells, and an antisense oligonucleotide against HER-2, which is a therapeutic target. (Sequence: 5'-TCC ATG GTG CTC ACT-3'), and may be a diagnostic contrast agent such as Gd-DTPA, an MRI contrast agent.
  • a diagnostic contrast agent such as Gd-DTPA, an MRI contrast agent.
  • the complex of the drug delivery system and the drug of the present invention can be prepared as a pharmaceutical composition in an oral formulation or parenteral formulation depending on the route of administration by a conventional method known in the art, including a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable carrier refers to a carrier or diluent that does not irritate living organisms and does not inhibit the biological activity and properties of the administered compound.
  • Acceptable pharmaceutical carriers in compositions formulated as liquid solutions include those that are sterile and biocompatible, such as saline solution, sterile water, Ringer's solution, buffered saline solution, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, ethanol, and One or more of these ingredients can be mixed and used, and other common additives such as antioxidants, buffers, and bacteriostatic agents can be added as needed.
  • the present invention relates to an anti-cancer immunological agent comprising a complex of the drug delivery system of the present invention and a drug as an active ingredient.
  • the present invention relates to a pharmaceutical composition for preventing or treating M2 tumor-related macrophage-mediated cancer, comprising a complex of the drug delivery system of the present invention and a drug as an active ingredient.
  • the pharmaceutical composition of the present invention can be used as a stand-alone therapy, but can also be used in combination with other conventional biological therapies, chemotherapy, or radiotherapy. When such combination therapy is performed, cancer can be treated more effectively.
  • chemotherapy agents that can be used with the composition include cisplatin, carboplatin, procarbazine, mechlorethamine, Cyclophosphamide, ifosfamide, melphalan, chlorambucil, bisulfan, nitrosourea, dactinomycin, daunoru daunorubicin, doxorubicin, bleomycin, plicomycin, mitomycin, etoposide, tamoxifen, taxol, transpla Includes transplatinum, 5-fluorouracil, vincristin, vinblastin and methotrexate.
  • Radiation therapy that can be used with the composition of the present invention includes X-ray irradiation and ⁇ -ray ir
  • prevention refers to all actions that inhibit or delay the occurrence, spread, and recurrence of cancer by administering the composition according to the present invention.
  • the therapeutically effective amount of the composition of the present invention may vary depending on various factors, such as administration method, target site, patient condition, etc. Therefore, when used in the human body, the dosage must be determined as appropriate by considering both safety and efficiency. It is also possible to estimate the amount used in humans from the effective amount determined through animal testing. These considerations in determining an effective amount include, for example, Hardman and Limbird, eds., Goodman and Gilman's The Pharmacological Basis of Therapeutics, 10th ed. (2001), Pergamon Press; and E.W. Martin ed., Remington's Pharmaceutical Sciences, 18th ed. (1990), Mack Publishing Co.
  • the pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount.
  • pharmaceutically effective amount refers to an amount that is sufficient to treat a disease with a reasonable benefit/risk ratio applicable to medical treatment and does not cause side effects, and the effective dose level is determined by the patient's Factors including health status, type and severity of disease, activity of drug, sensitivity to drug, method of administration, time of administration, route of administration and excretion rate, duration of treatment, drugs combined or used simultaneously, and other factors well known in the field of medicine. It can be decided depending on The composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered singly or multiple times. Considering all of the above factors, it is important to administer an amount that can achieve maximum effect with the minimum amount without side effects, and this can be easily determined by a person skilled in the art.
  • the pharmaceutical composition of the present invention may contain a carrier, diluent, excipient, or a combination of two or more commonly used in biological products.
  • a carrier diluent, excipient, or a combination of two or more commonly used in biological products.
  • pharmaceutically acceptable means that the composition exhibits non-toxic properties to normal cells or humans exposed to the composition.
  • the carrier is not particularly limited as long as it is suitable for in vivo delivery of the composition, for example, Merck Index, 13th ed., Merck & Co. Inc.
  • saline solution sterilized water, Ringer's solution, buffered saline solution, dextrose solution, maltodextrin solution, glycerol, ethanol, and one or more of these ingredients can be mixed and used, and if necessary, other ingredients such as antioxidants, buffers, and bacteriostatic agents. Normal additives can be added.
  • diluents, dispersants, surfactants, binders, and lubricants can be additionally added to formulate dosage forms such as aqueous solutions, suspensions, emulsions, etc., into pills, capsules, granules, or tablets.
  • it can be preferably formulated according to each disease or ingredient using an appropriate method in the art or a method disclosed in Remington's Pharmaceutical Science (Mack Publishing Company, Easton PA, 18th, 1990).
  • the pharmaceutical composition may be one or more formulations selected from the group including oral formulations, topical formulations, suppositories, sterile injectable solutions, and sprays.
  • composition of the present invention may also include carriers, diluents, excipients, or combinations of two or more commonly used in biological products.
  • Pharmaceutically acceptable carriers are not particularly limited as long as they are suitable for in vivo delivery of the composition, for example, Merck Index, 13th ed., Merck & Co. Inc.
  • the compounds described in, saline solution, sterilized water, Ringer's solution, buffered saline solution, dextrose solution, maltodextrin solution, glycerol, ethanol, and one or more of these ingredients can be mixed and used, and if necessary, other ingredients such as antioxidants, buffers, and bacteriostatic agents. Normal additives can be added.
  • diluents can be additionally added to formulate dosage forms such as aqueous solutions, suspensions, emulsions, etc., into pills, capsules, granules, or tablets.
  • dosage forms such as aqueous solutions, suspensions, emulsions, etc., into pills, capsules, granules, or tablets.
  • it can be preferably formulated according to each disease or ingredient using an appropriate method in the art or a method disclosed in Remington's Pharmaceutical Science (Mack Publishing Company, Easton PA, 18th, 1990).
  • composition of the present invention may additionally contain one or more active ingredients that exhibit the same or similar functions.
  • the composition of the present invention contains 0.0001 to 10% by weight of the protein, preferably 0.001 to 1% by weight, based on the total weight of the composition.
  • the pharmaceutical composition of the present invention may further include pharmaceutically acceptable additives, wherein the pharmaceutically acceptable additives include starch, gelatinized starch, microcrystalline cellulose, lactose, povidone, colloidal silicon dioxide, and calcium hydrogen phosphate. , lactose, mannitol, taffy, gum arabic, pregelatinized starch, corn starch, powdered cellulose, hydroxypropyl cellulose, Opadry, sodium starch glycolate, lead carnauba, synthetic aluminum silicate, stearic acid, magnesium stearate, aluminum stearate, Calcium stearate, white sugar, dextrose, sorbitol, and talc may be used.
  • the pharmaceutically acceptable additive according to the present invention is preferably contained in an amount of 0.1 to 90 parts by weight based on the composition, but is not limited thereto.
  • composition of the present invention can be administered parenterally (e.g., intravenously, subcutaneously, intraperitoneally, or topically) or orally depending on the desired method, and the dosage is determined by the patient's weight, age, gender, health condition, The range varies depending on diet, administration time, administration method, excretion rate, and severity of disease.
  • the daily dosage of the composition according to the present invention is 0.0001 to 10 mg/ml, preferably 0.0001 to 5 mg/ml, and it is more preferable to administer it once or several times a day.
  • Liquid preparations for oral administration of the composition of the present invention include suspensions, oral solutions, emulsions, syrups, etc., and in addition to the commonly used simple diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, fragrances, and preservatives are used. etc. may be included together.
  • Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried preparations, suppositories, etc.
  • the present invention relates to a composition for eliminating M2 tumor-related macrophages comprising the complex of the present invention as an active ingredient.
  • the composition is capable of removing only M2 macrophages without removing M1 macrophages.
  • the present invention relates to a marker for diagnosing solid tumors including the ITGB2 gene or a protein expressed from the gene.
  • the present invention provides a composition for diagnosing solid cancer, comprising an agent for measuring the expression level of ITGB2 at the mRNA or protein level.
  • the agent for measuring at the mRNA level includes a nucleic acid sequence of the gene, a nucleic acid sequence complementary to the nucleic acid sequence, a primer pair, a probe, or a primer that specifically recognizes a fragment of the nucleic acid sequence and the complementary sequence. It can be a primer pair and a probe, and the measurement can be performed using polymerase chain reaction, real-time RT-PCR, reverse transcription polymerase chain reaction, competitive polymerase chain reaction (Competitive RT-PCR), and nuclease protection assay. (RNase, S1 nuclease assay), in situ hybridization, nucleic acid microarray, Northern blot, DNA chip, multiplex PCR, or ddPCR.
  • the agent for measuring at the protein level is an antibody, antibody fragment, aptamer, avidity multimer, or peptidomimetic ( peptidomimetics), and the measurement may be Western blot, ELISA (enzyme linked immunosorbent assay), RIA (Radioimmunoassay), radioimmunodiffusion, immunoelectrophoresis, tissue immunostaining, and immunoprecipitation assay. ), complement fixation assay, FACS, mass spectrometry, or protein microarray.
  • the term “detection” or “measurement” means quantifying the concentration of a detected or measured object.
  • the term "primer” is a nucleic acid sequence with a short free 3-terminal hydroxyl group that can form a base pair with a complementary template and serves as a starting point for copying the template strand. It refers to a short nucleic acid sequence that functions as a point. Primers can initiate DNA synthesis in the presence of four different nucleoside triphosphates and reagents for polymerization (i.e., DNA polymerase or reverse transcriptase) in an appropriate buffer solution and temperature.
  • probe refers to a nucleic acid fragment such as RNA or DNA that is as short as a few bases or as long as several hundred bases, capable of forming a specific binding to mRNA, and is labeled to determine the presence or absence of a specific mRNA. You can check. Probes may be manufactured in the form of oligonucleotide probes, single stranded DNA probes, double stranded DNA probes, RNA probes, etc. In the present invention, hybridization is performed using a probe complementary to ITGB2, and the level of gene expression can be diagnosed based on hybridization. Selection of appropriate probes and hybridization conditions can be modified based on those known in the art, so the present invention is not particularly limited thereto.
  • Primers or probes of the present invention can be chemically synthesized using the phosphoramidite solid support method or other well-known methods. These nucleic acid sequences can also be modified using many means known in the art. Non-limiting examples of such modifications include methylation, capping, substitution of a native nucleotide with one or more homologues, and modifications between nucleotides, such as uncharged linkages (e.g., methyl phosphonate, phosphotriester, phosphoronucleotide). amidate, carbamate, etc.) or charged linkages (e.g. phosphorothioate, phosphorodithioate, etc.).
  • uncharged linkages e.g., methyl phosphonate, phosphotriester, phosphoronucleotide
  • charged linkages e.g. phosphorothioate, phosphorodithioate, etc.
  • suitable conditions for hybridizing a probe to a cDNA molecule can be determined through a series of processes through an optimization procedure. These procedures are performed as a series by those skilled in the art to establish protocols for use in the laboratory. For example, conditions such as temperature, concentration of components, hybridization and washing time, buffer components and their pH and ionic strength depend on various factors such as the length of the probe, the amount of GC, and the target nucleotide sequence. Detailed conditions for hybridization are described in Joseph Sambrook, et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2001); and M.L.M. Anderson, NucleicAcidHybridization, Springer-Verlag New York Inc.
  • the high stringency conditions are hybridization at 65°C in 0.5 M NaHPO4, 7% SDS (sodium dodecyl sulfate), and 1mM EDTA, and 68°C in 0.1 x standard saline citrate (SSC)/0.1% SDS. It means washing with conditions.
  • high stringency means washing at 48°C in 6 x SSC/0.05% sodium pyrophosphate.
  • Low stringency conditions mean, for example, washing at 42°C in 0.2 x SSC/0.1% SDS.
  • an antibody is a term known in the art and refers to a specific protein molecule directed to an antigenic site.
  • an antibody refers to an antibody that specifically binds to the ITGB2 protein, which is a marker of the present invention, and the antibody can be produced using a well-known method. This also includes partial peptides that can be made from the above proteins.
  • the form of the antibody of the present invention is not particularly limited, and as long as it is a polyclonal antibody, monoclonal antibody, or has antigen binding properties, a portion thereof is also included in the antibody of the present invention, and all immunoglobulin antibodies are included.
  • the antibodies of the present invention also include special antibodies such as humanized antibodies.
  • the present invention provides the steps of processing a candidate material to ITGB2 protein; And it relates to a method of screening an anticancer immunotherapy agent, including the step of selecting a candidate substance that binds to the ITGB2 protein.
  • a step of determining that a candidate substance bound to the ITGB2 protein is a substance with anti-cancer effect may be further included.
  • the present invention relates to a method for predicting reactivity or prognosis for an immunotherapy agent targeting M2 macrophages, which includes the step of confirming the expression level of ITGB2 in a sample treated with an immunotherapy agent.
  • a step of determining that the sample has good responsiveness to the anticancer immunotherapy agent may be further included.
  • the sample is blood, serum, plasma, amniotic fluid, saliva, ascites, bone marrow, tear fluid, bile, lung lavage fluid, cerebrospinal fluid, pleural fluid, synovial fluid, lymph, semen, urine, or tissue biopsy or protein extraction from cells. It may be a solution.
  • the present invention includes the steps of treating a sample separated from a subject with a candidate substance; A method for screening an anticancer agent targeting M2 macrophages, comprising the step of confirming the expression level of ITGB2 in the sample.
  • the step of determining the candidate substance as an anticancer agent may be further included.
  • the present invention includes the steps of confirming the expression level of ITGB2 in a sample isolated from a subject; Comparing the expression level of ITGB2 with the control group; It relates to a method of providing information necessary for the diagnosis of cancer, including classifying subjects in which ITGB2 is overexpressed compared to the control group.
  • a step of determining that a subject in which ITGB2 is overexpressed is more likely to have cancer compared to the control group may be further included.
  • the primary cancer is colon cancer, breast cancer, uterine cancer, cervical cancer, ovarian cancer, prostate cancer, brain tumor, head and neck cancer, melanoma, myeloma, leukemia, lymphoma, stomach cancer, lung cancer, pancreatic cancer, non-small cell lung cancer, and liver cancer.
  • esophageal cancer small intestine cancer, perianal cancer, fallopian tube carcinoma, endometrial carcinoma, vaginal carcinoma, vulvar carcinoma, Hodgkin's disease, bladder cancer, kidney cancer, ureteral cancer, renal cell carcinoma, renal pelvic carcinoma, bone cancer, skin cancer, head cancer, Cervical cancer, cutaneous melanoma, intraocular melanoma, endocrine cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, central nervous system (CNS) tumor, primary CNS lymphoma, spinal tumor, It may be any one or more selected from the group consisting of glioblastoma multiforme and pituitary adenoma, and is most preferably a solid tumor.
  • CNS central nervous system
  • the present invention relates to a method of classifying a subject with cancer having reactivity to a substance targeting M2 macrophages, comprising the step of confirming the expression level of ITGB2 in a sample isolated from the subject.
  • a step of determining that the method may be reactive to a substance targeting M2 macrophages may be further included.
  • the Melittin, TAMPep826, Melittin-dKLA, and TB511 peptides shown in Table 1 below were supplied to GenScript (Piscataway, NJ, USA) for synthesis, and were used in subsequent experiments by dissolving them in D-PBS at 5 mg/ml.
  • GenScript Procataway, NJ, USA
  • the binding of dKLA was conducted to confirm the ease of binding with various anticancer drugs, and was connected through an amide bond between peptides.
  • a linker consisting of 4 glycines and 1 serine was placed in the middle to separate both ends, and KLA was used in a non-L form to minimize degradation in the body.
  • the D-type isomer was used.
  • THP-1 tumor-associated macrophages
  • PMA phorbol 12-myristate 13-acetate
  • M0 macrophages differentiated M0 macrophages were treated with 100 nM of LPS and 20 ng/mL.
  • M1 macrophages M1 macrophages
  • M2 macrophages M2 macrophages
  • Differentiated cells were treated with 50 nM of FITC-conjugated melittin for 1 hour, and cells were detected with BD FACS CantoII instruments (BD biosciences, San Jose, CA, USA) and analyzed with FlowJo software (BD biosciences).
  • biotin-conjugated melittin (melittin-biotin) was synthesized. After differentiating THP-1 cells (5 ⁇ 10 6 cells) into M0, M1, and M2 macrophages, cells were collected using a scraper, washed by centrifugation (300 ⁇ g, 5 minutes), and cell membrane protein was extracted. Cell membrane proteins of macrophages were obtained using a membrane protein extraction kit (Thermo Fisher scientific).
  • melittin-biotin 200 ⁇ g
  • streptavidin resin streptavidin resin (Thermo Fisher scientific).
  • a protein bound to tin was obtained.
  • 100 ⁇ l each of 100% methanol, 0.1% formic acid, and 80% CAN were added to an 18 Micro Spin-Column to prepare the sample, and then the sample was loaded onto the prepared column and 50 ⁇ l of 0.1% formic acid was added to remove unnecessary salts. The material was removed. Afterwards, 100 ⁇ l of 80% CAN was added to elute the peptides.
  • the target protein was confirmed through LC-MS/MS proteomics by reacting TAMpep826-biotin with cell membrane proteins extracted from M0, M1, and M2 macrophages, respectively.
  • ITGB2 was found to be expressed at a higher level in M2 macrophages compared to M0 and M1 macrophages ( Figure 4), and was selected as the M2 macrophage binding target protein of TAMpep826.
  • Western blot analysis was performed to evaluate the protein expression level of ITGB2 in M0, M1, M2 macrophages and TAMs. Specifically, proteins were extracted from each cell differentiated into M0, M1, and M2 macrophages using Proprep solution (iNtRON), and the proteins were transferred to the membrane by electrophoresis. The membrane was blocked by soaking it in 5% BSA, then treated with primary antibody (ITGB2) at 4°C for 24 hours and secondary antibody at room temperature for 1 hour, followed by D-Plus TM ECL Pico System (Dongin LS). was processed and protein band photos were taken with the Davinch-Chemi TM imaging system (Intoxia).
  • IGB2 primary antibody
  • D-Plus TM ECL Pico System Dongin LS
  • Immunofluorescence analysis was performed to evaluate the protein expression level of ITGB2 in M0, M1, M2 macrophages and TAMs. Specifically, each cell differentiated into M0, M1, and M2 macrophages was fixed with 4% formalin, washed with PBS, blocked by soaking in 5% BSA, and incubated with primary antibody (ITGB2) at 4°C for 24 hours. and staining was performed by treating with secondary antibody at room temperature for 2 hours. Stained cells were photographed using LSM800 (Zeiss) after DAPI mounting.
  • the protein of ITGB2 showed a higher expression level in M2 macrophages and TAMs compared to M0 and M1 ( Figure 8).
  • TAMpep826 interacts with ITGB2 in M2 macrophages
  • the expression of ITGB2 was assessed through competitive reaction of TAMpep826 and biotinylated TAMpep826. Specifically, cells differentiated into M2 macrophages were collected with a scraper, washed by centrifugation (300 ⁇ g, 5 minutes), and cell membrane proteins of macrophages were obtained using a cell membrane protein extraction kit (Thermo Fisher scientific).
  • TAMpep826 or scramble peptide After pre-treating TAMpep826 or scramble peptide at different concentrations (0.001, 0.01, 0.1, 1, and 10 ⁇ g) for 1 hour, react with TAMpep826 (200 ⁇ g), which has biotin bound to the protein, at room temperature (20-24°C) for 1 hour. After this, the protein bound to TAMpep826 was obtained using Dynabeads TM M-280 Streptavidin (Thermo Fisher scientific). The expression of ITGB2 was confirmed by Western blot analysis of the protein.
  • TAMpep826 and ITGB2 were found to be concentratedly distributed in the cell membrane of M2 macrophages (FIG. 11).
  • ITGB2 protein was coated on a gold thin film sensor chip, then ITGB2 antibody and Melittin-dKLA were flowed as positive controls, respectively, and the reflected light was measured, and surface plasmon resonance (SPR) was used. Analysis was performed. Specifically, ITGB2 was immobilized on the surface of the sensor chip, and then immobilization was performed using amine coupling under the conditions shown in Table 4 below (FIG. 12).
  • a cell model in which the expression of ITGB2 was suppressed in M2 macrophages was created using Crispr/cas9. Specifically, cells differentiated into M2 macrophages were cultured in Opti-MEM medium. For Crispr/cas9, Cas9 protein V2 (Thermo Fisher scientific) and gRNA (Genscript; ITGB2; Table 6) from the CRISPRMAX TM Reagent kit (Thermo Fisher scientific) were mixed with Cas9 Plus TM Reagent (Thermo Fisher scientific), and CRISPRMAX TM Reagent was used.
  • Opti-MEM medium After diluting in Opti-MEM medium, everything was mixed and reacted for 5-10 minutes, then added to cells differentiated into M2 macrophages and cultured at 37°C for 2-3 days. Afterwards, the mRNA expression level of ITGB2 was confirmed.
  • Example 7-1 M2 macrophages in which ITGB2 expression was suppressed by Crispr/cas9 produced in Example 7-1. (1 ⁇ M) was treated for 24 hours, and cell viability was analyzed using the CCK-8 assay method. Specifically, ITGB2 knockdown macrophages prepared in Example 7-1 were reacted with Melittin-dKLA (1 ⁇ M) for 1 hour. After the reaction, the medium was replaced and cultured at 37°C for 24 hours. To check cell viability, CCK-8 reagent (Enzo Life Sciences) was added to 1/10 of the medium and reacted at 37°C for 3 hours. Absorbance was measured at 450 nm with a microplate leader (Molecular Devices).
  • TAMPep826-dKLA (TB511) induces apoptosis through ITGB2 in M2 macrophages
  • the TB511 was reacted with M2 macrophages in which ITGB2 expression was suppressed by Crispr/cas9.
  • M2 macrophages showed a cell viability of about 50% due to TB511, but cells in which ITGB2 expression was suppressed showed a significant increase in cell viability (FIG. 17).
  • M2 macrophages with suppressed ITGB2 expression prepared in Example 7-1 were pretreated with anti-ITGB2 antibody (1 ⁇ g) for 1 hour. Then TB511 was reacted.

Abstract

The present invention relates to a drug delivery system that specifically binds to ITGB2 on M2 tumor-associated macrophages, and, according to the present invention, the complex of a drug delivery vehicle comprising a targeting agent that specifically binds to ITGB2 on M2 tumor-associated macrophages and a drug bound to the vehicle can selectively (or specifically) act on the M2 tumor-associated macrophages, thereby reducing side effects of the drug and increasing the efficacy thereof, and can be useful as a drug delivery vehicle for apoptosis-inducing agents, anticancer agents, contrast agents, and the like.

Description

ITGB2 매개 약물 전달시스템ITGB2-mediated drug delivery system
본 발명은 ITGB2에 특이적으로 결합하는 약물 전달시스템에 관한 것이다.The present invention relates to a drug delivery system that specifically binds to ITGB2.
기존의 항암치료는 암세포를 직접 공격하거나 암세포를 공격하는 체내 면역세포의 활성을 강화시키는 방향으로 연구되어 왔다. 그러나 이러한 항암제는 암세포 외의 다른 정상세포 또한 공격하여 탈모, 오심, 구토등의 많은 부작용을 가져왔고, 면역세포의 과도한 증가로 인한 부가적 반응을 초래한다. 기존의 화학적 요법이나 방사선 치료방법에 비하여 부작용을 최소화할 수 있는 항암면역치료방법은 체내의 면역시스템을 이용하여 암을 치료하는 방법이며, 이러한 항암면역치료기법 중에는 치료용 면역세포인 T 세포 (CAR-T 포함), 수지상세포(Dendritic Cells), 자연살해세포(Natural Killer Cells) 등을 체외에서 활성화 시킨 후에 체내에 직접 주입하는 세포치료제방법과 암 항원과 면역활성화 물질을 체내에 주입함으로써, 체내에 존재하는 면역세포를 직접적으로 활성화함으로써 항암효능을 높이는 항암백신에 대한 방법 등이 활발하게 진행되고 있다. 하지만, 이러한 세포치료제나 암백신은 주로 혈액암 관련 질병에 주로 사용되고 있고, 고형암에서는 대부분 그 치료효능이 매우 낮다는 단점을 갖고 있다. 이러한 이유 중의 하나는 고형암 주위에서 면역기능을 억제하는 미세환경 요인에 기인한다. 실제로, 종양미세환경에서 면역세포의 기능을 저하시키는 세포 (MDSC: myeoloid-derived stromal cells, Treg: regulatory T cell, TAM: tumor-assocaited macrophages)나 면역억제유발 사이토카인, 대사체 등이 활발하게 작용함으로써, 면역활성화 물질과 치료용 면역세포의 활성을 급격하게 저하시키는 것이다. 따라서 종양세포 및 면역세포에 직접적인 영향없이 종양세포의 주변 미세환경만을 조절하여 종양세포로의 영양분 공급, 종양세포 주변의 혈관신생등을 차단하여 항암효과를 갖는 치료제 개발이 중요해지고 있다. 종양 미세 환경은 악성 세포의 증식 및 생존, 혈관 신생, 전이, 비정상 적응 면역 및 호르몬 및 화학요법제에 대한 반응 감소에 기여하여 치료 목표로 크게 고려되고 있다. Existing anti-cancer treatments have been studied to attack cancer cells directly or to strengthen the activity of the body's immune cells that attack cancer cells. However, these anticancer drugs also attack other normal cells in addition to cancer cells, resulting in many side effects such as hair loss, nausea, and vomiting, and cause additional reactions due to an excessive increase in immune cells. Anticancer immunotherapy, which can minimize side effects compared to existing chemotherapy or radiation treatment methods, is a method of treating cancer using the body's immune system. Among these anticancer immunotherapy methods, T cells (CAR), which are therapeutic immune cells, are used to treat cancer. -T included), dendritic cells, natural killer cells, etc. are activated outside the body and then directly injected into the body, and cancer antigens and immune-activating substances are injected into the body. Methods for anti-cancer vaccines that increase anti-cancer efficacy by directly activating existing immune cells are being actively developed. However, these cell therapies and cancer vaccines are mainly used for diseases related to blood cancer, and have the disadvantage of having very low therapeutic efficacy in most solid cancers. One of these reasons is due to microenvironmental factors that suppress immune function around solid tumors. In fact, cells that reduce the function of immune cells (MDSC: myeoloid-derived stromal cells, Tregs: regulatory T cells, TAM: tumor-associated macrophages), immunosuppression-inducing cytokines, and metabolites actively act in the tumor microenvironment. By doing so, the activity of immune-activating substances and therapeutic immune cells is drastically reduced. Therefore, it is becoming important to develop treatments that have anti-cancer effects by controlling the microenvironment surrounding tumor cells without directly affecting tumor cells and immune cells, thereby blocking the supply of nutrients to tumor cells and angiogenesis around tumor cells. The tumor microenvironment contributes to the proliferation and survival of malignant cells, angiogenesis, metastasis, abnormal adaptive immunity, and reduced response to hormonal and chemotherapy agents, and is therefore greatly considered as a therapeutic target.
종양 주변의 미세환경(microenvironment)은 내피세포, 염증성 세포 및 섬유아세포로 구성되어 있으며, 1970년대에 종양 관련 대식세포(tumor-associated macrophage, TAM)가 종양의 성장에 있어서 중요한 역할을 한다는 것이 밝혀졌다. 종양 관련 대식세포는 암의 성장, 전이 등 전반적인 종양 미세환경과 관련하여 중요한 역할을 담당하며, 종양억제 M1형 대식세포 또는 종양지지 M2형 대식세포의 두 가지 표현형으로 분류된다. M1형 대식세포는 항원을 제시하는 강력한 능력을 갖고, 일반적으로 인터페론-γ, 지방당류(LPS), 종양 괴사 인자(TNF)-α에 의하여 활성화 되며, 전염증 작용 및 살균 작용을 한다. M2형 대식세포는 다양한 세포 외 기질성분, 혈관 신생 및 주화성 인자를 방출함으로써 면역억제, 종양형성 및 혈관형성을 촉진하는 것으로 알려져 있다. 일반적으로, IL-4와 IL-13에 의해 유도되며, 아르기나제-1, mannose(MMR, CD206), 스캐빈저 수용체(SR-A, CD204), CD163, IL-10과 같은 마커를 발현함으로써 M1형 종양 관련 대식세포와 구별된다. 종양 주변에 존재하는 종양 관련 대식세포는 종양 세포의 성장, 전이와 밀접하게 관련이 되어 있으며, 암 환자에서 많은 숫자의 M2형 종양 관련 대식세포가 종양 주변에 존재하면 환자의 예후 및 생존률이 좋지 못한 것으로 보고되고 있다. M2형 대식세포는 암의 성장을 촉진하는 IL-10, TGFβ 및 CCL18과 같은 사이토카인을 생성하며, M2형 종양 관련 대식세포의 표면에 존재하는 PDL1 및 B7-1/2와 같은 수용체들은 T 세포, NK 세포의 항종양 활성을 억제하는 것으로 보고되고 있다. M2형 종양 관련 대식세포가 다량으로 존재하는 미세환경에서는 종양의 성장, 분화 및 전이가 활발하게 이루어지므로, M2형 종양 관련 대식세포를 표적으로 하는 표적 치료제의 개발이 필요하다.The microenvironment surrounding the tumor is composed of endothelial cells, inflammatory cells, and fibroblasts, and in the 1970s, it was discovered that tumor-associated macrophages (TAMs) play an important role in tumor growth. . Tumor-related macrophages play an important role in the overall tumor microenvironment, including cancer growth and metastasis, and are classified into two phenotypes: tumor suppressor M1 type macrophages or tumor supporting M2 type macrophages. M1 type macrophages have a strong ability to present antigens, are generally activated by interferon-γ, liposaccharide (LPS), and tumor necrosis factor (TNF)-α, and have pro-inflammatory and bactericidal effects. M2-type macrophages are known to promote immunosuppression, tumorigenesis, and angiogenesis by releasing various extracellular matrix components, angiogenic, and chemotactic factors. Generally, it is induced by IL-4 and IL-13 and expresses markers such as arginase-1, mannose (MMR, CD206), scavenger receptor (SR-A, CD204), CD163, and IL-10. This differentiates them from M1 type tumor-related macrophages. Tumor-related macrophages present around the tumor are closely related to the growth and metastasis of tumor cells. In cancer patients, if a large number of M2-type tumor-related macrophages are present around the tumor, the patient's prognosis and survival rate are poor. It is reported that M2-type macrophages produce cytokines such as IL-10, TGFβ, and CCL18 that promote cancer growth, and receptors such as PDL1 and B7-1/2 present on the surface of M2-type tumor-related macrophages stimulate T cells. , it has been reported to inhibit the anti-tumor activity of NK cells. Since tumor growth, differentiation, and metastasis occur actively in a microenvironment where large amounts of M2-type tumor-related macrophages exist, the development of targeted therapeutic agents targeting M2-type tumor-related macrophages is necessary.
본 발명의 목적은 약물 전달시스템을 제공하는 것이다. The object of the present invention is to provide a drug delivery system.
또한, 본 발명의 목적은 약물 전달시스템와 약물의 복합체를 제공하는 것이다. Additionally, an object of the present invention is to provide a complex of a drug delivery system and a drug.
또한, 본 발명의 목적은 면역항암제를 제공하는 것이다. Additionally, an object of the present invention is to provide an immuno-anticancer agent.
또한, 본 발명의 목적은 암의 예방 또는 치료용 약학적 조성물을 제공하는 것이다.Additionally, an object of the present invention is to provide a pharmaceutical composition for preventing or treating cancer.
또한, 본 발명의 목적은 M2 종양 관련 대식세포 제거용 조성물을 제공하는 것이다.Additionally, an object of the present invention is to provide a composition for eliminating M2 tumor-related macrophages.
또한, 본 발명의 목적은 고형암 진단용 마커 및 이를 포함하는 조성물을 제공하는 것이다.Additionally, an object of the present invention is to provide a marker for diagnosing solid cancer and a composition containing the same.
또한, 본 발명의 목적은 면역항암제를 스크리닝하는 방법을 제공하는 것이다.Additionally, an object of the present invention is to provide a method for screening immuno-anticancer agents.
또한, 본 발명의 목적은 면역항암제에 대한 반응성 예측 또는 예후 진단 방법을 제공하는 것이다.Additionally, the purpose of the present invention is to provide a method for predicting responsiveness to anti-cancer immunotherapy or diagnosing prognosis.
또한, 본 발명의 목적은 M2 대식세포를 표적화하는 항암제를 스크리닝하는 방법을 제공하는 것이다.Additionally, an object of the present invention is to provide a method for screening anticancer drugs targeting M2 macrophages.
아울러, 본 발명의 목적은 암의 진단에 필요한 정보를 제공하는 방법을 제공하는 것이다.In addition, the purpose of the present invention is to provide a method of providing information necessary for diagnosing cancer.
상기 목적의 달성을 위해, 본 발명은 ITGB2 매개 약물 전달시스템을 제공한다.To achieve the above object, the present invention provides an ITGB2-mediated drug delivery system.
또한, 본 발명은 상기 ITGB2 매개 약물 전달시스템에 약물이 결합한 복합체를 제공한다.Additionally, the present invention provides a complex in which a drug is bound to the ITGB2-mediated drug delivery system.
또한, 본 발명은 상기 복합체를 유효성분으로 포함하는 면역항암제를 제공한다.Additionally, the present invention provides an anti-cancer immunological agent containing the above complex as an active ingredient.
또한, 본 발명은 상기 복합체를 유효성분으로 포함하는 M2 종양 관련 대식세포 매개 암의 예방 또는 치료용 약학적 조성물을 제공한다.Additionally, the present invention provides a pharmaceutical composition for preventing or treating M2 tumor-related macrophage-mediated cancer, comprising the complex as an active ingredient.
또한, 본 발명은 상기 복합체를 유효성분으로 포함하는 M2 종양 관련 대식세포 제거용 조성물을 제공한다.Additionally, the present invention provides a composition for eliminating M2 tumor-related macrophages containing the complex as an active ingredient.
또한, 본 발명은 ITGB2 유전자 또는 상기 유전자로부터 발현된 단백질을 포함하는 고형암 진단용 마커 및 이를 포함하는 조성물을 제공한다.Additionally, the present invention provides a marker for diagnosing solid tumors containing the ITGB2 gene or a protein expressed from the gene and a composition containing the same.
또한, 본 발명은 면역항암제를 스크리닝하는 방법을 제공한다.Additionally, the present invention provides a method for screening immuno-anticancer agents.
또한, 본 발명은 면역항암제에 대한 반응성 예측 또는 예후 진단 방법을 제공한다.Additionally, the present invention provides a method for predicting responsiveness to anti-cancer immunotherapy or diagnosing prognosis.
또한, 본 발명은 M2 대식세포를 표적화하는 항암제를 스크리닝하는 방법을 제공한다.Additionally, the present invention provides a method for screening anticancer agents targeting M2 macrophages.
아울러, 본 발명은 암의 진단에 필요한 정보를 제공하는 방법을 제공한다.In addition, the present invention provides a method of providing information necessary for diagnosing cancer.
본 발명에서는 ITGB2가 M2 대식세포 매개 고형암에 특이적인 마커임을 확인하였으며, 본 발명의 ITGB2(Integrin beta 2)에 특이적으로 결합하는 표적화 물질을 유효성분으로 포함하는 ITGB2 매개 약물 전달시스템과 이에 약물이 결합한 복합체는 M2 종양 관련 대식세포에만 선택적으로(또는 특이적으로) 작용함으로써 약물 부작용은 경감되고 약물의 약효는 증대시킬 수 있는 효과를 가지므로, 세포사멸 유도제, 항암제, 조영제 등의 약물 전달체로서 유용하게 사용될 수 있다.In the present invention, ITGB2 was confirmed to be a specific marker for M2 macrophage-mediated solid cancer, and an ITGB2-mediated drug delivery system containing a targeting substance that specifically binds to ITGB2 (Integrin beta 2) of the present invention as an active ingredient and the drug thereto were developed. The combined complex has the effect of reducing drug side effects and increasing drug efficacy by acting selectively (or specifically) only on M2 tumor-related macrophages, so it is useful as a drug carrier for apoptosis inducers, anticancer agents, contrast agents, etc. It can be used effectively.
도 1은 TAMpep의 M2 대식세포에 대한 친화도를 확인한 도이다:Figure 1 is a diagram confirming the affinity of TAMpep for M2 macrophages:
A: 유세포 분석으로 확인한 TAMpep의 결합 친화도; 및A: Binding affinity of TAMpep determined by flow cytometry; and
B: 정량화한 TAMpep의 결합 친화도 그래프 (*P < 0.05 versus the M0.).B: Quantified binding affinity graph of TAMpep (* P < 0.05 versus the M0.).
도 2는 M2 대식세포에서 TAMpep과 결합하는 단백질을 식별한 도이다:Figure 2 is a diagram identifying proteins that bind to TAMpep in M2 macrophages:
A: M2/M0 및 M1/M0 비율 분석 벤다이어그램;A: M2/M0 and M1/M0 ratio analysis Venn diagram;
B: M2/M0 비율의 산점도 플롯(scatter plots) 분석 결과;B: Scatter plots analysis results of M2/M0 ratio;
C: M1/M0 비율의 산점도 플롯 분석 결과; 및C: Scatter plot analysis results of M1/M0 ratio; and
D: M2/M1 비율의 산점도 플롯 분석 결과.D: Scatter plot analysis results of M2/M1 ratio.
도 3은 M0, M1 및 M2 대식세포에서 각각 추출한 세포막 단백질과 TAMpep826-비오틴을 반응시켜 얻은 LC-MS 크로마토그램 데이터를 나타낸 도이다.Figure 3 is a diagram showing LC-MS chromatogram data obtained by reacting TAMpep826-biotin with cell membrane proteins extracted from M0, M1, and M2 macrophages, respectively.
도 4는 LC-MS/MS 프로테오믹스를 이용하여 TAMpep826의 타겟 단백질을 동정한 도이다.Figure 4 is a diagram showing the identification of the target protein of TAMPep826 using LC-MS/MS proteomics.
도 5는 Quantitative RT-PCR 조건을 나타낸 도이다.Figure 5 is a diagram showing quantitative RT-PCR conditions.
도 6은 M2 대식세포에서 ITGB2 mRNA 발현을 확인한 도이다.Figure 6 is a diagram confirming ITGB2 mRNA expression in M2 macrophages.
도 7은 M2 대식세포에서 ITGB2 단백질 발현을 확인한 도이다.Figure 7 is a diagram confirming ITGB2 protein expression in M2 macrophages.
도 8은 면역형광분석으로 M2 대식세포에서 ITGB2 단백질의 발현을 확인한 도이다.Figure 8 is a diagram confirming the expression of ITGB2 protein in M2 macrophages by immunofluorescence analysis.
도 9는 M2 대식세포의 세포막에서 ITGB2의 발현을 확인한 도이다.Figure 9 is a diagram confirming the expression of ITGB2 in the cell membrane of M2 macrophages.
도 10은 M2 대식세포에서 TAMpep826 및 ITGB2의 상호작용을 풀-다운 분석(Pull-down assay)으로 확인한 도이다.Figure 10 is a diagram confirming the interaction of TAMpep826 and ITGB2 in M2 macrophages by pull-down assay.
도 11은 면역형광분석으로 M2 대식세포에서 TAMpep826 및 ITGB2의 상호작용 확인한 도이다.Figure 11 is a diagram confirming the interaction between TAMpep826 and ITGB2 in M2 macrophages using immunofluorescence analysis.
도 12는 센서 칩 표면에 ITGB2을 고정화하고 확인한 도이다.Figure 12 is a diagram showing the immobilization and confirmation of ITGB2 on the surface of the sensor chip.
도 13은 항-ITGB2 항체와 ITGB2 단백질의 결합력을 SPR로 확인한 도이다.Figure 13 is a diagram confirming the binding force between anti-ITGB2 antibody and ITGB2 protein by SPR.
도 14는 Melittin-dKLA와 ITGB2 단백질의 결합력을 SPR로 확인한 도이다.Figure 14 is a diagram confirming the binding force between Melittin-dKLA and ITGB2 protein by SPR.
도 15는 M2 대식세포에서 Crispr/cas9을 통해 확립한 ITGB2 발현 억제 세포 모델에서 ITGB2의 mRNA 발현을 확인한 도이다.Figure 15 is a diagram confirming the mRNA expression of ITGB2 in a cell model that suppresses ITGB2 expression established through Crispr/cas9 in M2 macrophages.
도 16 M2 대식세포에서 ITGB2 발현 억제에 의한 Melittin-dKLA의 세포사멸 감소 효과를 확인한 도이다.Figure 16 is a diagram confirming the effect of Melittin-dKLA on reducing apoptosis by suppressing ITGB2 expression in M2 macrophages.
도 17은 M2 대식세포에서 ITGB2 발현 억제에 의한 TB511(TAMpep826-dKLA)의 세포사멸 유도 감소 효과를 확인한 도이다.Figure 17 is a diagram confirming the effect of TB511 (TAMpep826-dKLA) on reducing apoptosis induction by suppressing ITGB2 expression in M2 macrophages.
도 18은 M2 대식세포에서 ITGB2 항체에 의한 TB511의 세포사멸 유도 감소 효과를 확인한 도이다.Figure 18 is a diagram confirming the effect of reducing apoptosis induction of TB511 by ITGB2 antibody in M2 macrophages.
이하, 첨부된 도면을 참조하여 본 발명의 구현예로 본 발명을 상세히 설명하기로 한다. 다만, 하기 구현예는 본 발명에 대한 예시로 제시되는 것으로, 당업자에게 주지 저명한 기술 또는 구성에 대한 구체적인 설명이 본 발명의 요지를 불필요하게 흐릴 수 있다고 판단되는 경우에는 그 상세한 설명을 생략할 수 있고, 이에 의해 본 발명이 제한되지는 않는다. 본 발명은 후술하는 특허청구범위의 기재 및 그로부터 해석되는 균등 범주 내에서 다양한 변형 및 응용이 가능하다. Hereinafter, the present invention will be described in detail through embodiments of the present invention with reference to the attached drawings. However, the following embodiments are provided as examples of the present invention, and if it is judged that a detailed description of a technology or configuration well known to those skilled in the art may unnecessarily obscure the gist of the present invention, the detailed description may be omitted. , the present invention is not limited thereby. The present invention is capable of various modifications and applications within the description of the claims described below and the scope of equivalents interpreted therefrom.
또한, 본 명세서에서 사용되는 용어(terminology)들은 본 발명의 바람직한 실시예를 적절히 표현하기 위해 사용된 용어들로서, 이는 사용자, 운용자의 의도 또는 본 발명이 속하는 분야의 관례 등에 따라 달라질 수 있다. 따라서, 본 용어들에 대한 정의는 본 명세서 전반에 걸친 내용을 토대로 내려져야 할 것이다. 명세서 전체에서, 어떤 부분이 어떤 구성요소를 "포함"한다고 할 때, 이는 특별히 반대되는 기재가 없는 한 다른 구성요소를 제외하는 것이 아니라 다른 구성 요소를 더 포함할 수 있는 것을 의미한다.In addition, the terminology used in this specification is a term used to appropriately express preferred embodiments of the present invention, and may vary depending on the intention of the user or operator or the customs of the field to which the present invention belongs. Therefore, definitions of these terms should be made based on the content throughout this specification. Throughout the specification, when a part is said to “include” a certain element, this means that it may further include other elements rather than excluding other elements, unless specifically stated to the contrary.
본 발명에서 사용되는 모든 기술용어는, 달리 정의되지 않는 이상, 본 발명의 관련 분야에서 통상의 당업자가 일반적으로 이해하는 바와 같은 의미로 사용된다. 또한 본 명세서에는 바람직한 방법이나 시료가 기재되나, 이와 유사하거나 동등한 것들도 본 발명의 범주에 포함된다. 본 명세서에 참고문헌으로 기재되는 모든 간행물의 내용은 본 발명에 통합된다.All technical terms used in the present invention, unless otherwise defined, are used with the same meaning as commonly understood by a person skilled in the art in the field related to the present invention. In addition, preferred methods and samples are described in this specification, but similar or equivalent methods are also included in the scope of the present invention. The contents of all publications incorporated herein by reference are hereby incorporated by reference.
본 명세서 전반을 통하여, 천연적으로 존재하는 아미노산에 대한 통상의 1문자 및 3문자 코드가 사용될 뿐만 아니라 Aib(α-아미노이소부티르산), Sar(N-methylglycine) 등과 같은 다른 아미노산에 대해 일반적으로 허용되는 3문자 코드가 사용된다. 또한 본 발명에서 약어로 언급된 아미노산은 하기와 같이 IUPAC-IUB 명명법에 따라 기재되었다:Throughout this specification, the usual one- and three-letter codes for naturally occurring amino acids are used, as well as generally acceptable codes for other amino acids such as Aib (α-aminoisobutyric acid), Sar (N-methylglycine), etc. A three-character code is used. Additionally, amino acids referred to by abbreviations in the present invention are described according to the IUPAC-IUB nomenclature as follows:
알라닌: A, 아르기닌: R, 아스파라긴: N, 아스파르트산: D, 시스테인: C, 글루탐산: E, 글루타민: Q, 글리신: G, 히스티딘: H, 이소류신: I, 류신: L, 리신: K, 메티오닌: M, 페닐알라닌: F, 프롤린: P, 세린: S, 트레오닌: T, 트립토판: W, 티로신: Y 및 발린: V. Alanine: A, Arginine: R, Asparagine: N, Aspartic Acid: D, Cysteine: C, Glutamic Acid: E, Glutamine: Q, Glycine: G, Histidine: H, Isoleucine: I, Leucine: L, Lysine: K, Methionine : M, phenylalanine: F, proline: P, serine: S, threonine: T, tryptophan: W, tyrosine: Y and valine: V.
일 측면에서, 본 발명은 ITGB2(Integrin beta 2)에 특이적으로 결합하는 표적화 물질을 유효성분으로 포함하는 ITGB2 매개 약물 전달시스템에 관한 것이다.In one aspect, the present invention relates to an ITGB2-mediated drug delivery system comprising a targeting substance that specifically binds to ITGB2 (Integrin beta 2) as an active ingredient.
일 구현예에서, 표적화 물질은 M2 종양 관련 대식세포의 세포막에 존재하는 ITGB2 단백질에 특이적으로 결합할 수 있다.In one embodiment, the targeting agent can specifically bind to the ITGB2 protein present in the cell membrane of M2 tumor-related macrophages.
일 구현예에서, 표적화 물질은 ITGB2와 특이적으로 결합하는 소분자, 바이러스, 항체, 항체 단편, 압타머, 호르몬, 사이토카인, 케모카인, 리간드, 사이토카인의 일부 영역인 펩타이드 또는 리간드의 일부 영역인 펩타이드일 수 있다.In one embodiment, the targeting agent is a small molecule, virus, antibody, antibody fragment, aptamer, hormone, cytokine, chemokine, ligand, peptide that is a partial region of a cytokine, or a peptide that is a partial region of a ligand that specifically binds to ITGB2. It can be.
일 구현예에서, 상기 표적화 물질에 반감기 또는 안정성을 증가시키기 위한 특정 목적을 위해 설계된 표적화 서열, 태그, 표지된 잔기 또는 아미노산 서열을 추가로 포함할 수 있다.In one embodiment, the targeting agent may further include a targeting sequence, tag, labeled residue, or amino acid sequence designed for the specific purpose of increasing half-life or stability.
일 구현예에서, 상기 표적화 물질에 RNA, DNA, 항체, 이펙터, 약물, 전구약물, 독소, 펩티드 또는 전달 분자가 추가로 접합(conjugate)될 수 있다 (Shoari et al., Pharmaceutics 13:1391, pp. 1-32 (2021) 참조).In one embodiment, RNA, DNA, antibodies, effectors, drugs, prodrugs, toxins, peptides, or delivery molecules may be additionally conjugated to the targeting agent (Shoari et al., Pharmaceutics 13:1391, pp 1-32 (2021)).
본 발명에서, 표적화 물질은 표적 세포인 M2 종양 관련 대식세포에 ITGB2와의 결합을 통해 선택적인 결합이 가능하게 함으로써 표적 기능을 제공한다. 이 세포 표적화 물질은 표적세포의 표면에 존재하는 ITGB2와 특이적으로 결합하여 엔도시토시스(endocytosis)를 유도함으로써, 이와 결합하는 약물의 세포 내로의 이동을 가능하게 한다.In the present invention, the targeting agent provides a targeting function by enabling selective binding to M2 tumor-related macrophages, which are target cells, through binding to ITGB2. This cell-targeting substance specifically binds to ITGB2 on the surface of target cells and induces endocytosis, enabling the drug that binds to it to move into the cell.
본 발명에서, 항체, 압타머, 특정 세포에서 분비되어 표적세포인 다른 세포의 표면 수용체에 작용함으로써 세포 사이에서의 신호 전달의 역할을 하는 호르몬(예컨대 erythropoietin hormone), 사이토카인이나 케모카인(예컨대 IL13), 표적세포 표면 수용체 결합하는 VEGF(Vascular endothelial growth factor), BDNF(Brain-derived neurotrophic factor) 등의 생체 분자인 리간드, 수용체와의 특이적 결합 능력을 보유한 이들 인자의 일부 영역인 펩타이드 등이 이러한 세포 표적화 물질로 이용될 수 있다. 대표적으로 표적화 물질로 항체 또는 압타머가 사용될 수 있으며, 표적화 물질로서의 항체는 모노클로날 항체, 폴리클로날 항체뿐만 아니라 다중특이적 항체(즉 두 개 이상의 항원 또는 두 개 이상의 에피토프를 인식하는 항체로서 이특이적 항체 등을 말함)이거나, ITGB2와 특이적으로 결합할 수 있는 능력을 보유하는 한, 항체의 단편, 화학적으로 수식된 항체, 키메릭 항체(인간과 마우스의 키메릭 항체, 인간과 원숭이 키메릭 항체 등), 면원원성을 낮춘 인간화 항체나 인간 항체 등 임의의 항체일 수 있다. 또한 항체 단편, 화학적 수식된 항체의 다양한 형태들이 당업계에 공지되어 있으며, 예컨대 Fab, Fd, Fab', dAb, F(ab'), F(ab')2, scFv(single chain fragment variable), Fv, 단일쇄 항체, Fv 이량체, 상보성 결정 영역 단편, 인간화 항체, 키메라 항체 및 디아바디(diabody) 등이나 항체를 단백질 절단 효소 예컨대, 파파인, 펩신으로 처리하여 얻어진 항체 단편 등을 들 수 있다.In the present invention, antibodies, aptamers, hormones (e.g., erythropoietin hormone), cytokines or chemokines (e.g., IL13) that are secreted from specific cells and play a role in signal transmission between cells by acting on surface receptors of other cells that are target cells. , Ligands, which are biomolecules such as VEGF (Vascular endothelial growth factor) and BDNF (Brain-derived neurotrophic factor), that bind to target cell surface receptors, and peptides, which are some regions of these factors that have the ability to specifically bind to receptors, are used to target these cells. It can be used as a targeting agent. Typically, antibodies or aptamers can be used as targeting substances, and antibodies as targeting substances include not only monoclonal antibodies and polyclonal antibodies, but also multispecific antibodies (i.e., antibodies that recognize two or more antigens or two or more epitopes). target antibodies, etc.), or antibody fragments, chemically modified antibodies, chimeric antibodies (human and mouse chimeric antibodies, human and monkey chimeric antibodies) as long as they have the ability to specifically bind to ITGB2. It may be any antibody, such as a humanized antibody or a human antibody with reduced immunogenicity (antibodies, etc.). Additionally, various forms of antibody fragments and chemically modified antibodies are known in the art, such as Fab, Fd, Fab', dAb, F(ab'), F(ab') 2 , scFv (single chain fragment variable), Examples include Fv, single-chain antibodies, Fv dimers, complementarity-determining region fragments, humanized antibodies, chimeric antibodies, and diabodies, and antibody fragments obtained by treating antibodies with protein cleavage enzymes such as papain and pepsin.
항체의 제조 방법, 천연 항체에 인위적 변형을 가하여 표적 항원에 대한 특이성을 항상시키거나 면역원성을 개선시킨 항체 등과 관련해서는 당업계에 공지된 다양한 문헌 예컨대 미국 특허 4,444,887, 미국 특허 4,716,111, 미국 특허 5,545,806, 미국 특허 5,814,318, 국제 공개특허 WO 98/46645, 국제 공개특허 WO 98/50433, 국제 공개특허 WO 98/24893, 국제 공개특허 WO 98/16654, 국제 공개특허 WO 96/34096, 국제 공개특허 WO 96/33735, 문헌[Protein Eng 1994, 7(6):805-814], 문헌[Proc Natl Acad Sci U S A 1994, 91:969-973] 등을 참조할 수 있다.Regarding methods for producing antibodies and antibodies in which specificity for target antigens is increased or immunogenicity is improved by adding artificial modifications to natural antibodies, various documents known in the art, such as U.S. Patent 4,444,887, U.S. Patent 4,716,111, U.S. Patent 5,545,806, US Patent 5,814,318, International Publication Patent WO 98/46645, International Publication Patent WO 98/50433, International Publication Patent WO 98/24893, International Publication Patent WO 98/16654, International Publication Patent WO 96/34096, International Publication Patent WO 96/ 33735, Protein Eng 1994, 7(6):805-814, Proc Natl Acad Sci U S A 1994, 91:969-973, etc.
세포 표적화 영역으로서의 압타머는 단일가닥 DNA 압타머 또는 단일가닥 RNA 압타머일 수 있다. 압타머는 항체와 마찬가지로 표적 항원 등 표적 분자에 특이적으로 결합할 수 있는 핵산 리간드를 의미하는데, 표적분자와 특이적으로 결합할 수 있다면, 압타머는 이중가닥 DNA 또는 RNA 압타머일 수도 있다. 이러한 표적분자와의 특이적 결합할 수 있는 압타머의 제조, 선별 방법 등은 모두 당업계에 공지되어 있다. 압타머의 선별을 위한 구체적인 방법이나 적절한 시약, 재료 등의 사용에 대해서는 문헌[Methods Enzymol 267:275-301, 1996], 문헌[Methods Enzymol 318:193-214, 2000] 등을 참조할 수 있다. 압타머는 생체 내 반감기 향상을 위하여 당, 포스페이트 및/또는 염기에서 변형된 것일 수 있다. 이러한 당, 포스페이트 및/또는 염기에서 변형된 뉴클레오티드는 당업계에 그 제조방법을 포함하여 구체적으로 공지되어 있다. 예컨대 당에서 변형된 뉴클레오티드는, 그 당의 하이드록실 기(OH group)가 할로겐 기, 지방족 기, 에테르 기, 아민 기 등으로 수식된 것, 당인 리보스 또는 디옥시 리보오스 자체가 이를 대신할 수 있는 당 유사체 α-아노머 당(α-anomeric sugars), 아라비노스(arabinose), 자일로스(xyloses) 또는 릭소오스(lyxoses)와 같은 에피머 당(epimeric sugars), 피라노오스 당(pyranose sugars), 퓨라노오스 당(furanose sugars) 등으로 치환된 것을 들수 있다. 또한 예컨대 포스페이트에서의 변형은 포스페이트가 P(O)S(thioate), P(S)S(dithioate), P(O)NR2(amidate), P(O)R, P(O)OR', CO 또는 CH2(formacetal)로의 변형된 것 등을 들수 있다. 여기서 상기 R 또는 R'는 H 또는 치환되거나 치환되지 않은 알킬 등이며, 포스페이트에서 변형될 경우 그 연결기는 -O-, -N-, -S- 또는 -C-가 되어, 이러한 연결기를 통해 인접 뉴클레오티드가 서로 결합하게 된다.The aptamer as the cell targeting region can be a single-stranded DNA aptamer or a single-stranded RNA aptamer. Like an antibody, an aptamer refers to a nucleic acid ligand that can specifically bind to a target molecule such as a target antigen. If it can specifically bind to a target molecule, the aptamer may be a double-stranded DNA or RNA aptamer. Methods for producing and selecting aptamers capable of specifically binding to such target molecules are all known in the art. For specific methods for selecting aptamers and the use of appropriate reagents and materials, refer to the literature [Methods Enzymol 267:275-301, 1996], the literature [Methods Enzymol 318:193-214, 2000], etc. Aptamers may be modified in sugars, phosphates and/or bases to improve half-life in vivo. Nucleotides modified from such sugars, phosphates and/or bases are specifically known in the art, including methods for their preparation. For example, a nucleotide modified from a sugar is one in which the hydroxyl group (OH group) of the sugar is modified with a halogen group, aliphatic group, ether group, amine group, etc., and the sugar ribose or deoxyribose itself is a sugar analogue that can replace it. α-anomeric sugars, epimeric sugars such as arabinose, xyloses or lyxoses, pyranose sugars, furanos Examples include those substituted with furanose sugars, etc. Also, for example, modifications in phosphate can cause phosphate to form P(O)S(thioate), P(S)S(dithioate), P(O)NR2(amidate), P(O)R, P(O)OR', CO Or transformation into CH2 (formacetal), etc. Here, R or R' is H or substituted or unsubstituted alkyl, etc., and when modified from phosphate, the linking group becomes -O-, -N-, -S- or -C-, and the linking group becomes -O-, -N-, -S- or -C-, and the linking group is used to connect adjacent nucleotides. are combined with each other.
본 발명의 약물 전달시스템은 M2 종양 관련 대식세포의 세포막에 발현된 ITGB2에 특이적으로 직접 결합하므로 약물 효과가 비선택적으로 나타나는 부작용이 없이 표적분자인 ITGB2가 발현되는 표적세포 M2 종양 관련 대식세포에 대해서만 선택적으로 약물 효과가 나타내는 것을 가능하게 한다.The drug delivery system of the present invention specifically and directly binds to ITGB2 expressed on the cell membrane of M2 tumor-related macrophages, so that the drug effect is non-selective, without the side effects of the target cell, M2 tumor-related macrophages, expressing ITGB2. It allows the drug effect to be displayed only selectively.
일 측면에서, 본 발명은 ITGB2를 표적화하는 물질을 포함하는 M2 종양 관련 대식세포 표적화 조성물에 관한 것이다.In one aspect, the present invention relates to a composition targeting M2 tumor-associated macrophages comprising an agent targeting ITGB2.
일 측면에서, 본 발명은 약물 전달시스템에 약물이 결합한 복합체에 관한 것이다.In one aspect, the present invention relates to a complex in which a drug is bound to a drug delivery system.
일 구현예에서, 상기 복합체는 M2 종양 관련 대식세포의 세포막에 존재하는 ITGB2 단백질에 특이적으로 결합하여 M2 종양 관련 대식세포에 약물을 전달할 수 있다.In one embodiment, the complex can deliver a drug to M2 tumor-related macrophages by specifically binding to the ITGB2 protein present in the cell membrane of M2 tumor-related macrophages.
일 구현예에서, 약물은 전세포사멸성(pro-apoptotic) 펩타이드, 면역원성 세포사멸 유도제 또는 항암제일 수 있다.In one embodiment, the drug may be a pro-apoptotic peptide, an immunogenic apoptosis inducer, or an anticancer agent.
일 구현예에서, 전세포사멸성 펩타이드는 KLA, 알파-디펜신-1(alpha-defensin-1), BMAP-28, Brevenin-2R, 부포린 IIb(Buforin IIb), 세크로핀 A-마가이닌 2(cecropin A-Magainin 2, CA-MA-2), 세크로핀 A(Cecropin A), 세크로핀 B(Cecropin B), 크리소피신-1(chrysophsin-1), D-K6L9, 고메신(Gomesin), 락토페리신 B(Lactoferricin B), LLL27, LTX-315, 마가이닌 2(Magainin 2), 마가이닌 II-봄패신 결합체(Magainin II-bombesin conjugate, MG2B), 파르닥신(Pardaxin) 및 이들의 조합으로 이루어진 군에서 선택될 수 있다.In one embodiment, the pro-apoptotic peptide is KLA, alpha-defensin-1, BMAP-28, Brevenin-2R, Buforin IIb, cecropin A-magainin 2(cecropin A-Magainin 2, CA-MA-2), Cecropin A, Cecropin B, chrysophsin-1, D-K6L9, gomesin (Gomesin), Lactoferricin B, LLL27, LTX-315, Magainin 2, Magainin II-bombesin conjugate (MG2B), Pardaxin and It may be selected from the group consisting of combinations of these.
일 구현예에서, 면역원성 세포사멸 유도제는 안트라사이클린계열 항암제, 탁산 계열 항암제, 항-EGFR 항체, BK 채널 작용제, 보르테조밉(Bortezomib), 강심성 배당체(cardiac glycoside), 사이클로포스마이드 계열 항암제, GADD34/PP1 저해제, LV-tSMAC, Measles 바이러스, 블레오마이신(bleomycin), 미토잔트론(mitoxantrone), 옥살리플라틴(oxaliplatin) 및 이들의 조합으로 이루어진 군에서 선택될 수 있다.In one embodiment, the immunogenic apoptosis inducer is an anthracycline-based anticancer agent, taxane-based anticancer agent, anti-EGFR antibody, BK channel agonist, bortezomib, cardiac glycoside, cyclophosmide-based anticancer agent, GADD34 /PP1 inhibitor, LV-tSMAC, Measles virus, bleomycin, mitoxantrone, oxaliplatin, and combinations thereof.
일 구현예에서, 항암제는 세포독성 항암제일 수 있으며, 독소루비신(doxorubisin), 파클리탁셀(paclitaxel), 아지트로마이신(azithromycin), 에리트로마이신 (erythromycin), 빈블라스틴 (vinblastin), 블레오마이신(bleomycin), 닥티노마이신(dactinomycin), 다우노루비신(daunorubicin), 아이다루비신 (idarubicin), 미톡산트론 (mitoxantron), 플리카마이신(plicamycin), 미토마이신(mitomycin), 메토트렉세이트(Methotrexate), 엔티노스테트(Entinostat), 크라드리빈(Cladribine), 프랄라트렉세이트(Pralatrexate), 로라티닙(Lorlatinib), 메이탄시네 DM1(Maytansine DM1), 메이탄시네 DM3(Maytansine DM3), 메이탄시네 DM4(Maytansine DM4) 및 이들의 조합으로 이루어진 군에서 선택될 수 있다.In one embodiment, the anticancer agent may be a cytotoxic anticancer agent, such as doxorubisin, paclitaxel, azithromycin, erythromycin, vinblastin, bleomycin, Dactinomycin, daunorubicin, idarubicin, mitoxantron, plicamycin, mitomycin, methotrexate, entinosthate ( Entinostat, Cladribine, Pralatrexate, Lorlatinib, Maytansine DM1, Maytansine DM3, Maytansine DM4 and combinations thereof.
일 구현예에서, 약물 전달시스템의 ITGB2(Integrin beta 2)에 특이적으로 결합하는 표적화 물질과 약물은 링커를 매개로 공유적으로 결합하거나 링커의 매개없이 매개없이 비공유적으로 결합할 수 있다.In one embodiment, the targeting agent and the drug that specifically bind to Integrin beta 2 (ITGB2) of the drug delivery system may be covalently bound via a linker or non-covalently bound without the mediation of a linker.
일 구현예에서, 약물 전달 시스템의 ITGB2(Integrin beta 2)에 특이적으로 결합하는 표적화 물질과 약물은 링커를 매개로 연결/결합될 수 있으며, 링커는 펩타이드나 리간드, 항체, 항체 단편 등의 단백질의 아민기(amine group), 카르복시기(carboxyl group) 또는 설프히드릴기(sulfhydryl group)나 압타머 등의 핵산의 인산기(phosphate group, 히드록시기(hydroxyl group)를 통해 결합할 수 있는 작용기를 가진 임의의 링커를 사용할 수 있다. In one embodiment, the targeting agent and the drug that specifically bind to ITGB2 (Integrin beta 2) of the drug delivery system may be connected/combined via a linker, and the linker may be a protein such as a peptide, ligand, antibody, or antibody fragment. Any functional group that can bind through the amine group, carboxyl group, sulfhydryl group, or phosphate group or hydroxyl group of nucleic acids such as aptamers. A linker can be used.
일 구현예에서, 상기 링커는 약물에 따라 적절한 것을 선택하여 사용할 수 있다. 예컨대 약물에, 알데하이드 반응기를 가지는 링커를 연결하고 약물 전달 시스템의 표적화 물질의 항체(세포 표적화 영역)의 N 말단의 아미노기에 결합시킬 수 있다.In one embodiment, the linker may be selected and used appropriately depending on the drug. For example, a linker having an aldehyde reactive group can be connected to the drug and bound to the N-terminal amino group of the antibody (cell targeting region) of the targeting material of the drug delivery system.
이러한 링커의 작용기는 아이소티오시아네이트(isothiocyanate), 아이소시아네이트(isocyanates), 아실 아자이드(acyl azide), NHS 에스터(NHS ester), 설포닐 클로라이드(sulfonyl chloride), 알데하이드(aldehyde), 글리옥살(glyoxal), 에폭사이드(epoxide), 옥시레인(oxirane), 칼보네이트(carbonate), 아릴 할라이드(arylhalide), 이미도에스터(imidoester), 카보이미드(carbodiimide), 안하이드라이드(anhydride), 플루오로페닐 에스터(fluorophenyl ester), 히드록시메틸포스핀(hydroxymethyl phosphine), 말레이미드(maleimide), 할로아세틸(haloacetyl), 피리딜디설파이드(pyridyldisulfide), 티오술포네이트(thiosulfonate), 또는 비닐술폰(vinylsulfone) 등일 수 있다. 링커는 프로테아제에 의해서 절단 가능하거나, 산이나 염기 조건에서 절단 가능하거나, 고온이나 광조사에 의해서 절단 가능하거나 또는 환원 또는 산화 조건에서 절단 가능한 링커일 수 있고, 또는 이러한 조건들에서 절단 가능하지 않은 링커일 수도 있다. 절단 가능한 링커로서는 예컨대 산성 조건에서 절단되는 히드라존(hydrazone) 링커, 프로테아제에 의해 절단되는 펩타이드 링커, 환원 조건에서 절단되는 디설파이드(disulfide) 작용기를 갖는 링커 등을 들 수 있고, 절단 가능하지 않은 링커로서는 MCC(Maleimidomethyl cyclohexane-1-carboxylate) 링커, MC(maleimidocaproyl) 링커, 또는 그 유도체로서 석신이미딜-4-(N-말레이미도메틸)사이클로헥산-1-카르복실레이트(sMCC) 링커나 설포석신이미딜-4-(N-말레이미도메틸)사이클로헥산-1-카르복실레이트(sulfo-sMCC)를 들 수 있다. The functional groups of these linkers include isothiocyanate, isocyanates, acyl azide, NHS ester, sulfonyl chloride, aldehyde, and glyoxal ( glyoxal, epoxide, oxirane, carbonate, arylhalide, imidoester, carbodiimide, anhydride, fluorophenyl ester It may be (fluorophenyl ester), hydroxymethyl phosphine, maleimide, haloacetyl, pyridyldisulfide, thiosulfonate, or vinylsulfone. . The linker may be cleavable by protease, cleavable under acid or base conditions, cleavable by high temperature or light irradiation, cleavable under reducing or oxidizing conditions, or a linker that is not cleavable under these conditions. It may be. Examples of cleavable linkers include hydrazone linkers that are cleaved under acidic conditions, peptide linkers that are cleaved by proteases, and linkers with a disulfide functional group that are cleaved under reducing conditions. Examples of linkers that are not cleaved include: Maleimidomethyl cyclohexane-1-carboxylate (MCC) linker, maleimidocaproyl (MC) linker, or a derivative thereof such as succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sMCC) linker or sulfosuccine. and imidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sulfo-sMCC).
또한 링커는 자가 희생 링커(self-immolative linker) 또는 절단 후 흔적을 남기지 않는 링커(traceless linker)일 수 있다. 자가 희생 링커는 예컨대 발명의 명칭이 "Hydrophilic self-immolative linkers and conjugates thereof "인 미국 특허 제9,089,614호에 개시된 링커, 명칭이 "SELF-IMMOLATIVE LINKERS CONTAINING MANDELIC ACID DERIVATIVES, DRUG-LIGAND CONJUGATES FOR TARGETED THERAPIES AND USES THEREOF"인 국제공개 제WO2015038426호에 개시된 링커를 들 수 있으며, 절단 후 흔적을 남기지 않는 링커로서는 페닐하이드라지드 링커, 아릴-트리아젠 링커, 문헌[Blaney, et al., "Traceless solid-phase organic synthesis," Chem Rev. 102: 2607-2024 (2002)]에 개시된 링커 등일 수 있다.Additionally, the linker may be a self-immolative linker or a traceless linker that leaves no trace after cleavage. Self-immolative linkers include, for example, the linker disclosed in U.S. Pat. No. 9,089,614, entitled “Hydrophilic self-immolative linkers and conjugates thereof,” and the linker, entitled “SELF-IMMOLATIVE LINKERS CONTAINING MANDELIC ACID DERIVATIVES, DRUG-LIGAND CONJUGATES FOR TARGETED THERAPIES AND USES. THEREOF", the linker disclosed in International Publication No. WO2015038426, and linkers that leave no trace after cutting include phenylhydrazide linker, aryl-triazene linker, and the literature [Blaney, et al., "Traceless solid-phase organic synthesis," Chem Rev. 102: 2607-2024 (2002)], etc.
당업계에서는 상기 예시한 바의 링커 이외에도 본 발명에 적용 가능한 수많은 링커가 상당한 수의 문헌을 통해 공지되어 있다. 그러한 문헌으로서 구체적으로 문헌[Castaneda, et al, "Acid-cleavable thiomaleamic acid linker for homogeneous antibodydrug conjugation," Chem Commun. 49: 8187-8189 (2013)], 문헌[Lyon, et al, "Self-hydrolyzing maleimides improve the stability and pharmacological properties of antibody-drug conjugates," Nat Biotechnol. 32(10):1059-1062 (2014)], 문헌[Dawson, et al "Synthesis of proteins by native chemical ligation," Science 1994, 266, 776-779], 문헌[Dawson, et al "Modulation of Reactivity in Native Chemical Ligation through the Use of Thiol Additives," J Am Chem Soc. 1997, 119, 4325-4329] 문헌[Hackeng, et al "Protein synthesis by native chemical ligation: Expanded scope by using straightforward methodology," Proc Natl Acad Sci USA 1999, 96, 10068-10073], 문헌[Wu, et al "Building complex glycopeptides: Development of a cysteine-free native chemical ligation protocol," Angew Chem Int Ed 2006, 45, 4116-4125], 문헌[Geiser et al "Automation of solid-phase peptide synthesis" in Macromolecular Sequencing and Synthesis, Alan R Liss, Inc, 1988, pp 199-218], 문헌[Fields, G and Noble, R (1990) "Solid phase peptide synthesis utilizing 9-fluoroenylmethoxycarbonyl amino acids", Int J Peptide Protein Res 35:161-214] 등이나, 미국 특허 제6,884,869호, 미국 특허 제7,498,298호, 미국 특허 제8,288,352호, 미국 특허 제8,609,105호, 미국 특허 제8,697,688호, 미국 특허공개 제2014/0127239호, 미국 특허공개 제2013/028919호, 미국 특허공개 제2014/286970호, 미국 특허공개 제2013/0309256호, 미국 특허공개 제2015/037360호, 미국 특허공개 제2014/0294851호, 국제 특허공개 WO2015057699, 국제 특허공개 WO2014080251, 국제 특허공개 제WO2014197854, 국제 특허공개 WO2014145090, 국제 특허공개 WO2014177042 등을 참조할 수 있다.In the art, in addition to the linkers exemplified above, numerous linkers applicable to the present invention are known through a considerable number of literature. Such documents specifically include Castaneda, et al, “Acid-cleavable thiomaleamic acid linker for homogeneous antibodydrug conjugation,” Chem Commun. 49: 8187-8189 (2013), Lyon, et al, “Self-hydrolyzing maleimides improve the stability and pharmacological properties of antibody-drug conjugates,” Nat Biotechnol. 32(10):1059-1062 (2014), Dawson, et al "Synthesis of proteins by native chemical ligation," Science 1994, 266, 776-779, Dawson, et al "Modulation of Reactivity in Native Chemical Ligation through the Use of Thiol Additives," J Am Chem Soc. 1997, 119, 4325-4329] Hackeng, et al "Protein synthesis by native chemical ligation: Expanded scope by using straightforward methodology," Proc Natl Acad Sci USA 1999, 96, 10068-10073], Wu, et al “Building complex glycopeptides: Development of a cysteine-free native chemical ligation protocol,” Angew Chem Int Ed 2006, 45, 4116-4125, Geiser et al “Automation of solid-phase peptide synthesis” in Macromolecular Sequencing and Synthesis, Alan R Liss, Inc, 1988, pp 199-218], Fields, G and Noble, R (1990) "Solid phase peptide synthesis utilizing 9-fluoroenylmethoxycarbonyl amino acids", Int J Peptide Protein Res 35:161-214. etc., US Patent No. 6,884,869, US Patent No. 7,498,298, US Patent No. 8,288,352, US Patent No. 8,609,105, US Patent No. 8,697,688, US Patent Publication No. 2014/0127239, US Patent Publication No. 2013/028919. , US Patent Publication No. 2014/286970, US Patent Publication No. 2013/0309256, US Patent Publication No. 2015/037360, US Patent Publication No. 2014/0294851, International Patent Publication WO2015057699, International Patent Publication WO2014080251, International Patent Publication No. Reference may be made to WO2014197854, International Patent Publication WO2014145090, International Patent Publication WO2014177042, etc.
본 발명의 약물 전달 시스템의 표적화 물질과 약물은 생체 적합성 고분자를 매개로 또는 캐리어로 하여 결합될 수도 있다. 생체적합성 고분자는 생체조직 또는 혈액과 접촉하여 조직을 괴사시키거나 혈액을 응고시키지 않는 조직적합성(tissue compatibility) 및 항응혈성(blood compatibility)을 가지고 있는 고분자를 의미한다.The targeting material and drug of the drug delivery system of the present invention may be combined through a biocompatible polymer or carrier. Biocompatible polymers refer to polymers that have tissue compatibility and anticoagulant properties that do not cause tissue necrosis or blood coagulation in contact with biological tissue or blood.
상기 생체적합성 고분자로서의 합성 중합체는 폴리에스테르, 폴리하이드 록시알카노에이트(PHAs), 폴리(α-하이드록시액시드), 폴리(β-하이드록시액시드), 폴리(3-하이드로식부티레이트-co-발러레이트; PHBV), 폴리(3-하이드록시프로프리오네이트; PHP), 폴리(3-하이드록시헥사노에이트; PHH), 폴리(4-하이드록시액시드), 폴리(4-하이드록시부티레이트), 폴리(4-하이드록시발러레이트), 폴리(4-하이드록시헥사노에이트), 폴리(에스테르아마이드), 폴리카프로락톤, 폴리락타이드, 폴리글리코라이드, 폴리(락타이드-co-글리코라이드; PLGA), 폴리디옥사논, 폴리오르토에스테르, 폴리언하이드라이드, 폴리(글리콜산-co-트리메틸렌카보네이트), 폴리포스포에스테르, 폴리포스포에스테르 우레탄, 폴리(아미노산), 폴리사이아노아크릴레이트, 폴리(트리메틸렌 카보네이트), 폴리(이미노카보네이트), 폴리(타이로신 카보네이트), 폴리카보네이트, 폴리(타이로신 아릴레이트), 폴리알킬렌 옥살레이트, 폴리포스파젠스, PHA-PEG, 에틸렌 비닐 알코올 코폴리머(EVOH), 폴리우레탄, 실리콘, 폴리에스테르, 폴리올레핀, 폴리이소부틸렌과 에틸렌-알파올레핀 공중합체, 스틸렌-이소브틸렌-스틸렌 트리블록 공중합체, 아크릴 중합체 및 공중합체, 비닐 할라이드 중합체 및 공중합체, 폴리비닐 클로라이드, 폴리비닐 에테르, 폴리비닐 메틸에테르, 폴리비닐리덴 할라이드, 폴리비닐리덴 플루오라이드, 폴리비닐리덴 클로라이드, 폴리플루오로알켄, 폴리퍼플루오로알켄, 폴리아크릴로니트릴, 폴리비닐 케톤, 폴리비닐 아로마틱스, 폴리스틸렌, 폴리비닐 에스테르, 폴리비닐 아세테이트, 에틸렌-메틸 메타크릴레이트 공중합체, 아크릴로니트릴-스틸렌 공중합체, ABS 수지와 에틸렌-비닐 아세테이트 공중합체, 폴리아마이드, 알키드 수지, 폴리옥시메틸렌, 폴리이미드, 폴리에테르, 폴리아크릴레이트, 폴리메타크릴레이트, 폴리아크릴산-co-말레산 또는 폴리아미노아민이며, 천연 중합체는 키토산, 덱스트란, 셀룰로오스, 헤파린, 히알루론산, 알기네이트, 이눌린, 녹말 또는 글리코겐이다.Synthetic polymers as biocompatible polymers include polyester, polyhydroxyalkanoate (PHAs), poly(α-hydroxyacid), poly(β-hydroxyacid), and poly(3-hydrosybutyrate-co). -valerate; PHBV), poly(3-hydroxypropionate; PHP), poly(3-hydroxyhexanoate; PHH), poly(4-hydroxy acid), poly(4-hydroxy butyrate), poly(4-hydroxyvalerate), poly(4-hydroxyhexanoate), poly(esteramide), polycaprolactone, polylactide, polyglycolide, poly(lactide-co-glycoside) ride; PLGA), polydioxanone, polyorthoester, polyanhydride, poly(glycolic acid-co-trimethylene carbonate), polyphosphoester, polyphosphoester urethane, poly(amino acid), polycyano Acrylate, poly(trimethylene carbonate), poly(iminocarbonate), poly(tyrosine carbonate), polycarbonate, poly(tyrosine arylate), polyalkylene oxalate, polyphosphazene, PHA-PEG, ethylene vinyl Alcohol copolymers (EVOH), polyurethanes, silicones, polyesters, polyolefins, polyisobutylene and ethylene-alphaolefin copolymers, styrene-isobutylene-styrene triblock copolymers, acrylic polymers and copolymers, vinyl halide polymers and copolymers, polyvinyl chloride, polyvinyl ether, polyvinyl methyl ether, polyvinylidene halide, polyvinylidene fluoride, polyvinylidene chloride, polyfluoroalkene, polyperfluoroalkene, polyacrylonitrile, poly. Vinyl ketone, polyvinyl aromatics, polystyrene, polyvinyl ester, polyvinyl acetate, ethylene-methyl methacrylate copolymer, acrylonitrile-styrene copolymer, ABS resin and ethylene-vinyl acetate copolymer, polyamide, alkyd resin. , polyoxymethylene, polyimide, polyether, polyacrylate, polymethacrylate, polyacrylic acid-co-maleic acid or polyaminoamine, and natural polymers include chitosan, dextran, cellulose, heparin, hyaluronic acid, and alginate. , inulin, starch or glycogen.
약물 전달 시스템과 약물의 결합에 사용되는 링커는 표적세포 밖에서 안정적이어서 절단되지 않고, 표적세포 내 산성 조건인 엔도좀이나 리조솜에서도 절단되지 않는 링커인 것이 바람직할 것이다. 이러한 링커는 표적세포 밖에서 안정적이어서 절단되지 않음으로서 약물이 세포 내로 이동되도록 하고, 산성 조건인 엔도좀이나 리조좀에서 절단되지 않으로써 엔도좀이나 리조솜에서 세포질로 이동될 수 있도록 한다.It would be preferable that the linker used to combine the drug delivery system with the drug is stable outside the target cell and is not cleaved, nor is it cleaved even in endosomes or rhizosomes under acidic conditions within the target cell. This linker is stable outside the target cell and is not cleaved, allowing the drug to move into the cell, and is not cleaved in endosomes or rhizosomes under acidic conditions, allowing it to be moved from endosomes or rhizosomes to the cytoplasm.
본 발명의 약물 전달 시스템과 약물의 복합체에서, 약물은 약물 전달 시스템에 비공유적으로 결합할 수도 있다. 예컨대 핵산에 인터컬레이션(intercalation)되어 효과를 발휘하는, 항암제 일종인 독소루비신과 같은 인터컬레이터제(intercalator agents)는 약물 전달 시스템의 세포 표적화 영역으로서 압타머를 사용할 경우에 압타머에 비공유적으로 인터컬레이션(intercalation) 방식으로 결합될 수 있다. 압타머는 올리고뉴클레오타이드 분자이기 때문에, 뉴클레오타이드 염기들의 염기 스태킹(base stacking)이 있으며, 이 염기 스태킹 사이에 약물이 인터컬레이션(intercalation) 방식으로 결합할 수 있게 된다.In the complex of the drug delivery system and the drug of the present invention, the drug may be non-covalently bound to the drug delivery system. For example, intercalator agents such as doxorubicin, a type of anticancer drug that exerts its effect by intercalating with nucleic acids, are non-covalently attached to the aptamer when using the aptamer as the cell targeting portion of the drug delivery system. They can be combined in an intercalation manner. Since an aptamer is an oligonucleotide molecule, there is base stacking of nucleotide bases, and the drug can bind in an intercalation manner between this base stacking.
본 발명의 약물 전달 시스템과 약물의 복합체에서, 약물은 그것이 세포 내로 이동하여 효과를 발휘할 수 있는 약물이면 특별한 제한이 없다. 그러한 약물은 세포독성 항암제 등의 임의의 저분자 화합물로 된 약물, 재조합 단백질, siRNA 등의 임의의 바이오의약품일 수 있다. 또한 약물은 효능 면에서 항염증제, 진통제, 항관절염제, 진경제, 항우울증제, 항정신병약물, 신경안정제, 항불안제, 마약길항제, 항파킨스 질환 약물, 콜린성 아고니스트, 항암제, 혈관신생억제제, 면역억제제, 면역촉진제, 항바이러스제, 항생제, 식욕억제제, 진통제, 항콜린제, 항히스타민제, 항편두통제, 호르몬제, 관상혈관, 혈관 확장제, 피임약, 항혈전제, 이뇨제, 항고혈압제, 심혈관질환치료제, 조영제 등의 진단제 등일 수 있다.In the complex of the drug delivery system and the drug of the present invention, the drug is not particularly limited as long as it is a drug that can move into cells and exert its effect. Such drugs may be drugs made of any small molecule compounds such as cytotoxic anticancer drugs, recombinant proteins, or any biopharmaceuticals such as siRNA. In addition, in terms of efficacy, drugs include anti-inflammatory drugs, analgesics, anti-arthritis drugs, antispasmodics, antidepressants, antipsychotics, tranquilizers, anti-anxiety drugs, narcotic antagonists, anti-Parkinson's disease drugs, cholinergic agonists, anticancer drugs, angiogenesis inhibitors, immunosuppressants, etc. Immunostimulants, antivirals, antibiotics, appetite suppressants, painkillers, anticholinergics, antihistamines, anti-migraine drugs, hormones, coronary vasodilators, vasodilators, contraceptives, antithrombotic agents, diuretics, antihypertensive agents, cardiovascular disease treatments, contrast media, etc. It may be a diagnostic agent, etc.
본 발명에서 약물은 바람직하게는 세포독성 항암제이이다. 세포독성 항암제는 대사길항제(Antimetabolites), 미세관(microtubulin) 표적화제(Tubulin polymerase inhibitor 및 Tubulin depolymerisation), 알킬화제(Alkylating agents), 유사분열 억제제(Antimitotic Agents), DNA 절단제(DNA cleavage agent), DNA 가교제(DNA cross-linker agent), DNA 인터컬레이터제(DNA intercalator agents), DNA 토포아이소머라아제 억제제(DNA topoisomerase inhibitor) 등으로 대별될 수 있는데, 대사길항제로서는 메토트렉세이트(Methotrexate) 등의 폴산(Folic acid) 유도체, 클라드리빈(Cladribine) 등의 퓨린(Purine) 유도체, 아자시티딘(Azacitidine) 등의 피리미딘(pyrimidine) 유도체, 독시플루리딘(Doxifluridine), 플루오로우라실(Fluorouracil) 등이 당업계에 공지되어 있고, 미세관 표적화제로서는 모노메틸아우리스타틴 E(MMAE), 모노메틸아우리스타틴 F(MMAF), 돌라스타틴 등의 아우리스타틴 계열의 약물, 메이탄신(Maytansines) 등이 당업계에 공지되어 있으며, 알킬화제로서는 부설판(Busulfan), 트레오설판(Treosulfan) 등의 알킬 설포네이트(Alkyl Sulfonate) 제제, 벤다머스틴(Bendamustine) 등의 니트로젠 머스타드(Nitrogen Mustard) 유도체, 시스플라틴(Cisplatin), 헵타플라틴(Heptaplatin) 등의 플라티늄(Platinum) 제제 등이 당업계에 공지되어 있다. 또 유사분열 억제제(Antimitotic Agents)로서는 도세탁셀(Docetaxel), 파크리탁셀(Paclitaxel) 등의 탁산(Taxane) 제제, 빈플루닌(Vinflunine) 등의 빈카 알칼리드(Vinca alkalids), 에토포시드(Etoposide) 등의 포도필로톡신(Podophyllotoxin) 유도체 등이 당업계에 공지되어 있고, DNA 절단제(DNA cleavage agent)로서는 칼리채미신(Calicheamicins) 등이 공지되어 있으며, DNA 가교제(DNA cross-linker agent)로서는 PBD 이중체 등이 공지되어 있다. 또 DNA 인터컬레이터제로서는 독소루비신 등이 당업계에 공지되어 있으며 DNA 토포아이소머라아제 억제제로서는 SN-28 등이 당업계에 공지되어 있다.In the present invention, the drug is preferably a cytotoxic anticancer agent. Cytotoxic anticancer agents include antimetabolites, microtubulin targeting agents (Tubulin polymerase inhibitor and Tubulin depolymerisation), alkylating agents, antimitotic agents, DNA cleavage agents, and DNA. They can be roughly divided into DNA cross-linker agents, DNA intercalator agents, and DNA topoisomerase inhibitors. As metabolic antagonists, folic acid such as methotrexate acid derivatives, purine derivatives such as Cladribine, pyrimidine derivatives such as Azacitidine, Doxifluridine, Fluorouracil, etc. It is known in the industry, and microtubule targeting agents include monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), auristatin series drugs such as dolastatin, and maytansines. Known in the industry, alkylating agents include alkyl sulfonate agents such as Busulfan and Treosulfan, nitrogen mustard derivatives such as Bendamustine, and cisplatin. ), Platinum preparations such as Heptaplatin, etc. are known in the art. Also, as antimitotic agents, taxane preparations such as Docetaxel and Paclitaxel, Vinca alkalids such as Vinflunine, and Etoposide. Podophyllotoxin derivatives such as podophyllotoxin are known in the art, Calichamicins, etc. are known as DNA cleavage agents, and PBD is known as a DNA cross-linker agent. Duplexes and the like are known. Additionally, doxorubicin and the like are known in the art as DNA intercalator agents, and SN-28 and the like are known in the art as DNA topoisomerase inhibitors.
또한 약물은 유전자, 플라스미드 DNA, 안티센스 올리고뉴클레오티드, siRNA, 펩티드, 리보자임, 바이러스 입자, 면역조절제, 단백질, 조영제 등일 수도 있다. 보다 구체적으로 약물은 망막모세포종 종양 서프레서 유전자의 변이체인 Rb94를 코딩하는 유전자, 종양 세포에서만 세포자멸을 유도하는 아폽틴을 코딩하는 유전자일 수 있고, 치료 표적이 되는 HER-2 등에 대한 안티센스 올리고뉴클레오티드(서열: 5'-TCC ATG GTG CTC ACT-3')수 있으며, MRI 조영제인 Gd-DTPA 물질 등의 진단 조영제일 수도 있다.Additionally, the drug may be a gene, plasmid DNA, antisense oligonucleotide, siRNA, peptide, ribozyme, viral particle, immunomodulator, protein, contrast agent, etc. More specifically, the drug may be a gene encoding Rb94, a variant of the retinoblastoma tumor suppressor gene, a gene encoding apoptin, which induces apoptosis only in tumor cells, and an antisense oligonucleotide against HER-2, which is a therapeutic target. (Sequence: 5'-TCC ATG GTG CTC ACT-3'), and may be a diagnostic contrast agent such as Gd-DTPA, an MRI contrast agent.
본 발명의 약물 전달 시스템과 약물의 복합체는 약제학적으로 허용되는 담체를 포함하여 당업계에 공지된 통상의 방법으로 투여 경로에 따라 경구용 제형 또는 비경구용 제형의 약제학적 조성물로 제조될 수 있다. 여기서 "약학적으로 허용가능한 담체"는 생물체를 자극하지 않고 투여 화합물의 생물학적 활성 및 특성을 저해하지 않는 담체 또는 희석제를 말한다. 액상 용액으로 제제화되는 조성물에 있어서 허용되는 약학적 담체로는, 멸균 및 생체에 적합한 것으로서, 식염수, 멸균수, 링거액, 완충 식염수, 알부민 주사용액, 덱스트로즈 용액, 말토 덱스트린 용액, 글리세롤, 에탄올 및 이들 성분 중 1 성분 이상을 혼합하여 사용할 수 있으며, 필요에 따라 항산화제, 완충액, 정균제 등 다른 통상의 첨가제를 첨가할 수 있다.The complex of the drug delivery system and the drug of the present invention can be prepared as a pharmaceutical composition in an oral formulation or parenteral formulation depending on the route of administration by a conventional method known in the art, including a pharmaceutically acceptable carrier. Here, “pharmaceutically acceptable carrier” refers to a carrier or diluent that does not irritate living organisms and does not inhibit the biological activity and properties of the administered compound. Acceptable pharmaceutical carriers in compositions formulated as liquid solutions include those that are sterile and biocompatible, such as saline solution, sterile water, Ringer's solution, buffered saline solution, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, ethanol, and One or more of these ingredients can be mixed and used, and other common additives such as antioxidants, buffers, and bacteriostatic agents can be added as needed.
일 측면에서, 본 발명은 본 발명의 약물 전달 시스템과 약물의 복합체를 유효성분으로 포함하는 면역항암제에 관한 것이다.In one aspect, the present invention relates to an anti-cancer immunological agent comprising a complex of the drug delivery system of the present invention and a drug as an active ingredient.
일 측면에서, 본 발명은 본 발명의 약물 전달 시스템과 약물의 복합체를 유효성분으로 포함하는 M2 종양 관련 대식세포 매개 암의 예방 또는 치료용 약학적 조성물에 관한 것이다.In one aspect, the present invention relates to a pharmaceutical composition for preventing or treating M2 tumor-related macrophage-mediated cancer, comprising a complex of the drug delivery system of the present invention and a drug as an active ingredient.
본 발명의 약학적 조성물은 단독의 요법으로 이용될 수 있으나, 다른 통상적인 생물학적 요법, 화학 요법 또는 방사 요법과 함께 이용될 수도 있으며, 이러한 병행 요법을 실시하는 경우에는 보다 효과적으로 암을 치료할 수 있다. 본 발명을 암의 예방 및 치료에 이용하는 경우 상기 조성물과 함께 이용될 수 있는 화학 요법제는 시스플라틴(cisplatin), 카르보플라틴(carboplatin), 프로카르바진(procarbazine), 메클로레타민(mechlorethamine), 시클로포스파미드(cyclophosphamide), 이포스파미드(ifosfamide), 멜팔란(melphalan), 클로라부실(chlorambucil), 비술판(bisulfan), 니트로소우레아(nitrosourea), 디악티노마이신(dactinomycin), 다우노루비신(daunorubicin), 독소루비신(doxorubicin), 블레오마이신(bleomycin), 플리코마이신(plicomycin), 미토마이신(mitomycin), 에토포시드(etoposide), 탁목시펜(tamoxifen), 택솔(taxol), 트랜스플라티눔(transplatinum), 5-플루오로우라실(5-fluorouracil), 빈크리스틴(vincristin), 빈블라스틴(vinblastin) 및 메토트렉세이트(methotrexate) 등을 포함한다. 본 발명의 조성물과 함께 이용될 수 있는 방사 요법은 X-선 조사 및 γ-선 조사 등이다.The pharmaceutical composition of the present invention can be used as a stand-alone therapy, but can also be used in combination with other conventional biological therapies, chemotherapy, or radiotherapy. When such combination therapy is performed, cancer can be treated more effectively. When the present invention is used for the prevention and treatment of cancer, chemotherapy agents that can be used with the composition include cisplatin, carboplatin, procarbazine, mechlorethamine, Cyclophosphamide, ifosfamide, melphalan, chlorambucil, bisulfan, nitrosourea, dactinomycin, daunoru daunorubicin, doxorubicin, bleomycin, plicomycin, mitomycin, etoposide, tamoxifen, taxol, transpla Includes transplatinum, 5-fluorouracil, vincristin, vinblastin and methotrexate. Radiation therapy that can be used with the composition of the present invention includes X-ray irradiation and γ-ray irradiation.
본 발명에서, 용어 "예방"이란 본 발명에 따른 조성물의 투여에 의해 암의 발생, 확산 및 재발을 억제 또는 지연시키는 모든 행위를 의미한다.In the present invention, the term “prevention” refers to all actions that inhibit or delay the occurrence, spread, and recurrence of cancer by administering the composition according to the present invention.
본 발명의 조성물의 치료적으로 유효한 양은 여러 요소, 예를 들면 투여방법, 목적부위, 환자의 상태 등에 따라 달라질 수 있다. 따라서, 인체에 사용 시 투여량은 안전성 및 효율성을 함께 고려하여 적정량으로 결정되어야 한다. 동물실험을 통해 결정한 유효량으로부터 인간에 사용되는 양을 추정하는 것도 가능하다. 유효한 양의 결정시 고려할 이러한 사항은, 예를 들면 Hardman and Limbird, eds., Goodman and Gilman's The Pharmacological Basis of Therapeutics, 10th ed.(2001), Pergamon Press; 및 E.W. Martin ed., Remington's Pharmaceutical Sciences, 18th ed.(1990), Mack Publishing Co.에 기술되어있다.The therapeutically effective amount of the composition of the present invention may vary depending on various factors, such as administration method, target site, patient condition, etc. Therefore, when used in the human body, the dosage must be determined as appropriate by considering both safety and efficiency. It is also possible to estimate the amount used in humans from the effective amount determined through animal testing. These considerations in determining an effective amount include, for example, Hardman and Limbird, eds., Goodman and Gilman's The Pharmacological Basis of Therapeutics, 10th ed. (2001), Pergamon Press; and E.W. Martin ed., Remington's Pharmaceutical Sciences, 18th ed. (1990), Mack Publishing Co.
본 발명의 약학적 조성물은 약학적으로 유효한 양으로 투여한다. 본 발명에서 사용되는 용어, "약학적으로 유효한 양"은 의학적 치료에 적용 가능한 합리적인 수혜/위험 비율로 질환을 치료하기에 충분하며 부작용을 일으키지 않을 정도의 양을 의미하며, 유효용량 수준은 환자의 건강상태, 질환의 종류, 중증도, 약물의 활성, 약물에 대한 민감도, 투여 방법, 투여 시간, 투여 경로 및 배출 비율, 치료기간, 배합 또는 동시 사용되는 약물을 포함한 요소 및 기타 의학 분야에 잘 알려진 요소에 따라 결정될 수 있다. 본 발명의 조성물은 개별 치료제로 투여하거나 다른 치료제와 병용하여 투여될 수 있고, 종래의 치료제와 순차적으로 또는 동시에 투여될 수 있으며, 단일 또는 다중 투여될 수 있다. 상기한 요소들을 모두 고려하여, 부작용 없이 최소한의 양으로 최대 효과를 얻을 수 있는 양을 투여하는 것이 중요하며, 이는 당업자에 의해 용이하게 결정될 수 있다.The pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount. As used in the present invention, the term "pharmaceutically effective amount" refers to an amount that is sufficient to treat a disease with a reasonable benefit/risk ratio applicable to medical treatment and does not cause side effects, and the effective dose level is determined by the patient's Factors including health status, type and severity of disease, activity of drug, sensitivity to drug, method of administration, time of administration, route of administration and excretion rate, duration of treatment, drugs combined or used simultaneously, and other factors well known in the field of medicine. It can be decided depending on The composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered singly or multiple times. Considering all of the above factors, it is important to administer an amount that can achieve maximum effect with the minimum amount without side effects, and this can be easily determined by a person skilled in the art.
본 발명의 약학적 조성물은 생물학적 제제에 통상적으로 사용되는 담체, 희석제, 부형제 또는 둘 이상의 이들의 조합을 포함할 수 있다. 본 발명에서 사용되는 용어, "약학적으로 허용가능한"이란 상기 조성물에 노출되는 정상 세포나 인간에게 독성이 없는 특성을 나타내는 것을 의미한다. 상기 담체는 조성물을 생체 내 전달에 적합한 것이면 특별히 제한되지 않으며, 예를 들면, Merck Index, 13th ed., Merck & Co. Inc. 에 기재된 화합물, 식염수, 멸균수, 링거액, 완충 식염수, 덱스트로스 용액, 말토 덱스트린 용액, 글리세롤, 에탄올 및 이들 성분 중 1 성분 이상을 혼합하여 이용할 수 있으며, 필요에 따라 항산화제, 완충액, 정균제 등 다른 통상의 첨가제를 첨가할 수 있다. 또한, 희석제, 분산제, 계면활성제, 결합제 및 윤활제를 부가적으로 첨가하여 수용액, 현탁액, 유탁액 등과 같은 주이용 제형, 환약, 캡슐, 과립 또는 정제로 제제화할 수 있다. 더 나아가 당 분야의 적정한 방법으로 또는 Remington's Pharmaceutical Science(Mack Publishing Company, Easton PA, 18th, 1990)에 개시되어 있는 방법을 이용하여 각 질환에 따라 또는 성분에 따라 바람직하게 제제화할 수 있다.The pharmaceutical composition of the present invention may contain a carrier, diluent, excipient, or a combination of two or more commonly used in biological products. As used in the present invention, the term “pharmaceutically acceptable” means that the composition exhibits non-toxic properties to normal cells or humans exposed to the composition. The carrier is not particularly limited as long as it is suitable for in vivo delivery of the composition, for example, Merck Index, 13th ed., Merck & Co. Inc. The compounds described in, saline solution, sterilized water, Ringer's solution, buffered saline solution, dextrose solution, maltodextrin solution, glycerol, ethanol, and one or more of these ingredients can be mixed and used, and if necessary, other ingredients such as antioxidants, buffers, and bacteriostatic agents. Normal additives can be added. In addition, diluents, dispersants, surfactants, binders, and lubricants can be additionally added to formulate dosage forms such as aqueous solutions, suspensions, emulsions, etc., into pills, capsules, granules, or tablets. Furthermore, it can be preferably formulated according to each disease or ingredient using an appropriate method in the art or a method disclosed in Remington's Pharmaceutical Science (Mack Publishing Company, Easton PA, 18th, 1990).
일 구현예에서, 상기 약학 조성물은 경구형 제형, 외용제, 좌제, 멸균 주사용액 및 분무제를 포함하는 군으로부터 선택되는 하나 이상의 제형일 수 있다. In one embodiment, the pharmaceutical composition may be one or more formulations selected from the group including oral formulations, topical formulations, suppositories, sterile injectable solutions, and sprays.
본 발명의 조성물은 또한 생물학적 제제에 통상적으로 사용되는 담체, 희석제, 부형제 또는 둘 이상의 이들의 조합을 포함할 수 있다. 약학적으로 허용 가능한 담체는 조성물을 생체 내 전달에 적합한 것이면 특별히 제한되지 않으며, 예를 들면, Merck Index, 13th ed., Merck & Co. Inc. 에 기재된 화합물, 식염수, 멸균수, 링거액, 완충 식염수, 덱스트로스 용액, 말토 덱스트린 용액, 글리세롤, 에탄올 및 이들 성분 중 1 성분 이상을 혼합하여 이용할 수 있으며, 필요에 따라 항산화제, 완충액, 정균제 등 다른 통상의 첨가제를 첨가할 수 있다. 또한, 희석제, 분산제, 계면활성제, 결합제 및 윤활제를 부가적으로 첨가하여 수용액, 현탁액, 유탁액 등과 같은 주이용 제형, 환약, 캡슐, 과립 또는 정제로 제제화할 수 있다. 더 나아가 당 분야의 적정한 방법으로 또는 Remington's Pharmaceutical Science(Mack Publishing Company, Easton PA, 18th, 1990)에 개시되어 있는 방법을 이용하여 각 질환에 따라 또는 성분에 따라 바람직하게 제제화할 수 있다.The composition of the present invention may also include carriers, diluents, excipients, or combinations of two or more commonly used in biological products. Pharmaceutically acceptable carriers are not particularly limited as long as they are suitable for in vivo delivery of the composition, for example, Merck Index, 13th ed., Merck & Co. Inc. The compounds described in, saline solution, sterilized water, Ringer's solution, buffered saline solution, dextrose solution, maltodextrin solution, glycerol, ethanol, and one or more of these ingredients can be mixed and used, and if necessary, other ingredients such as antioxidants, buffers, and bacteriostatic agents. Normal additives can be added. In addition, diluents, dispersants, surfactants, binders, and lubricants can be additionally added to formulate dosage forms such as aqueous solutions, suspensions, emulsions, etc., into pills, capsules, granules, or tablets. Furthermore, it can be preferably formulated according to each disease or ingredient using an appropriate method in the art or a method disclosed in Remington's Pharmaceutical Science (Mack Publishing Company, Easton PA, 18th, 1990).
본 발명의 조성물에 추가로 동일 또는 유사한 기능을 나타내는 유효성분을 1종 이상 함유할 수 있다. 본 발명의 조성물은, 조성물 총 중량에 대하여 상기 단백질을 0.0001 내지 10 중량 %로, 바람직하게는 0.001 내지 1 중량 %를 포함한다. The composition of the present invention may additionally contain one or more active ingredients that exhibit the same or similar functions. The composition of the present invention contains 0.0001 to 10% by weight of the protein, preferably 0.001 to 1% by weight, based on the total weight of the composition.
본 발명의 약학적 조성물은 약제학적으로 허용 가능한 첨가제를 더 포함할 수 있으며, 이때 약제학적으로 허용 가능한 첨가제로는 전분, 젤라틴화 전분, 미결정셀룰로오스, 유당, 포비돈, 콜로이달실리콘디옥사이드, 인산수소칼슘, 락토스, 만니톨, 엿, 아라비아고무, 전호화전분, 옥수수전분, 분말셀룰로오스, 히드록시프로필셀룰로오스, 오파드라이, 전분글리콜산나트륨, 카르나우바 납, 합성규산알루미늄, 스테아린산, 스테아린산마그네슘, 스테아린산알루미늄, 스테아린산칼슘, 백당, 덱스트로스, 소르비톨 및 탈크 등이 사용될 수 있다. 본 발명에 따른 약제학적으로 허용 가능한 첨가제는 상기 조성물에 대해 0.1 중량부 내지 90 중량부 포함되는 것이 바람직하나, 이에 한정되는 것은 아니다.The pharmaceutical composition of the present invention may further include pharmaceutically acceptable additives, wherein the pharmaceutically acceptable additives include starch, gelatinized starch, microcrystalline cellulose, lactose, povidone, colloidal silicon dioxide, and calcium hydrogen phosphate. , lactose, mannitol, taffy, gum arabic, pregelatinized starch, corn starch, powdered cellulose, hydroxypropyl cellulose, Opadry, sodium starch glycolate, lead carnauba, synthetic aluminum silicate, stearic acid, magnesium stearate, aluminum stearate, Calcium stearate, white sugar, dextrose, sorbitol, and talc may be used. The pharmaceutically acceptable additive according to the present invention is preferably contained in an amount of 0.1 to 90 parts by weight based on the composition, but is not limited thereto.
본 발명의 조성물은 목적하는 방법에 따라 비 경구 투여(예를 들어 정맥 내, 피하, 복강 내 또는 국소에 적용)하거나 경구 투여할 수 있으며, 투여량은 환자의 체중, 연령, 성별, 건강상태, 식이, 투여시간, 투여방법, 배설률 및 질환의 중증도 등에 따라 그 범위가 다양하다. 본 발명에 따른 조성물의 일일 투여량은 0.0001 ~ 10 ㎎/㎖이며, 바람직하게는 0.0001 ~ 5 ㎎/㎖이며, 하루 일 회 내지 수회에 나누어 투여하는 것이 더욱 바람직하다. The composition of the present invention can be administered parenterally (e.g., intravenously, subcutaneously, intraperitoneally, or topically) or orally depending on the desired method, and the dosage is determined by the patient's weight, age, gender, health condition, The range varies depending on diet, administration time, administration method, excretion rate, and severity of disease. The daily dosage of the composition according to the present invention is 0.0001 to 10 mg/ml, preferably 0.0001 to 5 mg/ml, and it is more preferable to administer it once or several times a day.
본 발명의 조성물의 경구 투여를 위한 액상 제제로는 현탁제, 내용액제, 유제, 시럽제 등이 해당되는데, 통상적으로 사용되는 단순 희석제인 물, 액체 파라핀 이외에 다양한 부형제, 예컨대 습윤제, 감미제, 방향제, 보존제 등이 함께 포함될 수 있다. 비경구 투여를 위한 제제에는 멸균된 수용액, 비수성 용제, 현탁제, 유제, 동결건조 제제, 좌제 등이 포함된다.Liquid preparations for oral administration of the composition of the present invention include suspensions, oral solutions, emulsions, syrups, etc., and in addition to the commonly used simple diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, fragrances, and preservatives are used. etc. may be included together. Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried preparations, suppositories, etc.
일 측면에서, 본 발명은 본 발명의 복합체를 유효성분으로 포함하는 M2 종양 관련 대식세포 제거용 조성물에 관한 것이다.In one aspect, the present invention relates to a composition for eliminating M2 tumor-related macrophages comprising the complex of the present invention as an active ingredient.
일 구현예에서, 상기 조성물은 M1 대식세포는 제거하지 않으면서, M2 대식세포만을 제거할 수 있다.In one embodiment, the composition is capable of removing only M2 macrophages without removing M1 macrophages.
일 측면에서, 본 발명은 ITGB2 유전자 또는 상기 유전자로부터 발현된 단백질을 포함하는 고형암 진단용 마커에 관한 것이다.In one aspect, the present invention relates to a marker for diagnosing solid tumors including the ITGB2 gene or a protein expressed from the gene.
일 측면에서, 본 발명은 ITGB2의 발현양을 mRNA 또는 단백질 수준에서 측정하는 제제를 포함하는, 고형암 진단용 조성물In one aspect, the present invention provides a composition for diagnosing solid cancer, comprising an agent for measuring the expression level of ITGB2 at the mRNA or protein level.
일 구현예에서, mRNA 수준에서 측정하는 제제는, 상기 유전자의 핵산서열, 상기 핵산서열에 상보적인 핵산서열, 상기 핵산서열 및 상보적인 서열의 단편을 특이적으로 인식하는 프라미어 쌍, 프로브, 또는 프라이머 쌍 및 프로브일 수 있으며, 상기 측정은 중합효소연쇄반응, 실시간 RT-PCR (Real-time RT-PCR), 역전사 중합효소연쇄반응, 경쟁적 중합효소연쇄반응(Competitive RT-PCR), Nuclease 보호 분석(RNase, S1 nuclease assay), in situ 교잡법, 핵산 마이크로어레이, 노던블랏, DNA 칩, multiplex PCR 또는 ddPCR로 이루어진 군으로부터 선택되는 하나 이상의 방법으로 수행될 수 있다.In one embodiment, the agent for measuring at the mRNA level includes a nucleic acid sequence of the gene, a nucleic acid sequence complementary to the nucleic acid sequence, a primer pair, a probe, or a primer that specifically recognizes a fragment of the nucleic acid sequence and the complementary sequence. It can be a primer pair and a probe, and the measurement can be performed using polymerase chain reaction, real-time RT-PCR, reverse transcription polymerase chain reaction, competitive polymerase chain reaction (Competitive RT-PCR), and nuclease protection assay. (RNase, S1 nuclease assay), in situ hybridization, nucleic acid microarray, Northern blot, DNA chip, multiplex PCR, or ddPCR.
일 구현예에서, 단백질 수준에서 측정하는 제제는, 상기 유전자의 단백질 전장 또는 그 단편을 특이적으로 인식하는 항체, 항체단편, 앱타머(aptamer), 아비머(avidity multimer) 또는 펩티도모방체(peptidomimetics)일 수 있으며, 상기 측정은 웨스턴블랏, ELISA(enzyme linked immunosorbent assay), 방사선면역분석(RIA: Radioimmunoassay), 방사면역확산법(radioimmunodiffusion), 면역 전기영동, 조직면역염색, 면역침전 분석법(Immunoprecipitation assay), 보체 고정 분석법(Complement Fixation Assay), FACS, 질량분석 또는 단백질 마이크로어레이로 이루어진 군으로부터 선택되는 하나 이상의 방법으로 수행될 수 있다.In one embodiment, the agent for measuring at the protein level is an antibody, antibody fragment, aptamer, avidity multimer, or peptidomimetic ( peptidomimetics), and the measurement may be Western blot, ELISA (enzyme linked immunosorbent assay), RIA (Radioimmunoassay), radioimmunodiffusion, immunoelectrophoresis, tissue immunostaining, and immunoprecipitation assay. ), complement fixation assay, FACS, mass spectrometry, or protein microarray.
본 발명에서 사용된 용어, "검출" 또는 "측정"은 검출 또는 측정된 대상의 농도를 정량하는 것을 의미한다.As used herein, the term “detection” or “measurement” means quantifying the concentration of a detected or measured object.
본 발명에서 사용된 용어, "프라이머"는 짧은 자유 3말단 수산화기 (free 3 hydroxyl group)를 가지는 핵산 서열로 상보적인 템플레이트(template)와 염기쌍 (base pair)를 형성할 수 있고 템플레이트 가닥 복사를 위한 시작 지점으로 기능을 하는 짧은 핵산 서열을 의미한다. 프라이머는 적절한 완충용액 및 온도에서 중합반응 (즉, DNA 폴리머레이트 또는 역전사효소)을 위한 시약 및 상이한 4가지 뉴클레오사이드 트리포스페이트의 존재하에서 DNA 합성이 개시할 수 있다. As used in the present invention, the term "primer" is a nucleic acid sequence with a short free 3-terminal hydroxyl group that can form a base pair with a complementary template and serves as a starting point for copying the template strand. It refers to a short nucleic acid sequence that functions as a point. Primers can initiate DNA synthesis in the presence of four different nucleoside triphosphates and reagents for polymerization (i.e., DNA polymerase or reverse transcriptase) in an appropriate buffer solution and temperature.
본 발명에서 사용된 용어, "프로브"란 mRNA와 특이적 결합을 이룰 수 있는 짧게는 수 염기 내지 길게는 수백 염기에 해당하는 RNA 또는 DNA 등의 핵산 단편을 의미하며 라벨링 되어 있어서 특정 mRNA의 존재 유무를 확인할 수 있다. 프로브는 올리고 뉴클레오타이드(oligonucleotide) 프로브, 단쇄 DNA(single stranded DNA) 프로브, 이중쇄 DNA(double stranded DNA) 프로브, RNA 프로브 등의 형태로 제작될 수 있다. 본 발명에서는 상기 ITGB2와 상보적인 프로브를 이용하여 혼성화를 실시하여, 혼성화 여부를 통해 상기 유전자 발현 정도를 진단할 수 있다. 적당한 프로브의 선택 및 혼성화 조건은 통상의 기술분야에 공지된 것을 기초로 변형할 수 있으므로 본 발명에서는 이에 대해 특별히 한정하지 않는다.As used in the present invention, the term "probe" refers to a nucleic acid fragment such as RNA or DNA that is as short as a few bases or as long as several hundred bases, capable of forming a specific binding to mRNA, and is labeled to determine the presence or absence of a specific mRNA. You can check. Probes may be manufactured in the form of oligonucleotide probes, single stranded DNA probes, double stranded DNA probes, RNA probes, etc. In the present invention, hybridization is performed using a probe complementary to ITGB2, and the level of gene expression can be diagnosed based on hybridization. Selection of appropriate probes and hybridization conditions can be modified based on those known in the art, so the present invention is not particularly limited thereto.
본 발명의 프라이머 또는 프로브는 포스포르아미다이트 고체 지지체 방법, 또는 기타 널리 공지된 방법을 사용하여 화학적으로 합성할 수 있다. 이러한 핵산 서열은 또한 당해 분야에 공지된 많은 수단을 이용하여 변형시킬 수 있다. 이러한 변형의 비-제한적인 예로는 메틸화, 캡화, 천연 뉴클레오타이드 하나 이상의 동족체로의 치환및 뉴클레오타이드 간의 변형, 예를 들면, 하전되지 않은 연결체 (예: 메틸 포스포네이트, 포스소트리에스테르, 포스포로아미데이트, 카바메이트 등) 또는 하전된 연결체 (예: 포스포로티오에이트, 포스포로디티오에이트 등)로의 변형이 있다.Primers or probes of the present invention can be chemically synthesized using the phosphoramidite solid support method or other well-known methods. These nucleic acid sequences can also be modified using many means known in the art. Non-limiting examples of such modifications include methylation, capping, substitution of a native nucleotide with one or more homologues, and modifications between nucleotides, such as uncharged linkages (e.g., methyl phosphonate, phosphotriester, phosphoronucleotide). amidate, carbamate, etc.) or charged linkages (e.g. phosphorothioate, phosphorodithioate, etc.).
본 발명에서, 프로브를 cDNA 분자와 혼성화시키는 적합한 조건은 최적화 절차에 의하여 일련의 과정으로 결정될 수 있다. 이런 절차는 연구실에서 사용을 위한 프로토콜을 수립하기 위하여 당업자에 의하여 일련의 과정으로 실시된다. 예를 들어, 온도, 성분의 농도, 혼성화 및 세척 시간, 완충액 성분 및 이들의 pH 및 이온세기 등의 조건은 프로브의 길이 및 GC 양 및 타깃 뉴클레오타이드 서열 등의 다양한 인자에 의존한다. 혼성화를 위한 상세한 조건은 Joseph Sambrook, et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.(2001); 및 M.L.M. Anderson, NucleicAcidHybridization, Springer-Verlag New York Inc. N.Y.(1999)에서 확인할 수 있다. 예를 들어, 상기 엄격조건 중에서 고 엄격조건은 0.5 M NaHPO4, 7% SDS(sodium dodecyl sulfate),1mM EDTA에서 65℃ 조건으로 혼성화하고, 0.1 x SSC(standard saline citrate)/0.1% SDS에서 68℃ 조건으로 세척하는 것을 의미한다. 또는, 고 엄격조건은 6 x SSC/0.05% 소듐 파이로포스페이트에서 48℃ 조건으로 세척하는 것을 의미한다. 저 엄격조건은 예를 들어, 0.2 x SSC/0.1% SDS에서 42℃ 조건으로 세척하는 것을 의미한다.In the present invention, suitable conditions for hybridizing a probe to a cDNA molecule can be determined through a series of processes through an optimization procedure. These procedures are performed as a series by those skilled in the art to establish protocols for use in the laboratory. For example, conditions such as temperature, concentration of components, hybridization and washing time, buffer components and their pH and ionic strength depend on various factors such as the length of the probe, the amount of GC, and the target nucleotide sequence. Detailed conditions for hybridization are described in Joseph Sambrook, et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2001); and M.L.M. Anderson, NucleicAcidHybridization, Springer-Verlag New York Inc. It can be found in N.Y. (1999). For example, among the above stringent conditions, the high stringency conditions are hybridization at 65°C in 0.5 M NaHPO4, 7% SDS (sodium dodecyl sulfate), and 1mM EDTA, and 68°C in 0.1 x standard saline citrate (SSC)/0.1% SDS. It means washing with conditions. Alternatively, high stringency means washing at 48°C in 6 x SSC/0.05% sodium pyrophosphate. Low stringency conditions mean, for example, washing at 42°C in 0.2 x SSC/0.1% SDS.
본 발명에서 사용된 용어, "항체"란 당해 분야에서 공지된 용어로서 항원성 부위에 대해서 지시되는 특이적인 단백질 분자를 의미한다. 본 발명의 목적상, 항체는 본 발명의 마커인 ITGB2 단백질에 대해 특이적으로 결합하는 항체를 의미하며, 상기 항체의 제조방법은 널리 공지된 방법을 사용하여 제조할 수 있다. 여기에는 상기 단백질에서 만들어질 수 있는 부분 펩티드도 포함된다. 본 발명의 항체의 형태는 특별히 제한되지 않으며 폴리클로날 항체, 모노클로날 항체 또는 항원 결합성을 갖는 것이면 그것의 일부도 본 발명의 항체에 포함되고 모든 면역 글로불린 항체가 포함된다. 나아가, 본 발명의 항체에는 인간화 항체 등의 특수 항체도 포함된다.As used in the present invention, the term “antibody” is a term known in the art and refers to a specific protein molecule directed to an antigenic site. For the purpose of the present invention, an antibody refers to an antibody that specifically binds to the ITGB2 protein, which is a marker of the present invention, and the antibody can be produced using a well-known method. This also includes partial peptides that can be made from the above proteins. The form of the antibody of the present invention is not particularly limited, and as long as it is a polyclonal antibody, monoclonal antibody, or has antigen binding properties, a portion thereof is also included in the antibody of the present invention, and all immunoglobulin antibodies are included. Furthermore, the antibodies of the present invention also include special antibodies such as humanized antibodies.
일 측면에서, 본 발명은 ITGB2 단백질에 후보 물질을 처리하는 단계; 및 ITGB2 단백질과 결합하는 후보 물질을 선별하는 단계를 포함하는, 면역항암제를 스크리닝하는 방법에 관한 것이다.In one aspect, the present invention provides the steps of processing a candidate material to ITGB2 protein; And it relates to a method of screening an anticancer immunotherapy agent, including the step of selecting a candidate substance that binds to the ITGB2 protein.
일 구현예에서, ITGB2 단백질과 결합한 후보 물질을 항암 효과가 있는 물질로 판단하는 단계를 추가로 포함할 수 있다.In one embodiment, a step of determining that a candidate substance bound to the ITGB2 protein is a substance with anti-cancer effect may be further included.
일 측면에서, 본 발명은 면역항암제를 처리한 시료에서 ITGB2의 발현양을　확인하는 단계를 포함하는, M2 대식세포를 표적화하는 면역항암제에 대한 반응성 예측 또는 예후 진단 방법에 관한 것이다.In one aspect, the present invention relates to a method for predicting reactivity or prognosis for an immunotherapy agent targeting M2 macrophages, which includes the step of confirming the expression level of ITGB2 in a sample treated with an immunotherapy agent.
일 구현예에서, 면역항암제 처리 후 시료에서 ITGB2 단백질의 발현량이 감소한 경우 면역항암제에 대한 반응성이 좋은 것으로 판단하는 단계를 추가로 포함할 수 있다.In one embodiment, if the expression level of ITGB2 protein is reduced in the sample after treatment with the anticancer immunotherapy agent, a step of determining that the sample has good responsiveness to the anticancer immunotherapy agent may be further included.
일 구현예에서, 상기 시료는 혈액, 혈청, 혈장, 양수, 타액, 복수, 골수, 누액, 담즙, 폐세척액, 뇌척수액, 흉수, 활액, 림프, 정액, 뇨, 또는 조직 생검 또는 세포로부터 단백질을 추출한 용액일 수 있다.In one embodiment, the sample is blood, serum, plasma, amniotic fluid, saliva, ascites, bone marrow, tear fluid, bile, lung lavage fluid, cerebrospinal fluid, pleural fluid, synovial fluid, lymph, semen, urine, or tissue biopsy or protein extraction from cells. It may be a solution.
일 측면에서, 본 발명은 대상으로부터 분리된 시료에 후보 물질을 처리하는 단계; 상기 시료에서 ITGB2의 발현양을　확인하는 단계를 포함하는, M2 대식세포를 표적화하는 항암제를 스크리닝하는 방법.In one aspect, the present invention includes the steps of treating a sample separated from a subject with a candidate substance; A method for screening an anticancer agent targeting M2 macrophages, comprising the step of confirming the expression level of ITGB2 in the sample.
일 구현예에서, 후보 물질의 처리 전과 비교하여 처리 후에 ITGB2 단백질의 발현량이 감소한 경우 상기 후보 물질을 항암제로 판단하는 단계를 추가로 포함할 수 있다.In one embodiment, if the expression level of ITGB2 protein is reduced after treatment compared to before treatment of the candidate substance, the step of determining the candidate substance as an anticancer agent may be further included.
일 측면에서, 본 발명은 대상으로부터 분리된 시료에서　ITGB2의 발현양을　확인하는 단계; 대조군과 ITGB2의 발현양을 비교하는 단계; 및 ITGB2가 대조군에 비해 과발현된 대상을 분류하는 단계를 포함하는, 암의 진단에 필요한 정보를 제공하는 방법에 관한 것이다.In one aspect, the present invention includes the steps of confirming the expression level of ITGB2 in a sample isolated from a subject; Comparing the expression level of ITGB2 with the control group; It relates to a method of providing information necessary for the diagnosis of cancer, including classifying subjects in which ITGB2 is overexpressed compared to the control group.
일 구현예에서, ITGB2가 대조군에 비해 과발현된 대상을 암에 걸린 가능성이 높은 것으로 판단하는 단계를 추가로 포함할 수 있다.In one embodiment, a step of determining that a subject in which ITGB2 is overexpressed is more likely to have cancer compared to the control group may be further included.
일 구현예에서, 원발성 암은 대장암, 유방암, 자궁암, 자궁경부암, 난소암, 전립선암, 뇌종양, 두경부암, 흑색종, 골수종, 백혈병, 림프종, 위암, 폐암, 췌장암, 비소세포성폐암, 간암, 식도암, 소장암, 항문부근암, 나팔관암종, 자궁내막암종, 질암종, 음문암종, 호지킨병, 방광암, 신장암, 수뇨관암, 신장세포암종, 신장골반암종, 골암, 피부암, 두부암, 경부암, 피부흑색종, 안구내흑색종, 내분비선암, 갑상선암, 부갑상선암, 부신암, 연조직육종, 요도암, 음경암, 중추신경계(central nervous system; CNS) 종양, 1차 CNS 림프종, 척수종양, 다형성교모세포종 및 뇌하수체선종으로 구성된 군으로부터 선택되는 어느 하나 이상일 수 있으며, 고형암인 것이 가장 바람직하다.In one embodiment, the primary cancer is colon cancer, breast cancer, uterine cancer, cervical cancer, ovarian cancer, prostate cancer, brain tumor, head and neck cancer, melanoma, myeloma, leukemia, lymphoma, stomach cancer, lung cancer, pancreatic cancer, non-small cell lung cancer, and liver cancer. , esophageal cancer, small intestine cancer, perianal cancer, fallopian tube carcinoma, endometrial carcinoma, vaginal carcinoma, vulvar carcinoma, Hodgkin's disease, bladder cancer, kidney cancer, ureteral cancer, renal cell carcinoma, renal pelvic carcinoma, bone cancer, skin cancer, head cancer, Cervical cancer, cutaneous melanoma, intraocular melanoma, endocrine cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, central nervous system (CNS) tumor, primary CNS lymphoma, spinal tumor, It may be any one or more selected from the group consisting of glioblastoma multiforme and pituitary adenoma, and is most preferably a solid tumor.
일 측면에서, 본 발명은 대상으로부터 분리된 시료에서 ITGB2의 발현양을　확인하는 단계를 포함하는, M2 대식세포를 표적화하는 물질에 대한 반응성을 갖는 암에 걸린 대상을 분류하는 방법에 관한 것이다.In one aspect, the present invention relates to a method of classifying a subject with cancer having reactivity to a substance targeting M2 macrophages, comprising the step of confirming the expression level of ITGB2 in a sample isolated from the subject.
일 구현예에서, ITGB2 단백질의 발현량이 암에 걸리지 않은 대조군에 비해 높은 경우 M2 대식세포를 표적화하는 물질에 대한 반응성을 가질 것으로 판단하는 단계를 추가로 포함할 수 있다.In one embodiment, when the expression level of ITGB2 protein is higher than that of a control group that does not have cancer, a step of determining that the method may be reactive to a substance targeting M2 macrophages may be further included.
하기의 실시예를 통하여 본 발명을 보다 상세하게 설명한다. 그러나 하기 실시예는 본 발명의 내용을 구체화하기 위한 것일 뿐 이에 의해 본 발명이 한정되는 것은 아니다The present invention will be described in more detail through the following examples. However, the following examples are only for illustrating the content of the present invention and are not intended to limit the present invention.
제조예. 펩타이드 합성Manufacturing example. Peptide synthesis
하기 표 1의 멜리틴(Melittin), TAMpep826, Melittin-dKLA 및 TB511 펩타이드들을 GenScript (Piscataway, NJ, USA)에 합성 의뢰하여 공급받았으며, 이후 실험에서 D-PBS에 5 mg/ml로 녹여 사용되었다. 참고로, dKLA의 결합은 다양한 항암제 약물과의 결합 용이성을 확인하기 위해 실시하였으며, 펩타이드간 아미드 결합을 통해 연결하였다. 이 때, 멜리틴 또는 TAMpep826과 dKLA간의 상호작용 및 폴드를 최소화하기 위해 중간에 4개의 글리신 및 1개의 세린으로 구성된 링커를 배치하여 양단을 구분하였으며, KLA는 체내 분해를 최소화 하기 위해 L형이 아닌 D형 이성질체를 사용하였다.The Melittin, TAMPep826, Melittin-dKLA, and TB511 peptides shown in Table 1 below were supplied to GenScript (Piscataway, NJ, USA) for synthesis, and were used in subsequent experiments by dissolving them in D-PBS at 5 mg/ml. For reference, the binding of dKLA was conducted to confirm the ease of binding with various anticancer drugs, and was connected through an amide bond between peptides. At this time, in order to minimize the interaction and folding between melittin or TAMPep826 and dKLA, a linker consisting of 4 glycines and 1 serine was placed in the middle to separate both ends, and KLA was used in a non-L form to minimize degradation in the body. The D-type isomer was used.
[표 1][Table 1]
Figure PCTKR2022005783-appb-img-000001
Figure PCTKR2022005783-appb-img-000001
실시예 1. 멜리틴의 M2 종양 관련 대식세포(M2 TAM)에 대한 친화도(Affinity) 확인Example 1. Confirmation of affinity of melittin for M2 tumor-related macrophages (M2 TAM)
멜리틴이 M2 종양 관련 대식세포(M2 TAM)에 특이적으로 결합하는지 확인하기 위해, 인간 단핵구 세포주 THP-1를 M0, M1 및 M2 대식세포로 분화시키고, FITC 컨쥬게이트된 멜리틴을 처리하여 유세포 분석으로 확인하였다. 구체적으로, THP-1 세포는 100 nM의 PMA(phorbol 12-myristate 13-acetate)를 37℃에서 24시간 동안 처리하였고 (M0 대식세포), 분화된 M0 대식세포를 100 nM의 LPS 및 20 ng/ml의 IFN-γ와 37℃에서 72시간 동안 배양하여 M1 대식세포 (M1)로 분화시켰으며, 20 ng/ml의 IL-4 및 IL-13와 37℃에서 72시간 동안 배양하여 M2 대식세포 (M2)로 분화시켰다. 분화된 세포에 FITC 컨쥬게이트된 멜리틴 50 nM을 1시간 동안 처리하고 BD FACS CantoII instruments (BD biosciences, San Jose, CA, USA)로 세포를 검출하여 FlowJo software (BD biosciences)로 분석하였다. To determine whether melittin specifically binds to M2 tumor-associated macrophages (M2 TAMs), the human monocytic cell line THP-1 was differentiated into M0, M1, and M2 macrophages, treated with FITC-conjugated melittin, and flow cytometry was performed. This was confirmed by analysis. Specifically, THP-1 cells were treated with 100 nM of PMA (phorbol 12-myristate 13-acetate) at 37°C for 24 hours (M0 macrophages), and differentiated M0 macrophages were treated with 100 nM of LPS and 20 ng/mL. They were differentiated into M1 macrophages (M1) by culturing with ml of IFN-γ at 37°C for 72 hours, and by culturing with 20 ng/ml of IL-4 and IL-13 at 37°C for 72 hours to differentiate into M2 macrophages ( M2). Differentiated cells were treated with 50 nM of FITC-conjugated melittin for 1 hour, and cells were detected with BD FACS CantoII instruments (BD biosciences, San Jose, CA, USA) and analyzed with FlowJo software (BD biosciences).
그 결과, FITC 양성 세포는 M0 및 M2 대식세포에 비해 M2 대식세포에서 현저히 증가하였다 (도 1). 이를 통해, 멜리틴이 M2 대식세포에 우선적으로 결합하는 것을 확인하였다.As a result, FITC-positive cells were significantly increased in M2 macrophages compared to M0 and M2 macrophages (Figure 1). Through this, it was confirmed that melittin preferentially binds to M2 macrophages.
실시예 2. 멜리틴의 M2 대식세포 결합 표적 단백질 식별Example 2. Identification of M2 macrophage binding target protein of melittin
M2 대식세포의 어떤 단백질이 멜리틴과 M2 대식세포의 특이적 결합에 관련되었는지 확인하기 위해, 비오틴-컨쥬게이트된 멜리틴 (멜리틴-biotin)을 합성하였다. THP-1 세포 (5 Х 106 cells)를 M0, M1 및 M2 대식세포로 분화시킨 뒤, 스크래퍼를 이용하여 세포를 모아 원심분리 (300 Х g, 5분)를 통해 세포를 세척하고 세포막 단백질 추출 키트(membrane protein extraction kit) (Thermo Fisher scientific)을 사용하여 대식세포의 세포막 단백질을 얻었다. 멜리틴에 비오틴(biotin)이 결합된 멜리틴-비오틴 (200 μg)과 상온 (20~24℃)에서 1시간 동안 반응시킨 후 스트렙타비딘 레진(streptavidin resin) (Thermo Fisher scientific)을 이용하여 멜리틴과 결합한 단백질을 얻었다. 염을 제거하기 위해, 100% 메탄올, 0.1% 포름산 및 80% CAN을 각각 100㎕씩 18 Micro Spin-Column에 첨가하여 준비하고, 준비된 컬럼에 시료를 로딩하고 50 ㎕의 0.1% 포름산을 첨가하여 불필요한 물질을 제거하였다. 그 후, 80% CAN 100 ㎕를 첨가하여 펩타이드를 용리시켰다. 탈염이 끝나면 Speed-vac으로 건조시켜 용액을 모두 제거하고, 하기 표 2의 조건으로 UPLC-Exactive equipment (Thermo Fisher Scientific)를 이용하여 LC-MS/MS로 분석하였다. MaxQuant 및 MPP (Mass Profiler Professional)를 이용하여 M0 대식세포에 비해 M1 및 M2 대식세포에서 상향 조절된 단백질에 대해 데이터를 분석하였다. 데이터 분석을 위해 각 시표 별로 LC-MS/MS 데이터를 Proteome Discoverer를 이용하여 분석하였고, 데이터베이스는 Uniprot의 human database를 사용하여, LFQ(Label-Free Quantification)로 진행하였다. Modificaiton은 Oxidation(M), Carbamyl(N-term), Carbamidomethyl(C)를 추가하였고, Baseline option은 none으로 하였다.To determine which protein of M2 macrophages was involved in the specific binding of melittin and M2 macrophages, biotin-conjugated melittin (melittin-biotin) was synthesized. After differentiating THP-1 cells (5 Х 10 6 cells) into M0, M1, and M2 macrophages, cells were collected using a scraper, washed by centrifugation (300 Х g, 5 minutes), and cell membrane protein was extracted. Cell membrane proteins of macrophages were obtained using a membrane protein extraction kit (Thermo Fisher scientific). After reacting melittin-biotin (200 μg), which is biotin bound to melittin, at room temperature (20-24°C) for 1 hour, melittin was reacted using streptavidin resin (Thermo Fisher scientific). A protein bound to tin was obtained. To remove salts, 100 ㎕ each of 100% methanol, 0.1% formic acid, and 80% CAN were added to an 18 Micro Spin-Column to prepare the sample, and then the sample was loaded onto the prepared column and 50 ㎕ of 0.1% formic acid was added to remove unnecessary salts. The material was removed. Afterwards, 100 μl of 80% CAN was added to elute the peptides. After desalting, all of the solution was removed by drying with Speed-vac, and analyzed by LC-MS/MS using UPLC-Exactive equipment (Thermo Fisher Scientific) under the conditions shown in Table 2 below. Data were analyzed for proteins upregulated in M1 and M2 macrophages compared to M0 macrophages using MaxQuant and MPP (Mass Profiler Professional). For data analysis, LC-MS/MS data for each target was analyzed using Proteome Discoverer, and Uniprot's human database was used as the database, and LFQ (Label-Free Quantification) was performed. Modification was done by adding Oxidation (M), Carbamyl (N-term), and Carbamidomethyl (C), and the Baseline option was set to none.
[표 2][Table 2]
Figure PCTKR2022005783-appb-img-000002
Figure PCTKR2022005783-appb-img-000002
그 결과, M2/M0에서 53개의 단백질이, M1/M0에서 123개의 단백질이 상향조절되었다 (도 2A). M2 대식세포보다 더 많은 상향조절된 단백질들이 M1 대식세포와 상호작용하였으나, 세포막에 존재하는 단백질은 그렇지 않았다. 막 단백질들 중, ITGB2가 M2 대식세포에서 상향조절되는 것으로 나타났다 (도 2B). 또한, M1 대식세포의 마커로 알려진 IL-10 및 CXCL10와 같은 사이토카인들이 M1 대식세포에서 상향조절되었다 (도 2C). 또한, 단백질들이 M1에 비해 M2에서 상향조절된 단백질들이 확인되었으나, 막 단백질들 중 ITGB2만이 M2 대식세포에서 상향조절되는 것으로 확인되었다 (도 2D). 이를 통해, 멜리틴이 M2 대식세포의 막 단백질 ITGB2과 상호작용하고 결합하는 것을 확인하였다.As a result, 53 proteins were upregulated in M2/M0 and 123 proteins were upregulated in M1/M0 (Figure 2A). More upregulated proteins interacted with M1 macrophages than with M2 macrophages, but not proteins present in the cell membrane. Among the membrane proteins, ITGB2 was shown to be upregulated in M2 macrophages (Figure 2B). Additionally, cytokines such as IL-10 and CXCL10, known as markers of M1 macrophages, were upregulated in M1 macrophages (Figure 2C). In addition, proteins were confirmed to be upregulated in M2 compared to M1, but among the membrane proteins, only ITGB2 was confirmed to be upregulated in M2 macrophages (Figure 2D). Through this, it was confirmed that melittin interacts with and binds to the membrane protein ITGB2 of M2 macrophages.
실시예 3. TAMpep826의 M2 대식세포 결합 표적 단백질 식별Example 3. Identification of M2 macrophage binding target protein of TAMpep826
상기 실시예 2에서와 같이, M0, M1 및 M2 대식세포에서 각각 추출한 세포막 단백질과 TAMpep826-비오틴을 반응시켜 LC-MS/MS proteomics를 통해 타겟 단백질을 확인하였다. As in Example 2, the target protein was confirmed through LC-MS/MS proteomics by reacting TAMpep826-biotin with cell membrane proteins extracted from M0, M1, and M2 macrophages, respectively.
그 결과, 세포막 단백질 중 ITGB2가 M0 및 M1 대식세포에 비해 M2 대식세포에서 더 높은 수준으로 발현되는 것으로 나타나 (도 4), TAMpep826의 M2 대식세포 결합 표적 단백질로서 선별되었다.As a result, among cell membrane proteins, ITGB2 was found to be expressed at a higher level in M2 macrophages compared to M0 and M1 macrophages (Figure 4), and was selected as the M2 macrophage binding target protein of TAMpep826.
실시예 4. M2 대식세포에서 ITGB2 발현 확인Example 4. Confirmation of ITGB2 expression in M2 macrophages
4-1. ITGB2 mRNA 발현 확인4-1. Confirmation of ITGB2 mRNA expression
M0, M1, M2 대식세포 및 TAMs에서 ITGB2의 mRNA 발현 수준을 평가하기 위해 실시간 중합효소연쇄반응을 수행하였다. 구체적으로, M0, M1 또는 M2 대식세포로 분화된 세포에서 easy-BLUE total RNA Extraction Kit (iNtRON)를 이용하여 RNA를 추출하고, CycleScript-Reverse Transcriptase (Bioneer)를 이용하여 cDNA로 합성하였다. Actin 또는 ITGB2 유전자의 프라이머 (표 3)를 사용하고 도 5의 조건으로 real-time PCR (CFX96, Bio-rad)을 진행하여 Actin 대비 각 유전자의 발현량을 상대정량(Relative quantification)으로 비교 및 분석하였다. Real-time polymerase chain reaction was performed to evaluate the mRNA expression level of ITGB2 in M0, M1, M2 macrophages and TAMs. Specifically, RNA was extracted from cells differentiated into M0, M1, or M2 macrophages using the easy-BLUE total RNA Extraction Kit (iNtRON), and synthesized into cDNA using CycleScript-Reverse Transcriptase (Bioneer). Actin or ITGB2 gene primers (Table 3) were used and real-time PCR (CFX96, Bio-rad) was performed under the conditions of Figure 5 to compare and analyze the expression level of each gene compared to Actin through relative quantification. did.
[표 3][Table 3]
Figure PCTKR2022005783-appb-img-000003
Figure PCTKR2022005783-appb-img-000003
그 결과, ITGB2의 mRNA 발현은 M0 및 M1에 비해 M2 대식세포 및 TAM에서 유의하게 증가한 것으로 나타났다 (도 6).As a result, the mRNA expression of ITGB2 was found to be significantly increased in M2 macrophages and TAMs compared to M0 and M1 (Figure 6).
4-2. ITGB2 단백질 발현 확인4-2. Confirmation of ITGB2 protein expression
M0, M1, M2 대식세포 및 TAMs에서 ITGB2의 단백질 발현 수준을 평가하기 위해 웨스턴 블롯 분석을 수행하였다. 구체적으로, M0, M1 및 M2 대식세포로 분화된 각각의 세포에서 Proprep solution (iNtRON)를 이용하여 단백질을 추출하고, 전기영동법으로 멤브레인에 단백질을 트랜스퍼하였다. 멤브레인을 5% BSA에 담가 블로킹한 뒤, 1 차 항체 (ITGB2)를 24시간 동안 4℃에서 처리하고 2차 항체를 1시간 동안 상온에서 처리한 뒤, D-PlusTM ECL Pico System (동인LS)을 처리하고 Davinch-ChemiTM imaging system (Intoxia)으로 단백질 밴드 사진을 촬영하였다.Western blot analysis was performed to evaluate the protein expression level of ITGB2 in M0, M1, M2 macrophages and TAMs. Specifically, proteins were extracted from each cell differentiated into M0, M1, and M2 macrophages using Proprep solution (iNtRON), and the proteins were transferred to the membrane by electrophoresis. The membrane was blocked by soaking it in 5% BSA, then treated with primary antibody (ITGB2) at 4°C for 24 hours and secondary antibody at room temperature for 1 hour, followed by D-Plus TM ECL Pico System (Dongin LS). was processed and protein band photos were taken with the Davinch-Chemi TM imaging system (Intoxia).
그 결과, ITGB2의 단백질 발현은 M0 및 M1에 비해 M2 대식세포 및 TAM에서 유의하게 증가한 것으로 나타났다 (도 7).As a result, protein expression of ITGB2 was found to be significantly increased in M2 macrophages and TAMs compared to M0 and M1 (Figure 7).
4-3. 면역형광법을 통한 ITGB2 단백질 발현 확인4-3. Confirmation of ITGB2 protein expression through immunofluorescence
M0, M1, M2 대식세포 및 TAMs에서 ITGB2의 단백질 발현 수준을 평가하기 위해 면역형광분석을 수행하였다. 구체적으로, M0, M1 및 M2 대식세포로 분화된 각각의 세포를 4% 포르말린으로 고정시킨 뒤, PBS로 세척하고 5% BSA에 담가 블로킹한 뒤, 1 차 항체(ITGB2)를 24시간 동안 4℃에서 처리하고 2차 항체를 2시간 동안 상온에서 처리하여 염색을 수행하였다. 염색된 세포는 DAPI mounting 후 LSM800 (Zeiss)으로 형광 사진을 촬영하였다.Immunofluorescence analysis was performed to evaluate the protein expression level of ITGB2 in M0, M1, M2 macrophages and TAMs. Specifically, each cell differentiated into M0, M1, and M2 macrophages was fixed with 4% formalin, washed with PBS, blocked by soaking in 5% BSA, and incubated with primary antibody (ITGB2) at 4°C for 24 hours. and staining was performed by treating with secondary antibody at room temperature for 2 hours. Stained cells were photographed using LSM800 (Zeiss) after DAPI mounting.
그 결과, ITGB2의 단백질은 M0 및 M1에 비해 M2 대식세포 및 TAM에서 높은 발현 수준을 나타냈다 (도 8).As a result, the protein of ITGB2 showed a higher expression level in M2 macrophages and TAMs compared to M0 and M1 (Figure 8).
실시예 5. TAMpep826 및 ITGB2의 상호작용 확인Example 5. Confirmation of interaction between TAMpep826 and ITGB2
5-1. M2 대식세포 세포막에서 ITGB2의 발현 확인5-1. Confirmation of ITGB2 expression in M2 macrophage cell membrane
ITGB2이 M2 대식세포의 세포막에서 발현되는지 확인하기 위해, M2 대식세포에서 세포막 단백질 및 세포질(cytosol) 단백질을 각각 분리 추출하여 비오틴화된 TAMpep826과 반응시켜 ITGB2의 발현을 웨스턴 블롯 분석으로 평가하였다. 그 결과, ITGB2 단백질은 M2 대식세포의 세포막에서 발현되는 것으로 나타났다 (도 9).To confirm whether ITGB2 is expressed in the cell membrane of M2 macrophages, cell membrane proteins and cytosol proteins were separately extracted from M2 macrophages and reacted with biotinylated TAMpep826, and the expression of ITGB2 was evaluated by Western blot analysis. As a result, ITGB2 protein was found to be expressed in the cell membrane of M2 macrophages (Figure 9).
5-2. TAMpep826 및 ITGB2의 상호작용 확인5-2. Confirmation of interaction between TAMPep826 and ITGB2
M2 대식세포에서 TAMpep826이 ITGB2과 상호작용하는지 확인하기 위해, TAMpep826 및 비오틴화된 TAMpep826의 경쟁적인 반응을 통해 ITGB2의 발현을 평가하였다. 구체적으로, M2 대식세포로 분화된 세포를 스크래퍼로 모아 원심분리 (300 Х g, 5분)로 세포를 세척하고, 세포막 단백질 추출 키트 (Thermo Fisher scientific)를 사용하여 대식세포의 세포막 단백질을 얻었다. TAMpep826 또는 scramble 펩타이드를 농도별 (0.001, 0.01, 0.1, 1 및 10 μg)로 1시간 동안 전처리한 후 단백질에 비오틴이 결합된 TAMpep826 (200 μg)과 상온 (20~24℃)에서 1시간 동안 반응시킨 후 DynabeadsTM M-280 Streptavidin (Thermo Fisher scientific)을 이용하여 TAMpep826과 결합한 단백질을 얻었다. 단백질을 웨스턴 블롯 분석하여 ITGB2의 발현을 확인하였다.To determine whether TAMpep826 interacts with ITGB2 in M2 macrophages, the expression of ITGB2 was assessed through competitive reaction of TAMpep826 and biotinylated TAMpep826. Specifically, cells differentiated into M2 macrophages were collected with a scraper, washed by centrifugation (300 Х g, 5 minutes), and cell membrane proteins of macrophages were obtained using a cell membrane protein extraction kit (Thermo Fisher scientific). After pre-treating TAMpep826 or scramble peptide at different concentrations (0.001, 0.01, 0.1, 1, and 10 μg) for 1 hour, react with TAMpep826 (200 μg), which has biotin bound to the protein, at room temperature (20-24°C) for 1 hour. After this, the protein bound to TAMpep826 was obtained using Dynabeads TM M-280 Streptavidin (Thermo Fisher scientific). The expression of ITGB2 was confirmed by Western blot analysis of the protein.
그 결과, scrambled 펩타이드를 전처리하였을 때 비오틴화된 TAMpep826과 결합된 ITGB2의 발현은 영향이 없는 것으로 나타난 반면, TAMpep826으로 전처리하였을 때는 농도에 따라 ITGB2 발현이 억제되는 것으로 나타났다 (도 10). 이를 통해, TAMpep826이 M2 대식세포의 세포막에서 ITGB2과 상호작용하는 것을 확인할 수 있었다.As a result, when pretreated with the scrambled peptide, the expression of ITGB2 bound to biotinylated TAMpep826 appeared to be unaffected, whereas when pretreated with TAMpep826, ITGB2 expression was suppressed depending on the concentration (FIG. 10). Through this, it was confirmed that TAMpep826 interacts with ITGB2 in the cell membrane of M2 macrophages.
5-3. 면역형광법을 이용한 TAMpep826 및 ITGB2의 상호작용 확인5-3. Confirmation of interaction between TAMpep826 and ITGB2 using immunofluorescence
M2 대식세포의 세포막에서 ITGB2 및 TAMpep826이 co-localization하는지 확인하기 위해, FITC가 결합된 TAMpep826을 이용하여 면역형광분석을 수행하였다. 그 결과, TAMpep826과 ITGB2은 M2 대식세포의 세포막에 집중적으로 분포되어 있는 것으로 나타났다 (도 11).To confirm whether ITGB2 and TAMpep826 co-localize in the cell membrane of M2 macrophages, immunofluorescence analysis was performed using FITC-conjugated TAMpep826. As a result, TAMpep826 and ITGB2 were found to be concentratedly distributed in the cell membrane of M2 macrophages (FIG. 11).
실시예 6. Melittin-dKLA 및 ITGB2의 결합력 확인Example 6. Confirmation of binding force of Melittin-dKLA and ITGB2
ITGB2 및 Melittin-dKLA의 결합력을 확인하기 위해, ITGB2 단백질을 골드 박막 센서칩 위에 코팅한 후 양성 대조군으로 ITGB2 항체 및 Melittin-dKLA를 각각 흘려 반사광을 측정하고, 표면 플라즈몬 공명 (Surface plasmon resonance, SPR) 분석을 수행하였다. 구체적으로, 센서 칩 표면에 ITGB2을 고정시킨 후 아민 커플링(amine coupling)을 이용하여 하기 표 4의 조건으로 고정화(immobilization)를 수행하였다 (도 12). 그 후, ITGB2이 코팅된 센서칩에 여러 농도의 ITGB2 항체 및 Melittin-dKLA을 흘려 결합(association) 및 해리(dissociation) 구간을 관찰하고 센서그램을 통해 하기 표 5의 분자간 결합 속도상수 분석(kinetic evoluation) 조건으로 분자간 결합 속도상수(kinetic prarmeter)를 계산하였다.To confirm the binding force of ITGB2 and Melittin-dKLA, ITGB2 protein was coated on a gold thin film sensor chip, then ITGB2 antibody and Melittin-dKLA were flowed as positive controls, respectively, and the reflected light was measured, and surface plasmon resonance (SPR) was used. Analysis was performed. Specifically, ITGB2 was immobilized on the surface of the sensor chip, and then immobilization was performed using amine coupling under the conditions shown in Table 4 below (FIG. 12). Afterwards, various concentrations of ITGB2 antibody and Melittin-dKLA were flowed onto the ITGB2-coated sensor chip, the association and dissociation sections were observed, and the intermolecular association rate constant (kinetic evolution) was analyzed through sensorgram in Table 5 below. ) The intermolecular bonding rate constant (kinetic parameter) was calculated under the following conditions.
[표 4][Table 4]
Figure PCTKR2022005783-appb-img-000004
Figure PCTKR2022005783-appb-img-000004
[표 5][Table 5]
Figure PCTKR2022005783-appb-img-000005
Figure PCTKR2022005783-appb-img-000005
그 결과, 양성 대조군인 ITGB2 항체의 두 분자 사이의 결합력을 평가하는 해리속도상수 (kd; dissociation rate constant)를 결합속도상수 (ka; association rate constant)로 나누어 얻는 평형해리상수 (KD; equilibrium dissociation constant)가 1.14-8로 나타났고, Melittin-dKLA의 평형해리상수는 7.86-8로 나타났다 (도 13 및 14). 따라서, Melittin-dKLA이 ITGB2과 결합력을 가지고 있음을 확인하였다.As a result, the equilibrium dissociation constant (KD) obtained by dividing the dissociation rate constant (kd; dissociation rate constant), which evaluates the binding force between two molecules of the positive control ITGB2 antibody, by the association rate constant (ka) ) was found to be 1.14 -8 , and the equilibrium dissociation constant of Melittin-dKLA was found to be 7.86 -8 (Figures 13 and 14). Therefore, it was confirmed that Melittin-dKLA has binding affinity to ITGB2.
실시예 7. M2 대식세포에서 ITGB2을 통한 Melittin-dKLA의 세포사멸 확인Example 7. Confirmation of apoptosis by Melittin-dKLA through ITGB2 in M2 macrophages
7-1. ITGB2의 발현 억제 세포 모델 제작7-1. Production of cell model suppressing expression of ITGB2
M2 대식세포에서 ITGB2을 통해 세포사멸을 유도하는지 확인하는데 사용하기 위한 세포 모델로서, Crispr/cas9를 이용하여 M2 대식세포에서 ITGB2의 발현이 억제된 세포 모델을 제작하였다. 구체적으로, M2 대식세포로 분화된 세포를 Opti-MEM 배지에서 배양하였다. Crispr/cas9은 CRISPRMAXTM Reagent kit (Thermo Fisher scientific)의 Cas9 protein V2 (Thermo Fisher scientific)와 gRNA (Genscript; ITGB2; 표 6)를 Cas9 PlusTM Reagent (Thermo Fisher scientific)와 함께 섞고, CRISPRMAXTM Reagent를 Opti-MEM 배지에 희석한 후 모두 섞어 5 - 10분 동안 반응시킨 뒤, M2 대식세포로 분화된 세포에 첨가하여 37℃에서 2-3일간 배양하였다. 그 후, ITGB2의 mRNA 발현 수준을 확인하였다.As a cell model to be used to confirm whether apoptosis is induced through ITGB2 in M2 macrophages, a cell model in which the expression of ITGB2 was suppressed in M2 macrophages was created using Crispr/cas9. Specifically, cells differentiated into M2 macrophages were cultured in Opti-MEM medium. For Crispr/cas9, Cas9 protein V2 (Thermo Fisher scientific) and gRNA (Genscript; ITGB2; Table 6) from the CRISPRMAX TM Reagent kit (Thermo Fisher scientific) were mixed with Cas9 Plus TM Reagent (Thermo Fisher scientific), and CRISPRMAX TM Reagent was used. After diluting in Opti-MEM medium, everything was mixed and reacted for 5-10 minutes, then added to cells differentiated into M2 macrophages and cultured at 37°C for 2-3 days. Afterwards, the mRNA expression level of ITGB2 was confirmed.
[표 6][Table 6]
Figure PCTKR2022005783-appb-img-000006
Figure PCTKR2022005783-appb-img-000006
그 결과, ITGB2 sgRNA를 주입한 M2 대식세포는 ITGB2의 발현이 대조군에 비해 유의하게 감소되는 것으로 나타났다 (도 15).As a result, the expression of ITGB2 in M2 macrophages injected with ITGB2 sgRNA was found to be significantly reduced compared to the control group (FIG. 15).
7-2. ITGB2 발현 억제에 의한 Melittin-dKLA의 세포사멸 유도 감소 확인7-2. Confirmation of decreased induction of apoptosis by Melittin-dKLA by inhibiting ITGB2 expression
Melittin-dKLA가 M2 대식세포에서 ITGB2에 결합하여 세포자멸사(apoptosis)를 유도하는지 확인하기 위해, 상기 실시예 7-1에서 제작한 Crispr/cas9에 의해 ITGB2 발현이 억제된 M2 대식세포에 Melittin-dKLA (1 μM)를 24시간 동안 처리하고, CCK-8 분석 방법을 이용하여 세포 생존율을 분석하였다. 구체적으로, 상기 실시예 7-1에서 제작한 ITGB2 낙다운 대식세포에 Melittin-dKLA (1 μM)을 1시간 동안 반응시켰다. 반응 후, 배지를 교체하고 37℃에서 24시간 동안 배양하였다. 세포 생존능을 확인하기 위해 CCK-8 reagent (Enzo Life Sciences)를 배지의 1/10으로 넣고 37℃에서 3시간 동안 반응시켰다. 흡광도는 microplate leader (Molecular Devices)로 450 nm에서 측정하였다.To confirm whether Melittin-dKLA binds to ITGB2 in M2 macrophages and induces apoptosis, Melittin-dKLA was added to M2 macrophages in which ITGB2 expression was suppressed by Crispr/cas9 produced in Example 7-1. (1 μM) was treated for 24 hours, and cell viability was analyzed using the CCK-8 assay method. Specifically, ITGB2 knockdown macrophages prepared in Example 7-1 were reacted with Melittin-dKLA (1 μM) for 1 hour. After the reaction, the medium was replaced and cultured at 37°C for 24 hours. To check cell viability, CCK-8 reagent (Enzo Life Sciences) was added to 1/10 of the medium and reacted at 37°C for 3 hours. Absorbance was measured at 450 nm with a microplate leader (Molecular Devices).
그 결과, 대조군 M2 대식세포에서 Melittin-dKLA에 의해 세포 생존율이 현저히 감소하였으나, ITGB2가 낙다운된 M2 대식세포에서는 Melittin-dKLA에 의한 세포 생존율 감소가 억제되는 것으로 나타났다 (도 16). 따라서, 이를 통해, Melittin-dKLA가 M2 대식세포의 ITGB2와 특이적으로 결합함으로써 M2 대식세포의 세포자멸사를 유도하는 것을 확인할 수 있었다. As a result, cell viability was significantly reduced by Melittin-dKLA in control M2 macrophages, but the decrease in cell viability by Melittin-dKLA was suppressed in M2 macrophages in which ITGB2 was knocked down (FIG. 16). Therefore, through this, it was confirmed that Melittin-dKLA induces apoptosis of M2 macrophages by specifically binding to ITGB2 of M2 macrophages.
실시예 8. M2 대식세포에서 ITGB2을 통한 TAMpep826-dKLA의 세포사멸 확인Example 8. Confirmation of apoptosis of TAMpep826-dKLA through ITGB2 in M2 macrophages
8-1. ITGB2 발현 억제에 의한 TAMpep826-dKLA의 세포사멸 유도 감소 확인8-1. Confirmation of decreased induction of apoptosis by TAMpep826-dKLA by inhibiting ITGB2 expression
TAMpep826-dKLA(TB511)이 M2 대식세포에서 ITGB2을 통해 세포사멸을 유도하는지 확인하기 위해, 상기 실시예 7-1에서 제작한 Crispr/cas9에 의해 ITGB2 발현이 억제된 M2 대식세포에 TB511을 반응시켰다. 그 결과, M2 대식세포는 TB511에 의해 50% 정도의 세포 생존능을 나타냈으나, ITGB2 발현이 억제된 세포에서는 세포 생존능이 유의하게 증가하는 것을 나타냈다 (도 17).To confirm whether TAMPep826-dKLA (TB511) induces apoptosis through ITGB2 in M2 macrophages, the TB511 was reacted with M2 macrophages in which ITGB2 expression was suppressed by Crispr/cas9. As a result, M2 macrophages showed a cell viability of about 50% due to TB511, but cells in which ITGB2 expression was suppressed showed a significant increase in cell viability (FIG. 17).
8-2. ITGB2 항체에 의한 TAMpep826-dKLA의 세포사멸 유도 감소 확인8-2. Confirmation of reduced apoptosis induction of TAMpep826-dKLA by ITGB2 antibody
TB511이 M2 대식세포에서 ITGB2을 통해 세포사멸을 유도하는지 확인하기 위해, 상기 실시예 7-1에서 제작한 ITGB2 발현이 억제된 M2 대식세포에 anti-ITGB2 항체 (1 μg)을 1시간 동안 전처리한 후 TB511을 반응시켰다. To confirm whether TB511 induces apoptosis through ITGB2 in M2 macrophages, M2 macrophages with suppressed ITGB2 expression prepared in Example 7-1 were pretreated with anti-ITGB2 antibody (1 μg) for 1 hour. Then TB511 was reacted.
그 결과, M2 대식세포는 ITGB2 항체에 의해서는 세포 생존능에는 영향이 없었으며, TB511에 의해서는 세포 생존능이 유의하게 감소하였다. 반면에 ITGB2 항체를 전처리한 M2 대식세포에서는 TB511에 의한 세포 생존능이 유의하게 증가한 것으로 나타났다 (도 18).As a result, the cell viability of M2 macrophages was not affected by the ITGB2 antibody, and the cell viability was significantly reduced by TB511. On the other hand, in M2 macrophages pretreated with ITGB2 antibody, cell viability by TB511 was significantly increased (FIG. 18).

Claims (21)

  1. ITGB2(Integrin beta 2)에 특이적으로 결합하는 표적화 물질을 유효성분으로 포함하는 ITGB2 매개 약물 전달시스템.ITGB2-mediated drug delivery system containing a targeting substance that specifically binds to ITGB2 (Integrin beta 2) as an active ingredient.
  2. 제 1항에 있어서, 표적화 물질은 M2 종양 관련 대식세포의 세포막에 존재하는 ITGB2 단백질에 특이적으로 결합하는, ITGB2 매개 약물 전달시스템.The ITGB2-mediated drug delivery system according to claim 1, wherein the targeting agent specifically binds to the ITGB2 protein present in the cell membrane of M2 tumor-related macrophages.
  3. 제 1항에 있어서, 표적화 물질은 ITGB2와 특이적으로 결합하는 소분자, 바이러스, 항체, 항체 단편, 압타머, 호르몬, 사이토카인, 케모카인, 리간드, 사이토카인의 일부 영역인 펩타이드 또는 리간드의 일부 영역인 펩타이드인, ITGB2 매개 약물 전달시스템.The method of claim 1, wherein the targeting agent is a small molecule, virus, antibody, antibody fragment, aptamer, hormone, cytokine, chemokine, ligand, peptide or partial region of a cytokine that specifically binds to ITGB2. Peptide, ITGB2-mediated drug delivery system.
  4. 제 1항의 표적화 물질에 표적화 서열, 태그, 표지된 잔기 또는 아미노산 서열을 추가로 포함하는, ITGB2 매개 약물 전달시스템.An ITGB2-mediated drug delivery system further comprising a targeting sequence, a tag, a labeled residue, or an amino acid sequence to the targeting agent of claim 1.
  5. 제 1항의 표적화 물질에 RNA, DNA, 항체, 이펙터, 약물, 전구약물, 독소, 펩티드 또는 전달 분자가 추가로 접합(conjugate)된, ITGB2 매개 약물 전달시스템.An ITGB2-mediated drug delivery system in which RNA, DNA, antibody, effector, drug, prodrug, toxin, peptide or delivery molecule is additionally conjugated to the targeting agent of claim 1.
  6. 제 1항의 ITGB2 매개 약물 전달시스템에 약물이 결합한 복합체.A complex in which a drug is bound to the ITGB2-mediated drug delivery system of claim 1.
  7. 제 6항에 있어서, M2 종양 관련 대식세포의 세포막에 존재하는 ITGB2 단백질에 특이적으로 결합하는, 복합체.The complex according to claim 6, which specifically binds to the ITGB2 protein present in the cell membrane of M2 tumor-related macrophages.
  8. 제 6항에 있어서, 약물은 전세포사멸성(pro-apoptotic) 펩타이드, 면역원성 세포사멸 유도제 또는 항암제인, 복합체.The complex according to claim 6, wherein the drug is a pro-apoptotic peptide, an immunogenic apoptosis inducer, or an anticancer agent.
  9. 제 8항에 있어서, 전세포사멸성 펩타이드는 KLA, 알파-디펜신-1(alpha-defensin-1), BMAP-28, Brevenin-2R, 부포린 IIb(Buforin IIb), 세크로핀 A-마가이닌 2(cecropin A-Magainin 2, CA-MA-2), 세크로핀 A(Cecropin A), 세크로핀 B(Cecropin B), 크리소피신-1(chrysophsin-1), D-K6L9, 고메신(Gomesin), 락토페리신 B(Lactoferricin B), LLL27, LTX-315, 마가이닌 2(Magainin 2), 마가이닌 II-봄패신 결합체(Magainin II-bombesin conjugate, MG2B), 파르닥신(Pardaxin) 및 이들의 조합으로 이루어진 군에서 선택되는, 복합체.The method of claim 8, wherein the pro-apoptotic peptide is KLA, alpha-defensin-1, BMAP-28, Brevenin-2R, Buforin IIb, and cecropin A-mag. Cecropin A-Magainin 2 (CA-MA-2), Cecropin A, Cecropin B, chrysophsin-1, D-K6L9, high Gomesin, Lactoferricin B, LLL27, LTX-315, Magainin 2, Magainin II-bombesin conjugate (MG2B), Pardaxin and a complex selected from the group consisting of combinations thereof.
  10. 제 8항에 있어서, 면역원성 세포사멸 유도제는 안트라사이클린계열 항암제, 탁산 계열 항암제, 항-EGFR 항체, BK 채널 작용제, 보르테조밉(Bortezomib), 강심성 배당체(cardiac glycoside), 사이클로포스마이드 계열 항암제, GADD34/PP1 저해제, LV-tSMAC, Measles 바이러스, 블레오마이신(bleomycin), 미토잔트론(mitoxantrone), 옥살리플라틴(oxaliplatin) 및 이들의 조합으로 이루어진 군에서 선택되는, 복합체.The method of claim 8, wherein the immunogenic apoptosis inducing agent is an anthracycline-based anticancer agent, taxane-based anticancer agent, anti-EGFR antibody, BK channel agonist, bortezomib, cardiac glycoside, cyclophosmide-based anticancer agent, A complex selected from the group consisting of GADD34/PP1 inhibitor, LV-tSMAC, Measles virus, bleomycin, mitoxantrone, oxaliplatin, and combinations thereof.
  11. 제 8항에 있어서, 항암제는 세포독성 항암제인, 복합체.The complex according to claim 8, wherein the anticancer agent is a cytotoxic anticancer agent.
  12. 제 6항의 복합체를 유효성분으로 포함하는 면역항암제.An immunotherapy agent comprising the complex of claim 6 as an active ingredient.
  13. 제 6항의 복합체를 유효성분으로 포함하는 M2 종양 관련 대식세포 매개 암의 예방 또는 치료용 약학적 조성물.A pharmaceutical composition for preventing or treating M2 tumor-related macrophage-mediated cancer, comprising the complex of claim 6 as an active ingredient.
  14. 제 6항의 복합체를 유효성분으로 포함하는 M2 종양 관련 대식세포 제거용 조성물.A composition for eliminating M2 tumor-related macrophages comprising the complex of claim 6 as an active ingredient.
  15. ITGB2 유전자 또는 상기 유전자로부터 발현된 단백질을 포함하는 고형암 진단용 마커.A marker for diagnosing solid tumors containing the ITGB2 gene or a protein expressed from the gene.
  16. ITGB2의 발현양을 mRNA 또는 단백질 수준에서 측정하는 제제를 포함하는, 고형암 진단용 조성물.A composition for diagnosing solid cancer, comprising an agent for measuring the expression level of ITGB2 at the mRNA or protein level.
  17. ITGB2 단백질에 후보 물질을 처리하는 단계; 및Processing the candidate material into the ITGB2 protein; and
    ITGB2 단백질과 결합하는 후보 물질을 선별하는 단계를 포함하는, 면역항암제를 스크리닝하는 방법.A method of screening an immunotherapy agent, comprising the step of selecting a candidate substance that binds to the ITGB2 protein.
  18. 면역항암제를 처리한 분리된 시료에서 ITGB2의 발현양을　확인하는 단계를 포함하는, M2 대식세포를 표적화하는 면역항암제에 대한 반응성 예측 또는 예후 진단 방법.A method for predicting or prognosticating reactivity to an immunotherapy agent targeting M2 macrophages, comprising the step of confirming the expression level of ITGB2 in a separated sample treated with an immunotherapy agent.
  19. 대상으로부터 분리된 시료에 후보 물질을 처리하는 단계; 상기 시료에서 ITGB2의 발현양을　확인하는 단계를 포함하는, M2 대식세포를 표적화하는 항암제를 스크리닝하는 방법.Processing a candidate material on a sample separated from the target; A method for screening an anticancer agent targeting M2 macrophages, comprising the step of confirming the expression level of ITGB2 in the sample.
  20. 대상으로부터 분리된 시료에서 ITGB2의 발현양을　확인하는 단계를 포함하는, M2 대식세포를 표적화하는 물질에 대한 반응성을 갖는 암에 걸린 대상을 분류하는 방법.A method of classifying a subject with cancer having reactivity to a substance targeting M2 macrophages, comprising the step of confirming the expression level of ITGB2 in a sample isolated from the subject.
  21. 대상으로부터 분리된 시료에서　ITGB2의 발현양을　확인하는 단계; 대조군과 ITGB2의 발현양을 비교하는 단계; 및 ITGB2가 대조군에 비해 과발현된 대상을 분류하는 단계를 포함하는, 암의 진단에 필요한 정보를 제공하는 방법.Confirming the expression level of ITGB2 in a sample isolated from the subject; Comparing the expression level of ITGB2 with the control group; And a method of providing information necessary for the diagnosis of cancer, including the step of classifying subjects in which ITGB2 is overexpressed compared to the control group.
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XU HOUSHI; ZHANG ANKE; HAN XIAYING; LI YANNING; ZHANG ZEYU; SONG LIYING; WANG WEI; LOU MEIQING: "ITGB2 as a prognostic indicator and a predictive marker for immunotherapy in gliomas", CANCER IMMUNOLOGY IMMUNOTHERAPY, SPRINGER, BERLIN/HEIDELBERG, vol. 71, no. 3, 27 July 2021 (2021-07-27), Berlin/Heidelberg , pages 645 - 660, XP037694336, ISSN: 0340-7004, DOI: 10.1007/s00262-021-03022-2 *

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