ITGB2-mediated drug delivery system

Abstract

The present invention relates to a drug delivery system that specifically binds to ITGB2 of M2 tumor-associated macrophages. The drug delivery system of the present invention, which contains an ITGB2 binding moiety that specifically binds to ITGB2 (Integrin beta 2), has the effect of reducing drug side effects and increasing the efficacy of drugs by acting selectively (or specifically) only on M2 tumor-associated macrophages, and can therefore be usefully used as a drug delivery system for cell death inducers, anticancer agents, contrast agents, etc.

Description

[Technical field]

[0001] The present invention relates to a drug delivery system that specifically binds to ITGB2. [Background technology]

[0002] Existing anti-cancer treatments have been studied in the direction of directly attacking cancer cells or enhancing the activity of immune cells in the body that attack cancer cells. However, these anti-cancer drugs also attack other normal cells in addition to cancer cells, causing many side effects such as hair loss, nausea, and vomiting, and causing additional reactions due to excessive increase in immune cells. Anti-cancer immunotherapy, which can minimize side effects compared to existing chemotherapy and radiation therapy, is a method of treating cancer using the body's immune system. Among these anti-cancer immunotherapy methods, cell therapy in which therapeutic immune cells such as T cells (including CAR-T), dendritic cells, and natural killer cells are activated outside the body and then directly injected into the body, and anti-cancer vaccine methods that increase anti-cancer efficacy by directly activating immune cells present in the body by injecting cancer antigens and immune activating substances into the body are being actively developed. However, such cell therapy agents and cancer vaccines are mainly used for blood cancer-related diseases, and most of them have a disadvantage that their therapeutic efficacy in solid cancers is extremely low. One of the reasons is due to microenvironmental factors that suppress immune function around solid tumors. In fact, in the tumor microenvironment, cells that suppress the function of immune cells (MDSC: myeoloid-derived stromal cells, Treg: regulatory T cells, TAM: tumor-associated macrophages), immunosuppressive cytokines, metabolites, etc. act actively, rapidly reducing the activity of immune activators and therapeutic immune cells. Therefore, it is important to develop therapeutic agents that have anti-cancer effects by regulating only the microenvironment around tumor cells without directly affecting tumor cells and immune cells, supplying tumor cells with effects, blocking angiogenesis around tumor cells, etc. The tumor microenvironment contributes to the proliferation and survival of malignant cells, angiogenesis, metastasis, abnormal adaptive immunity, hormones, and reduced response to chemotherapy drugs, and is greatly considered as a therapeutic target.

[0003] The tumor microenvironment is composed of endothelial cells, inflammatory cells and fibroblasts. In the 1970s, it was discovered that tumor-associated macrophages (TAMs) play an important role in tumor growth. TAMs are associated with and play an important role in the overall tumor microenvironment, including cancer growth and metastasis, and are classified into two phenotypes: tumor-suppressive M1 macrophages or tumor-supportive M2 macrophages. M1 macrophages have a strong ability to present antigens and are generally activated by interferon-γ, lipopolysaccharide (LPS) and tumor necrosis factor (TNF)-α, resulting in proinflammatory and bactericidal effects. M2 macrophages are known to promote immunosuppression, tumorigenesis and angiogenesis by releasing a variety of extracellular matrix components, angiogenic and chemotactic factors. Generally, M2-type tumor-associated macrophages are differentiated from M1-type tumor-associated macrophages by expressing markers such as arginase-1, mannose (MMR, CD206), scavenger receptor (SR-A, CD204), CD163, and IL-10, induced by IL-4 and IL-13. Tumor-associated macrophages present around tumors are closely related to the growth and metastasis of tumor cells, and it has been reported that the presence of a large number of M2-type tumor-associated macrophages around tumors in cancer patients leads to poor prognosis and survival rates for patients. 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-associated macrophages have been reported to suppress the anti-tumor activity of T cells and NK cells. In a microenvironment where M2-type tumor-associated macrophages are abundant, tumor growth, differentiation, and metastasis are actively carried out, so it is necessary to develop targeted therapeutic agents that target M2-type tumor-associated macrophages. Summary of the Invention [Problem to be solved by the invention]

[0004] An object of the present invention is to provide a drug delivery system that targets M2 macrophages.

[0005] It is also an object of the present invention to provide a pharmaceutical composition for preventing or treating cancer.

[0006] Another object of the present invention is to provide a method for screening a substance that specifically delivers a drug to M2 tumor-associated macrophages.

[0007] Another object of the present invention is to provide a method for selecting patients having responsiveness to an anticancer agent that targets M2 macrophages.

[0008] It is also an object of the present invention to provide a method for treating cancer.

[0009] Another object of the present invention is to provide a use of the drug delivery system for the manufacture of a pharmaceutical composition for the prevention and treatment of cancer. [Means for solving the problem]

[0010] To achieve the above object, the present invention provides a drug delivery system comprising an ITGB2-binding moiety and a drug bound thereto.

[0011] The present invention also provides a pharmaceutical composition for preventing or treating cancer.

[0012] The present invention also provides a method for screening a substance that specifically delivers a drug to M2 tumor-associated macrophages.

[0013] The present invention also provides a method for selecting patients having responsiveness to an anticancer agent that targets M2 macrophages.

[0014] The present invention also provides a method for treating cancer, comprising administering a pharma- ceutically effective amount of the drug delivery system of the present invention to a subject suffering from cancer.

[0015] In addition, the present invention provides an application of the drug delivery system of the present invention for the manufacture of a pharmaceutical composition for the prevention and treatment of cancer. Effect of the Invention

[0016] In the present invention, it has been confirmed that ITGB2 is a marker specific to M2 macrophage-mediated cancer, and a drug delivery system comprising an ITGB2 binding moiety that specifically binds to ITGB2 (Integrin beta 2) of the present invention has the effect of reducing drug side effects and enhancing the efficacy of drugs by acting selectively (or specifically) only on M2 tumor-associated macrophages, and can therefore be usefully used as a drug delivery system for cell death inducers, anticancer agents, contrast agents, etc. [Brief description of the drawings]

[0017] [Figure 1a] FIG. 1 shows the affinity of TAMpep for M2 macrophages confirmed by flow cytometry. [Figure 1b] This is a graph quantifying the affinity of TAMpep for M2 macrophages (*P<0.05 versus the M0.). [Figure 2a] FIG. 1 shows Venn diagram analysis of M2 / M0 and M1 / M0 ratios to identify proteins that bind to TAMpep in M2 macrophages. [Figure 2b] FIG. 1 shows the identification of proteins that bind to TAMpep in M2 macrophages by scatter plot analysis of M2 / M0 ratios. [Figure 2c] FIG. 1 shows the identification of proteins that bind to TAMpep in M2 macrophages by scatter plot analysis of M1 / M0 ratios. [Figure 2d] FIG. 1 shows scatter plot analysis of M2 / M1 ratios to identify proteins that bind to TAMpep in M2 macrophages. [Diagram 3]FIG. 1 shows LC-MS chromatogram data obtained by reacting cell membrane proteins extracted from M0, M1, and M2 macrophages with TAMpep826-biotin. [Figure 4] FIG. 1 shows the identification of target proteins of TAMpep826 using LC-MS / MS proteomics. [Diagram 5] FIG. 1 shows quantitative RT-PCR conditions. [Figure 6] FIG. 1 shows ITGB2 mRNA expression confirmed in M2 macrophages. [Figure 7] FIG. 1 shows the results of confirming ITGB2 protein expression in M2 macrophages. [Figure 8] FIG. 13 shows the expression of ITGB2 protein in M2 macrophages confirmed by immunofluorescence analysis. [Figure 9] FIG. 13 is a diagram confirming the expression of ITGB2 in the cell membrane of M2 macrophages. [Figure 10] FIG. 1 shows the interaction between TAMpep826 and ITGB2 in M2 macrophages confirmed by pull-down assay. [Figure 11] FIG. 11 shows the interaction of TAMpep826 and ITGB2 in M2 macrophages confirmed by immunofluorescence analysis. [Figure 12] The binding sites of TB511 and ITGB2 were confirmed by docking simulation. [Figure 13] The binding sites of TB511 and ITGB2 were confirmed by docking simulation. [Figure 14] FIG. 1 is a schematic diagram showing that the anti-CD18 antibody used to prepare the ITGB2-binding ADC of the present invention specifically binds to the activated structure (extended-open form) of CD18 in M2TAM. [Figure 15] This is a diagram confirming the immobilization of ITGB2 on the surface of a sensor chip. [Figure 16]FIG. 1 shows the binding strength between anti-ITGB2 antibody and ITGB2 protein confirmed by SPR. [Figure 17] This is a diagram showing the binding strength between Melittin-dKLA and ITGB2 protein confirmed by SPR. [Figure 18] This figure shows that ITGB2 mRNA expression was confirmed in M2 macrophages in an ITGB2 expression-suppressed cell model established by Crispr / cas9. [Figure 19] FIG. 13 shows the effect of Melittin-dKLA in reducing cell death by suppressing ITGB2 expression in M2 macrophages. [Figure 20] FIG. 13 shows the effect of suppressing ITGB2 expression on reducing induction of apoptosis by TB511 (TAMpep826-dKLA) in M2 macrophages. [Figure 21] FIG. 1 shows the effect of ITGB2 antibody in reducing induction of apoptosis by TB511 in M2 macrophages. [Figure 22] This figure confirms the effect of TB511 in breast cancer 3D organoids. [Diagram 23] This figure confirms the effect of TB511 in breast cancer 3D organoids. [Figure 24] This figure confirms the effect of TB511 on lung cancer 3D organoids. [Diagram 25] This figure confirms the effect of TB511 on lung cancer 3D organoids. [Figure 26] This figure confirms the effect of M-DM1 in lung cancer 3D organoids. [Figure 27] This figure confirms the effect of TB511 on anticancer drug-resistant lung cancer 3D organoids. [Figure 28] This figure confirms the effect of TB511 on anticancer drug-resistant lung cancer 3D organoids. [Figure 29] This figure confirms the effect of TB511 on anticancer drug-resistant prostate cancer 3D organoids. [Diagram 30]This figure confirms the effect of TB511 on anticancer drug-resistant prostate cancer 3D organoids. [Diagram 31] This figure confirms the effect of CD18-DM1 in breast cancer 3D organoids. [Diagram 32] This figure confirms the effect of CD18-MMAE in breast cancer 3D organoids. [Diagram 33] This figure confirms the effects of ADC (CD18-DM1 and CD18-MMAE) in lung cancer 3D organoids. [Diagram 34] This figure confirms the effects of ADC (CD18-DM1 and CD18-MMAE) in lung cancer 3D organoids. [Diagram 35] This figure confirms the effects of ADC (CD18-DM1 and CD18-MMAE) on anticancer drug-resistant lung cancer 3D organoids. [Diagram 36] This figure confirms the effects of ADC (CD18-DM1 and CD18-MMAE) on anticancer drug-resistant lung cancer 3D organoids. [Figure 37] This figure confirms the effects of ADC (CD18-DM1 and CD18-MMAE) on anticancer drug-resistant prostate cancer 3D organoids. [Figure 38] This figure confirms the effects of ADC (CD18-DM1 and CD18-MMAE) on anticancer drug-resistant prostate cancer 3D organoids. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0018] Hereinafter, the present invention will be described in detail with reference to the accompanying drawings. However, the following embodiments are presented as examples of the present invention, and if it is determined that a detailed description of a well-known technology or configuration may unnecessarily obscure the gist of the present invention, the detailed description may be omitted and the present invention is not limited thereby. The present invention is subject to various modifications and applications within the scope of the claims and the scope of equivalents interpreted therefrom.

[0019] In addition, the terminology used in this specification is used to appropriately express the preferred embodiment of the present invention, and may vary depending on the intention of the user or operator, or the practice of the field to which the present invention belongs. Therefore, the definition of the term should be based on the overall content of this specification. Throughout the specification, when a part "includes" a certain element, this does not exclude other elements, but means that the part may further include other elements, unless otherwise specified to the contrary.

[0020] All technical terms used in the present invention are used in the same meaning as commonly understood by those of ordinary skill in the art in the field of the present invention unless otherwise specified. In addition, although the present specification describes a suitable method or sample, similar or equivalent methods and samples are also included in the scope of the present invention. The contents of all publications mentioned as references in this specification are incorporated herein.

[0021] Throughout this specification, the usual one-letter and three-letter codes for naturally occurring amino acids are used, as well as commonly accepted three-letter codes for other amino acids, such as Aib (α-aminoisobutyric acid), Sar (N-methylglycine), etc. Also, in the present invention, the amino acids referred to as abbreviations are written according to the IUPAC-IUB nomenclature as follows: 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.

[0022] In one aspect, the present invention relates to a drug delivery system comprising an ITGB2 (Integrin beta 2) binding moiety; and a drug conjugated thereto.

[0023] In one embodiment, ITGB2 may comprise the amino acid sequence of SEQ ID NO:7 or SEQ ID NO:8.

[0024] In one embodiment, the ITGB2 binding portion can specifically bind to ITGB2 protein present on the cell membrane of M2 tumor-associated macrophages, and can include small molecules, viruses, antibodies, antibody fragments, aptamers, hormones, cytokines, chemokines, ligands, peptides that are partial regions of cytokines, or peptides that are partial regions of ligands that specifically bind to ITGB2.

[0025] In some embodiments, the binding moiety may further comprise a targeting sequence, tag, labeled residue or amino acid sequence designed for a specific purpose to increase half-life or stability.

[0026] In one embodiment, the ITGB2 binding moiety can specifically bind to the extended active conformation of CD18.

[0027] In one embodiment, the ITGB2 binding moiety can bind to amino acids 449-617 of ITGB2.

[0028] In one embodiment, the ITGB2 binding moiety can bind to amino acids 466 to 565 of ITGB2.

[0029] In one embodiment, the ITGB2 binding moiety may comprise melittin, a variant thereof, or an analog thereof, and may comprise the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:2.

[0030] In one embodiment, the drug delivery system may include one or more selected from the group consisting of amino acid sequences of SEQ ID NOs: 3-6.

[0031] In one embodiment, the drug delivery system may be a peptide-drug conjugate (PDC), melittin-dKLA having the amino acid sequence of SEQ ID NO: 5, TB511 having the amino acid sequence of SEQ ID NO: 6, or M-DM1 in which the drug DM1 is conjugated to maleimide-modified melittin.

[0032] In one embodiment, TB511 can bind to amino acids 466 to 565 of ITGB2, LEU(6) of TB511 can bind to the LEU(528), THR(538), LEU(556), CYS(557) and PHE(558) sites of ITGB2, and TRP(12) of TB511 can bind to the GLU(466), ILE(469), CYS(470) and ARG(471) sites of ITGB2.

[0033] In one embodiment, the ITGB2 binding portion can bind to an epitope comprising amino acids 732 to 748 (SEQ ID NO: 9), an epitope comprising amino acids 504 to 508, an epitope comprising amino acids 534 to 546, an epitope comprising amino acids 449 to 617, or an epitope comprising amino acids 23 to 700 (Gln23-Asn700) of ITGB2 (CD18).

[0034] In one embodiment, the ITGB2 binding moiety may be an antibody or an immunologically active fragment thereof that binds to the epitope.

[0035] In one embodiment, the immunologically active fragment may be any one selected from the group consisting of Fab, Fd, Fab', dAb, F(ab'), F(ab')2, scFv (single chain fragment variable), Fv, single chain antibody, Fv dimer, complementarity determining region fragment, humanized antibody, chimeric antibody and diabody.

[0036] In one embodiment, the drug delivery system may be an antibody-drug conjugate (ADC).

[0037] In one embodiment, the ITGB2 binding moiety and the drug of the drug delivery system can be covalently bonded via a linker or non-covalently bonded without the use of a linker.

[0038] In one embodiment, the ITGB2 binding moiety of the drug delivery system and the drug may be linked / conjugated via a linker. The linker may be any linker having a functional group that can be linked via an amine group, carboxyl group, or sulfhydryl group of a protein such as a peptide, ligand, antibody, or antibody fragment, or a phosphate group or hydroxyl group of a nucleic acid such as an aptamer.

[0039] In one embodiment, the linker can be appropriately selected depending on the drug. For example, a linker having an aldehyde reactive group can be linked to the drug and then bound to the N-terminal amino group of an antibody (cell targeting domain), which is the ITGB2 binding portion of the drug delivery system.

[0040] The functional group of such a linker may be an isothiocyanate, an isocyanate, an acyl azide, an NHS ester, a sulfonyl chloride, an aldehyde, a glyoxal, an epoxide, an oxirane, a carbonate, an arylhalide, an imidoester, a carbodiimide, an anhydride, a fluorophenyl ester, a hydroxymethyl phosphine, a maleimide, a haloacetyl, a pyridyldisulfide, a thiosulfonate, or a vinylsulfone. The linker may be a linker that is cleavable by a protease, cleavable under acid or base conditions, cleavable by high temperature or light irradiation, cleavable under reducing or oxidizing conditions, or may be a linker that is not cleavable under such conditions. Examples of cleavable linkers include hydrazone linkers that are cleaved under acidic conditions, peptide linkers that are cleaved by proteases, and linkers having a disulfide functional group that are cleaved under reducing conditions. Examples of non-cleavable linkers include maleimidomethyl cyclohexane-1-carboxylate (MCC) linkers, maleimidocaproyl (MC) linkers, and derivatives thereof such as succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sMCC) linkers and sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sulfo-sMCC).

[0041] The linker may be a self-immolative linker or a traceless linker after cleavage. Examples of the self-immolative linker include the linker disclosed in U.S. Pat. No. 9,089,614, entitled "Hydrophilic self-immolative linkers and conjugates thereof," and the linker disclosed in International Publication No. WO2015038426, entitled "SELF-IMMOLATIVE LINKERS CONTAINING MANDELIC ACID DERIVATIVES, DRUG-LIGAND CONJUGATES FOR TARGETED THERAPIES AND USES THEREOF." Examples of the traceless linker after cleavage include a phenylhydrazide linker, an aryl-triazene linker, and a linker disclosed in the literature [Blaney, et al., "Traceless solid-phase organic synthesis," Chem Rev. 102: 2607-2024 (2002)].

[0042] In addition to the above-mentioned linkers, many other linkers applicable to the present invention are known in the art through a number of publications, including Castaneda, et al., "Acid-cleavable thiomaleamic acid linker for homogeneous antibody-drug 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, and Dawson, et al., "Modulation of Reactivity in Native Chemical Ligation through the Use of Thiol Additives," J Am Chem Soc.1997, 119, 4325-4329] References [HackenG, et al "Protein synthesis by native chemical ligation:Expanded scope by using straightforward methodology," Proc Natl Acad Sci USA 1999, 96, 10068-10073] References [Wu, et al "Building complex glycopeptides:Development of a cysteine-free native chemical ligation protocol," Angew Chem Int Ed 2006, 45, 4116-4125], Reference [Geiser et al "Automation of solid-phase peptide synthesis" in Macromolecular Sequencing and Synthesis, Alan R Liss, Inc, 1988, pp 199-218], Reference [Fields, G and Noble, R (1990) "Solid phase peptide synthesis utilizing 9-fluoroenylmethoxycarbonyl amino acids”, Int J Peptide Protein Res 35:161-214], but U.S. Patent No. 6884869, U.S. Patent No. 7498298, U.S. Patent No. 8288352, U.S. Patent No. 8609105, U.S. Patent No. 8697688, U.S. Patent Application Publication No. 2014 / 0127239, U.S. Patent Application Publication No. 2013 / 028919, U.S. Patent Application Publication No. 2014 / 286970, Reference can be made to U.S. Patent Application Publication No. 2013 / 0309256, U.S. Patent Application Publication No. 2015 / 037360, U.S. Patent Application Publication No. 2014 / 0294851, International Publication No. 2015 / 057699, International Publication No. 2014 / 080251, International Publication No. 2014 / 197854, International Publication No. 2014 / 145090, International Publication No. 2014 / 177042, and the like.

[0043] The ITGB2 binding moiety and the drug of the drug delivery system of the present invention can be bound via a biocompatible polymer or a carrier. A biocompatible polymer means a polymer that has tissue compatibility and antithrombotic properties that does not cause tissue necrosis or blood coagulation when in contact with biological tissue or blood.

[0044] The synthetic polymers as the biocompatible polymers include polyesters, polyhydroxyalkanoates (PHAs), poly(α-hydroxyacids), poly(β-hydroxyacids), poly(3-hydroxybutyrate-co-valerate; PHBV), poly(3-hydroxyproprionate; PHP), poly(3-hydroxyhexanoate; PHH), poly(4-hydroxyacids), poly(4-hydroxybutyrate), poly(4-hydroxyvalerate), poly(4-hydroxyhexanoate), poly(esteramides), polycaprolactone, polylactides, polyglycolides, poly(lactide-co-glycolides;PLGA), polydioxanones, polyorthoesters, polyanhydrides, poly(glycolic acid-co-trimethylene carbonate), polyphosphoesters, polyphosphoester urethanes, poly(amino acids), polycyanoacrylates, poly(trimethylene carbonate), poly(iminocarbonates), poly(tyrosine carbonate), polycarbonates, poly(tyrosine arylates), polyalkylene oxalates, polyphosphazenes, PHA-PEGs, ethylene vinyl alcohol copolymers (EVOH), polyurethanes, silicones, polyesters, polyolefins, polyisobutylene and ethylene-alpha olefin copolymers, styrene-isobutylene-styrene triblock copolymers, acrylic polymers and copolymers, vinyl halide polymers and copolymers, polyvinyl chloride, polyvinyl ethers, polyvinyl methyl ethers, polyvinylidene halides, polyvinylidene fluoride, polyvinylidene chloride, polyfluoroalkenes, polyperfluoroalkenes, polyacrylonitriles, polyvinyl ketones, polyvinyl aromatics, polystyrenes, polyvinyl esters, polyvinyl acetates, ethylene-methyl methacrylate copolymers, acrylonitrile-styrene copolymers, ABS resins and ethylene-vinyl acetate copolymers, polyamides, alkyd resins, polyoxymethylenes, polyimides, polyethers, polyacrylates, polymethacrylates, polyacrylic acid-co-maleic acid or polyaminoamines, and the natural polymers are chitosan, dextran, cellulose, heparin, hyaluronic acid, alginates, inulin, starch or glycogen;

[0045] The linker used to bind the ITGB2-binding portion to the drug is preferably a linker that is stable outside the target cell and therefore not cleaved, and is also not cleaved in endosomes or lysosomes, which are acidic conditions in the target cell. Such a linker allows the drug to be transferred into the cell by being stable outside the target cell and not cleaved in endosomes or lysosomes, which are acidic conditions, and allows the drug to be transferred to the cytoplasm in endosomes or lysosomes by not being cleaved in endosomes or lysosomes, which are acidic conditions.

[0046] In the drug delivery system of the present invention, the drug can also be non-covalently bound to the ITGB2 binding moiety. For example, intercalator agents such as doxorubicin, a type of anticancer drug that exerts its effect by intercalating into nucleic acids, can be non-covalently bound to the aptamer in an intercalation manner when the aptamer is used as the cell targeting region of the drug delivery system. Since the aptamer is an oligonucleotide molecule, there is base stacking of nucleotide bases, and the drug can be bound between the base stacks in an intercalation manner.

[0047] In one embodiment, the ITGB2 binding moiety may be further conjugated with RNA, DNA, an antibody, an effector, a drug, a prodrug, a toxin, a peptide or a signaling molecule (see Shoari et al., Pharmaceutics 13:1391, pp.1-32 (2021)).

[0048] In the drug delivery system of the present invention, the drug is not particularly limited as long as it can move into cells and exert its effect. Such drugs may be any low molecular weight drug such as cytotoxic anticancer drugs, recombinant proteins, siRNAs, and other biopharmaceuticals. In addition, the drug may be an anti-inflammatory drug, analgesic drug, anti-arthritic drug, antispasmodic drug, antidepressant, antipsychotic drug, neuroleptic drug, anxiolytic drug, narcotic antagonist, antiparkinsonian drug, cholinergic agonist, anticancer drug, angiogenesis inhibitor, immunosuppressant, immunostimulant, antiviral drug, antibiotic, appetite suppressant, analgesic drug, anticholinergic drug, antihistamine, antimigraine drug, hormone drug, coronary blood vessel, vasodilator, antiseptic drug, antithrombotic drug, diuretic drug, antihypertensive drug, cardiovascular disease treatment drug, diagnostic agent such as contrast agent, etc.

[0049] In one embodiment, the drug may be a chemical compound, RNA, DNA, an antibody, an effector, a prodrug, a toxin, a peptide, or a radionuclide.

[0050] In one embodiment, the drug may be an immunogenic cell death inducer, a pro-apoptotic peptide, a microtubulin structure formation inhibitor, a mitosis inhibitor, a topoisomerase inhibitor, a DNA intercalator, a toxin, or an anticancer drug.

[0051] In one embodiment, the pro-apoptotic peptide may be selected from the group consisting of KLA, alpha-defensin-1, BMAP-28, Brevenin-2R, Buforin IIb, Cecropin A-Magainin 2 (CA-MA-2), Cecropin A, Cecropin B, chrysophsin-1, D-K6L9, Gomesin, Lactoferricin B, LLL27, LTX-315, Magainin 2, Magainin II-bombesin conjugate (MG2B), Pardaxin, and combinations thereof.

[0052] In one embodiment, the immunogenic cell death inducer may be selected from the group consisting of anthracycline anticancer drugs, taxane anticancer drugs, anti-EGFR antibodies, BK channel agonists, bortezomib, cardiac glycosides, cyclophosphamide anticancer drugs, GADD34 / PP1 inhibitors, LV-tSMAC, Measles virus, bleomycin, mitoxantrone, oxaliplatin, and combinations thereof.

[0053] In one embodiment, the anticancer drug is SN-38 (7-Ethyl-10-hydroxy-camptothecin), daunorubicin, doxorubicin, epirubicin, idarubicin, pixantrone, sabarubicin, valrubicin, paclitaxel, docetaxel, mechloethamine, clopidogrel, or the like. Chlorambucil, phenylalanine, mustard, cyclophosphamide, ifosfamide, carmustine (BCNU), lomustine (CCNU), streptozotocin, busulfan, thiotepa, cisplatin, carboplatin, dactinomycin (actinomycin D), plicamycin, mitomycin C, vincristine, vinblastine, teniposide, topotecan, irinotecan, uramustine, melphalan, bendamustine, dacarbazine, temozolomide, altretamine, duocarmycin, nedaplatin, oxaliplatin, satraplatin, triplatin tetranitratetetranitrate, 5-fluorouracil, 6-mercaptopurine, capecitabine, cladribine, clofarabine, cystarbine, floxuridine, fludarabine, gemcitabine, hydroxyurea, methotrexate, pemetrexate The agent may be selected from the group consisting of pemetrexed, pentostatin, thioguanine, etoposide, mitoxantrone, ixabepilone, vindesine, vinorelbine, estramustine, maytansine, DM1 (mertansine), DM4, dolastatin, auristatin E, auristatin F, monomethyl auristatin E (MMAE), monomethyl auristatin F and derivatives thereof, and may be DM1 or MMAE.

[0054] In the present invention, the drug is preferably a cytotoxic anticancer drug. Cytotoxic anticancer drugs can be broadly classified into antimetabolites, microtubulin targeting agents (tubulin polymerase inhibitors and tubulin depolymerisation), alkylating agents, antimitotic agents, DNA cleavage agents, DNA cross-linker agents, DNA intercalator agents, DNA topoisomerase inhibitors, etc., and examples of antimetabolites include folic acid such as methotrexate. Examples of microtubule targeting agents well known in the art include auristatin drugs such as monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), dolastatin, and maytansines. Examples of alkylating agents well known in the art include alkyl sulfonate preparations such as busulfan and treosulfan, nitrogen mustard derivatives such as bendamustine, and platinum preparations such as cisplatin and heptaplatin.In addition, as the antimitotic agents, taxane preparations such as docetaxel and paclitaxel, vinca alkaloids such as vinflunine, podophyllotoxin derivatives such as etoposide, etc. are known in the art, as the DNA cleavage agents, calicheamicins, etc. are known, as the DNA cross-linker agents, PBD dimers, etc. are known, as the DNA intercalators, doxorubicin, etc. are known in the art, and as the DNA topoisomerase inhibitors, SN-28, etc. are known in the art.

[0055] Furthermore, the drug may be a gene, a plasmid DNA, an antisense oligonucleotide, an siRNA, a peptide, a ribozyme, a virus particle, an immunomodulator, a protein, a contrast agent, etc. More specifically, the drug may be a gene encoding Rb94, which is a mutant of the retinoblastoma tumor suppressor gene, a gene encoding apoptin that induces apoptosis only in tumor cells, an antisense oligonucleotide (sequence: 5'-TCC ATG GTG CTC ACT-3') against HER-2 or the like, which is a therapeutic target, or a diagnostic contrast agent such as Gd-DTPA, which is an MRI contrast agent.

[0056] The conjugate of the drug delivery system and the drug of the present invention can be prepared into a pharmaceutical composition in the form of an oral dosage form or a parenteral dosage form according to the administration route by a conventional method known in the art, including a pharma- ceutically acceptable carrier. Here, the term "pharma- ceutically acceptable carrier" refers to a carrier or diluent that does not stimulate the organism and does not inhibit the biological activity and properties of the administered compound. In the composition formulated in a liquid solution, acceptable pharmaceutical carriers include saline, sterile water, Ringer's solution, buffered saline, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, ethanol, and a mixture of one or more of these components, which are sterile and suitable for the body, and other conventional additives such as antioxidants, buffers, and bacteriostatic agents can be added as necessary.

[0057] In one embodiment, the drug delivery system of the present invention can specifically deliver the drug to M2 tumor-associated macrophages.

[0058] In the present invention, the ITGB2-binding portion provides a targeting function by enabling selective binding through binding to ITGB2 of target cells, i.e., M2 tumor-associated macrophages. This ITGB2-binding portion specifically binds to ITGB2 present on the surface of target cells and induces endocytosis, thereby enabling the transfer of the drug bound to it into the cells.

[0059] In the present invention, antibodies, aptamers, hormones (e.g., erythropoietin hormone), cytokines and chemokines (e.g., IL13) that are secreted from specific cells and act on surface receptors of other cells that are target cells, thereby playing a role in signal transduction between cells, ligands that are biological molecules such as VEGF (vascular endothelial growth factor) and BDNF (brain-derived neurotrophic factor) that bind to target cell surface receptors, and peptides that are partial regions of these factors that have specific binding ability to receptors can be used as such ITGB2 binding moieties. Typically, an antibody or an aptamer can be used, and the antibody as the ITGB2 binding portion can be not only a monoclonal antibody or a polyclonal antibody, but also a multispecific antibody (i.e., an antibody that recognizes two or more antigens or two or more epitopes, such as a bispecific antibody), or any antibody, such as an antibody fragment, a chemically modified antibody, a chimeric antibody (human-mouse chimeric antibody, human-monkey chimeric antibody, etc.), a humanized antibody with reduced immunogenicity, or a human antibody, as long as it has the ability to specifically bind to ITGB2. In addition, 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), Fv, single chain antibody, Fv dimer, complementarity determining region fragment, humanized antibody, chimeric antibody, and diabody, as well as antibody fragments obtained by treating an antibody with a protein-cleaving enzyme, so-called papain or pepsin.

[0060] With regard to a method for producing an antibody, an antibody in which a natural antibody is artificially modified to improve its specificity for a target antigen or improve its immunogenicity, etc., various documents known in the art, such as U.S. Pat. No. 4,444,887, U.S. Pat. No. 4,716,111, U.S. Pat. No. 5,545,806, U.S. Pat. No. 5,814,318, WO 98 / 46645, WO 98 / 50433, WO 98 / 24893, WO 98 / 16654, WO 96 / 34096, WO 96 / 33735, the document [Protein Eng 1994, 7(6):805-814], the document [Proc Natl Acad Sci USA 1994, 91:969-973], etc. may be referred to.

[0061] The aptamer as the cell targeting region may be a single-stranded DNA aptamer or a single-stranded RNA aptamer. An aptamer means a nucleic acid ligand capable of specifically binding to a target molecule such as a target antigen, as with an antibody, but the aptamer may be a double-stranded DNA or RNA aptamer as long as it can specifically bind to a target molecule. The production and selection methods of 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, materials, etc., reference can be made to literature [Methods Enzymol 267:275-301, 1996] and literature [Methods Enzymol 318:193-214, 2000]. The aptamer may be modified with sugar, phosphate, and / or base to improve the half-life in vivo. These sugar, phosphate, and / or base-modified nucleotides are specifically known in the art, including their production methods. For example, sugar-modified nucleotides include those in which the hydroxyl group (OH group) of the sugar is modified with a halogen group, an aliphatic group, an ether group, an amine group, etc., and those in which the sugar ribose or deoxyribose itself is replaced with a sugar analogue that can substitute for it, such as α-anomeric sugars, epimeric sugars such as arabinose, xylose, or lyxose, pyranose sugars, furanose sugars, etc. In addition, for example, modifications at the phosphate include those in which the phosphate is modified to P(O)S (thioate), P(S)S (dithioate), P(O)NR2 (amidate), P(O)R, P(O)OR', CO, or CH2 (formacetal). Here, R or R' is H or a substituted or unsubstituted alkyl, etc., and when modified with phosphate, the linking group becomes -O-, -N-, -S- or -C-, and adjacent nucleotides are bonded to each other via such linking group.

[0062] The drug delivery system of the present invention specifically and directly binds to ITGB2 expressed on the cell membrane of M2 tumor-associated macrophages, particularly ITGB2 (CD18) in the active conformation in M2TAM, thereby enabling the target molecule ITGB2 to be expressed without the side effects of non-selective drug effects, and enabling the drug effect to be selectively exerted only on activated M2 tumor-associated macrophages.

[0063] In one aspect, the present invention relates to a peptide-drug conjugate (PDC) comprising an ITGB2-binding moiety comprising melittin, a variant thereof, or an analog thereof; and a drug conjugated thereto.

[0064] In one embodiment, the PDC may bind to amino acids 466 to 565 of ITGB2, and may bind to LEU(528), THR(538), LEU(556), CYS(557), PHE(558), GLU(466), ILE(469), CYS(470) or ARG(471) of ITGB2.

[0065] In one embodiment, the PDC may be Melittin-dKLA having the amino acid sequence of SEQ ID NO: 5, TB511 having the amino acid sequence of SEQ ID NO: 6, or M-DM1 in which the drug DM1 is conjugated to maleimide-modified melittin.

[0066] In one embodiment, TB511 can bind to amino acids 466 to 565 of ITGB2, LEU(6) of TB511 can bind to the LEU(528), THR(538), LEU(556), CYS(557) and PHE(558) sites of ITGB2, and TRP(12) of TB511 can bind to the GLU(466), ILE(469), CYS(470) and ARG(471) sites of ITGB2.

[0067] In one aspect, the present invention relates to an antibody-drug conjugate (ADC) comprising an antibody that binds to ITGB2 or a fragment thereof having immunological activity, and a drug bound thereto.

[0068] In one embodiment, the ADC may bind to an epitope comprising amino acids 732-748, an epitope comprising amino acids 504-508, an epitope comprising amino acids 534-546, an epitope comprising amino acids 449-617, or an epitope comprising amino acids 23-700 of ITGB2.

[0069] In one embodiment, the ADC may further include an antibody-drug conjugate (ADC) linker, and the ADC linker may be 6-maleimidocaproyl (MC), maleimidopropanoyl (MP), valine-citrulline (val-cit), alanine-phenylalanine (ala-phe), p-aminobenzyloxycarbonyl (PAB), N-succinimidyl 4-(2-pyridylthio)pentanoate (SPP), N-succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), valine-citrulline-p-aminobenzyloxycarbonyl (val-cit-PAB), or N-succinimidyl (4-iodo-acetyl)aminobenzoate (SIAB).

[0070] In one embodiment, the antibody-drug conjugate may comprise an anti-CD18 antibody or a fragment thereof having immunological activity conjugated to a drug via an ADC linker.

[0071] The antibody includes not only whole antibody forms, but also functional fragments of antibody molecules. A whole antibody has a structure having two full-length light chains and two full-length heavy chains, and each light chain is linked to a heavy chain by a disulfide bond. A functional fragment of an antibody molecule means a fragment that retains an antigen-binding function, and examples of antibody fragments include: (i) a Fab fragment consisting of a light chain variable region (VL), a heavy chain variable region (VH), a light chain constant region (CL), and a first heavy chain constant region (CH1); (ii) an Fd fragment consisting of a VH and CH1 domain; (iii) an Fv fragment consisting of the VL and VH domains of a single antibody; and (iv) a dAb fragment consisting of a VH domain (Ward ES et al., Nature 2003, 144:1311-1315, 2003). 341:544-546 (1989)]; (v) isolated CDR regions; (vi) a F(ab')2 fragment, which is a bivalent fragment comprising two linked Fab fragments; (vii) a single-chain Fv molecule (scFv) joined by a peptide linker which joins the VH and VL domains to form an antigen-binding site; (viii) bispecific single-chain Fv dimers (PCT / US92 / 09965) and (ix) diabodies, which are multivalent or multispecific fragments produced by gene fusion (WO94 / 13804).

[0072] The antibody or immunologically active fragment thereof of the present invention may be selected from the group consisting of animal-derived antibodies, chimeric antibodies, humanized antibodies, human antibodies, and immunologically active fragments thereof. The antibody may be recombinantly or synthetically produced.

[0073] Antibodies derived from animals, which are produced by immunizing a target animal with a desired antigen, may generally cause immune rejection when administered to humans for therapeutic purposes. Chimeric antibodies have been developed to suppress such immune rejection. Chimeric antibodies are produced by replacing the constant region of an animal-derived antibody, which causes anti-isotype reactions, with the constant region of a human antibody using genetic optical techniques. Chimeric antibodies have improved the anti-isotype reaction to a large extent compared to antibodies derived from animals, but they still contain amino acids derived from animals in the variable region, which means that they have potential side effects due to anti-idiotypic reactions. Humanized antibodies have been developed to improve such side effects. They are produced by grafting the CDR (complementarity determining regions) site, which plays an important role in antigen binding, in the variable region of a chimeric antibody onto a human antibody framework.

[0074] In the CDR grafting technology for producing humanized antibodies, the most important thing is to select an optimized human antibody that can best accept the CDR site of the animal-derived antibody, and for this purpose, antibody databases, crystal structure analysis, molecular modeling technology, etc. are utilized. However, even if the CDR site of the animal-derived antibody is grafted onto the optimized human antibody framework, there are cases where the antigen binding cannot be preserved because there are cases where amino acids that affect antigen binding while located in the framework of the animal-derived antibody are present, and therefore it can be said that the application of further antibody engineering technology to restore antigen binding ability is essential.

[0075] The antibody or a fragment thereof having immunological activity may be one isolated from a living body (not present in a living body) or one that is non-naturally occurring, or may be one that is synthetically or recombinantly produced.

[0076] In the present invention, the term "antibody" refers to a substance produced in the immune system by the stimulation of an antigen, and the type of the substance is not particularly limited and may be obtained naturally or non-naturally (i.e., synthetically or recombinantly). Antibodies are highly stable and have a long half-life not only in vitro but also in vivo, making them advantageous for mass expression and production. In addition, antibodies essentially have a dimer structure, and therefore have very high avidity. A complete antibody has a structure having two full-length light chains and two full-length heavy chains, and each light chain is linked to a heavy chain by a disulfide bond. The constant region of an antibody is divided into a heavy chain constant region and a light chain constant region, the heavy chain constant region has gamma (γ), mu (μ), alpha (α), delta (δ) and epsilon (ε) types, with gamma 1 (γ1), gamma 2 (γ2), gamma 3 (γ3), gamma 4 (γ4), alpha 1 (α1) and alpha 2 (α2) subclasses, and the light chain constant region has kappa (κ) and lambda (λ) types. In the present invention, the term "heavy chain" refers to a variable region domain V that contains an amino acid sequence having sufficient variable region sequence to confer specificity to an antigen. H and three constant region domains C H1 , C H2 and C H3 The term "light chain" is intended to include a full-length heavy chain, including the heavy chain and hinge, and a fragment thereof. The term "light chain" is intended to include a variable region domain V that contains an amino acid sequence having sufficient variable region sequence to confer specificity to an antigen. L and constant region domain C L It is to be understood as meaning to include all full length light chains and fragments thereof.

[0077] In the present invention, the term "variable region" or "variable domain" refers to a portion of an antibody molecule that exhibits many variations in sequence while performing the function of specifically binding to an antigen, and the variable region contains complementarity determining regions CDR1, CDR2, and CDR3. Between the CDRs, there is a framework region (FR) portion that plays a role in supporting the CDR ring. The "complementarity determining region" is a cyclic region involved in antigen recognition, and the specificity of the antibody for the antigen is determined as the sequence of this region changes.

[0078] In the present invention, the term "single chain fragment variable (scFv)" refers to a single chain antibody produced by expressing only the variable region of an antibody through genetic recombination, and refers to an antibody in a single chain form in which the VH and VL regions of the antibody are linked by a short peptide chain. The term "scFv" includes scFv fragments including antigen-binding fragments, unless otherwise specified or understood differently in the context. This is obvious to those skilled in the art.

[0079] In the present invention, the term "complementarity determining region (CDR)" refers to the amino acid sequence of the hypervariable region of the heavy and light chains of an immunoglobulin. The heavy and light chains can each contain three CDRs (CDRH1, CDRH2, CDRH3 and CDRL1, CDRL2, CDRL3). The CDRs can provide key contact residues in binding of an antibody to an antigen or an antigenic determinant site.

[0080] In the present invention, the terms "specifically bind" and "specifically recognize" have the same meaning as commonly known to those skilled in the art, that is, an antigen and an antibody specifically interact with each other and undergo an immunological reaction.

[0081] In the present invention, the term "antigen-binding fragment" refers to a portion of a polypeptide that is a fragment of the entire immunoglobulin structure and contains a portion to which an antigen can bind. For example, it may be, but is not limited to, scFv, (scFv)2, scFv-Fc, Fab, Fab', or F(ab')2. Among the antigen-binding fragments, Fab contains the variable regions of the light chain and heavy chain, the constant region of the light chain, and the first constant region of the heavy chain (C H1 ) has one antigen-binding site. Fab' has a heavy chain C H1 It is different from Fab in that it has a hinge region containing one or more cysteine ​​residues at the C-terminus of the domain. F(ab')2 antibody is generated by disulfide bonding between cysteine ​​residues in the hinge region of Fab'. Fv is the smallest antibody fragment having only a heavy chain variable region and a light chain variable region, and recombinant techniques for generating Fv fragments are widely known in the art. In a two-chain Fv, the heavy chain variable region and the light chain variable region are linked by a non-covalent bond, and in a single-chain Fv, the heavy chain variable region and the single chain variable region are generally linked by a covalent bond or directly at the C-terminus via a peptide linker, so that it can form a dimer-like structure like a two-chain Fv. The linker may be a peptide linker consisting of any amino acid of 1 to 100 or 2 to 50, and suitable sequences are known in the art. The antigen-binding fragments can be obtained using proteolytic enzymes (for example, whole antibodies can be subjected to limited cleavage with papain to obtain Fab fragments, or pepsin to obtain F(ab')2 fragments) or can be produced through recombinant gene techniques.

[0082] In the present invention, the term "hinge region" refers to a region contained in the heavy chain of an antibody, H1 and C H2It means a region that exists between regions and functions to provide flexibility to the antigen-binding site in the antibody. The so-called hinge may be derived from a human antibody, specifically, from IgA, IgE, or IgG, so-called IgG1, IgG2, IgG3, or IgG4.

[0083] The antibody or immunologically active fragment of the present invention can be produced by any method known in the art. After coding the polypeptide sequence of the antibody or immunologically active fragment of the present invention, it is used to form a nucleic acid that can be cloned into a host cell, expressed, and assayed, if desired. Various methods for this are described in the literature (Molecular Cloning-A Laboratory Manual, 3rd Ed., Maniatis, Cold Spring Harbor Laboratory Press, New York, 2001; Current Protocols in Molecular Biology, John Wiley & Sons).

[0084] The nucleic acid encoding the antibody or immunologically active fragment of the present invention can be inserted into an expression vector for protein expression. The expression vector usually contains a protein operably linked, i.e., in a functional relationship, with a regulatory or control sequence, a selection marker, any fusion partner, and / or additional elements. The antibody or immunologically active fragment of the present invention can be produced by culturing a host cell transformed with a nucleic acid, preferably an expression vector containing a nucleic acid encoding the antibody or immunologically active fragment of the present invention, under appropriate conditions, and inducing protein expression. A variety of suitable host cells can be used, including, but not limited to, mammalian cells, bacteria, insect cells, and yeast. Methods for introducing exogenous nucleic acid into a host cell are known in the art and may vary depending on the host cell used. Preferably, the antibody or immunologically active fragment of the present invention is produced using E. coli, which has low production costs and high industrial utility.

[0085] In one aspect, the present invention relates to a chimeric antigen receptor (CAR) comprising, as an antigen-binding domain, an antibody that binds to ITGB2 of the present invention or a fragment thereof having immunological activity.

[0086] In one aspect, the present invention relates to a recombinant vector comprising a gene encoding the chimeric antigen receptor.

[0087] In one aspect, the present invention relates to a chimeric antigen receptor-expressing cell transformed with the recombinant vector.

[0088] In one embodiment, the chimeric antigen receptor-expressing cell may be a chimeric antigen receptor-expressing macrophage (CAR-macrophage), a chimeric antigen receptor-expressing T (CAR-T) cell, a chimeric antigen receptor-expressing gamma-delta T (CAR-Gamma-delta T) cell, or a natural killer (CAR-NK) cell.

[0089] In the present invention, the term "CAR (chimeric antigen receptor)" refers to a non-naturally occurring receptor that can confer specificity to a particular antigen to an immune effector cell. Usually, the CAR refers to a receptor used to transfer the specificity of a monoclonal antibody to a T cell. CAR is generally composed of an extracellular domain (Ectodomain), a transmembrane domain, and an intracellular domain (Ectodomain).

[0090] The extracellular domain includes an antigen-binding site, and the transmembrane domain of the CAR may be derived from a natural or synthetic source in a form linked to the extracellular domain. When derived from a naturally occurring source, it may be derived from a membrane-bound or membrane-permeable protein, and may be a portion derived from the membrane-permeable region of various proteins such as the alpha, beta or zeta chain of the T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CDS, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154 or CD8. The sequence of such a transmembrane domain can be obtained from, but is not limited to, documents known in the art that are known to include the membrane-permeable region portion of a membrane-permeable protein.

[0091] Furthermore, when the transmembrane domain is synthetic, it may mainly contain hydrophobic amino acid residues such as leucine and valine, for example, a triplet of phenylalanine, tryptophan and valine may be present in the synthetic transmembrane domain, but is not limited thereto. Sequence information for such a transmembrane domain may be obtained from, but is not limited to, literature known in the art for synthetic transmembrane domains.

[0092] In the CAR of the present invention, the intracellular domain is a part of the domain of the CAR present in the cell, and is linked to the transmembrane domain. The intracellular domain of the present invention can include an intracellular signaling domain that is characterized by causing T cell activation, preferably T cell proliferation, when an antigen binds to the antigen-binding site of the CAR. The intracellular signaling domain is not particularly limited in type as long as it is a part that transmits a signal that can cause T cell activation when an antibody binds to an antigen-binding site present outside the cell, and various types of intracellular signaling domains can be used, for example, an immunoreceptor tyrosine-based activation motif (Tyrosine-based activation motif) or ITAM, and the ITAM includes, but is not limited to, those derived from CD3 zeta (ξ, zeta), FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CDS, CD22, CD79a, CD79b, CD66d, or FcεRIγ.

[0093] CARs contain a single-chain fragment variable portion (scFv) of an antibody specific for a tumor-associated antigen (TAA) coupled to the cytoplasmic domain of a T-cell signaling molecule via a hinge and transmembrane region. Most common lymphocyte activation moieties contain a T-cell costimulatory (e.g., CD28, CD137, OX40, ICOS, and CD27) domain in tandem with a T-cell triggering (e.g., CD3ζ) moiety. CAR-mediated adoptive immunotherapy allows CAR-implanted cells to directly recognize the TAA on target tumor cells in a non-HLA-restricted manner.

[0094] In the present invention, the term "chimeric antigen receptor-expressing T (CAR-T) cells" refers to T cells expressing CAR. The chimeric antigen receptor-expressing T (CAR-T) cells have the advantages of i) being able to treat cancers that avoid the action of anticancer drugs by reducing HLA expression on the cell surface since they recognize cancer antigens in an HLA (human leukocyte antigen)-independent manner, ii) being able to be used in treatment regardless of the patient's HLA type since they are unrelated to HLA type, and iii) being able to produce a large amount of cancer-specific T cells in a short period of time, thereby exhibiting excellent anticancer effects.

[0095] The T cells are CD4 + T cells (helper T cells, TH cells), CD8 + T cells (cytotoxic T cells, CTL), memory T cells, regulatory T cells (Treg cells), natural killer T cells, etc., and in the present invention, the T cells into which the CAR is introduced are preferably CD8 + Examples include, but are not limited to, T cells.

[0096] In one embodiment, the present invention relates to a T-cell engager comprising an antibody or an immunologically active fragment thereof that binds to ITGB2.

[0097] In one embodiment, the T cell engager may be, for example, a bispecific T-cell engager (BiTE). BiTE is a class of artificial bispecific monoclonal antibodies, which are fusion proteins composed of amino acid sequences from four different genes or two single chain variable fragments (scFv) of different antibodies on a single peptide chain of about 55 kilodaltons. One of the scFvs binds to T cells via the CD3 receptor, and the other binds to tumor cells through a tumor-specific molecule. Similar to other bispecific antibodies, but unlike conventional monoclonal antibodies, BiTE forms a link between T cells and tumor cells. It allows T cells to exert cytotoxic activity on tumor cells independent of the presence of MHC I or costimulatory molecules by producing proteins such as perforin and granzymes. These proteins enter tumor cells and initiate cell death.

[0098] In one aspect, the present invention relates to a pharmaceutical composition for preventing or treating cancer, comprising the drug delivery system, peptide-drug conjugate or antibody-drug conjugate of the present invention as an active ingredient.

[0099] In one embodiment, the drug delivery system of the present invention relates to an immunological anticancer agent comprising a peptide-drug conjugate or an antibody-drug conjugate as an active ingredient.

[0100] In one embodiment, the cancer may be M2 tumor-associated macrophage-mediated cancer.

[0101] In one embodiment, the composition may be administered to a subject in which expression of ITGB2 is upregulated in M2 macrophages compared to M0 and M1 macrophages.

[0102] In one embodiment, the cancer is brain cancer, melanoma, myeloma, non-small cell lung cancer, oral cancer, liver cancer, gastric cancer, colon cancer, breast cancer, triple negative breast cancer (TNBC), lung cancer, bone cancer, pancreatic cancer, skin cancer, head or neck cancer, cervical cancer, ovarian cancer, colon cancer, small intestine cancer, rectal cancer, fallopian tube cancer, anal cancer, endometrial cancer, vaginal cancer, vulvar cancer, Hodgkin's disease, The cancer may be any one selected from the group consisting of esophageal cancer, lymph node cancer, bladder cancer, gallbladder cancer, endocrine gland cancer, thyroid cancer, parathyroid cancer, adrenal gland cancer, soft tissue sarcoma, urethral cancer, penile cancer, prostate cancer, chronic or acute leukemia, lymphocytic lymphoma, kidney or ureter cancer, renal cell carcinoma, renal pelvic cancer, central nervous system tumor, primary central nervous system lymphoma, spinal cord tumor, brain stem glioma, and pituitary adenoma, or may be an anticancer drug resistant cancer.

[0103] The pharmaceutical composition of the present invention can be used as a single therapy, but can also be used together with other conventional biological therapy, chemotherapy, or radiation therapy, and such concurrent therapy can be used to treat cancer more effectively. When the present invention is used for the prevention and treatment of cancer, chemotherapeutic agents that can be used together with the composition include cisplatin, carboplatin, procarbazine, mechlorethamine, cyclophosphamide, ifosfamide, melphalan, chlorambucil, busulfan, nitrosourea, dactinomycin, and the like. cin, daunorubicin, doxorubicin, bleomycin, plicamycin, mitomycin, etoposide, tamoxifen, taxol, transplatinum, 5-fluorouracil, vincristine, vinblastin, and methotrexate, etc. Radiation therapies that can be used with the compositions of the present invention include X-ray irradiation and gamma-ray irradiation, etc.

[0104] In the present invention, the term "prevention" refers to any action that inhibits or delays the onset, spread and recurrence of cancer by administering a composition according to the present invention.

[0105] The therapeutically effective amount of the composition of the present invention may vary depending on various factors, such as the administration method, the target site, and the condition of the patient. Therefore, the dosage when used in the human body should be determined as an appropriate amount, taking into consideration both safety and efficacy. It is also possible to estimate the amount used in humans from the effective amount determined through animal experiments. Such matters to be considered when determining the effective amount are described, for example, in Hardman and Limbird, eds., Goodman and Gilman's The Pharmacological Basis of Therapeutics, 10th ed.(2001), Pergamon Press; and EWMartin ed., Remington's Pharmaceutical Sciences, 18th ed.(1990), Mack Publishing Co.

[0106] The pharmaceutical composition of the present invention is administered in a pharmacologic effective amount. The term "pharmacologic effective amount" used in the present invention means an amount sufficient to treat a disease with a reasonable benefit / risk ratio applicable to medical treatment and without causing side effects, and the effective dose level can be determined by factors including the patient's health condition, type and severity of the disease, activity of the drug, sensitivity to the drug, administration method, administration time, administration route and excretion rate, treatment period, co-administered or co-used drugs, and other factors known in the medical field. The composition of the present invention can be administered as an individual therapeutic agent or in combination with other therapeutic agents, can be administered sequentially or simultaneously with conventional therapeutic agents, and can be administered in a single dose or multiple doses. Taking into account all of the above factors, it is important to administer an amount that provides maximum effect with minimal amount without side effects, which can be easily determined by those skilled in the art.

[0107] The pharmaceutical composition of the present invention may contain carriers, diluents, excipients, or combinations of two or more thereof that are commonly used in biological preparations. The term "pharmaceutical acceptable" as used in the present invention means that the composition exhibits properties that are not toxic 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, and may be, for example, a compound listed in the Merck Index, 13th ed., Merck & Co. Inc., saline, sterile water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol, or a mixture of one or more of these components, and other common additives such as antioxidants, buffers, bacteriostatic agents, etc. may be added as necessary. In addition, the composition may be formulated into an injectable dosage form such as an aqueous solution, suspension, emulsion, etc., pills, capsules, granules, or tablets by additionally adding a diluent, dispersant, surfactant, binder, and lubricant. Furthermore, the composition can be formulated appropriately depending on each disease or ingredient by using a suitable method in the art or the method disclosed in Remington's Pharmaceutical Sciences (Mack Publishing Company, Easton PA, 18th, 1990).

[0108] In one embodiment, the pharmaceutical composition may be in one or more dosage forms selected from the group including oral dosage forms, topical preparations, suppositories, sterile injection solutions, and sprays.

[0109] The composition of the present invention may also contain carriers, diluents, excipients, or combinations of two or more thereof that are commonly used in biological preparations. The pharma-ceutically acceptable carrier is not particularly limited as long as it is suitable for in vivo delivery of the composition, and may be, for example, a compound described in the Merck Index, 13th ed., Merck & Co. Inc., saline solution, sterile water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol, or one or more of these components may be mixed and used, and other common additives such as antioxidants, buffers, bacteriostatic agents, etc. may be added as necessary. In addition, diluents, dispersants, surfactants, binders, and lubricants may be added to the composition to form an injection preparation such as an aqueous solution, suspension, emulsion, etc., pills, capsules, granules, or tablets. Furthermore, the composition may be suitably formulated according to each disease or component by using a method appropriate in the art or a method disclosed in Remington's Pharmaceutical Science (Mack Publishing Company, Easton PA, 18th, 1990).

[0110] The composition of the present invention may further contain one or more active ingredients exhibiting the same or similar functions. The composition of the present invention contains the above protein in an amount of 0.0001 to 10% by weight, preferably 0.001 to 1% by weight, based on the total weight of the composition.

[0111] The pharmaceutical composition of the present invention may further contain a pharma- ceutically acceptable additive, and examples of the pharma-ceutically acceptable additive include starch, gelatinized starch, microcrystalline cellulose, lactose, povidone, colloidal silicon dioxide, calcium hydrogen phosphate, lactose, mannitol, candy, gum arabic, pregelatinized starch, corn starch, powdered cellulose, hydroxypropyl cellulose, opadry, sodium starch glycolate, carnauba wax, synthetic aluminum silicate, stearic acid, magnesium stearate, aluminum stearate, calcium stearate, sucrose, dextrose, sorbitol, and talc. The pharma-ceutically 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.

[0112] The composition of the present invention can be administered parenterally (e.g., intravenously, subcutaneously, intraperitoneally or topically) or orally according to the intended method, and the dosage range varies depending on the patient's body weight, age, sex, health condition, diet, administration time, administration method, excretion rate, severity of disease, etc. The daily dosage of the composition of the present invention is 0.0001 to 10 mg / ml, preferably 0.0001 to 5 mg / ml, and more preferably administered once or in divided doses per day.

[0113] Liquid preparations for oral administration of the composition of the present invention include suspensions, oral liquids, emulsions, syrups, etc., which may contain various excipients such as wetting agents, sweeteners, flavoring agents, preservatives, etc. in addition to water and liquid paraffin, which are commonly used simple diluents. Preparations for parenteral administration include sterilized aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried preparations, suppositories, etc.

[0114] In one aspect, the present invention relates to a method for screening a substance that specifically delivers a drug to M2 tumor-associated macrophages, the method comprising the steps of treating a candidate substance with ITGB2 (Integrin beta 2); and selecting a candidate substance that binds to ITGB2.

[0115] In one embodiment, the method may screen for candidate substances that specifically bind to CD18 in an extended active conformation.

[0116] In one aspect, the present invention relates to a method for selecting a patient having responsiveness to an anticancer agent targeting M2 macrophages, comprising the step of confirming the expression level of ITGB2 in an isolated sample.

[0117] In an embodiment, the method may further include identifying a patient as being responsive to an anticancer drug targeting M2 macrophages when the expression level of ITGB2 in M2 macrophages is higher than that in M0 macrophages and M1 macrophages.

[0118] In one aspect, the present invention relates to a marker for diagnosing solid cancer, comprising the ITGB2 gene or a protein expressed from said gene.

[0119] In one aspect, the present invention relates to a composition for diagnosing solid cancer, comprising a preparation for measuring the expression level of ITGB2 at the mRNA or protein level.

[0120] In one embodiment, the preparation to be measured at the mRNA level may be 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 pair and a probe that specifically recognize a fragment of the nucleic acid sequence and the complementary sequence, and the measurement may be performed by one or more methods selected from the group consisting of polymerase chain reaction, real-time RT-PCR, reverse transcription polymerase chain reaction, competitive RT-PCR, nuclease protection assay (RNase, S1 nuclease assay), in situ hybridization, nucleic acid microarray, Northern blot, DNA chip, multiplex PCR, or ddPCR.

[0121] In one embodiment, the preparation to be measured at the protein level may be an antibody, an antibody fragment, an aptamer, an avidity multimer, or a peptidomimetics that specifically recognizes the full-length protein or a fragment thereof of the gene, and the measurement may be performed by one or more methods selected from the group consisting of Western blot, enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA), radioimmunodiffusion, immunoelectrophoresis, tissue immunostaining, immunoprecipitation assay, complement fixation assay, FACS, mass spectrometry, or protein microarray.

[0122] In the present invention, the term "detecting" or "measuring" as used herein means quantifying the concentration of the object detected or measured.

[0123] In the present invention, the term "primer" refers to a short nucleic acid sequence that can form a base pair with a template complementary to a nucleic acid sequence having a short free 3-hydroxyl group at its end and serves as a starting point for template strand replication. A primer can initiate DNA synthesis in the presence of a reagent for polymerization reaction (i.e., DNA polymerase or reverse transcriptase) and four different nucleoside triphosphates at an appropriate buffer solution and temperature.

[0124] The term "probe" used in the present invention means a nucleic acid fragment such as RNA or DNA, which is short and has a few bases or long and has a few hundred bases, and can specifically bind to mRNA, and is labeled to confirm the presence or absence of a specific mRNA. The probe can be prepared in the form of an oligonucleotide probe, a single stranded DNA probe, a double stranded DNA probe, an RNA probe, etc. In the present invention, hybridization is performed using a probe complementary to the ITGB2, and the degree of expression of the gene can be diagnosed based on the hybridization. The selection of an appropriate probe and the hybridization conditions can be modified based on those known in the art, and the present invention is not particularly limited thereto.

[0125] The primer or probe of the present invention can be chemically synthesized using the phosphoramidite solid support method or other widely known methods. Such 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 one or more of the natural nucleotides with their analogs, and internucleotide modifications, such as uncharged linkages (e.g., methylphosphonates, phosphorotriesters, phosphoramidates, carbamates, etc.) or charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.).

[0126] In the present invention, the suitable conditions for hybridizing the probe with the cDNA molecule can be determined through a series of optimization procedures. Such procedures are carried out by those skilled in the art in a series of procedures to establish a protocol for use in a 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 and GC content of the probe and the target nucleotide sequence. Detailed conditions for hybridization can be found in Joseph Sambrook, et al., Molecular CloninG, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2001); and MLM Anderson, Nucleic Acid Hybridization, Springer-Verlag New York Inc. NY (1999). For example, among the stringency conditions, the high stringency condition means hybridization in 0.5 M NaHPO4, 7% SDS (sodium dodecyl sulfate), 1 mM EDTA at 65° C. and washing in 0.1×SSC (standard saline citrate) / 0.1% SDS at 68° C. Alternatively, the high stringency condition means washing in 6×SSC / 0.05% sodium pyrophosphate at 48° C. The low stringency condition means, for example, washing in 0.2×SSC / 0.1% SDS at 42° C.

[0127] In one aspect, the present invention relates to a method for predicting or prognosing responsiveness to an immune anticancer agent targeting M2 macrophages, comprising a step of confirming the expression level of ITGB2 in a sample treated with the immune anticancer agent of the present invention.

[0128] In one embodiment, the method may further include determining that the response to the immuno-anticancer agent is good if the expression level of ITGB2 protein is decreased in the sample after treatment with the immuno-anticancer agent.

[0129] In one embodiment, the sample may be blood, serum, plasma, amniotic fluid, saliva, peritoneal fluid, bone marrow, tears, bile, lung lavage fluid, cerebrospinal fluid, pleural fluid, synovial fluid, lymph, semen, urine, or a solution of protein extracted from tissue biopsy or cells.

[0130] In one aspect, the present invention relates to a method for providing information necessary for diagnosing cancer, comprising the steps of: confirming the expression level of ITGB2 in a sample isolated from a subject; comparing the expression level of ITGB2 with that of a control group; and classifying subjects in which ITGB2 is overexpressed compared to the control group.

[0131] In one embodiment, the method may further include determining that a subject in which ITGB2 is overexpressed compared to a control group is likely to be affected by cancer.

[0132] In one aspect, the present invention relates to a method for treating cancer comprising administering a pharma- ceutically effective amount of the drug delivery system of the present invention to a subject suffering from cancer.

[0133] In one embodiment, the subject may be a patient in which expression of ITGB2 is upregulated in M2 macrophages compared to M0 and M1 macrophages.

[0134] In one aspect, the present invention relates to the use of the drug delivery system of the present invention for the manufacture of a pharmaceutical composition for the prevention and treatment of cancer.

[0135] The present invention will be described in more detail with reference to the following examples, which are merely for illustrative purposes and are not intended to limit the scope of the present invention.

[0136] Example 1. Synthesis of M2 Tumor-Associated Macrophage (M2TAM) Targeting Peptides Melittin and TAMpep826 in Table 1 below were synthesized by GenScript (Piscataway, NJ, USA) and then dissolved in D-PBS at 5 mg / ml for use in the experiments.

[0137] [Table 1]

[0138] Example 2. Analysis of M2TAM targeting 2-1. Confirmation of the affinity of melittin for M2TAM To confirm whether the melittin prepared in Example 1 specifically binds to M2 tumor-associated macrophages (M2TAM), human monocyte cell line THP-1 was differentiated into M0, M1, and M2 macrophages, treated with FITC-conjugated melittin, and confirmed by flow cytometry. Specifically, THP-1 cells were treated with 100 nM PMA (phorbol 12-myristate 13-acetate) at 37°C for 24 hours (M0 macrophages), and the differentiated M0 macrophages were cultured with 100 nM LPS and 20 ng / ml IFN-γ at 37°C for 72 hours to differentiate into M1 macrophages (M1), and then cultured with 20 ng / ml IL-4 and IL-13 at 37°C for 72 hours to differentiate into M2 macrophages (M2). Differentiated cells were treated with 50 nM FITC-conjugated melittin for 1 h, and the cells were detected using BD FACS CantoII instruments (BD biosciences, San Jose, CA, USA) and analyzed using FlowJo software (BD biosciences).

[0139] As a result, FITC-positive cells were significantly increased in M2 macrophages compared with M0 and M2 macrophages (Fig. 1a and b), confirming that melittin preferentially binds to M2 macrophages.

[0140] 2-2. Identification of M2TAM-binding target proteins of melittin To determine whether a certain protein in M2 macrophages is involved in the specific binding of melittin to M2 macrophages, we synthesized biotin-conjugated melittin (melittin-biotin). 6After differentiating macrophages (18-well plate) into M0, M1 and M2 macrophages, the cells were collected using a scraper and washed by centrifugation (300 Х g, 5 min), and the cell membrane proteins of the macrophages were obtained using a membrane protein extraction kit (Thermo Fisher Scientific). After reacting with melittin-biotin (200 μg) at room temperature (20-24 °C) for 1 hour, the protein bound to melittin was obtained using streptavidin resin (Thermo Fisher Scientific). To remove salts, 100 μl each of 100% methanol, 0.1% formic acid and 80% CAN were added to an 18 Micro Spin-Column, and the sample was loaded onto the column, and 50 μl of 0.1% formic acid was added to remove unnecessary substances. Then, 100 μl of 80% CAN was added to elute the peptides. After desalting, the mixture was dried in a Speed-vac, the solution was completely removed, and the mixture was analyzed by LC-MS / MS using UPLC-Exactive equipment (Thermo Fisher Scientific) under the conditions in Table 2 below. Data was analyzed for proteins upregulated in M1 and M2 macrophages compared to M0 macrophages using MaxQuant and MPP (Mass Profiler Professional). To analyze the data, LC-MS / MS data for each index was analyzed using Proteome Discoverer, and the database was Uniprot's human database, and LFQ (Label-Free Quantification) was performed. Modifications included Oxidation (M), Carbamyl (N-term), and Carbamidomethyl (C), and the baseline option was none.

[0141] [Table 2]

[0142] As a result, 53 proteins were upregulated in M2 / M0 and 123 proteins in M1 / M0 (Fig. 2a). More upregulated proteins interacted with M1 macrophages than with M2 macrophages, but this was not limited to proteins present in the cell membrane. Among the membrane proteins, ITGB2 was shown to be upregulated in M2 macrophages (Fig. 2b). In addition, cytokines such as IL-10 and CXCL10, which are known as markers of M1 macrophages, were upregulated in M1 macrophages (Fig. 2v). In addition, proteins were identified that were upregulated in M2 compared to M1, but among the membrane proteins, only ITGB2 was confirmed to be upregulated in M2 macrophages (Fig. 2d). Through this, it was confirmed that melittin interacts with and binds to the membrane protein ITGB2 of M2 macrophages.

[0143] 2-3. Identification of M2TAM-binding target proteins of TAMpep826 As seen in Example 2-2, cell membrane proteins extracted from M0, M1 and M2 macrophages were reacted with TAMpep826-biotin, and the target proteins were identified through LC-MS / MS proteomics.

[0144] The results showed that, among cell membrane proteins, ITGB2 was expressed at higher levels in M2 macrophages than in M0 and M1 macrophages (Figure 4), and was selected as the M2 macrophage-binding target protein of TAMpep826.

[0145] Example 3. Confirmation of ITGB2 expression in M2 macrophages 3-1.Confirmation of ITGB2 mRNA expression To evaluate the mRNA expression level of ITGB2 in M0, M1, M2 macrophages and TAMs, real-time polymerase chain reaction was performed. Specifically, RNA was extracted from cells differentiated into M0, M1, or M2 macrophages using easy-BLUE total RNA Extraction Kit (iNtRON) and cDNA was synthesized using CycleScript-Reverse Transcriptase (Bioneer). Real-time PCR (CFX96, Bio-rad) was performed using primers for actin or ITGB2 genes (Table 3) under the conditions shown in Figure 5, and the expression levels of each gene compared to actin were compared and analyzed by relative quantification.

[0146] [Table 3]

[0147] The results showed that ITGB2 mRNA expression was significantly increased in M2 macrophages and TAMs compared with M0 and M1 (Figure 6).

[0148] 3-2.Confirmation of ITGB2 protein expression Western blot analysis was performed to evaluate the protein expression level of ITGB2 in M0, M1, M2 macrophages and TAMs. Specifically, in each cell differentiated into M0, M1 and M2 macrophages, proteins were extracted using Proprep solution (iNtRON) and transferred to a membrane by electrophoresis. After blocking by immersing the membrane in 5% BSA, the primary antibody (ITGB2) was treated at 4°C for 24 hours, and the secondary antibody was treated at room temperature for 1 hour, and then treated with D-Plus (trademark pending) ECL Pico System (Doning LS) and protein band photographs were taken with Davinch-Chemi (trademark pending) imaging system (Intoxia).

[0149] The results showed that ITGB2 protein expression was significantly increased in M2 macrophages and TAMs compared with M0 and M1 (Figure 7).

[0150] 3-3. Confirmation of ITGB2 protein expression by immunofluorescence Immunofluorescence analysis was performed to evaluate the protein expression level of ITGB2 in M0, M1, M2 macrophages and TAMs. Specifically, cells differentiated into M0, M1, and M2 macrophages were fixed with 4% formalin, washed with PBS, blocked by immersion in 5% BSA, and stained with a primary antibody (ITGB2) for 24 hours at 4°C and a secondary antibody for 2 hours at room temperature. After mounting with DAPI, the stained cells were photographed with an LSM800 (Zeiss).

[0151] As a result, ITGB2 protein was shown to have higher expression levels in M2 macrophages and TAMs compared to M0 and M1 (Figure 8).

[0152] Example 4. Confirmation of interaction between M2TAM targeting peptide and ITGB2 4-1. Confirmation of ITGB2 expression in the plasma membrane of M2 macrophages To confirm whether ITGB2 is expressed on the cell membrane of M2 macrophages, cell membrane proteins and cytosol proteins were isolated and extracted from M2 macrophages, reacted with biotinylated TAMpep826, and the expression of ITGB2 was evaluated by Western blot analysis. The results showed that ITGB2 protein is expressed on the cell membrane of M2 macrophages (Figure 9).

[0153] 4-2. Confirmation of the interaction between TAMpep826 and ITGB2 To confirm whether TAMpep826 interacts with ITGB2 in M2 macrophages, the expression of ITGB2 was evaluated through a 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 min), and the cell membrane proteins of macrophages were obtained using a cell membrane protein extraction kit (Thermo Fisher Scientific). After pretreatment with TAMpep826 or scramble peptide at different concentrations (0.001, 0.01, 0.1, 1 and 10 μg) for 1 hour, the cells were reacted with TAMpep826 (200 μg) with biotin bound to the protein at room temperature (20-24 ° C) for 1 hour, and then the protein bound to TAMpep826 was obtained using Dynabeads (trademark pending) M-280 Streptavidin (Thermo Fisher Scientific). Western blot analysis of proteins was performed to confirm the expression of ITGB2.

[0154] As a result, it was shown that pretreatment with scrambled peptide had no effect on the expression of ITGB2 bound to biotinylated TAMpep826, whereas pretreatment with TAMpep826 suppressed ITGB2 expression depending on the concentration (Figure 10). This confirmed that TAMpep826 interacts with ITGB2 on the cell membrane of M2 macrophages.

[0155] 4-3. Confirmation of the interaction between TAMpep826 and ITGB2 using immunofluorescence To confirm whether ITGB2 and TAMpep826 co-localize on the plasma membrane of M2 macrophages, we performed immunofluorescence analysis using FITC-conjugated TAMpep826. The results showed that TAMpep826 and ITGB2 were distributed intensively on the plasma membrane of M2 macrophages (Figure 11).

[0156] 4-4. Identification of the binding site of TB511 and ITGB2 Based on the sequence of the cysteine-rich portion of CD18 (ITGB2) (uniport_p05107, cysteine-rich tandem repeats region: 449-617), a tertiary structure was constructed using the trRosetta algorithm (https: / / yanglab.nankai.edu.cn / trRosetta / ). Then, a docking simulation of TB511 and CD18 was performed through the CABS-dock server (http: / / biocomp.chem.uw.edu.pl / CABSdock / ) using the CD18 PDB file created with trRosetta and the amino acid sequence of TB511. From the 10 models created as a result of the docking simulation, a representative model was selected based on the results of alanine substitution in the TB511 sequence (Figure 12).

[0157] As a result, LEU(6) of TB511 was predicted to bind to the LEU(528), THR(538), LEU(556), CYS(557) and PHE(558) sites of CD18, and TRP(12) of TB511 was predicted to bind to GLU(466), ILE(469), CYS(470) and ARG(471) sites of CD18. In addition, when LEU(6) and TRP(12) of TB511 were replaced with alanine, respectively, and the binding with CD18 was simulated, it was shown that neither of the two sites bind to any sequence of CD18. Therefore, LEU(6) and TRP(12) of TB511 are the key parts in the docking between TB511 and CD18, and TB511 can be predicted to bind to 466-565 of CD18 (Figure 13).

[0158] Example 5. Synthesis of ITGB2 binding moieties 5-1.Synthesis of ITGB2-binding peptide-drug conjugates (PDCs) The Melittin-dKLA and TB511 peptides in Table 4 below were synthesized and supplied by GenScript (Piscataway, NJ, USA), and then dissolved in D-PBS at 5 mg / ml for use in the experiments. dKLA is linked through an amide bond between peptides, and to minimize the interaction and folding between melittin or TAMpep826 and dKLA, a linker consisting of four glycines and one serine is placed in the middle, both ends are separated, and D-isomers, not L-isomers, were used for KLA to minimize degradation in the body. To prepare M-DM1, DM1 (Mertansine) (MedChem Express, Princeton) (1.1 equivalents, 10 mM in DMF) and DMF (dimethylformamide) were added to maleimide-modified melittin (GenScript, Beijing, China) (100 μM, 1 mL, 0.1 mmol) dissolved in 25 mM sodium borate buffer (25 mM NaCl, 1 mM EDTA, pH 8.0) and reacted at 37 °C for 1 h. The product was then filtered three times by ultrafiltration with Dulbecco's phosphate-buffered saline (DPBS; Welgene) to prepare PDC by conjugating DM1 to the N-terminus of maleimide-modified melittin M, which was named M-DM1. The prepared M-DM1 was characterized by reversed-phase high-performance liquid chromatography (HPLC) on a Poroshell 120 C18 column (2.7 μm, 3 × 50 mm, Agilent, Santa Clara, CA, USA) with 5 μL injected at a rate of 0.5 mL / min.

[0159] [Table 4]

[0160] 5-2.Synthesis of ITGB2-binding ADC (Antibody-drug conjugate) 5-2-1. Preparation of anti-CD18 antibody and ADC of DM1 To prepare an antibody (FIG. 14) that specifically binds to the active conformation of CD18 in M2TAM, which is one of the macrophage-1 antigens (Mac-1), the culture medium of hybridoma (MM18 / 2.a.8) cells secreting anti-CD18 antibody was collected and centrifuged at 2,500 rpm and 4°C for 10 minutes to obtain the supernatant, which was then purified by protein G-Sepharose column chromatography. The antibody solution was slowly passed through a protein G-Sepharose column (Pharmacia, Sweden) that had been pre-equilibrated with PBS, and the column was connected to a peristatic pump and thoroughly washed with PBS. After washing was completed, the antibody was eluted with 0.2 M glycine-HCl (pH 2.7). At this time, the eluate was buffered in a tube containing 1 M Tris (pH 9.0) that had been prepared in advance. The antibody was dialyzed against PBS before use. The amount of purified antibody was measured using a BCA protein assay kit (BCA (trademark pending) Protein Assay Kit, Pierce), and the binding ability of the antibody was confirmed through flow cytometry analysis. The purified anti-CD18 antibody was buffer-exchanged into PBS (pH 7.4), and the anti-CD18 antibody (1 mg / ml, 6.67 μM) was mixed with SMCC-DM1 (mertansine, 120 μM) (MedChemExpress) dissolved in DMSO at a ratio of 1:18 and reacted at room temperature for 4 to 20 hours. To remove unreacted DM1, CD18-DM1 was buffer-exchanged into PBS (pH 7.4) using a 10 KDa cutoff centrifugal filter, and filtered with a 0.2 μm syringe filter. The amount of DM1 (252 nm) bound to the anti-CD18 antibody (280 nm) was measured using UV-Vis spectroscopy, and the ratio of DM1 to the anti-CD18 antibody (drug antibody ratio, DAR) was calculated, and the DAR value was shown to be 5.8. The ADC thus prepared, in which the anti-CD18 antibody and DM1 were bound, was named CD18-DM1.

[0161] 5-2-2. Preparation of ADC of anti-CD18 antibody and MMAE (Monomethyl auristatin E) The anti-CD18 antibody purified in the above example was buffer-exchanged to PBS (pH 7.4), and in order to partially reduce the disulfide bond of the CD18 antibody, the anti-CD18 antibody (1 mg / ml, 6.67 μM) was mixed with 1 mM DTT (66.7 μM) at a ratio of 1:10, reacted at 37 °C for 1 to 2 hours, and buffer-exchanged to PBS (pH 7.4). The partially reduced anti-CD18 antibody (1 mg / ml, 6.67 μM) was mixed with 10 mM Suo-Val-Cit-PAB-MMAE (MedChemExpress) at a ratio of 1:20 to 1:40, reacted at 4 °C for 12 hours, and buffer-exchanged to PBS (pH 7.4). The amount of vc-MMAE (248 nm) bound to the anti-CD18 antibody (280 nm) was measured using UV-Vis spectroscopy, and the ratio of vc-MMAE to the anti-CD18 antibody (drug antibody ratio, DAR) was calculated, with the DAR value ranging from 2.8 to 5.4. The ADC thus prepared, in which the anti-CD18 antibody and MMAE were bound, was named CD18-MMAE.

[0162] Example 6. Confirmation of M2TAM binding of ITGB2-bound PDC To confirm the binding strength of ITGB2 and Melittin-dKLA, ITGB2 protein was coated on a gold thin film sensor chip, and then ITGB2 antibody and Melittin-dKLA were injected as positive controls, and the reflected light was measured to perform surface plasmon resonance (SPR) analysis. Specifically, ITGB2 was immobilized on the surface of the sensor chip, and then immobilization was performed using amine coupling under the conditions in Table 5 below (FIG. 15). Then, ITGB2 antibody and Melittin-dKLA of various concentrations were injected on the ITGB2-coated sensor chip, and the association and dissociation sections were observed, and the kinetic evoluation constant of intermolecular binding was calculated through the sensorgram under the kinetic evolution conditions in Table 6 below.

[0163] [Table 5]

[0164] [Table 6]

[0165] As a result, the equilibrium dissociation constant (KD) obtained by dividing the dissociation rate constant (kd) that evaluates the binding strength between two molecules of the ITGB2 antibody, which is the positive control, by the association rate constant (ka), was 1.14. -8 The equilibrium dissociation constant of Melittin-dKLA is 7.86. -8 16 and 17. Therefore, it was confirmed that Melittin-dKLA has the ability to bind to ITGB2.

[0166] Example 7. Confirmation of the M2TAM cell killing effect of ITGB2 in PDC 7-1. Creation of ITGB2 expression suppression cell model As a cell model to be used for confirming whether cell death is induced through ITGB2 in M2 macrophages, a cell model in which ITGB2 expression is suppressed in M2 macrophages was prepared using Crispr / cas9. Specifically, cells differentiated into M2 macrophages were cultured in Opti-MEM medium. Crispr / cas9 was prepared by mixing Cas9 protein V2 (Thermo Fisher scientific) and gRNA (Genscript; ITGB2; Table 7) of CRISPRMAX (trademark application) Reagent kit (Thermo Fisher scientific) with Cas9 Plus (trademark application) Reagent (Thermo Fisher scientific), diluting CRISPRMAX (trademark application) Reagent in Opti-MEM medium, mixing them all together and reacting for 5 to 10 minutes, and then adding them to cells differentiated into M2 macrophages and culturing them at 37 ° C for 2 to 3 days. Thereafter, the mRNA expression level of ITGB2 was confirmed.

[0167] [Table 7]

[0168] As a result, it was shown that M2 macrophages injected with ITGB2 sgRNA showed significantly reduced ITGB2 expression compared to the control group (Figure 18).

[0169] 7-2. Confirmation of M2TAM cell death by ITGB2 in Melittin-dKLA In order to confirm whether Melittin-dKLA binds to ITGB2 in M2 macrophages and induces cell apoptosis, M2 macrophages in which ITGB2 expression was suppressed by Crispr / cas9 prepared in Example 7-1 were treated with Melittin-dKLA (1 μM) for 24 hours, and cell viability was analyzed using CCK-8 analysis. Specifically, Melittin-dKLA (1 μM) was reacted with ITGB2-locked down macrophages prepared in Example 7-1 for 1 hour. After the reaction, the medium was replaced and cultured at 37°C for 24 hours. To confirm cell viability, CCK-8 reagent (Enzo Life Sciences) was added in 1 / 10 of the medium and reacted at 37°C for 3 hours. Absorbance was measured at 450 nm using a microplate leader (Molecular Devices).

[0170] As a result, in control M2 macrophages, cell viability was significantly reduced by Melittin-dKLA, but in ITGB2-locked down M2 macrophages, the reduction in cell viability caused by Melittin-dKLA was suppressed (Figure 19). Therefore, it was confirmed that Melittin-dKLA specifically binds to ITGB2 in M2 macrophages, thereby inducing apoptosis in M2 macrophages.

[0171] 7-3. Confirmation of M2TAM cell death by ITGB2 in TB511 To confirm whether TB511 (TAMpep826-dKLA) induces cell death by ITGB2 in M2 macrophages, TB511 was reacted with M2 macrophages in which ITGB2 expression was suppressed by Crispr / cas9, as prepared in Example 7-1. As a result, M2 macrophages showed about 50% cell viability by TB511, but cell viability was significantly increased from cells in which ITGB2 expression was suppressed (Figure 20).

[0172] 7-4. Confirmation of reduction in induction of TB511 cell death by ITGB2 antibody To confirm whether TB511 induces cell death through ITGB2 in M2 macrophages, M2 macrophages with suppressed ITGB2 expression prepared in Example 7-1 were pretreated with anti-ITGB2 antibody (Polyclonal Rabbit anti-Human ITGB2 / CD18 antibody) (LS-C312785; LS Bio) (1 μg) for 1 hour and then reacted with TB511 (1 μM) for 1 hour. Then, whether cell death was induced was confirmed by cell viability analysis using CCK-8 assay, protein expression of caspase-3, a cell death marker, by Western blot analysis, gene expression of caspase-3, caspase-8, and caspase-9 by real-time PCR, and induction of caspase-3 protein expression by immunofluorescence.

[0173] As a result, ITGB2 antibody had no effect on cell viability in M2 macrophages, but TB511 significantly decreased cell viability. On the other hand, TB511 significantly increased cell viability in M2 macrophages pretreated with ITGB2 antibody (Figure 21). Furthermore, TB511 significantly increased caspase-3 expression in M2 macrophages, whereas TB511 significantly decreased caspase-3 expression in M2 macrophages pretreated with ITGB2 antibody (Figure 21). Furthermore, TB511 significantly increased caspase-3, caspase-8, and caspase-9 expression in M2 macrophages, whereas TB511 significantly decreased caspase-3, caspase-8, and caspase-9 expression in M2 macrophages pretreated with ITGB2 antibody (Figure 21). In M2 macrophages, caspase-3 expression was increased by TB511, whereas in M2 macrophages pretreated with ITGB2 antibody, TB511-induced caspase-3 expression was decreased (Figure 21). Furthermore, the mitochondrial targeting of TB511 was confirmed, and co-localization of mitochondria and TB511 was decreased in M2 macrophages pretreated with ITGB2 antibody compared to cells not pretreated with ITGB2 (Figure 21).

[0174] Example 8. Confirmation of efficacy of PDC in 3D cancer organoids 8-1. Confirmation of the effect of TB511 on breast cancer 3D organoids To complement the limitations of in vitro and to replace in vivo, we created 3D organoids that mimicked the tumor microenvironment (TME) of breast cancer, and confirmed the effect of TB511 on them. Specifically, Matrigel was mixed to 3% in RPMI-1640 culture medium, and MDA-MB-231 human breast cancer cells, CAFs (Cancer-associated Fibroblasts), and THP-1 cells that were created to express GFP using Incucyte Nuclight Lentivirus Reagents (Sartorius) were counted and dispensed into a 96u-bottom 3-D culture plate (Sbio) at a ratio of 5:1:1 to make 200 μl. After culturing, the cells were centrifuged at 1200 rpm for 3 minutes, collected, and then cultured in a 37°C incubator for 48 hours to form spheroids. From the second day of spheroid formation, TB511 was treated at concentrations of 1 μM, 2 μM, 4 μM, and 8 μM every three days, and images were taken with a fluorescent microscope. After taking a bright-field image, the spheroid area was measured by measuring the diameter of the pellet. The average tumor spheroid diameter was measured using an open source, and the size was analyzed using ImageJ (software version 1.47m). After taking a bright-field image (20X), the average tumor spheroid diameter was measured for an average of three spheroids per spheroid, and analyzed using ImageJ (software version 1.47m). In addition, on the 10th day of drug treatment, the spheroids were fixed and reacted with an antibody (abcam) against the tumor growth factor ki-67 for immunofluorescence staining (control staining: DAPI). In the case of THP-1, no additional staining was performed, and images of the GFP fluorescence expressed in the cells were taken using a confocal microscope, and the number of cells expressing the fluorescence was counted.

[0175] As a result, in the drug-untreated group, the size of the spheroids gradually increased over time, and it was shown that when THP-1 cells, which are monocytes, are co-cultured with cancer cells and CAFs, they differentiate into TAMs by the cancer cells and TME, promoting tumor growth. On the other hand, in the TB511-treated group, it was confirmed that the size of the spheroids decreased in a concentration-dependent manner at a concentration of 1 μM on the 10th day (Figure 22), and it was shown that the number of THP-1 cells expressing GFP fluorescence decreased significantly in a concentration-dependent manner compared to the drug-untreated group ( *** p<0.001) (FIG. 23). Thus, it was confirmed that TB511, an ITGB2-binding PDC, selectively binds to THP-1 cells and induces cell death of MDA-MB-231 human breast cancer cells.

[0176] 8-2. Confirmation of the effect of TB511 on lung cancer 3D organoids A549 human lung cancer cells, CAF and THP-1 cells were mixed by the method of Example 8-1 to prepare lung cancer 3D spheroids, and then TB511 was treated at concentrations of 1 μM, 2 μM and 4 μM, respectively, and the effect was analyzed in the same manner as in Example 8-1. When preparing spheroids, 3D spheroids were also prepared by mixing only cancer cells or only cancer cells + CAF cells, and the size of the spheroids was compared.

[0177] The results showed that the size of spheroids formed with A549+CAF+THP-1 cells was significantly larger than that of spheroids formed with A549 cells alone or A549+CAF cells alone. In addition, the size of spheroids in the TB511-treated group was significantly reduced in a TB511 concentration-dependent manner compared to the TB511-untreated group ( *** p<0.001) (Figure 24). In addition, spheroids were fluorescently stained on the 10th day after drug treatment to confirm the related markers. Compared to the group not treated with TB511, the number of THP-1 cells was significantly decreased in a concentration-dependent manner, and the tumor growth factor ki-67 was also decreased in a concentration-dependent manner ( *** p<0.001) (Figure 25).

[0178] 8-3. Confirmation of the effect of M-DM1 on lung cancer 3D organoids To confirm the efficacy of M-DM1 (Mel-DM1) against lung cancer, A549 human lung cancer cells, CAFs and THP-1 cells were mixed to prepare lung cancer 3D spheroids by the method described in Example 8-1, and then treated with M-DM1 at concentrations of 1 μM, 2 μM and 4 μM, respectively, and the effects were analyzed in the same manner as in Example 8-1.

[0179] The results showed that the size of spheroids in the M-DM1-treated group was reduced in a concentration-dependent manner compared to the M-DM1-untreated group ( *** p<0.001) (Figure 26).

[0180] Example 9. Confirmation of efficacy of PDC in anticancer drug resistant 3D cancer organoids 9-1. Confirmation of the effect of TB511 on anticancer drug resistant lung cancer 3D organoids To confirm the efficacy of TB511 against carboplatin-resistant lung cancer, carboplatin-resistant A549 cells, CAFs and THP-1 cells were mixed to form 3D-spheroids according to the method of Example 8-1, and TB511 was treated at concentrations of 1 μM, 2 μM and 4 μM, respectively, and the effect was analyzed in the same manner as in Example 8-1.

[0181] The results showed that the size of spheroids formed from A549+CAF+THP-1 cells was significantly larger than that of spheroids formed from A549-resistant cells alone or A549+CAF cells alone, and that the size of spheroids in the TB511-treated group was significantly reduced in a TB511 concentration-dependent manner compared to the TB511-untreated group ( ***p<0.001) (Figure 27). In addition, spheroids were immunofluorescently stained on the 10th day after drug treatment to confirm the related markers. Compared to the TB511-untreated group, the number of THP-1 cells in the TB511-treated group was significantly reduced in a concentration-dependent manner, and the tumor growth factor ki-67 was also significantly reduced from 2 μM ( *** p<0.001) (Figure 28).

[0182] 9-2. Confirmation of the effect of TB511 on anticancer drug resistant prostate cancer 3D organoids To confirm the efficacy of TB511 against docetaxel-resistant prostate cancer, docetaxel-resistant PC3 prostate cancer cells, CAFs and THP-1 cells were mixed to form 3D-spheroids according to the method of Example 8-1, and TB511 was treated at concentrations of 1 μM, 2 μM or 4 μM, respectively, and the effect was analyzed in the same manner as in Example 8-1.

[0183] As a result, the size of spheroids formed from PC3+CAF+THP-1 cells was significantly larger than that of spheroids formed from PC3-resistant cells alone or PC3+CAF cells alone, and the size of spheroids in the TB511-treated group was significantly reduced in a TB511 concentration-dependent manner compared to the TB511-untreated group ( *** p<0.001) (Figure 29). In addition, spheroids were immunofluorescently stained 10 days after drug treatment to confirm the related markers. Compared to the TB511-untreated group, the number of THP-1 cells was significantly reduced in a concentration-dependent manner in the TB511-treated group, and the tumor growth factor ki-67 also showed a tendency to decrease ( ** p<0.01, *** p<0.001) (Figure 30).

[0184] Example 10. Confirmation of ADC efficacy in 3D cancer organoids 10-1. Confirmation of the effect of ADC on breast cancer 3D organoids To confirm the efficacy of ITGB2-binding ADC (antibody-drug conjugate) against breast cancer, MDA-MB-231 human breast cancer cells, CAFs and THP-1 cells were mixed to form 3D-spheroids by the method of Example 8-1, and treated with CD18-DM1 or CD18-MMAE, the ADC of the present invention, at concentrations of 10 μg and 20 μg, respectively, and the effects were analyzed in the same manner as in Example 8-1.

[0185] As a result, the size of breast cancer spheroids in the non-ADC treated group gradually increased over time, whereas the size of the spheroids in the ADC treated group decreased on day 8 (Figures 31 and 32). In addition, the number of GFP-positive THP-1 cells was strongly increased in the non-ADC treated group, whereas the number of GFP-positive THP-1 cells was decreased in a concentration-dependent manner in the CD18-DM1 or CD18-MMAE treated groups ( *** p<0.001) (Figures 31 and 32).

[0186] 10-2. Confirmation of the effect of ADC on lung cancer 3D organoids To confirm the efficacy of the ADC against lung cancer, A549 human lung cancer cells, CAFs, and THP-1 cells were mixed to form 3D-spheroids according to the method of Example 8-1, and treated with the ADC of the present invention, CD18-DM1 or CD18-MMAE, at a concentration of 10 μg, respectively, and the effect was analyzed in the same manner as in Example 8-1.

[0187] As a result, in the group not treated with ADC, the size of the spheroids gradually increased over time, whereas in the group treated with CD18-DM1 or CD18-MMAE, the size of the spheroids decreased on the 8th day (Figure 33). In addition, the spheroids were immunofluorescently stained on the 8th day after drug treatment to confirm the related markers, and it was shown that the number of THP-1 cells strongly increased in the group not treated with ADC, whereas it significantly decreased in the ADC-treated group ( *** p<0.001) (Figure 34).

[0188] Example 11. Confirmation of efficacy of ADC in anticancer drug resistant 3D cancer organoids 11-1. Confirmation of the effect of ADC on anticancer drug resistant lung cancer 3D organoids To confirm the efficacy of ADC against carboplatin-resistant lung cancer, carboplatin-resistant A549 cells, CAFs and THP-1 cells were mixed to form 3D-spheroids according to the method of Example 8-1, and treated with CD18-DM1 or CD18-MMAE at a concentration of 10 μg each, and the effects were analyzed in the same manner as in Example 8-1.

[0189] As a result, in the group not treated with ADC, the size of the spheroids gradually increased over time, whereas in the group treated with CD18-DM1 or CD18-MMAE, the size of the spheroids decreased on day 8 (Figure 35). In addition, the spheroids were immunofluorescently stained on day 8 after drug treatment to confirm related markers. Compared to the group not treated with ADC, the number of THP-1 cells was significantly reduced in the group treated with CD18-DM1 or CD18-MMAE, and the tumor growth factor Ki-67 was also reduced ( *** p<0.001) (Figure 36).

[0190] 11-2. Confirmation of the effect of ADC on anticancer drug-resistant prostate cancer 3D organoids To confirm the efficacy of ADC against docetaxel-resistant prostate cancer, docetaxel-resistant PC3 prostate cancer cells, CAFs and THP-1 cells were mixed to form 3D-spheroids according to the method of Example 8-1, and treated with CD18-DM1 or CD18-MMAE at a concentration of 10 μg each, and the effects were analyzed in the same manner as in Example 8-1.

[0191] As a result, in the group not treated with ADC, the size of the spheroids gradually increased over time, whereas in the group treated with CD18-DM1 or CD18-MMAE, the size of the spheroids decreased on the 8th day (Figure 37). In addition, the spheroids were immunofluorescently stained on the 8th day after drug treatment to confirm the related markers. In the group not treated with ADC, the fluorescence strongly increased, whereas in the group treated with CD18-DM1 or CD18-MMAE, the number of THP-1 cells was significantly decreased, and the tumor growth factor Ki-67 was also decreased ( *** p<0.001) (Figure 38).

Claims

1. A drug delivery system comprising an ITGB2 binding moiety; and a drug bound thereto.

2. The drug delivery system of claim 1, wherein the ITGB2 binding portion is a small molecule, a virus, an antibody, an antibody fragment, an aptamer, a hormone, a cytokine, a chemokine, a ligand, a peptide which is a partial region of a cytokine, or a peptide which is a partial region of a ligand that specifically binds to ITGB2.

3. The drug delivery system of claim 1 , wherein the ITGB2 binding moiety specifically binds to an extended active conformation of CD18.

4. The drug delivery system of claim 1 , wherein ITGB2 comprises the amino acid sequence of SEQ ID NO: 7 or 8.

5. The drug delivery system of claim 1, wherein the ITGB2 binding moiety binds to amino acids 449-617 of ITGB2.

6. The drug delivery system of claim 5 , wherein the ITGB2 binding portion comprises the amino acid sequence of SEQ ID NO: 1 or 2.

7. The drug delivery system of claim 5 , comprising a peptide comprising the amino acid sequence of SEQ ID NO: 5 or 6.

8. The drug delivery system of claim 1, wherein the ITGB2 binding moiety binds to amino acids 449-617 of ITGB2.

9. The drug delivery system of claim 1, wherein the ITGB2 binding moiety binds to amino acids 732-748, amino acids 504-508, amino acids 534-546, amino acids 449-617, or amino acids 23-700 of ITGB2.

10. The drug delivery system of claim 1, wherein the ITGB2 binding moiety comprises an anti-CD18 antibody or an immunologically active fragment thereof.

11. Immunologically active fragments include Fab, Fd, Fab', dAb, F(ab'), F(ab') ２ 11. The drug delivery system of claim 10, which is any one selected from the group consisting of a single chain antibody, a scFv (single chain fragment variable), an Fv, a single chain antibody, an Fv dimer, a complementarity determining region fragment, a humanized antibody, a chimeric antibody and a diabody.

12. The drug delivery system of claim 1 , wherein the drug is a chemical compound, RNA, DNA, an antibody, an effector, a prodrug, a toxin, a peptide or a radionuclide.

13. 2. The drug delivery system of claim 1, wherein the drug is an immunogenic cell death inducer, a pro-apoptotic peptide, a microtubulin structure formation inhibitor, a mitosis inhibitor, a topoisomerase inhibitor, a DNA intercalator, a toxin, or an anticancer drug.

14. Proapoptotic peptides include KLA, alpha-defensin-1, BMAP-28, Brevenin-2R, Buforin IIb, cecropin A-Magainin 2 (CA-MA-2), cecropin A, cecropin B, chrysophsin-1, D-K6L9, gomesin, lactoferricin B, LLL27, LTX-315, magainin 2, magainin II-bombesin conjugate, and cecropin A-Magainin 2 (CA-MA-2).

14. The drug delivery system of claim 13, wherein the drug is selected from the group consisting of: MG2B, MG2A conjugate, MG2B, Pardaxin, and combinations thereof.

15. The drug delivery system of claim 13, wherein the immunogenic cell death inducer is selected from the group consisting of anthracycline anticancer drugs, taxane anticancer drugs, anti-EGFR antibodies, BK channel agonists, bortezomib, cardiac glycosides, cyclophosphamide anticancer drugs, GADD34 / PP1 inhibitors, LV-tSMAC, Measles virus, bleomycin, mitoxantrone, oxaliplatin, and combinations thereof.

16. Anticancer drugs include SN-38 (7-ethyl-10-hydroxy-camptothecin, 7-Ethyl-10-hydroxy-camptothecin), daunorubicin, doxorubicin, epirubicin, idarubicin, pixantrone, sabarubicin, valrubicin, paclitaxel, docetaxel, mechlorethamine, chlorambucil, chlorambucil, phenylalanine, mustard, cyclophosphamide, ifosfamide, carmustine (BCNU), lomustine (CCNU), streptozotocin, busulfan, thiotepa, cisplatin, carboplatin, dactinomycin (actinomycin D), plicamycin, mitomycin C C), vincristine, vinblastine, teniposide, topotecan, irinotecan, uramustine, melphalan, bendamustine, dacarbazine, temozolomide, altretamine, duocarmycin, nedaplatin, oxaliplatin, satraplatin, triplatin tetranitratetetranitrate, 5-fluorouracil, 6-mercaptopurine, capecitabine, cladribine, clofarabine, cysturbine, floxuridine, fludarabine, gemcitabine, hydroxyurea, methotrexate, pemetrexate, Pemetrexed, pentostatin, thioguanine, etoposide, mitoxantrone, ixabepilone, vindesine, vinorelbine, estramustine, maytansine, DM1 (mertansine), DM4, dolastatin, auristatin E 14. The drug delivery system of claim 13, wherein the agonist is selected from the group consisting of monomethyl auristatin E, auristatin F, monomethyl auristatin E (MMAE), monomethyl auristatin F, and derivatives thereof.

17. The drug delivery system of claim 1 , which specifically delivers a drug to M2 tumor-associated macrophages.

18. An ITGB2 binding moiety comprising melittin, a mutant or analogue thereof; and a peptide-drug conjugate (PDC) comprising a drug bound thereto.

19. The peptide-drug conjugate of claim 18, which binds to amino acids 466 to 565 of ITGB2.

20. The peptide-drug conjugate of claim 18, which binds to LEU(528), THR(538), LEU(556), CYS(557), PHE(558), GLU(466), ILE(469), CYS(470) or ARG(471) of ITGB2.

21. An antibody that binds to ITGB2 or a fragment thereof having immunological activity; and an antibody-drug conjugate (ADC) comprising a drug bound thereto.

22. The antibody-drug conjugate of claim 21, which binds to an epitope comprising amino acids 732-748, 504-508, 534-546, 449-617, or 23-700 of ITGB2.

23. The antibody-drug conjugate of claim 21, further comprising an antibody-drug conjugate linker.

24. 24. The antibody-drug conjugate of claim 23, wherein the ADC linker is 6-maleimidocaproyl (MC), maleimidopropanoyl (MP), valine-citrulline (val-cit), alanine-phenylalanine (ala-phe), p-aminobenzyloxycarbonyl (PAB), N-succinimidyl 4-(2-pyridylthio)pentanoate (SPP), N-succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), valine-citrulline-p-aminobenzyloxycarbonyl (val-cit-PAB) or N-succinimidyl (4-iodo-acetyl)aminobenzoate (SIAB).

25. A pharmaceutical composition for preventing or treating cancer, comprising the drug delivery system according to claim 1, the peptide-drug conjugate according to claim 18, or the antibody-drug conjugate according to claim 21 as an active ingredient.

26. The pharmaceutical composition for preventing or treating cancer according to claim 25, which is administered to a patient in whom ITGB2 expression is upregulated in M2 macrophages compared to M0 macrophages and M1 macrophages.

27. A method for screening a substance that specifically delivers a drug to M2 tumor-associated macrophages, comprising the steps of treating a candidate substance with ITGB2 (Integrin beta 2); and selecting a candidate substance that binds to ITGB2.

28. A method for screening a substance that specifically delivers a drug to M2 tumor-associated macrophages according to claim 27, comprising selecting a candidate substance that specifically binds to CD18 in an extended active conformation.

29. A method for selecting a patient having responsiveness to an anticancer agent that targets M2 macrophages, comprising the step of confirming the expression level of ITGB2 in an isolated sample.

30. The method according to claim 29, further comprising the step of identifying a patient as having responsiveness to an anticancer agent that targets M2 macrophages when the expression level of ITGB2 in M2 macrophages is higher than that in M0 macrophages and M1 macrophages.

31. A method for treating cancer, comprising administering a pharma- tically effective amount of the drug delivery system of claim 1, the peptide-drug conjugate of claim 18, or the antibody-drug conjugate of claim 21 to an object suffering from cancer.

32. Use of the drug delivery system for use in the manufacture of a pharmaceutical composition for the prevention and treatment of cancer.

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