Novel peptide-based immunotherapy for cancer

Abstract

The present invention relates to a novel peptide-based immunological anticancer agent, and to the use of melittin, its variants or analogs thereof, or a complex of these linked to a drug as an immunological anticancer agent. In the present invention, melittin, its variants or analogs thereof specifically bind to ITGB2 expressed on the cell membrane of M2 tumor-associated macrophages, and kill M2 tumor-associated macrophages. A complex of melittin, its variants or analogs thereof linked to a pro-apoptotic peptide or an anticancer drug as a drug significantly increases the apoptotic effect of the M2 tumor-associated macrophages, and can be used as an anticancer composition for suppressing tumor growth and metastasis.

Description

[Technical field]

[0001] The present invention relates to a novel peptide-based immunological anti-cancer agent, and more particularly to the use of melittin, its mutants or analogs, or conjugates of these with drugs as immunological anti-cancer agents. [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 proliferation of 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 in the body by injecting cancer antigens and immune activating substances into the body are being actively developed. However, such cell therapy and cancer vaccines are mainly used for blood cancer-related diseases, and have the disadvantage that most of them have very low therapeutic efficacy for solid cancers. 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, blocking the supply of nutrients to tumor cells, angiogenesis around tumor cells, etc. The tumor microenvironment contributes to the proliferation and survival of malignant cells, angiogenesis, metastasis, abnormal adaptive immunity, and reduced response to hormones and 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. In general, 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. Type 2 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 a large number of M2-type tumor-associated macrophages are present, 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 pharmaceutical composition for eliminating tumor-associated macrophages in the tumor microenvironment.

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

[0006] It is also an object of the present invention to provide a method for treating cancer.

[0007] Another object of the present invention is to provide a use of the conjugate of the present invention for the manufacture of a pharmaceutical composition for the prevention and treatment of cancer. [Means for solving the problem]

[0008] To achieve the above-mentioned objective, the present invention provides a pharmaceutical composition for eliminating tumor-associated macrophages in a tumor microenvironment, comprising as an active ingredient a conjugate of melittin, a mutant thereof, or an analog thereof, and a pro-apoptotic peptide or an anti-cancer drug.

[0009] The present invention also provides a pharmaceutical composition for preventing or treating cancer, which comprises as an active ingredient a conjugate in which melittin, a mutant or analog thereof is bound to a proapoptotic peptide or an anticancer agent.

[0010] The present invention also provides a method for treating cancer, comprising administering to a subject suffering from cancer a pharma- tically effective amount of a conjugate of melittin, a variant thereof or an analog thereof linked to a pro-apoptotic peptide or an anti-cancer drug.

[0011] In addition, the present invention provides a use of a conjugate comprising melittin, a mutant or an analog thereof and a proapoptotic peptide or an anticancer drug for the manufacture of a pharmaceutical composition for the prevention and treatment of cancer. Effect of the Invention

[0012] In the present invention, melittin, its mutants or analogs thereof specifically bind to ITGB2 expressed on the cell membrane of M2 tumor-associated macrophages, killing the M2 tumor-associated macrophages, and a complex in which melittin, its mutants or analogs thereof are bound to a pro-apoptotic peptide or an anti-cancer drug as a drug significantly increases the apoptotic effect of the M2 tumor-associated macrophages, and can be used as an anti-cancer composition for suppressing tumor growth and metastasis. [Brief description of the drawings]

[0013] [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 scatter plot analysis of M2 / M0 ratios to identify proteins that bind to TAMpep in M2 macrophages. [Figure 2c] FIG. 1 shows scatter plot analysis of M1 / M0 ratios to identify proteins that bind to TAMpep in M2 macrophages. [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 extracted cell membrane proteins with TAMpep826-biotin from M0, M1, and M2 macrophages. [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. 13 is a graph confirming ITGB2 mRNA expression in M2 macrophages. [Figure 7] FIG. 13 is a graph 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 graph confirming the expression of ITGB2 on 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 immunofluorescence analysis confirming the interaction of TAMpep826 and ITGB2 in M2 macrophages. [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 M2 TAM. [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. [Figure 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

[0014] 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.

[0015] 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.

[0016] 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.

[0017] 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.

[0018] In one aspect, the present invention relates to a pharmaceutical composition for eliminating tumor associated macrophages (TAM) in the tumor microenvironment, comprising as an active ingredient a conjugate of melittin, a mutant thereof or an analog thereof linked to a proapoptotic peptide or an anticancer drug.

[0019] In one embodiment, the tumor-associated macrophages may be M2 tumor-associated macrophages that express IGBT2 (Integrin beta 2).

[0020] In one embodiment, the composition may deplete only M2 macrophages without depleting M1 macrophages.

[0021] In one embodiment, melittin may comprise the amino acid sequence of SEQ ID NO:1.

[0022] In one embodiment, the melittin mutant may be a peptide in which amino acids 1 to 7 are deleted in the amino acid sequence of SEQ ID NO:1, and may include the amino acid sequence of SEQ ID NO:2.

[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 melittin, a variant thereof, or an analog thereof may specifically bind to an extended active conformation of ITGB2 (CD18).

[0025] In one embodiment, melittin, a variant or analog thereof may bind to amino acids 466-565 of ITGB2.

[0026] In one embodiment, the composition may include a peptide comprising the amino acid sequence of SEQ ID NO:5 or SEQ ID NO:6.

[0027] In one embodiment, the conjugate in which a proapoptotic peptide or an anticancer drug is bound to melittin, a mutant thereof, or an analog thereof may be a peptide-drug conjugate (PDC), and 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.

[0028] In one embodiment, 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.

[0029] In one embodiment, melittin, a variant or analog thereof and a pro-apoptotic peptide or an anti-cancer drug can be covalently bonded via a linker or non-covalently bonded without the use of a linker.

[0030] In one embodiment, the linker may comprise the amino acid sequence of SEQ ID NO:3.

[0031] In one embodiment, melittin, its variants or analogs thereof may further comprise a targeting sequence, tag, labeling residue or amino acid sequence designed for the specific purpose of increasing half-life or stability.

[0032] In one embodiment, the pro-apoptotic peptide is 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, or a combination thereof. conjugate, MG2B), Pardaxin, and combinations thereof, more preferably D-isomer KLA, and may comprise the amino acid sequence of SEQ ID NO:4.

[0033] 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.

[0034] In one embodiment, the melittin, its variants or analogs and the pro-apoptotic peptide or anti-cancer drug may be linked by a chemical linker or a direct bond, and the chemical linker may be linked via an amine group, a carboxyl group or a sulfhydryl group on the melittin, its variants or analogs and the pro-apoptotic peptide or anti-cancer drug.

[0035] In one embodiment, the chemical linker may include a functional group selected from a carbodiimide group, an N-hydroxysuccinimide ester (NHS ester), an imidoester, a pentafluorophenyl ester, a hydroxymethyl phosphine, a maleimide, a haloacetyl, a pyridyldisulfide, a thiosulfonate, a vinylsulfone, and a combination thereof at both ends. Examples of the functional group include 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), N,N'-dicyclohexylcarbodiimide (DCC), succinimidyl ester (SATA), and combinations thereof. acetylthioacetate), Sulfo-SMCC (sulfosuccinimidyl-4-(NDmaleimidomethyl)cyclohexane-1-carboxylate), DMA (dimethyladipimidate 2HCl), DMP (dimethylpimelimidate 2HCl), DMS (dimethylSuberimidate 2HCl), DTBP (dimethyl 3,3'-dithiobispropionimidate) 2HCl), Sulfo-SIAB(sulfosuccinimidyl(4-iodoacetyl)aminobenzoate), SIAB(succinimidyl(4-iodoacetyl)aminobenzoate), SBAP(succinimidyl 3-(bromoacetamido)propionate), SIA(succinimidyl iodoacetate), SM(PEG)n(succinimidyl-([N-maleimidopropionamido]-#ethyleneglycolester、n=2、4、6、8、12または24)、SMCC(succinimidyl-4-(N-Dmaleimidomethyl)cyclohexane-1-carboxylate)、LCSMCC(succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxy-(6-amidocaproate))、スルホ-EMCS(N-εester)、EMCS(N-εスルホ-GMBS(N-γester)、GMBS(N-γester)、スルホ-KMUS(N-κester)、スルホ-MBS(m-maleimidobenzoyl-Nhydroxysulfosuccinimide ester)、MBS(m-maleimidobenzoyl-N-hydroxysuccinimide ester)、スルホ-SMPB(sulfosuccinimidyl 4-(p-maleimidophenyl)butyrate)、SMPB(succinimidyl 4-(pmaleimidophenyl)butyrate)、AMAS(N-α-maleimidoacet-oxysuccinimide ester)、BMPS(N-β-maleimidopropyloxysuccinimide ester)、SMPH(succinimidyl6-[(β-maleimidopropionamido)hexanoate])、PEG12-SPDP(2-pyridyldithiol-tetraoxaoctatriacontane-N-hydroxysuccinimide)、PEG4-SPDP、スルホ-LCSPDP(sulfosuccinimidyl6-[3’-(2-pyridyldithio)propionamido]hexanoate)、SPDP(succinimidyl 3-(2-pyridyldithio)propionate)、LC-SPDP(succinimidyl6-[3’-(2-pyridyldithio)propionamido]hexanoate)、SMPT(4-succinimidyloxycarbonyl-alpha-methyl-alpha(2-pyridyldithio)toluene)、DSS(disuccinimidylsuberate)、BS(PEG)5(bis(succinimidyl)penta(ethylene glycol))、BS(PEG)9(bis(succinimidyl)nona(ethylene glycol))、BS3(bis[sulfosuccinimidyl]suberate)、BSOCOES(bis[2-(succinimidooxycarbonyloxy)ethyl]sulfone)、PDPH(3-(2-pyridyldithio)propionyl hydrazide)、DSG(disuccinimidyl glutarate)、DSP(dithiobis[succinimidyl propionate])、BM(PEG)n(1,8-bismaleimido-ethyleneglycol、n=2または3)、BMB(1,4-bismaleimidobutane)、BMDB(1,4-bismaleimidyl-2,3-dihydroxybutane)、BMH(bismaleimidohexane)、BMOE(bismaleimidoethane)、DTME(dithiobismaleimidoethane)、TMEA(tris(2-maleimidoethyl)amine)、DSS(disuccinimidyl suberate)、DST(disuccinimidyl tartarate)、DTSSP(3,3’-dithiobis[sulfosuccinimidylpropionate])、EGS(ethylene glycol bis[succinimidylsuccinate])、スルホ-EGS(ethylene glycolThe sulfonyl ether derivative may be selected from bis[sulfosuccinimidylsuccinate] and tris-succinimidylaminotriacetate (TSAT), 1,5-difluoro-2,4-dinitrobenzene (DFDNB), and combinations thereof.

[0036] In the present invention, the peptide preferably has the above amino acid sequence, but is not limited thereto. According to a preferred embodiment of the present invention, the peptide preferably has a high ratio of the above amino acids, such as 50% or more, preferably 60% or more, more preferably 70% or more, more preferably 80% or more, even more preferably 90% or more, and most preferably 100%.

[0037] In the present invention, the peptides may also include targeting sequences, tags, labeled residues, and additional amino acid sequences specifically designed to increase the half-life or stability of the peptide. The peptides of the present invention may also be linked to coupling partners such as effectors, drugs, prodrugs, toxins, peptides, and transmitter molecules.

[0038] In the present invention, the peptide can be prepared in the form of a pharma- ceutically acceptable salt. Specifically, the salt can be formed by adding an acid, for example, inorganic acids (e.g., hydrochloric acid, hydrobromic acid, phosphoric acid, nitric acid, sulfuric acid, etc.), organic carboxylic acids (e.g., acetic acid, haloacetic acid such as trifluoroacetic acid, propionic acid, maleic acid, succinic acid, malic acid, citric acid, tartaric acid, salicylic acid), and organic sulfonic acids (e.g., methanesulfonic acid, p-toluenesulfonic acid) including acidic sugars (glucuronic acid, galacturonic acid, gluconic acid, ascorbic acid), acidic polysaccharides (e.g., hyaluronic acid, chondroitin sulfate, alginic acid), and sulfonic acid sugar esters such as chondroitin sulfate can be added to form a salt.

[0039] The term "conjugate" as used herein refers to a complex of melittin, its variants or analogue peptides with proapoptotic peptides or anticancer drugs, which can target M2-type tumor-associated macrophages. The conjugate binds to the targeted M2-type macrophages and damages the mitochondria of the macrophages, thereby inhibiting tumor growth and metastasis, and selectively inhibiting angiogenesis in the surrounding area, thereby inhibiting cancer.

[0040] That is, the conjugate of the present invention may have improved anticancer activity compared to an anticancer drug, but is not limited thereto.

[0041] In the present invention, the conjugate may be a conjugate in which melittin, its variant or analogue thereof is linked to a proapoptotic peptide via a GGGGS linker (SEQ ID NO: 3), or may be linked to an anticancer drug such as Doxorubicin, Methotrexate, Entinostat, Cladribine, Pralatrexate and Lorlatinib via a SPDP linker. In addition, among the anticancer drugs, Maytansine DM1, Maytansine DM3 and Maytansine DM4 may be directly linked and conjugated without a linker, but are not limited thereto.

[0042] That is, the conjugate of the present invention may be in a form in which melittin, a mutant thereof, or an analog thereof is linked to an anticancer drug via a chemical linker or directly, but is not limited thereto.

[0043] The term "melittin (MEL)" of the present invention is a peptide that constitutes the main component of bee venom. The term "bee venom (BV)" used in the present specification is a mixture of acidic and alkaline secretions produced in the abdomen of honeybees (Apismellifera), and takes the form of a bitter, colorless liquid. Its main components are peptides melittin, apamin, mast cell degranulating (MCD) peptide, and enzyme phospholipase A2 (PLA2), and also contain various other trace components. Therefore, the melittin of the present invention may be isolated from honeybee (Apismellifera) bee venom, but is not limited thereto.

[0044] The term "peptide" used in the present invention means an amino acid polymer, which can contain not only natural amino acids but also non-proteinogenic amino acids as components.

[0045] As used herein, the term "variant" refers to a corresponding amino acid sequence that contains at least one amino acid difference (substitution, insertion or deletion) when compared to a reference. In certain embodiments, a "variant" has high amino acid sequence homology and / or conservative amino acid substitutions, deletions and / or insertions when compared to a reference sequence.

[0046] As used herein, the term "peptide analog" includes analogs in which one or more other functional groups are substituted on the amino acid side chain or on the alpha-amino acid backbone. Examples of peptide analogs with modified side chains or backbones include, but are not limited to, hydroxyproline, in which the pyrrolidine ring is replaced with a hydroxy group, and N-methylglycine "peptoids." The types of peptide analogs are known in the art.

[0047] Protein variants according to the present invention are also intended to include variants in which amino acid residues are conservatively substituted at particular amino acid residue positions.

[0048] In the present invention, the term "conservative substitution" refers to a modification of a mutant that includes replacing one or more amino acids with amino acids having similar biochemical properties that do not cause the loss of biological or biochemical functions of the melittin mutant protein. A "conservative amino acid substitution" is a substitution in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Classes of amino acid residues having similar side chains are defined and widely known in the art. These classes include amino acids with basic side chains (e.g., lysine, arginine, histidine), amino acids with acidic side chains (e.g., aspartic acid, glutamic acid), amino acids with uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), amino acids with non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), amino acids with beta-branched side chains (e.g., threonine, valine, isoleucine) and amino acids with aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).

[0049] The peptides according to the invention can be produced by standard synthesis methods, recombinant expression systems, or any other method known in the art. Thus, the peptides according to the invention can be synthesized by a number of methods, including, for example, the following: (a) synthesizing peptides stepwise or by fragment assembly by means of solid-phase or solution-phase techniques, and isolating and purifying the final peptide product; or (b) expressing a nucleic acid construct encoding the peptide in a host cell and recovering the expression product from the host cell culture; or (c) carrying out cell-free in vitro expression of a nucleic acid construct encoding a peptide and recovering the expression product; or A method comprising obtaining peptide fragments by any combination of (a), (b) and (c), then ligating the fragments to obtain a peptide, and recovering the peptide. In one aspect, the present invention relates to a pharmaceutical composition for preventing or treating cancer, comprising as an active ingredient a conjugate of melittin, a mutant or analog thereof, and a proapoptotic peptide or an anticancer drug.

[0050] 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, The cancer may be any one or more selected from the group consisting of breast cancer, ovarian cancer, colon cancer, small intestine cancer, rectal cancer, fallopian tube cancer, anal cancer, endometrial cancer, vaginal cancer, vulvar cancer, Hodgkin's disease, 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, and may be an anticancer drug resistant cancer.

[0051] In one embodiment, the composition can be administered to a subject in which expression of ITGB2 is upregulated in M2 macrophages compared to M0 and M1 macrophages.

[0052] In one embodiment, the composition may be a targeted anti-cancer drug.

[0053] 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 cyclophosphamide. 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.

[0054] 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.

[0055] 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.

[0056] 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 can be administered in combination with other therapeutic agents, can be administered sequentially or simultaneously with conventional therapeutic agents, and can be administered in a single 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.

[0057] 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).

[0058] 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. 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).

[0059] 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.

[0060] 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.

[0061] 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.

[0062] 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.

[0063] In one aspect, the present invention relates to a method for removing M2 tumor-associated macrophages, comprising the step of treating M2 tumor-associated macrophages in a test tube with a composition of the present invention.

[0064] In one aspect, the present invention relates to a composition for diagnosing cancer, comprising the composition of the present invention.

[0065] In one aspect, the present invention relates to a cancer diagnostic kit comprising the cancer diagnostic composition of the present invention.

[0066] In the present invention, the term "cancer diagnostic kit" refers to a kit containing the cancer diagnostic composition of the present invention. Therefore, the expression "cancer diagnostic kit" can be used cross-sectionally or interchangeably with "cancer diagnostic composition". As used herein, the term "diagnosis" includes determining the susceptibility of an object to a particular disease or disorder, determining whether an object currently has a particular disease or disorder, determining the prognosis of an object suffering from a particular disease or disorder (e.g., identifying a pre-metastatic or metastatic cancer state, determining the stage of a cancer, or determining the response of a cancer to a treatment), or therametrics (e.g., monitoring the status of an object to provide information on the efficacy of a treatment).

[0067] In the present invention, melittin, its variants or analogs thereof specifically bind to ITGB2 expressed on the cell membrane of M2 tumor-associated macrophages, and thus they themselves or drugs (proapoptotic peptides or anticancer drugs) linked thereto kill M2 tumor-associated macrophages, thereby suppressing tumor growth and metastasis, and selectively suppressing angiogenesis around the tumor, thereby exhibiting anticancer activity. Since they specifically bind to M2 tumor-associated macrophages, they can be used as theranostic preparations for use in cancer diagnosis. They can also be used as anticancer adjuvants administered simultaneously, separately or sequentially with anticancer drugs. In addition, since melittin, its variants or analogs thereof specifically bind to M2 tumor-associated macrophages, they can also be used as drug delivery agents targeting the M2 tumor-associated macrophages.

[0068] In one aspect, the present invention relates to a method for providing information for cancer diagnosis, comprising contacting a biological sample isolated from a subject with a composition of the present invention; and comparing with a normal control sample.

[0069] In one embodiment, the method may further include determining that the test subject has cancer if the level of M2 tumor-associated macrophages in the biological sample isolated from the subject is higher than that in a normal control sample.

[0070] The term "sample" as used herein means a biological sample obtained from a subject or patient. The source of the biological sample may be fresh, frozen and / or preserved organ or tissue samples or solid tissue from biopsies or aspirates; blood or any blood components; cells at any time during the subject's pregnancy or development.

[0071] In one aspect, the present invention relates to a method for treating cancer, comprising administering to a subject suffering from cancer a pharma- tically effective amount of a conjugate of the present invention, a melittin, variant or analog thereof, linked to a pro-apoptotic peptide or an anti-cancer drug.

[0072] 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.

[0073] In one aspect, the present invention relates to the use of a conjugate of melittin, a variant or an analog thereof linked to a pro-apoptotic peptide or an anti-cancer drug for the preparation of a pharmaceutical composition for the prevention and treatment of cancer.

[0074] 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.

[0075] 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.

[0076] [Table 1]

[0077] Example 2. Analysis of M2 TAM Targeting 2-1. Confirmation of the affinity of melittin for M2 TAM To confirm whether the melittin prepared in Example 1 specifically binds to M2 tumor-associated macrophages (M2 TAM), 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).

[0078] 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.

[0079] 2-2. Identification of M2 TAM-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 the macrophages (18-well plate) into M0, M1, and M2 macrophages, the cells were collected using a scraper and washed by centrifugation (300Xg, 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 (200μg) at room temperature (20-24℃) for 1 hour, the proteins bound to melittin were obtained using streptavidin resin (Thermo Fisher Scientific). To remove salts, 100μl 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 50μl 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.

[0080] [Table 2]

[0081] 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.

[0082] 2-3. Identification of M2 TAM-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.

[0083] 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.

[0084] 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.

[0085] [Table 3]

[0086] The results showed that ITGB2 mRNA expression was significantly increased in M2 macrophages and TAMs compared with M0 and M1 (Figure 6).

[0087] 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, proteins were extracted from each cell differentiated into M0, M1 and M2 macrophages using Proprep solution (iNtRON) and transferred to a membrane by electrophoresis. After blocking by immersing the membrane in 5% BSA, the membrane was treated with a primary antibody (ITGB2) for 24 hours at 4°C, and a secondary antibody for 1 hour at room temperature, and then treated with D-Plus (trademark pending) ECL Pico System (Doning LS) and protein band photographs were taken with a Davinch-Chemi (trademark pending) imaging system (Intoxia).

[0088] The results showed that ITGB2 protein expression was significantly increased in M2 macrophages and TAMs compared to M0 and M1 (Figure 7).

[0089] 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).

[0090] As a result, ITGB2 protein was shown to have higher expression levels in M2 macrophages and TAMs compared to M0 and M1 macrophages (Figure 8).

[0091] Example 4. Confirmation of the interaction of M2 TAM targeting peptide with 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).

[0092] 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 (300Xg, 5 minutes), 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 protein analysis was performed to confirm the expression of ITGB2.

[0093] 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.

[0094] 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).

[0095] 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 CD18PDB file created by 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).

[0096] 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).

[0097] 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.

[0098] [Table 4]

[0099] 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 M2 TAM of CD18, 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 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 anti-CD18 antibody and DM1 are bound, was named CD18-DM1.

[0100] 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) conjugated 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 conjugated, was named CD18-MMAE.

[0101] Example 6. Confirmation of M2 TAM 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.

[0102] [Table 5]

[0103] [Table 6]

[0104] 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.

[0105] Example 7. Confirmation of the killing effect of M2 TAM cells by ITGB2 of 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, reacting for 5-10 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.

[0106] [Table 7]

[0107] 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).

[0108] 7-2. Confirmation of M2 TAM cell death by ITGB2 in Melittin-dKLA In order to confirm whether Melittin-dKLA binds to ITGB2 in M2 macrophages and induces cell apoptosis, the 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 the cell viability was analyzed using the CCK-8 analysis method. Specifically, the ITGB2 lockdown macrophages prepared in Example 7-1 were reacted with Melittin-dKLA (1 μM) for 1 hour. After the reaction, the medium was replaced and cultured at 37 ° C for 24 hours. To 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. The absorbance was measured at 450 nm using a microplate leader (Molecular Devices).

[0109] As a result, Melittin-dKLA significantly reduced cell viability in control M2 macrophages, but the reduction in cell viability caused by Melittin-dKLA was suppressed in ITGB2-locked M2 macrophages (Figure 19). Therefore, it was confirmed that Melittin-dKLA induces cell apoptosis in M2 macrophages by specifically binding to ITGB2 in M2 macrophages.

[0110] 7-3. Confirmation of M2 TAM 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).

[0111] 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 or not TB511 induces cell death 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.

[0112] 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).

[0113] 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, 3D organoids simulating the tumor microenvironment (TME) of breast cancer were prepared, and the effect of TB511 on them was confirmed. Specifically, Matrigel was mixed to 3% in RPMI-1640 culture medium, and MDA-MB-231 human breast cancer cells, CAF (Cancer-associated Fibroblast), and THP-1 cells prepared to express GFP using Incucyte (registered trademark) 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.

[0114] 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.

[0115] 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.

[0116] 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, 10 days after drug treatment, the spheroids were fluorescently stained 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).

[0117] 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.

[0118] 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).

[0119] 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.

[0120] 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).

[0121] 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.

[0122] 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).

[0123] 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.

[0124] 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).

[0125] 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.

[0126] 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).

[0127] 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.

[0128] 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).

[0129] 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.

[0130] 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 pharmaceutical composition for eliminating tumor associated macrophages (TAM) in a tumor microenvironment, comprising as an active ingredient a conjugate in which melittin, a mutant thereof, or an analog thereof is bound to a proapoptotic peptide or an anticancer drug.

2. The pharmaceutical composition for eliminating tumor-associated macrophages in a tumor microenvironment according to claim 1, wherein the tumor-associated macrophages are M2 tumor-associated macrophages that express IGBT2 (Integrin beta 2).

3. The pharmaceutical composition for eliminating tumor-associated macrophages in a tumor microenvironment according to claim 1, wherein melittin, a mutant thereof or an analog thereof specifically binds to an extended active conformation of CD18.

4. The pharmaceutical composition for eliminating tumor-associated macrophages in a tumor microenvironment according to claim 1, wherein melittin, a mutant thereof or an analog thereof binds to amino acids 449466-565 of ITGB2.

5. A pharmaceutical composition for eliminating tumor-associated macrophages in a tumor microenvironment as described in claim 1, wherein melittin comprises the amino acid sequence of SEQ ID NO:

1.

6. A pharmaceutical composition for eliminating tumor-associated macrophages in a tumor microenvironment as described in claim 1, wherein the melittin mutant comprises the amino acid sequence of SEQ ID NO:

2.

7. The pharmaceutical composition for eliminating tumor-associated macrophages in a tumor microenvironment according to claim 1 , wherein the pharmaceutical composition is linked by a linker.

8. The pharmaceutical composition for eliminating tumor-associated macrophages in a tumor microenvironment according to claim 7, wherein the linker comprises the amino acid sequence of SEQ ID NO:

3.

9. A pharmaceutical composition for eliminating tumor-associated macrophages in a tumor microenvironment according to claim 1, comprising a peptide having the amino acid sequence of SEQ ID NO: 5 or 6.

10. 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).

2. The pharmaceutical composition for eliminating tumor-associated macrophages in a tumor microenvironment according to claim 1, wherein the compound is selected from the group consisting of MG2B, MG2A conjugate, MG2B, Pardaxin, and combinations thereof.

11. 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 2. The pharmaceutical composition for eliminating tumor-associated macrophages in a tumor microenvironment according to claim 1, wherein the active ingredient is selected from the group consisting of monomethyl auristatin E (MMAE), auristatin F, monomethyl auristatin E (MMAE), monomethyl auristatin F and derivatives thereof.

12. A pharmaceutical composition for preventing or treating cancer, comprising as an active ingredient a conjugate in which melittin, a mutant or an analog thereof is bound to a proapoptotic peptide or an anticancer drug.

13. Cancers include brain cancer, melanoma, myeloma, non-small cell lung cancer, oral cancer, liver cancer, stomach 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, 13. The pharmaceutical composition for preventing or treating cancer according to claim 12, wherein the cancer is any one or more selected from the group consisting of cancer of the esophagus, 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.

14. The pharmaceutical composition for preventing or treating cancer according to claim 12, wherein the cancer is an anticancer drug resistant cancer.

15. The pharmaceutical composition for preventing or treating cancer according to claim 12, which is administered to a patient in whom ITGB2 expression is upregulated in M2 macrophages compared to M0 and M1 macrophages.

16. A method for treating cancer, comprising administering to a subject suffering from cancer a pharma- tically effective amount of a conjugate of melittin, a variant thereof or an analog thereof, and a proapoptotic peptide or an anticancer drug.

17. Use of a conjugate comprising melittin, a mutant or an analog thereof, and a proapoptotic peptide or an anticancer drug, for the manufacture of a pharmaceutical composition for the prevention and treatment of cancer.

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