Targeting of M2 tumor-associated macrophages using a melittin-based apoptosis-inducing peptide

Abstract

The present invention relates to a melittin-anticancer agent conjugate formed by combining melittin with an anticancer agent and a method for preparing the melittin-anticancer agent conjugate by connecting melittin and an anticancer agent. The conjugate of the present invention is an anticancer substance targeting M2 tumor-associated TAMs and has an excellent effect of selectively screening M2 tumor-associated TAMs. Therefore, it will be suitable for drug delivery targeting M2 tumor-associated macrophages in the future.

Description

Technical Field

[0001] The present invention relates to a melittin-anticancer agent conjugate formed by combining melittin with an anticancer agent, and more specifically, to a melittin-anticancer agent conjugate of the present invention which inhibits only M2 tumor-associated macrophages without affecting M1 tumor-associated macrophages and cancer cells, and a preparation method thereof. Background Art

[0002] Tumor-associated macrophages are important innate immune cells found in almost all tissues. They originate from the bone marrow and circulate in the blood, and differentiate in tissues through outflow from blood vessels. These tumor-associated macrophages are classified into two phenotypes: tumor-suppressing M1 macrophages and tumor-supporting M2 macrophages. M1 macrophages have a strong ability to indicate antigens, and are usually activated by interferon-γ, liposaccharide (LPS), and tumor necrosis factor (TNF)-α, and have pro-inflammatory and bactericidal effects.

[0003] M2 macrophages promote immunosuppression, tumor formation and angiogenesis by releasing a variety of extracellular matrix components, angiogenesis and chemokines. Usually, M2 macrophages are induced by IL-4 and IL-13, and express unique markers such as arginase-1, mannose (MMR, CD206), scavenger receptor (SR-A, CD204) that only M2 has, thus distinguishing them from M1 macrophages.

[0004] Melittin is a major component of the bee venom of the honey bee (Apis mellifera L.) and is an amphipathic peptide with 26 amino residues. Melittin has membrane perturbing effects such as pore formation, fusion and vesicle formation. Melittin is used in tumor-bearing mouse studies because of its ability to inhibit cytotoxicity and cell growth against tumor cells or induce cell apoptosis and necrosis (Cancer Immunol Immunother. 2004; 53: 411-421.).

[0005] Also, as conventional technologies utilizing melittin, a composition for treating arteriosclerosis containing melittin (Publication No. 10-2011-0117789) and a composition for inhibiting the activity of fibroblast-like synoviocytes containing melittin (Publication No. 10-2011-0117788) are known.

[0006] On the other hand, a pharmaceutical composition for selective apoptosis of M2 macrophages using bee venom peptide has been confirmed (Publication No.: 10-2019-0021765), but an M2-targeted pharmaceutical composition using it as a binding partner has not yet been disclosed. In response to this, the inventors prepared a conjugate formed by combining bee venom peptide with an anticancer agent, and confirmed that bee venom peptide only inhibited CD206+ tumor-associated macrophages as M2 tumor-associated macrophages in a tumor mouse model without affecting CD86+ tumor-associated macrophages and cancer cells as M1 tumor-associated macrophages, thereby completing the present invention in which the side effects caused by previous anticancer agents were significantly reduced. Summary of the invention

[0007] Technical issues

[0008] The object of the present invention is to provide a melittin-anticancer agent conjugate formed by combining melittin with an anticancer agent.

[0009] Another object of the present invention is to provide a method for preparing a melittin-anticancer agent conjugate by linking melittin and an anticancer agent.

[0010] Technical Solution

[0011] One embodiment of the present invention for solving the above-mentioned problems provides a melittin-anticancer agent conjugate in which melittin is conjugated to an anticancer agent.

[0012] The term "Melittin (MEL)" of the present invention is a peptide that constitutes the main component of bee venom. The above-mentioned term "bee venom (BV)" used herein is a mixture of acidic and alkaline secretions produced in the abdomen of honey bees (Apis mellifera), in the form of a colorless bitter liquid, and its main components are melittin, apamin, and mast cell degranulating (MCD) peptides as peptides and phospholipase A2 (phospholipase A2; PLA2) as an enzyme, etc., and also contain trace amounts of various components. Therefore, the melittin of the present invention can be isolated from the bee venom of honey bees (Apis mellifera), but is not limited to this.

[0013] In a specific embodiment of the present invention, it was confirmed that the treatment of M1 and M2 macrophages was performed by combining the apoptotic peptide dKLA with the MEL peptide targeting CD206+M2 macrophages through a GGGS linker, thereby removing only the M2 macrophages in the tumor stroma without affecting other eukaryotic cells ( Figures 1 to 3 ), and it was confirmed that the conjugate composed of MEL peptide and DM1 anticancer agent also only removed M2 macrophages in the tumor stroma ( Figure 4 ), confirming cell death caused by mitochondrial membrane disturbance by measuring cellular respiration ( Figure 5 to Figure 6 ), and confirmed by staining that the conjugate of the present invention was inserted into the mitochondria ( Figure 7 Furthermore, when mice were treated with MEL, dKLA, and MEL-dKLA, the MEL-dKLA conjugate reduced the size and weight of the tumor ( Figure 8 ), and inhibited the number of tumor nodules and intratumor metastasis ( Fig. 9 ), the conjugate inhibited breast cancer growth in experimental mice and confirmed by luminescence that it inhibited metastasis to the lungs and the whole body ( Figure 10 to Figure 11 ). In addition, it was confirmed that when the MEL-anticancer agent conjugate was treated, the expression of CD44, which is expressed in many cancers, CCL22, which is a marker of M2 macrophages, HIF-1α, which is a marker of angiogenesis, metastasis and infiltration, Ym1, which is a marker of M2 macrophages, and MMP-9, which intervenes in the migration and retention of tumor cells, was reduced ( Fig.12 ), and confirmed that only M2 TAMs were reduced by measuring the changes in the ratio of immune cells in the tumor through staining ( Fig.13 ), and selective cell death of M2 tumor-associated TAMs was confirmed by staining ( Fig.14 ). In addition, after treatment with MEL and MEL-dKLA, tumor endothelial cells were photographed using a laser scanning confocal microscope to confirm that the vascular density in the endothelial cells decreased ( Fig.15 ).

[0014] The melittin of the present invention has the effect of targeting M2 macrophages and can produce anticancer activity by delivering an anticancer agent bound to melittin to M2 macrophages, but the present invention is not limited thereto.

[0015] The term "anticancer agent" in the present invention is a general term for drugs used in chemotherapy for treating cancer. The anticancer agent may be a compound or a pro-apoptotic peptide, but is not limited thereto.

[0016] In the present invention, the term "cancer" refers to an abnormally growing tumor or a tumor-forming disease caused by the autonomous excessive growth of body tissues.

[0017] Specifically, the above-mentioned cancer can be lung cancer (e.g., non-small cell lung cancer, small cell lung cancer, malignant mesothelioma), mesothelioma, pancreatic cancer (e.g., pancreatic duct cancer, pancreatic endocrine tumor), pharyngeal cancer, laryngeal cancer, esophageal cancer, gastric cancer (e.g., papillary adenocarcinoma, mucinous adenocarcinoma, adenosquamous carcinoma), duodenal cancer, small intestine cancer, colorectal cancer (e.g., colon cancer, rectal cancer, anal cancer, familial colorectal cancer, hereditary non-polyposis colorectal cancer, gastrointestinal stromal tumor), breast cancer (e.g., invasive breast cancer, non-invasive breast cancer, inflammatory breast cancer), ovarian cancer (e.g., epithelial ovarian cancer, extratesticular germ cell tumor, ovarian germ cell tumor, ovarian malignant tumor), testicular tumor, prostate cancer (e.g., hormone-dependent prostate cancer, hormone-independent prostate cancer), liver cancer (e.g., hepatocellular carcinoma, primary liver cancer, extrahepatic bile duct cancer), The present invention relates to adenocarcinoma (e.g., medullary thyroid carcinoma), kidney cancer (e.g., renal cell carcinoma, transitional cell carcinoma of the renal pelvis and ureter), uterine cancer (e.g., cervical cancer, uterine body cancer, uterine sarcoma), brain tumor (e.g., medulloblastoma, glioma, pineal cell tumor, pilocytic astrocytoma, diffuse astrocytoma, degenerative astrocytoma, pituitary adenoma), retinoblastoma, skin cancer (e.g., basal cell carcinoma, malignant melanoma), sarcoma (e.g., rhabdomyosarcoma, leiomyosarcoma, soft tissue sarcoma), malignant bone tumor, bladder cancer, blood cancer (e.g., multiple myeloma, leukemia, malignant lymphoma, Hodgkin's disease, chronic myeloproliferative disease), cancer of unknown primary origin, etc. More specifically, it can be lung cancer, metastatic tumor or breast cancer. More specifically, the above-mentioned lung cancer can be Lewis lung cancer, but it is not limited to this.

[0018] In the present invention, the above-mentioned anticancer agent can be doxorubicin, methotrexate, entinostat, cladribine, pralatrexate, lorlatinib, maytansine DM1, maytansine DM3, maytansine DM4, etc., but is not limited thereto.

[0019] The term "pro-apoptosis" of the present invention refers to a process in which cells actively consume ATP as a bioenergy source and die. A typical apoptosis process is carried out by cell shrinkage, regular DNA severing, and cell membrane fragmentation. When cells cannot maintain normal functions due to abnormal cell division, radiation, ultraviolet rays, bacterial infection, or viral infection, apoptosis can be induced.

[0020] In the present invention, the pro-apoptotic peptide may be 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, but is not limited thereto.

[0021] The term "peptide" of the present invention refers to a polymer formed by amino acids linked by amide bonds (or peptide bonds). For the purpose of the present invention, it refers to a peptide that has a high selectivity for cancer cells and produces a strong anti-cancer activity.

[0022] In the present invention, preferably, the above-mentioned peptide has the above-mentioned amino acid sequence, but is not limited thereto. According to a preferred embodiment of the present invention, in the above-mentioned peptide, the ratio of the above-mentioned amino acids is 50% or more, preferably 60% or more, more preferably 70% or more, more preferably 80% or more, more preferably 90% or more, and most preferably up to 100%.

[0023] In the present invention, the above-mentioned peptides may also contain targeting sequences, tags, labeled residues, additional amino acid sequences proposed for specific purposes for increasing the half-life or stability of the peptides. In addition, the peptides of the present invention may be linked to partners such as effectors, drugs, prodrugs, toxins, peptides, and delivery molecules.

[0024] In the present invention, the above peptides can be obtained by various methods known in the art. Specifically, they can be prepared using gene recombination and protein expression systems or by chemical synthesis methods such as peptide synthesis in vitro and cell-free protein synthesis.

[0025] In the present invention, the peptide can be prepared into a pharmaceutically acceptable salt form. Specifically, the salt can be formed by adding an acid, for example, by adding an inorganic acid (e.g., hydrochloric acid, hydrobromic acid, phosphoric acid, nitric acid, sulfuric acid, etc.), an organic carboxylic acid (e.g., acetic acid, trifluoroacetic acid and other halogenated acetic acid, propionic acid, maleic acid, succinic acid, malic acid, citric acid, tartaric acid, salicylic acid), and an acidic sugar (glucuronic acid, galacturonic acid, gluconic acid, ascorbic acid), an acidic polysaccharide (e.g., hyaluronic acid, chondroitin sulfate, arginine), an organic sulfonic acid (e.g., methanesulfonic acid, p-toluenesulfonic acid) containing sulfonic sugar esters such as chondroitin sulfate, etc. to form a salt.

[0026] The term "conjugate" of the present invention refers to a conjugate formed by combining melittin with an anticancer agent, and can be a conjugate targeting M2 tumor-associated macrophages. The conjugate can damage the mitochondria of macrophages by combining with M2 macrophages targeted by drugs, thereby inhibiting tumor growth and metastasis, and can inhibit cancer by selectively inhibiting peripheral angiogenesis.

[0027] That is, the anticancer activity of the conjugate of the present invention can be improved compared to the anticancer agent, but the present invention is not limited thereto.

[0028] In the present invention, the above-mentioned conjugate can be a conjugate formed by connecting MEL (sequence 1; GIGAVLKVLTTGLPALISWIKRKRQQ) purchased from Piscataway, NJ, USA and dKLA (sequence 2; d[KLAKLAKKLAKLAK]) peptides through a GGGGS linker, or can be a conjugate formed by connecting anticancer agents such as doxorubicin, methotrexate, entinostat, cladribine, pralatrexate and lorlatinib through an SPDP linker. In addition, maytansine DM1, maytansine DM3 and maytansine DM4 can be prepared by combining without a linker, but are not limited thereto.

[0029] That is, the conjugate of the present invention may be in a form in which melittin and the anticancer agent are linked via a chemical linker or directly linked.

[0030] In the present invention, the term "chemical linker" can be combined through the amine, carboxyl or sulfhydryl groups on bee venom peptide and anticancer agents, but is not limited thereto. Specifically, the chemical linker can be EDC (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide), DCC (N,N'-dicyclohexylcarbodiimide), SATA (succinimidyl acetylthioacetate), sulfo-SMCC (sulfosuccinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate (salt)), DMA (dimethyl diimidate adipate·2HCl), DMP (dimethyl diimidate pimelate·2HCl), DMS (dimethyl diimidate suberate·2HCl), DTBP (3,3'-dithiobispropionimidate dimethyl ester·2HCl), sulfo-SIAB (sulfosuccinimidyl (4-iodoacetic acid) aminobenzoate), SIAB (succinimidyl (4-iodoacetic acid) aminobenzoate), SBAP (succinimidyl 3-(bromoacetyl) propionate), SIA (succinimidyl iodoacetate), SM(PEG)n (succinimidyl-[N-maleimidopropionamido]-#ethylene glycol ester, n=2, 4, 6, 8, 12 or 24), SMCC (succinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate), LCSMCC (succinimidyl-[4-(N-maleimidomethyl)]-cyclohexane-1-carboxylate- (6-aminohexanoate), sulfo-EMCS (6-maleimidohexanoic acid sulfosuccinimide ester), EMCS (6-(maleimido)hexanoic acid succinimide ester), GMBS (4-maleimidobutyric acid-N-succinimide), sulfo-KMUS (11-maleimidoundecanoic acid sulfo-N-succinimide ester), sulfo-MBS (m-maleimidobenzoyl-N-hydroxysulfosuccinimide ester), MBS (m-maleimidobenzoyl-N-hydroxysuccinimide ester), sulfo-SMPB (sulfosuccinimidyl 4-(p-maleimidophenyl)butyrate), SMPB (succinimidyl 4-(maleimidophenyl)butyrate) ester), AMAS (N-α-maleimidoacetyl-oxysuccinimide ester), BMPS (N-β-maleimidopropoxysuccinimide ester), SMPH (succinimidyl 6-[(β-maleimidopropionamido)hexanoate]), PEG12-SPDP (2-pyridyldithiol-tetraoxooctacyclodecane-N-hydroxysuccinimide), PEG4-SPDP, sulfo-LCSPDP (6-hexanoic acid sulfosuccinimidyl ester), SPDP (3-(2-pyridyldithio) propionate succinimidyl ester), LC-SPDP (6-adipate succinimidyl ester), SMPT (4-succinimidyloxycarbonyl-α-permethyl-yl (2-pyridyldithio) toluene), DSS (disuccinimidyl suberate), BS(PEG)5 (bis(succinimidyl)pentyl(ethylene glycol)), BS(PEG)9 (bis(succinimidyl)nonyl(ethylene glycol)), BS3 (bis[sulfosuccinimidyl]suberate), BSOCOES (bis(2-[succinimidyloxycarbonyloxy)ethyl)sulfone), PDPH (3-(2-pyridyldithio)propionylhydrazide), DSG (disuccinimidyl glutarate), DSP (bis[succinimidyl propionate] dithioate), BM(PEG)n (1,8-dimaleimido-ethylene glycol, n=2 or 3), BMB (1,4-dimaleimidobutane), BMDB (1,4-dimaleimido-2,3-dihydroxybutane), BMH (bismaleimidohexane), BMOE (bismaleimidoethane), DTME (disulfide bismaleimidoethane), TMEA (tris(2-maleimidoethyl)amine), DSS (disuccinimidyl suberate), DST (disuccinimidyl tartrate), DTSSP (3,3'-dithiobis[sulfosuccinimidyl propionate]), EGS (ethylene glycol bis(succinimidyl succinate)), sulfo-EGS (ethylene glycol-bis[succinimidyl succinate] disulfonate) and TSAT (trisuccinimidyl aminotriacetate), DFDNB (1,5-difluoro-2,4-dinitrobenzene) and combinations thereof, but are not limited thereto. ,

[0031] The term "tumor associated macrophage (TAM, Tumor associated tumor)" of the present invention refers to macrophages that play an important role in the overall tumor microenvironment such as the growth and metastasis of cancer. Tumor associated macrophages present around the tumor are closely related to the growth and metastasis of tumor cells. Tumor associated macrophages are classified into two phenotypes, namely tumor suppressor M1 or tumor supportive M2 macrophages. M2 tumor associated macrophages have the function of generating cytokines such as IL-10, TGFβ and CCL18 that promote the growth of cancer and inhibiting the anti-tumor activity of T cells and NK cells through surface receptors. Such tumor associated macrophages can be differentiated from monocytes and macrophages produced in the bone marrow, yolk sac or extramedullary hematopoiesis, especially in the spleen, and preferably, can be isolated from the bone marrow, but is not limited thereto.

[0032] Still another embodiment of the present invention for solving the above-mentioned problems provides a pharmaceutical composition for preventing or treating a disease mediated by tumor-associated macrophages.

[0033] In the present invention, the composition may be a pharmaceutical composition for preventing or treating the growth and metastasis of cancer by removing M2 tumor-associated macrophages, but is not limited thereto.

[0034] The term "tumor-associated macrophages" of the present invention is as described above.

[0035] The term "prevention" of the present invention refers to all actions of inhibiting or delaying the growth and metastasis of tumors by the conjugate of the present invention.

[0036] The term "treatment" of the present invention refers to all actions that improve or ameliorate the symptoms of tumor growth and metastasis through the above-mentioned conjugates.

[0037] In the present invention, the conjugate is preferably used in humans, but can also be used in livestock such as cattle, horses, sheep, pigs, goats, camels, antelopes, dogs or cats that develop inflammatory diseases or cancer and can suppress or reduce cancer by administering the peptide of the present invention.

[0038] In the cancer prevention or treatment composition of the present invention, the administration route and administration method of the above-mentioned composition are not particularly limited, and any administration route and administration method can be used as long as the above-mentioned composition can reach the desired corresponding part. Specifically, the above-mentioned composition can be administered by various routes of oral or parenteral administration, and as non-limiting examples of its administration route, it can be administered by eyeball, oral, rectal, topical, intravenous, intraperitoneal, intramuscular, intraarterial, transdermal, intranasal or inhalation. In addition, the above-mentioned composition can be administered by any device that can move the active substance to the target cell.

[0039] In the present invention, the pharmaceutical composition may further include a pharmaceutically acceptable carrier, excipient or diluent commonly used in the preparation of a pharmaceutical composition, and the carrier may include a non-naturally occurring carrier.

[0040] In the present invention, the term "pharmaceutically acceptable" refers to the property of having no toxicity to cells or humans exposed to the composition.

[0041] More specifically, the pharmaceutical composition can be formulated into oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, sprays, external preparations, suppositories, and sterile injections by conventional methods for use. However, any dosage form used in the art for the purpose of preventing or treating cancer may be used without restriction.

[0042] Specific examples of carriers, excipients and diluents that can be included in the above-mentioned pharmaceutical composition include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum arabic, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, polycaprolactone (PCL), polylactic acid (PLA), poly-L-lactic acid (PLLA), mineral oil, etc.

[0043] In the preparation of a drug, a filler, a bulking agent, a binder, a wetting agent, a disintegrant, a surfactant, or other diluent or excipient is usually used for formulation.

[0044] Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc. Such solid preparations can be prepared by mixing the extracts and fractions thereof with at least one excipient such as starch, calcium carbonate, sucrose or lactose, gelatin, etc. In addition, in addition to the excipients alone, lubricants such as magnesium stearate, talc, etc. can also be used.

[0045] Liquid preparations for oral administration include suspensions, internal liquids, emulsions, syrups, etc., which may contain various excipients such as wetting agents, sweeteners, aromatics, and preservatives in addition to the commonly used simple diluents of water and liquid paraffin. Preparations for parenteral administration may include sterilized aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried preparations, suppositories, etc. As non-aqueous solvents and suspensions, vegetable oils such as propylene glycol, polyethylene glycol, and olive oil, and injectable esters such as ethyl oleate, etc. may be used. As the base of suppositories, witepsol, polyethylene glycol, tween 61, cocoa butter, laurin, glyceryl gelatin, etc. may be used.

[0046] Another embodiment of the present invention for solving the above-mentioned problems provides a method for preparing a melittin-anticancer agent conjugate, comprising the step of linking melittin and an anticancer agent.

[0047] Another embodiment of the present invention provides a method for preventing or treating a tumor-associated macrophage-mediated disease, comprising the step of administering the above-mentioned conjugate or a pharmaceutical composition comprising the conjugate to a subject in need thereof.

[0048] Another embodiment of the present invention for solving the above-mentioned problems provides a tumor-associated macrophage-mediated "preventive or therapeutic use of a disease" of melittin-anticancer agent linked to melittin and an anticancer agent.

[0049] Technical Effects

[0050] The MEL-anticancer agent conjugate of the present invention is an anticancer substance targeting M2 tumor-associated TAMs and has an excellent effect of selectively screening M2 tumor-associated TAMs. Therefore, the method of combining MEL with an anticancer agent will be applicable to drug delivery targeting M2 tumor-associated macrophages in the future. BRIEF DESCRIPTION OF THE DRAWINGS

[0051] Figure 1 Part (a) is a graph showing the cytotoxicity of dKLA, MEL, and MEL-dKLA against M1 by MTS assay.

[0052] Figure 1Part (b) is a graph showing the cytotoxicity of dKLA, MEL, and MEL-dKLA against M2 by MTS assay.

[0053] Figure 1 Part (c) is a bar graph showing the analysis of the cell cycle measured by PI staining.

[0054] Figure 2 This is a curve diagram showing the comparative analysis of the changes of M1 in response to dKLA, MEL and MEL-dKLA by flow cytometry analysis after staining with Annexin VFITC and MItoTracker-Red CMXRos.

[0055] Figure 3 This is a curve diagram showing the comparative analysis of the changes of M2 in response to dKLA, MEL and MEL-dKLA by flow cytometry analysis after staining with Annexin VFITC and MItoTracker-Red CMXRos.

[0056] Figure 4 The graph shows the cytotoxicity of MEL-DM1 against M2 by MTS assay.

[0057] Figure 5 Part (a) is a graph showing changes in intracellular respiration for measuring cell death caused by mitochondrial membrane disturbance.

[0058] Figure 5 Part (b) is a graph showing changes in the intracellular responses to cell death caused by mitochondrial membrane disturbance.

[0059] Figure 5 Part (c) is a bar graph showing changes in basal respiration used to measure cell death caused by mitochondrial membrane perturbations.

[0060] Figure 5 Part (d) is a bar graph showing changes in cellular ATP production used to measure cell death caused by mitochondrial membrane perturbations.

[0061] Figure 5 Part (e) is a bar graph showing the maximum intracellular respiration change used to determine cell death caused by mitochondrial membrane disturbance.

[0062] Figure 6 Part (a) is a graph showing changes in the cellular energy phenotype measured by XF in order to measure cell death caused by mitochondrial membrane perturbations.

[0063] Figure 6 Part (b) is a bar graph showing changes in intracellular oxygen ratio (OCR) for measuring cell death caused by mitochondrial membrane disturbance.

[0064] Figure 6 Part (c) is a bar graph showing changes in the extracellular acidification rate (ECAR) for measuring cell death caused by mitochondrial membrane disturbance.

[0065] Figure 7 Part (a) is a staining photograph showing the locations of MEL, dKLA, and MEL-dKLA in mitochondria.

[0066] Figure 7 Part (b) shows the correlation coefficients of MEL, dKLA and MEL-dKLA in mitochondria.

[0067] Figure 8 Part (a) is a bar graph comparing tumor sizes in order to compare the anticancer effects of MEL-dKLA.

[0068] Figure 8 Part (b) is a bar graph comparing tumor weights in order to compare the anticancer effects of MEL-dKLA.

[0069] Figure 8 Part (c) is a bar graph showing the fold change in tumor size for comparison of the anticancer effects of MEL-dKLA.

[0070] Figure 8 Part (d) is a bar graph comparing the weight changes of experimental mice in order to compare the anti-cancer effects of MEL-dKLA.

[0071] Fig. 9 Part (a) is a photograph of the lungs of experimental mice for comparison of the anticancer effects of MEL-dKLA.

[0072] Fig. 9 Part (b) is a photograph showing the lungs of experimental mice stained for comparison of the cancer metastasis inhibitory effect of MEL-dKLA.

[0073] Fig. 9 Part (c) is a bar graph comparing the number of lung tumor nodules in order to compare the anticancer effects of MEL-dKLA.

[0074] Fig.10 Part (a) is a photograph showing the comparison of cancer growth in mice injected with luminescent factors in order to compare the anti-cancer effect of MEL-dKLA.

[0075] Fig.10 Part (b) is a bar graph comparing the luminescence intensity in mice in order to compare the anti-cancer effect of MEL-dKLA.

[0076] Fig.10Part (c) is a graph comparing tumor sizes in mice in order to compare the anti-cancer effects of MEL-dKLA.

[0077] Fig.10 Part (d) is a bar graph comparing the luminescence intensity of the entire metastatic area in mice in order to compare the anti-cancer effect of MEL-dKLA.

[0078] Fig.10 Part (e) is a bar graph comparing the areas of the entire metastatic region in mice in order to compare the anti-cancer effects of MEL-dKLA.

[0079] Fig.11 Part (a) shows photographs of comparing cancer metastasis in mice by injecting luminescent factors in order to compare the cancer metastasis inhibitory effect of MEL-dKLA.

[0080] Fig.11 Part (b) is a bar graph comparing the overall luminescence intensity in mice in order to compare the cancer metastasis inhibitory effect of MEL-dKLA.

[0081] Fig.12 Part (a) is a bar graph showing the comparison of the anticancer effect of MEL-dKLA by measuring the expression of CD44, a cancer expression marker.

[0082] Fig.12 Part (b) is a bar graph showing the comparison of the anticancer effect of MEL-dKLA by measuring the expression of CCL22, a marker for M2 macrophages.

[0083] Fig.12 Part (c) is a bar graph showing the comparison of the anticancer effect of MEL-dKLA by measuring the expression of HIF-α, a metastasis and infiltration marker.

[0084] Fig.12 Part (d) is a bar graph showing the comparison of the anticancer effect of MEL-dKLA by measuring the expression of Ym1, a macrophage marker.

[0085] Fig.12 Part (e) is a bar graph showing the comparison of the anticancer effect of MEL-dKLA by measuring the expression of MMP-9 that interferes with the migration and retention of tumor cells.

[0086] Fig.13 Part (a) is a graph showing the infiltration of M1 tumor-associated macrophages into the tumor stroma by CD45+F4 / 80+CD86+ staining in order to compare the selective reduction of the number of M2 tumor-associated TAM cells by MEL-dKLA.

[0087] Fig.13 Part (b) is a graph showing the infiltration of M2 tumor-associated macrophages into the tumor stroma by CD45+F4 / 80+CD206+ staining in order to compare the selective reduction of the number of M2 tumor-associated TAM cells caused by MEL-dKLA.

[0088] Fig.13 Part (c) is a bar graph showing the conversion of CD45+F4 / 80+CD86+ infiltrating into the tumor stroma of M1 tumor-associated macrophages in order to compare the selective reduction of the number of M2 tumor-associated TAM cells caused by MEL-dKLA.

[0089] Fig.13 Part (d) is a bar graph showing the conversion of CD45+F4 / 80+CD206+ infiltrating into the tumor stroma of M2 tumor-associated macrophages in order to compare the selective reduction of the number of M2 tumor-associated TAM cells caused by MEL-dKLA.

[0090] Fig.13 Part (e) is a bar graph for comparing the changes in the M1 / M2 ratio caused by MEL-dKLA.

[0091] Fig.13 Part (f) is a bar graph for comparing the changes in CD4 T cells caused by MEL-dKLA.

[0092] Fig.13 Part (g) is a bar graph for comparing the changes in CD8 T cells caused by MEL-dKLA.

[0093] Fig.13 Part (h) is a bar graph for comparing the changes in regulatory T cells caused by MEL-dKLA.

[0094] Fig.13 Part (i) is a bar graph for comparing the changes in dendritic cells caused by MEL-dKLA.

[0095] Fig.14 Part (a) is a graph showing the selective cell death of M2 tumor-associated TAMs induced by MEL-dKLA by staining M1.

[0096] Fig.14 Part (b) is a graph showing M2 staining for comparison of selective cell death of M2 tumor-associated TAMs induced by MEL-dKLA.

[0097] Fig.14Part (c) is a graph showing the ratio of stained M1 to M2 in order to compare the selective cell death of M2 tumor-associated TAMs caused by MEL-dKLA.

[0098] Fig.15 Part (a) shows immunofluorescence staining of LLC tumor endothelial cells for comparison of the anti-angiogenesis effects of MEL and MEL-dKLA.

[0099] Fig.15 Part (b) is a bar graph showing the density of blood vessels in each region in order to compare the anti-angiogenesis effects of MEL and MEL-dKLA. DETAILED DESCRIPTION

[0100] Preparation Example 1. Combination of multiple anticancer agents using MEL

[0101] 1-1.MEL-dKLA conjugate

[0102] In order to confirm the drug binding and ease of binding of multiple anticancer agents to MEL, the binding of dKLA was performed.

[0103] MEL (sequence 1) and dKLA (sequence 2) are short peptides, so the peptides can be linked by amide bonds. At this time, in order to minimize the interaction and folding between MEL and dKLA, a linker consisting of four glycines and one serine is placed in the middle to separate the two ends. In order to minimize in vivo decomposition, the D-isomer is used as KLA instead of the L-isomer.

[0104] 1-2.MEL-DM1 conjugate

[0105] In order to confirm the multiple anticancer drugs and the ease of conjugation of MEL, conjugation with DM1 was performed.

[0106] More specifically, bee venom peptide with a maleimide structure synthesized at the N-terminus of the amino acid sequence was purchased for use. Maleimide can form a covalent bond with the free-sulfhydryl group (-SH) of DM1. After bee venom peptide was reacted with DM1 in a boric acid buffer for 2 hours, the buffer exchange and unbound bee venom peptide were filtered using amicon ultra centrifugal filters (MerkMillipore) and PBS. Bee venom peptide has a molecular weight of about 3kDa, and if it is combined with DM1, it will have a molecular weight of more than 3.6kDa. The filter used filters substances above 3kDa, separates bee venom peptide bound to DM1, and confirms it by a Q-TOF mass analyzer.

[0107] Example 1-1. Delivery of peptide dKLA to induce apoptosis in M2 macrophages via MEL

[0108] In order to observe whether MEL-dKLA induces apoptosis in M2 macrophages, the cell survival rate was measured using various concentrations of dKLA, MEL and MEL-dKLA (0.1-1 μM).

[0109] More specifically, dKLA (sequence 2), MEL (sequence 1), MEL-dKLA (sequence 3; GIGAVLKVLTTGLPALISWIKRKRQQGGGGS-d[KLAKLAKKLAKLAK]) peptides and 5-carboxyl tetramethylrhodamine (TMR)-conjugated dKLA, MEL, and MEL-KLA peptides were purchased from GenScript (Piscataway, NJ, USA). TMR is conjugated to the amino acid located at the N-terminus of the peptide, and all peptides used were more than 95% purified. Murine Lewis lung carcinoma (LLC) cells and mouse macrophages RAW264.7 were cultured in Dulbecco's modified Eagle's medium (DMEM; Welgene, Korea) supplemented with 10% heat-inactivated fetal bovine serum (Welgene) and 100 U / mL penicillin and 100 μg / mL streptomycin (Invitrogen, CA, USA). For macrophages (RAW264.7) that differentiate into M2 type, IL-4 and IL-13 were treated in the culture medium for 24 hours. After treatment, the cells were cultured in a serum-deficient state for 24 hours. M1 type macrophages were treated with 1 ng / mL LPS for 24 hours to induce differentiation.

[0110] Cell viability was determined by MTS assay. RAW264.7 macrophages were differentiated into M1 or M2 macrophages and cultured in 96-well plates at 3 × 10 4 cells / well. The next day, PBS, dKLA, MEL, and MEL-dKLA were treated respectively. After 24 hours, the culture medium was replaced and 20 μL of MTS reaction solution (Promega, WI, USA) was treated to the cells. After reacting at 37°C, the fluorescence was measured at 490 nm to determine the cell survival rate.

[0111] The results, such as Figure 1As shown, dKLA will be used as a control group because it cannot interfere with the membrane of eukaryotic cells. When 0.6-0.5μM MEL-dKLA and 79-71% MEL were treated and reacted for 24 hours, the cell survival rate decreased by about 55-53%. The half-maximal inhibitory concentration (IC50) of MEL-dKLA for M2 macrophages is lower than the half-maximal inhibitory concentration when MEL is used alone (0.85μM MEL-dKLA / 0.6-0.8μM MEL). However, when treated with 0.6-0.8μM MEL-dKLA, the survival rate of M1 macrophages was 86-66%, and when treated with 0.6-0.8μM MEL, the survival rate was 74%. Therefore, in the IC50 test between the two substances, MEL and MEL-dKLA, using the above-mentioned macrophages as the object, no significant difference was confirmed ( Figure 1 (a) and (b) of the present disclosure).

[0112] Furthermore, in order to observe whether MEL-dKLA causes random cell death in tumor cells, the cell cycle was observed in vitro by PI staining.

[0113] More specifically, cells were fixed with 70% ice ethanol and stored at 20°C for 24 hours, then treated with a mixture of propidium iodide (PI) in PBS containing 0.1% Triton X-100 and 20 μg / ml RNase to a concentration of 50 μg / ml, and measured using flow cytometry.

[0114] As a result, 0.1-1 μM MEL-dKLA showed no cytotoxicity in LLC tumor cells ( Figure 1 (c) of the Regulations).

[0115] Example 1-2. Apoptosis of M2 macrophages by mitochondrial membrane perturbation of MEL-dKLA

[0116] Flow cytometry analysis was performed to confirm whether the death of M2 macrophages was caused by mitochondrial membrane disturbance due to MEL-dKLA treatment.

[0117] More specifically, cells were stained with Annexin V-fluoresceinisothiocyanate (FITC; BD Biosciences, CA, USA). 5Cells were inoculated at 100 μg / well and treated with 0.8 μM peptide on the next day. After treatment for 1 and 3 times and 6 hours, the cells were reacted with 250 nM MitoTracker in a serum-free medium for 1 hour. Afterwards, the cells were collected and reacted with Annexin V again. The reacted cells were measured with BD FACSCalibur and analyzed by FlowJo software (Treestar, Inc., CA, USA). MitoTracker can accumulate in mitochondria as the membrane charge passes through the plasma membrane. That is, staining can be confirmed in the mitochondrial membrane of living cells, but it is difficult to stain in cells with apoptosis caused by membrane interference.

[0118] The results, such as Figures 2 to 3 As shown in Figure 2, when MEL and MEL-dKLA peptides were treated for 1 and 3 hours, no effect was produced in M1 macrophages. When treated for 6 hours, MitoTracker staining decreased and Annexin V+ staining increased, but no significant changes were observed ( Figure 2 However, when MEL-dKLA was treated for 6 hours, considerable cell death was confirmed. In contrast, no significant difference was found when MEL and dKLA were used alone ( Figure 3 ).

[0119] Example 1-3. Delivery of the peptide anticancer agent DM1 via MEL to induce apoptosis in M2 macrophages

[0120] In order to observe whether MEL-dKLA induces apoptosis in M2 macrophages, cells were treated with DM1, MEL and MEL-DM1 to measure the cell survival rate.

[0121] The MEL-DM1 anticancer agent binding method and cell experiment method were as described above.

[0122] The results, such as Figure 4 As shown, when the MEL-DM1 conjugate was treated, a higher M2-selective apoptotic effect was observed compared to the separate treatments of MEL and DM1, and thus, it was confirmed that the MEL-anticancer agent conjugate had a more excellent effect.

[0123] Example 2. Determination of metabolic changes caused by MEL-dKLA

[0124] To determine whether MEL-dKLA affects mitochondrial metabolism, seahorse analysis was performed.

[0125] More specifically, metabolic effects were measured using an XF24 Extracellular Flux analyzer (Agilent, CA, USA). RAW264.7 cells differentiated into M2 were seeded on XF-24 plates (3×10 4 cells / well), and on the next day, in order to minimize potential stress caused by the change in conditions, 1 μM of each peptide was treated and cultured in an incubator at 37°C under 5% CO2. After the culture for temporary adaptation, the cells were inoculated in 500 μL of XF running medium (pH 7.4) supplemented with 4500 mg / L D-glucose (w / v), 1 mM sodium pyruvate, and 4 mM L-glutamine, and cultured at 37°C in the absence of carbon dioxide, and metabolic toxins (1 μM oligomycin, 0.5 μM carbonyl cyanide p-trifluoromethoxy-phenylhydrazone (FCCP), 0.5 μM rotenone, and antimycin A (Rot / AA)) were impregnated in the drug port. After that, drugs were added to measure the intracellular oxygen consumption rate (OCR) and the extracellular acidification rate (ECAR).

[0126] In addition, OCR was measured at 12 positions, basal state was measured three times, ATP-linked respiration after addition of oligomycin was measured three times, maximum respiration after addition of FCCP was measured three times, and the last three times, Rot / AA was measured to show non-mitochondrial respiration to measure the respiratory capacity of mitochondria. ECAR, which represents the ratio of this action, was also measured at the same points.

[0127] The results, such as Figure 5 to Figure 6 As shown in Figure 2, the basal respiration rate in the MEL-dKLA group was significantly lower than that in the PBS group. However, the basal OCR did not change when only dKLA or MEL was treated compared to PBS ( Figure 5 In addition, MEL-dKLA significantly reduced the production of ATP ( Figure 5 Part (a), (d)). In particular, when MEL-dKLA was treated, the maximum respiration was significantly decreased ( Figure 5 (a) and (e) of Figure 5). The basal response capacity did not change significantly, but when oligomycin was treated, all groups showed an upward trend ( Figure 5 (b)). In the energy standard model, MEL-dKLA confirmed a decrease in respiratory capacity in both the basal state and the stress state ( Figure 6(a) and (b)). However, the basal ECAR was not inhibited by the treatment with MEL-dKLA. On the contrary, in the group stressed by MEL-dKLA, the ECAR was slightly reduced compared with the dKLA group. However, no significant difference was found when comparing the results of the PBS group with the MEL-dKLA group ( Figure 6 (a) and (c) of the present document).

[0128] This suggests that the effect of MEL-dKLA was minimal in the corresponding action in the cytoplasm, and therefore, the dysfunction of mitochondrial respiration is caused by MEL-dKLA targeted to mitochondria.

[0129] Example 3. Determination of the selective mitochondrial permeability of MEL-dKLA

[0130] The penetration and location of MEL-dKLA in the mitochondria of M2 macrophages were confirmed by staining using a fluorescence microscope, and quantitative analysis was performed using PASCAL 5LSM image detection.

[0131] More specifically, RAW264.7 macrophages differentiated into M2 were cultured with 1 μM TMR-bound dKLA, MEL or MEL-dKLA for 2 hours. After washing away the unbound peptide, the cells were stained with 250 nM mitochondrial green fluorescent probe (MitoTrackergreen, Invitrogen). After staining, the cells were stained again with PBS supplemented with 4 μg / mL DAPI (4'6-diamidino-2-phenylidole, Sigma-Aldrich, MO, USA). The stained cells were measured by laser scanning confocal microscopy (Carl Zeiss, Germany), and the location of MEL-dKLA in mitochondria was confirmed by LSM5 image detector (CarlZeiss).

[0132] The results, such as Figure 7 As shown in FIG. 1 , the above image detection confirmed the presence of a considerable amount of MEL-dKLA in the mitochondria, but no MEL was confirmed, and a small amount of dKLA was confirmed to be bound to the cells ( Figure 7 In addition, only MEL-dKLA has a positive correlation with mitochondria, while dKLA and MEL have no correlation with mitochondria ( Figure 7 (b) of the Regulations).

[0133] This confirmed that MEL-dKLA can selectively react with mitochondria and permeate therein.

[0134] Example 4. Determination of the inhibitory effect of MEL-dKLA on the growth of tumor cells in mice

[0135] Example 4-1. Determination of the inhibitory effect of MEL-dKLA on lung cancer cells in mice

[0136] In order to compare the anticancer effects of MEL-dKLA and MEL in vivo, PBS, dKLA, MEL, or MEL-dKLA peptide were injected into mice bearing lung cancer cells, and the changes in tumors were examined.

[0137] More specifically, C57BL / 6 wild-type mice were purchased from DBL (Korea), and LLC tumor cells were mixed with Matrigel (Corning, NY, USA) and injected into the right rib cage of the mice (5×10 4 cells / mouse). Five days after the injection of tumor cells, recombinant dKLA, MEL, and MEL-dKLA peptides were injected intraperitoneally for 3 days (175 nmol / kg per body weight / weight), for a total of three injections. All tumor tissues were recovered 12 days after injection. The above animal experiments were approved by the Animal Care Committee of Kyung Hee University [KHUASP(SE)-17-087], and the animals were managed in a 12-hour light / dark cycle environment in the absence of pathogens, with water and food intake voluntarily.

[0138] The results, such as Figure 8 As shown in the figure, the tumors of mice injected with PBS and dKLA continued to grow. The growth of tumors in the group injected with MEL-dKLA was greatly suppressed compared with the control group injected with PBS. It was also confirmed that the size and weight of the tumors were greatly reduced compared with the group injected with dKLA ( Figure 8 (a) and (b)). It was also confirmed that the size of the tumor in the group injected with MEL was also greatly reduced. Importantly, the size and weight of the tumor in the group administered with MEL-dKLA were greatly reduced compared to the dKLA group ( Figure 8 In contrast, the body weight of mice did not change in all groups ( Figure 8 (d)

[0139] This confirmed that MEL-dKLA inhibits tumor growth and metastasis more effectively than other peptides and anticancer agents alone in lung cancer.

[0140] Example 4-2. Determination of the inhibitory effect of MEL-dKLA on breast cancer cells in mice

[0141] In order to determine the tumor suppressive and cancer metastasis suppressive effects of MEL-dKLA and MEL, tumors were identified, and the extent of metastasis to the lungs was confirmed by staining.

[0142] More specifically, 4T1 breast cancer cells (1×10 5 ) was injected into BALB / c mice 3 days later, 175 nmol / kg of PBS, dKLA, MEL, and MEL-dKLA were injected into each group at 3-day intervals. In order to observe the degree of metastasis and nodules on the lung surface, mice were sacrificed 15 days after the cancer cell injection to confirm the surface nodules of the lung.

[0143] The results, such as Fig. 9 As shown in Figure 2, the nodules on the lung surface were significantly reduced in the group treated with MEL-dKLA, but not in the group treated with MEL and dKLA alone ( Fig. 9 (a) of the Regulations).

[0144] Furthermore, H&E staining was performed by a conventional method to determine the extent of metastasis to the lung.

[0145] More specifically, in H&E staining, the tissue is immersed in a hematoxylin solution to stain the cell nucleus purple, and then the remaining stained part except the nucleus is removed with an acidic solution, and then immersed again in a solution added with eosin to stain the cytoplasm pink, thereby observing the structure of the cell.

[0146] The results, such as Fig. 9 As shown in the figure, in the groups treated with PBS and dKLA, metastasis of tumor cells was confirmed to be widespread. In contrast, in the groups treated with MEL and MEL-dKLA, metastasis was confirmed to be relatively small ( Fig. 9 However, MEL-dKLA significantly reduced the number of nodules, in contrast to the absence of a significant decrease in nodule number when MEL was treated alone ( Fig. 9 (c) of the Regulations).

[0147] Furthermore, after the fluorescent substance 4T1-luciferase was injected into mice, the effects of MEL-dKLA on tumor growth and metastasis were confirmed by fluorescence.

[0148] More specifically, first, to investigate the effect of MEL-dKLA on tumor growth and inhibition when breast cancer cells were orthotopically transplanted into the breast, 4T1-luciferase breast cancer cells (1×10 5) was injected into NOD-SCID mice as immunodeficient mice. Five days later, at the time point when tumor masses began to grow in the fourth mammary gland, 175nmol / kg of PBS, dKLA, MEL and MEL-dKLA were injected into each group once every 3 days. In order to measure the growth of the tumor, the size of the tumor was measured once every 3 days using a caliper device, and all tumor tissues were recovered 4 days after the injection. In the fourth week of tumor transplantation, in order to confirm the extent of tumor metastasis to the lymph nodes and lungs, luciferin (D-luciferin), a substrate for luciferase, was diluted to a concentration of 40mg / ml and administered to mice intraperitoneally at a concentration of 100μL each time. After about 15 minutes of reaction, luminescence was measured using NightOwl (Berthold Technologies), an in vivo imaging device, and analyzed together with the photos of the mice to analyze the extent of metastasis to the whole body and lungs. The animal experiments were approved by the Animal Care Committee of Kyung Hee University [KHUASP(SE)-18-133] and were conducted in the absence of pathogens with a 12-h light / dark cycle, allowing the animals to voluntarily consume water and food.

[0149] Furthermore, in order to confirm the metastatic potential of tumor cells, 4T1-luciferase breast cancer cells (1×10 5 ) was injected into the tail vein of BALB / c mice. Starting 3 days later, 175nmol / kg of PBS, dKLA, MEL and MEL-dKLA were injected into each group once every 3 days. 15 days after the injection of cancer cells, luciferin (D-luciferin), which is a substrate for luciferase, was diluted to a concentration of 40mg / ml and intraperitoneally administered to mice at a dose of 100μL each time. After about 15 minutes of reaction, NightOwl (Berthold Technologies) was used to analyze the retention and metastasis of cancer cells metastasized to the whole body and lungs together with the photos of the mice.

[0150] The results, such as Figure 10 to Figure 11 As shown, in the group treated with PBS, it can be confirmed that breast cancer masses on the breast grow rapidly. In the group treated with MEL-dKLA, the growth of breast cancer cells was effectively reduced, and in the group treated with MEL and dKLA alone, the effect of reducing breast cancer growth was not significant ( Fig.10(a) to (c)). In the results of confirming the extent of metastasis to the axillary lymph nodes and lungs, it was also confirmed that the area of ​​metastasis in the PBS group was wide and the luminescence value of the metastatic site was very high. In the dKLA or MEL group, there was no significant difference in the metastatic area from the PBS group, and there was a trend of decreasing luminescence values, but it was not significant. In MEL-dKLA, almost no metastasis was observed, and a significant decrease in the metastatic area and luminescence value was confirmed ( Fig.10 (d) and (e) of the Regulations).

[0151] Furthermore, in the experiment for confirming the metastatic potential of tumor cells, it was also confirmed that the luminescence values ​​of cancer cells in the whole body were high in the groups treated with PBS and dKLA, and metastasis was reduced when MEL was treated compared to PBS or dKLA. In the MEL-dKLA group, very low luminescence values ​​of cancer cells in the lungs were detected, and significantly lower metastasis was also observed in the whole body compared to MEL ( Fig.11 ).

[0152] This confirmed that MEL-dKLA inhibits tumor growth and metastasis more effectively than other peptides and anticancer agents alone in breast cancer.

[0153] Example 5. Determination of tumor metastasis gene expression level of MEL-dKLA

[0154] In order to determine the tumor metastasis inhibitory ability of MEL-dKLA, the expressions of CD44, which interacts with extracellular matrix ligands to promote metastasis and infiltration and is mostly expressed in cancer, CCL22, a marker of M2 macrophages, HIF-1α, a marker of angiogenesis, metastasis and infiltration, Ym1, a marker of M2 macrophages, and MMP-9, which interferes with the migration and retention of tumor cells, were determined by real-time fluorescence quantitative PCR (real-time PCR).

[0155] More specifically, RNA was extracted from lung tissue using the easy-BLUE RNA extraction kit (iNtRON Biotechnology, Korea), and cDNA was synthesized according to the reverse transcriptase (transcriptase, Bioneer, Korea) guide. The cDNA synthesis conditions were 95°C for 15 seconds, 55°C for 10 seconds, and 72°C for 10 seconds, with each reaction performed 3 times. Afterwards, CD44: forward, (sequence 4; 5'-TGGATCCGAATTAGC TGGA-3'); (sequence 5; reverse, 5'-GCTTTTTCTTCTGCCCACA-3'); CCL22: forward, (sequence 6; 5'-TCCCAGGGGAAGGAATAAA-3'); reverse, (sequence 7; 5'-GGTTTGGATCAA GCCCTTT-3'); HIF-1α: forward, (sequence 8; 5'-TCCCTTTTTCAAGCAGCAG-3'); reverse, (sequence 9; 5'-TGCCTTGTATGGGAGCATT-3'); Ym-1: forward, (sequence 10; 5'-CATTCAGTCAGTTATCAGATTCC-3'); reverse, (sequence 11; 5'-AGTGAGTAGCAGCCTTGG-3'); MMP-9: forward, (sequence 12; 5'-TGAATCAGCTGGCTTTTGTG-3'); reverse, (sequence 13; 5'-GTGGATAGCTCG GTGGTGTT-3'); primers, real-time fluorescence quantitative PCR was performed using the SensiFAST SYBR no-Rox kit (Bioline, Korea).

[0156] The results, such as Fig.12 As shown, compared with WT, the expression of CD44 in the PBS-treated group was significantly increased, and compared with the PBS, dKLA, and MEL-treated groups, the expression level of CD44 in the MEL-dKLA-treated group was significantly decreased ( Fig.12 (a)). It was confirmed that the expression of CCL22 and HIF-1α in the PBS-treated group was also increased compared with WT, but was significantly decreased in the MEL-dKLA-treated group. In contrast, it was confirmed that there was no significant difference between the dKLA- and MEL-treated groups and the PBS group ( Fig.12 It was confirmed that the expression of Ym1 was greatly increased in the dKLA group compared with the PBS group, while there was no significant difference between the MEL and MEL-dKLA treatment groups and the PBS group ( Fig.12(d)). It was confirmed that the expression level of MMP-9 was high in the PBS group and the dKLA group, and the expression level of MMP-9 was reduced in the MEL-treated group compared with the PBS group. In contrast, it was confirmed that the expression level of MMP-9 was lower in the MEL-dKLA-treated group compared with the MEL group ( Fig.12 (e) of the Regulations).

[0157] This confirmed that the tumor transcription factor expression inhibitory effect was more significant when MEL-dKLA was used than when MEL and dKLA were used alone.

[0158] Example 6. Analysis of MEL-dKLA targeting CD206+M2 tumor-associated TAMs by flow cytometry

[0159] In order to confirm in vivo whether the MEL-dKLA peptide can be used as a peptide targeting M2-type tumor-associated TAMs, tumor tissues were cultured and stained to analyze them individually, thereby confirming cell increase.

[0160] More specifically, tumor cells were crushed and separated in DMEM supplemented with DNase I (1 U / mL) and collagenase D (1 mg / mL). The cells were gently stirred at 37°C for 1 hour and separated using a 100-μM nylon mesh filter. Red blood cells were lysed in Phrmlyse buffer (BE bioscience). Individual cells were filtered through a 40-μM nylon mesh and stained with antibodies. CD4+T cells (CD45+CD4+CD8-), CD8+T cells (CD45+CD4-CD8+), Foxp3+ regulatory T cells (CD4+CD25+Foxp3+), dendritic cells (CD45+CD11b+CD11c+) and M1 (CD45+F4 / 80+CD86+) or M2 macrophages (CD45+F4 / 80+CD206+): anti-CD45-FITC, anti-CD4-phycoerythrin (PE), anti-CD8-allophycocyanin (APC), anti-CD4-FITC, anti-CD25-PE, anti-Foxp3-Alexa Fluor647, anti-CD11b-APC, anti-CD11c-APCcy7, anti-Gr1-PEcy7, anti-CD86-PEcy7 and anti-CD206-APC antibodies. To determine the rate of migration of macrophages, cells were pre-treated with Annexin-V.

[0161] The results, such as Fig.13As shown in the figure, it was confirmed that the F480+CD86+M1 tumor-associated macrophages in the MEL-administered group increased slightly compared with the PBS- and dKLA-administered groups, but there was no significant difference with the dKLA group. In contrast, it was confirmed that the F480+CD86+M1 tumor-associated macrophages in the MEL-dKLA-administered group increased significantly compared with the MEL group ( Fig.13 (a) and (c)). The ratio of M2 tumor-associated F4 / 80+CD206+TAM in CD45+ leukocytes in the PBS and dKLA groups was about 20%. It was confirmed that the M2 tumor-associated TAM cells in the MEL and MEL-dKLA groups were reduced by about half compared to the PBS group, with a ratio of about 10% ( Fig.13 (b) and (d) of the study). However, it was confirmed that the M1 / M2 ratio in the MEL-dKLA group was significantly higher than that in the MEL group. Although the M2 tumor-associated TAMs in the MEL and MEL-dKLA groups were significantly reduced compared to the PBS and dKLA groups ( Fig.13 (e) of the study), but other white blood cells such as CD4 T cells, Foxp3+ Tregs, CD8 T cells, and dendritic cells did not change, so it was confirmed that these cells had no effect ( Fig.13 (f) to (i) of the Regulations).

[0162] Furthermore, in order to confirm whether M2 tumor-associated TAM cells selectively undergo apoptosis in mice, M1 tumor-associated TAM (F4 / 80CD86+) and M2 tumor-associated TAM (F4 / 80CD206+) were stained with Annexin-V.

[0163] The results, such as Fig.14 As shown, compared with the control PBS group, there was no increase in cells stained with Annexin in CD86+M1 tumor-associated TAMs in all groups ( Fig.14 In addition, the number of M2 tumor-associated TAMs in the MEL and MEL-dKLA groups decreased, but a considerable amount of CD206+M2 tumor-associated TAM cell death was confirmed only in the MEL-dKLA-administered group ( Fig.14 (b)). Compared with M1-type tumor-associated TAMs, the apoptosis rate caused by the administration of MEL-dKLA in M2-type tumor-associated TAMs was significantly higher ( Fig.14 (c) of the Regulations).

[0164] This confirmed that the M1 / M2 ratio of MEL-dKLA was significantly increased compared to the treatment of MEL by selectively inducing apoptosis of M2-type tumor-associated TAMs.

[0165] Example 8. Relationship between the reduction of CD206+TAM in tumors and the anti-angiogenic effect

[0166] CD31 (PECAM-1) is known to be a marker that is actively secreted by the vascular endothelium and can predict angiogenesis. Tumor angiogenesis is an essential means for supplying oxygen and nutrients to oxygen-poor areas within the tumor, and is closely related to the growth and metastasis of cancer. M2-bipolar macrophages are the main precursors of angiogenesis factors, including cyclooxygenase-2, matrix metalloproteinase-9, and VEGF, and the density of macrophages is also related to angiogenesis. Therefore, in order to confirm whether the reduction of M2-type tumor-related TAMs in the tumor causes the reduction of angiogenesis, it was confirmed by immunostaining and laser scanning confocal microscopy.

[0167] More specifically, the tissue was dried with paraformaldehyde for 24 hours. Afterwards, it was cut into 4 μM thickness using a rotary microtome and restored by autoclaving with tri-sodium citrate buffer. The restored tissue sections were incubated with anti-mouse serum endothelial cell adhesion substance (PECAM; CD31) antibody (1:200; Santa Cruz Biotechnology, CA, USA) and anti-rabbit secondary antibody conjugated to Alexa-488 (1:500; Invitrogen) for visualization. The stained sections were embedded and analyzed under a laser scanning confocal microscope (Carl Zeiss). All images were taken in LSM5 PASCAL and the fluorescence values ​​were analyzed using ImageJ software.

[0168] The results, such as Fig.15 As shown in Figure 2, a significant decrease in CD31+ endothelial cells was observed in both the MEL and MEL-dKLA groups ( Fig.15 Part (a), Part (b).

[0169] This confirmed that the inhibition of angiogenesis was associated with a decrease in M2 tumor-associated TAMs.

[0170] The concentration of TAM in the tumor matrix is ​​closely related to the growth, metastasis and angiogenesis of the tumor. However, the method of reducing macrophages alone cannot effectively solve the problems such as the above-mentioned tumor growth and angiogenesis. It is believed that the anti-cancer effect improved by the present invention is related to the high ratio of M1 / M2. The MEL-dKLA of the present invention effectively increases the ratio of M1 / M2 by selectively reducing M2-type tumor-associated TAMs, and inhibits tumor growth and angiogenesis by inducing mitochondrial apoptosis. Therefore, MEL-dKLA can be used as a cancer therapeutic agent that effectively targets M2-type tumor-associated TAMs.

[0171] It can be understood from the above description that a person skilled in the art to which the present invention belongs can implement the present invention into other specific forms without changing the technical idea or essential features of the present invention. Accordingly, it should be understood that the embodiments described above are illustrative in all aspects, but not restrictive. It should be interpreted that, within the scope of the present invention, all changes or deformations derived from the meaning and scope of the invention claims not described in detail above and their equivalent concepts are included within the scope of the present invention. <110> Shuanghai Life Sciences Co., Ltd. <120> Targeting of M2 tumor-associated macrophages using a melittin-based apoptosis-inducing peptide <130> OPA19100-PCT <150> KR 10-2018-0051800 <151> 2018-05-04 <160> 13 <170> KoPatentIn 3.0 <210> 1 <211> 26 <212> PRT <213> Artificial Sequence <220> <223> Recombinant Peptides <400> 1 Gly Ile Gly Ala Val Leu Lys Val Leu Thr Thr Gly Leu Pro Ala Leu 1 5 10 15 Ile Ser Trp Ile Lys Arg Lys Arg Gln Gln 20 25 <210> 2 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> Recombinant Peptides <400> 2 Lys Leu Ala Lys Leu Ala Lys Lys Leu Ala Lys Leu Ala Lys 1 5 10 <210> 3 <211> 45 <212> PRT <213> Artificial Sequence <220> <223> Recombinant Peptides <400> 3 Gly Ile Gly Ala Val Leu Lys Val Leu Thr Thr Gly Leu Pro Ala Leu 1 5 10 15 Ile Ser Trp Ile Lys Arg Lys Arg Gln Gln Gly Gly Gly Gly Ser Lys 20 25 30 Leu Ala Lys Leu Ala Lys Lys Leu Ala Lys Leu Ala Lys 35 40 45 <210> 4 <211> 19 <212> PRT <213> Artificial Sequence <220> <223> Recombinant Peptides <400> 4 Thr Gly Gly Ala Thr Cys Cys Gly Ala Ala Thr Thr Ala Gly Cys Thr 1 5 10 15 Gly Gly Ala <210> 5 <211> 19 <212> PRT <213> Artificial Sequence <220> <223> Recombinant Peptides <400> 5 Gly Cys Thr Thr Thr Thr Thr Cys Thr Thr Cys Thr Gly Cys Cys Cys 1 5 10 15 Ala Cys Ala <210> 6 <211> 19 <212> PRT <213> Artificial Sequence <220> <223> Recombinant Peptides <400> 6 Thr Cys Cys Cys Ala Gly Gly Gly Gly Ala Ala Gly Gly Ala Ala Thr 1 5 10 15 Ala Ala Ala <210> 7 <211> 19 <212> PRT <213> Artificial Sequence <220> <223> Recombinant Peptides <400> 7 Gly Gly Thr Thr Thr Gly Gly Ala Thr Cys Ala Ala Gly Cys Cys Cys 1 5 10 15 Thr Thr Thr <210> 8 <211> 19 <212> PRT <213> Artificial Sequence <220> <223> Recombinant Peptides <400> 8 Thr Cys Cys Cys Thr Thr Thr Thr Thr Cys Ala Ala Gly Cys Ala Gly 1 5 10 15 Cys Ala Gly <210> 9 <211> 19 <212> PRT <213> Artificial Sequence <220> <223> Recombinant Peptides <400> 9 Thr Gly Cys Cys Thr Thr Gly Thr Ala Thr Gly Gly Gly Ala Gly Cys 1 5 10 15 Ala Thr Thr <210> 10 <211> twenty three <212> PRT <213> Artificial Sequence <220> <223> Recombinant Peptides <400> 10 Cys Ala Thr Thr Cys Ala Gly Thr Cys Ala Gly Thr Thr Ala Thr Cys 1 5 10 15 Ala Gly Ala Thr Thr Cys Cys 20 <210> 11 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> Recombinant Peptides <400> 11 Ala Gly Thr Gly Ala Gly Thr Ala Gly Cys Ala Gly Cys Cys Thr Thr 1 5 10 15 Gly Gly <210> 12 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Recombinant Peptides <400> 12 Thr Gly Ala Ala Thr Cys Ala Gly Cys Thr Gly Gly Cys Thr Thr Thr 1 5 10 15 Thr Gly Thr Gly 20 <210> 13 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Recombinant Peptides <400> 13 Gly Thr Gly Gly Ala Thr Ala Gly Cys Thr Cys Gly Gly Thr Gly Gly 1 5 10 15 Thr Gly Thr Thr 20

Claims

1. A melittin-anticancer agent conjugate, characterized in that: It is composed of bee venom peptide and an anticancer agent, wherein the anticancer agent is maytansine DM1. Wherein, the bee venom peptide is connected with the anticancer agent via a chemical linker.

2. The melittin-anticancer agent conjugate according to claim 1, characterized in that: The above conjugates target M2 tumor-associated macrophages.

3. The melittin-anticancer agent conjugate according to claim 1, characterized in that: The chemical linker is combined via the amino group, carboxyl group or sulfhydryl group on the melittin and the anticancer agent.

4. The melittin-anticancer agent conjugate according to claim 1, characterized in that: Both ends of the chemical linker contain a functional group selected from the group consisting of carbodiimide, N-hydroxysuccinimide ester, imide ester, pentafluorophenyl ester, hydroxymethylphosphine, maleimide, haloacetyl, pyridyl disulfide, sulfonate, vinyl sulfone and a combination thereof.

5. The melittin-anticancer agent conjugate according to claim 1, characterized in that: The above chemical linker is selected from SATA, sulfo-SMCC, DMA, DMP, DMS, DTBP, sulfo-SIAB, SIAB, SBAP, SIA, SM(PEG)n, SMCC, LCSMCC, sulfo-EMCS , EMCS, GMBS, sulfo-KMUS, sulfo-MBS, MBS, sulfo-SMPB, SMPB, AMAS, BMPS, SMPH, PEG12-SPDP, PEG4-SPDP, sulfo-LCSPDP , SPDP, LC-SPDP, SMPT, DSS, BS(PEG)5, BS(PEG)9, BS3, BSOCOES, PDPH, DSG, DSP, BM(PEG)n, BMB, BMDB, BMH, BMOE, DTME, TMEA, DST, DTSSP, EGS, sulfo-EGS, TSAT, DFDNB and a combination thereof, wherein n in SM(PEG)n is 2, 4, 6, 8, 12 or 24, and n in BM(PEG)n is 2 or 3.

6. A pharmaceutical composition for preventing or treating a tumor-associated macrophage-mediated disease, characterized in that: The invention comprises the melittin-anticancer agent conjugate according to any one of claims 1 to 5.

7. The pharmaceutical composition for preventing or treating tumor-associated macrophage-mediated diseases according to claim 6, characterized in that: The above diseases are lung cancer, metastatic tumors, and breast cancer.

8. The pharmaceutical composition for preventing or treating tumor-associated macrophage-mediated diseases according to claim 6, characterized in that: The above diseases are Lewis lung cancer or inflammatory diseases.

9. The pharmaceutical composition for preventing or treating tumor-associated macrophage-mediated diseases according to claim 6, characterized in that: The composition is a pharmaceutical composition for preventing or treating the growth and metastasis of cancer by removing M2 tumor-associated macrophages.

10. A method for preparing a melittin-anticancer agent conjugate, characterized in that: The method comprises the steps of connecting melittin and an anticancer agent, wherein the anticancer agent is maytansine DM1, and wherein the melittin and the anticancer agent are connected via a chemical linker.

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