Peptides that selectively bind to exosomes derived from cancer cells and their uses

Abstract

The present invention relates to a peptide that selectively binds to exosomes derived from cancer cells and its uses. The ExoPep (CRKVAKG) peptide was discovered using phage display technology for discovering peptides having the ability to specifically bind to exosomes derived from cancer cells. This peptide binds to exosomes derived from cancer cells that have the property of migrating to cancer sites and cancer metastasis sites, and thus may have the effect of delivering anticancer drugs to cancer metastasis sites.

Description

Technical Field

[0001] The present disclosure relates to a peptide that selectively binds to exosomes derived from cancer cells and uses thereof. Background Art

[0002] Exosomes are small vesicles secreted by cells and contain proteins, mRNAs, and miRNAs expressed by the cells, exhibiting the characteristics of the cells themselves. Exosomes participate in cell-cell communication by delivering substances to other cells. In particular, exosomes derived from cancer cells are present in high concentrations in the blood and are known to assist cancer metastasis by moving to the sites where cancer metastasis occurs. Many studies have shown that exosomes are secreted very actively from tumor cells and play a role in delivering substances essential for cancer metastasis and development to the metastatic sites.

[0003] Cancer is one of the most common diseases worldwide, and current treatments include surgery, radiation, and chemotherapy. Although the molecular mechanisms of cancer are being actively studied, most of the currently developed treatments rely on surgery. However, recently, various targeted therapeutic agents (e.g., small molecule inhibitors, monoclonal antibodies, and short targeting peptides targeting cancer cells) have been developed and applied as therapeutic agents. In particular, short targeting peptides have high tissue permeability and low toxicity and immune responses, thus ensuring high potential as effective anticancer agents.

[0004] Phage display of peptides and antibodies is a very useful method for identifying target cell-specific ligands and is widely used for discovering peptides and antibodies targeting cancer cells in vitro and in vivo. Therefore, the researchers of the present disclosure intended to discover peptides targeting exosomes by utilizing the property that exosomes derived from cancer cells move to the sites of cancer development and cancer metastasis when circulating in the blood, and thus apply them to the delivery of anticancer drugs and anticancer treatment. Summary of the Invention

[0005] Technical Problem

[0006] The present disclosure relates to a peptide that specifically binds to exosomes derived from cancer cells and uses thereof. Specifically, an object of the present disclosure is to provide a peptide that specifically binds to exosomes derived from cancer cells, the peptide having an amino acid sequence represented by SEQ ID NO: 1; a composition for diagnosing cancer; a composition for drug delivery; a composition for imaging cancer cells; and a composition for detecting exosomes derived from cancer cells, the composition containing the peptide as an active ingredient; a fusion peptide, wherein an apoptosis-inducing peptide having an amino acid sequence represented by SEQ ID NO: 2 binds to the peptide; a pharmaceutical composition for preventing or treating cancer, the pharmaceutical composition containing the fusion peptide as an active ingredient; and a pharmaceutical composition for preventing or treating cancer, the pharmaceutical composition containing the fusion peptide and an anticancer agent as active ingredients.

[0007] Technical solution

[0008] To solve the above problems, an exemplary embodiment of the present disclosure provides a peptide that specifically binds to exosomes derived from cancer cells, the peptide having an amino acid sequence represented by SEQ ID NO: 1.

[0009] In addition, an exemplary embodiment of the present disclosure provides a polynucleotide encoding the peptide, a recombinant vector containing the polynucleotide, and a transformant transformed with the recombinant vector.

[0010] In addition, an exemplary embodiment of the present disclosure provides a composition for diagnosing cancer, the composition containing the peptide as an active ingredient.

[0011] In addition, an exemplary embodiment of the present disclosure provides a composition for drug delivery, the composition containing the peptide as an active ingredient.

[0012] In addition, an exemplary embodiment of the present disclosure provides a fusion peptide, wherein an apoptosis-inducing peptide having an amino acid sequence represented by SEQ ID NO: 2 binds to the peptide; and a pharmaceutical composition for preventing or treating cancer, the pharmaceutical composition containing the fusion peptide as an active ingredient.

[0013] In addition, an exemplary embodiment of the present disclosure provides a pharmaceutical composition for preventing or treating cancer, the pharmaceutical composition containing the fusion peptide and an anticancer agent as active ingredients.

[0014] In addition, an exemplary embodiment of the present disclosure provides a composition for imaging cancer cells, the composition containing the peptide as an active ingredient.

[0015] In addition, an exemplary embodiment of the present disclosure provides a composition for detecting exosomes derived from cancer cells, the composition containing the peptide as an active ingredient.

[0016] Beneficial effects

[0017] Exemplary embodiments of the present disclosure relate to peptides that selectively bind to exosomes derived from cancer cells and their uses. The peptide ExoPep (CRKVAKG) was discovered using phage display technology for discovering peptides with the ability to specifically bind to exosomes derived from cancer cells. The peptide binds to exosomes derived from cancer cells having the property of migrating to cancer sites and cancer metastasis sites, and thus may have the effect of delivering anti-cancer drugs to cancer metastasis sites. Brief description of the drawings

[0018] Figure 1 Shows the results of analyzing the characteristics of exosomes isolated from various cell lines using an ultracentrifuge and ExoQuick reagent.

[0019] Figure 2 Shows the phage library screening process, the fluctuation of phage titer, and the sequences of selected candidate peptides for discovering peptides that bind to tumor-derived exosomes.

[0020] Figure 3 Shows the results of evaluating the binding ability of 10 selected phage clones to exosomes and exosome-producing cells by ELISA assay.

[0021] Figure 4 Shows the results of evaluating the binding ability of ExoPep peptide to exosomes derived from A549, MDA-MB231, MCF7, HEK293, and MCF10A cells and the cells producing each exosome using flow cytometry, and the results of evaluating the internalization of ExoPep peptide bound to exosomes into cells using confocal microscopy.

[0022] Figure 5 Shows the results of studying the inhibition of exosome internalization into cells using confocal fluorescence microscopy, which is carried out by reacting exosomes derived from A549 tumor cells with ExoPep peptide labeled with magnetic particles and then using a magnet to remove exosomes bound to the peptide.

[0023] Figure 6 Shows the results of analyzing the inhibition of exosome release from A549 cells by treatment with GW4869 (which is an exosome production inhibitor), the effect on cell survival, and the resulting fluctuation of internalization of ExoPep peptide by confocal microscopy.

[0024] Figure 7 Shows the results of analyzing the in vitro binding of ExoPep peptide by separating exosomes from the blood of A549 tumor-bearing mice and normal mice using flow cytometry; and the results of analyzing the internalization into cells by confocal microscopy.

[0025] Figure 8 The results of Western blot analysis showing the characteristics of exosomes isolated using streptavidin beads or CD63 antibody beads, respectively, after injecting biotin-ExoPep peptide into the blood of A549 tumor-bearing mice and normal mice; and the results of hemolytic activity according to the concentration of ExoPep peptide in red blood cells.

[0026] Figure 9 The results of images and histological analysis of the in vivo distribution in A549 tumor-bearing mice of exosomes derived from the blood of A549 tumor-bearing mice and exosomes derived from A549 cells, with or without binding to ExoPep peptide.

[0027] Figure 10 The results of determining cytotoxicity by reacting exosomes isolated from cancer cells (A549, MDA-MB231, Panc-1, HT29, HepG2) and normal HEK293 cells with ExoPep-KLA peptide, and then performing the same treatment on each of the cells that produced each exosome.

[0028] Figure 11 The results of measuring apoptosis induction by the phosphorescence intensity emitted from cells, by reacting exosomes isolated from A549 and MDA-MB231 cell lines with ExoPep-KLA peptide and then performing the same treatment on each of the cells that produced the exosomes.

[0029] Figure 12 The results of measuring A549 apoptosis induced by ExoPep-KLA peptide bound to exosomes derived from A549 cells and exosomes derived from the blood of A549 tumor-bearing mice, as a percentage of cells stained with Annexin V.

[0030] Figure 13 The results of analysis of the stability of ExoPep-KLA peptide in serum.

[0031] Figure 14 The results of analysis of the inhibition of tumor growth and metastasis by ExoPep-KLA peptide in an A549 human lung cancer cell mouse tumor model.

[0032] Figure 15 The results of analysis of the blood levels and liver and kidney functions after treatment with ExoPep-KLA peptide in an A549 human lung cancer cell mouse tumor model.

[0033] Figure 16 The results of analysis of the inhibition of tumor growth and metastasis by co-administering ExoPep-KLA peptide and doxorubicin in an A549 human lung cancer cell mouse tumor model.

[0034] Figure 17 Shows the analysis results of blood levels and liver and kidney functions after co - administration of doxorubicin and ExoPep - KLA peptide in a mouse tumor model of A549 human lung cancer cells.

[0035] Figure 18 Shows the analysis results of inhibiting tumor growth and metastasis by single administration of ExoPep - KLA peptide and co - administration with gemcitabine in a Panc - 1 pancreatic cancer mouse model. Detailed Description of the Invention

[0036] Exemplary embodiments of the present disclosure provide a peptide that specifically binds to exosomes derived from cancer cells, and the peptide has an amino acid sequence represented by SEQ ID NO: 1.

[0037] Preferably, the cancer cells can be lung cancer cells, breast cancer cells or pancreatic cancer cells, but are not limited thereto.

[0038] The peptides of the exemplary embodiments of the present disclosure can be easily prepared by chemical synthesis known in the art (Creighton, Proteins; Structures and Molecular Principles, W.H. Freeman and Co., NY, 1983). Typical methods may include liquid - phase or solid - phase synthesis, fragment condensation, and F - MOC or T - BOC chemical methods (Chemical Approaches to the Synthesis of Peptides and Proteins, Williams et al., eds., CRC Press, Boca Raton Florida, 1997; A Practical Approach, Atherton & Sheppard, eds., IRL Press, Oxford, England, 1989), but are not limited thereto.

[0039] In addition, the peptides of the exemplary embodiments of the present disclosure can be prepared by genetic engineering methods. First, a DNA sequence encoding the peptide is synthesized according to a conventional method. The DNA sequence can be synthesized by PCR amplification using appropriate primers. Alternatively, the DNA sequence can be synthesized by standard methods known in the art, such as using an automated DNA synthesizer (e.g., those sold by Biosearch or Applied Biosystems). The constructed DNA sequence is inserted into a vector containing one or more expression control sequences (e.g., promoters, enhancers, etc.), which are operably linked to the DNA sequence to control the expression of the DNA sequence, and thus the host cell is transformed with the recombinant expression vector thus prepared. The prepared transformant is cultured under appropriate culture media and conditions to express the DNA sequence, and thus a substantially pure peptide encoded by the DNA sequence is harvested from the culture. The harvesting can be carried out by methods known in the art (e.g., chromatography). The term "substantially pure peptide" as used herein can refer to a state in which the peptide according to the exemplary embodiments of the present disclosure substantially does not contain any other host-derived proteins.

[0040] In the exemplary embodiments of the present disclosure, the peptide having the amino acid sequence represented by SEQ ID NO: 1 is a concept including its functional variants. The term "functional variant" as used herein refers to all similar sequences in which substitutions of some amino acids occur at amino acid sites that do not affect the property of the peptide of the exemplary embodiments of the present disclosure specifically binding to exosomes derived from cancer cells.

[0041] In addition, the exemplary embodiments of the present disclosure provide polynucleotides encoding the peptides.

[0042] The term "polynucleotide" as used herein refers to a polymer of deoxyribonucleotides or ribonucleotides, which exists in single-stranded or double-stranded form. It includes RNA genomic sequences, DNA (gDNA and cDNA), and RNA sequences transcribed therefrom, and includes analogs of natural polynucleotides unless otherwise specified.

[0043] The polynucleotide not only includes the nucleotide sequence encoding the peptide, but also includes a sequence complementary to the sequence. The complementary sequence includes not only a perfectly complementary sequence, but also a substantially complementary sequence.

[0044] In addition, the polynucleotide can be modified. Such modifications include the addition, deletion, or non-conservative or conservative substitution of nucleotides. A polynucleotide encoding an amino acid sequence can be understood to include nucleotide sequences that exhibit substantial identity to the nucleotide sequence. Substantial identity can refer to sequences that exhibit at least 80% homology, at least 90% homology, or at least 95% homology, which are obtained by aligning the nucleotide sequence with any other sequence to the maximum correspondence and then analyzing the aligned sequences using algorithms commonly used in the art.

[0045] In addition, exemplary embodiments of the present disclosure provide a recombinant vector comprising the polynucleotide.

[0046] In addition, exemplary embodiments of the present disclosure provide a transformant (other than human) transformed with the recombinant vector.

[0047] In an exemplary embodiment of the present disclosure, the term "vector" as used herein refers to a self-replicating DNA molecule used to carry a cloned gene (or another cloned DNA segment).

[0048] In an exemplary embodiment of the present disclosure, the term "recombinant vector" refers to a plasmid, viral vector, or other vehicle known in the art that can express the inserted nucleic acid in a host cell, and can be a recombinant vector in which a polynucleotide encoding a peptide of an exemplary embodiment of the present disclosure can be operably linked to a conventional expression vector known in the art. The recombinant vector can include a polynucleotide encoding a peptide of an exemplary embodiment of the present disclosure operably linked to an origin of replication capable of general proliferation in a host cell, one or more expression control sequences (such as promoters, enhancers, etc.) for controlling expression, a selection marker, and an expression control sequence. The transformant can be a transformant transformed by the recombinant vector.

[0049] Preferably, the transformant can be obtained by introducing a recombinant vector comprising a polynucleotide encoding a peptide of an exemplary embodiment of the present disclosure into a host cell by methods known in the art, such as (but not limited to) transient transfection, microinjection, transduction, cell fusion, calcium phosphate precipitation, liposome-mediated transfection, DEAE-dextran-mediated transfection, polybrene-mediated transfection, electroporation, gene gun, and other known methods for introducing nucleic acids into cells (Wu et al., J. Bio. Chem., 267: 963-967, 1992; Wu and Wu, J. Bio. Chem., 263: 14621-14624, 1988).

[0050] In addition, the exemplary embodiments of the present disclosure provide a composition for diagnosing cancer, the composition comprising the peptide as an active ingredient.

[0051] Preferably, the peptide can specifically bind to exosomes derived from cancer cells. More preferably, the cancer may include lung cancer, breast cancer, pancreatic cancer, brain tumor, liver cancer, skin cancer, esophageal cancer, testicular cancer, kidney cancer, colorectal cancer, rectal cancer, gastric cancer, bladder cancer, ovarian cancer, cholangiocarcinoma, gallbladder cancer, uterine cancer, cervical cancer, prostate cancer, head and neck cancer, and squamous cell carcinoma, but is not limited thereto.

[0052] In the exemplary embodiments of the present disclosure, the term "diagnosis" as used herein refers to identifying the presence or characteristics of a pathological condition. For the purposes of the exemplary embodiments of the present disclosure, the diagnosis is to identify the presence or characteristics of cancer.

[0053] Diagnosis of cancer using the peptide of the exemplary embodiments of the present disclosure can be performed by reacting the peptide of the exemplary embodiments of the present disclosure with cells or tissues directly obtained from blood, urine, or biopsy and detecting the binding thereof.

[0054] In addition, in order to easily confirm, detect, and quantify whether the peptide of the exemplary embodiments of the present disclosure binds to cancer tissue, the peptide of the exemplary embodiments of the present disclosure can be provided in a labeled state. In other words, it can be provided by linking (e.g., covalently bonding or crosslinking) with a detectable label. The detectable label may include chromogenic enzymes (e.g., peroxidase, alkaline phosphatase), radioisotopes (e.g., 124 I, 125 I, 111 In, 99 mTc, 32 P, 35 S), chromophores, luminescent materials, or fluorescent materials (e.g., FITC, RITC, rhodamine, cyanine, Texas Red, fluorescein, phycoerythrin, and quantum dot).

[0055] Similarly, detectable labels can be antibody epitopes, substrates, cofactors, inhibitors, or affinity ligands. Such labels can be incorporated during the synthesis of the peptides of the exemplary embodiments of the present disclosure or can be added subsequently to peptides that have already been synthesized. If a fluorescent substance is used as the detectable label, cancer can be diagnosed by fluorescence mediated tomography (FMT). For example, the peptides of the exemplary embodiments of the present disclosure labeled with a fluorescent substance can circulate in the blood to observe the fluorescence of the peptides by fluorescence tomography. If fluorescence is observed, cancer is diagnosed.

[0056] Furthermore, the exemplary embodiments of the present disclosure provide a composition for drug delivery, the composition comprising the peptide as an active ingredient.

[0057] Preferably, the drug can be a peptide drug or an anticancer drug. More preferably, the peptide drug can be a cytotoxic peptide having the activity of inducing apoptosis or necrosis, but is not limited thereto.

[0058] The peptides according to the exemplary embodiments of the present disclosure can be used for intelligent drug delivery to selectively deliver drugs to exosomes derived from cancer cells. By using the peptides of the exemplary embodiments of the present disclosure to treat cancer by conjugating the peptides with conventional known drugs, since the drugs are selectively delivered to exosomes derived from cancer cells by the peptides of the exemplary embodiments of the present disclosure, the efficacy of the drugs can be improved while the side effects of the drugs on normal tissues can be significantly reduced.

[0059] The drug is an anti-cancer agent, and as an anti-cancer agent that can be linked to the peptides of the exemplary embodiments of the present disclosure, it can be used without limitation as long as it is used for conventional cancer treatment. Examples may include mertansine, doxorubicin, paclitaxel, vincristine, daunorubicin, vinblastine, actinomycin-D, docetaxel, etoposide, teniposide, bisantrene, homoharringtonine, STI-571, cisplatin, 5-fluorouracil, adriamycin, methotrexate, busulfan, chlorambucil, cyclophosphamide, melphalan, nitrogen mustard, and nitrosourea. The linkage between the anti-cancer agent and the peptides of the exemplary embodiments of the present disclosure can be carried out by methods known in the art (such as covalent bonding or cross-linking). For this purpose, if necessary, the peptides of the exemplary embodiments of the present disclosure can be chemically modified within the range where their activity is not degraded.

[0060] In addition, the exemplary embodiments of the present disclosure provide a fusion peptide, wherein an apoptosis-inducing peptide having the amino acid sequence represented by SEQ ID NO: 2 binds to the peptide.

[0061] The apoptosis-inducing peptide having the amino acid sequence represented by SEQ ID NO: 2 is "KLAKLAKKLAKLAK", which is abbreviated as "KLA" in this specification.

[0062] In addition, the exemplary embodiments of the present disclosure provide a pharmaceutical composition for preventing or treating cancer, the pharmaceutical composition comprising the fusion peptide as an active ingredient.

[0063] In addition, the exemplary embodiments of the present disclosure provide a pharmaceutical composition for preventing or treating cancer, the pharmaceutical composition comprising the fusion peptide and an anti-cancer agent as active ingredients.

[0064] Preferably, the anti-cancer agent may include mertansine, doxorubicin, paclitaxel, vincristine, daunorubicin, vinblastine, actinomycin-D, docetaxel, etoposide, teniposide, bisantrene, homoharringtonine, STI-571, cisplatin, 5-fluorouracil, adriamycin, methotrexate, busulfan, chlorambucil, cyclophosphamide, melphalan, nitrogen mustard, or nitrosourea, but is not limited thereto.

[0065] Preferably, the cancer may include lung cancer, breast cancer, pancreatic cancer, brain tumor, liver cancer, skin cancer, esophageal cancer, testicular cancer, kidney cancer, colorectal cancer, rectal cancer, gastric cancer, bladder cancer, ovarian cancer, bile duct cancer, gallbladder cancer, uterine cancer, cervical cancer, prostate cancer, head and neck cancer, and squamous cell carcinoma, but is not limited thereto.

[0066] The pharmaceutical composition of the exemplary embodiments of the present disclosure can be prepared by using pharmaceutically suitable and physiologically acceptable adjuvants in addition to the active ingredient, and solubilizers (such as excipients), disintegrants, sweeteners, binders, coating agents, swelling agents, polishing agents, lubricants, or flavoring agents can be used as adjuvants. The pharmaceutical composition of the exemplary embodiments of the present disclosure can preferably be formulated into a pharmaceutical composition by including one or more pharmaceutically acceptable carriers in addition to the active ingredient for administration. As an acceptable pharmaceutical carrier for the composition formulated into a liquid solution, the components should be sterile and biocompatible, and can be used by mixing saline, sterile water, Ringer's solution, buffered saline, albumin injection solution, dextran solution, maltodextrin solution, glycerol, ethanol, and one or more of these components. If necessary, other conventional additives, such as antioxidants, buffers, and bacteriostatic agents, can be added. In addition, diluents, dispersants, surfactants, binders, and lubricants can be additionally added to formulate injectable preparations (such as aqueous solutions, suspensions, and emulsions), pills, capsules, granules, or tablets.

[0067] The pharmaceutical formulation types of the pharmaceutical composition of the exemplary embodiments of the present disclosure may include granules, powders, coated tablets, tablets, capsules, suppositories, syrups, juices, suspensions, emulsions, drops, or injectable solutions, as well as sustained-release formulations of the active compound. The pharmaceutical composition of the exemplary embodiments of the present disclosure can be administered in a conventional manner via intravenous, intra-arterial, intraperitoneal, intramuscular, intra-arterial, intraperitoneal, intrasternal, transdermal, intranasal, inhalation, topical, rectal, oral, intraocular, or intradermal routes. The effective dose of the active ingredient of the pharmaceutical composition of the exemplary embodiments of the present disclosure refers to the amount required to prevent or treat the disease. Therefore, the dose can be adjusted according to various factors such as the type of the disease, the severity of the disease, the type and content of the active ingredient and other ingredients included in the composition, the type of the formulation, the age, weight, general health condition, gender, and diet of the patient, the administration time, the administration route, the secretion rate of the composition, the treatment period, and various factors including drugs used in combination therewith. Although not limited thereto, in the case of administering once to several times a day to an adult, the dose of the composition of the exemplary embodiments of the present disclosure can be, for example, 0.1 ng / kg - 10 g / kg in the case of the compound.

[0068] In addition, the exemplary embodiments of the present disclosure provide a composition for imaging cancer cells, the composition comprising the peptide as an active ingredient.

[0069] Preferably, the peptide may be labeled with a chromogenic enzyme, a radioisotope, a chromophore, a luminescent material, a fluorescent agent, a magnetic resonance imaging material, superparamagnetic particles, or ultrasuperparamagnetic particles, but is not limited thereto.

[0070] Cancer cell imaging and cancer diagnosis can be used not only for the purpose of initially diagnosing cancer diseases, but also for monitoring development, treatment, and response to therapeutic agents, but are not limited thereto. The peptide may be provided in a labeled state to facilitate the confirmation, detection, and quantification of binding, which is the same as described above.

[0071] In addition, the exemplary embodiments of the present disclosure provide a composition for detecting exosomes derived from cancer cells, the composition comprising the peptide as an active ingredient.

[0072] Examples

[0073] Hereinafter, exemplary embodiments will be described in detail to help understand the content of the present disclosure. However, the following exemplary embodiments are merely illustrative of the present disclosure, and the scope of the present disclosure is not limited to the following examples. The exemplary embodiments of the present disclosure are provided to more fully explain the content of the present disclosure to those of ordinary skill in the art.

[0074] <Example 1> Analysis of the characteristics of exosomes isolated from A549 tumor cells

[0075] Exosomes were isolated from A549 lung cancer cell culture medium using an ultracentrifuge or Exoquick (a commercial reagent). First, to analyze the characteristics of the isolated exosomes by Western blot analysis, the exosome extract was loaded onto an SDS polyacrylamide gel. The gel was electrophoresed and transferred to an NC membrane, and reacted with a blocking solution composed of Tris-buffered saline with Tween-20 (TBST) containing 5% skim milk for 1 hour to block non-specific reactions. Thereafter, the membrane was washed 3 times with TBST for 10 minutes each time, and reacted overnight at 4 °C with antibodies against CD63 and Alix (Abcam, USA). The next day, the membrane was washed several times with TBST at room temperature, and reacted with a horseradish peroxidase-conjugated secondary antibody at room temperature for 1 hour. Thereafter, analysis was performed using West Femto Maximum Sensitivity Substrate (ThermoFisher, USA) reagent and LAS.

[0076] As a result, exosomes derived from the A549, MDA-MB231, and HEK293 cell lines showed higher quantitative levels of exosomal markers (such as CD63, Alix, and Tsg 101) compared to cell extracts, and calnexin was expressed only in cell extracts( Figure 1 A). On the other hand, according to the cell lines, there was no significant difference in the levels of exosomal markers.

[0077] In addition, to measure the size of the isolated exosomes, the exosomes were analyzed using a NanoSight instrument. As a result, the sizes of exosomes isolated by ultracentrifugation and Exoquick were 193 nm and 103 nm, respectively, which were within the normal size range of exosomes( Figure 1 B).

[0078] In addition, exosomes captured by beads labeled with CD63 antibody (Invitrogen, USA) and exosomes captured by avidin magnetic beads after biotinylation were subjected to gel electrophoresis in the same manner as described above and western blotting using a CD63 antibody. As a result, the exosomal marker CD63 was well observed in both cases( Figure 1 C).

[0079] <Example 2> Discovery of Peptides Specifically Binding to Exosomes Derived from A549 Tumor Cells by Phage Screening

[0080] Phage screening was performed using a T7 phage hydrophobic amino acid library (1.3×10 10 pfu). The library has a CXXXXXXXC (X = random sequence) amino acid sequence, with cysteines at both ends and 7 random amino acids in the middle, and at least one of the 7 random amino acids has a hydrophobic amino acid. A549 was used as a lung cancer cell line. Exosomes were isolated from the tumor cell culture medium using an ultracentrifuge. To select T7 phages that selectively bind only to A549-derived exosomes, a process of removing phages that non-specifically bind to beads was first performed by reacting phages with beads labeled with CD63 antibody, and then phages that did not bind to the beads in the supernatant were collected. Then, the phages in the supernatant were reacted with exosomes captured on beads labeled with CD63 antibody( Figure 2 A).

[0081] Escherichia coli (E. coli) was used as a host to collect phages that bound to exosomes, and the titer of phages collected in each round was calculated by plaque assay. The remaining phages that were not subjected to plaque assay were amplified with E. coli to ensure the phages to be used in the next round. The same process was repeated up to 5 rounds.

[0082] As a result, the titer of the phages collected in each round was calculated. When the first round was performed, the titer of the collected phages was approximately 5.7×10 6 , and it was found that 2.06×10 7 phages were finally collected after 5 rounds. In other words, as a result of progressing from the first round to the fifth round, the titer of the phages increased by approximately 4-fold ( Figure 2 B).

[0083] To perform amino acid sequencing of the peptides displayed on the selected phages, 50 clones (100 in total) were collected from the products of the plaque assays used for titer measurement in the fourth and fifth rounds and stored in 10 μL of Tris buffer. The genes encoding the peptides inserted into these phages were amplified by PCR, the nucleotide sequences were analyzed, and amino acid sequencing was thereby performed. The peptide sequences were aligned and analyzed using the Clustal X program ( Figure 2 C). Among them, 10 phage clones representing peptides composed of 7 to 9 amino acids were selected for further study.

[0084] <Example 3> Evaluation of the selective binding ability of phage clones to exosomes derived from A549 tumor cells and the cells producing exosomes

[0085] To study the selective binding of phage clones to exosomes derived from A549 cells and the cells producing exosomes, phage-exosome binding ELISA and phage-cell binding ELISA were performed. For phage-exosome binding ELISA, the exosomes were first biotinylated and immobilized on magnetic beads labeled with monomeric avidin ( Figure 3 A). The phages bound to the exosomes were detected with an antibody against T7 phage conjugated to horseradish peroxidase, and the quantity was quantified based on the degree of the reaction and the color development of the substrate used for the enzyme.

[0086] As a result, it was found that three phage clones displaying the peptides with the sequences CTDTKIK (4R-3), CRLSKKS (5R-25), and CRKVAKG (5R-34) each bound more to the exosomes derived from A549 than the other clones ( Figure 3 B).

[0087] In addition, to measure the binding of phages to the cells producing exosomes by phage-cell binding ELISA, the cells were cultured on plates, and the phages bound to the cells were quantified using the antibody and substrate as described above. As a result, it was found that two clones displaying the peptides with the sequences CRKRPAL (4R-12) and CAVRRKL (5R-47) each bound more to the A549 cells producing exosomes than the other clones ( Figure 3 C).

[0088] <Example 4> Analysis of the Binding and Internalization of ExoPep Peptide to Exosomes Derived from A549 Tumor Cells

[0089] Based on Figure 3 the ELISA results shown in, five peptides (CTDTKIK, CRLSKKS, CRKVAKG, CRKRPAL, CAVRRKL) were synthesized. To study the binding of the candidate peptides by the FACS method, Peptron Co. (Daegeon, Korea) was requested to synthesize peptides in which fluorescein isothiocyanate (FITC, a fluorescent substance) was conjugated to the carboxyl terminus. Each peptide was synthesized by the standard Fmoc method and purified by mass spectrometry. In addition, the FITC-labeled NSSSVDK peptide was used as a control.

[0090] To observe the binding of the peptide to exosomes, biotin and exosomes were first complexed in the same manner as described in Figure 3 , and then the exosomes were immobilized by binding to avidin beads. The non-specific binding of the exosome-bead complex was blocked by treating with 1% bovine serum albumin (BSA) dissolved in phosphate buffer at room temperature for 30 minutes. After washing, the FITC-labeled peptide was bound to the exosome-bead complex at 4 °C for 30 minutes. After the reaction, the binding of the peptide to the exosomes was analyzed using a flow cytometer.

[0091] On the other hand, to observe the binding of the peptide to the cells that produce exosomes, first 1×10 6 cells were prepared. Each cell culture medium was treated with 1% BSA at 37 °C for 30 minutes. Thereafter, the FITC-labeled peptide was bound at 4 °C for 1 hour. After the reaction, the binding of the peptide to the cells was analyzed using a flow cytometer.

[0092] The results are as shown in Figure 4 A. Compared with each cell that produces exosomes, the peptide (sequence: CRKVAKG, name: ExoPep; SEQ ID NO: 1) of the exemplary embodiment of the present disclosure showed higher binding to exosomes derived from A549 lung cancer cells, H460 lung cancer cells, MDA-MB231 breast cancer cells, and Panc-1 pancreatic cancer cells. On the other hand, compared with each cell that produces exosomes, very low binding or no higher binding was observed in exosomes derived from normal cells (including HEK293 and MCF10A cells) and other types of tumor cells (including LLC mouse lung cancer cells, HT-29 colorectal cancer cells, and HepG2 liver cancer cells). On the other hand, due to the relatively high binding to normal cells, other types of peptides were excluded from the candidate group.

[0093] Figure 4Panel B shows a schematic diagram of the binding and internalization experiment of exosomes and ExoPep peptide. The culture media of A549, HEK293, and MDA-MB231 cells were collected, and exosomes were isolated by ultracentrifugation. The exosomes were labeled with DiD fluorescent reagent (red), and then reacted with ExoPep peptide (FITC-labeled, green). After the reaction, the cells were fixed, and the nuclei were stained with DAPI (blue) for analysis by confocal microscopy. More specifically, the isolated exosomes and DiD (diluted 1:200) were reacted on a stirrer at 37 °C for 30 minutes. After adding Exoquick reagent, the mixture was kept at 4 °C for 30 minutes, and then the DiD-labeled exosomes were precipitated by centrifugation at 13,000 rpm for 3 minutes. Thereafter, the mixture of 10 μM FITC-conjugated peptide and DiD-labeled exosomes was reacted with A549 cells at 37 °C for 1 hour. After washing three times, fixation was performed using 2% paraformaldehyde (PFA). After washing again, the cells were treated with 4'-6-diamidino-2-phenylindole (DAPI) to stain the nuclei. Finally, after mounting, observation was performed using a confocal microscope (Zeiss, Oberkochen, Germany).

[0094] As a result, in A549 cells, the ExoPep peptide was observed together with exosomes, but no control peptide (c.p) Figure 4 C) was observed. This indicates that the control peptide did not bind to exosomes derived from A549 cells, but the ExoPep peptide bound and was effectively internalized into the cells. On the other hand, exosomes derived from normal HEK293 cells were treated in the same manner as described above and observed under a confocal microscope (Zeiss, Oberkochen, Germany). As a result, the internalization of exosomes into HEK293 cells occurred effectively, and neither the control peptide nor the ExoPep peptide was observed in the cells Figure 4 C). This indicates that the ExoPep peptide did not bind to exosomes of normal cells.

[0095] In addition, an experiment was conducted on exosomes derived from MDA-MB231 cells. The cells were prepared to secrete exosomes that showed green fluorescence by expressing CD63-GFP. GFP exosomes (green) and 10 μM TAMARA-conjugated peptide (red) were reacted with MDA-MB231 cells at 37 °C for 1 hour. Thereafter, after fixation, DAPI nuclear staining, and mounting as described above, observation was performed using a confocal microscope (Zeiss, Oberkochen, Germany). As a result, the ExoPep peptide bound to exosomes derived from MDA-MB231 cells and then was internalized into the cells together with the exosomes. More of this peptide was observed in the cells compared to the control peptide Figure 4 D).

[0096] <Example 5> Inhibiting the internalization of exosomes into recipient cells by using the binding of ExoPep peptide to exosomes derived from A549 tumor cells

[0097] To further confirm the binding specificity of the ExoPep peptide to exosomes derived from A549 cells, the exosomes were labeled with DID fluorescent dye, reacted with biotin-labeled ExoPep peptide, and then conjugated with biotin by reacting the labeled avidin with magnetic particles. The exosome / biotin-peptide / avidin-magnetic particle complex prepared by the reaction was captured and removed using a magnet, and the remaining exosomes were processed to recipient cells ( Figure 5 A).

[0098] As a result, compared with the case of directly processing cell-derived exosomes to recipient cells or reacting exosomes and ExoPep peptide without undergoing the removal process, the internalization of exosomes into recipient cells was significantly inhibited by pretreating A549 cell-derived exosomes with ExoPep peptide and then removing the same exosomes ( Figure 5 B). On the other hand, pretreatment with a control peptide (sequence: NSSSVDK) did not reduce the internalization of exosomes into recipient cells ( Figure 5 B). In addition, when a similar experiment was performed on exosomes derived from normal HEK293 cells, the internalization of exosomes into recipient cells was not inhibited by the ExoPep peptide and the control peptide of the exemplary embodiments of the present disclosure ( Figure 5 C).

[0099] <Example 6> The internalization and exosome binding of ExoPep peptide are reduced due to the inhibition of the release of exosomes derived from A549 tumor cells

[0100] To further confirm the binding specificity of ExoPep to exosomes, GW4869 (a sphingomyelin inhibitor, Sigma-Aldrich), a drug that inhibits the release of exosomes extracellularly, was used to treat cells at different concentrations (2.5 μM, 5 μM, and 10 μM). Thereafter, the exosomes were isolated and subjected to electrophoresis and Western blotting with antibodies against CD63, Alix, Tsg101, and calnexin (Abcam) in the same manner as described in Figure 1 .

[0101] As a result, it was found that exosome markers (such as CD63, Alix, and Tsg101) were greatly reduced under the GW4869 inhibitor at a concentration of 10 μM compared with those at concentrations of 2.5 μM and 5 μM ( Figure 6 A).

[0102] On the other hand, to confirm the effect of the GW4869 inhibitor on cell viability, A549 cells (5×10 3 cells per well in a 96-well cell culture vessel) were cultured with different concentrations of GW4869 in serum-free medium at 37 °C for 3 hours. Thereafter, the medium was replaced with medium containing 10% fetal bovine serum (FBS), and the cells were cultured for 24 hours, and then the cytotoxicity was measured using a CCK-8 assay (Dojindo, Japan). As a result, even at high concentrations, GW4869 did not show toxicity ( Figure 6 B). Based on the above results, using 10 μM GW4869 inhibitor, the binding of ExoPep peptide to exosomes and its internalization were analyzed. As a result of immunofluorescence staining, when treated with 10 μM GW4869, due to the reduced exosome release, the internalization of exosomes and the peptide bound to them was significantly reduced ( Figure 6 C).

[0103] <Example 7> Analysis of the binding and internalization of ExoPep peptide to exosomes derived from the blood of A549 tumor-bearing mice and normal mice

[0104] A549 cells were injected into BALB / c nude mice, and after tumor growth, blood was collected and exosomes were isolated using an exosome isolation kit. To measure the binding of the peptide to exosomes by flow cytometry, exosomes were labeled with CD63 antibody beads. As a control group, exosomes isolated from the blood of normal mice were used. The exosome-bead complexes labeled with CD63 beads were blocked by reacting with 1% BSA / PBS at room temperature for 30 minutes to reduce non-specific binding. After washing, the FITC-labeled ExoPep peptide was bound to the exosome-bead complexes at 4 °C for 30 minutes. After washing again with PBS, the binding of the peptide to the exosome-bead complexes was analyzed using a flow cytometer (ThermoFisher Scientific, USA) ( Figure 7 A).

[0105] As a result, it was found that the ExoPep peptide bound better to exosomes derived from the blood of A549 tumor-bearing mice than to exosomes derived from the blood of normal mice ( Figure 7 B and Figure 7 C).

[0106] To observe the internalization of the peptide into cells, exosomes were labeled with DiD, reacted with the FITC-labeled peptide, and treated with A549 cells. The cells were fixed with 4% paraformaldehyde for 5 minutes, stained with DAPI, and then observed under a microscope.

[0107] As a result, it was found that compared with exosomes derived from the blood of normal mice, the ExoPep peptide of the exemplary embodiments of the present disclosure binds to exosomes derived from the blood of tumor-bearing mice and is more internalized into the recipient cell A549. In addition, fluorescence of the peptide (green) and tumor-derived exosomes (red) was observed together in the cells ( Figure 7 E).

[0108] <Example 8> Analysis of the binding of ExoPep peptide to exosomes circulating in the blood of A549 tumor-bearing mice

[0109] To examine the binding of the peptide to exosomes circulating in the blood of mice, biotin-labeled peptide was intravenously injected into A549 tumor-bearing mice and healthy mice and circulated. Then, serum was collected and reacted with CD63 antibody beads and streptavidin beads, respectively, 30 minutes after collection. The serum treated with CD63 antibody beads was reacted overnight, and the beads were precipitated with a magnet the next day. The exosomes bound to the beads were lysed using RIPA lysis buffer, and then western blot analysis was performed using CD63 and ALIX antibodies. The serum treated with streptavidin beads was reacted for 1 hour, and then precipitated with a magnet. The exosomes bound to the beads were eluted using elution buffer (2 mM D-biotin in PBS), and then western blot analysis was performed using CD63 and ALIX antibodies ( Figure 8 A).

[0110] As a result, much higher exosome markers were detected when isolated from the blood of tumor-bearing mice with CD63 antibody beads compared with healthy mice ( Figure 8 B, left). Even when isolated with streptavidin beads, higher exosome markers were detected in the blood of tumor-bearing mice, but the amount of exosome markers was relatively lower than that when isolated with CD63 beads ( Figure 8 B, right). This indicates that since the CD63 antibody binds to all exosomes in the blood, while the biotin-ExoPep peptide selectively binds to tumor-derived exosomes, the amount of isolated exosomes is relatively small. The above results indicate that the peptide of the exemplary embodiments of the present disclosure binds to exosomes in the circulating blood and, in particular, selectively binds to tumor-derived exosomes.

[0111] Then, in vitro hemolysis assay was performed. The in vitro hemolysis assay, as an indicator of red blood cell hemolysis after exposure to a drug or agent, was conducted by measuring the hemoglobin release in plasma. This is an accurate and sensitive method for predicting the hemolytic activity of a drug. Fresh blood was centrifuged at 500×g for 10 minutes, and the red blood cell pellet was washed three times and resuspended in 10 mM PBS at pH 7.4. Equal volumes of red blood cells were reacted with different concentrations of ExoPep peptide by stirring at 37 °C for 1 hour. Then the samples were centrifuged at 500×g for 10 minutes at 4 °C. RBC lysis was measured by analyzing the absorbance at OD540nm according to different peptide concentrations. Considering that the 1% Triton X 100 sample used as a control showed 100% hemolysis, the percentage of hemolysis was measured. The hemolytic activity of the peptide was calculated as a percentage using the following equation:

[0112] Equation: H = 100×(O P - O B ) / (O T - O B )

[0113] In the above equation, O P , O B and O T represent the optical density of the peptide solution at the indicated concentration, the optical density of the buffer, and the optical density of Triton X 100, respectively.

[0114] Therefore, among various concentrations of the peptide up to a final concentration of 200 μM, only low levels of hemolytic activity were shown ( Figure 8 C).

[0115] <Example 9> In vivo distribution of exosomes derived from A549 tumor mouse blood and exosomes derived from A549 cells in A549 tumor mice according to the binding of ExoPep peptide

[0116] DiD-labeled exosomes derived from A549 tumor mouse blood and exosomes derived from A549 cells were reacted with ExoPep peptide, and the in vivo distribution was monitored by imaging. Tumor xenograft mice were prepared by subcutaneously implanting a suspension of A549 cells (5×10 6 cells) together with PBS into the right flank of 5-week-old female BALB / c nude mice. When the tumor size reached approximately 100 - 200 mm 3When anesthetizing the mice, the volume was considered. DiD-labeled exosomes (mEXO) derived from the blood of A549 tumor-bearing mice and the product (mEXO+ExoPep, n = 3) obtained by reacting the exosomes (mEXO) with the peptide, as well as DiD-labeled exosomes (cEXO) derived from A549 cells and the product (cEXO+ExoPep, n = 3) obtained by reacting the exosomes (cEXO) with the peptide were intravenously administered to the A549 tumor-bearing mice. The inventors of the present disclosure used FITC-labeled control peptide and ExoPep (n = 3) as controls. The fluorescence images in vivo were analyzed at different time points (2, 4, 8, and 24 hours respectively) after the administration of DiD-labeled exosomes using an IVIS imaging system (Caliper Life Sciences, Massachusetts, USA). At 24 hours after the administration, the in vitro fluorescence images of the excised tumors and organs were collected. All major organs (liver, kidney, spleen, heart, and lung) with tumor tissues were separated, washed with PBS, and subjected to in vitro fluorescence analysis (n = 3).

[0117] As a result, compared with mEXO and cEXO, more accumulation of the combination of mEXO+ExoPep and cEXO+ExoPep was observed in the tumor tissue 4 hours after injection ( Figure 9 A). Statistical significance was also obtained in the results of analyzing the fluorescence intensity in the target regions (ROIs) throughout the body, especially at the tumor site ( Figure 9 C). At 24 hours after the injection, the organs were removed and the fluorescence was photographed ( Figure 9 B). As a result of measuring the intensity, there was no difference in the accumulation of mEXO+ExoPep in the tumor tissue compared with mEXO, but less accumulation was observed in the liver ( Figure 9 D). On the other hand, compared with cEXO, more accumulation of cEXO+ExoPep was shown in the tumor tissue and the liver ( Figure 9 D). The FITC-labeled control and the ExoPep peptide itself showed low signals ( Figure 9 A- Figure 9 D).

[0118] <Example 10> Cytotoxicity IC50 analysis of ExoPep-KLA peptide composed of ExoPep peptide and apoptosis-inducing peptide

[0119] Taking advantage of the fact that the ExoPep peptide can be well internalized into cells after binding to exosomes, a peptide (named ExoPep-KLA) was prepared by fusing with a peptide (KLA) that induces apoptosis by damaging the mitochondrial membrane in cells. To confirm the cytotoxicity of ExoPep-KLA in A549, MDA-MB231, HEK293, Panc-1, HT29 and HepG2 cells (5×10 3 cells per well in a 96-well cell culture vessel), the cells were cultured with different concentrations of ExoPep-KLA in serum-free medium at 37 °C for 3 hours. Thereafter, the medium was replaced with medium containing 10% FBS, the cells were cultured for 24 hours, and then the cytotoxicity was measured using the CCK-8 assay (Dojindo). Different concentrations of ExoPep-KLA were reacted with various exosomes (5 μg) isolated from A549, MDA-MB231, Panc-1, HT29, HepG2 and HEK293 cells, and then treated with the cells. As a control, either ExoPep-KLA alone or the product obtained by first treating the cells with ExoPep-KLA and then with exosomes was used.

[0120] As a result, compared with the group treated with the peptide alone and the group treated with the peptide followed by exosomes, a more effective cytotoxicity (i.e., a lower IC50 value) was exhibited in the group treated with the peptide and exosomes. In particular, the ExoPep-KLA peptide showed almost no cytotoxicity in normal HEK293 cells, HT-29 colorectal cancer cells and HepG2 liver cancer cells, but specifically showed higher cytotoxicity in A549 lung cancer cells, MDA-MB231 breast cancer cells and Panc-1 pancreatic cancer cells ( Figure 10 A- Figure 10 F). On the other hand, after reacting the exosomes of normal HEK293 cells with ExoPep-KLA and treating them with HEK293 cells, almost no cytotoxicity was observed. On the other hand, after reacting A549 cell exosomes with ExoPep-KLA and treating them with HEK293 cells, compared with the group treated with the peptide alone and the group treated with the peptide followed by exosomes, cytotoxicity to HEK293 cells was obtained when exosomes and the ExoPep-KLA peptide were treated together ( Figure 10 G). This indicates the binding specificity of the ExoPep-KLA peptide to exosomes derived from tumor cells.

[0121] <Example 11> Apoptosis-inducing effect of ExoPep-KLA

[0122] To study the induction of apoptosis by ExoPep-KLA, phosphorescent A549-luc and MDA-MB231-luc cells were seeded at a density of 1×10 4 cells / well in black 96-well ELISA plates. After reacting the cells with ExoPep-KLA and exosomes (5 μg) from each cell type for 24 h, 3 μL (3 mg / mL) of D-luciferin was added to each well, and the phosphorescence intensity (efflux activity) was measured using an IVIS imaging system (PerkinElmer, Waltham, MA, USA). As a result, compared with the group treated with ExoPep-KLA peptide alone and the group treated with exosomes after peptide treatment, as the treatment concentration and reaction time of ExoPep-KLA with exosomes increased, the apoptotic effect increased and the luc signal intensity decreased ( Figure 11 ).

[0123] <Example 12> Apoptotic effect of ExoPep-KLA (which has undergone reaction with exosomes derived from A549 cells and exosomes derived from the blood of A549 tumor-bearing mice) on A549 cells

[0124] To determine the apoptosis-inducing effect of ExoPep-KLA dependent on binding to exosomes derived from A549 cells, the inventors of the present disclosure treated A549 cells with 5 μg of ExoPep-KLA pre-reacted with cell-derived exosomes or unreacted ExoPep-KLA. The cells were stained with Annexin V-647 and analyzed using a flow cytometer (ThermoFisher Scientific, USA) to quantify apoptotic cells (Annexin+ / PI-). Compared with the group treated with ExoPep-KLA alone, the ExoPep-KLA group pre-reacted with exosomes (cEXO+ExoPep-KLA) showed a higher proportion of apoptotic cells, 62.9% and 20.3% respectively (p<0.05, Figure 12 A). The results showed that ExoPep-KLA bound to exosomes induced apoptosis more effectively than ExoPep-KLA alone.

[0125] In addition, to measure the apoptosis-inducing effect of ExoPep-KLA that depends on binding to exosomes derived from the blood of mice subcutaneously implanted with A549 tumors, the inventors of the present disclosure treated A549 cells with exosomes (5 μg) derived from the blood of mice implanted with A549 tumors, with or without pre-reaction with ExoPep-KLA. The cells were stained with Annexin V-647, and the cells were analyzed by flow cytometry (ThermoFisher Scientific, USA) to quantify apoptotic cells (Annexin+ / PI-). The group of ExoPep-KLA pre-reacted with exosomes (mEXO+ExoPep-KLA) showed a higher ratio of apoptotic cells than the group treated with ExoPep-KLA alone, 19.1% and 6.1% respectively, but the ratio itself was not high (p<0.05, Figure 12 B). The above results indicate that a larger number of ExoPep-KLA binds to exosomes derived from tumor cells compared to exosomes derived from the blood of tumor-implanted mice, and induces higher apoptosis than in A549 cells.

[0126] <Example 13> Analysis of the Stability of ExoPep-KLA in Serum

[0127] The inventors of the present disclosure attempted to confirm the stability of the ExoPep-KLA peptide in mouse serum. After reacting the ExoPep-KLA peptide with mouse serum for 24 hours, the amount of the remaining peptide in the serum was analyzed. The peptide peak was separated from the non-specific peaks of the serum, and the residual amount of the peptide in the serum was calculated by the peak area. The ExoPep-KLA peptide was hardly decomposed until 4 hours, and was partially decomposed at 8 hours, with a half-life of about 24 hours ( Figure 13 A and Figure 13 B). As a result of the mass spectrometry analysis of each peptide peak, it was found that the peak was the ExoPep-KLA peptide. The results indicate that the peptide is relatively stable in serum.

[0128] <Example 14> Inhibition of Tumor Growth and Metastasis by ExoPep-KLA Peptide in an A549 Tumor Mouse Model

[0129] To test the anti-cancer effect of using ExoPep-KLA in an A549 tumor nude mouse model, the ExoPep-KLA peptide was either used alone or pre-reacted with exosomes in vitro, and then administered systemically intravenously at the time points indicated in the protocol for 3 weeks, with doxorubicin administered once a week for 3 weeks ( Figure 14 A).

[0130] As a result, when phosphate buffer and exosomes derived from the blood of tumor-bearing mice (mEXO only) were administered, tumor growth was not inhibited. However, in the groups administered with doxorubicin, ExoPep-KLA, and mEXO+ExoPep-KLA prepared by pre-reacting exosomes (mEXO) and ExoPep-KLA, tumor growth was significantly inhibited( Figure 14 B). In particular, even when ExoPep-KLA was injected alone, tumor growth was inhibited at a similar level compared to the case where exosomes were injected after pre-reaction in vitro. This indicates that the ExoPep-KLA peptide binds to tumor-derived exosomes (mEXO) circulating in the blood and effectively migrates to the tumor tissue. During the treatment, the body weight of the mice did not change even with the continuous administration of the treatment groups( Figure 14 C). On the other hand, when treated with doxorubicin, ExoPep-KLA, and mEXO+ExoPep-KLA, the weight of the tumor decreased( Figure 14 D).

[0131] When A549 lung cancer cells were xenografted subcutaneously in nude mice, the number of tumor nodules caused by lung metastasis was low( Figure 14 E). In addition, in the mEXO+ExoPep-KLA group and the group administered ExoPep-KLA alone, the number of tumor nodules was slightly reduced compared to phosphate buffer and doxorubicin, but there was no significant difference( Figure 14 E). In addition, there was no significant difference in lung weight among other groups( Figure 14 F). However, when exosomes isolated from the blood of tumor-bearing mice (mExo only) were administered alone, the weight of the lung and the nodules metastasized to the lung increased significantly compared to other groups( Figure 14 E and Figure 14 F). This is an unexpected result, indicating that when exosomes circulating in the blood of tumor-bearing mice are administered after in vitro isolation, tumor metastasis is promoted.

[0132] On the other hand, as a result of measuring the liver weight, there was no significant difference among all groups( Figure 14 G). The survival rate was significantly prolonged in the groups treated with mEXO+ExoPep-KLA and ExoPep-KLA compared to phosphate buffer, the group treated with mEXO alone, and the group administered doxorubicin( Figure 14H). The results showed that the intravenously injected ExoPep-KLA peptide specifically bound to circulating exosomes in tumor-bearing mice, promoted internalization into tumor tissues and tumor cells, and subsequently induced cancer cell apoptosis. This also indicated that similar anti-cancer therapeutic effects were obtained when exosomes were bound to ExoPep, even when blood-derived exosomes were administered. After treatment, liver tissues were excised to examine the accumulation of exosomes (mEXO-DiD labeling), but no accumulation was observed in all treatment groups ( Figure 14 I).

[0133] <Example 15> Analysis of blood levels and liver and kidney functions after administration of ExoPep-KLA peptide in an A549 tumor mouse model

[0134] To investigate the systemic side effects caused by the treatment with ExoPep-KLA peptide, mouse blood was collected after the treatment in Example 14, and blood levels and the functional levels of the liver and kidney were analyzed.

[0135] As a result, there were no significant differences outside the normal range in blood levels (including white blood cell count) between the groups treated with ExoPep-KLA or mEXO+ExoPep-KLA peptide and healthy mice ( Figure 15 ). In addition, as a result of the functional tests of the liver and kidney in the treatment groups, no particular toxicity was shown ( Figure 15 ).

[0136] <Example 16> Inhibition of tumor growth and metastasis by co-administration of doxorubicin and ExoPep-KLA peptide in an A549 tumor mouse model

[0137] In an A549 tumor nude mouse model, ExoPep-KLA peptide was administered three times a week at the specified time points, and doxorubicin was administered once a week for three weeks. The times of single administration and co-administration are shown in the treatment protocol ( Figure 16 A).

[0138] As a result, in the groups treated with doxorubicin alone (2.5 mg / kg), ExoPep-KLA (5 mg / kg), and ExoPep-KLA (10 mg / kg), the size of the tumor was slightly reduced compared to the group treated with phosphate buffer. However, there were no significant differences ( Figure 16 B). On the other hand, when doxorubicin (5 mg / kg), doxorubicin + ExoPep-KLA (2.5 + 5 mg / kg), and doxorubicin + ExoPep-KLA (2.5 + 10 mg / mg) were co-administered, the tumors were more strongly inhibited. Despite the continuous administration of the exosome peptide and the combination with doxorubicin, the body weight of the mice did not change during the treatment ( Figure 16C). In addition, the co - administration of doxorubicin + ExoPep - KLA (2.5 + 10 mg / kg) and doxorubicin + ExoPep - KLA (2.5 + 5 mg / kg) significantly reduced the tumor weight. This effect was most obvious in the group with the doxorubicin + ExoPep - KLA (2.5 + 10 mg / kg) combination compared to other groups ( Figure 16 D). In addition, compared with the buffer - treated group, the metastatic nodules and micro - nodules were significantly reduced in the groups treated with doxorubicin (5 mg / kg), doxorubicin + ExoPep - KLA (2.5 + 5 mg / kg), and doxorubicin + ExoPep - KLA (2.5 + 10 mg / kg) ( Figure 16 E).

[0139] In addition, as a result of measuring the weight of the lungs, there was no significant difference among all groups ( Figure 16 F). In the result of measuring the liver weight, there was no significant difference among all groups ( Figure 16 G).

[0140] On the other hand, compared with the phosphate - buffered saline and single - treatment groups, the survival rate was significantly increased in the groups co - treated with doxorubicin + ExoPep - KLA (2.5 + 5 mg / kg) and doxorubicin + ExoPep - KLA (2.5 + 10 mg / kg) ( Figure 16 H). In addition, after treatment, the tumor tissues were sectioned and TUNEL staining (green) was observed in the tissues. As a result of immunohistochemical analysis, the treatment with doxorubicin + ExoPep - KLA (2.5 + 5 mg / kg) and doxorubicin + ExoPep - KLA (2.5 + 10 mg / kg) significantly increased the proportion of TUNEL - positive apoptotic cells in the tumor tissues ( Figure 16 I). The above results indicate that when ExoPep - KLA and doxorubicin are co - administered, the therapeutic effect of doxorubicin may increase, and its side effects may be weakened by reducing the dose later.

[0141] <Example 17> Analysis of blood levels and liver and kidney function levels after co - administration of ExoPep - KLA peptide and doxorubicin in an A549 tumor mouse model

[0142] At Figure 16After treatment in, blood was collected to analyze blood levels and liver and kidney function. As a result, there was no significant difference in blood levels (including white blood cell counts) beyond the normal range between the treated group and healthy mice, which supports the absence of systemic side effects due to co-treatment with ExoPep-KLA or doxorubicin + ExoPep-KLA (2.5 + 10 mg / kg). In addition, as a result of liver and kidney function tests, co-treatment with ExoPep-KLA or doxorubicin + ExoPep-KLA (2.5 + 10 mg / kg) showed no toxicity compared to healthy mice ( Figure 17 ).

[0143] <Example 18> Inhibition of tumor growth and metastasis by administration of ExoPep-KLA peptide alone and co-administration with gemcitabine in the Panc-1 pancreatic cancer mouse model

[0144] In the Panc-1 tumor model, the antitumor effects of ExoPep-KLA alone and in combination with gemcitabine were confirmed ( Figure 18 A). Systemic co-administration of ExoPep-KLA and gemcitabine significantly inhibited tumor growth, while the ExoPep-KLA administration group and the PBS group did not significantly inhibit tumor growth ( Figure 18 B). In order to confirm the biosafety of the exosome peptide and co-administration groups, the inventors of the present disclosure observed changes in body weight during administration. No changes in mouse body weight were observed during the administration of the exosome peptide ( Figure 18 C). Importantly, the group co-administered with ExoPep-KLA and gemcitabine reduced the growth of the primary tumor.

[0145] Compared with the ExoPep-KLA alone administration group and the PBS group, a decrease in tumor weight was observed in the co-administration group ( Figure 18 D). The ExoPep-KLA and gemcitabine co-administered group showed almost no metastatic nodules or micronodules compared to the PBS group ( Figure 18 E). In addition, lung weight was measured at the end of administration ( Figure 18 F). On the other hand, the weight of the primary tumor was reduced and the life span was increased in the co-administered group ( Figure 18 G). No systemic side effects due to administration were observed in all groups ( Figure 18 H).

[0146] Since the specific parts of the present disclosure have been described in detail above, it is obvious to those skilled in the art that these specific descriptions are only preferred exemplary embodiments, and the scope of the present disclosure is not limited thereby. Therefore, the essential scope of the present disclosure is intended to be defined by the appended claims and their equivalents. <110> KYUNGPOOK NATIONAL UNIVERSITY INDUSTRY-ACADEMIC COOPERATION FOUNDATION <120> Peptide Selectively Binding to Exosomes Derived from Cancer Cells and Use Thereof <130> AOP-2021-0024PCT / CN <150> KR 10-2020-0100354 <151> 2020-08-11 <150> KR 10-2021-0105103 <151> 2021-08-10 <160> 2 <170> KopatentIn 2.0 <210> 1 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Peptide <400> 1 Cys Arg Lys Val Ala Lys Gly 1 5 <210> 2 <211> 14 <212> PRT <213> Proapoptotic Peptide <400> 2 Lys Leu Ala Lys Leu Ala Lys Lys Leu Ala Lys Leu Ala Lys 1 5 10

Claims

1. A peptide that specifically binds to exosomes derived from cancer cells, the peptide consisting of the amino acid sequence shown in SEQ ID NO: 1, wherein, The cancer cells are lung cancer cells, breast cancer cells or pancreatic cancer cells.

2. A polynucleotide encoding the peptide according to claim 1.

3. A recombinant vector comprising the polynucleotide according to claim 2.

4. A transformant transformed with the recombinant vector according to claim 3.

5. A composition for diagnosing cancer, the composition comprising the peptide according to claim 1 as an active ingredient, wherein, The peptide specifically binds to exosomes derived from cancer cells, and wherein the cancer is lung cancer, breast cancer or pancreatic cancer.

6. A composition for drug delivery, the composition comprising the peptide according to claim 1 as an active ingredient, wherein, The drug is a cytotoxic peptide having apoptosis-inducing or necrosis-inducing activity, and wherein the drug is fused with the peptide according to claim 1.

7. A fusion peptide comprising an apoptosis-inducing peptide consisting of the amino acid sequence shown in SEQ ID NO: 2 and the peptide according to claim 1.

8. A pharmaceutical composition for treating cancer, the pharmaceutical composition comprising the fusion peptide according to claim 7 as an active ingredient, wherein, The cancer is lung cancer, breast cancer or pancreatic cancer.

9. A pharmaceutical composition for treating cancer, the pharmaceutical composition comprising the fusion peptide according to claim 7 and an anti-cancer agent as active ingredients, wherein, The cancer is lung cancer, breast cancer or pancreatic cancer, and wherein the anticancer agent is doxorubicin or gemcitabine.

10. A composition for imaging cancer cells, the composition comprising the peptide according to claim 1 as an active ingredient, wherein, The cancer cells are lung cancer cells, breast cancer cells or pancreatic cancer cells.

11. The composition according to claim 10, wherein, The peptide is labeled with any one selected from the group consisting of: chromogenic enzymes, radioisotopes and chromophores.

12. The composition according to claim 10, wherein, The peptide is labeled with any one selected from the group consisting of: fluorescent agents, superparamagnetic particles and super-superparamagnetic particles.

13. A composition for detecting exosomes derived from cancer cells, the composition comprising the peptide according to claim 1 as an active ingredient, wherein, The cancer cells are lung cancer cells, breast cancer cells or pancreatic cancer cells.

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