WO2016018134A1 - Polypeptide destiné à acheminer des biomolécules en direction de cellules et son utilisation - Google Patents

Polypeptide destiné à acheminer des biomolécules en direction de cellules et son utilisation Download PDF

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WO2016018134A1
WO2016018134A1 PCT/KR2015/008109 KR2015008109W WO2016018134A1 WO 2016018134 A1 WO2016018134 A1 WO 2016018134A1 KR 2015008109 W KR2015008109 W KR 2015008109W WO 2016018134 A1 WO2016018134 A1 WO 2016018134A1
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polypeptide
domain
cell
cells
seq
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PCT/KR2015/008109
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Korean (ko)
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김학성
유정현
조성민
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한국과학기술원
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/42—Proteins; Polypeptides; Degradation products thereof; Derivatives thereof, e.g. albumin, gelatin or zein
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression

Definitions

  • the present invention relates to a polypeptide for delivering a useful biomolecule into a cell, and more particularly, a polypeptide for delivering various molecules into a cell of a eukaryotic cell, a polynucleotide encoding the polypeptide, and a vector comprising the polynucleotide.
  • the present invention relates to a method for producing the polypeptide by culturing the recombinant main cell transformed with the vector and the recombinant host cell.
  • the cell membranes of animal cells do not pass many biomolecules. Although various biomolecules are used in fields such as basic science and medicine, the use of biomolecules inside cells has been limited due to cell membranes.
  • Protein is an important substance that is widely used in basic science and medicine fields, and can have a much more diverse and delicate function than a small molecule substance.
  • proteins do not cross cell membranes, there are significant limitations in their use in the cytoplasm.
  • Protein therapeutics are being actively developed for the effective treatment of diseases including cancer, but most of them are limited to disease targets outside the cell because proteins cannot cross the cell membrane. However, since there are more disease targets in cells, the importance of developing protein therapeutics for them is of great importance. Therefore, in order to develop a protein therapeutic agent having a high therapeutic effect against a wide range of diseases, development of a technology capable of safely delivering a therapeutic protein that targets intracellular disease targets into specific cells is required first.
  • PTD protein transduction domains
  • cell penetrating peptides such as cell penetrating peptides
  • cell penetrating peptides are conjugated to proteins and transferred into cells, or foreign proteins are attached to nanoparticles and transferred into cells, but low efficiency and cell specificity are the biggest. It remains a problem.
  • the present inventors have developed a polypeptide that can safely and efficiently deliver foreign biomolecules into specific cells based on the cell permeation domain of the toxin protein produced by bacteria, and completed the present invention.
  • An object of the present invention is to provide a polypeptide capable of safely and efficiently delivering biomolecules into a specific cell, a polynucleotide encoding the polypeptide, a vector comprising the polynucleotide, a recombinant microorganism into which the vector is introduced, and the recombinant microorganism. It provides a method for producing the polypeptide by culturing.
  • Another object of the present invention is to provide a composition for intracellular delivery containing the polypeptide and a method for intracellular delivery of biomolecules using the composition.
  • FIG. 1 is a schematic diagram of the present invention, which illustrates the polypeptide consisting of three domains and a endoplasmic reticulum residue sequence.
  • FIG. 2 is a schematic diagram illustrating the crystallization structure of a polypeptide that recognizes a Gb3 cell membrane receptor based on the basic concept of FIG. 1 and delivers eGFP into a cell.
  • FIG. 3 is a confocal laser scanning micrograph showing successful delivery of eGFP into Gb3-positive cells using the polypeptide of FIG. 2.
  • FIG. 4 is a schematic diagram illustrating the crystallization structure of a polypeptide that recognizes an EGFR cell membrane receptor based on the basic concept of FIG. 1 and delivers an eGFP into a cell.
  • FIG. 5 is a confocal laser scanning micrograph of eGFP delivered into EGFR positive and negative cells using the polypeptide of FIG. 4.
  • FIG. 6 is a graph illustrating the successful delivery of luciferase by a polypeptide that recognizes a Gb3 cell membrane receptor based on the basic concept of FIG. 1 and delivers luciferase into a cell.
  • FIG. X axis hour
  • Y axis relative activity of intracellular luciferase
  • blue the polypeptide
  • green polypeptide with domain 2 removed
  • red polypeptide with inactive B-subunit bound.
  • a polypeptide is prepared by using domain 2 of a toxin protein produced by P. aeruginosa as a cell permeation domain and binding a receptor recognition domain and a cargo domain to the N and C ends of the cell permeation domain, respectively. It was.
  • the present invention provides a biomolecule into a cell, wherein the cell permeation domain is a basic skeleton, and the receptor recognition domain and the cargo domain are respectively coupled to the N and C ends of the cell permeation domain. It relates to a polypeptide that can be delivered.
  • the "cell permeation domain” refers to a portion that allows the biomolecule to enter the cell through the cell membrane.
  • the domain may use a polypeptide derived from a bacterial toxin protein.
  • a part of Pseudomonas Exotoxin A, a part of Shiga like toxin of E. coli can be used.
  • the "receptor recognition domain” means a portion that recognizes a specific receptor in the cell membrane of the cell to be delivered.
  • the domain may be a variety of molecules that can recognize a specific receptor on the cell membrane, including the polypeptide.
  • natural proteins such as EGF, IL-6, Fc, monoclonal antibodies (scFv), lipids (Repebody), artificial proteins such as DARPin, Monobody, Nanobody, small molecule ligands such as RGD, folate, etc. can be used. have.
  • “cargo domain” refers to a biomolecule to be delivered into the cell.
  • the domain may be conjugated to various C-terminal end of the cell permeation domain in a variety of ways.
  • a nucleotide of a foreign protein can be used to be conjugated to the nucleotide of the polypeptide to produce one binding polypeptide form.
  • the cysteine cysteine
  • maleimide maleimide
  • biomolecules to be delivered may be targets for therapeutic and diagnostic proteins, single-stranded nucleic acids, double-stranded nucleic acids, small molecule drugs, and the like.
  • the polypeptide according to the present invention may be characterized in that the endoplasmic reticulum residue sequence is further connected to the C terminal of the cargo domain (biomolecule) in order to increase the intracellular delivery efficiency of the biomolecule.
  • the endoplasmic reticulum means a sequence that allows the polypeptide to remain in the endoplasmic reticulum of the cell to help foreign molecules penetrate into the cell.
  • the endoplasmic reticulum sequences include sequences such as KDEL and REDLK to help foreign molecules remain in the endoplasmic reticulum.
  • the N-terminal and the receptor recognition domain of the domain 2 may be connected by a linker represented by the amino acid sequence of SEQ ID NO: 4, but is not limited thereto.
  • the C terminal and the cargo domain of the domain 2 may be connected by a linker represented by the amino acid sequence of SEQ ID NO: 5, but is not limited thereto.
  • nucleic acids encoding the respective domains were amplified by PCR and then introduced into E. coli.
  • B-Subunit of cigatoxin was used as the N-terminal receptor recognition domain of domain 2
  • eGFP was used as the C-terminal cargo domain.
  • the expressed polypeptide was purified using Ni-NTA column and gel permeation chromatography (GPC) (SEQ ID NO: 10). 1 ⁇ M of the purified polypeptide was treated in HepG2 (KCTC, No. HC18302) and Vero cell (KCTC, No. AC28810) for 3 hours, and then confirmed by confocal laser scanning microscope. As a result, eGFP was delivered into the cells. It was confirmed that (Fig. 3).
  • hEGF was used as the receptor recognition domain, leaving the cargo domain intact to prove that the receptor recognition domain was interchangeable.
  • the completed gene was inserted into a pET-based vector for expression, and then introduced into E. coli and expressed.
  • the expressed polypeptide was purified using Ni-NTA column and gel permeation chromatography (GPC) (SEQ ID NO: 11). 1 ⁇ M of the purified polypeptide was treated with Hcc827 (ATCC, No. CRL-2868) and Vero cells for 6 hours, and then confirmed by confocal laser scanning. As a result, eGFP was delivered only in Hcc827 cells expressing EGFR. It could be confirmed that (Fig. 5).
  • renilla luciferase was used as the cargo domain while leaving the receptor recognition domain intact to prove that the cargo domain was replaceable.
  • the completed gene was inserted into a pET-based vector for expression, and then introduced into E. coli and expressed.
  • the expressed polypeptide was purified purely using Ni-NTA column and gel permeation chromatography (GPC) (SEQ ID NO: 12).
  • GPC gel permeation chromatography
  • the purified polypeptide was tested using HepG2 cells. As a result of measuring the activity of intracellular luciferase, it was confirmed that a sufficient amount of luciferase was delivered into the cell in 1 hour (FIG. 6).
  • the present invention a polynucleotide encoding the polypeptide;
  • the present invention relates to a recombinant microorganism into which the polynucleotide or the recombinant vector is introduced.
  • the polynucleotide means that thousands or more of the nucleotides, which are a unit in which sugar, phosphate, and base are bonded to each other. Nucleotides form long chains with diester bonds. At this time, the skeleton of sugar and phosphoric acid enters repeatedly, and the order of bases is irregularly arranged between. Irregularities and lengths of base placement produce key information that enters the gene. Polynucleotides are directional, with one end of the chain 5 'end and the other end of the chain necessarily 3' end. At this time, the nature of the terminal 5 ⁇ -phosphate group, 3 ⁇ -hydroxy group is displayed together. Both DNA and RNA are in polynucleotide form. Polynucleotides play an important role in protein biosynthesis.
  • vector refers to a DNA preparation containing a nucleotide sequence of a polynucleotide encoding the target protein operably linked to a suitable regulatory sequence so that the target protein can be expressed in a suitable host cell.
  • the regulatory sequence may comprise a promoter capable of initiating transcription, any operator sequence for regulating such transcription, a sequence encoding a suitable mRNA ribosomal binding site, and a sequence regulating the termination of transcription and translation, as desired It can be manufactured in various ways.
  • the promoter of the vector may be constitutive or inducible. After being transformed into a suitable host, the vector can replicate or function independently of the host genome and integrate into the genome itself.
  • the vector used in the present invention is not particularly limited as long as it can be replicated in a host cell, and any vector known in the art may be used.
  • Examples of commonly used vectors include natural or recombinant plasmids, phagemids, cosmids, viruses and bacteriophages.
  • pWE15, M13, ⁇ MBL3, ⁇ MBL4, ⁇ IXII, ⁇ ASHII, ⁇ APII, ⁇ t10, ⁇ t11, Charon4A, and Charon21A can be used as the phage vector or cosmid vector
  • pBR, pUC, and pBluescriptII systems are used as plasmid vectors.
  • pGEM-based, pTZ-based, pCL-based and pET-based and the like can be used.
  • the vector usable in the present invention is not particularly limited and known expression vectors can be used.
  • pACYC177, pACYC184, pCL, pECCG117, pUC19, pBR322, pMW118, pCC1BAC vector and the like can be used.
  • pACYC177, pCL, pCC1BAC vectors can be used.
  • a "recombinant microorganism” means that a vector having a polynucleotide encoding at least one target protein is introduced into a host cell, or a polynucleotide encoding at least one target protein is introduced at a microorganism so that the polynucleotide is incorporated into a chromosome.
  • a cell infected with a trait to express a protein and may be any cell, such as eukaryotic cells, prokaryotic cells, etc., but is not particularly limited thereto, bacterial cells such as E.
  • coli coli, Streptomyces, Salmonella typhimurium
  • Yeast cells Fungal cells such as Pchia pastoris; Insect cells such as Drozophila and Spodoptera Sf9 cells
  • Animal cells such as CHO, COS, NSO, 293, Bow Melanoma cells, and the like.
  • transformation refers to introducing a vector containing a polynucleotide encoding a target protein into a host cell or integrating a polynucleotide encoding a target protein into a chromosome of the host cell to complete the polynucleotide in the host cell. It means that the encoding protein can be expressed. Transformed polynucleotides include all of them, as long as they can be expressed in the host cell, whether they are inserted into or located outside the chromosome of the host cell. In addition, the polynucleotides include DNA and RNA encoding the target protein.
  • the polynucleotide may be introduced in any form as long as it can be expressed by being introduced into a host cell.
  • the polynucleotide may be introduced into a host cell in the form of an expression cassette, which is a gene construct containing all elements necessary for self-expression.
  • the expression cassette typically includes a promoter, transcription termination signal, ribosomal binding site and translation termination signal operably linked to the polynucleotide.
  • the expression cassette may be in the form of an expression vector capable of self replication.
  • the polynucleotide may be introduced into a host cell in its own form and operably linked to a sequence required for expression in the host cell.
  • the present invention provides a method for producing a polypeptide comprising: (a) culturing the recombinant microorganism to produce a polypeptide; And (b) relates to a method for producing a polypeptide that can deliver the biomolecule into the cell comprising the step of recovering the generated polypeptide.
  • the step of culturing the recombinant microorganism is not particularly limited thereto, but is preferably performed by a known batch culture method, continuous culture method, fed-batch culture method, and the like, and the culture conditions are not particularly limited thereto.
  • the culture conditions are not particularly limited thereto.
  • sodium hydroxide, potassium hydroxide or ammonia or acidic compounds (e.g. phosphoric acid or sulfuric acid) can be used to adjust the appropriate pH (pH 5-9, preferably pH 6-8, most preferably pH 6.8).
  • the incubation temperature can be maintained at 20 to 45 °C, preferably 25 to 40 °C, incubated for about 10 to 160 hours This is preferable.
  • the polypeptide produced by the culture may be secreted into the medium or remain in the cell.
  • the culture medium used may include sugars and carbohydrates (e.g. glucose, sucrose, lactose, fructose, maltose, molasses, starch and cellulose), fats and fats (e.g. soybean oil, sunflower seeds) as carbon sources.
  • sugars and carbohydrates e.g. glucose, sucrose, lactose, fructose, maltose, molasses, starch and cellulose
  • fats and fats e.g. soybean oil, sunflower seeds
  • fatty acids e.g. palmitic acid, stearic acid and linoleic acid
  • alcohols e.g. glycerol and ethanol
  • organic acids e.g. acetic acid
  • Nitrogen sources include nitrogen-containing organic compounds such as peptone, yeast extract, gravy, malt extract, corn steep liquor, soybean meal and urea, or inorganic compounds such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate and Ammonium nitrate) and the like can be used individually or in combination;
  • As a source of phosphorus, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, a corresponding sodium-containing salt, and the like can be used individually or in combination;
  • Other metal salts such as magnesium sulfate or iron sulfate, and essential growth-promoting substances such as amino acids and vitamins.
  • the method for recovering the polypeptide produced in the culturing step of the present invention is a method for recovering the desired polypeptide from the culture medium using a suitable method known in the art according to the culture method, for example, batch, continuous or fed-batch culture method, etc. Can be collected.
  • the core of the present invention is to design a non-toxic toxin from which the toxicity of host cells has been removed to use bacterial toxins as an intracellular delivery medium for foreign proteins, and to design the cell membrane receptors of host cells by using them as a basic skeleton.
  • biomolecule carriers including the "receptor recognition domain” that recognizes it as a target and the “cargo domain” to which foreign proteins are linked.
  • PCR was performed to synthesize a receptor recognition domain and a cargo domain, which are components of the template.
  • PCR conditions were 100 ⁇ M primers, 0.3 ⁇ l, 10X nPfu Buffer 5 ⁇ l, 2 mM dNTP 5 ⁇ l, 2 U nPfu Polymerase and template were added to make the final volume 50 ⁇ l.
  • the primer sets used to synthesize each domain are shown in SEQ ID NOs: 13-20 (Table 1).
  • PCR program is performed once in 95 °C 30 seconds in the first step, the second step (95 °C 30 seconds, 55 °C 30 seconds, 72 °C 30 minutes) is repeated 30 times, finally 72 °C 2 minutes It was performed once.
  • PCR products were cloned into pET21a vector using HindIII and NheI for cargo domain and NdeI and Eco RI for receptor binding domain. All genes were synthesized by E. coli transformation using a vector.
  • Transformation was performed using Origami B (DE3) strain ( E. coli ).
  • the vector was mixed into a competent cell at 4 ° C., heated for 90 seconds in a 42 ° C. constant temperature water bath, and then cooled on ice for 3 minutes. After incubation for 1 hour in a 37 °C shaking culture incubator at 37 °C was plated on a solid medium. The transformed cells were incubated in 500 mL LB medium to OD 0.5 and expressed at 18 ° C. for 24 hours.
  • Toxin proteins produced by bacteria such as intestinal hemorrhagic Escherichia coli or Pseudomonas aeruginosa bind to specific cell membrane receptors of the host and then move to the endoplasmic reticulum through the initial endosomes using the protein delivery system inherent in the host cell and finally penetrate into the cytoplasm Causes cell death.
  • bacteria such as intestinal hemorrhagic Escherichia coli or Pseudomonas aeruginosa
  • bind to specific cell membrane receptors of the host and then move to the endoplasmic reticulum through the initial endosomes using the protein delivery system inherent in the host cell and finally penetrate into the cytoplasm causes cell death.
  • intestinal hemorrhagic Escherichia coli or Pseudomonas aeruginosa bind to specific cell membrane receptors of the host and then move to the endoplasmic reticulum through the initial endosomes using the protein delivery system inherent in the host cell and
  • the system was optimally composed of the "receptor recognition domain", "domain 2", “cargo domain”, and finally the endoplasmic reticulum residual sequence.
  • Various tests of linkers between the receptor recognition domain and domain 2 showed that the use of a linker of about 12 amino acids (SEQ ID NO: 6) was optimal.
  • Tests between domain 2 and the cargo domain were performed in the same manner, and it was found to be optimal to use a linker of about eight amino acids (SEQ ID NO: 7). Since the endoplasmic reticulum sequence was optimally bound to the carboxy terminus of the cargo domain, templates were constructed at the gene level in this order.
  • the template is configured such that the receptor recognition domain and cargo domain can be easily inserted and replaced using restriction enzymes. Inserted into the pET-based vector for the convenience of expression and purification of the template was completed template construction of the basic system (Fig. 1).
  • Example 2 Based on the template constructed in Example 1, a polypeptide for delivering eGFP into cells through Gb3, a cell membrane receptor, was designed and tested.
  • B-subunit of cigatoxin was used as the receptor recognition domain.
  • B-subunit is a part of Shigatoxin and is known to recognize Gb3, a cell membrane receptor of animal cells.
  • genes of B-subunit and eGFP were used as receptor recognition domains and cargo domains, respectively (FIG. 2).
  • Example 2-1 1 ⁇ M of the polypeptide purified in Example 2-1 was treated with HepG2 (KCTC, No. HC18302) and Vero cell (KCTC, No. AC28810) for 3 hours, and then confirmed by confocal laser scanning microscope. It was confirmed that the eGFP was delivered (Fig. 3).
  • Example 3-1 Gene and Polypeptide Synthesis for Intracellular Delivery of eGFP Via EGFR
  • hEGF Human Epidermal Growth Factor
  • Example 3-2 Intracellular Delivery Evaluation of EGFR Mediated eGFP
  • Example 3-1 1 ⁇ M of the polypeptide purified in Example 3-1 was treated with Hcc827 (ATCC, No. CRL-2868) and Vero cells for 6 hours, and then confirmed by confocal laser scanning microscopy in Hcc827 cells expressing EGFR. Only eGFP could be confirmed that delivered (Fig. 5).
  • a polypeptide for delivering luciferase into cells through Gb3, a cell membrane receptor was designed and tested based on the template constructed in Example 1.
  • B-subunit of cigatoxin was used as in Example 2, and renilla luciferase was used as a cargo domain.
  • renilla luciferase was used as a cargo domain.
  • the expressed polypeptide was purified purely using Ni-NTA column and gel permeation chromatography (GPC) (SEQ ID NO: 12).
  • the purified polypeptide was tested using HepG2 cells. As a result of measuring the activity of intracellular luciferase, it was confirmed that a sufficient amount of luciferase was delivered into the cell in 1 hour (FIG. 6).

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Abstract

La présente invention concerne un polypeptide destiné à acheminer des biomolécules utiles en direction de cellules. Plus particulièrement, la présente invention concerne un polypeptide qui achemine diverses molécules en direction de cellules eucaryotes, un polynucléotide codant pour le polypeptide, un vecteur comprenant le polynucléotide, une cellule hôte recombinée transformée au moyen du vecteur et un procédé de préparation du polypeptide consistant à cultiver la cellule hôte recombinée. Un polypeptide, selon la présente invention, peut acheminer des biomolécules en direction de cellules particulières avec une grande efficacité et, par conséquent, est utile pour le développement et la préparation de médicaments, ainsi que dans le domaine de la recherche médicale et scientifique de base.
PCT/KR2015/008109 2014-08-01 2015-08-03 Polypeptide destiné à acheminer des biomolécules en direction de cellules et son utilisation WO2016018134A1 (fr)

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WO2018212540A2 (fr) * 2017-05-18 2018-11-22 한국과학기술원 Composite repebody-anticancéreux protéique à capacité de pénétration cellulaire améliorée, son procédé de préparation et utilisation
KR102098709B1 (ko) * 2017-05-18 2020-04-08 한국과학기술원 세포투과능이 향상된 리피바디-항암 단백질 약물 복합체, 그 제조방법 및 용도
KR102591441B1 (ko) * 2022-06-28 2023-10-23 정해관 세포외 소포체 탑재 유도 펩타이드 및 이를 포함하는 세포외 소포체

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KR20130098839A (ko) * 2012-02-28 2013-09-05 한양대학교 산학협력단 인간 ｌｐｉｎ３ 단백질 유래의 세포 투과 펩티드 및 이를 이용한 카고 전달 시스템

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