KR20220101559A - Exosome comprising overexpressed Fc receptor or its part and manufacturing method thereof - Google Patents

Abstract

The present invention relates to a method for preparing an exosome comprising an Fc receptor that exhibits a higher expression level, or a part thereof. The method for preparing an exosome according to the present invention comprises the steps of: preparing a DNA construct encoding a full-length Fc receptor or a part thereof without the fusion with a nucleic acid sequence encoding an exogenous transmembrane protein or a transmembrane domain thereof; transducing the DNA construct into a cell in which an FC receptor or a part thereof can not be naturally expressed; enabling the DNA construct to be expressed in the cell; and separating an exosome, which has been secreted or released, from a cell culture medium.

Description

Exosome comprising overexpressed Fc receptor or its part and manufacturing method thereof {Exosome comprising overexpressed Fc receptor or its part and manufacturing method thereof}

The present invention relates to an exosome comprising an overexpressed Fc receptor or a portion thereof and a method for preparing the same.

In addition, the present invention provides a gene construct encoding a full-length Fc receptor or a portion thereof, without fusion with a nucleic acid sequence encoding an exogenous transmembrane protein or a transmembrane domain thereof, the Fc receptor or a portion thereof naturally It relates to an exosome containing an overexpressed Fc receptor or a part thereof by transduction into a cell that cannot be expressed, and a method for producing the same.

Further, the present invention relates to the application of an exosome comprising an overexpressed Fc receptor or a portion thereof to the prevention, suppression, alleviation, improvement or treatment of immune-related diseases, cancer, inflammatory diseases, viral diseases, and various other diseases. .

A drug delivery system (DDS) is used to make a therapeutic agent administered to the human body work effectively and reduce side effects.

For example, when an antibody, protein, or polypeptide is exposed to an in vivo environment, its efficacy may be reduced or it may not be delivered to a target site to be treated. For example, a targeted anticancer agent has been developed to specifically act on cancer cells, but when administered into the human body, if it can act more selectively on a cancer tissue to be treated, the therapeutic effect can be maximized.

Liposomes or micelles developed as drug delivery systems so far have increased the retention time of the drug in vivo and have improved in terms of pharmacokinetics, but the ability to selectively target specific cells, for example, immune cells or cancer cells, is lacking. have. In addition, liposomes using synthetic materials may have biocompatibility problems.

On the other hand, recent studies have been reported that cell secretions contain various bioactive factors that control cell behavior. ' or 'extracellular vesicle' is included, so research on its components and functions is being actively conducted.

Cells release various membrane types of ERs to the extracellular environment, and these ERs are commonly referred to as extracellular vesicles (EVs). The extracellular vesicles are also called cell membrane-derived ERs, ectosomes, shedding vesicles, microparticles, exosomes, and the like, and in some cases, they are used separately from exosomes.

Exosomes are endoplasmic reticulum with a size of several tens to hundreds of nanometers having the same double phospholipid membrane as the structure of the cell membrane, and contain proteins, nucleic acids (mRNA, miRNA, etc.) called exosome cargo inside. Exosome cargo includes a wide range of signaling factors, and these signaling factors are known to be cell type-specific and differentially regulated according to the environment of secretory cells. Exosomes are intercellular signaling mediators secreted by cells, and various cellular signals transmitted through them regulate cell behavior, including activation, growth, migration, differentiation, dedifferentiation, apoptosis, and necrosis of target cells. is known Exosomes contain specific genetic material and bioactive factors according to the nature and state of the cell from which they are derived. In the case of proliferating stem cell-derived exosomes, it regulates cell behaviors such as cell migration, proliferation and differentiation, and reflects the characteristics of stem cells related to tissue regeneration (Nature Review Immunology 2002 (2) 569-579).

That is, exosomes called avatars of cells contain bioactive factors such as growth factors similar to cells, and serve as a carrier for transporting bioactive factors between cells, that is, communication between cells. Exosomes are known not only to be released from animal cells such as stem cells, immune cells, fibroblasts and cancer cells, but also from cells of various organisms such as plants, bacteria, fungi, and algae. .

When such an exosome is used as a drug carrier, it is biocompatible and has the advantage of being well absorbed while increasing the stability of the drug in vivo. However, the method of passively absorbing and loading drug cargo into exosomes naturally generated by using hydrophobic properties, etc. has a limitation in that manufacturing efficiency is low and sufficient drug cargo cannot be loaded in the exosomes.

Recently, in the gene construct production stage, the target protein or peptide is fused to the transmembrane protein of the exosome, expressed in the cell, and the exosome secreted or released into the cell culture medium is separated and loaded with the target protein or peptide (Exo). A method for obtaining an exosome in which a target protein or peptide is fused to a transmembrane protein of a small cell has been proposed (see WO 2013/084000 A2; WO 2014/168548 A2).

However, like other technical fields, the technical field to which the present invention belongs also requires continuous development of new technologies for effectively loading a target protein or peptide into an exosome and effectively delivering it to a target site to be treated.

On the other hand, it should be understood that the matters described as the above background art are only for improving understanding of the background of the present invention, and are not cited as approval that they can be used as "prior art" of the present invention.

Republic of Korea Patent Publication No. 10-2020-0098512 (2020.08.20) International Publication WO 2013/084000 A2 (2013.06.13) International Publication WO 2014/168548 A2 (2014.10.16)

It is an object of the present invention to provide an exosome comprising an overexpressed Fc receptor or a part thereof and a method for preparing the same.

Another object of the present invention is to convert a gene construct encoding a full-length Fc receptor or a portion thereof into a native Fc receptor or a portion thereof, without fusion with a nucleic acid sequence encoding an exogenous transmembrane protein or a transmembrane domain thereof. To provide an exosome containing an overexpressed Fc receptor or a part thereof by transduction into a cell that cannot be expressed as an exo, and a method for producing the same.

Another object of the present invention is to provide an application of an exosome containing an overexpressed Fc receptor or a part thereof to the prevention, suppression, alleviation, improvement or treatment of immune-related diseases, cancer, inflammatory diseases, viral diseases, and various other diseases. is doing

However, the problems of the present invention as described above are exemplary, and the scope of the present invention is not limited thereto. Further, other objects and advantages of the present invention will become more apparent by the following detailed description of the invention, claims and drawings.

In order to achieve the above object, as a result of repeated intensive research, the present inventors have developed a full-length Fc receptor or a part thereof without fusion with a nucleic acid sequence encoding an exogenous transmembrane protein or a transmembrane domain thereof. When the encoding gene construct is transduced into a cell that cannot naturally express the Fc receptor or a part thereof, the gene construct is expressed in the cell and the exosome secreted or released into the cell culture medium is isolated, contrary to expectations. It was confirmed that the content of the Fc receptor or a portion thereof per exosome is high (that is, the Fc receptor or part of the Fc receptor for the exosome has significantly excellent loading efficiency), and completed the present invention.

As used herein, the term "extracellular vesicles (EVs)" generally encompasses membrane vesicles, ectosomes, shedding vesicles, microparticles, or equivalents thereof. used in the sense that Depending on the isolation environment, conditions and methods, etc., the extracellular vesicle may have the same meaning as an exosome, and may have the same or similar size as the exosome, but may also have a meaning including nanovesicles that do not have the composition of the exosome. On the other hand, in the present specification, the term exosome used in relation to the loading of an Fc receptor or a portion thereof is used to encompass the extracellular vesicles.

As used herein, the term "exosomes" refers to endoplasmic reticulum having a size of several tens to hundreds of nanometers (preferably about 30 to 200 nm) having the same double phospholipid membrane as the structure of the cell membrane (provided that the separation target is Particle size of exosomes may vary depending on cell type, separation method and measurement method) (Vasiliy S. Chernyshev et al., "Size and shape characterization of hydrated and desiccated exosomes", Anal Bioanal Chem, (2015) DOI) 10.1007/s00216-015-8535-3). Exosomes contain proteins, nucleic acids (mRNA, miRNA, etc.) called exosome cargo. Exosome cargo includes a wide range of signaling factors, and these signaling factors are known to be cell type-specific and differentially regulated according to the environment of secretory cells. Exosomes are intercellular signaling mediators secreted by cells, and various cellular signals transmitted through them regulate cell behavior, including activation, growth, migration, differentiation, dedifferentiation, apoptosis, and necrosis of target cells. is known

On the other hand, the term "exosome" used herein has a nano-sized vesicle structure secreted from animal-derived cells and released into the extracellular space and has a composition similar to that of exosomes (eg, exosomes). -It means to include all similar vesicles). The type of the cell is not limited, but as an example that does not limit the present invention, it may be a HEK293 cell, a HEK293T cell, an Expi293F cell, a CHO cell, a stem cell, an immune cell, or a cancer cell, preferably a HEK293 cell, HEK293T cells or Expi293F cells.

In addition, as an example and not limiting the present invention, the animal-derived cells may be stem cells, immune cells, immortalized cells, or cancer cells. The stem cells may be embryonic stem cells, induced pluripotent stem cells (iPSCs), adult stem cells, embryonic stem cell-derived mesenchymal stem cells, or induced pluripotent stem cell-derived mesenchymal stem cells. The immune cells may be T cells, B cells, NK cells, cytotoxic T cells, dendritic cells or macrophages. The adult stem cells may be one or more adult stem cells selected from the group consisting of mesenchymal stem cells, human tissue-derived mesenchymal stromal cells, human tissue-derived mesenchymal stem cells, and pluripotent stem cells. The mesenchymal stem cells may be mesenchymal stem cells derived from one or more tissues selected from the group consisting of umbilical cord, umbilical cord blood, bone marrow, fat, muscle, nerve, skin, amniotic membrane, Wharton's jelly and placenta.

However, as long as it does not cause adverse effects on the human body, it is of course possible to use various animal cells that are used in the art or that can be used in the future. Therefore, the HEK293T cells used in the examples described below should be understood as an example of animal cells that can be used in the present invention, and the present invention is not limited thereto.

As used herein, the term "Fc receptor" refers to Fc alpha receptor (FcαR), Fc gamma receptor (FcγR), Fc epsilon receptor (FcεR), Fc mu receptor (FcμR), Fc alpha/mu receptor (Fcα/μR). , a broad concept including FcRn (neonatal Fc receptor). As an example not limiting the present invention, Fc alpha receptors include FcαRI (CD89), Fc gamma receptors include FcγRI (CD64), FcγRIIA (CD32), FcγRIIB1 (CD32), FcγRIIB2 (CD32), FcγRIIIA ( CD16A) and FcγRIIIB (CD16B), and Fc epsilon receptors include FcεRI and FcεRII (CD23).

The Fc alpha receptor is a receptor that binds to the Fc domain of IgA, the most abundant immunoglobulin in the human body. CD89 is expressed on cytotoxic immune effector cells including polymorphonuclear leukocytes (PMN), monocytes, macrophages, neutrophils and eosinophils. Ligand binding to CD89 triggers phagocytosis and antibody-mediated cytotoxicity of leukocytes and CD89-bearing cell lines. In addition, CD89 can enhance phagocytosis on target cells by cooperating with receptors for IgG on effector cells.

Fc gamma receptors are proteins that bind to an antibody portion called the Fc region or Fc domain of an IgG antibody and stimulate phagocytosis or cytotoxic activity through antibody-mediated phagocytosis or antibody-dependent cell-mediated cytotoxicity. For example, CD64 is a high affinity Fc gamma receptor, also called Fc gamma receptor I (FcyRI). The extracellular domain (ectodomain) of CD64 contains three immunoglobulin domains responsible for antibody binding. CD64 is expressed in monocytes, macrophages, dendritic cells and neutrophils, etc., and is involved in antibody-mediated phagocytosis and cytotoxic activation. CD16 is a low-affinity Fc gamma receptor, also called Fc gamma receptor III (FcγRIII). The extracellular domain (ectodomain) of CD16 contains two immunoglobulin domains responsible for antibody binding. There are two types of CD16: CD16A and CD16B. CD16A is expressed in monocytes, macrophages and NK cells, etc., and induces cytotoxic activity of NK cells. CD32 is a low-affinity Fc gamma receptor, also called Fc gamma receptor II (FcγRII). CD32 is present in monocytes, phagocytes, granulocytes, B cells, T cells, etc., and binds to complex or aggregated IgG.

Fc epsilon receptors are present on the surface of mast cells, basophils, eosinophils, monocytes, macrophages and platelets. FcεRI is a high-affinity Fc epsilon receptor, and FcεRII (CD23) is a low-affinity Fc epsilon receptor. IgE primes an IgE-mediated allergic response by binding to the Fc epsilon receptor present on the surface of mast cells and basophils.

The recirculating Fc neonatal receptor (FcRn) binds to antibodies of the IgG isotype and extends the in vivo half-life of IgG antibodies. However, FcRn does not bind IgA and IgM isotype antibodies. FcRn is known to prolong the half-life of IgG by effectively blocking the degradation of IgG in lysosomes.

Fc μ receptors (FcμR) and Fc α/μ receptors (Fcα/μR) bind to antibodies of the IgM isotype. Fcα/μR promotes uptake of antibodies and immune complexes bound to foreign substances by B cells and phagocytes. In addition, FcμR is important for B cell development and is known to affect IgM homeostasis, B cell survival, humoral immune response and autoantibody formation.

As used herein, the term "overexpression" refers to a protein or peptide that is not genetically engineered in wild-type cells and/or exosomes derived therefrom, or is expressed at low or normal levels, is genetically engineered. It means that it is expressed at a high level in cells and/or exosomes derived therefrom. In addition, the term "genetically engineered" refers to an act of introducing one or more genetic modifications into a cell or a cell made by such an act and/or exosomes derived therefrom .

As used herein, the term "transmembrane protein" refers to an extracellular domain existing outside the cell (ectodomain), a transmembrane domain existing in the cell membrane across the cell membrane, and an intracellular domain present inside the cell (endodomain). ) is composed of "Exogenous transmembrane protein or transmembrane domain thereof" means any transmembrane protein or a transmembrane domain thereof except for the transmembrane protein or its transmembrane domain possessed by the Fc receptor to be loaded into the exosome.

As used herein, a cell that does not naturally express an Fc receptor or a portion thereof may be, for example, a HEK293 cell, a HEK293T cell or an Expi293F cell. HEK293 cells, HEK293T cells or Expi293F cells do not express Fc receptors such as CD64, CD32 or CD16, and exosomes derived from HEK293 cells, HEK293T cells or Expi293F cells also do not have Fc receptors such as CD64, CD32 or CD16. The present invention is a technology capable of loading Fc receptors such as CD64, CD32 or CD16 into exosomes derived from HEK293 cells, HEK293T cells or Expi293F cells.

The method for producing an exosome comprising an overexpressed Fc receptor or a portion thereof of one embodiment of the present invention, without fusion with a nucleic acid sequence encoding an exogenous transmembrane protein or a transmembrane domain thereof, a full-length Fc receptor Or a portion thereof (provided that the step of preparing a gene construct encoding a transmembrane protein or a transmembrane domain of an Fc receptor), and the gene construct to a cell that does not naturally express the Fc receptor or a portion thereof Transducing, expressing the full-length Fc receptor or a portion thereof in the cell, and separating the exosomes containing the full-length Fc receptor or a portion thereof.

In the method for producing an exosome comprising an overexpressed Fc receptor or a portion thereof of one embodiment of the present invention, the exosome comprising the full-length Fc receptor or a portion thereof is secreted or released from the cell and contained in the culture medium of the cell. do. It is possible to separate the exosomes containing the full-length Fc receptor or a part thereof from the culture medium.

In the method for producing an exosome comprising an overexpressed Fc receptor or a portion thereof of one embodiment of the present invention, the full-length Fc receptor or a portion thereof is located on the surface of the exosome. In the present invention, the full-length Fc receptor or a portion thereof is presented on the exosome surface by a transmembrane protein or a transmembrane domain thereof having its own full-length Fc receptor or a portion thereof.

In the method for producing an exosome comprising an overexpressed Fc receptor or a portion thereof of one embodiment of the present invention, the Fc receptor may be CD64, CD32 or CD16. In addition, cells that do not naturally express the Fc receptor or a part thereof may be HEK293 cells, HEK293T cells or Expi293F cells.

As an example not to limit the present invention, the Fc receptors are FcαRI (CD89), FcγRI (CD64), FcγRIIA (CD32), FcγRIIB1 (CD32), FcγRIIB2 (CD32), FcγRIIIA (CD16A), FcγRIIIB (CD16B), FcεRI, FcεRII (CD23), FcμR, Fcα/μR, or FcRn.

In addition, the present invention provides an exosome comprising an overexpressed Fc receptor or a part thereof prepared according to the manufacturing method as described above, and comprises an exosome comprising the overexpressed Fc receptor or a part thereof as an active ingredient. A composition is provided. The composition may be a pharmaceutical composition or a cosmetic composition. For example, the pharmaceutical composition may be prepared as an injection.

The pharmaceutical composition of one embodiment of the present invention includes an exosome comprising the overexpressed Fc receptor or a portion thereof, and a pharmacologically acceptable carrier. For example, the pharmaceutical composition may be used for enhancing the anti-tumor effect or for enhancing the anti-cancer effect.

The pharmaceutical composition of one embodiment of the present invention may contain a cancer cell killing material. The cancer cell killing material may be located on the surface or inside the exosome, or may be located fused to the overexpressed Fc receptor or a portion thereof.

For example, the cancer cell killing material is IL-2, IL-7, IL-12, IL-15, IL-18, IL-21, such as cytokines, toxin proteins, gene damage inducing proteins, CRISPR-binding proteins, For immune checkpoint proteins such as apoptosis inducing protein, granzyme A, granzyme B, perforin, FAS protein, TRAIL (TNF-related apoptosis-inducing ligand) protein, PD-1, PD-L1, CTLA-4 Cancer-specific antibodies such as antibody, anti-CD47 antibody, anti-EGFR antibody, T cell receptor, immune cell surface protein such as NKG2D, Stimulator of Interferon Genes (STING), STING agonist ( STING agonist), albumin binding paclitaxel, actinomycin, alitretinoin, azacitidine, azathioprine, bevacizumab, bexatotene, bleomycin, botezomib, carboplatin, capecitabine, cetuximab, cisplatin , chlorambucil, cyclophosphamide, cytarabine, daunorubicin, docetaxel, doxyfluridine, doxorubicin, epirubicin, epothilone, erlotinib, etoposide, fluorouracil, gefitinib, gemsi Tabine, hydroxyurea, idarubicin, imatinib, ipilimumab, irinotecan, lapatinib, mechlorethamine, melphalan, mercaptopurine, methotrexate, mitoxantrone, oxrelizumab, ofatumumab, oxaliplatin, paclitaxel, Panitumumab, pemetrexed, rituximab, tafurposide, teniposide, thioguanine, topotecan, tretinoin, valrubicin, vemurafenib, vinblastine, vincristine, vindesine, vinorelbine, vorinostat , romidepsin, 5-fluorouracil (5-FU), 6-mercaptopurine (6-MP), cladribine, clofarabine, floxuridine, fludarabine, pentostatin, mitomycin, ixabe It may be at least one selected from the group consisting of phylon, estramustine, and the like. However, the present invention is not limited thereto, and of course, various anti-tumor or anti-cancer substances known in the art may be used.

Further, the present invention provides a drug delivery system comprising a pharmaceutical composition as described above.

The drug delivery system of one embodiment of the present invention may further include an antibody. The antibody may be administered in combination with the exosome.

In the pharmaceutical composition or drug delivery system of one embodiment of the present invention, the antibody is, for example, 3F8, 8H9, abagobumab, avelumab, abciximab, actozumab, adalimumab, adecatumumab, aducanu Mab, apelimomab, afutuzumab, alacizumab pegol, ALD518, alemtuzumab, alirocumab, altumomab pentetate, amatuximab, anatumomab mafenatox, aniprolumab, anlukinzumab, Apolizumab, arsitumomab, acelizumab, atinumab, atlizumab, atrolimumab, bafinuzumab, basiliximab, babituximab, bectumomab, belimumab, benralizumab, verr tilimumab, besilesumab, bevacizumab, bezlotoxumab, bicirumab, bimagrumab, bibatuzumab mertansine, blinatumomab, blosozumab, brentuximab vedotin, briakinumab , brodalumab, canakinumab, cantuzumab mertansine, cantuzumab rabtansine, caplacizumab, capromap pendetide, karlumab, catumaxomab, CC49, cBR96-doxorubicin immune complex, cedelizumab, sertoli Zumab pegol, cetuximab, Ch.14.18, situtuzumab bogatox, drinking watertumumab, clazakizumab, clenoliximab, civatuzumab tetraxetane, conatumumab, concizumab, crenezumab, CR6261 , dacetuzumab, daclizumab, dalotuzumab, daratumumab, dempsizumab, denosumab, detumomab, dolimomab aritox, drogitumab, duligotumab, dupilumab, dusigitumab, ecromeximab, eculizumab, edovacomab, edrecolomab, efalizumab, efungumab, eldelumab, elotuzumab, elcilimomab, enabatuzumab, enrimomab pegol, enokizumab, eno ticumab, encituximab, epitumomab cituxetan, epratuzumab, erlizumab, ertumaxomab, etaracizumab, etrolizumab, evolocumab, exbivirumab, panolesomab, paralimomab, Parletuzumab, Facinumab, FBTA05, Felbizumab, Pezakinumab, Piclatuzumab, Pigitumumab, Flanbotumab, Fontolizumab, Foralumab, Foravirumab, Presolimumab, Fulanumab, Putuximab, galiximab, ganitumab, gantenerumab, ganvirimomab, gemtuzumab ozogamicin, gevokizumab, girentuximab, gremba Tumumab Vedotin, Golimumab, Gomiliximab, Guselkumab, Ivalizumab, Ibritumomab Tucetan, Icrucumab, Igobumab, Ipilimumab, IMAB362, Imimumab, Imgatuzumab, In Clacumab, indatuximab rabtansine, infliximab, intetumumab, inolimomab, inotuzumab ozogamicin, ipilimumab, iratumumab, itolizumab, zekizumab, keliximab, Rabetuzumab, lambrolizumab, rampalizumab, lebrikizumab, remalesomab, lerdelimumab, lexatumumab, ribivirumab, rigelizumab, lintuzumab, lirilumab, rhodelcizumab, lorbotuzumab Mertansine, Lukatumumab, Lumiliximab, Mapatumumab, Margetuximab, Maslimomab, Mabrilimumab, Matuzumab, Mepolizumab, Metelimumab, Milatuzumab, Minretumomab, Mitumomab , mogamulizumab, morolimumab, motavizumab, moxetumomab fasodotox, muromonab-CD3, nacolomab tafenatox, namilumab, naftumomab estafenatox, narnatumab, natalizumab, Nebacumab, nesitumumab, nerelimomab, nesbacumab, nimotuzumab, nivolumab, nofetumomab merpentan, okaratuzumab, okrelizumab, odulimumab, ofatumumab, olalatumab, ol Lokizumab, omalizumab, onartuzumab, ontuxizumab, ofortuzumab monatox, oregobumab, orticumab, otelixizumab, otlertuzumab, oxelumab, ozanezumab, ozoralizumab , pagivacimab, palivizumab, panitumumab, pancomab, panovacumab, parsatuzumab, pascolizumab, pateclizumab, patritumab, femtumomab, perakizumab, pertuzumab, pexelli Zumab, pidilizumab, pinatuzumab vedotin, pintumomab, placulumab, folatuzumab vedotin, ponezumab, priliximab, pretoxaximab, pritumumab, PRO 140, quilizumab, Lacotumomab, radretumab, rafivirumab, ramucirumab, ranibizumab, laxibacumab, regavirumab, reslizumab, rilotumumab, rituximab, lovatumumab, loredumab, romosozumab, Rontalizumab, Lobelizumab, Luflizumab, Samalizumab, Sarilumab, Satumomab pendetide, Secukinumab, Cerivantumab, Setoxaximab, Savirumab, Cibrotuzumab, SGN-CD19A , SGN-CD33A, sifalimumab, siltuximab, simtuzumab, ciflizumab, sirucumab, solanezumab, solitomab, sonepsizumab, sontuzumab, stamulumab, sulesomab, suvizumab, tavalumab, tacatuzumab tet laxetan, tadozozumab, talizumab, tanezumab, taplitumomab paftox, tefivazumab, telimomab aritox, tenatumomab, teneliximab, teflizumab, teprotumumab, TGN1412, T Silimumab, tildrakizumab, tigatuzumab, TNX-650, tocilizumab, toralizumab, tositumomab, bexa, tovetumab, tralokinumab, trastuzumab, TRBS07, tregalizumab, tre Melimumab, tucotuzumab celmorleukin, tuvirumab, ublituximab, urerumab, urtoxazumab, ustekinumab, vantiktumab, bafaliximab, batelizumab, vedolizumab, veltu consisting of zumab, befalimomab, besenkumab, vicilizumab, voloxiximab, borcetuzumab mapodotin, botumumab, zalutumab, zanolimumab, zatuximab, jiralimumab and zolimomab aritox It may be at least one selected from the group. However, the present invention is not limited thereto, and of course, various antibodies known in the art may be used.

By way of example and not limitation of the present invention, the pharmaceutical composition or drug delivery system of one embodiment of the present invention may be in various oral or parenteral dosage forms.

The pharmaceutical composition or drug delivery system according to one embodiment of the present invention can be used to prevent, suppress, alleviate, ameliorate or treat immune-related diseases, cancer, inflammatory diseases, viral diseases, and/or various other diseases.

The pharmaceutical composition or drug delivery system of one embodiment of the present invention may include a pharmaceutically acceptable carrier, excipient or diluent. The carrier, excipient and diluent include lactose, dextrose, trehalose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium carbonate, calcium silicate, cellulose , methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil, but are not limited thereto. . The pharmaceutical composition or drug delivery system of one embodiment of the present invention is a formulation for oral administration such as powders, pills, tablets, capsules, suspensions, emulsions, syrups, granules, elixirs, aerosols, etc. according to a conventional method; It can be formulated and used in the form of external preparations, suppositories, or sterile injection solutions.

Administration of the pharmaceutical composition or drug delivery system of one embodiment of the present invention means introducing a predetermined substance to the patient by any suitable method, and the route of administration of the pharmaceutical composition or drug delivery system allows the drug to reach the target tissue. As long as there is, it can be administered through any general route. For example, the pharmaceutical composition or drug delivery system of one embodiment of the present invention may be administered orally or parenterally, and parenteral administration includes intratumoral administration, intra-articular administration, intra-articular administration, intrasynovial administration, intrasternal administration, intrathecal administration, intralesional and intracranial administration, transdermal administration, intraperitoneal administration, intravenous administration, intraarterial administration, intralymphatic administration, intramuscular administration, subcutaneous administration, intradermal administration, Topical administration, rectal administration, etc. may be mentioned. However, it is not limited thereto and does not exclude various administration methods known in the art. In addition, the pharmaceutical composition or drug delivery system of one embodiment of the present invention may be administered by any device capable of transporting an active substance to a target tissue or cell. In addition, the therapeutically effective amount of the pharmaceutical composition or drug delivery system of one embodiment of the present invention means an amount required for administration in order to expect a disease therapeutic effect.

The pharmaceutical composition or solid preparation for oral administration of the drug delivery system of one embodiment of the present invention may be prepared by mixing at least one excipient, for example, starch, calcium carbonate, sucrose, lactose, gelatin, and the like. In addition, the solid preparation for oral administration may include a lubricant (glidant) such as silica, stearic acid, magnesium stearate, calcium stearate, talc, and polyethylene glycol in addition to the excipients. The pharmaceutical composition or liquid formulation for oral administration of the drug delivery system of one embodiment of the present invention may contain various excipients, for example, a wetting agent, a sweetener, a fragrance, a preservative, etc. in addition to the simple diluent water and liquid paraffin.

The pharmaceutical composition or formulation for parenteral administration of the drug delivery system of one embodiment of the present invention may be a sterile aqueous solution, a non-aqueous solution, a suspension, an emulsion, a freeze-dried formulation, or a suppository. The pharmaceutical composition or formulation for parenteral administration of the drug delivery system of one embodiment of the present invention may also be prepared as an injection. The injection of one embodiment of the present invention may be an aqueous injection, a non-aqueous injection, an aqueous suspension injection, a non-aqueous suspension injection, or a solid injection used by dissolving or suspending, but is not limited thereto. The injection of one embodiment of the present invention is distilled water for injection, vegetable oil (for example, peanut oil, sesame oil, camellia oil, etc.), monoglyceride, diglyceride, propylene glycol, camphor, benzoate estradiol, bismuth salicylate, depending on the type , Arsenobenzol sodium, or at least one of streptomycin sulfate, and may optionally include a stabilizer or a preservative.

The compounding ratio of the pharmaceutical composition or drug delivery system of one embodiment of the present invention can be appropriately selected according to the type, amount, form, etc. of the additional components as described above. For example, based on the total amount of the injection, the pharmaceutical composition or drug delivery system for drug delivery of the present invention may be included in about 0.1 to 99% by weight, preferably about 10 to 90% by weight. In addition, a suitable dosage of the pharmaceutical composition or drug delivery system for drug delivery of one embodiment of the present invention is the patient's disease type, disease severity, formulation type, formulation method, patient's age, sex, weight, health condition, diet , excretion rate, administration time and administration method. For example, when administering the pharmaceutical composition or drug delivery system for drug delivery of one embodiment of the present invention to an adult, it may be administered at a dose of 0.001 mg/kg to 100 mg/kg in one to several divided doses per day.

On the other hand, when the composition of one embodiment of the present invention is prepared as a cosmetic composition, components commonly used in cosmetic compositions, for example, moisturizing agents, antioxidants, oily components, ultraviolet absorbers, Emulsifiers, surfactants, thickeners, alcohols, powder components, color materials, aqueous components, water, various skin nutrients, etc. can be appropriately blended as needed.

The cosmetic composition of one embodiment of the present invention is, for example, a patch, mask pack, mask sheet, cream, tonic, ointment, suspension, emulsion, paste, lotion, gel, oil, pack, spray, aerosol, mist, foundation, It can be applied to various forms such as powder and oil paper.

The cosmetic composition of one embodiment of the present invention may be prepared in any cosmetic formulation conventionally prepared in the art. For example, patch, mask pack, mask sheet, flexible lotion, nourishing lotion, astringent lotion, nourishing cream, massage cream, eye cream, cleansing cream, essence, eye essence, cleansing lotion, cleansing foam, cleansing water, sunscreen, lipstick , soap, shampoo, surfactant-containing cleansing, bathing agent, body lotion, body cream, body oil, body essence, body wash, hair dye, hair tonic, etc., but is not limited thereto.

The cosmetic composition of one embodiment of the present invention includes components commonly used in the cosmetic composition, for example, it may include conventional adjuvants and carriers such as antioxidants, stabilizers, solubilizers, vitamins, pigments and fragrances. In addition, in each formulation for the cosmetic composition, other components can be appropriately selected and formulated by those skilled in the art without difficulty depending on the type or purpose of use of the cosmetic composition.

According to the present invention, without fusion with a nucleic acid sequence encoding an exogenous transmembrane protein or a transmembrane domain thereof, exosomes are prepared using a gene construct encoding a full-length Fc receptor or a portion thereof. By fusion to an exogenous transmembrane protein or a transmembrane domain thereof, the Fc receptor or a portion thereof may be loaded into the exosome at a higher density than the conventional method of loading the Fc receptor or a portion thereof into the exosome. That is, the method for producing an exosome comprising an overexpressed Fc receptor or a portion thereof of the present invention is fused to an exogenous transmembrane protein or a transmembrane domain thereof to load the Fc receptor or a portion thereof into the exosome Fc receptor than the conventional method. Or a part thereof has the advantage of being able to load the exosomes with high density and high efficiency.

On the other hand, the scope of the present invention is not limited by the above-described effects.

1 shows a vector map (pEF6_fCD64_mGFP vector map) in which a gene encoding a fusion polypeptide of SEQ ID NO: 2 is fused to the C-terminus of full-length CD64 (SEQ ID NO: 1).
Figure 2 shows that the immunoglobulin kappa signal sequence is fused to the N-terminus of the extracellular domain of CD64 and the C-terminus of ITGB1 (exogenous transmembrane protein) fused to the C-terminus of the extracellular domain of CD64. A vector map (pEF6_IgK_ecto_TM_mGFP) into which the gene encoding the fusion polypeptide of SEQ ID NO: 3 is fused with mGFP is shown.
Figure 3 shows that the signal sequence of ITGB1 is fused to the N-terminus of the extracellular domain (ectodomain) of CD64, and mGFP is fused to the C-terminus of ITGB1 (exogenous transmembrane protein) fused to the C-terminus of the extracellular domain of CD64. The vector map (pEF6_IT_ecto_TM_mGFP) into which the gene encoding the fusion polypeptide of SEQ ID NO: 4 was fused is shown.
FIG. 4 is a fluorescence micrograph showing fluorescence observed in HEK293T cells transfected with pEF6_fCD64_mGFP vector, pEF6_IgK_ecto_TM_mGFP vector, and pEF6_IT_ecto_TM_mGFP vector, respectively.
Figure 5a is a flow cytometry result showing the CD64 content when CD64 is loaded into the exosome using the pEF6_fCD64_mGFP vector, and Figure 5b is a flow cytometry result showing the CD64 content when CD64 is loaded into the exosome using the pEF6_IgK_ecto_TM_mGFP vector Figure 5c is a flow cytometry result showing the CD64 content when CD64 is loaded into exosomes using the pEF6_IT_ecto_TM_mGFP vector.
FIG. 6 is a graph quantifying the flow cytometry results of FIGS. 5A to 5C as a relative value of the mean fluorescence intensity (MFI) of CD64 with respect to the mean fluorescence intensity (MFI) of the IgG isotype as a control. In FIG. 6 , a case in which CD64 was loaded into exosomes using a gene construct encoding full-length CD64 (pEF6_fCD64_mGFP vector) without fusion with a nucleic acid sequence encoding an exogenous transmembrane protein or a transmembrane domain thereof. It indicates that the content (or expression level) of CD64 per exosome is the highest.
Figure 7 shows that the exosome (fCD64 Exosome) containing overexpressed CD64 of the present invention is fluorescently stained with a fluorescently-labeled human-derived anti-EGFR antibody, that is, it binds to the Fc domain of a human-derived antibody.
8A is a standard curve used for quantification of doxorubicin loaded in exosomes, and is a standard curve showing the relationship between the concentration of doxorubicin and fluorescence intensity.
8B is a graph showing that when the concentration of doxorubicin is high and the concentration of exosomes is high, the absolute amount of doxorubicin loaded in the exosomes increases.
Figure 9a is a flow cytometry graph showing that the peak shifts to the right when the fCD64_mGFP exosome captured by Dynabead is reacted with an APC anti-human Fc fragment antibody, compared to the negative control (DPBS).
9B is a flow cytometry graph showing that the peak shifts to the right when the fCD64_mGFP exosome captured by Dynabead is reacted with an APC anti-human Fc fragment antibody, compared to the antibody untreated group.
Figure 10a shows the reaction of the APC anti-human Fc fragment antibody with the fCD64_mGFP exosome while fixing the concentration of the APC anti-human Fc fragment antibody and increasing the concentration (number of particles/mL) of the fCD64_mGFP exosome, the increase in the exosome concentration It is a graph showing that the mean fluorescence intensity (MFI) increases accordingly.
Figure 10b shows the reaction of APC anti-human Fc fragment antibody with fCD64_mGFP exosome while fixing the concentration of fCD64_mGFP exosome (number of particles/mL) and increasing the concentration of the APC anti-human Fc fragment antibody, according to the increase in antibody concentration It is a graph showing an increase in mean fluorescence intensity (MFI).
Figure 10c shows the reaction of APC anti-human EGFR antibody with fCD64_mGFP exosome while fixing the concentration of fCD64_mGFP exosome (number of particles/mL) and increasing the concentration of APC anti-human EGFR antibody, MFI ( It is a graph showing an increase in mean fluorescence intensity).
11 is a graph showing comparison of anti-tumor or anti-cancer effects (IC ₅₀ of cancer cells) when cancer cells are treated with doxorubicin alone and doxorubicin-loaded fCD64_mGFP exosomes loaded with the same amount.
Figure 12a is when MDA-MB-231, a cell expressing EGFR, and MCF-7, a cell not expressing EGFR, are co-cultured and treated with exosomes bound with a fluorescently labeled anti-EGFR antibody. , It is an optical micrograph and a fluorescence micrograph confirming that more exosomes were uptaken in MDA-MB-231.
Figure 12b is a graph showing the comparison of anti-tumor or anti-cancer effects (IC ₅₀ of cancer cells) when doxorubicin-fCD64_mGFP exosomes and doxorubicin-fCD64_mGFP exosomes-anti-EGFR are treated in cancer cells, respectively.

Hereinafter, the present invention will be described in more detail in the following examples. However, the following examples are merely illustrative of the content of the present invention and do not limit or limit the scope of the present invention. What can be easily inferred by those skilled in the art from the detailed description and examples of the present invention is construed as belonging to the scope of the present invention. The references cited herein are incorporated herein by reference.

Throughout the specification, when a part "includes" a certain component, it means that other components may be further included, rather than excluding other components, unless otherwise stated.

Example

Example 1: Preparation of a vector expressing the target fusion polypeptide

DNA was linearized by cutting the KpnI and XbaI portions of the multicloning site of the pEF6/V5-His A vector (#V96120, purchased from Invitrogen, USA) using KpnI and XbaI restriction enzymes, respectively. After amplifying the gene fragment encoding each of the fusion polypeptide having the amino acid sequence of SEQ ID NO: 2, the fusion polypeptide having the amino acid sequence of SEQ ID NO: 3, and the fusion polypeptide having the amino acid sequence of SEQ ID NO: 4 through PCR Each of the amplified gene fragments was subcloned into the pEF6/V5-His A vector (see FIGS. 1 to 3 ).

A linker (GGGGS) was inserted four times between the full-length CD64 (SEQ ID NO: 1) and mGFP constituting the fusion polypeptide having the amino acid sequence of SEQ ID NO: 2. In addition, a linker (GGGGS) was repeatedly inserted 4 times between the CD64 extracellular domain (ectodomain) of the fusion polypeptide having the amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 4 and the exogenous transmembrane protein (ITGB1), and the exogenous transmembrane A linker (GGGGS) was repeatedly inserted between the protein (ITGB1) and mGFP four times.

1 shows a vector (hereinafter referred to as "fCD64") into which a gene fragment encoding a fusion polypeptide (fusion polypeptide having the amino acid sequence of SEQ ID NO: 2) in which mGFP is fused to the C-terminus of full-length CD64 (hereinafter referred to as "fCD64") is inserted (hereinafter referred to as "fCD64"). , referred to as “pEF6_fCD64_mGFP”) is shown. In addition, in FIG. 2, an immunoglobulin kappa signal peptide (hereinafter, referred to as "IgK") is fused to the N-terminus of the extracellular domain of CD64 (hereinafter referred to as "ecto"). A fusion polypeptide (having the amino acid sequence of SEQ ID NO: 3) in which mGFP is fused to the C-terminus of ITGB1 (hereinafter referred to as "TM"), an exogenous transmembrane protein fused to the C-terminus of the extracellular domain of CD64 A vector (hereinafter, referred to as “pEF6_IgK_ecto_TM_mGFP”) into which a gene fragment encoding a fusion polypeptide) is inserted is shown. In addition, FIG. 3 shows an exogenous transmembrane protein fused to the N-terminus of the extracellular domain of CD64 with the signal sequence of ITGB1 (hereinafter referred to as “IT”) and fused to the C-terminus of the extracellular domain of CD64. A vector (hereinafter referred to as "pEF6_IT_ecto_TM_mGFP") into which a gene fragment encoding a fusion polypeptide (fusion polypeptide having the amino acid sequence of SEQ ID NO: 4) in which mGFP is fused to the C-terminus of ITGB1 is shown is shown.

Example 2: Cell culture and stabilization cell line preparation

HEK293T cells (purchased from Horizon Discovery) to be used for transduction were treated with 10% fetal bovine serum (FBS; purchased from ThermoFisher Scientific, USA) and 1% antibiotics-antimycotics; ThermoFisher Scientific as recommended by the supplier. ^. _{_}

In order to construct a cell line stably expressing each of the target fusion polypeptides of Example 1, each of the pEF6_fCD64_mGFP vector, pEF6_IgK_ecto_TM_mGFP vector and pEF6_IT_ecto_TM_mGFP vector constructed in Example 1 was treated with Effectene Transfection Reagent (Qiagen, Germany). purchased) was used to transduce HEK293T cells, and cultured for 48 hours after transduction. 10 μg/mL Blasticidin (purchased from Invivogen, USA) was added to the DMEM medium as described above and cultured for 3 weeks, and only cells stably expressing each target fusion polypeptide were selected.

Then, using Incucyte S3 (purchased from Sartorius, Germany), it was confirmed whether the target fusion polypeptide was expressed in the HEK293T cells selected as described above. Since all three vectors constructed in Example 1 contain the mGFP gene, fluorescence should be observed in HEK293T cells when each of these vectors is normally transduced into HEK293T cells and stably expressed in the cells. After culturing for 7 days, images were taken with a fluorescence microscope. As a result, fluorescence was observed in HEK293T cells transduced with pEF6_fCD64_mGFP vector, pEF6_IgK_ecto_TM_mGFP vector, and pEF6_IT_ecto_TM_mGFP vector (see FIG. 4). Therefore, it can be seen that each of the three vectors constructed in Example 1 was normally transduced into HEK293T cells, and each target fusion polypeptide encoded by each of these vectors was normally expressed in HEK293T cells.

Example 3: Exosome Isolation and CD64 Quantification

The HEK293T stabilized cell lines expressing the target fusion polypeptides of SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4 prepared in Example 2, respectively, were dispensed into 75T cell culture flasks, respectively, 3.75×10 ⁶ , and 5% CO ₂ , 37 ^o C was cultured under conditions. The next day, 1% exosome-depleted fetal bovine serum (exosome-depleted FBS; purchased from System Biosciences, USA), 1% antibiotic-antifungal, and 10 μg/mL Blasticidin (purchased from Invivogen, USA) were administered. The culture medium was replaced with the contained Opti-MEM (purchased from ThermoFisher Scientific, USA) and incubated at 5% CO ₂ , 37 ^° C for 48 hours.

Then, all of the cell culture supernatant was taken and filtered with a 0.22 μm syringe filter (product name: SLGVR33RB; purchased from Merck, Germany). Then, 5 mL of the supernatant was concentrated at 4° C. for 1 hour using an Amicon Ultra-15 Centrifugal 100 kDa Filter Unit (Amicon Ultra-15 Centrifugal 100 kDa Filter Unit; purchased from Merck, Germany). After the concentrate was suspended in DPBS (Dulbecco's Phosphate-Buffered Saline; purchased from Gibco, USA), 20% of the total volume of PEG8000 (500 mg/mL) solution was added, mixed, and stored at 4°C. After 24 hours, the concentrate mixed with PEG8000 was centrifuged at 10,000×g at 4°C, the supernatant was removed, and the exosome pellet was suspended in 200 μL DPBS for microBCA assay (ThermoFisher Scientific, USA). ) was used to quantify the protein concentration. Thereafter, DPBS was added to adjust the concentration of each exosome to 100 μg/mL and stored at -80°C until use.

In order to confirm the content (or expression level) of CD64 on the surface of each exosome, the exosomes were captured using CD81 Dynabead (purchased from ThermoFisher Scientific, USA). This is a method of capturing only the exosomes using 2.7 μm beads because the size of the exosomes is small and analysis with a general flow cytometer is impossible. "APC mouse IgG1, κ isotype control antibody" or "APC anti-human CD64 antibody" (purchased from BioLegend, USA) was used for staining at room temperature for 1 hour. Thereafter, flow cytometry was performed using a NovoCyte 2000R (Novocyte 2000R) flow cytometer (purchased from Agilent, USA) ( FIGS. 5A to 5C ). Figure 5a is a flow cytometry result showing the CD64 content when CD64 is loaded into exosomes using the pEF6_fCD64_mGFP vector, and Figure 5b is a flow cytometry result showing the CD64 content when CD64 is loaded into the exosomes using the pEF6_IgK_ecto_TM_mGFP vector. Figure 5c is a flow cytometry result showing the CD64 content when CD64 is loaded into the exosome using the pEF6_IT_ecto_TM_mGFP vector.

The flow cytometry results of FIGS. 5A to 5C were quantified as relative values of the mean fluorescence intensity (MFI) of CD64 with respect to the mean fluorescence intensity (MFI) of the IgG isotype as a control. As shown in FIG. 6 , exosomes isolated from a culture medium of a stabilized cell line expressing pEF6_fCD64_mGFP had higher CD64 content (or expression level) on the exosome surface compared to exosomes isolated from a culture medium of a stabilized cell line expressing pEF6_IgK_ecto_TM_mGFP or pEF6_IT_ecto_TM_mGFP. about twice as high. According to the present invention, CD64 was introduced into exosomes using a gene construct encoding full-length CD64 (pEF6_fCD64_mGFP vector) without fusion with a nucleic acid sequence encoding an exogenous transmembrane protein or a transmembrane domain thereof. Contrary to expectations, the loading case shows the highest CD64 content (or expression level) per exosome.

Example 4: Confirmation of Fc domain binding ability of exosomes

CD64, which is expressed on the surface of immune cells such as macrophages and dendritic cells, binds to the Fc domain consisting of an antibody constant region. This allows immune cells to recognize the antigen-bound antibody and trigger an antigen-specific immune response.

In addition to the exosome loading of CD64 confirmed in Example 3, it was confirmed whether CD64 loaded onto the exosome as described above exhibits an antibody-binding function. The exosomes isolated from the non-transformed HEK293T cell culture medium and the exosomes isolated from the stabilized cell line culture medium expressing pEF6_fCD64_mGFP (hereinafter referred to as “fCD64_mGFP exosomes”) were captured with CD81 Dynabead. Thereafter, human EGFR Alexa Fluor ^® 488-conjugated antibody (purchased from R&D Systems, USA) was used for staining at room temperature for 1 hour, followed by analysis with a NovoCyte 2000R (Novocyte 2000R) flow cytometer.

As shown in FIG. 7, exosomes isolated from untransformed HEK293T culture had no difference in FITC fluorescence intensity when stained with antibody and without staining (left graph in FIG. 7), but fCD64_mGFP exosomes were When stained with , about 48.9% of the exosomes were bound to the antibody, resulting in an increase in FITC fluorescence intensity (right graph of FIG. 7). This indicates that the fCD64_mGFP exosome can bind to the Fc domain of a human-derived antibody.

On the other hand, a target anticancer agent using an antibody binds to an antigen specific to cancer cells and exhibits an antitumor or anticancer effect. Exosomes comprising an overexpressed Fc receptor (eg, CD64, CD32, or CD16) or a portion thereof of the present invention are present in a higher density than conventional exosomes on the surface of the Fc receptor or a portion thereof. Accordingly, the exosome comprising the overexpressed Fc receptor or a portion thereof of the present invention on its surface has chemotaxis to the Fc domain of an antibody, and the Fc receptor or a portion thereof of the exosome binds specifically to the Fc domain of the antibody. will do Therefore, when an exosome containing an overexpressed Fc receptor or a part thereof and loaded with a cancer cell death substance is administered to a cancer patient, an antibody anticancer agent having an Fc domain acts because the Fc receptor or a part thereof is present at a high density on the surface of the exosome. It is possible to increase the anti-tumor or anti-cancer effect by enabling additional and target-specific attack on cancer cells.

Example 5: Preparation of doxorubicin-loaded exosomes

Anti-tumor efficacy can be enhanced by loading exosomes with chemotherapeutic drugs such as doxorubicin (DOX), a toxin commonly used for the treatment of solid tumors and various cancers, to target tumor cells. Doxorubicin can be loaded into the exosomes by incubating with the exosomes at room temperature. 20.4±0.5% by weight of doxorubicin out of 100% by weight of doxorubicin was finally loaded into the exosomes through the following process.

To load the exosomes with doxorubicin, 1,000 μg of fCD64_mGFP exosomes were mixed with doxorubicin hydrochloride at a concentration of 2,000 μg/mL overnight at room temperature. Since doxorubicin has a characteristic of forming a precipitate over time when mixed with various neutral buffers, in order to prevent this, a hulamixer sample mixer (purchased from Thermofisher, USA) was used and mixed overnight at various angles. . Thereafter, doxorubicin (unloaded doxorubicin) not loaded into the exosomes was removed using ultra-high-speed centrifugation, and the exosomes loaded with doxorubicin were filtered through a 0.1 μm syringe filter (product name: SLVVR33RS; purchased from Merck, Germany). It was used by mixing with one DPBS buffer.

The exosome loading of doxorubicin was measured by analyzing the fluorescence intensity (excitation wavelength of 480 nm and radiation wavelength of 590 nm) of doxorubicin. Unloaded doxorubicin was also analyzed and measured in the same way. A standard curve was created between the known concentration of doxorubicin and the measured fluorescence intensity (FIG. 8a), and the concentration of doxorubicin loaded in the exosomes was measured using this standard curve (FIG. 8b). As shown in FIG. 8B , it can be seen that when the concentration of doxorubicin is high and the concentration of exosomes is high, the absolute amount of doxorubicin loaded in the exosomes increases.

Example 6: Confirmation of antibody binding ability of fCD64_mGFP exosomes

In this example, after preparing an exosome containing overexpressed CD64, it was confirmed that CD64 was present on the surface of the prepared exosome, and it was confirmed that the antibody actually binds to the surface of the exosome. A large amount of CD64-loaded exosomes (fCD64_mGFP exosomes) prepared according to Examples 1 to 3 were collected and pooled to prepare a high concentration of exosomes, and then exosomes using CD81 dynabead (purchased from ThermoFisher Scientific, USA) The moth was captured, and the antibody binding ability of the captured exosome surface was confirmed.

"APC mouse IgG1, κ isotype control antibody", "APC anti-human EGFR antibody (purchased from R&D bioscience, USA)" and "APC anti-human Fc fragment antibody (purchased from Jackson immunoresearch, USA)" were each obtained from fCD64_mGFP exo It was reacted with moth and stained at room temperature for 1 hour. Thereafter, flow cytometry was performed using a NovoCyte 2000R (Novocyte 2000R) flow cytometer (purchased from Agilent, USA).

As shown in Figures 9a and 9b, when the fCD64_mGFP exosome captured by Dynabead was reacted with the APC anti-human Fc fragment antibody compared to the negative control (DPBS) and the antibody untreated group, it was confirmed that the peak shifted to the right. could These results indicate that CD64 artificially presented on the fCD64_mGFP exosome normally binds to the Fc domain of an APC-labeled antibody, and that the fCD64_mGFP exosome is labeled by APC through binding of CD64 and the Fc domain.

Meanwhile, after reacting the APC anti-human Fc fragment antibody with the fCD64_mGFP exosome while fixing the concentration of the APC anti-human Fc fragment antibody to 2 μg/mL and increasing the concentration (number of particles/mL) of the fCD64_mGFP exosome, NovoCyte 2000R (Novocyte 2000R) When flow cytometry was performed using a flow cytometer, the mean fluorescence intensity (MFI) from APC increased as the concentration of exosomes increased (see FIG. 10a ).

In addition, the concentration of fCD64_mGFP exosomes (number of particles/mL) was fixed at 1×10 ⁹ particles/mL and the concentration of APC anti-human Fc fragment antibody or APC anti-human EGFR antibody was increased while increasing the concentration of APC anti-human Fc fragment. After reacting the antibody or APC anti-human EGFR antibody with the fCD64_mGFP exosome, flow cytometry was performed using a NovoCyte 2000R (Novocyte 2000R) flow cytometer. As the concentration of the antibody increased, the MFI (mean fluorescence intensity) from APC was increased (see FIGS. 10b and 10c).

The above result means that the antibody binding capacity to the fCD64_mGFP exosome increases in a concentration-dependent manner of the antibody. In addition, the above result means that the binding capacity of an antibody having an Fc domain increases in a concentration-dependent manner because CD64 is present at a high density on the surface of the fCD64_mGFP exosome.

Example 7: Anti-tumor activity of doxorubicin-loaded exosomes

Example 7-1: Comparison of antitumor or anticancer efficacy of doxorubicin alone and doxorubicin-fCD64_mGFP exosomes

In this example, it was confirmed whether the fCD64_mGFP exosome loaded with doxorubicin had anti-tumor or anticancer efficacy as follows.

In vitro, MDA-MB-231 human breast cancer cell line (10,000 cells/well) was treated with doxorubicin alone and fCD64_mGFP exosomes loaded with doxorubicin in the same amount (hereinafter referred to as “doxorubicin-fCD64_mGFP exosomes”). Doxorubicin-fCD64_mGFP exosomes were treated in breast cancer cells at a concentration of 2.56×10 ¹¹ particles/mL.

As a result, both doxorubicin treatment alone and doxorubicin-fCD64_mGFP exosome treatment inhibited breast cancer cell growth. However, the group treated with the same amount of doxorubicin-loaded fCD64_mGFP exosomes (indicated as "Dox-exo" in FIG. 11) showed anti-tumor or anti-cancer effects even with a smaller amount compared to doxorubicin alone treatment (FIG. 11) . These results indicate that doxorubicin-fCD64_mGFP exosomes more effectively delivered doxorubicin to cancer cells than doxorubicin alone.

Example 7-2: doxorubicin-fCD64_mGFP exosome and anti-EGFR antibody co-administration

In this example, anti-tumor or anti-cancer effects were confirmed by co-administration of doxorubicin-fCD64_mGFP exosomes and an anti-EGFR antibody capable of targeting EGFR, a cancer cell specific target.

MDA-MB-231 human breast cancer cells strongly express EGFR on the surface. After culturing together MDA-MB-231, a cell expressing EGFR, and MCF-7, a cell not expressing EGFR, the cells were treated with fCD64_mGFP exosomes bound with a fluorescently-labeled anti-EGFR antibody, It was confirmed that more exosomes were uptaken in MDA-MB-231 (FIG. 12a). These results indicate that CD64 artificially presented on the fCD64_mGFP exosome normally binds to the Fc domain of an anti-EGFR antibody.

In addition, if anti-EGFR and doxorubicin-fCD64_mGFP exosomes are co-administered to exhibit more effective anti-tumor or anti-cancer effects, CD64 presented on the doxorubicin-fCD64_mGFP exosomes of the present invention effectively binds to the Fc domain of anti-EGFR, The complex of "doxorubicin-fCD64_mGFP exosome-anti-EGFR" means that it has excellent targeting efficiency against cancer cells. To confirm this, an experiment was performed as follows.

10 μg of anti-EGFR was mixed with doxorubicin-fCD64_mGFP exosomes and then reacted at 4° C. for 2 hours using a hulamixer sample mixer (purchased from Thermofisher, USA). Thereafter, the non-specific binding antibody was removed using ultra-high speed centrifugation, and the anti-EGFR antibody-bound exosome (hereinafter referred to as "doxorubicin-fCD64_mGFP exosome-anti-EGFR") was filtered with a 0.1 μm syringe filter (product name: SLVVR33RS (purchased from Merck, Germany) was used by mixing with DPBS buffer filtered.

MDA-MB-231 human breast cancer cell line (10,000 cells/well) was treated with doxorubicin-fCD64_mGFP exosomes and doxorubicin-fCD64_mGFP exosome-anti-EGFR in vitro. Doxorubicin-fCD64_mGFP exosome (indicated as “Dox-exo” in FIG. 12B) and doxorubicin-fCD64_mGFP exosome-anti-EGFR antibody (indicated as “Dox-exo+Ab” in FIG. 12) both 2.56×10 ¹¹ Particle count Breast cancer cells were treated at a concentration of /mL, and the amount of doxorubicin loaded on the doxorubicin-fCD64_mGFP exosome and the amount of doxorubicin loaded on the doxorubicin-fCD64_mGFP exosome-anti-EGFR were the same.

As a result of the test, both groups synergistically inhibited cancer cell growth, but it was confirmed that the group treated with doxorubicin-fCD64_mGFP exosome-anti-EGFR had better anti-tumor or anti-cancer effect ( FIG. 12b ). From these results, when mixed treatment with doxorubicin-fCD64_mGFP exosome and anti-EGFR antibody, CD64 presented on doxorubicin-fCD64_mGFP exosome effectively binds to the Fc domain of anti-EGFR, and "doxorubicin-fCD64_mGFP exosome-anti- It was confirmed that the complex of "EGFR" has excellent targeting efficiency against cancer cells.

In the above, the present invention has been described with reference to the above examples, but the present invention is not limited thereto. Those skilled in the art can make modifications and changes without departing from the spirit and scope of the present invention, and it will be appreciated that such modifications and changes also belong to the present invention.

<110> ExoCoBio Inc. <120> Exosome comprising overexpressed Fc receptor or its part and manufacturing method thereof <130> P21-0119KR <160> 4 <170> KoPatentIn 3.0 <210> 1 <211> 374 <212> PRT <213> Artificial Sequence <220> <223> full-length CD64 <400> 1 Met Trp Phe Leu Thr Thr Leu Leu Leu Trp Val Pro Val Asp Gly Gln 1 5 10 15 Val Asp Thr Thr Lys Ala Val Ile Thr Leu Gln Pro Pro Trp Val Ser 20 25 30 Val Phe Gln Glu Glu Thr Val Thr Leu His Cys Glu Val Leu His Leu 35 40 45 Pro Gly Ser Ser Ser Thr Gln Trp Phe Leu Asn Gly Thr Ala Thr Gln 50 55 60 Thr Ser Thr Pro Ser Tyr Arg Ile Thr Ser Ala Ser Val Asn Asp Ser 65 70 75 80 Gly Glu Tyr Arg Cys Gln Arg Gly Leu Ser Gly Arg Ser Asp Pro Ile 85 90 95 Gln Leu Glu Ile His Arg Gly Trp Leu Leu Leu Leu Gln Val Ser Ser Arg 100 105 110 Val Phe Thr Glu Gly Glu Pro Leu Ala Leu Arg Cys His Ala Trp Lys 115 120 125 Asp Lys Leu Val Tyr Asn Val Leu Tyr Tyr Arg Asn Gly Lys Ala Phe 130 135 140 Lys Phe Phe His Trp Asn Ser Asn Leu Thr Ile Leu Lys Thr Asn Ile 145 150 155 160 Ser His Asn Gly Thr Tyr His Cys Ser Gly Met Gly Lys His Arg Tyr 165 170 175 Thr Ser Ala Gly Ile Ser Val Thr Val Lys Glu Leu Phe Pro Ala Pro 180 185 190 Val Leu Asn Ala Ser Val Thr Ser Pro Leu Leu Glu Gly Asn Leu Val 195 200 205 Thr Leu Ser Cys Glu Thr Lys Leu Leu Leu Gln Arg Pro Gly Leu Gln 210 215 220 Leu Tyr Phe Ser Phe Tyr Met Gly Ser Lys Thr Leu Arg Gly Arg Asn 225 230 235 240 Thr Ser Ser Glu Tyr Gln Ile Leu Thr Ala Arg Arg Glu Asp Ser Gly 245 250 255 Leu Tyr Trp Cys Glu Ala Ala Thr Glu Asp Gly Asn Val Leu Lys Arg 260 265 270 Ser Pro Glu Leu Glu Leu Gln Val Leu Gly Leu Gln Leu Pro Thr Pro 275 280 285 Val Trp Phe His Val Leu Phe Tyr Leu Ala Val Gly Ile Met Phe Leu 290 295 300 Val Asn Thr Val Leu Trp Val Thr Ile Arg Lys Glu Leu Lys Arg Lys 305 310 315 320 Lys Lys Trp Asp Leu Glu Ile Ser Leu Asp Ser Gly His Glu Lys Lys 325 330 335 Val Ile Ser Ser Leu Gln Glu Asp Arg His Leu Glu Glu Glu Leu Lys 340 345 350 Cys Gln Glu Gln Lys Glu Glu Gln Leu Gln Glu Gly Val His Arg Lys 355 360 365 Glu Pro Gln Gly Ala Thr 370 <210> 2 <211> 632 <212> PRT <213> Artificial Sequence <220> <223> fCD64_mGFP vector <400> 2 Met Trp Phe Leu Thr Thr Leu Leu Leu Trp Val Pro Val Asp Gly Gln 1 5 10 15 Val Asp Thr Thr Lys Ala Val Ile Thr Leu Gln Pro Pro Trp Val Ser 20 25 30 Val Phe Gln Glu Glu Thr Val Thr Leu His Cys Glu Val Leu His Leu 35 40 45 Pro Gly Ser Ser Ser Ser Thr Gln Trp Phe Leu Asn Gly Thr Ala Thr G ln 50 55 60 Thr Ser Thr Pro Ser Tyr Arg Ile Thr Ser Ala Ser Val Asn Asp Ser 65 70 75 80 Gly Glu Tyr Arg Cys Gln Arg Gly Leu Ser Gly Arg Ser Asp Pro Ile 85 90 95 Gln Leu Glu Ile His Arg Gly Trp Leu Leu Leu Gln Val Ser Ser Arg 100 105 110 Val Phe Thr Glu Gly Glu Pro Leu Ala Leu Arg Cys His Ala Trp Lys 115 120 125 Asp Lys Leu Val Tyr Asn Val Leu Tyr Tyr Arg Asn Gly Lys Ala Phe 130 135 140 Lys Phe Phe His Trp Asn Ser Asn Leu Thr Ile Leu Lys Thr Asn Ile 145 150 155 160 Ser His Asn Gly Thr Tyr His Cys Ser Gly Met Gly Lys His Arg Tyr 165 170 175 Thr Ser Ala Gly Ile Ser Val Thr Val Lys Glu Leu Phe Pro Ala Pro 180 185 190 Val Leu Asn Ala Ser Val Thr Ser Pro Leu Leu Glu Gly Asn Leu Val 195 200 205 Thr Leu Ser Cys Glu Thr Lys Leu Leu Leu Leu Gln Arg Pro Gly Leu Gln 210 215 220 Leu Tyr Phe Ser Phe Tyr Met Gly Ser Lys Thr Leu Arg Gly Arg Asn 225 230 235 240 Thr Ser Ser Glu Tyr Gln Ile Leu Thr Ala Arg Arg Glu Asp Ser Gly 245 250 255 Leu Tyr Trp Cys Glu Ala Ala Thr Glu Asp Gly Asn Val Leu Lys Arg 260 265 270 Ser Pro Glu Leu Glu Leu Gln Val Leu Gly Leu Gln Leu Pro Thr Pro 275 280 285 Val Trp Phe His Val Leu Phe Tyr Leu Ala Val Gly Ile Met Phe Leu 290 295 300 Val Asn Thr Val Leu Trp Val Thr Ile Arg Lys Glu Leu Lys Arg Lys 305 310 315 320 Lys Lys Trp Asp Leu Glu Ile Ser Leu Asp Ser Gly His Glu Lys Lys 325 330 335 Val Ile Ser Ser Leu Gln Glu Asp Arg His Leu Glu Glu Glu Leu Lys 340 345 350 Cys Gln Glu Gln Lys Glu Glu Gln Leu Gln Glu Gly Val His Arg Lys 355 360 365 Glu Pro Gln Gly Ala Thr Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 370 375 380 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Met Ser Lys Gly Glu Glu 385 390 395 400 Leu Phe Thr Gly Val Val Pro Ile Leu Val Glu Leu Asp Gly Asp Val 405 410 415 Asn Gly His Lys Phe Ser Val Ser Gly Glu Gly Glu Gly Asp Ala Thr 420 425 430 Tyr Gly Lys Leu Thr Leu Lys Phe Ile Cys Thr Thr Gly Lys Leu Pro 435 440 445 Val Pro Trp Pro Thr Leu Val Thr Thr Leu Thr Tyr Gly Val Gln Cys 450 455 460 Phe Ser Arg Tyr Pro Asp His Met Lys Gln His Asp Phe Phe Lys Ser 465 470 475 480 Ala Met Pro Glu Gly Tyr Val Gln Glu Arg Thr Ile Phe Phe Lys Asp 485 490 495 Asp Gly Asn Tyr Lys Thr Arg Ala Glu Val Lys Phe Glu Gly Asp Thr 500 505 510 Leu Val Asn Arg Ile Glu Leu Lys Gly Ile Asp Phe Lys Glu Asp Gly 515 520 525 Asn Ile Leu Gly His Lys Leu Glu Tyr Asn Tyr Asn Ser His Asn Val 530 535 540 Tyr Ile Met Ala Asp Lys Gln Lys Asn Gly Ile Lys Val Asn Phe Lys 545 550 555 560 Ile Arg His Asn Ile Glu Asp Gly Ser Val Gln Leu Ala Asp His Tyr 565 570 575 Gln Gln Asn Thr Pro Ile Gly Asp Gly Pro Val Leu Leu Pro Asp Asn 580 585 590 His Tyr Leu Ser Thr Gln Ser Lys Leu Ser Lys Asp Pro Asn Glu Lys 595 600 605 Arg Asp His Met Val Leu Leu Glu Phe Val Thr Ala Ala Gly Ile Thr 610 615 620 His Gly Met Asp Glu Leu Tyr Lys 625 630 <210> 3 <211> 607 <212> PRT <213> Artificial Sequence <220> <223> IgK_ecto_TM_mGFP vector <400> 3 M et Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro 1 5 10 15 Gly Ser Thr Gly Gln Val Asp Thr Thr Lys Ala Val Ile Thr Leu Gln 20 25 30 Pro Pro Trp Val Ser Val Phe Gln Glu Glu Thr Val Thr Leu His Cys 35 40 45 Glu Val Leu His Leu Pro Gly Ser Ser Ser Thr Gln Trp Phe Leu Asn 50 55 60 Gly Thr Ala Thr Gln Thr Ser Thr Pro Ser Tyr Arg Ile Thr Ser Ala 65 70 75 80 Ser Val Asn Asp Ser Gly Glu Tyr Arg Cys Gln Arg Gly Leu Ser Gly 85 90 95 Arg Ser Asp Pro Ile Gln Leu Glu Ile His Arg Gly Trp Leu Leu Leu 100 105 110 Gln Val Ser Ser Arg Val Phe Thr Glu Gly Glu Pro Leu Ala Leu Arg 115 120 125 Cys His Ala Trp Lys Asp Lys Leu Val Tyr Asn Val Leu Tyr Tyr Arg 130 135 140 Asn Gly Lys Ala Phe Lys Phe Phe His Trp Asn Ser Asn Leu Thr Ile 145 150 155 160 Leu Lys Thr Asn Ile Ser His Asn Gly Thr Tyr His Cys Ser Gly Met 165 170 175 Gly Lys His Arg Tyr Thr Ser Ala Gly Ile Ser Val Thr Val Lys Glu 180 185 190 Leu Phe Pro Ala Pro Val Leu Asn Ala Ser Val Thr Ser Pro Leu Leu 195 200 205 Glu Gly Asn Leu Val Thr Leu Ser Cys Glu Thr Lys Leu Leu Leu Gln 210 215 220 Arg Pro Gly Leu Gln Leu Tyr Phe Ser Phe Tyr Met Gly Ser Lys Thr 225 230 235 240 Leu Arg Gly Arg Asn Thr Ser Ser Glu Tyr Gln Ile Leu Thr Ala Arg 245 250 255 Arg Glu Asp Ser Gly Leu Tyr Trp Cys Glu Ala Ala Thr Glu Asp Gly 260 265 270 Asn Val Leu Lys Arg Ser Pro Glu Leu Glu Leu Gln Val Leu Gly Leu 275 280 285 Gln Leu Pro Thr Pro Val Trp Phe His Gly Gly Gly Gly Ser Gly Gly 290 295 300 Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ile Ile Pro 305 310 315 320 Ile Val Ala Gly Val Val Ala Gly Ile Val Leu Ile Gly Leu Ala Leu 325 330 335 Leu Leu Ile Trp Lys Leu Leu Met Ile Ile His Asp Arg Gly Gly Gly 340 345 350 Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 355 360 365 Ser Met Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu 370 375 380 Val Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Val Ser Gly 385 390 395 400 Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile 405 410 415 Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr 420 425 430 Leu Thr Tyr Gly Val Gln Cys Phe Ser Arg Tyr Pro Asp His Met Lys 435 440 445 Gln His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu 450 455 460 Arg Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu 465 470 475 480 Val Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly 485 490 495 Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr 500 505 510 Asn Tyr Asn Ser His Asn Val Tyr Ile Met Ala Asp Lys Gln Lys Asn 515 520 525 Gly Ile Lys Val Asn Phe Lys Ile Arg His Asn Ile Glu Asp Gly Ser 530 535 540 Val Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly 545 550 555 560 Pro Val Leu Leu Pro Asp Asn His Tyr Leu Ser Thr Gln Ser Lys Leu 565 570 575 Ser Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu Leu Glu Phe 580 585 590 Val Thr Ala Ala Gly Ile Thr His Gly Met Asp Glu Leu Tyr Lys 595 600 605 <210> 4 <211> 607 <212> PRT <213> Artificial Sequence <220> <223> IT_ecto_TM_mGFP vector <400> 4 Met Asn Leu Gln Pro Ile Phe Trp Ile Gly Leu Ile Ser Ser Val Cys 1 5 10 15 Cys Val Phe Ala Gln Val Asp Thr Thr Lys Ala Val Ile Thr Leu Gln 20 25 30 Pro Pro Trp Val Ser Val Phe Gln Glu Glu Thr Val Thr Leu His Cys 35 40 45 Glu Val Leu His Leu Pro Gly Ser Ser Ser Thr Gln Trp Phe Leu Asn 50 55 60 Gly Thr Ala Thr Gln Thr Ser Thr Pro Ser Tyr Arg Ile Thr Ser Ala 65 70 75 80 Ser Val Asn Asp Ser Gly Glu Tyr Arg Cys Gln Arg Gly Leu Ser Gly 85 90 95 Arg Ser Asp Pro Ile Gln Leu Glu Ile His Arg Gly Trp Leu Leu Leu 100 105 110 Gln Val Ser Ser Arg Val Phe Thr Glu Gly Glu Pro Leu Ala Leu Arg 115 120 125 Cys His Ala Trp Lys Asp Lys Leu Val Tyr Asn Val Leu Tyr Tyr Arg 130 135 140 Asn Gly Lys Ala Phe Lys Phe Phe His Trp Asn Ser Asn Leu Thr Ile 145 150 155 160 Leu Lys Thr Asn Ile Ser His Asn Gly Thr Tyr His Cys Ser Gly Met 165 170 175 Gly Lys His Arg Tyr Thr Ser Ala Gly Ile Ser Val Thr Val Lys Glu 180 185 190 Leu Phe Pro Ala Pro Val Leu Asn Ala Ser Val Thr Ser Pro Leu Leu 195 200 205 Glu Gly Asn Leu Val Thr Leu Ser Cys Glu Thr Lys Leu Leu Leu Gln 210 215 220 Arg Pro Gly Leu Gln Leu Tyr Phe Ser Phe Tyr Met Gly Ser Lys Thr 225 230 235 240 Leu Arg Gly Arg Asn Thr Ser Ser Glu Tyr Gln Ile Leu Thr Ala Arg 245 250 255 Arg Glu Asp Ser Gly Leu Tyr Trp Cys Glu Ala Ala Thr Glu Asp Gly 260 265 270 Asn Val Leu Lys Arg Ser Pro Glu Leu Glu Leu Gln Val Leu Gly Leu 275 280 285 Gln Leu Pro Thr Pro Val Trp Phe His Gly Gly Gly Gly Ser Gly Gly 290 295 300 Gly Gly Ser Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ile Ile Pro 305 310 315 320 Ile Val Ala Gly Val Val Ala Gly Ile Val Leu Ile Gly Leu Ala Leu 325 330 335 Leu Leu Ile Trp Lys Leu Leu Met Ile Ile His Asp Arg Gly Gly Gly 340 345 350 Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 355 360 365 Ser Met Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu 370 375 380 Val Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly 385 390 395 400 Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile 405 410 415 Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr 420 425 430 Leu Thr Tyr Gly Val Gln Cys Phe Ser Arg Tyr Pro Asp His Met Lys 435 440 445 Gln His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu 450 455 460 Arg Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu 465 470 475 480 Val Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly 485 490 495 Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr 500 505 510 Asn Tyr Asn Ser His Asn Val Tyr Ile Met Ala Asp Lys Gln Lys Asn 515 520 525 Gly Ile Lys Val Asn Phe Lys Ile Arg His Asn Ile Glu Asp Gly Ser 530 535 540 Val Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly 545 550 555 560 Pro Val Leu Leu Pro Asp Asn His Tyr Leu Ser Thr Gln Ser Lys Leu 565 570 575 Ser Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu Leu Glu Phe 580 585 590Val Thr Ala Ala Gly Ile Thr His Gly Met Asp Glu Leu Tyr Lys 595 600 605

Claims (17)

encoding a full-length Fc receptor or a portion thereof (provided that it has a transmembrane protein or transmembrane domain of an Fc receptor) without fusion with a nucleic acid sequence encoding an exogenous transmembrane protein or transmembrane domain thereof preparing the genetic construct;
transducing the genetic construct into a cell that does not naturally express an Fc receptor or a portion thereof;
expressing the full-length Fc receptor or a portion thereof in the cell;
A method for producing an exosome comprising an overexpressed Fc receptor or a portion thereof, comprising the step of isolating the exosome containing the full-length Fc receptor or a portion thereof. According to claim 1,
The exosome comprising the full-length Fc receptor or a part thereof is secreted or released from the cell and is contained in the culture medium of the cell. 3. The method of claim 2,
A method for producing an exosome comprising an overexpressed Fc receptor or a portion thereof, characterized in that the exosome containing the full-length Fc receptor or a portion thereof is separated from the culture medium. 4. The method according to any one of claims 1 to 3,
The full-length Fc receptor or a portion thereof is characterized in that located on the surface of the exosome, the overexpressed Fc receptor or a method for producing an exosome comprising a portion thereof. 4. The method according to any one of claims 1 to 3,
The Fc receptor is CD64, CD32 or CD16, characterized in that the overexpressed Fc receptor or a method for producing an exosome comprising a portion thereof. 4. The method according to any one of claims 1 to 3,
Cells that do not naturally express the Fc receptor or a part thereof are HEK293 cells, HEK293T cells or Expi293F cells. A pharmaceutical composition comprising, as an active ingredient, an exosome comprising an overexpressed Fc receptor or a portion thereof prepared according to the method according to claim 1 . 8. The method of claim 7,
A pharmaceutical composition comprising an exosome comprising the overexpressed overexpressed Fc receptor or a portion thereof, and a pharmaceutically acceptable carrier. 9. The method of claim 8,
For enhancing anti-tumor effect or for enhancing anti-cancer effect, a pharmaceutical composition. 10. The method of claim 9,
A pharmaceutical composition comprising a cancer cell killing material. 11. The method of claim 10,
The cancer cell killing material is characterized in that located on the surface or inside the exosome, a pharmaceutical composition. 12. The method of claim 11,
The cancer cell killing material is characterized in that it is fused to the overexpressed Fc receptor or a portion thereof, a pharmaceutical composition. A drug delivery system comprising the pharmaceutical composition according to any one of claims 7 to 12. 14. The method of claim 13,
A drug delivery system, further comprising an antibody. 15. The method of claim 14,
The antibody is characterized in that co-administered with the exosome, drug delivery system. A cosmetic composition comprising, as an active ingredient, an exosome comprising an overexpressed Fc receptor or a portion thereof prepared according to the manufacturing method according to claim 1 . 17. The method of claim 16,
Shampoo, soap, rinse, surfactant-containing cleansing, cream, lotion, ointment, tonic, treatment, conditioner, suspension, emulsion, paste, gel, oil, wax, spray, aerosol, mist, or powder cosmetic composition .

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