WO2004014954A1 - Exosome containing exogenous antigen through gene transfection and method for utilizing the same - Google Patents
Exosome containing exogenous antigen through gene transfection and method for utilizing the same Download PDFInfo
- Publication number
- WO2004014954A1 WO2004014954A1 PCT/KR2003/001575 KR0301575W WO2004014954A1 WO 2004014954 A1 WO2004014954 A1 WO 2004014954A1 KR 0301575 W KR0301575 W KR 0301575W WO 2004014954 A1 WO2004014954 A1 WO 2004014954A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- antigen
- exosomes
- mucl
- exosome
- present
- Prior art date
Links
- 210000001808 exosome Anatomy 0.000 title claims abstract description 65
- 239000000427 antigen Substances 0.000 title claims abstract description 54
- 108091007433 antigens Proteins 0.000 title claims abstract description 54
- 102000036639 antigens Human genes 0.000 title claims abstract description 54
- 238000000034 method Methods 0.000 title description 7
- 238000012637 gene transfection Methods 0.000 title description 2
- 210000004027 cell Anatomy 0.000 claims abstract description 46
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 42
- 210000001723 extracellular space Anatomy 0.000 claims abstract description 12
- 102000004169 proteins and genes Human genes 0.000 claims description 26
- 230000002163 immunogen Effects 0.000 claims description 4
- 238000002955 isolation Methods 0.000 claims description 2
- 230000008859 change Effects 0.000 abstract description 4
- 210000000987 immune system Anatomy 0.000 abstract description 4
- 238000004519 manufacturing process Methods 0.000 abstract description 3
- 206010028980 Neoplasm Diseases 0.000 description 17
- 238000001262 western blot Methods 0.000 description 8
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 7
- 230000000890 antigenic effect Effects 0.000 description 6
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 description 5
- 230000028993 immune response Effects 0.000 description 5
- 239000002953 phosphate buffered saline Substances 0.000 description 5
- 238000012413 Fluorescence activated cell sorting analysis Methods 0.000 description 4
- 210000002487 multivesicular body Anatomy 0.000 description 4
- 239000002245 particle Substances 0.000 description 4
- 230000008569 process Effects 0.000 description 4
- 239000003981 vehicle Substances 0.000 description 4
- 230000003612 virological effect Effects 0.000 description 4
- 201000009030 Carcinoma Diseases 0.000 description 3
- 108010001336 Horseradish Peroxidase Proteins 0.000 description 3
- 108700018351 Major Histocompatibility Complex Proteins 0.000 description 3
- 210000001744 T-lymphocyte Anatomy 0.000 description 3
- 238000003556 assay Methods 0.000 description 3
- 239000013592 cell lysate Substances 0.000 description 3
- 210000000170 cell membrane Anatomy 0.000 description 3
- 210000002472 endoplasmic reticulum Anatomy 0.000 description 3
- 239000012528 membrane Substances 0.000 description 3
- 230000020382 suppression by virus of host antigen processing and presentation of peptide antigen via MHC class I Effects 0.000 description 3
- 238000005199 ultracentrifugation Methods 0.000 description 3
- 229960005486 vaccine Drugs 0.000 description 3
- 230000028973 vesicle-mediated transport Effects 0.000 description 3
- 102000002812 Heat-Shock Proteins Human genes 0.000 description 2
- 108010004889 Heat-Shock Proteins Proteins 0.000 description 2
- 102000018697 Membrane Proteins Human genes 0.000 description 2
- 108010052285 Membrane Proteins Proteins 0.000 description 2
- 101100346932 Mus musculus Muc1 gene Proteins 0.000 description 2
- 210000000612 antigen-presenting cell Anatomy 0.000 description 2
- 239000012228 culture supernatant Substances 0.000 description 2
- 238000012258 culturing Methods 0.000 description 2
- 238000001962 electrophoresis Methods 0.000 description 2
- 230000009454 functional inhibition Effects 0.000 description 2
- BRZYSWJRSDMWLG-CAXSIQPQSA-N geneticin Chemical compound O1C[C@@](O)(C)[C@H](NC)[C@@H](O)[C@H]1O[C@@H]1[C@@H](O)[C@H](O[C@@H]2[C@@H]([C@@H](O)[C@H](O)[C@@H](C(C)O)O2)N)[C@@H](N)C[C@H]1N BRZYSWJRSDMWLG-CAXSIQPQSA-N 0.000 description 2
- 230000006698 induction Effects 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 238000004806 packaging method and process Methods 0.000 description 2
- 230000037361 pathway Effects 0.000 description 2
- 102000004196 processed proteins & peptides Human genes 0.000 description 2
- 108090000765 processed proteins & peptides Proteins 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- 239000000243 solution Substances 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 210000004881 tumor cell Anatomy 0.000 description 2
- 208000023275 Autoimmune disease Diseases 0.000 description 1
- 238000009020 BCA Protein Assay Kit Methods 0.000 description 1
- 206010006187 Breast cancer Diseases 0.000 description 1
- 208000026310 Breast neoplasm Diseases 0.000 description 1
- 206010057248 Cell death Diseases 0.000 description 1
- 101710163595 Chaperone protein DnaK Proteins 0.000 description 1
- 206010009944 Colon cancer Diseases 0.000 description 1
- 101710178376 Heat shock 70 kDa protein Proteins 0.000 description 1
- 101710152018 Heat shock cognate 70 kDa protein Proteins 0.000 description 1
- 102000008949 Histocompatibility Antigens Class I Human genes 0.000 description 1
- 108091054437 MHC class I family Proteins 0.000 description 1
- 241001529936 Murinae Species 0.000 description 1
- 239000004677 Nylon Substances 0.000 description 1
- 210000003719 b-lymphocyte Anatomy 0.000 description 1
- 210000001772 blood platelet Anatomy 0.000 description 1
- 201000008275 breast carcinoma Diseases 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 238000002619 cancer immunotherapy Methods 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 239000006285 cell suspension Substances 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000010367 cloning Methods 0.000 description 1
- 208000029742 colonic neoplasm Diseases 0.000 description 1
- 230000009089 cytolysis Effects 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 210000004443 dendritic cell Anatomy 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 238000006911 enzymatic reaction Methods 0.000 description 1
- 210000003743 erythrocyte Anatomy 0.000 description 1
- 230000001747 exhibiting effect Effects 0.000 description 1
- 230000004927 fusion Effects 0.000 description 1
- 206010017758 gastric cancer Diseases 0.000 description 1
- 208000010749 gastric carcinoma Diseases 0.000 description 1
- 102000035122 glycosylated proteins Human genes 0.000 description 1
- 108091005608 glycosylated proteins Proteins 0.000 description 1
- 210000002288 golgi apparatus Anatomy 0.000 description 1
- 210000002865 immune cell Anatomy 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- 239000002502 liposome Substances 0.000 description 1
- 210000002540 macrophage Anatomy 0.000 description 1
- 230000035800 maturation Effects 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 229920001778 nylon Polymers 0.000 description 1
- 239000013612 plasmid Substances 0.000 description 1
- 230000035755 proliferation Effects 0.000 description 1
- 210000001995 reticulocyte Anatomy 0.000 description 1
- 230000004936 stimulating effect Effects 0.000 description 1
- 201000000498 stomach carcinoma Diseases 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 238000003151 transfection method Methods 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/0005—Vertebrate antigens
- A61K39/0011—Cancer antigens
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
- A61K2039/515—Animal cells
- A61K2039/5152—Tumor cells
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
- A61K2039/515—Animal cells
- A61K2039/5156—Animal cells expressing foreign proteins
Definitions
- the present invention in general, relates to an exosome carrying an exogenous protein and use thereof. More particularly, the present invention relates to an exosome carrying an exogenous antigen, which is secreted into the extracellular space by cells transfected with a gene encoding the exogenous antigen and stably expressing the antigen, and use thereof.
- Exosomes are small cup-shaped membrane vesicles with diameters ranging from 50 to 90 nm, which are secreted by a multitude of cell types. In particular, during reticulocyte maturation, unnecessary proteins are eliminated via such exosomes. Under an electron microscope, exosomes were found not to be directly pinched off from the plasma membrane but to be derived from endosomal compartments, collectively called multivesicular bodies (MVBs), and to be released into the extracellular space. That is, upon fusion of multivesicular bodies to the plasma membrane, vesicles are released into the extracellular space, which are then called exosomes. A mechanism associated with the production of exosomes is not clearly identified.
- antigen presenting cells present antigenic peptides to MHC (major histocompatibility complex) class II molecules via intracellular membrane- bound compartments including multivesicular bodies, and thus APC-derived exosomes bear antigenic peptide-MHC class II complexes.
- exosomes as vehicles for immunogens, are able to present antigenic peptides to CD4 + T lymphocytes, resulting in the induction of specific immune responses, such as proliferation of T lymphocytes, h addition, since exosomes accumulate molecules capable of stimulating the immune response, such as MHC class I molecules or heat shock proteins (HSPs), they are effective in autoimmune disease cancer immunotherapy by increasing or reducing the immune response.
- MHC class I molecules or heat shock proteins (HSPs) molecules capable of stimulating the immune response, such as MHC class I molecules or heat shock proteins (HSPs)
- Fig. 1 is a diagram schematically showing a process of preparing exosomes carrying an exogenous protein according to the present invention
- Figs. 2a and 2b show results of SDS-PAGE, in which protein composition of an established cell line according to the present invention is compared with that of exosomes derived from the cell line;
- Figs. 3a and 3b show results of FACS analysis, in which an exogenous tumor antigen is positioned on the cell surface of a target cell line transfected with a gene encoding the exogenous antigen according to the present invention;
- Figs. 4a and 4b show results of Western blotting, in which an exogenous tumor antigen is present in exosomes isolated from a target cell line transfected with a gene encoding the exogenous antigen according to the present invention
- Fig. 5 show a result of Western blotting, in which tumor antigens are present in exosomes isolated from a human carcinoma cell line naturally carrying the tumor antigens.
- the present invention provides an exosome carrying a specific antigen by introducing a gene encoding the antigen into a target cell line by a transfection method and stably expressing the antigen therein.
- the exosome according to the present invention may be utilized to isolate proteins that are difficult to isolate while maintaining their natural states without structural damage or functional inhibition.
- the exosome may be utilized as a vehicle for immunogens when intended to induce change of the immune system employing the exogenous antigen as an immunogen.
- Exosomes originate from endosomal compartments produced during the vesicular transport from the endoplasmic reticulum (ER) to the Golgi apparatus, where antigens bind to MHC molecules, and the resulting complexes are released into the extracellular space when the endosomal compartments are fused with the plasma membrane.
- ER endoplasmic reticulum
- cell surface molecules or cell-specific antigens are produced in the ER and transferred to the cell surface via the vesicular transport pathway, resulting in exposure to the extracellular space.
- antigenic proteins newly produced in the cell may be present on the cell surface via the vesicular transport pathway, or released to the extracellular space by means of exosomes.
- the present inventors investigated that the specific antigen is stably expressed in the cells and secreted by means of exosomes.
- an exosome carrying an exogenous protein is produced by a process comprising the steps of (1) cloning a human mucl gene into a BamHI site of a pLXIN plasmid to prepare a recombinant vector, (2) introducing the recombinant vector into a packaging cell line, PA317, to produce viral particles bearing the mucl gene, (3) infecting a target cell line with the viral particles, and (4) isolating exsomes by ultra-centrifuging the tansfected cells.
- the exosomes obtained by the aforementioned process were evaluated for expression of the introduced antigen by employing a fluorescent substance or electrophoresis.
- the antigenic protein was found to be stably expressed in the target cell line, present in exosomes, and released into the extracellular space by means of exosomes.
- the exosome according to the present invention of the present invention may be applied to a wide variety of areas by employing other tumor antigens as the exogenous antigen artificially introduced into the cells.
- the exogenous antigen carried in the exosome according to the present invention has its natural structure without conformational change or degradation, which is identical to the three dimensional structure of the antigen upon exposed to the immune system in the body where tumors develop. Therefore, when intended to induce change in the immune system using an exogenous antigen, the exosome according to the present invention may be useful as a vehicle for the exogenous antigen as an immunogen.
- exosome according to the present invention may be used in isolation and purification of cell surface proteins that are difficult to be isolated and purified, macro-molecular proteins, glycosylated proteins, or complex folding-undergoing proteins, without structural damage or functional inhibition.
- Example 1 Establishment of cell line stably expressing Mucl
- Human mucl gene was cloned into a BamHI site of pLXIN (Clontech), and the resulting vector was introduced into a packaging cell line, PA 317, by means of liposomes, resulting in the production of viral particles bearing the mucl gene.
- the murine colon cancer cell line CT26 was infected with the viral particles, and selected in the presence of 1200 ⁇ g/ml of geneticin. Among the formed colonies, one clone exhibiting high expression of Mucl was determined and designated "CT26 pLXL - ucl N1010".
- each protein sample was subjected to SDS-PAGE electrophoresis.
- the cell lysate from the CT26 pLXIN-mucl N1010 clone was found to differ from the exosomes isolated from the clone in protein composition (Fig. 2a). This result indicates that the introduced mucl gene is expressed in exosomes.
- 2xl0 5 of the CT26 pLXIN-mucl N1010 cells were suspended in 0.1% BSA-containing PBS (phosphate- buffered saline) and placed on ice.
- 2 ⁇ l of a primary antibody (mouse anti-mucl IgG) was added to the cell suspension, followed by incubation for 1 hr 30 min. The cells were washed with PBS to remove unbound antibodies. Then, the cells were treated with 1 ⁇ l of a secondary antibody (FITC-tagged anti-mouse IgG) on ice for 1 hr. After washing with PBS, the cells were treated with 200 ⁇ l of a cold fixing solution (2% PFA-containing PBS). Fluorescent intensity was assayed with a Coulter FACScan flow cytometer.
- the cell surface protein Mucl was found to be present on the cell surface of the CT26 pLXIN-mucl N1010 cells prepared by introducing pLXIN-Mucl into the CT26 cells (Fig. 3a).
- Exosomes were isolated from another stably transfected clone, CT26 pLSIN-mucl N1019, prepared by introducing mucl gene into CT26 cells in Example 1.
- the isolated exosomes were subjected to SDS-PAGE, FACS analysis and Western blotting, and the results are given Figs. 2a, 3a and 4a, respectively.
- Muc 1 protein was found to be present in the surface of exosomes.
- Mucl gene was introduced into TA3HA cells according to the same procedure as in
- Example 1 thus giving a stably transfected clone, TA3HA pLXIN-mucl 615.
- Exosomes were isolated from the clone, and subjected to SDS-PAGE, FACS analysis and Western blotting, and the results are given Figs. 2b, 3b and 4b, respectively. As a result, the Mucl protein was found to be present on the surface of the exosomes.
- exosomes were isolated from the culture supernatant by ultracentrifugation, and subjected to Western blotting. The results are given in Fig. 5. As shown in Fig. 5, the tumor antigens CEA and Mucl were found to be present in the exosomes.
- exosomes were isolated from the culture supernatant by ultracentrifugation, and subjected to Western blotting. The result is given in Fig. 5. As shown in Fig. 5, the tumor antigen Mucl were found to be present in the exosomes.
- the tumor antigen Mucl was found to be secreted into the extracellular space along with exosomes.
- a desired antigen such as a vaccine to be used for induction of the immune response
- the tumor antigens CEA and Mucl were released into the extracellular space via the exosomes secreted from the carcinoma cell lines naturally expressing the tumor antigens. Based on this finding, a variety of tumor antigens can be obtained from exosomes isolated from various carcinoma cell lines, and the obtained tumor antigens can be utilized for establishment of an antigen pool or antigen bank.
- the present invention provides an exosome carrying an exogenous antigen, which is secreted by tumor cells stably transfected with a gene encoding the exogenous antigen and expressing the antigen.
- antigenic proteins as vaccines should be synthesized, isolated and then purified.
- the exosome of the present invention is effective in improving such a time-consuming and uneconomical process.
- the exosome makes it possible to isolate and purify a desired protein at its natural state, thereby reducing harmful immune responses caused by modification of the desired protein and experimental errors.
Abstract
Disclosed is an exosome carrying an exogenous antigen. The exosome of the present invention is characterized by being secreted into the extracelluar space by a target cell line transfected with a gene encoding an exogenous antigen and stably expressing the antigen, leading to the release of the antigen into the extracellular space. Therefore, the exosome facilitates the production of a variety of exogenous antigen to be used as immunogens when intended to induce change in the immune system.
Description
EXOSOME CONTAINING EXOGENOUS ANTIGEN THROUGH GENE TRANSFECTION AND METHOD FOR UTILIZING THE
SAME
Technical Field
The present invention, in general, relates to an exosome carrying an exogenous protein and use thereof. More particularly, the present invention relates to an exosome carrying an exogenous antigen, which is secreted into the extracellular space by cells transfected with a gene encoding the exogenous antigen and stably expressing the antigen, and use thereof.
Background Art
Exosomes are small cup-shaped membrane vesicles with diameters ranging from 50 to 90 nm, which are secreted by a multitude of cell types. In particular, during reticulocyte maturation, unnecessary proteins are eliminated via such exosomes. Under an electron microscope, exosomes were found not to be directly pinched off from the plasma membrane but to be derived from endosomal compartments, collectively called multivesicular bodies (MVBs), and to be released into the extracellular space. That is, upon fusion of multivesicular bodies to the plasma membrane, vesicles are released into the extracellular space, which are then called exosomes. A mechanism associated with the production of exosomes is not clearly identified. However, in addition to erythrocytes, a broad range of immune cells, including B lymphocytes, T lymphocytes, dendritic cells, platelets and macrophages, and tumor cells have been reported to secrete exosomes in living states. In particular, antigen presenting cells (APCs) present antigenic peptides to MHC (major histocompatibility complex) class II molecules via intracellular membrane-
bound compartments including multivesicular bodies, and thus APC-derived exosomes bear antigenic peptide-MHC class II complexes. Therefore, exosomes, as vehicles for immunogens, are able to present antigenic peptides to CD4+ T lymphocytes, resulting in the induction of specific immune responses, such as proliferation of T lymphocytes, h addition, since exosomes accumulate molecules capable of stimulating the immune response, such as MHC class I molecules or heat shock proteins (HSPs), they are effective in autoimmune disease cancer immunotherapy by increasing or reducing the immune response.
Disclosure of Invention
It is therefore an object of the present invention to provide an exosome carrying an exogenous antigen, which is secreted into the extracellular space by cells stably transfected with a gene encoding the exogenous antigen and expressing the antigen. It is another object of the present invention to provide use of such an exosome in purification of proteins that are difficult to be purified or as a vehicle for various immunogens.
Brief Description of the Drawings
The above and other objects, features and other advantages of the present invention will be more clearly understood from the following detailed description taken in conjunction with the accompanying drawings, in which:
Fig. 1 is a diagram schematically showing a process of preparing exosomes carrying an exogenous protein according to the present invention;
Figs. 2a and 2b show results of SDS-PAGE, in which protein composition of an established cell line according to the present invention is compared with that of exosomes derived from the cell line;
Figs. 3a and 3b show results of FACS analysis, in which an exogenous tumor antigen is positioned on the cell surface of a target cell line transfected with a gene encoding the exogenous antigen according to the present invention;
Figs. 4a and 4b show results of Western blotting, in which an exogenous tumor antigen is present in exosomes isolated from a target cell line transfected with a gene encoding the exogenous antigen according to the present invention; and Fig. 5 show a result of Western blotting, in which tumor antigens are present in exosomes isolated from a human carcinoma cell line naturally carrying the tumor antigens.
Best Mode for Carrying Out the Invention
In order to achieve the aforementioned objects, the present invention provides an exosome carrying a specific antigen by introducing a gene encoding the antigen into a target cell line by a transfection method and stably expressing the antigen therein. In addition, the exosome according to the present invention may be utilized to isolate proteins that are difficult to isolate while maintaining their natural states without structural damage or functional inhibition.
Moreover, the exosome may be utilized as a vehicle for immunogens when intended to induce change of the immune system employing the exogenous antigen as an immunogen.
Exosomes originate from endosomal compartments produced during the vesicular transport from the endoplasmic reticulum (ER) to the Golgi apparatus, where antigens bind to MHC molecules, and the resulting complexes are released into the extracellular space when the endosomal compartments are fused with the plasma membrane. On the other hand, cell surface molecules or cell-specific antigens are produced in the ER and transferred to the cell surface via the vesicular transport
pathway, resulting in exposure to the extracellular space. Similarly, antigenic proteins newly produced in the cell may be present on the cell surface via the vesicular transport pathway, or released to the extracellular space by means of exosomes. In this regard, when introducing a gene encoding a specific antigen into cells and isolating exosomes from the transfected cells, the present inventors investigated that the specific antigen is stably expressed in the cells and secreted by means of exosomes.
In accordance with the present invention, an exosome carrying an exogenous protein is produced by a process comprising the steps of (1) cloning a human mucl gene into a BamHI site of a pLXIN plasmid to prepare a recombinant vector, (2) introducing the recombinant vector into a packaging cell line, PA317, to produce viral particles bearing the mucl gene, (3) infecting a target cell line with the viral particles, and (4) isolating exsomes by ultra-centrifuging the tansfected cells. The exosomes obtained by the aforementioned process were evaluated for expression of the introduced antigen by employing a fluorescent substance or electrophoresis. As a result, the antigenic protein was found to be stably expressed in the target cell line, present in exosomes, and released into the extracellular space by means of exosomes. In addition to the Mucl tumor antigen, the exosome according to the present invention of the present invention may be applied to a wide variety of areas by employing other tumor antigens as the exogenous antigen artificially introduced into the cells.
The exogenous antigen carried in the exosome according to the present invention has its natural structure without conformational change or degradation, which is identical to the three dimensional structure of the antigen upon exposed to the immune system in the body where tumors develop. Therefore, when intended to induce
change in the immune system using an exogenous antigen, the exosome according to the present invention may be useful as a vehicle for the exogenous antigen as an immunogen.
In addition, the exosome according to the present invention may be used in isolation and purification of cell surface proteins that are difficult to be isolated and purified, macro-molecular proteins, glycosylated proteins, or complex folding-undergoing proteins, without structural damage or functional inhibition.
The present invention will be explained in more detail with reference to the following example in conjunction with the accompanying drawings. However, it will be apparent to one skilled in the art that the following examples are provided only to illustrate the present invention, and the present invention is not limited to the examples.
Example 1 (1) Establishment of cell line stably expressing Mucl
Human mucl gene was cloned into a BamHI site of pLXIN (Clontech), and the resulting vector was introduced into a packaging cell line, PA 317, by means of liposomes, resulting in the production of viral particles bearing the mucl gene.
Thereafter, the murine colon cancer cell line CT26 was infected with the viral particles, and selected in the presence of 1200 μg/ml of geneticin. Among the formed colonies, one clone exhibiting high expression of Mucl was determined and designated "CT26 pLXL - ucl N1010".
(2) Assay for Mucl expression by SDS-PAGE and enzymatic reaction lxlO7 of the transfected cells were suspended in 500 μl of RD?A buffer and incubated on ice for 10 min to allow cyto lysis, followed by centrifugation, thus giving supernatant as a protein fraction. Also, exosomes were isolated from the
transfected cells by ultracentrifugation. In the supernatant from the cell lysate and exosomes, the total protein amount was measured using a BCA protein assay kit (PIERCE). 20 μg of each protein sample was incubated with mouse anti-mucl IgG as a primary antibody and then HRP (horseradish peroxidase)-conjugated anti-mouse antibody as a secondary antibody. When a HRP substrate was added into the solution, the color developed.
Also, each protein sample was subjected to SDS-PAGE electrophoresis. The cell lysate from the CT26 pLXIN-mucl N1010 clone was found to differ from the exosomes isolated from the clone in protein composition (Fig. 2a). This result indicates that the introduced mucl gene is expressed in exosomes.
(3) Assay for Mucl expression using a fluorescent substance
In order to investigate whether Mucl is present on the cell surface, 2xl05 of the CT26 pLXIN-mucl N1010 cells were suspended in 0.1% BSA-containing PBS (phosphate- buffered saline) and placed on ice. 2 μl of a primary antibody (mouse anti-mucl IgG) was added to the cell suspension, followed by incubation for 1 hr 30 min. The cells were washed with PBS to remove unbound antibodies. Then, the cells were treated with 1 μl of a secondary antibody (FITC-tagged anti-mouse IgG) on ice for 1 hr. After washing with PBS, the cells were treated with 200 μl of a cold fixing solution (2% PFA-containing PBS). Fluorescent intensity was assayed with a Coulter FACScan flow cytometer.
As a result, the cell surface protein Mucl was found to be present on the cell surface of the CT26 pLXIN-mucl N1010 cells prepared by introducing pLXIN-Mucl into the CT26 cells (Fig. 3a).
(4) Assay for Mucl expression by Western blotting The exosomes isolated from the CT26 pLXIN-mucl N1010 cells were evaluated for carrying the Mucl protein. From the cell lysate and the isolated exosomes, 20
μg of each protein sample was subjected to SDS-PAGE, and transferred onto a nylon membrane. The membrane was sequentially incubated with antibodies specifically recognizing Mucl and HSP70 proteins. The blot was developed with chemi- luminescent reagents. As a result, Mucl protein was found to be present on the surface of the exosomes isolated from the CT26 pLXIN-mucl N1010 clone (Fig. 4a).
Example 2
Exosomes were isolated from another stably transfected clone, CT26 pLSIN-mucl N1019, prepared by introducing mucl gene into CT26 cells in Example 1. In order to investigate whether Muc 1 protein is expressed in exosomes, the isolated exosomes were subjected to SDS-PAGE, FACS analysis and Western blotting, and the results are given Figs. 2a, 3a and 4a, respectively. As shown in Figs. 2a, 3a and 4a, Muc 1 protein was found to be present in the surface of exosomes.
Example 3
Mucl gene was introduced into TA3HA cells according to the same procedure as in
Example 1, thus giving a stably transfected clone, TA3HA pLXIN-mucl 615.
Exosomes were isolated from the clone, and subjected to SDS-PAGE, FACS analysis and Western blotting, and the results are given Figs. 2b, 3b and 4b, respectively. As a result, the Mucl protein was found to be present on the surface of the exosomes.
Example 4
From another stably transfected clone, TA3HA pLXIN-mucl 617, prepared in Example 3, exosomes were isolated, and subjected to SDS-PAGE, FACS analysis and
Western blotting, and the results are given Figs. 2b, 3b and 4b, respectively. As a
. _
result, the Mucl protein was found to be present on the surface of the exosomes.
Example 5
After culturing the human gastric carcinoma cell line KATO III expressing the tumor antigens CEA and Mucl, exosomes were isolated from the culture supernatant by ultracentrifugation, and subjected to Western blotting. The results are given in Fig. 5. As shown in Fig. 5, the tumor antigens CEA and Mucl were found to be present in the exosomes.
Example 6
After culturing the human breast carcinoma cell line MCF7 expressing the tumor antigen Mucl, exosomes were isolated from the culture supernatant by ultracentrifugation, and subjected to Western blotting. The result is given in Fig. 5. As shown in Fig. 5, the tumor antigen Mucl were found to be present in the exosomes.
Upon introducing Mucl as an exogenous antigen into a target cell line in Examples 1 and 4, the tumor antigen Mucl was found to be secreted into the extracellular space along with exosomes. In this way, a desired antigen, such as a vaccine to be used for induction of the immune response, can be produced easily while maintaining its natural state. In addition, in Examples 5 and 6, the tumor antigens CEA and Mucl were released into the extracellular space via the exosomes secreted from the carcinoma cell lines naturally expressing the tumor antigens. Based on this finding, a variety of tumor antigens can be obtained from exosomes isolated from various carcinoma cell lines, and the obtained tumor antigens can be utilized for establishment of an antigen pool or antigen bank.
Industrial Applicability
As described hereinbefore, the present invention provides an exosome carrying an exogenous antigen, which is secreted by tumor cells stably transfected with a gene encoding the exogenous antigen and expressing the antigen. Upon development of tumor vaccines, antigenic proteins as vaccines should be synthesized, isolated and then purified. The exosome of the present invention is effective in improving such a time-consuming and uneconomical process.
In addition, the exosome makes it possible to isolate and purify a desired protein at its natural state, thereby reducing harmful immune responses caused by modification of the desired protein and experimental errors.
The present invention has been described in an illustrative manner, and many modifications and variations of the present invention are possible in light of the above teachings. Therefore, it is to be understood that within the scope of the appended claims, the invention may be practiced otherwise than as specifically described.
Claims
1. An exosome carrying an exogenous protein, which is secreted into the extracellular space by a target cell line transfected with a gene encoding the exogenous antigen and stably expressing the exogenous antigen.
2. Use of the exosome of claim 1 in isolation of a protein that is difficult to be isolated while maintaining its natural structure and function.
3. Use of the exosome of claim 1 as a vehicle for an immunogen when intended to use an antigen as an immunogen.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| AU2003248150A AU2003248150A1 (en) | 2002-08-13 | 2003-08-06 | Exosome containing exogenous antigen through gene transfection and method for utilizing the same |
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| KR10-2002-0047779 | 2002-08-13 | ||
| KR10-2002-0047779A KR100519384B1 (en) | 2002-08-13 | 2002-08-13 | Manufacturing method of exosomes using gene transfection and use of the same |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| WO2004014954A1 true WO2004014954A1 (en) | 2004-02-19 |
Family
ID=31713115
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| PCT/KR2003/001575 WO2004014954A1 (en) | 2002-08-13 | 2003-08-06 | Exosome containing exogenous antigen through gene transfection and method for utilizing the same |
Country Status (3)
| Country | Link |
|---|---|
| KR (1) | KR100519384B1 (en) |
| AU (1) | AU2003248150A1 (en) |
| WO (1) | WO2004014954A1 (en) |
Cited By (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1322115C (en) * | 2005-07-06 | 2007-06-20 | 清华大学 | Ectobody loaded with exogenous ligand molecule and its preparation method and application |
| GB2437727A (en) * | 2006-05-04 | 2007-11-07 | Univ Open | Aptamers against MUC1 and their uses |
| WO2011080271A2 (en) | 2009-12-28 | 2011-07-07 | Centre De Recerca En Salut Internacional De Barcelona | Exosomes derived from reticulocytes infected with plasmodium sp., method for obtaining them and uses thereof |
| WO2015085096A1 (en) * | 2013-12-04 | 2015-06-11 | Board Of Regents, The University Of Texas System | Analysis of genomic dna, rna, and proteins in exosomes for diagnosis and theranosis |
| JP2017101012A (en) * | 2015-11-30 | 2017-06-08 | 義之 小山 | Immunotherapeutic formulation |
| US10233445B2 (en) | 2009-04-17 | 2019-03-19 | Oxford University Innovation Limited | Composition for delivery of genetic material |
| US10500231B2 (en) | 2013-03-13 | 2019-12-10 | University Of Miami | Method for isolation and purification of microvesicles from cell culture supernatants and biological fluids |
| US10959952B2 (en) | 2015-06-10 | 2021-03-30 | Board Of Regents, The University Of Texas System | Use of exosomes for the treatment of disease |
| US11103586B2 (en) | 2011-12-07 | 2021-08-31 | Oxford University Innovation Limited | Exosomes for delivery of biotherapeutics |
| WO2022040516A1 (en) | 2020-08-21 | 2022-02-24 | University Of Miami | Compositions and methods of treatment using microvesicles from bone marrow-derived mesenchymal stem cells |
| WO2023205158A1 (en) | 2022-04-19 | 2023-10-26 | University Of Miami | Compositions comprising microvesicles for use in the prevention and treatment of graft versus host disease |
| US11926824B2 (en) | 2013-03-15 | 2024-03-12 | Board Of Regents, The University Of Texas System | miRNA biogenesis in exosomes for diagnosis and therapy |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2018062973A1 (en) | 2016-09-30 | 2018-04-05 | Cellex Life Sciences, Incorporated | Compositions containing protein loaded exosome and methods for preparing and delivering the same |
| WO2022211473A1 (en) | 2021-03-30 | 2022-10-06 | (주)카이노스메드 | Fas-associated factor 1 (faf1)-loaded exosomes and use thereof as anti-cancer agent |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1997005900A1 (en) * | 1995-08-03 | 1997-02-20 | Rijksuniversiteit Te Leiden | Cell derived antigen presenting vesicles |
| WO1999003499A1 (en) * | 1997-07-16 | 1999-01-28 | Institut National De La Sante Et De La Recherche Medicale | Cellular vesicle called ''exosome'', preparation and use thereof in immune stimulation |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| FR2785543B1 (en) * | 1998-11-05 | 2003-02-28 | Inst Nat Sante Rech Med | MODIFIED EXOSOMES AND USES |
| US20040241176A1 (en) * | 2000-04-27 | 2004-12-02 | Ap Cells. Inc. | Method of producing membrane vesicles |
-
2002
- 2002-08-13 KR KR10-2002-0047779A patent/KR100519384B1/en active IP Right Grant
-
2003
- 2003-08-06 WO PCT/KR2003/001575 patent/WO2004014954A1/en not_active Application Discontinuation
- 2003-08-06 AU AU2003248150A patent/AU2003248150A1/en not_active Abandoned
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1997005900A1 (en) * | 1995-08-03 | 1997-02-20 | Rijksuniversiteit Te Leiden | Cell derived antigen presenting vesicles |
| WO1999003499A1 (en) * | 1997-07-16 | 1999-01-28 | Institut National De La Sante Et De La Recherche Medicale | Cellular vesicle called ''exosome'', preparation and use thereof in immune stimulation |
Non-Patent Citations (2)
| Title |
|---|
| DENZER K. ET AL.: "Exosome: from internal vesicle of the multivesicular body to intercellular signaling device", J. CELL SCI., vol. 113, 2000, pages 3365 - 3374 * |
| FORTIN A. ET AL.: "Trafficking of surface-linked and encapsulated liposomal antigens in macrophages: an immunocytochemical study", J. HISTOCHEM. CYTOCHEM., vol. 49, no. 11, 2001, pages 1407 - 1420 * |
Cited By (22)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1322115C (en) * | 2005-07-06 | 2007-06-20 | 清华大学 | Ectobody loaded with exogenous ligand molecule and its preparation method and application |
| GB2437727A (en) * | 2006-05-04 | 2007-11-07 | Univ Open | Aptamers against MUC1 and their uses |
| GB2437727B (en) * | 2006-05-04 | 2011-04-20 | Univ Open | Aptamers directed to MUC1 |
| US8039609B2 (en) | 2006-05-04 | 2011-10-18 | The Open University | Aptamers directed to MUC1 |
| US11230715B2 (en) | 2009-04-17 | 2022-01-25 | Oxford University Innovation Limited | Composition for delivery of genetic material |
| US10704047B2 (en) | 2009-04-17 | 2020-07-07 | Oxford University Innovation Limited | Composition for delivery of genetic material |
| US10233445B2 (en) | 2009-04-17 | 2019-03-19 | Oxford University Innovation Limited | Composition for delivery of genetic material |
| US10329561B2 (en) | 2009-04-17 | 2019-06-25 | Oxford University Innovation Limited | Composition for delivery of genetic material |
| WO2011080271A2 (en) | 2009-12-28 | 2011-07-07 | Centre De Recerca En Salut Internacional De Barcelona | Exosomes derived from reticulocytes infected with plasmodium sp., method for obtaining them and uses thereof |
| US11103586B2 (en) | 2011-12-07 | 2021-08-31 | Oxford University Innovation Limited | Exosomes for delivery of biotherapeutics |
| US10500231B2 (en) | 2013-03-13 | 2019-12-10 | University Of Miami | Method for isolation and purification of microvesicles from cell culture supernatants and biological fluids |
| EP3677271A1 (en) | 2013-03-13 | 2020-07-08 | University Of Miami | Method for isolation and purification of microvesicles from cell culture supernatants and biological fluids |
| EP4218774A1 (en) | 2013-03-13 | 2023-08-02 | University Of Miami | Method for isolation and purification of microvesicles from cell culture supernatants and biological fluids |
| US11730768B2 (en) | 2013-03-13 | 2023-08-22 | University Of Miami | Method for isolation and purification of microvesicles from cell culture supernatants and biological fluids |
| US11926824B2 (en) | 2013-03-15 | 2024-03-12 | Board Of Regents, The University Of Texas System | miRNA biogenesis in exosomes for diagnosis and therapy |
| US10598665B2 (en) | 2013-12-04 | 2020-03-24 | Board Of Regents, The University Of Texas System | Analysis of genomic DNA, RNA, and proteins in exosomes for diagnosis and theranosis |
| US9921223B2 (en) | 2013-12-04 | 2018-03-20 | Board Of Regents, The University Of Texas System | Analysis of genomic DNA, RNA, and proteins in exosomes for diagnosis and theranosis |
| WO2015085096A1 (en) * | 2013-12-04 | 2015-06-11 | Board Of Regents, The University Of Texas System | Analysis of genomic dna, rna, and proteins in exosomes for diagnosis and theranosis |
| US10959952B2 (en) | 2015-06-10 | 2021-03-30 | Board Of Regents, The University Of Texas System | Use of exosomes for the treatment of disease |
| JP2017101012A (en) * | 2015-11-30 | 2017-06-08 | 義之 小山 | Immunotherapeutic formulation |
| WO2022040516A1 (en) | 2020-08-21 | 2022-02-24 | University Of Miami | Compositions and methods of treatment using microvesicles from bone marrow-derived mesenchymal stem cells |
| WO2023205158A1 (en) | 2022-04-19 | 2023-10-26 | University Of Miami | Compositions comprising microvesicles for use in the prevention and treatment of graft versus host disease |
Also Published As
| Publication number | Publication date |
|---|---|
| AU2003248150A1 (en) | 2004-02-25 |
| KR100519384B1 (en) | 2005-10-06 |
| KR20040015508A (en) | 2004-02-19 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Delcayre et al. | Exosome display technology: applications to the development of new diagnostics and therapeutics | |
| Deres et al. | In vivo priming of virus-specific cytotoxic T lymphocytes with synthetic lipopeptide vaccine | |
| WO2004014954A1 (en) | Exosome containing exogenous antigen through gene transfection and method for utilizing the same | |
| EP1278825B1 (en) | Methods of producing membrane vesicles | |
| JP4603894B2 (en) | Assays to identify antibody producing cells | |
| Leifert et al. | Targeting plasmid‐encoded proteins to the antigen presentation pathways | |
| US7914792B2 (en) | Methods and compounds for raising antibodies and for screening antibody repertoires | |
| Gaubin et al. | Processing of filamentous bacteriophage virions in antigen-presenting cells targets both HLA class I and class II peptide loading compartments | |
| JP2010180216A (en) | Chimeric polypeptide comprising fragment b of shiga toxin and peptide of therapeutic interest | |
| JP7340639B2 (en) | Nucleotide sequences expressing exosome anchor proteins for use as vaccines | |
| Ruben et al. | Apoptotic blebs from leukemic cells as a preferred source of tumor-associated antigen for dendritic cell-based vaccines | |
| JP2011182702A (en) | Fused mhc molecule connecting magnetic fine particle, method for screening antigen peptide, recombinant vector and transformant of magnetic bacterium | |
| Pietersz et al. | Definition of MHC-restricted CTL epitopes from non-variable number of tandem repeat sequence of MUC1 | |
| Petrarca et al. | Isolation of MUC1-primed B lymphocytes from tumour-draining lymph nodes by immunomagnetic beads | |
| Neumann et al. | Identification of an HLA‐DR‐restricted peptide epitope with a promiscuous binding pattern derived from the cancer testis antigen HOM‐MEL‐40/SSX2 | |
| Kim et al. | In vitro induction of HLA-A2402-restricted and carcinoembryonic-antigen-specific cytotoxic T lymphocytes on fixed autologous peripheral blood cells | |
| Smyth et al. | Cd8 T‐cell recognition of human 5T4 oncofetal antigen | |
| Qin et al. | Melanoma B16-F1 cells coated with fusion protein of mouse calreticulin and virus G-protein coupled receptor induced the antitumor immune response in Balb/C mice | |
| Chikamatsu et al. | CD4+ T helper responses in squamous cell carcinoma of the head and neck | |
| Nazarkina et al. | Design of polyepitope DNA vaccine against breast carcinoma cells and analysis of its expression in dendritic cells | |
| Notter et al. | Tumor‐specific T‐cell clones recognize different protein determinants of autologous human malignant melanoma cells | |
| WO2022074098A1 (en) | Method for the identification of cancer neoantigens | |
| WO2001083783A2 (en) | In vivo loading of mhc | |
| Rodeberg et al. | Lack of effective T-lymphocyte response to the PAX3/FKHR translocation area in alveolar rhabdomyosarcoma | |
| US20090004212A1 (en) | Tumour vaccines for MUC1-positive carcinomas |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| AK | Designated states |
Kind code of ref document: A1 Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BY BZ CA CH CN CO CR CU CZ DE DK DM DZ EC EE ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX MZ NI NO NZ OM PG PH PL PT RO RU SC SD SE SG SK SL SY TJ TM TN TR TT TZ UA UG US UZ VC VN YU ZA ZM ZW |
|
| AL | Designated countries for regional patents |
Kind code of ref document: A1 Designated state(s): GH GM KE LS MW MZ SD SL SZ TZ UG ZM ZW AM AZ BY KG KZ MD RU TJ TM AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HU IE IT LU MC NL PT RO SE SI SK TR BF BJ CF CG CI CM GA GN GQ GW ML MR NE SN TD TG |
|
| 121 | Ep: the epo has been informed by wipo that ep was designated in this application | ||
| 122 | Ep: pct application non-entry in european phase | ||
| NENP | Non-entry into the national phase |
Ref country code: JP |
|
| WWW | Wipo information: withdrawn in national office |
Country of ref document: JP |