WO2018124378A1 - Cellules tumorales artificielles reconstituées, leur procédé de préparation et composition anticancéreuse en contenant - Google Patents

Cellules tumorales artificielles reconstituées, leur procédé de préparation et composition anticancéreuse en contenant Download PDF

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WO2018124378A1
WO2018124378A1 PCT/KR2017/001558 KR2017001558W WO2018124378A1 WO 2018124378 A1 WO2018124378 A1 WO 2018124378A1 KR 2017001558 W KR2017001558 W KR 2017001558W WO 2018124378 A1 WO2018124378 A1 WO 2018124378A1
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cancer cells
reconstituted artificial
cells
reconstituted
present application
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PCT/KR2017/001558
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Korean (ko)
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임용택
김선영
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성균관대학교산학협력단
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Liposomes
    • A61K9/1271—Non-conventional liposomes, e.g. PEGylated liposomes, liposomes coated with polymers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
    • A61K31/7048—Compounds having saccharide radicals and heterocyclic rings having oxygen as a ring hetero atom, e.g. leucoglucosan, hesperidin, erythromycin, nystatin, digitoxin or digoxin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/13—Tumour cells, irrespective of tissue of origin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Liposomes
    • A61K9/1271—Non-conventional liposomes, e.g. PEGylated liposomes, liposomes coated with polymers
    • A61K9/1272—Non-conventional liposomes, e.g. PEGylated liposomes, liposomes coated with polymers with substantial amounts of non-phosphatidyl, i.e. non-acylglycerophosphate, surfactants as bilayer-forming substances, e.g. cationic lipids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Liposomes
    • A61K9/1277—Processes for preparing; Proliposomes

Definitions

  • the present application relates to a reconstituted artificial cancer cell for anticancer immune activity, which plays an important role in the process of preventing and treating cancer, a method for producing the reconstituted artificial cancer cell, and an anticancer composition comprising the reconstituted artificial cancer cell.
  • the anti-cancer immunotherapy technique which is a technique for activating antigens for cancer cells into antigen-presenting cells such as dendritic cells and effectively activating T cells that can directly kill cancer cells, is of great interest in the field of anti-cancer treatment. It is becoming.
  • the technique for activating various immune cells that are responsible for the anticancer function in the living body, compared to the conventional chemotherapy or radiation therapy-based chemotherapy method has the advantage of minimizing side effects because of utilizing the patient's own immune system.
  • immune cells are in the spotlight as next-generation anticancer treatments in that they can selectively remove cancer cells having a small size or cancer cells that have spread to other tissues, which are difficult to diagnose with modern technology.
  • Korean Patent Laid-Open No. 2012-0002943 discloses a cancer treatment and cancer diagnosis method using microorganisms derived from bacteria.
  • the present application is to provide a reconstituted artificial cancer cell, a method for producing the reconstituted artificial cancer cells, and an anticancer composition comprising the reconstituted artificial cancer cells.
  • the present application is to provide an anticancer composition based on reconstituted artificial cancer cells having high antigenicity while including all of heterogeneity of antigens information.
  • a first aspect of the present invention is a reconstituted artificial cancer cells, lipids forming the outer wall of the reconstituted artificial cancer cells; Immunoactive substances; And lysates of cancer cells, wherein the lipids forming the outer wall of the reconstituted artificial cancer cells form the outer wall of the round or oval reconstituted artificial cancer cells, wherein the immunoactive substance and the lysate of the cancer cells are each independently.
  • the present invention provides a reconstituted artificial cancer cell, which is present in the outer wall of the reconstituted artificial cancer cell and / or inside the reconstituted artificial cancer cell.
  • a second aspect of the present application provides an anticancer composition comprising reconstituted artificial cancer cells according to the first aspect of the present application.
  • a method for producing reconstituted artificial cancer cells comprising: dissolving a lipid and an immunoactive substance that forms an outer wall of reconstituted artificial cancer cells in a first solvent; Evaporating the first solvent to form a lipid membrane comprising an immunoactive material; Dissolving said lipid membrane in a second solvent to form a first solution; And mixing and sonicating lysate of cancer cells in the first solution to provide reconstituted artificial cancer cells.
  • cancer cell-derived proteins having cancer cell properties obtained from the lysate of cancer cells and immunoactive substances are hybridized.
  • immunoactive substances eg, pathogen-derived cationic lipids and toll-like receptor ligands
  • reconstitution it is possible to prepare a tumosome, a nano-sized reconstituted artificial cancer cell, and to develop an anticancer immune cell activation technology using the reconstituted artificial cancer cell, tumosome.
  • the reconstructed artificial cancer cell according to an embodiment of the present application includes all of the heterogeneity of antigens information, which is an advantage of using an actual cancer cell lysate as an anticancer composition, but has a disadvantage of using an actual cancer cell lysate. It can be used as a new concept anticancer composition, which overcomes the low antigenicity of phosphorus.
  • the efficiency of delivering cancer antigens and immunoactive substances to immune cells can be improved by using reconstituted artificial cancer cells, and at the same time, the antigenicity of the antigens can be simultaneously enhanced.
  • the efficiency of delivering cancer antigens and immunoactive substances to immune cells can be improved by using reconstituted artificial cancer cells, and at the same time, the antigenicity of the antigens can be simultaneously enhanced.
  • by markedly increasing the expression of cytokines associated with the activation of anti-cancer immune cells it can exhibit excellent efficacy in the therapeutic effect of cancer.
  • Reconstituted artificial cancer cells according to an embodiment of the present application effectively induces activation of T cells, which play an important role in anticancer immunotherapy, and when applied to a prophylactic and therapeutic model of cancer, the therapeutic efficacy may be significantly improved.
  • Figure 1 in one embodiment of the present application, shows a method and model diagram of the synthesis of tumosome (remosome) that is a reconstituted artificial cancer cells.
  • the reconstituted artificial cancer cells are reconstituted with a hybridization structure of cancer cell-derived proteins having cancer cell characteristics and immunoactive substances (eg, pathogen-derived cationic lipids and TLR4 ligands).
  • FIG. 5 is a graph showing a comparison of T cell activation efficacy of tumosomes synthesized in various combinations in one embodiment of the present application.
  • Figure 6 in one embodiment of the present application, shows a manufacturing method and model diagram of the tumosome using the membrane or cytoplasmic proteins of cancer cells obtained from the excised cancer tissue.
  • Figure 7 (a) and (b), in one embodiment of the present application, is a schematic diagram of an experimental procedure for preventing the recurrence (tumor recurrence) of cancer using a tumosome prepared using the excised cancer tissue.
  • Figure 7 (a) is to grow the tumor to the C57BL / 6 mouse melanoma model after the tumor, leaving the tumor to less than about 1% through the surgical procedure to remove the remaining portion, and then suture using a pin (clip) 7 (b) shows the result of a week after suture and shows the growth of the remaining tumor.
  • FIG. 9 is a graph illustrating an anticancer treatment efficacy evaluation of tumosomes using a C57BL / 6 mouse melanoma model in one embodiment of the present application.
  • the term "combination (s) thereof" included in the expression of a makushi form refers to one or more mixtures or combinations selected from the group consisting of components described in the expression of makushi form, It means to include one or more selected from the group consisting of the above components.
  • a first aspect of the present invention is a reconstituted artificial cancer cells, lipids forming the outer wall of the reconstituted artificial cancer cells; Immunoactive substances; And lysates of cancer cells, wherein the lipids forming the outer wall of the reconstituted artificial cancer cells form the outer wall of the round or oval reconstituted artificial cancer cells, wherein the immunoactive substance and the lysate of the cancer cells are each independently.
  • the present invention provides a reconstituted artificial cancer cell, which is present in the outer wall of the reconstituted artificial cancer cell and / or inside the reconstituted artificial cancer cell.
  • the reconstituted artificial cancer cells may be named "tumosome" (tumosome).
  • the reconstituted artificial cancer cells are lysates of cancer cells which are cancer cell-derived proteins having cancer cell information; Lipids forming an outer wall of the reconstituted artificial cancer cell; And a reconstituted liposome form formed by hybridizing an immunoactive substance (eg, pathogen-derived cell membrane component, etc.) having an immune cell activating function, having a diameter of about 1,200 mm 3, for example, in the range of about 300 mm 3 to about 5,000 mm 3
  • an immunoactive substance eg, pathogen-derived cell membrane component, etc.
  • the phospholipid-binding cancer cell membrane protein and the protein present in the cancer cell cytoplasm may refer to membrane or cytoplasmic proteins isolated from the lysate of the cancer cell, that is, a membrane or an antigen based on the lysate of the cancer cell.
  • the cytoplasmic protein and the immunoactive substance may be reconstitution with lipids forming the outer wall of the reconstituted artificial cancer cells (FIG. 1).
  • the reconstituted artificial cancer cells may have a diameter in the range of about 300 mm 3 to about 5,000 mm 3.
  • This size is a condition optimized for recognizing reconstituted artificial cancer cells produced by the immune cells in the body as real cancer cells and effectively recognizing cancer antigens through intracellular uptake, for example, about 300 mm 3 to about 4,000 ⁇ , about 300 ⁇ to about 3,000 ⁇ , about 300 ⁇ to about 2,000 ⁇ , about 300 ⁇ to about 1,000 ⁇ , about 300 ⁇ to about 500 ⁇ , about 500 ⁇ to about 5,000 ⁇ , about 500 ⁇ to about 4,000 ⁇ From about 500 kPa to about 3,000 kPa, from about 500 kPa to about 2,000 kPa, from about 500 kPa to about 1,000 kPa, from about 1,000 kPa to about 5,000 kPa, from about 1,000 kPa to about 4,000 kPa, about 1,000 kPa to about 4,000 kPa, about 1,000 kPa to about 4,000
  • the intracellular delivery capacity of the cancer antigen and / or other immunoactive substances by phagocytosis or endocytosis can be improved.
  • the cancer cell has a variety of cancer cell-related information present in the cancer cell membrane and cytoplasm Was lysed for use as a cancer antigen.
  • the cancer cells may be obtained using cancer cell lines, or may be separated from cancer tissues present in the body.
  • the actual cancer tissue may be prepared by lysing cancer cells after inducing the production of proteins related to intracellular stress by applying an anticancer agent or radiation.
  • the membrane or cytoplasmic protein isolated from the lysate of the cancer cell is used herein because immune cells recognize the antigen in a particle form (particulate type) rather than a solution state in the process of recognizing the cancer antigen. Rather than using them as they were intended to be prepared in nanoparticle form.
  • protein antigens isolated from the lysate of cancer cells include membrane proteins having phospholipid binding properties and various protein components existing in the cytoplasm, it is difficult to implement nanoparticle forms having stable structures using only these components. .
  • the membrane or cytoplasmic proteins isolated from the lysate of the cancer cells are reconstituted with an immunoactive material and lipids forming the outer wall of the reconstituted artificial cancer cells, thereby having a nano-sized form Tumosomes, which are cancer cells, can be formed, resulting in various cancer antigen components present in the cancer cell membrane and / or cytoplasm that can be encapsulated within the outer wall and / or inside of the reconstituted artificial cancer cells (lower figure in FIG. 1). .
  • protein antigens and immunoactive substances isolated from the lysate of the cancer cell are reconstituted into reconstituted artificial cancer cells by cationic lipids in the immunoactive material, thereby providing various cancer protein antigens present in the lysate of the cancer cell.
  • the efficiency of delivery in immune cells of substances related to these is increased, so that immune cells can effectively recognize substances related to the cancer antigens.
  • the reconstituted artificial cancer cells include lipids forming a circular or oval outer wall, and include lysate and an immunoactive material of cancer cells independently in the outer wall and / or inside of the reconstituted artificial cancer cells. , Liposome structure.
  • various hydrophilic and / or lipophilic immunoactive substances may be loaded by the liposome structure of the complex liposome.
  • the immunoactive substance may be an immunoactive substance expressed in stressed cancer cells, for example, a heat-shock protein, or a substance inducing T cell activation. Can be.
  • the immunoactive substance is a toll-like receptor agonist (TLR), saponin, antiviral peptide, inflammasome inducer, NOD ligand (NOD) ligand), CDS ligand (cytosolic DNA sensor ligand), STING (stimulator of interferon genes) ligand, cationic lipids (cationic lipids), and combinations thereof, but may include a material selected from the group It may not be.
  • TLR toll-like receptor agonist
  • saponin saponin
  • antiviral peptide antiviral peptide
  • inflammasome inducer NOD ligand (NOD) ligand
  • CDS ligand cytosolic DNA sensor ligand
  • STING stirr of interferon genes
  • cationic lipids cationic lipids
  • combinations thereof but may include a material selected from the group It may not be.
  • the toll-like receptor agonist may be a natural toll-like receptor agonist or a synthetic toll-like receptor agonist.
  • the immunoactivator may comprise a combination of one or more toll-like receptor agonists, for example, CL401 (dual TLR2 and TLR7 agonists) or CL429 (dual TLR2 and NOD2 agonist), but may not be limited thereto.
  • CL401 dual TLR2 and TLR7 agonists
  • CL429 dual TLR2 and NOD2 agonist
  • the toll-like receptor agonist may be one that can cause a signaling response through TLR-1, for example, tri-acylated lipopeptide (LP); Phenol-soluble modulins; Cobacterium tuberculosis (Mycobacterium tuberculosis) lipopeptide; S- (2,3-bis (palmitoyloxy)-(2-RS) -propyl) -N-palmitoyl- (R) -Cys- (S) -Ser- (S) -Lys (4) -OH ; Bacterial lipopeptides from Borrelia burgdorfei; Trihydrochloride (Pam3Cys) lipopeptides that mimic the acetylated amino termini of OspA lipopeptides; And one or more materials selected from the group consisting of combinations thereof, but may not be limited thereto.
  • LP tri-acylated lipopeptide
  • Phenol-soluble modulins Cobacterium tub
  • the toll-like receptor agonist may include a TLR-2 agonist, for example, may include Pam3Cys-Lip, but may not be limited thereto.
  • the toll-like agonist may include a TLR-3 agonist, for example, Poly (I: C), Poly (ICLC), Poly (IC12U ), But may include, but are not limited to, Ampligen.
  • the toll-like agonist may be one comprising a TLR-4 agonist, for example, Shigella flexineri outer membrane protein preparation, AGP, CRX-527, MPLA, It may include, but is not limited to, one or more materials selected from the group consisting of PHAD, 3D-PHAD, GLA, and combinations thereof.
  • the toll-like agonist may include a TLR-5 agonist, for example, may include, but is not limited to, flagellin or a fragment thereof. .
  • the toll-like agonist may be one comprising a TLR-7 agonist or a TLR-8 agonist, for example, such as imiquimod, R837, resquimod, or R848.
  • Imidazoquinoline molecules VTX-2337; CRX642; Imidazoquinoline covalently bound to a phospholipid group or a phosphonolipid group; And one or more materials selected from the group consisting of combinations thereof, but may not be limited thereto.
  • the toll-like agonist may include a TLR-9 agonist, for example, may include an immunostimulatory oligonucleotide, but may not be limited thereto.
  • the immunostimulatory oligonucleotide may include one or more CpG motifs, but may not be limited thereto.
  • the saponin may be selected from the group consisting of QS21, Quil A, QS7, QS17, ⁇ -eskin, Digitonin, and combinations thereof, but may not be limited thereto.
  • the antiviral peptide may include KLK, but may not be limited thereto.
  • the influmersome inducer may be TDB (trehalose-6,6-dibehenate), but may not be limited thereto.
  • the NOD ligand may be M-TriLYS (NOD2 agonist-synthetic muramyl tripeptide) or NOD2 agonist (N-lycolylated muramyldipeptid), but may not be limited thereto.
  • the CDS ligand may be Poly (dA: dT), but may not be limited thereto.
  • the STING ligand may be cGAMP, di-AMP, or di-GMP, but may not be limited thereto.
  • the immunoactive material is Pam3Cys-Lip, Poly (I: C), CRX-527, MPLA (monophosphoryl lipid A), flagellin (flagellin), imiquimod, resqui Mode, CpG, QS21, M-TriLys (MurNAc-Ala-D-isoGln-Lys), trehalose-6,6-dibehenate (TDB), 8837, Poly (dA: dT), cGAMP, and combinations thereof
  • the MPLA is derived from Gram-negative bacteria, lipopolysaccharide (LPS), and is a cell signal transduction system, through toll-like receptor 4 (TLR4).
  • TLR4 signaling stimulates the secretion of proinflammatory cytokines that activate the immune response, which stimulates the proliferation, differentiation and activity of T and B cells.
  • activated T cells promote differentiation into cytotoxic T cells, Secretion of cytokines such as interferon-gamma is involved in cellular immune responses.
  • the immunoactive material including a functional group, such as a carboxyl group, an amine group, a thiol group, an aldehyde group, a biotin group
  • substances responsible for various functions may be conjugated (conjugated).
  • the cationic lipid in the immunoactive material, the negatively charged immunoactive material or biomaterials such as DNA, RNA may be effectively loaded into the reconstituted artificial cancer cells.
  • antigen cross-presentation is induced by the cationic lipid, the cancer antigen can be delivered into the cell.
  • antigen cross-presentation may be induced to transfer the cancer antigens into the cells, thereby resulting in an anticancer effect. It may not be limited.
  • the reconstituted artificial cancer cells have a nano size or a diameter size of angstroms, the cancer antigen and / or other immunoactive substances caused by pagocytosis or endocytosis Intracellular delivery ability of can be improved.
  • the reconstituted artificial cancer cells include an immunoactive material of cationic lipids
  • the cationic lipids induce an electrostatic attraction between the reconstituted artificial cancer cells and the anionic cell membrane, the cancer Intracellular delivery of antigens and / or other immunoactive agents may be further enhanced.
  • the cationic lipid is DC-cholesterol (3 ⁇ - [N- (N '(N', N'-dimethylaminoethane) -carbamoyl] cholesterol hydrochloride), DDA (dimethyldioctadecylammonium), DOTAP (1,2-dioleoyl -3-trimethylammoniumpropane), DOTMA (1,2-di-O-octadecenyl-3-trimethylammonium propane), EPC (1,2-dimyristoleoyl-sn-glycero-3-ethylphosphocholine), MVL5 (N1- [2-(( 1S) -1-[(3-aminopropyl) amino] -4- [di (3-amino-propyl) amino] butylcarboxamido) ethyl] -3,4-di [oleyloxy] -benzamide), DODAP (lipids), lipids, lipid
  • the lipid that forms the outer wall of the reconstituted artificial cancer cells are phosphatidylglycerols, phosphatidylcholines, phosphatidylethanolamines, phosphatidylserine, phosphatidyl inositol, egg yolk lecithin, soybean lecithin, dioleoyl phosphatidyl phospho Choline, dioleoyl phosphatidylethanolamine, 2,3-dioleoyloxy-N- [2 (sperminecarboxamido) ethyl] -N, N-dimethyl-1-propaneaminium trifluoroacetate, N, N , N, N, N-tetramethyl-N, N, N, N-tetrapalmitylspermine, 1,2-dimyristyloxypropyl-3-dimethyl-hydroxyethylammonium bromide, 3 ⁇ - [N- (N ′, N'
  • the reconstituted artificial cancer cells may be injected directly around the cancer cells, or may be injected to prevent the recurrence of cancer after removing the cancer tissue.
  • the cancer cells may include cancer cells of lung, colon, central nervous system, skin, ovary, kidney, breast, stomach, or colon, but may not be limited thereto.
  • the cancer cells may be cancer cells of the skin, for example, may include melanoma-derived cancer cells, or melanoma_B16-F10, but may not be limited thereto.
  • the lysate of the cancer cell may be one comprising a membrane or cytoplasmic protein, for example, the membrane or cytoplasmic protein, for example, recombinant protein membrane or cytoplasmic protein, subunit, split It may include, but is not limited to, membrane or cytoplasmic protein antigens.
  • a second aspect of the present application provides an anticancer composition comprising reconstituted artificial cancer cells according to the first aspect of the present application.
  • the anticancer composition according to the second aspect of the present application detailed descriptions of portions overlapping with the first side of the present application have been omitted, but the contents described in the first aspect of the present application may be The same may apply.
  • the reconstituted artificial cancer cells are lipids that form the outer wall of the reconstituted artificial cancer cells; Immunoactive substances; And lysate of cancer cells.
  • the reconstituted artificial cancer cells may comprise lipids forming an outer wall of the reconstituted artificial cancer cells; Immunoactive substances; And a membrane or cytoplasmic protein isolated from the lysate of the cancer cell, wherein the lipid forming the outer wall forms the outer wall of the reconstituted artificial cancer cell of circular or oval shape, wherein the immunoactive substance and the lysate of the cancer cell are each independently, It may be present in the outer wall of the reconstituted artificial cancer cells and / or inside the reconstituted artificial cancer cells.
  • the reconstituted artificial cancer cells include lipids forming a circular or oval outer wall, and the membrane or cytoplasmic protein and immunity separated from the lysate of cancer cells in the outer wall and / or inside the reconstituted artificial cancer cells Liposome structure, including the active substance.
  • the reconstituted artificial cancer cells may be named "tumosome" (tumosome).
  • the reconstituted artificial cancer cell is a reconstituted liposome form formed by hybridizing a lysate of cancer cells, which are cancer cell-derived proteins having cancer cell information, and an immunoactive substance having an immune cell activating function (eg, a pathogen-derived cell membrane component, etc.).
  • a novel form comprising a skeleton-shaped outer wall formed by lipids forming the outer wall of the reconstituted artificial cancer cell, and a phospholipid-binding membrane protein present in the outer wall of the cancer cell and a cytoplasmic protein contained within the cytoplasm of the cancer cell. It may mean a liposome having a hybridized structure of.
  • the phospholipid-binding cancer cell membrane protein and the protein present in the cancer cell cytoplasm may refer to membrane or cytoplasmic proteins isolated from the lysate of the cancer cell, that is, a membrane or an antigen based on the lysate of the cancer cell.
  • the cytoplasmic protein and the immunoactive substance may be reconstitution with lipids forming the outer wall of the reconstituted artificial cancer cells.
  • the cancer cells may be lysed to use a substance having various cancer cell-related information present in the cancer cell membrane and cytoplasm as a cancer antigen.
  • the cancer cells may be obtained using cancer cell lines, or may be separated from cancer tissues present in the body.
  • the actual cancer tissue may be prepared by lysing cancer cells after inducing the production of proteins related to intracellular stress by applying an anticancer agent or radiation.
  • the membrane or cytoplasmic proteins isolated from the lysate of the cancer cells are reconstituted artificial cancer cells having a nano-sized form by reconstituting the immunoactive material and lipids forming the outer wall of the reconstituted artificial cancer cells Tumosomes may be formed, such that various cancer antigen components present in the cancer cell membrane and / or cytoplasm may be encapsulated within the outer wall and / or inside the reconstituted artificial cancer cell.
  • protein antigens and immunoactive substances isolated from the lysate of the cancer cell are reconstituted into reconstituted artificial cancer cells by cationic lipids in the immunoactive material, thereby providing various cancer protein antigens present in the lysate of the cancer cell.
  • the efficiency of delivery in immune cells of substances related to these is increased, so that immune cells can effectively recognize substances related to the cancer antigens.
  • the diameter of the reconstituted artificial cancer cells may be from about 300 mm 3 to about 4,000 mm 3. This size is a condition optimized for recognizing reconstituted artificial cancer cells prepared by immune cells in the body as real cancer cells, and effectively recognizing cancer antigens through intracellular uptake.
  • the intracellular delivery capacity of the cancer antigen and / or other immunoactive substances by phagocytosis or endocytosis can be improved.
  • the anticancer composition comprising the reconstituted artificial cancer cells, may be injected directly around the cancer cells, or injected to prevent recurrence of cancer after removing the cancer tissue.
  • various hydrophilic and / or lipophilic immunoactive substances may be loaded by the liposome structure of the complex liposome.
  • the immunoactive substance may be an immunoactive substance expressed in stressed cancer cells, for example, a heat-shock protein, or a substance inducing T cell activation. Can be.
  • the immunoactive substance is a toll-like receptor agonist (TLR), saponin, antiviral peptide, inflammasome inducer, NOD ligand (NOD) ligand), CDS ligand (cytosolic DNA sensor ligand), STING (stimulator of interferon genes) ligand, cationic lipids (cationic lipids), and combinations thereof, but may include a material selected from the group It may not be.
  • TLR toll-like receptor agonist
  • saponin saponin
  • antiviral peptide antiviral peptide
  • inflammasome inducer NOD ligand (NOD) ligand
  • CDS ligand cytosolic DNA sensor ligand
  • STING stirr of interferon genes
  • cationic lipids cationic lipids
  • combinations thereof but may include a material selected from the group It may not be.
  • the toll-like receptor agonist may be a natural toll-like receptor agonist or a synthetic toll-like receptor agonist.
  • the immunoactivator may comprise a combination of one or more toll-like receptor agonists, for example, CL401 (dual TLR2 and TLR7 agonists) or CL429 (dual TLR2 and NOD2 agonist), but may not be limited thereto.
  • CL401 dual TLR2 and TLR7 agonists
  • CL429 dual TLR2 and NOD2 agonist
  • the toll-like receptor agonist may be one that can cause a signaling response through TLR-1, for example, tri-acylated lipopeptide (LP); Phenol-soluble modulins; Cobacterium tuberculosis (Mycobacterium tuberculosis) lipopeptide; S- (2,3-bis (palmitoyloxy)-(2-RS) -propyl) -N-palmitoyl- (R) -Cys- (S) -Ser- (S) -Lys (4) -OH ; Bacterial lipopeptides from Borrelia burgdorfei; Trihydrochloride (Pam3Cys) lipopeptides that mimic the acetylated amino termini of OspA lipopeptides; And one or more materials selected from the group consisting of combinations thereof, but may not be limited thereto.
  • LP tri-acylated lipopeptide
  • Phenol-soluble modulins Cobacterium tub
  • the toll-like receptor agonist may include a TLR-2 agonist, for example, may include Pam3Cys-Lip, but may not be limited thereto.
  • the toll-like agonist may include a TLR-3 agonist, for example, Poly (I: C), Poly (ICLC), Poly (IC12U ), But may include, but are not limited to, Ampligen.
  • the toll-like agonist may be one comprising a TLR-4 agonist, for example, Shigella flexineri outer membrane protein preparation, AGP, CRX-527, MPLA, It may include, but is not limited to, one or more materials selected from the group consisting of PHAD, 3D-PHAD, GLA, and combinations thereof.
  • the toll-like agonist may include a TLR-5 agonist, for example, may include, but is not limited to, flagellin or a fragment thereof. .
  • the toll-like agonist may be one comprising a TLR-7 agonist or a TLR-8 agonist, for example, such as imiquimod, R837, resquimod, or R848.
  • Imidazoquinoline molecules VTX-2337; CRX642; Imidazoquinoline covalently bound to a phospholipid group or a phosphonolipid group; And one or more materials selected from the group consisting of combinations thereof, but may not be limited thereto.
  • the toll-like agonist may include a TLR-9 agonist, for example, may include an immunostimulatory oligonucleotide, but may not be limited thereto.
  • the immunostimulatory oligonucleotide may include one or more CpG motifs, but may not be limited thereto.
  • the saponin may be selected from the group consisting of QS21, Quil A, QS7, QS17, ⁇ -eskin, Digitonin, and combinations thereof, but may not be limited thereto.
  • the antiviral peptide may include KLK, but may not be limited thereto.
  • the influmersome inducer may be TDB (trehalose-6,6-dibehenate), but may not be limited thereto.
  • the NOD ligand may be M-TriLYS (NOD2 agonist-synthetic muramyl tripeptide) or NOD2 agonist (N-glycolylated muramyldipeptid), but may not be limited thereto.
  • the CDS ligand may be Poly (dA: dT), but may not be limited thereto.
  • the STING ligand may be cGAMP, di-AMP, or di-GMP, but may not be limited thereto.
  • the immunoactive material is Pam3Cys-Lip, Poly (I: C), CRX-527, MPLA (monophosphoryl lipid A), flagellin (flagellin), imiquimod, resqui Mode, CpG, QS21, M-TriLys (MurNAc-Ala-D-isoGln-Lys), trehalose-6,6-dibehenate (TDB), 8837, Poly (dA: dT), cGAMP, and combinations thereof
  • the MPLA is derived from Gram-negative bacteria, lipopolysaccharide (LPS), and is a cell signal transduction system, through toll-like receptor 4 (TLR4).
  • TLR4 signaling stimulates the secretion of proinflammatory cytokines that activate the immune response, which stimulates the proliferation, differentiation and activity of T and B cells.
  • activated T cells promote differentiation into cytotoxic T cells, Secretion of cytokines such as interferon-gamma is involved in cellular immune responses.
  • the immunoactive material including a functional group, such as a carboxyl group, an amine group, a thiol group, an aldehyde group, a biotin group
  • substances responsible for various functions may be conjugated (conjugated).
  • the cationic lipid in the immunoactive substance, the other immunoactive substance having a negative charge or biomaterials such as DNA, RNA may be effectively loaded into the reconstituted artificial cancer cells.
  • antigen cross-presentation is induced by the cationic lipid
  • the cancer antigen can be delivered into the cell.
  • antigen cross-presentation is induced, and the cancer antigen can be delivered into the cell, thereby exhibiting an anticancer effect, but not limited thereto. It may not be.
  • the reconstituted artificial cancer cells have a nano size or a diameter size of angstroms, the cancer antigen and / or other immunoactive substances caused by pagocytosis or endocytosis Intracellular delivery ability of can be improved.
  • the reconstituted artificial cancer cells include an immunoactive material of cationic lipids
  • the cationic lipids induce an electrostatic attraction between the reconstituted artificial cancer cells and the anionic cell membrane, the cancer Intracellular delivery of antigens and / or other immunoactive substances may be further enhanced.
  • the cationic lipid is DC-cholesterol (3 ⁇ - [N- (N '(N', N'-dimethylaminoethane) -carbamoyl] cholesterol hydrochloride), DDA (dimethyldioctadecylammonium), DOTAP (1,2-dioleoyl -3-trimethylammoniumpropane), DOTMA (1,2-di-O-octadecenyl-3-trimethylammonium propane), EPC (1,2-dimyristoleoyl-sn-glycero-3-ethylphosphocholine), MVL5 (N1- [2-(( 1S) -1-[(3-aminopropyl) amino] -4- [di (3-amino-propyl) amino] butylcarboxamido) ethyl] -3,4-di [oleyloxy] -benzamide), DODAP (lipids), lipids, lipid
  • the lipid that forms the outer wall of the reconstituted artificial cancer cells is phosphatidylglycerols, phosphatidylcholines, phosphatidylethanolamines, phosphatidylserine, phosphatidyl inositols, egg yolk lecithin, soybean lecithin, dioleoylphosphatidyl phospho Choline, dioleoyl phosphatidylethanolamine, 2,3-dioleoyloxy-N- [2 (sperminecarboxamido) ethyl] -N, N-dimethyl-1-propaneaminium trifluoroacetate, N, N , N, N, N-tetramethyl-N, N, N, N-tetrapalmitylspermine, 1,2-dimyristyloxypropyl-3-dimethyl-hydroxyethylammonium bromide, 3 ⁇ - [N- (N ′, N'
  • the cancer cells may include cancer cells of lung, colon, central nervous system, skin, ovary, kidney, breast, stomach, or colon, but may not be limited thereto.
  • the cancer cells may be cancer cells of the skin, for example, may include melanoma-derived cancer cells, or melanoma_B16-F10, but may not be limited thereto.
  • the lysate of the cancer cell may be one comprising a membrane or cytoplasmic protein, for example, the membrane or cytoplasmic protein, for example, recombinant protein, membrane or cytoplasmic protein, subunit, It may include, but is not limited to, split protein antigens.
  • a method for producing reconstituted artificial cancer cells comprising: dissolving a lipid and an immunoactive substance that forms an outer wall of reconstituted artificial cancer cells in a first solvent; Evaporating the first solvent to form a lipid membrane comprising an immunoactive material; Dissolving said lipid membrane in a second solvent to form a first solution; And mixing and sonicating lysate of cancer cells in the first solution to provide reconstituted artificial cancer cells.
  • the sonication plays an important role in uniformly distributing the membranes and cytoplasmic proteins separated from the lysate of cancer cells on the outer wall of the lipid layer having amphiphilic properties, and can also increase the efficiency encapsulated therein.
  • the reconstituted artificial cancer cells lipids forming the outer wall of the reconstituted artificial cancer cells; Immunoactive substances; And lysates of cancer cells, wherein the lipids forming the outer wall of the reconstituted artificial cancer cells form the outer wall of the round or oval reconstituted artificial cancer cells, wherein the immunoactive substance and the lysate of the cancer cells are each independently. It may be present in the outer wall of the reconstituted artificial cancer cells and / or inside the reconstituted artificial cancer cells.
  • the reconstituted artificial cancer cells include lipids forming a circular or oval outer wall, and the membrane or cytoplasmic protein and immunity separated from the lysate of cancer cells in the outer wall and / or inside the reconstituted artificial cancer cells It may be a liposome structure named "tumosome", which contains the active substance.
  • the first solvent may be an organic solvent and a C1 to C6 alcohol.
  • the organic solvent may be a nonpolar solvent having a boiling point of about 40 ° C. to about 110 ° C., and may include a hydrocarbon, a halogen hydrocarbon, or an aromatic hydrocarbon.
  • the alcohol may be ethanol, and lipids forming the outer wall of the reconstituted artificial cancer cells may be dissolved in the ethanol to form a lipid membrane.
  • the second solvent may be selected from the group consisting of phosphate buffer solution (PBS), phosphate buffer, distilled water, and combinations thereof.
  • PBS phosphate buffer solution
  • phosphate buffer phosphate buffer
  • distilled water distilled water
  • the lysate of the cancer cell; Lipids forming an outer wall of the reconstituted artificial cancer cell; And the immunoactive material may be mixed in a weight ratio (or volume ratio) of about 0.01 to 10: 0.01 to 10: 0.1 to 100, but may not be limited thereto.
  • lysate of the cancer cells; Lipids forming the outer wall; And the immunoactive substance is about 0.01: 0.01: 0.1, about 0.01: 0.01: 100, about 0.01: 10: 0.1, about 0.01: 10: 100, about 0.01: 0.01: 0.1, about 10: 0.01: 0.1, about 10: 0.01: 100, about 10: 10: 0.1, about 10: 10: 100, or about 10: 0.01: 0.1, but may be mixed in a weight ratio (or volume ratio), but may not be limited thereto.
  • the reconstituted artificial cancer cells include lipids forming a circular or oval outer wall, and the membrane or cytoplasm separated from the lysate of cancer cells, each independently of the outer wall and / or inside the reconstituted artificial cancer cells Liposome structures, including proteins and immunoactive substances.
  • the reconstituted artificial cancer cells may be named "tumosome" (tumosome).
  • the reconstituted artificial cancer cell is a reconstituted liposome form formed by hybridizing a lysate of cancer cells, which are cancer cell-derived proteins having cancer cell information, and an immunoactive substance having an immune cell activating function (eg, a pathogen-derived cell membrane component, etc.).
  • a novel form comprising a skeleton-shaped outer wall formed by lipids forming the outer wall of the reconstituted artificial cancer cell, and a phospholipid-binding membrane protein present in the outer wall of the cancer cell and a cytoplasmic protein contained within the cytoplasm of the cancer cell. It may mean a liposome having a hybridized structure of.
  • the phospholipid-binding cancer cell membrane protein and the protein present in the cancer cell cytoplasm may refer to membrane or cytoplasmic proteins isolated from the lysate of the cancer cell, that is, a membrane or an antigen based on the lysate of the cancer cell.
  • the cytoplasmic protein and the immunoactive substance may be reconstitution with lipids forming the outer wall of the reconstituted artificial cancer cells.
  • the cancer cells may be lysed to use a substance having various cancer cell-related information present in the cancer cell membrane and cytoplasm as a cancer antigen.
  • the cancer cells may be obtained using cancer cell lines, or may be separated from cancer tissues present in the body.
  • the actual cancer tissue may be prepared by lysing cancer cells after inducing the production of proteins related to intracellular stress by applying an anticancer agent or radiation.
  • various hydrophilic and / or lipophilic immunoactive substances may be loaded by the liposome structure of the complex liposome.
  • the immunoactive substance may be an immunoactive substance expressed in stressed cancer cells, for example, a heat-shock protein, or a substance inducing T cell activation. Can be.
  • the reconstituted artificial cancer cells may have a diameter in the range of about 300 mm 3 to about 5,000 mm 3.
  • This size is a condition optimized for recognizing reconstituted artificial cancer cells produced by the immune cells in the body as real cancer cells and effectively recognizing cancer antigens through intracellular uptake, for example, about 300 mm 3 to about 4,000 ⁇ , about 300 ⁇ to about 3,000 ⁇ , about 300 ⁇ to about 2,000 ⁇ , about 300 ⁇ to about 1,000 ⁇ , about 300 ⁇ to about 500 ⁇ , about 500 ⁇ to about 5,000 ⁇ , about 500 ⁇ to about 4,000 ⁇ From about 500 kPa to about 3,000 kPa, from about 500 kPa to about 2,000 kPa, from about 500 kPa to about 1,000 kPa, from about 1,000 kPa to about 5,000 kPa, from about 1,000 kPa to about 4,000 kPa, about 1,000 kPa to about 4,000 kPa, about 1,000 kPa to about 4,000
  • the intracellular delivery capacity of the cancer antigen and / or other surface active material by pagocytosis or endocytosis can be improved.
  • the immunoactive substance is a toll-like receptor agonist (TLR), saponin, antiviral peptide, inflammasome inducer, NOD ligand (NOD) ligand), CDS ligand (cytosolic DNA sensor ligand), STING (stimulator of interferon genes) ligand, cationic lipids (cationic lipids), and combinations thereof, but may include a material selected from the group It may not be.
  • TLR toll-like receptor agonist
  • saponin saponin
  • antiviral peptide antiviral peptide
  • inflammasome inducer NOD ligand (NOD) ligand
  • CDS ligand cytosolic DNA sensor ligand
  • STING stirr of interferon genes
  • cationic lipids cationic lipids
  • combinations thereof but may include a material selected from the group It may not be.
  • the toll-like receptor agonist may be a natural toll-like receptor agonist or a synthetic toll-like receptor agonist.
  • the immunoactivator may comprise a combination of one or more toll-like receptor agonists, for example, CL401 (dual TLR2 and TLR7 agonists) or CL429 (dual TLR2 and NOD2 agonist), but may not be limited thereto.
  • CL401 dual TLR2 and TLR7 agonists
  • CL429 dual TLR2 and NOD2 agonist
  • the toll-like receptor agonist may be one that can cause a signaling response through TLR-1, for example, tri-acylated lipopeptide (LP); Phenol-soluble modulins; Cobacterium tuberculosis (Mycobacterium tuberculosis) lipopeptide; S- (2,3-bis (palmitoyloxy)-(2-RS) -propyl) -N-palmitoyl- (R) -Cys- (S) -Ser- (S) -Lys (4) -OH ; Bacterial lipopeptides from Borrelia burgdorfei; Trihydrochloride (Pam3Cys) lipopeptides that mimic the acetylated amino termini of OspA lipopeptides; And one or more materials selected from the group consisting of combinations thereof, but may not be limited thereto.
  • LP tri-acylated lipopeptide
  • Phenol-soluble modulins Cobacterium tub
  • the toll-like receptor agonist may include a TLR-2 agonist, for example, may include Pam3Cys-Lip, but may not be limited thereto.
  • the toll-like agonist may include a TLR-3 agonist, for example, Poly (I: C), Poly (ICLC), Poly (IC12U ), But may include, but are not limited to, Ampligen.
  • the toll-like agonist may be one comprising a TLR-4 agonist, for example, Shigella flexineri outer membrane protein preparation, AGP, CRX-527, MPLA, It may include, but is not limited to, one or more materials selected from the group consisting of PHAD, 3D-PHAD, GLA, and combinations thereof.
  • the toll-like agonist may include a TLR-5 agonist, for example, may include, but is not limited to, flagellin or a fragment thereof. .
  • the toll-like agonist may be one comprising a TLR-7 agonist or a TLR-8 agonist, for example, such as imiquimod, R837, resquimod, or R848.
  • Imidazoquinoline molecules VTX-2337; CRX642; Imidazoquinoline covalently bound to a phospholipid group or a phosphonolipid group; And one or more materials selected from the group consisting of combinations thereof, but may not be limited thereto.
  • the toll-like agonist may include a TLR-9 agonist, for example, may include an immunostimulatory oligonucleotide, but may not be limited thereto.
  • the immunostimulatory oligonucleotide may include one or more CpG motifs, but may not be limited thereto.
  • the saponin may be selected from the group consisting of QS21, Quil A, QS7, QS17, ⁇ -eskin, Digitonin, and combinations thereof, but may not be limited thereto.
  • the antiviral peptide may include KLK, but may not be limited thereto.
  • the influmersome inducer may be TDB (trehalose-6,6-dibehenate), but may not be limited thereto.
  • the NOD ligand may be M-TriLYS (NOD2 agonist-synthetic muramyl tripeptide) or NOD2 agonist (N-glycolylated muramyldipeptid), but may not be limited thereto.
  • the CDS ligand may be Poly (dA: dT), but may not be limited thereto.
  • the STING ligand may be cGAMP, di-AMP, or di-GMP, but may not be limited thereto.
  • the immunoactive material is Pam3Cys-Lip, Poly (I: C), CRX-527, MPLA (monophosphoryl lipid A), flagellin (flagellin), imiquimod, resqui Mode, CpG, QS21, M-TriLys (MurNAc-Ala-D-isoGln-Lys), trehalose-6,6-dibehenate (TDB), 8837, Poly (dA: dT), cGAMP, and combinations thereof It may include, but is not limited to, one or more materials selected from the group
  • the MPLA is derived from Gram-negative bacteria, lipopolysaccharide (LPS), and signals through Toll-like receptor 4 (TLR4), a cellular signaling system. Stimulated signaling of TLR4 leads to the secretion of proinflammatory cytokines (activating immune responses), which act to stimulate the proliferation, differentiation and activity of T cells and B cells. Activated T cells thus promote differentiation into cytotoxic T cells and are involved in cellular immune responses due to the secretion of cytokines such as interferon-gamma.
  • cytokines activating immune responses
  • the immunoactive material including a functional group, such as a carboxyl group, an amine group, a thiol group, an aldehyde group, a biotin group
  • substances responsible for various functions may be conjugated (conjugated).
  • the cationic lipid in the immunoactive substance, the other immunoactive substance having a negative charge or biomaterials such as DNA, RNA may be effectively loaded into the reconstituted artificial cancer cells.
  • antigen cross-presentation is induced by the cationic lipid
  • the cancer antigen can be delivered into the cell.
  • antigen cross-presentation is induced, and the cancer antigen can be delivered into the cell, thereby exhibiting an anticancer effect, but not limited thereto. It may not be.
  • the reconstituted artificial cancer cells have a nano size or a diameter size of angstroms, the cancer antigen and / or other immunoactive substances caused by pagocytosis or endocytosis Intracellular delivery ability of can be improved.
  • the reconstituted artificial cancer cells include an immunoactive material of cationic lipids
  • the cationic lipids induce an electrostatic attraction between the reconstituted artificial cancer cells and the anionic cell membrane, the cancer Intracellular delivery of antigens and / or other immunoactive substances may be further enhanced.
  • the cationic lipid is DC-cholesterol (3 ⁇ - [N- (N '(N', N'-dimethylaminoethane) -carbamoyl] cholesterol hydrochloride), DDA (dimethyldioctadecylammonium), DOTAP (1,2-dioleoyl -3-trimethylammoniumpropane), DOTMA (1,2-di-O-octadecenyl-3-trimethylammonium propane), EPC (1,2-dimyristoleoyl-sn-glycero-3-ethylphosphocholine), MVL5 (N1- [2-(( 1S) -1-[(3-aminopropyl) amino] -4- [di (3-amino-propyl) amino] butylcarboxamido) ethyl] -3,4-di [oleyloxy] -benzamide), DODAP (lipids), lipids, lipid
  • the lipid that forms the outer wall of the reconstituted artificial cancer cells is glycolipid trehalose phosphatidylglycerols, phosphatidylcholine, phosphatidylethanolamines, phosphatidylserine, phosphatidyl inositols, egg yolk lecithin, soybean lecithin, Dioleoyl phosphatidyl phosphocholine, dioleoyl phosphatidylethanolamine, 2,3-dioleyloxy-N- [2 (sperminecarboxamido) ethyl] -N, N-dimethyl-1-propaneamitrifluoro Roacetate, N, N, N, N-tetramethyl-N, N, N, N-tetrapalmitylspermine, 1,2-dimyristyloxypropyl-3-dimethyl-hydroxyethylammonium bromide, 3 ⁇ - [ N- (N ') phosphatidyl
  • the cancer cells may include cancer cells of lung, colon, central nervous system, skin, ovary, kidney, breast, stomach, or colon, but may not be limited thereto.
  • the cancer cells may be cancer cells of the skin, for example, may include melanoma-derived cancer cells, or melanoma_B16-F10, but may not be limited thereto.
  • the lysate of the cancer cell may be one comprising a membrane or cytoplasmic protein, for example, the membrane or cytoplasmic protein, for example, recombinant protein, membrane or cytoplasmic protein, subunit, It may include, but is not limited to, split protein antigens.
  • the effective intracellular delivery of tumor antigens using reconstituted artificial cancer cells activation of immune cells, efficacy and prevention of anticancer treatment, and activation of cytotoxic T cells were verified through small animal models.
  • Mouse-derived melanoma melanoma (melanoma_B16-F10) cells were cultured in DMEM medium (Dulbecco's modified eagle's medium) containing fetal bovine serum (10% FBS) and antibiotics (50 U / ml penicillin and 50 ug / ml streptomycin). And maintained in a 37 ° C. incubator with 5% carbon dioxide (CO 2 ). For cell membrane induction, 1.5 x 10 8 cells were harvested from the culture vessels using trypsin-EDTA. Harvested cells were centrifuged at 500 xg and washed three times with phosphate buffered saline (PBS).
  • PBS phosphate buffered saline
  • Cells suspended in cell lysis buffer were lysed 20 times using a homogenizer and pestle before centrifugation at 900 xg for 5 minutes.
  • the pellet was resuspended in the cell lysis buffer, and the above procedure was repeated: the supernatant collected through two repetitions was centrifuged at 10,000 x g for 20 minutes, then the pellet was discarded, and only the supernatant was again 30 minutes.
  • Figure 1 shows a tumosome production method and a model diagram of the present application.
  • 1,2-Dioleyl-sn-glycero-3-phosphocholine (1,2-dioleoyl-sn-glycero-3-phosphocholine, DOPC, 2 mg / mL, Sigma-Aldrich, USA), dimethyldiooctadecylammonium Bromide (dimethyldioctadecylammonium bromide, DDA, 2 mg / mL, Sigma Aldrich, USA), Cholesterol (cholesterol 0.5 mg / mL, Sigma Aldrich, USA), Monophosphoryl lipid A, MPLA, 0.2 mg / mL, Avan Avanti Polar Lipids, USA], 1 mL of ethanol.
  • 1,2-Dioleyl-sn-glycero-3-phosphocholine (1,2-dioleoyl-sn-glycero-3-phosphocholine,
  • phosphate buffer solution PBS, 0.0067M PO 4
  • the cancer antigen protein melanoma_B16-F10 tumor-associated antigen protein, 0.4 mg / mL
  • Example 1 2 mL of phosphate buffer solution (PBS, 0.0067M PO 4 ) containing the cancer antigen protein (melanoma_B16-F10 tumor-associated antigen protein, 0.4 mg / mL) isolated in Example 1 was added, followed by a stirrer.
  • the lipid membrane was dispersed in the solution at 600 rpm for 30 minutes at 60 °C.
  • the dispersed solution was transferred to a 4 mL vial and released using a ultrasonic tipsonicator to completely disperse the lipid membrane in phosphate buffer solution (PBS) for 1 minute.
  • PBS phosphate buffer solution
  • Figure 2 (a) and (b) is an image showing the structure of the manufactured tumosome, the structure of the tumosome is DLS (dynamic light scattering, Otsuga, Japan) and TEM (transmission electron microscope, high-resolution transmission electron microscopy). Through the image of the tumosome observed with a transmission electron microscope, it was confirmed that the spherical nano liposomes.
  • DLS dynamic Light Scattering, Otsuga, Japan
  • FT-IR infrared spectroscopy
  • amide I, II parts amide I: 1600-1690 cm -1 , Amide II: 1480-1575 cm -1
  • C O stretch and NH bending (bedding) confirmed that the tumor antigen protein obtained from the cancer cell lysate was successfully loaded in the tumosome.
  • DOPC (2 mg / mL, Sigma Aldrich, USA), Cholesterol (0.5 mg / mL, Sigma Aldrich, USA), MPLA [0.2 mg / mL, Avanti Polar Lipids, USA], QS21 (2 mg Desert King International, San Diego, USA) is dissolved in 1 mL of ethanol, the complete evaporation of ethanol using a rotary evaporator, a thin-film form is formed. Thereafter, tumosome-3 was prepared using the same method as in Example 2-1.
  • tumosome-4 was prepared using the same method as in Example 2-1.
  • tumosome-7 was prepared using the same method as in Example 2-1.
  • tumosome-8 was prepared using the same method as in Example 2-1.
  • DOPC 2 mg / mL, Sigma Aldrich, USA
  • MPLA 0.2 mg / mL, Avanti Polar Lipids, USA
  • DDA 2 mg / mL, Sigma Aldrich, USA
  • Cholesterol 0.5 mg / mL, Sigma Aldrich, USA
  • poly (I: C) (2 mg, Invivo Gen, San Diego, USA) was dissolved in 1 mL of ethanol, and then ethanol was completely removed using a rotary evaporator. Evaporation produces a thin-film form. Thereafter, tumosome-12 was prepared using the same method as in Example 2-1.
  • DOPC 2 mg / mL, Sigma Aldrich, USA
  • MPLA 0.2 mg / mL, Avanti Polar Lipids, USA
  • DDA (2 mg / mL, Sigma Aldrich, USA)
  • Cholesterol 0.5 mg / mL, Sigma-Aldrich, USA
  • CpG 2 mg, Invivo Gen, San Diego, USA
  • tumosome-13 was prepared using the same method as in Example 2-1.
  • the tumosome of Example 2 (tumosome-1 to tumosome-13) for 3 hours on bonemarrow-derived dendritic cells (BMDCs) )
  • BMDCs bonemarrow-derived dendritic cells
  • the activity of BMDCs by each of the tumosomes prepared in Example 2 was determined by the amount of proinflammatory cytokines (TNF- ⁇ , IL-6). It was analyzed using the ELISA test method.
  • MPLA monophosphoryl lipid A
  • the secretion of IL-6 which induces a Th1 response, increased with the concentration of the tumosome.
  • TNF- ⁇ which induces cell death and tumor suppression, was significantly increased after treatment of tumosomes with MPLA.
  • MPLA is known to induce dendritic cell maturation by stimulation of Toll-like-receptor 4.
  • Each tumosome prepared in Example 2 may be injected into the body in a number of ways.
  • 3 (a) to 3 (c) show an image guided surgery image using near infrared fluorescence.
  • Indocyanine green (ICG) can be used for cancer surgery because it can map the first lymph node, Sentinel lymph node (SLN), when tumors metastasize directly through lymph nodes. It is becoming.
  • SSN Sentinel lymph node
  • FIG. 3 first, after injection of ICG into mice, lymph nodes were visualized, and the prepared tumosome was subjected to image guide surgery using near infrared fluorescence. Co-localization was combined after infusion and confirmed by removing lymph nodes.
  • lymph nodes were removed to confirm the influx into cells through tissue fluorescence techniques.
  • tumosome FITC, antigen
  • DCs dendritic cells
  • mice were sacrificed by cervical dislocation and the abdominal cavity was dissected to extract the spleen.
  • Single cells were obtained from tissues using a wire-mesh, washed with PBS, and centrifuged (1500 rpm, 5 minutes).
  • the single cell suspension was a RPMI medium (Roswell Park Memorial Institute medium) containing 10% fetal bovine serum (FBS, fetal Bovine Serum, Sigma Aldrich), 100 U / ml penicillin, 100 ⁇ g / ml streptomycin (Life Technologies, USA). , Life Technology) was dispensed at 1 x 10 5 cells / well. Stimulation of cancer cell lysate was added to the culture, followed by incubation at 37 ° C.
  • T cell proliferation was significantly increased in the tumosome administration group compared to the control group.
  • secretion amount of IFN- ⁇ it was confirmed that the most secreted in the tumosome experimental group.
  • the tumosome prepared using the C57BL / 6 mouse melanoma model was administered by lymph node (intranodal injection) to confirm tumor healing ability.
  • 1 x 10 5 melanoma cells (B16F10) were injected subcutaneously into the right thigh of five female 6-week-old mice, and from the next day, the tumosome was injected into the inguinal lymph node three times every 10 ⁇ l. Injected, PBS was injected into the control. Then, the tumor volume was measured 2-3 times a week for 21 days after cancer cell administration.
  • the tumosome was administered after the cancer cell injection, it was observed that the proliferation of tumor cells was significantly suppressed compared to the control group. In addition, it was confirmed that it is effective in the survival rate.
  • the tumosome prepared in the mouse model was first administered and induced melanoma, and the anticancer effect on melanoma was analyzed.
  • 10 ⁇ l of tumosome was injected into both female furrow lymph nodes of 5 females of C57BL / 6 6-week-old mice, and PBS was injected into the control group.
  • the injection was repeated in the same manner, and after one week, subcutaneous injection of 1 ⁇ 10 5 melanoma cells (B16F10) was performed, followed by one week for 21 days from the day of tumor cell administration (day 0). Tumor volume was measured 2-3 times each.
  • the administration of tumosome showed a cancer prevention effect that significantly suppressed the production and proliferation of tumor cells compared to the control group.
  • the proliferation of tumor cells was significantly suppressed compared to the control group.
  • the survival rate was also confirmed to be effective.
  • Example 2 The in vivo T cell activity characteristics of the tumosome recombined with various immunoactivating substances and lipid compositions prepared in Example 2 were confirmed through animal experiments. Experiment was carried out in the same manner as in Example 7 and confirmed the secretion amount of IFN- ⁇ by various tumosomes (Fig. 5). In the tumosome group consisting of three component-based substances such as MPLA / DDA / CpG or MPLA / DDA / poly (I: C) rather than the tumosome consisting of two immunoactivating substances, T cell activation effect that can kill cancer cells It was found to increase.
  • tumosome group consisting of three component-based substances such as MPLA / DDA / CpG or MPLA / DDA / poly (I: C)
  • 6-week-old female C57BL / 6 mice (COREAC, South Korea) were injected subcutaneously with 5 x 10 5 melanoma cells (B16F10) in the right thigh, and after 2 weeks, when the tumors were about 12-13 mm in size, After removing the cancer cells by excision of the cancer tissue through surgery to separate the membrane or cytoplasmic protein in the same manner as in Example 1, it was used for further experiments.
  • FIG. 6 shows a method and a model diagram of a tumosome using a membrane or cytoplasmic protein of cancer cells obtained from the excised cancer tissue.
  • Tumosomes were prepared in the same manner as in Example 2, using the cancer cell membrane and cytoplasmic protein isolated in Example 11.
  • Example 13 Manufactured Using Resected Cancer Tissue Tumosome Test preparation to prevent cancer recurrence
  • FIG. 7 (a) shows that after administering the C57BL / 6 mouse melanoma model, the tumor was grown to less than 1% after the tumor was grown, and then a 7 mm sized clip was used. This is the image that was sealed.
  • Figure 7 (b) is an image showing the result confirmed after a week after the suture, the remaining tumor was grown again, it was confirmed that the tumor of the original size is produced again.
  • Example 14 Manufactured Using Resected Cancer Tissue Tumosome Intracellular Influence Effect Assessment
  • Figure 8 (a) is a graph showing the effect of influx in the cells evaluated by flow cytometry after treating the tumosome prepared in Example 12, dendritic cells (BMDCs) in the same manner as in Example 3. As shown in (a) of FIG. 8, it was confirmed that the influx of cells in BMDCs was 5.45 times higher than that of the tumosome prepared using the excised cancer tissues alone.
  • Figure 8 (b) is treated with the tumosome of Example 12 to dendritic cells (BMDCs) in the same manner as in Example 4, the amount of pro-inflammatory cytokines (TNF- ⁇ , IL-6) secreted EL It is analyzed using the ELISA test method.
  • TNF- ⁇ pro-inflammatory cytokines
  • MPLA is known to induce maturation of dendritic cells by stimulation of Toll-like-receptor 4.
  • results indicate that dendritic cell activity and maturity are higher when treated with tumosomes containing MPLA than with MPLA alone. This is due to the increased synergy-induced immune activity of both intracellular delivery efficiency and stimulation of Toll-like-receptor 4 by the tumosome hybridization structure.
  • Example 16 Manufactured using Resected Cancer Tissue On the tumosome Cellular maturity evaluation
  • FIG. 8 shows the results of labeling the concentrations of CD80, CD86 and CD40 expressed according to the degree of maturation of BMDCs after treating the tumosome of Example 12 to dendritic cells. As shown in (c) of FIG. 8, after the tumosome containing MPLA, the concentration was greatly increased. This is because the synergy induced by the synergy of both intracellular delivery efficiency and stimulation of Toll-like-receptor 4 is increased.
  • Example 17 Manufactured Using Resected Cancer Tissue On the tumosome Evaluation of efficacy of preventing cancer recurrence
  • the anti-cancer treatment efficacy evaluation of cancer recurrence was confirmed using the C57BL / 6 mouse melanoma model.
  • 5 x 10 5 mouse females were injected with 5 x 10 5 melanoma cells (B16F10) subcutaneously into the right thigh, and after 14 days, when the tumor size averaged 400 mm 3 , Example 13
  • the tumosome of Example 12 was injected three times at three days intervals and the control group was injected with PBS.
  • the red arrow represents the day of surgery
  • the blue arrow represents the day injected with tumosome.
  • Tumor volume was measured 2 to 3 times a week for 25 days after cancer cell administration. As a result, it was observed that in the experimental group to which the tumosome was administered after surgery, the proliferation of tumor cells was significantly suppressed as compared with the control group.
  • the present application relates to a reconstituted artificial cancer cell prepared to hybridize with a membrane and / or cytoplasmic protein of cancer cells and a lipid comprising an immunoactive material, wherein the reconstituted artificial cancer cells tumosomes are heterogeneity of antigens. While including all the information, it significantly increased the expression of cytokines associated with the activation of anticancer immune cells and showed excellent efficacy in the therapeutic effect of cancer.

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Abstract

La présente invention concerne : des cellules tumorales artificielles reconstituées présentant des activités immunitaires anticancéreuses, jouant un rôle très important dans les processus de prévention et de traitement du cancer ; un procédé de préparation des cellules tumorales artificielles reconstituées ; et une composition anticancéreuse contenant les cellules tumorales artificielles reconstituées.
PCT/KR2017/001558 2017-01-02 2017-02-13 Cellules tumorales artificielles reconstituées, leur procédé de préparation et composition anticancéreuse en contenant WO2018124378A1 (fr)

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WO2021101340A1 (fr) * 2019-11-22 2021-05-27 차의과학대학교 산학협력단 Composition d'aide à la thérapie anticancéreuse comprenant une composition de liposome et procédé d'administration de médicament l'utilisant

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