+

WO2009008666A2 - Cellule dendritique autologue médiatisée par une thérapie photodynamique ayant l'aptitude d'inhiber la croissance d'une tumeur - Google Patents

Cellule dendritique autologue médiatisée par une thérapie photodynamique ayant l'aptitude d'inhiber la croissance d'une tumeur Download PDF

Info

Publication number
WO2009008666A2
WO2009008666A2 PCT/KR2008/004042 KR2008004042W WO2009008666A2 WO 2009008666 A2 WO2009008666 A2 WO 2009008666A2 KR 2008004042 W KR2008004042 W KR 2008004042W WO 2009008666 A2 WO2009008666 A2 WO 2009008666A2
Authority
WO
WIPO (PCT)
Prior art keywords
cancer
cells
cancer cell
cell
dendritic cells
Prior art date
Application number
PCT/KR2008/004042
Other languages
English (en)
Other versions
WO2009008666A3 (fr
Inventor
Dae-Seog Lim
Ju-Ah Jeong
Hyun-Soo Lee
Yong-Soo Bae
Original Assignee
Creagene Inc.
Kang, Mi-Sun
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Creagene Inc., Kang, Mi-Sun filed Critical Creagene Inc.
Publication of WO2009008666A2 publication Critical patent/WO2009008666A2/fr
Publication of WO2009008666A3 publication Critical patent/WO2009008666A3/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/10—Cellular immunotherapy characterised by the cell type used
    • A61K40/19—Dendritic cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/20—Cellular immunotherapy characterised by the effect or the function of the cells
    • A61K40/24—Antigen-presenting cells [APC]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
    • A61K40/41—Vertebrate antigens
    • A61K40/42—Cancer antigens
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0639—Dendritic cells, e.g. Langherhans cells in the epidermis
    • 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
    • A61K2035/124—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells the cells being hematopoietic, bone marrow derived or blood cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K40/00
    • A61K2239/31—Indexing codes associated with cellular immunotherapy of group A61K40/00 characterized by the route of administration
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K40/00
    • A61K2239/38—Indexing codes associated with cellular immunotherapy of group A61K40/00 characterised by the dose, timing or administration schedule
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K40/00
    • A61K2239/46—Indexing codes associated with cellular immunotherapy of group A61K40/00 characterised by the cancer treated
    • A61K2239/49—Breast
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/20—Cytokines; Chemokines
    • C12N2501/22—Colony stimulating factors (G-CSF, GM-CSF)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/20—Cytokines; Chemokines
    • C12N2501/23—Interleukins [IL]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/20—Cytokines; Chemokines
    • C12N2501/24—Interferons [IFN]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/20—Cytokines; Chemokines
    • C12N2501/25—Tumour necrosing factors [TNF]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2502/00—Coculture with; Conditioned medium produced by
    • C12N2502/30—Coculture with; Conditioned medium produced by tumour cells

Definitions

  • the present invention relates to mature dendritic cells (DCs) having the ability to induce anti-cancer immune responses, and to a method for the preparation of the above-mentioned DCs through the procedure of pulsing immature DCs with lysates of cancer cells on which photodynamic therapy (PDT) has been conducted.
  • the present invention relates to cancer cell lysates for the use of inducing the maturation of immature DCs to mature DCs having the ability to inhibit the growth of cancer cells, and to the preparation method thereof.
  • Photodynamic therapy is a treatment that is based upon the differential uptake by cancerous cells of photosensitizing agents, followed by irradiation of the cells to cause a photochemical reaction that is believed to generate chemically disruptive species, such as singlet oxygen. These disruptive species in turn injure the cells through reaction with cell parts, such as cellular and nuclear membranes (U.S. Pat. No. 5,211,938).
  • PDT may play a role in the induction of immune responses in the subject. It is also suggested that if cancer cells are treated with PDT, pro-inflammatory cytokines and heat shock proteins are produced in around cancer cells and change the environment of cancer cells to increase the immunogenicity.
  • TNF tumor necrosis factor
  • IL interleukin
  • HSP heat shock protein
  • the immune responses of T cell are initiated by the effect of mature dendritic cells on naive T cell. Immature dendritic cells exert phagocytosis but cannot stimulate T cells. Accordingly, the maturation of dendritic cells is a key step in the induction of immune responses.
  • the maturation of dendritic cells comprises the steps of modification of dendritic cells to more effective antigen presenting cells, migration of dendritic cells to the regional lymph node and activation of T cells. It is also known that certain heat shock proteins play important roles in the initiation of immune responses of T cells and are evaluated as an effective vaccine adjuvant in the cancer vaccination.
  • the present inventors have discovered that where immature dendritic cells are matured by pulsing with lysates prepared from cancer cells on which photodynamic therapy has been conducted, the ability to induce anti-cancer immune responses of the matured dendritic cells has been significantly increased.
  • It is still another object of this invention to provide a method for treating cancer comprising administering to a subject suffering from cancer a pharmaceutical composition comprising mature dendritic cells that have a significantly improved ability to induce anti-cancer immune responses.
  • a method for preparing cancer cell lysates for pulsing dendritic cells which comprises the steps of: (a) culturing cancer cells in the presence of a photosensitizing agent; (b) activating the photosensitizing agent bound to the cancer cells by irradiating the cultured cancer cell with a light; and (c) obtaining cancer cell lysates from the cultured cancer cells.
  • the present inventors have discovered that where immature dendritic cells are matured by pulsing with lysates prepared from cancer cells on which photodynamic therapy has been conducted, the ability to induce anti-cancer immune responses of the matured dendritic cells has been significantly enhanced.
  • the first step of this invention is culturing cancer cells in the presence of a photosensitizing agent.
  • cancer cell used herein means a type of cell that has acquired an immortality to proliferate without any restriction. Cancer cell includes any type of cancer cell not limiting to the specific type of cancer cells. According to a preferred embodiment of this invention, the cancer cell is a renal cancer cell or breast cancer cell.
  • photosensitizing agent refers to a chemical compound that is able to bind to target cells, and when exposed to light of an appropriate wavelength, absorb the light, causing substance to be produced that impair or destroy the target cells.
  • a photosensitizing agent that may be used in the present invention may be found in Kreimer-Bimbaum, Seminars in Hematology (1989) 26(2):157-73[8].
  • Phtosensitizing agents of this invention includes, but are not limited to, chlorines, bacteriochlorins, phthalocyanines, porphyrins, purpurins, merocyanines, psoralens, benzoporphyrin derivatives (BPD), and porfimer sodium and pro-drugs such as delta-aminolevulinic acid, which can produce photosensitive agents such as protoporphyrin IX.
  • Other suitable photosensitive compounds include ICG, methylene blue, toluidine blue, texaphyrins and any other agent that absorbs light in a range of 500 nm - 1100 nm.
  • the photosensitizing agent is porphyrins.
  • a medium useful in the procedure of culturing cancer cells includes any conventional medium for culturing animal cells, preferably, a medium containing serum (e.g., fetal bovine serum, horse serum and human serum).
  • the medium used in this invention includes, for example, RPMI series (e.g., RPMI 1640), Eagles's MEM (Eagle's minimum essential medium, Eagle, H. Science 130:432(1959)), ⁇ -MEM (Stanner, CP. et al., Nat. New Biol. 230:52(1971)), Iscove's MEM (Iscove, N. et al., J. Exp. Med.
  • the medium may contain other components, for example, antioxidant (e.g., ⁇ -mercaptoethanol).
  • antioxidant e.g., ⁇ -mercaptoethanol
  • the culture medium used for culturing cancer cells is RPMI-1640 or DMEM.
  • the time for culturing cancer cell in the presence of a photosensitizing agent may be one sufficient for a photosensitizing agent to bind to cancer cell not being limited to the specific range.
  • the photosensitizing agent bound to cancer cells is activated by proper irradiation of a light.
  • the amount of energy of activating irradiation used in this invention may be regulated by selecting the appropriate wavelength, power, power density, energy density, and time of application.
  • the wavelength of the radiation can be any wavelength absorbed by the photosensitizing agent, or any other wavelength that mediates the desired biological response in the target cancer cells.
  • the source of the light that can be used in the present method may be any light source which is able to produce wavelength that can activate the photosensitizing agent.
  • the source of light in this invention includes laser, lamp, optoelectric magnetic device, diode, and diode-laser, but not limited to.
  • the source of the light energy is a laser or halogen lamp.
  • cancer cell lysates are prepared from the irradiated cultured cancer cells.
  • cancer cell lysates used herein means cancer cell lysis products that comprise any proteins from cancer cells including cancer antigens.
  • heat shock proteins may be contained in the cancer cell lysates.
  • the cancer cell lysates are obtained through the steps of collecting the irradiated cultured media containing cancer cells by using scrapper, centrifuging the collected media, and recovering the supernatant of them.
  • PDT photodynamic therapy
  • cancer cell lysates prepared by PDT treatment or "PDT- prepared cancer cell lysates” used herein refers to cancer cell lysates that are prepared through the procedure of the above described steps (a), (b) and (c).
  • a cancer cell lysates for pulsing dendritic cells prepared through the procedure of the steps (a), (b) and (c).
  • the cancer cell lysates contains heat shock proteins.
  • HSPs heat shock proteins
  • the term "heat shock proteins (HSPs)" used herein means a protein whose intracellular concentration increases when a cell is exposed to a stressful stimulus such as heat. It is capable of binding other proteins or peptides, and releasing the bound proteins or peptides in the presence of adenosine triphosphate (ATP) of low pH.
  • ATP adenosine triphosphate
  • Three major families of HSPs have been identified based on molecular weight. The families have been called Hsp 60, Hsp 70 and Hsp 90, and where the numbers reflect the approximate molecular weight of the stress proteins in kilodaltons.
  • the major HSPs can accumulate to very high levels in stressed cells, but they occur at low to moderate levels in cells that have not been stressed.
  • the highly inducible mammalian hsp70 is hardly detectable at normal temperatures but becomes one of the most actively synthesized proteins in the cell upon heat shock (Welch, et al., 1985, J. Cell. Biol. 101:1198-1211).
  • HSPs appear to induce an inflammatory reaction at the tumor site and ultimately cause a regression of the tumor burden in the cancer patients treated.
  • the cancer cell lysates prepared through PDT treatment for pulsing dendritic cells comprises heat shock protein 70 (Hsp 70).
  • Hsp 70 heat shock protein 70
  • a composition for inducing the maturation of immature dendritic cells comprising the cancer cell lysates prepared by the method of this invention as an active ingredient.
  • the expression "inducing the maturation of immature dendritic cells” refers to an induction of the maturation of immature dendritic cells to mature dendritic cells by contacting the immature dendritic cells with the specific antigens.
  • the resulting matured dendritic cells When immature dendritic cells are matured through a pulsing with cancer cell lysates that have been prepared by the method of this invention, the resulting matured dendritic cells have a remarkably increased activity to suppress the growth of tumor via the process of inducing immune responses against tumor.
  • a method for preparing mature dendritic cells having the ability to inhibit the growth of tumor which comprises the steps of; (a) culturing cancer cells in the presence of a photosensitizing agent; (b) activating the photosensitizing agent bound to the cancer cells by irradiating the cultured cancer cells with a light; (c) obtaining cancer cell lysates from the cultured cancer cells; and (d) inducing the maturation of immature dendritic cells by pulsing with the obtained cancer cell lysates.
  • step (d) of the method of this invention immature dendritic cells are matured by pulsing with the antigen of PDT-prepared cancer cell lysates, which have been prepared through the procedure of the steps (a) to (c).
  • PDT-mediated dendritic cells refers to dendritic cells matured by pulsing immature dendritic cells with PDT-prepared cancer cell antigens which have been prepared via the steps of (a) to (c).
  • the term "pulsing" used herein means contacting immature dendritic cells with the specific antigen.
  • dendritic cells When dendritic cells are contacted with antigens, they engulf and process the antigens, then present the processed antigens with MHC molecules on their surface to T cells. T cells that have been contacted with dendritic cells which present the processed specific antigens become to acquire the capability to induce immune responses to the specific antigens.
  • immature dendritic cells are matured by pulsing with the antigens in the PDT-prepared cell lysates of the specific cancer, the resulting matured dendritic cells have the increased ability to suppress the growth of tumor, since they have a remarkably enhanced potential to induce immune responses to the specific cancer cell.
  • the pulsing in the present invention may be carried out by contacting dendritic cells with the specific antigens for a sufficient time for the specific antigens in the cancer cell lysates to be presented on the surface of dendritic cells.
  • the contacting is co-culturing.
  • dendritic cells are pulsed with cancer cell lysates by co-culturing immature dendritic cells with PDT-prepared cancer ceil lysates.
  • dendritic cells refers to professional antigen-presenting cells that can internalize antigen and process the antigen such that the antigen or peptide derived from the antigen is presented in the context of both MHC (major histocompatibility complex) class I complex and the MHC class II complexes. Dendritic cells may be classified as immature or fully mature dendritic cells.
  • imDCs implant dendritic cells
  • CD83 and CD14 express low levels of CCR7 and the cytosolic protein DC-LAMP, and low levels of the costimulatory molecules CD40, CD80 and CD 86, and usually express CDIa and CCRl, CCR2, CCR5 and CXCRl.
  • mDCs mature dendritic cells
  • mDCs mature dendritic cells
  • Mature DCs typically express high levels of CCR7, and CXCR4 and low levels of CCRl and CCR5.
  • Suitable source for isolating immature dendritic cells is tissue that contains immature dendritic cells or their progenitors, and specifically include spleen, afferent lymph, bone marrow, blood, and cord blood, as well as blood cells elicited after administration of cytokines such as G-CSF or FLT-3 ligand.
  • a tissue source may be treated prior to culturing with substances that stimulate hematopoiesis, such as, for example, G-CSF, FLT-3, GM-CSF, M-CSF, TGF- ⁇ , and thrombopoietin in order to increase the proportion of dendritic cell precursors relative to other cell types.
  • tissue source may also remove cells which may compete with the proliferation of the dendritic cell precursors or inhibit their survival.
  • Pretreatment may also be used to make the tissue source more suitable for in vitro culture.
  • the method of treatment will likely depend on the particular tissue source. For example, spleen or bone marrow would first be treated so as to obtain single cells followed by suitable cell separation techniques to separate leukocytes from other cell types as described in U.S. Pat. Nos. 5,851,756 and 5,994,126 which are herein incorporated by references.
  • Treatment of blood would preferably involve cell separation techniques to separate leukocytes from other cell types including red blood cells (RBCs) which are toxic. Removal of RBCs may be accomplished by standard methods known in the art.
  • the tissue source is blood or bone marrow.
  • immature dendritic cells are derived from multipotent blood monocyte precursors (see WO 97/29182). These multipotent cells typically express CD14, CD32, CD68 and CDl 15 monocyte markers with little or no expression of CD83, or p55 or accessory molecules such as CD40 and CD86. When cultured in the presence of cytokines such as a combination of GM-CSF and IL-4 or IL-13 as described below, the multipotent cells give rise to the immature dendritic cells.
  • the immature dendritic cells can be modified, for example using vectors expressing IL-IO to keep them in an immature state in vitro or in vivo.
  • Cells obtained from the appropriate tissue source are cultured to form a primary culture, preferably, on an appropriate substrate in a culture medium supplemented with granulocyte/macrophage colony-stimulating factor (GM-CSF), a substance which promotes the differentiation of pluripotent cells to immature dendritic cells as described in U.S. Pat. Nos. 5,851,756 and 5,994,126 which are herein incorporated by references.
  • the substrate would include any tissue compatible surface to which cells may adhere.
  • the substrate is commercial plastic treated for use in tissue culture.
  • GM-CSF may be added to the culture medium which block or inhibit proliferation of non-dendritic cell types.
  • factors which inhibit non- dendritic cell proliferation include interleukin-4 (IL-4) and/or interleukin-13 (IL-13), which are known to inhibit macrophage proliferation.
  • IL-4 interleukin-4
  • IL-13 interleukin-13
  • an enriched population of immature dendritic cells can be generated from blood monocyte precursors by plating mononuclear cells on plastic tissue culture plates and allowing them to adhere. The plastic adherent cells are then cultured in the presence of GM-CSF and IL-4 in order to expand the population of immature dendritic cells.
  • Other cytokines such as IL-13 may be employed instead of using IL-4.
  • a medium useful in the procedure of obtaining immature dendritic cells includes any conventional medium for culturing animal cells, preferably, a medium containing serum (e.g., fetal bovine serum, horse serum and human serum).
  • the medium used in this invention includes, for example, RPMI series (e.g., RPMI 1640), Eagles's MEM (Eagle's minimum essential medium, Eagle, H. Science 130:432(1959)), ⁇ -MEM (Stanner, CP. et al., Nat. New Biol. 230:52(1971)), Iscove's MEM (Iscove, N. et al., J. Exp. Med. 147:923(1978)), 199 medium (Morgan et al., Proc. Soc. Exp. Bio. Med. 73:1(1950)), CMRL 1066, RPMI 1640 (Moore et al., 1 Amer. Med. Assoc.
  • RPMI series e.g., RPMI 1640
  • Eagles's MEM Eagle's minimum essential medium, Eagle, H. Science 130:432(1959)
  • ⁇ -MEM Stanner, CP. et al
  • McCoy's 5A McCoy, T.A., et al., Proc. Soc. Exp. Biol. Med. 100:115(1959)
  • MCDB series Ham, R.G. et al., In Vitro 14:11(1978) but not limited to.
  • the medium may contain other components, for example, antioxidant (e.g., ⁇ -mercaptoethanol).
  • antioxidant e.g., ⁇ -mercaptoethanol
  • immature dendritic cells are cultured in the presence of TNF (tumor necrosis factor)- ⁇ , IFN- ⁇ and poly(I) • poly(C) in order to induce the maturation of immature dendritic cells.
  • TNF tumor necrosis factor
  • IFN- ⁇ IFN- ⁇
  • poly(I) • poly(C) in order to induce the maturation of immature dendritic cells.
  • Mature dendritic cells exhibit expression of surface markers of CD83, DC-
  • LAMP LAMP, p55, CCR-7, and have high expression level of MHC class II and costimulatory molecule of CD86.
  • Immature dendritic cells are identified based on typical morphologies, low expression level of MHC class II and costimulatory molecules, and non expression level of markers of mature dendritic cells (e.g., CD83, CD-LAMP).
  • markers of mature dendritic cells e.g., CD83, CD-LAMP.
  • positive markers for immature dendritic cells for example, DC-SIGN, Langerin and CDIa can be used.
  • Antibodies may also be used to isolate or purify immature dendritic cells from mixed cell cultures by flow cytometry or other cell sorting techniques well known in the art.
  • the cancer cell in the step (a) of the procedure for preparing mature dendritic cells, is a renal cancer cell or breast cancer cell.
  • the photosensitizing agent is selected from the group consisting of chlorins, bacteriochlorins, phthalocyanines, porphyrins, purpurins, merocyanines, psoralens, benzoporphyrin derivatives (BPD), and porfimer sodium.
  • the photosensitizing agent is porphyrins.
  • the source of a light is selected from the group consisting of laser, lamp, optoelectric magnetic device, diode, and diode-laser.
  • the source of a light is a laser or halogen lamp.
  • a mature dendritic cell which has been prepared according to the method of this invention such that the mature dendritic cell has the ability to induce anti-cancer immune responses.
  • the present mature dendritic cell which has been prepared by pulsing with PDT-prepared cancer cell lysates as an antigen, has an enhanced ability to induce immune responses to the cancer cell such that it is able to effectively suppress the growth of the cancer cell.
  • the mature dendritic cell of this invention has increased expression level of MHC class II.
  • the mature dendritic cell of the present invention has the capability to induce the proliferation of CD8 T cells and activates the cancer cell specific cytotoxic T lymphocytes, and thus exerts the ability to suppress the growth of cancer cells.
  • the dendritic cell of the instant invention has the activity to stimulate ThI immune responses and suppress Th2 immune responses.
  • the dendritic cell of this invention promotes the secretion of IFN- ⁇ (ThI cytokine) and reduces the secretion of IL-4 (Th2 cytokine) so that they increase the ratio of Thl/Th2.
  • the dendritic cell prepared by the method of the present invention induces immune responses in vivo.
  • a pharmaceutical composition for treating a cancer which comprises (a) a pharmaceutically effective amount of the mature dendritic cell of claim 16; and (b) a pharmaceutically acceptable carrier.
  • the dendritic cell of this invention prepared by pulsing with PDT- prepared cancer cell lysates has acquired a capability to induce the cancer cell specific immune responses such that it is able to suppress the growth of cancer cell, and it can be used as an anti-cancer agent.
  • the dendritic cell in the pharmaceutical composition has the increased expression level of MHC class II.
  • the dendritic cell in the pharmaceutical composition has the capability to induce the proliferation of CD8 T cells and stimulated the activity of the cancer cell specific CTL (cytotoxic T lymphocyte).
  • the dendritic cell in the pharmaceutical composition stimulates Th 1 immune responses and suppresses Th2 immune responses.
  • the mature dendritic cell used in the pharmaceutical composition is an autologous or syngeneic dendritic cell.
  • autologous dendritic cells are employed in the present invention. Since the pharmaceutical composition of this invention contains an autologous dendritic cell which has been derived from a subject, it has advantages of little elicitation of immune responses to the injected dendritic cells.
  • the cancer that is a therapeutic target of the pharmaceutical composition of this invention is renal cancer or breast cancer.
  • the pharmaceutically acceptable carrier may be conventional one for formulation, including lactose, dextrose, sucrose, sorbitol, mannitol, starch, rubber arable, potassium phosphate, arginate, gelatin, potassium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrups, methyl cellulose, methylhydroxy benzoate, propylhydroxy benzoate, talc, magnesium stearate, and mineral oils, but not limited to.
  • the pharmaceutical composition according to the present invention may further include a lubricant, a humectant, a sweetener, a flavoring agent, an emulsifier, a suspending agent, and a preservative. Details of suitable pharmaceutically acceptable carriers and formulations can be found in Remington's Pharmaceutical Sciences (19th ed., 1995), which is incorporated herein by reference.
  • the pharmaceutical composition according to the present invention may be administered via the oral or parenterally. It is preferably administered via intravenous (percutaneous) or subcutaneous route.
  • a suitable dose of the pharmaceutical composition of the present invention may vary depending on pharmaceutical formulation methods, administration methods, the patient's age, body weight, sex, severity of diseases, diet, administration time, administration route, an excretion rate and sensitivity for a used pharmaceutical composition.
  • the pharmaceutical composition of the present invention is administered with a daily dose of 0.001-100 mg/kg (body weight).
  • the pharmaceutical composition may be formulated with pharmaceutically acceptable carrier and/or vehicle as described above, finally providing several forms including a unit dose form and a multi-dose form.
  • a method for treating or preventing cancer which comprises administering to a subject suffering from cancer a pharmaceutical composition comprising (a) a pharmaceutically effective amount of the mature dendritic cell of claim 16; and (b) a pharmaceutically acceptable carrier.
  • the mature dendritic cell used in the method of this invention is autologous.
  • the cancer that can be treated or prevented by the method of this invention is renal cancer or breast cancer.
  • the composition comprising the mature dendritic cell of this invention for the preparation of anti-cancer medicine.
  • the mature dendritic cell is autologous.
  • the cancer that is a target of the medicine is renal cancer or breast cancer.
  • the present invention provides cancer cell lysates for pulsing dendritic cells in order to prepare mature dendritic cells that have the ability to induce anticancer immune responses, and also provides the method for preparing thereof.
  • the instant invention provides PDT-mediated dendritic cells that have a remarkably enhanced activity to induce anti-cancer immune responses, and also provides the method for preparing thereof.
  • This invention provides a pharmaceutical composition comprising the dendritic cell that has a remarkable activity to suppress the growth of cancer cells.
  • PDT-prepared cancer cell lysates of the present invention can be used to manufacture dendritic cells whose potential to induce cancer cell specific immune responses has been significantly enhanced.
  • Ia shows immunological phenotypes of immature dendritic cells (imDC), mature dendritic cells (mDC), dendritic cells (PDT DC) pulsed with photodynamic therapy (PDT)-prepared cancer cell lysates, dendritic cells (FT DC) pulsed with freezing-thawing method (FT)-prepared cancer cell lysates.
  • imDC immature dendritic cells
  • mDC mature dendritic cells
  • PDT DC dendritic cells
  • FT DC dendritic cells pulsed with freezing-thawing method
  • Rg. Ib shows survival rates of PDT-DC pulsed with PDT-prepared cancer cell lysates, F/T-DC pulsed with FT-prepared cancer cell lysates, and mDC having no pulsing.
  • Fig. Ic shows amounts of secreted IL-12 from PDT-DC pulsed with PDT- prepared cancer cell lysates and F/T-DC pulsed with FT-prepared cancer cell lysates, and mDC having no pulsing.
  • Fig. 2 shows the result of western blotting exhibiting the expression levels of heat shock proteins, HSP70, HSP-27, and HO-I in EMT6 breast cancer cell that have been irradiated after being treated with the photosensitizing agent of porphyrins.
  • Fig. 3a shows tumor growth rate (-D-) in the breast cancer (EMT6) mouse model in which PDT-DCs that had been pulsed with PDT-prepared cancer cell lysates were administered 3 days after transplantation of cancer cells; tumor growth rate (- A-) in the breast cancer mouse model in which FT-DCs that had been pulsed with cancer cell lysastes prepared by freeze-thawing method were administered 3 days after transplantation of cancer cells; and tumor growth rate of control (- ⁇ -).
  • Fig. 3b shows tumor growth rate (-D-) in the renal cancer (RENCA) mouse model in which PDT-DCs that had been pulsed with PDT-prepared cancer cell lysates were administered 3 days after transplantation of cancer cell; tumor growth rate (- A-) in the renal cancer mouse model in which FT-DCs that had been pulsed with cancer cell lysastes prepared by freeze-thawing method were administered 3 days after transplantation of cancer cells; and tumor growth rate of control (- ⁇ -).
  • dendritic cells of this invention that have been prepared by pulsing with PDT-prepared cancer cell lysates significantly suppress the growth of breast cancer (EMT6) and renal cancer (RENCA) in the mouse model.
  • Rg. 3c shows survival rates (-D-) of the individual animal of breast cancer
  • EMT6 mouse model which was vaccinated with PDT-DCs that had been pulsed with
  • Rg. 3d shows survival rates (-D-) of individual animal of renal cancer
  • Rg. 3e is a photograph that shows the size of tumors in control, breast cancer mouse model (PDT-DC) that was administered with DCs pulsed with PDT-prepared cancer cell lysates, and breast cancer mouse model (FT-DC) that was administered with DCs pulsed with cancer cell lysastes prepared by freeze-thawing method.
  • PDT-DC breast cancer mouse model
  • FT-DC breast cancer mouse model
  • Rg. 4a shows that the proliferation of spleen CD8 T cells was significantly increased where the mouse was administered with PDT-DCs prepared by pulsing with PDT-prepared cancer cell lysastes.
  • the proliferation of spleen CD8 T cell was not greatly increased where the mouse treated with FT-DCs prepared by pulsing with cancer cell lysates prepared by freeze-thawing method.
  • EMT6 cancer cell specific CTL cytotoxic T lymphocyte
  • Fig. 4c shows that the level of ThI immune responses is remarkably increased after treatment with dendritic cells. From the result of Fig. 4c, it can be understood that the level of ThI immune responses is effectively increased and the level of Th2 immune response is effectively decreased by PDT-DCs pulsed with PDT-prepared cancer cell lysates. In addition, the effectiveness of FT-DCs pulsed with FT-prepared cancer cell lysates to stimulate ThI immune responses and to suppress Th2 immune response is not as strong as PDT-DCs.
  • Fig. 5a and Fig. 5b show suppression effect of tumor growth of PDT-DCs of this invention.
  • the tumor growth suppression effect of the treatment with PDT-DCs pulsed with PDT-prepared cancer cell lysates was superior to the treatment with FT-DCs pulsed with cancer cell lysates prepared freeze-thawing method.
  • Fig. 5c and Fig. 5d show survival rates of individual animal of mouse model.
  • the survival rates in the treatment with PDT-DCs pulsed with PDT-prepared cancer cell lysates was superior to the treatment with FT- DCs pulsed with cancer cell lysates prepared freeze-thawing method.
  • mice 6 to 7 weeks of age, were purchased from Orient Bio (Gyeonggi-do, SungNam-Si, Republic of Korea). The mice were maintained under standard conditions of temperature and light, and were fed standard laboratory chow and water. Every mouse had adaptation period before being used in the experimentation.
  • the culture medium used was DMEM and RPMI 1640 (GIBCO Laboratories, Grand Island, NY, USA) supplemented with 10% FBS (GIBCO Laboratories, Grand Island, NY, USA), 50 ⁇ M 2-mercaptoethanol (Life technologies, Gaithersburg, MD, USA), 100 ⁇ g/ml streptomycin, 100 U/ml penicillin, and 25 ⁇ g/ml amphotericin B (GIBCO Laboratories; Grand Island, NY, USA).
  • Recombinant mouse (rm) TNF- ⁇ was purchased from BD Bioscience (Mountain View, CA, USA) and polyriboinosinic polyribocytidylic acid, (PoIy(I) • PoIy(C)) was purchased from Amersham Biosciences (Piscataway, NJ, USA).
  • rmGM-CSF and IL-4 were purchased from CreaGene (Gyeonggi-do, SungNam-Si, Republic of Korea).
  • fluorescein isothiocyanate (FITC)- or phcoerythrin (PE)- conjugated monoclonal antibodies to MHC class II, CD80, CD86, CD54, CD40, CD14, CD4, CD8, IFN- ⁇ and CDlIc were purchased from BD PharMingen (San Diego, CA, USA).
  • FITC fluorescein isothiocyanate
  • PE phcoerythrin
  • Bone marrow prepared from Balb/c mice was flushed out of femurs with RPMI-1640 by use of a syringe and separated as a single cell in the media by using a screen mesh.
  • RBCs red blood cells
  • ACK lysis buffer Tris-buffered 0.15 M ammonium chloride solution
  • F/T prepared cancer cell lysates were prepared from cancer cell lines of EMT6, 4T-1, or RENCA through the procedure of freezing-thawing method using liquid nitrogen (-180 0 C) and incubator (37°C) and removing cell debris by centrifugation at 600 rpm.
  • PDT prepared cancer cell lysates were manufactured as follows: Cancer cell lines of EMT6, 4T-1, or RENCA (supplied by Yonsei University Medical School, Seoul, Republic of Korea) were incubated in the complete medium for 4 hr in the presence of 1 ⁇ g/ml porphyrins. The incubated cells were washed with serum-free media, transferred to IX PBS, and irradiated with a laser or halogen lamp (6 J/cm 2 , 20 mW/cm 2 ). After that, the PDT-treated cells were transferred to serum-free media and cultured in the incubator. After culturing, cells and supernatants were collected using scrapper and removed cell debris by centrifugation at 600 rpm for 20 min. The protein contents in the supernatant were measured by Bradford method. The F/T or PDT prepared cancer cell lysates were used as antigens for the induction of the maturation of dendritic cells.
  • Culturing myeloid dendritic cells isolated from mouse bone marrow monocytes was carried out by the method modified from Inaba et al [I]. Myeloid cells were separated from the isolated bone marrow monocytes, and incubated for 6 days in the presence of 10 ng/ml rmGM-CSF and 10 ng/ml rmIL-4 at the concentration of 1 x 10 6 cells/ml. On sixth day of incubation, cancer cell lysates 50 ug/ml were added and incubated for additional 8 hr.
  • dendritic cells In order to induce the maturation of dendritic cells, 20 ng/ml TNF- ⁇ , 100 U/ml IFN- ⁇ , 10 ug/ml PoIy(I) • PoIy(C) were added and incubated for additional 18 hr. Incubated dendritic cells were collected on seventh day of incubation and their phenotypes were analyzed. Dendritic cells for therapeutic injection were recovered, suspended in physiological saline solution and administered it to mouse subcutaneously within 2 hr (1 x 10 6 cells / 100 ul / mouse).
  • EMT6 and 4T-1 were collected and transplanted into subcutaneous tissue with the concentration of 10 s cells/ mouse, or RENCA cells were collected and transplanted into subcutaneous tissues with the concentration of 1 X 10 6 cells/mouse.
  • the administration of therapeutic dendritic cells (1 X 10 6 cells/ mouse) was done on seventh day after injection of cancer cells (7 day model) or on third day after injection of cancer cells (3 day model).
  • the dendritic cells were injected into subcutaneous tissues twice at the interval of one week.
  • the size of tumor growing in the subcutaneous tissues was measured twice per a week using caliper.
  • the change of systemic immune function was detected with spleen cells.
  • the phenotype determination of dendritic cells or spleen cells was performed in accordance with the conventional method of Yoon et al [2].
  • Spleen monocytes IxIO 5 cells/100 ul
  • FACS buffer PBS mixed with 0.1% sodium azide and 1% FBS
  • FBS fluorescence labeled antibodies
  • Cells were washed after culturing, suspended in 500 ul FACS buffer, and analyzed with flow cytometer (BD Bioscience, Mountain View, CA, USA).
  • the activity of tumor specific cytotoxic T cell (CTL) induced after dendritic cell injection therapy was analyzed in accordance with the method described by Liu SQ et al [3].
  • Target cells (IxIO 4 cells/lOOul) were mixed with effector cells in various concentrations, incubated in a 96 well plate for 24 hr, and then the number of viable target cells was determined by crystal violet assay. Crystal violet assay that measures the activity of CD8 + T cell to kill adherent target cells has a credible sensitivity.
  • the cells in the well were washed with PBS and fixed with 10% Formalin (100 ul/ well) for 1 hr at room temperature.
  • the cell was stained with Crystal violet solution (0.4% in water, 100 ul/ well) for 30 min at room temperature. Plate was washed with water and dried at room temperature. After 200 ul of 80% methanol was added into the respective wells, the survival rates of cells were determined by measuring an absorbance at 570 nm.
  • the measurement of cell survival rates of control group was done by measuring an absorbance at 570 nm in 100% target cells containing no effector cell.
  • Enzyme-Linked Immuno-Sorbent Assay In order to measure the expression level of ThI cytokine IFN- ⁇ and Th2 cytokine IL-4, ELISA was performed on the supernatant of culture media of mouse spleen cells. In addition, in order to characterize the nature of dendritic cells, ELISA was carried out on the supernatant of dendritic cell culture media so as to measure the expression level of IL-2, Th2 cytokine that can induce the migration of dendritic cells. The extent of secretion of cytokines was determined by ELJSA (R&D system, Abington, OX, UK) in accordance with instructions of manufacturer.
  • the immune responses of T cells initiate by the reaction of mature DCs with na ⁇ ve T cells.
  • immature dendritic cells have a phagocytic function, they are weak T cell stimulator.
  • the maturation of dendritic cells is an important process in the inducing mechanism of immune responses.
  • PDT-prepared cancer cell lysates have more effectiveness than F/T-prepared cancer cell lysates in the induction of the maturation of dendritic cells, and thus make it possible to manufacture more effective tumor vaccines.
  • immature dendritic cells were obtained from bone marrow cells that have been isolated from Balb/C and induced to be matured by culturing in the presence of PDT-prepared lysates or FT-prepared lysates.
  • the phenotype of DCs was examined with FACS analysis by use of surface markers of dendritic cells, CD80, CD86, CD54, D40, MHC class II and surface marker of monocytes, CD14 as a control. Although there was little difference in the expression level of phenotypic markers between dendritic cells that have been cultured with F/T prepared lysates and PDT prepared lysates, the expression level of MHC class II in DCs incubated with PDT prepared lysates was increased compared to DCs pulsed with F/T prepared lysates. Accordingly, it is shown that PDT-prepared lysates more effectively induce the maturation of phenotypes of dendritic cells (Fig. la).
  • IL-2 cytokine induces ThI response by activating T cells and NK cells, and stimulates migration of dendritic cells so as to increase the immune responses by T cells at the secondary immune organs.
  • IL-12 cytokine By the results of ELISA as to IL-12 cytokine, it was confirmed that there was little difference in the secretion level of IL-12 in the respective mDCs, F/T DCs and PDT DCs (Fig. Ic).
  • HSPs Heat shock proteins
  • the expression level of HSPs is increased by external stimulations induced from physiological causes (growth or differentiation of cells or tissues), pathological causes (infections, inflammatory reactions, tumors and autoimmunity), or environmental causes (heat shock, heavy metals, oxygen free radical) [4, 5]. It is reported that chemotherapy, radiotherapy, and hyperthermia in cancer cells can increase intracellular expression levels of HSP70 [6. 7]. After EMT6 cancer cells were treated with the photosensitizing agent of porphyrins and irradiation of a laser or halogen lamp, the expression level of HSP was measured by western blotting.
  • EMT6 cancer cells were treated with 1 ug/ml of the photosensitizing agent of porphyrins, incubated for 4 hr, transferred to serum- free medium and cultured for 1, 2, 4, 6 and 12 hr respectively.
  • the protein content in the supernatant was measured in accordance with Bradford method and expression levels of HSP70, HO-I, HSP27 were detected from supernatants that have the same protein content.
  • the expression level of HSP70 reached the maximum at 2 hr after treatment of PDT on EMT6 cancer cell and decreased after 2 hr.
  • the expression level of the other heat shock protein of HO-I (Hemeoxygenase-1) in the PDT prepared group was higher than in the F/T prepared group.
  • the expression level of HSP 27 in the F/T prepared group was higher than in the PDT treated group (Fig. 2).
  • the therapeutic effectiveness of dendritic cells pulsed with cancer cell lysastes was determined in the mouse model in which EMT6 cancer cells (5X10 5 cells/mouse) were administered into subcutaneous tissues. Two types of cancer cell lysates of PDT-prepared cancer cell lysates and F/T-prepared cancer cell lysastes were used as cancer antigens in the procedure to induce the maturation of dendritic cells. Primary injection of dendritic cells pulsed with PDT-prepared or F/T-prepared cancer cell lysates was done on third day after injection of EMT6 cancer cells, secondary injection of dendritic cells was done on one week after injection. The growth of cancer cells was inhibited where dendritic cells pulsed with cancer cell lysates were injected.
  • the changes in systemic immune responses were detected from mouse spleen cells on second weeks after the last administration of dendritic cells.
  • Tumor specific responses of lymphocytes that are expected to be induced by dendritic cells pulsed with cancer cell lysates were examined.
  • the proliferation of spleen CD8 T cells was remarkably increased after administration of dendritic cells (PDT-DCs) matured by the process of the present invention (Fig 4a).
  • About 10.2 folds of proliferation increase in the treated group with DCs pulsed with PDT-prepared cancer cell lysates was measured compared to the untreated group.
  • about 5.3 folds of proliferation increase in the treated group with DCs pulsed with F/T prepared cancer cell lysates was detected.
  • the increase in the CTL activity specific to EMT6 cancer cells was confirmed in dendritic cell treated group (Fig 4b).
  • ThI immune responses were effectively increased after treatment with dendritic cells. There was more increase in ThI cell immune responses and more decrease in Th2 cell immune responses in the group (PDT-DC) treated with DCs pulsed with PDT-prepared cancer cell lysates than the group (F/T-DC) treated with DCs pulsed with FT-prepared cancer cell lysates (Fig 4c).
  • the therapeutic effects of DCs pulsed with PDT-prepared cancer cell lysates were examined in the breast cancer mouse model having tumor developed from transplanted EMT6 or 4T-1 cells.
  • the size of tumor was measured after injection of IXlO 6 DCs with two times a week after one week of tumor generation.
  • the more remarkable inhibition effect of tumor growth in the group (PDT-DC) treated with DCs pulsed with PDT-prepared cancer cell lysates was detected compared to the group (FT-DC) treated with FT-prepared cancer cell lysates (Figs. 5a and 5b).

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • General Health & Medical Sciences (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Organic Chemistry (AREA)
  • Chemical & Material Sciences (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Genetics & Genomics (AREA)
  • Biotechnology (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • Animal Behavior & Ethology (AREA)
  • Epidemiology (AREA)
  • Cell Biology (AREA)
  • Microbiology (AREA)
  • Immunology (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • Hematology (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

La présente invention porte sur des cellules dendritiques (DC) matures ayant une aptitude améliorée d'induire des réponses immunitaires anti-tumeur, et sur un procédé pour la préparation des DC mentionnées ci-dessus par la procédure d'impulsion de DC immatures avec des lysats de cellules cancéreuses sur lesquelles une thérapie photodynamique (PDT) a été conduite. La cellule dendritique de cette invention acquiert une activité pour induire des réponses immunitaires anti-tumorales de telle sorte qu'elle exerce un effet remarquable pour inhiber la croissance d'une tumeur.
PCT/KR2008/004042 2007-07-09 2008-07-09 Cellule dendritique autologue médiatisée par une thérapie photodynamique ayant l'aptitude d'inhiber la croissance d'une tumeur WO2009008666A2 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
KR10-2007-0068524 2007-07-09
KR1020070068524A KR100954315B1 (ko) 2007-07-09 2007-07-09 종양성장 억제능을 갖는 광역동성 치료 매개 자가 유래수지상 세포

Publications (2)

Publication Number Publication Date
WO2009008666A2 true WO2009008666A2 (fr) 2009-01-15
WO2009008666A3 WO2009008666A3 (fr) 2009-03-19

Family

ID=40229274

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/KR2008/004042 WO2009008666A2 (fr) 2007-07-09 2008-07-09 Cellule dendritique autologue médiatisée par une thérapie photodynamique ayant l'aptitude d'inhiber la croissance d'une tumeur

Country Status (2)

Country Link
KR (1) KR100954315B1 (fr)
WO (1) WO2009008666A2 (fr)

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2012512629A (ja) 2008-12-17 2012-05-31 ディスカバリー テクノロジー インターナショナル，インク． 高トルク圧電モータ

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20020022032A1 (en) * 1999-04-23 2002-02-21 Curry Patrick Mark Immuno-adjuvant PDT treatment of metastatic tumors

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20020022032A1 (en) * 1999-04-23 2002-02-21 Curry Patrick Mark Immuno-adjuvant PDT treatment of metastatic tumors

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
GOLLNICK S.O. ET AL.: 'Generation of effective antitumor vaccines using photodynamic therapy' CANCER RES. vol. 62, 2002, pages 1604 - 1608 *
GOMER C.J. ET AL.: 'Photodynamic therapy-mediated oxidative stress can induce expression of heat shock proteins' CANCER RES. vol. 56, 1996, pages 2355 - 2360 *

Also Published As

Publication number Publication date
WO2009008666A3 (fr) 2009-03-19
KR100954315B1 (ko) 2010-04-21
KR20090005472A (ko) 2009-01-14

Similar Documents

Publication Publication Date Title
Liau et al. Treatment of intracranial gliomas with bone marrow—derived dendritic cells pulsed with tumor antigens
JP5707284B2 (ja) 腫瘍の処置のための、インビトロでの部分的に成熟した樹状細胞の投与
JP7364237B2 (ja) 癌治療のためのｔ細胞の活性化方法
US8673296B2 (en) Method and composition for producing a cellular allogeneic vaccine
AU748074B2 (en) Method of treating leukocytes, leukocyte compositions and methods of use thereof
US20030124091A1 (en) Endothelial cell derived hematopoietic growth factor
JP2021533798A (ja) エクソソームに基づく抗腫瘍ワクチン
JP2006510667A5 (fr)
BRPI0817535B1 (pt) método in vitro eficiente para obter células apresentadoras de antígenos ativadas (apcs), especialmente células dendríticas (dcs), úteis na preparação de vacinas para o tratamento do câncer
EP2606125B1 (fr) Cellules exprimant les caractéristiques et les propriétés cytolytiques de thl
WO2024057892A1 (fr) Amplificateur de réponse immunitaire anti-tumeur
TWI764896B (zh) 有效率的ｎｋｔ細胞活化技術
EP1959007A1 (fr) Procédé pour générer des cellules dendritiques à maturité
WO2009008666A2 (fr) Cellule dendritique autologue médiatisée par une thérapie photodynamique ayant l'aptitude d'inhiber la croissance d'une tumeur
BG107482A (bg) Лекарствено средство за имунотерапия на злокачествени тумори
JP2002069001A (ja) 樹状細胞を主成分とする細胞ワクチン
JP2023540827A (ja) 高純度及び高効率の自然殺害細胞の製造方法及びその用途
KR20030084826A (ko) 동종 종양-관련 항원이 탑재된 수지상세포, 이의 제조방법및 이를 유효성분으로 하는 암 치료용 백신 조성물
Perez et al. DNA associated with the cell membrane is involved in the inhibition of the skin rejection response induced by infusions of photodamaged alloreactive cells that mediate rejection of skin allograft
Lee Dendritic cells-based vaccine and immune monitoring for hepatocellular carcinoma
WO2001062092A1 (fr) Formulations et procedes d'utilisation desdites formulations pour provoquer une reaction immunitaire
KR100675761B1 (ko) 1,2-디옥타노일-ｓｎ-글리세롤3-포스페이트를 이용한 급성골수성 백혈병 세포의 수지상 세포로의 전이분화 유도 방법
JP2025072455A (ja) 高純度及び高効率の自然殺害細胞の製造方法及びその用途
JPH1067681A (ja) 癌ワクチン及びその製造法
KR20050113344A (ko) 악성 종양이 있는 환자의 혈구로부터 수지상 세포를 제조하는 방법 및 상기 수지상 세포를 유효 성분으로 함유하는 약학 조성물

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 08778702

Country of ref document: EP

Kind code of ref document: A2

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 08778702

Country of ref document: EP

Kind code of ref document: A2

点击 这是indexloc提供的php浏览器服务，不要输入任何密码和下载