WO2005083062A1 - Vaccins avec cellule - Google Patents

Vaccins avec cellule Download PDF

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Publication number
WO2005083062A1
WO2005083062A1 PCT/JP2005/002941 JP2005002941W WO2005083062A1 WO 2005083062 A1 WO2005083062 A1 WO 2005083062A1 JP 2005002941 W JP2005002941 W JP 2005002941W WO 2005083062 A1 WO2005083062 A1 WO 2005083062A1
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Prior art keywords
cell
cells
immunoreactive
fusion
antigen
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PCT/JP2005/002941
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English (en)
Japanese (ja)
Inventor
Yasufumi Kaneda
Kazuya Hiraoka
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Genomidea Inc.
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Priority to JP2006519367A priority Critical patent/JPWO2005083062A1/ja
Publication of WO2005083062A1 publication Critical patent/WO2005083062A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/461—Cellular immunotherapy characterised by the cell type used
    • A61K39/4615—Dendritic cells
    • 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
    • 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
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/76—Viruses; Subviral particles; Bacteriophages
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/462—Cellular immunotherapy characterized by the effect or the function of the cells
    • A61K39/4622—Antigen presenting cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/4644—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
    • C12N5/10—Cells modified by introduction of foreign genetic material
    • C12N5/12—Fused cells, e.g. hybridomas
    • C12N5/16—Animal 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 A61K39/46
    • A61K2239/46—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterised by the cancer treated
    • A61K2239/57—Skin; melanoma

Definitions

  • the present invention relates to a novel vaccine using cells. More specifically, the present invention relates to a cell vaccine (eg, an anti-cancer vaccine) using cells fused with a virus.
  • a cell vaccine eg, an anti-cancer vaccine
  • Vaccines are a method for preventing and treating diseases utilizing an immune response in the body, and have been widely used.
  • the most suitable gene therapy for suppressing cancer metastasis and preventing recurrence is immune gene therapy, but its effect has not yet been sufficiently enhanced worldwide.
  • One of the causes is the transfection method, particularly due to poor transfection efficiency into antigen presenting cells such as dendritic cells and insufficient transfection of target genes.
  • Another is that tumor immunity cannot be enhanced enough to be therapeutic.
  • Patent Document 1 There are already reports that a cancer antigen can be presented by fusion of dendritic cells and cancer cells to induce tumor immunity (Patent Document 1), but this method is not perfect for treating or preventing cancer
  • Patent Literature 1 JP-T 2003-519486
  • an object of the present invention is to provide a method, a medicine, a system, and the like for easily treating or preventing a desired disease, disorder or condition.
  • the present inventors have proposed a gene transfer vector and a gene expression method in order to achieve this object. And so on.
  • an effective disease eg, cancer
  • a virus such as inactivated HVJ
  • tumor immunity was induced by fusion of dendritic cells and X-irradiated cancer cells using inactivated HVJ having strong cell fusion activity, and CpG was used in combination with adjuvant.
  • Particularly favorable effects have been achieved by using a cancer vaccine with enhanced tumor immunity.
  • Cells fused by conventional fusion techniques are expected to be structurally different from cells fused by a virus such as the inactivated HVJ of the present invention. In particular, large differences in antigen-presenting ability appear to suggest structural differences directly related to function.
  • the main two technologies of the present invention include the following two.
  • the first is the use of cell fusion technology using a virus such as HVJ.
  • a virus such as HVJ.
  • cell fusion methods such as polyethylene glycol (PEG) and electroporation (also known as electroporation) are known.
  • PEG polyethylene glycol
  • electroporation also known as electroporation
  • cell fusion technology using a virus such as inactivated HVJ has never been used for this purpose, but it is not only inferior to the efficiency reported by existing methods, but it is also superior. It has therapeutic and / or prophylactic effects, and furthermore, has the advantage that it does not need to be fused before administration because it can cause cell fusion with higher reproducibility and can also fuse in living tissues.
  • the present invention also provides, secondly, enhancement of tumor immunity induction by administering CpG or the like as an adjuvant together with the fusion cell vaccine. This combination has not been reported so far. Adjuvants other than CpG can of course be used.
  • the present invention provides the following.
  • a fusion cell of an antigen presenting cell and at least one type of immunoreactive donor cell (1) A fusion cell of an antigen presenting cell and at least one type of immunoreactive donor cell.
  • the antigen-presenting cells include dendritic cells (DC), B cells, macrophages 2.
  • DC dendritic cells
  • B cells B cells
  • macrophages 2 The fused cell according to item 1, which is a cell or a precursor cell thereof.
  • the immunoreactive donor cells are cancers selected from melanoma cells, kidney cancer cells, colon cancer cells, liver cancer cells, lung cancer cells, cervical cancer cells, ⁇ cancer cells, brain cancer cells, and tongue cancer cells 2.
  • the fusion cell according to item 1 having at least one cell surface marker.
  • Such cell surface markers include the following.
  • a pharmaceutical composition comprising a fusion cell of an antigen-presenting cell and at least one type of immunoreactive donor cell.
  • the adjuvant is selected from the group consisting of aluminum hydroxide gel, alum, CpG, Freund's complete adjuvant, Freund's incomplete adjuvant, heat-killed pertussis, lipopolysaccharide, muramyl peptide, and starch bentonite.
  • the pharmaceutical composition according to 26 is selected from the group consisting of aluminum hydroxide gel, alum, CpG, Freund's complete adjuvant, Freund's incomplete adjuvant, heat-killed pertussis, lipopolysaccharide, muramyl peptide, and starch bentonite.
  • a pharmaceutical composition comprising an antigen-presenting cell, at least one type of immunoreactive donor cell, and an adjuvant.
  • the adjuvant is selected from the group consisting of aluminum hydroxide gel, alum, CpG, Freund's complete adjuvant, Freund's incomplete adjuvant, heat-killed pertussis, lipopolysaccharide, muramylbeptide and starch bentonite.
  • the pharmaceutical composition according to 30 is selected from the group consisting of aluminum hydroxide gel, alum, CpG, Freund's complete adjuvant, Freund's incomplete adjuvant, heat-killed pertussis, lipopolysaccharide, muramylbeptide and starch bentonite.
  • At least one immunoreactive donor cell At least one immunoreactive donor cell
  • a kit for treating or preventing a disease caused by the above immunoreactive supply cells comprising:
  • the antigen-presenting cell is a dendritic cell (DC), a B cell, a macrophage cell, or a precursor cell thereof.
  • DC dendritic cell
  • the antigen-presenting cells include CD1, CDl lc (integrin X), CD40, CD58 (LF A-3), CD86, HLA-DR, CD123 (anti-IL-3Ra), CD21, CD23, CD148, CD1 57,
  • the kit according to item 32 having at least one cell surface marker selected from the group consisting of CD80, MHC class I and MHC class II.
  • the immunoreactive donor cells are cancers selected from melanoma cells, kidney cancer cells, colon cancer cells, liver cancer cells, lung cancer cells, cervical cancer cells, ⁇ cancer cells, brain tumor cells, and tongue cancer cells 33.
  • the immunoreactive feeder cell is at least one cell surface marker selected from the group consisting of an oncogene product, an embryonic protein, a viral protein, a tissue-specific antigen, a mutated tumor suppressor protein, and an idiotypic peptide; 33.
  • the kit according to item 32 comprising:
  • a method for producing a fused cell comprising:
  • cell fusion step includes a method selected from the group consisting of a polyethylene glycol method and an electofusion.
  • immunoreactive donor cells include normal cells, cancer cells, or xenogeneic cells.
  • the adjuvant is selected from the group consisting of aluminum hydroxide gel, alum, CpG, Freund's complete adjuvant, Freund's incomplete adjuvant, heat-killed pertussis, lipopolysaccharide, muramyl peptide, and starch bentonite. 56. The method according to 56.
  • a method for treating or preventing a disease, disorder or condition caused by the above immunoreactive supply cells comprising:
  • the present invention achieves the following effects. For example, it is thought that this combination effect can overcome the problems of conventional cancer treatment for the following reasons.
  • immunoreactive supply cells for example, cancer cells
  • many immunoreactive supply cells often lack MHC class I, and the In some cases, presentation was not possible and dendritic cells did not recognize cancer antigens.
  • fusion of antigen presenting cells with immunoreactive donor cells increased the efficiency of antigen presentation.
  • cytotoxic T cells against cancer are activated, and it is possible to suppress diseases and disorders such as tumors for a long time.
  • the present invention can use the patient's own antigen-presenting cells and immunoreactive supply cells, it can be safely applied without serious side effects and other problems.
  • it compared to gene therapy currently under development, it has the effect of using no foreign nucleic acid (DNA) or virus, and of having no risk of side effects.
  • FIG. 1A shows the cell distribution of the fused cells.
  • FL2 left of upper left panel
  • FL1 right of upper left panel
  • FIG. 1B shows the results when the value of HAU was varied from 0 to 1000. From the right, 1 000 HAU, 500 HAU, 100HAU and OHAU (just a mixture) are shown. The upper right corner of the lower FACS panel shows the percentage of fused cells. [Garden 1C] Comparison of double staining in dendritic cells, melanoma cells and fused cells.
  • FIG. 2A shows the secretion of TNF ⁇ .
  • Mix indicates a simple mixture
  • FC indicates a fused cell
  • Mix + CpG indicates a mixture plus an adjuvant CpG
  • FC + CpG indicates a fused cell plus an adjuvant CpG
  • FCLnonCpG Shown is a non-CpG oligonucleotide that was not adjuvanted to the fused cells. As shown in the figure, the addition of the fusion cells and the addition of CpG significantly promoted the secretion of TNF.
  • FIG. 2B shows the secretion of IL_12p40.
  • Mix indicates a simple mixture
  • FC indicates a fused cell
  • Mix + CpG indicates a mixture plus an adjuvant CpG
  • FC + CpG indicates a fused cell with an adjuvant CpG
  • FC + LnonCpG indicates fusion cells plus non-CpG non-adjuvant oligonucleotides.
  • the addition of the fused cells and the addition of CpG significantly promoted the secretion of IL_12p40.
  • FIG. 3A shows the secretion of interferon ⁇ .
  • PBS indicates control
  • CpG indicates CpG only
  • Mix indicates mere mixture
  • FC indicates fused cells
  • Mix + CpG indicates mixture plus adjuvant CpG
  • FC + CpG Shows fusion cells to which adjuvant CpG was added.
  • the addition of fusion cells and the addition of CpG significantly promoted the secretion of interferon ⁇ .
  • the Cr / ⁇ (effector cell / target cell) ratio of 51 Cr specific release is shown (B16 cells).
  • PBS indicates control
  • CpG indicates CpG only
  • Mix indicates mixture
  • FC indicates fused cells
  • Mix + CpG indicates mixture plus adjuvant CpG
  • FC + CpG indicates fusion.
  • the figure shows the cells prepared with the adjuvant CpG.
  • FIG. 4C shows the E / T (effector cell / target cell) ratio of 51 Cr specific release (EL cells).
  • PBS indicates control, CpG indicates CpG only, Mix indicates a mixture, FC indicates fusion cells, Mix + CpG indicates a mixture obtained by adding adjuvant CpG to the mixture, and FC + C garden 4A] 3 shows changes in tumor volume. It is a change of the volume after inoculation of B16 cells.
  • PBS indicates control, CpG indicates CpG only, Mix indicates mixture, FC indicates fused cells, Mix + CpG indicates mixture plus adjuvant CpG, FC + CpG indicates mixture
  • Fig. 4B Shows the transition of survival rate. The survival rate after inoculation of B16 cells is shown.
  • PBS indicates control
  • CpG indicates CpG only
  • Mix indicates mixture
  • FC indicates fused cells
  • Mix + CpG indicates mixture plus adjuvant CpG
  • FC + CpG indicates mixture. Shown are the fusion cells plus the adjuvant CpG. The survival rate was significantly improved in the fused cells and the group to which CpG was added.
  • C + CpG refers to fused cells plus the adjuvant CpG.
  • the survival rate was significantly improved.
  • FIG. 5A shows the secretion of interferon ⁇ in Example 4.
  • PBS indicates control
  • CpG indicates adjuvant only
  • Mix indicates simple mixture
  • FC indicates fused cells
  • Mix + CpG indicates mixture plus adjuvant CpG
  • FC + CpG indicates fusion
  • the figure shows the cells prepared with the adjuvant CpG. As shown in the figure, the addition of the fused cells and the addition of CpG significantly promoted the secretion of interferon ⁇ .
  • C + CpG indicates a fusion cell prepared by adding the adjuvant CpG.
  • PBS indicates control
  • CpG indicates CpG only
  • Mix indicates mixture
  • FC indicates fused cells
  • Mix + CpG indicates mixture plus adjuvant CpG
  • C + CpG indicates a fusion cell prepared by adding the adjuvant CpG.
  • FC indicates fused cells
  • Mix + CpG indicates a mixture obtained by adding an adjuvant CpG to a mixture
  • FC + CpG indicates a mixed cell obtained by adding an adjuvant CpG.
  • FIG. 6 shows the protection rate of lung metastasis.
  • the effect of fused cells was also observed in the protective effect on embryo transfer, with the combination of fused cells and CpG being the most effective. Each point represents an individual individual and bars represent the average.
  • SEQ ID NO: 1 shows a CpG oligonucleotide (CpG1668).
  • SEQ ID NO: 2 shows a GpG oligonucleotide (GpG1668).
  • the gene vaccine of the present invention can be carried out using a technique known in the art. The production of such vaccines is performed by applying molecular biological, biochemical and genetic engineering techniques as described in detail elsewhere herein. be able to.
  • cell fusion means that a plurality of cells becomes a smaller number of cells as a result of fusion of adjacent cells and loss of the septum. Typically, when cells fuse, multinucleation occurs. Although it can occur in nature such as at the time of fertilization of germ cells, it is usually provided as a means of cell engineering. It can be produced by fusing two different cells chemically or physically and culturing them using a selective medium in which only the fused cells are grown. In 1957, Yoshio Okada and colleagues discovered that cell fusion was induced by HVJ (Sendai virus), whose infectivity was inactivated by ultraviolet light; viruses with cell fusion activity include the parainfluenza virus and Newcastle.
  • HVJ Sendai virus
  • Paramyxoviruses such as disease viruses are mainly known.
  • Cell fusion is also possible with chemical substances, such as lysolecithin, polyethylene glycol (PEG) 6000, and glycerol oleate.
  • the method using a chemical agent is said to be superior in that fusion of protoplasts of animal cells or plant cells without a virus receptor is possible, and there is no influence from the virus genome.
  • cell fusion electric fusion
  • virus refers to an infectious microstructure having either DNA or RNA as a genome and proliferating only in infected cells.
  • Viruses include retroviridae, togaviridae, coronaviridae, flaviviridae, noramyxoviridae, orthomyxoviridae, buniyaviridae, rhabdoviridae, boxviridae, herpesviridae, baculoviridae.
  • the virus used herein may be an influenza virus of the family Orthomyxoviridae or a Sendai virus. More preferably, the virus used is Sendai virus.
  • Sendai virus or "HVJ” (Hemagglutinating virus of Japan) is a virus which is used interchangeably and which belongs to the genus Paramyxovirus of the family Paramyxoviridae and has a cell fusion activity. . M. Kuroya et al. (1953) reported this as a Sendai virus. The genome is a negative strand RNA of about 15500 bases in length. Sendai wi Rus virus particles have an envelope and exhibit a polymorphism of 150-300 nm in diameter. Sendai virus has an RNA polymerase. It is unstable to heat, agglutinates almost all types of red blood cells, and is also hemolytic.
  • Sendai virus Proliferates in the cytoplasm of embryonated eggs and / or cultured cells from kidneys of various animals.
  • Sendai virus is susceptible to persistent infection when it infects cell lines. Because of its ability to fuse various cells, it is widely used for cell fusion such as formation of heterokaryons in cells and production of hybrid cells.
  • part of a virus refers to a part (for example, an envelope or a core) of a virus that retains the cell fusion ability of the virus. Such ability can be determined by preparing a plurality of arbitrary cells and determining whether or not the cells are fused when providing a portion to be tested.
  • (virus) envelope refers to a membrane structure based on a lipid bilayer surrounding a nucleocapsid present in a specific virus such as Sendai virus. Envelopes are usually found in viruses that mature by budding from cells.
  • the envelope consists of small projection structures, generally consisting of spike proteins encoded by viral genes, and host-derived lipids. This envelope may be used in the cell fusion technology used herein.
  • inactivation refers to inactivation of the genome when referring to a virus (eg, Sendai virus).
  • the inactivated virus is replication defective.
  • the inactivated virus usually retains cell fusion ability, but preferably has lost the pathogenicity of the virus. Inactivation is achieved by, for example, ultraviolet irradiation, X-ray irradiation, electron beam irradiation, ⁇ -ray irradiation, or treatment with an alkylating agent.
  • alkylation refers to an action of replacing a hydrogen atom of an organic compound with an alkyl group
  • an alkylating agent A compound that provides an alkyl group.
  • the alkylating agent include organometallic compounds such as alkyl halides, dialkyl sulfates, alkyl sulfonates, and alkyl zincs.
  • Preferred alkylating agents include / 3-propiolatatone, petit-mouthed ratatone, Yowi-Dani Methinole, iodide chill, propyl iodide, methyl bromide, bromide tyl, propyl bromide, dimethyl sulfate, getyl sulfate, and the like. But not limited to them.
  • "antigen presenting cell” also referred to as APC refers to a cell that can present an antigen on the cell surface. Representative examples include non-lymphoid coexisting cells necessary for the response of helper T cells to proliferate and differentiate in response to various cell division-inducing substances such as antigens.
  • MHC major histocompatibility complex
  • antigen degraded product of foreign protein, ie, a peptide consisting of more than ten amino acid residues
  • T cells T cells in a sandwiched form.
  • dendritic cells and Langerhans cells of the epidermis mainly perform this function, and activated macrophages and B cells also have the same function.
  • any antigen presenting cell can be used.
  • Antigen presenting cells do not merely present antigen to T cells.It is known that they interact with T cells via cell surface molecules (for example, B7 and CD28), and also stimulate by cytoforce. .
  • dendritic cells are also abbreviated as DCs, and refer to various tissues such as tampa-based tissues, and form dendrites in the intercellular spaces of each tissue. Take out the cells, let the cells go. They differ from macrophages in that they do not form connective tissue and have no or very low phagocytosis. It is derived from bone marrow hematopoietic stem cells and expresses class I and class II histocompatibility antigens on the cell surface and plays an important role in ⁇ cell activation at the onset of the immune response.
  • Epidermal Langerhans cells are also considered to be a type of dendritic cells, and in this specification, unless otherwise specified, dendritic cells also include these Langerhans cells. It is understood that progenitor cells of dendritic cells are also used herein.
  • immunoreactive supply cell refers to a cell that, when present in a certain organism having an immune system, can elicit an immune response against that organism. Therefore, usually, immunoreactive donor cells can express or express a force having a substance that is an antigen for a certain organism. Immunoreactive donor cells typically cause, eg, neoplastic (eg, cancer) infectious cells (eg, viruses, bacteria, protozoa, rickettsia, chlamydia, fungi, parasites, etc.), and autoimmune diseases. Or related cells. It is not limited to.
  • neoplastic eg, cancer
  • infectious cells eg, viruses, bacteria, protozoa, rickettsia, chlamydia, fungi, parasites, etc.
  • autoimmune diseases Or related cells. It is not limited to.
  • normal cell refers to any cell other than a cell that causes a disease (for example, causes a disease) that becomes a disease state. Thus, normal cells are ubiquitous in a normal host. On the other hand, cells in a disease state may be referred to as "affected cells.”
  • cancer or “cancer” is used interchangeably and can destructively infiltrate or metastasize into surrounding tissues where the growth is more atypical than normal cells.
  • cancers include, but are not limited to, solid cancers and hematopoietic tumors.
  • solid cancer is a concept that refers to a cancer having a solid form and is opposed to a hematopoietic tumor such as leukemia.
  • solid cancers include breast cancer, liver cancer, stomach cancer, lung cancer, head and neck cancer, cervical cancer, prostate cancer, retinoblastoma, malignant lymphoma, esophageal cancer, brain tumor, and bone tumor. It is not limited to.
  • Cancers specifically contemplated by the present invention include, but are not limited to, lung cancer, melanoma, gastric cancer, and the like.
  • cancer cell refers to a cell in a cancer state.
  • examples of cancer cells include melanoma, lung cancer, gastric cancer, adenocarcinoma, squamous cell carcinoma, adenosquamous cell carcinoma, thymic carcinoma, lymphoma, sarcoma, liver cancer, non-Hodgkin's lymphoma, Hodgkin's lymphoma, leukemia, uterine cancer , Breast, prostate, ovarian, ovarian, colorectal (colorectal), multiple myeloma, neuroblastoma, bladder, cervix, skin, breast, esophagus, kidney And cells such as brain tumors, but are not limited thereto.
  • cell surface marker is also referred to as "cell surface antigen” and refers to a protein or peptide that is characteristically expressed on the surface of a certain cell.
  • cell surface markers of antigen presenting cells include CD1, CDl lc (integrin X), CD40, CD58 (LFA-3), CD86, HLA-DR, CD123 (anti-IL-3Ra), CD21, CD23, Examples include, but are not limited to, CD148, CD157, CD80, MHC class I and MHC class II.
  • cell line markers include, but are not limited to, CD3, CD14, CD16, CD19, CD20, CD56, and the like.
  • tumor-specific antigen refers to any tumor that is specifically expressed in a certain tumor. Refers to an antigen. Therefore, it can be said that the tumor-specific antigen is a kind of cell surface marker. Such antigens are exemplified in Table A herein.
  • the term "inactivation” refers to an immunoreactive supply cell (for example, a cancer cell) and inactivates the pathogenicity of the immunoreactive supply cell. To be done. It is preferable that the inactivated immunoreactive supply cells have inactivated or removed a toxic factor. Inactivated immunoreactive donor cells are preferred because they do not cause disease when used as vaccines. Such inactivation can be achieved by, for example, ultraviolet irradiation, X-ray irradiation, electron beam irradiation, ⁇ -ray irradiation, or treatment with an alkylating agent.
  • Cells used in the present invention include cells derived from any organism (for example, multicellular organisms of any kind (for example, animals (for example, vertebrates, invertebrates)), plants (for example, monocotyledonous plants, Dicots etc.))).
  • organisms for example, multicellular organisms of any kind (for example, animals (for example, vertebrates, invertebrates)), plants (for example, monocotyledonous plants, Dicots etc.)).
  • cells derived from vertebrates are used, and more preferably, mammals (e.g., Pores, marsupials, oligodonts, skin wings, winged hands, carnivores, insectivores, longnoses, hoofs, artiodactyla, tubulars, scales, marine cattle, whales Eyes, primates, rodents, etc.) are used. More preferably, cells from primates (eg, chimpanzees, macaques, humans) are used. Most preferably, human-derived cells are used. As an antigen presenting cell, any animal cell having an immune system is used. As the immunoreactive supply cells, any cells that can adversely affect animals having an immune system can be used.
  • mammals e.g., Pores, marsupials, oligodonts, skin wings, winged hands, carnivores, insectivores, longnoses, hoofs, artiodactyla, tubulars, scales, marine cattle, whales
  • amino acid may be natural or unnatural as long as the object of the present invention is satisfied.
  • derivative amino acids or “amino acid analogs” refer to amino acids that differ from naturally occurring amino acids but have the same function as the original amino acids. Such derivative amino acids and amino acid analogs are well known in the art.
  • nucleotide may be natural or non-natural.
  • derivative nucleotide or “nucleotide analog” refers to a nucleotide different from a naturally occurring nucleotide but having the same function as the original nucleotide.
  • derivative nucleotides and nucleotide analogs are well known in the art. Examples of such derivative nucleotides and nucleotide analogs include phospho- Rotiates, phosphoramidates, methyl phosphonates, chiral methyl phosphonates, 2-O-methyl ribonucleotides, peptide-nucleic acid (PNA), but are not limited thereto.
  • PNA peptide-nucleic acid
  • any analog may be used as long as the gene product is expressed.
  • a genetic vaccine containing natural nucleotides is used. This is because the natural form is more likely to be translated into a peptide.
  • the present invention is useful for clinical or non-clinical applications.
  • the method for preparing the fused cells of the present invention is quick and effective.
  • the fused cells of the present invention are particularly useful in therapeutic regimens that utilize the immune system to treat or prevent disease (eg, cancer treatments, cancer prevention, etc.).
  • the fused cells of the present invention are also useful in preparing monoclonal antibodies, as in conventional hybridomas.
  • the fusion cells of the invention include antigen presenting cells lacking accessory components required for an immune response, and cells from an organ intended for transplantation of a patient. These cells can be used to induce tolerance to the transplanted cells. After inducing tolerance, it reduces the incidence of transplant rejection. Kits for producing the fusion cells of the present invention and performing the method of the present invention are also provided.
  • antibody refers to polyclonal, monoclonal, human, humanized, multispecific, chimeric, and anti-idiotypic antibodies, and fragments thereof, such as F (ab ') and Fab. Fragments, as well as other recombinantly produced
  • antibodies may be covalently linked or recombinantly fused to an enzyme such as alkaline phosphatase, horseradish peroxidase, ⁇ - galactosidase, and the like.
  • an enzyme such as alkaline phosphatase, horseradish peroxidase, ⁇ - galactosidase, and the like.
  • neutralizing antibody refers to an antibody that neutralizes biological activities of enzymes, toxins, bacteria, viruses, and the like.
  • the present invention particularly refers to an antibody that neutralizes a biological activity associated with an antigen.
  • the term "antigen” refers to any substrate that can be specifically bound by an antibody molecule.
  • immunogen refers to an antigen that can initiate lymphocyte activation that produces an antigen-specific immune response.
  • epitope refers to a group constituting a structure that determines an antigen. Thus, epitopes require a set of amino acid residues involved in recognition by a particular immunoglobulin, or, in the case of T cells, recognition by T cell receptor proteins and / or major histocompatibility complex (MHC) receptors.
  • MHC major histocompatibility complex
  • epitopes are molecular features (eg, primary, secondary or tertiary peptide structures and charges) that are recognized by immunoglobulins, T cell receptors or HLA molecules. To form a site. Epitopes containing peptides may contain more than two amino acids in a spatial conformation unique to the epitope. Generally, an epitope is composed of at least 5 such amino acids, typically at least 6, 7, 8, 9, or 10 such amino acids.
  • epitopes are generally preferred because they are more similar to the antigenicity of the original peptide, but may not always be so in view of conformation.
  • Methods for determining the spatial conformation of amino acids include, for example, X-ray crystallography, and two-dimensional nuclear magnetic resonance spectroscopy.
  • identification of the epitope in a given protein is readily accomplished using techniques well known in the art. See, eg, Geysen et al. (1984) Pro Natl. Acad. Sci. USA 81: 3998 (a general method for rapid peptide synthesis to determine the location of immunogenic epitopes on a given antigen); No.
  • a sequence having a length of at least 3 amino acids is necessary for use as an epitope containing a peptide.
  • this sequence has at least 4 amino acids, more preferably Pama 5 amino acids, 6 amino acids, 7 amino acids, 8 amino acids, 9 amino acids, 10 amino acids, 15 amino acids, 20 amino acids, 25 amino acids in length are required. possible.
  • virus inactivation When virus inactivation is necessary, it is typically carried out by ultraviolet irradiation or alkylation treatment as described below.
  • Ultraviolet irradiation method A lml suspension of virus (for example, HVJ) was placed in a 30 mm-diameter dish, and
  • a suspension of the virus (eg, HVJ) immediately after purification was supplemented with ⁇ -propiolatatone to a final concentration of 0.01%, and incubated on ice for 60 minutes. After that, the cells were incubated at 37 ° C for 2 hours. Dispense 10, OOOHAU per tube into an Eppendorf tube and centrifuge at 15, OOOrpm for 15 minutes to settle the precipitate at -20. Save with C.
  • the virus eg, HVJ
  • these pharmaceutical compositions and medicaments may contain suitable pharmaceuticals, including excipients or other compounds that facilitate processing of the envelope, to prepare a pharmaceutically usable formulation. It may include a chemically acceptable carrier. Further details on techniques for prescribing and administration can be found in, for example, the latest edition of the Japanese Pharmacopoeia and the latest supplement.
  • an “adjuvant” is a substance that increases or otherwise alters an immune response when mixed with an administered immunogen.
  • Adjuvants are classified as appropriate, for example, as mineral, bacterial, plant, synthetic or host products.
  • An adjuvant is a substance that enhances the immunogenicity of the hapten epitope, and in a more preferred embodiment, a modulating or potent non-toxic adjuvant is used.
  • Adjuvants are required to be used with conventional vaccines to elicit faster, more potent, or more prolonged responses. Such adjuvants also This is useful in cases where the supply of raw materials is limited or where production is costly.
  • Adjuvants are classified depending on the case, for example, as mineral, bacterial, plant, synthetic or host products.
  • the first class is mineral adjuvants, such as aluminum compounds.
  • the first use of aluminum compounds as adjuvants was described in 1926. Since that time, antigens precipitated with the anoreminium compound or mixed with or adsorbed to preformed aluminum compounds have been used to enhance the immune response in animals and humans.
  • Aluminum compounds and similar adjuvants seem to work through the following mechanism. Aluminum physically binds to the antigen to form particles and forms a reservoir for the antigen in the tissue after injection. It slows the secretion of the antigen and thus prolongs the time of interaction between the antigen and the cells that represent the antigen, for example, macrophages or follicular dendritic cells. In addition, immunocompetent cells are attached to the injection area and activated.
  • Aluminum particles are demonstrated 7 days after immunization in the regional lymph nodes of the egret, and other significant functions may be able to direct antigens to the T cell-containing regions of the nodes themselves.
  • Adjuvant efficacy was shown to correlate with draining lymph node information. Numerous studies have confirmed that antigen administered with aluminum leads to increased immunity in body fluids, but cell-mediated immunity appears to increase only slightly, as measured by delayed-type hypersensitivity . Aluminum has also been described as activating the complement pathway. This mechanism may play a role in the local inflammatory response as well as in immunoglobulin polysaccharide and bile duct memory.
  • Aluminum compounds are almost the only safe adjuvants currently used in humans. However, vaccines containing aluminum sometimes cause local reactions. Although the development of allergies is usually not a clinical problem, aluminum compounds also attract eosinophils to the injection area via a T-cell dependent mechanism and, when injected, trigger an IgE response after antigen priming. It is said to induce a population of carrier-specific cells with helper function in response to the IgE response. In addition, vaccines containing aluminum cannot be freeze-dried, thus requiring refrigerated transport and storage, creating a risk of contamination. Moreover, aluminum compounds provoke sustained protection from disease When you always succeed, you don't have to.
  • Bacterial adjuvants have been purified and synthesized (eg, muranyl dipeptide, lipid A) and host mediators have been closed (interleukin 1 and interleukin 2). Recently, Bordetella pertussis, lipopolysaccharide and Freund's complete adjuvant (FCA) are being used at the laboratory level.
  • Bacterial adjuvants have been purified and synthesized (eg, muranyl dipeptide, lipid A) and host mediators have been closed (interleukin 1 and interleukin 2).
  • Bordetella pertussis, lipopolysaccharide and Freund's complete adjuvant (FCA) are being used at the laboratory level.
  • Oligonucleotides are also used herein as adjuvants.
  • the method for preparing the oligonucleotide can be in accordance with the method for preparing a nucleic acid molecule described above, and a method well known in the art can be used.
  • a kit available from Sigma-Aldrich Japan Co., Ltd., Yamasa Shoyu, Fluka, or the like can be used.
  • the present invention can be provided as a medicament.
  • the present invention can be administered as a vaccine or as a therapeutic agent.
  • a "vaccine” can be administered into the body to produce active immunity, usually an infectious agent or some portion of an infectious agent, or to produce such an agent or portion.
  • a composition eg, suspension or solution
  • an agent eg, a gene sequence, etc.
  • the antigenic portion of a vaccine can be a microorganism (eg, a virus or a bacterium) or a natural, purified, or engineered protein, peptide, polysaccharide, or similar product purified from a microorganism, or a product thereof. It can be a nucleic acid molecule comprising a nucleic acid sequence encoding such a protein. Vaccines exert their effects by raising neutralizing antibodies.
  • Vaccines that can be used as medicaments herein include, but are not limited to, vaccines for cancer, acquired immunodeficiency syndrome, and the like. These vaccines can be present in the form of cells or compositions containing them in the medicament of the present invention.
  • the organism e.g. The immunity (humoral immunity, cellular immunity, or both) that is actively generated in the body of animals (vegetarians and vectors) prevents the transmission, transmission, and spread of pathogens.
  • the term "gene vaccine” refers to a factor (typically, a nucleic acid molecule) that is expressed in a patient to which the vaccine is administered and whose expression has the effect of the vaccine. Containing compositions (eg, suspensions or solutions).
  • An exemplary genetic vaccine can be a nucleic acid molecule (eg, vector, plasmid, naked DNA, etc.) that includes a nucleic acid sequence encoding a gene product having antigenicity.
  • cell vaccine refers to a vaccine administered in the form of cells.
  • the cell used here may be of any form, but in the present invention, a fused cell is used.
  • the immunological effect of the vaccine can be confirmed using any method known in the art.
  • a method include, but are not limited to, CTL precursor cell frequency analysis, ELISPOT method, tetramer method, real-time PCR method, and the like.
  • CTL precursor cell frequency analysis is performed by limiting dilution of peripheral blood lymphocytes or lymphocytes cultured in the presence of antigenic peptide and IL-12 and culturing them in the presence of IL-2 and feeder cells. Then, the proliferating wells are stimulated with the vaccine or its candidate, and the presence or absence of IFN- ⁇ production is measured by ELISA or the like.
  • the efficacy of the vaccine can be evaluated by calculating the frequency of CTL progenitor cells according to Poisson analysis of the positive cells.
  • the number of positive cells is the number of antigen-specific CTLs, and the greater the number, the higher the efficacy as a vaccine.
  • such a composition may further include a pharmaceutically acceptable carrier and the like.
  • Pharmaceutically acceptable carriers included in the medicament of the present invention include any substance known in the art.
  • the pharmaceutical compositions and medicaments of the present invention are administered in any sterile biocompatible pharmaceutical carrier, including, but not limited to, saline, buffered saline, dextrose, and water. obtain. Any of these molecules can be administered to a patient, alone or in combination with other agents, in a pharmaceutical composition mixed with suitable excipients, adjuvants, and / or pharmaceutically acceptable carriers. obtain.
  • the pharmaceutically acceptable carrier is pharmaceutically inert.
  • the present invention when provided as a vaccine, it can preferably include any composition that achieves an immune system capable of producing an immunologically favorable immune response when a disease is afflicted.
  • Vaccines usually contain an immune determinant (eg, antigen, cell, etc.) and an adjuvant that acts to rapidly enhance the response of the immune determinant, and the immunological determinant may not be used in combination with an adjuvant. It is suitable.
  • the immunostimulant is usually used, for example, Freund's complex adjuvant, Freund's non-complex adjuvant, and the like. The immunostimulant is not limited to these.
  • the vaccine of the present invention has efficacy even without using an adjuvant.
  • Pharmaceutically acceptable carriers that can be used in the compositions, vaccines and the like of the present invention include, for example, antioxidants, preservatives, coloring agents, flavors, and diluents, emulsifiers, suspending agents , Solvents, fillers, bulking agents, buffers, delivery vehicles, diluents, excipients and / or pharmaceutical adjuvants.
  • the medicament of the invention will be administered in the form of a composition comprising the vaccine, or a variant or derivative thereof, together with one or more physiologically acceptable carriers, excipients or diluents.
  • a suitable vehicle may be water for injection, physiological solution, or artificial cerebrospinal fluid, which may be supplemented with other materials common in compositions for parenteral delivery. .
  • an acceptable carrier, excipient, or stabilizer is non-toxic to recipients, and is preferably inert at the dosages and concentrations employed.
  • Such non-toxic and inert carriers include, for example, phosphates, citrates, or other organic acids; ascorbic acid, ⁇ -tocopherol; low molecular weight polypeptides; proteins (eg, serum Hydrophilic polymers (eg, polyvinylinolepyrrolidone); amino acids (eg, glycine, glutamine, asparagine, arginine or lysine); monosaccharides, disaccharides, and other carbohydrates (gnorecose, mannose) Or a dextrin); a chelate complex 1J (eg, EDTA); a sugar alcohol (eg, mannitol or sorbitol); a salt-forming counterion (eg, sodium); and / or a nonionic surfactant ( For example, Tween,
  • Exemplary carriers suitable for cellular vaccines include neutral buffered saline or saline mixed with serum albumin.
  • the product is formulated as a lyophilizate using appropriate excipients (eg, sucrose).
  • excipients eg, sucrose
  • Other standard carriers, diluents and excipients may be included as desired.
  • Other exemplary compositions include Tris buffer at pH 7.0-8.5 or acetate buffer at pH 4.0-5.5, which may further include sorbitol or a suitable alternative thereof. .
  • the present invention can also be provided as an animal drug composition, a quasi-drug, a marine drug composition, a food composition, a cosmetic composition, and the like. It can be understood that can also be produced by a known preparation method.
  • the pharmaceutical of the present invention may contain a physiologically acceptable carrier, excipient or stabilizing agent (Japanese Pharmacopoeia, 14th edition or its latest edition, Remington's Pharmaceutical Sciences, 18th Edition, AR Gennaro, ed., Mack Publishing Company, 1990, etc.) and can be prepared and stored in the form of an aqueous solution.
  • a physiologically acceptable carrier excipient or stabilizing agent
  • excipient or stabilizing agent Japanese Pharmacopoeia, 14th edition or its latest edition, Remington's Pharmaceutical Sciences, 18th Edition, AR Gennaro, ed., Mack Publishing Company, 1990, etc.
  • compositions for oral administration can be formulated using pharmaceutically acceptable carriers well known in the art in dosage forms suitable for administration. Such carriers enable the pharmaceutical compositions to be formulated into liquids, gels, syrups, slurries, suspensions, etc., suitable for consumption by a patient.
  • the administration of the cells themselves is intended, and it is preferable to administer parenterally instead of oral administration.
  • compositions for parenteral administration include aqueous solutions or suspensions of the active ingredients (eg, vaccines).
  • the pharmaceutical compositions of the invention may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks's solution, Ringer's solution, or buffered saline.
  • Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran.
  • suspensions of the active compounds may be prepared as appropriate oily injection suspensions.
  • Suitable lipophilic solvents or vehicles include fatty acids such as sesame oil, or synthetic fatty acid esters such as ethyl oleate or triglycerides, or ribosomes. If desired, suspensions may increase the solubility of the stabilizer or compound, which allow for the preparation of highly concentrated solutions. It may contain appropriate agents or reagents to increase it.
  • the fused cells, compositions, kits, medicaments and the like of the present invention may also contain a biocompatible material.
  • a biocompatible material include silicone, collagen, gelatin, a copolymer of glyconoleic acid and lactic acid, an ethylenebutyric acid copolymer, polyurethane, polyethylene, polytetrafluoroethylene, polypropylene, polyatalylate, and polymetallic. It may include at least one selected from the group consisting of rates. Silicone is preferred because it is easy to mold.
  • biodegradable polymers examples include collagen, gelatin, polyhydroxycarboxylic acids (eg, glyconoleic acid, lactic acid, hydroxybutyric acid, etc.), hydroxydicarboxylic acids (eg, malic acid, etc.) and hydroxytricarboxylic acid.
  • polyhydroxycarboxylic acids eg, glyconoleic acid, lactic acid, hydroxybutyric acid, etc.
  • hydroxydicarboxylic acids eg, malic acid, etc.
  • hydroxytricarboxylic acid examples include collagen, gelatin, polyhydroxycarboxylic acids (eg, glyconoleic acid, lactic acid, hydroxybutyric acid, etc.), hydroxydicarboxylic acids (eg, malic acid, etc.) and hydroxytricarboxylic acid.
  • the type of polymerization may be random, block, or graft.
  • ⁇ -hydroxycarboxylic acids, hydroxydicarboxylic acids, or hydroxytricarboxylic acids have an optically active center in the molecule, D-form, L-integral, DL- Any body can be used.
  • compositions of the present invention can be manufactured in a manner similar to that known in the art for cell preparation (eg, mixing, lysing, etc.).
  • compositions of the present invention include compositions wherein the cell vaccines of the present invention are contained in an amount effective to achieve the intended purpose.
  • a “prophylactically effective amount,” “therapeutically effective amount,” or “pharmacologically effective amount” is a term well-recognized by those skilled in the art and is intended for its pharmacological consequence (eg, prevention, treatment, prevention of relapse, etc.). Refers to the amount of drug effective to produce Thus, a therapeutically effective amount is an amount sufficient to reduce the symptoms of the disease to be treated.
  • a prophylactically effective amount is an amount sufficient to reduce the symptoms of the disease to be prevented.
  • An effective dose to prevent recurrence is an amount sufficient to prevent recurrence.
  • an effective amount eg, a therapeutically effective amount
  • the actual amount administered will depend on the individual to whom the treatment is to be applied, and will preferably be an amount that is optimized such that the desired effect is achieved without significant side effects. . Determination of an effective dose is well within the capability of those skilled in the art.
  • the effective dose can be estimated initially either in neutralizing antibody measurements, or in any suitable animal model. Animal models are also used to achieve the desired concentration range and route of administration. Such information can then be used to determine useful doses and routes for administration in humans.
  • Therapeutically effective, prophylactically effective amounts, etc. and toxicity can be determined by standard pharmaceutical procedures in cell cultures or experimental animals (eg, ED, doses that are therapeutically effective in 50% of the population;
  • the dose ratio between toxic effects is the therapeutic index, which can be expressed as the ratio ED / LD.
  • compositions that exhibit large therapeutic indices are preferred.
  • the data obtained from cell culture assays and animal studies is used in formulating a range of dosage for use in humans.
  • the dosage of such compounds lies preferably within a range of circulating concentrations that include the ED with little or no toxicity. This dose depends on the dosage form used, the patient
  • the dose is appropriately selected depending on the age and other conditions of the patient, the type of the disease, the type of the cells used, and the like.
  • a virus or envelope vector equivalent to at least 100 HAU, preferably at least 500 HAU, and more preferably equivalent to 1000 HAU per 6 X 10 6 cells is used for producing fused cells.
  • the ratio of cells to be fused is preferably, but not limited to, equivalent; typically, a range of 1: 100-100: 1 can be used.
  • a range within 1: 10—10: 1 is used, more preferably, a range within 1: 33: 1. More preferably, 1: 2 2: 1 or about 1: 1 can be employed.
  • HAU refers to the activity of a virus capable of aggregating 0.5% of chicken erythrocytes.
  • One HAU is equivalent to approximately 24 million innoles particles (Okada, Y. et al., Biken Journal 4, 209-213, 1961). If the above amount is used, it can be used once or several times.
  • the exact dose will be selected by the individual clinician, taking into account the patient to be treated. . Dosage and administration are adjusted to provide sufficient levels of the active moiety or to maintain the desired effect. Additional factors that may be considered include the severity of the disease state (eg, tumor size and location; patient age, weight, and gender; dietary time and frequency of administration, drug combinations, response sensitivity, and resistance to treatment) / Response). Depending on the half-life and clearance rate of the particular formulation, long acting pharmaceutical compositions may be administered every 34 days, every week, or once every two weeks. Guidance on specific dosages and methods of delivery is provided in the literature known in the art.
  • the fused cells, compositions, kits, medicaments and the like of the present invention can be provided in a sustained release form.
  • the sustained release dosage form can be any form known in the art as long as it can be used in the present invention. Examples of such a form include preparations such as a tablet (pellet, cylinder, needle, etc.), a tablet, a disc, a sphere, and a sheet. Methods for preparing sustained release forms are known in the art, and are described, for example, in the Japanese Pharmacopoeia, the United States Pharmacopeia, and in other countries.
  • Methods for producing a sustained-release preparation include, for example, a method using the release of a drug from a complex, a method using an aqueous suspension injection, an oily injection or an oily suspension. And a method for preparing an emulsion injection solution (o / w type, w / o type emulsion injection solution, etc.).
  • compositions and medicaments of the present invention are usually carried out under the supervision of a physician, but can be carried out without the supervision of a physician if permitted by the regulatory authority and law of the country.
  • the vaccine according to the present invention may be an emulsion containing various adjuvants.
  • adjuvants include Freund's adjuvant (complete or incomplete), Adjuvant 65 (including decocted oil, mannide monooleate and aluminum monostearate), and aluminum hydroxide and aluminum phosphate. Or mineral gels such as alum, oligonucleotides (CpG) and the like.
  • the vaccine according to the present invention includes a diluent, a fragrance, a preservative, an excipient, a disintegrant, a lubricant, a binder, an interface, and the like. It may contain one or more pharmaceutical additives selected from activators, plasticizers, etc. Les ,.
  • Parenteral delivery methods include local, intraarterial (eg, via the carotid), intramuscular, subcutaneous, intramedullary, intrathecal, intraventricular, intravenous, intraperitoneal, or intranasal administration Are mentioned. In the present invention, any route that reaches the treatment site may be used.
  • the route of administration of the vaccine according to the present invention is not particularly limited, but parenteral administration is preferred.
  • Parenteral administration to be exemplified includes, for example, intravenous, intraarterial, subcutaneous, intradermal, intramuscular or intraperitoneal administration.
  • the single dose of the vaccine according to the present invention depends on the purpose of administration, and whether the infection is primary or reinfected, and further depends on various factors such as the age and weight of the patient, the symptoms and the severity of the disease. It can be appropriately selected according to the conditions.
  • the dose of the vaccine according to the present invention may be about 0.1 Olng to 10 mg per kg body weight, more preferably about 0.1 lng to 1 mg. .
  • 10 6 cells to 10 8 cells preferably 5 ⁇ 10 6 cells to 5 ⁇ 10 7 cells, and the like can be mentioned, but are not limited thereto.
  • the number of times of administration of the vaccine according to the present invention varies depending on the above-described various conditions, and cannot be determined unconditionally. However, it is preferable to repeatedly administer the vaccine at intervals of a day to a week. Among them, it is preferable to administer several times, preferably about 112 times, at an interval of about 114 weeks. It is more preferable to determine the number of administrations (administration time) while monitoring the disease status by disease symptomatology or basic tests using antibody titers.
  • fusion cells use immunoreactive donor cells.
  • Immunoreactive donor cells refer to any cells capable of supplying immunoreactivity to a host.
  • any cell that has or is predicted to have a cell surface marker that can be an antigen falls within the scope of the immunoreactive donor cell.
  • Such a cell surface marker is in this case an antigen.
  • the cell surface marker is usually a force existing on the surface of a cell, a possible peptide or a variant or complex thereof (eg, a glycopeptide).
  • An amino acid sequence (for example, an epitope) capable of inducing specific recognition by T cells is usually a peptide having a length of at least about 5 amino acids, preferably at least about 8 amino acids.
  • CD8-positive and CD4-positive.CD8-positive CTLs are class I-restricted, and CD4-positive CTLs are class II-restricted. , GplO 0, Tyrosinase, NY-ESO-1).
  • Cell surface markers for example, when taken up by cancer cells, are carried to the endoplasmic reticulum (ER), where peptides exhibiting binding to HLA class I molecules form complexes with class I molecules. This complex is said to be expressed on the cell surface.
  • Sites of interest in binding to class I molecules are the second amino acid and the ninth (possibly the eighth or tenth) amino acid in the peptide.
  • the second amino acid depends on the type of HLA.
  • the second amino acid is often tyrosine (Y) or phenylalanine (F), and in the case of HLA-A2 it is leucine (L).
  • This characteristic sequence can be appropriately determined depending on the type of HLA.
  • the ninth (or eighth or tenth) amino acid is often leucine (L), isocyanate isine (I), or phenylalanine (F).
  • it is mouth insulin (L) or palin (V).
  • cell surface markers can vary depending on the HLA type of the patient of interest. Once formed, such a cell surface marker is recognized by the T cell receptor of CTL and triggers T cell degradation.
  • cell surface markers are preferably identified, but need not be identified. This is because, even if not identified, if it is confirmed that the cell is a fusion cell by another method, such a fusion cell exerts a therapeutic effect or a prophylactic effect according to the present invention. In particular, in the case of cancer cells, even if a tumor-specific marker has not been identified, the therapeutic effect can be achieved, and thus the usefulness of the present invention is high.
  • Such a cell surface marker is identified using a method known in the art. That can be S. Examples of such methods include, but are not limited to, cDNA expression cloning method, SEREX method, and the like.
  • a method known in the art that can be S. Examples of such methods include, but are not limited to, cDNA expression cloning method, SEREX method, and the like.
  • the Sampnole derived from a cDNA library prepared from cancer cells is transiently expressed together with HLA cDNA in COS-7 cells, and the expression is maximized.
  • the cells can be identified by performing a mixed culture with cancer-specific CTL overnight and measuring the amount of interferon- ⁇ in the supernatant by a measurement method such as ELISA.
  • a measurement method such as ELISA.
  • a clone having a cell surface marker can be identified, and thereby its coding sequence can also be identified.
  • a cDNA library is screened using an IgG antibody present in the serum of a patient.
  • a phage library derived from a target cell eg, a cancer cell
  • E. coli a protein produced by E. coli is transferred to a filter, hybridized with patient serum, detected with an anti-IgG antibody, and reacted with the antibody.
  • the desired gene can be cloned from the plaque.
  • cancer antigen refers to an antigen molecule that is newly expressed as normal cells become cancerous.
  • cancer antigens include, but are not limited to, for example:
  • Cancer virus-derived antigens eg, T antigens derived from DNA-type tumor viruses such as adenovirus, poliovirus, and SV40.
  • DNA-type tumor viruses such as adenovirus, poliovirus, and SV40.
  • the viral envelope protein is expressed on the cell surface.
  • TTA Tumor specific transplantation antigen
  • MHC major histocompatibility complex
  • a cancer antigen can be used in any form.
  • a form called a cancer-associated antigen is preferably used. This is because the association with MHC results in expression on the surface of cancer cells.
  • Representative cell surface markers include, for example, but are not limited to: In the table below, one-character display is adopted.
  • cell surface markers and tumor-specific markers to be listed include, for example, oncogene products, embryonic proteins, viral proteins, tissue-specific antigens, mutated tumor sub- Type epitope.
  • Embryonic protein MAGE family (melanoma, breast cancer, etc.)
  • Winorestan / Quick EBV (Parkit's lymphoma, Hodgkin's lymphoma, nasopharyngeal carcinoma)
  • HBV HBV
  • Antigen-specific antigen tyrosinase MART-1, gplOO (melanoma)
  • Idiotype Epitope Idiotype Ig B-cell lymphoma
  • T cell receptor type 1 ⁇ f diotype T cell lymphoma
  • markers can be identified by antibodies. Many of these can be easily amplified by PCR or the like because their gene sequences are known.
  • prophylaxis or prevention refers to treating a disease or disorder before such condition is caused so that such condition does not occur.
  • treatment refers to preventing the deterioration of a disease or disorder when such a condition occurs, preferably maintaining the status quo, more preferably Means reducing, more preferably reducing.
  • prevention of recurrence refers to a means for preventing a disease or disorder from recurring after it has become such a condition, recovered or cured.
  • Such therapeutic or prophylactic activity is preferably tested in vitro prior to use in humans and then in vivo.
  • in vitro assays to demonstrate the therapeutic or prophylactic utility of the gene vaccines of the present invention include the effect of specific binding of the vaccine to cell lines or patient tissue samples.
  • Such tests can be determined using techniques known to those of skill in the art (eg, immunological assays such as ELISA).
  • immunological assays such as ELISA
  • Examples include, but are not limited to, methods for testing for the ability to raise neutralizing antibodies.
  • patient refers to an organism to which the treatment of the present invention is applied, and is also referred to as “subject” or “subject”.
  • the patient can preferably be a human.
  • the present invention provides methods for treatment, inhibition and prevention by administering to a patient an effective amount of the gene vaccine of the present invention.
  • the genetic vaccine of the present invention can be substantially purified (eg, in a state where there is substantially no substance that limits its effect or produces undesirable side effects).
  • the animal targeted by the present invention may be any organism (eg, an animal (eg, a vertebrate, an invertebrate)) as long as it has an immune system or a similar system.
  • it is a vertebrate animal (for example, a metal eel, alfalfa, chondrichthyes, teleosts, amphibians, reptiles, birds, mammals, and the like), and more preferably, a mammal (for example, monops, marsupials) , Rodents, dermis, skin wings, carnivores, carnivores, carnivores, longnoses, ungulates, artiodactyla, tubulars, squamata, squids, cetaceans, primates, rodents Teeth, egrets).
  • a vertebrate animal for example, a metal eel, alfalfa, chondrichthyes, teleosts, amphibians, rep
  • Exemplary patients include, but are not limited to, animals such as, for example, horses, pigs, horses, chickens, cats, dogs, and the like. More preferably, primates (eg, chimpanzees, diphthora, humans) are targeted. Most preferably, humans are targeted.
  • animals such as, for example, horses, pigs, horses, chickens, cats, dogs, and the like. More preferably, primates (eg, chimpanzees, diphthora, humans) are targeted. Most preferably, humans are targeted.
  • compositions and the like of the present invention include, for example, aqueous solutions, ribosomes, microparticles, and microcapsules. Therefore, the vaccine of the present invention can be administered orally or parenterally.
  • administration methods include oral administration, parenteral administration (e.g., intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, mucosal administration (intranasal, vaginal, airway, oral, rectal mucosal) And intestinal mucosa), topical administration to the affected area, dermal administration, etc.).
  • the vaccines used in the present invention preferably do not contain pyrogens.
  • the preparation of such pharmaceutically acceptable compositions can be readily performed by those skilled in the art by considering pH, isotonicity, stability, and the like.
  • Formulations for such administration may be provided in any formulation.
  • Such formulations include, for example, solutions, injections, and sustained release agents.
  • Can be Examples of the method of introduction include, but are not limited to, administration as an oral preparation, inhalation (eg, lungs), syringe, catheter, injection using a tube, needleless injection, and a gene gun. In this case, it can be administered together with other biologically active agents.
  • the amount of vaccine used in the prophylactic method of the present invention can be easily determined by those skilled in the art in consideration of the purpose of use, the target disease (type, etc.), the age, weight, and medical history of the patient. Can be.
  • the frequency of applying the treatment method of the present invention to a patient (or a patient) also depends on the purpose of use, target disease (type, severity, etc.), age, weight, medical history, and course of the patient. Can be easily determined by those skilled in the art.
  • the frequency may include, for example, daily-once-monthly administration (eg, once a week, once a month), or once a year before the epidemic.
  • the administration amount of the vaccine or the like of the present invention varies depending on the age, body weight, symptoms or administration method of the patient, and is not particularly limited.
  • Administration of the vaccine in the present invention may be performed by any method, but it is preferable to use needleless injection. This is because the administration can be performed without imposing an excessive burden on the patient.
  • the needleless syringe according to the present invention refers to a device in which a piston is moved by gas pressure or elasticity of an elastic member to inject a drug solution onto skin without using a needle, and a drug component is more preferably subcutaneously. Or a medical device administered subcutaneously into cells.
  • a gene gun is a high-density material such as gold or tungsten coated with DNA. It is a medical / experimental device capable of in vivo gene transfer by accelerating particles using gas pressure such as helium.
  • the advantages of gene guns are that they allow efficient transfer of cells with a small amount of DNA and that stable results can be obtained with different operators.
  • Helios Gene Gun manufactured by Bio-Rad, USA, etc. is commercially available, and can be used for IJ. (See below)
  • Judgment of termination of the prophylactic treatment by the method of the present invention can be performed by confirming an antibody elicited by using a commercially available assay or a device.
  • the present invention also provides a pharmaceutical package or kit comprising a container containing the vaccine of the present invention.
  • a notice in the form of a government agency that regulates the manufacture, use or sale of pharmaceuticals or biological products may optionally accompany such containers, and may include such notice for manufacture, use or sale for human administration. Represents government approval for
  • the term “cell biologically active substance” or “physiologically active substance” refers to a substance that acts on cells or tissues. Such effects include, but are not limited to, for example, control or alteration of the cell or tissue.
  • Physiologically active substances include cytodynamics and growth factors.
  • the physiologically active substance may be a naturally occurring substance or a synthetic substance.
  • a physiologically active substance The quality may be that produced by the cell or that has a similar effect, but an altered effect.
  • a bioactive substance is used as an indicator of the effect of vaccination because it is induced by vaccination, and is also used together with vaccination to enhance the effects of vaccine prevention or treatment. Can be
  • Cytokines are generally proteins or polypeptides that regulate immune response, regulate the endocrine system, regulate the nervous system, have antitumor and antiviral effects, regulate cell proliferation, regulate cell differentiation. Etc.
  • growth factor or “cell growth factor” is used interchangeably herein and refers to a substance that promotes or controls cell growth. Growth factors are also called growth factors or growth factors. Growth factors can be added to the medium in cell or tissue culture to replace the action of serum macromolecules. Many growth factors have been shown to function as regulators of differentiation status in addition to cell growth.
  • cytokins include interleukins, chemokines, hematopoietic factors such as colony stimulating factors, tumor necrosis factors (TNFa, etc.), interferons (eg, interferon ⁇ ). Is included.
  • Typical growth factors include platelet-derived growth factor (PDGF), epidermal growth factor (EGF), fibroblast growth factor (FGF), hepatocyte growth factor (HGF), and vascular endothelial growth factor (VEGF).
  • PDGF platelet-derived growth factor
  • EGF epidermal growth factor
  • FGF fibroblast growth factor
  • HGF hepatocyte growth factor
  • VEGF vascular endothelial growth factor
  • Preferable exemplary sites for the present invention include, but are not limited to, those capable of inducing differentiation, proliferation and activation of immune cells, and include, for example, interleukin 1, interleukin 2, interleukin 4, interleukin 6, interleukin 7, IL_12, IL_15, IL_18, interferon, interferon / 3, interferon ⁇ , granulocyte macrophage, colony stimulating factor (GM_CSF), HGF, VEGF, FGF, etc. Growth factor (GF), tumor necrosis factor (TNF), tumor necrosis factor (TNF-a), tumor necrosis factor / 3 (TNF-) and the like, but are not limited thereto.
  • Physiologically active substances such as cytodynamics and growth factors generally have a function duplication phenomenon (redun dancy), the activity of the bioactive substance used in the present invention can be increased even if it is a cytokin or growth factor known by other names and functions (eg, cell killing ability, signal transduction ability, etc.). Can be used in the present invention.
  • the cytodynamics or growth factor when used supplementarily, may have the desired activity herein (eg, the ability to kill the desired pathogen) as long as it has the vaccine of the present invention. Or it can be used in a preferred embodiment of the medicament.
  • the present invention provides a fusion cell of an antigen-presenting cell and at least one immunoreactive supply cell.
  • the fused cells are fused with a virus or a part thereof, and have higher fusion efficiency (for example, 10% or more, preferably %, More preferably 25% or more), with good reproducibility and low cytotoxicity.
  • any cells can be used as long as they can be fused.
  • any cells can be used as long as they can provide immunoreactivity to the host to be administered.
  • immunoreactive supply cells include, but are not limited to, normal cells, cancer cells, xenogeneic cells, and autologous cells.
  • normal cells When normal cells are used, they are usually intended to be used as substitutes for ribonucleases and hybridomas.
  • cancer cells, xenogeneic cells and the like When cancer cells, xenogeneic cells and the like are used, they are intended to be used for preventing or treating diseases caused by them.
  • autologous cells they can be applied, for example, to the treatment of autoimmune diseases (for example, ARENOLOGY).
  • the immunoreactive donor cell used in the present invention is a cancer cell. Vesicles.
  • the cancer cells any cells can be used as long as they are fused.
  • Examples include cells of head cancer, skin cancer, breast cancer, esophagus cancer, nephroma, brain tumor, tongue cancer, sarcoma, mesothelioma, and the like.
  • the fused cells of the present invention exhibit the ability
  • the fusion cell of the present invention is fused with a virus or a part thereof.
  • Such fused cells provide cells to be fused (eg, one or more antigen presenting cells (eg, dendritic cells) and one or more immunoreactive donor cells (eg, cancer cells)).
  • a virus or a portion thereof eg, an envelope
  • the virus used when producing the fusion cells is an inactivated virus.
  • Inactivation is the power that can provide a safe vaccine for treatment or prevention.
  • Inactivated viruses usually do not have the ability to replicate. Therefore, whether or not the virus has been inactivated can be confirmed by examining the replication ability.
  • the virus is inactivated by an alkylating agent or ultraviolet irradiation.
  • Any alkylating agent can be used as the alkylating agent used in the present invention.
  • Preferred alkylidizing agents that can be used in the present invention include j3-propiolatatatone, petit-mouthed ratatone, Yowi-Dani Methinole, Iodo-Kako, Iyo-D-propyl, methyl bromide, bromo-, propyl-bromide, dimethyl sulfate, and getyl sulfate. Examples include, but are not limited to:
  • the immunoreactive donor cells used for the fusion cells of the present invention are preferably inactivated. Since immunoreactive donor cells are usually used as pathogenic cells, it is desirable to inactivate such pathogenicity for vaccine administration. Such inactivation can be achieved by any means, usually eliminating pathogenicity Any means (eg, ⁇ -ray irradiation, X-ray irradiation, etc.) can be used. Preferably, X-irradiated cells are used as the immunoreactive supply cells.
  • the antigen-presenting cells are preferably autologous cells of the host to be administered. This is because no immune rejection occurs and the ability to present the antigen in the host is usually retained.
  • any antigen presenting cell can be used, as long as the antigen can be presented and an immune rejection reaction does not occur. Alternatively, a similar effect can be obtained by a treatment that does not cause immune rejection.
  • the immunoreactive donor cells are autologous cells.
  • the immunoreactive donor cells may be cancer cells, cells causing autoimmune diseases, and the like.
  • autologous cells are also used when utilized for autoantibody production.
  • allogeneic cells may be used. If the immunoreactive donor cell is a non-self pathogen, such as a bacterium, a heterologous cell will be used.
  • Examples of the antigen-presenting cell used in the present invention include, but are not limited to, dendritic cells, dendritic cells, macrophage cells, or precursor cells thereof.
  • dendritic cells or their precursor cells are used as a source of the fused cells. More preferably, dendritic cells are used. This is because it is known that it has excellent antigen presentation ability.
  • immunoreactive donor cells used in the present invention include, but are not limited to, a cell capable of presenting a tumor-specific antigen or a cell having the ability. Whether the tumor-specific antigen is known or unknown, it can be identified.
  • the fused cells of the present invention preferably have the characteristics of cells before fusion.
  • Representative examples of such features include, but are not limited to, for example, the presence of cell surface markers.
  • the characteristics of the cell include, for example, the antigen presenting ability of an antigen presenting cell.
  • Supply capacity including but not limited to. The ability to provide such immunoreactivity can be the presentation of cell surface markers, particularly those associated with virulence.
  • the fusion cell of the present invention has a cell surface marker of an antigen presenting cell.
  • cell surface markers include, for example, CD1, CDllc (integrin X), CD40, CD58 (LFA-3), CD86, HLA-DR, CD123 (anti-IL-3Ra) derived from antigen-presenting cells , CD21, CD23, CD148, CD157, CD80, MHC class I and MHC class II, and the like.
  • CDllc is used. This is because it is specific to dendritic cells.
  • Such cell surface markers may be identified by a force, preferably a plurality of forces, for which it is sufficient to identify at least one.
  • a fluorescent cell sorter For marker identification, a fluorescent cell sorter (FACS), magnetic beads, or the like can be used.
  • FACS fluorescent cell sorter
  • Such negative, weakly positive, positive and strongly positive cell surface markers can preferably be based on the expression intensity employed in FACS or magnetic bead based techniques.
  • FACS or magnetic beads can be performed using procedures as exemplified herein, based on techniques well known in the art. Therefore, factors that can determine the degree of positivity with high sensitivity are preferred, such as highly specific monoclonal antibodies, specific ligands, or modified or fragmented fragments thereof. It is not limited to.
  • the cells to be sampled are collected, for example, from the bone marrow (femur, tibia, etc.), suspended in a buffer such as PBS, and passed through a filter such as a nylon filter (eg, 70 / im). Remove contaminating tissues such as cell clumps and muscle.
  • a buffer such as PBS
  • a filter such as a nylon filter (eg, 70 / im).
  • Antibodies that should be positive are labeled, for example, with PE, APC, FITC, and the like.
  • the fusion cell of the present invention preferably has a cell surface marker of an immunoreactive donor cell.
  • a cell surface marker provides an antigen to be possessed by the immunoreactive supply cell, so that it is presented as an antigen by the antigen presenting ability of the fused cells, and a vaccine effect is exerted.
  • Such cell surface markers include, for example, oncogene products, embryonic proteins, viral proteins, Tissue specific antigens, mutated tumor suppressor proteins and idiotype epitopes, or those described in Tables A and B can be used.
  • gplOO can be used for melanoma.
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising a fusion cell of an antigen-presenting cell and at least one immunoreactive supply cell.
  • a fused cell can take any form described in the above (fused cell).
  • the fused cells contained in the pharmaceutical composition of the present invention constitute at least 6%. Pharmaceutical compositions containing such fusion cells in such a ratio have not hitherto existed. It should be noted that the present invention has achieved a prophylactic and Z or therapeutic effect by including fused cells at such a high rate.
  • the fused cells to be included preferably constitute at least 10% of the cells contained in the pharmaceutical composition of the present invention, and more preferably at least 10% of the cells contained in the pharmaceutical composition of the present invention. 20%, more preferably at least 25% of the cells contained in the pharmaceutical composition of the present invention.
  • the pharmaceutical composition of the present invention further comprises an adjuvant.
  • an adjuvant By including such an adjuvant, the present invention further enhances the vaccine effect. Such enhancement of vaccine effect has not been reported for cell vaccines, and it is a surprising effect and its usefulness should be noted.
  • adjuvants that can be used in the present invention include mineral adjuvants (eg, aluminum hydroxide gel, alum), CpG, Freund's complete adjuvant, Freund's incomplete adjuvant, heat-killed pertussis, lipopolysaccharide, muramyl peptide, and the like. But not limited thereto, such as starch bentonite. In one preferred embodiment, CpG may be used as the adjuvant.
  • the pharmaceutical composition of the present invention comprises an antigen-presenting cell and at least one type of immune system.
  • a reactive feeder cell and an adjuvant In such a case, these three components can be provided as a mixture. This is because it has been found in the present invention that the adjuvant effect is exerted even when the antigen-presenting cells and the immunoreactive supply cells are not fused.
  • the adjuvant used may be a mineral adjuvant (eg, aluminum hydroxide gel, alum), CpG, Freund's complete adjuvant, Freund's incomplete adjuvant, heat-killed pertussis, lipopolysaccharide, muramyl peptide And starch bentonite, but are not limited thereto.
  • the present invention treats or prevents a disease caused by an immunoreactive supply cell, comprising an antigen-presenting cell, at least one type of immunoreactive supply cell, and a virus or a part thereof.
  • an immunoreactive supply cell comprising an antigen-presenting cell, at least one type of immunoreactive supply cell, and a virus or a part thereof.
  • the virus or a part thereof, the antigen-presenting cell and the immunoreactive supply cell to be used in the kit of the present invention are described in the section (fusion cell) in the present specification. It is understood that it can be used in any form.
  • the kit of the present invention further comprises instructions for instructing the fusion of the virus or a part thereof with the immunoreactive supply cell and the antigen-presenting cell.
  • a fusion method include, for example, mixing immunoreactive donor cells and antigen-presenting cells, adding a virus or a part thereof to the mixture, or administering the mixture to a host, and then administering the mixture to a host.
  • a virus or a part thereof is similarly administered, but is not limited thereto.
  • the present invention is characterized in that it is not always necessary to first perform fusion outside the body, and the kit of the present invention does not necessarily require that the immunoreactive supply cells and antigen-presenting cells be fused. It is understood that. Therefore, the virus used in the present invention is preferably inactivated. Viruses are often pathogenic. Inactivation can be achieved by any means, but can be effected by the use of alkylating agents or irradiation such as ultraviolet light.
  • the virus used in the kit of the present invention is preferably inactivated HVJ. This is because it has been found in the present invention that the cell fusion ability is excellent and the fused cells exhibit excellent prevention and / or therapeutic ability.
  • the instruction when the virus is provided in a non-inactivated state, in a preferred embodiment, the instruction further includes instructing to inactivate. Some may be further provided with an inactivator. Examples of such inactivating factors include, but are not limited to, alkylating agents and irradiation sources (eg, a source capable of irradiating X-rays, ultraviolet rays, and the like).
  • the invention further comprises the following steps: A) providing antigen presenting cells and at least one immunoreactive donor cell; and B) providing said antigen presenting cells and said at least one A method for producing a fused cell, comprising a step of mixing an immunoreactive donor cell with a virus or a part thereof.
  • the cells can be provided by any method.
  • an autologous cell it can be obtained from the host from which the cell is derived.
  • informed consent in advance.
  • animals it is also preferable to obtain them in an animal welfare manner.
  • the cell type and virus can take any of the forms described above (fused cells).
  • the method of the present invention further comprises a step of inactivating the immunoreactive donor cells. It is understood that inactivation of the cells can be performed using any of the techniques described herein in (Fused Cells) and other techniques known in the art. By introducing an inactivation step, it is expected that any pathogenicity derived from immunoreactive feeder cells will be eliminated.
  • the method of the present invention further comprises a step of confirming that the fusion cell of the present invention has cell surface markers of immunoreactive supply cells and antigen-presenting cells.
  • a confirmation step it can be confirmed that the fusion cells produced by the method of the present invention have desired properties.
  • the method of the present invention can be confirmed not only by confirming such cell surface markers but also by using other techniques.
  • the present invention may also include an additional cell fusion step in addition to cell fusion using the virus or a part thereof.
  • an additional cell fusion step in addition to cell fusion using the virus or a part thereof.
  • the technique used not only uses a virus or a part thereof, but may also use other techniques, for example, a polyethylene glycol method, an electofusion, and the like.
  • the present invention provides a disease caused by the immunoreactive supply cells, comprising the step of administering to a patient a fusion cell of the antigen-presenting cell and at least one type of immunoreactive supply cell.
  • Methods are provided for treating or preventing a disorder or condition.
  • the antigen-presenting cells, immunoreactive supply cells, and the like that can be used can take any form described in the section such as (fused cells) in the present specification.
  • the patient targeted by the present invention is any animal having an immune system, but preferably, the patient includes a mammal, and more preferably, the patient includes a human. Is understood.
  • the disease, disorder or condition caused by immunoreactive donor cells includes cancer (eg, melanoma, lung cancer, gastric cancer, adenocarcinoma, squamous cell carcinoma, adenosquamous cell carcinoma, thymic carcinoma, Lymphoma, sarcoma, liver cancer, non-Hodgkin's lymphoma, Hodgkin's lymphoma, leukemia, ovarian cancer, breast cancer, prostate cancer, ovarian cancer, victory cancer, colorectal cancer (colorectal cancer), multiple myeloma, neuroblastoma, Bladder cancer, cervical cancer, skin cancer, breast cancer, esophageal cancer, nephroma, brain tumor, etc., infectious diseases (eg, viruses, bacteria, fungi, parasites (protozoa, protozoa, leishmania, toxoplasma, etc.)) Disease), autoimmune diseases (eg, collagen disease, allergy (eg,
  • the method of the present invention is provided for prevention of a disease, disorder or condition caused by immunoreactive donor cells. This is because the fused cells provided by the method of the present invention act as a vaccine, and thus are expected to have a preventive effect. Alternatively, the methods of the invention are provided to prevent a recurrence of such a disease, disorder or condition.
  • the method of the present invention comprises an adjuvant (for example, alum, CpG, hydroxylamine oleoresinus mugenole, Freund's complete adjuvant, Freund's incomplete adjuvant, heat-killed pertussis, liposome).
  • an adjuvant for example, alum, CpG, hydroxylamine oleoresinus mugenole, Freund's complete adjuvant, Freund's incomplete adjuvant, heat-killed pertussis, liposome.
  • adjuvant for example, alum, CpG, hydroxylamine oleoresinus mugenole, Freund's complete adjuvant, Freund's incomplete adjuvant, heat-killed pertussis, liposome.
  • the process further includes: It is understood that such adjuvants can take any of the forms described herein, such as in (pharmaceutical compositions). Administration of the adjuvant can be before, simultaneously with, or after administration of the fusion cells or other
  • the antigen of the immunoreactive donor cell used in the method of the present invention may be unknown. This is because even if it is unknown, the present invention has achieved, for the first time, the effect of preventing and treating by using the method of the present invention.
  • the method of the present invention is provided for preventing cancer metastasis. This is because the effect of the present invention for preventing and treating cancer is also useful for preventing cancer metastasis.
  • a prophylactically, therapeutically or prognostically effective amount can be determined by one of ordinary skill in the art using techniques well known in the art, while taking into consideration various parameters. To determine such an amount, For example, it can be easily determined by those skilled in the art in consideration of the purpose of use, the target disease (type, severity, etc.), the patient's age, weight, medical history, etc. (for example, the “vaccine handbook”, The Institute of Health Sciences Gakuyukai (1994); Vaccination Guide, 8th Edition, Mikio Kimura, Munehiro Hirayama, Harumi Sakai, Modern Publishing (2000); Standards for Biological Preparations, Bacteria Preparation Association (1993)).
  • the present invention provides the following steps: A) a step of identifying a disease, disorder or condition; B) a step of identifying an immunoreactive supply cell associated with the disease, disorder or condition. C) providing a fused cell of the antigen-presenting cell and at least one of the immunoreactive donor cells; and D) administering the fused cell to a patient. And a method for treating or preventing a disease, disorder or condition caused by the above.
  • identification of a disease, disorder or condition can be performed by any method known in the art. For identification of such a disease, for example, a standard medical book (for example, the latest version of the Merck Manual) can be referred to.
  • Identification of cells associated with such a disease, disorder or condition can also be performed by any method known in the art.
  • the identification of such cells is described, for example, in standard medical books (e.g., M The latest version of the erck Manual) can be considered. It is understood that in this method of the invention, the provision and administration of the fused cells can also take any of the forms described elsewhere herein (eg, such as in the section (Fused Cells)). You.
  • the present invention provides a method for treating or preventing a disease, disorder or condition of a fusion cell of an antigen-presenting cell and at least one type of immunoreactive supply cell, which is caused by the immunoreactive supply cell.
  • a method of manufacturing a medicament of the present invention It is understood that the cells, viruses, and other components to be used herein should take any of the forms described herein for (fused cells), (pharmaceutical compositions). It is also understood that diseases, disorders and conditions should also take any form described herein, such as in (Prophylaxis / Therapeutic kits), (Prophylaxis' methods for treatment) and the like.
  • Example 1 Preparation of fused cells
  • Mouse dendritic cells and mouse melanoma B16BL6 X-irradiation were fused with HVJ (inactivated by ultraviolet light). About 20% of fused cells were obtained.
  • HVJ inactivated by ultraviolet light.
  • a and B the lipid of each cell was labeled with a fluorescent dye, and the fusion image of ⁇ cells and cancer cells was confirmed.
  • C was double-stained using antibodies recognizing the respective surface antigens (Figure 1A and Figure 1C). The procedure is described below.
  • HVJ (species Z) was purified by centrifugation from the chorioallantoic fluid of chicken eggs and the titer was determined using known techniques. HVJ seed virus is propagated and isolated using fertilized eggs of SPF (Specific pathogen free). 'Purified HVJ (species Z) is dispensed into a cell storage tube, and 10% DMS ⁇ is added to liquid nitrogen. Stored and prepared.
  • Seed virus (removed from liquid nitrogen) with a polypeptone solution (mixed with 1% polypeptone and 0.2% NaCl, adjusted to ⁇ 7.2 with 1M NaOH, sterilized in an autoclave, and stored at 2 ° C-6 ° C) was diluted 500-fold and placed at 2 ° C-6 ° C. Eggs were disinfected with isodine and alcohol, a hole was made through the virus injection site with a awl, and 0.1 ml of the diluted seed virus was injected using a 26-gage lml syringe with a needle into the chorioallantoic cavity. .
  • the melted paraffin (melting point 50-52 ° C) was placed over the hole using a Pasteur pipette and closed.
  • the eggs were placed in an incubator and kept for 3 days at 34-36.5 ° C and a humidity of 40% or more.
  • the inoculated eggs were then placed at 2 ° C-6 ° C overnight.
  • the next day divide the air chamber of the egg with tweezers, place a 10 ml syringe with an 18-gauge needle into the chorioallantoic membrane, aspirate the chorioallantoic acid, collect in a sterile bottle, 2 ° C 6 ° C Saved.
  • HVJ Concentration of HVJ was performed as necessary. Approximately 100 ml of the HVJ-containing chorioallantoic fluid obtained in the above process (collect the HVJ-containing chicken egg chorioallantoic acid and store at 2 ° C and 6 ° C) is placed in two 50-ml centrifuge tubes using a wide-mouth Komagome pipette. The eggs were centrifuged at 3,000 rpm for 10 minutes at 2 ° C-6 ° C (with the brake off) in a low-speed centrifuge to remove egg tissue fragments.
  • HVJ concentrate 0.1 ml was mixed with 0.9 ml of BSS, the absorbance at 540 nm was measured with a spectrophotometer, and the virus titer was converted to hemagglutinating activity (HAU).
  • HAU hemagglutinating activity
  • the virus was inactivated by ultraviolet irradiation (99 mj m 2 ). The inactivation was performed immediately before use, but need not be performed immediately before use.
  • the cell lines used are as follows:
  • CT26 colon adenocarcinoma H_2 d ) (CRL-2638). These were all purchased from the American 'Type Kaliya I. Collection (ATCC, Rockville, MD).
  • Mouse dendritic cells derived from bone marrow were used as antigen-presenting cells. The cells were isolated from the bone marrow of the mouse tibia and femur. After separation, place in RPMI1640 medium (containing 10% heat-inactivated fetal serum, 50 ⁇ 2_mercaptoethanol, lOngZml recombinant mouse granulocyte macrophage colony stimulating factor (GM-CSF; Genzyme-Techne, Minneapolis, ⁇ )) Transferred and cultured. Dendritic cells are fused one day before lipopolysaccharide (LPS) And matured. To increase the fusion efficiency, dendritic cells and cancer cells were labeled with red and green fluorescence respectively.
  • LPS lipopolysaccharide
  • PKH26 and PKH67a were used as fluorescent labels, respectively. Labeling was in accordance with the manufacturer's instructions (Zynaxis, Phoenixville, Pike Malvern, PA). Alternatively, dendritic cells and B16BL6 cells were isolated from a fluorescein'isothiosinate (FITC) conjugated mouse anti-mouse monoclonal antibody (mAb) specific for CDllc and a phycoerythrin (PEC) specific for gplOO, respectively. ) Labeled with conjugated mouse monoclonal antibody (BD PharMingen, San Diego, CA).
  • FITC fluorescein'isothiosinate
  • mAb mouse anti-mouse monoclonal antibody
  • PEC phycoerythrin
  • Itoda Tsukiha was irradiated with gamma-rays at a dose of lOOGy for Gammacell (Nordion International, Canada, Canada).
  • Cell fusion was performed at a cancer cell to dendritic cell ratio of 1: 2. Fusion was performed using HVJ prepared as described above.
  • dendritic cells (4 x 10 6 cells) were converted to a buffered physiological diet containing 2 mM CaCl.
  • the cells were suspended in 250 ⁇ l of saline (BSS; 10 mM Tris-Cl pH 7.5, 137 mM NaCl, 5.4 mM KC1).
  • BSS saline
  • Gamma-irradiated cancer cells (2 x 10 6 cells) were suspended in a 250 / i 1 BSS containing 2 mM CaCl.
  • HVJ HVJ (0-1, 000 HAU) (in 500 ⁇ BSS) in a 2 ml tube. After incubation at 0 ° C for 5 minutes, the mixture was incubated at 37 ° C for 15 minutes with shaking (120 rpm) in a water bath to induce cell fusion. After centrifugation at 1,200 rpm for 3 minutes at 4 ° C., the fusion product was washed twice with 1.5 ml of BSS to remove free HVJ. Thereafter, this fusion product was cultured overnight at 37 ° C. in 5% C ⁇ . After fusion 2
  • Example 1 Next, the fusion cells prepared in Example 1 were cultured for 24 hours, and cytokin in the culture supernatant was measured by ELISA.
  • Fusion products made using HVJ of 0 HAU (ie, a simple mixture) and 500 HAU (ie, fusion cells) were treated with 10 ⁇ g / ml CpG oligonucleotide for 24 hours. After incubation without, the supernatant was harvested from the cell culture and stored at _80 ° C. The concentrations of TNF_ct, IL_12p40 and interferon (IFN) _y in the supernatant were determined according to the manufacturer's instructions by ELISA ELISA kit for mouse cytoplasmin.
  • the fused cells of the present invention were inoculated intradermally into the mouse, and two weeks later, spleen lymphocytes were taken out and the secretion of interferon ⁇ ( ⁇ ) and cytotoxic ⁇ cells (CTL) against melanoma ( ⁇ ) were measured. The procedure is described below.
  • mice 10 mice Z group
  • mice 10 mice Z group
  • these vaccinations were carried out by injecting a total of 200 ⁇ l of PBS into the skin (id) on the bilateral hind flank near the base of the tail at a time (100 ⁇ ⁇ / side) Per abdomen).
  • the number of cells in the fusion product was described per cell number used in the dendritic cell-cancer cell fusion.
  • spleen cells were pooled from each mouse group (3 / group).
  • Cells (5 x 10 6 cells / well) with mitomycin C (MMC) -treated cancer cells in a 20: 1 ratio, 2 ml T cell culture medium (10% heat inactivated FBS, 100 units in RPMI1640 medium) per ml penicillin, 100 g Zml streptomycin, 50 ⁇ M 2-mercaptoethanol) in 5% CO 2 in 24 ⁇ l.
  • MMC mitomycin C
  • Harmful T lymphocytes were harvested on day 5 and used as effector cells in a standard 4 hour 51 Cr release assay to target effector cells against target cancer cells. Cytotoxic activity was tested. Briefly, target cancer cells (1 ⁇ 10 6 cells) were grown at 37 ° C. in 200 ⁇ l RPMI1640 (supplemented with 10% FBS) with 100 ⁇ Ci 51 Cr (Amersham Bioscience, Buckinghamshire England). Labeled for 90 minutes. Label target cells (1 ⁇ 10 4 cells / well) for 4 hours 37. C was incubated with effector cells in a 96-well microtiter plate (filled with 200 ⁇ l of T cell medium and cells were introduced at various effector / target ( ⁇ / ⁇ ) ratios).
  • CTLs Harmful T lymphocytes
  • the plate was then centrifuged and the radioactivity of the supernatant was measured and counted using a MicroBeta Trilux Scintillation Counter (Wallac, Gaitherburg, MD). Maximum or spontaneous release was defined as counts from samples incubated with 2% Triton X-100 or medium alone, respectively.
  • the cytotoxic activity was calculated by the following equation.
  • 51 Cr specific release rate (experimental release value-natural release) x 100 / (maximum release-natural release)
  • Atsushi went in duplicate. The natural release of all Atsushi was less than 20% of the maximum release.
  • mice were challenged by subcutaneous (sc) injection of 1 ⁇ 10 5 cells of B16BL6.
  • BALB / c mice were injected with 1 ⁇ 10 5 RENCA cells at a different but proximal back site than the two vaccinated sites.
  • mice were monitored daily and cancer incidence was considered positive when tumor length exceeded 3 mm.
  • Tumor size was measured with a vernier caliper every other day in a blinded fashion. The volume of the tumor was increased by the following equation.
  • Tumor volume (mm 3) Length X Width 2/2.
  • mice were euthanized when the case or size ulcerated exceeded 4000 mm 3. 60 days after tumor inoculation, 5 ⁇ 10 4 cells of EL4 or 1 ⁇ 10 5 cells of RENCA cells and 5 ⁇ 10 4 cells of CT26 cells were subcutaneously subcutaneously applied to tumor-free C57BL / 6 mice or BALB-free mice, respectively. Challenge by injection revealed the tumor specificity of vaccination in vivo. The details are shown in Table 1 below.
  • Table 1 shows the tumor re-inoculation experiments on tumor rejected mice. Again, mice that were tumor free for the first tumor challenge in two different tumor models Challenged with the same tumor cell or another syngeneic tumor cell. Mice were challenged subcutaneously with 1 ⁇ 10 5 B16BL6 or RENCA cells or 5 ⁇ 10 4 EL4 or CT26 cells. The ratio of the number of tumor-free mice on day 60 is the number of mice that were first challenged or re-challenged with tumor cells, and the percentages are shown in the table above. Immunity with fused cells and CpG provided complete protection against re-challenge with the same cells (B16 BL6 and RENCA). In other syngeneic tumor cells (EL4 and CT26) . Immunization alone with the fused cells also provided protection against a second tumor challenge with the same tumor cells (RENCA), but not completely. Repeating these experiments twice again gave almost the same results.
  • FIG. 4 shows the results.
  • the tumor volume is shown in FIG. 4A.
  • the mouse survival rate shown in FIG. 4B the effect of the fused cells was observed in the proportion of the tumor-free mice shown in FIG. 4C, and the combination of the fused cells and CpG was the most effective.
  • FIGS 5A to 5D show further results.
  • melanoma tumor-associated antigens are known, but vaccine effects were observed using renal cancer cells (RENCA), for which tumor-associated antigens were not identified.
  • RENCA renal cancer cells
  • the effect of fusion cells was observed, and the combination of fusion cells and CpG was most effective It was a target.
  • mice Ten days after the second vaccination, 50/11 of C57BL / 6 mice (8 mice / group) 5 ⁇ 10 5 B16BL6 cells suspended in 83 were injected subcutaneously (sc) into the right hind paw to initiate primary tumor growth.
  • the present invention is effective in disease models other than cancer.
  • the cells are killed by 0 / radiation.
  • the killed cells and dendritic cells are fused with HVJ and immunity is activated using CpG.
  • the procedure follows Example 1 in principle. In this way, a vaccine that is effective against viral or bacterial infection can be made.
  • the effect of the fused cells thus prepared is confirmed by the procedure described in Examples 2-5. It is understood that bacteria and viruses can be used to make cellular vaccines as well.
  • macrophages as other antigen-presenting cells, confirm whether they have the same vaccine effect. Since macrophages are known to be fused with HVJ, fused cells are prepared using macrophages instead of ⁇ cells according to the procedure described in Example 1. The effect of the fusion cells thus produced is confirmed by the procedure described in Examples 2-5. It is understood that macrophages can be used for cell vaccine production as well.
  • Dendritic cells and melanoma cells were fused (prepared as described in Example 1) using polyethylene glycol as described in Patent Document 1. However, the fusion efficiency was less than 5%. No therapeutic effect was observed.
  • the present invention has the utility of treating or preventing a disease associated with an antigen without specifying the antigen.
  • the present invention provides vaccines and related utilities that can be used for such treatment or prevention.

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Abstract

L'objectif est de prévoir un procédé, un médicament (en particulier un vaccine), un système etc. pour traiter ou prévenir facilement une maladie, un trouble ou condition souhaitée (en particulier un cancer). Un vaccin préventif efficace pour maladie (par exemple le cancer) obtenu en utilisant une technique de fusion de cellule avec l'utilisation d'un vaccin tel qu'un HVJ inactif et/ou un adjuvant, où les métastases et la récurrence de cancer peuvent être particulièrement inhibées. Notamment, une cellule fondue et/ou un adjuvant, où les métastases et la récurrence de cancer peuvent être particulièrement inhibées. Notamment une cellule fondu préparé á partir d'une cellule présentant un antigène avec au moins une cellule appliquant l'immunoréactivité et une technique s'y rapportant.
PCT/JP2005/002941 2004-02-26 2005-02-23 Vaccins avec cellule WO2005083062A1 (fr)

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JP (1) JPWO2005083062A1 (fr)
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JP2002510644A (ja) * 1998-04-03 2002-04-09 ユニバーシティ オブ アイオワ リサーチ ファウンデーション 免疫治療用オリゴヌクレオチドおよびサイトカインを用いる免疫系刺激のための方法および産物
WO2002032378A2 (fr) * 2000-10-20 2002-04-25 Tsuneya Ohno Cellules de fusion et compositions cytokiniques pour le traitement de maladies
JP2003524020A (ja) * 2000-02-27 2003-08-12 エーバーハルト−カルルス−ウニヴェルズィテート テュービンゲン ウニヴェルズィテートクリーニクム ハイブリッド細胞ワクチン

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CA2286873C (fr) * 1997-04-15 2010-07-13 Dana-Farber Cancer Institute, Inc. Hybrides de cellules dendritiques

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JP2002510644A (ja) * 1998-04-03 2002-04-09 ユニバーシティ オブ アイオワ リサーチ ファウンデーション 免疫治療用オリゴヌクレオチドおよびサイトカインを用いる免疫系刺激のための方法および産物
JP2003524020A (ja) * 2000-02-27 2003-08-12 エーバーハルト−カルルス−ウニヴェルズィテート テュービンゲン ウニヴェルズィテートクリーニクム ハイブリッド細胞ワクチン
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TREFZER U. ET AL.: "A phase I trial with a hybrid cell vaccine in patients with metastatic melanoma", ADV. EXP. MED. BIOL., vol. 451, 1998, pages 519 - 525, XP002987126 *

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