WO2003018784A1 - Procede de production de lymphocytes t humains specifiques d'un antigene et medicaments associes - Google Patents

Procede de production de lymphocytes t humains specifiques d'un antigene et medicaments associes Download PDF

Info

Publication number
WO2003018784A1
WO2003018784A1 PCT/JP2002/008499 JP0208499W WO03018784A1 WO 2003018784 A1 WO2003018784 A1 WO 2003018784A1 JP 0208499 W JP0208499 W JP 0208499W WO 03018784 A1 WO03018784 A1 WO 03018784A1
Authority
WO
WIPO (PCT)
Prior art keywords
cells
cancer
blood
patient
cell
Prior art date
Application number
PCT/JP2002/008499
Other languages
English (en)
Japanese (ja)
Inventor
Takashi Nishimura
Original Assignee
Toray Industries, Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Toray Industries, Inc. filed Critical Toray Industries, Inc.
Publication of WO2003018784A1 publication Critical patent/WO2003018784A1/fr

Links

Classifications

    • 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
    • A61K2239/48—Blood cells, e.g. leukemia or lymphoma
    • 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
    • A61K39/4611—T-cells, e.g. tumor infiltrating lymphocytes [TIL], lymphokine-activated killer cells [LAK] or regulatory T cells [Treg]
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/06—Antianaemics
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0636—T lymphocytes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K2035/124—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells the cells being hematopoietic, bone marrow derived or blood cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/515—Animal cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/57—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/20—Cytokines; Chemokines
    • C12N2501/22—Colony stimulating factors (G-CSF, GM-CSF)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/20—Cytokines; Chemokines
    • C12N2501/23—Interleukins [IL]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/20—Cytokines; Chemokines
    • C12N2501/24—Interferons [IFN]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2502/00—Coculture with; Conditioned medium produced by
    • C12N2502/11—Coculture with; Conditioned medium produced by blood or immune system cells

Definitions

  • the present invention relates to a method for producing human Th1 cells, cells produced by the method, and pharmaceutical uses thereof.
  • the human Thl cells of the present invention are particularly useful for immunotherapy of cancer, host versus graft reaction, and the like.
  • cancer Treatment of cancer is one of the most important medical issues. To date, a variety of surgical, chemo, and radiation therapies have been tried, but the outcome has improved, but is still not at a satisfactory level. Since the 1980s, immunotherapy has also been attempted and progressed with improvements, and is expected as a new cancer treatment. Originally, it is thought that cancer cells can be eliminated by the immune function of the host.However, in patients who have a large amount of cancer cells in the body and are in an immunosuppressed state, the patient's own immune system alone is no longer sufficient for cancer. The cells cannot be eliminated.
  • immunotherapy is intended to treat cancer by administering various cytotoxic agents or immunostimulating substances from outside the body, or by enhancing the active immunity in the body by injecting activated lymphocytes. It is. To date, various methods have been tried, but the effect is still limited and further improvement is required (Fujimimoto T. et al: J. I. uno l 158: 5619, 199) Fu, Patent No. 2530966 and "Current State of Cellular Immunotherapy", History of Medicine, vol. 195, No. 1, 2000).
  • dendritic cells play a very important role in antigen presentation, the fundamental response of the immune response (Inaba. K. et al: Proc. Nat I. Acad Sc. USA 80: 6041, 1983, Banchereau J. et al: Nature 392: 245, 1998).
  • vaccine therapy for cancer using dendritic cells is currently receiving attention, and some of its promises have been reported (Sato ⁇ et al: Int. Immunol. 12: 335, 2000, "The Present Situation of Cellular Immunotherapy", History of Medicine, vo 1.195, No. 1, 2000).
  • the dendritic cells are transfused into a patient, and the dendritic cells are further transferred to the patient.
  • This method involves inducing cytotoxic T cells (GTL) from peripheral blood lymphocytes of patients as stimulator cells and injecting them into cancer patients.
  • GTL cytotoxic T cells
  • Such immunotherapy can specifically sensitize antigen-presenting cells when sensitizing dendritic cells and other antigen-presenting cells with cancer antigen-cancer antigen-derived peptides in vitro. Then, how efficiently the required amount of GTL can be induced, and how efficiently the GTL can reach the lesion in the cancer patient, are important matters for improving the therapeutic effect.
  • the most rational antigen used to sensitize dendritic cells is the cancer cell antigen of the patient, but the cancer peptide antigen is the major histocompatibility complex of the patient.
  • MHG MHG
  • MHG restriction MHG restriction
  • the specific antigen used in this case is an MHG class I binding peptide (expressed on all nucleated cells and platelets, (A peptide derived from a cytoplasmic protein is presented to cytotoxic GD8-positive killer cells.)
  • MHG class I binding peptide expressed on all nucleated cells and platelets, (A peptide derived from a cytoplasmic protein is presented to cytotoxic GD8-positive killer cells.)
  • GTL class I-binding peptide alone
  • attempts to overcome this problem by using various adjuvants to enhance immunity have continued.
  • a practical method has not yet been established. For example, trials using interleukin 2 (IL-2), IL-12, etc. (Ribas A et al: Cancer Res 57: 2865,
  • MHG class I MHG class II
  • GD4-positive helper T cells peptides mainly expressed on antigen presenting cells and derived from plasma membrane and extracellular proteins
  • MHG class II-restricted antigen-specific killer T cells GD4 positive GT1J or GD4 positive helper. It is extremely important to create an environment in which Th1-driven systemic cellular immunity, which is the induction of Nokiller cells, is activated.
  • helper T cells are divided into two subtypes, TM and Th2, and that many immune responses are controlled in the mouse system ( Mosmann, TR: Ann. NY Acd. Sci. 664: 89, 1992). It was also revealed that a similar helper T cell subset exists in humans, and it has been speculated that the balance between Th1 and Th2 plays an important role in the development of a number of diseases including cancer (Sal game, P. et. Science, 254: 279, 1991, Abbas. AK et al. Nature 383: 787, 1996).
  • Th1 and Th2 are in a functional balance with each other, and when this balance is maintained, they are healthy, but when Th1 becomes excessive, autoimmune diseases, rheumatoid arthritis, etc. In multiple sclerosis and the like, it is said that excess Th2 causes cancer, immunodeficiency, allergic disease and the like.
  • This method of immunoregulation based on TM ZTh2 balance is also expected to lead to effective treatment of cancer, but no efficient method for inducing Thl cells and Th2 cells has been established, and specific clinical applications have not been established. Not yet done.
  • the concept of Th1 / Th2 balance was originally proposed in mouse experiments. It is necessary to verify whether they have the ability to induce T cells and produce cytokines that control cancer cytotoxicity and immune balance that have functions applicable to immunotherapy. This verification is not yet sufficient.
  • bone marrow transplantation and hematopoietic stem cell transplantation can also be considered as one of the immunotherapy using Th1 cells.
  • Bone marrow transplants are used to treat leukemia and aplastic anemia
  • hematopoietic stem cell transplants are used in solid tumors after high-dose chemotherapy or intense radiation therapy (myeloablative hematopoietic stem cell transplants) and in patients with solid cancers. After a small amount of chemotherapy or radiation therapy (non-myeloablative hematopoietic stem cell transplantation), 02 08499
  • HVGD host-graft reaction
  • GVHD weak graft-versus-host reaction
  • immunotherapy requires the isolation of antigen-presenting cells and antigen sensitization, followed by cultivation, and separate isolation and culturing of T cells, which requires a great deal of labor and high know-how. Desired.
  • there remain issues such as difficulties in obtaining antigens such as cancer cells and cancer peptides, complicated procedures for handling them, and adequate facilities.
  • the use of patient cells poses medical problems, such as the invasion and burden on patients, and the fact that it is virtually impossible to draw blood from patients who have become extremely anemic due to chemotherapy or radiation therapy. is there. Therefore, in the case of using blood cells derived from a patient, it is extremely important for practical use that the target cells are easily and efficiently induced from a small amount of sample.
  • immunotherapy has many challenges, including not only the concept verification but also the development of corresponding medical technologies, and simple and useful treatments including these have not yet been established. There is no present.
  • An object of the present invention is to provide a method for producing human Th1 cells, which solves these problems, and enables a new therapeutic method for cancer and immune diseases, a medicament comprising cells produced by the production method,
  • An object of the present invention is to provide a use for a disease that can be treated with cells produced by the production method.
  • the present inventors have made it possible to easily obtain MHG class II-restricted antigen-specific human Th1 cells having cytotoxic activity using exogenous cytokines from peripheral blood and bone marrow cells of patients.
  • the present inventors have found that the human Th1 cells can be used for cancer treatment, bone marrow transplantation, and the like, and have completed the present invention.
  • the present invention provides a method for inducing dendritic cells by using peripheral blood or bone marrow collected from a patient as a material, and culturing the cells in vitro without adding a disease-derived antigen from the outside and using an exogenous cytokine.
  • a major histocompatibility complex class 11-restricted antigen which comprises culturing CD4 positive T cells contained in a substance in the presence of the dendritic cells and inducing human Th1 cells having cytotoxic activity.
  • a method for producing a specific human Th1 cell is provided.
  • the present invention also provides a human Th1 cell produced by the method of the present invention, and provides a medicine or a pharmaceutical composition containing the human Th1 cell.
  • the present invention provides drugs for treating cancer, drugs for treating malignant diseases of the blood or hematopoietic system, and drugs for controlling host versus graft reactions.
  • therapeutic agents for malignant diseases of the blood or hematopoietic system include various therapeutic agents for leukemia, malignant lymphoma, multiple myeloma, myelodysplastic syndrome, or aplastic anemia.
  • the present invention provides a use of the above-mentioned cell of the present invention for producing a cancer therapeutic agent, a therapeutic agent for blood or hematopoietic malignancy, or a host versus graft reaction control agent.
  • the present invention provides a method for treating cancer, blood or hematopoietic malignant disease, which comprises administering an effective amount of the above-described cell of the present invention to each patient of cancer, immune disease, blood or hematopoietic malignant disease.
  • the present invention also provides a method for controlling a host-graft reaction, which comprises administering an effective amount of the above-described cell of the present invention to a patient who wants to control the host-graft reaction.
  • the antigen-specific Th1 cells obtained by the method of the present invention also produce cytokines, and can exert specific cytotoxic activity against target cells and precise cytotoxic activity against target cells, so that therapeutic effects on patients are obtained. Can induce a Th1-driven systemic cellular immune state with Moreover, since the obtained Th1 cells are derived from the T lymphocytes of the patient, even if they are returned to the body of the patient, no problem such as harmful rejection occurs. Therefore, effective immunotherapy for various cancers including leukemia can be performed. It is also useful as a method for controlling host-graft reactions. BRIEF DESCRIPTION OF THE FIGURES
  • FIG. 1 is a diagram showing a method for inducing activated T cells using leukemia cells as an example. The commonly used conventional method (A) and the method (B) of the present invention are shown.
  • FIG. 2 is a diagram showing the results of identification of lymphocyte surface antigens before and after culture in Example 1. After culturing, pure CD4-positive cells are obtained. GD4-positive and ZGD8-negative helper T cells, which had only 0.92% before culture (before culture), were induced to 94.66% after culture (after culture). It turns out that it is a pure CD4 positive helper killer cell population.
  • FIG. 3 shows the cytotoxicity of T lymphocytes against autologous cancer obtained by the method of the present invention (Th1 condition) or the method of the comparative example (Th0 condition and Th2 condition) in Examples 1 to 3.
  • ThO ThO-type helper T cell induction condition; Th0 / 1; ThO and Thl-type helper T cell induction condition; Th1; Th1-type helper T cell induction condition; Th2; Th2-type helper T cell induction condition
  • the expression is as follows, and it can be seen that the cytotoxicity is highest when induced by Th1 type.
  • FIG. 4 is a diagram showing the amounts of IFN-r production obtained in Examples 1 to 3 by the method of the present invention (Th1 condition) or the method of the comparative example (Th0 condition and Th2 condition).
  • ThO In the case of ThO type helper T cell induction condition, Thl; In the case of Th1 type helper T cell induction condition, Th2; In the case of Th2 type helper T cell induction condition,
  • FIG. 5 is a diagram showing MHG class II antigen restriction of Example 3. Inhibition of IFN-r production by autologous cancer cell stimulation and cytotoxicity against autologous cancer cells by antibodies against MHG class 11 antigens shown by Th1 cells induced from IM patients.
  • the MHG class 11 antibody inhibits IFN- production and cytotoxic activity and clearly shows MHG class 11 restriction.
  • the present invention preferably induces dendritic cells from granulocyte macrophage colony stimulating factor (GM-GSF) and IL-3 from a patient's bone marrow or peripheral blood without externally adding an antigenic substance. Then, by adding IL-2 to the culture system, a small amount of GD4-positive T cells were allowed to interact with dendritic cells and proliferated. Cytotoxic activity against autologous cancer cells and production of IFN- ⁇ can be obtained by culturing CD4-positive T cells with Th1-inducible cytokines such as IL-12 and interferon ⁇ (IFN-r). The following are the Th1 cell type GD4-positive helper killer cells and the method of inducing them.
  • Th1-type GD4-positive helper killer cells have strong cytotoxicity and high IFN-T production ability, so they elicit an immune response in the patient and induce a Th1-driven systemic cellular immune state You can expect the ability to do it. Since the immunotherapeutic activity is extremely high, it can be used to treat cancer and control host-graft reactions. Also, since the patient's original autologous cells are used, it is highly safe and can be expected to avoid serious side effects.
  • Fig. 1 shows an example of a conventional method for sensitizing cancer antigens followed by a method for inducing activated T cells (Fig. 1A), using leukemia as an example.
  • Fig. 1A shows the present invention (FIG. 1B) using killer cells.
  • cytokines are added over time in a single culture system that does not require a foreign cancer antigen, which is conventionally required, and is performed without mixing with another cell. This makes it possible to obtain MHG class II-restricted Th1-type CD4-positive helper killer cells very simply and efficiently.
  • the cells used here are not particularly limited as long as they are peripheral blood or bone marrow of various cancer patients, but can be preferably obtained from blood cancer patients such as leukemia and malignant lymphoma.
  • the peripheral blood or bone marrow may be any of a fresh sample, a cryopreserved sample, and a cryopreserved sample. Since the amount of the sample required for the present invention may be a very small amount of about a few mL of bone marrow or peripheral blood, it may be a small sample for examination or a residual sample, or may be a frozen sample. Therefore, it can be applied to cancer patients with extreme anemia and leukemia patients with few normal blood cells, and has the advantage that it can be applied to many target diseases with little burden on patients. For example, it is possible to induce 10 7 to 10 8 cells required for normal adoptive immunotherapy from 1 to 2 mL of leukemia blood.
  • peripheral blood whole blood may be cultured, or only white blood cell components may be separated and cultured, but the latter is more efficient and preferable.
  • mononuclear cells may be separated from leukocyte components.
  • the whole cells that make up the bone marrow are cultured.
  • mononuclear cells may be separated and cultured therefrom.
  • Peripheral blood and its leukocyte components and bone marrow cells include monocytes, dendritic cells, hematopoietic stem cells or immature dendritic cells, GD4-positive cells, and target cells such as blood cancer cells. Cancer cells are also included.
  • Th1-driven systemic cell-mediated immunity is not formed in which peripheral blood and bone marrow remain in a state where a sufficient therapeutic effect can be obtained. Is obtained.
  • Th1 cells can be prepared from blood from a very small amount, which was conventionally impossible or extremely difficult to prepare.
  • the ability to induce cells from frozen lymphocytes greatly reduces the burden on patients. For example, compared with the conventional method, where blood is collected at a time of about 20 mL and blood is collected once every two weeks, this method freezes lymphocytes with 5 mL of blood at a time. Store and make 5 servings per 1 mL. Patients with leukemia or solid cancer are often anemic, and frequent blood collections place a heavy burden on patients, but this law greatly reduces the burden.
  • cells provided from donors can be similarly cultured by this method, and the host-graft reaction can be appropriately controlled, thereby improving graft survival and treating underlying diseases.
  • the host-graft reaction can be appropriately controlled, thereby improving graft survival and treating underlying diseases.
  • the production method of the present invention can be carried out in a single step by adding the above-described various cytotoxic agents to a medium, or a first step of inducing dendritic cells, and GD4-positive T cells contained in a culture. Is cultured in the presence of the above-mentioned dendritic cells, and a second step of inducing human Th1 cells having cytotoxic activity can also be performed.
  • the cytodynamic force used for stimulating cells in the present invention is preferably at least GM-GSF, IL-3, IL-2 and IFN-.
  • Other cytokines, such as IL-12, may be used as needed.
  • culture period The period is not particularly limited as long as the required number of dendritic cells or Th1-type CD4-positive helper / killer T cells are induced, but is usually performed for 3 to 8 weeks.
  • the first step is performed in the presence of granulocyte macrophage colony stimulating factor and / or interleukin 3, and the second step is to add interleukin 2 to the culture obtained in the first step. And growing the CD4-positive T cells while interacting with the dendritic cells, and further adding interferon r and / or interleukin 12 to obtain human Th1 having cytotoxic activity. Inducing into cells.
  • the culture obtained in the first step is preferably interleukin 2 and granulocyte-macrophage coagulation stimulating factor or interleukin 3, more preferably Granulocyte macrophage colony stimulating factor and interleukin 3 may be further added to interleukin 2 to the culture obtained in the step, and the culture may be continued.
  • the second step can be carried out in one step or in two steps, but when it is carried out in two steps, the step of expanding GD4-positive T cells and human Th1 cells having cytotoxic activity Add the cytokines used in any of the steps of induction.
  • the culture period is not particularly limited as long as the required number of dendritic cells and Th1-type GD4-positive helper killer T cells are induced, but usually the first step is 1 to 7 days, preferably 2, The first stage of the second step is 2 to 6 weeks, preferably 2 to 3 weeks, and the second stage of the second step is 2 to 7 days, preferably 2 days.
  • any cytokine may be used, such as a natural type or a genetically modified type, as long as it has characteristics that have been confirmed to be safe and bioactive. However, it is preferable to use a standard that has been used for medical purposes and that has been assured of the required quality in the minimum amount required.
  • the concentration of the cytokine to be added is not particularly limited in any of the methods and steps described above, as long as dendritic cells and TM-type GD4-positive helper killer T cells are induced. The concentration is preferably about 10-1000 ng / mL, more preferably about 20-500 ng / mL.
  • the concentration of each cytokine is usually preferably 1 ng / nl or more.
  • Culture is white It can be performed using a well-known medium usually used for culturing blood cells.
  • the culture temperature is not particularly limited as long as leukocyte proliferation is possible, but the human body temperature of about 37 ° C is most preferable.
  • the gaseous environment in the culture is not limited especially as long as the proliferation of white blood cells, it is preferable to vent the 5% G0 2.
  • the equipment used for cell separation and culture can be appropriately used. It is preferable that safety is confirmed for medical use, and that the operation is stable and simple.
  • for cell culture devices irrespective of general containers such as petri dishes, flasks, bottles, etc., stacked containers, multi-stage containers, roller bottles, spinner bottles, bag type incubators, hollow fiber columns, etc. can also be used. .
  • the following procedure is considered as a specific treatment form. That is, blood is collected from a patient with the target disease, lymphocyte and leukocyte components are separated, culture is started, and the Th1-type GD4-positive helper killer T cells induced 2 to 4 weeks later are infused into the patient themselves .
  • the burden on the patient is basically only blood collection and transfusion, and the cancer cells are suppressed by the induced Th1-type GD4-positive helper killer T cells, and as a result are treated.
  • cells provided by donors are similarly cultured, and host-graft reactions are controlled, leading to improved graft survival and treatment of primary diseases be able to.
  • Thl cells When using the Th1 cells obtained by the present invention for therapy, it is preferable to administer Thl cells to peripheral blood by intravenous injection, infusion, or the like. In the case of solid cancer, it may be administered to peripheral blood or directly into cancer tissue.
  • the number of cells to be administered can be appropriately set depending on the patient's condition, etc., but when administered to peripheral blood, it is usually about 10 7 to 10 11 cells per adult patient, preferably 10 8 to 10 9 About one.
  • ALL Human acute lymphocytic leukemia separating mononuclear from bone marrow cells or peripheral blood of patients, 2 ⁇ 10 6 cells Roh including mL concentration in 10% human serum AIM-V medium (GI BG0 - BRL), seeded in a 12-well plate, seeded with GM-GSF (30 ng / mL) and IL-3 (30 ng / mL). ) was added and cultured for 2 days to induce dendritic cells. Thereafter, IL-2 (100 U / mL (10 ng / mD) was added to the culture solution, and cultured for 14 to 21 days to obtain a large amount of GD4-positive T cells.
  • GI BG0 - BRL human serum AIM-V medium
  • GM-GSF 30 ng / mL
  • IL-3 30 ng / mL
  • the cells were stained with GD4 antibody and Fluorescein isot hiocyanate-labeled anti-human GD8 antibody, and confirmed by flow cytometry (see Fig. 2) (Immunological Experiment Procedures, 1995, Namedo Co., Ltd.).
  • the GD4-positive T cells were further cultured for two days in a culture solution containing -12 (40 U / mL (4 ng / mL)) and! FN-r (30 ng / mL). If the volume exceeds 5 ⁇ 10 6 mL, collect the cells with a glass pipette while stirring, re-appear with approximately 1 ⁇ 10 6 mL of the above-mentioned cytokine-supplemented culture medium, and renew the plate.
  • the obtained CD4-positive T cells were collected in a centrifuge tube using a glass pipette with stirring, and centrifuged. Recovered.
  • Th2 conditions were added at the concentration of IL-4 (30 ng / mL) from the beginning of the culture, and no IL-12 and IFN-r were added at the final stage, or ThO conditions (ThO conditions).
  • ThO conditions ThO conditions
  • Cells or Th1 / 2 progenitor cells) at the final stage without -12 and IFN-r were also cultured and recovered in the same manner as above.
  • CIVIL chronic myelogenous leukemia
  • peripheral blood from mononuclear isolated minute
  • 2Kai10 6 pieces Roh including mL concentration in 10% human serum AIM-V medium (manufactured by GIBC0-BRL Co.)
  • the cells were opaque, seeded on a 12-well plate, added with GM-GSF (30 ng / mL) and IL-3 (30 ng / mL), and cultured for 2 days to induce dendritic cells. Thereafter, IL-2 (100 U / mL (10 ng / mD) was added to the culture solution, and cultured for 14 to 21 days to obtain a large amount of CD4-positive T cells.
  • Example 1 (4 ng / mD) and culture medium containing IFN-r (30 ng / mL) and continued culturing for another 2 days, and CD4-positive T cells were collected in the same manner as in Example 1.
  • the cells were cultured under Th2 conditions and ThO conditions in the same manner as in 1, and collected in the same manner as described above.
  • AMIL (M4) Human acute monocytic leukemia (AMoL (M4)) mononuclear than bone marrow cells or peripheral blood of a patient is separated, 2x10 containing six mL concentration in 10% human serum AIM-V medium (GIBG 0-BRL), seeded on a 12-well plate, added GM-GSF (30 ng / mL) and IL-3 (30 ng Ml), and cultured for 2 days to induce dendritic cells. Thereafter, -2 (100 U / mL (10 ng / mD) was added to the culture solution, and the mixture was cultured for 14 to 21 days to obtain a large amount of GD4-positive T cells.
  • GIBG 0-BRL human serum AIM-V medium
  • Example 1 mL (4 ng / mL)) and IFN-r (30 ng / mL) were further cultured for 2 days in a culture solution containing the same, and GD4-positive T cells were collected in the same manner as in Example 1. Therefore, the cells were cultured under Th2 and ThO conditions in the same manner as in Example 1. The cells were collected in the same manner as described above, and IL-12 (40 U / mL (4 ng / mD) and IFN-r (30 ng / mL) were continuously added, and CD4-positive T cells were collected in the same manner as described above. It was bad condition.
  • the cytotoxic activity of the CD4-positive T cells obtained in Examples 1 to 3 on leukemia cells of the patient was examined.
  • a specific method for examining the cytotoxic activity was as follows: the leukemia cells stored in a frozen state of the patient were frozen and thawed, then cultured, radiolabeled with 51 Cr according to a standard method during the experiment, and the GD4 positive obtained in the patient was obtained. T cells and radiolabeled leukemia cells were mixed and cultured at each effector cell-to-target cell ratio, and after 4 hours, the radioactivity released into the culture supernatant was measured to determine the cytotoxic activity. (Ishi 1995, Nankodo Co., Ltd.).
  • GD4-positive T cells obtained from each patient by the method of the present invention are high on leukemia cells of each patient and show cytotoxic activity.
  • the IFN-r producing ability of the GD4-positive T cells obtained in Examples 1 to 3 was examined. This was specifically performed as follows. Processing the leukemic cells obtained from patients with mitomycin C (50 g / mL) ( ( Ltd.) Kyowa Hakko), obtained from the My Bok mycin C treatment leukemia cells (5 ⁇ 10 4 cells) and patient The obtained CD4-positive ⁇ cells (2 ⁇ 10 5 ) were mixed and cultured, and 48 hours later, IFN-r produced in the culture supernatant was measured using an IFM-r ELISA kit (manufactured by R & D). Fig. 4 shows the results. As shown in FIG. 4, the GD4-positive T cells obtained by the method of the present invention (Th1 condition) showed IFN-r-producing ability, indicating that they were Th1-type cells.
  • the suppression of IFN- ⁇ production and cytotoxic activity against autologous cancer cells by antibodies against MHG class II by stimulation of autologous cancer cells exhibited by the Th1 cells of the present invention obtained in Experimental Example 3 was examined. This was specifically performed as follows. The test was performed in the same manner as in the test system performed in Example 4 and Example 5, and the antiserum against MHG class I or antiserum against class II was added to the mixed culture system at a dilution of 100-fold, and the test was performed. .
  • FIG. 5 shows the results.
  • Figure 5 shows that the production of IFM- ⁇ by stimulation of autologous cancer cells was completely suppressed by the addition of antiserum to MHC Class II (a), and the cytotoxic activity against autologous cancer cells was also reduced by the addition of antiserum to MHG Class II. As a result, it was reduced by about 40% (b). This indicates that the induced GD4-positive T cells are restricted to MHC class II.
  • OVA ovalbumin
  • IL-12 (20 U / mL)
  • IFN-r (1 ng / ml
  • mice 2 ⁇ 10 6 cancer cells (A20-0VA) expressing the OVA antigen by gene transfer were transplanted intradermally into mice, and when the 0VA antigen-expressing carcinoma grew to 6-8 mm, 2-10 7 induced 0V A antigen-specific Th1 cells were transferred to the tumor-bearing mice.
  • A20-0VA cancer cells expressing the OVA antigen by gene transfer were transplanted intradermally into mice, and when the 0VA antigen-expressing carcinoma grew to 6-8 mm, 2-10 7 induced 0V A antigen-specific Th1 cells were transferred to the tumor-bearing mice.
  • disappearance of cancer was observed in all cases, and a marked antitumor effect was observed by injection into antigen-specific Thl cells.
  • Reference example 1 The effect of Th1 cell control on transplant cell engraftment was investigated in a mouse bone marrow transplantation model. That is, the 5 X 1 0 7 or lymphocytes G57BL / 6 mice BDF1 mice transplanted with intravenous injection was measured the induced IFN-r. As a result, 5 to 7 days after transplantation, IFN-r was detected in the serum from 50 Opg / mL to 200 Opg / mL in serum, and 10 to 4 days later. All cells were replaced by cells from transplanted mice, whereas when IFN-r was 1 Opg / nt or less, transplanted lymphocytes did not survive.
  • graft-versus-host disease graft-versus-host disease
  • transplantation during allogeneic hematopoietic cell transplantation in patients with hematopoietic tumors, such as leukemia patients.
  • This is considered to be an important finding in controlling hematopoietic leukemia lymphoma (graft-versus-leukemia / lymphoma: GVL).

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Immunology (AREA)
  • General Health & Medical Sciences (AREA)
  • Cell Biology (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • Animal Behavior & Ethology (AREA)
  • Medicinal Chemistry (AREA)
  • Organic Chemistry (AREA)
  • Biomedical Technology (AREA)
  • Microbiology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Epidemiology (AREA)
  • Hematology (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Mycology (AREA)
  • Genetics & Genomics (AREA)
  • Oncology (AREA)
  • Biotechnology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • Diabetes (AREA)
  • Hospice & Palliative Care (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)

Abstract

La présente invention se rapporte à un procédé de production de lymphocytes Th1 humains spécifiques d'un antigène à reconnaissance limitée au complexe majeur d'histocompatibilité de classe II, qui permettent une nouvelle thérapie du cancer ou des maladies immunitaires. Ledit procédé consiste à prélever dans un premier temps du sang périphérique ou de la moelle osseuse chez un patient, à effectuer une culture in vitro du prélèvement au moyen d'une cytokine exogène sans ajout extérieur d'aucun antigène à l'origine d'une maladie, ceci provoquant l'induction de dendrocytes, et à effectuer la culture de lymphocytes T positifs CD4 contenus dans la culture et coexistants avec les dendrocytes mentionnés ci-dessus, ceci induisant la production des lymphocytes Th1 humain cytotoxiques. L'administration à des patients de ces lymphocytes Th1 humains cytotoxiques permet de traiter chez ces patients le cancer et des maladies immunitaires.
PCT/JP2002/008499 2001-08-24 2002-08-23 Procede de production de lymphocytes t humains specifiques d'un antigene et medicaments associes WO2003018784A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
JP2001-255095 2001-08-24
JP2001255095 2001-08-24

Publications (1)

Publication Number Publication Date
WO2003018784A1 true WO2003018784A1 (fr) 2003-03-06

Family

ID=19083132

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/JP2002/008499 WO2003018784A1 (fr) 2001-08-24 2002-08-23 Procede de production de lymphocytes t humains specifiques d'un antigene et medicaments associes

Country Status (1)

Country Link
WO (1) WO2003018784A1 (fr)

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2000143534A (ja) * 1998-11-13 2000-05-23 Asahi Chem Ind Co Ltd 樹状細胞ワクチン及びその製造方法
JP2002065263A (ja) * 2000-08-23 2002-03-05 Toyobo Co Ltd ｃＤＮＡの増幅方法及び標識ｃＤＮＡの調製方法
JP2002069001A (ja) * 2000-08-29 2002-03-08 Asahi Kasei Corp 樹状細胞を主成分とする細胞ワクチン

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2000143534A (ja) * 1998-11-13 2000-05-23 Asahi Chem Ind Co Ltd 樹状細胞ワクチン及びその製造方法
JP2002065263A (ja) * 2000-08-23 2002-03-05 Toyobo Co Ltd ｃＤＮＡの増幅方法及び標識ｃＤＮＡの調製方法
JP2002069001A (ja) * 2000-08-29 2002-03-08 Asahi Kasei Corp 樹状細胞を主成分とする細胞ワクチン

Similar Documents

Publication Publication Date Title
US10806777B2 (en) Method for allogeneic cell therapy
AU2019215034B2 (en) Method of producing natural killer cells and composition for treating cancer
TW585915B (en) Methods for activating natural killer (NK) cells
JPH11505512A (ja) マクロファージを調製する方法、ならびにそのためのキットおよび組成物
US20030124091A1 (en) Endothelial cell derived hematopoietic growth factor
WO2014161887A1 (fr) Thérapie immunitaire anticancéreuse ciblée
CN107488235B (zh) 一种新的增强型抗原联合多肽诱导肝癌特异性ctl细胞的制备及应用
Yannelli et al. The large scale generation of dendritic cells for the immunization of patients with non-small cell lung cancer (NSCLC)
WO2019152663A1 (fr) Procédé de production de cellules tueuses naturelles et composition pour le traitement du cancer
JP2001181205A (ja) 樹状細胞と腫瘍細胞を用いた腫瘍特異的抗腫瘍細胞性ワクチンの製造方法
EP1959007A1 (fr) Procédé pour générer des cellules dendritiques à maturité
KR100797050B1 (ko) 아토피성 피부염에 치료효과를 갖는 ｃｄ8 ｔ 세포
WO2003018784A1 (fr) Procede de production de lymphocytes t humains specifiques d'un antigene et medicaments associes
JP4106488B2 (ja) 同種移植片に対する免疫寛容の誘導および／または白血病処置のための、幹細胞およびｃｄ６枯渇幹細胞の使用
JP2002517409A (ja) 抗原提示細胞組成物の調製およびインビボ投与のための方法
KR100797049B1 (ko) 아토피성 피부염에 치료효과를 갖는 ｃｄ4 ｔ 세포
WO2009008666A2 (fr) Cellule dendritique autologue médiatisée par une thérapie photodynamique ayant l'aptitude d'inhiber la croissance d'une tumeur
Burgoyne Improved dendritic cell therapy for cancer by enhancing in vivo lymph node migration using a novel chemokine-based sorting method
JP2004248504A (ja) Ｔｈ２型またはＴｈ１型にシフトしたナチュラルキラーＴ細胞の増幅方法
JP5084012B2 (ja) イディオタイプ抗原用担体およびそれを用いたイディオタイプワクチン
US20020182231A1 (en) Methods of stem cell manipulation for immunotherapy
De Silvestro et al. Legislative and ethical aspects of administering granulocyte colony-stimulating factor to normal donors
CZ29538U1 (cs) Sada pro přípravu protinádorové vakcíny, protinádorová vakcína včetně posílení účinku
WO2002055103A1 (fr) Vaccins anti-tumoraux
JP2001026545A (ja) 免疫応答活性化製剤

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BY BZ CA CH CN CO CR CU CZ DE DK DM DZ EC EE ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX MZ NO NZ OM PH PL PT RO RU SD SE SG SI SK SL TJ TM TN TR TT TZ UA UG US UZ VC VN YU ZA ZM ZW

Kind code of ref document: A1

Designated state(s): AE AG AL AM AT AU AZ BA BB BG BY BZ CA CH CN CO CR CU CZ DE DM DZ EC EE ES FI GB GD GE GH HR HU ID IL IN IS JP KE KG KP KR LC LK LR LS LT LU LV MA MD MG MN MW MX MZ NO NZ OM PH PL PT RU SD SE SG SI SK SL TJ TM TN TR TZ UA UG US UZ VC VN YU ZA ZM

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): GH GM KE LS MW MZ SD SL SZ UG ZM ZW AM AZ BY KG KZ RU TJ TM AT BE BG CH CY CZ DK EE ES FI FR GB GR IE IT LU MC PT SE SK TR BF BJ CF CG CI GA GN GQ GW ML MR NE SN TD TG

Kind code of ref document: A1

Designated state(s): GH GM KE LS MW MZ SD SL SZ TZ UG ZM ZW AM AZ BY KG KZ MD RU TJ TM AT BE BG CH CY CZ DE DK EE ES FI FR GB GR IE IT LU MC NL PT SE SK TR BF BJ CF CG CI CM GA GN GQ GW ML MR NE SN TD TG

121 Ep: the epo has been informed by wipo that ep was designated in this application
NENP Non-entry into the national phase

Ref country code: JP

122 Ep: pct application non-entry in european phase