WO2003012080A2 - Selection ex vivo de lymphocytes t - Google Patents

Selection ex vivo de lymphocytes t Download PDF

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Publication number
WO2003012080A2
WO2003012080A2 PCT/EP2002/008052 EP0208052W WO03012080A2 WO 2003012080 A2 WO2003012080 A2 WO 2003012080A2 EP 0208052 W EP0208052 W EP 0208052W WO 03012080 A2 WO03012080 A2 WO 03012080A2
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Prior art keywords
cells
allogeneic
binding molecules
cell
combinations
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PCT/EP2002/008052
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German (de)
English (en)
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WO2003012080A3 (fr
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Ahmed Sheriff
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Genethor Gmbh
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Priority claimed from DE10135615A external-priority patent/DE10135615A1/de
Priority claimed from EP01121806A external-priority patent/EP1291414A1/fr
Application filed by Genethor Gmbh filed Critical Genethor Gmbh
Priority to AU2002321240A priority Critical patent/AU2002321240A1/en
Publication of WO2003012080A2 publication Critical patent/WO2003012080A2/fr
Publication of WO2003012080A3 publication Critical patent/WO2003012080A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0639—Dendritic cells, e.g. Langherhans cells in the epidermis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/14—Blood; Artificial blood
    • A61K35/16—Blood plasma; Blood serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/26—Lymph; Lymph nodes; Thymus; Spleen; Splenocytes; Thymocytes
    • 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
    • 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
    • A61K39/4621—Cellular immunotherapy characterized by the effect or the function of the cells immunosuppressive or immunotolerising
    • 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
    • A61K39/46434—Antigens related to induction of tolerance to non-self
    • 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
    • A61K39/464402—Receptors, cell surface antigens or cell surface determinants
    • A61K39/464411—Immunoglobulin superfamily
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0639—Dendritic cells, e.g. Langherhans cells in the epidermis
    • C12N5/064—Immunosuppressive 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
    • A61K2035/122—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells for inducing tolerance or supression of immune responses
    • 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
    • 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
    • C12N2510/00—Genetically modified cells

Definitions

  • the present invention relates to antigen-presenting cells, methods for producing antigen-presenting cells, medicaments containing antigen-presenting cells and use of the antigen-presenting cells.
  • Tissue transplantation to replace diseased organs is an important medical therapy today.
  • an adaptive immune system response to the graft poses the greatest threat to successful treatment.
  • Adaptive immune system responses are induced by antigen presenting cells by activating T helper lymphocytes.
  • the ABO and Rh blood group antigens When transfusing blood, which is the first and most commonly used graft, the ABO and Rh blood group antigens must be matched to avoid the rapid destruction of inappropriate erythrocytes.
  • the very polymorphic major histocompatibility complexes Tissue Compatibility Complexes, MHC
  • MHC tissue Compatibility Complexes
  • Leukemia is cancer of the blood cells. 50 out of 1 million people get leukemia every year. When leukemia develops, the body produces large amounts of abnormal blood cells. In most types of leukemia, the abnormal cells are white blood cells.
  • leukemia There are several types of leukemia, which essentially show two characteristics. One characteristic is how quickly the disease develops and worsens. The other is determined by the type of blood cell that is affected.
  • Leukemia is either acute or chronic. In acute leukemia, the abnormal blood cells are very immature blasts that cannot perform their normal functions. The number of blasts increases rapidly and the ailments quickly get worse. Some blasts are present in chronic leukemia, but generally these cells are more mature and can perform some of their normal functions. The number of blasts also rises more slowly than with acute leukemia. With chronic leukemia, the disease gradually gets worse.
  • Leukemia essentially appears in two main types of white blood cells, lymphoid cells or myeloid cells. Accordingly, leukemia occurs as lymphatic leukemia or . myeloid leukemia. The most common leukemias are:
  • ALL Acute lymphoblastic leukemia
  • AML Acute myeloid leukemia
  • ANLL acute non-lymphoblastic leukemia
  • CLL Chronic lymphoblastic leukemia
  • CML Chronic myeloid leukemia
  • Leukaemic cells are abnormal cells that cannot function as normal blood cells. They cannot help the body fight infections. Because of this, people with leukemia often develop infections and have a fever.
  • leukemic cells Like all blood cells, leukemic cells circulate in the body. Due to the number of abnormal cells and their location, patients with leukemia can show various symptoms. In acute leukemia, symptoms appear quickly and the patient's condition also deteriorates rapidly. Chronic leukemia appears
  • symptoms for a long time. When symptoms appear, they are usually mild at first and the patient's general condition only gradually worsens.
  • the abnormal cells can collect in the brain or spinal cord (central nervous system or CNS).
  • CNS central nervous system
  • the result can be headache, nausea, nausea, confusion, loss of muscle control and strokes.
  • Leukaemic cells can also collect in the testes and cause swelling there. Some patients develop painful eyes or skin. Leukemia can also affect the digestive tract, kidneys, lungs, or other parts of the body.
  • Chronic leukemia With chronic leukemia, the abnormal cells can gradually collect in different parts of the body. Chronic leukemia can affect the skin, CNS, digestive tract, kidneys and testicles. Treatment of leukemia:
  • Treating leukemia is complex. It varies with the type of leukemia and is not the same in all patients. Treatment also depends on
  • the leukemic cells certain characteristics of the leukemic cells, the extent of the disease, whether the leukemia has been treated before. The patient's age, symptoms and general health are also relevant factors.
  • Acute leukemia must be treated immediately. Patients with chronic leukemia who show no symptoms do not need to be treated immediately. Unfortunately, chronic leukemia can rarely be cured.
  • KMT bone marrow transplantation
  • BMT bone marrow transplantation
  • removal of the spleen can help.
  • patients receive full body radiation combined with chemotherapy to destroy the leukemia-producing bone marrow.
  • the healthy bone marrow can come from a donor or it can come from the patient. It is then removed and treated outside the body to remove leukemic cells before high dose treatment. After that, a hospital stay of several weeks is necessary so that the transplant grows and enough white blood cells (leukocytes), such as T, B lymphocytes, neutrophils, basophils and eosinophils, platelets (platelets), monocytes and other subpopulations for the reconstitution (restoration) of the hematopoietic hematopoietic) system can produce. In the meantime, patients need to be protected from infection.
  • the biological therapies include treatments with substances that influence the immune response to cancer. Cytokines, such as especially interferons, interleukins and colony stimulating factors (CSF) are used for example
  • GVHD graft versus host disease
  • BMT Allologic bone marrow transplantations
  • CML chronic myeloid leukemia
  • CML is characterized in the early chronic phase by a single transformative genetic abnormality identified. It is the t (9; 22) translocation (Philadelphia Chromosome, Ph) that the bcr-abl oncogene creates, the only factor that is essential for the development of the Disease is needed (Daley et al 1990). Compared to other tumor types in the chronic phase, CML patients have a relatively intact immune system (Lewalle et al 1996). CML is accessible for a tumor-specific immune response. In the past 20-30 years, anti-tumor
  • Allo-BMT is a fairly rough approach with significant graft-related morbidity and mortality. The risk of dying is 20-41% (Silver et al 1999). This form of immunotherapy also offers egg ne up to 70% leukemia-free survival rate for the transplant recipients (Clift &. Anasetti 1997). This GvHD overlays the desired anti-leukemic effect.
  • Bone marrow transplants are also used for other leukemias.
  • GvHD graffc versus host disease
  • mucous membranes and conjunctiva dry out and myositis.
  • immunosuppression is carried out with cyclosporin, possibly in combination with methotrexate and corticosteroids.
  • T lymphocytes in the graft reduces the risk of GvHD.
  • the risk of tumor recurrence increases because the donor T lymphocytes also mediate the favorable graft versus leukemia effect (GvL). If you want to maintain the GvL effect while reducing the GvHD risk, you have to partially deplete the T cells and gradually reinfuse the donor T lymphocytes at later times. _ g.
  • FasL is known as a potent apoptosis-inducing molecule (membrane protein). It communicates its effects via Fas, a receptor that occurs on T cells. If DCs are transfected with FasL, they induce apoptosis in
  • FasL-transfected DCs can cause an Ag-specific reduction in graft rejection. This is based on the interaction (apoposis induction) of FasL on DCs and Fas receptor on the T cells (Min et al 2000).
  • TRAIL tumor necrosis factor-related apoptosis-inducing ligand
  • DCs arise from circulating progenitor cells after a maturation period.
  • DCs can be characterized by a specific population of marker molecules. These include accessory, costimulatory gene products such as CD40, CD80, and CD86 as well as MHC class I and II.
  • the CD83 molecule in particular is a well-characterized marker for fully mature DCs, since CD83 cannot be detected on immature DC precursors.
  • the functional significance of CD83 is unknown. Its increasing presence during the ripening indicates an important function (Berchtold et al 1999).
  • Herpes simplex virus is able to suppress the expression of CD83 in an unexplained manner, while the production of other molecules typical of DCs such as CD25, CD40, CD80, CD86, CD95 and MHC of class I and class II remains unaffected (Kruse et al 2000b).
  • Inhibition of CD83 expression on the plasma membrane leads to a dramatic reduction in DC-mediated T cell stimulation. If the eukaryotic initiation factor 5A is hindered by a specific inhibitor, CD83 expression can be prevented, which also leads to a strong reduction in DC-mediated T cell stimulation (Kruse et al 2000a).
  • eIF-5A is a
  • Protein that is included in the export path of specific RNAs from the cell nucleus Protein that is included in the export path of specific RNAs from the cell nucleus.
  • eIF-5A is the only cellular protein known to contain the unusual amino acid hypusin. This modification appears to be necessary for cell division.
  • Hypusin modification is a spermidine-dependent post-translational reaction that is catalyzed by two enzymes. This includes the transfer of the aminobutyl group of spermidine to the ⁇ -NH 2 group of lysine at position 50 in eIF-5A by the deoxyhypusin synthase (Kruse et al 2000a).
  • eIF-5A is then hydroxylated by the deoxyhypusin hydroxylase. In this way the active form of eIF-5A is created.
  • eIF-5A was originally referred to as an "initiation factor"
  • recent in vitro and in vivo experiments have shown that eIF-5A is not an initiator of protein translation.
  • eIF-5A appears to be part of a specific export path from the cell nucleus, which is used, for example, by the Rev / Rex class of retroviral RNA transport factors. It was also shown that the eIF-5A protein accumulates on the nucleoplasmic side of the nuclear pore complex in order to interact with the general nuclear export receptor CRM1 and to migrate from the nucleus into the cytoplasm in mammalian cells.
  • eIF-5A is constitutively expressed in cell lines as well as various other tissues.
  • the eIF-5A gene is induced in primary lymphoid cells during the activation and / or proliferation of primary human blood cells. If an inhibitor of hypusin modification [GC7 (N 1 - guanyl-l, 7-diaminoheptan)], the expression of CD83 and thus the full stimulatory activity of mature DCs is inhibited (Kruse et al 2000a).
  • the cell according to the invention is preferably a dendritic cell or another antigen-presenting cell or one of the precursors of such a cell.
  • the APCs are the switching points of the adaptable immune system. Only they can trigger or stop a T cell-mediated immune response.
  • the allologic immune reactions cannot be maintained without the help and mediation of APCs and T-helper lymphocytes.
  • the cytotoxic T cells are also activated by APCs. They mediate a cellular immune response in which cells are killed directly by the cytotoxic T cells.
  • the molecular mechanisms of interaction - cell-cell contact - of APCs with the T helper and cytotoxic T lymphocytes are known in detail at the receptor level.
  • the antigen is e.g. a bacterial protein
  • the immune response is useful for the organism.
  • the antigen is a transplant antigen, one speaks of a (pathological) rejection reaction.
  • WO 01/29192 discloses a method of using antigen-presenting cells to prevent GvHD effects. However, the antigen-presenting cells are co-incubated with dead or dying cell parts of the transplant recipient in order to contain loaded antigen-presenting cells
  • the cell components should contain antigens for which a reduction in an immune response is desired. According to the invention, this step is not required since cells of the receiver are used. In addition, further changes are made or immature DCs are used to reduce a repulsive immune response.
  • the problem on which the invention is based is, inter alia, to make available therapeutically usable products for the reduction of allologic immune reactions in bone marrow transplants.
  • Figure 1 shows the recovery of non-alloreactive T cells with mature DC.
  • Figure 2 shows the extraction of non-alloreactive T cells with immature DC.
  • Figure 3 shows the treatment scheme for DC from Balb / c mice.
  • Figure 4 shows the first and second incubation of T cells with alloglogic cells.
  • Figure 5 shows CD86 expression of the Balb / c-DC before starting the first incubation.
  • Figure 6 shows the cytotoxicity of the CD8 + T cells, which were incubated in the first incubation with differently treated DC.
  • the problem is solved by cells of the recipient which are antigen-presenting cells or are cells from which antigen-presenting cells can be generated.
  • the antigen-presenting cells are the dendritic cells, but also B cells.
  • the antigen-presenting cells are to be incubated with allologic bone marrow beam planets. These grafts contain lymphocytes.
  • the antigen-presenting cells can can also be incubated alone with the lymphocytes or T cells (T lymphocytes).
  • T lymphocytes T lymphocytes
  • T-lymphocytes T cells
  • the T cells are of particular interest because they are important reactions for the recipient / patient
  • the useful response is anti-leukemic GvL
  • the harmful response is GvHD.
  • the incubation of the antigen-presenting cells with the transplant and here in particular with the T cells occurring therein is said to switch off the GvHD reaction and to maintain only the GvL reaction.
  • the antigen-presenting cells are selected or treated in such a way that they can no longer activate T cells, but can still present the recipient's alloantigens. If an alloreactive T cell encounters such an antigen-presenting cell, it is permanently shut down, or sent to programmed cell death (apoptosis), or converted into a regulatory T cell.
  • a regulatory T cell is understood to be a T cell that prevents immune responses to specific antigens, here alloantigens. The following are ways to solve the problem:
  • the cell according to the invention which originates from the recipient of the transplant and is characterized in that one of the functions of co-stimulatory receptors, such as a CD83 receptor and / or B7 and / or CD40 receptor, is suppressed and / or CTLA4-binding molecules are added which bind CTLA4 to T cells of the transplant and / or Fas binding molecules are added which bind the Fas to T cells of the transplant and / or TRAIL receptor binding molecules are added, bind the TRAIL receptors to T cells of the graft, or interleukin 10 (IL-10) is added.
  • co-stimulatory receptors such as a CD83 receptor and / or B7 and / or CD40 receptor
  • CTLA4-binding molecules are added which bind CTLA4 to T cells of the transplant and / or Fas binding molecules are added which bind the Fas to T cells of the transplant and / or TRAIL receptor binding molecules are added, bind the TRAIL receptors to T cells of the graft, or
  • T cells are switched off and / or they are driven into programmed cell death (apoptosis) and / or they differentiate into regulatory T cells if the T cells simultaneously present an antigen with their T cell receptor detect.
  • Molecules that bind Fas and / or TRAIL receptors to T cells in the transplant can cause T cells to enter programmed cell death (apoptosis)
  • the binding molecules are crosslinked, which e.g. by other binding molecules, e.g. Antibodies can happen, which in turn bind the binding molecules against CD83, CD80, CD86, CD40, CTLA4, Fas, TRAIL receptors. Networking promotes the desired effect.
  • the cells according to the invention can also be represented in whole or in part by immature dendritic cells (DC; e.g. those derived in vitro). Due to the lack of costimulation in immature DCs, T cells (as in A.) are removed
  • the cell according to the invention can also have at least one gene for a
  • T cells (as in A.) can be forced to programmed cell death (apoptosis).
  • the cell according to the invention can also contain at least one gene for a tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) receptor.
  • TRAIL tumor necrosis factor-related apoptosis-inducing ligand
  • TRAIL receptor binding molecules on the plasma membrane of dendritic cells can force T cells (as in A.) to programmed cell death (apoptosis).
  • the cell according to the invention can also be added by adding aspirin, la, 25 dihydroxyvitamin D3, la, 25 (OH) 2-16-ene-23-yne-26,27-hexafluoro-19-nor-vitamin D3 (D3 analog), vitamin D3, glucocorticoids are treated (Adorini et al 2001; Griffin et al 2001; Griffin et al 2000; Ralphstein et al 2001; Penna & Adorini 2000; Penna & Adorini 2001) (WO 01/24818).
  • dendritic cells remain in a state which limits the immunostimulatory properties of the dendritic cells.
  • the reduced costimulation switches off T cells (as in A.) and / or drives them to programmed cell death (apoptosis) and / or differentiates them into regulatory T cells.
  • the cell according to the invention can also be transfected with at least one gene for a CTLA4 binding molecule.
  • CTLA4 binding molecules By expressing CTLA4 binding molecules on the plasma membrane of dendritic cells, T cells can be switched off and / or driven into programmed cell death (apoptosis) and / or differentiated into regulatory T cells. G.
  • the cell according to the invention can also be transfected with at least one gene for a CD83 binding molecule and / or for B7 (CD80 / CD86) binding molecules and / or for a CD40 binding molecule.
  • a CD83 binding molecule and / or for B7 (CD80 / CD86) binding molecules and / or for a CD40 binding molecule By expressing CD83 binding molecules and / or B7 (CD80 / CD86) binding molecules and / or CD40 binding molecules in the endoplasmic reticulum and / or Golgi and / or extracellularly and / or on the plasma membrane of dendritic cells, T cells can be switched off and on / or driven into programmed cell death (apoptosis) and / or differentiated into regulatory T cells.
  • the cell according to the invention can also be transfected with at least one gene for IL-10.
  • Expression of IL-10 from dendritic cells prevents the dendritic cells from activating T cells (Corinti et al 2001), so that T cells can be switched off and / or driven into programmed cell death (apoptosis) and / or be differentiated into regulatory T cells.
  • the cell according to the invention can be induced by incubation with the interferons (IFNa or IFN ⁇ or IFNß) to synthesize tumor necrosis factor-related apoptosis inducing ligand (TRAIL).
  • IFNa or IFN ⁇ or IFNß interferons
  • TRAIL- on the plasma membrane of dendritic cells can force T cells (as in A.) to programmed cell death (apoptosis) (Liu et al 2001; Fanger et al 1999).
  • One of the procedures (A.-I.) and / or any sequence of these procedures (A.-I.) is used to treat the cell according to the invention in vitro with allogeneic stem cells (eg including lymphocytes) or with allogeneic T cells or incubate with allogeneic lymphocytes from a foreign stem cell transplant donor (e.g. bone marrow).
  • allogeneic stem cells eg including lymphocytes
  • allogeneic T cells e.g. allogeneic T cells
  • allogeneic lymphocytes e.g. bone marrow
  • the cells mentioned which come from the recipient of the transplant and in cancer patients, esp especially those with leukaemias that can contain cancer cells can be treated in a preferred embodiment of the method according to the invention before incubation with the transplant in such a way that these cells will not proliferate. This is preferably achieved by irradiating these cells.
  • the cell according to the invention can also be transfected with at least one gene for IL-10.
  • Expression of IL-10 from dendritic cells prevents the dendritic cells from activating T cells (Corinti et al 2001), so that T cells can be switched off and / or driven into programmed cell death (apoptosis) and / or to be differentiated into regulatory T cells.
  • the cell according to the invention can be induced by incubation with the interferons (IFNa or IFN ⁇ or IFNß) to synthesize tumor necrosis factor-related apoptosis inducing ligand (TRAIL).
  • IFNa or IFN ⁇ or IFNß interferons
  • TRAIL tumor necrosis factor-related apoptosis inducing ligand
  • Expression of TRAIL- on the plasma membrane of dendritic cells can force T cells (as in A.) to programmed cell death (apoptosis) (Liu et al 2001; Fanger et al 1999).
  • One of the procedures (A.-I.) and / or any sequence of these procedures (A.-I.) is used to treat the cell according to the invention in vitro with allogeneic stem cells (eg including lymphocytes) or with allogeneic T cells or incubate with allogeneic lymphocytes from a foreign stem cell transplant donor (e.g. bone marrow).
  • allogeneic stem cells eg including lymphocytes
  • allogeneic T cells e.g. allogeneic T cells
  • allogeneic lymphocytes e.g. bone marrow
  • the cells mentioned which originate from the recipient of the transplant and can contain cancer cells in cancer patients, in particular those with leukemia, can be found in ei ner preferred embodiment of the method according to the invention are treated before incubation with the graft so that proliferation of these cells will not occur. This is preferably achieved by irradiating these cells.
  • the non-alloreactive, but allologic T-cells positively selected by one of these procedures and / or any sequence of these procedures are presented to the patient together with the allologic stem cells (e.g. bone marrow)
  • graft-versus-leukemia effect (GvL) is obtained, but none, or a significantly reduced graft-versus-host disease (GvHD).
  • the CD83 binding molecules can preferably be antibodies, monoclonal antibodies, F (ab) 2 , single chain antibodies (scFv), and / or Fab fragments.
  • the proteins which have affine structures for B7 receptors can preferably be CTLA4, CTLA4 derivatives (eg CTLA4Ig), CD28, antibodies, F (ab) 2 , scFv and / or Fab fragments.
  • the proteins which have structures affinity to CD40 receptors can preferably be CD40L, CD40L derivatives, antibodies, F (ab) 2 , scFv and / or Fab fragments.
  • the proteins which have affine structures for CTLA4 (CD152) receptors can preferably be antibodies, F (ab) 2 , scFv and / or Fab fragments.
  • the proteins which have affine structures for Fas ligands can preferably be FasL, FasL derivatives, antibodies, F (ab) 2 , scFv and / or Fab fragments.
  • the proteins which have affine structures for TRAIL receptors can preferably be TRAIL, TRAIL derivatives, antibodies, F (ab) 2 , scFv and / or Fab fragments.
  • the method according to the invention can be based on genetic engineering interventions on the patient's own cells (pre-cells: stem cells, monocytes, DCs, CD34 + stem cells, CD34 + progenitor stem cells and / or B cells).
  • the interventions take place by means of suitable probes and bring about the reduction of CD83 molecules and possibly B7 molecules and / or CD40 molecules by hindering or preventing the formation of the molecule on the surface of the antigen-presenting cells and / or CTLA4 binding Molecules, preferably antibodies, and / or the production of Fas binding molecules, preferably FasL and / or the production of TRAIL receptor binding molecules, preferably TRAIL and / or the production of IL-10.
  • Transfection of the pre-cells causes expression of Fas binding molecules and / or TRAIL receptor binding molecules and / or IL-10 and / or B7 binding molecules and / or CTLA4 binding molecules and / or CD83 binding molecules and / or CD40 binding molecules.
  • the nucleic acids can be DNA, RNA, oligonucleotides, polynucleotides, ribozymes or peptide nucleic acids (PNA).
  • the DNA contains regulatory elements such as enhancers, promoters, polyA-coding 3'-ends for the transcription of the DNA into RNA.
  • the RNA in turn should contain regulatory elements for translating the RNA into protein.
  • the nucleic acids are provided with a signal sequence which effects the transport of the expression products to the plasma membrane.
  • nucleic acids encode a signal sequence which causes the expression products to remain in the respective cell compartments.
  • the cells mentioned can be transfected ex vivo by treatment with viruses, viral vectors, bacterial vectors, plasmids which are suitable by viral gene transfer, electroporation techniques, iontophoresis, ballistic methods and / or other techniques for introducing molecules into eukaryotic cells.
  • Said cell can be treated with viruses, viral vectors, bacterial vectors, plasmids by viral gene transfer, electroporation techniques, iontophoresis, ballistic methods and / or other techniques for introducing molecules into a cell with increased production of Fas binding molecule and / or TRAIL receptor binding molecule and / or CD83 binding molecules, CD40 binding molecules, B7 (CD80 / CD86) binding molecules, CTLA4 binding molecule, IL-10 are transfected, whereby T cells which have allologic antigens, for example on MHC -Molecules are presented to the antigen- bind presenting cell, be switched off.
  • viruses viral vectors, bacterial vectors, plasmids by viral gene transfer, electroporation techniques, iontophoresis, ballistic methods and / or other techniques for introducing molecules into a cell with increased production of Fas binding molecule and / or TRAIL receptor binding molecule and / or CD83 binding molecules, CD40 binding molecules, B7 (CD80
  • Molecules such as antibodies, proteins, peptides, peptidomimetics, CTLA4, CTLA4 derivatives such as CTLA4Ig, CD28, CD40L and / or constituents and / or combinations of these molecules, which bind, for example, B7-1, B7-2, CD83, CD40, can be used as binding molecules , serve. These molecules hinder stimulation and / or co-stimulation of T cells which take place in the presence of an alloantigen presentation and are brought into contact with the cell according to the invention.
  • the molecules can be generated by vehicles such as liposomes, hydrogels,
  • Nucleic acids can be transferred in particular by viruses, viral vectors, bacterial vectors, plasmids, which are transferred into the monoantigenic antigen-presenting cell and / or the antigen-presenting cell by electroporation techniques, iontophoresis, ballistic methods and / or other techniques for introducing molecules.
  • the APC or pre-cell which consists of the group consisting of stem cells, monocytes, DCs, CD34 + stem cells, CD34 + progenitor stem cells and / or B-
  • a medicament containing at least one cell according to the invention is also claimed according to the invention.
  • the medicament according to the invention is preferably formulated as an infusion solution for intravenous or intraperitoneal application. The formulation is chosen so that when the drug is administered there is no significant impairment of the effectiveness of the antigen-presenting cell according to the invention.
  • physiological saline is preferred as the infusion solution.
  • other solutions with a pH of 5.5 to 8.5 are also suitable.
  • Serum for example human serum, autologous serum or serum of other species, solutions with plasma substitutes, such as polyvinylpyrrolidone, are also suitable.
  • plasma substitutes such as polyvinylpyrrolidone
  • 0.5 ml to 500 ml should be applied.
  • the antigen-presenting cell according to the invention can be used in particular for the production of a medicament for the treatment of immune reactions against allologic tissue features or for the prevention of GvHD.
  • the immune reactions to be treated are connected with allologic tissue characteristics, their gene sequences and / or partial sequences ⁇ , in particular major histocompatibility complexes, MHC I, MHC II, rhesus factor, minor histocompatibility antigens (minor histocompatibility antigens)
  • a use is claimed according to the invention in which the binding molecules are crosslinked in a preferred embodiment, which is achieved by other binding molecules, e.g. Antibodies, which in turn bind the molecules against CD83, CD80,
  • Bind CD86, CD40, CTLA4, Fas, TRAIL receptors Such cross-linking enhances the effect of these binding molecules.
  • the ex vivo selected cells are infused into the patient and the patient is then subjected to an in vivo treatment with IL-2.
  • the cells selected ex vivo can also be incubated ex vivo with IL-2 or other stimulants such as phytohemaglutinin (PHA) and then infused into the patient. It has been shown that the GvL effect can be enhanced in this way.
  • PHA phytohemaglutinin
  • helper CD4 + T cells and cytotoxic CD8 + T cells C3H mouse
  • target cells such as A20 cells (lymphoma cells from Balb / c) and Balb / c-DC after they were incubated with differently treated DC.
  • the CD8 + T cells are referred to as effector cells.
  • A20 cells and Balb / c-DC are the target cells.
  • the cytotoxicity assay consists of two incubations. The first is used to incubate allologic T cells (C3H) with "autologous" DC (Balb / c). In the second incubation (the actual cytotoxicity assay), the T cells are removed from the first incubation and incubated with target cells for 4 h.
  • the target cells are lysed by allologic, cytotoxic CD8 + T cells, to an extent that depends on how many allologic T cells react against the foreign target cells.
  • Mature and immature DC were used as DC. These should act differently on the T cells. Mature DC stimulate T cells, while immature DC turn off T cells. Mature DCs blocked in Kostimulus B7 turn off T cells. The experimental approach is shown in Fig. 1 and Fig. 2. Execution:
  • Immature DC (imDC from immature DC) were obtained from bone marrow from Balb / c-
  • the spleens were prepared from two C3H mice and the T cells isolated using MACS.
  • the isolated T cells were sown with a cell density of 5xl0 5 / 500 ⁇ l complete medium in a well of a 24-well plate and thus above sea level. incubated at 37 ° C in the incubator.
  • the complete medium had the following composition:
  • the complete medium was set up as follows:
  • 100 ml of 10 x RPMI were diluted with 870 ml of water.
  • 30 ml of NaHCO 3 , 20 ml of L-glutamine and 10 ml of sodium pyruvate were pipetted.
  • the finished medium was mixed by swirling and could then be portioned. Then the appropriate amount of fetal calf serum and 1 ml of 50 mM mercaptoethanol are added.
  • the DCs were prepared the day before C3H T cells with a cell density of lxl0 4/500 .mu.l of complete medium (per well of a 24-well plate) was added.
  • the DC were incubated with T cells in a ratio of 1:50 (in cell numbers 1x10 4 : 5x10 5 per well of a 24-well plate) for 6 days (FIG. 4).
  • the cells After the first incubation, the cells (all cells -> also the Balb / c-DC) were removed from the well and incubated for 4 hours with the target cells A20 cells or Balb / c-DC (Fig. 4). 5xl0 3 target cells and lxlO 5 effector cells were used for each approach. The total volume was 200 ⁇ l complete medium.
  • A20 cells were from a continuous cell culture. They are a Balb / c B cell lymphoma line. BALB / c DC
  • Balb / c-DC were differentiated from bone marrow cells and used as target cells in the immature stage on day 7.
  • TruCount beads Becton
  • the batches were stained with propidium iodide (PI) shortly before the measurement.
  • PI propidium iodide
  • the cells that were CFSE / PI double positive were considered for the evaluation.
  • the target cells that died without the influence of T cells were determined as a control.
  • the effectiveness of the B7 blockade was determined using anti-CD86 (B7-2, aCD86) monoclonal antibodies. If the CD86 signal becomes smaller in the FACS, there are fewer unblocked B7-2 molecules on the cell surface of the DC. Fig. 5 shows that the TNF ⁇ treated and therefore mature DC have a higher CD86 expression than the immature DC. CD86 could be blocked in mature and immature DC. Irradiation had no significant effect on CD86 expression.
  • Fig. 6 shows the percentage of the lysed target cells as a function of the irradiated / unirradiated or blocked / unblocked DC (during the first incubation), which were incubated with the effector cells.
  • immature DC blocked and unblocked
  • DC blocked mature DC
  • Unmodified DC on the other hand, have promoted the activation of alloantigen-specific T cells.
  • TRAIL tumor necrosis factor-related apoptosis-inducing ligand

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Abstract

La présente invention repose sur une réduction de réactions immunitaires allogéniques. Selon le présent procédé, les propriétés régulatrices de cellules présentant les antigènes sont utilisées pour supprimer ex vivo les réactions du greffon contre l'hôte.
PCT/EP2002/008052 2001-07-21 2002-07-19 Selection ex vivo de lymphocytes t WO2003012080A2 (fr)

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AU2002321240A AU2002321240A1 (en) 2001-07-21 2002-07-19 Ex-vivo selection of t-cells with reduced alloreactivity and use thereof in bone marrow transplantations

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DE10135615A DE10135615A1 (de) 2001-07-21 2001-07-21 Ex vivo Selektion von T-Zellen
DE10135615.3 2001-07-21
EP01121806.2 2001-09-11
EP01121806A EP1291414A1 (fr) 2001-09-11 2001-09-11 Sélection ex vivo de celulles T présentant une alloréacitvité diminuée (effet greffon-versus-hôte dimimué et effet greffon-versus-leucémie augmenté) et utilisation de ces cellues dans des greffes de moelle osseuse

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Non-Patent Citations (7)

* Cited by examiner, † Cited by third party
Title
APPELBAUM FREDERICK R: "Haematopoietic cell transplantation as immunotheraphy." NATURE (LONDON), Bd. 411, Nr. 6835, 2001, Seiten 385-389, XP002187722 ISSN: 0028-0836 *
APPLEMAN LEONARD J ET AL: "Helper T cell anergy: From biochemistry to cancer pathophysiology and therapeutics." JOURNAL OF MOLECULAR MEDICINE (BERLIN), Bd. 78, Nr. 12, 2001, Seiten 673-683, XP002187721 ISSN: 0946-2716 *
BLAZAR BRUCE R ET AL: "CD4+ T cells tolerized ex vivo to host alloantigen by anti-CD40 ligand (CD40L:CD154) antibody lose their graft-versus-host disease lethality capacity but retain nominal antigen responses." JOURNAL OF CLINICAL INVESTIGATION, Bd. 102, Nr. 3, 1. August 1998 (1998-08-01), Seiten 473-482, XP002187718 ISSN: 0021-9738 *
CHAMPLIN RICHARD ET AL: "Harnessing graft-versus-malignancy: Non-myeloablative preparative regimens for allogeneic haematopoietic transplantation, an evolving strategy for adoptive immunotherapy." BRITISH JOURNAL OF HAEMATOLOGY, Bd. 111, Nr. 1, Oktober 2000 (2000-10), Seiten 18-29, XP002187720 ISSN: 0007-1048 *
GUINAN EVA C ET AL: "Transplantation of anergic histoincompatible bone marrow allografts." NEW ENGLAND JOURNAL OF MEDICINE, Bd. 340, Nr. 22, 9. Juni 1999 (1999-06-09), Seiten 1704-1714, XP002187717 ISSN: 0028-4793 *
LU L ET AL: "ADENOVIRAL DELIVERY OF CTLA4LG INTO MYELOID DENDRITIC CELLS PROMOTES THEIR IN VITRO TOLEROGENICITY AND SURVIVAL IN ALLOGENEIC RECIPIENTS" GENE THERAPY, MACMILLAN PRESS LTD., BASINGSTOKE, GB, Bd. 6, Nr. 4, 1999, Seiten 554-563, XP000946114 ISSN: 0969-7128 *
ZELLER ET AL: "Induction of CD4+ T Cell Alloanitgen-Specific Hyporesponsiveness by Il-10 and TGF-beta" THE JOURNAL OF IMMUNOLOGY, Bd. 163, 1999, Seiten 3684-3691, XP002187719 *

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