WO1999046365A1 - Methodes et compositions de developpement selectif de lymphocytes t gamma/delta - Google Patents

Methodes et compositions de developpement selectif de lymphocytes t gamma/delta Download PDF

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WO1999046365A1
WO1999046365A1 PCT/US1999/005355 US9905355W WO9946365A1 WO 1999046365 A1 WO1999046365 A1 WO 1999046365A1 US 9905355 W US9905355 W US 9905355W WO 9946365 A1 WO9946365 A1 WO 9946365A1
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cells
cell
population
gamma
cultures
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PCT/US1999/005355
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Edmund K. Waller
Richard Lopez
Robert Negrin
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Emory University
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Priority to EP99911325A priority Critical patent/EP1062320A4/fr
Priority to CA002323080A priority patent/CA2323080A1/fr
Priority to AU29992/99A priority patent/AU746531B2/en
Publication of WO1999046365A1 publication Critical patent/WO1999046365A1/fr

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    • 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
    • A61K2239/31—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterized by the route of administration
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/38—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterised by the dose, timing or administration schedule
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/20—Interleukins [IL]
    • A61K38/208—IL-12
    • 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
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • 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
    • A61K39/462—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
    • A61K39/464—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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2806—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against CD2
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2809—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against the T-cell receptor (TcR)-CD3 complex
    • 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
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/20—Cytokines; Chemokines
    • C12N2501/23—Interleukins [IL]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/20—Cytokines; Chemokines
    • C12N2501/24—Interferons [IFN]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/50—Cell markers; Cell surface determinants
    • C12N2501/599—Cell markers; Cell surface determinants with CD designations not provided for elsewhere
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/52—Assays involving cytokines
    • G01N2333/54—Interleukins [IL]

Definitions

  • the present invention relates to methods for expanding gamma-delta ( ⁇ ) T cells in hematolymphoid cell populations for the purpose of generating cell populations enriched for ⁇ T cells.
  • enriched populations can be used in a variety of applications, including therapeutic uses such as treatment of cancer and infectious diseases, promotion of wound healing and enhancement of bone marrow engraftment, as well as for functional and structural studies of ⁇ T cells and their interactions with other components of the immune system.
  • ⁇ T cells The subset of T cells known as ⁇ T cells is normally present in hematolymphoid cell populations as a very small fraction.
  • HSC hematopoietic stem cell
  • BMT allogeneic bone marrow transplant
  • MHC major histocompatibility complex
  • ⁇ T cells can play a beneficial role in the control of infectious disease, in inhibiting tumor growth and in promoting wound healing.
  • PB peripheral blood
  • BM bone marrow
  • ⁇ T cells are extremely sensitive to TCR/CD3 engagement (especially in the presence of IL-2) and undergo apoptosis upon receiving mitogenic stimuli through the TCR (27).
  • IL-2 interleukin-2
  • studies have shown that human ⁇ T cell clones readily undergo apoptosis when stimulated simultaneously by anti- CD3/TCR MAb plus exogenous IL-2 (28,29). This mechanism of activation-induced cell death (AICD) has not previously been overcome, thereby significantly limiting the usefulness of the ⁇ T cells expanded as previously described.
  • AICD activation-induced cell death
  • ⁇ T cells have a very limited life span (about 1-2 weeks) and by the time a sufficient number of ⁇ T cells could be isolated and pooled from primary cultures, most of the cells will have died or would be very near death. Such cells would not be useful for administration to a subject for the various clinical applications described herein because the ⁇ T cells would not survive long enough in the subject to facilitate engraftment, inhibit an infectious process, inhibit tumor growth or promote wound healing. Thus, what is needed is a method of expanding ⁇ T cells which can survive both ex vivo for a period of time sufficient to produce sufficiently large numbers of ⁇ T cells for clinical use and in vivo for a period of time sufficient to impart their intended clinical effect.
  • the present invention overcomes previous shortcomings in the art by providing methods for increasing the percentage of ⁇ T cells, which can survive for prolonged periods, in a population of hematolymphoid cells and for administering hematolymphoid cell populations which are enriched for these ⁇ T cells to subjects to treat cancer, treat infections, promote wound healing and enhance transplant 3
  • the present invention also provides populations of hematolymphoid cells having increased percentages of ⁇ T cells which can survive for prolonged periods.
  • the present invention provides a method of increasing the percentage of gamma-delta T cells in a population of hematolymphoid cells and which gamma-delta T cells can survive for a prolonged period, comprising: a) contacting a population of hematolymphoid cells with interleukin 12 and a ligand of CD2 which induces responsiveness to interleukin 12; and b) contacting the cells of step (a) with an antibody to CD3 and interleukin-2.
  • Also provided is a method of screening a ligand of CD2 for the ability to induce responsiveness to interleukin 12 comprising: a) contacting a population of hematolymphoid cells with the ligand and interleukin 12; b) contacting the cells of step (a) with an antibody to CD3 and interleukin 2; c) maintaining the cells of step (b) in culture for at least seven days; and d) determining the percentage of viable gamma delta T cells in the population of cells of step (c), whereby greater than 10% viable gamma delta T cells identifies a ligand of CD2 having the ability to induce responsiveness to interleukin- 12.
  • hematolymphoid cells having greater than 10% gamma delta T cells and which gamma-delta T cells can survive for a prolonged period.
  • the present invention also provides a method of treating cancer, treating an infection, promoting wound healing and enhancing bone marrow engraftment in a subject comprising administering to the subject an effective amount of the cells of this invention.
  • a or “an” can mean multiples.
  • a cell can mean at least one cell or more than one cell.
  • the present invention is based on the surprising discovery that the percentage of ⁇ T cells in a population of hematolymphoid cells can be increased to percentages never before achieved by the administration to the cell population of a particular combination of cytokines and mitogenic stimuli in a specific order.
  • this invention provides the discovery that ⁇ T cells can be expanded in a population of hematolymphoid cells by first administering IL-12 and a ligand of CD2, followed by administering IL-2 and T cell mitogenic stimulus.
  • a further unexpected discovery is that the expanded ⁇ T cells are capable of surviving for a prolonged period of time, making them very useful as source populations for studying the functional and structural aspects of ⁇ T cells, for identifying additional cytokines and/or other substances which activate ⁇ T cells and for analyzing their interactions with other immune components, as well as for developing clinical uses for these cells.
  • the present invention provides a method of increasing the percentage of gamma-delta T cells in a population of hematolymphoid cells and which gamma-delta T cells can survive for a prolonged period, comprising: a) contacting a population of hematolymphoid cells with interleukin 12 and a ligand of CD2 which induces responsiveness to interleukin 12; and b) contacting the cells of step (a) with a T cell mitogen and interleukin-2.
  • the method of this invention can further comprise the step of contacting the cells of step (a) with interferon- ⁇ .
  • ⁇ T cells in the population of hematolymphoid cells has increased as a result of this method can be determined according to standard methods well known in the art and as described herein for determining percentages of various cell types in a mixed population of cells.
  • the percentage of ⁇ T cells can be measured in a population of hematolymphoid cells by fluorescence activated cell sorting (FACS) as described in the Examples herein.
  • FACS fluorescence activated cell sorting
  • ⁇ T cells "which can survive for a prolonged period” means ⁇ T cells which can survive under the culture conditions described herein for a period of time which is greater than the period of time during which hematolymphoid cells in primary culture normally survive.
  • a prolonged period of time for survival of ⁇ T cells means survival of ⁇ T cells for greater than three weeks and more preferably, for greater than six weeks and most preferably, for greater than eight weeks, under the culture conditions described herein. It is contemplated that the ⁇ T cells of this invention could be kept alive and functional for the methods described herein indefinitely upon subsequent restimulation of the ⁇ T cells according to the methods described herein.
  • a ligand of CD2 which induces responsiveness to interleukin 12 means any natural or synthetic molecule, including antibodies to CD2, that, upon interaction with CD2 itself, results in the generation of the cellular and molecular events of signal transduction, thus increasing a cell's responsiveness to IL- 12.
  • responsiveness to IL-12 is meant that the cells which bind the ligand of CD2 are enhanced in their ability to bind and/or respond to IL-12.
  • a ligand of CD2 which can be used in the methods of this invention can include, but is not limited to, an antibody or antibody fragment which specifically binds CD2, CD58, a natural or synthetic homologue of CD58, a receptor-binding fragment of CD58, CD48, a natural or synthetic homologue of CD48 and a receptor-binding fragment of CD48, any of which induce responsiveness to IL-12.
  • a ligand of CD2 can be screened for the ability to induce responsiveness to interleukin- 12 according to the methods described herein.
  • the ligand of CD2 can be the monoclonal antibody S5.2 (mouse IgG2a, Becton Dickinson).
  • IL-12 and IL-2 can include fragments of IL-12 or IL-2, respectively, which retain the binding and signal transducing activity of an entire IL-12 or IL-2 molecule.
  • the T cell mitogen of this invention can be any substance, now known or later identified to have a mitogenic effect on T cells.
  • the T cell mitogen of this invention can be, but is not limited to, an antibody to CD3, pokeweed mitogen, ionomycin, phorbol myristate acetate (PMA), a superantigen (e.g., substances, such as bacterial products, which activate the T cell receptor nonspecifically by binding to less polymorphic domains) and any other T cell mitogen now known or later identified to be a T cell mitogen.
  • a superantigen e.g., substances, such as bacterial products, which activate the T cell receptor nonspecifically by binding to less polymorphic domains
  • the present invention further provides a method of screening a ligand of CD2 for the ability to induce responsiveness to interleukin 12 comprising: a) contacting a population of hematolymphoid cells with the ligand and interleukin 12; b) contacting the cells of step (a) with an antibody to CD3 and interleukin 2; c) maintaining the cells of step (b) in culture for at least seven days; and d) determining the percentage of viable gamma delta T cells in the population of cells of step (c), whereby greater than 10% viable gamma delta T cells identifies a ligand of CD2 having the ability to induce responsiveness to interleukin- 12.
  • the screening method of this invention can further comprise the step of contacting the cells of step (a) with interferon gamma.
  • the percentage of viable ⁇ T cells can be determined according to the methods provided in the Examples herein and as are well known in the art.
  • the ⁇ T cells can be selectively separated from other cell types by FACS, as described herein and the viability of the ⁇ T cells can be determined by trypan blue staining, as also described herein.
  • hematolymphoid cells means any cells derived from bone marrow precursor cells, which comprise myeloid cells, erythroid cells, lymphoid cells, platelets and the like, as is well known in the art.
  • the hematolymphoid cells which can be used in the methods of this invention can be bone marrow cells, peripheral blood mononuclear cells and/or cord blood cells, as well as cells from any tissue in which ⁇ T cells and/or their precursors can be found (e.g., skin, intestinal epithelium, thymus, liver, spleen, fetal tissues such as fetal liver and fetal thymus).
  • the hematolymphoid cells of this method can be from any animal which produces ⁇ T cells, which can be any mammal and in a preferred embodiment, is a human.
  • the hematolymphoid cells used in the methods of this invention can be cells which are removed from a subject and returned to the subject (autologous). Additionally, the hematolymphoid cells can be removed from a donor and administered to a recipient of the same species, that is a genetically different (allogeneic); removed from a donor and administered to a recipient that is genetically identical (syngeneic), and/or removed from a donor and administered to a recipient of a different species (xenogeneic).
  • the subject, donor and/or recipient of the present invention can be diagnosed with cancer (e.g, leukemia, lymphoma and solid tumors) tissue injury (e.g., due to trauma, burns, graft-versus-host disease or autoimmune destructive processes) and/or an infection by a pathogen (e.g., human immunodeficiency virus or other pathogenic virus, pathogenic bacteria, parasites, mycoplasma, pathogenic fungi, etc.).
  • cancer e.g, leukemia, lymphoma and solid tumors
  • tissue injury e.g., due to trauma, burns, graft-versus-host disease or autoimmune destructive processes
  • an infection by a pathogen e.g., human immunodeficiency virus or other pathogenic virus, pathogenic bacteria, parasites, mycoplasma, pathogenic fungi, etc.
  • the hematolymphoid cells used in the methods of this invention can be removed from a donor and administered to a transplant recipient.
  • the transplant recipient can be diagnosed with cancer, tissue injury and/or an infection by a pathogen.
  • the cells of this invention can be used to enhance engraftment of the recipient's transplanted tissue and/or treat the recipient's cancer, infection and/or tissue injury.
  • the population of hematolymphoid cells in which the percentage of ⁇ T cells is increased by the methods described herein can be further enriched for ⁇ T cells to yield a population of hematolymphoid cells having any percentage of ⁇ T cells, up to 100% ⁇ T cells.
  • Such enriched cell populations can be administered, in a pharmaceutically acceptable carrier, to a subject for a variety of therapeutic treatments, as described herein.
  • the method of increasing the percentage of ⁇ T cells in a population of hematolymphoid cells as described herein can also comprise the step of further increasing the percentage of ⁇ T cells in the population of hematolymphoid cells of step (c) of the above-described method by procedures well known in the art and as described herein, such as selective separation of ⁇ T cells by fluorescence activated cell sorting (FACS), affinity column chromatography, immunomagnetic separation, 8
  • FACS fluorescence activated cell sorting
  • affinity column chromatography affinity column chromatography
  • immunomagnetic separation 8
  • the ⁇ T cells are selectively separated by FACS with negative staining methods, as described in the Example section herein, to avoid stimulation of the ⁇ T cells.
  • the present invention also provides a population of hematolymphoid cells having greater than 10% ⁇ T cells and which ⁇ T cells s can survive for a prolonged period, as defined herein.
  • the population of hematolymphoid cells of this invention can have greater than 20% ⁇ T cells, greater than 30% ⁇ T cells, greater than 40% ⁇ T cells, greater than 50%> ⁇ T cells, greater than 60% ⁇ T cells, greater than 70% ⁇ T cells, greater than 80% ⁇ T cells and greater than 90% ⁇ T cells and can have any percentage of ⁇ T cells up to 100% ⁇ T cells.
  • the hematolymphoid cells having the percentages of ⁇ T cells listed above can be bone marrow cells, peripheral blood mononuclear cells and/or cord blood cells, as well as cells from any tissue in which ⁇ T cells and/or their precursors can be found, as described herein.
  • the other cell types included in the population of hematolymphoid cells can include but are not limited to, red blood cells, platelets, white blood cells (e.g., neutrophils, eosinophils, basophils, monocytes, lymphocytes), tissue cells (e.g, macrophages, epithelial cells, endothelial cells, dendritic cells, mast cells) and the like as would be well known to an artisan.
  • the hematolymphoid cells of this invention can be from any animal which produces ⁇ T cells, which can be any mammal and in a preferred embodiment is a human.
  • the cells of this invention can be administered to a subject to treat various disorders as well as to enhance transplant engraftment.
  • the present invention provides a method of treating cancer in a subject comprising administering to a subject diagnosed with cancer an effective amount of the cells of this invention.
  • the cancer of this invention can be any malignant blood disorder, acute and/or chronic leukemia, multiple myeloma, lymphoma, solid tumors (e.g., breast cancer, pancreatic cancer).
  • the cells of this invention can be administered to a subject to treat aplastic anemia, bone marrow failure and any other hematological disorder and/or immunological disorder which can be treated by administration of the cells of this invention.
  • a method of treating an infection in a subject comprising administering to a subject diagnosed with an infection an effective amount of the cells of this invention.
  • a method of promoting wound healing in a subject comprising administering to a subject having a wound an effective amount of the cells of this invention.
  • a method of enhancing bone marrow engraftment in a bone marrow recipient comprising administering to the recipient an effective amount of the cells of this invention.
  • the hematolymphoid cells of this invention can be removed from a subject or donor and treated according to the methods described herein to increase the percentage of ⁇ T cells in the hematolymphoid cell population and administered to the same subject or to a recipient by ex vivo methods for removing cells, maintaining the cells in culture and re-administering them, as well known in the art. Standard methods are known for removal of cells from a subject or donor (e.g., phlebotomy, apheresis) and infusion of cells into a subject or recipient.
  • the percentage of ⁇ T cells can be increased in a population of hematolymphoid cells in vivo.
  • the ligand of CD2, the T cell mitogen, IL-12 and IL-2 can be administered to the subject in a pharmaceutically acceptable carrier.
  • an anti-CD2 antibody which induces responsiveness to IL-12 e.g., MAb S5.2
  • IL-12 can be administered intravenously to a subject in a dosage range of 100 to 1000 nanograms/kg of body weight (41)
  • an anti-CD3 antibody e.g., OKT3
  • IL-2 can be administered intravenously to a subject in a range of 200,000 to 800,000 international units (IU) (manufacturer's package insert and Physician's Desk Reference, latest edition).
  • IU international units
  • the exact amount of these substances can vary from subject to subject, depending on the species, age, weight and general condition of the subject, the severity of the disorder being treated, the particular substance being used, its mode of administration and the like. Thus, it is not possible to specify an exact amount for every substance in every subject. However, an appropriate amount can be determined by one of ordinary skill in the art using only routine experimentation given the teachings herein (see, e.g., Remington 's Pharmaceutical Sciences).
  • 0.1 mg/kg of anti-CD2 and 500 nanogram/kg of IL- 12 is administered in a pharmaceutically acceptable carrier as an intravenous infusion to the subject.
  • 600,000 IU of IL-2 is administered in a pharmaceutically acceptable carrier as a 15 minute intravenous infusion and 5 mg of anti-CD3 as a single dose is administered in a pharmaceutically acceptable carrier, intravenously as a rapid injection.
  • the peripheral blood of the subject can be analyzed by FACS before and after administration to determine if the percentage of ⁇ T cells has increased.
  • the time intervals for such measurement of the peripheral blood can be hours, days, weeks and/or months after administration of the substances listed herein.
  • Other clinical parameters which can be monitored to determined the efficacy of administration of these substances can include measurement of 1) disease progression or response, 2) rate of graft failure, 3) graft-versus-host disease assessment, 4) time to engraftment after bone marrow transplant, 5) rate of infectious complications and/or 6) objective parameters of epithelial mucosal tissue injury, as would be known to one of skill in the art.
  • the present invention further provides a method for 1) treating cancer in a subject; 2) treating an infection in a subject; 3) promoting wound healing in a subject; 11
  • enhancing engraftment of a transplant in a subject by increasing the percentage of ⁇ T cells in the subject comprising: a) administering to the subject on day 0 an effective amount of a ligand of CD2 which induces responsiveness to IL-12, in a pharmaceutically acceptable carrier and an effective amount of IL-12; and b) administering to the subject on day 1 an effective amount of a T cell mitogen in a pharmaceutically acceptable carrier and an effective amount of IL-2 in a pharmaceutically acceptable carrier, whereby the administration of the substances of steps (a) and (b) increases the percentage of ⁇ T cells in the subject.
  • the cells and/or substances administered to a subject in vivo can be in a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable is meant a material that is not biologically or otherwise undesirable, i.e., the material may be administered to a subject, along with the cells or substances, without causing any undesirable biological effects or interacting in a deleterious manner with any of the other components of the pharmaceutical composition in which it is contained.
  • the carrier would naturally be selected to minimize any degradation of the active ingredient and to minimize any adverse side effects in the subject, as would be well known to one of skill in the art.
  • the cells of the present invention and the substances which increase the percentage of ⁇ T cells in vivo are typically administered parenterally and are most typically administered by intravenous injection, although other parenteral routes of administration, such as intramuscular, intradermal, subcutaneous, intraperitoneal administration, etc., is also contemplated.
  • injectables can be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for solution of suspension in liquid prior to injection, or as emulsions.
  • a more recently revised approach for parenteral administration involves use of a slow release or sustained release system, such that a constant level of dosage is maintained. See, e.g., U.S. Patent No. 3,710,795, which is incorporated by reference herein. 12
  • PBMC adherent cell-depleted PBMC and CD14 + monocytes.
  • PBMC replete with monocytes, were isolated from healthy, human volunteers by Ficoll gradient centrifugation of peripheral blood anticoagulated with heparin.
  • PBMC were depleted of monocytes by removal of plastic-adherent cells, as previously described (15).
  • CD14+ monocytes were purified directly from PBMC by sorting FITC- CD14 (Leu-M3) cells, as described below.
  • PBMC or monocyte-depleted PBMC were initiated at a cell density of 1 x 10 6 cells/mL in 24-well flat-bottom tissue culture trays (Costar, Cambridge, MA) and were maintained in 5 % CO 2 at 37° C in complete medium consisting of RPMI-1640 (Applied Scientific, San Francisco, CA), 10% autologous human plasma, 2 mM L-glutamine, 100 U/mL penicillin, 100 U/mL streptomycin and 50 ⁇ M 2-ME (GIBCO, Grand Island, NY).
  • human rIFN- ⁇ Boehringer Mannheim, Indianapolis, IN was added at a concentration of 1,000 U/mL.
  • rIL-12 commercially prepared recombinant IL-12 (Genetics Institute, Cambridge, MA) was used at a final concentration of 10 U/mL and was added as a single dose to appropriate cultures on the day of initiation.
  • a neutralizing polyclonal anti-IL-12 antibody, a neutralizing monoclonal anti-IL-12 antibody, or an irrelevant isotype control antibody were used at concentrations of 20 ⁇ g/mL and were added as a single dose to cultures on the day of 13
  • PE-directly conjugated MAbs recognizing CD3, CD14, CD19, CD20 or CD56 (Becton- Dickinson, San Jose, CA). Directly-conjugated isotype-matched irrelevant antibodies served as controls.
  • Cells were stained for 30 min at 4° C in staining buffer consisting of Hank's buffered saline solution (HBSS, Mediatech, Herndon, VA) containing 2% autologous human plasma. Excess Ab was removed by dilution with 10 volumes of staining buffer, followed by centrifugation at 500x g.
  • staining buffer consisting of Hank's buffered saline solution (HBSS, Mediatech, Herndon, VA) containing 2% autologous human plasma. Excess Ab was removed by dilution with 10 volumes of staining buffer, followed by centrifugation at 500x g.
  • Stained cells were immediately analyzed using a FACSCAN flow cytometer (BDIS, San Jose, CA), or sterile-sorted using a FACSVANTAGE cell sorter (BDIS, San Jose, CA).
  • List-mode data were acquired using forward- and side-scatter gates appropriate for viable lymphoid cells or monocytes. Data analysis was performed using CellQuestTM or LYSIS-II software (BDIS, San Jose, CA).
  • IL-12 by ELISA Monocytes, B lymphocytes, T lymphocytes and NK cells were isolated directly from fresh PBMC by sorting the respective CD 19 CD14 + CD14 ⁇ CD19 + , CD3 + CD56 ⁇ and CD3 " CD56 + cell populations. Equivalent numbers (1 x 10 ) of each sorted population were cultured separately as described above, with or without the addition of IFN- ⁇ on the day of culture initiation. Supernatants were harvested after 72 hours, centrifuged to remove cellular debris and stored at -20° C. Detection of IL-12 in culture supernatants was performed using an enzyme-linked immunoassay following the manufacturer's instructions (IL-12 QuantikineTM Assay, R&D Systems, Minneapolis, MN).
  • Anti-CD2 MAb-induced ⁇ T cell proliferation was augmented by the addition of exogenous rIL-12. If both anti-CD2 MAb and rIL-12 (10 U/mL) were included at the initiation of cultures, an even greater percentage of ⁇ T cells was detected after 14 days in culture (32%). Addition of IL-12 with only the isotype control for anti-CD2 resulted in a minimal increase of ⁇ T cell percentage. If anti-CD2 MAb was added to culture but neutralizing MAb to IL-12 was also added at the initiation of culture, ⁇ T cell proliferation was completely inhibited, indicating that the CD2-mediated ⁇ T cell proliferation is dependent upon the presence of endogenous IL-12 in these cultures.
  • Endogenous IL-12 exerts an important effect upon ⁇ T cell expansion in this culture system.
  • Monocytes are an important cellular source of IL-12 and interferon- gamma (IFN ⁇ ) stimulates the production of IL-12 by monocytes.
  • IFN ⁇ interferon- gamma
  • IFN ⁇ plays a role in the stimulation of IL-12 production in these conditions, it is also possible that IFN ⁇ induces the expression of other soluble factors or surface structures which may also contribute to the observed ⁇ T cells expansion.
  • Antibodies to be tested were added to cultures at the time of initiation (day 0) at equivalent concentrations (5 mcg/mL). These antibodies included 6F10.3 (mouse IgGl, Immunotech); 39C1.5 (rat IgG2a, Immunotech) and LT-2 (mouse IgG2b, Serotech). None of these reagents tested 15
  • Anti-CD2 MAb S 5.2 is a commercially available preparation, available in a low endotoxin azide-free form.
  • IL-12 antibody 25 mcg/mL, R&D Systems. Isotype control for anti-human IL-12 antibody had no effect.
  • ⁇ T cells Sorting of ⁇ T cells and ⁇ T cells by negative selection.
  • ⁇ T cells were isolated by negative sorting.
  • ⁇ T cells were identified and sorted on the basis of being TCR ⁇ - and CD5+. (Greater than 96% of CD3+ cells also stained for CD5). All cells sorted were taken from the PI- gate to assure viability. Briefly, ⁇ T cells were stained using FITC- or PE-directly conjugated MAbS recognizing CD5 and TCR ⁇ (Becton-Dickinson, San Jose, CA). Directly-conjugated isotype-matched irrelevant antibodies served as controls.
  • ⁇ T cells sorted from 3 week old short-term cultures proliferate to a greater degree than ⁇ T cells.
  • Cultures of human PBMC were initiated as described above, receiving on day 0: IFN- ⁇ , IL-12 and anti-CD2 mAb S5.2.
  • OKT3 and IL-2 were added on day 1. Cultures were maintained at a cell density of 1-2 17
  • ⁇ T cell and ⁇ T cell populations were sorted from these initial cultures by negative selection as described above and were replated at equivalent cell density (5,000 cells/well) in 96-well micro-titer trays, receiving either IL-2 at 100 U/mL or nothing. After 24 hours, sorted cells were labeled and harvested as above. In these cultures, ⁇ T cells proliferated to a greater extent in response to IL- 2 when compared to ⁇ T cells arising from identical cultures, in contrast to the above data obtained in fresh PBMC cultures.
  • ⁇ T cells have been reported to be highly sensitive to TCR/CD3 engagement (especially in the presence of IL-2) and undergo apoptosis upon receiving mitogenic stimuli through the TCR (10), an interpretation of these data is that ⁇ T cells found in these longer-term cultures represent the outgrowth of a subset of cells which upon engagement of CD2 were imparted a survival advantage, i.e., became resistant to apoptosis or activation-induced cell death (AICD) caused by engagement of TCR CD3.
  • AICD activation-induced cell death
  • ⁇ T cells present in longer-term cultures represented the out-growth of a population of cells more resistant to apoptosis (either through inherent or acquired means).
  • PBMC peripheral blood mononuclear cells
  • OKT3 and IL-2 on day 1.
  • Cultures were maintained at a cell density of 1-2 x 10 6 cells/mL for 3 weeks with the addition of fresh media and IL-2 (100 U/mL) as needed.
  • highly pure ⁇ T cell populations and ⁇ T cell populations were sorted from these initial cultures by negative selection as described above.
  • AICD activation-induced cell death
  • AICD programmed cell death
  • initiation of programmed cell death likely involves the transduction of death-initiating signals generated by engagement of the Fas (CD95/Apo-l) antigen present on T cells and that apoptosis triggered by TCR CD3 signaling is not restricted to CD4+CD8+ immature thymocytes or transformed leukemic T cell lines but can also occur in IL-2-dependent normal ⁇ T cells (10,19- 21). It has also been shown that Fas/CD95 engagement and subsequent mobilization of intracellular Ca 2+ are causally related to apoptosis occurring in human ⁇ T cells clones (22).
  • Flow cytometry Flow cytometry using a four-color dual laser configuration (FACS Calibur flow cytometer or FACS Vantage cell sorter, Becton Dickinson) allows a discrete population of cells (such as ⁇ T cells) to first be defined by surface phenotype from within a heterogeneous population of cells. Simultaneously, biologically relevant processes related to activation, proliferation, cytokine production or apoptosis can be examined, provided the proper reagents and methods are employed.
  • Annexin V conjugated to FITC has been used to detect apoptosis in a variety of cell types.
  • Annexin V binds with high affinity to phosphatidylserine which is normally confined to the inner plasma membrane leaflet of live, non-apoptotic cells. Phosphatidylserine externalization is an early and widespread event associated with apoptosis in a variety of human cell types, regardless of the initiating stimulus.
  • Annexin V-FITC can be used to examine apoptosis in a phenotypically defined subpopulation of cells in heterogeneous cell cultures.
  • PI propidium iodide
  • ⁇ T cells at rest are more prone to apoptosis than ⁇ T cells.
  • T cell subsets from fresh PBMC isolated from several different individuals were first characterized for their "baseline" apoptotic tendencies, ⁇ - and ⁇ T cells were first identified and gated on the basis of surface staining with directly conjugated antibodies recognizing CD3 and TCR ⁇ . Gated populations of ⁇ - and ⁇ T cell populations were then analyzed with respect to Annexin-FITC and PI. Statistics were expressed as the percentage of cells appearing in the corresponding dot-plot quadrants.
  • apoptotic and necrotic regions were defined using the Jurkat T cell line treated with the anti-Fas (CD95/Apo-l) monoclonal antibody CH-11 (Kamiya Biomedical) or isotype control IgM. 20
  • day 0 stimuli were designated as "rescue” signals and day 1 stimuli (a standard mitogenic combination) were designated as the "death" signals.
  • PBMC peripheral blood by Ficoll density centrifugation. Cultures were initiated receiving either day 1 "death” signals alone; day 0 "rescue” signals alone or both day 0 + day 1 signals, i.e., "standard” conditions. At 18 hours after receiving the day 1 signals (mitogen), cultures were analyzed for 21
  • ⁇ T cells were highly sensitive to AICD in response to day 1 ("death") signals alone. As stated previously, close to 60 % of resting ⁇ T cells were viable (AnnexinVPI " ). However, after receiving mitogenic stimuli, this percentage falls to 5%. In contrast, ⁇ T cells in the same cultures demonstrated no such sensitivity to these mitogenic conditions, with close to 90% of these cells remaining viable.
  • ⁇ T cells serve as a control, indicating that it is the ⁇ T cell compartment which is most affected by these manipulations.
  • PBMC peripheral blood by Ficoll density centrifugation. Cultures were initiated, receiving either day 1 "death" signals alone or both day 0 + day 1 signals, i.e., "standard” conditions. Cultures receiving no mitogenic stimulation via OKT3 and IL-2 ("rescue" signals alone) failed to proliferate. One week after receiving the day 1 signals (mitogen), cultures were analyzed for apoptosis by four-color flow cytometry as described above.
  • the cell cultures described herein which have been treated by standard conditions and which have been shown to contain up to 80% viable ⁇ T cells after one week, have been maintained in culture for a time period exceeding eight weeks. These cultures can be perpetuated indefinitely by cell passage and addition of fresh medium as needed and restimulation of the cells by administering the substances described herein for standard conditions at regular intervals, as can be determined by routine methods. 23
  • PBMC peripheral blood mononuclear cells
  • PBMC peripheral blood mononuclear cells
  • RPMI-1640 Mediatech, Herdnon, VA
  • 10% fetal bovine serum HyClone, Logan, UT
  • 2 mM L-glutamine 100 U/mL penicillin, 100 U/mL streptomycin and 50 ⁇ M 2-ME (GIBCO, Grand Island, NY).
  • neutralizing monoclonal anti-human IL-12 antibody, or an irrelevant isotype control antibody were added as a single dose at a final concentration of 25 ⁇ g/mL on the day of culture initiation.
  • Neutralizing monoclonal anti-human CD58 MAb clone L306 (mouse IgG2 a , Camfolio, Becton Dickinson, San Jose, CA) or IgG2 a isotype control antibody were added to cultures where indicated, at a final concentration of 5 ⁇ g/mL. In all cultures, cell density was maintained at 1 to 2 x 10 cells/mL, with the addition of fresh media and transfer to larger tissue culture flasks as required. Fresh, complete medium with 10 U/mL IL-2 was added to cultures every 7 days.
  • MAbs at a concentration of 10 ⁇ g/mL in PBS were placed in 24 well flat-bottom tissue culture trays (Costar), making certain to coat the entire surface of each well. After incubating at 37° C for 2 hours or at 4°C overnight, wells were washed 3 times with ice cold PBS followed by gentle suction aspiration of well contents.
  • [ 3 H]thymidine proliferation assay Highly pure ⁇ and ⁇ T cells were plated at equivalent cell numbers (5,000 cells/well) in 96-well micro-titer trays, receiving either IL-2 at 100 U/mL or PBS only. After 24 hours, sorted cells were incubated with 1 ⁇ Ci [ 3 H]thymidine and after an additional 18 hours, cells were harvested onto glass fiber filters using a standard cell harvester (PHD Cell Harvester, Cambridge, MA); radioactivity was measured using a liquid scintillation counter. All samples were assayed in triplicate with data presented as mean CPM.
  • IX annexin binding buffer provided by manufacturer (Apoptosis Detection Kit, R&D Systems). Cells were then resuspended in 100 ⁇ L of binding buffer to which the appropriate volume of annexin V-FITC and PI were added, as determined by titration. Cells were incubated for 15 minutes at room temperature in the dark after which time 300 ⁇ l IX annexin binding buffer was added. Without washing, cells were immediately analyzed utilizing a dual laser, four color FACSCalibur flow cytometer (BDIS, San Jose, CA). At this point, cells were kept on ice to prevent the capping and internalization of surface-bound MAbs.
  • BDIS color FACSCalibur flow cytometer
  • Mitogenic stimulation of PBMC in the presence of anti-CD2 MAb S5.2 results in a large expansion of ⁇ T cells.
  • Culture of PBMC in the presence of mitogenic anti- CD3 MAb OKT3 and IL-2 is a well known method for the induction of in vitro T cell proliferation, particularly ⁇ T cells (31-33).
  • IL-12-dependent expansion of human CD56+ ⁇ T cells arising in OKT3/IL-2 -stimulated PBMC cultures, particularly if these cultures were first primed with IFN- ⁇ 24 hours prior to stimulation with mitogens has been described (2,3).
  • monocytes serve as an important cellular source of both endogenous IL-12 and a contact-dependent factor in the form of CD58/LFA-3, both of which are critical for the in vitro expansion of these CD56+ ⁇ T cells.
  • ⁇ T cell expansion induced by anti-CD2 MAb S5.2 in mitogen-stimulated PBMC cultures requires the presence ofIL-12.
  • Mitogen-stimulated PBMC cultures were initiated as described above, primed first with IFN- ⁇ then stimulated 24 hours later with mitogenic OKT3 and IL-2. After 7 to 10 days, cultures were analyzed by FACS for the percentage of ⁇ T cells.
  • anti-CD2 MAb S5.2 (but not its isotype control) can induce ⁇ T cell expansion in cultures to which no exogenous IL-12 is added
  • the addition of exogenous IL-12 to identical cultures containing MAb S5.2 results in a further increase in the percentage of ⁇ T cells (24% to 32%).
  • IL-12 alone cannot significantly induce ⁇ T cell expansion in mitogen-stimulated 27
  • anti-CD2 MAb S5.2 was added at the indicated concentration ( ⁇ g/mL). After 14 days, absolute numbers of both ⁇ and ⁇ T cells present in cultures were determined by multiplying the total cell number present in culture by the percentage of ⁇ and ⁇ T cells as measured by FACS. Data are presented as fold expansion over starting ⁇ and ⁇ T cell number.
  • Anti-CD2 MAb S5.2 induces ⁇ T cell expansion via an agonistic and not a blocking interaction with CD2.
  • the existence of "accessory" or “alternate” CD2 signaling pathways triggered by MAbs to CD2 which function exclusively in ⁇ T cells have previously been suggested by several investigators (28,34). While the majority of anti-CD2 MAbs capable of delivering a proliferative signal to either ⁇ or ⁇ T cells appear to do so only if combined with a second anti-CD2 MAb recognizing a separate CD2 epitope (28,34,35), single epitope-binding anti-CD2 MAbs have been reported which appear to stimulate only ⁇ T cells (28,34).
  • anti-CD2 MAb S5.2 functions to induce ⁇ T cell expansion in heterogeneous PBMC cultures.
  • the following experiments were performed to show that MAb S5.2 functions in an agonistic and not a blocking capacity, thereby initiating rather than inhibiting CD2 signal transduction events leading to the observed IL-12-dependent ⁇ T cell expansion.
  • CD58 LFA-3 and CD48 have each been shown to serve as ligands for CD2; in humans, however, only CD58 has been shown to interact with CD2 on T cells in a functionally significant manner (20, 36-38).
  • anti-CD2 MAb S5.2 were inducing ⁇ T cell expansion by blocking an inhibitory interaction between CD2 and CD58, then the effect of a neutralizing anti-CD58 MAb would be the same, i.e., enhancement of ⁇ T cell expansion. This was shown not to be the case.
  • Cultures of fresh PBMC were initiated as described receiving IFN- ⁇ initially (day 0) and OKT3 and IL-2 the next day (day 1).
  • mice IgG 2a mouse anti-human-CD58 MAb L066.4 (mouse IgG 2a ), or mouse IgG 2a isotype control were added separately to identical cultures. After 14 days, cultures were analyzed by FACS to determine the percentage of ⁇ T cells present. Under these conditions, whereas addition of anti-CD2 MAb S5.2 resulted in the expansion of ⁇ T cells, addition of a blocking MAb to CD58 did not cause the same.
  • MAb S5.2 is not causing ⁇ T cell expansion by disrupting a putative inhibitory CD2-CD58 interaction.
  • ⁇ T cells can be induced to expand significantly in mitogen-stimulated cultures by immobilized but not soluble anti-human CD2 MAb S5.2.
  • Immobilized or soluble IgG 2a (isotype control for MAb S5.2) similarly had minimal effect on ⁇ T cell expansion.
  • Cultures maintained for longer periods (up to 35 days) similarly display a preferential expansion of ⁇ T cells induced by immobilized but not soluble MAb S5.2.
  • Enhanced ⁇ T cell expansion induced by MAb S5.2 does not occur simply as a consequence of increased ⁇ T cell proliferation.
  • Data derived in clones clearly show that some anti-CD2 MAbs can preferentially induce proliferation of ⁇ T cells compared to ⁇ T cells (34,35). If the increased ⁇ T cell expansion observed in S5.2- treated cultures was occurring simply on the basis of a preferential proliferation induced by MAb S5.2, then it is likely that ⁇ T cells isolated from these cultures would incorporate [ 3 H]thymidine to a greater degree than ⁇ T cells isolated from identical cultures.
  • IL-12 -dependent MAb S 5.2 -mediated signaling through CD2 protects ⁇ T cells from activation-induced cell death.
  • Janssen et al. have shown that ⁇ T cells areakily sensitive to TCR CD3 engagement (especially in the presence of IL-2) and undergo apoptosis upon receiving mitogenic stimuli through the TCR (27).
  • CD2 engagement by MAb S5.2 in the presence of IL-12 provides a signal to a subset of ⁇ T cells which protects them from activation-induced cell death (AICD) caused by mitogenic OKT3 and IL-2. It is then these apoptosis-resistant ⁇ T cells which eventually come to be represented in larger numbers in S5.2-stimulated cultures.
  • AICD activation-induced cell death
  • Annexin V binds with high affinity to phosphotidylserine (PS) which is normally confined to the inner plasma membrane leaflet of live, non- apoptotic cells; appearance of PS on the outer plasma membrane leaflet is an early and widespread event associated with apoptosis in a variety of human cell types, regardless of the initiating stimulus.
  • day 0 stimuli IFN- ⁇ , IL-12 and anti-CD2 MAb S5.2
  • putative "protective” signals IFN- ⁇ , IL-12 and anti-CD2 MAb S5.2
  • PBMC cultures were initiated as described above. Those receiving day 0 rescue signals were defined as protected; those receiving no day 0 signals (PBS only) were defined as unprotected. All cultures received OKT3 and IL-2 24 hours later (day 1).
  • ⁇ and ⁇ T cell populations within protected and unprotected cultures were simultaneously analyzed for apoptosis using four color flow cytometry.
  • ⁇ and ⁇ T cell populations were first delineated by electronically gating on the corresponding ⁇ and ⁇ T cells defined by anti-CD3-APC and anti-TCR- ⁇ -PE MAbs.
  • Apoptosis occurring in ⁇ and ⁇ T cell populations was then determined examining the uptake of Annexin V-FITC and PI in the respective gated events.
  • Sorted populations were plated at equivalent cell densities and after 24 to 36 hours, apoptosis in each population was determined by two color FACS employing annexin V-FITC and PI only.
  • purified ⁇ T cells from both mitogen-stimulated protected and unprotected cultures were found to be viable (Annexin-/PI-) to an equivalent degree (routinely at least 94%, not shown). More importantly, and in complete agreement with the four color analysis of unsorted ⁇ T cells, unprotected compared to protected ⁇ T cells were confirmed to be far more sensitive to apoptosis induced by mitogen stimulation (54% viable versus 80% viable).
  • IL-2 100 U/mL was added to equivalent numbers of cells from both protected and unprotected PBMC cultures. After overnight incubation, apoptosis in ⁇ and ⁇ T cell populations was determined (day 8) by measuring the uptake of Annexin V-FITC and PI in the respective gated populations.
  • Agonistic mouse anti -human CD95/Fas MAb CHI 1 (mouse IgM) or mouse IgM isotype control were included in identical cultures as controls.
  • cytotoxic activity of these highly purified ⁇ and ⁇ T cells was then tested against 51 Cr-labeled human melanoma cell lines SK-MEL-3, SK- MEL-5 and SK-MEL-28 at various effector to target ratios. Data were evaluated as 34
  • Either ⁇ T cells expanded ex vivo according to the methods of this invention or the substances described herein for increasing the percentage of ⁇ T cells in vivo can be administered to a subject diagnosed with a malignancy, according to the dosage regimens described herein, with the intent of exploiting the anti-neoplastic (anti-tumor or anti-leukemic effect) activity of these cells. Efficacy of this treatment would be determined by assessing a response of the malignancy as measured by tumor regression or failure of tumor progression.
  • ⁇ T cells expanded ex vivo can be administered as an adjuvant to an allogeneic bone marrow transplant in a clinical setting where graft failure would be a likely complication (such as where the donor is an HLA-mismatched sibling or a matched unrelated donor).
  • Ex vivo expanded donor or recipient ⁇ T cells can be administered prior to, in conjunction with or following the administration to the recipient of a donor bone marrow stem cell product which is first depleted of all T cells (which is done in an attempt to minimize the likelihood of graft- versus-host disease).
  • ex vivo expanded ⁇ T cells with the transplanted cells in the recipient would be to facilitate engraftment of donor-derived hematopoietic stem cells in the transplant recipient.
  • Efficacy of this treatment can be determined in a clinical setting by measuring a number of clinical indices as would be well known to the clinician, such as time to full engraftment, or decreased incidence of graft failure.
  • the substances described herein can be administered to the 35
  • bone marrow transplant recipient according to the dosage regimens described herein for increasing the percentage of the recipient's ⁇ T cells in vivo to impart the same beneficial effects as described herein for the direct administration of ex vivo expanded ⁇ T cells. Efficacy of treatment by this in vivo stimulation method would also be assessed according to the same parameters as described herein for the administration of ex vivo expanded ⁇ T cells.
  • the ex vivo expanded ⁇ T cells of this invention can be administered as an adjuvant to standard therapy (which can include, but is not limited to, antibiotics), to a subject diagnosed with an infectious process, including, but not limited to, infection with viral pathogens (such as HIV), bacterial pathogens or other infectious agents. Efficacy would be measured by assessing the subject's ability to effectively clear infectious organisms according to standard protocols well known in the art. Alternatively, the substances described herein can be administered to the subject to treat an infectious process according to the dosage regimens described herein for increasing the percentage of the recipient's ⁇ T cells in vivo to impart the same beneficial effects as described herein for the direct administration of ⁇ T cells. Efficacy of treatment by this in vivo stimulation method would also be assessed according to the same parameters as described herein for the administration of ⁇ T cells.
  • the ex vivo expanded ⁇ T cells of this invention can be administered as an adjuvant to subjects diagnosed with a tissue injury, which can include but is not limited to, tissue trauma, burns, graft-versus-host disease or autoimmune destructive processes, ⁇ T cells appear to contribute to wound healing by the elaboration of a number of important factors including, but not limited to, keratinocyte growth factor (KGF), also known as FGF-7.
  • KGF keratinocyte growth factor
  • the substances described herein can be administered to the subject to treat a tissue injury according to the dosage regimens described herein for increasing the percentage of the recipient's ⁇ T cells in vivo to impart the same beneficial effects as described herein for the direct administration of ⁇ T cells. Efficacy of treatment by this in vivo stimulation method would also be assessed according to the same parameters as described herein for the administration of ⁇ T cells.
  • ⁇ T cells are first expanded ex vivo according to the methods described herein, typically from either an autologous or allogeneic source and delivered intravenously at a dose effective in mediating a biologically significant process, as determined according to the methods described herein.
  • the amount of cells administered to a subject can be in the range of 1 X 10 5 to 1 X 10 8 ⁇ T cells/kg body weight.
  • Negrin RS A novel population of expanded human CD3+CD56+ cells derived from T cells with potent in vivo antitumor activity in mice with severe combined immunodeficiency. J Immunol 153:1687, 1994. 37
  • NKSF natural killer cell stimulatory factor
  • NK Natural killer (NK) cell stimulatory factor or IL-12 has differential effects on the proliferation of TCR- ⁇ , TCR- ⁇ T lymphocytes, and NK cells. J Immunol 149:3495, 1992.
  • Trinchieri G Interleukin- 12: A cytokine produced by antigen-presenting cells with immunoregulatory functions in the generation of T-helper cells type I and cytotoxic lymphocytes. Blood 84:4008, 1994.
  • Tax WJM Hermes FFM, Willems RW, Capel PJA, Koene RAP: Fc receptors for mouse IgGl on human monocytes: polymo ⁇ hisms and role in antibody-induced T cell proliferation. J Immunol 133:1185, 1984.
  • T cell receptor/CD3-signaling induces death by apoptosis in human T cell receptor gamma delta + T cells. Journal of Immunology 146:35-9.
  • Monocytes provide a novel costimulatory signal to T cells that is not mediated by the CD28/B7 interaction. Journal of Immunology 152:429-437.
  • OKT3 A monoclonal anti-human T lymphocyte antibody with potent mitogenic properties. Journal of Immunology 124:2708.
  • CD48 A soluble multimeric recombinant CD2 protein identifies CD48 as a low affinity ligand for human CD2: divergence of CD2 ligands during the evolution of humans and mice. Journal of Experimental Medicine 177:1439-50.
  • CD48 is a counter-receptor for mouse CD2 and is involved in T cell activation. Journal of Experimental Medicine 176:1241-9.

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Abstract

La présente invention concerne une méthode permettant d'accroître le pourcentage de lymphocytes T gamma-delta dans une population de cellules hématolymphoïdes, lesdits lymphocytes T gamma-delta pouvant survivre sur une durée prolongée, consistant: a) à mettre une population de cellules hématolymphoïdes au contact de l'interleukine 12 et d'un ligand de CD2 induisant la faculté de réponse à l'interleukine 12; et b) à mettre les cellules de l'étape a) au contact d'un anticorps dirigé contre CD3 et l'interleukine-2. La présente invention concerne également une méthode de traitement du cancer ou d'une infection, une méthode favorisant la cicatrisation et facilitant la greffe de moelle osseuse chez un sujet, consistant à administrer au sujet une dose efficace des cellules de l'invention.
PCT/US1999/005355 1998-03-12 1999-03-12 Methodes et compositions de developpement selectif de lymphocytes t gamma/delta WO1999046365A1 (fr)

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Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000026347A1 (fr) * 1998-11-04 2000-05-11 Hemosol Inc. METHODES DE PRODUCTION DE LYMPHOCYTES T TCRη$g(d)
WO2001075072A2 (fr) * 2000-04-03 2001-10-11 Hemosol Inc. Production de lymphocytes t gamma delta
EP1147186A1 (fr) * 1999-01-28 2001-10-24 Palmetto Health Alliance d/b/a Palmetto Richland Memorial Hospital Lymphocytes gamma delta actives in vitro
EP1231840A1 (fr) * 1999-11-24 2002-08-21 Ernest G. Hope Agent cellulaire anti-angiogenique destine au traitement du cancer
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US6537812B1 (en) 1998-11-04 2003-03-25 Hemosol Inc. Methods for the production of TcRγδ+ T cells
WO2000026347A1 (fr) * 1998-11-04 2000-05-11 Hemosol Inc. METHODES DE PRODUCTION DE LYMPHOCYTES T TCRη$g(d)
EP1147186A1 (fr) * 1999-01-28 2001-10-24 Palmetto Health Alliance d/b/a Palmetto Richland Memorial Hospital Lymphocytes gamma delta actives in vitro
US7078034B2 (en) 1999-01-28 2006-07-18 Palmetto Health Alliance In vitro activated γ δ lymphocytes
EP1147186A4 (fr) * 1999-01-28 2002-05-15 Palmetto Health Alliance D B A Lymphocytes gamma delta actives in vitro
AU2005239674B2 (en) * 1999-11-24 2009-02-26 Ernest G Hope Anti-angiogenic cellular agent for cancer therapy
EP1231840A1 (fr) * 1999-11-24 2002-08-21 Ernest G. Hope Agent cellulaire anti-angiogenique destine au traitement du cancer
EP1231840A4 (fr) * 1999-11-24 2004-09-29 Ernest G Hope Agent cellulaire anti-angiogenique destine au traitement du cancer
WO2001075072A3 (fr) * 2000-04-03 2002-04-25 Hemosol Inc Production de lymphocytes t gamma delta
WO2001075072A2 (fr) * 2000-04-03 2001-10-11 Hemosol Inc. Production de lymphocytes t gamma delta
EP3018200A1 (fr) 2014-11-07 2016-05-11 Fondazione Matilde Tettamanti e Menotti de Machi Onlus Procédé amélioré pour la production de cellules génétiquement modifiées
US11572542B2 (en) 2014-11-07 2023-02-07 Fondazione Matilde Tettamanti E Menotti De Marchi Onlus Method for the generation of genetically modified cells
WO2020065584A1 (fr) 2018-09-27 2020-04-02 Phosphogam, Inc. Procédés et compositions pour la multiplication et l'utilisation de lymphocytes t gamma/delta allogéniques

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CA2323080A1 (fr) 1999-09-16

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