EP0804550A1 - Activation ex vivo des cellules immunes - Google Patents

Activation ex vivo des cellules immunes

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Publication number
EP0804550A1
EP0804550A1 EP95906781A EP95906781A EP0804550A1 EP 0804550 A1 EP0804550 A1 EP 0804550A1 EP 95906781 A EP95906781 A EP 95906781A EP 95906781 A EP95906781 A EP 95906781A EP 0804550 A1 EP0804550 A1 EP 0804550A1
Authority
EP
European Patent Office
Prior art keywords
sample
cells
t3cs
cell
concentration
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP95906781A
Other languages
German (de)
English (en)
Other versions
EP0804550A4 (fr
Inventor
Bruce P. Babbitt
Zhengyi J. Zhang
Michael E. Osband
Joseph James Goodman
Barry Inn Caplan
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Cellcor Inc
Original Assignee
Cellcor Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from US08/214,400 external-priority patent/US5569585A/en
Application filed by Cellcor Inc filed Critical Cellcor Inc
Publication of EP0804550A1 publication Critical patent/EP0804550A1/fr
Publication of EP0804550A4 publication Critical patent/EP0804550A4/fr
Withdrawn legal-status Critical Current

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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
    • C12N5/0636—T lymphocytes
    • 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
    • A61K39/461—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
    • A61K39/46433—Antigens related to auto-immune diseases; Preparations to induce self-tolerance
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/4644—Cancer antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • 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
    • A61K39/464838—Viral antigens
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • 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
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/46—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterised by the cancer treated
    • A61K2239/56—Kidney
    • 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
    • C12N2501/01—Modulators of cAMP or cGMP, e.g. non-hydrolysable analogs, phosphodiesterase inhibitors, cholera toxin
    • 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
    • C12N2501/515—CD3, T-cell receptor 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
    • C12N2502/00—Coculture with; Conditioned medium produced by
    • C12N2502/11—Coculture with; Conditioned medium produced by blood or immune system cells

Definitions

  • This invention relates to immunotherapy.
  • TIL tumor infiltrating lymphocytes
  • the invention provides a novel, safe, and cost- effective method of nonspecifically enhancing a cell- mediated immune response to specific antigens or foreign substances in the body, including cancer cells.
  • the process involves removing a patient's mononuclear cells and exposing the cells in vitro to substances which enhance the immune function of the cells.
  • the ex vivo activated (EVA) cells are then reinfused into the patient to enhance the patient's immune responses and to treat various forms of cancer, infectious diseases, autoimmune diseases, or immune deficiency diseases.
  • the invention features a process of producing a population of immunoreactive cells by (a) contacting a sample of mononuclear.cells derived from a patient, e.g., peripheral blood mononuclear cells (PBMC) , with OKT3 at or below 37°C to produce an OKT3-derived culture supernatant (T3CS) ; (b) removing the T3CS from the sample of patient-derived mononuclear cells; (c) determining the concentration of OKT3 in the T3CS, and if required, supplementing the T3CS with additional OKT3 to achieve a concentration of at least 0.1 ng/ml; (d) providing a second sample of mononuclear cells derived from the patient; and, (e) contacting the second sample of cells with the previously-generated T3CS for a period of time sufficient to yield a population of immunoreactive cells.
  • PBMC peripheral blood mononuclear cells
  • a sample of patient-derived mononuclear cells may contain T cells, B cells, monocytes and macrophages as well as other immune cells such as polymorphonuclear leukocytes, neutrophils, eosinophils, natural killer cells, and stem cells.
  • Mononuclear cells may be derived from the peripheral blood of the patient, or other sites, e.g., a tumor or tumor-draining lymph node.
  • T3CS is a conditioned medium containing a mixture of autologous cytokines together with OKT3.
  • the autologous cytokines mixture preferably promotes the growth and differentiation of Thl-type T cells, rather than Th2-type T cells.
  • the OKT3 which is preferably in solution phase catalyzes the polyclonal activation of T cells, while the cytokines act synergistically as co- stimulants to optimize the overall degree of activation.
  • the presence of both OKT3 and cytokines prevents the generation of T cells that are anergic or apoptotic and overcomes signal transduction defects in mononuclear cells derived from patients with cancer or chronic infectious diseases.
  • immunoreactive cells polyclonal T cells that exist in a primed state of activation.
  • Primed cells are multifunctional, i.e., they possess an enhanced capacity to proliferate and produce cytokines upon further stimulation.
  • the primed state of activation of the immunoreactive cells induced by culture in the 0KT3-autologous cytokine mixture can be identified by measuring the stable biochemical changes, e.g., expression of growth, differentiation, and activation markers, which occur both on the cell surface and intracellularly.
  • Immunoreactive cells of the invention have enhanced immunologic effector function, e.g., helper activity (CD4 + T cells) or cytotoxicity (CD8 + T cells) , compared to unprocessed patient-derived mononuclear cells.
  • Immunoreactive cells have a low spontaneous level of immune function following processing, but are highly sensitized to respond to low doses of second signals upon further culture, or in vivo.
  • the immunoreactive cells of the invention therefore require further exposure to an immune stimulant, such as an antigen; target cell, e.g., a tumor cell or virus-infected cell; an inflammatory molecule; an adhesion molecule; an immune cell, e.g., an accessory cell; a cytokine; or any combination thereof, to achieve full immunologic effector function.
  • the immunoreactive cells of the invention are multifunctional, polyclonally-activated T cells which have been generated independent of disease-specific antigens utilizing a mixture of nonspecific lymphocyte activators, i.e., autologous cytokines, and a mouse monoclonal antibody, i.e, OKT3, as synergistic stimulants.
  • a mixture of nonspecific lymphocyte activators i.e., autologous cytokines
  • a mouse monoclonal antibody i.e, OKT3, as synergistic stimulants.
  • Suppressor cells in a population of patient- derived mononuclear cells may be inactivated by contacting the second sample with a suppressor cell inhibitory compound, e.g., cimetidine, indomethacin, cyclophosphamide, ranitidine, pepsid, or any combination thereof.
  • a suppressor cell inhibitory compound e.g., cimetidine, indomethacin, cyclophosphamide, ranitidine, pepsid, or any combination thereof.
  • Other histamine type-2 receptor blockers may also be used alone or in combination with the compounds listed above.
  • Cimetidine and indomethacin are preferably used together at concentrations of 5 x 10 ⁇ 5 M (+ 2-fold) and 0.8 x 10 ⁇ 8 M (+ 2-fold), respectively.
  • the concentration of 0KT3 used in the process of the invention is an amount that promotes activation of the patient's cells, but leaves minimal surface-bound 0KT3 on the activated cell product. Minimizing the amount of surface-bound 0KT3 on the immunoreactive cells in turn minimizes human anti-mouse antigen (HAMA) immune responses and rapid clearance of the immunoreactive cells from the circulation of a patient undergoing therapy with the immunoreactive cells of the invention.
  • the concentration of 0KT3 is preferably greater than 0.1 ng/ l but less than 25 ng/ml, more preferably 1-25 ng/ml, and most preferably 10-15 ng/ml.
  • any compound that binds to the T cell receptor or the T cell receptor-associated CD3 molecule on the cell surface may be used to stimulate the first sample of patient-derived mononuclear cells to produce T3CS.
  • Culture of the first sample with OKT3 is carried out for a period of time sufficient to produce a mixture of nonspecific lymphocyte activators capable of promoting the OKT3-catalyzed activation and differentiation of the second sample of patient-derived mononuclear cells into a population of immunoreactive cells.
  • the culture period may range from 1 to 7 days and is preferably 3 days.
  • the length of culture of the first sample may be adjusted, e.g.
  • a nonspecific lymphocyte activator e.g., a cytokine, e.g., tumor necrosis factor-alpha (TNF ⁇ ) or interleukin-2 (IL-2) , in the T3CS.
  • a nonspecific lymphocyte activator e.g., a cytokine, e.g., tumor necrosis factor-alpha (TNF ⁇ ) or interleukin-2 (IL-2)
  • TNF ⁇ tumor necrosis factor-alpha
  • IL-2 interleukin-2
  • Culture of the second sample of patient-derived cells with T3CS is carried out for a period of time sufficient to produce a population of immunoreactive cells.
  • the immunoreactive cells are in a primed state of activation, i.e., the cells are no longer in a resting state but require an additional stimulus, e.g., exposure to an antigen or other immune stimuli, to achieve a fully activated state characterized by enhanced immune function compared to unprocessed cells.
  • Fully activation of immunoreactive cells may be measured by expression of new cell surface markers, e.g., CD25, secretion of lymphokines, e.g., IFN ⁇ , GM-CSF, or TNF ⁇ , cellular proliferation, or cellular differentiation into effector cells, e.g., cytolytic T cells or helper T cells.
  • new cell surface markers e.g., CD25
  • lymphokines e.g., IFN ⁇ , GM-CSF, or TNF ⁇
  • cellular proliferation e.g., cytolytic T cells or helper T cells.
  • Culture of patient-derived mononuclear cells with T3CS may be carried out for a period of 1 to 30 days, preferably 5 days.
  • the length of the culture period may be as short as 1-3 days; in the presence of antigen, the cells may be co-cultured for a period of 1 to 30 days.
  • the length of culture may be adjusted, e.g., prolonged, to achieve the desired level of activation of the immunoreactive cells.
  • the invention also features a process of producing a population of immunoreactive cells by (a) providing a first sample of mononuclear cells derived from a patient; (b) determining the concentration of Fc- receptor positive accessory cells in the first sample, and if required, suppCVementing the first sample with a second sample of mononuclear cells derived from the same patient to achieve a concentration of 0.1-50% Fc-receptor positive accessory cells in the first sample; (c) contacting the first sample with OKT3 at or below 37°C to produce a T3CS; (d) removing the T3CS from the first sample; (e) determining the concentration of OKT3 in the T3CS, and if required, supplementing the T3CS with 0KT3 to achieve a concentration of at least 0.1 ng/ml; (f) providing a third sample of mononuclear cells derived from the same patient; (g) contacting the third sample with T3CS for a period of time sufficient to activate the third sample in vitro to
  • the Fc-receptor positive cells are preferably monocytes, but may be granulocytes or dendritic cells.
  • the concentration of patient-derived monocytes is preferably 0.1-50%, more preferably 1-30%, more preferably 5-15%, and most preferably 10% of the cells in the sample.
  • the second sample of patient- derived mononuclear cells may be enriched for monocytes using cell fractionation techniques known in the art, e.g, panning or FACS, prior to augmenting the concentration of monocytes in the first sample.
  • the process is carried out by: (a) contacting a first sample of mononuclear cells derived from a patient with 0KT3 at or below 37°C to produce a T3CS; (b) removing the T3CS from the first sample; (c) determining the concentration of tumor necrosis factor-alpha (TNF ⁇ ) in the T3CS, and if required, supplementing the T3CS with TNF ⁇ to achieve a concentration of at least 5 pg/ml; (d) providing a second sample of mononuclear cells derived from the same patient; (e) inactivating suppressor cells in the second sample; and, (f) contacting the second sample with T3CS and for a period of time sufficient to activate the second sample in vitro to yield a population of immunoreactive cells.
  • TNF ⁇ tumor necrosis factor-alpha
  • the concentration of TNF ⁇ is preferably in the range of 100 pg/ml to 100 ng/ml, more preferably in the range of 100 pg/ml to 3000 pg/ml, and most preferably in the range of 500 pg/ml to 1000 pg/ml.
  • the concentration of a cytokine, e.g., TNF ⁇ may be augmented by (1) altering the length of the T3CS- generation step, (2) supplementing T3CS with purified non-recombinant cytokine, (3) supplementing T3CS with a recombinant cytokine,. or (4) increasing the concentration of a cytokine-producing patient-derived mononuclear cell.
  • the T3CS generation step (step (a) ) of the process may be carried out for 1-7 days and is preferably carried out for 3 days.
  • the process requires after step (b) , determining the concentration of both TNF ⁇ and 0KT3, and if required, supplementing the T3CS with TNF ⁇ to achieve a concentration of at least 5 pg/ml TNF ⁇ and with 0KT3 to achieve a concentration of at least 0.1 ng/ml OKT3.
  • the levels of interferon-gamma (IFN- ⁇ ) , interleukin-4 (IL-4) , and interleukin-10 (IL-10) in T3CS may be adjusted to achieve optimal generation of a particular type of immunoreactive cell, e.g., a Thl-type T cell.
  • the process may include the steps of: (a) contacting a first sample of mononuclear cells derived from a patient with OKT3 at or below 37°C to produce a T3CS; (b) removing the T3CS from the first sample; (c) determining the concentration of IFN- ⁇ , IL-4, and IL-10 in the T3CS, and if required, adjusting the concentration of IFN- ⁇ to at least 500 pg/ml, the concentration of IL-4 to less than or equal to 20 pg/ml, and the concentration of IL-10 to less than or equal to 20 pg/ml; (d) providing a second sample of mononuclear cells derived from the same patient; (e) inactivating suppressor cells in the second sample; and, (f) contacting the second sample with T3CS and for a period of time sufficient to activate the second sample in vitro to yield a population of immunoreactive cells.
  • the process of the invention may also include a step in which the state of activation of the immunoreactive cells is evaluated prior to administration of the cells to the patient.
  • the cells may be evaluated phenotypically or functionally, i.e., by measuring the expression of a cell surface marker indicative of cell activation and/or differentiation or by measuring cell proliferation in response to an additional immune stimulus, e.g, antigen or phorbol myristate acetate (PMA) .
  • PMA phorbol myristate acetate
  • the number of CD25-positive cells in the population of immunoreactive T cells may be measured and the cells discarded if the number is less than 10% of the total number of T cells in the population.
  • the number of CD25 + cells is at least 20% of the total number of T cells in the population.
  • the level of activation of the immunoreactive cells may also be evaluated by measuring proliferation in response to further stimulation by immune stimulants.
  • the cells are discarded if the level of proliferation, e.g., the amount of 3 H-thymidine incorporated into cellular DNA, is less than twice the level of proliferation of a sample of unprocessed mononuclear cells.
  • the level of proliferation of the immunoreactive cells is at least twice that of unprocessed cells and preferably is 5-fold greater than that of unprocessed patient-derived cells.
  • the level of activation of the immunoreactive cells may be adjusted, e.g., to a higher level of activation, by altering the length of the patient-derive mononuclear cell-T3CS co-culture period to generate the immunoreactive cells, or alternatively, by adding fresh T3CS.
  • the mixture of nonspecific autologous lymphocyte activators and 0KT3, i.e., T3CS preferably contains: interleukin-1-alpha (IL-l ⁇ ) , interleukin-1-beta " (IL-10) , interleukin-6 (IL-6) , interleukin-8 (IL-8) , TNF ⁇ , tumor necrosis factor-beta (TNF0) , interferon-gamma (IFN ⁇ ) , granulocyte macrophage-colony stimulating factor (GM- CSF) , monocyte/macrophage colony stimulating factor (M- CSF) and OKT3.
  • IL-1-alpha IL-l ⁇
  • IL-10 interleukin-1-beta
  • IL-6 interleukin-6
  • IL-8 interleukin-8
  • TNF ⁇ tumor necrosis factor-beta
  • TNF0 tumor necrosis factor-beta
  • IFN ⁇ interferon-gamma
  • T3CS contains 12.7 ng/ml ( ⁇ 10-40%) OKT3 in addition to autologous cytokines in the following amounts ( ⁇ 10%-40%) : IL-l ⁇ (105 pg/ml), IL-1/3 (1433 pg/ml), IL-6 (808 pg/ml), IL-8 (213 ng/ml), TNF ⁇ (570 pg/ml), TNF3 (171 pg/ml), IFN ⁇ (14350 pg/ml), M-CSF (1193 pg/ml), and GM-CSF (840 pg/ml).
  • IL-2, interleukin-3, IL-4, interleukin-7, IL-10, IL-12, T cell growth factor-beta (TGF3) , and granulocyte- colony stimulating factor may each be present in T3CS at a concentration of less than 20 pg/ml.
  • these cytokines are present at a concentration of less than 5 pg/ml.
  • At least a 20% increase in the number of CD25 + T cells in the first sample of patient-derived mononuclear cells following a T3CS-generation culture compared to a sample of uncultured mononuclear cells is predictive of sufficient production of autologous cytokines.
  • the concentration of IL-2 must be less than 100 units/ml.
  • the concentration of IL-2 in T3CS is preferably less than 50 units/ml, and more preferably in the range of 10-20 units/ml, and most preferably in the range of 1-5 ng/ml (1 unit of IL-2 is approximately equal to 250 pg/ml) .
  • T3CS is preferably carried out at a temperature greater than 29°C but less than 37°C, e.g., 35°C, for a period of 2 days.
  • Co-culture of patient-derived mononuclear cells with T3CS to generate the immunoreactive cells may also be carried out at sub-physiologic temperature, e.g., a temperature greater than 29°C but less than 37°C.
  • the cells may be removed from the T3CS and contacted with IL- 2, preferably in an amount which is sufficient to bind to at least 25% of the IL-2 receptors on the surface of the immunoreactive cells; more preferably, the amount of IL-2 is sufficient to saturate the IL-2 receptors on the surface of the immunoreactive cells.
  • Contacting the immunoreactive cells with IL-2 is preferably done at 4°C, e.g., during storage or delivery of the cells prior to administration to the patient.
  • the invention also features a process of producing a population of antigen-specific polyclonal T cells by (a) contacting a first sample of mononuclear cells derived from a patient with OKT3 at or below 37°C to produce a T3CS; (b) removing the T3CS from the first sample; (c) determining the concentration of 0KT3 in the T3CS, and if required, supplementing the T3CS with additional OKT3 to achieve a concentration of at least 0.1 ng/ml; (d) providing a second sample of mononuclear cells derived from the same patient; (e) contacting the second sample with T3CS and an antigen for a period of time, e.g., 1-30 days, sufficient to activate the second sample in vitro to yield a population of antigen-specific polyclonal T cells.
  • the concentration of OKT3 in the T3CS is preferably 0.1-1 ng/ml.
  • T3CS may be supplemented with OKT3, or OKT3 may be removed from T3CS using methods known in the art, such as chromatography, antibody-mediated depletion, or filtration.
  • the antigen may be in the form of a natural or synthetic peptide, cell extract, a purified antigen, or a recombinantly expressed antigen and may be a tumor antigen, bacterial antigen, viral antigen, or autoantigen.
  • the invention provides an immunoreactive mononuclear cell produced by the inventive process.
  • the cell is preferably a T cell, more preferably a Thl-type T cell.
  • the T cell preferably expresses at least 10%, more preferably at least 75%, and most preferably at least 100% more cell-surface CD25 than an unprocessed mononuclear cell, e.g., a mononuclear cell in a resting state.
  • the cell of the invention is preferably in a primed state, i.e., the cell proliferates at a rate that is at least twice that of an unprocessed T cell when contacted with an immune stimulant.
  • the invention also includes a mixture of immunoreactive cells produced by the inventive process at least 75% of which are T lymphocytes, e.g., Thl-type T cells.
  • the cells of the invention can be used to treat any condition characterized by sub-optimal immune responsiveness.
  • Another aspect of the invention features a process for producing a mixture of autologous nonspecific lymphocyte activators by collecting mononuclear cells from the blood of a patient afflicted with cancer or an infectious disease, inactivating suppressor T cells in the sample of mononuclear cells, and contacting the mononuclear cells with a compound that binds to the T cell receptor or the T cell receptor-associated CD3 molecule at or below 37°C, e.g., 29-36°C, e.g., 35°C for 2 days.
  • the T cell receptor-binding compound may bind to the alpha chain or beta chain of a T cell receptor (or alternatively to the gamma or delta chain) .
  • the CD3-binding compound is preferably soluble OKT3, but may be any ligand that binds to the CD3 molecule on the surface of the cell.
  • the cells may be contacted with the CD3-binding compound in the absence or in the presence of an antigen; the CD3-binding compound may be removed from the cell culture supernatant following the production of the mixture of autologous cytokines.
  • the mixture contains autologous cytokines in the following amounts ( ⁇ 10%-40%) : IL-l ⁇ (105 pg/ml) , IL-13 (1433 pg/ml) , IL-6 (808 pg/ml) , IL-8 (213 ng/ml) , TNF ⁇ (570 pg/ml), TNF0 (171 pg/ml), IFN ⁇ (14350 pg/ml), M-CSF (1193 pg/ml) , and GM-CSF (840 pg/ml) , either in the presence or absence of 12.7 ng/ml ( ⁇ 10-40%) OKT3.
  • the invention also includes the immunoreactive cells of the invention together with a pharmaceutically acceptable carrier or diluent for patient administration.
  • the invention features a method of treating a tumor or a viral pathogen in a patient by administering to the patient the immunoreactive cells of the invention.
  • a suppressor cell inhibiting compound e.g., cimetidine, indomethacin, or both, may be concurrently administered to the patient.
  • the method may be used to treat any type of cancer including both solid tumors and hematologic tumors, e.g., renal cell carcinoma, breast carcinoma, prostate carcinoma, colo-rectal carcinoma, pancreatic carcinoma, ovarian carcinoma, melanoma and non-small cell carcinoma of the lung as well as leukemias and lymphomas.
  • hematologic tumors e.g., renal cell carcinoma, breast carcinoma, prostate carcinoma, colo-rectal carcinoma, pancreatic carcinoma, ovarian carcinoma, melanoma and non-small cell carcinoma of the lung as well as leukemias and lymphomas.
  • the methods and compositions of the invention represent a promising approach to tumors not treatable by conventional forms of therapy such as chemotherapy, radiation therapy, or surgery.
  • Mononuclear cells taken from a patient afflicted with a complex chronic viral disease may also be processed according to the invention to yield immunoreactive cells which can then be returned to the patient to augment the patient's immune response to the pathogen.
  • hepatitis B virus hepatitis C virus
  • recurrent herpesvirus herpes simplex virus, varicella zoster virus, cytomegalovirus
  • papilloma virus Epstein Barr viurs
  • HIV HIV-1 and HIV-2
  • pathogenic viruses e.g., hepatitis B virus, hepatitis C virus, recurrent herpesvirus (herpes simplex virus, varicella zoster virus, cytomegalovirus) , papilloma virus, Epstein Barr viurs and HIV (HIV-1 and HIV-2)
  • Fig. 1A is a flow cytometric histogram showing the level of cell surface CD25 on T lymphocytes cultured in culture media alone.
  • Fig. IB is a flow cytometric histogram showing the level of cell surface CD25 on T lymphocytes activated by OKT3 alone.
  • Fig. 1C is a flow cytometric histogram showing the level of cell surface CD25 on T lymphocytes activated with 25% (vol/vol) T3CS (patient #1) .
  • Fig. ID is a flow cytometric histogram showing the level of cell surface CD25 on T lymphocytes activated with 25% (vol/vol) T3CS (patient #2).
  • Fig. IE is a flow cytometric histogram showing the level of cell surface CD25 on T lymphocytes activated with 25% T3CS (patient #3).
  • Fig. 2A is a bar graph showing cell surface CD25 (IL-2 receptor) expression of peripheral blood mononuclear cells (PBMC) obtained from 7 metastatic renal cell carcinoma (mRCC) patients following a 5-day culture with either 25% T3CS, OKT3-depleted T3CS, or OKT3 alone.
  • PBMC peripheral blood mononuclear cells
  • mRCC metastatic renal cell carcinoma
  • Fig. 2B is a bar graph showing PMA-induced proliferation of PBMC following a 5-day culture with 25% T3CS, OKT3-depleted T3CS, or OKT3 alone.
  • Fig. 3A is a line graph showing the enhanced capacity of chimpanzee EVA cells (EVA #2) to proliferate upon stimulation with PMA compared to unprocessed chimpanzee-derived mononuclear cells (PBMCs) .
  • Fig. 3B is a line graph showing the enhanced capacity of chimpanzee EVA cells (EVA #3) to proliferate upon stimulation with PMA compared to unprocessed chimpanzee-derived mononuclear cells (PBMCs) .
  • Fig. 4 is a line graph showing the enhanced capacity of EVA cells to proliferate and produce cytokines (IFN ⁇ ) upon stimulation with PMA compared to unprocessed mononuclear cells (PBMC) .
  • IFN ⁇ cytokines
  • Fig. 5 is a line graph showing the enhanced capacity of EVA cells to proliferate upon stimulation with recombinant IL-2 compared to unprocessed mononuclear cells (PBMC) .
  • Fig. 6 is a line graph showing the enhancement of PBMC proliferation in response to a recall antigen (influenza) due to the addition of irradiated EVA cells as helper cells.
  • Fig. 7 is a bar graph showing the enhanced cytolytic function of EVA cells compared to unprocessed mononuclear cells (PBMC) . Processing of peripheral blood mononuclear cells
  • Mononuclear cells to be processed according to the invention can be obtained from patients, e.g., those afflicted with a malignant tumor or an infectious disease such as hepatitis B.
  • Peripheral blood or a mononuclear cell-enriched population of cells obtained using known methods, e.g., apheresis
  • an anticoagulant e.g., heparin, sodium citrate, ethylenediaminetetraacetic acid, sodium oxalate.
  • the blood-anticoagulant mixture then is diluted in a physiologically acceptable solution such as sodium chloride or phosphate buffered solution.
  • Mononuclear cells are recovered by layering the Talood- anticoagulant composition onto a centrifugation separation medium such as Ficoll-Hypaque (Pharmacia Corporation) or Lymphocyte Separation Medium (Litton Bionetics Corporation) .
  • a centrifugation separation medium such as Ficoll-Hypaque (Pharmacia Corporation) or Lymphocyte Separation Medium (Litton Bionetics Corporation) .
  • the layered mixture then is centrifuged, and the interface containing the mononuclear cells is collected and washed.
  • the suppressor cells in the mononuclear cell population may be functionally inactivated by contacting the mononuclear cells with an agent that has a specific affinity for or effect upon suppressor cells.
  • a particularly suitable composition for inactivating suppressor cells is an H2 receptor antagonist, such as cimetidine; a suitable composition for inactivating the suppressor activity of monocytes is indomethacin.
  • the mononuclear cells are suspended in a culture medium containing a mitogenic compound which binds to the T cell receptor or the T cell receptor-associated CD3 molecule, e.g., a CD3- binding compound, e.g., OKT3, to produce T3CS.
  • a mitogenic compound which binds to the T cell receptor or the T cell receptor-associated CD3 molecule e.g., a CD3- binding compound, e.g., OKT3, to produce T3CS.
  • Cimetidine may be used in the inventive process to inactivate suppressor cells.
  • the addition of cimetidine to the medium when PBMC are cultured in the T3CS resulted in increased activation of T cells as measured by enhanced proliferative responses of immunoreactive cells upon further stimulation with PMA
  • the concentration of mononuclear cells can be in the range of about 0.5 - 5.0 x IO 6 cells/ml, preferably 1-2 x IO 6 cells/ml.
  • the cells are preferably cultured under serum-free conditions at 37°C using a standard tissue culture medium, e.g., AIM V medium available from Gibco-BRL, Grand Island, NY.
  • Mononuclear cells are generally cultured with OKT3 for a period of 3 days to generate T3CS.
  • the T3 " CS may be used immediately or stored frozen and then thawed for use.
  • the concentration of cytokines, e.g., TNF ⁇ , or OKT3 concentration in the T3CS may be measured by any conventional means such as radioimmunoassay or enzyme- linked immunosorbent assay (ELISA) using antibodies specific for those components.
  • ELISA enzyme- linked immunosorbent assay
  • PBMC Peripheral blood mononuclear cells
  • AIM V medium Gibco-BRL, Grand Island, NY
  • 0KT3 Orthoclone OKT3; Ortho Pharmaceutical Corporation, Raritan, NJ
  • cimetidine To inhibit suppressor cell activity, 50 ⁇ M cimetidine (Tagamet®; Smith Kline Beecham Pharmaceutical, Cidra, PA) and 10 nM indomethacin (Indocin®; Merck Sharp & Dohme, West Point, PA) were also added to the culture medium. At the end of the culture, the culture bags were centrifuged at 1100 x g for 20 min at room temperature. and the supernatants were collected, aliquoted, and stored at -70°C.
  • composition of the T3CS was determined by ELISA analysis using Quantikine kits from R&D Systems (Minneapolis, MN) for IL-l ⁇ , IL-2, 3, 4, 6, 7, 8, TNF ⁇ , TNF3, GM-CSF, TGF3 and G-CSF, IFN ⁇ kits from Endogen (Boston, MA) and Gibco (Grand Island, NY) , IL-10 kits from Biosource International (Camarillo, CA) , and IL-1,9 kits from Cistron Biotechnology (Pine Brook, NJ) .
  • IL-12 was determined by a bioassay (phytohemagglutinin (PHA) blast proliferation) .
  • the amount of OKT3 present in T3CS was determined by ELISA using the following reagents purchased from Vector Laboratories, Inc. (Burlingame, CA) : Horse anti-mouse IgG to capture the OKT3 mouse mAb, biotinylated horse anti-mouse IgG to detect the captured mAb and ABC reagent consisting of avidin-conjugated horseradish peroxidase to amplify the signal.
  • Ortho-phenylenediamine dihydrochloride (Sigma Chemical Co., St. Louis, MO) was used as a substrate.
  • EVA cells cultures were carried out under the same conditions as used in processing patients-derived cells except on a smaller scale (5 ml) .
  • Excess PBMC obtained from mRCC patients were isolated by ficoll density gradient and were cultured at 2 x 10 6 /ml for 5 days in complete AIM V medium (containing 50 M cimetidine and 10 nM indomethacin) with either 25% (vol/vol) T3CS, or various concentrations of OKT3.
  • Cells cultured with medium alone served as control. After incubation, the cells were washed and resuspended at a concentration of 10 x 10 6 /ml in "infusion medium" (1% human serum albumin and 0.5% dextrose in Lactated Ringers solution) .
  • the cells were then stored overnight at 4°C prior to analysis (to simulate overnight storage for final QA/QC and shipping to clinical sites) .
  • Cells were resuspended at 2 x 10 6 /ml in AIM V medium and aliquoted to 100 ⁇ l per tube. Two to four ⁇ ls of labeled mAbs were added to the cells according to the manufacturers' recommendation. The cells were then incubated with the Abs at 4°C for 30 min, washed once with 500 ⁇ l of cold PBS, and resuspended at a concentration of 4 x 10 s cells/ml in PBS for immediate analysis using a Coulter Epics Profile II flow cytometer.
  • PBMC or EVA cells were resuspended to 1 x IO 6 cells/ml in AIM V medium with or without l ng/ml PMA, and cultured in triplicate in 96-well flat-bottom plates (Costar,
  • T3CS Depletion of 0KT3 or Cytokines From T3CS
  • T3CS was divided into 6 ml aliquots, and incubated with the appropriate neutralizing antibody (Goat anti-human, from R&D Systems) at 37°C for one hour.
  • the amount of antibody used for depletion was determined by the level of the particular cytokine known to be present and the antibody activity required for neutralization as specified by the manufacturer. Specifically, 125 ⁇ l of antibody was added for the depletion of IL-1,9 or TNF ⁇ , 20 ⁇ l for IL-6, 60 ⁇ l for IFN, 100 ⁇ l for GM-CSF, and 250 ⁇ l for IL-8.
  • the cytokine profile indicated that the cells involved in the PBMC activation process were predominantly monocytes and Thl-type T cells (Seder et al., 1994, Ann. Rev. Immunol. 12:635-673) or T cells that had differentiated into Thl-type cytokine producers during the cultures.
  • kinetic studies indicated that significant amounts of IL-2 (15-433 pg/ml) were present in all samples of T3CS analyzed during the first 24 hours of the culture, the level of IL-2 dropped sharply by day 2 (0-132 pg/ml) . IL-2 levels became undetectable by day 3 when the culture supernatant was harvested. The drop in IL-2 is likely to be due to active consumption of the cytokine during cell culture.
  • T3CS contains significantly lower levels of IL-2 than conventional conditioned media, e.g., PHA-generated cell supernatants, Concanavalin-A-generated cell supernatants, or mixed lymphocyte culture supernatants.
  • Cytokine levels in T3CS from the majority patients fell into acceptable ranges, i.e., sufficient amounts of the key ' cytokines were present to produce immunoreactive cells in subsequent PBMC cultures.
  • T3CS from a small number of patients contained low levels of certain cytokines.
  • the concentration of cytokines may be augmented, if necessary, by carrying out the T3CS generating step for a longer period of time or alternatively, by adding recombinant or nonrecombinant cytokines.
  • the concentration of patient-derived monocytes in a sample of patient-derived mononuclear cells is preferably 0.1-50%, more preferably 1-30%, more preferably 5-15%, and most preferably 10%.
  • monocytes were depleted from patient-derived mononuclear cells using the well-known methods of adherence, e.g., to plastic plates, or incubation with L-leucine methyl ester. Incubation of a monocyte-depleted sample of patient-derived mononuclear cells with T3CS failed to yield the immunoreactive cells of the invention.
  • Fc-receptor positive accessory cells i.e., monocytes, granulocytes or dendritic cells, are required to generate immunoreactive cells using the methods of the invention when OKT3 is used in solution phase.
  • the sample of patient-derived mononuclear cells may be enriched for monocytes, granulocytes, or dendritic cells.
  • the OKT3 used in the process of the invention is preferably in solution phase rather than solid phase.
  • One advantage of using soluble OKT3 in the inventive process is that soluble OKT3 mediates a more physiological interaction between Fc-receptor-bearing accessory cells, e.g., monocytes, and T cells. By forming a bridge between these cells, a full complement of costimulatory signals is initiated, thus minimizing the potential for the generation of incompletely (anergic) or aberrantly (apoptotic) activated T cells.
  • cytokines in the induction of T cell activation was compared to that of OKT3 alone.
  • PBMC from seven mRCC patients were cultured for 5 days in AIM-V medium containing either 2.5 ng/ml OKT3 alone or 25% (vol/vol) T3CS that was first depleted of 0KT3 and then reconstituted with 2.5 ng/ml of fresh antibody. This depletion/reconstitution approach was undertaken to assure that the amount of biologically active OKT3 in the T3CS would be identical to the OKT3 control.
  • a pool made from three different mRCC patients was used to stimulate the PBMC from all seven patients.
  • Figs. 1A-1E are representative immunofluorescence histograms that demonstrate that CD25 expression on T cells stimulated with the autologous cytokine-OKT3 mixture was significantly higher than that on T cells stimulated with OKT3 alone.
  • the degree of T cell activation was also assessed functionally by measurement of the proliferative response of EVA cells upon further stimulation by PMA, a protein kinase C activator.
  • Fig. 4 shows the results of an experiment in which immunoreactive cells were contacted with an immune stimulant, e.g., various concentrations of PMA.
  • an immune stimulant e.g., various concentrations of PMA.
  • immunoreactive cells displayed very little spontaneous proliferation or cytokine secretion when cultured at 37°C in AIM V medium alone.
  • immunoreactive cells (but not unprocessed PBMC) displayed an enhanced capacity to proliferate and produce IFN ⁇ (as well as other Thl-type cytokines such as TNF ⁇ , TNF3, and GM-CSF (data not shown) ) .
  • cytotoxicity was determined using a conventional chromium-51 release assay with the K562 leukemia cell line and allogeneic human renal carcinoma cell line 769P as targets.
  • EVA cells i.e., T cells which have been polyclonally-activated independent of tumor-associated antigens according to the invention, possessed a greatly enhanced cytotoxicity toward both tumor lines compared to unprocessed PBMC.
  • helper T Cells In addition to enhanced cytolytic function, the immunoreactive cells of the invention were found to have enhanced helper function.
  • the ability of T3CS to support the generation of EVA cells with helper cell function was determined by measurement of the ability of irradiated EVA cells to enhance the proliferative response of unprocessed mononuclear cells upon stimulation by a recall antigen, e.g., an influenza antigen.
  • a recall antigen e.g., an influenza antigen.
  • EVA cells added to cultures at low levels (5-10%) provided helper signals to unprocessed patient-derived mononuclear cells (presumably through the secretion of Thl-type cytokines) resulting in a significant increase in proliferation.
  • EVA cells proliferate and to produce a variety of cytokines (IL-2, GM-CSF, IFN ⁇ , TNF ⁇ ) in vitro in response to further stimulation by such agents as PMA and IL-2, as well as to lyse tumor cell targets, is greatly enhanced compared to the PBMC from which they were derived.
  • the lowered activation threshold of • the EVA cells exhibited in vitro suggests that once they are reinfused into patients, they are likely to demonstrate enhanced responsiveness to immunological signals, such as weakly immunogenic tumor antigens which normally are non-stimulatory to unprocessed cells.
  • EVA cells Following short term (5-day) culture of patient- derived mononuclear cells in the autologous cytokine mixture/OKT3-containing conditioned medium, the resulting EVA cells were analyzed for the expression of cell surface antigens. EVA cells expressed enhanced levels of a variety of activation and/or differentiation markers on their cell surface including cytokine receptors, e.g., CD25, major histocompatibility complex (MHC) antigens, e.g., MHC class II, adhesion molecules/homing receptors, e.g., CD44/Leu8, costimulatory molecules, e.g., CD28, and markers of primed or memory T cells, e.g. , CD45RO, as shown in Table 5.
  • cytokine receptors e.g., CD25
  • MHC major histocompatibility complex
  • adhesion molecules/homing receptors e.g., CD44/Leu8
  • costimulatory molecules e.g., CD28
  • CD3(+)/CD45RO(+) - T CELLS (PRIMED 49% 69% "MEMORY” CELLS)
  • CD3(+)/CD44(+) - T CELLS (EARLY 6.0* 8.3* ACTIVATION MARKER - CELL ADHESION MOLECULE)
  • N 19 mRCC Patient PBMC & EVA Cell Products
  • OKT3 was depleted from the T3CS in order to determine the relative effects of the anti-CD3 monoclonal antibody and the autologous cytokines on the induction of CD25 expression on the surface of T lymphocytes (Fig. 2A) and the proliferation of EVA cells upon further stimulation by PMA (Fig. 2B) . Following depletion of OKT3 from the T3CS, there was little or no measurable activation of T cells, i.e., the autologous cytokines were not capable of stimulating resting PBMC in the absence of OKT3.
  • cytokines in the T3CS have the greatest effects on T cell activation.
  • the roles of 5 major cytokines (IL-13, IL-6, TNF ⁇ , IFN- ⁇ , GM-CSF) were analyzed utilizing various combinations of recombinant cytokines together with OKT3.
  • Table 2 shows the results of a series of experiments in which PBMC from 7 mRCC cancer patients were cultured in an allogeneic T3CS, OKT3 alone, or various 0KT3-multiple recombinant cytokine mixtures.
  • T3CS complete conditioned medium
  • PBMC from seven individual mRCC patients were cultured for 5 days with OKT3 alone, or OKT3 plus various combinations of cytokines, or with 25% of an allogeneic conditioned medium.
  • the cells were stored at 4°C overnight and then analyzed for CD25 expression by flow cylom ⁇ try.
  • IL-1b 250 pg/ml
  • IL-6 100 pg/ml
  • TNFa 100pg/ml
  • IFNg 250pg/ml
  • GM-CSF 250pg/ml
  • "25% conditioned medium (WP35) contains- OKT3 2.4 ng/ml.
  • TNFa Is an Important Co-stimulant for T Cell Activation
  • Table 3 shows the results from a series of experiments in which the contributions of various cytokines present in the T3CS to the overall T cell activation process was analyzed.
  • the level of induction of CD25 on PBMC cultured in T3CS was compared to that achieved when cytokines were selectively depleted from aliquots of the same T3CS preparation.
  • TNF ⁇ serves a key costimulatory function in enhancing 0KT3-catalyzed polyclonal T cell activation.
  • PBMC from seven different mRCC patients were stimulated with 25% allogeneic conditioned media either complete or depleted with one of the cytokines listed.
  • the cells were harvested 5 days later and examined for surface IL-2 receptor (CD25) expression by flow cytometry the next day.
  • CD25 surface IL-2 receptor
  • the effect of cytokine depletion Is expressed as % of control which Is the total IL-2R expression (% CD25 positive cells x mean channel fluorescence Intensity) on cells activated by the complete, undepleted conditioned media.
  • T3CS was utilized as a nonspecific stimulant in the ex vivo generation of polyclonally activated T cells for the treatment of metastatic renal cell carcinoma.
  • T3CS produced according to the invention contains a wide variety of monokines and lymphokines together with low levels of OKT3.
  • OKT3 functions synergistically with the cytokines in the T3CS. Removal of OKT3 from the T3CS resulted in a substantial decrease of T cell activation demonstrating the role of anti-CD3 monoclonal antibody as a catalyst while the cytokines provide the costimulatory T cell activation signals.
  • Serum-free culture is particularly desirable for the generation of EVA cells used in adoptive immunotherapy.
  • the claimed process employs aliquots of the T3CS • ⁇ generated in advance to be used as a stimulant in the secondary cultures.
  • This approach ensures that a full complement of costimulatory signals are available at the initiation of each activation culture and therefore minimizes the probability of generating anergic or apoptotic T cells.
  • use of the pre-manufactured and quality-assured autologous cytokine-OKT3 mixture as a stimulant decreases the dependence upon de novo synthesis of cytokines during the early stage of the subsequent T cell activation cultures. Such decreased dependency is especially important under the conditions such as off-site cell processing that require shipping and storage of the cells, and when dealing the with cells from patients of various clinical stages.
  • 0KT3-induced T cell activation for one patient's cells may strongly be affected by a given cytokine while cells from another patient are not affected at all by the same cytokine. This variation is due both to the inherent diversity of human lymphocytes and accessory cells, and to the fluctuation caused by the process prior to the activation such as collection of the cells by apheresis. Overnight storage and shipping may affect accessory cell activity which in turn affects the dependence of the T cells on exogenous cytokine present in the culture.
  • the methods of the invention require evaluation of various key components of the T3CS, e.g., OKT3 and TNF ⁇ concentration, and the concentration of accessory cells, e.g., monocytes.
  • the methods may also require the evaluation of the activation state, i.e., PMA responsiveness, of the processed cells to predict their clinical effectiveness.
  • T3CS IL-2-Independent Generation of Immunoreactive Cells
  • T3CS as a stimulant for mononuclear cells, it is possible to generate T cells expressing high levels of CD25/IL-2 receptors, independent of the inclusion of high levels of exogenous IL-2 in the culture medium.
  • low levels of IL-2 are present in both the T3CS and EVA cultures at early timepoints, there is no detectable ( ⁇ 6 pg/ml) autologous IL-2 present in the T3CS when the EVA culture is initiated. Consequently, in contrast to LAK and TIL cells which are both cultured in high levels of IL-2, the immunoreactive cells generated by the inventive process are presumably less dependent upon systemic administration of high dose IL-2 for therapeutic efficacy.
  • the lack of IL-2 in the T3CS allows the production of immunoreactive cells, i.e., multifunctional, polyclonal T cells containing both CD8 + /cytotoxic T cells and a high percentage of CD4 + /helper cells, in contrast to PBMC grown in high dose IL-2 which are highly enriched in CD8 + /cytotoxic T cells.
  • immunoreactive cells of the invention have broader functional capacities than PBMC cultured in IL-2.
  • T3CS containing low levels, e.g., up to 10 units/ml, of autologous IL-2 is desired to further enhance the generation of immunoreactive cells
  • IL-2 production may be increased and IL-2 consumption simultaneously decreased by modification of the culture conditions, such as the kinetics and temperature of the T3CS generation process.
  • the immunoreactive cells may be exposed to IL-2 at 4°C such that IL-2 binds to at least 25% of the cell surface IL-2 receptors.
  • the cell surface IL-2 receptors are saturated, i.e., 100% of the IL-2 receptors on the surface of a cell are bound to IL-2, with IL-2 prior to infusion into a patient.
  • Immunoreactive cells with cell surface bound IL-2 are likely to have an enhanced ability to expand in vivo and a decreased dependence upon helper cell-mediated IL-2 production, an activity which may be lacking or depressed in immunosuppressed cancer patients.
  • administration of IL-2 to a patient in a cell-bound form avoids the toxicity and other clinical complications often associated with intravenous or subcutaneous co-administration of high dose IL-2 to support cell therapy.
  • OKT3 Intravenous OKT3 administration, utilized for the suppression of transplant graft rejection in humans, induces rapid (within 24 hours) removal of a high percentage of CD3 + T cells from the circulation due to the presence of the foreign antibody molecules on the lymphocyte cell surface (Vigeral et al., 1986, Transplantation 41:730; Chatenoud et al., 1986, J. Immunol. 137:830-838). Excessive amounts of OKT3 bound to an EVA cell surface may accelerate the clearance of the T cells, resulting in decreased therapeutic efficacy.
  • T3CS as a stimulant minimizes this clinical problem because T cell activation can be achieved with a minimal concentration of OKT3 in the presence of autologous cytokines compared to the concentration required in the absence of autologous cytokines.
  • T3CS T3CS to stimulate PBMC allows a high level of T cell activation to be catalyzed by a minimal amount of 0KT3 (1-4 ng/ml) .
  • the inventive methods leave very little detectable surface-bound antibody on the activated T cell product, and therefore decrease the probability of the EVA cells being rapidly removed from the circulation.
  • the low level of surface-bound 0KT3 allows more CD3-TCR complexes on the surface of the activated T cells to remain unoccupied so that key molecules such as MHC Class I and II molecules, tumor antigens, and immunogenic peptides may bind to and stimulate the T-cell receptor following re-infusion of the cells into patients.
  • T3CS Conditions under which patient-derived mononuclear cells are cultured with OKT3 to generate T3CS were analyzed.
  • T3CS cells were cultured for a period of 1 to 5 days.
  • table 4 it is possible to manipulate the time course and/or temperature at which T3CS is generated in order to selectively increase or decrease the levels of certain autologous cytokines.
  • Human peripheral blood cells are collected by apheresis, and the PBMC are isolated using a Ficoll gradient.
  • the PBMC are incubated at 37°C at a concentration of IO 6 cells/ml in AIM V medium containing cimetidine (5 x IO "5 mol/1) , indomethacin (IO' 8 mol/1) , and 25 ng/ml OKT3 (Ortho Biotech, Raritan, NJ) .
  • the activity of suppressor T cells and of suppressor monocytes is inactivated by culturing the PBMC with cimetidine and indomethacin.
  • the culture supernatant i.e., T3CS
  • T3CS the culture supernatant
  • a second sample of PBMC is collected from a patient, and suspended at approximately 2 x IO 6 cells/ml in AIM V medium containing 25% v/v T3CS, cimetidine (5 x IO "5 mol/1) and indomethacin (10 "8 mol/1) .
  • the cells are harvested following a five day culture at 37°C in a- moist-air incubator containing 5% C0 2 , then resuspended at IO 7 cells/ml.
  • the levels of one or more of the cytokines may be increased as follows.
  • One way to increase the amount of a cytokine, e.g., TNF ⁇ , is to adjust, e.g., increase, the time of culture before harvesting the T3CS.
  • the concentration of monocytes (which produce TNF ⁇ ) may be increased in the T3CS-generating culture, or the temperature at which the T3CS-generating step is carried out may be adjusted to optimize TNF ⁇ production.
  • Another way to increase the amount of a cytokine is to increase the percentage of T3CS added to the second sample of patient-derived mononuclear cells, i.e., by adding greater than 25% T3CS.
  • the therapeutic methods of the invention which utilize EVA cells may be used to treat mRCC and other types of metastatic cancers, as well as infectious diseases, autoimmune diseases, and immunodeficiency diseases.
  • Clinical outcome may be assessed by such measures as length of patient survival, quality of life measurements, changes in any indicators of medical function such as clinical chemistries, size of tumors, changes in load of virus, bacteria, fungus, or parasite, toxicity of the therapy, or delay in time of recurrence of the disease, or other assessments.
  • Clinical outcome may also be evaluated by monitoring changes in immune status or time to recurrence of a tumor in a cancer patient.
  • Viral hepatitis refers to an infection of the liver caused by a small group of hepatotropic viruses that may be differentiated serologically. Of the three serotypes, A, B and C; B and C cause far more serious infections than hepatitis A.
  • the methods of the invention were used to produce immunoreactive cells from mononuclear cells derived from chimpanzees infected with hepatitis B, a primate model of chronic viral infection.
  • the chimpanzee model was chosen over the woodchuck or mouse model as the most relevant model of the human disease because chimpanzees can be experimentally infected with the human hepatitis B virus.
  • Mononuclear cells from chimpanzees with chronic hepatitis B were polyclonally activated with 0KT3 to produce a T3CS.
  • a second sample of mononuclear cells was cultured with T3CS to obtain EVA cells.
  • Chimpanzee- derived mononuclear cells processed according to the invention yielded immunoreactive cells.
  • the immunoreactive cells were in a primed state of activation, i.e., they proliferated upon stimulation with PMA whereas unprocessed chimpanzee- derived mononuclear cells did not.
  • the spectrum of phenotypic markers on the chimpanzee EVA cells was found to be similar to that on EVA cells produced from patient-derived mononuclear cells (data not shown) .
  • These data indicate that the methods of the invention can be used to generate immunoreactive cells from animals, e.g., patients, with chronic viral infections. These immunoreactive cells can then be re- infused into the patient to augment the patient's immune response to the pathogenic virus.
  • T3CS may also be used as a formulation for the in vivo delivery of cytokines.
  • T3CS which has been depleted of OKT3 may be encapsulated in an appropriate slow release matrix, e.g., a liposome or biocompatible polymer, in order to maintain a critical level of cytokine over time and to minimize the toxicity associated with bolus-delivery of cytokines.
  • In vivo delivery of T3CS may also be targeted to a particular site in the body for optimal effectiveness.
  • T3CS In vivo administration of T3CS offers several advantages over conventional cytokine therapy, i.e., administration of one cytokine.
  • T3CS is a unique mixture of both monokines and lymphokines which initiate and promote activation and/or recruitment of several different immune cell types. Thus, T3CS can induce a full spectrum of immune responses in a patient.
  • Other embodiments are within the following claims.

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Abstract

l'invention se rapporte à un procédé d'activation de cellules mononucléaires prélevées sur un patient et consistant à exposer les cellules in vitro à des substances afin de générer des cellules immnoréactives. Les cellules activées ex vivo sont ensuite réintroduites dans le corps du patient afin de renforcer son système immun, et de traiter diverses formes du cancer, des maladies infectieuses, des maladies autoimmunes ou des maladies immunodéficitaires.
EP95906781A 1994-01-27 1995-01-06 Activation ex vivo des cellules immunes Withdrawn EP0804550A4 (fr)

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