WO2005003335A2 - Lymphocytes t resistant a la rapamycine et leurs utilisations therapeutiques - Google Patents

Lymphocytes t resistant a la rapamycine et leurs utilisations therapeutiques Download PDF

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WO2005003335A2
WO2005003335A2 PCT/US2004/018609 US2004018609W WO2005003335A2 WO 2005003335 A2 WO2005003335 A2 WO 2005003335A2 US 2004018609 W US2004018609 W US 2004018609W WO 2005003335 A2 WO2005003335 A2 WO 2005003335A2
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cells
rapamycin
cell
resistant
subset
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PCT/US2004/018609
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WO2005003335A3 (fr
WO2005003335A9 (fr
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Daniel H. Fowler
Unsu Jung
Ronald E. Gress
Bruce Levine
Carl June
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Government Of The United States Of America, As Represented By The Secretary, Department Of Health And Human Services
The Trustees Of The University Of Pennsylvania
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Priority to CA2529244A priority Critical patent/CA2529244C/fr
Publication of WO2005003335A2 publication Critical patent/WO2005003335A2/fr
Publication of WO2005003335A3 publication Critical patent/WO2005003335A3/fr
Publication of WO2005003335A9 publication Critical patent/WO2005003335A9/fr
Priority to US11/298,313 priority patent/US7718196B2/en
Priority to US12/750,374 priority patent/US8075921B2/en

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    • 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
    • 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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K2035/122—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells for inducing tolerance or supression of immune responses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K2035/124—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells the cells being hematopoietic, bone marrow derived or blood cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/26—Universal/off- the- shelf cellular immunotherapy; Allogenic cells or means to avoid rejection
    • 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
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/04—Immunosuppressors, e.g. cyclosporin, tacrolimus
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/20—Cytokines; Chemokines
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/50—Cell markers; Cell surface determinants
    • C12N2501/51—B7 molecules, e.g. CD80, CD86, CD28 (ligand), CD152 (ligand)
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    • 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

Definitions

  • T cell-based therapies for treatment of medical conditions such as cancer, disease due to infectious disease organisms such as viruses, autoimmune diseases and Graft Nersus Host Disease (“GNHD”) are provided.
  • enriched populations selected for Till, Th2, Tel or Tc2 functions are selected for and controlled when administered to a patient in vivo.
  • HSCT HSCT
  • new immunosuppressive agents and improvements in histocompatibility matching, organ procurement, and surgical techniques are gradually improving the outcome of clinical transplantation (Hariharan et al, 2000. N. Engl J. Med. 342:605-12).
  • chronic allograft rejection remains the prime determinant of long-term graft survival (Paul. L. C, 1999, Kidney International 56:783-793).
  • stem cell graft rejection typically limits the application of allogeneic HSCT to those patients having an HLA-matched sibling donor, which represents a minority of all patients that might benefit from allogeneic HSCT therapy.
  • Tissue transplantation between genetically non-identical individuals results in immunological rejection of the tissue through T cell-dependpnt mechanisms.
  • immunosuppressive agents such as calcineurin phosphatase inhibitors and glucocorticosteroids which directly or indirectly interfere with IL-2 signaling are administered to transplant recipients (see, e.g., Morris, P. J., 1991, Curr. Opin. Immunol. 3:748-751; Sigal et al., 1992, Ann. Rev. Immunol 10:519-560; and L'Azou et al, 1999, Arch. Toxicol 73:337-345).
  • the most commonly used immunosuppressive agents today are the calcineurin inhibitors cyclosporin A and FK506, which act relatively indiscriminately by impairing T cell receptor (“TCR”) signal transduction.
  • a third primary immune suppression drug, rapamycin which has recently received FDA approval for prevention of organ transplant rejection, acts through a distinct mechanism of inhibition of the protein mammalian target of rapamycin (mTOR).
  • mTOR protein mammalian target of rapamycin
  • the biological effect of these three immunosuppressive agents is short-lasting, and as such, transplant recipients normally require life-long treatment of immunosuppressive agents to prevent transplant rejection.
  • these immunosuppressive agents have many serious adverse effects.
  • the a ⁇ -n inistration of cyclosporin A or FK506 to a transplant recipient results in degenerative changes in renal tubules.
  • Transplant recipients receiving long-term immunosuppressive treatment have a high risk of developing infections and tumors. For example, patients receiving immunotherapy are at higher risk of developing lymphomas, skin tumors and brain tumors (see, e.g., Fellstrom et aL, 1993, Immunol. Rev. 134:83-98).
  • immune T cells In addition to graft rejection, immune T cells also mediate the primary cause of lethality after allogeneic HSCT, graft- versus-host disease (GVHD).
  • GVHD which is primarily initiated by donor CD4 + T cells expressing a Thl cytokine phenotype characterized by IL-2 and IFN- ⁇ secretion, manifests clinically as damage to the skin, intestine, liver, and immune system.
  • immune suppression therapy involving either cyclosporin A or FK506 is typically administered, often in combination with other immune suppression agents such as methotrexate.
  • T cell activation requires both TCR-mediated signal transduction and simultaneously delivered costimulatory signals. These costimulatory signals are contributed, in part, by the activation of the costimulatory molecule CD28, which is expressed on resting T cells, by CD80 (B7-1) or CD86 (B7-2) expressed on antigen presenting cells ("APCs").
  • CD40L CD40 ligand
  • CTLA-4 is normally expressed as a membrane-bound receptor on T cells and, similar to CD28, binds to B7-1 and B7-2 molecules on APCs; however, signaling of T cells via CTLA-4 downregulates T cells.
  • the administration of soluble CTLA-4Ig is believed to prevent allograft rejection by competing with CD28 for B7-1 and B7-2.
  • Soluble CTLA-4Ig has been administered to transplant recipients to disrupt the CD28/B7 interaction so that T cell costimulation is blocked and allograft rejection does not occur (Zheng et al., 1999, J. Immunol. 162:4983-4990; Lenschow et al., 1996, Ann. Rev. Immunol. 14:233-258).
  • CTLA-4Ig has variable efficacy, and typically does not prevent development of chronic rejection.
  • Anti-CD40L (anti-CD 154) monoclonal antibodies have also been administered to transplant recipients to prevent allogaft rejection. These antibodies function by blocking the interaction of CD40 on antigen presenting cells (APC) and CD40L on activated T cells. It has recently been shown that graft survival achieved through the use of anti-CD40L monoclonal antibodies results in a significant inhibition of Thl type cytokines (i.e., IL-2, IL-12, " TNF- ⁇ , and IFN- ⁇ ), and an increase in the levels of the Th2 type cytokines (i.e., IL-4, and IL-10) in the graft sections (Hancock et al., 1996, Proc. Natl. Acad. Sci.
  • Thl type cytokines i.e., IL-2, IL-12, " TNF- ⁇ , and IFN- ⁇
  • Th2 type cytokines i.e., IL-4, and IL-10
  • Immunodeficient patients such as AIDS and SCID patients, are also at high risk from these opportunistic pathogens.
  • patients undergoing bone marrow transplantation (BMT) are severely immunocompromised until their immune systems reconstitute.
  • BMT bone marrow transplantation
  • these patients are susceptible to serious, and sometimes fatal, virus infections caused by normally benign viruses such as adenovirus, cytomegalovirus (CMV), and Epstein-Barr virus (EBV).
  • normally benign viruses such as adenovirus, cytomegalovirus (CMV), and Epstein-Barr virus (EBV).
  • CMV cytomegalovirus
  • EBV Epstein-Barr virus
  • CTLs cytotoxic T lymphocytes
  • antigens for presentation by class I MHC involves a complex cellular process (Berzofsky and Berkower, Fundamental -Immunology, Third Edition, Paul (ed.), Raven Press, Ltd.: New York, pp. 258-259 (1993).
  • antigen presented by class I MHC generally must be synthesized endogenously and processed by a nonendosomal pathway into peptides.
  • exogenous antigens can enter the cytoplasm for processing by the nonendosomal pathway and presentation by class I MHC.
  • immune T cells of autologous or allogeneic source may play a beneficial role in mediating anti-tumor effects and anti- infectious disease effects, including against viral, bacterial, and fungal processes.
  • This T cell biology offers the possibility that adoptive transfer of e vivo generated T cell populations might be utilized in the therapy of cancer or infection.
  • full realization of this possibility is limited by a general inability to amplify a potent autologous immune response against cancer or infectious disease antigens in vivo.
  • immune T cell therapy in the allogeneic setting is limited by allogeneic T cell attack against normal host tissues, which is manifested as GVHD.
  • the allogeneic anti-tumor immune therapy setting the graft-versus-leukemia (GVL) or graft-versus- tumor (GVT) effect is reduced by the -.mmune suppression drugs cyclosporine A, FK-506, corticosteroids, and methotrexate that are utilized to prevent or treat GVHD. Avoidance of standard GVHD prevention or treatment agents through rapamycin administration post-transplant will predictably facilitate improved GVL and GVT effects, resulting in improved rates of cancer cure.
  • Thl/Tcl and Th2/Tc2 functions we have now found methods and systems for generating highly enriched Thl/Tcl and Th2/Tc2 functions in a subject. These methods and systems allow for the preferential selection of either Thl/Tcl or Th2/Th2 functions, administration of the selected functions to a patient and subsequent control of these functions once administered.
  • T cells generated in rapamycin also express molecules that improve immune T cell function such as CD28 and CD62L.
  • the invention provides a method for selecting and expanding enriched T cell subsets, comprising co-stimulating isolated T lymphocytes in vitro by adding cytokines for selecting a T cell subset followed by expansion of the T cell subset in the presence of rapamycin or a rapamycin derivative compound:
  • the subset of T cells is selected by culturing T cell subsets with cytokines.
  • a Thl/Tcl subset of T lymphocytes is selected by culturing the lymphocytes in the presence of IL-12, and a Th2/Tc2 subset of T lymphocytes can be preferably generated by addition of IL-4.
  • the T lymphocytes are co-stimulated.
  • the co-stimulation of T lymphocytes suitably comprises initiating one or more intracellular signaling events.
  • the intracellular signaling events can be initiated by culturing the T lymphocytes with one or more antibodies, polypeptides, polynucleotides, small molecules, or combinations thereof
  • the intracellular signaling events are initiated by solid phase anti-CD3 and anti-CD28 antibodies binding to their respective ligands.
  • a subset of T lymphocytes is selected based on the disease to be treated.
  • the T lymphocytes are cultured with cytokines and rapamycin to select for either a Thl/Tcl or Th2/Tc2 subset.
  • the desired subset is expanded and re-infused into a patient suffering from or susceptible to a disease.
  • the T lymphocytes are autologous lymphocytes from a patient, and/or they can be derived from an allogeneic donor, which may represent an HLA-matched sibling donor, an HLA-matched donor from a non-family member, or a partially matched family member, such as a haplo-identical donor (parent or child).
  • an allogeneic donor which may represent an HLA-matched sibling donor, an HLA-matched donor from a non-family member, or a partially matched family member, such as a haplo-identical donor (parent or child).
  • a patient to be treated is suffering from, or is susceptible to, cancer or infectious disease organisms, such as a virus.
  • the preferred rapamycin resistant subset of lymphocytes that are infused into the patient are the Thl/Tcl subset.
  • the patient to be treated is suffering from, or is susceptible to graft-versus-host-disease (GVHD).
  • GVHD graft-versus-host-disease
  • the donor T cells of preference would be rapamycin resistant T cells of Th2/Tc2 phenotype, which are typically associated with reduced GVHD.
  • T cells from the donor would be harvested prior to transplantation, in vitro expanded in rapamycin or a rapamycin derivative compound to generate a Th2/Tc2 phenotype, and subsequently administered in the setting of the allogeneic HSCT to allow for a beneficial allogeneic T cell effect, such as the mediation of GVL or GVT effects, or the prevention of stem cell graft rejection, with reduced GVHD.
  • a beneficial allogeneic T cell effect such as the mediation of GVL or GVT effects, or the prevention of stem cell graft rejection, with reduced GVHD.
  • the selected T cell subsets are preferably cultured in at least about 0.01 ⁇ M rapamycin or a rapamycin derivative compound, more preferably the T cell subsets are cultured in at least about 0.1 ⁇ M rapamycin or a rapamycin derivative compound, most preferably the T cell subsets are cultured in at least about or up to 1.0 ⁇ M, 2.0 ⁇ M, 4.0 ⁇ M, 6.0 ⁇ M, or 10.0 ⁇ M rapamycin or a rapamycin -derivative compound. It is preferred that the rapamycin resistant T cell subset populations express surface markers such as CD28, and preferably CD62L.
  • methods for preventing and/or treating GVHD in a mammal comprise, harvesting allogeneic cells from the transplant donor; selecting for a subset of rapamycin resistant CD4 + T cells and CD8 + T cells in vitro; and, administering to the mammal rapamycin resistant T cells concomitantly with rapamycin.
  • the subset of rapamycin resistant T cells that are administered to a mammal is a Th2/Tc2 subset.
  • the rapamycin resistant Th2 cell subset express CD4 and the Tc2 cell subset express CD8.
  • the rapamycin resistant Th2/Tc2 cells express CD62L and secrete cytokines, preferably type II cytokines.
  • references to rapamycin resistant T cells in inclusive of T cells that are resistant to rapamycin or a rapamycin derivative compound are resistant to rapamycin or a rapamycin derivative compound.
  • T cells that are resistant to rapamycin or a rapamycin derivative compound also will be resistant to rapamycin.
  • rapamycin or a rapamycin derivative compound is co-administered with rapamycin resistant T cells to a mammal in need of therapy.
  • the dosage of rapamycin or a rapamycin derivative compound to be administered to the mammal will be tailored to each recipient based on serum monitoring of rapamycin drug levels.
  • rapamycin exposed T cells will have a selective advantage in such an in vivo state, the achievement of rapamycin levels at the higher side of the therapeutic range is desirable.
  • rapamycin and any derivative, salt, ethers and the like can be used.
  • the invention provides methods for treating a patient suffering from or susceptible to cancer, comprises, harvesting autologous cells from the mammal; selecting for a subset of rapamycin resistant CD4 + T cells and CD8 + T cells in vitro; and, administering to the mammal rapamycin resistant T cells concomitantly with rapamycin or a rapamycin derivative compound.
  • the subset of rapamycin resistant T cells for treating a patient suffering from or susceptible to cancer is preferably a Thl/Tcl subset and the Thl/Tcl subset expresses CD62L.
  • the Thl cells express CD4 and the Tel cells express CD8.
  • the rapamycin resistant Thl /Tel cellular subset secretes type I cytokines.
  • the invention provides a use of T cells as disclosed herein for the treatment of a targeted disease or disorder, including for the treatment of undesired cell proliferation including cancer, infectious diseases, reduction of graft versus host disease (GVHD) and the like.
  • a targeted disease or disorder including for the treatment of undesired cell proliferation including cancer, infectious diseases, reduction of graft versus host disease (GVHD) and the like.
  • GVHD graft versus host disease
  • the invention provides a use for the preparation of a therapeutic composition of T cells as disclosed herein for treatment of a targeted disease or disorder, including for the treatment of undesired cell proliferation including cancer, infectious diseases, reduction of graft versus host disease (GVHD) and the like.
  • a therapeutic composition of T cells as disclosed herein for treatment of a targeted disease or disorder, including for the treatment of undesired cell proliferation including cancer, infectious diseases, reduction of graft versus host disease (GVHD) and the like.
  • GVHD graft versus host disease
  • Preferred methods of the invention including identifying and/or selecting a subject (e.g. a mammal, particularly a human) that is susceptible to or suffering from a condition as disclosed herein such as cancer, an infectious diseases, reduction of graft versus host disease (GVHD) and the like; and thereafter a ⁇ -lministering to the identified and selected subject a T cell composition as disclosed herein.
  • a subject e.g. a mammal, particularly a human
  • a condition as disclosed herein such as cancer, an infectious diseases, reduction of graft versus host disease (GVHD) and the like
  • the invention also includes pharmaceutical compositions that comprise rapamycin resistant T cells optionally admixed with a pharmaceutically acceptable carrier and optionally packaged together with instructions (e.g. written) for use of the composition for a condition as disclosed herein.
  • the invention also includes rapamycin resistant T cells, e.g. as maybe obtainable as disclosed herein such as by treating a sample of isolated T cells (mammalian, preferably human) with rapamycin or a rapamycin derivative compound and selecting a subset of rapamycin resistant T cells particularly rapamycin resistant CD4 + T cells and/or CD8 + T cells, typically in vitro. Other aspects of the invention are discussed infra.
  • infectious agent refers to an organism wherein growth/multiplication leads to pathogenic events in humans or animals.
  • infectious agent refers to an organism wherein growth/multiplication leads to pathogenic events in humans or animals. Examples of such agents are: bacteria, fungi, protozoa and viruses.
  • a "pharmaceutically acceptable” component is one that is suitable for use with humans and/or animals without undue adverse side effects (such as toxicity, irritation, and allergic response) commensurate with a reasonable benefit/risk ratio.
  • safe and effective amount refers to the quantity of a component which is sufficient to yield a desired therapeutic response without undue adverse side effects (such as toxicity, irritation, or allergic response) commensurate with a reasonable benefit/risk ratio when used in the manner of this invention.
  • therapeutically effective amount is meant an amount of a compound of the present invention effective to yield the desired therapeutic response.
  • an amount effective to delay the growth of or to cause a cancer either a sarcoma or lymphoma, or to shrink the cancer or prevent metastasis.
  • the specific safe and effective amount or therapeutically effective amount will vary with such factors as the particular condition being treated, the physical condition of the patient, the type of mammal or animal being treated, the duration of the treatment, the nature of concurrent therapy (if any), and the specific formulations employed and the structure of the compounds or its derivatives.
  • a "pharmaceutical salt” include, but are not limited to, mineral or organic acid salts of basic residues such as amines; alkali or organic salts of acidic residues such as carboxylic acids.
  • the salts are made using an organic or inorganic acid.
  • These prefened acid salts are chlorides, bromides, sulfates, nitrates, phosphates, sulfonates, formates, tartrates, maleates, malates, citrates, benzoates, salicylates, ascorbates, and the like.
  • the most preferred salt is the hydrochloride salt.
  • cancer refers to all types of cancer or neoplasm or malignant tumors found in mammals, including, but not limited to: leukemias, lymphomas, melanomas, carcinomas and sarcomas.
  • Examples of cancers are cancer of the brain, breast, pancreas, cervix, colon, head and neck, kidney, lung, non-small cell lung, melanoma, mesothelioma, ovary, sarcoma, stomach, uterus and Medulloblastoma.
  • leukemia refers broadly to progressive, malignant diseases of the blood- forming organs and is generally characterized by a distorted proliferation and development of leukocytes and their precursors in the blood and bone marrow. Leukemia is generally clinically classified on the basis of (1) the duration and character of the disease-acute or chronic; (2) the type of cell involved; myeloid (myelogenous), lymphoid (lymphogenous), or monocytic; and (3) the increase or non-increase in the number of abnormal cells in the blood-leukemic or aleukemic (subleukemic).
  • the present invention includes a method of treating leukemia, and, preferably, a method of treating acute nonlymphocytic leukemia, chronic lymphocytic leukemia, acute granulocytic leukemia, chronic granulocytic leukemia, acute promyelocytic leukemia, adult T-cell leukemia, aleukemic leukemia, a leukocythemic leukemia, basophylic leukemia, blast cell leukemia, bovine leukemia, chronic myelocytic leukemia, leukemia cutis, embryonal leukemia, eosinophilic leukemia, Gross' leukemia, hairy-cell leukemia, hemoblastic leukemia, hemocytoblastic leukemia, histiocytic leukemia, stem cell leukemia, acute monocytic leukemia, leukopenic leukemia, lymphatic leukemia, lymphoblastic leukemia, lymphocytic leukemia, lymphogenous leukemia
  • sarcoma generally refers to a tumor which is made up of a substance like the embryonic connective tissue and is generally composed of closely packed cells embedded in a fibrillar or homogeneous substance.
  • sarcomas which can be treated with the compositions disclosed herein, and optionally a potentiator and/or chemotherapeutic agent include, but not limited to a chondrosarcoma, fibrosarcoma, lymphosarcoma, melanosarcoma, myxosarcoma, osteosarcoma, Abemethy's sarcoma, adipose sarcoma, liposarcoma, alveolar soft part sarcoma, ameloblastic sarcoma, botryoid sarcoma, chloroma sarcoma, chorio carcinoma, embryonal sarcoma, Wilms' tumor sarcoma, endometrial sarcoma, strom
  • melanoma is taken to mean a tumor arising from the melanocytic system of the skin and other organs.
  • Melanomas which can be treated with the compositions disclosed herein, and optionally a potentiator and/or another chemotherapeutic agent include but not limited to, for example, acral-lentiginous melanoma, amelanotic melanoma, benign juvenile melanoma, Cloudman's melanoma, S91 melanoma, Harding-Passey melanoma, juvenile melanoma, lentigo maligna melanoma, malignant melanoma, nodular melanoma, subungal melanoma, and superficial spreading melanoma.
  • Carcinoma refers to a malignant new growth made up of epithelial cells tending to infiltrate the surrounding tissues and give rise to metastases.
  • Carcinomas which can be treated with the compositions disclosed herein, and optionally a potentiator and/or a chemotherapeutic agent include but not limited to, for example, acinar carcinoma, acinous carcinoma, adenocystic carcinoma, adenoid cystic carcinoma, carcinoma adenomatosum, carcinoma of adrenal cortex, alveolar carcinoma, alveolar cell carcinoma, basal cell carcinoma, carcinoma basocellulare, basaloid carcinoma, basosquamous cell carcinoma, bronchioalveolar carcinoma, bronchiolar carcinoma, bronchogenic carcinoma, cerebriform carcinoma, cholangiocellular carcinoma, chorionic carcinoma, colloid carcinoma, comedo carcinoma, corpus carcinoma, cribriform carcinoma, carcinoma en cuirasse, carcinoma cutaneum, cylindrical carcinoma, cylindrical cell carcinoma, duct carcinoma, carcinoma durum, embryonal carcinoma, ence
  • Additional cancers which can be treated with the methods and compositions according to the invention include, for example, Hodgkin's Disease, Non-Hodgkin's Lymphoma, multiple myeloma, neuroblastoma, breast cancer, ovarian cancer, lung cancer, rhabdomyosarcoma, primary thrombocytosis, primary macroglobulinemia, small-cell lung tumors, primary brain tumors, stomach cancer, colon cancer, malignant pancreatic insulanoma, malignant carcinoid, urinary bladder cancer, premalignant skin lesions, testicular cancer, lymphomas, thyroid cancer, neuroblastoma, esophageal cancer, genitourinary tract cancer, malignant hypercalcemia, cervical cancer, endometrial cancer, adrenal cortical cancer, and prostate cancer.
  • Diagnostic or “diagnosed” means identifying the presence or nature of a pathologic condition. Diagnostic methods differ in their sensitivity and specificity.
  • the "sensitivity” of a diagnostic assay is the percentage of diseased individuals who test positive (percent of "true positives”). Diseased individuals not detected by the assay are “false negatives.” Subjects who are not diseased and who test negative in the assay, are termed “true negatives.”
  • the "specificity" of a diagnostic assay is 1 minus the false positive rate, where the "false positive” rate is defined as the proportion of those without the disease who test positive. While a particular diagnostic method may not provide a definitive diagnosis of a condition, it suffices if the method provides a positive indication that aids in diagnosis.
  • patient or “individual” are used interchangeably herein, and refers to a mammalian subject to be treated, with human patients being preferred.
  • the methods of the invention find use in experimental animals, in veterinary application, and in the development of animal models for disease, including, but not limited to, rodents including mice, rats, and hamsters; and primates.
  • sample is used herein in its broadest sense.
  • a sample comprising polynucleotides, polypeptides, peptides, antibodies and the like may comprise a bodily fluid; a soluble fraction of a cell preparation, or media in which cells were grown; a chromosome, an organelle, or membrane isolated or extracted from a cell; genomic DNA, RNA, or cDNA, polypeptides, or peptides in solution or bound to a substrate; a cell; a tissue; a tissue print; a fingerprint, skin or hair; and the like.
  • Treatment is an intervention performed with the intention of preventing the development or altering the pathology or symptoms of a disorder. Accordingly, “treatment” refers to both therapeutic treatment and prophylactic or preventative measures. Those in need of treatment include those already with the disorder as well as those in which the disorder is to be prevented.
  • a therapeutic agent may directly decrease the pathology of tumor cells, or render the tumor cells more susceptible to treatment by other therapeutic agents, e.g., radiation and/or chemotherapy.
  • ameliorated refers to a symptom which approaches a normalized value (for example a value obtained in a healthy patient or individual), e.g., is less than 50% different from a normalized value, preferably is less than about 25% different from a normalized value, more preferably, is less than 10% different from a normalized value, and still more preferably, is not significantly different from a normalized value as determined using routine statistical tests.
  • amelioration or treatment of a patient suffering from an infectious disease organism such as for example, Hepatitis B Virus
  • neoplastic disease or neoplastic cells refers to an amount of rapamycin resistant T cells, described throughout the specification and in the Examples which follow, capable of invoking one or more of the following effects: (1) inhibition, to some extent, of tumor growth, including, (i) slowing down and (ii) complete growth arrest; (2) reduction in the number of tumor cells; (3) maintaining tumor size; (4) reduction in tumor size; (5) inhibition, including (i) reduction, (ii) slowing down or (iii) complete prevention, of tumor cell infiltration into peripheral organs; (6) inhibition, including (i) reduction, (ii) slowing down or (iii) complete prevention, of metastasis; (7) enhancement of anti-tumor immune response, which may result in (i) maintaining tumor size, (ii) reducing tumor size, (Hi) slowing the growth of a tumor, (iv) reducing, slowing or preventing invasion and/or (8) relief, to some extent, of the severity or number of one or more symptoms associated with the disorder
  • Treatment of an individual suffering from an infectious disease organism refers to a decrease or elimination of the disease organism from an individual. For example, a decrease of viral particles as measured by plaque forming units or other automated diagnostic methods such as ELIS A, etc., may be used to monitor efficacy of treatment.
  • "Treatment of an individual suffering from graft-versus-host-disease or GVHD” refers to a decrease or cessation of symptoms associated with GVHD. For example, an amelioration of lacy, livid maculopapular rash, jaundice, diarrhea, abdominal pain, hepatosplenomegaly, alopecia, bullae, desquamation of skin. Treatment or amelioration of GVHD results in clinical downgrading of the disease.
  • liver GVHD is staged from none (stage 0; bilirubin ⁇ 2 mg/dl) to severe (stage 4; bilirubin > 15 mg/dl) based on serum bilirubin level.
  • Skin GVHD is staged based upon the percent body surface area that the rash involves, with stage 0 having no rash and stage 4 having rash of up to 100 % body surface area with bullae or desquamation.
  • Intestinal GVHD is staged based upon the volume of daily liquid stool output, with stage 0 being no diarrhea and stage 4 being > 1500 ml liquid stool per day with abdominal pain or ileus.
  • Chronic GVHD which typically occurs after day 100 post-transplant and can last several years post-transplant, is typically scored based upon number of organ sites that the chronic GVHD involves (limited chronic GVHD, one site; extensive chronic GVHD, two or more sites). Chronic GVHD involves the same organs as acute GVHD, but in addition, chronic GVHD may also affect the mucous glands in the eyes, salivary glands in the mouth, and glands that lubricate the stomach lining and intestines.
  • an ameliorated symptom or “treated symptom” refers to a symptom which is approaches a normalized value, e.g., is less than 50% different from a normalized value, preferably is less than about 25% different from a normalized value, more preferably, is less than 10% different from a normalized value, and still more preferably, is not significantly different from a normalized value as determined using routine statistical tests.
  • Cells of the immune system or “immune cells” as used herein, is meant to include any cells of the immune system that may be assayed, including, but not limited to, B lymphocytes, also called B cells, T lymphocytes, also called T cells, natural killer (NK) cells, natural killer T (NKT) cells, lymphokine-activated killer (LAK) cells, monocytes, macrophages, neutrophils, granulocytes, mast cells, platelets, Langerhans cells, stem cells, dendritic cells, peripheral blood mononuclear cells, tumor-infiltrating (TIL) cells, gene modified immune cells including hybridomas, drug modified immune cells, and derivatives, precursors or progenitors of the above cell types.
  • B lymphocytes also called B cells
  • T lymphocytes also called T cells
  • NK natural killer
  • NKT natural killer T
  • LAK lymphokine-activated killer
  • monocytes monocytes
  • macrophages neutrophils
  • granulocytes mast cells
  • Immuno effector cells refers to cells capable of binding an antigen and which mediate an immune response selective for the antigen. These cells include, but are not limited to, T cells (T lymphocytes), B cells (B lymphocytes), monocytes, macrophages, natural killer (NK) cells and cytotoxic T lymphocytes (CTLs), for example CTL lines, CTL clones, and CTLs from tumor, inflammatory, or other infiltrates.
  • Immunorelated molecules refers to any molecule identified in any immune cell, whether in a resting ("non-stimulated") or activated state, and includes any receptor, ligand, cell surface molecules, nucleic acid molecules, polypeptides, variants and fragments thereof.
  • T cells or "T lymphocytes” are a subset of lymphocytes originating in the thymus and having heterodimeric receptors associated with proteins of the CD3 complex (e.g., a rearranged T cell receptor, the heterodimeric protein on the T cell surfaces responsible for antigen MHC specificity of the cells).
  • T cell responses may be detected by assays for their effects on other cells (e.g., target cell killing, activation of other immune cells, such as B-cells) or for the cytokines they produce.
  • allogeneic is used to refer to immune cells derived from non- self major histocompatibility complex donors.
  • HLA haplotypes/allotypes vary from individual to individual and it is often helpful to determine the individual's HLA type.
  • the HLA type may be determined via standard typing procedures.
  • host compatible or “autologous” cells means cells that are of the same or similar haplotype as that of the subject or “host” to which the cells are administered, such that no significant immune response against these cells occurs when they are transplanted into a host.
  • CD4 is a cell surface protein important for recognition by the T cell receptor of antigenic peptides bound to MHC class II molecules on the surface of an APC.
  • Thl cells are primarily involved in activating macrophages with respect to cellular immunity and the inflammatory response, whereas “Th2 cells” or “helper T cells” are primarily involved in stimulating B cells to produce antibodies (humoral immunity).
  • Effector molecules for Thl cells include, but are not limited to, IFN- ⁇ , GM-CSF, TNF- ⁇ , CD40 ligand, Fas ligand, IL-3, TNF- ⁇ , and IL-2.
  • Effector molecules for Th2 cells include, but are not limited to, IL-4, IL-5, CD40 ligand, IL-3, GS-CSF, DL-10, TGF- ⁇ , and eotaxin.
  • Activation of the Thl type cytokine response can suppress the Th2 type cytokine response, and reciprocally, activation of the Th2 type cytokine response can suppress the Thl type response.
  • a “chemokine” is a small cytokine involved in the migration and activation of cells, including phagocytes and lymphocytes, and plays a role in inflammatory responses.
  • a "cytokine” is a protein made by a cell that affect the behavior of other cells through a "cytokine receptor” on the surface of the cells the cytokine effects. Cytokines manufactured by lymphocytes are sometimes termed “lymphokines.” Cytokines are also characterized as Type I (e.g. IL-2 and IFN- ⁇ ) and Type II (e.g. IL-4 and IL-10).
  • modulate it is meant that any of the mentioned activities, are, e.g., increased, enhanced, increased, agonized (acts as an agonist), promoted, decreased, reduced, suppressed blocked, or antagonized (acts as an agonist). Modulation can increase activity more than 1-fold, 2-fold, 3-fold, 5-fold, 10-fold, 100-fold, etc., over baseline values. Modulation can also decrease its activity below baseline values.
  • An “epitope”, as used herein, is a portion of a polypeptide that is recognized (i.e., specifically bound) by a B-cell and/or T-cell surface antigen receptor.
  • Epitopes may generally be identified using well known techniques, such as those summarized in Paul, Fundamental Immunology, 3rd ed., 243-247 (Raven Press, 1993) and references cited therein. Such techniques include screening polypeptides derived from the native polypeptide for the ability to react with antigen-specific antisera and/or T-cell lines or clones. An epitope of a polypeptide is a portion that reacts with such antisera and/or T- cells at a level that is similar to the reactivity of the full length polypeptide (e.g., in an ELISA and or T-cell reactivity assay). Such screens may generally be performed using methods well known to those of ordinary skill in the art, such as those described in Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988. B-cell and T-cell epitopes may also be predicted via computer analysis.
  • Substrate refers to any rigid or semi-rigid support to which nucleic acid molecules or proteins are bound and includes membranes, filters, chips, slides, wafers, fibers, magnetic or nonmagnetic beads, gels, capillaries or other tubing, plates, polymers, and microparticles with a variety of surface forms including wells, trenches, pins, channels and pores.
  • Immunoassay is an assay that uses an antibody to specifically bind an antigen
  • the immunoassay is characterized by the use of specific binding properties of a particular antibody to isolate, target, and/or quantify the antigen.
  • a transplant includes any cell, organ, organ system or tissue which can elicit an immune response in a recipient subject mammal.
  • a transplant includes an allograft or a xenograft cell, organ, organ system or tissue.
  • An allograft refers to a graft (cell, organ, organ system or tissue) obtained from a member of the same species as the recipient.
  • a xenograft refers to a graft (cell, organ, organ system or tissue) obtained from a member of a different species as the recipient.
  • immune rejection is intended to refer to immune responses involved in transplant rejection, as well as to the concomitant physiological result of such immune responses, such as for example, interstitial fibrosis, chronic graft artheriosclerosis, or vasculitis.
  • immune rejection is also intended to refer to immune responses involved in autoimmune disorders, and the concomitant physiological result of such immune responses, including T cell-dependent infiltration and direct tissue injury; T cell-dependent recruitment and activation of macrophages and other effector cells; and T cell-dependent B cell responses leading to autoantibody production.
  • transplant rejection refers to T cell-mediated rejection of transplant cells, organs, organ systems or tissue. In general, such transplant rejection generally includes accelerated, acute and chronic rejection. It is intended that the term, as used herein, also refer to GVHD, and the physiological results of such a disorder.
  • reducing immune rejection is meant to encompass prevention or inhibition of immune rejection, as well as delaying the onset or the progression of immune rejection.
  • the term is also meant to encompass prolonging survival of a transplant in a subject mammal, or reversing failure of a transplant in a subject.
  • ameliorating a symptom of an immune rejection including, for example, ameliorating an immunological complication associated with immune rejection, such as for example, interstitial fibrosis, chronic graft atherosclerosis, or vasculitis.
  • induction of tolerance in a subject mammal that has undergone a transplant is also meant to encompass induction of tolerance in a subject mammal that has undergone a transplant.
  • the term "tolerance,” as used herein, refers to a state wherein the immune system of a transplant recipient subject mammal is non-responsive to the transplant. This state is considered donor transplant-specific, and, as such, is distinguished from nonspecific immunosuppression. Operatively, the term as used herein, refers to permanent acceptance of a graft without ongoing immunosuppression, wherein, for example, challenge with a second graft of donor origin (especially when the second graft is of the same tissue as the first graft) should be accepted, and challenge with a third party graft should be rejected.
  • autoimmune rejection refers to immune responses involved in autoimmune disorders, and the concomitant physiological result of such immune responses.
  • activated T cell refers to a T cell that expresses antigens indicative of T-cell activation (that is, T cell activation markers).
  • T cell activation markers include, but are not limited to, CD25, CD26, CD30, CD38, CD69, CD70, CD71, ICOS, OX-40 and 4-1BB.
  • the expression of activation markers can be measured by techniques known to those of skill in the art, including, for example, western blot analysis, northern blot analysis, RT-PCR, immunofluorescence assays, and fluorescence activated cell sorter (FACS) analysis.
  • FACS fluorescence activated cell sorter
  • resting T cell refers to a T cell that does not express T-cell activation markers. Resting T cells include, but are not limited to, T cells which are CD25 “ , CD69 “ , ICOS “ , SLAM “ , and 4- IBB “ . The expression of these markers can be measured by techniques known to those of skill in the art, including, for example, western blot analysis, northern blot analysis, RT-PCR, immunofluorescence assays, and fluorescence activated cell sorter (FACS) analysis.
  • FACS fluorescence activated cell sorter
  • T cell activator refers to any compound or factor that is a T cell receptor stimulatory factor, that is, induces T cell receptor signaling.
  • T cell activators include, but are not limited to, anti-CD3, antibodies (preferably monoclonal antibodies) either alone or in conjunction with anti-CD28 antibodies (preferably monoclonal antibodies), or mitogens such as, for example, phorbol 12-myristate 13-acetate (PMA), phytohemagglutinin (PHA) or concanavalin-A (Con-A).
  • PMA phorbol 12-myristate 13-acetate
  • PHA phytohemagglutinin
  • Con-A concanavalin-A
  • Figure 2 is a graph showing supernatant ELISA results for the type II cytokines IL-4 and IL-10.
  • Figure 3 is a graph showing that addition of CSA, and in particular, FK506, results in Thl cells with significantly diminished capacity for both IL-2 and IFN- ⁇ secretion.
  • Figure 4 is a graph showing that at a high dose of rapamycin, co-stimulation and cytokine supplementation allowed for the expansion of either Thl or Th2 subsets without any apparent reduction in numbers of CD4-expressing cells.
  • Figure 5 is a graph showing that Thl cells in each of the rapamycin concentrations had similarly high secretion of both IL-2 and IFN- ⁇ .
  • Figure 6 is a graph showing that Th2 cells propagated in the 0.1 ⁇ M rapamycin concentration had preservation of capacity for secretion of the type II cytokines IL-4, IL-5, and IL-10. .
  • Figure 7 is a graph showing Th2, and Thl cell expansion was greatly reduced relative to CD3, CD28 co-stimulated control Thl/Th2 cultures.
  • Figure 8 is a graph showing CD3, CD28 generated Th2 cell expansion from day 0 to 6 of culture at rapamycin concentrations ranging from 0.1 ⁇ M to 10.0 ⁇ M.
  • Figure 9 is a graph showing the increase in numbers of CD4+ expressing cells in the high dose rapamycin cultures after day 6.
  • Figure 10 is a graph showing that at the highest rapamycin concentration, the Th2 cells had an enhanced Th2 polarity on the basis of abrogation of contaminating DL- 2 secretion and modest reduction in IFN- ⁇ secretion.
  • Figure 11 is a graph showing Thl or Th2 cells re-stimulated in the presence of 0.1 ⁇ M rapamycin, and 10 ⁇ M rapamycin.
  • Figure 12 is a graph showing that CD28 was greatly increased on the CD4+ cells that were propagated in high dose ( 10 ⁇ M) rapamycin.
  • Figure 13 is a graph showing that Th2 cells expanded in high dose rapamycin, had an increased expression of CD62L.
  • Figure 14 is a graph showing shows median cell volume changes during Thl , Th2, Tel, or Tc2 expansion in the presence or absence of either 0.1 or 10.0 ⁇ M rapamycin.
  • Figure 15 is a graph showing CD8 + Tcl/Tc2 expansion after CD3, CD28 co- stimulation in the presence of 0.1 ⁇ M and 10 ⁇ M of rapamycin.
  • Figure 16 is a graph showing CTL assays using Tc2 effectors generated in the presence or absence of rapamycin.
  • Figure 17 is a graph showing Tel cells expanded in the high dose of rapamycin lost their capacity for IFN- ⁇ secretion and had reduced capacity for IL-2 secretion.
  • Figure 18 is a graph showing that CD8 + cell expansion in high-dose rapamycin was associated with a more na ⁇ ve T cell phenotype, as evidenced by increased CD62L expression.
  • Figure 19 is a graph showing that recipients of allogeneic splenic T cell inocula had a significant number of alloreactive CD8 + T cells capable of IFN- ⁇ secretion at day 7 post-BMT.
  • Figure 20 is a graph showing that recipients of splenic CD4 + and CD8 + T cells underwent weight loss consistent with acute GVHD.
  • Figure 21 is a graph showing survival results in recipients of Th2 cells generated under either low dose or high dose rapamycin.
  • Figure 22 is a graph showing that administration of rapamycin-generated Th2 cells and in vivo rapamycin resulted in a greater number of Th2 cells in the day +7 spleens than cell administration and CSA or vehicle administration.
  • Figure 24 is a graph showing the growth curves of CD4 + cell in the presence of rapamycin.
  • Figure 25 is a graph showing CD3, CD28 re-stimulation of rapamycin-generated Th2 cells with or without 0.01 ⁇ M rapamycin.
  • Figure 26 is a graph showing that cells propagated under Th2 conditions and rapamycin had an increased Th2 cytokine purity, as evidenced by reduction in capacity for IFN- ⁇ secretion.
  • Figure 27 is a graph showing that rapamycin-generated Th2 cells had an increased capacity for secretion of the type II cytokines IL-4 and IL-13.
  • Figure 28 is a graph showing that rapamycin-generated Th2 cells had increased expression of CD62L relative to control Th2 cells; the increase in CD62L was most marked in Th2 cultures that were continuously exposed to rapamycin.
  • Figure 29 is a graph showing rapamycin-generated Th2 cells indeed had an increased capacity for rhodamine dye exclusion.
  • Figure 30 is a graph showing that rapamycin generated Th2 cells increased MDR function.
  • Figure 31 is a graph showing purified na ⁇ ve or memory human CD4 cells co- stimulated either with or without rapamycin.
  • rapamycin generated T cells were selectively resistant to the inhibitory effects of rapamycin in vivo.
  • in vivo administration of rapamycin-resistant Thl, Th2, Tel or Tc2 cells with concomitant administration of rapamycin drug inhibits non-cultured T cells that may not possess the desired function and at the same time allow preferential expansion of the in vitro cultured T cell of optimal function.
  • This new immune therapy strategy greatly amplifies the in vivo effects of immune therapeutic T cells of a selected function.
  • T cell subset generation are growth in in vitro T cell culture conditions comprising the immune suppression drug rapamycin to generate rapamycin-resistant cells having a desired T, lymphocyte function, such as for example, T helper cells (Thl or Th2 function) and/or cytotoxic T cells (Tel or Tc2 function).
  • the desired T cell subset is selected for, activated, and co-administered with rapamycin to a patient in need of therapy.
  • T cells are harvested from the patient, activated and expanded in the identified conditions with rapamycin to generate rapamycin-resistant Thl or Tel cells, then re-infused to the patient with simultaneous administration of rapamycin drug.
  • This expands the therapeutic T cell against cancer or infection, and inhibits any non-cultured T cell in the body that might otherwise adversely affect the therapeutic T cell response.
  • T cells would be harvested from the patient, expanded in conditions containing rapamycin to generate, preferably, rapamycin resistant Th2 or Tc2 cells, and then re-infused to the patient with simultaneous administration of the rapamycin drug.
  • the immune therapeutic T cells are expanded to prevent GVHD or treat autoimmune disease, and inhibit any non-cultured T cell in the body that may otherwise promote GVHD or autoimmune disease.
  • therapeutic T cell refers to the rapamycin resistant T cell subsets, for example, Thl/Tcl and Th2/Tc2.
  • rapamycin refers to rapamycin and/or structurally modified rapamycin compounds (such structurally modified rapamycin compounds sometimes referred to herein as rapamycin derivatives).
  • the unmodified compound is the macrolide antibiotic that can be produced by Streptomyces hyhoscopius having the stracture as disclosed e.g. in J.B. McAlpine et al. J. Antibiotics (1991) 44:688 and S.L. Schrieber et al., J. Am. Chem. Soc, (1991) 113:7433.
  • That unmodified rapamycin is in general a preferred rapamycin compound and is the compound referred to in the examples which follow.
  • Additional suitable and preferred stracturally modified rapamycin compounds can be identified through simple testing.
  • suitable rapamycin derivatives for identifying resistant cells can be evaluated using in vitro assays as described in detail in the Examples which follow. Briefly, cells such as for example T cells are stimulated and cultured in the presence of cytokines till the cells reach a desired concentration, such as for example 2 x 10 6 cells.
  • Candidate rapamycin derivative compounds are added to the cell culture in varying concentrations such as at least about 0.004 ⁇ M up to about 0.02 ⁇ M.
  • Viable cells as determined by microscopic observations or dye exclusion assays are counted by a Multi-Sizer Instrument (Coulter), and the cellular expansion, for example, CD4 expansion is plotted, as shown in Figure 1. If a candidate rapamycin compound results in decrease in cell populations as compared to normal controls and controls incubated with rapamycin, then the compound is considered suitable for use in the methods and compositions of the invention.
  • Candidate rapamycin compounds include, but are not limited to, tetrazole containing rapamycin analogs disclosed in US Patent No. 6,329,386; acyl derivatives of rapamycin disclosed in US Patent No.
  • the number of cells of desired function, administered to the patient will vary depending on various factors such as the disease or condition to be treated, the condition of the patient, which should be determined via consideration of all appropriate factors by the practitioner.
  • about cells of desired function are administered to a patient, more preferably about lxl 0 8 to about lxlO 11 cells of desired function are administered to a patient, and even more preferably, about lxl0 9 to about lxl0- 10 cells of desired function are administered to an adult human.
  • the number of cells administered are about 2.5x 10 9 cells.
  • Methods of re-introducing cellular components are known in the art and include procedures such as those exemplified in U.S. Pat. No. 4,844,893 to Honsik, et al. and U.S. Pat. No. 4,690,915 to Rosenberg.
  • administration of CD8 + cells of Thl/Tcl function via intravenous infusion is appropriate.
  • T cells from patients are, preferably activated ex vivo, either by soluble anti-CD3 antibody, or most preferably, are co-activated by using anti-CD3 and anti-CD28 monoclonal antibodies, either by soluble or immobilized on a solid support.
  • a preferred solid support are plastics, or any surface upon which antibodies can be immobilized, or beads, such as, for example, Dynal beads.
  • Particularly prefened surface antigens for optimal co-stimulation are CD3 and/or CD28 and particular secreted cytokines (like IL-2, IL-4, IL-10, IFN- ⁇ ).
  • the present invention is also useful as the activation is conducted in vitro and the activated helper or cytotoxic T-cells are reinfroduced into the patient.
  • Activation is achieved by the crosslinking of the T cell receptor complex (anti-CD3 and anti-CD28 antibodies) which increase the effectiveness of the activation.
  • Cross linking of the TCR with anti-CD3 triggers a signaling cascade resulting in T cell proliferation, cytokine synthesis, and immune responses.
  • optimal activation and proliferation requires costimulation of CD28 receptors on T cells with anti-CD28 or B7 molecules (CD80 and CD86).
  • MHC major histocompatibility complex
  • MHC major histocompatibility complex
  • the MHC genes which are also referred to as the HLA (human leukocyte antigen) complex, are located on chromosome 6 in humans.
  • the molecules encoded by MHC genes are present on cell surfaces and are largely responsible for recognition of tissue- transplants as "non-self. Thus, membrane-bound MHC molecules are intimately involved in recognition of antigens by T-cells.
  • MHC products are grouped into three major classes, referred to as I, II, and EL T-cells that serve mainly as helper cells express CD4 and primarily interact with Class II molecules, whereas CD8-expressing cells, which mostly represent cytotoxic effector cells, interact with Class I molecules.
  • transplantation antigen is used to refer to antigenic molecules that are expressed on the cell surface of transplanted cells, either at the time of transplantation, or at some point following transplantation. Generally these antigenic molecules are proteins and glycoproteins.
  • the primary transplantation antigens are products of the major histocompatibility complex (MHC), located on chromosome 6 in humans.
  • MHC major histocompatibility complex
  • HLA human leukocyte antigen
  • MHC antigens are divided into MHC class I antigens (in humans, this class includes HLA- A, -B, and -C antigens) and MHC class II antigens (in humans, this class includes HLA-DP, -DQ, and -DR antigens).
  • MHC-II antigens MHC class II antigens
  • MHC class II transplantation antigens MHC class II transplantation antigens
  • MHC class II transplantation antigens MHC class II transplantation antigens
  • MHC class II transplantation antigens include cell surface molecules other than MHC class I and II antigens.
  • antigens include the following: (1) the ABO antigens involved in blood cell recognition; (2) cell adhesion molecules such as ICAM, which is involved in leukocyte cell-cell recognition; and (3) ⁇ 2 -microglobulin, a polypeptide associated with the 44 kd heavy chain polypeptide that comprises the HLA-I antigens but is not encoded by the MHC complex.
  • GVHD frequently results. It has been suggested that GVHD results, in those instance, from alloaggression due to minor histocompatibility antigen differences for which many authors have suggested the depletion of donor T cells as a means to avoid GVHD.
  • this strategy of T cell depletion may avoid GVHD, such patients are at increased risk for tumor relapse, infection, and graft rejection, and as such, T cell depletion has both positive and negative consequences.
  • Class I molecules are membrane glycoproteins with the ability to bind peptides derived primarily from intracellular degradation of endogenous proteins. Complexes of MHC molecules with peptides derived from viral, bacterial and other foreign proteins comprise the ligand that triggers the antigen responsiveness of T-cells. In contrast, complexes of MHC molecules with peptides derived from normal cellular products play a role in "teaching" the T-cells to tolerate self-peptides, in the thymus. Class I molecules do not present entire, intact antigens; rather, they present peptide fragments "loaded” onto their "peptide binding groove".
  • the presentation of Class I MHC molecules bound to peptide alone has generally been ineffective in activating CD8 cells.
  • the CD8 cells are activated by antigen-presenting cells, such as, for example, dendritic cells, which present not only a peptide-bound Class I MHC molecule, but also a costimulatory molecule.
  • antigen-presenting cells such as, for example, dendritic cells, which present not only a peptide-bound Class I MHC molecule, but also a costimulatory molecule.
  • costimulatory molecules include B7 which is now recognized to be two subgroups designated as B7.1 and B7.2. It has also been found that cell adhesion molecules such as integri ⁇ s assist in this process.
  • Dendritic cells are antigen-presenting cells that are found in all tissues and organs, including the blood.
  • dendritic cells present antigens for T lymphocytes, i.e., they process and present antigens, and stimulate responses from naive and memory T cells. In addition to their role in antigen presentation, dendritic cells directly communicate with non-lymph tissue and survey non-lymph for an injury signal (e.g., ischemia, infection, or inflammation) or tumor growth. Once signaled, dendritic cells initiate the immune response by releasing IL-1 which triggers lymphocytes and monocytes.
  • an injury signal e.g., ischemia, infection, or inflammation
  • the CD8 T-cell When the CD8 T-cell interacts with an antigen-presenting cell, such as a dendritic cells, having the peptide bound by a Class I MHC and costimulatory molecule, the CD8 T-cell is activated to proliferate and becomes an effector T-cell. See, generally, Janeway and Travers, Immunobiology, published by Current Biology Limited, London (1994), incorporated by reference.
  • an antigen-presenting cell such as a dendritic cells, having the peptide bound by a Class I MHC and costimulatory molecule
  • rapamycin resistant T cells co-administered with rapamycin ameliorate GVHD as determined by the change in stage of GVHD.
  • graft-versus-host-disease is ameliorated by at least about 50%, more preferably by at least about 75%, most preferably about at least 90%, 95%, 98%, 99%, 99.9% or 100%.
  • autologous T cells from the patient are cultured in rapamycin and/or a rapamycin derivative and under conditions to generate a Th2 response.
  • Prefened conditions include the addition of cytokines such as IL-4 and IL-2, and rapamycin and/or a rapamycin derivative, alone. Specific conditions are described in the Examples which follow.
  • allogeneic donor Th2 cells are used to supplement the allotransplant.
  • the allogeneic Th2 cells increase in number with a concomitant decrease in GVHD.
  • immune T cell therapy is utilized for the treatment of a wide range of medical conditions such as cancer, disease due to infectious disease organisms such as viruses, autoimmune diseases, immunosuppressed individuals, burn victims and Graft versus Host Disease.
  • the invention provides for pharmaceutical compositions comprising rapamycin and/or a rapamycin derivative compound and/or rapamycin resistant T cells, rapamycin resistant stem cells and or rapamycin resistant dendritic cells.
  • the invention provides use of a rapamycin resistant T cell; a rapamycin resistant stem cell; a rapamycin resistant dendritic cell; composition for the treatment or prevention (including prophylactic treatment) of a disease or condition as disclosed herein, including acute GVHD, chronic GVHD, lacy, livid maculopapular rash, jaundice, dianhea, abdominal pain, hepatosplenomegaly, alopecia, bullae, desquamation of skin; prolonging survival of a transplant in a subject mammal, or reversing failure of a transplant in a subject and ameliorating disorders and symptoms such as associated with immune rejection, including, for example, interstitial fibrosis, chronic graft atherosclerosis, or vasculitis; freatment of cancers such as, leukemias, lymphomas, melanomas, carcinomas and sarcomas; diseases caused by or otherwise associated with a virus such as viruses of the herpes family,
  • Examples of clinical conditions which are caused by such virases include herpetic keratitis, herpetic encephalitis, cold sores and genital infections (caused by herpes simplex), chicken pox and shingles (caused by varicella zoster) and CMN-pneumonia and retinitis, particularly in immunocompromised patients including renal and bone manow transplant patients and patients with Acquired Immune Deficiency Syndrome (AIDS).
  • Epstein-Barr virus can cause infectious mononucleosis, and is also suggested as the causative agent of nasopharyngeal cancer, immunoblastic lymphoma and Burkitt's lymphoma.
  • refroviral infections which may be suitably treated in accordance with the invention include human refroviral infections such as HTV-1, HIV-2, and Human T-cell Lymphotropic Viras (HTLV) e.g. HTLV-I or HTLV- ⁇ infections.
  • human refroviral infections such as HTV-1, HIV-2, and Human T-cell Lymphotropic Viras (HTLV) e.g. HTLV-I or HTLV- ⁇ infections.
  • HTLV Human T-cell Lymphotropic Viras
  • the invention provides use of a rapamycin resistant T cell; a rapamycin resistant stem cell; a rapamycin resistant dendritic cell; composition for the preparation of a medicament for the freatment or prevention (including prophylactic treatment) of a disease or condition as disclosed herein, including acute GVHD, chronic GVHD, lacy, livid maculopapular rash, jaundice, dianhea, abdominal pain, hepatosplenomegaly, alopecia, bullae, desquamation of skin; prolonging survival of a transplant in a subject mammal, or reversing failure of a transplant in a subject and ameliorating disorders and symptoms such as associated with immune rejection, including, for example, interstitial fibrosis, chronic graft atherosclerosis, or vasculitis; treatment of cancers such as, leukemias, lymphomas, melanomas, carcinomas and sarcomas; diseases caused by or otherwise associated with a virus such as
  • Examples of clinical conditions which are caused by such virases include herpetic keratitis, herpetic encephalitis, cold sores and genital infections (caused by herpes simplex), chicken pox and shingles (caused by varicella zoster) and CMN-pneumonia and retinitis, particularly in immunocompromised patients including renal and bone marrow transplant patients and patients with Acquired Immune Deficiency Syndrome (AIDS).
  • Epstein-Barr viras can cause infectious mononucleosis, and is also suggested as the causative agent of nasopharyngeal cancer, immunoblastic lymphoma and Burkitt's lymphoma.
  • refroviral infections which may be suitably treated in accordance with the invention include human refroviral infections such as HIV-1, HIV-2, and Human T-cell Lymphotropic Viras (HTLV) e.g. HTLV-I or HTLV-ff infections.
  • human refroviral infections such as HIV-1, HIV-2, and Human T-cell Lymphotropic Viras (HTLV) e.g. HTLV-I or HTLV-ff infections.
  • HTLV Human T-cell Lymphotropic Viras
  • Prefened methods of the invention including identifying and/or selecting a subject (e.g. a mammal, particularly human) that is susceptible to or suffering from a condition disclosed herein, and thereafter administering to the identified and selected subject one or more compounds of the invention, particularly a subject that is identified and selected as being susceptible to or suffering from acute GVHD, chronic GVHD, lacy, livid maculopapular rash, jaundice, dianhea, abdominal pain, hepatosplenomegaly, alopecia, bullae, desquamation of skin; prolonging survival of a transplant in a subject mammal, or reversing failure of a transplant in a subject and ameliorating disorders and symptoms such as associated with immune rejection, including, for example, interstitial fibrosis, chronic graft atherosclerosis, or vasculitis; treatment of cancers such as, leukemias, lymphomas, melanomas, carcinomas and sarcomas; diseases caused by or otherwise associated with a virus such
  • herpetic keratitis examples include herpetic keratitis, herpetic encephalitis, cold sores and genital infections (caused by herpes simplex), chicken pox and shingles (caused by varicella zoster) and CMV-pneumonia and retinitis, particularly in immunocompromised patients including renal and bone manow transplant patients and patients with Acquired Immune Deficiency Syndrome (AIDS).
  • Epstein-Barr viras can cause infectious mononucleosis, and is also suggested as the causative agent of nasopharyngeal cancer, immunoblastic lymphoma and Burkitt's lymphoma.
  • refroviral infections which may be suitably treated in accordance with the invention include human refroviral infections such as HIV-1, HIV-2, and Human T-cell Lymphotropic Virus (HTLV) e.g. HTLV-I or HTLV-II infections:
  • HTLV Human T-cell Lymphotropic Virus
  • T cell function is selected based on the cell type that is generated by the immune system in response to that disease. For example, the immune system effectively responds to a viral, bacterial, and fungal infection by generating a Thl/Tcl cell subset; an effective immune response to other infections may require the generation of a Th2/Tc2 response.
  • both a Thl/Tcl and Th2/Tc2 immune response may be optimal in some treatment settings, so as to invoke both cellular and antibody arms of the immune response.
  • Mossman and Coffinan Manton T. R., Coffinann R. L.: Thl and Th2 cells: Different patterns of lymphokine secretion lead to different functional properties.
  • growth factors known as cytokines produced by T helper or CD4 + T cells in both human and murine systems were classified into two subsets, Thl and Th2. These were characterized by their functions in regulating various types of immune responses.
  • Cytokines produced by Thl cells [interleukin (IL)-2, interferon-alpha, interferon-gamma, tumor necrosis factor-alpha (TNF- ⁇ ), IL-12] stimulated strong cellular immunity whereas Th2 cytokines [IL-4, IL-5, IL-6, IL-10, IL- 13] were important for eliciting humoral (antibody) responses in vivo.
  • Cytokines produced by non-CD4 + T cells have been shown to be important in in vivo responses.
  • the cytotoxic or CD8 + T cells can also be subdivided into two subgroups, Tel and Tc2, which conespond to the same subsets in T helper cells (Carter L. L., Dutton R.
  • T cell cytotoxic assays are well known to those skilled in the art. In general, cytotoxicity is measured in a 5 hr 51 Sodium chromate ( 51 Cr ) release assay. Target cells, that is cells that are recognized by the T cells are plated in flat-bottomed microtiter plates and incubated at 37°C overnight.
  • the targets are washed and labeled the next day with 51 Cr at 37°C.
  • 51 Cr is taken up by the target cells, either by endocytosis or pinocytosis, and is retained in the cytoplasm.
  • the wells containing target cells are washed, and then T cells, refened to as "effector cells” are plated at different E:T ratios and incubated overnight at 37°C. Cytolysis is a measure of the 51 Cr released from the target cells into the supernatant due to destruction of the target cells by the effector cells.
  • microtiter plates are centrifuged at 1000 rpm for 10 minutes and an aliquot of about 50 ⁇ l to about 100 ⁇ l is removed and the level of radioactivity is measured the next day by a gamma counter and the percent specific lysis calculated.
  • Percent specific lysis is measured by using the formula: ( 51 Cr released from the target cells) - (spontaneous 5I Cr released from the target cells)/ (maximum 5, Cr released from the target cells) - (spontaneous 5I Cr released from the target cells) x 100
  • the spontaneous 51 Cr released from the target cells is measured with tumor cells to which no effector cells have been added.
  • Maximum 51 Cr released from the target cells is obtained by adding, for example, 1M HC1 and represents the total amount of 51 Cr present in the cytoplasm of the target cell.
  • 3 H-TdR tritiated thymidine
  • 3 H-TdR is taken up by target cells into the nucleus of the cell. Release of 3 H-TdR is a measure of cell death by DNA fragmentation.
  • the assay is conducted as above except the incubation period is at least about 48 hours and 50 ⁇ l to about 100 ⁇ l of the supernatant is measured by a beta- counter in the presence of at least about 1 ml of scintillation fluid. Calculation of percent specific lysis is performed using the above formula.
  • T cell proliferation assays are used to determine class II MHC antigen recognition. Briefly, target cells are inadiated so that they do not proliferate. The source of the target cells can be allogeneic or autologous cells. CD4 + T cells are incubated with the inadiated target cells in the presence of H-TdR. The CD4 T cells react against the Class II MHC by proliferating. Proliferation is measured by the amount of 3 H-TdR that is taken up by the proliferating T cells as compared to normal control cells. The search for additional immunosuppressive agents for preventing transplant rejection and for the treatment of autoimmune and inflammatory disorders occupies considerable attention in the pharmaceutical industry.
  • cytokines such as interferon-gamma and tumor necrosis factor-alpha play a critical role in transplant rejection and in the pathophysiology of autoimmune disorders, much effort has been invested in the development of agents that suppress their production, secretion and/or end-organ effect.
  • the methods described herein which ameliorate organ transplant rejection and/or GVHD, are believed due to an increase in Th2/Tc2 function. This is surprising and contrary to the teachings in the prior art whereby, immunosuppressants are used to suppress immune responses, thereby, preventing prevent organ rejection or GVHD.
  • one potential mechanism that may contribute to the observed rapamycin-associated changes in human Th2 cell generation is preferential utilization of the multi-drag resistance (MDR) pump in cells of more na ⁇ ve phenotype. That is, previous data indicates that human na ⁇ ve CD45RA + cells express increased MDR, and to this extent, such cells may be intrinsically more resistant to rapamycin effects.
  • MDR multi-drag resistance
  • Th2 cells expanded with or without rapamycin were evaluated for their ability to exclude an MDR substrate, rhodamine. This evaluation was performed by flow cytometry in the presence or absence of an MDR pump inhibitor (results in Figure 29). As this figure shows, rapamycin-generated Th2 cells indeed had an increased capacity for rhodamine dye exclusion. This enhanced MDR function in the rapamycin-generated Th2 cells was significantly abrogated by the MDR blocking agent.
  • graft-versus host disease the reaction of the donor immune system in allogeneic transplantation against the- tissue of the recipient, is initiated by a T-cell reaction.
  • T cells in addition to causing GVHD, can also mediate a beneficial graft-versus- leukemia lymphoma (GVL) effect or graft-versus-tumor (GVT) effect to eradicate the malignant clone.
  • GVHD occurs at 3 different time points after transplantation, involving different organs and with different clinical and histopathological pictures.
  • Hyper-acute and acute GVHD develop during and after engraftment till day + 100 post- transplant; an acute inflammation of the recipient's tissue especially involving skin, soft tissue of the whole gastro-intestinal-tract, liver and biliary fract system.
  • amount of dianhea and the value of ALT/AST and bilirabin four different grades are defined as 0-IV.
  • Acute GVHD > II typically needs an intensification of the immunosuppressive therapy and grade TO/TV are often refractory to high dose immunosuppression.
  • Chronic GVHD typically develops after day +100, and usually ensues directly from acute GVHD or during the reduction of the immunosuppression.
  • the tissue of chronic GVHD shows no inflammation but does show a fibrotic or sclerotic appearance.
  • Skin, liver and the GI- tract tissue are involved and additionally: eyes, sino bronchial-system, lung, pancreas or vagina.
  • a reduced quality of life is the result of decreased organ functions. Therefore, avoiding refractory acute and chronic GVHD is the main goal of the rapamycin resistant T cell based therapy before and after transplantation.
  • the added advantage is that the associated increase of risk of infection is not observed as is the case with freatment with immunosuppressive agents, as the rapamycin resistant T cells are fully functional. (See the examples which follow).
  • the rapamycin resistant T cells express CD62L.
  • CD62L mediates lymphocyte homing to high endothelial venules of peripheral lymphoid tissue and leukocyte rolling on activated endothelium at inflammatory sites.
  • CD62L is expressed on the surfaces of most peripheral blood B cells, T cells, monocytes and granulocytes express CD62L.
  • some NK cells express CD62L.
  • CD62L CD62L
  • some spleen lymphocytes, bone manow lymphocytes, bone manow myeloid cells and thymocytes express CD62L
  • certain hematopoietic malignant cells express CD62L.
  • T cells at different stages of maturation or differentiation express surface molecules indicative of that stage or differentiation.
  • memory T cells express CD45RO + .
  • Memory T cells can be expanded (proliferated) without the need of specific antigenic stimulation to maintain the clonal size.
  • Na ⁇ ve T cell repertoires express CD45RA + .
  • CD45RO + CD4 + resting T cells can be cultured with IL- 2 alone or in combination with TNF- ⁇ and EL-6, in the presence of autologous inadiated macrophages and anti-DR antibodies to prevent a ⁇ toreactive responses.
  • Systemic memory T cells are characterized according to the cell surface expression of certain known antigens. Typically, these cells are positive for CD4, and lack expression of CD45RA, and integrin ⁇ 4 ⁇ 7. They are further characterized by expression of CCR4. A subset of cells of interest are common leukocyte antigen positive (CLA*). Verification of the identity of the cells of interest may be performed by any convenient method, including antibody staining and analysis by fluorescence detection, ELISA, etc., reverse transcriptase PCR, transcriptional amplification and hybridization to nucleic acid microarrays, etc. Some memory T cells associated with the skin are known to express CLA. Thus, any type of cell can be identified when necessary. Other systemic memory cells are triggered to adhere to endothelial ICAM-1, by
  • systemic memory T cells are killed by the co- administration of rapamycin to a patient that has received an organ, tissue or cell transplant. Without, wishing to be bound by theory, removal of memory T cells decreases a cell mediated immune rejection of an allograft.
  • rapamycin or a rapamycin derivative compound can be administered together with other agents such as for example, CCR4 blocking agents that prevents triggering of LFA-1 mediated adhesion is useful in the inhibition of graft rejection by preventing the accumulation of memory T cells at the site of graft implantation; preventing infra-islet infiltration by T cells to inhibit development of insulin-dependent diabetes mellitus; blocking infiltration of T cells into the central nervous system to treat multiple sclerosis and other demyelinating diseases; blocking the accumulation of T cells in the synovial joints of patients suffering from rheumatoid arthritis; accumulation of memory T cells to influence immune responsiveness, and the like.
  • CCR4 blocking agents that prevents triggering of LFA-1 mediated adhesion is useful in the inhibition of graft rejection by preventing the accumulation of memory T cells at the site of graft implantation; preventing infra-islet infiltration by T cells to inhibit development of insulin-dependent diabetes mellitus; blocking infiltration of T cells into the central nervous
  • Immune cells express a variety of cell surface molecules which can be detected with either monoclonal antibodies or polyclonal antisera. Immune cells that have undergone differentiation or activation can also be enumerated by staining for the presence of characteristic cell surface proteins by direct immunofluorescence in fixed smears of cultured cells.
  • T lymphocytes at whichever stage of maturity and cell differentiation expressing CD62L can be identified.
  • one such method is by measuring cell phenotypes.
  • the phenotypes of immune cells and any phenotypic changes can be evaluated by flow cytometry after immunofluorescent staining using monoclonal antibodies that will bind membrane proteins characteristic of various immune cell types.
  • a second means of assessing cell differentiation is by measuring cell function. This may be done biochemically, by measuring the expression of enzymes, mRNA's, genes, proteins, or other metabolites within the cell, or secreted from the cell. Bioassays may also be used to measure functional cell differentiation or measure specific antibody production directed at a patient's tumor, tumor cell lines or cells from fresh tumors.
  • rapamycin or a rapamycin derivative enhances the generation of other therapeutic cells such as, for example, dendritic cells, pluripotent stem cells, or hematopoietic stem cells.
  • Rapamycin-generated dendritic cells would, for example, improve cellular immune therapy strategies, as the dendritic cells can be pulsed with tumor or infectious disease antigens to more optimally generate an effective T cell immune response.
  • Purified dendritic cells can be pulsed with (exposed to) antigen, to allow them to take up the antigen in a manner suitable for presentation to other cells of the immune systems. Antigens are classically processed and presented through two pathways.
  • dendritic cells Numerous methods of pulsing dendritic cells with antigen are known; those of skill in the art regard development of suitable methods for a selected antigen as routine experimentation.
  • the antigen is added to cultured dendritic cells under conditions promoting viability of the cells, and the cells are then allowed sufficient time to take up and process the antigen, and express antigen peptides on the cell surface in association with either Class I or Class II MHC, a period of about 24 hours (from about 18 to about 30 hours, preferably 24 hours).
  • Dendritic cells may also be exposed to antigen by transfecting them with DNA encoding the antigen. The DNA is expressed, and the antigen is presumably processed via the cytosolic/Class I pathway.
  • the present invention provides methods of using therapeutic compositions comprising activated, antigen-pulsed dendritic cells.
  • the use of such cells in conjunction with soluble cytokine receptors or cytokines, or other immunoregulatory molecules is also contemplated.
  • the inventive compositions are administered to stimulate an allogeneic immune response, and can be given by bolus injection, continuous infusion, sustained release from implants, or other suitable technique.
  • the cells will be administered in the form of a composition comprising the antigen-pulsed, activated dendritic cells in conjunction with physiologically acceptable carriers, excipients or diluents. Such carriers will be nontoxic to recipients at the dosages and concentrations employed.
  • ex vivo culture and expansion comprises: (1) collecting CD34 + hematopoietic stem and progenitor cells from a patient from peripheral blood harvest or bone manow explants; and (2) expanding such cells ex vivo.
  • ex vivo culture and expansion comprises: (1) collecting CD34 + hematopoietic stem and progenitor cells from a patient from peripheral blood harvest or bone manow explants; and (2) expanding such cells ex vivo.
  • flt3-L Stem or progenitor cells having the CD34 marker constitute only about 1 % to 3% of the mononuclear cells in the bone manow.
  • the amount of CD34 + stem or progenitor cells in the peripheral blood is approximately 10- to 100-fold less than in bone manow.
  • Cytokines such as flt3-L may be used to increase or mobilize the numbers of dendritic cells in vivo. Increasing the quantity of an individual's dendritic cells may facilitate antigen presentation to T cells for antigen(s) that already exists within the patient, such as a tumor antigen, or a bacterial or viral antigen.
  • cytokines may be administered prior to, concurrently with or subsequent to administration of an antigen to an individual for immunization purposes.
  • Peripheral blood cells are collected as described in the Examples which follow or, alternatively, can be using procedures known in the art such as, for example, apheresis procedures. See, for example, Bishop et al., Blood, vol. 83, No. 2, pp. 610- 616 (1994). Briefly, peripheral blood progenitor cells (PBPC) and peripheral blood stem cells (PBSC) are collected using conventional devices, for example, a Haemonetics Model V50 apheresis device (Haemonetics, Braintree, Mass.).
  • PBPC peripheral blood progenitor cells
  • PBSC peripheral blood stem cells
  • MNCVkg mononuclear cells
  • the cells are suspended in standard media and then centrifuged to remove red blood cells and neutrophils. Cells located at the interface between the two phases (the buffy coat) are withdrawn and resuspended in HBSS. The suspended cells are predominantly mononuclear and a substantial portion of the cell mixture are early stem cells.
  • a variety of cell selection techniques are known for identifying and separating CD34 + hematopoietic stem or progenitor cells from a population of cells.
  • monoclonal antibodies or other specific cell binding proteins
  • Several such markers or cell surface antigens for hematopoietic stem cells i.e., flt-3, CD34, My-10, and Thy-1 are known in the art, as are specific binding proteins.
  • antibodies or binding proteins are fixed to a surface, for example, glass beads or flask, magnetic beads, or a suitable chromatography resin, and contacted with the population of cells.
  • the stem cells are then bound to the bead matrix.
  • the binding proteins can be incubated with the cell mixture and the resulting combination contacted with a surface having an affinity for the antibody- cell complex. Undesired cells and cell matter are removed providing a relatively pure population of stem cells.
  • the specific cell binding proteins can also be labeled with a fluorescent label, e.g., chromophore or fluorophore, and the labeled cells separated by sorting. Preferably, isolation is accomplished by an immunoaffinity column.
  • Immunoaffinity columns can take any form, but usually comprise a packed bed reactor.
  • the packed bed in these bioreactors is preferably made of a porous material having a substantially uniform coating of a substrate.
  • the porous material which provides a high surface area-to- volume ratio, allows for the cell mixture to flow over a large contact area while not impeding the flow of cells out of the bed.
  • the substrate should, either by its own properties, or by the addition of a chemical moiety, display high-affinity for a moiety found on the cell-binding protein.
  • Typical substrates include avidin and streptavidin, while other conventional subsfrates can be used.
  • monoclonal antibodies that recognize a cell surface antigen on the cells to be separated are typically further modified to present a biotin moiety.
  • the affinity of biotin for avidin thereby removably secures the monoclonal antibody to the surface of a packed bed (see Berenson, et al., J. Immunol. Meth., 91:11, 1986).
  • the packed bed is washed to remove unbound material, and target cells are released using conventional methods.
  • Immunoaffinity columns of the type described above that utilize biotinylated anti-CD34 monoclonal antibodies secured to an avidin- coated packed bed are described for example, in WO 93/08268.
  • An alternative means of selecting the quiescent stem cells is to induce cell death in the dividing, more lineage-committed, cell types using an antimetabolite such as 5- fluorouracil (5-FU) or an alkylating, agent such as 4-hydroxycyclophosphamide (4- HC).
  • the non-quiescent cells are stimulated to proliferate and differentiate by the addition of growth factors that have little or no effect on the stem cells, causing the non- stem cells to proliferate and differentiate and making them more vulnerable to the cytotoxic effects of 5-FU or 4-HC. See Berardi et al., Science, 267:104 (1995), which is incorporated herein by reference.
  • Isolated stem cells can be frozen in a controlled rate freezer (e.g., Cryo-Med, Mt.
  • a variety of growth and culture media can be used for the growth and culture of dendritic cells (fresh or frozen), including serum- depleted or serum-based media.
  • Useful growth media include RPMI, TC 199, Iscoves modified Dulbecco's medium (Iscove, et al., F. J. Exp. Med., 147:923 (1978)), DMEM, Fischer's, alpha medium, NCTC, F-10, Leibovitz's L-15, MEM and McCoy's.
  • Particular nutrients present in the media include serum albumin, fransferrin, lipids, cholesterol, a reducing agent such as 2-merca ⁇ toethanoI or monothioglycerol, pyruvate, buryrate, and a glucocorticoid such as hydrocortisone 2-hemisuccinate.
  • the standard media includes an energy source, vitamins or other cell-supporting organic compounds, a buffer such as HEPES, or Tris, that acts to stabilize the pH of the media, and various inorganic salts.
  • a variety of serum-free cellular growth media is described in WO 95/00632, which is incorporated herein by reference.
  • the collected CD34 + cells are cultured with suitable cytokines, for example, as described herein.
  • CD34 cells then are allowed to differentiate and commit to cells of the dendritic lineage. These cells are then further purified by flow cytometry or similar means, using markers characteristic of dendritic cells, such as CDla, HLA DR, CD80 and/or CD86.
  • the cultured dendritic cells are exposed to an antigen, for example, an allogeneic class I HLA molecule, allowed to process the antigen, and then cultured with an amount of a CD40 binding protein to activate the dendritic cell.
  • the dendritic cells are transfected with a gene encoding an allogeneic HLA class I molecule or immune related receptors, and then cultured with an amount of a CD40 binding protein to activate the antigen- presenting dendritic cells.
  • the activated, antigen-carrying dendritic cells are them administered to an individual in order to stimulate an antigen-specific immune response.
  • the dendritic cells can be administered prior to, concurrently with, or subsequent to, antigen administration.
  • T cells may be collected from the individual and exposed to the activated, antigen-carrying dendritic cells in vitro to stimulate antigen-specific T cells, which are administered to the individual.
  • Rapamycin-generated pluripotent stem cells would have particular application for stem cell therapy, which includes for example, the treatment of a wide variety of diseases such as Parkinson's Disease, post cerebral vascular accident neurological deficiency, type I diabetes mellitus, and post myocardial infarction deficiency. Rapamycin-generated hematopoietic stem cells would have particular application to the use of hematopoietic stem cell transplantation, which includes therapeutic application for the freatment of immune deficiency syndromes, auto-immune disease, hematologic malignancy, and solid tumors, hi each of these embodiments detailed in this invention, the relevant starting cell population is, for example, precursor monocytes or hematopoietic stem cells in the case of dendritic cell therapy.
  • highly purified pluripotent stem cells if the desired cell is for use in stem cell therapy.
  • CD34+ hematopoietic stem cells are used in the case of hematopoietic stem cell therapy.
  • the cells are placed into in vitro culture conditions, described herein, in the presence of rapamycin.
  • the cell culture in the presence of rapamycin is performed in the presence of suitable cytokines.
  • the dendritic cells are preferably propagated in cytokines such as IL-4 and GM-CSF.
  • cytokine additives to the culture comprise, for example, recombinant stem cell factor, IL-3, IL-6, GM-CSF, G-CSF, IL-7, or other recombinant cytokines.
  • a precursor cell population includes cells of a mesodermal derived cellular lineage, more particularly of hematopoietic lineage, endothelial lineage, muscle cell lineage, epithelial cell lineage and neural cell lineage.
  • a “precursor cell” can be any cell in a cell differentiation pathway that is capable of differentiating into a more mature cell.
  • the term “precursor cell population” refers to a group of cells capable of developing into a more mature cell.
  • a precursor cell population can comprise cells that are totipotent, cells that are pluripotent and cells that are stem cell lineage restricted (i.e. cells capable of developing into less than all hematopoietic lineages, or into, for example, only cells of erythroid lineage).
  • the term “totipotent cell” refers to a cell capable of developing into all lineages of cells.
  • the term “totipotent population of cells” refers to a composition of cells capable of developing into all lineages of cells.
  • pluripotent cell refers to a cell capable of developing into a variety (albeit not all) lineages and are at least able to develop into all hematopoietic lineages (e.g., lymphoid, erythroid, and thrombocytic lineages).
  • hematopoietic lineages e.g., lymphoid, erythroid, and thrombocytic lineages.
  • a pluripotent cell can differ from a totipotent cell by having the ability to develop into all cell lineages except endothelial cells.
  • a "pluripotent population of cells” refers to a composition of cells capable of developing into less than all lineages of cells but at least into all hematopoietic lineages.
  • a totipotent cell or composition of cells is less developed than a pluripotent cell or compositions of cells.
  • the terms “develop”, “differentiate” and “mature” all refer to the progression of a cell from the stage of having the potential to differentiate into at least two different cellular lineages to becoming a specialized cell. Such terms can be used interchangeably for the purposes of the present application.
  • the term “population” refers to cells having the same or different identifying characteristics.
  • the term “lineage” refers to all of the stages of the development of a cell type, from the earliest precursor cell to a completely mature cell (i.e. a specialized cell).
  • Preferred cells within a stem cell population of the present invention include cells of at least one of the following cellular lineages: hematopoietic cell lineage, erythroid lineage, endothelial lineage, leukocyte lineage, thrombocyte lineage, erythroid lineage (including primitive and definitive erythroid lineages), macrophage lineage, neutrophil lineage, mast cell lineage, megakaryocyte lineage, natural killer cell lineage, eosinophil lineage, T cell lineage, endothelial cell lineage and B cell lineage.
  • Monoclonal antibodies are particularly useful for identifying markers associated with particular cell lineages and/or stages of differentiation.
  • a large proportion of terminally differentiated cells may be removed by initially using a "relatively crude” separation.
  • magnetic bead separations may be used initially to remove large numbers of lineage committed cells. Desirably, at least about 80%, usually at least 70% of the total hematopoietic cells will be removed.
  • Procedures for separation may include but are not limited to, magnetic separation, using antibody-coated magnetic beads, affinity chromatography, cytotoxic agents joined to a monoclonal antibody or used in conjunction with a monoclonal antibody, including but not limited to, complement and cytotoxins, and "panning" with antibody attached to a solid matrix, e.g., plate, elutriation or any other convenient technique.
  • Techniques providing accurate separation include but are not limited to, flow cytometry, which can have varying degrees of sophistication, e.g., a plurality of color channels, low angle and obtuse light scattering detecting channels, impedance channels, etc.
  • cells are isolated and purified cell from a sample, patient or donor individual and are used in functional assays to determine any properties of the cells.
  • appropriate functional assays known in the art can be conducted. For example, if the population of cells are T cells specific for a desired antigen such as a tumor antigen, cytotoxic T cell assays, T cell proliferation assays, cytokine profiles, determination of surface antigens for T cell maturity or memory T cells, etc., can be carried out.
  • peripheral blood mononuclear cells can be obtained from a subject and isolated by density gradient centrifugation, e.g., with Ficoll/Hypaque.
  • Specific cell populations can be depleted or enriched using standard methods.
  • monocytes/macrophages can be isolated by adherence on plastic.
  • T cells or B cells can be enriched or depleted, for example, by positive and/or negative selection using antibodies to T cell or B cell surface markers, for example by incubating cells with a specific primary monoclonal antibody (mAb), followed by isolation of cells that bind the mAb using magnetic beads coated with a secondary antibody that binds the primary mAb.
  • mAb primary monoclonal antibody
  • Peripheral blood or bone manow derived hematopoietic stem cells can be isolated by similar techniques using stem cell-specific mAbs (e.g., anti-CD34 mAbs). Specific cell populations can also be isolated by fluorescence activated cell sorting according to standard methods. Monoclonal antibodies to cell-specific surface markers known in the art and many are commercially available. If desired, a large proportion of terminally differentiated cells may be removed by initially using a "relatively crude" separation. For example, magnetic bead separations maybe used initially to remove large numbers of lineage committed cells. Desirably, at least about 80%, usually at least 70% of the total hematopoietic cells can be removed.
  • Procedures for separation may include but are not limited to, magnetic separation, using antibody-coated magnetic beads, affinity chromatography, cytotoxic agents joined to a monoclonal antibody or used in conjunction with a monoclonal antibody, including but not limited to, complement and cytotoxins, and "panning" with antibody attached to a solid matrix, e.g., plate, elutriation or any other convenient technique.
  • Techniques providing accurate separation include but are not limited to, flow cytometry, which can have varying degrees of sophistication, e.g., a plurality of color channels, low angle and obtuse light scattering detecting channels, impedance channels, etc.
  • rapamycin resistant allogeneic cells are administered to a patient.
  • Allogeneic cells may be derived from any person and comprise both CD4 + and CD8 + T cells. Cells are treated with the desired cytokines and rapamycin prior to administering to a patient.
  • An advantage of the present invention is that the peripheral pool of memory T cells (CD45RO 4 ) are susceptible to rapamycin or a rapamycin derivative compound and are inhibited, thereby decreasing the risk of GVHD. Conversely, the naive T cell repertoire (CD45RA 4 ) is maintained. For example, to evaluate the frequency of resting T cells with memory phenotype that could be stimulated by cytokines to grow, limiting dilution experiments can be performed. CD45RO + CD4 + resting T cells can be cultured with E -2 alone or in combination with TNF- ⁇ and IL-6, in the presence of autologous inadiated macrophages and anti-DR antibodies to prevent autoreactive responses.
  • the in vitro expansion of immune T cells with a more na ⁇ ve phenotype may be particularly applicable to the therapy of autoimmune disease.
  • patients with autoimmune disease may undergo apheresis to isolate T cells, have T cells expanded in rapamycin to enrich for a na ⁇ ve T cell phenotype, receive immune depleting chemotherapy to eliminate autoreactive T cell clones in vivo, and then receive infusion of in vitro generated T cells from immune reconstitution with a T cell source less likely to reconstitute autoimmunity (T cells with characteristics more typical of na ⁇ ve T cells; i.e., CD28 + , CD62L 4 ).
  • the allogeneic cells contained in the medicament of the invention may assume any formation.
  • the allogeneic cells suspended in an adequate solution may be used.
  • the solution containing the allogeneic cells can desirably be used as an injection or drip-feed solution.
  • an injection or drip-feed solution which is prepared by suspending the allogeneic cells in physiological saline and so on containing about 0.01% to 5% of human serum albumin.
  • the allogeneic cells or the preparations containing them may be frozen and kept in their frozen state so as to be used for remedying or preventing various disease. Cryopreservation should be performed under liquid nitrogen conditions, preferably in solutions that preserve immune T cell function, such as reduced DMSO concentrations of 5% and addition of cryopreseryant molecules such as pentastarch.
  • the medicaments according to the invention can desirably be administered to a patient by an intravenous drip, arterial injection, local injection and the like.
  • the desirable dosage of the medical solution varies in accordance with the way or place of the administration thereof. However, it is commonly desirable to administer at least about 50 to about 500 ml of the medical solution containing the allogeneic cells in the aforesaid ratio to the patient. It is preferable that the medical solution is administered one time a day to one time a month. In any event, at least one administration of the medicament comprising the allogeneic cells should be made.
  • the T cells are administered at the time of the HSCT (within 24 hours of stem cell infusion), and can be administered at the time of any other donor T cell infusion, for example, at the time of a donor lymphocyte infusion (DLI).
  • HSCT single cell infusion
  • DAI donor lymphocyte infusion
  • the dosage of the allogeneic cells contained, as the main ingredient, in the medicament of the invention may be arbitrarily decided in accordance with the condition of the patient and/or the clinical procedure. In general, about 1x10 to about IxlO 9 allogeneic cells per kilogram of patient's weight may be used.
  • the extraction of cells from the donor may be performed any way, for example, by blood collection, pheresis, or other possible operations. It is desirable to draw blood from the vein of the donor, and add heparin or citric acid to the blood thus drawn to prevent blood coagulation.
  • the blood of the order of 0.01 ml to 100 ml is generally drawn in one blood extraction operation, but the amount of the blood to be drawn is not limited in the invention. Taking into consideration the physical burden of the donor, labors involved in collecting the blood, and troublesome operations for separating the lymphocyte cells, it is desirable to draw the blood by 5 ml to 10 ml, preferably 10 ml to 20 ml in one blood extraction operation. For most clinical applications, harvest of sufficient numbers of autologous or allogeneic T cells will require an outpatient apheresis procedure.
  • the operation for separating the lymphocyte cells from the blood drawn in the aforementioned manner may be accomplished by a known method for separating lymphocyte cells such as a discontinuous density gradient centrifugation method which is performed by using sucrose or lymphocyte separating agents on the market.
  • the apheresis product can be subjected to counterflow centrifugal elutriation as a mechanism to enrich for lymphocyte populations.
  • lymphocytes can be enriched for the desired T cell subset by negative or positive selection using antibodies and selection beads or selection columns.
  • the type of the anti-CD3 antibodies- used in the invention is not limited to a specific antibody, as far as the antibody makes for proliferation and activation of the desired lymphocyte cells.
  • the anti-CD3 antibodies used for stimulating the lymphocyte cells are possibly yielded in organisms or organic cells by use of refined CD3 molecules.
  • the culture medium solution for cultivating the desired cells there may be used a culture medium derived from a living organism or a culture medium composed by mixing amino acid, vitamins, nucleic acid base and the like with equilibrium salt solution.
  • the culture medium RPMI-1640, AJM-V, DMEM, IMDM, X-Vivo 15, or X-Vivo 20 or the like are preferable.
  • the culture medium of X-Vivo 20 is particularly recommended for expansion of human T cells under the conditions identified here.
  • Such media is further supplemented by the addition of 5% autologous plasma, or 5% human A B serum.
  • the cultivation of the desired cells may be fulfilled by common cell-cultivating methods. For example, it can be carried out in a CO 2 -incubator at a CO 2 concentration of about 1% to about 10%, preferably about 5%, at a temperature of 30° C. to 40° C, most prefened at about 37° C.
  • the number of days which the cultivation takes place is not specifically restricted, but it is desirable to allow about 2 to about 20 days. For the human condition, a period of about 20 days appears sufficient to achieve the desired T cell cytokine phenotype and to achieve clinically relevant T cell numbers.
  • Such cells appear to be stable, with appropriate re-stimulation with anti-CD3 and anti-CD28 molecules, for several weeks after day 20, and such an expansion may prove valuable in some circumstances that require increased cell number or further in vitro modifications.
  • the proliferation of the cells does not appreciably take place within about 1 to about 2 days after commencement of the cultivation, but is generally observed about 3 to six days after the commencement.
  • the color of the culture medium solution will be changed from orange to yellow.
  • the culture medium is supplemented at about 0.1 to about 5 times the culture solution initially given. It is prefened to monitor the cell number and median cell volume by Coulter Multisizer evaluation daily, as this approach allows accurate determination of T cell expansion and T cell activation.
  • Murine CD4 + splenic T cells from C57B1/6 mice were purified to > 98% purity by negative selection using anti-macrophage, anti-B cell, anti-CD8 cell, and anti- granulocyte antibodies (StemCell Technologies; murine CD4+ T cell enrichment procedure).
  • Murine CD4 cells were plated at a concentration of 0.2 x 10 6 cells/ml in RPMI-1640 media supplemented with 10% fetal calf serum (Gemini Bioproducts).
  • CD4 + cells were stimulated with magnetic beads (tosylated beads; Dynal) that were coated with anti-murine CD3 (PharMingen) and anti-murine CD28 (PharMingen) at a T cell to bead ratio of 1 :3.
  • magnetic beads tosylated beads; Dynal
  • anti-murine CD3 PharMingen
  • anti-murine CD28 PharMingen
  • Media in the Thl condition consisted of recombinant murine IL-12 (2.5 ng/ml; R and D Systems), anti-murine IL-4 neutralizing antibody (clone 11B11; 10 micrograms/ml), recombinant human IL-2 (20 LUJml; Chiron), recombinant human IL-7 (20 ng/ml; Peprotech), and the anti-oxidant N-acetyl cysteine (NAC; 3.3 ⁇ M).
  • murine IL-12 2.5 ng/ml; R and D Systems
  • anti-murine IL-4 neutralizing antibody clone 11B11; 10 micrograms/ml
  • recombinant human IL-2 (20 LUJml; Chiron
  • recombinant human IL-7 (20 ng/ml; Peprotech
  • NAC N-acetyl cysteine
  • Media in the Th2 condition consisted of recombinant murine IL-4 (10001.UJml; Peprotech), recombinant human IL-2 (20 LUJml), recombinant human IL-7 (20 ng/ml), and 3.3 ⁇ M NAC.
  • Immune suppression molecules cyclosporine A (CSA), FK506, and rapamycin were purchased from Sigma and reconstituted according to the manufacturers instructions, with rapamycin and FK506 being tested at 0.004 ⁇ M and 0.02 ⁇ M concentrations and CSA being tested at 0.04 ⁇ M and 0.2 ⁇ M concentrations.
  • Media containing IL-2, IL-7, NAC, and the particular immune suppression agent was added to maintain cell concenfration at between 0.2 and 1.0 x 10 6 cells/ml throughout the culture interval. Cells were counted by a Multi-Sizer Instrument (Coulter), and CD4 expansion is plotted, as shown in Figure 1.
  • the donor undergoes a 2 to 5 liter apheresis procedure using a CS-3000 or an equivalent machine.
  • the apheresis product is subjected to counterflow centrifugal elutriation by standard operating procedures of the NIH Department of Transfusion Medicine, Cell Processing Section.
  • the lymphocyte fraction of the elutriation product (120 to 140 fraction) is depleted of B cells by incubation with an anti-B cell antibody (anti-CD20; Nexell) and an anti-CD8 antibody (Nexell) and sheep anti-mouse magnetic beads (Dynal; obtained through Nexell) by standard operating procedures using the MaxCep Device (Nexell).
  • Flow cytometry will be performed to document that CD8 + T cell contamination is ⁇ 1%.
  • the resultant CD4-enriched donor lymphocyte product can be cryopreserved in aliquots of 50 to 200 x 10 6 cells/vial. Sterility of the population is not tested at this early stage of the Th2 cell generation procedure; such testing occurs after final co-culture of donor CD4 cells.
  • the donor will receive filgrastim as an outpatient (10 ⁇ g/kg/day each morning; subcutaneously) for 5, 6, or 7 days.
  • the donor should take the filgrastim as early as possible upon awakening in the morning. This is especially important on days 5, 6, and 7 of the injections.
  • Apheresis is typically performed on days 5 and 6 of this regimen. On some occasions, sufficient numbers of CD34 + cells might be obtained with a single apheresis on day 5; on other occasions, it may be necessary to perform apheresis on days 5, 6, and 7 to reach the target CD34 + cell number f 4 x 10 6 per kg).
  • the donor is instructed to take filgrastim for the complete 7 day period, unless notified by the transplant team that adequate CD34 cells were harvested before day 7. If > 3 x 10 CD34+ cells per kg are harvested after apheresis on days 5, 6, and 7, no further mobilization or apheresis is performed, and the patient is eligible to receive the stem cell transplant with that dose of CD34+ cells.
  • the apheresis procedure typically uses ACD-A anti-coagulant; alternatively, partial anti-coagulation with heparin may be utilized.
  • the apheresis product can be cryopreserved and stored at -180 degrees Celsius in a solution containing Plasmalyte A, Pentastarch, human serum albumin, DMSO, and preservative free heparin (10 U/ml).
  • the concentration of CD34 + cells in the apheresis product is determined by flow cytometry, and the number of CD34 + cells in each cryopreserved bag calculated. If the donor and host are ABO incompatible, red blood cells will be depleted from the stem cell product by standard protocols.
  • donor CD4 + T cells are resuspended to a concentration of 0.3 x 10 6 cells per ml.
  • Media consist of X-Vivo 20 supplemented with 5% heat-inactivated autologous plasma.
  • the donor CD4 + T cells are cultured in filtered flasks at 37° C in 5% CO 2 humidified incubators.
  • T cells are stimulated with anti-CD3/anti-CD28 coated magnetic beads (3 to 1 ratio of beads to T cells).
  • the following reagents are added: recombinant human IL-4 (Shering IL-4; 1000 LU.
  • IL-2 purchased from Chiron Therapeutics; 20 LU. per ml
  • cells are maintained at a concentration of 0.25 to 1.0 x 10 6 cells per ml by the addition of fresh X-Vivo 20 media supplemented with autologous plasma (5%), IL-2 (20 LUJml), and IL-4 (1000 LUJml).
  • the median cell volume is determined using a Multisizer II instrument (Coulter).
  • the T cell volume approaches 500 fl (acceptable range of 650 to 350)
  • the T cells are restimulated with anti-CD3/anti-CD28 beads; typically, this time of restimulation will be after 8 to 12 days of culture.
  • Bead restimulation is at a bead to T cell ratio of 3:1.
  • T cell concentration is 0.2 x 10 cells/ml.
  • Media consists of X-Vivo 20 supplemented with autologous plasma (5%), IL-2 (20 LUJml), and IL-4 (10001.U./ml).
  • CD4 cells are maintained at a concentration of 0.25 to 1.0 x 10 6 cells per ml by the addition of fresh X-Vivo 20 media supplemented with autologous plasma (5%), IL-2 (20 LUJml), and IL-4 (1000 LUJml).
  • Rapamycin (commercially available oral solution; Sirolimus, Wyeth-Ayerst) is added to the Th2 culture condition at day 0 at a concenfration of 1 micromolar. For some donors who are particularly sensitive to the effects of rapamycin, it may be necessary to initiate culture in lower doses of rapamycin, such as 0.01 to 0.1 micromolar.
  • the Th2 culture media is expanded for the purposes of cytokine addition or maintenance of cell concentration at 0.2 to 1.0 x 10 6 cells/ml, the media added to culture should be replete with rapamycin, and contain a concenfration of rapamycin between 0.01 and 1.0 micromolar. The highest concentration of rapamycin that allows CD4 Th2 cell expansion should be utilized.
  • Th2 generation in rapamycin it is typically not necessary to restimulate the CD4 cells with anti-CD3 and anti-CD28, as the cells have attained a purified Th2 phenotype after only one round of CD3, CD28 co- stimulation.
  • This methodology therefore allows rapid and uncomplicated generation of Th2 cells and represents a technical advance that allows Th2 generation with reduced reagent utilization and reduced labor.
  • the CD4 cell mean cell volume approaches 500 fl (acceptable range of 650 to 350), the cells are harvested and cryopreserved.
  • Th2 Cell Administration a) On day 1 of the transplant procedure, the cryopreserved donor Th2 cells are thawed and immediately administered intravenously. b) No steroids are allowed in the management of DMSO-related toxicities (chills, muscle aches) that may occur immediately after cellular infusion (diphenhydramine and meperidine are allowed). c) The determination of whether a Th2 cell infusion is safe will be based on the presence or absence of hyperacute GVHD and of any grade 4 or 5 toxicity attributable to the Th2 cells that occurs in the first 14 days post-transplant.
  • Hyperacute GVHD is defined as a severe level of acute GNHD (grade IH or IV) that occurs within the first 14 days post-transplan e)
  • Th2 cell dose level #1 (5 x 10 6 Th2 cells/kg). If no hyperacute GVHD or grade 4 or 5 toxicity attributable to the Th2 cells is observed in these initial three patients, then it will be determined that this dose level is safe, and accraal to dose level #2 will commence. If hyperacute GVHD or grade 4 or 5 toxicity attributable to the Th2 cells is observed in any of the initial three patients, then accraal to dose level #1 will be expanded to include a total of six patients.
  • Th2 cell dose level #2 If it is determined that Th2 cell dose level #2 is safe, accrual to the final dose level #3 will start (Th2 cell dose of 1.25 x 10 8 cells/kg). Six patients in total will be evaluated on dose level #3. If more than one patient on dose level #3 develops hyperacute GVHD or grade 4 or 5 toxicity attributable to the Th2 cells, then accrual to dose level #3 will stop. h) In the phase II component of this study, eighteen (18) additional patients will receive Th2 cells at either dose level #2 or level #3. To help ensure that the TJ- ⁇ cells continue to be safely administered in this expanded cohort, the same accrual and stopping rules pertaining to severe toxicity attributed to the Th2 cells will be continued.
  • Example I CD4 + Thl /Th2 Modulation.
  • Use of the calcineurin inhibitors cyclosporine A or FK506 is a standard component of immune suppression after allogeneic PBSCT. Given the known role of Thl/Th2 biology in the modulation of immunity post-SCT, it is an important goal to identify any differential influence of these two agents on the Thl/Th2 balance.
  • rapamycin is an immune suppression agent that has been studied in murine models, and more recently, in clinical trials of allogeneic PBSCT.
  • Rapamycin by binding to the mammalian target of rapamycin, controls multiple aspects of T cell metabolism, including phosphorylation of Rb protein with subsequent regulation of cyclin dependent kinases and control of protein franslation via the 14-3-3 pathway.
  • the mechanism of action of rapamycin stands in stark contrast to that of CSA and FK506, which work primarily through inhibition of cytokine and other molecule mRNA transcription.
  • CSA and FK506 work primarily through inhibition of cytokine and other molecule mRNA transcription.
  • FIG. 1 results are shown that illustrate the differential effect of CSA, FK506, and rapamycin on the generation of murine CD4 + Thl and Th2 cells.
  • Murine CD4 cells were purified, stimulated in a polyclonal manner with anti-CD3 and anti- CD28 antibodies, and propagated in culture conditions that promote either Thl or Th2 differentiation.
  • media was supplemented with IL-12, antibody to IL- 4, IL-2, IL-7, and the cell death inhibitor N-acetyl cysteine;
  • Th2 cultures media was supplemented with IL-4, IL-2, IL-7, and NAC.
  • control Thl and Th2 cell cultures expanded approximately 2 to 3 logs in six days.
  • Thl or Th2 expansion in the presence of either 0.004 ⁇ M or 0.02 ⁇ M of FK506 was associated with a dramatic reduction in CD4 cell expansion under these optimized conditions; in both Thl or Th2 conditions, there was only a two to three fold increase in CD4 cell number in the presence of FK506.
  • Presence of CSA at either 0.04 ⁇ M or 0.2 ⁇ M concentrations yielded a similar inhibition of Thl and Th2 expansion. As such, there did not appear to be any preferential Th2 or Thl generation with the calcineurin inhibitors.
  • Example 2 Evaluation of Immunosuppressive agents on Th2 Responses.
  • CSA CSA, FK506, and rapamycin
  • the CD4 cells were harvested from culture, washed, normalized to a concentration of 0.5 x 10 cells/ml, and re- stimulated with anti-CD3 and anti-CD28 for supernatant generation.
  • T cells were harvested, washed, normalized to a concentration of 0.5 x 10 cells/ml, and re-stimulated with anti-CD3 and anti-CD28 (3:1 bead to T cell ratio) for 24 hours to generate a supernatant.
  • Th2 expansion in the presence of rapamycin resulted in Th2 cells with similar IL-4 and IL-10 secretion relative to the control.
  • rapamycin, but not CSA or FK506, facilitated or maintained Th2 cell generation, both with regards to CD4 + cell expansion and effector Th2 cytokine production.
  • Example 3 Evaluation of Immunosuppressive agents on Thl Responses.
  • CSA CSA, FK506, and rapamycin
  • the Thl cultured cells were also re-stimulated with anti-CD3, anti- CD28, and the supernatant was tested for the type I cytokines IL-2 and IFN- ⁇ .
  • Murine CD4 + cells were expanded under the Thl culture condition using anti-CD3, anti-CD28 co-stimulation.
  • T cells were harvested, washed, normalized to a concentration of 0.5 x 10 6 cells/ml, and re-stimulated with anti-CD3 and anti-CD28 (3:1 bead to T cell ratio) for 24 hours to generate a supernatant.
  • Culture supematants were tested for IL-2 and IFN- ⁇ content by a ' two site ELISA (BioSource).
  • Experimental samples scored relative to a standard curve generated from evaluation of recombinant murine IL-2 and IFN- ⁇ .
  • Cell culture labels along the x-axis represent cytokine and immune suppression agent conditions during the initial six days of T cell generation; there were no cytokines or immune suppression agents added during the time of 24 hour supernatant generation.
  • CSA and in particular, FK506, resulted in Thl cells with significantly diminished capacity for both IL-2 and IFN- ⁇ secretion.
  • Thl expansion in the presence of rapamycin resulted in a dramatic increase in Thl cell capacity for IFN- ⁇ secretion, and a nominal increase in IL-2 secretion capacity.
  • rapamycin, but not CSA or FK506, facilitated or maintained Thl cell generation, both with regards to CD4 + expansion and effector Thl cytokine production.
  • rapamycin although it has been associated with a type II cytokine immune shift upon in vivo administration, does not appear to induce a Thl to Th2 shift directly upon CD4 + cells. This observation implies that rapamycin induced type II promotion may operate indirectly, for example, through its actions on APC modulation.
  • rapamycin unexpectedly preserved or enriched for Thl/Th2 polarity
  • a five-fold higher concentration of rapamycin, 0.1 ⁇ M was evaluated.
  • Murine CD4 + cells were co-stimulated with anti-CD3 and anti-CD28 coated magnetic beads under Thl or Th2 conditions at previously tested rapamycin concentrations (0.008 ⁇ M to 0.02 ⁇ M), and a relatively high concenfration (0.1 ⁇ M).
  • Cell expansion was monitored over the six day culture by Multi-Sizer evaluation, and plotted on a log scale.
  • FIG 4 shows that even at this higher dose of rapamycin, the method of optimized co-stimulation and cytokine supplementation disclosed herein, allowed for the expansion of either Thl or Th2 subsets without any apparent reduction in CD4 cell yield.
  • Example 4 Evaluation of Thl and Th2 Responses Generated in Rapamycin. The Thl or Th2 populations generated in the 0.1 ⁇ M rapamycin concenfration were also evaluated.
  • Murine CD4 + cells were expanded with anti-CD3, anti-CD28 coated magnetic beads under the Thl or the Th2 culture conditions in the absence or presence of rapamycin (0.008 ⁇ M to 0.1 ⁇ M), as denoted on the x-axis of this figure. On day 6 of culture, the T cells were harvested, washed, and restimulated with fresh
  • CD3, CD28 coated beads (3:1 bead to T cell ratio) in media not containing cytokines or immune suppression agent.
  • a 24 hour culture supernatant was generated and tested for IL-2 and IFN- ⁇ cytokine content by two site ELISA (BioSource) in reference to a standard curve.
  • Thl cells in each of the rapamycin concentrations had similarly high secretion of both IL-2 and IFN- ⁇ .
  • Th2 cell expansion in rapamycin it was observed that expansion in the 0.1 ⁇ M rapamycin concenfration was associated with elimination of the "contaminating" quantities of IL-2 secretion that were present in the lower dose rapamycin cultures and the control Th2 cultures.
  • the higher dose of rapamycin was associated with an improved Th2 phenotype, as defined by reduced IL-2 secretion.
  • FIG. 6 shows that Th2 cells propagated in the 0.1 ⁇ M rapamycin concenfration had preservation of capacity for secretion of the type II cytokines IL-4, IL-5, and DL-10. Thl cells propagated in 0.1 ⁇ M rapamycin did not have an increased capacity for type II cytokine secretion.
  • murine CD4 + cells were expanded with anti-CD3, anti-CD28 coated magnetic beads under the Thl or the Th2 culture conditions in the absence or presence of rapamycin (0.008 ⁇ M to 0.1 ⁇ M), as denoted on the x-axis of this figure.
  • the T cells were harvested, washed, and restimulated with fresh CD3, CD28 coated beads (3:1 bead to T cell ratio) in media not containing cytokines or immune suppression agent.
  • a 24 hour culture supernatant was generated and tested for type II cytokine content (IL-4, IL-5, and IL-10) by two site ELISA (BioSource) in reference to a standard curve.
  • Example 5 Cytokine Production after Rapamycin Exposure without Co-stimulation
  • CD28 signaling perhaps through up-regulation of survival molecules such as bcl-2 family members or activation of the AKT pathway, might account for the observed capacity to overcome the expected rapamycin immune T cell suppression effect
  • the experiments were performed evaluating the polarizing cytokine conditions and rapamycin exposure after activation without co-stimulation through use of beads conjugated with only anti- CD3 antibodies.
  • Murine CD4 + T cells were expanded with magnetic beads conjugated with only the T cell receptor activating antibody anti-CD3 or with beads conjugated with both anti-CD3 and anti-CD28 (denoted in figure by Thl or Th2 condition).
  • the condition receiving only anti-CD3 stimulation was performed either with or without the addition of rapamycin (0.02 ⁇ M concentration).
  • Cell expansion was monitored over the six day culture by Multi-Sizer evaluation, and plotted on a log scale.
  • Figure 7 shows, Th2, and in particular, Thl cell expansion was greatly reduced relative to CD3, CD28 co-stimulated control Thl/Th2 cultures.
  • addition of rapamycin to "signal 1 only" generated Thl or Th2 cultures did not result in a significant decrease in CD4 + cell yield. This result suggests, at least at the 0.02 ⁇ M rapamycin concentration tested, that CD28 in this system may not provide a specific T cell activation or survival signal for the abrogation of the expected rapamycin T cell inhibition effects.
  • Example 6 Evaluation of Thl/ Th2 Differentiation Generated in High Doses of Rapamycin. Higher dose levels of rapamycin during Thl Th2 differentiation were evaluated.
  • murine CD4 + cells were expanded in the Th2 culture condition using anti-CD3 and anti-CD28 coated magnetic beads, with culture performed either in the absence or presence of rapamycin (0.1 ⁇ M to 10 ⁇ M). Cell expansion was monitored over the six day culture by Multi-Sizer evaluation, and plotted on a log scale.
  • CD4 + cells in each of the Th2 culture conditions were replated with normalization of T cell concentrations, and further expanded in media containing both the Th2 culture condition additives and rapamycin at the same concentrations as during culture initiation. Cell expansion was monitored from day 6 to day 9 of culture by Multi-Sizer evaluation, and plotted on a log scale.
  • FIG 8 shows CD3, CD28 co-stimulation, generated Th2 cell expansion from day 0 to 6 of culture at rapamycin concentrations ranging from 0.1 ⁇ M to 10.0 ⁇ M (left panel).
  • Th2 cell expansion was reduced approximately one log relative to the control culture.
  • cultures were each normalized for cell number on day 6 of culture, and propagated an additional three days in cytokine replete media (no further CD3, CD28 re-stimulation; Figure 8, right panel).
  • the Th2 cultures initiated and continued in the higher concentration of rapamycin had an increase in CD4 + cell expansion during day 6 to day 9 culture interval relative to the confrol Th2 culture.
  • the increased CD4 + expansion in the high dose rapamycin cultures was also observed after day 6 anti-CD3 and anti-CD28 re-stimulation of cultures (with ongoing cytokine and rapamycin addition; result in Figure 9).
  • Th2 cultures generated in concentrations of rapamycin ranging from 0.1 to 10.0 ⁇ M were also evaluated for their Thl/Th2 cytokine secretion capacity (Figure 10).
  • the Th2 cells had an enhanced Th2 polarity on the basis of abrogation of contaminating IL- 2 secretion and modest reduction in IFN- ⁇ secretion.
  • Such high-dose rapamycin generated Th2 cells had full preservation of the Th2-type cytokines associated with more proximal Th2 effector function, namely IL-4 and IL-5.
  • Th2-type cytokines associated with more distal Th2 effector function, IL-10 and EL-13 were significant reduction in the Th2-type cytokines associated with more distal Th2 effector function, IL-10 and EL-13.
  • these results indicate that the high-dose rapamycin facilitated generation of a Th2 cell of enhanced purity (less Thl contaminating elements) that was more proximal in its state of Th2 differentiation.
  • rapamycin To address the possibility that high dose rapamycin might be selecting for a more na ⁇ ve CD4 cell in culture, an evaluation of surface markers characteristic of na ⁇ ve vs. memory function was conducted.
  • One such functional marker is CD28 itself, which is present on nearly all na ⁇ ve CD4 + cells, only to be reduced during the end stages of memory CD4 + effector differentiation.
  • Figure 12 demonstrates that CD28 indeed was greatly increased on the CD4 + cells propagated in high dose rapamycin. This result demonstrates that rapamycin, and in particular, the high dose rapamycin conditions, select for a more na ⁇ ve CD4 + cell phenotype that expands during CD3, CD28 co-stimulation and thereby attains an increased purity of Th2 polarity.
  • CD62L that functionally helps determine na ⁇ ve vs. memory CD4 cell function.
  • CD62 which is primarily expressed by more na ⁇ ve CD4 cells, dictates T cell lymph node homing capacity rather than tissue-based effector function.
  • Th2 cells expanded in rapamycin, in particular, high dose rapamycin had an increased expression of CD62L.
  • Another cell surface molecule evaluated in these cultures was CD40L.
  • CD40L is not so much a characteristic of na ⁇ ve vs. memory status, but rather is a marker for Thl/Th2 polarity. That is, in our prior results, Thl-type cells have significantly increased CD40L relative to Th2-type cells.
  • T cell activation patterns of murine CD4 + and CD8 + T cells under Thl/Th2 or Tcl/Tc2 differentiation conditions were evaluated.
  • median cell volume as measured by Coulter counting, is a surrogate marker for T cell activation as it conelates with other events such" as CD25 and CD69 up-regulation.
  • Figure 14 shows median cell volume changes during Thl, Th2, Tel, or Tc2 expansion in the presence or absence of either 0.1 or 10.0 ⁇ M rapamycin. Without rapamycin, each T cell subset has a dramatic increase in median cell volume after CD3, CD28 co-stimulation.
  • CD3 CD28 co-stimulation was nominally reduced at the 0.1 ⁇ M rapamycin concentration, with more significant reductions occurring at 10 ⁇ M of rapamycin (Figure 15).
  • Example 8 Effects of Rapamycin on Cytotoxic T cells
  • chromium release assays were performed (Figure 16 shows CTL assays using Tc2 effectors generated in the presence or absence of rapamycin).
  • the Tc2 cells propagated in high dose rapamycin had reduced lytic capacity through the fas pathway, as evidenced by their reduced capacity to.
  • Tc2 cells propagated in high dose rapamycin had a reduced capacity to lyse the P815 tumor target in a heteroconjugate assay in calcium-replete conditions, an assay that reflects granule- mediated killing function, h sum, Tc2 cells generated in high dose rapamycin had reduced granule and fasL killing function relative to Tc2 cells propagated in low-dose or no rapamycin.
  • the purity of Tc2 cells can be increased (on the basis of reduction in contaminating type I cytokine secretion) by high dose rapamycin exposure. It is interesting to note that loss of IFN- ⁇ secretion in the rapamycin-generated Tel culture was not associated with induction of Tel cell type II cytokine secretion, and therefore does not indicate a simple rapamycin-associated Tl to T2 shift in polarity. Similar to the case with CD4 + Th2 cells, CD8 expansion in high-dose rapamycin was associated with a more na ⁇ ve T cell phenotype, as evidenced by increased CD62L expression (Figure 18).
  • GVHD GVHD
  • rapamycin enhanced Th2 purity of co- stimulated donor Th2 cells it is likely that rapamycin-generated Th2 cells may have enhanced in vivo capacity to modulate GVHD.
  • the rapamycin- generated cells maintained resistance to rapamycin inhibition relative to unmanipulated T cells it was hypothesized that in vivo rapamycin may allow selective expansion of the Th2 cells relative to other, unmanipulated donor T cells.
  • IFN- ⁇ cytokine secretion
  • Th2 recipients were also evaluated for IFN- ⁇ secretion from the expanded Th2 cells 7 days after in vivo transfer in the GVHD/GVT model (Th2 cells were identified by flow cytometry on the basis of their expression of the congenic marker, Ly5.1).
  • Th2 cells propagated in rapamycin had a reduced capacity for JFN- ⁇ secretion after in vivo transfer relative to conventional co-stimulated Th2 cells.
  • This reduced Th2 cell BFN- ⁇ secretion in rapamycin-generated Th2 recipients was observed with syngeneic DC re-stimulation, which likely reflects trae in vivo activation in the GVHD model, and with potential for IFN- ⁇ secretion upon allogeneic DC stimulation.
  • Th2 cells maintained a more pure Th2 function in vivo in the GVHD model as determined by reduced IFN- ⁇ secretion.
  • conventional Th2 cells or Th2 cells expanded in low dose (0.1 ⁇ M) or high dose (10 ⁇ M) rapamycin were evaluated in the same GVHD/GVT model, with weight loss, histology, and survival as experimental endpoints.
  • recipients of splenic CD4 + and CD8 + T cells underwent weight loss consistent with acute GVHD.
  • the TS/A control group that received tumor and no donor splenic T cells; weight loss in this group is thus attributed to tumor (pulmonary metastasis).
  • each recipient of carcinoma cells and no donor T cells (TS/A control) died of tumor within one month post-BMT.
  • the GVHD control group receiving splenic T cells did not undergo lethality in spite of the dramatic pattern of progressive GVHD-induced weight loss.
  • this treatment cohort there was a significant GVT effect based on increased survival, with deaths in this group attributed to GVHD.
  • Th2 modulation of acute GVHD it was evaluated whether rapamycin-generated Th2 cells might be preferentially expanded by in vivo rapamycin administration.
  • the GVHD/GVT model was utilized, with splenic T cell inoculate supplemented with rapamycin-generated donor Th2 cells (0.1 ⁇ M concentration).
  • recipient mice were injected daily with either rapamycin, cyclosporin A, or CMC vehicle from day 0 to day +7 post-BMT.
  • Example 11 Evaluation of Rapamycin in Human CD4 Cells To evaluate whether a similar rapamycin biology exists in human CD4 cells, and to initiate a translation of rapamycin-generated Th2 cells into clinical trials, experiments of human CD4 cell co-stimulation in the presence or absence of rapamycin were performed.
  • Example 12 Purity ofThl/Th2 cells
  • the human Th2 cultures were additionally evaluated for issues of Thl/Th2 purity.
  • FIG 26 shows, cells propagated under Th2 conditions and rapamycin had an increased Th2 cytokine purity, as evidenced by reduction in capacity for IFN- ⁇ secretion.
  • This increased Th2 purity was observed simply by an initial day 0 to day 6 rapamycin exposure, and was more fully realized by continued presence of rapamycin in the Th2 culture.
  • rapamycin generated Th2 cells had a dramatic reduction in IL-2 secretion, with this effect occurring during the initial six days of rapamycin exposure. Concomitant with these reductions in type I cytokine contaminations, Figure 27 demonstrates that rapamycin- generated Th2 cells had an increased capacity for secretion of the type II cytokines IL-4 and IL-13. In sum, rapamycin enhanced the ability of CD28 co-stimulation and cytokines to generate human Th2 cells, both on the basis of reducing Thl cytokines and increasing Th2 cytokines.
  • rapamycin-generated Th2 cells in the human system displayed a more na ⁇ ve CD4 cell phenotype relative to conventionally co-stimulated Th2 cells.
  • rapamycin-generated Th2 cells had increased expression of CD62L relative to control Th2 cells; the increase in CD62L was most marked in Th2 cultures that were continuously exposed to rapamycin.
  • human rapamycin-generated Th2 cells also expressed significantly reduced CD40L relative to confrol Th2 cells; because CD40L is a molecule preferentially expressed on Thl cells, this observation further supports the conclusion that rapamycin facilitates generation of a human Th2 cell with enhanced purity.
  • Example 13 Rapamycin Treated Na ⁇ ve CD4 + T cells
  • na ⁇ ' ve CD4 cells would be more resistant to rapamycin, and would therefore exhibit a higher cloning efficiency after co-stimulation during rapamycin exposure.
  • na ⁇ ve or memory human CD4 cells were purified by flow sorting, and co-stimulated either with or without rapamycin (results in Figure 31).
  • na ⁇ ve sorted CD4 cells had only a nominal reduction in CD4 expansion in rapamycin relative to the control culture ( ⁇ 25% reduction in CD4 yield).
  • CD45RO + sorted cells not only had reduced expansion to CD28 co-stimulation, but also had a more significant degree of rapamycin-associated reduction in CD4 + T cell expansion ( ⁇ 50% reduction).
  • na ⁇ ve CD4 + T cells are more resistant to rapamycin inhibition, perhaps in part through their increased expression of MDR molecules, which results in co-stimulation of CD4 + T cells that have a more na ⁇ ve effector phenotype and a greater capacity for Th2 polarization.
  • Example 14 Reduction of GVHD by Th2 cells.
  • the lymphocyte fraction of the elutriation product (120 to 140 fraction) will be depleted of B cells by incubation with an anti-B cell antibody (anti-CD20; Nexell) and an anti-CD8 antibody (Nexell) and sheep anti-mouse magnetic beads (Dyrial; obtained through Nexell) by a standard operating procedure of the NIH DTM using the MaxCep Device (Nexell). Flow cytometry will be performed to document that CD8+ T cell contamination is ⁇ 1%.
  • the resultant CD4-enriched donor lymphocyte product will be cryopreserved using an NIH DTM protocol in aliquots of 50 to 200 x 10 6 cells/vial. Sterility of the population will not be tested at this early stage of the Th2 cell generation procedure; such testing will occur after final co-culture of donor CD4 cells.
  • the donor will receive filgrastim as an outpatient (10 ⁇ g/kg/day each morning; subcutaneously) for 5, 6, or 7 days.
  • the donor should take the filgrastim as early as possible upon awakening in the morning. This is especially important on days 5, 6, and 7 of the injections, b) Apheresis will typically be performed on days 5 and 6 of this regimen.
  • sufficient numbers of CD34+ cells might be obtained with a single apheresis on day 5; on other occasions, it may be necessary to perform apheresis on days 5, 6, and 7 to reach the target CD34+ cell number (> 4 x 10 6 per kg).
  • the donor will be instructed to take filgrastim for the complete 7 day period, unless notified by the transplant team that adequate CD34+ cells were harvested before day 7. c) If > 3 x 10 6 CD34+ cells per kg are harvested after apheresis on days 5, 6, and 7, no further mobilization or apheresis will be performed, and the patient will be eligible to receive the stem cell fransplant with that dose of CD34+ cells. d) In the event that less than 3 x 10 6 CD34+ cells per kg are harvested after apheresis on days 5, 6, and 7, the donor will be given two weeks of rest, and then will be re-treated with filgrastim followed by repeat peripheral blood stem cell harvesting.
  • a 15 to 25 liter large volume whole blood pheresis will be performed in the NIH DTM via a 2-armed approach or via a temporary central venous catheter in the femoral position using the Baxter CS3000Plus, Cobe Spectra, or an equivalent instrument. This procedure typically takes 4 to 6 hours.
  • Apheresis procedure will typically use ACD-A anti-coagulant; alternatively, partial anti-coagulation with heparin may be utilized.
  • the apheresis product will be cryopreserved and stored at -180 degrees Celsius in a solution containing Plasmalyte A, Pentastarch, human serum albumin, DMSO, and preservative free heparin (10 U/ml).
  • the concentration of CD34+ cells in the apheresis product will be determined by flow cytometry, and the number of CD34+ cells in each cryopreserved bag calculated, i) If the donor and host are ABO incompatible, red blood cells will be depleted from the stem cell product by standard DTM protocols.
  • Donor CD4+ Th2 Cells a) Cryopreserved donor CD4+ T cells will be resuspended to a concentration of 0.3 x 10 6 cells per ml. Media will consist of X-Vivo 20 supplemented with 5% heat- inactivated autologous plasma. b) The donor CD4+ T cells will be cultured in filtered flasks at 37° C in 5% CO2 humidified incubators.
  • T cells will be stimulated with anti-CD3/anti-CD28 coated magnetic beads (3 to 1 ratio of beads to T cells), c)
  • the following reagents will be added: recombinant human IL-4 (obtained through cross-filing on CTEP IND of Shering IL-4; 1000 LU. per ml), and recombinant human IL-2 (purchased from Chiron Therapeutics; 20 LU. per ml).
  • Bead restimulation will be at a bead to T cell ratio of 3 : 1.
  • T cell concentration will be 0.2 x 10 6 cells/ml.
  • Media will again consist of X-Vivo 20 supplemented with autologous plasma (5%), IL-2 (20 UJml), and IL-4 (1000 LUJml).
  • CD4 cells will be maintained at a concentration of 0.25 to 1.0 x 106 cells per ml by the addition of fresh X-Vivo 20 media supplemented with autologous plasma (5%), IL-2 (20 LUJml), and IL-4 (1000 LUJml).
  • the cells will be harvested and cryopreserved by the NIH DTM method in protocol-relevant quantities for administration on study. It is anticipated that the total time of CD4 cell culture will be 15 to 20 days. i) If an adequate numbers of CD4 cells is obtained, then such cells may be available for administration on this protocol as a Th2 infusion. j) The following will be the minimal phenotypic requirements of any particular
  • Th2 cell culture to qualify for cryopreservation with subsequent administration:
  • cryopreserved product will be tested for sterility with both fungal and bacterial cultures, through the ongoing testing done on cell products processed in the NIH Department of Transfusion Medicine.
  • CD4 Th2 cell product will be tested for endotoxin content by the limulus assay. Cell products positive for fungal, bacterial, or endotoxin content will be discarded.
  • Pre-transplant Induction Chemotherapy a) After cell products have been harvested from the patient, chemotherapy will be administered as an outpatient. All patients will receive at least one cycle of induction chemotherapy, even if their CD4 count is less than 50 cells per ⁇ l at the.time of study entry. At this point in the protocol or earlier at the time of cell harvesting, placement of permanent central venous access may be requested.
  • Fludarabine will be administered i.v. at a dose of 25 mg/m2 per day for three days (days 1, 2, and 3). Fludarabine will administered over a 30 minute interval. Steroids should not be used as an anti-emetic during this chemotherapy regimen, d) Cyclophosphamide will be administered i.v. at a dose of 600 mg/m2 over 30 minutes on day 4. d) Etoposide will be administered at a dose of 50 mg/m2 per day by continuous intravenous infusion for three days (days 1, 2, and 3). e) Doxorubicin will be administered at a dose of 10 mg/m2 per day by continuous intravenous infusion for three days (days 1, 2, and 3).
  • Vincristine will be administered at a dose of 0.5 mg m2 per day by continuous intravenous infusion for three days (days 1, 2, and 3).
  • Prednisone will be administered at a dose of 60 mg m2 per day orally for four days (days 1, 2, 3, and 4).
  • Filgrastrim will be initiated on day 5 at a dose of 10 ⁇ g/kg/day; G-CSF will be continued until the ANC is greater than 1000 cells per ⁇ l on two consecutive days.
  • Cycle 2 and Cycle 3 Dose Escalation a) If the first cycle of induction chemotherapy does not reduce the CD4 count to a value below 50 cells per ⁇ l and does not result in febrile neutropenia or prolonged neutropenia as evidenced by two consecutive bi-weekly ANC values less than 500 cells per ⁇ l, then the next cycle of induction chemotherapy may be dose escalated. b) Dose escalation will consist of a 20% escalation in the daily dose of fludarabine, etoposide, adriamycin, and cyclophosphamide.
  • Transplant Procedure Preparative Regimen a) On day 22 after the final cycle of induction chemotherapy, patients will be eligible to receive the following transplant preparative regimen. Therefore "day 22 of the final induction chemotherapy cycle will be transplant day -6. However, in cases where additional recovery time is required (for example, due to prolonged neutropenia, documented infection, or other medical compUcations of the induction regimen), an additional two weeks of recovery time may be utilized prior to initiation of the transplant preparative regimen.
  • Fludarabine will be administered i.v. over 15 to 30 minutes at a dose of 30 mg/m 2 /day on days -6, -5, -4, and -3.
  • Cyclophosphamide will be administered at a dose of 1200 mg/m 2 /day over a two hour infusion on days -6, -5, -4, and -3.
  • Mesna will be administered at a dose of 1200 mg m 2 per day by continuous i.v. infusion on days -6, -5, -4, and -3. The mesna should be started one hr prior to the cyclophosphamide.
  • Bag #1 of the mesna will be 150 mg/m 2 in 250 ml over a 3 hr infusion (thus stopping when cyclophosphamide ends). Then, mesna will be given at 1200 mg/m 2 in 500 ml over 24 hour infusion, for four days (days -6, -5, -4, and -3).
  • CSA dose may be modified to achieve adequate steady-state CSA levels. Once this intravenous dose is established and the patient is able to tolerate oral feedings (typically by day 14 post-transplant), then CSA will be switched to the oral formulation. Conversion of CSA to the oral formulation is typically performed by multiplying the adequate i.v. dose by a factor of 1.5 to 2.0. Patients will then be maintained on oral CSA on a 12 hour schedule, with a goal to achieve steady state frough CSA levels of 200 ng/ml CSA (acceptable range: 150 to 250 ng/ml).
  • hyperacute GVHD will be defined as a severe level of acute GVHD (grade III or IV) that occurs within the first 14 days post-transplant, e)
  • the initial three patients will be enrolled to Th2 cell dose level #1 (5 x 10 6 Th2 cells/kg). If no hyperacute GVHD or grade 4 or 5 toxicity attributable to the Th2 cells is observed in these initial three patients, then it will be determined that this dose level is safe, and accraal to dose level #2 will commence. If hyperacute GVHD or grade 4 or 5 toxicity attributable to the Th2 cells is observed in any of the initial three patients, then accrual to dose level #1 will be expanded to include a total of six patients.
  • Th2 cell dose level #2 If it is determined that Th2 cell dose level #2 is safe, accrual to the final dose level #3 will start (Th2 cell dose of 1.25 x 10 8 cells/kg). Six patients in total will be evaluated on dose level #3. If more than one patient on dose level #3 develops hyperacute GVHD or grade 4 or 5 toxicity attributable to the Th2 cells, then accrual to dose level #3 will stop. h) In the phase II component of this study, eighteen (18) additional patients will receive Th2 cells at either dose level #2 or level #3. To help ensure that the Th2 cells continue to be safely administered in this expanded cohort, the same accraal and stopping rales pertaining to severe toxicity attributed to the Th2 cells will be continued.
  • DLI may be administered alone or after chemotherapy administration.
  • Donor lymphocytes will be collected by apheresis, either in steady state (no donor therapy) or after G-CSF mobilization.
  • the donor product may be enriched for lymphocytes by Ficoll-Hypaque procedure as per NIH DTM protocol.
  • the donor product may be administered without lymphocyte purification.
  • DLI may be sequentially administered, with initial dosing at 1 x 10 6 CD3+ T cells per kg, and with subsequent dose increases to 1 x 10 7 and 1 x 10 8 per kg.
  • persistent or progressive disease may be treated with any approved therapy thought to be in the best standard care of the patient, such as chemotherapy, cytokine therapy, or monoclonal antibody therapy. Alternatively, patients with relapse may receive therapy on other NCI protocols.
  • Th2 dose level #2 The incidence of grade II to IV acute GVHD at Th2 dose level #2 is 2/6; results from T----2 dose level #3 are not known, as accrual to this cohort has just now been initiated. If Th2 dose level #3 is associated with unacceptable toxicity (more than 1/6 incidence of severe toxicity) or significant GVHD (more than 2/6 cases of grade II to IV acute GVHD), the additional 18 patients will be treated on Th2 dose level #2. If recipients of Th2 dose level #3 have 0/6 or 1/6 cases of severe toxicity and 0/6, 1/6, or 2/6 cases of grade II to IV acute GVHD, the additional 18 patients will be treated at dose level #3.
  • the phase II component of accraal may be initiated at dose level #2.
  • the incidence and severity of acute GVHD in the cohort of 24 patients receiving Th2 cells at dose level #2 or #3 will be determined, and compared to the initial protocol cohort of 19 patients receiving fransplantation without Th2 cells. In this protocol, we hypothesize that recipients of the Th2 cells will have reduced GVHD relative to non- Th2 recipients. In the cohort of non-Th2 recipients, the incidence of grade II to grade IV acute GVHD was 12/19. Based on this experience, one can conclude that the trae rate of grade II to IV GVHD without Th2 cells is approximately 60%.
  • grade II to IV acute GVHD The incidence of grade II to IV acute GVHD in non-Th2 recipients was 12/19, or 63%. In the expanded cohort of Th2 recipients, the incidence of grade II to IV acute GVHD will be calculated on an ongoing basis and reviewed at the weekly protocol meeting. If at any point in protocol implementation the incidence of grade II to IV acute GVHD in Th2 recipients is 60% or greater, then further accraal to the protocol will be stopped. Up to 2/6 cases of grade II to IV acute GVHD will be allowed for expansion of Th2 accrual to the phase II component. Therefore, it is possible that the phase II component of the Th2 accraal may be stopped after 4 patients (in the event that each develops grade II to IV acute GVHD).
  • this design allows for a safety evaluation for administering donor Th2 cells in a dose range that we hypothesize would be associated with a reduction in GVHD (a 1 :1 ratio of unmodified donor CD4 + T cells to donor Th2 cells).
  • GVHD a 1 :1 ratio of unmodified donor CD4 + T cells to donor Th2 cells.
  • Allogeneic PBSCT in the Treatment of Leukemia and Lymphoid Neoplasia represents a potentially curative treatment for patients with multiple hematologic and lymphoid malignancies.
  • the allogeneic graft-versus-leukemia effect contributes to disease remission in acute lymphocytic leukemia, acute myelogenous leukemia, chronic lymphocytic leukemia, chronic myelogenous leukemia, indolent and high-grade non-Hodgkin's lymphoma, Hodgkm's lymphoma, multiple myeloma, and myelodysplastic syndrome.
  • EPOCH Because the EPOCH regimen has an established response rate in patients with chemotherapy- refractory lymphoid malignancy, such patients will be eligible for this protocol.
  • the addition of fludarabine to EPOCH may further improve the anti-tumor effects of this regimen.
  • the activity of fludarabine and EPOCH chemotherapy in patients with leukemia is not known. As such, patients with leukemia (AML, myelodysplasia, ALL, and CML) will be candidates for this protocol.
  • the imtial patient entered a pathologic complete remission from refractory bulky lymphoma, but died of DIG and shock at day 22 post-SCT (had grade II clinical GVHD).
  • Th2 recipients have had rapid recovery of hematopoiesis, with full donor chimerism; the Th2 cells thus do not appear to impair engraftment.
  • Th2 dose level #2 cohort has achieved alloengraftment with documented anti-tumor responses and limited GVHD (2/6 grade II-IV acute GVHD), this cohort is a candidate for evaluation in the phase II aspect of the protocol.
  • Table 2 Overall Study Design: 616 HLA-matched (a) Apheresis with Elutriation (lymphocyte Sibling Donor harvest) (b) In Vitro Th2 Generation, cryopreservation (c G-CSF treatment, stem cell harvest
  • Transplant Recipient Induction Chemotherapy (1 to 3 cycles; 21 d cycles; cycles 2 and 3 may be administered in a dose-escalated fashion, as detailed on page 16-18 of protocol)
  • Th2 Cell Administration (Day 1 of PBSCT) Phase I Th2 Phase I Th2 Phase I Th2 Dose Level 1 Dose Level 2 Dose Level 3 Phase II Th2 Arm (5 x 10 6 /kg) (25 x l0 6 /kg) (125 x l0 6 /kg) (25 oorr 112255 xx 1100 66 /kg) 3 patients 6 patients 6 patients 18 patients
  • Phase II Th2 Arm (5 x 10 6 /kg) (25 x l0 6 /kg) (125 x l0 6 /kg) (25 oorr 112255 xx 1100 66 /kg) 3 patients 6 patients 6 patients 6 patients 18 patients

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Abstract

L'invention concerne des méthodes de génération de fonctions Th1/Tc1 et Th2/Tc2 hautement enrichies. En particulier, la génération de ces fonctions est obtenue par l'addition d'un médicament immunodépresseur, la rapamycine ou un composé dérivé de rapamycine. En plus d'une pureté accrue de la fonction lymphocytes T, les lymphocytes T générés dans la rapamycine expriment également des molécules améliorant la fonction lymphocytes T immune telle que CD28 et CD62L. Lesdits sous-ensembles de lymphocytes T fonctionnels générés par rapamycine peuvent avoir une application dans la prévention ou dans le traitement de la réaction de greffe contre hôte GVHD après transplantation de cellules souches hématopoïétiques allogéniques, le traitement de l'auto-immunité ou la thérapie d'infections ou du cancer.
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Cited By (9)

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Publication number Priority date Publication date Assignee Title
US8562974B2 (en) * 2005-02-25 2013-10-22 Fondazione Telethon Method for expanding Cd4+ Cd25+ T regulator cells
US20100068193A1 (en) * 2006-02-15 2010-03-18 Invitrogen Dynal As Methods and materials for the generation of regulatory t cells
US20100129340A1 (en) * 2006-02-15 2010-05-27 Life Technologies Corporation Methods and materials for the generation of regulatory t cells
US9119807B2 (en) 2006-02-15 2015-09-01 Life Technologies As Methods and materials for the generation of regulatory T cells
EP2375897A1 (fr) * 2009-01-14 2011-10-19 Health Research, INC. Procédés et compositions contenant des inhibiteurs de la mtor pour améliorer les réponses immunitaires
JP2012515213A (ja) * 2009-01-14 2012-07-05 ヘルス リサーチ インコーポレイテッド 免疫応答を増強するための、ｍＴＯＲ阻害剤を含有する方法及び組成物
EP2375897A4 (fr) * 2009-01-14 2013-05-15 Health Research Inc Procédés et compositions contenant des inhibiteurs de la mtor pour améliorer les réponses immunitaires
CN111454903A (zh) * 2020-05-06 2020-07-28 青岛瑞思德生物科技有限公司 免疫细胞体外培养、诱导、激活、冻存方法及其细胞库建立
CN111454903B (zh) * 2020-05-06 2023-10-20 青岛瑞思德生物科技有限公司 免疫细胞体外培养、诱导、激活、冻存方法及其细胞库建立

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