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WO2006109300A1 - Traitement de pre-transplantation de cellules donneuses permettant le controle de la maladie du greffon contre l’hote (gvhd) chez des receveurs transplantes - Google Patents

Traitement de pre-transplantation de cellules donneuses permettant le controle de la maladie du greffon contre l’hote (gvhd) chez des receveurs transplantes Download PDF

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WO2006109300A1
WO2006109300A1 PCT/IL2006/000452 IL2006000452W WO2006109300A1 WO 2006109300 A1 WO2006109300 A1 WO 2006109300A1 IL 2006000452 W IL2006000452 W IL 2006000452W WO 2006109300 A1 WO2006109300 A1 WO 2006109300A1
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cells
donor
cpg
gvhd
ifa
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WO2006109300A9 (fr
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Shoshana Morecki
Shimon Slavin
Yelena Yacovlev
Yael Gelfand
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Hadasit Medical Research Services and Development Co
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Hadasit Medical Research Services and Development Co
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0648—Splenocytes
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/05—Dipeptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/12—Cyclic peptides, e.g. bacitracins; Polymyxins; Gramicidins S, C; Tyrocidins A, B or C
    • A61K38/13—Cyclosporins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/164—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0636—T lymphocytes

Definitions

  • the invention relates, in general, to GVHD (graft-versus-host disease). Specifically, the invention relates to treating donor cells pre -transplantation in order to avoid GVHD.
  • GVHD graft-versus-host disease
  • Allogeneic bone marrow transplantation has been a potentially curative therapy for patients with a variety of diseases, including hematological malignancies [Bortin M.M. and Rimm A.A. (1986) Transplantation 42: 229-234].
  • GVHD graft-versus-host disease
  • GVHD graft versus host disease
  • GVHD The pathophysiology of GVHD is complex and involves donor T cell responses to host antigens and inflammatory cytokine effectors [Ferrara J.L.M. and Deeg H.J. (1991) N. Engl. J. Med. 324:667-674; Hill G. et al., (1998) Hematology 2:423—434].
  • cytokine dysregulation results as a consequence of synergistic interactions between cells of both myeloid and lymphoid lineages [Krenger W. et al., (1997) Transplantation 64:553-558].
  • cytokines produced by donor T cells in response to host alloantigens "prime" monocytes and macrophages to secrete cytopathic amounts of inflammatory cytokines (e.g., TNF- ⁇ and IL-I) and consequently ThI and Th2 cytokines secretion becomes deregulated
  • inflammatory cytokines e.g., TNF- ⁇ and IL-I
  • ThI and Th2 cytokines secretion becomes deregulated
  • Immunoregulators capable of affecting this complex network may modulate the development of GVHD. Regulation of the disturbances in the ThI and Th2 cytokines secretion in experimental autoimmune disease is efficiently controlled by immunomodulators like complete Freund's adjuvant (CFA) [Weber F. and Hempel K. (1987) Int. Arch. Allergy Appl. Immunol. 83: 174-177; Qin H.Y. et al., (1993) J. Immunol. 150: 2072-2080; Matthys P. et al., (1999) J. Immunol. 163: 3503-3510].
  • CFA complete Freund's adjuvant
  • CFA non-obese diabetic
  • TLR Toll-like receptors
  • GVH reaction involves a complex immune deregulation in the host, and therefore, it is the intention of the inventor to avoid further immune disturbance of the host's immune system by triggering the donor's immune system in vivo and modulating the cell source itself.
  • the donor's immune system can be triggered with the intact mycobacterium tuberculosis present in the CFA or with some bacterial components, such as cell wall lipopolysaccharides (LPS) and bacteria-derived oligodeoxynucleotides containing a CpG motif (CpG-ODN) emulsified in incomplete Freund's adjuvant.
  • CpG motifs are known for their ability to stimulate TLRs, evoke inflammatory response and induce signals for controlling adaptive immunity [Iwasaki A. and Medzhitov R. (2004) Nat. Immunol. 5:987-995].
  • the TLR9 toll-like receptor found in a subset of dendritic and B cells recognizes a specific nucleotide pattern, known as CpG DNA, commonly present in bacteria and viruses, but uncommon in the human DNA.
  • CpG DNA a specific nucleotide pattern
  • Synthetic CpG sequences that mimic those found in pathogens are capable of binding to and activating TLR9.
  • CpGs emulsified in IFA are already approved for application in clinical trials of tumor cell vaccines.
  • In vivo pre-transplant treatment of the donor with such agents may provide immune modulated cell populations capable of minimizing GVHD in the immune-compromised recipient mice while preserving the GVL effect when used as source for allogeneic cells therapy.
  • the present invention provides a novel approach for transplants and immunotherapy protocols of malignant and non-malignant diseases. Specifically, the present invention provides treatment of the donor (or its cells) pre-transplant with immuno-modulating agents, avoiding the development of GVHD.
  • the present invention involves the preparation of donor cells for transplantation into a recipient, wherein said cells have reduced GVHD activity, said method comprising the steps of: a. contacting said cells, in vivo or ex vivo, with an effective amount of an immune modulator agent; and b. harvesting said cells between 0 and 30 days following step (a).
  • Said donor cells are selected from the group consisting of bone marrow cells, peripheral blood mononuclear cells, cells used for hematopoietic transplantation, cells used for immunotherapy, mobilized blood cells, cord blood or embryonic stem cells including naive or activated lymphocytes, and na ⁇ ve or activated lymphocytes with no stem cells.
  • the donor cells prepared by the above-described method make up, therefore, a population of donor cells which are characterized by having reduced GVHD activity.
  • said preparation is preferably effected ex vivo.
  • Preferred immune modulator agents are CpG, CpG in IFA, CFA, IFA, LPS in IFA, Montanide, muramil dipeptide, QS21, aluminum salts (alum), LPS, Mycobacterium Tuberculosis components, difteria toxin, and a biological or synthetic factor that affects Thl/Th2 cytokine balance, used in vivo or ex vivo accordingly, as specified further in the description.
  • the present invention provides a pharmaceutical composition for reducing, alleviating or abrogating GVHD, comprising as active agent the donor cells or the population of donor cells above-described, further optionally comprising a pharmaceutically acceptable carrier, excipient or diluent.
  • Said donor cells or population of donor cells is also used in the preparation of said pharmaceutical composition.
  • the present invention presents a method of treating, alleviating or abrogating GVHD in a subject in need of one of cell immunotherapy, adoptive transfer immunotherapy, stem cell transplant, and stem cell transplantation for induction of tolerance to donor-derived allograft, said method comprising administering to said subject a therapeutically effective amount of the above- described composition or donor cells with reduced GVHD activity.
  • said subject suffers from a malignant or non-malignant disorder, particularly one of a hematopoietic cell deficiency disorder, a congenital or acquired immune deficiency, a genetic disorder causing hemoglobinopathy, an enzyme deficiency disease; a hematological malignancy, cancer, a metastatic solid tumor or an autoimmune disease.
  • a malignant or non-malignant disorder particularly one of a hematopoietic cell deficiency disorder, a congenital or acquired immune deficiency, a genetic disorder causing hemoglobinopathy, an enzyme deficiency disease; a hematological malignancy, cancer, a metastatic solid tumor or an autoimmune disease.
  • said hematological disorder is refractory to chemotherapy.
  • said donor cells further present increased graft- versus -leukemia or graft- versus-tumor activity.
  • the above method may further comprise administering to said subject in need an additional antineoplastic or immunosuppressant agent before, together with, or after administration of said donor cells, in an amount effective to allow engraftment and prevent rejection of the said donor cells.
  • Additional antineoplastic or immunosuppressant agents are selected from the group consisting of an adjunctive agent, an alkylating agent, an antimetabolite, a hormone, a miscellaneous antineoplastic drug or an immunosuppressant agent selected from the group of Mycophenolate Mofetil, Cyclosporine, Azathioprine, Cyclosporine analogues, Prednisone, Tacrolimus, Sirolimus, Cyclophosphamide, FTY 720, ionizing radiation, anti-lymphocytic agents, anti-costimulatory molecules, and 2CdA.
  • an adjunctive agent selected from the group consisting of an adjunctive agent, an alkylating agent, an antimetabolite, a hormone, a miscellaneous antineoplastic drug or an immunosuppressant agent selected from the group of Mycophenolate Mofetil, Cyclosporine, Azathioprine, Cyclosporine analogues, Prednisone, Tacrolimus, Sirolimus
  • the donor cells are preferably administered to said subject in need in the form of a peripheral blood mononuclear cell preparation.
  • the present invention provides a method for reducing the probability of graft- versus-host disease following bone marrow transplantation whilst maintaining or promoting graft-versus-tumor effect and graft-versus- leukemia effect, said method comprising administering to said subject a therapeutically effective amount of the above-described donor cells with reduced GVHD activity or composition comprising thereof.
  • An additional aspect of the present invention involves a method for induction of transplantation tolerance in a recipient subject in- need of a tissue or organ transplant, which, may be an allograft or xenograft, said method comprising administering to said subject a therapeutically effective amount of the above- described donor cells with reduced GVHD activity or a composition comprising thereof. Said administration of said cells or composition further prevents relapse of said tumor.
  • said cells or the composition comprising thereof are preferably administered intravenously, parenterally, intratechally or intra- tumor.
  • the present invention provides the use of an immune modulator agent substance as herein in the preparation of a composition comprising donor cells for transplantation into a recipient, wherein said cells, upon treatment with said substance, have reduced GVHD activity, and said composition is for the treatment of any one of a malignant or non-malignant disorder, particularly one of a hematopoietic cell deficiency disorder, a congenital or acquired immune deficiency, a genetic disorder causing hemoglobinopathy, an enzyme deficiency disease, a hematological malignancy, a metastatic solid tumor or an autoimmune disease.
  • a malignant or non-malignant disorder particularly one of a hematopoietic cell deficiency disorder, a congenital or acquired immune deficiency, a genetic disorder causing hemoglobinopathy, an enzyme deficiency disease, a hematological malignancy, a metastatic solid tumor or an autoimmune disease.
  • said hematological disorder involves neoplastic proliferation of hematopoietic cells, and is selected from the group consisting of lymphoblastic leukemia, acute or chronic myelogenous leukemia, Hodgkin's lymphoma, Non-Hodgkin's lymphoma, myelodysplastic syndrome, multiple myeloma, and chronic lymphocytic leukemia.
  • CFA Complete Freund's adjuvant
  • IFA Incomplete Freund's Adjuvant
  • CpG and its non-CpG control given with IFA
  • Figure 2A-2D Effect of donor pre-treatment on induction of GVHD as measured by body weight
  • Sub-lethally irradiated recipient (BALB/CXC57BL/6) Fl mice were inoculated with 30X10 6 splenocytes derived from either na ⁇ ve or C57 donor mice treated 10 days previously with CFA or IFA (0.2ml) (Fig. 2A), CpG alone or non-CpG alone (lOO ⁇ g) (Fig. 2B), LPS (40 ⁇ g) or LPS (40 ⁇ g)-emulsified in IFA (Fig. 2C); and CpG or non-CpG (lOO ⁇ g) emulsified in IFA (Fig. 2D).
  • Figure 3A-3D Effect of donor pre-treatment with CpG on GVHD related death
  • Sub-lethally irradiated recipient (BALB/CxC57BL/6)Fl mice were inoculated with 30X10 6 splenocytes derived from either na ⁇ ve or C57 donor mice treated 6 or 10 days previously with IFA (0.2ml) (Fig. 3A), CpG alone or non-CpG alone (lOO ⁇ g) given 6 days prior to harvesting (Fig. 3B), CpG alone or non-CpG alone (lOO ⁇ g) given 10 days prior to harvesting (Fig. 3C); and CpG or non- CpG (lOO ⁇ g) emulsified in IFA 10 days prior .to harvesting (Fig. 3D).
  • Figure 4A-4C Phenotypic analysis of spleen cells following treatment with CFA or CpG
  • Sub-lethally irradiated recipient (BALB/CxC57BL/6)Fl mice were inoculated with 30X10 6 splenocytes derived from either na ⁇ ve or C57 donor mice treated 10 days previously with CFA or IFA (0.2ml) (Fig. 4A), 6 days previously with CpG alone or non-CpG alone (lOO ⁇ g) (Fig. 4B), and 10 days previously with CpG or non-CpG (lOO ⁇ g) emulsified in IFA (Fig. 4C). Percentages of positive cells determined by FACS analysis are shown for those surface markers (CD) in which a change in the number of positive cells was observed as compared to the relevant controls.
  • CD surface markers
  • FIG. 5 Survival of (BALB/CXC57BL/6) Fl mice inoculated with B cell leukemia (BCLl)
  • the values represent a summary of two separate experiments, in which in one, 11 out of 13 mice that received splenocytes treated with CpG in IFA were disease-free (no GVHD and no leukemia) for more than 160 days, and in the second (which was still ongoing at the time of filing this application), 7 out of 13 mice were disease-free (no GVHD and no leukemia) for more than 90 days.
  • the present invention relates to a method of preparing donor cells for transplantation into a recipient, wherein said cells have reduced GVHD activity, said method comprising the steps of: a. contacting said cells with an effective amount of an immune modulator agent; and b. harvesting said cells between 0 and 30 days following step (a), preferably harvesting between 5 and 20 days.
  • the cells may be optionally phenotyped, through the identification of expressed cell markers, and still further selected prior to injection.
  • the present invention intends to modify the potential immune reactivity of a biological transplant by treating either the donor subject (in vivo) or the transplant (the donor cells) itself (ex vivo) with an immune modulator agent prior to transplantation into a recipient.
  • reduced GVHD activity it is to be understood that, upon transplantation, the donor cells are inhibited or are incapable of generating a graft versus host condition.
  • the immune modulator agent used in the present invention is preferably selected from the group consisting of CFA, CpG, CpG in IFA, LPS in IFA and IFA, Montanide, muramil dipeptide (MDP), difteria toxin, and QS21 for the donor in vivo treatment.
  • the immune modulator agent may be an adjuvant which further comprises an infectious substance that stimulates the immune system.
  • Immuno modulators for treating the donor cells, or transplant, ex vivo are selected from the group consisting of CpG, LPS, Mycobacterium Tuberculosis components, difteria toxin, and a biological or synthetic factor that affects the Thl/Th2 cytokine balance, as well as aluminum salts.
  • transplantation it is meant transferring a healthy cell, tissue or organ to replace a damaged one (cell, tissue or organ).
  • an immune modulator agent or “immune modulator agent” are defined as an agent that affects, enhances, or suppresses the immune system.
  • Some accepted immunomodulators are gamma interferon, interleukin 1, interleukin 2, interleukin 12, interleukin 18, as well as tumor necrosis factor (TNF) and colony stimulatory factors (CSFs).
  • an immune modulator agent is a substance which, when applied to donor cells either in vivo or in vitro, is capable of rendering these cells resistant or inhibited to develop a GVHD response in the recipient, upon transplant.
  • An adjuvant is also an immune modulator agent of the present invention.
  • Said immuno modulator agent is delivered to the donor via sub-cutaneous, intravenous, parenteral, intramuscular, or in any other appropriate way, according to the agent.
  • adjuvant taken from the Latin word adjuvans means to help, particularly to reach a goal.
  • adjuvant relates to substances that augment, stimulate, activate, potentiate, or modulate the immune response at either the cellular or humoral level.
  • the classical agents (Freund's adjuvant, BCG or Corynebacterium parvum) contain bacterial antigens. Some are endogenous, e.g., histamine, interferon, transfer factor, tuftsin and interleukin-1.
  • An adjuvant enhances the pharmacological effect of a drug or increases the ability of an antigen to stimulate the immune system. It enhances or modifies the immune-stimulating properties of a vaccine. As an ingredient in a prescription or solution, it facilitates or modifies the action of the principal ingredient by accelerating or improving its action (auxiliary remedy).
  • CpG oligodeoxynucleotides (also referred herein as "CpG” or “CpG ODN”) is also considered an adjuvant, being the preferred immune modulator agent in the present invention.
  • CpG oligodeoxynucleotides are characterized by the presence of CpG motifs. Oligonucleotides containing CpG motifs are also known in the literature as ISS (immunostimulatory sequences) have been described as potent adjuvants of type 1 immune response when coadministered with protein or peptide vaccines [Roman, M. et al. (1997) Nat. Med. 3: 849].
  • ISS immunonostimulatory sequences
  • the present invention presents CpG as a safe and clinically proved immunoregulator for the treatment of donor or donor cells.
  • adjuvants are considered agents which modify the effect of other agents while having few if any direct effects when given by themselves.
  • the commonly used Freund's Adjuvant is an adjuvant made from an industrial emulsifier in combination with killed versions of the bacterium that causes tuberculosis (Mycobacterium tuberculosis) in the case of CFA, or without in the case of IFA.
  • adjuvants and their respective analogs are suitable for use in the present invention, in the treatment of donor cells in order to make them less prone or resistant to trigger GVHD.
  • Said adjuvants include, but are not limited to, Freund's (complete and incomplete), mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, and potentially useful human adjuvants such as BCG (bacille Calmette-Guerin), corynebacterium parvum, difteria toxin, muramyl peptide (MDP), MF59, QS21, and immunostimulating complexes (ISCOMs) comprised by a saponin, a sterol and optionally a phospholipid.
  • BCG Bacille Calmette-Guerin
  • corynebacterium parvum corynebacterium parvum
  • difteria toxin muramyl
  • the present invention also offers a method of obtaining any kind of cells used for hematopoietic transplantation or cell immunotherapy, which cells have reduced GVHD reactivity.
  • an effective amount or “sufficient amount” mean an amount necessary to achieve a selected result, which at present, involves the amount of an immunomodulating agent necessary for treating donor cells to make them unable to trigger GVHD.
  • Donor cells which are employed in the present invention are selected from the group consisting of bone marrow cells, peripheral blood mononuclear cells, cells used for hematopoietic transplantation, cells used for immunotherapy, mobilized blood cells, cord blood or embryonic stem cells including na ⁇ ve or activated lymphocytes, and na ⁇ ve or activated lymphocytes with no stem cells.
  • Cells used for hematopoietic transplantation or for immunotherapy are bone marrow cells, peripheral blood stem cells, cord blood cells, activated or expanded lymphocytes, hematopoietic stem cells and peripheral blood mononuclear cells (PBMC).
  • PBMC peripheral blood mononuclear cells
  • Bone marrow is a soft, spongy tissue found in the center of many bones where blood cells are produced. It contains the precursor stem cells of red blood cells, platelets, polymorphonuclear leukocytes, macrophages and lymphocytes. The different precursor stem cells disperse from- the bone marrow and undergo further differentiation to perform specific functions.
  • a bone marrow stem cell is a parent cell that grows and divides to produce red blood cells, white blood cells, and platelets.
  • a stem cell is primarily found in the bone marrow, but also in the peripheral blood.
  • PBSC Peripheral blood stem cells
  • the present invention provides a population of donor cells used in transplantation, said cells being characterized by having reduced GVHD activity, wherein said population is obtained through contacting said cells in vivo or in vitro with an effective amount of immune modulator agent, and harvesting said cells between o and 30 days, preferably between 5 and 20 days, following said contact with said immune modulator agent.
  • said donor cells are selected from the group consisting of bone marrow cells, peripheral mononuclear cells, cells used for hematopoietic transplantation, cells used for immunotherapy, mobilized blood cells, cord blood or embryonic stem cells including na ⁇ ve or activated lymphocytes, and na ⁇ ve or activated lymphocytes with no stem cells.
  • Donor cells may also be originated from an organ or tissue, e.g. placenta, intestine, etc.
  • the data described in the Examples below indicates the presence of a cell population, which functionally suppressed allogeneic response (i.e., MLK in vitro, or GVHD in vi ⁇ ). Phenotypically, these cells could be ascribed to a non-T cell subpopulation that has an increased potential to undergo apoptosis upon the right stimulus, due to elevated levels of CD95 expression as measured by FACS analysis. Thus, the inventors describe the generation of a population, or a sub-population of cells which when used as donor cells and transplanted into the recipient, inhibit a GVHD response in the host.
  • the present invention provides a pharmaceutical composition for reducing, alleviating or abrogating GVHD, comprising as active agent the donor cells prepared by the method as described above, or the above- described population of donor cells.
  • the present invention also provides a method of treating, alleviating or abrogating GVHD in a subject in need of one of cell immunotherapy, adoptive transfer immunotherapy, stem cell transplant, intestine transplant, and stem cell transplantation for induction of tolerance to donor-derived allograft, said method comprising administering to said subject a therapeutically effective amount of the pharmaceutical composition as described above, or donor cells with reduced GVHD activity, wherein said cells were produced by the method described herein.
  • the invention also describes a method of treating a subject suffering from a malignant or non-malignant disorder by subjecting said subject to cell immunotherapy or adoptive transfer immunotherapy comprising transplanting donor cells previously treated ex vivo with an immune modulator agent, selected from the group consisting of CpG, LPS, Mycobacterium Tuberculosis components, aluminum salts (alum), difteria toxin, and a biological or synthetic factor that affect Thl/Th2 cytokine balance or with cells that were obtained from a donor who was treated in vivo with a immune modulator agent selected from the group consisting of CpG, CFA, IFA, CpG in IFA, LPS in IFA, Montanide, Muramil dipeptide, difteria toxin, and QS21, prior to cell harvesting.
  • an immune modulator agent selected from the group consisting of CpG, LPS, Mycobacterium Tuberculosis components, aluminum salts (alum), difteria toxin, and
  • treat, treating or treatment mean ameliorating one or more clinical indicia of disease activity in a patient having a malignant or non-malignant disease.
  • Treatment refers to therapeutic treatment.
  • patient or “subject in need” is meant any mammal for which cell immunotherapy or adoptive transfer immunotherapy treatment is desired in order to overcome said malignant or non-malignant disease.
  • immunotherapy refers to the treatment, or prevention of a disease, achieved through manipulation of the patient's immune system.
  • “Adoptive immunotherapy” is a type of passive immunotherapy (treatment performed the body) which involves the transfer of immune cells into a patient.
  • said donor cells are partially or completely mismatched allogeneic or xenogeneic bone marrow cells, mobilized blood cells including na ⁇ ve or activated lymphocytes, or na ⁇ ve or activated lymphocytes with no stem cells, or any mixture thereof.
  • allogeneic refers to "taken from different individuals of the same species". Two or more individuals are said to be allogeneic to one another when the genes at one or more loci are not identical.
  • An "allogeneic transplant” is a transplant from a donor who is not an identical genetic match.
  • an "allogeneic stem cell transplant” is a procedure in which bone marrow or peripheral blood stem cells from a donor (usually but not necessarily related) are collected, stored, and infused into a patient (recipient) following conditioning with chemotherapy and/or radiation therapy.
  • lograft refers to an allogeneic stem cell transplant.
  • a "xenograft” is a transplant of tissue or cells from a donor of one species to a recipient of another species.
  • heterograft and heterotransplant are also sometimes used, while the term homograft refers to a same-species transplant.
  • a "bone marrow transplant” is a procedure in which bone marrow is collected from a donor subject, stored, and infused (i.e., transferred, administered or injected) into a recipient subject, generally a subject or a patient in need of such treatment, usually following chemotherapy and/or radiation therapy.
  • a "stem cell transplant” is a therapeutic procedure in which stem cell- containing bone marrow or peripheral blood stem cells are collected from a donor subject, stored, and infused (i.e., transferred, administered or injected) into a recipient subject, generally a subject or a patient in need of such treatment, usually following chemotherapy and/or radiation therapy, in order to restore blood cell production.
  • PBSC transplant is the procedure in which blood containing mobilized stem cells is collected by aphaeresis, stored, and infused following chemotherapy and/or radiation therapy.
  • Activated cells or activated lymphocytes are cells or lymphocytes, respectively, obtained from a patient or a donor and which are activated in vitro with antibodies, cytokines or any other activating factors, e.g. IL-2, IL-7, IL-12, IL-18 or anti-CD3, with the purpose of increasing lymphocyte activity.
  • Lymphocyte activity is usually assessed by e.g. cell proliferation, which is measured through e.g. thymidine uptake or ELISA assays. Alternatively, activity may be evaluated by killing activity, measured through cytotoxic test such as e.g. 51 Cr release assay.
  • a “therapeutically effective amount” is determined by the severity of the disease in conjunction with the preventive or therapeutic objectives, the route of administration and the patient's general -condition (age, sex, weight and other considerations known to the attending physician).
  • the above described method is intended to be used to treat subjects suffering from hematopoietic cells deficiency disorders, congenital or acquired immune deficiencies, genetic disorders causing hemoglobinopathies, enzyme deficiency diseases, hematological malignancies, cancer, metastatic solid tumors and autoimmune diseases.
  • disorder refers to a condition in which there is a disturbance of normal functioning.
  • a “disease” is any abnormal condition of the body or mind that causes discomfort, dysfunction, or distress to the person affected or those in contact with the person.
  • the term is used broadly to include injuries, congenital malformations, disabilities, syndromes, symptoms, deviant behaviors, and atypical variations of structure and function, chronic or permanent health defects resulting from disease.
  • said method is intended to be administered to subjects suffering from hematopoietic cells deficiency disorders such as Severe Aplastic Anemia and osteopetrosis.
  • Aplastic anemia is not a single disease, but a group of closely related disorders characterized by the failure of the bone marrow to produce all three types of blood cells: red blood cells, white blood cells and platelets.
  • red blood cells red blood cells
  • white blood cells white blood cells
  • platelets red blood cells
  • the exact cause of aplastic anemia is unknown, although it has been linked to exposure to chemicals and radiation. It is also believed that some cases of aplastic anemia are inherited or are due to a viral infection.
  • Osteopetrosis is also known as Albers-Scho ⁇ berg Disease, Generalized Congenital Osteosclerosis, Ivory Bones, Marble Bones, Osteosclerosis Fragilis Generalisata. Osteopetrosis is a congenital disease characterized in each of its forms by defective osteoclast function. Osteopetrosis is a rare congenital disorder (present at birth) in which the bones become overly dense. There are several types of osteopetrosis of varying severity. Symptoms can include fractures, frequent infections, blindness, deafness, and stroke.
  • the method of treatment of the invention is also suitable for subjects suffering from congenital or acquired immune deficiencies.
  • Primary immune deficiency diseases are disorders in which part of the body's immune system is missing or does not function properly.
  • secondary immune deficiency disease in which the immune system is compromised by factors outside the immune system, such as viruses or chemotherapy, the primary immune deficiency diseases are caused by intrinsic or genetic defects in the immune system.
  • primary immune deficiencies There is a wide variety of primary immune deficiencies. Nearly 100 primary immune deficiency diseases have been identified, including X-linked agammaglobulinemia (Bruton's Disease), Common Variable Immune Deficiency Disease, Selective IgA Deficiency, Severe Combined Immune Deficiency (SCID, boy-in-the-bubble disease), Chronic Granulomatous Disease, Wiskott-Aldrich Syndrome, X-Linked Hyper IgM Syndrome, DiGeorge Syndrome, IgG Subclass Deficiency and Ataxia Telangiectasia. Some disorders, such as Selective IgA Deficiency are quite common, while others, such as Severe Combined Immune Deficiency, are very rare. Untreated primary immune deficiencies are characterized by frequent life-threatening infections and debilitating illnesses.
  • said method of treatment of the invention is also suitable for subjects suffering from genetic disorders causing hemoglobinopathies such as beta major thalassemia and sickle cell anemia.
  • a "hemoglobinopathy” is a genetic defect that results in abnormal structure of one of the globin chains of the hemoglobin molecule. Most of the hemoglobinopathies are not clinically apparent, and very few produce serious disease. The genetic defect may be due to substitution of one amino acid for another (as with the very common Hb S and Hb C and the great majority of the other abnormal hemoglobins), deletion of a portion of the amino acid sequence (Hb Gun Hill), abnormal hybridization between two chains (Hb Lepore), or abnormal elongation of the globin chain (Hb Constant Spring).
  • the abnormal chain that results may be the ⁇ chain.
  • Hb GpMkdeiphia ⁇ chain
  • Hb S, Hb C ⁇ chain
  • Hb F ⁇ e ⁇ as ⁇ chain
  • Hb A2Fiatbush ⁇ chain
  • Thalassemia is a genetic defect that results in production of an abnormally low quantity of given hemoglobin chain or chains.
  • the defect may affect the a, ⁇ , ⁇ or ⁇ chain, or may affect some combination of the ⁇ , ⁇ , and ⁇ chain in the same patient (but never the ⁇ and ⁇ chain together).
  • Sickle cell anemia is an inherited autosomal recessive condition that causes abnormal hemoglobin in blood cells, leading to infections and organ damage.
  • said method of treatment is suitable for subjects suffering from hematological disorders involving neoplastic proliferation of hematopoietic cells or hematological malignancies, metastatic solid tumors and autoimmune diseases such as Multiple sclerosis (MS), Rheumatoid Arthritis (RA), Systemic Lupus Erythematosus (SLE), Psoriasis and Psoriatic arthritis.
  • MS Multiple sclerosis
  • RA Rheumatoid Arthritis
  • SLE Systemic Lupus Erythematosus
  • Psoriasis Psoriatic arthritis.
  • a group of hematological disorders recommended to be treated with said method includes lymphoblastic leukemia, acute or chronic myelogenous leukemia, Hodgkin's lymphoma, Non-Hodgkin's lymphoma, myelodysplastic syndrome, multiple myeloma, and chronic lymphocytic leukemia, and said hematological disorder may be refractory to chemotherapy.
  • a "refractory disease” is a disease, for example a myeloma, which does not respond to initial therapy, as well as relapsed disease that does not respond to subsequent treatment. In this last instance, the disease may also be referred to as relapsed and refractory disease.
  • Another method of the invention involves treating immune-compromised subjects in need of adoptive transfer of donor immunity or passive immunity cell therapy against infectious agents.
  • the donor or donor immune competent cells are immunized against specific infectious agents, for example Hepatitis B, and said immunized cells or blood lymphocytes are transplanted into a recipient who is immune deficient as result of allogeneic stem cell transplantation, an organic cause, a genetic cause or an intentional treatment.
  • infectious agent is also called a “biological agent” and includes viruses, bacteria and parasites.
  • infectious agents against which the passive immunity cell therapy might be desired are Hepatitis B, HIV and CMV viruses, diarrhea causing agents, Tuberculosis, Malaria, Measles, Pertussis, Tetanus, poliomyelitis, diphtheria, Meningitis, Gram Negative bacteria and Tropical diseases causing agents.
  • Specific immune reactivity can be achieved by immunizing the donor with infectious agent antigenic peptides or with cancer antigens.
  • Adoptive donor immunity transfer using donor cells treated with an immune modulator agent may prevent GVHD while providing immune protection to the immune compromised recipient.
  • Cytotoxic T cell therapy consists of subjecting a lymphocyte population isolated from a patient or a donor, to sensitization for several times with a target, which may be an infectious agent, tumor tissues or peptide fragments of protein(s) specifically found in said target. This 1 sensitization produces a T-cell population with specific cytotoxic activity. This cell population is then returned or transplanted into the patient for destruction of the targeted infectious agent or cancer cells.
  • a target which may be an infectious agent, tumor tissues or peptide fragments of protein(s) specifically found in said target.
  • This 1 sensitization produces a T-cell population with specific cytotoxic activity.
  • This cell population is then returned or transplanted into the patient for destruction of the targeted infectious agent or cancer cells.
  • the invention also relates to a method for the prevention, treatment and prevention of recurrence of proliferative diseases by administering to a subject in need matched, partially or completely mismatched allogeneic or xenogeneic cell transplant wherein said cells were treated ex vivo with CpG, LPS, Mycobacterium Tuberculosis components, diffceria toxin, aluminum salts (alum), and a biological or synthetic factor that affectsThl/Th2 cytokine balance prior to transplantation or were obtained from a donor who was treated with a substance selected from the group consisting of CpG, CFA, IFA, CpG in IFA, LPS in IFA, Montanide, Muramil dipeptide, difteria toxin, QS21, and aluminum salts (alum), prior to cell harvesting.
  • an “in vivo” treatment refers to a process that takes place within a living organism.
  • An “ex vivo” treatment relates to a process taking place outside of a living organism or body, e.g. the treatment of cells, which treated cells are returned to the same or to a different living organism.
  • Said method may be applied to treat malignant or non-malignant proliferative diseases and consists of transplanting any kind and source of cells commonly used in immunotherapy for example, donor lymphocyte infusion (DLI) or adoptive transfer of donor immunity, preferably selected from the group of bone marrow cells, mobilized blood cell, cord blood or embryonic stem cells transplant including na ⁇ ve or activated lymphocytes and na ⁇ ve or activated lymphocytes transplant with no stem cells or any mixture thereof.
  • DLI donor lymphocyte infusion
  • donor immunity preferably selected from the group of bone marrow cells, mobilized blood cell, cord blood or embryonic stem cells transplant including na ⁇ ve or activated lymphocytes and na ⁇ ve or activated lymphocytes transplant with no stem cells or any mixture thereof.
  • allografts come from a healthy individual other than the patient, they have the benefit of not containing tumor cells.
  • a potential benefit of allogeneic transplants is their ability to help the patient fight against the tumor. Just as immune cells in the allograft may attack the patient's tissue, they also help attack the tumor, a phenomenon referred to as a graft-versus-tumor (GVT) and graft-versus-leukemia (GVL) effect. This effect may account in part for the lower relapse rates seen following allogeneic transplants compared to autologous transplants.
  • GVT graft-versus-tumor
  • VTL graft-versus-leukemia
  • Allogeneic stem cell transplants are more effective in preventing cancer recurrences than autologous transplants because the donor cells recognize the cancer as foreign and kill the cancer cells.
  • the present method of treatment is also suitable for treating cancer in a subject in need without causing any acute GVHD effect, comprising administering to said subject a therapeutically effective dose of immune system donor cells which have been pre-treated with an immune stimulatory agent, preferably an immune modulator agent prior to administration to said subject.
  • GVHD graft- versus -host disease
  • transplants When donor immune T cells recognize the recipient's cells as foreign and mounting an attack against the host's tissues. Graft-versus-host disease is seen most often in cases where the donor is unrelated to the patient or when the donor is related to the patient but not a perfect histocompatibility match.
  • acute GVHD which occurs soon after the transplant (during the first three months) when the number of white cells increases.
  • the tissues affected are skin, liver, stomach, and/or intestines.
  • Chronic GVHD develops after the third month post-transplant, and in this condition glands may also be affected. Chronic GVHD is more common in patients whose donor is unrelated or whose marrow is not perfectly matched.
  • the method of the invention may also be applied to subjects suffering from malignant disorders such as breast cancer, bladder cancer, lung cancer, prostate cancer, thyroid cancer, leukemias, multiple myeloma, lymphomas, colon cancer, glioma, seminoma, liver cancer, pancreatic cancer, renal cancer, cervical cancer, testicular cancer, head and neck cancer, ovarian cancer, neuroblastoma and melanoma or a metastatic solid tumor.
  • malignant disorders such as breast cancer, bladder cancer, lung cancer, prostate cancer, thyroid cancer, leukemias, multiple myeloma, lymphomas, colon cancer, glioma, seminoma, liver cancer, pancreatic cancer, renal cancer, cervical cancer, testicular cancer, head and neck cancer, ovarian cancer, neuroblastoma and melanoma or a metastatic solid tumor.
  • CML chronic myeloid leukemia
  • ALL acute lymphocytic leukemia
  • AML acute myeloid leukemia
  • PML promyeloid leukemia
  • the method may further comprise administering to the recipient subject an additional antineoplastic or immunosuppressant agent before or after introduction of the said donor cells, in an amount effective to control acute GVHD and prevent rejection of the said donor cells.
  • Additional anti-neoplastic or immunosuppressant agents are adjunctive agents, alkylating agents, antimetabolites, hormones, miscellaneous antineoplastic drugs or immunosuppressant agents selected from the group of Mycophenolate Mofetil, Cyclosporine, Azathioprine, Cyclosporine analogues, Prednisone, Tacrolimus, Sirolimus, Cyclophosphamide, FTY 720, ionizing radiation, anti-lymphocytic agents, anti-costimulatory molecules, and 2CdA (2- chloro-2'deoxyadenosine).
  • an “immunosuppressive agent” is a drug or treatment given to suppress a patient's immune system, such as one given to prevent rejection of transplanted tissue
  • analogue refers to compounds derived or obtained from another and containing essential elements of the parent substance, capable of functioning or producing the same intended action or effect as said parent substance.
  • the invention also describes a method of preventing or ameliorating graft- versus-host disease which comprises administering to a mammal in need of cell transplant treatment, a therapeutically effective amount of donor cells treated with an immunomodulator agent selected from the group consisting of CpG, LPS, Mycobacterium Tuberculosis components, aluminum salts (alum), and a biological or synthetic factor that might affect Thl/Th2 cytokine balance ex ⁇ i ⁇ o or with IFA or CFA or treated with one of CpG , CpG in IFA, LPS in IFA, Montanide, Muramil dipeptide, difteria toxin, QS21 or aluminum salts (alum) in vivo, wherein said treatment is applied to the donor before said harvest or to donor cells post-harvest and said treatment is also effective in maintaining or promoting graft-versus-tumor and graft-versus-leukemia effects in said mammal.
  • an immunomodulator agent selected from the group consisting of CpG
  • graft-versus-tumor and "graft-versus-leukemia” relate to the beneficial effect of allogeneic transplants resulting from the donor cells mounting an attack on the recipient's tumor or leukemia cells.
  • Another method of the invention is a method for reducing the probability of graft-versus-host disease following bone marrow transplantation whilst maintaining or promoting graft-versus-tumor effect and graft-versus-leukemia effect, which comprises administering to a mammal in need of bone marrow transplantation a therapeutically effective amount of donor cells treated with IFA, CFA, CpG, CpG in IFA, LPS in IFA, Montanide, Muramil Dipeptide, difteria toxin, QS21 or aluminum salts (alum) in ⁇ i ⁇ o or ex- ⁇ i ⁇ o with CpG, LPS, Mycobacterium Tuberculosis components, difteria toxin, a biological or synthetic factor that might affect Thl/Th2 cytokine balance, or aluminum salts (alum) prior to transplantation.
  • the graft-versus-tumor effect and the graft- versus-leukemia effect are manifested by the elimination of cancer cells
  • the donor cells used in the methods described above are pre-treated between 0 to 30 days prior to the day of the transplant, preferably between 5 to 20 days, or even 6, 10 or 12 days prior to transplant.
  • Said donor cells may be allogeneic cells derived from an organ donor or bone -marrow donor partially or completely mismatched allogeneic or xenogeneic bone marrow cells, mobilized blood cells, cord blood or embryonic stem cells including na ⁇ ve or activated lymphocytes, or naive or activated lymphocytes with no stem cells or any mixture thereof.
  • donated stem cell grafts especially those obtained from umbilical cord blood, or when the donor is a young individual, contain insufficient number of stem cells for preparing the donor cells for transplant.
  • these may be cultivated in a culture system outside the body.
  • the procedure for growing stem cells outside the body is called expansion, and comprises culturing the donor cells in the presence of at least one of hormones, growth factors and/or cytokines, which induce said cells to divide and multiply.
  • Stem cells normally circulate in the blood in very small quantities. Cytokines administration causes substantial increase in the number of circulating blood stem cells for collection. The process of delivering a cytokine or growth factor for the purpose of collecting stem cells is referred to as "stem cell mobilization".
  • T cell depletion Removal of T-lymphocytes from bone marrow stem cell collections, also known as T cell depletion, reduces the severity or incidence of graft-versus-host disease in patients undergoing allogeneic stem cell transplant. However, it also associated with increased graft failure. In contrast, as shown by the present inventors, engrafting was successful when the transplant, i.e. the donor cells were treated with immune modulator agents as proposed by this invention (data not shown).
  • the treated donor cells of the invention used for transplantation and cell therapy generally originate from the same species as the transplanted subject.
  • donor cells are selected from allogeneic lymphocytes obtained from a family member, a matched unrelated donor or an intentionally mismatched related or unrelated donors.
  • the methods of the invention may be efficient to treat mammalian subjects.
  • "Mammal” or “mammalian” for purposes of treatment refers to any animal classified as a mammal including, human, research animals, domestic and farm animals in particular pigs, and zoo, sports, or pet animals, such as dogs, horses, cats, cows, etc.
  • the mammal is human.
  • the treated donor cells of the invention may be administered to patients in the form of a peripheral blood mononuclear cell preparation.
  • Cells are usually collected via apheresis, followed by Ficoll-Hypaque gradient, in order to obtain PBMC.
  • the PBMCs may be injected as is, or following activation/expansion.
  • cells are resuspended in physiological solution, e.g. saline, and injected usually intravenously or intra-thecal.
  • donor lymphocyte infusion between 10 3 to 10 9 cells/kg (of recipient) are injected.
  • CD34+ cells are injected, usually >10 4 cells/kg.
  • Suggested doses for CpG oligonucleotide treatment are usually between 10- 2000 ⁇ g/dose, preferably between 50-1000 ⁇ g/dose, more preferably 100- 700 ⁇ g/dose, in one or multiple doses.
  • 500 ⁇ g/dose of CpG is administered in one dose, 0 to 30 days prior to harvest, preferably between 5 to 20 days.
  • IFA or its substitutes Montanide, Muramil Dipeptide and QS21
  • one or multiple doses of 50-500 ⁇ l/dose preferably 300 ⁇ l/dose are generally used.
  • CFA is administrated in one or multiple doses of 50-600 ⁇ l/dose, preferably 300 ⁇ l/dose containing 0.3 mg of mycobacterium.
  • Doses of detoxified LPS tolerated in humans consist of 25 micrograms given intramuscularly or three 1.5 mg doses of LPS extracted from bacteria given intranasal into each nostril. All of these substances are diluted in a solution suitable for in ⁇ i ⁇ o injection, e.g saline, PBS or water for injection.
  • treatment of the donor cells, or more specifically of the donor subject may be effected with only one administration of the immune modulator agent, e.g., CpG, which makes the method of preparing donor cells provided in the present invention much more feasible and compatible with donor compliance. ' .
  • the immune modulator agent e.g., CpG
  • the invention also relates to a method for induction of transplantation tolerance in a subject in need of a tissue or organ transplant, comprising the step of treating donor with IFA, CFA, CpG, CpG in IFA, LPS in IFA, Montanide, Muramil dipeptide, difteria toxin, QS21, and aluminum salts (alum) in vivo or donor stem cells with CpG, LPS, Mycobacterium Tuberculosis components, a biological or synthetic factor that affects Thl/Th2 cytokine balance, and aluminum salts (alum) ex vivo and transplanting them into recipient to induce donor- specific tolerance to all organ and tissue.
  • Said tolerance should be induced when the transplant is an allograft or a xenograft.
  • One further advantage of the method of the invention is that, as shown in the Examples below, and especially in Figure 3, pre-treatment of the donor resulted in a high proportion of animals with long-term GVHD-free survival. As shown, recipients survived for more than 200 days following treatment. It should be appreciated considering the normal lifespan of mice (the model system used herein), 200 days is a significant amount of time.
  • Figure 5 shows disease-free (no GVHD and no leukemia) survival for at least 90 days and even for 160 days, ensuring the anti r leukemia effect conferred by the donor cells prepared by the method of the invention.
  • the administration of the treated donor cells to the recipient is performed before, together with or after transplantation of the organ or tissue to which tolerance is induced.
  • “Tolerance”, “immunotolerance”, “immunological tolerance”, or “immune tolerance” is the acquired inability to respond with an immune reaction to an antigen to which the organism would normally r'espond. Such tolerance may be induced by exposing an animal to the antigen at a very early stage of life, prior to maturation of the immune system, or, in adults, by exposing the animal to repeated low doses of a weak protein antigen (low-zone tolerance), or to a large amount of an antigen (high-zone tolerance). Transplantation tolerance to major histocompatibility (MHC) antigens can be induced after conditioning the host with chemo- and/or radiotherapy.
  • MHC major histocompatibility
  • immune modulator agent-treated stem cell transplantation to induce donor-specific tolerance to organs and tissue allografts is relevant for transplants which originate from living related donors or cadavers which are kept alive artificially for a few days. Donors might be treated before the stem cell and organ harvest.
  • Induction of tolerance by said method may be pertinent when other mammals, for example pigs, will serve as donors.
  • Another aspect of the invention relates to a composition for cellular immunotherapy (DLI) and adoptive transfer of donor immunity therapy for treating hematological disorders, immune deficiencies, autoimmune diseases and malignant or non-malignant proliferative disorders in .
  • a mammalian subject in need comprising as an active ingredient donor cells pre-treated with CpG, LPS, Mycobacterium Tuberculosis components, difteria toxin, and a biological or synthetic factor that affects Thl/Th2 cytokine balance or aluminum salts (alum) ex vivo or cells harvested from a donor treated with IFA, GFA, CpG, CpG in IFA, LPS in IFA, Montanide, Muramil dipeptide, difteria toxin, QS21, or aluminum salts (alum) in vivo, wherein said composition prevents relapse of a tumor without triggering GVHD, and may further comprise a pharmaceutically acceptable carrier, excipient or diluent.
  • compositions are well known in the art and has been described in many articles and textbooks, see e.g., Remington's Pharmaceutical Sciences, Gennaro A. B. ed., Mack Publishing Co., Easton, PA, 1990, and especially pp. 1521-1712 therein, fully incorporated herein by reference.
  • composition of the invention is suitable for administration directly to the subject to be treated.
  • Formulations typically comprise at least one active ingredient (e.g. the donor cells), as defined above, together with one or more acceptable carriers thereof.
  • Each carrier should be both pharmaceutically and physiologically acceptable in the sense of being compatible with the other ingredients and not injurious to the patient.
  • compositions must be stable and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi.
  • compositions of the invention generally comprise a buffering agent, an agent that adjusts the osmolarity thereof, and optionally, one or more pharmaceutically acceptable carriers, excipients and/or additives as known in the art.
  • Supplementary active ingredients can also be incorporated into the compositions.
  • acceptable carrier includes any and all solvents, dispersion media, antibacterial and antifungal agents and the like. The use of such media and agents for pharmaceutical active substances is well known in the art.
  • the preferred dosage would be individualized to the patient following good laboratory practice and standard medical practice.
  • the decision as to the particular dosage to be employed (and the number of times to be administered per day) is within the discretion of the physician, and may be varied by titration of the dosage to the particular circumstances of this invention to produce the desired therapeutic effect.
  • the dose will depend on weight, age, sex, severity of the disease and tolerability, and will be determined by the attending physician.
  • the therapeutic agent (the donor cells or a composition comprising thereof) should be delivered in a sufficient dose as defined herein.
  • Preferred means of administration are intravenous, parenteral, intratechal or intra-tumor. Administration may, depending on the case, also be done by organ perfusion, catheterization through blood vessels to the target organ, or through direct injection into an organ.
  • composition of the invention may be administered alone, or in combination with other active ingredients that improve the therapeutic effect, whether administered in combination, serially or simultaneously.
  • the invention also describes the use of an immuno modulator substance selected from the group consisting of CpG, IFA, CFA, CpG in IFA, LPS in IFA, LPS, Mycobacterium Tuberculosis components, Montanide, Muramil Dipeptide, difteria toxin, QS21, aluminum salts (alum) and a biological or synthetic factor that affects Thl/Th2 cytokine balance, in the preparation of a composition for the pre-transplantation treatment of donor or cell transplants prior to transplantation into a recipient, wherein said treatment modulates immune responses of said donor cell.
  • an immuno modulator substance selected from the group consisting of CpG, IFA, CFA, CpG in IFA, LPS in IFA, LPS, Mycobacterium Tuberculosis components, Montanide, Muramil Dipeptide, difteria toxin, QS21, aluminum salts (alum) and a biological or synthetic factor that affects Thl/Th2 cytokine
  • a cell As used in the specifications and the appended claims and in accordance with long-standing patent Law practice, the singular forms “a” “an” and “the” generally mean “at least one”, “one or more”, and other plural references unless the context clearly dictates otherwise.
  • a cell For example “a cell”, “a peptide” and “an immune modulator agent” include mixture of cells, one or more peptides and a plurality of adjuvants of the type described.
  • mice Female BALB/c H-2 ⁇ * (BALB), C57BL/6 ⁇ .-2* (C57) and (BALB/c x C57BL/6) Fi H-2 d/b (F 1 ) mice aged 10-12 weeks, weighing 22-24 grams were used in this study. All mice were purchased from Harlan, Israel, and maintained in the animal facility of the Hadassah University Hospital with sterilized food and water ad libitum, in full compliance with the regulations for the protection of animal rights.
  • BCLi B cell leukemia/lymphoma
  • Recipient Fi mice were conditioned with non-lethal total body irradiation (TBI) of 4Gy, using a 6MEV linear accelerator at a dose rate of 1.9 Gy/min.
  • TBI non-lethal total body irradiation
  • 6MEV linear accelerator at a dose rate of 1.9 Gy/min.
  • Non- lethally irradiated recipients were inoculated (IV) with 3OxIO 6 C57BL/6 splenocytes 24-48 hours later.
  • Recipient F 1 mice were conditioned with TBI- (4Gy) 24h prior to intravenous inoculation with 10 4 BCLi cells. On the following day, Fi mice were injected intravenously with 3OxIO 6 na ⁇ ve C57BL/6 splenocytes or with C57BL/6 splenocytes treated 4 or 10 days pre -transplant as specified in each experiment.
  • CFA Complete Freund's adjuvant
  • IFA incomplete Freund's adjuvant
  • LPS Lipopolysaccharide
  • Splenocytes were harvested as described in each experiment, and applied for GVHD induction.
  • Hematopoietic stem cells e.g. obtained from bone marrow, were also used as donor cells (data not shown).
  • mice were checked daily for the appearance of GVHD symptoms such as hunched posture, ruffled fur, diarrhea and cachexia.
  • mice inoculated with BCL 1 cells were tracked for leukemia development by serial blood counts and enlarged spleen.
  • MLR Mixed Lymphocyte Reaction
  • One million responding cells isolated from normal peripheral lymph nodes were cultured in flat-bottom 96 microculture wells (Nunc A/S, Denmark) with IxIO 6 stimulator lymphocytes derived from normal spleens, in a total volume of 0.25ml. Stimulator cells were inactivated by a single in vitro exposure to 50 Gy from a radioactive cesium-137 source.
  • One million irradiated spleen cells from CFA treated C57BL/6 mice or 1 x 10 6 spleen cells from naive mice were added to MLR cultures in order to check for the presence of suppressive activity.
  • Co- cultured cells were inactivated by 15 Gy prior to culturing, to eliminate proliferative responses directed to the responding or stimulating cells.
  • the culture medium consisted of RPMI 1640 medium supplemented with 10% human AB serum, L-glutamine (2mM), 2-mercaptoethanol (5xl0" 5 M), penicillin (100 U/ml), and streptomycin (lOO ⁇ g/ml).
  • Tissue culture media and reagents were purchased from Biological Industries, Beit Haemek, Israel. Cells were incubated at 37 0 C with 5% CO2/air mixture in a humidified incubator for 72 hours, and then pulsed for 16-18 hours with l ⁇ Ci of tritiated thymidine (5.0 Ci/immol; 185 GBQ/mmol, Amersham Biosciences, UK) per well.
  • splenocytes 5X105
  • FITC Fluorescein Isothiocyanate
  • Gr-I Fluorescein Isothiocyanate
  • PE R-Phycoerythrin
  • DX5 anti-Pan NK
  • PC61 anti-Fas
  • PC61 PerCP-Cy5.5 labeled anti-CDllb
  • GVHD was induced in (BALB/c x C57BL/6)F1 recipients by inoculation with parental C57 splenocytes derived from either na ⁇ ve or pre-treated donors.
  • Na ⁇ ve or IFA pre-treated donor derived splenocytes caused marked GVHD- related body weight loss of about 5 grams in the Fl recipients.
  • Pre-treatment of donor mice with CFA or LPS led to a slight, transient reduction in body weight ( ⁇ 2 grams) of the Fl hosts.
  • a MLR was carried out by culturing IXlO 6 lymph node-derived cells as responders with IXlO 6 irradiated (50Gy) splenocytes as stimulators, for 4 days.
  • Irradiated (1.5Gy) spleen cells (IXlO 6 ) derived either from na ⁇ ve C57 mice or from C57 mice treated with CFA 10 days before cell harvesting, were added to the MLR as co-cultured cells.
  • a MLR was carried out by culturing IXlO 6 T cells as responders with IXlO 6 irradiated (50Gy) splenocytes as stimulators, for 4 days.
  • Irradiated (1.5Gy) spleen cells (IXlO 6 ) derived from either na ⁇ ve C57 mice or from C57 mice treated with CpG 6 days before cell harvesting, or with CpG+IFA 10 days before cell harvesting, were added as co- cultured cells to the MLR.
  • IXlO 6 Irradiated (1.5Gy) spleen cells
  • c % suppression was calculated, after subtraction of the syngeneic response, by the following formula: 100- [cpm of allogeneic response in the presence of CpG treated co- cultured cells / na ⁇ ve co-cultured cells XlOO].
  • C57 splenocytes harvested 4 days following CFA treatment or 10 days following IFA treatment were much less effective in preventing GVHD, and only 29% (10/34) and 21% (24/112) mice, respectively, remained GVHD-free (Table 3).
  • Pre-transplant treatment of C57 donor mice with soluble LPS led to severe GVHD with 50% (5/10) mortality in sub-lethally irradiated Fl.
  • IFA, CFA and CpG+IFA were injected subcutaneously to Fl host mice 10 days before TBI (4-5Gy). 24h later, 30X10 6 C57 splenocytes were inoculated intravenously.
  • Sub-lethally irradiated (4Gy) Fl (BALB/C .x 057 Bl/6) mice were inoculated intravenously with 10 4 BGLi cells one day following irradiation. 24h later, naive or CFA treated C57BL/6 donor splenocytes (3OxIO 6 ) were inoculated intravenously. CFA was injected sub-cutaneously into donor mice on day 10 before spleen harvesting.
  • mice inoculated with either BCLi alone or inoculated with BCLi and treated with na ⁇ ve C57BL/6 splenocytes died of leukemia (median 29 days) or early acute GVHD (median 17 days).
  • CFA- treated donor splenocytes were able to prevent early GVHD but could not prevent late GVHD (23/27 mice died of late GVHD in a median of 62 days).
  • CFA treatment of donor mice induced an efficient GVL effect in the primary hosts (1/27 mice died of leukemia) and also proved effective in the adoptive transfer experiments shown in Table 6.
  • Adoptive Transfer experiments were performed in order to verify the anti-leukemia effect of the treated donor cells, while by-passing GVHD.
  • Table 6 Effect of pre-transplant donor treatment with CFA on graft versus leukemia (GVL) effect in adoptive transfer (AT) experiments
  • CFA- treated donor splenocytes harvested 14 or 21 days following BCLi inoculation did not cause leukemia in secondary BALB hosts for up to >230 days period, while splenocytes from control mice harvested 14 or 21 days following inoculation of BCLi alone, caused leukemia-related death in all secondary BALB hosts within a median of 42 and 24 days, respectively. It is worth noting that none of the spleens of primary hosts treated with na ⁇ ve C57BL/6 cells were available for adoptive transfer experiments carried out 21 days following BCLi inoculation, due to early acute" GVHD mortality, while the spleens of CFA-treated C57BL/6 donor mice in similar adoptive transfer experiments showed an efficient GVL effect.

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Abstract

L’invention se rapporte ci-après à des cellules donneuses qui, après transplantation, ont une capacité réduite ou abrogée à générer une réponse GVHD chez le receveur. Un procédé de préparation desdites cellules est décrit, ainsi qu’une composition les comprenant. En outre, lesdites cellules sont adaptées à un grand nombre de maladies, en particulier un trouble lié à une déficience des cellules hématopoïétiques, une déficience congénitale ou un déficit immunitaires acquis, une anomalie génétique génératrice de l’hémoglobinopathie, un déficit enzymatique, des malignités hématologiques, un cancer, des tumeurs solides métastatiques ou des maladies auto-immunes. Lesdites cellules donneuses sont essentiellement adaptées pour être administrées chez tout sujet exigeant une immunothérapie cellulaire ou une transplantation d’immunothérapie adoptive.
PCT/IL2006/000452 2005-04-14 2006-04-10 Traitement de pre-transplantation de cellules donneuses permettant le controle de la maladie du greffon contre l’hote (gvhd) chez des receveurs transplantes Ceased WO2006109300A1 (fr)

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