WO2006109300A1 - Pre-transplantation treatment of donor cells to control graft versus host disease (gvhd) in transplant recipients - Google Patents

Abstract

Described herein are donor cells which, upon transplant, have reduced or abrogated capacity to generate a GVHD response in the recipient. A method for preparing said cells is described, as well as a composition comprising thereof. Furthermore, said cells are suitable for treatment of a number of diseases, particularly hematopoietic cell deficiency disorder, congenital or acquired immune deficiency, a genetic disorder causing hemoglobinopathy, an enzyme deficiency disease, hematological malignancies, cancer, metastatic solid tumors or autoimmune diseases. Essentially, said donor cells are suitable for administration to any subject in need of cell immunotherapy or adoptive transfer immunotherapy transplant.

Description

PRE-TRANSPLANTATION TREATMENT OF DONOR CELLS

TO CONTROL GRAFT VERSUS HOST DISEASE (GVHD)

IN TRANSPLANT RECIPIENTS

Field of the Invention

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.

Background of the Invention

All publications mentioned throughout this application are fully incorporated herein by reference, including all references cited therein.

Allogeneic bone marrow transplantation (allo-BMT) 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]. However, the major obstacle in allo-BMT is acute graft-versus-host disease (GVHD), which is caused by T cells present in the donor marrow graft [Lum L.G. (1987) Blood 69: 369-380].

Graft versus host disease (GVHD) is one of the major complications associated with allogeneic stem cell transplantation and donor lymphocyte infusion (DLI), which are the treatments of choice for a large number of malignant and non- malignant diseases [Sullivan K.M. et al., (1989) Blood 73: 1720-1728 and Erratum in: (1989) Blood 74: 1180; Horowitz MM. (1990) Blood 75: 555-562; Slavin S. et al., (1996) Blood 87: 4011-4014; KoIb H.J. et al., (1990) Blood 76: 2462-2465; Childs R. Et al., (2000) N. Engl. J. Med. 343: 750-758; Slavin S. (2001) Lancet Oncol. 2: 491-498]. Morbidity and mortality related to GVHD limit the clinical application of allogeneic cell therapy (alloCT) which aims to induce graft versus leukemia (GVL) or graft versus tumor (GVT) effects [Slavin S. (2001) Lancet Oncol. 2: 491-498; Slavin S. et al., (2002) J. Hematother Stem Cell Res. 11: 265-276, Cohen P. Et al., (1993) J. Immunol. 151: 4803-4810; Morecki S. et al., (1998) Cancer Res. 58: 3891-3895; Burroughs L. et al., (2005) Curr. Opin. Hematol. 12: 45-54]. Therefore, controlling GVHD while securing the ability of the allograft to complete hematopoietic reconstitution and to exert an anti-tumor effect, is of great therapeutic importance.

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]. During GVHD, 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].

After transplantation, 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-α and IL-I) and consequently ThI and Th2 cytokines secretion becomes deregulated [Fowler D.H. and Gress R.E. (2000) Leuk. Lymphoma 38: 221-234; Nikolic B. Et al., (2000) J. Clin. Invest. 105: 1289-1298; Liu J. et al., (2001) Blood 98: 3367-3375; Devetten M.P. and Vose J.M. (2004) Biol. Blood Marrow Transplant. 10: 815-825]. 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]. A single injection of CFA downregulates auto-reactive cytotoxic T cells and stimulates cytotoxic NK cells in the non-obese diabetic (NOD) mice experimental autoimmune diabetes model [Lee LF. et al., (2004) J. Immunol. 172: 937-942]. The mycobacterial component in the CFA appears to play a key role in the immunoregulatory process, since no regulatory activity is usually attributed to the incomplete Freund's adjuvant (IFA) constituent [Qin H.Y. et al., (1993) J. Immunol. 150: 2072-2080].

Bacterial and other infectious agents are detected and recognized by Toll-like receptors (TLR). TLRs also recognize endogenous ligands which are induced during the inflammatory response and trigger the host's innate and adaptive immune system [Akira S. et al., (2001) Nat. Immunol. 2: 675-680; Iwasaki A and Medzhitov R. (2004) Nat. Immunol. 5: 987-995].

Attempts to alleviate GVHD development while preserving the hematopoietic stem cells and potential GVL/GVT effects, usually rely on host treatment or, ex vivo donor cells pre-transplant treatment [Sykes M. et al., (19910) Proc. Natl. Acad. Sci. USA 87: 5633-5637; Sykes M. et al., (1995) Blood 86: 2429-2438; Reddy P. et al., (2001) J. Exp. Med. 194: 1433-1440; Murphy W.J. et al., (1998) J. Clin. Invest. 102: 1742-1748; Morecki S. and Slavin S. (2000) J. Hematother Stem Cell Res. 9: 355-366; Naparstek E. et al., (1999) Exp. Hematol. 27: 1210- 1218; Champlin R.E. et al., (2000) Blood 95: 3996-4003; Grcevic D. et al., (1999) Bone Marrow Transplant. 23: 1145-1152]. In a recent study, interleukin 18 (IL-18) was used for the treatment of the donor prior to the harvest of the transplant. IL-18 treatment modulated GVHD and GVL responses, via induction of Th2-polarization. However, survival follow-up of the mice studied was only 30-50 days, and the treatment included a series of ten injections to the donor [Reddy P. et al., (2003) Blood 101: 2877-2885].

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]. For example, 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. Synthetic CpG sequences that mimic those found in pathogens are capable of binding to and activating TLR9. Moreover, 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.

It is the intention of the present invention to provide 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.

Summary of the Invention

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 naϊve 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.

When necessary, 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.

Further, 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.

In addition, 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.

In the above method, 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.

In certain cases, said hematological disorder is refractory to chemotherapy. Another feature of the present invention is that 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.

The donor cells are preferably administered to said subject in need in the form of a peripheral blood mononuclear cell preparation.

Even further, 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.

In the present invention, said cells or the composition comprising thereof are preferably administered intravenously, parenterally, intratechally or intra- tumor.

Finally, 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. In particular, 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..

Brief Description of the Figures

Figure 1: Effect of CFA and CpG treatments on spleen size

Complete Freund's adjuvant (CFA), Incomplete Freund's Adjuvant (IFA) or CpG and its non-CpG control given with IFA, were injected subcutaneously into C57 mice. Ten days later, spleens were harvested and number of cells determined. Number of spleen cells harvested from C57 mice inoculated with CpG and its non-CpG control given without IFA, were determined on day 6 (*) and 10 following inoculation. All CFA and CpG treatments resulted in a statistically significant increase in number of spleen cells as compared to their paired controls (p<0.03). There was no statistically significant difference between spleens harvested on day 6 versus day 10 following treatment with CpG (p>0.05). Results are expressed as a mean ±se of cell number per spleen, calculated on the basis of at least 25 spleens in 4 separate experiments.

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 30X10⁶ splenocytes derived from either naϊve or C57 donor mice treated 10 days previously with CFA or IFA (0.2ml) (Fig. 2A), CpG alone or non-CpG alone (lOOμg) (Fig. 2B), LPS (40μg) or LPS (40μg)-emulsified in IFA (Fig. 2C); and CpG or non-CpG (lOOμg) 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 30X10⁶ splenocytes derived from either naϊve or C57 donor mice treated 6 or 10 days previously with IFA (0.2ml) (Fig. 3A), CpG alone or non-CpG alone (lOOμg) given 6 days prior to harvesting (Fig. 3B), CpG alone or non-CpG alone (lOOμg) given 10 days prior to harvesting (Fig. 3C); and CpG or non- CpG (lOOμg) emulsified in IFA 10 days prior .to harvesting (Fig. 3D). All compared pairs in this Figure showed statistically significant differences (p<0.05): p=0.016 for the comparison of CpG alone given 6 days versus CpG alone given 10 days pre-transplant; p=0.006 for the comparison of CpG in emulsion of IFA versus CpG alone given 10 days pre-transplant. No statistically significant difference was observed in the comparison of CpG alone given 6 days pre-harvesting and CpG in emulsion of IFA given 10 days pre- harvesting (p=0.466).

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 30X10⁶ splenocytes derived from either naϊve 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 (lOOμg) (Fig. 4B), and 10 days previously with CpG or non-CpG (lOOμg) 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.

Figure 5: Survival of (BALB/CXC57BL/6) Fl mice inoculated with B cell leukemia (BCLl)

Graph showing percentage survival of disease-free (no GVHD and no leukemia) of three groups of mice following BCLl inoculation: BCLl alone, BCLl+naϊve cells, and BCL1+C57 cells treated with CpG in IFA. 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. Detailed Description of the Invention

The following abbreviations are used herein:

AT Adoptive Transfer

BALB BALB/c

BCG Bacillus Calmette-Guerin

BCLl B-cell leukemia

BMT Bone Marrow Transplant

C57 C57BL/6

CFA Complete Freund's adjuvant

CpG DNA sequences commonly present in bacteria and viruses

DLI Donor Lymphocyte Infusion

Fl BALB/C X C57BL/6

GVHD Graft versus Host Disease

GVL Graft versus Leukemia

GVT Graft versus Tumor

IFA Incomplete Freund's adjuvant

IV Intravenous

LPS Bacterial cell wall Lipopolysaccharides

MDP Muramil dipeptide

MLR Mixed Lymphocyte Reaction

PBSC Peripheral blood stem cells

TLR Toll-like receptors

TNF Tumor Necrosis Factor

In an attempt to overcome the event of GVHD in patients that undergo cellular immunotherapy aimed at eradicating malignant or non-malignant pathological disorders, the present inventors have developed a novel approach, which involves the treatment of donor cells in order to make these resistant or unable of triggering an immune reaction against the recipient.

As shown herein below in the Examples, treatment of parental C57 donor mice with mycobacteria, LPS emulsified in IFA, or synthetic ODN containing the CpG motif, before inoculating their splenocytes into irradiated Fl host mice, successfully abrogated their capability to induce GVHD. Most of the Fl mice inoculated with these splenocytes had a slight and transitional loss of body weight that was fully reconstituted whereas the control mice died of acute GVHD. Following inoculation of splenocytes derived from G57 mice pre-treated with CpG+IFA, CpG alone, CFA+IFA and LPS+IFA, 95%, 89%, 77% and 70% of Fl hosts, respectively, remained GVHD-free and maintained a normal health profile for >200 days.

Thus, in a first aspect, 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.

Further to harvesting the cells may be optionally phenotyped, through the identification of expressed cell markers, and still further selected prior to injection.

In essence, 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.

By the term "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.

Recommended immune 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.

By "transplantation" it is meant transferring a healthy cell, tissue or organ to replace a damaged one (cell, tissue or organ).

The terms "immunomodulator 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). More specifically in the present invention, 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.

The term "adjuvant" taken from the Latin word adjuvans means to help, particularly to reach a goal. In immunology the term "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. Their mode of action is either non-specific, resulting in increased immune responsiveness to a wide variety of antigens, or antigen- specific, i.e., affecting the immune response restricted to a narrow group of antigens. The therapeutic efficacy of many biological response modifiers is related to their antigen-specific immunoadjuvanticity.

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).

For its known abilities to stimulate the immune system, 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]. Interestingly, as shown in the Examples below, the present inventors show that CpG is a potent modulator of immune response also when administered alone to the donor.

The present invention presents CpG as a safe and clinically proved immunoregulator for the treatment of donor or donor cells.

In medicine, 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.

Various 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. Such adjuvants are also well known in the art, e.g. in US 6,861,410.

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.

The terms "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 naϊve or activated lymphocytes, and naϊve 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).

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.

Peripheral blood stem cells (PBSC) are stem cells collected from the blood. The term "peripheral" means that the cells come from outside the bone marrow. The procedure of obtaining stem cells from peripheral blood is called aphaeresis.

In another aspect 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.

As specified above, 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 naϊve or activated lymphocytes, and naϊve or activated lymphocytes with no stem cells. Donor cells may also be originated from an organ or tissue, e.g. placenta, intestine, etc.

Reports from the late 1970s showed that immunization of donor with host derived H-2 antigens mixed with CFA could suppress antigen cytotoxicity and splenomegaly as measures of GVHD ■ symptoms in hematopoietic transplantation across H-2 non-identical combinations [Nagino, H. et al. (1978) Int. Archs. Allergy Appl Immunol. 56: 48-56; Miyazaki, S. et al. (1978) Int. Archs. Allergy Appl. Immunol. 56: 57-64]. In these studies, CFA was used for increasing immunogenicity of the H-2 antigens, in order to exhaust all anti- host reactive cells. However, the control of CFA treatment alone was not included.

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 viυό). 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.

Further characterization of cell subset populations carried out following CFA donor treatment revealed an increase in Gr-I and Mac-1 positive cells and a decrease in CD 3 and Thyl positive cells (Fig. 4A). Treatment with CpG or CpG emulsified in IFA did not affect the number of Gr-I or Mac-1 positive cells, but there was a perceptible decrease in cell subsets expressing T cell surface markers (Fig. 4C).

In a further aspect, 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.

The terms "treat, treating or treatment" as used herein 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.

By "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.

The term "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.

In the present invention, said donor cells are partially or completely mismatched allogeneic or xenogeneic bone marrow cells, mobilized blood cells including naϊve or activated lymphocytes, or naϊve or activated lymphocytes with no stem cells, or any mixture thereof.

The term "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.

Similarly, 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.

In general, the term "allograft" refers to an allogeneic stem cell transplant.

A "xenograft" (xenotransplant) is a transplant of tissue or cells from a donor of one species to a recipient of another species. The terms 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.

A "peripheral blood stem cell (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. ⁵¹Cr release assay.

Usually, 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.

As used herein, the term "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. Sometimes 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.

The terms "disease", "disorder", "condition" and "illness" are equally used herein.

More specifically, 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. 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-Schoήberg 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. In contrast to 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.

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.

In addition, 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 α chain. (Hb GpMkdeiphia), β chain (Hb S, Hb C), γ chain (Hb Fτ_eχas), or δ chain (Hb A2Fiatbush)-

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, β,γ or δ chain, or may affect some combination of the β, γ, and δ chain in the same patient (but never the α and β chain together).

Sickle cell anemia is an inherited autosomal recessive condition that causes abnormal hemoglobin in blood cells, leading to infections and organ damage.

Other genetic disorders that result in enzyme deficiency diseases like Gaucher's disease, metachromatic leukodystrophy and Hurler's disease may also be treated by said method.

Furthermore, 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. 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. For such treatment, 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.

An "infectious agent" is also called a "biological agent" and includes viruses, bacteria and parasites. Examples of 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. This¹ 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.

To provide a "preventive treatment" or "prophylactic treatment" is acting in a protective manner; to defend against or prevent something, especially a condition or disease. :

An "in vivo" treatment, as used herein, 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 naϊve or activated lymphocytes and naϊve or activated lymphocytes transplant with no stem cells or any mixture thereof.

Since 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.

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.

Despite the graft-versus-tumor reaction, many patients with allogeneic transplants still experience high mortality as a result of GVHD, which may be avoided by treating the patients with the donor cells prepared as described herein, and as demonstrated in Example 5. 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.

"Graft- versus -host disease" (GVHD) occurs following allogeneic 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. There are two forms of GVHD: an early form called 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.

In particular, it may be useful for the treatment of different leukemias like chronic myeloid leukemia (CML), acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML), and promyeloid leukemia (PML). In the below described results, the treatment of B-cell leukemia is exemplified (Example 5, and especially Tables 5 and 6 and Fig. 5).

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

The term "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 υiυo 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.

The terms "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 υiυo or ex-υiυo 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, as demonstrated in Example 5.

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 naϊve or activated lymphocytes, or naive or activated lymphocytes with no stem cells or any mixture thereof.

Sometimes 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. In order to increase the number of stem cells for transplantation, 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".

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. Preferably, 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. Preferably, 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. For injection, cells are resuspended in physiological solution, e.g. saline, and injected usually intravenously or intra-thecal. In donor lymphocyte infusion (DLI), between 10³ to 10⁹ cells/kg (of recipient) are injected. In stem cell transplantation, CD34+ cells are injected, usually >10⁴ cells/kg.

Suggested doses for CpG oligonucleotide treatment are usually between 10- 2000 μg/dose, preferably between 50-1000 μg/dose, more preferably 100- 700 μg/dose, in one or multiple doses. Generally, 500 μg/dose of CpG is administered in one dose, 0 to 30 days prior to harvest, preferably between 5 to 20 days. For IFA or its substitutes (Montanide, Muramil Dipeptide and QS21), one or multiple doses of 50-500 μl/dose, preferably 300 μl/dose are generally used. CFA is administrated in one or multiple doses of 50-600 μl/dose, preferably 300 μl/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 υiυo injection, e.g saline, PBS or water for injection.

It is one major advantage of the present invention that 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. '_.

Further, 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.

In addition Figure 5 shows disease-free (no GVHD and no leukemia) survival for at least 90 days and even for 160 days, ensuring the anti_rleukemia 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.

The use of 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.

The preparation of compositions is 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.

The 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.

The compositions must be stable and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi.

The 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. As used herein "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.

It is understood by the skilled artisan that 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.

The 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.

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. Thus, 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.

Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

The contents of all publications quoted to herein are fully incorporated by reference.

The following examples are representative of techniques employed by the inventors in carrying out aspects of the present invention. It should be appreciated that while these techniques are exemplary of preferred embodiments for the practice of the invention, those of skill in the art, in light of the present disclosure, will recognize that numerous modifications can be made without departing from the spirit and intended scope of the invention. Examples

Materials and Methods Mice

Female BALB/c H-2<* (BALB), C57BL/6 Η.-2* (C57) and (BALB/c x C57BL/6) Fi H-2^d/b (F₁) 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.

Tumor

A murine B cell leukemia/lymphoma (BCLi) model was used. BCLi arose spontaneously in a BALB/c female mouse as described before [Slavin S. and

Strober S. (1978) Nature 272: 624-626]. Stocked BCLi cells derived from frozen single cell suspension of spleens were inoculated intravenously (IV) into BALB or Fi mice causing splenomegaly and high lymphocytosis (>10⁸ cells/ml) in the blood.

Experimental design for GVHD induction

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. Non- lethally irradiated recipients were inoculated (IV) with 3OxIO⁶ C57BL/6 splenocytes 24-48 hours later.

Experimental design for GVL induction

Recipient F₁ mice were conditioned with TBI- (4Gy) 24h prior to intravenous inoculation with 10⁴ BCLi cells. On the following day, Fi mice were injected intravenously with 3OxIO⁶ naϊve C57BL/6 splenocytes or with C57BL/6 splenocytes treated 4 or 10 days pre -transplant as specified in each experiment.

Donor pre-treatment

Complete Freund's adjuvant (CFA) or incomplete Freund's adjuvant (IFA) (Difco Laboratories, Detroit, Michigan, USA) was injected sub-cutaneously into C57BL/6 mice (0.2 ml). Lipopolysaccharide (LPS) (Sigma Saint Louis, Missouri, USA) was injected sub-cutaneously into C57 mice (40μg/0.1ml) either alone or in emulsion of IFA. CpG (ODN#1826; TCC ATG ACG TTC CTG ACG TT, fully phosphorothioate backbone; SEQ. ID. No.l) or a non-CpG control (ODN#2138; TCC ATG AGC TTC CTG AGC TT, fully phosphorothioate backbone; SEQ .ID. No.2) (Coley Pharmaceutical Group, Kanata, Canada) was injected sub-cutaneously into C57BL/6 mice (lOOμg/O.lml) either alone or in emulsion of IFA.

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).

Follow-up

Mice were checked daily for the appearance of GVHD symptoms such as hunched posture, ruffled fur, diarrhea and cachexia.

Mice inoculated with BCL₁ cells were tracked for leukemia development by serial blood counts and enlarged spleen.

Survival was monitored and GVHD or leukemia-related death was determined on the basis of the aforementioned symptoms and post mortem examination. Mixed Lymphocyte Reaction (MLR)

One million responding cells isolated from normal peripheral lymph nodes (LN) were cultured in flat-bottom 96 microculture wells (Nunc A/S, Denmark) with IxIO⁶ 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 10⁶ 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"⁵M), penicillin (100 U/ml), and streptomycin (lOOμg/ml). Tissue culture media and reagents were purchased from Biological Industries, Beit Haemek, Israel. Cells were incubated at 37⁰C with 5% CO2/air mixture in a humidified incubator for 72 hours, and then pulsed for 16-18 hours with lμCi of tritiated thymidine (5.0 Ci/immol; 185 GBQ/mmol, Amersham Biosciences, UK) per well. Cells were subsequently collected on paper filters using a Filtermate Harvester Unifilter- 96 and counted in a Top Count NXT™ Microplate Scintillation & Luminescence Counter (both from Packard Bio Science Company, IL, USA). Background proliferation was assessed in each experiment by adding irradiated spleen cells syngeneic to responding cells. Counts per minute (cpm) of allogeneic MLR were divided by cpm of syngeneic response and identified as stimulation index (SI). Suppressive activity was calculated according to the formulas presented in Table 1. Flow Cytometry Analysis

The following antibodies were applied for the staining of splenocytes (5X105): Fluorescein Isothiocyanate (FITC) labeled anti-CD3, anti-CD4, anti-CD8a, and anti-Ly-6G (Gr-I), or R-Phycoerythrin (PE) labeled anti-Thy-1.2 (Southern Biotech Birmingham, Alabama, USA), anti-Pan NK ( DX5), anti-Fas, and anti- CD25 (PC61), or PerCP-Cy5.5 labeled anti-CDllb (mac-1). Unless otherwise stated, all antibodies were purchased from BD Bioscience Pharmigen, San Diego, CA, USA. All samples were pre -incubated for 10 minutes on ice with unlabeled rat anti-mouse CD32/16 antibodies to prevent non-specific staining through the Fc fraction of the labeled antibodies. Spleen cells were incubated later with the specific labeled antibodies for 30 minutes on ice and washed twice with cold staining buffer (phosphate-buffered saline containing 1% bovine serum albumin and 0.03% sodium azide). Samples were analyzed by fluorescein-activated cell sorting (FACS; FACStar Plus, Becton-Dickinson, San Jose, CA, USA).

Statistical analysis

Body weights were presented as mean ± SE (standard error). The Kaplan- Meier method was used to calculate the probability of survival as a function of time after cell inoculation for GVHD induction [Kaplan and Meier (1958) J. Am. Stat. Assoc. 53:457-482]. The statistical significance of survival between pairs of Kaplan-Meier curves was evaluated by the log rank test [Mantel, E. (1966) Cancer Chemother. 50: 163-170]. The statistical significance of differences in the number of spleen cells of control versus treated mice was evaluated by the standard two-tailed, unpaired student t-test. A value of p<0.050 was considered statistically significant. Example 1 Effect of CFA and CpG treatment on spleen size

Injection of CFA or CpG emulsified in IFA led to enlarged spleens, which were harvested 10-12 days following treatment. As shown in Figure 1, a significantly larger number of cells (p<0.008) was observed in these spleens than in spleens derived from normal or IFA treated mice (132 X 10⁶ or 140 X 10⁶ respectively, versus 87 X 10⁶ cells). A significant increase (p<0.035) in spleen cell number was also observed on day 6 or 10 following inoculation of CpG alone (276 X 10⁶ and 148 X 10⁶ cells, respectively) compared to control mice treated 6 or 10 days previously with free non-CpG or inoculated 10 days previously with non-CpG emulsified in IFA (72X10⁶ or 82XlO⁶ and 76 XlO⁶ cells, respectively).

Example 2

Effect of CFA, LPS and CpG on GVHD symptoms

GVHD was induced in (BALB/c x C57BL/6)F1 recipients by inoculation with parental C57 splenocytes derived from either naϊve or pre-treated donors. Naϊve 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. Splenocytes derived from donors pretreated with CpG with or without IFA and LPS emulsified in IFA, efficiently prevented loss of body weight in the host mice (Figure 2).

Example 3

Induction of suppressive activity in CFA or CpG treated mice

CFA treated spleen cells of C57 mice totally suppressed one way MLR of both C57 lymphocytes responding to BALB derived stimulator cells (99% inhibition) and BALB derived splenocytes responding to C57 cells (97% inhibition) (Table 1). Table 1 - Effect of CFA-treated splenocytes on one way mixed lymphocyte reaction (MLR)^a

^a MLR was carried out by culturing IXlO⁶ lymph node-derived cells as responders with IXlO⁶ irradiated (50Gy) splenocytes as stimulators, for 4 days. Irradiated (1.5Gy) spleen cells (IXlO⁶) derived either from naϊve C57 mice or from C57 mice treated with CFA 10 days before cell harvesting, were added to the MLR as co-cultured cells. ^{b 3}H-Thymidine uptake (triplicates) ^c % suppression was calculated, after subtraction of syngeneic response, by the following formula: 100- [cpm of allogeneic response in the presence of CFA co-cultured cells / naϊve co-cultured cells XlOO]

Inoculation of CpG alone or CpG emulsified in IFA also induced suppressive MLE, activity (87 or 81 % inhibition, respectively), compared to controls of non- CpG emulsified or not emulsified in IFA (11 or 63 % inhibition, respectively), as shown in Table 2.

Table 2 - Effect of CpG treated splenocytes on one way mixed lymphocyte reaction (MLE)^a

^a MLR was carried out by culturing IXlO⁶ T cells as responders with IXlO⁶ irradiated (50Gy) splenocytes as stimulators, for 4 days. Irradiated (1.5Gy) spleen cells (IXlO⁶) derived from either naϊve 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. ^{b 3}H-Thymidine uptake (triplicates). ^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 / naϊve co-cultured cells XlOO].

Example 4

Effect of CFA or LPS treatment on GVHD induction

Spleen cells of naϊve C57 mice inoculated into sub-lethally irradiated (BALB/c x C57BL/6)F1 recipients led to development of severe GVHD with 99% mortality (77/78 mice) (Table 3). In contrast, CFA-treated C57 splenocytes harvested 10 days following CFA treatment, caused GVHD-related death in only 23% of Fl recipients (14/61), and 77% of .the mice (47/61) remained healthy and GVHD-free for >200 days (Table 3). 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. LPS emulsified in IFA given to C57 mice 10 days pre-transplant, caused GVHD-related death in only 6/20 Fl recipients (30%), while the majority of the mice (14/20) (20%) remained healthy and GVHD-free for >200 days (Table 3).

Table 3 - Effect of allogeneic cells pre-treated with CFA or LPS on GVHD induction

Sub-lethally irradiated (4Gy) (BALB/C x C57 BL/6) Fl mice were inoculated intra-venous, one day following irradiation, with C57BL/6 donor splenocytes (3OxIO⁶) derived from naive or donor mice treated with IFA, CFA or LPS (40μg) with or without IFA. All treatments were given subcutaneously to donor mice on day 4 or 10 prior to spleen harvesting. P=0.000 for the comparison of IFA versus naϊve, CFA day -4 versus naϊve, CFA day -10 versus naϊve or versus IFA or versus CFA day-4. P=0.908.for the comparison of IFA versus CFA day-4. p<0.001 for the comparison of LPS or LPS+IFA treatment versus naϊve cells but there was no statistically significant difference between each of the treatments (p>0.050). p=0.000 for the comparison of LPS+IFA treatment versus IFA treatment. In contrast to the apparent inhibition of GVHD caused by pre-transplant CFA treatment of the donor, no such inhibitory effect was observed when the recipients were treated with CFA 10 days or 1 day prior to induction of GVHD (Table 4). All mice died of severe GVHD within a median of 15-19 days.

Table 4 - Effect of pre-transplant treatment of recipient mice with CFA or CpG on GVHD induction

¹ IFA, CFA and CpG+IFA were injected subcutaneously to Fl host mice 10 days before TBI (4-5Gy). 24h later, 30X10⁶ C57 splenocytes were inoculated intravenously.

² CFA was given to host one day before irradiation

Example 5

Effect of CFA and CpG treatment on graft versus leukemia (GVL)

C57BL/6 donor splenocytes treated with CFA 10 days prior to harvesting were injected into Fi recipients inoculated with BCLi cells. The effect of CFA donor treatment on survival of leukemia bearing mice is shown in Tables 5 and 6. The effect of CpG donor treatment on survival of leukemia bearing mice is shown in Figure 5. Table 5 - Effect of pre-transplant CFA donor treatment with on Graft-versus- Leukemia (GVL)

Sub-lethally irradiated (4Gy) Fl (BALB/C .x 057 Bl/6) mice were inoculated intravenously with 10⁴ BGLi cells one day following irradiation. 24h later, naive or CFA treated C57BL/6 donor splenocytes (3OxIO⁶) were inoculated intravenously. CFA was injected sub-cutaneously into donor mice on day 10 before spleen harvesting.

All control mice inoculated with either BCLi alone or inoculated with BCLi and treated with naϊve C57BL/6 splenocytes, died of leukemia (median 29 days) or early acute GVHD (median 17 days). In contrast, 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

10⁵ splenocytes were adoptively transferred into naϊve secondary BALB hosts 14 or 21 days following BCLi inoculation into sublethally (4Gy) irradiated (BALBXC57) Fi mice that were treated with naϊve or CFA pre-treated C57 splenocytes. ^a Mice treated with naϊve C57B1/6 splenocytes died of acute GVHD before the AT experiment that was carried out on day 21 following BCLi inoculation.

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 naϊve 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. As shown in Tables 5 and 6, it is clear that CFA pre-transplant treatment of the donor does not hamper the ability of the allogeneic cells to exert a GVL effect. Allogeneic cell transplant (AlIoCT) of CFA-treated C57BL/6 splenocytes into F₁ mice inoculated with B cell leukemia/lymphoma (BCL₁) cells, inhibited the development of leukemia for at least 3 weeks and prevented early, but not late, GVHD. These results suggest that immunoregulation of donor cells prior to alloCT cell may augur a new strategy for cellular immunotherapy of malignant and non-malignant diseases while avoiding or minimizing the risk of GVHD.

An experiment to test the anti-leukemic effect of splenocytes treated with CpG in IFA is presented in Figure 5. Fl mice were inoculated intravenously with 10⁴ BCL₁ cells 24 hours following total body irradiation (6Gy). 24h later, naive or CpG in IFA-treated (10 days previously) C57BL/6 donor splenocytes (3OxIO⁶) were inoculated intravenously. This experiment was repeated twice. In the first experiment, 11 out of 13 mice who received donor cells CpG-IFA treated donor cells survived disease-free (no leukemia and no GVHD) for at least 160 days, while in the second experiment 7 out of 13 mice survived disease-free (no leukemia and no GVHD) for at least 90 days. The graph in Figure 5 summarizes both experiments.

Example 6

Effect of CpG treatment on GVHD induction

Similarly to the CFA treatment of recipients, treatment of recipients with CpG alone, IFA, or CpG emulsified in IFA 10 days or 1 day before induction of GVHD, did not prevent GVHD induction in recipient mice (Table 4), nor did treatment of recipients with CpG alone on the day of cell inoculation (data not shown). Inoculation of C57 donor cells treated with non-CpG.6 and 10 days prior to transplantation resulted in 69% (20/29) and 100%(19/19) GVHD- related mortality, respectively, while treatment of C57 donor mice with CpG alone 6 or 10 days pre-transplant, led to GVHD with 11% (3/28) and 41% (11/27) mortality, respectively. Non-CpG emulsified in IFA given to C57 control donors 10 days pre-transplant led to 100% GVHD-related mortality in Fl recipients (13/13), whereas CpG emulsified in IFA given to C57 mice 10 days pre-transplant caused GVHD-related death in only 5% (1/20) of Fl recipients, the other 95% (19/20) remaining healthy and GVHD-free for >200 days (Figure 3).

Example 7

Effect of CpG and CFA on cell surface markers

The effect of CFA and CpG treatment on various cell subset populations is presented in Figure 4. Phenotypic analysis carried out on donor splenocytes treated with CFA 10 days before harvesting for transplant revealed an increased number of cells expressing myeloid cell surface markers Gr-I and Mac-1, compared to controls of splenocytes derived either from naϊve or IFA treated mice. In comparison to naϊve mice, IFA treated mice displayed a reduced number of cells expressing T cell markers CD3 and Thy-1.2, which were further reduced following CFA treatment. Following CpG treatment given 6 days prior to harvesting, an increased number of CD95 positive cells were observed, compared to non-CpG treatment or to naϊve splenocytes. A clear decrease in T cell subset markers CD4 and CD8 or CD3 and Thy-1.2 was seen in the CpG treated splenocytes compared to non-CpG derived splenocytes or naϊve cells. Treatment of donor mice with CpG emulsified in IFA 10 days before harvesting, resulted in a remarkable increase in the number of CD95 positive cells and a decrease in CD3, CD4 and; CD 8 positive spleen cells, in comparison to splenocytes derived from naϊve mice. While this invention has been described in terms of some specific examples, many modifications and variations are possible. It is therefore understood that within the scope of the appended claims, the invention may be realized otherwise than as specifically described.

Claims

Claims

1. 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).

2. The method of claim 1, wherein said step (a) is effected in vivo or ex vivo.

3. A method of ex vivo preparing donor cells for transplantation into a recipient, wherein said cells have reduced GVHD activity, said method comprising the steps of: a. providing a sample of donor cells, 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).

4. The method of any one of claims 1 to 3, wherein 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 naϊve or activated lymphocytes, and naϊve or activated lymphocytes with no stem cells.

5. The method of any one of the preceding claims, wherein said preparation is effected in vivo, and said immune modulator agent is selected from the group consisting of CpG, CpG in IFA, CFA, IFA, LPS in IFA, Montanide, Muramil dipeptide, QS21, difteria toxin and ahiminum salts.

6. The method of any one of claims 1 to 4, wherein said preparation is effected ex vivo, and said immune modulator agent is selected from the group consisting of CpG, LPS, Mycobacterium Tuberculosis components, aluminum salts, difteria toxin and a biological or synthetic factor that affects Thl/Th2 cytokine balance.

7. 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 0 and 30 days following said contact with said immune modulator agent.

8. The population of donor cells of claim 7, wherein said 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 naϊve or activated lymphocytes, and naϊve or activated lymphocytes with no stem cells.

9. The population of donor cells of any one of claims 7 or 8, wherein said cells are contacted with said immune modulator agent in vivo, and said immune modulator agent is selected from the group consisting of CpG, CpG in IFA, CFA, IFA, LPS in IFA, Montanide, Muramil dipeptide, QS21, difteria toxin and aluminum salts. .^"

10. The population of donor cells of any one of claims 7 or 8, wherein said cells are contacted with said immune modulator agent ex vivo, and said immune modulator agent is selected from the group consisting of CpG, LPS, Mycobacterium Tuberculosis components, aluminum salts, difteria toxin, and any biological or synthetic factor that affects Thl/Th2 cytokine balance.

11. A pharmaceutical composition for reducing, alleviating or abrogating GVHD, comprising as active agent the donor cells prepared by the method of any one of claims 1 to 6, or the', population of donor cells of any one of claims 7 to 10.

12. The composition of claim 11, further comprising a pharmaceutically acceptable carrier, excipient or diluent.

13. Use of the population of donor cells as defined in any one of claims 7 to 10, in the preparation of a pharmaceutical composition for reducing, alleviating or abrogating GVHD.

14. 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 one of the composition of claims 11 or 12, or donor cells with reduced GVHD activity, wherein said cells were produced by the method of any one of claims 1 to 6.

15. The method of claim 14, wherein 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.

16. The method of claim 14, wherein said subject in need suffers from a hematological disorder involving neoplastic proliferation of hematopoietic cells.

17. The method of claim 16, wherein said hematological disorder 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.

18. The method of claims 15 and 16, wherein said hematological disorder is refractory to chemotherapy.

19. The method of any one of claims 16 to 18, wherein said donor cells further present increased graft- versus -leukemia or graft-versus-tumor activity.

2O.The method of claim 15, wherein said cancer is one of breast cancer, bladder cancer, lung cancer, prostate cancer, thyroid cancer, leukemias, lymphomas, colon cancer, glioma, seminoma, liver cancer, pancreatic cancer, renal cancer, cervical cancer, testicular cancer, head and neck cancer, ovarian cancer, neuroblastoma arid melanoma or a metastatic solid tumor.

21. The method of claim 20, wherein said cancer is leukemia, particularly chronic myeloid leukemia (CML), acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML), or promyeloid leukemia (PML).

22. The method of any one of claims 14 to 21, further comprising 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 engraffcment and prevent rejection of the said donor cells.

23. The method of claim 22, wherein said additional antineoplastic or immunosuppressant agent is 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.

24. The method of any one of claims 14 to 25, wherein said donor cells are administered to said subject in need in the form of a peripheral blood mononuclear cell preparation.

25.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 one of the composition of claims 11 or 12, or donor cells with reduced GVHD activity, wherein said cells were produced by the method of any one of claims 1 to 6.

26.A method for induction of transplantation tolerance in a recipient subject in need of a tissue or organ transplant, said method comprising administering to said subject a therapeutically effective amount of one of the composition of claims 11 or 12, or donor cells with reduced GVHD activity, wherein said cells were produced by the method of any one of claims 1 to 6.

27. The method of claim 26, wherein said transplant is an allograft or a xenograft.

28. The method of any one of claims 26 or 27, wherein said donor cells are administered to said recipient before, together with, or after transplantation of said organ or tissue.

29. The method of claim 14, wherein said administration of said cells or said composition further prevents relapse of said tumor.

30. The method of any one of claims 14 to 29, wherein said cells or said composition are administered intravenously, parenterally, intratechally or intra-tumor.

31. Use of an immune modulator agent substance, said immune modulator agent being selected from the group comprised of CpG, CpG in IFA, IFA, CFA, LPS in IFA, LPS, Mycobacterium Tuberculosis components, Montanide, Muramil dipeptide, QS21, difteria toxin, a biological or synthetic factor that might affect Thl/Th2 -cytokine balance, or any mixture thereof, 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.

32. The use of claim 31, wherein said hematological disorder involves neoplastic proliferation of hematopoietic cells.

33. The use of claim 32, wherein said hematological disorder 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..