NZ627272B2 - A combination therapy for a stable and long term engraftment - Google Patents

Abstract

Disclosed is the use of a dose of T cell depleted immature hematopoietic cells and a therapeutically effective amount of cyclophosphamide in the manufacture of a medicament for treating a subject in need of a non-syngeneic cell, tissue graft or immature hematopoietic cell transplantation.

Description

A COMBINATION THERAPY FOR A STABLE AND LONG TERM TMENT RELATED APPLICATION/S US. Provisional Patent Application No. ,917 filed on December 22, 2011, which is hereby incorporated by reference as if fully set forth herein.

FIELD AND BACKGROUND OF THE INVENTION The present ion, in some embodiments thereof, relates to a combination therapy for attaining a stable and long term cell or tissue transplantation.

The use of full—haplotype mismatched haploidentical donors as an alternative source for hematopoietic stem cell transplantation (HSCT) is highly attractive since virtually all patients have a readily available haploidentical family member that can serve as an HSCT donor. Early ts to avoid fatal graft versus host disease (GVHD) risk and to apply haploidentical rigorously T cell depleted bone marrow transplantation (TDBMT) in leukemia patients revealed that the absence of donor T cells within the graft leads to a high rate of graft rejection, mediated by residual radiotherapy and chemotherapy ant host—derived T cells (HTC). To overcome this obstacle, a ’mega dose’ of TDBM cells was contemplated which can overcome this HTC mediated immune barrier and be engrafted successfully even when using fully mismatched murine strain combinations [Bachar-Lustig E et al., Nat Med. (1995) 1:1268—1273].

Subsequently, it was demonstrated that in humans, as in rodents, CD34+ hematopoietic stem cell dose escalation may be used to overcome genetic barriers, enabling satisfactory survival rates ing purified haploidentical HSCT [Reisner Y and Martelli ME. Immunol Today. (1995) -440 and US. Patent No. 5,806,529].

While the use of a purified ’mega dose’ of CD34+ HSCT has enabled haploidentical transplantation in leukemia patients, one major drawback, common to all T cell ed transplants, is the slow recovery rate of the recipient’s immune .

This is attributed to ive immune ablating conditioning protocols prior to transplantation, the low numbers of donor T cells infused within the graft and to the decreased thymic function of adult recipients. Thus, in adult recipients of a haploidentical CD34+ stem cell graft, a significant rate of transplant d mortality (TRM) is caused by opportunistic infections.

Several approaches are being developed to address this challenge. This includes novel modalities to improve thymic function, post—transplant ve transfer of anti- viral specific T cells, transfer of partially polyclonal host—non—reactive allo-depleted T cells or transfer of fully polyclonal T cells transfected with inducible e genes. An alternative and additional approach to preserve host immunity is the use of reduced intensity ioning (RIC). This non-myeloablative approach spares a substantial level of host immune cells and thus may reduce TRM by both improving post—transplant immune reconstitution and reducing the toxicity associated with the conditioning agents. Haploidentical transplantation under RIC is even more intricate due to the substantial immunological barrier presented by the surviving host T cells. Recent attempts to overcome this barrier, largely made use of non—T cell depleted grafts, which enable a high rate of engraftment, but in the expanse of increased rates of GVHD.

Another ch for applying haploidentical transplantation under RIC uses CD3/CD19 depleted grafts, which not only contain CD34+ stem cells but also CD34 negative progenitors, NK, graft facilitating cells and dendritic cells, however, this too is at the expanse of increased rates of GVHD and TRM.

In the 1970’s George Santos demonstrated in rodents that a short course of high— dose cyclophosphamide (CY) soon after bone marrow transplant (BMT) targeted activated donor or host active T cells [Owens AH Jr and GW. S. Transplantation. (1971) 11:378—382]. Cyclophosphamide was observed to be non—toxic to hematopoietic stem cells e of their high expression of the detoxifying enzyme de dehydrogenase, and Slavin et al. further demonstrated that administration of high dose hosphamide can reduce GVHD and graft rejection in mice, without adverse ix.) LII effects on stem cell engraftment [Brodsky RA and R]. J. Lancet. (2005) 365:1647— 1656]. Clinical trials by the John s and Fred Hutchinson Cancer Research Center groups, evaluated a non-myeloablative protocol of cyclophosphamide, abine and ZGy TBI, and post~transplant GVHD prophylaxis With hosphamide (50 mg/kg days +3 and +4), MMF (days +5 to +35) and tacrolimus (days +5 to +180) [Luznik L et al., Biology of blood and marrow transplantation: l of the American Society for Blood and Marrow Transplantation. (2008) 14:641]. According evident from their teachings, this protocol resulted in a high relapse rate, which was probably due to poor disease debulking by the non-myeloablative conditioning and to lack of GVHD related graft versus ia (GVL) effect [Munchel A et al., ric Reports (2011) 3:43—47].

Additional ches for achieving stable engraftment of allogeneic poietic stem cells have been attempted, some are described in U.S. Patent Application No. 10909, U.S. Patent Application No. 18142, U.S. Patent Application No. 20070098693, U.S. Patent No. 5,876,692, U.S. Patent No. 5,514,364, U.S. Patent No. 6,217,867, U.S. Patent No. 5,635,156, U.S. Patent Application No. 20060140912, U.S. Patent Application No. 20040005300, U.S. Patent Application No. 41027, U.S. Patent Application No. 20030017152, U.S. Patent Application No. 20030165475 and U.S. Patent Application No. 20010009663.

SUMMARY OF THE ION In a first aspect of the invention there is provided the use of a dose of T cell depleted immature hematopoietic cells, wherein said T cell depleted immature hematopoietic cells comprise less than 5 X 105 CD3+ cells per kilogram body weight of a subject, and wherein said dose comprises at least about 5 x 106 CD34+ cells per kilogram body weight of the subject, and a therapeutically effective amount of cyclophosphamide, wherein said therapeutically effective amount comprises 25—200 mg per kilogram body weight of the subject, and wherein said cyclophosphamide is for administration to the subject following transplantation of a cell or tissue graft, in the manufacture of a medicament for treating a subject in need of a non-syngeneic cell or tissue graft, wherein said subject has a malignant disease.

In another aspect of the present invention there is provided the use of a dose of T cell depleted immature hematopoietic cells, wherein said T cell depleted immature hematopoietic cells comprise less than 5 x 105 CD3+ cells per kilogram body weight of a subject, and wherein said dose comprises at least about 5 x 106 CD34+ cells per kilogram body weight of the subject, and a therapeutically ive amount of cyclophosphamide, wherein said therapeutically effective amount comprises 25-200 mg per am body weight of the subject, and wherein said hosphamide is for administration to the subject following transplantation of a cell or tissue graft, in the manufacture of a (followed by page 3a) medicament for treating a subject in need of a non-syngeneic cell or tissue graft, wherein said subject has a non-malignant disease.

In a further aspect of the invention there is provided the use of a dose of T cell depleted immature poietic cells, wherein said T cell depleted immature hematopoietic cells comprise less than 5 X 105 CD3+ cells per kilogram body weight of a subject, and wherein said dose comprises at least about 5 x 106 CD34+ cells per kilogram body weight of the subject, and a therapeutically effective amount of cyclophosphamide, wherein said therapeutically effective amount comprises 25—200 mg per kilogram body weight of the subject, and wherein said cyclophosphamide is for administration to the subject following transplantation of immature hematopoietic cells in the manufacture of a medicament for treating a conditioned subject in need of an re hematopoietic cell transplantation, and wherein said subject has a malignant disease .

In a still r aspect of the invention there is provided the use of a dose of T cell depleted immature poietic cells, wherein said T cell depleted immature hematopoietic cells se less than 5 x 105 CD3+ cells per kilogram body weight of a subject, and wherein said dose comprises at least about 5 X 106 CD34+ cells per kilogram body weight of the subject, and a therapeutically effective amount of hosphamide, wherein said therapeutically effective amount comprises 25-200 mg per kilogram body weight of the t, and wherein said cyclophosphamide is for administration to the subject following transplantation of immature hematopoietic cells in the manufacture of a medicament for treating a conditioned subject in need of an re hematopoietic cell transplantation, and n said subject has a nonmalignant disease .

In another aspect of the invention there is provided the use of a reduced intensity ioning protocol, wherein said reduced intensity conditioning comprises a total body irradiation (TBI) and a chemotherapeutic agent; a dose of T cell depleted immature poietic cells, wherein said T cell depleted immature hematopoietic cells comprise less than 5 x 105 CD3+ cells per kilogram body weight of a t, and wherein said dose comprises at least about 5 x 106 CD34+ cells per kilogram body weight of the subject, and a therapeutically effective amount of cyclophosphamide, wherein said therapeutically effective amount comprises 25—200 mg per kilogram body weight of the subject, and wherein said cyclophosphamide is for administration to the subject following (followed by page 3b) transplantation of immature hematopoietic cells in the manufacture of a medicament for ng a subject in need of an immature hematopoietic cell transplantation, and wherein said subject has a malignant disease.

In yet another aspect of the invention there is provided the use of a reduced ity conditioning protocol, wherein said reduced intensity conditioning comprises a total body irradiation (TBI) and a chemotherapeutic agent; a dose of T cell depleted immature hematopoietic cells, wherein said T cell depleted immature hematopoietic cells se less than 5 x 105 CD3+ cells per kilogram body weight of a subject, and wherein said dose comprises at least about 5 x 106 CD34+ cells per kilogram body weight of the t; and a therapeutically ive amount of cyclophosphamide, wherein said therapeutically effective amount comprises 25-200 mg per kilogram body weight of the subject, and wherein said cyclophosphamide is for administration to the subject following transplantation of immature hematopoietic cells in the manufacture of a medicament for treating a subject in need of an immature hematopoietic cell transplantation, and wherein said subject has a non-malignant disease.

In a still r aspect of the invention there is provided the use of a dose of T cell depleted immature hematopoietic cells from a non-syngeneic donor, n said T cell depleted immature hematopoietic cells comprise less than 5 x 105 CD3+ cells per kilogram body weight of a subject, and wherein said dose comprises at least about 5 x 106 CD34+ cells per kilogram body weight of the subject; and a therapeutically effective amount of cyclophosphamide, n said therapeutically ive amount comprises 25—200 mg per kilogram body weight of the t, and wherein said cyclophosphamide is for administration to the subject following transplantation of a cell or tissue graft in the manufacture of a medicament for inducing donor specific tolerance in a subject in need of a ngeneic cell or tissue graft, wherein said subject has a malignant disease.

In another aspect of the invention there is provided the use of a dose of T cell depleted immature hematopoietic cells from a non-syngeneic donor, wherein said T cell depleted re hematopoietic cells comprise less than 5 X 105 CD3+ cells per am body weight of a subject, and wherein said dose comprises at least about 5 x 106 CD34+ cells per kilogram body weight of the subject; and a therapeutically effective amount of cyclophosphamide, wherein said therapeutically effective amount ses 25—200 mg (followed by page 30) per kilogram body weight of the subject, and wherein said cyclophosphamide is for administration to the subject following transplantation of a cell or tissue graft in the manufacture of a medicament for inducing donor specific tolerance in a t in need of a ngeneic cell or tissue graft, wherein said subject has a non-malignant disease.

According to an aspect of some embodiments of the present invention there is provided a method of treating a subject in need of a non—syngeneic cell or tissue graft, the method comprising: (a) transplanting into a t a dose of T cell depleted immature hematopoietic cells, n the T cell depleted immature hematopoietic cells comprise less than 5 x 105 CD3Jr T cells per kilogram body weight of the subject, and wherein the dose comprises at least about 5 x 106 CD34+ cells per kilogram body weight of the subject; and uently (b) administering to the subject a therapeutically effective amount of cyclophosphamide, n the therapeutically effective amount comprises 25- 200 mg per kilogram body weight, thereby ng the subject.

According to an aspect of some embodiments of the present invention there is provided a method of treating a subject in need of an immature hematopoietic cell transplantation, the method comprising: (a) transplanting into a conditioned subject a dose of T cell depleted re hematopoietic cells, wherein the T cell depleted immature hematopoietic cells comprise less than 5 x 105 CD3+ T cells per kilogram body weight of the subject, and wherein the dose ses at least about 5 x 106 CD34+ cells per kilogram body weight of the subject; and subsequently (b) administering to the subject a therapeutically effective amount of cyclophosphamide, wherein the therapeutically effective amount comprises 25-200 mg per kilogram body , thereby treating the subject.

{FOLLOWED BY PAGE 4] According to an aspect of some ments of the present invention there is provided a method of treating a subject in need of an immature hematopoietic cell transplantation, the method comprising: (a) conditioning a subject under a reduced ity conditioning protocol, wherein the d intensity conditioning ses a total body ation (TBI) and a chemotherapeutic agent; (b) transplanting into the subject a dose of T cell ed immature hematopoietic cells, wherein the T cell depleted re poietic cells comprise less than 5 X 105 CD3+ T cells per kilogram body weight of the subject, and wherein the dose ses at least about 5 X 106 CD34+ cells per kilogram body weight of the subject; and subsequently (c) administering to the subject a eutically effective amount of hosphamide, wherein the therapeutically effective amount comprises 25~200 mg per kilogram body weight, thereby treating the subject.

According to an aspect of some embodiments of the present invention there is provided a method of inducing donor specific tolerance in a subject in need of a non- eic cell or tissue graft, the method comprising: (a) transplanting into a subject a dose of T cell depleted re hematopoietic cells obtained from a non-syngeneic donor, wherein the T cell depleted immature hematopoietic cells comprise less than 5 x 105 CD3+ T cells per kilogram body weight of the subject, and wherein the dose comprises at least about 5 x 106 CD34+ cells per kilogram body weight of the subject; and subsequently (b) administering to the subject a therapeutically effective amount of cyclophosphamide, wherein the therapeutically effective amount comprises 25~200 mg per kilogram body weight, thereby inducing donor specific tolerance in the subject.

According to some embodiments of the invention, the method further comprises conditioning the subject under reduced intensity conditioning prior to step (a).

According to some embodiments of the invention, the method further comprises conditioning the t with in-vivo T cell debulking prior to step (a).

According to some embodiments of the invention. the dose of the T cell depleted immature hematopoietic cells comprises 5 - 40 x 106 CD34+ cells per kilogram body weight of the subject.

According to some embodiments of the invention, the dose of the T cell depleted immature hematopoietic cells comprises at least about 10 x 106 CD34+ cells per kilogram body weight of the subject.

WO 93920 According to some embodiments of the invention, the T cell depleted immature hematopoietic cells are selected from the group consisting of T cell depleted bone marrow cells, T cell depleted G-CSF mobilized peripheral blood progenitor cells, T cell depleted cord blood, ed CD34+ cells attained by positive selection from bone marrow and/or from G—CSF mobilized eral blood progenitor cells, and eX-vivo expanded CD34+ cells.

According to some embodiments of the invention, the T cell depleted immature hematopoietic cells comprise less than 1 X 106 CD8+ TCRu/B' cells per kilogram body weight of the subject.

According to some embodiments of the invention, the T cell depleted immature hematopoietic cells are obtained by T cell debulking.

According to some embodiments of the invention, the T cell debulking is effected by antibodies.

According to some embodiments of the ion, the antibodies are selected from the group consisting of an anti-CD8 antibody, an antinCD4 antibody, an anti—CD3 antibody, an anti—CD2 antibody and an anti-TCRa/B dy.

According to some embodiments of the invention, the antibodies comprise an anti—CD3 antibody. ing to some embodiments of the invention, the immature hematopoietic cells are d by B cell debulking, According to some embodiments of the invention, the B cell debulking is effected by an anti—CD19 antibody or by anti—CD20 antibody.

According to some embodiments of the invention, the T cell depleted immature hematopoietic cells are obtained from a non—syngeneic donor.

According to some embodiments of the invention, the non—syngeneic donor is allogeneic or xenogeneic with respect to the subject.

According to some embodiments of the invention, the allogeneic donor is selected from the group consisting of an HLA d sibling, an HLA d unrelated donor, an HLA haploidentical related donor and a donor displaying one or more disparate HLA determinants.

According to some embodiments of the invention, the subject is a human subject.

According to some embodiments of the invention, the iii-viva T cell debulking is effected by antibodies. ing to some embodiments of the invention, the antibodies comprise an anti—CD8 dy, an anti—CD4 antibody, or both.

According to some embodiments of the invention, the antibodies comprise anti- yte in (ATG) dies, anti—CD52 antibodies or anti—CD3 (OKT3) antibodies.

According to some embodiments of the invention, the reduced ity conditioning comprises a eloablative conditioning.

According to some embodiments of the invention, the non—myeloablative conditioning comprises at least one of a total body irradiation (TBI), a total. lymphoid irradiation (TLI), a chemotherapeutic agent and/or an antibody immunotherapy.

According to some embodiments of the ion, the TBI comprises a single or fractionated irradiation dose within the range of 1—7.5 Gy.

According to some embodiments of the invention, the TBI comprises a single or fractionated irradiation dose within the range of 1-3.5 Gy.

According to some embodiments of the invention, the TBI comprises a single or fractionated irradiation dose within the range of 2 Gy. ing to some embodiments of the invention, the TBI is effected in a single dose 2 days prior to step (b).

According to some embodiments of the invention, the herapeutic agent comprises at least one of Busulfan, Fludarabine, Melphalan and Thiotepa.

According to some ments of the invention, the antibody comprises at least one of an anti-CD52 antibody, anti—thymocyte globulin (ATG) antibody or anti—CD3 (OKT3) antibody.

According to some embodiments of the invention, the concentration of the cyclophosphamide is about 100 - 200 mg per kg body weight.

According to some embodiments of the invention, the concentration of the cyclophosphamide is about 100 mg per kg body weight.

According to some embodiments of the invention, the cyclophosphamide is administered in a single dose.

WO 93920 According to some embodiments of the invention, the cyclophosphamide is administered in two doses.

According to some embodiments of the invention, each of the two doses comprises a concentration of about 50 mg per kg body weight.

According to some embodiments of the invention, each of the two doses is administered on days 3 and 4 following step (a).

According to some embodiments of the invention, the subject has a malignant disease.

According to some embodiments of the invention, the malignant disease is a hematopoietic cancer.

According to some embodiments of the invention, the hematopoietic cancer comprises a leukemia or lymphoma. ing to some ments of the invention, the hematopoietic cancer is selected from the group ting of acute lymphoblastic leukemia (ALL), acute myelocytic leukemia (AML), acute nonlymphoblastic leukemia (ANLL), Chronic lymphocytic leukemia (CLL), chronic myelocytic leukemia (CML), Hodgkin's Lymphoma, non- Hodgkin's Lymphoma, Extranodal natural killer/T-cell lymphoma, Cutaneous T—cell lymphoma, Enteropathy type T-cell lymphoma, Angioimmunoblastic T—cell lymphoma, Anaplastic large T/null—cell lymphoma, Subcutaneous panniculitis- like T-cell lymphoma, Unspecified T~cell lymphoma, e large B—cell lymphoma, B— cell chronic lymphocytic leukemia (B—CLL)/chronic lymphoid leukemia (CLL), Chronic cytic leukemia/small lymphocytic lymphoma, Extranodal marginal zone B—cell lymphomas - mucosa—associated lymphoid tissue lymphomas, Follicular ma, Mantle cell ma, Nodal al zone B-cell lymphoma, Burkitt lymphoma, Hairy cell ia, Primary central nervous system lymphoma, Splenic marginal zone B—cell lymphoma, plasmocytic lymphoma, Primary mediastinal B-cell lymphoma and multiple myeloma.

According to some embodiments of the invention, the subject has a non— malignant disease.

According to some embodiments of the ion, the non-malignant disease is a genetic disease or disorder, an autoimmune disease or a metabolic disorder.

WO 93920 According to some embodiments of the invention, the non-malignant disease is selected from the group consisting of sickle cells disease, a congenital neutropenia, a ocytopenia, an aplastic anemia, a myelodysplastic syndrome, a monosomy 7, an osteopetrosis, a Gaucher's disease, a Hurler's disease, a romatic leukodystrophy, an adrenal leukodystrophy, a thalassemia, a congenital or genetically—determined hematopoietic abnormality, lupus, mune hepatitis, celiac disease, type I diabetes mellitus, Grave's disease, Guillain—Barr syndrome, Myasthenia gravis, Rheumatoid arthritis, scleroderma and psoriasis.

According to some embodiments of the invention, the cell or tissue graft comprises immature hematopoietic cells.

According to some embodiments of the invention, the cell or tissue graft is selected from the group consisting of a liver, a pancreas, a spleen, a kidney, a heart, a lung, a skin, an intestine and a lymphoid/hematopoietic tissue or organ.

According to some embodiments of the invention, the cell or tissue graft is transplanted into the subject prior to, concomitantly with or following the transplanting the dose of T cell depleted immature hematopoietic cells into the subject. ing to some embodiments of the ion, the cell or tissue graft comprises a co—transplantation of several organs.

According to some embodiments of the invention, the cell or tissue graft and the T cell depleted re poietic cells are obtained from the same donor.

According to some embodiments of the invention, the conditioned subject has been conditioned under reduced ity conditioning.

According to some embodiments of the invention, the reduced intensity conditioning is effected l—lO days prior to the transplanting. ing to some embodiments of the invention, the conditioned subject has been conditioned with in-vivo T cell debulking.

According to some embodiments of the invention, the in—vivo T cell debulking is effected 4-7 days prior to the lanting. ing to some embodiments of the invention, the chemotherapeutic agent comprises Fludarabine.

According to some embodiments of the invention, the Fludarabine is ed at a dose of 30 mg/mZ/day.

According to some embodiments of the ion, the Fludarabine is administered daily on days 3 to 7 prior to step (b).

According to some embodiments of the invention, the T cell depleted immature hematopoietic cells comprise T cell depleted G—CSF mobilized peripheral blood itor cells.

It will be appreciated that the present teachings can be used with other tolerance inducing protocols such as bed in PCT publication Nos. W0 2001/49243, WO 2007/023491 and , which are herein incorporated by reference in their entirety.

Unless otherwise defined, all cal and/or scientific terms used herein have the same meaning as ly understood by one of ordinary skill in the art to which the invention pertains. Although methods and materials similar or equivalent to those bed herein can be used in the practice or testing of embodiments of the invention, exemplary methods and/or materials are described below. In case of conflict, the patent specification, including definitions, will control. In addition, the materials, s, and examples are illustrative only and are not intended to be necessarily limiting.

BRIEF DESCRIPTION OF THE DRAWINGS Some embodiments of the invention are herein described, by way of example only, with reference to the accompanying drawings. With specific reference now to the drawings in detail, it is stressed that the particulars shown are by way of example and for purposes of illustrative sion of embodiments of the invention. In this regard, the descn’ption taken with the gs makes apparent to those skilled in the art how embodiments of the ion may be practiced.

In the drawings: FIGS. lA-B are graphs illustrating durable engraftment of mismatched donor bone marrow (BM) following lantation of 'mega dose' rigorously T cell depleted BM and posttranplantation cyclophosphamide. Mice were conditioned with T cell debulking (TCD), using anti-CD4 and anti—CD8 antibodies, on day -6, and by exposure to 2.0 Gy total body ation (TBI) on day -1. High dose Cyclophosphamide (CY, 100 mg/kg) was administered on days +3 and +4 post transplant. Donor type chimerism was evaluated 35 days (Figure 1A) and 95 days (Figure 18) post transplant.

FIGS. 2AeC are dot plot graphs illustrating a typical FACS ism analysis.

Figure 2C depicts that mixed chimerism was achieved in recipients that were transplanted with ‘mega dose’ (25 x 106) rigorously T cell depleted BM and were treated with high dose CY. In contrast, recipient mice that received only the conditioning protocol (Figure 2A) or which were inoculated with only 5 x 106 BM cells and CY did not exhibit donor type chimerism (Figure 2B). is a graph illustrating e mixed chimerism 180 and 225 days post transplant in recipient mice that were transplanted with ‘mega dose’ (25 x 106) T cell depleted BM and were treated with high dose CY. Of note, mice which were inoculated with 5 x 106 T cell depleted BM and CY did not exhibit mixed chimerism.

FIGS. 4A—B illustrate the transplantation of donor type or 3rd party skin grafts in chimeric mice. Figure 4A is a graft illustrating acceptance d by “+”) or rejection (marked by “—“) of donor type (Balb/c) or 3rd party (C57BL/6) skin grafts in ents of regular dose (5 x 106) or ‘mega dose’ (25 x 106) T depleted BM, treated with high dose CY on days +3 and +4 post transplant. Figure 4B is a photograph of donor type (Balb/c) skin graft (white fur) or 3rd party (C57BL/6) skin graft (black fur) in recipients of ‘mega dose’ (25 x 106) T depleted BM, treated with high dose CY on days +3 and +4 post transplant. is a graph illustrating the effect of different doses of irradiation on donor type ism in recipient mice of ‘mega dose’ (25 x 106) T depleted BM and treated with high dose CY post transplant. is a graph illustrating the effect of increased doses of Cyclophosphamide (CY) on donor type chimerism in recipients of ‘mega dose’ (25 X 106) T depleted BM and 2 Gy TBI. is a graph illustrating engraftment of mismatched donor BM achived by combining ‘mega dose’ CD8+ T cell depleted EM and post trasplant CY. Of note, the depletion of residual CD8+ T cells from the BM preparation did not have any adverse impact on the level of chimerism achieved when combing ‘mega dose’ T cell ed BM cells with post transplant CY.

WO 93920 DESCRIPTION OF SPECIFIC EMBODIMENTS OF THE INVENTION The present invention, in some embodiments thereof, relates to a combination therapy for attaining a stable and long term cell or tissue transplantation.

The ples and operation of the present invention may be better understood with reference to the drawings and accompanying descriptions.

Before explaining at least one ment of the invention in detail, it is to be understood that the invention is not necessarily limited in its application to the s set forth in the following description or exemplified by the Examples. The invention is e of other embodiments or of being practiced or carried out in s ways.

Also, it is to be understood that the phraseology and terminology employed herein is for the purpose of description and should not be regarded as limiting.

Application of neic hematopoietic stem cell transplantation (HSCT) has been limited by the lack of available tched donors within the family or in the international ries of ted volunteer donors. Conversely, virtually all patients in need for a transplant have a full-haplotype ched family donor.

The major obstacles to bone marrow transplantation from full-haplotype mismatched related donors were graft versus host disease (GVHD) and graft rejection.

The use of very large numbers of hematopoietic stem cells with minimal residual T cell contamination and an aggressive immunosuppressive and myeloablative regimen has resulted in high rates of engraftment with little severe GVHD. However, immune reconstitution has been delayed and incomplete after this approach and a significant rate of transplant related mortality (TRM) is caused by opportunistic infections.

While reducing the t invention to practice, the present inventors have uncovered that a successful engraftment of mismatched bone marrow can be achieved by transplantation of rigorously T cell depleted “mega dose’ bone marrow and subsequently administering to the subject a high-dose cyclophospamide early after transplantation.

The present inventors have shown that such a regimen requires only a short myeloablative conditioning regimen. The present inventors have further shown that such a transplantation ure leads to a long and stable chimerism and that tolerance has been achieved.

As is shown hereinbelow and in the Examples section which follows, the present inventors have uncovered through laborious experimentation that the combination of ‘mega dose’ T cell depleted bone marrow transplantation (TDBMT) and post transplant high dose cyclophosphamide (CY) allows for a durable engraftment of mismatched donor bone marrow (see Figures lA-B and 2A-C). Durable mixed chimerism was ted for prolonged periods of time after transplantation (180 and 225 days post transplant in mice, see Figure 3). Importantly, the combination of ‘mega dose’ TDBMT and high dose CY following transplantation allowed hematopoietic stem cell engraftment under reduced intensity conditioning (see Figure 5) and ed in tolerance ion, as indicated by acceptance of donor skin grafts (see Figure 4B).

Thus, according to one aspect of the present invention there is provided a method of treating a subject in need of a non—syngeneic cell or tissue graft, the method comprising: (a) transplanting into a subject a dose of T cell ed immature hematopoietic cells, wherein the T cell depleted immature hematopoietic cells comprise less than 5 x 105 CD34r T cells per kilogram body weight of the subject, and wherein the dose comprises at least about 5 X l06 CD34+ cells per am body weight of the subject; and subsequently (b) administering to the subject a therapeutically effective amount of cyclophosphamide, wherein the therapeutically effective amount comprises —200 mg per kilogram body weight, thereby treating the subject.

As used herein, the term ing” includes abrogating, substantially ting, slowing or reversing the progression of a condition, substantially ameliorating clinical or aesthetical symptoms of a condition or substantially preventing the appearance of clinical or aesthetical symptoms of a condition.

As used , the term "subject" or “subject in need thereof” refers to a mammal, preferably a human being, male or female at any age that is in need. of a cell or tissue transplantation. Typically the subject is in need of cell or tissue transplantation (also referred to herein as recipient) due to a disorder or a pathological or undesired condition, state, or syndrome, or a al, morphological or physiological abnormality which is amenable to treatment via cell or tissue transplantation.

According to an embodiment the subject is in need of tissue regeneration (solid or soft ) such as due to aging, trauma, wound or any ogical condition which results in loss of organ functionality.

According to one embodiment the subject has a malignant disease.

According to one ment the ant disease is a hematopoietic cancer. 2012/050542 Exemplary hematopoietic cancers include, but are not limited to, acute lymphoblastic leukemia (ALL), T-cell acute lymphocytic leukemia (T-ALL), acute myelocytic leukemia (AML), acute nonlymphoblastic leukemia , chronic cytic leukemia (CLL), chronic myelocytic leukemia (CML), T—cell prolymphocytic leukemia, B—cell prolymphocytic ia, Juvenile myelomonocytic leukemia, n's Lymphoma, non- Hodgkin's Lymphoma, Extranodal natural killei'flT-cell lymphoma, Cutaneous T—cell lymphoma, Enteropathy type T-cell lymphoma, Angioimmunoblastic T—cell lymphoma, Anaplastic large T/null~cell lymphoma, Subcutaneous ulitis—like T-cell lymphoma, Unspecified T—cell lymphoma, Diffuse large B—cell lymphoma (DLBCL), B—cell chronic lymphocytic leukemia (B-CLL)/chr0nic id leukemia (CLL), Chronic lymphocytic leukemia/small lymphocytic lymphoma, Extranodal marginal zone B-cell lymphomas - mucosa—associated id tissue lymphomas, Follicular lymphoma, Mantle cell lymphoma, Nodal marginal zone B-cell lymphoma, Burkitt lymphoma, Hairy cell leukemia, Primary central s system lymphoma, c marginal zone B-cell lymphoma, Lymphoplasmocytic lymphoma, Primary mediastinal B—cell lymphoma, precursor T-cell leukemia/lymphoma, MALT lymphoma, Mycosis fungoides and multiple myeloma.

According to one embodiment the hematopoietic cancer comprises a leukemia or a lymphoma.

According to one embodiment the subject has a non—malignant disease.

According to one embodiment the non-malignant disease is a genetic disease or disorder, an autoimmune e or a metabolic disorder.

Exemplary non—malignant diseases e, but are not limited to, severe combined immunodeficiency syndromes (SCID), sickle cell disease (sickle cell anemia), congenital neutropenia, thrombocytopenia, aplastic anemia (e. g. severe aplastic anemia), myelodysplastic syndrome, monosomy 7, osteopetrosis, Gaucher's disease, Hurler's disease, metachromatic leukodystrophy, adrenal ystrophy, thalassemia, congenital or genetically—deteimined hematopoietic ality, adenosine deaminase (ADA), lupus, autoimmune hepatitis, celiac disease, type I diabetes us, Grave’s disease, Guillain-Barr syndrome, Myasthenia gravis, Rheumatoid arthritis, derma and psoriasis.

According to one embodiment the subject of the present invention may suffer from any of a cardiovascular disease, a rheumatoid disease, a glandular disease, a intestinal disease, a cutaneous disease, a hepatic disease, a neurological disease, a muscular disease, a nephric e, a connective tissue disease, a systemic disease and/or a disease related to reproduction, treatable by cell or tissue transplantation.

As used herein, the phrase “cell or tissue graft” refers to a bodily cell (eg. a single cell or a group of cells) or tissue (eg. solid tissues or soft tissues, which may be transplanted in full or in part). Exemplary tissues which may be transplanted according to the present teachings include, but are not d to, liver, pancreas, spleen, kidney, heart, lung, skin, ine and lymphoid/hematopoietic tissues (e. g. lymph node, Peyer’s s, thymus or bone marrow). Exemplary cells which may be lanted according to the present teachings include, but are not limited to, immature hematopoietic cells ing stem cells. The present invention also contemplates transplantation of whole organs, such as for example, , heart, lung, liver, pancreas or spleen.

According to one embodiment, the cell or tissue graft comprises immature poietic cells.

According to one embodiment, the method is effected using a cell or , which is non—syngeneic with the subject.

Depending on the ation, the method may be effected using a cell or tissue graft which is allogeneic or xenogeneic with the subject.

As used herein, the term “allogeneic” refers to a cell or tissue which is derived from a donor who is of the same s as the subject, but which is substantially non— clonal with the subject. Typically, outbred, non~zygotic twin mammals of the same species are allogeneic with each other. It will be appreciated that an allogeneic donor may be HLA cal or HLA non~identical (i.e. displaying one or more disparate HLA determinants) with respect to the subject.

According to one embodiment, the allogeneic donor is an HLA matched sibling, an HLA matched unrelated donor, an HLA haploidentical related donor or a donor displaying one or more disparate HLA determinants.

As used herein, the term “xenogeneic” refers to a cell or tissue which substantially expresses antigens of a different species relative to the species of a substantial proportion of the lymphocytes of the subject. Typically, outbred mammals of different species are xenogeneic with each other.

The present ion envisages that xenogeneic cells or tissues are derived from a variety of species such as, but not limited to, bovines (e. g., cow), equines (e.g, horse), porcines (e. g. pig), ovids (e.g., goat, sheep), felines (e.g., Felis ica), s (e.g., Canis domestica), rodents (e. g., mouse, rat, , guinea pig, gerbil, hamster) or primates (e.g., chimpanzee, rhesus monkey, e monkey, marmoset).

Cells or tissues of xenogeneic origin (e.g. porcine origin) are preferably obtained from a source which is known to be free of zoonoses, such as porcine endogenous retroviruses. Similarly, human—derived cells or tissues are preferably obtained from substantially pathogen~free sources.

According to an embodiment of the present invention, both the subject and the donor are humans.

Depending on the application and available sources, the cell or tissue graft of the present invention may be obtained from a prenatal organism, postnatal organism, an adult or a r donor. Moreover, depending on the application needed, the cell or tissue graft may be naive or genetically modified. Determination of the type of cell or tissue graft to be used is well within the ability of one of ordinary skill in the art.

Furthermore, any method known in the art may be employed to obtain a cell or tissue graft (e. g. for transplantation).

As mentioned, a dose of T cell depleted hematopoietic cell or tissue comprising re hematopoietic cells (including e.g. CD345, are transplanted into a subject.

According to one embodiment, the T cell depleted re hematopoietic cells are non—syngeneic (e. g. allogeneic or xenogeneic) with the t. ing to one embodiment, the T cell depleted immature hematopoietic cells and the cell or tissue graft are syngeneic (e. g. obtained from the same donor).

As used herein the phrase “immature hematopoietic cells” refers to a poietic tissue or cell preparation comprising precursor hematopoietic cells. Such tissue/cell preparation includes or is derived from a biological sample, for e, bone marrow, mobilized peripheral blood (e. g. zation of CD34 cells to enhance their concentration), cord blood (eg. umbilical cord), fetal liver, yolk sac and/or placenta.

Additionally, purified CD34+ cells or other hematopoietic stem cells such as CDl3l+ cells can be used in accordance with the present teachings, either with or without ex-vivo ion. ing to one embodiment, the immature poietic cells complise T cell depleted immature hematopoietic cells.

As used herein the phrase “T cell depleted immature hematopoietic cells” refers to a population of hematopoietic cells which are depleted of T lymphocytes. The T cell depleted immature hematopoietic cells, may include e.g. CD34+, CD33+ and/or CD56+ cells. The T cell depleted re hematopoietic cells may be depleted of CD3+ cells, CD2+ cells, CD8+ cells, CD4+ cells, (ll/[3 T cells and/or 7/5 T cells.

According to one embodiment, the immature hematopoietic cells complise T cell depleted G—CSF mobilized blood cells enriched for CD34+ immature poietic cells.

According to one embodiment, the immature hematopoietic cells are depleted of CD3+ T cells.

According to an embodiment, the T cell depleted immature hematopoietic cells comprise less than 50 x lo5 CD3+ T cells, 40 x 105 CD3+ T cells, 30 x 105 CD3+ T cells, x lo5 CD3+ T cells, 15 x lo5 CD3+ T cells, 10 x lo5 CD3+ T cells, 9 x lo5 CD3+ T cells, 8 x 105 CD3+ T cells, 7 x 105 CD3+ T cells, 6 x lo5 CD3+ T cells, 5 x 105 c133+ T cells, 4 x 105 CD3+ T cells, 3 x lo5 CD3+ T cells, 2 x lo5 cm+ T cells or 1 x lo5 cos+ T cells per kilogram body weight of the subject.

According to a specific embodiment, the T cell depleted immature hematopoietic cells comprise less than 5 X 105 CD3+ T cells per kilogram body weight of the subject.

According to a specific embodiment the T cell depleted immature hematopoietic cells comprise less than 20 x 105 CD3+ T cells but more than 10 CD3+ T cells. ing to an embodiment, the T cell depleted immature poietic cells comprise at least 1 x 103 — l x 105 CD3+ Tcells.

According to one embodiment, the immature hematopoietic cells are depleted of CD8+ cells.

According to an embodiment, the T cell ed immature hematopoietic cells comprise less than 1 x 104— 4 x 105 CD8+ cells per kilogram body weight of the subject.

According to an embodiment, the T cell depleted immature hematopoietic cells comprise less than 50 x 105 (:le+ cells, 25 x lo5 cps+ cells, 15 x 105 CD8+ cells, 10 x lo5 CD8+ cells, 9 x lo5 cps+ cells, 8 x lo5 CD8+ cells, 7 x 105 CD8+ cells, 6 x lo5 CD8+ cells, 5 x 105 CD8+ cells, 4 x 105 CD8+ cells, 3 x 105 CD8" cells, 2 x lo5 cps+ cells, 1 x lo5 CD8+ cells, 9 x lo" CD8+ cells, 8 x 10“ CD8+ cells, 7 x lo4 CD8+ cells, 6 x lo4 CD8+ cells, 5 x 10“ CD8+ cells, 4 x 10“ CD8+ cells, 3 x lo4 CD8+ cells, 2 x 10“ CD8+ cells or 1 x 104 CD8+ cells per kilogram body weight of the subject.

According to a specific ment, the T cell depleted immature hematopoietic cells comprise less than 4 x 105 CD8+ cells per kilogram body weight of the subject.

According to a specific embodiment the T cell depleted immature hematopoietic cells comprise less than 4 x 105 CD8+ cells but more than 10 CD8+ cells.

According to an embodiment, the T cell depleted immature hematopoietic cells comprise less than 1 X 106 CD8+ TCRa/B- cells per am body weight of the t.

According to an ment, the T cell depleted immature hematopoietic cells comprise less than l x lo6 CD8+ TCRa/B’ cells, 0.5 x lo6 CD8+ TCRa/B‘ cells, 1 x lo5 CD8+ TCRa/B‘ cells, 0.5 x 105 CD8+ TCRd/B' cells, 1 x 104 CD8+ TCRo/B' cells, 0.5 x lo4 CD8+ 'l‘CRcl/B~ cells, 1 x lo3 CD8+ TCRa/B‘ cells or 0.5 x 103 CD8+ TCRo/B' cells per kilogram body weight of the t.

According to a specific embodiment, the T cell depleted immature hematopoietic cells comprise less than 1 x 106 CD84r TCROl/B~ cells per kilogram body weight of the subject.

According to a specific embodiment the T cell depleted immature hematopoietic cells comprise less than 1 x 106 CD8+ TCRa/B' cells but more than 10 CD8+ TCRO/B’ cells.

According to one embodiment, the immature poietic cells are depleted of B cells.

According to an ment, the immature hematopoietic cells are depleted of B cells (CD19+ and/or CD20+ B cells).

According to an embodiment, the re hematopoietic cells comprise less than 50 x 105 B cells, 40 x 105 B cells, 30 x 105 B cells, 20 x 105 B cells, 10 x 105 B cells, 9 x lo5 B cells, 8 x lo5 B cells, 7 x 105 B cells, 6 x lo5 B cells, 5 x lo5 B cells, 4 x 105 B cells, 3 X 105 B cells, 2 x 105 B cells or 1 x 105 B cells per kilogram body weight of the subject.

WO 93920 According to a specific embodiment, the re hematopoietic cells comprise less than 4 x 105 B cells per kilogram body weight of the subject. According to a specific embodiment the immature hematopoietic cells comprise less than 50 x 105 B cells but more than 10 B cells.

Depletion of T cells, e.g. CD3+, CD2+, TCRU/B+, CD4+ and/or CD8+ cells, or B cells, e. g. CDl9+ and/or CD20+ cells, may be carried out using any method known in the art, such as by eradication (e. g. killing) with specific antibodies or by affinity based purification e.g. such as by the use of magnetic cell separation techniques, FACS sorter and/01' capture ELISA labeling.

Such methods are described herein and in THE HANDBOOK OF EXPERIMENTAL IMMUNOLOGY, Volumes 1 to 4, (D.N. Weir, editor) and FLOW CYTOMETRY AND CELL SORTING (A. Radbruch, editor, Springer Verlag, 1992).

For e, cells can be sorted by, for example, flow cytometry or FACS. Thus, fluorescence activated cell sorting (FACS) may be used and may have varying degrees of color channels, low angle and obtuse light scattering detecting channels, and impedance channels. Any ligand-dependent separation techniques known in the art may be used in ction with both positive and negative separation techniques that rely on the al properties of the cells rather than antibody affinity, including but not limited to ation and y nt centrifugation.

Other s for cell sorting include, for e, panning and separation using affinity techniques, including those ques using solid supports such as plates, beads and columns. Thus, biological samples may be ted by "panning" with an antibody attached to a solid matrix, e.g. to a plate.

Alternatively, cells may be sorted/separated by magnetic separation techniques, and some of these methods utilize magnetic beads. Different magnetic beads are available from a number of sources, including for example, Dynal (Norway), Advanced Magnetics (Cambridge, MA, USA), lmmuncon (Philadelphia, U.S.A.), lmmunotec (Marseille, France), Invitrogen, Stem cell Technologies (USA) and Cellpro (USA).

Alternatively, dies can be biotinylated or conjugated with digoxigenin and used in conjunction with avidin or anti-digoxigenin coated affinity columns. ing to an embodiment, different depletion/separation methods can be combined, for example, magnetic cell sorting can be combined with FACS, to increase the separation quality or to allow sorting by multiple ters.

According to one embodiment, the T cell depleted immature hematopoietic cells are obtained by T cell ing (TCD).

T cell debulking may be effected using antibodies, including e.g. anti—CD8 antibodies, anti-CD4 antibodies, anti—CD3 antibodies, anti—CD2 antibodies, anti—TCRtx/B antibodies and/or anti—TCRy/B antibodies.

According to one ment, depletion of B cells is effected by B cell IO debulking.

B cell debulking may be effected using antibodies, including e.g. anti-CD19 or D20 antibodies. Alternatively, debulking iii—viva of B cells can be attained by infusion of anti—CD20 antibodies.

Alternatively, positive ion of CD34+ or CD13l+ stem cells may be cairied out using e. g. magnetic cell separation techniques, FACS sorter and/or e ELISA labeling as described in r detail above.

As mentioned, T cell or B cell debulking may be effected in-vitro or in-vivo (eg. in a donor prior to acquiring re hematopoietic cells therefrom).

According to one ment, the T cell depleted immature hematopoietic cells (e. g. comprising CD34+ cells) comprise T cell depleted bone marrow cells, T cell depleted mobilized peiipheral blood progenitor cells (e. g. mobilized by G—CSF), T cell depleted cord blood/fetal liver/yolk sac and/or, pun'fied CD34+ cells (harvested from all the sources mentioned above e.g. from bone marrow and/or from G-CSF mobilized peripheral blood itor cells) and ed by positive selection (e.g. with magnetic beads using an anti—CD34 antibody). In addition purified CD34+ cells expanded ex—vivo to increase cell numbers are also contemplated by the present methods.

According to an embodiment of the present invention, the subject is administered with a dose of T cell depleted immature hematopoietic cells comprising at least about, 4 x 106, 4.5 x 106, 5 x 106, 5.5 x 106, 6 x 106, 6.5 x 106, 7 x 106, 7.5 x 106, s x 106, 8.5 x 106, 9 x 106, 9.5 x 106., 10 x 106, 12.5 x106, 15 x 106, 20 x 106, 25 x 106, 30 x 106, 35 x 106, 40 x 106, 45 x 106, 50 x 106, 60 x 106, 70 x 106’ 80 x 106, 90 x 106 CD34+ cells per am body weight.

According to a specific embodiment, the subject is administered a dose of T cell ed immature hematopoietic cells comprising at least about 10 x 106 CD34+ cells per kilogram body .

According to a specific ment, the subject is administered a dose of T cell depleted immature hematopoietic cells comprising at least about 5 X 106 CD34+ cells per kilogram body weight.

According to one embodiment, the subject is administered a dose of T cell depleted immature poietic cells comprising a range of about 4—30 X 106, 4—40 x 106, 4-50 x 106,460 x 106, 4-70 x 106, 4-80 x 106, 4-90 x 10": 4-100 x 106, 5-10 x 106, 5- 20 x 106, 5-30 x 1.06, 5-40 x 106, 5-50 x 106, 5-60 x 106, 5-70 x 106, 5-80 x 106, 5-90 x 106, 5-100 x 106, 10-20 x 106, 10-30 x 106,10-40 x 106, 10-50 x 106,10—60 x 106,10-70 x 106, 10-80 x 10", 10-90 x 106, 10-100 x 106, 20-30 x 106, 20-40 x 106, 20-50 x 106, 20-60 x 106, 20-70 x 106, 20—80 x 106, 20-90 x 106, 20-100 x 106, 30-40 x 106, 30-50 x 106, 30- 60 x 106, 30-70 x 106, 30-80 x 106, 30-90 x 106, 30-100 x 106, 40-50 x 106, 40-60 x 106, 40-70 x 106, 40-80 x 106, 40-90 x 106, 40100 x 106, 50-60 x 106, 50-70 x 106, 50-80 x 106, 50-90 x 106, 50100 x 106, 60-70 x 106, 60-80 x 106, 60-90 x 106, 60-100 x 106. 70-80 x 106, 70-90 x 106, 70-100 x 106, 80—90 x 106, 80-100 x 106 CD34+ cells per kilogram body weight of the subject.

According to a c embodiment, the subject is administered a dose of T cell depleted immature hematopoietic cells sing a range of about 5—40 x 106 CD34+ cells per kilogram body weight.

The T cell depleted immature hematopoietic cells of the present invention be transplanted into a recipient using any method known in the art for cell transplantation, such as but not limited to, cell infusion (e.g. I.V.), via an intraperitoneal route or via an intrabone route.

As mentioned, the subject of the instant invention may r be transplanted with a cell or tissue graft (e. g. liver, pancreas, spleen, kidney, heart, lung, skin, intestine and/or lymphoid/hematopoietic tissues).

Transplanting the cell or tissue into the subject may be effected in numerous ways, depending on various parameters, such as, for example, the cell or tissue type; the type, stage or severity of the ent's disease (cg. organ failure); the physical or logical parameters specific to the subject; and/or the desired therapeutic outcome.

Transplanting a cell or tissue graft of the present invention may be effected by transplanting the cell or tissue graft into any one of various anatomical locations, depending on the ation. The cell or tissue graft may be transplanted into a homotopic anatomical location (a normal anatomical location for the lant), or into U] an ectopic ical on (an abnormal anatomical location for the transplant).

Depending on the application, the cell or tissue graft may be advantageously implanted under the renal capsule, or into the kidney, the ular fat, the sub cutis, the omentum, the portal vein, the liver, the spleen, the heart cavity, the heart, the chest cavity, the lung, the skin, the pancreas and/or the intra abdominal space.

For example, a liver tissue according to the t teachings may be transplanted into the liver, the portal vein, the renal capsule, the sub—cutis, the omentum, the spleen, and the abdominal space. Transplantation of a liver into various anatomical locations such as these is commonly practiced in the art to treat diseases amenable to treatment via c transplantation (eg. hepatic failure). Similarly, transplanting a pancreatic tissue according to the present invention may be advantageously ed by transplanting the tissue into the portal vein, the liver, the pancreas, the testicular fat, the sub-cutis, the omentum, an intestinal loop (the subserosa of a U loop of the small intestine) and/or the intra-abdominal space. Transplantation of pancreatic tissue may be used to treat diseases amenable to treatment via pancreatic transplantation (eg. diabetes). Likewise, transplantation of tissues such as a kidney, a heart, a lung or skin tissue may be d out into any anatomical location described above for the purpose of treating recipients suffering from, for example, renal e, heart failure, lung failure or skin damage (e.g., burns).

Optionally, when transplanting a cell or tissue graft of the present invention into a subject having a defective organ, it may be advantageous to first at least partially remove the failed organ from the subject so as to enable optimal development of the transplant, and ural/functional integration thereof with the anatomy/physiology of the subject.

The method of the present invention also envisions co-transplantation of several organs (e.g. heart and lung, liver and spleen, pancreas and bone marrow e.g. poietic stem cells, kidney and bone marrow e.g. hematopoietic stem cells, etc.) in case the subject may be beneficially affected by such a procedure. ing to one embodiment, the co-transplantation comprises transplantation of re hematopoietic cells and a solid tissue/organ or a number of solid organs/tissues. ing to one ment, the immature hematopoietic cells and the solid Ul organ are obtained from the same donor.

According to one embodiment, the cell or tissue graft (e.g. solid organ) is transplanted into the subject prior to, concomitantly with or following transplanting of the T cell depleted immature hematopoietic cells (e.g. sing CD34+ cells) into the subject.

Following transplantation of the cell or tissue graft into the subject, it is advisable, according to standard medical practice, to monitor the growth functionality and immuno—compatability of the organ according to any one of various standard art techniques. For example, the functionality of a atic tissue transplant may be monitored following transplantation by standard pancreas function tests (eg. analysis of serum levels of insulin). se, a liver tissue transplant may be monitored following lantation by standard liver function tests (e.g. analysis of serum levels of albumin, total protein, ALT, AST, and bilirubin, and is of blood-clotting time). Structural development of the cell or tissue graft may be monitored via computerized tomography, or ultrasound imaging.

Regardless of the transplant type, in order to reduce, by at least about 30 %, 4O %, 50 %, 6O %, 7O %, 80 %, 9O % or 95 %, or preferably avoid graft rejection and/or graft versus host disease (GVHD), the present invention plates post transplant administration of cyclophosphamide.

According to one embodiment, the t invention further contemplates administration of cyclophosphamide prior to transplantation (e.g. on days 4, 3 or 2 prior to transplantation, i.e. "D4, —3 or O.) in addition to the administration following transplantation as described herein.

Of note, the date of transplantation (of the cell or tissue graft) is considered Tzzero.

As used herein, the term ”cyclophosphamide" refers to the nitrogen mustard alkylating agent which specifically adds an alkyl group (CnH2n+1) to DNA (also known as cytophosphane). In a specific embodiment, the cyclophosphamide refers to the 2012/050542 lar a C7H15CBNZOZP'HZO and the chemical name 2-[bis(2— chloroethyl)amino]tetrahydro—ZH-1,3,2-oxazaphosphorine '2-oxide monohydrate.

Cyclophosphamide is commercially available from e.g. Zydus (German Remedies), Roxane Laboratories ehringer eim, Bristol~Myers Squibb Co — Mead Johnson and Co, and Pfizer — Pharmacia & Upjohn, under the brand names of n, Cytoxan, Neosar, Procytox and une.

A therapeutically effective amount of cyclophosphamide is typically administered to the subject following transplantation of the cell or tissue graft.

Without being bound to theory, a therapeutically effective amount is an amount of cyclophosphamide efficient for killing ted donor or host alloreactive T cells without being toxic to the subject.

For example, in case of cell or tissue graft, the therapeutic effective amount of cyclophosphamide comprises about 1—25 mg, 1—50 mg, 1—75 mg, 1—100 mg, 1—250 mg, 1—500 mg, l—750 mg, 1—1000 mg, 5-50 mg, 575 mg, 5400 mg, 5—250 mg, 5-500 mg, 5— 750 mg, 5—1000 mg, 10-50 mg, 10-75 mg, 10—100 mg, 10—250 mg, 10-500 mg, 10-750 mg, 10-1000 mg, 25—50 mg, 25-75 mg, 25-100 mg, 25—125 mg, 25—200 mg, 25-300 mg, -400 mg, 25-500 mg, 25-750 mg, 25-1000 mg, 50-75 mg, 50-100 mg, 50—125 mg, 50— 150 mg, 50—175 mg, 50—200 mg, 50—250 mg, 50-500 mg, 50—1000 mg, 75-100 mg, 75- 125 mg, 75~150 mg, 75~250 mg, 75—500 mg, 75—1000 mg, 100—125 mg, 100-150 mg, 100-200 mg, 100—300 mg, 100—400 mg, 100—500 mg, 100—1000 mg, 125—150 mg, 125- 250 mg, 125-500 mg, 125—1000 mg, 150—200 mg, 150—300 mg, 150-500 mg, 150-1000 mg, 200-300 mg, 200-400 mg, 200600 mg, 200—750 mg, 200—1000 mg, 250-500 mg, 250-750 mg, 250—1000 mg per kilogram body weight of the subject.

According to a specific embodiment, the therapeutic effective amount of cyclophosphamide is about 25200 mg per kilogram body weight of the subject.

As illustrated in the Examples section which follows, the present inventors have shown that administration of two doses of cyclophosphamide post transplant (on days 3 and 4 post transplant) allows for a e engraftment and tolerance of ‘mega dose’ T cell depleted mismatched donor bone marrow.

According to one embodiment, cyclophosphamide is administered in a single dose.

According to one embodiment, hosphamide is administered in multiple doses, e.g. in 2, 3, 4, 5 doses or more.

According to a specific embodiment, cyclophosphamide is administered in two doses.

According to one embodiment, cyclophosphamide is administered daily such as once a day or twice a day.

The dose of each cyclophosphamide administration may comprise about 5 mg, 7.5 mg, 10 mg, 20 mg, 30 mg, 40 mg, 50 mg, 60 mg, 70 mg, 80 mg, 90 mg, 100 mg, 110 mg, 120 mg, 130 mg, 140 mg, 150 mg, 160 mg, 170 mg, 180 mg, 190 mg, 200 mg, 210 mg, 220 mg, 230 mg, 240 mg, 250 mg, 260 mg, 270 mg, 280 mg, 290 mg, 300 mg, 350 mg, 400 mg, 450 mg or 500 mg per kilogram body weight of the subject.

According to a specific embodiment, the dose ophosphamide is 50 mg per kilogram body weight of the subject.

As mentioned, cyclophosphamide is administered post transplantation. Thus, for example, cyclophosphamide may be administered to the t 1, 2, 3, 4, 5, 6, 7, 8, 9, days or more post transplant (i.e., T+1, +2, +3, +4, +5, +6, +7, +8, +9, +10).

According to a c embodiment, cyclophosphamide is administered to the subject in two doses 3 and 4 days post transplant.

According to an embodiment, cyclophosphamide is administered prior to transplantation and post lantation. Thus, for e, cyclophosphamide may be administered to the subject 3 days prior to transplantation (T-3) and then post transplantation (e. g. on days T+3, +4, etc.).

The number of administrations and the therapeutically effective amount of cyclophosphamide may be adjusted as needed taking into account the type of k)U: transplantation and the subject's response to the regimen. Determination of the number of administrations and the therapeutically effective amount is well within the capability of those skilled in the art, especially in light of the detailed disclosure provided herein.

In order to facilitate engraftment of the cell or tissue graft, the method may further advantageously comprise ioning the subject with an additional immunosuppressive drug and/or immunosuppressive irradiation prior to, concomitantly with or following transplantation of the cell or tissue graft.

It will be appreciated that in situations in which the cell or tissue graft (e. g. solid organ) is transplanted prior to the T cell depleted immature hematopoietic cells, it is advisable to use general immune suppressive agents (eg. cyclosporine A, as described in further detail below) in order to avoid organ rejection. Once the T cell depleted immature hematopoietic cells are transplanted and chimerisrn is ed, the general immune suppression agents may be tapered down and subsequently stopped. In contrast in situations in which the cell or tissue graft (e. g. solid organ) is lanted uent to the T cell depleted immature hematopoietic cells, after chimerism induction, the use of general immune suppression may not required.

Ample guidance for selecting and administering suitable immunosuppressive ns for transplantation is ed in the ture of the art (for example, refer to: Kirkpatrick CH. and Rowlands DT Jr., 1992. JAMA. 268, 2952; Higgins RM. et al., 1996. Lancet 348, 1208; Suthanthiran M. and Strom TB., 1996. New Engl. J. Med. 331, 365; Midthun DE. et al., 1997. Mayo Clin Proc. 72, 175; Morrison VA. et al., 1994. Am J Med. 97, 14; Hanto DW., 1995. Annu Rev Med. 46, 381; Senderowicz AM. et al., 1997. Ann Intern Med. 126, 882; Vincenti F. et a1., 1998. New Engl. J. Med. 338, 161; Dantal J. et a1. 1998. Lancet 351, 623).

Thus, according to an embodiment of the present invention, the subject is conditioned under reduced intensity conditioning prior to transplantation of a cell or tissue graft, According to an embodiment, the reduced intensity ioning is effected for up to 2 weeks (e.g. 1—10 or l~7 days) prior to transplantation of the cell or tissue graft.

Thus, for example, the subject may be treated with a myeloablative or non- myeloablative conditioning. Such conditioning may comprise, for example and as described in detail in the Examples section which s, in-vivo T cell debulking e.g. by D4 dy, anti-CD8 antibody, anti—CD3 (OKT3) antibodies, anti—CD52 antibodies (e.g. CAMPATH) and/or anti—thymocyte globulin (ATG) antibody (e.g. 6 days prior to transplantation at a therapeutic effective dose of about 300 n g each).

The conditioning may additionally or alternatively comprise total body ation (TBI), total lymphoid irradiation (TLI, i.e. exposure of all lymph nodes, the thymus, and spleen), a chemotherapeutic agent and/or an antibody immunotherapy.

WO 93920 Thus, according to one embodiment, the TBI comprises a single or fractionated ation dose within the range of 0.5-1 Gy, 0.5-1.5 Gy, 0.5-2.5 Gy, 0.5—5 Gy, 0.5-7.5 Gy, 0.5—10 Gy, 0.5-15 Gy, 1—1.5 Gy, 1-2 Gy, 1-2.5 Gy, 1—3 Gy, l-3.5 Gy, 1-4 Gy, 1—4.5 Gy, 1—1.5 Gy, 1—7.5 Gy, l-lO Gy, 2—3 Gy, 2—4 Gy, 2-5 Gy, 2—6 Gy, 2-7 Gy, 2—8 Gy, 2—9 Ur Gy, 2-10 Gy, 3—4 Gy, 3—5 Gy, 3—6 Gy, 3—7 Gy, 3—8 Gy, 3—9 Gy, 3-10 Gy, 4—5 Gy, 4-6 Gy, 4—7 Gy, 4—8 Gy, 4-9 Gy, 4—10 Gy, 5—6 Gy, 5—7 Gy, 5—8 Gy, 5—9 Gy, 5—10 Gy, 6—7 Gy, 6—8 Gy, 6—9 Gy, 6—10 Gy, 7—8 Gy, 7-9 Gy, 7-10 Gy, 8—9 Gy, 8—10 Gy, 10—12 Gy or 10—15 Gy.

According to a specific ment, the TBI comprises a single or fractionated irradiation dose within the range of 1—3.5 Gy.

According to an embodiment, TBl treatment is administered to the subject 1—10 days (e.g. 1—3 days) prior to transplantation. According to one embodiment, the subject is conditioned once with TBI l or 2 days prior to transplantation.

According to a specific embodiment, the TLI comprises an irradiation dose within the range of 0.5-1 Gy, 0.5—1.5 Gy, 0.5—2.5 Gy, 0.5—5 Gy, 0.5-7.5 Gy, 0.5—10 Gy, 0.5-15 Gy, 1—1.5 Gy, 1-2 Gy, 1—2.5 Gy, 1-3 Gy, 1—3.5 Gy, 1-4 Gy, 1-4.5 Gy, 1—1.5 Gy, l- 7.5 Gy, l-lO Gy, 2-3 Gy, 2—4 Gy, 2-5 Gy, 2-6 Gy, 2-7 Gy, 2—8 Gy, 2-9 Gy, 2-10 Gy, 3-4 Gy, 3—5 Gy, 3-6 Gy, 3-7 Gy, 3—8 Gy, 3-9 Gy, 3-10 Gy, 4-5 Gy, 4—6 Gy, 4-7 Gy, 4-8 Gy, 4—9 Gy, 4—10 Gy, 5—6 Gy, 5—7 Gy, 5-8 Gy, 5—9 Gy, 5-10 Gy, 6—7 Gy, 6—8 Gy, 6—9 Gy, 6— Gy, 7—8 Gy. 7—9 Gy, 7—10 Gy, 8-9 Gy, 8—10 Gy, 10—12 Gy, 10—15 Gy, 10—20 Gy, 10— 30 Gy, 10—40 Gy, 10—50 Gy, 0.5—20 Gy, 0.5—30 Gy, 0.5—40 Gy or 05—50 Gy. ing to a specific embodiment, the TLI ses a single or fractionated irradiation dose within the range of 1—3.5 Gy. ing to an embodiment, TLI treatment is administered to the subject 1—10 days (e. g. 1—3 days) prior to transplantation. According to one embodiment, the subject is conditioned once with TL12—7 days prior to transplantation.

According to one embodiment, the conditioning comprises a chemotherapeutic agent. Exemplary chemotherapeutic agents include, but are not limited to, Busulfan, Myleran, Busulfex, Fludarabine, Melphalan and Thiotepa and cyclophosphamide. The chemotherapeutic agent/s may be administered to the subject in a single dose or in several doses 6g. 2, 3, 4, 5, 6, 7, 8, 9, 10 or more doses (cg. daily doses) prior to transplantation. ing to one embodiment, the subject is administered a WO 93920 chemotherapeutic agent (e. g. Fludarabine e.g. at a dose of about 30 mg/mZ/day) for 5 consecutive days prior to transplantation (e. g. on days -7 to -3).

According to one embodiment, the ioning comprises an antibody immunotherapy. Exemplary antibodies include, but are not limited to, an anti—CD52 antibody (e.g. Alemtuzumab sold under the brand names of e.g. h, MabCampath, Campath—lH and Lemtrada) and an anti-thymocyte globulin (ATG) agent [c.g.

Thymoglobulin (rabbit ATG, rATG, available from Genzyme) and Atgam (equine ATG, eATG, available from Pfizer)]. Additional antibody immunotherapy may se anti— CD3 (OKT3), anti~CD4 or anti-CD8 . According to one embodiment, the antibody is administered to the subject in a single dose or in several doses e.g. 2, 3, 4, 5, 6, 7, 8, 9, or more doses (eg. daily doses) prior to transplantation (e.g. 6 days prior to transplantation).

According to one embodiment, the subject is not treated chronically (e.g. for a prolonged period of time, e.g. for more than 10 days) with GVHD prophylaxis post transplant.

According to one embodiment, in case of relapse after hematopoietic stem cell transplantation, the subject may be further treated by donor lymphocyte infusions (DLIs). For example, the t may be administered with graded doses of T-cells as previously described by Dazzi et al [Dazzi, Szydlo et al., Blood, (2000) 96: 2712—6] fully incorporated herein by reference.

According to one embodiment, the subject may be treated by on of about 0.5 - 5 x 104 CD3+ lymphocytes per kg recipient body weight (eg. 1 x 104 CD3+ lymphocytes, e.g. unmanipulated CD3+ lymphocytes, per kg recipient body weight) for the treatment of relapse following T cell depleted haploidentical transplantation.

According to one embodiment, a patient with early molecular and/or hematological relapse will further be treated with a first dose of about 1 x 104 CD3+ cells per Kg recipient body . In the absence of GVHD, the second on of about 1 x 105 CD3+ cells per kg recipient body weight will lly be given about 45 days later followed 2 months later by a third dose of about 1 x 106 CD3“ cells per kg recipient body weight. It will be appreciated that donors typically undergo a leukoapheresis to collect cytes prior to mobilization of hematopoietic cells (e. g. for transplantation). The frozen products are thawed as needed and infused quickly over WO 93920 a period of 5—10 minutes. ts ting acute GVHD or who fail to trate hematological engraftment lly will not receive any DLI.

According to one embodiment, a patient with relapsing B cell non-Hodgkin lymphoma will lly be further treated with rituximab (e.g. 375 mg/m2 weekly for about 4 weeks) with DLI concomitant with the second rituximab dose.

According to one embodiment, a patient with relapsing multiple myeloma will be further treated with bortezomib (e.g. 1.3 mg/sqm on days 1, 4, 8 and ll) before starting DLI. ing to one embodiment, no post—DLl immunosuppressive agents will be used along with the present methods.

According to an aspect of the present invention, there is provided a method of treating a subject in need of a T cell depleted immature hematopoietic cell transplantation, the method comprising: (a) transplanting into a conditioned subject a dose of T cell depleted immature poietic cells, wherein the T cell depleted re hematopoietic cells comprise less than 5 X 105 CD3+ cells per kilogram body weight of the subject, and n the dose comprises at least about 5 x 106 CD34+ cells per kilogram body weight of the subject; and subsequently (b) administering to the subject a therapeutically effective amount of cyclophosphamide, wherein the therapeutically effective amount comprises 25—200 mg per kilogram body weight, thereby treating the subject.

According to an aspect of the present invention, there is provided a method of treating a subject in need of an immature hematopoietic cell transplantation, the method comprising: (a) conditioning a subject under a reduced intensity conditioning ol, wherein the reduced intensity conditioning comprises a total body irradiation (TBI) and a chemotherapeutic agent; (b) transplanting into the t a dose of T cell depleted immature hematopoietic cells, n the T cell depleted immature hematopoietic cells comprise less than 5 x 105 CD3+ cells per kilogram body weight of the subject, and wherein the dose comprises at least about 5 x 106 CD34+ cells per kilogram body weight of the subject; and subsequently (c) administering to the subject a therapeutically effective amount of cyclophosphamide, wherein the therapeutically effective amount comprises 25-200 mg per kilogram body weight, thereby treating the subject. 2012/050542 According to an aspect of the present invention, there is provided a method of inducing donor specific tolerance in a t in need of a ngeneic cell or tissue graft, the method comprising: (a) transplanting into a t a dose of T cell depleted immature hematopoietic cells obtained from a non—syngeneic donor, n the T cell depleted immature hematopoietic cells comprise less than 5 X 105 CD3+ cells per kilogram body weight of the subject, and wherein the dose comprises at least about 5 x 106 CD34+ cells per kilogram body weight of the subject; and subsequently (b) administering to the subject a therapeutically effective amount of cyclophosphamide, wherein the therapeutically effective amount comprises 25—200 mg per kilogram body weight, thereby treating the t.

As used herein, the term “donor specific tolerance” as used herein refers to a condition in which there is a decreased responsiveness of the recipient's cells (e.g. recipient's T cells) when they come in t with the s cells (cg. donor hematopoietic cells) as compared to the responsiveness of the recipient's cells in the absence of such a treatment method.

Tolerance induction enables lantation of a cell or tissue graft (as described in further detail hereinabove) with reduced risk of graft rejection or GVI—ID.

According to one embodiment of the present invention, patients with early molecular and/or hematological e may receive donor lymphocyte infusions (DLI).

According to one embodiment of the present invention, DLI may comprise 1 x 103 - 1 x 106 CD3+ T cell/Kg recipient body weight.

According to one embodiment, patients with early molecular and/or hematological relapse may receive a single dose or several doses (two, three, four, five or more doses) of DLI.

Thus, for example. patients with early molecular and/or hematological relapse may receive a first dose of l x 104 CD3“ T cell/Kg recipient body weight. In the absence of graft versus host disease (GVHD), a second infusion of l x 105 CD3+ T cell/kg recipient body weight may be given e. g. 45 days later followed e.g. 2 months later by a third dose of l x 106 CD3+ T cell/kg recipient body weight.

According to one embodiment, patients with early molecular and/or logical relapse may receive total body irradiation (TBI), total id irradiation (TLI), a chemotherapeutic agent and/or an antibody immunotherapy.

Thus, for e, patients with relapsing B cell non—Hodgkin lymphoma may receive rituximab (e.g. at a dose of 375 mg/m2 weekly) for about 4 weeks with DLI concomitant with the second rituximab dose.

Thus, for example, patients with relapsing multiple myeloma may be treated with bortezomib (eg. at a dose of 1.3 mg/sqm on days 1, 4, 8 and 11) before starting DLI.

As used herein the term “about” refers to i 10 %.

The terms ises", "comprising", ”includes", "including", “having” and their conjugates mean "including but not limited to".

The term “consisting of means “including and limited to”.

The term ”consisting essentially of” means that the composition, method or structure may include additional ingredients, steps and/or parts, but only if the additional ingredients, steps and/0r parts do not materially alter the basic and novel characteristics of the claimed composition, method or structure.

As used herein, the singular form ”a“, 'an” and "the" include plural references unless the t clearly dictates ise. For example, the term "a compound" or "at least one compound" may include a plurality of compounds, including mixtures thereof.

Throughout this application, s embodiments of this invention may be presented in a range format. It should be understood that the ption in range format is merely for convenience and brevity and should not be ued as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically sed all the possible subranges as well as individual numerical values within that range. For e, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers Within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.

Whenever a numerical range is indicated herein, it is meant to include any cited numeral (fractional 0r integral) within the indicated range. The phrases “ranging/ranges between” a first indicate number and a second indicate number and “ranging/ranges from” ‘9 a first indicate number “to‘ a second indicate number are used herein interchangeably and are meant to include the first and second indicated numbers and all the fractional and integral numerals etween.

As used herein the term "method" refers to manners, means, techniques and procedures for accomplishing a given task including, but not limited to, those manners, means, techniques and procedures either known to, or readily developed from known manners, means, techniques and procedures by practitioners of the chemical, pharmacological, ical, biochemical and medical arts.

It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the ion, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination or as suitable in any other described embodiment of the invention. Certain features described in the context of various ments are not to be considered essential features of those ments, unless the ment is inoperative without those elements.

Various embodiments and aspects of the present invention as delineated above and as claimed in the claims section below find experimental support in the following examples.

EXAMPLES Reference is now made to the following examples, which together with the above ptions, illustrate the invention in a non limiting fashion. lly, the nomenclature used herein and the tory procedures utilized in the present invention include molecular, biochemical, microbiological and recombinant DNA techniques. Such techniques are thoroughly explained in the literature. See, for example, "Molecular Cloning: A laboratory Manual" Sambrook et al., ; "Current Protocols in Molecular y" Volumes I—Ill Ausubel, R. M., ed.

; Ausubel et al., "Current Protocols in Molecular Biology”, John Wiley and Sons, Baltimore, Maryland (1989); Perbal, "A Practical Guide to Molecular Cloning", John Wiley & Sons, New York (1988); Watson et al., "Recombinant DNA", ific American Books, New York; Birren et al. (eds) "Genome Analysis: A Laboratory Manual Series", Vols. 1-4, Cold Spring Harbor Laboratory Press, New York (1998); methodologies as set forth in US. Pat. Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and 5,272,057; ”Cell y: A Laboratory Handbook", Volumes I-III Cellis, J. E., ed. (1994); ”Current Protocols in Immunology" Volumes 1-111 Coligan 1. 13., ed. (1994); Stites et al. (eds), "Basic and Clinical Immunology" (8th Edition), Appleton & Lange, Norwalk, CT (1994); Mishell and Shiigi (eds), "Selected s in Cellular Immunology", W. H. Freeman and Co., New York (1980); available immunoassays are ively bed in the patent and scientific ture, see, for example, US. Pat.

Nos. 3,791,932; 3,839,153; 3,850,752; 3,850,578; 3,853,987; 3,867,517; 3,879,262; 3,901,654; 3,935,074; 3,984,533; 3,996,345; 4,034,074; 4,098,876; 219; 5,011,771 and 5,281,521; ”Oligonucleotide Synthesis” Gait, M. 1., ed. ; “Nucleic Acid Hybridization” Hames, B. D., and Higgins S. 1., eds. (1985); "Transcription and Translation" Hames, B. D., and Higgins S. 1., Eds. (1984); "Animal Cell Culture” Freshney, R. 1., ed. ; ”Immobilized Cells and Enzymes" IRL Press, ; "A Practical Guide to Molecular Cloning" Perbal, B., (1984) and "Methods in Enzymology" Vol. 1—317, Academic Press; "PCR Protocols: A Guide To Methods And Applications", Academic Press, San Diego, CA (1990); Marshak et al., "Strategies for Protein Purification and Characterization — A Laboratory Course " CSHL Press (1996); all of which are incorporated by reference as if fully set forth herein. Other l references are provided throughout this document. The procedures therein are believed to be well known in the art and are provided for the convenience of the reader.

All the information contained therein is incorporated herein by reference.

EXAMPLE 1 Stable engraflment ofHLA mismatched bone marrow following transplantation of 'mega dose’ bone marrow andpost transplantation cyclophosphamide MATERIALS AND EXPERIMENTAL PROCEDURES Animals Mice used in these studies were 6-12 week old female mice. Balbfc—Nude (H-2d) and C3H/Hen (H—2k) were purchased from Harlan Israel (Rehovot, Israel). All mice were kept in small cages (5 animals in each cage) and fed sterile food and acid water.

These studies were approved by the Weizmann Institute of Science, Institutional Animal Care and Use Committee.

Transplantation protocol Low (5 x 106) and high dose (25 x 106) Balb/c—Nude BM cells (providing a U: source of BM depleted of T cells) were transplanted into neic recipients en) on day 0 following in—vivo T cell debulking (TCD) with anti—CD4 (clone GK1.5) and anti—CD8 (clone 53.6.72) dies (300 pg each; Bio X Cell, NH, USA) red on day —6, and exposure to 2.0 Gy total body ation (TBI) on day —1.

High dose hosphamide (CY, 100 mg/kg, Baxter Oncology, Germany) was administered on days +3 and +4 post transplant and donor type chimerism was evaluated 35 and 95 days post transplant using fluorescein anti-host and donor H—2 antibodies (e.g. FlTC labeled anti—l—l-2Dd antibody specific for donor type cells and PE labeled anti-H-ZKk antibody c for host type cells).

Skin graft ol Donor (Balb/c) and 3rd party (C57BU6) skin grafts were transplanted to the mixed chimeric recipients as bed above [i.e. to those mice which were previously transplanted with mega dose (25 x 106) T cell depleted EM and were treated with high dose CY] and to the recipient mice that were inoculated with a regular (5 x 106) T cell depleted BM cell dose and were treated with high dose CY.

RESULTS To test the potential synergy between “mega dose’ T cell depleted bone marrow transplant (BMT) and high dose cyclophosphamide (CY) post transplant, after reduced intensity conditioning (RIC) of the recipient mice, the following experiments were carried out.

Recipient mice (C3H/Hen) were treated with a conditioning protocol prior to transplantation of a T cell depleted bone marrow transplant. Specifically, mice were in— vivo treated with T cell debulking (TCD) using anti—CD4 and anti—CD8 antibodies delivered on day -6 and by exposure to 2.0 Gy total body irradiation (TBI) on day ~l.

Next, low (5 x 106) or high dose (25 x 106) Balb/c—Nude BM cells (providing a source of BM depleted of T cells, as Nude mice have miniscule numbers of mature T cells) were transplanted into allogeneic recipients (C3H/Hen) on day 0. High dose cyclophosphamide (CY, lOO mg/kg) was administered on days +3 and +4 post transplant. Evaluation of bone marrow cell engraftment was evaluated by donor type chimerism at 35 and 95 days post transplant.

As shown in Figures lA—B and Figures 2A-B, chimerism analysis on day 35 and day 95 ed that none of the control mice (conditioned with TCD, 2 Gy TBI and optionally CY, but did not receive BM) expressed donor type chimerism. Similarly, none of the BM mice recipients that were transplanted with a regular dose of 5 x 106 T cell depleted BM, in the ce or absence of cyclophosphamide treatment, expressed donor type chimeiism. However when the dose of T cell ed BM was increased to x 106 cells, durable mix chimerisrn was achieved in 4 out of 7 mice that were also treated with cyclophosphamide on days +3 and +4 post lant (see Figures lA-B and Figure 2C).

Further follow-up of these recipient mice at 180 and 225 days post transplant revealed that the chimeiism induced was stable and e (Figure 3). As illustrated in Figure 3, the number of donor type chimeric recipients and the level of donor chimeiism remained unchanged 225 days post transplant, suggesting that tolerance has been achieved. nce induction was measured by transplantation of donor (Balb/c) and 3rd party (C57BL/6) skin grafts to the mixed chimeric recipients that were transplanted with mega dose (‘25 x 106) T cell depleted BM and were treated with high dose CY (as 2O described above), in comparison to the ents that were inoculated with a regular (5 x 106) T cell depleted BM cell dose (as described above).

As shown in Figures 4A~B, three out of 4 chimeric mice that were transplanted with 25 x 106 T cell depleted BM accepted the donor graft and rejected the 3rd party skin grafts. In contrast, recipient mice that were inoculated with 5 X 106 T cell depleted BM cells and CY rejected both the donor and 3rd party skin grafts e 4A).

These results illustrate that the combination of mega dose T cell depleted BM and high dose Cyclophosphamide treatment allows the successful engraftment of hematopoietic stem cells, under reduced intensity conditioning, along with tolerance ion.

Encouraged by these s a set of calibration ments were initiated in order to determine the Optimal irradiation and Cyclophosphamide dose to improve chimerism induction by this approach.

WO 93920 EXAMPLE 2 The efiect ofdifferent doses of total body ation (TBI) on chimerism MATERIALS AND EXPERIMENTAL PROCEDURES Animals As described in Example 1, hereinabove.

Transplantation ol High dose (25 x 106) Balb/c—Nude BM cells (providing a source of BM depleted of T cells) were transplanted into allogeneic recipients (C3l-l/l-len) on day 0 following in—vivo T cell ing (TCD) with anti-CD4 (clone GK1.5) and anti—CD8 (clone 53.6.72) antibodies (300 5.1g each; Bio X Cell, NH, USA) delivered on day —6, and exposure to different doses of irradiation ranging from 1 to 3.5 Gy TBI on day -1. High dose Cyclophosphamide (CY, 100 mg/kg, Baxter Oncology, Germany) was administered on days +3 and +4 post transplant and donor type chimerism was evaluated 30 days post transplant using fluorescein anti~host and donor H—2 antibodies (eg. FITC labeled anti—H-ZDd antibody specific for donor type cells and PE labeled anti—H—ZKk dy specific for host type cells).

RESULTS In this experiment, the minimal irradiation dose was defined. ‘Mega dose’ (25 x 106) —Nude T cell depleted BM was transplanted into 5 groups of allogeneic recipients (C3H/Hen) on day 0 following T cell debulking (with anti-CD4 and anti—CD8 antibodies) on day —6, and different doses of irradiation (ranging from 1 to 3.5 Gy TBI) on day ~1. High dose Cyclophosphamide (CY) was administered on days +3 and +4 post transplant and donor type chimerism was ted at 30 days post transplant.

As can be seen in Figure 5, all the recipient mice that were ated with 2.5, 3 or 3.5 Gy TBI (6/6) were ic, exhibiting donor type chimerism ranging between 58 — 83 %. Similarly, 87 % ) of the mice treated with 2 Gy TBI exhibited donor type chimeiism ranging between 56 - 85 %.

Further reduction of the irradiation dose to 1.0 Gy caused a small reduction in the percentage of chimeric mice, namely 83 % (5/6), however the donor type chimerism range was significantly reduced to 14.5 — 58 %.

EXAMPLE 3 The efiect of ent Cyclophosphamide (CY) doses on chimerism MATERIALS AND EXPERIMENTAL PROCEDURES Animals As described in Example 1, hereinabove.

Transplantation protocol High dose (25 X 106) Balb/C~Nude BM cells ding a source of BM depleted of T cells) were lanted into allogeneic recipients (CSH/Hen) on day 0 following in—vivo T cell debulking (TCD) with anti—CD4 (clone GK1.5) and anti—CD8 (clone 53.6.72) antibodies (300 ug each; Bio X Cell, NH, USA) delivered on day —6, and exposure to 2.0 Gy total body irradiation (TBI) on day —l. Different doses of Cyclophosphamide (CY, 100 mg/kg, 125 mg/kg or 150 rug/kg, Baxter gy, Germany) were administered on days +3 and +4 post transplant and donor type chimerism was evaluated 30 days post lant using fluorescein anti—host and donor H-2 antibodies (e.g. FlTC labeled anti-H—2Dd antibody specific for donor type cells and PE labeled anti-H-2Kk antibody specific for host type cells).

RESULTS In this experiment, the optimal dose of CY post transplant was defined. ‘Mega dose’ (25 x 106) Balb/c—Nude BM cells were transplanted into 3 groups of allogeneic ents (C3H/Hen) on day 0 following T cell debulking (TCD) with anti—CD4 and anti—CD8 antibodies on day -6, and 2 Gy TBl on day -1. Different doses of Cyclophospharnide (CY), 100 mg/kg, 125 mg/kg or 150 nag/kg, were administered on days +3 and +4 post transplant and donor type chimerism was performed 30 days post transplant.

As can be seen in Figure 6, increasing CY dose to 125 mg/kg or 150 mg/kg did not provide a significant enhancement of chimeiism. Thus, the recipient mice that were d with 100 tug/kg, 125 mg/kg or 150 nag/kg CY exhibited an average of 57.5 i .8, 66.5 i 20.6 or 67.4 i 27.4 donor type chimerism, tively. No statistical icance was found when the recipients treated with 100 mg/kg were compared to those treated with 125 mg/kg or 150 mg/kg (P=O.5 and p=0.469 respectively).

EXAMPLE 4 C08+ Non-T cells are not importantfor attaining chimerism by combining ‘mega dose’ T cell depleted BM with CYpost transplant MATERIALS AND EXPERIMENTAL PROCEDURES Animals As bed in Example 1, hereinabove.

Transplantation protocol High dose (25 x 106) of CD8 depleted and non-depleted Balb/c—Nude BM cells were transplanted into 2 cohorts of allogeneic recipients (C3H/Hen) on day 0 following in~vivo T cell debulking (TCD) with anti-CD4 (clone GK1.5) and anti-CD8 (clone 53.6.72) antibodies (300 ug each; Bio X Cell, NH, USA) delivered on day -6, and exposure to 2.0 Gy total body irradiation (TBI) on day —1. High dose Cyclophosphamide (CY, 100 , Baxter Oncology, Germany) was administered on days +3 and +4 post lant and donor type chimeiism was evaluated 30 days post transplant using fluorescein anti-host and donor H-Z antibodies (e. g. FITC labeled anti- H-2Dd antibody specific for donor type cells and PE labeled anti—H-2Kk antibody specific for host type cells).

The BM source in these experiments was —Nude mice. Moreover, the lanted mice in these experiments were athyrnic and as such they lacked T cells.

However in order to refute the possibility that the effect was a contribution of residual non—T CD8 cells, the BM ation from Balb/c—Nude mice was vely sorted for CD8 cells using a cell sorting system (e. g. anti—CD8 magnetic beads or FACS sorter).

As Ildstad et al. previously taught that a subset of CDSJr TCR‘ BM cells are critical for achieving donor type chimerism [Fugier-Vivier I] et al., J Exp Med (2005) 201:373-383; Grimes HL et al., Exp Hematol. (2004) 32:946—954; Huang Y et al., Blood (2011) 117:2494-2505; Kaufman CL et al., Blood (1994) 84:2436-2446; Leventhal J et al., BMC Med (2012) 10:48; Leventhal J et al., Sci Trans! Med. (2012) 4:1241‘al28], the present inventors ed residual CD8+ cells from the ——Nude ‘mega dose’ BM preparation, and measured chimerism induction compared to control non-CD8+ depleted Nude BM cells.

As can be seen in Figure 7, ion of CD8+ T cells from the BM preparation did not have any adverse impact on the level of chimeiism achieved when combing ‘mega dose’ T cell ed BM cells with post transplant CY.

EXAMPLE 5 Clinical protocol STUDY DESIGN This is a prospective, observational, phase l/ll multicenter study. Ten patients with logical disorders will be enrolled over a one year period.

The primary endpoint of the study is engraftment and 10 evaluable patients (i.e. patients surviving beyond day 28) will be entered. An acceptable primary graft failure or rejection rate is approximately 10 %.

Sludv duration The primary analysis will be conducted using 6 and 12 months follow—up data.

Patients will be followed—up until 48 months after lantation.

Definitions Stable sustained engraftment is defined as phils, more than lOOO/ul for three consecutive days, and ets, more than ZOOOO/ul for three consecutive days, 2O without transfusion.

Graft rejection is d as rapid decline of neutrophils, less than lOO/nl after documented neutrophil engraftment, with or without increase of lymphocytes.

Graft failure is defined as failure to reach more than l neutrophils for three consecutive days and more than 20000/lll platelets for three consecutive days without transfusion at day +28.

The secondary endpoint of the study is the incidence of grade II—IV acute GVHD. An acceptable incidence of grade II~lV acute GVHD is approximately 10 %.

For acute GVHD grading criteria is indicated in Tables lA—B, below.

Table 1A: Clinical g of acute GVHD SKIN LIVER GUT Rash more than 25 % Bilirubin = 2—3 mg/dl Diarrhea 500- 1000 ml Rash 25-50 % Bilirubin = 3—6 mg/dl Diarrhea 1000—1500 n11 Generalized Diarrhea more than 1500 Bilirubin = 6-15 mg/dl erythroderrna ml Desquamation and Bilirubin more than 15 Pain or ileus bullae mg/dl Table 1B: Clinical grading of acute GVHD ++ to +++ IV / life ening ++ to ++++ ++ to ++++ Statistical considerations The time intervals for tment, survival, disease-free survival, relapse rate and risk of transplant—related mortality will be calculated from the day of stem cell transplantation. Actuarial curves will be ated according to the Kaplan—Meier method.

ELIGIBILITY CRITERIA Inclusion Criteria - Patient — Age ~ more or equal to 18 and less or equal to 70 years old — CLL patients with toriness to fludarabine or other chemotherapy due to the p53 loss by 17p deletion and/or TP53 mutation - Follicular lymphoma with either unfavorable cytogenetics such as complex karyotype, dell7p, del 6q23-26, mutations in TP53, minus 1p — Hodgkin's Lymphoma relapsed after autologous transplantation, not eligible for immunotherapy with anti-CD30 — Multiple myeloma relapsing after gous lantation, with rable cytogenetics in either partial or complete remission - Severe Aplastic Anemia relapsing after immunotherapy — Absence of fully HLA-matched or one locus HLA—mismatched family donor — Absence of matched unrelated donor or ineligibility for donor search in the donor registry (lBMDR) — Presence of haploidentical family donor and a back—up of patient gous stem cells - Stable clinical ions and life expectancy of more than 12 weeks — Karnofsky — more than 70 ‘70 — Written informed consent Pre-treatment tion — complete clinical history and examination and determination of mance status and body surface area. - complete blood count — blood group, red blood cells subgroups, anti~A and/or anti—B agglutinin titration — creatinine nce, uric acid, ferritin, LDH, beta 2 microglobulin, protein electrophoresis, SGOT, SGPT, urine test, blood glucose, blood nitrogen, immunoglobulin levels, Coombs tests. — pregnancy test — HIV-ab, HBsAg, HBVDNA, HCV—ab, HCVRNA, CMV—ab, Toxoplasma~ab, HSVab — ECG and measurement of ejection fraction by ultrasound or scintigraphic tCSt. - chest X ray. — lung CT scan, brain CT scan, maxillary sinus CT scan. - dental X ray and examination. — biopsy and bone marrow aspirate for morphologic and cytogenetic analysis, search for a lar marker (if not known) and FACS analysis (according to underlying disease). — neurologic examination and lumbar puncture in patient at risk. - radiologic scan (CT, NMR) of the known disease localization. ~ complete serologic and molecular HLA typing, ML cultures and cytotoxicity test with the selected donors. — cytotoxic anti HLA antibodies.

- Abdominal echography IO Exclusion criteria - Patient — History of central nervous system disease localization — Positivity for HIV, HCV, HCVRNA, HBsAg, HBVDNA — Active and documented nia of any kind, fungal tissue infection, viral positive cultures of respiratory secretion or blood - bilirubin of more than 2 times normal - blood creatinine nce less than 50 ml/min - DLCO less than 50 % of the predicted value — ejection fraction less than 45 % (or myocardial stroke in the last year) — pregnancy or lactation — psychiatric disorders Eligibility Criteria - Donor ~ Absence of poietic or marrow function related disease that interferes with the collection of sufficient numbers of normal progenitor cells. - e of any medical condition that would pose a serious health risk by undergoing peripheral blood stem cell harvest ~ Negative HIV, HTLV—l tests - Any healthy family member will be considered for hematopoietic stem cell donation. Selection of a donor will be based on typing of HLA-A, B, C, DR loci to be carried out on the recipient, siblings, parents and ly other family members such as aunts, uncles and cousins. A prospective d donor must be at least genotypically HLA-A, B, C, DR haploidentical to the patient, but can differ for 2-3 HLA alleles on the ed haplotype. 2012/050542 - Donor will be selected preferentially on the basis of the donor—versus— recipient NK alloreactivity.

Donor Evaluation — te history, physical examination and examination of physical veins by the pheresis service for determination of suitability for pheresis via eral veins.

- Blood tests: WBC, FLT, Hb, PT, PTT, total n, albumin, electrolytes, glucose, SGOT/SGPT. alkaline phosphatase, bilirubin, LDH, acid uric, creatinine.

— CMV, EBV, HSV, VZV, Hepatitis B + C, HIV, Toxoplasma serology.

— Complete red blood cell typing — Serology for Syphilis, CMV, EBV, HSV, VZV, Hepatitis B + C, HTLV— 1, HIV, Toxoplasmosis.

— Transfusion transmitted disease g must be performed between 30 and 7 days prior to stem cell collection — Chest X-ray - EKG - VNTR analysis by PCR — Donors will be piioritized on the basis of r age, better health, and being CMV~negative for CMV—negative recipients.

Exclusion Criteria — Donor ~ A positive HIV or HTLV—l test or evidence of active/persistent viral hepatitis infection will exclude the donor from participation in this study. — ce of any medical condition that would pose a serious health risk by undergoing peripheral biood stem cell harvest (i.e. insulin—dependent diabetes, cardiovascular disorders, chronic inflammatory diseases).

TREATMENT PROCEDURES Mobilization of donor HSC and Graft processing.

Patients are required to have a family donor (aged 18 to 60 yrs), g and e of donating filgrastim/lenogastrim-stimulated peripheral blood hematopoietic cells. Donors will be screened according to Blood Bank general miles. It is advisable to perform an se EKG testing in donors above 50 yrs of age. Normal donors will receive filgrastim or lenogastrim 5 meg/kg subcutaneously every 12 hours; on day 5 the leukapheresis will be started. Filgrastim/Lenogastrim dosage will be adjusted to maintain white blood cells below 60 x 109/L. On the 4th day of filgrastim/lenograstim treatment, if the circulating CD34+ cell count is more than 40/uL, the donor will start leukapheresis. Daily leukapheresis will be continued for a planned 3 days, with a maximum of 4 days, to collect a target cell dose of more than 10 x 106 CD34+ cells/kg.

If the target is reached early, collection can continue for 3 total days in order to give the largest possible dose. If the donor does not tolerate the procedure in any of its component parts, an alternative donor may be used if available. If a site is unable to t more than 10 x 106 CD34+ cells/kg from an appropriate donor, ts may not d on study. PBPCs will be depleted of donor T and B cells by selection of CD3+ and/or CD19+ cells using a cell sorting system (cg. anti-CD3/l9 magnetic beads or FACS sorter). Target value of CD34-positive cells will be at least 10 x 106/kg of the recipient body weight (b.w.).

The apheresis will be med through the antecubital veins.

Table 2: Conditioning regimen day -7 i Fludarabine 301ng/sqm day 6 E Fludarabine 30mg/sq1n day -5 E Fludarabine 3O mg/sqm day 4 l Fludarabinc 30 mg/sqm day —3 Fludarabine 3O mg/sqm day -2 § TBI 2 Gy single fraction day -l l Rest day 0 l Graft day +1 l Rest day +2 l Rest day +3 ; CY 50 rug/kg day +4 I CY 50 trig/kg As described in Table 2, above, fludarabine will be administered intravenously daily on 5 sequential days, -7, -6, -5, —4, and —3, at a dose of 30 mg/m2. Each dose will be d over 30 s. TBI 200 cGy will be given on day —l in a single fraction.

On day 0, CD3'/CD19' immunoselected HSCs will be thawed, washed and infused h a central access.

CY will be administered intravenously in one hour on days +3 and +4 post— transplantation at 50 mg/kg/day.

Special management orders a. a double—lumen central venous line will be placed before conditioning regimen; b. for urate prophylaxis allopurinol 300 mg per 08 will be given; c. antiemetic therapy will be given according to single center guidelines; d. transfusion of filtered and irradiated blood products. Keep hemoglobin level more than 8 g/L and platelets more than 15000/ ttL in absence of fever or bleeding signs; Patient monitoring during treatment a. daily full blood count and ential b. serum nine, Na+, K+, Ca++, bilirubin daily during chemotherapy and hyper-hydration c. liver function tests, albumin, coagulation tests with antitrombin III, cytomegalovirus nemia and PCR twice a week. d. surveillance cultures according to center guidelines TOXICITY EVALUATION Toxicity will be ted according to WHO criteria, as indicated in Table 3, below.

Table 3: WHO toxicity criteria —GradeO Grade 1 -_—— 2 11.0 g/dl 95—109 g/dl 4 g/dl 6.5~7.9 g/dl < 6.5 g/dl Hen10g 01 bin 2 6-8 5.6-6.7 mmol/l 4.9—5.6 mmol/l 4.0-4.9 mmol/l < 4.0 mmol/l mmol/l ytes 2 4-0 339 2.0 2 9 1.0—1.9 (IOOOfmm) Granulocytes 2 2.0 1.5—1.9 l 0—1.4 0.5-0.9 < 0 5 (1000/mm) Platelets (lOOO/unn) 2 100 75—99 50-74 25—49 PCTlIL2012/050542 Mild blood Gross blood Hemorrhage Petechiae Debilitating loss loss blood loss Gastrointestinal Transaminases 126—25 X N* (SGOT SGP’T) Alkaline 1.26~2.5 X N* otase Erythema, Ulcers : Soreness/ Alimentation NO change ulcers: can eat requires liquid erythema not possible solids diet onl Transient Vomiting lntractable /vomiting requiring vomiting therav vomiting Intolerable.

Transient < 2 Tolerable, but Diarrhoea Hemorrhagic requiring ation Blood urea or $1.25X 126m5xNn 2%: creatinine Ny 1+ 23+ Proteinuria No change < 0.3 g % Nephrotic 0&10g% < 3 g/l 340 0/1 syndrome Obstructive Heinaturia No change Microscopic Gross + clots uro - ath_ Mild onal Complete Pulmonary No change Dyspnoea at bed rest symptoms ea rest re 0 uired Fever with drug None Fever < 38 °C Fever 38-40 ° Fever > 40 “C h notenswn Bronchospasm: Bronchospas No change Oedema parenteral : no parenteral Anaphylaxis therapy therapy neede re uired Exfoliative Dry dermatitis: Most Cutaneous necrOSis No change Erythema mation, desquamation,. vesiculation, . . ulceration. reqmring pruritus surgical. intervention Moderate, Complete Non- Minimal hair No change patchy loss alopecia but reversible alo n ecia reversible alo ecia Major with Infection (specify Minor Moderate None Major site) infection. , infection infection hypotension Sinus Unifocal PVC Multifocal Ventricular Rhythm tachycardia, atrial arrh thmia tachycardia > 110 at rest Transient . Symptomatic Asymptomatic,. . Symptomatic matic , . dysfunction Function. but abnormal . ction dysfunction: . non- cardiac s1gn. . swe to . no therapy responsive to . therapy re urred thera .

Tamponade: Pericarditis No change Asymptomatic Symptomatic: Tamponade: effusron. . . surgery no tap require tap ed re uired State of Transient . 50 % of Coma conscrousness therapy waking hours Paresthesias Severe Intolerable and or hesias paresthesias Peripheral decreased and or mild and or marked Paralysis tendon reflexe weakness motor loss Constipation. . xx Abdominal Distension - Moderate. distenSion. . and vomitino. .

Pain Moderate Intractable N* upper limit of normal value of population under study.

** This does not include constipation resultant from narcotics + Only treatment-related pain is considered, not disease-related pain.

UI Use of narcotics may be helpful in grading pain ing on the patient's tolerance.

SUPPORTIVE CARE Monitoring and treatment of bacterial and fungal infections ts are cared for in isolation rooms with laminar airflow or fficiency air-particulate filtration. Liposomal Amphotericin is given at 1 mg/kg/day from day —5 to engraftment as antifungal prophylaxis. Bacterial infections are monitored by swabs and blood es . Intravenous otic therapy is started on the basis of clinical signs of infection (fever of unknown origin) or positive blood cultures. If the patient is still febrile after 72 hours, empiric antifungal therapy is started using either L— AMB 3 mg/kg/day or Voriconazole 8 mg/kg/day i.v. Vancomycin is added after an additional 72 hours of fever, or in the presence of Gram+ sepsis, or positive blood culture.

Prophylaxis, ring and treatment of cytomegalovirus infections In recipients who are seropositive for CMV antibody, CMV prophylaxis consists of ganciclovir (10 mg/kg/day) between the tenth and second day before stem cell on. Ganciclovir is oduced as preemptive therapy from day +21 until day +360. CMV antigenemia/PCR is determined weekly in blood samples. If CMV antigenemiaJPCR develops, patients will treated with ganciclovir (10 mg/kg/day) or foscarnet (180 mg/kg/day).

The blood products are irradiated (30 Gy) before transfusion.

Post-Transplant Laboratory Evaluation: 1. Daily te hemograms until granulocytes and platelets are self— sustaining, then three times/week until discharge; at least every week post discharge to day 100 and then every 2 weeks to 12 months. 2. ing profile with liver and renal function tests twice weekly for the first 30 days, then weekly to discharge; more frequently if clinically indicated. 3. Bone marrow aspirates for morphology analysis of chimerism by FISH (sex—mismatched grafts) or cytogenetics will be done at approximately 1, 3, 6, 12 months, and every 4 months thereafter for approximately 3 years. onal analysis will be done as clinically indicated. Patients with CML will be also monitored for bcr/abl evidence of recurrence 4. Immunological reconstitution will be monitored by in vitro assays, including ypic analysis of ating lymphocytes, assessment of natural killer and lymphokine activated killer cell function, lymphocyte transformation responses to T-cell and B—cell mitogens and immuneglobulin levels.

Follow-up Until day +90 complete blood counts, nemia and PCR for CMV, reactive protein C, complete liver and renal function will be assess twice a week.

Every two weeks until +90 peripheral blood phenotype (CD3, CD4, CD8, CD19, CD56, CD57, HLADR), chest Xi'ay.

Every two weeks from +90 till +180: complete blood counts, antigenemia and PCR for CMV, reactive protein C, complete liver and renal function.

Monthly: immunoglobulin , n electrophoresis, after +90 peripheral blood phenotype (CD3, CD4, CD8, CD19, CD56, CD57, HLADR), chest Xray. after + 180 complete blood counts, antigenemia and PCR for CMV, reactive protein C, complete liver and renal on.

Complete restaging of disease will be performed 2, 4, 6, 8, l2, l8 and 24 months after transplantation then annually, this will include assessment of donor chimerism by PCR analysis of HLA on eral blood and bone marrow cells.

For Performance Status grading criteria see Table 4, below.

Table 4: Karnofsky Performance scale FUNCTIONAL STATUS GROUP SCORES Normal. No complaints. No ce of disease.

Able to carry on normal ty. Minor signs or symptoms of e. Rehabilitated Normal activity with effort. Some signs or symptoms (80+) of disease.

Cares for self. Unable to carry on normal ty or Self-care only do active work. (70-90) Requires occasional assistance, but able to care for most needs.

Requires considerable assistance and frequent medical Requires Caretaker care. (40-69) Disabled. Requires special care and assistance.

Severely disabled. Hospitalisation is indicated, although death is not imminent.

Very sick, Hospitalisation ary. 2O institutionalisation Moribund. Fatal processes progressing. 10 ( 1-39) Dead.

PROGRAMMED INFUSIONS of DONOR LYMPHOCYTES Donor lymphocyte ons (DLls) are effective to treat relapses after allogeneic HSCT. Nevertheless, the success of DLI has been limited to some extent by the morbidity and mortality ated with GVHD. Graded doses of T—cells are less likely to produce GVHD than a single large infusion and appear to be as effective to induce remission [Dazzi, Szydlo et al., Blood, (2000) 96: 2712—6]. A recent dose finding study has shown that l x 104 unmanipulated CD3+ lymphocyte/kg recipient b.w. can be safely infused in patients who have received a T cell depleted haploidentical transplantation [Lewalle P. et al. Bone Marrow Transplant (2002) 29 (suppl 2): S26, 0164a].

Patients with early lar and/0r hematological relapse will receive a first dose of 1 x 104 CD3+ cell/Kg recipient b.w.; in the absence of GVHD, the second infusion of 1 x 105 CD3+ cell/kg will be given 45 days later followed 2 months later by a third dose of 1 X 106 CD3+ g. Donors will undergo a leukoapheresis to collect lymphocytes prior to mobilization of hematopoietic cells because it has been shown that G—CSF has an immune—modulatory effect on some T cyte subsets, decreasing their responsiveness to allogeneic stimuli. The frozen products will be thawed and d y over a period of 5—10 minutes. Patients with acute GVHD or who fail to demonstrate hematological tment will not receive any DLI.

Patients with relapsing B cell non-Hodgkin lymphoma will receive rituximab 375 mg/m2 weekly for 4 weeks with DLI concomitant with the second rituximab dose.

Patients with relapsing multiple myeloma will be treated with bortezomib (1.3 mg/sqm on days 1, 4, 8 and 11) before starting DLI.

No LI immunosuppressive agents will be used gh the invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications and variations will be apparent to those skilled in the art. Accordingly, it is intended to embrace all such alternatives, modifications and variations that fall within the range and broad scope of the appended claims.

All publications, patents and patent applications ned in this specification are herein incorporated in their entirety by into the cation, to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated herein by reference. In addition, citation or identification of any reference in this application shall not be construed as an admission that such reference is available as prior art to the present invention. To the extent that section headings are used, they should not be construed as necessarily limiting.

Claims (76)

WHAT IS CLAIMED IS:

1. Use of a dose of T cell depleted immature hematopoietic cells, wherein said T cell ed immature poietic cells comprise less than 5 x 105 CD3+ cells per kilogram body weight of a subject, and wherein said dose comprises at least about 5 x 106 CD34+ cells per am body weight of the subject, and a therapeutically effective amount of cyclophosphamide, wherein said therapeutically effective amount ses 25~200 mg per kilogram body weight of the subject, and n said cyclophosphamide is for administration to the subject following transplantation of a cell or tissue graft, in the manufacture of a medicament for treating a subject in need of a non-syngeneic cell or tissue graft, wherein said subject has a malignant disease.

2. Use of a dose of T cell depleted immature hematopoietic cells, wherein said T cell depleted immature hematopoietic cells comprise less than 5 x 105 CD3+ cells per kilogram body weight of a subject, and wherein said dose comprises at least about 5 x 106 CD34+ cells per kilogram body weight of the subject, and a therapeutically effective amount of hosphamide, wherein said therapeutically effective amount comprises 25—200 mg per kilogram body weight of the subject, and wherein said hosphamide is for administration to the subject following lantation of a cell or tissue graft, in the manufacture of a medicament for treating a subject in need of a non-syngeneic cell or tissue graft, wherein said subject has a non—malignant disease.

3. The use of claim 1 or 2, wherein the medicament is adapted for administration with a reduced intensity conditioning protocol.

4. The use of claim 1 or 2, wherein the medicament is adapted for stration with an in—vz’vo T cell debulking conditioning protocol.

5. The use of claim 1 or 2, wherein a dose of said T cell depleted immature hematopoietic cells comprises 5 - 40 X 106 CD34+ cells per kilogram body weight of the subject.

6. The use of claim 5, wherein a dose of said T cell depleted immature hematopoietic cells comprises at least about 10 x 106 CD34+ cells per kilogram body weight of the subject.

7. The use of claim 1 or 2, wherein said T cell depleted immature poietic cells are selected from the group consisting of T cell depleted bone marrow cells, T cell depleted G-CSF mobilized peripheral blood progenitor cells, T cell depleted cord blood, purified CD34+ cells attained by positive selection from bone marrow and/or from G-CSF mobilized peripheral blood progenitor cells, and o expanded CD34+ cells.

8. The use of claim 1 or 2, wherein said T cell depleted re hematopoietic cells comprise less than 1 x 106 CD8+ TCRoz/H cells per am body weight of the subject.

9. The use of claim 1 or 2, wherein said T cell depleted immature hematopoietic cells are obtained by T cell debulking.

10. The use of claim 9, wherein said T cell ing is effected by antibodies.

11. The use of claim 10, wherein said antibodies are selected from the group consisting of an anti-CD8 antibody, an anti—CD4 dy, an anti-CD3 antibody, an anti— CD2 antibody and an anti—TCRoz/B antibody.

12. The use of claim 1 or 2, wherein said immature hematopoietic cells are treated by B cell ing.

13. The use of claim 12, wherein said B cell debulking is effected by an anti— CD19 antibody or by anti-CD20 antibody.

14. The use of claim 1 or 2, n said T cell depleted immature hematopoietic cells are from a non-syngeneic donor.

15. The use of claim 14, wherein said non-syngeneic donor is allogeneic or xenogeneic with respect to the subject.

16. The use of claim 15, wherein said neic donor is selected from the group consisting of an HLA matched sibling, an HLA matched unrelated donor, an HLA haploidentical related donor and a donor displaying one or more disparate HLA determinants.

17. The use of claim 1 or 2, wherein the subject is a human t.

18. The use of claim 4, wherein said o T cell debulking is effected by antibodies.

19. The use of claim 18, wherein said antibodies comprise an anti-CD8 antibody, an anti—CD4 antibody, or both.

20. The use of claim 18, wherein said antibodies comprise anti-thymocyte globulin (ATG) antibodies, D52 antibodies or anti-CD3 (OKT3) dies.

21. The use of claim 3, wherein said reduced ity conditioning comprise a non-myeloablative conditioning.

22. The use of claim 21, wherein said non—myeloablative conditioning comprise at least one of a total body irradiation (TBI), a total lymphoid irradiation (TLI), a chemotherapeutic agent and/or an antibody immunotherapy.

23. The use of claim 22, wherein said TBI comprises a single or fractionated irradiation dose within the range of 1-7.5 Gy.

24. The use of claim 23, wherein said TBI comprises a single or fractionated irradiation dose Within the range of 1—3 .5 Gy.

25. The use of claim 22, wherein said chemotherapeutic agent comprises at least one of Busulfan, Fludarabine, Melphalan and Thiotepa.

26. The use of claim 22, wherein said antibody comprises at least one of an D52 antibody, an anti—thymocyte globulin (ATG) dy and anti-CD3 (OKT3) antibody.

27. The use of claim 1 or 2, wherein said concentration of said cyclophosphamide is about 100 — 200 mg per kg body weight of the subject.

28. The use of claim 27, wherein said concentration of said cyclophosphamide is about 100 mg per kg body weight of the subject.

29. The use of claim 1 or 2, wherein said cyclophosphamide is ed in a single dose.

30. The use of claim 1 or 2, wherein said cyclophosphamide is effected in two doses.

31. The use of claim 30, wherein each of said two doses comprises a concentration of about 50 mg per kg body weight of the subject.

32. The use of claim 30, wherein each of said two doses is for administration on days 3 and 4 ing said transplantation of said cell or tissue graft.

33. The use of claim 1 or 2, wherein said cell or tissue graft ses immature hematopoietic cells.

34. The use of claim 1 or 2, wherein said cell or tissue graft is ed from the group consisting of a liver, a pancreas, a , a kidney, a heart, a lung, a skin, an intestine and a lymphoid/hematopoietic tissue or organ.

35. The use of claim 34, wherein said cell or tissue graft is effected prior to, concomitantly with or following said dose of T cell depleted immature hematopoietic cells.

36. The use of claim 34, wherein said cell or tissue graft comprises a co— transplantation of several organs.

3?. The use of claim 1 or 2, wherein said cell or tissue graft and said T cell depleted immature poietic cells are from the same donor.

38. Use of a dose of T cell depleted immature hematopoietic cells, wherein said T cell depleted immature hematopoietic cells comprise less than 5 x 105 CD3+ cells per kilogram body weight of a subject, and wherein said dose comprises at least about 5 x 106 CD34+ cells per kilogram body weight of the subject, and a therapeutically effective amount of cyclophosphamide, wherein said therapeutically effective amount ses 25—200 mg per kilogram body weight of the subject, and wherein said hosphamide is for administration to the subject following transplantation of immature hematopoietic cells in the manufacture of a medicament for treating a conditioned subject in need of an immature hematopoietic cell transplantation, and wherein said subject has a malignant disease .

39. Use of a dose of T cell depleted immature hematopoietic cells, n said T cell depleted re hematopoietic cells comprise less than 5 x 105 CD3+ cells per kilogram body weight of a subject, and wherein said dose comprises at least about 5 x 106 CD34+ cells per kilogram body weight of the t, and a therapeutically ive amount of cyclophosphamide, wherein said therapeutically ive amount comprises 25-200 mg per kilogram body weight of the subject, and wherein said cyclophosphamide is for administration to the subject following transplantation of immature hematopoietic cells in the manufacture of a medicament for treating a conditioned subject in need of an immature hematopoietic cell transplantation, and wherein said subject has a non- ant disease .

40. The use of claim 38 or 39, wherein said conditioned subject has been conditioned under reduced intensity conditioning protocol.

41. The use of claim 40, wherein said reduced intensity conditioning protocol is effected 1—10 days prior to lantation of immature hematopoietic cells.

42. The use of claim 40, wherein said reduced intensity conditioning comprises a non-myeloablative ioning protocol.

43. The use of claim 42, wherein said non-myeloablative conditioning protocol comprises at least one of a total body irradiation (TBI), a total id irradiation (TLI), a herapeutic agent and/or an antibody immunotherapy.

44. The use of claim 43, wherein said TBI comprises a single or fractionated irradiation dose within the range of 1—7.5 Gy.

45. The use of claim 44, wherein said TBI comprises a single or fractionated irradiation dose within the range of 1—3.5 Gy.

46. The use of claim 43, wherein said chemotherapeutic agent comprises at least one of Busulfan, Fludarabine, Melphalan and Thiotepa.

47. The use of claim 43, wherein said antibody comprises at least one of an anti-CD52 dy, an anti-thymocyte globulin (ATG) antibody and D3 (OKT3) antibody.

48. The use of claim 38 or 39, wherein said conditioned subject has been conditioned with in—vivo T cell debulking.

49. The use of claim 48, wherein said in—vivo T cell debulking is effected 4-7 days prior to transplantation of immature hematopoietic cells.

50. The use of claim 48, n said in—vivo T cell ing is effected by dies.

51. The use of claim 50, wherein said antibodies comprise an anti—CD8 dy, an anti—CD4 antibody or both.

52. The use of claim 50, wherein said dies comprise anti-thymocyte globulin (ATG) antibodies, an anti-thymocyte globulin (ATG) antibody and anti-CD3 (OKT3) antibody.

53. The use of claim 38 or 39, wherein a dose of said T cell depleted immature hematopoietic cells comprises at least about 5 - 40 X 106 CD34+ cells per kilogram body weight of the subject.

54. The use of claim 53, wherein a dose of said T cell depleted immature hematopoietic cells comprises at least about 10 x 106 CD34+ cells per kilogram body weight of the subject.

55. The use of claim 38 or 39, wherein said T cell depleted immature hematopoietic cells are selected from the group consisting of T cell depleted bone marrow cells, T cell depleted G—CSF mobilized peripheral blood progenitor cells, T cell depleted cord blood, purified CD34+ cells attained by positive selection from bone mairow and/or from G-CSF mobilized peripheral blood itor cells, and o expanded CD34+ cells.

56. The use of claim 38 or 39, wherein said T cell depleted immature hematopoietic cells comprise less than 1 X 106 CD8+ TCROl/B’ cells per kilogram body weight of the subject.

57. The use of claim 38 or 39, wherein said T cell depleted immature hematopoietic cells are ed by T cell debulking.

58. The use of claim 57, wherein said T cell debulking is effected by antibodies.

59. The use of claim 58, wherein said dies are selected from the group consisting of an anti-CD8 antibody, an anti-CD4 antibody, an anti-CD3 antibody, an anti- CD2 antibody and an anti-TCRodfi antibody.

60. The use of claim 38 or 39, wherein said immature hematopoietic cells are treated by B cell debulking.

61. The use of claim 60, wherein said B cell debulking is effected by an anti- CD19 antibody or anti—CD20 antibody.

62. The use of claim 38 or 39, wherein said T cell depleted immature hematopoietic cells are from a non—syngeneic donor.

63. The use of claim 62, n said non-syngeneic donor is allogeneic or xenogeneic with respect to the subject.

64. The use of claim 38 or 39, wherein the subject is a human subject.

65. The use of claim 38 or 39, wherein said concentration of said cyclophosphamide is about 100 - 200 mg per kg body weight of the subject.

66. The use of claim 65, wherein said concentration of said cyclophosphamide is about 100 mg per kg body weight of the subject.

67. The use of claim 38 or 39, wherein said cyclophosphamide is effected in a single dose.

68. The use of claim 38 or 39, n said hosphamide is effected in two doses.

69. The use of claim 68, wherein each of said two doses comprises a concentration of about 50 mg per kg body weight of the subject.

70. The use of claim 68, wherein each of said two doses is for administration on days 3 and 4 following said transplantation of said cell immature hematopoietic cells.

71. The use of claim 1 or 38, wherein said malignant disease is a hematopoietic cancer.

72. The use of claim 71, wherein said hematopoietic cancer comprises a leukemia or lymphoma.

73. The use of claim 71, wherein said hematopoietic cancer is selected from the group consisting of acute lymphoblastic leukemia (ALL), acute myelocytic ia (AML), acute nonlymphoblastic leukemia (ANLL), Chronic lymphocytic leukemia (CLL), chronic myelocytic leukemia (CML), Hodgkin's Lymphoma, non— n's Lymphoma, Extranodal natural killer/T-cell ma, ous T-cell lymphoma, Enteropathy type T-cell lymphoma, Angioimmunoblastic T—cell lymphoma, Anaplastic large -cell lymphoma, Subcutaneous panniculitis-like T—cell lymphoma, Unspecified T-cell lymphoma, Diffuse large B-cell lymphoma, B-cell chronic lymphocytic ia (B-CLL)/chronic lymphoid leukemia (CLL), Chronic lymphocytic leukemia/small lymphocytic ma, Extranodal marginal zone B-cell lymphomas - mucosa-associated lymphoid tissue lymphomas, Follicular lymphoma, Mantle cell lymphoma, Nodal marginal zone B—cell lymphoma, Burkitt lymphoma, Hairy cell leukemia, Primary central nervous system ma, Splenic marginal zone B-cell lymphoma, Lymphoplasmocytic lymphoma, y mediastinal B-cell lymphoma and multiple myeloma.

74. The use of claim 2 or 39, wherein said non-malignant disease is a genetic disease or disorder, an autoimmune disease or a metabolic er.

75. The use of claim 2 or 39, wherein said non-malignant disease is selected from the group consisting of sickle cells e, a congenital neutropenia, a thrombocytopenia, an aplastic anemia, a myelodysplastic syndrome, a monosomy 7, an osteopetrosis, a Gaucher’s e, a Hurler's disease, a metachromatic leukodystrophy, an adrenal ystrophy, a thalassemia, a congenital or genetically-detennined hematopoietic abnormality, lupus, autoimmune hepatitis, celiac disease, type I diabetes mellitus, Grave's disease, Guillain-Barr syndrome, Myasthenia , Rheumatoid arthritis, scleroderma and psoriasis.

76. The use of any one of claims 1 to 75, substantially as herein described with reference to any one of the Examples and/or